Oleic acid vesicles: a new approach for topical delivery of antifungal agent

Abstract

The occurrence of topical fungal infections is increasing nowadays. Cutaneous fungal infections like cutaneous candidiasis are more prominent in patients associated with AIDS. Current available strategies for the treatment of cutaneous fungal infections are creams or gels which show various adverse effects on skin along with systemic absorption. These drawbacks can be overcome by using various novel drug delivery systems. So, the present investigation aims at exploring the potential of fatty acid vesicles (ufasomes) for the topical delivery of clotrimazole. Oleic acid was employed as a fatty material for the preparation of vesicles. Clotrimazole-loaded oleic acid vesicles were prepared using a thin film hydration method. Prepared vesicles were characterized for size, size distribution, shape, thermal behaviour (differential scanning calorimetry), in vitro release, in vitro antifungal activity, in vitro skin permeation and retention studies and for in vivo antifungal activity. Transmission electron microscopic (TEM) images confirmed the formation of vesicular dispersion (ufasomes) of clotrimazole. Oleic acid vesicles possessed high drug entrapment (49.5 ± 1.0%) and optimum size (455 ± 22 nm) along with good colloidal characteristics (polydispersity index = 0.210 ± 0.035 & zeta potential = − 22.45 ± 0.25 mV) at 4:6 drug-to-oleic acid ratio. In vitro drug release study showed sustained release of drug from the vesicular dispersion. Skin permeation and skin retention studies suggested accumulation of drug in the epidermal part of the skin. In vivo study confirmed prolonged release of drug from oleic acid vesicle up to five days indicating its usefulness for long-term therapy. So, it can be concluded from the present study that fatty acid vesicle may be a good approach to treat topical fungal infections.

Keywords::

• colloidal
• cutaneous
• fatty acid
• fungal infection
• oleic acid
• sustained release

Introduction

Skin is the outermost covering of human body performing various important functions, but its protective role is perhaps the most demanding. Stratum corneum is the outermost epidermal layer of skin acting as a principal regulatory barrier to the transcutaneous traffic of water and exogenous substances like bacteria or fungus (Downing 1992). There is alteration in the structure of stratum corneum when skin is attacked by external agent like fungus leading to change in permeability of skin (Golden et al. 1978). The incidences of fungal infections are increasing nowadays especially in the patients that are immunocompromised. This increase in the occurrence of topical/systemic fungal infection is due to coupling of causative fungus with the diseases like AIDS or due to excessive use of immunosuppressive drugs in this period of time when there is a technological advancement in the field of solid organ transplantation medicines, stem cell transplantation and neonatology (Li et al. 2007). Topical fungal infections are mainly found in the subcutaneous tissue. They may be invasive in nature and can also reach deep into epidermis (Kaur and Kakkar 2010). Oleic acid is generally employed as a penetration enhancer for delivery of different bioactives into the skin. Oleic acid induces penetration into skin due to subcutaneous lipid fluidization and phase separation (Naik et al. 1995). It has been reported that fatty acids like oleic acid and linoleic acid have tendency to form vesicular structures in the aqueous environment (Gebicki and Hicks 1973). Methotrexate-entrapped deformable liposomes prepared from phosphatidylcholine and oleic acid were compared with those of methotrexate-entrapped conventional liposomes prepared from phosphatidylcholine and cholesterol for skin permeation. Liposomes containing oleic acid showed high transdermal permeation as compared with the other (Srisuk et al. 2012). Oleic acid vesicles containing 5-flourouracil were developed for topical delivery. Results of the study showed that drug-loaded oleic acid vesicles effectively penetrated stratum corneum and formed drug depot in epidermal part of skin for localized delivery of drug (Dhillon et al. 2011). Methotrexate was entrapped into oleic acid vesicles (ufasomes) for the effective topical delivery against psoriasis. The methotrexate amount permeated through rat skin was three- to four-fold higher using oleic acid compared to that from plain drug solution or carbopol gel (Sharma and Arora 2012). Clotrimazole is a potent antifungal agent used against topical fungal infections. One of the most common problems with this drug is that patients often stop using it before the infection is completely removed, leading to re-infection in the body. It also shows absorption into the systemic circulation on its frequent dosing (Souto and Muller 2006).

