Intravaginal administration of metformin HCL loaded nanoemulgel for treatment of PCOS: in-vitro and in-vivo studyAbstract
Metformin HCL (MET-HCL), is a Biguanide derivative, most effective agent in treatment of PCOS. The focus of the present study was on developing and optimizing a nanoemulsion formulation. of MET-HCL and convert into intravaginal MET-HCL Nanoemulsion Gel (MET-HCLNE GEL) To enhance the therapeutic efficacy in managing PCOS. The nanoemulsion was formulated using water titration method and evaluated for droplet size, zeta potential, polydispersity index (PDI), drug content, viscosity, and morphology. Then incorporated into gel and evaluated for pH, drug content, viscosity, in-vitro release & in-vivo study. The formulation MET-HCLNE 1 showed the desired droplet size (45.60 ± 1.7 nm), PDI (0.198 ± 0.2), zeta potential (22.4 ± 0.3), drug content (89.19 ± 0.2 %) and viscosity (42.85 ± 2.1 cp). In in-vitro release, MET-HCLNEGEL showed significant prolonged release over MET-HCL solution. Intravaginal MET-HCLNE GEL showed significant efficiency against PCOS compared to oral administration in female Wistar rats. No significant changes were found in the values of viscosity, drug content and pH over the period of 120 days. Thus, MET-HCLNE GEL was found to have good potential to function as an Intravaginal formulation & having great PCOS activity and good storage stability.
Keywords:
Metformin hydrochloride; Nanoemulsion gel; In-vitro release; In-vivo study; Intravaginal delivery; Polycystic ovary syndrome (PCOS).
INTRODUCTION
Polycystic ovary syndrome (PCOS), an endocrine disorder which include reproductive hormonal abnormality. This illness affects an estimated 4-21% of reproductive-age females globally and leading causes of infertility (Heidarpour et al., 2024; Larik et al., 2024). Reduced levels of FSHFollicle Stimulating Hormone and elevated levels of LHLuteinizing Hormone are associated with PCOS. In parallel, an increase level of LH induces release of testosterone estrogen. This leads to the development of cysts in the ovary (Saini et al., 2016). The exact cause of PCOS is unknown, it is considered a complicated multi-genetic condition caused by abnormal gonadotropin release. PCOS women exhibit abnormalities such as obesity, insulin resistance, risk of cardiovascular diseases (Yakubu et al., 2015). Metformin, an insulin sensitizer, has been suggested as a first-line medicine for the treating PCOS. Metformin helps people with PCOS improve their metabolic and reproductive problems (Peng et al., 2023). Metformin reduces metabolic imbalances and PCOS related menstrual irregularities. (Stoica et al., 2019). MTF-HCl work by activating AMP-dependent kinase (AMPK-alpha) pathway, which lowers glucose production, promotes fatty acid oxidation, thus increasing absorption of glucose in tissues (Tang et al., 2024). Metformin HCL administered orally results in reduced absorption (50-60%) and needs frequent dosage 500 mg twice-three times daily (Siavash et al., 2017; Zhao et al., 2021). To optimize its therapeutic efficacy, metformin HCL must be administered in a tailored dose form.
The intravaginal route of drug delivery considers to be best alternative to overcome drawbacks associated with oral administration as vaginal drug delivery offers numerous advantages. Large vascularized mucosal surface area allows both systemic and local systemic administration, avoid first-pass effects, complemented by low dose requirements (De Lima et al., 2017). There are some disadvantages associated with intravaginal medication delivery, such as limited mucosal contact time, frequent vaginal discharge washing-out, and significant anatomical and physiological diversity across individuals (Kawarkhe, Poddar, 2010; Smoleński et al., 2021). Hence, there is a necessity for innovative formulations development, like micro and nanoemulsion-based drug delivery (Mallipeddi, Rohan, 2010; Bassi, Kaur, 2012; Machado et al.,2013). Unlike traditional hydrophilic formulations, nanoemulsion allow the hydrophilic and lipophilic substances administration into the vagina (Matanović, Kristl, Grabnar, 2014). Furthermore, nanoemulsion coversion into gel prolong retention at application site and controlled release of drug through vaginal mucosa (Srikrishna, Cardozo, 2013). HPMC k100 gelling agent consider to be best for vaginal drug delivery as it is biocompatible and biodegradable (Cook, Brown, 2018; Patel, Joshi, 2012). Based on these facts, the current study was aimed to develop the nanoemulsion of MET-HCL and convert into appropriate intravaginal nanoemulsion gel to enhance therapeutic action, better dispersity, and storage stability in treatment of PCOS (Smoleński et al., 2021). The therapeutic effectiveness of prepared nanoemulsion gel was observed in female Wistar rats through vaginal route of administration (Yakubu et al.,2015).
MATERIAL AND METHODS
Material
Metformin Hydrochloride (MET-HCL) was obtained from vasuchem lifescience, Pune, India. HPMC K100 was obtained from lobachem, Mumbai, India. Sesame oil, Coconut oil, castor oil and peppermint oil were purchased from S.D Fine Chemical, Mumbai, India. Tween 80, tween 20, transcutol P, chremophore RH 40 received from Mohini organics Mumbai, India as gift sample. All other agents utilized in the study were of analytical grade.
