**Sustained Release of Linezolid from Prepared Hydrogels with Polyvinyl Alcohol and Aliphatic Dicarboxylic Acids of Variable Chain Lengths**

#### **Gustavo Carreño 1,2, Adolfo Marican 1,2, Sekar Vijayakumar 3, Oscar Valdés 4, Gustavo Cabrera-Barjas 5, Johanna Castaño <sup>6</sup> and Esteban F. Durán-Lara 2,7,\***


Received: 10 September 2020; Accepted: 15 October 2020; Published: 17 October 2020

**Abstract:** A series of hydrogels with a specific release profile of linezolid was successfully synthesized. The hydrogels were synthesized by cross-linking polyvinyl alcohol (PVA) and aliphatic dicarboxylic acids, which include succinic acid (SA), glutaric acid (GA), and adipic acid (AA). The three crosslinked hydrogels were prepared by esterification and characterized by equilibrium swelling ratio, infrared spectroscopy, thermogravimetric analysis, mechanical properties, and scanning electron microscopy. The release kinetics studies of the linezolid from prepared hydrogels were investigated by cumulative drug release and quantified by chromatographic techniques. Mathematical models were carried out to understand the behavior of the linezolid release. These data revealed that the sustained release of linezolid depends on the aliphatic dicarboxylic acid chain length, their polarity, as well as the hydrogel crosslinking degree and mechanical properties. The in vitro antibacterial assay of hydrogel formulations was assessed in an *Enterococcus faecium* bacterial strain, showing a significant activity over time. The antibacterial results were consistent with cumulative release assays. Thus, these results demonstrated that the aliphatic dicarboxylic acids used as crosslinkers in the PVA hydrogels were a determining factor in the antibiotic release profile.

**Keywords:** hydrogel; polyvinyl alcohol; aliphatic dicarboxylic acids; sustained release; linezolid; equilibrium swelling ratio; accumulative release; thermogravimetric analysis

#### **1. Introduction**

Multidrug-resistant (MDR) bacteria or "superbugs" represent one of the most important challenges to public health and pose a huge economic burden on global health care [1,2]. Indeed, antibacterial resistance causes 700,000 deaths per year worldwide [3]. The World Health Organization (WHO) has classified *Enterococcus faecium* as one of the primary drug-resistant pathogens posing the most significant risk to public health. This bacterium causes urinary tract infections, hospital-acquired bloodstream

infections, abdominal and pelvic abscesses, endocarditis, and chronic periodontitis. The importance of this pathogen in these types of infection is reinforced by their intrinsic and acquired resistance to various antimicrobial agents, which renders them challenging to treat [4]. Linezolid is among the few available antibiotics that can treat bacterial resistance. This antibiotic is the first clinically useful oxazolidinone antibacterial agent [5]. This chemotherapeutic agent has been approved for the treatment of complicated skin and skin-structure tissue infections principally caused by vancomycin-resistant *E. faecium* [6]. Recent studies indicate that the effectiveness of antibacterial agents is better when they are released through drug delivery systems. Furthermore, these systems could slow down the progression of bacterial resistance to antibiotics [7,8]. In this context, hydrogels appear to be excellent candidates as antibiotic delivery platforms [9]. Hydrogels are a form of 3D porous material; these biomaterials consist of polymer chains with physical or chemical crosslinking [10]. Hydrogels can be of natural and/or synthetic origin [11]. Polyvinyl alcohol (PVA) hydrogels have been deeply explored due to their excellent biocompatibility properties and have been FDA (Food and Drug Administration) approved [12]. Hydrogels have received growing attention as drug delivery systems over the last decade due to their exclusive properties, such as high biocompatibility, tunable release rate, and versatility to be loaded with different molecules [13,14]. The most relevant characteristics of hydrogels are their porosity, pore size, and physicochemical environment of the matrix, which is tunable by modifying the crosslink density and/or varying the crosslinker type in their network. Therefore, the crosslinking agents such as aliphatic dicarboxylic acids (ADAs) could play a key role in the drug release profile from the hydrogel. In the hydrogel network, the pore size, swelling capacity, affinity with the drug, and subsequent sustained release have a very close relationship with the chemical structure and length of ADAs [14,15]. New antibacterial therapy strategies based on the sustained or prolonged release of linezolid could be an effective solution to fight pathogens. Thus, the purpose of this study was to develop hydrogels with ADAs of variable chain lengths as crosslinker agents featuring the tunable sustained release of linezolid.

#### **2. Materials and Methods**

#### *2.1. Materials*

Polyvinyl alcohol (PVA) 30–60 KDa, succinic acid (SA), glutaric acid (GA) adipic acid (AA), NaHCO3, acetonitrile (HPLC grade), and linezolid analytical standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). HCl, methanol (HPLC grade), K2HPO4, and H3PO4 were purchased from Merck (Darmstadt, Germany). All solutions were prepared using MilliQ water. *Enterococcus faecium* ATCC® 19434 bacterial strain, brain heart infusion (BHI) agar, Luria–Bertani (LB), and peptone water were purchased from Merck (Darmstadt, Germany). Distilled water was utilized for the preparation of all the solutions in the antibacterial study. The mouse fibroblast cell line L929 (ATCC® CCL-1™) was purchased from ATCC (Manassas, VA, USA). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco®, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, Gibco®, Grand Island, NY, USA) and antibiotics (100 U penicillin and 100 U/mL streptomycin, Gibco®, Grand Island, NY, USA) under 5% CO2 at 37 ◦C. Cells were harvested after reaching confluence by using 0.05% trypsin–EDTA (Gibco®, Grand Island, NY, USA).

#### *2.2. Synthesis of Hydrogels Based on PVA, ADAs, and Linezolid Loading*

For this study, three hydrogels based on PVA and ADAs were synthetized. The methodology for preparing the hydrogels was performed through the esterification of PVA with ADAs according to the method from Rodríguez Nuñez et al. with minor modifications [16]. Briefly, the esterification reactions were performed by mixing an aqueous solution of PVA with an aqueous solution of a specific ADA (20 wt%) using HCl (1 <sup>×</sup> 10−<sup>1</sup> mol·L−1) and temperature as catalysts of crosslinking. Then, each reaction was performed under reflux at ~90 ◦C in a necked flask with magnetic agitation in the presence of air. After 3 h, each pre-hydrogel solution was poured into a new flask, and 8 mg of linezolid was added for its encapsulation, as depicted in Table 1. Then, each solution was vigorously stirred

for 1 h and then sonicated for another hour until a homogenized solution was reached. After that, each pre-hydrogel-linezolid solution was put in an oven at 45 ◦C overnight until the crosslinking was complete. The hydrogel of PVA cross-linked with SA is named PSAH, the hydrogel of PVA cross-linked with GA named PGAH, and the hydrogel of PVA cross-linked with AA named PAAH. Afterward, the PSAH, PGAH, and PAAH hydrogels with the encapsulated linezolid were partially neutralized with NaHCO3 to remove the excess acid and increase water uptake [17]. Then, the linezolid-loaded hydrogels were lyophilized to obtain the xerogel. The linezolid-loaded PSAH, linezolid-loaded PGAH, and linezolid-loaded PAAH were termed PSAH-Li, PGAH-Li, PAAH-Li, respectively. At the same time, three hydrogels were prepared without linezolid to perform the following characterizations: ESR, FT-IR, TGA, mechanical analysis, and SEM.

**Table 1.** Specifications of prepared hydrogels and the amount of linezolid loading.


\* % *w*/*w* respect to hydrogel; succinic acid (SA); glutaric acid (GA), and adipic acid (AA).

#### *2.3. Swelling Behavior*

The swelling behavior was calculated by the equilibrium swelling ratio (% ESR) at desired time intervals. Each xerogel film was immersed in phosphate buffer saline (PBS) [18] and acetate buffer (pH 3.0) at 25 ◦C for 21 h until swelling equilibrium was attained. The weight of the wet sample (*W*<sup>w</sup> (g)) was obtained after carefully eliminating moisture on the surface with an absorbent paper. The weight of the dried sample (*W*<sup>d</sup> (g)) was acquired after freeze-drying the hydrogel sample. The ESR of the hydrogel samples was obtained as follows:

$$\text{ESR} \left( \% \right) = \frac{\text{W}\_w - \text{W}\_d}{\text{W}\_d} \times 100\% \tag{1}$$

#### *2.4. FT-IR Analysis*

The freeze-dried samples were ground into small fragments. After that, the PSAH, PGAH, and PAAH were analyzed in KBr (potassium bromide) disks by Fourier transform infrared spectroscopy (Nicolet Nexus 470 spectrometer, Thermo Scientific, Waltham, MA, USA). The wavenumber range scanned was 4000–500 cm<sup>−</sup>1; 32 scans of 2 cm−<sup>1</sup> resolution were signal-averaged and stored. The films utilized in this analysis were sufficiently thin to obey the Beer–Lambert law.

#### *2.5. Thermogravimetric Analysis*

The thermal stability of PVA crosslinked films was evaluated using a thermobalance Cahn-2000 (Ventron Corp., CA, USA). Thermal analysis was carried out by heating samples (10 mg) from 25 to 600 ◦C at a heating rate of 10 ◦C/min under a nitrogen atmosphere (50 mL/min). The sample weight loss was recorded as a function of temperature.

#### *2.6. Mechanical Properties*

The tensile strength (TS), tensile modulus (E), and elongation at break (eB) of the hydrogels were measured according to American Society for Testing Materials (ASTM) D 882 test methods using an Autograph AGS-X Universal Tester (Shimadzu, Kyoto, Japan). The tensile samples were cut into rectangular shapes with dimensions of 100 mm in length and 10 mm in width. The gauge length was fixed at 50 mm, and the speed of the moving clamp was 5 mm·min−1. Three samples were tested, and the average values were taken as the reported results.

#### *2.7. Scanning Electron Microscopy Analysis*

Scanning Electron Microscopy (SEM) studies were carried out for all three formulations. The formulations morphology was evaluated using a scanning electron microscope (JEOL-JSM 6380, JEOL, Tokyo, Japan) operated at 15 kV. Surface views of cryogenically fracture films were examined. All samples were sputtered with a gold layer around 40 nm in thickness prior to the examination.

#### *2.8. Release Kinetics Studies*

The conformation of each proposed hydrogel is described in Table 1. Each linezolid-loaded hydrogel with a weight of 400 mg was placed into a 10 mL tube, and 5 mL of PBS [18] was poured over the formulation as a release medium. The tubes were transferred to an orbital shaking water bath (Faraz teb, Tehran, Iran) at 33.5 ± 0.1 ◦C [19] and shaken at 35 ± 2 rpm. At specific time intervals, the PBS was removed and replaced with an equal volume of PBS to maintain sink conditions throughout the study. For the quantification of linezolid, a stock solution (3 mg/mL) was prepared in methanol and stored at −18 ◦C. Standard solutions of the antibiotic were prepared with PBS (pH 7.4) in the range of 0.01–50 mg L−1. The chromatographic system consisted of a Perkin Elmer series 200 HPLC system (Norwalk, CT, USA) with a UV–vis detector and a C-18 Kromasil 100-5-C18 (250 mm × 4.6 mm i.d. × 5 μm) column. Fifty microliters of the sample were injected into the HPLC apparatus. Isocratic elution with methanol/water (50:50, *v*/*v*) at a constant flow rate of 1.0 mL min−<sup>1</sup> was utilized as the mobile phase. The analytical wavelength was 254 nm at room temperature The release rate of linezolid-loaded hydrogels was acquired by applying the concentration of released linezolid to the following correlation (Equation (2)):

$$\text{Cumulative Li release } (\%) = \text{Cumulative amount of Li released} \times \frac{100}{\text{Initial amount of Li}} \qquad (2)$$

Linezolid release kinetics were performed by employing different mathematical models of drug release equations, such as zero-order (Equation (3)), first-order (Equation (4)), Hixson–Crowell (Equation (5)), Higuchi (Equation (6)), Korsmeyer–Peppas (Equation (7)), and Peppas–Sahlin (Equation (8)) [20,21]:

$$Q\_t / Q\_0 = K\_0 t \tag{3}$$

$$\ln Q\_l / Q\_0 = K\_1 t \tag{4}$$

where *Qt* is the amount of linezolid released at time t, and *Q*<sup>0</sup> is the original linezolid concentration in the formulation.

$$\mathbf{C}\_0 \mathbf{^{1/3}} - \mathbf{C}\_l \mathbf{^{1/3}} = \mathbf{K}t \tag{5}$$

where *Ct* is the amount of drug released in time *t*, *C*<sup>0</sup> is the initial amount of linezolid in the formulation, and *K* is the rate constant.

$$Q = K t^{1/2} \tag{6}$$

where *Q* is the cumulative linezolid release, *K* is the Higuchi release constant, and *t* is the time.

$$\frac{M\_t}{M} = Kt^n \tag{7}$$

where *Mt*/*M* is the cumulative linezolid release, *K* is the release constant, *t* is the time, and n is the release exponent.

$$\frac{M\_t}{M\infty} = \mathcal{K}dt^n + \mathcal{K}rt^{2n} \tag{8}$$

where *Mt* and *M*<sup>∞</sup> are the absolute cumulative amounts of linezolid release at time *t* and at infinite time, respectively.

#### *2.9. Antibacterial Activity*

The studies were performed according to Oscar Forero-Doria et al. [8]. First, 50 mg of each linezolid-loaded hydrogel was placed into a tube with 5 mL of PBS [18] as "release medium". Concurrently, a tube with 5 mL of PBS loaded with 1 mg of linezolid was prepared and utilized as a control. After that, the tubes were placed into an orbital shaking water bath (Farazteb, Iran) at 37 ± 0.1 ◦C. Depending on the assay, at certain time intervals (1, 3, 6, 24, and 48 h) 200 μL of release medium was taken and replaced with an equal volume of PBS to maintain sink conditions throughout the study. Lastly, the samples of each tube were evaluated by screening the antimicrobial activity and quantitatively testing the antibacterial activity utilizing the following protocols.

#### *2.10. Assessment of Antimicrobial Activity of Proposed Hydrogels against E. faecium*

To estimate the inhibitory activity against *E. faecium*, a qualitative test with a ring-diffusion method was implemented. With the aim of assessing the antibacterial activity of the prepared hydrogels, the Gram-positive strain *E. faecium* ATCC® 19434 was used as a model pathogen. The bacteria were grown overnight in MRS (de Man Rogosa Sharpe) broth at 37 ◦C. The inoculum (100 μL) containing *E. faecium* (adjusted to <sup>∼</sup>1.0 <sup>×</sup> 10<sup>6</sup> CFU·mL<sup>−</sup>1) was seeded previously on the agar. Next, wells (8 mm in diameter) were made on an agar plate and filled with 100 μL of release medium for the specific interval times from Section 2.9. Additionally, two internal controls were treated with linezolid (10 and 15 <sup>μ</sup>g·mL−1, respectively). The plates were incubated at 37 ◦C for 24 h, and the antibacterial activity was calculated by the formation of bacterial inhibition zones surrounding the film disks. All tests were performed in duplicate.

#### *2.11. Quantitative Assay of the Antibacterial Activity of Proposed Hydrogels against E. faecium*

In this analysis, *E. faecium* ATCC® 19434 (concentration range of 1.0 <sup>×</sup> 106 CFU·mL−1) was inoculated in 1 mL of LB broth at 37 ◦C until reaching turbidity equivalent to a 0.5 McFarland standard. Afterward, 150 μL of release medium from the samples and controls of Section 2.8 was added to 2 mL of the previous inoculation solution and then shaken at 200 rpm for 24 h at 37 ◦C. Afterward, each culture was tested; serial dilutions were made in 0.1% sterile peptone water. From each of these dilutions, 100 μL aliquots were collected, which were placed in plate count agar and incubated at 37 ◦C for 24 h. Then, viable cell counts were performed. All trials were performed in triplicate.

#### *2.12. Cytotoxicity and Cell Viability*

The cytotoxicity of the proposed hydrogels was evaluated on fibroblast cells. For this goal, the viability of the cells was assessed using the MTT technique according to the protocol of Mossman et al. [22]. Briefly, the cells were seeded in 24-well plates (5 <sup>μ</sup>L, 1.6 <sup>×</sup> 104 cells per well) and 150 <sup>μ</sup>L of Dulbecco's Modified Eagle Medium (DMEM)-High medium was added and incubated for 24 h at 37 ◦C in 5% CO2. Afterward, the medium was replaced by 100 μL of fresh DMEM-High per well, which containing three diverse concentrations of PSAH, PGAH, and PAAH (500 <sup>μ</sup>g·mL−1, 1500 <sup>μ</sup>g·mL−1, and 2500 <sup>μ</sup>g·mL−<sup>1</sup> per hydrogel). Fresh medium without a sample was utilized as a control. Cell viability was assessed after 24 h by the MTT technique. Briefly, 5 μL of MTT solution (3 mg·mL−<sup>1</sup> in PBS) and 50 <sup>μ</sup>L of fresh medium were added to the respective sample and incubated for 4 h in the dark at 37 ◦C; formazan crystals were then dissolved in 100 μL of DMSO and incubated for 18 h. Supernatant optical density (o.d.) was analyzed at 570 nm (Spectrophotometer, Packard Bell, Meriden, CT, USA). Unprocessed fibroblast cells were taken as control with 100% viability. The hydrogels cytotoxicity was depicted as the relative viability (%), which correlates with the number of viable cells compared with the negative cell control (100%).

#### *2.13. Statistical Analysis*

In this work, all experiments were performed in triplicate. The SPSS 9.0 statistical software (IBM, Chicago, IL, USA, 1999) was used to perform the ANOVA analysis and Tukey's test (*p* < 0.05) to determine the statistical significance in some experiments such as the mechanical properties, ESR analysis, cumulative release test, quantitative test of antibacterial activity, and MTT assay. Graphs of the study results were designed by utilizing GraphPad Prism 6. Statistical significance was set at *p* < 0.05.

#### **3. Results and Discussion**

#### *3.1. Synthesis and Load of Hydrogels*

In Figure 1 the preparation of the hydrogels is depicted. Each hydrogel was prepared by esterification between PVA with a specific ADA (SA, GA, and AA). Once the pre-hydrogel was formed, the linezolid was added for its encapsulation (see Table 1). By this simple methodology, it is possible to achieve over 99% retention of the drug. Considering previous studies, a crosslinking degree of 10:2 of PVA:ADA was prepared, which was kept constant due to its good characteristics, such as porosity, mechanical properties, among others [14,16,23].

**Figure 1.** Synthesis and proposed structures of the linezolid-loaded hydrogels.

#### *3.2. Swelling Behavior*

This analysis allowed the confirmation of network formation in the three hydrogels. Since it is desirable to study the release of the antibiotic under physiological pH conditions, the ESR was evaluated at pH 7.4. Moreover, as a comparative analysis of swelling behavior, this analysis was also assessed at pH 4.0. Figure 2 shows the ESR for all formulations. An increase in the swelling index rate of the three hydrogels over time was observed at both pH values. In the beginning, the hydrogel swelling ratio increased fast and then slowed down to reach an equilibrium. This swelling behavior is characteristic of the hydrogel matrix obtaining the maximum swelling capacity. Specifically, PSAH, PGAH, and PAAH reached swelling equilibrium (zero-order) at approximately 3–4 h. On the other hand, a significant difference (*p* < 0.05) in all the cases was observed between the two pH models. For example, PSAH showed a better swelling degree at pH 7.4 with a value of approximately 600%, while at pH 4.0 the swelling degree was about 500%. For the other samples, PGAH and PAAH showed a swelling degree at pH 7.4 around 440% and 230%, and at pH 4.0 about 210% and 180%, respectively. The ESR difference at both pH values is due to the protonation degree of the free aliphatic carboxylic acids into the hydrogel network, which has different types of pKa [24]. Particularly, PGAH showed a higher difference in swelling behavior between pH values; such behavior can be attributed to the dissociation degree and ionization process of free COO- groups. These results are coherent, considering that the glutaric acid has the lowest pKa2 (5.22) compared to that of succinic and adipic acid (pKa2: 5.64 and 5.41), respectively. Therefore, in the network of PSAH more free carboxylic acid groups are susceptible to ionization by pH change.

**Figure 2.** The swelling ratio of the hydrogels at 24–25 ◦C as a function of time, pH, and crosslinker nature. Data are shown as mean ± SD (*n* = 3).

When correlating the pH and Time variables for each formulation, it was found that for PSAH, the *p*-value < 0.05 (0.0140) in the ANOVA, therefore, there is a statistically significant relationship between the variables at the 95% confidence level. The highest *p*-value on the independent variables is 0.1175, belonging to pH. Thus, since the *p*-value> 0.05, pH is not statistically significant at the 95% or higher confidence level. In the case of PGAH, the *p*-value< 0.05 (0.0045) in the ANOVA, there is a statistically significant relationship between the variables at the 95% confidence level. On the other hand, the highest P-value on the independent variables is 0.0415 (*p*-value< 0.05), belonging to Time. In this context, Time is statistically significant at the 95% confidence level. Finally, in the case of PAAH, given that the *p*-value> 0.05 (0.1243) in the ANOVA, there is not a statistically significant relationship between the variables at the 95% or higher confidence level. The highest *p*-value on the independent variables is 0.9926 (*p*-value> 0.05), belonging to pH. Because of this, pH is not statistically significant at the 95% or higher confidence level. The correlation models are presented below:

PSAH correlation model:

$$\text{1\% ESR} = 16\text{7.86} + 6.5042t \text{ (R}^2 = 35.85\text{)}\tag{9}$$

PGAH correlation model:

$$\text{V@\_5ESR} = 44.5875 + 37.3718 \text{pH} + 2.867t \text{ (R}^2 = 44.84) \tag{10}$$

PAAH correlation model:

$$\% \text{ ESR} = 202.421 \, + 1.54335t \, \text{(} \text{R}^2 = 14.17\text{)}\tag{11}$$

#### *3.3. FT-IR Analysis*

The FT-IR analysis was conducted in the range from 4000 to 500 cm−<sup>1</sup> to confirm the effectiveness of the crosslinking reaction between the PVA and different ADAs (SA, GA, and AA). Figure 3 shows the FT-IR spectra of hydrogel films (PSAH, PGAH, and PAAH). For PVA, all FT-IR spectra showed most of the characteristic infrared absorption bands (spectra not shown). The spectrum showed a broad band at around 3270 cm<sup>−</sup>1, which was attributed to inter- and intramolecular hydrogen bonds of -OH groups in PVA [25]. After crosslinking, this band showed a significant shift to 3400 cm−<sup>1</sup> caused by the chemical reaction, demonstrating a polymer structural change. Other FT-IR bands appeared between 2840 and 3000 cm−1, around 1688 cm−1, and between 1150 and 1085 cm−1, corresponding to the vibrations of the -CH2, C=O, and C-O-C groups, respectively [26]. In addition, the evidence of ester formation between the PVA hydroxyl groups and diacids carboxylic groups is the shifting of the ADA's -C=O absorption peak from 1691 to 1704 cm<sup>−</sup>1. This result was in agreement with that previously reported in [27]. It the intensity difference for the -CH stretching vibration (between 2840 and 3000 cm<sup>−</sup>1) between crosslinked films could also be noticed. The band intensity increased along with the crosslinker agent carbon numbers, as shown in Figure 1. It is important to note that other significant absorption bands recorded for PSAH, PGAH, and PAAH samples demonstrated the acid compounds in the hydrogel structure [14]. Table 2 summarizes the leading characteristics bands and their assignment for neat PVA and hydrogel samples. Finally, the spectral changes obtained in FT-IR analysis demonstrated the success of the crosslinking reaction between the hydroxyl group of PVA and the carboxylic groups from ADAs.

**Figure 3.** FT-IR spectra of PSAH, PGAH, and PAAH.

**Table 2.** Vibration modes and band frequencies in polyvinyl alcohol (PVA) and PVA crosslinked with SA, GA, and AA.



**Table 2.** *Cont*.

#### *3.4. Thermogravimetric Analysis*

Thermogravimetric (TG) analysis has become a frequently used technique for studying the thermal stability of complex materials. In this work, the thermal properties of the prepared hydrogels based on PVA crosslinked with different ADAs are investigated using TG. The TG and Derivative thermogravimetry (DTG) curves of hydrogel samples are presented in Figure 4, and the analysis results are summarized in Table 3.

**Figure 4.** TG (**A**) and DTG (**B**) curves of the PSAH, PGAH, and PAAH recorded at 10 ◦C/min in the N2 atmosphere.



It is known the degradation of neat PVA is observed over three temperature regions, which are 80–250 ◦C, 275–450 ◦C, and 475–525 ◦C peaking at 142, 287, and 440 ◦C, respectively [27]. For all formulations, a first thermal effect with a maximum decomposition rate from 137 to 142 ◦C and an associated mass loss from 8.8% to 12.7% were observed. This effect is related to the evaporation of the bound and unbound water of the films [25]. The thermal degradation of crosslinked films occurred in three temperature regions for PGAH and PAAH, but in four regions for PSAH. The second degradation step showed two stages for PSAH; the first TPeak appeared at 224 ◦C, and the second at 297 ◦C. The first process could be due to the thermal degradation of the free SA remaining in the hydrogel and showed a weight loss of 8.8%, whereas the second loss was 17.8%. In the case of PGAH and PAAH, the second effect was peaking at 245 and 273 ◦C with associated weight losses of 12.5% and 27.9%, respectively. All those peaks could correspond to a shift in PVA decomposition temperature (287 ◦C) (data not shown). In this thermal effect, the scission of partially esterified but still uncrosslinked PVA chains were co-occurring with polymer cyclization [27]. The third decomposition stage belonged to crosslinked PVA chain decomposition. The maximum decomposition rate appeared from 345 to 369 ◦C, demonstrating an increase in the thermal stability of formed hydrogels. Due to the formation of multiple interand intrachain ester bonds, an interpolymeric network that modifies the PVA structure was created. After crosslinking, an increase in the number of covalent bonds and hydrogen bonding between the polymer chains occurred, making them more thermally stable. This finding agrees with results from other authors that crosslinked PVA with AA and GA [28]. This thermal effect was more relevant for the PGAH sample, showing 39.7% of weight loss. The last thermal effect showed a maximum decomposition rate in the temperature interval of 435–449 ◦C and mass loss associated from 27.8% to 34.8%. Several authors reported that in this process the complete degradation of the PVA backbone occurs and cyclized chains turn them into charring residue [25,27].

#### *3.5. Mechanical Properties*

The influence of the ADA's chain length on the mechanical properties of crosslinked PVA hydrogel films is summarized in Table 4. Tensile strength (TS), tensile modulus (E), and elongation at break (eB) were included in the evaluated tensile properties.


