Development of a Novel Red Clay-Based Drug Delivery Carrier to Improve the Therapeutic Efficacy of Acyclovir in the Treatment of Skin Cancer

1. Introduction

Cancer is a group of complex diseases and one of the major causes of death worldwide. Among the several types of cancers, skin cancer specifically refers to the abnormal growth of skin cells. It commonly develops in areas where the skin is exposed to UV radiation, such as the scalp, face, ears, neck, arms and hands. According to the GLOBOCAN report, around 0.32 million individuals were diagnosed with skin cancer worldwide in 2020, with the highest overall rate of skin melanoma being reported in the New Zealand and Australian populations [1]. In India, the north and northeastern regions have been reported to show the highest incidences for both males and females [2]. Various treatment strategies are employed to treat melanoma, including surgery, photodynamic therapy, immunotherapy, virotherapy, targeted therapy, drug therapy, radiation therapy and chemotherapy. The drugs are generally applied as topical ointments for skin cancers. However, the current drugs used for the treatment of skin cancer lack efficacy as a result of toxicity and drug resistance. Therefore, it is crucial to find a suitable candidate that is more site-specific and less toxic to improve treatment outcomes.

The US FDA is increasingly focusing on drug repurposing due to the high risk, cost and slow pace of the development of new drugs. Recently, in the COVID-19 pandemic, many existing drugs were repurposed and tested for their efficacy in SARS-CoV-2 infection [3]. Researchers are now exploring the use of antiviral drugs to treat various cancers, as they have shown potential in suppressing cancer cell proliferation [4]. Acyclovir (ACV), an anti-viral drug commonly used to treat oral sores caused by the herpes virus [5], is being repurposed to treat skin cancer. Shaimerdenova et al. reported that ACV decreases the proliferation of cancer cells and upregulates the apoptotic pathway via cytokine caspase-3 [6]. Although ACV has various biological effects and is marketed in various dosage forms, it has a very low bioavailability (15–30%). It is classified as a BCS class III drug and has a poor permeability, which is attributed to its low bioavailability and slightly water-soluble nature [7,8]. Its existence in six different forms (four anhydrous and two hydrous) further limits its aqueous solubility. Hence, it is of paramount importance to develop a drug delivery system that can enhance the poor physicochemical properties of ACV and similar active pharmaceutical ingredients (APIs) to improve its therapeutic efficiency.

Among the various drug delivery systems, clay-based drug delivery systems have gained the attention of researchers due to the excellent properties exhibited by clay minerals, such as a low density, high porosity, large surface area, biocompatibility, etc. In particular, nanoclays and their composites with other polymers have been shown to have versatile applications in novel drug delivery systems. These composites offer a range of advantages, such as a high payload, extended stability, stimulus-responsive drug release and enhanced biodegradation. Due to the various interaction patterns between clay and API, like hydrogen bonding, cationic exchange, electrostatic interaction and hydrophobic affinity, nanoclay-based drug delivery systems offer high drug loading. Among the different types of clay, the cationic clay montmorillonite has a higher adsorption capacity than illite and kaolinite due to its specific surface area, basal interlayer spacing and greater cation exchange capacity [9]. Red clay (RC), a natural material rich in minerals, like iron, aluminum and silica [10], has not been extensively explored in drug delivery systems. However, it has been used for centuries for medicinal purposes due to its antibacterial, antifungal and anti-inflammatory properties [11]. Other clays have been well studied in treating or managing many diseases, including diabetes, colitis, inflammation, cancers, etc. Their application in melanoma has been extensively researched, and hence this study aimed to explore the anticancer activity of a red clay–sucrose stearate-based complex.

Moreover, among many natural hydrophilic polymers, chitosan, a polycation, forms bonds with anionic clay through electrostatic interactions [12], enabling high payloads of APIs, like ACV. The amino groups in chitosan can be exploited for a controlled drug release and mucoadhesion [13], allowing it to deliver drugs at the skin’s pH [14]. The complexation between RC, SS and ACV with chitosan was analyzed using in silico methods, and the results are reported here. Therefore, this study focused on formulating a novel combination of ACV, SS and RC that is more effective than ACV alone against melanoma.

