In situ Forming Nanoemulgel for Diabetic Retinopathy: Development, characterization, and in vitro efficacy assessment

Abstract

Diabetic retinopathy, the most common microvascular complication of diabetes mellitus, is the leading cause of vision impairment worldwide. Flavonoids with antioxidant properties have been shown to slow its progression. Myricetin, a flavonoid polyphenolic compound, possesses antioxidant properties, but its clinical use in ocular delivery is limited by poor aqueous solubility, stability, and bioavailability. Recently, in situ gels have gained interest as ocular drug delivery vehicles due to their ease of installation and sustained drug release. This study aimed to develop a myricetin-loaded thermoresponsive in situ nanoemulgel to enhance its efficacy in treating diabetic retinopathy. Nanoemulsions were developed via aqueous phase titration using Sefsol 218 as the oil phase, Kolliphore RH40 as the surfactant, and PEG 400 as the co-surfactant. Physicochemical evaluations identified formulation batch ISG17, consisting of 10% oil phase, 30% S_mix (1:2), and 60% distilled water, as the optimal formulation. The developed in situ nanoemulgel showed significant enhancement in corneal permeation and retention, which was further confirmed by fluorescence microscopy. Ocular tolerability was demonstrated through corneal hydration tests and histopathology investigations. The antioxidant potential of the myricetin-loaded nanoemulgel was assessed using the DPPH assay. Myricetin was found to be an efficient antioxidant, as indicated by its IC₅₀ values compared to ascorbic acid. The MTT cell viability assay results showed that the developed formulation effectively inhibits the proliferation of Y79 retinoblastoma cells, demonstrating comparable efficacy to the standard marketed preparation Avastin (Bevacizumab injection). In conclusion, the nanoemulsion formulation containing a thermoresponsive polymer for in situ gelling presents a promising drug delivery system, offering superior therapeutic efficacy and better patient compliance for the treatment of diabetic retinopathy.

Keywords

antidiabetic drugs - drug delivery - apoptosis

Introduction

Diabetic retinopathy (DR) is a significant late-stage complication of diabetes, leading to vision loss in many diabetic patients [1]. The combined impact of hyperglycemia and hypertension accelerates DR in individuals with type II diabetes mellitus. Hyperglycemia, oxidative stress, and altered redox homeostasis are major contributors to the development of DR [2]. Persistent high glucose levels in retinal microvessels increase oxidative stress through reactive oxygen species (ROS), inflammation, protein kinase C (PKC) activation, and pathways such as hexosamine and polyol, along with the formation of advanced glycation end products (AGEs). These metabolic changes and oxidative stress lead to the degeneration of capillary cells in the retinal microvasculature [3] [4].

Research suggests that certain antioxidants and dietary supplements can slow the progression of DR by enhancing the body’s antioxidant defenses. Flavonoids, particularly myricetin, have shown potential in reducing the advancement of DR [3]. Myricetin, a natural flavanol found in various fruits, vegetables, and medicinal plants, is known for its strong antioxidant properties and its protective effects on retinal cells, retinal ganglion cells, and corneal epithelial cells [5]. However, its clinical application is limited by its poor solubility, instability, and low bioavailability. The unique characteristics of ocular tissues, along with the anatomical and physicochemical barriers of the eye, make drug penetration particularly challenging [6] [7]. Traditional formulations achieve less than 5% corneal penetration due to precorneal loss from nasolacrimal drainage and high tear fluid turnover, resulting in low bioavailability [8] [9].

To overcome these limitations and enhance ocular drug bioavailability, nanoemulsions are considered a promising option for ocular drug delivery [10]. Nanoemulsions are pharmacokinetically stable emulsions with droplet sizes ranging from 10 to 100 nm [11] [12]. This system can sustain drug delivery in the ocular region, reduce dosing frequency, and minimize side effects. Smaller droplet sizes also improve corneal absorption and deeper ocular structure penetration. In situ gels offer another technique to increase retention time at the action site and enhance the sustaining properties of ocular dosage forms [10]. These polymeric solutions transition from sol to gel in response to physiological stimuli, such as body temperature, pH, and ionic strength of biological fluids, combining accurate dosing and easy administration of eye drops with prolonged retention and sustained drug delivery [13]. In situ gelling systems also serve as effective vehicles for intraocular and periocular injections, creating depots in the vitreous humor or periocular tissues for sustained drug release to the posterior segment of the eye [14].

Present study aimed to develop a thermoresponsive in situ nanoemulsion-based gel containing myricetin and assess its in vitro efficacy for treating diabetic retinopathy. This approach combines the advantages of a drug carrier with in situ gelling delivery systems to address the issues of low ocular bioavailability due to myricetin’s poor aqueous solubility and rapid drug clearance by ocular defense mechanisms [13] [14] [15]. Thermoresponsive gelling systems, which transform into semisolid gels in response to temperature changes, are well-suited for use as injectable solutions or eye drops to provide sustained drug delivery. In our study, HPMC 4KM was used as the thermoresponsive polymer due to its proven ability to form stable hydrogels and its well-documented thermoresponsive properties. It also provides an ideal balance of gelation temperature and mechanical strength, which was suitable for our formulation needs.

