Vitamin E TPGS based nanogel for the skin targeting of high molecular weight anti-fungal drug: development and in vitro and in vivo assessment

Abstract

The molecules having a molecular weight greater than 500 Da are considered to be ineffective in the treatment of skin diseases due to their low skin permeation. The present study entails the development of a Vitamin E TPGS nanoemulsion based nanogel formulation for the high molecular weight drug, amphotericin B (AmB 923 Da), intended for the effective treatment of cutaneous fungal infections. The nanoemulsion formulations were prepared in strengths similar to the marketed topical formulation and optimized with respect to the S_mix ratio (surfactant : co-surfactant), type of surfactant, co-surfactant, globule size, size distribution, percent transmittance, dispersibility, viscosity and rheology. The optimized nanoemulsion formulation was further incorporated into a gel base to form the AmB nanogel formulation. When compared to the marketed topical formulation, the optimized nanogel exhibited a 3.9 fold higher skin deposition through porcine ear skin. Confocal laser scanning microscopy studies (CLSM) showed the specific distribution of AmB in different parts of the skin and confirmed the permeation of the nanogel to the deeper layers of the skin. The antifungal activity of the optimized nanogel formulation was tested using microdilution method and found to be 2.0 fold higher against Aspergillus niger and Candida albicans in contrast to the marketed formulation. The results show that the prepared nanogel formulation is a better alternative for effective topical delivery of AmB.

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1 Introduction

Intensifying incidences of skin mycosis caused by different species of yeast, fungi and dermatophytes in both immunodeficient and immune-competent patients remain an imperative and adequately addressed medical problem.¹ It affects more than 25% of the world's population.² It may be life-threatening and can have debilitating effects on a patient's quality of life especially in immunocompromized patients suffering from diseases like AIDS, diabetes and cancer or may become invasive (systemic) and in some cases it may also spread to other people.³ Cutaneous fungal infections (Cutaneous candidiasis, burn fungemia, Cutaneous leishmaniasis) are more rampant in sub-tropical countries like India, Africa and Sri Lanka where the climatic conditions are warm and humid, which further accelerates the occurrence, growth and spread of dermatomycosis.⁴ Candida species are one of the major causes associated with skin membrane (Cutaneous candidiasis) and fingernail (candidiasis or thrush) related infections.⁵ These infections are generally difficult to diagnose and treat because they are often misguided for other disorders such as eczema or psoriasis.⁶

Amphotericin B (AmB), a membrane-active polyene is considered the most effective gold standard antifungal and is clinically used for the treatment of systemic and local fungal infections.⁷ This drug also has antiprotozoal activity and is used for the treatment of visceral and cutaneous leishmaniasis.⁸ AmB can be administered both intravenously and topically.⁹ Presently, the preferred route for the administration of AmB for the above mentioned disease conditions is parenteral. However, AmB is a highly hydrophobic drug and the conventional parenteral formulation uses sodium deoxycholate as a solubilizer in very high concentration (65% w/w). The conventional IV formulation has severe and potentially lethal side effects such as nephrotoxicity, cardiotoxicity, hepatotoxicity, anemia, high fever, shaking chills, hypotension, anorexia, nausea, vomiting, headache, dyspnea and cardiotoxicity.^10,11

Recently, a topical route has been explored for the safe and efficacious delivery of AmB for the treatment of surface fungal infections, for instance, skin and nail infections caused by dermatophytes,¹² burn related fungemia⁷ and Cutaneous leishmaniasis.¹³ Currently, two types of topical formulations of AmB have been developed and are commercially available (Fungizone cream/lotion and Fungisome gel). However, these formulations suffer from the drawbacks of poor skin permeation and limited clinical effectiveness. The reason behind the ineffectiveness of the topical therapy of AmB is its high molecular weight and bulky structure, which comprise hydrophobic heptane chains and a hydroxyl rich hydrophilic chain that are responsible for its poor solubility in both aqueous and hydrophobic systems thus leading to the poor skin partitioning resulting in poor permeation of drug.¹⁴ Moreover, it represents both amphoteric and amphiphilic nature,¹⁵ which is the main obstacle in the development of an effective topical drug delivery system. As a result, with existing conventional formulations, it is difficult to effectively treat topical fungal infections, which provides a rationale to explore the potential of novel carrier based formulations for topical delivery.

Nanocarrier based formulations are increasingly gaining importance in terms of topical drug delivery for the effective management of skin diseases. The various advantages offered by these systems include their nanometric size, high surface area, ability to act as a solubilizing agent for poorly soluble drug, high skin permeation and deposition potential, non-irritant nature and easy method of preparation.¹⁶ Therefore, in view of the attractive benefits offered by these systems, the present study proposes the use of a Vitamin E TPGS based nanoemulsion as a carrier system for the effective topical delivery of AmB. Vitamin E TPGS NF (TPGS, D-α-tocopheryl polyethylene glycol 1000 succinate) has been selected as a surfactant for the preparation of a nanoemulsion formulation as its chemical properties have suggested its wide use as a solubilizer, an emulsifier, an intestinal absorption enhancer for increasing the oral bioavailability of poorly water soluble drugs like paclitaxel¹⁷ and vancomycin,¹⁸ as skin permeation enhancer for drugs like griseofulvin^19,20 and estradiol,²¹ and also as stabilizer.²² In a recent study conducted by Bao et al., a TPGS based prodrug overcame the multidrug resistance (MDR) in a paclitaxel resistant human ovarian cell line by inhibiting P-glycoprotein leading to the enhanced efficacy of paclitaxel.²³ Furthermore, TPGS's property of being soluble in both water soluble and water insoluble compounds has helped many drugs to solve the problem of solubility by incorporating them into its micelles, which ultimately stabilized most of the drugs and made them soluble in nature.²⁴ The lucrative properties of Vitamin E TPGS have made it a widely used surfactant in innovative drug delivery systems like prodrugs, nanoparticles, micelles, liposomes, nanocrystals, nanosuspensions, solid dispersions, anticancer systems and nutritional supplements.^25,26

Despite the vast potential that it holds, no study has been reported stating the use of Vitamin E TPGS for the preparation of nanoemulsions and its use as a solubilizer and skin permeation enhancer for topical drug delivery. For this reason, in the present investigation, a detailed study was performed for the preparation, in vitro and in vivo characterization of Vitamin E TPGS based nanoemulsion of AmB as well as to explore its possible role in dermal drug delivery. The low viscosity of the nanoemulsion restrains its clinical applications. Therefore, an attempt has also been made to incorporate the nanoemulsion into a gel base to form a nanogel and study the effect of the gel on the physical stability, skin permeation, thixotropic and rheological behaviour of the nanogel formulation. The nanogel can aid in creating a uniform dispersion of nano-carriers in the matrix and increase the contact time, which could result in the enhanced skin penetration of the drug. Shah et al. proved that a nanogel consisting of nanoparticles leads to an improvement in the skin permeation of spantide II and ketoprofen by translocating the nanoparticles across the deeper skin layers via increasing the skin contact time and hydration by forming a thin layer on the skin surface.²⁷ Furthermore, these nanocarrier based nanogel formulations have also been exposed for the skin targeting of anti-cancer drugs like 5-FU to achieve enhanced topical delivery by increasing the local bioavailability.²⁸ Thus, in order to obtain prolonged skin retention and controlled release for the desired therapeutic effect, it is appropriate to incorporate a nanocarrier formulation into a proper gel matrix forming a nanogel system.