2. Materials and Methods

2.1. Materials

RC was purchased online (Amazon, India). ACV with 98% purity, SS with 97% purity and all other solvents of an analytical grade were purchased from Sigma Aldrich, Bengaluru, India.

2.2. Methodology

2.3. Statistical Analysis

The values are expressed as mean ± SD. The statistical comparisons were performed with a one-way analysis of variance (ANOVA) followed by a Duncan’s Multiple Range Test (DMRT), using SPSS version 12.0 for Windows (SPSS Inc. Chicago, IL, USA; http://www.spss.com (accessed on 30 May 2023). The values were considered statistically significant if the p-value was less than 0.05.

3. Results

Clay molecules have been extensively used for various purposes in pharmaceutical industries due to their versatile functions. For the first time, red clay was analyzed for its drug carrier property and the results are reported here.

3.1. Characterization of the Formulation

The particle size and zeta potential analysis of RC particles showed a mean hydrodynamic diameter of 938.0 ± 97.5 nm and zeta potential of −19.0 ± 7.6 mV, respectively . The negative charge may be attributed to the presence of higher hydroxides present in the red clay. The diameter was reduced to 426.0 ± 31 nm in nanocomplex F on the addition of SS and ACV to the suspension. Moreover, the nanocomplex suspension showed a zeta potential of −22.2 ± 5.1 mV followed by the addition of negatively charged SS and ACV. The % encapsulation efficiency, % drug loading and % yield of nanocomplex F1 were found to be 98.3 ± 6.6%, 31.10 ± 2.7% and 59.0 ± 8.2%, respectively.

The FTIR results of the RC samples shown in Figure 1C revealed multiple peaks confirming clay minerals. The stretching at 912 cm⁻¹ indicates the presence of Al-OH and Fe-OH; in addition, the existence of Al-Si-O was confirmed by the peak at 460 cm⁻¹. The stretching at 1092 cm⁻¹ and 692 cm⁻¹ corresponds to the Si-O bond. The band at 3395 cm⁻¹ showing a broad and low intensity may be due to the presence of water traces. In the FT-IR spectrum of ACV (Figure 1A), peaks at 3520 cm⁻¹ were attributed to –OH stretching vibration. Peaks at 1631 and 3181 cm⁻¹ were due to C=O stretching and -NH₂, respectively. The C=N and C–N stretching vibrations were confirmed by the peaks at 1484 and 1182 cm⁻¹, respectively. In the case of sucrose stearate, peaks at 3468 cm⁻¹ and 1739 cm⁻¹ were assigned to –OH stretching due to valence vibrations of the ester (νC=O), respectively (Figure 1B). The FT-IR of nanocomplex F1 (Figure 1D) was similar to the FT-IR spectrum of red clay and did not show any peaks of ACV. This confirms the effective encapsulation of ACV within the clay complex.

Figure 1. FTIR spectra of ACV (A), SS (B), RC (C) and nanocomplex F1 (D).

In Figure 2A, the XRD of ACV shows a 2θ value at 8.5, 11.6, 14.56, 19.7, 22.0, 23.4, 28.74, 34.2, 40.0 and 49.05°. The RC showed peaks at 6, 27.5, 30.2, 35.5 and 37° (Figure 2B). SS revealed a peak at 20° corresponding to the peak of stearate (Figure 2C). The nanoformulation showed peaks similar to that of red clay, confirming the effective encapsulation of ACV within the RC-SS carrier. The decreased peak intensity indicated the addition of ACV on the interlayers of the RC-SS complex (Figure 2). Moreover, it provides evidence for the incorporation of drug molecules into RC through an ion exchange mechanism.

