GQD—Graphene quantum dots, GQD-DOX—Graphene quantum dots-Doxorubicin, QDs-PEG—Quantum dots-polyethylene glycol, PTT—Photothermal therapy, PDT—Photodynamic therapy.

#### **4. Types of Targeting Moieties**

Various targeting moieties are used for targeted delivery in cancer therapy, target moieties are commonly incorporated on surfaces of transporters by physical absorption or chemical reaction. Peptides, proteins, nucleic acids and small molecules (carbohydrates or vitamins) are examples of targeting moieties.

#### *4.1. Aptamer-Based Targeting*

Nucleic acid-containing ligands are known as aptamers that can bind to highly precise sites for drug molecule delivery. These aptamers can be identified by the ligand known as SELEX ligand. An example of aptamer-based targeting is the delivery of cisplatin to prostate cancer cells by using an aptamer conjugated on the surfaces of nanocarriers [130].

One of the most well-known aptamers for cancer treatment is AS1411 (single strand aptamer). It was shown to effectively limit the growth of a variety of human tumour cell lines, including prostate cancer, breast cancer and lung cancer. For effective cellular transport of AS1411, nanocarriers such as Apt-AuNS (aptamer conjugated gold nanoparticles) were used to increase the bioactivity of AS1411 [131].

#### *4.2. Small Molecule-Based Targeting*

Small compounds are inexpensive to create, used for targeting and have a limitless number of structures and properties. Folate is the most commonly investigated small molecules for drug delivery. Folate is an aqueous soluble vitamin B6 that is essential for men's cell growth and division, particularly during embryonic development [2,132]. Riboflavin is a required nutrient for the cell metabolic process and a riboflavin carrier protein (RCP) has been found to be substantially increased in active tumour cells. An endogenous RCP ligand, flavin mononucleotide (FMN), was employed as a small molecule that targets the ligand in active tumour or endothelial cells [2].

Lactose-doxorubicin (Lac-DOX) based nanocarriers were developed and used for targeting cancer cells. The developed formulation exhibits improved anticancer activity and weak adverse effects by passive and active tumour targeting. Lac-DOX nanoparticles have extremely low toxicity in vivo, as seen by decreased uptake in normal body weights, key organs and normal blood biochemistry indices [133].

#### *4.3. Peptide Based Targeting*

They are ideal for targeting molecules due to their small low production cost, size and minimal immunogenicity. These peptides are derived from the binding areas of the protein of interest. A common example is ANGIO PEP-2, a peptide sequence and its complementary ligand is receptor-related protein (LRP), a type of low-density lipoprotein that is expressed in multiforme glioblastoma and blood–brain barrier (BBB), which is not an operable type of pituitary tumour. When coupled, the peptide sequence ANGIO PEP-2 will penetrate the BBB in sufficient quantities to target glioma in the brain [134,135].

Albumin fused chimeric polypeptide conjugated with self-assembled micelles were created by Parisa Y et al. and micelles are loaded with doxorubicin. When compared to conventional DOX, this formulation provides complete tumour inhibition with greater pharmacokinetics and dosage tolerance [136].

#### *4.4. Antibody-Based Targeting*

In recent decades, ligand manufacturing has been focused on the antibody's classes. Within a single molecule that contains two binding epitopes and the target of interest has an unusually high level of affinity and selectivity. Rituximab is an antibody approved by FDA for non-lymphoma Hodgkin's treatment [137]. Bevacizumab, is an anti-vascular endothelial growth factor (VEGF) monoclonal antibody used to treat metastatic rectal, breast and colon cancer, stops angiogenesis by sequestering soluble VEGF and inhibiting antibodies targeting different epitopes of the same protein from binding to VEGFR-2 [138].

Triple single chain antibodies were coupled to magnetic iron oxide nanoparticles to target pancreatic cancer for imaging and therapy were studied by Zou et al. Both in vitro and in vivo studies shows that triple single chain antibodies have clinical potential in both cancer therapy and imaging [139].

#### **5. Stimulus for Drug Release**

The two types of stimuli are endogenous and exogenous. Exogenous stimulation is defined as an extra-corporal signal that causes medications to be released from smartnanocarriers, such as a temperature change, an electric field, ultrasonic waves, or magnetic field. An endogenous stimulus is a signal created from within the body that causes the release of anti-cancer medications. Endogenous stimuli include pH changes, enzyme transformations, temperature changes and redox reactions [50].

#### *5.1. Endogenous Stimulus*

Intrinsic stimulus, also known as endogenous stimulation, is a type of stimulus that originates from the body. The triggering signal is generated by the body's internal enzyme activity, pH level and redox activity in the case of endogenous stimulation. The following are detailed information on the many types of endogenous stimuli [140].

#### 5.1.1. pH-Responsive Stimulus DDS

The Warburg effect states that tumour cells produce the majority of their energy in the cytosol via increased glycolysis followed by lactic acid fermentation [141]. This increased acid production causes cancer cells to have a lower PH. As pH levels differ from organ to organ and even tissue to tissue, the pH-responsive medicine delivery mechanism is unique. Tumours have an acidic pH compared to a slightly basic intracellular (pH 2). The inflammatory and extracellular tissues of tumours have a pH of about 6.5, while normal tissues have a pH of 7.4. The cytoplasm or organelles have lower pH, for-example lysosomes (pH 4–5), endosomes (pH 5–6) and the Golgi complex (pH 6.4). In conclusion, the pH differences between normal and cancer cells offers a solid foundation for creating a stimulisensitive drug delivery system [142,143]. The delivery system construction techniques fall into two categories based on the changes in the pH gradient outside and within the cells: One example is the polymer's variations in conformation or dissolution behaviour under different pH environments [144–147]. The other possibility is that the delivery systems will dissolve due to the breakage of groups that are acid-stimuli in the nanocarriers, and as a result, targeted delivery at certain locations is possible [148–151].

Liu et al. have developed a mesoporous silica nanoparticle conjugated with chitosan. Chitosan is a smart drug delivery system, and this system releases the drug at narrow pH. Ibuprofen release was higher at pH 6.8 than pH 7.4 and pH-stimulus drug release of Ibuprofen for breast cancer has been accomplished [152].

#### 5.1.2. Redox-Sensitive Stimulus DDS

Reductive compounds found in the human body include glutathione (GSH), vitamin E and vitamin C [153–156]. Based on the properties of these compounds, several redoxsensitive nanocarriers are produced and used in the controlled release of genes, proteins and anti-cancer medicines, targeted delivery and also for ultrasound imaging [157–160]. Zhao et al. (2015) used surface modification technology to create a redox responsive nanocapsule that could hold two functional molecules, one of which is encoded via disulphide bonds in the shell of the capsule and the other of which is enclosed in the capsule's core. The redox reaction trigger could cause a cascade release of the loaded medication [161].

Sun et al. have developed an amphiphilic conjugate coupled heparosan with deoxycholic acid via disulfide bond self-assembled into stable micelles to deliver doxorubicin into cancer tissues. This formulation exhibited good loading capacity and glutathione-triggered drug release behaviour [162].

#### 5.1.3. Enzyme Responsive Stimulus DDS

Phosphor esters, polymers and inorganic materials, among other nanomaterials, have previously been employed to develop enzyme responsive drug delivery systems [163–167]. In pathological conditions such as tumours or inflammations, the peptide structure or ester bonds of the stimuli-responsive carriers may be broken down by various enzymes, allowing the loaded medications or proteins to be released at specific sites to exhibit therapeutic effects [168,169]. The protein and peptides are degraded by an enzyme known as proteases, are an excellent choice for drug release from liposomes [3].

Lee et al. have prepared doxorubicin (Dox) loaded GLFG (Gly Leu-Phe-Gly) liposomes. These liposomes are degraded by cathepsin B enzyme, which is overexpressed in several cancer cells types and exhibits an effective anticancer effect on Hep G2 cells in vitro and inhibit cancer cell proliferation in a zebrafish model [170].

#### *5.2. Exogenous Stimulus*

Ultrasound, temperature, magnetic field and light are the most common exogenous physical stimulus. Drug releases can happen quickly when these signals interact with nanocarriers that respond to external stimuli [171–175].

#### 5.2.1. Temperature Responsive Stimulus DDS

Liposomes, nanoparticles and polymer micelles are common temperature-responsive carriers. When the ambient temperature exceeds the polymer's critical solution temperature (CST), the hydrophilic–hydrophobic equilibrium breaks and the polymer chain dehydrates, causing the drug-delivering carrier's structure to change and the contents packed in the system to be released [176].

Allam et al. have developed camptothecin loaded superparamagnetic nanoparticles (spions) coated with 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and L-αdipalmitoylphosphatidyl glycerol (DPPG). This thermo-responsive nanocomposite has shown improved solubility and stability due to magnetic hyperthermia and also highly cytotoxicity towards cancer cells than the free camptothecin [177].

#### 5.2.2. Light-Responsive Delivery Systems

The precise drug release is achieved in light-responsive drug delivery systems when exposed to exogenous light (such as visible, infrared light or ultraviolet) [178–182].

For example, the doxorubicin-loaded gold nanocarrier has increased drug release under 808 nm illumination [183].

For chemophotothermal treatment in breast cancer, A. Zhang et al. have produced polyethylene glycol (PEG) linked liposomes (PEG-liposomes) coated doxorubicin-loaded mesoporous carbon nanocomponents. The study was carried in the presence and absence of NIR irradiation. The presence of NIR irradiation triggers the drug release from the formulation compared to the absence of NIR irradiation. The created system was able to transport the drug to breast cancer cells and cell toxicity viability tests revealed that the drug-loaded system had no cytotoxicity to normal cells [184].

#### 5.2.3. Magnetic Field Responsive DDS

An extracorporeal magnetic field is employed in magnetically induced systems to collect drug-loaded nanocarriers in tumour locations following nanocarrier injection. Magnetic stimulus candidates include core-shell shaped nanoparticles coated with magneto liposome (maghemite nanocrystals enclosed in liposomes), polymer or silica [185,186].

For siRNA delivery to breast cancer cells, superparamagnetic iron oxide nanoparticles coated with calcium phosphate and PEG-PAsp were developed by Dalmina et al. These systems efficiently carried siRNA and delivered the siRNA in breast cancer cells under an external magnetic field. This research vocation signifies VEGF (vascular endothelium growth factor) silencing being effective in breast cancer cells without causing cytotoxicity [187].

#### 5.2.4. Ultra-Sound Responsive DDS

Due to its non-ionizing irradiation, non-invasiveness and deep penetration into the body ultrasound are being studied extensively for medication release from nanocarriers [188]. Ultrasound can be used to create both mechanical and thermal effects in nanocarriers, allowing the loaded medicine to be released in 2007, Dromi et al. utilized highintensity focused ultrasound waves to study temperature-sensitive liposomes for the drug release [189–191].

For hepatocellular carcinoma (HCC), Yin et al. have developed siRNA and paclitaxel (PTX) ultrasound sensitive nanobubbles (NBs). Encapsulating both anti-cancer drug paclitaxel (PTX) and siRNA into liposomes. When the low-frequency ultrasound was exposed, this system exhibits cell apoptosis decreases the tumour volume. As a result, new ways for co-administration of siRNA and PTX using ultrasound responsive polymer for hepatocellular carcinoma treatment have been created [192,193].

