Preparation and Optimization of Clotrimazole

Transdermal Gel of Nanosize Transfersome with Different Non-Ionic Surfactant

Pallavi Chavan, Kedar Bavaskar*, Rutuja Sawant , Dr. Ashish Jain

Department of Pharmaceutics, Shri. D.D. Vispute College of Pharmacy and Research Centre, Panvel, Maharashtra, India

*Correspondence: cpallavi53@gmail.com

INTRODUCTION

A major cause of skin illness in the world is fungus. About 40 million people in emerging and underdeveloped countries have a fungal infection, according to reports. The vast majority of the time, fungi penetrate the skin through the surface prior to using desquamation to spread deeper into the skin. One of the fungi most commonly responsible for superficial cutaneous infections is the Candida species.¹ Dermatophytes that can penetrate the stratum corneum and keratinized tissues, such as Trichophyton, Microsporum, and Epidermophyton, are the principal causes underlying skin fungal infections. Non-dermatophyte fungi less frequently cause skin fungal infections.² Cutaneous mycoses are a type of fungal infection that manifests in the deeper layers of the skin. Dermatophytes is a synonym for fungal skin diseases. The term "subcutaneous mycosis" indicates a fungus infection that has already spread to deeper skin tissue.³ However, applying antifungal drugs topically may have unfavourable skin side effects, like an allergic reaction and irritation. Standard formulations also call for repeated administration of high doses, which raises the possibility of local and systemic adverse effects.⁴ To Decrease local side effects and improve the efficacy of treatment, a novel drug delivery system is envisioned. Novel drug delivery system (NDDS) have created a lot of attention in pharmaceutical research as one of the themes of topical formulation .⁵ The fluidity and integrity of the fungal cell membrane are probably maintained in part by ergosterol. The effective treatment of cutaneous dermatophytosis should include topical antifungal formulations with high skin penetration. Azoles' antifungal efficacy is connected to their capacity to prevent the formation of ergosterol. Determined by factors such as particle size, surface charge, and lipophilicity, penetration depth into various skin layers can be determined. To cure cutaneous dermatophytosis, it is thought that negatively charged nanostructured lipid carriers with sizes between 200 and 300 nm have a stronger penetration into the deep skin layers.⁶ Novel topical formulation techniques include solid-lipid nanoparticles, liposomes, niosomes, Transfersomes, microemulsions, and nano emulsion appear to be more effective for penetrating the skin permeability barrier.⁷

A broad-spectrum imidazole antifungal medication called clotrimazole is mostly used to treat skin infections like candidiasis. The BCS class (II) drug clotrimazole exhibits high permeability and rate-limited release characteristics. Clotrimazole prevents the proliferation of fungal cells by inhibiting the formation of ergosterol. Transfersomes exhibit increased deformability as a result of edge activators weakening their lipid bilayers.  Due to their greater deformability and ability to easily pass through holes that are 5 times smaller than their diameter, transfersomes have greater skin penetration than ordinary liposomes.  transfersomes have been created to address the issue of release permeability and side effects, avoiding the issue with conventional dosage forms and reducing the side effects associated with antifungal medications like Clotrimazole.⁸ However, increasing skin penetration too much runs the risk. after significant penetration, medication molecules may enter the bloodstream and be bad for treating skin fungal infections locally. ⁹

In 1991, Gregor Ceve created the Transferosome. The complex system known as a transferosome is composed of an ultra-deformable, highly flexible, stress-responsive composite lipid bilayer surrounding the aqueous center.¹⁰ Transferosomes push against the internal sealing lipid of the stratum corneum to penetrate the skin's barrier to penetration. Transferosome membranes are flexible when the proper combination of surface-active molecules is used in the proper ratios. There are numerous ways to prepare transferosomes. For the preparation of transfersomes, further techniques include reverse phase evaporation, rotary film evaporation, modified hand shaking, vortex/sonication, freeze-thaw, and ethanol injection.¹¹ Transferosomes penetrate the skin's barrier by breaking through the internal sealing lipids of the stratum corneum. The mechanism for transfersome penetration can be triggered by water evaporating as a lipid solution is applied to the skin's surface. This results in the formation of an osmotic gradient. Due to their high bilayer deformability, transfersomes have a greater capacity for creating in and holding onto water.¹² It penetrates the skin barrier when applied to the non-occluded skin surface and hydrates the deeper strata (an area rich in water).¹³ Transferosomes serve as penetration enhancers by disrupting the tightly organized intercellular lipids of the stratum corneum, allowing drug molecules to enter and pass through the stratum corneum.¹⁴

The purpose of this research was to create Clotrimazole transfersomes, which were subsequently added to an appropriate hydrogel foundation for maximum drug loading and improved penetration properties. Through the thin film hydration approach, clotrimazole transfersomes were created utilizing phosphatidylcholine and the edge activators tween 80 and span 60. Then, characteristics such as drug entrapment, size, charge, morphology, and stability of the transfersomes were determined. Carbopol 934 and Xanthan gum, two natural and artificial gelling agents, were used in the formulation of the clotrimazole hydrogel. Additionally, the drug concentration, pH, viscosity, in vitro release, spreadability, extrudability, and in vitro antifungal efficacy of the transfersomal hydrogel have been evaluated.