Materials and Methods

Materials

Myricetin was purchased from Aktin Chemical, Inc., China. HPMC 4KM was furnished by Colorcon Asia Pvt. Ltd., India. Tween-80 was provided by SD Fine Chemicals, India. PEG400 was obtained from Fisher Scientific, India. Triacetin was supplied by Qualigens Fine Chemicals, India. Kolliphor RH40 and Brij 35 were procured from BASF Chemical Company, India. Sefsol was sourced from Nikko, Japan. Ascorbic acid was acquired from Parchem Fine & Speciality Chemicals, India. DPPH was sourced from Cosmic Crop Solutions Pvt. Ltd., India. Avastin (Bevacizumab injection) was purchased from Cytonova Labs International Pvt. Ltd., India. All materials were of pharmaceutical grade, and double distilled water was used throughout the experiment.

Methods

Solubility Study

The solubility of myricetin was determined by equilibrium solubility method. For the assessment of solubility various Oils (Castor oil, Sunflower oil, Olive oil, Sefsol 218, Corn oil, Oleic acid, Triacetin), Solvents (Water, Acetone, DMSO, Ethanol, Methanol, Chloroform), Surfactants (Kolliphor RH40, Tween 80, Tween 20, Span 80, Span 20), and Co-surfactants (Ethanol, PEG 400, Brij 35, 1,2-Propanediol, Glycerin, Isopropanol) were selected. Myricetin was added in excess to 2 mL of each vehicle in 5 mL stoppered vials and mixed with a vortex mixer. The vials were then incubated at 25±2°C in an isothermal shaker for 72 h. Once equilibrium was achieved, the samples were centrifuged at 1968 G for 15 minutes, and the supernatants were filtered through a 0.45 µm syringe membrane filter. The concentration of myricetin in each filtrate was then measured spectrophotometrically [16] [17].

Construction of Pseudo Ternary Phase Diagrams

Based on the findings of solubility studies, Sefsol 218 was chosen as the oil phase for the nanoemulsion. Kolliphor RH40/Tween 80, PEG 400, and double-distilled water were used as the surfactant, co-surfactant, and aqueous phase, respectively. The surfactant and co-surfactant were mixed in various volume ratios (1:0, 1:1, 1:2, 2:1) to explore different phase diagrams, analyzing the effect of increasing surfactant concentration relative to the co-surfactant and vice versa. Oil and specific S_mix ratios were combined in volume ratios ranging from 1:20 to 1:5. Ten combinations (1:20, 1:9, 1:5.3, 1:3, 1:3.2, 1:16, 1:6, 1:5, 1:10, and 1:8) were prepared to map the phase boundaries accurately. Phase diagrams were created using an aqueous titration method, where the aqueous phase was gradually added to each oil and S_mix ratio. For optimization, the co-surfactant was initially kept constant, then both the surfactant and co-surfactant were mixed. The physical state of the nanoemulsion was documented on a pseudo-three-component phase diagram, with one axis representing the aqueous phase, another for oil, and the third for the surfactant and co-surfactant mixture at fixed volume ratios. Formulations from the nanoemulsion region of each diagram were chosen to include a single dose of the drug in the oil phase. Four S_mix ratios (1:1, 1:2, 1:3, and 2:1) were screened, with clarity observed in the 1:1 and 1:2 ratios in the ternary phase diagrams [17] [18]. Formulations were selected based on criteria including a 10 mg dose for nanoemulsion development, ensuring the oil could dissolve 10 mg of myricetin, and choosing the formula with the minimum S_mix concentration needed to form the nanoemulsion [18].

Thermodynamic Stability Studies

To determine the stability of nanoemulsions and discard unstable or metastable ones, placebo nanoemulsions underwent the following stability tests:

Freeze-Thaw Cycle

Nanoemulsions were stored at − 20°C for 24 h and then brought to room temperature. Stable nanoemulsions returned to their original state within 2–3 minutes. This cycle was repeated 2–3 times [18].

Centrifugation Studies

Following the freeze-thaw cycle, nanoemulsions were centrifuged at 158.85 x g for 30 minutes. Stable formulations showed no phase separation or turbidity [19].

Heating-Cooling Cycle

Six cycles between refrigerator temperature (4°C) and 40°C, with each cycle involving 48 h storage, were performed. Formulations that remained stable at these temperatures were selected for further studies [19].

