AAPS PharmSciTech

, Volume 19, Issue 3, pp 1297–1307 | Cite as

Nanolipid Gel of an Antimycotic Drug for Treating Vulvovaginal Candidiasis—Development and Evaluation

Research Article
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Abstract

This paper focuses on the development and evaluation of mucoadhesive vaginal gel of fluconazole using nanolipid carriers to enhance tissue deposition in treating vulvovaginal candidiasis. Treatment of vulvovaginal candidiasis includes antimycotic agents prescribed for 1 to 7 days or longer, in relapse either orally or topically. The delivery of fluconazole as nanolipid carriers in vaginal gel can be proposed as suitable alternative to the existing conventional formulations to improve the patient acceptability, compliance and localized drug action. The nanolipid carriers of fluconazole were prepared by phase inversion temperature technique and incorporated into Carbopol 974P as gelling polymer. GRAS excipients selected and optimized were Precirol ATO 5, oleic acid and Kolliphor RH 40 to produce nanolipid dispersions. Stable nanolipid dispersions were developed using sodium dodecyl sulfate as the charge inducer. The optimized nanolipid dispersion of fluconazole had particle size, polydispersity index and zeta potential value of 158.33 ± 2.55 nm, 0.278 ± 0.003 and − 27.33 ± 0.40 mV, respectively and the average entrapment of fluconazole in the lipid carriers was found to be 67.24 ± 0.87%. The optimized vaginal gel had satisfactory mucoadhesive strength and rheological properties to facilitate vaginal application. The fluconazole release from the gel was sustained showing 30.69 ± 1.02% drug deposition in the porcine vaginal mucosa at the end of 8 h with improved antifungal activity against Candida albicans during well diffusion studies. The optimized gel was non-irritant to the vaginal mucosa of female Wistar rats with no signs of erythema or edema.

KEY WORDS

nanolipid carriers fluconazole phase inversion temperature technique sodium dodecyl sulfate 

INTRODUCTION

During the childbearing years, about three fourths of women experience at least one episode of vulvovaginal candidiasis (VVC) possibly with relapse, during the third and fourth decades of life. Candida albicans is the species which is the major causative organism in most cases of VVC with rarely, involvement of other species of Candida such as C. glabrata or C. tropicalis. The colonization by opportunistic pathogenic Candida species contributes to the deterioration of vaginal mucosal surfaces which can serve as reservoir for further chronic infections and serious consequences such as sterility, infertility and immunosuppression. The treatment of vulvovaginal candidiasis includes antimycotic agents prescribed for 1 to 7 days or longer during relapse, either orally or topically (1, 2, 3). Though the oral route of drug delivery is most popular, it is supplemented by peripheral side effects especially when localized delivery of drugs at the desired site would be beneficial. In this case, delivery of the drugs to vaginal tissue would circumvent the unnecessary drug exposure to other regions of the body. Vaginal delivery of antimycotic agents by conventional methods, viz, creams, gels, lotions and emulsions limits the effectiveness of drug therapy as the active ingredients are provided in relatively high concentrations but for a shorter duration. This may allow proliferation of the microorganisms, causing deterioration of vaginal surfaces. Additionally, it may also lead to development of resistance due to cycles of short-term overmedication followed by long-term undermedication depending upon the frequency of usage (4). This highlights the need to develop vaginal drug delivery systems that would provide high mucosal tissue levels with lower systemic exposure for prolonged periods of time in treating vaginal candidiasis. Nanoparticulate carriers, owing to their small size, can effectively traverse and localize in many biological barriers and may also cause alteration in the microstructure of mucus, thereby enhancing drug delivery to the mucosal surfaces (5). The tunable size of these systems greatly influences the release profiles of entrapped drugs at the site of action that can be controlled, thereby reducing the frequency of usage for improving patient compliance. Nanolipid carriers contain mixtures of biocompatible lipids with emulsifiers and are in the size range of less than 500 nm. The presence of liquid lipids confers long-term colloidal stability and greater drug encapsulation and loading due to imperfect crystal lattices (6, 7, 8, 9). Vaginal gels are easily miscible with vaginal secretions as compared to creams and ointments and patients are known to tolerate them better than inserts or ointments (10). However, the vagina is a mucosal tissue and due to the self-cleansing action of the vaginal tract, the residence time of the delivery systems can be reduced (11,12). This can be challenging in achieving therapeutic drug concentrations in the vagina. To overcome the limitation of short residence time, an extended and intimate contact of the drug delivery system with the vaginal mucosa can be achieved using mucoadhesive polymers. Among the azole class of antifungals used for the treatment and prophylaxis of invasive fungal infections, fluconazole (FLZ) is available in the local market as tablets and conventional gels but not as nanocarriers incorporated in gels (13,14). Also, administration of oral azole antifungals during pregnancy is not recommended, when the vagina is particularly susceptible to Candida infections. Hence, topical vaginal delivery would limit most of the antifungal drugs in the vagina preventing its distribution into systemic circulation. Literature reports the safest course as first-line therapy for vaginal candidiasis during pregnancy is the use of topical delivery and no adverse effects on the human fetus have been attributed (15,16). Thus, the lipid nanocarriers-based gel of fluconazole (0.5% w/w) can be proposed to show enhanced therapeutic effects in combating vulvovaginal candidiasis.