2 Materials and methods

2.1 Materials

AmB was received as a gift sample from Kwality Pvt. Ltd., Amritsar, India. Oleic acid, DMSO, isopropyl alcohol, propylene glycol and polyethylene glycol were purchased from Hi media, Mumbai, India. Transcutol and rhodamine were purchased from Sigma Aldrich Pvt Ltd., Mumbai, India. Vitamin E TPGS was received as a gift sample from V.B. Medicare Pvt. Ltd., Tamil Naidu, India. Olive oil was purchased from Figaro, Deoleo S.A, Spain. Carbopol 940P and Carbopol 934P were purchased from Titan Biotech Ltd., Rajasthan, India. Methanol was purchased from Finar Ltd., Ahmedabad, India. The marketed AmB formulation (0.1% w/w Life care Innovations Pvt. Ltd., Gurgaon, India) was purchased from a local pharmacy for research purposes. All other reagents used were of AR grade.

2.2 Solubility studies

The solubility of the drug was determined individually in different oils, surfactants and co-surfactants and in their combinations. An excess amount of drug was added into 5 mL of oil, surfactant and co-surfactant in vials, which were tightly stoppered and stirred on a water bath shaker for 24 h. The samples were then centrifuged at 3000 rpm and the supernatant was analysed for their drug content using an HPLC assay (Agilent Technology, USA).

2.3 Screening of surfactants and co-surfactants using a pseudoternary phase diagram

Surfactants were selected on the basis of the solubility of drug, miscibility of the surfactant with oils and required HLB value. After the selection of the surfactant, it was combined with co-surfactants, such as isopropyl alcohol, propylene glycol and polyethylene glycol 400, and selected from the construction of phase diagrams. Pseudoternary phase diagrams were mapped in order to optimize the surfactant to co-surfactant ratio and type of co-surfactant that could result in a large existence area of nanoemulsion. They were constructed using an aqueous titration method for various surfactant/co-surfactant ratios, i.e., an S_mix ratio of 1 : 1, 2 : 1, 1 : 2, 3 : 1 and 1 : 3, and at specific S_mix, the ratio of oil to the S_mix were varied at 1 : 9, 2 : 8, 3 : 7, 4 : 6, 5 : 5, 6 : 4, 7 : 3, 8 : 2 and 9 : 1 in order to cover the maximum ratios to delineate the boundaries of the phases precisely formed in the phase diagrams.

2.4 Preparation of the nanoemulsions

Nanoemulsion formulations with suitable components and concentration selected from the phase diagram were prepared using a high energy method.²⁹ For the preparation, aqueous and oil phases were prepared and heated separately. To prepare the oil phase, the drug was dissolved in a mixture of olive oil, oleic acid, transcutol and DMSO under stirring. The aqueous phase was prepared by dissolving hydrophilic surfactants and co-surfactants at 60 ± 5 °C. The oil phase was then added dropwise to the aqueous phase under continuous stirring for 3–4 h. It was then ultrasonicated at 25 °C for 10 min to achieve the desired size range of the dispersed globules.

2.5 In vitro characterization of the nanoemulsions

2.5.1 Globule size, shape and size distribution. The globule size of the nanoemulsion formulations was determined using a particle size analyzer (Malvern Zetasizer Nano ZS90 UK). The intensity distribution and polydispersity index (PDI), which are indicators of uniformity in size distribution are also attributable to particle size analysis. The visualization of the morphology and structure of the globules after staining negatively with phosphotungstic acid was carried out using transmission electron microscopy (TEM).

2.5.2 Zeta potential measurement. For zeta potential analysis, samples were diluted with deionized water and analysed using a Zetasizer (Malvern Zetasizer, Nano ZS90, UK).

2.5.3 Drug content. The drug content of the formulation was determined by diluting 1 mL of the nanoemulsion with 20 mL of methanol. About 1 mL of the above mentioned solution was withdrawn and diluted up to 5 mL with the mobile phase (a mixture of acetonitrile : methanol : 0.01 M sodium dihydrogen orthophosphate in ratio of 41 : 10 : 49)³⁰ and the drug content was analysed using an HPLC assay.³¹

2.5.4 Dispersibility. 5 mL of each formulation was added to 500 mL of distilled water in a USP Type II Dissolution apparatus (Lab India DS 8000) at 37 ± 0.5 °C and 50 rpm. After 30 min, the formulations were visually examined for water dispersibility using the following grading system: grade A: rapidly forming (<1 min) nanoemulsion, having a clear appearance. Grade B: rapidly forming, slightly less clear emulsion. Grade C: fine milky emulsion that formed within 2 min. Grade D: dull white emulsion having slightly oily appearance that is slow to emulsify (>2 min). Grade E: formulation, exhibiting either poor or minimal emulsification with large oil globules present on the surface.

2.5.5 Percentage transmittance. The nanoemulsions were subjected for the measurement of the %transmittance using a UV spectrophotometer (U-2800, HITACHI, Japan) against water as a blank at λ_max 540 nm.

2.5.6 Thermodynamic stability. In order to overcome the stability issues, the formulations firstly underwent thermodynamic stability studies, in which the formulations were subjected to different temperature conditions (heating–cooling cycle) and centrifugation and then assessed visually. In the case of the heating–cooling cycle, the formulations were stored at 4 ± 1 °C and 45 ± 1 °C for not less than 48 h and examined. Similarly, they were also centrifuged at 4000 rpm for 20 min and assessed for any phase separation.

2.6 Preparation of the nanogel formulation

To prepare the gel base, accurately weighed Carbopol 934P and Carbopol 940P were soaked separately in distilled water and were allowed to swell and hydrate for 3–4 h. The developed Carbopol dispersions were found to be acidic, which were then neutralized with the help of triethanolamine. After that, the gels were stirred using a mechanical stirrer at 2500–3000 rpm at room temperature for 45 min until clear gels were obtained, which were further left for 24 h at room temperature for equilibration until further use. The nanoemulsion was then mixed with the prepared Carbopol gels under mechanical stirring for 1 h to form the nanogel formulations.³²

2.6.1 In vitro characterization of the nanogel formulations. The developed nanogels were evaluated for clarity, color, uniformity, drug content, presence of particulate matter and grittiness. The smears of the gels were prepared on glass slides and observed under a microscope for the presence of any particulate matter. The pH of the prepared nanogel was determined using a digital pH meter (Equiptronics Model EQ611, Delhi, India).

2.6.2 Extrudability measurements. To measure the extrudability, a closed collapsible tube containing nanogel was pressed firmly at the crimped end and a clamp was applied to prevent any roll back. The cap of the tube was removed and the gel was extruded to determine the ease with which the formulation comes out of the collapsible tube.³²

2.6.3 Rheological behavior of the nanogels. The rheological properties of the nanogels and marketed formulations were determined using a rheometer (Rheolab QC, Anton paar, Germany). Viscosity profiles for the different nanogels and marketed gel at a constant shear rate (40 s⁻¹) and varying shear rate (0–50 s⁻¹) were determined at room temperature. The yield stress of the gel formulation was determined using the Bingham equation. The thixotropic behavior of the formulations at a shear rate of 80 s⁻¹ was also determined to evaluate the strength recovery ratio of the prepared nanogels as well as the marketed gel.