**Table 4.** Mechanical properties of PVA crosslinked films \*.

\* Different letters next to the standard deviation, in each column, indicate statistically significant differences using Tukey HSD (honestly significant difference), at 95% confidence.

The results showed interesting mechanical properties of the hydrogel films prepared with ADAs. We hypothesized that the formation of the ester bond between the ADAs and the PVA matrix would play an important role in the mechanical properties, dependent on the chain length of the ADAs used. Thus, the PGAH hydrogel exhibits better mechanical properties (TS: 25 MPa; eB: 126%) than the PSAH and PAAH hydrogels. It was significantly different in TS values with the PSAH hydrogel and for eB values with the PSAH and PAAH samples, respectively. On the other hand, the PAAH hydrogel showed a significant difference in E values compared to the other ones, which means it is the stiffer sample. This result could be due to the degree of crosslinking obtained between the PVA matrix and glutaric acid chains during the formation of the hydrogel network. This fact agrees with the SEM analysis (Figure 5) of this sample, which showed a rough and homogeneous surface that improves mechanical strength. The mechanical properties of the materials are associated with their chemical nature and the interactions among the forming components [29]. The results reveal that crosslinking agents with intermediate chain lengths (GA) favor a higher chemical interaction with the PVA matrix.

Moreover, thermal analysis results confirm this finding because a higher crosslinking degree was found in this sample. Furthermore, previous hydrogels based on PVA found a linear relationship between mechanical strength and the crosslinking degree [30]. These results are in agreement with the FT-IR analysis regarding the ester band appearance around 1704 cm<sup>−</sup>1. On the other hand, the PAAH hydrogel showed mechanical properties similar to the stiff and brittle materials with high tensile modulus and low elongation at break. This behavior could be associated with the porosity and the highly compact structure found in the fractured surface of hydrogel films observed by the SEM (Figure 5). The lower mechanical behavior of the hydrogel films prepared with PAAH was associated with the crosslinker characteristics, consisting of a larger molecule that restricts the formation of crosslinking density in the hydrogel [31]. The tensile strength and tensile modulus indicate the toughness, and the elongation at break indicates the elasticity of the materials, suggesting their possible applications. The hydrogels of this study can be used for drug delivery applications such as antibiotic release because they have adequate (strong and flexible) mechanical properties [32,33]. Thus, several authors [34,35] reported the following values for the mechanical properties of wound dressing hydrogel films (TS = 18 MPa; E = 98; eB = 200%) and drug delivery films (TS = 13–35; eB = 44–112%), respectively.

#### *3.6. SEM Analysis*

The hydrogel formulations with different crosslinkers were observed using Scanning Electron Microscopy (SEM) to understand surface properties. As depicted in Figure 5, for all samples, a rough surface is observed. In the case of PGAH (Figure 5B), a more uniform structure than those in the PSAH sample could be observed (Figure 5B). Finally, the PAAH micrograph (Figure 5C) revealed a different and more compact morphology with hollows fractures on the surface. It seems that in hydrogel formulations, the surface morphology was highly influenced by the crosslinker chemical structure, being stiff and dense when the ADA's chain length is longer. Then the hydrogel supramolecular structure could affect the drug release behavior.

**Figure 5.** SEM micrographics of PSAH (**A**), PGAH (**B**), and PAAH (**C**).

#### *3.7. Release Kinetics Studies*

The linezolid release profile analysis was carried out by HPLC with a 400 mg hydrogel charged with linezolid. In vitro release kinetics of linezolid from each hydrogel were obtained under physiological conditions (37 ◦C, PBS at pH 7.4). The cumulative percent released of the antibiotic was monitored over time and results are shown in Figure 6.

For all loaded hydrogels (PSAH-Li, PGAH-Li, and PAAH-Li) a rapid antibiotic release into the medium was observed. The cumulative release of PSAH was significantly higher than the other samples from 2 h. From 6 h onward, all hydrogels showed a significantly different drug release profile. At this time, 51%, 40%, and 29% of the linezolid had been released from PSAH-Li, PGAH-Li, and PAAH-Li, respectively. The PAAH-Li revealed a slower release profile than the other two formulations, and PSAH-Li a higher one. After 6 h, the formulations exhibited a significantly lower and continuous antibiotic release into the medium. For PSAH-Li, PGAH-Li and PAAH-Li, the average rapid-release phase was 0.68, 0.53, and 0.39 mg/h of linezolid, respectively. This rate changed after 6 h for all cases, and the average of the slow-release phase was 0.06, 0.05, 0.04 mg/h of linezolid, respectively. In Table 5, all the average release values of the antibiotic are shown. According to these results and the graph depicted in Figure 6, the linezolid release profile follows the next order: PSAH-Li > PGAH-Li > PAAH-Li.

**Figure 6.** Release profile of linezolid from prepared hydrogels in phosphate buffer saline (PBS) at 33.4 ◦C; mean Scanning Electron Microscopy (SEM) (*n* = 3). Different letters next to the standard deviation on each point indicate statistically significant differences using Tukey HSD, at 95% confidence.

**Table 5.** Release profile of linezolid-loaded hydrogels.


\* The rapid phase occurred over 6 h. \*\* The release rate was calculated in a specific time frame because, until 72 h, the formulation still released antibiotic. Different letters next to the standard deviation, in each row, indicate statistically significant differences using Tukey HSD, at 95% confidence.

The release patterns of each formulation depend on the structure of the crosslinker agent, the intermolecular interactions between the linezolid drug and hydrogel network [12], and mostly on the swelling degree. Therefore, in light of the obtained outcomes, it could be deduced that the release rate of PAAH-Li was slower because the expansion of the compact network was minimal, as revealed by the water uptake process (% ESR). One of the reasons for this result is that the size of AA aliphatic chain is larger than SA and GA, respectively, which could contribute to a more apolar environment. Moreover, in concordance with the mechanical studies, PAAH showed a stiffer structure with high tensile modulus and low elongation at break. This performance has a direct relation with its highly compact morphology observed by SEM. The lower mechanical behavior of PAAH could be associated with the larger crosslinker agent that limits the formation of crosslinking density in the hydrogel. With a lesser crosslinking degree, there are less carboxylic groups potentially ionizable. Therefore, there are fewer charges of the electrostatic repulsion between chains from networks and, consequently, less capacity to generate an uptake of solvent into the matrix. This result is consistent with swelling behavior. On the contrary, for the case of PSAH and PGAH with a higher crosslinking degree, the increased swelling ability of the hydrogels contributes to the destruction of hydrogen bonding between the polymer molecules, resulting in an increase in chain mobility and network expansion [21]. The above mentioned results can explain the faster release of linezolid from PSAH-Li. In contrast, in a lesser expanded, more compacted, and stiff hydrogel (PAAH-Li), the encapsulated drug is released slower.

The average release profiles of samples were fitted through several mathematical models to elucidate the mechanism of linezolid release. The coefficients of correlation (*R*) and release exponents (*n*) are shown in Table 6. According to *R2* value obtained, among all the studied models, the Korsmeyer–Peppas model was the best fit for PSAH-Li and PGAH-Li with *R2* values of 0.9967 and 0.9845, respectively. In contrast, an with *R<sup>2</sup>* value of 0.9013, the Higushi model is the best fitted to PAAH-Li. On the other hand, for the case of PSAH-Li and PGAH-Li the release mechanism for linezolid was Fickian diffusion. For the case of PAAH-Li the mechanism for linezolid release was pseudo-Fickian.

**Table 6.** Linezolid release kinetics and correlation coefficient values from Fick, Hixon–Crowell, Higushi and Korsmeyer–Peppas models.


#### *3.8. Antibacterial Studies*

Some studies indicate that the effectivity of antibacterial agents is better when they are applied through sustained release. Furthermore, this approach could lower bacterial resistance incidence [7,8,36]. Regarding the limitations presented by PSAH (faster release) and PAAH (reduced mechanical properties), PGAH was selected to carry out the antibacterial analyses. The results obtained here are consistent with the acquired data by HPLC. For instance, linezolid antibacterial activity was significantly higher over time compared to the control, as shown in Figure 7. In this context, it is concluded that a sustained release at a relatively constant dose from PGAH-Li maintains antibiotic integrity, comprising better activity over time, as shown in Figure 7B,C. On the contrary, in the control sample, linezolid displayed higher activity in the first hour; however, it loses effectiveness over time, as depicted in Figure 7A,C. The control inhibition zone in the first hour was close to ~30 mm, but over time was decreasing, reaching ~8 mm at 48 h. In contrast, PGAH-Li started with an inhibition zone of ~11 mm and progressively rose to complete an inhibition zone of 23 mm at 48 h. These results are in concordance with the quantitative analysis of antibacterial activity against the *E. faecium* that is exhibited in Figure 7D. The data revealed that, in the control, the bacterial colony forming unit (CFU) increases over time, demonstrating that the antibiotic loses its activity until 72 h. Conversely, the assay with the PGAH-Li release medium significantly inhibits bacterial proliferation, suggesting that the linezolid acted as a bacteriostatic agent against *Enterococcus faecium* [37]. An additional experiment with PGAH without linezolid was performed. However, the antibacterial activity was not observed (data not shown). These data suggest that the hydrogel could improve the bioavailability of linezolid.

#### *3.9. Cytotoxicity Studies*

As potential biomaterials, it is pivotal that the designed formulations be innocuous. Therefore, the cytotoxicity of each hydrogel was evaluated on fibroblast cells. The biocompatibility of the sterilized PSAH, PGAH, and PAAH was investigated by a cell viability assay using L929 fibroblast cells after 24 h. Figure 8 displayed cell viability after exposure to three different concentrations of the respective formulation (a concentration range of 500–2500 <sup>μ</sup>g·mL<sup>−</sup>1). As specified in Figure 8, at 500 <sup>μ</sup>g·mL−1, the fibroblast cell viability is nearly 100% for three hydrogels. When drastically increasing the hydrogel concentration from three to five-fold, the fibroblast cell viability only declines vaguely. That is to say, for all cases, the cell viability was not less than 87%. The results revealed that the prepared hydrogels have minimum toxicity to the fibroblast cell. These data could guarantee that these hydrogels can be potential candidates for medical applications.

**Figure 7.** Screening of the antibacterial effect of PGAH-Li. Control (**A**); PGAH-Li (**B**); T0: 0 h, T1: 1 h, T3: 3 h, T4: 6 h, T24: 24 h, T48: 48 h; the antibacterial effect was expressed as the inhibition area against *E. faecium* (**C**); quantitative test of antibacterial activity against *E. faecium* (**D**). (Equal letters above the bars indicate that there are no statistically significant differences using Tukey's HSD procedure, at 95% confidence level). C1 and C2 in Figure 7B are positive controls of 15 and 10 <sup>μ</sup>g·mL−<sup>1</sup> linezolid, respectively.

**Figure 8.** Percentage of cell viability obtained from the MTT assay of the L929 fibroblast cells compared to that of a negative control (without hydrogel). Each bar indicates mean ± relative standard deviations (RSD) of three replications. Bars not labeled by the same letter represent statistically significant differences with the negative control at *p* ≤ 0.05 using ANOVA followed by Tukey's HSD test.

#### **4. Conclusions**

A series of hydrogels based on PVA and ADAs of variable chain lengths with sustained release of linezolid properties were successfully synthesized. The hydrogels were prepared by crosslinking of PVA and different ADAs of varying chain lengths, such as SA, GA, and AA, respectively. The swelling response, FT-IR, TGA, mechanical properties, and SEM analysis validate the formation of the three hydrogels. The swelling index data evidenced that all the proposed hydrogels are responsive to pH. Moreover, the swelling index depends on the type of ADA and crosslinking degree. The series of hydrogels showed a sustained release rate of linezolid according to the results shown in the chromatographic analysis. The three hydrogels displayed significant differences regarding the release rate of linezolid. This difference seems to be ruled by the intermolecular interactions between linezolid and hydrogel matrix morphology, crosslinking degree, and mechanical properties. These mentioned features have a direct relation with ADA type used as crosslinker. ADAs can confer unique physicochemical and mechanical properties based on their specific structure. Therefore, the ADAs could play a key role in the release profile of the drug. The linezolid release kinetic of PSAH-Li and PGAH-Li were found to follow the Korsmeyer–Peppas release model, and the release mechanism in both cases was Fickian diffusion. On the contrary, the Higushi model was the best fit for PAAH-Li, and their mechanism for linezolid release was pseudo-Fickian. The antibacterial assays confirmed that the sustained release of linezolid from PGAH-Li has a better antibacterial activity compared with the conventional release. This suggests that the hydrogel has the capability to improve the bioavailability of linezolid. The set of proposed hydrogels showed good biocompatibility with L929 mouse connective tissue fibroblasts. The results exhibited viability over 87%. In conclusion, drug delivery platforms based on hydrogels of PVA and specific crosslinker agents such as ADAs could be potentially utilized as an antibiotic delivery system in potential infectious processes. Furthermore, this approach could become a strategy to help stop bacterial resistance.

**Author Contributions:** G.C.-B., A.M., S.V., O.V., G.C.-B., J.C., and E.F.D.-L. contributed to the conceptualization, methodology, validation, formal analysis, and investigation. E.F.D.-L., S.V., and G.C. contributed to the writing—original draft preparation. E.F.D.-L. contributed to supervision, writing—review, and editing, project administration, resources, and funding acquisition. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research work was financially supported by ANID FONDECYT project No. 11170155 (E.F.D.-L.), project No. 11170008 (O.V.), project No. 11180059 (A.M.), and ANID CONICYT PIA/APOYO CCTE AFB170007 (G.C.-B.).

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

#### **References**


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### *Review* **Polymeric Nanomaterials for Efficient Delivery of Antimicrobial Agents**

**Yin Wang <sup>1</sup> and Hui Sun 2,\***


**Abstract:** Bacterial infections have threatened the lives of human beings for thousands of years either as major diseases or complications. The elimination of bacterial infections has always occupied a pivotal position in our history. For a long period of time, people were devoted to finding natural antimicrobial agents such as antimicrobial peptides (AMPs), antibiotics and silver ions or synthetic active antimicrobial substances including antimicrobial peptoids, metal oxides and polymers to combat bacterial infections. However, with the emergence of multidrug resistance (MDR), bacterial infection has become one of the most urgent problems worldwide. The efficient delivery of antimicrobial agents to the site of infection precisely is a promising strategy for reducing bacterial resistance. Polymeric nanomaterials have been widely studied as carriers for constructing antimicrobial agent delivery systems and have shown advantages including high biocompatibility, sustained release, targeting and improved bioavailability. In this review, we will highlight recent advances in highly efficient delivery of antimicrobial agents by polymeric nanomaterials such as micelles, vesicles, dendrimers, nanogels, nanofibers and so forth. The biomedical applications of polymeric nanomaterial-based delivery systems in combating MDR bacteria, anti-biofilms, wound healing, tissue engineering and anticancer are demonstrated. Moreover, conclusions and future perspectives are also proposed.

**Keywords:** antimicrobial agent; polymeric nanomaterial; self-assembly; antimicrobial peptide; silver nanoparticle; anti-biofilm; wound healing; multidrug resistance

#### **1. Introduction**

Infectious diseases induced by bacteria, virus and fungi have been considered as one of the biggest enemies that threatened the lives of human beings for a long time [1]. Since the discovery of penicillin in 1928, antibiotics have played an unprecedented role in saving lives of human beings and caused revolutionary changes in medicine. However, with overuse and improper use of antibiotics, the emergence of bacterial drug resistance is becoming a severe problem. In particular, combating MDR bacteria such as methicillinresistant *Staphylococcus aureus* (*S. aureus*) (MRSA) has drawn wide attention and efforts [2,3]. Non-antibiotic antimicrobial agents such as AMPs [4–6], silver nanoparticles (AgNPs) [7–9], metal oxides [10–12], antimicrobial peptoids [13,14] and polymers [15–18] are alternatives for treating infectious diseases that kill bacteria in a physical manner and avoid the generation of drug resistance. For instance, cationic compounds including AMPs, antimicrobial peptoids and polymers, as well as their corresponding nanostructures, strongly interacted with the negatively charged cell membrane of bacteria, resulting in the disruption of the cell membrane and outflow of the content of bacteria [19,20]. Metal (oxide) nanoparticles such as widely studied AgNPs kill bacteria via heavy metal ions induced by the denaturation of proteins or genetic materials, while ZnO and TiO2 nanoparticles eliminate bacteria by reacting with reactive oxygen species (ROS) generated from photocatalytic process [12,21].

**Citation:** Wang, Y.; Sun, H. Polymeric Nanomaterials for Efficient Delivery of Antimicrobial Agents. *Pharmaceutics* **2021**, *13*, 2108. https://doi.org/10.3390/ pharmaceutics13122108

Academic Editor: Umile Gianfranco Spizzirri and Patrick J. Sinko

Received: 5 November 2021 Accepted: 3 December 2021 Published: 7 December 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 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/).

Moreover, emerging antimicrobial agents including gases, photothermal sensitizers and carbon materials were also developed to combat bacterial infections [17,22–24].

The efficient delivery of antimicrobial agents to the target preventing the defense system of bacteria including efflux pump, degrading enzymes and resistance genes is critical for reducing the emergence of drug resistance [25–27]. Polymeric nanomaterials are promising vehicles for the efficient delivery of antimicrobial agents due to their tailorable chemical compositions, microstructures and biological properties for a wide range of biomedical applications [28–30]. For instance, low dimensional nanostructures including dendrimers [31,32], polymeric nanoparticles [33,34], micelles [35,36], vesicles [37,38] and nanogels [39,40] have shown superiorities in the delivery of antimicrobial agents to the areas of infections and on-demand release. Polymeric nanofibers and hydrogels are beneficial for the long-term release of antimicrobial agents and wound coverage [41,42]. Very recently, metal organic frameworks (MOFs) have attracted attention as emerging carriers for the efficient delivery of metal ions, metal nanoparticles, antibiotics and enzymes due to their highly porous structures [43–46]. There are several advantages of using polymeric nanomaterials as carriers to accomplish the on-demand delivery of antimicrobial agents: (i) reduced dosage and drug resistance; (ii) increased in vivo circulation stability; (iii) enhanced penetration ability; (iv) prolonged antimicrobial performance; and (v) improved bioavailability. Therefore, apart from the wide attentional broad spectrum antimicrobial properties, biomedical applications including combating MDR bacteria, anti-biofilm, anticancer, wound healing and tissue engineering based on the polymeric antimicrobial agent delivery systems have been rapidly developed [47–50].

In this review, we aim to present the state of the art of polymeric nanomaterials as carriers for the efficient delivery of antimicrobial agents from the following aspects: (1) classification of polymeric nanoparticles based on their nanostructures; (2) the structural features and corresponding advantages in delivery of antimicrobial agents; and (3) biomedical applications benefiting from the constructed delivery systems, as illustrated in Figure 1.

**Figure 1.** Schematic illustration of polymeric nanomaterials for efficient delivery of antimicrobial agents and their biomedical applications.

#### **2. Efficient Delivery of Antimicrobial Agents by Diverse Polymeric Nanostructures** *2.1. Self-Assembled Polymeric Nanoparticles*

Polymer self-assembly has been recognized as one of the most versatile strategies for preparing soft nanomaterials with various morphologies and functionalities from small building blocks [51–56]. Typically, polymer micelles and vesicles are the most easily obtained and widely studied nano-objects due to their well-organized structures [57–60]. Polymer micelles are formed by the regular arrangement of building blocks with hydrophobic components forming the cores and hydrophilic polymer chains covering the surface. Moreover, the hydrophobic cores facilitated the efficient encapsulation of hydrophobic drugs [61,62], while polymer vesicles are hollow bilayer nanostructures with hydrophobic membranes, hydrophilic coronas and interior cavities, endowing them with superiorities in loading and delivering hydrophobic, hydrophilic and large-sized cargoes [63–66]. The design of polymer vesicles for meeting the requirements of various applications mainly focuses on the chemical composition and structure of coronas and membranes, such as permeability and homogeneity of the membrane, symmetricity of the corona and so forth [67–69]. Despite the wide applications of polymer micelles and vesicles in cancer therapy, gene delivery and cell imaging, they also exhibit considerable potentials in the efficient delivery of antimicrobial agents [36,70,71].

Polymer micelles, core-shell nanostructures that usually self-assembled from amphiphilic block copolymers, are regarded as one of the most extensively studied nanostructures for antimicrobial agent delivery [35,72]. Typically, hydrophobic antibiotics, AMPs and AgNPs can be loaded in the hydrophobic core, and amphiphilic antimicrobial molecules are usually decorated on the surface of polymer micelles by covalent bonding or electrostatic interaction [73–76]. For instance, a poor water-soluble anti-fungi agent, amphotericin B, could be encapsulated in the core of micelles and showed ultra-long sustained release for 150 h, resulting in reduced hemotoxicity and comparable anti-fungi activity compared with free amphotericin B [77]. Xiong and coworkers [78] functionalized terpyridine on the surface of polymer micelles; after chelating with Fe(II), the micelles displayed excellent biofilm inhibition activity up to 99.9% at a concentration of 128 μM. Recently, Lee et al. [79] prepared AMP-covered micelles by the co-assembly of chimeric antimicrobial lipopeptide and a biodegradable amphiphilic polymer (poly-(lactic-*co*-glycolic acid)-*b*-poly(ethylene glycol), (PLGA-*b*-PEG)). The chimeric peptide HnMc and PEG formed the shell of the micelles in which PEG protected HnMc from proteolytic degradation. Moreover, HnMc on the surface could help micelles in preferentially binding and killing bacteria. Due to the synergy between HnMc and PEG, the micelles targeted a wide range of bacteria preferentially including *Escherichia coli* (*E. coli*), *Listeria monocytogenes*, *Pseudomonas aeruginosa* (*P. aeruginosa*) and *S. aureus* instead of mammalian cells. Moreover, in vivo experiments also demonstrated superior anti-inflammatory effects of the micelles in a mouse model of drug-resistant *P. aeruginosa* lung infection with highly targeted abilities, as shown in Figure 2.

Very recently, Wooley and coworkers [80] fabricated spherical micelles, cylinders and nanoplates derived from the crystallization-driven self-assembly (CDSA) of an amphiphilic block copolymer composed of zwitterionic poly(-glucose carbonate) and semicrystalline poly(Ï-lactide) segments (PDGC-*b*-PLLA). As illustrated in Figure 3, fluorescent molecule and cysteine were modified on the polymer in order to afford tracing ability and to chelate with silver ions, respectively. The morphology of the nanostructures could be well controlled by the hydrophilic-to-hydrophobic ratios, which exhibited negligible cytotoxicity, immunotoxicity and cytokine adsorption. However, the nanostructures offered substantial silver ion loading capacity, extended release and in vitro antimicrobial activity. Compared with spherical micelles, the cylinders and nanoplates exhibited enhanced association with uroepithelial cells due to their high aspect ratio, resulting in improved inhibition of the growth of *E. coli* in recurrent urinary tract infections.

Compared with polymer micelles, polymer vesicles are closed hollow spheres with more complicated structures usually acting as simple mimics of biological cells [81]. There are three compartmentalized regions that should be considered for realizing different functions, namely the inner hydrophilic cavity, hydrophobic membrane and hydrophilic corona in contact with external environments [82]. Therefore, both hydrophilic and hydrophobic compounds and even nanoparticles could be encapsulated in the interior cavity or membrane of vesicles, respectively. Moreover, hydrophilic molecules could also be linked onto the coronas of polymer vesicles by covalent bonding. Considering the structural feasibility of polymer vesicles, a large variety of antimicrobial agents could be loaded and delivered to combat bacteria with high loading efficiency, controlled release manner, targeting capability

and improved bioavailability [83,84]. For example, Du and coworkers [35] deposited ultrafine AgNPs with a diameter of 1.9 ± 0.4 nm on the membrane of polymer vesicles by in situ reduction of silver ions to inhibit the growth of Gram-negative and Gram-positive bacteria. Battaglia et al. [71] reported the intracellular delivery of metronidazole or doxycycline to *P. gingivalis*-infected oral epithelial cells by polymer vesicles, which were disassembled in early endosomes due to the acidic condition, resulting in the release of loaded cargoes.

**Figure 2.** Targeted antibacterial activity of HnMc micelles in the mouse model of drug resistant *P. aeruginosa* lung infection. (**a**) Fluorescent imaging of infected mice after intravenous injection of fluorescent IR820-loaded HnMc micelles. (**b**) Survival rate of infected mice after administration of HnMc micelles. (**c**) Number of remaining cells in the infected lungs after administration of HnMc micelles. Inhibitory effects of HnMc micelles on the expression of TNF-α (**d**) and nitric oxide (**e**) in the blood of infected mice. (**f**) H&E-stained lung tissues. (**i**): control; (**ii**): *P. aeruginosa* + PBS; (**iii**): *P. aeruginosa* + gentamicin (5 mg kg<sup>−</sup>1); (**iv**): *P. aeruginosa* + scrambled HnMc micelle (5 mg kg<sup>−</sup>1); (**v**): *P.* aeruginosa + HnMc micelle (2.5 mg kg<sup>−</sup>1); and (**vi**): *P. aeruginosa* + HnMc micelle (5 mg kg<sup>−</sup>1) (reproduced with permission from Park et al. [79], ACS Applied Materials & Interfaces; published by American Chemical Society, 2020).

Recently, Liu and coworkers [85] designed enzyme-responsive polymer vesicles for bacterial strain-selective delivery of antimicrobials, as shown in Figure 4. Both hydrophilic and hydrophobic antimicrobials including vancomycin, gentamicin, quinupristin and dalfopristin could be encapsulated either in the interior cavity or membrane of the polymer vesicles with high efficiency. The PEG chains covered on the surface of the vesicle could reduce cytotoxicity and improve biocompatibility, while the self-immolative side chains could be degraded by penicillin Gamidase (PGA) and *β*-lactamase (Bla), which are overexpressed by drug resistant bacterial strains. Without the trigger by PGA and Bla, the encapsulated antimicrobials were well protected by vesicles. Upon being exposed to drug-resistant bacteria, the membrane of the vesicle was degraded, resulting in the sustained release of antimicrobials, as well as the elimination of bacteria. Considering that Bla is the main cause of bacterial resistance to *β*-lactam antibiotic drugs that are secreted by MRSA, selective antimicrobial activity of the antimicrobials-loaded vesicles was achieved.