MATERIALS AND METHODS

Materials

Fluconazole (FLZ) was received as a gift sample from Glenmark Research Center (Taloja, India). Precirol ATO 5, Compritol ATO 888, Apifil, Gelucire 50/13, Gelot 64, Labrafil M 1944 CS, Labrafac, Capryol 90, Labrasol and Transcutol HP were obtained as gift samples from Gattefosse India Pvt. Ltd. (Mumbai, India). Stearic acid, Kolliphor RH 40, Kolliphor PS 20, Kolliphor PS 60 and Tween 80 were obtained as gift samples from Signet Chemical Corporation (Mumbai, India). Imwitor 491, Miglyol 812 and Softigen 767 were obtained from Cremer Oleodivison (Germany). Sefsol 228 was obtained from Nikkol (Japan) and Captex 355 was obtained from Abitec Corporation (USA). Various grades of Carbopol were obtained from Lubrizol (India). The other chemicals of analytical grade were purchased from S.D.Fine Chemicals Ltd. (Mumbai, India).

Methods

Analytical Method Development

The UV-Visible spectrophotometric method (double beam UV-Visible spectrophotometer (Jasco V-530 Japan)) was developed and validated in Methanol AR for estimation of drug content and in simulated vaginal fluid (pH 4.2) for drug release studies from the developed formulations.

Screening of Solid Lipids

Various solid lipids such as stearic acid, Compritol 888 ATO, Imwitor 491, Precirol ATO 5, Apifil, Gelucire 50/13 and Gelot 64 were screened for their solubilizing capacity of fluconazole. Fifty milligrams of drug was taken; various solid lipids were then added in increments of 10 milligrams and maintained at 5–7°C above the melting point of lipids till complete solubilization of drug occurred. The melting points of the lipids studied are given in Table I. Optical microscopic observation of the drug lipid melt was also carried out to confirm the absence of drug crystals (15).
Table I

Melting Points of Solid Lipids

Solid lipids

Melting points (°C)

Gelot 64

55.5–62.5

Gelucire 50/13

46–51

Precirol ATO 5

50–60

Imwitor 491

66–77

Compritol 888 ATO

65–77

Stearic acid

69–71

Apifil

59–70

Screening of Liquid Lipids, Surfactants and Co-surfactants

For the selection and optimization of liquid lipids, surfactants and co-surfactants, excess of drug was added individually to known amounts of liquid lipids such as oleic acid, Captex, Labrafil M 1944 CS, Sefsol, Labrafac, Miglyol 812 and Capryol 90 and surfactants and co-surfactants (10% w/w in distilled water) such as Softigen 767, Labrasol, Transcutol HP, polyethylene glycol 400, Kolliphor RH 40, Kolliphor PS 20, Kolliphor PS 60 and Tween 80. The mixtures were shaken for about 72 h in a water bath shaker at 32 ± 2°C and the amount of drug dissolved in each excipient was determined by a UV-Visible spectrophotometer at λmax 260 nm (16).

Optimization of Composition of Lipid Blend

Miscibility test was carried out using various ratios of optimized solid and liquid lipids based on solubility studies. The lipid mixture was heated to temperature 5–7°C above the melting point of the selected solid lipid. The mixture was then cooled until complete solidification occurred and a filter paper was pressed over the solidified lipid melt and immiscibility of liquid lipid in the blend was indicated as oil stains on the filter paper. After optimizing solid to liquid lipid ratio, fluconazole was incorporated and depending upon its solubility in lipid melt, the optimum lipid blend was selected for drug loading.

Optimization of Charge Inducer

To obtain stable nanocarriers, different charge inducers such as oleic acid, lactic acid, cetyl trimethyl ammonium bromide and sodium dodecyl sulfate were tried in different concentrations and optimized for zeta potential to produce stable nanodispersions.