#### **6. Nanomedicines: Development, Cost-Effectiveness and Commercialization**

Though the nanotechnology and nanocarriers-based drug delivery approaches have gained much attention and popularity in today's world and hold great potential from

the application perspective, still there exists a lag between the development of excellent technology and its efficient commercialization. Presently, the commercialization of the majority of nanotherapeutics is either start-ups or small/medium-level enterprises driven. For emerging nanotherapeutics, there is a low interest of investment by big pharma firms. Hence, for the small nanomedical firms, it is an enormously difficult task to find a suitable major pharma firm for partnering; which will be willing to license and bring into the market their established nanotherapeutic technology [194]. Moreover, the firms dealing with nanomedicines are subject to suggestively higher per-unit costs. Subsequently, the prevailing diseconomies in the field for scale-up of nanomanufacturing ends in huge acquisition costs for nanotherapeutics; which ultimately hamper nanotherapeutics success and restricts their implication in day-to-day clinical practice [195]. Owing to low financial rewards allied with nanotherapeutic products, companies/firms developing and marketing such products find it difficult to recover their research and development costs. This signifies a major hurdle in the way of viable nanotherapeutics commercialization, thus undermining their future success in the market.

The unceasingly increasing healthcare costs are a prime challenge for both privately owned and governmental payers and development firms in the developed nations. At present, there is much pressure for delivering public services with utmost efficiency. Thus, medical developments in the future must not only be safe and efficacious, but should also have to be very cost-effective [196]. However, novel approaches that contain growing healthcare costs simultaneously maintaining clinical efficacy, seem to be almost inevitable [197]. However, the 'expensive' nanotherapeutics market uptake can be significantly increased by implying comprehensive standardized cost-effectiveness analyses [198]. Presently, such studies in the nanomedicine field are still in their infancy. The use of cost-effectiveness analyses and studies are indeed the vital missing link that could significantly improve the nanotherapeutics market introduction. Chiefly, it could be more crucial during the times when the healthcare sector is dealing with a shrinking budget [199]. On proper evaluation, the initially perceived 'unattractive' nanotherapeutic products, via their high acquisition costs, could turn into the ideal product for reimbursement.

Nanotherapeutics could offer affordable care, offsetting their high acquisition cost elsewhere. The major plus is the lack of adverse effects that strongly favour novel encapsulated nanotherapeutics; resulting not only in savings the medical procedures to be undertaken, but also reducing hospitalization days and personnel costs, and permitting continuity of work by the patients [199,200]. This is a very valuable boon for society. These cost savings will be pivotal for the development of overall cost-effective nanotherapeutic products [201,202]. Thus, the implication of standardized cost-effectiveness studies is one unique way of making the nanomedicine market more fascinating and likely attracting huge investments from big pharmaceutical firms. Lately, a comprehensive study on nanotherapeutics cost-effectiveness indicated that nanomedicines for ovarian cancer therapy are not only quite cost-effective, but also cost-saving for society [200]. Thus, to accomplish a smooth introduction of nanotherapeutics into the market, many of such cost-effectiveness studies focusing on a range of nanotherapeutics are needed to be undertaken; which in turn will support higher reimbursements and efficient commercialization.

#### **7. Future Perspectives in Cancer Treatment**

Cancer nanomedicines have extensively advanced in recent years. As a result, nanoparticles with the potential of targeted drug delivery when combined with customizable triggering capabilities will have a considerable influence on cancer therapy [203]. Cancer is a diverse, heterogeneous, and mysterious disorder; hence, some of the cancer types and allied aetiology are yet unknown. Furthermore, the pathophysiology and physical characteristics of cancer differ from person to person. Thus, demanding for personalized and customizable anti-cancer therapy; which in itself is a great challenge [3]. Stimuli-sensitive nanostructures and DNA-based nanostructures have a wide range of applications in tumour treatment and diagnostics. The DNA nanostructures that are stimulus sensitive and hybrid in nature; offers excellent specificity and numerous functionalities in drug delivery [204]. Such DNA nanostructures and stimuli-sensitive nanocarriers have been extensively studied nowadays and hold great future in terms of their application in augmenting cancer treatment effectiveness with decreased instances of unwanted effects on normal cells.

Additionally, cancer immunotherapy has proven to be a viable option for achieving a variety of immunomodulatory activities and as an alternative to currently available conventional immunotherapies [205]. In turn, the development of cancer vaccines based on tailored polymeric nanoparticles—which activate a variety of anti-tumour immune responses—would be an adequate alternative to replace existing therapy modalities. Thus, the encouraging features of polymeric nanoparticles and tailored polymeric nanostructures for next-generation cancer immunotherapy modulations would be a viable approach in customised cancer treatment.

#### **8. Conclusions**

Nanocarriers, being a current scientific sensation, have an imperative part in biological applications, particularly in the delivery of anticancer drugs. Nanotechnology is a rapidly expanding and advancing field with the potential to scan, track, identify and transfer medications to specific tumour target cells. When compared to traditional cancer chemotherapy, nanocarriers have shown a considerable improvement in drug therapeutic efficacy with a few adverse reactions. Nanocarriers provide an extended therapeutic circulation lifetime, repeated therapeutic delivery and regulated and targeted drug release under-stimulation. However, in order to overcome the side effects of nanocarriers, surface modification techniques and nano-formulation finetuning must be used to continuously improve their characteristics. Smart nanocarriers must be stable, biodegradable, non-toxic and capable of releasing suitable amounts of drugs to target the tumour location for an extended period of time in order to provide the most effective and safest treatment. Considering this, the nanocarriers are neatly constructed to release the medication at the desired site before being completely degraded. Nanocarriers-mediated diagnostic and therapeutic approaches hold great promises for augmented cancer therapy and hence, with further advancements, these systems will be extensively adopted for facilitated cancer therapy. In this article, the significance of the various categories of smart nanocarriers and their promising potential for site-specific drug delivery applications has been outlined in great detail.

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

**Funding:** This work was funded by the Deanship of Scientific Research (DSR) at King Khalid University through research group programs under grant number RGP-2, 168–42 and the APC was funded by DSR.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** This study did not report any data.

**Acknowledgments:** The authors are thankful to the Deanship of Scientific Research at King Khalid University for funding this work through the grant number RGP-2, 168–42.

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

**Sample Availability:** Not applicable.

## **References**


## *Review* **Exploring the Potential of Natural Product-Based Nanomedicine for Maintaining Oral Health**

**Rajeev Kumar 1,†, Mohd A. Mirza 2,† , Punnoth Poonkuzhi Naseef <sup>3</sup> , Mohamed Saheer Kuruniyan <sup>4</sup> , Foziyah Zakir 1,\* and Geeta Aggarwal 1,\***


**Abstract:** Oral diseases pose a major threat to public health across the globe. Diseases such as dental caries, periodontitis, gingivitis, halitosis, and oral cancer affect people of all age groups. Moreover, unhealthy diet practices and the presence of comorbidities aggravate the problem even further. Traditional practices such as the use of miswak for oral hygiene and cloves for toothache have been used for a long time. The present review exhaustively explains the potential of natural products obtained from different sources for the prevention and treatment of dental diseases. Additionally, natural medicine has shown activity in preventing bacterial biofilm resistance and can be one of the major forerunners in the treatment of oral infections. However, in spite of the enormous potential, it is a less explored area due to many setbacks, such as unfavorable physicochemical and pharmacokinetic properties. Nanotechnology has led to many advances in the dental industry, with various applications ranging from maintenance to restoration. However, can nanotechnology help in enhancing the safety and efficacy of natural products? The present review discusses these issues in detail.

**Keywords:** dental diseases; essential oils; herb; natural products; nanotechnology; regulations

### **1. Introduction**

Dental diseases are a major public health concern and they severely impact the quality of life of individuals. They represent a very important health problem in several countries and create distress among individuals during their lifetimes, causing pain, uneasiness, deformity, and even death. According to WHO, oral diseases affect approximately 3.5 billion people globally (https://www.who.int/news-room/fact-sheets/detail/oral-health, accessed on 5 January 2022).

The most common dental diseases are dental caries (tooth decay), oral cancer, periodontitis (gum disease), noma, and trauma to the oral cavity. Globally, oral cancer is the most prevailing type of cancer. Additionally, with the increase in the consumption of processed and sweet foods, high in free sugars, such as chocolates, candies, and other confectionaries, the problem has worsened. Children are more exposed to this seriousproblem. Soft drinks come into contact with the surfaces of the teeth, causing demineralization. Chewing gums slowly release sugar content in the mouth, which promotes tooth decay. Moreover, diseases such as obesity, diabetes, cancer, chronic respiratory conditions, and cardiovascular complications are also associated with oral diseases. Furthermore, high consumption of tobacco and alcohol also contributes to dental problems. The human mouth

**Citation:** Kumar, R.; Mirza, M.A.; Naseef, P.P.; Kuruniyan, M.S.; Zakir, F.; Aggarwal, G. Exploring the Potential of Natural Product-Based Nanomedicine for Maintaining Oral Health. *Molecules* **2022**, *27*, 1725. https://doi.org/10.3390/ molecules27051725

Academic Editor: Ildiko Badea

Received: 6 January 2022 Accepted: 1 February 2022 Published: 7 March 2022

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

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

is already home to several bacteria, fungi, viruses, and protozoa species, which together constitute the oral microbiome. These microorganisms are determinants of oral health, and infection occurs when the equilibrium is interrupted, which allows the invasion of pathogens [1]. Moreover, consumption of a high-carbohydrate diet disturbs the acid mantle in the oral cavity. The microorganisms convert the carbohydrates into acids, which degrades the hydroxyapatite in the tooth enamel. This promotes contamination with bacteria and the formation of dental caries [2,3]. Nowadays, people are more prone to oral diseases because they remain indoors, which causes vitamin D deficiency. Given that vitamin D is associated with the absorption of calcium, a lack of this vitamin can lead to hypoplasia, which can also contribute to dental caries [4]. Developing and underdeveloped nations are more prone to such problems due to poor health hygiene, lack of awareness, and improper health facilities. It is also a fact that dental treatment is expensive in developed countries, accounting for approximately 5% of the total health expenditure, which is mostly borne by the individuals [5]. Therefore, with current lifestyle choices, maintaining oral hygiene is essential and cannot be neglected.

It is believed that with traditional diet practices (with low sugar content), most of the dental diseases can be avoided [6]. Further, doctors advise the use of fluoride-based mouthwashes, toothpastes, and gels to prevent dental caries [7,8]. However, synthetic products should not be used in the long term. Overuse can cause oral or systemic adverse reactions such as irritation, swelling, itching, and dry mouth [9]. Many over-the-counter (OTC) medications contain ingredients such as chloral hydrate, nitrites, etc., which are consumed by oral pathogens and release products, which causes halitosis [10]. Long-term use of antiplaque agents has been known to be associated with staining of teeth and taste alterations. Furthermore, dental infections are progressively linked with the formation of biofilms. Bacterial/fungal biofilms promote drug resistance against antimicrobials, which makes the infection difficult to treat. Additionally, many challenges, such as side effects/adverse reactions and poor bioavailability issues, may lead to withdrawal because of the inconvenience of long-term therapy.

Herbal products have been used since antiquity for the prevention of diseases and to promote well-being. The Vedic age in India documented the use of herbal remedies in Rigveda and Charaka Samhita [11]. The use of twigs from the *Salvadora persica* tree (known as miswak) for teeth cleaning was reported 7000 years ago in Arabic culture. Studies have proven that miswak possesses antibacterial activity, which prevents the formation of dental plaque (https://clinicaltrials.gov/ct2/show/NCT04561960, accessed on 5 January 2022). In, 1986, miswak was recommended by WHO for oral hygiene. Following this, extracts from *Salvadora persica* were added to toothpastes. Ayurvedic texts also mention the traditional practice of oil pulling. A teaspoon of coconut oil, when swirled in the mouth for around 10–20 min, is believed to improve oral health [12]. Similarly, clove oil has been used for centuries as an analgesic for toothache.

