Materials
Terbinafine hydrochloride was a gifted sample by Dr. Reddy’s Laboratories (Ameerpet, Hyderabad, Telangana, India), glyceryl tri(oleate-1-13C) was purchased from Sigma-Aldrich (Bangalore, India), Sephadex® G-50 was purchased from Sigma-Aldrich (Bangalore, India), and dialysis tubing cellulose membrane with flat width 10 mm (0.4 in.) was purchased from Sigma-Aldrich (Bangalore, India). The fungal strain Candida albicans (Robin) Berkhout (ATCC® 10231™) was a gift sample from RK University (Rajkot, India), synthetics non-human skin called Strat-M® membrane arranged from Millipore (Canada), and the rest of the samples were analytical grades and purchased from Rankem Laboratories (Gurugram, Haryana, India)
Preparation of terbinafine hydrochloride ufasomes
By using a thin-film hydration method, the terbinafine hydrochloride ufasomes were prepared in a round bottom flask. After slide modification of the Sharma et al. [14] procedure, new preparation steps were incorporated [24]. Initially, terbinafine hydrochloride and glyceryl oleate were dissolved in dichloromethane and kept in a round-bottomed flask for vacuum drying using a rotatory evaporator (IKA-RV 3 V-C Digital Rotatory Evaporator, Germany). The assembly was set for 50 rpm at 45 °C. After overnight vacuum drying, a dried thin film formed, indicating complete removal of residual solvent. The thin film was further hydrated for 12 h with freshly prepared pH 5.5 phosphate buffer solution. The mixture was further sonicated in 1.5 L Athena Ultrasonic Bath Sonicator for 15 min to form uniform vesicular dispersion. Also, optimization is performed by changing the ratios of terbinafine hydrochloride and glyceryl oleate. The manual optimization procedure followed, where different ratios of terbinafine hydrochloride and glyceryl oleate were screen for the best optimization batch. A total of five (UF1–UF5) formulations batches was prepared, and subsequently, the best formulation batch has been selected after suitable statistical screening (p < 0.05). The unentrapped drug was separated from the formulations using OMNISEC Gel permeation Chromatography (Malvern Panalytical, UK), considering borate buffer as eluent.
Vesicular size, zeta potential, and morphological characterization of glyceryl oleate vesicles
Delsa Nano C Particle Size and Zeta Potential Analyzer (Beckman Coulter, USA) was used for accurate vesicular size and zeta potential measurement [25]. For measuring the internal morphology and surface characterization of the vesicles, transmission electron microscopy (JEOL Transmission Electron Microscopes, USA) and mitotic compound microscope (BA210 LE, Germany) were used.
Entrapment efficacy
Entrapment efficacy was measured after exposing the vesicles in 1 N sodium hydroxide [26]. The release of the terbinafine hydrochloride was measured spectrophotometrically (UV-1280-UV-VIS Spectrophotometer, Shimadzu Life Science, Japan) at 283 nm. The following equation measured the entrapment efficacy:
$$ \mathrm{Entrapment}\ \left(\%\right)=\mathrm{Entrapped}\ \mathrm{drug}/\mathrm{Total}\ \mathrm{drug}\ \mathrm{incorporated}\times 100 $$
Differential scanning electron microscopy
To understand the thermal behavior of drug, drug-loaded vesicles, and blank vesicles, differential scanning electron microscopy was performed (Q-10, TA instrument) [22]. Calibration of heat flow is compulsory before initializing the process. Sample purging was done using inert nitrogen gas at a flow rate of 30 ml/min. The temperature was raised at 20 °C/min increments.
In vitro drug release studies
The dialysis tubing cellulose membrane with flat width 10 mm (0.4 in) was used for the in vitro dialysis of glyceryl oleate ufasomes. For proper execution of this study, 5 mL glyceryl oleate ufasomes were taken into dialysis tubing and both ends were tied using nylon thread and dispersed into a conical flask comprising of 50 mL pH 5.5 phosphate buffer solution containing 0.01% sodium lauryl sulfate. The conical flask was further incubated into an incubated shaker for 15 min in 37 °C at 60 rpm. Also, a 5-mL sample was withdrawn from the bottle, and 5 mL of fresh buffer was added into the conical flask. The withdrawn sample was filtered and determined spectrophotometrically at 283 nm.
In vitro antifungal activity of glyceryl oleate ufasomes
The cup plate method was incorporated to estimate the antifungal activity of glyceryl oleate ufasomes. Initially, Candida albicans was first grown overnight and inoculated into Sabouraud dextrose agar (SDA) media plates. Using cork borer, 10-mm diameter, three wells were introduced after solidification of Candida albicans in media plates. In the first good glyceryl oleate, 30% w/w ufasomes was placed as a test group; in the second well, plain 2% w/w terbinafine hydrochloride was placed in third pH 5.5 phosphate buffer solution was placed, which is considered a controlled group. The plates were incubated for 48 h at 37 °C. At different time intervals, the zone of inhibition was measured for the control, plan drug, and test sample.
In vitro skin permeation study for glyceryl oleate ufasomes
Using Franz diffusion cell (Dolphin Pharmacy Instruments Private Limited, India) in vitro skin permeation study for glyceryl oleate ufasomes (sample A), marketed gel (B-Fine, Cian Healthcare Limited, Mumbai, India) and plain drug of terbinafine hydrochloride (gift sample, Dr. Reddy’s Laboratories) were carried out. The diffusion area for the Franz diffusion cell was 4.5 cm2, and the 60-mL volume was kept inside. The penetration study was done using a synthetic non-animal-based model called 47 mm 60/pk Strat-M® membrane [27] (Millipore, Canada), which is specifically designed to mimic human skin. The most significant advantages of this synthetic skin are no need for an animal model, deficient human to animal skin variability alternative (CV = 8%), almost around 1.38 correlation to human skin, and longer self-life with no specific storage requirement. In the experimental procedure, the disk-shaped Strat-M® membrane was plucked using forceps and socked into a Petri dish, continuing 10 mL of saline water.