Preparation and Characterization of Nanolipid Dispersions of Fluconazole

The optimized quantities of lipids and surfactant were heated to the temperature 10°C above the phase inversion temperature of selected surfactant (17,18). 0.5% w/w of fluconazole was then added to the lipid surfactant phase. The aqueous phase containing the charge inducer was also heated to the same temperature. Then, the aqueous phase was added to the lipid surfactant phase under stirring at 700 rpm. The resultant emulsion was then cooled to 60°C and three temperature cycles (80–60°C) were followed, finally with rapid cooling in an ice bath to achieve better distribution of surfactant at the interface.

Particle Size and Zeta Potential Measurements

The obtained fluconazole nanolipid dispersions were visually checked for homogeneity while particle size and zeta potential measurements were carried out using Zetasizer (Nano ZS Malvern instruments, UK). All the samples were diluted ten times, dispersed in double-distilled water and analyzed, using disposable polystyrene cells.

Entrapment Efficiency

The entrapment efficiency of fluconazole in nanolipid carriers was determined by indirect method (19). Briefly, the drug-loaded lipid carriers were separated from unentrapped fluconazole by ultra-centrifugation of nanolipid dispersion at 80,000 rpm for 60 min at 4°C using Optima Max XP ultracentrifuge (Beckman Coulter, USA). The clear supernatant after appropriate dilution was analyzed by UV spectrophotometry at λmax of 260 nm.
$$ \mathrm{EE}=\frac{\left({\mathrm{W}}_{\mathrm{L}}\right)\hbox{--} \left({\mathrm{W}}_{\mathrm{F}}\right)}{{\mathrm{W}}_{\mathrm{L}}}\times 100 $$
(1)

wherein WL = total amount of fluconazole added and WF = amount of fluconazole in the supernatant.

In Vitro Release Studies

Ten grams of nanolipid dispersion containing 50 mg drug was transferred to a dialysis bag (molecular cutoff 12–14 kD, Himedia, India) and immersed in 100-ml simulated vaginal fluid (pH 4.2) maintained at 32 ± 0.5°C and stirred at 50 rpm. Appropriate aliquots were withdrawn at time intervals of 0.5, 1, 2, 3, 4, 5, 6, 7 and 8 h and analyzed by validated UV spectroscopy method at λmax of 260 nm.

Transmission Electron Microscopy

The lipid dispersion was appropriately diluted with filtered double-distilled water and then few drops were placed on copper grid (3 mm) with the aid of micropipette and allowed to dry for about 20 min under IR lamp. The grid with sample was then loaded on a probe and bombarded with electrons accelerated at 120 kV. The particle size of the sample was measured and its morphology was observed on the computer interface (20,21).

Preparation and Characterization of Fluconazole Nanolipid Gel

Various mucoadhesive polymers such as Carbopol 971 P, Noveon AA-1 Polycarbophil and Carbopol 974 P in different concentrations (0.5–1% w/w) were tried as gelling polymers for vaginal delivery by incorporating them in the nanolipid dispersions to produce stable gels (22). Fluconazole nanolipid dispersions were prepared and the gelling polymer, methyl paraben and disodium EDTA were added, allowed to hydrate for 8 h and neutralized with triethanolamine to pH 4.2–4.5 producing nanolipid vaginal gels and optimized formula is shown in Table II.
Table II

Optimized Formula for Fluconazole Nanolipid Vaginal Gel

Ingredients

Quantity (% w/w)

Fluconazole

0.5

Precirol ATO 5

3.5

Oleic acid

1.5

Kolliphor RH 40

2.0

Carbopol 974 P

1.0

Methyl paraben

0.02

Disodium EDTA

0.1

Purified water

qs 100 g

Triethanolamine

qs

qs quantum sufficit

Particle Size and Zeta Potential Measurements

The gel was diluted ten times and the particle size and zeta potential measurements were carried out using Zetasizer (Nano ZS Malvern instruments, UK).

pH and Drug Content

The pH of the optimized gel was measured using a digital pH meter (UD—95 Universal Enterprises, Mumbai). For determining the drug content, weighed amounts of gel, in triplicates, were extracted in Methanol AR by sonicating for 30 min. After appropriate dilutions, the drug content was determined by UV spectrophotometry at λmax of 260 nm.