Development of In-Situ Nanoemulgel

Myricetin-loaded in-situ nanogel (ISG) was prepared using the cold method. Initially, the thermoresponsive polymer (1% w/w HPMC 4KM) was dissolved in distilled water using a mechanical stirrer. Once fully dissolved, the mixture was stored at 4°C for 12 h. Drug-loaded ISG solutions (0.1% w/v) were created by incorporating the optimized nanoemulsion (containing 0.1% w/v of the drug) into the polymer solution. Methylparaben and sodium chloride were added as antimicrobial and isotonic agents, respectively. The final volume was adjusted to 50 mL with distilled water and stored at 4°C for 24 h [18] [19].

Evaluation and Characterization of In-Situ Nanoemulgel

Visual Appearance and Clarity

The formulations were visually inspected for appearance and clarity against a white background at room temperature (20°C) [20].

pH and Viscosity Measurement

The pH of the gel was measured using a pH meter at 25°C. The viscosity was assessed with a Brookfield viscometer, equipped with spindle number CC14, operating at 5 rpm for 10 sec at 37°C [20] [21].

Drug Content Determination

To estimate the drug content, 1 mL of the ISG formulations was diluted in 50 mL of freshly prepared Simulated Tear Fluid (STF; pH 7.4). A 5 mL sample was taken and further diluted with STF. The myricetin content was quantified using a UV-Visible spectrophotometer at 328 nm.

Flowability and Gelation Temperature

The phase behavior and gelation temperature were assessed using the tube inversion method. Test tubes containing 1 g of in-situ nanoemulgel were incubated at 5±1°C (storage temperature), 25±1°C (average room temperature), and 35±1°C (precorneal temperature) and shaken at 50 rpm. Samples were analyzed under two conditions: one with STF dilution at a 40:7 ratio (ISG, v/v) and one without. Experiments were conducted in triplicate [21] [22].

Sterility Testing

The sterility of the optimized formulation was tested against aerobic and anaerobic bacteria, as well as fungi, using fluid thioglycolate and soybean casein digest media, respectively, according to Indian Pharmacopoeia guidelines. Sterility testing was carried out using the direct inoculation method, where approximately 10 mL of the test sample was added to 100 mL of culture medium and sealed with a cotton swab. After inoculation, the media were sterilized at 121°C and 15 psi for 20 minutes, incubated at 32°C for 7 days, and monitored for microbial growth [18] [23].

Isotonicity Test

The isotonicity value was determined using the hemolytic method by observing the effects of different drug solutions on red blood cells (RBCs). RBCs were suspended in solutions of 2%, 0.2%, 0.9% sodium chloride, and the optimized formulation (ISG 17). The appearance of the RBCs was monitored for swelling, bursting, shrinking, and wrinkling. The amount of oxyhemoglobin released in hypertonic solutions correlates with the number of hemolyzed cells, while in isotonic solutions, RBCs maintain their morphology [20] [21].

Zeta Potential and Polydispersity index

The size distribution of oil droplets in the formulation was assessed using photon correlation spectroscopy, which evaluates light scattering fluctuations due to particle Brownian motion with a Zetasizer (Malvern Instrument). The polydispersity index measures droplet size uniformity in the nanoemulsion, with higher values indicating lower uniformity [20].

In Vitro Release Study

Preparation of Simulated tear fluid (STF)

Simulated tear fluid (STF) with a pH of 7.4 was prepared for drug release and transcorneal permeation studies. The STF was composed of sodium bicarbonate (0.200 g), sodium chloride (0.670 g), calcium chloride hydrate (0.008 g), and purified water q.s. to 100 g [24].

In-vitro release experiment In-vitro release studies of the nanoemulsion and in-situ nanoemulgel were conducted using a Franz diffusion cell with a diffusion area of 1.31 cm². A dialysis membrane (0.22 μm pore size) was pre-soaked overnight in the dissolution medium at room temperature before being mounted between the donor and receptor compartments. The receptor compartment was filled with 50 mL of freshly prepared STF (pH 7.4), and 1 mL of the formulation was placed in the donor compartment. The setup was maintained on a magnetic stirrer at 50 rpm and 37°C. At specified intervals, 5 mL aliquots of the receptor medium were withdrawn and replaced with fresh STF for up to 8 hours. The samples were analyzed using a UV spectrophotometer at 328 nm [21] [24].

Ex Vivo Transcorneal Permeation Studies

The optimized nanoemulsion and in situ nanoemulgel formulation were subjected to transcorneal permeation studies.

Corneal Treatment

For transcorneal permeation studies, fresh whole goat eyeballs, obtained from a local butcher, were used. The eyeballs were transported in cold normal saline (4°C). The cornea, along with 2–4 mm of surrounding scleral tissue, was excised and thoroughly washed with cold normal saline until the washings were protein-free, as confirmed by Folin’s Phenol reagent and zero UV absorbance using 0.9% normal saline as a blank. Special care was taken to avoid physical damage to the tissue [24] [25].

Transcorneal Permeation Experiment

A Franz diffusion cell with a diffusion area of 0.80 cm² was used for permeation studies. The freshly excised cornea was mounted between the donor and receptor compartments of the diffusion cell, with the epithelial surface facing the donor compartment. The formulation was placed in the donor compartment, and the receptor compartment was filled with 15 mL of STF (pH 7.4). The assembly was maintained at 37°C and stirred continuously at 50 rpm using a magnetic stirrer.

