Optimization of Nanostructured Lipid Carrier Using Central Composite Design for Ocular Delivery of Pirfenidone

Optimization of Nanostructured Lipid Carrier Using Central Composite Design for Ocular Delivery of Pirfenidone | 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 Optimization of Nanostructured Lipid Carrier Using Central Composite Design for Ocular Delivery of Pirfenidone Vivek Basudkar, Sankalp Gharat, Afiya Baig, Munira Momin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7107100/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Jan, 2026 Read the published version in Journal of Pharmaceutical Innovation → Version 1 posted You are reading this latest preprint version Abstract Purpose: Pirfenidone (PFD) possesses significant anti-inflammatory and anti-fibrotic properties, making it a promising therapeutic agent for ocular fibrotic conditions. However, its clinical application is limited due to a short half-life (< 19 minutes) in corneal tissue and poor ocular bioavailability. This study aimed to develop and characterize a nanostructured lipid carrier (NLC) based in situ gel formulation for enhancing the ocular delivery and therapeutic efficacy of PFD. Methods: NLCs were prepared via hot melt emulsification followed by probe sonication and incorporated into a gellan gum-based in situ gel system. A central composite design was used for formulation optimization. The optimized NLC formulation included Compritol® 888 ATO (1.64%), Capmul® MCM (0.32%), Poloxamer 188 (0.5%), and Tween 80 (0.5%). The formulation was characterized for particle size, polydispersity index (PDI), zeta potential, entrapment efficiency, in vitro drug release, ex vivo permeation, cytotoxicity, bioadhesion, and biocompatibility using HET-CAM assay. Stability studies were also conducted. Results: The optimized PFD-loaded NLCs showed a particle size of 90.15 ± 10.2 nm, PDI of 0.155 ± 0.014, zeta potential of -11.4 ± 1.2 mV, and entrapment efficiency of 90.43 ± 2.14%. In vitro release studies demonstrated sustained drug release (84.39 ± 3.41% over 12 hours). Ex vivo corneal and scleral permeation were 82.97 ± 3.01% and 77.01 ± 1.98%, respectively. Biocompatibility, cytotoxicity, and stability assessments confirmed the safety and robustness of the formulation. Conclusion: The developed NLC-based in situ gel offers a promising strategy for enhancing the ocular bioavailability and therapeutic potential of PFD, potentially overcoming the limitations of conventional topical administration. Pirfenidone Nanostructured lipid carrier Cornea Central Composite Design Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. INTRODUCTION The transparent cornea, which makes up the anterior surface of the eye, protects the eye's internal structures and produces two thirds of its refractive power. Corneal transparency plays a significant role in optimal vision and effective transmission of incident light into the eye. However, the anatomical location of cornea increases the risk of injury to chemical, burns or surgery. Long-term effects of corneal injury causes corneal haze which can lead to permanent vision loss. Corneal haze is a fibrotic condition in which the cornea loses its transparency and becomes opaque and cloudy, which leads to corneal blindness 1 . This can have a substantial impact on quality life of humans 2 . Irreparable loss of corneal transparency is one of the major cause of blindness 3 . Corneal injury can occur due to alkali burn or post-operative glaucoma surgery 4 , 5 . Transforming growth factor beta 1 (TGFβ1), as a key inflammatory cytokine, promotes post injuries corneal fibrosis. TGFβ1 and platelet-derived growth factor (PDGF) secreted from corneal inflammatory epithelial cells, stimulate and accelerate the proliferation of stromal cells and their differentiation to myofibroblasts 6 , 7 . Myofibroblasts with alpha‐smooth muscle actin (α‐SMA) feature by scattering the incident light, may cause corneal haze propagation 8 . Additionally, TGFβ1 increases the secretion of collagen type І and fibronectin from myofibroblasts and fibroblasts. This may serve as another cause of light scattering and corneal haze after injuries 9 . Corneal neovascularization is an additional reason for corneal opacity after deep injury due to an imbalance between angiogenic (e.g., vascular endothelial growth factor [VEGF]) and antiangiogenic (e.g., thrombospondin 1) factors 10 . Pirfenidone (PFD) (5-methyl-1-phenyl-2-[1H]-pyridone) is therapeutic agent that exhibits anti-inflammatory, antifibrotic effect and modulates cellular oxidation in pre-clinical as well clinical studies in idiopathic pulmonary fibrosis, renal fibrosis, multiple sclerosis 3 , 11 . Its antifibrotic action is primarily attributed to its antagonism of fibroblast proliferation and migration and the reduction of extracellular matrix deposits 12 . PFD exhibits anti-inflammatory activity by inhibiting TGF-β, TNF-α synthesis, collagen I and III production, and fibroblast proliferation. PFD also stimulates release of matrix metalloproteinases and collagenases, which promotes the anti-fibrotic activity 13 . However, when it was investigated the pharmacokinetics of 0.5% pirfenidone as a topically administered solution in eyes of rabbits, it exhibited a short half-life of less than 19 min in cornea tissue which may result in low bioavailability. This can be overcome by incorporating PFD in nano formulation like NLC due to its high drug loading capacity 14 . Nanostructured lipid carrier (NLC) are lipidic nano drug delivery system formulated by replacing solid lipids with liquid lipids in solid lipid nanoparticles (SLN). Compared to rest of the conventional carriers, NLC has numerous advantages, involving high drug bioavailability, high drug loading, biodegradability, good tolerance. Moreover, they are comparatively easy to scale up on a large manufacturing than other nanoformulations 14 , 15 , 26 . NLCs are primarily aqueous dispersions, resulting in a low precorneal retention time which can be overcome by in situ gels. Ophthalmic in situ gels are sol-gel preparations that are converted into gel after administration due to changes in the environment. This leads to prolong drug residence time and decrease in frequency of drug administration 16 , 26 . An ophthalmic formulation combining the benefits of NLCs with the benefits of an in-situ gel system was developed. In the present study, PFD loaded NLCs were formulated using hot melt emulsification followed by probe sonication. Central composite design (CCD) was used to systematically optimize the NLCs. Optimized PFD-NLCs were developed as in situ gel. The developed formulation was sterilized using membrane filtration and evaluated on various parameters like Hen’s egg test using choriollantoic membrane (HET CAM’s) test, bioadhesion and in vitro cytotoxicity by MTT assay etc. 2. MATERIALS AND METHODS 2.1 Materials Pirfenidone was obtained as a gift sample from Cipla Ltd., Mumbai India; Compritol ATO 888 was obtained as a gift sample from Gattefosse India Pvt. Ltd.; Glyceryl monostearate and Isopropyl Myristate was provided as a gift sample by Arihant Innochem Pvt. Ltd., Mumbai India; Cetyl palmitate, Tween 20, Tween 40, Tween 60, Tween 80, Span 20, Span 60, Span 80, were received as gift sample from Mohini Organics Pvt. Ltd., Mumbai, India; Captex® 200, Captex® 355 and Capmul® MCM, were received as a gift sample by Abitec Corporation, Mumbai India; Sesame oil and Soyabean oil were gifted by Croda India Company Pvt. Ltd.; Kolliphor® ELP, Kolliphor® RH 40, Kolliphor® HS 15, and Poloxamer 188 were gifted by BASF Pvt. Ltd., Mumbai India; Castor oil was purchased from Jayant Agro-Organics Limited, Mumbai India; Ethyl oleate, Oleic acid and Transcutol P purchased from Otto Chemie Pvt. Ltd, Mumbai India, All solvents were purchased form S.D. Fines Pvt Ltd, India. 2.2 Methods 2.2.1 Screening of solid lipids, liquid lipids, and surfactants Solubility studies of pirfenidone (PFD) were conducted in various solid lipids, liquid lipids, and surfactants (n = 3). To assess PFD solubility in solid lipids (Compritol® 888 ATO, glyceryl monostearate, and cetyl palmitate), each lipid (1 g) was melted separately in vials at a temperature 10°C above its melting point. PFD was incrementally added until the lipid reached saturation. The solubility of PFD in liquid lipids (Capmul® MCM, Captex® 200, Captex® 355, castor oil, ethyl oleate, isopropyl myristate, oleic acid, sesame oil, and soybean oil) and surfactants (Kolliphor® ELP, Kolliphor® HS 15, Kolliphor® RH40, Poloxamer 188, Span 20, Span 60, Span 80, Transcutol® P, Tween 20, Tween 40, Tween 60, and Tween 80) was determined using saturation solubility studies. Briefly, a known quantity of PFD was added to 1 g of each liquid lipid and surfactant in separate vials. The mixtures were vortexed, incubated in an orbital shaker for 72 hours, and subsequently centrifuged at 7500 rpm for 15 minutes. The supernatant was diluted with methanol and analyzed using a UV spectrophotometer at 317 nm to quantify the solubility of PFD. 2.2.2 Determination of compatibility between Solid lipids and liquid lipids: Solid and liquid lipids exhibiting maximum solubility for pirfenidone (PFD) were combined in varying ratios (6:4, 7:3, 8:2, and 9:1) to evaluate their physical compatibility. Accurately weighed quantities (0.5 g) of each solid and liquid lipid were transferred into glass vials and heated to a temperature 10°C above the melting point of the solid lipid to ensure complete melting. The mixtures were then allowed to cool to ambient temperature, and phase separation was assessed after 24 hours. 17 . 2.2.3 Preparation of Pirfenidone loaded Nanostructured lipid carrier (PFD-NLCs) PFD-NLCs were prepared using the hot melt emulsification technique followed by probe sonication 18 . A predetermined quantity of solid lipid, liquid lipid, and pirfenidone (lipid phase) was heated in a beaker to a temperature 10°C above the melting point of the solid lipid. Simultaneously, the aqueous phase was prepared by dissolving a specified amount of surfactant in water and heating it to the same temperature (78–82°C). The aqueous phase was then gradually added to the lipid phase under continuous stirring at 750 rpm for 30 minutes, maintaining the temperature at 78–82°C to facilitate emulsification. The resulting emulsion was further homogenized using probe sonication at 70% amplitude for 7 minutes to achieve nanoscale particle size. 2.2.4 Optimization of PFD-NLCs using Central composite design (CCD) Various Design of Experiments (DoE) approaches have proven valuable in evaluating the impact of individual process variables on the performance of selected formulations. In this study, a two-level full factorial central composite design (CCD) was employed to optimize the formulation of pirfenidone-loaded nanostructured lipid carriers (PFD-NLCs). The CCD was generated using Minitab software (version 20.2.0). Based on preliminary screening of various formulation and process parameters, critical process parameters that significantly influenced the performance of PFD-NLCs were identified. The CCD consisted of factorial design points, axial (star) points, and center points. The concentration of solid lipid and liquid lipid were selected as independent variables, while particle size (PS), zeta potential (ZP), polydispersity index (PDI), and percentage entrapment efficiency (%EE) were designated as dependent variables. 2.2.5 Characterization and evaluation of PFD-NLC The developed NLC were characterized by PS, PDI, (ZP) and %EE. The morphological characterization of the optimized formulation was evaluated by Transmission electron microscopy analysis. 2.2.6 Particle size (PS), polydispersity index (PDI)and zeta potential (ZP) The particle size was determined using Zetasizer (Nano-ZS, Malvern) Instrument. The test samples were diluted with purified water and measured at a 90° scattering angle at 25°C in triplicate. Zeta potential was determined using the same instrument based on electrophoretic mobility. For zeta potential measurement, the test samples were similarly diluted with purified water to ensure appropriate conductivity and analyzed under identical conditions 19 , 20 . 2.2.7 Transmission electron microscopy (TEM) Morphological characterization of the nanostructured lipid carriers (NLCs) was performed using transmission electron microscopy (TEM) (Model-JEM-2100, JEOL). The NLCs, previously diluted with double-distilled water, were placed onto carbon-coated copper grids. The samples were stained with 1% (w/w) phosphotungstic acid and allowed to stain for 10 minutes before examination under the TEM. 2.2.8 In vitro drug diffusion release from PFD-NLC In vitro drug diffusion studies of the optimized formulation were conducted using a dialysis membrane with a molecular weight cutoff of 150 Daltons (Himedia) to quantify the release of pirfenidone (PFD). The Franz diffusion apparatus was used, with the receptor compartment filled with 22 mL of simulated tear fluid (STF) at pH 7.4. The temperature of the diffusion cell was maintained at 37 ± 0.5°C under continuous stirring at 100 rpm. A 1 g aliquot of the formulation was placed in the donor compartment and covered. At predetermined intervals (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 hours), samples were withdrawn, filtered, and analyzed for drug content using UV spectrophotometry. The cumulative percentage of drug released was plotted against time to evaluate the release profile. 2.2.9 Preparation of in situ gel loaded PFD-NLC A gellan gum solution was prepared by dispersing the required amount of gellan gum in an appropriate volume of purified water under continuous stirring until complete dissolution was achieved. Sodium chloride and benzalkonium chloride were then added at fixed concentrations to the solution, followed by filtration through a 0.22 µm polyvinylidene fluoride (PVDF) membrane filter. The filtered solution was subsequently incorporated into the polymer dispersion under continuous stirring to obtain a uniform and clear solution. Pirfenidone (PFD)-loaded nanostructured lipid carriers (NLCs) were then gradually dispersed into the prepared medium under continuous stirring at 750 rpm to ensure homogeneous dispersion. 2.2.10 Characterization of in situ gel loaded PFD-NLC The physicochemical properties of the developed PFD-NLC-based in situ gel were evaluated by assessing its physical appearance, pH, viscosity, and bioadhesive strength. In vitro and ex vivo drug diffusion studies were conducted using a Franz diffusion apparatus, employing simulated tear fluid (STF) at pH 7.4 as the receptor medium. A dialysis membrane (Himedia 150 Da) was utilized to assess the permeation characteristics of the formulation under controlled experimental conditions. 