Mucoadhesive Strength

Mucoadhesion was measured by modified two pan balance fabricated in our laboratory. A Teflon block of 3.8 cm in diameter was kept inside the glass container, which was then placed below the hanging cylinder on the left-hand setup of the balance. The two sides of pan were balanced using calibrated weights. The mucin films were prepared by placing 20 μl of 3% w/v porcine mucin in simulated vaginal fluid (pH 4.2) on glass slips. The films were air dried and used for adhesion studies. During the experiment, mucin films were hydrated with simulated vaginal fluid (pH 4.2) for 1 min. The cover slips were attached to the protrusion on the Teflon block and to the hanging cylinder each, with mucin containing sides facing each other. Fifty milligrams of the formulation was placed on the lower mucin film and 5-g weight from the right pan was removed. This lowered the Teflon cylinder with mucin film onto the formulation with weight of 5 g. The balance was kept in this position for a period of 3 min. The weights were then added to the right-hand side pan until the detachment of the two mucin surfaces occurred and the mucoadhesion force was calculated as
$$ F=W\times g $$

where F is the mucoadhesion force (dyne/cm2), W is the minimum weight required to break the mucoadhesion bond and g is the acceleration due to gravity (cm/s2) (23).

Viscosity and Spreadability

Viscosity of gel was determined by using a Brookfield viscometer RVT model. Dial readings were recorded at ambient temperature using a spindle number 6 at 1, 2.5, 5, 10 and 20 rpm, respectively. The viscosity in centipoises was calculated and rheogram of rpm versus viscosity was plotted to predict the rheology of the optimized gel (24,25). Spreadability was determined by apparatus suggested by Mutimar (26). A ground glass slide was fixed on the wooden block. About 1 g of gel was placed on this ground slide and then sandwiched between the ground slide and second glass slide having same dimensions as that of the fixed ground slide. Weight of 500 g was placed on the top of two slides for 5 min. The increase in the diameter due to spreading of the gel was noted.

Differential Scanning Calorimetry

The DSC thermograms were obtained using Perkin-Elmer Pyris 1 DSC (USA). Briefly, about 10 mg of drug-loaded gel sample was placed in aluminum sample pan and sealed. The sample was heated from 30 to 300°C at a heating rate of 10°C/min using nitrogen as purge gas (20 ml/min). Blank and drug gel were also treated in the same manner.

In Vitro Release Studies

Ten grams of nanolipid gel containing 50 mg fluconazole was transferred to a dialysis bag (molecular cutoff 12–14 kD, Himedia, India) and immersed in 100-ml simulated vaginal fluid (pH 4.2) maintained at 32 ± 0.5°C and stirred at 50 rpm (27,28). Appropriate aliquots were withdrawn at time intervals 0.5, 1, 2, 3, 4, 5, 6, 7 and 8 h and analyzed by validated UV spectroscopy method at λmax of 260 nm. The mechanism of drug release from the gel was determined using suitable mathematical models.

In Vitro Permeation Studies

In vitro drug permeation from 1-g nanolipid vaginal gel containing 5 mg fluconazole was determined using 20 ml of simulated vaginal fluid (pH 4.2) using cellulose acetate membrane (47 mm, 0.45 μm) (Pall Corporation) at 32 ± 0.5°C using Franz diffusion cells. Appropriate aliquots were withdrawn at time intervals of 0.5, 1, 2, 3, 4, 5, 6, 7, and 8 h and analyzed by validated UV spectroscopy method at λmax 260 nm.

Ex Vivo Permeation and Tissue Deposition Studies Using Porcine Vaginal Mucosa

Porcine vaginal mucosa was obtained from a local slaughterhouse after sacrificing and porcine tissues were separated. The vaginal mucosa was preserved in simulated vaginal fluid (pH 4.2) during transportation and was used within 2 h after the animals were slaughtered (29,30).

The ex vivo permeation studies were carried out using Franz diffusion cells. The vaginal tissue of suitable size was cut and then placed between the donor and the receptor compartments of the cells. The drug permeation from 1-g nanolipid vaginal gel containing 5 mg fluconazole was determined using 20 ml of simulated vaginal fluid (pH 4.2) at 32 ± 0.5°C. Appropriate aliquots were withdrawn at time intervals of 0.5, 1, 2, 3, 4, 5, 6, 7 and 8 h and analyzed by validated UV spectroscopy method at λmax of 260 nm. The cumulative amount of drug permeated from the gel through the porcine vaginal mucosa was plotted as a function of time. At the end of 8 h, the gel present on vaginal tissue was wiped off and the tissue was washed with filtered distilled water. It was then cut into fine pieces and sonicated in Methanol AR for 30 min to extract the deposited drug (31). The extract was filtered through a 0.45-μm Millipore filter, diluted suitably and was analyzed for drug deposition by UV spectroscopy at λmax of 260 nm using appropriate blank.

