**Multifunctional Application of Biopolymers and Biomaterials**

Editors

**Swarup Roy Valentina Siracusa**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Editors* Swarup Roy Food Technology and Nutrition Lovely Professional University Phagwara India

Valentina Siracusa Chemical Science University of Catania Catania Italy

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *International Journal of Molecular Sciences* (ISSN 1422-0067) (available at: www.mdpi.com/journal/ ijms/special\_issues/mul\_biopoly\_biomat).

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© 2023 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications.

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## **Contents**


Recent Advances of Chitosan Formulations in Biomedical Applications Reprinted from: *Int. J. Mol. Sci.* **2022**, *23*, 10975, doi:10.3390/ijms231810975 . . . . . . . . . . . . . **139**


## **About the Editors**

## **Swarup Roy**

Dr. Swarup Roy is currently working as an Assistant Professor in the Department of Food Technlogy and Nutrition, Lovely Professional University. He previously worked as an Assistant Professor at the School of Bioengineering and Food Technology, Faculty of Applied Sciences and Biotechnology, Shoolini University, Solan, India. He has completed his Ph.D. in Biochemistry (2016) from the University of Kalyani, West Bengal. He holds more than five years of postdoctoral research experience from various institutes Kyung Hee University (3 years) and Inha University (1 year), Republic of Korea, and Indian Institute of Technology Indore (1.5 years). His academic contributions include several international scientific papers, reviews, book chapters, published by ACS, Elsevier, Springer, Taylor & Francis, RSC, Wiley, etc. He has published a total of 111 SCI publications, 2 book chapters, filed 2 patents, and 30 other papers with 4700 citations, 40 h-index, and 83 i10-index (Google Scholar). At present, Dr. Roy is involved in research work based on the fabrication of blend biopolymers-based food packaging films/coatings. His research work is focused on the preparation and application of biopolymer-based functional active and smart food packaging film and coatings for food preservation applications.

### **Valentina Siracusa**

Siracusa Valentina received her degree in Industrial Chemistry from the University of Catania (Italy) at 23 years old. She completed her PhD and post-PhD study working on the synthesis and characterization of innovative polyesters used in the engineering field. After a period as a lecturer for "Chemistry and Materials" for Engineering, from 2006, she has been an Associate Professor in Chemistry for Engineering at University of Catania (Italy) and Invited Professor on "Life Cycle Assessment Study (LCA)" courses at University of Bologna (Italy). She collaborates on several research projects, both for academic than industrial interest, on topic such as recycle, ambient, food packaging, graphene for packaging application, nanoparticles, and polymer drug delivery. She collaborates with national and international research groups on biopolymers used in the field of food packaging, for modified atmosphere packaging of fresh foods, with also Life Cycle Assessment study (with SimaPro software). She is author of more than 110 papers in high-impact-factor scientific journals, she is author of several book chapters for Wiley, Springer, Elsevier, she is author of articles for special Module of Elsevier Encyclopedia and she is Editorial Board Member and Lead Guest Editor of several International Journals. Her research interest includes: synthesis and full characterization of biodegradable and bio-based polymers; gas barrier behavior in standard as well as in moisture condition, at different temperature; Life Cycle Assessment (LCA) study of polymers; thermal and photo degradation behavior of packaging materials analyzed during food shelf-life study.

## *Editorial* **Multifunctional Application of Biopolymers and Biomaterials**

**Swarup Roy 1,\* and Valentina Siracusa <sup>2</sup>**


Biopolymers and biomaterials are two interconnected key topics, which have recently drawn significant attention from researchers across all fields, owing to the emerging potential in multifunctional use. Biopolymers are commonly used to fabricate many biomaterials and, moreover, the combination of biomaterials and biopolymers can produce a new generation of materials for versatile applications. Biopolymers are a topic of interest nowadays, not only due to their potential applications in food, pharmaceuticals, textiles, medicals and other sectors but also to address the challenges of upward environmental pollution [1]. The unorganized use of readily available and cost-effective synthetic plastic has already caused severe damage to the environment and, now, these plastics are becoming a serious threat to all living beings on the planet [2,3]. The tremendous use of synthetic plastic in all daily used items is becoming a serious threat to us and, thus, there is a need for an instant alternative, and, in this context, bio-based sustainable and degradable polymers have high potential to replace the petrochemical-derived synthetic polymers. There are various biopolymers, such as protein, polysaccharides or their combinations, that are commonly used to develop bioplastics. Biopolymer-based polymers have comparable properties like their synthetic counterparts [4,5]. The addition of functional materials, such as nanomaterials, essential oils, phytochemicals, bioactive components, etc., helps in further improvements in both physical and functional properties of the biopolymer-based materials [6–8]. Recent research has shown that biopolymer-based functional packaging (active and intelligent packaging) film and coatings have good potential to improve the life span of packed food items [9,10]. Moreover, biopolymers have also been used as hydrogel and dressing materials to treat wounds in the biomedical sector [11,12]. Even biomaterials are commonly used to fabricate medical devices [13].

Biomaterials include, but are not limited to, synthetic or natural polymers, ceramics and composites, which can interact with biological matter. Recently, biomaterials have been increasingly used and, day by day, they are substituting the conventional polymeric materials in agriculture, textile, medical, food, cosmetics and pharmaceutical sectors [14–17]. The medical sector is the most used market for biomaterials [18]. Nowadays, in the pharmaceutical and textile sectors, biomaterials are also readily available. Many biopolymer-based biomaterials are used as micro- and nanofibers in the textile sector [19]. Biomaterials are frequently used in drug delivery systems in the pharmaceutical industry. Moreover, there is growing public concern for the environment and health; indeed, many folds replicate the use of biomaterials in various sectors. Even though biomaterials and biopolymers are emerging very rapidly, as already discussed, more research is still needed for further development in this field. Nevertheless, the complete conversion of conventional materials to biomaterials is expected to take more time.

Biomaterials and biopolymers are promising for making sustainable materials, but there are many concerns, which need more attention before further progress. The cost of these materials is higher than the traditionally used ones, which restricts the use in many sectors. More in-depth knowledge about biopolymers and biomaterials is required to

**Citation:** Roy, S.; Siracusa, V. Multifunctional Application of Biopolymers and Biomaterials. *Int. J. Mol. Sci.* **2023**, *24*, 10372. https:// doi.org/10.3390/ijms241210372

Received: 12 June 2023 Accepted: 19 June 2023 Published: 20 June 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

produce futuristic materials. Furthermore, there is a need for improvements in the physical properties of the biomaterials to meet the requirement in industrial-level applications. The ongoing and future research on this topic is anticipated to help in developing more sustainable materials.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


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## *Article* **Oxygen Nanocarriers for Improving Cardioplegic Solution Performance: Physico-Chemical Characterization**

**Maria Tannous 1,2,3 , Gjylije Hoti <sup>1</sup> , Francesco Trotta 1,\*, Roberta Cavalli <sup>2</sup> , Takanobu Higashiyama <sup>4</sup> , Pasquale Pagliaro <sup>3</sup> and Claudia Penna <sup>3</sup>**


**Abstract:** Nanocarriers for oxygen delivery have been the focus of extensive research to ameliorate the therapeutic effects of current anti-cancer treatments and in the organ transplant field. In the latter application, the use of oxygenated cardioplegic solution (CS) during cardiac arrest is certainly beneficial, and fully oxygenated crystalloid solutions may be excellent means of myocardial protection, albeit for a limited time. Therefore, to overcome this drawback, oxygenated nanosponges (NSs) that can store and slowly release oxygen over a controlled period have been chosen as nanocarriers to enhance the functionality of cardioplegic solutions. Different components can be used to prepare nanocarrier formulations for saturated oxygen delivery, and these include native α-cyclodextrin (αCD), αcyclodextrin-based nanosponges (αCD-NSs), native cyclic nigerosyl-nigerose (CNN), and cyclic nigerosyl-nigerose-based nanosponges (CNN-NSs). Oxygen release kinetics varied depending on the nanocarrier used, demonstrating higher oxygen release after 24 h for NSs than the native αCD and CNN. CNN-NSs presented the highest oxygen concentration (8.57 mg/L) in the National Institutes of Health (NIH) CS recorded at 37 ◦C for 12 h. The NSs retained more oxygen at 1.30 g/L than 0.13 g/L. These nanocarriers have considerable versatility and the ability to store oxygen and prolong the amount of time that the heart remains in hypothermic CS. The physicochemical characterization presents a promising oxygen-carrier formulation that can prolong the release of oxygen at low temperatures. This can make the nanocarriers suitable for the storage of hearts during the explant and transport procedure.

**Keywords:** oxygen delivery; cardioplegic solution; α-cyclodextrin; cyclic nigerosyl-nigerose; nanosponges; prolonged release; hypothermia; organ transplantation; organ explantation

## **1. Introduction**

The safe capture, storage, and adsorption of large quantities of gas are some of the major technological and scientific challenges facing scientists today. Gas storage is a topic that affects several research fields, as well as climate change, energy production, and medical applications. In particular, oxygen assumes a great therapeutic value in the treatment of solid tumors. The latter is characterized by a highly hypoxic environment that greatly limits current therapies such as chemotherapy, radiotherapy, photodynamic therapy, and even immunotherapy. Different approaches are used to increase the oxygen concentration in tumor tissues. In particular, perfluorocarbons capable of solubilizing large quantities of oxygen are used. However, they cause severe inflammation, release oxygen in an uncontrolled manner, and, above all, are immiscible in both hydrophilic and hydrophobic environments. For these reasons, it is essential to find materials capable of transporting

**Citation:** Tannous, M.; Hoti, G.; Trotta, F.; Cavalli, R.; Higashiyama, T.; Pagliaro, P.; Penna, C. Oxygen Nanocarriers for Improving Cardioplegic Solution Performance: Physico-Chemical Characterization. *Int. J. Mol. Sci.* **2023**, *24*, 10073. https:// doi.org/10.3390/ijms241210073

Academic Editors: Valentina Siracusa and Swarup Roy

Received: 10 May 2023 Revised: 8 June 2023 Accepted: 8 June 2023 Published: 13 June 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

oxygen and releasing it in a controlled way that is biocompatible, non-toxic, and easily synthesized and therefore easily transferable to industrial production [1–4]. Cyclodextrins (CDs) are natural cyclic oligosaccharides with a lipophilic central cavity and a hydrophilic outer surface consisting of (α-1,4-)-linked α-D-glucopyranose units. They contain six (αCD), seven (βCD), and eight (γCD) D-glucose units. The presence of numerous reactive hydroxyl groups on CDs enables their chemical modification, using a wide variety of bi- or polyfunctional chemicals and thus tailoring their structure. Consequently, water-soluble and insoluble cyclodextrin-based polymers are produced. The insoluble cyclodextrin-based polymers or cyclodextrin-based nanosponges (CD-based NSs) are chemically cross-linked polymers obtained by reacting the parent CDs with an appropriate cross-linking agent such as dianhydrides, active carbonyl compounds, carboxylic acids, epoxides, diisocyanates, etc. CD-based NSs are three-dimensional polymer networks with remarkable adsorption properties due to their extensive nanometer-sized porosity. The most outstanding feature of CD-based NSs is their capability to form inclusion complexes with a wide range of liquid, solid, and gaseous compounds through a molecular complexation. CD-based NSs have shown more benefits than CDs to improve the stability, release, and bioavailability of complex guest molecules. This is because the CDs' cross-linking agent molar ratio affects the nanochannels produced [5–11].

Various parameters, such as chemical composition, pore size, and thus surface area and pore volume, can be effectively modulated, even in theoretical simulation models, to optimize the gas adsorption capacity of nanosponges (NSs).

The ability of CD-based NSs to bind compounds reversibly, even in the gas phase, does not require high pressures and low temperatures and thus avoids the hazards of handling high-pressure compressed gases and the technological constraints associated with the low temperatures of liquefied gas. This is possible via physical absorption mechanisms, which may lead to the development of a new technology for the efficient storage of significant amounts of gas in strikingly small volumes [12,13].

CD-based NSs, particularly those prepared based on alpha cyclodextrins (αCD), have proven themselves to be highly suitable for the storage of gases. The ring that makes up αCD resembles a conical cylinder, or truncated cone, in the shape of a crown and includes six glucopyranose units that form a cavity, which is lined by hydrogen atoms that form glycosidic oxygen bridges. The non-bonding electron pairs of these oxygen glycosidic bridges point towards the internal part of the cavity, yielding high electron density and providing it with some basic Lewis properties. Its internally hydrophobic and externally hydrophilic nature means that αCD is principally used for stabilizing reactive intermediates and trapping small molecules (Figure 1) [14,15]. *Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 3 of 18

**Figure 1.** Chemical structures of (**a**) cyclic nigerosyl-1,6-nigerose (CNN) and (**b**) α-cyclodextrin **Figure 1.** Chemical structures of (**a**) cyclic nigerosyl-1,6-nigerose (CNN) and (**b**) α-cyclodextrin (αCD).

(αCD). Numerous potential applications for sugar-based NSs have been investigated in several technological fields, including pharmaceuticals, cosmetics, catalysis, gas storage, On the other hand, cyclic nigerosyl-1,6-nigerose (CNN), also known as cyclo tetra glucose, is also worth considering for gas storage. It is naturally found in sake (the sediment formed during rice-wine production) and *Saccharomyces cerevisiae* cells. Indeed, CNN is a

agriculture, and polymer additives. One of the most promising areas of investigation is the application of sugar-based NSs as novel drug-delivery systems. The use of oxygenated cardioplegic solution (CS) during cardiac arrest may be beneficial. Indeed, cold, fully

However, the forced oxygenation of a cardioplegic solution, while maximizing its oxygen content, may result in it becoming highly alkaline. The equilibration of a bicarbonatecontaining solution with 100 percent oxygen decreases the amount of dissolved carbon

Altering the pH of a CS can undoubtedly affect the functional recovery of an explanted organ. Furthermore, the concentration of hydrogen ions alters with cooling, as the dissociation constant of any salt solution is a function of temperature. Therefore, when introducing a gas into a cardioplegic solution, it is necessary to control both the temperature of the gas and the temperature at which the solution is delivered, as they

Oxygenated nanosponges that can store and slowly release oxygen over a controlled time have been chosen as nanocarriers to enhance the functionality of cardioplegic solutions for this purpose. Different formulations are used to prepare nanocarriers for oxygen delivery, and these include native αCD, α-cyclodextrin nanosponges (αCD-NSs), native CNN, and cyclic nigerosyl-nigerose nanosponges (CNN-NSs), among others, which have proven to be innovative tools for controlled and prolonged oxygen delivery. The addition of nanocarriers with desirable versatility to cold (4 °C) cardioplegic solution, which can be sterilized to reduce the risk of infections, can be naturally decomposed to release more oxygen, thereby improving the functional recovery and/or prolong the amount of time that the heart stays in static hypothermic cardioplegic solution (so-called "static cold storage", SCS). Thus, it can be theoretically possible to extend the time in hypothermia and to use organs explanted in facilities far away from the recipient's

interact to determine the final pH and concentration of the gas [18,19].

location, thus exceeding the 4–5 h that have canonically been tolerated so far [20].

temperature can be considered the sub-normothermic state [21].

oxygen into the cardioplegic solution for a more prolonged period.

Because temperature greatly influences the solubility of a gas in a liquid, the ability of oxygen-loaded formulations to store and slowly release oxygen over time was evaluated at 4 °C, at room temperature (RT, 23 °C), and 37 °C. The chosen temperatures were the ones utilized in laboratory and clinical practice. Specifically, 4 °C mimics the hypothermic situation in which organ metabolism is low (i.e., SCS), while room

This investigation aimed to compare two diverse biocompatible nanodevices (CNN-NSs and αCD-NSs) as oxygen reservoirs. This study displays for the first time the ability of synthesized nanosponges (CNN-NSs and αCD-NSs) to encapsulate, store, and release

dioxide and consequently that of carbonic acid [17].

natural, novel, non-reducing carbohydrate in which four glucopyranose units are bound by alternating α-1,6 and α-1,3 glycosidic linkages.

Numerous potential applications for sugar-based NSs have been investigated in several technological fields, including pharmaceuticals, cosmetics, catalysis, gas storage, agriculture, and polymer additives. One of the most promising areas of investigation is the application of sugar-based NSs as novel drug-delivery systems. The use of oxygenated cardioplegic solution (CS) during cardiac arrest may be beneficial. Indeed, cold, fully oxygenated crystalloid solutions can be an excellent means of myocardial protection [16]. However, the forced oxygenation of a cardioplegic solution, while maximizing its oxygen content, may result in it becoming highly alkaline. The equilibration of a bicarbonatecontaining solution with 100 percent oxygen decreases the amount of dissolved carbon dioxide and consequently that of carbonic acid [17].

Altering the pH of a CS can undoubtedly affect the functional recovery of an explanted organ. Furthermore, the concentration of hydrogen ions alters with cooling, as the dissociation constant of any salt solution is a function of temperature. Therefore, when introducing a gas into a cardioplegic solution, it is necessary to control both the temperature of the gas and the temperature at which the solution is delivered, as they interact to determine the final pH and concentration of the gas [18,19].

Oxygenated nanosponges that can store and slowly release oxygen over a controlled time have been chosen as nanocarriers to enhance the functionality of cardioplegic solutions for this purpose. Different formulations are used to prepare nanocarriers for oxygen delivery, and these include native αCD, α-cyclodextrin nanosponges (αCD-NSs), native CNN, and cyclic nigerosyl-nigerose nanosponges (CNN-NSs), among others, which have proven to be innovative tools for controlled and prolonged oxygen delivery.

The addition of nanocarriers with desirable versatility to cold (4 ◦C) cardioplegic solution, which can be sterilized to reduce the risk of infections, can be naturally decomposed to release more oxygen, thereby improving the functional recovery and/or prolong the amount of time that the heart stays in static hypothermic cardioplegic solution (so-called "static cold storage", SCS). Thus, it can be theoretically possible to extend the time in hypothermia and to use organs explanted in facilities far away from the recipient's location, thus exceeding the 4–5 h that have canonically been tolerated so far [20].

Because temperature greatly influences the solubility of a gas in a liquid, the ability of oxygen-loaded formulations to store and slowly release oxygen over time was evaluated at 4 ◦C, at room temperature (RT, 23 ◦C), and 37 ◦C. The chosen temperatures were the ones utilized in laboratory and clinical practice. Specifically, 4 ◦C mimics the hypothermic situation in which organ metabolism is low (i.e., SCS), while room temperature can be considered the sub-normothermic state [21].

This investigation aimed to compare two diverse biocompatible nanodevices (CNN-NSs and αCD-NSs) as oxygen reservoirs. This study displays for the first time the ability of synthesized nanosponges (CNN-NSs and αCD-NSs) to encapsulate, store, and release oxygen into the cardioplegic solution for a more prolonged period.

These naturally proposed nanocarriers can be sterilized, infused into the organ before explantation, and added to the cardioplegic solution. CNN-NSs presented as a more controlled oxygen release than CD-NSs. These findings will serve such a perspective for the further investigation of indicated nanodevices with pre-clinical studies and thereafter clinically as cardioprotective agents.

#### **2. Results**

The oxygenation of a cardioplegic solution (CS) can likely improve explanted heart vitality and functions.

Several strategies have been studied to increase the concentration of CS oxygen from red blood cells to artificial oxygen carriers, such as hemoglobin-based oxygen carriers, extracellular vesicles, per-fluorocarbon emulsions, and nanoparticulate systems.

In the present work, two glucose derivatives, αCD and CNN, and two cross-linked polymers between them that form nanoparticles called nanosponges (NS) have been investigated in an assessment of their oxygen-carrier capacity in the National Institutes of Health (NIH) CS. Both αCD and CNN are cyclic oligosaccharides with a central cavity (see Figure 1) that can encapsulate molecules, including gases, which can be entrapped and stored in the α-CD and CNN cavities and the nanoporous matrices of αCD and CNN nanosponges, as previously demonstrated. Oxygen-loaded αCD- and CNN-based nanosponges have recently been demonstrated to protect against hypoxia/reperfusion (H/R)-induced cell death in cell experiments [22,23].

In this work, the effect of the addition of oxygenated αCD, CNN, αCD-NS, and CNN-NS on the oxygenation of NIH CS (National Institutes of Health Cardioplegic Solution) was evaluated at different temperatures and concentrations to increase and prolong the oxygen content over time.

First, the αCD- and CNN-based nanosponges were successfully synthesized according to a previously tuned procedure, and their stability in NIH CS was evaluated.

Four oxygenated formulations were then prepared with the addition of αCD, CNN, αCD-NS, and CNN-NS to NIH CS and loaded with oxygen. The formulations were characterized, and their physicochemical parameters (Table 1) were determined.

**Table 1.** Physicochemical characterization of NIH CS with αCD and CNN loaded with oxygen.


The basic pH value of the cardioplegic solution is due to the presence of bicarbonate in its composition. The presence of the two compounds slightly decreased the pH of NIH CS. Interestingly, the addition of O<sup>2</sup> maintained the basic pH value. The viscosity increased only in the presence of CNN oligosaccharide, but the solution is still suitable for organ perfusion.

Table 2 reports the physicochemical characteristics of NIH CS in the presence of the two types of oxygen-loaded nanosponges. The two formulations are nanosuspensions, which, from the physical point of view, are αCD- and CNN-based nanosponges and consequently solid water-insoluble nanoparticles.

**Table 2.** Physicochemical characteristics of NIH CS with the two types of oxygen-loaded NSs.


Both the oxygen-loaded CNN-NSs and αCD-NSs were around 500 nm in size and had a negative surface charge, with zeta potential values high enough to avoid aggregation phenomena in NIH CS.

The sizes and surface charges of the two NSs formulations in NIH CS are similar to those measured in water. Only the PDI showed a slight increase, and this can be correlated to the salts present in the solution.

The addition of oxygen-loaded NSs did not increase the pH value, as the oxygen is stored in the nanocarriers and not immediately available in the solution. This behavior can overcome the limitations of the formation of more alkaline solutions because of the low buffering capacity of the bicarbonate concentration present.

The viscosity-value increase in NIH CS was negligible in the presence of αCD-NSs, while it reached a value of 1.56 mPAs with the addition of CNN-NSs. Nevertheless, the two values are suitable for the potential clinical application of the formulations.

The production of Reactive Oxygen Species (ROS), which can induce cell damage and apoptosis, is a limitation in oxygen delivery. The amount of ROS in oxygenated NIH CS formulations was determined and compared with formulations that are not saturated with oxygen. The results are reported in Table 3. Interestingly, the addition of αCD and αCD-NSs did not significantly increase the ROS concentration compared to the value of NIH CS.


**Table 3.** Reactive Oxygen Species (ROS) concentration in the oxygenated NIH CS formulations.

Because temperature remarkably influences the solubility of a gas in a liquid, the capability of the oxygen-loaded formulations to store and slowly release oxygen over time was evaluated at 4 ◦C, at room temperature (RT, 23 ◦C), and 37 ◦C. *Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 6 of 18

> Figure 2 reports the oxygen profiles of gas diffusion from the oxygenated NIH CS at three different temperatures for up to 24 h.

**Figure 2.** Oxygen diffusion from NIH-oxygen-saturated cardioplegic solutions at different **Figure 2.** Oxygen diffusion from NIH-oxygen-saturated cardioplegic solutions at different temperatures.

temperatures. Slight differences in oxygen content were observed between room temperature and Slight differences in oxygen content were observed between room temperature and 37 ◦C (Table 4) after the first hours.

Indeed, as the temperature increases (4 °C, room temperature (RT), and 37 °C), the

gas to expand [24,25]. Moreover, the presence of salts in the NIH CS can play a crucial role

**Table 4.** The concentration of oxygen in oxygenated NIH CS was recorded after 30 min at different

**Cardioplegic Solution 4 °C RT 37 °C** 

Henry's law states that "at a constant temperature, the amount of a given gas that dissolves in a given type and volume of liquid is directly proportional to the partial

The oxygen concentration in NIH CS depends on Henry's law and, after 24 h, the

The oxygen-saturated NIH CS was considered as a reference in testing the ability of the oxygen-carrier-containing formulations to store and prolong the release kinetics of

Figure 3 reports the kinetics of oxygen release from oxygenated NIH CS that contained CNN, which was added at a concentration of 1.3 g/L, at different temperatures. The graph also confirms the effect of temperature on the amount of dissolved oxygen in this NIH CS formulation, although the presence of CNN markedly affects the amount of dissolved oxygen in the CS solution and produced sustained release over time.

(mg/L) 29.60 ± 3.01 20.38 ± 2.50 18.64 ± 2.42

37 °C (Table 4) after the first hours.

**Oxygen Concentration in NIH** 

temperatures.

oxygen over time.

in favoring the liberation of gas from solution.

pressure of that gas in equilibrium with that liquid".

amount of oxygen reaches equilibrium with the atmospheric content.

Specifically, the oxygen-carrier capability of CNN is evident at 4 °C.


**Table 4.** The concentration of oxygen in oxygenated NIH CS was recorded after 30 min at different temperatures.

Indeed, as the temperature increases (4 ◦C, room temperature (RT), and 37 ◦C), the solubility of a gas decreases from 29.60 mg/L to 18.64 mg/L because of the tendency of a gas to expand [24,25]. Moreover, the presence of salts in the NIH CS can play a crucial role in favoring the liberation of gas from solution.

Henry's law states that "at a constant temperature, the amount of a given gas that dissolves in a given type and volume of liquid is directly proportional to the partial pressure of that gas in equilibrium with that liquid".

The oxygen concentration in NIH CS depends on Henry's law and, after 24 h, the amount of oxygen reaches equilibrium with the atmospheric content.

The oxygen-saturated NIH CS was considered as a reference in testing the ability of the oxygen-carrier-containing formulations to store and prolong the release kinetics of oxygen over time.

Figure 3 reports the kinetics of oxygen release from oxygenated NIH CS that contained CNN, which was added at a concentration of 1.3 g/L, at different temperatures. The graph also confirms the effect of temperature on the amount of dissolved oxygen in this NIH CS formulation, although the presence of CNN markedly affects the amount of dissolved oxygen in the CS solution and produced sustained release over time. Specifically, the oxygen-carrier capability of CNN is evident at 4 ◦C. *Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 7 of 18

**Figure 3.** In-vitro release kinetics of oxygen from CNN in cardioplegic solution CS at a concentration of 1.3 g/L at different temperatures. **Figure 3.** In-vitro release kinetics of oxygen from CNN in cardioplegic solution CS at a concentration of 1.3 g/L at different temperatures.

The addition of CNN played an important role in sustaining oxygen release for a longer time in the NIH CS solution, as it can encapsulate oxygen in the inner cavity of the The addition of CNN played an important role in sustaining oxygen release for a longer time in the NIH CS solution, as it can encapsulate oxygen in the inner cavity of the molecule.

molecule. The prolonged oxygen release is demonstrated by the greater O2 concentration at 4 °C, which can be compared to the marked gas-concentration drop in NIH CS after 6 h. Moreover, the oxygen concentration is still higher after 24 h. This behavior may be The prolonged oxygen release is demonstrated by the greater O<sup>2</sup> concentration at 4 ◦C, which can be compared to the marked gas-concentration drop in NIH CS after 6 h. Moreover, the oxygen concentration is still higher after 24 h. This behavior may be promising for the enhancement of heart storage before transplant in static cold storage [17].

promising for the enhancement of heart storage before transplant in static cold storage [17]. Interestingly, the capability of CNN to sustain oxygen release was enhanced with the Interestingly, the capability of CNN to sustain oxygen release was enhanced with the addition of CNN-NSs in NIH CS.

The benefits that the CNN-NSs display over the CNN oligosaccharide in the NIH CS

at least over the first 6 h of the recorded oxygen release. This is in contrast to the quick decrease in oxygen concentration in the CNN-containing cardioplegic solution. The entrapment of oxygen is also evident at 37 °C (Figure 4), and this behavior underscores the crucial role played by the polymer network in the matrix and by the CNN cavities, as

addition of CNN-NSs in NIH CS.

reported in Figure 5.

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NSs at a concentration of 1.3 g/L at different temperatures.

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**Figure 4.** The kinetics of in-vitro oxygen release from saturated cardioplegic solution with CNN-

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The benefits that the CNN-NSs display over the CNN oligosaccharide in the NIH CS are underlined by a slower and more controlled release achieved using the nanosponges, at least over the first 6 h of the recorded oxygen release. This is in contrast to the quick decrease in oxygen concentration in the CNN-containing cardioplegic solution. The entrapment of oxygen is also evident at 37 ◦C (Figure 4), and this behavior underscores the crucial role played by the polymer network in the matrix and by the CNN cavities, as reported in Figure 5. are underlined by a slower and more controlled release achieved using the nanosponges, at least over the first 6 h of the recorded oxygen release. This is in contrast to the quick decrease in oxygen concentration in the CNN-containing cardioplegic solution. The entrapment of oxygen is also evident at 37 °C (Figure 4), and this behavior underscores the crucial role played by the polymer network in the matrix and by the CNN cavities, as reported in Figure 5.

*Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 7 of 18

**0**

molecule.

[17].

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of 1.3 g/L at different temperatures.

addition of CNN-NSs in NIH CS.

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**Figure 3.** In-vitro release kinetics of oxygen from CNN in cardioplegic solution CS at a concentration

The addition of CNN played an important role in sustaining oxygen release for a longer time in the NIH CS solution, as it can encapsulate oxygen in the inner cavity of the

The prolonged oxygen release is demonstrated by the greater O2 concentration at 4 °C, which can be compared to the marked gas-concentration drop in NIH CS after 6 h. Moreover, the oxygen concentration is still higher after 24 h. This behavior may be promising for the enhancement of heart storage before transplant in static cold storage

Interestingly, the capability of CNN to sustain oxygen release was enhanced with the

The benefits that the CNN-NSs display over the CNN oligosaccharide in the NIH CS

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**Figure 4.** The kinetics of in-vitro oxygen release from saturated cardioplegic solution with CNN-**Figure 4.** The kinetics of in-vitro oxygen release from saturated cardioplegic solution with CNN-NSs at a concentration of 1.3 g/L at different temperatures.

NSs at a concentration of 1.3 g/L at different temperatures.

**Figure 5.** Comparison of the kinetics of the in-vitro release of oxygen from CNN and CNN-NSs in CS at the same concentration at room temperature. **Figure 5.** Comparison of the kinetics of the in-vitro release of oxygen from CNN and CNN-NSs in CS at the same concentration at room temperature.

The capability of αCD to act as an oxygen nano reservoir is also demonstrated. The capability of αCD to act as an oxygen nano reservoir is also demonstrated.

The kinetics of oxygen release from αCD in CS at different temperatures is shown in Figure 6. The kinetics of oxygen release from αCD in CS at different temperatures is shown in Figure 6.

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concentration of 1.3 g/L at different temperatures.

nanoformulations, as presented in Figure 7.

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**Figure 6.** The kinetics of in-vitro oxygen release from saturated CS with αCD oligosaccharide at a

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Similar to the CNN results, the αCD, dissolved at a concentration of 1.3 g/L in NIH CS and loaded with oxygen, increased oxygen concentration in the solution. Moreover, αCD displayed faster release at higher temperatures compared to the αCD-NSs

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**Oxygen released from CNN and CNN\_NS at RT**

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**Figure 5.** Comparison of the kinetics of the in-vitro release of oxygen from CNN and CNN-NSs in

The capability of αCD to act as an oxygen nano reservoir is also demonstrated.

The kinetics of oxygen release from αCD in CS at different temperatures is shown in

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**Figure 6.** The kinetics of in-vitro oxygen release from saturated CS with αCD oligosaccharide at a concentration of 1.3 g/L at different temperatures. **Figure 6.** The kinetics of in-vitro oxygen release from saturated CS with αCD oligosaccharide at a concentration of 1.3 g/L at different temperatures.

Similar to the CNN results, the αCD, dissolved at a concentration of 1.3 g/L in NIH CS and loaded with oxygen, increased oxygen concentration in the solution. Moreover, αCD displayed faster release at higher temperatures compared to the αCD-NSs Similar to the CNN results, the αCD, dissolved at a concentration of 1.3 g/L in NIH CS and loaded with oxygen, increased oxygen concentration in the solution. Moreover, αCD displayed faster release at higher temperatures compared to the αCD-NSs nanoformulations, as presented in Figure 7. *Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 9 of 18

**Figure 7.** The kinetics of in-vitro oxygen release from saturated CS with αCD-NS at a concentration of 1.3 g/L at different temperatures. **Figure 7.** The kinetics of in-vitro oxygen release from saturated CS with αCD-NS at a concentration of 1.3 g/L at different temperatures.

The kinetics of oxygen release from the oxygen-loaded αCD-NSs displays a marked difference compared to results with αCD oligosaccharide. Oxygen encapsulation was greater in the nanosponges due to the presence of the polymer network caused by the presence of a cross-linking agent in the NSs matrix and the cooperation of the αCD cavities The kinetics of oxygen release from the oxygen-loaded αCD-NSs displays a marked difference compared to results with αCD oligosaccharide. Oxygen encapsulation was greater in the nanosponges due to the presence of the polymer network caused by the presence of a cross-linking agent in the NSs matrix and the cooperation of the αCD cavities (Figure 8), as previously observed with CNN-NSs.

**Figure 8.** Comparison of the in-vitro release kinetics of oxygen from αCD, αCD-NSs, and CS at room

CS released a higher concentration of oxygen over the first six hours (Figure 8). However, 24 h later, the αCD-NSs had a higher oxygen concentration than that of αCD

The results showed that oxygen-release kinetics were slower for CNN in CS than that for αCD since CNN is a tetraglucose that has a smaller cavity and is more polar than αCD, which consists of six glucose units; the smaller cavity of CNN favors oxygen

(Figure 8), as previously observed with CNN-NSs.

temperature.

alone or CS.

encapsulation.

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(Figure 8), as previously observed with CNN-NSs.

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**Figure 7.** The kinetics of in-vitro oxygen release from saturated CS with αCD-NS at a concentration

The kinetics of oxygen release from the oxygen-loaded αCD-NSs displays a marked difference compared to results with αCD oligosaccharide. Oxygen encapsulation was greater in the nanosponges due to the presence of the polymer network caused by the presence of a cross-linking agent in the NSs matrix and the cooperation of the αCD cavities

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**Figure 8.** Comparison of the in-vitro release kinetics of oxygen from αCD, αCD-NSs, and CS at room temperature. **Figure 8.** Comparison of the in-vitro release kinetics of oxygen from αCD, αCD-NSs, and CS at room temperature.

CS released a higher concentration of oxygen over the first six hours (Figure 8). However, 24 h later, the αCD-NSs had a higher oxygen concentration than that of αCD CS released a higher concentration of oxygen over the first six hours (Figure 8). However, 24 h later, the αCD-NSs had a higher oxygen concentration than that of αCD alone or CS.

alone or CS. The results showed that oxygen-release kinetics were slower for CNN in CS than that for αCD since CNN is a tetraglucose that has a smaller cavity and is more polar than αCD, The results showed that oxygen-release kinetics were slower for CNN in CS than that for αCD since CNN is a tetraglucose that has a smaller cavity and is more polar than αCD, which consists of six glucose units; the smaller cavity of CNN favors oxygen encapsulation.

which consists of six glucose units; the smaller cavity of CNN favors oxygen encapsulation. The effect of oxygen-loaded nanosponge concentration on the gas storage and delivery capability was then investigated (Table 5). The NSs retained more oxygen at 1.3 g/L than when diluted to 0.13 g/L and showed no significant increase at a concentration of 13.0 g/L, with this effect being more marked at 37 ◦C.

**Table 5.** Oxygen concentration in NIH CS with increasing nanocarrier concentration, was recorded at 37 ◦C for 12 h.


This behavior suggests that small amounts of nanosponge can be used to obtain a suitable oxygen concentration.

The histograms (Figures 9–13) highlight the oxygen percentages recorded at a certain time over the 24 h release study. CNN-NSs and αCD-NSs are more adequate nanocarriers than the parent CNN and αCD, thus validating the concept of storing oxygen in the cardioplegic solution for at least 12 h after the loading procedure. The tuning of the flux time, concentration, storage conditions, and controlled environment are areas for further study and can strengthen the rationale and support the results.

study and can strengthen the rationale and support the results.

study and can strengthen the rationale and support the results.

of 13.0 g/L, with this effect being more marked at 37 °C.

of 13.0 g/L, with this effect being more marked at 37 °C.

*Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 10 of 18

**The Concentration of Prepared Nanosuspension (g/L)** 

**The Concentration of Prepared Nanosuspension (g/L)** 

suitable oxygen concentration.

suitable oxygen concentration.

different temperatures.

at 37 °C for 12 h.

at 37 °C for 12 h.

**Figure 9.** Histogram showing the variation of oxygen concentration in cardioplegic solution at **Figure 9.** Histogram showing the variation of oxygen concentration in cardioplegic solution at different temperatures. different temperatures.

The effect of oxygen-loaded nanosponge concentration on the gas storage and delivery capability was then investigated (Table 5). The NSs retained more oxygen at 1.3 g/L than when diluted to 0.13 g/L and showed no significant increase at a concentration

The effect of oxygen-loaded nanosponge concentration on the gas storage and delivery capability was then investigated (Table 5). The NSs retained more oxygen at 1.3 g/L than when diluted to 0.13 g/L and showed no significant increase at a concentration

**Table 5.** Oxygen concentration in NIH CS with increasing nanocarrier concentration, was recorded

**Table 5.** Oxygen concentration in NIH CS with increasing nanocarrier concentration, was recorded

This behavior suggests that small amounts of nanosponge can be used to obtain a

The histograms (Figures 9–13) highlight the oxygen percentages recorded at a certain

This behavior suggests that small amounts of nanosponge can be used to obtain a

The histograms (Figures 9–13) highlight the oxygen percentages recorded at a certain

time over the 24 h release study. CNN-NSs and αCD-NSs are more adequate nanocarriers than the parent CNN and αCD, thus validating the concept of storing oxygen in the cardioplegic solution for at least 12 h after the loading procedure. The tuning of the flux time, concentration, storage conditions, and controlled environment are areas for further

time over the 24 h release study. CNN-NSs and αCD-NSs are more adequate nanocarriers than the parent CNN and αCD, thus validating the concept of storing oxygen in the cardioplegic solution for at least 12 h after the loading procedure. The tuning of the flux time, concentration, storage conditions, and controlled environment are areas for further

αCD 5.39 ± 0.31 6.01 ± 0.50 6.12 ± 1.50 CNN 5.63 ± 0.25 5.25 ± 0.32 6.81 ± 1.02 αCD-NSs 6.02 ± 1.03 8.63 ± 1.23 7.38 ± 0.43 CNN-NSs 8.57 ± 1.51 14.52 ± 2.43 11.33 ± 1.73

αCD 5.39 ± 0.31 6.01 ± 0.50 6.12 ± 1.50 CNN 5.63 ± 0.25 5.25 ± 0.32 6.81 ± 1.02 αCD-NSs 6.02 ± 1.03 8.63 ± 1.23 7.38 ± 0.43 CNN-NSs 8.57 ± 1.51 14.52 ± 2.43 11.33 ± 1.73

**The Concentration of Oxygen (mg/L) 0.13 1.30 13.0** 

**The Concentration of Oxygen (mg/L) 0.13 1.30 13.0** 

**Figure 10.** Histogram showing the variation of oxygen concentration in cardioplegic solution + CNN monomer (at a concentration of 1.3 g/L) at different temperatures. monomer (at a concentration of 1.3 g/L) at different temperatures.

**Figure 11.** Histogram showing the variation of oxygen concentration in cardioplegic solution + **Figure 11.** Histogram showing the variation of oxygen concentration in cardioplegic solution + CNN-NSs (at a concentration of 1.3 g/L) at different temperatures.

**Figure 12.** Histogram showing the variation of oxygen concentration in cardioplegic solution + αCD

**Figure 13.** Histogram showing the variation of oxygen concentration in cardioplegic solution + αCD-

monomer (at a concentration of 1.3 g/L) at different temperatures.

NSs (at a concentration of 1.3 g/L) at different temperatures.

CNN-NSs (at a concentration of 1.3 g/L) at different temperatures.

CNN-NSs (at a concentration of 1.3 g/L) at different temperatures.

CNN-NSs (at a concentration of 1.3 g/L) at different temperatures.

**Figure 12.** Histogram showing the variation of oxygen concentration in cardioplegic solution + αCD monomer (at a concentration of 1.3 g/L) at different temperatures. **Figure 12.** Histogram showing the variation of oxygen concentration in cardioplegic solution + αCD monomer (at a concentration of 1.3 g/L) at different temperatures. monomer (at a concentration of 1.3 g/L) at different temperatures.

**Figure 12.** Histogram showing the variation of oxygen concentration in cardioplegic solution + αCD

**Figure 10.** Histogram showing the variation of oxygen concentration in cardioplegic solution + CNN

**Figure 10.** Histogram showing the variation of oxygen concentration in cardioplegic solution + CNN

**Figure 11.** Histogram showing the variation of oxygen concentration in cardioplegic solution +

**Figure 11.** Histogram showing the variation of oxygen concentration in cardioplegic solution +

monomer (at a concentration of 1.3 g/L) at different temperatures.

monomer (at a concentration of 1.3 g/L) at different temperatures.

*Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 11 of 18

**Figure 13.** Histogram showing the variation of oxygen concentration in cardioplegic solution + αCD-NSs (at a concentration of 1.3 g/L) at different temperatures. **Figure 13.** Histogram showing the variation of oxygen concentration in cardioplegic solution + αCD-NSs (at a concentration of 1.3 g/L) at different temperatures.

#### **Figure 13.** Histogram showing the variation of oxygen concentration in cardioplegic solution + αCD-NSs (at a concentration of 1.3 g/L) at different temperatures. **3. Discussion**

Nanocarriers as a tool to improve the delivery and storage of oxygen have been the focus of extensive research in the field of organ transplants. The static cold storage (SCS) of the donor's heart after brain death remains the clinical standard. The main advantage of hypothermia is to "prevent" ischemic damage when the cardioplegic flow is interrupted. This advantage is achieved by a reduction in the temperature-dependent rate of myocardial metabolism, which results in a slowing of the rate of ischemic damage. However, even when drastically reduced by cold cardioplegia, the heart continues to have a minimally oxygen-demanding metabolism [26,27].

The automated machine perfusion (MP) of a donor's heart is being evaluated as an alternative approach to donor-organ management and as a means to expand the donor pool and/or increase the utilization rate. However, technical and biological issues (e.g., machine malfunction, user error, infections, and costs) limit its large-scale use, and further well-designed studies are needed to draw clear conclusions.

Previous studies have demonstrated the ability of αCD-NSs and CNN-NSs to accommodate and convey oxygen, thus addressing growth problems in several diseases, from inflammation to cancer, and improving tumor treatment. The αCD-based formulations can be used for the treatment of cardiovascular diseases as oxygen nanocarriers that can limit ischemic reperfusion injury (IRI) via direct injection into the myocardial wall before starting full-blood reperfusion. Similarly, the proposed CNN-based nanocarriers have shown marked efficacy in controlled oxygenation and have effectively protected cellular models (e.g., cardiomyocytes and endothelial cells) from simulated IRI, thus reducing cell mortality [13,22,23,28,29]. The polymerization reactions lead to a successful synthesis of carbonate NSs. The resulting products were further investigated to understand the

difference between CNN and α-CD as building blocks. When the CNN and α-CD react with CDI, two OH groups are completely esterified, and the reaction results in imidazole formation. The cross-linking process is confirmed via carbonate formation. The formed product is more resistant to hydrolysis than, for example, the synthesized-based NSs based on carboxylic acid esters [8].

In this novel study, two different biocompatible low-temperature oxygen-releasing nanodevices (CNN-NSs and αCD-NSs) that can be easily sterilized, infused into the organ before explantation and added to the cardioplegic solution under SCS conditions, where they release oxygen over a long period, are evaluated. Under these conditions, the organs to be transplanted can be theoretically kept under oxygenated conditions in SCS for a longer time, with the belief that these nanodevices can be first verified experimentally with pre-clinical studies and later clinically as cardioprotective agents.

The two kinds of nanocarriers were capable of playing the relevant roles of oxygen reservoirs in this evaluation of their oxygen-encapsulation capability. The two NSs were compared with their building units, i.e., αCD and CNN. Interestingly, the NSs nanostructure can affect oxygen storage and release, and CNN-NSs have been demonstrated to be a better oxygen reservoir than CD-NSs.

This behavior may be related to the different nanostructures of the two nanocarriers. Compared with CNN, αCD has a much larger and more hydrophobic cavity. CNN has two inward-oriented hydroxyl moieties, so only small molecules can be included within them. Nevertheless, similarly to CD, CNN-NSs have two types of nanopores: the interstitial spaces between the CD units and the internal hydrophobic CD cavities. The spaces between the CD can be more or less hydrophilic, depending on cross-link density and cross-linking agent polarity. Due to a large number of reactive hydroxyl groups, starch derivatives can act as polyfunctional monomers and be cross-linked using a wide array of chemicals (e.g., active carbonyl compounds, diisocyanates, dianhydrides, epoxides, carboxylic acids with two or more functionalities, etc.), thus yielding three-dimensional, insoluble polymers [8], with specific characteristics and capability for molecule encapsulation.

As it is known, the formation of an inclusion complex involves partial or complete coverage of the host molecule. This provides, on the one hand, protection from evaporative degradation and oxidation and allows for the stabilization of the host, while on the other, it alters many of the physicochemical properties of the molecule. These modifications facilitate the use of characterization techniques to verify that the host is indeed contained in the cavity. αCD and CNN may maintain some of their ability to form hydrogen bonds with other molecules, allowing them to stay, at least partially, soluble in water in the form of an inclusion complex [30]. Furthermore, αCD and CNN cross-linking insoluble nanodevices are obtained with increased oxygen encapsulation capacity.

In addition, the storage of oxygen in nanodevices and the subsequent slow and constant delivery avoid the formation of high ROS species that can damage heart cells in the NIH CS. It is a key point to the limitation of free radical species in transplantation to maintain organ function.

Oxygen-release kinetics at low temperatures varied according to the nanocarrier used, suggesting that they may be appropriate and tuned for typical SCS temperatures (4 ◦C). Nevertheless, this aspect requires verification via the use of a transplanted heart and clinical applications.

Unquestionably, either a slight or severe oxygen deficiency can lead to cell death, but a wealth of research suggests that fluctuating oxygen levels rather than persistently low pO2 are the most harmful aspects [31]. The most intriguing aspect of oxygen consumption by the heart is that it can switch from low to high consumption levels in response to metabolic demand. As a result, we can suggest that the intracellular compartment experiences a range of oxygen tensions, from high to low, depending on the balance between supply and demand. A pro-oxidative environment is created by a positive balance. In this context, the changes in oxygen levels (dysoxia) switching from hypothermia to normothermia can be crucial. Indeed, interactions of oxygen with other gases, such as nitric oxide, can either aggravate or protect ischemic organs, depending on several circumstances and including the relative level of the two gases. Additionally, the effects of dysoxia are crucial for iron metabolism. Therefore, only future studies can reveal the real effects of controlled oxygen delivery by nanodevices into transplanted organs that are first stored at low temperatures and then transplanted into a warm body.

#### **4. Materials and Methods**

#### *4.1. Chemicals*

α-Cyclodextrin (α-CD, M<sup>w</sup> = 972.846 g/mol) was a gift from Roquette Frères SA (Lestrem, France). Cyclic nigerosyl-1-6-nigerose (CNN, M<sup>w</sup> = 648.564 g/mol) was obtained from Hayashibara (Tokyo, Japan). CNN and α-CD were desiccated in an oven at 80 ◦C up to constant weight before their usage to remove any traces of absorbed moisture. Additionally, 1,10 -Carbonyldiimidazole (CDI, ≥97.0%), 2<sup>0</sup> ,70 -dichlorodihydrofluorescein diacetate (H2DCFDA fluorescent probe), and 20 ,70 -dichlorofluorescein (DCF), N,N-dimethylformamide (DMF, ≥99.8%), acetone (≥99% (GC)), and ethanol (96.0–97.2%) were purchased from Sigma-Aldrich (Munich, Germany). Deionized and MilliQ® water was obtained using a Millipore Direct-QTM 5 production system. All other chemicals used to prepare the cardioplegic solution (CS) were commercially available as analytical-grade products.

#### *4.2. CNN-Based Nanosponge Synthesis*

CNN-based nanosponges (CNN-NSs) were successfully synthesized following an existing procedure in literature with minor modifications [22]. The synthesis was performed by dissolving 5.00 g (7.70 mmol) of anhydrous CNN (Figure 1) powder in 30 mL DMF at room temperature in a round bottom flask, using a hotplate stirrer equipped with thermoregulation and heat-on block. Subsequently, 4.99 g (30.70 mmol) of CDI as a cross-linking agent was added, and to observe a clear solution, the temperature was increased up to 80 ◦C until the gel formation. Additionally, the formed gel was kept at 90 ◦C for around 5 h until a solid product was obtained. The stoichiometric molar ratio between CNN and CDI was 1:4. The acquired monolithic block was then broken up and manually ground in a mortar. The product was further purified with an excess of deionized water and recovered using a Buchner filtration system using filter paper (Whatman No. 1, Whatman, Maidstone, UK). The by-products were completely removed through Soxhlet extraction using ethanol for around 24 h. Finally, the CNN-NSs were air-dried, milled, and exploited for characterization as a white homogeneous powder. *Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 14 of 18 increased up to 80 °C until the gel formation. Additionally, the formed gel was kept at 90 °C for around 5 h until a solid product was obtained. The stoichiometric molar ratio between CNN and CDI was 1:4. The acquired monolithic block was then broken up and manually ground in a mortar. The product was further purified with an excess of deionized water and recovered using a Buchner filtration system using filter paper (Whatman No. 1, Whatman, Maidstone, UK). The by-products were completely removed through Soxhlet extraction using ethanol for around 24 h. Finally, the CNN-NSs were airdried, milled, and exploited for characterization as a white homogeneous powder.

#### *4.3. αCD-Based Nanosponge Synthesis 4.3. αCD-Based Nanosponge Synthesis*

*4.4. Preparation of Cardioplegic Solution* 

**Table 6.** Composition of NIH cardioplegic solution.

modification [32].

*\*—*Lidocaine is reported in mg/L.

The same synthesis, purification, and recovery procedures, as previously described for CNN-NSs, were utilized to obtain αCD-based nanosponges (αCD-NSs). Briefly, 5.00 g (5.14 mmol) of αCD and 3.33 g (20.54 mmol) of CDI were dissolved in 30 mL DMF. The stoichiometric molar ratio between α-CD and CDI was 1:4. A schematic representation of the synthesis of αCD-NSs is shown in Figure 14. The same synthesis, purification, and recovery procedures, as previously described for CNN-NSs, were utilized to obtain αCD-based nanosponges (αCD-NSs). Briefly, 5.00 g (5.14 mmol) of αCD and 3.33 g (20.54 mmol) of CDI were dissolved in 30 mL DMF. The stoichiometric molar ratio between α-CD and CDI was 1:4. A schematic representation of the synthesis of αCD-NSs is shown in Figure 14.

**Figure 14.** The schematic representation of the reaction of αCD with CDI at certain conditions. **Figure 14.** The schematic representation of the reaction of αCD with CDI at certain conditions.

The National Institutes of Health (NIH) cardioplegic solution (CS) used throughout

Cardioplegic Protection protocol. The formulation is reported in Table 6 with a slight

**NIH CS Components mmol/L**  Na 98.9 Cl 107.8 K 30.0 Ca 1.0 HCO3 22.0 Glucose 152.6 Mannitol 68.6 Lidocaine, mg/L \* 20.0

#### *4.4. Preparation of Cardioplegic Solution*

The National Institutes of Health (NIH) cardioplegic solution (CS) used throughout our experiments was freshly prepared according to the National Institute of Health for Cardioplegic Protection protocol. The formulation is reported in Table 6 with a slight modification [32].


**Table 6.** Composition of NIH cardioplegic solution.

\*—Lidocaine is reported in mg/L.

#### *4.5. Preparation of Nanoformulations*

The defined amounts of CNN and αCD powders were weighed and dissolved in the prepared NIH cardioplegic solution to obtain solutions with different concentrations. A weighed amount of finely milled NSs powder was first added to the cardioplegic solution and then homogenized with a high-shear mixer (Ultra-Turrax®, Konigswinter, Germany) at 24,000 rpm for 10 min to reduce the size and obtain well-distributed suspensions with a uniform particle size. The aqueous suspension was later subjected to an ultrasonic sonicator for 20 min to achieve homogenization. The obtained nanosuspensions were then purified and stored in the fridge. All nanoformulations were stable after one week at 4 ◦C.

#### *4.6. Characterization of Nanoformulations*

The different NSs formulations were further characterized in vitro to evaluate their size and surface charge. The average diameter and polydispersity index of the formulations were measured using photon correlation spectroscopy (PCS) with a 90 Plus instrument (Brookhaven, NY, USA) at a fixed angle of 90 and a temperature of 25 ◦C after dilution with filtered water. The zeta potential was determined using a 90 Plus instrument (Brookhaven, NY, USA). For zeta-potential determination, diluted samples of nanoformulations were placed in an electrophoretic cell, to which a rounded 15 V/cm electric field was applied. The pH was recorded at room temperature using an Orion 420A pH meter.

#### *4.7. Oxygen-Loading Procedure*

To prepare oxygen-loaded nanoformulations, different quantities of αCD, CNN, αCD-NSs, and CNN-NSs were weighed and placed into a vial with the prepared NIH cardioplegic solution. After shear homogenization and ultrasound sonication, NSs dispersions were transferred into a three-neck round-bottom flask and saturated with an oxygen purge at a flux of 4 L/min under stirring, while the gas concentration was monitored up to 35 mg/L in the external aqueous phase. The stability of the oxygen-encapsulating NSs was evaluated over time at different temperatures by measuring the oxygen concentration inside sealed falcon tubes using the oximeter electrode mounted inside.

#### *4.8. Oxygen-Release Profile*

The dissolved oxygen (DO) concentration was recorded using an oxygen bench meter (HI5421, Hanna Instruments Inc., Woonsocket, RI, USA). The bench meter is supplied with a probe for laboratory use and a built-in temperature sensor (HI76483 Hanna Instruments Inc. Woonsocket, RI, USA) was inserted inside a sealed falcon tube. This allowed the

measurements of DO to be carried out in a closed system and thus avoided the interferences from the oxygen in the environment. A fixed volume of 45 mL of oxygen-loaded NIH CS solution/nanosuspension was purged with oxygen flux for 30 min. After a predetermined incubation and equilibration time, the cap of the falcon tube was removed and replaced with the electrode for DO measurement. The oxygen concentration was logged every 30 min for 24 h. To minimize fluctuations in the readings caused by environmental and instrumentation factors, both a thermostat and a cryo-compact circulator (JULABO GmbH, Seelbach, Germany) were used to regulate the temperature (4–37 ◦C) of the medium in the falcon containing the solution and the probe. All measurements were carried out in triplicate under determined conditions.

#### *4.9. Determination of Reactive Oxygen Species (ROS)*

The fluorescence method was used, based on the oxidation of the H2DCFDA fluorescent probe (20 ,70 -dichlorodihydrofluorescein diacetate), for the determination of the oxygen free radicals present in the cardioplegic-solution samples.

For this purpose, a 2 mL ethanolic solution of H2DCFDA (10 mM) was added to 0.5 mL of 0.01 M NaOH to hydrolyze H2DCFDA into DCFH (non-fluorescent compound). The hydrolysis product was maintained at room temperature for 30 min and neutralized by adding 10 mL of PBS (50 mM, pH 7.2). The presence of ROS in the DCFH compound is rapidly oxidized to 20 ,70 -dichlorofluorescein (DCF).

The green fluorescence color of DCF was measured using a fluorimeter (EnSightTM automated multimode plate reader, PerkinElmer, Inc., Waltham, MA 02451, USA) with the excitation wavelength set at 485 nm and emission at 530 nm. The concentration of ROS was determined using a calibration curve built by analyzing a series of standard solutions of DCF in the 0.001–0.500 µM concentration range. Standard solutions were prepared by diluting a stock solution of DCF (500 µM) with phosphate buffer saline solution (PBS).

A linear calibration curve was obtained in the concentration range between 0.001 and 0.500 µM, with an R<sup>2</sup> value of 0.998.

#### *4.10. Quantitative Determination of ROS in Blank/Oxygen-Loaded Samples*

The safety profiles of the αCD-NSs and CNN-NSs have been thoroughly investigated as candidates for several pharmaceutical applications [33–36]. Their cytotoxicity on anaplastic thyroid cancer cells and internalized in the A2780 cell line have been evaluated in previous studies [28,37].

For the determination of ROS, the blank cardioplegic solution CS, the prepared solutions, and the nanosuspensions with the αCD, CNN, αCD-NSs, and CNN-NSs (concentration 10 mg/mL) were loaded with oxygen for 30 min before being tested. In separate vials, 2 mL of each sample was pipetted, and 25 µL of the prepared DCFH was added to reach a final concentration of 5 µM. The samples were left at room temperature and protected from light for 20 min to achieve reaction completion. The fluorescence intensity was then measured with a fluorimeter (excitation 485 nm, emission 530 nm) for 60 min. The analyses were performed in triplicate on each sample.

#### **5. Conclusions**

In this study, two oligosaccharides, i.e., CNN and αCD, and their cross-linked polymers, namely CNN-NSs, and αCD-NSs were extensively studied as artificial oxygen nanocarriers. Oxygen-release kinetics were slower for CNN than for αCD, and the results showed that the polymer network can play a key role in oxygen storage and release. CNN-NSs and αCD-NSs display the ability to encapsulate, store, and release oxygen into the cardioplegic solution for a more prolonged period.

The proposed nanocarriers have considerable versatility; they can be sterilized by their addition to the cardioplegic solution which reduces the risk of infections, and they can be naturally decomposed so that they release oxygen. This leads to potentially prolonging the amount of time that the heart stays in the hypothermic cardioplegic solution. Furthermore, the nanocarriers can be used for distant organ transport, thus exceeding the current canonically tolerated time (around 6 h). These findings are promising for the tuning of an oxygen-carrier formulation that can prolong the storage of the heart during the explantation and transport procedures.

**Author Contributions:** Conceptualization, P.P., C.P., F.T. and R.C.; methodology, M.T. and G.H.; investigation, G.H. and M.T.; writing—original draft preparation, G.H.; writing—review and editing, F.T., C.P. and T.H.; validation, T.H., supervision, F.T. and P.P.; funding acquisition, P.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Fondo Beneficenza Intesa San Paolo, Project ID: B/2021/0159 to P.P.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The authors confirm that the data supporting the findings of this study are available within the article.

**Acknowledgments:** This research acknowledges support from Project CH4.0 under the MIUR program "Dipartimenti di Eccellenza 2023–2027".

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


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## *Article* **Application of Box–Behnken Design to Optimize Phosphate Adsorption Conditions from Water onto Novel Adsorbent CS-ZL/ZrO/Fe3O4: Characterization, Equilibrium, Isotherm, Kinetic, and Desorption Studies**

**Endar Hidayat 1,2 , Nur Maisarah Binti Mohamad Sarbani 1,2, Seiichiro Yonemura 1,2, Yoshiharu Mitoma 1,2 and Hiroyuki Harada 1,2,\***


**Abstract:** Phosphate (PO<sup>4</sup> <sup>3</sup>−) is an essential nutrient in agriculture; however, it is hazardous to the environment if discharged in excess as in wastewater discharge and runoff from agriculture. Moreover, the stability of chitosan under acidic conditions remains a concern. To address these problems, CS-ZL/ZrO/Fe3O<sup>4</sup> was synthesized using a crosslinking method as a novel adsorbent for the removal of phosphate (PO<sup>4</sup> <sup>3</sup>−) from water and to increase the stability of chitosan. The response surface methodology (RSM) with a Box–Behnken design (BBD)-based analysis of variance (ANOVA) was implemented. The ANOVA results clearly showed that the adsorption of PO<sup>4</sup> 3− onto CS-ZL/ZrO/Fe3O<sup>4</sup> was significant (*p* ≤ 0.05), with good mechanical stability. pH, dosage, and time were the three most important factors for the removal of PO<sup>4</sup> <sup>3</sup>−. Freundlich isotherm and pseudo-second-order kinetic models generated the best equivalents for PO<sup>4</sup> <sup>3</sup><sup>−</sup> adsorption. The presence of coexisting ions for PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal was also studied. The results indicated no significant effect on PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal (*<sup>p</sup>* <sup>≤</sup> 0.05). After adsorption, PO<sup>4</sup> <sup>3</sup><sup>−</sup> was easily released by 1 M NaOH, reaching 95.77% and exhibiting a good capability over three cycles. Thus, this concept is effective for increasing the stability of chitosan and is an alternative adsorbent for the removal of PO<sup>4</sup> 3− from water.

**Keywords:** phosphate adsorption; zeolite; chitosan; ZrO; Fe3O<sup>4</sup> ; Box–Behnken design; mechanical stability

#### **1. Introduction**

Phosphate (PO<sup>4</sup> <sup>3</sup>−) is a macronutrient needed for plant growth and is frequently applied as a fertilizer on agricultural lands. The increasing demands of food supply nowadays have led to the excessive application of fertilizer. However, excessive fertilizer use can cause PO<sup>4</sup> <sup>3</sup><sup>−</sup> to leach into waterways, leading to eutrophication and harmful algal bloom. These blooms diminish oxygen levels [1–3], interfere with aquatic life, and adversely affect the quality of drinking water (taste and odor) [4]. According to [5], PO<sup>4</sup> <sup>3</sup><sup>−</sup> decontamination must be performed efficiently while having a minimal impact on the surrounding ecosystem. Many methods have been reported to be effective in removing PO<sup>4</sup> <sup>3</sup><sup>−</sup> from water, including biological [6] methods, electrochemical [7,8] methods, precipitation [9], ion exchange [10], and adsorption [11,12]. Each strategy has advantages and disadvantages. Biological techniques are more economical; however, the residue of dead bacteria left behind after the process is inconvenient [13]. Electrochemical techniques are expensive but have a lower effectivity toward PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal [14]. The precipitation process is simple

**Citation:** Hidayat, E.; Mohamad Sarbani, N.M.B.; Yonemura, S.; Mitoma, Y.; Harada, H. Application of Box–Behnken Design to Optimize Phosphate Adsorption Conditions from Water onto Novel Adsorbent CS-ZL/ZrO/Fe3O4: Characterization, Equilibrium, Isotherm, Kinetic, and Desorption Studies. *Int. J. Mol. Sci.* **2023**, *24*, 9754. https://doi.org/ 10.3390/ijms24119754

Academic Editors: Swarup Roy and Valentina Siracusa

Received: 11 May 2023 Revised: 31 May 2023 Accepted: 1 June 2023 Published: 5 June 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and effective for chemical treatment but is inefficient for sewage sludge and waste disposal [15]. Ion exchange may also be used to remove anions by exchanging sulfates (SO<sup>4</sup> <sup>2</sup>−) for PO<sup>4</sup> <sup>3</sup><sup>−</sup> ions; however, this would make the solution more corrosive, and it requires a costly clean-up (Blaney et al. [16]). Adsorption is the best option and is the most widely used method for water contaminants including PO<sup>4</sup> <sup>3</sup><sup>−</sup> ions [17,18]. This is because the technique is environmentally safe, the operation is easy and fast, and the technology is highly efficient.

Chitosan is currently gaining popularity as a potential adsorbent for water contaminants because it contains hydroxyl (–OH) and amino (–NH2) functional groups, which can easily react with other materials and are environmentally friendly [19]. This material, which cannot be accessed readily from nature, is synthesized through the chemical deacetylation of chitin. However, because of its low tensile strength and dissolution under acidic conditions, the use of chitosan directly in wastewater treatment technologies is not recommended. Therefore, chitosan must be modified to increase its chemical stability and adsorption capability [20]. The selection of an appropriate modification method and modifying agent is crucial for assessing the quality and functionality of the product created during the modification process. Crosslinking is one of the most frequently used procedures to enhance the physicochemical characteristics of chitosan [21,22]. Crosslinking is the process of combining two or more molecules via covalent bonds.

Zeolites are crystalline aluminum silicate (Al2O3·2SiO2) minerals with a porous and highly stable structure, and they could enhance the adsorption of chitosan onto their surface, leading to the improved stability of chitosan. These materials can be obtained from natural sources, such as shrimp, or can be synthesized using various methods [23]. Several reports have proven the use of chitosan and zeolite to remove dyes [24,25], pharmaceuticals [26], nitrate [27], and humic acid [28]. On the other hand, the fabrication of chitosan–metal oxides has attracted the attention of a lot of scientists owing to their numerous beneficial characteristics, such as chemical stability, a large surface area, and favorable adsorptive characteristics [29]. Magnesium oxide (MgO) [30], titanium oxide (TiO) [31], zinc oxide (ZnO) [32,33], zirconium oxide (ZrO) [34], and copper oxide (CuO) [35] are examples of metal oxides. ZrO was selected for this study owing to its strong affinity for anions [36].

The separation of the adsorbents is another issue of concern since the usual separation procedures result in the loss of the adsorbents as well as possible dangers to the environment [37,38]. Magnetite (Fe3O4) is one of the most magnetic particles that can be used in the manufacture of magnetic adsorbents for water purification because of its biodegradability, thermal stability, and large surface area [39,40]. The use of the crosslinking method to combine magnetite, zeolite, ZrO, and chitosan is a viable strategy. This is because the magnetic particles allow for easy separation when subjected to an external magnetic field, while the chitosan, zeolite, and ZrO provide many adsorption sites [41]. Therefore, the amalgamation of chitosan/zeolite/ZrO, and Fe3O<sup>4</sup> (CS-ZL/ZrO/Fe3O4) may result in the development of novel composite materials with multifunctional constituents.

This study synthesized CS-ZL/ZrO/Fe3O<sup>4</sup> with the target of using it as a novel adsorbent for PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal from water. The response surface methodology (RSM) with the Box–Behnken design (BBD) optimization strategy was used to acquire insight into the effect of process factors such as pH, adsorbent dosage, temperature, and time to achieve the maximal adsorptive removal of PO<sup>4</sup> <sup>3</sup>−. This process was performed to obtain the highest PO<sup>4</sup> <sup>3</sup><sup>−</sup> adsorptive removal. The adsorption isotherms and kinetic models were also calculated to figure out the adsorption mechanism.

### **2. Results and Discussion**

## *2.1. Characterization of CS-ZL/ZrO/Fe3O<sup>4</sup>*

The experimental results of BBD are listed in Table 1. It can be concluded that a pH of 2 offers the best conditions for PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal. The pHZPC findings revealed that, at a pH of 2, the surface of CS-ZL/ZrO/Fe3O<sup>4</sup> had a positive charge (pH < pHzpc) (Figure 1a). This might indicate the protonation of the -NH<sup>2</sup> groups to -NH<sup>3</sup> <sup>+</sup> groups on the surface. These attract negatively charged H2PO<sup>4</sup> − ions to CS-ZL/ZrO/Fe3O4, resulting in the construction of a surface complex between PO<sup>4</sup> <sup>3</sup><sup>−</sup> ions and CS-ZL/ZrO/Fe3O4. This study was similar to that reported by [42,43], which used SCBC-La and leftover coal, respectively, for PO<sup>4</sup> 3− removal under acidic conditions. The other possible reaction that could occur is shown in Equation (1).

$$\text{Fe}\_3\text{O}\_4 + 4\text{Zr(OH)}\_4 + 6\text{H}\_2\text{PO}\_4 \longrightarrow \text{FeZr(PO}\_4)\_3 + 12\text{H}\_2\text{O} \tag{1}$$

**Table 1.** Experimental data results from 4 factors of BBD for PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal onto CS-ZL/ZrO/Fe3O<sup>4</sup> .


27 7 0.06 45 35 53.47

57.08, and 62.78 [44]. Figure 1c shows a photograph of CS-ZL/ZrO/Fe3O4. It can be seen

that the adsorbent is attached to the external magnet.

**Figure 1.** (**a**) pHzpc of CS-ZL/ZrO/Fe3O4, (**b**) XRD spectra of CS-ZL/ZrO/Fe3O4, and (**c**) photograph of CS-ZL/ZrO/Fe3O4 (taken by phone). **Figure 1.** (**a**) pHzpc of CS-ZL/ZrO/Fe3O<sup>4</sup> , (**b**) XRD spectra of CS-ZL/ZrO/Fe3O<sup>4</sup> , and (**c**) photograph of CS-ZL/ZrO/Fe3O<sup>4</sup> (taken by phone).

The SEM-EDS characterization of CS-ZL/ZrO/Fe3O4 was carried out to explore the surface properties and chemical components of the material. Figure 2 and Table 3 compare Table 2 summarizes the physical characteristics of these adsorbents. The results show that the BET-specific surface area was 88.1 m2/g, with a pore volume of 0.572 mL/g, an

the SEM images and EDS data before and after PO43− adsorption. Before adsorption, the surface morphology of the adsorbent was sticky, rough, and porous. The surface became

trapped on the adsorbent surface. The primary objective of the EDS data analysis was to identify the components of the adsorbent materials. The weight percentages of Zr and Fe were the highest at 50.68 and 38.92%, respectively. The N value was derived from the chitosan materials [45–47]. Al, Si, and Fe were derived from zeolite and magnetite, respectively. Furthermore, the presence of P after the adsorption process indicates that PO43− was

Figure 3 shows the functional groups in CS-ZL/ZrO/Fe3O4 before and after PO43− adsorption through an FTIR-ATR analysis. A CS-ZL/ZrO/Fe3O4 band was detected following PO43− adsorption from 3326 cm−1 to 3320 cm−1. This shows that PO43− ions interact with the stretching vibrations of hydrogen and amine in chitosan [48]. After PO43− adsorption, a decrease in the peak from 1634 to 1627 cm−1 was observed, which is associated with carboxyl groups (–COOCH3) [49]. An increased peak and a more curved and newer peak appeared after PO43− adsorption from 951 to 1006 cm−1 and at 2161 cm−1, which were assigned to Si-O-Al, Fe-O-Si, or Zr-O-Fe and CN stretching, respectively. This indicated a

successfully adsorbed.

strong interaction with PO43− ions.

average diameter of 43.9 µm, and a porosity of 59%. These parameters show that the adsorbent had a substantial surface area for the adsorption of PO<sup>4</sup> <sup>3</sup><sup>−</sup> ions.

**Table 2.** Physical properties of the adsorbent.


Figure 1b shows the XRD data used to verify the crystalline structure of the composite material. The XRD pattern shows a huge hump around 2θ = 21.22, which is a chitosanspecific peak [20]. Furthermore, the sharp peaks at 30.11, 35.49, 43.21 are mostly composed of crystalline phases, such as quartz, hematite, and alumina, which are all formed from zeolite- and zirconium-based materials. Magnetite corresponds to the peaks at 53.52, 57.08, and 62.78 [44]. Figure 1c shows a photograph of CS-ZL/ZrO/Fe3O4. It can be seen that the adsorbent is attached to the external magnet.

The SEM-EDS characterization of CS-ZL/ZrO/Fe3O<sup>4</sup> was carried out to explore the surface properties and chemical components of the material. Figure 2 and Table 3 compare the SEM images and EDS data before and after PO<sup>4</sup> <sup>3</sup><sup>−</sup> adsorption. Before adsorption, the surface morphology of the adsorbent was sticky, rough, and porous. The surface became smooth and compact after PO<sup>4</sup> <sup>3</sup><sup>−</sup> adsorption, and this indicates that PO<sup>4</sup> <sup>3</sup><sup>−</sup> ions were trapped on the adsorbent surface. The primary objective of the EDS data analysis was to identify the components of the adsorbent materials. The weight percentages of Zr and Fe were the highest at 50.68 and 38.92%, respectively. The N value was derived from the chitosan materials [45–47]. Al, Si, and Fe were derived from zeolite and magnetite, respectively. Furthermore, the presence of P after the adsorption process indicates that PO<sup>4</sup> <sup>3</sup><sup>−</sup> was successfully adsorbed. *Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 5 of 20

**Figure 2.** SEM images before (**a**), and after (**b**) PO43<sup>−</sup> adsorption. **Figure 2.** SEM images before (**a**), and after (**b**) PO<sup>4</sup> <sup>3</sup><sup>−</sup> adsorption.



Figure 3 shows the functional groups in CS-ZL/ZrO/Fe3O<sup>4</sup> before and after PO<sup>4</sup> 3− adsorption through an FTIR-ATR analysis. A CS-ZL/ZrO/Fe3O<sup>4</sup> band was detected following PO<sup>4</sup> <sup>3</sup><sup>−</sup> adsorption from 3326 cm−<sup>1</sup> to 3320 cm−<sup>1</sup> . This shows that PO<sup>4</sup> <sup>3</sup><sup>−</sup> ions interact with the stretching vibrations of hydrogen and amine in chitosan [48]. After PO<sup>4</sup> <sup>3</sup><sup>−</sup> adsorption, a decrease in the peak from 1634 to 1627 cm−<sup>1</sup> was observed, which is associated with carboxyl groups (–COOCH3) [49]. An increased peak and a more curved and newer peak appeared after PO<sup>4</sup> <sup>3</sup><sup>−</sup> adsorption from 951 to 1006 cm−<sup>1</sup> and at 2161 cm−<sup>1</sup> , which were assigned to Si-O-Al, Fe-O-Si, or Zr-O-Fe and CN stretching, respectively. This indicated a strong interaction with PO<sup>4</sup> <sup>3</sup><sup>−</sup> ions. *Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 6 of 20

**Figure 3.** FTIR-ATR before, and after PO43<sup>−</sup> adsorption. **Figure 3.** FTIR-ATR before, and after PO<sup>4</sup> <sup>3</sup><sup>−</sup> adsorption.

#### *2.2. Mechanical Stability 2.2. Mechanical Stability*

sites did not change significantly.

deviation (error bars).

The mechanical stability of the CS-ZL/ZrO/Fe3O4 composite was determined through the percentage of the initial mass that was preserved after drying. Figure 4a shows that increasing the concentration of the solution led to a higher WR%. Compared to the HClcontaining solution, the H2SO4-containing solution exhibited a higher WR%. Consequently, the crystalline structure of CS-ZL/ZrO/Fe3O4 was deformed, indicating that H2SO4 had significant contact with the chitosan group. Figure 4b shows the IR spectra after treatment. The positions of the peaks were consistent for all the samples. According to [50], the broad band visible at 3176–3345 cm−1 is assigned to the -NH2 groups changing to –NH3+ groups. The peaks between 1611 and 1630 cm−1, which were ascribed to the carboxyl (–COOCH3) and –NH2 groups, were generated through H+ generation by HCl and H2SO4. The peak shifted to 1068 cm−1, and expansion occurred when treated with 0.1 M H2SO4. SO42− ions have been shown to be associated with Si, Al, Fe, and Zr [51]. According to these results, the physical and chemical characteristics of the CS-ZL/ZrO/Fe3O4 compo-The mechanical stability of the CS-ZL/ZrO/Fe3O<sup>4</sup> composite was determined through the percentage of the initial mass that was preserved after drying. Figure 4a shows that increasing the concentration of the solution led to a higher WR%. Compared to the HClcontaining solution, the H2SO4-containing solution exhibited a higher WR%. Consequently, the crystalline structure of CS-ZL/ZrO/Fe3O<sup>4</sup> was deformed, indicating that H2SO<sup>4</sup> had significant contact with the chitosan group. Figure 4b shows the IR spectra after treatment. The positions of the peaks were consistent for all the samples. According to [50], the broad band visible at 3176–3345 cm−<sup>1</sup> is assigned to the -NH<sup>2</sup> groups changing to –NH<sup>3</sup> <sup>+</sup> groups. The peaks between 1611 and 1630 cm−<sup>1</sup> , which were ascribed to the carboxyl (–COOCH3) and –NH<sup>2</sup> groups, were generated through H<sup>+</sup> generation by HCl and H2SO4. The peak shifted to 1068 cm−<sup>1</sup> , and expansion occurred when treated with 0.1 M H2SO4. SO<sup>4</sup> <sup>2</sup><sup>−</sup> ions have been shown to be associated with Si, Al, Fe, and Zr [51]. According to these results, the physical and chemical characteristics of the CS-ZL/ZrO/Fe3O<sup>4</sup> composites did not change significantly.

treatment. Solution a (0.01 M HCl), b (0.1 M HCl), c (0.01 M H2SO4), and d (0.1 M H2SO4). Standard

Table 4 shows the results of the statistical analysis, using the ANOVA test to evaluate the relationship between the input effective variables (A, B, C, and D) and their responses

*2.3. ANOVA and Equations for Fitting Empirical Models* 

**Figure 3.** FTIR-ATR before, and after PO43<sup>−</sup> adsorption.

*2.2. Mechanical Stability* 

sites did not change significantly.

**Figure 4.** WR of CS-ZL/ZrO/Fe3O4. (**a**) Percentage WR, and (**b**) FTIR-ATR of CS-ZL/ZrO/Fe3O4 after treatment. Solution a (0.01 M HCl), b (0.1 M HCl), c (0.01 M H2SO4), and d (0.1 M H2SO4). Standard deviation (error bars). **Figure 4.** WR of CS-ZL/ZrO/Fe3O<sup>4</sup> . (**a**) Percentage WR, and (**b**) FTIR-ATR of CS-ZL/ZrO/Fe3O<sup>4</sup> after treatment. Solution a (0.01 M HCl), b (0.1 M HCl), c (0.01 M H2SO<sup>4</sup> ), and d (0.1 M H2SO<sup>4</sup> ). Standard deviation (error bars).

The mechanical stability of the CS-ZL/ZrO/Fe3O4 composite was determined through the percentage of the initial mass that was preserved after drying. Figure 4a shows that increasing the concentration of the solution led to a higher WR%. Compared to the HClcontaining solution, the H2SO4-containing solution exhibited a higher WR%. Consequently, the crystalline structure of CS-ZL/ZrO/Fe3O4 was deformed, indicating that H2SO4 had significant contact with the chitosan group. Figure 4b shows the IR spectra after treatment. The positions of the peaks were consistent for all the samples. According to [50], the broad band visible at 3176–3345 cm−1 is assigned to the -NH2 groups changing to –NH3+ groups. The peaks between 1611 and 1630 cm−1, which were ascribed to the carboxyl (–COOCH3) and –NH2 groups, were generated through H+ generation by HCl and H2SO4. The peak shifted to 1068 cm−1, and expansion occurred when treated with 0.1 M H2SO4. SO42− ions have been shown to be associated with Si, Al, Fe, and Zr [51]. According to these results, the physical and chemical characteristics of the CS-ZL/ZrO/Fe3O4 compo-

#### *2.3. ANOVA and Equations for Fitting Empirical Models 2.3. ANOVA and Equations for Fitting Empirical Models*

Table 4 shows the results of the statistical analysis, using the ANOVA test to evaluate the relationship between the input effective variables (A, B, C, and D) and their responses Table 4 shows the results of the statistical analysis, using the ANOVA test to evaluate the relationship between the input effective variables (A, B, C, and D) and their responses (Y). Table 4 shows that the F-value of the quadratic model was 16.68 and that the *p*-value was <0.0001, indicating that this model was significant. Models A, B, D, A<sup>2</sup> , C<sup>2</sup> , D<sup>2</sup> , A × B, A × D, and C × D, marked with an asterisk (\*), were found to be significant parameters of the model. The statistical variables obtained from the ANOVA test (Equation (2)) provide a full quadratic regression model for PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal (%).

PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal (%) = 99.2 <sup>−</sup> 1.72 A + 63 B <sup>−</sup> 1.478 C <sup>−</sup> 0.472 D + 0.2333 A<sup>2</sup> + 840 B<sup>2</sup> + 0.01575 C<sup>2</sup> + 0.00661 D<sup>2</sup> <sup>−</sup> 17.23 A\*B − 0.0123A\*C − 0.02107 A\*D − 1.02 B\*C + 2.098 B\*D + 0.00343 C\*D



\* Significant.

The coefficients in the equation with positive and negative signs describe the additive and multiplicative effects of the variables on the response. The "Lack of Fit" was determined by comparing the residual error to the pure error in close proximity to the repeatedly designed points. F = 3.05 and *p* = 0.272 for the "Lack of Fit" revealed that it was not significant for the model (*p* < 0.05). It can be assumed that the model was adequately fitted and that there was no lack of fit.

The R<sup>2</sup> value of the calculated second-order response model was 95.11, which is also known as the coefficient of determination. Consequently, it can be applied to reliably calculate the response at any given parameter level regardless of their values. In addition, a regression model was used to calculate the standardized influence of the independent factors on PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal. A response surface plot was generated to investigate the influence of two components at initial the PO<sup>4</sup> <sup>3</sup><sup>−</sup> concentration of 20 mg/L (Figure 5). This plot was used to determine the standardized effects of all the independent variables. A surface plot is an easier way to display the response behavior that occurs when two parameters are simultaneously altered at the same time. It is more beneficial to select the quantity of various ingredients to obtain the desired response. Figure 5a displays a Pareto chart that compares the relative magnitude and the corresponding main, square, and interaction effects of the selected variables. The square effects of all four factors were found to be very significant (*p* ≤ 0.05) in addition to the main effects of the factors, which were also found to be highly significant (*p* ≤ 0.05). The ANOVA results reported in Table 4 led to the same conclusions. PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal continuously increased in response to the pH, adsorbent dosage, and time. Figure 5b,c show that pH is a key factor in the removal of PO<sup>4</sup> <sup>3</sup>−, and Figure 5d shows that increasing the contact time increases the percentage removal. *Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 8 of 20 Total 26 882.908 R2 95.11 R2 adj 89.41 \* Significant.

**Figure 5.** (**a**) Pareto chart for the standardized effect of various factors on PO43− removal by adsorbent, (**b**) pH and dosage of adsorbent response surface's effect on PO43<sup>−</sup> removal (%), (**c**) pH and time response surface's effect on PO43<sup>−</sup> removal (%), and (**d**) pH and dosage of adsorbent and time response surface's effect on PO43<sup>−</sup> removal (%). **Figure 5.** (**a**) Pareto chart for the standardized effect of various factors on PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal by adsorbent, (**b**) pH and dosage of adsorbent response surface's effect on PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal (%), (**c**) pH and time response surface's effect on PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal (%), and (**d**) pH and dosage of adsorbent and time response surface's effect on PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal (%).

The effects of the initial PO43− concentrations ranging from 20 to 500 mg/L, 0.06 g of adsorbent (CS-ZL/ZrO/Fe3O4), and pH (2.0) were investigated. Figure 6 shows that the PO43− adsorption capacity rose from 30.64 to 682.31 mg/g; however, the percentage of PO43<sup>−</sup>

concentration because the total number of molecules increased. Consequently, the mass transfer resistance of adsorbate decreased. As a result, the percentage of removal de-

*2.4. Initial Concentration and Isotherm Studies* 

creased [52].

#### *2.4. Initial Concentration and Isotherm Studies*

(error bars).

favorable.

The effects of the initial PO<sup>4</sup> <sup>3</sup><sup>−</sup> concentrations ranging from 20 to 500 mg/L, 0.06 g of adsorbent (CS-ZL/ZrO/Fe3O4), and pH (2.0) were investigated. Figure 6 shows that the PO<sup>4</sup> <sup>3</sup><sup>−</sup> adsorption capacity rose from 30.64 to 682.31 mg/g; however, the percentage of PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal decreased from 91.91% to 81.88%. The adsorption capacity increased with the concentration because the total number of molecules increased. Consequently, the mass transfer resistance of adsorbate decreased. As a result, the percentage of removal decreased [52]. *Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 9 of 20

**Figure 6.** Effect of initial concentration on PO43<sup>−</sup> removal onto CS-ZL/ZrO/Fe3O4. Standard deviation **Figure 6.** Effect of initial concentration on PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal onto CS-ZL/ZrO/Fe3O<sup>4</sup> . Standard deviation (error bars).

Adsorption isotherms are necessary to assess the capabilities of an adsorbent and the interactions between an adsorbate (a solution containing PO43− ions) and an adsorbent (CS-ZL/ZrO/Fe3O4). The acquired isotherm parameters can be used to conduct an accurate analysis while constructing an effective adsorption system. Both the equilibrium concentration and the adsorption capacity were estimated. The Langmuir model describes the monolayer adsorption processes at the designated homogenous surfaces on the adsorbent (Equation (3)). The essential property of the Langmuir model can be described as a dimensionless constant also known as the separation factor (RL), which is shown in Equation (4). By contrast, the Freundlich model is based on heterogeneous surfaces and multilayer Adsorption isotherms are necessary to assess the capabilities of an adsorbent and the interactions between an adsorbate (a solution containing PO<sup>4</sup> <sup>3</sup><sup>−</sup> ions) and an adsorbent (CS-ZL/ZrO/Fe3O4). The acquired isotherm parameters can be used to conduct an accurate analysis while constructing an effective adsorption system. Both the equilibrium concentration and the adsorption capacity were estimated. The Langmuir model describes the monolayer adsorption processes at the designated homogenous surfaces on the adsorbent (Equation (3)). The essential property of the Langmuir model can be described as a dimensionless constant also known as the separation factor (RL), which is shown in Equation (4). By contrast, the Freundlich model is based on heterogeneous surfaces and multilayer sorption (Equation (5)). This is a linear equation, which is shown as follows:

$$\mathbf{C\_e/q\_e} = \left(\frac{\mathbf{C\_e}}{\mathbf{q\_{max}}}\right) + 1/(\mathbf{K\_l q\_{max}}) \tag{3}$$

$$\mathbf{R}\_{\rm L} = (\frac{1}{1 + \mathbf{b} \mathbf{C}\_{\rm o}}) \tag{4}$$

$$\mathrm{Ln\,q} = \mathrm{lnK}\_{\mathrm{f}} + \frac{1}{\mathrm{n}} \times \ln \mathrm{C}\_{\mathrm{e}} \tag{5}$$

Ln q = lnKf + 1 n x lnCe (5) qe (mg/g) is the adsorption capacity; Kl (L/mg) is the equilibrium constant of adsorption; qmax (mg/g) is the maximal adsorption capacity; Ce (mg/L) is the equilibrium concentraq<sup>e</sup> (mg/g) is the adsorption capacity; K<sup>1</sup> (L/mg) is the equilibrium constant of adsorption; qmax (mg/g) is the maximal adsorption capacity; C<sup>e</sup> (mg/L) is the equilibrium concentration; C<sup>o</sup> (mg/L) is the initial concentration; R<sup>L</sup> is the separation factor; 0 < R<sup>L</sup> is favorable; R<sup>L</sup> > 1 is unfavorable; R<sup>L</sup> = 1 is linear; and K<sup>f</sup> (mg/g) and 1/n are the adsorption capacity and the intensity of adsorption, respectively.

tion; Co (mg/L) is the initial concentration; RL is the separation factor; 0 < RL is favorable; RL > 1 is unfavorable; RL = 1 is linear; and Kf (mg/g) and 1/n are the adsorption capacity

responding to these curves. The Freundlich model's linear correlation coefficient (R2) was 0.9970, indicating that it provided the best fit compared to the other models. More importantly, the Langmuir and Freundlich parameters, known as RL and 1/n, indicate that the PO43− ion has a type of < 1. According to these data, the PO43− adsorption method is

and the intensity of adsorption, respectively.

