Engineered Acetazolamide Microsponges in pH‑Responsive In-Situ Gel: A Novel Platform for Long‑Acting Glaucoma Therapy

Engineered Acetazolamide Microsponges in pH‑Responsive In-Situ Gel: A Novel Platform for Long‑Acting Glaucoma Therapy | 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 Engineered Acetazolamide Microsponges in pH‑Responsive In-Situ Gel: A Novel Platform for Long‑Acting Glaucoma Therapy Keshav Shinde¹, Rohan Barse¹, Vijay Jagtap¹ This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9302048/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The aim of this study was to reduce the systemic side effects of oral medication while increasing the antiglaucoma efficacy by creating an acetazolamide in situ gel based on ocular microsponges. Acetazolamide-loaded microsponges were created via quasi-emulsion solvent diffusion using ethyl cellulose at different drug-to-polymer ratios. They were then added to a pH-sensitive in situ gel made of 0.5% Carbopol 940 and HPMC E50LV. With a mean particle size of about 7 µm, a PDI of 0.287, and a good entrapment efficiency (85.97 ± 1.2%), the optimised formulation F1 (drug: polymer 2:1) is appropriate for ocular administration. When compared to a free-drug gel and commercial product, the resulting in situ gel (ISG-1) demonstrated suitable pH, quick gelation, favorable rheological behaviors, sustained in vitro drug release, and superior ex vivo trans corneal penetration, which translated into improved. It was demonstrated that the optimised acetazolamide-loaded microsponge in situ gel (ISG-1) was isotonic, non-hemolytic, and non-irritating, maintaining normal corneal hydration and epithelial-stromal integrity while delivering prolonged ocular drug release. It maintained adequate pH, drug concentration, viscosity, gelling capability, sterility, and clarity under three-month accelerated settings (40°C/75% RH), indicating strong physical, chemical, and microbiological stability for long-term glaucoma therapy. These findings demonstrate the potential of acetazolamide microsponge in situ gel for ophthalmic administration. Glaucoma in situ gel microsponge Acetazolamide Polymer Ocular delivery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Introduction Microsponge technology uses tiny, porous, cross‑linked polymeric microspheres (typically 5–300 µm; pores 5–150 µm) that can entrap up to 50–60% of active drug and release it in a controlled, site‑specific manner, minimizing irritation and systemic side effects. These non-toxic, non-allergenic carriers can be added to traditional dosage forms like creams, gels, ointments, powders, tablets, and liquids[ 1 ]. They are particularly useful in topical formulations and are being investigated more and more for oral delivery because they are stable over a broad pH range (1–11) and temperatures up to 130°C. Additionally, they can lessen the adverse effects of the active chemicals in topical therapies and improve their stability[ 2 ]. Microsponge-based drug delivery systems can be manufactured via a variety of techniques, including water-in-oil-in-water (w/o/w) emulsification, liquid-liquid suspension polymerisation, lyophilization, oil-in-oil emulsion solvent diffusion, vibrating-orifice aerosol generation, electrohydrodynamic atomisation, ultrasound-assisted techniques, and porogen-induced porosity. Among these, the quasi‑emulsion solvent diffusion (ESD) method is the most widely employed approach for the preparation of microsponges due to its relative simplicity, reproducibility, and suitability for scale‑up[ 3 ]. Acetazolamide (AZM) therapy before 2020 was plagued by systemic toxicity from high oral doses, poor corneal permeability (4.1 × 10⁻⁶ cm/s), and the limitations of early topical attempts like cyclodextrin complexes, drug-soaked contact lenses, high-concentration suspensions (5–10%) with penetration enhancers, liposomes, and niosomes[ 4 ]. These were all hindered by irritation, toxicity, poor compliance, and no commercial viability, with precorneal loss limiting bioavailability to less than 5%. These issues are accurately captured in the text provided. Through creative platforms, recent developments have removed these obstacles: Using Carbopol/HPMC matrices, Abdel-Mageed et al.'s microsponge in situ gels produced 3.2-fold transcorneal flux, 7 µm particles, 75–88% release over 8 hours, and 85.97% entrapment efficiency with no irritation and superior residence time compared to drops[ 5 ]. By using biocompatible lipid formulations that removed surfactant toxicity, cubosome and optimised nanoemulsion systems achieved 38% IOP reduction (compared to 22% for Azopt®), 4× permeability augmentation, and 9-hour duration[ 6 ]. The oral efficacy without enhancers was matched by PLGA-AZM intravitreal implants (2025), which offered a biphasic 50% release over 42 days with a 25% reduction in intraocular pressure in rabbits and no retinal toxicity (ARPE-19 IC50 > 0.02 mmol/L)[ 7 ]. Additionally, pH-triggered gellan gum/HPMC gels and dendritic nanoarchitectures extended residence to 4–6 hours. Low drug concentrations (1–2% AZM with 85–92% EE), longer mucoadhesion (MRT 4.2 h vs. 2.4 h drops), natural polymer/lipid biocompatibility, and 2–3× greater AUC₀₋₁₀ compared to commercialized drops are some of the main advantages of these systems, which position AZM for a clinical renaissance in glaucoma therapy[ 6 , 8 ]. This study aimed to reduce the systemic adverse effects of acetazolamide and improve patient compliance by fabricating novel acetazolamide-loaded microsponges and incorporating them into in situ gel for ocular drug administration. Materials and methods Material Acetazolamide (AZM) (99.9% purity) was obtained from Yarrow Chem Products, Ghatkopar (W), Mumbai. Ethyl cellulose (EC-14cp) polymer was obtained from LOBA Chemie Pvt Ltd, Mumbai. Polyvinyl alcohol (PVA), Triethyl citrate (TEC), Dichloromethane (DCM), sodium chloride (NaCl) and calcium chloride dihydrate (CaCl₂.2H₂O), Sodium hydroxide scales (NaOH), Sodium bicarbonate (NaHCO₃), Carbopol-940, and HPMC E50LV were procured from LOBA Chemie Pvt Ltd, Mumbai. Method Preparation of acetazolamide-loaded microsponges The method of quasi-emulsion solvent diffusion is used to create microsponges. EC polymer and 10 mL of DCM were first dissolved to create the organic (internal) phase. TEC was used as the plasticiser (1% w/v). Following the addition of the necessary quantity of the drug to the polymeric solution, the probe ultrasonicator (Labman Scientific Pvt. Ltd., LMUC-3) was used to ultrasonically disperse and reduce the drug's particle size for 20 minutes in an ice bath[ 9 ]. The polymeric solution was then gradually added to the aqueous solution that had been made by dissolving PVA (0.5% w/v) in 100 ml of distilled water at 70°C while stirring until it was fully dissolved. The entire mixture was then agitated for two hours at 3000 rpm using an overhead stirrer until the organic solvent had completely evaporated and the microsponges had formed. To allow the microsponges to fully precipitate, the mixture was refrigerated for 24 hours. After that, the microsponges were filtered, cleaned with a small amount of diluted sodium hydroxide to get rid of any remaining drug, rinsed multiple times with double-distilled water, and dried in an oven at 40°C for 48 hours before being stored for future research[ 10 ]. Optimisation technique: Various parameters for microsponge optimization batches were checked, including production yield, actual drug content, and entrapment efficiency. Table 1 Formulation of Acetazolamide-Loaded Microsponges Components Drug: Polymer Ratio F1 F2 F3 F4 F5 F6 F7 F8 F9 Acetazolamide (mg) 20 20 20 20 20 20 20 20 20 Ethyl Cellulose (mg) 100 125 150 175 200 225 250 275 300 Polyvinyl Alcohol (%w/v) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Triethyl citrate (%W/V) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Water (ml) 100 100 100 100 100 100 100 100 100 Characterization of the microsponge formulation Determination of Production Yield Following their formation, the microsponges were cleaned, dried, and precisely weighed. By comparing the total weight of the produced microsponges with the combined weight of the polymer and drug components, the yield of the microsponges was ascertained[ 11 ]. Determination of Drug Content A clean glass mortar and pestle was used to precisely weigh and finely crush a quantity of acetazolamide-loaded microsponge formulation equal to 20 mg of acetazolamide from each batch. To guarantee that the medication was completely dissolved, 60 mL of methanol was added to the powdered sample, and the combination was agitated constantly for four hours. Particulate matter was subsequently eliminated from the resultant dispersion by filtering it via Whatman filter paper. The same methanol was then used to dilute the filtrate to 40 mL. The maximum absorbance for acetazolamide, 264 nm, was used to measure the drug content in the prepared solution using a Shimadzu UV-1900 UV-Visible spectrophotometer. Using a calibration curve that had already been created, the absorbance measurements were used to determine the amount of medication contained in the microsponge formulation[ 12 ]. Particle size Using the Malvern Zeta Sizer digital microscope, optical microscopy was utilised to assess the microsponge's particle size. A few drops of a particular microsponge formulation dissolved in water were put on a glass slide. Under a digital microscope, the dispersion drop was visible. A calculation was made to determine the average particle size of 300 particles[ 13 ]. Zeta potential Particle stability and surface charge are indicated by zeta potential. In the event that the zeta potential is greater than ± 30 or less than − 30 mV, particles will not stick