In Vitro Antifungal Activity

The antifungal activity of fluconazole nanolipid vaginal gel, marketed fluconazole gel, fluconazole solution in polyethylene glycol 400 and blank nanolipid gel was evaluated using well diffusion method. Candida albicans MTCC 4748 was used as the fungal strain on Sabouraud dextrose agar medium. The growth medium seeded with C. albicans was plated and wells of diameter 5 mm were punched aseptically. Appropriately diluted samples of the same volume of drug and formulations were placed in the wells such that the amount of fluconazole in each well was 45 μg. The plates were allowed to stand for complete diffusion of solutions and then incubated at 25 °C for 48 h. The zones of inhibition produced were measured and the in vitro antifungal activity was determined (32).

Primary Vaginal Mucosal Irritation Study

The experimental protocol for the study was approved by the Animal Ethics Committee of Mumbai Veterinary College (MVC/IAEC/12/2016). The test was carried out using healthy female Wistar rats. Animals were divided into two groups of three animals each. One group of animals was applied the control blank gel and the second group was applied the optimized nanolipid gel. All the formulations were applied intravaginally at a dose of approximately 0.15 μg drug. After single vaginal application, the vaginal cavity was observed visually for 3 days for any signs of possible irritation of vaginal mucosal tissue such as erythema, edema and related mucosal reactions (24,33). The animals were then sacrificed and vaginal mucosal tissues were isolated which were kept for fixing in 10% v/v formalin solution. Further, studies were carried out at a local laboratory to determine any histopathological changes in the vaginal mucosa due to formulation exposure.

Stability Studies of Optimized Formulation

The optimized gel was packed in lacquered aluminum tubes. The stability testing of optimized formulation was carried out for 3 months at 25°C/60% RH, 30°C/65% RH, and 40°C/75% RH respectively, as per ICH guidelines.

RESULTS

Analytical Method Development

A validated analytical method was developed for fluconazole using UV spectroscopy with regression coefficient of 0.9999 and linearity range of 80–320 ppm in Methanol AR while in simulated vaginal fluid pH 4.2, the regression coefficient and linearity range were found to be 0.9994 and 80–360 ppm, respectively.

Screening and Optimization of Solid Lipids, Liquid Lipids, Surfactants, and Co-surfactants

Various solid lipids, liquid lipids, surfactants and co-surfactants were screened and optimized for development of nanolipid carriers (Fig. 1a–c). Precirol ATO 5 and oleic acid in ratio of 7:3 were selected as optimized composition of solid and liquid lipid, respectively, on the basis of mutual miscibility. The drug solubility in selected lipid ratio was found to be 175 mg/g of lipid blend which was suitable for optimum drug loading. Kolliphor RH 40 over Labrasol was chosen as the surfactant as the method of choice for producing lipid nanoparticles was the phase inversion temperature method. Kolliphor RH 40 at 2% w/w concentration was able to produce stable nanolipid dispersions containing 5% w/w of lipids.
Fig. 1

Screening of solid lipids (a), liquid lipids (b), surfactants, and co-surfactants (c)

Preparation and Characterization of Nanolipid Dispersion of Fluconazole

The nanolipid dispersions were prepared by the phase inversion temperature method and were found to be stable during freeze thaw cycles (− 4 to 40°C) of 24 h for a period of 1 week.

Optimization of Charge Inducer

Among the various charge inducers, sodium dodecyl sulfate (SDS) was selected at minimum concentration of 0.002% (Fig. 2a) to produce stable drug-loaded nanodispersions as indicated by zeta potential values.
Fig. 2

a Effect of charge inducer on zeta potential. b TEM image of fluconazole nanolipid dispersion

Particle Size and Zeta Potential Measurements

The optimized drug-loaded nanodispersions were found to have average particle size of 158.33 ± 2.55 nm indicating narrow particle size distribution and polydispersity index of 0.278 ± 0.003 showing homogeneity of the sample. Incorporation of SDS as charge inducer in the drug-loaded nanodispersions showed zeta potential of − 27.33 ± 0.40 mV indicating stability whereas the nanodispersions without charge inducer showed zeta potential of − 14.19 ± 0.78 mV indicating possibility of phase separation during storage.

Entrapment Efficiency

The average entrapment of fluconazole in the developed nanolipid carriers was found to be 67.24 ± 0.87%.