Methods
Standard Calibration Curve of Metformin HCl in Simulated Vaginal Fluid (SVF)
10 mg of Metformin HCl was weighed and dissolved in 5 mL of SVF, sonicated for 10 minutes and the final volume was made up to 10 mL in volumetric flask with SVF, resulting in a solution containing 1000 µg/mL of Metformin HCl. Then 1 mL of this primary stock solution was diluted to 10 mL with SVF to form the secondary stock solution. The resulting solution was then scanned for absorbance. To prepare dilutions, 0.2, 0.4, 0.6, 0.8, and 1mL of the secondary stock solution were diluted to 10 mL with SVF in separate volumetric flasks, resulting in standard solutions of 2, 4, 6, 8, 10 µg/mL, respectively. The absorbance of the resulting solutions was measured at 232 nm against SVF as the blank.
Solubility Study
Solubility tests are known to be a basic criterion for selecting oils, co-surfactants, and surfactants in the formulation of nanoemulsions, to determine the solubility of metformin HCL (MET-HCL), in an experiment on saturated solubility, an excessive of the drug was dissolved in predetermined amount of certain oils, co-surfactants, and surfactants. In 2 ml of the selected oil, co-surfactant and surfactant, an excess amount of MET-HCL added. The mixture was vortexed for 30 sec. For 72h again mixed on mechanical shaker to attain equilibrium. These mixtures then centrifuged for 10 min at 10,000 rpm. The supernatant was collected and diluted with methanol. Using UV-spectrophotometer (JASCO, V-730, Japan) at 232 nm the samples were analyzed (Khan et al., 2022).
Screening of surfactants for emulsification efficiency
The selection process of surfactants (tween 80, tween 20, cremophore RH 40, labrasol) was based on the transparency percentage and how easily the substances could be emulsified. To conduct the emulsification study, the oil and surfactant were gently mixed for 2 minutes in a 1:1 ratio by weight at a temperature of 50 degrees celsius and the mixture was then mixed further to ensure uniformity. The oil surfactant mixture was combined using distilled water in a glass stoppered flask at a ratio of 1:100. The stoppered flasks were turned upside down multiple times, and the number of flask inversions required to achieve a homogeneous microemulsion (free of cloudiness or phase separation) was noted. Emulsions were left to settle for 2 hours for the percentage transmittance study, and their percentage transmittance at 650nm was measured using a UV spectrophotometer (V-730, JASCO, Japan). Three replicates were used to calculate the transmittance % for each emulsion. The surfactant that produced a clear emulsion with minimal inversions and a higher percentage of transmittance was chosen for further investigation. (Nilsuwan, Benjakul, Prodpran, 2016).
Screening of co-surfactants for emulsification efficiency
The surfactant and oil phase chosen were utilized to screen various co-surfactants (PEG 400, PEG 200, Transcutol P, propylene glycol) for further evaluation. The emulsification efficiency was evaluated by preparing a mixture of 200ml, 400ml, and 600ml of co-surfactant, selected surfactant, and chosen oil, respectively. After allowing the emulsions to settle for 2 hours, the percentage of light passing through at 650nm was measured using a UV spectrophotometer (V-730, JASCO, Japan). The percent light transmitted was measured for each emulsion in three different experiments. The surfactant that created a clear emulsion with fewer inversions and a higher percentage of light transmitted was selected for further study. (Nasr, Gardouh, Ghorab, 2016).
Preparation and Screening of Nanoemulsion using Pseudo-ternary Phase Diagram
In the solubility analysis, cremophore RH 40 (surfactant), virgin coconut oil (oil phase) and transcutol P(co-surfactant) showed the highest solubility for MET-HCL. In transmittance efficiency analysis, cremophore RH 40, transcutol P showed higher percent transmittance hence, different ratios (1:1, 1:2, 1:3) of surfactant and co-surfactant (Smix) were prepared whereas, in different glass vials, coconut oil and Smix at a specific ratio (1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1) were mixed. Pseudo ternary phase diagram of coconut oil, Smix and distilled water was created using the water titration method (spontaneous emulsification method). Transparent and easily flowable o/w nanoemulsions were visually observed. (Jhawat, Gulia, Sharma, 2021). A pseudo-ternary phase diagram has the nanoemulsion area marked on it (Chemix school software, Arne Standnes, Bergen, Norway) as represented in.
Preparation of metformin HCL nanoemulsion
Metformin HCL nanoemulsion (MET-HCLNE) The spontaneous emulsification technique was used to achieve the formulation and optimize it. The O/W nanoemulsion formulation of MET-HCL was created by adding the oil phase drop by drop into the aqueous phase. For oil phase right amounts of virgin coconut oil and MET-HCL was homogenized, then gradually adding transcutol P as a co-surfactant and homogenizing further to reduce surface tension. The water phase was created by combining deionized water with cremophore RH 40, a non-ionic surfactant. In order to make NE, the two phases were combined by slowly adding the oil phase to the aqueous phase while constantly stirring to create a pre-emulsion. This pre-emulsion mixture was ultra sonicated using probe sonicator for 15 min to achieve clear, stable and homogenous formulation (Ghosh, Mukherjee, 2013). composition details to formulate nanoemulsion are given in (Table I).
Characterization of Nanoemulsion
Droplet size distribution and zeta potential analysis
The size of MET-HCLNE droplets was assessed by utilizing dynamic light scattering with a Zetasizer Nano ZS. (Malvern Instruments Ltd., Worcester, UK). To ensure homogeneity, MET-HCLNE was dispersed evenly in double distilled water at a ratio of 1:50 and vortexed for 1 minute. The droplet size and polydispersity index (PDI) were both measured. Furthermore, the zeta potential was measured utilizing a Zetasizer Nano ZS. The information is presented as the average ± standard deviation of three repeated measurements (Al-Suwayeh et al.,2023).
