A 3² full factorial design for development and characterization of a nanosponge-based intravaginal in situ gelling system for vulvovaginal candidiasis

Abstract

Clotrimazole (CTZ) is a Biopharmaceutics Classification System (BCS) Class II drug having a limited therapeutic potential because of its poor aqueous solubility and relatively short half-life. The rationale behind the present research effort was to enhance the solubility and efficacy of CTZ by having it form a complex with hydroxypropyl β-cyclodextrin (HP-β-CD) nanosponges. Nanosponges (NSs) are hyper-cross linked cyclodextrin polymer-based colloidal structures with three-dimensional networks. Herein, NSs were prepared using dimethyl carbonate as a cross linker, suitably gelled, and were assessed for in vitro release, in vitro bioadhesion, in vivo antifungal activity and in vivo irritation using female Wistar albino rats. Nine formulations were prepared based on a 3² full factorial design using different Pluronic F-127: Pluronic F-68 ratios. The prepared CTZ-HP-β-CD NS samples were characterized by carrying out SEM, TEM, and FT-IR spectroscopy studies, as well as DSC and XRPD studies. The average particle size of loaded NS (N6) was found to be 455.6 nm. This sample displayed the lowest polydispersity index of the samples tested, and displayed a high zeta potential (−21.32 ± 1.3 mV), indicative of a stable colloidal nanosuspension. The optimized CTZ NS-based in situ gel (F-10) demonstrated prolonged drug release (up to 15 h), considerably longer than that of the conventional in situ gel, whose drug release only lasted for less than 6 h. The CTZ-NS gel showed higher in vivo antifungal activity and in vitro bioadhesion than did the conventional in situ gel. Furthermore, in vivo irritation studies showed the optimized CTZ NS gel formulation to be a non-irritant. All of these results signified the promising applicability of the formulated CTZ NS gel as a novel delivery system for the local treatment of vaginal candidiasis and other similar infections.

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1. Introduction

Vaginal candidiasis is a clinical condition which is prevalent in about 75% of women for at least once in their lifetime. About 80% of these cases are caused by the most pathogenic species of the genus Candida, i.e., Candida albicans.^1,2 C. albicans primarily exists as a dimorphic fungus that is propagated through its blastophore phenotype also known as ‘blastoconidia’. It is capable of transforming into various forms such as hyphae, yeast and pseudohyphae upon recognition environmental signals, which are important for its virulence.^3,4 The outermost cell wall layer of Candida comprises mannoproteins with N-glycosylated polysaccharide and O-glycosylated oligosaccharide moieties. Both carbohydrate moieties have been shown to be vital in host–fungal interactions and virulence.^5–7 The N-glycosylated polysaccharide has a comb-like structure with an α-1,6-linked backbone moiety and an oligomannose side chain chiefly consisting of α-1,2-, α-1,3-, and β-1,2-linked mannose residues with a trivial number of phosphate groups. Three types of β-1,2-linkage-containing manno-oligosaccharides are observed in mannan. One of these is located in a phosphodiesterified oligosaccharide moiety and functions as a common epitope of C. albicans serotypes A.^8,9 Candida albicans strain J-1012 (serotype A) is one the main organisms that cause vaginal candidiasis.^1,2 Vulvovaginal candidiasis can be initially treated locally with antifungal agents. Such local vaginal delivery provides a site-specific cure and avoids toxic side effects of antifungal agents administered orally. Acute vulvovaginal candidiasis needs to be treated for five to seven days, although patients tend to prefer and better comply with shorter treatment schedules. Longer treatments, however, are needed to effectively combat recurrent vaginal infections.¹⁰ Antifungal imidazole derivatives are the most commonly used drugs to treat fungal infections. Imidazole derivatives, however, display relatively poor aqueous solubility, because of their hydrophobic structures, and thus poor efficacy along with high formulation tribulations, when taken orally.¹¹ Therefore, applying the imidazole derivative clotrimazole (CTZ) topically is the most commonly recommended regimen for vaginal candidiasis.

CTZ displays a wide range of anti-mycotic activities, and hence offers a broadly efficient local treatment with a minimum risk of side effects.¹² It acts by inhibiting the fungal cytochrome P450 3A enzyme (lanosine 14 α-demethylase) that regulates the conversion of lanosterol to ergosterol (the chief sterol comprising the fungal cell membrane). CTZ is available in numerous conventional dosage forms such as ovules, gels, tablets, creams and pessaries for vaginal application. However, these conventional dosage forms of CTZ are not active for a long period of time, because of their short residence times in the genitourinary tract. This issue compromises the efficiency of CTZ treatment by requiring multiple and frequent administrations of the drug.¹³ Moreover, CTZ is a poorly water-soluble drug, which interferes with its local availability, thereby limiting the effective antifungal treatment.¹² To overcome these limitations, various delivery vehicles such as microemulsions, polycarbophil gels, liposomes, microspheres, nano-structured lipid carriers, cyclodextrin inclusion complexes, and sustained release bioadhesives have been proposed.^14–19 However, nanosponges (NSs) have yet to be explored for the delivery of CTZ for improvised candidiasis therapy.

NSs are made up of microscopic particles containing cavities that are a few nanometers wide.^20,21 These recently proposed nano-sized colloidal carriers specially designed for drug delivery can solubilize drugs that are insoluble in water, offer prolonged release, and by altering pharmacokinetic parameters they improve drug bioavailability.^21,22 In the pharmaceutical field, cyclodextrins (CDs) are used to form inclusion complexes with drug molecules in order to enhance the aqueous solubility of the drugs, mask their unwanted characteristics, reduce their side effects, and increase their photostability or aqueous stability. CDs have also been shown to control the release of certain active ingredients.^13,20 The CD-based NS constitutes a hyper-crosslinked system produced by the use of compounds such as carbonyl or carboxylate compounds to crosslink different CD molecules.²³ The resulting systems formed are crosslinked polymers that have intriguing properties such as the inclusion or absorption of chemicals, swelling, and the proficient release of active agents. When compared to other nanocarriers, the NS offers high drug loading as well as having great potential for addressing concerns related to bioavailability, solubility, the controlled release of a range of therapeutic agents, and stability.²² Besides, prepared NSs can be assimilated into conventional dosage forms such as ointments, gels, creams, lotions and powders for beneficial applications.