At predetermined intervals, 1 mL samples were withdrawn from the receptor compartment through the sampling port and immediately replaced with fresh receptor solution. This process was repeated for up to 8 h. The drug content in the samples was analyzed spectrophotometrically at 328 nm. Experiments were conducted in triplicate. Post-experiment, the cornea’s integrity was microscopically checked to ensure no pores or tears. [24] [25].

Data Analysis

Cumulative drug permeation data were plotted, and apparent permeability (Papp) and drug flux (J) were calculated using the following equations [26]:

Papp = dQ/dt x 1/ACo (1)

whereP_app= Apparent permeability; dQ/dt=cumulative drug permeated over time) the steady-state flux (mol/s); C₀ is Initial drug concentration in the donor chamber at each time interval (mol/mL); and A is the surface area of the filter (cm²).

The drug flux (J) was calculated using the following equation [26]:

J = Papp × C0 (2)

Corneal Retention Study

Fluorescence microscopy was used to evaluate the retention and permeation of nanoemulsion and in situ gel formulations in the cornea. The formulations were tagged with fluorescein dye for visualization purposes. Corneal samples underwent permeation studies with the fluorescent dye formulations and were subsequently fixed for microscopic examination. A control sample, consisting of a dye solution in water, was also evaluated. Before examination, all corneal sample slides were treated with 10% formic acid to remove excess dye from the surface. The prepared slides were then assessed under the microscope [24].

Transmission Electron Microscopy (TEM) Analysis

The structure of the optimized ISG formulation was examined using a transmission electron microscope (TEM, JEM-2100Plus Electron Microscope, JEOL Ltd.). A diluted drop of the nanoemulsion was placed on a 300-mesh copper grid and allowed to stand for 1 minute. After inverting the grid, a drop of phosphotungstic acid (PTA) was applied for 10 seconds. Excess PTA was blotted with filter paper, and the grid was analyzed using the Morgagni 268D (FEI Company, Hillsboro, USA) at 60–80 kV and 1550x magnification [19] [20].

Toxicity Studies

To determine the safety of the formulations, several toxicity tests were conducted to assess potential adverse effects on the cornea:

Corneal Hydration Test

Goat corneas from the permeation studies were used to measure corneal hydration. At the end of the experiment, each cornea was weighed, soaked in 1 mL of methanol, dried overnight at 90°C, and reweighed. The difference in weight was used to calculate corneal hydration [21] [25].

Histological Studies

The potential toxic effects of the optimized formulation were investigated through histological examinations. Goat corneas were treated with the formulation, a simple drug sample, normal saline (negative control), and saturated KCl solution (positive control). After treatment, the corneas were fixed in a 10% formalin solution, and properly fixed and stained slides were prepared for microscopic evaluation to detect any signs of cell disruption or toxicity [25].

Stability Studies

Storage ability of optimized nanoemulgel was assessed by short-term accelerated stability study following the International Conference on Harmonization (ICH) guidelines. Test samples were stored in amber vials sealed with aluminum foil at conditions of 25°C±2°C/60±5% RH and 40°C±2°C/65±5% RH for three months. Monthly analyses included assessments of clarity, pH, gelling ability, rheological behaviour, drug content, and % drug release [19] [20].

DPPH Assay

The antioxidant activity of ISG17 was evaluated using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. This method assesses ISG17’s ability to donate hydrogen, which reduces the DPPH radical and results in a color change from purple to pale yellow. Different concentrations of ISG17 (2–10 µl/mL) were combined with 5 mL of 0.1 mM DPPH solution and incubated for 20 minutes at 27°C. The absorbance was recorded at 520 nm, with ascorbic acid used as a reference standard [26]. The IC50 value, representing the concentration of ISG17 required to reduce the DPPH radical by 50%, was derived from the data. The DPPH scavenging activity was calculated using the following formula:

Scavenging activity (%) = I0−I1 / I0 × 100 (3)

where, I₀ and I₁ are the absorbances of the control and test samples, respectively.

MTT Assay

The formulation’s potential effectiveness against diabetic retinopathy was evaluated using a cell viability assay with Y79 retinoblastoma cells.

Cell Viability The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was employed to determine the viability of myricetin-loaded nanoemulgel, following standard protocols [27]. The cytotoxic effects on the Y79 cell line were evaluated.

Cell Culture Conditions Y79 cells (10,000 cells/well) were seeded in 96-well plates and cultured for 24 h in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotics at 37°C with 5% CO2. To simulate diabetic conditions, cells were exposed to high glucose levels (25 mM) and then treated with different concentrations of the formulations (1 to 1000 µM) in incomplete medium. After 24 h of treatment, MTT solution (final concentration 250 µg/mL) was added and incubated for another 2 h. The supernatant was removed, the cell matrix dissolved in 100 µL of dimethyl sulfoxide (DMSO), and absorbance measured at 540 nm and 660 nm using an Elisa plate reader [27] [28].

