Development and Optimization of Puerarin-loaded in situ liquid crystal gels for ocular delivery in dry eye disease

Development and Optimization of Puerarin-loaded in situ liquid crystal gels for ocular delivery in dry eye disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development and Optimization of Puerarin-loaded in situ liquid crystal gels for ocular delivery in dry eye disease Pallavi Desale, Amol S. Rakte, Sanjay R. Arote This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9256829/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objectives: To design and optimize puerarin-loaded LCGs for controlled ocular delivery in DED, elucidating the drawbacks inherent to conventional ophthalmic vehicles regarding corneal bioavailability and precorneal elimination. Methods: Nine different formulations (PF1–PF9) were developed via a modified fusion-vortex method where glyceryl monooleate (GMO) and Soluplus® were used as independent variables in a 3 2 full factorial design. The formulations were examined for physicochemical characteristics, drug release kinetics, ex vivo corneal permeation, ocular irritation (HET-CAM), cytotoxicity (MTT assay in HCE-T cells), and stability according to ICH Q1A(R2) guidelines. Results: The optimization was carried out by the desirability function approach using Design Expert® v.13. Acceptable pH (7.14–7.26), drug content (96.4–98.6%) and entrapment efficiency (82.3–94.3%) were achieved by all the formulations. Corneal permeation and viscosity were well described by quadratic (R 2 adj = 0.9977) and linear models (R 2 adj = 0.9954), respectively. The optimum formulation PF6 (GMO 60% w/w, Soluplus® 15% w/w) possessed the highest desirability (D = 1), cumulative corneal permeation of 76.4 ± 2.43% in 8 hours with flux of 52.34 ± 1.78 μg/cm 2 /h and precursor viscosity of 121.4 ± 3.7 mPa·s with non-Fickian anomalous transport qualified as the best fit for drug release profile (R 2 = 0.9997). HET-CAM non-irritant classification was confirmed (IS = 0.38 ± 0.04), and IC 50 of PF6 (182.4 ± 4.1 µg/mL) was greater than free puerarin (148.6 ± 3.2 µg/mL). Less than 3.5% variation was found in the stability studies over three months. Conclusion: PF6 exhibited superior corneal permeation, prolonged release, and good ocular safety, indicating its potential as a viable candidate therapeutic platform for DED treatment worth further in vivo validation. Dry eye disease Puerarin In situ liquid crystal gel Glyceryl monooleate Soluplus® Factorial design Corneal permeation Ocular drug delivery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Necessitating the need for a treatment strategy in patients suffering from dry eye disease (DED), a multifactorial ocular surface disorder with tear film instability and hyperosmolarity and chronic ocular surface inflammation, affecting an estimated 344 million individuals worldwide[ 1 ]. Its prevalence in different populations varies from 5% to 50%, with an increased incidence among older adults, postmenopausal women, and patients using digital devices for prolonged periods[ 2 ]. Beyond discomfort, the global burden of DED includes substantial visual impairment, decreased quality of life, and loss of workplace productivity[ 3 ]. The economic burden is high, with annual health care costs of more than USD 3.84 billion in the United States alone, as well as increased expenses due to recurrent clinic visits, costly diagnostic tests, and chronic therapeutic management[ 4 ]. Although lubricating eye drops, cyclosporine A emulsions and lifitegrast solutions are widely available pharmacotherapy, remain limited by poor patient compliance, transient symptom relief, suboptimal corneal bioavailability and need for frequent dosing[ 5 ]. When a conventional ophthalmic formulation is instilled into the eye, most of it drains within minutes through the nasolacrimal duct; only 1–5% reaches its target site due to ocular bioavailability. Although DED encompasses a wide spectrum from mild to severe, the worldwide increase of screen time, aging populations and environmental pollution are expected to increase DED prevalence in the near future that drives an urgent demand for new therapeutic strategies with efficacy and patient-friendliness[ 6 ]. Puerarin (4′,7-dihydroxyisoflavone-8-C-glucoside; molecular formula: C 21 H 20 O 9 ; MW: 416.38 g/mol) is a naturally occurring C-glycosylated isoflavone phytoestrogen primarily obtained from the roots of Pueraria lobata (Willd.) Ohwi[ 7 ]. The molecular architecture of puerarin includes hydroxyl groups at the C-4′ and C-7 positions, as well as a glucose moiety at the C-8 site, conferring significant water solubility compared with other isoflavones[ 8 ]. Puerarin has highly relevant pleiotropic pharmacological actions in the context of DED pathophysiology, including potent antiinflammatory activity by inhibiting NF-κB and MAPK signaling pathways as well as decreasing pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), antioxidant activity via Nrf2 pathway activation; and stimulation of goblet cell proliferation to enhance mucin secretion. In preclinical studies, this agent has been shown to restore tear secretion and increase conjunctival goblet cell density while reducing corneal epithelial apoptosis in several experimental models of DED[ 9 ]. In addition, puerarin has a relatively favorable safety profile with low systemic toxicity, which can be regarded as an excellent candidate for treatment of the ocular surface. However, its clinical ocular use is limited due to rapid precorneal elimination, moderate corneal permeability and lack of suitable ophthalmic delivery platform for maintaining therapeutic concentrations at the ocular surface[ 10 ]. In situ liquid crystal gels (LCGs) are an advanced drug delivery platform based on a phase transition of a low-viscosity solution into a structured liquid crystalline mesophase stimulated by contact with physiological triggers such as components in tear fluid, temperature or dilution-induced phase transitions[ 11 ]. These nanostructured systems cubic, hexagonal, or lamellar mesophases are made of biocompatible lipid-based materials such as glyceryl monooleate, phytantriol and poloxamer-based block copolymers. These excipients are amphiphilic in nature which self-assemble on the formation of ordered nanoarchitectures bearing separate hydrophilic channels and hydrophobic domains providing sustained release for both drug types i.e., hydrophilic & hydrophobic drugs[ 12 ]. In situ LCGs provide many advantageous characteristics for ophthalmic delivery including extended precorneal retention time (> 4 hours), lower dosing frequency, improved corneal permeation via close bioadhesive contact with the cornea, protection of sensitive active agents from enzymatic degradation and good biocompatibility with ocular tissues. Recent innovations have shown that cubic and hexagonal mesophases greatly improve bioavailability of less permeable compounds without compromising ocular tolerability[ 13 ]. In addition, the liquid-like precursor state enables easy instillation as traditional eye drops, immediately changing into a viscous mucoadhesive depot upon lacrimal dilution. These features make in situ LCGs a theoretically sound and clinically practical carrier for extended ocular release of puerarin in the treatment of DED[ 14 ]. The current study focuses on the design and optimization of puerarin-loaded in situ liquid crystal gels for sustained ocular delivery to dry eye disease. Particular aims comprise physicochemical characterization and in vitro drug release profiling, as well as ex vivo corneal permeation studies and in vitro cytotoxicity evaluation. This study aims to develop a new ocular drug delivery system with better pharmacological properties and patient compliance. MATERIALS AND METHODS MATERIALS Puerarin (purity: 98% and MW: 416.38 g/mol) was acquired at Sciquaint Innovations Pvt. ltd. (Pune, India). Soluplus 118,000 g/mol (MW) and glyceryl monooleate (pharmaceutical grade) were purchased at Sciquaint Chemicals (Pune, India). epoxy resin was purchased as propylene glycol (USP grade), whereas benzalkonium chloride was purchased as benzalkonium chloride (pharmaceutical grade), both obtained in Research Lab Fine Chem Industries (Mumbai, India). Ethanol, methanol and acetinitrile (all of HPLC grade) were bought at Neeta Chemicals (Pune, India). MTT reagent and dialysis membrane (cutoff 12,000-14 000Da) were obtained with Sciquaint Chemicals (Pune, India). Sodium lauryl sulfate (analytical grade) was bought in Research Lab Fine Chem Industries (Mumbai, India). All other chemicals and reagents were of analytical grade and not subjected to any additional purification. METHODS Calibration Curve of Puerarin The linear relationship between absorbance and puerarin (purity ≥ 98%) concentration in ethanol was established by means of a calibration curve. 10 mL of puerarin was weighed out and dissolved in ethanol in a 10 mL volumetric flask to prepare the primary stock solution (1000 μg/mL), and six working standard solutions (5, 10, 15, 20, 25 and 30 μg/mL) were prepared by transferring aliquots of 0.05, 0.10, 0.15, 0.20, 0.25 and 0.30 mL of the stock solution into separate 10 mL volumetric flasks using an adjustable micropipette (Tarsons India) followed by dilution to volume with ethanol at ambient temperature (25 ±2°C); absorbance values for each solution were measured at λ max against blank made from ethanol as solvent using a double-beam UV-visible spectrophotometer (Systronics UV-2202 India), employing quartz cuvettes of path length =1 cm. The mean absorbance was plotted against the concentration (μg/mL) to obtain a calibration graph in which the equation of linear regression analysis and correlation coefficient (r 2 ) ≥ 0.999 confirmed linearity as per ICH Q2(R1). All measurements were conducted in triplicate (n = 3) and presented as mean ± SD[15]. Solubility Study of Puerarin The solubility profile of puerarin in physiologically relevant and organic solvents was characterized to inform dosage form development. From this, an excess amount of puerarin (~50 mg) was suspended separately in 10 mL each of distilled water, phosphate buffer pH 7.4, simulated tear fluid (STF), artificial tear fluid, methanol, ethanol and acetonitrile inside a series of stoppered 25mL conical flasks that were capped tightly to minimize evaporation and placed on an orbital shaker (Remi Instruments; India) at 25 ± 2°C and agitation at a speed of roughly 100 rpm for 24 hours to equilibrate saturation. After 30 min of settling followed by filtration through Whatman filter paper No. 41, the supernatant was appropriately diluted and the absorbance measured at λmax using double-beam UV-visible spectrophotometer (Systronics UV-2202, India) with quartz cuvettes of path length 1 cm. Solubility was determined from the regression equation y = mx + c and was expressed as mg/mL. All measurements were conducted in triplicate (n = 3) and reported as mean ± SD[16]. Differential Scanning Calorimetry (DSC) Thermal investigation, crystallinity and drug-excipient compatibility of puerarin was studied using differential scanning calorimetry (DSC) instrument (Mettler Toledo DSC 822e; Switzerland). Samples of pure puerarin and its physical mixture with the excipients (3–5 mg each) were accurately weighed and placed individually in standard aluminum pans, sealed hermetically with aluminum lids by a crimping press, and analyzed, using an empty sealed aluminum pan as reference. To avoid oxidative decomposition, samples were heated from 30°C to 300°C at a scanning rate of 10 °C/min under an inert nitrogen atmosphere and a purge gas flow rate of 20 mL/min. The thermograms obtained were examined for characteristic endothermic or exothermic peaks, onset temperature, peak temperature and changes in enthalpy (ΔH), while shifts or disappearances of peaks were interpreted as a sign of drug-excipient interactions or changes in crystallinity. All assays were conducted in triplicate (n = 3) and values are expressed as mean ± SD[17,18]. Fourier Transform Infrared Spectroscopy (FTIR) Functional groups contained in puerarin and possible chemical interactions between the drug and excipients by changes in characteristic absorption bands were analyzed using infrared spectroscopy (IR). FTIR spectrophotometer (Shimadzu IRSpirit, Japan) was used to record the spectra using KBr pellet technique. Prior to use, KBr powder was dried for 2 hours at 40–50°C to remove moisture. A reasonable amount of (~200 mg) dried KBr was triturated separately with 2–3 mg pure puerarin and its physical mixture with excipients in clean mortar and pestle for 5–10 minutes to make sure the blend was homogeneously mixed as well as uniform particle size reduction. The resultant mixture was then pressed into a thin, transparent, self-supporting disc with the aid of an hydraulic press at ~10 tons for 3–5 min. Immediately after preparation, the KBr disc was mounted in the sample holder, and spectra were collected over 4000–400 cm -1 at a resolution of 4 cm -1 and with 32 scans per sample, using an air background reference. Absorption spectra were also examined for characteristic absorption bands associated with functional groups, and any pronounced shifts, disappearances of peaks or appearance of new peaks suggestive of drug-excipient interactions. All experiments were done in triplicate (n = 3) and results reported as mean ± SD[19,20]. Experimental Design A 3 2 full factorial design was used to screen puerarin-loaded in situ liquid crystal gels, wherein two independent variables were each examined at three levels (low, medium, high). The selected independent variables were glyceryl monooleate (GMO; X 1 ) concentration, as the primary amphiphilic lipid that promoted lacrimal dilution-triggered production of liquid crystalline mesophase, and Soluplus® (polyvinyl caprolactam–polyvinyl acetate–polyethylene glycol graft copolymer; X 2 ) concentration, incorporated as a novel amphiphilic polymeric co-matrix agent to achieve concomitant improvement in puerarin solubilization stability and control over mesophases structure both of which had not been explored simultaneously within the context of lipidic ophthalmic in situ liquid crystal gel systems. A total of nine experimental preparations (F1–F9) were generated as per the complete factorial matrix. The dependent variables were the percent cumulative drug release at 8 hours (Y 1 ; target: maximize and ≥ 70%) and viscosity of the precursor solution (Y 2 ; target minimize to ≤ 150 mPa·s for instillability) as given in table 1. Multiple linear regression was performed using Design Expert® software v.13 (Stat-Ease Inc., USA), and response surface plots as well as polynomial equations were produced. For each response, we can write the general polynomial equation: Y = b 0 + b 1 X 1 + b 2 X 2 + b 12 X 1 X 2 + b 11 X 1 2 + b 22 X 2 2 where Y is the dependent variable, b 0 is the intercept, b 1 and b 2 are linear coefficients, b 12 is the interaction coefficient, and b 11 , b 22 are quadratic coefficients[21,22]. Table 1: Independent and Dependent Variables in the 3 2 Full Factorial Design Variable Level −1 (Low) Level 0 (Medium) Level +1 (High) Independent Variables X₁: Glyceryl monooleate (% w/w) 50 60 70 X₂: Soluplus® (% w/w) 5 10 15 Dependent Variables Goal Y₁: Cumulative drug release at 8 h (%) Maximize Y₂: Precursor viscosity (mPa·s) Minimize Table 2: Formulation Composition of Puerarin-Loaded In Situ Liquid Crystal Gels (F1–F9) Excipient PF1 PF2 PF3 PF4 PF5 PF6 PF7 PF8 PF9 Puerarin (% w/w) 1 1 1 1 1 1 1 1 1 Glyceryl monooleate (% w/w) 50 50 50 60 60 60 70 70 70 Soluplus® (% w/w) 5 10 15 5 10 15 5 10 15 Propylene glycol (% w/w) 10 10 10 10 10 10 10 10 10 Benzalkonium chloride (% w/w) 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 Simulated tear fluid (STF) q.s. to 100 q.s. to 100 q.s. to 100 q.s. to 100 q.s. to 100 q.s. to 100 q.s. to 100 q.s. to 100 q.s. to 100 (All values are % w/w; STF composed of NaCl 0.67%, NaHCO 3 0.20%, KCl 0.14%, pH 7.4) Preparation of Puerarin-Loaded In Situ Liquid Crystal Gels Puerarin- loaded in situ liquid crystal gels were formulated utilizing a modified fusion-vortex method based on Wu et al. (2021) and Wang et al. (2019). Weigh the exact quantities of glyceryl monooleate (Table 2) and melt in a water bath (Equitron, India) at 45 ± 2°C until it became clear homogeneous melt. In parallel, Soluplus® was stirred with propylene glycol in a magnetic stirrer (Remi, India) at 300 rpm for 15 minutes at 40°C. Puerarin (1% w/w) was dispersed into the Soluplus®–propylene glycol solution and stirred, at 500 rpm and 25 ± 2°C for 30 minutes until complete dissolution. List of things to do on the day of arrival until departure. Benzalkonium chloride (0.01% w/w) was dissolved in a minimum volume of simulated tear fluid and the total weight adjusted to 100% w/w with STF under continuous vortexing for an additional 3 minutes. All formulations were kept at 4 ± 2°C in sterile amber glass vials until further characterization[23,24]. Characterization of Puerarin-Loaded In Situ Liquid Crystal Gels Visual Appearance and pH All formulations were visually inspected for clarity, color and phase homogeneity. The pH of individual precursor solutions was measured at 25 ± 2 °C employing calibrated digital pH meter (SystronicsμpH System 362, India) that has been standardized with suitable buffer solutions (Merck, India): pH 4.0 and pH 7.0. Data were recorded as mean ± SD (n = 3); the acceptable ophthalmic pH range is 6.5–8.0 as per IP 2022 guidelines[25] Viscosity of Precursor Solution The viscosity measurements of the precursor (pre-gelation) formulations were performed at 25 ± 0.5°C using a Brookfield DV-II+ Pro viscometer (Brookfield Engineering Company, Middleboro, MA USA), fitted with spindle no. S-62 and set to 10 rpm. After equilibration for 2 min, the measurements were conducted. Mean ± SD (n = 3) is given for viscosity (mPa·s). Conventional instillability of the optimized formulations was ensured by targeting a precursor viscosity of ≤ 150 mPa·s[26]. In Situ Gelling Capacity and Gel Strength Each formulation's gelling capacity upon contacting STF was qualitatively evaluated by applying 50 µL of the precursor solution onto a glass slide maintained at 35 ± 0.5 °C and observing whether/when its phase transition behavior occurred. The gel strength was measured by an inverted tube method: 2 mL formulation into a pre-weighed vial, 0.1 mL STF was mixed at 35 °C, then recording the average weight (g) required to insert a stainless-steel probe (10 mm diameter) into the formed gel up to a depth of 5 mm using a texture analyzer (TA-HD plus, Stable Micro Systems, UK). Results were presented as mean ± SD (n = 3)[27]. Polarized Light Microscopy (PLM) for Liquid Crystalline Phase Identification The observed internal liquid crystalline mesophase structure of the resulting gel (pseudocylindrical) was investigated using polarized light microscopy, utilizing a Carl Zeiss Axiolab 5 polarizing microscope with cross-polarizers (Carl Zeiss, Germany). Each formulation (~5 mg) was spread on a clean glass slide and submerged into excess water (5 µL) at 35°C, covered with coverslip and examined under polarized light at ×40 magnification for birefringent textures associated with cubic or hexagonal mesophases. Photomicrographs were obtained with a built-in digital camera. All experiments were performed in triplicate (n = 3)[28]. Drug Content In all of the formulations, 100 mg was dissolved in 10 mL of ethanol under vortex mixing (2500 rpm, 5 min) and then diluted with ethanol to achieve an expected concentration of 10 µg/mL puerarin. Absorbance was recorded at λ max versus blank of the related placebo formulation on double beam UV-visible spectrophotometer (Systronics UV-2202, India) using 1 cm quartz cuvettes. Drug content (%) was obtained from the regression equation. Results represent mean ± SD (n = 3); acceptable limits correlate with ICH Q6A: 95–105% of the labeled claim[29]. Entrapment Efficiency Entrapment efficiency (EE%) was calculated by ultracentrifugation at 15,000 rpm for 45 minutes at 4°C (Remi C-24 Plus, India) to separate unentrapped free drug and liquid crystal matrix. The supernatant was separated, diluted in ethanol as appropriate and absorbance at λ max recorded using Systronics UV-2202 spectrophotometer (India). EE% was calculated using: EE (%) = [(Total drug − Free drug) / Total drug] × 100 Results were expressed as mean ± SD (n = 3)[30]. In Vitro Drug Release In vitro drug release was carried out by dialysis bag diffusion method as described by Wu et al. (2021). A dialysis membrane (MW cutoff 12,000–14,000 Da; Hi-Media, India) that was pre-soaked in dissolution medium for 12 hr was employed. Exact amounts of test formulation equivalent to 1 mg puerarin were carefully introduced into the membrane and sealed, before being suspended in 50 mL of STF (pH 7.4) in a USP dissolution apparatus Type II (paddle; Electrolab EDT-08Lx, India) operating at 100 rpm and at temperature maintained at 37 ± 0.5°C. At predetermined time intervals (0.5, 1, 2, 4, 6, 8, 12 and 24 h), aliquots of 2 mL were taken out and replaced with the same volume of fresh medium to sustain sink conditions. Absorbance values were measured at λmax after filtering the samples with a 0.45 µm PVDF syringe filter and dilution to proper absorbance level. The cumulative drug release (%) was calculated from the calibration curve. Release kinetics were evaluated fitting the data to zero order, first order, Higuchi and Korsmeyer–Peppas models; model selection was done based on higher r 2 and lower AIC. Data are shown as mean ± standard deviation (SD) (n = 3)[31]. Ex Vivo Corneal Permeation Study The freshly excised goat corneas were collected from a local abattoir within 2 hours of slaughter following the ethical guidelines laid down by CPCSEA, India and was used for the ex vivo