2.2.11 In vitro drug diffusion from in situ gel loaded PFD-NLC The in vitro drug diffusion study of the optimized formulation was conducted to evaluate the extent of drug permeation across a dialysis membrane (150 Da). The study was performed using a Franz diffusion apparatus, with the receptor compartment filled with simulated tear fluid (STF) at pH 7.4. The system was maintained at a constant temperature of 37 ± 0.5ºC and continuously stirred at 100 rpm to simulate physiological conditions. A precisely weighed 1 g aliquot of the formulation was placed in the donor compartment and securely covered to prevent evaporation or contamination. At predetermined intervals (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 hours), aliquots of the receptor medium were withdrawn, filtered, and analyzed for drug content using UV spectrophotometry. The cumulative percentage of drug released over time was plotted to assess the release kinetics of the formulation. 2.2.12 Ex vivo corneal and scleral drug diffusion profile of in situ gel loaded PFD-NLC The ex vivo drug permeation study was conducted using goat cornea to evaluate the transcorneal diffusion of PFD from the optimized PFD-NLC based in situ gel. Fresh goat eyeballs were procured from a local slaughterhouse and transported to the laboratory under cold conditions, ensuring constant saline immersion to maintain tissue viability. The cornea, along with the surrounding scleral tissue, was carefully excised and thoroughly rinsed with sterile saline to remove any residual debris. The diffusion study was performed using a Franz diffusion apparatus, where the excised goat cornea was mounted between the donor and receptor compartments with the epithelial side facing the donor chamber. The receptor compartment was filled with 22 mL of simulated tear fluid (STF, pH 7.4) and maintained at 37 ± 0.5ºC under continuous stirring at 100 rpm to mimic physiological ocular conditions. Precisely weighed 1 g of the formulation was placed in the donor compartment, ensuring uniform contact with the corneal membrane, and the setup was securely covered to prevent evaporation or contamination. At predetermined time intervals (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 hours), aliquots were withdrawn from the receptor compartment, filtered, and analyzed for drug content using UV spectrophotometry. The cumulative percentage of drug permeated across the cornea was plotted against time to assess the permeation profile. A similar experimental procedure was followed for ex vivo scleral permeation studies. 2.2.13 Flux and Permeability coefficient The steady-state flux (Jss) was determined by calculating the slope (Q) from the linear portion of the plot representing the cumulative amount of drug permeated per unit surface area of the membrane as a function of time (µg/cm 2 vs. h) 21 . The permeation coefficient (Kp​) was determined using the equation: Kp = Jss/C0 where C0 represents the initial concentration of the drug in the donor compartment. The flux and permeation coefficient were used to assess the permeability characteristics of the developed NLC-based in situ gel for ocular drug delivery. 2.2.14 Bioadhesive strength The bioadhesive strength of the developed NLC-based in situ gel was assessed using a modified bioadhesion apparatus. Freshly excised goat corneas were procured and carefully isolated for the study. One corneal section was securely affixed to a wooden platform mounted on a digital weighing balance using an inert adhesive, ensuring stable attachment. The second corneal section was mounted below the pan of the balance. A predetermined amount of the in situ gel formulation was applied between the two corneal sections to facilitate adhesion. Incremental weights were gradually added to the opposing pan until complete detachment of the corneal sections was observed. The bioadhesive strength was calculated using the following equation: Bioadhesive strength = Weight required (in kgs)/ Area (cm 2 ) 2.2.15 HEN’S Egg Test-Chorioallantoic Membrane (HET-CAM) study HET-CAM is a rapid and sensitive procedure to predict skin and ocular irritancy by evaluation of the changes in the CAM of the fertilized eggs. This is an alternative ex vivo toxicity evaluation technique for the in vivo Draize test on rabbit’s eye 22 , 23 . CAM comprises of complete laminate vascular system with arteries, veins and capillaries that are sensitive to harmful and corrosive substances with an inflammatory process. As per the ICCVAM-Recommended Test Method Protocol, the irritation potential of PFD loaded NLCs formulations were evaluated by the HET- CAM assay (ICCVAM 2010). Fertile White Leghorn chicken eggs weighing 50 to 60 grams were obtained from Central Poultry Development Organization, Mumbai. Nine-day old, fertilized eggs which were incubated in at 37.5 ± 0.5℃ and 62.5% ± 7.5% RH. Forceps were used to carefully detach the egg's shell from the air cell. After 5 min of saline moistening, the inner membrane directly in contact with the CAM was carefully removed using forceps. The experimentation method was validated using 0.1 N NaOH (negative control), 0.9% NaCl (positive control). 0.3mL of test samples were applied to the CAM and the irritation effect was studied visually for 5min. After adding each sample, the duration and severity of injuries were noted, and the irritation score (IS) was determined using the formula below.: \(\:IS\) = \(\:\frac{\left(301-tH\right)*5}{300}\) + \(\:\frac{\left(301-tL\right)*7}{300}\) + \(\:\frac{\left(301-tC\right)*9}{300}\) Where tH, tL and tC are time (in seconds) required for the occurrence of hemolysis, lysis and coagulation, respectively. The experimentation was performed in triplicate. 2.2.16 In vitro cytotoxicity studies The potential cytotoxic effects of the developed PFD-NLC formulation were evaluated using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. This assay measures cell viability by assessing mitochondrial activity, providing insights into possible adverse effects such as reduced cell membrane integrity, cell lysis, and apoptosis. The cytotoxicity study was conducted on Statens Seruminstitut Rabbit Cornea (SIRC) cell lines. Briefly, 200 µL of a cell suspension (1×10⁵ cells per well) was seeded into a 96-well plate (Sigma, Germany) and incubated at 37°C in a 5% CO₂ atmosphere for 24 hours to allow cell attachment. Subsequently, the cells were exposed to varying concentrations (50, 150, 250, 500, 800, 1000, and 1200 µg/mL) of pure pirfenidone (PFD) and PFD-loaded NLCs (PFD-NLC) and incubated for an additional 12 hours. Following exposure, the cells were treated with 0.5 mg/mL MTT reagent and incubated for 4 hours to facilitate formazan crystal formation. After incubation, the MTT reagent was carefully removed, and 100 µL of dimethyl sulfoxide (DMSO) was added to each well to solubilize the formazan crystals. The absorbance of the resulting solution was measured at 570 nm using a 96-well microplate reader (BioTek Synergy H1 Multimode Reader, USA). The IC₅₀ values were calculated to determine the concentration at which 50% of the cells remained viable, providing a quantitative measure of cytotoxicity. 2.2.17 Stability studies Stability studies were conducted in accordance with ICH Q1A (R2) guidelines to assess the physicochemical stability of the developed formulations over a period of three months under different storage conditions. The formulations were stored in sealed glass containers and subjected to low-temperature conditions (5°C ± 3°C), long-term stability conditions (25°C ± 2°C / 60% RH ± 5% RH), and accelerated stability conditions (40°C ± 2°C / 75% RH ± 5% RH). Following the storage period, the formulations were evaluated for any physical changes, variations in pH, and drug content to determine their stability and integrity under different environmental conditions. 3. RESULTS 3.1 Screening of solid lipids, liquid lipids and surfactants A solubility study was conducted to evaluate the capacity of various lipids to solubilize pirfenidone (PFD). The solubility profile of all excipients is presented in Figure. 1. Initially, solid lipids, liquid lipids, and surfactants were selected based on a literature review, followed by experimental screening. Among the solid lipids, cetyl palmitate exhibited a solubility of 12.2 ± 0.5 mg/g, whereas Compritol® 888 ATO and glyceryl monostearate demonstrated PFD solubility exceeding 100 mg/g. Capmul® MCM showed solubility greater than 100 mg/g, followed by oleic acid (84.3 ± 1.6 mg/g), while castor oil solubilized 2.1 ± 0.4 mg/g of PFD. Kolliphor® HS 15, Transcutol® P, and Span® 60 exhibited PFD solubility exceeding 100 mg/g, whereas Poloxamer 188 solubilized 3.2 ± 0.7 mg/g. Based on literature findings and solubility data, Compritol® 888 ATO and glyceryl monostearate were selected as solid lipids, Capmul® MCM as the liquid lipid, and Tween® 80 along with Poloxamer 188 as surfactants for the formulation of PFD-loaded nanostructured lipid carriers (PFD-NLC). 3.2 Solid lipid - Liquid lipid compatibility Compatibility studies were performed to evaluate the physical compatibility between selected solid and liquid lipids. Solid and liquid lipids were mixed in various ratios, heated, and subsequently maintained at ambient temperature for 24 hours. No phase separation was observed in any of the tested ratios, indicating physical stability. These findings suggest that Compritol® 888 ATO and glyceryl monostearate are compatible with Capmul® MCM, making them suitable lipid components for formulation development. 3.3 Formulation and optimization of Pirfenidone loaded Nanostructured lipid carrier Preliminary batches of nanostructured lipid carriers (NLCs) were formulated using the hot melt emulsification method, followed by probe sonication for homogenization. The preliminary studies indicated that the combination of solid lipid (Compritol® 888 ATO), liquid lipid (Capmul® MCM), and surfactants (Tween® 80 and Poloxamer 188) resulted in a stable formulation. Additionally, the processing parameters, including an amplitude of 70% and a sonication time of 7 minutes, were identified as optimal for NLC homogenization. Further investigations revealed that variations in the concentration of Compritol® 888 ATO (1.2–1.8%) and Capmul® MCM (0.8–0.2%) had a significant impact on particle size (PS), polydispersity index (PDI), zeta potential (ZP), and entrapment efficiency (%EE) of NLCs. To optimize the formulation, a Central Composite Design (CCD) was employed as the design of experiment (DoE). Based on CCD, 13 experimental batches were formulated, and the corresponding results are presented in (Table 1 ). Table 1 CCD generated by Minitab software with measured responses. B. No Comp ATO 888 (A) % w/w Capmul MCM (B) % w/w PSA (nm) PDI Zeta(mV) Entrapment EE (%) 1 1.200 0.200 67.80 ± 5.89 0.232 ± 0.029 -13.3 ± 0.55 81.97 ± 0.04 2 1.800 0.200 84.98 ± 0.59 0.197 ± 0.002 -11.3 ± 0.52 91.64 ± 0.95 3 1.200 0.800 289.40 ± 4.25 0.390 ± 0.014 -9.13 ± 0.62 89.08 ± 1.05 4 1.800 0.800 390.00 ± 6.50 0.276 ± 0.013 -6.84 ± 0.44 90.22 ± 1.66 5 1.075 0.500 246.30 ± 5.21 0.474 ± 0.006 -9.97 ± 0.91 78.86 ± 0.65 6 1.924 0.500 260.40 ± 2.44 0.440 ± 0.052 -9.2 ± 0.43 90.18 ± 1.22 7 1.500 0.075 71.75 ± 1.48 0.199 ± 0.007 -13.6 ± 1.04 79.44 ± 1.33 8 1.500 0.924 370.60 ± 2.77 0.374 ± 0.015 -7.3 ± 0.45 88.83 ± 0.82 9 1.500 0.500 137.16 ± 2.68 0.160 ± 0.014 -10.8 ± 0.70 89.21 ± 0.80 10 1.500 0.500 137.16 ± 2.68 0.160 ± 0.014 -10.8 ± 0.70 89.21 ± 0.80 11 1.500 0.500 137.16 ± 2.68 0.160 ± 0.014 -10.8 ± 0.70 89.21 ± 0.80 12 1.500 0.500 137.16 ± 2.68 0.160 ± 0.014 -10.8 ± 0.70 89.21 ± 0.80 13 1.500 0.500 137.16 ± 2.68 0.160 ± 0.014 -10.8 ± 0.70 89.21 ± 0.80 The influence of Compritol® 888 ATO and Capmul® MCM concentrations, along with their interactions, on the dependent variables was systematically evaluated. To establish a quantitative relationship between the independent factors and the responses, polynomial equations were generated. Additionally, contour plots were constructed (Figure. 2) to visually represent the effects of varying Compritol® 888 ATO and Capmul® MCM concentrations on PS, PDI, ZP, and %EE. 3.3.1 Effect of Compritol® 888 ATO and Capmul® MCM on Particle size The results of the Central Composite Design (CCD) indicated that the model was statistically significant, with particle size exhibiting a linear relationship with the selected factors. The concentration of Capmul® MCM was identified as a statistically significant factor influencing the particle size of NLCs, with an R² value of 97.06%, indicating a strong model fit. Analysis of the contour plot revealed that a decrease in the concentration of Capmul® MCM resulted in a reduction in particle size. Additionally, when the concentration of Compritol® 888 ATO was maintained between approximately 1.2% and 1.9%, the resulting NLCs exhibited a particle size below 100 nm. The polynomial equation describing the effect of Compritol® 888 ATO and Capmul® MCM on particle size is as follows: Equation 1: PSA = 1393–1751 comp 888 ATO − 338 capmul MCM + 564 comp 888 ATO *comp 888 ATO + 386 capmul MCM*capmul MCM + 232 comp 888 ATO *capmul MCM 3.3.2 Effect of Compritol® 888 ATO, Capmul® MCM on PDI The results demonstrated that the model was statistically significant, with the PDI exhibiting a linear relationship with the selected factors. The concentration of Capmul® MCM was identified as a statistically significant factor influencing the PDI of NLCs, with an R² value of 87.71%, indicating a good model fit. Analysis of the contour plot revealed that at lower concentrations of Capmul® MCM (< 0.7%), the PDI remained below 0.2. A similar trend was observed when the concentration of Compritol® 888 ATO was maintained between 1.3% and 1.7%, suggesting optimal conditions for achieving a monodisperse system. The polynomial equation describing the effect of Compritol® 888 ATO and Capmul® MCM on PDI is as follows: Equation 2: PDI = 3.225–4.106 comp 888 ATO + 0.100 capmul MCM + 1.378 comp 888 ATO *comp 888 ATO + 0.431 capmul MCM*capmul MCM − 0.219 comp 888 ATO *capmul MCM 3.3.3 Effect of Compritol® 888 ATO, Capmul® MCM on Zeta Potential It was observed that the model was statistically significant, and zeta potential exhibited a linear relationship. The concentrations of Compritol® 888 ATO and Capmul® MCM were statistically significant in determining the zeta potential of NLCs, with an R² value of 97.11%. The contour plot indicated that the zeta potential remained higher than − 13.5 when the concentration of Capmul® MCM was below 0.1% and the concentration of Compritol® 888 ATO ranged between 1.2% and 1.5%. The polynomial equation describing the effect of Compritol® 888 ATO and Capmul® MCM on zeta