Cell Morphology Study

Changes in the morphology of Y79 cells were observed after 24 h of treatment with varying concentrations of myricetin-loaded nanoemulgel. The cells were examined using an inverted phase-contrast microscope. Avastin (Bevacizumab) was used as a standard for comparison.

Statistical Analysis

Data were presented as mean±standard deviation (SD). Comparisons between groups were performed using one-way ANOVA followed by Tukey’s post hoc test, with a significance threshold of P<0.05.

Results

Solubility Study

The results of the solubility studies are presented in [Fig. 1].

Fig. 1 Solubility assessment in various solvents, oils, surfactants and co-surfactants. Results are reported as the average of three determination±standard deviation (SD).

Selection of Oil, Surfactant, and Cosurfactant

Selecting the appropriate components for the nanoemulsion involves evaluating the solubility of the poorly soluble drug in the oil phase, which is critical for maintaining the drug in a solubilized form. Sefsol 218 was chosen as the oil phase due to its high solubility for the drug. Kolliphor RH40 was selected as the surfactant and PEG 400 was taken as cosurfactant, based on their solubility profiles. Double-distilled water was used as the aqueous phase.

Construction of Pseudo-Ternary Phase Diagrams

Pseudo-ternary phase diagrams were created using the aqueous titration method, involving oil, S_mix (a blend of surfactant and cosurfactant), and distilled water. The surfactant and cosurfactant were mixed in various volume ratios to adjust their concentrations relative to each other. Separate pseudo-ternary phase diagrams were generated for each S_mix ratio. As depicted in [Fig. 2], increasing the surfactant ratio in S_mix slightly modified the nanoemulsion isotropic region. The size of the nanoemulsion region in the ternary phase diagrams depends on the ability of the specific S_mix to solubilize the oil phase. Clear regions were observed at surfactant to cosurfactant ratios of 1:1 and 1:2 in the ternary phase diagrams.

Fig. 2 Pseudo-ternary phase diagrams for the ratios 1:1 and 1:2.

Thermodynamic Stability Studies

Nanoemulsions are kinetically stable systems created using precise concentrations of oil, surfactant, and water, and they do not exhibit phase separation, creaming, or cracking. To assess their robustness, the optimized nanoemulsions were subjected to various stress stability tests, including heating-cooling cycles, centrifugation, and freeze-thaw cycles.

During these stress tests, the optimized formulation displayed no signs of Ostwald ripening and passed all conditions successfully. Specifically, formulations with S_mix ratios of 1:1 and 1:2 successfully passed the heating-cooling cycle, centrifugation studies, and freeze-thaw cycle, indicating their stability. However, formulations with S_mix ratios of 1:3 and 2:1 failed to pass these stress tests, showing signs of instability under the applied conditions.

Samples passing the freeze–thaw cycle were examined for particle size, PDI and zeta potential.

The zeta potential and the polydispersity index (PDI) was falling within the specified range as illustrated in [Table 1]. The PDI value indicates the homogeneity of the dispersed system, while the zeta potential reflects the stability of the formulation.

Table 1 Particle size, PDI and zeta potential.

Formulation Batch code	Particle size	PDI	Zeta potential (mV)
ISG16 (Smix ratio 1:1)	242.2±0.52	0.236±0.21	− 11.6±4.23
ISG17 (Smix ratio 1:2)	190.6±1.67	0.201±0.03	− 28.5±5.31

*Results are reported as Mean±SD, n=3.

Selection and Evaluation of In Situ Nanoemulgel Formulations

Based on the thermodynamic stability studies, two ratios, 1:1 and 1:2, were chosen for developing the in situ nanoemulgel formulations: ISG16 (S_mix ratio 1:1) and ISG17 (S_mix ratio 1:2). These formulations were assessed for various parameters including appearance, clarity, pH, viscosity, drug content, gelling ability, rheological properties, and sterility.

Visual Appearance and Clarity

The developed in-situ gels were light yellow and clear.

pH and Viscosity

The pH values of the formulations were nearly neutral, indicating their suitability for ocular drug delivery without causing irritation ([Table 2]). Viscosity was measured using a Brookfield viscometer to predict in vivo retention behavior and structural integrity. The sol-to-gel transition occurred at 35°C, indicating an increase in viscosity at this temperature. This suggests that the formulation is likely to be more persistently retained on the corneal surface as viscosity increases.

Table 2 Evaluation of optimized formulations.

Formulation Batch code	Appearance	Clarity	pH	Gelling capacity test (s)	Gelation temperature	Drug content (%)
ISG16 (Smix ratio 1:1)	Clear	√	7.1±0.31	60	35±0.43°C	94.86±0.68
ISG17 (Smix ratio 1:2)	Clear	√	6.9±0.36	60	35±0.23°C	95.93±0.73

*Results are reported as Mean±SD, n=3.