**Figure 3.** Polymer micelles, cylinders and nanoplates derived from the CDSA of PDGC-*b*-PLLA and their antimicrobial activity (\* *p* < 0.05 and \*\* *p* < 0.01 by *t* test) (reproduced with permission from Song et al. [80], Nano Letters; published by American Chemical Society, 2021).

**Figure 4.** Enzyme-responsive polymer vesicles for bacterial strain-selective delivery of antimicrobials (reproduced with permission from Li et al. [85], Angewandte Chemie International Edition; published by Wiley, 2015).

Loading bioactive enzymes by polymer vesicles to generate antimicrobial active species triggered by external stimuli is another effective method for combating bacteria. For example, Blackman et al. [86] prepared glucose oxidase-loaded semipermeable polymer vesicles by polymerization-induced self-assembly inspired by honey. Hydrogen peroxide, an effective antimicrobial agent, could be generated in response to glucose to switch on antimicrobial activity of the vesicles. In the absence of glucose, the vesicles were completely nontoxic to bacteria, while the vesicles showed seven-log reduction in bacterial growth at high glucose concentrations against a range of Gram-negative and Gram-positive bacterial pathogens including *S. aureus*, *S. epidermidis*, *E. coli* and *Klebsiella pneumoniae* (*K. pneumonia*), even the MRSA clinical isolate. More importantly, the toxicity of the vesicle toward human fibroblasts at different dosage and glucose concentrations was also

evaluated, demonstrating that the optimal concentration of the vesicle was 0.69 mg mL−<sup>1</sup> at physiological blood glucose level to effectively eliminate bacteria while preserving good compatibility to mammalian cells.

#### *2.2. Dendrimers*

Dendrimers are highly branched, globular macromolecules with many arms emanating from a central core, which have shown unique structural properties such as high degree of branching, multivalency, globular architecture and well-defined molecular weight, rendering them promising scaffolds for drug delivery [87,88]. Many commercial drugs with anticancer and antimicrobial activity have been successfully loaded within dendrimers including poly(amidoamine) (PAMAM), poly(propylene imine) (PPI) and poly(etherhydroxylamine) (PEHAM), either via physical interactions or by chemical bonding to improve their water solubility [89]. Dendrimers themselves could be used as effective antimicrobial agents [90]. For instance, those with positively charged surfaces usually have strong interaction with negatively charged bacterial cell membranes, while those with metal cores can release active antimicrobial agents such as metal ions and ROS, resulting in the death of bacteria [91].

Moreover, antimicrobial agents including antibiotics, AMPs, AgNPs and metal oxide nanoparticles could be also effectively loaded by dendrimers [89,92]. For example, Tang et al. [93] prepared silver-dendrimer nanocomposites by loading AgNPs in low generation poly(amido amine) dendrimers. The AgNPs were formed by an in situ reduction of silver ions enriched by the amine groups of dendrimers. The factors that influenced the size of AgNPs were discussed, and the average diameter of the AgNPs could be controlled from 7.6 to 16.2 nm. The synthesized silver-dendrimer nanocomposite was used as antimicrobial agent in the fabrication of cotton fabrics, which exhibited excellent antimicrobial activity against both of *E. coli* and *S. aureus*. Recently, Huang and coworkers [94] reported PLGA nanoparticles and PAMAM dendrimers in order to effectively encapsulate and deliver platensimycin, a potent inhibitor for the synthesis of bacterial fatty acid, respectively, to combat MDR bacteria. Benefiting from the improved pharmacokinetics, both the platensimycin-loaded PLGA nanoparticles and PAMAM dendrimers showed enhanced antimicrobial activity and reduced cytotoxicity compared with free platensimycin, resulting in an efficient inhibition of *S. aureus* biofilm formation and the full survival of MRSA-infected mice.

Dendrimers are ideal platforms for compacting and delivering deoxyribonucleic acids (DNAs) and ribonucleic acids (RNAs) for gene therapy due to their hyperbranched structure and strong positive charges, especially PAMAM [95,96]. Recently, antisense therapy strategy has been developed to treat bacterial infections facilitated by the dendrimersbased antisense delivery system [97]. For example, the G3 PAMAM dendrimer has good antimicrobial activity, as shown in Figure 5. However, the cytotoxicity of the G3 PAMAM dendrimer toward mammalian cells is also high. Luo et al. [98] conjugated LED209, a specific inhibitor of quorum sensor QseC of Gram-negative bacteria, onto the surface of G3 PAMAM to generate PAMAM-LED209 in order to reduce cytotoxicity to mammalian cells while retaining the excellent antibacterial activity of the G3 PAMAM dendrimer. In addition, PAMAM-LED209 also inhibited the virulence gene expression of Gram-negative bacteria and prevented the generation of drug resistance. As shown in Figure 5, compared with the control group (Figure 5A), *entero-hemorrhagic E. coli* (*EHEC*) were severely damaged after being treated with G3 PAMAM and G3 PAMAM-LED209 for 300 min (Figure 5B,C), demonstrating that G3 PAMAM-LED209 retained strong antibacterial activity toward resistant Gram-negative bacteria after functionalization of LED209. The induction of the resistance of G3 PAMAM-LED209 was also evaluated after 15 reproductions of bacteria, as illustrated in Figure 5D. The minimal inhibition concentration (MIC) of G3 PAMAM-LED209 barely changed, while the MIC values of classical antimicrobials, including ceftazidime, ampicillin and levofloxacin, increased by 8-fold to 64-fold. The cytotoxicity and antibacterial activity of terminally modified PAMAM are related to the

conjugated ligand and degree of modification, as shown in Figure 5E. With an increase in modification ratio, the cytotoxicity of G3 PAMAM-PEG and G3 PAMAM-LED209 decreased dramatically to being almost nontoxic and then increased, while the antimicrobial activity of the G3 PAMAM-PEG and G3 PAMAM-LED209 decreased with an increase in modification ratio due to the shielding of positive charges. Therefore, there is an optimal modification ratio range for balancing cytotoxicity and antimicrobial activity, as pointed out by the arrow in Figure 5E. Moreover, the antibacterial potency of G3 PAMAM-LED209 is also higher than that of G3 PAMAM-PEG, which is indicated by area A and B in Figure 5E, demonstrating better biocompatibility and higher antibacterial potency than compared to G3 PAMAM-PEG.

**Figure 5.** The morphology of EHEC was investigated by transmission electron microscopy (TEM) after different treatments for 300 min. (**A**) Control; (**B**) 75 μg mL−<sup>1</sup> of G3 PAMAM; and (**C**) 150 μg mL−<sup>1</sup> of G3 PAMAM-LED209. (**D**) Induction of resistance to G3 PAMAM-LED209. (**E**) The influence of the conjugated ligand and degree of modification on cytotoxicity and antibacterial activity of terminally modified G3 PAMAM dendrimer. Area A and B showed increased antibacterial activity and reduced cytotoxicity with respect to G3 PAMAM-LED209 and G3 PAMAM-PEG, respectively (reproduced with permission from Xue et al. [98], Nanomedicine: Nanotechnology, Biology and Medicine; published by Elsevier, 2015).

#### *2.3. Polymer Nanofibers*

Polymer nanofibers are one dimensional nanostructures with large aspect ratio and high surface area and have shown significant potential for delivering antimicrobial agents locally into an infected area, especially in wound healing [42,99]. Typically, there are several methods for preparing nanofibers including self-assembly [100], template synthesis [101], phase separation [102] and electrospinning [103], among which electrospinning is a superior technique for preparing nanofibers with desired chemical compositions and diameters due to its simplicity and versatility [104–106]. Antimicrobial agents including antibiotics, AMPs, AgNPs and metal oxide nanoparticles could be incorporated into nanofibers by mixing with polymer precursors followed by electrospinning or attaching onto the surface of the nanofibers by noncovalent interactions or chemical bonds [107]. For instance, Schiffman et al. [108] immobilized zeolites nanoparticles with high silver ion change capability onto the surface of chitosan nanofibers. After ion exchange, silver ions were loaded in the zeolites to function as molecular delivery vehicles, and their ion release profiles and ability to inhibit *E. coli* were evaluated as a function of time. Interestingly, the zeo-

lites immobilized on the nanofibers showed significantly enhanced antibacterial activity 11-times greater than that of the pure zeolites due to high porosity and hydrophilicity of the nanofibers.

Recently, Tu and coworkers [109] reported the in situ deposition of AgNPs on gold/polydopamine core-shell nanoparticles encapsulated by poly(lactic acid) (PLA) nanofibers (PLA-Au@PDA@Ag), which could be applied to biological coatings for bacteriostatic functionality. The schematic illustration of the preparation and antimicrobial capability of the PLA-Au@PDA@Ag is presented in Figure 6. Chloroauric acid was reduced by ascorbic acid to afford gold nanoparticles. Following the polymerization of dopamine on the surface, Au@PDA core-shell nanoparticles formed, which were then mixed with PLA solution to produce PLA-Au@PDA hybrid nanofibers by electrospinning. Later, PLA-Au@PDA hybrid nanofibers were immersed in silver nitrate solution for in situ reduction of adsorbed silver ions into AgNPs to yield PLA-Au@PDA@Ag nanofibers. The hydrophilicity of the PLA-Au@PDA@Ag nanofibers significantly improved compared to that of PLA nanofibers, resulting in the promoted release of silver ions. Benefiting from the synergy between AuNPs, PDA and AgNPs, including AuNPs providing effective contact with microorganisms, PDA as binder was used to immobilize AgNPs and facilitated the release of silver ions; the PLA-Au@PDA@Ag nanofibers showed significant antibacterial ability against both of *E. coli* and *S. aureus*.

**Figure 6.** Schematic diagram illustrating the preparation of PLA-Au@PDA@Ag nanofibers and their antibacterial capacity (reproduced with permission from Zhang et al. [109], Colloids and Surfaces B: Biointerfaces; published by Elsevier, 2019).

Due to their large exposed surface area and nanoporosity, polymer nanofiber meshes have shown distinct advantages in wound healing compared with hydrogels, films and foams [110]. The extracellular matrix (ECM) mimicking the structure of nanofibers facilitated the interaction with cells in the wound bed. Moreover, small molecules such as water, oxygen, nutrients and metabolic wastes could be efficiently exchanged due to the highly porous structure of nanofibers [111]. In order to promote the healing rate and elimination of bacteria, functional agents including enzymes, drugs and antimicrobial agents have been incorporated in polymer nanofibers. Rath et al. [112] loaded ZnO nanoparticles and cefazolin in the gelatin nanofibers to accelerate wound healing and prevented infection concurrently. Cefazolin was used to inhibit bacterial reproduction, while zinc cations could be released from ZnO nanoparticles to raise re-epithelialization, reduce inflammation and inhibit bacterial growth. Moreover, ROS was also produced by ZnO nanoparticles, thereby

optimizing cell adhesion, proliferation and migration via growth factor mediated pathways, promoting the regeneration of the ECM.

#### *2.4. Polymer Nanogels*

Polymer nanogels are a class of nanoparticles composed of nanosized physically or chemically cross-linked hydrophilic or amphiphilic polymer networks [113]. They are of wide interest in various fields including drug delivery due to their flexible nanosize, good stability and high loading capacity, etc. [114]. As their analogues, polymer hydrogels have been widely used in antimicrobial applications due to their high water content, three-dimensional structure and stimuli-responsive sol-gel transition behavior [115]. There are several reviews summarizing the recent advances of antimicrobial polymer hydrogels [116–118]. Therefore, we will not discuss this part and focus on the nanogels as carriers for antimicrobial agent delivery in this section.

The stimuli-responsive swelling and collapsing of nanogels triggered by external stimuli including pH, temperature, enzymes or ionic strength render them ideal candidates in on-demand delivery and release of antimicrobial agents [119]. For instance, AMPs could be encapsulated in nanogels with high loading content via strong electrostatic interaction with negatively charged polymer chains, and they can be released when triggered by salt ions in physiological conditions [120,121]. El-Feky et al. [122] loaded silver sulfadiazine in alginate coated chitosan nanogels to heal burn wounds, and the nanogels showed a release profile of an initial burst followed by a slow and continuous release, resulting in excellent in vivo therapeutic efficacy.

In addition, loading and delivery of antimicrobials including berberine, cyclodextrin, tetracycline hydrochloride and lincomycin hydrochloride by nanogels to combat bacteria and MDR bacteria were widely studied by Paunov, Schaefer and so forth [123–126]. Wang and coworkers [127] designed a lipase-sensitive polymeric triple-layered nanogel (TLN) formed by a cross-linked polyphosphoester core, poly(*ε*-caprolactone) (PCL) fence and PEG shell to encapsulate and deliver vancomycin, as illustrated in Figure 7. In aqueous solutions, hydrophobic PCL segments collapsed and covered the core to form a densely packed molecular fence to prevent the leakage of vancomycin. Once TLN was exposed to lipase secreting bacteria, the PCL chains were degraded to trigger the release of vancomycin, resulting in the inhibition of bacterial growth. They found that all encapsulated vancomycins were released within 24 h in the presence of *S. aureus*. Moreover, lipase secreting bacteria inside the cells could also be inhibited by TLN, demonstrating the versatility of the strategy of lipase-induced on-demand delivery and release of antimicrobials.

Recently, Knowles et al. [128] synthesized hybrid organic/inorganic AgNPs loaded nanofibrillar silk microgels to effectively eradicate bacteria by a two-step mechanism including bacterial adherence and consequent eradication. Compared with conventional AgNPs and silver ions, the hemolysis and cytotoxicity of hybrid microgels toward mammalian cell lines were significantly reduced due to the protection of the silk matrix. van Rijn and coworkers [129] prepared injectable nanogels loaded with hydrophobic triclosan in hydrophobic domains inside the nanogel networks through intraparticle self-assembly of aliphatic chains, which enhanced antimicrobial efficiency of triclosan up to 1000 times. As shown in Figure 8, a three-stage antimicrobial mechanism of the nanogels was proposed. Firstly, the nanogels attached onto the surfaces of the bacteria via electrostatic interaction to disturb the balance of charge density of the cell membranes. Secondly, bacterial cell membranes were destroyed by the insertion of hydrophobic aliphatic chains. Thirdly, loaded triclosan was released from the hydrophobic domains inside the nanogels and injected into the bacterial cell membranes, resulting in the death of bacteria. This approach dramatically increases the effective concentration of triclosan inside the bacteria. Moreover, both the MIC and minimal bactericidal concentration (MBC) against Gram-positive *S. aureus* and *S. epidermidis* decreased by three orders of magnitude compared with free triclosan, resulting in a decrease in the dosage of triclosan and reduction in drug resistance.

**Figure 8.** Schematic illustration of the bactericidal mechanism of Triclosan-loaded nanogels (adapted from Zu et al. [129], ACS Applied Polymer Materials; published by American Chemical Society, 2020).

#### *2.5. Hybrid Delivery Systems*

Incorporating polymer nanoparticles including dendrimers, micelles and vesicles with high dimensional polymeric nanomaterials such as nanofibers, hydrogels and coatings as hybrid delivery systems could combine the advantages of both and achieve the hierarchical release of antimicrobial agents [130–133]. For example, Zhang and coworkers [130] developed a bioadhesive nanoparticle-hydrogel hybrid in order to enhance localized antimicrobial drug delivery. The antimicrobials ciprofloxacin was loaded in polymer nanoparticles that were embedded in hydrogels adhering to biological surfaces. Hydrogel network

properties could be tailored independently for adhesion, which maintained controlled and prolonged ciprofloxacin release profiles from nanoparticles. Imae et al. [131] immobilized AgNPs-loaded amine-terminated fourth generation poly(amido amine) dendrimers onto the viscose rayon cellulose fibers, which exhibited excellent biocidal activity against *E. coli* with low weight percentage of silver of 0.2%. Du and coworkers [132] embedded penicillin encapsulated polypeptide polymersomes in the hydrogels to achieve quick and long-term antibacterial capability in which penicillin could be released from the hydrogel networks for quick bacteria elimination while the intrinsic antibacterial property of the polymersomes ensured long-term antibacterial activity. However, despite the advantages of hybrid delivery systems, the development of incorporation of different polymeric nanostructures as hybrid delivery platforms is still in its infancy, which may bring new opportunities in efficient loading and delivery of antimicrobial agents.

#### **3. Biomedical Applications of Polymeric Nanomaterials Based Antimicrobial Agent Delivery Systems**

#### *3.1. Combating MDR Bacteria*

The generation of drug resistance of pathogens is typically caused by the accumulation of drug resistant genes through mutation with the long-term use, especially overuse and improper use of antibiotics [25]. Therefore, the exploration of highly efficient delivery system to reduce dosage and improve bioavailability of antibiotics, as well as the delivery of non-antibiotic antimicrobial agents including AMPs, AgNPs, metal oxides, gases and so forth, is a promising strategy for reducing drug resistance [134,135]. Polymeric nanomaterial-based antimicrobial agent delivery systems have widely been used in combating MDR bacteria [136,137]. For instance, Liu et al. [138] conjugated quercetin and acetylcholine on the surface of selenium nanoparticles to combat MDR bacteria, which could effectively eliminate MRSA by destroying the membrane due to the synergy between quercetin, acetylcholine and selenium nanoparticles. Cationic polymeric star-shaped nanoparticles or dendrimers have also shown excellent antimicrobial activity against MDR bacteria even without loading antimicrobial agents [139–141], demonstrating the great potentials of polymeric nanomaterials in combating MDR bacteria.

Hu et al. [142] prepared polyprodrug antimicrobials to combat MRSA by membrane damage and concurrent drug release, as shown in Figure 9. Triclosan was covalently linked with acrylic acid to produce a triclosan prodrug monomer (TMA). Then, TMA was copolymerized with quaternized *N*,*N*-dimethylaminoethyl methacrylate (QDMA), affording PQDMA-*b*-PTMA, which could self-assemble into prodrug micelles with positively charged surfaces. The hydrophilic–hydrophobic balance of the prodrug micelles was optimized to enhance interaction with bacterial cell membranes, resulting in improved antimicrobial activity. They proposed that the antimicrobial mechanism was as follows: (1) the prodrug micelles attached onto the surface of MRSA due to strong electrostatic interaction; (2) the prodrug micelles fused with and inserted into the cell membrane of MRSA; (3) the cell membrane of MRSA was damaged due to charge disorder, and prodrug micelles were encapsulated into the cell; (4) prodrug micelles were disassembled, and the linkage between triclosan and acrylic acid was broken due to the reductive milieu environment, resulting in the in situ release of triclosan and death of MRSA. It was noteworthy that no detectable resistance was observed due to the synergistic antibacterial mechanism, and prodrug micelles exhibited remarkable bacterial inhibition and low hemolysis toward red blood cells compared with commercial triclosan and vancomycin.

The combination of different classes of antimicrobial agents such as antimicrobials and AgNPs could afford synergistic effects, resulting in the efficient inhibition of MDR bacteria that is far better than its individual components [143,144]. Webster and coworkers [145] prepared polymer vesicles to co-deliver ampicillin and AgNPs simultaneously in the hydrophilic cavity and hydrophobic membrane, respectively. The AgNPs-embedded polymersomes exhibited potent antibacterial activity against *E. coli* transformed with a gene for ampicillin resistance in a dose-dependent fashion, while the free ampicillin, Ag-NPs decorated polymersomes without ampicillin and ampicillin loaded polymersomes

without AgNPs had no effect on bacterial growth. TEM images in Figure 10 revealed that the interactions between vesicles, AgNPs and bacterial cells might result in the deformation and disruption of bacterial envelopes and consequently result in the death of bacteria. Later, the same group [146] functionalized proline-rich AMP PR-39 on the corona of polymer vesicles with AgNPs embedded in the membrane to combat MRSA with a AMP/AgNPs ratio-dependent behavior. A ratio of AgNPs-to-AMP of 1:5.8 corresponding to 11.6 <sup>μ</sup>g mL−<sup>1</sup> of AgNPs and 14.3 × <sup>10</sup>−<sup>6</sup> M of AMP exhibited the best MRSA inhibition activity, demonstrating the potentials of binary or ternary antimicrobial agent co-delivery systems in combating MDR bacteria.

**Figure 9.** The polyprodrug antimicrobials with optimized hydrophilic–hydrophobic balance for efficiently eradicating MRSA with remarkable membrane damage and concurrent drug release profiles (reproduced with permission from Cao et al. [142], Small; published by Wiley, 2018).

**Figure 10.** TEM images of bacteria-polymer vesicles interactions. Scale bars are 100 nm for (**A**–**D**,**F**–**H**) and 500 nm for (**E**), respectively. White arrows pointed out the indentation of bacterial cell membrane in regions of AgNPs loaded polymer vesicles; yellow arrows implied the polarization indicative of hydrophobic interactions of AgNPs inside the vesicles; black arrows revealed that the regions of the outer membrane with little AgNPs loaded polymer vesicles contact appeared morphologically normal (reproduced with permission from Geilich et al. [145], Nanoscale; published by Royal Society of Chemistry, 2015).

#### *3.2. Anti-Biofilm*

Biofilms are matrix-enclosed communities of bacteria that show increased drug resistance and capability to evade the immune system [47]. It has been widely recognized that bacteria exist in the form of biofilms in many instances, which is hard to eliminate due to the protection of extracellular polymeric substances (EPS), a complex matrix composed of proteins, nucleic acids, phospholipids, polysaccharides, blood components and humic substances produced by bacteria [147]. Therefore, it is difficult for antimicrobials to penetrate the EPS to kill bacteria, resulting in the occurrence of drug resistance. The efficient delivery of antimicrobial agents by polymeric nanomaterials is considered a promising strategy for penetrating the biofilm and delivering antimicrobial agents to the deep end of the matrix to kill pathogens [148–150]. For example, Deoxyribonuclease I functionalized ciprofloxacin-loaded PLGA nanoparticles were prepared to target and disassemble the *P. aeruginosa* biofilm by degrading extracellular DNA that stabilizes the biofilm matrix and released ciprofloxacin inside the biofilm to effectively eliminate *P. aeruginosa*, as reported by Torrents and coworkers [151].

Webster et al. [152] prepared bifunctional polymersomes with methicillin encapsulated in the hydrophilic cavity and superparamagnetic iron oxide nanoparticles (SPIONs) embedded in the membrane, as illustrated in Figure 11. The iron oxide-encapsulated polymersomes (IOPs) penetrated into the *S. epidermidis* biofilm with high efficiency, promoted by external magnetic field. Comparing with individual SPIONs, methicillin and SPION co-encapsulated polymersomes showed enhanced penetration capability up to 20 μm due to the improved relaxivity and magneticity (Figure 11c). Thus, methicillin could be released into the deep end of the biofilm, resulting in the effective eradication of pathogens. The confocal microscopy images and the 3D reconstructions of z-stacks of the bacterial biofilm revealed the capability of IOPs to eradicate biofilms with and without methicillin, as shown in Figure 11d. When there was no methicillin, only bacteria in the bottom layer of the biofilm were killed. On the contrary, all bacteria throughout the biofilm were eliminated by the methicillin loaded IOPs. These organic/inorganic hybrid nanocarriers showed great promise as new weapons for eradicating persistent biofilm or drug-resistant bacteria.

Recently, Du and coworkers [153] reported the treatment of periodontitis by efficiently disrupting biofilms using a dual corona antimicrobials-loaded polymer vesicle with stealthy poly(ethylene oxide) (PEO) corona to penetrate the biofilm and antibacterial polypeptide corona to provide intrinsic antimicrobial activity, as shown in Figure 12. The dual corona polymer vesicles were prepared by the co-assembly of two polymers PCL-*b*-poly(lysine*stat*-phenylalanine) [PCL-*b*-P(Lys-*stat*-Phe)] and PEO-*b*-PCL with the same hydrophobic biodegradable PCL segment and different hydrophilic chains. Ciprofloxacin could be efficiently encapsulated in the cavity of the vesicles. Due to the protein-repelling ability of PEO, dual corona polymer vesicles penetrated the EPS of the biofilms with high efficiency, while the positive charged P(Lys-*stat*-Phe) allowed the vesicle to target and kill bacteria via electrostatic interaction. In addition, the encapsulated ciprofloxacin could be released as the polymer vesicle reached the deep end of the biofilm, resulting in a reduced dosage of the antimicrobials up to 50% to eradicate *E. coli* or *S. aureus* biofilms. In vivo experiment results demonstrated excellent performance of the dual corona vesicles in reducing dental plaque and alleviating inflammation using a rat periodontitis model.

Despite the strategy of delivering antibiotics to the deep end of biofilms by polymeric nanocarriers in order to reduce dosage and enhance antimicrobial activity, the efficient delivery of non-antibiotic antimicrobial agents including AMPs, AgNPs, photosensitizers and so forth for eliminating biofilms was also widely studied [154–156]. For instance, Haldar et al. [157] fabricated biodegradable polymer-coated AgNPs nanocomposite to eradicate biofilms, which reduced MRSA burden both on the catheter (>99.99% reduction) and in tissues surrounding the catheter (>99.999% reduction) in a mice model. Ji and coworkers [158] developed targeted photodynamic therapy strategies by using a supramolecular delivery system for the treatment of biofilms. The photosensitizer Chlorin e6 was grafted onto *α*-cyclodextrin, and the targeting group AMP Magainin I was covalently

bound with PEG. Taking advantage of supramolecular recognition between *α*-cyclodextrin and PEG, targeting supramolecular micelles loaded with Chlorin e6 were formed, which exhibited excellent bacterial targeting effects and enhanced biofilm eradication ability against *P. aeruginosa* biofilm and MRSA biofilm. These results proved the versatility and great potential of polymeric nanomaterial-based antimicrobial agent delivery systems for eradicating biofilms.

**Figure 11.** (**a**) Synthesis of IOPs loaded with SPION and methicillin. (1) 5 nm monodisperse hydrophobic SPIONs are combined with mPEG-PDLLA co-polymer in organic solvent and ultrasonicated to create a uniform suspension. (2) This organic phase is injected through an atomizer into an actively stirring aqueous phase containing PBS and methicillin. (3) The mixture is dialyzed against pure PBS to remove the organic solvent and unencapsulated drug to yield (4) highly stable polymersome solution. (**b**) TEM image of SPIONs loaded polymersomes. (**c**) Magnetic field induced treatment of biofilm using SPIONs and/or antimicrobials. (**d**) Confocal microscopy images of LIVE/DEAD staining of *S. epidermidis* biofilms treated with IOPs with an external magnetic field (reproduced with permission from Geilich et al. [152], Biomaterials; published by Elsevier, 2017).

**Figure 12.** Schematic model showing the treatment of periodontitis by efficiently disrupting biofilms via antimicrobials -loaded multifunctional dual corona vesicles (reproduced with permission from Xi et al. [153], ACS Nano; published by American Chemical Society, 2019).