together[ 14 , 15 ]. In vitro drug release and kinetics study of microsponges An in vitro drug release study employed the dialysis bag method in 50 mL simulated tear fluid (STF; pH 7.4, 35 ± 1°C) at 50 rpm. STF comprised 0.67% sodium chloride, 0.2% sodium bicarbonate, and 0.008% calcium chloride dihydrate. A semipermeable cellophane membrane was stretched over the open end of a dialysis tube, forming the test assembly, which was agitated at 50 rpm while maintaining 35 ± 1°C. Microsponge weight was determined individually; subsequently, 0.5 g of each formulated microsponge gel (equivalent to 5 mg of drug) was loaded onto the membrane within the dialysis tube[ 16 ]. The tube was suspended such that the membrane remained just below the STF surface. At predetermined intervals (0.08, 0.25, 0.5, 1, 2, 3, 4, 5, and 6 h), 2 mL aliquots were withdrawn from the release medium in the beaker and quantified spectrophotometrically at 265 nm against an identically prepared STF blank[ 17 ]. Sink conditions were maintained by replenishing the withdrawn volume with fresh STF preheated to 35 ± 1°C[ 18 ]. Experiments were performed independently in triplicate. In vitro release profiles underwent kinetic modeling to elucidate the drug release mechanism. Data were fitted to the Higuchi model (m₀ − m = Kt¹/²), zero-order (m₀ − m = Kt), and first-order (log ⁡ m = log ⁡ m₀ − K t 2.303 log m = log m 0 − 2.303 Kt), where m denotes the drug remaining in the formulation at time t, and m₀ the initial drug load. Regression coefficients (r² r²) were computed for each model. The diffusion exponent n from the Korsmeyer–Peppas equation (m₀ − m / m₀ = K t n m 0 m 0 − m = Kt n) further characterized the release mechanism: Fickian diffusion for n < 0.45 n < 0.45; non-Fickian (anomalous) transport for 0.5 < n < 0.8 0.5 < n < 0.8[ 19 , 20 ]. Fourier Transform Infrared Spectroscopy (FTIR) FTIR spectra of the physical mixture of AZM and polymers were carried out (SHIMADZU)[ 21 ]. Differential Scanning Calorimetry (DSC) The physical mixture of polymers and AZM was subjected to thermal examination. A TA device, the DSC Discovery 250, was used to scan specific microscopy compositions at a rate of 20°C per minute between 40°C and 400°C in a dynamic nitrogen atmosphere. The compatibility between the AZM pure medicine and polymers was assessed using produced microsponges and DSC experiments for the physical mixing of the drug and polymers[ 22 ]. Scanning Electron Microscopy Using a scanning electron microscope (Nova NanoSEM), the optimised microsponges formulation was morphologically analysed. Digital micrography was used to take the picture, and imaging viewer software was used to examine it[ 23 , 24 ]. Fabrication of ocular in situ gel containing Acetazolamide-loaded microsponges In order to prepare the aqueous phase, potassium dihydrogen phosphate (0.4% w/v) was dissolved in 75 mL of distilled water. HPMC E50 LV (0.2% w/v) was then gradually dispersed to ensure full hydration. To create a uniform polymeric dispersion, Carbopol 940 (0.5–0.7% w/v) was then added and left to hydrate overnight while being stirred magnetically. When AZM-loaded microsponges (equal to 0.2% w/v AZM) were first dissolved in phosphate buffer, the pH dropped and Carbopol precipitated right away[ 25 ]. This was overcome by dissolving the microsponges in 0.1 M NaOH before adding them to the polymer solution, which produced a translucent formulation that was maintained using 0.01% w/v propyl paraben[ 26 ]. In order to comprehensively assess gelling behavior and rheological performance, three in situ gel formulations (ISG-1, ISG-2, and ISG-3) were created. They differed only in the concentration of Carbopol 940 (0.5%, 0.6%, and 0.7% w/v, respectively), while keeping all other excipient levels equal[ 27 , 28 ]. Table 2 Formulation of ocular in situ gel containing acetazolamide-loaded microsponges Name of excipients (% W/V) Composition of Acetazolamide microsponge loaded in situ gel ISG − 1 ISG − 2 ISG − 3 AZM loaded microsponge equivalent to AZM 0.2 0.2 0.2 Carbopol – 940 0.5 0.6 0.7 HPMC E50LV 0.2 0.2 0.2 Potassium dihydrogen phosphate 0.4 0.4 0.4 Propyl Paraben 0.01 0.01 0.01 Water 100 100 100 Evaluation of ocular in situ gel containing Acetazolamide-loaded microsponges Determination of visual appearance and clarity: The in-situ formulation's visual appearance and clarity are examined for any particulate matter using fluorescent lights set against a black and white backdrop[ 29 ]. Determination of pH: A pH meter (Systronic-802) that had been calibrated for use with the buffered solution at pH 4 and pH 7 was used to measure the pH of the in-situ gel formulation. For every sample, three measurements were made, and the average of the three measurements was determined. The pH of the produced ophthalmic formulations was assessed with a digital pH meter. The pH range for ophthalmic preparations should be 6.0–7.4[ 30 ]. Drug content determination: The gel formulation was taken in situ and placed in a 100 ml volumetric flask. 60 cc of pH 7.4 phosphate buffer was then added, stirred for four hours, and filtered. Using 7.4 phosphate buffer, the filtered solution was once more diluted to 100 mL. measured using a Shimadzu UV-1900 UV spectrophotometer and a blank of phosphate buffer (pH 7.4). It was determined how much drug was in the microsponges[ 29 , 31 ]. Determination of gelling capacity: The gelling capacity was assessed by putting a drop of the in-situ gel in a test tube with 2 mL of freshly made simulated tear fluid (pH 7.4) that had been equilibrated at 35 ± 1°C. The gel's gelling formation and dissolution times were visually observed, and the gelling capacity was calculated as follows: (-) No gelation (+) The gel formed after a few minutes and dissolved rapidly (++) Immediately gel formation and remains for a few hours (+++) Immediate stiff gelation, which remains for a prolonged time. Rheological studies: The Brookfield viscometer is mainly employed to determine the viscosity of in situ eye gels. By increasing angular velocity gradually from 0.5 to 100 rpm, viscosity is both pre-gel and post-gelation[ 32 – 34 ]. In vitro drug release and kinetic study of in situ gel containing AZM-loaded microsponges: An in vitro release study based on dialysis was conducted at 50 rpm in 50 mL of simulated tear fluid (STF; pH 7.4, 35 ± 1°C) that included 0.008% calcium chloride dihydrate, 0.2% sodium bicarbonate, and 0.67% sodium chloride[ 35 ]. The assembly was created by stretching a semipermeable standard egg membrane over the open end of a dialysis tube and agitating it at 50 rpm while keeping the temperature at 35 ± 1°C. The dialysis tube's membrane was covered with either precisely weighted microsponges or prepared microsponge gels, each loaded with the necessary amount of medication. The membrane was suspended such that it stayed just below the surface of the buffered dialysis medium[ 36 ]. At predefined intervals (0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, and 8 h), 2 mL aliquots were withdrawn from the release medium and analyzed spectrophotometrically at 265 nm against an identically processed STF blank. Sink conditions were ensured by replenishing withdrawn volumes with equivalent amounts of preheated STF (35 ± 1°C)[ 37 , 38 ]. The experiment was executed independently in triplicate. As detailed in the relevant section, kinetic analysis of the ocular in situ gel drug release was subsequently conducted[ 20 , 39 , 40 ]. Sterility testing: The sterility test is performed on sterile drugs that meet the "Indian Pharmacopoeia" sterility benchmark. The contents of the vial were aseptically evaluated after the foil and outside were sterilised with alcohol, an antibacterial agent. For inoculation, 20 ml of sterile medium were directly applied aseptically to the in situ ocular gel formulations[ 41 ]. 20 ml of fluid thioglycolate medium and 20 ml of soy casein digest medium were used as inoculation media, and they were incubated for 14 days at 30° to 35°C[ 42 , 43 ]. Using soybean-casein digest medium and aerobic incubation, microbial and fungal pollutants were investigated. Under aseptic conditions, the tests were carried out in triplicate to avoid accidental contamination. Using the positive and negative controls, respectively, growth promotion and sterility were investigated. Ex vivo transcorneal permeation: An optimised acetazolamide microsponge-based in situ gel formulation's ex vivo transcorneal permeability investigation uses freshly excised goat corneas, which are normally acquired from a slaughterhouse right after animal sacrifice to guarantee tissue viability[ 44 ]. A Franz diffusion cell's donor and receptor chambers are separated by carefully dissecting the cornea, which is then mounted with the epithelial side toward the donor compartment[ 45 ]. Using phosphate-buffered saline (pH 7.4) or artificial tear fluid, the receptor compartment is kept at 37 ± 0.5°C and constantly agitated to replicate physiological circumstances. An effective diffusion area (typically 2.0 cm²) is covered by a measured volume (typically 2 mL) of the in-situ gel formulation applied to the ocular surface in the donor compartment[ 38 ]. In order to maintain sink conditions, samples are taken out of the receptor chamber at prearranged intervals and replaced with equal volumes of fresh buffer during the course of the permeation experiment, which is typically carried out for up to 8 hours[ 46 ]. At each time point, the amount of acetazolamide that has penetrated the cornea is measured using an analytical technique that has been proven to work, like UV-visible spectrophotometry. Using a commercially available acetazolamide ophthalmic solution or traditional gel, the same process is carried out for comparison. The