MATERIALS AND METHODS

Materials

Clotrimazole was a gift sample from Jk Chemicals Valsad, Gujrat Soya Lecithin, Tween 80, Span 60, Carbopol 934, and Xanthan gum were obtained from Research-Lab Fine Chem Industries, Mumbai.

Ultraviolet-Visible (UV-Vis) Spectrophotometry

Preparation of clotrimazole standard stock solution (in methanol). A 100 ml volumetric flask had been loaded with 10 mg of Clotrimazole after it had been precisely weighed. To create 100 g/ml of standard stock solution, it was dissolved and diluted with solvent until the desired strength was reached. Scanners used spectrophotometry to calculate the drug's maximum effective dose. 1 ml of standard stock solution was pipette out and diluted up to 10 ml by using suitable solvent and scanned between 200 nm and 400 nm in wavelength. ^15,25

FT-IR Spectroscopy

The interaction of the drugs and excipients could be determined by contrasting the spectra. By using FTIR, the IR spectra of the pure drug (clotrimazole), excipients, and mixtures of the two were recorded.

Preparation of Transfersomes by Thin film hydration Method

The thin film hydration methodology was used to develop transfersomal formulations using clotrimazole, soy lecithin, span 60, and Tween 80. The organic solvent Methanol was used to weigh out and completely dissolve the drug, phospholipid, and surfactant. Using a rotary flash evaporator, the solvent was evaporated for 30 to 40 minutes at reduced pressure and 60 revolutions per minute. Dry After the formation of the dry film, a transfersomal dispersion made up of multi-lamellar vesicles was produced using PBS pH 7.4 and gentle shaking. The Prepared vesicles were sonicated to create tiny vesicles at room temperature.¹⁶ Formulation batches of different ratios of drug and non-ionic surfactant are shown in Table 01.

Characterization of Clotrimazole transfersomes

Visual inspection was done by putting the transfersomal dispersion in clear containers, turbidity, flocculation, and sedimentation were examined. Using a digital pH meter that had been previously calibrated by standard solutions with pH of 4, 7, and 9.2, respectively.¹⁸

Entrapment efficiency

A volume of 2 ml from each formulation was centrifuged using microcentrifugation tubes at 10000 rpm for 20 min. The amount of free (unencapsulated) Clotrimazole in the filtrate was measured using a UV spectrophotometer (Shimadzu®, Japan) set to measure at 260 nm.¹⁹ The EE % was calculated by following formula,

EE%= [total amount of drug added−free drug total amount of drug added] ×100

Preparation of Transfersomal gel by dispersion method

The gel base made with various concentrations of carbopol 934 was given the best transfersome formulation to be added. Gelling agent was measured out and added to the distilled water while being constantly stirred. It was irrigated for two hours while being immersed. Propylene glycol (10% w/w), for example, was continuously swabbed and mixed after the necessary amount of the optimum transferosomes formulation was introduced. The gel was neutralized with triethanolamine (TEA) to a pH range of 5 to 7, and the final weight adjusted by adding water. The gel was left undisturbed for the remainder of the day after being sonicated for 30 minutes on a bath sonicator to remove trapped air. ^20,21 Preparation batches of Carbopol 934 and Xanthan gum transfersomal gel are shown in Table 02.

Evaluation of Transfersomal gel

Spreadability

Glass slides and wooden block measuring tools were used to determine it.  An excess sample was put between two glass slides to measure spreadability, and it was then compacted to a consistent thickness.  50 grams of weight were put into the pan.  The spreadability (S) was calculated as the time it took to move the upper glass slide across the bottom plates or as the time it took to separate the two slides.

Extrudability

A Pfizer hardness tester was used for the extrudability test.  The metal tube was collapsible and contained 15g of gel. After adjusting the plunger to hold the tube securely, 1 kg/cm2 of pressure was applied for 30 seconds. Weighing was done on the amount of gel that was extruded.

Viscosity

A Brookfield viscometer was used to measure the viscosity. Using a Brookfield viscometer (Model DV2T) with T-94 Spindle, at ambient temperature (25–27°C), viscosity measurements were made.