#### *3.3. Wound Healing*

Wound infections induced by pathogens have become one of the main problems in wound care management systems, which impede the healing process and may result in life threatening complications. One of the approaches for treating wound infection is the use of wound dressings with antibacterial agents possessing broad-spectrum antimicrobial activity [159]. Typically, the moisture environment provided by the dressing has been shown to promote ulcer healing and to reduce pain experienced by patients [160]. Moreover, there are other requirements for wound dressings such as separating the wound with external environments and providing good breathability to promote wound healing. Polymeric nanomaterial-based delivery systems have shown considerable potentials in wound healing, especially polymer nanofibers and hydrogels [99,161]. For example, Lakshminarayanan et al. [162] prepared polydopamine crosslinked polyhydroxy antimicrobials loaded gelatin nanofiber mats for advanced wound dressings with long-term antimicrobial activity up to 20 days. The morphology of the nanofiber mats was retained for 1 month in an aqueous environment and showed comparable wound closure compared to commercially available silver-based dressings. Cai and coworkers [163] prepared composite hydrogels embedded with copper nanoparticles that could effectively convert NIR laser irradiation energy into localized heat for photothermal therapy. The synergistic effect of photothermal performance and rapid release of copper ions upon laser irradiation were responsible for excellent antimicrobial activity, reduced inflammatory response and promoted angiogenesis ability.

Antimicrobial agents including AMPs [49], antibiotics [164], AgNPs [165], metal oxide such as ZnO [166], photothermal sensitizers including porphyrin [167] and heavy metal ions [163] are usually used to improve the antimicrobial activity of polymeric wound dressings by covalent linkage, physical interaction or encapsulation. For example, Liu et al. [168] decorated chloramine on the surface of chitosan films by electrostatic interaction to heal MRSA infected wounds. Zhou and coworkers [167] prepared porphyrin containing alternating copolymer vesicles for the disinfection of drug-resistant bacteria infected wounds via photothermal effect. Fahimirad et al. [169] loaded recombinant LL37 AMP into chitosan nanoparticles for the elimination of MRSA infection during wound healing process with ultrahigh encapsulation efficiency of 78.52% and improved the activity and stability of LL37 AMP under thermal, salts and acidic pH treatments. Guo et al. [170] prepared injectable antimicrobial conductive quaternized chitosan hydrogels by loading graphene oxide via covalent bond for drug resistant bacterial disinfection and infectious wound healing, and the hybrid hydrogels showed excellent performance in the treatment of MRSA infected full-thickness defect mouse model.

Very recently, polymer vesicles loaded with antimicrobials have been explored as dressings in promoting wound healing by spraying onto wounds [167,171–173]. Du and coworkers [173] reported bifunctional polymer vesicles loaded with antimicrobials and antioxidant for healing infected diabetic wounds, as presented in Figure 13. As one of the chronically infected wounds, the diabetic wounds are difficult to heal due to high ROS concentration and recurrent infections, resulting in the occurrence of diabetic ulcers and chronic diabetic complications with very high mortality rate. Therefore, scavenging ROS is very important in the treatment of diabetic wounds. In this study, well-dispersed ceria nanoparticles were deposited on the membrane of ciprofloxacin-loaded polymer vesicles (CIP-Ceria-PVs). The CIP-Ceria-PVs could inhibit peroxide free radicals up to 50% at extremely low cerium concentrations of 1.25 μg mL<sup>−</sup>1, protecting normal L02 cells from the damage of peroxide free radicals. Moreover, CIP-Ceria-PVs exhibited enhanced antimicrobial activity compared with free ciprofloxacin due to scavenging ROS. In vivo studies in Figure 13b demonstrated the excellent wound healing capability of CIP-Ceria-PVs, and the diabetic wound was completely healed within 14 days. At the same time, they developed a H2S delivery polymer vesicle, which was capable of long-term H2S generation to promote the proliferation, migration of epidermal and endothelial cells and angiogenesis, accelerating the complete healing of diabetic wounds [172].

**Figure 13.** *Cont*.

**Figure 13.** (**a**) Illustration of the preparation of CIP-Ceria-PVs and the combined antioxidantantimicrobials treatment of infected diabetic wounds. (**b**) Digital images of infected diabetic wounds at different time intervals under treatment. Scale bar: 2 cm (reproduced with permission from Wang et al. [173], ACS Nano; published by American Chemical Society, 2021).

#### *3.4. Tissue Engineering*

The regeneration of adult tissue following an injury or degeneration is quite a limited process. Usually, the injury site is vulnerable to bacterial infections, which causes complications and delay of the regeneration of tissues [174]. Therefore, the prerequisite of tissue regeneration is to eliminate localized bacterial infections, followed by the delivery of bioactive molecules such as growth factor to the defected tissues. Antimicrobial polymer coatings on the surface of implants can provide appropriate biointerfaces to promote the regeneration of tissues. For instance, ZnO nanoparticles embedded PLA was dip coated on magnesium alloy, which helped to control the degradation and increase antibacterial activity [175]. Suteewong et al. [176] deposited polymethylmethacrylate (PMMA)/chitosansilver hybrid nanoparticles on rubber substrate, which exhibited enhanced antibacterial activity toward *E. coli* and *S. aureus* and reduced cytotoxicity to L-929 fibroblast cells, demonstrating the potential of this hybrid nanoparticle coating at soft substrates. In addition, antimicrobial agents loaded with polymer nanomaterials can be used as bioadhesives to repair damaged soft tissues. Gu and coworkers [177] developed fast and high strength bioadhesives based on polysaccharides and peptide dendrimers with inherent hemostatic ability and antibacterial properties. Moreover, the bioadhesive showed a remarkable 5-fold increase in adhesion strength comparing with commercial bioadhesive Coseal.

Biocompatible polymeric nanoparticles have been investigated as delivery vehicles for various tissue engineering applications [178]. For example, Du and coworkers [179] prepared antibacterial peptide-mimetic alternating copolymers (PMACs) vesicles loaded with growth factor for bone regeneration. They designed a series of PMACs with different repeating units, and the PMAC with a repeating unit of 14 exhibited the best antibacterial activity against both *E. coli* and *S. aureus* with ultralow MICs of 8.0 μg mL−1. After self-assembling into vesicles in pure water, the antimicrobial activity of the vesicles was well-preserved. Growth factor could be encapsulated in antimicrobial vesicles and released during the long-term antibacterial process to promote the regeneration of bone with a 20 mm defect model in rabbits. Micro-CT, bone mineral content and BMD were used to evaluate the repair of bone defects with scaffolds at 4 weeks and 6 weeks after implantation. After 6 weeks, the defect in the rabbit bone was completely repaired, demonstrating the excellent bone repair capability of antimicrobial growth factor-loaded vesicles.

#### *3.5. Anticancer*

The anticancer application of antimicrobial agents is an attracting field since cancers are often accompanied by inflammation, and the drug resistance of cancer cells is becoming increasingly concerning [180]. Theoretically, antimicrobial agents that kill bacteria via non-selective behaviors such as damage of the cell membrane [181], elevating temperature [182] and induced degeneration of proteins and genetic materials [183] can also kill cancer cells. For instance, Shim et al. [183] prepared AgNPs loaded chitosan-alginate composite, exhibiting broad-spectrum antimicrobial activity and high toxicity toward breast cancer cell line MDA-MB-231; Jothivenkatachalam and coworkers [184] fabricated chitosan-copper nanocomposite for the inhibition of various microorganisms and A549 cancer cells by photocatalytic effect. In addition, AMPs with specific sequences and proper positive charge densities have shown anticancer and antiviral activities, such as cecropin A and B, magainins, melittin, defensins, lactoferricin and so forth, as summarized by Hoskin's and Franco's group, respectively [181,185]. However, the AMPs are vulnerable to enzymes and can easily cause immune responses; thus, the delivery system is critical for in vivo applications of AMPs. Hazekawa et al. [186] conjugated antimicrobial human peptide, LL-37 peptide fragment analog, with a PLGA copolymer. The formed micellar system significantly improved the permeability of the peptide to cancer cells, and the proliferation, migration and invasion in various cancer cell lines were effectively exhibited. The intracellular delivery of peptides by polymer carriers in oncology applications has been summarized by Pun et al. very recently [187].

Another strategy for eliminating cancer cells using antimicrobial delivery systems is the co-delivery of antimicrobial and anticancer agents simultaneously or loading anticancer drugs with antimicrobial carriers [188,189]. For instance, Du and coworkers [190] proposed the concept of "armed" carrier to co-deliver anticancer and antiepileptic drugs with antibacterial polypeptide-grafted chitosan-based nanocapsules. Mahkam et al. [191] designed pH-responsive antibacterial clay/polymer nanocomposite as a carrier to deliver anticancer drug methotrexate and antibacterial agent ciprofloxacin with an ultrahigh efficiency of >90%, which showed enhanced antimicrobial and anticancer activity compared with free methotrexate and ciprofloxacin, demonstrating the potential of antibacterial nanocarriers in cancer therapy. Lei and coworkers [192] developed a class of multifunctional polymeric hybrid micelles (PHM) with high antibacterial activity for the efficient delivery of siRNA to cancer cells, as illustrated in Figure 14. The PHM was prepared by the co-assembly of EHP-FA and EHE, for which their structures were presented in Figure 14A. Due to the existence of positively charged poly(ethylene imine) (PEI) and poly-ε-L-lysine (EPL), the PHM showed high antibacterial activity against *S. aureus* in vitro and in vivo. On the contrary, PHM exhibited good hemocompatibility and lower cytotoxicity toward A549, HeLa, HepG2 and C2C12 cells benefiting from the shield effect of PEG. siRNA could be complexed onto PHM by electrostatic interaction, and PHM with folic acid decorated on the surface could effectively target FA receptor overexpressed HeLa cells and other low-expressed cancer cells, resulting in the targeted delivery of siRNA. In vitro experiments revealed that the PHM showed a high p65 gene silencing efficiency above 90% in various cancer cells, which is significantly higher than EHP-FA and EHE, demonstrating the potential of PHM as a safe and effective siRNA vector with high antibacterial activity for multifunctional gene therapy.

**Figure 14.** Scheme showing the synthesis and potential application of the PHM copolymer in siRNA delivery. (**A**) EHP and EHP-FA were synthesized by Michael addition and esterification reaction, respectively; PHM micelles were prepared by mixing EHP-FA and EHE copolymer; (**B**) schematic illustration of the application of PHM in siRNA delivery (reproduced with permission from Zhou et al. [192], Nanoscale; published by Royal Society of Chemistry, 2018).

#### **4. Conclusions and Future Perspectives**

In summary, the recent progress of efficient loading, delivery and controlled release of antimicrobial agents in vivo or in vitro by polymeric nanomaterial-based delivery systems have been concluded. A large diversity of antimicrobial agents including antibiotics, AMPs, AgNPs, metal nanoparticles, metal oxides, gases, photosensitizers and so forth could be loaded and delivered by polymeric nanomaterials either by physical interactions or covalent bonding while maintaining the intrinsic antimicrobial activity of these antimicrobial agents. In order to fit the physiochemical properties of different kinds of antimicrobial agents to construct highly efficient delivery systems with superiorities such as high loading content and efficiency, good stability and on-demand release, polymeric nanomaterials with different chemical compositions and nanostructures including micelles, vesicles, dendrimers, nanofibers and nanogels etc. are developed. Benefiting from the versatility of polymeric nanomaterials, the antimicrobial agent delivery systems have shown significant potentials in a wide variety of biomedical applications, such as combating MDR bacteria, anti-biofilm, wound healing, tissue engineering and anticancer. Despite the rapid development of this field, the in vivo and intracellular delivery of antimicrobial agents is still in its early stage, and there are numerous challenges that should be considered in the

future, which may bring new opportunities in the biomedical applications of antimicrobial agent-based delivery systems.

Non-covalent interactions such as hydrogen bonding, π-π stacking and coordination should be introduced to enhance the interactions between antimicrobial agents and the polymeric nanocarriers to increase loading content and efficiency. The strong interactions could also prevent the leakage of cargoes before reaching the target and enhance the stability of the delivery system. Modulation of the properties of different kinds of antimicrobial agents and the structural features of carriers may maximize the efficiency of the loaded antimicrobial agents. Targeting the infected area and high selectivity toward bacteria rather than mammalian cells should always be considered, which is very important for the reduction in side effects and drug resistance. Moreover, external stimuli, especially non-invasive stimuli-triggered release of loaded antimicrobial agents (in other words, the switchable antimicrobial activity of the delivery system), are also helpful for the reduction in side effects and drug resistance. However, the spatial and temporal sensitivity of the stimuli-triggered response still needs to be improved to meet practical applications. Furthermore, the generations of antimicrobial active species such as ROS or change of the micro-circumstance including elevating temperature triggered by stimuli or chemicals secreted by bacteria are also effective methods for eliminating bacteria without the generation of drug resistance. Regardless of the generation of drug resistance, taking advantage of the synergistic effect of multiple antibacterial agents is an effective strategy for eradicating MDR bacteria. In addition, the combination of antibacteria and anticancer simultaneously will be of great significance in cancer therapy.

The biosafety of polymeric nanomaterial-based delivery systems has always been selectively ignored in previous studies. Although many biodegradable polymers have been used, the cytotoxicity and hemolytic activity of the polymeric carriers, especially those with positively charged surfaces, should be evaluated systematically. In addition, the word "biocompatibility" is a comprehensive evaluation of in vivo delivery systems. If we claim that the carrier is biocompatible, numerous parameters should be evaluated more than cytotoxicity and hemolytic activity. The in vivo delivery of antimicrobial agents has been reported in many studies. However, very few investigated the stability of the delivery system in physiological conditions and the interactions between the carriers and proteins, salts, glucose, fatty acids, antigens and so forth. Moreover, the immune response of the delivery systems is also hardly investigated. Considering the complexity of the physiological condition, it is necessary to reveal the stability and true circulation behavior of the delivery systems in vivo and not only borrowing the results of in vitro experiments. Furthermore, the full life-cycle assessment of polymeric carriers should be conducted to explore blood circulation behavior, biodistribution, metabolism and organic accumulations, etc., which will be very valuable for the instructive design of polymer carriers to promote the clinical applications of polymeric nanomaterials-based antimicrobial delivery systems.

**Author Contributions:** Conceptualization, supervision and writing—review and editing, H.S.; resources and visualization, Y.W.; writing—original draft preparation, H.S. and Y.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Natural Science Foundation of Ningxia, grant number 2020AAC03003 and 2021AAC03026.

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

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** This work was supported by the Natural Science Foundation of Ningxia (2020AAC03003 and 2021AAC03026). H.S. thanks the Ningxia Youth Talent Support Project of Science and Technology and Young Scholars of Western China of CAS.

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

#### **References**


### *Review* **Nanotechnologies: An Innovative Tool to Release Natural Extracts with Antimicrobial Properties**

**Umile Gianfranco Spizzirri 1,\*, Francesca Aiello 1, Gabriele Carullo 2, Anastasia Facente <sup>1</sup> and Donatella Restuccia <sup>1</sup>**


**\*** Correspondence: g.spizzirri@unical.it; Tel.: +39-0984-493298

**Abstract:** Site-Specific release of active molecules with antimicrobial activity spurred the interest in the development of innovative polymeric nanocarriers. In the preparation of polymeric devices, nanotechnologies usually overcome the inconvenience frequently related to other synthetic strategies. High performing nanocarriers were synthesized using a wide range of starting polymer structures, with tailored features and great chemical versatility. Over the last decade, many antimicrobial substances originating from plants, herbs, and agro-food waste by-products were deeply investigated, significantly catching the interest of the scientific community. In this review, the most innovative strategies to synthesize nanodevices able to release antimicrobial natural extracts were discussed. In this regard, the properties and structure of the starting polymers, either synthetic or natural, as well as the antimicrobial activity of the biomolecules were deeply investigated, outlining the right combination able to inhibit pathogens in specific biological compartments.

**Keywords:** nanotechnologies; plant extracts; agro-food-wastes; antimicrobial agents; polymeric nanocarriers

#### **1. Introduction**

Nanotechnology involves different strategies by using natural and synthetic materials in nanoscale dimensions to fabricate devices widely employed in the electronic and food industries, as well as in the pharmaceutical and biomedical fields [1]. Polymeric nanocarriers, due to their high surface area and small dimension (1–100 nm), are able to increase permeability and solubility of the enclosed molecules, making them available for several health applications, including diagnosis, disease treatments, and imaging [2–4]. In addition, effectively modifying the key features of nanocarriers, i.e., size, constituents, shape, and surface properties, it is possible to tune their mechanical, biological, and physicochemical characteristics [5]. In particular, nanotechnologies have gained outstanding consideration in the development of smart and effective pharmaceutical systems able to transport and deliver bioactive components in a specific site, avoiding, at the same time, deterioration due to enzymatic activity and pH values [6]. Among bioactive molecules, natural compounds have always represented the most widely employed substances for their unique therapeutic properties against several diseases [7]. In fact, natural bioactive extracts from plants, herbals, or agro-food by-products represent a rich source of compounds (polyphenols, anthocyanins, flavonoids, and many others) useful in the treatment of various diseases, thus suggesting their addition to pharmaceutical and cosmetic formulations, as well as to nutraceutical supplies [8]. In particular, many nanodevices (i.e., nanofibers and nanoparticles) have been developed to serve as antimicrobial agents to avoid pathogens' proliferation. In literature, a large number of articles can be found, describing the transport of antimicrobial agents, mainly, but not only, to the skin compartment in the wound treatment, in order to prevent infections and/or to accelerate the healing process [9–11].

**Citation:** Spizzirri, U.G.; Aiello, F.; Carullo, G.; Facente, A.; Restuccia, D. Nanotechnologies: An Innovative Tool to Release Natural Extracts with Antimicrobial Properties. *Pharmaceutics* **2021**, *13*, 230. https://doi.org/10.3390/ pharmaceutics13020230

Academic Editor: Clive Prestidge Received: 29 December 2020 Accepted: 3 February 2021 Published: 6 February 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 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/).

In this review, highly innovative nanotechnology-based delivery systems loaded with bioactive molecules recovered from natural matrices and showing antimicrobial activity were described. The referenced papers were selected through the articles published from the year 2010 and sorted based on the specific type of nanocarrier.

#### **2. Natural Extracts with Antimicrobial Activity**

The search for new therapeutically active compounds has spurred researchers over the years to investigate natural compounds [12–17]. In particular, food and plant wastes represent interesting sources of biologically active molecules [18–24] and have been proposed as indigenous remedies [25,26]. Specifically, secondary metabolites from plants represent valuable bioactive ingredients [27–30] with remarkable antibacterial properties [31,32], useful in the treatment of several diseases. Table 1 summarizes the main natural extracts proposed for their antimicrobial features.

The valuable therapeutic power of *Glycyrrhiza glabra* L. var cordara is well known: its extracts showed a panel of antibacterial features against various bacterial strains (128 < minimal inhibitory concentration (MIC < 512 μg/mL), the activity being mainly related to the pinocembrin, recovered in the extract as free and fatty acids-conjugated form [14]. Similarly, the nutritional properties of the male date palm flower, via defining its antibacterial actions, were also scouted [33]. The chromatographic analysis identified the presence of several phenolic compounds. Among them, quinic acid was recovered as the main component (84.52% *w/w*) and significantly influenced the nutraceutical and pharmacological properties of the extract.

Thin-layer chromatography (TLC) micro-fractionation of the organic extracts of *Ferula ferulioides* [34], a traditional medicinal plant, served as a guiding tool to isolate two compounds (dalpanitin and vicenin-3), with remarkable antimicrobial activity against drug-resistant *Staphylococcus aureus* [35].

Antimicrobial activity of essential oil (EO) of Cyprus *Citrus aurantium* L. flowers was analyzed, and the recorded minimum inhibitory concentrations (MIC) against Amoxycillinresistant *Bacillus cereus* was 1.562 mg/mL [36]. Moreover, the extracts of *Psidium* sp., *Mangifera* sp., and *Mentha* sp. and its mixtures displayed antimicrobial effects and strongly reduced *Streptococcus mutans* [37].

The phytochemical study of the aerial part of *Pulicaria undulata* L. led to the isolation of nine compounds. The organic extracts (methanol, ethyl acetate, and dichloromethane) of the aerial parts were assayed by in vitro antimicrobial activity against a panel of sensitive microorganisms [38]. Similarly, the phenolic compounds in the solvent extracts of *Genista saharae* were analyzed. The chloroform extract, containing high amounts of quercetin and naringenin, revealed the antioxidant potential and antibacterial activity against the bacterial strains (MIC 0.02 mg/mL). These results seemed to indicate a high contribution of quercetin and naringenin in the antimicrobial and antioxidant activities recorded [39].

The prevalence of different types of chronic wounds due to the aging population and the increasing incidence of diseases is a worldwide clinical emergency. Various medicinal plants used in folk medicine and showing wound healing and antimicrobial properties have been widely assessed [40].

As known, some mushrooms and numerous other fungi exhibit innovative properties, including antimicrobial features against bacteria, fungi, and protozoans. In particular, in a study, 316 species of 150 genera from 64 fungal families were analyzed, showing antibacterial activity against different bacteria and fungi [41].

The main component of extracts from white guava (*Psidium guajava* L. cv. Pearl) was quercetin-glycosides. In particular, the micro-morphology of both *Escherichia coli* and *S. aureus* was changed with a flavonoids concentration of 5.00 mg/mL and 0.625 mg/mL, extracted from white guava leaves [42].


Plant

endowed

with

antimicrobial


**Table 1.** *Cont.* Inhibition zone; minimal inhibitory concentration (MIC), Minimum bactericidal concentration (MBC) (mg/mL); cell-surface hydrophobicity of bacteria (%); inhibition dimension (IZD) (mm).

The traditional use of *Polyscias scutellaria* Fosberg to treat body odor suggested that this plant shows antibacterial properties. Most of the microorganisms hosted by human skin are harmless and even useful against pathogenic bacteria. Furthermore, *Acinetobacter* sp., formerly known as commensal bacteria, evolved into pathogenic bacteria and caused outbreaks in the intensive care unit. In this context, the antibacterial activity of *P. scutellaria* Fosberg extracts against *Acinetobacter* sp. isolated from healthy human armpit was investigated [43].

The ethyl acetate fraction from the leaves of *Schismus fasciculatus* contained kaempferol, quercetin, and agathist flavone, which showed moderate antibacterial activity against different tested strains (IC50 0.9 mg/mL) [44]. In addition, both cardamom (*Elettaria cardamomum*) fruit and seed extracts exerted remarkable antibacterial effect against *Aggregatibacter actinomycetemcomitans*, *Fusobacterium nucleatum*, *Porphyromonas gingivalis*, and *Prevotella intermedia* [45].

The antimicrobial properties of oregano (*Origanum vulgare*), sage (*Salvia officinalis*), and thyme (*Thymus vulgaris*) essential oils (EO) were assayed against *Klebsiella oxytoca* (MIC of 0.9 mg/mL for oregano EO and 8.1 mg/mL for thyme EO) [46].

The water and ethanolic extracts of *Myristica fragrans* (Myristicaceae) wood displayed interesting antimicrobial, anti-inflammatory, and antioxidant activities [47].

The phenolic composition, antimicrobial activities, and antioxidant activity of *Euphorbia tirucalli* L. extracts were evaluated by agar dilution methods, and MIC values were recorded. In all samples, ferulic acid resulted as the main phenolic compound identified and quantified through LC-UV. The extracts demonstrated inhibitory potential against *Staphylococcus epidermidis* and *S. aureus* [48].

The methanol leaf extract of *Tradescantia zebrina* showed the highest antioxidant content and activity, exhibiting antibacterial activity against six species of Gram-positive and two species of Gram-negative bacteria in a range of 5–10 mg/mL [49].

The aqueous extracts of *Adiantum caudatum* leaves, obtained by Soxhlet extraction, resulted as more powerful than the hexanoic one against *Pseudomonas aeruginosa* [50]. The methanol extracts from the flowers of *Agastache rugosa* (Korean mint) showed high antibacterial activities [51]. Finally, the carotenoid fucoxanthin was observed to have a significantly stronger impact on Gram-positive than Gram-negative bacteria [52,53].

#### **3. Agro-Food Wastes as Antimicrobials**

The processing of agro-products generates huge amounts of waste materials every year in the form of peels, seeds, and oilseed meals, thus representing serious environmental concerns. Besides, the cost of drying, storage, or transportation poses a severe financial limitation to wastes utilization. To support the transformation and exploitation of these by-products, there is a growing interest in recycling waste biomass of agro-products in particular, considering their therapeutic properties (Table 2).

For example, the major wastes for industrial apple juices are the seeds. After a Soxhlet extraction, oil was recovered, and this apple seed oil was completely active against bacteria, showing MIC values ranged from 0.3–0.6 mg/mL [54].

About a quarter of the total tomato production undergoes processing, leading to derivatives like sauces, canned tomatoes, ketchup, or juices, largely consumed worldwide. At the same time, the tomato industry generates huge quantities of wastes, up to 5–0% of the total production. These by-products are used as livestock feed or discarded in landfills, creating many environmental problems. However, considering that valuable phytochemicals, such as carotenoids, polyphenols, tocopherols, some terpenes, and sterols, resist industrial treatment, tomato by-products represent also a precious resource. An interesting experimental work reported that the most active peel tomato extract against *S. aureus* and *Bacillus subtilis* (MIC: 2.5 mg tomato peels/mL) belonged to the ¸Tărăne¸sti roz variety, owing to its high carotenoid amount [55].



*Pharmaceutics* **2021**, *13*, 230

**Table 2.** *Cont.*

(mm); c μg GAE/mL; d IZ (inhibition zone mm).

Fennel and carrot, two species belonging to the Apiaceae family, are, like many others (e.g., tomatoes, potatoes, and onions), the most commonly consumed vegetables worldwide. They are aromatic and have been used as spices and condiments. Their EO, related to the fruits, is well characterized, whereas the chemical composition of the leaves, a by-product, is poor. Wiem Chiboub and co-workers performed a hydrodistillation of fresh leaves of carrot and Daucus carota subsp. sativus orange roots and yellow roots and *F. vulgare* subsp. vulgare var. azoricum and *F. vulgare* subsp. vulgare var. latina. The recorded results showed that the Daucus carota subsp. sativus yellow roots oil was significantly more effective against Gram-negative than Gram-positive bacteria, and the MIC values were in the range 6.25–50 mg/mL [56].