potential was found to be: Equation 3: Zeta Potential = -2.41–17.37 comp 888 ATO + 4.50 capmul MCM + 6.40 comp 888 ATO *comp 888 ATO + 1.60 capmul MCM*capmul MCM + 0.81 comp 888 ATO *capmul MCM 3.3.4 Effect of Compritol® 888 ATO, Capmul® MCM on %Entrapment Efficiency It was observed that the model was statistically significant, and % entrapment efficiency exhibited a linear relationship. Entrapment efficiency was determined by Direct and Indirect estimation. Direct estimation: NLC dispersion was centrifuged and the supernatant was decanted. The settled mass at the bottom was dissolved in methanol and sonicated for 30 minutes. The resulting methanolic solution was filtered and the solution was analyzed by UV-spectrophotometry using the developed analytical method. Indirect method: NLC dispersion was centrifuged and the supernatant was decanted. The decanted solution was analyzed by developed UV spectrophotometric method for the amount of drug present. The concentrations of Compritol® 888 ATO and Capmul® MCM were statistically significant in determining the % entrapment efficiency of NLCs, with an R² value of 81.42%. The contour plot indicated that the % entrapment efficiency remained above 90% when the concentration of Compritol® 888 ATO ranged between 1.5% and 1.9%, and the concentration of Capmul® MCM ranged between 0.2% and 0.9%. The polynomial equation describing the effect of Compritol® 888 ATO and Capmul® MCM on % entrapment efficiency was found to be: Equation4: %Entrapment Efficiency = 12.1 + 68.7 comp 888 ATO + 60.8 capmul MCM − 15.2 comp 888 ATO *comp 888 ATO − 17.4 capmul MCM*capmul MCM − 23.7 comp 888 ATO *capmul MCM 3.3.5 Central composite design solution batch Based on the CCD results, a solution batch for the NLC formulation was developed. The objectives were to reduce particle size and PDI, increase the surface electric charge of the particles towards more negative values, and enhance drug loading and entrapment efficiency. The optimal parameters of the independent variables were predicted based on the degree of desirability assigned to each response. The solution batch was formulated and evaluated for particle size (PS), zeta potential (ZP), polydispersity index (PDI), and % entrapment efficiency (%EE). It was observed that all the values were close to the predicted values from the CCD. 3.4 Characterization and evaluation of PFD-NLC The particle size of the optimized NLC formulation was 90.15 ± 10.2 nm. The optimized batch exhibited a PDI of 0.155 ± 0.014, indicating uniform particle size distribution within the nanoformulation. The zeta potential was measured at -11.4 ± 1.2 mV, which could be attributed to the presence of non-ionic surfactants on the NLC surface 24 , 25 . Table 2 presents the predicted values alongside the experimentally obtained characterization data for the developed formulation. Table 2 Evaluation of Optimized batch suggested by CCD. Predicted Actual Compritol 888 ATO 1.64% - Capmul MCM 0.32% - Particle size 93.212 90.15 ± 10.2nm PDI 0.159 0.155 ± 0.014 Zeta Potential -11.60 -11.4±-1.2mV Entrapment Efficiency (%) 89.14 90.43 ± 2.14% The particle size and zeta potential of the optimized batch are illustrated in Figure. 3. The entrapment efficiency and drug loading were found to be 90.43 ± 2.14% and 97.04 ± 1.9%, respectively. The morphology of the NLC was further examined using transmission electron microscopy (TEM) at a magnification of 50 nm, as shown in Figure. 4. 3.5 Preparation of in situ gel loaded PFD-NLC PFD-loaded NLCs were formulated using the hot melt emulsification method. A gellan gum solution was prepared by dispersing 0.6% w/w gellan gum in a sufficient amount of purified water under constant stirring until complete solubilization was achieved. Sodium chloride (0.9% w/w) and benzalkonium chloride (0.2% w/w) were added at fixed concentrations to the solution, which was then filtered through a 0.22 µm PVDF membrane filter. The filtered solution was subsequently added to the polymer dispersion and stirred continuously until a uniform, clear solution was obtained. The prepared NLCs loaded with PFD were then dispersed in the gellan gum-containing medium. 3.6 Evaluation of PFD-NLC loaded in situ gel The optimized batch of PFD-NLC-loaded in situ gel was evaluated for various physicochemical parameters. The formulation appeared white to off-white in color. The pH ranged from 6.8 to 7.1, and the gelation time was observed to be between 5 and 8 seconds. The gel remained stable for up to 12 hours. The viscosity of the formulation was measured at 771 ± 2.5 cP, while the drug content was found to be 96.4%. The bioadhesive strength was determined to be 0.207 ± 0.07 N. 3.7 In vitro and ex vivo drug release The Franz diffusion cell apparatus was utilized for both in vitro and ex vivo drug diffusion studies (Figure. 5). In vitro drug diffusion studies of PFD-NLC demonstrated that 89.29 ± 4.01% of the drug was released over 12 hours in simulated tear fluid (STF) at pH 7.4. In contrast, the in vitro drug release from the PFD-NLC in situ gel was observed to be 84.39 ± 3.41%, indicating a reduction in drug release, which could be attributed to the formation of the sol-gel system. Ex vivo drug diffusion studies of the PFD-NLC in situ gel were conducted using goat cornea and sclera. After 12 hours, 82.97 ± 3.01% and 77.01 ± 1.98% of the drug permeated through the goat cornea and sclera, respectively, in STF (pH 7.4). The permeation flux was calculated to be 0.581 µg/cm²/hr for the cornea and 0.556 µg/cm²/hr for the sclera. With more than 84% drug release in vitro and more than 77% drug penetration through corneal and scleral tissues ex vivo over 12 hours, the formulation assures stable drug absorption and extended ocular residence time, which lessens the need for frequent dossing. When it comes to getting past ocular obstacles and sustaining therapeutic medication levels, this continuous release is especially helpful. Pharmacokinetically speaking, this type of delivery method improves absorption, shields the medication from fast metabolism, prolongs drug retention by lowering excretion through tear drainage, and assures targeted distribution within ocular tissues, all of which contribute to increased therapeutic efficacy and patient compliance. 3.8 Hen’s Egg Test-chorioallantoic Membrane (HET-CAM) Study The ocular mucosal layer is highly sensitive to drug administration; therefore, it is crucial to evaluate the developed formulation for potential irritation. Given the similarity between the vascular tissue of the chorioallantoic membrane (CAM) and the human conjunctiva, the Hen’s Egg Test-Chorioallantoic Membrane (HET-CAM) assay serves as a reliable alternative for assessing ocular irritation, including hyperemia, hemorrhages, and coagulation. The results indicated that the negative control exhibited moderate toxicity, while the API solution showed mild toxicity. In contrast, the positive control, in situ gel, NLC-loaded drug, and NLC-loaded in situ gel formulations were classified as non-irritating. The HET-CAM assay results are presented in Figure. 6. 3.9 In vitro cytotoxicity studies The cytotoxicity of pure PFD and PFD-loaded NLCs was assessed using the MTT assay against SIRC cells. Typically, cell viability above 50% is considered non-toxic, whereas viability below 50% is indicative of potential irritation. In this study, it was observed that pure PFD and PFD-NLC exhibited cell viability below 50% at concentrations of 1000 µg/mL and 1200 µg/mL, respectively, indicating a superior safety margin for the nano formulation. Formulation with 50% viability level is widely accepted in the preliminary screening of ophthalmic formulations as an upper threshold of tolerability under static in vitro conditions 27 . In the dynamic in vivo ocular environment where factors such as tear turnover, blinking, and dilution are present the actual cytotoxic impact is likely to be significantly attenuated. The observed enhancement in biocompatibility is attributable to the encapsulation of PFD within the lipid matrix, which facilitates sustained drug release while reducing peak drug concentrations at the cellular interface. This mitigates acute cytotoxic responses commonly associated with free drug exposure 27 . Compared to currently available ocular therapies, which often suffer from limitations such as poor retention time, frequent administration, and ocular irritation, the PFD-NLC system offers a notable improvement in both tolerability and therapeutic potential. The elevated LD₅₀ of the NLC formulation, in conjunction with non-irritant classification in the HET-CAM assay, underscores its suitability for ocular application, particularly in managing corneal fibrosis where prolonged drug residence and minimal epithelial disruption are critical for therapeutic success. These findings suggest that PFD-NLC demonstrated slightly lower cytotoxicity compared to pure PFD, potentially due to the controlled release and protective effects of the nanocarrier system. 3.10 Stability studies The final formulation was subjected to stability testing for three months under different storage conditions: 5ºC ± 3ºC, 25ºC ± 2ºC / 60% RH ± 5% RH, and 40ºC ± 2ºC / 75% RH ± 5% RH. The formulation was evaluated for physical appearance, pH, and drug content. As presented in Table 3 , the formulation maintained its white to off-white appearance with no visible changes. The pH remained stable, ranging between 6.79 and 6.84, while the drug content was found to be between 95.01% and 97.21%. These findings indicate that the developed NLC in situ gel formulation exhibits good stability under the tested conditions. Table 3 Stability studies observation and results data Sr. No. Parameters Day 0 After 3 months 5 ºC ± 3 ºC 25 ºC ± 2 ºC/ 60% RH ± 5% RH 40 ºC ± 2 ºC/ 75% RH ± 5% RH 1 Physical appearance White to off white White to off white White to off white White to off white 2 pH 6.83 6.81 6.84 6.81 3 Drug content 97.24% 96.11% 97.21% 95.01% 4. DISCUSSION Pirfenidone (PFD) is an antifibrotic drug primarily marketed for the treatment of pulmonary fibrosis. However, emerging evidence suggests its potential in mitigating corneal fibrosis. Given the anatomical and physiological barriers of the eye, effective ocular drug delivery remains a challenge, necessitating the development of an optimized formulation that enhances drug retention, bioavailability, and therapeutic efficacy. In this study, PFD was incorporated into nanostructured lipid carriers (NLCs) to improve ocular delivery by leveraging the advantages of high bioavailability and superior drug-loading capacity inherent to NLC formulations. A systematic preformulation study was conducted to select appropriate lipids and surfactants based on their solubilizing capacity. Saturation solubility studies, a critical preformulation parameter, were performed to assess the ability of selected excipients to encapsulate PFD effectively. Among the tested excipients, Compritol® 888 ATO, glyceryl monostearate, Capmul® MCM, Poloxamer 188, and Tween 80 demonstrated optimal solubilization and were selected for further formulation development. During preliminary formulation trials, solvent evaporation and hot melt emulsification techniques were explored for NLC preparation. However, the solvent evaporation method failed to produce a stable emulsion, necessitating the use of hot melt emulsification followed by homogenization. Probe sonication was employed as the homogenization technique, ensuring efficient size reduction. Initial studies indicated that NLCs formulated with glyceryl monostearate exhibited instability across varying concentrations and process parameters. In contrast, formulations containing solid lipid (Compritol® 888 ATO), liquid lipid (Capmul® MCM), and surfactants (Poloxamer 188 and Tween 80) displayed greater stability. However, the concentration of solid and liquid lipids significantly influenced the physicochemical characteristics of NLCs, warranting further optimization. Pirfenidone (PFD), when administered orally as Esbriet® (267–801 mg/day), offers systemic delivery but is often linked to gastrointestinal side effects and hepatotoxicity. Topical contrast, PFD-loaded nanostructured lipid carriers (NLCs) provide high drug loading with sustained release and improved permeation. Incorporation into an in situ gel further enhances retention and bioavailability, making it a promising approach for ocular delivery. Formulation Type Dosage (mg) Drug Loading (%) Delivery Route Key Outcomes Marketed Oral Tablet (e.g., Esbriet®) 267–801 mg/day - Oral systemic Systemic delivery; associated with side effects like GI disturbances & hepatotoxicity. Topical PFD Solution (0.5%) in studies ~ 5 mg/mL ~ 0.5% Topical ocular Rapid clearance from eye; short corneal half-life (< 19 min); poor bioavailability. PFD-NLC (Optimized Batch) ~ 10 mg/g ~ 97.04% Topical ocular (NLC) High drug loading; sustained release (89.29% over 12 hrs); improved permeation. PFD-NLC in situ gel ~ 10 mg/g ~ 97.04% Topical ocular (gel) Controlled release (84.39% over 12 hrs); enhanced retention & bioavailability. To optimize the formulation, a two-level full-factorial central composite design (CCD) was employed using Minitab software, with Compritol® 888 ATO and Capmul® MCM concentrations as independent variables. The dependent variables included particle size (PS), polydispersity index (PDI), zeta potential (ZP), and entrapment efficiency (%EE). Based on CCD analysis, the optimal formulation was predicted, prepared, and evaluated, yielding a particle size of 90.15 nm, PDI of 0.155, ZP of -11.4 mV, and %EE of 90.43%. The observed particle size of < 100 nm is favorable for ocular drug delivery, as it enhances bioavailability by increasing surface area and promoting better corneal permeation. The low PDI (0.155) suggests a narrow size distribution and uniformity within the formulation. Although the zeta potential was relatively low (-11.4 mV), the formulation remained stable, which can be attributed to the use of non-ionic surfactants. Non-ionic surfactants typically exhibit minimal charge, which may reduce electrostatic repulsion but still confer stability through steric hindrance. The optimized NLC batch was formulated using 1.64% Compritol® 888 ATO, 0.32% Capmul® MCM, 0.5% Poloxamer 188, and 0.5% Tween 80, with homogenization performed at 70% amplitude for 7 minutes. Transmission electron microscopy (TEM) analysis (Figure. 