Figure 7 shows the isotherm model curves, and Table 5 shows the fitting results corresponding to these curves. The Freundlich model's linear correlation coefficient (R<sup>2</sup> ) was 0.9970, indicating that it provided the best fit compared to the other models. More importantly, the Langmuir and Freundlich parameters, known as R<sup>L</sup> and 1/n, indicate that the PO<sup>4</sup> <sup>3</sup><sup>−</sup> ion has a type of <1. According to these data, the PO<sup>4</sup> <sup>3</sup><sup>−</sup> adsorption method is favorable. *Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 10 of 20

**Figure 7.** Linear curves of PO43− adsorption isotherm models. (**a**) Langmuir, and (**b**) Freundlich models. Standard deviation (error bars). **Figure 7.** Linear curves of PO<sup>4</sup> <sup>3</sup><sup>−</sup> adsorption isotherm models. (**a**) Langmuir, and (**b**) Freundlich models. Standard deviation (error bars).


**Table 5.** Isotherm model parameters for PO43<sup>−</sup> removal onto CS-ZL/ZrO/Fe3O4. **Table 5.** Isotherm model parameters for PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal onto CS-ZL/ZrO/Fe3O<sup>4</sup>

#### *2.5. Adsorption Kinetic Studies*

*2.5. Adsorption Kinetic Studies*  This study investigated the influence of the contact time (35–2880 min) on PO43− adsorption at 30 °C. Figure 8 shows that the percentage of PO43− removal and the capacity for adsorption increased rapidly from 35 to 60 min and then gradually increased up to 90 min. This is because the adsorbent includes carboxyl, amine, hydrogen, and magnetite groups, all of which cause the adsorbent surface to become active and trap PO43− ions. Subsequently, the adsorption capacity decreased and increased, resulting in fast/slow PO43− adsorption, and it finally reached equilibrium at 1440 min, with an adsorption ca-This study investigated the influence of the contact time (35–2880 min) on PO<sup>4</sup> 3− adsorption at 30 ◦C. Figure 8 shows that the percentage of PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal and the capacity for adsorption increased rapidly from 35 to 60 min and then gradually increased up to 90 min. This is because the adsorbent includes carboxyl, amine, hydrogen, and magnetite groups, all of which cause the adsorbent surface to become active and trap PO<sup>4</sup> <sup>3</sup><sup>−</sup> ions. Subsequently, the adsorption capacity decreased and increased, resulting in fast/slow PO<sup>4</sup> <sup>3</sup><sup>−</sup> adsorption, and it finally reached equilibrium at 1440 min, with an adsorption capacity and percent removal of 732.56 mg/g and 87.91%, respectively.

pacity and percent removal of 732.56 mg/g and 87.91%, respectively. Adsorption kinetic studies are important because they deliver information on the adsorption mechanism, which is necessary to assess the effectiveness of the process [53]. Two kinetic models were used in this study: pseudo-first-order (PFO) (Equation (6)) and pseudo-second-order (PSO) (Equation (7)) models were investigated. The linear form can be obtained by calculating the following equation.

$$\text{Log}(\mathbf{q}\_{\text{e}} - \mathbf{q}\_{\text{t}}) = \log \mathbf{q}\_{\text{e}} - \mathbf{K}\_{\text{1}} \mathbf{t} \tag{6}$$

.

$$\mathbf{t}/\mathbf{q}\_{\rm t} = 1/\left(\mathbf{K}\_2 \mathbf{q}\_{\rm e}^2\right) + \mathbf{t}/\mathbf{q}\_{\rm e} \tag{7}$$

0

20

PO4340

60

80

100

where k<sup>1</sup> (min−<sup>1</sup> ) is the rate constant of the PFO model, t (min) is the time, and a linear plot of log t against log (q<sup>e</sup> − qt) and t against t/q<sup>t</sup> was used to determine K<sup>1</sup> and K<sup>2</sup> from the slope of the linear plots, respectively. *Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 11 of 20 0 200

400

qe

(mg/g)

600

800

*Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 11 of 20

**Figure 8.** The effect of contact time on PO43<sup>−</sup> removal onto CS-ZL/ZrO/Fe3O4. Standard deviation (error bars). **Figure 8.** The effect of contact time on PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal onto CS-ZL/ZrO/Fe3O<sup>4</sup> . Standard deviation (error bars). of log t against log (qe – qt) and t against t/qt was used to determine K1 and K2 from the slope of the linear plots, respectively.

Adsorption kinetic studies are important because they deliver information on the adsorption mechanism, which is necessary to assess the effectiveness of the process [53]. Two kinetic models were used in this study: pseudo-first-order (PFO) (Equation (6)) and pseudo-second-order (PSO) (Equation (7)) models were investigated. The linear form can Figure 9 shows the fitting curves for the kinetic models, and Table 6 lists the fitting results corresponding to those curves. The findings confirm that the PSO model provided better results than the PFO model in terms of the linear correlation coefficient R<sup>2</sup> value (0.9979). These findings imply that chemical processes control the adsorption rate. Figure 9 shows the fitting curves for the kinetic models, and Table 6 lists the fitting results corresponding to those curves. The findings confirm that the PSO model provided better results than the PFO model in terms of the linear correlation coefficient R2 value (0.9979). These findings imply that chemical processes control the adsorption rate.

**Figure 9.** Linear curves of PO43<sup>−</sup> adsorption kinetic studies. (**a**) Pseudo-first-order (PFO) and (**b**) pseudo-second-order (PSO) models. Standard deviation (error bars). **Figure 9.** Linear curves of PO<sup>4</sup> <sup>3</sup><sup>−</sup> adsorption kinetic studies. (**a**) Pseudo-first-order (PFO) and (**b**) pseudo-second-order (PSO) models. Standard deviation (error bars).

**Table 6.** Kinetic model parameters for PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal onto CS-ZL/ZrO/Fe3O<sup>4</sup> .


#### *2.6. Effect of Anions and Cations on PO<sup>4</sup> <sup>3</sup>*<sup>−</sup> *Removal onto CS-ZL/ZrO/Fe3O<sup>4</sup> 2.6. Effect of Anions and Cations on PO43− Removal onto CS-ZL/ZrO/Fe3O4*

*Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 12 of 20

**Table 6.** Kinetic model parameters for PO43<sup>−</sup> removal onto CS-ZL/ZrO/Fe3O4.

**Kinetics Parameters Value** 

Wastewater contains various substances, including anions and cations, which can affect the adsorption process [54]; it is essential to investigate the effect of ionic strength in an aqueous solution. This is because wastewater is made up of numerous components that might be found together. Figure 10 depicts the effect of different anions and cations on the PO<sup>4</sup> <sup>3</sup><sup>−</sup> adsorption capacity of CS-ZL/ZrO/Fe3O4. The experimental data indicate that there was no significant influence on PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal. It revealed that the fabrication of CS-ZL/ZrO/Fe3O<sup>4</sup> was effective in eliminating PO<sup>4</sup> <sup>3</sup><sup>−</sup> from water. Wastewater contains various substances, including anions and cations, which can affect the adsorption process [54]; it is essential to investigate the effect of ionic strength in an aqueous solution. This is because wastewater is made up of numerous components that might be found together. Figure 10 depicts the effect of different anions and cations on the PO43− adsorption capacity of CS-ZL/ZrO/Fe3O4. The experimental data indicate that there was no significant influence on PO43− removal. It revealed that the fabrication of CS-ZL/ZrO/Fe3O4 was effective in eliminating PO43− from water.

qe 2.5165 K1 1.42857 × 10−<sup>6</sup> R2 1.00 × 10−<sup>4</sup>

qe 510,204.1 K2 0.000119 R2 0.9979

**Figure 10.** The effect of coexisting ions on PO43<sup>−</sup> removal onto CS-ZL/ZrO/Fe3O4. Standard deviation (error bars). A: no significant effect (*p* ≤ 0.05). **Figure 10.** The effect of coexisting ions on PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal onto CS-ZL/ZrO/Fe3O<sup>4</sup> . Standard deviation (error bars). A: no significant effect (*p* ≤ 0.05).

#### *2.7. Desorption Studies*

ure 11c).

PFO

PSO

*2.7. Desorption Studies*  Figure 11a shows the desorption percentage of PO43− at different NaOH concentrations from 0.01 M to 1 M for 30 min at 30 °C. The results indicate that increasing the concentration increased the desorption percentage to 95.77%. Then, subsequent experiment at different contact times, from 30 to 150 min, using 1 M NaOH (Figure 11b). The desorption percentage increased and then decreased up to 150 min, which is similar to the results of the adsorption studies. The highest desorption percentage was observed after 30 min. The desorption mechanism may cause the hydroxide ions (OH-) in the sodium hydroxide solution to react with the CS-ZL/ZrO/Fe3O4-P surface and replace the PO43− groups, resulting in the release of PO43− into the liquid solution (Equation (8)). The reusability studies of PO43− adsorption onto CS-ZL/ZrO/Fe3O4 showed good performance for three cycles (Fig-Figure 11a shows the desorption percentage of PO<sup>4</sup> <sup>3</sup><sup>−</sup> at different NaOH concentrations from 0.01 M to 1 M for 30 min at 30 ◦C. The results indicate that increasing the concentration increased the desorption percentage to 95.77%. Then, subsequent experiment at different contact times, from 30 to 150 min, using 1 M NaOH (Figure 11b). The desorption percentage increased and then decreased up to 150 min, which is similar to the results of the adsorption studies. The highest desorption percentage was observed after 30 min. The desorption mechanism may cause the hydroxide ions (OH-) in the sodium hydroxide solution to react with the CS-ZL/ZrO/Fe3O4-P surface and replace the PO<sup>4</sup> <sup>3</sup><sup>−</sup> groups, resulting in the release of PO<sup>4</sup> <sup>3</sup><sup>−</sup> into the liquid solution (Equation (8)). The reusability studies of PO<sup>4</sup> <sup>3</sup><sup>−</sup> adsorption onto CS-ZL/ZrO/Fe3O<sup>4</sup> showed good performance for three cycles (Figure 11c).

$$\mathrm{H\_2PO\_4}^- + \mathrm{OH}^- \rightarrow \mathrm{HPO\_4}^{2-} + \mathrm{H\_2O} \tag{8}$$

**Figure 11.** The percentage of desorption. (**a**) Different NaOH concentrations and (**b**) different contact times using 1 M NaOH, and (**c**) in recycle studies on PO43− adsorption capacity. Standard deviation (error bars). **Figure 11.** The percentage of desorption. (**a**) Different NaOH concentrations and (**b**) different contact times using 1 M NaOH, and (**c**) in recycle studies on PO<sup>4</sup> <sup>3</sup><sup>−</sup> adsorption capacity. Standard deviation (error bars).

H2PO4− + OH– → HPO42− + H2O (8)

#### *2.8. Adsorption Performance Comparison*

*2.8. Adsorption Performance Comparison*  Table 7 compares the equilibrium and maximum adsorption capacity of CS-ZL/ZrO/Fe3O4 with those of various adsorbents. It can be seen that the pH is one of the main factors for PO43− removal onto the adsorbent, and the surface charge can become either positive or negative over a wide pH range, which influences the interaction between the adsorbent and PO43− ions. It is clear that the CS-ZL/ZrO/Fe3O4 adsorbent has a higher capacity than the other adsorbents. It is feasible to conclude that these adsorbents are vi-Table 7 compares the equilibrium and maximum adsorption capacity of CS-ZL/ZrO/ Fe3O<sup>4</sup> with those of various adsorbents. It can be seen that the pH is one of the main factors for PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal onto the adsorbent, and the surface charge can become either positive or negative over a wide pH range, which influences the interaction between the adsorbent and PO<sup>4</sup> <sup>3</sup><sup>−</sup> ions. It is clear that the CS-ZL/ZrO/Fe3O<sup>4</sup> adsorbent has a higher capacity than the other adsorbents. It is feasible to conclude that these adsorbents are viable alternatives for removing PO<sup>4</sup> <sup>3</sup><sup>−</sup> from water.

able alternatives for removing PO43− from water. **Table 7.** List comparing PO<sup>4</sup> <sup>3</sup><sup>−</sup> adsorption amounts.




#### **3. Materials and Methods**

#### *3.1. Materials*

Chitosan (CH) (C6H11NO4) with molecular weight of 100,000–300,000 Da was bought from Acros Organics, Belgium. Zeolite (ZL) (Al2O3·2SiO2) was obtained from Tosoh Co. Ltd., Japan. Sodium hydroxide (NaOH), acetic acid (CH3COOH), disodium hydrogen phosphate (Na2HPO4), ferric chloride (FeCl3), ferrous sulfate (Fe2SO4), ammonium molybdate ((NH4)6Mo7O24·4H2O)), antimony potassium tartrate (K2Sb2(C4H2O6)2), ascorbic acid (C6H8O6), hydrochloric acid (HCl), and sulfuric acid (H2SO4) were bought from Kanto Chemical Co., Inc., Tokyo, Japan. ZrClO was purchased from Fujifilm Wako Chemical, Tokyo, Japan.

## *3.2. Synthesis of CS-ZL/ZrO/Fe3O<sup>4</sup>*

CS-ZL/ZrO/Fe3O<sup>4</sup> was synthesized through crosslinking method; chitosan (1 g) was dissolved in 100 mL of acetic acid (1%), and the resulting viscous solution was maintained at ambient temperature (25–30 ◦C) with magnetic stirring for 24 h (Equation (9)). Subsequently, 25 mL of the resulting chitosan solution was mixed with 0.5 g of zeolite and 20 mL of 1 M FeCl<sup>3</sup> + 0.5 M Fe2SO<sup>4</sup> + 0.5 M ZrClO. The mixture solution was then heated to 60 ◦C and was stirred for 1 h. The pH of the solution was adjusted to 10 using 3 M NaOH over 24 h with magnetic stirring at ambient temperature (25–30 ◦C), and the solution was filtered and washed multiple times with acetone and distilled water (DW) to remove any remaining NaOH. Subsequently, the materials were dried for 48 h in an oven at 60 ◦C (Equation (13)). The adsorbents are referred to as CS-ZL/ZrO/Fe3O4.

$$\text{(CH}\_3\text{COOH)}\text{n} + \text{(C}\_6\text{H}\_{11}\text{NO}\_4\text{)}\text{m} \rightarrow \text{(CH}\_3\text{COO}^-\text{)}\text{n(C}\_6\text{H}\_{11}\text{NO}\_4\text{H}^+\text{)}\text{m} \tag{9}$$

$$\text{(C}\_6\text{H}\_{11}\text{NO}\_4\text{H}^+\text{)}\text{n} + \text{Al}\_2\text{O}\_3\text{-}2\text{SiO}\_2 \rightarrow \text{(C}\_6\text{H}\_{11}\text{NO}\_4 - \text{Al}\_2\text{O}\_3\cdot2\text{SiO}\_2\text{)}\text{n} + 2\text{H}\_2\text{O} \tag{10}$$

$$\text{5FeCl}\_3 + \text{15Fe}\_2(\text{SO}\_4)\_3 + \text{12NaOH} \rightarrow \text{5Fe}\_3\text{O}\_4 + \text{15Na}\_2\text{SO}\_4 + \text{6H}\_2\text{O} + \text{36NaCl} \tag{11}$$

$$\text{FeCl}\_3 + 3\text{Fe}\_2\text{(SO}\_4\text{)}\_3 + 2\text{ZrOO} + 14\text{NaOH} \rightarrow 5\text{Fe}\_3\text{O}\_4 + 2\text{r(OH)}\_4 + 2\text{Na}\_2\text{SO}\_4 + 6\text{NaCl} + 7\text{H}\_2\text{O} \tag{12}$$

2(CH3COO−)n(C6H11NO4H<sup>+</sup> )m + 3Al2O3·2SiO<sup>2</sup> + 3FeCl<sup>3</sup> + Fe2SO<sup>4</sup> + ZrClO<sup>4</sup> + 14NaOH → [3Al2O3·2SiO<sup>2</sup> − (C6H11NO4)]2m/3·Fe3O4·xH2O + 3Fe(OH)<sup>3</sup> + 2Zr(OH)<sup>4</sup> + 6NaCl + (2n + 2m)CH3COONa + (2n + m)H2O (13)

> Following this reaction, the negatively charged surface of the zeolite (Al2O3.2SiO2) may interact with the positively charged chitosan to produce chitosan–aluminosilicate complex. Electrostatic interactions between Fe3+ and Zr4+ ions and chitosan are another mechanism by which chitosan combines with metal ions to form chitosan–metal complexes. Fe(OH)<sup>3</sup> and Fe3O<sup>4</sup> are formed when Fe2+ and Fe3+ ions react with hydroxide ions (OH−) from NaOH.

#### *3.3. The Design of the Experiment*

Experiments were conducted using response surface methodology (RSM) in combination with Box–Behnken design (BBD), and statistical analysis was performed using Minitab 21.3.1 software. (A) The pH (2–10), (B) dosage (0.02–0.1 g), (C) temperature (30–60 ◦C),

and (D) contact time (10–60 min) were the independent variables examined in the BBD, with three levels and four parameters (Table 8). In total, 27 different sets of experiments were performed to determine the optimal conditions for PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal. The data obtained were assessed using an equation for a quadratic polynomial response surface, which was calculated using Equation (14), to identify the relationships between independent variables and response.

$$\mathbf{Y} = \mathbf{E}\_0 + \mathbf{E}\_1 \mathbf{A} + \mathbf{E}\_2 \mathbf{B} + \mathbf{E}\_3 \mathbf{C} + \mathbf{E}\_4 \mathbf{D} + \mathbf{E}\_{11} \mathbf{A}^2 + \mathbf{E}\_{22} \mathbf{B}^2 + \mathbf{E}\_{33} \mathbf{C}^2 + \mathbf{E}\_{12} \mathbf{A} \mathbf{B} + \mathbf{E}\_{13} \mathbf{A} \mathbf{C} + \mathbf{E}\_{23} \mathbf{B} \mathbf{C} + \varepsilon \tag{14}$$

**Table 8.** Variables and levels.


The coefficients of the polynomial model are represented as follows: E0 is constant expression, E1–E3 are linear effects, E11–E33 are second-order effects, E12–E23 are interactive effects, and ε is error term. An analysis of variance (ANOVA) was performed to calculate the F- and *p*-values of the model to measure its statistical significance and appropriateness. The statistical significance of the model is shown through the model's F-value and *p*-value, and a lack-of-fit study of the proposed model was executed using Minitab 21.3.1 software. In addition, a 3D response surface plot and Pareto chart of standardized effects were developed to figure out the cooperative quantitative impact of the independent variables on the response and overall value of the model [63].

#### *3.4. Batch Adsorption Study and Response Determination (PO<sup>4</sup> <sup>3</sup>*<sup>−</sup> *Removal %)*

To evaluate the efficiency of PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal, batch adsorption approach was used in this study. In total, 100 mL of PO<sup>4</sup> <sup>3</sup><sup>−</sup> (20 mg/L) was placed in a 300 mL conical flask. After the adsorption procedure was completed, external magnetite was placed in the conical flask to separate the adsorbent and adsorbate. PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal was calculated using Equation (15).

$$\text{PO}\_4^{3-} \text{ removal } \%= \frac{\text{C}\_{\text{o}} - \text{C}\_{\text{e}}}{\text{C}\_{\text{o}}} \times 100 \tag{15}$$

where C<sup>o</sup> and C<sup>e</sup> are the initial and equilibrium PO<sup>4</sup> <sup>3</sup><sup>−</sup> concentrations (mg/L), respectively.

The data from run 17 of the BBD were used for subsequent experiments (isotherm and kinetic models). However, 30 min was not used because the results were far from equilibrium. The amount of PO<sup>4</sup> <sup>3</sup><sup>−</sup> adsorbed was determined using Equation (16).

$$\mathbf{q}\_{\mathbf{e}} = \frac{\mathbf{C}\_{\mathbf{o}} - \mathbf{C}\_{\mathbf{e}}}{\mathbf{W}} \times \mathbf{V} \tag{16}$$

where q<sup>e</sup> (mg/g) is the adsorption capacity, W (g) is the amount of CS-ZL/ZrO/Fe3O4, and V (L) is the volume of adsorbate (PO<sup>4</sup> <sup>3</sup><sup>−</sup> solution).

#### *3.5. Adsorption Isotherm Studies*

The isotherm model was studied with PO<sup>4</sup> <sup>3</sup><sup>−</sup> solutions ranging from 20 mg/L to 500 mg/L with pH of 2. These examinations were performed for 60 min at 30 ◦C, and adsorbent dosage of 0.06 g was placed in the flask. In this work, Langmuir and Freundlich models were used to assess PO<sup>4</sup> <sup>3</sup><sup>−</sup> adsorption onto CS-ZL/ZrO/Fe3O<sup>4</sup> [64].

#### *3.6. Adsorption Kinetic Studies*

Pseudo-first-order (PFO) and pseudo-second-order (PSO) models were used to investigate the model of adsorption kinetics. The following parameters were used in the

experiment: an adsorption temperature of 30 ◦C, an initial PO<sup>4</sup> <sup>3</sup><sup>−</sup> concentration of 500 mg/L at pH of 2, an adsorbent dosage of 0.06 g, and contact time ranging from 35 to 2880 min.

#### *3.7. Influence of Coexisting Ionic Strength*

The experiment was conducted under optimum conditions with a dosage of 0.06 g, an initial PO<sup>4</sup> <sup>3</sup><sup>−</sup> concentration of 500 mg/L, and a contact time of 1440 min at 30 ◦C. The coexisting ion was prepared with cationic and anionic ions at a concentration of 20 mg/L (Mg2+, Ca2+, CO<sup>3</sup> <sup>2</sup>−, SO<sup>4</sup> <sup>−</sup>, and Na<sup>+</sup> ).

#### *3.8. Desorption and Reusability Studies*

In most practical applications, it is essential to employ adsorbents with high level of reusability. NaOH was chosen as desorbing agent to release PO<sup>4</sup> <sup>3</sup><sup>−</sup> ion from CS-ZL/ZrO/Fe3O4. Firstly, 0.06 g of CS-ZL/ZrO/Fe3O<sup>4</sup> was loaded with 500 mg/L of PO<sup>4</sup> 3− ion at pH of 2.0, which was called CS-ZL/ZrO/Fe3O4-P. Then, 0.01 g of CS-ZL/ZrO/Fe3O4- P was dispersed in 60 mL of NaOH at 30 ◦C. The desorption capacity and desorption percentage are shown in Equations (17) and (18), respectively. Reusability was assessed using the same treatment as described above.

$$\mathbf{q}\_{\text{des}} = \frac{\mathbf{C}}{\mathbf{W}} \times \mathbf{V} \tag{17}$$

$$\% \text{ Desorption} = \frac{\mathbf{q\_{des}}}{\mathbf{q\_e}} \times 100\tag{18}$$

where qdes (mg/g) is the desorption capacity; C (mg/L) is the PO<sup>4</sup> <sup>3</sup><sup>−</sup> concentration of desorption; % Desorption (%) is the percentage desorption; and W, V, and q<sup>e</sup> are the same as above.

#### *3.9. PO<sup>4</sup> <sup>3</sup>*<sup>−</sup> *Measurements*

PO<sup>4</sup> <sup>3</sup><sup>−</sup> ions were measured using the molybdate blue method. A total of 12 g of (NH4)6Mo7O24·4H2O was mixed with 100 mL of DW. K2Sb2(C4H2O6)<sup>2</sup> (0.277 g) was added followed by 140 mL of 18 M H2SO4. Afterward, it was adjusted to 1 L with distilled water (solution A). A total of 1.06 g of C6H8O<sup>6</sup> was added to and mixed with 100 mL of solution A, 25 mL of 4 N H2SO<sup>4</sup> was added, and the solution was adjusted to 1 L with DW (solution B). Note: This solution must be prepared in every experiment. The procedure for the mixed solution was as follows: 2 mL of liquid sample/standard was mixed with 10 mL of solution B. Afterwards, we waited for 30 min and then analyzed the solution using a UV-Vis spectrophotometer (Jasco V-530) at a wavelength of 693 nm. A standard curve for PO<sup>4</sup> <sup>3</sup><sup>−</sup> was constructed using Na2HPO4.

#### *3.10. Mechanical Stability*

The mechanical stability of the CS-ZL/ZrO/Fe3O<sup>4</sup> composite was evaluated based on the responses of the samples to a water bath shaker at 80 ◦C. For one hour, dried CS-ZL/ZrO/Fe3O<sup>4</sup> was soaked in HCl and H2SO<sup>4</sup> concentrations ranging from 0.01 to 0.1 M. Following that, the sample was dried in an oven at 60 ◦C for twenty-four hours. The calculation of the dry weight retention (WR) was performed using Equation (19).

$$\text{WR } \text{(\%)} = \frac{\text{w}\_{\text{i}}}{\text{w}\_{\text{a}}} \times 100 \tag{19}$$

where w<sup>i</sup> and w<sup>a</sup> are the dry weights of CS-ZL/ZrO/Fe3O<sup>4</sup> before and after treatment, respectively.

## *3.11. Characterization of CS-ZL/ZrO/Fe3O<sup>4</sup>*

The crystalline structure of CS-ZL/ZrO/Fe3O<sup>4</sup> was analyzed using a powder X-ray diffractometer (XRD) equipped with Cu/Kα radiation (Hypix-3000). Fourier transform infrared spectra (FTIR) of CS-ZL/ZrO/Fe3O<sup>4</sup> were measured before and after PO<sup>4</sup> 3− adsorption using a Thermo Scientific Nicolet iS10 instrument (Thermo Fisher Scientific Inc., Waltham, MA, USA). The ATR-FTIR approach was used to analyze samples with a resolution of 4 cm−<sup>1</sup> throughout the wavenumber spectrum spanning 400–4000 cm−<sup>1</sup> . To determine the specific surface area (SSA), the BET approach was combined with a surface area analyzer (MicroActive AutoPore V 9600 2.03.00, Micromeritics, Norcross, GA, USA). SEM-EDS (JIED-2300, Shimadzu, Kyoto, Japan) was used to examine the SEM images and the elemental distributions of CS-ZL/ZrO/Fe3O4. The initial (pHi) and final (pHf) pH values of the solutions were measured to determine the surface charge over a range of pH values (pHzpc). The pHi was adjusted from 2.0 to 10.0 in 0.01 M NaCl solution. Following that, 0.1 g of CS-ZL/ZrO/Fe3O<sup>4</sup> was added and stirred for 24 h at 30 ◦C, and pHf was measured. A plot of ∆pH = pHf − pHi vs. pHi was used to determine pHpzc, which corresponds to the neutral surface charge.

#### *3.12. Data Analysis*

All results were noted and edited using Microsoft Excel. The effects of coexisting ions on PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal were examined using a completely randomized design (CRD). Data were analyzed using ANOVA with Tukey's test (*p* ≤ 0.05) using Minitab 21.3.1.

#### **4. Conclusions**

In this study, a novel adsorbent, CS-ZL/ZrO/Fe3O4, was prepared from chitosan (CS), zeolite (ZL), ZrO, and magnetite (Fe3O4) via a crosslinking approach. The Box–Behnken design (BBD) and the response surface methodology (RSM), with their corresponding four separate factors (pH, dosage, temperature, and time), were used to develop the best experimental conditions for PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal. Weight retention (WR) was measured in a batch reactor under acidic conditions (HCl and H2SO4) at 80 ◦C for 1 h to determine the mechanical stability. The results indicate that CS-ZL/ZrO/Fe3O<sup>4</sup> was stable and did not change in the functional group peak area after treatment. The best conditions were at a pH of 2.0, with an adsorption capacity and percentage removal of 732.56 mg/g and 87.91%, respectively. The Freundlich isotherm and pseudo-second-order (PSO) kinetic models were fitted to PO<sup>4</sup> <sup>3</sup><sup>−</sup> removal, indicating heterogeneous and chemical sorption. In addition, the results suggest that PO<sup>4</sup> <sup>3</sup><sup>−</sup> adsorption occurred via the electrostatic interactions between the positive charge of CS-ZL/ZrO/Fe3O<sup>4</sup> and the negative charge of H2PO4<sup>−</sup> as well as ion exchange and hydrogen bonding. The presence of coexisting ions (Mg2+, Ca2+, CO<sup>3</sup> <sup>2</sup>−, SO<sup>4</sup> <sup>2</sup>−, and Na<sup>+</sup> ) had no effect on the removal of PO<sup>4</sup> <sup>3</sup><sup>−</sup> (*<sup>p</sup>* <sup>≤</sup> 0.05). The desorption studies revealed that 1 M NaOH was better at releasing PO<sup>4</sup> <sup>3</sup>−, reaching 95.77% after 30 min of treatment at 30 ◦C. The reusability of CS-ZL/ZrO/Fe3O<sup>4</sup> showed good performance over three cycles. These findings imply that CS-ZL/ZrO/Fe3O<sup>4</sup> is the best way to improve the stability of chitosan under acidic conditions, and it is a good adsorbent for removing PO<sup>4</sup> 3− and other potential water pollutants from water.

**Author Contributions:** Conceptualization, E.H.; Methodology, E.H.; Validation, Y.M.; Formal analysis, E.H.; Investigation, E.H. and Y.M.; Data curation, S.Y.; Writing—original draft, E.H.; Writing review & editing, E.H. and N.M.B.M.S.; Visualization, H.H.; Supervision, S.Y., Y.M. and H.H.; Project administration, H.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The author (E.H.) would like to express gratitude to the MEXT Scholarship for the funding received while studying at the Prefectural University of Hiroshima in Japan.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


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## *Review* **The Osteogenic Properties of Calcium Phosphate Cement Doped with Synthetic Materials: A Structured Narrative Review of Preclinical Evidence**

**Siti Sarah Md Dali <sup>1</sup> , Sok Kuan Wong 1,\* , Kok-Yong Chin <sup>1</sup> and Fairus Ahmad <sup>2</sup>**


**Abstract:** Bone grafting is commonly used as a treatment to repair bone defects. However, its use is challenged by the presence of medical conditions that weaken the bone, like osteoporosis. Calcium phosphate cement (CPC) is used to restore bone defects, and it is commonly available as a bioabsorbable cement paste. However, its use in clinical settings is limited by inadequate mechanical strength, inferior anti-washout characteristics, and poor osteogenic activity. There have been attempts to overcome these shortcomings by adding various natural or synthetic materials as enhancers to CPC. This review summarises the current evidence on the physical, mechanical, and biological properties of CPC after doping with synthetic materials. The incorporation of CPC with polymers, biomimetic materials, chemical elements/compounds, and combination with two or more synthetic materials showed improvement in biocompatibility, bioactivity, anti-washout properties, and mechanical strength. However, the mechanical property of CPC doped with trimethyl chitosan or strontium was decreased. In conclusion, doping of synthetic materials enhances the osteogenic features of pure CPC. The positive findings from in vitro and in vivo studies await further validation on the efficacy of these reinforced CPC composites in clinical settings.

**Keywords:** biomimetic materials; bone defect; chemical elements; polymers

### **1. Introduction**

Bone grafts are used to fill missing bone segments, improve skeletal projection, and provide mechanical support in bone defects by promoting osseous ingrowth, providing a structural substrate, and acting as a vehicle for controlled drug delivery in bone healing [1,2]. Although bone grafting is a widely utilised treatment to rebuild bone, the management of bone defects remains a great challenge, especially in individuals with medical conditions which compromise the bone healing process, such as osteoporosis, diabetes, and hypothyroidism [3]. An excellent bone graft should meet the characteristics of bioactive, biocompatible, osteoinductive, osteoconductive, resorbable, and high mechanical strength [4].

Autogenous bone graft represents the gold standard for treating bone defects because it does not cause immunoreaction and has osteoconductive properties. However, the use of autogenous bone grafts in clinical practice is restricted by limited bone grafts that are readily available, as well as the high incidence of complications at the donor and recipient sites [5]. Other alternatives used to optimise treatment include allografts and xenografts. Donor site morbidity issues are avoided by using these alternatives. However, they require sterilisation and purification, do not produce osteoconductive signals, and lack living cells. Additionally, they have the potential to cause bacterial or viral infections as well as a host

**Citation:** Md Dali, S.S.; Wong, S.K.; Chin, K.-Y.; Ahmad, F. The Osteogenic Properties of Calcium Phosphate Cement Doped with Synthetic Materials: A Structured Narrative Review of Preclinical Evidence. *Int. J. Mol. Sci.* **2023**, *24*, 7161. https://doi.org/10.3390/ ijms24087161

Academic Editors: Swarup Roy and Valentina Siracusa

Received: 23 March 2023 Revised: 7 April 2023 Accepted: 8 April 2023 Published: 12 April 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

tissue immunological reaction after implantation [6]. Herein, artificially produced bone grafts have been considered for the reasons of unlimited supply, minimal risk of disease transmission or immunoreaction, easy sterilisation and storage, as well as the availability of different shapes and sizes for surgical applications [7]. as well as a host tissue immunological reaction after implantation [6]. Herein, artificially produced bone grafts have been considered for the reasons of unlimited supply, minimal risk of disease transmission or immunoreaction, easy sterilisation and storage, as well as the availability of different shapes and sizes for surgical applications [7].

lack living cells. Additionally, they have the potential to cause bacterial or viral infections

*Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 2 of 17

Calcium phosphate cement (CPC) is a synthetic self-setting material serving as an alternative to treat bone defects. CPC comprises a liquid phase and a calcium phosphatesolid phase in which they react chemically to form hydroxyapatite when combined. CPC offers the advantages of biocompatibility, injectability, mouldability, and hardening in situ, allowing optimal bone tissue-implant contact even in irregular defect dimensions and minimally invasive surgery, thus making it a highly appealing bone substitute in surgical applications [8]. However, the major drawbacks of CPC are brittleness, low mechanical strength, inferior anti-washout, and the lack of osteogenic ability, which decreases the implant stability that limits its application to non-stress bearing locations. To overcome these shortcomings, many studies have been conducted to design and fabricate CPCs enhanced by biological and synthetic materials with superior mechanical strength and osteogenic properties. Calcium phosphate cement (CPC) is a synthetic self-setting material serving as an alternative to treat bone defects. CPC comprises a liquid phase and a calcium phosphatesolid phase in which they react chemically to form hydroxyapatite when combined. CPC offers the advantages of biocompatibility, injectability, mouldability, and hardening in situ, allowing optimal bone tissue-implant contact even in irregular defect dimensions and minimally invasive surgery, thus making it a highly appealing bone substitute in surgical applications [8]. However, the major drawbacks of CPC are brittleness, low mechanical strength, inferior anti-washout, and the lack of osteogenic ability, which decreases the implant stability that limits its application to non-stress bearing locations. To overcome these shortcomings, many studies have been conducted to design and fabricate CPCs enhanced by biological and synthetic materials with superior mechanical strength and osteogenic properties. A recent review has been published to summarise the incorporation of CPC with ma-

A recent review has been published to summarise the incorporation of CPC with materials derived from living organisms (including bone-related transcription factors, proteins, polysaccharides, and blood components) in treating bone defects [9]. Herein, the current review confers a comprehensive overview of the CPC reinforced by synthetic materials on their physical, mechanical, and biological properties (Figure 1). The available evidence indicates the strategies are reinforcement with synthetic polymers, biomimetic materials, chemical elements or compounds, and the combination of several synthetic materials. terials derived from living organisms (including bone-related transcription factors, proteins, polysaccharides, and blood components) in treating bone defects [9]. Herein, the current review confers a comprehensive overview of the CPC reinforced by synthetic materials on their physical, mechanical, and biological properties (Figure 1). The available evidence indicates the strategies are reinforcement with synthetic polymers, biomimetic materials, chemical elements or compounds, and the combination of several synthetic materials.

**Figure 1.** Conceptual framework of the review. **Figure 1.** Conceptual framework of the review.

#### **2. Literature Search 2. Literature Search**

Literature acquisition was performed using the PubMed and Scopus databases with the search string: (enhancement OR improvement OR reinforcement) AND (calcium phosphate cement) AND (bone OR osteoporosis OR fracture OR osteoblast OR osteoclast OR osteocyte). From the search, we obtained 815 and 348 records from inception until 15 January 2023 from PubMed and Scopus, respectively. Duplicate articles (*n* = 222) were excluded. The titles and abstracts were initially screened to exclude reviews, non-English, articles, books, book chapters, commentaries, conference papers, letters to the editor, Literature acquisition was performed using the PubMed and Scopus databases with the search string: (enhancement OR improvement OR reinforcement) AND (calcium phosphate cement) AND (bone OR osteoporosis OR fracture OR osteoblast OR osteoclast OR osteocyte). From the search, we obtained 815 and 348 records from inception until 15 January 2023 from PubMed and Scopus, respectively. Duplicate articles (*n* = 222) were excluded. The titles and abstracts were initially screened to exclude reviews, non-English, articles, books, book chapters, commentaries, conference papers, letters to the editor, metaanalyses, and irrelevant articles. Subsequently, the full-text articles were screened based on the inclusion and exclusion criteria. The main objective of this review is to summarise the characteristics of CPC enhanced with synthetic materials, defined as materials made by humans through chemical synthesis. The exclusion criteria of this review are (a) the

reinforcement of CPC with biological materials derived from living organisms; (b) original research articles not reporting bone parameters as the primary outcomes; and (c) original research articles with the absence of in vitro and in vivo experimental methods. A total of 30 relevant original research articles were included in this review. The evidence collection framework is summarised in Figure 2. the reinforcement of CPC with biological materials derived from living organisms; (b) original research articles not reporting bone parameters as the primary outcomes; and (c) original research articles with the absence of in vitro and in vivo experimental methods. A total of 30 relevant original research articles were included in this review. The evidence collection framework is summarised in Figure 2.

meta-analyses, and irrelevant articles. Subsequently, the full-text articles were screened based on the inclusion and exclusion criteria. The main objective of this review is to summarise the characteristics of CPC enhanced with synthetic materials, defined as materials made by humans through chemical synthesis. The exclusion criteria of this review are (a)

**Figure 2.** Evidence collection framework. **Figure 2.** Evidence collection framework.

#### **3. The Enhancement of CPC Using Synthetic Materials 3. The Enhancement of CPC Using Synthetic Materials**

*Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 3 of 17

#### *3.1. Synthetic Polymers 3.1. Synthetic Polymers*

Synthetic polymers refer to macromolecules artificially produced in laboratories through repetitive bonding of multiple monomers [10,11]. Several polymers, including poly(lactic-co-glycolic acid) (PLGA), PEGylated poly(glycerol sebacate) (PEGS), and so-Synthetic polymers refer to macromolecules artificially produced in laboratories through repetitive bonding of multiple monomers [10,11]. Several polymers, including poly(lactic-co-glycolic acid) (PLGA), PEGylated poly(glycerol sebacate) (PEGS), and sodium polyacrylate (PAAS), have been incorporated into CPC to enhance its properties (Table 1).

dium polyacrylate (PAAS), have been incorporated into CPC to enhance its properties (Table 1). PLGA is produced by the co-polymerisation of glycolic acid and lactic acid. It was commonly used as an enhancer for CPC as it has excellent biocompatibility, and the degradation time can be controlled. A study by Lu et al. showed that the addition of PLGA with different particle morphologies influenced the characteristics of CPC. Their findings indicated that CPC incorporated with dense spherical and irregularly shaped PLGA had proper setting time and improved compressive strength, whereas porous PLGA prolonged final setting time and decreased compressive strength of CPC. However, PLGA favoured cell proliferation of mouse bone mesenchymal stem cells regardless of their morphologies [12]. In another study, the addition of PLGA fibres electrospun on CPC exhibited higher flexural strength and work-of-fracture as compared to the CPC without PLGA fibres. The seeding of human umbilical cord mesenchymal stem cells (hUCMSCs) on CPC incorporated with PLGA fibres showed rapid cell proliferation and mineralisation. The expressions of alkaline phosphatase (ALP), osteocalcin (OCN), and collagen type I (COL1) were higher in hUCMSCs cultured on CPC containing PLGA [13]. Likewise, good cell proliferation and growing ALP activity were detected using rat bone marrow mesenchymal stem cells (rMSCs) seeded on PLGA/CPC scaffolds [14]. In the same study, the implantation of CPC with PLGA on bone defects at the femora of New Zealand white rabbits showed that the implant was gradually replaced by the host's new bones and numerous PLGA is produced by the co-polymerisation of glycolic acid and lactic acid. It was commonly used as an enhancer for CPC as it has excellent biocompatibility, and the degradation time can be controlled. A study by Lu et al. showed that the addition of PLGA with different particle morphologies influenced the characteristics of CPC. Their findings indicated that CPC incorporated with dense spherical and irregularly shaped PLGA had proper setting time and improved compressive strength, whereas porous PLGA prolonged final setting time and decreased compressive strength of CPC. However, PLGA favoured cell proliferation of mouse bone mesenchymal stem cells regardless of their morphologies [12]. In another study, the addition of PLGA fibres electrospun on CPC exhibited higher flexural strength and work-of-fracture as compared to the CPC without PLGA fibres. The seeding of human umbilical cord mesenchymal stem cells (hUCMSCs) on CPC incorporated with PLGA fibres showed rapid cell proliferation and mineralisation. The expressions of alkaline phosphatase (ALP), osteocalcin (OCN), and collagen type I (COL1) were higher in hUCMSCs cultured on CPC containing PLGA [13]. Likewise, good cell proliferation and growing ALP activity were detected using rat bone marrow mesenchymal stem cells (rMSCs) seeded on PLGA/CPC scaffolds [14]. In the same study, the implantation of CPC with PLGA on bone defects at the femora of New Zealand white rabbits showed that the implant was gradually replaced by the host's new bones and numerous osteoblasts after 16 weeks [14]. A study done by Maenz et al. also showed improved bone microstructure, bone mineral density (BMD), bone biomechanical compression strength, and static bone histomorphometry in ageing osteopenic female sheep with lumbar vertebrae bone defects and subjected to implantation using CPC incorporated with PLGA after 3 and 9 months [15].

PEGS is a biodegradable elastomer formed from the polycondensation of glycerol and sebacic acid. It has been developed to address the drawbacks of CPC in enhancing mechanical robustness, biocompatibility, bioactivity, and osteogenic activity for bone regeneration [16,17]. Ma et al. conducted in vitro and in vivo experiments to investigate the effects of PEGS-modified CPC scaffold for bone regeneration using rMSCs and rat calvarial defect model. The results indicated an increase in cell viability, cell proliferation, cell attachment, and osteogenic differentiation of rMSCs on PEGS/CPC scaffolds. New bone formation, greater mineralisation rate, higher bone volume/total volume (BV/TV), and promoted osteogenesis were also detected in rats subjected to two critical-sized (5 mm) calvarial defects and implanted with PEGS/CPC scaffold [16].

PAAS is a cross-linked polymer containing sodium with a super-absorbing ability. It is non-toxic and biocompatible, suggesting that it is frequently used as a food additive and has a potential application in drug delivery. When dissolved in aqueous solutions, PAAS has a high viscosity, strong hydrophilicity, and shape retention properties, thus rendering it a potential enhancer to enhance the anti-washout property of CPC [18]. A previous study demonstrated that the incorporation of PAAS into CPC enhanced the anti-washout, injectability, and compressive strength of the cement paste while retaining the setting time and material microstructure. The mouse mesenchymal stem cells were well-adhered, spread, and proliferated when incubated on PAAS/CPC in vitro [18].

**Table 1.** Bone-sparing properties of CPC enhanced by polymers.


Abbreviations: ALP, alkaline phosphatase; BV/TV, bone volume/total volume; COL1, collagen type I; hUCMSC, human umbilical cord mesenchymal stem cell; PAAS, sodium polyacrylate; PEGS, PEGylated poly (glycerol sebacate); PLGA, poly(lactic-co-glycolic acid); rMSCs, rat bone marrow mesenchymal stem cells; Tb.Th, trabecular thickness; OCN, osteocalcin; ↑, increase; ↓, decrease; ↔, no change.

#### *3.2. Biomimetic Materials*

Biomimetic materials are known as artificial synthetic materials which imitate biological substances of living organisms [19]. A variety of biological signalling cues are

required to provide an optimal environment for the physiological bone healing process. Thus, biomimetic materials can be alternatives to replicate the configuration of the microenvironment in natural bone tissue. Synthetic collagen I mimetic P-15, trimethyl chitosan, and chondroitin sulphate are examples of biomimetic materials incorporated into CPC (Table 2).

Synthetic collagen I mimetic P-15 is a synthetic 15-amino-acid sequence that is identical to the alpha I chain of COL1. It can bind with an inorganic bone matrix (hydroxyapatite), creating ideal conditions of biocompatibility, biodegradability and osteoconduction [20]. Since both CPC and P-15 have similar beneficial properties of osteoinductivity, osteoconductivity, and resorbability, their combination offers synergistic features in maintaining the stability of composite compared to the primary material [21]. A study using human mesenchymal stem cells seeded on chamber slides coated with CPC containing synthetic collagen I mimetic P-15 showed that the osteogenic differentiation was increased, evidenced by higher ALP, osteopontin (OPN), Runt-related transcription factor 2 (Runx-2), COL1, osteonectin, and OCN [21]. In addition, calcium deposits were detected in cells cultured on CPC with synthetic collagen I mimetic P-15 as an enhancer. Using the vertebrae of non-osteoporotic and osteoporotic sheep, the same group of researchers found that the bone augmented with CPC containing synthetic collagen I mimetic P-15 exhibited greater pull-out strength after pedicle screw insertion [21].

Trimethyl chitosan is a quarternised hydrophilic derivative of chitosan, which outperformed the parent molecule by its superior water solubility, biodegradable, biocompatible, and bioadhesive [22]. As it solves the well-known solubility drawback of chitosan, trimethyl chitosan is used as a reinforcing agent added to liquid CPC. The trimethyl chitosan-modified CPC had a longer setting time, improved wettability, and increased load-bearing capacity while maintaining the elasticity and bending strength compared to CPC without trimethyl chitosan additive. In vitro, the osteoblastic-like (MG-63) cells showed increased cell viability on CPC added with trimethyl chitosan, indicating good biocompatibility of the materials [23].

Chondroitin sulphate is a predominant glycosaminoglycan made up of alternating glucuronic acid and N-acetylgalactosamine disaccharide units [24]. It is an important structural component of the extracellular matrix network in bone and cartilage, incorporating fibronectin and growth factors to facilitate cell adhesion, migration, proliferation, and differentiation. Chondroitin sulphate can be animal-derived or manufactured synthetically. It has been introduced into CPC to enhance its osteogenic and integration abilities. Shi et al. (2019) reported that the setting time was prolonged, injectability was improved, and the fibronectin adsorption amount favouring cell adhesion was increased in the CPC paste reinforced with chondroitin sulphate relative to CPC per se. The bone mesenchymal stem cells also showed higher cell proliferation, cell differentiation, as well as osteogenic expression of ALP and OPN when cultured on the chondroitin sulphate-added CPC [25].

**Table 2.** Bone-sparing properties of CPC enhanced by biomimetic materials.



**Table 2.** *Cont.*

Abbreviations: ALP, alkaline phosphatase; BMD, bone mineral density; BV/TV, bone volume/total volume; COL1, collagen type I; OCN, osteocalcin; OPN, osteopontin; Runx-2, Runt-related transcription factor 2; ↑, increase; ↓, decrease; ↔, no change.

#### *3.3. Chemical Elements and Compounds*

Chemical elements are defined as substances that cannot be decomposed into simpler materials by the normal chemical process. On the other hand, chemical compounds refer to substances that contain two or more chemical elements held together by chemical bonds. Both chemical elements and compounds, such as strontium, selenium, iron, zinc, copper, magnesium, lithium, silicon, and calcium silicate, have been incorporated into CPC as enhancers (Table 3).

Similar to calcium, strontium is located in group 2 of the periodic table, indicating the comparable chemical properties and biological functions between these elements. Strontium exists as a trace element in the human body, and it concentrates in the bones [26]. With the distinct ability to intensify osteoblastogenesis and suppress osteoclastogenesis, strontium-releasing CPC might be a promising material for the regeneration of bone defects. The reinforcement of CPC by strontium resulted in no significant difference in the setting time but greater compressive modulus in comparison to CPC alone [27,28]. Two in vitro studies demonstrated higher cell proliferation and ALP activity in MG-63 cells [28] and primary human mesenchymal stromal cells seeded on strontium-doped CPC [27]. The performance of strontium-loaded CPC on defect at the non-load bearing (distal femoral condyle) and load-bearing sites (proximal tibia metaphysis) of female merino sheep were evaluated. After 26 weeks of implantation, the bone area and proportion of materials covered with bone were increased, but material degradation and osteoclast formation were not affected [29]. The clinical applicability of strontium-modified CPC in balloon kyphoplasty was tested in an 80-year-old male cadaver via vertebral body reconstruction, in which the sample was injected into the vertebral bodies of a human cadaver. The findings revealed that the strontium-containing CPC had higher viscosity, thus a lower tendency to leak out into the surrounding tissue during treatment compared to the control, PMMA cement [27]. In another study, male Sprague-Dawley rats with calvarial defect and implanted with nanostrontium-loaded CPC had increased bone formation, as well as higher expressions of bone morphogenetic protein-2 (BMP-2), OCN, and osteoprotegerin (OPG) in comparison to those implanted with CPC without nanostrontium [30]. Strontium ranelate is one of the anti-osteoporosis medications in postmenopausal women. The addition of strontium ranelate into CPC caused an increase in cell spreading area, cell proliferation, and expression of COLI, ALP, OCN, and Runx-2 after the culture of mouse bone marrow mesenchymal stem cells [31]. The expression of osteoclastogenesis-related genes coding for tartrate-resistant acid phosphatase (TRAP), cathepsin K (CTSK), matrix metalloproteinase 9 (MMP-9), and carbonic anhydrase II (Car2) was downregulated in the murine macrophage (RAW264.7) cells cultured on strontium ranelate-containing CPC [31]. Although the combination of strontium ranelate and CPC is a promising formula to stimulate

osteogenesis and new bone formation, the potential cardiovascular risks of strontium ranelate should not be neglected.

Selenium is an essential trace mineral required for the synthesis of selenoproteins. The skeletal-promoting effects of selenium have been widely established in vivo, whereby selenium deficiency was associated with impaired bone health which can be reversed through selenium supplementation [32]. Given the potential beneficial effects of selenium on bone, selenium-based biomaterials have been developed to promote bone tissue regeneration. An ovariectomised rat model with bone defect at the femoral epiphysis was utilised to study the effects of selenium in enhancing the efficacy of CPC in the treatment of osteoporotic bone defect for 12 weeks. Micro-computed tomography analysis pointed out that the animals treated with selenium-added CPC exhibited higher BMD, BV/TV, trabecular number (Tb.N), connectivity density (Conn.D), trabecular thickness (Tb.Th), but lower trabecular separation (Tb.Sp). The implantation of selenium-added CPC also resulted in greater new bone formation, mineral apposition rate (MAR), and biomaterial biodegradation at the defect site. The levels of superoxide dismutase (SOD), glutathione peroxidase (GPX), and OPG were raised, whereas the expression of catalase (CAT) and receptor activator of nuclear factor-kappa B ligand (RANKL) were decreased. These findings indicated that the rapid bone-repairing ability of this material might be in part achieved through suppression of oxidative stress and receptor activator of nuclear factor-kappa B (RANK)/RANKL/OPG pathway [33].

Iron is a mineral naturally present in many types of foods and available as a dietary supplement. It is a necessary component of haemoglobins, enzymes, and cytochromes. Accumulating evidence suggested that iron deficiency exerted a negative impact on bone. Female rats fed on an iron-restricted diet had compromised trabecular bone microstructure at lumbar vertebrae [34,35]. Hence, iron sufficiency plays an important role in bone regeneration. Zhang et al. reported that the setting time was shortened, but the injectability and compressive strength were increased in the iron-doped CPC relative to CPC alone. The effects of these materials on osteogenesis and angiogenesis were tested using two different cells, mouse bone marrow stromal cells and human umbilical vein endothelial cells (HUVECs). The cell proliferation of mouse bone marrow stromal cells and expression of ALP, COL1, OPN, and Runx-2 were elevated when incubated with iron-modified CPC. Higher levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) were also detected in HUVECs when cultured on CPC containing iron [36]. Moreover, iron-incorporated CPC demonstrated good biocompatibility with new bone formation as well as no sign of inflammation and necrosis at the defect site in female sheep after implantation [37].

Zinc is a crucial mineral for the growth, development, and maintenance of healthy bones [38]. A meta-analysis conducted by Ceylan et al. pinpointed that serum zinc concentration was lower in osteoporotic patients than in healthy subjects, and zinc supplementation was effective in increasing bone formation markers and BMD [39]. Based on the beneficial role of zinc in maintaining normal physiological bone growth, the use of zinc as an additive to reinforce CPC for bone tissue regeneration is hypothesised. Two in vitro studies found higher cell viability, cell proliferation, ALP activity, and osteogenic gene expression in murine mesenchymal stem cells and MC3T3-E1 cells seeded on zinc-modified CPC as compared to the cement without zinc [40,41].

Magnesium is the second most abundant intracellular cation in the human body after potassium. Approximately 60% of magnesium is present in bone. Magnesium deficiency negatively impacts bone health in different ways: (a) it directly increases osteoclastic activity and decreases osteoblastic activity; (b) it indirectly induces hormonal changes (such as parathyroid hormone and vitamin D) as well as promotes inflammatory response and oxidative stress, leading to bone loss [42]. Apart from its physiological role in maintaining musculoskeletal health, magnesium is a degradable metal with potential use as an alternative for non-resorbable materials in the fabrication of implantable medical devices [43]. The bone cell response to CPC doped with magnesium has been investigated in vitro and

in vivo by two groups of investigators. Zhang et al. revealed higher fibronectin adsorption, cell attachment, integrin α5β1, ALP activity, COL1 and OCN expression in bone marrow stromal cells cultured on CPC reinforced with magnesium than pure CPC [44]. Another study also showed higher cell proliferation of MG-63 cells cultured on CPC mixed with magnesium [45]. Besides, both studies showed increased osteogenesis and new bone formation in in vivo studies using the calvarial bone defect model in rats and rabbits.

Copper is a vital trace element in the human body for the proper functioning of organs and metabolic processes. Adequate serum copper level is important in maintaining good bone health, whereby lower concentration was associated with decreased BMD, and higher concentration was associated with increased fracture risk [46]. Considering the potential bone-protecting effects of copper, its application can be broadened to the repairing of bone defects. With the addition of copper, there were increases in setting time, compressive strength, and injectability of the CPC. However, there was no significant difference in the porosity of the CPC after the addition of copper. Mouse bone marrow mesenchymal stem cells seeded on CPC containing copper displayed higher adhesion activity, cell proliferation, and osteogenic expression of COL1, OCN, and ALP. HUVECs cultured on the combination of CPC and copper ions showed higher expression of angiogenesis-related genes [including eNOS, VEGF, basic fibroblast growth factor (bFGF), and nitric oxide] compared to pure CPC [47].

Apart from its use in the treatment of bipolar disorder, lithium has received much attention for its osteoprotective properties [48]. Lithium is a well-known inhibitor of glycogen synthase kinase-3 beta (GSK3β), a protein kinase that modulates the canonical Wingless (Wnt)/beta (β)-catenin pathways via phosphorylation of β-catenin as the downstream target [49]. Hence, lithium can be an excellent candidate to be incorporated into CPC to enhance bone regeneration. A lithium chloride-doped CPC was developed and tested for bone regenerative effects in MC3T3-E1 cells and ovariectomised rats with bone defects. The in vitro experiment showed higher cell proliferation, cell differentiation, mineralisation, and osteogenic differentiation after the incubation of MC3T3-E1 cells on a lithium/CPC scaffold. Mechanistically, the osteogenic properties of lithium-modified CPC were mediated through activation of the Wnt/β-catenin pathway, indicated by a higher level of phosphorylated GSK3β and a lower level of phosphorylated β-catenin. In in vivo study, lithium-modified CPC was implanted on bone defect created at the medial tibial shaft of female Sprague-Dawley rats. The findings showed higher BV/TV and increased new bone formation around the defect site implanted with lithium and CPC [50].

Silicon carbide whiskers are fibre-like materials produced by mixing and sintering silicon carbide fibres and alumina powder. It has excellent elasticity, strength, hardness, and chemical stability (such as wear, corrosion, and temperature resistance), thus, is commonly used as a reinforcement material for ceramics, metals, and plastics for a wide range of industrial applications [51]. With these features, silicon carbide whiskers were fused into CPC to overcome its brittleness, resulting in superior strength and toughness for weight-bearing applications. An early study found that the compressive strength, flexural strength, and elastic modulus of CPC were elevated after the addition of silicon carbide whiskers. However, the MC3T3-E1 cells cultured on pure CPC and silicon carbide whiskers/CPC composite showed similar live cell density, cell adhesion, cell viability, and cell proliferation [52].

Calcium silicate is a compound synthesised by reacting calcium oxide and silica at different ratios [53]. Both calcium and silica have a wide application for bone tissue engineering, mainly attributed to their role in promoting osteogenic differentiation and bone calcification, respectively [54]. The outstanding bioactivity and biocompatibility of calcium silicate make it a promising bioceramic in the field of dentistry and orthopaedics [53]. Zhao and colleagues proved that the MC3T3-E1 cells and HUVECs cultured on CPC incorporated with calcium silicate had higher cell proliferation and ALP activity [55].


**Table 3.** Bone-sparing properties of CPC enhanced by chemical elements/compounds.


#### **Table 3.** *Cont.*

Abbreviations: ALP, alkaline phosphatase; B.Ar/T.Ar, bone area/total area; BMD, bone mineral density; BMP-2, bone morphogenetic protein-2; BV/TV, bone volume/total volume; Car2, carbonic anhydrase II; CAT, catalase; COL1, collagen type I; Conn.D, connectivity density; CTSK, cathepsin K; eNOS, endothelial nitric oxide synthase; GPX, glutathione peroxidase; MAR, mineral apposition rate; MMP-9, matrix metalloproteinase 9; OCN, osteocalcin; OPG, osteoprotegerin; OPN, osteopontin; p-GSK3β, phosphorylated glycogen synthase kinase-3 beta; RANKL: receptor activator of nuclear factor-kappa B ligand; Runx-2, Runt-related transcription factor 2; SOD, superoxide dismutase; Tb.N, trabecular number; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness; TRAP, tartrateresistant acid phosphatase; VEGF, vascular endothelial growth factor; ↑, increase; ↓, decrease; ↔, no change.

#### *3.4. Combination between Two or More Synthetic Materials*

Two or more synthetic materials can be combined/mixed to enhance CPC (Table 4). PLGA has been incorporated into the CPC as an individual enhancer or in combination with other synthetic materials, such as wollastonite, perfluorocarbon, silicon/zinc, simvastatin/strontium, and alendronate.

Wollastonite is a naturally occurring mineral composed of calcium, silicon, and oxygen. It possesses outstanding performance in apatite mineralisation, biocompatibility, biodegradability, non-toxicity, mechanical properties, osteogenesis, vascularisation, and the ability to release bioactive silicon ions, suggesting its significant application in bone tissue regeneration [56]. Wollastonite has a rapid rate of decomposition, causing a rise in the pH level in the local environment and, subsequently, the release of excessive silicon ions, which can be harmful to cells. For this reason, wollastonite is not typically employed as a bone graft individually [57]. Qian et al. revealed that the flexibility was increased when wollastonite was mixed with PLGA as the enhancer for CPC. The cell attachment, cell proliferation, and expression of Runx-2, COL1, and BSP of mouse bone mesenchymal stem cells on wollastonite/PLGA/CPC composite were improved. Implanted material with wollastonite/PLGA/CPC on bone defect at the femoral condyle of New Zealand rabbits also showed increases in new bone formation, bone matrix, new blood vessels, and a decrease in material residual [57].

Perfluorocarbons are synthetic colourless, odourless, non-flammable, and unreactive compounds consisting of fluorine and carbon. It can dissolve oxygen, thus playing a crucial role in the delivery of oxygen for organ preservation [58]. Recent evidence reported the bone fracture healing properties of nanoscale perfluorocarbon in a rabbit model with radial fractures, as shown by the elevations in soft callus formation, collagen synthesis, as well as the expression of VEGF, MMP-9, and OCN [59]. Perfluoro-15-crown-5-ether (PFCE) is a commonly used perfluorocarbon, which is chemically and biologically inert, temperature and storage stable, as well as posing no infectious risk. In a study conducted by Mastrogiacomo et al., a novel composite was created by combining PFCE, PLGA, and gold nanoparticles, followed by incorporation into CPC. This composite was tested in vivo using a rat femoral condyle defect model and the increase in new bone formation was seen [60].

Various trace elements are present in the natural physiological extracellular environment to facilitate osteogenesis. Therefore, mimicking the bone microenvironment would require more than one trace element as the doping material. Silicon stimulates collagen synthesis and vascularisation, whereas zinc promotes bone growth and mineralisation. A study by Liang et al. showed increases in initial and final setting time, injectability, and compressive strength when PLGA microsphere and silicon/zinc dual elements were presented in CPC. The osteoimmunomodulatory effects of PLGA/silicon/zinc/CPC scaffold were proven to be superior to either one of the materials mixed with CPC, indicated by higher production of BMP-2 in the rMSCs. The immunomodulatory effects of the composites were tested using the RAW 264.7 macrophages. Better cell adhesion and spreading, lower proinflammatory cytokines [tumour necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6)], higher anti-inflammatory mediators [interleukin-10 (IL-10) and transforming growth factor-1 beta (TGF-1β)], as well as raised vascular genes [VEGF and platelet-derived growth factor-BB (PDGF-BB)] were detected in the RAW 264.7 cells seeded on PLGA/silicon/zinc/CPC composite. The implantation of PLGA/silicon/zinc/CPC scaffold on bone defect at the femur of male Sprague-Dawley rats also resulted in higher new bone formation, bone ingrowth, and BV/TV with a lower residual material area as early as week 4 [61].

Simvastatin is a hypolipidemic medication that can promote bone regeneration. Statin exerts pleiotropic effects, which include hypocholesterolemic and bone protective actions via the inhibition of the mevalonate pathway by blocking 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, an enzyme catalysing the conversion of HMG-CoA to mevalonic acid. Subsequently, the downstream synthesis of cholesterol is suppressed, whereas the expression of bone morphogenetic protein-2 is promoted via inhibition of protein prenylation using isoprenoid intermediates as substrates, such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) [62]. The combination of PLGA microsphere, simvastatin, and strontium improved CPC features. For instance, (a) PLGA microsphere provides a good delivery system due to its biodegradation potential; (b) statin enhances the stability of PLGA microspheres; and (c) strontium affects the reactivity of CPC by inducing osteoblastic activity and suppressing osteoclastic activity [30]. The implantation of PLGA/simvastatin/strontium/CPC scaffold for 8 weeks into parietal bone defects in rabbits showed superior osteogenic activities and biocompatibility [30].

Alendronate is a bisphosphonate medication to treat osteoporosis. The anti-resorptive effects of alendronate are mediated through several mechanisms: (a) it directly prevents the development and recruitment of osteoclast progenitors and promotes apoptosis of osteoclasts [63]; (b) it interferes with the mevalonate pathway by inhibiting farnesyl pyrophosphate synthase enzyme and reduced prenylated protein, thus inducing osteoclast apoptosis [64]. A study by van Houdt et al. incorporated alendronate and PLGA into CPC. The findings showed that the increase in alendronate content gradually increased the initial and final setting time but decreased the compressive strength of the composite. The biological performance of the composite was evaluated in vivo using ovariectomised female Wistar rats subjected to femoral condyle bone defect. Micro-computed tomography analysis showed the implanted material was in contact with surrounding bone tissue. In addition, the animals implanted with alendronate and PLGA-loaded CPC displayed stimulated bone formation and raised bone density along with the reduction of material remnants from week 4 to week 12 [3].

Laponite® is a synthetic nanoclay which has been described as a biocompatible diskshaped silicate [65]. The particle size on Laponite® is 1 nm in thickness and 25–30 nm in

diameter, with a negative charge on the surface and a positive charge at the edge. Laponite® displays non-cytotoxic and osteogenic effects on human bone marrow stromal cells. It also has wide application in the fabrication of composites to enhance mechanical properties [66]. On the other hand, dexamethasone is an anti-inflammatory and immunosuppressive medication used to treat inflammatory conditions. Utilising an in vivo ectopic bone formation model, the muscle of rats implanted with scaffolds containing dexamethasone and BMP-2 had higher bone formation than those implanted with scaffolds containing BMP-2 only, reiterating that the presence of dexamethasone enhanced the osteogenic capability of BMP-2, thus potentially reducing the required dosage of BMP-2 for clinical application [67]. Both Laponite® and dexamethasone were used in combination and added into CPC to enhance its properties. Higher compressive strength and modulus but shorter setting time were noted in CPC encapsulated with dexamethasone-loaded Laponite® nanoplates. When tested in vitro, the proliferation of MG-63 cells was improved after being cultured on dexamethasone/Laponite® nanoplates/CPC composite [68]. Although the osteogenic capability of dexamethasone has been reported, the adverse effects of glucocorticoid in inducing bone loss should be under careful consideration for its suitability to be used as an enhancer for bone tissue engineering.

**Table 4.** Bone-sparing properties of CPC enhanced by the combination of two or more synthetic materials.


Abbreviations: BMD, bone mineral density; BMP-2, bone morphogenetic protein-2; BSP, bone sialoprotein; BV/TV, bone volume/total volume; COL1, collagen type I; IL-6, interleukin-6; IL-10, interleukin-10; PDGF-BB, plateletderived growth factor-BB; PFCE, perfluoro-15-crown-5-ether: PLGA, poly(lactic-co-glycolic acid); rMSCs, rat bone marrow mesenchymal stem cells; Runx-2, Runt-related transcription factor 2; TGF-1β, transforming growth factor-1 beta; TNF-α, tumour necrosis factor-alpha; VEGF, vascular endothelial growth factor; ↑, increase; ↓, decrease.

#### **4. Perspectives**

The current review presented evidence on CPC enhancement by several synthetic materials in bone defect healing. The incorporation of various synthetic materials in CPC has been scientifically proven to resolve the limitations of CPC and/or influence the characteristics of CPC in terms of physical, mechanical, and biological properties. The addition of synthetic polymers (such as PLGA and PAAS) resulted in increased setting time, biomechanical strength, and osteogenic properties of CPC. For PLGA, the setting time and compressive strength of the composite were highly dependent on its particle morphology, whereby incorporating dense PLGA leads to proper setting time and increased compressive strength, whereas introducing porous PLGA into CPC prolonged the setting time and decreased compressive strength. Based on the previous evidence, biomimetic materials (such as synthetic collagen and chondroitin sulfate) have been proven to be a potential enhancer for CPC as the reinforced CPC exhibited more superior characteristics of injectability, raised mechanical strength, osteoconductivity, and osteogenic properties than pure CPC, satisfying the requirements for a good bone graft. However, trimethyl chitosan did not improve the compressive strength of the CPC after their combination. Strontium, iron, and zinc reduced, but copper increased the setting time of the incorporated CPC. Most chemical elements and compounds increased biomechanical properties and promoted osteogenic differentiation after addition into CPC. For strontium, both the setting time and compressive strength decreased with increasing content of strontium in the composite. The combination of PLGA with wollastonite or silicon/zinc as an enhancer seemed to increase the flexibility and injectability, which was not observed in the PLGA/CPC composite. The setting time of the composites was also increased when CPC was enhanced by the combination of PLGA and silicon/zinc or alendronate. In addition, alendronate was found to compromise the increased compressive strength conferred by PLGA. The overall characteristics of CPC after the addition of synthetic polymers, biomimetic materials, chemical elements/compounds, or in combination have been summarised (Table 5).


**Table 5.** The characteristics of CPC upon enhancement by different types of synthetic enhancers.

Abbreviations: PLGA, poly(lactic-co-glycolic acid); ↑, increase; ↓, decrease.

Several limitations of current evidence need to be acknowledged. Young osteoporotic animals have been widely used as a model in most of these studies. The presence of other medical conditions may inhibit bone regeneration and thus should be tested in research using a bone defect model in animals with various types of diseases. In addition, small animals (such as rats, mice, and rabbits) were used as research models for bone defects, which may have different prognoses than large animals such as sheep and bovine. It may take a longer time to exert similar outcomes, understand the mechanisms involved, and validate the mechanisms of the components involved to be translated into clinical settings. The synthetic materials used to enhance CPC summarised in the current review were heterogenous, indicating the lack of original evidence to allow the direct comparison between pure CPC and those enhanced by single or multiple synthetic materials in the same experimental setting.

#### **5. Conclusions**

In summary, the incorporation of synthetic materials into CPC can help to overcome the drawbacks of the conventional powder-liquid type of cement. The composites displayed enhanced properties in the aspects of mechanical strength and osteogenic activities while retaining CPC's injectability, mouldability, osteoconductivity, biocompatibility and biodegradability; the various composites possess great potential to be used as materials for implantation in bone defect via minimally invasive surgical techniques. It is recommended to validate the effectiveness of synthetically-enhanced cement in human trials and investigate its effects in more challenging conditions such as the coexistence of infection or disease, poor blood supply, and critical bone defects.

**Author Contributions:** Conceptualisation, S.K.W.; methodology, S.S.M.D. and S.K.W.; validation, S.K.W. and K.-Y.C.; writing—original draft preparation, S.S.M.D. and S.K.W.; writing—review and editing, S.K.W., K.-Y.C. and F.A.; visualisation, S.K.W. and K.-Y.C.; supervision, S.K.W., K.-Y.C. and F.A.; project administration, S.K.W.; funding acquisition, S.K.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Universiti Kebangsaan Malaysia, grant number GUP-2021-034.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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## *Article* **Prospect of Bioactive Curcumin Nanoemulsion as Effective Agency to Improve Milk Based Soft Cheese by Using Ultrasound Encapsulation Approach**

**Uday Bagale 1,\* , Ammar Kadi <sup>1</sup> , Mostafa Abotaleb <sup>2</sup> , Irina Potoroko <sup>1</sup> and Shirish Hari Sonawane <sup>3</sup>**

<sup>1</sup> Department of Food and Biotechnology, South Ural State University, Chelyabinsk 454080, Russia

<sup>2</sup> Department of System Programming, South Ural State University, Chelyabinsk 454080, Russia

<sup>3</sup> Department of Chemical Engineering, National Institute of Technology Warangal, Telangana 506004, India

**\*** Correspondence: bagaleu@susu.ru; Tel.: +7-(351)-267-93-80

**Abstract:** The aim of this paper was to determine the effect of stabilized curcumin nanoemulsions (CUNE) as a food additive capable of directionally acting to inhibit molecules involved in dairy products' quality and digestibility, especially cheese. The objects were cheeses made from the milk of higher grades with addition of a CUNE and a control sample. The cheeses were studied using a scanning electron microscope (SEM) in terms of organoleptic properties, such as appearance, taste, and aroma. The results show that the addition of CUNEs improved the organoleptic properties compared to the control cheese by 150% and improved its shelf life. The SEM study shows that formulation with CUNE promotes the uniform distribution of porosity. The CUNE-based cheese shows a better sensory evaluation compared to the emulsion without curcumin. CUNE-processed cheese provided better antioxidant and antimicrobial analysis than the control sample and offers added value to the dairy sector.

**Keywords:** curcumin nanoemulsion; cheese; ultrasound; SEM; self-life and sensory analysis

**1. Introduction**

Dairy products are an important sector of the food industry. Daily dairy consumption varies from 150 to 500 g per capita in different countries and is steadily increasing [1]. Processed cheese has gained more success, owing to a combination of economical available ingredients and better functional properties than other cheese [2,3]. However, processed cheeses have a problem with storage and shelf life. This problem can be minimized by the fortification of cheese with bio-active compounds. Bioactive compounds, such as essential oils, medical plants, and fruit extracts, have resulted in better versions of cheese [4–7]. The addition of bioactive compounds in processed cheese also affects the taste and the consistency [7].

A common antioxidant is curcumin, extracted from turmeric root [7–11]. Curcumin is a polyphenol, characterized by more than one phenolic group per molecule. Turmeric contains a wide variety of vitamins and other substances essential for the human organism. In addition, curcumin has antioxidant, anticarcinogenic, immunomodulatory, antifungal, and anti-inflammatory properties, which make it suitable in the medical and food industries [9–15]. However, the poor water solubility of curcumin limits its direct use. This poor solubility also leads to low absorption, fast metabolism, and quick systematic elimination [16,17]. To enhance the bioavailability and biological activity of curcumin, structural modifications are required [18].

Most research reported that stability can be improved by scaling to nanosize particles using high or low energy methods, such as ultrasound, high speed homogenization, or solvent evaporation [9,10,17,19]. Curcumin nanoparticles have a higher surface charge and surface area, are more hydrophobic, and have greater antioxidant activity than untreated

**Citation:** Bagale, U.; Kadi, A.; Abotaleb, M.; Potoroko, I.; Sonawane, S.H. Prospect of Bioactive Curcumin Nanoemulsion as Effective Agency to Improve Milk Based Soft Cheese by Using Ultrasound Encapsulation Approach. *Int. J. Mol. Sci.* **2023**, *24*, 2663. https://doi.org/10.3390/ ijms24032663

Academic Editors: Valentina Siracusa and Swarup Roy

Received: 23 December 2022 Revised: 27 January 2023 Accepted: 29 January 2023 Published: 31 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

curcumin. Curcumin nanoparticles have higher water solubility and suspension, which improves their antimicrobial activity [20–22].

The use of nanoemulsions is a promising technique for incorporating these bioactive complexes into foods [23–25]. Nanoscale curcumin provides superior properties in comparison to micron-scale curcumin, allowing it to mix effectively with other food ingredients to reduce the biological and enzymatic reactions, and allowing innovative products to be fabricated [26]. Nanoemulsions can be exploited in the food industry, since there are different approaches for their preparation, and they are stable systems for the encapsulation of bioactive substances [24–26]. Ultrasound techniques produce bioactive nanoemulsions with a higher yield and encapsulation efficiency [9,15,17,26,27].

The present study investigated the incorporation of curcumin in cheese in order to improve its dietary value. In this work, with the aid of ultrasound technology and polysorbate 20 as an emulsifier, a stable curcumin nanoemulsion (CUNE) was created. Data on particle size, encapsulation effectiveness, and stability were used to characterize the nanoemulsions. These were incorporated into the cheese which was checked, using a scanning electron microscope, against a control sample for sensory evaluation, antimicrobial activity.

#### **2. Results and Discussion**

Initially, we stabilized the CUNE containing edible oil and Tween 20 by using a sonochemical approach. To obtain stable CUNE, we optimized the oil and emulsifier concentration along with curcumin concentration. In the nanoemulsion, the curcumin concentration was encapsulated in the oil phase from 0.15 g to 0.75 g. We tried different inner and outer phase combinations to make a stable nanoemulsion, but only a few were found to be stable on centrifuge and heating at 80 ◦C for 30 min.

#### *2.1. Characterization of Nanoemulsion*

In the current study, we first used a sonochemical method to stabilize the curcumin nanoemulsion made up of edible oil and Tween 20. The curcumin concentration ranged from 0.75 g to 0.15 g in the oil phase of the current o/w nanoemulsion technology. Only a few of the inner and outer phase combinations we explored to create a stable nanoemulsion were discovered to be stable after centrifugation and 30 min of heating at 80 ◦C.

Particle Size Distribution, Polydispersity Index (PDI), and Optical Microscope for Stable Nanoemulsion

Nanotrac software was used to analyze the diameter size of the nanoemulsion and its polydispersity index (PDI) based on dynamic light scattering. Stable nanoemulsion results for the particle size and PDI are shown in Table 1, while stable particle size distribution is shown in Figure 1. With a PDI smaller than 0.4, stable nanoemulsions have a limited particle size distribution. According to Ahmed et al. (2012), an emulsion will not be stable if the PDI is higher than 0.5. In terms of their release phenomena, they also proposed that medium chain triglyceride-based nanoemulsions offer superior stability and improve curcumin's bio-availability. According to Bagale et al., stable nanoemulsion with 50:50 (long-chain triglyceride: short-chain triglyceride) oil ratio has polydispersity less than 0.4 and an average droplet size of 200 nm. CUNE with medium-chain triglyceride palm oil has less water solubility than short-chain triglyceride oil, which shows a polydispersity index in the range of 0.3–0.357.

Figure 2a,b show the optical microscope image of CUNE with concentrations of 0.15 and 0.75 g curcumin. We can observe the encapsulation of curcumin in the oil in water morphology. The dark spots present in micelles are curcumin particles, which are surrounded by a layer of oil droplets in a stable form without any aggregation. There are some micelles without curcumin. The optical microscope images show the particle size distribution of the oil–water droplets. The droplet sizes are approximately 10–20 nm, calculated by using TEM and confirmed through light scattering. These oil droplets are tiny and disperse thoroughly in the nanoemulsion. The total phenolic content method used

the normal correction curve of gallic acid at variance with an R<sup>2</sup> value of 0.9894 to calculate encapsulation efficiency. CUNE has a higher encapsulation efficiency because of the small surface tension among droplets; flocculation, and accumulation are eluded, cultivating curcumin solubility. According to Bagale et al. (2022), who optimized the sonication period, polydispersity gives insight into the homogeneity of the size distribution and was low (0.3) for all samples, indicating the creation of monodisperse systems. This suggests that sonication would increase the effectiveness of encapsulation. Tween 20 concentrations that promote curcumin stability also increase the effectiveness of encapsulation [28].


CUNE 4 5.55 15 135 ± 3 625 ±10 0.43 −18

**Table 1.** pH and gravitational stability of nanoemulsions data. *Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 3 of 16

**Figure 1.** Stability data of curcumin nanoemulsion sample in term of PSD. **Figure 1.** Stability data of curcumin nanoemulsion sample in term of PSD.

**Transmission (%)**

tions that promote curcumin stability also increase the effectiveness of encapsulation [28]. (**a**) CuNE2 (**b**) CuNE3

Figure 3 shows the FTIR spectra of free curcumin and CUNE. The spectra of CUNE

Figure 4 shows that the nanoemulsion has a particle size of 14 nm and is spherical in shape. According to Biswas et al. [20], the noble assets and functionality of the nanoparticles are owing to his higher ratio surface area/volume and nanoscale size and hydrophobicity. They also reported that CUNE with a particle size of approximately 5–50 nm shows

ing (1348.24 cm−1). Due to the presence of O-C=O (2953.03 cm−1), O-H (2852.72 cm−1), and C-H (2922.16 cm−1) groups in the curcumin-loaded nanoemulsion, the other bands in the

**Figure 2.** Optical image for curcumin encapsulation in different nanoemulsion sample. **Figure 2.** Optical image for curcumin encapsulation in different nanoemulsion sample.

**Figure 3.** FTIR spectra Free curcumin and Curcumin nanoemulsion**.** 

**Wavelength (cm-1)**

**3900 3400 2900 2400 1900 1400 900 400**

*2.2. Transmission Electron Microscope Analysis of Nanoemulsion* 

higher antimicrobial potential than its natural form.

area of 2965–2855 cm−1 were provided by the C-H stretching vibration.

**CuNE Pure Curcumin**

Figure 3 shows the FTIR spectra of free curcumin and CUNE. The spectra of CUNE resembled that of pure curcumin, which contained all the normal absorption peaks. The two most significant functional groups were C=O stretching (1741.72 cm−<sup>1</sup> ) and O-H bending (1348.24 cm−<sup>1</sup> ). Due to the presence of O-C=O (2953.03 cm−<sup>1</sup> ), O-H (2852.72 cm−<sup>1</sup> ), and C-H (2922.16 cm−<sup>1</sup> ) groups in the curcumin-loaded nanoemulsion, the other bands in the area of 2965–2855 cm−<sup>1</sup> were provided by the C-H stretching vibration. Figure 3 shows the FTIR spectra of free curcumin and CUNE. The spectra of CUNE resembled that of pure curcumin, which contained all the normal absorption peaks. The two most significant functional groups were C=O stretching (1741.72 cm−1) and O-H bending (1348.24 cm−1). Due to the presence of O-C=O (2953.03 cm−1), O-H (2852.72 cm−1), and C-H (2922.16 cm−1) groups in the curcumin-loaded nanoemulsion, the other bands in the area of 2965–2855 cm−1 were provided by the C-H stretching vibration.

**Figure 2.** Optical image for curcumin encapsulation in different nanoemulsion sample.

*Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 4 of 16

(**a**) CuNE2 (**b**) CuNE3

**Figure 3.** FTIR spectra Free curcumin and Curcumin nanoemulsion**. Figure 3.** FTIR spectra Free curcumin and Curcumin nanoemulsion.

#### *2.2. Transmission Electron Microscope Analysis of Nanoemulsion 2.2. Transmission Electron Microscope Analysis of Nanoemulsion*

Figure 4 shows that the nanoemulsion has a particle size of 14 nm and is spherical in shape. According to Biswas et al. [20], the noble assets and functionality of the nanoparticles are owing to his higher ratio surface area/volume and nanoscale size and hydrophobicity. They also reported that CUNE with a particle size of approximately 5–50 nm shows higher antimicrobial potential than its natural form. Figure <sup>4</sup> shows that the nanoemulsion has a particle size of 14 nm and is spherical inshape. According to Biswas et al. [20], the noble assets and functionality of the nanoparticles are owing to his higher ratio surface area/volume and nanoscale size and hydrophobicity. They also reported that CUNE with a particle size of approximately 5–50 nm shows higher antimicrobial potential than its natural form. *Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 5 of 16

which can cause disruptions in the functions of the cell membrane **Figure 4.** *Cont*.

**Figure 4.** Transmission electron microscope analysis of CUNE sample.

The outcomes of the antibacterial potential of CUNE are revealed in Table 2 (Figure 5). CUNE with a concentration of 100 and 50 μg/mL showed the highest antibacterial activity against S. aureus and E. coli. At a concentration of 25 μg/mL, the nanoemulsion showed reasonable antibacterial activity for both strains; however, the lowest antimicrobial inhibition level was at a diameter of 5 mm. An earlier study found that CUNE had better aqueous-phase solubility and dispersibility than pure curcumin and hence had antibacterial activity. Any nanoscale particle's antibacterial potential will depend on its physicochemical characteristics (size, shape, and surface qualities), as well as the quantity used. According to Naghadri et al. [29] and Wang et al. [30], nanoparticles smaller than 100 nm have a higher adhesion to the surface of cell membranes than larger nanoparticles,

*2.3. Antimicrobial Activity Nanoemulsion* 

**Figure 4.** Transmission electron microscope analysis of CUNE sample. **Figure 4.** Transmission electron microscope analysis of CUNE sample.

#### *2.3. Antimicrobial Activity Nanoemulsion 2.3. Antimicrobial Activity Nanoemulsion*

The outcomes of the antibacterial potential of CUNE are revealed in Table 2 (Figure 5). CUNE with a concentration of 100 and 50 μg/mL showed the highest antibacterial activity against S. aureus and E. coli. At a concentration of 25 μg/mL, the nanoemulsion showed reasonable antibacterial activity for both strains; however, the lowest antimicrobial inhibition level was at a diameter of 5 mm. An earlier study found that CUNE had better aqueous-phase solubility and dispersibility than pure curcumin and hence had antibacterial activity. Any nanoscale particle's antibacterial potential will depend on its physicochemical characteristics (size, shape, and surface qualities), as well as the quantity used. According to Naghadri et al. [29] and Wang et al. [30], nanoparticles smaller than 100 nm have a higher adhesion to the surface of cell membranes than larger nanoparticles, which can cause disruptions in the functions of the cell membrane The outcomes of the antibacterial potential of CUNE are revealed in Table 2 (Figure 5). CUNE with a concentration of 100 and 50 µg/mL showed the highest antibacterial activity against S. aureus and E. coli. At a concentration of 25 µg/mL, the nanoemulsion showed reasonable antibacterial activity for both strains; however, the lowest antimicrobial inhibition level was at a diameter of 5 mm. An earlier study found that CUNE had better aqueous-phase solubility and dispersibility than pure curcumin and hence had antibacterial activity. Any nanoscale particle's antibacterial potential will depend on its physicochemical characteristics (size, shape, and surface qualities), as well as the quantity used. According to Naghadri et al. [29] and Wang et al. [30], nanoparticles smaller than 100 nm have a higher adhesion to the surface of cell membranes than larger nanoparticles, which can cause disruptions in the functions of the cell membrane.

#### *2.4. Curcumin Nanoemulsion in Cheese Formulation and Its Analysis*

Based on the above result, we took the curcumin nanoemulsion (CUNE 2) for further study in terms of its addition in cheese formulation.


**Table 2.** Antimicrobial activity of CUNE.

Concentration of CUNE are in percent as follows 100 \* = 100 µg NC/mL, 50 \* = 50 µg NC/mL, 25 \* = 25 µg NC/mL, and 12.5 \* = 12.5 µg NC/mL. Note: All analysis measurement was conducted in triplets and have ± SEM (*p* < 0.05).

E. Coli 18 15 13 8.2 S. Aureus 12 9 6 3

**Figure 5.** Antimicrobial activity of curcumin nanoemulsion for (**a**) *E. coli* and (**b**) *S. aureus*. **Figure 5.** Antimicrobial activity of curcumin nanoemulsion for (**a**) *E. coli* and (**b**) *S. aureus*.

#### *2.4. Curcumin Nanoemulsion in Cheese Formulation and Its Analysis*  2.4.1. Sensory Analysis of Cheese

**Table 2.** Antimicrobial activity of CUNE.

**Microorganism Zone Inhibition Diameter (mm)** 

plets and have ± SEM (*p* < 0.05).

Based on the above result, we took the curcumin nanoemulsion (CUNE 2) for further study in terms of its addition in cheese formulation. 2.4.1. Sensory Analysis of Cheese The number of foods enriched with bioactive compounds are increasing in the dairy The number of foods enriched with bioactive compounds are increasing in the dairy and food industries. As bioactive compounds show better antioxidant activity, these food products promote health and wellness. Figure 6 provides an evaluation of the CUNEenriched effect on the organoleptic properties of cheese compared to the control and emulsion cheeses during a 60-day period (at 4 ◦C).