In order to reuse agro-wastes, the betel leaf stalk extract was found to be a potent antimicrobial agent, showing activity against Gram-positive and Gram-negative bacteria. The MIC values were in the range 25–250 μg/mL, measured against ciprofloxacin as a standard [57].

Among agro-food wastes, those derived from the olive oil production represent the most representative, especially in the Mediterranean area. Their composition was found to be rich in hydroxytyrosol and secoiridoids derivatives, important for their healthy properties. Inass et al. investigated the in vitro antimicrobial potential of olive mill wastewater and olive cake extracts. Oleuropein and verbascoside, already pointed out in various studies for their important antimicrobial potential, were also detected in these extracts. Furthermore, the elenolic acid, the main fragment of the oleuropein degradation, was mostly found in the olive cake extract. It can be considered as an important antimicrobial and antiviral agent, justifying the reuse of this kind of wastes [58].

Besides, the fruits belonging to the Citrus sinensis family produce large amounts of wastes, mostly seeds and peels, endowing suitable biological value. Seed oil demonstrated better activities than peel oil, with remarkable inhibitions obtained against *S. aureus* and *Candida albicans* at a concentration as low as 2.5 mg/mL [59]. Furthermore, the orange peel of 12 cultivars of Citrus sinensis from central-eastern was extracted through steam distillation and using hexane. In all the cultivars, the main component was D-limonene (73.9–97%). The antimicrobial activity was investigated against *S. aureus*, *Listeria monocytogenes*, and *P. aeruginosa*. 'Sanguinello' and 'Solarino Moro' essential oils were significantly active against *L. monocytogenes*, while 'Valencia' hexanoic extract against all the tested microorganisms [60].

In this context, the winemaking process is also involved in smart wastes management. Grape seeds are the by-products of the fruit juice and wine industries. Nowadays, more attention is devoted to the valorization of these kinds of wastes due to the valuable phytochemicals content. A study performed on different varieties of grape seeds, extracted with 70% ethanol, showed that all the tested varieties possessed a considerable antibacterial activity. Particularly, the variety Shiraz showed a large zone of inhibition (17 mm) [61]. Different fractions of wine residue (pomace, including seed and skin, seeds, or skin) from two red varieties of *Vitis vinifera* grapes (Pinot noir and Pinot Meunier) grown in New Zealand were extracted using different water-organic solvent mixtures. It was found that all the extracts exhibited antibacterial and antifungal effects, with MIC values ranging between 0.195 and 100 mg/mL [62].

Similarly, lavender and melissa wastes were proved to be rich in polyphenols (especially rosmarinic acid) and exhibited high antimicrobial activity [63]. The processing of jackfruit (*Artocarpus heterophyllus* Lam) yields large amounts of bio-wastes. The ethyl acetate extracts obtained from the peel, fiber, and core of the jackfruit showed antibacterial activity against *Xanthomonas* axonopodis pv. manihotis [64].

Apple and Sabine mango kernel extracts exhibited significantly high inhibition zones of 1.93 and 1.73 compared to Kent and Ngowe with 1.13 and 1.10, respectively, against *E. coli*. For *C. albicans,* the inhibition of Kent mango kernel extract, 1.63, was significantly lower than that of Ngowe, Apple, and Sabine with 2.23, 2.13, and 1.83, respectively [65].

The *Vaccinium meridionale* Swartz pomace is a source of bioactive compounds with remarkable antibacterial activity. Quercetin derivatives represented 100% of the total flavonols in the extracts, and *S. aureus* was the most sensitive strain [66].

The walnut green husk is an agro-forest waste obtained during walnut (*Juglans regia L.*) processing; its aqueous extracts were found to be able to inhibit the growth of Grampositive bacteria [67]. Another study reported the antioxidant and antimicrobial activities of extracts of pecan nutshell. The MIC and minimum bactericidal concentration (MBC) values against *L. monocytogenes*, *Vibrio parahaemolyticus*, *S. aureus*, and *B. cereus* were significantly lower (*p* < 0.05) for the extract obtained through infusion, followed by atomization in a spray dryer when compared to the other extracts [68]. Water, methanol, ethanol, and 50% (*v/v*) aqueous solutions of methanol and ethanol extracts of disposed garlic husk displayed antimicrobial activity against Gram-positive bacteria when applied at different concentrations (1–10 mg/mL). These interesting biological properties could be attributed to specific phenolic compounds, such as caffeic, p-coumaric, ferulic, and di-ferulic acids [69].

Extracts prepared from mangosteen bark or fruit pericarp exhibited strong pH-dependent bacteriostatic and bactericidal effects against *L. monocytogenes* and *S. aureus* [70]. Ethyl acetate extract of Newhall orange peel showed the best antimicrobial effect due to the presence of sinensetin, 4 ,5,6,7-tetramethoxyflavone, nobiletin, 3,3 ,4 ,5,6,7-hexamethoxyflavone, and narirutin [71]. Punica granatum peels (Bhagwa) furnished extracts, endowing interesting antibacterial properties. LC analysis of the extract recorded the punicalagin (163.52 mg/g of waste) as a major ellagitannin compound. Peel extract exerted high antibacterial activity against both Gram-positive and Gram-negative bacteria [72].

Extracts from brewer's spent grain, the major by-product of the brewing industry, proved to be a rich source of bioactive compounds with antimicrobial activity (especially against *C. albicans*) [73].

The phytochemical screening of the aqueous extract of the Agave sisalana Perrine juice (waste) revealed the presence of saponins, glycosides, phlobatannins, terpenoids, tannins, flavonoids, and cardiac glycosides and had the potential to be used against pathogenic organisms [74].

The antimicrobial activity of three abundantly available fruits peel waste (orange, yellow lemon, and banana) was evaluated on a wide range of microorganisms. Methanol, ethyl acetate, ethanol, and distilled water were used for extraction, and the results showed that, among the used solvents, the extracts exhibiting better performances were in decreasing order: Distilled water > Methanol > Ethanol > Ethyl acetate, reflecting the suitability of solvent for fruit peel extraction. Additionally, the effectiveness of fruit peel extracts was evaluated, showing Yellow lemon > Orange > Banana peel. It was observed that Gram-negative bacteria were more sensitive to the extracts, and, among them, *Klebsiella pneumoniae* showed the highest sensitivity against the extract of yellow lemon peel with the highest zone of inhibition [75]. Beet stalk, peanut peel, Pinot Noir grape marc, Petit Verdot grape seed and marc, red grapes fermentation lees, and guava bagasse wastes showed antimicrobial activity against *S. aureus* and *L. monocytogenes*. Analyses by GC-MS identified relevant concentrations of compounds exhibiting antimicrobial activity, such as caffeic, gallic, ferulic, and r-coumaric acids, and flavonoids quercetin, myricetin, and epicatechin. This study confirmed that agro-industrial wastes from wine and food industries could be used in the research about new antimicrobial compounds to be used as natural preservatives in the food and beverage industry with promising applications also in the pharmaceutical and biomedical fields [76].

With a growing world production, mango represents one of the most important tropical fruits produced worldwide. India is one of the most important producers where any mango-based products are also commonly consumed. Mango is mostly used in food processing industries, such as juice, jam, jelly, and pickle industries. This processed food leads to an enormous generation of mango peel as a waste product. It needs a huge capital to decompose these peels and to make sure that it does not pollute the environment. The EO of both mango indica cultivars pulp and peel showed a wide range of antibacterial and antifungal activities [77].

The peel of Camu-camu (*Myrciaria dubia* (Kunth) McVaugh) displayed the richest phenolic profile as well as the most significant antibacterial activity (MICs recorded were in the range 0.625–10 mg/mL) [78].

Interesting results showed that the fraction with the highest content of phenolic and secoiridoid compounds from crude olive mill wastewater had a relevant antibacterial activity against a large panel of strains with a strain-dependent character [79].

The natural food colors market is trying to fit the consumer needs by increasingly replacing synthetic additives with natural ones. Besides being a natural product, the biological relevance of the carotenoids is related to their potential antioxidant and antimicrobial features, boosting their wide application in the food and pharmaceutical industry. The production of carotenoids by microorganisms using agricultural waste has been reported, employing coffee pulp and husk using a non-conventional yeast [80]. Despite the high antimicrobial activity exhibited by these extracts, their bioavailability is poor when used as such. In this context, the inclusion of nanocarriers seems to be an innovative tool to overcome this limit.

#### **4. Nanofibers as Carriers of Antimicrobial Natural Products**

During the last years, an increasing interest in the biomedical use of polymeric nanofibers was recorded due to their high porosity, outstanding mechanical strength, and simplicity of fabrication [10]. In particular, nanofibers have been proposed as systems for the delivery of bioactive molecules [81] or in regenerative medicine [82] and also as wound dressings devices [83]. Usually, polymeric nanofibers can be fabricated by template synthesis [84], self-assembly [85], phase separation [86], and electrospinning [87]. Among these, the electrospinning technique is the most used strategy for applications in tissue engineering and drug delivery due to its high-throughput, easy handling, and reproducibility (Figure 1).

**Figure 1.** Electrospinning process and the different experimental parameters affecting the diameters of the produced nanofibers. Reproduced with permission from [10], Elsevier, 2020.

Additionally, electrospun meshes significantly increase adhesion and drug loading due to the structural similarity to the extracellular environment of the living tissues [88]. Literature data clearly indicate that electrospun nanofibers have the remarkable potential to amplify effectively the biological properties of medicinal plant extracts, essential oils, or pure single components with antimicrobial features. For this reason, they were largely employed as bioactive molecules to fabricate delivery systems, tissue engineering scaffolds, and regenerative medicine devices (Figure 2) [11].

**Figure 2.** Schematic representation for the use of extracts isolated from medicinal plants and their nanofibers fabrication useful for pharmaceutical and biomedical applications. Reproduced with permission from [11]; Elsevier, 2020.

Specifically, in order to obtain polymeric nanofibers able to protect bioactive molecules and to ensure their controlled and site-specific delivery, both synthetic (polyesters, polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), or polyacrylates) and/or natural (polysaccharides or proteins) macromolecular structures have been proposed [89,90]. Synthetic constituents are usually cheaper and stronger, more easily electrospinnable, and show a precise structure [91]. Among the synthetic polymers used in the preparation of the nanofibers, PVA, PVP, and polyesters, such as polylactic, polylactic-*co*-glycolic acid, polyurethane, and polycaprolactone, were widely used with significant results (Table 3). On the contrary, natural polymers can be obtained by environment-friendly sources, offer good biocompatibility, thus reducing the adverse effects that could be observed when introduced to the human body [65] (Table 4). However, synthetic polymers are often required to strengthen the weak mechanical resistance of natural polymers [66].




Dandelion

polysaccharide

*cordifolia* extracts

*Litsea cubeba* essential oil

*S. aureus*

engineering

Antimicrobial

 activity

 [125]

 scaffolds


**Table 4.** *Cont*.

#### *4.1. Synthetic Polymer-Based Nanofibers*

#### 4.1.1. Polyacrylates

Among polyacrylates, polyacrylonitrile (PAN) has been well documented to be a very important material for easy manufacturing of synthetic fibers with unique thermal and mechanical stability, as well as excellent resistance to the solvents [144,145]. In the biomedical field, PAN nanofibers loaded with moringa leaf extracts, rich in phytochemicals, such as zeatin, quercetin, amino acids, and phenolic compounds, have been proposed for wound healing applications [92,146]. The antibacterial properties of the loaded nanofibers were evaluated against *E. coli* and *S. aureus*. Results displayed that the activity of the device strictly depended on the concentration of the extract in the PNA nanofibers, confirming its promising potential as an effective wound dressing. Alternatively, electrospinning PNA nanofibers loaded with *Syzygium aromaticum* oil, containing unexplored natural bioactive molecules (eugenol and caryophyllene), were found to be highly effective against both Gram-positive and Gram-negative bacteria in in vitro delivery studies [93,147]. The release profile of *Syzygium aromaticum* was characterized by an initial burst, followed by controlled diffusion kinetics. Similarly, PNA fibers were loaded with EO of lavender (*Lavendula angustifolia*) [94]. This natural product is largely exploited in aromatherapy as a holistic relaxant and has been exploited for its excellent antimicrobial features. Lavender oil, highly encapsulated in PAN fibers with a loading content equal to 13.6%, displayed a release profile with an initial burst effect, reaching a 35% of active release after 24 h in a buffer medium (pH 7.4). The loaded polymeric device showed also in vitro antibacterial activity after an incubation time of 24 h (MIC equal to 100 mg·mL<sup>−</sup>1).

#### 4.1.2. Polyesters

Poly (lactic acid) (PLA) is an outstanding polymeric material largely employed in the pharmaceutical and biomedical sectors due to its excellent biodegradability and biocompatibility [148]. PLA represents the most extensively used synthetic polymer in the fabrication of nanofibers [149]. Electrospinning of PLA fibers containing EO derived from *Leptospermum scoparium* and *Melaleuca alternifolia* at different concentrations has been proposed for the treatment of microbial infections induced by *S. epidermidis* [95], a bacterium abundant on human skin and often responsible for infections and formation of biofilms on the medical devices [150]. The antibacterial activity of *Leptospermum scoparium* EO was mainly ascribed to triketone species, such as flavesone and leptospermone [151], while terpinen-4-ol and α-terpinene determined the antimicrobial activity of *Melaleuca alternifolia* EO. Although *Melaleuca alternifolia* oil showed remarkable activity against *S. epidermidis*, its efficacy was largely limited due to the low amount of active molecules available after the electrospinning process. On the contrary, the antimicrobial activity of the PLA-*Leptospermum scoparium* oil system was preserved during the electrospinning process.

In order to improve its water solubility, PLA was successfully modified by hyperbranched polyglycerol, a highly hydrophilic polymer with excellent biocompatibility and water solubility [152]. Nanofibrous dressing with PLA and the hyperbranched polyglycerol blend was fabricated using electrospinning technique and loaded with curcumin, a polyphenolic biologically active ingredient of turmeric isolated from the dry rhizomes of *Curcumin Longa* L. [153]. Curcumin release profiles, recorded at 37◦C in PBS and displayed as the increased hydrophilicity of the device, allowed to achieve a complete delivery in 72 h, suggesting its use as potential wound patch dressing for chronic and acute wound diseases.

Alternatively, fibers based on poly(D,L-lactide-*co*-glycolide) (PLGA) have been successfully proposed as delivery systems due to their high biodegradability. In particular, the electrospinning technique was employed to fabricate ultrafine PLGA fiber mats containing a methanolic extract of *Grewia mollis* (7.5% *w/w*). The antimicrobial effect of the device was tested against pathogenic bacteria, such as *E. coli* and *S. aureus*, suggesting its use in the treatment of dermal bacterial infections or as a wound dressing agent [96]. Garcia-Orue and coworkers developed a PLGA-based nanofibrous membrane in which the antibacterial

properties of the natural extract and the ability of the epidermal growth factor to enhance fibroblast proliferation allowed the fabrication of an efficient device against *S. aureus* and *S. epidermidis*, reducing, at the same time, the wound healing time [97].

Polycaprolactone (PCL) represents a biodegradable aliphatic polyester, largely employed to produce nanofibers by electrospinning techniques. The incorporation of suitable natural extracts allowed the fabrication of devices with remarkable antimicrobial properties. Specifically, crude extract of *biophytum sensitivum* was loaded, and experimental release displayed a controlled delivery of the bioactive molecules (59% in 72 h) [98]. The antimicrobial performances of the device were evaluated against some common wound bacteria, such as *S. aureus* (27 mm inhibition) and *E. Coli* (47 mm inhibition). Similarly, electrospun PCL-based nanofiber mats containing natural leaves extracts of *Gymnema sylvestre* [99] or *Clerodendrum phlomidis* [100] were proposed as a wound dressing with remarkable antimicrobial characteristics. The cumulative delivery profiles depicted an intense burst effect, mainly due to bioactive desorption release mechanisms, while a constant delivery of the therapeutic was reached after 24 h.

*Althea officinalis* extract is also well known as a traditional herbal drug with noteworthy wound healing capacity and antimicrobial properties. In particular, *Althea officinalis* extract was loaded in nanofibers based on PCL and gelatin [101]. The blending of synthetic and natural macromolecules was proposed as an efficient strategy to optimize the mechanical properties of the scaffolds. In addition, the superior viscoelastic properties of PCL with respect to other polyesters made it more compatible with natural macromolecules, allowing the construction of multilayer structures. Cumulative release experiments highlighted a release of bioactive molecules proximately to 100% after 24 h. Similarly, the PCL layer was deposited onto the gelatin films by electrospinning to obtain complex structures able to vehicle black pepper oleoresin bioactive components. Strong antimicrobial properties of the multilayer were recorded over time (10 days) against *S. aureus* [102].

Blended polymer-based electrospun nanofibers have gained great attention because of the mechanical properties of the matrices and easy monitoring of the release profiles, depending on the ratio of the components in the polymer chains. By coupling the strength of PCL with polyvinyl pyrrolidone (PVP), PCL/PVP nanofibers containing crude bark extract of *Tecomella undulata* were fabricated, and their antimicrobial behavior was evaluated against *S. aureus*, *E.coli,* and *P. aeruginosa* [103]. Recently, in order to improve antimicrobial properties of PCL-nanofibers loaded with *Nephelium lappaceum* extract, they were decorated with silver nanoparticles [154], providing a multi-component device with synergistic antibacterial properties [104]. The antimicrobial activity was evaluated against *E. coli*, *S. aureus*, and *P. aeruginosa,* and the results suggested a synergistic effect between silver nanoparticles and extract-loaded nanofibers.

Polyurethane (PU) is a biocompatible hydrophobic polymer, largely used for biomedical and pharmaceutical scopes, owing to its excellent oxygen permeability and good barrier and mechanical properties [155]. Nanofibers based on PU were successfully prepared to load emu oil, a natural mixture derived from the emu (*Dromaius novaehollandiae*), which originated in Australia and positively tested against both Gram-positive (*B. subtilis*) and Gram-negative (*E. coli*) pathogens [105]. Similarly, PU nanofibers were proposed as polymeric carriers of propolis [84], a resinous substance produced by bees from plant exudates, containing different bioactive compounds [156]. In addition, the adhesive properties of propolis afford the point-bonding to the PU nanofibers, improving their mechanical issues. The antimicrobial properties of propolis/PU nanofibers were evaluated against *E. coli*, and the results showed that the bacterial inhibition zone was progressively increased with increasing the propolis amount in the nanocomposite. More recently, herbal extracts of *Agrimonia eupatoria*, *Satureja hortensis*, and *Hypericum perforatum* were loaded on several PU composite nanofibers, and their antimicrobial properties were evaluated against *S. aureus* and *P. aeruginosa* [110]. Finally, antibacterial and elastic nanofibers from thermoplastic PU were produced by coating process with *Syzgium aromaticum* extract, gained by Soxhlet

extraction of clove oil [107]. All coated nanofibers (2–10 mg cm−2) showed antibacterial activity against *S. aureus* and *E. coli*.

Nanomaterials to be employed as antimicrobial clothing materials or bedding materials for allergic patients often required a lamination process. In this regard, the nanofibers of PU/*Juniperus chinensis* extracts were prepared by laminating electrospraying PU adhesive resin on polyethylene terephthalate-based fabric and an electrospun PU nanofiber web [108]. The antibacterial experiments performed at 110 and 130◦C against *S. aureus* and *K. pneumoniae* confirmed that the combination between laminating and electrospinning represents an interesting way to project useful composites to be employed in the biomedical field. Finally, in order to improve the absorption ability of wound exudates, hydrophilic blend materials based on a different weight ratio between PU and carboxymethyl cellulose (CMC) and containing *Malva sylvestris* extract were prepared. The new materials proved to efficiently deliver the bioactive compounds to diabetic wounds [109]. The release profile of the active compounds resulted in a complete delivery in 85 h, and strong antibacterial activity against *E. coli* and *S. aureus* was recorded.

#### 4.1.3. Poly(Vinyl Alcohol)

Synthetic biodegradable polymers, such as PLA, PLGA, and poly(glycolic acid), are poorly soluble in water. It follows that the production of nanofiber mats often requires the use of organic solvents, incompatible with the preparation of carriers for pharmaceutical and biomedical uses. On the contrary, biocompatible, biodegradable, and no-toxic poly(vinyl alcohol) (PVA) can be easily employed to prepare nanofibers in aqueous environments [157]. Different natural extracts, as sources of antimicrobial molecules, were enclosed in PVA nanofibers. In particular, *Lawsonia inermis* leaves' hydroalcoholic (ethanol/water 90/10 *v/v*) extracts were loaded to achieve antibacterial nanofibers (2.8% *w/w*) with bacteriostatic action towards *E. coli*. and bactericidal efficiency against *S. aureus* [110]. Similarly, the methanolic extracts of *Tridax procumbens* leaves [111] and the aqueous extracts of *Coptis chinensis* [112] were employed to prepare nanofibrous mats with antimicrobial activity. PVA/*Tridax procumbens* nanofibers showed an outstanding zone of inhibitions and improved resistivity power against *S. aureus* and *E. coli*, whereas PVA/*Coptis chinensis* nanofibers, evaluated against different Gram-positive bacteria, displayed the highest antibacterial activity against *S. epidermidis*. The ethyl acetate *Rhodomyrtus tomentosa* extract, rich in myricetin and rhodomyrtosone, was involved in the fabrication of electrospun nanofibers; the antimicrobial experiments against the common human pathogens (*E. coli*, *P. aeruginosa*, *B. subtilis,* and *E. faecalis*) displayed a clear inhibition zone (7–12 mm) by loading the extract in the concentration range of 1.5–2.5% (*w/w*) [113]. Finally, nanofibers containing *Coptidis rhizoma* extracts (10, 20, and 30% *w/w*) were fabricated using PVA as a carrier [114]. The release experiments performed at physiological pH of 5.5, set by acetate buffer solution, displayed an initial fast release, followed by a gradual release for 48 h. In addition, high antimicrobial activity was recorded against *S. aureus* (max inhibition zone 17.0 mm) and *S. epidermidis* (max inhibition zone 6.2 mm).

In addition, PVA was employed in combination with a natural polymer to achieve bio-nanocomposites fibrous mats. Specifically, a PVA-based device enclosing nanocellulose from pineapple was loaded with *Stryphodedron barbatimao* extract (water-alcohol solution 96:4 *v/v*) [115], a well-known mixture employed in medicine as an antiseptic, antiinflammatory, and anti-bacterial remedy [158]. More recently, PVA/guar gum composite nanofibers were proposed as a carrier of alcoholic extract of *Acalypha indica*, a traditionally acclaimed plant for wound healing [116]. The release profiles displayed a constant and slow delivery throughout the experimental time, while a complete release was observed after 38 h. The antimicrobial experiments against *E. coli, B. subtilis, S. aureus, P. fluorescens* highlighted bacteriostatic action against all microbial strains. Finally, several EOs (cinnamon, clove, and lavender at a concentration of 0.5, 1, and 1.5% *w/w*) were involved in the synthesis of PVA/sodium alginate (SA) polymeric nanofibers, allowing the preparation

of devices able to efficiently inhibit *S. aureus* [117]. Cinnamon oil (1.5% *w/w*)/PVA/SA nanofibers exhibited the best results (inhibition zone of 2.7 cm).

#### 4.1.4. Polyvinylpyrrolidone

Polyvinylpyrrolidone (PVP) exhibits unique properties because it is biocompatible, water-soluble, and non-toxic, allowing its application in the biomedical area [159].

*Sophora flavescens* extract was incorporated into PVP nanofibers, and the device was proposed as antimicrobial air filtration [118]. Filtration and antimicrobial performances of the PVP-based nanofiber filter were evaluated, employing *S. epidermidis* bioaerosols as test airborne particles; the results displayed excellent antimicrobial activity and highly effective air filters (99.99% filtration efficiency).

Nutraceutical properties of the cinnamon EO were exploited by enclosing the oil in the PVP-based nanofibers by oil-in-water emulsion electrospinning, and its antimicrobial properties were recorded against *S. aureus, E. coli, P. aeruginosa, C. albicans* [119]. The outstanding antimicrobial activity of the device was proved to be related to the size of the fibers and mainly ascribed to the high eugenol concentration in the cinnamon oil. Experimental tests were highlighted as the antimicrobial activity was recorded with cinnamon EO concentration into PVP in the range 2–4% (*w/w*).

#### *4.2. Natural Polymer-Based Nanofibers*

#### 4.2.1. Polysaccharides

Materials based on cellulose acetate (CA) have been largely employed in the biopharmaceutical processing industry as wound dressings, tissue engineering scaffolds, and drug delivery systems [160]. Electrospun CA nanofibers encapsulating lemongrass, cinnamon, and peppermint EOs were employed to fabricate scaffold devices able to inhibit bacteria growth. Results demonstrated the complete inhibition of *E. coli*, while *C. albicans* yeast showed a remarkable resistance, mainly due to its diameter more than four times larger than *E. coli* [120]. More recently, CA-based nanofibers enclosing rosemary and oregano EOs were evaluated against *S. aureus*, *E. coli,* and *C. albicans* [121]. Nanofibers loaded with the oregano oil (containing a high concentration of carvacrol and thymol) displayed the best anti-biofilm and antimicrobial performances. Electrospun nanofibrous mats based on carboxymethyl cellulose (CMC) and SA were proposed as functional wound dressing materials able to load olive leaf extracts [122]. The antibacterial properties of oleuropein and hydroxytyrosol contained in this aqueous extract allowed to fabricate a device able to inhibit pathogens mostly responsible for infections by skin wounds. The analysis of the release profile from the nanofibers depicted a substantial burst effect (50%) and a complete delivery within 24 h.

Nanofibers have also been proposed as reinforcing agents to improve the mechanical and physical performances of other polymeric devices, such as films, hydrogels, and sponges [161]. Nanocomposites based on CMC and cellulose nanofibers (CNF) were loaded with *Salvadora persica* hydro-alcoholic extract to obtain a device with good antimicrobial properties against both *S. aureus* and *E. coli*. [123]. The incorporation of CNF (5% *w/w*) into the CMC matrix caused a significant improvement of the mechanical properties in comparison with CMC. Similarly, composite biosponges of SA reinforced with CNF were prepared [124], determining a noteworthy enhancement in the mechanical performances and the thermal stability for SA-based composite biosponges. Moreover, the sponges loading with *Oryza sativa* and *Tinospora cordifolia* extracts imparted additional antibacterial functionality to the composite against *E. coli* and *P. aeruginosa*.