Prior studies have shown the CD-based NS to serve as a carrier for paclitaxel, acyclovir, curcumin, camptothecin, itraconazole and other drugs.^24–29 Furthermore, the ability of NSs to increase the solubility and bioavailability of diverse molecules including resveratrol and tamoxifen have also been reported.^21,24

A vital challenge in the vaginal drug delivery is patient’s pliability during administration of dosage forms and following up with repeated-dose therapeutic regimen.¹³ Among the various conventional formulations, gels have critical advantages such as versatility, safety, high bioavailability and being economical. Gels are better tolerated by patients than any other dosage forms, and vaginal therapy can be considerably improved if the delivery system can hold the drug at the site of administration for an extended period of time.^10,13 In situ gelling drug delivery systems release drugs in response to environmental signals as a result of stimulus-dependent changes in the rheological properties of the polymer platform.^30–36 Hence, such systems can be formulated to deliver an adequate coverage of the vagina and retain the formulation on much of the mucosal tissue. Pluronic F-127 (PF-127) and Pluronic F-68 (PF-68) are synthetic copolymers of poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) and exhibit thermoreversible behavior in aqueous solutions. The hydrophilicity and hydrophobicity of the Pluronics mainly depend on the polyethylene oxide (PEO) to polypropylene oxide (PPO) ratio. PF-127, with an average molar mass of 12 600 g mol⁻¹, contains PEO/PPO in the ratio of 7 : 3; copolymer PF-68, with a molar mass of ∼8400 g mol⁻¹, contains an 8 : 2 ratio of PEO/PPO.³¹ The thermoreversible behavior of Pluronics is due to the change in their micellar properties as a function of both concentration of the polymers and ambient temperature, and a reversible gelation may occur at physiological temperature.^37,38 By using such systems, many benefits like high spreadability and ease of application at temperatures that are below sol–gel temperature and rheological structuring are offered to the vagina for topically administering therapeutic agents and therefore, vaginal retention at body temperature is enriched.

In this perspective, we studied the vaginal delivery of a mucoadhesive and thermosensitive CTZ-HP-β-CD NS-based in situ gel formulation to attempt to achieve a longer residence time at the infection site, and thereby provide a satisfactory release profile of CTZ for an improved and efficient vulvovaginal candidiasis therapy.

2. Materials and methods

CTZ (MW: 344.837 g mol⁻¹, purity >98%) was kindly provided as a gift sample from Glenmark Pharmaceuticals Ltd., Mumbai, India. (2-Hydroxypropyl)-β-cyclodextrin (∼1480 g mol⁻¹) and dimethylcarbonate (90.08 g mol⁻¹) were purchased from Alfa Aesar, England and Loba Chemie, Mumbai, India, respectively. Pluronic F-127 (PF-127, 12 600 g mol⁻¹) and Pluronic F-68 (PF-68, ∼8400 g mol⁻¹) were purchased from Sigma-Aldrich, St. Louis, USA. All other chemicals and reagents used were of analytical grade. Ultra-purified water was used for all experiments.

2.1. Synthesis of the HP-β-CD NS

HP-β-CD-based NS were prepared using dimethylcarbonate (DMC) as a cross-linking agent. Different molar ratios of HP-β-CD to DMC (1 : 2, 1 : 4 and 1 : 8 for batches ‘A’, ‘B’ and ‘C’, respectively) were used to prepare NS via method previously reported by Swaminathan et al.²⁷ In brief, anhydrous HP-β-CD was added to DMC at about 90 °C under magnetic stirring and allowed to react for 5 h. The reaction mixture was left to cool and the resulting solid was recovered by filtration. Later, this obtained solid mass was broken up via gentle mortar grounding, and Soxhlet assembled extraction using ethanol was carried out in order to remove unreacted DMC and other impurities. An excess amount of cross linker was used to carry out the reaction and the resulting NS were stored at 25 °C after purification for further usage.

2.2. Incorporation of CTZ into the NS

Accurately weighed amounts of CTZ were dispersed in aqueous NS suspensions in diverse weight ratios (1 : 1A, 1 : 1B, 1 : 1C, 1 : 2A, 1 : 2B, 1 : 2C) and stirred magnetically for 24 h (Table 1). These suspensions were then centrifuged at 2000 rpm for 10 min to separate out the non-complexed drug as a residue below the colloidal supernatant. The colloidal supernatants were then subjected to freeze-drying using a Modulyo freeze drier (Edwards, UK) to obtain the CTZ-loaded NS. Finally, the acquired NSs were stored in a covered vacuum desiccator at ambient temperature for further studies.^23,24

Table 1 Formulation batches and entrapment efficiency of CTZ-loaded NSs

CTZ-NS batch code	Drug (CTZ) : blank NS batch (A/B/C)^a	Entrapment efficiency^b (%)
a A, B and C stands for blank NSs prepared using 1 : 2, 1 : 4 and 1 : 8 (HP-β-CD : dimethylcarbonate) ratios respectively.b Mean ± SD, n = 3.
N1	1 : 1A	15.34 ± 0.49
N2	1 : 1B	51.46 ± 0.91
N3	1 : 1C	74.23 ± 0.74
N4	1 : 2A	39.76 ± 0.83
N5	1 : 2B	62.89 ± 1.19
N6	1 : 2C	85.12 ± 0.61

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2.3. Solubilization efficiency

The CTZ solubilization efficiency values of NS batches A, B, and C were determined using the shake-flask method to evaluate to what extent each of these NS batches solubilize CTZ.¹² Briefly, an excess quantity of CTZ was suspended in 20 mL ultra-purified water in one glass vial, and excess CTZ was added into another glass vial containing a fixed quantity of the HP-β-CD NS in 20 mL ultra-purified water. Both vials were sealed and shaken using a mechanical shaker (Remi, India) at the ambient temperature for 24 h. Resultant suspensions were then subjected to centrifugation (5000 rpm for 15 min using a Research Compufuge, Remi PR-24 Centrifuge, Remi, India), supernatants were filtered through a membrane filter (0.45 μm, 13 mm, Pall Life Sciences, Mumbai, India) and filtrates were analyzed for the concentration of dissolved CTZ by carrying out UV-spectrophotometry using light at a wavelength of 264 nm (Pharmaspec 1700, Schimadzu, Japan). The experiments were performed in triplicate (n = 3).

2.4. Entrapment efficiency

Entrapment or loading efficiency of CTZ was determined by quantitatively estimating the amount of drug loaded into the NSs. A mass of 10 mg of the CTZ-HP-β-CD NS was dissolved in methanol, sonicated for 15 min to break up the complex, and centrifuged; the resulting filtered supernatant was diluted sufficiently for the subsequent UV-spectrophotometric analysis at 264 nm.^25,39 The entrapment efficiency (%) of the NS was determined using eqn (1).

Entrapment efficiency = C_e/C_th × 100 (1)

In this equation, C_e denotes the entrapped drug concentration, and C_th the theoretical drug concentration.

2.5. Characterization of CTZ HP-β-CD NS

2.5.1. Fourier transform infrared (FT-IR) spectroscopy. FT-IR spectroscopy (Shimadzu 8400 S, Japan) was used to detect any interaction of CTZ with NS. The spectra were obtained using KBr pellets within the range 4000 cm⁻¹ to 400 cm⁻¹.

