Antiacne Gel Containing Aloe vera and Clindamycin Phosphate: Design, Characterization, and Optimization Using Response Surface Methodology

Clindamycin phosphate is a topical antibiotic agent used to treat acne vulgaris, while Aloe vera has both antimicrobial and anti-in ﬂ ammatory properties. The current study is aimed at formulating an antiacne gel with antioxidant and antimicrobial e ﬀ ects. The antiacne gels were prepared by using polymer HPMC K15M by cold dispersion method. Unveiling the intricacies of gel design, our research harnessed the power of Design Expert 11 to optimize critical parameters — viscosity, spreadability, and permeability. In vitro characterization tests, including pH, spreadability, viscosity, permeability, antimicrobial activity, antioxidant activity, and stability of the gels, were performed. The results of in vitro characterization tests showed that the gels had a mint-like odor, a pH of 6.8, and a spreadability of 21.5gcm/sec. The gels had a viscosity of 34.2Pas and drug content ranging within 90%-110%, as per USP standards. Notably, in vitro permeation assays reveal an exceptional 86% drug release, showcasing the e ﬃ cacy of our formulation. The uniqueness of our study lies not only in the robust optimization process but also in the multifaceted characterization. Our gel emerges as a promising candidate, exhibiting not only desired antimicrobial and antioxidant properties against acne vulgaris but also demonstrating stability under varied conditions. As we advance toward in vivo studies, our research paves the way for a nuanced understanding of the safety and e ﬃ cacy of this distinctive antiacne gel.


Introduction
Acne vulgaris is an ailment of the skin that involves oil glands situated at the base of hair follicles [1].A large per-centage of teenagers (up to eighty-five percent) suffer from chronic acne inflammation, which affects their social life and psychological health [2].Four processes can play a pivotal part in the onset of acne lesions: release of inflammatory mediators in the skin, keratinization process alteration, increased sebum production due to increased androgen receptor sensitivity, and follicular colonization induced by Cutibacterium acnes [3].Follicles containing sebum provide a lipid-rich and anaerobic environment for the Cutibacterium acnes to flourish, which directly augments lipogenesis.Different strains of Cutibacterium acnes have different resistance profiles and tend to colonize in pilosebaceous units leading to inflammation due to the activation of macrophages and keratinocyte receptors [4,5].
Topical treatments, including antibiotics, retinoids, azelaic acid, benzoyl peroxide, and a combination of these agents, are used effectively as the first choice in mild-tomoderate acne situations [6,7].Using multiple synthetic agents poses the risk of moderate-to-severe side effects such as erythema, skin peeling, photosensitivity, dryness, and eczematous irritation.Effective acne treatment is observed with clindamycin, tetracycline, and erythromycin [8,9].Clindamycin phosphate is a common topical antibacterial used in the treatment of acne in adults [10].Clindamycin belongs to the family of antibiotics called lincomycins.It is a 7-deoxy-7-chloro derivative of lincomycin antibiotic [11].It acts by the inhibition of chemotactic factors by Cutibacterium acnes as well as by the blockage of protein synthesis of the bacteria by the inhibitory action of the 50S ribosomal subunit [12].
The conventional treatments available are not well tolerated by many due to the risk of severe side effects.The use of herbal medicines and natural remedies dates a thousand years back.The increasing side effects, costs for treatment, and resistance to antimicrobial agents have led to an increasing interest in medicinal herbs [13].The benefits of Aloe vera (A.vera) have been increasing in recent years in both medicines and cosmetics [14,15].It has been reported to exhibit antibacterial, anti-inflammatory, and skin-healing activities [16,17].A. vera is a typical xerophyte that is promoted for a variety of skin conditions.Research has suggested that the various bioactive phytochemicals of the ethanolic extracts of A. vera impart antioxidant and healing properties to it [18].
In this study, we used clindamycin phosphate [19] and A. vera powder [20] as active ingredients for their antimicrobial and anti-inflammatory actions.HPMC K15M is used as a polymer to form the gel.Rheological tests showed that HPMC gel formulations showed a pseudoplastic behavior, a characteristic feature of topical gel [21].Oleic acid is used as a surfactant and permeation enhancer.Oleic acid-based surfactants showed an ability to self-aggregate into micelles and were readily biodegradable as compared to conventional surfactants [22][23][24].Eucalyptus oil was used as flavorant as well as penetration enhancer.Studies have shown that it is an essential penetration enhancer.It alters the permeability of certain drugs by the disruption of the lipid bilayer of the stratum corneum resulting in the modification of diffusivity via the stratum corneum [25].Glycerin was used as a plasticizer.It has been seen that glycerin assists in maintaining moisture within the gel structure thus preventing it from drying.This quality is also beneficial for the skin as when the gel is applied topically, the skin remains hydrated [26].
Propylene glycol is used as a solvent.Propylene glycol is a regular solvent that can enhance the solubility of active ingredients [27].Methylparaben and propylparaben were used as preservatives [28].
Our primary aim was to develop an innovative antiacne gel merging clindamycin phosphate and Aloe vera utilizing the cold dispersion method and Design Expert 11 for precise optimization.The secondary aim was to investigate distinctive in vitro characteristics, emphasizing exceptional drug permeation, to pave the way for subsequent in vivo studies exploring the safety and efficacy of the novel formulation.Mathematical modeling for variables and calculated responses was done by the Design Expert using the quadratic model.
The concentration of active ingredients and polymer was kept constant, while the surfactant (oleic acid), plasticizer (glycerin), and permeation enhancer (eucalyptus oil) were selected as variables (Table 1).