The encapsulation into nanofibers of the EOs gained from natural plants often requires a strong electric field and high-voltage. This could lead to adverse reactions, frequently involving the species able to impart pharmacological and nutraceutical properties to the extracts. To overcome this inconvenience, the employment of the β-cyclodextrin represents an endearing strategy [162]. *Litsea cubeba* EO was first encapsulated in the cavity of the βcyclodextrin, and the inclusion complex was then loaded by electrospinning into nanofibers

based on the dandelion polysaccharide [125], a natural polymer used for the preparation of active nanofibers [163]. The release profiles of the EO in PBS solution (pH 7.2) showed a good sustained release (about 70% after 100 h), ensuring also a long-lasting antibacterial effect against *S. aureus*.

Biomedical devices based on chitosan (CT) were exploited for developing nanofibrous wound dressing materials [164]. Additionally, CT is also known for its strong antibacterial activity against different fungi, bacteria, and viruses [165–167]. However, CT viscosity, as well as the high charge of the polysaccharide chains, made difficult the electrospinning process, and the application of highly acidic and toxic solvents was required [168]. These drawbacks can be overcome by co-spinning CT with other spun polymers, such as poly(ethylene oxide) (PEO), PCL, and PVA [169].

CT-ethylenediaminetetraacetic acid and PVA were selected for the preparation of nanofibrous mats to be employed as carriers of extracts gained by maceration (acetone/water 70/30 *v/v*) of *Garcinia mangostana* fruit hull [126], known to exert important antimicrobial properties due to the presence of the α-mangostin (13.20% *w/w*) [170]. α-Mangostin release experiments from the nanofiber mats loaded with the extracts revealed that active molecules were rapidly released, reaching 80% within 1 h. Additionally, the fiber mats loaded with the natural extracts exhibited antibacterial activity against *S. aureus* and *E. coli*. More recently, CT and PVA were successfully used to fabricate electrospun nanofibers containing *Bidens pilosa* extract. Composite nanofibers were effective against *E. coli* (growth inhibition 91% and MIC 10 mg/mL) and *S. aureus* (growth inhibition 86% and MIC 10 mg/mL) bacteria due to the combined effect of CT and natural extract [127]. Polymeric devices with multi-antibacterial activity were prepared by electrospinning of CT and honey, a carbohydrate-rich syrup, showing a relevant wound healing activity and remarkable antibacterial performances [171]. The synthesis of honey-based nanofibers was frequently hard due to the viscosity of the honey that permits its use in the electrospinning process only at small concentrations (<10%) [172]. Electrospinning of CT and honey at high concentration, with PVA and using eco-friendly solvents, was performed, and the antimicrobial activity of the fiber mats was evaluated against *E. coli* and *S. aureus* [128,129]. In addition, two aqueous extracts (*Cleome droserifolia* and *Allium sativum*) were loaded within honey, PVA, and CT nanofibers; in vitro antimicrobial experiments revealed a complete inhibition of *E. coli* and *S. aureus* [130]. However, only the fibers simultaneously loaded with both the extracts exhibited some antimicrobial activity against methicillin-resistant *S. aureus*. In addition, preliminary in vivo study (wound closure rates in mice and histological examination of the wounds) revealed the beneficial effects of the extract-loaded device on the wound healing process in comparison with the untreated control.

Alternatively, CT-based nanofibers can be fabricated employing PEO, which allows the use of solutions at pH 4, thus not requiring an extremely acid environment. CT/PEO solutions were successfully electrospun, by oil-in-water emulsion technique, into fibrous mats and loaded with cinnamaldehyde (0.5 and 5.0% *w/w*), a volatile EO derived from cinnamon bark [131]. The delivery/antimicrobial experiments of cinnamaldehyde EO from the CT/PEO nanofibers displayed a strong inactivation of *P. aeruginosa* (81% after 180 min). Finally, a green tea extract was proposed as an antibacterial enhancer to be enclosed in the electrospinning process involving CT/PEO chains [132]. In vitro tests revealed that this device had an antibacterial effect against *E. coli* and *S. aureus*.

#### 4.2.2. Proteins

Gelatin (GL) is a hydrophilic biopolymer, which has been widely used in the biomedical field [173]. Nanofibrous material based on GL was proposed as a carrier of active ingredients gained from natural sources. Specifically, electrospinning of GL in the presence of a small amount of the *Phaeodactylum tricornutuma* extracts provided a device with remarkable antimicrobial properties against *E. coli* and multidrug-resistant *S. aureus* [133,174]. Similarly, *Centella asiatca*, a traditional herbal medicine able to facilitate the wound-repair process, was involved in the fabrication of GL-based nanofibrous mats [175]. This device

exhibited remarkable antimicrobial properties against *S. aureus*, *E. coli,* and *P. aeruginosa* and dermal wound-healing activity in the rat model. More recently, GL fibers loaded with *Curcuma comosa* Roxb. extract displayed antibacterial activities against *S. aureus* (Inhibition zone of 7.77 mm) and *S. epidermidis* (Inhibition zone of 7.73 mm) [135], while the loading of *Chromolaena odorata* crude extract [136] allowed the fabrication of a system with excellent antimicrobial activity against *S. aureus* (100% of inhibition). However, nanofibers only based on natural polymers, such as GL, are often not useful for biomedical applications due to their low mechanical strength and high rate of degradation [103]. Baghersad et al., (2018) fabricated hybrid scaffolds, based on GL, PCL, and aloe vera, as an active extract, displaying good antibacterial activity against *E. coli* (inhibition 85.63%) and *S. aureus* (inhibition > 99%) [137].

Silk fibroin (SF) nanofibers were largely employed in the biomedical field due to their valuable properties, such as biocompatibility, electrospinnability, low inflammatory response, and therapeutic features [176]. SF/PEO nanofibers, incorporating Manuka honey [177], have been successfully fabricated by electrospinning and evaluated as a potential antimicrobial tissue engineering scaffold [138]. Manuka honey/SF nanofibers exhibited antibacterial properties against methicillin-resistant *S. aureus*, *P. aeruginosa*, *E. coli,* and *S. aureus*. Similarly, SF/PVP nanofibers loaded with baicalein, a Chinese herbal extract, were prepared by electrospinning technique and proposed as wound healing devices [139]. Experimental release displayed that almost 65% of baicalein was delivered within 24 h, reaching the lag phase after 48 h. In addition, in vitro antibacterial test against *S. aureus* displayed complete inhibition of the pathogen, while in vivo experiments in mice treated with SFP/PVP/baicalein exhibited a significant acceleration of the wound closure process. SF was employed with hyaluronic acid to fabricate nanofibers by coaxial electrospinning [140], an innovative synthetic methodology able to produce nanofibers with sheath/core morphology, utilizing two needles that, coaxially placed, allowed to feed core and shell solutions throughout two different channels. Specifically, the device was structured by a shell of SF, while hyaluronic acid and olive leaf extract, a source of bioactives, formed the core. The analysis of the release profiles showed an almost complete delivery (>90%) in nine days, reaching the lag time after two weeks. Olive leaf extractloaded nanofibers also revealed remarkable antibacterial features against *S. aureus* and *E. coli* bacteria. The same synthetic methodology was proposed to fabricate core-shell nanofibers from zein (core) and tragacanth gum (shell) [141] for the encapsulation of saffron extract [175,178]. Release values in the range 16.1–43.9% after 2 h were recorded in saliva, water, and in media, simulating gastric and intestinal fluids. Zein nanofiber mats loaded with ethanol propolis extracts were also effectively fabricated by the classic electrospinning method, and their antimicrobial activities were investigated against various microorganisms, highlighting that the nanofibers were able to mainly inhibit the growth of the Gram-positive species [142].

Finally, electrospun nanocomposite fibers were fabricated, employing soy protein isolate, PEO, and raspberry extract, a natural mixture containing high levels of anthocyanin, displaying a significant growth inhibition of *S. epidermidis* [173].

#### **5. Nanoparticles as Carriers of Antimicrobial Natural Products**

Several examples are present in the literature, describing the involvement of nanoparticles in chemical reactions to form new effective carriers to deliver antimicrobial agents from natural sources in order to eliminate pathogens without introducing chemical undesirable preservatives (Table 5) [179,180].

The different methodology can be involved in the fabrication of nanoparticles with suitable properties, including soft lithography, mechanical stretching, microfluidics, or self-assembly, using suitable starting materials, such as small molecules or polymeric structures. When loading bioactive molecules into nanoparticle carriers with the aim to control pathogens growth and/or to prevent infection, different physicochemical and

mechanical parameters should be considered, including size, shape, surface, and interior properties (Figure 3).

**Figure 3.** Size, shape, surface, and interior properties of nanoparticles important for their use in infection control. Reproduced with permission from [5], RSC, 2019.

Nanoparticles size plays a crucial role in their penetration into biofilm-bacteria, ensuring to overcome the inconvenience usually related to the multidrug-resistant strains. Ideal nanoparticle sizes to fight bacterial infections range between 5 and 500 nm. Generally, to improve the nanocarriers' efficiency, their size should not exceed the dimensions of water-filled channels into biofilms. In addition, nanoparticles sizes above 500 nm can be easily recognized by the immune system and eliminated from the bloodstream [5]. Surface properties should be also considered during nanoparticle design to ensure the inhibition of bacteria proliferation. Specifically, stealth transport through the bloodstream can be accomplished by decorating nanoparticles with suitable uncharged (polyethylene glycol) or zwitterionic hydrophilic polymers. Contact-killing capacity is strongly influenced by the nanoparticle shape that affects local adhesion forces and subsequently the extent of damage to the bacterial membrane [5]. In this regard, nano-blades or nano-knives by puncturing bacteria membranes determine the dispersion of intracellular components and cell death. Finally, the hydrophobic/hydrophilic balance into the interior of the nanoparticles should be designed to guarantee high loading capacity towards antimicrobial substances, avoiding the unnoticed loss of the cargo on their way to the infection site.

Cinnamon bark extract was embedded in PLGA/PVA nanoparticles synthesized by an emulsion evaporation method using different ratios of lactide/glycolide (65:35 and 50:50); TEM images revealed that nanoparticles were all spherical with a darker perimeter on the edge of the spheres attributed to the PVA. These products were able to gradually release active molecules and effectively inhibit *S. enterica* serovar Typhimurium and *L. monocytogenes* after 24 and 72 h at concentrations ranging from 224.42–549.23 μg/mL (Table 4) [181]. Acerola, guava, and passion fruit by-product extracts were also embedded in PLGA/PVA nanoparticles with a spherical shape and smooth surface. The antimicrobial performances against *L. monocytogenes* Scott A and *E. coli* K12 were concentration and timedependent, with MIC values ranging from 200–1000 μg/mL, higher than the corresponding isolated extracts (from 500–3000 μg/mL) [182]. Passion fruit by-products (seed and cake) extracts were encapsulated into nanospheres synthesized by emulsion solvent evaporation method, involving different PLGA (lactide to glycolide ratios equal to 50:50 and 65:35) as organic phase and PVA aqueous solution (0.5 % *w/w*). The antimicrobial properties of the systems were evaluated against *E. coli* and *L. innocua*, and PLGA 65:35 particles showed a MIC value of 188 μg/mL against *L. innocua*, while the best PLGA against *E. coli* was PLGA 50:50 particle with a MIC of 226 μg/mL. In all the cases, the extracts were derived from cake extracts [183].

Similarly, PLGA/PVA nanoparticles (size range from 145–162 nm) were encapsulated with guabiroba extract (GE), a rich source of polyphenols and carotenoids. Release experiments, performed at 37◦C and neutral pH, displayed an initial burst effect, more pronounced decreasing the lactide/glycolide ration in the PLGA chain, followed by a slower release rate of carotenoids. Nanoparticles based on PLGA 65:35 released 77% of the enclosed molecules after 6 h, while PLGA 50:50 displayed a marked burst effect (92% after 1 h). This behavior was a consequence of the highest lactide content able to delay the diffusion of the lipophilic carotenoids through the polymeric chains. Nanoparticles showed the growth inhibition of *L. innocua* within the concentration range tested (<1200 mg/mL), not displayed by the free extract [184]. The same authors improved the synthetic methodologies to achieve polymeric nanoparticles, proposing a modified emulsion-evaporation encapsulation method, allowing the synthesis of delivery devices able to preserve the extract's phenolic content for a prolonged time during release. In this study, GE showed improved antioxidant efficacy and an interesting MIC value of 2.251 μg/mL for PLGA 65:35 nanoparticles against *L. innocua* [185].

In the search for new antibacterial materials, silica mesoporous nanoparticles (SMN) have also attracted burgeoning attention due to high surface area, good biodegradability, high biocompatibility, tunable pore/particle size, and easy surface functionalization [186]. In addition, the presence of a significant porous structure allows designing high-performance devices to be applied as a carrier in the pharmaceutical and biomedical fields. Sol-gel chemistry is the synthetic strategy usually proposed to prepare SMN, mainly involving two reaction steps: (i) synthesis of silica precursor around a template by reactions of hydrolysis and condensation; (ii) removing of the template by solvent extraction and calcination [187]. The synthetic strategy deeply determined physicochemical and the morphological properties of the SMNs, such as size, porosity, and surface properties, as well as their cytotoxicity and biocompatibility, which represent important peculiarity to their employment in biological fluids. In this regard, SMNs are hydrolyzed to the nontoxic silicic acid, easily and safely excreted, and/or absorbed by the human body [188].

By using tetra-alkylammonium and pluronic surfactants with different molecular weights, it was possible to easily tune SMN pore sizes to better control loading capacity and cargo release rate. In addition, a relevant concentration of silanol groups on the SMNs surface permitted the preparation of hybrid inorganic/organic nanodevices, mainly through condensation reactions, grafting methodology, or direct incorporation of organic moieties into the silica wall [189].

MCM-41 represents a member of SMN with specific morphological and structural properties: uniform hexagonal array and channels with pores ranging from 2–50 nm [190].

This structure was proposed as a carrier of red propolis [191], showing remarkable antibacterial, antioxidant, and anticancer properties [192]. MCM-41 nanocarrier was fabricated by the co-condensation method using n-cetyl-n,n,n-trimethyl ammonium bromide as a template. In this view, different amounts of red propolis were embedded in silica mesoporous nanoparticles and tested against *S. aureus* at concentrations of 1050, 750, 500, 375, 225, and 150 μg/mL, finding an inhibition zone of approximately 19, 20, 21, 17, 16, and 17 mm in diameter, respectively.


**Table 5.** Synthetic polymers employed in the fabrication of nanoparticles for the delivery of antimicrobial natural extracts.

MCM-41 was also synthesized by the sol-gel method assisted by hydrothermal treatment and employing tetraethyl orthosilicate and n-cetyl-n,n,n-trimethyl ammonium bromide as silica source and template, respectively. Polyphenolic extracts of *Salvia officinalis* L. and *Thymus serpyllum* L. were encapsulated in these matrices and tested against *S. enterica*, *S. flexneri serotype 2b*, *E. faecalis*, *E. coli*, *P. aeruginosa*, *S. aureus*, *S. pneumoniae*, *S. pyogenes*, *B. fragilis*, *C. albicans,* and *C.n parapsilosis*, although showing milder improved antibacterial properties with respect to the parent extracts [193].

#### **6. Conclusions and Future Perspectives**

Nanotechnologies have been proposed as a valuable tool to overcome the problems frequently related to the employment of natural products with pharmacological activities. Indeed, most of the clinical trials involving natural products fail due to their poor water solubility, unsuitable molecular weight, and low lipophilicity, which produce unstable structures undergoing high metabolic rate and fast clearance. In addition, if the bioactive molecules are accumulated in non-targeted tissues and organs, significant side effects can be detected.

Currently, different studies proposed polymeric nanocarriers as favorable devices to increase bioavailability and activities of natural products, representing a plausible approach for the treatment of a variety of diseases. In particular, the perspectives of a multidisciplinary approach, expecting the combination between nanotechnology-based delivery systems and antimicrobial features from natural extracts, are promising in the infection control in order to produce multi-component devices able to avoid the multidrugresistance, usually associated with the infections. Nanodevices, such as nanofibers or

nanoparticles, were effectively proposed as carriers of extracts from plants, herbals, and agro-food by-products with remarkable antimicrobial properties.

Electrospun nanofibers, usually fabricated by single-fluid electrospinning, represent a valuable approach to produce high performing nanodevices. This goal was reached by employing both natural and synthetic polymers. However, the best results were recorded, fabricating blended macromolecular structures able to conjugate the feature of synthetic constituents (cheapness, mechanical resistance, and electrospinnability) with the highest biocompatibility of the natural polymers, reducing the adverse effects, potentially affecting the different biological compartments. In addition, the loading of several bioactive molecules within nanofibers required the optimization of different key factors, such as burst release reduction and/or low delivery efficiency. Recently, coaxial electrospinning was proposed as an innovative tool to fabricate core-shell nanostructures able to optimize the distribution of bioactive molecules according to the required function. In this way, the burst effect was highly reduced, and the antimicrobial effect against both Gram-positive and Gram-negative pathogens was recorded.

Alternatively, polymeric nanoparticles represent a valuable device with tailored physicochemical features to become a therapeutic revolution against human pathogens. They have shown to be biocompatible, non-toxic, safe, biodegradable, and easily eliminated, providing many advantages to release natural molecules with antimicrobial activity.

However, the transfer of these polymeric systems from the laboratory to practical healthcare applications remains a significant obstacle. In general, rigorous protocols of validation of in vitro and in vivo procedures are required to simplify translation from the bench to the clinical trials. Nowadays, some formulations are in clinical or preclinical stages in order to verify long-term toxicity, as well as degradation and metabolism. In the future, the clinical potential of these complex nanostructures should be deeply investigated, employing effective devices in the pharmaceutical and biomedical fields as delivery systems and/or tissue engineering scaffolds, useful to ensure infection control, to speed up skin regeneration, enhancing, in this way, patient's quality of life.

Similarly, the challenges for large scale fabrication necessitate novelty from engineers and chemists, and regulatory policies have to facilitate access to trials and patients. Thus, employing these nanocarriers in clinical practice remains challenging and will represent a major focus in the next decades.

**Author Contributions:** Conceptualization, U.G.S.; software, G.C.; validation, F.A. and D.R.; investigation, U.G.S. and F.A.; resources, D.R.; data curation, U.G.S.; writing-original draft preparation, U.G.S., G.C., F.A. and D.R.; writing-review and editing, U.G.S., F.A., A.F. and D.R.; All authors have read and agreed to the published version of the manuscript.

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

**Acknowledgments:** Authors acknowledge Ministero dell'Istruzione, dell'Università e della Ricerca for the "Department of Excellence 2018–2022" grant (Italina Law n. 232/2016).

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

#### **References**


### *Review* **Bromelain and Nisin: The Natural Antimicrobials with High Potential in Biomedicine**

**Urška Janˇciˇc <sup>1</sup> and Selestina Gorgieva 1,2,\*f**


**Abstract:** Infectious diseases along with various cancer types are among the most significant public health problems and the leading cause of death worldwide. The situation has become even more complex with the rapid development of multidrug-resistant microorganisms. New drugs are urgently needed to curb the increasing spread of diseases in humans and livestock. Promising candidates are natural antimicrobial peptides produced by bacteria, and therapeutic enzymes, extracted from medicinal plants. This review highlights the structure and properties of plant origin bromelain and antimicrobial peptide nisin, along with their mechanism of action, the immobilization strategies, and recent applications in the field of biomedicine. Future perspectives towards the commercialization of new biomedical products, including these important bioactive compounds, have been highlighted.

**Keywords:** bromelain; nisin; bioactivity; antimicrobial agent; biomedicine; carrier

#### **1. Introduction**

One of the tremendous burdens on human health worldwide is infectious diseases [1], where antibiotics act as first-line therapy in treating infections caused by bacteria. Still, their widespread use, over-utilization and improper consumption in humans and animals cause an increase in the number of resistant bacterial strains. Furthermore, one pathogen organism is gaining resistance to more than one antibiotic, leading to the development of multidrug resistance strains for various species, such as *Staphylococcus aureus* (*S. aureus*), *Pseudomonas aeruginosa* (*P. aeruginosa*), *Salmonella* spp., *Enterococcus faecium* (*E. faecium*), *Campylobacter*, *Neisseria gonorrhoeae* (*N. gonorrhoeae*), *Streptococcus pneumonia* (*S. pneumonia*) [2], etc. Consequently, the cost of hospitalization and healthcare, together with morbidity and death are increasing [3]. According to World Health Organization and Organisation for Economic Co-operation and Development at least 700,000 patients die every year from infections caused by resistant microorganisms [2] and approximately 2.4 million people in Europe, North America, and Australia are expected to die due to diseases caused by drug-resistant pathogens over the next 30 years, which means \$3.5 billion in economic cost per year [4]. Furthermore, multidrug resistance of cancer cells against conventional chemotherapeutic agents [5] is another problem that needs to be solved. Therefore, it is necessary to search for innovative alternative therapies and new drug candidates [6]. Various studies exhibit promising results when natural antimicrobial peptides and proteins are used as therapeutics [7], especially since their conjunction with conventional chemotherapeutic agents promotes effectiveness, decreases antibiotics use and possibly reduces instances of chemotherapy resistance [8].

This review gives a comprehensive overview on two compounds obtained from two different natural sources, i.e., nisin as a bacterial origin representative and bromelain as a plant origin representative. With nearly 50 years of safe usage in the food industry, and very little evidence of cross-resistance compared with that of conventional antibiotics [7,8],

**Citation:** Janˇciˇc, U.; Gorgieva, S. Bromelain and Nisin: The Natural Antimicrobials with High Potential in Biomedicine. *Pharmaceutics* **2022**, *14*, 76. https://doi.org/10.3390/ pharmaceutics14010076

Academic Editor: Umile Gianfranco Spizzirri

Received: 22 November 2021 Accepted: 23 December 2021 Published: 29 December 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 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/).

non-toxicity and low immunogenicity [9], researchers have begun to explore the nisin, an antimicrobial peptide with a broad-spectrum of antibacterial activity [6] as a potential alternative agent for infectious diseases [7]. On the other hand, the demand for medicinal plants with therapeutic agents has been rising [10] as natural plant products are increasingly recognized as non-toxic, side-effect free, readily available and affordable [1]. Among them, pineapple has been identified to possess valuable qualities for medical purposes, especially its proteolytic enzyme bromelain due to its antimicrobial, anti-inflammatory, antithrombotic, fibrinolytic and anti-cancer functions [11]. The present review comprehensively discusses the structure, isolation and suggested bioactivity mechanisms, as well as immobilization strategies and application of nisin and bromelain in the last 10 years. Published reports were collected using the Web of Science and Scopus databases, with search terms "bromelain", "nisin", "bioactive", "antimicrobial", "anticancer", "anti-inflammatory", "toxicity", "immobilization", "adsorption", "encapsulation", "entrapment" and "carrier". Our aim is to emphasize the importance and relevance of these bioactive compounds, where the researchers and relevant stakeholders may gain the latest fundamental knowledge to explore the new possibility of bromelain- or nisin-based products in biomedicine and pharmacy. Moreover, giving comprehensive information for two different origin bioactive compounds can allow direct comparison of their ultimate properties and action, giving the ease of selecting the suitable candidate for a particular biomedical application. Relating to this, we also point to very limited clinical trials (and even fewer approved products) involving bromelain and nisin, as contradictory to the potential they hold in this segment. As a hypothetically written, future perspective, the possibility to combine both bioactive components in an attempt to merge and even boost their multiple bioactivities, utilising diverse immobilization routes, have been brought forward.

#### **2. Bromelain**

#### *2.1. Structural and Biological Properties*

Bromelain is a protein purified from a crude aqueous extract of pineapples (*Bromeliaceae* family) [1]. Pineapple is a common name of *Ananas comosus*, also known as *Ananassa sativa*, *A. sativus*, *Bromelia ananas* or *B. comosa*, grown in several (sub)tropical countries such as Costa Rica, Philippines, Brazil, Thailand, China, Indonesia, India, Malaysia, Hawaii and Kenya [1,12]. In the pineapple plant, bromelain acts as a defensive protein; it protects the pineapple throughout the development, maturation and ripening process [13,14].

Bromelain was identified for the first time in 1891 by Vicente Marcano, a Venezuelan chemist, while its isolation and analysis started in 1894. However, its commercial production began in 1957 with Heinecke's discovery that the pineapple fruit contains less bromelain than the pineapple stem [15], making a waste by-product stem bromelain more commercialized [13].

Bromelain belongs to the class of proteases also known as proteinases or peptidases, a group of enzymes that catalyzes proteolytic reactions where the breakdown of proteins into smaller polypeptides or single amino acids occurs [13,16,17]. More specifically, it is classified as cysteine proteinase (EC 3.4.22, CP, also known as thiol proteinase) due to the cysteine thiol in its active site [1,13]. Crude bromelain (crude extract of the pineapple) contains various cysteine endopeptidases and other components, including phosphatases, glucosidase, peroxidases, cellulases, glycoproteins, carbohydrates, ribonucleases, protease inhibitors and organically bound calcium [1,12,15]. Among them, the specific activity of proteases is the highest, e.g., the specific activity of protease, peroxidase, acid phosphatase, alkaline phosphatase and amylase studied in the crude bromelain extracted from pineapple crown leaf was 45 U/mg, 2.19 U/mg, 1.12 U/mg, 0.98 U/mg, 0.65 U/mg, respectively [18]. At least four evolutionarily and structurally related cysteine endopeptidases can be synthesized from crude bromelain: stem bromelain (EC 3.4.22.32), fruit bromelain (EC 3.4.22.33), ananain (EC 3.4.22.31) and comosain (Table 1) [1,13,15]. Stem bromelain is the major protease present in the stem of the pineapple plant, and fruit bromelain is the major protease in the pineapple fruit [1,19]. Ananain and comosain were detected only in minor quantities

in stem pineapple [1]. All the endopeptidases of the pineapple plant have generally been referred to as "the bromelains" and the name "bromelain" was originally used to describe any protease of the *Bromeliaceae* family [15].