corneal permeation studies. The cornea was placed between modified Franz diffusion cell (PermeGear, USA) systems with receptor compartment containing 15 mL of STF (pH 7.4), maintained at a temperature of 35 ± 0.5°C and continuous stirring at a rate of 300 rpm. The donor compartment was treated with 50 µL (containing 1% puerarin) of formulation. At 1, 2, 4, 6, 8 and 12 h samples (1 mL) were removed and STF was replaced with fresh STF. Puerarin was measured spectrophotometrically at λmax Papp (cm /s ) and cumulative amount permeated (µg/cm 2 ) were calculated. As a control, puerarin solution (1% w/v in STF) was used. Results are shown as mean ± SD (n = 3)[32]. Ocular Irritation (HET-CAM Assay) According to ICCVAM guidelines, HET-CAM was used as an ethical alternative for testing ocular irritation potential (i.e., the Draize rabbit eye test). Gelatinase activity was determined using the gelatinase assay according to Dani et al. [36], as well as protein extraction from chicken egg white, for which fertilized white Leghorn eggs (Venkateshwara Hatcheries, India) were incubated at 37 ± 0.5°C with 60% relative humidity in a humidified egg incubator (Igenix, India) for 9 d. After 9 days of incubation, the eggs were carefully cracked open on the shell over the air sac to expose the membrane and 300 µL of each formulation was placed directly onto the CAM. Over 300 seconds, hemorrhage (H), vascular lysis (L) and coagulation took place. Irritation score (IS), calculated according to an established formula; IS < 0.9 = non-irritant, 0.9–4.9 = slight irritant. Physiological saline (0.9% NaCl) and sodium lauryl sulfate (1% w/v) were used as the negative and positive controls, respectively. Results were shown as mean ± SD (n = 3)[33,34]. In Vitro Cytotoxicity (MTT Assay) The cytotoxicity of the optimized formulation was assessed in human corneal epithelial cells (HCE-T cell line, ATCC) by performing MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] test. DMEM/F12 medium supplemented with 10% FBS was used to seed the cells in 96 well plates at a density of 1 × 10 4 cells/well.for 24 hours with incubation at 37°C/5% CO 2 . Cells were subsequently treated with serial dilutions of the optimized formulation and free puerarin (1–200 µg/mL) for 24 h. MTT solution (5 mg/mL in PBS; 20 µL/well) was added and incubated at 37°C for 4 hours, then formazan crystals were dissolved in DMSO (150 µL/well). Absorbance was measured at 570 nm using microplate reader (BioradiMark, India). Percentage viability (%) and IC 50 values were calculated. Values are mean ± SD (n = 3); statistical comparison by one-way ANOVA with Tukey's post-hoc test (p < 0.05)[35,36]. Stability Study Stability study of optimized in situ liquid crystal gel formulation (PF6) loaded with puerarin was performed according to ICH Q1A(R2) guidelines. The formulation was filled in sterile amber glass vials and stored under three separate conditions: an accelerated condition (40°C ± 2°C / 75% RH), a room temperature condition (25°C ± 2°C / 60% RH), and freezing conditions (−20°C ± 2°C) for three months. Samples were withdrawn at fixed time intervals (0 (baseline),1,2, and 3 months) and reviewed for physical appearance, pH, precursor viscosity, drug content, entrapment efficiency and cumulative corneal permeation after 8 hours. All analyses were conducted in triplicate (n = 3) and data shown are mean ± SD. The degree of variation during the storage period was calculated using the percentage change between each parameter as compared to the initial value[37,38]. Statistical Analysis Results were expressed as mean ± SD (n = 3). Statistical comparisons were conducted by one-way ANOVA and Tukey’s post-hoc test (p < 0.05) with GraphPad Prism v.9.0 (GraphPad Software, USA). Response surface methodology utilizing Design Expert® v.13 (Stat-Ease Inc, USA) 3 2 full factorial design was applied for formulation optimization and adjusted R 2 , predicted R 2 , p-value of lack-of-fit and F-values were utilized to test the adequacy of the model. Sample coverage was determined for the best combination of three distinct function types: touch, contact, and incubation, using a desirability function approach for simultaneous optimization of dependent variables[39]. RESULTS AND DISCUSSION RESULTS Calibration Curve Determination A calibration curve for puerarin in ethanol was established over a concentration range of 5–30 µg/mL. Excellent linearity (y = 0.0334x + 0.005, r 2 = 0.9996) was observed with ICH Q2(R1) compliance reported here. The relationship between absorbance and concentration for the span studied was linear as indicated by the high r 2 value. The calibration curve shown in Figure 1 confirms the applicability of the developed spectrophotometric method, allowing for accurate and reproducible quantification of puerarin in all further analyses. Results of Solubility Study Table 3 shows the solubility of puerarin in different solvents. Puerarin was poorly soluble in distilled water (0.82 ± 0.15 mg/mL), and slightly soluble in physiologically relevant media, such as simulated tear fluid (7.35 ± 0.32 mg/mL) and phosphate buffer pH 7.4 (7.56 ± 0.28 mg/mL). The highest solubility value was reported in ethanol (26.28 ± 1.45 mg/mL) and methanol (25.12 ± 1.84 mg/mL). The solubility in aqueous and tear fluid was limited, confirming the need for a lipid-based liquid crystal carrier system to improve ocular targeting of puerarin. Table 3: Solubility Profile of Puerarin in Different Solvents Sr. No. Solvent Solubility (mg/mL) Inference 1 Distilled Water 0.82 ± 0.15 Very slightly soluble 2 Phosphate Buffer pH 7.4 7.56 ± 0.28 Slightly soluble 3 Simulated Tear Fluid (STF) 7.35 ± 0.32 Slightly soluble 4 Artificial Tear Fluid 7.28 ± 0.26 Slightly soluble 5 Methanol 25.12 ± 1.84 Sparingly soluble 6 Ethanol 26.28 ± 1.45 Sparingly soluble 7 Acetonitrile 2.15 ± 0.38 Slightly soluble All values are expressed as mean ± SD Differential Scanning Calorimetry (DSC) Analysis The DSC thermogram of pure puerarin showed a sharp characteristic endothermic peak at a melting temperature of 185.93°C, confirming its crystalline appearance (Figure 2A). In the thermogram of physical mixture (Figure 2B), two endotherms were observed at 155.73°C and 185.44°C; the appearance of an additional peak at 155.73°C as well as shifting of puerarin melting peak from 185.93°C to 185.44°C seen in this thermogram confirmed that some type of interaction had occurred between puerarin and excipients which reduced crystallinity to some extent but there was no notable incompatibility indicating suitability for formulation development. Fourier Transform Infrared Spectroscopy (FTIR) Analysis Characteristic absorption bands were exhibited in the FTIR spectrum of pure puerarin (Figure 3A), including those at 3473.30 and 3397.38 cm -1 (O–H stretching), 2936.34 cm -1 (C–H stretching), 1649.26 cm -1 (C=O stretching), 1579.07 cm -1 (C=C aromatic stretching) and 1035.17 cm⁻¹ (C–O–C stretching), confirming structural integrity of puerarin[14]. In the spectrum of physical mixture (Figure 3B), all characteristic peaks of puerarin were retained and only slightly shifted, such as O–H stretching at 3435.23 cm -1 and C=O at 1647.16 cm -1 , and no great peak disappearance was noticed after mixing with formulation excipients, proving that there is no significant chemical incompatibility between puerarin and those formulation excipients. Characterization of puerarin-loaded in situ liquid crystal gel formulations Visual Appearance The nine puerarin-loaded in situ liquid crystal gel formulations (PF1–PF9) were also pale yellow liquid with characteristic odor and a homogeneous phase as listed in Table 3. PF1, PF2 and PF4 containing lower concentrations of GMO or Soluplus® appeared clear and free-flowing, but increasing concentrations of each excipient progressively increased viscosity and opalescence; PF9 had a turbid highly viscous appearance. All formulations showed phase homogeneity [showing all prepared formulations were successfully formulated with drug uniformly distributed in the liquid crystal matrix]. Table 4.Visual characteristics of puerarin-loaded in situ liquid crystal gel formulations (PF1–PF9) F. Code Appearance Color Clarity Phase Homogeneity Odor PF1 Clear, free-flowing liquid Pale yellow Clear Homogeneous Characteristic PF2 Clear, slightly viscous liquid Pale yellow Clear Homogeneous Characteristic PF3 Slightly opalescent, viscous Pale yellow Slightly turbid Homogeneous Characteristic PF4 Clear, free-flowing liquid Pale yellow Clear Homogeneous Characteristic PF5 Clear, moderately viscous Pale yellow Clear Homogeneous Characteristic PF6 Opalescent, viscous liquid Pale yellow Slightly turbid Homogeneous Characteristic PF7 Clear, viscous liquid Pale yellow Clear Homogeneous Characteristic PF8 Opalescent, viscous liquid Pale yellow Slightly turbid Homogeneous Characteristic PF9 Opalescent, highly viscous Pale yellow Turbid Homogeneous Characteristic Physicochemical Characterization, Drug Content, Entrapment Efficiency, Gelling Capacity and Gel Strength All formulations had a pH in the ophthalmic range of 6.5–8.0 (Table 5) at 25°C, ranging from 7.14 to 7.26. Precursor viscosity progressively increased from 48.3 mPa·s (PF1) to 148.3 mPa·s (PF9), reflecting the rising concentrations of GMO and Soluplus®, while all formulations remained below the ≤150 mPa·sinstillability threshold. Drug contents were acceptable (96.4–98.6%) and entrapment efficiency was also within limits (82.3–94.3%). Gelling abilities increased with higher excipient concentrations, reaching fully rigid PF6–PF9 gels within 30 seconds. Gel strength ranged progressively from 18.4 g (PF1) to 38.1 g (PF9), indicating increased matrix strength with increasing levels of GMO and Soluplus®. Table 5.Physicochemical characterization, drug content, entrapment efficiency, gelling capacity, gel strength, and in vitro drug release of puerarin-loaded in situ liquid crystal gel formulations (PF1–PF9) F. Code pH Precursor Viscosity (mPa·s) Drug Content (%) Entrapment Efficiency (%) Gelling Capacity Gel Strength (g) PF1 7.21 ± 0.04 48.3 ± 1.8 96.4 ± 0.9 82.3 ± 1.4 ++ 18.4 ± 0.7 PF2 7.18 ± 0.03 67.6 ± 2.1 97.1 ± 0.7 86.7 ± 1.2 ++ 19.8 ± 0.9 PF3 7.14 ± 0.05 89.4 ± 2.6 97.8 ± 0.8 89.2 ± 1.6 +++ 21.3 ± 1.1 PF4 7.24 ± 0.03 74.2 ± 2.3 96.8 ± 1.1 85.4 ± 1.3 +++ 24.6 ± 0.8 PF5 7.19 ± 0.04 96.8 ± 3.1 97.6 ± 0.9 89.8 ± 1.5 +++ 26.9 ± 1.2 PF6 7.16 ± 0.06 121.4 ± 3.7 98.2 ± 0.6 92.1 ± 1.4 ++++ 29.4 ± 1.4 PF7 7.26 ± 0.05 108.7 ± 3.4 97.2 ± 1.0 87.6 ± 1.7 +++ 31.2 ± 1.3 PF8 7.22 ± 0.04 131.6 ± 4.2 98.0 ± 0.8 91.4 ± 1.6 ++++ 34.7 ± 1.6 PF9 7.17 ± 0.07 148.3 ± 4.8 98.6 ± 0.7 94.3 ± 1.8 ++++ 38.1 ± 1.9 All values expressed as mean ± SD (n = 3), Gelling capacity: ++ = moderate gel formed within 60 s; +++ = firm gel within 45 s; ++++ = rigid gel within 30 s Results of Ex Vivo Corneal Permeation Figure 4 and Table 3a summarises the cumulative ex vivo corneal permeation profiles of all formulations across goat cornea, for an 8 hours period. All the formulations showed time-dependent permeation that was observed in a range from 49.7% (PF1) to 83.1% (PF9) at the end of the study (8 hours). PF6 demonstrated permeation of 76.4 ± 2.43% for the period of 8 hours, where flux was recorded as52.34 ± 1.78 µg/cm 2 /h and corresponding permeability coefficient was found to be5.23 ×10 -3 cm/h (Table 6), indicative of an excellent compromise between enhancement in permeation & viscosity supported (Vishwakarma et al.,nam.*). The observed progressive enhancement of permeation with increasing concentrations of both GMO and Soluplus® supports the notion that these excipients act to enhance corneal drug transport. Flux and Permeability Table 6 presents the flux and permeability coefficient values for the different formulations. The flux values were in the range of 34.93 ± 1.24 µg/cm 2 /h (PF1) to 56.69 ± 1.94 µg/cm 2 /h (PF9), and the permeability coefficient was in the range of 3.49 × 10 -3 cm/h (PF1) to 5.67 × 10 -3 cm/h (PF9) and increased proportionately with increase in concentration of GMO and Soluplus® PF6's flux (52.34 ± 1.78 µg/cm 2 /h) and Kp (5.23 × 10 -3 cm/h) values indicated the best corneal permeation enhancement among the SFPs studied. As seen for all three formulations, the increasing trend confirms that in addition to aforementioned effect of GMO, we could make use of contribution of it along with Soluplus® which led to a synergistic affect on augmenting transcorneal drug transport. Table 6: Flux and Permeability Coefficient of PF1–PF9 Formulation Flux J (µg/cm 2 /h) Kp (×10 -3 cm/h) PF1 34.93 ± 1.24 3.49 ± 0.12 PF2 39.39 ± 1.37 3.94 ± 0.14 PF3 43.95 ± 1.52 4.40 ± 0.15 PF4 40.55 ± 1.41 4.06 ± 0.14 PF5 47.03 ± 1.63 4.70 ± 0.16 PF6 52.34 ± 1.78 5.23 ± 0.18 PF7 44.37 ± 1.54 4.44 ± 0.15 PF8 50.32 ± 1.71 5.03 ± 0.17 PF9 56.69 ± 1.94 5.67 ± 0.19 All values expressed as mean ± SD (n = 3) Drug Release kinetics As represented in Figure 5, the in vitro drug release data of optimized formulation PF6 was applied to zero order, first order, Higuchi, Hixson–Crowell and Korsmeyer–Peppas models. The highest R 2 value observed was that of Hixson–Crowell model which was found to be the best fitted having a slope of (0.2207) with all models; followed by first order model (R² = 0.9959) and Higuchi model (R² = 0.9980) given in kinetic parameters. An R² value of 0.9937 with a slope of 0.8798 obtained using the Korsmeyer–Peppas model confirms a non-Fickian mode of anomalous diffusion transport, validating that drug release from the liquid crystal matrix was through a combined mechanism governed by diffusion and erosion controlled mechanisms. Optimization of Puerarin-Loaded In Situ Liquid Crystal Gel Response 1: Cumulative Corneal Permeation at 8 h (Y 1 ) According to the model building criteria of Design Expert® v.13, the quadratic model was the best fit for Y 1 . Table 8 indicates that the model achieved an adjusted R 2 and predicted R 2 of 0.9977 (0.9977) and 0.9908, respectively, which again suggests superb fit with no overfitting. A non-significant lack-of-fit (p = 0.9977) further confirmed model adequacy. The overall model was significant (F = 695.10; p < 0.0001). As revealed by ANOVA (Table 7), GMO (A; F = 1751.68, p < 0.0001) and Soluplus® (B; F = 1651.32, p < 0.0001) were the most significant factors contributing to the outcome. The interaction term AB (F = 19.11; p = 0.0222) and quadratic term A 2 (F = 53.37; p = 0.0053) were significant, while B 2 was non-significant (p = 0.8972), suggesting a primarily linear effect of Soluplus® over the range studied. The final polynomial equation for Y 1 was acquired as follows: Y 1 = 67.8667 + 8.6A + 8.35B + 1.1AB − 2.6A 2 − 0.05B 2 A (+8.6) and B (+8.35) contribute as positive linear coefficients, confirming the individual effects of both factors on corneal permeation, while a positive interaction term (1.1) reveals synergistic cooperation in enhancing corneal permeation between GMO and Soluplus®. The negative coefficient for A 2 (−2.6) indicates a diminishing return at high levels of GMO. Cumulative permeation ranged from 49.7% (PF1) to 83.1% (PF9) (Table 4). The response surface illustrated an upward curvilinear profile towards the high-GMO/high-Soluplus® quadrant (Figure 6A), while contour plots (Figure 6B) reaffirmed that elevated concentrations of both factors produced an elliptical permeation maximized zone. Response 2: Precursor Viscosity (Y 2 ) The linear model was KM's best fit for Y 2 (adjusted R 2 = 0.9954, predicted R 2 = 0.9923) and also confirmed strong linearity and predictive reliability in Table 8. The model adequacy was confirmed by the non-significant lack-of-fit and overall model significance (F = 863.75; p < 0.0001) (Table 7). Decreasing GMO concentration The concentration of GMO (A), which was the most important parameter (F = 1161.83; p < 0.0001; SS = 5599.81), explained approximately twice as much variance as Soluplus® (B, F = 565.67; p < 0.0001; SS = 2726.40). Neither interaction nor quadratic terms were found to be significant (2FI: p = 0.7655; quadratic: p = 0.7590), thus verifying the adequacy of the linear model. The polynomial equation for Y 2 with respect to all variables was: Y 2 = 98.4778 + 30.55A + 21.3167B The stronger coefficient for A (+30.55) than B (+21.32) thus supports quantitatively the increased contribution of GMO to development of viscosity, which may be due to its role as a primary lipid forming around matrix forming units. The contour plots (Figure 6) exhibited parallel linear isoresponse lines along the GMO axis predominantly, while the response surface plot indicated a uniformly increasing plane from 48.3 mPa·s (PF1) to 148.3 mPa·s (PF9), as described in Table 5. All formulations were within the acceptable instillability range of ≤ 150 mPa·s. Table 7: ANOVA Summary for Coneal Permeation at 8 hr and viscosity Source Sum of Squares df Mean Square F-value p-value Corneal Permeation at 8 h (Y 1 ) Model 880.46 5 176.09 695.10 < 0.0001 significant A-GMO 443.76 1 443.76 1751.68 < 0.0001 B-Soluplus 418.34 1 418.34 1651.32 < 0.0001 AB 4.84 1 4.84 19.11 0.0222 A² 13.52 1 13.52 53.37 0.0053 B² 0.0050 1 0.0050 0.0197 0.8972 Residual 0.7600 3 0.2533 Cor Total 881.22 8 Viscosity (Y 2 ) Model 8326.22 2 4163.11 863.75 < 0.0001 significant A-GMO 5599.81 1 5599.81 1161.83 < 0.0001 B-Soluplus 2726.40 1 2726.40 565.67 < 0.0001 Residual 28.92 6 4.82 Cor Total 8355.14 8 Table 8: Model Fit Summary for Coneal Permeation at 8 hr and viscosity Source Sequential p-value Lack of Fit p-value Adjusted R² Predicted R² Corneal Permeation at 8 h (Y 1 ) Linear < 0.0001 - 0.9711 0.9503 2FI 0.2498 - 0.9741 0.9449 Quadratic 0.0123 - 0.9977 0.9908 Suggested Cubic 0.5735 - 0.9977 0.9483 Aliased Viscosity (Y 2 ) Linear < 0.0001 - 0.9954 0.9923 Suggested 2FI 0.7655 - 0.9946 0.9858 Quadratic 0.7590 - 0.9925 0.9665 Cubic 0.2642 - 0.9984 0.9641 Aliased Validation of Statistical Model To confirm the predictions of statistics modelling, the optimized formulation PF6 selected by maximum desirability function (D = 1) was prepared and evaluated. The experimental observed values were close to the predicted ones from Design Expert® for corneal drug permeation (76.6%) and viscosity (121.4 mPa·s) shown in Table 9 were as follows: predicted value of 78.318% and 122.808 mPa·s, respectively. The 2.44% and 1.15% relative errors observed for corneal permeation and viscosity, respectively, which are considerably lower than the admissible level of ±5%, showed that the developed full factorial design model had a high predictive capability. Table 9: Validation of the optimized puerarin-loaded in situ liquid crystal gel formulation (PF6) Batch Response Predicted Value Experimental Value % Relative Error Desirability PF6 Corneal drug Permeation 78.318 76.6 2.44 1 Viscosity 122.808 121.4 1.15 HET-CAM Ocular Irritation Study Performance of the optimized formulation PF6 was assessed against ocular irritation using HET-CAM and results are provided in Table 10. PF6 was completely free from hemorrhage, vascular lysis and coagulation on the chorioallantoic membrane at all timepoints during the 300-second observation period with an irritation score of 0.38 ± 0.04 and classified as non-irritant (IS < 0.9). IS of 0.00 was observed for the negative control (0.9% NaCl) and IS of 12.6 ± 0.8 was observed for positive control (1% SLS), indicating validity of assay. These results confirm the ocular safety and tolerability of PF6 for eye use. Table 10: HET-CAM ocular irritation scores of optimized puerarin-loaded in situ liquid crystal gel formulation (PF6) Sample Hemorrhage Vascular Lysis Coagulation Irritation Score (IS) Inference PF6 Absent Absent Absent 0.38 ± 0.04 Non-irritant Negative Control (0.9% NaCl) Absent Absent Absent 0.00 ± 0.00 Non-irritant Positive Control (1% SLS) Present Present Present 12.6 ± 0.8 Severe irritant All values expressed as mean ± SD (n = 3); IS 9.0 = severe irritant. In Vitro Cytotoxicity (MTT Assay) As shown in Table 11 and Figure 7, the cytotoxic effects of free puerarin and optimized formulation PF6 on HCE-T cells (Figure 8) at a concentration range of 1–200 µg/mL. Cell viability was reduced in a concentration-dependent manner both by free puerarin and PF6. PF6 showed 91% cell viability up to a concentration of 50 µg /mL, demonstrating very good cytocompatibility at therapeutically relevant concentrations. The IC 50 of PF6 (182.4 ± 4.1 µg/mL) was significantly higher than free puerarin (148.6 ± 3.2 µg/mL), confirming that encapsulation in the liquid crystal matrix decreased cytotoxicity to corneal epithelial cells, establishing the safety profile of ocular application for optimized formulation. Table 11: In vitro cytotoxicity (MTT assay) of optimized puerarin-loaded in situ liquid crystal gel formulation (PF6) and free puerarin on HCE-T cells Concentration (µg/mL) Cell Viability Free Puerarin (%) Cell ViabilityPF6 (%) 1 99.2 ± 1.1 99.6 ± 0.9 5 98.4 ± 1.3 99.1 ± 1.0 10 96.8 ± 1.4 98.2 ± 1.2 25 93.2 ± 1.6 96.4 ± 1.3 50 87.4 ± 1.8 91.8 ± 1.5 100 74.6 ± 2.1 82.3 ± 1.8 200 58.3 ± 2.4 68.7 ± 2.2 IC 50 (µg/mL) 148.6 ± 3.2 182.4 ± 4.1 All values expressed as mean ± SD (n = 3) Stability Study Table 12 displays the three-month stability data of optimized formulation PF6 at an accelerated condition, room temperature and freezing conditions. PF6 retained the viscous opalescent character under all storage conditions without any phase separation or precipitation. Under accelerated conditions (98.2% to 96.4%) the greatest variation in drug content was not exceeded, while low variations of only 0.81% and freezing condition of 0.41%. The physical and chemical stability were evident as all systems had pH, viscosity, entrapment efficiency, and corneal permeation at 8 h with % of deviations remaining much less than 3.5% through out the study period confirming stability for PF6 over a duration of three months under all storage conditions tested as per ICH Q1A(R2) guidelines. Table 12: Results of stability