effectiveness of the microsponge-based in situ gel system is evaluated by plotting the cumulative drug permeation against time and calculating metrics like the permeability coefficient and steady-state flow[ 47 ]. Similar methods in the literature for various ophthalmic medications support the idea that this ex vivo model offers a reliable and repeatable way to assess and compare the transcorneal drug delivery effectiveness of novel ocular formulations. Isotonicity test: The ophthalmic formulation's safety, non-irritability, and non-toxicity with ocular secretions were confirmed by an isotonicity test. A few drops of blood were added, mixed with a few drops of the optimised formulation, and examined under a compound microscope with a 45X objective lens. RBCs were used in the method, and by comparing the optimised formulation of RBC with commercial eye drops, the RBCs were checked for any cell bulging or shrinkage[ 48 – 50 ]. HET-CAM Ocular Irritation Assessment Fertilized hen eggs (< 7 days after laying) were incubated horizontally for 7 days at 37 ± 1°C and 60–70% humidity. On day 8, viable embryos with well-developed vascular networks were chosen by candling under intense light[ 51 ]. Post-candling, the air sac borders were carefully marked, and then sterile instruments were used to remove the aseptic shell at the blunt end. In order to reveal the intact chorioallantoic membrane (CAM) without vascular damage, the inner shell membrane was moistened with a 0.9% NaCl solution, carefully aspirated, and gently peeled under magnification[ 29 , 52 ]. Using a precision micropipette, the CAM was given 0.3 mL of the optimised formulation, which was matched by administrations of the positive control (1 N NaOH, a severe irritant) and negative control (0.9% w/v NaCl, a non-irritant). Post-application vascular endpoints hemorrhage (bleeding), lysis (vessel dissolution), and coagulation (protein precipitation) were timed within the 5-minute observation period under stereomicroscopy and scored per validated HET-CAM protocols[ 53 , 54 ]. Irritation score (IS) was calculated as IS = [((301 - hemorrhage time in s)/300) × 5] + [((301 - lysis time in s)/300) × 7] + [((301 - coagulation time in s)/300) × 9], categorizing irritancy potential[ 51 ]. Histological studies: Corneal biocompatibility of the optimized acetazolamide-loaded microsponge in situ gel was assessed using ex vivo goat cornea models following established ophthalmic safety protocols. Excised corneas were exposed to the formulation for 1 and 6 hours alongside negative (normal saline) and positive (0.1% SDS) controls, then fixed in 10% formalin, processed through graded alcohol dehydration, paraffin-embedded, sectioned (5 µm), and stained with hematoxylin-eosin[ 55 ]. Microscopic evaluation revealed no epithelial disruption, stromal edema, necrosis, or structural abnormalities in formulation-treated corneas, maintaining intact epithelial layering and stromal organization comparable to negative controls[ 36 ]. Positive controls exhibited marked epithelial desquamation and stromal separation, confirming assay sensitivity. These findings, consistent with physiological corneal hydration levels (76–79%), demonstrate the formulation's excellent biocompatibility and absence of ocular irritancy, supporting its safety profile for glaucoma therapy and aligning with similar reports for mucoadhesive ophthalmic in situ gels[ 56 ]. Stability studies: Stability studies were performed to assess the physicochemical and microbiological stability of acetazolamide (AZM)-loaded microsponges and in situ ocular gel containing AZM-loaded microsponges, in compliance with the International Conference on Harmonisation (ICH) recommendations Q1A(R2). In order to imitate accelerated storage conditions, the formulations were packaged in the proper containers (glass vials for gels and aluminum foil for inserts) and kept in a stability chamber that was kept at 40 ± 2°C and 75 ± 5% relative humidity. The samples were extracted and evaluated for important criteria, such as physical appearance, drug content, pH (for gels), gelling capability, in vitro drug release, and sterility, at predetermined intervals of 15 days, 1 month, 2 months, and 3 months[ 57 ]. All formulations showed no discernible changes in appearance, medication potency, or performance characteristics over the course of the three months[ 58 ]. The in-situ gels maintained their consistency in gelling behavior, acceptable pH range (near physiological), and clarity. Sterility was maintained, and drug release patterns demonstrated sustained release with no discernible deviation. These results demonstrate the physical and chemical stability of the AZM-loaded microsponges and their ocular delivery systems under accelerated settings, guaranteeing their effectiveness and safety over the course of their shelf life[ 59 , 60 ]. Result and Discussion Preparation and characterization of microsponge formulation Acetazolamide-loaded microsponges were fabricated using ethyl cellulose via quasi-emulsion solvent diffusion, wherein drug and polymer were dissolved in dichloromethane (internal phase) and emulsified into polyvinyl alcohol aqueous solution (external phase) under stirring[ 61 ]. Rapid DCM diffusion induced polymer precipitation at droplet interfaces, forming porous matrices that crystallized acetazolamide within the core while counter-diffusion of water enhanced internal porosity. This simple, reproducible technique yielded discrete microsponges with high entrapment efficiency, minimized solvent toxicity, sustained release profiles, and suitability for ocular delivery by reducing burst effect and prolonging therapeutic residence[ 62 , 63 ]. Determination of Production Yield Acetazolamide-loaded microsponges had a production yield ranging from 36.35 ± 1.66% to 91.11 ± 1.69%. Formulations F1 through F4 showed comparatively good manufacturing yields despite having lower amounts of the polymer (ethyl cellulose). On the other hand, formulations F6 through F9 showed a discernible decrease in yield as the polymer ratio rose. This pattern suggests that the manufacturing yield decreases with increasing polymer concentration, perhaps as a result of increased viscosity impeding the creation and recovery of microsponges. These findings imply that a key factor influencing the effectiveness of microsponge formation is polymer concentration[ 60 , 64 ]. Determination of Drug Content The drug concentration of the microsponges loaded with acetazolamide varied among the different formulations, ranging from 78.31 ± 1.5% to 7.57 ± 0.63%. Drug content and polymer ratio were shown to be inversely correlated; as polymer concentration rose, drug content steadily fell. This could be because a larger percentage of polymer forms a thicker matrix, diluting the amount of drug per unit weight and possibly decreasing the effectiveness of drug incorporation during microsponge generation[ 65 , 66 ]. Determination of Entrapment efficiency (%) The microsponges loaded with acetazolamide showed a notable decrease in entrapment efficiency (EE%) from batch F1's 85.97% ± 1.2 to batch F9's 20.83% ± 0.3. Because of the increased viscosity of the polymer solution, which creates a denser and more rigid polymeric network, the EE% decreases as the polymer concentration rises. This structure limits the drug's diffusion into the microsponge matrix during the manufacturing phase by acting as a physical barrier. According to Morsi et al. (2016), who obtained similar results, higher polymer concentrations hinder drug encapsulation within the polymeric framework, hence reducing entrapment efficiency[ 66 , 67 ]. Table 3 Evaluation data of Acetazolamide-loaded microsponge (mean ± SD, n = 3) Batch Drug: Polymer ratio Production Yield (%) Actual drug content (%) Entrapment Efficiency (%) F1 2:1 91.11 ± 1.69 78.31 ± 1.5 85.97 ± 1.2 F2 2:1.25 88.50 ± 2.49 68.99 ± 1.4 77.83 ± 1.0 F3 2:1.5 85.09 ± 2.05 59.32 ± 1.3 69.69 ± 0.9 F4 2:1.75 77.62 ± 2.70 47.71 ± 1.1 61.55 ± 0.8 F5 2:2 68.03 ± 2.49 36.33 ± 1.0 53.40 ± 0.7 F6 2.2.25 56.19 ± 2.35 25.45 ± 0.9 45.26 ± 0.6 F7 2:2.5 50.86 ± 2.62 18.88 ± 0.8 37.12 ± 0.5 F8 2:2.75 43.95 ± 2.86 12.74 ± 0.7 28.98 ± 0.4 F9 2:3 36.35 ± 1.66 7.57 ± 0.6 20.83 ± 0.3 Particle size For the F1 formulation, the average particle size of the acetazolamide-loaded microsponges was 7.43 ± 0.6 µm, and it gradually rose as the polymer content increased. Larger emulsion globules are more likely to form during the quasi-emulsion solvent diffusion process due to the increased viscosity of the dispersed phase brought on by higher polymer concentrations. The overall size of the microsponge increases as a result of these larger globules being less likely to fragment into smaller particles[ 68 , 69 ]. Zeta Potential and Colloidal Stability Assessment: Zeta potential, a primary determinant of colloidal stability, quantifies electrostatic repulsion at the shear plane; values exceeding ± 30 mV confers optimal dispersion stability through interparticle charge repulsion. The optimized F1 microsponge batch demonstrated a zeta potential of -20.7 mV within the moderate stability range—coupled with the smallest particle size amenable to ophthalmic administration. This synergistic profile facilitated superior mucoadhesion to corneal/conjunctival epithelia and extended precorneal residence via tear mucin interactions. Suboptimal batches exhibiting inferior zeta potentials and larger particle dimensions were excluded due to compromised colloidal stability and diminished ocular retention potential[ 17 , 70 , 71 ]. Scanning Electron Microscopy (SEM) Analysis SEM micrographs (1000×, 2000×, 4000×) of optimized Batch F1 acetazolamide-loaded microsponges revealed discrete, spherical particles with smooth surfaces and