2.5.2. Differential scanning calorimetry (DSC). Differential scanning calorimetry (DSC) was performed for CTZ, the physical mixture, the blank NS sample and optimized NS formulation (N6) using a Shimadzu DSC 60 apparatus. All calorimetric measurements were completed using a high-purity alpha alumina disc (empty cell) as the reference. High-purity indium metal was adopted as a standard for instrument calibration. Dynamic scans were taken in the temperature range 10–300 °C in nitrogen atmosphere with a heating rate of 10 °C min⁻¹.

2.5.3. X-ray powder diffraction (XRPD). XRD patterns of CTZ, HP-β-CD, and of the unloaded and CTZ-loaded HP-β-CD NS samples were recorded at an ambient temperature using a diffractometer (Rigaku, Japan). Diffraction patterns were recorded using Ni-filtered CuKα radiation (λ = 1.5418 Å), 40 kV voltage, a 20 mA current and a 0.02° step size and 2 s per step with a scan speed 0.01° s⁻¹ in the interval 2θ = 10–60°.

2.5.4. Particle size, zeta potential, polydispersity. The average particle size and distribution, zeta potential and polydispersity index (PI) of the NSs were determined using a Malvern Instrument (DTS Ver.5.10, MAL1031371, Malvern, UK). The experiments were performed using a clear disposable zeta cell, and using water as a dispersant, which has refractive index (RI)-1.330 and viscosity (cP)-0.73 at a constant 25 °C temperature.

2.5.5. Scanning electron microscopy (SEM). The morphology and surface topography of the prepared NSs were examined using a scanning electron microscope (Zeiss EVO LS 15, Smart SEM 5.05, Germany) operating at an acceleration voltage 15 kV and suitable magnification at room temperature. In brief, samples were mounted onto 5 mm silicon wafers and sputter coating was done with Au under an argon atmosphere.

2.5.6. Transmission electron microscopy (TEM). The average size and shape of the NSs were evaluated by using a transmission electron microscope (Philips CM 10) assembled with NIH image software. Suspensions (with 0.5% w/v concentration) of CTZ-loaded or CTZ-unloaded NSs were sprayed uniformly on Formwar-coated copper grids, and were observed after complete air-drying.

2.6. Formulation of NS-based CTZ gel

PF-127 and PF-68 served as gelling agents and the gel was prepared according to the classical ‘cold method’.⁴⁰ The Pluronic F-127 (18–22% w/w) and Pluronic F-68 (5–15% w/w) polymers were added into pre-cooled ultra-purified water (4 °C) with continuous agitation. The solutions were then kept in a refrigerator overnight (4 °C) to ensure complete dissolution and the pH was adjusted to neutral using triethanolamine. CTZ NSs (drug equivalent to 200 mg) were incorporated into the gel (10 g) to attain 2% w/w CTZ. A conventional gel was made in the same way; instead of NS, free CTZ was incorporated. The prepared gels were packed in glass vials and sealed until further use.

2.7. Preparation of simulated vaginal fluid (SVF)

SVF was prepared from 3.51 g L⁻¹ sodium chloride, 1.40 g L⁻¹ potassium hydroxide, 0.222 g L⁻¹ calcium hydroxide, 2 g L⁻¹ lactic acid, 0.018 g L⁻¹ bovine serum albumin, 0.4 g L⁻¹ urea, 1 g L⁻¹ acetic acid, 0.16 g L⁻¹ glycerol and 5 g L⁻¹ glucose. The pH of the mixture was adjusted to 4.5 ± 0.02 using 0.1 M hydrochloric acid.¹⁹

2.8. Experimental design

Experimental design is a systematic and scientific approach to study the relationship and interaction between independent and dependent variables. A 2-factor, 3-level full factorial design (3²) was employed for optimizing CTZ-HP-β-CD NS-based in situ gels using DESIGN EXPERT® (version 9.0.5) software available from Stat-Ease Inc., Minneapolis, MN. The concentrations of PF-127 (A) and PF-68 (B) were optimized by using Design of Experiment (DoE) at three different levels: low (−1), medium (0) and high (+1). Gelation temperature (°C) (R1), gelation time (s) (R2) and in vitro drug release (% cumulative drug release) (R3) were selected as response variables (Table 2). A statistical model incorporating interactive and polynomial terms was utilized to evaluate the formulation responses according eqn (2).

Y = b₀ + b₁A + b₂B + b₃AB + b₄A² + b₅B² (2)

Table 2 Different combinations of CTZ NS-based in situ gels using 3² factorial designs

Formulation code	Coded factor levels
	A	B
F-1	−1	−1
F-2	0	−1
F-3	−1	1
F-4	1	−1
F-5	1	1
F-6	−1	0
F-7	0	0
F-8	0	1
F-9	1	0

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Factors and their coded levels	−1	0	1
A: PF-127 amount (%)	18	20	22
B: PF-68 amount (%)	5	10	15

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In this equation, Y is the response, and b₀ is the arithmetic mean response of the 9 runs. The responses in the above equation Y are the quantitative effect of the formulation components or independent variables A and B; b₀ is the arithmetic mean response; b₁, b₂, b₃, b₄ and b₅ are the estimated coefficient for the factors A and B. Details of the factorial design are given in Table 2. The prepared gels were characterized and evaluated for sol–gel transition, gelation temperature, gelation time, in vitro drug release, pH, drug content, viscosity, spreadability, in vitro bioadhesion, in vivo antifungal activity and in vivo irritation.

2.9. Characterization of the CTZ NS gel

2.9.1. Gelation temperature. The gelation temperature of the thermosensitive NS-based in situ gel (5 mL) was estimated by heating it in a thin-walled glass tube (internal diameter-10 mm, length-82 mm, thickness-0.6 mm) placed in a temperature-controlled water bath with gentle shaking until it converted to a gel. The temperature of the water was increased at a constant rate of 2 °C/5 min. Gel formation was taken as the point where there was no flow when the test tube was inverted. This temperature was noted as the gelation temperature.⁴¹

2.9.2. Gelation time. Gelation time of the NS-based in situ gel was determined by using the tube inversion method. A volume of 5 mL of the NS-based in situ gel was added into a thin-walled glass tube having the same geometry/specifications as mentioned above. The gelation time was measured at the respective gelation temperature noted earlier. The filled glass tube was immersed in a temperature-controlled water bath adjusted to the respective gelation temperature. The test tube was taken out at regular intervals and inverted to observe the physical state of the sample. The gelation time was determined by a flow or no-flow criterion with the test tube inverted. The amount of time it took for the system to gel, i.e. to stop flowing, was noted as the gelation time.⁴²

2.9.3. In vitro release studies. The in vitro drug release studies were performed using a Franz diffusion cell (Perme Gear Inc., Bethlehem, PA) with the donor chamber and water-jacketed receptor chamber (20 mL) maintained at 37 °C. A commercial semipermeable cellophane membrane (Fischer Scientific Co., London, England; pore size 0.45 μm) was used as the permeation barrier, which was soaked overnight in SVF before the study. One gram of gel was placed carefully on the cellophane membrane, which was placed between the donor and receptor compartments. The receptor compartment contained 20 mL SVF, while the donor compartment was empty and open to atmosphere. The contents of the receptor section were kept at 37 ± 5 °C with continuous stirring at rate of 25 rpm, using a magnetic stirrer. Aliquots (5 mL) were withdrawn at regular intervals from the receptor compartment and an equal volume of fresh receptor medium (at 37 ± 5 °C) was replaced to maintain the sink condition. Withdrawn samples were analyzed by using a UV-spectrophotometer at 264 nm. The study was conducted in triplicate.