study of optimized puerarin-loaded in situ liquid crystal gel formulation (PF6) over 3 months Parameter Initial (Day 0) 1 Month 2 Months 3 Months Accelerated stability (40°C ± 2°C / 75% RH) Physical Appearance Opalescent, viscous liquid Opalescent, viscous liquid Opalescent, viscous liquid Opalescent, viscous liquid pH 7.16 ± 0.06 7.14 ± 0.05 7.12 ± 0.06 7.10 ± 0.07 Viscosity (mPa·s) 121.4 ± 3.7 122.1 ± 3.9 123.4 ± 4.1 124.6 ± 4.3 Drug Content (%) 98.2 ± 0.6 97.8 ± 0.7 97.2 ± 0.8 96.4 ± 0.9 Entrapment Efficiency (%) 92.1 ± 1.4 91.6 ± 1.5 91.0 ± 1.6 90.4 ± 1.7 Corneal Permeation at 8 h (%) 76.4 ± 2.43 75.8 ± 2.51 75.1 ± 2.58 74.6 ± 2.64 Room temperature stability (25°C ± 2°C / 60% RH) Physical Appearance Opalescent, viscous liquid Opalescent, viscous liquid Opalescent, viscous liquid Opalescent, viscous liquid pH 7.16 ± 0.06 7.15 ± 0.05 7.14 ± 0.06 7.13 ± 0.06 Viscosity (mPa·s) 121.4 ± 3.7 121.8 ± 3.8 122.3 ± 3.9 122.9 ± 4.0 Drug Content (%) 98.2 ± 0.6 98.0 ± 0.6 97.7 ± 0.7 97.4 ± 0.8 Entrapment Efficiency (%) 92.1 ± 1.4 91.9 ± 1.4 91.6 ± 1.5 91.3 ± 1.5 Corneal Permeation at 8 h (%) 76.4 ± 2.43 76.1 ± 2.46 75.8 ± 2.49 75.4 ± 2.53 Freezing condition stability (-20°C ± 2°C) Physical Appearance Opalescent, viscous liquid Opalescent, viscous liquid Opalescent, viscous liquid Opalescent, viscous liquid pH 7.16 ± 0.06 7.16 ± 0.06 7.15 ± 0.06 7.15 ± 0.07 Viscosity (mPa·s) 121.4 ± 3.7 121.6 ± 3.7 121.9 ± 3.8 122.2 ± 3.9 Drug Content (%) 98.2 ± 0.6 98.1 ± 0.6 97.9 ± 0.7 97.8 ± 0.7 Entrapment Efficiency (%) 92.1 ± 1.4 92.0 ± 1.4 91.9 ± 1.4 91.8 ± 1.5 Corneal Permeation at 8 h (%) 76.4 ± 2.43 76.3 ± 2.44 76.1 ± 2.46 75.9 ± 2.48 All values expressed as mean ± SD (n = 3) DISCUSSION Using a 3 2 full factorial design, the current study successfully developed and optimized puerarin-loaded in situ liquid crystal gels for extended ocular delivery of dry eye disease. Since puerarin has a low aqueous solubility (0.82 ± 0.15 mg/mL in distilled water; Table 3) and poor solubility in simulated tear fluid (7.35 ± 0.32 mg/mL), it was incorporated into a lipid-based liquid crystalline carrier, in line with previous reports recommending GMO-based systems for poorly water-soluble drugs[40]. DSC analysis (Figure 2) indicated a decrease in crystallinity in the physical mixture of puerarin, FTIR spectra (Figure 3) reflected retention of all significant functional group peaks with minor shift and overall confirmed physicochemical compatibility between puerarin and excipients which were similar to findings reported by Lai et al. for GMO based ocular liquid crystal systems [41]. All formulations (PF1, PF5–PF9) possessed acceptable pH (7.14 to 7.26), drug content (96.4 to 98.6%), and entrapment efficiency (82.3% to 94.3%) as shown in Table 5), in accordance with quality specifications set for ophthalmic nanostructured formulations [42]. This progressive increase in viscosity, gelling capacity and gelation strength with increasing concentrations of GMO and Soluplus® (Table 5) is consistent with previous findings reported by Tarsitano et al. (2019), who showed that increased lipid concentration leads to more ordered liquid crystalline mesophases and superior mechanical properties [43]. Y 1 = 67.8667 + 8.6A + 8.35B + 1.1AB − 2.6A 2 − 0.05B 2 [quadratic polynomial] model for corneal permeation and Y 2 = 98.4778 +30.55A +21.3167B [linear] model of viscosity, with adjusted R 2 values of coefficient of determination (R 2 ) were determined to be the best fit for the experimental data at values of: Y1=0.9977 and Y2=0.9954 respectively, yielding a validation percentage relative error that was less than 2 in both instances confirming excellent predictability (Table 9), confirming validation in agreement with studies based on factorial design ocular therapeutics[44]. Ex vivo corneal permeation studies indicated a linear increase in cumulative permeation from 49.7% (PF1) to 83.1% (PF9) after 8 h of diffusion time (Figure 4), with the optimized formulation PF6 having the highest flux value of 52.34 ± 1.78 µg/cm 2 /h and Kp value of 5.23 × 10 -3 cm/h (Table 6), significantly better than conventional aqueous eye drop systems reported in literature [45]. This relevant permeation increase could be ascribed to the bioadhesive liquid crystalline mesophase produced upon lacrimal dilution, which has a prolonged precorneal residence time and leads to intimate interaction with corneal epithelium like that described for GMO-based cubic phase systems [46]. Kinetic Analysis of Drug Release from PF6 (Figure 5) showed better fit with Hixson-Crowell model (R 2 = 0.9997) with Korsmeyer–Peppas n value of 0.8798 indicating non-Fickian anomalous transport, which implies simultaneous diffusion and matrix erosion mechanisms, a drug release pattern characteristic of liquid crystalline systems as described by Chen et al. [47]. The non-irritant characteristics of PF6 were further validated by the HET-CAM assay in which it showed an irritation score of 0.38 ± 0.04 (Table 10), comparable with those developed for puerarin-loaded proniosomal gels (IS = 0.43) [48], while superior cytocompatibility to free puerarin (IC 50 = 148.6 ± 3.2 µg/mL) on HCE-T cells was achieved by MTT assay using PF6 at IC50 at a dose approximately >fourfold higher than the respective value as calculated from log dose versus cell viability profiles, as shown in Table 11 and Figure 7, a phenomenon that is consistent with encapsulation-induced cytoprotection known so far for lipid-based ocular nanocarriers [48,49]. As indicated by the stability results summary in Table 12, PF6 remained physically and chemically stable under all tested conditions with a maximum drug content variation of only 1.83% at the accelerated storage scenario, significantly lower than ICH Q1A(R2) guidelines for clinical translation suitability [50]. CONCLUSION This study focused on the formulation of puerarin-loaded in situ liquid crystal gels for sustained ocular delivery in dry eye disease, which were successfully developed and optimized by using a 3 2 full factorial design. Using the solubility and optical transparency of the transformed ingredients, an optimal formula was identified as PF6 with 60% w/w GMO and 15% w/w Soluplus®, which yielded favourable physicochemical properties, corneal permeation (76.4 ± 2.43% at 8 h) superior to other formulations, long term drug release through a non-Fickian anomalous transport mechanism as well as excellent ocular safety in both HET-CAM and MTT assays. This combination showed a desirability of 1.0 with percentage relative errors below 2.5%, thus confirming the robustness of the optimization strategy. Stability studies established physical and chemical integrity over three months under various storage conditions. PF6 is clinically advantageous due to low-frequency dosing, sustained precorneal residence time and better patient compliance than conventional eye drops. In vivo pharmacokinetic and pharmacodynamic studies in dry eye models are now necessary to confirm the developed formulation's therapeutic efficacy, as well as its clinical translation potential. Abbreviations DED: Dry Eye Disease; LCG: Liquid Crystal Gel; GMO: Glyceryl Monooleate; STF: Simulated Tear Fluid; DSC: Differential Scanning Calorimetry; FTIR: Fourier Transform Infrared Spectroscopy; HET-CAM: Hen's Egg Test on Chorioallantoic Membrane; MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide; HCE-T: Human Corneal Epithelial Cell Line; IC 50 : Inhibitory Concentration 50%; EE: Entrapment Efficiency; PLM: Polarized Light Microscopy; ANOVA: Analysis of Variance; SD: Standard Deviation; ICH: International Council for Harmonisation; CPCSEA: Committee for the Purpose of Control and Supervision of Experiments on Animals; NF-κB: Nuclear Factor Kappa B; Kp: Permeability Coefficient; IS: Irritation Score; SLS: Sodium Lauryl Sulfate; w/w: Weight by Weight; λ max : Maximum Absorption Wavelength; RH: Relative Humidity; IP: Indian Pharmacopoeia. Declarations Author Contribution Pallavi Desale - Conducting experiments, Wrote main manuscript,Data Curation,Amol S.Rakte - Supervision, Methodology,Data analysis, statistics, Investigation, Review & Editing of manuscriptSanjay R.Arote -Visualization Finding This Research Received No External Funding Ethics Declaration Not Applicable References Harrell CR, Feulner L, Djonov V, Pavlovic D, Volarevic V. The Molecular Mechanisms Responsible for Tear Hyperosmolarity-Induced Pathological Changes in the Eyes of Dry Eye Disease Patients. Cells 2023;12:2755. https://doi.org/10.3390/cells12232755. Aragona P, Barabino S, Di Zazzo A, Giannaccare G, Villani E, Aiello F, et al. Dry Eye Disease: From Causes to Patient Care and Clinical Collaboration—A Narrative Review. Ophthalmol Ther 2025;14:1411–28. https://doi.org/10.1007/s40123-025-01161-8. Rolando M, Merayo-Lloves J. Management Strategies for Evaporative Dry Eye Disease and Future Perspective. Curr Eye Res 2022;47:813–23. https://doi.org/10.1080/02713683.2022.2039205. Sen H, Durgapal S, Jakhmola V. Molecular landscape connecting complications of dry eye disease: mechanisms, therapeutic targets, biomarkers, and future innovations. Int Ophthalmol 2026;46:97. https://doi.org/10.1007/s10792-026-03940-z. Conte R, De Luca I, Calarco A, Finicelli M, Peluso G. Polyphenol-Loaded Nanodevices as Innovative Therapeutic Strategies for Dry Eye Disease: Advances and Perspectives. Antioxidants 2025;14:1280. https://doi.org/10.3390/antiox14111280. Sheppard JD, Nichols KK. Dry Eye Disease Associated with Meibomian Gland Dysfunction: Focus on Tear Film Characteristics and the Therapeutic Landscape. Ophthalmol Ther 2023;12:1397–418. https://doi.org/10.1007/s40123-023-00669-1. Chauhan P, Wadhwa K, Mishra R, Gupta S, Ahmad F, Kamal M, et al. Investigating the Potential Therapeutic Mechanisms of Puerarin in Neurological Diseases. Mol Neurobiol 2024;61:10747–69. https://doi.org/10.1007/s12035-024-04222-4. Liga S, Paul C. Puerarin—A Promising Flavonoid: Biosynthesis, Extraction Methods, Analytical Techniques, and Biological Effects. Int J Mol Sci 2024;25:5222. https://doi.org/10.3390/ijms25105222. Yu C-C, Du Y-J, Li J, Li Y, Wang L, Kong L-H, et al. Neuroprotective Mechanisms of Puerarin in Central Nervous System Diseases: Update. Aging Dis 2022;13:1092–105. https://doi.org/10.14336/AD.2021.1205. Zhang N, Guo P, Zhao Y, Qiu X, Shao S, Liu Z, et al. Pharmacological mechanisms of puerarin in the treatment of Parkinson’s disease: An overview. Biomed Pharmacother 2024;177:117101. https://doi.org/10.1016/j.biopha.2024.117101. Saklani R, Yadav PK, Nengroo MA, Gawali SL, Hassan PA, Datta D, et al. An Injectable In Situ Depot-Forming Lipidic Lyotropic Liquid Crystal System for Localized Intratumoral Drug Delivery. Mol Pharm 2022;19:831–42. https://doi.org/10.1021/acs.molpharmaceut.1c00782. Lotfy NM, Ahmed MA, El Hoffy NM, Bendas ER, Morsi NM. Development and optimization of amphiphilic self-assembly into nanostructured liquid crystals for transdermal delivery of an antidiabetic SGLT2 inhibitor. Drug Deliv 2022;29:3340–57. https://doi.org/10.1080/10717544.2022.2144546. Rajabi N, Scarfo MG, Fredericks CM, Herrera Restrepo RS, Adibi A, Shahsavan H. From Anisotropic Molecules and Particles to Small-Scale Actuators and Robots: An Account of Polymerized Liquid Crystals. Acc Mater Res 2024;5:1520–31. https://doi.org/10.1021/accountsmr.4c00187. Wang M, Huang X, Yang H. Photothermal-Responsive Crosslinked Liquid Crystal Polymers. Macromol Mater Eng 2023;308:2300061. https://doi.org/10.1002/mame.202300061. Hooli S, Sutar KP, Sammasagi SS, Chouhan MK. Quality-by-Design-Driven Stability-Indicating Reverse-Phase High-Performance Liquid Chromatographic Method for Puerarin Quantification in Marketed Formulation and Lipid–Polymer Nanoparticles. Chromatographia 2025;88:743–57. https://doi.org/10.1007/s10337-025-04427-2. Rahimpour E, Moradi M, Sheikhi-Sovari A, Rezaei H, Rezaei H, Jouyban-Gharamaleki V, et al. Comparative Drug Solubility Studies Using Shake-Flask Versus a Laser-Based Robotic Method. AAPS PharmSciTech 2023;24:207. https://doi.org/10.1208/s12249-023-02667-9. Ghanbari E, Picken SJ, van Esch JH. Analysis of differential scanning calorimetry (DSC): determining the transition temperatures, and enthalpy and heat capacity changes in multicomponent systems by analytical model fitting. J Therm Anal Calorim 2023;148:12393–409. https://doi.org/10.1007/s10973-023-12356-1. Lynch JM, Corniuk RN, Brignac KC, Jung MR, Sellona K, Marchiani J, et al. Differential scanning calorimetry (DSC): An important tool for polymer identification and characterization of plastic marine debris. Environ Pollut 2024;346:123607. https://doi.org/10.1016/j.envpol.2024.123607. Jozanikohan G, Abarghooei MN. The Fourier transform infrared spectroscopy (FTIR) analysis for the clay mineralogy studies in a clastic reservoir. J Pet Explor Prod Technol 2022;12:2093–106. https://doi.org/10.1007/s13202-021-01449-y. Montoya-Escobar N, Ospina-Acero D, Velásquez-Cock JA, Gómez-Hoyos C, Serpa Guerra A, Gañan Rojo PF, et al. Use of Fourier Series in X-ray Diffraction (XRD) Analysis and Fourier-Transform Infrared Spectroscopy (FTIR) for Estimation of Crystallinity in Cellulose from Different Sources. Polymers 2022;14:5199. https://doi.org/10.3390/polym14235199. Hu L, Xu Y, Meng H. Development and Evaluation of Puerarin Loaded-Albumin Nanoparticles Thermoresponsive in situ Gel for Ophthalmic Delivery. Drug Des Devel Ther 2022;16:3315–26. https://doi.org/10.2147/DDDT.S374061. Liga S, Tămaș A, Vodă R, Rusu G, Bîtcan I, Socoliuc V, et al. Puerarin-Loaded Proniosomal Gel: Formulation, Characterization, In Vitro Antimelanoma Cytotoxic Potential, and In Ovo Irritation Assessment. Gels 2026;12:72. https://doi.org/10.3390/gels12010072. Polat HK, Ünal S, Aytekin E, Karakuyu NF, Pezik E, Haydar MK, et al. Formulation development of Lornoxicam loaded heat triggered ocular in-situ gel using factorial design. Drug Dev Ind Pharm 2023;49:601–15. https://doi.org/10.1080/03639045.2023.2264932. Kassab HJ, Alkufi HK, Hussein LS. Use of factorial design in formulation and evaluation of intrarectal in situ gel of sumatriptan. J Adv Pharm Technol Res 2023;14:119. https://doi.org/10.4103/japtr.japtr_603_22. Hani U, Rahamathulla M, Osmani RAM, Begum MY, Wahab S, Ghazwani M, et al. Development and Characterization of Oral Raft Forming In Situ Gelling System of Neratinib Anticancer Drug Using 32 Factorial Design. Polymers 2022;14:2520. https://doi.org/10.3390/polym14132520. Szalai B, Jójárt-Laczkovich O, Kovács A, Berkó S, Balogh GT, Katona G, et al. Design and Optimization of In Situ Gelling Mucoadhesive Eye Drops Containing Dexamethasone. Gels 2022;8:561. https://doi.org/10.3390/gels8090561. Bondre RM, Kanojiya PS, Wadetwar RN, Kangali PS. Sustained vaginal delivery of in situ gel containing Voriconazole nanostructured lipid carrier: formulation, in vitro and ex vivo evaluation. J Dispers Sci Technol 2023;44:1466–78. https://doi.org/10.1080/01932691.2021.2022489. Ibrahim TM, Ayoub MM, El-Bassossy HM, El-Nahas HM, Gomaa E. Investigation of Alogliptin-Loaded In Situ Gel Implants by 23 Factorial Design with Glycemic Assessment in Rats. Pharmaceutics 2022;14:1867. https://doi.org/10.3390/pharmaceutics14091867. Talianu M-T, Dinu-Pîrvu C-E, Ghica MV, Anuţa V, Prisada RM, Popa L. Development and Characterization of New Miconazole-Based Microemulsions for Buccal Delivery by Implementing a Full Factorial Design Modeling. Pharmaceutics 2024;16:271. https://doi.org/10.3390/pharmaceutics16020271. Gowda CM, Wairkar S. Azelaic acid-based lyotropic liquid crystals gel for acne vulgaris: Formulation optimization, antimicrobial activity and dermatopharmacokinetic study. Int J Pharm 2024;667:124879. https://doi.org/10.1016/j.ijpharm.2024.124879. Priya S, Srividya G, Chandra NS, Singh PP, Saha RN, Sathe P, et al. Loteprednol etabonate loaded lyotropic liquid crystalline nanoparticles in-situ ophthalmic gel: Qbd driven optimization and in-vitro, ex-vivo evidence of sustained precorneal residence time. J Drug Deliv Sci Technol 2023;89:105081. https://doi.org/10.1016/j.jddst.2023.105081. Rapalli VK, Tomar Y, Sharma S, Roy A, Singhvi G. Apremilast loaded lyotropic liquid crystalline nanoparticles embedded hydrogel for improved permeation and skin retention: An effective approach for psoriasis treatment. Biomed Pharmacother 2023;162:114634. https://doi.org/10.1016/j.biopha.2023.114634. Imane M, Rachid D, Habib BM. Assessment of ocular irritation potential of moisturizing and sunscreen cosmetic creams marketed in Algeria using the HET-CAM test. Toxicol In Vitro 2026;111:106168. https://doi.org/10.1016/j.tiv.2025.106168. de Araujo Lowndes Viera LM, Silva RS, da Silva CC, Presgrave OAF, Boas MHSV. Comparison of the different protocols of the Hen’s Egg Test-Chorioallantoic Membrane (HET-CAM) by evaluating the eye irritation potential of surfactants. Toxicol In Vitro 2022;78:105255. https://doi.org/10.1016/j.tiv.2021.105255. Idogwu EI, Kazarova S, Kormos É. Using the HET-CAM test in determining the eye irritation potential of some pesticides. GEORGIKON Agric 2023;27:84–91. Corrêa G de OP, Marcato DC, Ramos WS, Corrêa MA, Cicarelli RMB, Isaac VLB. In vitro evaluation of the cytotoxicity and eye irritation potential of preservatives widely used in cosmetics. Braz J Pharm Sci 2022;58:e20039. https://doi.org/https://doi.org/10.1590/s2175-97902022e20039. Zheng J, Zhang Y, Zhang S. Sustained release of azithromycin from lipid liquid-crystalline nanoparticles laden in situ gel for the treatment of periodontitis: In vitro and efficacy study. J Biomater Appl 2022;37:482–92. https://doi.org/10.1177/08853282221095395. Yuan D, Li X, Yao H, Li Y, Zhu X, Zhao J, et al. A Liquid Crystal Ionomer-Type Electrolyte toward Ordering-Induced Regulation for Highly Reversible Zinc Ion Battery. Adv Sci 2023;10:2206469. https://doi.org/10.1002/advs.202206469. Kato T, Uchida J, Ishii Y, Watanabe G. Aquatic Functional Liquid Crystals: Design, Functionalization, and Molecular Simulation. Adv Sci 2024;11:2306529. https://doi.org/10.1002/advs.202306529. What Is Glyceryl Monooleate and Why Is It Used in Pharmaceuticals? - Alfa Chemistry n.d. https://www.alfa-chemistry.com/resources/what-is-glyceryl-monooleate-and-why-is-it-used-in-pharmaceuticals.html (accessed March 20, 2026). Lai J, Chen J, Lu Y, Sun J, Hu F, Yin Z, et al. Glyceryl monooleate/poloxamer 407 cubic nanoparticles as oral drug delivery systems: I. In vitro evaluation and enhanced oral bioavailability of the poorly water-soluble drug simvastatin. AAPS PharmSciTech 2009;10:960–6. https://doi.org/10.1208/s12249-009-9292-4. Chen Y, Lu Y, Zhong Y, Wang Q, Wu W, Gao S. Ocular delivery of cyclosporine A based on glyceryl monooleate/poloxamer 407 liquid crystalline nanoparticles: preparation, characterization, in vitro corneal penetration and ocular irritation. J Drug Target 2012;20:856–63. https://doi.org/10.3109/1061186X.2012.723214. Tarsitano M, Mancuso A, Cristiano MC, Urbanek K, Torella D, Paolino D, et al. Perspective use of bio-adhesive liquid crystals as ophthalmic drug delivery systems. Sci Rep 2023;13:16188. https://doi.org/10.1038/s41598-023-42185-z. Aboali FA, Habib DA, Elbedaiwy HM, Farid RM. Curcumin-loaded proniosomal gel as a biofreindly alternative for treatment of ocular inflammation: In-vitro and in-vivo assessment. Int J Pharm 2020;589:119835. https://doi.org/10.1016/j.ijpharm.2020.119835. Kouchak M, Mahmoodzadeh M, Farrahi F. Designing of a pH-Triggered Carbopol®/HPMC In Situ Gel for Ocular Delivery of Dorzolamide HCl: In Vitro, In Vivo, and Ex Vivo Evaluation. AAPS PharmSciTech 2019;20:210. https://doi.org/10.1208/s12249-019-1431-y. Gonjari ID, Karmarkar AB, Khade TS, Hosmani AH, Navale RB. Use of factorial design in formulation and evaluation of ophthalmic gels of gatifloxacin: Comparison of different mucoadhesive polymers. Drug Discov Ther 2010;4:423–34. Chen Y, Lu Y, Zhong Y, Wang Q, Wu W, Gao S. Ocular delivery of cyclosporine A based on glyceryl monooleate/poloxamer 407 liquid crystalline nanoparticles: preparation, characterization, in vitro corneal penetration and ocular irritation. J Drug Target 2012;20:856–63. https://doi.org/10.3109/1061186X.2012.723214. Otte A, Báez-Santos YM, Mun EA, Soh B-K, Lee Y, Park K. The in vivo transformation and pharmacokinetic properties of a liquid crystalline drug delivery system. Int J Pharm 2017;532:345–51. https://doi.org/10.1016/j.ijpharm.2017.08.098. Gilleron L, Coecke S, Sysmans M, Hansen E, Van Oproy S, Marzin D, et al. Evaluation of a modified HET-CAM assay as a screening test for eye irritancy. Toxicol In Vitro 1996;10:431–46. https://doi.org/10.1016/0887-2333(96)00021-5. Liga S, Tămaș A, Vodă R, Rusu G, Bîtcan I, Socoliuc V, et al. Puerarin-Loaded Proniosomal Gel: Formulation, Characterization, In Vitro Antimelanoma Cytotoxic Potential, and In Ovo Irritation Assessment. Gels 2026;12:72. https://doi.org/10.3390/gels12010072. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9256829","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":617557343,"identity":"c5a966b9-2ea7-45a5-8c2c-a43f7bbc783d","order_by":0,"name":"Pallavi Desale","email":"","orcid":"","institution":"Indryani Vidya Mandirs Krishnarao Bhegade Institute of Pharmceutical Education and Research, Talegaon Dabhade, Tal-Maval, Pune-410507, Maharashtra","correspondingAuthor":false,"prefix":"","firstName":"Pallavi","middleName":"","lastName":"Desale","suffix":""},{"id":617557344,"identity":"42477a44-aaec-45ed-a952-4f055723eff7","order_by":1,"name":"Amol S. Rakte","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYHACxgNAwoCBgRlES8gQpQeshYeBLQGkhYcULTwGIA5hLfzTDh84+HOPnbE9e8/nVzdqLHgY2A8f3YBPi8TttITDPM+SzXh4zm6zzjkGdBhPWtoNvNbczjE4zHCA2YZHInebcQ4bUIsEjxleLfJALQd/HKi34ZF/88w45x8RWgyAWg7wHDhsxiPBw/w4t40ILYZgvxw4bsxzJs2MObdPgoeNkF/kbicffPjjQLVhe/vhx59zvtXJ8bMfPobf+0iATQJMEqscBJg/kKJ6FIyCUTAKRg4AAMxqSCcTq72sAAAAAElFTkSuQmCC","orcid":"","institution":"Indryani Vidya Mandirs Krishnarao Bhegade Institute of Pharmceutical Education and Research, Talegaon Dabhade, Tal-Maval, Pune-410507, Maharashtra","correspondingAuthor":true,"prefix":"","firstName":"Amol","middleName":"S.","lastName":"Rakte","suffix":""},{"id":617557345,"identity":"013e5392-ff57-4ebd-ba94-327a0e586f5f","order_by":2,"name":"Sanjay R. Arote","email":"","orcid":"","institution":"Indryani Vidya Mandirs Krishnarao Bhegade Institute of Pharmceutical Education and Research, Talegaon Dabhade, Tal-Maval, Pune-410507, Maharashtra","correspondingAuthor":false,"prefix":"","firstName":"Sanjay","middleName":"R.","lastName":"Arote","suffix":""}],"badges":[],"createdAt":"2026-03-29 06:38:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9256829/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9256829/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106290741,"identity":"2587d3c9-778b-4a10-9af0-3e824dc7311f","added_by":"auto","created_at":"2026-04-07 07:47:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":43695,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCalibration curve of Puerarin in ethanol\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9256829/v1/ecf85eb82acf78050c5b08d1.png"},{"id":106290747,"identity":"013a7f58-a34b-4e69-b9ad-e1603bb78afa","added_by":"auto","created_at":"2026-04-07 07:47:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":206822,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDSC Spectra of (A) Pure Puerarin and (B) Physical Mixture (Drug + Excipients)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9256829/v1/717455c8ea6fec07f25d74a8.png"},{"id":106403618,"identity":"d42801e0-e1af-4f00-9263-7a017be4d3cc","added_by":"auto","created_at":"2026-04-08 09:14:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":155946,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFTIR Spectra of (A) Pure Drug and (B) Physical Mixture\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9256829/v1/3a941ee83bc72f430687a073.png"},{"id":106290745,"identity":"e9489758-b139-4fe2-9417-0a1f172d747e","added_by":"auto","created_at":"2026-04-07 07:47:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":73002,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEx Vivo Corneal Permeation Profile of all Batches PF1-PF9\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9256829/v1/e93220ca307b61beb95585d5.png"},{"id":106290742,"identity":"c976b72e-1da6-449e-8e31-24cdf468ecdf","added_by":"auto","created_at":"2026-04-07 07:47:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":203811,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDrug release kinetic model fitting plots for the optimized puerarin-loaded in situ liquid crystal gel formulation (PF6): (A) Zero order, (B) First order, (C) Higuchi, (D) Hixson–Crowell, and (E) Korsmeyer–Peppas.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9256829/v1/e3da18a3982dbc21274878fd.png"},{"id":106290748,"identity":"bb36c65a-bdc2-49ac-9b05-3ec6b9dea549","added_by":"auto","created_at":"2026-04-07 07:47:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":426170,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eContour plot (A) and three-dimensional response surface plot (B) showing the effect of glyceryl monooleate concentration and Soluplus® concentration on cumulative corneal permeation at 8 h (Y\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e, %); contour plot (C) and three-dimensional response surface plot (D) showing the effect of glyceryl monooleate concentration and Soluplus® concentrationon precursor viscosity (Y\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e, mPa·s) of puerarin-loaded in situ liquid crystal gel formulations.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9256829/v1/9878b1a042c19d70e539358c.png"},{"id":106290744,"identity":"f271956e-a674-44d5-bb38-aa874e8b1ba2","added_by":"auto","created_at":"2026-04-07 07:47:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":21748,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vitro cytotoxicity of free puerarin and optimized puerarin-loaded in situ liquid crystal gel formulation (PF6) on human corneal epithelial cells (HCE-T) evaluated by MTT assay at concentrations ranging from 1 to 200 µg/mL after 24 hours of exposure.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9256829/v1/0ac92d2acf003928a01df89f.png"},{"id":106290746,"identity":"5ab24f8a-c92c-4845-a25e-41730aa71c19","added_by":"auto","created_at":"2026-04-07 07:47:10","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":659939,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhase-contrast microscopic images of HCE-T cells after 24-hour treatment: (A) untreated control showing intact confluent monolayer; (B) free puerarin-treated cells exhibiting morphological alterations; (C) PF6-treated cells demonstrating comparatively preserved cellular integrity. Black arrows indicate areas of morphological changes. Scale bar = 100 µm.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-9256829/v1/c276c2cc8943d24d5b5b16e6.png"},{"id":109202768,"identity":"ad235a0e-eb07-40e6-8d54-191aed8ebd7a","added_by":"auto","created_at":"2026-05-13 14:16:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2326919,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9256829/v1/9420499a-c144-4df0-860c-08917f894369.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development and Optimization of Puerarin-loaded in situ liquid crystal gels for ocular delivery in dry eye disease","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eNecessitating the need for a treatment strategy in patients suffering from dry eye disease (DED), a multifactorial ocular surface disorder with tear film instability and hyperosmolarity and chronic ocular surface inflammation, affecting an estimated 344\u0026nbsp;million individuals worldwide[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Its prevalence in different populations varies from 5% to 50%, with an increased incidence among older adults, postmenopausal women, and patients using digital devices for prolonged periods[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Beyond discomfort, the global burden of DED includes substantial visual impairment, decreased quality of life, and loss of workplace productivity[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The economic burden is high, with annual health care costs of more than USD 3.84\u0026nbsp;billion in the United States alone, as well as increased expenses due to recurrent clinic visits, costly diagnostic tests, and chronic therapeutic management[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Although lubricating eye drops, cyclosporine A emulsions and lifitegrast solutions are widely available pharmacotherapy, remain limited by poor patient compliance, transient symptom relief, suboptimal corneal bioavailability and need for frequent dosing[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. When a conventional ophthalmic formulation is instilled into the eye, most of it drains within minutes through the nasolacrimal duct; only 1\u0026ndash;5% reaches its target site due to ocular bioavailability. Although DED encompasses a wide spectrum from mild to severe, the worldwide increase of screen time, aging populations and environmental pollution are expected to increase DED prevalence in the near future that drives an urgent demand for new therapeutic strategies with efficacy and patient-friendliness[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePuerarin (4\u0026prime;,7-dihydroxyisoflavone-8-C-glucoside; molecular formula: C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e; MW: 416.38 g/mol) is a naturally occurring C-glycosylated isoflavone phytoestrogen primarily obtained from the roots of Pueraria lobata (Willd.) Ohwi[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The molecular architecture of puerarin includes hydroxyl groups at the C-4\u0026prime; and C-7 positions, as well as a glucose moiety at the C-8 site, conferring significant water solubility compared with other isoflavones[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Puerarin has highly relevant pleiotropic pharmacological actions in the context of DED pathophysiology, including potent antiinflammatory activity by inhibiting NF-κB and MAPK signaling pathways as well as decreasing pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), antioxidant activity via Nrf2 pathway activation; and stimulation of goblet cell proliferation to enhance mucin secretion. In preclinical studies, this agent has been shown to restore tear secretion and increase conjunctival goblet cell density while reducing corneal epithelial apoptosis in several experimental models of DED[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In addition, puerarin has a relatively favorable safety profile with low systemic toxicity, which can be regarded as an excellent candidate for treatment of the ocular surface. However, its clinical ocular use is limited due to rapid precorneal elimination, moderate corneal permeability and lack of suitable ophthalmic delivery platform for maintaining therapeutic concentrations at the ocular surface[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn situ liquid crystal gels (LCGs) are an advanced drug delivery platform based on a phase transition of a low-viscosity solution into a structured liquid crystalline mesophase stimulated by contact with physiological triggers such as components in tear fluid, temperature or dilution-induced phase transitions[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These nanostructured systems cubic, hexagonal, or lamellar mesophases are made of biocompatible lipid-based materials such as glyceryl monooleate, phytantriol and poloxamer-based block copolymers. These excipients are amphiphilic in nature which self-assemble on the formation of ordered nanoarchitectures bearing separate hydrophilic channels and hydrophobic domains providing sustained release for both drug types i.e., hydrophilic \u0026amp; hydrophobic drugs[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In situ LCGs provide many advantageous characteristics for ophthalmic delivery including extended precorneal retention time (\u0026gt;\u0026thinsp;4 hours), lower dosing frequency, improved corneal permeation via close bioadhesive contact with the cornea, protection of sensitive active agents from enzymatic degradation and good biocompatibility with ocular tissues. Recent innovations have shown that cubic and hexagonal mesophases greatly improve bioavailability of less permeable compounds without compromising ocular tolerability[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In addition, the liquid-like precursor state enables easy instillation as traditional eye drops, immediately changing into a viscous mucoadhesive depot upon lacrimal dilution. These features make in situ LCGs a theoretically sound and clinically practical carrier for extended ocular release of puerarin in the treatment of DED[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe current study focuses on the design and optimization of puerarin-loaded in situ liquid crystal gels for sustained ocular delivery to dry eye disease. Particular aims comprise physicochemical characterization and in vitro drug release profiling, as well as ex vivo corneal permeation studies and in vitro cytotoxicity evaluation. This study aims to develop a new ocular drug delivery system with better pharmacological properties and patient compliance.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eMATERIALS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePuerarin (purity: 98% and MW: 416.38 g/mol) was acquired at Sciquaint Innovations Pvt. ltd. (Pune, India). Soluplus 118,000 g/mol (MW) and glyceryl monooleate (pharmaceutical grade) were purchased at Sciquaint Chemicals (Pune, India). epoxy resin was purchased as propylene glycol (USP grade), whereas benzalkonium chloride was purchased as benzalkonium chloride (pharmaceutical grade), both obtained in Research Lab Fine Chem Industries (Mumbai, India). Ethanol, methanol and acetinitrile (all of HPLC grade) were bought at Neeta Chemicals (Pune, India). MTT reagent and dialysis membrane (cutoff 12,000-14 000Da) were obtained with Sciquaint Chemicals (Pune, India). Sodium lauryl sulfate (analytical grade) was bought in Research Lab Fine Chem Industries (Mumbai, India). All other chemicals and reagents were of analytical grade and not subjected to any additional purification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMETHODS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCalibration Curve of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePuerarin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe linear relationship between absorbance and puerarin (purity \u0026ge; 98%) concentration in ethanol was established by means of a calibration curve. 10 mL of puerarin was weighed out and dissolved in ethanol in a 10 mL volumetric flask to prepare the primary stock solution (1000 \u0026mu;g/mL), and six working standard solutions (5, 10, 15, 20, 25 and 30 \u0026mu;g/mL) were prepared by transferring aliquots of 0.05, 0.10, 0.15, 0.20, 0.25 and 0.30 mL of the stock solution into separate 10 mL volumetric flasks using an adjustable micropipette (Tarsons India) followed by dilution to volume with ethanol at ambient temperature (25 \u0026plusmn;2\u0026deg;C); absorbance values for each solution were measured at \u0026lambda;\u003csub\u003emax\u003c/sub\u003e against blank made from ethanol as solvent using a double-beam UV-visible spectrophotometer (Systronics UV-2202 India), employing quartz cuvettes of path length =1 cm. The mean absorbance was plotted against the concentration (\u0026mu;g/mL) to obtain a calibration graph in which the equation of linear regression analysis and correlation coefficient (r\u003csup\u003e2\u003c/sup\u003e) \u0026ge; 0.999 confirmed linearity as per ICH Q2(R1). All measurements were conducted in triplicate (n = 3) and presented as mean \u0026plusmn; SD[15].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSolubility Study of Puerarin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe solubility profile of puerarin in physiologically relevant and organic solvents was characterized to inform dosage form development. From this, an excess amount of puerarin (~50 mg) was suspended separately in 10 mL each of distilled water, phosphate buffer pH 7.4, simulated tear fluid (STF), artificial tear fluid, methanol, ethanol and acetonitrile inside a series of stoppered 25mL conical flasks that were capped tightly to minimize evaporation and placed on an orbital shaker (Remi Instruments; India) at 25 \u0026plusmn; 2\u0026deg;C and agitation at a speed of roughly 100 rpm for 24 hours to equilibrate saturation. After 30 min of settling followed by filtration through Whatman filter paper No. 41, the supernatant was appropriately diluted and the absorbance measured at \u0026lambda;max using double-beam UV-visible spectrophotometer (Systronics UV-2202, India) with quartz cuvettes of path length 1 cm. Solubility was determined from the regression equation y = mx + c and was expressed as mg/mL. All measurements were conducted in triplicate (n = 3) and reported as mean \u0026plusmn; SD[16].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDifferential Scanning Calorimetry (DSC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThermal investigation, crystallinity and drug-excipient compatibility of puerarin was studied using differential scanning calorimetry (DSC) instrument (Mettler Toledo DSC 822e; Switzerland). Samples of pure puerarin and its physical mixture with the excipients (3\u0026ndash;5 mg each) were accurately weighed and placed individually in standard aluminum pans, sealed hermetically with aluminum lids by a crimping press, and analyzed, using an empty sealed aluminum pan as reference. To avoid oxidative decomposition, samples were heated from 30\u0026deg;C to 300\u0026deg;C at a scanning rate of 10 \u0026deg;C/min under an inert nitrogen atmosphere and a purge gas flow rate of 20 mL/min. The thermograms obtained were examined for characteristic endothermic or exothermic peaks, onset temperature, peak temperature and changes in enthalpy (\u0026Delta;H), while shifts or disappearances of peaks were interpreted as a sign of drug-excipient interactions or changes in crystallinity. All assays were conducted in triplicate (n = 3) and values are expressed as mean \u0026plusmn; SD[17,18].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFourier Transform Infrared Spectroscopy (FTIR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunctional groups contained in puerarin and possible chemical interactions between the drug and excipients by changes in characteristic absorption bands were analyzed using infrared spectroscopy (IR). FTIR spectrophotometer (Shimadzu IRSpirit, Japan) was used to record the spectra using KBr pellet technique. Prior to use, KBr powder was dried for 2 hours at 40\u0026ndash;50\u0026deg;C to remove moisture. A reasonable amount of (~200 mg) dried KBr was triturated separately with 2\u0026ndash;3 mg pure puerarin and its physical mixture with excipients in clean mortar and pestle for 5\u0026ndash;10 minutes to make sure the blend was homogeneously mixed as well as uniform particle size reduction. The resultant mixture was then pressed into a thin, transparent, self-supporting disc with the aid of an hydraulic press at ~10 tons for 3\u0026ndash;5 min. Immediately after preparation, the KBr disc was mounted in the sample holder, and spectra were collected over 4000\u0026ndash;400 cm\u003csup\u003e-1\u003c/sup\u003e at a resolution of 4 cm\u003csup\u003e-1\u003c/sup\u003e and with 32 scans per sample, using an air background reference. Absorption spectra were also examined for characteristic absorption bands associated with functional groups, and any pronounced shifts, disappearances of peaks or appearance of new peaks suggestive of drug-excipient interactions. All experiments were done in triplicate (n = 3) and results reported as mean \u0026plusmn; SD[19,20].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 3\u003csup\u003e2\u003c/sup\u003e full factorial design was used to screen puerarin-loaded in situ liquid crystal gels, wherein two independent variables were each examined at three levels (low, medium, high). The selected independent variables were glyceryl monooleate (GMO; X\u003csub\u003e1\u003c/sub\u003e) concentration, as the primary amphiphilic lipid that promoted lacrimal dilution-triggered production of liquid crystalline mesophase, and Soluplus\u0026reg; (polyvinyl caprolactam\u0026ndash;polyvinyl acetate\u0026ndash;polyethylene glycol graft copolymer; X\u003csub\u003e2\u003c/sub\u003e) concentration, incorporated as a novel amphiphilic polymeric co-matrix agent to achieve concomitant improvement in puerarin solubilization stability and control over mesophases structure both of which had not been explored simultaneously within the context of lipidic ophthalmic in situ liquid crystal gel systems. A total of nine experimental preparations (F1\u0026ndash;F9) were generated as per the complete factorial matrix. The dependent variables were the percent cumulative drug release at 8 hours (Y\u003csub\u003e1\u003c/sub\u003e; target: maximize and \u0026ge; 70%) and viscosity of the precursor solution (Y\u003csub\u003e2\u003c/sub\u003e; target minimize to \u0026le; 150 mPa\u0026middot;s for instillability) as given in table 1. Multiple linear regression was performed using Design Expert\u0026reg; software v.13 (Stat-Ease Inc., USA), and response surface plots as well as polynomial equations were produced. For each response, we can write the general polynomial equation:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eY = b\u003csub\u003e0\u003c/sub\u003e + b\u003csub\u003e1\u003c/sub\u003eX\u003csub\u003e1\u003c/sub\u003e + b\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e2\u003c/sub\u003e + b\u003csub\u003e12\u003c/sub\u003eX\u003csub\u003e1\u003c/sub\u003eX\u003csub\u003e2\u003c/sub\u003e + b\u003csub\u003e11\u003c/sub\u003eX\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e + b\u003csub\u003e22\u003c/sub\u003eX\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ewhere Y is the dependent variable, b\u003csub\u003e0\u003c/sub\u003e is the intercept, b\u003csub\u003e1\u003c/sub\u003e and b\u003csub\u003e2\u003c/sub\u003e are linear coefficients, b\u003csub\u003e12\u003c/sub\u003e is the interaction coefficient, and b\u003csub\u003e11\u003c/sub\u003e, b\u003csub\u003e22\u003c/sub\u003e are quadratic coefficients[21,22].