In Vitro Release Studies

The in vitro studies of fluconazole nanolipid dispersions showed an initial burst release due to the presence of drug in the aqueous surfactant phase and then sustained release for 8 h due to drug partitioning between the lipid matrix and the surrounding aqueous surfactant phase (Fig. 3).
Fig. 3

In vitro drug release of fluconazole nanolipid dispersion and gel

Transmission Electron Microscopy

TEM studies were carried out to understand the morphology of nanoparticles and the photomicrographs indicated the presence of almost spherical lipidic particles in nanometric range (Fig. 2b).

Preparation and Characterization of Fluconazole Nanolipid Gel

To improve the residence time of the nanolipid dispersion in the vaginal cavity, gel was prepared using Carbopol 974P (1% w/w) as mucoadhesive polymer.

Particle Size and Zeta Potential Measurements

The particle size and polydispersity index of the optimized vaginal gel were 179.2 ± 1.58 nm and 0.285 ± 0.003 respectively, while the zeta potential was found to be − 26.33 ± 0.41 mV.

pH and Drug Content

The pH of the optimized vaginal gel was found to be about 4.2–4.5 and the drug content was 98.05 ± 0.5377%.

Mucoadhesive Strength

Mucoadhesive strength of the optimized gel was determined using modified two pan balance apparatus as 3.01 (dyne/cm2) and was 1.5 times that of the marketed gel indicating improved adherence.

Viscosity and Spreadability

The pseudoplastic flow of gel was indicated from the concave nature of the rheogram (Fig. 4) inclined towards shear rate axis with the viscosity of 100,800 ± 2.959 cps at 5 rpm. The diameter due to spreading of the gel using spreadability apparatus was found to be 7.1 cm.
Fig. 4

Rheogram of optimized fluconazole nanolipid vaginal gel

Differential Scanning Calorimetry

The DSC thermograms of fluconazole, blank gel, and fluconazole nanolipid vaginal gel as in Fig. 5a–c show the absence of drug endotherm in the gel indicating solubilization of the drug in the nanolipid dispersion.
Fig. 5

DSC thermograms of fluconazole (a), blank gel (b), and fluconazole nanolipid vaginal gel (c)

In Vitro Release Studies

The in vitro drug release of nanolipid gel was as shown in Fig. 3 indicating sustained drug release for 8 h. The mechanism of drug release was determined by mathematical modeling of release data and Table III reveals the regression parameters (R2) obtained after fitting various release kinetic models to the in vitro release data of fluconazole nanolipid vaginal gel. The drug release kinetics of fluconazole nanolipid vaginal gel was found to follow Higuchi model based on goodness of fit.
Table III

Mathematical Model of In Vitro Release Data of the Fluconazole Vaginal Gel

Formulation

Zero order (R2)

First order (R2)

Higuchi model (R2)

Korsmeyer-Peppas (R2)

Fluconazole nanolipid vaginal gel

0.9007

0.5188

0.9963

0.8681

In Vitro Permeation Studies

In vitro permeation study from the nanolipid gel through cellulose acetate membrane was investigated to determine the permeation of fluconazole. About 50.36 + 2.22% drug permeated through the cellulose acetate membrane in 8 h as shown in Fig. 6 which indicated sustained drug release.
Fig. 6

In vitro permeation and ex vivo permeation of fluconazole nanolipid vaginal gel

Ex Vivo Permeation and Tissue Deposition Studies Using Porcine Vaginal Mucosa

The cumulative percentage of drug permeated through porcine vaginal mucosa was 19.93 ± 0.50% as shown in Fig. 6. In ex vivo studies, the amount of drug deposited in porcine vaginal mucosa was 30.69 ± 1.02% indicating higher drug deposition proposing localized effects in treating vaginal infections.

In Vitro Antifungal Activity

Antifungal activity of fluconazole nanolipid vaginal gel, marketed fluconazole gel, fluconazole in polyethylene glycol 400 as solution and blank vaginal gel was evaluated using well diffusion method and zones of inhibitions were measured. The diameters of the zones of inhibition of fluconazole-loaded nanolipid vaginal gel were found to be greater as compared to those of the other formulations (Fig. 7a) indicating better antifungal activity due to nanosized carriers.
Fig. 7

a Comparative diameters of zones of inhibition of various formulations. b Histopathology of control gel. c Histopathology of fluconazole nanolipid vaginal gel

Primary Vaginal Mucosal Irritation Study

Primary vaginal mucosal irritation studies were carried out to evaluate the tolerability of fluconazole nanolipid gel after vaginal application. After a single vaginal application of the gel, the vaginal cavity was observed visually for 3 days for any signs of possible irritation of vaginal mucosal tissue such as erythema, edema and mucosal reactions. The results indicated that the developed nanolipid gel of fluconazole was non-irritant to the rat vagina and results were comparable to those of the control gel. After animal sacrificing, the histopathological studies of the exposed vaginal tissue showed normal cell lining without any damage to the vaginal mucosa as shown in (Fig. 7b, c).