It has been shown that double layer membranes possess fusogenic characteristics because they reduce phase transition temperature of the lipids in the biological membrane system. When this vesicular membrane comes into contact with skin, it shows its fusion with skin lipid bilayers and releases its content. So, it is considered that fatty acid vesicles are very effective carriers for enhancing the penetration of drug molecules through the stratum corneum with the reduction of toxicity. Fatty acid vesicles are cheaper in cost and their method of preparation is very easy. So in the present study we tried to develop an effective vesicular drug delivery system (ufasomes) of clotrimazole for targeting fungus in deeper epidermal layer of skin and to provide a local effect of drug in the skin with reduction of dose. For achieving the goal, oleic acid vesicles containing clotrimazole were prepared using thin film hydration method. The effects of drug-to-fatty acid ratio on size and entrapment efficiency of vesicles were studied. Vesicular dispersion of drug (Sample B) was compared with marketed formulation (Sample A) for in-vitro skin permeation and in-vivo antifungal activity. Results of in-vivo studies revealed the prolonged effect of clotrimazole ufasomes for 5 days in the treatment of experimentally induced cutaneous candidiasis. Thus, vesicular dispersion (ufasomes) of clotrimazole was found to be more effective as compared to marketed product

Materials and methods

Materials

Clotrimazole was provided as a gift sample by Edifice Labs (Ludhiana, India). Oleic acid was purchased from CDH (New Delhi, India). SephadexG-50 and dialysis membrane − 70 (LA393-10MT) was purchased from Himedia (Mumbai, India). Fungal strain Candida albicans was purchased from MTCC – Chandigarh, India. All other solvents used were of analytical grade and purchased from CDH (New Delhi, India).

Preparation of oleic acid vesicles

Oleic acid vesicles were prepared using film hydration method, as reported earlier (Sharma and Arora 2012, Murakami 1996). Briefly, oleic acid and clotrimazole were dissolved in methanol in a round-bottomed flask followed by evaporation of solvent under vacuum using a rotary evaporator (Perfit equipments, Ambala, India) to remove even the last traces of organic solvent. The completely dried film in rota-evaporator was left overnight for the removal of any possible traces of methanol and also to prevent the formation of emulsion due to the residual organic solvent. The dried film formed was then hydrated at ambient temperature for 2 h with phosphate buffer (pH 5.5). The prepared vesicular dispersion was sonicated to form the uniform size vesicular dispersion. Optimization was performed by varying the ratios of oleic acid to clotrimazole. Unentrapped drug was separated from the vesicle dispersion using gel chromatography (Sephadex G-50 minicolumn) and borate buffer as an eluant.

Entrapment efficiency

The entrapment efficiency was determined by disrupting the vesicles using sodium hydroxide solution (1 M) and subsequence estimation of released clotrimazole, as entrapped drug. Entrapment efficiency (%) was calculated using following formula:

Vesicle size, zeta potential and morphology of oleic acid vesicles

Vesicle sizes and zeta potentials of different formulations were determined using particle size analyzer (DelsaTM Nano C, Beckman Coulter). Shape and surface morphology of oleic acid vesicles was examined using transmission electron microscope (TEM) (HRTEM, H47500 Hitachi Ltd., Tokyo, Japan).

Differential Scanning Calorimetry

Thermal behaviour of drug, drug-loaded vesicles and blank vesicles were studied using differential scanning calorimeter (DSC) (Q-10, TA Instrument waters). Before adding sample for analysis, calibration of heat flow scale was done. The samples were purged with dry nitrogen at a flow rate of 20 ml/min. The temperature was raised at a rate of 10°C/min (Dhillon et al. 2011).