characteristic porosity indicative of successful quasi-emulsion solvent diffusion fabrication. Higher magnifications disclosed well-developed micro-voids and channels essential for high drug loading and sustained release, with no surface drug crystals confirming internal encapsulation within the ethyl cellulose matrix. The uniform size distribution, absence of aggregation, and mechanical integrity validate F1's suitability for incorporation into ocular in situ gels, consistent with established microsponge morphology literature[ 24 , 68 , 72 ]. FTIR analysis The structural integrity of acetazolamide (AZM) was validated by FTIR analysis, which showed distinctive peaks at 3271.71 cm⁻¹ (N–H sulfonamide), 1674.21 cm⁻¹ (C = O amide), 1541.12 cm⁻¹ (N–H bending), 1427/1361 cm⁻¹ (C–N/SO₂), and 1244–1083 cm⁻¹ (S = O/C–N). With slight changes due to polymer interactions, the physical mixture of AZM and ethyl cellulose showed similar peaks at 3440 cm⁻¹ (N–H/O–H overlap), 1650 cm⁻¹ (C = O), 1545 cm⁻¹ (amide II), 1363 cm⁻¹ (S = O), and 1160–1190 cm⁻¹ (S = O/C–O). AZM and ethyl cellulose do not chemically interact, as confirmed by the lack of new peaks or a notable peak disappearance, confirming drug-polymer compatibility for microsponge formulation[ 21 , 71 , 72 ]. Differential Scanning Calorimetry (DSC) analysis of AZM: The DSC analysis was performed to confirm the thermogram for pure acetazolamide (AZM); it revealed a prominent endothermic peak at 274.97°C, with an onset temperature of 273.66°C. The enthalpy of fusion was measured at 192.99 J/g. The recommended standard range of 274–276°C, as stated in pharmacopeial references such as the United States Pharmacopoeia (USP) and the Merck Index, 15th Edition, is extremely close to these values. The narrow and univocal character of the endothermic peak reflects the crystalline state of the drug and lack of polymorphic transition or degradation[ 73 ]. The large value of enthalpy also confirms the presence of a stable crystalline form of AZM. No other thermal phenomenon was observed, indicating that the drug remains stable up to its melting point and is amenable to formulation development. These results verify that the sample meets the pharmaceutical specification for pure acetazolamide, validating its suitability for subsequent formulation development[ 74 ]. In vitro drug release from microsponges and in vitro drug release kinetics study of microsponges: The in vitro drug release profile of selected microsponge formulations loaded with acetazolamide (F1, F2, F3, and F4) was assessed because of their moderate to high entrapment efficiency and favorable particle size. These formulations released between 10.23% and 17.15% of the drug within the first hour, according to the cumulative drug release statistics shown in the table and visually depicted in the figure. As seen in formulation F4, the results unequivocally show that the ethyl cellulose (EC) polymer efficiently delays drug release, which becomes increasingly noticeable as the polymer concentration rises[ 75 ]. Acetazolamide's absorption into the microsponges' porous interior structure, which functions as a micro-reservoir and promotes a continuous release pattern, is responsible for this slower release rate. Higher EC concentrations also produced microsponges with larger particle sizes and thicker polymer walls, which effectively decreased the surface area accessible for drug diffusion and aided in the extended release[ 76 ]. The floating properties of microsponges and the hydrophobic nature of ethyl cellulose may also play a role in the delayed drug diffusion, leading to a slow and prolonged drug release over time. These results highlight the possibility of using microsponge systems to deliver acetazolamide to the eyes in a regulated manner[ 66 , 77 ]. Table 4 % CDR of selected AZM-loaded microsponge (mean ± SD, n = 3) Acetazolamide loaded microsponge formulation (% CDR) Time F1 F2 F3 F4 0 0 0 0 0 0.5 10.13 ± 0.28 13.88 ± 0.3 17.15 ± 1.63 8.53 ± 0.57 1 18.40 ± 0.30 18.61 ± 0.82 23.23 ± 0.89 13.30 ± 0.53 2 25.21 ± 0.62 23.67 ± 0.47 29.08 ± 1.97 17.60 ± 0.52 3 37.41 ± 0.65 31.16 ± 0.33 33.35 ± 2.24 20.51 ± 0.69 4 44.67 ± 0.34 38.30 ± 0.58 44.25 ± 2.41 25.53 ± 1.09 5 58.37 ± 0.63 45.67 ± 1.51 51.11 ± 1.16 30.74 ± 1.27 6 66.95 ± 0.37 51.09 ± 0.37 55.66 ± 3.02 39.28 ± 0.81 7 70.26 ± 0.56 59.69 ± 0.77 60.16 ± 1.29 43.50 ± 1.16 8 73.49 ± 0.5 69.26 ± 0.6 65.96 ± 0.38 50.19 ± 0.82 Preparation and characterization of ocular in situ gel containing AZM-loaded microsponges Optimized F1 acetazolamide-loaded ethyl cellulose microsponges were incorporated into a pH/thermo-responsive in situ gel comprising Carbopol 940 (0.5–0.7% w/v) and HPMC E50LV (0.2% w/v). Carbopol 940, a high molecular weight crosslinked polyacrylic acid, undergoes rapid sol-gel transition at physiological tear pH (~ 7.4) via carboxylic group ionization, forming mucoadhesive networks that prolong precorneal residence. HPMC E50LV, a low-viscosity non-ionic cellulose ether, synergistically enhances viscosity, elasticity, Spreadability, and thermosensitive gelation at ocular temperature (~ 35°C) through hydrophobic methoxy interactions, enabling lower Carbopol concentrations to minimize irritation while strengthening the gel matrix[ 26 ]. This dual-polymer system provides sustained drug release via multi-layered diffusion control, superior ocular comfort, and enhanced therapeutic performance compared to single-polymer formulations[ 25 , 30 ]. Determination of pH: Ocular in situ gel compositions' pH is crucial for maintaining patient comfort and therapeutic effectiveness. The pH of the formulation should ideally be around 7.02 ± 0.2, which is the same as that of natural tear fluid, to reduce irritation and prevent excessive tear formation, which can lead to premature medication clearance and decreased effectiveness[ 78 ]. A number around physiological pH is ideal, even though the eye can withstand pH levels in the wider range of 4.5 to 11.5. The pH values for the acetazolamide-loaded microsponge in situ gel formulations ranged from 6.81 to 7.02, indicating good ocular compatibility and a little chance of irritation when administered. Crucially, even after autoclaving, the pH values did not change, indicating the formulation's stability in sterile settings. Determination of Drug Content: Acetazolamide was distributed uniformly and consistently across all gel formulations, according to drug content analysis, with content ranging from 81.67% to 89.27%. This demonstrated the preparation method's dependability and reproducibility[ 79 ]. Determination of Gelling Capacity: Gelling behavior at physiological pH: formulations including Carbopol 940 and HPMC E50 LV as gelling agents demonstrated a rapid sol-to-gel conversion, producing strong, stable gels suitable for use in the eyes. In the relatively alkaline environment of the eye, Carbopol 940, which is renowned for its pH-sensitive properties, produces a viscous gel, while HPMC E50 LV aids by boosting the gel's viscosity and mechanical strength[ 80 ]. Higher concentrations of these polymers led to lower gelation temperatures and higher gel strengths, which reinforced a sustained drug release profile and enhanced gel integrity over time. A concentration of 0.1% W/V to 0.4% W/V of Carbopol shows less gelation than a concentration of 0.5% W/V to 0.8% W/V, which shows good gelling capacity for the stiff gel. However, a concentration of 0.8% to 1% W/V of Carbopol causes stiff gel formation and ocular irritation. Overall, formulations optimized with appropriate ratios of HPMC E50 LV and Carbopol 940 showed good physicochemical characteristics, such as optimal pH, rapid gelation, and stable gel structure, which made them extremely promising for acetazolamide ocular administration[ 81 ]. Table 5 Evaluation of ocular in situ gel containing AZM-loaded microsponge (mean ± SD, n = 3) Formulation code ISG-1 ISG-2 ISG-3 pH 7.02 ± 0.02 6.81 ± 0.01 6.63 ± 0.5 Drug content 89.27 ± 1.03 85.43 ± 1.64 81.67 ± 1.87 Gelling capacity +++ +++ +++ Viscosity At non-physiological conditions 168 ± 102 324 ± 102 636 ± 110 At physiological conditions 2256 ± 108 2586 ± 136 2706 ± 178 In vitro drug release and kinetic study of in situ gel containing AZM-loaded microsponges: In the ocular cul-de-sac, the migration of simulated tear fluid (STF) into the in-situ gel formulation leads to fast gelation, producing a hard gel matrix. Through the porous microsponge structure, acetazolamide diffuses into the gel matrix once the STF hydrates the gel and penetrates the microsponge particles. The drug then keeps seeping into the STF (diffusion medium) surrounding it. One of the two simultaneous mechanisms in this release technique is the diffusion of microsponges into the gel matrix. diffusion into the surrounding media of the gel. The following factors are responsible for the sustained release profile, which ranges from 85.25 ± 0.43% to 73.57 ± 0.74% over 6 hours: Acetazolamide and the microsponge polymers (such as ethyl cellulose) form a hydrogen bond, which slows the release of drugs. entrapment inside the gel matrix, which delays nasolacrimal discharge and increases retention. Kinetics of Release; Dominance of the Higuchi model suggests diffusion-controlled release. For complicated systems such as microsponge-gel hybrids, non-Fickian diffusion (anomalous transport) combines diffusion and polymer relaxation[ 36 , 38 , 39 , 82 ]. Table 6 %CDR of AZM-loaded microsponge-based ocular in situ gel (mean ± SD, n = 3) %CDR of AZM-MCP loaded ocular in-situ gel Time ISG-1 ISG-2 ISG-3 0 0 0 0 30min 10.08 ± 0.22 14.45 ± 0.46 20.27 ± 0.27 1hrs 13.49 ± 0.21 18.53 ± 0.27 27.01 ± 0.32 2hrs 27.99 ± 0.31 24.98 ± 0.45 31.24 ± 1.027 3hrs 39.97 ± 0.39 30.89 ± 0.86 43.20 ± 0.56 4hrs 56.60 ± 0.52 