Figure 2. XRD pattern of ACV (A), RC (B), SS (C) and nanocomplex F1 (D).

Figure 3 shows the thermal behavior of ACV, RC, SS and nanocomplex F1. The thermogram of ACV revealed an endothermic peak at 253.30 °C followed by an exothermic peak proximately, which confirmed the initiation of thermal degradation at 295.34 °C with a weight loss of 31.10%. The thermogram of RC indicated an endothermic peak at 520.36 °C corresponding to dehydration with a weight loss of about 8.47% and no degradation peak was evidenced. The sucrose stearate thermogram was characterized by an endothermic peak at 222.35 °C and degradation occurred at high temperatures with a weight loss of about 67.85%. The thermogram of nanocomplex F1 showed an exothermic peak at 368.16 °C, confirming the increase in the degradation temperature of the ACV-loaded RC-SS complex from 295.34 °C and its thermal stability. The presence of RC enhanced the thermal stability of nanocomplex F1 with a weight loss of only about 7.66%.

Figure 3. TGA overlay showing thermal degradation (weight loss (A) and heat flow (B)) of ACV, RC, SS and nanocomplex F1.

3.2. In Vitro Permeation Study of ACV-RC-SS Complex

The in vitro study was carried out at pH 5.5 to determine the rate of the drug release after permeation through the membrane using a Franz diffusion cell. The results are given in Figure 4. The nanoclay complex showed an immediate release of ACV up to 1 h followed by a sustained release. The release slowed down as the time increased and around 90% of the drug was released after permeation at the end of 120 min. This represents an enhanced permeation and release of ACV, a BCS class III drug. The drug release after permeation at acidic conditions provides a further conclusion that the release of the drug is due to ion exchange and due to alteration in the pH.

Figure 4. In vitro release profile of ACV from nanocomplex F1.

3.3. Drug Release Kinetics

The release kinetics of ACV release (i.e., the order and the mechanism of release) from nanocomplex F1 could be inferred with different kinetic models such as the Zero-order, First-order, Hixson–Crowell, Korsmeyer–Peppas and Higuchi’s model, as given in Figure 5. From the results, it is clear that the First-order plot exhibited a good linearity (R² = 0.9874) in comparison to the Zero-order plot (R² = 0.8785) and ACV release from nanocomplex F also showed the best fit with the Hixson–Crowell plot with a good linearity value (R² = 0.9834), supporting the dissolution of the carrier. The slope value of the Korsmeyer–Peppas plot is 0.7553, which is between 0.45 and 0.89, indicating that the drug release followed a non-fickian transport mechanism. Moreover, the Higuchi plot with a good linearity (R² = 0.9532) suggested that the drug release is owing to a diffusion mechanism. Hence, combined mechanisms of diffusion, dissolution and erosion were suggested for the ACV release.

Figure 5. Release kinetics of ACV from the nanocomplex (A) Zero-order, (B) First-order, (C) Hixson–Crowell, (D) Krosmeyer–Peppas, (E) Higuchi model.

3.4. Interaction Study with In Silico Docking

The forcite run results showed that the geometry of the molecule and the other components was optimized and minimized to the local minimum level. The initial total energy of ACV was found to be 6683.74 kcal/mol and it was further minimized to an energy of 34.59 kcal/mol. Similarly, other molecules were also minimized to form a stable complex. The energy graph showed that the lowest confirmation molecule energy was attained at the 100th step of the process (Figure 6).

Figure 6. Forcite geometric optimization of acyclovir using compass forcefield.

3.5. Cytotoxicity Study Using MTT

The MTT assay gave valuable insight into RC acting as a cytotoxic agent either alone or along with ACV for topical drug delivery. It not only acts as a carrier molecule but also aids ACV permeation to produce its action. The results were compared with doxorubicin (DOX) as a standard drug. Figure 12 displays the cytotoxicity of the free drug and its formulation against SK-MEL-3 cells. The IC₅₀ value of RC, ACV, RC+ACV and DOX was found to be 35, 20, 25 and 16 µg/mL, respectively.