Stability Studies of Optimized Formulation

The developed gel was found to be stable for a period of 3 months as per ICH guidelines.

DISCUSSION

Fluconazole, the primary treatment option for vulvovaginal candidiasis is available locally only as oral tablets and conventional gels but not as nanocarriers-based gels. In spite of having potential for improved therapeutic effects, high local tissue levels and lower systemic exposure of drugs when applied as nanocarriers for vaginal delivery, literature reports are not available for vaginal gels containing fluconazole as nanolipidic particles. However, literature reports vaginal films, microemulsion-based gels and thermosensitive and mucoadhesive gels of fluconazole (24,34,35). Hence, the aim of the current research was to develop and evaluate nanolipid carriers based gel of fluconazole for vaginal delivery. Such an approach can provide localized and sustained drug delivery for combating vaginal infections and reducing peripheral side effects. During the study, validated analytical methods were developed for quantification of fluconazole using UV spectroscopy. Lipid screening was carried out to optimize the suitable lipid which provided the highest solubility of drug and thus enhanced drug entrapment efficiency in nanocarriers. Though Apifil and Compritol 888 ATO showed the highest solubility for fluconazole, these were not miscible with the liquid lipid, Capryol 90 which also showed maximum fluconazole solubility. Hence, Precirol ATO 5 and oleic acid in ratio of 7:3 were selected as optimized composition of solid and liquid lipid, respectively, depending upon mutual solubility and optimum drug loading. Although Labrasol showed the highest solubility of drug, it could not emulsify the selected lipids and hence, Kolliphor RH 40 was chosen as surfactant at 2% w/w concentration for producing nanolipid dispersion containing 5% w/w of lipids. This surfactant was suitable as the choice of method for preparing nanoparticles was the phase inversion temperature technique. The charge on the colloidal particles has considerable effects on the zeta potential and plays a significant role in the stability of the dispersion. Among the various charge inducers, sodium dodecyl sulfate (SDS) was selected at a minimum concentration of 0.002% to avoid potential irritation to the vaginal mucosa and also produce stable nanodispersions as indicated by the value of zeta potential. Blank and nanolipid dispersions of fluconazole were prepared by the phase inversion temperature method. To achieve stable nanodispersions, various parameters like drug loading, type of lipid, concentration of surfactant, particle size and entrapment efficiency were optimized and freeze thaw cycles were carried out to assess the stability of nanodispersions. The drug-loaded nanolipid dispersion was found to be homogeneous with particle size and polydispersity index of 158.33 ± 2.55 nm and 0.278 ± 0.003, respectively. Zeta potential greater than ± 25 mV helped to stabilize colloidal particles due to electric repulsion between the particles as indicated by the freeze thaw studies. Fluconazole nanolipid dispersions showed zeta potential of − 27.33 ± 0.40 mV with SDS and − 14 mV without charge inducer indicating stabilization effect of SDS. The average entrapment of fluconazole in the developed nanolipid particles was found to be 67.24 ± 0.87% which was due to the lipophilic nature of fluconazole improving its entrapment in lipids. The in vitro release studies of the nanocarriers showed initial rapid release due to the presence of drug in the aqueous surfactant phase and then sustained release for 8 h due to drug partitioning between the lipid matrix and the surrounding aqueous surfactant phase. TEM studies of the fluconazole nanocarriers indicated the presence of spherical lipidic particles with no evidence of drug precipitation. Carbopol 974P, a mucoadhesive polymer, was selected to achieve optimum viscosity and improve the residence time of gel in the vaginal cavity. The optimized gel had particle size and polydispersity index of 179.2 ± 1.58 nm and 0.285 ± 0.003, respectively, while the zeta potential was found to be − 26.33 mV ± 0.41. The pH of the gel was 4.2–4.5 suitable for use within the vaginal cavity and the percent drug content was found to be 98.05 ± 0.5377. Mucoadhesive strength of the optimized gel was 1.5 times greater than that of the conventional gel and the flow behavior was found to be pseudoplastic. The spreading of the gel using spreadability apparatus was found to be 7.1 cm indicating satisfactory spreadability. The DSC thermogram of the gel showed absence of drug peak indicating drug entrapment in the lipid matrix. The in vitro drug release studies indicated initial rapid release due to presence of drug in the aqueous surfactant phase. The drug release was later sustained due to partitioning and slow leaching out of the entrapped drug from the lipid matrix and the mechanism of drug release was found to follow Higuchi kinetics. About 50.36 + 2.22% fluconazole permeated from the vaginal gel through the cellulose acetate membrane in 8 h. This indicated sustained release of the drug from the nanocarriers-based gel to combat vaginal fungal infections for a prolonged period without development of resistance. Ex vivo permeation of fluconazole through porcine vaginal mucosa was 19.93 ± 0.50% while vaginal tissue deposition of drug was 30.69 ± 1.02% indicating greater deposition due to colloidal carriers that can be useful in treating localized fungal vaginal infections without peripheral side effects. The evaluation parameters of nanolipid dispersion and nanolipid gel are indicated in Table IV. Antifungal activity of fluconazole nanolipid vaginal gel was greater than that of the marketed fluconazole gel, fluconazole in polyethylene glycol 400 as solution and blank vaginal gel, respectively, during well diffusion studies. This showed that fluconazole nanolipid vaginal gel shows better antifungal activity as compared to other formulations due to nanometric size and hence, better permeability into the fungal membranes for growth inhibition. After a single vaginal application of the gel, the vaginal cavity showed no signs of possible irritation to vaginal mucosal tissue such as erythema, edema and mucosal reactions. It was observed that blank vaginal gel and fluconazole nanolipid vaginal gel were well tolerated by rats and no signs of erythema and/or edema were seen even after 72 h. Histopathological findings concluded that there was no vaginal mucosal irritation as indicated by normal cell lining without any damage to the vaginal mucosa. The optimized gel was found to be stable for a period of 3 months.
Table IV