In vitro drug release study

In vitro release study of oleic acid vesicles was carried out using dialysis bag method (Dhillon et al. 2011). For this study, 5 ml of vesicular dispersion was added to dialysis membrane-70 (Himedia, LA393-10MT). Dialysis membrane was taken into 50 ml of phosphate buffer saline pH 5.5containing 0.01% SLS (Sodium lauryl sulphate), in a conical flask. The flask was kept in an incubator shaker and the speed of the shaker maintained was 60 rpm at 37°C. Samples (5 ml) were withdrawn and filtered. Samples were collected after different time intervals. Same volume (5 ml) of the phosphate buffer, pH 5.5, containing 0.01% SLS was replaced after each sampling. The drug content in the sample was determined spectrophotometrically at 265 nm.

In vitro antifungal activity of vesicular dispersion

In vitro antifungal activity of vesicular dispersion was determined using cup plate (cylinder plate) method. The overnight grown culture of Candida albicans was inoculated into the sterilized Sabouraud dextrose agar (SDA) media plates. After solidification, in each plate, three wells, each 10 mm in diameter, were bored with a cork borer. In each plate, vesicular dispersion in phosphate buffer of pH 5.5, plain drug or control was placed in one of the wells in appropriate amount and the plates were incubated at 37°C for 48 h. Phosphate buffer of pH 5.5 was used as a control. Zone of inhibitions of drug, control and vesicular dispersion were measured at different time intervals (El laithy and El-Shaboury 2002).

In vitro skin permeation studies of vesicular dispersion

In vitro skin permeation studies of vesicular dispersion (Sample B) and marketed gel (Sample A) (Candid-V, Glenmark) were carried out using Franz diffusion cell (Electrolab Ltd., Mumbai, India) with a diffusion area of 3.3 cm² and volume of 60 ml. Penetration was studied using abdominal skin of guinea pig. Hairs from the abdominal part of skin were carefully removed, and skin was excised from abdomen using surgical blade (Satturwar et al. 2005). Dermal side of skin was carefully cleaned. Dermis part of the skin was washed with a cotton swab soaked in isopropanol for the removal of fatty material (Bhatia et al. 2004). Later on skin samples were washed with normal saline and cut into appropriate sizes. Skin was placed on donor side of Franz diffusion cell, maintained at 37°C. Optimized vesicular formulation and marketed gel (equivalent to 7.37 mg of clotrimazole) were placed in the skin on donor side of Franz diffusion cell. A volume of 0.5 ml of sample was removed from the acceptor media at regular time interval for a period of 24 h and replaced with the same amount of buffer to maintain sink condition. Samples were analysed at 265 nm using UV spectrophotometry (Pierre et al. 2001).

Skin retention of vesicular dispersion

After performing skin permeation studies, skin was carefully removed from the Franz diffusion cell. Remaining formulation was washed with cotton swab soaked in phosphate buffer of pH 5.5. The cleaned skin piece was mashed, and 50 ml of methanolic PBS (4:6) of pH 5.5 was added to the meshed mass and mechanically shaken in a water shaker bath at 37°C for 1 h for the complete extraction of the drug. After extraction, the resulting solution was filtered and the amount of drug content in filtrate was determined using UV spectrophotometer at 265 nm (Agarwal and Katare 2002). Skin retention (in percentage) was calculated using the following formula:

In vivo studies

All animal studies were performed according to the guidelines compiled by the Committee for the Purpose of Control and Supervision of Experiments on Animal (CPCSEA, Ministry of Culture, Government of India). All the study protocols were approved by the animal ethical committee of the Abhilashi College of Pharmacy, Mandi (Himachal Pradesh), India.

In vivo antifungal activity of gel

Animals. Male guinea pigs weighing 400–450 g were used in the study. They were immunosuppressed by giving prednisolone injection subcutaneously (30 mg/kg of body weight). Prednisolone was administered four times (2 days and immediately before, and 2 and 4 days after the inoculation of fungal suspension) (Maebashi et al. 1995).