Drug Content

The drug content of both selected formulations, ISG16 and ISG17, was nearly identical, as shown in [Table 2]. This indicates that incorporating the drug-loaded nanoemulsion into the nanoemulgel system resulted in negligible drug loss.

Flowability and Gelation Temperature

The nanoemulgel system remains in a liquid state at lower temperatures (approximately 5°C) and converts to a gel state at body temperature (around 35°C), demonstrating its potential for ocular use. The flowability outcomes for various samples are detailed in [Table 3]. The gelling time recorded was 60 seconds, as indicated in [Table 2]. Samples that remained liquid at 5±1°C and 25±1°C but solidified at 35±1°C within 30 seconds were considered optimal thermoresponsive in situ nanoemulgels. Upon dilution with STF, the ISG formulation displayed fluid-like properties at 25°C and transitioned into a gel at body temperature, as shown in [Fig. 3].

Fig. 3 (a) Nanoemulgel before gelation (b) Nanoemulgel after gelation.

Table 3 Flowability assessment at different temperatures.

Formulation batch code	Flowability
	5°C	25°C	35°C
ISG 16	+++	++	++
ISG 17	+++	+++	++

*Flowability:+++very good;++good.

Sterility Testing

Sterility testing was conducted on the optimized formulations. After a 7-day incubation period, there were no signs of precipitation observed. The growth medium remained transparent, indicating the absence of microbial growth. Findings of the sterility study are presented in [Table 4].

Table 4 Sterility Testing.

Formulation batch code	Incubation period (Days)
	1	2	3	4	5	6	7
ISG 16	-	-	-	-	-	-	-
ISG 17	-	-	-	-	-	-	-

*(- sign indicates no growth).

Isotonicity Test

The morphological results of the isotonicity test are depicted in [Fig. 4], provides a clear indication of the isotonic nature of the formulation. The absence of significant morphological changes, such as swelling (indicative of a hypotonic solution) or shrinking (indicative of a hypertonic solution), suggests that the formulation maintains osmotic balance with the biological system.

Fig. 4 Appearance of RBCs indicating swelling, shrinking, or no change during isotonicity measurement.

In Vitro Release Study

Following the physicochemical evaluations, ISG17 (composed of 10% oil phase, 30% S_mix 1:2, and 60% distilled water) was identified as the best formulation for in vitro drug release study. The study was performed by Franz diffusion cell using simulated tear fluid (STF; pH 7.4) as the dissolution medium.

As depicted in [Fig. 5], the drug release profile showed distinct behaviors for the nanoemulsion and nanoemulgel formulations. The nanoemulsion displayed a continuous increase in drug release over time. In contrast, the nanoemulgel exhibited a biphasic release pattern: it started with a slow release phase, followed by a rapid release, and finally, a sustained release phase. The initial slow release phase occurred as the drug moved through the oil layer to reach the aqueous phase. Once the drug exited the oil layer, the release rate increased due to the polymer’s pre-hydration with water, which facilitated the initial release. Ultimately, the polymer controlled the release, resulting in a sustained release pattern. The nanoemulsion achieved a cumulative drug release of 76.74% after 8 hours, whereas the ISG17 formulation reached 64.98%. This variation might be due to the lower viscosity of the formulation, allowing it to dilute more easily with simulated tear fluid.

Fig. 5 Cumulative percentage drug release from nanoemulsion and in situ nanoemulgel (Mean±SD, n=3).

Corneal Permeation Study

The transcorneal permeation profile of the optimized formulation was compared to that of the nanoemulsion, as illustrated in [Fig. 6]. The in situ nanoemulgel formulation exhibited a permeation rate of 87.16% (Apparent permeability Papp=1711.56 cm/h; flux J=17.12 mg/cm²/h), which was higher than the nanoemulsion’s rate of 80.23% (Apparent permeability Papp=626.67 cm/h; flux J=12.53 mg/cm²/h). This suggests the in situ nanogel formulation enhances permeation.

Fig. 6 Cumulative percentage transcorneal permeation from nanoemulsion and in situ nanoemulgel (Mean±SD, n=3).

Corneal Retention Study

After 24 h of continuous homogenization, the in situ nanoemulgel formulation showed 8% corneal retention, compared to 3% for the nanoemulsion in simulated tear fluid (STF). Fluorescent dye solution initially showed fluorescence that gradually faded. As shown in [Fig. 7], the dye solution showed limited retention on the goat cornea when using the nanoemulsion. In contrast, the in situ nanogel significantly prolonged the retention time of the dye in corneal tissues. However, the in situ nanogel increased the dye’s retention time in corneal tissues. The ISG17 in situ nanogel demonstrated strong retention capacity, potentially extending myricetin retention in the eye and reducing dosing frequency.

Fig. 7 Photomicrographs of corneal retention: (a) cornea with simple dye, (b) cornea with dye and nanoemulsion, (c) cornea with dye and in situ nanoemulgel.