Further, the Strat-M® membrane was placed into the donner compartment of the Franz diffusion cell using forceps and maintain temperature around 35–37 °C. Thirty percent w/w optimized glyceryl oleate ufasomes, 1% w/w B-Fine Marketed gel (2.91 mg equivalent of terbinafine hydrochloride), and 2.91 mg of terbinafine hydrochloride were placed in the donner compartment of three different Franz diffusion cell. Nearly 0.5 mL of samples were withdrawn from the acceptor compartments for various time alternatives within 24 h using digital micropipettes (Thermo Fisher Scientific India Private Limited, Mumbai, India). The same amount of buffer solutions was added to the maintained sink condition in the acceptor compartment. The filtered samples were analyzed using UV-1280-UV-VIS Spectrophotometer (Shimadzu Life Science, Japan) at 283 nm.
Skin retention of vesicular dispersion
After performing in vitro skin permeation studies in three Strat-M® membranes [28], the utilized membranes were carefully removed from the Franz diffusion cell and subsequently chopped into small pieces. Now, 50 mL of pH 5.5 methanolic phosphate buffer (6:4) was added into the cut pieces and placed in shakers incubators with Vortexes Stirrer (Athena Technology, Mumbai, India) for 1 h at 37 °C. This procedure completely extracts drug from the embedded Strat-M® membrane. After extraction, the resultant solutions were filtered using a compact cooling centrifuge (Remi Elektrotechnik Limited, Mumbai, India) and using UV-1280-UV-VIS Spectrophotometer at 283 nm the embedded amount of drug estimated.
The skin retention was calculated using the following formula:
$$ \mathrm{Skin}\ \mathrm{retention}\ \left(\%\right)=\frac{amount\ of\ drug\ embedded\ in\ Strat-M\circledR Membranes}{Total\ drug\ incorporated}x\ 100 $$
In vivo studies
While performing animal studies, the Committee for Control and Supervision of Experiments on Animals (CPCSEA) guidelines issued by the Government of India followed. The animal ethical committee approved all the study protocol of ISF College of Pharmacy, Moga, Punjab, India.
In vivo antifungal activity of gel
In developing a model of immunocompromised animals, 5 mg/kg/bodyweight of Cyclosporine, a calcineurin Inhibitor, the injection was administered subcutaneously in 450–500 g weighted male Guinea pig. Cyclosporine was applied five times, i.e., 3 days before the injection of fungal suspension and 2 days after the inoculation of the suspension.
Preparation of Candida albicans inoculums
Candida albicans (Robin) Berkhout (ATCC® 10231™) was utilized to inoculate fungal infections in the Guinea pig. Initially, it is necessary to prepare a culture strain. For this purpose, sabouraud dextrose agar solution was used. The Candida albicans were inoculated in it for 48 h for 37 °C. The expressed cells were collected and resuspended in sterile saline. It was making sure that the final concentration in the sterile saline must be 107 colony-forming unit/mL (cfu/mL)
Initiation of fungal infection in the skin
Before shaving the animals back, it is necessary to anastasis the animal using 5–10 mg/k IM Ketamine. The back was shaved using NOVA Electric Trimmer Clipper. The total surface area trimmed was 3.0 cm2. Each animal body back surface area was inoculated with 107 cfu/mL of Candida albicans dispersion using a sterile cotton swab (socked in 70%v/v ethanol). The swab was rubbed on the surface of anesthetized animals unless and until no fluid certain in the animal body surface. Further, animals were caged separately. Animals were observed from time to time, and the sign of intense erythema was investigated into the site on the animal body.
Treatment of Candida albicans-induced infection
Once the symptoms of infections were palpable, the treatment would begin. The animals were devised into four groups. Each group contains four animals. The first group was treated with marketed B-Fine gel, equivalent to 5.8 mg of terbinafine hydrochloride (sample A); the second group was treated with terbinafine hydrochloride-loaded glyceryl oleate ufasomes, equivalent to 5.8 mg of terbinafine hydrochloride (sample B); the third group was treated with 5.5 mg of terbinafine hydrochloride, which was sprinkled on the surface of exposed skin (sample C); and finally the fourth group was exposed with the pH 5.5 phosphate buffer solution, which is considered a controlled group (sample D). Before applying samples from A to D in animal skin, one animal from each group was sacrificed to check the initial colony count (day 0), as prescribed in Aggarwal and Goindi [29]. For the 3 alternative days, animals were ingested with treatment once in a day. For the measurement of the colony count, 24 h after the last treatment, each animal from each group was sacrificed (day 1), subsequently after 72 h (day 3) and 120 h (day 5); the same procedure was followed for each and every group. Further, the treatment skin was separated from the animal body and carefully soaked in saline solution and homogenized. An aliquoted portion of homogenate was placed in sabouraud dextrose agar (SDS) plats for 48 h at 37 °C, and colony-forming unit (cfu) value was recorded using digital colony counter (J. S. Enterprises, New Delhi, India).
Statistical analysis
Using analysis of variance (ANOVA), the treatment group was analyzed with a controlled group. The multiple comparison test of ANOVA was performed in GraphPad Prism v.7.0 (San Diego, Canada). All the results were expressed in mean ± SD. The p value < 0.05 was considered significant.