All four cysteine endopeptidases possess distinguished physicochemical properties, as summarized in Table 1. Fruit bromelain is an acidic protein, unlike stem bromelain, which is alkaline (isoelectric point 4.6 and ≥9.5, respectively). Generally, the molecular weight of stem and fruit bromelain is from 23.8 to 37.0 kDa and 23.0 to 32.5 kDa, respectively. This heterogeneity in molecular weight may be due to heterogeneity of the amino acid sequence and the glycosylation pattern [20], both being a consequence of the formation of various forms of bromelain isolated from crude bromelain [21]. Furthermore, different purification methods and several purification steps could also contribute to molecular weight heterogeneity. The optimum temperature range for stem bromelain is between 40 and 60 ◦C (37–70 ◦C for fruit bromelain) and its optimum pH range is 4–8 (3–8 for fruit bromelain) [1,13,15,22–24]. However, its activity is no longer susceptible to the effect of the pH once it is combined with a substrate [1]. Bromelain preferentially cleaves glycyl, alanyl and leucyl peptide bonds [25]. Its activity can be determined using different substrates, including casein [16,26–28], gelatin [1], azocasein [19,29], azoalbumin, hemoglobin, sodium caseinate [23,30], and synthetic peptide substrates (Nα-CBZ-ι-Lysine p- nitrophenyl ester, Z-Arg-Arg-pNa, Bz-Phe-Val-Arg-pNA, H-Val-Ala-pNA, Suc-Ala-Ala-Val-pNA, Suc-Ala-Pro-Leu-Phe-pNA, Suc-Phe-Leu-Phe-pNA, Z-Phe-Arg-pNA and Z-Phe-pNA) [31,32]. The value of Michaelis–Menten constant (Km) vary significantly when different substrates (azoalbumin, azocasein, sodium caseinate, casein and hemoglobin) are used for fruit bromelain activity determination, being the lowest (0.026 mM) for azoalbumin and the highest (0.165 mM) for hemoglobin [33]. The most suitable substrate for the fruit bromelain activity is azocasein, followed by azoalbumin, casein, sodium caseinate and hemoglobin according to the enzyme catalytic power parameter (Vmax/Km ratio), being 0.104, 0.096, 0.022, 0.020 and 0.014, respectively [33]. Bromelain inactivation rate follows first-order kinetics at 55 ◦C and 60 ◦C, but not above 70 ◦C, while its thermal deactivation is entirely irreversible and follows a two-stage mechanism, including the formulation of an intermediate between native and denatured states [15]. Bromelain retains more than 50% of its original proteolytic activity after 30 min incubation at 60 ◦C, from 9% to 22% after 15 min incubation at 70 ◦C, and becomes utterly inactive when heated for 10 min at 100 ◦C [34]. Aqueous proteolytic activity of bromelain decreases rapidly at 21 ◦C, while its concentrated forms (>50 mg/mL) are stable for one week at room temperature and can be repeatedly frozen and thawed [35].

**Table 1.** Physiochemical properties of cysteine endopeptidases derived from pineapple plants [1,13,15,22–24].


The activation energy of bromelain is 41.7 kcal/mol [23], and same can be activated by many chemical agents, including calcium chloride, cysteine, sodium cyanide, bisulfate salt, hydrogen sulfide, sodium sulfide and benzoate [13,36,37]. Stem bromelain is reversibly inhibited during reaction with organic mercury, ions of mercury and tetrathionate. Its irreversible inhibition occurs by reacting with *N*-ethylmaleimide, *N*-(4-dimethyl3,5-dinitrophenyl) maleimide, monoiodoacetic acid and 1,3-dibromine acetone due to alkylation of the thiol group, an essential group for the activity of the enzyme [15].

Until now, several different (fruits or stem) bromelain amino acids sequences have been deposited in the National Center for Biotechnology Information (NCBI) Genbank database with around 90–100% similarity. Alanine, glycine and serine are the most abundant amino acids in stem and fruit bromelains, while histidine is present in the lowest amount [13]. Bromelain amino acid sequence is highly similar to papain, actinidin, proteinase Ω and chymopapain [24]. A single polypeptide chain constitutes the primary structure of bromelain with amino acids folded into two structure domains: α-helix domain (domain cathepsin propeptide inhibitor—I29) and antiparallel β-sheet domain (domain peptidase C1) (Figure 1). Mainly, the I29 domains are located between amino acids number 1 and 100 of the N-terminal sites. The structure domains are stabilized by disulfide bridges and numerous hydrogen bonds. Stem bromelain differs from the fruit bromelain in the number of polar amino acids (arginine and lysine), and acidic amino acids (aspartate and glutamate). The stem bromelain contains more polar amino acids, and the fruit bromelain has more acidic amino acids, leading to a difference in isoelectric point (4.6 and ≥9.5 for fruit and stem bromelain, respectively). The active site is located on the surface molecules between domains and the proposed catalytic residues for the modeled BAA21848 structure is composed of three amino acids Cys-121, His-254 and Asn-275; for CAA08861 structure Cys-147, His-281 and Asn-302 are proposed, which fall into approximately the exact locations as in papain catalytic residues (Cys-25, His-159 and Asn-175) [13,38].

**Figure 1.** Model domain organisation of (**a**) fruit bromelain (sequences with the accession number of BAA21848 in the NCBI Genbank database and 352 amino acids) and (**b**) stem bromelain (sequences with the accession number of CAA08861 in the NCBI Genbank database and 357 amino acids). αhelix domain (domain I29 at the N-terminal region) is colored in green, β-sheet domain (domain peptidase C1 at the C-terminal region) is colored in orange. The catalytic amino acids of both models are represented as sticks (Reproduced with permission from [13], Elsevier, Amsterdam, The Netherlands, 2018).

#### *2.2. Isolation, Extraction and Purification*

Bromelain can be isolated from all parts of the pineapple plant (stem, core, peel, crown and leaves), which affect the concentration and composition. The stem and pineapple fruit allow the production of high amounts of bromelain, while the pineapple core, peel and leaves contain smaller quantities, yet, together with pineapple stem and crown, they represent up to 50% (*w/w*) of the total pineapple waste [16], making extraction of bromelain from pineapple waste economically and environmental attractive [28]. Consequently, the most commercially available bromelain is usually obtained from pineapple stem, which is also therapeutically more effective and shows higher proteolytic activity than fruit bromelain [17].

Numerous strategies have been developed for the extraction and purification of bromelain. The bromelain production process consists of several sequential steps, as depicted in Figure 2. Fresh pineapple stem parts or any other parts of the pineapple are washed, cut into small pieces, crushed in an industrial blender to disrupt the plant cells and separate the enzyme from the cells, filtered to remove the fibrous material and centrifugated to remove insoluble materials [1,38–40]. The obtained supernatant is called crude extract and is further purified as impurities and by-products (e.g., proteins, pigments, polysaccharides) can react with bromelain and inhibit its activity [17]. Purification can be done using chromatographic processes (among them ion-exchange chromatography with prior precipitation by adding ammonium sulfate is the most relevant), a two-phase aqueous system (e.g., PEG/K2SO4, PEG/MgSO4, PEG/poly(acrylic acid), PEG/(NH4)2SO4) or a reverse micellar system [1,17,41], the selection primarily depends on the application. Purification can also be performed by membrane-based processes (microfiltration, ultrafiltration) [40] or precipitation, followed by centrifugation and solubilization in phosphate buffer [38]. The residual specific activity of crude pineapple extract purified by fractionation using ammonium sulfate at 20–50% saturation level is 70 U/mg with the total activity of 167.3 U, total protein content of 2.39 mg and the purity level of 5.3 fold compared to the crude enzyme extract [16]. When acetone (50–80% saturation) was used as fractionating agent, the residual specific activity of bromelain fraction was 19.7 U/mg [16]. The crude bromelain of pineapple fruit purified by high-speed counter-current chromatography coupled with the reverse micelle solvent system yielded 3.01 g of bromelain from 5.00 g crude extract in 200 min [42]. The choice of a purification method determines the purity of the enzyme and the enzyme production cost [40]. Commercially available bromelain is produced by a lengthy and costly purification method that yields bromelain in varying degrees of purity [32]. The purification steps correspond to 70–90% of the total production costs [38], implying the need to develop innovative, cost-efficient methods for pure bromelain production in fewer steps [39].

Isolation of bromelain from pineapple fruit and its various parts is not the only way to obtain bromelain; researchers are also trying to clone the bromelain gene in multiple hosts, such as *E. coli* BL21-AI [32,43,44], *E. coli* BL21-CodonPlus(DE3) [45], *E. coli* BL21 DE3pLysS [14], *Pichia pastori* [46] and Chinese cabbage (*Brassica rapa*) [47], leading to recombinant bromelain—an intracellular enzyme abundant in the cytoplasm of the host cell, meaning that the host cell wall needs to be disrupted using homogenization, chemical lysis, sonication with lysozyme or freeze-thawing to release the bromelain [44]. Amid et al. [32] reported about higher specific activity of recombinant bromelain (1.231 U/mg) in comparison to commercial bromelain (0.846 U/mg) when the release of p-nitrophenol from a synthetic substrate Nα-CBZ-ι-Lysine p-nitrophenyl ester was monitored. The recombinant bromelain obtained in a single step immobilized metal affinity chromatography was purified 41-fold and showed optimum activity at pH 4.6 and 45 ◦C [32]. In contrast, George and co-workers [14] reported a higher protease activity of native bromelain obtained from Sigma (a purified form of crude stem bromelain) in comparison to recombinant bromelain when casein was used as a substrate. Crude bromelain showed even higher proteolytic activity than native bromelain due to its composition of a mixture of protease complexes which can cleave substrate even more effectively. However, the effectiveness of the extraction of the (recombinant) bromelain and its residual activity are related to the choice of buffer, presence of chelating agents (ethylenediaminetetraacetic acid (EDTA), cyclohexane-1,2-diaminoetetraacetic acid (CDTA), hydroxyethyl ethylenediamine triacetic acid (HEDTA)), reducing agents and protease inhibitors [44].

**Figure 2.** Scheme of a typical bromelain production.

#### *2.3. Bioactivity*

Bromelain has been a valuable compound in traditional medicine in Southeast Asia, Kenya, India, and China for a long time [28,48] due to its numerous therapeutic effects (Figure 3), including antimicrobial [16,49,50], anti-inflammatory [30,51], anticoagulant [52], anticancer [53,54], antiplaque [55,56], and antiulcer properties [50]. Furthermore, it is also beneficial for wound healing [57–60], dermatological disorders [19], post-surgery recovery, enhanced antibiotic absorption [1], treatment of osteoarthritis [61], sinusitis and

diarrhea [17]. Recently, bromelain is suggested as an antiviral agent against COVID-19 due to the inhibition of different versions of SARS-CoV-2 [62]. Some of its therapeutic mechanisms are discussed below.

**Figure 3.** Overview of the bromelain bioactivity.

The mechanism behind the antimicrobial activity of bromelain is not well known, yet, is believed that bromelain may hinder bacterial growth by hydrolyzing some peptide bonds in the bacterial cell wall [14]. When bromelain digests the surface proteins, the cell wall is damaged, allowing the cell to leak, swell, and open [1]. Bromelain also inhibits the growth of some bacteria by preventing bacterial adhesion to specific glycoprotein receptors on the surface [1,48]. Furthermore, bromelain inhibits enterotoxin production of *Escherichia coli* (*E. coli*) and prevents diarrhea caused by *E. coli* [17]. Bromelain shows antimicrobial activity against both Gram-positive and Gram-negative bacteria, including *E. coli*, *Aggregatibacter actinomycetemcomitans* (*A. actinomycetemcomitans*), *Porphyromonas gingivalis* (*P. gingvalis*), *Streptococcus mutans* (*S. mutans*) [56], *Bacillus subtilis* (*B. subtilus*), (*S. aureus*), *Pseudomonas aeruginosa* (*P. aeruginosa*), *Proteus* spp., *Acinetobacter* spp., ... [1,63]. Additionally, synergistic use of bromelain and antibiotics increases the antibacterial effect due to increased absorption of antibiotics induced by bromelain, leading to better drug distribution in the microbes [1,17]. Bromelain has also been reported to act as an inhibitor of fungal pathogens [39,64].

Inflammation is the body's attempt to protect itself [28]. It is a complex biological mechanism primarily regulated by the disruption of tissue homeostasis [17]. Most often, non-steroidal anti-inflammatory drugs are prescribed to combat the classic signs of inflammation (heat, pain, redness and swelling), leading to severe damage to the gastrointestinal tract and numerous side effects. In such cases, the bromelain can be used as an alternative [28] due to its anti-inflammatory activity mediated by (Figure 4):


**Figure 4.** Anti-inflammatory and anticoagulant mechanisms of action of bromelain (Adapted with permission from [65], MDPI, Basel, Switzerland, 2021).

Because of these actions, bromelain is potentially effective in several conditions and diseases associated with inflammation, including rheumatoid arthritis, osteoarthritis, cardiovascular diseases, skin wounds and burns, perioperative sports injuries and chronic rhinosinusitis [65]. Furthermore, inflammation is also associated with cancer; suppressing chronic inflammation may inhibit cancer progression due to reduced PGE-2 and prostaglandin-endoperoxide synthase 2 (COX-2) after bromelain administration [17]. The anti-inflammatory effect of bromelain is also the most traditional and established one [17].

Bromelain affects blood clotting by increasing the fibrinolytic capacity of serum and inhibiting the synthesis of the blood-clotting protein fibrin (Figure 4). It also decreases prekallikrein—a proenzyme that must be converted to kallikrein to help in coagulation. Consequently, it inhibits the generation of bradykinin, leading to pain and edema reduction, and increased circulation on the side of the injury [17,28,39].

The molecular mechanisms of bromelain's anticancer activity are also not fully understood [11]. However, some research has suggested that the bromelain anticancer mechanism is mainly attributed to its protease components and proteolysis [11,35]. One of the described anti-tumor mechanisms of bromelain includes induced differentiation of leukemic cells, leading to apoptosis of tumor cells [1]. Bromelain inhibits the growth of cancer cells by increasing the expression of two activators of apoptosis in mouse skin—p53 and Bax [66]. It also decreases the activity of cell survival regulators such as Akt and Erk, promoting apoptotic cell death in tumors. Expression of promoters of cancer progression—nuclear factor kappa B (NF-κB) and Cox-2 are also inhibited by bromelain in mouse papillomas and models of skin tumorigenesis [1,11].

Bromelain is well tolerated and considered a safe nutraceutical with no serious adverse effects [30,65]. It has already received FDA approval for clinical use as an orally administered anti-inflammatory and anticoagulant therapeutic [52]. Its oral administration

is well tolerated even in high doses (up to 3 g/day) for prolonged therapy periods, even up to several years [11]. It has a very low level of toxicity [48]. The lethal dose (LD50) for intraperitoneal administration is 37 mg/kg and 85 mg/kg for mice and rabbits, respectively, and 30 mg/kg and 20 mg/kg for intravenous administration [65], with no immediate toxic reactions [25]. Daily oral administration of 500 mg/kg of bromelain did not provoke any alteration in food intake, growth, histology of the heart, kidney and spleen, or hematological parameters in rats [25]. After daily bromelain administration up to 750 mg/kg no toxicity was observed in dogs after 6 months [17]. No relevant side effects have been observed in humans at doses of up to 2000 mg/kg, even with prolonged oral administration [65]. However, clinical trials have reported some side effects, mainly gastrointestinal (i.e., diarrhea, nausea and flatulence), headache, tiredness, dry mouth, allergic reactions, and bleeding risk, especially in individuals treated with other anticoagulant drugs [17,61,65].

#### *2.4. Immobilization Strategies*

One of the issues related to enzymes (such as bromelain) utilization is a decline of their activity with time or after processing. Indeed, enzymes, isolated from their natural environments, are susceptible to process conditions, such as pH, temperature, strong acids and bases, and non-aqueous solvents, which may affect their activity [67], health benefits and pharmaceutical applicability [68]. A promising strategy to secure their efficiency is immobilization [69], which requires selecting supporting material (inorganic components, synthetic polymers or natural polymers) with suitable surface chemistry for controlled enzymatic attachment. The next step is optimizing the immobilization process towards desired immobilization yield, activity retention of even amplification, stability and reusability [69] (Figure 5). Successful immobilization requires thorough knowledge and control of the interactions between the carrier and the enzyme [70]. The choice of immobilization method and carrier depends on the nature of the immobilized compound and the goal of immobilization (resistance against high temperature, pH, controlling the release, preventing negative interactions . . . ) [71].

**Figure 5.** Steps for immobilization of bioactive enzymes.

Immobilization methods and carriers utilized for immobilization of bromelain in the last 10 years are summarized in Table 2 and Figure 6. Bromelain has been combined mostly with nanoparticles, hydrogels, fibers and matrices with the aim to improve the properties of the final formulation [29]. Baker and co-workers [72] encapsulated bromelain in silica nanosphere aggregates, using sodium metasilicate as a silica precursor and ethyleneamines (diethylenetriamine (DETA), triethylenetetramine (TETA), tetraethylenepentamine (TEPA), and pentaethylenehexamine (PEHA)) of different chain lengths as initiators. They found out that increased loading mass of bromelain resulted in the increased activity of bromelain, being 61.7% when 10 mg of bromelain was encapsulated in silica and only 12.1% when 2 mg of bromelain was used. The encapsulation also increases the thermostability with maximum activity at 40 ◦C for free bromelain and at 50 ◦C for encapsulated bromelain. At 70 ◦C free bromelain lost its activity while encapsulated bromelain retained approximately 30% of its activity [72]. Chitosan-methyl cellulose hydrogel [73], freeze-dried chitosan nanoparticles [29], chitosan microspheres [74], poly(lactide-co-glycolic) acid nanoparticles [75] and katira gum nanoparticles [76] have also been studied for encapsulation of bromelain, showing various immobilization yield and bromelain activity. Esti et al. [77] covalently immobilized stem bromelain on chitosan beads by direct mechanism, involving the bromelain carboxyl groups of Asp or Glu residues and the amino groups of the chitosan. Ataied et al. [78] studied bacterial nanocellulose as a support material for physical adsorption of bromelain and reported about 9-times increased antimicrobial activity of adsorbed bromelain. Holyavka et al. [70] also used the adsorption method for immobilization of cysteine proteases onto chitosan and observed significant loss of the bromelain catalytic activity due to: (a) nonspecific binding, (b) structural changes of bromelain upon interaction with the carrier, and (c) diffusional and steric limitations, leading to impeded access of the active bromelain center [70]. All the studies clearly show the influence of the carrier and immobilization method on bromelain's immobilization yield, residual activity, and thermal stability. By choosing a suitable carrier and immobilization method, it is possible to significantly reduce the influence of the carrier on the structural and functional properties of the bromelain [70], enhance its stability and activity upon exposure to a wide range of pH and high temperatures and improve its antimicrobial and anti-inflammatory activity. However, there is not yet a standard, highly efficient immobilization approach for bromelain delivery [29].

**Table 2.** Review of bromelain immobilization methods.



**Table 2.** *Cont.*


**Table 2.** *Cont.*

**Figure 6.** Schematic illustration and SEM micrographs of immobilization methods of bromelain: (**a**) entrapment into hydrogels (Reproduced with permission from [86], Elsevier, Amsterdam, The Netherlands, 2018); (**b**) adsorption onto chitosan matrix (Reproduced with permission from [70], Elsevier, Amsterdam, The Netherlands, 2021); (**c**) covalent immobilization (Reproduced with permission from [81], Elsevier, Amsterdam, The Netherlands, 2018); (**d**) entrapment into nanoparticles (Reproduced with permission from [29], Elsevier, Amsterdam, The Netherlands, 2021); (**e**) SEM micrographs of encapsulated silica nanoparticles formed without bromelain (left) and with bromelain (right) (Reproduced with permission from [72], John Wiley and Sons, Hoboken, NJ, USA, 2014).

#### *2.5. Applications*

Bromelain finds widespread applications in several areas, including medicine, health, food, and cosmetics [15]. In the food industry, it is used for meat tenderization [90–92] (together with papain representing 95% of the enzymes used to tenderize meat in the

USA [32]), baking process [93], protein hydrolysate production [94], as a food supplement [95–97] and as an anti-browning agent in fruit juices [98]. Furthermore, bromelain also shows antimicrobial activity against *Alicyclobacillus acidoterrestris* (*A. acidoterrestris*), Gram-positive bacteria often related to the deterioration of acidic products (citrus juices, iced tea, isotonic drinks and tomato extract) [99]. Still, its main application continues to be in the pharmaceutical industry [24].

Several experimental data and clinical studies showed better burns and wound healing under the influence of bromelain due to its proteolytic, anti-inflammatory, antibacterial, and anti-edematogenic effects [58,59,73,86,100–102]. Recently, Chen et al. demonstrated reduced inflammation and improved wound healing rate in a rat model when treated with bromelain-immobilized electrospun poly(ε-caprolactone) fibres [100]. These fibres also effectively prevented wound infections due to their antibacterial activity against Grampositive bacteria *S. aureus*, dominant in the initial stage of chronic wound formation, and Gram-negative bacteria *E. coli* [100]. Aichele et al. confirmed the effect of bromelain on myofibroblast reduction, resulting in attenuated fibrotic development [58]. Topical application of bromelain is effective in the eschar removal (debridement) of uncomplicated gunshot wounds when used as an adjunct to a simple wound incision and simplifies the conventional wound excision treatment [103]. Bromelain treatment has a characteristic of attacking mainly necrotic tissue, while healthy tissue seems unaffected [58]. One example is bromelain-based enzymatic debridement product NexoBrid (produced by MediWound Ltd., Yavne, Israel), which reduced infection, blood loss, length of hospital stays, and the need for skin grafting in treating deep partial and full-thickness burns due to early non-surgical eschar removal without harming surrounding viable tissue (Figure 7) [59,101,104,105]. The NexoBrid, a topically-applied concentrate of proteolytic enzymes enriched in bromelain, was clinically approved in 2012 by the European Medicines Agency (EMA) to remove dead tissue in severe skin burns, and until now is the only clinical-approved application of bromelain [106]. Moreover, EscharEx (MediWound Ltd., Yavne, Israel) is another bromelainbased enzymatic debridement currently in development for chronic wounds [107,108]. Several researchers have also incorporated bromelain into various hydrogels [73,86,87,102] to create a dressing that ensures a moist environment around the wound and provides a barrier against infection [87].

Bromelain has clinical potential for the treatment of skin problems such as acne owing to its antimicrobial activity against microbial flora that is often associated with acne infection, including *P. acne*, *S. aureus*, *C. diphtheria* and *E. coli*, among which *S. aureus* was the most susceptible organism to the action of bromelain extracts, followed by *P. acne* [16,19].

In addition, bromelain can be used to inhibit the growth of bacteria that causes dental caries due to the intense antimicrobial activity against *P. gingvalis* (diameter of clear zone of 21 mm) [56]. The minimum inhibitory concentration (MIC) of bromelain against microorganisms associated with periodontal diseases was also determined by Praveen and co-workers, being 2 mg/mL, 4.15 mg/mL, 16.6 mg/mL and 31.25 mg/mL for *S. mutans*, *P. gingivalis*, *A. actinomycetemcomitans* and *Enterococcus fecalis* (*E. fecalis*), respectively [50]. The minimum bactericidal concentration (MBC) of crude bromelain of pineapple fruit to multidrug-resistance Gram-negative *P. aeruginosa* is 0.75 g/mL [109]. *P. aeruginosa* is a leading cause of nosocomial infections, responsible for 10% of hospital-acquired infections [109]. Crude bromelain, extracted from pineapple fruit, exhibited a 12 mm zone of inhibition against *Streptococcus pneumoniae* (*S. pneumoniae*), *P. aeruginosa* and *S. aureus* at a concentration of 1.0 g/mL [63]. Crude bromelain extracted from pineapple crown leaf (aqueous extract of pineapple crown leaf) showed 70–95% inhibition of microbial growth with MIC range of 1.65–4.95 mg/mL against laboratory strain *Saccharomyces cerevisiae* (*S. cerevisiae*) and *E. coli* XL1 blue, type strain *S. aureus*, drug-resistant strain *E. coli* DH5α pet16b Amp<sup>r</sup> and two pathogenic strain *B. subtilis* and *Candida albicans* (C. *albicans*) [18]. It is also hypothesized that bromelain inhibits the development and progression of periodontitis through the elimination of important cell surface molecules (CD25) in leucocytes (proteolytic activity of bromelain), decreased growth of periodontal microorganisms (anti-adhesion property), reduced migration of neutrophils to periodontal sites (the hyperactivity of the neutrophils leads to damage of the periodontium), downregulating of inflammatory mediators (COX-2, tumor necrosis factor (TNF)), decreased osteoclastogenesis process with reduction in alveolar bone loss (Figure 8a,b) [110,111]. A clinical study conducted by Odresi et al. confirmed the anti-edematous action of bromelain in third molar surgery. The group treated with bromelain showed a reduced inflammatory response compared to the control group [112].

**Figure 7.** Bromelain-based treatment (BBT): (**a**) venous insufficiency ulcer; 1—pre-existing for 5 months, 2—after first BBT 4-h application, 3—after fourth BBT 4-h application (16 h total exposure to bromelain-based debridement), 4—one week post-split-thickness skin grafting, 5—seven weeks post-split-thickness skin grafting (Adapted with permission from [108], John Wiley and Sons, Hoboken, NJ, USA, 2018); (**b**) large venous leg ulcers; 1—venous leg ulcer pre-existing 10 weeks, 2—after 7 BBT, and 3—two months after split-thickness skin grafting (Reproduced with permission from [107], John Wiley and Sons, Hoboken, NJ, USA, 2021); (**c**) hand burn; 1—before BBT, 2—after BBT, 3—outcome 38 days post-burn (Reproduced with permission from [104], Baoshideng Publishing Group Inc., Pleasanton, CA, USA, 2017).

The anticarcinogenic effect of bromelain has been investigated through in vitro studies involving various cancer cell lines [66]. It can inhibit the growth and proliferation of mouse breast carcinoma 4T1, human breast adenocarcinoma GI-101A and MCF7, human prostate carcinoma PC3 and human gastric carcinoma AGS in a dose-dependent manner [43,113–115]. Bromelain concentration >75 μg/mL remarkably decreased cell viability in MCF7, PC3 and AGS human cell lines as a single therapy [113]. Moreover, it is also effective as an anticancer agent against cell lines of melanoma (A375), epidermoid carcinoma (A431) [116], gastric carcinoma (KATO-III and MKN45) [117], colorectal cancer (human colon adenocarcinoma (Caco-2)) [118], ovarian cancer (A2780), colon cancer (HT29) [119], lung cancer [120], pancreatic [121] and liver cancer (hepatocellular carcinoma HepG2) [10]. The absorption and efficiency of chemotherapy drugs (5-fluorouracil, vincristine, cisplatin, idarubicin, doxorubicin), antibiotics (amoxicillin and tetracycline) or blood pressure medication (captopril and lisinopril) [17,122–124] can be potentiated when combined with oral, subcutaneous or intramuscular administration of bromelain [17]. Higashi et al. [121] investigated whether bromelain could be used to degrade the barrier of dense extracellular matrix (ECM), a characteristic inhibitor of penetration of anticancer drugs in the treatment of pancreatic cancer. Due to the short half-life of the bromelain in the blood, they prepared reversibly PEGylated bromelain using "self-assembly PEGylation retaining activity (SPRA)" technology, thus retaining

high bromelain activity and causing ECM degradation and increase of anticancer drugs in tumor tissue of pancreatic cancer (Figure 8c) [121]. Encapsulated bromelain also enables slow delivery, thus being favorable for cancer treatment [66].