**100 \* 50 \* 25 \* 12.5 \*** 

Concentration of CUNE are in percent as follows 100 \* = 100 μg NC/mL, 50 \* = 50 μg NC/mL, 25 \* = 25 μg NC/mL, and 12.5 \* = 12.5 μg NC/mL. Note: All analysis measurement was conducted in tri-

and food industries. As bioactive compounds show better antioxidant activity, these food products promote health and wellness. Figure 6 provides an evaluation of the CUNE-enriched effect on the organoleptic properties of cheese compared to the control and emulsion cheeses during a 60-day period (at 4 °C). With a decrease in the particle size, there is an increase in the surface-to-volume ratio, and a reduction of the concentration at which compounds are added to food products. This does not hamper its sensory analysis compared to the control sample. The data show that lower concentrations can have more strength than otherwise [31], such as a droplet delivering the identical sensory profile [32] or an increase in sensory perception [33]. It is suggested that a larger particle surface causes an increase in the saliva dissolution rate and, possibly, a stronger taste perception. Figure 6 shows that there were no significant differences (*p* > 0.05) in color, smell, appearance, acceptability, or taste between the control and CUNE-enriched samples. However, when compared to the control and CUNE samples, the emulsion without curcumin samples had the lowest (*p* < 0.05) sensory values. This is owing to the structural change in the cheese matrix caused by the larger particle size of the emulsion affecting the organoleptic values. While the outer layer of a nanoemulsion can be designed to be resistant to the environment during the first stages With a decrease in the particle size, there is an increase in the surface-to-volume ratio, and a reduction of the concentration at which compounds are added to food products. This does not hamper its sensory analysis compared to the control sample. The data show that lower concentrations can have more strength than otherwise [31], such as a droplet delivering the identical sensory profile [32] or an increase in sensory perception [33]. It is suggested that a larger particle surface causes an increase in the saliva dissolution rate and, possibly, a stronger taste perception. Figure 6 shows that there were no significant differences (*p* > 0.05) in color, smell, appearance, acceptability, or taste between the control and CUNE-enriched samples. However, when compared to the control and CUNE samples, the emulsion without curcumin samples had the lowest (*p* < 0.05) sensory values. This is owing to the structural change in the cheese matrix caused by the larger particle size of the emulsion affecting the organoleptic values. While the outer layer of a nanoemulsion can be designed to be resistant to the environment during the first stages of ingestion, preventing consumers from experiencing any unpleasant tastes or odors, nanoencapsulation may offer a potential mechanism to physically trap the compounds that cause these unpleasant tastes and odors. The product's quality and acceptability are directly tied to the raw material and production quality. Consumers are showing a preference for minimally processed foods with the highest natural constituents, additives with health benefits such as antimicrobials, and naturally occurring antioxidants. In order to meet this demand, nanotechnology allows for the development of many ingredients' functionality by lowering the concentration of substances, altering their solubility, and enhancing or controlling their effectiveness [31,32].

ing or controlling their effectiveness [31,32].

of ingestion, preventing consumers from experiencing any unpleasant tastes or odors, nanoencapsulation may offer a potential mechanism to physically trap the compounds that cause these unpleasant tastes and odors. The product's quality and acceptability are directly tied to the raw material and production quality. Consumers are showing a preference for minimally processed foods with the highest natural constituents, additives with health benefits such as antimicrobials, and naturally occurring antioxidants. In order to meet this demand, nanotechnology allows for the development of many ingredients' functionality by lowering the concentration of substances, altering their solubility, and enhanc-

<sup>(</sup>**c**)

**Figure 6.** *Cont*.

(**e**)

**Figure 6.** Sensory analysis of cheese sample with addition of curcumin nanoemulsion and emulsion: (**a**) Odor, (**b**) Taste, (**c**) Shade, (**d**) Appearance, and (**e**) Acceptability. **Figure 6.** Sensory analysis of cheese sample with addition of curcumin nanoemulsion and emulsion: (**a**) Odor, (**b**) Taste, (**c**) Shade, (**d**) Appearance, and (**e**) Acceptability.

#### 2.4.2. Physicochemical Parameters for Cheese 2.4.2. Physicochemical Parameters for Cheese

The physicochemical features of nutriments play a part in defining their effectiveness and shelf life. Table 3 shows that the physicochemical properties of the cheese treated with two types of emulsifiers (CUNE and emulsion) compared to the control sample stored at 4 °C and kept for 30 and 60 days. With the fresh sample as a control, the CUNE- and emulsion-based cheeses do not have significant differences (*p* > 0.05) for dry matter, ash, and protein content. However, after 30 days, there are significant difference (*p* < 0.05) observed in the percentage of dry matter for CUNE and emulsion compared to the control, which increased in dry matter content. As dry matter content increases, the total phenolic content decreases and shows less antioxidant activity. Whereas the CUNE sample has less dry matter content over time, which maintains its integrity in the cheese matrix, as the nanoemulsion, with a smaller particle size, is better distributed within the cheese matrix. Fresh samples from each group did not differ significantly from one another in the content of ash, fat, or protein (*p* > 0.05), while the control samples showed significant differences (*p* < 0.05) after 30 and 60 days of storage. The percentage of fat, protein, and ash increased in the control sample, while the CUNE and emulsion samples showed no significant differences [34,35]. The presence of emulsifiers in the treated samples reduced the increase The physicochemical features of nutriments play a part in defining their effectiveness and shelf life. Table 3 shows that the physicochemical properties of the cheese treated with two types of emulsifiers (CUNE and emulsion) compared to the control sample stored at 4 ◦C and kept for 30 and 60 days. With the fresh sample as a control, the CUNE- and emulsion-based cheeses do not have significant differences (*p* > 0.05) for dry matter, ash, and protein content. However, after 30 days, there are significant difference (*p* < 0.05) observed in the percentage of dry matter for CUNE and emulsion compared to the control, which increased in dry matter content. As dry matter content increases, the total phenolic content decreases and shows less antioxidant activity. Whereas the CUNE sample has less dry matter content over time, which maintains its integrity in the cheese matrix, as the nanoemulsion, with a smaller particle size, is better distributed within the cheese matrix. Fresh samples from each group did not differ significantly from one another in the content of ash, fat, or protein (*p* > 0.05), while the control samples showed significant differences (*p* < 0.05) after 30 and 60 days of storage. The percentage of fat, protein, and ash increased in the control sample, while the CUNE and emulsion samples showed no significant differences [34,35]. The presence of emulsifiers in the treated samples reduced the increase of dry matter and thus reduced moisture loss compared to the control sample.

of dry matter and thus reduced moisture loss compared to the control sample. Thus, the nanoemulsion is recommended for fortifying cheese. For the pH, there was no noticeable

change in the pH values with a storage period of 30 or 60 days.

Thus, the nanoemulsion is recommended for fortifying cheese. For the pH, there was no noticeable change in the pH values with a storage period of 30 or 60 days.


**Table 3.** Physicochemical analysis of cheese.

#### *2.5. Scanning Electron Microscope Analysis of Cheese* 60 days 5.69 ± 0.09 41.84 ± 0.22 6.04 ± 0.05 11.74 ± 0.14 19.41 ± 0.12

SEM was used to study the structural morphology of the cheese matrix with the addition of CUNE and the emulsion in comparison to the control. Three-dimensional images were obtained to facilitate the identification of substances. The photograph of the sample (control samples 1, 2) is shown in Figure 7. There are fewer variances in the protein structure between the CUNE samples compared to the control. There is some free fat present, and the cheese fat and protein clusters are tightly packed. The control sample's protein and fat clusters look smaller than those in the CUNE sample. The control sample has substantially more free fat globules than those seen in the other samples. The fat-free globules in the control sample are smaller than those in the emulsion sample. The hardness was reduced, and the casein network structure softened when CUNE was added, showing that the incorporation of CUNE transformed the structure of the protein–fat network. CUNE was added together with emulsifying salts, which increased the dispersion of fat globules. It is possible to infer that the cheese produced with the CUNE supplement has particle-filled gel networks, where fat globules serve as filler molecules in the protein network. According to [31], foods containing nanoemulsions enhance the functionality of the formulations' constituents. Nanoemulsions can also be utilized to modify texture. Depending on the internal-phase percentage, oil composition, stabilizer (type and concentration), and droplet size, nanoemulsions may exhibit rheological characteristics that are different from those of viscous liquids in viscoelastic solids. *2.5. Scanning Electron Microscope Analysis of Cheese*  SEM was used to study the structural morphology of the cheese matrix with the addition of CUNE and the emulsion in comparison to the control. Three-dimensional images were obtained to facilitate the identification of substances. The photograph of the sample (control samples 1, 2) is shown in Figure 7. There are fewer variances in the protein structure between the CUNE samples compared to the control. There is some free fat present, and the cheese fat and protein clusters are tightly packed. The control sample's protein and fat clusters look smaller than those in the CUNE sample. The control sample has substantially more free fat globules than those seen in the other samples. The fat-free globules in the control sample are smaller than those in the emulsion sample. The hardness was reduced, and the casein network structure softened when CUNE was added, showing that the incorporation of CUNE transformed the structure of the protein–fat network. CUNE was added together with emulsifying salts, which increased the dispersion of fat globules. It is possible to infer that the cheese produced with the CUNE supplement has particlefilled gel networks, where fat globules serve as filler molecules in the protein network. According to [31], foods containing nanoemulsions enhance the functionality of the formulations' constituents. Nanoemulsions can also be utilized to modify texture. Depending on the internal-phase percentage, oil composition, stabilizer (type and concentration), and droplet size, nanoemulsions may exhibit rheological characteristics that are different from those of viscous liquids in viscoelastic solids.

(**A**) **Control Cheese** (**B**) **CUNE cheese** 

(**C**) **Normal Emulsion** 

**Figure 7.** Photo study experienced samples prepared using scanning electron microscopy (total magnification of ×5000) Cheese matrix: (**A**) Control, (**B**) CUNE (**C**) Normal emulsion. **Figure 7.** Photo study experienced samples prepared using scanning electron microscopy (total magnification of ×5000) Cheese matrix: (**A**) Control, (**B**) CUNE (**C**) Normal emulsion.

#### *2.6. Antioxidant Activity of Curcumin Nanoemulsion 2.6. Antioxidant Activity of Curcumin Nanoemulsion*

As shown in Table 4, the antioxidant capacity of the control sample and samples 1 and 2 is expressed as μg of Trolox equivalents (TE) per g of nanoemulsion. Regardless of surfactant content, the antioxidant capacity of the nanoemulsion as determined by the ferric reducing antioxidant power (FRAP) assay did not show any significant changes. The antioxidant capacity values reported by the FRAP assay, however, were considerably lower than those obtained by DPPH. In this respect, it follows that if one species is reduced, another must be oxidized. Curcumin's three active sites can be oxidized through hydrogen abstraction and electron transfer. Test samples differed in antioxidant activity depending on the additives introduced into the formulation. Samples 1 and 2, compared with the control sample, had a higher antioxidant activity. The CUNE cheese (sample 2) showed the most significant antioxidant activity (2.3 times the control sample). CUNE contributes to maintaining and enhancing BAS (curcumin) encapsulated in the nanoemulsion with sonication. This is due to the fact that the antioxidant capabilities of the nanoemulsions containing Tween 20 differed depending on the surfactant concentration and the assay for curcumin measurement utilized. Although based on our findings that high Tween 20 concentrations resulted in a slow release of curcumin, it has been reported that an excess of this surfactant can form micelles capable of encasing the bioactive compound, which enhances the protection of encapsulated curcumin and thereby boosts the system's antioxidant capacity. The sonication method, which compactly embeds curcumin in the nanoemulsion and controls its solubility, is one of the main reasons for the increase in antioxidant activity. **Table 4.** AOA prototypes cheese. As shown in Table 4, the antioxidant capacity of the control sample and samples 1 and 2 is expressed as µg of Trolox equivalents (TE) per g of nanoemulsion. Regardless of surfactant content, the antioxidant capacity of the nanoemulsion as determined by the ferric reducing antioxidant power (FRAP) assay did not show any significant changes. The antioxidant capacity values reported by the FRAP assay, however, were considerably lower than those obtained by DPPH. In this respect, it follows that if one species is reduced, another must be oxidized. Curcumin's three active sites can be oxidized through hydrogen abstraction and electron transfer. Test samples differed in antioxidant activity depending on the additives introduced into the formulation. Samples 1 and 2, compared with the control sample, had a higher antioxidant activity. The CUNE cheese (sample 2) showed the most significant antioxidant activity (2.3 times the control sample). CUNE contributes to maintaining and enhancing BAS (curcumin) encapsulated in the nanoemulsion with sonication. This is due to the fact that the antioxidant capabilities of the nanoemulsions containing Tween 20 differed depending on the surfactant concentration and the assay for curcumin measurement utilized. Although based on our findings that high Tween 20 concentrations resulted in a slow release of curcumin, it has been reported that an excess of this surfactant can form micelles capable of encasing the bioactive compound, which enhances the protection of encapsulated curcumin and thereby boosts the system's antioxidant capacity. The sonication method, which compactly embeds curcumin in the nanoemulsion and controls its solubility, is one of the main reasons for the increase in antioxidant activity.



20 (DIY ECO Cosmetic, 3.2 fat% milk (Prostokvashino) and NaCl were procured from a

#### **3. Materials and Methods**

Native lab grade curcumin (Magiya vostoka), Safflower oil (FAO Code: 0281), Tween 20 (DIY ECO Cosmetic, 3.2 fat% milk (Prostokvashino) and NaCl were procured from a local market in Chelyabinsk, Russian Federation. In all experiments, a 440 W U-sonic ultrasound at 80% power, 22 ± 1.65 kHz, and with a tip diameter of 22 mm. Distilled water was used for the experiment.

#### *3.1. Ultrasound Assisted Curcumin Encapsualtion 3.1. Ultrasound Assisted Curcumin Encapsualtion*

was used for the experiment.

*Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 11 of 16

Initially, to prepare a lipid phase of the emulsion, curcumin powder (0.05 g/mL) was dissolved in Safflower oil at 50 ◦C [15]. The aqueous phase was created by dissolving Tween 20 in distilled water at concentrations of 0.2 and 0.3 g/mL oil. The oil phase was added slowly to the water phase and stirred to make a coarse emulsion (15 min at 2000 rpm), then sonochemically at 80% amplitude of the total power (400 W). A digital thermometer was used to track the temperature during the emulsification and to keep it below 50 ◦C. A cool water jacket encircled the reactor, and the sonication time was divided into four cycles of 3 min each to reduce hot spot creation during sonication. Once the sample was prepared under vacuum, the emulsion was freeze dried (Figure 8). Initially, to prepare a lipid phase of the emulsion, curcumin powder (0.05 g/mL) was dissolved in Safflower oil at 50 °C [15]. The aqueous phase was created by dissolving Tween 20 in distilled water at concentrations of 0.2 and 0.3 g/mL oil. The oil phase was added slowly to the water phase and stirred to make a coarse emulsion (15 min at 2000 rpm), then sonochemically at 80% amplitude of the total power (400 W). A digital thermometer was used to track the temperature during the emulsification and to keep it below 50 °C. A cool water jacket encircled the reactor, and the sonication time was divided into four cycles of 3 min each to reduce hot spot creation during sonication. Once the sample was prepared under vacuum, the emulsion was freeze dried (Figure 8).

local market in Chelyabinsk, Russian Federation. In all experiments, a 440 W U-sonic ultrasound at 80% power, 22 ± 1.65 kHz, and with a tip diameter of 22 mm. Distilled water

#### *3.2. Physicochemical Analysis of Nanoemulsions 3.2. Physicochemical Analysis of Nanoemulsions*

#### Nanoemulsion Stability

Nanoemulsion Stability Nanoemulsion stability was confirm using a technique defined in Kumar et al. 2016 [28]. The 10 mL of nanoemulsions samples were kept in a hot water bath at 80 °C for 30 min, then moved to a freeze for 15 min, and monitored by centrifugation (Hettich Zentrifugen, Mikro 22R) at 5000 rpm for 30 min. The whole volume (WV) and emulsion phase volume (EPV) of the nanoemulsion in the centrifuge tube were measured. The nanoemul-Nanoemulsion stability was confirm using a technique defined in Kumar et al. 2016 [28]. The 10 mL of nanoemulsions samples were kept in a hot water bath at 80 ◦C for 30 min, then moved to a freeze for 15 min, and monitored by centrifugation (Hettich Zentrifugen, Mikro 22R) at 5000 rpm for 30 min. The whole volume (WV) and emulsion phase volume (EPV) of the nanoemulsion in the centrifuge tube were measured. The nanoemulsion stability was calculated using the formula below (ES):

$$\text{Nanomulus stability} \left( \% \right) = \frac{\text{Volume of nonoemulus phase}}{\text{Total volume of nonoemulus}} \times 100\tag{1}$$

Using Nanotrac FLEX, the average particle size of the fresh nanoemulsion was evaluated. For analysis, the nanoemulsion sample was diluted in water to measure the particle size through dynamic light scattering. The total phenolic content of the nanoemulsion was used to calculate encapsulation efficiency. The Folin–Ciocalteu reagent was used to determine the total phenolic content of the nanoemulsion. The percentage oxidation inhibition and encapsulation effectiveness of the CUNE were measured before and 30 min after the centrifugation at 5000 rpm. Encapsulation efficiency was determined using the method in Using Nanotrac FLEX, the average particle size of the fresh nanoemulsion was evaluated. For analysis, the nanoemulsion sample was diluted in water to measure the particle size through dynamic light scattering. The total phenolic content of the nanoemulsion was used to calculate encapsulation efficiency. The Folin–Ciocalteu reagent was used to determine the total phenolic content of the nanoemulsion. The percentage oxidation inhibition and encapsulation effectiveness of the CUNE were measured before and 30 min after the centrifugation at 5000 rpm. Encapsulation efficiency was determined using the method in [36] with some modifications. For instance, 15 mL of the nanoemulsion was centrifuged at 5000 rpm at 5 ◦C for 30 min after being passed through a filter membrane. Following the collection of centrifuged permeate, the UV (Shimadzu UV-2700, Japan) absorbance at 520 nm wavelength was measured. Triplets were performed for all the measurements. For optical imaging of the nanoemulsion, a sample on a slide was dried and checked at magni-

sion stability was calculated using the formula below (ES):

fications of 40<sup>×</sup> to 60×. CUNE and free curcumin scanned in the range of 400–4000 cm−<sup>1</sup> wavelength using a Fourier-transform infrared (FTIR) spectrometer. wavelength using a Fourier-transform infrared (FTIR) spectrometer. *3.3. Transmission Electron Microscope (TEM) of Nanoemulsion Samples* 

[36] with some modifications. For instance, 15 mL of the nanoemulsion was centrifuged at 5000 rpm at 5 °C for 30 min after being passed through a filter membrane. Following the collection of centrifuged permeate, the UV (Shimadzu UV-2700, Japan) absorbance at 520 nm wavelength was measured. Triplets were performed for all the measurements. For optical imaging of the nanoemulsion, a sample on a slide was dried and checked at magnifications of 40× to 60×. CUNE and free curcumin scanned in the range of 400–4000 cm−<sup>1</sup>

#### *3.3. Transmission Electron Microscope (TEM) of Nanoemulsion Samples* The magnitude and nature of the CUNE were resolute by TEM (JEOL-100 CX). An

*Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 12 of 16

The magnitude and nature of the CUNE were resolute by TEM (JEOL-100 CX). An aqueous solution of nanoemulsion sample (1:4 ratio) was sonicated for 10 min. A droplet of the sample was placed on a 200-mesh carbon-coated copper grid at room temperature and dried, before 2% uranyl acetate was added at 37 ◦C and grid mounted for TEM inspection. aqueous solution of nanoemulsion sample (1:4 ratio) was sonicated for 10 min. A droplet of the sample was placed on a 200-mesh carbon-coated copper grid at room temperature and dried, before 2% uranyl acetate was added at 37 °C and grid mounted for TEM inspection.

#### *3.4. Antimicrobial Activity of CuNE 3.4. Antimicrobial Activity of CuNE*

For the antimicrobial potential of CUNE, we used two different strains: one positive strain, *Staphylococcus aureus*, and one negative strain, *Escherichia coli*. To check antimicrobial activity, we performed a zone inhibition test. The bacteria culture was added to a Petri dish that contained nutrient agar. On the nutrient agar, was a sample that had been antimicrobially treated. The Petri dish was then kept at 36 ◦C for 18 to 24 h. For the antimicrobial potential of CUNE, we used two different strains: one positive strain, *Staphylococcus aureus*, and one negative strain, *Escherichia coli*. To check antimicrobial activity, we performed a zone inhibition test. The bacteria culture was added to a Petri dish that contained nutrient agar. On the nutrient agar, was a sample that had been antimicrobially treated. The Petri dish was then kept at 36 °C for 18 to 24 h.

#### *3.5. Preparation of Cheese with Nanoemulsion 3.5. Preparation of Cheese with Nanoemulsion*

The ultrafiltrate (UF) milk retentate was pasteurized (72 ◦C, 15 s) and then a 5% nanoemulsion with and without curcumin were enriched for cheese production at 32 ◦C. A standard white cheese was produced and used as a reference. The schematic formation of the cheese is shown in Figure 9. The ultrafiltrate (UF) milk retentate was pasteurized (72 °C, 15 s) and then a 5% nanoemulsion with and without curcumin were enriched for cheese production at 32 °C. A standard white cheese was produced and used as a reference. The schematic formation of the cheese is shown in Figure 9.

**Figure 9.** Schematic diagram for addition of curcumin nanoemulsion during cheese making. **Figure 9.** Schematic diagram for addition of curcumin nanoemulsion during cheese making.

Physical and Chemical Analysis of Nanoemulsion-Enriched Cheese Physical and Chemical Analysis of Nanoemulsion-Enriched Cheese

The pH of the nanoemulsion-based cheese was restrained through an electrode (model HI98103, Hanna Instruments, Romania) inserted into grated cheese after calibration with the standard buffers pH 4 and 7 from 22 to 31 ◦C. Titratable acidity was measured (g/100 g of lactic acid) using the technique in [37]. Dry matter (DM) content of cheese samples was analyzed using the oven drying method at 102 ± 2 ◦C. The Kjeldahl method determined total nitrogen (TN), WSN, and NPN (Jalilzadeh et al. 2020). The cheese was cut into cylinders at a height of 20 mm and a diameter of 20 mm, using a stainless-steel cylinder knife, and kept at room temperature (20 ◦C). The ash content of the cheese samples was determined using the method described in [38].

#### *3.6. Field Emission Scanning Electron Microscope of Cheese*

Cheese samples of 5 mm<sup>2</sup> had their exterior microstructure and morphology determined by FE-SEM. The cheese was immersed into liquid nitrogen to remove any moisture content and then ruptured. Using an ion sputter, gold was sucked into the punctured cheese. Finally, the sample was imaged using FE-SEM at 5 kV (Jeol JSM-7001F, Moscow).

### *3.7. Organoleptic Analysis of Cheese*

We invited 15 students (aged 25–40) from the food and biotechnology department of South Ural State University, Chelyabinsk for the organoleptic analysis of the cheeses. Each evaluator received the three kinds of cheese simultaneously, each with a different number assigned to it. The panel consisted of 15 experts who rated the smell, color, appearance, mouth feel, and taste in two consecutive sessions using shapeless scales with anchors at the split ends. A nine-point system was used (1 = extremely dislike, 3 = moderately dislike, 5 = neither like nor dislike, 7 = moderately like, and 9 = extremely like).

#### *3.8. Antioxidant of Processed Cheese*

A 0.1 mm DPPH radical solution was made to test the antioxidant activity of the cheese samples with CUNE. Using a UV spectrophotometer, the solution's absorbance was measured at 515 nm. Cheese samples were prepared by soaking 10 g in 90% ethanol for 45 min at 150 rpm in a LOIP LS-120 laboratory shaker. The mixture was then centrifuged for 10 min, and the supernatant was collected for testing. Each sample received 280 µL of DPPH radical solution and 20 µL of supernatant in a microplate. After 30 min of incubation, the samples were examined, and the absorbance was calculated using a 517 nm reference wavelength.

#### *3.9. Statistical Analysis*

For each group, the data are shown as the standard error of mean (SEM). GraphPad Prism software version 8.0 was used for statistical analysis (GraphPad software, 2019). The column values in Tables 1–4 were with significance of difference (*p* < 0.05).

#### **4. Conclusions**

The current work demonstrates the successful formation of stable curcumin encapsulation in a nanoemulsion with a high percent of loading (0.05 g/mL) containing Tween 20 surfactant using the ultrasound approach and its incorporation in cheese. CUNE shows good antimicrobial activity for *S. aureus* and *E. coli* (12- and 18-mm zone inhibition diameters). Based on the data, we can conclude that there is no negative impact of CUNE on cheese, as it maintain its overall sensory analysis and physicochemical properties in comparison with a control sample. A normal emulsion without curcumin shows reasonable sensory analysis compared to control emulsion.

**Author Contributions:** U.B. was responsible for conceptualization, methodology, validation, formal analysis, investigation, resources, data curation, writing—original draft preparation, and writing review, and editing; A.K. and M.A. were responsible for formal analysis and validation; I.P. and S.H.S. were responsible for supervision project administration.. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors acknowledge the RSF grant 22-76-10049 support for experimental and characterization of material for this manuscript.

**Institutional Review Board Statement:** There are no competing interests and there is no study on animals conducted for this manuscript. However, on human studies we have taken approval from the Ethics Committee of the Federal State Autonomous Educational Institution of Higher Education South Ural State University (NRU) with MU 28-1/2406.

**Informed Consent Statement:** There are no competing interests and there is no study on animals conducted for this manuscript. However, on human studies we have taken approval from the Ethics Committee of the Federal State Autonomous Educational Institution of Higher Education South Ural State University (NRU) with MU 28-1/2406.

**Data Availability Statement:** The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

**Acknowledgments:** The authors acknowledge the Department of Food and Biotechnology, South Ural State University, Chelyabinsk, Russian Federation, and RSF grant 22-76-10049 for financial support.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

## **References**


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## *Review* **Natural Gum-Based Functional Bioactive Films and Coatings: A Review**

**Arushri Nehra <sup>1</sup> , Deblina Biswas <sup>1</sup> , Valentina Siracusa 2,\* and Swarup Roy 1,\***


**Abstract:** Edible films and coatings are a current and future food packaging trend. In the food and envi-ronmental sectors, there is a growing need to understand the role of edible packaging and sus-tainability. Gums are polysaccharides of natural origin that are frequently utilized as thickeners, clarifying agents, gelling agents, emulsifiers, and stabilizers in the food sector. Gums come in a variety of forms, including seed gums, mucilage gums, exudate gums, and so on. As a biodegradable and sustainable alternative to petrochemical-based film and coatings, gums could be a promising option. Natural plant gum-based edible packaging helps to ensure extension of shelflife of fresh and processed foods while also reducing microbiological alteration and/or oxidation processes. In this review, the possible applications of gum-based polymers and their functional properties in development of edible films and coatings, were comprehensively dis-cussed. In the future, technology for developing natural gum-based edible films and coatings might be applied commercially to improve shelf life and preserve the quality of foods.

**Keywords:** natural gums; polysaccharides; edible polymers; coating and film; active packaging

#### **1. Introduction**

Balanced nutrition is essential to maintaining optimal health in an individual, and can be obtained through food. Hence, food plays an important role in everyone's lives. In this current era of globalization, food packaging has emerged as a most exciting and pro-ductive aspect of the food industry [1,2]. Food waste is currently a key concern, as it con-tributes to a high amount of food loss and has a negative influence on national resources and economic progress. Furthermore, food oxidation plays a significant role in degrading food quality due to its deteriorating effects. It reduces the nutritional quality and flavor of food, raises toxicity, and alters textural qualities. Thus, a primary focus of the food indus-try is to preserve food quality and wholesomeness, thus gaining in consumer acceptability. One viable alternative to dealing with food waste and plastic packaging problems would be through incorporating edible film- and coating-based food packaging [3–6].

The global production of packaging materials rises by roughly 8% every year. Over 90% of used plastics are accumulating in landfills, while <5% are recycled, resulting in a huge environmental threat [7,8]. Inventors have focused on the development of appropri-ate tools to resolve these concerns. Attempts have involved various tactics, such as in-creased safety, preservation quality, and recycling [9,10]. Biodegradable packaging is an attractive alternative to traditional plastics due to its sustainability, renewability, and nontoxicity. Thus, a variety of biopolymers have been used to produce materials for envi-ronmentally friendly food packaging [11–15]. Recently, demands have increased for food packaging that does not contribute to pollution, that is produced using environmentally friendly methods, and that remains affordable. Coatings and edible films are key packag-ing types for edible materials [4,16–19].

Food packaging extends shelf life by reducing unwanted variations in food and safeguarding food from microbiological infection, moisture loss, and exterior damage. In this

**Citation:** Nehra, A.; Biswas, D.; Siracusa, V.; Roy, S. Natural Gum-Based Functional Bioactive Films and Coatings: A Review. *Int. J. Mol. Sci.* **2023**, *24*, 485. https:// doi.org/10.3390/ijms24010485

Academic Editor: Francesco Trotta

Received: 16 November 2022 Revised: 19 December 2022 Accepted: 23 December 2022 Published: 28 December 2022

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

context, active food packaging (AP) technology is a promising approach [20–23]. Edible films and coatings can be used as postharvest treatments to protect the quality of foods while lowering the amount of nonbiodegradable packaging materials used. As a result, edible films and coatings fabricated from hydrocolloids (e.g., chitosan, carrageenan, pullulan, hydroxyl propyl methyl cellulose, alginate, etc.) are commonly used to maintain the quality of foods [6,24–28]. To make edible films, natural gums could be a promising alter-native, as they are biocompatible, inexpensive, nontoxic, and readily available [29,30]. Natural gums have attracted considerable interest recently due to their unusual rheologi-cal qualities and structural variety. Gum hydrocolloids, often called gum polysaccharides, are common and adaptable polymers utilized to collect materials with varying structural and functional characteristics [29]. Several novel gums have been reported in the litera-ture in recent years from various sources [31,32].

Gum-based edible packaging is already known to preserve the quality of fruits and vegetables after harvest [33]. Plants, microbes, and animal tissues are all sources of gums. However, plant-derived gums are the most popular [34]. Plant-derived gums are readily available. Furthermore, two or more gums can be mixed together to offer synergistic benefits [29]. In gums, plant seed gums like guar, locust bean, tara, and tamarind were used to extract or isolate polysaccharides [35]. Polysaccharides-based gums are generated from the endosperm of many plant seeds (mostly leguminosae). The majority of polysaccharides gums are galactomanans. These are polysaccharides that are primarily made up of the monosaccharides mannose and galactose. Depending on the plant origin, mannose components from linear chains are coupled with galactopyranosyl residues as sidechains [36].

Gums can bind water and produce gels. Mucilage gums, seed gums, exudate gums, and other types of gums exist [32,37]. Gums come from a variety of plant parts, e.g., seed epidermis, leaves, and bark [37]. Some plant gum exudates, such as karaya, Ghatti, and tragacanth, have been well documented in recent decades. Gum Arabic has been used for over 5000 years [4,38]. Natural gums have generated interest in fruit and vegetable coat-ing applications [39].

There have been few literature reviews on gum, gum extraction methods, or methods of dealing with gum structural modification. There have been studies on gum-based film and coatings and their application in food systems, however [35]. Previously, Khezerlou et al. reviewed fabrication of edible films and coatings using plant gums [35]. The use of gum coating on food products has been reviewed [29]. Recently, gums have been used in active and intelligent food packaging, i.e., film and coating [32,37,40,41]. Although some review articles have been published on natural plant gum-based films and coatings, there were, as of this review, no up-to-date reports available regarding the use of gums in food packaging. Therefore, in this review, we introduced various types of gums, their sources and properties, and finally, their utilization in food packaging film and coatings. Consequent-ly, the application of various gum-based bioactive functional composite film and coating in fruits, vegetables, and animal food product packaging was discussed comprehensively.

#### **2. Types of Plant Gums**

There are various types of plant-based gums. Gums and mucilage are mainly polysaccharides. Gums are known as polyuronides and contain various salts of potassium, magnesium, calcium, and so on [35]. Mucilages are mainly sulfuric acid esters of polysaccharides. Galactose and arabinose are commonly found sugars in gums and mucilage [29]. The details of various types of gums will be briefly discussed below.

#### *2.1. Seed Gum and Mucilage*

Gums are derived from a variety of plant components. Some gums come from the seed epidermis, whereas others are derived from plant leaves. Gums are pathogenic constituents that are produced by plant impairment or unfavorable situations. Mucilage is a naturally metabolic product generated in the cell. Mucilage is insoluble in water. Almost all plants and certain microbes create mucilage, a thick, sticky material. Both gums and

mucilage are plant hydrocolloids; hence, they have certain similarities. They are likewise made up of a combination of transparent amorphous polymers and monosaccharide polymers, as well as uronic acid [37]. Mucilage is widely used in the food industry, owing to its exceptional functional properties (e.g., antimicrobial, antioxidant, water-holding, oil holding, etc.) which are beneficial for food packaging film and coatings [35,37]. However, the films formed from mucilage films are fragile and have poor mechanical properties [35].

#### 2.1.1. Guar Gum

Guar gum is a galactomannan originating from the seed of the plant Cyamopsis tetragonolobus [42,43]. Guar gum is made by separating the endosperm from the hull and germ [44]. It is a high molecular weight, odorless polysaccharide attained from the guar plant that has a white to yellowish-white color. The guar plant grows to a height of 0.6 m, with pods ranging in length from 5 to 12.5 cm [31]. This plant is a largely sun-loving plant that can withstand high temperatures, but it is vulnerable to cold. Guar gum powder is the most common form used as a food ingredient. Guar gum is similar to locust bean gum in that it is made up mostly of the complex carbohydrate polymer galactose and mannose. However, the amounts of these two sugars differ in these gums. India produces 80% of the world's guar, with Rajasthan accounting for 70% of the crop. Guar is cultivated in north-ern provinces of India, including Rajasthan, Gujrat, Haryana, and Punjab, and India is the global leader in guar production [45]. Guar gum-based films are known for their great mechanical strength, good barrier qualities, and antibacterial or germ resistance [31]. Because of its long polymeric chain and high molecular weight relative to other forms of gums, guar gum is an excellent choice for making edible coatings. It is a galactomannan with a mannose backbone ((14)-linked -D-mannopyranose) and galactose side groups ((16)-linked -D-galactopyranose).

#### 2.1.2. Locust Bean Gum

Locust bean gum (LBG) is made from the seed endosperm of the carob tree's fruit pods. These are botanically known as *Ceratonia siliqua* L. and are found in Mediterranean countries. As a result, locust bean gum is often referred to as carob gum. The husk, endosperm, and germ are the three sections of the carob seed. LBG is used in food, paper, textiles, oil well drilling, and in the cosmetics sector [45]. It is a neutral polysaccharide comprised of mannose and galactose. The seeds are primarily made of galactomannan, which accounts for around 80% of the total weight. The remaining 20% is proteins and impurities. The protein concentration of LBG is roughly 32% albumin and globulin, and the rest is glutelin and impurities (ash and acid-insoluble materials) [46]. Among natural polysaccharides, LBG is a promising alternative for food packaging. LBG is only weakly soluble in cold water. In order to attain full hydration and maximal viscosity, an LBG solution must be heated up to a certain temperature. It can produce films and coatings with high mechanical and water vapor barrier characteristics. LBG has been explored for its potential applications in film production and coating applications [47]. It can also be used to stabilize dispersion and emulsion in the food sector as a fat replacement in several dairy products [34].

#### 2.1.3. Tara Gum

Tara gum, commonly known as Peruvian carob, is obtained from the seed endosperm of the *Caesalpinia spinosa* tree [48]. The primary constituent of this gum is galactomannan polysaccharides [49]. Tara gum is a commonly used food additive [50]. It is also used as a thickener and binding agent. The water-binding ability of Tara gum makes it perfect for rapid hydration-forming thick colloidal solutions. It can be used as a thickening agent or viscosity modifier. Additionally, it is used as a polymer matrix for food packaging applications.

#### 2.1.4. Basil Gum

Basil (*Ocimum basilicum* L.) is widely grown in India's Himalayan states of Jammu and Kashmir. In aqueous conditions, the seeds of this plant create mucilage. Mucilage is produced and is closely linked to the seed core, providing a large amount of polysaccharides and soluble fiber [51]. Basil is an aromatic plant that is commonly used to give food a distinct aroma and taste. To use as a spice, the leaves can be used fresh or dried. Food additives can be made from essential oils taken from fresh plants and flowers. Basil seed gum (BSG) has great potential in the food sector as a gelling, foaming, thickening, binding, fat replacement, and reducing agent [52].

#### 2.1.5. Fenugreek Gum

Natural fenugreek gum is produced by the *Trigonellafoenum graecum* plant (Family Leguminosae). Northern Africa, Canada, Western Asia, and India are among the countries that grow it. It can be used as a spice, a vegetable, or a medicinal herb, among other things [53]. The leaves, seeds (whole and gum), chemical components (such as hydroxy isoleucine), and immature shoots are known to possess antioxidant activities [54]. It is also applied as a food binder, glue, and emulsifier in food products [53]. More significantly, it has been used to create healthy, nutritious extruded and baked products. Because of its dietary fiber, protein, and gum content, it is utilized as a food stabilizer, glue, and emulsifying agent. Fenugreek is made up of 23–26% fenugreek, protein, 6–7% fat, and 58% carbohydrates (approximately 25% of which is nutritional fiber) [55]. Dietary fiber, particularly soluble fiber, may be found in a variety of foods and beverages, including cereal bars, yogurts, and nutritious beverages. Soluble fiber powder or total dietary fiber powder can be mixed with fruit juices, spices, and other spice blends [56].

#### *2.2. Exudate Gum*

Plant exudate-based gums originate from the bark and branches of trees to protect them from environmental and microbiological harm. The earliest forms include exudates gums, which are regularly utilized as thickeners, stabilizers, rheology modifiers, soluble fiber, and fat replacers [44].

#### 2.2.1. Gum Ghatti

The extruded gum from the *Anogeissus latifolia* tree, commonly known as India Gum, is known as gum ghatti (GG). Gotifilia is the term given to a newly produced GG, a spraydried powder made from specially selected high-grade GG. It has a uniform color and is extremely soluble. The high emulsification characteristics of GG is a key attribute. GG's capacity to emulsify is superior to that of gum Arabic or any other natural gum. It may form stable emulsions at concentrations as low as 25% of those of gum Arabic [57]. Due to its component glycoprotein, GG could potentially be useful in manufacture of films. It is commonly used in the food industry as a thickener, stabilizer, and emulsifier [58]. It has been used in India since ancient times due to medicinal and related characteristics that make it beneficial for use in food items. It is found in numerous writings such as Indian (Ayurveda) and Greek (Unani) medical systems. It is used as a wellness product in some parts of India, or as a mark of status and richness [32].

#### 2.2.2. Persian Gum

Persian gum (PG) is derived from the *Amygdalus scoparia*. Since PG is less expensive than other natural hydrocolloids, it is a possible alternative to other gums. It is comprised of approximately 90% polysaccharides, mostly galactopyranosyl and arabinofuranosyl, based on the dry weight. PG can normally be dissolved in water at 25–30% which makes it a good chemical for the production of film-forming solutions [59]. Currently, it has the potential to be used as a suspending or emulsifying agent in foods, medicines, and other sectors [44].

### 2.2.3. Tragacanth Gum

Tragacanth gum (TG) is produced by wounds in some plants. *Astragalus gummifer* and other *Asiatic Astragalus* species generate the gum as a dry exudate from their stems and branches [40]. It is commonly utilized as a natural thickening agent and emulsifier. Additionally, it possesses outstanding thermal stability, great solubility, and good rheological behavior [60]. It is an excellent emulsifier which enhances aqueous phase viscosity [61]. TG is known to be used as a thickening agent in a variety of foods, including sauce, ice cream, jelly, salad dressing, syrup, confectionery, and mayonnaise [40]. In the presence of water, TG expands and forms a polymeric molecular network. This eventually stabilizes the aqueous and serum phases of food items and increases their viscosity. TG is also utilized as a binder in a variety of confectionary products (i.e., candies). Subsequently, it is used in ice cream to achieve a smooth texture as well as to hinder ice crystal development during storage [32]. In Table 1, types of gums used in fabrication of bio-based edible film are presented.

**Table 1.** Types of natural gums used in fabrication solution casting films.


### **3. Preparation of Natural Gum-Based Films and Coatings**

The fabrication of natural gum-based films is primarily performed by relatively simple solution casting techniques. Various stages of film casting processes are schematically represented in Figure 1, while an overview of preparation process of films or coatings for active food packaging application is illustrated in Figure 2. Furthermore, the preparation of various natural gum-based films is briefly described herein, and some of the results are tabulated in Table 2.

tabulated in Table 2.

tabulated in Table 2.

*Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 6 of 20

of various natural gum-based films is briefly described herein, and some of the results are

of various natural gum-based films is briefly described herein, and some of the results are

**Figure 1.** Schematic representation of various stages in a film casting process (Modified from Yong **Figure 1.** Schematic representation of various stages in a film casting process (Modified from Yong & Liu et al., 2021 [66]). **Figure 1.** Schematic representation of various stages in a film casting process (Modified from Yong & Liu et al., 2021 [66]).

**Figure 2.** Preparation process of films or coatings for active food packaging application (Modified from Ribeiro et al. (2021) [67]).


**Table 2.** Various plant originated gum based functional films and coatings.


### **Table 2.** *Cont.*

#### *3.1. Guar Gum*

A total of 150 mL of film-forming solutions were put onto Teflon plates (15 cm × 15 cm) lying on a flat surface to make the films. The dried films were peeled away from the surface of the casting. In a controlled temperature and humidity chamber, films were equilibrated at 23 ◦C and relative humidity (RH) of 50% to measure barrier and mechanical characteristics [62].

#### *3.2. Locust Bean Gum*

For complete solubilization of LBG in an aqueous solution, it was heated for 1 h at 80 ◦C using a magnetic stirrer. The gum solutions were left to stand overnight at 4 ◦C after dissolution. Before casting into plates, film-forming solutions were centrifuged to eliminate air bubbles. Each gum solution was poured into Teflon plates and dried in an air oven. They were then carefully peeled off the plates and stored with saturated Mg (NO3)<sup>2</sup> at 23 ◦C until they reached a constant weight at RH of 52.80 ± 0.20% [47].

#### *3.3. Tara Gum*

TG (0.75%) solution was made in distilled water using agitation at 45 ◦C. The plasticizer (a 1:1 combination of sorbitol and glycerol) was then added. After that, ultrasonic treatment was used to eliminate any remaining air bubbles in the solution and it was cast in petri plates [63].

#### *3.4. Basil Seed Gum*

A total of 70 g of mucilage was mixed with 30 g kg−<sup>1</sup> (based on BSM weight) glycerol as a plasticizer to make basil seed mucilage (BSM) film. Then, depending on the original weight of BSM, 30 g kg−<sup>1</sup> of TA, MA, and SA were added. At 60 ◦C, the solution was agitated continuously for 45 min. BSM was made by putting the mixture onto a polypropylene plate and drying it for 10 h at 40 ◦C in a hot air oven. To complete crosslinking, the BSM film was baked in the oven at 150 ◦C for 10 min. Before characterization, the dry BSM film was peeled off and stored [64].

#### *3.5. Fenugreek Gum*

FSG was dissolved in distilled water containing glycerol for 3 h at 1000 rpm at 65 ◦C. Nano clay (2.5, 5, and 7.5%) was then gently mixed into the FSG solution. The nanocomposite films were made by pouring the film forming solutions onto Teflon plates and drying them for 24 h at 45 ◦C [55].

#### *3.6. Ghatti Gum*

GG films were made by mixing 0.75 and 1.0% GG in water for 15 min at 25 ± 2 ◦C with steady stirring. In a thermostatic bath water, they were heated at 40 ◦C for 1 h. After cooling to room temperature, plasticizer was mixed, and then cast on Petri dishes [58]. Finally, dried films were immersed in crosslinking solutions (1.5% CaCl<sup>2</sup> + 1% citric acid) for 5 min to yield crosslinked films.

#### *3.7. Tragacanth and Persian Gum*

Tragacanth and Persian gum granules were mixed in water and heated to 60 ◦C for 30 min until the particles were completely dissolved. Following that, glycerol was added and agitated for an additional 15 min and then cast to make film [65].

#### *3.8. Mucilage*

Various types of mucilage were already used for the fabrication of film [77–80]. Recently biodegradable film was developed based on *Pereskia aculeata* Miller mucilage [81]. The film-forming solution was made by dissolving 1.5, 1.8, and 2.0% of the mucilage in water and kept for 12 h for hydration. Thereafter homogenization was used at 12,000 rpm for half an hour and then varying content of glycerol (20–25% (*w*/*w*)) was mixed and again stirred for 10 min. The film forming solution was casted in Teflon coated surface to fabricate the film.

#### **4. Various Film Forming Properties of Natural Gums**

Primarily, there are two key types of packaging: edible films & coatings. The films are mainly utilized for edible packaging, wrapping over the food surface whereas the coating is directly used on the food surfaces [11]. Casting and extrusion are the most commonly used tools to fabricate film. On the other hand, there are many methods, such as dipping, panning, spraying, etc., for coatings [13].

The film casting process uses a wet chemical method for developing film. It is the most commonly used method for making film at lab scale. In this method, the biopolymers are solubilized in a solvent, and in this regard, water and ethyl alcohol are most commonly used. The completely soluble biopolymer solution is poured into the mold and dried until all the solvent is removed to make a film [22]. The primary advantage of this method is easy handling without any equipment and cost effectiveness. However, it has many limitations, including a long processing time, limited forms, etc. On the other hand, the extrusion technique is a commercially-applicable dry method for making film. In this method, generally, no solvent is used. It includes melting of biopolymers and mixing with other ingredients to produce film of the prescribed thickness and shape. The extrusion process in temperature sensitive and as a result, biopolymers which are highly sensitive to temperature cannot be used in the extruder [13]. Extrusion is an efficient and high-performance method for commercial purposes but in this process, only temperature-tolerant polymers can be used. Additionally, the cost of equipment is high, another major drawback of this method.

In coating, dipping is the most commonly-used method in lab scale. It includes immersion of the food system in the coating formulation and generation of a thin layer on the food product [28]. A thick coating is sometimes disadvantageous for respiration of food. Another widely-applied technique is spraying, in which liquid solution is sprayed in the form of small droplets on the surface of the food items. There are different types of spraying methods, such as air spray atomization, pressure atomization, etc. [28]. This method produces uniformly thick coating on the food surface but the highly viscous coating formulation is difficult to spray. Panning is another suitable and efficient method for coating of foods, where in a large pan of food is coated while spinning. Using this method, large quantities of food items can be coated easily [28].

While edible films and coatings lack in several essential packaging characteristics (e.g., mechanical strength and water barrier capabilities, lack of functionalities), they provide certain unique features (e.g., biodegradability, sustainability) to food packaging. There are solutions to extant problems. Mixing of bioactive components like essential oils (EOs) in the gum-based edible film can be useful to improve physical and functional properties (e.g., antibacterial and antioxidant activities). The release of oil-soluble compounds from the edible film into the mobile lipid phase of fatty meals might provide extra nutritional advantages while also preventing oxidative rancidity and microbiological deterioration. A decrease in film hydrophilicity was predicted with the addition of essential oil, crosslinking, or bioactive compounds. The addition of orange oil, along with curcumin, induced antibacterial properties in GG films [68].

In a study, Martins et al. developed LBG- and kappa-carrageenan (k-car)-based edible films with specific qualities [70]. The mixing of k-car to LBG increased the films' barrier characteristics, resulting in a reduction in WVP. Furthermore, as compared to carrageenan and LBG films, the carrageenan/LBG blend films had a higher tensile strength (TS). The same authors studied the effects of organically modified clay Cloisite 30B (C30B) on the same composition of film [69]. The authors reported that, with the increase in clay content, strength and flexibility were improved remarkably. Moreover, the authors reported that, as quantity of C30B in the film formulation was increased, the antibacterial activity toward *L. monocytogenes* improved. Carrageenan/LBG–C30B showed antibacterial action.

Gahruie et al., observed that, in the presence of *Z. multiflora* essential oils nanoemulsion, the mechanical characteristics of the film improved and they showed increased antibacterial activity by reducing the particle size of the nanoemulsion [72]. *Z. multiflora* essential oils (ZMEO) were effectively added to basil seed gum films in order to fabricate next-generation active packaging materials with enhanced antimicrobial properties. In another study, when oregano essential oil was added to basil seed gum edible film, it resulted in a film with potential antibacterial and antioxidant properties [71]. Memis et al. used a nano clay to fabricate FSG films [55]. The nano clay improved the barrier properties of the films. FSG-based nanocomposite films have good mechanical characteristics and antibacterial activities, which could be promising for use in food packaging.

A combination of sodium alginate and GG was investigated in order to enhance the performance of biodegradable SA film [73]. The film had improved mechanical and barrier properties. In addition, the mix film's light barrier qualities were increased by 65.17%. Khezerlou et al. reported a film made from sodium caseinate (SC), *Zingiber officinale* extract (ZOE), and PG [74]. The addition of ZOE resulted in a considerable improvement in tensile strength. However, the presence of ZOE and the PG reduced elongation at break (EB). Furthermore, the addition of ZOE improved hydrodynamic properties, but the presence of PG increased opacity. Tonyali et al., studied the effects of whey protein isolate (WPI) on tragacanth gum-based film [75]. The results showed that combining WPI and TG in film formulation resulted in elastic, less soluble films with reduced water permeability and less transparency. The addition of Plantago seed mucilage in carrageenan gum led to reduction in mechanical strength and increase in flexibility of the film. The antioxidant activity and crystallinity of the carrageenan-based film was improved [76]. The blending of chia seed mucilage (2.5%) on levan-based film was studied [82]. The author reported that the presence of mucilage had a good impact on the mechanical and barrier properties of the levan-based film. The transparency of the film was reduced, but the antioxidant activity and antimicrobial performance of the film were significantly enhanced.

The biodegradable film fabricated using *Pereskia aculeata* Miller mucilage was opaque and the films mechanical properties was low (TS = ~1–5 Mpa) [81]. The moisture barrier properties of the films based on mucilage were higher compared to synthetic film and the water solubility is low. The film also showed good thermal stability. The authors inferred that both mucilage and glycerol content affected the physical properties such as mechanical, barrier, and thermal properties of the film. In another work, quince seed mucilage (1%) based functional packaging film was developed adding thyme essential oil [83]. The film exhibited good mechanical and barrier characteristics excellent antioxidant activity and inhibited the growth of *Shewanella putrefaciens, Listeria monocytogenes* and *Staphylococcus aureus*.

Dick et al., 2015 studied the fabrication of Chia seed mucilage edible film [84]. 1% mucilage and varying content of glycerol (25, 50, 75% (*w*/*w*)) was used to produce the film. The fabricated films presented high solubility, transparency, and strong UVlight barrier properties. With increasing glycerol content, the mechanical strength of the film was reduced while the elongation at break and water vaper permeability increased. The blend film of chia seed mucilage in whey protein isolate (WPI) was studied by Muñoz et al., 2012 [85]. The authors used chia mucilage in blend (1:3 or 1:4) with WPI for the development of film. They reported the formed film showed good mechanical properties and high water vapor barrier properties. In another work edible and biodegradable film was developed using mucilage from *Opuntia ficus-indica* [86]. The mucilage-based films' physical properties were reported to improve by adding pectin. Araújo et al., studied the Okra mucilage based edible packaging film and reported the characteristics of the film was analogous to the other polysaccharide-based film [87]. The physical properties of the film such as water vapor permeability, solubility, thermal and mechanical properties, etc. were improved by mixing with starch. Very recently the cactus (*Cylindropuntia fulgida*) mucilage has also been used to fabricate biopolymer based antimicrobial packaging film [88]. The authors used Cactus mucilage and gelatine as biopolymers while *Euphorbia caducifolia* extract as an antimicrobial agent. The various physical properties such as water solubility, the water vapor barrier properties, flexibility as well as antimicrobial activity of the films were meaningfully improved in presence of 20% extract.

#### **5. Application in Food Preservation**

Various gum-based edible coating and films are effective to delay aging and extend the storage life of various foods. Natural hydrocolloid-based edible coatings and films provide extra protection to foods.

Dipping, coacervation, and spraying are a few of the available methods for applying edible coatings on food [89]. Each method has a number of reported benefits and drawbacks, and the success of each method is greatly determined by the traits and qualities of the items to be coated, as well as the coating's physical characteristics (viscosity, surface tension, density) [90]. For instance, in a study, while using the dipping approach, the outer layer of the meal was seen to be diluted by coating suspensions. Consequently, functional properties of the coating are reduced by fruits and vegetables. Edible finishes are often applied in single layer coatings on food using the dipping technique [91]. Natural gum edible coatings provide a promising way to expand the quality of foods while also extending their shelf life [29]. The use of natural gum-based films and coating on food preservation is briefly presented in Table 3.

GG was used to delay the ripening of Roma tomatoes by reducing their respiration rate [92]. The results indicated that GG coating not only preserved firmness but also enhanced postharvest quality when stored at room temperature. The GG coating was transparent and stuck effectively to the surface of the Roma tomato. During the 20-day storage period, all tomato fruits shrunk, but the coated ones shrunk more slowly than the uncoated ones. The GG coating was biodegradable, easy to apply, and less costly than other hydrocolloids and commercial waxes. Therefore, it could be applied large-scale to extend the shelf-life of Roma tomatoes (Figure 3).


**Table 3.** Application of gum-based film films and coatings on food preservation.

**Figure 3.** Effect of storage time (day 1 and day 15) on Roma tomatoes coated with guar gum (C) and uncoated (UC) at room temperature (22± 2 °C) (Modified from Ruelas-Chacon et al., 2017 [92]). **Figure 3.** Effect of storage time (day 1 and day 15) on Roma tomatoes coated with guar gum (C) and uncoated (UC) at room temperature (22± 2 ◦C) (Modified from Ruelas-Chacon et al., 2017 [92]).

Mangoes are seasonal fruits with a short post-harvest life. Their storage life is mostly determined by the mango fruit variety chosen and the storage conditions. When kept at 13 °C, the shelf life might be as long as a week. Mango fruit losses of 20–30% are recorded Mangoes are seasonal fruits with a short post-harvest life. Their storage life is mostly determined by the mango fruit variety chosen and the storage conditions. When kept at 13 ◦C, the shelf life might be as long as a week. Mango fruit losses of 20–30% are recorded

annually, amounting to 3000 tons (~28 million USD), owing to incorrect handling, insufficient storage and poor strategies after harvest [93]. Various techniques have been used to

coated with GG infused with EOs had a 24-day shelf life. The hardness of the mangoes diminished as the storage period progressed. However, the delicious scent of the raw mangoes remained. The fruits' skin color altered from greenish to yellowish, indicating

The use of LBG coating on sausages led to a prominent reduction in moisture loss [94]. Coating lowered the rate of respiration, reduced the oil content, and prolonged the life span of the meat. The coated samples could be stored for up to two weeks during cold storage at 5 °C. TG reduced mass loss, maintained firmness, reduced color change, and inhibited mold and yeast development in peaches [95]. The best results were obtained using Tara gum in combination with citric and ascorbic acids, as well as sodium chloride. Tara gum reduced mass loss, maintained firmness, reduced color change, and inhibited mold and yeast development. As a result, this gum showed great potential an edible coat-

The use of BSG coating as a polysaccharide-based coating on fresh strawberries improved their physicochemical, sensory, and microbiological qualities during cold storage [96]. Furthermore, adding echinacea extract to the BSG coating composition improved the quality of fresh strawberries in a synergetic manner. Microbial counts (yeast and molds)

Recently it was reported that the optimal coating composition was able to increase the postharvest quality of guava fruit [97]. The response surface approach was shown to

decreased as the content of Echinacea extract increased.

that they were fully ripe.

ing substance.

annually, amounting to 3000 tons (~28 million USD), owing to incorrect handling, insufficient storage and poor strategies after harvest [93]. Various techniques have been used to increase the life span of mangoes in normal and cold storage environments. Mangoes coated with GG infused with EOs had a 24-day shelf life. The hardness of the mangoes diminished as the storage period progressed. However, the delicious scent of the raw mangoes remained. The fruits' skin color altered from greenish to yellowish, indicating that they were fully ripe.

The use of LBG coating on sausages led to a prominent reduction in moisture loss [94]. Coating lowered the rate of respiration, reduced the oil content, and prolonged the life span of the meat. The coated samples could be stored for up to two weeks during cold storage at 5 ◦C. TG reduced mass loss, maintained firmness, reduced color change, and inhibited mold and yeast development in peaches [95]. The best results were obtained using Tara gum in combination with citric and ascorbic acids, as well as sodium chloride. Tara gum reduced mass loss, maintained firmness, reduced color change, and inhibited mold and yeast development. As a result, this gum showed great potential an edible coating substance.

The use of BSG coating as a polysaccharide-based coating on fresh strawberries improved their physicochemical, sensory, and microbiological qualities during cold storage [96]. Furthermore, adding echinacea extract to the BSG coating composition improved the quality of fresh strawberries in a synergetic manner. Microbial counts (yeast and molds) decreased as the content of Echinacea extract increased.

Recently it was reported that the optimal coating composition was able to increase the postharvest quality of guava fruit [97]. The response surface approach was shown to be a useful statistical tool for separating the interacting effects of independent variables. Weight loss and TSS were considerably decreased when edible coatings based on FG and Guar galactomannan were used. Furthermore, during storage, the covered fruit was fresher, firmer, and lower in TA. Weight loss was found to be 1.71% and 2.11%, firmness to be 0.72% and 2.14%, TSS to be 1.02% and 1.44%, pH to be 0.83 and 1.36, and acidity to be 1.03% and 1.44% in edible coatings, respectively. Coated guava showed a significant reduction in weight loss and maximal firmness retention. TSS enhanced in all treatments up to a specific storage duration and then declined as the storage period extended. However, pH increased while acidity decreased significantly. The edible coating of guava may be enhanced significantly by integrating Guar galactomannan and FG.

Grapes were coated with chitosan-based formulas with and without GG to increase postharvest quality [57]. Weight loss, acidity, pH, etc., of grapes were all improved when gum-ghatti was added to the chitosan solution. During two months of cold storage, coatings slowed down variations in ascorbic acid and enhanced polyphenol oxidase antioxidant enzyme activity. The chitosan-gum-ghatti-based coatings retained the bioactive components in grapes. The appropriate sort of chitosan-GG coating on grapes could help to improve its life span during transportation.

In comparison to untreated controls, the use of Kurdi Gum (KG) and PG solutions considerably enhanced the sensory features of bananas, and the addition of 0.25 and 0.5% *Prosopis farcta* extract enhanced the sensory features even more [98]. As a result, using KG and FG coatings supplemented with *P. farcta* extract should help boost banana commercialization during long-term storage. Tragacanth gum is commonly utilized in the food sector as a polysaccharide food covering. Reduced respiration, dehydration, and enzymatic browning are achieved by using TG as a food coating. Because fruits like bananas deteriorate quickly, TG has also been used as an edible covering material. In comparison to uncoated samples, coated dried banana slices showed reduced shrinkage, higher quality, low water loss, and improved rehydration [60].

The flaxseed mucilage (0.75%, 1% and 1.25%) and xanthan gum (0.5%) was used as a coating material for preservation of Cheddar cheese and it was reported that the coating showed substantial effect on the storage shelf-life of Cheddar cheese [99]. In another work, the effect of varying content of plantego mucilage (15, 20 and 25%) was added to the Arabic

gum solution and used as a coating material for chicken breast storage [100]. The meat specimen coated with 25% Plantago showed the lowest value of lipid peroxidation and total bacterial count. The coating significantly improved the life span of chicken breast during storage for 3 weeks.

Taghinia et al. 2021, recently developed intelligent packaging film using *lallemantia iberica* seed mucilage and curcumin for Shrimp freshness indicator [101]. They found that mechanical strength of the film was meaningfully improved while the flexibility reduced in presence of curcumin. The functional film also showed good antioxidant activity as well as antibacterial/mold activity. Moreover, the film showed good pH dependent color indicator properties which used to detect the freshness of Shrimp. It was reported that good correlation between TVBN content of shrimp and color change of the film during storage. The quince seed mucilage-based edible films functionalized with essential oil (oregano or thyme) was used for preservation of rainbow trout fillets [102]. It was reported that the thyme oil incorporated film effectively reduced the microbiological count in the fish fillets during refrigerated storage. Kang et al. 2020, developed okra mucilage and polyvinyl alcohol-based smart color indicator packaging label using anthocyanin [103]. The film showed distinctive color change in pH 2–12 range. The color indicator film was efficiently screen shrimp freshness in real time and the changes in color of the film were clearly identified. *Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 15 of 20

> Edible coatings and films on the surface of food act as a semipermeable membrane barrier which in turn restricts the exchange of gas and moisture [104,105]. The gas and moisture adjust the internal atmosphere of the food, affecting food qualities such as color, sensory quality, firmness, antioxidant activity, etc. Apparently, the different food quality parameters greatly influence food shelf-life [105]. Employing coating and film on food surfaces can effectively reduce the gas and moisture exchange of the food. The inclusion of antioxidant and antimicrobial agents into the food packaging matrix (coating or film) also help improve food shelf life by restricting the growth of unwanted foodborne pathogens, as well as by lowering the oxidation of food [28,104]. The mechanism of an active packaging system is schematically presented in Figure 4. The gas and moisture scavengers are absorbed by the functional active packaging system. The gas scavenger is used to restrict the browning reaction of foods. parameters greatly influence food shelf-life [105]. Employing coating and film on food surfaces can effectively reduce the gas and moisture exchange of the food. The inclusion of antioxidant and antimicrobial agents into the food packaging matrix (coating or film) also help improve food shelf life by restricting the growth of unwanted foodborne pathogens, as well as by lowering the oxidation of food [28,104]. The mechanism of an active packaging system is schematically presented in Figure 4. The gas and moisture scavengers are absorbed by the functional active packaging system. The gas scavenger is used to restrict the browning reaction of foods.

**Figure 4.** Mechanism of active packaging (Reproduced from Ahmed et al., 2017 [106]). aging purposes, but neat gum-based film exhibited very low mechanical properties and **Figure 4.** Mechanism of active packaging (Reproduced from Ahmed et al., 2017 [106]).

The food sector has struggled to preserve and extend the life span of chopped fresh

The manufacturing of bio-based edible coatings and films based on plant-derived natural gums has received enormous attention in recent years. Several researchers have studied and evaluated the potential of plant gums as a possible replacement for synthetic packaging. By altering the proportion of plasticizers and bioactive compounds, edible films with preferred physical characteristics and antioxidant/antimicrobial activities may be developed. Plant-derived, gum-based functional films and coatings are effective in food preservation as they delay ripening, lower respiration, reduce oxidation, hinder microbial development, and carry antioxidants and antimicrobial chemicals that eventually

Natural gum-based bioactive films and coatings could be beneficial for active pack-

Therefore, attempts to reduce microbial contamination in fresh fruits and vegetables are inevitable. In this context, applying various coatings on chopped fruits and vegetables could effectively reduce the spread of microbial illnesses. Edible gum-based coatings provide a reductive packaging approach to enhance the life span of foods and prevent post-

harvest illnesses [107].

improve the foods' life span.

**6. Conclusions and Future Prospective** 

The food sector has struggled to preserve and extend the life span of chopped fresh fruits and vegetables. Thus, various food preservation methods have been investigated. Foodborne pathogens have been identified as one of the leading causes of human illness. Therefore, attempts to reduce microbial contamination in fresh fruits and vegetables are inevitable. In this context, applying various coatings on chopped fruits and vegetables could effectively reduce the spread of microbial illnesses. Edible gum-based coatings provide a reductive packaging approach to enhance the life span of foods and prevent postharvest illnesses [107].

#### **6. Conclusions and Future Prospective**

The manufacturing of bio-based edible coatings and films based on plant-derived natural gums has received enormous attention in recent years. Several researchers have studied and evaluated the potential of plant gums as a possible replacement for synthetic packaging. By altering the proportion of plasticizers and bioactive compounds, edible films with preferred physical characteristics and antioxidant/antimicrobial activities may be developed. Plant-derived, gum-based functional films and coatings are effective in food preservation as they delay ripening, lower respiration, reduce oxidation, hinder microbial development, and carry antioxidants and antimicrobial chemicals that eventually improve the foods' life span.

Natural gum-based bioactive films and coatings could be beneficial for active packaging purposes, but neat gum-based film exhibited very low mechanical properties and high water affinity compared to synthetic plastic film—which is a primary concern in making active packaging film. Moreover, these gum-based films are not effective for all types of food products owing to their permeable acid, base and water—which can be addressed by mixing with other additives and biopolymers. The mechanical properties of neat natural gum-based films are lower compared to conventional packaging. However, these mechanical (and other physical) properties could be improved by incorporating other biopolymers, bioactive materials, or additives. One of the major tasks facing researchers is to find an appropriate recipe for plant gum biopolymers and additives to achieve optimum parameters for the packaging of food products. Moreover, in most cases, wet methods are produced to develop packaging, which makes commercialization troublesome. Even though natural gum-based materials are promising, further research should emphasize the use of optimum combinations of edible covering materials to improve the nutritional value of foods. Since gum-based edible coatings and films are in the developmental stage, future research should focus on development of prototypes. It is anticipated that researchers will soon be able to address key challenges and advance appropriate skills that will aid industries in upscaling the fabrication of edible films and coatings for food products.

**Author Contributions:** Conceptualization, A.N. and S.R.; software, A.N. and D.B.; validation, A.N., D.B., V.S. and S.R.; writing—original draft preparation, A.N. and S.R.; writing—review and editing, D.B., V.S. and S.R.; visualization, D.B., S.R. and V.S.; supervision, S.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


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## *Article* **Silver Nanoparticles and** *Glycyrrhiza glabra* **(Licorice) Root Extract as Modifying Agents of Hydrogels Designed as Innovative Dressings**

**Magdalena K˛edzierska <sup>1</sup> , Magdalena Ba ´nkosz <sup>2</sup> , Anna Drabczyk 2,\*, Sonia Kudłacik-Kramarczyk <sup>2</sup> , Mateusz Jamrozy˙ 3,\* and Piotr Potemski <sup>1</sup>**


**Abstract:** The interest in the application of plant extracts as modifiers of polymers intended for biomedical purposes is constantly increasing. The therapeutical properties of the licorice root, including its anti-inflammatory and antibacterial activity, make this plant particularly promising. The same applies to silver nanoparticles showing antibacterial properties. Thus the main purpose of the research was to design hydrogel dressings containing both licorice root extract and nanosilver so as to obtain a system promoting wound regeneration processes by preventing infection and inflammation within the wound. The first step included the preparation of the plant extract via the solid-liquid extraction using the Soxhlet extractor and the synthesis of silver nanoparticles by the chemical reduction of silver ions using a sodium borohydride as a reducing agent. Subsequently, hydrogels were synthesized via photopolymerization and subjected to studies aiming at characterizing their sorption properties, surface morphology via scanning electron microscopy, and their impact on simulated physiological liquids supported by defining these liquids' influence on hydrogels' structures by FT-IR spectroscopy. Next, the tensile strength of hydrogels and their percentage elongation were determined. Performed studies also allowed for determining the hydrogels' wettability and free surface energies. Finally, the cytotoxicity of hydrogels towards L929 murine fibroblasts via the MTT reduction assay was also verified. It was demonstrated that developed materials showed stability in simulated physiological liquids. Moreover, hydrogels were characterized by high elasticity (percentage elongation within the range of 24–29%), and their surfaces were hydrophilic (wetting angles below 90◦ ). Hydrogels containing both licorice extract and nanosilver showed smooth and homogeneous surfaces. Importantly, cytotoxic properties towards L929 murine fibroblasts were excluded; thus, developed materials seem to have great potential for application as innovative dressings.

**Keywords:** hydrogel dressings; licorice root extract; silver nanoparticles; wettability; cytotoxicity; tensile strength; sorption ability

### **1. Introduction**

*Glycyrrhiza glabra* (licorice) root is widely applied as a sweetener and flavoring agent in candies, sweets, and other food products. The sweet taste of licorice root results from the presence of glycyrrhizin [1,2]. However, despite its use in the food industry, this plant is also applied as a medicinal herb [3]. Licorice root has been used in Chinese medicine for over 1000 years. Its pharmacological properties result from the fact that it consists of a huge amount of active chemical compounds, including isoflavonoids, chalcones, amino acids, lignins, amines, gums, and volatile oils. It was demonstrated that the composition of licorice

**Citation:** K˛edzierska, M.; Ba ´nkosz, M.; Drabczyk, A.; Kudłacik-Kramarczyk, S.; Jamrozy, M.; ˙ Potemski, P. Silver Nanoparticles and *Glycyrrhiza glabra* (Licorice) Root Extract as Modifying Agents of Hydrogels Designed as Innovative Dressings. *Int. J. Mol. Sci.* **2023**, *24*, 217. https://doi.org/10.3390/ ijms24010217

Academic Editors: Valentina Siracusa and Swarup Roy

Received: 17 November 2022 Revised: 4 December 2022 Accepted: 20 December 2022 Published: 22 December 2022

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

root contains over 20 triterpenoids and 300 flavonoids [4,5]. Such an extensive chemical composition makes this plant an extremely interesting research material in terms of the possibilities of its pharmaceutical applications. Biologically active licorice compounds are used in many diseases due to their anti-inflammatory, antibacterial, and antiviral properties [6–8]. Furthermore, licorice also shows anti-carcinogenic and neuroprotective activity (it may prevent or delay the degradation of neurons) [9]. It has also been proven that licorice may be applied as an effective therapeutic agent in the treatment of diabetes [10]. As has been demonstrated by Yang et al., licorice extracts show excellent anti-diabetic activity both in vitro and in vivo. They affect the mechanisms of insulin receptor site sensitivity, increase the use of glucose in various tissues and organs, and correct metabolic disorders improving microcirculation in the body simultaneously [11]. Subsequently, it has also been reported that glabrydin occurring in the licorice root may be an alternative therapeutic agent in thrombogenic disorders due to the impact of this compound on the prevention of platelet aggregation [12]. *Glycyrrhiza* L. root may also be successfully used in the treatment of infectious hepatitis and bronchitis [13]. Feng Yeh et al. proved the effective activity of licorice extract against HRSV infection on airway epithelial cells. Bioactive compounds present in this plant extract prevented virus transfer and internalization. Moreover, it has been demonstrated that under the influence of *Glycyrrhiza* L., mucosal cells are stimulated to release IFN-β, counteracting the same viral infection [14]. It has also been stated that the flavonoids extracted from licorice root reduced the inflammation of acute pneumonia induced in vivo in mice by lipopolysaccharide [15]. Studies on the application of licorice extracts in the treatment of respiratory diseases, including COVID-19, were also presented by Gomaa et al. [16] and li Ng et al. [17]. Additionally, the possibility of the application of licorice extracts in the treatment of alcoholic liver injury was described in [18]. Furthermore, the attention of scientists was focused not only on the therapeutic effect of the bioactive compounds included in licorice but also on their ability to enhance the action of the other medicinal compounds by forming supermolecular complexes with them [19]. Due to its antioxidant, anti-inflammatory, and antibacterial properties, many studies have been on the use of licorice in the treatment of inflammation accompanying certain skin diseases [20]. It has been shown that the preparations based on licorice extract may be successfully applied in the treatment of erythema [21], vitiligo [22,23], atopic dermatitis [24,25], as well as baldness [26].

The wide spectrum of the activity of the compounds included in the licorice root corresponds to the great application potential of this plant. Thus considering its properties desirable in terms of skin inflammation treatment, the main purpose of the presented research was to develop hydrogel dressing material modified with licorice root extract. Many commercially available preparations occur in the form of a gel of an ointment wherein such forms may cause some of the active substance to rub into clothing; thus, the introduction of such a substance into the hydrogel dressing may prevent this phenomenon. In this work, the results of the research aimed at developing hydrogel dressing materials containing bioactive substances extracted from licorice root. Furthermore, developed materials were additionally incorporated with silver nanoparticles showing antibacterial properties. The main hypothesis of the research was to develop a multifunctional dressing material that could simultaneously fulfill protective functions, absorb wound exudate, and show anti-inflammatory and antibacterial properties due to the presence of the modifiers: licorice root extract and nanosilver. To our knowledge, such a combination is innovative and has not yet been presented. The base forming the hydrogel matrix consisted of natural polymers such as gelatin and chitosan, demonstrating high biocompatibility. In the first step of the research, the synthesis of silver nanoparticles was performed, while for this purpose, the chemical reduction was employed. Next, licorice extract was prepared via the Soxhlet extraction. Both these materials were subsequently used as modifiers of chitosan/gelatin-based hydrogel dressings. The hydrogels were obtained by means of UV radiation and next subjected to the detailed physicochemical characteristic. In the course of the research, sorption properties of hydrogels important in terms of potential sorption

of the wound exudate were determined. Next, incubation of hydrogels in simulated physiological liquids supported by determining their impact on hydrogels' structure via FT-IR spectroscopy was also performed. Hydrogels' surface morphology, wettability as well as mechanical properties, including the tensile strength and the percentage elongation, were also characterized. Finally, the cytotoxicity of hydrogels towards L929 murine fibroblasts via the MTT reduction assay was also verified. wettability as well as mechanical properties, including the tensile strength and the percentage elongation, were also characterized. Finally, the cytotoxicity of hydrogels towards L929 murine fibroblasts via the MTT reduction assay was also verified. **2. Results and Discussion** *2.1. Characteristic of Nanosilver Suspension*

modifiers of chitosan/gelatin-based hydrogel dressings. The hydrogels were obtained by means of UV radiation and next subjected to the detailed physicochemical characteristic. In the course of the research, sorption properties of hydrogels important in terms of potential sorption of the wound exudate were determined. Next, incubation of hydrogels in simulated physiological liquids supported by determining their impact on hydrogels' structure via FT-IR spectroscopy was also performed. Hydrogels' surface morphology,

#### **2. Results and Discussion** 2.1.1. The Particle Size Analysis via DLS Technique


*Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 3 of 23

Below in Figure 1, the results of the DLS analysis are presented.

**Figure 1.** The particle size distribution in obtained silver nanoparticles suspension.

**Figure 1.** The particle size distribution in obtained silver nanoparticles suspension. Based on the performed analysis, it may be reported that in the tested suspension, the particles with sizes within the range of 20–200 nm occurred, wherein the most common population of the particles showed the size with an average hydrodynamic diameter within the range of 40–80 nm. Thus the results of the DLS analysis clearly indicated the Based on the performed analysis, it may be reported that in the tested suspension, the particles with sizes within the range of 20–200 nm occurred, wherein the most common population of the particles showed the size with an average hydrodynamic diameter within the range of 40–80 nm. Thus the results of the DLS analysis clearly indicated the presence of nanoparticles in the suspension.

#### presence of nanoparticles in the suspension. 2.1.2. Characterization of Optical Properties of Nanosilver Suspension

2.1.2. Characterization of Optical Properties of Nanosilver Suspension UV–Vis spectrum of the suspension obtained as a result of the chemical reduction of UV–Vis spectrum of the suspension obtained as a result of the chemical reduction of silver ions is presented below in Figure 2. The analysis allowed us to verify whether silver nanoparticles were obtained as a result of the method applied.

silver ions is presented below in Figure 2. The analysis allowed us to verify whether silver nanoparticles were obtained as a result of the method applied. In order to confirm the presence of the silver nanoparticles, the suspension obtained as a result of the procedure described in Section 3.2. of the paper was subjected to UV–Vis spectroscopy. Noble metal nanoparticles, including silver ones, are characterized by specific optical properties as well as the ability to absorb radiation within the range of visible and ultraviolet. Thus, UV–Vis spectroscopy is one of the most frequently used methods aimed at confirming the presence of silver nanoparticles [27,28]. On the UV–Vis spectrum visible in Figure 3, the absorption band with a maximum at a wavelength of approximately 432 nm, typical exactly for silver nanoparticles, was observed. The absorption band at a similar wavelength demonstrating the presence of nanosilver was also presented by Agustina et al. [29] and Alim-Al-Razy et al. [30]. This, in turn, indicates the consistency of the results of the performed analysis with previously reported studies.

Swelling ratio, %

of silver ions.

0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1

Absorbance

*Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 4 of 23

**Figure 2.** UV–Vis spectrum of the particle suspension obtained as a result of the chemical reduction **Figure 2.** UV–Vis spectrum of the particle suspension obtained as a result of the chemical reduction of silver ions. consistency of the results of the performed analysis with previously reported studies.

350 400 450 500 550 600

**Figure 3.** Results of investigations on sorption properties of hydrogels in distilled water (**a**), SBF (**b**), Ringer liquid (**c**), and artificial saliva (**d**) (n = 3, n—number of repetitions). **Figure 3.** Results of investigations on sorption properties of hydrogels in distilled water (**a**), SBF (**b**), Ringer liquid (**c**), and artificial saliva (**d**) (n = 3, n—number of repetitions).

#### *2.2. Results of Hydrogels' Swelling Ability Measurements 2.2. Results of Hydrogels' Swelling Ability Measurements*

Swelling ratios of tested hydrogels are presented below in Figure 3, wherein the results are compiled separately for each liquid. The study was conducted in triplicates, and the results are shown as average values with corresponding standard deviations (SD, presented as error bars). Swelling ratios of tested hydrogels are presented below in Figure 3, wherein the results are compiled separately for each liquid. The study was conducted in triplicates, and the results are shown as average values with corresponding standard deviations (SD, presented as error bars).

Studies on the swelling properties of the materials are aimed at determining the ability of the material to interact with aqueous solutions, which means its ability to their absorption. Such ability may be expressed as the swelling ratio (with unit %) after the ap-

swelling at a specific time. So based on such calculations for performed experiments, it may be concluded that tested hydrogels showed a sorption capability of about 200–300%. Importantly, the highest values of this parameter were reported for hydrogels swelling in distilled water, wherein the lowest ones were in the case of materials tested in SBF. High values of swelling ratio in selected liquid mean that the samples absorbed this liquid to the greatest extent. Distilled water, contrary to the rest of the tested media, does not contain any ions which could negatively affect the sorption process of the tested material. For example, divalent ions such as calcium ions may interact with the polymer network, resulting in an increase in the crosslinking density of polymer chains. This, in turn, may result in a decrease in the swelling properties of such polymer. This dependence is clearly visible in the case of the results presented in Figure 3b–d, where the calculated swelling

Results of performed studies also allow for determining the impact of the modifiers licorice root extract and nanosilver suspension—on the sorption properties of hydrogels. Thus, referring to the first mentioned modifier, it may be reported that the presence of the plant extract in the hydrogel matrix did not affect the swelling properties of tested materials. Due to the fact that hydrogels showing high swelling properties are considered beneficial in terms of their biomedical application as dressings, the lack of the negative impact of the licorice root extract on this property is determined as a positive aspect. Thus it may be concluded that the mentioned extract does not affect the swelling ability and, importantly, does not reduce this ability compared to the swelling properties of hydrogel without this additive. Another situation was observed in the case of the modification of hydrogels with nanosilver. A clear increase in the swelling ratios of hydrogels containing this modifier was reported. Probably this resulted from the interactions between the aqueous suspension of silver nanoparticles and the hydroxyl groups included in the medium in which the swelling samples were placed. These interactions cause the attraction of the water molecules and thus contribute to the higher values of the swelling ratios observed on the y-axis. From the application viewpoint, such a phenomenon is most desirable.

ratios are lower than the ones presented in Figure 3a.

Studies on the swelling properties of the materials are aimed at determining the ability of the material to interact with aqueous solutions, which means its ability to their absorption. Such ability may be expressed as the swelling ratio (with unit %) after the appropriate calculation, including the mass of a dry hydrogel and a mass of hydrogel after swelling at a specific time. So based on such calculations for performed experiments, it may be concluded that tested hydrogels showed a sorption capability of about 200–300%. Importantly, the highest values of this parameter were reported for hydrogels swelling in distilled water, wherein the lowest ones were in the case of materials tested in SBF. High values of swelling ratio in selected liquid mean that the samples absorbed this liquid to the greatest extent. Distilled water, contrary to the rest of the tested media, does not contain any ions which could negatively affect the sorption process of the tested material. For example, divalent ions such as calcium ions may interact with the polymer network, resulting in an increase in the crosslinking density of polymer chains. This, in turn, may result in a decrease in the swelling properties of such polymer. This dependence is clearly visible in the case of the results presented in Figure 3b–d, where the calculated swelling ratios are lower than the ones presented in Figure 3a.

Results of performed studies also allow for determining the impact of the modifiers—licorice root extract and nanosilver suspension—on the sorption properties of hydrogels. Thus, referring to the first mentioned modifier, it may be reported that the presence of the plant extract in the hydrogel matrix did not affect the swelling properties of tested materials. Due to the fact that hydrogels showing high swelling properties are considered beneficial in terms of their biomedical application as dressings, the lack of the negative impact of the licorice root extract on this property is determined as a positive aspect. Thus it may be concluded that the mentioned extract does not affect the swelling ability and, importantly, does not reduce this ability compared to the swelling properties of hydrogel without this additive. Another situation was observed in the case of the modification of hydrogels with nanosilver. A clear increase in the swelling ratios of hydrogels containing this modifier was reported. Probably this resulted from the interactions between the aqueous suspension of silver nanoparticles and the hydroxyl groups included in the medium in which the swelling samples were placed. These interactions cause the attraction of the water molecules and thus contribute to the higher values of the swelling ratios observed on the y-axis. From the application viewpoint, such a phenomenon is most desirable. Nonetheless, it should be emphasized that the appropriate selection of the components of the hydrogel matrix allows for controlling the sorption properties of the tested materials.

#### *2.3. Results of Incubation Studies*

Results of the measurements of the pH and the temperature of incubation media in the presence of the hydrogel samples are presented in Figures 4–7. The study was performed in triplicates wherein the results are given as average values with corresponding standard deviations (SD, shown as error bars).

The occurrence of rapid changes in pH may indicate the degradation of hydrogel materials or the release of unreacted reagents, such as a crosslinking agent or photoinitiator from the matrix, which is an undesirable phenomenon. In the case of all tested materials, such rapid and significant changes in the values of tested parameters have not been observed. This, in turn, indicates the stability of the hydrogels in tested environments. However, some slight changes of various natures—i.e., decreases or increases—in measured pH values of the incubation media were noticed. Nonetheless, these changes were very slight, i.e., by a maximum of one pH unit. The hydrogel swells under the influence of the incubation medium; this phenomenon has been described in more detail in Section 2.2. As a result of liquid sorption, the loosening of the polymer network may take place. This, in turn, may lead to the occurrence of various interactions between the components of the polymer and the incubation medium. As a result, numerous compounds (both acidic and alkaline in nature) included in the licorice root extract acting as a modifier of the hydrogel may release from the polymer matrix. Finally, slight changes in pH of the incubation

media in which such a release takes place may be observed. Alternating changes in pH values (i.e., their slight decreases or increases) may be caused by constant interactions of the incubation material with the liquid. Such a phenomenon indicating the gradual release of active compounds present in licorice root extract is beneficial due to the fact that it gives information on the development of the materials with active substance delivery function. Results of the measurements of the pH and the temperature of incubation media in the presence of the hydrogel samples are presented in Figures 4–7. The study was performed in triplicates wherein the results are given as average values with corresponding standard deviations (SD, shown as error bars).

*Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 6 of 23

*2.3. Results of Incubation Studies*

**Figure 4.** pH (**a**) and temperature (**b**) measurements of distilled water during hydrogels' incubation (n = 3, n—number of repetitions). **Figure 4.** pH (**a**) and temperature (**b**) measurements of distilled water during hydrogels' incubation (n = 3, n—number of repetitions).

**Figure 5.** pH (**a**) and temperature (**b**) measurements of SBF during hydrogels' incubation (n = 3, n number of repetitions). **Figure 5.** pH (**a**) and temperature (**b**) measurements of SBF during hydrogels' incubation (n = 3, n—number of repetitions).

**Figure 6.** pH (**a**) and temperature (**b**) measurements of Ringer liquid during hydrogels' incubation (n = 3, n—number of repetitions). **Figure 6.** pH (**a**) and temperature (**b**) measurements of Ringer liquid during hydrogels' incubation (n = 3, n—number of repetitions).

**Figure 7.** pH (**a**) and temperature (**b**) measurements of artificial saliva during hydrogels' incubation (n = 3, n—number of repetitions). **Figure 7.** pH (**a**) and temperature (**b**) measurements of artificial saliva during hydrogels' incubation (n = 3, n—number of repetitions).

#### The occurrence of rapid changes in pH may indicate the degradation of hydrogel materials or the release of unreacted reagents, such as a crosslinking agent or photoiniti-*2.4. The Impact of the Incubation Studies on Hydrogels' Chemical Structure Verified via FT-IR Spectroscopy*

ator from the matrix, which is an undesirable phenomenon. In the case of all tested materials, such rapid and significant changes in the values of tested parameters have not been observed. This, in turn, indicates the stability of the hydrogels in tested environments. FT-IR spectra of hydrogels are presented in Figure 8. The spectra were compiled in such a way as to compare the structure of particular samples before and after the incubation in tested media.

However, some slight changes of various natures—i.e., decreases or increases—in measured pH values of the incubation media were noticed. Nonetheless, these changes were very slight, i.e., by a maximum of one pH unit. The hydrogel swells under the influence of the incubation medium; this phenomenon has been described in more detail in Section 2.2. As a result of liquid sorption, the loosening of the polymer network may take place. This, in turn, may lead to the occurrence of various interactions between the components The performed spectroscopic analysis allowed for verifying the presence of characteristic groups for compounds included in developed polymer matrices. The study was performed both for samples before and after the incubation in simulated physiological liquids. On the one hand, the disappearance of characteristic absorption bands may suggest the degradation of the tested material. On the other hand, it may be reported that such a degradation did not occur because this process would be reflected in significant pH changes

of the polymer and the incubation medium. As a result, numerous compounds (both

of incubation media, and such ones have not been observed. Thus such disappearance or decrease in the intensity of some absorption bands may also indicate the release of the modifiers—i.e., licorice root extract or silver nanoparticles—which, located within the polymer network, may obscure some characteristic groups (as a result, the signal deriving from them is limited). *Spectroscopy* FT-IR spectra of hydrogels are presented in Figure 8. The spectra were compiled in such a way as to compare the structure of particular samples before and after the incubation in tested media.

delivery function.

*Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 10 of 23

the hydrogel may release from the polymer matrix. Finally, slight changes in pH of the incubation media in which such a release takes place may be observed. Alternating changes in pH values (i.e., their slight decreases or increases) may be caused by constant interactions of the incubation material with the liquid. Such a phenomenon indicating the gradual release of active compounds present in licorice root extract is beneficial due to the fact that it gives information on the development of the materials with active substance

*2.4. The Impact of the Incubation Studies on Hydrogels' Chemical Structure Verified via FT-IR* 

**Figure 8.** FT-IR spectra showing the impact of the incubation on the structure of sample 0/0 nanoAg (**a**), 5/0 nanoAg (**b**), 0/1 nanoAg (**c**), and 5/1 nanoAg (**d**). **Figure 8.** FT-IR spectra showing the impact of the incubation on the structure of sample 0/0 nanoAg (**a**), 5/0 nanoAg (**b**), 0/1 nanoAg (**c**), and 5/1 nanoAg (**d**).

The performed spectroscopic analysis allowed for verifying the presence of characteristic groups for compounds included in developed polymer matrices. The study was performed both for samples before and after the incubation in simulated physiological liquids. On the one hand, the disappearance of characteristic absorption bands may suggest the degradation of the tested material. On the other hand, it may be reported that such a degradation did not occur because this process would be reflected in significant pH changes of incubation media, and such ones have not been observed. Thus such disappearance or decrease in the intensity of some absorption bands may also indicate the release of the modifiers—i.e., licorice root extract or silver nanoparticles—which, located In Figure 8a, it is possible to observe the absorption bands characteristic of the structure of two polymers used for the synthesis of hydrogel matrix, i.e., chitosan and gelatin [31–33]. Additionally, on FT-IR spectra of the sample after incubation in artificial saliva, an occurrence of more absorption bands characteristic for these polymers—invisible on the spectrum of the material prior to incubation—was observed (Figure 8a). This may suggest that the polymer chains in the tested material after the drying process were arranged in a way that allowed the disclosure and detection of more groups characteristic for components forming the polymer matrix. Such a dependence was also observed for other tested materials, i.e., in Figure 8b–d). This probably results from the chemical composition of the artificial saliva and the interactions occurring between the functional groups of the polymers and ions included in this incubation liquid.

An interesting dependence was also observed in the case of the hydrogel modified with licorice root extract. In Figure 8b), an increase in the intensity of the absorption band characteristic for the –C–O–C– group deriving from polysaccharides included in this extract may be observed (this has been marked via the blue frame in Figure 8b). Thus, this demonstrates that the presence of the additive in the form of the plant extract is indicated by the increase in the intensity of the selected absorption band in the FT-IR spectra.

Another equally interesting dependence observable during the performed study may be noticed in the FT-IR spectra of hydrogels incorporated with silver nanoparticles. In

Figure 8c,d, the spectra of materials before incubation may be observed (the absorption bands have been marked via the frames—pink one in Figure 8c and green one in Figure 8d), wherein in the case of the material without the licorice root extract (Figure 8c), the spectrum is very blurry and differs significantly in the intensity from the spectrum visible in Figure 8d. In the case of the hydrogel with nanosilver, these nanoparticles are probably located between the polymer chains (in free spaces between them), which makes it difficult to identify groups deriving from polymers. However, a release of nanosilver or its elution probably takes place as a result of the incubation, which in turn results in exposing the characteristic groups giving the same appropriate absorption bands on FT-IR spectra. In the case of the hydrogels modified both with licorice root extract and nanosilver, such a phenomenon was not observed. This is probably caused by the different placement of metallic nanoparticles within the polymer structure. Additionally, some absorption bands derive also from the polysaccharides included in the plant extract. Figure 8c,d, the spectra of materials before incubation may be observed (the absorption bands have been marked via the frames—pink one in Figure 8c and green one in Figure 8d), wherein in the case of the material without the licorice root extract (Figure 8c), the spectrum is very blurry and differs significantly in the intensity from the spectrum visible in Figure 8d. In the case of the hydrogel with nanosilver, these nanoparticles are probably located between the polymer chains (in free spaces between them), which makes it difficult to identify groups deriving from polymers. However, a release of nanosilver or its elution probably takes place as a result of the incubation, which in turn results in exposing the characteristic groups giving the same appropriate absorption bands on FT-IR spectra. In the case of the hydrogels modified both with licorice root extract and nanosilver, such a phenomenon was not observed. This is probably caused by the different placement of metallic nanoparticles within the polymer structure. Additionally, some absorption bands derive also from the polysaccharides included in the plant extract.

within the polymer network, may obscure some characteristic groups (as a result, the sig-

In Figure 8a, it is possible to observe the absorption bands characteristic of the structure of two polymers used for the synthesis of hydrogel matrix, i.e., chitosan and gelatin [31–33]. Additionally, on FT-IR spectra of the sample after incubation in artificial saliva, an occurrence of more absorption bands characteristic for these polymers—invisible on the spectrum of the material prior to incubation—was observed (Figure 8a). This may suggest that the polymer chains in the tested material after the drying process were arranged in a way that allowed the disclosure and detection of more groups characteristic for components forming the polymer matrix. Such a dependence was also observed for other tested materials, i.e., in Figure 8b–d). This probably results from the chemical composition of the artificial saliva and the interactions occurring between the functional groups of the

An interesting dependence was also observed in the case of the hydrogel modified with licorice root extract. In Figure 8b), an increase in the intensity of the absorption band characteristic for the –C–O–C– group deriving from polysaccharides included in this extract may be observed (this has been marked via the blue frame in Figure 8b). Thus, this demonstrates that the presence of the additive in the form of the plant extract is indicated by the increase in the intensity of the selected absorption band in the FT-IR spectra.

Another equally interesting dependence observable during the performed study may be noticed in the FT-IR spectra of hydrogels incorporated with silver nanoparticles. In

#### *2.5. Results of SEM Imaging of Hydrogels 2.5. Results of SEM Imaging of Hydrogels*

The surface morphology of hydrogels was characterized by means of scanning electron microscopy. Obtained SEM images are presented in Figure 9. The surface morphology of hydrogels was characterized by means of scanning electron microscopy. Obtained SEM images are presented in Figure 9.

*Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 11 of 23

polymers and ions included in this incubation liquid.

nal deriving from them is limited).

**Figure 9.** SEM images of hydrogel materials: sample: 0/0 nanoAg (**a**); 5/0 nanoAg (**b**); 0/1 nanoAg (**c**); 5/1 nanoAg (**d**). **Figure 9.** SEM images of hydrogel materials: sample: 0/0 nanoAg (**a**); 5/0 nanoAg (**b**); 0/1 nanoAg (**c**); 5/1 nanoAg (**d**).

Based on the presented SEM images, it may be reported that the highest impact on the surface morphology of the hydrogels has the presence of the plant extract. The samples without this additive (whose images are presented in Figure 9a,b) are characterized by heterogeneous and undulating surfaces. In the case of hydrogels modified with the men-Based on the presented SEM images, it may be reported that the highest impact on the surface morphology of the hydrogels has the presence of the plant extract. The samples without this additive (whose images are presented in Figure 9a,b) are characterized

tioned extract, their surface is homogeneous and smooth (Figure 9b,d). The plant extract

Next, in order to verify the hydrophilicity or hydrophobicity of the surfaces of developed hydrogels, their wetting angles were determined. The results of performed investigations supported by the images showing the first contact of the liquid of distilled water with the hydrogel sample are presented in Table 1, wherein the results of the statistical

**with Water**

*2.6. Wettability of Hydrogels Supported by Determining Their Surface Free Energy*

**Sample Name Total Surface Free Energy, mJ/m<sup>2</sup> Contact Angle, ° Image of Hydrogel during Its First Contact** 

oped materials' surface morphology was not observed.

analysis are shown in Table 2.

0/0 nanoAg 55.22 42.85 ± 0.68

3/0 nanoAg 60.58 35.15 ± 1.15

5/0 nanoAg 67.67 29.17 ± 0.93

**Table 1.** Results of hydrogels' wettability analysis.

by heterogeneous and undulating surfaces. In the case of hydrogels modified with the mentioned extract, their surface is homogeneous and smooth (Figure 9b,d). The plant extract introduced into the polymer matrix probably fills the cavities on the polymer surface, thus making it smoother. On the other hand, any significant impact of nanosilver on the developed materials' surface morphology was not observed. tioned extract, their surface is homogeneous and smooth (Figure 9b,d). The plant extract introduced into the polymer matrix probably fills the cavities on the polymer surface, thus making it smoother. On the other hand, any significant impact of nanosilver on the developed materials' surface morphology was not observed. introduced into the polymer matrix probably fills the cavities on the polymer surface, thus making it smoother. On the other hand, any significant impact of nanosilver on the developed materials' surface morphology was not observed. *2.6. Wettability of Hydrogels Supported by Determining Their Surface Free Energy* oped materials' surface morphology was not observed. *2.6. Wettability of Hydrogels Supported by Determining Their Surface Free Energy* Next, in order to verify the hydrophilicity or hydrophobicity of the surfaces of developed hydrogels, their wetting angles were determined. The results of performed investi-

gations supported by the images showing the first contact of the liquid of distilled water

**Figure 9.** SEM images of hydrogel materials: sample: 0/0 nanoAg (**a**); 5/0 nanoAg (**b**); 0/1 nanoAg

**Figure 9.** SEM images of hydrogel materials: sample: 0/0 nanoAg (**a**); 5/0 nanoAg (**b**); 0/1 nanoAg

**Figure 9.** SEM images of hydrogel materials: sample: 0/0 nanoAg (**a**); 5/0 nanoAg (**b**); 0/1 nanoAg

Based on the presented SEM images, it may be reported that the highest impact on the surface morphology of the hydrogels has the presence of the plant extract. The samples without this additive (whose images are presented in Figure 9a,b) are characterized by heterogeneous and undulating surfaces. In the case of hydrogels modified with the men-

Based on the presented SEM images, it may be reported that the highest impact on the surface morphology of the hydrogels has the presence of the plant extract. The samples without this additive (whose images are presented in Figure 9a,b) are characterized by heterogeneous and undulating surfaces. In the case of hydrogels modified with the mentioned extract, their surface is homogeneous and smooth (Figure 9b,d). The plant extract

Based on the presented SEM images, it may be reported that the highest impact on the surface morphology of the hydrogels has the presence of the plant extract. The samples without this additive (whose images are presented in Figure 9a,b) are characterized by heterogeneous and undulating surfaces. In the case of hydrogels modified with the mentioned extract, their surface is homogeneous and smooth (Figure 9b,d). The plant extract introduced into the polymer matrix probably fills the cavities on the polymer surface, thus making it smoother. On the other hand, any significant impact of nanosilver on the devel-

#### *2.6. Wettability of Hydrogels Supported by Determining Their Surface Free Energy 2.6. Wettability of Hydrogels Supported by Determining Their Surface Free Energy* Next, in order to verify the hydrophilicity or hydrophobicity of the surfaces of devel-Next, in order to verify the hydrophilicity or hydrophobicity of the surfaces of developed hydrogels, their wetting angles were determined. The results of performed investiwith the hydrogel sample are presented in Table 1, wherein the results of the statistical

*Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 12 of 23

*Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 12 of 23

*Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 12 of 23

(**c**) (**d**)

(**c**) (**d**)

(**c**) (**d**)

(**c**); 5/1 nanoAg (**d**).

(**c**); 5/1 nanoAg (**d**).

(**c**); 5/1 nanoAg (**d**).

Next, in order to verify the hydrophilicity or hydrophobicity of the surfaces of developed hydrogels, their wetting angles were determined. The results of performed investigations supported by the images showing the first contact of the liquid of distilled water with the hydrogel sample are presented in Table 1, wherein the results of the statistical analysis are shown in Table 2. oped hydrogels, their wetting angles were determined. The results of performed investigations supported by the images showing the first contact of the liquid of distilled water with the hydrogel sample are presented in Table 1, wherein the results of the statistical analysis are shown in Table 2. gations supported by the images showing the first contact of the liquid of distilled water with the hydrogel sample are presented in Table 1, wherein the results of the statistical analysis are shown in Table 2. **Table 1.** Results of hydrogels' wettability analysis. analysis are shown in Table 2. **Table 1.** Results of hydrogels' wettability analysis. **Sample Name Total Surface Free Energy, mJ/m<sup>2</sup> Contact Angle, ° Image of Hydrogel during Its First Contact with Water**

**with Water**


**Table 1.** Results of hydrogels' wettability analysis. **Table 1.** Results of hydrogels' wettability analysis. **Sample Name Total Surface Free Energy, mJ/m<sup>2</sup> Contact Angle, ° Image of Hydrogel during Its First Contact** 

> **Table 2.** Results of the statistical analysis of contact angle measurements performed via the two-**Table 2.** Results of the statistical analysis of contact angle measurements performed via the twoway analysis of variance (ANOVA) (*p* indicates the statistical significance). **Table 2.** Results of the statistical analysis of contact angle measurements performed via the twoway analysis of variance (ANOVA) (*p* indicates the statistical significance). **Table 2.** Results of the statistical analysis of contact angle measurements performed via the two-way analysis of variance (ANOVA) (*p* indicates the statistical significance).


Based on the performed analysis, it was reported that as the content of the modifiers in the polymer matrices increased, the values of their wetting angles decreased. In the case Based on the performed analysis, it was reported that as the content of the modifiers in the polymer matrices increased, the values of their wetting angles decreased. In the case of the unmodified hydrogel sample, its wetting angle was 42°, wherein the value of this in the polymer matrices increased, the values of their wetting angles decreased. In the case of the unmodified hydrogel sample, its wetting angle was 42°, wherein the value of this Based on the performed analysis, it was reported that as the content of the modifiers in the polymer matrices increased, the values of their wetting angles decreased. In the

Based on the performed analysis, it was reported that as the content of the modifiers

of the unmodified hydrogel sample, its wetting angle was 42°, wherein the value of this parameter determined for the sample containing both these additives (i.e., 5 mL of the

parameter determined for the sample containing both these additives (i.e., 5 mL of the plant extract and 1 mL of nanosilver suspension) was 26°. Hydrophilic surfaces are defined as the ones for which the value of their wetting angle is lower than 90° [34]. Thus it

parameter determined for the sample containing both these additives (i.e., 5 mL the plant extract and 1 mL of nanosilver suspension) was 26°. Hydrophilic surfaces are de-

fined as the ones for which the value of their wetting angle is lower than 90° [34]. Thus it may be concluded that all analyzed materials showed a hydrophilic surface wherein the larger the amount of the modifiers, the higher hydrophilicity. Due to the presence of the chemical compounds included in licorice root extract in the modified materials, interactions in the form of hydrogen bonds between functional groups from these compounds and the water molecules may occur, thus increasing the wettability of such materials' surface. Moreover, in the case of the presence of nanosilver, which was introduced into the polymer matrix in the form of an aqueous suspension, such interactions between modified hydrogels and the drop of liquid may occur, which also translates into the decrease of the wetting angle. The surface wettability is strictly correlated with the value of its surface free energy. Along with the decreasing values of the contact angle, the increase in the value of the surface free energy, which may be defined as a measure of the attractive force of the tested substrate, is observed. The highest value of the total surface free energy was reported in the case of the samples characterized by the lowest wetting angle and thus the most hydrophilic surface, i.e., sample 5/1 nanoAg (modified with the highest amounts of

larger the amount of the modifiers, the higher hydrophilicity. Due to the presence of the chemical compounds included in licorice root extract in the modified materials, interactions in the form of hydrogen bonds between functional groups from these compounds and the water molecules may occur, thus increasing the wettability of such materials' surface. Moreover, in the case of the presence of nanosilver, which was introduced into the polymer matrix in the form of an aqueous suspension, such interactions between modified hydrogels and the drop of liquid may occur, which also translates into the decrease of the wetting angle. The surface wettability is strictly correlated with the value of its surface free energy. Along with the decreasing values of the contact angle, the increase in the value of the surface free energy, which may be defined as a measure of the attractive force of the tested substrate, is observed. The highest value of the total surface free energy was reported in the case of the samples characterized by the lowest wetting angle and thus the most hydrophilic surface, i.e., sample 5/1 nanoAg (modified with the highest amounts of

may be concluded that all analyzed materials showed a hydrophilic surface wherein the larger the amount of the modifiers, the higher hydrophilicity. Due to the presence of the chemical compounds included in licorice root extract in the modified materials, interactions in the form of hydrogen bonds between functional groups from these compounds and the water molecules may occur, thus increasing the wettability of such materials' surface. Moreover, in the case of the presence of nanosilver, which was introduced into the polymer matrix in the form of an aqueous suspension, such interactions between modified hydrogels and the drop of liquid may occur, which also translates into the decrease of the wetting angle. The surface wettability is strictly correlated with the value of its surface free energy. Along with the decreasing values of the contact angle, the increase in the value of the surface free energy, which may be defined as a measure of the attractive force of the tested substrate, is observed. The highest value of the total surface free energy was reported in the case of the samples characterized by the lowest wetting angle and thus the most hydrophilic surface, i.e., sample 5/1 nanoAg (modified with the highest amounts of

The material's surface, its roughness, wettability, topography, as well as surface free energy constitute the very important parameters characterizing the biomaterial. The initial cell adhesion, which depends to a large extent on the aforementioned parameters, is of key importance for further cell proliferation and their regenerative processes [35]. As it

The material's surface, its roughness, wettability, topography, as well as surface free energy constitute the very important parameters characterizing the biomaterial. The initial cell adhesion, which depends to a large extent on the aforementioned parameters, is of key importance for further cell proliferation and their regenerative processes [35]. As it

The material's surface, its roughness, wettability, topography, as well as surface free energy constitute the very important parameters characterizing the biomaterial. The initial cell adhesion, which depends to a large extent on the aforementioned parameters, is of key importance for further cell proliferation and their regenerative processes [35]. As it

vorable for effective cell adhesion, growth, and proliferation [36]. This is consistent with other works where it was demonstrated that cells show better adhesion to hydrophilic surfaces [37]. Considering the obtained results, it may be concluded that developed

vorable for effective cell adhesion, growth, and proliferation [36]. This is consistent with other works where it was demonstrated that cells show better adhesion to hydrophilic surfaces [37]. Considering the obtained results, it may be concluded that developed

vorable for effective cell adhesion, growth, and proliferation [36]. This is consistent with other works where it was demonstrated that cells show better adhesion to hydrophilic surfaces [37]. Considering the obtained results, it may be concluded that developed

is the most fa-

is the most fa-

is the most fa-

was reported by Majhy et al., a moderate surface free energy of 70 mJ/m<sup>2</sup>

was reported by Majhy et al., a moderate surface free energy of 70 mJ/m<sup>2</sup>

was reported by Majhy et al., a moderate surface free energy of 70 mJ/m<sup>2</sup>

Interaction 0.99443

licorice root extract and nanosilver).

licorice root extract and nanosilver).

licorice root extract and nanosilver).

case of the unmodified hydrogel sample, its wetting angle was 42◦ , wherein the value of this parameter determined for the sample containing both these additives (i.e., 5 mL of the plant extract and 1 mL of nanosilver suspension) was 26◦ . Hydrophilic surfaces are defined as the ones for which the value of their wetting angle is lower than 90◦ [34]. Thus it may be concluded that all analyzed materials showed a hydrophilic surface wherein the larger the amount of the modifiers, the higher hydrophilicity. Due to the presence of the chemical compounds included in licorice root extract in the modified materials, interactions in the form of hydrogen bonds between functional groups from these compounds and the water molecules may occur, thus increasing the wettability of such materials' surface. Moreover, in the case of the presence of nanosilver, which was introduced into the polymer matrix in the form of an aqueous suspension, such interactions between modified hydrogels and the drop of liquid may occur, which also translates into the decrease of the wetting angle. The surface wettability is strictly correlated with the value of its surface free energy. Along with the decreasing values of the contact angle, the increase in the value of the surface free energy, which may be defined as a measure of the attractive force of the tested substrate, is observed. The highest value of the total surface free energy was reported in the case of the samples characterized by the lowest wetting angle and thus the most hydrophilic surface, i.e., sample 5/1 nanoAg (modified with the highest amounts of licorice root extract and nanosilver).

The material's surface, its roughness, wettability, topography, as well as surface free energy constitute the very important parameters characterizing the biomaterial. The initial cell adhesion, which depends to a large extent on the aforementioned parameters, is of key importance for further cell proliferation and their regenerative processes [35]. As it was reported by Majhy et al., a moderate surface free energy of 70 mJ/m<sup>2</sup> is the most favorable for effective cell adhesion, growth, and proliferation [36]. This is consistent with other works where it was demonstrated that cells show better adhesion to hydrophilic surfaces [37]. Considering the obtained results, it may be concluded that developed materials, due to the surfaces' hydrophilic nature and the high surface free energy values, show the desired features in terms of supporting regeneration processes.

## *2.7. Results of Mechanical Investigations including Determining the Hydrogels' Tensile Strength and Percentage Elongation*

Results of studies on the tensile strength of hydrogels are presented in Figure 10, the values of their percentage elongation are shown in Figure 11, and the results of the statistical analysis are shown in Tables 3 and 4.

Materials, which are considered for applications for biomedical purposes, should meet a number of requirements. Apart from biocompatibility and relatively simple and quick synthesis methodology, their mechanical properties are extremely important [38]. One of the most popular tools for characterizing the mechanical properties of hydrogels is the static tensile test which provides information about the hydrogels' tensile strength and the possibility of their elongation [39]. Tensile strength may be defined as the maximum stress that a material is able to withstand before its breakage [40].

Based on the results presented in Figure 11, it may be observed that the highest tensile strength—i.e., 0.112 MPa—was reported for unmodified hydrogel. Next, as the amount of the licorice root extract increased, the hydrogel tensile strength decreased, wherein the lowest value of this parameter—i.e., 0.072 MPa—was calculated for the sample containing 5 mL of the plant extract and 1 mL of nanosilver suspension (sample 5/1 nanoAg). The decrease in the value of the tensile strength results from the introduction into the material of additional modifying substances while simultaneously maintaining the same amount of crosslinking agent. The introduction of the plant extract and nanosilver suspension results in the dilution of the reaction mixture, while the use of the same amount of the crosslinker may result in the preparation of the material with a lower crosslinking density. However, it should be emphasized that in the case of the percentage elongation, which indicates the hydrogels' elasticity, such changes in its values were not so visible. The elasticity of modified

hydrogels compared to the elasticity of unmodified materials decreased slightly. The sample containing the highest amounts of the modifying substances—sample 5/1 nanoAg—is characterized by a percentage elongation of approximately 24.5%, which in the case of the application of such a material as dressing, is beneficial and consistent with previously presented research [41–43]. Results of studies on the tensile strength of hydrogels are presented in Figure 10, the values of their percentage elongation are shown in Figure 11, and the results of the statistical analysis are shown in Tables 3 and 4. 0.14

materials, due to the surfaces' hydrophilic nature and the high surface free energy values,

*2.7. Results of Mechanical Investigations Including Determining the Hydrogels' Tensile Strength* 

materials, due to the surfaces' hydrophilic nature and the high surface free energy values,

*2.7. Results of Mechanical Investigations Including Determining the Hydrogels' Tensile Strength* 

Results of studies on the tensile strength of hydrogels are presented in Figure 10, the values of their percentage elongation are shown in Figure 11, and the results of the statis-

show the desired features in terms of supporting regeneration processes.

show the desired features in terms of supporting regeneration processes.

*Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 14 of 23

*Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 14 of 23

*and Percentage Elongation*

tical analysis are shown in Tables 3 and 4.

*and Percentage Elongation*

**Figure 10.** Results of tensile strength measurements of hydrogels (number of repetitions n = 3).

Sample

**Figure 10.** Results of tensile strength measurements of hydrogels (number of repetitions n = 3).

0/0nanoAg 3/0nanoAg 5/0nanoAg 0/1nanoAg 3/1nanoAg 5/1nanoAg Sample**Figure 11.** Results of percentage elongation measurements of hydrogels (number of repetitions n = **Figure 11.** Results of percentage elongation measurements of hydrogels (number of repetitions n = 3).

**Figure 11.** Results of percentage elongation measurements of hydrogels (number of repetitions n =

0

3).

3).


**Table 3.** Results of the statistical analysis of hydrogels' tensile strength measurements performed via the two-way analysis of variance (ANOVA) (*p* indicates the statistical significance).

**Table 4.** Results of the statistical analysis of hydrogels' percentage elongation measurements performed via the two-way analysis of variance (ANOVA) (*p* indicates the statistical significance).


### *2.8. In Vitro Biological Analysis of Hydrogels via MTT Reduction Assay*

In vitro cytotoxicity analysis was performed in line with EN ISO 10993-5:2009 standard [44]. Results of the MTT assay performed using L929 murine fibroblasts are presented in Figure 12, wherein the results of the statistical analysis are shown in Table 5. The study was performed in triplication, wherein the results are presented as average values with corresponding standard deviations (SD, given as error bars). *Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 16 of 23

**Figure 12.** Results of in vitro cytotoxicity analysis of hydrogels via the MTT reduction assay. **Table 5.** Results of the statistical analysis of MTT reduction assay conducted via the two-way anal-**Table 5.** Results of the statistical analysis of MTT reduction assay conducted via the two-way analysis of variance (ANOVA) (*p* indicates the statistical significance).


In accordance with the guidelines of the previously indicated standard, the material

Chitosan (high molecular weight, deacetylation degree 75–85%), gelatin (obtained from porcine skin, Type A, gel strength 300), 2-hydroxy-2-methylpropiophenone (photoinitiator, d = 1.077 g/mL, 97%), diacrylate poly(ethylene glycol (crosslinking agent, d = 1.120 g/mL, average molecular weight Mn = 700 g/mol), and polyvinylpyrrolidone (average molecular weight 10,000 g/mol) were bought in Merck (Darmstadt, Germany). Silver nitrate (99.9%, pure p.a.) and sodium borohydride (NaBH4, 98%, pure p.a.) were

bated for 24 h in its presence above 70% (this cell viability has been marked in Figure 12 via the pink dotted line). Thus, in the case of all tested materials, this requirement was met, which confirms the lack of cytotoxic activity of developed hydrogels against the L929 murine fibroblasts. As it was demonstrated in Figure 12, in the case of samples containing silver nanoparticles, the cell viability slightly increased. This effect may be attributed to the antibacterial activity of nanosilver [45,46]. Developed materials that are applicable for biomedical uses were obtained in strictly controlled conditions, ensuring the highest possible sterility. However, during the synthesis, transport, or investigations, their slight contamination will occur; then, the presence of silver nanoparticles showing antibacterial properties may result in a slight increase in cell survival. This, in turn, constitutes an ad-

Interaction 0.03264

ditional advantage of developed materials.

**3. Materials and Methods**

*3.1. Materials*

In accordance with the guidelines of the previously indicated standard, the material is defined as non-cytotoxic in the case when the viability of the selected cell line is incubated for 24 h in its presence above 70% (this cell viability has been marked in Figure 12 via the pink dotted line). Thus, in the case of all tested materials, this requirement was met, which confirms the lack of cytotoxic activity of developed hydrogels against the L929 murine fibroblasts. As it was demonstrated in Figure 12, in the case of samples containing silver nanoparticles, the cell viability slightly increased. This effect may be attributed to the antibacterial activity of nanosilver [45,46]. Developed materials that are applicable for biomedical uses were obtained in strictly controlled conditions, ensuring the highest possible sterility. However, during the synthesis, transport, or investigations, their slight contamination will occur; then, the presence of silver nanoparticles showing antibacterial properties may result in a slight increase in cell survival. This, in turn, constitutes an additional advantage of developed materials.

#### **3. Materials and Methods**

#### *3.1. Materials*

Chitosan (high molecular weight, deacetylation degree 75–85%), gelatin (obtained from porcine skin, Type A, gel strength 300), 2-hydroxy-2-methylpropiophenone (photoinitiator, d = 1.077 g/mL, 97%), diacrylate poly(ethylene glycol (crosslinking agent, d = 1.120 g/mL, average molecular weight Mn = 700 g/mol), and polyvinylpyrrolidone (average molecular weight 10,000 g/mol) were bought in Merck (Darmstadt, Germany). Silver nitrate (99.9%, pure p.a.) and sodium borohydride (NaBH4, 98%, pure p.a.) were purchased from Avantor Performance Materials Poland S.A. (Gliwice, Poland). *Glycyrrhiza glabra* (Licorice) root was bought in Natur-Sklep (Wrocław, Poland). *Int. J. Mol. Sci.* **2023**, *24*, x FOR PEER REVIEW 17 of 23 purchased from Avantor Performance Materials Poland S.A. (Gliwice, Poland). *Glycyrrhiza glabra* (Licorice) root was bought in Natur-Sklep (Wrocław, Poland).

#### *3.2. Preparation of Glycyrrhiza glabra (Licorice) Root Extract 3.2. Preparation of Glycyrrhiza glabra (Licorice) Root Extract*

In order to obtain bioactive components of licorice, the solid–liquid extraction using the Soxhlet extractor was performed. This is the first-choice type of extraction in the case of isolating organic compounds from plant materials. The scheme of this process is presented below in Figure 13. In order to obtain bioactive components of licorice, the solid–liquid extraction using the Soxhlet extractor was performed. This is the first-choice type of extraction in the case of isolating organic compounds from plant materials. The scheme of this process is presented below in Figure 13.

The procedure of the extraction was as follows: firstly, the licorice root was placed in a thimble while the distilled water was placed in the round bottom flask. Then, the solvent

Silver nanoparticles were prepared via chemical reduction in which a silver nitrate was used as a source of silver while sodium borohydride was used as a reducing agent. Firstly, 0.039 g AgNO<sup>3</sup> (so 250 ppm Ag) was dissolved in a 3% aqueous PVP solution (mixture I). Next, a solution of NaBH<sup>4</sup> in 3% PVP solution was prepared and introduced dropwise to mixture I. Such process was performed at constant stirring and at ambient temperature. After dropping, obtained mixture was maintained at constant stirring for 15 min. Next, it was centrifuged (13,000 rpm) for 20 min. The supernatant was decanted,

The size of the particles obtained via the chemical reduction was verified using dy-

a mild boiling state. Obtained aqueous extract of licorice root was subsequently used as a

**Figure 13.** Licorice root extraction scheme. namic light scattering (DLS technique). For this purpose, a Zetasizer Nano ZS Malvern **Figure 13.** Licorice root extraction scheme.

modifying agent of hydrogels.

wherein the residue was suspended in distilled water.

*3.4. Characterization of Silver Nanoparticle Suspension* 3.4.1. The Particle Size Analysis via DLS Technique

*3.3. Synthesis of Silver Nanoparticles via the Chemical Reduction Process*

The procedure of the extraction was as follows: firstly, the licorice root was placed in a thimble while the distilled water was placed in the round bottom flask. Then, the solvent was heated to the boiling point. Such a process was performed for 4 h while maintaining a mild boiling state. Obtained aqueous extract of licorice root was subsequently used as a modifying agent of hydrogels.

#### *3.3. Synthesis of Silver Nanoparticles via the Chemical Reduction Process*

Silver nanoparticles were prepared via chemical reduction in which a silver nitrate was used as a source of silver while sodium borohydride was used as a reducing agent. Firstly, 0.039 g AgNO<sup>3</sup> (so 250 ppm Ag) was dissolved in a 3% aqueous PVP solution (mixture I). Next, a solution of NaBH<sup>4</sup> in 3% PVP solution was prepared and introduced dropwise to mixture I. Such process was performed at constant stirring and at ambient temperature. After dropping, obtained mixture was maintained at constant stirring for 15 min. Next, it was centrifuged (13,000 rpm) for 20 min. The supernatant was decanted, wherein the residue was suspended in distilled water.

#### *3.4. Characterization of Silver Nanoparticle Suspension*

#### 3.4.1. The Particle Size Analysis via DLS Technique

The size of the particles obtained via the chemical reduction was verified using dynamic light scattering (DLS technique). For this purpose, a Zetasizer Nano ZS Malvern apparatus (Malvern Panalytical Ltd., Malvern, UK) was employed, wherein the measurements were performed at ambient temperature.

#### 3.4.2. Analysis of the Optical Properties of Nanosilver Suspension

Suspension of silver nanoparticles was also subjected to UV–Vis spectroscopy. The study aimed to determine the ability of nanoparticles to absorb light within the UV–Vis range. The analysis was conducted using a ThermoScientific Evolution 220 UV–Vis spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) at room temperature.

#### *3.5. Synthesis of Hydrogel Polymers via the Photopolymerization Process*

In order to prepare hydrogel materials, the UV-induced photopolymerization process was employed. This method allows for obtaining hydrogels in a quick, waste-free, and low energy-demand manner. As a source of UV radiation, an EMITA VP-60 lamp (power 180 W, λ = 320 nm) was applied. Firstly, a 3% chitosan solution in 0.05% acetic acid solution and 2% gelatin solution was prepared. Next, adequate amounts of these solutions were mixed with adequate amounts of Glycyrrhiza glabra (licorice) root extract, nanosilver suspension, crosslinking agent, and photoinitiator. The mixtures obtained were thoroughly mixed, poured into the Petri dishes, and treated with UV radiation for 120 s. Detailed compositions of all prepared hydrogels are given below in Table 6.


**Table 6.** Compositions of prepared hydrogels.

After the synthesis, hydrogels were dried at 37 ◦C for 24 h and investigated to characterize their physicochemical and biological properties. The main attention was focused on determining the impact of the modifiers—licorice root extract and nanosilver suspension—on

hydrogels' properties. Moreover, the discussion over the results of performed experiments also included their evaluation in terms of their application as dressing materials.

#### *3.6. Assessment of the Swelling Properties of Hydrogels*

High swelling properties are one of the most characteristic features of hydrogels. Swelling ability of these materials is particularly important in terms of their potential use as dressings with wound exudate sorption function. Thus the hydrogels' swelling capacity was verified in the artificial saliva, SBF, Ringer liquid, and distilled water. The procedure was as follows: dry hydrogel samples (with a diameter of 2 cm) were firstly accurately weighed and then placed in tested liquids (50 mL) for 1 h. Next, the materials were separated from the liquids, the excess liquid (unbound with the sample) was removed via the paper towel, and samples were weighed again. Subsequently, the hydrogels were placed again in the same liquids, and the procedure was repeated after 24 h and 72 h. The swelling ability of hydrogels was defined in each tested liquid and after each swelling period via the swelling ratio (*Q*) calculated by means of the following Equation (1):

$$Q = \frac{m\_{\rm s} - m\_{\rm d}}{m\_{\rm d}} \times 100\% \tag{1}$$

where: *Q*—swelling ratio, %; *m*s—weight of hydrogel after swelling for a specific time period (i.e., after 1 h, 24 h, or 72 h), g; *m*d—weight of dry sample (before swelling), g.

The swelling studies were performed at ambient temperature and in triplicates for each sample.

#### *3.7. Analysis of the Influence of Hydrogels on Simulated Physiological Fluids (Incubation Studies)*

The incubation studies consisted of introduction of dry hydrogel samples (weighing approximately 1.0 g and with a diameter of 2.0 cm) into selected liquids for 12 days and measurement every two days of the pH and the temperature of the incubation medium. In terms of the potential application of tested materials for biomedical purposes (as dressing materials), the following liquids were selected for incubation: artificial saliva solution, Ringer liquid (infusion liquid used to restore the body's water-electrolyte balance), simulated body fluid (SBF, isotonic to human blood plasma) and distilled water (as a reference liquid). The study aimed to verify whether hydrogel affects the parameters of the liquids. The measurements were performed using the multifunctional ELMETRON CX-701 (Elmetron, Zabrze, Poland) meter. In order to simulate conditions occurring in the human body to a greater extent, the incubation was performed at 37 ◦C. The study was conducted in triplicates for each sample.

## *3.8. Evaluation of the Impact of Hydrogels' Incubation on Their Chemical Structure via FT-IR Spectroscopy*

Hydrogel samples, after incubation in simulated physiological liquids, were subjected to FT-IR spectroscopy to verify potential changes in their structures resulting from the incubation. Such changes could indicate, e.g., the degradation of the hydrogels. The spectroscopic analysis was carried out using the Nicolet iS5 Thermo Scientific (Thermo Fischer Scientific, Waltham, MA, USA) spectrometer, wherein the spectra were recorded within the range 4500–500 cm−<sup>1</sup> and at a resolution of 4.0 cm−<sup>1</sup> .

#### *3.9. Analysis of the Surface Morphology Using SEM Technique*

The next step in the research involved characterization of hydrogels' surface morphology. For this purpose, dry hydrogel samples (with dimensions 1 cm × 1 cm) were sputtered with gold and subjected to the analysis using scanning electron microscopy, wherein the study was conducted by means of a Jeol 5510 LV (Jeol Ltd., Tokyo, Japan) microscope. The imaging was conducted at ambient temperature.

#### *3.10. Studies on the Wettability of Hydrogels Supported by Determining the Surface Free Energy*

The hydrogels were also subjected to the analysis of their wettability. For this purpose, hydrogel samples were treated with a drop of double distilled water dispensed from a syringe. The procedure was conducted with simultaneous recording of the behavior of the drop of the wetting liquid during its first contact with the tested material. Therefore, as a result of the study, the wetting angle for each sample, as well as the images showing the placement of the drop on its surface, were obtained. The measurements were performed at ambient temperature using the Drop Shape Analyzer Kruss DSA100 M measuring instrument (Gmbh, Hamburg, Germany). The whole procedure of the analysis was described in more detail in our previous paper [47]. Importantly, the analysis also enabled the calculation of the surface free energy via the Owens-Wendt method [48].

## *3.11. Characteristics of the Mechanical Properties of Hydrogels*

Hydrogels were also subjected to the analysis of their mechanical properties, including determining their percentage elongation and tensile strength. The study was performed according to the ISO 37 type 2 and ISO 527-2 type 5A standards, wherein the universal testing machine (Shimadzu, Kyoto, Japan) was applied for the measurements. Firstly, after the synthesis, the paddle-shaped hydrogel samples were prepared using the ZCP020 manual blanking press, and they were next dried under pressure (to keep the shape) at 37 ◦C for 24 h. Then, the measurements were performed, during which hydrogel samples were placed between the jaws of the machine. During the analysis, the jaws moved apart, proceeding with simultaneous sample stretching. The procedure was carried on until the sample breakage. The measurements were performed at ambient temperature. Such an analysis allowed to determine the hydrogels' tensile strength (*Rm*) using Equation (2) and the percentage elongation (*A*) using Equation (3). Both equations are presented below:

$$R\_m = \frac{Fm}{S\_0} \tag{2}$$

$$A = \frac{(l\_{\rm ll} - l\_0)}{l\_0} \times 100\% \tag{3}$$

where: *Fm*—maximum hydrogel's strength; *S*0—cross-sectional area of sample in its initial state (before the analysis); *Iu*—measuring length after sample breakage; *I*0—measuring length of sample in its initial state (before the analysis).

#### *3.12. In Vitro MTT Reduction Assay Using L929 Murine Fibroblasts*

In addition to characterizing the physicochemical properties of hydrogels, the key aspect was to verify their cytotoxicity towards selected cell lines. For this purpose, in vitro MTT reduction assay was employed, wherein, as tested cell lines, L929 murine fibroblasts were selected. Conducting this type of preliminary biological investigation provides information on whether the chosen synthesis methodology, as well as the composition of the developed materials, leads to the preparation of materials that could be considered for more advanced biological experiments. When such an assay indicates cytotoxicity of the hydrogels, then the synthesis methodology or the amounts of individual reagents applied during the synthesis needs to be modified. The principle of MTT reduction assay is to check the cell viability by determining their metabolic activity. For this purpose, the MTT reagent (i.e., 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; tetrazolium salt) is added to the medium with tested cell lines (here: L929 murine fibroblasts). Metabolically active cells secrete into the culture medium mitochondrial dehydrogenase, and this enzyme converts the MTT reagent into formazan. The blue crystals of formazan are next dissolved in the organic solvent (e.g., in dimethyl sulfoxide (DMSO)), and obtained solution may be next analyzed via UV–Vis spectroscopy. The absorbance of the solution corresponds to its concentration and, thus, to the amount of the enzyme present in the tested medium. In turn, the amount of the enzyme provides information on the number of viable cells. The procedure of MTT reduction assay, as well as L929 murine fibroblast culture, were described more precisely in our previous publication [49].

## *3.13. Statistical Analysis*

The results of the research were subjected to statistical analysis wherein the statistical importance was determined by means of the two-way analysis of variance (ANOVA) (*α* = 5%). The calculations were performed in the case of the results of the mechanical studies, contact angle measurements, and biological studies (MTT reduction assay). The statistical analysis was carried out to verify the importance of the modifying factors—i.e., licorice root extract and nanosilver suspension. All experiments were performed in triplicates, and their results are provided, including the average value and the standard deviation (SD).

## **4. Conclusions**


**Author Contributions:** Conceptualization, M.J. and S.K.-K.; methodology, M.J. and S.K.-K.; software, M.J.; validation, M.J., A.D. and M.B.; formal analysis, M.J., S.K.-K., A.D. and M.B.; investigation, M.J., S.K.-K., A.D. and M.B.; resources, M.J., S.K.-K., A.D. and M.B.; data curation, M.J., S.K.-K., A.D. and M.B.; writing—original draft preparation, M.J., S.K.-K., A.D. and M.B.; writing—review and editing, M.J., S.K.-K., A.D. and M.B.; visualization, M.K. and P.P.; supervision, M.K.; project administration, S.K.-K., A.D. and M.B.; funding acquisition, M.K. and P.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data sharing is not applicable to this article.

**Conflicts of Interest:** The author declares no conflict of interest.

#### **Abbreviations**

FT-IR spectroscopy, Fourier transform infrared spectroscopy; MTT, 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide; HRSV, human respiratory syncytial virus; UV, ultraviolet; DLS, dynamic light scattering; UV–Vis, ultraviolet–visible, SD, standard deviation; SEM, scanning electron microscopy; EN ISO, English International Organization for Standardization; PVP, polyvinylpyrrolidone; SBF, simulated body fluid; DMSO, dimethyl sulfoxide.

#### **References**


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## *Article* **Colourimetric Plate Assays Based on Functionalized Gelatine Hydrogel Useful for Various Screening Purposes in Enzymology**

**Karolina Labus \* and Halina Maniak**

Department of Micro, Nano and Bioprocess Engineering, Faculty of Chemistry, Wroclaw University of Science and Technology, Wybrzeze Wyspia ´nskiego 27, 50-370 Wrocław, Poland ˙ **\*** Correspondence: karolina.labus@pwr.edu.pl; Tel.: +48-71-320-3314

**Abstract:** Hydrogels are intensively investigated biomaterials due to their useful physicochemical and biological properties in bioengineering. In particular, naturally occurring hydrogels are being deployed as carriers for bio-compounds. We used two approaches to develop a plate colourimetric test by immobilising (1) ABTS or (2) laccase from *Trametes versicolor* in the gelatine-based hydrogel. The first system (1) was applied to detect laccase in aqueous samples. We investigated the detection level of the enzyme between 0.05 and 100 µg/mL and pH ranging between 3 and 9; the stability of ABTS in the solution and the immobilised form, as well as the retention functional property of the hydrogel in 4 ◦C for 30 days. The test can detect laccase within 20 min in the concentration range of 2.5–100 µg/mL; is effective at pH 3–6; preserves high stability and functionality under storage and can be also successfully applied for testing samples from a microbial culture. The second system with the immobilised laccase (2) was tested in terms of substrate specificity (ABTS, syringaldazine, guaiacol) and inhibitor (NaN<sup>3</sup> ) screening. ABTS appeared the most proper substrate for laccase with detection sensitivity CABTS > 0.5 mg/mL. The NaN<sup>3</sup> tested in the range of 0.5–100 µg/mL showed a distinct inhibition effect in 20 min for 0.5 µg/mL and total inhibition for ≥75 µg/mL.

**Keywords:** gelatine hydrogel; laccase detection; inhibitor screening; substrate specificity screening; storage stability; microbial cultivation

## **1. Introduction**

Currently, a progressive degeneration of the natural environment is being observed due to expansive human activity [1–4]. In addition to large-scale mining and refining processes, as well as the daily consumption of material goods by humankind, the manufacturing industry has the largest share in the global generation of waste and pollution of all types. In particular, the food, pharmaceutical, and chemical sectors produce millions of tons of by-products, and waste effluents contributing to the high carbon footprint [4–9]. Intensive multi-directional research is constantly being carried out to minimise these adverse environmental effects [5,6,10,11]. In this respect, one of the most promising trends is the replacement of classical chemical processes with sustainable biocatalytic transformations [12–15]. The leading benefit of enzyme use is the possibility of carrying out chemical reactions under mild processing conditions with high selectivity. Moreover, biocatalysts are used not only at the stage of synthesis/conversion of various compounds, but they are also efficient tools for the detection and neutralisation of harmful substances [16–19]. Hence, it seems reasonable to undertake research in the development of more effective functional biocatalytic systems for various industrial applications. Due to great diversity and relatively easy handling of cultivation, microorganisms are most often used as a rich source of enzymes with valuable catalytic activities, high selectivity, and process stability [20–22]. The displacement of polymers and plastics from industrial trade in favour of biomaterials has also a very significant impact on reducing the global formation of nuisance post-production and post-consumer wastes [23–27]. In this case, the main advantage

**Citation:** Labus, K.; Maniak, H. Colourimetric Plate Assays Based on Functionalized Gelatine Hydrogel Useful for Various Screening Purposes in Enzymology. *Int. J. Mol. Sci.* **2023**, *24*, 33. https://doi.org/ 10.3390/ijms24010033

Academic Editors: Swarup Roy and Valentina Siracusa

Received: 1 December 2022 Revised: 18 December 2022 Accepted: 19 December 2022 Published: 20 December 2022

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

of biomaterials is their easy degradability to environmentally harmless, low-molecularweight compounds, which can be used as ready-to-use products [27–29]. Different types of biopolymers such as polysaccharides or proteins are not only renewable source of valuable mono- and oligomers, but also serve as carriers of bioactive molecules, e.g., drugs, enzymes, agrochemicals, nanoparticles, stem cells, and antibodies [30–36]. To this extent, more and more attention is being paid to hydrogels. These materials are being intensively researched due to their favourable functional properties, such as biodegradability, biocompatibility, swelling capacity, porosity, semi-permeability, and the ability to create stable multilayer structures [37–39]. In particular, they found applications in bioengineering sectors including medicine, pharmacy, tissue engineering, food processing, agriculture, wastewater treatment, environmental protection, biocatalytic processes, and many others [38–45]. Specifically, over the past few years, interest in hydrogels has increased in the context of their use as chemosensors in colourimetric assays. These systems contain an immobilised enzyme or chromogenic/fluorogenic substrate or functional groups in a hydrogel matrix, which react with a target analyte in the sample to give a coloured product. They have been developed mainly as potential diagnostic tools with applications for the detection of molecules, such as ions, low-molecular-weight compounds, and macromolecules. For instance, colourimetric assays were applied for sensing hydrogen [46], nitrite [47,48], nitrate, phosphate [47], and heavy metal ions [49]. In the group of low-molecular-weight compounds, we found cholesterol [50], melanin [51], urea [52], dopamine [53], and bisphenol A [54]. The tests for detecting macromolecules concerned RNA [55] and proteins of specific activity such as streptavidin alkaline phosphatase [56], galactosidase, and glucuronidase [57] or horseradish peroxidase [58]. Both synthetic and natural-originated hydrogel matrices were used in the examples discussed, which are polyalcohols [47,52,54–56], polysaccharides [46,49,53,57], and gelatine [44,59,60]. The cited reports do not exhaust the already published colourimetric assays for the detection of specific analytes; however, it should be noted, that only individual examples of the colourimetric research with immobilised laccase in hydrogels were described in the literature so far. In such studies, laccase was used for the reduction of persistent compounds such as bisphenol A [46,47] and synthetic dye [48], but not strictly for detection purposes. Laccase was immobilised on cellulose/alginate composite hydrogel [46], in poly(ethylene glycol) [47], or alginate/gelatine hydrogel [48]. The other usage of the colourimetric test was the entrapment of a substrate in the solid agar medium and used for identifying the microbes producing laccase [49,50]. Apart from immobilisation in hydrogel matrices, another example was the application of the ABTS-impregnated paper discs for the determination of laccase activity. Such a non-hydrogel assay was applied during the purification of this enzyme on chromatographic columns [51], but practical application, possibilities, and limitations have not been presented in detail.

The system we propose is also based on the use of a substrate that in the presence of a particular enzyme undergoes visible conversion to a coloured product. However, we show the broader applicability of such a system in two opposite modes. In our approach, one of the bioactive compounds (depending on the application: substrate or enzyme) was immobilised within the naturally originated hydrogel matrix to obtain the stable testing kit. For this purpose, we used gelatine, which is a readily available, cheap compound obtained as a side product of commercial animal and fish processing [27,52,53]. Importantly, it is certified by U.S. Food and Drug Administration as a GRAS compound (Generally Recognised As Safe) [54] and is commonly used as a gelling agent for food [27,52,55,56], cosmetics [57,58], medicines [39,56,58,59], pharmaceuticals [54,56,58], and in other branches [39,60]. Furthermore, gelatine has favourable physicochemical properties as a hydrogel matrix. It provides suitable reaction conditions for enzyme activity and is easily biodegradable to non-toxic, low-molecular-weight compounds [36,39,43,44,61]. The selection of gelatine was additionally supported by the results obtained in our previous work, where the properties of alginate and gelatine hydrogel as enzyme carriers were compared [62]. In this case, the gelatine-based hydrogel enzymatically cross-linked with microbial transglutaminase

(mTGase) was a more efficient support for the permanent immobilisation of the given enzyme. transglutaminase (mTGase) was a more efficient support for the permanent immobilisation of the given enzyme. We would like to underline that our concept of a colourimetric detection test based

biodegradable to non-toxic, low-molecular-weight compounds [36,39,43,44,61]. The selection of gelatine was additionally supported by the results obtained in our previous work, where the properties of alginate and gelatine hydrogel as enzyme carriers were compared [62]. In this case, the gelatine-based hydrogel enzymatically cross-linked with microbial

*Int. J. Mol. Sci.* **2023**, *24*, 33 3 of 21

We would like to underline that our concept of a colourimetric detection test based on hydrogel matrices enriched with bioactive ingredients can be applied to enzymes with various catalytic activities. We have already started considering the development of such a solution for selected enzymes in previous research for *β*-galactosidase [63,64], and tyrosinase [65]. However, it was only applied in the case of detecting their presence in the tested samples, whereas in the current study, we would like to present the multifunctionality of using gelatine hydrogel matrices containing targeted active compounds in enzymological screening studies. To precisely analyse the possibilities of the practical application of the provided test, we used the laccase (EC 1.10.3.2) as a model enzyme, and its substrate, 2,20 -azino-bis(3-ethylbenzothiazoline-6- sulfonate) sodium salt (ABTS). on hydrogel matrices enriched with bioactive ingredients can be applied to enzymes with various catalytic activities. We have already started considering the development of such a solution for selected enzymes in previous research for *β*-galactosidase [63,64], and tyrosinase [65]. However, it was only applied in the case of detecting their presence in the tested samples, whereas in the current study, we would like to present the multifunctionality of using gelatine hydrogel matrices containing targeted active compounds in enzymological screening studies. To precisely analyse the possibilities of the practical application of the provided test, we used the laccase (EC 1.10.3.2) as a model enzyme, and its substrate, 2,2′-azino-bis(3-ethylbenzothiazoline-6- sulfonate) sodium salt (ABTS). **2. Results and Discussion** 

#### **2. Results and Discussion** For convenience and readability of the experiments undertaken, we have schemati-

For convenience and readability of the experiments undertaken, we have schematically presented the particular stages of the research considered (Figure 1). Briefly, the first stage, highlighted in blue, involves the preparation of the hydrogel matrices enriched with (1) ABTS (left side) and (2) laccase (right side). The second step, marked in red, includes the example tests and applications of both types of hydrogel matrices developed in the current study. cally presented the particular stages of the research considered (Figure 1). Briefly, the first stage, highlighted in blue, involves the preparation of the hydrogel matrices enriched with (1) ABTS (left side) and (2) laccase (right side). The second step, marked in red, includes the example tests and applications of both types of hydrogel matrices developed in the current study.

**Figure 1.** Scheme of research on the development of hydrogel-based colourimetric tests for various screening purposes in enzymology using laccase as the model biocatalyst. **Figure 1.** Scheme of research on the development of hydrogel-based colourimetric tests for various screening purposes in enzymology using laccase as the model biocatalyst.

#### *2.1. Hydrogel-Based Test for Colourimetric Detection of Laccase*

A quick and simple test that allows the effective detection of a biocatalyst with a specific activity is a desirable analytical tool to improve the preliminary enzymatic screening in both microbiological cultures and other multicomponent mixtures.

In our research, we focused on preparing a diagnostic test which was based on gelatine hydrogel matrices enriched with a substrate (2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonate) sodium salt, ABTS) of laccase for the detection of its activity in a sample. The following functional parameters for detection sensitivity, operating pH range, and storage stability were determined. Validation of the proposed solution was performed using a commercial laccase preparation from *Trametes versicolor*. In the next stage, the suitability of the developed assay for screening laccase in culture fluids during the cultivation of *Cerrena unicolor* was tested.

#### 2.1.1. Development of the Test for the Detection of Laccase in Aqueous Solutions of the developed assay for screening laccase in culture fluids during the cultivation of *Cerrena unicolor* was tested.

A quick and simple test that allows the effective detection of a biocatalyst with a specific activity is a desirable analytical tool to improve the preliminary enzymatic screening

In our research, we focused on preparing a diagnostic test which was based on gelatine hydrogel matrices enriched with a substrate (2,2′-azino-bis(3-ethylbenzothiazoline-6 sulfonate) sodium salt, ABTS) of laccase for the detection of its activity in a sample. The following functional parameters for detection sensitivity, operating pH range, and storage stability were determined. Validation of the proposed solution was performed using a commercial laccase preparation from *Trametes versicolor*. In the next stage, the suitability

*Int. J. Mol. Sci.* **2023**, *24*, 33 4 of 21

*2.1. Hydrogel-Based Test for Colourimetric Detection of Laccase* 

in both microbiological cultures and other multicomponent mixtures.

The typical substrates used for measurements of laccase activity were presented in Figure 2. The colourimetric test for laccase detection proposed in this study was based on gelatine hydrogel matrices enriched with 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonate) sodium salt (ABTS)—one of the well-known synthetic substrate of this enzyme [49,51,66–69]. Syringaldazine (SNG) [70–75] and guaiacol [49,50,67,76] are other substrates frequently used in the determination of laccase activity. Unfortunately, their application in hydrogel-based tests has some limitations which were discussed in Section 2.2.1. In the assay with ABTS, the presence of laccase was indicated by the appearance of a green-blue product resulting from the biocatalytic oxidation of ABTS to its radical cation (Figure 2b). 2.1.1. Development of the Test for the Detection of Laccase in Aqueous Solutions The typical substrates used for measurements of laccase activity were presented in **Figure 2**. The colourimetric test for laccase detection proposed in this study was based on gelatine hydrogel matrices enriched with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) sodium salt (ABTS)—one of the well-known synthetic substrate of this enzyme [49,51,66–69]. Syringaldazine (SNG) [70–75] and guaiacol [49,50,67,76] are other substrates frequently used in the determination of laccase activity. Unfortunately, their application in hydrogel-based tests has some limitations which were discussed in Section 2.2.1. In the assay with ABTS, the presence of laccase was indicated by the appearance of a green-blue product resulting from the biocatalytic oxidation of ABTS to its radical cation (Figure 2b).

**Figure 2.** Laccase catalysed reactions with typical synthetic substrates. (**a**). The oxidation of ABTS (upper), syringaldazine (SNG, middle), and guaiacol (lower) to colourful products: green-blue, pink-violet, and orange-brown, respectively. (**b**). The scheme of substrate oxidation by laccase is part of the red-ox reaction where the enzyme undergoes reduction by substrate oxidation and in turn, it returns to its native oxidised form by transferring the electrons to the oxygen molecule which is a final electron acceptor. **Figure 2.** Laccase catalysed reactions with typical synthetic substrates. (**a**). The oxidation of ABTS (upper), syringaldazine (SNG, middle), and guaiacol (lower) to colourful products: green-blue, pinkviolet, and orange-brown, respectively. (**b**). The scheme of substrate oxidation by laccase is part of the red-ox reaction where the enzyme undergoes reduction by substrate oxidation and in turn, it returns to its native oxidised form by transferring the electrons to the oxygen molecule which is a final electron acceptor.

The proposed colourimetric assay for visual detection of laccase is based on the use of ABTS entrapped in gelatine-based hydrogel introduced into a 96-well plate. The methodology proceeds as follows: a small amount of aqueous solution potentially containing this enzyme was applied to the surface of the ABTS-enriched hydrogel matrix and the colour change from transparent to green-blue was monitored over time. The appearance of colour indicated the presence of laccase in the test solution. Whereas, the intensity of the colour that appeared after a defined time allowed us to estimate the approximate enzyme concentration. Applying this procedure, we performed detailed studies to determine the functional properties of the proposed diagnostic test. Firstly, we have taken into consideration its detection sensitivity. For this purpose, the laccase solutions of different concentrations (0.05–100 µg/mL) were prepared and added on the surface of gelatine hydrogel matrices, as shown in Figure 3.

drogel matrices, as shown in Figure 3.

*Int. J. Mol. Sci.* **2023**, *24*, 33 5 of 21

The proposed colourimetric assay for visual detection of laccase is based on the use of ABTS entrapped in gelatine-based hydrogel introduced into a 96-well plate. The methodology proceeds as follows: a small amount of aqueous solution potentially containing this enzyme was applied to the surface of the ABTS-enriched hydrogel matrix and the colour change from transparent to green-blue was monitored over time. The appearance of colour indicated the presence of laccase in the test solution. Whereas, the intensity of the colour that appeared after a defined time allowed us to estimate the approximate enzyme concentration. Applying this procedure, we performed detailed studies to determine the functional properties of the proposed diagnostic test. Firstly, we have taken into consideration its detection sensitivity. For this purpose, the laccase solutions of different concentrations (0.05–100 µg/mL) were prepared and added on the surface of gelatine hy-

**Figure 3.** The visual response obtained after a different time (0–1440 min) of hydrogel-based test with immobilised 1.5 mg/mL ABTS performed for laccase from *Trametes versicolor* solutions with a concentration range of 0.05–100 µg/mL, left—top view, right—side view. **Figure 3.** The visual response obtained after a different time (0–1440 min) of hydrogel-based test with immobilised 1.5 mg/mL ABTS performed for laccase from *Trametes versicolor* solutions with a concentration range of 0.05–100 µg/mL, left—top view, right—side view.

As early as 5 min after the samples were applied, a distinct change in the gel colour was observed for laccase in the concentration between 10 and 100 µg/mL (Figure 3, side view). Prolonging the assay time to at least 20 min increased the sensitivity of the test which enabled the detection of the laccase concentration even at 2.5 µg/mL. Further extension of the time to 120 min resulted in a colour response in the ABTS test for two successively lower concentrations of this enzyme (1.0 and 0.5 µg/mL). Moreover, the results after 24 h (Figure 3, last row), revealed the presence of laccase activity as low as 0.25 µg/mL. Concluding this part of the research, the satisfactory results of visual laccase detection in the test are obtained for enzyme concentrations between 2.5 and 100 µg/mL and higher. In this case, the required duration of the analysis is at least 20 min. Nevertheless, it is also possible to detect lower concentrations of laccase (starting from 0.25 µg/mL), but then the test time should be extended to 24 h. Based on the literature review, laccases of different origins may have a varying range As early as 5 min after the samples were applied, a distinct change in the gel colour was observed for laccase in the concentration between 10 and 100 µg/mL (Figure 3, side view). Prolonging the assay time to at least 20 min increased the sensitivity of the test which enabled the detection of the laccase concentration even at 2.5 µg/mL. Further extension of the time to 120 min resulted in a colour response in the ABTS test for two successively lower concentrations of this enzyme (1.0 and 0.5 µg/mL). Moreover, the results after 24 h (Figure 3, last row), revealed the presence of laccase activity as low as 0.25 µg/mL. Concluding this part of the research, the satisfactory results of visual laccase detection in the test are obtained for enzyme concentrations between 2.5 and 100 µg/mL and higher. In this case, the required duration of the analysis is at least 20 min. Nevertheless, it is also possible to detect lower concentrations of laccase (starting from 0.25 µg/mL), but then the test time should be extended to 24 h.

of preferential pH [77,78]. Therefore, in the next step, we verified the possibility of detecting this enzyme under different pH conditions. For that purpose, we used the hydrogel Based on the literature review, laccases of different origins may have a varying range of preferential pH [77,78]. Therefore, in the next step, we verified the possibility of detecting this enzyme under different pH conditions. For that purpose, we used the hydrogel with immobilised ABTS and laccase solutions in one fixed concentration, prepared in buffers at pH ranging from 3.0 to 9.0. Such samples were subjected to colourimetric detection according to the developed procedure. The results of the experiment are shown in Figure 4.

As could be observed after just 2 min, the change in gel colour became rapidly visible for enzyme solutions at a pH from 3.0 to 6.0. Prolongation of the analysis time resulted in a deepening blue-green colour in this pH range, and 60 min was required to visualise the presence of the laccase activity at pH 7.0. Based on these results, it was concluded that the activity of commercial laccase from *Trametes versicolor* in the proposed assay conditions certainly could not be determined at pH 8.0 and above; however, one should take into account that the test results reflected the pH range at which the enzyme exhibited activity. In fact, the pH range at which the laccase from *T. versicolor* preserves the ability to oxidise ABTS is between 3 and 6 [79,80] with the optimum falling at around pH 5 [69,80,81]. Thus, this colourimetric test is also a suitable tool for qualitative diagnosis of the spectrum of enzyme activity, depending on the pH applied.

4.

*Int. J. Mol. Sci.* **2023**, *24*, 33 6 of 21

with immobilised ABTS and laccase solutions in one fixed concentration, prepared in buffers at pH ranging from 3.0 to 9.0. Such samples were subjected to colourimetric detection according to the developed procedure. The results of the experiment are shown in Figure

**Figure 4.** Visual response of the assays in a hydrogel matrix with immobilised ABTS (1.5 mg/mL) obtained after exposure time between 0 and 60 min for laccase solution (100 µg/mL) prepared in the citrate-phosphate buffer with pH ranging from 3.0 to 9.0. **Figure 4.** Visual response of the assays in a hydrogel matrix with immobilised ABTS (1.5 mg/mL) obtained after exposure time between 0 and 60 min for laccase solution (100 µg/mL) prepared in the citrate-phosphate buffer with pH ranging from 3.0 to 9.0.

As could be observed after just 2 min, the change in gel colour became rapidly visible for enzyme solutions at a pH from 3.0 to 6.0. Prolongation of the analysis time resulted in a deepening blue-green colour in this pH range, and 60 min was required to visualise the presence of the laccase activity at pH 7.0. Based on these results, it was concluded that the activity of commercial laccase from *Trametes versicolor* in the proposed assay conditions certainly could not be determined at pH 8.0 and above; however, one should take into account that the test results reflected the pH range at which the enzyme exhibited activity. In fact, the pH range at which the laccase from *T. versicolor* preserves the ability to oxidise ABTS is between 3 and 6 [79,80] with the optimum falling at around pH 5 [69,80,81]. Thus, this colourimetric test is also a suitable tool for qualitative diagnosis of the spectrum of The next step in the development of the hydrogel test with immobilised ABTS was to determine the sensibility of laccase detection at different pH values. The enzyme was prepared in the concentration range of 1–100 µg/mL and fixed pH values between 3 and 9. As shown in Figure 5, regardless of the concentration used, the presence of the colour was observed in the pH between 4.0 and 5.2. At the lowest enzyme concentration (1 µg/mL), the effect of ABTS oxidation was also detected at pH 3.0 and 6.0, but the colour of the product was visible at the perceptual border. As mentioned above, these pH values overlapped the pH range at which laccase from *T. versicolor* preserves the ABTS oxidation activity [79,80]. *Int. J. Mol. Sci.* **2023**, *24*, 33 7 of 21


**Figure 5.** The results of the colourimetric test for the different concentrations of laccase in phosphatecitrate buffer with fixed pH values. The comparison of the detection level of enzymatic activity was examined for laccase concentrations of 100, 10, and 1 µg/mL and in the pH range of 3.0–9.0. Test conditions: hydrogel gelatine matrices containing ABTS at a concentration of 1.5 mg/mL; test time: 60 min. **Figure 5.** The results of the colourimetric test for the different concentrations of laccase in phosphate-citrate buffer with fixed pH values. The comparison of the detection level of enzymatic activity was examined for laccase concentrations of 100, 10, and 1 µg/mL and in the pH range of 3.0–9.0. Test conditions: hydrogel gelatine matrices containing ABTS at a concentration of 1.5 mg/mL; test time: 60 min.

To summarise this part of the research, it may be concluded that the proposed colourimetric assay based on immobilised ABTS in a gelatine matrix was effective in detecting laccase at its relatively low concentrations. A key aspect was the pH of the tested solutions. The values of pH should coincide with the pH range of the enzyme's catalytic activity. In the case of the commercial enzyme used here, the level of detection of laccase activity was observed even at its lowest concentration, i.e., 1 µg/mL, in the pH range of 3– 6. To summarise this part of the research, it may be concluded that the proposed colourimetric assay based on immobilised ABTS in a gelatine matrix was effective in detecting laccase at its relatively low concentrations. A key aspect was the pH of the tested solutions. The values of pH should coincide with the pH range of the enzyme's catalytic activity. In the case of the commercial enzyme used here, the level of detection of laccase activity was observed even at its lowest concentration, i.e., 1 µg/mL, in the pH range of 3–6.

A very important functional parameter that determines the commercial applicability of any type of chemical or enzymatic test is its storage stability. An economically desirable characteristic is the longest possible shelf life of the proposed product. Therefore, our next experiments were concerned with the determination of the storage stability of both the substrate (ABTS) chosen to measure enzymatic activity as well as the ABTS-containing gelatine hydrogel matrix. The ABTS was examined in the form of a buffer solution and immobilised in a hydrogel matrix. The results of ABTS storage in particular form was A very important functional parameter that determines the commercial applicability of any type of chemical or enzymatic test is its storage stability. An economically desirable characteristic is the longest possible shelf life of the proposed product. Therefore, our next experiments were concerned with the determination of the storage stability of both the substrate (ABTS) chosen to measure enzymatic activity as well as the ABTS-containing gelatine hydrogel matrix. The ABTS was examined in the form of a buffer solution and

shown in Figure 6a. The buffer solution of ABTS underwent slow but visible auto-oxidation after only 5 days of storage at 4 °C. While the ABTS entrapped in the gelatine hydrogel

**Figure 6.** Evaluation of storage stability for ABTS—a substrate used for determination of laccase activity in a gelatine hydrogel-based colourimetric test. ABTS was tested in its native form in a phosphate-citrate buffer solution at pH 5.2 (**a**), and as immobilised in a hydrogel matrix after 0–20 days (**b**). Storage stability of gelatine hydrogel test with immobilised ABTS was performed after 30 days. Laccase detection assays were compared after 30 min of colourimetric test applying enzyme con-

When considering the storage stability of the entire system tested, a similar procedure was applied. The gelatine matrices containing ABTS were left in closed well plates at 4 °C for 30 days. During this time, the determination of storage stability was performed for the hydrogel matrix at days 10 and 30 and for the matrix at the start of the test (control). The results of the colourimetric response of the test for time t = 0 min and after 30 min for

centration of 100 µg/mL. Experimental conditions: 4 °C, and with 1.5 mg/mL of ABTS.

(**a**) (**b**)

immobilised in a hydrogel matrix. The results of ABTS storage in particular form was shown in Figure 6a. The buffer solution of ABTS underwent slow but visible auto-oxidation after only 5 days of storage at 4 ◦C. While the ABTS entrapped in the gelatine hydrogel was stable for 20 days of storage under similar conditions. gelatine hydrogel matrix. The ABTS was examined in the form of a buffer solution and immobilised in a hydrogel matrix. The results of ABTS storage in particular form was shown in Figure 6a. The buffer solution of ABTS underwent slow but visible auto-oxidation after only 5 days of storage at 4 °C. While the ABTS entrapped in the gelatine hydrogel was stable for 20 days of storage under similar conditions.

**Figure 5.** The results of the colourimetric test for the different concentrations of laccase in phosphatecitrate buffer with fixed pH values. The comparison of the detection level of enzymatic activity was examined for laccase concentrations of 100, 10, and 1 µg/mL and in the pH range of 3.0–9.0. Test conditions: hydrogel gelatine matrices containing ABTS at a concentration of 1.5 mg/mL; test time:

To summarise this part of the research, it may be concluded that the proposed colourimetric assay based on immobilised ABTS in a gelatine matrix was effective in detecting laccase at its relatively low concentrations. A key aspect was the pH of the tested solutions. The values of pH should coincide with the pH range of the enzyme's catalytic activity. In the case of the commercial enzyme used here, the level of detection of laccase activity was observed even at its lowest concentration, i.e., 1 µg/mL, in the pH range of 3–

A very important functional parameter that determines the commercial applicability of any type of chemical or enzymatic test is its storage stability. An economically desirable characteristic is the longest possible shelf life of the proposed product. Therefore, our next experiments were concerned with the determination of the storage stability of both the substrate (ABTS) chosen to measure enzymatic activity as well as the ABTS-containing

*Int. J. Mol. Sci.* **2023**, *24*, 33 7 of 21

60 min.

6.

**Figure 6.** Evaluation of storage stability for ABTS—a substrate used for determination of laccase activity in a gelatine hydrogel-based colourimetric test. ABTS was tested in its native form in a phosphate-citrate buffer solution at pH 5.2 (**a**), and as immobilised in a hydrogel matrix after 0–20 days (**b**). Storage stability of gelatine hydrogel test with immobilised ABTS was performed after 30 days. Laccase detection assays were compared after 30 min of colourimetric test applying enzyme concentration of 100 µg/mL. Experimental conditions: 4 °C, and with 1.5 mg/mL of ABTS. When considering the storage stability of the entire system tested, a similar proce-**Figure 6.** Evaluation of storage stability for ABTS—a substrate used for determination of laccase activity in a gelatine hydrogel-based colourimetric test. ABTS was tested in its native form in a phosphate-citrate buffer solution at pH 5.2 (**a**), and as immobilised in a hydrogel matrix after 0–20 days (**b**). Storage stability of gelatine hydrogel test with immobilised ABTS was performed after 30 days. Laccase detection assays were compared after 30 min of colourimetric test applying enzyme concentration of 100 µg/mL. Experimental conditions: 4 ◦C, and with 1.5 mg/mL of ABTS.

dure was applied. The gelatine matrices containing ABTS were left in closed well plates at 4 °C for 30 days. During this time, the determination of storage stability was performed for the hydrogel matrix at days 10 and 30 and for the matrix at the start of the test (control). The results of the colourimetric response of the test for time t = 0 min and after 30 min for When considering the storage stability of the entire system tested, a similar procedure was applied. The gelatine matrices containing ABTS were left in closed well plates at 4 ◦C for 30 days. During this time, the determination of storage stability was performed for the hydrogel matrix at days 10 and 30 and for the matrix at the start of the test (control). The results of the colourimetric response of the test for time t = 0 min and after 30 min for the storage period considered were presented in Figure 6b. A visual comparison of the results of the laccase detection test indicated that there were no differences in the response of the system, and the colour intensity for the storage times (day 0, 10, and 30) was comparable. In conclusion, the experimental results justified that immobilisation of the substrate in the hydrogel matrix as a key step in the development of a colourimetric test. Furthermore, the developed hydrogel-based assay was stable throughout the storage period, which confirms the validity of the approach considered in the current study. This approach provided suitable conditions for the determination of laccase activity and ensured the long-term stability of the developed test. *Int. J. Mol. Sci.* **2023**, *24*, 33 8 of 21 the storage period considered were presented in Figure 6b. A visual comparison of the results of the laccase detection test indicated that there were no differences in the response of the system, and the colour intensity for the storage times (day 0, 10, and 30) was comparable. In conclusion, the experimental results justified that immobilisation of the substrate in the hydrogel matrix as a key step in the development of a colourimetric test. Furthermore, the developed hydrogel-based assay was stable throughout the storage period, which confirms the validity of the approach considered in the current study. This approach provided suitable conditions for the determination of laccase activity and ensured

Another important parameter defining the reliability of the proposed colourimetric assay is the repeatability of the detection response obtained for a given concentration of the enzyme. As shown in Figure 7, the colour intensity is visually comparable in all 8 replicates performed for each of the tested solutions. the long-term stability of the developed test. Another important parameter defining the reliability of the proposed colourimetric assay is the repeatability of the detection response obtained for a given concentration of the enzyme. As shown in Figure 7, the colour intensity is visually comparable in all 8 replicates performed for each of the tested solutions.

**Figure 7.** Repeatability of the visual responses of the hydrogel-based test performed for the different concentrations of laccase in phosphate-citrate buffer pH 5.2. Test conditions: hydrogel gelatine matrices containing ABTS at a concentration of 1.5 mg/mL; test time: 60 min. **Figure 7.** Repeatability of the visual responses of the hydrogel-based test performed for the different concentrations of laccase in phosphate-citrate buffer pH 5.2. Test conditions: hydrogel gelatine matrices containing ABTS at a concentration of 1.5 mg/mL; test time: 60 min.

These results demonstrate that the test developed in our study is reliable over the entire range of laccase concentrations used (1–100 µg/mL) and can be applied effectively

Microbiological cultures are long-term processes usually occupying several to dozens of days. During this time, studies are conducted on the growth of the microorganism, the products released and the substrate consumed. Analytical methods used in monitoring changes in microbial cultures require special equipment, reagents, and specific physico-chemical conditions. Samples for these analyses often require dilution to contain a specific concentration of an analyte. Some analyses take several hours (determination of dry weight), while others require elevated temperatures or specialised reagents and developed standard curves. Therefore, any opportunity to reduce the duration of sample prep-

To verify the applicability of a quick test based on the colour reaction of the investigated sample with the substrate entrapped in the hydrogel matrix, a microbial cultivation experiment was planned. We employed a fungus *Cerrena unicolor* that produces an extracellular laccase secreted into the culture medium. The detailed characteristics and process parameters that resulted from microbiological culture may be found in Supporting Materials. The general assumption of the experiment was to quantitatively analyse laccase activity by spectrophotometric measurement of the ABTS oxidation rate expressed in U/mg

2.1.2. Application of the Hydrogel-Based Colourimetric Assay in Microbiological Cul-

for various screening research in enzymology.

aration and analysis is highly desirable.

tures

These results demonstrate that the test developed in our study is reliable over the entire range of laccase concentrations used (1–100 µg/mL) and can be applied effectively for various screening research in enzymology.

#### 2.1.2. Application of the Hydrogel-Based Colourimetric Assay in Microbiological Cultures

Microbiological cultures are long-term processes usually occupying several to dozens of days. During this time, studies are conducted on the growth of the microorganism, the products released and the substrate consumed. Analytical methods used in monitoring changes in microbial cultures require special equipment, reagents, and specific physicochemical conditions. Samples for these analyses often require dilution to contain a specific concentration of an analyte. Some analyses take several hours (determination of dry weight), while others require elevated temperatures or specialised reagents and developed standard curves. Therefore, any opportunity to reduce the duration of sample preparation and analysis is highly desirable.

To verify the applicability of a quick test based on the colour reaction of the investigated sample with the substrate entrapped in the hydrogel matrix, a microbial cultivation experiment was planned. We employed a fungus *Cerrena unicolor* that produces an extracellular laccase secreted into the culture medium. The detailed characteristics and process parameters that resulted from microbiological culture may be found in Supporting Materials Table S1. The general assumption of the experiment was to quantitatively analyse laccase activity by spectrophotometric measurement of the ABTS oxidation rate expressed in U/mg units and compare it with the analytical results obtained in hydrogel assays. *Cerrena unicolor* cultivation results were presented in Figure 8. Briefly, the monitoring of changes in biomass (X), glucose (S), specific laccase activity (U/mg of protein), and pH was conducted for 16 days. *Int. J. Mol. Sci.* **2023**, *24*, 33 9 of 21 units and compare it with the analytical results obtained in hydrogel assays. *Cerrena unicolor* cultivation results were presented in Figure 8. Briefly, the monitoring of changes in biomass (X), glucose (S), specific laccase activity (U/mg of protein), and pH was conducted for 16 days.

**Figure 8.** The course of *Cerrena unicolor* cultivation. The legend: yellow diamond—fungal dry mass [g], red circle—glucose (substrate) mass [g], green triangle—specific laccase activity [U/mg], blue square—pH in culture medium. The set of similar points was linked with a dotted line for clarity. The starting glucose mass was 0.63 g, on the 4th day the culture was induced with 10 µM of pyro-**Figure 8.** The course of *Cerrena unicolor* cultivation. The legend: yellow diamond—fungal dry mass [g], red circle—glucose (substrate) mass [g], green triangle—specific laccase activity [U/mg], blue square—pH in culture medium. The set of similar points was linked with a dotted line for clarity. The starting glucose mass was 0.63 g, on the 4th day the culture was induced with 10 µM of pyrogallol.

gallol. Spectroscopic measurements reflecting the course of laccase activity (green triangles) showed that the production of the enzyme appeared on day 4 of culture and increased rapidly reaching a maximum on days between 6 and 8. The analysis of the course for the specific laccase activity revealed another peak of maximum activity recorded on day 11. This phenomenon is known and typical for the white-rot fungi cultures and could be attributed to the production of laccase isoforms at different stages of growth [82–84]. After Spectroscopic measurements reflecting the course of laccase activity (green triangles) showed that the production of the enzyme appeared on day 4 of culture and increased rapidly reaching a maximum on days between 6 and 8. The analysis of the course for the specific laccase activity revealed another peak of maximum activity recorded on day 11. This phenomenon is known and typical for the white-rot fungi cultures and could be attributed to the production of laccase isoforms at different stages of growth [82–84]. After day 11, a decrease in specific laccase activity was observed, associated with substrate

day 11, a decrease in specific laccase activity was observed, associated with substrate depletion and culture collapse. Figure 9 showed the results of a colourimetric hydrogel-

depletion and culture collapse. Figure 9 showed the results of a colourimetric hydrogelbased test performed for analogous samples measured spectrophotometrically. *Int. J. Mol. Sci.* **2023**, *24*, 33 10 of 21

> **Figure 9.** The visual response of colourimetric assay measurements performed for *Cerrena unicolor* culture samples (day 1–16) measured at different times (0–1440 min) using a hydrogel-based assay with immobilised ABTS (1.5 mg/mL); left—top view, right—side view. **Figure 9.** The visual response of colourimetric assay measurements performed for *Cerrena unicolor* culture samples (day 1–16) measured at different times (0–1440 min) using a hydrogel-based assay with immobilised ABTS (1.5 mg/mL); left—top view, right—side view.

> Determination of laccase activity with a colourimetric test allowed detection of the enzyme activity as early as 5 min after the sample was applied on the hydrogel surface. At this time, it was possible to determine the samples with the most concentrated active protein that was for samples corresponding to days 6–16 (Figure 9, side view). At the 20 min of the test, the substantial effect was visible on days 6–8 and 11. These visual response corresponded to the maxima of laccase activity determined spectrophotometrically (Figure 8) and were reflected by the more intense colour of the oxidised ABTS in the hydrogel matrix. After 2 and 3 h, it was possible to detect even the smallest amount of laccase in samples corresponding to the initial days of culture—days 2 and 3. Such determinations were not observed in the spectrophotometric measurement of enzyme activity. Based on the results, it appeared that laccase from *Cerrena unicolor* could be detected with the hydrogel test even in a culture solution having a slightly alkaline pH, such as pH 8.1–8.8 (Figure 8, blue square). A comparison of the above results with a commercial preparation, for which detection was possible up to pH 6 (Figure 5), showed that pH is an important factor for determining enzyme activity in samples of different origins. Laccases from *C. unicolor* show higher pH tolerance [82,83], when compared to laccase from *T. versicolor*, since *C. unicolor* production medium, reaches the alkaline pH [85] and therefore laccase activity could be detected by the colourimetric test. To sum up the results of the experiment, it should be claimed that assays based on Determination of laccase activity with a colourimetric test allowed detection of the enzyme activity as early as 5 min after the sample was applied on the hydrogel surface. At this time, it was possible to determine the samples with the most concentrated active protein that was for samples corresponding to days 6–16 (Figure 9, side view). At the 20 min of the test, the substantial effect was visible on days 6–8 and 11. These visual response corresponded to the maxima of laccase activity determined spectrophotometrically (Figure 8) and were reflected by the more intense colour of the oxidised ABTS in the hydrogel matrix. After 2 and 3 h, it was possible to detect even the smallest amount of laccase in samples corresponding to the initial days of culture—days 2 and 3. Such determinations were not observed in the spectrophotometric measurement of enzyme activity. Based on the results, it appeared that laccase from *Cerrena unicolor* could be detected with the hydrogel test even in a culture solution having a slightly alkaline pH, such as pH 8.1–8.8 (Figure 8, blue square). A comparison of the above results with a commercial preparation, for which detection was possible up to pH 6 (Figure 5), showed that pH is an important factor for determining enzyme activity in samples of different origins. Laccases from *C. unicolor* show higher pH tolerance [82,83], when compared to laccase from *T. versicolor*, since *C. unicolor* production medium, reaches the alkaline pH [85] and therefore laccase activity could be detected by the colourimetric test.

> the determination of laccase activity in a gelatin hydrogel matrix enriched with ABTS are tests ready to use since they do not require the preparation of any additional reagents and specific reaction conditions. An important advantage of the test is the size of the sample introduced on the surface of the matrix, which is in the range of 50–250 µL, this preserves a significant amount of material for testing and performing other additional analyses. Furthermore, a distinctive feature of this assay is the rapid response in the hydrogel matrix, which becomes visible in the form of a coloured stripe, ensuring ease of reading. The intensity of this colour increases over time and enables one to conclude which sample contains significant amounts of the enzyme in a relatively short time (in 5 min). Finally, the To sum up the results of the experiment, it should be claimed that assays based on the determination of laccase activity in a gelatin hydrogel matrix enriched with ABTS are tests ready to use since they do not require the preparation of any additional reagents and specific reaction conditions. An important advantage of the test is the size of the sample introduced on the surface of the matrix, which is in the range of 50–250 µL, this preserves a significant amount of material for testing and performing other additional analyses. Furthermore, a distinctive feature of this assay is the rapid response in the hydrogel matrix, which becomes visible in the form of a coloured stripe, ensuring ease of reading. The

intensity of this colour increases over time and enables one to conclude which sample contains significant amounts of the enzyme in a relatively short time (in 5 min). Finally, the analyses are performed at room temperature so they do not require the application of any additional equipment. analyses are performed at room temperature so they do not require the application of any additional equipment.

*Int. J. Mol. Sci.* **2023**, *24*, 33 11 of 21

#### *2.2. Gelatine Hydrogels Containing Immobilised Laccase for Various Purposes in Enzymology 2.2. Gelatine Hydrogels Containing Immobilised Laccase for Various Purposes in Enzymology*

Following the success achieved in the case of developing a detection test for laccase using ABTS-enriched gelatine matrices, it was decided to use the potential of such enzyme/substrate/support system in a reversed mode. In this approach, the main functional element of the test was laccase immobilised by entrapment in a gelatine-based hydrogel. In this approach, such type of assay was examined in terms of suitability for screening potential substrates and inhibitors of the tested enzyme. Following the success achieved in the case of developing a detection test for laccase using ABTS-enriched gelatine matrices, it was decided to use the potential of such enzyme/substrate/support system in a reversed mode. In this approach, the main functional element of the test was laccase immobilised by entrapment in a gelatine-based hydrogel. In this approach, such type of assay was examined in terms of suitability for screening potential substrates and inhibitors of the tested enzyme.

#### 2.2.1. Hydrogel-Based Assay for Colourimetric Screening a Substrate Specificity of Laccase 2.2.1. Hydrogel-Based Assay for Colourimetric Screening a Substrate Specificity of Lac-

Laccase-enriched gelatine matrices served as a colourimetric assay to determine the substrate specificity of this enzyme. To demonstrate the application potential of laccaseenriched gelatine matrices for substrate testing, we used three compounds most commonly used to determine the activity of this enzyme—ABTS, syringalazine (SNG) and guaiacol. As mentioned earlier, their common feature is that they are all oxidised into coloured products by laccase (Figure 2a,b), which is essential for an effective visual response in the assay. The tests were conducted for different concentrations of each compound. For ABTS and guaiacol, the range was 0.5–10 mg/mL. Whereas, due to the low solubility of syringaldazine, the concentrations were one order lower (0.05–1.0 mg/mL). Colourimetric results obtained after different test times for ABTS, guaiacol, and SNG were depicted in Figures 10–12 respectively. case Laccase-enriched gelatine matrices served as a colourimetric assay to determine the substrate specificity of this enzyme. To demonstrate the application potential of laccaseenriched gelatine matrices for substrate testing, we used three compounds most commonly used to determine the activity of this enzyme—ABTS, syringalazine (SNG) and guaiacol. As mentioned earlier, their common feature is that they are all oxidised into coloured products by laccase (Figure 2a,b), which is essential for an effective visual response in the assay. The tests were conducted for different concentrations of each compound. For ABTS and guaiacol, the range was 0.5–10 mg/mL. Whereas, due to the low solubility of syringaldazine, the concentrations were one order lower (0.05–1.0 mg/mL). Colourimetric results obtained after different test times for ABTS, guaiacol, and SNG were depicted in Figures 10, 11, and 12, respectively.


**Figure 10.** The visual response obtained after a different time of hydrogel-based test with immobilised laccase from T*rametes versicolor* (final concentration 200 µg/mL) was performed for ABTS solutions with a concentration in the range of 0.5–10 mg/mL. **Figure 10.** The visual response obtained after a different time of hydrogel-based test with immobilised laccase from *Trametes versicolor* (final concentration 200 µg/mL) was performed for ABTS solutions with a concentration in the range of 0.5–10 mg/mL.