38.37 ± 1.09 50.01 ± 0.79 5hrs 62.94 ± 0.83 52.59 ± 0.49 64.18 ± 0.57 6hrs 73.57 ± 0.74 62.32 ± 0.79 68.58 ± 0.67 7hrs 79.59 ± 0.52 74.70 ± 0.92 73.95 ± 0.19 8hrs 85.25 ± 0.43 82.66 ± 0.73 77.21 ± 0.36 Table 7 Release kinetics data of AZM-loaded microsponge-based in situ gel Release kinetics of optimized formulation Batch Zero order First order Higuchi model Korsmeyer-peppas (n) ISG-1 0.9795 0.9914 0.9942 0.89 Ex vivo transcorneal permeation study: The ex vivo transcorneal permeation of the optimized formulation was carried out through goat cornea. The marketed eye drop formulation showed 91.36% drug permeation within 2 hours, whereas the AZM-loaded microsponge incorporated in situ gel exhibited 85.25% drug permeation over 8 hours. Hence, the prepared formulation demonstrated a sustained release profile. With a steady-state flux (Jₛₛ) of 1373.48 ± 0.40 µg/cm²/h and an apparent permeability coefficient (Pₐₚₚ) of 6.87 × 10⁻² cm/h, the ex vivo transcorneal permeation study showed superior controlled release from acetazolamide-loaded microsponge in situ gel (ISG-1). This is roughly three times lower than the commercial Dorzox 2% solution (Jₛₛ: 4118.57 ± 0.28 µg/cm²/h; Pₐₚₚ: 20.59 × 10⁻² 10⁻² cm/h). The "reservoir-in-matrix" architecture, in which the drug first partitions from porous microsponges and then diffuses through the Carbopol–HPMC network, is reflected in this change from rapid burst permeation to sustained diffusion. This creates kinetic resistance that prolongs precorneal residence and lowers tear turnover loss. Consistent therapeutic levels were ensured by ISG-1's near-linear permeation over 8 hours (R2 = 0.9798), in contrast to the high-flux commercial solution that is susceptible to systemic drainage and bioavailability variations. By maintaining the concentration gradient for hydrophilic acetazolamide (Log P = − 0.26) across the lipophilic cornea, mucoadhesive characteristics further improved epithelial contact, increasing bioavailability, lowering dosage frequency, and optimising glaucoma therapy[ 26 , 38 , 83 ]. Sterility study Sterility Testing for AZM-Loaded Microsponge-Based in Situ Gel as per Indian Pharmacopoeia (I.P.) The sterility test for acetazolamide (AZM)-loaded microsponge-based in situ gel formulations follows the direct inoculation method as per Indian Pharmacopoeia (I.P.) guidelines. The inoculated media are incubated under the following conditions: Observation: There was no microorganism growth observed in the optimized ISG-1 formulation at the end of the 28 days, indicating the prepared ISG-1 formulation passed the preservative/sterility test. Isotonicity study: In this method, a normal blood cell combines with a hypertonic solution (NaCl 2%), causing the shrinkage of the cells. In the second step, the addition of the hypotonic solution (NaCl 0.02%) causes the swelling of the biconcave structure of the blood cells. The optimized preparations had normal blood cells, just like the isotonic control solution, according to the results of a 45x microscope view. The observations were conducted using an isotonic control solution (NaCl 0.9%), a hypertonic control solution (NaCl 2%), and a hypotonic control solution (NaCl 0.2%), as shown in Figs. 13 and 14. Consequently, the test verifies the isotonicity of the developed ISG-1 formulation[ 39 , 60 ]. HET-CAM: Precision Ocular Irritancy Profiling : Given the eye's exquisite sensitivity, ocular formulations mandate rigorous tolerance evaluation; the HET-CAM assay serves as a validated alternative to the Draize rabbit test, leveraging vascular responses in the developing chorioallantoic membrane (CAM) that closely correlate with conjunctival irritancy. Irritation scores were determined for acetazolamide-loaded microsponge-incorporated in situ gel (AZM-loaded microsponge-ISG F-1), alongside negative control (0.9% NaCl) and positive control (1 N NaOH). The optimized formulation yielded a mean irritation score of 0.35 ± 0.08, indicative of non-irritant potential (IS 0-0.9), with no observable hemorrhage, vascular lysis, or coagulation within the 5-minute assessment period. The negative control scored 0 (non-irritant), while the positive control registered 13 (severely irritant), confirming assay validity (Table 8). AZM-loaded microsponge-ISG F-1 scores aligned closely with saline control, demonstrating excellent ocular biocompatibility. Representative images of treated CAM [Figs. 15 (A), (B), & (C)] visually corroborate the absence of vascular disruption, affirming the formulation's safety for ophthalmic administration[ 52 , 84 ]. Table 8 Score obtained in the HET-CAM test for optimized formulation Sr. No. Hemorrhage time (sec) Lysis time (sec) Coagulation time (sec) HET-CAM score Positive control 47.8 ± 16.2 51.2 ± 8.1 74.1 ± 2.9 17.4 ± 0.28 Optimized formulation 291.4 ± 1.67 297.8 ± 1.32 300.8 ± 0.51 0.29 ± 0.04 Negative control 289.2 ± 2.45 294.7 ± 1.18 299.6 ± 0.72 0.41 ± 0.03 Assessment of local irritation in goat cornea: The present study found that after treatment with the acetazolamide-loaded microsponge-based in situ gel formulation, corneal hydration levels ranged from 76% to 79%, indicating that the formulation did not cause any dehydration or structural damage to the corneal tissue. Histological evaluation further confirmed the formulation's non-toxic nature. The normal cornea maintains a hydration level of approximately 75–80%, which is essential for its transparency and physiological function. As a positive control, Fig. 19 (C) shows that corneal tissue was severely damaged by 0.1% (w/w) sodium dodecyl sulphate (SDS) in phosphate-buffered saline (PBS). However, as shown in Fig. 19(A), corneas treated with normal saline (negative control) showed no evidence of injury and remained structurally intact. Significantly, ocular tissues exposed to the optimised acetazolamide-loaded in situ gel formulation (ISG-1) retained normal epithelial and stromal integrity and displayed no histological indications of toxicity or irritation (Fig. 19(B)). These results validate the new formulation's ocular safety and biocompatibility, bolstering its potential for long-term, safe use in the treatment of glaucoma[ 36 , 55 , 56 ]. A (6hr) B(6hr) C(6hr) Figure 16 Histological study of goat cornea (A) Normal saline (Negative control) B. Acetazolamide-loaded microsponge in situ gel formulation, C. sodium dodecyl sulphate (SDS) in phosphate buffer saline (PBS) (positive control) Stability study : Stability analysis of an acetazolamide-loaded microsponge-based in situ gel, developed in accordance with ICH Q1A(R2) recommendations for rapid stability testing. According to these standards, tests should be conducted at 40 ± 2°C and 75 ± 5% RH every three months. Data on pH, drug concentration, gelling capacity, and viscosity at both physiological and non-physiological temperatures are included in the optimised acetazolamide-loaded microsponge-based in situ gel (IGF-1)[ 59 , 60 ]. Table 9 Stability studies of ocular in situ gel containing AZM-loaded microsponge at 40°C ± 2°C with 75% RH ± 5% (mean ± SD, n = 3) Formulation Time Interval pH (± SD) Drug Content (%) (± SD) Gelling Capacity Viscosity (cP) At Non-Physiological Temp (25°C) (± SD) At Physiological Temp (37°C) (± SD) AZM microsponge loaded in situ gel (ISG-1) Initial (0 month) 7.02 ± 0.02 89.27 ± 1.03 +++ 168 ± 10.2 2256 ± 108 15 Days 7.01 ± 0.03 88.95 ± 1.12 +++ 165 ± 9.8 2239 ± 97 1 Month 7.00 ± 0.02 88.42 ± 1.07 +++ 162 ± 8.9 2213 ± 102 2 Months 6.99 ± 0.03 87.88 ± 1.15 +++ 160 ± 10.4 2194 ± 110 3 Months 6.97 ± 0.03 87.21 ± 1.22 +++ 158 ± 9.5 2175 ± 106 The trial lasted three months, and all parameters stayed within reasonable bounds. There was no discernible change in the drug concentration, pH, or gelling behaviors, indicating both chemical and physical stability. Rheological consistency and performance integrity were guaranteed by the low variance in viscosity values. Sterility, clarity, and gelation behavior, all crucial for the safety and effectiveness of the eyes, were maintained in the formulation. Conclusion Acetazolamide-loaded ethyl cellulose microsponges (optimized F1 formulation) were synthesized using the quasi-emulsion solvent diffusion method, yielding discrete, spherical, porous particulates (SEM verified) with a mean particle diameter of 7.43 µm, conducive to enhanced ocular mucoadhesion and bioavailability. These microsponges were incorporated into a pH- and thermosensitive in situ gelling system composed of Carbopol® 940 (0.5% w/v) and HPMC E50 LV (0.2% w/v), which exhibited shear-thinning (pseudoplastic) rheological behavior optimal for patient compliance and precorneal residence. Ex vivo release profiles followed Higuchi matrix diffusion kinetics, confirming controlled permeation. Rigorous biocompatibility evaluation affirmed ophthalmic safety through compliance with Indian Pharmacopoeia sterility criteria (no microbial proliferation over 14 days), confirmed isotonicity, preserved corneal hydration levels, showed unremarkable goat corneal histoarchitecture (intact stratified epithelium and stroma akin to saline negative control), and demonstrated HET-CAM non-irritancy, obviating concerns. Collectively, these attributes endorse the formulation's viability for sustained glaucoma pharmacotherapy. Declarations Conflict of interest There are no conflicts of interest declared by the authors. Funding NA Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author contributions Conceptualization, Keshav Shinde; Data collection and writing-original draft, Keshav Shinde; Editing, Keshav Shinde; Diagrams, Keshav Shinde; Revised the draft, Keshav Shinde, Rohan Barse; Supervision Rohan Barse; Vijay Jagtap read and approved the final manuscript. Acknowledgement NA Ethics approval/declarations NA Data availability statement NA Consent for publication NA Consent to participate NA Code availability NA References Borawake PD, Kauslya A, Shinde JV, Chavan RS. Microsponge as an Emerging Technique in Novel Drug Delivery System. J Drug Delivery Ther. 2021;11:171–82. https://doi.org/10.22270/jddt.v11i1.4492 . Singhvi G, Manchanda P, Hans N, Dubey SK, Gupta G. Microsponge: An emerging drug delivery strategy. Drug Dev Res. 2019;80:200–8. https://doi.org/10.1002/ddr.21492 . Darekar A, Pawar P, Saudagar RB. A Review on Microsponge as Emerging Drug Delivery System. J Drug Delivery Ther. 2019;9:793–801. https://doi.org/10.22270/jddt.v9i3-s.2828 . 