2.9.4. pH. The pH of the prepared 2% (w/w) gel was determined using a digital pH meter (Mettler Toledo MP 220, Greifensee, Switzerland) at 25 °C in triplicate.

2.9.5. Drug content. The drug content was determined by placing 1 g of the NS-based in situ gel into a 100 mL volumetric flask, and then dissolving the gel in methanol. After appropriate dilutions, the solution was analyzed by UV-spectrophotometry as described earlier.^39,43

2.9.6. Viscosity studies. Viscosity measurements of the formulated NS-based in situ gel were taken using a Brookfield viscometer (DV-II, LV model, Brookfield, USA) using a small volume adaptor with a thermostatted water jacket and SC4-18 spindle. The viscosity was measured (n = 3) at three temperature values, viz. at 4 °C, 25 °C and 37 °C, at different rotational speeds from 0.5–20 rpm with a torque of nearly 100%. The samples were equilibrated for 10 min before the measurement; also the instrument was equipped with a temperature-control unit.^31,44

2.9.7. Spreadability. One of the criteria for an ideal gel is that it should display good spreadability, which is the term that denotes the extent of the area of the application site on which the gel readily spreads. The spreadability was determined using a method previously reported by Bachhav et al.³⁹ The spreadability was evaluated by placing 0.5 g of gel within a premarked circle of 1 cm diameter on a glass plate that was 5 mm thick and had an area of 15 cm². Another glass plate with similar dimensions was placed over the gel; care had to be taken to avoid air bubbles from being trapped between the two slides. A mass of 500 g was kept on the upper glass plate for 5 min to cause the gel to spread uniformly. The increase in the diameter due to spreading of the gel was noted as an indicator of spreadability.

2.9.8. In vitro bioadhesion studies. The in vitro bioadhesive potential of the CTZ-loaded NS gel was evaluated and compared with the plain CTZ gel, as reported previously by Bachhav et al.¹² Briefly, an agar plate (1%, w/w) was prepared in pH 4.5 citrate phosphate buffer, and a test sample (50 mg) was placed at its center. After five minutes, the plate was assembled with a USP disintegration test apparatus and ran in SVF (pH 4.5) at 37 ± 1 °C. The motion of instrument arm was such that the sample on the plate was immersed into the solution at the lowest point and was taken out of solution at the highest point. The residence time of the test samples on the plate was noted by visual observation.

2.9.9. In vivo antifungal activity. In vivo experimental procedures, including handling of the animals used in our experiments, were approved by the Institutional Animal Ethics Committee (Registration No. 144/2013) and complied with the guidelines set out by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Animal Welfare Division, Ministry of Environment and Forests, Government of India, India.

Eighteen healthy female Wistar albino rats (150–200 g, 6–8 weeks) were used for the assessment of in vivo performance of the CTZ NS based in situ gel. Two days prior to vaginal inoculation, all animals were maintained under pseudoestrus by subcutaneous injection of estradiol valerate (25 mg kg⁻¹). Before starting the experiments, vaginal cultures of all animals were performed and no Candida sp. were noted. In these oophorectomized rats, C. albicans (10⁷ blastoconidia per mL in 20 μL of sterile saline solution) were inoculated. Administration was done using a tuberculin syringe devoid of a needle.

From each animal, vaginal fluid was taken using a sterile swab after every two days. These swabs were then streaked on a Sabouraud dextrose agar (SDA) plates and were incubated (LHC-78-Labhospmake, India) at 35 °C for 72 h. At least one vaginal sample was evaluated for each animal and inoculum viability was established by counting the number of colony-forming units (CFUs) via a serial-dilution method. The infected animal subjects were divided into three groups: a control group with no treatment (GF1), a group that received the plain CTZ gel (GF2), and a group that received an optimized NS gel formulation (F3) designed to treat vaginal candidiasis (GF3).

The respective gel samples (for GF2 and GF3) were individually administered into the respective vaginas using an injector without a needle.

For evaluating vaginal burden, samples were collected by rolling a sterile cotton swab over the vaginal cavity. Swabs were then streaked over Sabouraud dextrose agar (SDA) plates and incubated (LHC-78-Labhospmake, India) for three days at 35 °C prior to analysis. The vaginal swabs were collected on the 1^st, 3^rd, 5^th, 7^th, 14^th and 21^st days after administering the formulations intravaginally. After 21 days, the animals were sacrificed by administering an excess dose of pentobarbital and vaginal tissues were excised. The vaginal tissues were fixed in 10% neutral buffered formalin, dehydrated in a graded alcohol series, cleared with methyl benzoate and embedded in paraffin wax. Histopathological studies were carried out using light microscopy by taking tissue sections (4 μm) stained with haematoxylin and eosin (HE), and vaginal inflammation was investigated by observing neutrophil accumulation. Grading scores were assigned as low, moderate or extensive depending on inflammatory infiltrate, fibrosis and lamina propria infiltration by inflammatory cells.

2.9.10. In vivo irritation studies. The vaginal irritation potential of plain CTZ and CTZ NS based in situ gels were evaluated by a method reported by Francois et al.⁴⁵ Nine healthy female Wistar albino rats (150–200 g, 6–8 weeks) were used for the in vivo irritation studies and were evaluated for vaginal/vulval irritation, bleeding or discharge from the vagina prior to studies. Animals were divided into three groups (n = 3): control group with no treatment (Group GI1), a group that was administered the plain CTZ in situ gel (Group GI2), and a group that was administered the CTZ NS in situ gel (Group GI3).

The formulations were administered (0.25 mL kg⁻¹) by using a 1 mL plastic syringe. After application, the vaginal cavity was observed for any signs of possible irritation of the vaginal mucosa (i.e., erythema and edema) and related mucosal reactions for three days. The mean erythemal scores were recorded (0–4) on the basis of degree of erythema as: 0 = no erythema, 1 = slight erythema (barely perceptible light pink), 2 = moderate erythema (dark pink), 3 = moderate to severe erythema (light red) and 4 = severe erythema (extreme redness). The experimental animals were sacrificed and a transverse section of the vaginal tissue was scrutinized by an experienced pathologist for severity of atrophy and epithelial loss.