Evaluation of Fluconazole Nanolipid Dispersion and Nanolipid Gel

Evaluation parameters

Nanolipid dispersion

Nanolipid gel

Appearance

Milky white dispersion

Milky white gel

Particle size (nm)

158.33 ± 2.55

179.20 ± 1.58

Polydispersity index (nm)

0.278 ± 0.003

0.285 ± 0.003

Zeta potential (mV)

− 27.33 ± 0.40

− 26.33 ± 0.41

Entrapment efficiency (%)

67.24 ± 0.87

In vitro release study (%)

64.15 ± 0.57

58.60 ± 1.14

pH

4.2–4.5

Mucoadhesion strength determination (dyne/cm2)

3.01

Drug content (%)

98.05 ± 0.5377

Viscosity (centipoises)

100,000

Spreadability (cm)

7.1

In vitro permeation study (%)

50.36 ± 0.57

Ex vivo permeation study (%)

19.93 ± 0.51

Tissue deposition (%)

30.69 ± 1.025

CONCLUSIONS

Fluconazole nanolipid carriers were prepared using the phase inversion temperature technique. The nanolipid dispersion of fluconazole showed optimum particle size and polydispersity index while the charge inducer provided zeta potential to yield stable formulation. Transmission electron microscopy showed spherical particles in nanometric range. These lipid nanoparticles could be effectively formulated as vaginal gel using Carbopol 974P as mucoadhesive polymer. The developed vaginal mucoadhesive gel showed optimum particle size, polydispersity index and zeta potential indicating stability of the formulation during accelerated testing. The optimized vaginal gel of fluconazole showed sustained drug release up to 8 h in in vitro release studies. This might be due to partitioning of the drug between the nanolipid matrix and aqueous surfactant phase. Ex vivo permeation studies through porcine vaginal mucosa showed appreciable tissue deposition suggesting suitability in treating localized vaginal infections. Animal studies in female Wistar rats showed no vaginal mucosal irritation, which was further confirmed by histopathological evaluation of the excised vaginal tissue. Stability studies indicated stability of the developed gel for 3 months at accelerated conditions. Thus, nanolipid carrier-based gel of fluconazole can be used for vaginal application in the treatment of vulvovaginal candidiasis for prolonged and localized action with reduced systemic side effects.

Notes

Acknowledgements

The authors wish to acknowledge Glenmark Research Center (Taloja, India), Gattefosse India Pvt. Ltd (Mumbai, India), Signet Chemical Corporation (Mumbai, India), Cremer Oleodivison (Germany), Nikkol (Japan), Abitec Corporation (USA) and Lubrizol (India) for providing the gift samples of drug and excipients.

Compliance with Ethical Standards

The experimental protocol for the study was approved by the Animal Ethics Committee of Mumbai Veterinary College (MVC/IAEC/12/2016).

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Copyright information

© American Association of Pharmaceutical Scientists 2018

Authors and Affiliations

  1. 1.Department of PharmaceuticsBombay College of PharmacyMumbaiIndia

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