However, with the progression of science, evidence has become a problem for herbal remedies. For this reason, herb-based natural treatments were confined to only a few regions of the world where they have been practiced for a long time, although, with categories such as dietary supplements, neutraceuticals, and botanicals, herbals could be placed into the market. Therefore, now, with the availability of sophisticated technologies and regulatory guidelines, healthcare companies are beginning to take advantage of the opportunities associated with herbal products. The importance of herbal products in the pharmaceutical industry can be demonstrated by the fact that 50% of the drugs approved during the last 20 years were derived from plant sources [13]. Due to cost-effectiveness, cultural acceptability, and minimal adverse drug reactions, 75–80% of the world population relies on herbal drug products. Thus, the paradigm in oral healthcare is also witnessing a shift towards herbal remedies. Presently, various organizations across the world, including WHO, are promoting herbal products for better health. In fact, developed countries have also embraced herbal products as complementary and alternative medicine (CAM) [14]. Herbal medicines are supposed to be safe if not adulterated and quality standards are maintained. With the increasing awareness of the effectiveness and benefits of herbal products, financial aid is also being offered by different research supporting bodies. Nevertheless, this potential has not been exploited to the maximum. It cannot be ignored that even herbal products present some shortcomings. This review details these limitations and discusses the strategies that can be adopted to improve their acceptability in the dental care product industry.

#### **2. Herbal Remedies for Dental Diseases**

A great deal of research has been carried out that proves the activity of herbal ingredients against several dental diseases. Rosemary and *Bougainvillea glabra* essential oil show anti-inflammatory activity that is modulated by the inhibition of histamine and prostaglandin signals [15,16]. This suggests that essential oils with anti-inflammatory activity can be used for the treatment of gum diseases [17].

Treatment of dental diseases often requires topical antioxidants in the form of toothpastes, gels, and mouth rinses. There are numerous factors, such as stress, disease, or dental procedures, that can increase the levels of free radicals; bacterial infections also trigger immune responses, which add to free radical formation. Prolonged infection can result in inflammation, which, if left untreated, can lead to chronic stress. Although salivary antioxidants can control free radicals, this is often insufficient during oral/systemic infection. Therefore, additional antioxidant supplements are required to fight inflammation [18]. Consumers are now becoming aware of the harmful effects of synthetic antioxidants. Essential oils from rosemary and lavender were tested for their IC<sup>50</sup> values, which demonstrated their antioxidant activity [19].

A clinical trial study was conducted on 60 subjects, where the antimicrobial effect of neem extract was investigated. It was found that liquid neem extract significantly (*p* < 0.05) reduced the *Lactobacillus* and *S. mutans* counts, thus suggesting activity against gingivitis and dental plaque [20]. In another study, the antimicrobial effect of Triphala powder against *S. mutans* was tested. The results showed complete inhibition of bacterial growth in 6 min with an MIC of 3.125 mg/mL, which was comparable to the MIC of 0.2 µg/mL exhibited by 0.2% chlorhexidine [21]. Thomas et al. [22] proposed that mouthrinses containing extracts of garlic and lime have significant antibacterial and antifungal activity against lactobacilli, *S. mutans* (*p* = 0.001), and *C. albicans* (*p* < 0.001). Chlorhexidine and fluoride are the main constituents of chemical-based mouthwashes due to their antibacterial activity. The study showed the effective antimicrobial activities exhibited by herbal ingredients when compared with synthetic mouthwashes, suggesting their potential to be used as a substitute for synthetic mouthwashes. Some authors have claimed the anti-cariogenic potential of dentifrices containing clove oil, extracts of black pepper, mint, long pepper, pomegranate, babool, and miswak [23,24]. Most essential oils have demonstrated antimicrobial properties, which is the reason for the rise in their popularity in the treatment of dental infections. A significant amount of research has been carried out to prove that the MIC values of synthetic antibacterial agents are considerably reduced by different essential oils. Their antimicrobial activity has been demonstrated against both Gram-positive and Gram-negative bacteria, fungi, and yeasts [25]. For this reason, many oral hygiene products contain mixtures of essential oils, which serve as antimicrobial agents, control bad smells, and reduce oral bacteria. In another study, a product containing peppermint oil, lemon oil, and tea tree oil was used to treat bad oral smell in 32 intensive care unit patients. After 5 min of essential oil treatment, the strength of the bad smell was significantly lowered. This study showed that, besides the antimicrobial activity, essential oils can also control bad oral smell [26].

According to a report, herbal ingredients from clove, miswak, neem, propolis, and aloe vera exhibit multiple activities, such as anti-inflammatory, antibacterial, antioxidant, and so on, which suggest their role in the treatment of dental plaque and gingivitis [27].

Additionally, herbal formulations have the advantage of being sugar- and alcohol-free. Natural sweeteners such as stevia extracts and xylitol are added to prevent the problem of halitosis [24].

There are increasing numbers of reports that suggest the biofilm disruption activity of herb extracts [28]. In a study by Ramalingam et al. [29], mixtures of *Acacia arabica* and triphala extracts were tested for their biofilm disruption activity against *A. viscosus*, *C. albicans*, *L. casei*, and *S. mutans*. The results revealed that the extracts, at a concentration of 150 µg/mL, not only reduced the biofilm by 91–99% but also prevented bacterial adhesion, thus stressing that they can act as effective anti-caries agents. Nonetheless, even essential oils have recorded biofilm disruption activity. For instance, in a study, the activity of *Allium sativum* essential oil was tested against fluconazole-resistant *C. albicans* biofilms. It was found to be effective at a concentration of <1 mg/mL, which suggested its possible use to prevent denture stomatitis [30]. A similar study carried out suggested the possible role of *Cymbopogon citratus* essential oil against polymicrobial biofilms. The study proved its inhibitory and cytotoxic activity against different species responsible for dental caries, with the added advantage of inhibiting the adhesion of biofilms to dental enamel [31].

Among dental disorders, oral cancer is the major cause of death worldwide. Considering the toxicity of anticancer agents coupled with the emergence of resistance, it has become imperative to search for low-risk therapies for cancer treatment [32]. Studies have suggested that *Lawsonia inermis* essential oil has the potential to be used as an adjuvant in cancer treatment [33]. In another study, a cocktail of extracts of *Ganoderma lucidum*, *Antrodia camphorata*, and Antler showed an IC50 of 15 mg in 72 h during an MTT assay. Further, it inhibited the proliferation and migration of cancer cells without any toxicity/adverse events [34]. Curcumin causes apoptosis of cancer cells via the production of reactive oxygen species and suppression of p53 protein [35]. Simultaneously, curcumin has been found to possess anti-inflammatory and antioxidant properties, which are modulated by preventing lipooxyenase- and cyclooxygenase-mediated inflammation [36]. This property acts synergistically in cancer treatment. Research has shown that *Cryptomeria japonica* essential oil induces apoptosis of human oral epithelial carcinoma cell lines such as KB cells, which may suggest its potential as a chemotherapeutic agent [37]. In a similar study, *Thymus caramanicus* essential oil has shown anti-proliferative and cytotoxic properties on KB cells [38]. Another recent study has shown that essential oil from *P. rivinoides* exhibits cytotoxic activity in oral squamous cell carcinoma cell lines [39]. Additionally, it has been shown that herbal ingredients not only act as chemopreventive and chemotherapeutic agents but also have beneficial effects on chemotherapy-induced side effects. Herbal medicines such as Rikkunshito, Hangeshashinto, and Goshajinkigan have been known to ameliorate side effects such as oral mucositis, diarrhea, anorexia, neurotoxicity, etc. [40].

The many benefits associated with herbal remedies have promoted the use of herbalbased products in the oral health industry.

The potential applications of different essential oils and herbal ingredients investigated for different dental diseases are enumerated in Table 1.

In addition to standalone products, herbal ingredients can be used in synergistic combinations. This approach is known as "herbal shotgun" [41]. For instance, mixtures of extracts of neem, aloe, eucalyptus, hibiscus, rose, and tulsi are useful for the inhibition of most periodontal pathogens and the treatment of dental caries [42]. It is believed that this strategy can offer a multi-targeted effect with maximum benefits and lower potential to develop drug resistance.

However, regarding natural products, there are many shortcomings that cannot be ignored. The dental industry must address these in order to succeed in the global sector.


**Table 1.** List of different phytoconstituents obtained from herbal sources along with their potential pharmacological activity in oro-dental diseases.


#### **Table 1.** *Cont.*

#### **3. Challenges of Herbal Therapies**

Herbal products are now widely present in the market in different regulatory categories. They are also becoming in-demand products for primary healthcare treatment over the conventional medicinal system, due to their fewer side effects and better acceptance. Despite the many advantages, the delivery of herbal ingredients is a challenge (Figure 1), which is discussed in a subsequent section. For instance, essential oils are volatile in nature, which limits their application. Further, when used topically, they can cause irritation/sensitivity to the oral mucosa, which restricts their use [25]. Consequently, other challenges of herbal therapies, such as low solubility, low permeability, long duration of treatment, poor bioavailability, and other challenges (discussed in subsequent sections), limit their potential.

**Figure 1.** Limitations of herbal medicines restricting application in dental industry.

#### *3.1. Safety Issues with Herbal Products*

As we have discussed, medicinal plants contain many potential ingredients that can be used to treat oro-dental diseases. Due to the long history of effectiveness of herbal ingredients against dental diseases, people use them without caution. It is generally believed that herbal remedies are relatively safe when compared to allopathic treatments. However, the claim that herbal medicine does not have any toxic or side effects is not true in all cases. Allergic reactions to essential oils cannot be ignored. Studies have shown that essential oils from sandalwood, lavender, tea tree, and clove are most likely to cause irritation and inflammation. The principle components responsible are benzyl alcohol, geraniol, eugenol, hydroxyl-citronellal, etc. Subsequently, the use of high concentrations/doses of essential oils can trigger adverse reactions [26]. Various factors, such as the amount of

biological content, source of the material, and the route of exposure, should also be taken into consideration with regard to irritation potential.

The latest research studies show that extracts of herbal preparations may have adverse/side effects even if the preparation is used in low doses. There are a few plants reported in research studies that are well known for their medicinal value and are currently used for treatment but exhibit toxic effects too [58]. Moreover, synergistic combinations of herbal ingredients are used for better therapeutic outcomes, which would mean that the content of chemical constituents may be several times greater and thus linked with increased risks of toxicity ("National policy on traditional medicine and regulation of herbal medicines", Report of a WHO global survey, World Health Organization, 2005). However, increased side effects do not mean that the use of herbal medicinal preparations should be avoided. Judicious use can be ensured by pharmacological screening and evaluation of the components in the preparation [58].

#### *3.2. Patient Acceptance*

Although the good therapeutic efficacy of herbal products has been demonstrated, patient acceptability is another important criterion that cannot be overlooked. Since the product is meant for the treatment of dental diseases, good taste and smell, besides other organoleptic properties, are also essential. Essential oils cannot be ingested orally, and can only be used for local application in the form of gargles, mouthwashes, and ointments.

The main problem with essential oils is their strong odor. Tea tree oil (*Melaleuca alternifolia*) has shown antimicrobial properties when tested in 34 patients. However, when the organoleptic properties were tested against Colgate toothpaste, an unpleasant taste was experienced [59]. Similarly, mouthwashes containing tea tree oil have exhibited poor taste and a stinging sensation in the mouth [60]. Although most of the essential oils, due to their strong smell, are used to mask odor in oral diseases, they are nonetheless often not accepted by the consumers. Eucalyptus oil and tea tree oil are more commonly known essential oils with a strong odor that are poorly acknowledged [26].

#### *3.3. Poor Bioavailability*

During the formulation of herbal drug products, the permeability of drug molecules across the epithelial mucosal barrier must be achieved for better therapeutic action of the drug product. Variations in the permeability of a drug across different locations in the oral mucosa can be observed. The keratinized regions contain ceramides, which act as a barrier for hydrophilic drugs, whereas non-keratinized areas limit the permeation of hydrophobic drugs.