3. Results and discussion

3.1. Solubilization efficiency

The solubilization efficiency studies revealed the potential of the HP-β-CD NS in enhancing CTZ solubility. The percentage of CTZ that was solubilized by the NS batches was observed in the order C(1 : 8) > B(1 : 4) > A(1 : 2) as depicted in Fig. 1. The solubilization enhancement factor observed for the NS batches A, B, and C was 9 ± 0.03, 17 ± 0.02 and 32 ± 0.06, respectively. The increase in the solubility of CTZ was due to matrix entrapment and formation of inclusion complexes. The major factor affecting the extent of inclusion complex formation was the degree of crosslinking between NS and CTZ. The higher the degree of this crosslinking, the higher will be the drug entrapment/loading and inclusion complexation, consequently leading to enhanced solubilization.⁴⁶ Moreover, increased solubilization by NSs could be attributed to possible masking of CTZ hydrophobic groups, decreased crystallinity and increased wetting behavior as reported in earlier findings.^23,29

Fig. 1 Comparison of CTZ solubilization by NS batches with different HP-β-CD: crosslinker ratios – A (1 : 2), B (1 : 4) and C (1 : 8).

3.2. Entrapment efficiency

The entrapment efficiency of the NS formulations was found to be in the range of 15.34 ± 0.49 to 85.12 ± 0.61 (Table 1). The entrapment efficiency was observed in the order N6 > N3 > N5 > N2 > N4 > N1. That the N6 NS formulation displayed a higher loading (at 85.12%) than did the other formulations; which could be attributed to the highest degree of crosslinking between HP-β-CD and DMC, which permits the encapsulation of CTZ in the inner structure of the NS. The difference in entrapment of CTZ between the different batches showed that the degree of cross-linking affected the capacity of the NS to form complexes. As observed in N1, the lower amount of crosslinker formed a network with incomplete cyclodextrin cross-linking, and hence with a decreased number sites for complexing CTZ; thus, CTZ was not incorporated in high amounts. However, in N6, the presence of more cross-linker yielded a greater extent of HP-β-CD cross-linking, and consequently more interactions between CTZ and HP-β-CD cavities, leading to better entrapment as reported previously by Ansari et al.⁴⁷ and Shende et al.⁴⁸ Based on its promising solubilization and entrapment efficiency, NS formulation N6 was chosen as the optimized formulation and was used for further studies.

3.3. Characterization of CTZ-HP-β-CD NS

3.3.1. FT-IR studies. The comparison of FT-IR spectra of pure CTZ, the unloaded NS sample and the CTZ-loaded HP-β-CD NS sample is shown in Fig. 2. The CTZ spectrum (Fig. 2A) showed all characteristic absorption peaks, including at 3057 cm⁻¹ (aromatic C–H stretch), 1597 cm⁻¹ (aromatic C=N stretch of imidazole ring), 1423 cm⁻¹ (aromatic C=C stretch of imidazole ring), 1082 cm⁻¹ and 1016 cm⁻¹ (aromatic C–N stretch of imidazole ring), 1215 cm⁻¹ and 1278 cm⁻¹ (in-plane C–H bend) and 912 cm⁻¹, 845 cm⁻¹ and 767 cm⁻¹ (out-of-plane C–H bend).¹⁹ The FT-IR spectrum of the unloaded HP-β-CD NS sample (Fig. 2B) showed the characteristic absorption band at 3400–2800 cm⁻¹ due to O–H stretching vibrations of HP-β-CD and the presence of a carbonate bond, which produced a peak at about 1728 cm⁻¹, and which indicates formation of a CD-based NS, as previously reported.^27,49 In addition, other characteristics peaks of the unloaded NS spectrum were found at 2929 cm⁻¹, which was due to C–H stretching vibration, at 1361 cm⁻¹ due to C–H bending vibration, and 1018 cm⁻¹ corresponding to C–O stretching vibration of primary alcohol.

Fig. 2 FT-IR spectra of (A) CTZ, (B) the unloaded HP-β-CD NS sample and (C) the CTZ-loaded HP-β-CD NS sample.

In the CTZ-loaded HP-β-CD NS spectrum (Fig. 2C), due to the interaction of CTZ with NS, all peaks belonging to the HP-β-CD NS appeared and were sharp, but were shifted to higher or lower wave number, i.e., 1726–1734 cm⁻¹, 1424–1401 cm⁻¹, 1027–1030 cm⁻¹ and only few characteristics peak of CTZ were observed, at 1064 cm⁻¹, 1020 cm⁻¹, 962 cm⁻¹, 750 cm⁻¹ and 528 cm⁻¹, confirming the interaction between CTZ and NS.

3.3.2. DSC studies. DSC is an extremely valuable tool for investigating the thermal behavior of NSs and it offers both qualitative and quantitative information about the physicochemical state of a drug loaded in the NS. The DSC thermograms of CTZ, the CTZ NS physical mixture, the unloaded HP-β-CD NS sample and the CTZ-loaded HP-β-CD NS sample are shown in Fig. 3. DSC analysis of pure CTZ (Fig. 3A) showed a gradual enthalpy change, producing a sharp endothermic peak at temperature 147.47 °C which is indicative of its melting temperature.⁵⁰ This endothermic peak was also observed in the NS physical mixture thermogram (Fig. 3B), but with a lower intensity. In contrast, in the case of the blank NS thermogram, a broad peak appeared at about 95.27 °C, corresponding to HP-β-CD (Fig. 3C) as reported earlier by Sinha et al.⁵¹ However, the DSC thermogram of the CTZ-loaded HP-β-CD NS sample (Fig. 3D) did not show the melting peak due to formation of inclusion and non-inclusion complexes between the NSs and CTZ. The lack of a DSC peak of the drug was mainly due to the drug having become encapsulated, molecularly dispersed in the NS structure and unable to crystallize, which confirmed the interaction between CTZ and NS, as previously alluded to by Rao et al.⁵²

Fig. 3 DSC thermograms of (A) CTZ, (B) physical mixture of CTZ and HP-β-CD, (C) the blank HP-β-CD NS sample and (D) the CTZ-loaded HP-β-CD NS sample.