Formulation Method.
The formulations were made using the cold dispersion method of gel preparation.3% HPMC was soaked overnight in 100 ml distilled water.The active ingredients, 1% clindamycin phosphate and 2% A. vera, were added to the gel after dissolving in 7.5% propylene glycol.Oleic acid was dissolved in propylene glycol [29] and then, along with glycerin and eucalyptus oil, 5 International Journal of Polymer Science incorporated into the gel.0.01% propylparaben and 0.1% methylparaben were added as preservatives by dissolving in 10 ml distilled water [30].The prepared gels were stored in air-tight plastic containers.

pH Determination.
The pH of all 14 gel formulations was evaluated by a digital pH meter after calibration with standard buffers [32].
2.3.3.Spreadability.The spreadability of the gels was determined by the fixed slide method using the following formula: where M is the weight of the upper slide, L is the slide length, and T is the time taken for the separation of slides [33].Test samples (5 ml) were taken at set time intervals: 15 min, 30 min, 1 hr, and up to 8 hrs.The consolidated % drug permeation was determined after spectrophotometric evaluation [35].

Chemical Compatibility of Ingredients of Formulation.
FTIR was conducted by placing the sample on KBr discs and observing the wavelengths using IR spectroscopy [36].
2.3.9.Antimicrobial Activity.The cup plate method was used to determine the antimicrobial action of gels against cultures of Cutibacterium acne [38].The zones of inhibition were noted after a period of incubation of 48 hrs [39].

Results and Discussion
3.1.Organoleptic Evaluation.The topical antiacne gels had a whitish appearance and minty odor due to the addition of eucalyptus oil.The active ingredient, 1,8-cineole of eucalyptus oil, is responsible for imparting this sharp, minty, and camphorous odor [43].The white color of the gel was because of clindamycin phosphate, which is white in color [44].The consistency of the gel is one of the most critical features of anti-inflammatory and analgesic topical preparations [45].Topical gels prepared in the current study had a good consistency owing to the addition of plasticizer glycerin.2.
The predicted outcomes of all responses for the optimized formulation are stated in Table 3.

pH.
The pH of the formulations was found to be in the range of 6.1 to 7.2 (Figure 1), which is desirable because studies show that the pH of the topical formulations must be close to that of the skin as very low pH can cause skin irritation.Lambers et al. studied the pH effect on the adhesion of resident microflora on the skin and suggested that an acidic skin pH helps the bacterial flora to remain attached to the skin.In contrast, basic pH promotes their eradication from the skin [47].[48].This leads to more viscous preparation, thus inferring that glycerin can increase a gel's viscosity [49].Oleic acid can also impart a higher significant viscosity to the gelling systems by inducing network formation between the molecules [50,51].In the current study, the gels with a higher concentration of oleic acid and glycerin were found to have a higher viscosity.Studies have shown that the increased percentages of glycerin can increase the viscosity of the gels.In addition, with the addition of glycerin, the stability of gels also increases [52].Sant et al. studied the effects of oleic acid, a lipid penetration-enhancing agent, on the in vitro release of lumiracoxib from poloxamer-based gel systems.They reported that the addi-tion of oleic acid in the gel systems increased their viscosity significantly.This could be due to the reduction of water content in the gels due to the incorporation of oleic acid which being a viscous liquid contributes to this effect [50].
3.5.Spreadability.The spreadability laid between 16.07 and 25 g cm/sec (Figure 3).This range was found to be compatible with the studies performed by Bhalekar et al., who studied different gelling agents for clindamycin phosphate gel [33].
3.6.Drug Content.The drug contents of all gel formulations were in the specified range, i.e., 90%-110%, showing that the active content ranges of the gels followed the USP specifications.The IR spectrum of the formulated topical gels showed peaks at the following locations: at 3500 and 3700 cm −1 due to O-H stretching, 2800 to 3000 cm −1 indicating a strong N-H stretching, and between 2000 and 2400 cm −1 due to strong O≡C≡O stretching.The peak at 1500 cm −1 showed the presence of a strong N-O bond.The peak between 1566 and 1650 cm −1 indicated medium C≡C stretching.Peaks were also observed at 1650 cm −1 due to C=O stretching, at 1085 and 1150 cm −1 due to C-O aliphatic ether stretching, and at 885 to 895 cm −1 and at 665 to 730 cm −1 due to C=C bending (Figure 4).