Drug Content
Nanoemulsion formulation (1ml) was properly diluted with SVF (simulated vaginal fluid pH 4.2) to obtain the desired drug concentration, Absorbance was determined by using a UV-visible spectrophotometer. (UV-1700, Pharmaspec, Shimadzu Ltd, Japan) at 232 nm (Kumar, Pathak, Misra, 2009).
Viscosity
Using a Brookfield cone and plate viscometer (Brookfield Engineering Laboratories, Inc., Middleboro, MA) at 25±0.5°C, the viscosity of the nanoemulsions was measured.
In vitro drug release
The optimized MET-HCLNE underwent in vitro release study using Franz diffusion cells. based on the droplet size, visual assessment, zeta potential, PDI & drug content. The Franz diffusion cell apparatus consists of two chambers: the receptor chamber and the donor chamber. Prior to commencing the study, the appropriate size of dialysis membrane was soaked in SVF of pH 4.2 for 24 hours, as the in-vitro drug diffusion of MET-HCLNE was conducted at pH 4.2. The choice of suitable release media for intravaginal formulation is determined by the solubility of MET-HCl in particular media and the pH of the vaginal mucosa. Vaginal pH typically falls between 3.8 to 4.5. Hence, it is recommended to choose a buffering system within this pH range in order to avoid causing irritation in the vaginal area. In the study, the receptor compartment, which has a capacity of 20 mL, was filled with SVF at a pH of 4.2. The system maintained a constant temperature of 37 ± 0.5 OC in order to mimic in vivo conditions. The pre-soaked dialysis membrane then kept in between the donor and receptor chambers. After that, 3 mL of MET-HCLNE was added to the donor chamber to assess drug penetration across the dialysis membrane. 3 mL sample was taken from the receptor chamber at intervals of 30, 60, 120, 180, 240, 300, and 360 minutes, and replaced with a 3 mL blank sample of SVF pH 4.2 The sink condition was maintained in order to retain it. After that, the sample that was withdrawn was diluted with the SVF. (pH 4.2) and examined using the UV spectrophotometer at 232 nm. The measurements were conducted three times to ensure accuracy (n = 3).
Transmission Electron Microscopy (TEM)
Optimized nanoemulsion was confirmed by studying the morphological characteristic utilizing transmission electron microscopy (Philips CM-10, USA). The nanoemulsion was applied to a carbon-coated grid with 2% phosphotungstic acid after being diluted 1:100 with distilled water. The images were then captured by scanning the grid. (Anjali et al.,2012).
Preparation of MET-HCL Nanoemulsion Gel
The best formulation, A1, was selected after characterization, and then it was transformed into a gel by utilizing HPMC K100 as a mucoadhesive gelling agent. (Valamla et al.,2022). In 20 ml of the nanoemulsion formulation (~250 mg of MET-HCL), added 1%w/v HPMC K100 slowly with the help of a magnetic stirrer (Remi Mechanical Stirrer, Mumbai) for 1 hr for proper swelling to achieve a homogenous mixture. The pH of the gel was maintained in between 4.2 with dilute acetic acid. So as to avoid vaginal irritation. The formulation was properly stored for further study in sealed glass container.
Characterization of MET-HCL nanoemulsion gel
pH
The pH values of the formulated gel preparation were determined using a pH meter (Schott CG 840, Germany), 24 h after preparation.
Rheological study
Viscosity
The viscosity of gel measured using Brookfield viscometer (Brookfield Engineering, Middleboro, MA) C50-1 spindle in triplicate (n = 3) at 25.0±0.5°C.
Spreadability
To test the spreadability of the formulations, two glass slides (7.5 cm x 2.5 cm) were used. One slide was fixed, and the other was moveable with a pulley and weight. The formulation was placed between the slides, and a 100 g weight was applied for 1-2 minutes to remove trapped air and form a uniform layer. After removing the weight, a 30 g weight was used to pull the top slide. The time it took for the slide to cover a set distance was measured in seconds. Spreadability was calculated using the formula S =M L/T, where M is the weight attached to the higher slide, L is the length of the glass slides, and T is the time required to separate the slides.
Drug content
MET-HCL Nanoemulsion gel (1ml) was suitably diluted with SVF pH 4.2 to achieved required drug concentration and UV-visible spectrophotometer (UV-1700, Pharmaspec, Shimadzu Ltd, Japan) was used to measure absorbance at 232 nm.
Simulated vaginal fluid (SVF) was prepared (Owen and Katz 1999). To prepare the simulated vaginal fluid medium, the following ingredients were mixed together: glycerin (0.16 g/L), sodium chloride (3.51 g/L), urea (0.4 g/L), glucose (5 g/L), potassium hydroxide (1.4 g/L), calcium hydroxide (0.22 g/L), lactic acid (2 g/L), acetic acid (1 g/L), and bovine serum albumin (0.018 g/L) (14,15). Distilled water was used as the solvent. This mixture was adjusted to pH 4.2 using HCl or NaOH.