The washing action of saliva in the mouth also contributes as a barrier against adequate delivery [61]. Further, the instability of herbal active compounds in the gastric region cannot be ignored.

Most of the herbal constituents isolated are hydrophobic in nature, and thus poorly soluble, making them less bioavailable, which needs to be taken into account for efficient therapeutic action [62]. This would mean that higher doses will be required, which can result in adverse effects and poor patient compliance. Additionally, phenolic-based plant constituents are water-soluble, which restricts their absorption across the lipid membrane. Further, improper molecular size is again a challenge thatcontributes to poor absorption. Chinese medicines comprise larger molecules that are difficult to absorb and this affects other phyisco-chemical attributes [63]. On the other hand, regarding essential oils, although they are small molecules that are able to permeate and absorb, the faster metabolism and short half-life lead to low bioavailability [64]. Many of the marketed products, such as curcumin and ellagic acid, have poor bioavailability because of their lower solubility in aqueous media and extensive metabolism. In a study carried out on rats, no curcumin was found in biological fluid/plasma when 400 mg of curcumin was administered via the oral route; however, a very small amount of curcumin was found in the portal blood [65].

It is for these reasons that most of the plant-based drugs have shown promising potential during invitro studies but under-perform in the clinical stage due to poor bioavailability.

#### *3.4. Long Duration of Treatment*

In most herbal medicinal products, the short duration of action represents a major limitation. Formulation scientists have to keep in mind that the dosing frequency of the dosage form should be minimal. Scientists are still working on improving the duration of action as well the onset of action of herbal medicinal products.

#### *3.5. Lack of Harmonized Regulations*

Herbal products are marketed in different product categories in different parts of the world. Currently, many regulatory categories exist for herbal medicinal products thatcomprise over-the-counter drugs, prescription drugs, traditional medicinal products, and dietary supplements. There is a need for the establishment of strict global and regional regulatory mechanisms for the monitoring of herbal medicinal products [66]. The magnitude of quality, safety, and efficacy data requirements for product registration varies from region to region. There should be a harmonized data requirement throughout. Furthermore, most of the herbal products available in the market lack evidence of their safety and efficacy. Improper cultivation and harvesting techniques and improper storage conditions create an urgent need to standardize herbal preparations. Another problem is contamination with heavy metals, which occurs during the cultivation stage. Adulteration of herbal ingredients is also a major quality concern.

WHO has been pioneer in setting the parameters for the quality, safety, and efficacy of herbal medicinal products to meet the basic criteria for evaluation. A set of basic parameters for the evaluation of herbal drug products have also been added in pharmacopeial monographs. Scientists are still performing research on herbal medicines to deliver them with maximum bioavailability and concentration to target cells [65]. Therefore, a suitable delivery system has to be developed to realize the full potential of natural products.

#### **4. Nanotechnology in Herbal Dentistry**

Although herbal ingredients have shown extensive potential in the treatment of dental diseases, one of the major limitations is their unfavorable physicochemical and pharmacokinetic properties, which contribute towards inadequate performance. Another problem is their instability in the biological milieu. Furthermore, the physical stability of active compounds cannot be overlooked. Environmental conditions, processing, and handling of plant materials can lead to degradation due to oxidation and dehydrogenation reactions, which ultimately affect the organoleptic properties [67].

Many approaches have been used to enhance their absorption, stability, and pharmacokinetic profile. One suitable method would be to encapsulate the phytoconstituents in a suitable carrier system, which will help in realizing the full potential of the herbal active moiety (Figure 2). The solubility profile can be improved by forming salts with weak acids/bases. However, the salt formation technique cannot be applied to all the phytoconstituents.

Nanotechnology has already produced some promising outcomes in the delivery of phytoconstituents. The technique has been found useful to assist in overcoming low systemic bioavailability and inadequate solubility. The drug delivery potential of nanoformulations has received a great deal of attention recently, with polymeric nanoparticles and lipid-based delivery systems such as phytosomes, ethosomes, liposomes, transferosomes, and nano-emulsions all attracting much interest [68].

**Figure 2.** Applications of nano-herbal technology in diverse dental domains.

#### *4.1. Nanotechnology to Enhance Solubility of Natural Bioactives*

It is well proven that reducing the size of the herbal bioactives can enhance the solubility and dissolution. Depending upon the intended site of action, the size of the formulation can be regulated to facilitate transport across the biomembrane. Since the bioavailability of a poorly soluble drug is limited by dissolution, even a minute increment in solubility will have a significant impact on the bioavailability [69]. For instance, curcumin, with very good anti-inflammatory activity if used as a powder or in other conventional delivery systems, shows low oral absorption due to its hydrophobic nature. Therefore, nanomicelles were prepared, which entrapped curcumin in a hydrophobic core, rendering them miscible with water. The delivery system enhanced the solubility, which proved to be successful in reducing inflammation in gingivitis and mild periodontitis [70]. Various approaches, such as the formulation of nanosuspensions, nanoemulsions, nanocrystals, etc., have been used, where the particle size of the delivery system is reduced, which ultimately enhances the solubility/dissolution (Table 2).


**Table 2.** Nanoparticle formulations of phytoconstituents with regard to dental diseases that show improved physicochemical and therapeutic properties.


**Table 2.** *Cont.*


**Table 2.** *Cont.*

#### *4.2. Nanotechnology to Enhance Permeability of Natural Bioactives*

There are a number of ways through which the permeation of herbal ingredients can be facilitated. Surface coating with hydrophilic surfactants/polymers or otherwise lipophilic polymers can be done to assist the transport if the hydrophilicity/lipophilicity of the molecule is a barrier. Consequently, mucoadhesive formulations can be prepared by using bioadhesive polymers. This will enhance the residence time of the formulation in the oral cavity, which will provide ample time for sufficient permeation. Further, encapsulating the herbal active moiety into a nanodelivery system will not only ensure better permeation but also provide stability to the molecule [89]. Herbal extracts from *Trypterygium wilfordii* have shown good potential as anticancer agents, but they exhibit insolubility and poor intestinal absorption. Lipid-based nanocarriers such as lipid nanoparticles [90] and lipid nanospheres [91] were developed to enhance the solubility and permeability. In another study, phospholipid-based phytosomes functionalized with protamine and loaded in chitosan sponges were prepared. The delivery system provided mucoadhesive properties coupled with enhanced permeation through the buccal mucosa to provide a 244% increase in bioavailability [92]. Tonglairoum et al. [93] reported the complexation of clove oil and betel oil with cyclodextrins to enhance the solubility. It was further incorporated into nanofibers, which provided fast release of the oil and enhanced the antifungal activity against Candida sp. The study proved that the formulation can be useful for the treatment of denture stomatitis.

#### *4.3. Nanotechnology to Enhance the Therapeutic Performance of Natural Products*

The concept of utilizing nanotechnology to enhance the therapeutic performance is not new. There are reports that the nanosizing of the formulation enhances the permeation and bioavailability of phytoconstituents [94]. In one study, the authors prepared microspheres of zedoaryoil obtained from turmeric. The small size of the delivery system facilitated better invivo absorption and improved the bioavailability by 135.6% [94]. Further, the sustained release prevented adverse effects and reduced the dosing frequency. In another study, a nanocrystal of baicalein was formulated and the results revealed enhanced solubility and bioavailability by 1.67 times [95]. Nanotechnology has also proven beneficial to enhance the stability of essential oils. It is shown to protect essential oils from oxidation, hydrolysis, photodegradation, thermal degradation and reduce volatility. Low aqueous solubility and high volatility prevents the use of bare essential oils, making encapsulation into delivery

systems a necessity [96]. Curcumin has been found to be photoreactive, which decreases its potency by 70%. Onoue et al. [73] prepared solid dispersions of curcumin to enhance the physical stability and only 17% degradation was observed. This can enhance the clinical acceptability of natural products.

Among the most important consequences of nanotechnology-based pharmaceuticals is cancer treatment, which otherwise entails many adverse effects and high costs. Certain unique innovative drug delivery methods have recently been developed using nanoparticles loaded with triclosan, which might be a turning point in preventing periodontal disease progression [97]. Peppermint oil hasbeen found to possess good anticancer properties against oral cancer; however, poor solubility limits its application. Tubtimsri et al. [98] developed a peppermint oil-loaded nanoemulsion whereby the droplet size was reduced to approx. 100 nm and further incorporation into a hydrophobic core with the exterior aqueous phase rendered them water-soluble. Further, the authors proposed the herbal shotgun approach, where synergistic combinations of peppermint oil and virgin coconut oil loaded in a nanoemulsion revealed promising cytotoxic properties against oral squamous cell carcinoma cell lines.

#### **5. Role of Nano-Herbal Technology in Biofilm Resistance**

Dental caries and periodontitis represent the most common oral infectious diseases. On studying the pathophysiology, it was found that invasion by pathogenic bacteria is the main etiology of the disease. These bacteria hide within the extracellular matrix, which prevents the entry of antimicrobial agents and forms biofilms. Herbal ingredients have shown activity in biofilm resistance. The main mechanism of action is preventing the synthesis of glucans, which are responsible for adherence, thus preventing the formation of biofilms [99]. Further, essential oils can play a pivotal role in the treatment of dental infections. Essential oils directly damage the integrity of cell membranes, which results in microbial growth inhibition. Infact, studies have shown that essential oils can be used as a substitute for synthetic antibacterials. For instance, the zone of inhibition against *S. aureus* and *E. coli* was found to be 9.94 ± 0.29 mm and 8.10 ± 0.31 mm, respectively, with doxycycline gel. A eugenol nanoemulsion gel was prepared and tested and the zone of inhibition was 8.82 ± 0.28 mm and 7.58 ± 0.31 mm, respectively, which is close to the antibacterial affect exhibited by doxycycline [100]. The relation between the lipophilicity of essential oils and their antimicrobial activity has driven researchers to examine the antibacterial properties of some biological components, such as *Citrus Aurantifolia*, *Thymus vulgaris*, and *Origanum vulgare* essential oils, against cariogenic oral bacteria [101]. Essential oils, due to their lipidic nature, interact with the hydrophobic bacterial cell membrane, causing the destabilization and leakage of ions, which is responsible for cell death.

However, a problem arises when the bacterial cells are protected by a hydrophilic extracellular matrix that is impermeable to essential oils. To combat the problem of resistance, nanotechnology has been successfully used, where enhanced antibacterial activity has been found [102] (Table 3). Poly(D,L-lactide-co-glycolide)(PLG) nanoparticles loaded with *H. madagascariensis* extract were prepared and tested for their antibacterial properties against Gram-positive and Gram-negative strains. The minimum bactericidal concentration (MBC) was considerably reduced for the nanoparticle formulation (1.875 <sup>×</sup> <sup>10</sup><sup>2</sup> mg/L), compared to 5–7 <sup>×</sup> <sup>10</sup><sup>2</sup> mg/L exhibited by extracts in ethyl acetate. The bioadhesive property of the PLG polymer allowed the attachment of nanoparticles to the bacterial cells while facilitating the controlled release of the extract and maintaining the concentration [103].

Biofilms are hydrophilic in nature, and so hydrophobic essential oils can be converted into nanoemulsions with a size of less than 300 nm, which facilitates the penetration of the active ingredient into the biofilm matrix. Cinnamon oil loaded in nanoemulsions inhibited a *S. mutans* biofilm by 86%, compared to 60% observed by an ethanolic oil solution [104]. Synergistic effects can be observed when essential oils are encapsulated in lipid-based nanodelivery systems. The nanosize facilitates higher diffusion into bacterial cell membranes. Researchers have suggested that micro-/nanoemulsions give more favorable outcomes in

terms of bacterial resistance [105]. The presence of surfactants in the formulation, coupled with the nanosize, provides high surface tension and wetting ability to the delivery system. This allows fusion with the cell membranes of microorganisms and eventually kills them.