3.8.
In Vitro Permeation Study.The release profiles of all fourteen formulations made using HPMC elicited about 80 to 90% release from the formulation within 8 hrs (Figures 5  and 6).The in vitro permeation was quite encouraging as an acceptable amount of drug diffused into the skin from the gels.Among the formulations, F10 showed better release characteristics compared to other formulations.This could be due to the higher amount of permeation enhancers, eucalyptus oil, and oleic acid.
A lot of studies show that when penetration enhancers are used in combination, they can cause synergistic action, which promotes permeation through the skin producing better results compared to a single penetration enhancer [53].Widely used penetration enhancers and vehicles known as cosolvents have been used in transdermal formulations.They not only increase the solubility of the drug, but they also change the skin structure and enhance the rate of penetration through the skin.So, they improve drug release and permeation as well [54].
Percutaneous absorption of the drug has been improved through the use of numerous fatty acids, such as oleic acid (OA).In this study, the use of a combination of OA plus eucalyptus oil manifests success in a lot of transdermal formulations [55].
Essential oils, along with their constituents, enhance penetration through the skin, and they are widely used in pharmaceutical products.Eucalyptus oil has been proven to be a very famous penetration enhancer in transdermal and dermal formulations [56].
3.9.Characterization of Optimized Gels.Table 4 shows the results obtained from characterization tests on the optimized formulation.
3.9.1.In Vitro Drug Permeation Study.The drug permeation through an optimized gel was found to be 86% (Figure 7).We also found that eucalyptus oil and oleic acid used in the formulation significantly enhanced the permeability of the active ingredients.Our findings were consistent with other studies; for instance, Herman A. and Herman A.P. reported that drug penetration through the skin barrier was proportional to the concentration of eucalyptus oil used in the formulation [57].Similarly, eucalyptol-a monoterpenoid that constitutes about 90% of eucalyptus oil-was also associated with increased drug permeation through the skin [58].In another study, Abd et al. reported that eucalyptol and oleic acid greatly enhanced the permeation of active ingredients [59].
3.9.2.Antioxidant Activity.The total antioxidant activity (TAA) of the gels (test formulations) was estimated by plotting a graph between percentage inhibition (%) and concentration (μg/ml), as shown in Figure 8, while the TTA of ascorbic acid (standard) is shown in Figure 9. Upon comparison, both the test formulations and standards followed a similar trend.A. vera-the primary active ingredient in the formulated gels-was responsible for the optimum antioxidant activity of the gels.
Furthermore, 2,2-diphenyl-1-picrylhydrazyl assays were also performed to confirm the antioxidant activity of the antiacne gels.The results of the 2,2-diphenyl-1-picrylhydrazyl assay confirmed that both standard (ascorbic acid) and gels (test formulations) significantly reduced the 2,2-diphenyl-1-picrylhydrazyl free radical in a concentrationdependent manner.Studies showed that nonflavonoid polyphenols in A. vera account for the majority of its total polyphenolic content, which is responsible for its antioxidant action [60].Our findings were consistent with another study that reported high antioxidant activity of A. vera gel against 2,2-diphenyl-1-picrylhydrazyl and ABTS (free radicals) in a concentration-dependent manner [61].Therefore, our study also suggests the potential applications of A. vera gels in treating various skin conditions [62].
3.9.3.Antimicrobial Activity.We tested the antimicrobial activity of a single ingredient A. vera (A) and compared it with a marketed clindamycin phosphate formulation (Clindacin) (Std) and a gel containing both A. vera and clindamycin phosphate (C).The zones of inhibition for A with singleingredient A. vera, C with clindamycin phosphate and A. vera, and Std (standard) were measured and are shown in Table 5.
Our findings suggested a significant inhibitory effect of the gels (test formulations) on the bacterial strain Cutibacterium acnes as shown in Figure 10.The inhibitory effect of single-ingredient A. vera gel (A) was comparable to that of the marketed clindamycin phosphate formulation (Std).The inhibitory effect of gels containing both A. vera and clindamycin phosphate (C) was estimated to be higher than the standard and single-ingredient A. vera gel (A).
Since the antimicrobial activity of active ingredients is crucial in the treatment of acne vulgaris [19], our study suggests the potential application of A. vera gels in the treatment of acne.Moreover, A. vera gels also contain specific polysaccharides and hydroxylated phenols such as pyrocatechol that exhibit antimicrobial properties [63].Studies indicate that the saponins, phenols, flavonoids, terpenes, and tannins present in A. vera impart antibacterial and anti-inflammatory properties to this plant [64].Moreover, the comparison of conventional treatments (antibiotics) with A. vera has shown that A. vera shows higher inhibitory effects against acne-causing bacteria [65].