One can notice that in all cases, a positive response was visible in the form of a colour stripe after only 10 min for all the solutions used; however, in the case of ABTS, the colour change for each concentration was the most apparent (Figure 10). This was one of the main reasons for using ABTS as a reference substrate in all the studies presented in this paper. The obtained results indicate the high potential of laccase-enriched gelatine hydrogel matrices as the screening test for substrates of a given enzyme. We would like to emphasise that also other enzyme-substrate systems can be analysed using this concept of the detection assay. There are only two restrictions: (i) the enzyme should retain its


properties after immobilisation in the hydrogel matrix and (ii) the analysed compounds should be converted into coloured products upon contact with the biocatalyst. *Int. J. Mol. Sci.* **2023**, *24*, 33 12 of 21 *Int. J. Mol. Sci.* **2023**, *24*, 33 12 of 21

> **Figure 11.** The visual response obtained after a different time of hydrogel-based test with immobilised laccase from T*rametes versicolor* (final concentration 200 µg/mL) performed for guaiacol solutions with a concentration in the range of 0.5–10 mg/mL. **Figure 11.** The visual response obtained after a different time of hydrogel-based test with immobilised laccase from *Trametes versicolor* (final concentration 200 µg/mL) performed for guaiacol solutions with a concentration in the range of 0.5–10 mg/mL. **Figure 11.** The visual response obtained after a different time of hydrogel-based test with immobilised laccase from T*rametes versicolor* (final concentration 200 µg/mL) performed for guaiacol solutions with a concentration in the range of 0.5–10 mg/mL.


lised laccase from T*rametes versicolor* (final concentration 200 µg/mL) performed for syringaldazine solutions with a concentration in the range of 0.05–1.0 mg/mL. **Figure 12.** The visual response obtained after a different time of hydrogel-based test with immobilised laccase from T*rametes versicolor* (final concentration 200 µg/mL) performed for syringaldazine solutions with a concentration in the range of 0.05–1.0 mg/mL. **Figure 12.** The visual response obtained after a different time of hydrogel-based test with immobilised laccase from *Trametes versicolor* (final concentration 200 µg/mL) performed for syringaldazine solutions with a concentration in the range of 0.05–1.0 mg/mL.

**Figure 12.** The visual response obtained after a different time of hydrogel-based test with immobi-

One can notice that in all cases, a positive response was visible in the form of a colour stripe after only 10 min for all the solutions used; however, in the case of ABTS, the colour One can notice that in all cases, a positive response was visible in the form of a colour 2.2.2. Hydrogel-Based Assay for Colourimetric Screening Potential Inhibitors of Laccase

change for each concentration was the most apparent (Figure 10). This was one of the main reasons for using ABTS as a reference substrate in all the studies presented in this paper. The obtained results indicate the high potential of laccase-enriched gelatine hydrogel matrices as the screening test for substrates of a given enzyme. We would like to emphasise that also other enzyme-substrate systems can be analysed using this concept of the detection assay. There are only two restrictions: (i) the enzyme should retain its properties after immobilisation in the hydrogel matrix and (ii) the analysed compounds should be converted into coloured products upon contact with the biocatalyst. stripe after only 10 min for all the solutions used; however, in the case of ABTS, the colour change for each concentration was the most apparent (Figure 10). This was one of the main reasons for using ABTS as a reference substrate in all the studies presented in this paper. The obtained results indicate the high potential of laccase-enriched gelatine hydrogel matrices as the screening test for substrates of a given enzyme. We would like to emphasise that also other enzyme-substrate systems can be analysed using this concept of the detection assay. There are only two restrictions: (i) the enzyme should retain its properties after immobilisation in the hydrogel matrix and (ii) the analysed compounds should be converted into coloured products upon contact with the biocatalyst. The next possible application of gelatine matrices containing immobilised laccase as a bioactive agent is screening potential inhibitors of this enzyme. Studies on the identification of effective enzyme inhibitors are particularly time-consuming and require a considerable number of experiments and analyses [74,75]. Preliminary studies require the testing of a large group of compounds in different concentrations. Therefore the use of a rapid colourimetric assay with immobilised enzyme significantly reduces the time of prescreening and allows the selection of potentially active inhibitors for further kinetic studies in a short time. In order to demonstrate the feasibility of such test in practice, sodium azide (NaN3) was

used as a well-known laccase inorganic inhibitor. In the study, a colourimetric assay was performed using a fixed concentration ABTS mixture (1.5 mg/mL) and various concentrations of sodium azide (0.5–100 µg/mL). The potency of this inhibitor was investigated by observing the colour change of individual samples over time with relation to a reference sample containing only the substrate solution (ABTS). As expected, as the concentration of NaN<sup>3</sup> increased, the intensity of the green-blue colour decreased (Figure 13). This means that by applying the colourimetric assay presented here, it is possible not only to effectively screen potential laccase inhibitors, but also to preliminarily estimate the concentration of this compound that rapidly inhibits catalytic activity. ies in a short time. In order to demonstrate the feasibility of such test in practice, sodium azide (NaN3) was used as a well-known laccase inorganic inhibitor. In the study, a colourimetric assay was performed using a fixed concentration ABTS mixture (1.5 mg/mL) and various concentrations of sodium azide (0.5–100 µg/mL). The potency of this inhibitor was investigated by observing the colour change of individual samples over time with relation to a reference sample containing only the substrate solution (ABTS). As expected, as the concentration of NaN3 increased, the intensity of the green-blue colour decreased (Figure 13). This means that by applying the colourimetric assay presented here, it is possible not only to effectively screen potential laccase inhibitors, but also to preliminarily estimate the concentration of this compound that rapidly inhibits catalytic activity.

2.2.2. Hydrogel-Based Assay for Colourimetric Screening Potential Inhibitors of Laccase The next possible application of gelatine matrices containing immobilised laccase as a bioactive agent is screening potential inhibitors of this enzyme. Studies on the identification of effective enzyme inhibitors are particularly time-consuming and require a considerable number of experiments and analyses [74,75]. Preliminary studies require the testing of a large group of compounds in different concentrations. Therefore the use of a rapid colourimetric assay with immobilised enzyme significantly reduces the time of prescreening and allows the selection of potentially active inhibitors for further kinetic stud-

*Int. J. Mol. Sci.* **2023**, *24*, 33 13 of 21

**Figure 13.** The visual response obtained after a different time of hydrogel-based test with immobilised laccase from T*rametes versicolor* (final concentration 200 µg/mL) performed for a fixed concentration of ABTS (1.5 mg/mL) containing different concentrations of sodium azide (0.5–100 µg/mL). **Figure 13.** The visual response obtained after a different time of hydrogel-based test with immobilised laccase from *Trametes versicolor* (final concentration 200 µg/mL) performed for a fixed concentration of ABTS (1.5 mg/mL) containing different concentrations of sodium azide (0.5–100 µg/mL).

#### *2.3. Results Discussion 2.3. Results Discussion*

Current research has resulted in the development of multifunctional colorimetric assays based on ABTS or laccase immobilised in gelatine hydrogel matrices. Through the use of this biopolymer support enriched with bioactive compounds, long-lasting, sensitive test kits for various enzymological screening studies were provided. In our study, we proposed two approaches for this enzyme/substrate/hydrogel system. The first one uses hydrogel matrix containing ABTS, which enables the sensitive detection of laccase in a Current research has resulted in the development of multifunctional colorimetric assays based on ABTS or laccase immobilised in gelatine hydrogel matrices. Through the use of this biopolymer support enriched with bioactive compounds, long-lasting, sensitive test kits for various enzymological screening studies were provided. In our study, we proposed two approaches for this enzyme/substrate/hydrogel system. The first one uses hydrogel matrix containing ABTS, which enables the sensitive detection of laccase in a relatively short time (20 min) in a wide range of concentrations (2.5–100 µg/mL) and pH (3.0–6.0). While the second is based on the reverse mode (laccase immobilised in a gelatine support) and enables effective screening for substrates already within 10 min (ABTS and guaiacol in the range of 0.5–10 mg/mL; syringaldazyne 0.05–1.0 mg/mL) and potential inhibitors within 30 min (sodium azide 0.5–100 µg/mL). To the best of our knowledge, the solutions demonstrating the versatility of using the hydrogel/laccase/ABTS system for the development of rapid visual screening tests proposed in our study are novel and not described in detail in the available literature. In previous reports, one can only find examples of using hydrogel-immobilised laccase for the removal of various compounds from aquous solutions (e.g., bisphenol A [46,47], synthetic dye [48]). In turn, the use of ABTS retained on some support for the detection of laccase has been described only for

paper discs impregnated with this compound [51], and due to incomplete data in the source article (laccase concentration in the tested samples was not given), the results cannot be directly compared with each other.

#### **3. Materials and Methods**

#### *3.1. Materials*

Potato dextrose agar was purchased from Merck (Warsaw, Poland), glucose test was from Biomaxima (Lublin, Poland), 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonate) sodium salt (ABTS), laccase from *Trametes versicolor* (EC 1.10.3.2), bovine serum albumin, porcine skin gelatine, sodium azide (NaN3) and Lowry reagent were from Sigma-Aldrich (Poznan, Poland). Other chemicals were purchased from POCh (Gliwice, Poland). Transglutaminase (mTGase) Activa®WM was kindly donated by Ajinomoto (Tokyo, Japan). All the chemicals used were of analytical grade.

#### *3.2. Methods*

3.2.1. Preparation of Gelatine Hydrogel Matrices Containing Reactive Compounds

Gelatine hydrogel matrices with immobilised 2,20 -azino-bis(3-ethylbenzothiazoline-6- sulfonate) sodium salt (ABTS) or a commercial preparation of laccase from *Trametes versicolor* were prepared according to a modified procedure described in the previous study [86]. Briefly, a weighted portion of gelatine (15% *w*/*v*) was dissolved in 0.1 M citrate-phosphate buffer (pH 5.2) in a glass beaker thermostated at 80 ◦C for approximately 30 min. Next, the solution was cooled to 45 ◦C and incubated at this temperature for a few minutes. In parallel, a buffer solution of ABTS or laccase with given concentration was prepared. Then, after total dissolution, a weighted portion of cross-linking agent (microbial transglutaminase, mTGase) was added to obtain its concentration of 3% *w/v*. The cross-linking step began by mixing the ABTS/mTGase or laccase/mTGase solution with gelatine at the volume ratio of 1:2. The blend thus obtained was pipetted into the 96-well plate (85.4 × 127.6 × 14.4 mm) with 200 µL for each hole (diameter: 6.5 mm; height: 10.8 mm) and stored until use at 4 ◦C. The gelatine hydrogel and the 96-well plate used are shown in Figure S1. In our study, concentrations of 4.5 mg/mL ABTS and 600 µg/mL commercial preparation of laccase were used for immobilisation purposes.

#### 3.2.2. Hydrogel-Based Assay for Colourimetric Detection of Laccase

The test for laccase detection was based on the monitoring of the colour change of the gelatin matrix containing the immobilised ABTS from transparent to green-blue. The appearance of the colour indicates the presence of laccase in the tested sample, and the intensity of the green-blue colour enables us to estimate the concentration of laccase.

The test was performed by applying 100 µL of an aqueous solution potentially containing laccase to the surface of a hydrogel containing ABTS placed on a microlitre well plate with a volume of 200 µL and monitoring the appearance of the colour in time from transparent to green-blue, or lack thereof.

## 3.2.3. Determination of Laccase Concentration Range Effectively Detected by the Hydrogel-Based Assay

First, solutions of laccase from *Trametes versicolor* with various concentrations (0.05–100 µg/mL) were prepared in 0.1 M citrate-phosphate buffer (pH 5.2). The protein concentrations in the enzyme solutions were determined by using the Lowry method [87]. Then, a 100 µL of each laccase preparation was applied to the gelatine-based hydrogel matrix containing ABTS, and the progress of the change in gel colour from transparent to green-blue was monitored in time.

3.2.4. Determination of the pH Range in which Laccase Is Effectively Detected by the Hydrogel-Based Assay

First, solutions of laccase from *Trametes versicolor* in a given concentration (200 µg/mL) were prepared in 0.1 M citrate-phosphate buffer with different pH (range of 3.0–9.0) Then, a 100 µL of each laccase preparation was applied to the gelatine-based hydrogel matrix containing ABTS (1.5 mg/mL), and the progress of the change in gel colour from transparent to green-blue was monitored in time.

3.2.5. Determination of Storage Stability of 2,20 -Azino-bis(3-ethylbenzothiazoline-6-sulfonate) Sodium Salt

The ABTS is known to auto-oxidise, therefore its stability in native and immobilised form was verified after storage of the samples on the well plate at 4 ◦C for a specified time (0–20 days). The result for each ABTS formulation (solution or entrapped in hydrogel) stored for a particular time was compared with the results determined on day 0 (control sample).

#### 3.2.6. Determination of Storage Stability of the Hydrogel-Based Test

The stability of gelatine hydrogel matrices containing ABTS was verified by performing a detection test for a given concentration of laccase from *Trametes versicolor* by using matrices that were previously stored at 4 ◦C for a specified time (0–30 days) in closed well plates. The result for each hydrogel matrix stored for a particular time was compared with the results determined on day 0 (control sample). For this purpose, the intensity of the colour change after 30 min was compared with that at the start of the laccase detection test. The hydrogel assay was classified as stable under storage conditions if the effect of the change in gel colour from transparent to green-blue determined 30 min after the application of the enzyme solution at the same concentration (100 µg/mL) was similar to that obtained for the control test performed on day 0.

#### 3.2.7. Hydrogel-Based Assay for Colourimetric Screening a Substrate Specificity of Laccase

The test for screening the substrate specificity of laccase was based on the monitoring of the colour change of the solution potentially containing a substrate that was dropped on the gelatine matrix containing immobilised laccase (200 µg/mL). The appearance of the colour indicates that laccase effectively converts the given substrate to the colourful product. The intensity of the colour enables us to estimate the affinity strength of the laccase for the given substrate. It should be noted that this test is limited to substrates converted by the enzyme to colourful products. In our study, ABTS, guaiacol, and syringaldazine (SNG) were used as substrates that give green-blue, red-brown, and pink products, respectively. The ABTS and guaiacol were used as buffer solutions in the concentration range of 0.5–10 mg/mL. Syringaldazine was used as a methanolic solution in a lower concentration range (0.05–1.0 mg/mL) due to the poor solubility in water. The test was performed in a microlitre well plate filled with 200 µL hydrogel containing immobilised laccase by applying 100 µL of the tested solution potentially containing the substrate on the hydrogel surface and monitoring the appearance of the colour or lack thereof.

### 3.2.8. Hydrogel-Based Assay for Colourimetric Screening Potential Inhibitors of Laccase

The test for screening potential inhibitors of laccase was based on the monitoring of the colour change of the ABTS solution potentially containing an inhibitor that was dropped on the gelatine matrix containing immobilised laccase. In the presented studies, sodium azide (NaN3) was used as a model inhibitor. The appearance of the deep green-blue colour indicated that laccase effectively converted the ABTS (1.5 mg/mL); however, the lower intensity of the colour or lack thereof was observed for solutions consisting of ABTS (1.5 mg/mL) and sodium azide at different concentrations (range of indicate the presence of inhibitor) in the analysed samples. The test was performed on a microlitre well plate. One hundred microliters of ABTS solution potentially containing the inhibitor was applied

on the surface of a hydrogel with the immobilised laccase (200 µL). The differences in colour appearance between the solution potentially containing laccase inhibitor and control solution of ABTS itself were observed.

#### 3.2.9. Microorganism and Cultivation Conditions

A white-rot fungus *Cerrena unicolor* (Bull.ex.Fr.) Murr, strain no. 139 originated from the culture collection of the Department of Biochemistry, University of Lublin (Poland). The stock culture was maintained on potato dextrose agar at 4 ◦C and periodically transferred to a fresh medium. The fungus cultivation and laccase production were monitored for 16 days according to [2,88] with changes. On the fourth day, the culture was induced with pyrogallol dissolved in methanol to a final concentration of 10 µM [89]. The changes in pH, substrate, biomass, protein concentration, and laccase activity were determined by analysing the cultivation medium in a single flask corresponding to one day of cultivation. A detailed description of the culture cultivation can be found in Supporting Materials.

### 3.2.10. Analytical Procedures

The mass of mycelium was determined by its separation from the cultivation medium through a paper filter, washing with distilled water, and drying at 85 ◦C to a constant mass. In the filtrate, pH, glucose, protein content, and laccase activity were determined. The pH measurements were performed with Crison Basic 20 pH-meter and a Crison 52 09 pH electrode at room temperature. The glucose amount was determined using the enzymatic test kit according to the procedure provided by a supplier (Biomaxima) and glucose as a standard. Protein content was determined with Lowry's method [87] and albumin serum bovine as a standard. The analytical measurements were performed in triplicate with a standard deviation (SD) of less than 5%.

#### 3.2.11. Determination of Laccase Catalytic Activity

Laccase activity was determined based on the spectrophotometric measurements (λ = 420 nm, Shimadzu UV-1800) of the oxidation reaction rate using 1.5 mg/mL ABTS as a substrate in 0.1 M citrate-phosphate buffer (pH 5.2, 25 ◦C). The proportion of the enzyme to the substrate was 1:2 (*v*/*v*). The specific activity unit (U/mg) was defined as the amount of the enzyme (1 mg) that oxidises ABTS to the 1 µmole of the product (ε<sup>420</sup> = 36 000 M−<sup>1</sup> cm−<sup>1</sup> ) per minute at 25 ◦C.

#### **4. Conclusions**

In this article, we have reported the preparation and application of a quick and effective colourimetric test based on a hydrogel matrix for the determination of enzyme catalytic properties. The assay components were the laccase from *Trametes versicolor*, its substrate ABTS, and a gelatine hydrogel. We presented two approaches for using this test. The first concept concerned a hydrogel matrix with an immobilised substrate (ABTS) for application in the monitoring of laccase production in microbiological culture by detecting its oxidation activity. The second approach used immobilised laccase in the hydrogel for the determination of its substrate specificity for ABTS, syringaldazine, and guaiacol as well as evaluation of the inhibitor influence on enzyme activity with NaN<sup>3</sup> as an example. The work was additionally supported by giving the characteristic parameters for the test, namely the detection sensitivity of the enzyme amount and pH range of examined samples, storage stability, and repeatability of the visual response. Despite the numerous advantages of this colourimetric test, there are some key requirements for its application: (i) the enzyme should retain its catalytic properties after immobilisation in the hydrogel matrix, (ii) the reaction product should be visually detectable, and (iii) the hydrogel matrix-substrate system should maintain a high level of enzyme detection for several weeks during storage at 4 ◦C.

We would like to emphasise that the examples given in this paper do not exhaust the application potential of the colourimetric test considered in the case of laccase. Other

possible usages are studies of decolourisation processes, detoxification of wastewater from the textile industry, and detection of polyphenols or amines. The proposed well plate assay can be also prepared in such a way that it enables the simultaneous detection of enzymes with different biocatalytic properties. In particular, this property can be very useful for the selective screening of given biocatalysts in complex mixtures of microbial culture fluids. These examples offer a further research challenge for the development of new effective colourimetric tests based on different enzymes, substrates, and hydrogel matrices.

**Supplementary Materials:** The supporting information can be downloaded at: https://www.mdpi. com/article/10.3390/ijms24010033/s1.

**Author Contributions:** Conceptualisation, K.L.; methodology, K.L. and H.M.; validation, K.L. and H.M.; formal analysis, K.L. and H.M.; investigation, K.L. and H.M.; resources, K.L. and H.M.; data curation, K.L. and H.M.; writing—original draft preparation, K.L. and H.M.; writing—review and editing, K.L. and H.M.; visualisation, K.L. and H.M.; project administration, K.L.; funding acquisition, K.L. and H.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** This article was created as part of the Authors' statutory research activity at the Department of Micro, Nano and Bioprocess Engineering, Faculty of Chemistry, Wrocław University of Science and Technology (Poland).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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## *Review* **Recent Advances of Chitosan Formulations in Biomedical Applications**

**Mohammed A. S. Abourehab 1,2,\* , Sheersha Pramanik <sup>3</sup> , Mohamed A. Abdelgawad <sup>4</sup> , Bassam M. Abualsoud <sup>5</sup> , Ammar Kadi <sup>6</sup> , Mohammad Javed Ansari <sup>7</sup> and A. Deepak 8,\***


**Abstract:** Chitosan, a naturally abundant cationic polymer, is chemically composed of cellulose-based biopolymers derived by deacetylating chitin. It offers several attractive characteristics such as renewability, hydrophilicity, biodegradability, biocompatibility, non-toxicity, and a broad spectrum of antimicrobial activity towards gram-positive and gram-negative bacteria as well as fungi, etc., because of which it is receiving immense attention as a biopolymer for a plethora of applications including drug delivery, protective coating materials, food packaging films, wastewater treatment, and so on. Additionally, its structure carries reactive functional groups that enable several reactions and electrochemical interactions at the biomolecular level and improves the chitosan's physicochemical properties and functionality. This review article highlights the extensive research about the properties, extraction techniques, and recent developments of chitosan-based composites for drug, gene, protein, and vaccine delivery applications. Its versatile applications in tissue engineering and wound healing are also discussed. Finally, the challenges and future perspectives for chitosan in biomedical applications are elucidated.

**Keywords:** chitosan; natural polymer; biomedical applications; drug delivery; tissue engineering; wound healing

## **1. Introduction**

Carbohydrates, the most common natural polymers, join their monomeric units through glycosidic linkages. Some of the beneficial polysaccharides in the biomedical field include starch, cellulose, chitin, pectin, and so on [1]. Scientists in the polymeric field have regarded chitin and chitosan as important biopolymers in the biomedical, electronic, and pharmaceutical fields [2]. Representing one of the most abundant natural polymers, the polycationic biopolymer, chitosan, has several applications such as sewage purification [3], cell entrapment coacervation [4], and seed coating for higher crop yields [5], and also as a food packaging material [6].

Chitosan is a straight-chain polymer of a (1→4)-linked 2-amino-2-deoxy-D-glucopyranose with some residual D-glucosamine units, that can be readily obtained by N-deacetylation of the highly crystalline heteropolymer, chitin [7]. It is naturally found in the cell walls of filamentous fungi, particularly the *Zygomycetes* class [8]. It refers to a heterogenous

**Citation:** Abourehab, M.A.S.; Pramanik, S.; Abdelgawad, M.A.; Abualsoud, B.M.; Kadi, A.; Ansari, M.J.; Deepak, A. Recent Advances of Chitosan Formulations in Biomedical Applications. *Int. J. Mol. Sci.* **2022**, *23*, 10975. https://doi.org/10.3390/ ijms231810975

Academic Editors: Valentina Siracusa and Swarup Roy

Received: 24 August 2022 Accepted: 13 September 2022 Published: 19 September 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

collection of oligomers and polymers which are distinct in the various degrees of polymerization, portions of acetylation, and the arrangements of acetylation [9]. It is industrially manufactured by hydrolysis of the amino acetyl functional groups of chitins. Chitosan is more relevant industrially than chitin because of its reactive amino and hydroxyl groups, its low crystallinity which makes it more receptive to reagents, and its solubility in most organic acidic solutions below its pKa of 6.5 [10].

Chitosan is used in a variety of industries, including treating effluents (removal of metallic ions, dyes, and as a membrane in contaminant expulsion), the food manufacturing sector (fat binding and cholesterol-lowering, food additives, packaging, and preservatives, farming (seed and fertilizer coatings, controlled agrochemical discharge), paper manufacturing (surface treatment, adhesive paper), cosmetic products (skincare products, face creams, etc.), tissue regeneration, and wound repair [11]. Gels, nanofibers, membranes, beads, microparticles, nanoparticles, sponges, and scaffolds could all be manufactured readily from chitin and chitosan [12].

Among the different biopolymers available in nature, chitosan especially has garnered attention because of its unique properties, such as inherent antimicrobial properties, natural abundance, versatility, non-toxicity, and biodegradability. Its degradation product consists of an innocuous amino sugar that could be absorbed by human tissues. The availability of reactive functional groups on the chitosan backbone makes it convenient to tailor it using physical or chemical means to fabricate desirable scaffolds for biological purposes [13,14]. For example, chitosan exhibits functional properties such as mucoadhesion, colon targeting, efflux pump inhibition, permeation improvement, in situ gelation, transfection, and other properties owing to its amino functionality in the chitosan structure [15].

There has been a rise in the number of publications reviewing the various facets of the extraction, preparation, properties, and applications of chitosan in recent years [14,16–19]. This review highlights the most current and significant advances in utilizing chitosan and its nanocarriers as drug delivery systems, tissue engineering scaffolds, wound dressings, and vaccine delivery carriers. The scope of the present work is to highlight the various aspects of chitosan and its derivatives, sometimes in combination with other biomaterials, in biomedical research areas. The Section 1 gives a general overview of chitosan, and its structural, physical, and chemical properties detailing the extraction, properties, and various methods for its modification and the implications in the desired biomedical applications. The Section 2 focuses on the current trends in chitosan applications in each biomedical domain. The Section 13 delves into the limitations and future potentialities of chitosan as a versatile biopolymer.

## **2. Properties of Chitosan**

#### *2.1. Physical Properties*

Hydrophilic polymeric scaffolds derived from chitosan have a three-dimensional cross-linked structure. Due to their physicochemical and biochemical properties, chitosan hydrogels are manufactured via chemical or physical cross-linking amongst the polymer backbone. They are used in therapeutic applications. Because of their capacity to regulate drug release using pH-responsive and temperature-responsive release techniques, as well as the networks that can carry active pharmaceutical compounds, chitosan hydrogels are helpful in drug delivery. Chitosan hydrogels are also a good alternative for wound healing due to their strong antibacterial properties, ability to provide humidity and heat to the wound, cytocompatibility, etc. Additionally, the swelling ratio, porosity, and mechanical behavior of these hydrogels make them an excellent choice for use as a tissue regeneration scaffold [20].

#### 2.1.1. Solubility

Chitosan is perhaps the most significant chitin derivative and the inclusion of numerous functional units on the polysaccharide backbone of this material, such as the hydroxyl and amine groups, allows for the creation of molecularly imprinted polymers and morphological changes [21]. Each D-glucosamine monomer has a free amino position, which could also become positively charged and provide essential features to chitosan, including solubility and antibacterial activity [22,23]. These moieties form excellent chelating ligands that can bind to several metal ions and electrostatically precipitate the dye anions. Furthermore, these amino units may be protonated, resulting in chitosan's solubility in a dilute acid medium [24,25].

Chitosan is water-insoluble and insoluble in most liquid organic media; nevertheless, it is soluble in various aqueous acidic media below its pKa (pH = 6.5), including lactic acids, acetic acid, formic acid, and citric acid, as well as 10-camphor sulfonic acid, p-toluene sulfonic acid, and dimethyl sulfoxide. Carboxymethylation, quaternization, and phosphorylation of chitosan are structural modifications that enhance the polymer's solubility in various solvent systems at atmospheric temperatures [26].

Chitosan's solubility may be improved by lowering its molecular mass. The concentration of N-acetylglucosamine chains in chitosan is affected by molecular weight, which has intramolecular and intermolecular effects, leading to diverse chitosan morphologies. Nevertheless, regulating the deacetylation improves solubility at the expense of yield [27]. Breaking down the chitosan crystalline structure expands the spectrum of chitosan solubility. The researchers looked at both the physical as well as chemical ways of increasing chitosan solubility. Re-acetylation enhanced chitosan's solubility until pH = 7.4 in their chemical method. The physical strategy included the utilization of admixtures with the ability to disturb the intra- and intermolecular hydrogen bond interactions, including urea and guanidine hydrochloride [28]. Chitosan's solubility can be dramatically increased by adding smaller reactive groups to its structure, including alkyl (hydroxypropyl chitosan or carboxymethyl groups) [29]. Implementing a series of low MW chitosan compounds, a quick and efficient method of manufacturing solubilized chitosan (half N-acetylated chitosan) was devised [30].

## 2.1.2. Viscosity

According to Kramer [η]Kra and Huggins [η]Hug, the intrinsic viscosity of chitosan in a buffered aqueous solution (0.3/0.3 M CH3COOH/CH3COONa) was 646 and 637, respectively [12]. Kasaai et al. studied the relationship between intrinsic viscosity and the molecular weight of shrimp shell-derived chitosan in 0.25 M acetic acid/0.25 M sodium acetate solution. The Mark–Houwink–Sakurada equation (MHS) was suggested for chitosan between molecular weight ranges of 35–2220 kDa. The exponent α in the MHS indicated that chitosan behaved as a flexible chain in the solvent composition. The α and K are inversely proportional and depend upon the degree of deacetylation, pH, and ionic strength of the solvents [31].

### *2.2. Chemical Properties*

The degradation products of chitosan called chito-oligosaccharides are water-soluble, have no cytotoxicity to organisms, are easily absorbable through the intestines, and are eliminated through the kidneys. Chito-oligosaccharides (COS) offer a plethora of biological properties, such as cholesterol-reducing activity, anticancer activity, and immunomodulatory activity [32].

### *2.3. Biodegradability*

Many studies have looked at the biodegradability of chitosan. Under certain conditions, lysozyme [33], proteases [34], and porcine pancreatic enzymes were discovered to be capable of degrading chitosan [35]. The *Aspergillus niger* pectinase isozyme was further demonstrated to degrade chitosan at low pH, leading to lower MW chitosan [36,37]. Connell et al. employed human feces to show that chitosan-based films, glutaraldehyde polymerized films, and tripolyphosphate crosslinked films degraded significantly [38]. According to Brenner et al., many of the enzymatic catalysts appear to have an effect against chitosan, particularly in vitro; however, variants could result in indigestible compounds. To be therapeutically effective, these chemical particles must be sufficiently tiny to be eliminated by the kidneys (< 42 Å for neutral compounds). The bio-distribution is controlled by the mode of delivery, dose form, and chitosan properties, i.e., due to the physical qualities, the film/dense matrices will exist at the location of the application. The deterioration of the matrix can be controlled by changing the amine units' crosslinking. Particle size (less than 100 nm) can affect intravenous diffusion, while molecular mass can dictate particle lifespan [39].

#### *2.4. Toxicity*

Chitosans' toxicity was studied in several ways, such as in guinea pigs, frogs, human nasal palate tissues, and the nasal mucosa of rats. Toxicity was minimal in each experiment [40–42]. Ribeiro et al. investigated the in vitro cytotoxic effects of chitosan-based hydrogels using rat-skin dermal fibroblasts. The hydrogel, as well as its breakdown byproducts, were shown to be non-cytotoxic in a cell viability assay. The total absence of a reactive or granuloma-forming inflammatory reaction in skin infections treated with chitosan biomaterials and no pathological irregularities in the organs corroborated the hydrogel's systemic and local histocompatibility [43].

The intravenous injection of chitosan (4.5 mg/kg/day for 11 days) to rabbits resulted in no aberrant alterations, according to in vivo chronic toxicological tests. Data are generally scarce on chitosan's toxic effects from human research [44]. According to Gades et al., the human participants who consumed more than 4.5 g of chitosan per day did not experience any harmful consequences. Even greater oral doses of up to 6.75 g were found to be safe [45]. Brief human testing lasting up to 12 weeks has revealed no clinically substantial effects, such as no signs of an allergic reaction [46]. The safety of chitosan mouthwash was determined using the Ames, MTT, and V79 chromosomal abnormality studies. The chitosan-based mouthwash was less toxic and had more decisive antibacterial action than the conventional mouthwash. Moreover, unlike commercialized mouthwash, this was found to suppress two pathogenicity indicators (streptococcus and enterococcus) while causing no significant changes in the survival of the typical oral microbiota [47].

Ravindranathan et al. examined pure, mild endotoxin chitosan and found that the viscosity/molecular weight and deacetylation degree within the limits of 20–600 CP and 80– 97 percent, correspondingly, have not affected the immunogenicity of chitosan. Endotoxin exposure was expected to have a significant impact on immunogenicity. Only endotoxin concentration, deacetylation degree, or viscosity impacted on the chitosan-induced immune reactions, according to their findings. Their findings also showed that lower endotoxin chitosan (0.01 EU/mg) with viscosities of 20–600 cP and deacetylation levels of 80–97% is largely innocuous. This work emphasized the importance of more thorough identification and purification of chitosan in laboratory development before being employed in clinical trials [48].

According to Baldrick, chitosan could be employed as a non-parenteral and non-blood constitutive medicinal excipient. The appropriate use of chitosan as an injectable excipient is not apparent depending on the existing data. The material's hemostatic physiological nature allows it to be used as a medical device to stop hemorrhaging, and investigations have shown local findings, such as blood clotting, thrombosis development, and platelet adhesion [49]. Despite some cytotoxicity observations in vitro, there are still instances of non-toxic chitosan used in medicines, such as to halt blood loss [50–52].

#### **3. Methods of Preparation**

Chitosan is naturally derived from the polysaccharide chitin, which is the second most abundant bio polysaccharide, generally seen in the shells of lobsters, shrimps, crabs, tortoises, and even insects [53]. Chitosan is produced by the physical or chemical deacetylation of chitin, and even though an established definition of chitosan does not exist, it is generally accepted that 70% deacetylated chitin is chitosan [54]. However, in commercial applications, a degree of deacetylation (DD) of 70–90% or even higher may be desirable by

undergoing subsequent deacetylation steps [55]. However, this process may also lead to polymer degradation and increased chances of reacetylation. Hence, chitosan's molecular weight is generally dependent on its DD. The lower the DD, the higher the MW, which imparts more significant chemical and mechanical stability but also decreases its solubility in most solvents in regular use. This deacetylation reaction is ideally carried out in a nitrogen-rich environment or by adding it to a mixture of NaOH and sodium borohydride to avoid any side reactions from taking place. In this way, the chitosan produced has an average molecular weight of 1.2 <sup>×</sup> <sup>10</sup><sup>5</sup> gmol−<sup>1</sup> [55].

Throughout the past decades, scientists have explored and implemented several ways of extracting chitosan from the shells of various crustaceans, insects, and fungi [56]. Chitosan biopolymers can also be produced from *Labeo rohita's* discarded scales [57]. To produce chitosan from chitin, two techniques with differing degrees of acetylation had proved to be widely employed. The first is the heterogeneous deacetylation of dry chitin, while the second is the uniform deacetylation of pre-swollen chitin in an aqueous solution in a vacuum [58]. The deacetylation procedure requires strong alkali treatments and extended operating durations in both of the circumstances. The production time is determined by whether the circumstances are heterogeneous or homogeneous and can range from 1 to over 80 h. Alternative manufacturing procedures have been devised to lessen the relatively long processing time and significant volume of alkali. Employing thiophenol in DMSO for sequential alkali processes [59]; thermo-mechanical methods employing a cascade reactor maintained at a low alkali percentage [60]; flash procedures under saturated steam [61]; microwave dielectric heating [62]; and periodic water washing [63,64] are other instances of alternative manufacturing procedures. Previous studies have revealed various sophisticated chitosan recovery strategies involving high-energy bombardment. Microwave radiation is a common alternative energy source that can transmit power directly and fast into the substrates, enhancing reaction efficiency [65]. Furthermore, microwave treatment can minimize the number of chemical compounds employed in the chitosan extraction method; nevertheless, the DD of chitosan obtained is not convincing. Rashid et al. described a –irradiation methodology for making chitosan from prawn shells that substantially increased chitin's DD while using a low alkali quantity [56].

#### **4. Extraction**

#### *4.1. Deproteinization*

Desiccated crustacean shells will be initially washed with an alkaline solution (e.g., NaOH, KOH, etc.) to eliminate the proteins. The alkali-insoluble portion is then separated by centrifugation, accompanied by repeated washings with distilled water till the pH reaches neutrality [53].

### *4.2. Desulfurization*

Next, the mineral contaminants are removed from deproteinized shells using a dilute mineral acid (e.g., HCl). The acid-insoluble portion is then separated using centrifugation. The acid is rinsed out of the isolated fraction using distilled water. The chitin, which is somewhat pink in color, evaporates to dryness overnight [53].

#### *4.3. Decolorization*

The chitin is decolorized by reacting with an oxidizing agent such as potassium permanganate, hydrogen peroxide, or other oxidizing agents, then washed with an oxalic acid solution. Purified chitin is the name given to the end product [53].

#### *4.4. Deacetylation*

The decolorized chitin would then be treated with highly alkaline solutions over many hours to deacetylate it and transform it into chitosan. Centrifugation separates the alkaline fraction of the combination, and excessive alkali is discharged with a rinse of distilled water till the pH becomes neutral. The chitosan portion recovered is then dried

and kept at ambient temperature. Raw chitosan is diluted with aqueous 2 percent (*w*/*v*) acetic acid to get the optimum product. The insoluble substance is then filtered, yielding a precise supernatant mixture neutralized with NaOH solution, yielding a pure chitosan specimen as a colorless precipitate. The chitosan appears in flakes that range in color from white to yellow and can be made into beads or powder form. To create medicinal and pharmaceutical-grade chitosan, more purification may be necessary [53].

#### **5. Modifications of Chitosan**

In recent years, chitosan has been increasingly utilized in the pharmaceutical and biomedical fields because of its many advantages, such as low immunoreactions, good biocompatibility, easy biological degradability, excellent mucoadhesion, and its natural abundance [66]. The versatile biological and physicochemical characteristics of chitosan are possible because of the availability of different functional groups. These groups can be functionalized by a variety of processes, such as the Schiff's base chemical reaction of aldehydes or ketones with the -NH<sup>2</sup> functional group, carboxymethylation, acetylation, quaternization, chelation with metals, alkylation, and sulfonation, etc. [2,67].

In its native form, chitosan has three terminal functional groups at distinct places: the C6-OH group, C3-OH group, and the C2-NH2 group, of which the C2-NH2 and the C6-OH groups could be easily modified, but the modification at the C3-OH site is not favorable because of the higher steric hindrance [68]. Naturally, the amino groups are the most common modifications made because of the feasible reactivity of the C2-NH2 group, making the grafting reactions much simpler. These amino groups are slightly more reactive with nucleophiles; nonetheless, both the hydroxyl and amino groups can readily react in electrophilic reactions with reagents such as acyl chlorides, acids, and alkanes, which can lead to the functionalization of the OH and NH2 groups in a non-selective manner [68]. Several reactive functional groups allow chitosan to interact with proteins and gain a cationic character, enhancing the adherence and differentiation of cells. Apart from this, the poor aqueous solubility of chitosan is an issue, which limits chitosan's biomedical utility, especially in physiological conditions, wherein it is poorly soluble and thus becomes a poor absorption promoter [69]. In addition, chitosan's efficiency of transfection is relatively low, and many beneficial functionalities are absent in chitosan, severely limiting its applications. Hence, it becomes imperative to modify the chemical characteristics of chitosan or to add some desirable functionalities, which will increase the water solubility for various therapeutic applications and offer a potential resource for endorsing novel biochemical actions while enhancing its material properties [70]. Astonishingly, it has been seen that modifying the structural features alone did not cause any significant change in chitosan's basic properties, but it does give them new properties. Chitosan has a unique structure that allows it to undergo various reactions, such as halogenation, oxidation, reduction, cross-linking, complexation, phosphorylation, and acylation, which impart new properties to these derivatives [68]. Thus, chitosan, along with its derivatives, has been famous for its tunable biological and chemical characteristics. Compared to native chitosan, their functionalized analogs have quicker gel-formation properties, greater aqueous solubility, and the capability of forming self-assembling nanostructures. Additionally, the design of hydrophobic equivalents with amphiphilic properties and chemical groups with a wide range of medicinal and active compounds; improved DNA complexing characteristics; and improved bioactivity [71].

Quaternized chitosan-modified black phosphorus nanosheets (BP-QCS) were prepared by Zhang et al. by electrostatic adsorption, wherein BP-QCS had more chemical stability and dispersiveness than plain BP in an aqueous medium. BP-QCS also had good biocompatibility, and photothermal/pharmacologic combination antibacterial action under NIR radiation (98% *S. aureus* inactivation in vivo) [72].

#### **6. Antimicrobial Properties of Chitosan**

Researchers have been quite interested in the antibacterial properties of chitosan as well as its analogs. Chitosan was shown to have antimicrobial inhibitory activity against bacteria, filamentous fungus, and yeast strains. Chitosan has also been identified as an antibacterial agent, although its ability to work in this manner is still unknown, as various distinct processes have been ascribed to its character. According to one theory, chitosan stimulates the migration of Ca++ from anionic locations of the membranes when subjected to the bacterial cell walls, leading to cell damage. It additionally has antiplaque efficacy against *Porphyromonomas gingivalis, Prevotella intermedia*, and *Actinobacillus actinomycetemcomitans*, among other oral infections [73–75].

Chitosan has a broad spectrum of action and high mortality towards gram-positive and gram-negative microorganisms, while being relatively nontoxic to mammals. Chitosan's bactericidal properties are said to be based on its molecular size. No et al. tested the antimicrobial property of six chitosan oligomers with wildly differing molecular masses against gram-positive and gram-negative bacteria to verify this theory. They discovered that chitosan significantly slowed the development of most bacteria examined, albeit the inhibitory effects varied depending on molecular size and strain. For gram-positive bacteria, chitosan had a more significant bactericidal impact than gram-negative bacteria. The explanation for this disparity is unknown; however, Y. J. Jeon et al. discovered that oligosaccharides and chitosan had stronger inhibitory activity against gram-positive microorganisms than gram-negative ones. As a result, it is understandable that chitosan has a strong inhibitory effect on gram-positive lactobacilli [28,76].

#### **7. Applications in Drug Delivery**

In recent years, nanocarrier-based drug delivery systems have become increasingly popular for the administration of active compounds to the desired site of action [77–84]. Numerous pieces of research have been conducted to determine chitosan's effectiveness as an orally administered vehicle [75,85]. The use of such medication carriers reduces the risks of systemic delivery [86]. Chitosan-based composite materials can be used to create reliable local drug carriers with the necessary mechanical behavior, retention time, and extendedrelease pattern [87]. Chitosan microspheres were designed to actively deliver therapeutics in disease areas [88,89]. Chitosan is non-toxic when taken orally and approved as a food additive by the Food and Drug Administration (FDA). It has also been investigated as a drug carrier for various macromolecules, including DNA, siRNA, growth regulators, and a variety of therapeutics [90,91].

Over the years, chitosan has been utilized in nanotechnology-based formulations, such as nanoparticles for drug, protein, and gene delivery through various routes of administration such as oral, topical, and parenteral routes [11]. To enhance the stability of chitosan nanoparticles (NPs), Saeed et al. used polyphosphoric acid or hexametaphosphate for concurrent ionotropic/covalent crosslinking with chitosan NPs. The resultant NPs showed considerable stability under CaCl2, 10% fetal bovine serum, and harsh pH conditions [92]. Similarly, Yu et al. synthesized octenyl succinic anhydride-modified chitosan nanoparticles to improve the anti-inflammatory and antioxidant properties of two model drugs: quercetin and curcumin. The as-prepared NPs exhibited pH-dependent release with faster drug release achieved at around pH 6.0 [93]. Thus, the versatile physicochemical properties and the tunable nature of chitosan renders it as a great candidate for nanotherapeutic drug delivery.

In a study by Barbosa et al. quercetin was delivered using novel polymeric nanoparticles derived from fucoidan and chitosan. Fucoidan/chitosan (F/C) nanoparticles, having three distinct weight fractions (1/1, 3/1, and 5/1), were produced by Barbosa et al. using the polyelectrolyte self-assembly approach. On increasing the mass ratio of fucoidan to chitosan inside the nanoparticles, the amount of quercetin in the fucoidan/chitosan nanoparticles ranged from 110 ± 3 to 335 ± 4 mgmL-1. With the size of the nanoparticles in the 300–400 nm region and membrane potential of more than +30 mV for the 1F/1C

proportion nanoparticles and about 30 mV for the 3F/1C and 5F/1C ratio nanoparticles, the physicochemical characteristics of stable nanoparticles were developed. As the pH rose from 2.5 to 7.4, the 1F/1C ratio nanoparticles grew larger and much more unstable, but the 3F/1C and 5F/1C nanoparticles remained unchanged. This showed that the latter nanoparticles remained stable throughout the digestive tract. Within simulated gastrointestinal conditions (particularly for the 3F/1C and 5F/1C combinations), the quercetin-loaded fucoidan/chitosan nanoparticles demonstrated significant antioxidant potential and controlled delivery, limiting quercetin deterioration and improving its oral absorption [94].

The fabrication of a film containing chitosan, sodium alginate (SA), and ethyl cellulose (EC) for buccal mucosa delivery was described in a study by Wang et al. (as depicted in Figure 1a). Utilizing self-made equipment, an interfacial reactive solvent-drying process was used to create a film of CS-SA unilateral releasing drug-loaded water-repellent layer EC. When matched to CS-EC and SA-EC films, the CS-SA-EC film had excellent tensile qualities. FT-IR acknowledged the formation of the amide linkage. DSC revealed that the active ingredient was distributed in an amorphous state inside the carrier system. The model compounds from the CS-SA-EC films had superior release qualities, according to the in vitro drug release study. All of the combinations of the drug release pathways are best described by the Ritger–Peppas theory. The films' permeation properties were tested using the TR146 cells culture and rabbit buccal mucosae through immunofluorescence and Western blotting. The prototype drugs' aggregate permeation levels were dramatically boosted. The film suppressed the translation of ZO-1 protein, and the ZO-1 protein expression pattern agreed with the results of the in vitro penetration tests. In rat models, the bioavailability of the drug-loaded films was assessed and compared to oral delivery. Compared to oral delivery, the relative absorption of the model medicines was 246.00 percent (Zolmitriptan) and 142.12 percent (Etodolac). The findings of this investigation show that the CS-SA-EC carrier has the capacity to transport drugs to the mucosal layer [95].

Chemotherapy is presently employed for most cancer therapies, but one of the substantial drawbacks of this approach is that it harms the body's normal tissues [96]. As a result, a few of the globe's most significant problems are developing new mechanisms for the smart and targeted delivery of these medications in tumor tissue. As a result, substantial money is now being spent on developing innovative drug delivery systems (DDS) featuring targeted delivery (as shown in Figure 1b). In a study by Kheiri et al., glutaraldehyde was employed to produce chitosan-polyacrylic acid-encapsulated Fe3O4 magnetic nanogelic core-shell (Fe3O4@CS-PAA) for carrying anticancer 5-fluorouracil (5-FU) medication. Then, the drug carrier assays were performed in an in vitro setting that mimicked a physiologic microenvironment and tumor tissue parameters. The Fe3O4@CS-PAA improved the rates of 5-FU release from nanogelic core-shell under tumor tissue settings (pH 4.5) compared to physiological fluids (pH 7.4). A variety of models were also employed to examine the drug release process. The process of 5-FU release from Fe3O4@CS-PAA was governed by Fickian diffusion [97].

Classic chemotherapy medicines for lung carcinoma have several drawbacks, including harsh side-effects, unpredictable drug release, low absorption, and resistant strains [98–102]. To overcome the constraints of unloaded drugs and enhance treatment outcomes, Zhu et al. created novel T7 peptide-based nanoparticles (T7-CMCS-BAPE, CBT) premised on carboxymethyl chitosan (CMCS), which were also likely to bind to the transferrin receptor (TfR) presented on lung carcinoma cells and accurately control the drug release depending on the pH and reactive oxygen species (ROS) levels. The drug-load content of docetaxel (DTX) and curcumin (CUR) was nearly 7.82 percent and 6.48 percent, respectively. Substantial biosafety was achieved even at concentrations as high as 500 g/mL. Notably, the T7-CMCS-BAPE-DTX/CUR (CBT-DC) combinations outperformed DTX solo therapy and other nanostructures loaded with DTX and CUR alone in vitro and in vivo studies. Additionally, they discovered that CBT-DC could improve the immunosuppressive surroundings, promoting tumor inhibitory activity (as illustrated in Figure 1c). These findings help set the groundwork for multimodal anticancer therapy [103].

p-mercaptobenzoic acid-embedded N, N, N-trimethyl chitosan nanoparticles (MT NPs) were effectively synthesized by Zhang et al. to carry anticancer medicines, genes, and immunological agents in a homogeneous nanoparticulate platform. Paclitaxel (PTX) was entrapped in the hydrophobic cavity of the MT NPs, while the hydrophilic exterior of the MT NPs had been loaded with survivin shRNA-expressing plasmids (iSur-pDNA) and recombinant human interleukin-2 (rhIL-2). The large quantity of glutathione induced a fast release of PTX due to the redox sensitivity of MT NPs. The MT/PTX/pDNA/rhIL-2 NPs were provided with higher anticancer therapeutic efficacy and enhanced tumor-induced immune responses resulting from the combined effects of PTX (1.5 mg/kg), iSur-pDNA (1.875 mg/kg), and rhIL-2 (6 <sup>×</sup> <sup>10</sup><sup>5</sup> IU/kg) at low dosages. The co-delivery of PTX, iSurpDNA, and rhIL-2 by amphipathic chitosan-derived NPs with redox sensitivity could be a potential mechanism in the therapy of malignancies [104]. 1c). These findings help set the groundwork for multimodal anticancer therapy [103]. p-mercaptobenzoic acid-embedded N, N, N-trimethyl chitosan nanoparticles (MT NPs) were effectively synthesized by Zhang et al. to carry anticancer medicines, genes, and immunological agents in a homogeneous nanoparticulate platform. Paclitaxel (PTX) was entrapped in the hydrophobic cavity of the MT NPs, while the hydrophilic exterior of the MT NPs had been loaded with survivin shRNA-expressing plasmids (iSur-pDNA) and recombinant human interleukin-2 (rhIL-2). The large quantity of glutathione induced a fast release of PTX due to the redox sensitivity of MT NPs. The MT/PTX/pDNA/rhIL-2 NPs were provided with higher anticancer therapeutic efficacy and enhanced tumor-induced immune responses resulting from the combined effects of PTX (1.5 mg/kg), iSurpDNA (1.875 mg/kg), and rhIL-2 (6 × 105 IU/kg) at low dosages. The co-delivery of PTX, iSur-pDNA, and rhIL-2 by amphipathic chitosan-derived NPs with redox sensitivity could be a potential mechanism in the therapy of malignancies [104].

outcomes, Zhu et al. created novel T7 peptide-based nanoparticles (T7-CMCS-BAPE, CBT) premised on carboxymethyl chitosan (CMCS), which were also likely to bind to the transferrin receptor (TfR) presented on lung carcinoma cells and accurately control the drug release depending on the pH and reactive oxygen species (ROS) levels. The drug-load content of docetaxel (DTX) and curcumin (CUR) was nearly 7.82 percent and 6.48 percent, respectively. Substantial biosafety was achieved even at concentrations as high as 500 g/mL. Notably, the T7-CMCS-BAPE-DTX/CUR (CBT-DC) combinations outperformed DTX solo therapy and other nanostructures loaded with DTX and CUR alone in vitro and in vivo studies. Additionally, they discovered that CBT-DC could improve the immunosuppressive surroundings, promoting tumor inhibitory activity (as illustrated in Figure

*Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 9 of 48

**Figure 1.** (**a**) Interfacial reaction solvent-drying method for EC-SA-CS polyelectrolyte film via selfmade equipment [95]; (**b**) Preparation and characterization of chitosan-based magnetic nanohydrogels for 5-fluorouracil drug administration and a kinetic assessment. Reproduced with permission from [97], copyright Elsevier 2022; (**c**) (B) Tumor-infected animals were given Cy5.5-labeled nanoparticles intravenously. The biodistribution of various compositions in vivo at 2 and 6 h, (C) ex vivo imaging of main organs and tumors. Reproduced with permission from [103], copyright Elsevier 2021. **Figure 1.** (**a**) Interfacial reaction solvent-drying method for EC-SA-CS polyelectrolyte film via selfmade equipment [95]; (**b**) Preparation and characterization of chitosan-based magnetic nanohydrogels for 5-fluorouracil drug administration and a kinetic assessment. Reproduced with permission from [97], copyright Elsevier 2022; (**c**) (B) Tumor-infected animals were given Cy5.5-labeled nanoparticles intravenously. The biodistribution of various compositions in vivo at 2 and 6 h, (C) ex vivo imaging of main organs and tumors. Reproduced with permission from [103], copyright Elsevier 2021.

In addition to the above, several other studies that were carried out to facilitate drug delivery by chitosan-based carriers include aluminum-modified mesoporous silica nanoparticles (H/Al-MSN)/curcumin/chitosan/mesalamine [105]; chitosan/polyvinyl pyrrolidone (PVP)/5-Fluorouracil [106]; quaternized chitosan/thiolated carboxymethyl chitosan [107]; chitosan/aptamer/mesoporous silica nanoparticles/doxorubicin [108]; norborene functionalized chitosan (CsNb)/polyacrylic acid (PAA)/5-Amino salicylic acid [109]; chitosan/pectin/5-Fluorouracil [110]; chitosan/dopamine/inulin aldehyde/indomethacin [111]; chitosan/magnetic alginate/amoxicillin [112]; chitosan/PVP/α-Fe2O3/doxorubicin [113]; and chitosan/mesoporous silica/methotrexate [114].

#### **8. Applications in Gene Delivery**

In the recent decade, the applications of RNA-interfering agents in gene therapy have been developed to exponential levels. Nevertheless, the tumor-targeting potential of these small interfering RNAs (siRNAs) is still a bottleneck [115–117]. Cancer progression involves various stages; caspases-linked anti-apoptotic factors inhibit apoptotic protein expression. One such gene, the survivin gene, is implicated in several physiological processes, such as regulating the cell cycle, cellular protection, and apoptosis suppression; these processes ensure that the cancerous cells survive [118,119]. Chitosan and polyethylene glycol (PEG) are known to aid the synthesis of cationic oligonucleotide nanoparticles [120,121]. Polyethyleneimine (PEI), an efficient gene carrier owing to its proton-sponge effect, functions as a buffer surrounding the endosome and delivers substances into the cytoplasmic space [122]. PEG helps reduce PEI toxicity, facilitates the formation of stable colloids, and prevents the deposition of nanoparticles [123]. The nanoparticles are coated by the chitosan, thus stabilizing them and preventing agglomeration. Hence, Arami et al. fabricated Fe3O4-PEG-LAC-chitosan-PEI nanoparticulate carriers, which were sufficiently cationic to react with siRNA. In vitro, they transferred the survivin siRNA to human breast cancer cells (MCF-7) and human chronic myelogenous leukemia cells (K562) using the nanoparticulate carrier. They found that the Fe3O4-PEG-LAC-chitosan-PEI nanoparticles combined sufficiently with siRNA, their sub-nanomolar size made them suitable gene carriers, and the survivin siRNA therapy was biocompatible and non-toxic to healthy cells [124].

Chitosan has shown potential for protecting siRNA from plasma denaturation and delivery into cancer cells by promoting the deposition of antineoplastic agents and biomolecules in the solid tumor tissues via the enhanced permeability retention (EPR) pathway [125]. Even though chitosan can be endocytosed by a ligand-receptor-mediated mechanism [126], the mono-ligand uptake of NPs is limited due to the saturation of membrane receptors [127]. RNA interference as a gene delivery method is limited by its low ability for targeted therapy and cellular absorption of small interfering RNA (siRNA). Hence, Zheng et al. fabricated chitosan-based dual-ligand nanoparticles (NPs) (GCGA) loaded with siPAK1 (GCGAsiPAK1), as shown in Figure 2a, whereby the targeting activity was influenced by the ligand molecules of glycyrrhetinic acid (GA) and galactose of lactobionic acid (LA). The NP targetability and siPAK1 cellular uptake were enhanced in the hepatocellular carcinoma by the GCGA-siRNA system [128]. *Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 11 of 48

**Figure 2.** (**a**) Delivery of siRNA via Dual-Targeting Nanoparticle-based Gene Therapy for Hepatocellular Carcinoma [128]; (**b**) A systematic approach for rheumatoid arthritis treatment using PLGA/PCADK hybrid microspheres encapsulating hyaluronic acid–chitosan siRNA nanoparticles. Reproduced with permission from [129], copyright Elsevier 2021; (**c**) Improved gene delivery with lipid-enveloped chitosan-DNA nanoparticles. Reproduced with permission from [130], copyright Elsevier 2018. In another study, Zhao et al. utilized the cationic chitosan's ability as a siRNA vector **Figure 2.** (**a**) Delivery of siRNA via Dual-Targeting Nanoparticle-based Gene Therapy for Hepatocellular Carcinoma [128]; (**b**) A systematic approach for rheumatoid arthritis treatment using PLGA/PCADK hybrid microspheres encapsulating hyaluronic acid–chitosan siRNA nanoparticles. Reproduced with permission from [129], copyright Elsevier 2021; (**c**) Improved gene delivery with lipid-enveloped chitosan-DNA nanoparticles. Reproduced with permission from [130], copyright Elsevier 2018.

modified using a targeting molecule such as hyaluronic acid (HA), which has an affinity toward CD44 receptors expressed on activated macrophages. They developed a sustained-release composite MP system using poly (cyclohexane-1, 4-diylacetone dimethylene ketal) (PCADK)-loaded HCNPs and PLGA-based siRNA therapy for rheuma-

lation method as a barrier for nuclease-mediated siRNA degradation and were then loaded as the aqueous phase into 20% PCADK and PLGA MPs, forming an NP-in-MP composite system (NiMPs) without disturbing the pH microenvironment inside the MPs. However, the introduction of HCNPs made the repulsion into attraction because of the surface positive charge on chitosan and changed the siRNA distribution from the periphery into a uniform distribution. In vitro release of siRNA was sustained release of 70% in 15 days. In vivo experiments on rat models revealed that siRNA had a relatively similar concentration in blood for up to 8 days and the pharmacodynamic effects were the same

Chitosan offers the benefit of a biodegradable and highly biocompatible biopolymer when used as a polycationic non-viral vector for gene transfer. Nevertheless, owing to its poor ability to successfully transfect under biological settings, it is of little value as a genetic delivery device without arduous chemical alterations to its composition. To solve this issue, Baghdan et al. created lipochitoplexes, which are liposome-encapsulated chitosan nanoparticles (LCPs), as shown in Figure 2c. The ionotropic gelation process was used to develop chitosan nanoparticles (CsNPs). A polyanionic tripolyphosphate was used for cross-linking the low molecular weight chitosan with a high DD, resulting in the effective trapping of plasmid DNA (pDNA) within the nanoparticles. The chitosan nanoparticles were incubated with anionic liposomes (DPPC/Cholesterol) to make LCPs. In physiological environments, the LCPs provided excellent pDNA protection, lower cytotoxicity, and a twofold improvement in transfection efficiency. In the chorioallantoic

as HCNPs [129].

In another study, Zhao et al. utilized the cationic chitosan's ability as a siRNA vector modified using a targeting molecule such as hyaluronic acid (HA), which has an affinity toward CD44 receptors expressed on activated macrophages. They developed a sustainedrelease composite MP system using poly (cyclohexane-1, 4-diylacetone dimethylene ketal) (PCADK)-loaded HCNPs and PLGA-based siRNA therapy for rheumatoid arthritis (as depicted in Figure 2b). The HCNPs were prepared by the ionotropic gelation method as a barrier for nuclease-mediated siRNA degradation and were then loaded as the aqueous phase into 20% PCADK and PLGA MPs, forming an NP-in-MP composite system (NiMPs) without disturbing the pH microenvironment inside the MPs. However, the introduction of HCNPs made the repulsion into attraction because of the surface positive charge on chitosan and changed the siRNA distribution from the periphery into a uniform distribution. In vitro release of siRNA was sustained release of 70% in 15 days. In vivo experiments on rat models revealed that siRNA had a relatively similar concentration in blood for up to 8 days and the pharmacodynamic effects were the same as HCNPs [129].

Chitosan offers the benefit of a biodegradable and highly biocompatible biopolymer when used as a polycationic non-viral vector for gene transfer. Nevertheless, owing to its poor ability to successfully transfect under biological settings, it is of little value as a genetic delivery device without arduous chemical alterations to its composition. To solve this issue, Baghdan et al. created lipochitoplexes, which are liposome-encapsulated chitosan nanoparticles (LCPs), as shown in Figure 2c. The ionotropic gelation process was used to develop chitosan nanoparticles (CsNPs). A polyanionic tripolyphosphate was used for cross-linking the low molecular weight chitosan with a high DD, resulting in the effective trapping of plasmid DNA (pDNA) within the nanoparticles. The chitosan nanoparticles were incubated with anionic liposomes (DPPC/Cholesterol) to make LCPs. In physiological environments, the LCPs provided excellent pDNA protection, lower cytotoxicity, and a twofold improvement in transfection efficiency. In the chorioallantoic membrane model (CAM), the efficacy of the delivery vector was also demonstrated in vivo. The LCPs could transfect the CAM without causing any damage to the nearby vascular capillaries. This unique biocompatible hybrid framework, free of chemical alterations, organic solvents, or harsh manufacturing processes, was deemed an ideal gene delivery mechanism for in vivo studies, revealing new information about non-viral therapeutics [130].

Other such studies related to gene delivery using chitosan are PEGylated chitosan/CRISPR-Cas9 dry powder [131]; carbonized chitosan/zeolite imidazolate nanoparticles/luciferaseexpressing plasmid (Pgl3)/splice correction oligonucleotides (SCO) [132]; methyl methacrylatebased chitosan/DNA [133]; cell-penetrating peptide-loaded chitosan-based iron oxide nanoparticles/plasmid pGL3/small interfering RNA/splice correction oligonucleotides [134]; thiolated trimethyl aminobenzyl chitosan/methylated 4-N,N-dimethyl aminobenzyl N,O carboxymethyl chitosan/thiolated trimethyl chitosan/plasmid DNA [135]; alkylaminemodified chitosan/p53 [136]; chitosan nanoparticles/polyethylene glycol/poly lactic acid/ nerve growth factor/acteoside/plasmid DNA [137]; chitosan/5-Amino-tetrazole(3-Chloropropyl) trimethoxysilane/Fe3O4 [138]; chitosan/starch polyplexes/plasmid DNA [139]; and chitosan/gelatin/oxidized sucrose/timolol maleate [140].

## **9. Applications in Protein Delivery**

Protein therapies have gained much traction in the medical business to fight diseases such as cancer, digestive problems, and autoimmune conditions. Protein distribution in vitro and in vivo, on the other hand, is hampered by protein degradation and a shorter lifetime. As a carrier system for protein, Rebekah et al. developed magnetic nanoparticles coated with graphene oxide chitosan hybrid (Fe-GO-CS), as shown in Figure 3A. To investigate the integrity and function of the produced nanocarrier, bovine serum albumin (BSA) was used as a protein. After 30 min and 3 h of exposure to Fe-GO-BSA and Fe-GO-CS-BSA solutions in trypsin, the SDS-PAGE examination revealed no significant changes. When relative to the Fe-GO composites, the Fe-GO-CS has a better drug load and release pattern, and the carrier system preserves the peptide from proteolytic action. As a result,

the Fe-GO-CS composite provides a superior nanocarrier that can be used in therapeutic settings [141].

Salivary proteins, such as histatins (HTNs), have been shown to have important physiological activities in dental homeostasis and avoiding periodontal disease. HTNs, on the other hand, are vulnerable to the mouth environment's considerable proteolytic activity. To preserve peptides from hydrolytic enzymes at a normal salivary pH, pHsensitive chitosan nanoparticles (CNs) have been proposed as possible carriers in a study by Zhu et al. The optimized formulations had a batch-to-batch consistency of 144 ± 6 nm, a polydispersity value of 0.15 ± 0.04, and a zeta potential of 18 ± 4 mV at a maximum pH of 6.3. Cationic polyacrylamide gel electrophoresis was used to examine HTN3 entrapment and release characteristics. HTN3 was successfully entrapped by the CNs, which were swollen exclusively at lower pH to aid HTN3 release. In diluted whole saliva, the stability of HTN3 against proteolysis was examined. Compared to unbound HTN3, HTN3 enclosed in CNs showed a longer lifetime. Biofilm density and microbial vitality were likewise lowered by CNs both in the presence and absence of HTN3. The study's findings showed that CNs are suitable as prospective protein carriers for oral purposes, particularly in the case of the difficulties that emerge under acidic circumstances [142].

Because of their versatility, supramolecular hydrogels are considered attractive drug vehicles for tissue regeneration [143]. Chitosan hydrogels lacking chemical cross-linkers are low in cytotoxicity and offer a high distribution potential, but they have poor mechanical qualities for injectable hydrogels. Jang et al. used click chemistry to create novel chitosan analogs for constructing supramolecular hydrogels with greater structural rigidity under mild circumstances (as depicted in Figure 3B). A sulfur–fluoride exchange process was used to synthesize the chitosan derivatives effectively, and the resulting chitosanmPEG/Pluronic-F127 (CS-mPEG/F127) bonded with -cyclodextrin (-CD) to produce a supramolecular hydrogel through a host–guest interaction. The proportion of chitosanmPEG and F127 could influence gelation kinetics, hydrogel characteristics, and bovine serum albumin (BSA) release. Thus, supramolecular hydrogels represent potential longterm tissue regeneration protein carriers [144].

The effective therapy of irritable bowel syndrome can benefit from the targeted administration of bioactive molecules such as proteins to the colonic region. Hence, Cao et al. used a single-step electro-spraying approach to make alginate/chitosan microcapsules (Alg/Chi/IL-1Ra MC) encapsulating IL-1Ra, as shown in Figure 3C. The pH-stimulation of the microcapsule and the in vitro release pattern was evaluated as critical efficacy considerations. The therapeutic efficacy of the Alg/Chi/IL-1Ra microcapsules was assessed using the dextran sodium sulfate (DSS)-induced colitis murine model, and the findings revealed that the Alg/Chi microcapsules shielded IL-1Ra from the hostile conditions of the upper gastrointestinal region. This was due to the microcapsule's pH-sensitive reaction, which enabled the targeted delivery of IL-1Ra in the colon. The Alg/Chi/IL-1Ra microcapsules reduced DSS-induced colitis in mice as measured by DAI, colonic length, colorectal tissue shape, histological injury ratings, and comparative protein concentrations (MPO, TNF-, and IL-1). This study indicated a viable method for oral protein administration, in situ colon release, and the potential use of IL-1Ra in treating autoimmunity and inflammatory illnesses [145].

Injectable hydrogels have long been a popular biomaterials subject. Unfortunately, due to tissue secretions, they are easily distributed during gelatinization in vivo, resulting in controlled-release drug delivery inability. To address this issue, Huang et al. described a new natural polymer-based injectable hydrogel made from aldehyde-modified xanthan (Xan-CHO) and carboxymethyl-modified chitosan (NOCC) that self-crosslinks, as illustrated in Figure 3D). The molar ratio of Xan-CHO and NOCC was adjusted to enhance the physical characteristics. Studies indicated that this composite material had self-healing, anti-enzymatic hydrolysis, biological compatibility, and biodegradability features. The release curve showed that BSA-FITC released in liquids was stable after 10 h. There was an association between this biopolymer and the hosts after integration with a vascular

*Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 13 of 48

term tissue regeneration protein carriers [144].

inflammatory illnesses [145].

supramolecular hydrogel through a host–guest interaction. The proportion of chitosanmPEG and F127 could influence gelation kinetics, hydrogel characteristics, and bovine serum albumin (BSA) release. Thus, supramolecular hydrogels represent potential long-

The effective therapy of irritable bowel syndrome can benefit from the targeted administration of bioactive molecules such as proteins to the colonic region. Hence, Cao et al. used a single-step electro-spraying approach to make alginate/chitosan microcapsules (Alg/Chi/IL-1Ra MC) encapsulating IL-1Ra, as shown in Figure 3C. The pH-stimulation of the microcapsule and the in vitro release pattern was evaluated as critical efficacy considerations. The therapeutic efficacy of the Alg/Chi/IL-1Ra microcapsules was assessed using the dextran sodium sulfate (DSS)-induced colitis murine model, and the findings revealed that the Alg/Chi microcapsules shielded IL-1Ra from the hostile conditions of the upper gastrointestinal region. This was due to the microcapsule's pH-sensitive reaction, which enabled the targeted delivery of IL-1Ra in the colon. The Alg/Chi/IL-1Ra microcapsules reduced DSS-induced colitis in mice as measured by DAI, colonic length, colorectal tissue shape, histological injury ratings, and comparative protein concentrations (MPO, TNF-, and IL-1). This study indicated a viable method for oral protein administration, in situ colon release, and the potential use of IL-1Ra in treating autoimmunity and

Injectable hydrogels have long been a popular biomaterials subject. Unfortunately, due to tissue secretions, they are easily distributed during gelatinization in vivo, resulting in controlled-release drug delivery inability. To address this issue, Huang et al. described a new natural polymer-based injectable hydrogel made from aldehyde-modified xanthan (Xan-CHO) and carboxymethyl-modified chitosan (NOCC) that self-crosslinks, as illustrated in Figure 3D). The molar ratio of Xan-CHO and NOCC was adjusted to enhance the physical characteristics. Studies indicated that this composite material had self-healing, anti-enzymatic hydrolysis, biological compatibility, and biodegradability features. The release curve showed that BSA-FITC released in liquids was stable after 10 h. There was an association between this biopolymer and the hosts after integration with a vascular endo-

endothelial growth factor, which expedited the rebuilding of the abdomen wall in rats. As a result, from a carrier perspective, this injectable hydrogel could minimize drug eruption in a range of circumstances and serve as a tissue-building scaffold [146]. thelial growth factor, which expedited the rebuilding of the abdomen wall in rats. As a result, from a carrier perspective, this injectable hydrogel could minimize drug eruption in a range of circumstances and serve as a tissue-building scaffold [146].

**Figure 3.** (**A**) A graphene oxide–chitosan combination with magnetic nanoparticles as a proteindelivering vehicle. Reproduced with permission from [141], copyright Elsevier 2021. (**B**) Fabrication of Chitosan–PEG conjugates and its Supramolecular Hydrogels for Protein Delivery Using Sulfur (VI) Fluoride Exchange (SuFEx) [144] (**C**) In situ application of the protein interleukin-1 receptor antagonist (IL-1Ra) for the therapy of dextran sulfate sodium (DSS)-induced colitis in a murine model using alginate/chitosan microcapsules. Reproduced with permission from [145], copyright Elsevier 2019. (**D**) For localized drug delivery, a unique in situ forming hydrogel consisting of xanthan and chitosan re-gelifying in liquids. Reproduced with permission from [146], copyright Elsevier 2018.

Other studies conducted for chitosan-based protein delivery were fluorinated chitosanchlorine e6/catalase [147]; chitosan/tripolyphosphate nanoparticles/ selenomethionine [148]; chitosan/ multiwalled carbon nanotubes/arginine-glycine-aspartic acid (RGD)/ urokinase [149]; mannose-anchored quaternized chitosan/thiolated carboxymethyl chitosan [107]; glycol chitosan/telechelic difunctional poly(ethylene glycol)/doxorubicin/gemcitabine [150]; chitosan/mesoporous silica/oxidized sodium carboxymethyl cellulose/sodium hyaluronate/ cytarabine/methotrexate [151]; chitosan/poly(N-isopropylacrylamide) (PNIPAAm)/ methotrexate [152]; chitosan/graphene oxide/folic acid [153]; chitosan/Fe3O4 nanoparticles/oxaliplatin/irinotecan [154]; and chitosan/acrylic acid [155].

#### **10. Vaccine Delivery**

Traditionally, vaccines were considered for large-dose administration, with limited targetability and noticeable immunogenicity. Thus, they necessitated using immunitymodifying adjuvants to improve their specific immunity [156,157]. The vaccine technology has since come a long way, and now nano-vaccines have demonstrated some added advantages such as higher antigenic retention ability, convenient administration, bettertargeted delivery, and enhanced bioavailability. Since chitosan has desirable properties, including its high biodegradability, non-toxicity, immunostimulatory activity, targeting potential, and provision for controlled vaccine release, it shows good potential for carrier material in nano-vaccines and their adjuvants. Chitosan-based materials also function as

adjuvating agents in vaccines and impart both immunostimulatory and immunotherapy functions. In addition, chitosan-based nanogels demonstrate improved penetrability and controlled drug release features; additionally, they show antigen-storing ability, antigen presentation, and immunoregulation.

The nanoparticles containing chitosan having a net positive surface charge can improve the adhesiveness of antigenic substances to the nasal mucosa and thus augment its absorption, which is essential for intranasal vaccine administration. To this end, Gao et al. developed chitosan NPs via a gelation method and functionalized the chitosan NPs using mannose by a hybridization technique. Using bovine serum albumin (BSA) as the model antigen, they prepared an intranasal vaccine using an optimization-based design of experiments (DOE). It was found that the mannose-based chitosan NPs (Man-BSA-CS-NPs) demonstrated good modification ability and an average particle size distribution and surface charge of 156 nm and +33.5 mV, respectively. The release of BSA from the systems displayed no irreversible deterioration or agglomeration. Additionally, the fluorescence analysis revealed an excellent binding constant between BSA and CS, indicating that BSA had good stability. In vitro studies also show that the Man-BSA-CS-NPs were non-toxic and biocompatible. Moreover, the Man-modified BSA-FITC-CS-NPs promoted the endocytic uptake and internalization of BSA-FITC when tested in DC2.4 cells. More importantly, the Man-BSA-CS-NPs demonstrated significant immunogenic enhancement of the BSA-specific IgG titer and the highest BSA-specific IgA response in the nasal lavage samples of in vivo mice models. Thus, this study shows how modifying chitosan with sugars and proteins and loading into NPs could be useful in vaccine delivery [158].

Wei et al. evaluated the vaccine delivery potential of a hydrophilic ph-responsive phosphorylated CS (PCS) incorporating ovalbumin (OVA) antigen and tested it in vivo on mice models. PCS solution in the mice formed a dense gel-like OVA network, resulting in enhanced immunity. This was hypothesized to be because of the sustained and controlled release of OVA, giving lengthier immune protection. Moreover, hydrogels' aqueous solubility enabled a convenient and hassle-free vaccine administration. Hence, because of the biocompatibility and non-toxicity of pH-responsive CS, hydrogels could be a potential platform for vaccine delivery [157]. However, their instability in solution and in vivo systems limits their overall applicability. Researchers suggest that modifying chitosan's hydrophilic surface moieties, deacetylation, or reduction in its MW may improve its water solubility. Otherwise, chemical alterations to its structure could be attempted to prevent its in vivo degradation [157].

Zho et al. developed a thermoresponsive hydrogel comprising N-(2-hydroxypropyl)- 3-trimethylammonium chitosan chloride (HTCC) and α-β-glycerophosphate (α-β-GP) as the vaccine carrier system for the *C. psittaci* antigen against avian influenza. The intranasal mucosal route of administration of vaccine gave the highest immune response in chickens [159].

Since the positively charged chitosan can easily interact with cellular membranes, contributing to its high biodegradability and formability, Zhang et al. prepared a chitosanbased PLGA nanoparticle vaccine encapsulating the recombinant protein OmpAVac (Vo) (VoNPs) against the *Escherichia coli* K1 caused meningitis in mice. The freeze-dried VoNPs were immunomodulatory in mice even after 180 days of storage [160].

Table 1 focuses on recent investigations on chitosan formulations for delivering active pharmaceutical compounds.


#### **Table 1.** Recent studies on chitosan focusing on delivering therapeutic compounds.

#### **11. Tissue Engineering**

A recently blooming field of research for regenerating injured/damaged tissues utilized versatile biomaterials encouraging cell adhesion and proliferation in tissue engineering (TE) [171]. Tissue engineering is a technique for developing biomaterials that can be used to repair, maintain, or regain tissue functionalities or entire organs. Tissue engineering has been a benefit to science. It has the capability of taking the place of traditional treatments, such as xenotransplantation and implanted devices. It is a technique for recovering or regenerating damaged tissues utilizing a combination of scaffolds, cells, and growth regulators. The scaffold components for tissue repair have been developed from various natural polymeric materials. Because of the desirable features, such as good biodegradability, biological activity, and biocompatibility, chitosan has been the most widely recognized biopolymer for constructing tissue frameworks among numerous biopolymers [172]. Table 2 describes the recent studies on chitosan formulations utilized for tissue engineering.

#### *11.1. Bone*

Bone tissue engineering (BTE) is a rapidly growing subject of research because it circumvents the limitations in therapeutic bone therapies, such as allogeneic or autologous bone transplantation, and irreversible prostheses' implants [173]. Using scaffolds, biomolecules, and implantable devices, BTE plays a critical role in the healing and regeneration of bone. When the injury is severe and has developed, bone tissue loses its potential to self-heal [174–176]. Nevertheless, this technique causes issues such as cellspecific bioactivity, the formation of massive structures, and a less inter-linked system [177]. As a result, nanoparticles and nanostructured materials are used to resolve this issue [178]. Chitosan has been explored for bone tissue regeneration because of its easy fabrication, chemical adjustments, compatibility with tissues, as well as other biomaterials, and its non-toxic nature.

Chitosan is reported to promote cell growth, suppress inflammatory reactions, and cause wound healing. In the early 2000s, Park et al. fabricated CS scaffolds loaded with platelet-derived growth factor (PDGF) for tissue regeneration in rat calvarial bones. Bernardi et al. showed that CS-derived scaffolds act as an independent stimulant for osteogenic maturation and do not merely act as a cell carrier. Nonetheless, native CS hydrogel has insufficient mechanical strength and a tendency for in vivo degradation. Thus, Zhang et al. incorporated short-chain chitosan (CS) into a semi-interpenetrating composite hydrogel (CG) in a covalently bound tetra-armed poly (ethylene glycol) network, as shown in Figure 4a. Acetylsalicylic acid (ASA) was encapsulated in the network by chain intermeshing and electrostatic attraction and obtained sustained release for more than 14 days and promoted osteogenic differentiation and growth of periodontal ligament stem cells (PDLSC) in a mouse calvarial osteogenic-defect model. This could be due to the expression of monocyte chemoattractant protein-1 on host mesenchymal stem cells and PDLSCs, which led to the stimulation of M2 macrophages and in situ polarization, demonstrating its potential for BTE [179].

Biopolymer-based nanomaterials have lately been used in therapeutic applications such as suture components, therapeutic delivery systems, tissue scaffolds, and inner bone stabilization implant devices. The biofilms formed by polymer-derived implants, on the other hand, are very sensitive to microbiological adherence. Chitosan biopolymer has good flexibility but poor mechanical properties; hence, when combined in composite films or nanoparticles, it leads to increased surface porosity and mechanical properties [180,181]. To this end, Prakash et al. created Chitosan/Polyvinyl alcohol/Graphene oxide/Hydroxyapatite/gold film materials for possible orthopedic applications. The graphene oxide/hydroxyapatite/gold hybrid (GO/HAP/Au) was made using a facile hydrothermal process, and the GO/HAP/Au composite integrated polymer film was made using the gel casting process. The biofilms were revealed to be compatible with murine mesenchymal cells (3.74% RBC lysis) and promoted osteoblast development, as indicated

by higher alkaline phosphatase enzymatic activity within the cells. As an outcome of these findings, it appeared that the biocomposite films created have osteogenic capabilities for managing bone-related disorders. High mechanical strength was seen, with tensile strength values ranging from 35.2% to 36.4% for composite films. According to the microbiological investigation, these films had high inhibitory regions against gram-positive and gram-negative microorganisms (*Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus mutans*, and *Escherichia coli*). As a result, the biocomposite biofilms developed were extremely biocompatible and could be employed for bone tissue regeneration [182].

Immune-modifying biomaterials have rapidly evolved as critical new systems for bone tissue regeneration. Eliciting macrophages to develop into the M2 subtype can lower inflammatory and immunological responses and speed the tissue healing following implants. Two biological compounds, bone morphogenetic protein-2 (BMP-2) and interleukin-4 (IL-4), were loaded and delivered in a controlled way in an inter-penetrable network hydrogel made of graphene oxide (GO)-carboxymethyl chitosan (CMC)/poly (ethylene glycol) diacrylate (PEGDA) to stimulate macrophage differentiation into M2 variety and improve bone growth in a study by Zou et al. (as depicted in Figure 4b). These two components were loaded with GO before being incorporated into a CMC/PEGDA hydrogel for longterm delivery. The hydrogel had improved mechanical rigidity, hardness, and stability. In vitro, the hydrogel containing IL-4 and BMP-2 strongly stimulated macrophage M2-type development and bone-marrow mesenchymal stem-cell bone formation. Moreover, in vivo investigations revealed that 8 weeks after insertion, the implantation of this hydrogel significantly lowered the local inflammatory reaction while increasing bone growth. Overall, the findings implied that IL-4- and BMP-2-loaded hydrogels synergistically impact on bone repair. A system such as this for the initiation and immunomodulatory reaction could be a potential method for further bone immunological management and tissue regeneration [183].

Similarly, Lu et al. used electrospun nanofibers of chitosan (CS ENFs) and modified them with fucoidan (Fu) and a CuS NPs polyelectrolyte complex involving genipin-based cross-linking, as shown in Figure 4c. This CuS–ENF composite provided antibacterial effects via photocatalytic and photothermal activities. Moreover, the composite could effectively promote the osteoblastic cells' alkaline phosphatase activity and the growth of capillary tubes within the endothelium. Thus, this novel CS ENF modification strategy could obtain scaffolds for BTE and antibacterial activity [184].

The foundation for generating nano-hydroxyapatite particles integrated in Chitosan/ carrageenan polyelectrolyte complexes (nHAp/CHI/-CGN) nanostructures with physically cross-linked constituents was presented by Zia and coworkers, as illustrated in Figure 4d. In the SBF investigation, the synthesized hybrid composites were shown to have lattice parameters and tensile characteristics similar to native bone and a rough texture, creating a thick apatite-like covering. It was also cytocompatible, biodegradable, and effective for protein adhesion. The nHAp/CHI/-CGN composites were highlighted as a promising contender for a BTE framework [185].

**Figure 4.** (**a**) Production of a biocompatible composite gel with long-lasting aspirin release for bone tissue regeneration [179]; (**b**) An interpenetrating network hydrogel with a GO-based controlled release mechanism induced M2-type macrophage differentiation for defective bone healing. Reproduced with permission from [183], copyright Wiley Online Library 2021; (**c**) CuS and fucoidan-modified chitosan nanofibers for antimicrobial and bone tissue regeneration. Reproduced with permission from [184], copyright Elsevier 2022; (**d**) Nanocomposite biomaterials for bone regeneration produced from nano-hydroxyapatite impregnated in Chitosan/κ-Carrageenan. Reproduced with permission from [185], copyright Wiley Online Library 2022. **Figure 4.** (**a**) Production of a biocompatible composite gel with long-lasting aspirin release for bone tissue regeneration [179]; (**b**) An interpenetrating network hydrogel with a GO-based controlled release mechanism induced M2-type macrophage differentiation for defective bone healing. Reproduced with permission from [183], copyright Wiley Online Library 2021; (**c**) CuS and fucoidan-modified chitosan nanofibers for antimicrobial and bone tissue regeneration. Reproduced with permission from [184], copyright Elsevier 2022; (**d**) Nanocomposite biomaterials for bone regeneration produced from nano-hydroxyapatite impregnated in Chitosan/κ-Carrageenan. Reproduced with permission from [185], copyright Wiley Online Library 2022.

Similarly, other composite scaffolds that researchers developed include chitosan/decellularized Alstroemeria flower stem [186]; chitosan/gelatin electrospun fibers [187]; chitosan thermo- and pH-responsive hydrogels [182,188]; chitosan/regenerated cellulose nanofibers [189]; copper(II)-chitosan/strontium-substituted hydroxyapatite [190]; chitosan/montmorillonite [191]; chitosan/silver polymeric scaffold [192]; carboxymethyl chitosan/polycaprolactone nanofibers [193]; and chitosan/collagen/hyaluronic acid oligosaccharides [194]. Similarly, other composite scaffolds that researchers developed include chitosan/ decellularized Alstroemeria flower stem [186]; chitosan/gelatin electrospun fibers [187]; chitosan thermo- and pH-responsive hydrogels [182,188]; chitosan/regenerated cellulose nanofibers [189]; copper(II)-chitosan/strontium-substituted hydroxyapatite [190]; chitosan/montmorillonite [191]; chitosan/silver polymeric scaffold [192]; carboxymethyl chitosan/polycaprolactone nanofibers [193]; and chitosan/collagen/hyaluronic acid oligosaccharides [194].

#### *11.2. Cartilage 11.2. Cartilage*

Chondrocytes create the extracellular matrix (ECM) protein molecules found in cartilages [195]. Articular cartilage provides crucial biomechanical activities to bone structures, including abrasion tolerance, load-carrying, and shock attenuation [196]. Since cartilage tissues are usually devoid of blood vessels, have a complicated structure, have quite a small density of cells, and have significant variability, it is harder to treat them [197]. Furthermore, they work in a rigorous atmosphere. So, when the thickness of the damage exceeds 4 mm, the ability for impulsive self-repair is reduced. Mosaicplasty, autologous chondrocyte injections, and micro-fracture are common therapies for cartilage tissue injuries, although they are not always the same structurally as natural tissue [198]. As a result, bioengineering has emerged as a viable option for osteochondral regeneration. Chondrocytes create the extracellular matrix (ECM) protein molecules found in cartilages [195]. Articular cartilage provides crucial biomechanical activities to bone structures, including abrasion tolerance, load-carrying, and shock attenuation [196]. Since cartilage tissues are usually devoid of blood vessels, have a complicated structure, have quite a small density of cells, and have significant variability, it is harder to treat them [197]. Furthermore, they work in a rigorous atmosphere. So, when the thickness of the damage exceeds 4 mm, the ability for impulsive self-repair is reduced. Mosaicplasty, autologous chondrocyte injections, and micro-fracture are common therapies for cartilage tissue injuries, although they are not always the same structurally as natural tissue [198]. As a result, bioengineering has emerged as a viable option for osteochondral regeneration.

Given its ability to be employed in various ways, such as fibers, sponges, and hydrogels, chitosan is often utilized in cartilage tissue regeneration [15,199,200]. Chitosan's resemblance to the GAGs present in ECM is also another significant aspect that renders it a desired substance in this domain [201]. Variable electrostatic exchanges with cytokines, receptors, and cell adhesion factors are notable features of GAGs. GAGs can also promote Given its ability to be employed in various ways, such as fibers, sponges, and hydrogels, chitosan is often utilized in cartilage tissue regeneration [15,199,200]. Chitosan's resemblance to the GAGs present in ECM is also another significant aspect that renders it a desired substance in this domain [201]. Variable electrostatic exchanges with cytokines, receptors, and cell adhesion factors are notable features of GAGs. GAGs can also promote

cartilage chondrogenesis. Chitosan can promote chondrogenesis, cartilage-specific protein production, and binding by electrostatic interactions with oppositely charged GAGs [202]. As a result of chitosan's ability to assist in or encourage the production of cartilage's unique GAGs, chitosan-derived composite scaffolds have now become attractive for osteochondral regeneration [203,204].