3.6. Detection of Apoptosis with AO/EB Staining

Figure 13 displays the AO/EB staining of control SK-MEL-3 cells and cells treated with IC₅₀ concentrations of RC, ACV, RC+ACV and DOX. The yellow and orange color in the cells treated with ACV, RC+ACV and DOX confirmed that the cells endured apoptosis. Morphological changes like cell shrinkage, detachment, membrane blebbing and a distorted shape were observed in treated cells.

4. Discussion

ACV is a well-known antiviral medication used to treat herpes simplex virus infections. However, recent studies have sparked an interest in exploring its potential for repurposing against other viral infections and even certain non-viral diseases such as cancer. Studies recently reported have demonstrated the anticancer potential of ACV towards particular cancers, making it a potential candidate for treating some types of cancer like glioblastoma. Another essential aspect of ACV repurposing is that it is an approved drug with a well-documented safety profile. Hence, it could be brought into the market faster and at a lower cost than a new drug developed from scratch. Moreover, innovative drug delivery systems like clay-based nanodrug delivery systems can address the poor physicochemical properties of ACV, limiting its applications.

Red clay-based drug delivery systems have garnered considerable attention as a promising approach for treating skin cancer. Red clay, customarily considered to be aluminosilicates rich in iron oxides, can be used as a drug delivery system with exceptional properties, including a low toxicity, better biocompatibility, biomolecule adhesion, high surface area, good absorption capacity and high ion-exchange capacity, with wide pharmaceutical applications [9]. One recently published study investigated the potential of clay as a carrier for 5-fluorouracil (5-FU) for skin cancer. The researchers prepared an Alg-CS/5-FU/Mt nanocomposite by incorporating 5-FU into red clay and evaluated its physicochemical properties and drug release behavior [17]. The findings indicated that the red clay/5-FU nanocomposites exhibited a high drug loading capacity and sustained drug release profile, thereby improving the therapeutic efficacy while minimizing the side effects of 5-FU.

The particle size analysis of clay particles showed a mean hydrodynamic diameter of 938.0 ± 97.5. The reduction in size 426.0 ± 31 nm in the final formulation, followed by the addition of SS and ACV to the suspension, confirmed the interaction between sucrose stearate and the minerals present in the red clay—further confirmed by the in silico interaction studies. Zhang et al. reported that the intercalation of kaolinite using organic solvents reduces its thickness without changing its morphology [18]. The negative charge on the surface of the nanocomplex obtained from the zeta potential value may be attributed to higher hydroxides in the RC along with negatively charged SS. RC also has various other known absorption mechanisms on its surface due to the presence of interlayers and cation exchange mechanisms [19].

The peak at 3620 cm⁻¹ in FTIR indicates the presence of free OH groups in the sample. The presence of a distinct transmittance at 912 cm⁻¹ indicates that nanocomplex F1 did not interact with SS or ACV. The slight stretch at 2301 cm⁻¹ indicates the presence of SS on the RC surface. This leads to the further understanding that ACV binding could be in interlayers of the RC surface due to the ion exchange capacity of clay molecules. ACV is a positively charged amine group (when dissolved in dilute acid during formulation). When ACV comes into contact with clay minerals, the positively charged amine group on the drug molecule interacts with the negatively charged sites on the clay surface. This interaction leads to the exchange of ions between the clay and ACV. In the case of pure SS, a good emulsifier, the amorphous to the liquid crystalline state is attributed to the breakage of the intramolecular H-bond of sugar molecules at 222.35 °C. The liquid-to-isotropic conversion occurs at high temperatures. It helps in the solubility and permeation enhancement of ACV; hence, its poor physicochemical properties are improved by loading it into the drug delivery carrier. The nanocomplex thermogram showed a slight peak at ~280 °C and no peaks were seen at high temperatures corresponding to pure SS. This might be attributed to the role of SS in the entanglement of ACV within its crystalline liquid state and enhanced physicochemical properties of ACV [20,21].