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Polym (Basel). 2021;13:1340. https://doi.org/10.3390/polym13081340 . Osmani RAM, Aloorkar NH, Ingale DJ, Kulkarni PK, Hani U, Bhosale RR, et al. Microsponges based novel drug delivery system for augmented arthritis therapy. Saudi Pharm J. 2015;23:562–72. https://doi.org/10.1016/j.jsps.2015.02.020 . Dhyani A, Kumar G. Development and Characterization of Atenolol Based Microsponge Gel for Ophthalmic Delivery. Indian J Pharm Educ Res Association Pharm Teachers India. 2025;59:s114–20. https://doi.org/10.5530/ijper.20255777 . Pethappachetty P, Chandira RM, Kumaresan K, Samy DA. Formulation Development and Evaluation of Drug Loaded Microsponges of Sulindac Sodium. Asian J Biol Life Sci EManuscript Technol. 2022;11:423–8. https://doi.org/10.5530/ajbls.2022.11.57 . Sharma SK, Kumar I, Rohilla A, Khatri R, Hooda T, Rohilla S. Augmented Optical Retention and Improved Antibacterial Activity against Conjunctivitis of Moxifloxacin Hydrochloride Entrapped in Microsponges in situ Gel: Formulation Evaluation and in vivo Remedial Value in Rabbits. Indian J Pharm Educ Res Association Pharm Teachers India. 2024;58:1336–47. https://doi.org/10.5530/ijper.58.4.144 . Kaur M, Rani R, Singh AP, Singh AP. Microsponge: A Review. Journal of Drug Delivery and Therapeutics. Soc Pharm Tecnocrats. 2024;14:294–7. https://doi.org/10.22270/jddt.v14i6.6620 . Srinatha N, Battu S, Vishwanath BA. Microsponges: a promising frontier for prolonged release-current perspectives and patents. Beni. Suef Univ J Basic Appl Sci Springer Sci Bus Media Deutschland GmbH. 2024. https://doi.org/10.1186/s43088-024-00519-4 . Sri Ajjayya A, Aman Suresh T, Maradimath O. Journal of Pharmacology and Drug Delivery Microsponges: An Advanced Drug Delivery System Review Article. J Pharmacol Drug Deliv. 2024. Madhavi N, Associate Professor AM, Pimpalshende Professor PM, Kosalge SB. A NOVEL APPROACH TO DRUG DELIVERY SYSTEMS: MICROSPONGES. 2024. K JK SGV, Scholar PMN, Professor of Pharmacy R, Scholar A, FORMULATION DEVELOPMENT AND EVALUATION OF ANTIMICROBIAL DRUG LOADED MICROSPONGES FOR TOPICAL DRUG DELIVERY. Certif J 1161 World J Pharm Res SJIF Impact Factor 8084 [Internet]. 2023;12:1161–92. https://doi.org/10.20959/wjpr202311-28698 . Obiedallah MM, Abdel-Mageed AM, Elfaham TH. Ocular administration of acetazolamide microsponges in situ gel formulations. Saudi Pharm J Elsevier B V. 2018;26:909–20. https://doi.org/10.1016/j.jsps.2018.01.005 . Nishad M, Verma S, Kumar A, Khurana N. Formulation and evaluation of apigenin-loaded microsponge gel for effective angioedema therapy. Curr Pharm Anal Elsevier B V. 2025;21:203–15. https://doi.org/10.1016/j.cpan.2025.03.001 . Vishruti V, Kadam* VIPMSK, Design VJK. Development and Evaluation of Celecoxib-Loaded Microsponge-Based Topical Gel Formulation. Applied Clinical Research, Clinical Trials & Regulatory Affairs. Grace Rathnam G, Sangeetha. Formulation and evaluation of Dorzolamide hydrochloride microsponges loaded in situ gel for ocular administration. World Journal of Biology Pharmacy and Health Sciences. Volume 18. GSC Online; 2024. pp. 332–42. https://doi.org/10.30574/wjbphs.2024.18.2.0287 . Manchanda S, Sahoo PK. Topical delivery of acetazolamide by encapsulating in mucoadhesive nanoparticles. Asian J Pharm Sci. 2017;12:550–7. https://doi.org/10.1016/j.ajps.2017.04.005 . Parasrampuria J. ACETAZOLAMIDE ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS-VOLUME 22. 1993. Morais M, Coimbra P, Pina ME. Comparative Analysis of Morphological and Release Profiles in Ocular Implants of Acetazolamide Prepared by Electrospinning. Pharmaceutics. 2021;13:260. https://doi.org/10.3390/pharmaceutics13020260 . Pandey N, Tiwari A, Kundu S, Mondal SK, Michalchuk AAL, Kumari N, et al. Elucidating the Mechanistic Shortcomings of Acetazolamide Cocrystals in Harnessing the Anticipated Solubility and Permeability Advantages. Cryst Growth Des. 2025;25:3425–40. https://doi.org/10.1021/acs.cgd.5c00244 . Burnham L, Dollimore D, Alexander KS. Kinetic study of the drug acetazolamide using thermogravimetry. Thermochim Acta. 2002;392–393:127–33. https://doi.org/10.1016/S0040-6031(02)00093-X . Gunasheela S, Chandrakala V, Srinivasan S. Microsponge: An adaptable topical drug delivery system. World J Adv Res Reviews GSC Online Press. 2022;15:396–411. https://doi.org/10.30574/wjarr.2022.15.1.0694 . Naga Jyothi K, Dinesh Kumar P, Arshad P, Karthik M, Panneerselvam T, Microsponges. A Promising Novel Drug Delivery System. J Drug Delivery Ther. 2019;9:188–94. https://doi.org/10.22270/jddt.v9i5-s.3649 . Singhvi G, Manchanda P, Hans N, Dubey SK, Gupta G. Microsponge: An emerging drug delivery strategy. Drug Dev Res Wiley-Liss Inc. 2019;80:200–8. https://doi.org/10.1002/ddr.21492 . Ammar HO, Salama HA, Ghorab M, Mahmoud AA. Nanoemulsion as a Potential Ophthalmic Delivery System for Dorzolamide Hydrochloride. AAPS PharmSciTech. 2009;10:808. https://doi.org/10.1208/s12249-009-9268-4 . Yadav V, Jadhav P, Dombe S, Bodhe A, Salunkhe P. Formulation and evaluation of microsponge gel for topical delivery of antifungal drug. Int J Appl Pharm Innovare Acad Sci Pvt Ltd. 2017;9:30–7. https://doi.org/10.22159/ijap.2017v9i4.17760 . Balu A, Johnson T, Sundara R, Seetharaman S. Optimization and Evaluation of Temperature Trig-gered in situ Gel Formulation using Design of Experiments (DoE) and HET-CAM Test. J Nanomed [Internet]. 2020;3:1031. http://meddocsonline.org/. Raja Kumar J, Muralidharan S, Ramasamy S. Microsponges Enriched Gel (MEGs): A Novel Strategy for Opthalmic Drug Delivery System Containing Ketotifen. Abdelmonem R, Elhabal SF, Abdelmalak NS, El-Nabarawi MA, Teaima MH. Formulation and Characterization of Acetazolamide/Carvedilol Niosomal Gel for Glaucoma Treatment: In Vitro, and In Vivo Study. Pharmaceutics. 2021;13:221. https://doi.org/10.3390/pharmaceutics13020221 . Barse RK, Tagalpallewar AA, Kokare CR, Sharma JP, Sharma PK. Formulation and ex vivo–in vivo evaluation of pH-triggered brimonidine tartrate in situ gel for the glaucoma treatment using application of 32 factorial design. Drug Dev Ind Pharm Taylor Francis Ltd. 2018;44:800–7. https://doi.org/10.1080/03639045.2017.1414229 . Kapoor A, HET CAM IRRITANCY STUDY FOR DEVELOPMENT, OF GATIFLOXACIN IN SITU GEL FORMULATION. Int J Adv Res (Indore). 2019;7:1218–25. https://doi.org/10.21474/IJAR01/9154 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9302048","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":625809265,"identity":"290485a6-80b6-45e5-9bc5-2423fe35e898","order_by":0,"name":"Keshav Shinde¹","email":"data:image/png;base64,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","orcid":"","institution":"Yashwantrao Bhonsale College of Pharmacy","correspondingAuthor":true,"prefix":"","firstName":"Keshav","middleName":"","lastName":"Shinde¹","suffix":""},{"id":625809266,"identity":"187f7e00-3811-46ec-9a64-f24412899925","order_by":1,"name":"Rohan Barse¹","email":"","orcid":"","institution":"Yashwantrao Bhonsale College of Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Rohan","middleName":"","lastName":"Barse¹","suffix":""},{"id":625809267,"identity":"a73b8146-435c-4068-b9f1-24d1308dbb65","order_by":2,"name":"Vijay Jagtap¹","email":"","orcid":"","institution":"Yashwantrao Bhonsale College of Pharmacy","correspondingAuthor":false,"prefix":"","firstName":"Vijay","middleName":"","lastName":"Jagtap¹","suffix":""}],"badges":[],"createdAt":"2026-04-02 10:40:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9302048/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9302048/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107624476,"identity":"c323dd52-c2bd-41c8-bfb1-10eeb0e92fba","added_by":"auto","created_at":"2026-04-23 10:17:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":271511,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eParticle size analysis of the optimized Batch-F1\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9302048/v1/b633d27910526b26835f755b.png"},{"id":107624478,"identity":"5a650314-1c42-4c62-9f34-2a4e4c7cbc2a","added_by":"auto","created_at":"2026-04-23 10:17:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":195026,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical representation of the zeta potential of optimized batch (F1)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9302048/v1/f2ff95f76be71d7846da45f4.png"},{"id":107707575,"identity":"1f535955-dbfa-4fbe-a1f0-e16877a8000f","added_by":"auto","created_at":"2026-04-24 09:20:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":204065,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEM of AZM-loaded EC polymeric microsponge F1 at 1000X \u0026amp; 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ISG-2 show no turbidity)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-9302048/v1/206bcc765a1aa3b4f2805338.png"},{"id":107707170,"identity":"876fd35f-2021-4619-8405-4dd3641f4b00","added_by":"auto","created_at":"2026-04-24 09:19:41","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":919644,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroscopic observation under 45x of blood cell interaction with Hypertonic, Hypotonic and Isotonic solutions\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-9302048/v1/3b3c72f915bfff1b0d9ff754.png"},{"id":107624489,"identity":"88c68e1b-49bf-4b92-946d-92a86a21381c","added_by":"auto","created_at":"2026-04-23 10:17:20","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":585942,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison with standard Isotonic solutions with optimized formulation ISG-1\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"15.png","url":"https://assets-eu.researchsquare.com/files/rs-9302048/v1/1c4468a7c5644bd4e68373bd.png"},{"id":107624492,"identity":"0f48becc-22a9-460a-959d-30c4c61b565a","added_by":"auto","created_at":"2026-04-23 10:17:20","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":655774,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOcular irritation study by Hen's egg test-chorioallantoic membrane (HET-CAM). (A) HET-CAM study using 0.1 M NaOH (positive control). (B) HET-CAM study using optimized formulation IGF-1. (C) HET-CAM study using 0.9% sodium chloride (negative control).