In the production of multipurpose microhabitats for cultured cells and tissue construction, bioinspired hydrogels are produced. Several mixtures of chitosan (CH) and hyaluronic acid (HA) derivatives with opposing ions were produced in the investigation by Davachi et al. To improve the communication among these constituents, phenolic moieties were supplemented on the backbones of CH (CHPH) and HA (HAPH) through a carbodiimide-based condensation, and further enzyme-assisted cross-linking in the availability of horseradish peroxidase was used to form a robust microenvironment for cell intercalation and tissue regeneration, as depicted in Figure 5a. The hydrogels' viscoelastic and structural properties revealed that a modest amount of HAPH produces the most remarkable outcomes in the structure. Cellular experiments revealed optimal cell survival and multiplication on the optimal hybrid hydrogel surfaces compared to plain hydrogels. In addition, the composite hydrogels showed better features for developing chondrocyte biomarkers and a greater tendency for MSCs to develop into cartilage-like cells. Altogether, the findings imply that in three-dimensional cartilage tissue regeneration, the optimal composite hydrogel can create an improved biological milieu for chondrocytes [205].

Three-dimensional (3D) printed hydrogel composites containing ceramics have shown promise for cartilage tissue engineering, but their mechanical and biological qualities remain unsatisfactory. In a study by Sadeghi et al. the production of chitosan/alginate-based scaffolds with nano-hydroxyapatite (nHA) using a combination of 3D printing and impregnating processes resulted in a combination, yet new, scaffold architecture for cartilage tissue engineering, as illustrated in Figure 5b. The introduction of nHA raised the Young's modulus of the scaffolds. In addition, the live/dead assay revealed that nHA significantly impacted the ATCD5 cell adhesion and scaffold survival. Moreover, the scaffolds containing nHA embedded in alginate hydrogels improved the cell survival and adhesion. Additionally, the chitosan scaffolds had good antibacterial properties, which were further increased by the nHA-based scaffolds. Overall, the chitosan/HA/alginate composite scaffolds are potential for cartilage tissue engineering, as well as the methodologies developed to generate hybrid scaffolds using 3D printing and impregnation methods, which could be applied to fabricate scaffolds for other applications [206].

For biological purposes, cryogel offers a highly porous architecture with mechanical strength and injectability. Three-dimensional (3D) printing is a form of customized manufacturing. Unfortunately, there is have been limited investigations into cryogel 3D printing. Chen et al. created a 3D-printable chitosan-based cryogel by employing polyfunctional polyurethane nanoparticles as the crosslinking agent, which interacted with chitosan at 4 ◦C for 4 h to build a rigid pre-cryogel for 3D printing, as shown in Figure 5c. To make 3D-printed chitosan cryogel, the generated pre-cryogel was frozen at 20 ◦C. The 3D-printed cryogel had features comparable to bulk cryogel, including high compression, elasticity modulus, and 3200 percent water absorption. The cell tests revealed that the 3D-printed chitosan cryogel frameworks offered good mechanical properties for human adipose-tissue adult stem-cell propagation and chondrocyte differentiation. The injectable and shaperecoverable 3D-printed chitosan-based cryogel scaffolds are viable substrates for tailored tissue regeneration and less surgical intervention [207].

Engineering a multiple-layered chitosan-based scaffold for osteochondral injury healing required a novel method. A highly porous, bioresorbable framework with a unique pore size distribution (mean = 160–275 m) was created by Pitrolino et al. using a hybrid of freeze-drying and porogen-leaching techniques. The inclusion of 70-weight percent nanohydroxyapatite (nHA) in the bone-like layer strengthened it. The scaffolding displayed fast mechanical restoration during compression loads and did not undergo delamination under tensile load capacity. The scaffold allowed human MSCs to adhere and proliferate,

exhibiting adherent cell morphology on the bony layer vs. the spherical cell shape on the chondrocyte layer. In vitro, the unique pore gradients and material constitution favored the osteogenic and chondrogenic development of MSCs in specific layers of the platform. This scaffold offered the ability to rebuild injured bone and cartilage and was an excellent option for noninvasive arthroscopic administration in the clinical setting [208]. shape on the chondrocyte layer. In vitro, the unique pore gradients and material constitution favored the osteogenic and chondrogenic development of MSCs in specific layers of the platform. This scaffold offered the ability to rebuild injured bone and cartilage and was an excellent option for noninvasive arthroscopic administration in the clinical setting [208].

proliferate, exhibiting adherent cell morphology on the bony layer vs. the spherical cell

When integrated alone without the inclusion of a chemical crosslinker, a new category of composite hydrogels obtained from chitosan (CS)/hyaluronic acid (HA) and silanizedhydroxypropyl methylcellulose (Si-HPMC) (CS/HA/Si-HPMC) have been synthesized and evaluated as injectable hydrogels for cartilage regeneration by Hu et al. (as shown in Figure 5d). The mechanical investigations revealed that, as the amount of Si-HPMC in the hydrogel grew, the swelling ratio and rheological characteristics rose, the compression strength fell, and the decomposition rate accelerated, so that particularly those containing 3.0% (*w*/*v*) Si-HPMC and 2.5/4.0% (*w*/*v*) CS/HA were feasible for cartilage tissue regeneration. The rate of regeneration was around 79.5% on the 21st day of the in vitro studies on cartilage ECM and was suitable for repairing joint cartilage tissue [209]. When integrated alone without the inclusion of a chemical crosslinker, a new category of composite hydrogels obtained from chitosan (CS)/hyaluronic acid (HA) and silanized-hydroxypropyl methylcellulose (Si-HPMC) (CS/HA/Si-HPMC) have been synthesized and evaluated as injectable hydrogels for cartilage regeneration by Hu et al. (as shown in Figure 5d). The mechanical investigations revealed that, as the amount of Si-HPMC in the hydrogel grew, the swelling ratio and rheological characteristics rose, the compression strength fell, and the decomposition rate accelerated, so that particularly those containing 3.0% (w/v) Si-HPMC and 2.5/4.0% (w/v) CS/HA were feasible for cartilage tissue regeneration. The rate of regeneration was around 79.5% on the 21st day of the in vitro studies on cartilage ECM and was suitable for repairing joint cartilage tissue [209].

*Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 21 of 48

**Figure 5.** (**a**) Enzymatic synthesizing chitosan/hyaluronic acid hydrogel scaffolds towards cartilage regeneration. Reproduced with permission from [205], copyright Elsevier 2022; (**b**) 3D printing and loading techniques were used to create chitosan/alginate/hydroxyapatite composite scaffolds for prospective cartilage tissue engineering. Reproduced with permission from [206], copyright Elsevier 2022; (**c**) Chitosan cryogel 3D printing as injectable and shape-restorable tissue engineering scaffolds. Reproduced with permission from [207], copyright Elsevier 2022; (**d**) The production steps for the Si-HPMC integrated CS/HA injectable hydrogel are depicted schematically [209]. **Figure 5.** (**a**) Enzymatic synthesizing chitosan/hyaluronic acid hydrogel scaffolds towards cartilage regeneration. Reproduced with permission from [205], copyright Elsevier 2022; (**b**) 3D printing and loading techniques were used to create chitosan/alginate/hydroxyapatite composite scaffolds for prospective cartilage tissue engineering. Reproduced with permission from [206], copyright Elsevier 2022; (**c**) Chitosan cryogel 3D printing as injectable and shape-restorable tissue engineering scaffolds. Reproduced with permission from [207], copyright Elsevier 2022; (**d**) The production steps for the Si-HPMC integrated CS/HA injectable hydrogel are depicted schematically [209].

Other studies for chondral tissue regeneration involve chitosan/xanthan/amniotic fluid stem cells [210], and thiolated chitosan/silk fibroin [211]. Other studies for chondral tissue regeneration involve chitosan/xanthan/amniotic fluid stem cells [210], and thiolated chitosan/silk fibroin [211].

#### *11.3. Blood Vessel 11.3. Blood Vessel*

Artificial grafts, which allow cells to create viable regenerative tissue, are extensively utilized in vascular tissue engineering [212]. Nevertheless, its usability is frequently restricted due to various host-cell invasions, calcification, or inadequate remodeling. Size incompatibility, supply shortage, and previous arterial disorders are all problems with Artificial grafts, which allow cells to create viable regenerative tissue, are extensively utilized in vascular tissue engineering [212]. Nevertheless, its usability is frequently restricted due to various host-cell invasions, calcification, or inadequate remodeling. Size incompatibility, supply shortage, and previous arterial disorders are all problems with

vascular autografts. Because the biomimetic implants or patches are proximate with the bloodstream, they must be non-toxic. Inflammatory responses and calcification, as well as post-surgical loss, might result from incompatibility. Biomaterial deterioration must be regulated so that it does not disintegrate too quickly or inadequately. The transplant is prone to form failure if it deteriorates too quickly. Cell growth and incorporation are hampered if it diminishes too gradually. As a result, effectiveness in blood vascular tissue engineering hinges on developing biodegradable polymer grafts, which can sustain cell uptake and multiplication while also undergoing fast remodeling. Based on its porosity and gel-forming capabilities, chitosan would not just be highly biocompatible and degradable but could simply be conveniently tweaked to show desirable qualities. Moreover, chitosan is a glucosamine and an N-acetyl glucosamine biopolymer. Glycosaminoglycans, having a striking resemblance to chitosan, make up the ECM of vasculature tissue. Mixtures of chitosan and other polymeric materials have demonstrated promising improvements in structural rigidity, cell attachment, and growth in this setting [212–214].

Wang et al. made a hybrid scaffold out of gelatin methacryloyl (GelMA) and carboxymethyl chitosan (CMCS), demonstrating its ability to stimulate revascularization. Compared to the pure GelMA scaffold, the composite GelMA/CMCS scaffolds implanted with BMSCs displayed outstanding mechanical characteristics, CD31 induction, and expression of vasculogenic genetic factors. The BMSC function in 3D-printed GelMA/CMCS scaffolds also revealed the potential of bioprinting cells impregnated with GelMA/CMCS hybrid scaffolds. Thus, GelMA/CMCS hybrid scaffolds have the potential to be used in vascular tissue regeneration [215].

Because a discrepancy in mechanical parameters between vascular patches and natural blood vasculature might lead to post-operative failures, vascular patches that mirror the biomechanics of natural blood capillaries should be designed. They created a bioinspired vascular patch by treating a decellularized scaffold (DCS) with a poly (ethylene glycol) (PEG) membrane and altering its surfaces with a heparin–chitosan polyelectrolyte multilayer (PEM). The PEM-functionalized PEG/DCS vascular patches had mechanical properties similar to natural blood vessels. They efficiently repelled platelet attachment, lowered hemolysis, enhanced coagulation time in vitro, and encouraged endothelial cell attachment and development. Additionally, the customized patches preserved the arteries' patency for an extended period (5 months) in vivo [216].

An electrospinning procedure was employed to build a scaffold for blood vessel regeneration using Poly-L-Lactic acid (PLLA) combined with chitosan and collagen in a study by Fiqrianti et al. The conduit's shape, chemical bonding, compressive strength, bursting pressure, hemocompatibility, and cellular viability were all studied using different amounts of chitosan in the mixture. In vitro tests revealed that adding chitosan–collagen to a cell culture could enhance cell survival and hemostasis. The conduit containing 10% PLLA, 0.5 percent chitosan, and 1 percent collagen had optimal results. The compressive modulus was 2.13 MPa, and the burst pressure was 2593 mmHg, both of which were within the biological blood vessel parameters threshold. The vascular graft component met the requirements of high hemocompatibility and lower toxicity with a hemolytic rate of 1.04 percent and a survival rate of 86.2 percent. The findings are encouraging for future research into vascular graft applications [217].

In a study by Soriente et al., they investigated how well the multifunctional chitosan (CS)/Poly (ethylene glycol) diacrylate (PEGDA) related scaffolds might promote revascularization in vitro. The substrates were bioactivated using both organic (BMP-2 peptide) and inorganic (hydroxyapatite nanoparticles) stimuli. In vitro angiogenic assays, focused on cell growth and differentiation, were used to investigate the qualities of the substances regarding physiological response stimulation on human umbilical vein endothelial cells (HUVECs). Their findings showed that the functional signals on the top of the CS/PEGDA scaffolds positively impacted angiogenesis responses, as measured by angiogenic marker production (CD-31) and endothelial tissue development (tube generation). Thus, the

bioactive CS/PEGDA scaffolds could be new inserts for encouraging the angiogenesis of tissue-engineered constructions in the domain of tissue engineering [218].

The grafting failure of medically useful synthetic tissues is caused by defects in the creation of microvascular systems, which bring oxygen and other nutrients to the cells. The vascularization of synthetic tissues can be aided by inflammation and immunomodulatory reactions. Endothelial progenitor cells (EPCs) and RAW264.7 macrophages were used as model cells in a capillary structure made of a gelatin methacrylate-derived cell-laden hydrogel scaffold complexed with interleukin-4 (IL-4)-loaded alginate-chitosan (AC) microspheres. Through electrostatic attachment, the AC microspheres retained and directed the EPCs, allowing for the creation of microvascular connections. The IL-4-loaded microspheres encouraged macrophage polarization into the M2 type, which resulted in a decrease in pro-inflammatory markers and an increase in vasculature [219].

#### *11.4. Corneal*

Ocular tissue engineering is a vital branch of bioengineering. The corneal epithelium oversees keeping the cornea clear by pushing ophthalmic fluid into the eyes. Since human corneal epithelial cells cannot renew, visual impairment occurs when they are lost due to aging, injury, or illness. Blindness develops when the body's cells decrease dramatically [220]. According to the World Health Organization, corneal degeneration and cataracts are among the reasons for visual impairment worldwide [221]. Corneal tissue engineering for retinal cell therapy is crucial because of the growing market for, and scarcity of, corneal donations.

The corneal tissue is a non-vascularized tissue that provides visibility. Corneal wounds are frequently repaired with amniotic membrane transplantation, which can be infected and immuno-rejected [222]. In the realm of cornea tissue regeneration, scaffold or membranes created from chitosan, gelatin, genipin, and other polymers are being investigated. Chitosan is popular because of its cytocompatibility and anti-inflammatory properties; however, its scaffolds have limitations, such as low structural rigidity. Polymers are combined with chitosan to improve scaffold qualities, similar to those of existing tissue engineering applications. Cornea tissue supports should have tensile and visual features identical to those of the corneal membrane and the capacity to sustain cells and adhesiveness. Because optical clarity is a critical quality for corneal prostheses, special care must be taken while choosing the material and production procedures. Under surgical treatment, the scaffolds or films should also be malleable and adaptive. The amniotic membranes now employed in clinical practice are not thick enough, disintegrate quickly, and have hygienic storage concerns [223,224].

By integrating chitosan nanoparticles (CSNPs) into chitosan/polycaprolactone (PCL) films, Tayebi et al. created a biodegradable transparent framework for growing corneal endothelium using the solvent casting process. The CSNP/PCL ratio increased, enhancing clarity and surface water sorption. The CSNP/PCL 50/25, which has the lowest WCA, demonstrated similar transparency with human acellular corneal stroma. According to the MTT experiment, the scaffold was non-cytotoxic and enhanced HCEC development. The HCECs were adequately attached to the framework and produced a dense monolayer. From the perspective of transparency and cytocompatibility, the scaffold is appropriate for corneal endothelium repair [225].

Clear, biologically compatible, and in situ formed biomimetic substrates are ideal for ocular bioengineering because they can profoundly fill irregularly shaped corneal stroma abnormalities and facilitate tissue repair. To this end, Feng et al. created a new class of corneal frameworks using oligoethylene glycol (OEG)-based dendronized chitosan (DCs), which have fascinating sol–gel shifts induced by temperatures close to physiological conditions, culminating in very clear, translucent hydrogels. These hydrogels' gelation points can be easily adjusted, and their tensile performance can be significantly increased when introduced into PBS at 37 ◦C rather than in pure water. In vitro experiments showed that these DC hydrogels have excellent biocompatibility and could enhance keratocyte differentiation and proliferation. In situ-produced DC hydrogels benefitted potential tissue repair in rabbits' eyes with cornea stromal deficiencies. Given their high biocompatibility and exceptional thermo-responsiveness, these thermo-gelling DCs have a lot of potential as ocular tissue replacements [226].

A hybrid membrane was created using carboxymethyl chitosan (CMCTS), gelatin, and hyaluronic acid in a study by Xu et al. Primary rabbit corneal epithelial cells (CEpCs) were implanted on it, and it was observed to be translucent, biodegradable, and ideal for CEpC adhesion and growth, as well as maintaining CEpC synthesis of epithelial cell-like proteins. The CEpCs/CMCTS membranes were utilized to treat the alkali-induced corneal injury in rabbits, and the membrane had the potential to enhance corneal epithelial restoration dramatically and regain corneal clarity and thickness [227].

There is no appropriate scaffold for transplanting limbal stem cells (LSCs) into the cornea to stimulate corneal regeneration following corneal alkali-induced burns. To this end, Xu et al. created a new alginate-Chitosan hydrogel (ACH) for LSC implantation in situ. Periodate-involved sodium alginate oxidation yielded sodium alginate dialdehyde (SAD), a physiological crosslinker. SAD quickly crosslinked carboxymethyl chitosan via Schiff's base reaction between the accessible aldehyde and amino residues. Self-crosslinking causes the ACH to develop rapidly on the wound surface without requiring any chemical cross-linking components. The in situ hydrogel was found to be remarkably transparent, gelled rapidly, bio-friendly, and noncytotoxic. The stem marker p63 was displayed by LSCs cultivated in vitro, while the differentiated epithelial biomarkers cytokeratin 3 and 12 were not. Moreover, the hydrogel encasing LSCs was proved to dramatically increase the epithelium restoration when applied to an alkali-induced burn site on the corneal region. Altogether, such an innovative in situ hydrogel-LSC grafting approach could be a quick and efficient way to cure corneal wounds [228].

Shahin et al. created a chitosan/gelatin hyaline film containing NHS and EDC crosslinking agents to transplant the corneal epithelial cells. Before crosslinking, the two gelatin and chitosan solutions were uniformly combined in proportions of 20/80, 30/70, 40/60, and 50/50 (Gel/Chi). They were dried in the oven for 24 h. It was found that rising chitosan concentrations enhanced the transparency and cytocompatibility of generated samples, greater water penetration and degradation rate of samples, and dramatically improved cell growth and vitality [229].

Other similar studies include chitosan/polycaprolactone [230]; carboxymethyl chitosan/gelatin/potassium acetate [231]; and chitosan/keratocyte spheroids [232].

#### *11.5. Periodontal*

With its biological properties, antibacterial properties, cytocompatibility, and capability to integrate with other substances, chitosan is evaluated as a viable contender for dental purposes. It works just as well as a single element and, in many circumstances, outperforms it when paired with other synthetic or natural components [75]. Chitosan-based materials are employed in a variety of applications, including enamel remineralization and development [233], dentin bonding [234], tooth repair material [235], and surface coatings for dental implants [236].

Meanwhile, periodontal diseases are among the critical challenges in medical research since it causes irreparable erosion of periodontal tissues, resulting in loss of teeth. In the advent of periodontitis, an inflammatory response condition, the hostile oral microenvironment becomes even more unfavorable. As a result, scientists have been looking for a viable biomaterial substitute. Various types and mixtures of chitosan have already been explored and examined; however, very few publications have been published in the last ten years. Many studies focused primarily on chitosan, while others looked at its ability to mix with several other organic and inorganic compounds. In most cases, the resulting material displayed potential dental action [53].

Chitosan, a promising polymer, is now being utilized in dental implant coating. Nevertheless, there is a paucity of studies on coating materials for implants apart from commercially purified titanium. As a result, Alnufaiy et al. studied the impact of chitosan

with two degrees of deacetylation (DDA) as coverings for laser surface microtopographic implants. In the chitosan-functionalized samples, the production of osteogenic biomarkers increased significantly. A high DDA of chitosan aided an enhanced bone mineralization and osteoblast development. As a result, the mixture of laser surfaces and chitosan may improve dental implant recovery and osseointegration procedures [237].

Given its superior biocompatibility, biodegradation by naturally occurring enzymes, suitable physicochemical attributes, and optimum molecular size, chitosan can be employed as a framework for the healing of periodontium (in the therapy of periodontal pockets). Furthermore, pluripotent dental mesenchymal stem cells, including stem cells from human exfoliated deciduous teeth (SHED) and human periodontal ligament cells (HPLCs), can be seeded into chitosan frameworks. Chitosan could be utilized to efficiently regenerate periodontal tissue by stimulating cementoblasts and osteoblasts to produce new tissues. Sukpaita et al. in their study using chitosan/dicarboxylic acid (CS/DA) with seeded HPLCs, found that within 6–12 weeks, significant in vivo bone growth was observed in calvarial-defects mice models [238].

The root canal network is chemo-mechanically debrided, and inflammatory or decaying pulp contaminated by bacteria is removed during root canal therapy. Several studies on the antimicrobial property of chitosan nanoparticles (CSNPs) towards pathogens such as P. gingivali, S. mutans, and E. faecalis have been previously reported. CSNPs could be used with calcium hydroxide as endodontic sealants or for temporary root canal filling. Regenerative endodontic therapies have also used chitosan. Bioactive compounds and growth stimulators can be added to chitosan-derived porous scaffolds. Dentin sialophosphoprotein, alkaline phosphate, and dentin matrix acidic phosphoprotein are odontoblastic indicators that promote the synthesis of secreted signaling chemicals, causing dental pulp stem cells (DPSCs) to proliferate and differentiate into odontoblasts [239].

Similarly, some other studies for dental tissue engineering composed of chitosan-based scaffolds are chitosan/PNIPAAm/graphene oxide [240]; chitosan/Ca-SAPO-34 monometallic or bimetallic nanoparticles [241]; chitosan/calcium [242]; chitosan/nanofluorohydroxyapatite or nanohydroxyapatite [243]; chitosan/gelatin/nanohydroxyapatite [244]; chitosan/PLAnanofibers [245]; chitosan/collagen/bone morphogenetic protein-7 [246]; chitosan/ gelatin [247]; chitosan biguanide/carboxymethyl cellulose [248]; and chitosan/polyurethane nanofibrous membrane/AgNPs [249].

#### *11.6. Miscellaneous*

#### 11.6.1. Skin

The skin represents the physical barrier between the surrounding environment and the physiological body [250,251]. Damage to the skin involves conditions such as burns, infections, and acute and chronic disorders such as psoriasis [252–255]. In such instances, TE provides advantages over conventional treatments because of its superior efficiency, fewer chances of donor morbidity, and immuno-compatibility reactions [256]. Additionally, the TE based on biocompatible biomaterials and their composites offer a substitute for fabricating tissue scaffolds that are physiochemically and biologically identical to natural tissues [257,258]. An essential requirement in skin TE is designing novel biopolymeric films or scaffolds resembling the extracellular matrix by combining biological, material chemistry principles and engineering [182], displaying features such as biodegradability, biocompatibility, and material characteristics [259].

Contemporary regenerative therapy is concerned with hypoxia and sepsis. The goal of oxygen-generating biopolymers with antibacterial properties is to address these issues. Oxygen deprivation at the implantation surface causes superoxide radicals, which slow the healing process. In addition, sepsis in the wound leads to a delayed healing process. As a result, antimicrobial and oxygen-producing scaffolds have demonstrated their ability to aid wound healing. The oxygen-releasing, ciprofloxacin-encapsulated collagen-chitosan scaffold used in this work was constructed for long-term oxygen supply. Biochemical oxygen was provided by calcium peroxide (CPO). The oxygenation pattern showed a

consistent diffusion of oxygen together with homogeneous CPO accumulation on the scaffold. Ciprofloxacin was released in a sustained manner. Cell culture experiments showed that the scaffold has good cell adhesion and motility capabilities for the fibroblasts. In the in vivo investigations in the skin, the flip model revealed that wound healing was improved, and necrosis was reduced. Histopathological investigations revealed that tissue structure was preserved, and collagen was deposited. The findings indicated that the suggested CPO-coated ciprofloxacin-based collagen–chitosan scaffold could be a suitable skin tissue regeneration alternative [260].

The tissue regeneration capability of the nanofibrous framework incorporating proteins and polysaccharides appears promising. Hence, Mohamad Pezeshki-Modaress investigated the influence of chitosan in chitosan/gelatin nanofibrous scaffolds created using an improved electrospinning method. The culture of dermal fibroblasts (HDF) on nanofibers in respect of adhesion, shape, and growth was investigated to see how the chitosan concentration affected the bioactivity of the electrospun chitosan/gelatin scaffolds for tissue regeneration. According to morphological observations, HDF cells were adhered to and propagated successfully on extremely porous chitosan/gelatin nanofibrous scaffolds with spindle-like forms and stretching. Electrospun gelatin/chitosan scaffolds in culture media kept their fibrous morphologies for 7 days. The MTS assay was used to measure cell proliferation on electrospun gelatin/chitosan scaffolds, revealing a beneficial influence of chitosan concentration (about 30%) and the nanofibrous pattern on scaffolds' biocompatibility (differentiation and adhesion) [261].

#### 11.6.2. Cardiac Tissue

Injectable biomaterials are a viable therapeutic method for cardiac tissue repair in the treatment of myocardial infarction-related chronic heart failure. Because of the ionized amino acid moieties, chitosan exhibits mucoadhesion, is hemostatic, and is effective in attaching to cellular membranes. Chitosan can also be used to create well-connected scaffolds with enough porosity to ensure cell survival by providing a constant oxygenated blood and nutrient supply [262]. Controlled release of loaded bioactive substances and growth factors becomes another essential aspect of a chitosan-derived scaffold. This makes it an excellent choice for cardiac tissue regeneration. Chitosan is a biocompatible platform that works as an ECM, allowing immobilized angiogenic growth factors to drive endothelial cell migration and proliferation, allowing for the development of a new vasculature to be facilitated [263]. The pig ECM is cross-linked with genipin and chitosan, according to investigations. This aids in maintaining ECM physiological components while also increasing the injected scaffolds' tensile stability. Before employing non-clinical ECM as a scaffold material for tissue engineering, it must be decellularized to minimize the hypersensitivity of the material [262].

#### 11.6.3. Connective Tissue

The 3D printing of the chitosan hydrogel is difficult because of its poor mechanical properties and weak formation capability. To this end, Zhang et al. fabricated acrylate substituted (DS 1.67) maleic chitosan (MCS) and thiol-terminated poly (ethylene glycol) (TPEG) through a step-chain growth photolytic polymerization technique, which helps in overcoming the formidable oxygen inhibitory effect. A strong intermolecular interaction between MCS and TPEG, and as compared to unmodified chitosan hydrogel, the prepared hydrogel showed a 10-fold and 2-fold enhancement in compression strength and gelling rate, respectively. Thus, 3D-printed chitosan hydrogel could be prepared by concurrent extrusion deposition and acrylate-thiol photopolymerization, having good printing efficiency and enhanced stability of the scaffold. The 3D-printed hydrogel demonstrated no cytotoxicity and supported L929 cell growth [264].

So far, only a handful of studies have been undertaken using carboxylated chitosan as a biomaterial for producing porous scaffolds and gels; hence, Yang et al. developed soft chitosan hydrogels in a hybrid composite with recombinant human collagen (RHC-CHI) through crosslinking-induced gelation method for use as soft-tissue scaffolds for tissue regeneration. They showed tunable mechanical properties by adjusting either the polymer amount or the RHC-to-chitosan ratio. Beyond a specific concentration, increasing chitosan's concentration decreased the hydrogel's tensile strength and started causing degradation. The prepared hydrogels were non-cytotoxic and promoted the adhesion and growth of NIH-3T3 cells. The in vivo tests also revealed that the hydrogels could rapidly infiltrate cells and cause wound closure; thus, they are good candidates for soft-tissue regeneration [265].


**Table 2.** Recent studies focusing on tissue engineering applications of chitosan formulations.


## **Table 2.** *Cont.*

#### **12. Wound Healing**

Apart from its applications in drug delivery and tissue regeneration, some antifungal and antibacterial activity have also been discovered with chitosan and its quaternary derivatives [277,278]. It offers additional benefits compared to other synthetic derivatives, such as its higher killing efficiency, broader antibacterial spectrum, and lower mammalian toxicity [279].

Modifications of chitosan with amino acids have had tremendous advantages for wound healing applications. Several approaches may be carried out for modifying native chitosan with amino acids, including physical and chemical methods, such as like blending, compositing, grafting, cross-linking, etc. Since the amino groups of chitosan have two distinct types of properties, alkaline as well as nucleophilic characteristics, chitosan can form imines and amides and can also lead to the formation of salt derivatives. It has been reported that the functionalization of chitosan with amino acids offers enhanced cell adhesiveness, re-epithelialization potential, rapid angiogenesis, and collagen formation [280].

Chitosan alone has quite limited tissue adhesiveness in a wet environment; hence, catechol-based chitosan was explored because of its ability to form strong covalent interactions between the tissue's thiol or amino group and oxidized catechol moieties. The catechol-functionalized chitosan also showed good anti-infective and tissue adhesive activity. To preserve the anticoagulant effect of chitosan, hydrophobically modified chitosan (hmCS) consisting of alkyl side chains was used as an amphiphilic analog of chitosan. Du et al. developed a hmCS lactate, and hydrocaffeic acid-modified chitosan (CS-HA)-based hydrogel via a two-step procedure, as shown in Figure 6A, which demonstrated good anti-infective activity against *P. aeruginosa* and *S. aureus*. The CS-HA/hmCS hydrogel was non-cytotoxic to 3T3 fibroblast cells and was capable of sutureless wound healing in a rat full-thickness skin model due to the remarkable tissue interfacial adhesiveness of the optimum Gel3, which could maintain the incision structure and promote wound healing [281].

In civil and wartime situations, producing an anti-infective shape-memory hemostasis sponge capable of guiding in situ tissue repair for noncompressible hemorrhages represents a problem. Chitosan has applications in producing hemostats because of its anti-infectivity, biocompatibility, non-toxicity, hemostasis, and so on [282]. However, in situations where severe hemorrhage or microbial infections occur, its hemostatic potential is quite limited [281]. To this end, Du et al. used a combination of 3D-printed microfiber leaching, freeze-drying, and surface-active modifications to create hemostatic chitosan sponges featuring strongly interconnective micro-channels. They showed that the micro-channeled alkylated chitosan

**Device Type Model** 

**Drug/Drug** 

sponge (MACS) could absorb water and blood while also restoring its structure quickly. In potentially deadly, healthy, and heparinized rats and pig hepatic-perforated wound specimens, the MACS provided better pro-coagulant and hemostatic capabilities than the clinically utilized gauze, gelatin sponge, CELOXTM (Crewe, UK), and CELOXTM gauze. In a rat liver damage model, they showed that it has anti-infective efficacy against *S. aureus* and *E. coli* and promotes liver parenchymal cell migration, vasculature, and tissue incorporation. Therefore, the MACS showed promise as a therapeutic translational tool for treating deadly noncompressible bleeding and promoting healing [283]. *Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 31 of 48

**Figure 6.** (**A**) Sutureless wound closure with anti-infective and pro-coagulator chitosan-mediated hydrogel tissue adhesives, reproduced with permission from [281], copyright ACS publications 2020; (**B**) Enhanced wound healing with electrospun ZnO-impregnated chitosan/PCL bilayer membranes featuring a spatially tailored structure. Reproduced with permission from [284], copyright Elsevier 2022; (**C**) Graphene-reinforced electrospinning-based chitosan/gelatin nanocomposite scaffolds having antibacterial and wound-healing properties [285]; (**D**) Chitosan-based hydrogels for wound repair that are injectable, self-healing, and antimicrobial. Reproduced with permission from [286], copyright Elsevier 2022. **Figure 6.** (**A**) Sutureless wound closure with anti-infective and pro-coagulator chitosan-mediated hydrogel tissue adhesives, reproduced with permission from [281], copyright ACS publications 2020; (**B**) Enhanced wound healing with electrospun ZnO-impregnated chitosan/PCL bilayer membranes featuring a spatially tailored structure. Reproduced with permission from [284], copyright Elsevier 2022; (**C**) Graphene-reinforced electrospinning-based chitosan/gelatin nanocomposite scaffolds having antibacterial and wound-healing properties [285]; (**D**) Chitosan-based hydrogels for wound repair that are injectable, self-healing, and antimicrobial. Reproduced with permission from [286], copyright Elsevier 2022.

Similar studies exploring the antibacterial wound healing abilities of chitosan were chitosan-based quaternary ammonium salt/ gentamicin sulphate hydrogel films [287]; chitosan/polyvinyl alcohol hydrogel/ZnO nanoparticles [288]; chitosan/pectin/lidocaine hydrogel [289]; quaternized chitosan/Matrigel/polyacrylamide hydrogels [290]; chitosan/carboxymethyl chitosan/AgNPs polyelectrolyte complex [291]; chitosan/PVA/starch electrospun mats [292]; carboxymethyl chitosan/polyurethane/helatin hydrolysate [293]; oxidized chitosan/amidated pectin hydrogel [294]; chitosan/PVA/copper [295]; and chitosan/PVA/HKUST-1 electrospun mats [296]. Table 3 depicts the latest investigations on chitosan formulations for wound healing **Table 3.** Recent studies focusing on the wound healing applications of chitosan formulation. **Polymer Formula-Preparation**  Similarly, Zhou et al. created a versatile multilayer membrane with electrospun chitosan (CS) and activated ZnO nanoparticles (as illustrated in Figure 6B). The bilayer membrane's external surface was made up of ZnO-loaded poly(-caprolactone) (PCL) fine fibers in an irregularly oriented arrangement, giving it many antimicrobial properties. The internal layer consisted of CS fibers with a core design that might perform as an anti-inflammatory and efficient cell interaction guide. Notably, the composite CS/PCL electrospun membranes containing 1.2 wt. percent ZnO nanoparticles had improved mechanical properties and a clear inhibiting zone against *E. coli* and *S. aureus*, as well as being non-cytotoxic to fibroblast cells. In addition, the bi-layered membranes allowed for the attainment of substantial ZnO nanoparticle bioavailability and coordination with the oriented structural characteristic of CS fibers, which reduced inflammation, encouraged cell motility, and allowed for re-epithelialization in vivo [284].

**tion Method Wound Site Effects/Results References**  Traditional DPPH scavenging experiments re-Ali et al. created electrospun chitosan/gelatin nanofibrous scaffolds that were strengthened with various amounts of graphene nanosheets and could be employed as antimicrobial and wound-healing constructs, as shown in Figure 6C. The different manufactured scaffolds

> vealed remarkable antioxidant characteristics. CS-GA formed hydrogels by cross-linking by undergoing oxidation at a physiological state. High cytocompatibility and hemocompatibility were observed in vivo

[297]

technology

were fully characterized before being tested for antibacterial activity, cytotoxicity, and cellular migration potential against *Escherichia coli* and *Staphylococcus aureus.* Nanostructures combined with 0.15 percent graphene nanosheets had the smallest width (106 ± 30 nm) and the most significant porosity (90 percent), along with good renewability and swellability. Nevertheless, increasing the number of graphene nanosheets by too great a degree resulted in beaded nanofibers with lower porosity, swelling ability, and degradability. Nanostructures reinforced using 0.15 percent graphene nanosheets, on the other hand, inhibited *E. coli* and *S. aureus* development by 50 and 80 percent, accordingly. When adult fibroblasts were cultivated with either non-reinforced or reinforced nanomaterials, the in vitro cytotoxicity experiments revealed negligible damage. After 24 and 48 h, cell movement was greater in the strengthened nanofibers compared to unmodified nanofibers, which is due mainly to the significant impact of graphene nanosheets on cellular migratory capability. After 48 h, cell migration outcomes for reinforced and unreinforced nanofibers were up to 93.69 and 97 percent, correspondingly [285].

A crucial healthcare concern is developing a cost-effective and readily available substance for better skin tissue regeneration. Hence, Deng et al. created injectable, self-healing adenine-functionalized chitosan (AC) hydrogels meant to expedite the healing process significantly without the need for medicinal agents and were inspired by the notion of wet wound repair, as shown in Figure 6D. In aqueous solutions, various AC derivatives with degrees of substitution (DS) ranging from 0.21 to 0.55 were synthesized, and AC hydrogels were constructed using a facile heating/cooling technique. AC hydrogels showed remarkable self-healing capability, low swelling rates, cytocompatibility, cell proliferation promotion, and hemostatic action. The hydrogels were proved to possess antibacterial properties against gram-negative and gram-positive bacteria, fungus, and bacteria with antibiotic resistance. Furthermore, full-thickness skin lesion model investigations revealed that AC hydrogels could greatly minimize inflammatory cell invasion and speed the wound repair. The hydrogel has the potential to revolutionize the design of multipurpose wound dressings [286].

Similar studies exploring the antibacterial wound healing abilities of chitosan were chitosan-based quaternary ammonium salt/gentamicin sulphate hydrogel films [287]; chitosan/polyvinyl alcohol hydrogel/ZnO nanoparticles [288]; chitosan/pectin/lidocaine hydrogel [289]; quaternized chitosan/Matrigel/polyacrylamide hydrogels [290]; chitosan/ carboxymethyl chitosan/AgNPs polyelectrolyte complex [291]; chitosan/PVA/starch electrospun mats [292]; carboxymethyl chitosan/polyurethane/helatin hydrolysate [293]; oxidized chitosan/amidated pectin hydrogel [294]; chitosan/PVA/copper [295]; and chitosan/PVA/HKUST-1 electrospun mats [296].

Table 3 depicts the latest investigations on chitosan formulations for wound healing


**Table 3.** Recent studies focusing on the wound healing applications of chitosan formulation.


#### **Table 3.** *Cont.*

Despite its various advantages, chitosan and chitosan-based formulations do suffer various drawbacks. The variations due to the source and preparation methods of chitosan have a direct influence on their mechanical and biological properties [307]. Moreover, due to its high solubility at an acidic pH, the ability of chitosan to control the release rate and stability of drugs is limited, requiring an additional coating of acid-resistant anionic polymers, such as alginate [308]. In gene delivery applications, the transfection efficiency of chitosan as a non-viral carrier depends on the media pH, degree of acetylation, cell type, molecular weight, etc. Most reports focus on the in vitro gene delivery efficacy, however, extensive research on in vivo models is still scarce [309].

#### **13. Conclusion and Future Perspectives**

Chitosan displays remarkable physicochemical attributes and good biocompatibility and links with human proteins, cells, and organs. The terminal amino groups present in its skeleton facilitate the formation of polycations in an acidic medium when the amine groups undergo protonation; this enables interactions with anionic polymers in various shapes and geometry. It is expected that chitosan and chitosan-based derivatives may be used to fabricate tissue repair scaffolds for a plethora of tissues, such as bone, cartilage, skin, cornea, blood vessel, and so on, with suitable properties. Such matrices are beneficial because of their natural resemblance to host tissues and similarities in structure and function to biological molecules.

As humans become more aware of the intricate biological reactions to the presently available biomaterials, as well as with the expanding knowledge about the human anatomy and physiology, organ and tissue damage, disease proliferation, and cellular changes in protein functions during tissue injury, a co-operative endeavor involving polymer chemists, engineering minds, biologists, and physicians could be undertaken for developing novel polymeric biomaterials specially tailored for each application. Currently, chitosan stands as one of the most reliable and convenient biomaterials for various kinds of biomedical applications, mainly due to its accessibility and peculiarities. Nonetheless, added efforts need to be taken to enhance the tissue scaffold for tailored properties for different types of tissues. Along with in vitro studies, the same scaffolds must also be tested in vivo to determine their clinical utility in humans. Several studies have shown that the in vitro benefits of chitosan composites for tissue and wound healing do not translate as expected in the in vivo animal models. A closer look into the reasons and modifications required must be encouraged.

With the remarkable progress of tissue engineering science such as novel stem cell sources, microfluidics' devices, versatile and tailored biomaterials, etc., some challenges remain, such as engineering blood vessels into the tissue scaffolds, the immunogenicity of the scaffolds, and regulatory issues. The 3D printing platform provides the flexibility to print complex structures, such as cells and scaffolds, while also giving good control of pore size and size distribution. Consequently, recent publications depict the utility of chitosan in printing constructs for bone, skin, vascular, and cartilage tissue engineering. However, their practical applications and clinical translation are still being investigated. Recent reports have suggested that chitosan and its quaternized derivative can have immunomodulatory effects by activating the antigen-presenting cells and inducing cytokine production. Hence, their role as vaccine adjuvants could open new paradigms in vaccine formulation delivery.

Although there have been definitive strides in technological advancements, more profound research is warranted for evaluating cell-specific intercommunications, in vivo understanding, and replication of bioactivity, long-term stability, and biocompatibility studies to make chitosan a more widely used biomaterial.

**Author Contributions:** Conceptualization and supervision: M.A.S.A., A.D. and M.J.A.; Resources: M.A.S.A., S.P., M.A.A., B.M.A., A.K. and M.J.A.; Literature review and writing—original draft preparation: M.A.S.A., S.P., B.M.A., M.A.A. and M.J.A. writing—review and editing: M.A.S.A., S.P., M.A.A., B.M.A., M.J.A., A.K. and A.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** M.A. would like to thank the Deanship of Scientific Research at Umm Al-Qura University for supporting this work by Grant Code: (22UQU4290565DSR78).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** M.A. would like to thank the Deanship of Scientific Research at Umm Al-Qura University for supporting this work by Grant Code: (22UQU4290565DSR78). S.P. would like to thank Ramesh Parameswaran and Vignesh Muthuvijayan for their continuous support and motivation. Author S.P. would like to thank the Indian Institute of Technology Madras, Ministry of Human Resource Development for providing financial assistance. Author M.J.A. acknowledges the support of the Deanship of Scientific Research at Prince Sattam bin Abdulaziz University.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**


## **References**


## *Article* **Electrochemical and Ion Transport Studies of Li<sup>+</sup> Ion-Conducting MC-Based Biopolymer Blend Electrolytes**

**Elham M. A. Dannoun <sup>1</sup> , Shujahadeen B. Aziz 2,3,\* , Mohamad A. Brza <sup>4</sup> , Sameerah I. Al-Saeedi <sup>5</sup> , Muaffaq M. Nofal <sup>6</sup> , Kuldeep Mishra <sup>7</sup> , Ranjdar M. Abdullah <sup>2</sup> , Wrya O. Karim <sup>8</sup> and Jihad M. Hadi <sup>9</sup>**


**Abstract:** A facile methodology system for synthesizing solid polymer electrolytes (SPEs) based on methylcellulose, dextran, lithium perchlorate (as ionic sources), and glycerol (such as a plasticizer) (MC:Dex:LiClO<sup>4</sup> :Glycerol) has been implemented. Fourier transform infrared spectroscopy (FTIR) and two imperative electrochemical techniques, including linear sweep voltammetry (LSV) and electrical impedance spectroscopy (EIS), were performed on the films to analyze their structural and electrical properties. The FTIR spectra verify the interactions between the electrolyte components. Following this, a further calculation was performed to determine free ions (FI) and contact ion pairs (CIP) from the deconvolution of the peak associated with the anion. It is verified that the electrolyte containing the highest amount of glycerol plasticizer (MDLG3) has shown a maximum conductivity of 1.45 <sup>×</sup> <sup>10</sup>−<sup>3</sup> S cm−<sup>1</sup> . Moreover, for other transport parameters, the mobility (*µ*), number density (*n*), and diffusion coefficient (*D*) of ions were enhanced effectively. The transference number measurement (TNM) of electrons (*t*el) was 0.024 and 0.976 corresponding to ions (*t*ion). One of the prepared samples (MDLG3) had 3.0 V as the voltage stability of the electrolyte.

**Keywords:** biopolymer blend electrolyte; EIS and FTIR; ion transport parameters; complex permittivity; LSV and TNM measurements

## **1. Introduction**

Due to demand for high-energy consumption, for instance, to power laptops and mobile devices, the usage of energy storage devices is widespread. In order to produce low-cost and safe energy storage systems, the design of high-performance electrochemical devices has been extensively studied [1,2]. It is essential to use polymer electrolytes (PEs) for electrochemical devices because of their common advantages including qualities such as wide electrochemical windows, leakage-free, ability to form thin films, lightweight, flexibility, ease of handling, transparency, good conductivity, and solvent-free feature compared to commercial liquid electrolytes (LEs) [3,4]. In the PEs of the energy storage

**Citation:** Dannoun, E.M.A.; Aziz, S.B.; Brza, M.A.; Al-Saeedi, S.I.; Nofal, M.M.; Mishra, K.; Abdullah, R.M.; Karim, W.O.; Hadi, J.M. Electrochemical and Ion Transport Studies of Li<sup>+</sup> Ion-Conducting MC-Based Biopolymer Blend Electrolytes. *Int. J. Mol. Sci.* **2022**, *23*, 9152. https://doi.org/10.3390/ ijms23169152

Academic Editors: Swarup Roy and Valentina Siracusa

Received: 16 July 2022 Accepted: 11 August 2022 Published: 15 August 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

devices, the host polymer is often divided into two types: natural and synthetic polymers [5]. Non-biodegradable synthetic polymers deplete petroleum resources and introduce disposal difficulties [6]. As a result, biopolymers may be employed as the host polymer to investigate energy storage devices and minimize plastic waste pollution. These polymers, which derive from natural resources, have distinct advantages over synthetic ones, including low cost, wide compatibility with a wide range of solvents, abundance, and high film formation efficiency [7,8]. In PE investigations, starch, cellulose, chitosan, dextran, and carrageenan are the most- often employed biopolymers [9–13].

The search for novel ion-conducting PEs for lithium-based energy devices continues incessantly [14–17]. To replace the LEs in lithium-ion batteries, PEs that are linked to lithium salts and integrated into neutral or ion-conducting polymers have been suggested [18]. In contrast to manufactured polymers, which are durable, natural biopolymers degrade with time [19]. Cellulose is nature's most abundant organic polymer, making it an excellent source of renewable energy [8]. As a natural polymer, cellulose is seen as a potential replacement for petrochemical polymers [20]. Cyanoacrylate is one of the most often used and lowest-priced types of cellulose. A biodegradable polymer that has excellent filmforming capabilities may be transparent and possesses superior mechanical and electrical properties that can be made from alkali cellulose. Methylcellulose (MC) is one of these cellulose derivatives [6]. By adding dimethyl sulfate or methyl chloride to alkali-based cellulose, a polymer with a 1,4 glycosidic link is created, known as MC [21]. Through a dative connection, ions create a complexation with polymer-host-oxygen-containing functional groups. Ion conduction in MC is facilitated by functional groups possessing lone-pair electrons, including hydroxyl, glycosidic link, and mexthoxy groups [22]. When it comes to film-forming and dissolving qualities, MC is a standout because of its strong mechanical, thermal, and chemical stabilities [23]. Glass transition temperature (Tg) for microcrystalline MC is between 184 and 200 ◦C, making it an excellent material for hightemperature applications [22]. Leuconostoc mesenteroides bacteria produce dextran, a non-toxic and biodegradable polysaccharide that has lone-pair electrons of heteroatoms, such as oxygen, which is essential for dissolving inorganic salts [13]. The polymer blend approach has been reported to generate a polymer mix host with higher ionic conduction sites [24]. A reduced glass transition temperature and degree of crystallinity may be achieved by mixing polymers [25]. A PE based on lithium salts is able to perform well overall in terms of crucial features, such as electrochemical window stability and ionic conductivity [26]. Various plasticizing agents were identified to further improve the above-mentioned properties. The loading of glycerol provided a conductivity of (1.32 <sup>±</sup> 0.35) <sup>×</sup> <sup>10</sup>−<sup>3</sup> S cm−<sup>1</sup> for the chitosan-PS-LiCF3SO<sup>3</sup> system [27].

In this study, glycerol (contains three OH groups) as an eligible plasticizer has been used in an effort to pick up the conductivity of the blended polymer system. It causes weakening of the attraction force between the polymer chains and cations and anions of the salts [4]. The objective of this study is to enhance the conductivity of the prepared SPEs by adding glycerol as more ions are dissociated to increase conductivity. The electrochemical tests indicate the films are convenient for applications.

#### **2. Results and Discussion**

#### *2.1. FTIR Results*

To study polymer-mix developments, several scientists have turned to FTIR. Intermolecular interactions may be studied using FTIR spectroscopy, which analyzes spectra based on the stretching or bending vibrations of specific bonds. Figure 1 showed the spectra of the electrolytes at the 400 to 4000 cm−<sup>1</sup> . A wide band of 3353 cm−<sup>1</sup> was observed in the FTIR spectra for MC: Dext, indicating the presence of OH groups [28,29]. The bands, due to -OH bending and -OH stretching, can be found at 1253–1503 cm−<sup>1</sup> in a sharp peak and 3703–3149 cm−<sup>1</sup> in a broad-peak form, respectively, by glycerol loading [30,31]. A peak at 1334 cm−<sup>1</sup> came from -OH bending. The -CH asymmetrical and -CH symmetrical stretching are found at 3049 to 2849 cm−<sup>1</sup> [32,33]. As the concentration of glycerol rises,

the intensity of the -CH bands increases, indicating a complicated interplay between the glycerol and MC-Dex-LiClO<sup>4</sup> [34]. As glycerol concentrations increase, the position of the electrolyte carboxamide and amine band shifts somewhat to 1749–1519 cm−<sup>1</sup> . The impact of increasing the content of glycerol on the strength of the interaction between the components of the polymer blend is proved where additional ions are interacting with the oxygen atoms and nitrogen atoms [27]. The range of carboxamide and amine bands can be recognized straightforwardly as reported by Aziz et al. [35] and Shukur et al. [36]. Interestingly, a sharp peak lies between 901 and 1203 cm−<sup>1</sup> that comes from the C-O stretching, which is in accordance with the findings of the study documented by Mejenom et al. [37]. This band peak widens as the glycerol concentration increases. The insertion of LiClO<sup>4</sup> salt into MC: Dex resulted in a significant shift in the strength of the bands, which is fascinating. The changes in the macromolecular arrangement have a direct effect on the intensity of these bands. The spectra of the complexes may show more and less organized structures, which might be the cause of these bands [38]. between the glycerol and MC-Dex-LiClO4 [34]. As glycerol concentrations increase, the position of the electrolyte carboxamide and amine band shifts somewhat to 1749–1519 cm−1. The impact of increasing the content of glycerol on the strength of the interaction between the components of the polymer blend is proved where additional ions are interacting with the oxygen atoms and nitrogen atoms [27]. The range of carboxamide and amine bands can be recognized straightforwardly as reported by Aziz et al. [35] and Shukur et al. [36]. Interestingly, a sharp peak lies between 901 and 1203 cm−1 that comes from the C-O stretching, which is in accordance with the findings of the study documented by Mejenom et al. [37]. This band peak widens as the glycerol concentration increases. The insertion of LiClO4 salt into MC: Dex resulted in a significant shift in the strength of the bands, which is fascinating. The changes in the macromolecular arrangement have a direct effect on the intensity of these bands. The spectra of the complexes may show more and less organized structures, which might be the cause of these bands [38].

The bands, due to -OH bending and -OH stretching, can be found at 1253–1503 cm−1 in a sharp peak and 3703–3149 cm−1 in a broad-peak form, respectively, by glycerol loading [30,31]. A peak at 1334 cm−1 came from -OH bending. The -CH asymmetrical and -CH symmetrical stretching are found at 3049 to 2849 cm−1 [32,33]. As the concentration of glycerol rises, the intensity of the -CH bands increases, indicating a complicated interplay

*Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 3 of 17

**Figure 1.** FTIR spectra for (i) MDLG1, (ii) MDLG2, and (iii) MDLG3 in the region 400–4000 cm<sup>−</sup>1. **Figure 1.** FTIR spectra for (i) MDLG1, (ii) MDLG2, and (iii) MDLG3 in the region 400–4000 cm−<sup>1</sup> .

Many useful qualities, such as peak resolution, noise removal, and checking for interconnections between deconvolution parameters, are provided by the FTIR deconvolution, which is used in support of the conductivity findings [39]. When using this method, the deconvolution FTIR spectra may be used to determine the ion fraction Many useful qualities, such as peak resolution, noise removal, and checking for interconnections between deconvolution parameters, are provided by the FTIR deconvolution, which is used in support of the conductivity findings [39]. When using this method, the deconvolution FTIR spectra may be used to determine the ion fraction that conducts electricity. Ramelli et al. noted that FTIR spectra might be deconvoluted, allowing one to isolate existing peaks and modify both intensity and wavenumber [40].

A peak for ClO<sup>4</sup> localizes from 650 to 600 cm−<sup>1</sup> and is regularly utilized in the investigation of ion–ion interactions in the PE and LiClO<sup>4</sup> salt addition [41,42]. The ClO<sup>4</sup> bands are featured by two peaks extending from 610 to 630 cm−<sup>1</sup> , which indicates that, at most, two dissimilar sorts of ClO<sup>4</sup> anions are present in this material.

Salomon et al. documented that the presence of Li+1 is attached to the ClO<sup>4</sup> band located at 610–630 cm−<sup>1</sup> . CIP ClO<sup>4</sup> <sup>−</sup><sup>1</sup> anions were observed at lower than 610 cm−<sup>1</sup> , while free ClO<sup>4</sup> anions were observed at about 610–630 cm−<sup>1</sup> [42]. Figure 2a–c show the deconvoluted FTIR spectra for the prepared electrolytes. The free ClO<sup>4</sup> peaks are larger than the peaks of contact-ion pairs, as shown in Figure 2. Glycerol plasticizer helps dissolve LiClO<sup>4</sup> salt in the MC: Dex matrix; therefore, this is what happens when the two mixtures are combined. Percentageof CIP(%) ×100% <sup>+</sup> <sup>=</sup> *f c c A A A* (2) where *Af* is the area of the FIP and *Ac* is the area of the CIP. The percentages of FI and CIP are shown in Table 1.

that conducts electricity. Ramelli et al. noted that FTIR spectra might be deconvoluted, allowing one to isolate existing peaks and modify both intensity and wavenumber [40]. A peak for ClO4 localizes from 650 to 600 cm−1 and is regularly utilized in the investigation of ion–ion interactions in the PE and LiClO4 salt addition [41,42]. The ClO4 bands are featured by two peaks extending from 610 to 630 cm−1, which indicates that, at

Salomon et al. documented that the presence of Li+1 is attached to the ClO4 band located at 610–630 cm−1. CIP ClO4−1 anions were observed at lower than 610 cm−1, while free ClO4 anions were observed at about 610–630 cm−1 [42]. Figure 2a–c show the deconvoluted FTIR spectra for the prepared electrolytes. The free ClO4 peaks are larger than the peaks of contact-ion pairs, as shown in Figure 2. Glycerol plasticizer helps dissolve LiClO4 salt in the MC: Dex matrix; therefore, this is what happens when the two

The free ions and contact ion pairs were measured using the area of the FTIR bands

Percentageof FI(%) ×100%

<sup>+</sup> <sup>=</sup> *f c f A A A*

(1)

*Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 4 of 17

most, two dissimilar sorts of ClO4 anions are present in this material.

mixtures are combined.

by the equations below [4]:

**Figure 2.** Deconvoluted FTIR spectra for (**a**) MDLG1, (**b**) MDLG2, and (**c**) MDLG3. **Figure 2.** Deconvoluted FTIR spectra for (**a**) MDLG1, (**b**) MDLG2, and (**c**) MDLG3.

**Table 1.** The percentages of ions. The free ions and contact ion pairs were measured using the area of the FTIR bands by the equations below [4]:

$$\text{Percentage of FI } (\%) \, = \frac{A\_f}{A\_f + A\_c} \times 100\% \tag{1}$$

$$\text{Percentage of CIP } (\%) = \frac{A\_c}{A\_f + A\_c} \times 100\% \tag{2}$$

<sup>×</sup> <sup>=</sup> (3)

= (4)

= (5)

(6)

dissociate from LiClO4 salts. There is a strong correlation between the conductivity and the proportion of free ions, according to Aniskari and colleagues [43]. The calculation of where *A<sup>f</sup>* is the area of the FIP and *A<sup>c</sup>* is the area of the CIP. The percentages of FI and CIP are shown in Table 1.

( *freeion*%) *<sup>V</sup>*

*ne* σ

*e kT <sup>D</sup>* μ

In Table 2 the *D*, *μ* and *n* values increase as the glycerol increases. The improvement of *D* and *μ* can be interpreted according to the increase in polymer chain flexibility upon

**Glycerol %** *n* **(cm−3)** *µ* **(cm2 V−1 s)** *D* **(cm2 s−1)**  MDLG1 2.32 × 1022 4.0 × 10−8 1.04 × 10−<sup>9</sup> MDLG2 5.92 × 1022 1.09 × 10−7 2.83 × 10−<sup>9</sup> MDLG3 1.13 × 1023 1.10 × 10−7 2.86 × 10−<sup>9</sup>

The relationship between the ionic conductivity of the electrolyte films and the ionic

η*q*μ

*<sup>A</sup>* ×

the number density (*n*), ionic mobility (*μ*), and diffusion coefficient (*D*) for each electrolyte can be calculated from Equations (3)–(5). In these equations, *M* stands for the molecular weight of glycerol and *e* is the electron charge, and *NA* is the Avogadro's constant. A polymer electrolyte has a total volume of VTotal. The calculated values of *n*, *μ*, and *D* are

*M N*

*Total*

μ

**Table 2.** The *n*, *D*, and *μ* at ambient temperature from FTIR approach.

mobility is well-acknowledged and mathematically stated as follows:

σ<sup>=</sup>

shown in Table 2.

the addition of the glycerol [1].



The rise in ionic conductivity might also be attributed to the rise in Li<sup>+</sup> ions that dissociate from LiClO<sup>4</sup> salts. There is a strong correlation between the conductivity and the proportion of free ions, according to Aniskari and colleagues [43]. The calculation of the number density (*n*), ionic mobility (*µ*), and diffusion coefficient (*D*) for each electrolyte can be calculated from Equations (3)–(5). In these equations, *M* stands for the molecular weight of glycerol and *e* is the electron charge, and *N<sup>A</sup>* is the Avogadro's constant. A polymer electrolyte has a total volume of *VTotal*. The calculated values of *n*, *µ*, and *D* are shown in Table 2.

$$m = \frac{M \times N\_A}{V\_{Total}} \times (frecion\%)\tag{3}$$

$$
\mu = \frac{\sigma}{ne} \tag{4}
$$

$$D = \frac{\mu kT}{e} \tag{5}$$



In Table 2 the *D*, *µ* and *n* values increase as the glycerol increases. The improvement of *D* and *µ* can be interpreted according to the increase in polymer chain flexibility upon the addition of the glycerol [1].

The relationship between the ionic conductivity of the electrolyte films and the ionic mobility is well-acknowledged and mathematically stated as follows:

$$
\sigma = \sum \eta q \mu \tag{6}
$$

where *σ* denotes the ionic conductivity, *η* represents the charge carrier density, and *q* stands for the single charge. From the equation above, it can be observed that the ionic conductivity improves with the increment of the ionic mobility as well as charge carrier density.

#### *2.2. Impedance Study*

Figure 3 shows the Cole-Cole plots used to estimate the impedance parameters of the electrolytes used in this study. An appropriate equivalent circuit model, with series connections for the resistor and the capacitor given by bulk resistance (*R<sup>b</sup>* ) and the constant phase element (*CPE*), is shown in the inset figure for each Cole-Cole plot. Ions flow via a resistor, whereas polymer chains remain immobile in a capacitor [44]. Because of the charge buildup and capacitive elements in the electrolytes, there is a spike in the plots created by the diffusion process inside the system [17,45]. This graph also shows how polarization and blocking electrodes in the Pes affect the inclination [46,47].

**Figure 3.** EIS spectra of (**a**) MDLG1, (**b**) MDLG2, and (**c**) MDLG3 electrolytes**. Figure 3.** EIS spectra of (**a**) MDLG1, (**b**) MDLG2, and (**c**) MDLG3 electrolytes.

The impedance of *CPE* (*ZCPE*) is written as follows [48,49]:

$$Z\_{\rm CPE} = \frac{1}{\mathcal{C}\omega^p} \left[ \cos\left(\frac{\pi p}{2}\right) - i \sin\left(\frac{\pi p}{2}\right) \right] \tag{7}$$

where *ω* denotes the angular frequency, *p* indicates the deviation of the plot from the axis, and *C* refers to the capacitance of *CPE* component. The spectra that involve only a spike and *R<sup>b</sup>* are in series with *CPE*, and the real and the imaginary parts of impedance, *Z<sup>r</sup>* and *Zi* , are based on the following mathematical relationships.

$$Z\_r = R + \frac{\cos\left(\frac{\pi p\_2}{2}\right)}{\mathcal{C}\_2 \omega^{p^2}}\tag{8}$$

$$Z\_i = \frac{\sin\left(\frac{\pi p\_2}{2}\right)}{\mathcal{C}\_2 \omega^{p2}}\tag{9}$$

The determined *R<sup>b</sup>* and *CPE* for each electrolyte are listed in Table 3. The *CPE* values increase while the *R<sup>b</sup>* values fall when glycerol concentrations increased. There are more ions in a solution, resulting in a higher capacitance value, which results in the greater mobility and dissociation of ions, thereby increasing conductivity [50,51]. The ionic conductivity (*σ*) measured using Equation (10) and also shown in Table 3 demonstrates this.

$$
\sigma\_{dc} = \left(\frac{1}{R\_b}\right) \times \left(\frac{t}{A}\right) \tag{10}
$$

**Table 3.** EEC fitting parameters for each sample.


Here, *t* refers to the electrolyte's thickness; *A* denotes the SS electrodes area.

This study demonstrated that the MC:Dex:LiClO4:glycerol combination is more flexible and mobile because of the glycerol [52]. The conductivity of 1.99 <sup>×</sup> <sup>10</sup>−<sup>3</sup> S cm−<sup>1</sup> , achieved by MDLG3, is close to that achieved by Amran et al. [27] and Shukur et al. [30], and they also used glycerol as a plasticizer in their studies. It is also comparable with our previous studies of the biodegradable-blend-polymer electrolytes incorporated with ammonium salts [53,54]. The conductivity that was achieved in this study bodes well for future applications in energy devices [31].

As the samples have only a spike, *D*, *µ*, and *n* are measured by below equations [2]: *D* is measured using Equations (11) and (12) [1]:

$$D = D\_{\rm o} \exp\left\{-0.0297 \left[\ln D\_{\rm o}\right]^2 - 1.4348 \ln D\_{\rm o} - 14.504\right\} \tag{11}$$

where the following is the case.

$$D\_0 = \left(\frac{4k^2l^2}{R\_b4\omega\_{\rm min}^3}\right) \tag{12}$$

Here, *ω*min and *l* correspond to the angular frequency that is based on the minimum *Z<sup>i</sup>* and the electrolyte thickness, respectively. *µ* is measurable from the relationship shown in Equation (13):

$$
\mu = \left(\frac{eD}{\mathcal{K}\_b T}\right) \tag{13}
$$

where *K<sup>b</sup>* and *T* are the Boltzmann constant possess normal meanings. The conductivity can be measured using Equation (6).

Thus, the number *n* is measured using Equation (14):

$$m = \left(\frac{\sigma\_{dc} K\_b T \tau\_2}{\left(e K\_2 \varepsilon\_o \varepsilon\_r A\right)^2}\right) \tag{14}$$

In Table 4, *D*, *µ*, and *n* increased when glycerol increased. This is caused by increasing the polymer chain's flexibility when the glycerol is loaded [2]. The outcome shows how the concentration of glycerol affects the values of the ion number density, the ionic mobility, and the diffusion coefficient. This increase in *D*, *µ*, and *n* values can cause an increase in conductivity [4]. It is interesting to observe that when glycerol concentration increases, the number of ions (*n*) tends to increase continuously. Glycerol enhances the dissociation of salts to free ions; thus, *n* increases correspondingly. Meanwhile, ionic mobility (*µ*) and diffusion coefficient (D) are observed to follow the same trend of ionic conductivity, as shown in Table 3. The value of the free ion, which gradually increased by adding glycerol to the system, indicates that the ionic conductivity of the present system increased by the increasing the (*n*) value. These results from EIS and the FTIR deconvolution are in agreement.

**Table 4.** The values of ion transport parameters of each film from impedance approach.


*2.3. Dielectric Properties*

According to current research, dielectric material qualities may be defined in multiple ways. There were many ways to increase the accuracy and sensitivity of material characterization [55–59]. Impedance studies at various frequencies have been shown to be a good approach for studying the molecular mobility of dielectric materials [60]. Dielectric studies may be used to examine the conductivity trend. Different amounts of glycerol at ambient temperature affect the dielectric constant (*ε* 0 ) and the dielectric loss (*ε* 00), as observed in Figures 4 and 5, respectively. *ε* 0 and *ε* 00 are measured using the equations below [61–63]:

$$\varepsilon' = \left[\frac{Z''}{\omega \mathbb{C}\_0 (Z'^2 + Z''^2)}\right] \tag{15}$$

$$\mathbf{y}'' = \left[\frac{Z'}{\omega \mathbb{C}\_o (Z'^2 + Z''^2)}\right] \tag{16}$$

where *C*<sup>o</sup> is the vacuum capacitance, which is equivalent to *ε*0*A*/*t* in which *ε*<sup>0</sup> is the vacuum permittivity; the angular frequency is denoted by *ω* (*ω* = 2πf); the frequency is denoted by f.

*ε*

The conductivity of a polymer electrolyte is determined by its dielectric constant [64]. Real dielectric permittivity (*ε* 0 ) is used to determine the polarization or dipole alignment, which is measured by capacitance. Similarly to *ε* 00, which indicates dielectric loss, conductance reflects the energy needed to align dipoles in a dielectric medium [65]. An important consideration in electrical conductivity testing is the identification of neutral ion pairs produced by the interaction of dissolved ion pairs [59]. In EIS measurements, it was shown that by adding more glycerol, the DC's conductivity significantly increased. *ε* 0 and *ε* 00 at low frequencies are higher (Figures 4 and 5), which show variations for the films. Charge carriers or space charge polarization build up at the electrode/electrolyte contact point, causes this phenomenon [60]. Increasing the frequency reduces the dielectric property (bulk property). As a result, *ε* 0 and *ε* 00 increase as a result of a decrease in the frequency of

the applied electric field [66]. As a result of the quick reversal of the electric field frequency, there is no new ion diffusion that takes place along its route, and polarization is reduced. Eventually, the peak shrinks to the point where it is no longer frequency dependent [48]. In a comparison to other samples, the system containing 42 wt.% of glycerol had a greater dielectric constant. Dielectric loss (*ε* 00) and constant (*ε* 0 ) are strongly impacted by the conductivity in the system [51,67]. *Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 10 of 17 *Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 10 of 17

**Figure 4.** *ε*′ spectra versus frequency for MC:Dex:LiClO4:Glycerol electrolytes. **Figure 4.** *ε* 0 spectra versus frequency for MC:Dex:LiClO<sup>4</sup> :Glycerol electrolytes. **Figure 4.** *ε*′ spectra versus frequency for MC:Dex:LiClO4:Glycerol electrolytes.

**Figure 5.** *ε*″ spectra versus frequency for MC:Dex:LiClO4:Glycerol electrolytes. **Figure 5.** *ε*″ spectra versus frequency for MC:Dex:LiClO4:Glycerol electrolytes. **Figure 5.** *ε* 00 spectra versus frequency for MC:Dex:LiClO<sup>4</sup> :Glycerol electrolytes.

*2.4. TNM Study*  In order to ensure the purely ionic nature of the PE system, the ion transport number *2.4. TNM Study*  In order to ensure the purely ionic nature of the PE system, the ion transport number It has previously been observed that the dielectric constant (*ε* 0 ) and the density of the charge carriers (*n<sup>i</sup>* ) were formulated by the following relationship:

(*tion*) has been measured for the optimized MC:Dex: LiClO4:Glycerol composition using the DC polarization technique [69]. The curve obtained for the SS|Polymer electrolyte|SS cell (SS: stainless-steel) is shown in Figure 6. An initial current (*Ii*) of 128 A and the total of

(*tion*) has been measured for the optimized MC:Dex: LiClO4:Glycerol composition using the DC polarization technique [69]. The curve obtained for the SS|Polymer electrolyte|SS cell (SS: stainless-steel) is shown in Figure 6. An initial current (*Ii*) of 128 A and the total of

$$n\_i = n\_o \exp\left(-\mathcal{U}/\varepsilon^\prime \mathcal{K}\_b T\right) \tag{17}$$

where *U* is the dissociation energy.

DC's conductivity, as well as dielectric constant values, can be manipulated successfully [68]. The dielectric constants of polymer electrolytes may be used to determine the conductivity of certain materials and, hence, their electrical properties. A drop in dielectric constant is accompanied with a decrease in capacitance (*ε* 0 = *C*/*C*o). The plots show that the *ε* 00 value is higher than the *ε* 0 , as shown in Figures 4 and 5. DC conduction processes and dielectric polarization processes both have an impact on dielectric loss [51]. *Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 11 of 17 ionic and electronic currents were delivered by the cell. Since the SS electrode is ionblocking in nature, the current declines quickly and is saturated at the residual electronic

#### *2.4. TNM Study* current (*I*e) of 3 µA. The electrolyte system's ionic composition is thought to be responsible

In order to ensure the purely ionic nature of the PE system, the ion transport number (*tion*) has been measured for the optimized MC:Dex: LiClO4:Glycerol composition using the DC polarization technique [69]. The curve obtained for the SS|Polymer electrolyte|SS cell (SS: stainless-steel) is shown in Figure 6. An initial current (*I<sup>i</sup>* ) of 128 A and the total of ionic and electronic currents were delivered by the cell. Since the SS electrode is ion-blocking in nature, the current declines quickly and is saturated at the residual electronic current (*I*e) of 3 µA. The electrolyte system's ionic composition is thought to be responsible for the abrupt reduction in current levels. The *tion* and *tel* values of the electrolyte film, obtained using the Equations (18) and (19), are found to be 0.976 and 0.024, respectively. These results show how ionic the electrolyte system is and how it will protect the electrodes of the energy storage device from each other since it is close to 1, which means it is close to the ideal value of unity. Ions are the key charge carriers in this system of methylcellulose-dextran-LiClO4:Glycerol [70–72]. The result obtained in this study is observed to be high compared to our previous study for methylcellulose-based polymer electrolytes impregnated with potassium iodide [73]. for the abrupt reduction in current levels. The *tion* and *tel* values of the electrolyte film, obtained using the Equations (18) and (19), are found to be 0.976 and 0.024, respectively. These results show how ionic the electrolyte system is and how it will protect the electrodes of the energy storage device from each other since it is close to 1, which means it is close to the ideal value of unity. Ions are the key charge carriers in this system of methylcellulose-dextran-LiClO4:Glycerol [70–72]. The result obtained in this study is observed to be high compared to our previous study for methylcellulose-based polymer electrolytes impregnated with potassium iodide [73]. Equations (17) and (18) are used to measure *tion* and *tel*. *i i ss ion I I I <sup>t</sup>* <sup>−</sup> <sup>=</sup> (18) *el ion t* = 1− *t* (19) In Equations (18) and (19), the starting and the steady-state current are expressed as *Ii* and *Iss*, respectively.

**Figure 6.** Chronoamperometric profile of for the MDLG3 electrolyte. **Figure 6.** Chronoamperometric profile of for the MDLG3 electrolyte.

The electrochemical stability window (ESW) is an important parameter for an electrolyte, which determines the working voltage range of the energy storage device. The Equations (17) and (18) are used to measure *tion* and *tel*.

$$t\_{\rm ion} = \frac{I\_{\rm i} - I\_{\rm ss}}{I\_{\rm i}} \tag{18}$$

~3 V. The present values increase sharply after the aforementioned potential. A

$$t\_{el} = 1 - t\_{ion} \tag{19}$$

In Equations (18) and (19), the starting and the steady-state current are expressed as *I<sup>i</sup>* and *Iss*, respectively.

The electrochemical stability window (ESW) is an important parameter for an electrolyte, which determines the working voltage range of the energy storage device. The ESW of the optimized MC:Dextran:40 wt.% LiClO4:48 wt.% Glycerol composition is obtained using linear sweep voltammetry (LSV). The LSV curve, shown in Figure 7, displays a plateau of negligible current without any anodic/cathodic current peak up to ~3 V. The present values increase sharply after the aforementioned potential. A considerable ESW of 3 V is shown, making the electrolyte film acceptable for supercapacitor use. In order for the film to be used in energy storage devices, the stability of the plasticized methylcellulosedextran-LiClO<sup>4</sup> system has been shown to be up to 3 V. The interesting observation in this study is the eligibility of the MDLG3 electrolyte for energy storage device utilization. This is caused by the satisfactory voltage breakdown of the sample at almost 1.0 V [74,75]. The decomposition voltage attained in this study is relatively high compared to our previous studies [76,77]. This could be due to the presence of LiClO<sup>4</sup> as an ionic source, which has higher stability than ammonium salts. Moreover, a protic ionic liquid electrolyte was utilized for lithium-ion batteries as documented by Bockenfeld et al. [78]. They demonstrated that the highest potential stability was 2.65 V for their electrolyte that incorporated 0.5 M lithium nitrate (LiNO3) in propylene carbonate-pyrrolidinium nitrate. *Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 12 of 17 supercapacitor use. In order for the film to be used in energy storage devices, the stability of the plasticized methylcellulose-dextran-LiClO4 system has been shown to be up to 3 V. The interesting observation in this study is the eligibility of the MDLG3 electrolyte for energy storage device utilization. This is caused by the satisfactory voltage breakdown of the sample at almost 1.0 V [74,75]. The decomposition voltage attained in this study is relatively high compared to our previous studies [76,77]. This could be due to the presence of LiClO4 as an ionic source, which has higher stability than ammonium salts. Moreover, a protic ionic liquid electrolyte was utilized for lithium-ion batteries as documented by Bockenfeld et al. [78]. They demonstrated that the highest potential stability was 2.65 V for their electrolyte that incorporated 0.5 M lithium nitrate (LiNO3) in propylene carbonate-pyrrolidinium nitrate.

**Figure 7.** LSV for the MDLG3 film of SPE. **Figure 7.** LSV for the MDLG3 film of SPE.

*3.1. Materials* 

#### **3. Materials and Methods 3. Materials and Methods**

#### *3.1. Materials*

MC polymer (*M*w avg = 10,000–220,000), LiClO4 (*M*w = 106.39 g/mol) and glycerol (*M*<sup>w</sup> = 92.09382 g/mol) were purchased from Sigma-Aldrich (Kuala Lumpur, Malaysia). MC polymer (*M*w avg = 10,000–220,000), LiClO<sup>4</sup> (*M*<sup>w</sup> = 106.39 g/mol) and glycerol (*M*<sup>w</sup> = 92.09382 g/mol) were purchased from Sigma-Aldrich (Kuala Lumpur, Malaysia).

#### *3.2. Electrolyte Preparation*

The synthesis of MC-Dex-blend polymer was performed by stirring and dissolving 40 wt.% of Dex (0.4 g) and 60 wt.% of MC (0.6 g) individually, each in a 1% solution of 30 mL acetic acid, for almost 2 h at room temperature. Then, the two solutions were stirred and blended using a magnetic stirrer for around 4 h until reaching a homogenous-blend solution. Then, with respect to the above solution, 40 wt.% (0.666 g) of LiClO4, MC-Dex-LiClO<sup>4</sup> formed. Ultimately, in the step of 14 wt.%, 14, 28, and 42 wt.% of glycerol were added to the MC-Dex-LiClO<sup>4</sup> solution followed by continuous stirring until the synthesis of plasticized SPEs was achieved. The labelling of the series of the samples was conducted as follows: MDLG1, MDLG2, and MDLG3 for the MC-Dex-LiClO<sup>4</sup> loading 14, 28, and 42 wt.% of glycerol, respectively as shown in Table 5. The casting of the series of sample solutions was carried out in the Petri dishes, followed by leaving them at room temperature to evaporate the solvent gradually. The free solvent sample films were kept in a desiccator.


**Table 5.** The identification and composition for the MC-Dex-LiClO4–glycerol systems.

#### *3.3. Methods of Characterizations*

#### 3.3.1. FTIR and EIS Measurements

The FTIR spectra of the blended polymer systems were acquired using FTIR Spectrophotometer (Malvern Panalytical Ltd., Malvern, UK), ranging from 4000 to 400 cm−<sup>1</sup> with a resolution of 2 cm−<sup>1</sup> . The EIS samples spectra were acquired using the EIS (3532-50 LCR HiTESTER (HIOKI), Nagano, Japan) within 50 Hz and 5,000,000 Hz of frequency. The circle film had a geometric circle shape (diameter of 2 cm), which was sandwiched between stainless steel (SS) electrodes using a spring force during electrochemical measurements. The cell was hyphenated with a computer to measure real and imaginary (Z0 and Z00) parts of the complex impedance spectra (Z\*).

#### 3.3.2. TNM and LSV

The ion (*tion*) and electron (*tel*) transference numbers were measured precisely. The cell (SS|MDLG3|SS) was connected to the UNI-T UT803 multimeter and A&V Instrument DP3003 digital DC power supply. By applying a voltage of 0.2 V to the cell, the polarization of the cell was obtained over a sufficient amount of time at room temperature. To obtain the potential stability of the MDLG3, LSV was used by applying 10 mV s−<sup>1</sup> within 0.0 and 4.0 V. The cell was the three-electrode type, and the working, counter, and reference electrodes were used by utilizing the Digi-IVY DY2300 potentiostat. The current changes over the mentioned potential were obtained.

#### **4. Conclusions**

In this study, SPEs based on MC:Dex:LiClO<sup>4</sup> plasticized with glycerol were synthesized by the solution-cast method. The conductivity increased to 1.45 <sup>×</sup> <sup>10</sup>−<sup>3</sup> S cm−<sup>1</sup> due to the doping of glycerol. The FTIR method showed that there was an interaction of LiClO<sup>4</sup> and glycerol with the MC and Dex by changing FTIR absorption peaks. The FTIR deconvolution of CLO<sup>4</sup> − anions showed that the free ion percentages increased when glycerol increased, while the percentages of contact ion pairs decreased. Further proof of *DC* conductivity trends was emphasized from the dielectric measurement. The addition of glycerol was effective in increasing the number density (*n*), diffusion coefficient (*D*), and mobility (*µ*). Additionally, the mass transport improvements of the electrolytes originate from the increase in chain flexibility. The values of measured *tion* and *tel* indicate the ion's responsibility for conduction in the polymer-electrolyte system. The stability voltage range of the electrolyte system is satisfactory, meaning that the SPE is eligible for utilization at large scales in electrochemical energy storage devices.

**Author Contributions:** Conceptualization, S.B.A. and S.I.A.-S.; formal analysis, S.B.A. and M.A.B.; funding acquisition, E.M.A.D., S.I.A.-S. and M.M.N.; investigation, M.A.B.; methodology, S.B.A. and M.A.B.; project administration, E.M.A.D., S.B.A., S.I.A.-S., M.M.N., K.M. and R.M.A.; resources, E.M.A.D.; supervision, S.B.A.; validation, K.M., R.M.A., W.O.K. and J.M.H.; writing—original draft, S.B.A.; writing—review and editing, E.M.A.D., S.I.A.-S., M.M.N., K.M., R.M.A., W.O.K. and J.M.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We would like to acknowledge all support for this study by the University of Sulaimani, Prince Sultan University, and Komar University of Science and Technology. The authors express their gratitude for the support of Princess Nourah bint Abdulrahman University Researchers, Supporting Project number (PNURSP2022R58), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. The authors would like to acknowledge the support of Prince Sultan University for paying the Article Processing Charges (APC) of this publication and for their financial support.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


## *Review* **Alginate as a Promising Biopolymer in Drug Delivery and Wound Healing: A Review of the State-of-the-Art**

**Mohammad A. S. Abourehab 1,2,\* , Rahul R. Rajendran <sup>3</sup> , Anshul Singh <sup>4</sup> , Sheersha Pramanik 5,\* , Prachi Shrivastav 6,7 , Mohammad Javed Ansari <sup>8</sup> , Ravi Manne <sup>9</sup> , Larissa Souza Amaral <sup>10</sup> and A. Deepak <sup>11</sup>**


**Abstract:** Biopolymeric nanoparticulate systems hold favorable carrier properties for active delivery. The enhancement in the research interest in alginate formulations in biomedical and pharmaceutical research, owing to its biodegradable, biocompatible, and bioadhesive characteristics, reiterates its future use as an efficient drug delivery matrix. Alginates, obtained from natural sources, are the colloidal polysaccharide group, which are water-soluble, non-toxic, and non-irritant. These are linear copolymeric blocks of α-(1→4)-linked l-guluronic acid (G) and β-(1→4)-linked d-mannuronic acid (M) residues. Owing to the monosaccharide sequencing and the enzymatically governed reactions, alginates are well-known as an essential bio-polymer group for multifarious biomedical implementations. Additionally, alginate's bio-adhesive property makes it significant in the pharmaceutical industry. Alginate has shown immense potential in wound healing and drug delivery applications to date because its gel-forming ability maintains the structural resemblance to the extracellular matrices in tissues and can be altered to perform numerous crucial functions. The initial section of this review will deliver a perception of the extraction source and alginate's remarkable properties. Furthermore, we have aspired to discuss the current literature on alginate utilization as a biopolymeric carrier for drug delivery through numerous administration routes. Finally, the latest investigations on alginate composite utilization in wound healing are addressed.

**Keywords:** alginate; drug delivery system; formulations; administration route; controlled release; wound healing; extraction methods

**Citation:** Abourehab, M.A.S.; Rajendran, R.R.; Singh, A.; Pramanik, S.; Shrivastav, P.; Ansari, M.J.; Manne, R.; Amaral, L.S.; Deepak, A. Alginate as a Promising Biopolymer in Drug Delivery and Wound Healing: A Review of the State-of-the-Art. *Int. J. Mol. Sci.* **2022**, *23*, 9035. https:// doi.org/10.3390/ijms23169035

Academic Editors: Valentina Siracusa and Swarup Roy

Received: 20 July 2022 Accepted: 9 August 2022 Published: 12 August 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction 1. Introduction**

In the past, there had been a hurdle in the investigation to reveal naturally derived polymers with exceptional physicochemical characteristics and a high magnitude of compatibility for applications in drug delivery. Escorted by the advancement in pharmaceutical discovery methods in recent years, several therapeutically active substances have come to notice. Nevertheless, the curative agent's delivery to the intentional site has been a severe hurdle in addressing many ailments. These novel pharmaceuticals need exquisite drug delivery systems (DDSs) that could be employed to improve their pharmacokinetics and pharmacodynamics characteristics, thereby advancing cell/tissue specificity along with their biocompatible properties. Therefore, the blooming of an effective DDS that can transport and administer an active accurately and safely to the desired site of action has to become the "bourne" of scientists. In the past, there had been a hurdle in the investigation to reveal naturally derived polymers with exceptional physicochemical characteristics and a high magnitude of com‐ patibility for applications in drug delivery. Escorted by the advancement in pharmaceuti‐ cal discovery methods in recent years, several therapeutically active substances have come to notice. Nevertheless, the curative agent's delivery to the intentional site has been a se‐ vere hurdle in addressing many ailments. These novel pharmaceuticals need exquisite drug delivery systems (DDSs) that could be employed to improve their pharmacokinetics and pharmacodynamics characteristics, thereby advancing cell/tissue specificity along with their biocompatible properties. Therefore, the blooming of an effective DDS that can transport and administer an active accurately and safely to the desired site of action has to become the "bourne" of scientists.

*Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 2 of 60

A drug delivery system (DDS) refers to a system that carries curative substances inside the body to accomplish a required remedial outcome. Two principal classes of DDSs are identified, namely, conventional drug delivery systems and novel drug delivery systems (NDDSs) [1]. The actives are supplied by the conventional method via different routes such as oral, buccal/sublingual, rectal, intravenous, subcutaneous, and intramuscular. The conc. of therapeutic actives is not persistent during the therapy and demands continual dosage management in the conventional route [2]. Hence, this describes the prompt enhancement in the level of the drugs in the blood beyond the toxicity limit after individual administration and later declines to a sub-therapeutic level until the following administration [3]. The enhancement in actives conc. beyond the toxicity limit leads to perniciousness in the body. Moreover, the increase of repeated administration might sum up to the remedial non-compliance upon the sufferer [4]. A drug delivery system (DDS) refers to a system that carries curative substances in‐ side the body to accomplish a required remedial outcome. Two principal classes of DDSs are identified, namely, conventional drug delivery systems and novel drug delivery sys‐ tems (NDDSs) [1]. The actives are supplied by the conventional method via different routes such as oral, buccal/sublingual, rectal, intravenous, subcutaneous, and intramus‐ cular. The conc. of therapeutic actives is not persistent during the therapy and demands continual dosage management in the conventional route [2]. Hence, this describes the prompt enhancement in the level of the drugs in the blood beyond the toxicity limit after individual administration and later declines to a sub‐therapeutic level until the following administration [3]. The enhancement in actives conc. beyond the toxicity limit leads to perniciousness in the body. Moreover, the increase of repeated administration might sum up to the remedial non‐compliance upon the sufferer [4].

To surmount the aforementioned limitations of the conventional approach, the progressive approach, NDDS, was prepared and included dosage forms. Consequently, the drug rate is sustained within the therapeutic-efficient level with controlled release of actives in both speed and period. Moreover, the NDDS transports actives to the particular action site with optimal dose and diminished toxic effect in contrast to the traditional drug delivery systems [4,5]. The convenient features of the NDDS (as pictured in Figure 1) encompass actives' controlled release, the capability to utilize different administrative ways, improved active guard and efficiency, the improved substrate solubility showing low solubility, and a novel business market prospective to retrieve pharmaceuticals that have been unsuccessful throughout the traditional drug delivery approaches [1,6,7]. To surmount the aforementioned limitations of the conventional approach, the pro‐ gressive approach, NDDS, was prepared and included dosage forms. Consequently, the drug rate is sustained within the therapeutic‐efficient level with controlled release of ac‐ tives in both speed and period. Moreover, the NDDS transports actives to the particular action site with optimal dose and diminished toxic effect in contrast to the traditional drug delivery systems [4,5]. The convenient features of the NDDS (as pictured in Figure 1) en‐ compass actives' controlled release, the capability to utilize different administrative ways, improved active guard and efficiency, the improved substrate solubility showing low sol‐ ubility, and a novel business market prospective to retrieve pharmaceuticals that have been unsuccessful throughout the traditional drug delivery approaches [1,6,7].