In vitro drug release
In vitro release study was carried out on metformin HCL nanoemulsion gel (MET-HCLNE GEL) was compared with the MET-HCL solution. In vitro release study was conducted on Franz diffusion cell apparatus. Before starting this study, dialysis membrane soaked in SVF of pH 4.2 for 24 hr because the in-vitro drug diffusion study of MET-HCLNE GEL & MTE-HCL solution was conducted at pH 4.2. The cellulose acetate membrane having moderate permeability with a pore size of 2.4-3.5 nm and thickness of 20-30 μm, was used.
The receptor compartment was filled with 20 mL of SVF (pH 4.2) (Shapiro et al.,2022). The temperature was maintained at 37 ± 0.5 OC to mimic in vivo condition. Previously soaked dialysis membrane was positioned between the receptor and donor chambers. 3 mL of MET-HCLNE GEL & MET-HCL solution was kept in donor chamber and drug permeation via dialysis membrane at specific time interval of 30, 60, 120, 180, 240, 300 and 360 min was determined. From receptor chamber 3 mL of sample taken out and then filled with a 3 mL of blank sample of SVF pH 4.2 to create sink condition. The sample that was withdrawn was then diluted with the SVF (pH 4.2) and analyzed using UV spectrophotometer at 232 nm. Every measurement was made three times. (n = 3).
Drug release kinetics
Four models were used to calculate release kinetics, were first order (log cumulative amount (%) of drug released with time), zero order (cumulative amount (%) of drug released with time) Higuchi (cumulative amount (%) of drug released with the square root of time), Korsemeyer Peppas (log cumulative amount (%) of drug released with log time). The in vitro release curves were analyzed using linear regression to determine the release kinetics. A mathematical model with the high coefficient of determination (R2) was selected as the one that best represented the kinetic release profile (Paarakh et al.,2018).
Differential Scanning Calorimetry (DSC)
Thermal analysis for METHCI, HPMCK100, MET-HCLNE & MET-HCLNE GEL was conducted using DSC (SW STARe, Mettler Toledo, USA). Weighing 5-6 mg, the samples were kept in a pan and sealed with lead. The temperature range for all samples was set at 30-300 0C, with a consistent temperature increase of 10 0C/min under a nitrogen atmosphere. (Shukat, Bourgaux, Relkin, 2012).
Fourier Transform Infrared Spectroscopy (FTIR)
FTIR analysis was done to deetrmine the structure of metformin HCl. Using The IR affinity 8400 Shimadzu equipment. The samples studied were Drug, physical mix (coconut oil, transcutol P, cremophore RH 40, drug) and MET-HCLNE GEL. The sample compartment was prepared for background measurement by wiping it with ethanol. After that, the samples were kept in the sample compartment and a rotating pressure tower was installed for analysis. Liquid sample pH was measured before the IR study due to sensor sensitivity. (Shaikh et al.,2019).
In vivo study
The therapeutic effectiveness of MET-HCLNE GEL was observed in PCOS induced experimental animal model. The Institutional Animal Ethics Committee (IAEC) had approved the study. Four groups of six each (n = 6), total 24 female wistar rats weighing 200-250 gm were made. The rats had unrestricted access to water and were fed standard lab chow. All animal studies were conducted in accordance with the requirements of the Committee for the Purpose of Control and Supervision of Experimental Animals (CPCSEA), Ministry of Forests and Culture, Government of India, India. Then animal in the group I was administered normal saline solution per oral (p.o) for 13 days (this group denote as negative control). The animals in group II were administered 20 mg/kg body weight Mifepristone solution via oral route (positive control) (Divyashree et al.,2019). The animals in group III & IV were administered 20 mg/kg body weight Mifepristone solution orally for 13 days to induce PCOS, followed by treatment of Metformin HCl solution (equivalent to 8.33 mg/kg) 1 time a day for 7 days, 2 times a day for next 7 days and 3 times a day for last 7 days via oral route, and nanoemulsion gel containing Metformin HCl administered intravaginally at a dosage of 4.16mg/kg as part of the treatment regimen for PCOS. At last, the blood, ovary, and uterus of the rats were isolated. To prevent clotting, the blood was kept in microcentrifuge tubes with 10% w/v sodium citrate. Serum was centrifuged at 4500 rpm for 15 minutes and kept at -20°C for subsequent analysis. The uterus and ovary were weighed independently. Prior to histopathological investigation, the ovary was preserved in a 4% paraformaldehyde solution. Blood samples were analyzed for progesterone, estrogen, and testosterone levels using a competitive enzyme-linked immunosorbent assay (ELISA), and non-competitive ELISA was used to measure serum FSH and LH levels. The isolated ovary undergone histopathological evaluation using a standardized hematoxylin-eosin staining technique.
Stability study
The stability investigation was conducted on optimized MET-HCLNE GEL at 4°C. Several parameters were used to assess gel stability over 0, 15, 30, 60, and 90 days. The drug concentration, pH, and viscosity were tested according to the procedure provided in the section characterization of MET-HCLNE GEL.
Statistical analysis
With column statistics in Graph Pad Prism04 software, the mean ± S.D. for n = 3 was computed to express the results. To determine the statistically significant variance between the mean values of the two groups, an unpaired ‘t’ test was performed. The different groups were compared using one-way and two-way ANOVA testing.
RESULTS
Standard Calibration Curve of Metformin HCl in Simulated Vaginal Fluid (SVF)
The calibration curve of Metformin HCl was plotted in SVF at 232 nm. The equation obtain was y
= 0.678x - 0.0474 and the correlation coefficient was found to be R2 = 0.9972. (Figure 1). Validation details of the UV method are listed in Table II.