Preparation of fungal inoculums. Candida albicans (C. albicans MTCC Code-1637) fungus was used to induce fungal infection in guinea pigs. A culture of fungal strain was grown on Sabouraud dextrose agar (SDA) for 48 h at 37°C. Grown cells from SDA plates were collected and re-suspended in sterile saline. Final concentration adjusted in saline was 10⁷ colony-forming unit/ml (cfu/ml) (Gupta and Vyas 2012).

Induction of fungal infection in skin. Each animal's back was shaved using an electric clipper, and two hairless patches of area 2.0 cm² were developed there. Every patch was inoculated with 10⁷ cfu/ml fungal dispersion by using a sterile cotton swab. Dispersion was rubbed into skin with a sterile cotton swab until no visible fluid appears on skin. Assessment of topical fungal infection was done by checking sign of more intense erythema at inoculation site on animal body (Maebashi et al. 1995).

Treatment of fungal infection. Treatment began after 24 h of assessment of infection. Animals were divided into three groups each containing four animals. First group was treated with marketed formulation (Sample A) (Candid-V Gel, Glenmark) (equivalent to 6.52 mg of clotrimazole). Second group was treated with clotrimazole-loaded oleic acid vesicles (Sample B) (equivalent to 6.52 mg of clotrimazole) and third group served as a control. Phosphate buffer of pH 5.5 was used as a control. One animal from each group was sacrificed just before applying formulation to determine initial colony count before applying formulation (Day 0). Treatment was given once in a day for three consecutive days. After 24 h of last treatment, one animal from each group was sacrificed for colony count (Day 1). After 72 h (Day 3) and 120 h (Day 5) of last treatment, similar process was carried out. For colony count, infected skin of animal was excised and homogenized in sterile saline solution. A portion of homogenate was cultured in Sabouraud dextrose agar (SDA) plates for 48 h at 37 ± 1°C, and cfu values were recorded using digital colony counter (Maebashi et al. 1995, Gupta and Vyas 2012).

Statistical analysis

All the results are expressed as mean ± standard deviation. The treated groups were compared with control using analysis of variance (ANOVA) thanks to GraphPad PRISM software (GraphPad Software Corp., San Diego, CA). The p value < 0.05 was considered as significant.

Results and discussions

Shape and morphology of clotrimazole-loaded oleic acid vesicles

Clotriamzole oleic acid vesicles were prepared effectively used thin film hydration method. TEM image of clotrimazole-loaded vesicles is shown in Figure 1. This image clearly indicates that prepared fatty acid vesicles were spherical in shape and of size below 500 nm.

Figure 1. Transmission electron microscopic (TEM) image of optimized clotrimazole-loaded oleic acid vesicles at 80 kV and 50,000 ×.

Size, drug entrapment and colloidal behavior of vesicular dispersion

Prepared vesicular system was further characterized for size, drug entrapment, PDI and zeta potential. Table I represents optimization of oleic acid vesicles (Murakami 1996). It is clear from the table that maximum entrapment efficiency (49.55 ± 1.02) was found for formulation F4 having drug-to-oleic acid ratio of 4:6. It was seen that the drug-bearing capacity of the oleic acid vesicles depends upon the molar ratio of oleic acid to clotrimazole. The entrapment efficiency increased up to a drug/oleic acid molar ratio of 4:6, beyond this ratio further increase in the amount of drug reduced the degree of drug entrapment inside the vesicles. This may be due to the drug saturation in the bilayer domain. Further addition of drug could have destabilized the vesicle membrane leading to the leakage of drug . Formulation F4 was having least particle size as compared to other formulations which was considered good for the skin penetration (Sharma and Arora 2012). Formulation F4 showed good colloidal characteristics compared to other formulations so it was taken as optimized formulation for further studies.

Table I. Optimization of oleic acid vesicles. Values are expressed as mean ± standard deviation (n = 3).