**Figure 1. Figure 1.** The ideal characteristics of nano delivery systems. The ideal characteristics of nano delivery systems.

Out of diverse mechanisms of delivery, "controlled drug delivery" and "targeted drug delivery" have been sighted as some of the utmost challenging and fast-progressing investigational areas in the past four years. It provides myriad benefits in contrast to conventional systems, e.g., it improves the absorption rate and biocompatible properties, enhances the actives protection against proteolytic enzyme degradation, cell and tissuespecific active targeting, and helps to regulate active levels within the body, inside the therapeutic level range, over a more prolonged time [8,9]. However, despite the advantages of controlled releases that were formerly persuasive, potential shortcomings, for example, toxicity inside the body, complicated synthetic pathways and the resulting degradation byproducts, and operative methods required to explant systems that are non-biodegradable, persist as severe impediments [10,11].

In NDDSs, the active carrier is a base that permits actives to be carried to the intended location, delivering the actives in a controlled manner, thereby enhancing the active bioavailability [12]. Nanoparticles, liposomes, microspheres, polymeric micelles, etc., are some of the significant actives carriers utilized in NDDSs [13–18].

Nonetheless, due to their suitable, variable characteristics, polymeric biomaterials are the most alluring opportunity for delivering drugs in a controlled and directed manner. They can be produced on an industrial scale and readily customized to meet the required applications [19]. But the polymer selection utilized for the drug carrier preparation performs a critical function in the process of actives delivery. The two kinds of polymers that are obtainable in the market are natural and synthetic polymers. Natural polymers (for example, chitosan, alginate, and bacterial cellulose), as well as many synthetic polymers such as poly(lactic-co-glycolic acid) (PLGA), poly-L-lysine (PLL), polycaprolactone (PCL), etc., are used as carriers for drug delivery. These polymers have less toxicity, are biocompatible, and are biodegradable, by which they are degraded via the action of enzymes [20,21]. Therefore, natural polymers, such as polysaccharides, polypeptides, or phospholipids, are generally used to prepare a cornerstone. From all of these, alginate (ALG), an anionic polysaccharide, enticed a growing appreciation for actives delivery with its extraordinary physical and biological characteristics. Among different ALGs, sodium alginate (SA) remains one of the most researched in the pharmaceutical area for applications in drug delivery.

#### **2. The Purview of the Present Review**

This review article comprises state of the art ALG-based preparation in the field of actives delivery. The first segment highlights the exceptional ALG characteristics, trailed by its most recent application in carrying therapeutically active substances. The review acknowledges the research field concentrating on pretty substantial advancement in the past period in medicinal delivery employing ALG and its derivatives as a carrier through various administration routes. Lastly, the review addresses the latest trends in the utilization of ALG composites in wound healing applications.

#### **3. Sources of Extraction and Properties of Alginate**

ALG, a naturally abundant linear and anionic polysaccharide, is generally obtained from the cell wall of brown seaweed belonging to the class Phaeophyceae [22], including *Ascophyllum nodosum*, *Laminaria hyperborea*, *Laminaria digitata*, *Laminaria japonica*, and *Microcystis pyrifera* [23], and many bacterial strains, including *Acetobacter* and *Pseudomonas* spp. Although it can be created from bacterial origins, it is commercially accessible from algae as SA in its salt form [24]. They are a class of linearly arranged biopolymers comprising 1,4-linked-β-D-mannuronic acid (M-blocks) and 1,4-α-L-guluronic acid (G-blocks) residues ordered in sequences of identical (MM, GG) or heterogeneous (MG) blocks (as portrayed in Figure 2) [25]. Divalent cations, for example, Ba2+ and Ca2+, can rapidly construct egg-box systems with G block to build ALG hydrogels via the procedure of gelation [26]. Increasing the molecular weight and G-block length dramatically increases the mechanical properties of ALG. Commercially available ALG has an average molecular

weight varying between 32,000 and 400,000 g/mol. The ALG solutions have a maximum viscosity at pH 3.0–3.5 because of the hydrogen bonding of the carboxylate groups forming the ALG backbone [23]. weight varying between 32,000 and 400,000 g/mol. The ALG solutions have a maximum viscosity at pH 3.0–3.5 because of the hydrogen bonding of the carboxylate groups form‐ ing the ALG backbone [23]. viscosity at pH 3.0–3.5 becauseof the hydrogen bonding of the carboxylate groups form‐ ing the ALG backbone [23].

[26]. Increasing the molecular weight and G‐block length dramatically increases the me‐ chanical properties of ALG. Commercially available ALG has an average molecular

[26]. Increasing the molecular weight and G‐block length dramatically increases the me‐ chanical properties of ALG.Commercially available ALG has an average molecular weight varying between 32,000 and 400,000 g/mol. The ALG solutions have a maximum

*Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 4 of 60

*Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 4 of 60

**Figure 2.** The monomer's conformation and blocks distribution of ALG salt [27]. **Figure 2.** The monomer's conformation and blocks distribution of ALG salt [27].

Recently, a multistage extraction process (from brown seaweeds, as illustrated in Fig‐ ure 3) is being carried out, which involves acid pretreatment of the seaweed extract, fol‐ lowed by aqueous alkali treatment (mainly sodium hydroxide) in which different salt forms of natural ALG are modified into aqueous‐soluble SA [28]. Subsequently, the fil‐ tered extract is incorporated with sodium or calcium chloride, and ALG gets precipitated. Then, dilute HCL is added, and salts of ALG get converted to alginic acid; following fur‐ ther purification and modification, a powder form of water‐soluble SA is prepared [29]. Recently, a multistage extraction process (from brown seaweeds, as illustrated in Figure 3) is being carried out, which involves acid pretreatment of the seaweed extract, followed by aqueous alkali treatment (mainly sodium hydroxide) in which different salt forms of natural ALG are modified into aqueous-soluble SA [28]. Subsequently, the filtered extract is incorporated with sodium or calcium chloride, and ALG gets precipitated. Then, dilute HCL is added, and salts of ALG get converted to alginic acid; following further purification and modification, a powder form of water-soluble SA is prepared [29]. Recently, a multistage extraction process (from brown seaweeds, as illustrated in Fig‐ ure 3) is beingcarried out, which involves acid pretreatment of the seaweed extract, fol‐ lowed by aqueous alkali treatment (mainly sodium hydroxide) in which different salt forms of natural ALG are modified into aqueous‐soluble SA [28]. Subsequently, the fil‐ tered extract is incorporated with sodium or calcium chloride, and ALG gets precipitated. Then, dilute HCL is added, and salts of ALG get converted to alginic acid; following fur‐ ther purification and modification, a powder form of water‐soluble SA is prepared [29].

**Figure 3.** The extraction technique of ALG from brown seaweeds. **Figure 3.** The extraction technique of ALG from brown seaweeds. **Figure 3.** The extraction technique of ALG from brown seaweeds.

The ALG production from bacterial biosynthesis has definite physical characteristics and chemical structures different from the extracted ALG from brown seaweed. The dif‐ ferent steps of ALG biosynthesis are (1) synthesis of the precursor substrate, (2) transfer polymerization and cytoplasmic membrane, (3) transport and alteration of the periplas‐ The ALG production from bacterial biosynthesis has definite physical characteristics and chemical structures different from the extracted ALG from brown seaweed. The dif‐ ferent steps of ALG biosynthesis are (1) synthesis of the precursor substrate, (2) transfer polymerization and cytoplasmicmembrane, (3) transport and alteration of the periplas‐ mic membrane, and (4) conveying via the outer membrane [30]. The ALG production from bacterial biosynthesis has definite physical characteristics and chemical structures different from the extracted ALG from brown seaweed. The different steps of ALG biosynthesis are (1) synthesis of the precursor substrate, (2) transfer polymerization and cytoplasmic membrane, (3) transport and alteration of the periplasmic membrane, and (4) conveying via the outer membrane [30].

mic membrane, and (4) conveying via the outer membrane [30]. Table 1 denotes the literature survey of various extraction methods of ALG. Table 1 denotes the literature survey of various extraction methods of ALG. Table 1 denotes the literature survey of various extraction methods of ALG.


**Table 1.** Various methods for the extraction of ALG.


**Table 1.** *Cont.*

ALG's biocompatibility, rheological properties [40,41], biodegradability, marginal toxicity, and chemical versatility [42] are prominent, along with its exceptional characteristics in producing stable gel in aqueous conditions and a mild environment by adding multivalent cations, making ALG beneficial for drug delivery [43,44]. Furthermore, ALGs can be readily created into various semi-solid or solid frameworks under a moderate environment due to their exceptional sol/gel transition capability. Hence, ALGs are also frequently utilized as viscosity-enhancing substances and thickening agents in the pharma industry [44].

The percentage of the three sorts of blocks—MM, GG, and MG—specifies the ALG's physical properties. In having a higher percentage of G, ALGs have higher gelling characteristics, while in having a high M content, ALGs have greater viscosity. Determining the M/G ratio is also essential for ALGs, those with a high ratio of M/G produce elastic gels, while those with small M/G ratios produce brittle gels [33,45]. The ALG-based formulation's mechanical characteristics rigorously rely on the count and conc. of G and M units. If G residues beat M, the formulation exhibits higher mechanical rigidity. Thus, by changing the content of G and M, it is possible to modify the elastic modulus [46].

ALG undergoes hydration at low pH, which leads to the development of "acid gels", which are highly viscous. The pH sensitivity of ALG can be attributed to acidic pendant groups that accept or release protons due to intermolecular binding when the pH is changed. As a result, the water molecules enter the ALG matrix and get physically entrapped within them but are still free to migrate. This ability is essential in the formation of ALG gels for cell encapsulation [47]. The ALG's capability to produce two different classes depending on the pH, i.e., acid gel at low pH and ionotropic gel at higher pH, makes it unique compared to neutral molecules [48].

ALG has excellent mucoadhesive characteristics due to the existence of free carboxyl moieties, enabling the biopolymer to attach to mucin via hydrogen bonding as well as electrostatic interaction. On the other hand, ALG solubility is largely dependent on environmental pH and, accordingly, affects their mucoadhesive property since only ionized carboxyl groups are proficient in engaging with tissues of the mucosa. Moreover, soluble ALG assists the penetration of solvent through the polymer matrix, forming a highly

viscous and cohesive gel framework for enhancing the mucoadhesive bond strength. In contrast, excessive and exorbitant ALG matrix hydration in physiological solution could diminish mucoadhesive properties due to the weakening of ALG functional groups accessible for mucosal tissue interactions [49,50].

ALGs can be tailored to fulfill the requirements of either pharmaceutical or biomedical applications. Owing to their high water uptake, sustained release, enhanced porosity, and non-immunogenicity, ALGs have found widespread applications in wound dressings [51]. ALG-based composites offer great utility in bioremediation by removing heavy metals, dyes, antibiotics, and other contaminants from wastewater [52]. Based on the types of cross-linkers and the cross-linking approaches used, materials ranging from small drug substances to macromolecular proteins can be designed as controlled drug delivery systems [53].

Biocompatibility is one more vital factor to be studied as the extraction of ALG obtained from nature is accompanied by the existence of numerous impurities capable of inciting allergic responses. In effect, an immune reaction has been stated in industrial-grade ALG; nonetheless, the multi-stage extraction method for eliminating metallic impurities and polyphenolic substances permits the acquisition of ALG of substantially high purity for use in biomedical applications [54].

ALG's antioxidant and anti-inflammatory actions have also been noticed. It has been reported that ALG oligosaccharides reduce nitric oxide, reactive oxygen species (ROS), and eicosanoids, such as prostaglandin E2 and cyclooxygenase COX-2 production [55–57]. Thus, ALG's exceptional characteristics have unlocked the doorways in its widespread actives delivery applications.

#### **4. Hydrogel Formation Methods**

In biomedicine, ALG is commonly utilized in the form of a hydrogel for wound healing, medicament delivery, and tissue regeneration applications. Hydrogels refer to highly cross-linked 3D networks comprising hydrophilic polymers. Because hydrogels are physically comparable to biomacromolecular constituents, they are frequently biocompatible and can be administered into the body by non-invasive administration. Hydrogels are commonly formed from hydrophilic polymers by chemical and/or physical cross-linking; their physicochemical characteristics depend on the type of cross-linking, the density of the cross-linker, and the polymers' chemical composition and molecular weight [58]. We present a review of different methods for cross-linking ALG sequences to generate gels and the effects of these methods on the hydrogel features crucial in the biomedical field.

#### *4.1. Ionic Cross-Linking*

Combining an aqueous ALG mixture with ionically cross-linking reagents, particularly divalent cations (i.e., Ca2+), is the most frequent approach to preparing hydrogels. It is thought that divalent cations attach distinctly to the guluronate block polymers of the ALG chains because the guluronate blocks' structure permits a good extent of divalent ion coordination. Acknowledged as the egg-box fashion of cross-linking, the guluronate units of one ALG chain form bonds with the guluronate blocks of neighboring polymer chains, culminating in a gel structure [59]. Calcium chloride (CaCl2) is a commonly utilized ionic cross-linking agent for ALG. Given the high solubility in aqueous solutions, it often drives fast and poorly regulated gelation. One option is to use a phosphate-rich buffer (e.g., sodium hexametaphosphate) to restrict and control gel formation. These phosphate molecules throughout the buffer compete with the ALG's carboxylate groups to interact with calcium cations, thus slowing the gelation process [60].

When utilizing divalent cations, the rate of gelation plays an important role in affecting gel strength and consistency; a higher gelation rate results in more homogenous structures and enhanced mechanical character [61]. The temperature at which gelation occurs impacts the gelation speed and the gels' material performance; the typical reactivity of ionic cross-linkers (e.g., Ca2+) is lowered at lower temperatures and consequently hampers the cross-linking. The cross-linked framework that results has more order, which ameliorates mechanical characteristics [46]. Furthermore, determined by the ALGs' chemical composition, the mechanical properties of ionically cross-linked ALG hydrogels might vary dramatically. For instance, ALG hydrogels made from a large concentration of G blocks are stiffer than those made from ALG with a lower number of G blocks [62].

Ionotropically cross-linked ALG hydrogels incorporating S-nitroso-mercaptosuccinic acid (S-nitroso-MSA), a NO donor, and AgNPs produced from green tea were prepared by Urzedo et al. using glycerol as a plasticizer, which enhanced the flexibility of the hydrogel. Rheological analysis revealed that G0 (elastic modulus) > G00 (viscous modulus) at all temperatures, indicating the prevalence of a solid-like gel structure with thermal stability. Relative to control ALG hydrogel, ALG hydrogels with AgNPs had a modest drop in elastic modulus (G0 ), leading to a lowering of the elastic response of the resulting nanocomposite gels, manifesting that the addition of AgNPs reinforces the hydrogel network while also preventing the creation of cross-linked junctions between ALG chains. As a consequence, the degree of stress required to break the elastic framework may be reduced. Because the ALG hydrogels were physically in the form of gels, the inclusion of MSA and AgNPs had no substantial effect on their rheological properties [63].

Bruchet and Melman demonstrated a reductive cation exchange mechanism for the synthesis of calcium-cross linked hydrogels from iron (III) ALG hydrogels while still maintaining the shape of the initial hydrogel. Hydrogels prepared by the traditional ionic cross-linking method are heterogeneous and difficult to control. They previously reported the selective oxidation of iron (II) cations, which dissolve homogeneously in SA, to iron (III) cations, triggering the formation of hydrogels; the obtained hydrogel films dissolve on electrochemical/photochemical reduction. Patterned films or coatings can be subsequently prepared from the as-prepared hydrogels [64].

Gattás-Asfura et al. studied various 1-methyl-2-diphenylphosphino-terephthalate (MDT) end groups, polar charged groups, polymer size, and material constitution to alter the physical characteristics of the cross-linkers and increase the capability for covalent stabilization of Alg-N3 beads by Staudinger ligation. The breakdown of cross-linkers after bead production was found to be reduced by branched poly(ethylene glycol) (PEG) polymers, albeit this effect was minimized as polymer miscibility was increased. Due to the elevated miscibility and absence of pre-incubation time for the synthesis of covalently stabilized beads, the results suggested that an ALG-based cross-linker gave the most stable and homogenous beads [65].

In another investigation, Awasthi et al. aimed to make a prolonged-release repaglinide encapsulated double cross-linked ALG-pectin bead matrix utilizing Ca2+ ions and the bifunctional alkylating agent epichlorohydrin. The dual cross-linked beads showed higher surface smoothness than single-cross-linked ALG beads owing to the higher cross-linking degree. On the other hand, ALG-pectin beads demonstrated a decrease in mucoadhesive strength due to the inability of solvent penetration owing to the rigid matrix [66].

#### *4.2. Covalent Cross-Linking*

Covalent cross-linking is being studied extensively to enhance the stability of ALG hydrogels for a variety of biomedical applications, with its carboxylate group being the main site of covalent bonding interactions. The cross-links disintegrate and reorganize in another location, and water is removed from the hydrogel, and the stress applied to it relaxes, resulting in plastic deformation. Although water migration can cause stress relaxation in covalently cross-linked gels, the incapability of dissolving and re-establishing bond formation causes considerable elastic deformity. Covalent cross-linking reagents, on the other hand, could be hazardous, and unreacted compounds may require elimination entirely from gels.

The covalent cross-linking of ALG with variable molecular weight poly(ethylene glycol)-diamines was initially studied with the purpose of generating gels with a diverse extent of mechanical behavior. As the elastic modulus grew steadily with the increase in the crosslinking density or weight fraction of PEG in the gel, it then declined as the molecular weight between cross-links (Mc) became lower than that of the softer PEG [67]. By exploring the use of various types of cross-linking agents and regulating the cross-linking densities, it was later proved that ALG hydrogels' mechanical characteristics and swelling might be finely controlled. As one may imagine, the chemical constitution of the cross-linking chains has a considerable impact on the swelling ability of the hydrogel. The addition of hydrophilic cross-linking compounds (e.g., PEG) as a supporting macromolecule can rationalize the hydrogel's loss of hydrophilicity due to the cross-linking reaction [68]. Gao et al. employed dual cross-linked methacrylated ALG hydrogel to overcome ionic crosslinked hydrogels' tendency to be prone to aqueous decomposition. The covalent cross-links between the methacrylate groups prevented the fracture of dual cross-linked chains of the hydrogels prepared under UV irradiation [69].

Basu et al. designed a nanocomposite DNA-based hydrogel via the establishment of reversible imine bonds that were crosslinked with oxidized alginate (OA). Storage moduli, yield stress, yield strain, and swift restoration following the withdrawal of cyclic stress were all highest in formulations containing OA with a higher oxidation degree. Owing to the reversibility of the covalent imine linkages generated between the aldehyde groups of OA and the amine groups found in the DNA molecules, the hydrogel preparations displayed self-recovering and shear-thinning capabilities. The improved hydrogel was effective in enhancing simvastatin's prolonged release for more than seven days [70].

In another investigation, Xing et al. reported covalently cross-linked carbohydratebased ALG/chitosan (CS) hydrogel inserted ALG microspheres encapsulating bovine serum albumin (BSA). The gelation occurred due to the Schiff-base reaction between the amino and aldehyde groups of N-succinyl CS (N-Chi) and OA. Higher content of OAlg in the hydrogels resulted in the creation of stable hydrogels, lowered swelling degree, and higher compression strength [71].

#### *4.3. Photo Crosslinking*

Photocross-linking is a novel method of in situ gelation that takes advantage of covalent cross-linking. With suitable chemical precursors, photocrosslinking can be done in mild reaction circumstances, even immediately contacting drugs and cells. ALG hydrogels are transparent and flexible when treated with methacrylate and cross-linked with a laser (argon-ion laser, 514 nm) for 30 s with eosin and triethanolamine treatment [72]. Rouillard et al. employed photocross-linking of methacrylate-modified ALG by the photoinitiator VA-086 in order to get high cell viability scaffolds (>85%) [73]. Bonino et al. monitored the reaction kinetics of methacrylate-modified SA hydrogels cross-linked by UV radiation in the presence of a photoinitiator by in-situ dynamic rheology [74].

In a similar study, Jeon et al. created a bioadhesive with adjustable material characteristics, adhesiveness, and biodegradation rate using a dual crosslinked oxidized methacrylated ALG/8-arm PEG amine (OMA/PEG) hydrogel framework. ALG had been chemically modified by reacting aldehydic groups via oxidation with PEG amino groups, and a proportion of the ALG carboxylate groups was further transformed with 2aminoethyl methacrylate (AEMA) using carbodiimide chemistry to enable photocross-linking of the methacrylate by UV light. When cultivated in the presence of human bone marrow-derived mesenchymal stem cells, the functionalized OMA/PEG hydrogels exhibited cytocompatibility. Furthermore, the adhesiveness of the hydrogels proved to be better than that of commercially available fibrin glue, which can be controlled by altering the oxidation level of ALG and assessed on a pig skin model [75].

#### *4.4. Click Chemistry Reactions*

Most of the strategies for preparing covalently cross-linked ALG hydrogels employ hazardous chemicals and catalysts that impact the biocompatibility of the hydrogels [76]. Hence, in recent times, copper-free "click" chemical procedures are effectively used to make hydrogels based on biopolymers without any need for catalysts or activators, with few side reactions, and are suitable for physiological environments even under mild conditions [77]. The most typical example is 1,3-dipolar cycloadditions, a copper (I)-catalyzed reaction of azides with alkynes, which has recently been used to generate biodegradable peptidemodified ALG hydrogels exhibiting usefulness as artificial analogous to extracellular matrix in tissue regeneration [78].

Anugrah et al. prepared novel near-infrared light-sensitive ALG hydrogels via click cross-linking by inverse electron demand Diels–Alder reaction between norbornenemodified ALGs and tetrazine cross-linkers consisting of diselenide bonds. Indocyanine green (ICG) produced reactive oxygen species upon NIR light irradiation that dissolved diselenide linkages in the hydrogel network, causing the gel-sol transition followed by the release of encapsulated DOX [79]. García-Astrain et al. synthesized cross-linked ALG hydrogels by means of Diels–Alder (DA) click chemistry. Furan groups were added to ALG by an amidation reaction using furfuryl amine. The ALG-containing furan was subsequently cross-linked utilising a DA reaction and the polymer poly(propylene oxide) b-poly(ethylene oxide)-b-poly(propylene oxide). As a bifunctional crosslinking agent, bismaleimide was used. The hydrogels as-prepared displayed rapid pH responsiveness and pulsatory activity between acidic and alkaline conditions, both of which were important characteristics for drug delivery of the model drug vanillin [80]. Coupling DA click chemistry with the thiol–ene reaction, antibacterial SA hydrogels SA/PEG–HHC10 were developed and manufactured by Wang et al. The cysteinyl-terminated antibacterial polypeptide HHC10–CYS (HHC10) was seeded employing the thiol–ene mechanism between the oxy-norbornene group and the thiol group after the hydrogels were made through DA click chemistry with high mechanical properties. The antibiotic hydrogels had a significant antibacterial effect (sterilization rate after 24 h was almost 100%) and excellent biocompatibility [81]. Lückgen et al. rendered the norbornene-tetrazine click cross-linked ALG hydrogels hydrolytically-degradable by oxidation of the backbone with sodium periodate in order to regulate the rheological, physicomechanical, and degradation properties. The produced hydrogels were suitable for cell seeding in 2D and encapsulating in 3D, as evidenced by cell number constancy and excellent viability preservation [82]. In a different study, Pérez-Madrigal et al. fabricated strong ALG/hyaluronic acid (HA) thio-yne clickhydrogel tissue engineering scaffolds which exhibited remarkable mechanical properties (the maximum compressive strength was 1.4 ± 0.55 MPa, with the strain at the break being around 97%; after 7 days, the hydrogels swelled to 198 ± 5.3%) and cytocompatibility [83].

#### *4.5. Thermal Gelling*

As a result of their customizable temperature-responsive swelling capabilities, thermosensitive hydrogels have recently been intensively explored in various pharmaceutical applications. This allows for pro re nata control of drug delivery from the gels [84]. The most widely used thermo-sensitive gels are poly(N-isopropyl acrylamide) (PNIPAAm) hydrogels, which undergo a reversible phase change in aqueous solutions at body temperature (low critical solution temperature near 32 ◦C). Notwithstanding the significance of thermo-responsive hydrogels in biomedical pertinence, several ALG-based systems have been documented thus far because ALG is not intrinsically thermo-sensitive. In situ copolymerization of N-isopropyl acrylamide (NIPAAm) with poly(ethylene glycol)-copoly(-caprolactone) (PEG-co-PCL) macromer by the addition of SA was used to create semi-interpenetrating polymer network (semi-IPN) framework. At a fixed temperature, the swelling capacity of the gels rose with the amount of SA and reduced with a temperature rise. The introduction of SA in semi-IPN structures enhanced the mechanical properties and BSA release from the hydrogels, suggesting that it could be advantageous in drug delivery [85]. Bezerra et al. prepared furosemide-loaded sericin/ALG beads via ionic gelation and then subjected them to thermal or covalent cross-linking using proanthocyanidin as the cross-linker to achieve gastro-resistant sustained release diuretic particles [86].

#### *4.6. Cell Cross-Linking*

Whereas a variety of chemical as well as physical ways of forming ALG gels have been documented, the potential of cells to promote gel production has largely been overlooked. Despite the lack of chemical cross-linkers, the ability of cells to attach multiple polymer chains can result in longer, reversible network development when ALG is altered with cell adhesion ligands. Cells introduced into an arginine–glycine–aspartic acid (Arg–Gly– Asp, RGD) functionalized ALG solution produce a uniform dispersion, and this system then forms the cross-linked networks without the use of any extra cross-linking chemicals through specialized receptor–ligand associations [23].

Yu et al. produced peptide-modified ALG microspheres encapsulating human mesenchymal stem cells for delivery into injured myocardium. Cell–ECM interface dynamics have an influence on both cell-matrix adhesion but also cellular processes, including migration, growth, maturation, and cytokine and growth factor signaling. In vitro data reveal that hMSCs adhere to the RGD-functionalized ALG surfaces more firmly than the un-modified ALG. Furthermore, compared to the non-modified cohort, the FGF2 expression level on the RGD-treated surface was considerably higher [87].

Similarly, in a study by Fonseca et al., ALG was modified by partial cross-linking with a matrix metalloproteinase cleavable peptide (proline–valine–glycine–leucine–isoleucine– glycine) by carbodiimide chemistry and co-incorporated into cell-adhesive RGD-ALG hydrogels. Matrix metalloproteinase-2 (MMP-2) function was enhanced in MSC grown in ALG functionalized with MMP-sensitive polypeptide; thus, this approach increased their role as ECM analogs in a more flexible and physiological 3-D cell milieu [88].

Other novel methods for ALG gelation include cryogelation, where growing crystals form interconnecting pores, generating solid materials and expelling swelling agents from gels via freeze-drying [89].

In the non-solvent induced phase separation technique, when the solubility of polymers decreases caused by the presence of a non-solvent, a polymeric solution segregates into polymer-heavy and polymer-lean phases. As an outcome, a lyogel is formed, which can be "hardened" by removing the parent solvent further [90,91].

At normal room temperature, a suspension of metal carbonate or hydroxycarbonate (Ca, Sr, Ba, Zn, Cu, Ni, or Co) is exposed to pressured carbon dioxide (30–50 bar) in a process called carbon dioxide-induced gelation. One of the driving forces behind the establishment of CO2-induced gelation is to eliminate some of the processes in aerogel manufacture as much as is feasible by merging gelation, solvent exchange, and SC-drying into a single procedure [92].

Another upcoming process of ALG gelation is the so-called carboxylic acid-induced gelation. Gelation was shown to be quick with oxalic, citric, and maleic acids, allowing the formation of gel beads by expelling ALG solution (4% *w*/*v*) into the relevant acidic solution (0.5 M). These findings demonstrate that the acid and ALG chains have such a strong interaction that gel breakdown was not observed after several washes of carboxylic acid-induced crosslinked gels in an aqueous medium at pH 7 [93,94].

Aerogels are an upcoming class of carriers for therapeutic drug delivery, having low density (0.005–0.50 g/cm<sup>3</sup> ), high specific surface area (300–1000 m2/g), and high porosity (upto 99%) [95–97]. Polymers such as starch, cellulose, pectin, ALG, etc., can be used to produce highly adsorption-efficient aerogel microparticles. Lovskaya and Menshutina prepared ALG-based microparticles loaded with three model drugs (loratadine, nimesulide, and ketoprofen) using the supercritical adsorption process. The release rate of drugs in the ALG-based aerogels was found to increase compared to pure drugs [98]. Similarly, Athamneh et al. used aerogel-based microspheres prepared by ALG and ALG-HA hybrids by the emulsion gelation method as carriers for pulmonary drug delivery. These ALG-based microspheres showed an in vitro aerodynamic diameter of 5 µm, indicating the favorable properties of ALG-based microspheres for pulmonary delivery [99,100].

#### **5. ALG Particles Formation Methods**

ALG gelation occurs via one of two mechanisms: (1) external gelation, in which cations enter the ALG system from the outside, or (2) internal gelation, in which cations escape the ALG structure. ALG-based particles of a variety of dimensions can be produced and categorized into various categories: (1) macroparticles, such as that of drug or vitamin tablets, may be seen by the naked eye; (2) microparticles vary significantly in size from a few microns to several millimeters; and (3) nanoparticles have a diameter ranging between 1 to 100 nm [101–104]. The most popular method for producing ALG particles is the drop-by-drop expulsion of an ALG solution through a needle in a cationic bath. External gelation is used to create ALG particles with diameters between 500–5000 µm [105–107]. The ALG particle size can be lowered, and particles having a smaller size distribution could be generated using several ways depending on this external gelation process, including coaxial laminar airflow at the nozzle, electrostatic fields, and vibrating nozzles [107–110]. In their study, Patel et al. described a method detailing the impact of ionotropic gelation residence time (IGRT) on the extent of cross-linking of ALG particles with Ca2+ ions. They found that to prevent the dissolution of the drug into the bulk cross-linking solution, the IGRT must be kept short which also enhances drug loading [111].

An emulsification–gelation process can also be used to make ALG spheres. Gelled spheres are made by emulsifying an ALG solution in an organic phase, followed by the gelation of the ALG emulsion droplets. External gelating of the ALG emulsion droplets leads to emulsion cracking; however, the spheres aggregate and, as a result, numerous emulsion droplets (W/O/W) are formed [112,113]. Membrane emulsification may yield ALG particles with a quite small size distribution; however, particle production is confined to the micro range (>1 µm), and clogging of the membrane layer, as well as the feasibility of the process scale-up, remains a concern. Mechanical emulsification is the most practical for industry, as it produces ALG particles of all sizes. Nevertheless, the size variation of the ALG particles produced is relatively wide. ALG nanoparticles can be made in various ways, although the majority of them rely on complexation, either through interactions with ions like calcium or negatively charged polyelectrolytes like CS [114] or poly-L-lysine [115]. Complexation is used in other processes as well, such as emulsification, to strengthen nanoparticles. In most cases, complexation will be required as a supplement to obtain the particles. The incorporation of calcium chloride into the ALG solution produces nanoaggregates; generally, the drug is typically added before this step. The size of nanoparticles varies greatly depending on a variety of factors, such as the manufacturing process, preparation parameters, crosslinking reagent, ALG molecular mass, and so on. Because ALG is a negatively charged polyelectrolyte, nanoparticles can range in size from several tens to nanometers, and the zeta potential is generally negative [116].

#### **6. Current Advancements in ALG Formulations in Drug Delivery**

A substantial number of therapeutic actives have been unveiled over the past few years. Numerous of them grasp magnificent therapeutic properties; nevertheless, they manifest poor bioavailability and poor pharmacokinetics, causing detrimental systemic impact after the administration [117]. Therefore, DDS has evolved into an essential device to enhance remedial effectiveness by employing delivery carriers (as illustrated in Figure 4) such as microspheres, hydrogels, beads, liposomes, nanoparticles, nanofibers, etc., to deliver drugs [118–120].

Different approaches (as shown in Figure 5) are employed to develop the aforementioned drug delivery devices.

Below are a few vital employments of ALG-mediated systems for enhanced actives delivery through various administration routes.

Different approaches (as shown in Figure 5) are employed to develop the aforemen‐

**Figure 4.** Diagrammatic representation of ALG formulations into diverse forms. **Figure 4.** Diagrammatic representation of ALG formulations into diverse forms. tioned drug delivery devices.

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**Figure 5.** Various approaches for preparing ALG‐based particulate carrier matrix. **Figure 5.** Various approaches for preparing ALG-based particulate carrier matrix.

#### Below are a few vital employments of ALG‐mediated systems for enhanced actives *6.1. Oral Drug Delivery*

**Figure 5.** Various approaches for preparing ALG‐based particulate carrier matrix. Below are a few vital employments of ALG‐mediated systems for enhanced actives delivery through various administration routes. *6.1. Oral Drug Delivery* Oral drug delivery is the first choice route of drug delivery for any therapeutic sub‐ stance due to its simplicity, patient compliance, cost‐effectiveness, easy bulk manufactur‐ ing, sustained and controlled release, and generation of immune response in case of vac‐ delivery through various administration routes. *6.1. Oral Drug Delivery* Oral drug delivery is the first choice route of drug delivery for any therapeutic sub‐ stance due to its simplicity, patient compliance, cost‐effectiveness, easy bulk manufactur‐ ing, sustained and controlled release, and generation of immune response in case of vac‐ cination [121–123]. However, some limitations exist, such as variable pH of the gastroin‐ testinal tract (the stomach is highly acidic while the small intestine is alkaline), first‐pass metabolism in the liver, the occurrence of hydrolytic enzymes, and an absorption barrier Oral drug delivery is the first choice route of drug delivery for any therapeutic substance due to its simplicity, patient compliance, cost-effectiveness, easy bulk manufacturing, sustained and controlled release, and generation of immune response in case of vaccination [121–123]. However, some limitations exist, such as variable pH of the gastrointestinal tract (the stomach is highly acidic while the small intestine is alkaline), first-pass metabolism in the liver, the occurrence of hydrolytic enzymes, and an absorption barrier in the liver intestine. This limits the extent of the therapeutic action of many drugs given by the oral route. Nevertheless, due to their non-toxicity, biocompatibility, and biodegradability, ALG hydrogels can be safely administered orally [124].

cination [121–123]. However, some limitations exist, such as variable pH of the gastroin‐ testinal tract (the stomach is highly acidic while the small intestine is alkaline), first‐pass metabolism in the liver, the occurrence of hydrolytic enzymes, and an absorption barrier in the liver intestine. This limits the extent of the therapeutic action of many drugs given by the oral route. Nevertheless, due to their non‐toxicity, biocompatibility, and biodegra‐ dability, ALG hydrogels can be safely administered orally [124]. in the liver intestine. This limits the extent of the therapeutic action of many drugs given by the oral route. Nevertheless, due to their non‐toxicity, biocompatibility, and biodegra‐ dability, ALG hydrogels can be safely administered orally [124]. Recently, Ilgin et al. used SA hydrogels as the carrier for diclofenac sodium to obtain controlled drug delivery at a specific pH. The porous structure made them valuable as a drug delivery system. The researchers prepared SA pH-responsive semi-interpenetrating hydrogels by altering the physical and biological properties of the biocompatible and biodegradable ALG via surface functionalization with the aid of monomers such as HEMA, MAPTAC, and MA. Polyvinyl alcohol (PVA) was employed to provide chemical stability,

and nMBA was the copolymer. The resultant hydrogels were evaluated for their swelling, drug loading, drug release, and antimicrobial activity. PVA and HEMA proved to be good polymeric systems because they enhanced biocompatibility and mechanical properties. Maximum swelling (38 gwater/ggel), drug loading (22.8%), and release profiles were observed at pH 7.0, where 95% release of drug was observed at the end of 2 h, as compared to 4.5% at pH 1.5. Antibacterial activity was observed against *E. coli*, *B. subtilis*, and *S. aureus* by the disk diffusion method, and it displayed excellent inhibition of the growth of all three microorganisms. Thus, ALG is confirmed to be a great, intelligent drug release platform for oral drug delivery, providing a controlled release [125].

To obtain colon-specific drug delivery for inflammatory bowel diseases such as Crohn's disease, it is challenging to move through the different pH conditions of the GI tract undisturbed. Hence, Ayub et al. tried to enhance the delivery of Paclitaxel to colonic cancer cells by formulating a self-assembled cysteamine-based disulfide cross-linked biodegradable thiolated SA-derived nanoparticles via a layer-by-layer assembly approach (as shown in Figure 6A). SA was oxidized using sodium periodate before adding cysteamine hydrochloride to alter its backbone. The disulfide bonds prevented the leakage of the drug before it reached its target site. The encapsulation efficiency of P3DL/PAH/PSCCMA was found to be 77.1%, with a cumulative-drug release of 45.1% after 24 h (as displayed in Figure 6B). The nanospheres demonstrated their maximum size at pH 7.0, thus signifying their efficacy in selectively delivering the actives to the colon. The MTT assay revealed the high viability (86.7%) of HT-29 cells (as shown in Figure 6C). More than 70% of the nanospheres were detected in human colon cancer adenocarcinoma HT-29 cells, indicating their high cellular uptake, as displayed in Figure 6D). Stability studies indicated that most nanospheres changed slightly in size and PDI but were stable with negligible differences in zeta potential. Thus, this approach could be employed for colon-targeted drug delivery with minimal toxic effects [126].

In order to furnish controlled drug delivery via the oral route, Cong et al. prepared an ALG hydrogel/CS micelle composite system encapsulating emodin. The research group prepared cross-linked micelles because, unlike conventional polymeric micelles, crosslinked micelles prevent drug leakage and protect them from dissociating quickly. Emodin was chosen as a model drug because it has poor water solubility and undergoes extensive first-pass metabolism. It was loaded onto CS micelles and then mixed with SA hydrogels developed by cross-linking with Ca2+ ions and β-GP to form hydrogel/micelle beads. Based on response surface methodology, the optimized biopolymer concentrations were determined. The 1:1 hydrogel/micelle beads showed sustained drug delivery, while the 3:1 ratio provided colon-specific delivery. The morphological, swelling, and degradation studies were carried out. The diameter of micelles increased from 80 nm in aqueous solution to 100–200 nm in the hydrogel, likely due to electrostatic interaction between the amino group of the CS chain and the carboxylate group of the ALG chain. The swelling of micelles was reduced at pH 1.2 due to SA hydrogel, whose swelling ratio is at acidic pH, thus preventing the release and degradation of CS chains. The initial release amount was 28% in SGF, and a final release amount of 85% for 1:1 micelle/hydrogel systems was reported. Thus, this pH-responsive hydrogel/micelle system could be an encouraging candidate for sustained-release or site-targeted actives delivery for unstable or hydrophobic actives [127].

Amphotericin-B is the first-choice drug for many fungal infections, including leishmaniasis. However, it has limited solubility, is susceptible to gastric pH, and its oral bioavailability is low; it is generally administered intravenously [128]. Hence, to overcome this issue, Senna et al. utilized nanostructured lipid carriers (NLC) combined with stimuli-sensitive ALG polymers using a high-pressure homogenization technique with calcium chloride as the cross-linker, providing a dual-benefit of oral drug delivery and protection from gastric pH without structural degradation and promoting drug release at intestinal pH. Nanostructured lipid carriers are systems in which hydrophilic drugs are dispersed in polymeric matrices, forming nanometric droplets, thus enhancing adsorption and preventing enzymatic degradation [129]. AmpB was solubilized by solid lipid glyceryl

monostearate (GSM). Studies using the trypan blue exclusion test showed that the NLCs showed low cytotoxicity on Vero cells (ATCC® no. CCL-81™), very high specificity, and their drug release profiles were equivalent to ALG swelling degree profiles, indicating that the drug delivery was primarily due to the polymer's swelling rate. In the responsive pH range, the carboxylic acid groups of ALGs became ionized and acquired a negative charge, resulting in electrostatic repulsion and allowing water molecules to enter. The NLC particles maintained their framework even after rehydration; thus, the aforementioned system proved promising for the delivery of AmpB orally [130]. their efficacy in selectively delivering the actives to the colon. The MTT assay revealed the high viability (86.7%) of HT‐29 cells (as shown in Figure 6C). More than 70% of the nano‐ spheres were detected in human colon cancer adenocarcinoma HT‐29 cells, indicating their high cellular uptake, as displayed in Figure 6D). Stability studies indicated that most nanospheres changed slightly in size and PDI but were stable with negligible differences in zeta potential. Thus, this approach could be employed for colon‐targeted drug delivery with minimal toxic effects [126].

Recently, Ilgin et al. used SA hydrogels as the carrier for diclofenac sodium to obtain controlled drug delivery at a specific pH. The porous structure made them valuable as a drug delivery system. The researchers prepared SA pH‐responsive semi‐interpenetrating hydrogels by altering the physical and biological properties of the biocompatible and bi‐ odegradable ALG via surface functionalization with the aid of monomers such as HEMA, MAPTAC, and MA. Polyvinyl alcohol (PVA) was employed to provide chemical stability, and nMBA was the copolymer. The resultant hydrogels were evaluated for their swelling, drug loading, drug release, and antimicrobial activity. PVA and HEMA proved to be good polymeric systems because they enhanced biocompatibility and mechanical properties. Maximum swelling (38 gwater/ggel), drug loading (22.8%), and release profiles were ob‐ served at pH 7.0, where 95% release of drug was observed at the end of 2 h, as compared to 4.5% at pH 1.5. Antibacterial activity was observed against *E. coli*, *B. subtilis*, and *S. aureus* by the disk diffusion method, and it displayed excellent inhibition of the growth of all three microorganisms. Thus, ALG is confirmed to be a great, intelligent drug release

To obtain colon‐specific drug delivery for inflammatory bowel diseases such as Crohn's disease, it is challenging to move through the different pH conditions of the GI tract undisturbed. Hence, Ayub et al. tried to enhance the delivery of Paclitaxel to colonic cancer cells by formulating a self‐assembled cysteamine‐based disulfide cross‐linked bio‐ degradable thiolated SA‐derived nanoparticles via a layer‐by‐layer assembly approach (as shown in Figure 6A). SA was oxidized using sodium periodate before adding cysteamine hydrochloride to alterits backbone. The disulfide bonds prevented the leakage of the drug before it reached its target site. The encapsulation efficiency of P3DL/PAH/PSCCMA was found to be 77.1%, with a cumulative‐drug release of 45.1% after 24 h (as displayed in Figure 6B). The nanospheres demonstrated their maximum size at pH 7.0, thus signifying

*Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 14 of 60

platform for oral drug delivery, providing a controlled release [125].

**Figure 6.** Biocompatible disulfide cross‐linked SA derivative nanoparticles for oral colon‐targeted drug delivery (**A**) P3DL/PAH/PSSCMA (a) TEM and (b) FeSEM pictures at magnifications of 110,000 and 100,000, respectively. (**B**) For 170 h in a simulated gastrointestinal medium, the cumulative % drug release of P3DL/PAH/PSSCMA was measured. \*\* means *p* < 0.01. (**C**) The effects of P3/PAH/PSSCMA, P3DL/PAH/PSSCMA, PCX and untreated P3/PAH/PSSCMA on (a) HT‐29 and (b) CRL 1790. Data marked with the same letters show significant difference between the samples. \* Indicates *p* < 0 .05 compared to the untreated samples. (**D**) P3DL/PAH/PSSCMA nanospheres tagged with rhodamine 110 are taken up by HT‐29 cells. Reproduced with permission from [126], copyright Taylor & Francis Online, 2019. **Figure 6.** Biocompatible disulfide cross-linked SA derivative nanoparticles for oral colon-targeted drug delivery (**A**) P3DL/PAH/PSSCMA (a) TEM and (b) FeSEM pictures at magnifications of 110,000 and 100,000, respectively. (**B**) For 170 h in a simulated gastrointestinal medium, the cumulative % drug release of P3DL/PAH/PSSCMA was measured. \*\* means *p* < 0.01. (**C**) The effects of P3/PAH/PSSCMA, P3DL/PAH/PSSCMA, PCX and untreated P3/PAH/PSSCMA on (a) HT-29 and (b) CRL 1790. Data marked with the same letters show significant difference between the samples.\* Indicates *<sup>p</sup>* < 0 .05 compared to the untreated samples. (**D**) P3DL/PAH/PSSCMA nanospherestagged with rhodamine 110 are taken up by HT-29 cells. Reproduced with permission from [126], copyright Taylor & Francis Online, 2019.

In order to furnish controlled drug delivery via the oral route, Cong et al. prepared an ALG hydrogel/CS micelle composite system encapsulating emodin. The research group prepared cross‐linked micelles because, unlike conventional polymeric micelles, cross‐linked micelles prevent drug leakage and protect them from dissociating quickly.

extensive first‐pass metabolism. It was loaded onto CS micelles and then mixed with SA hydrogels developed by cross‐linking with Ca2+ ions and β‐GP to form hydrogel/micelle beads. Based on response surface methodology, the optimized biopolymer concentrations were determined. The 1:1 hydrogel/micelle beads showed sustained drug delivery, while the 3:1 ratio provided colon‐specific delivery. The morphological, swelling, and degrada‐ tion studies were carried out. The diameter of micelles increased from 80 nm in aqueous solution to 100–200 nm in the hydrogel, likely due to electrostatic interaction between the amino group of the CS chain and the carboxylate group of the ALG chain. The swelling of micelles was reduced at pH 1.2 due to SA hydrogel, whose swelling ratio is at acidic pH, thus preventing the release and degradation of CS chains. The initial release amount was 28% in SGF, and a final release amount of 85% for 1:1 micelle/hydrogel systems was re‐ ported. Thus, this pH‐responsive hydrogel/micelle system could be an encouraging can‐ didate for sustained‐release or site‐targeted actives delivery for unstable or hydrophobic

actives [127].

Due to its simplicity, non-invasiveness, patient compliance, and economy, oral drug delivery is the most favored route. Particles having a size between 20–100 nm are readily absorbed from the cells while also avoiding renal clearance [131], while particles below 5 nm are quickly cleared via renal clearance. Thus, Thomas et al. prepared ALG-cellulose nanocrystal hybrid nanoparticles by a green method to achieve controlled-drug delivery of the drug rifampicin, an anti-tubercular agent with poor water solubility, which necessitates the search for alternate drug delivery routes. Cellulose nanocrystals (CNCs) were used to improve the mechanical stability, durability, and high diffusion rates due to ALG's highly porous structure. CNCs have been reported to reduce the voids in the gelatin structure [132]. In addition, CNCs have been shown to enhance the ALG bead's mechanical strength and improve drug release [133]. Ionotropic gelation was utilized to synthesize ALG-CNC NPs using water as the only solvent. The optimum nanoparticle preparation was found to be with 1% surfactant and a 1:6 ratio of ALG CNC. The drug entrapment efficiency (EE) was between 43–69%. The ALG-CNC-NPs swelled rapidly at pH 6.8 to 7.4. It showed 15% drug release in 2 h at pH 1.2, while at pH 7.4, almost 100% of the drug was released in 12 h, demonstrating its controlled-release profile. It has a negative zeta potential (−15 to −20 mV) due to free carboxylic groups, thus facilitating penetration into epithelial cells. CNCs and ALG-CNC NP's cytotoxicity was investigated via MTT assay using the L929 fibroblast. It showed 100% cell viability, thus suggesting that these nanoparticles could be a good candidate for drug delivery [134].

The researchers have prepared many formulations and systems to promote actives delivery efficiency via oral routes to beat oral drug delivery limitations. The recent literature includes preparations such as cationic cyclodextrin/ALG/CS nanoflowers/5 fluorouracil [135], hydroxyethylacryl CS/SA hydrogel/paracetamol [136], pH-sensitive nanocomposite using SA/pectin/tannic acid(TA)/silver(Ag)/propranolol [137], Cyperus esculentus starch-ALG/ibuprofen [138], calcium alginate (CA)/SWCNT-GI/curcumin [139], thiol-modified SA microspheres/bovine serum albumin [140], vitamin B12 modified amphiphilic SA nanoparticles/insulin [141], thiolated SA nanoparticles/docetaxel [142], CA beads/cetuximab/octreotide [143], ALG/barium ion/methotrexate [144], CS/CA/ liraglutide [145], nano pol cellulose (CMC)/ALG/CS [146].

#### *6.2. Ocular Drug Delivery*

Topical instillation in the eye is the most preferred non-invasive administration route of actives for various anterior and posterior segment diseases, for instance, glaucoma, uveitis, cataract, and age-related macular degeneration. It is best-loved due to its comfort in administration and being patient-friendly. Nevertheless, the eye has several protective anatomical barriers that limit drug absorption through this route. The lacrimal fluid drains the drug quickly from the ocular area. Hence, ocular drug delivery has the challenge of maintaining the conc. of the drug at the site of action for the necessary time [147,148]. To enhance the delivery of topical ocular therapeutics, researchers have principally highlighted two methods: (i) to improve the period of residence of the cornea by employing viscosity enhancers, mucoadhesive, particulate, and/or in situ gelling systems; and (ii) to enhance the permeability of the cornea using (a) penetration enhancers, (b) prodrugs, and (c) colloidal systems (like NPs and liposomes) [149].

There is a probability of permanent visual damage or blindness due to retinal diseases such as macular degeneration, uveitis, macular edema, etc., and hence, in such cases, immunosuppressant drugs must be provided on a long-term basis to sustain the functioning of the eye. Encapsulated-cell therapy, however, provides a modern approach with longlasting delivery of newly synthesized protein-rich drugs, eliminating the need for surgical treatment and is potentially removable by surgery. Some limitations pertaining to ECT use are its poor mechanical strength, inadequate biocompatibility, and lack of termination mechanisms [150]. Hence, Wong et al. synthesized an injectable ALG collagen (CAC) hydrogel with an inducible termination switch as the encapsulation matrix for glial-derived neurotrophic factor for safer ocular delivery. Here, collagen acts to enhance the cell viability

of ALG. The CAC ECT gels were developed by ionotropic gelation employing calcium chloride, where they used a Tet-on-pro-Casp8 switch mechanism for the effective termination of ECT systems. An oral DOX treatment was sufficient for the termination of the gel system. The therapeutic delivery was evaluated in pink-eyed dystrophic RCS/lav rats, a recessive RP model characterized by a progressive loss of photoreceptors and electrophysiological response. The retinal function of rats acquiring GDNF-secreting gels was found to be better than that of the control group. The ECT gel system was mechanically stable, viable, and functional in vivo. Stability studies revealed that following six months of implantation, the system was mechanically stable, strong, and assisted the growth of various types of cells, viz., HEK293 and ARPE-18 cells. Thus, this approach may be utilized to address a range of posterior eye disorders [151].

Nepafenac is a non-steroidal anti-inflammatory agent utilized to address post-surgery pain in the cornea, such as cataracts. Because of its water insolubility, it is only formulated as a suspension, which leads to irritation in the eye, severe lacrimation, and consequently decreased drug's residence time due to rapid drainage into the systemic circulation, limiting the conc. of the drug at the action site [152]. Hence, Shelley et al. utilized the ion-activated in-situ gel formation of ALG with Ca2+ ions in the lacrimal fluid to accomplish a sustained release of the actives. HPBCD was the actives solubilizer and permeation enhancer, and the retention time and permeation parameters were observed in the ex-vivo porcine perfusion eye model. The in-situ gel formulation showed a considerably increased diffusion rate and permeation rate than the placebo, with a sustained release of over 24 h with release kinetics following the Korsmeyer-Peppas equation [153].

In another investigation, Nagarwal et al. prepared CS-coated SA-CS nanoparticles for the intra-ocular delivery of 5-FU used for corneal carcinoma. It demonstrated an encapsulation efficiency (almost 27%) and drug loading capacity (around 19%). Moreover, the in-vitro and in-vivo drug-release profiles showed a sustained release (8 h) in contrast to the 5-FU solution and also showed good tolerability when tested by the Draize test on the rabbit eye [154].

Conventional ocular drug dosage forms, for example, solutions, suspensions, and ointments, possess certain disadvantages that cause poor absorption of the drug, such as the sweeping activity of eyelids, tears washing away the drug, impermeable endothelium, and blood barrier. In-situ gelling systems are an effective drug delivery and absorption method since the less viscous solution undergoes gelling in the presence of stimuli such as pH, ionic strength, and temperature. Hence, Noreen et al. developed ALG in situ gelling systems using gum obtained from Terminalia arjuna bark with moxifloxacin HCl as the drug for ophthalmic delivery. It undergoes a sol to gel transition at the pH of tear fluid. The preservative was methylparaben, and the osmolarity was adjusted with sodium chloride. The drug was stable and provided sustained release for 12 h. The ex vivo transcorneal penetration was found to be 4.76 ± 0.27%, and corneal hydration was 78.85 ± 0.19%. No ocular irritation was observed [155]. Thus, the overall results demonstrated the aforementioned system as a promising candidate for ocular drug delivery.

In another investigation, Polat et al. formulated nanofibrous ocular inserts for the therapy of bacterial keratitis incorporating the drug Besifloxacin HCl or BH-hydroxypropylbeta-cyclodextrin (HP-β-CD) complex comprising PCL/PEG fibrous inserts coated with mucoadhesive polymers such as SA or thiolated sodium alginate (TSA) based on the electrospinning method. The coating with SA and TSA increased the insert's bioadhesion. The preparation demonstrated an initial burst release followed by a slow release for two days, and the thickness and diameter of the inserts were comparable to commercial formulations. Drug loading efficiency was over 90%. Even after seven days of incubation, the inserts did not attain acidic pH values, indicating that the inserts did not cause eye irritation. In vitro studies on ARPE-19 cells on exposure to the ocular inserts demonstrated no cytotoxicity, and their antibacterial activity was comparable to that of commercial formulations. Ex vivo transport research showed that HP-β-CD enhanced solubility and corneal permeability, and

the actives delivery was equivalent to commercial formulations [156]. Hence, the overall results reported that the newly prepared system was suitable for ocular drug delivery.

Different researchers have studied various ALG-based preparations for enhanced ocular delivery of drugs. For example, CS/ALG multilayers/diclofenac [157], CS/ALG/ daptomycin [158], CS/ALG/acetamiprid [159], ALG/CS/levofloxacin [160], SA/glycerin/ flurbiprofen [161], ALG/peppermint phenolic extract [162], SA/methyl cellulose/ sparfloxacin [163], ALG/calcium gluconate(CaG)/tryptophan [164], SA dialdehyde/ carboxymethyl CS/limbal stem cells (LSC) [165], SA/methyl cellulose/CMC/carbopol/ pilocarpine [166], CS/SA/azelastine [167], SA/butyl methacrylate/lauryl methacrylate/ linezolid [168] are few of the current investigations which portrayed excellent ocular actives delivery.

## *6.3. Pulmonary Drug Delivery*

The lungs are an appealing site for the pulmonary administration of actives via diverse DDSs [169,170]. Moreover, the pulmonary route provides numerous benefits over traditional per oral administration, for instance, greater surface area with fast absorption owing to the high vascularization and evasion of the first-pass metabolism [171]. This selectivity enables targeted actives delivery and, therefore, diminishes the side effects [169]. Nevertheless, the pulmonary route is challenging to deliver the actives deep to the alveolar regions of the lung owing to diverse respiratory obstacles, including mechanical, chemical, pathological, and immunological hindrance [172]. The mechanical barrier employs mucociliary clearance to remove particles (having a mean diameter of >6 µm) [173]. When clearance takes place quicker than absorption, in particular of poorly soluble drugs, the availability of actives in the lungs might be restricted. Additionally, under pathologic states like asthma and COPD, excessive mucus is accumulated in the lung, which inhibits the deeper penetration of actives [173,174]. Furthermore, the chemical barrier containing proteolytic enzymes can degrade the breathed materials, which leads to the destruction of functions of actives and/or delivery carriers. Ultimately, the immunological barrier comprising primarily alveolar macrophages assembles the immunological reactions in the intense lung that eradicate all foreign materials with no difference between potential detrimental materials and advantageous ones. Thus, pulmonary bioavailability and systemic bioavailability for actives provided by maximum traditional pulmonary products are less. Therefore, the progress of unique, more effective DDSs is pivotal to alleviating the consequences of these obstacles [175].

The FDA-approved pulmonary dry powder inhalers are predominantly fast-releasing. To obtain sustained delivery to the lungs, Athamneh et al. developed an aerogel microsphere formulation for suitable delivery to the lower pulmonary tract using the ALG-HA hybrid system by the emulsion gelation method followed by drying with supercritical CO2. Combining these approaches resulted in highly porous Alg-HA microspheres with very low density and a high BET-specific surface area. The addition of HA to Alg successfully reduced the particle agglomeration and enhanced its biodegradation, possibly due to the formation of a hydrogen bond between ALG's carboxylate groups and N-acetylglucosamine amide in the hybrid aerogel. In addition, HA was employed to improve the physical characteristics of ALG, such as gelling efficiency and lung tolerance. This led to achieving the desired particle size for pulmonary deposition and improving biodegradability. However, more research using active pharmaceutical ingredients needs to be carried out in this area [100].

Drugs administered to the lungs to treat COPD and cancer suffer from low bioavailability due to distribution to other body tissues, thus requiring multiple dosing and increasing the risk of adverse effects [176]. Mahmoud et al. formulated microparticles based on ALG using the emulsified nano spray-drying process for the pulmonary carrier of roflumilast, a selective inhibitor of the phosphodiesterase-4 enzyme in lung cells. Isopropyl myristate was used as an oil, Tween 80 was used as a surfactant, and calcium beta-glycerophosphate was used as a cross-linking agent. The developed microparticles were assessed based on

encapsulation efficiency, particle size, and in vitro release of actives. The aerodynamic data showed that the actives could be deposited deep into the lungs at the bronchi or bronchial tube (as shown in Figure 7a). The formulation with the spherical-shaped microparticles swelled within 3 h at pH 7.4. Furthermore, it showed a remarkable cytotoxic effect on A-549 tumor cell lines (2.5-fold decrease in IC<sup>50</sup> compared to the pure drug) (as demonstrated in Figure 7b) and reduced pro-inflammatory cytokines (TNF-alpha, IL-6, and IL-10) in contrast to pure actives (as exhibited in Figure 7c). In addition, CD-based microparticles showed higher sustained bronchodilation on human volunteers than marketed Ventolin®HFA (as shown in Figure 7d). Thus, CD-based microparticles could be a propitious approach for delivering roflumilast in humans [177]. assessed based on encapsulation efficiency, particle size, and in vitro release of actives. The aerodynamic data showed that the actives could be deposited deep into the lungs at the bronchi or bronchial tube (as shown in Figure 7a). The formulation with the spherical‐ shaped microparticles swelled within 3 h at pH 7.4. Furthermore, it showed a remarkable cytotoxic effect on A‐549 tumor cell lines (2.5‐fold decrease in IC50 compared to the pure drug) (as demonstrated in Figure 7b) and reduced pro‐inflammatory cytokines (TNF‐al‐ pha, IL‐6, and IL‐10) in contrast to pure actives (as exhibited in Figure 7c). In addition, CD‐based microparticles showed higher sustained bronchodilation on human volunteers than marketed Ventolin®HFA (as shown in Figure 7d). Thus, CD‐based microparticles could be a propitious approach for delivering roflumilast in humans [177].

HA hybrid system by the emulsion gelation method followed by drying with supercritical CO2. Combining these approaches resulted in highly porous Alg‐HA microspheres with very low density and a high BET‐specific surface area. The addition of HA to Alg success‐ fully reduced the particle agglomeration and enhanced its biodegradation, possibly due to the formation of a hydrogen bond between ALG's carboxylate groups and N‐acetyl‐ glucosamine amide in the hybrid aerogel. In addition, HA was employed to improve the physical characteristics of ALG, such as gelling efficiency and lung tolerance. This led to achieving the desired particle size for pulmonary deposition and improving biodegrada‐ bility. However, more research using active pharmaceutical ingredients needs to be car‐

Drugs administered to the lungs to treat COPD and cancer suffer from low bioavail‐ ability due to distribution to other body tissues, thus requiring multiple dosing and in‐ creasing the risk of adverse effects [176]. Mahmoud et al. formulated microparticles based on ALG using the emulsified nano spray‐drying process for the pulmonary carrier of roflumilast, a selective inhibitor of the phosphodiesterase‐4 enzyme in lung cells. Isopro‐ pyl myristate was used as an oil, Tween 80 was used as a surfactant, and calcium beta‐ glycerophosphate was used as a cross‐linking agent. The developed microparticles were

*Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 19 of 60

ried out in this area [100].

**Figure 7.** Design and characterization of emulsified spray‐dried ALG microparticles as a carrier for the dually acting drug roflumilast (**a**) In an ethanolic phosphate buffer saline solution (30% *v*/*v*; pH 7.4), release patterns of roflumilast and emulsified spray‐dried ALG microparticles were studied. **Figure 7.** Design and characterization of emulsified spray-dried ALG microparticles as a carrier for the dually acting drug roflumilast (**a**) In an ethanolic phosphate buffer saline solution (30% *v*/*v*; pH 7.4), release patterns of roflumilast and emulsified spray-dried ALG microparticles were studied. (**b**) The effects of roflumilast, a medicated CD formulation, and a non-medicated CD formulation on the proliferation of A-549 tumor cells. (**c**) TNF-alpha, interleukin-6, and interleukin-10 levels were reduced in A-549 tumor cells by roflumilast and CD formulation. (**d**) FEV1/FVC%—time curve in healthy human volunteers upon inhaling the chosen CD formulation vs. Ventolin® HFA. Reproduced with permission from [177], Copyright Elsevier 2018.

The pulmonary route has also administered anti-cancer drugs to treat lung cancer due to their faster onset of action and evasion of the first-pass metabolism. One of them, cisplatin, shows toxicity in rat liver, lungs, and kidneys, due to which Alsmadi et al. developed CS-ALG nanoporous aerogel carriers loaded with cisplatin for the treatment of lung cancer using an emulsion–gelation method followed by supercritical fluid extraction. The drug exhibited excellent drug loading (76%) while retaining its crystal structure and gave a sustained release of over 6 h. The cisplatin aerogels, thus prepared, decreased the lung toxicity in rats and prevented weight loss [178].

In another investigation, Iglesias et al. prepared ALG aerogel by thermal inkjet technology followed by supercritical drying to encapsulate salbutamol sulfate, which is used to treat asthma attacks and COPD. The current pulmonary drug delivery system suffers from limitations in particle size uniformity and the presence of surfactants. This can be potentially used for personalized medicine. However, the gel precursor concentrations severely restrict the printable region. The process was highly compatible with the active ingredient (salbutamol sulfate), having a homogenous texture in the nanoporous range. The SS-loaded aerogel was well deposited in the bronchi and bronchioles owing to their nano-particle size. Thus, these aerogels may pave the way for pulmonary drug delivery and personalized medicine, fulfilling the demands of nanostructured, miniaturized, highresolution product designs [179].

Liposomes, micelles, and polymeric drug particles have been delivered by pulmonary route using biocompatible, biodegradable, and flexible biopolymers such as CS, ALG, gelatin, and other natural polymers. Blends of CS-ALG nanoparticles and microparticles find their applications in controlled drug delivery systems, with calcium chloride often acting as the cross-linking agent. However, researchers have found that using calcium chloride for pulmonary particles induces inflammatory responses. Hence, Alnaief et al. formulated hybrid ALG-CS aerogels via the emulsion-gelation technique without any cross-linking agent. CS solution (2%) was put into the oil phase containing liquid paraffin and 4% surfactant (Span 80 or Span 85), followed by the addition of 1% ALG solution. The resultant hybrid particles were isolated from the oil phase by the process of centrifugation. The pore liquid was replaced with ethanol by a solvent exchange method. The prepared aerogels were later dried with supercritical carbon dioxide. The polymer addition order throughout the gelling procedure and the kind of surfactant employed greatly affected the properties of the hydrogel. Samples prepared using span 85 showed high positive zeta potentials (35.4 ± 5.37 mV), indicating that CS was surrounding the ALG core, whereas those prepared using span 80 showed high negative zeta potentials (−2.15 ± 3.70 to −5.98 ± 5.37 mV), suggesting that ALG was surrounding the CS core. The particle size ranged from 70 nm to 4.17 µm, whereas the aerodynamic diameter ranged from 0.17 to 2.29 µm [180]. Thus, the entire results revealed the developed system is a promising candidate for pulmonary actives delivery.

For the treatment of cystic fibrosis, a potentially life-threatening disease characterized by mutations in the cystic fibrosis transmembrane conductance regulator (CTFR), antibiotics (colistimethane sodium) are generally prescribed. Due to the loss of Cl-channel activity, the lungs become more susceptible to bacterial infections. Of these, Pseudomonas aeroginosa survives the antibiotic treatment and emerges as the predominant infecting organism over time [181–183]. Tobramycin is the drug of choice for such infections that are deteriorating with regular colistimethane sodium. One issue associated with tobramycin therapy is the non-compliance of patients due to twice-daily dosing, which leads to the failure of antibiotic treatment. Another concern is the potential for ototoxicity and nephrotoxicity associated with nearly all aminoglycosides, although there is less evidence of ototoxicity and nephrotoxicity in clinical trials with inhaled tobramycin [184,185]. Hence, Hill et al. developed ALG/CS particles using CaCl<sup>2</sup> as the crosslinker. During the optimization studies, increasing the concentration of cations in the formulation resulted in a more significant aggregation of the particles. The optimal formulation was ALG: CS: tobramycin 9:1:1.5, giving high drug loading and narrow size distribution. Tobramycin release was evaluated, depicting a biphasic release with 18.9% of the entrapped drug released within 24 h. In vitro antibacterial studies showed that the drug-loaded optimal formulation showed a dosedependent activity against P. aeroginosa with a MIC of 6.25 µg/mL, while the unloaded formulation demonstrated no action. Tobramycin alone had a MIC of 1.5 µg/mL, possibly due to the drug diffusion rate. The Zeta potential was found to be 2.16 ± 0.07% mV and the %EE was 44.5%. SLPI conjugation enhanced the mucoadhesive properties [186]. Hence, the prepared system could help deliver drugs via the pulmonary route.

Moreover, investigators have developed various formulations to intensify pulmonary DDSs. For example, SA/CS/Tween 80/rifampicin [187], ALG/poly(N-isopropyl acryl amide (PNIPAAm)/theophylline [188], ALG/ciprofloxacin [189], ALG/CS/lapazine [190], ALG/CaCO3/levofloxacin/DNAse [191], ALG/HA/naproxen [99], ALG/CS/DOX/ paclitaxel [192], ALG modified PLGA nanoparticles/amikacin/moxifloxacin [193], ALG

particles encapsulating live Bacille Calmette– Guérin (BCG) and *Mycobacterium indicus pranii* (MIP) [194], CS/ALG/BSA gel/DOX [195] are some of the recent formulations showing excellent delivery of drugs via pulmonary delivery.

#### *6.4. Vaginal Drug Delivery*

Lately, the vagina has been considered a potential administration route replacing the parenteral route for actives delivery, bestowed with systemic effects that cannot be favorably administered per os due to the hepatic or GI degradation or to the commencement of adverse consequences in the GI. The significant constraint of such an administration route is described by the physiological elimination mechanisms, which are active in the vagina's lumen and are responsible for an unsatisfactory residence time of the conventional formulated system at the targeted site, following an unsteady actives dissemination onto the mucosa [196]. The vagina, a prominent female reproductive organ, allows for local and systemic actives delivery due to its large surface area and high blood supply. However, it is also associated with variations in the size of the endometrium during menstruation, which may alter the drug absorption properties [197,198].

Vaginal dosage forms should ideally be easy to administer, requiring fewer doses, and patient-compliant. However, it is challenging to formulate small water-soluble drugs through the vaginal route. Hence, Meng et al. prepared spray-dried microparticles (MPs) using thiolated-CS coated SA by the layer-by-layer method for the vaginal delivery of HIV microbicides. They were studied for their cytotoxicity and pre-clinical safety on human vaginal (VK2/E6E7) and endocervical (End1/E6E7) epithelial cell lines and in vivo on female mice. The outcomes showed that TCS-coated Ag-based multilayer microparticles had 20–50 fold more adhesion than native AGMPs [199]. Hence, the developed formulation could be useful for vaginal drug delivery.

Urogenital infections affect about a billion women worldwide. It is caused due to *Escherichia coli* and other enterobacteria commonly found in the vaginal tract. The chances of UTIs are increased in post-menopausal women, which account for 25% of all bacterial infections. Cystitis, the most common UTI, usually affects young women, with *E. coli* being the cause of most diseases. The treatment of UTIs is still a challenge because of frequent recurrence, the association of co-morbidities, and high prevalence. Cefixime, a third-generation cephalosporin, is usually given for such infections. However, its poor water solubility, limited oral bioavailability, and incomplete absorption limit its use. Hence, Maestrelli et al. developed a bioadhesive vaginal dosage form of cefixime that can overcome many drawbacks. They prepared CS-coated CA microspheres using an ionotropic gelation method, and their mean weight, diameter, %EE, and loading capacity were evaluated. All MS batches showed prolonged adhesion greater than 2 h on the excised porcine vaginal mucosa; %EE enhanced with increasing drug concentration. In vitro studies revealed the direct relationship between CFX drug release and % inhibition in *E. coli* metabolic activity [200]. Thus, the overall results demonstrated the developed system as an auspicious candidate for vaginal delivery of actives.

Despite having several benefits, for example, enormous surface area, evasion of the first-pass metabolism, and the ability to optimize drug absorption for systemic effects, it also has certain drawbacks, such as the restoration action on vaginal fluids and its acidic environment (pH 4.0–4.5), which hampers the local delivery of drugs due to low residence time and stability. Hence, Ferreira et al. formulated CA hydrogels based on the earlier formation of polyelectrolyte complexes (PECs) for the vaginal delivery of polymyxin B. First, PECs were formed between ALG and PMX, ensued by cross-linking with calcium chloride. The aforementioned system displayed a pore size of between 100–200 µm and adequate syringability; in vitro tests demonstrated mucoadhesiveness. The drug release was found to be pH-dependent with a sustained release of six days. A burst release was noticed at pH 7.4, and the drug was released by anomalous transport. At pH 4.5, actives release followed the Weibull model and actives transport was through Fick's diffusion [201]. Thus, the formulated system could be suitable for vaginal drug delivery.

Vulvovaginal candidiasis caused by the fungus Candida albicans is generally treated using azole antifungals like fluconazole given orally. However, fluconazole is reported to have severe side effects like nausea, vomiting, diarrhea, and abdominal pain [202,203]. Hence, Darwesh et al. developed vaginal inserts using PEC based on anionic ALG and cationic CS. The mucoadhesion was highest for Na–Alg-based vaginal inserts (pKa 3.21) as compared to carbopol (pKa 5.0) because the mucoadhesion depends on the no. of hydrogen bonds (-OH, -COOH) concerned in the mucoadhesion interaction. The ALG: CS (5:5) PEC revealed controlled release of fluconazole (RE6h ranged from 56.46 ± 3.42 to 79.38 ± 3.42%), good mucoadhesion, and therefore suitable vaginal retention. Moreover, it demonstrated excellent antifungal action against Candida albicans both in vitro (MIC for fluconazole vaginal inserts was 31 ± 0.4 mm and that of fluconazole solution was 22 ± 0.4 mm) and in vivo (complete healing after seven days in rats) with reduced inflammatory cells [204]. Thus, the overall results depicted that the formulated system could be a promising candidate for vaginal drug delivery.

In another investigation, Soliman et al. developed an in situ thermosensitive bioadhesive gel for the vaginal delivery of sildenafil citrate as a prospective therapy for endometrial thinning caused by the administration of clomiphene citrate for ovulation initiation in women with type II gonadotrophic anovulation. They were developed using various grades of Pluronic® (PF-68 and PF-127) grades, into which mucoadhesive polymers such as SA and hydroxyethylcellulose were incorporated in different concentrations. The thermosensitive gels were developed by the cold method. The acceptable range of Tsol-gel of 28–37 ◦C was achieved by decreasing PF-127 concentration and modulating the addition of PF-68. There was increased gel viscosity and mucoadhesive force when the concentration of Pluronic® was raised, but there was a reduction in drug release rate during in vitro evaluation in a standard semi-permeable cellophane membrane at pH 4.5 using citrate buffer to mimic the vaginal fluid. Due to its rapid swelling property, SA aided the formation of adhesive interaction between ALG and mucosa, which led to increased mucosal retention. The in situ gels substantially enhanced endometrial thickness and uterine blood flow, thus potentially improving conception chances in anovulatory women with clomiphene citrate failure [205].

Researchers also synthesized ALG/CS/P4/Pluronic® F-127/progesterone [206], CS/ SA/polycarbophil/metronidazole [207], SA/CS/*α*,*β*-glycerophosphate/Bletilla striata/ tenofovir [208], SA loaded with anise/fluconazole β-cyclodextrin inclusion complexes [209], CS/ALG/metronidazole [210], HPMC/SA/abacavir [211], dextran/ALG nanofibers/ clotrimazole [212], Polaxomer 407/SA/*Lactobacillus crispatus* [213], ALG/CMC/clove essential oil [214], and ALG/CS/metronidazole [215] formulations which enhanced the vaginal drug delivery.

#### *6.5. Nasal Drug Delivery*

Nasal actives delivery includes the inhalation of drugs into the extremely vascularized mucosal layer of the nasal epithelium, which ultimately reaches the systemic circulation [216]. The nose has an enormous surface area for actives absorption, quicker onset of action, and evasion of first-pass metabolism [217]. The curative action of the majority of the drugs is impacted owing to the BBB's existence. Therefore, nasal route delivery favors therapeutics straightaway reaching the central nervous system (CNS). Furthermore, nasal delivery of actives shows benefits from brain-targeting, fewer side effects, and easy administration [218].

Rhinosinusitis, inflammation of the nasal cavities characterized by nasal polyp growth, typically extending beyond the nasal valve, is generally treated using nasal corticosteroids since oral ones show systemic side effects. However, its long-term use causes an increase in intraocular pressure and changes in serum cortisol levels. Hence, Dukovski et al. formulated dexamethasone-loaded lipid/ALG nanoparticles dispersed in pectin solution based on an in-situ gelling process that undergoes a sol-gel transformation after contact with the Ca2+ ions in the nasal mucosa. The in vitro biocompatibility experiments utilizing colon

carcinoma Caco-2 cell lines suggested no effect on cell viability; however, in-vivo studies are required to confirm this observation [219].

It becomes feasible to administer vaccines in dry powder form during influenza outbreaks due to their ease of transport, patient compliance, and stability. As compared to conventional vaccines, dry powder vaccines provide both humoral and cellular immunity adequately. Thus, Dehghan et al. formulated ALG NPs using the ionotropic gelation method encapsulating whole inactivated influenza viruses and administered them to rabbits, which showed significantly higher IgG titers when short single-stranded cytosine triphosphate and guanine triphosphate-containing synthetic oligodeoxynucleotides (CpG ODN) were used as the adjuvant (as exhibited in Figure 8c,d). ALG NPs released 65.87% of CpG ODN, 35.68% of QS, and 34.00% of influenza antigen within 4 h (as displayed in Figure 8a). In addition, the XTT test demonstrated safety for in-vivo applications (as shown in Figure 8b), making the formulation useful for nasal drug delivery [220]. *Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 24 of 60

**Figure 8.** Preparation, characterization, and immunological evaluation of ALG nanoparticles loaded with whole inactivated influenza virus: Dry powder formulation for nasal immunization in rabbits (**a**) % of viral protein, QS, and CpG ODN released in vitro from ALG NPs over four hours. (**b**) After 2‐ and 24‐h exposure with varying concentrations of each formulation, the effect of ALG NPs and influenza virus suspension on cell viability in Calu‐6 cell lines (**c**) HAI antibody titers in each vac‐ cination group on day zero (control), day 45 (after prime dose), day 60 (after the second dose), day 75 (after the third dose), and day 90 (after the final booster) (**d**) IgG titers in blood samples taken from each vaccinated cohort on days zero (negative control), 45 (prime dose), 60 (second dose), 75 (third dose), and 90 (after the final booster). \* means *p* < 0.05, \*\* means *p* < 0.01, \*\*\* means *p* < 0.001. Reproduced with permission from [220], Copyright Elsevier 2019. In a different study, Rao et al. formulated the anti‐Parkinson drug Ropinirole as a **Figure 8.** Preparation, characterization, and immunological evaluation of ALG nanoparticles loaded with whole inactivated influenza virus: Dry powder formulation for nasal immunization in rabbits (**a**) % of viral protein, QS, and CpG ODN released in vitro from ALG NPs over four hours. (**b**) After 2- and 24-h exposure with varying concentrations of each formulation, the effect of ALG NPs and influenza virus suspension on cell viability in Calu-6 cell lines (**c**) HAI antibody titers in each vaccination group on day zero (control), day 45 (after prime dose), day 60 (after the second dose), day 75 (after the third dose), and day 90 (after the final booster) (**d**) IgG titers in blood samples taken from each vaccinated cohort on days zero (negative control), 45 (prime dose), 60 (second dose), 75 (third dose), and 90 (after the final booster). \* means *p* < 0.05, \*\* means *p* < 0.01, \*\*\* means *p* < 0.001. Reproduced with permission from [220], Copyright Elsevier 2019.

thermoreversible in situ nasal gel. The rationale for the nasal delivery of Parkinson's pa‐ tients is that it becomes difficult forthem to swallow oral solid dosage forms due to muscle rigidity. It was prepared by the cold method using PF 127 and HPMC K4M as the ther‐ moreversible polymer. They observed an astounding five‐fold enhancement in the bioa‐ vailability of ropinirole in contrast to intravenous drug delivery. Furthermore, in vitro evaluation of sheep nasal mucosa demonstrated that the in situ gel had a more protective impact on nasal mucosa than the plain drug, which caused mucosal damage [221]. In a different study, Rao et al. formulated the anti-Parkinson drug Ropinirole as a thermoreversible in situ nasal gel. The rationale for the nasal delivery of Parkinson's patients is that it becomes difficult for them to swallow oral solid dosage forms due to muscle rigidity. It was prepared by the cold method using PF 127 and HPMC K4M as the thermoreversible polymer. They observed an astounding five-fold enhancement in the bioavailability of ropinirole in contrast to intravenous drug delivery. Furthermore, in vitro

In another investigation, Youssef et al. developed SA nanoparticles for the anti‐mi‐ graine drug Almotriptan utilizing the w/o/w double emulsion solvent evaporation proce‐ dure. It is a water‐soluble drug; hence, SLNs were prepared to help pass the drug through

dium CMC, and carbopol. In addition, the gelling temperature, gelling time, viscosity, gel strength, pH, %EE, and in vitro mucoadhesion were evaluated [222]. The overall results

Vaccines typically include adjuvant substances to enhance the humoral or cellular response to an antigen. The most common antigen, aluminum, instigates a Th2 antibody response; hence, aluminum‐based vaccines are unsuitable for intracellular pathogens and

demonstrated the prepared formulation to be suitable for nasal drug delivery.

evaluation of sheep nasal mucosa demonstrated that the in situ gel had a more protective impact on nasal mucosa than the plain drug, which caused mucosal damage [221].

In another investigation, Youssef et al. developed SA nanoparticles for the antimigraine drug Almotriptan utilizing the w/o/w double emulsion solvent evaporation procedure. It is a water-soluble drug; hence, SLNs were prepared to help pass the drug through the lipophilic BBB. Poloxamer 407 was used as a stabilizer and evaluated for its physicochemical properties by combining various mucoadhesive polymers, for example, SA, sodium CMC, and carbopol. In addition, the gelling temperature, gelling time, viscosity, gel strength, pH, %EE, and in vitro mucoadhesion were evaluated [222]. The overall results demonstrated the prepared formulation to be suitable for nasal drug delivery.

Vaccines typically include adjuvant substances to enhance the humoral or cellular response to an antigen. The most common antigen, aluminum, instigates a Th2 antibody response; hence, aluminum-based vaccines are unsuitable for intracellular pathogens and chronic diseases. Currently, usable vaccines through the intramuscular (i.m.) or subcutaneous (s.c.) routes fail to elicit a mucosal immune response. Nasal immunization, a relatively simple, non-invasive route, can provide mucosal immunity [223–227]. However, an acceptable mucosal immune response depends on the mucociliary clearance, the tolerogenic character of the mucosal epithelium, and the huge size of the antigen. To circumvent this problem, the antigens can be incorporated into mucoadhesive polymeric NPs, extending the duration of antigen residence and conferring protection against enzymes [228,229]. Hence, Mosafer et al. developed SA-coated CS and trimethyl CS nanoparticles incorporated with the PR8 influenza virus for nasal administration. The zeta potential was −29.6 mV. The vaccine was evaluated in BALB/c mice, wherein the PR8-TMC-ALG formulation manifested a more excellent IgG2a/IgG1 ratio than PR8-TMC, PR8-CHT, and PR8-ALG. Thus, ALG-NPs could be used as immunoadjuvants for nasal immunization. The ALG-coated NPs generated an excellent immune reaction compared to uncoated NPs [230].

The recent nasal formulations include SA/Polaxomer 407/gellan gum/timosaponin BII [231], SA in situ gels based on agomelatine (AGM) [232], ALG based magnetic short nanofibres 3D composite hydrogel encapsulating human olfactory mucosa stem cells [233], OA-dopamine conjugate [234], ALG nanoparticles/venlafaxine (VLF-AG-NPs) [235], *Mycobacterium bovis* Bacille Calmette-Guérin (BCG)-loaded microparticles using ALG/CS [236], ALG/CS/attenuated Androctonus australis hector (Aah) venom [237], CS/ALG nanoparticles/SpBMP-9 (growth factor) [238], ALG/trimethyl CS liposomes/lipopeptide subunit vaccine [239], SA microspheres/*Lactobacillus casei* [240].

#### *6.6. Transdermal Drug Delivery*

Transdermal drug delivery relates to the delivery of actives via the skin for systemic or local absorption. They are advantageous over the conventional administration routes due to their capability to deliver controlled release of drugs, reducing the first-pass metabolism, less systemic side effects, effective control of drug plasma profile, and patient compliance [241]. However, its applications are limited owing to the extensive skin hindrance, particularly the stratum corneum. Consequently, few actives can penetrate the skin and reach the blood at a therapeutic concentration [242].

Insulin is usually given hypodermically via s.c. injection for the therapy of diabetes mellitus. But it generates biohazardous waste; furthermore, patient compliance is also a challenge, especially in children. Several approaches have come into the picture to resolve the issue, such as inhalational microparticles, oral administration of nanoparticles, and needle-free high-pressure injection systems [243–245]. Among these, transdermal patches have attracted much attention owing to minimal pain and tissue injury [246,247]. In this context, Yu et al. fabricated a dissolving polymer microneedle patch comprising 3-aminophenyl boronic acid-modified ALG (ALG-APBA) and HA that can quickly dissolve in the interstitial fluid of the skin. Alginate was chemically modified by adding 3-aminophenyl boronic acid to form ALG-APBA, and HA was crosslinked to form MNs in the presence of Ca2+ ions. The MNs thus prepared were evaluated for their mechanical strength, degradation, ex vivo skin insertion, stability of insulin, in vivo transdermal delivery to SD rats, and pharmacokinetic and pharmacodynamic activity in SD rats. In addition, the resistance of MNs to static and dynamic forces was calculated, and it was found that no change in the tips was observed even after the addition of 10–100 g weight on the microneedle patch, with no breaks in the needle after the addition of 500 g, demonstrating its excellent mechanical strength. The encapsulated insulin has comparable pharmacological activity to an s.c. injection with the same insulin dose, with RPA and RBA at 90.5 ± 6.8% and 92.9 ± 7.0%, respectively [248]. Overall results made the aforementioned system advantageous for nasal drug delivery.

In a different investigation, Lefnaoui et al. fabricated transdermal films based on ALG-CS PECs for the antiasthmatic drug ketotifen fumarate (KF). Polyethylene glycol was used as a plasticizer, and Span 20, and Tween 80 were utilized as permeability enhancers. The films were prepared by the film casting method and evaluated for their uniformity in weight, thickness, folding endurance, loss of moisture, and absorption of moisture. Furthermore, actives release and permeation through the rat abdomen mounted on the Franz diffusion cell were characterized. The transdermal films made by a 1:1 ratio of CS and ALG yielded smooth, flexible, strong, bioadhesive, biocompatible films. The drug release research showed that the KF was released in a controlled way over a long period (99.88% release after 24 h) to treat asthma, allergic rhinitis, and conjunctivitis [249]. The results revealed that the prepared formulation is favorable for transdermal drug delivery.

In another study, Abebeet et al. developed a self-adhesive hydrogel for strainresponsive transdermal delivery using gallic acid (GA) modified ALG as the mucoadhesive polymer. The model drug, caffeine, was encapsulated in the hydrogels prepared by the one-pot synthesis method. The diffusion kinetics were controlled by Fickian diffusion. The developed hydrogels had a 25% increase in tensile strength and twice the transdermal release of the drug. A powerful adhesion of 100 kPa was reported on a glass substrate. A close to 800% strain was observed, and it was attributed to the free movement and adhesion of ALG and polyacrylic acid. It could withstand body movement as well as skin stretching when tested on human skin. Thus, GA hydrogel seems to be a promising route for strain-controlled TDD [250].

In another investigation, Abnoos et al. prepared a CS-ALG nanocarrier for the transdermal delivery of pirfenidone (PFD) in idiopathic pulmonary fibrosis, a disease characterized by progressive dyspnea and pulmonary function loss [251,252]. CS-SA nanoparticles were developed by the pre-gelation technique, and the drug Pirfenidone, an anti-inflammatory and antifibrotic agent, was encapsulated with 94% efficiency. The prepared nanoparticles were evaluated using SEM, TEM, and DLS. These studies showed that particle morphology was spherical with an average size of 80 nm. FTIR spectra observed the complex formation between CS and ALG. The drug release studies show that PFD had undergone an initial burst release of 12% after 5 h and was later released in a sustained manner for 24 h. Ex vivo studies revealed that skin permeation of PFD was improved by using CS-SA nanoparticles compared to standard PFD solution. The permeation was also observed through fluorescent microscopic images labeled with FITC. SEM studies reveal that the nanoparticles could remain stable for six months [253]. Thus, the results proved that the prepared formulation is useful for transdermal drug delivery.