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1: Independent and Dependent Variables in the 3\u003csup\u003e2\u003c/sup\u003e Full Factorial Design\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 264px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariable\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLevel \u0026minus;1 (Low)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLevel 0 (Medium)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLevel +1 (High)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eIndependent Variables\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 264px;\"\u003e\n \u003cp\u003eX₁: Glyceryl monooleate (% w/w)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 264px;\"\u003e\n \u003cp\u003eX₂: Soluplus\u0026reg; (% w/w)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" style=\"width: 453px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDependent Variables\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGoal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" style=\"width: 453px;\"\u003e\n \u003cp\u003eY₁: Cumulative drug release at 8 h (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003eMaximize\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" style=\"width: 453px;\"\u003e\n \u003cp\u003eY₂: Precursor viscosity (mPa\u0026middot;s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 148px;\"\u003e\n \u003cp\u003eMinimize\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2: Formulation Composition of Puerarin-Loaded In Situ Liquid Crystal Gels (F1\u0026ndash;F9)\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eExcipient\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePF1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePF2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePF3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePF4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePF5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePF6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePF7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePF8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePF9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePuerarin (% w/w)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGlyceryl monooleate (% w/w)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSoluplus\u0026reg; (% w/w)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePropylene glycol (% w/w)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBenzalkonium chloride (% w/w)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSimulated tear fluid (STF)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eq.s. to 100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eq.s. to 100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eq.s. to 100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eq.s. to 100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eq.s. to 100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eq.s. to 100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eq.s. to 100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eq.s. to 100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eq.s. to 100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e(All values are % w/w; STF composed of NaCl 0.67%, NaHCO\u003csub\u003e3\u003c/sub\u003e 0.20%, KCl 0.14%, pH 7.4)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of Puerarin-Loaded In Situ Liquid Crystal Gels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePuerarin- loaded in situ liquid crystal gels were formulated utilizing a modified fusion-vortex method based on Wu et al. (2021) and Wang et al. (2019). Weigh the exact quantities of glyceryl monooleate (Table 2) and melt in a water bath (Equitron, India) at 45 \u0026plusmn; 2\u0026deg;C until it became clear homogeneous melt. In parallel, Soluplus\u0026reg; was stirred with propylene glycol in a magnetic stirrer (Remi, India) at 300 rpm for 15 minutes at 40\u0026deg;C. Puerarin (1% w/w) was dispersed into the Soluplus\u0026reg;\u0026ndash;propylene glycol solution and stirred, at 500 rpm and 25 \u0026plusmn; 2\u0026deg;C for 30 minutes until complete dissolution. List of things to do on the day of arrival until departure. Benzalkonium chloride (0.01% w/w) was dissolved in a minimum volume of simulated tear fluid and the total weight adjusted to 100% w/w with STF under continuous vortexing for an additional 3 minutes. All formulations were kept at 4 \u0026plusmn; 2\u0026deg;C in sterile amber glass vials until further characterization[23,24].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization of Puerarin-Loaded In Situ Liquid Crystal Gels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVisual Appearance and pH\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll formulations were visually inspected for clarity, color and phase homogeneity. The pH of individual precursor solutions was measured at 25 \u0026plusmn; 2 \u0026deg;C employing calibrated digital pH meter (Systronics\u0026mu;pH System 362, India) that has been standardized with suitable buffer solutions (Merck, India): pH 4.0 and pH 7.0. Data were recorded as mean \u0026plusmn; SD (n = 3); the acceptable ophthalmic pH range is 6.5\u0026ndash;8.0 as per IP 2022 guidelines[25]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eViscosity of Precursor Solution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe viscosity measurements of the precursor (pre-gelation) formulations were performed at 25 \u0026plusmn; 0.5\u0026deg;C using a Brookfield DV-II+ Pro viscometer (Brookfield Engineering Company, Middleboro, MA USA), fitted with spindle no. S-62 and set to 10 rpm. After equilibration for 2 min, the measurements were conducted. Mean \u0026plusmn; SD (n = 3) is given for viscosity (mPa\u0026middot;s). Conventional instillability of the optimized formulations was ensured by targeting a precursor viscosity of \u0026le; 150 mPa\u0026middot;s[26].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn Situ Gelling Capacity and Gel Strength\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach formulation\u0026apos;s gelling capacity upon contacting STF was qualitatively evaluated by applying 50 \u0026micro;L of the precursor solution onto a glass slide maintained at 35 \u0026plusmn; 0.5 \u0026deg;C and observing whether/when its phase transition behavior occurred. The gel strength was measured by an inverted tube method: 2 mL formulation into a pre-weighed vial, 0.1 mL STF was mixed at 35 \u0026deg;C, then recording the average weight (g) required to insert a stainless-steel probe (10 mm diameter) into the formed gel up to a depth of 5 mm using a texture analyzer (TA-HD plus, Stable Micro Systems, UK). Results were presented as mean \u0026plusmn; SD (n = 3)[27].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePolarized Light Microscopy (PLM) for Liquid Crystalline Phase Identification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe observed internal liquid crystalline mesophase structure of the resulting gel (pseudocylindrical) was investigated using polarized light microscopy, utilizing a Carl Zeiss Axiolab 5 polarizing microscope with cross-polarizers (Carl Zeiss, Germany). Each formulation (~5 mg) was spread on a clean glass slide and submerged into excess water (5 \u0026micro;L) at 35\u0026deg;C, covered with coverslip and examined under polarized light at \u0026times;40 magnification for birefringent textures associated with cubic or hexagonal mesophases. Photomicrographs were obtained with a built-in digital camera. All experiments were performed in triplicate (n = 3)[28].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDrug Content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn all of the formulations, 100 mg was dissolved in 10 mL of ethanol under vortex mixing (2500 rpm, 5 min) and then diluted with ethanol to achieve an expected concentration of 10 \u0026micro;g/mL puerarin. Absorbance was recorded at \u0026lambda;\u003csub\u003emax\u003c/sub\u003e versus blank of the related placebo formulation on double beam UV-visible spectrophotometer (Systronics UV-2202, India) using 1 cm quartz cuvettes. Drug content (%) was obtained from the regression equation. Results represent mean \u0026plusmn; SD (n = 3); acceptable limits correlate with ICH Q6A: 95\u0026ndash;105% of the labeled claim[29].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEntrapment Efficiency\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEntrapment efficiency (EE%) was calculated by ultracentrifugation at 15,000 rpm for 45 minutes at 4\u0026deg;C (Remi C-24 Plus, India) to separate unentrapped free drug and liquid crystal matrix. The supernatant was separated, diluted in ethanol as appropriate and absorbance at \u0026lambda;\u003csub\u003emax\u003c/sub\u003e recorded using Systronics UV-2202 spectrophotometer (India). EE% was calculated using:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEE (%) = [(Total drug \u0026minus; Free drug) / Total drug] \u0026times; 100\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResults were expressed as mean \u0026plusmn; SD (n = 3)[30].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn Vitro Drug Release\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn vitro drug release was carried out by dialysis bag diffusion method as described by Wu et al. (2021). A dialysis membrane (MW cutoff 12,000\u0026ndash;14,000 Da; Hi-Media, India) that was pre-soaked in dissolution medium for 12 hr was employed. Exact amounts of test formulation equivalent to 1 mg puerarin were carefully introduced into the membrane and sealed, before being suspended in 50 mL of STF (pH 7.4) in a USP dissolution apparatus Type II (paddle; Electrolab EDT-08Lx, India) operating at 100 rpm and at temperature maintained at 37 \u0026plusmn; 0.5\u0026deg;C. At predetermined time intervals (0.5, 1, 2, 4, 6, 8, 12 and 24 h), aliquots of 2 mL were taken out and replaced with the same volume of fresh medium to sustain sink conditions. Absorbance values were measured at \u0026lambda;max after filtering the samples with a 0.45 \u0026micro;m PVDF syringe filter and dilution to proper absorbance level. The cumulative drug release (%) was calculated from the calibration curve. Release kinetics were evaluated fitting the data to zero order, first order, Higuchi and Korsmeyer\u0026ndash;Peppas models; model selection was done based on higher r\u003csup\u003e2\u003c/sup\u003e and lower AIC. Data are shown as mean \u0026plusmn; standard deviation (SD) (n = 3)[31].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEx Vivo Corneal Permeation Study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe freshly excised goat corneas were collected from a local abattoir within 2 hours of slaughter following the ethical guidelines laid down by CPCSEA, India and was used for the ex vivo corneal permeation studies. The cornea was placed between modified Franz diffusion cell (PermeGear, USA) systems with receptor compartment containing 15 mL of STF (pH 7.4), maintained at a temperature of 35 \u0026plusmn; 0.5\u0026deg;C and continuous stirring at a rate of 300 rpm. The donor compartment was treated with 50 \u0026micro;L (containing 1% puerarin) of formulation. At 1, 2, 4, 6, 8 and 12 h samples (1 mL) were removed and STF was replaced with fresh STF. Puerarin was measured spectrophotometrically at \u0026lambda;max Papp (cm\u003csup\u003e/s\u003c/sup\u003e) and cumulative amount permeated (\u0026micro;g/cm\u003csup\u003e2\u003c/sup\u003e) were calculated. As a control, puerarin solution (1% w/v in STF) was used. Results are shown as mean \u0026plusmn; SD (n = 3)[32].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOcular Irritation (HET-CAM Assay)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to ICCVAM guidelines, HET-CAM was used as an ethical alternative for testing ocular irritation potential (i.e., the Draize rabbit eye test). Gelatinase activity was determined using the gelatinase assay according to Dani et al. [36], as well as protein extraction from chicken egg white, for which fertilized white Leghorn eggs (Venkateshwara Hatcheries, India) were incubated at 37 \u0026plusmn; 0.5\u0026deg;C with 60% relative humidity in a humidified egg incubator (Igenix, India) for 9 d. After 9 days of incubation, the eggs were carefully cracked open on the shell over the air sac to expose the membrane and 300 \u0026micro;L of each formulation was placed directly onto the CAM. Over 300 seconds, hemorrhage (H), vascular lysis (L) and coagulation took place. Irritation score (IS), calculated according to an established formula; IS \u0026lt; 0.9 = non-irritant, 0.9\u0026ndash;4.9 = slight irritant. Physiological saline (0.9% NaCl) and sodium lauryl sulfate (1% w/v) were used as the negative and positive controls, respectively. Results were shown as mean \u0026plusmn; SD (n = 3)[33,34].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn Vitro Cytotoxicity (MTT Assay)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cytotoxicity of the optimized formulation was assessed in human corneal epithelial cells (HCE-T cell line, ATCC) by performing MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] test. DMEM/F12 medium supplemented with 10% FBS was used to seed the cells in 96 well plates at a density of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well.for 24 hours with incubation at 37\u0026deg;C/5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were subsequently treated with serial dilutions of the optimized formulation and free puerarin (1\u0026ndash;200 \u0026micro;g/mL) for 24 h. MTT solution (5 mg/mL in PBS; 20 \u0026micro;L/well) was added and incubated at 37\u0026deg;C for 4 hours, then formazan crystals were dissolved in DMSO (150 \u0026micro;L/well). Absorbance was measured at 570 nm using microplate reader (BioradiMark, India). Percentage viability (%) and IC\u003csub\u003e50\u003c/sub\u003e values were calculated. Values are mean \u0026plusmn; SD (n = 3); statistical comparison by one-way ANOVA with Tukey\u0026apos;s post-hoc test (p \u0026lt; 0.05)[35,36].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStability Study\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStability study of optimized in situ liquid crystal gel formulation (PF6) loaded with puerarin was performed according to ICH Q1A(R2) guidelines. The formulation was filled in sterile amber glass vials and stored under three separate conditions: an accelerated condition (40\u0026deg;C \u0026plusmn; 2\u0026deg;C / 75% RH), a room temperature condition (25\u0026deg;C \u0026plusmn; 2\u0026deg;C / 60% RH), and freezing conditions (\u0026minus;20\u0026deg;C \u0026plusmn; 2\u0026deg;C) for three months. Samples were withdrawn at fixed time intervals (0 (baseline),1,2, and 3 months) and reviewed for physical appearance, pH, precursor viscosity, drug content, entrapment efficiency and cumulative corneal permeation after 8 hours. All analyses were conducted in triplicate (n = 3) and data shown are mean \u0026plusmn; SD. The degree of variation during the storage period was calculated using the percentage change between each parameter as compared to the initial value[37,38].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResults were expressed as mean \u0026plusmn; SD (n = 3). Statistical comparisons were conducted by one-way ANOVA and Tukey\u0026rsquo;s post-hoc test (p \u0026lt; 0.05) with GraphPad Prism v.9.0 (GraphPad Software, USA). Response surface methodology utilizing Design Expert\u0026reg; v.13 (Stat-Ease Inc, USA) 3\u003csup\u003e2\u003c/sup\u003e full factorial design was applied for formulation optimization and adjusted R\u003csup\u003e2\u003c/sup\u003e, predicted R\u003csup\u003e2\u003c/sup\u003e, p-value of lack-of-fit and F-values were utilized to test the adequacy of the model. Sample coverage was determined for the best combination of three distinct function types: touch, contact, and incubation, using a desirability function approach for simultaneous optimization of dependent variables[39].\u003c/p\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003e\u003cstrong\u003eRESULTS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCalibration Curve Determination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA calibration curve for puerarin in ethanol was established over a concentration range of 5\u0026ndash;30 \u0026micro;g/mL. Excellent linearity (y = 0.0334x + 0.005, r\u003csup\u003e2\u003c/sup\u003e = 0.9996) was observed with ICH Q2(R1) compliance reported here. The relationship between absorbance and concentration for the span studied was linear as indicated by the high r\u003csup\u003e2\u003c/sup\u003e value. The calibration curve shown in Figure 1 confirms the applicability of the developed spectrophotometric method, allowing for accurate and reproducible quantification of puerarin in all further analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults of Solubility Study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable 3 shows the solubility of puerarin in different solvents. Puerarin was poorly soluble in distilled water (0.82 \u0026plusmn; 0.15 mg/mL), and slightly soluble in physiologically relevant media, such as simulated tear fluid (7.35 \u0026plusmn; 0.32 mg/mL) and phosphate buffer pH 7.4 (7.56 \u0026plusmn; 0.28 mg/mL). The highest solubility value was reported in ethanol (26.28 \u0026plusmn; 1.45 mg/mL) and methanol (25.12 \u0026plusmn; 1.84 mg/mL). The solubility in aqueous and tear fluid was limited, confirming the need for a lipid-based liquid crystal carrier system to improve ocular targeting of puerarin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3: Solubility Profile of Puerarin in Different Solvents\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"width: 100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSr. No.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSolvent\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSolubility (mg/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eInference\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eDistilled Water\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.82 \u0026plusmn; 0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eVery slightly soluble\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePhosphate Buffer pH 7.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.56 \u0026plusmn; 0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSlightly soluble\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSimulated Tear Fluid (STF)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.35 \u0026plusmn; 0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSlightly soluble\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eArtificial Tear Fluid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.28 \u0026plusmn; 0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSlightly soluble\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eMethanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e25.12 \u0026plusmn; 1.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSparingly soluble\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eEthanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e26.28 \u0026plusmn; 1.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSparingly soluble\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAcetonitrile\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.15 \u0026plusmn; 0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSlightly soluble\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAll values are expressed as mean\u0026nbsp;\u0026plusmn;\u0026nbsp;SD\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDifferential Scanning Calorimetry (DSC) Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe DSC thermogram of pure puerarin showed a sharp characteristic endothermic peak at a melting temperature of 185.93\u0026deg;C, confirming its crystalline appearance (Figure 2A). In the thermogram of physical mixture (Figure 2B), two endotherms were observed at 155.73\u0026deg;C and 185.44\u0026deg;C; the appearance of an additional peak at 155.73\u0026deg;C as well as shifting of puerarin melting peak from 185.93\u0026deg;C to 185.44\u0026deg;C seen in this thermogram confirmed that some type of interaction had occurred between puerarin and excipients which reduced crystallinity to some extent but there was no notable incompatibility indicating suitability for formulation development.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFourier Transform Infrared Spectroscopy (FTIR) Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCharacteristic absorption bands were exhibited in the FTIR spectrum of pure puerarin (Figure 3A), including those at 3473.30 and 3397.38 cm\u003csup\u003e-1\u003c/sup\u003e (O\u0026ndash;H stretching), 2936.34 cm\u003csup\u003e-1\u003c/sup\u003e (C\u0026ndash;H stretching), 1649.26 cm\u003csup\u003e-1\u003c/sup\u003e (C=O stretching), 1579.07 cm\u003csup\u003e-1\u003c/sup\u003e (C=C aromatic stretching) and 1035.17 cm⁻\u0026sup1; (C\u0026ndash;O\u0026ndash;C stretching), confirming structural integrity of puerarin[14]. In the spectrum of physical mixture (Figure 3B), all characteristic peaks of puerarin were retained and only slightly shifted, such as O\u0026ndash;H stretching at 3435.23 cm\u003csup\u003e-1\u003c/sup\u003e and C=O at 1647.16 cm\u003csup\u003e-1\u003c/sup\u003e, and no great peak disappearance was noticed after mixing with formulation excipients, proving that there is no significant chemical incompatibility between puerarin and those formulation excipients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003epuerarin-loaded in situ liquid crystal gel formulations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVisual Appearance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe nine puerarin-loaded in situ liquid crystal gel formulations (PF1\u0026ndash;PF9) were also pale yellow liquid with characteristic odor and a homogeneous phase as listed in Table 3. PF1, PF2 and PF4 containing lower concentrations of GMO or Soluplus\u0026reg; appeared clear and free-flowing, but increasing concentrations of each excipient progressively increased viscosity and opalescence; PF9 had a turbid highly viscous appearance. All formulations showed phase homogeneity [showing all prepared formulations were successfully formulated with drug uniformly distributed in the liquid crystal matrix].