Formulation code	Drug: Oleic acid	Size (nm)	Percentage of entrapment	PDI	Zeta Potential (mV)
F1	1:9	538 ± 15	38.2 ± 0.8	0.367 ± 0.078	‐32.68 ± 0.65
F2	2:8	495 ± 12	43.4 ± 1.3	0.390 ± 0.056	‐28.67 ± 0.34
F3	3:7	481 ± 18	47.9 ± 0.8	0.229 ± 0.076	‐30.78 ± 0.67
F4	4:6	455 ± 22	49.5 ± 1.0	0.210 ± 0.035	‐22.45 ± 0.25
F5	5:5	554 ± 15	17.3 ± 0.9	0.765 ± 0.045	‐32.43 ± 0.49

Differential Scanning Calorimetry

Thermal behaviour of drug, blank oleic acid vesicles and clotrimazole-loaded oleic acid vesicles was checked using DSC analysis. DSC thermograms for drug, blank oleic acid vesicles and clotrimazole-loaded oleic acid vesicles are shown in Figure 2 (A, B and C, respectively).

Figure 2. DSC thermograms of pure drug (A), blank oleic acid vesicles (B) and drug-loaded oleic acid vesicles (C). Sharp endothermic peak in thermogram A clearly indicates the melting point of the drug (146.05°C). A less sharp peak was observed at 285.54°C in the case of blank oleic acid vesicles (B). No peak related to drug was found in the drug-loaded vesicles(C).

Sharp endothermic peak in thermogram A clearly indicates the melting point of drug (146.05°C). A less sharp peak was observed at 285.54°C in case of blank vesicles (B). No peak related to drug was found in drug-loaded vesicles (C). These results clearly indicate that the drug was entrapped inside the microspheres. These results were in accordance with the previous findings (Dhillon et al. 2011).

In vitro drug release study

Drug release study was carried out using dialysis bag method. Results of in vitro drug release studies are shown in Figure 3. It is clear from the graph that all the vesicular formulations released 30–40% drug within 24 h. Kinetics of drug release of various formulations is shown in Table II. R2 value was found maximum in the case of zero-order graphs, which clearly indicates that the formulations follow zero-order release. The Korsmeyer–Peppas release exponent (n) was found in the range of 0.354–0.408, which confirmed diffusion as the principle mechanism of drug release.

Figure 3. In vitro drug release profiles of different vesicular formulations. Values are expressed as mean ± standard deviation (n = 3).

Table II. Kinetics of drug release of different formulations.

Formulation code	Higuchi (R^2)	Zero order (R^2)	First order (R^2)	Korsmeyer–Peppas release exponent (n)
F1	0.302	0.946	0.941	0.358
F2	0.299	0.935	0.932	0.367
F3	0.289	0.928	0.923	0.354
F4	0.318	0.980	0.953	0.408
F5	0.303	0.959	0.944	0.396

In vitro antifungal activity of vesicular dispersion

Results of in vitro antifungal activity are shown in Figure 4. No zone of inhibition was observed in case of control at different time intervals, while initially there was a sharp increase in zone of inhibition in case of plain drug as compared to vesicular dispersion. This is because oleic acid vesicles released drug in a controlled fashion and less in amount. But after 48 h, zone of inhibition was more in case of vesicular dispersion clearly indicating that there was a continuos controlled release of drug from dispersion up to 48 h.

Figure 4. In vitro antifungal activity of different formulations (plain dug and drug loaded oleic acid vesicles) against C. albicans. Values are expressed as mean ± standard deviation (n = 3).

In vitro skin permeation studies of vesicular dispersion

In vitro skin permeation studies were performed on hairless guinea pig skin using Franz diffusion cell. Results of permeation studies are shown in Figure 5. It is clear from the graph that amount of drug permeated from marketed gel was very high as compared to vesicular dispersion. Cumulative amount of drug permeated in 24 h from marketed gel (Sample A) was 53.67 ± 1.98% while from oleic acid vesicles (Sample B) it was near about 14.63 ± 1.53%. Drug permeated from vesicular dispersion was low because clotrimazole-loaded oleic acid vesicles increased the accumulation of drug in epidermal part of skin and thus decreased the permeation of drug through skin. Drug permeated from marketed gel was very high because drug in this formulation was in free form for penetration.