Solubility Study and Selection of Excipients
It is recommended that the components used in nanoemulsion (NEs) formulation should have high drug solubilization capacity. The solubility of MET-HCL in different oils, co-surfactants, and surfactants was examined by dissolving an excess of the drug in a predetermined amount of particular oils, co-surfactants, and surfactants. Virgin Coconut oil (VCO) was selected as the oil phase to formulate NEs. Because MET-HCL exhibit highest solubility in VCO, i.e. 61.65 mg/mL and it is also considered as best for females with PCOS. It contains Medium Chain Fatty Acids & variety of antioxidants that help to maintain blood sugar, insulin secretion level, endocrine hormonal balance. Coconut oil also be easily digested and thus serves as a quick energy boost as well. This synergistic impact improves the formulation’s preparation as well as functioning (Chellapa, Eid, Elmarzugi, 2015). In case of surfactant cremophore RH 40 showed higher solubility, 13.05 mg/ mL, for MET-HCL than other. Similarly, Cremophor RH40’s non-ionic nature offers less toxic effects and irritation thus consider to be suitable for intravaginal drug delivery (Pathan, Setty, 2011). In case of co-surfactant, MET-HCL exhibit highest solubility in transcutol P, 11.22 mg/mL, thus for further study transcutol P was selected as cosurfactant.
Screening of surfactants and co-surfactants for emulsification efficiency
Basic parameters for selecting surfactants for nanoemulsion formulations include drug solubility and emulsifying efficiency. Various surfactants were evaluated for their emulsifying capability in relation to specific types of oil. The surfactant’s % UV transmittance indicated how well it could form stable emulsions. Based on this principle, high transmittance aqueous dispersions were thought to be optically clear, whereas oil droplets were thought to be in a state of micro-dispersion. The % transmittance of different dispersions is listed in Table III. The highest percentage of UV transmission was observed in cremophore RH 40 (95.17±2.1) over other surfactants and Transcutol P showed highest emulsification efficiency (90.09±0.9) over other co-surfactants. Hence, cremophore RH40 was selected as the surfactant and Transcutol P as co-surfactant for more research due to its remarkable micro-emulsification efficiency.
Pseudo ternary phase diagrams
The grey area in the pseudo ternary phase diagram (Figure 2) indicates the area of microemulsions (Moghimipour, Salimi, Leis, 2012). Figure 2A, to solubilize 10% of the oil, a 2:1 ratio (Smix) system requires a surfactant mixture of more than 60%. The reduction in the surfactant volume was observed in 3:1 ratio (Figure 2B), but the microemulsion’s area did not alter significantly in comparison to 2:1 ratio. As seen in Figure 2C, the microemulsion system operates at a 1:1 ratio to produce the area with the maximum microemulsion coverage. The 50% surfactant mixture is capable of incorporating more than 10% of oil. Thus, 1:1 ratio microemulsion system was selected for further batchs preparation.
Evaluation of Nanoemulsions
Particle size, PDI, zeta potential
The formulations were assesed for droplet size, PDI, drug content, viscosity determination & morphological studies (Deore, Surawase, Maru, 2019). The results recorded are represented in (Table IV). The droplet size of all 9 formulations was studied. The results indicated the direct relations between the droplet size & oil concentration. As concentration of oil increases, droplet size of nanoemulsion also increases. The droplet size range for the MET-HCLNE 1 to ME-HCLNE 9 (45.60 ± 1.7 to 107.3 ± 2.5 nm) was found to increase with the oil concentrations of (5 to 15%) respectively. Smaller droplet sizes in the emulsion have been found to exhibit greater kinetic stability, which results in a greater reduction in creaming, a faster rate of Brownian diffusion, and a larger stearic stabilization effect for the emulsion droplets. The sharp and elongated peak observed in droplet size analysis, confirmed the uniform nano size and dispersity (Figure 3A). The PDI values for all formulation showed in (Table IV). Low PDI values were observed for almost all the formulation except MET-HCLNE 9 (0.424±0.9) formulation representing a lower value of (0.198±0.2). The PDI value less than 0.3 revealed that nanoemulsion globules were homogenous and had uniform dispersity of particles. Zeta potential is thought to be a important factor of the stability of microemulsions. A formulation with a high zeta potential value is more stable and helps keep molecules from aggregating together. The zeta potential of all Formulations ranging from (22.4±0.3 Mv to 6.9±1.5 Mv). Highest zeta potential was found in MET-HCLNE1 (Figure 3B).
A: Droplet size analysis of optimized formulation (MET-HCLNE1). B: Zeta potential analysis of optimized formulation (MET-HCLNE1).
Drug content
The drug content (Table IV) ranged from 89.19 ± 0.2% to 78.03 ± 0.2% in all the formulations demonstrating the uniform distribution of MET-HCL in the developed formulations. Maximum drug content was observed in batch MET-HCLNE 1.
Viscosity studies
To verify the formulation’s stability, the viscosity of the mixture was examined. The viscosity of the nanoemulsion showed directly relationship with the concentration of oil. MET-HCLNE1 formulation has less viscosity (42.85± 2.1 cP) as compared to other. Viscosity was increased from 42.85± 2.1 to 120.4± 3.4 cP in other formulations, this may be associated to the higher concentration of oil (5% to 15%). Consequently, the outcomes demonstrated that the prepared batches had an isotropic nature and were chemically stable. By comparing result of 9 formulation, it can be concluded that formulation MET-HCLNE1 with Smix ratio 1:1 shows more promising nanoemulsion region with globule size (45.60±1.7 nm), PDI (0.198±0.2), zeta potential (22.4±0.3 mV), drug content (89.19±0.2), viscosity (42.85±2.1). Therefore, it considered as best optimized batch for further study.