3.3.3. XRPD studies. To characterize the physical nature of CTZ within the NS and to evaluate the mode of interaction between CTZ and NS, X-ray powder diffraction (XRPD) data of CTZ, HP-β-CD, the CTZ NS physical mixture and the CTZ-loaded HP-β-CD NS sample were acquired. Encapsulation of the drug in the HP-β-CD NS alters the crystallinity of the drug by causing it to adopt an amorphous state and hence lose its crystallinity, as previously reported by Shende et al.⁴⁸ The XRPD pattern of pure CTZ (Fig. 4A) showed intense, sharp diffraction peaks, indicative of its crystalline nature, at 2θ values of 10.3°, 12.4°, 18.6°, 19.5°, and 20.7° and several minor peaks at 14.2°, 16.7°, 18.8°, 19.9°, 24.4°, 27.5°, and 28.2°, as reported earlier.¹⁹ The XRPD pattern of HP-β-CD (Fig. 4B) did not have any sharp diffraction peaks but instead a broad peak in the 2θ range 20–30°; confirming its amorphous structure as previously reported by Wang et al.⁵³ Furthermore, the appearance of sharp peaks corresponding to the CTZ drug indicated the retention of its crystalline structure in the NS physical mixture (Fig. 4C). However, some of the peaks due to CTZ were less intense here, which could be due to the diluting effect of HP-β-CD.¹⁹ The crystallinity of CTZ became reduced when it was encapsulated by the NS, which consequently led to its amorphous nature and thus the absence of crystalline peaks in the XRPD pattern of the CTZ-loaded HP-β-CD NS sample (Fig. 4D), as described earlier by Rao et al.⁵² These XRPD findings showed that the formation of complexes by the drug was not just due to the mechanical mixing of components, consistent with the FT-IR and DSC results.

Fig. 4 X-ray diffractograms of (A) CTZ, (B) HP-β-CD, (C) the physical mixture of CTZ and HP-β-CD, and (D) the CTZ-HP-β-CD NS sample.

3.3.4. Particle size, zeta potential, polydispersity. The average diameter, polydispersity index and zeta potential of the CTZ-HP-β-CD NSs (for batch N6) were found to be 455.6 ± 11 nm, 0.143 and −21.32 ± 1.3 mV, respectively (Table 3). The zeta potential of the CTZ NS sample was found to be sufficiently high, probably due to the carbonate groups in its structure promoting electrostatic repulsion between the NS particles and hence ensuring their physical stability by avoiding aggregation.⁵⁴

Table 3 Average particle size and zeta potential of NSs

Formulation	Average diameter ± SD^a (nm)	Zeta potential ± SD^a (mV)	Polydispersity index
a Mean ± SD, n = 3.
HP-β-CD NS	449.3 ± 14	−21.61 ± 2.1	0.141
CTZ loaded HP-β-CD NS (N6)	455.6 ± 11	−21.42 ± 1.3	0.143

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3.3.5. SEM and TEM analysis. The NS size and surface morphology were further investigated using SEM and TEM analysis. NSs were uniformly found to be roughly spherical in shape with a spongy nature.^39,48 The SEM micrograph (Fig. 5) showed the formed NS having numerous fine surface voids, probably as a result of solvent diffusion. Also no residual, intact crystals of CTZ were observed on the NS surface, indicative of the NS matrix formed by CTZ-HP-β-CD. The TEM results (Fig. 6) showed that the regular size and shape of the blank HP-β-CD NS were unaffected even after CTZ loading. Furthermore, as explained by Shende et al., the extraporous nature of the NS might be due to its encapsulation of the drug.⁵⁴

Fig. 5 Scanning electron micrograph of the CTZ-HP-β-CD NS sample (A) at 1000× magnification and (B) at 2000× magnification.

Fig. 6 TEM images of (A) the blank HP-β-CD NS sample and (B) CTZ-loaded HP-β-CD NS sample at 46 000×.

3.4. Characterization of the CTZ NS gel

An NS-embedded thermosensitive in situ gel was prepared to increase the viscosity and residence time and to improve the localization of CTZ in the vaginal cavity. Nine formulations of CTZ-HP-β-CD NS-based in situ gels (F-1 to F-9) were prepared as per a full factorial design (Table 2) by changing two independent variables: the concentrations of PF-127 (A) and PF-68 (B). Table 4 shows the response of gelation temperature (°C) (R1), gelation time (s) (R2), and in vitro drug release (% cumulative drug release) (R3), and these response values were subjected to multiple regression analyses to yield polynomial equations whose coefficient values indicate the effect of changing the individual variable; 3D response surface graphs and contour plots were constructed.

Table 4 3² factorial design layout and responses noted for CTZ NS-based in situ gels

Formulation code	A^a	B^a	R1^a^,^b (°C)	R2^a^,^b (s)	R3^a^,^b (% cumulative release)
a A: concentration of PF-127, B: concentration of PF-68, R1: gelation temperature, R2: gelation time and R3: in vitro drug release.b Mean ± SD, n = 3.
F-1	−1	−1	38.8 ± 0.23	61 ± 1	97.81 ± 1.22
F-2	0	−1	35.2 ± 0.22	55 ± 1	84.93 ± 2.13
F-3	−1	1	34.1 ± 0.43	52 ± 2	79.82 ± 1.67
F-4	1	−1	31.4 ± 0.11	49 ± 1	73.38 ± 1.43
F-5	1	1	26.9 ± 0.33	43 ± 1	61.21 ± 2.03
F-6	−1	0	36.6 ± 0.57	58 ± 1	91.33 ± 1.39
F-7	0	0	33.1 ± 0.46	52 ± 1	79.65 ± 0.42
F-8	0	1	30.9 ± 0.34	46 ± 2	67.47 ± 0.92
F-9	1	0	29.1 ± 0.64	46 ± 1	66.87 ± 3.32

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3.4.1. Gelation temperature. The prepared CTZ NS-based in situ gels exhibited a temperature-dependent reversible sol-to-gel transition. These in situ gel systems were observed to transform from a sol to a gel at a temperature less than body temperature, specifically between 26.9 ± 0.33 °C and 38.8 ± 0.23 °C. Based on 3² factorial designs, the factor combinations of A and B resulted in different response variables for gelation temperature (R1). The equation derived by a mathematical best fit to relate the response R1 to factors A and B was R1 = +32.900 − 3.683A − 2.250B. ANOVA of the equation suggested the model F value to be 1829.07, with a P value < 0.0001, indicating that the model is significant. The increase or decrease in the effects of the response on different levels of combinations of independent variables was indicated by a positive or negative sign of the polynomial terms. As the predicted r² of 0.9963 is nearly the same as the adjusted r² of 0.9978, the above polynomial equation showed a good fit of the response of the variables at different levels. A 3D surface plot of R1 is shown in Fig. 7A and indicated a significant decrease in the gelation temperature with an increase in the concentrations of factors A and B. The gelation temperature of the developed system was found to be concentration dependent; as the concentrations of PF-127 and PF-68 were increased, the thermoresponsivity of was observed to increase. There was no significant effect of the interaction between factor A and factor B on the gelation temperature.

Fig. 7 Response surface plots of (A) R1: gelation temperature (°C); (B) R2: gelation time (s) and (C) R3: in vitro drug release (% cumulative drug release) up to 12 h at different levels of factor A and B.