2.6.4 Texture profile analysis. The mechanical properties of the prepared nanogels and marketed formulations were evaluated using a texture analyser (TA/TX2-plus, Stable Micro Systems, Surrey, UK) with probe no. 6. The probe was inserted in the texture vial containing the gel at a defined rate to a desired depth, allowing a pre-defined recovery period rate between the end of the first and the beginning of the second compression. The pre, post and test speed were 1 mm s⁻¹, the probe insertion distance was 10 mm and trigger force was 1 g. From the resultant force–time curve, the mechanical parameters, for instance, hardness, adhesiveness, cohesiveness, and springiness were calculated.³³

2.7 Skin permeation study

The in vitro permeation of AmB from different nanogels and marketed formulations was studied using the Franz diffusion cell. The effective permeation area through which a drug diffuses into the receptor phase was 2.303 cm². The receptor fluid used was composed of Isotonic Palitzsch buffer (IPB), methanol and tween 80 in a ratio of 79.5 : 20 : 0.5. The capacity of the receptor compartment was 22.5 mL. The temperature of the donor compartment was maintained at 37 ± 2 °C. Porcine ear skin was freshly obtained from a nearby slaughter house, cleaned under cold running water and any visible hairs were trimmed short. Before conducting the permeation experiment, extra fat layers were removed using a blunt dissection method to obtain an epidermal membrane, which was equilibrated by soaking it in the receptor phase for one hour. Then, the skin was sandwiched between the donor and receptor compartment such that the dermis had full contact with the receptor solution. The stirring was maintained at 100 rpm. The skin was run blank without the application of any formulation for 3 h. Then, the receptor fluid was replaced with fresh media. The nanogel and marketed formulations were applied to the epidermal skin surface. Samples (1.0 mL) were withdrawn from the sampling port at predetermined time intervals of 0, 1, 2, 3, 4, 5, 8, 10, 12, 20 and 24 h. The volume of the receptor fluid was maintained by replacing with fresh receptor fluid.³⁴ The samples were analyzed using an HPLC assay.

2.8 Drug deposition study

The skin deposition studies were carried out to determine the amount of drug deposited into the skin from the various formulations applied. The porcine ear skin mounted on the Franz diffusion cell for permeation studies was carefully removed after 24 h. The amount of formulation, which was still adhered to skin was washed using 50% v/v methanol and distilled water solution. Then, the washed skin was cut into small pieces and immersed in 10 mL of 0.05% trypsin solution. These samples were placed on a water shaker for 24 h at 100 rpm at 37 °C.³² After filtration, the samples were analyzed using HPLC.

2.9 Mechanistic study to evaluate the percutaneous permeation enhancement of the nanogel formulations

Albino rats were divided into different groups each comprising three rats. The first group served as the control and did not receive any topical formulation. The second, third and fourth groups received topical applications of a drug solution, marketed formulation and nanogel formulation, respectively. The formulations were applied non-occlusive to the abdomen side of the rat over an area of 1 cm². The treated rats were killed after 6 h of application of the formulation. The skin was immediately removed and was fixed at 4 °C in Karnovsky's fixative overnight, followed by 1% w/v osmium tetraoxide for 2 h, and then in ruthenium tetraoxide 0.2% w/v and K₃Fe(CN)₆ 0.25% w/v for 1 h. After fixation, the samples were dehydrated in a range of ethanolic solutions 70%, 90%, 95%, and 100% v/v and coated with a gold coater. The coated samples were then visualized under a scanning electron microscope. The treated skin samples were also analyzed using ATR-FTIR (PerkinElmer spectrum 400) and the spectrum was generated between 4000 and 400 cm⁻¹.³⁵

2.10 Skin localization index

The skin localization index was determined using CLSM (confocal laser scanning microscopy) and was carried out according to the procedure described by Jain et al.³⁶ Albino rats were divided into two groups each comprising three rats. The first group served as a control and received topical application of fluorescence marker Rhodamine 123 (0.16% w/v) loaded in the plain gel base. The second group was treated with the optimized nanogel loaded with fluorescence marker Rhodamine 123. The formulation was applied to the dorsal portion of rats. After the treatment, the animals were caged individually and sacrificed after 24 h. The treated skin was removed and cut into small pieces and washed with phosphate buffer solution (PBS) (pH 7.4). This skin was blotted dry and fixed in Carny's fluid. The fixed skin sections were then viewed under CLSM at various wavelengths (633, 488 and 405 nm) for determining the quantitative estimation of the depth and extent of marker penetration.³⁴

2.11 Determination of MIC

The minimal inhibitory concentrations (MIC) for the AmB formulations were evaluated by a microtiter plate dilution assay using different concentrations (200, 100, 50, 25, 12.5, 6.25 and 3.125 μg mL⁻¹) of the formulations. Test strains were grown under optimal conditions and the test was performed in triplicate. In 96 well microplates, 100 μL of a fungal spore suspension prepared in potato dextrose broth (PDB) (1 × 10⁵ spores per mL) and candidal suspension in yeast malt broth (YMB) (O.D 0.5) were mixed with 100 μL of the formulations. The control well consisted of 100 μL of the suspension of test organisms and 100 μL of DMSO. Control blanks consist of 100 μL of the formulations with 100 μL of PDB and YMB and the other consisted of 200 μL of PDB and YMB only. The microtiter plates were incubated at 28 ± 2.0 °C and the optical density was measured at 600 nm after 24 and 48 h using an ELISA microplate reader (Bio-rad, Model 680XR). The lowest concentration that inhibited the growth of the test strain and did not show any increase in absorption after 48 h was considered as the MIC.

2.12 Determination of the antifungal and anticandidal activity

The antifungal activity of the formulations was investigated against Aspergillus niger and anti-candidal activity against Candida albicans using an agar well diffusion assay.^9,37 The test cultures (100 μL) were inoculated into freshly prepared PDA plates (A. niger; 1 × 10⁵ spores per mL) and YMA plates (C. albicans; OD equivalent to MacFarland standard 0.5) using sterile cotton swabs. Then, the wells of about 6 mm diameter were made on inoculated plates using a sterile cork borer and each well was filled with 100 μL of formulations. The inoculated plates were incubated at 28 ± 2.0 °C. The diameter of the resultant zone of inhibition was measured in mm after 24, 48 and 72 h of incubation for the yeast and fungal strains.

2.13 Skin irritation study

A skin irritation study for the different formulations was determined on albino rats (190–200 g) in order to check the irritation potential of the formulations upon topical use. It was carried out according to the method described by Draize et al.³⁸ The animals were housed in an air conditioned room and the hair on their back was trimmed short 24 h before the study. Three squares were drawn on the backside of each rat. The first group did not receive any treatment and acted as a sham control, the second group received a topical application of 20% SLS solution acting as a positive control. The third, fourth and fifth group received a topical application of a drug solution, the nanogel and the marketed formulation, respectively.³⁹ The skin irritation was scored for erythema and oedema on a grade of 0–4 at 1, 3, 7, 14, 21 and 28 days for the toxicity study.³⁸ All investigations were performed after approval from the Institutional Animal Ethics Committee of Guru Nanak Dev University, Amritsar and in accordance with the disciplinary principles and guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

2.14 Statistical analysis

Data are expressed as the mean ± standard deviation (SD) for the obtained results. Statistical analysis of the data was performed using the analysis of variance followed by Tukey's multiple range test (ANOVA) (Sigma stat 3.5, STATCON, Witzenhausen, Germany). A value of p < 0.05 was considered statistically significant.