Transmission Electron Microscopy (TEM) Analysis

TEM analysis was conducted to examine the morphology and distribution of droplets in the nanoemulsion systems. The optimal ISG17 nanoemulgel formulation was evaluated using TEM imaging. The TEM micrograph ([Fig. 8]) revealed that the optimized in situ gel formulation consisted of individual nanostructures. The particle size ranged from 32 to 42 nm, with a spherical shape and homogeneous distribution.

Fig. 8 TEM images showcasing the optimal (ISG17) in situ nanoemulgel

Toxicity Studies

Corneal hydration levels within the normal range of 75–80% (76 to 79%) indicated no corneal damage. No toxic responses were observed with the drug sample or the ISG17 formulation. Histological studies further confirmed the lack of toxicity. KCl solution (positive control) caused marked corneal tissue damage ([Fig. 9]), whereas normal saline (negative control) and the ISG17 formulation did not cause any damage.

Fig. 9 Photomicrographs of toxicity study: (a) cornea with drug sample, (b) cornea with ISG17 formulation, (c) cornea with negative control, (d) cornea with positive control.

Stability Studies

The stability study of the selected formulation (ISG17) was conducted following the ICH guidelines. Results indicated that the thermoresponsive in-situ nanoemulgel demonstrated optimal stability at room temperature (25°C±2°C and 60±5% RH), compared to its stability at elevated temperatures (40°C±2°C and 65±5% RH).

DPPH Assay

The antioxidant activity of ascorbic acid and myricetin was assessed using the DPPH assay. The IC₅₀ value of ascorbic acid (standard) was determined to be 11.06±0.23 µg/mL, while for myricetin, it was 11.24±0.18 µg/mL. This indicates that higher concentrations of the compounds effectively captured more free radicals generated by DPPH, leading to a decline in absorbance and an increment in the IC₅₀ value ([Fig. 10]).

Fig. 10 Comparative representation of IC₅₀ value for DPPH assay (Mean±SD, n=3).

Cell Viability Assessment using MTT assay

A cytotoxic effect of myricetin was observed in ISG17 when pre-treated for 2 h against induced oxidative stress in Y79 cells ([Fig. 11]). Both ISG17, the myricetin-loaded nanoemulgel, and the marketed formulation exhibited a decrease in cell viability. This cytotoxicity is likely attributed to myricetin’s antioxidative properties [29]. Consequently, these findings suggest that ISG17 holds promise for the management of diabetic retinopathy.

Fig. 11 Comparative representation of cell viability of MTT assay in ISG17 and marketed formulation (Mean±SD, n=3).

Cell Morphology Study

The cellular morphology of the Y79 cell line was examined using an inverted phase-contrast microscope. Cell morphology images of formulation ISG17 ([Fig. 12]) and marketed formulation ([Fig. 13]) consistently displayed the same pattern across the samples. No cell death was observed in the control group, while both the nanoemulgel and the marketed formulation exhibited comparable results.

Fig. 12 Morphological observations of cell viability at different concentrations of ISG17.

Fig. 13 Morphological observations of cell viability at different concentrations of marketed formulation.

Discussion

This study aimed to develop and characterize an in situ forming nanoemulgel for treating diabetic retinopathy, using myricetin as the active ingredient. The formulation was designed to enhance drug solubility, improve ocular bioavailability, and provide sustained drug release.

The solubility study was crucial in selecting the appropriate components for the formulation. Myricetin, a poorly soluble drug, showed high solubility in Sefsol 218, making it the chosen oil phase to maintain the drug in a solubilized form within the nanoemulsion. Additionally, Kolliphor RH40 and PEG 400 were selected as the surfactant and cosurfactant, respectively, based on their solubilization capabilities. Pseudo-ternary phase diagrams were constructed to optimize the ratios of oil, surfactant, and cosurfactant for stable nanoemulsions, providing insights into the formulation’s phase behavior and helping select the most suitable ratios for further evaluation. The clarity observed in the nanoemulsion region of the diagrams indicated stable formulations [16] [18]. Thermodynamic stability studies confirmed the stability of the optimized nanoemulsion formulations under various stress conditions, such as freeze-thaw cycles, centrifugation, and heating-cooling cycles, with no signs of instability observed [17].

The in situ nanogel was prepared by incorporating the optimized nanoemulsion into a hydrogel matrix using a cold method. The resulting nanoemulgel exhibited desirable characteristics, including clarity, suitable pH, and controlled viscosity, indicating its potential for ocular application. Drug content analysis demonstrated efficient incorporation of myricetin into the nanoemulgel system [19]. Physicochemical characterization revealed crucial attributes of the nanoemulgel formulations, including their flow characteristics, gelling behavior, and rheological properties. The sol-to-gel transition observed at physiological temperature suggested enhanced retention on the ocular surface, facilitating prolonged drug release [17]. Isotonicity is essential for preventing hemolysis or crenation of RBCs, which could lead to adverse effects such as irritation, inflammation, or systemic toxicity. The findings of isotonicity study confirm that the developed formulation is isotonic and can be safely administered without compromising cellular integrity.