3.9.4.Stability Study.We subjected the formulated gels to stability testing (45 °C/75% RH) to assess any changes in their characteristics after six months, and the results are shown in Table 6.The physical appearance (color, homogeneity, and texture) of all formulations was intact after week 24 (when compared to week 0) at the given conditions.The viscosity of each formulation was estimated to be between 30 Pa s and 40 Pa s, and we found no significant changes in their viscosities at week 24 (when compared to week 0).The prolonged stability of the gels can also be due to the antimicrobial action of paraben preservatives used in the formulation [66].Moreover, the antimicrobial and antioxidant properties of A. vera gels can also contribute to the prolonged shelf-life of these topical gels [67].
3.10.Response Surface Methodology.The different responses, including the permeation of active ingredients, the viscosity of gels, and the spreadability of the gels, were studied by response surface methodology.Contours and 3D graphs illustrating the effects of different variables, i.e., oleic acid, eucalyptus oil, and glycerin, were generated by Design Expert.
3.10.1.In Vitro Permeation of Active Ingredients.The contour and 3D graphs of the factors affecting the permeation of active ingredients clindamycin phosphate (Figure 11) and A. vera (Figure 12) indicated that both surfactant oleic acid and permeation enhanced; eucalyptus oil significantly increased the permeation of drugs from the topical gels.Glycerin also increased the permeation of drugs, but its effect was lower in comparison to other factors.Moreover, the comparison of the influence of surfactant and permeation enhancer on drug permeation revealed that the surfactant had a better impact on increasing permeability.
Anwar et al. [68] also reported that oleic acid acted as a permeation enhancer in transdermal drug delivery.About 75% of eucalyptus oil is made of 1,8-cineole.The cineole is a cyclic terpene that generates liquid pools in the stratum corneum and changes its lipid structure.So this facilitates the penetration of polar and nonpolar drugs [69].A study conducted in 2020 shows that adding glycerin can enhance drug penetration, explained through the value of flux.The value of flux increases with the increase in the concentration of glycerin.The mechanism of action of glycerin is that it hydrates the stratum corneum and increases the solubility of drugs.It can also increase the value of patches for hygroscopic moisture content [70].
Tables 7 and 8 show the analysis of variance of permeation of clindamycin phosphate and A. vera, respectively.3.10.2.Viscosity.The developed graphs showed the various effects of variables on the viscosity of the topical gels (Figure 13).They indicated that the viscosity of the gels was increased by factors of oleic acid and glycerin while decreased by eucalyptus oil.A broadly used nonaqueous hydrogen bonding solvent, glycerol, is mainly used due to its good physical properties, which makes it useful for many industrial formulations.A study conducted by Matthews et al. shows that the material properties of solvents are more in glycerol than water as it follows the trend of hydrogen bonding capacity [71].This H-bonding results in network formation that increases the viscosity of the gel to which glycerin may be added [72].Table 9 shows the analysis of variance of viscosity of the gel formulation.
3.10.3.Spreadability.The impact of the variables on the spreadability of gels, as indicated by the graphs (Figure 14), showed that the overall response was constructive.Eucalyptus oil was seen to have a positive impact on spreadability.In contrast, oleic acid and glycerin seemed to decrease the spreadability.Table 10 shows the analysis of the variance of spreadability of gel formulation.The mathematical models obtained by the Design Expert for permeation showed positive values indicating that the overall response was constructive.All three factors enhanced the permeation of both active ingredients.The polynomial equation indicated that the viscosity of the gels was increased by factors X 1 and X 3 while decreased by X 2 .The positive value of X 0 indicated that the overall response was positive.Oleic acid and glycerin improved the viscosity as they had a thickening effect on the gels, whereas it was observed that eucalyptus oil decreased the viscosity of the topical gels.
The impact of the variables on the spreadability of gels, as indicated by the mathematical models, showed that the response was constructive.Factor X 2 had a positive impact on spreadability.In contrast, X 1 and X 3 decreased the spreadability.This could be explained by the fact that the spreadability and viscosity are inversely proportional.So, the factors reducing the viscosity of the gels will, in turn, increase their spreadability.