3 Results and discussion

3.1 Preparation of nanoemulsion formulations

A topical marketed formulation of AmB is available at 0.1% w/w strength, so the minimum required solubility of drug to make a formulation similar in strength to the marketed formulation is 1 mg mL⁻¹. However, AmB is a highly lipophilic drug and practically insoluble in water.⁴⁰ Therefore, in order to prepare formulations in a clinically effective dose range, the solubility of AmB in different oils individually, in a mixture of oils, surfactants, and co-surfactants was determined and the results summarized in Table 1. The maximum drug solubility of 12.50 ± 0.08 mg mL⁻¹ was found in olive oil, oleic acid and DMSO mixture in a ratio of 2 : 2 : 1 and same was selected as the oil phase for the preparation of the nanoemulsion formulation. After selection of the oil phase, different trials were taken employing varying concentrations of surfactant (Vitamin E TPGS, Transcutol, Tween 20, Tween 80, Span 20 and Span 80) and co-surfactant (PEG 400, propylene glycol, isopropyl alcohol) and the preliminary results for the miscibility of different surfactants, co-surfactants and oil phase are depicted in Table 2. The combination of excipients that formed a clear and transparent mixture was selected for further development as the miscibility of the excipients in each other is a pre-requisite for the development of stable nanoemulsion formulations. It was found that the oil phase is miscible with Vitamin E TPGS, Transcutol, Span 20, Tween 80 selected as surfactants and PEG 400, propylene glycol as co-surfactants and formed a transparent solution. Table 3 summarizes the composition of the different nanoemulsion formulations of AmB. The different surfactants and co-surfactants were selected on the basis of their non-irritant nature to skin, pharmaceutical acceptability and GRAS status. In addition, the components were also selected on the basis of their skin permeation enhancement effects. For instance, oleic acid, which is used as a part of oil phase, is known to act as skin penetration enhancer by increasing the fluidity of intercellular lipid barriers in the stratum corneum.⁴¹

Table 1 Solubility of AmB in different oils, their combinations, surfactants and co-surfactants^a

Oils	Solubility (mg mL^−1)	Observations
a Values are represented as the mean ± standard deviation (n = 3).
Soyabean oil	0.238 ± 0.01	Clear single phase
Oleic acid	0.282 ± 0.02	—
Olive oil	0.617 ± 0.03	—
Isopropyl myristate	0.424 ± 0.01	—
Sesame oil	0.0024 ± 0.01	—
Castor oil	0.0219 ± 0.01	—
Arachis oil	0.0069 ± 0.001	—
Rapeseed oil	0.157 ± 0.005	—
Oleic acid : olive oil (1 : 1)	0.385 ± 0.004	—
Oleic acid : olive oil (1 : 2)	0.562 ± 0.007	—
Oleic acid : olive oil (2 : 1)	0.329 ± 0.004	—
Olive oil : oleic acid : DMSO (2 : 2 : 1)	Stable (12.50 ± 0.08 mg mL^−1)	—
Olive oil : IPM : DMSO (2 : 2 : 1)	—	Phase separation
Olive oil : oleic acid : IPM (2 : 2 : 1)	2.315 ± 0.08	—
Oils with DMSO
IPM : DMSO (5 : 1)	—	Phase separation
Olive oil : DMSO (5 : 1)	—	Phase separation
Olive oil : IPM : DMSO (2 : 2 : 1)	—	Phase separation
Olive oil : oleic acid : DMSO (2 : 2 : 1)	12.5 ± 0.08 mg mL^−1	Stable
Oleic acid : IPM : DMSO (2 : 2 : 1)	—	Phase separation
Surfactants & co-surfactants
Tween 20	0.332 ± 0.01	Clear single phase
Tween 80	0.399 ± 0.01	Clear single phase
Span 20	0.216 ± 0.06	Clear single phase
Span 80	0.205 ± 0.05	Clear single phase
Transcutol	0.407 ± 0.04	Clear single phase
PEG 400	0.346 ± 0.03	Clear single phase
Propylene glycol	0.541 ± 0.03	Clear single phase
Isopropyl alcohol	0.375 ± 0.01	Clear single phase
Vitamin E TPGS (10 mg mL^−1)	0.424 ± 0.01	Clear single phase

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Table 2 Results of the miscibility study of selected oil phase, surfactants and co-surfactants

Group	Oil	Surfactants	Co-surfactants	Remarks
I	Olive oil : oleic acid : DMSO	Vitamin E TPGS, Span 20	PEG 400, propylene glycol	Translucent
II	Olive oil : oleic acid : DMSO	Vitamin E TPGS, Span 80	PEG 400, propylene glycol	Translucent
III	Olive oil : oleic acid : DMSO	Vitamin E TPGS, Span 20, tween 20	PEG 400, propylene glycol	Transparent
IV	Olive oil : oleic acid : DMSO	Vitamin E TPGS, Span 20, tween 80	PEG 400, propylene glycol	Transparent
V	Olive oil : oleic acid : DMSO	Vitamin E TPGS, Span 20, tween 80, transcutol	PEG 400, propylene glycol	Transparent

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Table 3 Composition of various AmB nanoemulsion formulations

Formulation Code	Oil^a (%)	Smix (%)	Smix ratio^b	Water (%)	Drug (%)
a Combination of olive oil, oleic acid and DMSO in a ratio of 2 : 2 : 1.b Surfactant : co-surfactant ratio.
ANE1	11	30	2 : 1	58.9	0.1
ANE2	11	32	2 : 1	56.9	0.1
ANE3	11	34	2 : 1	54.9	0.1
ANE4	11	36	2 : 1	52.9	0.1
ANE5	11	38	2 : 1	50.9	0.1
ANE6	11	40	2 : 1	48.9	0.1
ANE7	11	42	2 : 1	46.9	0.1
ANE8	11	44	2 : 1	44.9	0.1
ANE9	11	46	2 : 1	42.9	0.1

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Vitamin E TPGS is a water-soluble derivative of natural Vitamin E, which is formed by the esterification of Vitamin E succinate with polyethylene glycol 1000 and possesses surfactant properties. Considering its biocompatibility, amphiphilicity and low molecular weight, Vitamin E TPGS was selected for the preparation of the nanoemulsion formulation as it will act as a surfactant and as well as skin permeation enhancer. The choice of co-surfactant is also as critical as that of the surfactant because the co-surfactant enhances the penetration of the oil phase in the hydrophobic zone of surfactant monomers, reducing interfacial tension and in turn increases the entropy of the system.⁴² Three co-surfactants, namely, isopropyl alcohol, polyethylene glycol (PEG), and propylene glycol were selected in the study. The ratio of surfactant to co-surfactant and their type were optimized on the basis of a phase diagram study. Pseudoternary phase diagrams were constructed for the surfactant and co-surfactant at different ratios (1 : 1, 1 : 2, 2 : 1, 3 : 1 and 1 : 3), as shown in Fig. 1A–E. The nanoemulsion region was highest in the case of a 2 : 1 ratio of surfactant/co-surfactant, thus the same was optimized leading to formation of a clear, transparent and stable nanoemulsion with a good water dispersibility. The type of co-surfactant was also optimized on the basis of phase diagram study, as shown in Fig. 2A–C. It was observed that the percentage area of nanoemulsion region in the phase diagrams was widespread (i.e., it resulted in large existence area of nanoemulsion) for propylene glycol, which was in turn selected as the co-surfactant.

Fig. 1 Pseudoternary phase diagrams for the optimization of the S_mix ratio (A) 1 : 1 (B) 1 : 2 (C) 2 : 1 (D) 1 : 3 (E) 3 : 1.

Fig. 2 Pseudoternary phase diagrams for the optimization of the type of co-surfactant (A) isopropyl alcohol (B) PEG (C) propylene glycol.