**Table 3.** Nanodelivery systems of phytoconstituents and their role in microbial biofilm resistance.


#### **6. Synergistic Combinations of Phytoconstituents and Drugs in Nanotechnology**

It is believed that synergistic combinations of antibiotics with herbal ingredients can potentiate the antibacterial effects, which can help to overcome bacterial resistance. A study conducted by Saquib et al. [114] suggested that the use of phytoconstituents with antibiotics is effective against periodontal infections. For instance, use of a combination of *C. zeylanicum* with azithromycin exhibited strong antibacterial activity against *T. denticola* and *T. forsythia*. A synergistic combination of *S. presica* and tetracycline showed significantly reduced MICs against most periodontal pathogens. In another study by Dera et al. [115], the efficacy of thymoquinone with different macrolide and aminoglycoside antibiotics was tested and an enhanced antibacterial effect was witnessed. It is believed that efflux pumps acting within the bacterial cells are responsible for drug resistance. Studies have shown that herbal active constituents inhibit efflux pumps and also display antibacterial activity of their own, mostly through reducing the production of acids or preventing adhesion [114,116]. Therefore, combination with antibiotics significantly enhances the antibacterial efficacy. Further details are available in Table 4.

Hydroxyapatite has been found to possess bone formation properties and is therefore used as a bone substitute in dental implants. A study has shown that hydroxyapatite nanocrystals morphologically resemble apatite crystals and promote bone remineralization, but only in the outer enamel layer [117]. In a study by Huang et al. [118], superior bone remineralization and deposition was found on teeth at a depth of 40–140 µm by using a combination of nanohydroxyapatite and Gallachinensis extracts compared to single treatments. G. chinensis is a potential anti-caries agent that favors mineralization while simultaneously inhibiting demineralization.

**Table 4.** Studies showing potential of synergistic combinations of herbal ingredients and synthetic drugs in dental diseases.


#### **7. Regulatory and Commercial Manufacturing Challenges**

The challenges for herbal ingredients associated with dental products are as follows:


Furthermore, if the herbal products have been approved through the route of indigenous medicine, i.e., Ayurveda, Siddha, and Unani (ASU), they can make therapeutic claims; otherwise, they cannot be associated with any such claims. Other than ASU, dental care products fall within the categories of cosmetics (CDSCO, India), OTC drug products (USFDA), cosmetics (EMA), drug and health products (Health Canada), and cosmetics (TGA, Australia). If the product has to be marketed in the category of dietary supplements, no prior approval from the USFDA for manufacturing/selling is required. It becomes the responsibility of the manufacturer to ensure the safety of their products. The various quality attributes have to be checked by the manufacturer. The standard quality parameters of toothpaste can be obtained from the following documents:


The general quality parameters could be as follows:


Similarly, other quality guidance documents can be found. However, the challenges do not end here: the standardization of herbals is another major challenge. The efficacy of a natural preparation depends on the growth conditions, collection, and processing techniques of the raw materials. Heavy metal and microbial contamination is a persistent problem if proper harvesting is not carried out. Intentional adulteration is another issue.

Stringent regulatory agencies carry out marker-based identification of raw materials. However, this remains a challenge for underdeveloped nations due to high costs. Moreover, a lack of availability of reference standards for most of the herbal ingredients makes it impractical. All these factors contribute to the poor quality of natural preparations, often leading to limited acceptability and recognition by health practitioners.

#### **8. Patent Analysis**

There are a number of herbal products that are available on the market for the treatment of oral diseases. However, the use of nano-herbal technology in dental diseases is a relatively new concept. A patent analysis was carried out to determine the number of patents in the area and much literature cannot be found (Table 5). Therefore, nano-herbal dentistry needs further exploration.


**Table 5.** A snapshot of patents highlighting the use of nanotechnology in herbal dentistry.

### **9. Future Prospects of Herbal and Essential Oil-Based Formulations in the Treatment of Dental Diseases**

Phytochemical screening has already established the pharmacological properties of several biological actives. During screening studies, it was found that ingredients such as flavonoids, terpenes, and terpenoids are responsible for therapeutic effects. Invitro studies have proven that herbal remedies have potential in the treatment of dental diseases. However, the problem lies in reproducing the results invivo, which often becomes difficult. This due to the previously discussed issues, such as poor lipid solubilization and improper

molecular size of herbal active molecules. In order to achieve the desired therapeutic effects of herbal ingredients, researchers are continuously working to achieve the delivery of herbal active molecules at the desired concentrations in the blood. The greatest challenge in the development of herbal formulations is to cross the membrane with an enhanced pharmacokinetic profile and therapeutic efficacy. Lipid-based and oil-based carriers can be used to resolve these challenges. Bioactive molecules with a greater half-life have a long duration of action and long rate of elimination too as compared to molecules with a shorter half-life. The elimination rate and renal filtration determine the bioavailability of herbal drugs. The greater the elimination rate and renal filtration, the lower the bioavailability of herbal drugs in blood plasma. Novel delivery systems such as nanoemulsions are used successfully to deliver herbal drug molecules to the blood at maximum therapeutic value with minimum adverse effects [122]. Moreover, encapsulation in nanodelivery systems has overcome the existing physicochemical limitations of essential oils. Sustained and controlled release systems of oils into the cells of bacteria can be achieved by attaining the chemical stability, solubility in water, and encapsulation of oils, which can enhance the antimicrobial action. Additionally, the concept of the herbal shotgun has shown a tremendous surge with the application of nanotechnology in herbal industry. Previously, simultaneously using two or more ingredients in a single formulation was difficult as the actives were incompatible with other components in the formulation. The new drug delivery systems have made it possible to improve the efficacy of natural ingredients. Additionally, constituents that were disregarded previously due to their undesired properties have now come into the fore.

Nonetheless, there are many setbacks in the nano-herbal industry. The main challenge is to scaleup the development of nanotechnology-based herbal bioactive molecules at a commercial level. Further, geographical conditions, cultivation factors, and processing conditions affect the quality and quantity of active constituents. Additionally, isolation and purification is another challenge as it is a time-consuming and costly process. Pharmaceutical industries have to collect and screen the herbal actives themselves, which is seen as an impediment and discourages the use of natural ingredients. The lack of standardization of herbal ingredients and dire agricultural practices are significant setbacks in the herbal industry. Global harmonization in regulatory guidelines related to herbal products is the need of the hour.

The global herbal medicinal market size was valued at USD 85 billion in 2019 and is expected to increase at a rate of 20%. Two new herb-based NDAs were approved by the USFDA in 2006 and 2012 for the drugs sinecatechins and crofelemer, respectively. The current challenge is to bring newer nanotechnology-based herbal products into the market with the possibility of scaling up and complying with the international standards of safety and toxicology.

#### **10. Conclusions**

The goal of this review was to look back over the last ten years at the possibilities of natural medicine for treating dental disorders. Abundant evidence has been found thatproves that phytoconstituents present in herbal extracts or essential oils have the potential to be used as preventative or therapeutic therapies for oral disorders. Due to various drawbacks, natural medicine has not been explored sufficiently. While herbal medicinal products are leading to new formulations, further research is required to determine their therapeutic benefits, along with their safety and efficacy. Single or combination therapies in the form of a suitable delivery system can be used to reduce the global burden of oro-dental diseases.

**Author Contributions:** Conceptualization, F.Z. and G.A.; resources, R.K. and M.A.M.; data collection, P.P.N. and M.S.K.; writing, R.K. and F.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University, Saudi Arabia for funding this work through the Research Group Program under Grant No. RA.KKU/128/43.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

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

## **References**


## *Review* **Quality by Design Approach in Liposomal Formulations: Robust Product Development**

**Walhan Alshaer 1,† , Hamdi Nsairat 2,†, Zainab Lafi <sup>2</sup> , Omar M. Hourani <sup>3</sup> , Abdulfattah Al-Kadash <sup>1</sup> , Ezaldeen Esawi <sup>1</sup> and Alaaldin M. Alkilany 4,\***


**Abstract:** Nanomedicine is an emerging field with continuous growth and differentiation. Liposomal formulations are a major platform in nanomedicine, with more than fifteen FDA-approved liposomal products in the market. However, as is the case for other types of nanoparticle-based delivery systems, liposomal formulations and manufacturing is intrinsically complex and associated with a set of dependent and independent variables, rendering experiential optimization a tedious process in general. Quality by design (QbD) is a powerful approach that can be applied in such complex systems to facilitate product development and ensure reproducible manufacturing processes, which are an essential pre-requisite for efficient and safe therapeutics. Input variables (related to materials, processes and experiment design) and the quality attributes for the final liposomal product should follow a systematic and planned experimental design to identify critical variables and optimal formulations/processes, where these elements are subjected to risk assessment. This review discusses the current practices that employ QbD in developing liposomal-based nano-pharmaceuticals.

**Keywords:** drug delivery; nanomedicine; liposomes; quality by Design (QbD); nano-pharmaceuticals; pharmaceutical industry

#### **1. Introduction**

Nanomedicine and nanoparticle-based therapeutics are gaining increasing interest in both academia and industry. Currently, there are many FDA-approved nanomedicine products with proven clinical outcomes [1]. Liposomes are spherical vesicles of a continuous three-dimensional phospholipids bilayer wrapping an aqueous core [2]. Liposomes have been used to deliver a wide range of therapeutics [3]. For example, liposomes have been successfully loaded with the anticancer agent, doxorubicin, and showed enhanced therapeutic efficacy and decreased unwanted side effects [4]. Moreover, they have been widely investigated as carriers of nucleic acid-based therapies, such as siRNA [5] and DNA, enabling enhanced penetration in targeted cells and protecting drugs from degradation [5]. Liposomes were one of the first nanotechnology-platforms that entered the market early in 1995 and is still one of the major nano-platforms [1]. It is worth mentioning that the first FDA-approved mRNA vaccine for COVID-19 was approved in 2020 utilizes lipidic/liposomal nanocarriers as a delivery system [6]. Despite the outstanding properties of liposomes, the complexity in their formulations, product development and manufacturing are clearly challenging. The explanation of increased complexity in the case of nano-formulations/nanomanufacturing is associated with the unique physics and chemistry at the nanoscale and thus a higher number of variables needed to be understood

**Citation:** Alshaer, W.; Nsairat, H.; Lafi, Z.; Hourani, O.M.; Al-Kadash, A.; Esawi, E.; Alkilany, A.M. Quality by Design Approach in Liposomal Formulations: Robust Product Development. *Molecules* **2023**, *28*, 10. https://doi.org/10.3390/ molecules28010010

Academic Editor: Faiyaz Shakeel

Received: 11 November 2022 Revised: 10 December 2022 Accepted: 11 December 2022 Published: 20 December 2022

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

and optimized [7]. Lack of this understanding and optimization is the reason behind the common sensitivity and poor reproducibility in nano-preparations and manufacturing. For these systems, an experimental approach that facilitates the identification of critical parameters and help in understanding their contributions to the characteristics/quality of the final product is certainly beneficial. For this purpose, the quality by design (QbD) has been proposed and recommended by various industries and regulatory agencies [8,9]. QbD starts by identifying the quality target product profile (QTPP), which is a summary of the quality attributes (QA) of the final product to ensure its efficacy and safety. QA is dependent on critical attributes related to the material attributes (CMA) and process parameters (CPP). QbD follows by identifying and optimizing CMA and CPP and setting their target specifications to ensure the QA and ultimately QTPP for the final product [9–11]. Proper experimental design is used to link CMA and CPP to QA [8,12], which then facilitate the establishment of targeted specifications for materials, processes and the final product. Moreover, QbD enables the evaluation of the effect of more than one factor at a time on the QTPP. Additionally, risk assessments are used to prioritize QA [13]. Considering the potent liposomal-based drug products in clinical use and the diverse clinical and preclinical applications, there is an unmet need for strategic and systematic development of liposomes as potent drug delivery systems that enable better therapeutic efficacy of the loaded therapies. Although applying QbD liposomal drug delivery systems development have been described in several research, there is more and more need to understand and describe current advances in using QbD in liposomal formulation developments to guarantee liposomal-based drug delivery systems with higher therapeutic outcomes and possible industrial development. Therefore, this review highlights the main strategic points of developing liposomes according to the QbD to reduce the obstacles of using such vehicles in clinical applications in the future.