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4.Visual characteristics of puerarin-loaded in situ liquid crystal gel formulations (PF1\u0026ndash;PF9)\u003c/strong\u003e\u003c/p\u003e\n\u003ctable\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eF. Code\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAppearance\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eColor\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eClarity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePhase Homogeneity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eOdor\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eClear, free-flowing liquid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePale yellow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eClear\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eHomogeneous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCharacteristic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eClear, slightly viscous liquid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePale yellow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eClear\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eHomogeneous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCharacteristic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSlightly opalescent, viscous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePale yellow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSlightly turbid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eHomogeneous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCharacteristic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eClear, free-flowing liquid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePale yellow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eClear\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eHomogeneous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCharacteristic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eClear, moderately viscous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePale yellow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eClear\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eHomogeneous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCharacteristic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eOpalescent, viscous liquid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePale yellow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSlightly turbid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eHomogeneous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCharacteristic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eClear, viscous liquid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePale yellow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eClear\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eHomogeneous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCharacteristic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eOpalescent, viscous liquid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePale yellow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSlightly turbid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eHomogeneous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCharacteristic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eOpalescent, highly viscous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePale yellow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eTurbid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eHomogeneous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCharacteristic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003ePhysicochemical Characterization, Drug Content, Entrapment Efficiency, Gelling Capacity and Gel Strength\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll formulations had a pH in the ophthalmic range of 6.5\u0026ndash;8.0 (Table 5) at 25\u0026deg;C, ranging from 7.14 to 7.26. Precursor viscosity progressively increased from 48.3 mPa\u0026middot;s (PF1) to 148.3 mPa\u0026middot;s (PF9), reflecting the rising concentrations of GMO and Soluplus\u0026reg;, while all formulations remained below the \u0026le;150 mPa\u0026middot;sinstillability threshold. Drug contents were acceptable (96.4\u0026ndash;98.6%) and entrapment efficiency was also within limits (82.3\u0026ndash;94.3%). Gelling abilities increased with higher excipient concentrations, reaching fully rigid PF6\u0026ndash;PF9 gels within 30 seconds. Gel strength ranged progressively from 18.4 g (PF1) to 38.1 g (PF9), indicating increased matrix strength with increasing levels of GMO and Soluplus\u0026reg;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5.Physicochemical characterization, drug content, entrapment efficiency, gelling capacity, gel strength, and in vitro drug release of puerarin-loaded in situ liquid crystal gel formulations (PF1\u0026ndash;PF9)\u003c/strong\u003e\u003c/p\u003e\n\u003ctable\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eF. Code\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003epH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePrecursor Viscosity (mPa\u0026middot;s)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eDrug Content (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eEntrapment Efficiency (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGelling Capacity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eGel Strength (g)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.21 \u0026plusmn; 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e48.3 \u0026plusmn; 1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e96.4 \u0026plusmn; 0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e82.3 \u0026plusmn; 1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e18.4 \u0026plusmn; 0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.18 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e67.6 \u0026plusmn; 2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e97.1 \u0026plusmn; 0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e86.7 \u0026plusmn; 1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e19.8 \u0026plusmn; 0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.14 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e89.4 \u0026plusmn; 2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e97.8 \u0026plusmn; 0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e89.2 \u0026plusmn; 1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e21.3 \u0026plusmn; 1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.24 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e74.2 \u0026plusmn; 2.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e96.8 \u0026plusmn; 1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e85.4 \u0026plusmn; 1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e24.6 \u0026plusmn; 0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.19 \u0026plusmn; 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e96.8 \u0026plusmn; 3.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e97.6 \u0026plusmn; 0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e89.8 \u0026plusmn; 1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e26.9 \u0026plusmn; 1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.16 \u0026plusmn; 0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e121.4 \u0026plusmn; 3.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e98.2 \u0026plusmn; 0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e92.1 \u0026plusmn; 1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e++++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e29.4 \u0026plusmn; 1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.26 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e108.7 \u0026plusmn; 3.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e97.2 \u0026plusmn; 1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e87.6 \u0026plusmn; 1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e31.2 \u0026plusmn; 1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.22 \u0026plusmn; 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e131.6 \u0026plusmn; 4.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e98.0 \u0026plusmn; 0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e91.4 \u0026plusmn; 1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e++++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e34.7 \u0026plusmn; 1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.17 \u0026plusmn; 0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e148.3 \u0026plusmn; 4.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e98.6 \u0026plusmn; 0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e94.3 \u0026plusmn; 1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e++++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e38.1 \u0026plusmn; 1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAll values expressed as mean \u0026plusmn; SD (n = 3), Gelling capacity: ++ = moderate gel formed within 60 s; +++ = firm gel within 45 s; ++++ = rigid gel within 30 s\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults of Ex Vivo Corneal Permeation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 4 and Table 3a summarises the cumulative ex vivo corneal permeation profiles of all formulations across goat cornea, for an 8 hours period. All the formulations showed time-dependent permeation that was observed in a range from 49.7% (PF1) to 83.1% (PF9) at the end of the study (8 hours). PF6 demonstrated permeation of 76.4 \u0026plusmn; 2.43% for the period of 8 hours, where flux was recorded as52.34 \u0026plusmn; 1.78 \u0026micro;g/cm\u003csup\u003e2\u003c/sup\u003e/h and corresponding permeability coefficient was found to be5.23 \u0026times;10\u003csup\u003e-3\u003c/sup\u003e cm/h (Table 6), indicative of an excellent compromise between enhancement in permeation \u0026amp; viscosity supported (Vishwakarma et al.,nam.*). The observed progressive enhancement of permeation with increasing concentrations of both GMO and Soluplus\u0026reg; supports the notion that these excipients act to enhance corneal drug transport.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlux and Permeability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable 6 presents the flux and permeability coefficient values for the different formulations. The flux values were in the range of 34.93 \u0026plusmn; 1.24 \u0026micro;g/cm\u003csup\u003e2\u003c/sup\u003e/h (PF1) to 56.69 \u0026plusmn; 1.94 \u0026micro;g/cm\u003csup\u003e2\u003c/sup\u003e/h (PF9), and the permeability coefficient was in the range of 3.49 \u0026times; 10\u003csup\u003e-3\u003c/sup\u003e cm/h (PF1) to 5.67 \u0026times; 10\u003csup\u003e-3\u003c/sup\u003e cm/h (PF9) and increased proportionately with increase in concentration of GMO and Soluplus\u0026reg; PF6\u0026apos;s flux (52.34 \u0026plusmn; 1.78 \u0026micro;g/cm\u003csup\u003e2\u003c/sup\u003e/h) and Kp (5.23 \u0026times; 10\u003csup\u003e-3\u003c/sup\u003e cm/h) values indicated the best corneal permeation enhancement among the SFPs studied. As seen for all three formulations, the increasing trend confirms that in addition to aforementioned effect of GMO, we could make use of contribution of it along with Soluplus\u0026reg; which led to a synergistic affect on augmenting transcorneal drug transport.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 6: Flux and Permeability Coefficient of PF1\u0026ndash;PF9\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"width: 100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eFormulation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eFlux J (\u0026micro;g/cm\u003csup\u003e2\u003c/sup\u003e/h)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eKp (\u0026times;10\u003csup\u003e-3\u003c/sup\u003e cm/h)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e34.93 \u0026plusmn; 1.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.49 \u0026plusmn; 0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e39.39 \u0026plusmn; 1.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.94 \u0026plusmn; 0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e43.95 \u0026plusmn; 1.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.40 \u0026plusmn; 0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e40.55 \u0026plusmn; 1.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.06 \u0026plusmn; 0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e47.03 \u0026plusmn; 1.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.70 \u0026plusmn; 0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e52.34 \u0026plusmn; 1.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.23 \u0026plusmn; 0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e44.37 \u0026plusmn; 1.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.44 \u0026plusmn; 0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e50.32 \u0026plusmn; 1.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.03 \u0026plusmn; 0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e56.69 \u0026plusmn; 1.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.67 \u0026plusmn; 0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAll values expressed as mean \u0026plusmn; SD (n = 3)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDrug Release kinetics\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs represented in Figure 5, the in vitro drug release data of optimized formulation PF6 was applied to zero order, first order, Higuchi, Hixson\u0026ndash;Crowell and Korsmeyer\u0026ndash;Peppas models. The highest R\u003csup\u003e2\u003c/sup\u003e value observed was that of Hixson\u0026ndash;Crowell model which was found to be the best fitted having a slope of (0.2207) with all models; followed by first order model (R\u0026sup2; = 0.9959) and Higuchi model (R\u0026sup2; = 0.9980) given in kinetic parameters. An R\u0026sup2; value of 0.9937 with a slope of 0.8798 obtained using the Korsmeyer\u0026ndash;Peppas model confirms a non-Fickian mode of anomalous diffusion transport, validating that drug release from the liquid crystal matrix was through a combined mechanism governed by diffusion and erosion controlled mechanisms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOptimization of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePuerarin-Loaded In Situ Liquid Crystal Gel\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResponse 1: Cumulative Corneal Permeation at 8 h (Y\u003csub\u003e1\u003c/sub\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to the model building criteria of Design Expert\u0026reg; v.13, the quadratic model was the best fit for Y\u003csub\u003e1\u003c/sub\u003e. Table 8 indicates that the model achieved an adjusted R\u003csup\u003e2\u003c/sup\u003e and predicted R\u003csup\u003e2\u003c/sup\u003e of 0.9977 (0.9977) and 0.9908, respectively, which again suggests superb fit with no overfitting. A non-significant lack-of-fit (p = 0.9977) further confirmed model adequacy. The overall model was significant (F = 695.10; p \u0026lt; 0.0001). As revealed by ANOVA (Table 7), GMO (A; F = 1751.68, p \u0026lt; 0.0001) and Soluplus\u0026reg; (B; F = 1651.32, p \u0026lt; 0.0001) were the most significant factors contributing to the outcome. The interaction term AB (F = 19.11; p = 0.0222) and quadratic term A\u003csup\u003e2\u003c/sup\u003e (F = 53.37; p = 0.0053) were significant, while B\u003csup\u003e2\u003c/sup\u003e was non-significant (p = 0.8972), suggesting a primarily linear effect of Soluplus\u0026reg; over the range studied. The final polynomial equation for Y\u003csub\u003e1\u003c/sub\u003e was acquired as follows:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eY\u003csub\u003e1\u003c/sub\u003e = 67.8667 + 8.6A + 8.35B + 1.1AB \u0026minus; 2.6A\u003csup\u003e2\u003c/sup\u003e \u0026minus; 0.05B\u003csup\u003e2\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA (+8.6) and B (+8.35) contribute as positive linear coefficients, confirming the individual effects of both factors on corneal permeation, while a positive interaction term (1.1) reveals synergistic cooperation in enhancing corneal permeation between GMO and Soluplus\u0026reg;. The negative coefficient for A\u003csup\u003e2\u003c/sup\u003e (\u0026minus;2.6) indicates a diminishing return at high levels of GMO. Cumulative permeation ranged from 49.7% (PF1) to 83.1% (PF9) (Table 4). The response surface illustrated an upward curvilinear profile towards the high-GMO/high-Soluplus\u0026reg; quadrant (Figure 6A), while contour plots (Figure 6B) reaffirmed that elevated concentrations of both factors produced an elliptical permeation maximized zone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResponse 2: Precursor Viscosity (Y\u003csub\u003e2\u003c/sub\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe linear model was KM\u0026apos;s best fit for Y\u003csub\u003e2\u003c/sub\u003e (adjusted R\u003csup\u003e2\u003c/sup\u003e = 0.9954, predicted R\u003csup\u003e2\u003c/sup\u003e = 0.9923) and also confirmed strong linearity and predictive reliability in Table 8. The model adequacy was confirmed by the non-significant lack-of-fit and overall model significance (F = 863.75; p \u0026lt; 0.0001) (Table 7). Decreasing GMO concentration The concentration of GMO (A), which was the most important parameter (F = 1161.83; p \u0026lt; 0.0001; SS = 5599.81), explained approximately twice as much variance as Soluplus\u0026reg; (B, F = 565.67; p \u0026lt; 0.0001; SS = 2726.40). Neither interaction nor quadratic terms were found to be significant (2FI: p = 0.7655; quadratic: p = 0.7590), thus verifying the adequacy of the linear model. The polynomial equation for Y\u003csub\u003e2\u003c/sub\u003e with respect to all variables was:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eY\u003csub\u003e2\u003c/sub\u003e = 98.4778 + 30.55A + 21.3167B\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe stronger coefficient for A (+30.55) than B (+21.32) thus supports quantitatively the increased contribution of GMO to development of viscosity, which may be due to its role as a primary lipid forming around matrix forming units. The contour plots (Figure 6) exhibited parallel linear isoresponse lines along the GMO axis predominantly, while the response surface plot indicated a uniformly increasing plane from 48.3 mPa\u0026middot;s (PF1) to 148.3 mPa\u0026middot;s (PF9), as described in Table 5. All formulations were within the acceptable instillability range of \u0026le; 150 mPa\u0026middot;s.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 7: ANOVA Summary for Coneal Permeation at 8 hr and viscosity\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"width: 100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSum of Squares\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003edf\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eMean Square\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eF-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\n \u003cp\u003e\u003cstrong\u003eCorneal Permeation at 8 h (Y\u003csup\u003e1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eModel\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e880.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e176.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e695.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003esignificant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eA-GMO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e443.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e443.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1751.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB-Soluplus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e418.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e418.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1651.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e19.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.0222\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eA\u0026sup2;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e13.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e13.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e53.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.0053\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB\u0026sup2;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.0050\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.0050\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.0197\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.8972\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eResidual\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.7600\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.2533\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eCor Total\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e881.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\n \u003cp\u003e\u003cstrong\u003eViscosity\u003c/strong\u003e\u003cstrong\u003e(Y\u003csup\u003e2\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eModel\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8326.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4163.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e863.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003esignificant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eA-GMO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5599.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5599.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1161.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB-Soluplus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2726.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2726.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e565.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eResidual\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e28.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eCor Total\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8355.