3.2 In vitro characterization of the nanoemulsions

Table 4 summarizes the different parameters for the in vitro characterization of the nanoemulsion formulations. Particle size has been found to affect the transparency, stability, creaming of emulsion and degree of drug absorption through skin. The smaller the droplet size, the larger the interfacial area for percutaneous absorption and more is the stability; moreover, it was found to be in the range between 21.54 ± 0.8 and 105.41 ± 1.5 nm. Low values of the polydispersity index, ranging from 0.201 to 0.328 indicated the uniformity, stability and homogeneity of the nanoemulsions. Zeta potential is a very good index of the magnitude of the interactions between the colloidal particles and its measurement is commonly used to assess the stability of a nanoemulsion. The value for the zeta potential was found to be between −30.7 and −34.2 mV. As the surfactant concentration increased, there was a decrease in the average globule size of the nanoemulsion, which might be due to the effective interfacial activity of the surfactant that influences the droplet size. The optimized concentration of the S_mix ratio was found to be 42% v/v for the preparation of a transparent thermodynamically stable nanoemulsion with small globule size. Further increase in the surfactant concentration beyond 42% v/v produced nanoemulsions with grade B water dispersibility.

Table 4 Characterization of the AmB nanoemulsion formulations^a

Form. code	%Transparency	Globule size (nm)	Zeta pot. (mV)	PDI	Drug content (%)	Viscosity (cp)	Centrifugation (10 000 rpm)	Freeze–thaw cycle	Dispersibility
a Values are represented as the mean ± standard deviation (n = 3).
ANE1	80.6 ± 3.9	105.41 ± 1.5	−30.7 ± 1.8	0.313	98.5 ± 1.4	50.2 ± 1.2	Stable	Pass	Grade A
ANE2	85.2 ± 4.2	85.34 ± 1.8	−31.8 ± 2.1	0.201	97.2 ± 1.9	95.8 ± 2.6	Stable	Pass	Grade A
ANE3	89.5 ± 4.6	72.15 ± 0.6	−30.6 ± 1.5	0.328	99.1 ± 2.1	162.3 ± 2.9	Stable	Pass	Grade A
ANE4	90.5 ± 4.7	64.62 ± 0.8	−31.9 ± 2.2	0.215	96.3 ± 1.6	228.4 ± 3.2	Stable	Pass	Grade A
ANE5	91.3 ± 3.5	48.38 ± 0.9	−32.1 ± 1.7	0.244	98.2 ± 1.5	261.3 ± 3.0	Stable	Pass	Grade A
ANE6	93.6 ± 4.6	36.43 ± 0.7	−33.7 ± 1.2	0.217	95.7 ± 1.9	243.3 ± 2.8	Stable	Pass	Grade A
ANE7	99.3 ± 3.2	21.64 ± 0.8	−32.6 ± 1.1	0.204	97.3 ± 1.4	254.2 ± 2.9	Stable	Pass	Grade A
ANE8	98.5 ± 4.1	23.76 ± 0.5	−32.1 ± 1.5	0.211	99.5 ± 1.3	1025 ± 11	Stable	Pass	Grade B
ANE9	98.6 ± 4.5	21.54 ± 0.9	−34.2 ± 1.1	0.235	98.8 ± 1.8	1664 ± 14	Stable	Pass	Grade B

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Table 4 also summarizes the results of the dilution integrity test for the nanoemulsion formulations. Dilution integrity is an important characteristic of a nanoemulsion formulation as there is every possibility of phase separation, leading to the precipitation of a poorly soluble drug as the nanoemulsion are formed at a particular concentration of oil, surfactant and water. The process is thermodynamically driven by the requirement of the surfactant to maintain an aqueous phase concentration equivalent to its CMC.⁴³ All the nanoemulsion formulations were found to have a grade A dispersibility, which may be attributed to the lower interfacial tension between the oil and aqueous phase, and the formation of a thermodynamically stable emulsion.⁴⁴

All the prepared nanoemulsions remained stable and no physical change was observed after subjecting them to stress conditions of heating, cooling and freeze thaw cycles in the thermodynamic stability tests followed by centrifugation. No significant difference (P < 0.05) in particle size and polydispersity index was found after testing. The results of TEM indicated the spherical shape of the globules, as shown in Fig. 3. From the above mentioned findings, the nanoemulsion ANE7 composed of S_mix (42% v/v), oil (11% v/v), water (45.9% v/v) and drug (0.1% w/v) was optimized depending upon its superior properties, such as small globule size, polydispersity index, in vitro dispersibility, good flow properties and thermodynamic stability, for the preparation of an nanoemulsion based nanogel formulation.

Fig. 3 TEM morphological observation of the prepared nanoemulsion (ANE7). (Magnification 200 000×).

3.3 Preparation and characterization of an nanoemulsion based bioadhesive nanogel formulation

For the effective treatment of localized cutaneous fungal infections, it is mandatory for a formulation to be retained on the infected site for a sufficient period of time in order to get the maximum therapeutic effect. Nanoemulsions possess very low viscosity so their topical application suffers from drawbacks such as poor retention at the application and disease area, loss of drug, inconvenience in application and inaccurate dosing. Therefore, for proper topical application of an nanoemulsion formulation with good adhesiveness and retention at the infected site, it is vital to use a gel base for the preparation of the nanoemulsion based nanogel. The optimized nanoemulsion formulation ANE7 with superior properties, such as small globule size, polydispersity index, good dispersibility, good flow properties and thermodynamic stability, was selected for the preparation of the nanoemulsion based nanogel formulation. Carbopol 934P and Carbopol 940P at six different concentrations from 0.25 to 1.75% were selected as the gel base due to their ability to from a transparent, pharmaceutically elegant bioadhesive gel, surfactant properties and non-irritant nature. Table 5 summarizes the composition and in vitro characterization of the nanoemulsion based nanogel formulations. Nanogel formulations were characterized for drug content, appearance, pH, extrudability and detail rheological characteristics. Morphological analysis showed that the nanogel made with Carbopol 940P is more transparent in nature in comparison to Carbopol 934P. The pH of all prepared nanogels was measured and was found to be within neutral range, i.e., 6.7 to 6.9, which is close to the normal pH of skin. The prepared topical nanogels were also investigated for drug content, appearance, extrudability and transparent nature.

Table 5 Composition and characterization of the AmB nanoemulsion based nanogel formulations^a

Form. code	Type of gelling agent	Gelling agent con. (%)	Description	pH	Drug content (%)	Viscosity (cp)	Extrudability
a Values are represented as the mean ± standard deviation (n = 3). * Good, ** very good, ***excellent.
ANG1	Carbopol 934P	0.5	Translucent & having low viscosity	6.8 ± 0.02	98.5 ± 0.5	1415 ± 11	**
ANG2	—	0.75	Translucent & having low viscosity	6.7 ± 0.01	97.4 ± 0.7	2626 ± 19	***
ANG3	—	1	Translucent & having low viscosity	6.9 ± 0.01	98.5 ± 0.2	3946 ± 17	***
ANG4	—	1.25	Translucent & having good viscosity and good consistency	6.8 ± 0.03	98.3 ± 0.8	4922 ± 20	***
ANG5	—	1.50	Translucent & having good viscosity and excellent consistency	6.9 ± 0.02	99.3 ± 0.5	6160 ± 36	***
ANG6	—	1.75	Gel is slightly hard	6.7 ± 0.03	98.6 ± 0.8	7062 ± 27	**
ANG11	Carbopol 940P	0.5	Transparent & having low viscosity	6.7 ± 0.02	96.7 ± 0.6	1781 ± 14	**
ANG12	—	0.75	Transparent & having low viscosity	6.6 ± 0.03	98.4 ± 0.4	3330 ± 21	***
ANG13	—	1	Transparent & having low viscosity	6.3 ± 0.04	97.7 ± 0.7	5556 ± 28	***
ANG14	—	1.25	Transparent & having good viscosity and good consistency	6.9 ± 0.08	99.1 ± 0.5	6489 ± 34	***
ANG15	—	1.50	Transparent & having good viscosity and excellent consistency	6.8 ± 0.04	98.8 ± 0.6	7174 ± 39	***
ANG16	—	1.75	Gel in slightly hard	6.7 ± 0.06	97.4 ± 0.8	8259 ± 35	**
Marketed gel			Opaque gel	6.8 ± 0.02	100.4 ± 0.4	6279 ± 23	**