7) confirmed the spherical morphology of the NLCs, further validating the structural integrity of the formulation. The in vitro drug release study demonstrated a cumulative release of 89.29 ± 4.01% over 12 hours, indicating sustained drug release. However, one of the major challenges in ocular drug delivery is rapid precorneal elimination, leading to a low retention time. To address this limitation, the optimized PFD-NLC formulation was incorporated into an in situ gel system containing gellan gum to enhance retention time and prolong therapeutic efficacy. The in vitro drug release from the in situ gel was 84.39 ± 3.41%, slightly lower than the NLC suspension, suggesting a controlled release mechanism due to gelation. Ex vivo drug permeation studies using goat cornea and sclera revealed that 82.97 ± 3.01% and 77.01 ± 1.98% of the drug permeated through the cornea and sclera, respectively, over 12 hours. Pirfenidone's anti-inflammatory and anti-fibrotic effects in the eye, specifically its suppression of fibroblast proliferation and inhibition of TGF-β and TNF-α, necessitate long-term local drug concentrations at the corneal stroma site of action. Enhancing local (ocular) bioavailability is essential for treatment success since corneal haze and fibrosis are localized main pathologies. Poor ocular penetration and the possibility of off-target effects make systemic injection ineffective and undesirable. However, lacrimation, blinking, and nasolacrimal drainage cause traditional topical PFD formulations to be quickly removed from the precorneal region, resulting in tissue levels that are below therapeutic values. This is addressed by the developed PFD-NLC in situ gel technology, which enhances localized drug administration by facilitating regulated release and improves precorneal retention. These findings suggest that the developed PFD-NLC in situ gel enhances ocular bioavailability while providing sustained drug release, making it a promising approach for the therapeutic management of corneal fibrosis. 5. CONCLUSION The present study demonstrated the potential of PFD-loaded NLCs as an ocular drug delivery system to enhance bioavailability. The optimized PFD-NLC formulation exhibited a particle size of 90.15 ± 10.2 nm, with a spherical morphology and uniform size distribution (PDI: 0.155 ± 0.014). In vitro drug release studies showed that PFD-NLC and PFD-NLC in situ gel released 89.29 ± 4.01% and 84.39 ± 3.41% of the drug, respectively, over 12 hours. Ex vivo studies revealed corneal and scleral drug permeation of 82.97 ± 3.01% and 77.01 ± 1.98%, respectively. Further evaluations confirmed the bio adhesive nature of the formulation. The HET-CAM assay indicated that the formulation was non-irritant, and stability studies demonstrated its physicochemical stability under different storage conditions. These findings suggest that PFD-NLC in situ gel holds significant potential as a promising ocular drug delivery system for the treatment of corneal fibrosis. Declarations Consent for publication : Yes Data availability statement: The authors attest that the information in the paper supports the study's conclusions. 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Benefits at the nanoscale: a review of nanoparticle-enabled processes favouring microbil growth and functionality. Environ Microbiol. 2020;22(9):3633-3649. Astrid Subrizi et.al, Design principles of ocular drug delivery systems: importance of drug payload, release rate, and material properties. Drug Discovery Today . 2019; 24(8):1457 Additional Declarations No competing interests reported. Supplementary Files Graphicalabstract.docx Cite Share Download PDF Status: Published Journal Publication published 06 Jan, 2026 Read the published version in Journal of Pharmaceutical Innovation → 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. 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12:51:27","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":250464,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7107100/v1/8f659cc134f2710a2e3d198d.png"},{"id":94351755,"identity":"ada0905d-4e29-44d4-8073-6966a3521358","added_by":"auto","created_at":"2025-10-27 12:51:14","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":935,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7107100/v1/450a561ecfd8535a6083faa3.png"},{"id":94351833,"identity":"e2396023-fc0f-4fef-9a60-398b59c42841","added_by":"auto","created_at":"2025-10-27 12:51:21","extension":"xml","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":113764,"visible":true,"origin":"","legend":"","description":"","filename":"775f49723b7b48b9b1c9e1a433be4e731structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7107100/v1/916fe144028571427c78f72e.xml"},{"id":94351610,"identity":"36612021-40d9-437c-8166-ed5e95a96e36","added_by":"auto","created_at":"2025-10-27 12:50:59","extension":"html","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":120576,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7107100/v1/b79590363949478c29cdebc7.html"},{"id":94351615,"identity":"a72f6111-8a94-4b8d-a29c-6bb952478f0f","added_by":"auto","created_at":"2025-10-27 12:50:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":41705,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSolubility of PFD in solid lipids, liquid lipids and surfactants\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7107100/v1/6b4aab2f15ef564d5da0fc17.png"},{"id":94351943,"identity":"fe2075bf-5732-4cd6-b9c0-df06d4c0a459","added_by":"auto","created_at":"2025-10-27 12:51:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":181500,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure. 3: 3a) Particle size and PDI of optimized batch. 3b) Zeta potential of optimized batch.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7107100/v1/760b3050ff29d1624dca47ca.png"},{"id":94352083,"identity":"0e7652c4-6056-49da-808e-6b633231e35d","added_by":"auto","created_at":"2025-10-27 12:51:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":211157,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure. 4: TEM image under 50nm magnification.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7107100/v1/195ae5a7d7008cb5b7763036.png"},{"id":94489363,"identity":"e06a3e2b-dfd7-40e8-be76-3c1ffebd5a67","added_by":"auto","created_at":"2025-10-27 17:04:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":58405,"visible":true,"origin":"","legend":"\u003cp\u003eFigure legend not provided with this version\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7107100/v1/b8335eb4477e9ef71b5c38b2.png"},{"id":94351884,"identity":"bb200120-3663-4099-8e95-df9f393ca2fd","added_by":"auto","created_at":"2025-10-27 12:51:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":626606,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure. 6: HET CAM’s images after application a) Positive control b) Negative control c) Pure PFD d) \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein situ\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e drug e) NLC drug loaded and f) NLC \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein situ\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e drug loaded.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7107100/v1/b9a7ccfd3c546364e3e565fc.png"},{"id":94351760,"identity":"6e396bbd-55ad-4461-b138-ace353ec4e36","added_by":"auto","created_at":"2025-10-27 12:51:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":24489,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure. 7: Cytotoxicity studies of PFD and PFD-NLC on SIRC ocular cells.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7107100/v1/87627607985b8dd97761dc83.png"},{"id":100069233,"identity":"1368eb29-6898-4019-92cf-9e8e7d9aef95","added_by":"auto","created_at":"2026-01-12 16:11:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3468198,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7107100/v1/201c532f-4d61-4b76-b144-c798db3246df.pdf"},{"id":94351750,"identity":"9586be71-c4db-44e9-a468-7f6607313ebf","added_by":"auto","created_at":"2025-10-27 12:51:14","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":440179,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-7107100/v1/0421045a8802b72a04f8545e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eOptimization of Nanostructured Lipid Carrier Using Central Composite Design for Ocular Delivery of Pirfenidone\u003c/p\u003e","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eThe transparent cornea, which makes up the anterior surface of the eye, protects the eye's internal structures and produces two thirds of its refractive power. Corneal transparency plays a significant role in optimal vision and effective transmission of incident light into the eye. However, the anatomical location of cornea increases the risk of injury to chemical, burns or surgery. Long-term effects of corneal injury causes corneal haze which can lead to permanent vision loss. Corneal haze is a fibrotic condition in which the cornea loses its transparency and becomes opaque and cloudy, which leads to corneal blindness\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. This can have a substantial impact on quality life of humans\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Irreparable loss of corneal transparency is one of the major cause of blindness\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eCorneal injury can occur due to alkali burn or post-operative glaucoma surgery\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Transforming growth factor beta 1 (TGFβ1), as a key inflammatory cytokine, promotes post injuries corneal fibrosis. TGFβ1 and platelet-derived growth factor (PDGF) secreted from corneal inflammatory epithelial cells, stimulate and accelerate the proliferation of stromal cells and their differentiation to myofibroblasts\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Myofibroblasts with alpha‐smooth muscle actin (α‐SMA) feature by scattering the incident light, may cause corneal haze propagation\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Additionally, TGFβ1 increases the secretion of collagen type І and fibronectin from myofibroblasts and fibroblasts. This may serve as another cause of light scattering and corneal haze after injuries\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Corneal neovascularization is an additional reason for corneal opacity after deep injury due to an imbalance between angiogenic (e.g., vascular endothelial growth factor [VEGF]) and antiangiogenic (e.g., thrombospondin 1) factors\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePirfenidone (PFD) (5-methyl-1-phenyl-2-[1H]-pyridone) is therapeutic agent that exhibits anti-inflammatory, antifibrotic effect and modulates cellular oxidation in pre-clinical as well clinical studies in idiopathic pulmonary fibrosis, renal fibrosis, multiple sclerosis\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Its antifibrotic action is primarily attributed to its antagonism of fibroblast proliferation and migration and the reduction of extracellular matrix deposits\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. PFD exhibits anti-inflammatory activity by inhibiting TGF-β, TNF-α synthesis, collagen I and III production, and fibroblast proliferation. PFD also stimulates release of matrix metalloproteinases and collagenases, which promotes the anti-fibrotic activity\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. However, when it was investigated the pharmacokinetics of 0.5% pirfenidone as a topically administered solution in eyes of rabbits, it exhibited a short half-life of less than 19 min in cornea tissue which may result in low bioavailability. This can be overcome by incorporating PFD in nano formulation like NLC due to its high drug loading capacity\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eNanostructured lipid carrier (NLC) are lipidic nano drug delivery system formulated by replacing solid lipids with liquid lipids in solid lipid nanoparticles (SLN). Compared to rest of the conventional carriers, NLC has numerous advantages, involving high drug bioavailability, high drug loading, biodegradability, good tolerance. Moreover, they are comparatively easy to scale up on a large manufacturing than other nanoformulations\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. NLCs are primarily aqueous dispersions, resulting in a low precorneal retention time which can be overcome by \u003cem\u003ein situ\u003c/em\u003e gels. Ophthalmic \u003cem\u003ein situ\u003c/em\u003e gels are sol-gel preparations that are converted into gel after administration due to changes in the environment. This leads to prolong drug residence time and decrease in frequency of drug administration\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAn ophthalmic formulation combining the benefits of NLCs with the benefits of an \u003cem\u003ein-situ\u003c/em\u003e gel system was developed. In the present study, PFD loaded NLCs were formulated using hot melt emulsification followed by probe sonication. Central composite design (CCD) was used to systematically optimize the NLCs. Optimized PFD-NLCs were developed as \u003cem\u003ein situ\u003c/em\u003e gel. The developed formulation was sterilized using membrane filtration and evaluated on various parameters like Hen\u0026rsquo;s egg test using choriollantoic membrane (HET CAM\u0026rsquo;s) test, bioadhesion and \u003cem\u003ein vitro\u003c/em\u003e cytotoxicity by MTT assay etc.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials\u003c/h2\u003e\u003cp\u003ePirfenidone was obtained as a gift sample from Cipla Ltd., Mumbai India; Compritol ATO 888 was obtained as a gift sample from Gattefosse India Pvt. Ltd.; Glyceryl monostearate and Isopropyl Myristate was provided as a gift sample by Arihant Innochem Pvt. Ltd., Mumbai India; Cetyl palmitate, Tween 20, Tween 40, Tween 60, Tween 80, Span 20, Span 60, Span 80, were received as gift sample from Mohini Organics Pvt. Ltd., Mumbai, India; Captex\u0026reg; 200, Captex\u0026reg; 355 and Capmul\u0026reg; MCM, were received as a gift sample by Abitec Corporation, Mumbai India; Sesame oil and Soyabean oil were gifted by Croda India Company Pvt. Ltd.; Kolliphor\u0026reg; ELP, Kolliphor\u0026reg; RH 40, Kolliphor\u0026reg; HS 15, and Poloxamer 188 were gifted by BASF Pvt. Ltd., Mumbai India; Castor oil was purchased from Jayant Agro-Organics Limited, Mumbai India; Ethyl oleate, Oleic acid and Transcutol P purchased from Otto Chemie Pvt. Ltd, Mumbai India, All solvents were purchased form S.D. Fines Pvt Ltd, India.