3.4.2. Gelation time. Along with the sol–gel transition occurring at physiological conditions, another prerequisite of an effective in situ vaginal gel drug delivery system is that the gel forms within an optimum time period. A fast sol–gel transition is required to keep the in situ NS gel and hence the drug in the vaginal cavity, which is important for prolonging the localized release of the drug. The results of gelation time analysis indicated that the prepared CTZ NS-based in situ gel quickly responded to variations in gelation temperature. The gelation temperature and time were mainly evaluated for comparing the prepared batches and to optimize the final formulation. The formulations were subjected to their respective gelation temperatures, which resulted in quick gelations of less than 61 ± 1 s. The gelation times of all the formulations were between 43 ± 1 and 61 ± 1 s. Based on 3² factorial designs, different combinations of the factors A and B resulted in different response variables for gelation time (R2). The equation derived from the best fit of the response R2 to the independent variables was R2 = +51.333 − 5.500A − 4.000B. ANOVA of the equation suggested a model F value of 128.08, and a P value of <0.0001, indicating that the model was significant. Also, the predicted r² of 0.9442 is similar to the adjusted r² of 0.9695. The gelation time was significantly influenced by factors A and B. Increase in the concentrations of the polymers led to decreased gelation time of about 18 s. A three-dimensional response (3D) plot of R2 (Fig. 7B) showed a significant decrease in gelation time with increasing concentrations of factors A and B. This relationship might be due to the increased viscosity of the system at increased levels of factors A and B.

3.4.3. In vitro release studies. In vitro drug release studies provide vital information for predicting the performance of the formulation in vivo. The results of in vitro release studies indicated that drug release was prolonged. The equation derived for R3 by the best fit mathematical model was R3 = +78.052 − 11.250A − 7.936B with a predicted r² of 0.9490, which is in reasonable agreement with the adjusted r² of 0.9715. The effect of factor A was found to be more significant than the effect of factor B, with an increase in the factor A release rate of CTZ from in situ vaginal gels significantly decreased. Prolonged CTZ release from formulations up to 12 h was mainly influenced by factor A. In addition, factor B yielded a quicker gelation time and lower gelation temperature, and hence in these regards a better performance than did factor A. ANOVA of the equation suggested a model F value of 137.32, with a P value < 0.0001, indicative of the significance of the model. As shown in the dimensional response (3D) plot of R3 in Fig. 7C, there was a significant decrease in drug release with an increase in A and B.

All formulations exhibited prolonged release of CTZ devoid of any burst release. Drug release from the NS was due to gradual erosion of the NS and concomitant diffusion of the drug into the external polymer matrix. The prolonged release from NS gels is of great interest for enhancing vaginal drug delivery and maintaining the required concentration for overall treatment of vulvovaginal candidiasis.

3.4.4. Checkpoint analysis and optimization of design. To optimize all the responses with different targets, a multi-criterion decision approach (a numerical optimization technique using the desirability function and a graphical optimization technique using the overlay plot) was deployed (Fig. 8A). The optimized in situ gel formulation (F-10) was obtained by applying constraints such as R1 = 34 °C, R2 = 53 s, and R3 = 82% on the responses. These constraints were common for all the formulations. Recommended concentrations of the factors were calculated by the DoE from above plots, which has highest desirability near 1.0. The optimum values of selected variables obtained using DoE were 19.448% for A and 9.811% for B.

Fig. 8 (A) Overlay plot for optimization of the CTZ NS-based in situ gel and (B) contour plot represent overall desirability function of optimized formulation (F-10).

The desirability and overlay plot of the DoE provided optimum values of both factors (factor A: concentration of PF-127 and factor B: concentration of PF-68), from which the final formulation was prepared. The optimized gel formulation (F-10) was prepared for checkpoint analysis and evaluated for gelation temperature (°C), gelation time (s), and in vitro drug release (% cumulative drug release) up to 12 h, which showed response variables of R1 = 33.7 ± 0.61 °C, R2 = 52 ± 1 s, and R3 = 80.37 ± 0.59%. There is a close agreement between predicted and observed values (Table 5) as indicated by the desirability value of 0.991 with low relative errors (Fig. 8B). These results demonstrated the reliability of the optimization procedure followed in the present study to prepare formulations according to the 3² factorial designs. Factors A and B at concentrations of 19.5% and 9.9% were suitable for drug delivery in the NS-based in situ vaginal gel, and thus chosen for CTZ NS-based in situ gel delivery in this study.

Table 5 Checkpoint analysis of optimized formulation (F-10) of CTZ NS-based in situ gels

Parameters	A	B	R1 (°C)	R2 (s)	R3 (%)	Desirability
Predicted	19.448	9.811	34	53	81.45	0.991
Observed	19.5	9.9	33.7 ± 0.61	52 ± 1	80.37 ± 0.59	—
Relative error	0.052	0.089	0.3	1	1.08	—

---

Other evaluation parameters
Viscosity (cps)			Drug content (%)
At 4 °C	At 25 °C	At 37 °C
326	1143	201 700	97.83 ± 0.26

---

Fig. 9A shows the in vitro release profiles of the plain CTZ gel and optimized CTZ NS-based in situ gel (F-10). The release profile indicated that the drug encapsulated in the NS gel was not released completely at the end of 12 h (80.37%). The release data from the NS-based in situ gel was fitted into various kinetic models. The value of r² was found to be highest for the Higuchi model (r² = 0.92), which indicated that the test product followed matrix-diffusion-based release kinetics. No initial burst release was observed in the release profile of the optimized CTZ NS gel, which confirmed that CTZ was not adsorbed on the NS surface and was instead well encapsulated within the nanostructure. These results were in good agreement with earlier findings of Swaminathan et al.²⁷ A similar sustained release profile was obtained with camptothecin polyrotaxane-based delivery systems assembled using CDs.⁵⁵

Fig. 9 (A) In vitro release profiles of plain CTZ and CTZ-loaded HP-β-CD NS-based in situ vaginal gels in SVF (mean ± S.D, n = 3). (B) Fungal clearance kinetics of C. albicans infection in infected oophorectomized rats; each curve represents the mean of six rats. Marked differences in the fungal clearance kinetics of CTZ-HP-β-CD NS gels and plain CTZ gels have been noted (P < 0.003).

3.4.5. pH and drug content. The pH of the optimized formulation (F-10) was found to be 4.57, which is the same as vaginal pH. The drug content of the formulation (F-10) was found to be 97.83 ± 0.26% of the theoretical value (2% w/w), implying that a significant amount of the drug was loaded in the NS-based in situ gel.