The drug-loaded interlayer of clay is the reason for the effective encapsulation of ACV within clay nanocarriers and it is crucial for the success of clay-based drug delivery systems. The encapsulation efficiency and drug loading capacity of clay nanocarriers can be influenced by several factors, such as the type of clay used, the preparation method and the physicochemical properties of the drug. Here, the use of RC and its mineral composition is one of the key factors for a good encapsulation. Similar to our study, the anticancer drug methotrexate (MTX) was loaded within halloysite clay nanotubes (HNTs) using a solvent exchange method by Massaro et al., 2022 [22]. The researchers attributed the successful encapsulation of MTX within the HNTs to the unique structure and surface chemistry of the clay nanotubes, which allowed for an efficient drug adsorption and sustained drug release. But in our case, the release was found to be immediate and an enhanced solubility of ACV using clay and SS could be attributed to it. Moreover, in acidic conditions, the electrostatic interaction between a drug and clay is weakened, aiding in the release of the drug. At an acidic pH, the partial protonation of smectite particles may create a positive charge and reduce the surface charge of red clay and therefore the electrostatic interaction between the drug and nanoclay cleaves to aid the immediate drug release [23]. During the ion exchange process, the positively charged ions associated with the clay surface, such as sodium (Na+), calcium (Ca²⁺) or magnesium (Mg²⁺), are displaced by the ACV molecules (when exposed to an aqueous buffer, they attain a negative charge). The drug molecules bind to the clay surface through electrostatic interactions, resulting in the formation of a complex between the clay and acyclovir. The extent of ion exchange and the stability of the RC-ACV complex depend on various factors such as the pH, concentration, temperature and specific properties of RC and ACV. Hence, the exchange process is reversed, and ACV molecules are released from the clay surface under certain conditions for further action.

Clay-based drug delivery systems can provide an immediate drug release through various mechanisms, such as diffusion, swelling and erosion of the clay particles. Parallel to our study, one study investigated the immediate release of an anticancer drug, DOX, from a clay-based drug delivery system. The results showed that the DOX release from the MMT particles was controlled and reached approximately <30% within 24 h. The researchers attributed the controlled release of DOX to the layered PEG-CS/MMT sheets, which allowed the release through diffusion [24]. Furthermore, the drug release of the clay-based drug delivery system could be controlled by adjusting the concentration of the drug and the clay particles. An immediate release can be modulated to a sustained release by increasing the clay particle concentration. The reduced drug release rate indicates that the clay particles could serve as a sustained drug release platform.

When a drug is released from clay particles, it can penetrate cancer cells and exert its therapeutic effect. The drug release profile of clay-based drug delivery systems can improve the efficacy of treatment by maintaining the concentration of the drug at the tumor site. The mechanism of action of clay-based drug delivery systems in skin cancer involves several steps. First, the clay particles can penetrate the skin and reach the tumor site due to their small size and high surface area. Once they reach the site, the clay particles can adsorb or physically entrap the drug, protecting it from degradation and increasing its local concentration. In addition, the unique physicochemical properties of clay, such as its cation exchange capacity and ability to interact with proteins, can enhance the drug’s bioavailability and uptake by the cancer cells. One recently published study investigated clay nanoparticles as a potential carrier for curcumin, a natural compound with anticancer properties, for the treatment of melanoma skin cancer. The researchers prepared clay nanoparticles (Na-montmorillonite and palygorskite) loaded with curcumin that showed a good biocompatibility and were able to penetrate the skin to reach the tumor site. The researchers concluded that clay nanoparticles could be a promising platform for the delivery of anticancer drugs for the treatment of skin cancer [25].

The geometry of ACV and other components of the mixture was optimized using the COMPASS forcite protocol. Molecules at the lowest energy level (ground state) attain a stable configuration and thus form stable complexes [26]. The energy of ACV was brought down by modifying its torsional angle from 6683.74 kcal/mol to 34.59 kcal/mol in 100 iteration steps.