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Methods\u003c/h2\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e2.2.1 Screening of solid lipids, liquid lipids, and surfactants\u003c/h2\u003e\u003cp\u003eSolubility studies of pirfenidone (PFD) were conducted in various solid lipids, liquid lipids, and surfactants (n\u0026thinsp;=\u0026thinsp;3). To assess PFD solubility in solid lipids (Compritol\u0026reg; 888 ATO, glyceryl monostearate, and cetyl palmitate), each lipid (1 g) was melted separately in vials at a temperature 10\u0026deg;C above its melting point. PFD was incrementally added until the lipid reached saturation. The solubility of PFD in liquid lipids (Capmul\u0026reg; MCM, Captex\u0026reg; 200, Captex\u0026reg; 355, castor oil, ethyl oleate, isopropyl myristate, oleic acid, sesame oil, and soybean oil) and surfactants (Kolliphor\u0026reg; ELP, Kolliphor\u0026reg; HS 15, Kolliphor\u0026reg; RH40, Poloxamer 188, Span 20, Span 60, Span 80, Transcutol\u0026reg; P, Tween 20, Tween 40, Tween 60, and Tween 80) was determined using saturation solubility studies. Briefly, a known quantity of PFD was added to 1 g of each liquid lipid and surfactant in separate vials. The mixtures were vortexed, incubated in an orbital shaker for 72 hours, and subsequently centrifuged at 7500 rpm for 15 minutes. The supernatant was diluted with methanol and analyzed using a UV spectrophotometer at 317 nm to quantify the solubility of PFD.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.2.2 Determination of compatibility between Solid lipids and liquid lipids:\u003c/h2\u003e\u003cp\u003eSolid and liquid lipids exhibiting maximum solubility for pirfenidone (PFD) were combined in varying ratios (6:4, 7:3, 8:2, and 9:1) to evaluate their physical compatibility. Accurately weighed quantities (0.5 g) of each solid and liquid lipid were transferred into glass vials and heated to a temperature 10\u0026deg;C above the melting point of the solid lipid to ensure complete melting. The mixtures were then allowed to cool to ambient temperature, and phase separation was assessed after 24 hours.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.2.3 Preparation of Pirfenidone loaded Nanostructured lipid carrier (PFD-NLCs)\u003c/h2\u003e\u003cp\u003ePFD-NLCs were prepared using the hot melt emulsification technique followed by probe sonication\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. A predetermined quantity of solid lipid, liquid lipid, and pirfenidone (lipid phase) was heated in a beaker to a temperature 10\u0026deg;C above the melting point of the solid lipid. Simultaneously, the aqueous phase was prepared by dissolving a specified amount of surfactant in water and heating it to the same temperature (78\u0026ndash;82\u0026deg;C). The aqueous phase was then gradually added to the lipid phase under continuous stirring at 750 rpm for 30 minutes, maintaining the temperature at 78\u0026ndash;82\u0026deg;C to facilitate emulsification. The resulting emulsion was further homogenized using probe sonication at 70% amplitude for 7 minutes to achieve nanoscale particle size.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.2.4 Optimization of PFD-NLCs using Central composite design (CCD)\u003c/h2\u003e\u003cp\u003eVarious Design of Experiments (DoE) approaches have proven valuable in evaluating the impact of individual process variables on the performance of selected formulations. In this study, a two-level full factorial central composite design (CCD) was employed to optimize the formulation of pirfenidone-loaded nanostructured lipid carriers (PFD-NLCs). The CCD was generated using Minitab software (version 20.2.0). Based on preliminary screening of various formulation and process parameters, critical process parameters that significantly influenced the performance of PFD-NLCs were identified. The CCD consisted of factorial design points, axial (star) points, and center points. The concentration of solid lipid and liquid lipid were selected as independent variables, while particle size (PS), zeta potential (ZP), polydispersity index (PDI), and percentage entrapment efficiency (%EE) were designated as dependent variables.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.2.5 Characterization and evaluation of PFD-NLC\u003c/h2\u003e\u003cp\u003eThe developed NLC were characterized by PS, PDI, (ZP) and %EE. The morphological characterization of the optimized formulation was evaluated by Transmission electron microscopy analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.2.6 Particle size (PS), polydispersity index (PDI)and zeta potential (ZP)\u003c/h2\u003e\u003cp\u003eThe particle size was determined using Zetasizer (Nano-ZS, Malvern) Instrument. The test samples were diluted with purified water and measured at a 90\u0026deg; scattering angle at 25\u0026deg;C in triplicate. Zeta potential was determined using the same instrument based on electrophoretic mobility. For zeta potential measurement, the test samples were similarly diluted with purified water to ensure appropriate conductivity and analyzed under identical conditions \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.2.7 Transmission electron microscopy (TEM)\u003c/h2\u003e\u003cp\u003eMorphological characterization of the nanostructured lipid carriers (NLCs) was performed using transmission electron microscopy (TEM) (Model-JEM-2100, JEOL). The NLCs, previously diluted with double-distilled water, were placed onto carbon-coated copper grids. The samples were stained with 1% (w/w) phosphotungstic acid and allowed to stain for 10 minutes before examination under the TEM.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.2.8 \u003cem\u003eIn vitro\u003c/em\u003e drug diffusion release from PFD-NLC\u003c/h2\u003e\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e drug diffusion studies of the optimized formulation were conducted using a dialysis membrane with a molecular weight cutoff of 150 Daltons (Himedia) to quantify the release of pirfenidone (PFD). The Franz diffusion apparatus was used, with the receptor compartment filled with 22 mL of simulated tear fluid (STF) at pH 7.4. The temperature of the diffusion cell was maintained at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C under continuous stirring at 100 rpm. A 1 g aliquot of the formulation was placed in the donor compartment and covered. At predetermined intervals (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 hours), samples were withdrawn, filtered, and analyzed for drug content using UV spectrophotometry. The cumulative percentage of drug released was plotted against time to evaluate the release profile.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.2.9 Preparation of \u003cem\u003ein situ\u003c/em\u003e gel loaded PFD-NLC\u003c/h2\u003e\u003cp\u003eA gellan gum solution was prepared by dispersing the required amount of gellan gum in an appropriate volume of purified water under continuous stirring until complete dissolution was achieved. Sodium chloride and benzalkonium chloride were then added at fixed concentrations to the solution, followed by filtration through a 0.22 \u0026micro;m polyvinylidene fluoride (PVDF) membrane filter. The filtered solution was subsequently incorporated into the polymer dispersion under continuous stirring to obtain a uniform and clear solution. Pirfenidone (PFD)-loaded nanostructured lipid carriers (NLCs) were then gradually dispersed into the prepared medium under continuous stirring at 750 rpm to ensure homogeneous dispersion.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e\u003cb\u003e2.2.10 Characterization of\u003c/b\u003e \u003cb\u003ein situ\u003c/b\u003e \u003cb\u003egel loaded PFD-NLC\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eThe physicochemical properties of the developed PFD-NLC-based in situ gel were evaluated by assessing its physical appearance, pH, viscosity, and bioadhesive strength. In vitro and ex vivo drug diffusion studies were conducted using a Franz diffusion apparatus, employing simulated tear fluid (STF) at pH 7.4 as the receptor medium. A dialysis membrane (Himedia 150 Da) was utilized to assess the permeation characteristics of the formulation under controlled experimental conditions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e2.2.11 \u003cem\u003eIn vitro\u003c/em\u003e drug diffusion from \u003cem\u003ein situ\u003c/em\u003e gel loaded PFD-NLC\u003c/h2\u003e\u003cp\u003eThe in vitro drug diffusion study of the optimized formulation was conducted to evaluate the extent of drug permeation across a dialysis membrane (150 Da). The study was performed using a Franz diffusion apparatus, with the receptor compartment filled with simulated tear fluid (STF) at pH 7.4. The system was maintained at a constant temperature of 37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026ordm;C and continuously stirred at 100 rpm to simulate physiological conditions. A precisely weighed 1 g aliquot of the formulation was placed in the donor compartment and securely covered to prevent evaporation or contamination. At predetermined intervals (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 hours), aliquots of the receptor medium were withdrawn, filtered, and analyzed for drug content using UV spectrophotometry. The cumulative percentage of drug released over time was plotted to assess the release kinetics of the formulation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e\u003cb\u003e2.2.12\u003c/b\u003e \u003cb\u003eEx vivo\u003c/b\u003e \u003cb\u003ecorneal and scleral drug diffusion profile of\u003c/b\u003e \u003cb\u003ein situ\u003c/b\u003e \u003cb\u003egel loaded PFD-NLC\u003c/b\u003e\u003c/h2\u003e\u003cp\u003e\u003cem\u003eThe ex vivo\u003c/em\u003e drug permeation study was conducted using goat cornea to evaluate the transcorneal diffusion of PFD from the optimized PFD-NLC based in situ gel. Fresh goat eyeballs were procured from a local slaughterhouse and transported to the laboratory under cold conditions, ensuring constant saline immersion to maintain tissue viability. The cornea, along with the surrounding scleral tissue, was carefully excised and thoroughly rinsed with sterile saline to remove any residual debris.\u003c/p\u003e\u003cp\u003eThe diffusion study was performed using a Franz diffusion apparatus, where the excised goat cornea was mounted between the donor and receptor compartments with the epithelial side facing the donor chamber. The receptor compartment was filled with 22 mL of simulated tear fluid (STF, pH 7.4) and maintained at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026ordm;C under continuous stirring at 100 rpm to mimic physiological ocular conditions. Precisely weighed 1 g of the formulation was placed in the donor compartment, ensuring uniform contact with the corneal membrane, and the setup was securely covered to prevent evaporation or contamination. At predetermined time intervals (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 hours), aliquots were withdrawn from the receptor compartment, filtered, and analyzed for drug content using UV spectrophotometry. The cumulative percentage of drug permeated across the cornea was plotted against time to assess the permeation profile. A similar experimental procedure was followed for ex vivo scleral permeation studies.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e2.2.13 Flux and Permeability coefficient\u003c/h2\u003e\u003cp\u003eThe steady-state flux (Jss) was determined by calculating the slope (Q) from the linear portion of the plot representing the cumulative amount of drug permeated per unit surface area of the membrane as a function of time (\u0026micro;g/cm\u003csup\u003e2\u003c/sup\u003e vs. h) \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe permeation coefficient (Kp​) was determined using the equation:\u003c/p\u003e\u003cp\u003eKp\u0026thinsp;=\u0026thinsp;Jss/C0\u003c/p\u003e\u003cp\u003ewhere C0 represents the initial concentration of the drug in the donor compartment. The flux and permeation coefficient were used to assess the permeability characteristics of the developed NLC-based in situ gel for ocular drug delivery.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e2.2.14 Bioadhesive strength\u003c/h2\u003e\u003cp\u003eThe bioadhesive strength of the developed NLC-based in situ gel was assessed using a modified bioadhesion apparatus. Freshly excised goat corneas were procured and carefully isolated for the study. One corneal section was securely affixed to a wooden platform mounted on a digital weighing balance using an inert adhesive, ensuring stable attachment. The second corneal section was mounted below the pan of the balance. A predetermined amount of the in situ gel formulation was applied between the two corneal sections to facilitate adhesion. Incremental weights were gradually added to the opposing pan until complete detachment of the corneal sections was observed. The bioadhesive strength was calculated using the following equation:\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eBioadhesive strength\u0026thinsp;=\u0026thinsp;Weight required (in kgs)/ Area (cm\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\u003ch2\u003e2.2.15 HEN\u0026rsquo;S Egg Test-Chorioallantoic Membrane (HET-CAM) study\u003c/h2\u003e\u003cp\u003eHET-CAM is a rapid and sensitive procedure to predict skin and ocular irritancy by evaluation of the changes in the CAM of the fertilized eggs. This is an alternative \u003cem\u003eex vivo\u003c/em\u003e toxicity evaluation technique for the \u003cem\u003ein vivo\u003c/em\u003e Draize test on rabbit\u0026rsquo;s eye\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. CAM comprises of complete laminate vascular system with arteries, veins and capillaries that are sensitive to harmful and corrosive substances with an inflammatory process. As per the ICCVAM-Recommended Test Method Protocol, the irritation potential of PFD loaded NLCs formulations were evaluated by the HET- CAM assay (ICCVAM 2010). Fertile White Leghorn chicken eggs weighing 50 to 60 grams were obtained from Central Poultry Development Organization, Mumbai. Nine-day old, fertilized eggs which were incubated in at 37.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5℃ and 62.5% \u0026plusmn; 7.5% RH. Forceps were used to carefully detach the egg's shell from the air cell. After 5 min of saline moistening, the inner membrane directly in contact with the CAM was carefully removed using forceps. The experimentation method was validated using 0.1 N NaOH (negative control), 0.9% NaCl (positive control). 