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3.4 Rheological behavior of the nanogels

Table 6 shows the detailed rheological characterization of the nanogels wherein two gel formers, Carbopol 934P and Carbopol 940P, in a concentration range from 0.5 to 1.75% w/w were selected. Rheological characterization is an important quality control test for topical gel formulations. In the present study, complete rheological characterization of the nanogels has been performed with respect to parameters, such as viscosity, yield stress and thixotropy, and the results are summarized in Table 6. The viscosity determined at constant SR and variable SR suggested the non-Newtonian shear thinning flow behavior of the nanogels characterized by a decrease in viscosity with an increase in shear rate. The shear thinning property is a desirable characteristic for semi solid topical formulations, which permit a maximum area coverage and easy application upon topical administration.⁴⁵ The appropriate adhesion and retention on an application site depends on the viscosities of the gels. While evaluating the viscosities, the Carbopol 934P and 940P based formulation showed a comparable viscosity to that found for the marketed gel at a concentration of 1.25% and 1.5% w/w, respectively.

Table 6 Rheological and texture behavior of the nanogels and marketed gel formulations^a

Form. code	Viscosity at constant SR(40)	Viscosity at variable SR(0–50)	Yield stress (Pa)	Thixotrophy (%recovery)	Cohesiveness	Gumminess	Springiness	Adhesiveness	Hardness
a Values are represented as the mean ± standard deviation (n = 3).
ANG1	1415 ± 11	1826 ± 13	1.28 ± 0.1	99.12 ± 1.2	0.9455 ± 0.01	0.0128 ± 0.0002	1.0093 ± 0.008	−0.033 ± 0.001	0.0135 ± 0.0001
ANG2	2626 ± 19	3487 ± 19	62.95 ± 1.1	98.99 ± 1.5	0.9486 ± 0.02	0.0229 ± 0.0003	1.01860 ± 0.009	−0.117 ± 0.001	0.0241 ± 0.0005
ANG3	3946 ± 17	5316 ± 22	84.21 ± 1.4	99.73 ± 1.6	0.9518 ± 0.03	0.0312 ± 0.0005	0.9771 ± 0.006	−0.217 ± 0.002	0.0328 ± 0.0007
ANG4	4922 ± 20	6580 ± 36	97.60 ± 1.7	99.51 ± 1.9	0.8962 ± 0.02	0.0377 ± 0.0004	0.9752 ± 0.005	−0.28 ± 0.003	0.0421 ± 0.0009
ANG5	6160 ± 36	8248 ± 38	112.08 ± 2.0	100.75 ± 1.3	0.8999 ± 0.02	0.0439 ± 0.0006	0.9506 ± 0.007	−0.319 ± 0.005	0.0499 ± 0.0008
ANG6	7062 ± 27	9364 ± 48	156.96 ± 2.6	99.91 ± 1.6	0.8719 ± 0.01	0.0446 ± 0.0006	0.9609 ± 0.006	−0.31 ± 0.004	0.0512 ± 0.0009
ANG11	1781 ± 14	2221 ± 19	61.51 ± 1.0	99.15 ± 1.4	0.9396 ± 0.03	0.0203 ± 0.0002	0.9789 ± 0.007	−0.105 ± 0.001	0.022 ± 0.0004
ANG12	3330 ± 21	4509 ± 24	81.58 ± 1.3	98.52 ± 1.3	0.9430 ± 0.04	0.0279 ± 0.0003	0.9796 ± 0.008	−0.188 ± 0.001	0.030 ± 0.0006
ANG13	5556 ± 28	7641 ± 37	99.41 ± 1.9	99.21 ± 1.8	0.9206 ± 0.02	0.0386 ± 0.0004	0.9536 ± 0.006	−0.302 ± 0.002	0.042 ± 0.0007
ANG14	6389 ± 34	8966 ± 39	109.51 ± 2.1	99.80 ± 1.6	0.9497 ± 0.03	0.0424 ± 0.0005	0.9685 ± 0.007	−0.317 ± 0.002	0.045 ± 0.0005
ANG15	7174 ± 39	9705 ± 46	126.83 ± 2.5	101.37 ± 1.9	0.9242 ± 0.02	0.0504 ± 0.0006	0.9501 ± 0.006	−0.365 ± 0.003	0.055 ± 0.0007
ANG16	8259 ± 35	11 010 ± 41	147.51 ± 2.6	99.50 ± 1.2	0.9203 ± 0.04	0.0503 ± 0.0004	0.9483 ± 0.005	−0.357 ± 0.003	0.057 ± 0.0008
Marketed	6279 ± 23	8277±	73.84 ± 1.7	89.84 ± 1.1	0.8961 ± 0.01	0.0411 ± 0.0003	0.9696 ± 0.007	−0.3 ± 0.002	0.0459 ± 0.0004

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The prepared nanogels were also evaluated for yield stress and thixotrophic behavior, as the yield stress reflects the firmness of the gel and the force required for a gel to flow out of a container whereas the thixotrophic behavior, which was also determined, reflects the recovery of the gel when sheared, i.e., the viscosity of the gel decreases with an increase in shear stress. After the removal of shear stress, the viscosity regains its formal state under isothermal conditions.⁴⁶ Like the shear thinning property of gels, thixotropy is also a key parameter for the effective application and maximum therapeutic effect of a topical formulation. This is because when the gel is subjected to shear force, its network structure collapses leading to a gradual decrease in viscosity in order to spread on the skin. However, on the removal of shear force, the viscosity recovers slowly and the increase in viscosity prolongs the contact time of the preparation at the affected site by staying on the skin.⁴⁵

During thixotrophic analysis, the percentage recovery for the nanogel was found to be 99.80 ± 1.6%, while the marketed gel showed a value of 89.84 ± 1.1% indicating the better recovery of the nanogel as compared to the marketed gel upon stressed conditions. The yield stress value of the nanogel, i.e., 109.51 ± 2.1 Pa indicated the retention of the gel in the container when placed in an inverted position and it matches the reported ideal value for topical gels of 110 Pa.⁴⁷

The mechanical properties of the nanogels were also evaluated using a texture analyzer and the results summarized in Table 6. The adhesiveness of the gel increased with an increase in the gel former concentration reflecting the constant contact of the preparation with human skin and was found to be −0.317 for the nanogel and −0.3 for the marketed gel. The hardness of a gel indicates the force required for a probe of the texture analyzer to easily penetrate into the gel without obstruction and reflects the compactness of the gel. The cohesiveness and hardness values for the nanogels and marketed gel were found to be 0.9497 ± 0.03, 0.045 ± 0.0005 and 0.8961 ± 0.01, 0.0459 ± 0.0004, indicating comparable values. Thus, the gel former selected for the nanogel preparation showed good rheological behavior as well as mechanical properties.