In another investigation, Anirudhan et al. formulated bio-polymer matrix films acquired from CMC, SA, and PVA to transdermally deliver diltiazem, a calcium channel blocking drug used for cardiac failure. The oral bioavailability of DTZ is only 30–40%. Hence, alternative routes of drug delivery are being explored. Since diltiazem (DTZ) is hydrophilic in nature, a hydrophilic matrix consisting of polyethylene glycol coated vinyl trimethoxy silane-g-CS (PEG@VTMSg-CS) with matrices like Na-ALG, CMC, and PVA was developed. A dispersion of the matrix was prepared to avoid the undesirable effect of shrinkage of the polymer network when the drug is being eluted out of the matrix. The drug release studies were based on Franz diffusion cells. SA films showed more significant swelling than PVA films. The ALG films displayed a thickness value of

0.052 <sup>±</sup> 0.01 mm and a water permeation of 0.07 g cm2/24 h. DTZ permeation was less due to less film thickness. The in vitro skin penetration research of DTZ on rat skin revealed the effectiveness of the films with more than 49% viability in HaCaT and PBMC cell lines with no histological alterations on the skin. The present formulation could deliver 70% of the drug in 24 h [254]. Thus, the overall results demonstrated that the system is promising for transdermal drug delivery.

The currently investigated formulations include ALG/PVA/ciprofloxacin electrospun composite nanofibers [255], SA/propylene glycol/metoclopramide films [256], SA/ glucosamine sulphate [257], ALG/maltose composite microneedles/insulin [258], ALG/CS/ rabeprazole [259], oleic acid/SA/Na CMC/2,3,5,40-tetrahydroxystilbene 2-O-β-D-glucoside (THSG) [260], SA/CS/piplartine [261], SA/polyvinyl alcohol/quercetin [262], ALG/Hidrox-6/Hydroxytyrosol [263], sodium L-cysteine ALG/isopropyl myristate/ropinirole hydrochloride [264], ALG coated aminated nanodextrin/CS coated folate decorated aminated β-CD nanoparticles/curcumin/5-flurouracil [265] which intensified transdermal drug delivery.

### *6.7. Mucosal Drug Delivery*

In order to evade parenteral routes and beat biological impediments, one more DDS, specifically the mucosal delivery system, appeared in the limelight. Mucus gel layers line the vagina, lungs, and gastrointestinal tract (GIT). GIT represents a complicated condition, including several digestive enzymes and a changeable pH (acidic pH-stomach and basic pH-intestine). Different drugs necessitate being guarded against the circumstances referred to above to evade degradation by proteolytic enzymes. These pharmaceuticals exhibit inadequate permeability in gastric mucosa and also bear the first-pass effect. To preserve the actives of interest facing such conditions, ALG serves as one of the most satisfactory polymers with mucoadhesive characteristics and improved permeation results, and acts as a defensive envelope for such actives through mucosal delivery [266]. Mucoadhesion refers to the capability of materials to bind to the mucosal membrane and offer some retention. Mucoadhesive drug delivery provides various benefits over other routes of administration, for example, faster distribution to the local blood vessels, avoidance of first-pass metabolism, reduced dose frequency, and rapid relief [267]. Thus, the formulated system might be helpful for mucoidal drug delivery.

Oral cancer, one of the most common cancers, is usually treated with surgery and chemotherapy [268], but they have the drawback of alteration of facial morphology followed by surgical procedures. Hence, to provide an alternative approach, Shtenberg et al. formulated a mucoadhesive ALG/liposome paste. DOX-loaded liposomes were prepared, and ALG-fluorescein was synthesized using FITC and ethylenediamine; on homogenization, a cross-linked hybrid paste was formed. It showed the liposome's sustained release from ALG cross-linked paste (release index of 8). Furthermore, in vitro toxicity studies on human cell lines acquired from tongue SCC (CAL 27) showed viability of 15% after 48 h. Thus, polymers released from the liposomes were active and effective. Therefore, this approach could be used for potential oral cancer therapy [269].

Oral mucosal vaccine delivery has the disadvantage of significant enzymatic degradation of the antigen in the GI tract, and these have difficulty in uptaking from the intestine. To overcome this issue, HbsAg-loaded CS nanoparticles with an ALG coating were prepared by Saraf et al., in which lipopolysaccharides acted as an adjuvant. It showed elevated levels of sIgA in intestinal (3.32 mIU/mL) secretions compared to non-ALG coated HB-CNPs (as displayed in Figure 9c). Thus, ALG-coated CS NPs (CNPs) could entrap HbsAg effectively (as shown in Figure 9b). The release profile of protein showed that 84% of the drug was released at 0.5 h. owing to CNPs' loose binding that led to increased desorption at the acidic pH, but 95.5% of the drug was released at 48 h (as displayed in Figure 9a). Coating with ALG stabilized increased the stability of CNPs and provided a sustained release of 51.42% after 48 h. In addition, the ALG-coated NPs adhered better than uncoated NPs. The cell viability of RAW 264.7 cell lines was 70.45%; nevertheless, the cell viability decreased

to 66.45% after 48 h. This time-dependent decrease in cytotoxicity could be due to contact between positively charged nanoparticles and negatively charged membranes. Mucosal M-cell-specific ALG-coated CS nanoparticles elicited a significant immunological response in mice (as demonstrated in Figure 9d). Hence, overall results showed the aforementioned system is favorable for mucosal drug delivery [270]. *Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 28 of 60

**Figure 9.** Lipopolysaccharide‐derived ALG coated Hepatitis B antigen‐loaded CS nanoparticles for oral mucosal immunization (**a**) Release profile of produced nanoparticles in vitro. (**b**) HBsAg release as determined by SDS‐PAGE: Lane 1: Molecular weight markers; Lane 2: HBsAg solution; Lane 3: HBsAg loaded CS nanoparticles, Lane 4: HBsAg‐loaded ALG coated CS nanoparticles produced from LPS. (**c**) The levels of sIgA in the fluid secretions of mice immunized with different formula‐ tions. (**d**) Anti‐HBsAg IgG levels in mice inoculated orally with various formulations. Reproduced with permission from [270], Copyright Elsevier, 2020. **Figure 9.** Lipopolysaccharide-derived ALG coated Hepatitis B antigen-loaded CS nanoparticles for oral mucosal immunization (**a**) Release profile of produced nanoparticles in vitro. (**b**) HBsAg release as determined by SDS-PAGE: Lane 1: Molecular weight markers; Lane 2: HBsAg solution; Lane 3: HBsAg loaded CS nanoparticles, Lane 4: HBsAg-loaded ALG coated CS nanoparticles produced from LPS. (**c**) The levels of sIgA in the fluid secretions of mice immunized with different formulations. (**d**) Anti-HBsAg IgG levels in mice inoculated orally with various formulations. Reproduced with permission from [270], Copyright Elsevier, 2020.

In another investigation, Ghumman et al. utilized linseed mucilage‐ALG mucoad‐ hesive microspheres loaded with Metformin HCl developed by an ionotropic gelation procedure for mucosal actives delivery. Drug encapsulation efficiency was up to 92% with sustained release for 12 h. The results revealed that an optimized formulation (FM‐4) could furnish sustained release for up to 12 h and hold the level of blood glucose by reg‐ ulating and enhancing the absorption of metformin systemically. Therefore, LSM, a natu‐ ral emerging mucoadhesive agent, proved to be preferable for controlled release mucoad‐ hesive microspheres designed for oral utilization [271]. In periodontitis, there is inflammation and destruction of tooth tissues, and the path‐ In another investigation, Ghumman et al. utilized linseed mucilage-ALG mucoadhesive microspheres loaded with Metformin HCl developed by an ionotropic gelation procedure for mucosal actives delivery. Drug encapsulation efficiency was up to 92% with sustained release for 12 h. The results revealed that an optimized formulation (FM-4) could furnish sustained release for up to 12 h and hold the level of blood glucose by regulating and enhancing the absorption of metformin systemically. Therefore, LSM, a natural emerging mucoadhesive agent, proved to be preferable for controlled release mucoadhesive microspheres designed for oral utilization [271].

ogen forms a biofilm around the inflamed tissues. Currently, treatment approaches in‐ clude mechanical removal of the biofilm aided by antibiotics whose systemic side effects limit the effectiveness of treatment. Hence, Kilicarslanet al. used clindamycin phosphate‐ loaded ALG/CS PEC film to overcome this issue. CS and ALG, being oppositely charged biopolymers, form a PEC by cross‐linking with each other. It was observed that increased ALG concentrations in the polymer mixture increased adhesiveness, making it valuable for mucosal drug delivery [272]. In another investigation, Gonçalves et al. developed highly porous ALG/carrageenan In periodontitis, there is inflammation and destruction of tooth tissues, and the pathogen forms a biofilm around the inflamed tissues. Currently, treatment approaches include mechanical removal of the biofilm aided by antibiotics whose systemic side effects limit the effectiveness of treatment. Hence, Kilicarslanet al. used clindamycin phosphateloaded ALG/CS PEC film to overcome this issue. CS and ALG, being oppositely charged biopolymers, form a PEC by cross-linking with each other. It was observed that increased ALG concentrations in the polymer mixture increased adhesiveness, making it valuable for mucosal drug delivery [272].

aerogel nanoparticles for drug delivery of powdered model drugs quercetin and keto‐ profen. Biopolymer aerogels have low toxicity and are biocompatible, with large surface In another investigation, Gonçalves et al. developed highly porous ALG/carrageenan aerogel nanoparticles for drug delivery of powdered model drugs quercetin and ketoprofen.

areas with accessible pores or drug loading. The emulsion gelation method was used to produce the hybrid biopolymer nanoparticles, followed by drying using supercritical

Biopolymer aerogels have low toxicity and are biocompatible, with large surface areas with accessible pores or drug loading. The emulsion gelation method was used to produce the hybrid biopolymer nanoparticles, followed by drying using supercritical CO2. Pectin and ALG had better interaction, with an increased degree of cross-linking between the two polysaccharides giving NPs of higher specific surface area (>300 m<sup>2</sup> g –1) and lower shrinkage. The drug-loading capacity was 17 ± 2% for ketotifen and 3.8% for quercetin. The cytotoxicity test using the MTS assay on human carcinoma Caco-2 cell line showed almost 100% cell viability, thus demonstrating the prepared nanoparticles to be highly non-toxic. The drug release from ALG/κ-carrageenan aerogel was quicker than from ALG/pectin [273]. Thus, the developed system was found to be suitable for drug delivery.

In another research, Martín et al. developed effective antifungal mucoadhesive drug delivery systems to treat oral candidiasis using ALG microspheres containing the drug nystatin. Oral candidiasis is caused by the opportunistic pathogen Candida albicans, especially in immunocompromised, diabetic patients undergoing chemotherapy. Nystatin, a polyene antifungal antibiotic, is indicated to treat mucocutaneous fungal infections caused by C. albicans. However, nystatin possesses a huge lactone ring and numerous double bonds, which render it amphiphilic and amphoteric and thus render its formulation difficult. Consequently, it has been formulated as micellar gels, liposomes, intralipids, niosomes, and various other dosage forms [274–276]. Thus, Martin et al. developed nystatin microspheres based on the emulsification/internal gelation procedure with some modifications [277]. Evaluation of particle size, zeta potential, swelling behavior, loading content, encapsulation efficiency, rheology, mucoadhesive force, drug release studies, antimicrobial activity, and in vivo studies were carried out. It was found that the mean size of the microspheres was 85–135 um, and the viscosity was that of a Newtonian fluid. The drug release was in two steps; first, an initial burst release was followed by a more sustained drug release, with about 60% of the drug released in the first 2 h. The prepared microspheres showed marked antifungal activity with a drastic reduction in C. albicans colonies. In vivo research on the buccal mucosa of experimental animals demonstrated that the retained amount, which is 4 to 6 times higher than the MIC, was enough to elicit the therapeutic response [278].

Researchers have also formulated lysozyme mucoadhesive tablets/Ca2+ cross-linked ALG with HPMC [279], silybin/nanocrystals-in-microspheres PEC/ALG/CS [280], co-delivery of ketorolac and lidocaine/polymeric wafers/2:1 SA:PVP −25 and 10% glycerol [281], HPMC/ SA/nicotine [282], ALG modified with maleimide-PEG/ibuprofen sodium [283], silicone sheet/dexamethasone/ALG [284], ALG/sterculia gum/citicoline [285], ALG/ghatti gum/ montmorillonite/flubiprofen [286], pectin/ALG/repaglinide [66], polyacrylamide-g/locust bean gum/ALG/ketoprofen [287], CS/CA/*Lactobacillus casei* [288] for improved mucosal actives delivery.

#### *6.8. Intravenous Drug Delivery*

ALG, owing to its biocompatibility and safety in vivo, has been explored for intravenous drug delivery in order to increase the bioavailability of drugs that the conventional oral route cannot deliver.

Hydrophobic medicines can be entrapped in hydrophilic nanoparticles like CA and delivered to their site of action. Curcumin and resveratrol are both polyphenolic molecules found in nature that have anti-cancer properties. Their low water solubility and bioavailability unfortunately limit their therapeutic utility. In a study by Saralkar et al., the emulsification and cross-linking procedures were used to make curcumin and resveratrol-based CA nanoparticles. Particle size, zeta potential, moisture content, physicochemical stability, and %EE were all measured in the nanoparticles. For the combined estimation of curcumin and resveratrol, the UPLC methodology was designed and validated. The in vitro efficiency of ALG nanoformulation on DU145 prostate cancer cells was investigated. Curcumin and resveratrol had entrapment efficiencies of 49.3 ± 4.3% and 70.99 ± 6.1%, respectively. In 24 h, resveratrol had a greater release than curcumin (87.6 ± 7.9% against 16.3 ± 3.1%). Curcumin, both in solution and as nanoparticles, was found to be taken up by cells. Resveratrol

was poorly absorbed by cells. On DU145 cells, the drug-loaded nanoparticles cause cytotoxicity. The drug solution was more hazardous than nanoparticles at high concentrations. The intravenous administration of the ALG nanoformulation was proven to be safe [289].

Over the last 10 years, multifunctional theranostics have created some intriguing novel possibilities for chemotherapy and tumor detection. In a study by Yang et al., the photosensitizer chlorin e6 (Ce6) and the anticancer medication DOX were subsequently adsorbed onto the magnetic mesoporous silica nanoparticles (M-MSNs) to produce a pH-sensitive drug release and for adsorbing P-glycoprotein short hairpin RNA (P-gp shRNA) for preventing multidrug resistance, ALG/CS polyelectrolyte multilayers (PEM) were constructed on the M-MSNs (M-MSN(DOX/Ce6)/PEM/P-gp shRNA). Upon laser illumination, the nanoparticles with a mean diameter of 280 nm showed a pH-responsive drug release profile and increased singlet oxygen formation in tumor cell lines. The delivery mechanism only released 12 percent Ce6 after 30 h at pH 7.4 but >95 percent after 36 h at pH 4.0. In terms of DOX release, roughly 30% of it was released from M-MSN(DOX/Ce6)/PEM nanocomposites in 32 h at physiological pH (7.4), compared to a 46% release of DOX at pH 4.0. The multifunctional nanocomplexes greatly boosted apoptosis in vitro, as demonstrated by the CCK-8 assay and calcein-AM/PI co-staining. Using cancerous Balb/c mice for the animal studies, researchers used a combination of photodynamic treatment and chemotherapy to achieve a synergistic anti-cancer activity in vivo. Additionally, the cores of the bifunctional Fe3O4-Au nanoparticles in the multifunctional nanocomplexes allowed dual-modal MR and CT imaging, revealing high uptake into tumor-bearing animals via intravenous injection. This study demonstrates the excellent performance of magnetic mesoporous silica nanocomposites as a multipurpose delivery system for imaging-guided cancer synergistic therapy [290].

For cancer treatment, nanocarrier drug delivery systems (NDDSs) have received more attention than traditional drug delivery methods. The rapid evacuation of activated macrophages from the bloodstream, however, hinders efficacy. In a study by Wang et al., glycyrrhizin (GL) was loaded into ALG nanogel particles (NGPs) to create a versatile delivery mechanism to reduce activated macrophage clearance and improve anticancer activity with GL and DOX combined therapy. GL-ALG NGPs could not only prevent eliciting macrophage immuno-inflammatory responses, but they could also reduce macrophage phagocytosis. DOX/GL-ALG NGPs increased DOX bioavailability by 13.2 times compared to free DOX in the blood. The use of mice with normal immune systems instead of nude mice in the construction of tumor-bearing mice further revealed that NGPs are biocompatible. In vitro and in vivo, GL-mediated ALG NGPs had an exceptional hepatocellular carcinoma targeting effect [291].

Arsenic trioxide (ATO) is efficacious in managing acute promyelocytic leukemia (APL) and late-stage primary hepatic cancer, although it has serious adverse effects. Hence, Lian et al., in order to address these issues, synthesized red blood cell membrane-camouflaged ATO-loaded SA nanoparticles (RBCM-SA-ATO-NPs, RSANs). Ion crosslinking was used to make ATO-loaded SA nanoparticles (SA-ATO-NPs, SANs), wherein RBCM was deposited over the surface to make RSANs. RSANs had a mean particle size of 163.2 nm with an entire shell-core bilayer arrangement and a 14.31 percent encapsulation efficiency. When relative to SANs, a decreased phagocytosis in RAW 264.7 macrophages was observed RSANs by 51%, and the in vitro cumulative release rate was 95% at 84 h, indicating a notable sustained release. Moreover, RSANs were found to have reduced cytotoxicity than natural 293 cells and to have anti-cancer activity on both NB4 and 7721 cells. In vivo investigations also revealed that ATO can induce minor organ damage, whereas RSANs can minimize toxicity and boost anti-tumor efficacy [292].

Curcumin administration by nanocarriers is an appealing strategy for overcoming its limited bioavailability and rapid metabolism in the liver. Karabasz et al. developed AA-Cur, a blood-compatible ALG-curcumin combination that produced colloidally stable micelles of around 200 nm and displayed high cytotoxic effects against mouse cancer cell lines, as previously demonstrated. In their study, they investigated AA-toxicity and

anticancer efficacy in two different animal tumor models. In the first study, C57BL/6 mice were administered with colon cancer MC38-CEA cells subcutaneously. Breast tumor 4T1 cells were administered orthotopically, that is, into the mammary adipose tissue of BALB/c mice in the second study. Investigations of blood biochemistry, histology, morphology, DNA integrity (comet assay), and cytokine screening were used to assess the toxicity of intravenously injected AA-Cur (flow cytometry). The anticancer effects of AA-Cur were determined by comparing the development of colon MC38-CEA- or orthotopically injected breast 4T1 tumor cells in untreated and AA-Cur-treated mice. Four injection dosages of AA-Cur revealed no toxicity, showing that the conjugate is safe to use. The anti-cancer efficacy of AA-Cur was moderate in colon MC38-CEA and breast 4T1 carcinomas [293].

Other applications of ALG in intravenous drug delivery include ALG/deferoxamine conjugates [294], ALG Microparticles/Amphotericin B [295], ALG/poly(amidoamine)/ MC3T3-E1 pre-osteoblasts hybrid hydrogel [296], octanol grafted ALG nanoparticles/ propofol [297], pH-Responsive ALG/CS multilayer coating on Mesoporous Silica Nanoparticles encapsulating DOX [298], ALG-glycyl-prednisolone conjugate nanogel [299].

## *6.9. Others*

The buccal mucosa is an extremely appealing route of actives delivery for actives with low bioavailability, poor gastric stability, and vulnerability to first-pass metabolisms such as proteins and peptides, by carrying the actives straightaway into the bloodstream. The oral mucosa also lacks Langerhan cells, making the oral mucosa tolerant to various allergens [300]. Aphthous stomatitis, a type of inflammation in the intraoral cavity whose causative agent is still unknown, is currently treated symptomatically using corticosteroids. Ambroxol is gaining popularity as an upcoming agent to treat chronic inflammation. Laffleur and Küppers developed a buccal dosage form by anchoring sulfhydryl groups of the amino acid cysteine onto the ALG adhesive backbone, incorporating ambroxol as the antisecretory drug. Mucoadhesive studies show that ALG-SH had an 11.56-fold increase in adhesion time due to the binding of the sulfhydryl group to the cysteine-rich mucus glycoprotein, leading to prolonged residence time in comparison to the weak van der Waals and hydrogen bonding in native polymers. Permeation studies on freshly excised buccal mucosa showed a 1.89-fold increase in permeation of ALG-SH as compared to native ALG. The mechanism of permeation enhancement was by tyrosine kinase inhibition by disulfide bond formation between the sulfhydryl group of the polymer and the cysteine group of the protein. Ambroxol release from ALG-SH showed a 1.4-fold-controlled release compared to native ALG, possibly due to inter and intra-crosslinking formation, thus providing stability. Therefore, sulfhydryl-anchored ALG could be used for the effective therapy of aphthae [301].

Periodontal diseases affect the gums and bones of the teeth due to bacterial infections. The desirable properties of drug delivery systems for periodontal illnesses include low toxicity, biodegradability, and the ability to treat bacterial infections. Hence, Prakash et al. utilized a controlled-release formulation of amoxicillin using PVA/ALG/hydroxyapatite (HAp) films by wet precipitation to treat periodontal infections. SEM studies show HAp NPs were effectively blended and embedded with amoxicillin irrespective of the annealing temperature. The in-vitro analysis, cell viability assay (70%), fluorescent staining, and hemolysis assay provided conclusive evidence for the suitability of this composite film in treatment. It gave a sustained release, with 87% of the actives released by day 10. The swelling ratio was almost 80% for all films annealed at different temperatures. The tensile strength was more significant than the standard PVA/SA patch, and it increased with increasing annealing temperature. The fabricated films exhibit high anti-bacterial action against *Escherichia coli*, *Staphylococcus aureus*, *Enterococcus faecalis*, and *Pseudomonas aeruginosa*. The fabricated films are also highly biocompatible and hemocompatible, as evidenced by in vitro analysis, cell viability studies, fluorescent staining, and hemolysis assay. Annealing at different temperatures ranging from 300, 500, and 700 ◦C, respectively, gave films that can encapsulate and release the drug at different

extents. HAp also helps in the regeneration of damaged bone segments, in addition to its drug matrix properties. Thus, PVA/SA/HAp/amoxicillin films are good candidates for treating periodontal defects, orthopedic implants, and bone grafting [302].

The sublingual route of vaccine delivery is attracting a lot of attention because of the ease of self-administration of vaccines as well as an abundance of antigen-presenting cells in the sublingual mucosa. In contrast to intravenous and subcutaneous vaccines, sublingual vaccines confer mucosal immunity as well against pathogens like SARS, HIV, and HPV [303]. However, they suffer from a lack of adhesion and absorption through the sublingual epithelium. Hanson et al. prepared a biopolymer platform based on mucoadhesive ALG and CMC polymer wafers loaded with HIV gp14 protein for the delivery via sublingual route and protection of protein vaccines. The wafers were prepared by dissolving ALG:CMC polymers along with NaCl in deionized water. Microstructural analysis of the wafers revealed that CMC wafers had huge pores with thick strands, while the pores of ALG were smaller and smoother. This may be due to CMC being phased out of water during freezing while amorphous ALG chains were more flexible, hence more extensive entanglement. In addition, it was found that wafers with high ALG content showed high mechanical stability as well as protection from impairment owing to lyophilization and exorbitant heat, partly due to their network-forming capability and poor crystallinity. In contrast, a large number of CMC incorporated wafers were extremely mucoadhesive to sublingual mucosa tissue and could endure washing, leading to enhanced protein permeation into the tissue. Compared to liquid gp140 solution, the vaccines produced comparable T-cell and B-cell mediated immune reactions in mice and comparable IgA and IgG levels in blood, vagina, and saliva. The optimum formulation (CMC:ALG ratio of 1:1) could be safely stored and carried without a cold chain while also preserving its immunogenicity following vaccination in mice via a sublingual route. Thus, this novel platform could be used for the potential delivery of sublingual vaccines [304].

ALG-based formulations are presently in the limelight owing to their exceptional characteristics. They are researched meticulously to obtain the utmost benefits of drug delivery (shown in Tables 2 and 3).













#### **7. Recent Advances in ALG Formulations in Wound Healing**

Skin represents the largest human organ that functions as a protective barrier against dehydration, pathogens, environmental stresses, etc. [335]. Skin injuries can be acute or chronic and can occur in arterial insufficiency, diabetes mellitus, immunological disorders, and other infections. Rarely does complete re-epithelialization happen in repairing skin defects; hence, increased attention is diverted to preparing wound dressings to promote wound healing and reduce scars while also protecting from microbial infections and dehydration at the wound site [336,337].

Wound dressings play a vital part in wound management as they are applied to a variety of burns and wounds to aid in the repair and renewal of injured tissues, as well as to encourage healing and minimize the infection risk. The optimum wound dressing should decrease the recovery period and discomfort, increase tissue regeneration and recovery, absorb excess exudate from the wounds, encourage healing, and prevent infectious complications. For several decades, scientists have aspired to develop a specialized dressing to treat injuries. Traditionally, dressings are made from plant-based fibers, honey, animal fats, etc. Currently, biopolymeric materials are used as wound dressings as they offer unique properties, such as antibacterial, re-epithelializing, antioxidizing, and antiinflammatory properties, that significantly promote wound healing [338,339].

Recent developments in wound dressings also enable the release of therapeutic agents to restore skin homeostasis and integrity [336]. Biopolymers are attractive wound healing materials as they can maintain a moist wound bed due to water sorption; they can also absorb any tissue exudates and allow oxygen permeation across the wound [340]. Furthermore, because of the hydrophilic nature and structural features of these wound dressings, a sustained release of the encased bioactive substances could be achieved [341]. Both synthetic and natural polymers are useful for wound dressing, including heparin, CS, HA, dextrans, ALGs, and β-glucans, because of their desirable physical and biochemical properties [338,342].

ALG is a biopolymer widely used for tissue engineering applications, particularly in wound healing [343]. It has been used as a food additive for ages; hence, it is considered a biocompatible polymer. It has found applications in tissue regeneration and bioactive delivery because of its biodegradability and slow dissolution in the biological fluids when cross-linked with exchangeable cations. This rate could be adjusted by controlling the oxidation [344] and reducing ALG's molecular weight [345]. ALG composites are used for wound healing and soft tissue regeneration to strengthen the capacity and material characteristics of native ALG to adapt to different biomedical applications [346–348]. Calcium ions are released when water-insoluble CA encounters wound exudates as a function of calcium ions being replaced by sodium ions in bodily fluids, which can operate to achieve hemostasis. The SA-based fibers absorb a massive amount of exudates and transform into a gel-like substance, which maintains the moist barrier on the wound surface [349,350]. Some recent investigations of ALG composites in wound healing applications have been discussed in Table 4.

Diabetes-induced wounds currently have no effective treatment and thus represent a challenge in wound healing. Reports suggest that bacteria-caused inflammations due to the alkaline pH of ulcer wounds and incomplete blood flow to the wounds due to slow angiogenesis may be responsible for delayed diabetic wound healing. Hence, Wang et al. developed a novel, pH-sensitive CA-based hydrogel loaded with protamine nanoparticles and hyaluronan oligosaccharides (protamine NP/HAO CA hydrogel) by the ionotropic gelation method. The loading efficiency of HAO was 85.4 ± 6.25%, and 44.5% of the drug was released in 8 h at pH 3.0. At pH 8.0, which mimics the diabetic wound state, CA hydrogel swelled by the absorption of water, and a faster drug release of NPs and HAO took place. Compared to plain CA hydrogels, the protamine-loaded ones showed improved antibacterial activity against *E. coli* and *S. aureus*, enhanced adherence to wound tissue sites, and absorption of wound fluid into the hydrogels. Wounds closed with rates of up to 96.8% after 14 days without ulceration and festering at the wound site. Re-epithelialization was

observed, indicated by the formation of fibroblasts and collagen. Moreover, the composite hydrogels reduced inflammation and encouraged angiogenesis [351].

PECs produced from the polysaccharides ALG and CS at defined flow rates, pH, and agitation speeds give rise to slender, stable, and transparent sponges or films possessing excellent swelling ability in physiological medium. Unfortunately, these PECs have inadequate mechanical properties, which may negatively affect their performance in articulated regions such as knees, shoulders, and elbows. To overcome this, Pires et al. fabricated CS-ALG-based wound dressings with the addition of poly (dimethylsiloxane) to improve the flexibility and sorption capacity in contact with biological fluids. The optimum amount of poly(dimethylsiloxane) per gram of PEC was found to be 0.1 g, wherein the formulation exhibited high stability, a tensile strength of 12 MPa, non-hemolysis, and induction of thrombus formation. Moreover, when beta-carotene and thymol were loaded onto the formulations via the supercritical carbon dioxide impregnation/deposition (SSI/D) technique at 45 ◦C and 250 bar for 14 h, higher bioactive loading capacities were observed using SSI/D and a high depressurization rate (10 bar/min). A substantial amount of Thy and Bc were preserved in the matrix structure with the SSI/SD method, which functioned as a reservoir system. Thus, the combination of ALG, CS, and silicone gel could be a potentially successful wound dressing in the case of less articulated body sites and low extruding wounds [352].

The combination of ALG with proteins holds a lot of promise in enhancing the cellular interactions of ALG and for tailoring the biodegradation of the composite materials in tissue regenerating applications. Elastin, a highly flexible protein abundant in the extracellular matrix, is a good candidate for composites because it renders the tissues elastic enough to withstand continuous cycles of deformation/recovery without rupturing. Hence, Bergonzi et al. fabricated ALG/human elastin-like polypeptide (HELP) hybrid films by the solvent casting method with loaded curcumin to provide antioxidant activity. Strong intermolecular hydrogen bonding between the N-H amide bonds of HELP and the carboxylate group of ALG leads to close network formation due to molecular associations, thus giving higher tensile strength and Young's modulus, which is representative of its higher elasticity. Swelling capacity was higher in HALCur than in ALCur (as shown in Figure 10a). The incorporation of HELP was not only instrumental for obtaining a controlled release of curcumin (as depicted in Figure 10b), which helped in a higher antioxidant effect (as illustrated in Figure 10d), but also in enhancing the cytocompatibility of the final biomaterial, as shown in Figure 10c). With more in vivo studies, it may be possible to design customized bioplatforms for biomedical applications [353].

Full-thickness healing requires extensive healing time, which increases the risks of infections, wound ulcers, necrosis, and even fatal complications. To tackle this, a hybrid hydrogel composed of amine-functionalized fish collagen and OA was prepared by Feng et al. by a simple Schiff base reaction, and antimicrobial peptides bacitracin and Polymyxin B (AC/OSA-PB) were loaded into the hydrogels without the need for catalysts. These hydrogels illustrated modifiable gelation time, stable rheology, and a strain resembling that of human skin. Moreover, they could effectively cause inhibition of *S. aureus* and *E.coli* growth, promoting angiogenesis and cell proliferation in vitro. Similar results were observed in vivo too, with enhanced full-thickness wound healing ability by promoting granular tissue formation and deposition of collagen and accelerating neovascularization and re-epithelialization [354].

In a similar study related to full-thickness wound healing by Chaudhary et al., CA nanoparticles (CA-NPs) were used as hemostatic agents along with antimicrobial AgNPs in a CS-based hydrogel network. Herein, the fresh blood of the subjects was used in the hydrogels to substitute the growth factors required for wound healing. The CA-NPs had mean hydrodynamic sizes of 1037 nm and 120.56 nm, respectively, and a zeta potential of less than −30 mV indicated their negligible electrostatic repulsion tendency. The prepared hydrogels exhibited good spreadability and viscoelasticity. Moreover, they showed bacterial inhibition against both gram-positive and gram-negative strains and also

illustrated remarkable scar-free healing in vivo for up to 15 days by aiding in collagen deposition and acting as a protective sheath against microbial contamination for diabetesinduced wounds. Thus, the proposed composite films could be an exciting wound dressing for patients who have chronic diabetes [355]. *Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 39 of 60

**Figure 10.** ALG/human elastin‐like polypeptide composite films with antioxidant properties for potential wound healing applications. (**a**) swelling (A) and stability (B) graphs of ALCur, HALCur films encapsulating 0.1% curcumin. (**b**) % curcumin release characteristics of ALCur, HALCur films encapsulating 0.1% curcumin. (**c**) Cell viability assay of ALG/HELP composites on human fibroblast cell lines. (**d**) DPPH assay of ALCur and HALCur films indicating antioxidant activities. # *p* <0.05. Reproduced with permission from [353], copyright Elsevier 2020. **Figure 10.** ALG/human elastin-like polypeptide composite films with antioxidant properties for potential wound healing applications. (**a**) swelling (A) and stability (B) graphs of ALCur, HALCur films encapsulating 0.1% curcumin. (**b**) % curcumin release characteristics of ALCur, HALCur films encapsulating 0.1% curcumin. (**c**) Cell viability assay of ALG/HELP composites on human fibroblast cell lines. (**d**) DPPH assay of ALCur and HALCur films indicating antioxidant activities. # *p* <0.05. Reproduced with permission from [353], copyright Elsevier 2020.

Full‐thickness healing requires extensive healing time, which increases the risks of infections, wound ulcers, necrosis, and even fatal complications. To tackle this, a hybrid hydrogel composed of amine‐functionalized fish collagen and OA was prepared by Feng et al. by a simple Schiff base reaction, and antimicrobial peptides bacitracin and Poly‐ myxin B (AC/OSA‐PB) were loaded into the hydrogels without the need for catalysts. These hydrogels illustrated modifiable gelation time, stable rheology, and a strain resem‐ bling that of human skin. Moreover, they could effectively cause inhibition of *S. aureus* and *E.coli* growth, promoting angiogenesis and cell proliferation in vitro. Similar results were observed in vivo too, with enhanced full‐thickness wound healing ability by pro‐ moting granular tissue formation and deposition of collagen and accelerating neovascu‐ larization and re‐epithelialization [354]. In a similar study related to full‐thickness wound healing by Chaudhary et al., CA nanoparticles (CA‐NPs) were used as hemostatic agents along with antimicrobial AgNPs in a CS‐based hydrogel network. Herein, the fresh blood of the subjects was used in the hydrogels to substitute the growth factors required for wound healing. The CA‐NPs had In another study, Sharma et al. loaded rifampicin into ALG-gelatin fibers through a physical cross-linking reaction by the extrusion-gelation method and then embedded it into transdermal films for wound healing applications. The tensile strength of the fibers was between 2.32 <sup>±</sup> 0.45 to 14.32 <sup>±</sup> 0.98 N/mm<sup>2</sup> , and the extensibility was between 15.2 ± 0.98% to 30.54 ± 1.08%. The range of moisture absorption was low (up to 14.68%), which is essential for transdermal films. Other parameters such as the swelling ratio and water vapor transmission rate demonstrated the extensive gelation properties of the polymer and the reduced channel pore size. Antibacterial activities against *E. coli* and *S. aureus* were observed for the transdermal films. In vivo animal studies exhibited close to 83 degrees of contraction of the wound, marginally less than the commercial formulation (91.87 ± 3.72%). The drug release from the films followed sustained release; this could be due to sufficient contact of the wound dressing with the wound layer with hair growth signs evident from the 10th day onwards. Thus, the proposed fiber-in-films could be excellent carriers for drug delivery and wound healing purposes [356].

mean hydrodynamic sizes of 1037 nm and 120.56 nm, respectively, and a zeta potential of less than −30 mV indicated their negligible electrostatic repulsion tendency. The prepared hydrogels exhibited good spreadability and viscoelasticity. Moreover, they showed bac‐ terial inhibition against both gram‐positive and gram‐negative strains and also illustrated remarkable scar‐free healing in vivo for up to 15 days by aiding in collagen deposition Other studies where ALG composites showed promising results in wound healing include SA/HA films/sulfadiazine/silver nanoparticles [357], ALG/gelatin fibers/ curcumin [358], SA/xanthan gum film/pycnogenol [359], ALG/CS/maltodextrin/pluronic F127/pluronic P123/tween 80/curcumin polymeric micelles [360], SA/pectin/cefazolin nanoparticles [361], CA/ibuprofen hydrogels [362], ALG/cannabidiol [363], SA/polyvinyl

and acting as a protective sheath against microbial contamination for diabetes‐induced wounds. Thus, the proposed composite films could be an exciting wound dressing for alcohol/curcumin/graphene [364], ALG/polyhexanide/AgNPs [365], ALG/carboxymethyl CS/Kangfuxin sponges [366].


**Table 4.** Recent investigations of ALG-based composites for wound healing applications.

#### **8. Conclusions**

ALG emerges as a prospective naturally derived biomaterial in NDDSs due to the biocompatible, degradable characteristics and gel-forming capability of ALG. Controlled and targeted actives delivery via ALG carrier can be accomplished via well-created formulation as well as accurate parameters of synthesis concerned with the process of fabrication. Hence, this article provides an extensive review of the recent advances of ALG and its advancement in actives delivery. The most significant characteristics of ALG encompass safety, biocompatibility, and ease of preparation. Due to its biocompatible, biodegradable, and non-toxic characteristics, it is employed in diverse drug-delivery technologies. A significant challenge that persists is the preparation of environmentally friendly procedures for the NPs formation having a narrow size distribution, high mechanical and chemical stability, and practicality to scale up to the volumes of industrial-scale production. Moreover, it is critical to assess the toxic effects on cells, immune response, and biodegradability of such formulations in drug delivery.

As we look towards the future, the ALG-based composites utilized in pharmaceutical applications are possibly going to develop significantly. Although ALG composites are already used clinically for wound healing, they perform quite a passive function. Forthcoming dressings will probably perform a considerably more active role. One or more bioactives that assist wound healing can be loaded into ALG-based dressings, as such gels have displayed usefulness in preserving local conc. of biological factors (for example, proteins) for a prolonged period. In wound healing, and more usually, actives delivery, accurate control

over the single vs. multiple drugs delivery or drug release in sustained vs. sequential manner in response to exterior environmental modifications is immensely advisable.

Therefore, investigators are required to modernize the ALG-associated composite's advancement, and this review is the origin of advice for forthcoming investigation.

**Author Contributions:** Conceptualization and supervision: M.A.S.A., A.S., M.J.A. and S.P.; Resources: M.A.S.A., M.J.A., R.R.R., R.M. and S.P.; Literature review and writing—original draft preparation: M.A.S.A., R.R.R., A.S., S.P., P.S., R.M. and M.J.A.; writing—review and editing: M.A.S.A., R.R.R., A.S., L.S.A., A.D., M.J.A. and S.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** MA would like to thank the Deanship of Scientific Research at Umm Al-Qura University for supporting this work by Grant Code: (22UQU4290565DSR53).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** MA would like to thank the Deanship of Scientific Research at Umm Al-Qura University for supporting this work by Grant Code: (22UQU4290565DSR53). Author RR would like to acknowledge the financial support by the Department of Mechanical Engineering and Mechanics at Lehigh University. He would also like to thank his mentor, Arindam Banerjee, for continuous support and motivation. SP would like to thank Ramesh Parameswaran and Vignesh Muthuvijayan for their guidance in preparing the manuscript. Author SP would like to thank the Indian Institute of Technology Madras, Ministry of Human Resource Development for providing financial assistantship. Author MJA acknowledges the support of the Deanship of Scientific Research at Prince Sattam bin Abdulaziz University.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**




## **References**


## *Article* **Valorization of Berries' Agro-Industrial Waste in the Development of Biodegradable Pectin-Based Films for Fresh Salmon (***Salmo salar***) Shelf-Life Monitoring**

**Janira Romero <sup>1</sup> , Rui M. S. Cruz 2,3, Alexandra Díez-Méndez <sup>1</sup> and Irene Albertos 4,\***

<sup>1</sup> Faculty of Sciences and Art, Universidad Católica de Ávila (UCAV), Calle Canteros s/n, 05005 Ávila, Spain

<sup>2</sup> Department of Food Engineering, Institute of Engineering, Campus da Penha, Universidade do Algarve, 8005-139 Faro, Portugal


**Abstract:** The healthy properties of berries are known; however, red fruits are very perishable, generating large losses in production and marketing. Nonetheless, these wastes can be revalued and used. The main objective of this study was the development of biodegradable pectin films with berry agro-industrial waste extracts to monitor salmon shelf-life. The obtained extracts from blueberries, blackberries, and raspberries wastes were evaluated in terms of flavonols, phenols and anthocyanins contents, and antioxidant capacity. Then, pectin films with the extracts of different berries were developed and characterized. The results showed that the blueberry extract film was thicker (0.248 mm), darker (L\* = 61.42), and opaquer (17.71%), while the highest density (1.477 g/cm<sup>3</sup> ) was shown by the raspberry films. The results also showed that blueberries were the best for further application due to their composition in bioactive compounds, antioxidant capacity, and color change at different pHs. The salmon samples wrapped in blueberry films showed lower values of pH and deterioration of fish during storage compared to the control and pectin samples. This study contributes to the valorization of berries agro-industrial waste by the development of eco-friendly films that can be used in the future as intelligent food packaging materials contributing to the extension of food shelf-life as a sustainable packaging alternative.

**Keywords:** biopolymers; intelligent food packaging; bioactive natural compounds; biodegradable; shelf-life; sustainability

## **1. Introduction**

Food packaging is an essential part of the food sector as it guarantees the quality and safety of products, helps in the transport process, provides stable storage, prevents damage and losses, and ensures greater safety for the consumer [1,2]. Traditionally, the packaging is made from petroleum derivatives; however, they generally contain chemicals considered harmful, such as bisphenol and phthalates [3]. In addition, the long life and resistance of plastics to degradation have generated an accumulation of waste that has a great negative impact on the environment [4]. For this reason, we are currently looking for plastic reduction systems and the development of sustainable alternatives. Some of these alternatives are bio-based polymers, e.g., pectin [1] that can generate biodegradable packaging materials. Likewise, these new materials can be incorporated with different compounds, improving characteristics such as permeability or optical features, which allow the production of smart packaging systems [5].

Intelligent packaging is one type of smart packaging and is defined as one capable of making decisions to increase the shelf-life of the food, inform the consumer, and improve

**Citation:** Romero, J.; Cruz, R.M.S.; Díez-Méndez, A.; Albertos, I. Valorization of Berries' Agro-Industrial Waste in the Development of Biodegradable Pectin-Based Films for Fresh Salmon (*Salmo salar*) Shelf-Life Monitoring. *Int. J. Mol. Sci.* **2022**, *23*, 8970. https://doi.org/10.3390/ijms23168970

Academic Editors: Valentina Siracusa and Swarup Roy

Received: 20 July 2022 Accepted: 9 August 2022 Published: 11 August 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the quality of the product through various methods such as pH indicators through color changes [6]. The containers with pH indicators may incorporate extracts of various fruits or vegetables that have different properties and allow color changes associated with the variation in the pH of the product. Some of these extracts can be obtained from berries such as blueberries, raspberries, and blackberries, which possess antioxidant capacity thanks to their bioactive compounds, such as phenolic compounds and anthocyanins [7]. Berries are a product with increasing attractiveness, and their consumption has increased in recent years due to their beneficial properties for health such as antioxidants, anticancer substances, vitamins, minerals, fibers, and other essential nutrients [8,9]. However, berries are very perishable products since they have a high water content, which makes them susceptible to mechanical damage, contamination, freezing, or dehydration, generating large losses [10]. These losses generate serious nutritional, economic, and environmental problems [11]. To circumvent these problems, the fruits are normally processed, for example, for obtaining juices, from which numerous by-products can be achieved [12]. Food waste is already a global problem that endangers the long-term food supply chain, with 1.300 million tons being discarded every year [13]. For this reason, the Sustainable Development Goals (SDGs) require, by 2030, a halving of food waste per capita in the supply and consumption chain [13]. More than 1.748 million tons of wasted food correspond to fruits and vegetables [13], so there are already several reviews on the possible applications of these residues. Bayram et al., 2021 [14] studied the possible use of biopolymers, biocomposites, smart packaging, and edible films or coatings. In the processing of fruits and vegetables, by-products consisting of seeds and shells are produced in large quantities, which present a large concentration of bioactive components such as antioxidants, pigments, proteins, essential oils, enzymes, and dietary fibers [13].

The residues resulting from processing, called pomace, also in the case of berries can be reused in the food industry as ingredients or natural additives due to the bioactive compounds [11]. As previously referred to, the waste generated represents a great loss of valuable nutrients. For this reason, the biotransformation of waste is receiving increasing attention since it can be used as a resource to obtain useful products with added value [8]. There are numerous biopolymers used in the development of packaging materials, with poly (lactic acid) (PLA), cellulose, starch, and chitosan being more widely used [15]. PLA is a biodegradable polymer with mechanical properties very similar to those of thermoplastics. Starch is also biodegradable and is a polymer that is easily found; however, it has a strong hydrophilic behavior, making it sensitive to moisture [15]. On the other hand, cellulose is one of the most abundant renewable materials being used as a filler or host polymer in packaging [15]. Chitosan contains antimicrobial properties, so it can be used as a host and antimicrobial agent [15]. As previously referred to, another polymer that can be used in the development of packaging films is pectin, in which plant extracts can be easily added to generate active ingredients [16]. Pectin is a good component thanks to its ability as a gelling and emulsifying agent, generating water-soluble films with low opacity [16]. Essential oils can be added to pectin films, the most common being cinnamon, rosemary, oregano, cloves, thyme, lemon, and orange. Other elements that can be incorporated into pectin are agricultural residues such as banana, orange, and lemon peels among others [16]. Plant extracts rich in phenolic compounds are also added to pectin to increase the antioxidant capacity of the films, although there is less research in this area compared to in essential oils [16].

On the other hand, blueberry residues have multiple uses such as biofuel, biogas, biochar, or biocrude oil production [17]. There are also various uses in food as additives; for example, Rai et al., 2021 [18] observed that blueberry residues produce certain ferments that help to improve intestinal microbiota and intestinal function, corroborating its potential use as a functional food [18–20]. Different studies used blueberry residues to develop smart packaging as films based on different biopolymers such as chitosan, starch, and gelatin [21–27]. However, no subsequent application was carried out as smart packaging on food products such as meat or fish.

Fresh fish is considered key in the diet as it provides 17% of the animal protein ingested [28]. Specifically, salmon (*Salmo salar*) is one of the most consumed products worldwide, representing 93% of production [29]. Its consumption has been increasing thanks to the fact that it contains beneficial components for health such as omega-3 longchain polyunsaturated fatty acids (LC-PUFA) [28] as well as its color, taste, protein content, vitamins, and antioxidants [30]. However, salmon and other fishery products are very perishable due to their high-water activity, almost neutral pH, and other specific components that favor biochemical, physical, and microbial deterioration during the production chain, specifically the deterioration that begins immediately after capture [28].

To our knowledge, there is little information on the use of residues of other berries such as raspberries and blackberries for the development of smart packaging materials, as well as its comparison with blueberries and their subsequent use in fish products, particularly in salmon. In addition, few studies in the literature reported the use of pectin as a matrix to develop intelligent packaging. For this reason, this study aimed to develop and characterize biodegradable pectin films with the incorporation of blueberry, raspberry, and blackberry waste extracts and to study their effect on fresh salmon shelf-life.

#### **2. Results and Discussion**

#### *2.1. Characterization of the Extracts*

The results (Table 1) indicated significant differences (*p* < 0.05) in flavonol content among the different extracts. The blueberry extract was the one with the highest flavonols content (13.65 ± 0.01 mg of quercetin equivalents per g of extract), followed by the blackberry extract. Similar results were obtained in other studies [31,32].


**Table 1.** Characterization of the antioxidant properties of the extracts.

The values (mean ± standard deviation) followed by different lowercase letters in the same column, for each parameter, are significantly different (*p* < 0.05).

Blackberry and blueberry extracts showed the highest level of anthocyanins. The raspberry extract (Table 1) presented the lowest level of anthocyanins compared with the blackberry extract (*p* < 0.05). Different studies reported significant differences between anthocyanins obtained from blackberries and raspberries, being the latter of lower content [32,33]. Sariburun et al., 2010 [32] reported that the content of anthocyanins in extracts using water was more effective as they have a high solubility in water. In a variety of raspberry called "Rubin", anthocyanins obtained from an extract with water were 60.3 ± 0.7 mg GAE/100 g, while in an extract with methanol they were 24.1 ± 0.4 mg GAE/100 g. In the case of the blackberries, extraction with water proved to be more effective, showing values of 54.8 ± 0.8 mg GAE/100 g while with methanol the values were lower (41.3 ± 0.3 mg GAE/100 g) [32]. In short, a higher anthocyanin content was shown in blackberries, as in our study.

Blueberry extract showed the highest content of total polyphenols, differing significantly from raspberry and blackberry extracts, although their levels of polyphenols were also high. Other authors reported a higher polyphenol content in blackberry extract (24.85 <sup>±</sup> 0.11 mg g−<sup>1</sup> ) while that of cranberry extract was significantly lower (6.08 ± 0.04 mg g −1 ) [23] due to the extraction method in water to ensure food and environmental safety. In addition, phenols are an important indicator of antioxidant capacity [23]. In our study, the

highest antioxidant capacity in both methods was obtained in blueberry extract (Table 1), while no significant differences (*p* > 0.05) were observed between the antioxidant capacity of blackberry and raspberry extracts. This shows the expected positive correlation between total polyphenol content and antioxidant capacity. The use of two different methods to measure the antioxidant capacity under different reaction conditions allowed us to establish a more precise view of the antioxidant capacity for the developed films. Günde¸sli et al., 2019 [34] reported the highest antioxidant capacity in blackberries, followed by raspberries. Unlike other berries, blueberries have the greatest antioxidant capacity in the early stages of ripening, which is related to the high levels of flavonols and hydroxycinnamic acids in the pre-ripening stage [35].

On the other hand, the antioxidant capacity of raspberries was 40% lower than that of blueberries.

#### *2.2. Characterization of the Developed Films*

#### 2.2.1. Thickness, Density, and Hardness

The results for thickness, density, and hardness are presented in Table 2. Significant differences (*p* < 0.05) were obtained in the thicknesses of all films, with the pectin films being thinner while the blueberry films were thicker. In either case, it is shown that the addition of fruit powder significantly increased the thickness of the films compared to the control (pectin). For blueberries, similar values were recorded in the literature in the studies reported by Luchese et al., 2018 [24] and Andretta et al., 2019 [26] in cassava starch films. However, in other studies, Luchese et al., 2018 [25] and Jamróz et al., 2019 [36] obtained significantly lower thickness values, even at higher percentages of blueberry and yerba mate extracts. This decrease in thickness is possibly due to the particle size of the powder, which is within 100–300 µm. In the study by Luchese et al., 2018 [25], a lower thickness was observed in the films made from blueberry powder with smaller particle sizes. On the other hand, similar results to those obtained in the thickness of the films with blackberry extract (0.2 mm) were observed in the studies of Gutiérrez, 2018 [37] and Sganzerla et al., 2021 [38]. However, the films made with raspberry powder were thicker than those reported in Yang et al., 2021 [39], possibly due to differences in powder concentration.


**Table 2.** Thickness, density, and hardness of the developed films.

The values (mean ± standard deviation) followed by different superscript letters in the same column, for each parameter, are significantly different (*p* < 0.05).

Table 2 shows also the density in the different films; the film with raspberry extract was the one with the highest value (1.477 g/cm<sup>3</sup> ) followed by the film with blueberry extract. However, the results obtained were lower than those of Yang et al., 2021 [39], possibly due to the different matrices used. In our study, the films were produced with pectin while Yang et al., 2021 [39] used a matrix comprised of pectin, sodium alginate, and xanthan gum. This could be due to the intermolecular interactions between anthocyanins and the matrix, directly influencing their properties and, therefore, the films that contain them [40], and thus, hydrogen bonds benefit from a regular arrangement of the matrix chain in the film, generating a higher density [41–43].

On the other hand, there was also a trend of increasing hardness from pectin films to the films incorporated with the berries' extracts. The film with the blackberry extracts presented the highest hardness while the film with the raspberry extracts the lowest. Probably, the intermolecular hydrogen bonds were formed between the hydroxyl groups

of the raspberry extract and the matrix, reducing the crosslinking of water and the matrix, thus decreasing the hardness of the film [39].

#### 2.2.2. Color

The color and opacity of the films are shown in Table 3. Significant differences were observed in all the color parameters between the different films, except for the L\* parameter, where there were no significant differences (*p* > 0.05) between the film with blackberries and the film with blueberries. However, the pectin film showed the highest luminosity (L\*) and hue (h\*). It is worth noting a clear tendency h\* decrease from pectin film (85.19) to the film with blackberries (26.19). Despite the absence of differences in the L\* value between the films with blackberry and blueberry extracts, these presented different results in the other parameters. The film with blackberry extract was more reddish compared will all the other films. This may be due to the different composition of anthocyanins in the different extracts. The trend to the red/purple is the result of the presence of cyanidin, which is purple at neutral pH [23]. The results of the parameters a\* and b\* coincide with the ones given in the study of Kurek et al., 2018 [23], where the same trend was observed among films developed with blackberries and blueberry extracts. Of the films incorporated with berry extracts, the film with raspberry extract was the one that presented the greatest luminosity. The luminosity of the films with raspberry extract (74.75) was similar (77.76) to the raspberry films (0.5 g/L raspberry films) reported by Yang et al., 2021 [39].

**Table 3.** Color and opacity of the developed films.


The values (mean ± standard deviation) followed by different superscript letters in the same column, for each parameter, indicate significant differences (*p* < 0.05).

#### 2.2.3. Opacity

The protective action against light is an important feature in the packaging of food since UV radiation and light are powerful lipid oxidizers [44]. Regarding opacity, there was a clear trend of increase in the incorporation of extracts (Table 3). The film with blueberry extract showed higher opacity than the film with blackberry extract, followed by the raspberry one.

Conversely, Kurek et al., 2018 [23] reported that blackberry extract films turned out to be opaquer than blueberry extract films.

### 2.2.4. Color Changes at Different pH

The color parameters were evaluated in solutions with different pHs to determine whether color changes at an environmental pH can be shown. The highest color variability according to pH changes was presented in the films with blueberry and raspberry extracts (Figure 1).

In the case of blueberries, significant differences (*p* < 0.05) were determined in the R value from pH 2 to 12. Yellowish brown hues of pH 2–5 were obtained, turning to a slightly darker brown up to pH 12. However, the film with raspberry extract showed very few color variations at different pHs, apart from some points showing significant differences (*p* < 0.05) in R, G, and B values between pH 2 and 6. In fact, these changes were not noticeable in the visual assessment (Figure 1).

**Figure 1.** Films color changes at different pH value. RB (Raspberry), BK (Blackberry), and BB (Blueberry). Values followed by different superscript letters in the same row, for each parameter, indicate significant differences (*p* < 0.05). **Figure 1.** Films color changes at different pH value. RB (Raspberry), BK (Blackberry), and BB (Blueberry). Values followed by different superscript letters in the same row, for each parameter, indicate significant differences (*p* < 0.05).

In the case of blueberries, significant differences (*p* < 0.05) were determined in the R value from pH 2 to 12. Yellowish brown hues of pH 2–5 were obtained, turning to a slightly darker brown up to pH 12. However, the film with raspberry extract showed very few color variations at different pHs, apart from some points showing significant differences (*p* < 0.05) in R, G, and B values between pH 2 and 6. In fact, these changes were not noticeable in the visual assessment (Figure 1). Other authors such as Kurek et al. 2018 [23] determined color parameters L, a, and b in chitosan films with blueberry and blackberry residue in a pH range of 2–12. Red colors from pH 2 to 4; blue/green at pH 5, 6, and 7; and dark green at pH 10 and 12 were observed for blueberries [23]. Separately, a red color was displayed at pH 2 and 4; violet at pH 5, 6, and 7; and dark blue at pH 10 and 12 for blackberries. In contrast, in a film of carboxymethylcellulose (CMC) and blackberries, the colors obtained in a pH range of 1 to 13 varied from pink in an acid medium to yellowish green in a basic medium [38], possibly due to the influence of the CMC matrix. Luchese et al. 2017 [22] determined the color changes in chitosan films with blueberries, on a pH scale of 2–12, obtaining results like those of Kurek et al. 2018 [23]. In addition, the process of prior blanching of the fruits caused the luminosity values to be lower, obtaining darker films [22]. This procedure was also performed in our study, which is why darker films were obtained such as those of Other authors such as Kurek et al., 2018 [23] determined color parameters L, a, and b in chitosan films with blueberry and blackberry residue in a pH range of 2–12. Red colors from pH 2 to 4; blue/green at pH 5, 6, and 7; and dark green at pH 10 and 12 were observed for blueberries [23]. Separately, a red color was displayed at pH 2 and 4; violet at pH 5, 6, and 7; and dark blue at pH 10 and 12 for blackberries. In contrast, in a film of carboxymethylcellulose (CMC) and blackberries, the colors obtained in a pH range of 1 to 13 varied from pink in an acid medium to yellowish green in a basic medium [38], possibly due to the influence of the CMC matrix. Luchese et al., 2017 [22] determined the color changes in chitosan films with blueberries, on a pH scale of 2–12, obtaining results like those of Kurek et al., 2018 [23]. In addition, the process of prior blanching of the fruits caused the luminosity values to be lower, obtaining darker films [22]. This procedure was also performed in our study, which is why darker films were obtained such as those of Luchese et al., 2017 [22]. This fact is because in the production of films with unblanched fruits there is a greater degradation of anthocyanins [22]. Andretta et al., 2019 [26] observed in their study with blueberries a color variation from red/orange at acid pH and green/ yellowish at basic pH. Yun et al., 2019 [45] developed films with starch-based blueberries, obtaining colors from pale violet to intense red when exposed to hydrogen chloride. However, in exposure to ammonia, the violet turned blue and then green.

Luchese et al. 2017 [22]. This fact is because in the production of films with unblanched fruits there is a greater degradation of anthocyanins [22]. Andretta et al. 2019 [26] observed in their study with blueberries a color variation from red/orange at acid pH and green/ yellowish at basic pH. Yun et al. 2019 [45] developed films with starch-based blueberries, obtaining colors from pale violet to intense red when exposed to hydrogen chloride. However, in exposure to ammonia, the violet turned blue and then green. In the case of raspberry, variability was also observed, going from a yellowish rose In the case of raspberry, variability was also observed, going from a yellowish rose at pH 2, 3, and 4 to a stronger rose at pH 5 and then to a yellowish rose again in the rest of the pH. In the study of Yang et al., 2021 [39], the color variation was from red to pink from pH 1 to 6, from pink to violet-blue from pH 7 to 10, and green from pH 11 to 13. These colors are because in the acidic media, the red flavylium cation form of the anthocyanins predominates, which changes its structure as the pH becomes basic until it becomes a yellow chalcone [39,40].

at pH 2, 3, and 4 to a stronger rose at pH 5 and then to a yellowish rose again in the rest of the pH. In the study of Yang et al. 2021 [39], the color variation was from red to pink from pH 1 to 6, from pink to violet-blue from pH 7 to 10, and green from pH 11 to 13. These colors are because in the acidic media, the red flavylium cation form of the antho-Therefore, the films with blueberry extract were selected in our study for the second phase of the study to monitor the shelf-life of salmon fillets due to their higher polyphenol content and second-best in anthocyanin content. In addition, the films were opaquer and showed visible color changes at different pHs.

#### cyanins predominates, which changes its structure as the pH becomes basic until it becomes a yellow chalcone [39,40]. 2.2.5. Biodegradation Properties

Soil

Therefore, the films with blueberry extract were selected in our study for the second phase of the study to monitor the shelf-life of salmon fillets due to their higher polyphenol content and second-best in anthocyanin content. In addition, the films were opaquer and showed visible color changes at different pHs. The films showed no changes in their structure after 24 h, although presented some water absorption (from the wet soil). After the seventh day, the films started to show some changes in their structure due to the solubility in water (Figure 2A).

changes in their structure due to the solubility in water (Figure 2A).

2.2.5. Biodegradation Properties

Soil

**Figure 2.** (**A**) Biodegradation test in soil; (**B**) biodegradation test in seawater: (**a**) pectin film (control), (**b**) film with raspberry extract, (**c**) film with blackberry extract, and (**d**) films with blueberry extract. **Figure 2.** (**A**) Biodegradation test in soil; (**B**) biodegradation test in seawater: (**a**) pectin film (control), (**b**) film with raspberry extract, (**c**) film with blackberry extract, and (**d**) films with blueberry extract.

The films showed no changes in their structure after 24 h, although presented some water absorption (from the wet soil). After the seventh day, the films started to show some

Moreover, the organic matter and the availability of phosphorus in the soil contribute to a higher load of fungi also responsible for biodegradation [46]. A study in which cassava starch films were developed showed similar results, with signs of biodegradation after 6 days and greater changes in the degradation of the films after 12 days [47]. Moreover, the organic matter and the availability of phosphorus in the soil contribute to a higher load of fungi also responsible for biodegradation [46]. A study in which cassava starch films were developed showed similar results, with signs of biodegradation after 6 days and greater changes in the degradation of the films after 12 days [47].

Norcino et al. 2020 [48] developed pectin-based films with copaiba oil. In this study, the films were practically biodegraded in the soil after 28 days. Norcino et al., 2020 [48] developed pectin-based films with copaiba oil. In this study, the films were practically biodegraded in the soil after 28 days.

In a recent study reported by Ren et al. 2022 [49], pectin-based films also biodegraded significantly after three weeks and were biodegraded completely after five weeks. In a recent study reported by Ren et al., 2022 [49], pectin-based films also biodegraded significantly after three weeks and were biodegraded completely after five weeks.

The degradation can occur firstly from different physical and biological processes, including wetting/drying, heating/cooling, or freezing/thawing. These processes contribute to the cracking of the polymeric materials. In this study, the initial breakdown of the films occurred due to the presence of water. Then, during the degradation process, the The degradation can occur firstly from different physical and biological processes, including wetting/drying, heating/cooling, or freezing/thawing. These processes contribute to the cracking of the polymeric materials. In this study, the initial breakdown of the films occurred due to the presence of water. Then, during the degradation process, the extracellular enzymes from the microorganisms break down the polymer and the depolymerization occurs. Short chains or smaller molecules, such as oligomers, dimers, and monomers, pass the semipermeable outer bacterial membranes and are then converted to carbon dioxide, water, and biomass as the final products of the biodegradation [50,51]. The European Standard EN 13432 [52] indicates 90% as the value for packaging to be considered biodegradable by biological action in 6 months. Thus, it is possible to affirm that the developed films can be considered biodegradable.

#### Seawater

The films showed several changes during the biodegradation test in seawater (Figure 2B). After the second day, the films with the addition of berry extract kept their initial appearance but showed some loss of color. The results presented by Alvarez-Zeferino et al., 2015 [53] and Pereira et al., 2021 [54] are in agreement with the ones obtained in this study showing low levels of biodegradation in the first days of testing.

On the 30th day, the films' loss of color was more evident, and the seawater in which they were submerged began to show signs of clouding due to the transfer of pigments (i.e., anthocyanins) from the films to the seawater. On the 45th day, it was possible to verify that all films started to be fragmented, and the clouding of the water was also noticeable. On the 60th day, greater changes were observed in the films' structure, as they started to fragment into small pieces and dissolve considerably in the seawater. These changes are related to the swelling of the film, as both the swelling and the solubility of the film can directly affect the water-resistance properties of the film, particularly if it occurs in a humid environment [55,56].

After 90 days, all films lost their initial rectangular shape and were quite fragmented, presenting a "flaky" appearance. Several factors influence the rate of biodegradation of the films, such as the swelling, the movement/agitation of the seawater, the existence of oxygenation, the presence of microorganisms in the seawater, and the ratio volume of seawater/film [53,56,57].

In general, biodegradation in soil was practically obtained in a short period, with an average degradation rate of 3.6% per day while the biodegradation in seawater took more time, showing an average degradation rate of 1% per day. These results are of extreme importance since they quantify and prove the fast degradation of these types of biodegradable materials against plastic or even paper packaging. Paper and plastic degradation are very slow processes, and they can take several years to be fully degraded, depending on the type of plastic or paper and the used conditions [58].

For example, brown newspapers started to degrade by the 10th–12th week of exposure in soil, remaining small pieces of the papers, while plastic bags had thinned off and become transparent [59]. In another study, low-density polyethylene (LDPE) bags were estimated to decompose by 50% after 4.6 years in inland (buried) and 3.4 years in marine environments [60].

## *2.3. Effect of Films Monitoring Freshness of Salmon Fillets*

2.3.1. pH

The results (Table 4) showed a clear upward trend in the pH for both control and pectin treatment over storage. On the other hand, the pH of the fish samples treated with the film with blueberry extract showed a tendency to be maintained until day 4 with a subsequent rise on day 7. In addition, significant differences (*p* < 0.05) were observed among the treatments from day 2.

**Table 4.** pHs of salmon fillets during storage with different treatments.


The values followed by different lowercase superscript letters in the same column, for each parameter, are significantly different (*p* < 0.05). The values followed by different capital superscript letters in the same row, for each parameter, are significantly different (*p* < 0.05).

The initial pH values in salmon (pH > 6) for all samples with the different treatments were similar to those reported by Ambrosio et al., 2022 [28]. The increase in pH may be due to the production of ammonia and amines generated by the autolysis of nitrogenous compounds of bacteria that proliferate the decomposition process [28,61–64]. The films with the blueberry extract prevented the pH increase by avoiding the degradation of proteins and, therefore, the release of alkaline compounds during the first days of storage [65].

Therefore, the results showed a faster degradation in the control samples during the storage time than in the treated samples, especially for the film with the blueberry extract. The films composed of blueberry extract exerted a greater positive effect against deterioration since, being rich in antioxidants, these compounds can migrate to the fish surface, reducing the oxidation reactions [63,66].

#### 2.3.2. Moisture

In the first days of storage, there were no significant differences in the humidity of the samples between each treatment. From the fourth day, it is possible to observe significant differences *(p* < 0.05) between all the treatments (Table 5). A tendency of humidity decrease was also obtained throughout the storage in the treatments compared to the control samples. However, this trend only showed significance in the blueberry treatment. As in our trial, other authors obtained a reduction of humidity in the samples with the different films, possibly due to the absorption of water, which can be beneficial since it can favor the control of microbial growth [65] or due to drip loss.

**Table 5.** Moisture (%) of salmon fillets during storage with different treatments.


The values followed by different lowercase superscript letters in the same column, for each parameter, are significantly different (*p* < 0.05). The values followed by different capital superscript letters in the same row, for each parameter, are significantly different (*p* < 0.05).

#### 2.3.3. Fish Color

For the color analysis, a two-way ANOVA was performed, thus obtaining the global changes regarding the treatment and storage time.

The RGB color model is based on the mixing of red, green, and blue with different intensities to generate a color. Therefore, the color is presented as an RGB triplet. Each of the three colors can vary from zero to the maximum value (in this case 1023). The black color is represented as (000, 000, 000) and the white (1023, 1023, 1023) [67].

The color of fish is one of the most important qualities since it has a great influence on the consumer at the time of purchase [28]. Regarding the color in the salmon samples, significant differences (*p* < 0.05) were observed in all measurement parameters (R, G, and B) between the control samples and the samples with the film with blueberry extract (Figure 3).

The R value (red) obtained the highest values in the pectin treatment and on day 7 of storage. On day 7, the fish samples with a greater reddish color were observed in the samples with blueberry treatment, possibly due to the migration of the compound from the film to the fish. The same was observed in the study of Rico et al., 2020 [65], where color migration occurred from the fennel components to the sample.

On the other hand, authors such as Albertos et al., 2015 [68] obtained a significant decrease in luminosity (L\*) in trout (*Trachurus trachurus*), due to the discoloration and reduction of redness during storage, as in our control samples. This reduction of reddish coloration in salmonids may be due to the oxidation of hemoproteins (hemoglobin, and myoglobin), since in their oxidized ferric form they present a brown color [68]. In the treated fish, the reddish color remained better, possibly due to the antioxidant effect of the film.

ure 3).

the film.

migration occurred from the fennel components to the sample.

**Figure 3.** (**A**) Global salmon color changes: (**1**) global color for each treatment; (**2**) global color during storage. (**B**) Global pectin and blueberry film color changes: (**1**) global color for each treatment; (**2**) global color during storage. Different capital letters indicate significant differences (*p* < 0.05). **Figure 3.** (**A**) Global salmon color changes: (**1**) global color for each treatment; (**2**) global color during storage. (**B**) Global pectin and blueberry film color changes: (**1**) global color for each treatment; (**2**) global color during storage. Different capital letters indicate significant differences (*p* < 0.05).

significant differences (*p* < 0.05) were observed in all measurement parameters (R, G, and B) between the control samples and the samples with the film with blueberry extract (Fig-

The R value (red) obtained the highest values in the pectin treatment and on day 7 of storage. On day 7, the fish samples with a greater reddish color were observed in the samples with blueberry treatment, possibly due to the migration of the compound from the film to the fish. The same was observed in the study of Rico et al. 2020 [65], where color

On the other hand, authors such as Albertos et al. 2015 [68] obtained a significant decrease in luminosity (L\*) in trout (*Trachurus trachurus*), due to the discoloration and reduction of redness during storage, as in our control samples. This reduction of reddish coloration in salmonids may be due to the oxidation of hemoproteins (hemoglobin, and myoglobin), since in their oxidized ferric form they present a brown color [68]. In the treated fish, the reddish color remained better, possibly due to the antioxidant effect of

#### 2.3.4. Film Color

2.3.4. Film Color Clear differences were obtained in the treatment with blueberries compared to the treatment with pectin and without any treatment (Figure 3). In the case of G, a small re-Clear differences were obtained in the treatment with blueberries compared to the treatment with pectin and without any treatment (Figure 3). In the case of G, a small reduction was observed between treatments, although it was not significantly different.

duction was observed between treatments, although it was not significantly different. Blueberry film obtained a brownish to more yellowish coloration throughout the storage time. Zhai et al. 2017 [69] showed similar results in a study to control carp fresh-Blueberry film obtained a brownish to more yellowish coloration throughout the storage time. Zhai et al., 2017 [69] showed similar results in a study to control carp freshness using colorimetric films with different anthocyanin concentrations.

ness using colorimetric films with different anthocyanin concentrations. The film with the lowest concentration changed from a purple color, going through green, to yellow in a period of about 6 days, while the film with the highest concentration showed no color differences until the third day. In the case of Wu et al., 2019 [70], the color changes were observed at 24 h of storage, going from purple to grayish-blue or brown depending on the anthocyanin concentration of the film.

These results were probably related to the increase of TVB-N (total volatile basic nitrogen). The antioxidant capacity of anthocyanins migrating to fish would inhibit the formation of volatile substances with nitrogen in storage [70], and their effects could vary depending on whether they are in contact with fish. In our case, the film itself simply by being on the surface of the food reduced exposure to oxygen and thus oxidation. In addition, the salmon used in our study has muscle tissue with a reddish coloration due to its high myoglobin content in comparison to Zhai et al., 2017 [69] and Wu et al., 2019 [70], which may affect the color measurement of the films covering the samples.

#### *2.4. Sensorial Analysis*

The control samples presented the highest degree of fishy odor established by the panelists (Figure 4), while the lowest value corresponded to the treatment with the film with blueberry extracts. Albertos et al., 2015, 2018 [68,71] performed a sensory analysis in

which similar results were obtained concerning the control of fish odor, rancid odor, and ammonia, which reflects the deterioration of fish meat. In addition, the introduction of red fruit extract prevented the formation of unpleasant odors (fishy smell, rancid smell, and ammonia smell) in the film-covered samples, detecting a slightly fruity aroma. The same happened in the study of Albertos et al., 2018 [71], where an "herbaceous" aroma was found in films incorporated with olive leaf powder. which similar results were obtained concerning the control of fish odor, rancid odor, and ammonia, which reflects the deterioration of fish meat. In addition, the introduction of red fruit extract prevented the formation of unpleasant odors (fishy smell, rancid smell, and ammonia smell) in the film-covered samples, detecting a slightly fruity aroma. The same happened in the study of Albertos et al. 2018 [71], where an "herbaceous" aroma was found in films incorporated with olive leaf powder.

The film with the lowest concentration changed from a purple color, going through green, to yellow in a period of about 6 days, while the film with the highest concentration showed no color differences until the third day. In the case of Wu et al. 2019 [70], the color changes were observed at 24 h of storage, going from purple to grayish-blue or brown

These results were probably related to the increase of TVB-N (total volatile basic nitrogen). The antioxidant capacity of anthocyanins migrating to fish would inhibit the formation of volatile substances with nitrogen in storage [70], and their effects could vary depending on whether they are in contact with fish. In our case, the film itself simply by being on the surface of the food reduced exposure to oxygen and thus oxidation. In addition, the salmon used in our study has muscle tissue with a reddish coloration due to its high myoglobin content in comparison to Zhai et al. 2017 [69] and Wu et al. 2019 [70],

The control samples presented the highest degree of fishy odor established by the panelists (Figure 4), while the lowest value corresponded to the treatment with the film with blueberry extracts. Albertos et al. 2015, 2018 [68,71] performed a sensory analysis in

which may affect the color measurement of the films covering the samples.

*Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 11 of 19

depending on the anthocyanin concentration of the film.

**Figure 4.** Sensorial analysis on day 2. Aromatic (A), rot (R), oxidation (Ox), dehydration (D), general acceptability (GA), fishy odor (FO), rancid odor (RO), ammonia odor (AO), and others (O). **Figure 4.** Sensorial analysis on day 2. Aromatic (A), rot (R), oxidation (Ox), dehydration (D), general acceptability (GA), fishy odor (FO), rancid odor (RO), ammonia odor (AO), and others (O).

The control samples showed variability in the different parameters such as fish odor, putrefactive odor, degree of dehydration, ammonia odor... etc., over storage. On the other hand, in the treatment with the film with the blueberry extract, the values remained constant. This would indicate the effectiveness of films in preserving the fish characteristics. The control samples showed variability in the different parameters such as fish odor, putrefactive odor, degree of dehydration, ammonia odor . . . etc., over storage. On the other hand, in the treatment with the film with the blueberry extract, the values remained constant. This would indicate the effectiveness of films in preserving the fish characteristics.

#### **3. Materials and Methods**

#### *3.1. Materials*

*2.4. Sensorial Analysis*

All the chemicals used in the formulation of films were food-grade quality Panreac products (Panreac Química, Barcelona, Spain). Other reagents were purchased from Sigma-Aldrich (Sigma Aldrich Chemical Co., Steinheim, Germany), and pectin was supplied by Guinama (Guinama S.L.U., Valencia, Spain).

#### *3.2. Raw Materials*

Blackberry, raspberry, and blueberry wastes were obtained from Viveros Campiñas (Chañe, Segovia, Spain). Berry wastes were disinfected with sodium hypochlorite (12 ◦C) for 15 min and immediately dried at room temperature. Afterward, blackberry, raspberry, and blueberry wastes were steamed at 100 ◦C for 3 min. Then, they were frozen at −83 ◦C until berry extract preparation.

Gutted salmon (*Salmon salar*) was provided by Gallega de Distribuidores de Alimentación (GADISA) (Ávila, Spain). Fish was captured in the north of Galicia. The salmon was stored at 4 ◦C and immediately processed.

#### *3.3. Development of Films with Berry Extracts*

#### 3.3.1. Preparation of Berry Extracts

Berries wastes were lyophilized (Lyoquest-55, Azbil Telstar Technologies SLU, Terrasa, Spain) and then milled and sieved (100–300 µm) to obtain the residue in powder form.

Berry extracts were then dissolved in distilled water (12.5% *w*/*v*) for 2 h of stirring at room temperature. After, the suspension was centrifugated at 6000 rpm for 10 min and filtered through Whatman grade number 1 filter paper. The extracts were then stored at −80 ◦C until use.

## 3.3.2. Development of the Films Pectin Films

Low methoxy amidated pectin (3% *w*/*v*) was dissolved in water and stirred at 80 ◦C to obtain a homogenous solution. Afterward, glycerol (3%/biopolymer) was added as a plasticizer for 2 h to achieve complete dispersion. The films were obtained by casting 20 mL in 90 mm-diameter Petri dishes and dried at room temperature for 24 h. Before analyses, the films were peeled-off and conditioned in desiccators over a saturated solution of KBr (58% relative humidity) (Figure 5). *Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 13 of 19

**Figure 5.** Developed films: (**a**) pectin (control); (**b**) raspberry; (**c**) blackberry; and (**d**) blueberry. **Figure 5.** Developed films: (**a**) pectin (control); (**b**) raspberry; (**c**) blackberry; and (**d**) blueberry.

Films Incorporated with Berry Extracts Films Incorporated with Berry Extracts

Berry extracts (10% *w*/*v*) were completely dissolved in water before pectin addition. From this point, the films were prepared as previously referred (Figure 5). Berry extracts (10% *w*/*v*) were completely dissolved in water before pectin addition. From this point, the films were prepared as previously referred (Figure 5).

#### *3.4. Characterization of the Antioxidant Properties of the Extracts 3.4. Characterization of the Antioxidant Properties of the Extracts*

#### 3.4.1. Total Phenols (TPs) Content 3.4.1. Total Phenols (TPs) Content

Total phenols were measured using the Folin–Ciocalteu method [72]. Results were expressed as mg gallic acid equivalents (GAE) per g of extract using a calibration curve with gallic acid (Sigma Aldrich Co., Steinheim, Germany) as the standard (9.8 μM to 70 Total phenols were measured using the Folin–Ciocalteu method [72]. Results were expressed as mg gallic acid equivalents (GAE) per g of extract using a calibration curve with gallic acid (Sigma Aldrich Co., Steinheim, Germany) as the standard (9.8 µM to 70 µM).

#### μM). 3.4.2. Total Flavonols Content

3.4.2. Total Flavonols Content Total flavonols content was analyzed with Neus reagent according to Arnous, Total flavonols content was analyzed with Neus reagent according to Arnous, Markis, and Kefalas, 2002 [73]. Results were expressed as mg of quercetin equivalents per g of extract.

Markis, and Kefalas, 2002 [73]. Results were expressed as mg of quercetin equivalents per

#### g of extract. 3.4.3. Total Anthocyanin Content

3.4.3. Total Anthocyanin Content Total anthocyanin content was determined using the pH differential method [74]. Dilutions were prepared in 50 mL volumetric flasks with buffers pH 1.0 and pH 4.5.

Total anthocyanin content was determined using the pH differential method [74]. Dilutions were prepared in 50 mL volumetric flasks with buffers pH 1.0 and pH 4.5. Ab-

The effect of antioxidant activity on DPPH was estimated according to the procedure described by Brand-Williams, Cuvelier, and Berset 1995 [75]. Results were expressed as a

The analysis was carried out according to the method reported by Re et al. 1999 [76]; 100 μL of diluted samples was mixed with 1000 μL of ABTS and working solution in an Eppendorf tube. The decay in absorbance at 734 nm was recorded over 30 min with a

percentage of the inhibition of DPPH radical.

3.4.5. TEAC (Trolox Equivalent Antioxidant Capacity)

3.4.4. DPPH (1,1-Diphenyl-2-picrylhydrazyl) Radical Scavenging Activity

Absorbance was then recorded at 520 nm (Thermo Fisher Scientific, Genesys 150, Madison, WI, USA). Results were expressed as mg of malvidin-3-glucoside per L of extract.

#### 3.4.4. DPPH (1,1-Diphenyl-2-picrylhydrazyl) Radical Scavenging Activity

The effect of antioxidant activity on DPPH was estimated according to the procedure described by Brand-Williams, Cuvelier, and Berset 1995 [75]. Results were expressed as a percentage of the inhibition of DPPH radical.

#### 3.4.5. TEAC (Trolox Equivalent Antioxidant Capacity)

The analysis was carried out according to the method reported by Re et al., 1999 [76]; 100 µL of diluted samples was mixed with 1000 µL of ABTS and working solution in an Eppendorf tube. The decay in absorbance at 734 nm was recorded over 30 min with a spectrophotometer (Thermo Fisher Scientific, Genesys 150, Madison, WI, USA). Trolox was used as the standard (7.5–240 µM). Results were corrected for moisture and expressed as µmol TE 100 g−<sup>1</sup> d.m.

#### *3.5. Characterization of the Developed Films*

#### 3.5.1. Film Thickness and Density

The film thickness was measured using a digital micrometer (Mitutoyo, model IDC 112, Kawasaki, Japan). The results were expressed as the average of 10 replicates of samples taken from different locations on the film surface.

Density was determined from rectangular samples of the developed films with the following dimensions: 30 mm × 20 mm. Then, each film was weighted, and for volume calculation, the thickness of each film was used.

#### 3.5.2. Hardness

The films' hardness (g force) was determined according to the method of Bamdad et al., 2006 [77], with some modifications. Samples were measured in six different areas, using a texturometer (Brookfield, LFRA 1500, Middleborough, MA, USA), a stainless-steel probe with 4 mm diameter (TA44), with a target value of 6 mm of penetration and a test speed of 0.5 mm/s.

### 3.5.3. Color

Color measurements were performed using a Minolta CR-400 colorimeter (Minolta Inc, Tokyo, Japan) with D65 as illuminant and 10◦ observer angle. The instrument was calibrated with a white tile standard (L\* = 93.97, a\* = −0.88 and b\* = 1.21). To measure the color of films, a white surface was used as background. The L\* parameter (lightness index scale) ranges from 0 (black) to 100 (white). The a\* parameter measures the degree of red (+a) or green (−a) color and the b\* parameter measures the degree of yellow (+b) or blue (−b) color. Three measurements were taken from each sample, and six samples from each film were tested.

#### 3.5.4. Opacity

The opacity of the samples was calculated based on the method reported by Martins et al., 2010 [78], as the relationship between the opacity of each sample in a black standard (Yb) and the opacity of each sample in a white standard (Yw), as can be seen in Equation (1):

$$\text{Operity} \left( \% \right) = \text{Yb} / \text{Yw} \times 100 \tag{1}$$

#### 3.5.5. Color Changes at Different pHs

The films were cut into pieces of 2 cm<sup>2</sup> and were immersed in different pH buffers to adjust pH values to 2, 3, 4, 5, 6, 7, 8, 9, 10, and 12. Afterward, the color was determined with a colorimeter on solids PCE-RGB (PCE Ibérica S.L., Albacete, Spain).

## *3.6. Biodegradation Tests* 3.6.1. Soil

The biodegradation test in soil was based on the methodology used by Pereira et al., 2021 [54]. The films were cut (3 cm × 2 cm) and placed inside a perforated polyethylene net (5 cm × 4 cm; mesh opening 4 mm). Then, the films were buried in soil (Eco grow: nitrogen = 80–150 mg L−<sup>1</sup> ; phosphorus = 80–150 mg L−<sup>1</sup> ; potassium = 80–150 mg L−<sup>1</sup> ; organic Matter = >70%; pH = 5.5–6.5; humidity = 50–60%; conductivity = 0.2–1.2 EC) at a distance of 11 cm from the surface in a rectangular vase (71 cm × 26 cm × 25.5 cm) and with a distance of 5 cm between each film. The soil was watered with 500 mL of water every 7 days at 25 ◦C. The films' appearance was photographed, and the area of biodegradation was measured during the time of the experiment. This test was carried out in triplicate for each sample.

### 3.6.2. Seawater

The biodegradation test in seawater was based on the methodology used by Pereira et al., 2021 [54]. The films were cut (3 cm × 2 cm) and submerged in 300 mL of seawater (Faro, Portugal, pH = 7.20). The samples were shaken at 150 rpm (Edmund Bühler, KL2 shaker, Tübingen, Germany) and 25 ◦C. The films' appearance was photographed during the time of the experiment. This test was carried out in triplicate for each sample.

### *3.7. Monitoring the Shelf-Life of Salmon Fillets*

### 3.7.1. Preparation and Treatments of Salmon Samples

Gutted salmon (*Salmon salar*) was skinned and filleted, then cut into pieces of 5.7 × 2.5 cm (weighing approximately 10 g) and randomly allocated into 3 batches: fish without film (control), pectin film (pectin), and pectin film with blueberry extract. Each fish sample was individually wrapped with 90 mm-diameter films (control, pectin, and blueberry) with the help of tweezers under hygienic conditions. Afterward, all treatments were stored at 4 ◦C for 7 days. The assay was run in duplicate. All analyses were performed in triplicate.

#### 3.7.2. pH

Each fish sample (10 g) was homogenized in 100 mL of distilled water, and the mixture was filtered. The pH (pH-meter model basic 20, Crison, Barcelona, Spain) of the filtrate was measured at room temperature.

#### 3.7.3. Moisture

The moisture content of each sample was gravimetrically determined [79]: 10 g of fish were prepared until constant weight in an air oven at 100 ◦C for about 24 h. The moisture was expressed in percentage.

#### 3.7.4. Color Changes

Color changes of films and fish surface without film were measured using a colorimeter on solids PCE-RGB (PCE Ibérica S.L., Albacete, Spain) in the same conditions as previously referred to in Section 3.5.5.

#### 3.7.5. Sensorial Analysis

Samples were subjected to a descriptive test. A trained panel consisting of 10 panelists, formed by 5 men and 5 women, with ages within 25–30, were recruited from the Catholic University of Ávila, for their previous experience in sensory analysis.

In the descriptive test, the panelist scored the following different attributes: fishy (off-odors, putrefaction) odor intensity, aromatic odor intensity, ammonia odor intensity, other odors, drip loss, color without film (oxidation), and general acceptability. The scores ranged between 1 and 10.

## *3.8. Statistical Analysis*

Data were analyzed by a one-way ANOVA. Fisher's LSD (Least Significant Difference) test was applied at a significance level of 0.05 for determining group differences. In the color changes of salmon fillets, a two-way ANOVA (treatment, time) was performed. Kruskal–Wallis test was used to examine differences in the sensorial analysis. The software Statgraphics Centurion XVI was employed for carrying out the statistical analysis.

#### **4. Conclusions**

Berries are a source of anthocyanins and can be used as an effective method for controlling the quality and freshness of food as pH indicators. The concentration of anthocyanins in the developed films was a key factor influenced by both the intensity and the rate of color change. The addition of berry extracts to pectin films contributed to changing the films' properties. Moreover, the pectin films' supplement with blueberry extracts not only showed biodegradability properties in soil and seawater but also protected the salmon samples from deterioration due to their anthocyanin content and antioxidant capacity, increasing the salmon's shelf-life. This study contributes to the valorization of berries' agro-industrial waste by the development of eco-friendly films that can be used in the future as food packaging materials to meet the market demands. However, more studies are needed to evaluate different microbiological and quality parameters.

**Author Contributions:** Conceptualization, I.A.; formal analysis, J.R., R.M.S.C., A.D.-M. and I.A.; investigation, J.R., R.M.S.C., A.D.-M. and I.A.; resources, J.R., R.M.S.C., A.D.-M. and I.A.; writing original draft preparation, J.R., R.M.S.C., A.D.-M. and I.A.; writing—review and editing, J.R., R.M.S.C., A.D.-M. and I.A.; supervision, J.R., R.M.S.C., A.D.-M. and I.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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