In vitro release study
Based on the results of particle size, zeta potential, drug content, four nanoemulsion formulation were selected for invitro release study namely MET-HCLNE1, MET-HCLNE2, MET-HCLNE3, MET-HCLNE4. The high solubility of MET-HCL in cremophor RH40 and transcutol P might result in high drug release in MET-HCLNE. Additionally, a larger interfacial surface area is provided as droplet size decreases, leading to more drug release. Greater drug release (86.20± 0.80%) was observed with MET-HCLNE 1 after 9 h compared to 76.56±1.5%, 79.69±0.1%, and 80.50±0.3% of MET-HCLNE2, MET-HCLNE3, MET-HCLNE4, respectively (Figure 4A). Highest drug release was showed by MET-HCLNE1. Hence MET-HCLNE1 used as optimized batch for further study.
A: Invitro release profile of formulation (MET-HCLNE1 to MET-HCLNE4). B: Invitro release study of MET-HCLNE GEL compared with pure MET-HCL solution.
Transmission electron microscopy (TEM)
To visualize the morphological structure of optimized MET-HCLNE1, TEM was used. The TEM image represented in (Figure 5). The nanoemulsions were observed to have a spherical shape and there was no indication of coalescence. The size of the droplet was discovered to be comparable to the size determined by Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, UK). The droplets observed were uniform & non-aggregated without any signs of precipitation indicates physical stability of nanoemulsion.
Evaluation of nanoemulsion gel
PH
The results of various parameters of nanoemulsion gel are shown in the accepted pH range for intravaginal preparations is in between The pH of 3.8 to 4.5. MET-HCLNE GEL was found to be 4.2 ± 0.52. Therefore, the pH of the gel was within the acceptable limit and safe for intravaginal application.
Rheological studies
Viscosity
The nanoemulsion gel’s viscosity was measured and found to be 224.1 ± 50 cP. (Gupta, 2020). The result indicates that MET-HCLNE GEL shows optimum viscosity, for better applicability into the vaginal cavity.
Spreadability
Spredability is an essential characteristic for gel formulation. The spredability of the nanoemulsion gel was found to be 15.52 ± 1.5 cm2. The result indicates that MET-HCLNE GEL shows optimum spredability, allow easy spreading during application, thus increase patient compliance and ensure optimal contact between the formulation and the vaginal mucosa.
Drug content
The MET-HCLNE GEL contains a drug content of 84.63± 0.05 % which indicate that the drug is homogeneously distributed in gel matrix.
In Vitro Drug Release
Using a Franz-type diffusion cell device, the prepared nanoemulsion gel’s in vitro drug release study was assessed. As showed in (Figure 4B), pure metformin, due to its high-water solubility, was quickly released in the dissolution medium (SVF pH 4.2) with more than 90 % of the drug was released within 1 to 2 hours. The sustained drug release lasted for up to 9 hours with the metformin loaded nanoemulsion gel (Diwedi, Alexandar, Chandrasekar, 2012). The metformin-loaded nanoemulsion gel exhibited a drug release of approximately 81.11±0.08% over a 360-minute period. The droplet size of nanoemulsion, surfactant-oil and drug-oil interactions, drug solubility in oil, use of HPMCK100 as gelling agent were discovered to significantly affect the release of drugs from nanoemulsion gels.
Drug release kinetics
The zero-order kinetic model provides the best fit to the profile of MET-HCL release from nanoemulsion gel. Plot’s maximum linearity was observed. (R2 = 0.9942). It indicates that the active substance in the nanoemulsion gel is gradually released at a constant rate, regardless of the initial concentration of the drugs (Sreelakshmi et al., 2018). Releasing the drug steadily over the time can lower possible side effects by decreasing the need for frequent drug administration. The most optimal way to release the medication from the vagina is through an intravaginal prolonged action. HPMCK100 and coconut oil are effective carriers for the controlled release of MET-HCL.
DSC study
The drug’s possible interactions with other formulation ingredients are characterized and the physical condition of MET-HCL in the formulations is evaluated using the DSC data. The melting point of MET-HCL was indicated by a sharp endothermic characteristic peak on the DSC thermogram, which was observed at 231.90 °C (Figure 6a). The melting point for MET-HCL was reported as 223-242°C (Balpande et al.,2013). In Case of MET-HCLNE, a diffuse endothermic peak (Figure 6c) occurred at 98.40 °C, which was due to the loss of water (dehydration and/or desolvation process). However, the endothermic peak for MET-HCL was not observed in DSC thermogram of MET-HCLNE & MET-HCLNE GEL formulations (Figure 6 c, d). The DSC thermograms of all the formulations did not show the characteristic peak of MET-HCL, indicating that MET-HCL was molecularly dispersed in the oil phase.