## **2. Quality by Design (QbD)**

#### *2.1. QbD in Pharmaceutical Products*

The production of quality pharmaceutical products is the major goal of pharmaceutical industry [14]. The quality of the pharmaceutical products covers all aspects that may have an impact on the prescribed products which will consequently affect the health of the patients. Previously, the quality by testing method (QbT) was the common method to ensure quality of the manufactured products. QbT is based on an in-process testing of input materials, intermediates and the final product [15]. However, the pharmaceutical quality sectors call for an alternative practice that can ensure the quality before manufacturing in addition to maintaining the required quality control testing suggested by QbT. To this end, the current pharmaceutical industry and regulation firms switch toward what is now known as the QbD, which ensures that pharmaceutical products will be developed and manufactured as per pre-defined quality attributes, thus QbD is expected to minimize intensive testing during or after manufacturing as well as improve reproducibility, manufacturability, efficacy and safety [16]. Therefore, QbD can be defined as a prospective approach to improve product quality [17]. ICH, US FDA and EMA have specified thoroughly the outlines of the QbD key elements to ensure the consistency of high-quality pharmaceutical products (Figure 1), reflecting a continuous interest in QbD implementation by various international regulatory bodies [16,18].

### *2.2. Tools and Key Elements of QbD*

Generally, there are four key elements of the QbD: (i) the quality target product profile (QTPP), (ii) the critical quality attributes (CQAs), (iii) the critical material attributes (CMAs) and (iv) the critical process parameters (CPPs) [12,19,20]. All of these elements are collaborating in a step-by-step approach to draw the framework of the QbD strategy. The recruitment of these key elements in the QbD method needs well-defined experimental design combined to proper statistical analysis (Figure 2) [16,21].

ICH guidelines define QTPP as "a prospective summary of the quality characteristics of a drug product that ideally will be achieved to ensure the desired quality, taking into account safety and efficiency of the drug product" [16,18]. To identify QTPPs and define the desired performance of the product, the manufacturer should consider complex variables, such as drug pharmacokinetic parameters, product stability, sterility and drug release [22]. The critical quality attributes (CQAs) were defined by ICH Q8 guideline as "physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality." In light of this definition, the CQAs are derived from the QTPP, regulatory requirements, or available literature knowledge. Thus, the critical Quality attribute (CQA) of the drug product and its QTPP is the basis of its dosage form, excipient and manufacturing process selection [23]. *Molecules* **2023**, *28*, x FOR PEER REVIEW 3 of 20

**Figure 1.** The pharmaceutical development guidelines suggested by ICH, US FDA and EMA to outline the QbD key elements to ensure the consistency of high-quality pharmaceutical products. **Figure 1.** The pharmaceutical development guidelines suggested by ICH, US FDA and EMA to outline the QbD key elements to ensure the consistency of high-quality pharmaceutical products.

*2.2. Tools and Key Elements of QbD*  Generally, there are four key elements of the QbD: (i) the quality target product profile (QTPP), (ii) the critical quality attributes (CQAs), (iii) the critical material attributes (CMAs) and (iv) the critical process parameters (CPPs) [12,19,20]. All of these elements are collaborating in a step-by-step approach to draw the framework of the QbD strategy. The recruitment of these key elements in the QbD method needs well-defined experimental design combined to proper statistical analysis (Figure 2) [16,21]. The critical process parameters (CPPs) are the process-related parameters that significantly affect the QTPP [16]. The identification of CPPs, an in-depth understanding of the developed standards/specifications, and linking CMAs and CPPs to CQAs are crucial to ensure quality products [24]. Furthermore, both critical material attributes (CMAs) and critical process parameters (CPPs) are generally defined as "A material or process whose variability has an impact on a critical quality attribute and should be monitored or controlled to ensure the desired drug product quality" [23]. It is worth mentioning that CMAs are for the input materials including drug substances, excipients, in-process materials, while CQAs are for output materials, i.e., the product.

Implementing a risk assessment is vital to identify formulations, ingredients, or process parameters that can impact CQAs after the risk analysis appraises the impact of these parameters on the CQAs. Additionally, a qualitative or quantitative scale is used to rate the risk of each identified factor for the desired CQAs. For this reason, a risk assessment scale has to be established based on the severity and dubiety of the impact on efficacy and safety. Effect analysis and the Failer mode can be used to identify CQAs. After the risk evaluation

process a few of these parameters become potentially critical for the CMAs, which must have certain properties and must be selected within a reasonable range to guarantee the CQAs of the final product [25,26]. *Molecules* **2023**, *28*, x FOR PEER REVIEW 4 of 20

**Figure 2.** QbD roadmap, and QTPP and CQAs key elements. **Figure 2.** QbD roadmap, and QTPP and CQAs key elements.

#### ICH guidelines define QTPP as "a prospective summary of the quality characteristics **3. Development of Liposomes Using QbD**

of a drug product that ideally will be achieved to ensure the desired quality, taking into *3.1. QbD in Liposomal Formulation*

process selection [23].

account safety and efficiency of the drug product" [16,18]. To identify QTPPs and define the desired performance of the product, the manufacturer should consider complex variables, such as drug pharmacokinetic parameters, product stability, sterility and drug release [22]. The critical quality attributes (CQAs) were defined by ICH Q8 guideline as "physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality." In light of this definition, the CQAs are derived from the QTPP, regulatory require-The quality of liposomal pharmaceutical products is affected by their contents, preparation, properties and manufacturing key variables [12]. Therefore, QbD involves designing the final liposomal products by optimizing input material and manufacturing processes to acquire a pharmaceutical product with superior quality [27]. Moreover, QbD classifies and translates the critical parameters and key variables to produce a high-quality drug product with the most desired characteristics [28]. Indeed, several liposomal products have been developed using QbD approach, as summarized in Table 1.

ments, or available literature knowledge. Thus, the critical Quality attribute (CQA) of the drug product and its QTPP is the basis of its dosage form, excipient and manufacturing

developed standards/specifications, and linking CMAs and CPPs to CQAs are crucial to

The critical process parameters (CPPs) are the process-related parameters that signif-


**Table 1.** Examples of pharmaceutical liposomes developed by QbD.

To certify the desired quality of the final pharmaceutical product, a quality target product profile (QTPP) should be established [27]. QTPP is usually performed based on the available scientific data and proper in vivo significance [25]. To identify the QTPP and the process key parameters that can influence the liposomal product's quality attributes (CQAs), the following principal CQAs generally should be recognized/optimized: average particle size, particle size distribution, zeta potential, drug content, in vivo stability and drug release [25,38].

Although there are many benefits of applying QbD to liposomal-based products, there are many challenges that limit the application of QbD liposomal-based product development. Benefits and challenges are summarized in Table 2.

**Table 2.** Benefit and challenges of applying QbD in of liposomal-based products.

#### Benefits


Challenges


#### *3.2. QbD Process Key Parameters for Liposomal Products*

#### 3.2.1. Lipid Type and Content

The integrity and stability of liposomes mainly rely on the lipid type. Lipids with unsaturated fatty acids are susceptible to degradation by hydrolysis or oxidation, while saturated fatty acids are more stable and have higher transition temperature (Tm) [39]. Moreover, liposomes fluidity, permeability and surface charge also count on the lipid type and the liposomal lipidic composition [40]. For example, cholesterol typically increases liposome stability but should be optimized and not exceed 50% [41]. Generally, the carbon chain length of the formulated lipids may affect the drug encapsulation efficiency of both hydrophilic and hydrophobic drugs [40]. For example, a large aqueous core can be obtained using short fatty acid lipids that can enable a high internal volume for hydrophilic drugs. In contrast, long carbon chain lipids are more suitable to encapsulate the hydrophobic drugs within the hydrophobic lipid bilayer [42,43]. Furthermore, the loaded material has a great influence on the morphological features of the particles. The concentration of nucleic acids impacts the change from a multilamellar to an electron-dense morphology in lipidic-based particles [44].

Since 1978, liposomes have been used for the selective insertion of exogenous RNA into cells [45]. Many liposomes have been optimized and fabricated to encapsulate nucleic acids with low toxicity and high efficiency [46]. However, ionized lipids, especially cationic lipids, are still the most used for this purpose [47,48]. Unfortunately, cationic lipids produce many changes in the cell and proteins, such as cell shrinking, reduction in mitoses and changes in protein kinase C and cytoplasm vacuoles [49,50]. On the other hand, compared with viral vectors for gene delivery into cells, cationic lipids are easy to fabricate, simple and possess lower immunogenicity [51]

Both hydrophobic and hydrophilic parts of the cationic lipids have a toxic effect, especially if they contain a quaternary amine that acts as a protein kinase C inhibitor [52]. A new approach to decrease the effect of the positive charge was proposed to spread the charge by delocalizing it into a heterocyclic ring imidazolium [53] and a pyridinium [54]. Chang et. al., developed cationic lipids with a cyclen headgroup and revealed that this novel lipid is safer and possesses lower cytotoxicity than the commonly used lipid to deliver gene therapy [55].

#### 3.2.2. Manufacturing Process 3.2.2. Manufacturing Process The most commonly used manufacturing process for liposome preparation is the

liver gene therapy [55].

in lipidic-based particles [44].

simple and possess lower immunogenicity [51]

*Molecules* **2023**, *28*, x FOR PEER REVIEW 7 of 20

saturated fatty acids are more stable and have higher transition temperature (Tm) [39]. Moreover, liposomes fluidity, permeability and surface charge also count on the lipid type and the liposomal lipidic composition [40]. For example, cholesterol typically increases liposome stability but should be optimized and not exceed 50% [41]. Generally, the carbon chain length of the formulated lipids may affect the drug encapsulation efficiency of both hydrophilic and hydrophobic drugs [40]. For example, a large aqueous core can be obtained using short fatty acid lipids that can enable a high internal volume for hydrophilic drugs. In contrast, long carbon chain lipids are more suitable to encapsulate the hydrophobic drugs within the hydrophobic lipid bilayer [42,43]. Furthermore, the loaded material has a great influence on the morphological features of the particles. The concentration of nucleic acids impacts the change from a multilamellar to an electron-dense morphology

Since 1978, liposomes have been used for the selective insertion of exogenous RNA into cells [45]. Many liposomes have been optimized and fabricated to encapsulate nucleic acids with low toxicity and high efficiency [46]. However, ionized lipids, especially cationic lipids, are still the most used for this purpose [47,48]. Unfortunately, cationic lipids produce many changes in the cell and proteins, such as cell shrinking, reduction in mitoses and changes in protein kinase C and cytoplasm vacuoles [49,50]. On the other hand, compared with viral vectors for gene delivery into cells, cationic lipids are easy to fabricate,

Both hydrophobic and hydrophilic parts of the cationic lipids have a toxic effect, especially if they contain a quaternary amine that acts as a protein kinase C inhibitor [52]. A new approach to decrease the effect of the positive charge was proposed to spread the charge by delocalizing it into a heterocyclic ring imidazolium [53] and a pyridinium [54]. Chang et. al., developed cationic lipids with a cyclen headgroup and revealed that this novel lipid is safer and possesses lower cytotoxicity than the commonly used lipid to de-

The most commonly used manufacturing process for liposome preparation is the thin-film hydration method (Figure 3) [56,57]. Other approaches such as reverse-phase evaporation, ethanol injection and emulsification have also been applied [58]. The thin-film hydration method produces multilamellar structure liposomes with an average diameter in micrometers [42]. Thus, resizing liposomes to less than 200 nm is required to improve the surface area to volume ratio for superior encapsulation and drug loading efficiency. Improving the size distribution of the prepared liposomes, extrusion, sonication (probe or bath) and freeze–thaw cycling have been used for liposomes size reduction [59]. thin-film hydration method (Figure 3) [56,57]. Other approaches such as reverse-phase evaporation, ethanol injection and emulsification have also been applied [58]. The thinfilm hydration method produces multilamellar structure liposomes with an average diameter in micrometers [42]. Thus, resizing liposomes to less than 200 nm is required to improve the surface area to volume ratio for superior encapsulation and drug loading efficiency. Improving the size distribution of the prepared liposomes, extrusion, sonication (probe or bath) and freeze–thaw cycling have been used for liposomes size reduction [59].