Figure 5. In vitro skin permeation of different formulations (marketed gel and drug-loaded oleic acid vesicles). Values are expressed as mean ± standard deviation (n = 3).

Skin retention of vesicular dispersion

Oleic acid vesicles showed drug reservoir effect in skin so percent deposition of clotrimazole in skin was also calculated and compared with marketed formulation. Amount of drug retained in skin after 24 h was 42.71 ± 1.31% for vesicular dispersion (Sample B) whereas for marketed gel (Sample A) (Candid-V, Glenmark), it was found to be 10.90 ± 1.54%. High drug retention in case of microspheres gel may be due to formation of drug reservoir in epidermis due to deposition of clotrimazole oleic acid vesicles. Figure 6 shows results of skin retention studies.

Figure 6. Percent drug retention of different formulations (marketed gel and drug-loaded oleic acid vesicles). Values are expressed as mean ± standard deviation (n = 3).

In vivo studies

In vivo antifungal activity of microsphere gel was determined by inducing fungal infection in animal with C. albicans. It is clear from the Figure 7 that vesicular dispersion possessed high therapeutic effectiveness as compared to other formulation for prolonged period of time. On Day 0 (24 h prior to treatment), fungal colony count in all three group appeared almost similar indicating that fungal infection was induced effectively in dermal layer. After the assessment of this colony count, treatment was started for three consecutive days.

Figure 7. In vivo antifungal activity of formulations (marketed gel and drug-loaded oleic acid vesicles). Values are expressed as mean ± standard deviation (n = 3).

On Day 1 (after 24 h of last treatment), control group showed a slight increase in fungal count. Animal group treated with marketed gel (Sample A) showed a significant reduction in fungal colony count on Day 1 which was due to immediate release of drug from the marketed formulation. Excess drug released from this formulation reduced fungal colony to a very less amount as compared to control. In case of vesicular dispersion (Sample B), reduction in colony count was less as compared to marketed formulation which may be due to less amount of drug released from it because oleic acid vesicles retarded the drug release.

Further, after 72 h of last treatment (Day 3) colony count was found to increase in case of marketed gel. This increase in colony count may be due to lack of drug on skin after 4 days because whole drug from marketed gel released on Day 1 and no drug was available for further action (P < 0.05). However, in case of vesicular dispersion, the colony count was low because of sustained release of drug. After 120 h (Day 5), colony count was very high for marketed gel compared to oleic acid vesicular dispersion indicating that the drug was still available in vesicular dispersion for further action (P < 0.01). So, there was a significant reduction in colony count in the case of oleic acid vesicular dispersion even after the fifth day of application. Furthermore, the results may be due to reservoir effect of oleic acid vesicles in dermal skin layer. So, the therapeutic efficacy of vesicular dispersion was found to be better as compared to marketed gel.

Conclusions

From the present study, it is clear that oleic acid vesicles can be used as an effective carrier for the delivery of antifungal agent for the treatment of localized fungal infections. These delivery system has different advantages like cost-effectiveness, therapeutic viability and reduction in the dose of drug. These delivery systems also showed good sustained release behaviour of drug and skin retention properties which proved their effectiveness for a long-term drug therapy. Oleic acid vesicles can easily distributed in skin and may form depots in the skin because of its better permeation properties in skin. All these studies proved the effectiveness of clotrimazole-loaded oleic acid vesicle for treating cutaneous skin fungal infections, and thus, it can be taken as a preferred choice of carrier system for drug delivery.

Acknowledgement

The authors want to acknowledge the help of Dr. R. K. Abhilashi, Chairman, Abhilashi College of Pharmacy, Himachal Pradesh, for providing excellent research facilities.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.