0.3mL of test samples were applied to the CAM and the irritation effect was studied visually for 5min. After adding each sample, the duration and severity of injuries were noted, and the irritation score (IS) was determined using the formula below.:\u003c/p\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:IS\\)\u003c/span\u003e\u003c/span\u003e = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\left(301-tH\\right)*5}{300}\\)\u003c/span\u003e\u003c/span\u003e + \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\left(301-tL\\right)*7}{300}\\)\u003c/span\u003e\u003c/span\u003e + \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\left(301-tC\\right)*9}{300}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003cp\u003eWhere tH, tL and tC are time (in seconds) required for the occurrence of hemolysis, lysis and coagulation, respectively. The experimentation was performed in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\u003ch2\u003e2.2.16 \u003cem\u003eIn vitro\u003c/em\u003e cytotoxicity studies\u003c/h2\u003e\u003cp\u003eThe potential cytotoxic effects of the developed PFD-NLC formulation were evaluated using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. This assay measures cell viability by assessing mitochondrial activity, providing insights into possible adverse effects such as reduced cell membrane integrity, cell lysis, and apoptosis. The cytotoxicity study was conducted on Statens Seruminstitut Rabbit Cornea (SIRC) cell lines. Briefly, 200 \u0026micro;L of a cell suspension (1\u0026times;10⁵ cells per well) was seeded into a 96-well plate (Sigma, Germany) and incubated at 37\u0026deg;C in a 5% CO₂ atmosphere for 24 hours to allow cell attachment. Subsequently, the cells were exposed to varying concentrations (50, 150, 250, 500, 800, 1000, and 1200 \u0026micro;g/mL) of pure pirfenidone (PFD) and PFD-loaded NLCs (PFD-NLC) and incubated for an additional 12 hours. Following exposure, the cells were treated with 0.5 mg/mL MTT reagent and incubated for 4 hours to facilitate formazan crystal formation. After incubation, the MTT reagent was carefully removed, and 100 \u0026micro;L of dimethyl sulfoxide (DMSO) was added to each well to solubilize the formazan crystals. The absorbance of the resulting solution was measured at 570 nm using a 96-well microplate reader (BioTek Synergy H1 Multimode Reader, USA). The IC₅₀ values were calculated to determine the concentration at which 50% of the cells remained viable, providing a quantitative measure of cytotoxicity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\u003ch2\u003e2.2.17 Stability studies\u003c/h2\u003e\u003cp\u003e Stability studies were conducted in accordance with ICH Q1A (R2) guidelines to assess the physicochemical stability of the developed formulations over a period of three months under different storage conditions. The formulations were stored in sealed glass containers and subjected to low-temperature conditions (5\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026deg;C), long-term stability conditions (25\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C / 60% RH\u0026thinsp;\u0026plusmn;\u0026thinsp;5% RH), and accelerated stability conditions (40\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C / 75% RH\u0026thinsp;\u0026plusmn;\u0026thinsp;5% RH). Following the storage period, the formulations were evaluated for any physical changes, variations in pH, and drug content to determine their stability and integrity under different environmental conditions.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Screening of solid lipids, liquid lipids and surfactants\u003c/h2\u003e\u003cp\u003eA solubility study was conducted to evaluate the capacity of various lipids to solubilize pirfenidone (PFD). The solubility profile of all excipients is presented in Figure. 1.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eInitially, solid lipids, liquid lipids, and surfactants were selected based on a literature review, followed by experimental screening. Among the solid lipids, cetyl palmitate exhibited a solubility of 12.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 mg/g, whereas Compritol\u0026reg; 888 ATO and glyceryl monostearate demonstrated PFD solubility exceeding 100 mg/g. Capmul\u0026reg; MCM showed solubility greater than 100 mg/g, followed by oleic acid (84.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 mg/g), while castor oil solubilized 2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 mg/g of PFD. Kolliphor\u0026reg; HS 15, Transcutol\u0026reg; P, and Span\u0026reg; 60 exhibited PFD solubility exceeding 100 mg/g, whereas Poloxamer 188 solubilized 3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 mg/g. Based on literature findings and solubility data, Compritol\u0026reg; 888 ATO and glyceryl monostearate were selected as solid lipids, Capmul\u0026reg; MCM as the liquid lipid, and Tween\u0026reg; 80 along with Poloxamer 188 as surfactants for the formulation of PFD-loaded nanostructured lipid carriers (PFD-NLC).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Solid lipid - Liquid lipid compatibility\u003c/h2\u003e\u003cp\u003eCompatibility studies were performed to evaluate the physical compatibility between selected solid and liquid lipids. Solid and liquid lipids were mixed in various ratios, heated, and subsequently maintained at ambient temperature for 24 hours. No phase separation was observed in any of the tested ratios, indicating physical stability. These findings suggest that Compritol\u0026reg; 888 ATO and glyceryl monostearate are compatible with Capmul\u0026reg; MCM, making them suitable lipid components for formulation development.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Formulation and optimization of Pirfenidone loaded Nanostructured lipid carrier\u003c/h2\u003e\u003cp\u003ePreliminary batches of nanostructured lipid carriers (NLCs) were formulated using the hot melt emulsification method, followed by probe sonication for homogenization. The preliminary studies indicated that the combination of solid lipid (Compritol\u0026reg; 888 ATO), liquid lipid (Capmul\u0026reg; MCM), and surfactants (Tween\u0026reg; 80 and Poloxamer 188) resulted in a stable formulation. Additionally, the processing parameters, including an amplitude of 70% and a sonication time of 7 minutes, were identified as optimal for NLC homogenization.\u003c/p\u003e\u003cp\u003eFurther investigations revealed that variations in the concentration of Compritol\u0026reg; 888 ATO (1.2\u0026ndash;1.8%) and Capmul\u0026reg; MCM (0.8\u0026ndash;0.2%) had a significant impact on particle size (PS), polydispersity index (PDI), zeta potential (ZP), and entrapment efficiency (%EE) of NLCs. To optimize the formulation, a Central Composite Design (CCD) was employed as the design of experiment (DoE). Based on CCD, 13 experimental batches were formulated, and the corresponding results are presented in (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCCD generated by Minitab software with measured responses.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eB. No\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eComp ATO 888 (A)\u003c/p\u003e\u003cp\u003e% w/w\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCapmul MCM\u003c/p\u003e\u003cp\u003e(B)\u003c/p\u003e\u003cp\u003e% w/w\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePSA (nm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePDI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eZeta(mV)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eEntrapment EE (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e67.80\u0026thinsp;\u0026plusmn;\u0026thinsp;5.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.232\u0026thinsp;\u0026plusmn;\u0026thinsp;0.029\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e-13.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e81.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.800\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e84.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.197\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e-11.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e91.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.800\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e289.40\u0026thinsp;\u0026plusmn;\u0026thinsp;4.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.390\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e-9.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e89.08\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.800\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.800\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e390.00\u0026thinsp;\u0026plusmn;\u0026thinsp;6.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.276\u0026thinsp;\u0026plusmn;\u0026thinsp;0.013\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e-6.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e90.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.66\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.075\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e246.30\u0026thinsp;\u0026plusmn;\u0026thinsp;5.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.474\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e-9.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e78.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.924\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e260.40\u0026thinsp;\u0026plusmn;\u0026thinsp;2.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.440\u0026thinsp;\u0026plusmn;\u0026thinsp;0.052\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e-9.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e90.18\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.075\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e71.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.199\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e-13.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e79.44\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.924\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e370.60\u0026thinsp;\u0026plusmn;\u0026thinsp;2.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.374\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e-7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e88.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e137.16\u0026thinsp;\u0026plusmn;\u0026thinsp;2.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.160\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e-10.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e89.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e137.16\u0026thinsp;\u0026plusmn;\u0026thinsp;2.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.160\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e-10.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e89.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e137.16\u0026thinsp;\u0026plusmn;\u0026thinsp;2.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.160\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e-10.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e89.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e137.16\u0026thinsp;\u0026plusmn;\u0026thinsp;2.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.160\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e-10.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e89.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e137.16\u0026thinsp;\u0026plusmn;\u0026thinsp;2.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e0.160\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e-10.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e\u003cp\u003e89.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe influence of Compritol\u0026reg; 888 ATO and Capmul\u0026reg; MCM concentrations, along with their interactions, on the dependent variables was systematically evaluated. To establish a quantitative relationship between the independent factors and the responses, polynomial equations were generated. Additionally, contour plots were constructed (Figure. 2) to visually represent the effects of varying Compritol\u0026reg; 888 ATO and Capmul\u0026reg; MCM concentrations on PS, PDI, ZP, and %EE.\u003c/p\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003e3.3.1 Effect of Compritol\u0026reg; 888 ATO and Capmul\u0026reg; MCM on Particle size\u003c/h2\u003e\u003cp\u003eThe results of the Central Composite Design (CCD) indicated that the model was statistically significant, with particle size exhibiting a linear relationship with the selected factors. The concentration of Capmul\u0026reg; MCM was identified as a statistically significant factor influencing the particle size of NLCs, with an R\u0026sup2; value of 97.06%, indicating a strong model fit. Analysis of the contour plot revealed that a decrease in the concentration of Capmul\u0026reg; MCM resulted in a reduction in particle size. Additionally, when the concentration of Compritol\u0026reg; 888 ATO was maintained between approximately 1.2% and 1.9%, the resulting NLCs exhibited a particle size below 100 nm. The polynomial equation describing the effect of Compritol\u0026reg; 888 ATO and Capmul\u0026reg; MCM on particle size is as follows:\u003c/p\u003e\u003cp\u003eEquation 1: PSA\u0026thinsp;=\u0026thinsp;1393\u0026ndash;1751 comp 888 ATO \u0026minus;\u0026thinsp;338 capmul MCM\u0026thinsp;+\u0026thinsp;564 comp 888 ATO *comp 888 ATO\u0026thinsp;+\u0026thinsp;386 capmul MCM*capmul MCM\u0026thinsp;+\u0026thinsp;232 comp 888 ATO *capmul MCM\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003e3.3.2 Effect of Compritol\u0026reg; 888 ATO, Capmul\u0026reg; MCM on PDI\u003c/h2\u003e\u003cp\u003eThe results demonstrated that the model was statistically significant, with the PDI exhibiting a linear relationship with the selected factors. The concentration of Capmul\u0026reg; MCM was identified as a statistically significant factor influencing the PDI of NLCs, with an R\u0026sup2; value of 87.71%, indicating a good model fit. Analysis of the contour plot revealed that at lower concentrations of Capmul\u0026reg; MCM (\u0026lt;\u0026thinsp;0.7%), the PDI remained below 0.2. A similar trend was observed when the concentration of Compritol\u0026reg; 888 ATO was maintained between 1.3% and 1.7%, suggesting optimal conditions for achieving a monodisperse system. The polynomial equation describing the effect of Compritol\u0026reg; 888 ATO and Capmul\u0026reg; MCM on PDI is as follows:\u003c/p\u003e\u003cp\u003eEquation 2: PDI\u0026thinsp;=\u0026thinsp;3.225\u0026ndash;4.106 comp 888 ATO\u0026thinsp;+\u0026thinsp;0.100 capmul MCM\u0026thinsp;+\u0026thinsp;1.378 comp 888 ATO *comp 888 ATO\u0026thinsp;+\u0026thinsp;0.431 capmul MCM*capmul MCM \u0026minus;\u0026thinsp;0.219 comp 888 ATO *capmul MCM\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section3\"\u003e\u003ch2\u003e3.3.3 Effect of Compritol\u0026reg; 888 ATO, Capmul\u0026reg; MCM on Zeta Potential\u003c/h2\u003e\u003cp\u003eIt was observed that the model was statistically significant, and zeta potential exhibited a linear relationship. The concentrations of Compritol\u0026reg; 888 ATO and Capmul\u0026reg; MCM were statistically significant in determining the zeta potential of NLCs, with an R\u0026sup2; value of 97.11%. The contour plot indicated that the zeta potential remained higher than \u0026minus;\u0026thinsp;13.5 when the concentration