**Figure 3.** Liposomes preparation via thin-film hydration extrusion technique [57]. **Figure 3.** Liposomes preparation via thin-film hydration extrusion technique [57].

Various parameters can be optimized to achieve a uniform multilamellar thin film followed by proper size reduction [43]. Rota-evaporator temperature, rotation speed and Various parameters can be optimized to achieve a uniform multilamellar thin film followed by proper size reduction [43]. Rota-evaporator temperature, rotation speed and gradual pressure reduction, in addition to membrane pore size, can result in unilamellar, monodispersed liposomes with high encapsulation efficiency [33,60].

#### 3.2.3. Average Particle Size and Nanoparticles Distribution

Average particle size and nanoparticle distribution are considered the main CQAs for all nano-formulations [61]. These parameters play major roles in determining the nanoparticle in vivo distribution, drug loading ability, drug release and targeting capacity [62]. For better biodistribution, the ideal nanocarrier particle size should be in the range of 10 to 100 nm to avoid kidney elimination, escape the reticuloendothelial system (RES) and provide an effective enhanced permeability and retention (EPR) effect [63,64]. A small particle size means a high surface area to volume ratio. This leads to fast drug release due to more drugs close to the surface of the nanoparticles compared to larger ones [65]. However, it is important to keep in mind that for inhaled drug particles to be therapeutically useful, they should be smaller than 2 µm, which is most suitable for deposition in the alveolar [66]. Moreover, liposome delivery through the skin is dependent on size. Liposomes up to 600 nm penetrate through the skin easily, whereas liposomes larger than 1000 nm remain interiorized in the stratum corneum [67]. The polydispersity index (PDI) reflects the homogeneity and size distribution of the nano-dispersions. PDI values of less than 0.3 indicate homogeneous, stable and well-dispersed liposomes [68]. Generally, increasing lipids concentrations can lead to increased liposomal size and PDI values simultaneously [69].

#### 3.2.4. Zeta Potential (ZP)

ZP evaluates the nano-dispersion stability. Neutral nanoparticles have decreased stability and tend to aggregate [70]. A charge greater than +30 or less than −30 mV indicates good stability due to the high electrostatic repulsions [71]. The ZP of the nanosystem affects their systemic circulation, interactions with body tissues and cell recognition. For example, the cellular uptake of cationic liposomes is higher compared than anionic liposomes due to the negatively charged cell membrane [72]. Moreover, charged liposomes

can exhibit a high encapsulation efficiency for drugs with opposite charges [73]. In order to control the ZP values to achieve maximum stability, fatty acids and hydrophilic polymers of varying change can be incorporated into the liposome formulations [40].

#### 3.2.5. Drug Content

Liposomal drug content can be expressed in three ways: weight per volume (*w*/*v*); percentage encapsulation efficiency (EE%, weight of drug entrapped into the liposomes compared to the initial amount of drug used %); and drug loading (DL%, the amount of drug entrapped into the liposomes relative to the initial mass of the lipid used; drug-to-lipid ratio) [62,74]. Improved EE% preserves high concentrations of the precious pharmaceutical agent in liposomes and may reduce the manufacturing cost, thus resulting in enhanced pharmacokinetics and improved patient compliance [75].

Several parameters may influence the drug EE, such as the lipid-to-drug ratio, nature of phospholipids, cholesterol molar ratio and the manufacturing process parameters [76,77]. Increasing the lipid-to-drug ratio leads to an increase in the number of nano-vesicles that are able to entrap more hydrophilic drugs in their aqueous cores [78]. Cholesterol and unsaturated lipids create more pockets within the lipid bilayer, thereby entrapping more hydrophobic drugs [79,80]. Freeze–thaw resizing cycles have also been proven to enhance the EE [81]. Moreover, remote loading approaches into preformed liposomes have been able to raise the EE of ionizable drugs compared to conventional passive loading [82,83].

#### 3.2.6. In Vivo Stability

The hydrophobic/hydrophilic characteristics of the liposomes surface affect liposome interaction with blood components [84]. These interactions are responsible for the in vivo stability of liposomes. Liposomal in vivo stability causes prolonged drug release and enhanced drug localization in the targeted tissue [42]. For example, hydrophobic nanoparticles are easily cleared from blood circulation due to their high ability to bind blood proteins [38]. Moreover, stealth liposomes, usually coated with hydrophilic polymers, show higher in vivo stability with prolonged circulation time that leads to improved therapeutic potential of the encapsulated drug [70].

### 3.2.7. Drug Release Kinetics

The kinetics of releasing drugs from liposomes is a critical parameter for liposome formulation design and considered a key factor to accomplish optimal efficacy and to minimize drug toxicity [85]. The optimal therapeutic activity of the drug can be achieved when the whole drug delivery system enters the target cells via endocytosis or the drug is released at the proper rate at the site of action for enough time [86]. Furthermore, the liposomes surface can be functionalized with targeting ligands for active drug targeting [87]. These targeting ligands can selectively bind to certain receptors or biomarkers that are overexpressed on cancerous or diseased tissues. These ligands could be antibodies, peptides, oligonucleotides, small carbohydrates, or small organic molecules [88].

Triggered drug release from liposomes could be achieved by incorporating sensitive excipients within liposome structures [89]. These excipients produce a liposomal destabilizing effect upon exposure to specific stimuli, such as light, temperature, radiation or different pH [90,91].

#### *3.3. Product and Process Design Space*

For the effective implementation of QbD in liposomal formulation, QTPP should be first defined, then the formulae and manufacturing processes can be selected and designed to ensure achievement of the pre-defined QTPP. Identification of CQAs and CPPs is achieved by an experimental design that is capable of assessing their contribution to the CQAs [62].

DS is performed to assure a high-quality product through demonstrating a range of process and/or formulation parameters [62,75]. DS involves the product and process DS. The product DS is established with the products CQAs as scopes, while the process DS is presented as CQAs related to CPPs [92].

The DS for liposome preparation is established by understanding and controlling the formulations, materials and manufacturing variables. Alina et al. established a DS for lyophilized liposomes with the drug simvastatin [32]. Their DS approach was based on both formulation factors and CPPs. Their results showed that cholesterol molar ratio, the PEG proportion, the cryoprotectant to phospholipids amounts and the number of extrusion cycles were designated as the most significant factors for lyophilized liposome CQAs [32]. These parameters were proven to directly affect the QTPP, including proper particle size, high drug entrapment, proper lyophilization process and minimum changes in phospholipid transition temperature. This DS approach was validated and considered a valuable approach for designing stable high-quality lyophilized liposomes [32].

This DS methodology was also applied to the prednisolone-loaded long-circulating liposomes using the thin-film hydration-extrusion method. The selected formulation parameters were drug concentration and PEG ratio in the bilayer membrane, and the process parameters were the number of extrusion cycles, temperature and rotation speed [33]. The same DS strategy was used to encapsulate tenofovir into liposomes with high EE [62]. Pandey et al. established a DS for chitosan-coated nanoliposomes using the ethanol injection method as a function of drug and chitosan concentration, and the organic phase-to-aqueous phase ratio to achieve the best design, in terms of average particle size, EE and coating efficiency [60].

Several factors may affect CQAs in the DS strategy. For example, the co-encapsulation of two drugs in the same liposome expands the studied attributes that are related to both drugs which are usually independent of each other. These variations may not lead to enhanced product quality [37]. Moreover, liposome drying process parameters are considered major CQAs that should be involved in the DS process study to obtain longterm stable liposomes [93]. Drying steps, such as pre-freezing, lyophilization and/or spray drying or even the type and ratio of the used cryoprotectants should be managed to reach a high drug content after lyophilization, maintaining the same particle size and ZP with minimal moisture content [94]. For example, the DS for the freeze-drying process of pravastatin-loaded long-circulating liposomes was developed as a function of the freezing rate and the shelf temperature during the initial drying. The two processing factors were found to have a great influence on the product's CQAs [34].

#### *3.4. The Control Strategy*

Although liposomes have been shown to have many advantages as a stable and effective drug delivery system, they present many challenges in analytical and bioanalytical characterization due to their distinctive preparation processes and complex physicochemical properties. According to the FDA guidelines, numerous critical quality attributes (CQAs) have been reported that need full characterization for liposome drug products (Table 3).


**Table 3.** Critical quality attributes (CQAs) needed for full liposome drug product characterization.

#### 3.4.1. Lipid Content Identification and Quantification

The quality of the ultimate product is affected by the source of lipids and also by the nature of the lipids: synthetic, semi-synthetic or natural. Phospholipids are the major lipid component of liposome formulations. These lipids can be identified by nuclear magnetic resonance (NMR). <sup>31</sup>P-NMR can differentiate phospholipid types according to their unique <sup>31</sup>P shifts [95]. <sup>1</sup>H- and <sup>13</sup>C-NMR can also be used to clarify the molecular chemical structures of alkyl chains and lipid polar head groups. NMR analysis usually requires expensive instruments [96]. Liquid chromatography (LC) coupled with mass spectrometry (MS) is widely used for lipid identification and profiling [136]. MS is a powerful tool to determine the molecular mass of lipids especially when soft ionization approaches such as electrospray ionization (ESI) MS are used [137]. Raman spectroscopy can be used to characterize the vibrational modes of the lipid carbon skeleton. They are characterized by the C-C backbone vibrations (1000−1150 cm−<sup>1</sup> ) and C-H stretching (2800−2900 cm−<sup>1</sup> ) [138].

Liquid chromatography techniques have been widely applied in quantitative lipid analysis [139]. First, liposomes should be disrupted using organic solvents followed by chromatographic separation; then, lipids can be sensed and quantified by different detectors, including diode array ultraviolet (UV), refractive index (RI) [97], evaporative light scattering detector (ELSD) [98] and charged aerosol detector (CAD) [99]. Singh et al. quantified the phospholipids and cholesterol from six different liposomal preparations using isocratic, reversed-phase liquid chromatography (RP-HPLC) with UV and ELSD detectors [100].

Gas chromatography (GC) has also been applied for lipid analysis [102]. Lipid fatty acids should be first converted into volatile methyl esters prior to GC analysis [140]. Recently, supercritical fluid chromatography (SFC) has also been used for lipid analysis [103,141].