Drug content

In 100 ml of methanol, 1g of the formulation was sonicated for 30 minutes. The syringe filter was then used to filter the solution. Spectrophotometric analysis was used to assess the filtrate's drug content at 260 nm (UV-2450, Shimadzu, Japan). ^22,24

 Drug release studies from transfersomal gel

To study the rate at which the medication is released from the formulation, a set of Franz diffusion cells were used in an in-vitro diffusion investigation using Transfersomal gels. Receptor medium (PBS pH 7.4) was poured into the receptor chamber. Throughout the experiment, the temperature of the receptor medium was maintained at 37°C. A dialysis membrane was clamped between two chambers after being soaked in receptor media for 12 hours. The donor cell received 1g of the formulation. Then, after a set period of time, 1 ml samples of the receptor cell were obtained. To keep the volume constant, an equal amount of new medium was added after each collection. Fresh receptor media was used as the blank for the spectrophotometric examination, and the extracted samples were diluted as necessary. An equation created from standard calibration was used to calculate the drug concentration delivered at a specific time interval.^22,24

 Drug release kinetic modelling

The Results of in-vitro diffusion investigations were fitted with several kinetics models to determine the release mechanism. Commonly used analytical definitions of the Qt include the Higuchi-matrix, Peppas-Korsmeyer model, zero-order definition, and first-order definition .²²

 In vitro antifungal study

The tests were done on Candida albicans as the test microorganisms to determine the biological activity of the Transfersomal formulation in comparison to commercially available clotrimazole gel and plain clotrimazole transfersomal gel. The 'Cup plate' method of an agar diffusion test is used to determine this. In the Petri plates, a layer of Sabouraud's dextrose agar media containing the test microorganisms was allowed to set up. With the use of a sterile borer, cups were formed on the hardened agar layer. One cup is filled with the Transfersomal gel solution (1% of the drug), and the second cup is filled with commercialized gel (1% of the medication). ^8,23

Stability study

According to the International Council for Harmonization's (ICH) requirements, the stability study of the formulation was completed. According to ICH rules, freshly prepared formulations were separated into groups and stored under specific conditions.²²

RESULTS AND DISCUSSION

Ultraviolet-visible (UV-Vis) spectrophotometry

The maximum absorbance (max) of the drug, clotrimazole, was identified to be the peak with the greatest intensity in the UV-Vis spectrum analysis at 260 nm. The value of R² was found to be 0.999, indicating the relation between Drug concentration and absorbance was linear in selected range.

FTIR Spectroscopy

 By comparing the spectra, it was determined whether the drug and excipients were compatible with each other. The IR spectra of the pure drug and excipients as well as the mixture of drug and excipients have been captured by using FTIR. The spectrum of pure Drug (Clotrimazole) Presented characteristic Bands at 701.73,823,907.12 cm^-1 for the Aromatic C-H Bending group. For C-N stretching group peaks were noted on 1044.04,1078.28,1209.29 cm^-1.for C=N stretching peak was noted on 1557.51 cm^-1. Aromatic C-H stretching group type of vibration with frequency 2886.81,3063.67 and 2978.09 cm^-1. Aromatic C-Cl stretching group type of vibration with frequency 633.27 and 667.50 cm^-1.

The infrared spectra of the mixture of Soya Lecithin, a Non-ionic surfactant (Tween 80 and Span 60) ingredient showed Peak at the C-O group with frequency 1072 cm-1 and C=O group with frequency 1635.60 cm-1 and 1735 cm-1 which indicates Soya lecithin is present in Transfersome. Also, the C-H group shows a peak of 2816.07 cm-1. The Hydroxyl group shows a peak of 3425.58 which indicates the presence of Tween 80. Carboxylic acid shows a peak on 2312.65 cm-1 and Aromatics Stretching group peaks are noted on 717.52 cm-1 and 879.54 cm-1, it indicates the presence of Span 60. For the C-N group peaks were noted on 1072.42 cm-1 and 1211.30 cm-1.C-Cl group shows a peak on 725.23,1559.91 and 694.37 cm-1 which indicate the presence of the Drug in Transfersomes which shown in Figure 2.