3.5 Ex vivo skin permeation and drug deposition studies

The skin permeation parameters of the nanogel were determined using a porcine ear skin. Porcine skin has been selected as it revealed to have similar histological and physiological properties to that of human skin, which suggested it as a good model for the skin permeation study.⁴⁸ Steady state transdermal flux value was found to be 4.78 ± 0.03 and 1.51 ± 0.03 μg cm⁻² h⁻¹ for the nanogel and marketed gel formulations, respectively. The value of the permeability coefficient was found to be 9.6 ± 0.7 and 3.0 ± 0.5 cm h⁻¹ × 10³ for the nanogel and marketed gel formulation, respectively. The drug deposition study accounts for the drug retained in the skin layers after topical application. For the antifungal drug to be highly effective and therapeutically potent, it should have a trait to deposit in the layers of the skin and be available for a longer period of time. Table 7 depicts the percentage of drug deposited from the nanogel as well as the marketed gel. The enhancement ratio of transdermal flux and the amount of drug deposited from formulation ANG14 to the marketed formulation was found to be 3.2 and 3.9, respectively. This significant enhancement in skin permeation flux and deposition potential is due to the synergistic effect of using the nanoemulsion as the drug carrier, the permeation enhancement effect of the components in the nanoemulsion such as Vitamin E TPGS and the prolonged retention of drug on the application site due to the bioadhesive nature of Carbopol. The nanometric size of the nanoemulsion droplet, large surface area, presence of the drug in a solubilized form and sustained release of the drug facilitates better transport of the drug from the vehicle and across the skin, which contributes to the enhanced skin permeation and deposition of the nanogel formulation.

Table 7 Skin permeation and deposition of the different AmB formulations across porcine ear skin^a

Form. code	Jss^1* (μg cm^−2 h)	LT^2* (h)	P^3* (cm h^−1) × 10^3	D^4* (cm^2 h^−1) × 10^4	Drug deposited (μg)	ER^1	ER^2	ER^3	ER^4
a Values are represented as the mean ± standard deviation (n = 3). Jss^1* = steady state transdermal flux calculated from the slope of the Cartesian plot of the cumulative amount of drug present in the receptor compartment versus time. LT^2* = lag time, P^3* = permeability coefficient calculated using the formula Jss/cd (cd is the amount of drug applied in the donor compartment), D^4* = diffusion coefficient (cm^2 h^−1) calculated using the formula Jss = dk/h × cd (h is the thickness of rat skin), ^5* = drug solution in DMSO. ER^1 = enhancement ratio, it is the ratio of steady state transdermal flux from the formulation to the marketed formulation, ER^2 = enhancement ratio, it is the ratio of steady state transdermal flux from the formulation to the marketed formulation, ER^1 = enhancement ratio, it is the ratio of amount of drug deposited into from the formulation to the marketed formulation, ER^2 = enhancement ratio, it is the ratio of amount of drug deposited onto the skin from the formulation to the marketed formulation.
ANE7	5.89 ± 0.04	1.9 ± 0.3	11.8 ± 0.8	19.7 ± 1.8	263 ± 2.6	16.5	3.9	20.2	3.6
ANG	4.78 ± 0.03	2.1 ± 0.2	9.6 ± 0.7	17.9 ± 1.5	291 ± 2.4	13.4	3.2	22.4	3.9
Marketed cream	1.51 ± 0.03	2.8 ± 0.2	3.0 ± 0.5	13.4 ± 1.1	74 ± 1.5	4.2	—	5.7	—
^5*Drug solution	0.36 ± 0.02	3.2 ± .0.4	0.7 ± 0.2	11.7 ± 0.9	13 ± 0.6	—	0.2	—	0.2

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AmB is a high molecular weight, highly hydrophobic drug and is very slowly permeated by a passive diffusion process. In the conventional marketed formulation, the drug is dispersed in an ointment and gel base. As the drug itself has very poor skin permeation due to its physicochemical properties, the marketed gel base does not contribute much towards increasing its skin permeation. In the nanoemulsion, the drug is present in a solubilized form and is thereby thermodynamically more active. Moreover, it contains Vitamin E TPGS that also acts as a skin permeation enhancer, by which its stratum corneum lipid perturbation effect results in the increased skin permeation of AmB.

3.6 Mechanistic study

FTIR and SEM analyses were carried out to shed some insight into the mechanism of the improved skin permeation of the nanogel formulation. Fig. 4 shows the FTIR spectra of skin treated with phosphate buffered saline (pH 7.4) as a control, the drug solution, nanogel and marketed AmB gel formulation. Distinguishing peaks of hydrocarbon chains of SC were obtained at 2950 cm⁻¹ and 2850 cm⁻¹ owing to the asymmetric and symmetric C–H vibrations, respectively. The extraction of skin lipids and lipid perturbation effects can be described by variations in the height, area and displacement of these two peaks. Application of the nanogel formulation considerably abridged the extent of these peaks demonstrating the extraction of skin lipids and perturbation effects. In addition, peaks at 1637 and 1550 cm⁻¹ represented amide 1 (C=O stretching) and amide 2 (C–N stretching) linkages of the helical secondary structure found in epidermal protein keratin, present mainly in corneocyte cells of stratum corneum and described the confirmation of skin proteins. A notable reduction in the intensity of these peaks at 1637 and 1550 cm⁻¹ was observed with nanogel formulation in comparison to the marketed formulation and drug solution. Therefore, the enhanced skin permeability may be a consequence of either the disruption of highly ordered structure of stratum corneum lipid or the interactions with intracellular proteins. These outcomes clearly indicate the permeation enhancement effect of Vitamin E TPGS and the other components in the nanogel formulation. These results are well correlated with study of Hathout et al., in which they also confirmed skin lipid perturbation and extraction effects with Vitamin E TPGS application as a skin permeation enhancer.⁴⁹

Fig. 4 FTIR spectra of rat skin treated with the control, drug solution, marketed and nanogel formulations.

To further confirm the findings of the FTIR study, scanning electron microscopy was carried out for the skin samples treated with the different formulations. Fig. 5 shows the SEM photomicrographs of treated skin with phosphate buffered saline (pH 7.4) as a control, drug solution, nanogel and marketed AmB gel formulation. The nanogel formulation was found to affect the normal histology of the skin surface. Nanogel formulation showed the maximum lipid perturbation effect and interlamellar widening of surface lipid along with pore formation. It further revealed that the untreated and marketed formulation treated skin has closely attached cells in the stratum corneum, while the nanogel treated skin showed prominent loosening and creation of significant pores in the stratum corneum leading to a pathway for permeation of AmB from the nanogel through into the skin.

Fig. 5 SEM photomicrographs of untreated rat skin as the control (A) and skin treated with the drug solution (B) nanogel (C) marketed gel (D). (Magnification 150 000×).

3.7 CLSM study

Deep penetration of the nanogel in different layers of the skin was also assessed using CLSM. CLSM study has inimitable advantages in the determination of a quantitative estimation of depth and extent of penetration of a marker. It clearly shows the specific distribution of a marker in the different layers of the skin. In the present study, rhodamine 123 was selected as a fluorescence marker and the CLSM images of the treated skin samples were taken at four different wavelengths, as depicted in Fig. 6. Fig. 6 shows red color imaging as a result of a helium neon laser, which analysed the specimen at 633 nm. The intensity graph depicted by red color shows the highest fluorescence intensity (4000) with deepest skin permeation (500 μm) with the nanogel formulation. The penetration of the nanogel into the skin and the fluorescence intensity was 4 fold higher when compared to a simple gel formulation (130 μm). The value for the enhancement ratio found in the CLSM study is well correlated with results of the ex vivo skin deposition studies.