Many colorimetric assays have been stated to evaluate phospholipids. A blue-color is produced when reacting phosphorus with molybdate. Diphenylhexatriene (DPH) is usually used to identify bilayer membranes. Moreover, DPH fluorescence-based detection has improved the phospholipid concentration detection limits [101]. Additionally, several commercial kits have been designed to quantify unsaturated phospholipids based on the sulfo-phospho-vanillin reaction [142] or based on enzymatic assay [143,144].

#### 3.4.2. Quantification of Drug Encapsulation

Liposomes provide lipid bilayers and an aqueous core to entrap hydrophobic and/or hydrophilic drugs, respectively. To evaluate the drug encapsulation, the unloaded drug is first removed from the nanocarriers through ultrafiltration, ultracentrifugation, dialysis or solid-phase extraction. The loaded or unloaded drug amount can then be quantified with respect to the total drug amount, yielding the percent drug encapsulation [99].

RP-HPLC has shown high efficiency for both the separation and quantification of free drugs and drug-loaded liposomes [104]. RP-HPLC connected to a UV-detector has been used for fast quantification of doxorubicin-loaded into Doxil® with a linear correlation [105,106]. Capillary electrophoresis (CE) has also been used to separate loaded drugs into liposomes of different change [107]. Oxaliplatin-loaded, anionic PEGylated liposomes have been purified from unloaded oxaliplatin and calculated for EE using a CE-UV detector [108]. Moreover, cisplatin has also been analyzed from loaded liposomes using CE connected to inductively coupled plasma mass spectrometry (ICP-MS) [109]. Flow-based field-flow fractionation (FFF) has been developed to overcome the restrictions of traditional chromatography [110,111]. Size exclusion chromatography (SEC) has also been used to separate unloaded drugs from drug-loaded liposomes based on their size differences [112].

#### 3.4.3. Liposomes Size and Morphology Characterization

Direct particle size and morphology can be evaluated by electron microscopy, such as scanning or transition electron microscopy (SEM and TEM, respectively) [113]. Cryogenic TEM (Cryo-TEM) does not require a drying process because it solidifies the aqueous sample by rapid freezing and thus drying-related artifacts are minimal. Cryo-TEM has been developed to provide high-resolution morphology and comprehensive structural information about the lipid layers and encapsulation mechanisms (Figure 4) [114,145]. SEM can penetrate the particle surfaces and is not commonly used for liposomal imaging due to the destructive manner of sample preparation. In addition, atomic fluoresce microscopy (AFM) has also been used to explore the three-dimensional structure of liposomes [115]. *Molecules* **2023**, *28*, x FOR PEER REVIEW 12 of 20

**Figure 4.** High-resolution Cryo-TEM images of liposomes [146]. **Figure 4.** High-resolution Cryo-TEM images of liposomes [146].

Liposome lamellarity can be evaluated by 31P-NMR [116]. Phospholipids in unilamellar liposomes can be characterized by a narrow-line spectrum, whereas multilamellar liposomes displayed wider peaks due to the restricted anisotropic molecular motions within multiple lipid layers [117]. Liposome lamellarity can be evaluated by <sup>31</sup>P-NMR [116]. Phospholipids in unilamellar liposomes can be characterized by a narrow-line spectrum, whereas multilamellar liposomes displayed wider peaks due to the restricted anisotropic molecular motions within multiple lipid layers [117].

Dynamic light scattering (DLS) has been applied to characterize nanoparticle size distribution. DLS has become the conventional strategy for the simple quantitative analysis of nanoparticle size distributions [118]. DLS measures time-dependent fluctuations in the scattered light from particles in Brownian motions. Variable sample parameters for DLS measurements include temperature, solvent viscosity and solvent refractive index, should all be pre-determined to precisely estimate the hydrodynamic particle size [119]. Dynamic light scattering (DLS) has been applied to characterize nanoparticle size distribution. DLS has become the conventional strategy for the simple quantitative analysis of nanoparticle size distributions [118]. DLS measures time-dependent fluctuations in the scattered light from particles in Brownian motions. Variable sample parameters for DLS measurements include temperature, solvent viscosity and solvent refractive index, should all be pre-determined to precisely estimate the hydrodynamic particle size [119].

tor is most often expressed by the ZP [120,121]. It is an important physicochemical property that is responsible for the strength of liposome interactions, adsorption and therefore nanoparticle stability. ZP can be determined from the electrophoretic mobility of particles measured by the phase analysis light scattering (PALS) or electrophoretic light scattering (ELS) technique [122]. Significant medium properties including the phase nature, refractive index, and viscosity, as well as temperature, all have to be pre-determined to obtain exact measurements. ZP values outside ±30 mV maintain sufficient stable nanosuspensions [123]. The surface potential of liposomes can also be determined by several techniques including fluorescent labeling [124], electron paramagnetic resonance [125] and the

The physical and chemical stability of liposome formulations should be examined to meet the criteria for high product quality [147]. Spectroscopic methods and DLS measurements provide simple tests to measure liposome fusion and aggregation, respectively, while liposome disruption can be determined by chromatographic methods equipped with suitable detectors [42]. Liposomal fusion has been examined mainly using differential scanning calorimetry (DSC) and fluorescence-based lipid mixing assays [127]. Liposome aggregation can be envisaged by microscopic techniques and quantified by UV–Vis spectroscopy or DLS [128]. Lipid degradation rates can be affected by lipid composition, storage temperature, buffers and pH. The precursor lipid classes and their hydrolyzed derivatives can be separated and measured by several chromatographic approaches [129].

Several in vitro release testing methods to predict the in vivo behaviors of liposome formulations have been developed [130]. These methods can be classified into sampling and separate (SS), dialysis membrane (DM) and continuous flow (CF) [131,132]. The SS

second harmonic generation from optical analyses [126].

3.4.5. Physical and Chemical Stability

3.4.6. In Vitro Drug Release

3.4.4. Nanoparticle Surface Charge (Zeta Potential, ZP)

#### 3.4.4. Nanoparticle Surface Charge (Zeta Potential, ZP)

Liposomal surface charges are usually reflected by the polar head groups of the phospholipids, tertiary amines or negatively charged carboxylate functional groups. This factor is most often expressed by the ZP [120,121]. It is an important physicochemical property that is responsible for the strength of liposome interactions, adsorption and therefore nanoparticle stability. ZP can be determined from the electrophoretic mobility of particles measured by the phase analysis light scattering (PALS) or electrophoretic light scattering (ELS) technique [122]. Significant medium properties including the phase nature, refractive index, and viscosity, as well as temperature, all have to be pre-determined to obtain exact measurements. ZP values outside ±30 mV maintain sufficient stable nanosuspensions [123]. The surface potential of liposomes can also be determined by several techniques including fluorescent labeling [124], electron paramagnetic resonance [125] and the second harmonic generation from optical analyses [126].

#### 3.4.5. Physical and Chemical Stability

The physical and chemical stability of liposome formulations should be examined to meet the criteria for high product quality [147]. Spectroscopic methods and DLS measurements provide simple tests to measure liposome fusion and aggregation, respectively, while liposome disruption can be determined by chromatographic methods equipped with suitable detectors [42]. Liposomal fusion has been examined mainly using differential scanning calorimetry (DSC) and fluorescence-based lipid mixing assays [127]. Liposome aggregation can be envisaged by microscopic techniques and quantified by UV–Vis spectroscopy or DLS [128]. Lipid degradation rates can be affected by lipid composition, storage temperature, buffers and pH. The precursor lipid classes and their hydrolyzed derivatives can be separated and measured by several chromatographic approaches [129].

#### 3.4.6. In Vitro Drug Release

Several in vitro release testing methods to predict the in vivo behaviors of liposome formulations have been developed [130]. These methods can be classified into sampling and separate (SS), dialysis membrane (DM) and continuous flow (CF) [131,132]. The SS method involves incubating the samples in the release media, sampling and separating the released drug from integral liposomes, usually by stand-alone ultracentrifugation or filtration, followed by drug quantification [42,133]. Low-efficiency ultracentrifugation or filtration separation process for submicron nanoparticles has been observed upon using this method. DM is more common for studying the in vitro drug release of most nanoformulations. DM approaches mainly include dialysis sac (regular or tube dialysis) and reverse dialysis [134]. The dialysis sac keeps nano-formulations inside, attaining simultaneous release and separation, and then quantifying the released drug. Key factors for this approach include the type and cut-off of the dialysis membrane, volume ratios between the sample and release solvent, and mixing procedures [135].

#### 3.4.7. Liposomes Safety and Toxicity

The fact that liposomes are biocompatible, biodegradable and relatively easy to fabricate have led to an exponential increase in their use [148]. However, liposomes as a vehicle for drugs might be vulnerable to safety issues related to their lipid type, charge and concentrations. One of the most toxic effects of liposomes is the activation of the immune system of the patient that leads to drug sequestering in the mononuclear phagocytic system which might influence the function of the liver and spleen [149]. Therefore, strategies to improve the safety should be developed in the early stages of product design. Many strategies to improve drug safety and decrease the toxicity of the nanocarriers have been developed, such as increasing the encapsulation efficiency of drug into liposomes to decrease the lipid concentration needed to give the patient the recommended therapeutic dose [149]. The liposomes particle size, morphology, lipid content, charge, polydispersity and cholesterol content are key factors in toxicity. Consequently, precise design of all these factors will

increase the loading capacity of liposomes and decrease the toxicity [150]. Recently approved were the PEGylated and surface-engineered liposomes having a lesser effect on the immune system. The combination of lipids with polymers should be designed and optimized. Therefore, the type of materials used for liposomal functionalization and their concentration should be minimized [148,151].

Finally, as the risk assessment is the backbone of the QbD process connecting all the key elements together, the liposomal biocompatibility and toxicity should be assessed using in vitro cell lines, ex vivo and in animals [152]. Many in vitro approaches have been used to test nanoparticle toxicity, including liposomes such as two-dimensional monolayer cell culture [153] and three-dimensional cell culture [154]. Additionally, ex vivo models are valuable tests systems in which slices of complete tissue can be used similar to organ slice cultures [155]. Finally, the most relevant evaluation is in vivo [156]. In conclusion, to minimize liposomal toxicity, it is important to start with the safety by design approach to ensure a low toxicity and voluble drug delivery system.

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

The application of QbD in pharmaceutical manufacturing has become an essential approach for the pharmaceutical industry to ensure the efficacy and safety of pharmaceutical products. The implementation of commercial nanomedicines as drug delivery systems to the site of action with limited systemic toxicities is an emerging concept that unfortunately, has not reached its full potential yet. Nano-pharmaceuticals are still in the initial stages of their development. Therefore, the implementation of QbD could create great value and benefits. Particularly, nano-pharmaceuticals is faced with many challenges related to structural stability and the lack of in-depth understanding of the manufacturing processes.

Liposomes are biocompatible and biodegradable drug delivery systems that have shown important successes in their clinical use. However, there are a lot of regulatory and technical challenges connected with the production and quality control strategies of liposomal products. There is a wide range of variability in liposomal preparations that include their morphology, size, fabricating materials, spatial configuration and manufacturing methods. Consequently, the application of a QbD approach in developing liposomes is critical and challenging compared to traditional dosage forms. Therefore, for the successful development of quality liposomal products, manufacturers need to consider employing QbD to identify and classify product attributes as well as material/process parameters with a deeper understanding of their complex interplay using proper experimental design and statistical analysis. QbD implementation is vital to ensure the final product attributes and the intended therapeutic and safety profiles.

**Author Contributions:** Conceptualization, W.A. and A.M.A.; writing—original draft preparation, W.A., H.N., Z.L., O.M.H., A.A.-K. and E.E.; writing—review and editing, W.A. and A.M.A. All authors have read and agreed to the published version of the manuscript.

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

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

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

#### **References**


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