Clay minerals can interact with pharmaceutical organic compounds using several mechanisms. These molecular interactions are important as they can be used by formulation scientists to control the biopharmaceutical and pharmacological profile of a drug. These properties may help in increasing or decreasing the dissolution rate and immediate, delayed, extended or site-specific drug release, to reduce unwanted side effects, improve stability and for masking taste [27]. Molecular docking studies indicated that the formulated preparation was stable as the various components of RC (P₂O₅, SiO₂, Al₂O₃), the additive, the polymer and the drug ACV formed inter- and intra-molecular non-bonding interactions. RC mineral components produced intra-molecular interactions via multiple π-cationic interactions. ACV interacted with Al₂O₃ ions via hydrogen bonds between NH₂ and C=O groups in addition to van der Wall interactions (hydrophobic interactions) between the alkyl group and the neutral site on the clay. Kalonite with a composition of Al₂Si₂O₅(OH)_4, also forms hydrogen-bonding and van der Waals interactions [28]. RC through Si atoms was seen to form non-bonding interactions with additive SS, making the system colloidal. The complex is further stabilized by polymer chitosan, which can interact with both the drug as well as P₂O₅ and SiO₂ atoms of red clay.

The cytotoxic effects of the formulation on SK-MEL-3 cells were investigated with an MTT assay. The results showed a concentration-dependent reduction in the intensity of the purple color, confirming the dose-dependent decrease in cell viability, which is supported by previous reports. A previous report by Pajaniradje et al. (2014) supports our findings; they obtained a decrease in the intensity of a purple color produced as the result of the reduction in tetrazolium salts by mitochondrial enzymes [29]. ACV has an advantage over other drugs since the resistance is far less compared to other antiviral therapies for melanoma, and it produces lesser side effects. With additional red clay synergistic action, it could prove to be an effective option for the treatment of melanoma. To confirm the apoptosis, we stained the cells with AO/EB, after treatment with the IC₅₀ dose of the formulation. The results showed several cells with orange, fragmented nuclei, while the untreated cells were comparatively green in color. This indicates the apoptosis induced by the formulation. Live cells appear bright green, and early to late apoptotic cells appear with yellow, orange and red nuclei [16].

5. Conclusions

In conclusion, our study primarily focused on the development of a drug delivery strategy specifically tailored to utilize ACV in topical treatment. Considering that ACV is the preferred antiviral medication for the treatment of a herpes virus infection, which is a major contributor to primary skin cancer leading to melanoma, the current research findings described herein hold significant implications as they can contribute to enhancing the therapeutic potential of ACV and expanding its applications in cancer treatment. Moreover, ACV also exhibited minimal side effects when compared to conventional chemotherapeutic agents like DOX.

Clay-based nanodrug delivery systems for anticancer drugs hold great promise for enhancing patient outcomes and improving the overall management of skin cancer. Overall, our findings provide a solid foundation for future research and development efforts in the field of cancer therapeutics. It can be suggested that the clay-based nanoformulation of ACV could be a better alternative to currently used conventional chemotherapeutic agents because of its ability to mitigate resistance in cancer cells.