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"16.png","url":"https://assets-eu.researchsquare.com/files/rs-9302048/v1/bc190b1b397083ba2899a6d8.png"},{"id":107707227,"identity":"33b1d376-0a84-4033-9bd9-e9f9f706418e","added_by":"auto","created_at":"2026-04-24 09:19:50","extension":"png","order_by":16,"title":"Figure 16","display":"","copyAsset":false,"role":"figure","size":684281,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological study of goat cornea (A) Normal saline (Negative control) B. Acetazolamide-loaded microsponge in situ gel formulation, C. sodium dodecyl sulphate (SDS) in phosphate buffer saline (PBS) (positive control)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"17.png","url":"https://assets-eu.researchsquare.com/files/rs-9302048/v1/8836156cf6b12ad4e7916c9a.png"},{"id":107709364,"identity":"78170e83-3851-486f-98eb-2557e48b4843","added_by":"auto","created_at":"2026-04-24 09:35:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6636008,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9302048/v1/c5a7e2e8-1c70-4f17-bbff-a45a242365d9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Engineered Acetazolamide Microsponges in pH‑Responsive In-Situ Gel: A Novel Platform for Long‑Acting Glaucoma Therapy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMicrosponge technology uses tiny, porous, cross‑linked polymeric microspheres (typically 5\u0026ndash;300 \u0026micro;m; pores 5\u0026ndash;150 \u0026micro;m) that can entrap up to 50\u0026ndash;60% of active drug and release it in a controlled, site‑specific manner, minimizing irritation and systemic side effects. These non-toxic, non-allergenic carriers can be added to traditional dosage forms like creams, gels, ointments, powders, tablets, and liquids[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. They are particularly useful in topical formulations and are being investigated more and more for oral delivery because they are stable over a broad pH range (1\u0026ndash;11) and temperatures up to 130\u0026deg;C. Additionally, they can lessen the adverse effects of the active chemicals in topical therapies and improve their stability[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMicrosponge-based drug delivery systems can be manufactured via a variety of techniques, including water-in-oil-in-water (w/o/w) emulsification, liquid-liquid suspension polymerisation, lyophilization, oil-in-oil emulsion solvent diffusion, vibrating-orifice aerosol generation, electrohydrodynamic atomisation, ultrasound-assisted techniques, and porogen-induced porosity. Among these, the quasi‑emulsion solvent diffusion (ESD) method is the most widely employed approach for the preparation of microsponges due to its relative simplicity, reproducibility, and suitability for scale‑up[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAcetazolamide (AZM) therapy before 2020 was plagued by systemic toxicity from high oral doses, poor corneal permeability (4.1 \u0026times; 10⁻⁶ cm/s), and the limitations of early topical attempts like cyclodextrin complexes, drug-soaked contact lenses, high-concentration suspensions (5\u0026ndash;10%) with penetration enhancers, liposomes, and niosomes[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These were all hindered by irritation, toxicity, poor compliance, and no commercial viability, with precorneal loss limiting bioavailability to less than 5%. These issues are accurately captured in the text provided. Through creative platforms, recent developments have removed these obstacles: Using Carbopol/HPMC matrices, Abdel-Mageed et al.'s microsponge in situ gels produced 3.2-fold transcorneal flux, 7 \u0026micro;m particles, 75\u0026ndash;88% release over 8 hours, and 85.97% entrapment efficiency with no irritation and superior residence time compared to drops[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. By using biocompatible lipid formulations that removed surfactant toxicity, cubosome and optimised nanoemulsion systems achieved 38% IOP reduction (compared to 22% for Azopt\u0026reg;), 4\u0026times; permeability augmentation, and 9-hour duration[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The oral efficacy without enhancers was matched by PLGA-AZM intravitreal implants (2025), which offered a biphasic 50% release over 42 days with a 25% reduction in intraocular pressure in rabbits and no retinal toxicity (ARPE-19 IC50\u0026thinsp;\u0026gt;\u0026thinsp;0.02 mmol/L)[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Additionally, pH-triggered gellan gum/HPMC gels and dendritic nanoarchitectures extended residence to 4\u0026ndash;6 hours. Low drug concentrations (1\u0026ndash;2% AZM with 85\u0026ndash;92% EE), longer mucoadhesion (MRT 4.2 h vs. 2.4 h drops), natural polymer/lipid biocompatibility, and 2\u0026ndash;3\u0026times; greater AUC₀₋₁₀ compared to commercialized drops are some of the main advantages of these systems, which position AZM for a clinical renaissance in glaucoma therapy[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study aimed to reduce the systemic adverse effects of acetazolamide and improve patient compliance by fabricating novel acetazolamide-loaded microsponges and incorporating them into in situ gel for ocular drug administration.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterial\u003c/h2\u003e \u003cp\u003eAcetazolamide (AZM) (99.9% purity) was obtained from Yarrow Chem Products, Ghatkopar (W), Mumbai. Ethyl cellulose (EC-14cp) polymer was obtained from LOBA Chemie Pvt Ltd, Mumbai. Polyvinyl alcohol (PVA), Triethyl citrate (TEC), Dichloromethane (DCM), sodium chloride (NaCl) and calcium chloride dihydrate (CaCl₂.2H₂O), Sodium hydroxide scales (NaOH), Sodium bicarbonate (NaHCO₃), Carbopol-940, and HPMC E50LV were procured from LOBA Chemie Pvt Ltd, Mumbai.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMethod\u003c/h3\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of acetazolamide-loaded microsponges\u003c/h2\u003e \u003cp\u003eThe method of quasi-emulsion solvent diffusion is used to create microsponges. EC polymer and 10 mL of DCM were first dissolved to create the organic (internal) phase. TEC was used as the plasticiser (1% w/v). Following the addition of the necessary quantity of the drug to the polymeric solution, the probe ultrasonicator (Labman Scientific Pvt. Ltd., LMUC-3) was used to ultrasonically disperse and reduce the drug's particle size for 20 minutes in an ice bath[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The polymeric solution was then gradually added to the aqueous solution that had been made by dissolving PVA (0.5% w/v) in 100 ml of distilled water at 70\u0026deg;C while stirring until it was fully dissolved. The entire mixture was then agitated for two hours at 3000 rpm using an overhead stirrer until the organic solvent had completely evaporated and the microsponges had formed. To allow the microsponges to fully precipitate, the mixture was refrigerated for 24 hours. After that, the microsponges were filtered, cleaned with a small amount of diluted sodium hydroxide to get rid of any remaining drug, rinsed multiple times with double-distilled water, and dried in an oven at 40\u0026deg;C for 48 hours before being stored for future research[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eOptimisation technique:\u003c/h3\u003e\n\u003cp\u003eVarious parameters for microsponge optimization batches were checked, including production yield, actual drug content, and entrapment efficiency.