Conclusions
In this study, we successfully developed and evaluated topical gels containing Aloe vera and clindamycin phosphate, focusing on their antimicrobial and antioxidant properties.Our comprehensive assessment included tests for organoleptic properties, pH, spreadability, in vitro permeability, skin irritation, and stability.The findings reveal that the formulated antiacne gels exhibit potent antimicrobial activity against acne vulgaris and significant antioxidant action.Notably, the gels demonstrated consistent stability and reproducibility, underscoring their potential as effective treatments for acne and other inflammatory skin conditions.The correlation of our results with existing studies further validates the efficacy of Aloe vera-containing topical gels in dermatological applications.While the current findings are promising, we acknowledge the necessity for further in vivo studies to fully ascertain the therapeutic potential of these gels.Such studies will provide deeper insights into their efficacy and safety profiles, contributing to the development of more effective acne treatments.In conclusion, our research contributes to the growing body of evidence supporting the use of Aloe vera in dermatological applications, particularly in the treatment of acne.We anticipate that these findings will pave the way for future research, potentially leading to innovative and more effective acne treatment solutions.International Journal of Polymer Science

Figure 7 :Figure 8 :
Figure 7: Illustrating the in vitro drug permeation of optimized gel.

Figure 10 :
Figure 10: Illustrating antimicrobial activity of gels.A: with singleingredient A. vera; C: with clindamycin phosphate and A. vera; Std: standard.

Figure 12 :
Figure 12: Illustrating the effect of variables on the permeation of Aloe vera: (a) EUO and OA, (b) GC and OA, and (c) GC and EUO.

Figure 13 :
Figure 13: Illustrating the effect of variables on viscosity: (a) EUO and OA, (b) GC and OA, and (c) GC and EUO.

Figure 14 :
Figure 14: Illustrating the effect of variables on spreadability: (a) EUO and OA, (b) GC and OA, and (c) GC and EUO.

Table 1 :
Formulation design by Design Expert.

Table 2 :
Composition of optimized gel formulation.

Table 3 :
Predicted outcomes of optimized gel formulation.

Table 4 :
Results obtained from characterization tests on the optimized formulation.
[34]4.Viscosity.The viscosity of the gel formulations was evaluated by Brookfield viscometer by spindle no. 4 and speed of 12 rpm[34].2.3.5.Drug Content.Drug content was determined spectrophotometrically by making a 0.9 mg/ml dilution of the topical gel.The percentage content of clindamycin phosphate and A. vera was determined at 210 nm and 330 nm.2.3.6.In Vitro Drug Permeation Study.The Franz diffusion cell was used for this study.The antiacne gel was put on a SpectraPor dialysis membrane (pore size: 0.45 microns) of donor and receptor parts.A phosphate buffer of pH 7.4 was used as media.A temperature of 37 °C (body temperature) and a phosphate buffer of 7.4 pH were used.

Table 5 :
Antimicrobial activity of topical gels.
Formulation Zone of inhibition (cm) Standard gel (Clindacin) 2.4 Gel with A. vera 2.8 Gel with clindamycin phosphate and A. vera 3.4

Table 6 :
Results of stability studies after 6 months.

Table 7 :
Analysis of variance of permeation of clindamycin phosphate.

Table 8 :
Analysis of variance of permeation of A. vera.

Table 9 :
Analysis of variance of viscosity.

Table 10 :
Analysis of variance of spreadability.