3.4.6. Viscosity studies. For thermosetting gels, the viscosity at various conditions is an important rheological parameter that influences its utilization and in vivo performance. For instance, instillation of gel is difficult if the viscosity is too high, but viscosity that is too low causes increased drainage. An ideal in situ vaginal gel should be a less viscous liquid at room temperature so as to allow easy administration into the target site, but should undergo at this site in situ phase transition to form a strong gel with enhanced viscosity.^31,56 PF-68 alone caused formation of weaker gel bases, while the high concentration of PF-127 polymer yielded the hardest gel. Solutions containing less than 15% PF-127 did not form gels, whereas a PF-127 concentration higher than 25% led to difficulty in preparation and administration.^56–58 Thus, a combination of PF-127 and PF-68 was used in the present study.

The viscosity of the CTZ NS-based in situ gel was measured at 4 °C, 25 °C and 37 °C, representing the storage, room and body temperatures, respectively. These studies revealed a temperature-dependent increase in viscosity of the gel. The viscosity values of the optimized formulation at 4 °C and 25 °C were measured to be 326 cP and 1143 cP; however, a significant increase in viscosity (to 201 700 cP) was noted at 37 °C, which can be attributed to a sol–gel conversion. Pluronics being non-ionic PPO triblock copolymers aggregate into micelles at 37 °C, resulting from the dehydration of polymer blocks with increasing temperature. The formation of gels has been shown to result from enlargement of micelles, and hence they cannot be easily separated from each other, which accounts for the greater viscosity and rigidity of gels.^31,59,60 Gelation temperature (T_sol–gel) is the temperature at which the liquid phase makes a transition to gel with a sudden increase in viscosity. In the present study, the optimized formulation exhibited a gelation temperature of 33.7 ± 0.61 °C.

3.4.7. Spreadability. Spreadability is an important property of semisolid formulations, affecting the ease of application and patient compliance. An in situ gel with a good spreadability takes less time to spread, and is ultimately easier to administer. In the present study, spreadability was expressed in terms of the increase in the diameter (6.6 cm) of 0.5 g of gel, which was placed inside a previously drawn circle (of 1 cm in diameter) as a result of applied weight (as described in Section 2.9.7.). In the case of the optimized gel (F-10), the overall increase in diameter was found to be 7.6 cm, reflecting good spreadability.

3.4.8. In vitro bioadhesion studies. When two materials (whether both are biological, or one is biological and the other synthetic) are held together by interfacial forces for an extended period of time, they display ‘bioadhesion’.⁶¹ This term usually refers to when the interaction occurs between any polymer and the epithelial surface, but for bioadhesive drug delivery systems, bioadhesion typically refers to the adhesion between soft tissues and polymers, whether natural or synthetic.^62,63 Evaluation of bioadhesion is important to ensure that the adhesion offered by formulations is sufficient to ensure prolonged retention of the gel at the site of application, but not excessively so, as excessive retention may be associated with damage to the mucous membrane. The bioadhesive potentials of the CTZ NS-based vaginal gel and the plain CTZ gel were evaluated by using an in vitro method and were found to be 53 ± 2.5 min and 38 ± 1.2 min respectively (n = 3). The bioadhesive potential of formulated in situ gels could be attributed to the fact that Pluronics with hydrophilic oxide groups bind to oligosaccharide chains of the mucosal membrane.^57,64,65 Higher concentrations of PF-127 could therefore provide more hydrophilic oxide groups for binding oligosaccharide chains, which is likely to prolong the residence time at the absorption site. Addition of PF-68, which is a homologue of PF-127, enhanced the bioadhesive force, since 80% of PF-68 is made up of hydrophilic oxide groups. Moreover, the retention time displayed by the NS gel was found to be higher than that of the plain CTZ gel. These results suggest that the CTZ-HP-β-CD NS-based vaginal gel resides in the vaginal cavity for a longer period of time than does the plain gel. Similar results were previously reported for the bioadhesion of econazole nitrate by Esra et al.¹⁰

3.4.9. In vivo antifungal activity. The mechanism of action of CTZ involves inhibiting the fungal cytochrome P-450 enzyme and hence hindering the biosynthesis of ergosterol, which damages the integrity of the fungal cell wall.⁶⁶ A graph of the pattern of fungal clearance kinetics is shown in Fig. 9B. The results were found to be encouraging for the prepared CTZ NS-based in situ gel, when compared to the results for the plain CTZ in situ gel. The vaginal swab samples were taken on 1^st, 3^rd, 5^th, 7^th, 14^th and 21^st days after treatment. The vaginal swabs of the untreated control group of animals were observed to be positive for C. albicans up to the 21^st day, but a striking disparity between the fungal clearance kinetics of the plain CTZ gel and the CTZ NS-based gel was observed. The CTZ NS-based in situ gel showed an accelerated clearance, while the least clearance was noted with the plain CTZ in situ gel.

The biocompatibility of the CTZ NS in situ gel was assessed by histopathological studies. The fungal infection was observed to be consistent with time in the control group (GF1). In the case of the plain CTZ in situ gel (GF2), there was moderate evidence of inflammation, whereas the optimized CTZ NS in situ gel (GF3) showed few signs of inflammation. The inflammation scores for GF1, GF2 and GF3 were noted to be extensive inflammation (10/10), moderate inflammation (6/10) and low inflammation (1/10), respectively. The inflammatory and non-inflammatory micrographs of vaginal mucosa are shown in Fig. 10.

Fig. 10 Representative hematoxylin and eosin (H and E)-stained histological slides of vaginal mucosa from female Wistar albino rats. (A) Inflammatory condition with extensive neutrophil accumulation in lamina propria of the vaginal squamous epithelium and (B) non-inflammatory vaginal mucosa in a high-magnification (200×) view.

3.4.10. In vivo irritation studies. In vivo vaginal irritation studies were carried out for evaluating the tolerability of the CTZ NS-based in situ gel after administration. It was observed that plain CTZ (GI2) and the CTZ NS-based in situ gel (GI3) were well tolerated by the rats with no signs of erythema and/or edema (erythemal score 0) even after 3 days.^67,68 However, the control group (GI1) showed severe erythema (erythemal score 4).

4. Conclusions

Our study proved that the solubility and efficacy of CTZ was enhanced by loading it in biocompatible cross-linked cyclodextrin polymeric NSs. All characterization results confirmed the interaction and formation of an inclusion complex of CTZ with the NS. Incorporation of CTZ NSs in an in situ vaginal gel executed therapeutically better effects than did the conventional formulation. The higher solubilization and prolonged release of CTZ from our CTZ NS sample justified the purpose of synthesizing the NSs. Moreover, observed in vitro bioadhesion and in vivo antifungal activities revealed that the CTZ NS gel displayed a higher residence time and a greater potential to effectively alleviate vaginal infections than did the conventional formulation. The prepared cyclodextrin-based CTZ-loaded NS in situ gel is therefore a promising carrier for the effective local treatment of vulvovaginal candidiasis.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

The authors express a deep sense of gratitude for the Central Food Technological Research Institute (CFTRI), Mysuru (a constituent laboratory of CSIR, New Delhi) and JSS University, Mysuru for providing the necessary facilities to carry out the research.

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