 The IR spectrum of Polymer (Carbopol 934) showed characteristic peaks at 1172.72 and 1234.44 cm-1 for group C-O. for Group C=O peak showed at 1699.29 cm-1 .C-H stretching showed peak on 2945.30 cm-1. In the case of Final gel formulation, Infrared spectra of the mixture of Clotrimazole, transfersome, and polymer (Carbopol 934) showed similar characteristic peaks at the appropriate wavelengths, indicating Clotrimazole’s compatibility with different excipients. The IR spectrum of xanthan Gum as a polymer showed characteristic peaks at 3803.63 and 3302.13 cm-1 for group O-H. The peaks for group C=O showed peaks on 1717.12 cm-1. -COO group shows a frequency of 1419.61 cm-1. The Carboxyl group shows peaks on 1111.60 cm-1 and B glycoside showed frequency on 702.09 and 725.23 cm-1. The IR spectrum of Final Gel formulation showed similar peaks to Clotrimazole, Transfersomes, and xanthan gum so it indicates compatibility with each other by increasing its frequency shown in figure 3.

Entrapment efficiency

Clotrimazole transfersomal dispersion was prepared with different ratios by using soya lecithin and two different non-ionic surfactants (Tween 80 and span 60). based on Entrapment efficiency F1 batch was selected as the optimized batch. According to figure 4, F1 formulation with a ratio of 90:10 shows the greatest entrapment efficiency .it shows 91.91%. so, it concluded that Span 60 shows better Entrapment efficiency than Tween 80 in Figure 4.

Tween 80 (water-soluble surfactant) showed Lower drug entrapment within transfersome vesicles as compared to Span 60 because of the HLB value. The Tween HLB value is 15 and the Span HLB value is 4.7. Higher the HLB value lower the entrapment efficiency in the case of Transfersomes. Formulation F1 (91.91%) has a higher entrapment effectiveness due to the fact that is a greater concentration of phospholipid available for coating an aqueous volume. because of the higher Span 60 surfactant content, which causes micelle formation as well as less effective trapping.

Zeta potential and Polydispersity index

Zeta potential was revealed by particle size analysis, and the optimized batch's PDI was found to be 0.314 with an average size of 62.87 nm. The particles were homogeneously distributed and of uniform size, as evidenced by the decreased polydispersity. Transfersomes with Clotrimazole loaded had a surface charge of -25.8 mV, according to measurements. The graph of Zeta potential and PDI is shown in figure 5a and 5b.

Electronic microscopy

A transmission electron microscopy study was done of optimized clotrimazole transfersome batch F1. It displays the general shape and interior of the recognized spherical vesicles. The average size of the Transfersome was found to be 61 nm. TEM images display in figure 6

Evaluation of Clotrimazole Transfersomal gel

Xanthan gum and carbopol 934, the synthetic and natural polymers with varying quantities that are listed in Table 02, were used to create transfersomal gel. Tables 03 and 04 present the results of all the metrics used to evaluate Transfersomal gels, including homogeneity, pH, viscosity, spreadability, extrudability, and drug content uniformity.

Better extrudability is shown by more Transfersomal gel being extruded in a given amount. 1% Carbopol gel and 1% Xanthan Gum gel were shown to have superior extrudability than other formulations. Both formulations demonstrated excellent extrudability. Carbopol 934 was considered to have excellent spreadability since it spread over a shorter period of time than xanthan Gum at all concentrations. The effectiveness of gels as a treatment depends on how they are applied. Because it helps with the consistent distribution of the gel to the skin, the created gels must be easily spreadable and fulfil the perfect specifications for transdermal application. This is also considered to be a crucial component of patient adherence to medication.

The drug content of Carbopol transfersomal gel is better than the xanthan gum transfersomal gel. Carbopol 1% gel shows 90.86 % while 1% xanthan gum gel shows 85.96%. so, it concludes that Synthetic polymer shows better drug content than Natural polymer. Carbopol 934 gel shows low viscosity than xanthan gum gel. Viscosity also increases as the gelling agent concentration increases. The viscosity of gel compositions often reflects uniformity.

This behavior was chosen because, in high-shear circumstances, it has a low flow resistance. These gels become less viscous as the rate of shear increases, as demonstrated by non-Newtonian flow (shear thinning). the ability to spread widely due to a drop in viscosity caused by the application of a given force, while also maintaining its location at the application site without draining.  so, carbopol 934 Transfersomal gel showed the best evaluation result than xanthan gum transfersomal gel.

 In vitro Diffusion study

A diffusion study was also conducted between 1% carbopol gel, 1% xanthan gum gel, and 1 % Canesten® cream (marketed product). A 24-hour in vitro drug release investigation was carried out. In vitro drug release of transfersomal gel batches was examined in Phosphate Buffer solution with pH 7.4, as clotrimazole is a weak acid by nature and enters into the bloodstream in unionized form. Diffusion studies were conducted for 24-hour.

The maximum drug release was seen with the 1% carbopol 934 clotrimazole transfersomal gel because it allowed the drug to be released more gradually and for a longer period of time than the other two products.