FTIR study
FTIR spectra helps to assess the interaction between drug & excipients. The drug’s functional group vanishes, indicating the existence of an interaction. Two distinct standard bands were visible in the MET-HCL infrared spectrum observed at 3369 cm-1 and 3294 cm-1 that correspond to the primary stretching vibration of N-H, along with a band at 3155 cm-1 originating from the secondary stretching of N-H. Additionally, there are distinctive bands present at 1626 cm-1 and 1567 cm-1, which are associated with C-N stretching. (Gundogdu, Cetin, 2014). The prepared physical mixture exhibited all of the drug’s characteristic peaks. The absence of interfering peaks in MET-HCLNE GEL suggests that MET-HCL and the excipients do not interact (Figure 7). The spectra of the nanoemulsion gel formulation showed the presence of all functional groups, confirming that the prepared nanoemulsion was stable and did not exhibit any chemical interactions.
In vivo study
In a PCOS experimental animal model, the therapeutic efficacy of formulated nanoemulsion gel was examined. The Institutional Animal Ethical Committee (IAEC) (MMCOP/IAEC/10/2024) had approved the study. PCOS was induced in female wistar rats using mifepristone solution and the therapeutic efficiency of MET-HCLNE GEL was observed. Comparison is made between the administration of gel through the vaginal route and the oral administration of a pure drug solution. Using ELISA kit, the levels of various hormones that control ovulation in the body were measured. Rats in which PCOS is induced showed 40 ± 0.7 ng/ml serum level of testosterone which was significantly (Unpaired t-test, P < 0.05) higher than 22.1 ± 0.9 ng/ml normal control group treated with vehicle (Table V). The serum testosterone level was normalized to 25.2 ± 1.6 ng/ml in rats with PCOS which were treated with MTF-HCl solution by oral route and to 24.6 ± 1.5 ng/ml in those treated with intravaginal MTF-HClNE GEL. There was no significant difference (One-way ANOVA, P > 0.05) in comparison with the control group treated with the vehicle. Similarly, the formulated GEL also restored the serum concentrations of LH and estrogen in rats with PCOS to levels similar the normal control group treated with the vehicle (Table V). The serum FSH level in PCOS rats significantly decreased to 3.4 ± 1.6 mlU/ml compared to the 8.1 ± 0.8 mlU/ml in the normal control group treated with vehicle (Table V) as confirmed by an Unpaired t test (P < 0.05). Treatment with MTF-HCl solution via oral route and MTF-HClNE GEL via intravaginal route, the serum FSH level in PCOS rats normalized to 7.4 ± 1.5 mlU/ml and 7.2 ± 1.3 mIU/ml, respectively. There was no significant difference compared to the vehicle-treated normal control group, as determined by One-way ANOVA (P > 0.05). The level of progesterone in the serum of rats with PCOS was effectively regulated by tailored MET-HCLNE gel in the same way as the normal control group treated with vehicle. (Table V). The analysis of morphology and histopathology revealed a significant increase in the weight of the uterus and ovary in PCOS rats compared to the control group treated with vehicle, as indicated by the unpaired t test (P < 0.001) (Table V). Nevertheless, administration of MTF-HCl solution via oral route and MTF-HClNE GEL via intravaginal to PCOS rats resulted in the normalization of uterus and ovary weight compared to the normal control group treated with vehicle (Table V). The evaluation of the ovary tissue from the normal control group indicated the presence of normal, healthy ovaries without any signs of cyst formation. (Prajapati, Patel, Dharamsi, 2022) (Figure 8A). On other hand, the ovaries taken from rats treated with mifepristone displayed various changes, including the development of cysts in the ovaries and the filling of these cysts with fluid (Figure 8B). The orally administered MTF-HCl solution and intravaginally administered MTF-HCLNE GEL resulted in significant improvement in PCOS rats’ treatment, like decreased the cyst and, in polycystic ovaries, generated a normal follicle in less than 21 days. (Figure 8C, 8D). Thus, in vivo study obtained result indicated that intravaginal drug delivery of MET-HCL loaded coconut oil nanoemulsion gel is pharmacodynamically effective.
Histopathology images of rats ovaries obtained from (A) Normal vehicle treated ovaries (B) Mifepristone treated ovaries (C) Mifepristone treated ovaries followed by treatment of oral MET-HCL solution (D) Mifepristone treated ovaries followed by treatment of intravaginal MET-HCLNE GEL.
Stability study
The MET-HCLNE GEL formulation underwent a stability study for 15, 30, 60, and 90 days at 40°. According to the results (Table VI), the drug content, pH, and viscosity values did not change significantly. Stability obtained might be due to presence of Coconut oil, as it plays key role in improving the stability of nanoemulsion. The instability of nanoemulsion originates from droplet coalescence. This problem is overcome by converting nanoemulsion into a gel. In conclusion, the good stability of nanoemulsion gel should be credited to selecting components.