References

1. Skin Cancer Statistics. In WCRF International. Available online: https://www.wcrf.org/cancer-trends/skin-cancer-statistics/ (accessed on 4 May 2023).
2. Labani, S.; Asthana, S.; Rathore, K.; Sardana, K. Incidence of melanoma and nonmelanoma skin cancers in Indian and the global regions. J. Cancer Res. Ther. 2021, 17, 906–911. [Google Scholar] [CrossRef] [PubMed]
3. Zhan, P.; Yu, B.; Ouyang, L. Drug repurposing: An effective strategy to accelerate contemporary drug discovery. Drug Discov. Today 2022, 27, 1785–1788. [Google Scholar] [CrossRef] [PubMed]
4. Pfab, C.; Schnobrich, L.; Eldnasoury, S.; Gessner, A.; El-Najjar, N. Repurposing of Antimicrobial Agents for Cancer Therapy: What Do We Know? Cancers 2021, 13, 3193. [Google Scholar] [CrossRef]
5. Taylor, M.; Gerriets, V. Acyclovir; StatPearls Publishing: Treasure Island, FL, USA, 2023. [Google Scholar]
6. Shaimerdenova, M.; Karapina, O.; Mektepbayeva, D.; Alibek, K.; Akilbekova, D. The effects of antiviral treatment on breast cancer cell line. Infect. Agent Cancer 2017, 12, 18. [Google Scholar] [CrossRef] [PubMed]
7. Alqahtani, M.S.; Kazi, M.; Alsenaidy, M.A.; Ahmad, M.Z. Advances in Oral Drug Delivery. Front. Pharmacol. 2021, 12, 62. [Google Scholar] [CrossRef] [PubMed]
8. Papich, M.G.; Martinez, M.N. Applying Biopharmaceutical Classification System (BCS) Criteria to Predict Oral Absorption of Drugs in Dogs: Challenges and Pitfalls. AAPS J. 2015, 17, 948–964. [Google Scholar] [CrossRef]
9. Dong, J.; Cheng, Z.; Tan, S.; Zhu, Q. Clay nanoparticles as pharmaceutical carriers in drug delivery systems. Expert Opin. Drug Deliv. 2021, 18, 695–714. [Google Scholar] [CrossRef]
10. Rakhila, Y.; Ezzahi, A.; Elmchaouri, A.; Mestari, A. Synthesis and Characterization of a Red Clay Based New Composite Ceramic Material. Adv. Mater. Phys. Chem. 2018, 8, 295–310. [Google Scholar] [CrossRef]
11. Roy A French Red Clay Benefits—Skin Care Ingredients—Mirah Belle. In Magento2 Store. 2020. Available online: https://mirahbelle.com/blog/post/french-red-clay/# (accessed on 4 May 2023).
12. Aranaz, I.; Alcántara, A.R.; Civera, M.C.; Arias, C.; Elorza, B.; Caballero, A.H.; Acosta, N. Chitosan: An Overview of Its Properties and Applications. Polymers 2021, 13, 3256. [Google Scholar] [CrossRef]
13. Bernkop-Schnürch, A.; Dünnhaupt, S. Chitosan-based drug delivery systems. Eur. J. Pharm. Biopharm. 2012, 81, 463–469. [Google Scholar] [CrossRef]
14. Ojeda-Hernández, D.D.; Canales-Aguirre, A.A.; Matias-Guiu, J.; Gomez-Pinedo, U.; Mateos-Díaz, J.C. Potential of Chitosan and Its Derivatives for Biomedical Applications in the Central Nervous System. Front. Bioeng. Biotechnol. 2020, 8, 389. [Google Scholar] [CrossRef]
15. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55–63. [Google Scholar] [CrossRef]
16. Arjunan, A.; Pajaniradje, S.; Francis, A.P.; Subramanian, S.; Chandramohan, S.; Parthasarathi, D.; Sajith, A.M.; Padusha, M.S.A.; Mathur, P.P.; Rajagopalan, R. Epigenetic modulation and apoptotic induction by a novel imidazo-benzamide derivative in human lung adenocarcinoma cells. Daru 2021, 29, 377–387. [Google Scholar] [CrossRef]