of Capmul\u0026reg; MCM was below 0.1% and the concentration of Compritol\u0026reg; 888 ATO ranged between 1.2% and 1.5%. The polynomial equation describing the effect of Compritol\u0026reg; 888 ATO and Capmul\u0026reg; MCM on zeta potential was found to be:\u003c/p\u003e\u003cp\u003eEquation 3: Zeta Potential = -2.41\u0026ndash;17.37 comp 888 ATO\u0026thinsp;+\u0026thinsp;4.50 capmul MCM\u0026thinsp;+\u0026thinsp;6.40 comp 888 ATO *comp 888 ATO\u0026thinsp;+\u0026thinsp;1.60 capmul MCM*capmul MCM\u0026thinsp;+\u0026thinsp;0.81 comp 888 ATO *capmul MCM\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section3\"\u003e\u003ch2\u003e3.3.4 Effect of Compritol\u0026reg; 888 ATO, Capmul\u0026reg; MCM on %Entrapment Efficiency\u003c/h2\u003e\u003cp\u003eIt was observed that the model was statistically significant, and % entrapment efficiency exhibited a linear relationship. Entrapment efficiency was determined by Direct and Indirect estimation. Direct estimation: NLC dispersion was centrifuged and the supernatant was decanted. The settled mass at the bottom was dissolved in methanol and sonicated for 30 minutes. The resulting methanolic solution was filtered and the solution was analyzed by UV-spectrophotometry using the developed analytical method. Indirect method: NLC dispersion was centrifuged and the supernatant was decanted. The decanted solution was analyzed by developed UV spectrophotometric method for the amount of drug present. The concentrations of Compritol\u0026reg; 888 ATO and Capmul\u0026reg; MCM were statistically significant in determining the % entrapment efficiency of NLCs, with an R\u0026sup2; value of 81.42%. The contour plot indicated that the % entrapment efficiency remained above 90% when the concentration of Compritol\u0026reg; 888 ATO ranged between 1.5% and 1.9%, and the concentration of Capmul\u0026reg; MCM ranged between 0.2% and 0.9%. The polynomial equation describing the effect of Compritol\u0026reg; 888 ATO and Capmul\u0026reg; MCM on % entrapment efficiency was found to be:\u003c/p\u003e\u003cp\u003eEquation4: %Entrapment Efficiency\u0026thinsp;=\u0026thinsp;12.1\u0026thinsp;+\u0026thinsp;68.7 comp 888 ATO\u0026thinsp;+\u0026thinsp;60.8 capmul MCM \u0026minus;\u0026thinsp;15.2 comp 888 ATO *comp 888 ATO \u0026minus;\u0026thinsp;17.4 capmul MCM*capmul MCM \u0026minus;\u0026thinsp;23.7 comp 888 ATO *capmul MCM\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec30\" class=\"Section3\"\u003e\u003ch2\u003e3.3.5 Central composite design solution batch\u003c/h2\u003e\u003cp\u003eBased on the CCD results, a solution batch for the NLC formulation was developed. The objectives were to reduce particle size and PDI, increase the surface electric charge of the particles towards more negative values, and enhance drug loading and entrapment efficiency. The optimal parameters of the independent variables were predicted based on the degree of desirability assigned to each response. The solution batch was formulated and evaluated for particle size (PS), zeta potential (ZP), polydispersity index (PDI), and % entrapment efficiency (%EE). It was observed that all the values were close to the predicted values from the CCD.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Characterization and evaluation of PFD-NLC\u003c/h2\u003e\u003cp\u003eThe particle size of the optimized NLC formulation was 90.15\u0026thinsp;\u0026plusmn;\u0026thinsp;10.2 nm. The optimized batch exhibited a PDI of 0.155\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014, indicating uniform particle size distribution within the nanoformulation. The zeta potential was measured at -11.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 mV, which could be attributed to the presence of non-ionic surfactants on the NLC surface\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e presents the predicted values alongside the experimentally obtained characterization data for the developed formulation.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEvaluation of Optimized batch suggested by CCD.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePredicted\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eActual\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCompritol 888 ATO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.64%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCapmul MCM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.32%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParticle size\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e93.212\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e90.15\u0026thinsp;\u0026plusmn;\u0026thinsp;10.2nm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePDI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.159\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.155\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eZeta Potential\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e-11.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-11.4\u0026plusmn;-1.2mV\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEntrapment Efficiency (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e89.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e90.43\u0026thinsp;\u0026plusmn;\u0026thinsp;2.14%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe particle size and zeta potential of the optimized batch are illustrated in Figure. 3.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe entrapment efficiency and drug loading were found to be 90.43\u0026thinsp;\u0026plusmn;\u0026thinsp;2.14% and 97.04\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9%, respectively. The morphology of the NLC was further examined using transmission electron microscopy (TEM) at a magnification of 50 nm, as shown in Figure. 4.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Preparation of \u003cem\u003ein situ\u003c/em\u003e gel loaded PFD-NLC\u003c/h2\u003e\u003cp\u003ePFD-loaded NLCs were formulated using the hot melt emulsification method. A gellan gum solution was prepared by dispersing 0.6% w/w gellan gum in a sufficient amount of purified water under constant stirring until complete solubilization was achieved. Sodium chloride (0.9% w/w) and benzalkonium chloride (0.2% w/w) were added at fixed concentrations to the solution, which was then filtered through a 0.22 \u0026micro;m PVDF membrane filter. The filtered solution was subsequently added to the polymer dispersion and stirred continuously until a uniform, clear solution was obtained. The prepared NLCs loaded with PFD were then dispersed in the gellan gum-containing medium.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec33\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Evaluation of PFD-NLC loaded \u003cem\u003ein situ\u003c/em\u003e gel\u003c/h2\u003e\u003cp\u003eThe optimized batch of PFD-NLC-loaded in situ gel was evaluated for various physicochemical parameters. The formulation appeared white to off-white in color. The pH ranged from 6.8 to 7.1, and the gelation time was observed to be between 5 and 8 seconds. The gel remained stable for up to 12 hours. The viscosity of the formulation was measured at 771\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 cP, while the drug content was found to be 96.4%. The bioadhesive strength was determined to be 0.207\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 N.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec34\" class=\"Section2\"\u003e\u003ch2\u003e3.7 \u003cem\u003eIn vitro\u003c/em\u003e and \u003cem\u003eex vivo\u003c/em\u003e drug release\u003c/h2\u003e\u003cp\u003eThe Franz diffusion cell apparatus was utilized for both in vitro and ex vivo drug diffusion studies (Figure. 5). In vitro drug diffusion studies of PFD-NLC demonstrated that 89.29\u0026thinsp;\u0026plusmn;\u0026thinsp;4.01% of the drug was released over 12 hours in simulated tear fluid (STF) at pH 7.4. In contrast, the in vitro drug release from the PFD-NLC in situ gel was observed to be 84.39\u0026thinsp;\u0026plusmn;\u0026thinsp;3.41%, indicating a reduction in drug release, which could be attributed to the formation of the sol-gel system. Ex vivo drug diffusion studies of the PFD-NLC in situ gel were conducted using goat cornea and sclera. After 12 hours, 82.97\u0026thinsp;\u0026plusmn;\u0026thinsp;3.01% and 77.01\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98% of the drug permeated through the goat cornea and sclera, respectively, in STF (pH 7.4). The permeation flux was calculated to be 0.581 \u0026micro;g/cm\u0026sup2;/hr for the cornea and 0.556 \u0026micro;g/cm\u0026sup2;/hr for the sclera. With more than 84% drug release in vitro and more than 77% drug penetration through corneal and scleral tissues ex vivo over 12 hours, the formulation assures stable drug absorption and extended ocular residence time, which lessens the need for frequent dossing. When it comes to getting past ocular obstacles and sustaining therapeutic medication levels, this continuous release is especially helpful. Pharmacokinetically speaking, this type of delivery method improves absorption, shields the medication from fast metabolism, prolongs drug retention by lowering excretion through tear drainage, and assures targeted distribution within ocular tissues, all of which contribute to increased therapeutic efficacy and patient compliance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec35\" class=\"Section2\"\u003e\u003ch2\u003e3.8 Hen\u0026rsquo;s Egg Test-chorioallantoic Membrane (HET-CAM) Study\u003c/h2\u003e\u003cp\u003eThe ocular mucosal layer is highly sensitive to drug administration; therefore, it is crucial to evaluate the developed formulation for potential irritation. Given the similarity between the vascular tissue of the chorioallantoic membrane (CAM) and the human conjunctiva, the Hen\u0026rsquo;s Egg Test-Chorioallantoic Membrane (HET-CAM) assay serves as a reliable alternative for assessing ocular irritation, including hyperemia, hemorrhages, and coagulation. The results indicated that the negative control exhibited moderate toxicity, while the API solution showed mild toxicity. In contrast, the positive control, in situ gel, NLC-loaded drug, and NLC-loaded in situ gel formulations were classified as non-irritating. The HET-CAM assay results are presented in Figure. 6.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec36\" class=\"Section2\"\u003e\u003ch2\u003e3.9 \u003cem\u003eIn vitro\u003c/em\u003e cytotoxicity studies\u003c/h2\u003e\u003cp\u003eThe cytotoxicity of pure PFD and PFD-loaded NLCs was assessed using the MTT assay against SIRC cells. Typically, cell viability above 50% is considered non-toxic, whereas viability below 50% is indicative of potential irritation. In this study, it was observed that pure PFD and PFD-NLC exhibited cell viability below 50% at concentrations of 1000 \u0026micro;g/mL and 1200 \u0026micro;g/mL, respectively, indicating a superior safety margin for the nano formulation. Formulation with 50% viability level is widely accepted in the preliminary screening of ophthalmic formulations as an upper threshold of tolerability under static in vitro conditions\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In the dynamic in vivo ocular environment where factors such as tear turnover, blinking, and dilution are present the actual cytotoxic impact is likely to be significantly attenuated. The observed enhancement in biocompatibility is attributable to the encapsulation of PFD within the lipid matrix, which facilitates sustained drug release while reducing peak drug concentrations at the cellular interface. This mitigates acute cytotoxic responses commonly associated with free drug exposure\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Compared to currently available ocular therapies, which often suffer from limitations such as poor retention time, frequent administration, and ocular irritation, the PFD-NLC system offers a notable improvement in both tolerability and therapeutic potential. The elevated LD₅₀ of the NLC formulation, in conjunction with non-irritant classification in the HET-CAM assay, underscores its suitability for ocular application, particularly in managing corneal fibrosis where prolonged drug residence and minimal epithelial disruption are critical for therapeutic success. These findings suggest that PFD-NLC demonstrated slightly lower cytotoxicity compared to pure PFD, potentially due to the controlled release and protective effects of the nanocarrier system.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec37\" class=\"Section2\"\u003e\u003ch2\u003e3.10 Stability studies\u003c/h2\u003e\u003cp\u003eThe final formulation was subjected to stability testing for three months under different storage conditions: 5\u0026ordm;C\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026ordm;C, 25\u0026ordm;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026ordm;C / 60% RH\u0026thinsp;\u0026plusmn;\u0026thinsp;5% RH, and 40\u0026ordm;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026ordm;C / 75% RH\u0026thinsp;\u0026plusmn;\u0026thinsp;5% RH. The formulation was evaluated for physical appearance, pH, and drug content. As presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the formulation maintained its white to off-white appearance with no visible changes. The pH remained stable, ranging between 6.79 and 6.84, while the drug content was found to be between 95.01% and 97.21%. These findings indicate that the developed NLC in situ gel formulation exhibits good stability under the tested conditions.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eStability studies observation and results data\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSr. No.