DISCUSSION
MTF-HCl may have the ability to control the metabolic issues associated with PCOS in females. However, clinical trials’ review shows that MTF-HCl is a patient-convenient medicine that is safe and effective in treating PCOS. But MET-HCL has inadequate oral bioavailability and lactic-acidosis side-effect. Thus, to overcome these limitations, the current study looked into the possible vaginal delivery route of MET-HCl for PCOS patients. However, effective drug delivery via the vaginal route remains difficult, because there is limited absorption via the vaginal epithelium. The process of drug absorption through vaginal administration involves first dissolving in the lumen of vagina and then penetrating across the mucosal membrane. Hence, enhancing the absorption of drugs through the layers of vaginal mucosa requires the use of customized drug delivery methods. In the present study, MET-HCl nanoemulsion was prepared by water titration method followed by probe sonication. This approach linked with high encapsulation efficiency of drug or active moiety in colloidal system. To develop effective MET-HCLNE formulation containing MET-HCL, they need to be sufficiently soluble in various excipients like oil, surfactant, cosurfactant. The solubility studies showed that MET-HCL has maximum solubility in coconut oil hence used as oil phase. Similarly, MET-HCL has high solubility in transcutol P and cremophore RH40 hence selected as co-surfactant and surfactant. To identify the nano emulsification area and for the creation of NE, ternary phase diagrams were developed using solubility results to determine the right oil and Smix ratio. The area of nanoemulsion was displayed on a ternary diagram, illustrating the concentration of virgin coconut oil, Transcutol P and Cremophor RH40 for the creation of NE. The Smix ratio of 1:1 resulted in a large nanoemulsion region, allowing the 50% surfactant mixture to accommodate more than 10% of oil. Nanoemulsion droplet size plays crucial role to improve drug absorption. The large interfacial surface area was a result of the smaller droplet size, leading to an increase in drug release and permeation. There is direct relation between droplet size and oil concentration. As concentration of oil increases, droplet size of nanoemulsion also increases. Smaller droplet sizes in the emulsion demonstrated greater kinetic stability, which results in a greater reduction in creaming, faster brownian diffusion, and greater stearic stabilization effects, all of which contribute to the development of stable nanoemulsions. The PDI value less than 0.3 revealed that nanoemulsion globules were homogenous and had uniform dispersity of particles. In the same way, the charge of particles is essential for maintaining the stability of formulations. When the Zeta Potential (ZP) has high positive or negative values, it signifies the stabilization of the NE system. This happens when higher concentrations of comparable electrical charges cause electrostatic repulsion between the particles. As a result, the system exhibits reduced particle aggregation. The viscosity of the nanoemulsion was directly proportional to the concentration of oil, as oil concentration increases viscosity increases. The MET-HCLNE 1 batch showed particle size 45.60 ± 1.7nm, PDI 0.198 ± 0.2, ZP 22.4 ± 0.3mV, viscosity 42.85 ± 2.1 Cp this result indicates that developed formulation is stable. Due to such promising result MET-HCLNE 1 was optimized and converted to gel. To achieved sustained prolonged release of drug HPMCK 100 uses as gelling agent because of its mucoadhesive nature it remains in contact for prolong period of time with vaginal mucosa and thus help to achieved sustained release of drug. Conducting in vitro drug release tests helps to anticipate the enhanced therapeutic effectiveness of the developed gel formulation. In SVF (pH ∼4.2), MTF-HClNE Gel exhibited a prolong drug release than MTF-HCl solution. The study findings showed that the nanoemulsion gel significantly improved the drug’s permeation over a prolonged duration compared to the free drug solution. The effectiveness of MET-HCLNE Gel in treating PCOS induced by mifepristone was noted in female Wistar rats. Imbalanced levels of plasma hormones, polycystic ovarian morphology & histopathological analysis of ovary indicated PCOS induction. Serum hormone levels were measured biochemically, and it was found that MET-HCLNE Gel helped to regulate hormones including testosterone, FSH, progesterone, estrogen, and LH. Mifepristone administration to female rats increased LH levels, inhibited ovulation, and caused metabolic impairments. After analyzing the data, we have concluded that rats with PCOS which were treated with oral MTF-HCl solution and intravaginal MET-HCLNE Gel as treatment leads improvement in pathophysiology. Histopathological analysis showed a remarked recovery of the ovarian cysts with treatment of oral MTF-HCl solution. There is enough data from intravaginal MET-HCLNE Gel to support the advantages of the vaginal route over the oral route for the administration of MTF-HCl in PCOS patients.
In Conclusion, the MET-HCLNE were formulated and were characterized to confirm the efficacy and stability of the formulation. Desired droplet size (45.60 ± 1.7 nm), PDI (0.198 ± 0.2), zeta potential (22.4 ± 0.3) showed by A1 formulation. In in-vitro release study, MET-HCLNE GEL showed sustained and prolonged release of MET-HCL as compared pure MET-HCL solution. There were no significant changes in the medication content, pH, and viscosity readings during the period of 120 days. In vitro and in vivo studies demonstrated that MET-HCLNE GEL presented an analogous advantage as compared to oral MET-HCL solution in managing PCOS with a reduced dosage schedule and fewer side effects. Based on the comprehensive analysis, it can be interpreted that the MET-HCL nanoemulsion gel that was created has superior therapeutic efficacy, good dispersity, and better storage stability to used intravaginally in the treatment of PCOS.
ACKNOWLEDGMENTS
Authors are thankful to Marathwada Mitra Mandal’s College of Pharmacy & Hon. Dr. M. J. Patil sir for providing the platform for the conduction of experiments. Bhagyashri Sopan Binawade contributed to the data curation, investigation, methodology, manuscript preparation. Prachi Jadhav & Supriya Bhangare contributed to the execution of animal study, final editing & review of manuscript. Dr. M. P. Ratnaparkhi contributed to the supervised experiment, validation of data, final reviewing of manuscript. Dr. Manoj Aswar contributed to the supervised animal study, validated data. All the animals used in the experiments were sanctioned from Institutional Animal Ethics Committee (IAEC) as per the rules and regulations of CPCSEA, New Delhi.The authors received no extramural funding for the study.
DATA AVAILABILITY STATEMENT
All data is available within the article or its supplementary materials.
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Associate Editor:
Rose Mary Naal