(**a**)

**Figure 8.** (**a**) Comparison of the control group (normal gingiva), periodontitis group and group treated with bromelain 15 mg/kg (arrow shows the first molar and the letter T shows the tongue). Group treated with bromelain indicates improvement of gingival papilla staining, reduction in edema, absence of bleeding and moderate bone loss (Reproduced with permission from [111], John Wiley and Sons, Hoboken, NJ, USA, 2020); (**b**) morphometric analyses of alveolar bone height; \* *p* < 0.05 indicates the Periodontitis groups versus the Control group and # *p* < 0.05 indicates the Periodontitis groups versus the Bromelain group (Reproduced with permission from [111], John Wiley and Sons, Hoboken, NJ, USA, 2020); (**c**) the scheme of the SPRA-bromelain suggested a mechanism of ECM-degradation in pancreatic cancer (Reproduced with permission from [121], ACS Publications, Washington, DC, USA, 2020).

Bromelain effectively reduces the risk of clots-associated problems, including stroke or heart attack [15,17,25] due to the breaking down of the blood-clotting protein fibrin [125]. Bromelain has been shown to be effective in treating rheumatoid arthritis [86], exerciseinduced muscle injuries [125] and edema caused by post-surgical trauma [19]. It was also used in treating patients with osteoarthritis, where it worked similarly to diclofenac treatments [126]. In combination with *Boswellia serrata* (*B. serrata*)*,* bromelain improved the quality of life of patients suffering from different forms of osteoarthritis [96].

188

#### **3. Nisin**

#### *3.1. Structural and Biological Properties*

Antimicrobial peptides (AMPs) are cationic, hydrophobic or amphipathic natural antibiotics, consisting of amino acid residues of varying lengths (up to 100) in a linear or cyclic arrangement [127], derived from bacteria, insects, plants, birds, amphibians, fish, and mammals [128–130]. AMPs have attracted much attention because of their potent antibacterial activity against a broad spectrum of microorganisms, multiple modes of action, a low bacterial resistance rate, ability to destroy target cells rapidly and low cytotoxicity [127,131–134], therefore showing potential to overcome the growing problems of antibiotic resistance [135,136]. An example of AMPs is also an odorless, colorless, tasteless substance—nisin [131]. It is a cationic, amphiphilic, antimicrobial polypeptide [137,138], ribosomally synthesized and posttranslationally modified to its biologically active form [139]. It is a member of bacteriocins, classified as a Type A (I) lantibiotic [140], identified in 1928 in fermented milk cultures [6]. It contains the hydrophobic residues at the N—terminus and hydrophilic residues at the C—terminus (Figure 9a) [138], five thioether rings and four amino acids, usually not found in nature: lanthionine (Lan), β-methyl lanthionine (MeLan), and two dehydrated amino acids—dehydroalanine (Dha) and dehydrobutyrine (Dhb) (Figure 9b,c) [141,142]. These amino acids result from posttranslational modification of serine, threonine, and cysteine [143]. Moreover, the thioether rings give nisin unique properties, including nanomolar antimicrobial activity, resistance against proteolytic degradation and high heat stability [135]. The first two thioethers rings can bind lipid II, the flexible hinge region together with the last two thioethers rings can flip into the membrane and create a pore [3]. Unmodified prenisin contains 57 amino acids: the first 23 from the leader peptide and the last 34 residues from the core peptide [144]. The leader peptide renders the propeptide inactive and must be cleaved for a nisin to gain antimicrobial activity [142]. Therefore, active nisin consists of only 34 amino acids [3].

#### (**a**)

**Figure 9.** *Cont.*

**Figure 9.** (**a**) Primary structure of nisin Z with highlighted residues involved in crucial aspects of the antimicrobial activity (Adapted with permission from [145], Elsevier, Amsterdam, The Netherlands, 2018); (**b**) chemical formula of dehydroalanine (Dha), dehydrobutyrine (Dhb), lanthionine (Lan), and β-methyl lanthionine (MeLan) (Adapted with permission from [145], Elsevier, Amsterdam, The Netherlands, 2018); (**c**) chemical structure of nisin A (Reproduced with permission from [146], RSC, Cambridge, UK, 2012).

Nisin is mainly produced by Gram-positive bacteria that include *Lactococcus* and *Streptococcus* species [7] (e.g., *Lactococcus lactis* (*L. lactis*) [137], *Streptococcus hyointestinalis* (*S. hyointestinalis*) [147], ... ). Various production strains also lead to different naturally occurring variants of nisin (nisin A, nisin Z, nisin F, ... ). The molecular weight of nisin depends on the production strain; usually, it is between 3.0 and 3.5 kDa [147]. This polypeptide has an amphipathic property [140], is cationic at neutral pH and has an isoelectric point above 8.5 [148]. Nisin has no absorbance at 280 nm due to the absence of aromatic amino acids [149].

Nisin has been approved by the Joint Food and Agriculture Organization World Health Organization (FAO/WHO, 1969), the US Food and Drug Administration (FDA, 1988) [7], the European Food Safety Authority (acceptable uptake of 0.13 mg/kg/day/person [150]) and the Food Standards Australia New Zealand [151]. It was generally regarded as safe (GRAS) [151,152]. So far, it is the only bacteriocin in the market allowed to be used as a food additive [153].

#### *3.2. Isolation*

Since the first discovery of nisin (nisin A) in fermented milk cultures, several natural and bioengineered variants of nisin have been identified [7,147], which differ in their structure and properties (solubility, chemical reactivity, and spectra) [154]. Up to now, there are eleven reported natural occurring nisin analogues: nisin A, nisin Z, nisin F, nisin Q, nisin H, nisin O A1-A3, nisin O A4, nisin U, nisin U2, nisin P, nisin J (Table 3), isolated from various bacterial genera such as *Lactococcus*, *Streptococcus*, *Staphylococcus*, and *Blautia*, located in dairy products, human gastrointestinal tract, bovine mammary secretions, human skin microflora, porcine intestine, an alimentary tract of ruminants, fish gut and river water in Japan [7,155]. Nisin analogues from the same genera are more like each other than analogues from different genera. Nisin A and nisin Z are both isolated from *L. lactis*, found in dairy products, and differ only in one amino acid at position 27; histidine (His) in nisin A is substituted with asparagine (Asp) in nisin Z (Table 3, highlighted in yellow). This substitution mainly affects the solubility of the polypeptide. It causes nisin Z to be more soluble at neutral pH than nisin A due to a more polar side chain of the Asp in comparison to His at neutral pH; it has minimal effect on antimicrobial activity, resistance to pH changes, sensitivity to proteolytic enzymes and thermal stability [149,155]. Nisin F differs from nisin A due to Asp and valine (Val) at positions 27 and 30. Nisin Q differs in comparison to nisin A in three amino acids at four positions: valine (Val, in position 15 and 30), leucine (Leu, in position 21), and asparagine (Asp, in position 27) [155]. Nisin O (A1-A3 and A4), nisin U and U2, and nisin P are shorter than previously described nisin analogues; they contain 33, 32, and 31 amino acids, respectively. With 35 amino acids, nisin J is the longest natural nisin analogue identified to date [147].

**Table 3.** Primary structures of nisin natural analogues. The changes in amino acids compared to nisin A are highlighted in yellow (not valid for Nisin J).


Aside from natural nisin analogues, the bioengineered forms of nisin have been developed in the last twenty years by genetic modification tools [156], with an attempt to alter the solubility, stability and efficiency of nisin. A large number of generated bioengineered

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

forms of nisin revealed that modifying amino acids at the hinge-region (three amino acids asparagine-methionine-lysine at position 20–21–22 in the center of the peptide, Figure 9a) and at position 29, respectively, displayed an essential role in enhancing activity against Gram-negative bacteria, and both Gram-positive and Gram-negative pathogens [7,156]. Nisin A K22T, A N20P, A M21V, A K22S, A S29A, A S29D, A S29E, A S29G, Z N20K and Z M21K are some genetically modified nisin derivatives with changes in those positions and more significant activity against foodborne and clinical pathogens [6,7,156]. The names indicate the substitution position and the replaced amino acid; for example, nisin A K22T means that the amino acid sequence is the same as in Nisin A, the only difference is at position 22, where lysine (K) is substituted with threonine (T). Nisin derivative Z N20K and Z M21K showed enhanced activity against Gram-negative bacteria, including *Shigella*, *Pseudomonas* and *Salmonella* species, and displayed more significant thermal stability and solubility at neutral or alkaline pH [7]. Nisin A K22T exhibit enhanced activity against human and bovine pathogen *Streptococcus agalactiae* (*S. agalactiae*). Nisin A M21V showed enhanced antimicrobial activity against medically significant pathogens, including heterogenous Vancomycin intermediate *S. aureus* (hVISA), methicillin-resistant *S. aureus* (MRSA), *Clostridium difficile* (*C. difficile*), *S. agalactiae* and *Listeria monocytogenes* (*L. monocytogenes*). The S29G and S29A nisin variants showed enhanced activity against Gram-positive and Gram-negative pathogens, differentiating them from all nisin derivatives generated to date [156].

#### *3.3. Bioactivity*

Nisin is known for its broad-spectrum of antibacterial activity against a wide range of Gram-positive bacteria [7,140], even better than conventional antibiotics [157], due to its stability at a higher temperature, tolerance to low pH, and dual-mode of antimicrobial activity [6]. The latter includes binding of nisin molecule to an essential precursor for bacterial wall biosynthesis (the lipid II) through electrostatic interaction between the positively charged nisin and the negatively charged membrane phospholipids. This results in the formation of the complex within the bacterial cell membrane, which creates 2 nm wide pores, thus preventing the growth of the peptidoglycan network and increased membrane permeability, leading to leakage of essential cellular components, and eventually to cell death (Figure 10) [71,139,149,158,159].

Nisin is active against a wide variety of Gram-positive *Lactococcus*, *Enterococcus*, *Streptococcus*, *Staphylococcus*, *Listeria* and *Micrococcus* bacterial strains, as well as the vegetative forms and outgrowing spores of *Bacillus* and *Clostridium* species [138,142,158]. The Gramnegative bacteria (e.g., *E. coli*) are usually resistant to nisin due to their outer lipopolysaccharide membranes, which act as a barrier/shield and impede its access to the cytoplasmic membrane [160,161]. Additionally, nisin shows no inhibitory activity against yeast cells, filamentous fungi and viruses [149]. However, many studies [6,7,142,156,158,160,162–164] demonstrate that bioengineered variants of nisin, high purity nisin, nisin-antibiotics, nisinchelating agents (e.g., EDTA), nisin-inorganic nanoparticles (silver, gold, magnesium oxide, ... ) or other outer membrane destabilizing component/processes (e.g., heat treatment, freezing) could also be effective against Gram-negative bacteria.

Required nisin concentration for efficient bacteria inhibition depends on several parameters, such as pH, heat treatment intensity, storage time and storage conditions. Aqueous solubility and structural stability of nisin are also pH dependent. The antimicrobial activity, solubility, and thermal stability of nisin are higher at acidic pH and deactivate under alkaline conditions due to irreversible structural changes of the nisin molecule. Nisin has higher antimicrobial activity in a liquid medium than a solid medium. Nisin is highly stable at low temperatures (e.g., during freezing), but undergoes a loss of activity during long-time heating. Proteolytic enzymes such as pancreatin, α-chymotrypsin and ficin can inactivate nisin due to their ability to break down the peptide chain of nisin. Other enzymes such as trypsin, pepsin and carboxypeptidase have no significant effect on its antimicrobial effect. The antimicrobial activity of nisin is also inhibited by the titanium dioxide and sodium metabisulphite due to the oxidation of disulfide bridges in the nisin molecule [149,165].

**Figure 10.** Schematic representation of the bactericidal mechanism of nisin: (**a**) nisin reaches the bacterial membrane; (**b**) adsorption of nisin to docking molecule (lipid II) via electrostatic interactions; (**c**) stable transmembrane orientation of nisin (cationic region of nisin interact with the negatively charged phospholipid heads, while the hydrophobic region of nisin interacts with the membrane core); (**d**) assembly of nisin-lipid II pore complex (consisting of 4 lipids II and 8 nisin molecules) (Reproduced with permission from [71,149], Elsevier, Amsterdam, The Netherlands, 2019 and Taylor & Francis, Abingdon, UK, 2016).

#### *3.4. Immobilization Strategies*

Various immobilization methods have been developed to protect nisin from environmental stresses, degradation by biological fluids or biocomponents (i.e., proteolytic enzymes) or deactivation under alkaline conditions [4,165], including covalent immobilization, encapsulation, entrapment, adsorption and co-culture fermentation, summarized in Table 4 and Figure 11. Most of the reported strategies for nisin immobilization required special pre-treatment of used support material/carrier, chemical modifications, crosslinking agents (carbodiimide/N-hydroxysuccinimide (EDC/NHS), hexamethylene diisocyanate, glutaraldehyde, ... ) or a variety of other spacer molecules to obtain a composite with optimal, target-directed antimicrobial action against pathogenic bacteria [4,9,161]. In recent years, great emphasis has been placed on developing innovative nano-engineered approaches and nanostructured materials with enhanced antimicrobial activity in comparison to free nisin, including lipid-based nanoencapsulated nanoparticles (nanoliposomes, nanoemulsions, nanomicelles, solid lipid nanoparticles and nanostructured lipid carriers, Figure 12a), polymeric-based nanoencapsulated nanoparticles (nanocapsule and nanosphere, Figure 12b) and nanofibers [71,166]. Natural and synthetic materials studied as carrier or support material for immobilization of nisin includes liposomes [164,167], silica xerogels [168], polystyrene sheets [138], polyethylene oxide brush layer [169], soy lecithin liposomes [170], bacterial cellulose nanocrystals [151], chitosan nanoparticles [171], alginate beads [172] or a mixture of pectin-chitosan microcapsules [165], alginate-starch microcapsules [173], alginate-pectin microbeads [174] or chitosan-alginate microparticles [175],... having antimicrobial activity against various Gram-positive and Gram-negative bacteria (Table 4).

**Figure 11.** *Cont.*

**Figure 11.** Schematic illustration of immobilization methods of nisin: (**a**) covalent immobilization onto multi-walled carbon nanotubes with PEG1000 as a linker and hexamethylene diisocyanate as a crosslinking agent (Adopted with permission from [176], RSC, 2011); (**b**) co-culture fermentation of nisin-producing (*Lactococcus lactis* N8) and bacterial cellulose-producing (*Enterobacter* sp. FY-07) bacteria (Adopted with permission from [153], Elsevier, Amsterdam, The Netherlands, 2021); (**c**) covalent immobilization onto plasma-treated, EDC/NHS ester functionalized polystyrene sheets (Adopted with permission from [138], RSC, 2017); (**d**) covalent immobilization onto plasma-treated polystyrene sheets (Adopted with permission from [138], RSC, 2017); (**e**) nisin loaded chitosanpoly-γ-glutamic acid nanoparticles (encapsulation) (Reproduced with permission from [179], RSC, Cambridge, UK, 2016); (**f**) adsorption of nisin on blank and HGFI-coated polystyrene surface together with antimicrobial activity of both surfaces (Adopted with permission from [9], Elsevier, Amsterdam, The Netherlands, 2021).

**Figure 12.** Scheme of (**a**) lipid-based nanoparticles and (**b**) biopolymeric nanoparticles for encapsulation of nisin (Adopted with permission from [71], Elsevier, Amsterdam, The Netherlands, 2019).


**Table 4.** Immobilization methods of nisin and antimicrobial activity against Gram-positive and Gram-negative bacteria.


**Table 4.** *Cont.*

However, different hydrophilic/hydrophobic surface properties of these carriers affect the orientation of the nisin (Figure 13). It is proposed that the hydrophobic region of the nisin binds to the hydrophobic surface, leading to the reduced number of hydrophobic regions available to interact with the bacterial cell membrane. Similarly, the hydrophilic region of nisin binds to the hydrophilic surface, allowing the hydrophobic region to interact with the bacterial cell membrane [138]. Furthermore, nisin reacts with EDC/NHS functionalized surface through its amine group at the N-terminus, which could cause inefficient adsorption to the carrier due to steric barriers of the hydrophobic region [138].

#### *3.5. Applications*

Nisin's properties, such as inhibitory efficiency against a wide range of microorganisms, low probability of developing microbial resistance, no effect on the normal microbiota of the intestine, non-toxicity, colourless and tasteless, enable its use in both the biomedicine and food industry [137,189], especially in the second segment, where use as food bio preservative is already much exhausted [190]. Nisin is used to preserve pasteurized milk, aged cheeses, canned soups, juice, meat and vegetables [71,149]. It shows a better choice for prolonging the shelf life of meat (Tan sheep meat) in comparison to preservative potassium sorbate due to reduced nutrient loss [191]. Furthermore, it can be combined with other pasteurization preservation treatments to increase inhibition effectiveness against heat-resistant spore-former and extend the food shelf life [149]. As a food additive, it is assigned as E234 [149] and has been approved for use in over 60 countries around the

world as a natural agent to prevent food spoilage due to its low toxicity or non-toxicity, high efficiency [153], thermal stability, and colourless and tasteless properties [157]. Saini et al. [150] studied covalent immobilization of nisin on the surface of TEMPO-oxidized CNF and thus developed antimicrobial films, which could be used as active food packaging. Nisin was also studied to develop impedimetric label-free biosensors for bacterial contamination detection of *Salmonella* spp. [192].

**Figure 13.** Proposed orientation of nisin on (**a**) hydrophobic surface, (**b**) hydrophilic surface and (**c**) with EDC/NHS functionalized surface (Adopted with permission from [138], RSC, 2017).

In light of biomedical potential, the nisin already demonstrates promising results as an alternative to traditional antimicrobial therapeutics due to its activity against specific (antibiotic-resistant bacterial) pathogens and disease conditions, particularly concerning mastitis in lactating women and dairy cows (inhibition of *S. aureus* and *S. epidermidis* [193–198]), respiratory infections (inhibition of *S. aureus* [199]) and skin infections, e.g., atopic dermatitis [200] and MRSA skin infections (inhibition of *S. aureus*) [147,201–204]. It can be used either as a single agent or in combination with other agents [7,157,189,201,205]. Furthermore, it showed potential in oral diseases, such as caries and periodontal diseases, due to inhibition of oral bacteria, including *Streptococcus sanguinis* (*S. sanguinis*), *Streptococcus sobrinus* (*S. sobrinus*), *Streptococcus gordonii* (*S. gordonii*), *P. gingivalis*, *Prevotella intermedia* (*P. intermedia*), *A. actinomycetemcomitans* and *Treponema denticola* (*T. denticola*) [140,206,207]. Shin and coworkers [140] studied nisin's antimicrobial efficiency against the formation of saliva-derived multi-species oral biofilms. They reported on reduced biofilm biomass in a dose-dependent manner (Figure 14); no apoptotic changes of human oral cells were observed at nisin concentration <200 μg/mL [140]. Nisin also has the potential to control periodontal disease in dogs [208].

Additionally, nisin has been studied as a possible anticancer agent due to the multidrug resistance of cancer cells and drastic side effects of traditional chemotherapeutics [209,210]. Hosseini and co-workers reported a significant decrease in the growth rate of SW480 colorectal cancer cell line after being treated with nisin [211]. Similar conclusions are reported by Tavakoli et al. [212]. Nisin also showed a significant efficiency as an adjuvant to conventional chemotherapeutic agents. Preet et al. studied synergism between doxorubicin, a chemotherapeutic drug traditionally used to treat breast cancer, lymphoma, bladder cancer, acute lymphocytic leukemia [8], and nisin against skin carcinogenesis [209]. They reported on augmented anticancer activities when both these agents were used in conjunction with each other [209]. Rana and colleagues studied the possible use of a 5 fluorouracil-nisin combination as a topically applied chemotherapeutic drug against skin cancer [210]. They observed faster clearance of tumors and a reduced dose of 5-fluorouracil when a 5-fluorouracil-nisin combination was used [210]. Joo et al. reported on increased cell apoptosis and decreased cell proliferation at head and neck squamous cell carcinoma by nisin treatment [159]. Furthermore, nisin A has been demonstrated to have a potential for treating nonhealing wounds, as it increases the mobility of skin cells, dampens the effect

of lipopolysaccharide and proinflammatory cytokines, and decreases bacterial load in the wound [157].

**Figure 14.** Confocal microscopy images of the influence of nisin concentration on oral biofilm formation under the controlled microfluidic model system. A green signal indicates viable live cells (Syto 9) and a red signal indicates damaged/dead cells (propidium iodide). No biofilm was observed at a nisin concentration of 4 μg/mL (Reproduced with permission from [140], Meta UCL, 2015).

#### **4. Combination of Bioactive Compounds**

Simultaneous use of (bio)active agents is common practice to collect multiple activities and even augment their efficiency to a higher level than their simple sum. The use of enzymatic mixtures, comprising enzymes with wide diversity in the reactions they are catalyzing, is one frequent case of simultaneous use of multiple bioactive compounds. Moreover, the ˝crude enzymatic cocktails˝ (as crude bromelain itself) are more frequently present in nature than a single, specific type. Aside from simple mixtures, more than one enzyme's co-immobilization was found very efficient in terms of product yield and thermal stability increment, as present in the triple enzyme system [213]. Another example is antibiotics, where the combined therapy utilizing more than one antibiotic at the time is practiced in particular cases in order to broaden the antibacterial spectrum, to treat the polymicrobial infections, to obtain synergistic effect bringing higher efficiency at lower doses and finally, to tackle the emergence of bacterial resistance [214].

The bacteriocin nisin offers a range of advantageous features that include protease and heat stability; its efficacy can be further boosted via combination with other antimicrobials or membrane-active substances. Nisin demonstrates synergistic activity with the antibiotics colistin and clarithromycin against *P. aeruginosa* [215] with ramoplanin and other-β-lactam antibiotics against many strains of MRSA and VRE [216] with penicillin, streptomycin, chloramphenicol and rifampicin against *Pseudomonas fluorescens* [217]. Combinations of derivatives nisin V + penicillin or nisin I4V + chloramphenicol had an enhanced inhibitory effect against *S. aureus* SA113 and *S. pseudintermedius* DSM21284, respectively, compared to the equivalent nisin A + antibiotic combinations or when each antimicrobial was administered alone [218].

Reported studies demonstrate that such mixtures boost the antimicrobial action, but the same does not introduce new bioactive functions. One-pot (co-immobilisation, simultaneous immobilisation), or successive immobilization of bioactive compounds, together with diverse immobilisation strategies, all together present modalities to be used in obtaining a multi-active system including different types of bioactive compounds. Such an example is a two-step polydopamine-based surface modification strategy, used to co-immobilize an antimicrobial peptide Palm and an enzyme targeting an important component of biofilm matrix (DNase I). This immobilization approach imparted polydimethylsiloxane surfaces with both anti-adhesive and antimicrobial properties against the adhesion of relevant bacteria as single and dual-species, with excellent stability and biocompatible and antibiofilm properties, holding, therefore, great potential in the development of catheters able to prevent the catheter-associated infections [219].

To date, the co-immobilization of bromelain and nisin as proteolytic enzymes and protease-resistant antimicrobial peptide, respectively, has not been trialled. Aside from obstacles anticipated to such an experimental design, the potential success may offer a merge of an extensive portfolio of bioactive functions brought by both components. Both components are complementary in many terms, including the type of bacteria they are acting against, i.e., Gram-positive for nisin and Gram-negative for bromelain.

#### **5. Conclusions and Prospects**

Bromelain and nisin are undoubtedly among more perspective, natural bioactive components with outstanding potential in biomedicine due to diverse therapeutic benefits, demonstrated by several research groups in the recent decade. In vitro studies of bromelain and nisin show their potential in human medicine and healthcare, in the treatment of skin infections, caries, periodontal diseases, and many other conditions. Importantly, the bromelain shows promise within several in vitro studies involving cancer cell lines, yet, the clinical trials in this segment are in a premature stage, with only two examples at the moment (one for treatment of solid tumors in advanced stage of lung, breast, colon, ovary, cervix, uterus, prostatic, and liver and second for treatment of Pseudomyxoma Peritonei, Peritoneal Cancer, Mucinous Adenocarcinoma and Mucinous Tumor) [220]. The plant extract bromelain interacts with several biological processes that lead to its multi-action bioactivity, including antimicrobial, anti-inflammatory, anticarcinogenic and antithrombotic activity. Unlike bromelain, which has already gained FDA approval in topical product NexoBrid, the nisin is only approved as a food additive despite its effectiveness against drug-resistant organisms also in biomedical research. Nonetheless, much effort has been devoted to widening the nisin efficiency from Gram-positive bacteria towards Gram-negative bacteria, where biotechnological approaches or combination with other components (antibiotics, inorganic nanoparticles, chelating agents, ... ) have been applied, which paves its way towards use in more demanding clinical set-ups. Further, the production of different variants (from native and gene-modified bacterial species) with a high degree of purity, securing the safeness of final products are evidencing recognized the potential of this bioactive compound.

To the best of our knowledge, the synergistic action of both bioactive components is yet to be explored as an attractive topic. Before going ahead with a cost-demanding clinical translation of bromelain- or nisin-containing materials developed in a lab, much remains to be learned, particularly about different variants and combinations with conventional antibiotics and cancer drugs, their complex mechanism of action on the human body and pathogens, consequences of long-term clinical trials and choosing suitable optimized immobilization method with high immobilization yield and secured activity/efficiency. As said, most data for bromelain and nisin demonstrated an in vitro efficiency, and the extrapolation of in vitro to in vivo outcome is not that straightforward, yet, same present a solid background, important in future translation in a clinic. With all this, it will be possible to offer novel, safe and efficient natural therapeutic solutions to our society without significant risks to developing resistance in pathogenic organisms and cells.

**Author Contributions:** Conceptualization, S.G. and U.J.; methodology, S.G.; writing—original draft preparation, S.G. and U.J.; writing—review and editing, S.G. and U.J.; funding acquisition, S.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Slovenian Research Agency, young researcher program (P2- 0118/0795), the Textile Chemistry Programme (P2-0118) and project J2-2487. The APC was funded by Slovenian Research Agency, project J2-2487.

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

**Informed Consent Statement:** Not applicable.

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

#### **Abbreviations**


#### **References**


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