Fig. 6 CLSM images and fluorescence intensity spectra of rat skin treated with a fluorescence marker applied in nanogel (A) and simple gel base (B), respectively. Red colour image and line show the fluorescence intensity and imaging using a helium neon laser at 633 nm; blue colour image and line show the fluorescence intensity and imaging using a blue diode laser at 405 nm; green colour image and line show the fluorescence intensity and imaging using an argon laser at 488 nm; violet colour line shows fluorescence intensity and imaging using a violet diode laser at 405 nm; white and black are phase contrast images.

3.8 In vivo localization index

An in vivo local bioavailability measurement study was also conducted in rats to determine the permeation and deposition potential of the prepared nanogel by analyzing the distribution of drug applied on the skin. The amount of drug deposited in the different layers of skin and remaining on the outer surface of skin was measured and the results summarized in Table 8. The skin localization index of the nanogel was found to be 48.71%, whereas the marketed formulation showed a value of 14.92%, which confirmed the depot forming ability of the nanogel in higher percentage in the skin, which is a requisite for enhanced therapeutic fungicidal activity as cutaneous fungal infections affect the different layers of the skin.

Table 8 In vivo skin localization index for the AmB formulations^a

Form. code	%Fraction of applied dose found on the surface of the skin	%Fraction of applied dose found deposited onto skin (%)
a Values are represented as the mean ± standard deviation (n = 3).
Drug solution	89.94 ± 1.4	10.06 ± 0.2
Nanogel ANG14	46.29 ± 0.9	53.71 ± 0.8
Marketed cream	82.41 ± 1.1	15.19 ± 0.2

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3.9 Antifungal activity

The microdilution and zone of inhibition methods were performed to determine the antifungal activity of the nanogel, commercial formulation and drug solution against Aspergillus niger and Candida albicans, and the results are summarized in Table 9. The results using the microdilution method depicted an MIC of 3.12 μg and 6.25 μg by AmB nanogel against Aspergillus niger and Candida albicans, respectively, and in comparison the plain drug solution of AmB showed a value of 6.25 μg and 12.5 μg against Aspergillus niger and Candida albicans, respectively. Fig. 7 depicts a graphical comparison of the antifungal activity as determined by a zone of inhibition method. The zone of inhibition against Aspergillus niger was found to be significantly (p < 0.05) higher in the case of the nanogel when compared to the marketed formulation and drug solution, whereas the control and dummy formulations did not show any zone of inhibition. The present results shows that a lower amount of AmB is required to impede the growth of fungi by including it in a carrier based nanogel formulation in contrast to the marketed formulation and drug solution. Consequently, a lower dose of AmB will be required, which will further diminish the usual side effects associated with the use of AmB.

Table 9 Minimum inhibitory concentration (MIC) of the different formulations of AmB against Candida albicans and Aspergillus niger^a

S. no.	Formulation	MIC (μg) against Aspergillus niger	MIC (μg) against Candida albicans
a Values are represented as the mean (n = 3).
1	Nanogel ANG14	3.12	6.25
2	Marketed gel	6.25	12.5
3	Drug solution	6.25	12.5

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Fig. 7 Observed zone of inhibition for different the formulations of AmB against Candida albicans (A) and Aspergillus niger (B).

The probable reason for the observed enhanced anti-fungal activity of AmB in the nanogel formulation is the extended contact and improved intracellular uptake of the drug from the formulation. Most nail, ear, skin surface, mouth, vaginal and burn wound fungal infections are caused by Aspergillus niger and Candida albicans. Demographic data demonstrated that these are the more prevalent surface fungal infections. AmB is not used as a primary drug for the treatment of these infections due to its poor skin permeation. The results of the present study demonstrate that the prepared nanogel formulation could be used as an effective treatment of these surface fungal infections.

3.10 Skin irritation study

The skin irritation potential of the developed nanogel formulation compared to the marketed formulation was determined by an acute and 28 day repeated dose application skin toxicity assay. Skin toxicity was evaluated in terms of edema and erythema using the scale described by Draize et al.³⁸ and the results presented in Table 10. SLS (20%) application was taken as a positive control, which resulted in dryness and hardening of the skin; the skin underneath became red, proving that SLS is an irritant.³¹ The application of the nanogel did not result in any dryness or redness of skin, in contrast, the marketed formulation showed slight erythema and edema, after 3, 7, 14, 21 and 28 days of application. Therefore, it can be concluded that the developed nanogel is non-sensitizing and safe for long-term use, as repeated application of an AmB topical formulation is required for the effective management of surface fungal diseases.

Table 10 Acute and sub-acute 28 days skin toxicity assay of the AmB formulations^a

Formulation code	Erythema						Edema
	1 day	3 day	7 day	14 day	21 day	28 day	1 day	3 day	7 day	14 day	21 day	28 day
a Values are represented as the mean (n = 3). Scores are defined as 0 = no erythema, 1 = very slight erythema (light pink), 2 = well defined erythema (dark pink), 3 = moderate to severe erythema (light red), 4 = severe erythema (extreme redness) and are similarly defined for edema.
Control	0	0	0	0	0	0	0	0	0	0	0	0
20% SLS (positive control)	0.66	2.66	3	3	4	4	1.66	3	3	3	4	4
Nanoemulsion ANE7	0	0	0	0.33	0.33	0.33	0	0	0	0	0	0
Nanogel ANG14	0	0	0	0.33	0.33	0.33	0	0	0	0	0	0
Drug solution	0.33	0.66	0.66	0.66	1.66	1.66	0	0	0	0.33	0.33	0.33
Marketed gel	0	0.33	0.66	0.66	1.33	1.33	0	0	0	0	0.33	0.33

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4 Conclusion

Due to its clinical effectiveness, AmB is widely used for treating both cutaneous and systemic infections and besides its prolonged use, not so many fungal resistance cases have been reported. However, the clinical use of AmB topically is limited due to insufficient skin permeation required to obtain a therapeutic effect. The combined application of a nanoemulsion and Vitamin E TPGS in the present research has significantly enhanced the skin permeation and deposition potential of the prepared nanoemulsion based nanogel when compared to the marketed topical formulation. The improved in vivo and ex vivo performance of the developed nanogel can be attributed to the presence of Vitamin E TPGS. An in vivo localization study confirmed the depot forming ability and sustained release of the nanogel in the layers of skin, which is a requisite for enhanced therapeutic fungicidal activity as cutaneous fungal infections affect the layers of the skin. It can be corroborated from the above findings that the use of Vitamin E TPGS in the nanoemulsion can dramatically increase the skin permeation and deposition of AmB in the skin leading to an improved therapeutic effect with a minimization of systemic side effects. Hence, the developed nanogel can be a better substitute for the marketed topical formulation.

Conflicts of interest

The authors confirm that this article content has no conflicts of interest.

Acknowledgements

The authors are thankful to the UGC, New Delhi, for providing financial assistance (Scheme no. 42-673/2013 (SR)). The authors are grateful to the UGC, New Delhi for issuing the UPE (University with potential for excellence) status to the University and providing the grant for the establishment of the center of emerging life sciences (equipped with sophisticated instruments).

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