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 8: Model Fit Summary for Coneal Permeation at 8 hr and viscosity\u003c/strong\u003e\u003c/p\u003e\n\u003ctable\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSequential p-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eLack of Fit p-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAdjusted R\u0026sup2;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePredicted R\u0026sup2;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003eCorneal Permeation at 8 h (Y\u003csup\u003e1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eLinear\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.9711\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.9503\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e2FI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.2498\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.9741\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.9449\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eQuadratic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.0123\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.9977\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.9908\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSuggested\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCubic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.5735\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.9977\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.9483\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAliased\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003eViscosity\u003c/strong\u003e\u003cstrong\u003e(Y\u003csup\u003e2\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eLinear\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt; 0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.9954\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e0.9923\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSuggested\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e2FI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.7655\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.9946\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.9858\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eQuadratic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.7590\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.9925\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.9665\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCubic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.2642\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.9984\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.9641\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAliased\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eValidation of Statistical Model\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo confirm the predictions of statistics modelling, the optimized formulation PF6 selected by maximum desirability function (D = 1) was prepared and evaluated. The experimental observed values were close to the predicted ones from Design Expert\u0026reg; for corneal drug permeation (76.6%) and viscosity (121.4 mPa\u0026middot;s) shown in Table 9 were as follows: predicted value of 78.318% and 122.808 mPa\u0026middot;s, respectively. The 2.44% and 1.15% relative errors observed for corneal permeation and viscosity, respectively, which are considerably lower than the admissible level of \u0026plusmn;5%, showed that the developed full factorial design model had a high predictive capability.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 9: Validation of the optimized puerarin-loaded in situ liquid crystal gel formulation (PF6)\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"width: 100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eBatch\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eResponse\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePredicted Value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eExperimental Value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e% Relative Error\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eDesirability\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003ePF6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCorneal drug Permeation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e78.318\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e76.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eViscosity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e122.808\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e121.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eHET-CAM Ocular Irritation Study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePerformance of the optimized formulation PF6 was assessed against ocular irritation using HET-CAM and results are provided in Table 10. PF6 was completely free from hemorrhage, vascular lysis and coagulation on the chorioallantoic membrane at all timepoints during the 300-second observation period with an irritation score of 0.38 \u0026plusmn; 0.04 and classified as non-irritant (IS \u0026lt; 0.9). IS of 0.00 was observed for the negative control (0.9% NaCl) and IS of 12.6 \u0026plusmn; 0.8 was observed for positive control (1% SLS), indicating validity of assay. These results confirm the ocular safety and tolerability of PF6 for eye use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 10: HET-CAM ocular irritation scores of optimized puerarin-loaded in situ liquid crystal gel formulation (PF6)\u003c/strong\u003e\u003c/p\u003e\n\u003ctable\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSample\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eHemorrhage\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eVascular Lysis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eCoagulation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eIrritation Score (IS)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eInference\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePF6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAbsent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAbsent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAbsent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.38 \u0026plusmn; 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNon-irritant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eNegative Control (0.9% NaCl)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAbsent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAbsent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAbsent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.00 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNon-irritant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePositive Control (1% SLS)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePresent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePresent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ePresent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e12.6 \u0026plusmn; 0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSevere irritant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAll values expressed as mean \u0026plusmn; SD (n = 3); IS \u0026lt; 0.9 = non-irritant; 0.9\u0026ndash;4.9 = slight irritant; 5.0\u0026ndash;8.9 = moderate irritant; \u0026gt; 9.0 = severe irritant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn Vitro Cytotoxicity (MTT Assay)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Table 11 and Figure 7, the cytotoxic effects of free puerarin and optimized formulation PF6 on HCE-T cells (Figure 8) \u0026nbsp;at a concentration range of 1\u0026ndash;200 \u0026micro;g/mL. Cell viability was reduced in a concentration-dependent manner both by free puerarin and PF6. PF6 showed 91% cell viability up to a concentration of 50 \u0026micro;g /mL, demonstrating very good cytocompatibility at therapeutically relevant concentrations. The IC\u003csub\u003e50\u003c/sub\u003e of PF6 (182.4 \u0026plusmn; 4.1 \u0026micro;g/mL) was significantly higher than free puerarin (148.6 \u0026plusmn; 3.2 \u0026micro;g/mL), confirming that encapsulation in the liquid crystal matrix decreased cytotoxicity to corneal epithelial cells, establishing the safety profile of ocular application for optimized formulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 11: In vitro cytotoxicity (MTT assay) of optimized puerarin-loaded in situ liquid crystal gel formulation (PF6) and free puerarin on HCE-T cells\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"width: 100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eConcentration (\u0026micro;g/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eCell Viability\u003c/strong\u003e\u003cstrong\u003eFree Puerarin (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eCell ViabilityPF6 (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e99.2 \u0026plusmn; 1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e99.6 \u0026plusmn; 0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e98.4 \u0026plusmn; 1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e99.1 \u0026plusmn; 1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e96.8 \u0026plusmn; 1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e98.2 \u0026plusmn; 1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e93.2 \u0026plusmn; 1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e96.4 \u0026plusmn; 1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e87.4 \u0026plusmn; 1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e91.8 \u0026plusmn; 1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e74.6 \u0026plusmn; 2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e82.3 \u0026plusmn; 1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e58.3 \u0026plusmn; 2.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e68.7 \u0026plusmn; 2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eIC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;g/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e148.6 \u0026plusmn; 3.2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e182.4 \u0026plusmn; 4.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAll values expressed as mean \u0026plusmn; SD (n = 3)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStability Study\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable 12 displays the three-month stability data of optimized formulation PF6 at an accelerated condition, room temperature and freezing conditions. PF6 retained the viscous opalescent character under all storage conditions without any phase separation or precipitation. Under accelerated conditions (98.2% to 96.4%) the greatest variation in drug content was not exceeded, while low variations of only 0.81% and freezing condition of 0.41%. The physical and chemical stability were evident as all systems had pH, viscosity, entrapment efficiency, and corneal permeation at 8 h with % of deviations remaining much less than 3.5% through out the study period confirming stability for PF6 over a duration of three months under all storage conditions tested as per ICH Q1A(R2) guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 12: Results of stability study of optimized puerarin-loaded in situ liquid crystal gel formulation (PF6) over 3 months\u003c/strong\u003e\u003c/p\u003e\n\u003ctable\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eParameter\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eInitial (Day 0)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e1 Month\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e2 Months\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e3 Months\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e\n \u003cp\u003e\u003cstrong\u003eAccelerated stability (40\u0026deg;C \u0026plusmn; 2\u0026deg;C / 75% RH)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePhysical Appearance\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eOpalescent, viscous liquid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eOpalescent, viscous liquid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eOpalescent, viscous liquid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eOpalescent, viscous liquid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003epH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.16 \u0026plusmn; 0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.14 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.12 \u0026plusmn; 0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.10 \u0026plusmn; 0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eViscosity (mPa\u0026middot;s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e121.4 \u0026plusmn; 3.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e122.1 \u0026plusmn; 3.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e123.4 \u0026plusmn; 4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e124.6 \u0026plusmn; 4.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eDrug Content (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e98.2 \u0026plusmn; 0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e97.8 \u0026plusmn; 0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e97.2 \u0026plusmn; 0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e96.4 \u0026plusmn; 0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eEntrapment Efficiency (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e92.1 \u0026plusmn; 1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e91.6 \u0026plusmn; 1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e91.0 \u0026plusmn; 1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e90.4 \u0026plusmn; 1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCorneal Permeation at 8 h (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e76.4 \u0026plusmn; 2.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e75.8 \u0026plusmn; 2.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e75.1 \u0026plusmn; 2.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e74.6 \u0026plusmn; 2.64\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e\n \u003cp\u003e\u003cstrong\u003eRoom temperature stability (25\u0026deg;C \u0026plusmn; 2\u0026deg;C / 60% RH)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePhysical Appearance\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eOpalescent, viscous liquid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eOpalescent, viscous liquid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eOpalescent, viscous liquid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eOpalescent, viscous liquid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003epH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.16 \u0026plusmn; 0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.15 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.14 \u0026plusmn; 0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.13 \u0026plusmn; 0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eViscosity (mPa\u0026middot;s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e121.4 \u0026plusmn; 3.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e121.8 \u0026plusmn; 3.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e122.3 \u0026plusmn; 3.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e122.9 \u0026plusmn; 4.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eDrug Content (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e98.2 \u0026plusmn; 0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e98.0 \u0026plusmn; 0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e97.7 \u0026plusmn; 0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e97.4 \u0026plusmn; 0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eEntrapment Efficiency (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e92.1 \u0026plusmn; 1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e91.9 \u0026plusmn; 1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e91.6 \u0026plusmn; 1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e91.3 \u0026plusmn; 1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCorneal Permeation at 8 h (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e76.4 \u0026plusmn; 2.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e76.1 \u0026plusmn; 2.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e75.8 \u0026plusmn; 2.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e75.4 \u0026plusmn; 2.53\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e\n \u003cp\u003e\u003cstrong\u003eFreezing condition stability (-20\u0026deg;C \u0026plusmn; 2\u0026deg;C)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePhysical Appearance\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eOpalescent, viscous liquid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eOpalescent, viscous liquid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eOpalescent, viscous liquid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eOpalescent, viscous liquid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003epH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.16 \u0026plusmn; 0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.16 \u0026plusmn; 0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.15 \u0026plusmn; 0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e7.15 \u0026plusmn; 0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eViscosity (mPa\u0026middot;s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e121.4 \u0026plusmn; 3.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e121.6 \u0026plusmn; 3.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e121.9 \u0026plusmn; 3.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e122.2 \u0026plusmn; 3.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eDrug Content (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e98.2 \u0026plusmn; 0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e98.1 \u0026plusmn; 0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e97.9 \u0026plusmn; 0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e97.8 \u0026plusmn; 0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eEntrapment Efficiency (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e92.1 \u0026plusmn; 1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e92.0 \u0026plusmn; 1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e91.9 \u0026plusmn; 1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e91.8 \u0026plusmn; 1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCorneal Permeation at 8 h (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e76.4 \u0026plusmn; 2.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e76.3 \u0026plusmn; 2.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e76.1 \u0026plusmn; 2.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e75.9 \u0026plusmn; 2.48\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAll values expressed as mean \u0026plusmn; SD (n = 3)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDISCUSSION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing a 3\u003csup\u003e2\u003c/sup\u003e full factorial design, the current study successfully developed and optimized puerarin-loaded in situ liquid crystal gels for extended ocular delivery of dry eye disease. Since puerarin has a low aqueous solubility (0.82 \u0026plusmn; 0.15 mg/mL in distilled water; Table 3) and poor solubility in simulated tear fluid (7.35 \u0026plusmn; 0.32 mg/mL), it was incorporated into a lipid-based liquid crystalline carrier, in line with previous reports recommending GMO-based systems for poorly water-soluble drugs[40]. DSC analysis (Figure 2) indicated a decrease in crystallinity in the physical mixture of puerarin, FTIR spectra (Figure 3) reflected retention of all significant functional group peaks with minor shift and overall confirmed physicochemical compatibility between puerarin and excipients which were similar to findings reported by Lai et al. \u0026nbsp;for GMO based ocular liquid crystal systems [41]. All formulations (PF1, PF5\u0026ndash;PF9) possessed acceptable pH (7.14 to 7.26), drug content (96.4 to 98.6%), and entrapment efficiency (82.3% to 94.3%) as shown in Table 5), in accordance with quality specifications set for ophthalmic nanostructured formulations [42]. This progressive increase in viscosity, gelling capacity and gelation strength with increasing concentrations of GMO and Soluplus\u0026reg; (Table 5) is consistent with previous findings reported by Tarsitano et al. (2019), who showed that increased lipid concentration leads to more ordered liquid crystalline mesophases and superior mechanical properties [43]. Y\u003csub\u003e1\u003c/sub\u003e = 67.8667 + 8.6A + 8.35B + 1.1AB \u0026minus; 2.6A\u003csup\u003e2\u003c/sup\u003e \u0026minus; 0.05B\u003csup\u003e2\u003c/sup\u003e [quadratic polynomial] model for corneal permeation and Y\u003csub\u003e2\u003c/sub\u003e = 98.4778 +30.55A +21.3167B [linear] model of viscosity, with adjusted R\u003csup\u003e2\u003c/sup\u003e values of coefficient of determination (R\u003csup\u003e2\u003c/sup\u003e) were determined to be the best fit for the experimental data at values of: Y1=0.9977 and Y2=0.9954 respectively, yielding a validation percentage relative error that was less than 2 in both instances confirming excellent predictability (Table 9), confirming validation in agreement with studies based on factorial design ocular therapeutics[44].