In-vitro release studies demonstrated sustained drug release from the nanoemulgel formulations, with the in-situ formulation showing superior performance compared to the nanoemulsion alone. This sustained release profile can help maintain therapeutic drug levels in the eye over an extended period [21]. Ex vivo permeation studies revealed enhanced transcorneal permeation of the optimized nanoemulgel formulation, indicating its potential for effective drug delivery to the target site. In situ forming gels offer unique advantages for enhancing the residence time of ocular drug delivery systems, promoting improved ocular penetration and therapeutic efficacy [19]. Typically administered in a liquid state, these gels undergo gelation upon contact with ocular fluids or physiological conditions. This transformation increases the viscosity of the formulation, allowing it to adhere better to the ocular surface and prolonging its contact time with the target tissues [30]. The increased viscosity prevents rapid drainage from the eye and facilitates sustained release of the drug payload [20].

The in situ forming gel encapsulates the drug within the gel matrix, enabling controlled release over an extended period. The gel structure acts as a reservoir, gradually releasing the drug into the tear film and ocular tissues, ensuring prolonged exposure to therapeutic drug concentrations and maximizing bioavailability and therapeutic effect [21] [30]. The inclusion of HPMC 4KM provides mucoadhesive properties, allowing the formulation to adhere firmly to the mucus layer on the ocular surface [24]. This mucoadhesive characteristic forms a barrier that shields the encapsulated drug from rapid clearance pathways such as tear dilution and drainage via the lacrimal ducts [31]. Consequently, the formulation is securely anchored to the ocular surface, prolonging its residence time and enhancing drug absorption. This localized depot of the drug at the administration site mitigates systemic absorption and reduces the frequency of dosing required to achieve therapeutic efficacy [20] [24]. Moreover, the formulation employs biocompatible polymers that are well tolerated by ocular tissues, minimizing irritation or inflammation. Toxicity studies confirmed the safety of the nanoemulgel formulation, with no adverse effects observed on corneal tissues [24] [31].

Additionally, the antioxidant activity of the formulation, assessed by the DPPH assay, highlighted its potential in scavenging free radicals, which is particularly relevant for diabetic retinopathy [32]. Cell viability assays demonstrated the cytotoxic potential of the myricetin-loaded nanoemulgel formulation against Y79 retinoblastoma cells, suggesting its efficacy in combating diabetic retinopathy [27] [29].

Conclusion

In this study, we successfully developed and characterized an in-situ forming nanoemulgel loaded with myricetin for the potential treatment of diabetic retinopathy. The formulation was meticulously designed and optimized through a series of physicochemical characterization tests to ensure its stability, efficacy, and safety for ocular application. Our findings from solubility studies guided the selection of appropriate components for the nanoemulsion formulation. Pseudo-ternary phase diagrams helped identify the optimal ratios of oil, surfactant, and cosurfactant for nanoemulsion development. Thermodynamic stability studies confirmed the optimized formulations’ stability under various stress conditions.

The developed in situ nanoemulgel exhibited desirable characteristics, including clarity, suitable pH, and appropriate viscosity for ocular application. The formulation demonstrated a sol-to-gel transition at physiological temperature, indicating potential for easy instillation and prolonged retention on the ocular surface. Sterility testing ensured the formulation’s safety for ocular use. Furthermore, ex vivo studies showed enhanced transcorneal permeation and retention of myricetin with the in situ nanoemulgel compared to the nanoemulsion alone. This suggests the potential of our formulation to deliver therapeutically effective concentrations of myricetin to the retina for managing diabetic retinopathy. Toxicity studies revealed the formulation’s safety, with no observable damage or toxic responses in corneal tissues. Additionally, the formulation exhibited promising antioxidant activity and cytotoxic effects against Y79 retinoblastoma cells, further supporting its potential efficacy in managing diabetic retinopathy.

In conclusion, the developed in-situ forming nanoemulgel loaded with myricetin holds promise as an effective therapeutic strategy for the treatment of diabetic retinopathy. Further studies including in vivo efficacy and safety evaluations are warranted to validate its clinical potential.

Authors contributions

PK contributed significantly to the conception and design of the study. SS and SG were involved in data analysis and interpretation. SS handled data acquisition and drafted the manuscript, with all authors providing input. All authors reviewed and approved the final version of the manuscript.

Conflicts of Interest

The authors declare no financial or other conflicts of interest.

Acknowledgement

The authors express their gratitude to the Integral University Faculty of Pharmacy for providing the necessary facilities for this research (Manuscript Communication Number: IU/R&D/2024-MCN0002475).

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Publication History

Received: 23 August 2024

Accepted: 27 December 2024

Article published online:
07 February 2025