In Vitro Antifungal study

The anti-fungal efficacy of 1% Canesten® cream (standard) and 1% clotrimazole transfersomal gel (test) against Candida albicans was compared using the cup plate method. A response from zone of inhibition was interpreted as the comparison of standard and test preparations. Figure 8 showed the results for the zone of inhibition, and it was clear that 33mm zone of inhibition for the 1% Clotrimazole Transfersomal gel shown large zone of inhibition than 26mm zone for the 1% Canesten® cream. The ability of the Transfersome to get through the fungal cell wall and limit ergosterol biosynthesis, which in turn led to cell death and membrane lysis, may have contributed to this variation in the zone of inhibition. Picture of Zone of inhibition shown in figure 8.

Drug release kinetic model

According to the findings of our kinetics investigation, Prepared batches were best suited in the Zero order release kinetic model, which had the greatest R² value of all the kinetic models. Among all the formulations suggested as having the greatest fit for zero order kinetics, F4TG4 demonstrated the highest R2 value. It means that the medicine is released from the transfersomal gel at a constant rate per unit of time. Table 6 displays R² values.

CONCLUSIONS

In the present research, soya lecithin, non-ionic surfactant Tween 80, and span 60 were combined in different ratios to formulate transfersomes. The entrapment efficiency of the prepared transfersomes ranged from 91.91% to 50.9%, and they were negatively charged, nanosized vesicles (61 nm) in size. Due to span 60's low HLB value, span 60 exhibits the best entrapment efficiency. It concluded that among all formulations 1% carbopol 934 as a synthetic polymer shows better results than 1% Xanthan gum natural polymer. so, we can conclude that carbopol 934 is the best gelling agent for the preparation of transfersomal gel. as it shows better drug release as compared to the other two formulations. According to our research, the Transfersomal Formulation provided a sustained and prolonged pharmaceutical delivery with improved patient compliance and enhanced bioavailability. The transfersomal formulation's transdermal delivery method may be a useful dosage form for reducing the unfavorable effects of the oral delivery method. The preparation process is straightforward and practical from an industrial standpoint. Transfersomes may therefore be thought of as an efficient means of delivering clotrimazole via the transdermal route to treat deeper-layer fungal skin infections.

ACKNOWLEDGEMENTS

We appreciate the excellent guidance provided by Dr. Ashish Jain, the principal of Shri. D.D. Vispute college of Pharmacy and Research center. We also want to express our gratitude to Mr. Kedar Bavaskar for his help. A special thanks goes out to JK Chemicals Valsad, Gujarat for supplying the Clotrimazole sample as a gift. The authors also expressed gratitude to Mumbai's Research-Lab Fine Chem Industries for offering a variety of excipients

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Table 01. Formulation batches of clotrimazole transfersome.

Table 02: Batches for preparation of Clotrimazole transfersomal transdermal gel using Carbopol 934 and Xanthan gum

Table 03. Evaluation of Carbopol 934 transfersomal gel

Table 04.  Evaluation of xanthan gum transfersomal gel

Table 05. In vitro drug release of clotrimazole Transfersomal gel and marketed product

Table 06. R² values of drug release kinetic model of different formulations

Figure 1: Represents The Uv-vis Spectra of clotrimazole in methanol. The peak with greatest intensity in Uv vis spectrum analysis at 260 nm

Figure 2: Describes the FTIR of Transfersomal suspension with Drug and Excipients and final formulation of Transfersomal gel (Carbopol 934). Infrared spectra of the mixture of Clotrimazole, transfersome, and polymer (Carbopol 934) showed similar characteristic peaks at the appropriate wavelengths, indicating Clotrimazole’s compatibility with different excipients.

Figure 3: Describes the FTIR of Transfersomal suspension with Drug and Excipients and final formulation of Transfersomal gel (Xanthan gum). Infrared spectra of the mixture of Clotrimazole, transfersome, and polymer showed similar characteristic peaks at the appropriate wavelengths, indicating Clotrimazole’s compatibility with different excipients.

Figure 4: The figure shows entrapment efficiency of clotrimazole Transfersome in two different non-ionic surfactants like Tween 80 and Span 60.

Figure 5: a) Represents The polydispersity index of Clotrimazole containing transfersomes.

Figure 6: The above image shows Transmission electron microscopy of Transfersomes.

Figure 7: Represents In vitro Drug release of clotrimazole Transfersomal gel and marketed product up to 24 hrs. by using Franz cell diffusion apparatus

Figure 8: Shows In Vitro Antifungal study Clotrimazole containing Transfersomal gel and 1%   Canesten® cream against Candida albicans using the cup plate technique