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eParameters\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eDay 0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e\u003cp\u003eAfter 3 months\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5 \u0026ordm;C\u0026thinsp;\u0026plusmn;\u0026thinsp;3 \u0026ordm;C\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e25 \u0026ordm;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u0026ordm;C/ 60% RH\u0026thinsp;\u0026plusmn;\u0026thinsp;5% RH\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e40 \u0026ordm;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u0026ordm;C/ 75% RH\u0026thinsp;\u0026plusmn;\u0026thinsp;5% RH\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePhysical appearance\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWhite to off white\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWhite to off white\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWhite to off white\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eWhite to off white\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6.81\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDrug content\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e97.24%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e96.11%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e97.21%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e95.01%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003ePirfenidone (PFD) is an antifibrotic drug primarily marketed for the treatment of pulmonary fibrosis. However, emerging evidence suggests its potential in mitigating corneal fibrosis. Given the anatomical and physiological barriers of the eye, effective ocular drug delivery remains a challenge, necessitating the development of an optimized formulation that enhances drug retention, bioavailability, and therapeutic efficacy. In this study, PFD was incorporated into nanostructured lipid carriers (NLCs) to improve ocular delivery by leveraging the advantages of high bioavailability and superior drug-loading capacity inherent to NLC formulations.\u003c/p\u003e\u003cp\u003eA systematic preformulation study was conducted to select appropriate lipids and surfactants based on their solubilizing capacity. Saturation solubility studies, a critical preformulation parameter, were performed to assess the ability of selected excipients to encapsulate PFD effectively. Among the tested excipients, Compritol\u0026reg; 888 ATO, glyceryl monostearate, Capmul\u0026reg; MCM, Poloxamer 188, and Tween 80 demonstrated optimal solubilization and were selected for further formulation development. During preliminary formulation trials, solvent evaporation and hot melt emulsification techniques were explored for NLC preparation. However, the solvent evaporation method failed to produce a stable emulsion, necessitating the use of hot melt emulsification followed by homogenization. Probe sonication was employed as the homogenization technique, ensuring efficient size reduction. Initial studies indicated that NLCs formulated with glyceryl monostearate exhibited instability across varying concentrations and process parameters. In contrast, formulations containing solid lipid (Compritol\u0026reg; 888 ATO), liquid lipid (Capmul\u0026reg; MCM), and surfactants (Poloxamer 188 and Tween 80) displayed greater stability. However, the concentration of solid and liquid lipids significantly influenced the physicochemical characteristics of NLCs, warranting further optimization. Pirfenidone (PFD), when administered orally as Esbriet\u0026reg; (267\u0026ndash;801 mg/day), offers systemic delivery but is often linked to gastrointestinal side effects and hepatotoxicity. Topical contrast, PFD-loaded nanostructured lipid carriers (NLCs) provide high drug loading with sustained release and improved permeation. Incorporation into an in situ gel further enhances retention and bioavailability, making it a promising approach for ocular delivery.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFormulation Type\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDosage (mg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDrug Loading (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDelivery Route\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eKey Outcomes\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMarketed Oral Tablet (e.g., Esbriet\u0026reg;)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e267\u0026ndash;801 mg/day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOral systemic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSystemic delivery; associated with side effects like GI disturbances \u0026amp; hepatotoxicity.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTopical PFD Solution (0.5%) in studies\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e~\u0026thinsp;5 mg/mL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e~\u0026thinsp;0.5%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTopical ocular\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eRapid clearance from eye; short corneal half-life (\u0026lt;\u0026thinsp;19 min); poor bioavailability.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePFD-NLC (Optimized Batch)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e~\u0026thinsp;10 mg/g\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e~\u0026thinsp;97.04%\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTopical ocular (NLC)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eHigh drug loading; sustained release (89.29% over 12 hrs); improved permeation.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePFD-NLC in situ gel\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e~\u0026thinsp;10 mg/g\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e~\u0026thinsp;97.04%\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTopical ocular (gel)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eControlled release (84.39% over 12 hrs); enhanced retention \u0026amp; bioavailability.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTo optimize the formulation, a two-level full-factorial central composite design (CCD) was employed using Minitab software, with Compritol\u0026reg; 888 ATO and Capmul\u0026reg; MCM concentrations as independent variables. The dependent variables included particle size (PS), polydispersity index (PDI), zeta potential (ZP), and entrapment efficiency (%EE). Based on CCD analysis, the optimal formulation was predicted, prepared, and evaluated, yielding a particle size of 90.15 nm, PDI of 0.155, ZP of -11.4 mV, and %EE of 90.43%. The observed particle size of \u0026lt;\u0026thinsp;100 nm is favorable for ocular drug delivery, as it enhances bioavailability by increasing surface area and promoting better corneal permeation. The low PDI (0.155) suggests a narrow size distribution and uniformity within the formulation. Although the zeta potential was relatively low (-11.4 mV), the formulation remained stable, which can be attributed to the use of non-ionic surfactants. Non-ionic surfactants typically exhibit minimal charge, which may reduce electrostatic repulsion but still confer stability through steric hindrance. The optimized NLC batch was formulated using 1.64% Compritol\u0026reg; 888 ATO, 0.32% Capmul\u0026reg; MCM, 0.5% Poloxamer 188, and 0.5% Tween 80, with homogenization performed at 70% amplitude for 7 minutes. Transmission electron microscopy (TEM) analysis (Figure. 7) confirmed the spherical morphology of the NLCs, further validating the structural integrity of the formulation.\u003c/p\u003e\u003cp\u003eThe in vitro drug release study demonstrated a cumulative release of 89.29\u0026thinsp;\u0026plusmn;\u0026thinsp;4.01% over 12 hours, indicating sustained drug release. However, one of the major challenges in ocular drug delivery is rapid precorneal elimination, leading to a low retention time. To address this limitation, the optimized PFD-NLC formulation was incorporated into an in situ gel system containing gellan gum to enhance retention time and prolong therapeutic efficacy. The in vitro drug release from the in situ gel was 84.39\u0026thinsp;\u0026plusmn;\u0026thinsp;3.41%, slightly lower than the NLC suspension, suggesting a controlled release mechanism due to gelation.\u003c/p\u003e\u003cp\u003eEx vivo drug permeation studies using goat cornea and sclera revealed that 82.97\u0026thinsp;\u0026plusmn;\u0026thinsp;3.01% and 77.01\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98% of the drug permeated through the cornea and sclera, respectively, over 12 hours. Pirfenidone's anti-inflammatory and anti-fibrotic effects in the eye, specifically its suppression of fibroblast proliferation and inhibition of TGF-β and TNF-α, necessitate long-term local drug concentrations at the corneal stroma site of action. Enhancing local (ocular) bioavailability is essential for treatment success since corneal haze and fibrosis are localized main pathologies. Poor ocular penetration and the possibility of off-target effects make systemic injection ineffective and undesirable. However, lacrimation, blinking, and nasolacrimal drainage cause traditional topical PFD formulations to be quickly removed from the precorneal region, resulting in tissue levels that are below therapeutic values. This is addressed by the developed PFD-NLC in situ gel technology, which enhances localized drug administration by facilitating regulated release and improves precorneal retention. These findings suggest that the developed PFD-NLC in situ gel enhances ocular bioavailability while providing sustained drug release, making it a promising approach for the therapeutic management of corneal fibrosis.\u003c/p\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003eThe present study demonstrated the potential of PFD-loaded NLCs as an ocular drug delivery system to enhance bioavailability. The optimized PFD-NLC formulation exhibited a particle size of 90.15\u0026thinsp;\u0026plusmn;\u0026thinsp;10.2 nm, with a spherical morphology and uniform size distribution (PDI: 0.155\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014). In vitro drug release studies showed that PFD-NLC and PFD-NLC in situ gel released 89.29\u0026thinsp;\u0026plusmn;\u0026thinsp;4.01% and 84.39\u0026thinsp;\u0026plusmn;\u0026thinsp;3.41% of the drug, respectively, over 12 hours. Ex vivo studies revealed corneal and scleral drug permeation of 82.97\u0026thinsp;\u0026plusmn;\u0026thinsp;3.01% and 77.01\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98%, respectively. Further evaluations confirmed the bio adhesive nature of the formulation. The HET-CAM assay indicated that the formulation was non-irritant, and stability studies demonstrated its physicochemical stability under different storage conditions. These findings suggest that PFD-NLC in situ gel holds significant potential as a promising ocular drug delivery system for the treatment of corneal fibrosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e: Yes\u003c/p\u003e\n\u003cp\u003eData availability statement: The authors attest that the information in the paper supports the study's conclusions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure statement:\u003c/strong\u003e There are no conflicting interests to disclose, according to the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eNo funding was received for this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSimirskii VN. 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Benefits at the nanoscale: a review of nanoparticle-enabled processes favouring microbil growth and functionality. \u003cstrong\u003eEnviron Microbiol.\u003c/strong\u003e 2020;22(9):3633-3649.\u003c/li\u003e\n \u003cli\u003eAstrid Subrizi et.al, Design principles of ocular drug delivery systems: importance of drug payload, release rate, and material properties. \u003cstrong\u003eDrug Discovery Today\u003c/strong\u003e\u003cstrong\u003e. 2019;\u0026nbsp;\u003c/strong\u003e24(8):1457\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"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":"Pirfenidone, Nanostructured lipid carrier, Cornea, Central Composite Design","lastPublishedDoi":"10.21203/rs.3.rs-7107100/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7107100/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose:\u003c/h2\u003e\u003cp\u003ePirfenidone (PFD) possesses significant anti-inflammatory and anti-fibrotic properties, making it a promising therapeutic agent for ocular fibrotic conditions. However, its clinical application is limited due to a short half-life (\u0026lt;\u0026thinsp;19 minutes) in corneal tissue and poor ocular bioavailability. This study aimed to develop and characterize a nanostructured lipid carrier (NLC) based in situ gel formulation for enhancing the ocular delivery and therapeutic efficacy of PFD.\u003c/p\u003e\u003ch2\u003eMethods:\u003c/h2\u003e\u003cp\u003eNLCs were prepared via hot melt emulsification followed by probe sonication and incorporated into a gellan gum-based in situ gel system. A central composite design was used for formulation optimization. The optimized NLC formulation included Compritol\u0026reg; 888 ATO (1.64%), Capmul\u0026reg; MCM (0.32%), Poloxamer 188 (0.5%), and Tween 80 (0.5%). The formulation was characterized for particle size, polydispersity index (PDI), zeta potential, entrapment efficiency, in vitro drug release, ex vivo permeation, cytotoxicity, bioadhesion, and biocompatibility using HET-CAM assay. Stability studies were also conducted.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e\u003cp\u003eThe optimized PFD-loaded NLCs showed a particle size of 90.15\u0026thinsp;\u0026plusmn;\u0026thinsp;10.2 nm, PDI of 0.155\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014, zeta potential of -11.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 mV, and entrapment efficiency of 90.43\u0026thinsp;\u0026plusmn;\u0026thinsp;2.14%. In vitro release studies demonstrated sustained drug release (84.39\u0026thinsp;\u0026plusmn;\u0026thinsp;3.41% over 12 hours). Ex vivo corneal and scleral permeation were 82.97\u0026thinsp;\u0026plusmn;\u0026thinsp;3.01% and 77.01\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98%, respectively. Biocompatibility, cytotoxicity, and stability assessments confirmed the safety and robustness of the formulation.\u003c/p\u003e\u003ch2\u003eConclusion:\u003c/h2\u003e\u003cp\u003eThe developed NLC-based in situ gel offers a promising strategy for enhancing the ocular bioavailability and therapeutic potential of PFD, potentially overcoming the limitations of conventional topical administration.\u003c/p\u003e","manuscriptTitle":"Optimization of Nanostructured Lipid Carrier Using Central Composite Design for Ocular Delivery of Pirfenidone","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-24 13:47:33","doi":"10.21203/rs.3.rs-7107100/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":"dcc8d7f9-71de-4060-a43f-6d6ab8db63e6","owner":[],"postedDate":"October 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-12T16:02:07+00:00","versionOfRecord":{"articleIdentity":"rs-7107100","link":"https://doi.org/10.1007/s12247-025-10306-y","journal":{"identity":"journal-of-pharmaceutical-innovation","isVorOnly":false,"title":"Journal of Pharmaceutical Innovation"},"publishedOn":"2026-01-06 15:57:35","publishedOnDateReadable":"January 6th, 2026"},"versionCreatedAt":"2025-10-24 13:47:33","video":"","vorDoi":"10.1007/s12247-025-10306-y","vorDoiUrl":"https://doi.org/10.1007/s12247-025-10306-y","workflowStages":[]},"version":"v1","identity":"rs-7107100","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7107100","identity":"rs-7107100","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}