\u003c/p\u003e\n\u003cp\u003eEx vivo corneal permeation studies indicated a linear increase in cumulative permeation from 49.7% (PF1) to 83.1% (PF9) after 8 h of diffusion time (Figure 4), with the optimized formulation PF6 having the highest flux value of 52.34 \u0026plusmn; 1.78 \u0026micro;g/cm\u003csup\u003e2\u003c/sup\u003e/h and Kp value of 5.23 \u0026times; 10\u003csup\u003e-3\u003c/sup\u003e cm/h (Table 6), significantly better than conventional aqueous eye drop systems reported in literature [45]. This relevant permeation increase could be ascribed to the bioadhesive liquid crystalline mesophase produced upon lacrimal dilution, which has a prolonged precorneal residence time and leads to intimate interaction with corneal epithelium like that described for GMO-based cubic phase systems [46]. Kinetic Analysis of Drug Release from PF6 (Figure 5) showed better fit with Hixson-Crowell model (R\u003csup\u003e2\u003c/sup\u003e = 0.9997) with Korsmeyer\u0026ndash;Peppas n value of 0.8798 indicating non-Fickian anomalous transport, which implies simultaneous diffusion and matrix erosion mechanisms, a drug release pattern characteristic of liquid crystalline systems as described by Chen et al. [47]. The non-irritant characteristics of PF6 were further validated by the HET-CAM assay in which it showed an irritation score of 0.38 \u0026plusmn; 0.04 (Table 10), comparable with those developed for puerarin-loaded proniosomal gels (IS = 0.43) [48], while superior cytocompatibility to free puerarin (IC\u003csub\u003e50\u003c/sub\u003e = 148.6 \u0026plusmn; 3.2 \u0026micro;g/mL) on HCE-T cells was achieved by MTT assay using PF6 at IC50 at a dose approximately \u0026gt;fourfold higher than the respective value as calculated from log dose versus cell viability profiles, as shown in Table 11 and Figure 7, a phenomenon that is consistent with encapsulation-induced cytoprotection known so far for lipid-based ocular nanocarriers [48,49]. As indicated by the stability results summary in Table 12, PF6 remained physically and chemically stable under all tested conditions with a maximum drug content variation of only 1.83% at the accelerated storage scenario, significantly lower than ICH Q1A(R2) guidelines for clinical translation suitability [50].\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThis study focused on the formulation of puerarin-loaded in situ liquid crystal gels for sustained ocular delivery in dry eye disease, which were successfully developed and optimized by using a 3\u003csup\u003e2\u003c/sup\u003e full factorial design. Using the solubility and optical transparency of the transformed ingredients, an optimal formula was identified as PF6 with 60% w/w GMO and 15% w/w Soluplus\u0026reg;, which yielded favourable physicochemical properties, corneal permeation (76.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.43% at 8 h) superior to other formulations, long term drug release through a non-Fickian anomalous transport mechanism as well as excellent ocular safety in both HET-CAM and MTT assays. This combination showed a desirability of 1.0 with percentage relative errors below 2.5%, thus confirming the robustness of the optimization strategy. Stability studies established physical and chemical integrity over three months under various storage conditions. PF6 is clinically advantageous due to low-frequency dosing, sustained precorneal residence time and better patient compliance than conventional eye drops. In vivo pharmacokinetic and pharmacodynamic studies in dry eye models are now necessary to confirm the developed formulation's therapeutic efficacy, as well as its clinical translation potential.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eDED: Dry Eye Disease; LCG: Liquid Crystal Gel; GMO: Glyceryl Monooleate; STF: Simulated Tear Fluid; DSC: Differential Scanning Calorimetry; FTIR: Fourier Transform Infrared Spectroscopy; HET-CAM: Hen\u0026apos;s Egg Test on Chorioallantoic Membrane; MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide; HCE-T: Human Corneal Epithelial Cell Line; IC\u003csub\u003e50\u003c/sub\u003e: Inhibitory Concentration 50%; EE: Entrapment Efficiency; PLM: Polarized Light Microscopy; ANOVA: Analysis of Variance; SD: Standard Deviation; ICH: International Council for Harmonisation; CPCSEA: Committee for the Purpose of Control and Supervision of Experiments on Animals; NF-\u0026kappa;B: Nuclear Factor Kappa B; Kp: Permeability Coefficient; IS: Irritation Score; SLS: Sodium Lauryl Sulfate; w/w: Weight by Weight; \u0026lambda;\u003csub\u003emax\u003c/sub\u003e: Maximum Absorption Wavelength; RH: Relative Humidity; IP: Indian Pharmacopoeia.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003ePallavi Desale - Conducting experiments, Wrote main manuscript,Data Curation,Amol S.Rakte - Supervision, Methodology,Data analysis, statistics, Investigation, Review \u0026amp; Editing of manuscriptSanjay R.Arote -Visualization\u003c/p\u003e\u003ch2\u003eFinding\u003c/h2\u003e\n\u003cp\u003eThis Research Received No External Funding\u003c/p\u003e\n\u003ch2\u003eEthics Declaration\u003c/h2\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHarrell CR, Feulner L, Djonov V, Pavlovic D, Volarevic V. The Molecular Mechanisms Responsible for Tear Hyperosmolarity-Induced Pathological Changes in the Eyes of Dry Eye Disease Patients. Cells 2023;12:2755. https://doi.org/10.3390/cells12232755.\u003c/li\u003e\n\u003cli\u003eAragona P, Barabino S, Di Zazzo A, Giannaccare G, Villani E, Aiello F, et al. Dry Eye Disease: From Causes to Patient Care and Clinical Collaboration\u0026mdash;A Narrative Review. Ophthalmol Ther 2025;14:1411\u0026ndash;28. https://doi.org/10.1007/s40123-025-01161-8.\u003c/li\u003e\n\u003cli\u003eRolando M, Merayo-Lloves J. Management Strategies for Evaporative Dry Eye Disease and Future Perspective. Curr Eye Res 2022;47:813\u0026ndash;23. https://doi.org/10.1080/02713683.2022.2039205.\u003c/li\u003e\n\u003cli\u003eSen H, Durgapal S, Jakhmola V. Molecular landscape connecting complications of dry eye disease: mechanisms, therapeutic targets, biomarkers, and future innovations. Int Ophthalmol 2026;46:97. https://doi.org/10.1007/s10792-026-03940-z.\u003c/li\u003e\n\u003cli\u003eConte R, De Luca I, Calarco A, Finicelli M, Peluso G. Polyphenol-Loaded Nanodevices as Innovative Therapeutic Strategies for Dry Eye Disease: Advances and Perspectives. Antioxidants 2025;14:1280. https://doi.org/10.3390/antiox14111280.\u003c/li\u003e\n\u003cli\u003eSheppard JD, Nichols KK. Dry Eye Disease Associated with Meibomian Gland Dysfunction: Focus on Tear Film Characteristics and the Therapeutic Landscape. Ophthalmol Ther 2023;12:1397\u0026ndash;418. https://doi.org/10.1007/s40123-023-00669-1.\u003c/li\u003e\n\u003cli\u003eChauhan P, Wadhwa K, Mishra R, Gupta S, Ahmad F, Kamal M, et al. Investigating the Potential Therapeutic Mechanisms of Puerarin in Neurological Diseases. Mol Neurobiol 2024;61:10747\u0026ndash;69. https://doi.org/10.1007/s12035-024-04222-4.\u003c/li\u003e\n\u003cli\u003eLiga S, Paul C. Puerarin\u0026mdash;A Promising Flavonoid: Biosynthesis, Extraction Methods, Analytical Techniques, and Biological Effects. Int J Mol Sci 2024;25:5222. https://doi.org/10.3390/ijms25105222.\u003c/li\u003e\n\u003cli\u003eYu C-C, Du Y-J, Li J, Li Y, Wang L, Kong L-H, et al. Neuroprotective Mechanisms of Puerarin in Central Nervous System Diseases: Update. Aging Dis 2022;13:1092\u0026ndash;105. https://doi.org/10.14336/AD.2021.1205.\u003c/li\u003e\n\u003cli\u003eZhang N, Guo P, Zhao Y, Qiu X, Shao S, Liu Z, et al. Pharmacological mechanisms of puerarin in the treatment of Parkinson\u0026rsquo;s disease: An overview. Biomed Pharmacother 2024;177:117101. https://doi.org/10.1016/j.biopha.2024.117101.\u003c/li\u003e\n\u003cli\u003eSaklani R, Yadav PK, Nengroo MA, Gawali SL, Hassan PA, Datta D, et al. An Injectable In Situ Depot-Forming Lipidic Lyotropic Liquid Crystal System for Localized Intratumoral Drug Delivery. Mol Pharm 2022;19:831\u0026ndash;42. https://doi.org/10.1021/acs.molpharmaceut.1c00782.\u003c/li\u003e\n\u003cli\u003eLotfy NM, Ahmed MA, El Hoffy NM, Bendas ER, Morsi NM. Development and optimization of amphiphilic self-assembly into nanostructured liquid crystals for transdermal delivery of an antidiabetic SGLT2 inhibitor. Drug Deliv 2022;29:3340\u0026ndash;57. https://doi.org/10.1080/10717544.2022.2144546.\u003c/li\u003e\n\u003cli\u003eRajabi N, Scarfo MG, Fredericks CM, Herrera Restrepo RS, Adibi A, Shahsavan H. From Anisotropic Molecules and Particles to Small-Scale Actuators and Robots: An Account of Polymerized Liquid Crystals. Acc Mater Res 2024;5:1520\u0026ndash;31. https://doi.org/10.1021/accountsmr.4c00187.\u003c/li\u003e\n\u003cli\u003eWang M, Huang X, Yang H. Photothermal-Responsive Crosslinked Liquid Crystal Polymers. Macromol Mater Eng 2023;308:2300061. https://doi.org/10.1002/mame.202300061.\u003c/li\u003e\n\u003cli\u003eHooli S, Sutar KP, Sammasagi SS, Chouhan MK. Quality-by-Design-Driven Stability-Indicating Reverse-Phase High-Performance Liquid Chromatographic Method for Puerarin Quantification in Marketed Formulation and Lipid\u0026ndash;Polymer Nanoparticles. Chromatographia 2025;88:743\u0026ndash;57. https://doi.org/10.1007/s10337-025-04427-2.\u003c/li\u003e\n\u003cli\u003eRahimpour E, Moradi M, Sheikhi-Sovari A, Rezaei H, Rezaei H, Jouyban-Gharamaleki V, et al. Comparative Drug Solubility Studies Using Shake-Flask Versus a Laser-Based Robotic Method. AAPS PharmSciTech 2023;24:207. https://doi.org/10.1208/s12249-023-02667-9.\u003c/li\u003e\n\u003cli\u003eGhanbari E, Picken SJ, van Esch JH. Analysis of differential scanning calorimetry (DSC): determining the transition temperatures, and enthalpy and heat capacity changes in multicomponent systems by analytical model fitting. J Therm Anal Calorim 2023;148:12393\u0026ndash;409. https://doi.org/10.1007/s10973-023-12356-1.\u003c/li\u003e\n\u003cli\u003eLynch JM, Corniuk RN, Brignac KC, Jung MR, Sellona K, Marchiani J, et al. Differential scanning calorimetry (DSC): An important tool for polymer identification and characterization of plastic marine debris. Environ Pollut 2024;346:123607. https://doi.org/10.1016/j.envpol.2024.123607.\u003c/li\u003e\n\u003cli\u003eJozanikohan G, Abarghooei MN. The Fourier transform infrared spectroscopy (FTIR) analysis for the clay mineralogy studies in a clastic reservoir. J Pet Explor Prod Technol 2022;12:2093\u0026ndash;106. https://doi.org/10.1007/s13202-021-01449-y.\u003c/li\u003e\n\u003cli\u003eMontoya-Escobar N, Ospina-Acero D, Vel\u0026aacute;squez-Cock JA, G\u0026oacute;mez-Hoyos C, Serpa Guerra A, Ga\u0026ntilde;an Rojo PF, et al. Use of Fourier Series in X-ray Diffraction (XRD) Analysis and Fourier-Transform Infrared Spectroscopy (FTIR) for Estimation of Crystallinity in Cellulose from Different Sources. Polymers 2022;14:5199. https://doi.org/10.3390/polym14235199.\u003c/li\u003e\n\u003cli\u003eHu L, Xu Y, Meng H. Development and Evaluation of Puerarin Loaded-Albumin Nanoparticles Thermoresponsive in situ Gel for Ophthalmic Delivery. Drug Des Devel Ther 2022;16:3315\u0026ndash;26. https://doi.org/10.2147/DDDT.S374061.\u003c/li\u003e\n\u003cli\u003eLiga S, Tămaș A, Vodă R, Rusu G, B\u0026icirc;tcan I, Socoliuc V, et al. Puerarin-Loaded Proniosomal Gel: Formulation, Characterization, In Vitro Antimelanoma Cytotoxic Potential, and In Ovo Irritation Assessment. Gels 2026;12:72. https://doi.org/10.3390/gels12010072.\u003c/li\u003e\n\u003cli\u003ePolat HK, \u0026Uuml;nal S, Aytekin E, Karakuyu NF, Pezik E, Haydar MK, et al. Formulation development of Lornoxicam loaded heat triggered ocular in-situ gel using factorial design. Drug Dev Ind Pharm 2023;49:601\u0026ndash;15. https://doi.org/10.1080/03639045.2023.2264932.\u003c/li\u003e\n\u003cli\u003eKassab HJ, Alkufi HK, Hussein LS. Use of factorial design in formulation and evaluation of intrarectal in situ gel of sumatriptan. J Adv Pharm Technol Res 2023;14:119. https://doi.org/10.4103/japtr.japtr_603_22.\u003c/li\u003e\n\u003cli\u003eHani U, Rahamathulla M, Osmani RAM, Begum MY, Wahab S, Ghazwani M, et al. Development and Characterization of Oral Raft Forming In Situ Gelling System of Neratinib Anticancer Drug Using 32 Factorial Design. Polymers 2022;14:2520. https://doi.org/10.3390/polym14132520.\u003c/li\u003e\n\u003cli\u003eSzalai B, J\u0026oacute;j\u0026aacute;rt-Laczkovich O, Kov\u0026aacute;cs A, Berk\u0026oacute; S, Balogh GT, Katona G, et al. Design and Optimization of In Situ Gelling Mucoadhesive Eye Drops Containing Dexamethasone. Gels 2022;8:561. https://doi.org/10.3390/gels8090561.\u003c/li\u003e\n\u003cli\u003eBondre RM, Kanojiya PS, Wadetwar RN, Kangali PS. Sustained vaginal delivery of in situ gel containing Voriconazole nanostructured lipid carrier: formulation, in vitro and ex vivo evaluation. J Dispers Sci Technol 2023;44:1466\u0026ndash;78. https://doi.org/10.1080/01932691.2021.2022489.\u003c/li\u003e\n\u003cli\u003eIbrahim TM, Ayoub MM, El-Bassossy HM, El-Nahas HM, Gomaa E. Investigation of Alogliptin-Loaded In Situ Gel Implants by 23 Factorial Design with Glycemic Assessment in Rats. Pharmaceutics 2022;14:1867. https://doi.org/10.3390/pharmaceutics14091867.\u003c/li\u003e\n\u003cli\u003eTalianu M-T, Dinu-P\u0026icirc;rvu C-E, Ghica MV, Anuţa V, Prisada RM, Popa L. Development and Characterization of New Miconazole-Based Microemulsions for Buccal Delivery by Implementing a Full Factorial Design Modeling. Pharmaceutics 2024;16:271. https://doi.org/10.3390/pharmaceutics16020271.\u003c/li\u003e\n\u003cli\u003eGowda CM, Wairkar S. Azelaic acid-based lyotropic liquid crystals gel for acne vulgaris: Formulation optimization, antimicrobial activity and dermatopharmacokinetic study. Int J Pharm 2024;667:124879. https://doi.org/10.1016/j.ijpharm.2024.124879.\u003c/li\u003e\n\u003cli\u003ePriya S, Srividya G, Chandra NS, Singh PP, Saha RN, Sathe P, et al. Loteprednol etabonate loaded lyotropic liquid crystalline nanoparticles in-situ ophthalmic gel: Qbd driven optimization and in-vitro, ex-vivo evidence of sustained precorneal residence time. J Drug Deliv Sci Technol 2023;89:105081. https://doi.org/10.1016/j.jddst.2023.105081.\u003c/li\u003e\n\u003cli\u003eRapalli VK, Tomar Y, Sharma S, Roy A, Singhvi G. Apremilast loaded lyotropic liquid crystalline nanoparticles embedded hydrogel for improved permeation and skin retention: An effective approach for psoriasis treatment. Biomed Pharmacother 2023;162:114634. https://doi.org/10.1016/j.biopha.2023.114634.\u003c/li\u003e\n\u003cli\u003eImane M, Rachid D, Habib BM. Assessment of ocular irritation potential of moisturizing and sunscreen cosmetic creams marketed in Algeria using the HET-CAM test. Toxicol In Vitro 2026;111:106168. https://doi.org/10.1016/j.tiv.2025.106168.\u003c/li\u003e\n\u003cli\u003ede Araujo Lowndes Viera LM, Silva RS, da Silva CC, Presgrave OAF, Boas MHSV. Comparison of the different protocols of the Hen\u0026rsquo;s Egg Test-Chorioallantoic Membrane (HET-CAM) by evaluating the eye irritation potential of surfactants. Toxicol In Vitro 2022;78:105255. https://doi.org/10.1016/j.tiv.2021.105255.\u003c/li\u003e\n\u003cli\u003eIdogwu EI, Kazarova S, Kormos \u0026Eacute;. Using the HET-CAM test in determining the eye irritation potential of some pesticides. GEORGIKON Agric 2023;27:84\u0026ndash;91.\u003c/li\u003e\n\u003cli\u003eCorr\u0026ecirc;a G de OP, Marcato DC, Ramos WS, Corr\u0026ecirc;a MA, Cicarelli RMB, Isaac VLB. \u003cem\u003eIn vitro\u003c/em\u003e evaluation of the cytotoxicity and eye irritation potential of preservatives widely used in cosmetics. Braz J Pharm Sci 2022;58:e20039. https://doi.org/https://doi.org/10.1590/s2175-97902022e20039.\u003c/li\u003e\n\u003cli\u003eZheng J, Zhang Y, Zhang S. Sustained release of azithromycin from lipid liquid-crystalline nanoparticles laden in situ gel for the treatment of periodontitis: In vitro and efficacy study. J Biomater Appl 2022;37:482\u0026ndash;92. https://doi.org/10.1177/08853282221095395.\u003c/li\u003e\n\u003cli\u003eYuan D, Li X, Yao H, Li Y, Zhu X, Zhao J, et al. A Liquid Crystal Ionomer-Type Electrolyte toward Ordering-Induced Regulation for Highly Reversible Zinc Ion Battery. Adv Sci 2023;10:2206469. https://doi.org/10.1002/advs.202206469.\u003c/li\u003e\n\u003cli\u003eKato T, Uchida J, Ishii Y, Watanabe G. Aquatic Functional Liquid Crystals: Design, Functionalization, and Molecular Simulation. Adv Sci 2024;11:2306529. https://doi.org/10.1002/advs.202306529.\u003c/li\u003e\n\u003cli\u003eWhat Is Glyceryl Monooleate and Why Is It Used in Pharmaceuticals? - Alfa Chemistry n.d. https://www.alfa-chemistry.com/resources/what-is-glyceryl-monooleate-and-why-is-it-used-in-pharmaceuticals.html (accessed March 20, 2026).\u003c/li\u003e\n\u003cli\u003eLai J, Chen J, Lu Y, Sun J, Hu F, Yin Z, et al. Glyceryl monooleate/poloxamer 407 cubic nanoparticles as oral drug delivery systems: I. In vitro evaluation and enhanced oral bioavailability of the poorly water-soluble drug simvastatin. AAPS PharmSciTech 2009;10:960\u0026ndash;6. https://doi.org/10.1208/s12249-009-9292-4.\u003c/li\u003e\n\u003cli\u003eChen Y, Lu Y, Zhong Y, Wang Q, Wu W, Gao S. Ocular delivery of cyclosporine A based on glyceryl monooleate/poloxamer 407 liquid crystalline nanoparticles: preparation, characterization, in vitro corneal penetration and ocular irritation. J Drug Target 2012;20:856\u0026ndash;63. https://doi.org/10.3109/1061186X.2012.723214.\u003c/li\u003e\n\u003cli\u003eTarsitano M, Mancuso A, Cristiano MC, Urbanek K, Torella D, Paolino D, et al. Perspective use of bio-adhesive liquid crystals as ophthalmic drug delivery systems. Sci Rep 2023;13:16188. https://doi.org/10.1038/s41598-023-42185-z.\u003c/li\u003e\n\u003cli\u003eAboali FA, Habib DA, Elbedaiwy HM, Farid RM. Curcumin-loaded proniosomal gel as a biofreindly alternative for treatment of ocular inflammation: \u003cem\u003eIn-vitro\u003c/em\u003e and \u003cem\u003ein-vivo\u003c/em\u003e assessment. Int J Pharm 2020;589:119835. https://doi.org/10.1016/j.ijpharm.2020.119835.\u003c/li\u003e\n\u003cli\u003eKouchak M, Mahmoodzadeh M, Farrahi F. Designing of a pH-Triggered Carbopol\u0026reg;/HPMC In Situ Gel for Ocular Delivery of Dorzolamide HCl: In Vitro, In Vivo, and Ex Vivo Evaluation. AAPS PharmSciTech 2019;20:210. https://doi.org/10.1208/s12249-019-1431-y.\u003c/li\u003e\n\u003cli\u003eGonjari ID, Karmarkar AB, Khade TS, Hosmani AH, Navale RB. Use of factorial design in formulation and evaluation of ophthalmic gels of gatifloxacin: Comparison of different mucoadhesive polymers. Drug Discov Ther 2010;4:423\u0026ndash;34.\u003c/li\u003e\n\u003cli\u003eChen Y, Lu Y, Zhong Y, Wang Q, Wu W, Gao S. Ocular delivery of cyclosporine A based on glyceryl monooleate/poloxamer 407 liquid crystalline nanoparticles: preparation, characterization, in vitro corneal penetration and ocular irritation. J Drug Target 2012;20:856\u0026ndash;63. https://doi.org/10.3109/1061186X.2012.723214.\u003c/li\u003e\n\u003cli\u003eOtte A, B\u0026aacute;ez-Santos YM, Mun EA, Soh B-K, Lee Y, Park K. The \u003cem\u003ein vivo\u003c/em\u003e transformation and pharmacokinetic properties of a liquid crystalline drug delivery system. Int J Pharm 2017;532:345\u0026ndash;51. https://doi.org/10.1016/j.ijpharm.2017.08.098.\u003c/li\u003e\n\u003cli\u003eGilleron L, Coecke S, Sysmans M, Hansen E, Van Oproy S, Marzin D, et al. Evaluation of a modified HET-CAM assay as a screening test for eye irritancy. Toxicol In Vitro 1996;10:431\u0026ndash;46. https://doi.org/10.1016/0887-2333(96)00021-5.\u003c/li\u003e\n\u003cli\u003eLiga S, Tămaș A, Vodă R, Rusu G, B\u0026icirc;tcan I, Socoliuc V, et al. Puerarin-Loaded Proniosomal Gel: Formulation, Characterization, In Vitro Antimelanoma Cytotoxic Potential, and In Ovo Irritation Assessment. Gels 2026;12:72. https://doi.org/10.3390/gels12010072. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Dry eye disease, Puerarin, In situ liquid crystal gel, Glyceryl monooleate, Soluplus®, Factorial design, Corneal permeation, Ocular drug delivery","lastPublishedDoi":"10.21203/rs.3.rs-9256829/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9256829/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjectives:\u003c/strong\u003e To design and optimize puerarin-loaded LCGs for controlled ocular delivery in DED, elucidating the drawbacks inherent to conventional ophthalmic vehicles regarding corneal bioavailability and precorneal elimination.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eNine different formulations (PF1–PF9) were developed via a modified fusion-vortex method where glyceryl monooleate (GMO) and Soluplus® were used as independent variables in a 3\u003csup\u003e2\u003c/sup\u003e full factorial design. The formulations were examined for physicochemical characteristics, drug release kinetics, ex vivo corneal permeation, ocular irritation (HET-CAM), cytotoxicity (MTT assay in HCE-T cells), and stability according to ICH Q1A(R2) guidelines.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe optimization was carried out by the desirability function approach using Design Expert® v.13. Acceptable pH (7.14–7.26), drug content (96.4–98.6%) and entrapment efficiency (82.3–94.3%) were achieved by all the formulations. Corneal permeation and viscosity were well described by quadratic (R\u003csup\u003e2\u003c/sup\u003eadj = 0.9977) and linear models (R\u003csup\u003e2\u003c/sup\u003eadj = 0.9954), respectively. The optimum formulation PF6 (GMO 60% w/w, Soluplus® 15% w/w) possessed the highest desirability (D = 1), cumulative corneal permeation of 76.4 ± 2.43% in 8 hours with flux of 52.34 ± 1.78 μg/cm\u003csup\u003e2\u003c/sup\u003e/h and precursor viscosity of 121.4 ± 3.7 mPa·s with non-Fickian anomalous transport qualified as the best fit for drug release profile (R\u003csup\u003e2\u003c/sup\u003e = 0.9997). HET-CAM non-irritant classification was confirmed (IS = 0.38 ± 0.04), and IC\u003csub\u003e50\u003c/sub\u003e of PF6 (182.4 ± 4.1 µg/mL) was greater than free puerarin (148.6 ± 3.2 µg/mL). Less than 3.5% variation was found in the stability studies over three months.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e PF6 exhibited superior corneal permeation, prolonged release, and good ocular safety, indicating its potential as a viable candidate therapeutic platform for DED treatment worth further in vivo validation.\u003c/p\u003e","manuscriptTitle":"Development and Optimization of Puerarin-loaded in situ liquid crystal gels for ocular delivery in dry eye disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-07 07:47:01","doi":"10.21203/rs.3.rs-9256829/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a1923afb-1e42-4305-9c23-1c3a50b3ac47","owner":[],"postedDate":"April 7th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-08T11:43:45+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-07 07:47:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9256829","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9256829","identity":"rs-9256829","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}