17. Farshi Azhar, F.; Olad, A. A study on sustained release formulations for oral delivery of 5-fluorouracil based on alginate-chitosan/montmorillonite nanocomposite systems. Appl. Clay Sci. 2014, 101, 288–296. [Google Scholar] [CrossRef]
18. Zhang, Y.; Long, M.; Huang, P.; Yang, H.; Chang, S.; Hu, Y.; Tang, A.; Mao, L. Intercalated 2D nanoclay for emerging drug delivery in cancer therapy. Nano Res. 2017, 10, 2633–2643. [Google Scholar] [CrossRef]
19. Williams, L.B.; Haydel, S.E. Evaluation of the medicinal use of clay minerals as antibacterial agents. Int. Geol. Rev. 2010, 52, 745–770. [Google Scholar] [CrossRef]
20. Sun, Q.; Zhang, Y.; Zhang, Y.; He, H. Thermal effect on fluorine emission in coal and clay minerals. Environ. Earth Sci. 2017, 76, 579. [Google Scholar] [CrossRef]
21. Ng, S.P.; Khor, Y.P.; Lim, H.K.; Lai, O.M.; Wang, Y.; Wang, Y.; Tan, C.P. Improved thermal properties and flow behavior of palm olein-based diacylglycerol: Impact of sucrose stearate incorporation. Processes 2021, 9, 604. [Google Scholar] [CrossRef]
22. Massaro, M.; Poma, P.; Cavallaro, G.; García-Villén, F.; Lazzara, G.; Notarbartolo, M.; Muratore, N.; Sánchez-Espejo, R.; Iborra, C.V.; Riela, S. Prodrug based on halloysite delivery systems to improve the antitumor ability of methotrexate in leukemia cell lines. Colloids Surf. B Biointerfaces 2022, 213, 112385. [Google Scholar] [CrossRef]
23. Leal, D.A.; Kuznetsova, A.; Silva, G.M.; Tedim, J.; Wypych, F.; Marino, C.E.B. Layered materials as nanocontainers for active corrosion protection: A brief review. Appl. Clay Sci. 2022, 225, 106537. [Google Scholar] [CrossRef]
24. Huang, H.J.; Huang, S.Y.; Wang, T.H.; Lin, T.-Y.; Huang, N.-C.; Shih, O.; Jeng, U.-S.; Chu, C.-Y.; Chiang, W.-H. Clay nanosheets simultaneously intercalated and stabilized by PEGylated chitosan as drug delivery vehicles for cancer chemotherapy. Carbohydr. Polym. 2023, 302, 120390. [Google Scholar] [CrossRef] [PubMed]
25. Abduljauwad, S.N.; Ahmed, H.U.R.; Moy, V.T. Melanoma treatment via non-specific adhesion of cancer cells using charged nano-clays in pre-clinical studies. Sci. Rep. 2021, 11, 2737. [Google Scholar] [CrossRef] [PubMed]
26. Senthilvel, C.K.; Karuppaiyan, K.; Pothumani, A.; Vedharethinam, A.; Jose, A.W.; Mohamed, J.M.M.; El Sherbiny, M.; Ebrahim, H.A.; El Shafey, M.; Dejene, M. Development of Atorvastatin Calcium Biloaded Capsules for Oral Administration of Hypercholesterolemia. Evid.-Based Complement. Alternat. Med. 2022, 2022, 4995508. [Google Scholar] [CrossRef]
27. Aguzzi, C.; Cerezo, P.; Viseras, C.; Caramella, C. Use of clays as drug delivery systems: Possibilities and limitations. Appl. Clay Sci. 2007, 36, 22–36. [Google Scholar] [CrossRef]
28. Brigatti, M.F.; Galán, E.; Theng, B.K.G. Structure and Mineralogy of Clay Minerals. In Handbook of Clay Science Part A. Fundamentals, 2nd ed.; Bergaya, F., Lagaly, G., Eds.; Elsevier: Amsterdam, The Netherlands, 2013; pp. 21–81. [Google Scholar]
29. Pajaniradje, S.; Mohankumar, K.; Pamidimukkala, R.; Subramanian, S.; Rajagopalan, R. Antiproliferative and apoptotic effects of Sesbania grandiflora leaves in human cancer cells. BioMed Res. Int. 2014, 2014, 474953. [Google Scholar] [CrossRef] [PubMed]