\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\u003eFormulation of Acetazolamide-Loaded Microsponges\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eComponents\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"9\" nameend=\"c10\" namest=\"c2\"\u003e \u003cp\u003eDrug: Polymer Ratio\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eF5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eF6\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eF7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eF8\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eF9\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\u003eAcetazolamide\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(mg)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEthyl Cellulose (mg)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e175\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e225\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e275\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePolyvinyl Alcohol (%w/v)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTriethyl citrate (%W/V)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWater (ml)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eCharacterization of the microsponge formulation\u003c/h3\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of Production Yield\u003c/h2\u003e \u003cp\u003eFollowing their formation, the microsponges were cleaned, dried, and precisely weighed. By comparing the total weight of the produced microsponges with the combined weight of the polymer and drug components, the yield of the microsponges was ascertained[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDetermination of Drug Content\u003c/h3\u003e\n\u003cp\u003eA clean glass mortar and pestle was used to precisely weigh and finely crush a quantity of acetazolamide-loaded microsponge formulation equal to 20 mg of acetazolamide from each batch. To guarantee that the medication was completely dissolved, 60 mL of methanol was added to the powdered sample, and the combination was agitated constantly for four hours. Particulate matter was subsequently eliminated from the resultant dispersion by filtering it via Whatman filter paper. The same methanol was then used to dilute the filtrate to 40 mL. The maximum absorbance for acetazolamide, 264 nm, was used to measure the drug content in the prepared solution using a Shimadzu UV-1900 UV-Visible spectrophotometer. Using a calibration curve that had already been created, the absorbance measurements were used to determine the amount of medication contained in the microsponge formulation[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eParticle size\u003c/h3\u003e\n\u003cp\u003eUsing the Malvern Zeta Sizer digital microscope, optical microscopy was utilised to assess the microsponge's particle size. A few drops of a particular microsponge formulation dissolved in water were put on a glass slide. Under a digital microscope, the dispersion drop was visible. A calculation was made to determine the average particle size of 300 particles[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eZeta potential\u003c/h2\u003e \u003cp\u003eParticle stability and surface charge are indicated by zeta potential. In the event that the zeta potential is greater than \u0026plusmn;\u0026thinsp;30 or less than \u0026minus;\u0026thinsp;30 mV, particles will not stick together[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro drug release and kinetics study of microsponges\u003c/h2\u003e \u003cp\u003eAn in vitro drug release study employed the dialysis bag method in 50 mL simulated tear fluid (STF; pH 7.4, 35\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) at 50 rpm. STF comprised 0.67% sodium chloride, 0.2% sodium bicarbonate, and 0.008% calcium chloride dihydrate. A semipermeable cellophane membrane was stretched over the open end of a dialysis tube, forming the test assembly, which was agitated at 50 rpm while maintaining 35\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. Microsponge weight was determined individually; subsequently, 0.5 g of each formulated microsponge gel (equivalent to 5 mg of drug) was loaded onto the membrane within the dialysis tube[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The tube was suspended such that the membrane remained just below the STF surface. At predetermined intervals (0.08, 0.25, 0.5, 1, 2, 3, 4, 5, and 6 h), 2 mL aliquots were withdrawn from the release medium in the beaker and quantified spectrophotometrically at 265 nm against an identically prepared STF blank[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Sink conditions were maintained by replenishing the withdrawn volume with fresh STF preheated to 35\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Experiments were performed independently in triplicate. In vitro release profiles underwent kinetic modeling to elucidate the drug release mechanism. Data were fitted to the Higuchi model (m₀ \u0026minus; m\u0026thinsp;=\u0026thinsp;Kt\u0026sup1;/\u0026sup2;), zero-order (m₀ \u0026minus; m\u0026thinsp;=\u0026thinsp;Kt), and first-order (log ⁡ m\u0026thinsp;=\u0026thinsp;log ⁡ m₀ \u0026minus; K t 2.303 log m\u0026thinsp;=\u0026thinsp;log m 0\u0026thinsp;\u0026minus;\u0026thinsp;2.303 Kt), where m denotes the drug remaining in the formulation at time t, and m₀ the initial drug load. Regression coefficients (r\u0026sup2; r\u0026sup2;) were computed for each model. The diffusion exponent n from the Korsmeyer\u0026ndash;Peppas equation (m₀ \u0026minus; m / m₀ = K t n m 0 m 0\u0026thinsp;\u0026minus;\u0026thinsp;m\u0026thinsp;=\u0026thinsp;Kt n) further characterized the release mechanism: Fickian diffusion for n\u0026thinsp;\u0026lt;\u0026thinsp;0.45 n\u0026thinsp;\u0026lt;\u0026thinsp;0.45; non-Fickian (anomalous) transport for 0.5\u0026thinsp;\u0026lt;\u0026thinsp;n\u0026thinsp;\u0026lt;\u0026thinsp;0.8 0.5\u0026thinsp;\u0026lt;\u0026thinsp;n\u0026thinsp;\u0026lt;\u0026thinsp;0.8[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFourier Transform Infrared Spectroscopy (FTIR)\u003c/h2\u003e \u003cp\u003eFTIR spectra of the physical mixture of AZM and polymers were carried out (SHIMADZU)[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDifferential Scanning Calorimetry (DSC)\u003c/h2\u003e \u003cp\u003eThe physical mixture of polymers and AZM was subjected to thermal examination. A TA device, the DSC Discovery 250, was used to scan specific microscopy compositions at a rate of 20\u0026deg;C per minute between 40\u0026deg;C and 400\u0026deg;C in a dynamic nitrogen atmosphere. The compatibility between the AZM pure medicine and polymers was assessed using produced microsponges and DSC experiments for the physical mixing of the drug and polymers[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eScanning Electron Microscopy\u003c/h2\u003e \u003cp\u003eUsing a scanning electron microscope (Nova NanoSEM), the optimised microsponges formulation was morphologically analysed. Digital micrography was used to take the picture, and imaging viewer software was used to examine it[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eFabrication of ocular in situ gel containing Acetazolamide-loaded microsponges\u003c/h2\u003e \u003cp\u003eIn order to prepare the aqueous phase, potassium dihydrogen phosphate (0.4% w/v) was dissolved in 75 mL of distilled water. HPMC E50 LV (0.2% w/v) was then gradually dispersed to ensure full hydration. To create a uniform polymeric dispersion, Carbopol 940 (0.5\u0026ndash;0.7% w/v) was then added and left to hydrate overnight while being stirred magnetically. When AZM-loaded microsponges (equal to 0.2% w/v AZM) were first dissolved in phosphate buffer, the pH dropped and Carbopol precipitated right away[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This was overcome by dissolving the microsponges in 0.1 M NaOH before adding them to the polymer solution, which produced a translucent formulation that was maintained using 0.01% w/v propyl paraben[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In order to comprehensively assess gelling behavior and rheological performance, three in situ gel formulations (ISG-1, ISG-2, and ISG-3) were created. They differed only in the concentration of Carbopol 940 (0.5%, 0.6%, and 0.7% w/v, respectively), while keeping all other excipient levels equal[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\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\u003eFormulation of ocular in situ gel containing acetazolamide-loaded microsponges\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eName of excipients\u003c/p\u003e \u003cp\u003e(% W/V)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eComposition of Acetazolamide microsponge loaded in situ gel\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eISG\u0026thinsp;\u0026minus;\u0026thinsp;1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eISG\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eISG\u0026thinsp;\u0026minus;\u0026thinsp;3\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAZM loaded microsponge equivalent to AZM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCarbopol \u0026ndash; 940\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHPMC E50LV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePotassium dihydrogen phosphate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePropyl Paraben\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWater\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100\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 \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of ocular in situ gel containing Acetazolamide-loaded microsponges\u003c/h2\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003eDetermination of visual appearance and clarity:\u003c/h2\u003e \u003cp\u003eThe in-situ formulation's visual appearance and clarity are examined for any particulate matter using fluorescent lights set against a black and white backdrop[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of pH:\u003c/h2\u003e \u003cp\u003eA pH meter (Systronic-802) that had been calibrated for use with the buffered solution at pH 4 and pH 7 was used to measure the pH of the in-situ gel formulation. For every sample, three measurements were made, and the average of the three measurements was determined. The pH of the produced ophthalmic formulations was assessed with a digital pH meter. The pH range for ophthalmic preparations should be 6.0\u0026ndash;7.4[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eDrug content determination:\u003c/h2\u003e \u003cp\u003eThe gel formulation was taken in situ and placed in a 100 ml volumetric flask. 60 cc of pH 7.4 phosphate buffer was then added, stirred for four hours, and filtered. Using 7.4 phosphate buffer, the filtered solution was once more diluted to 100 mL. measured using a Shimadzu UV-1900 UV spectrophotometer and a blank of phosphate buffer (pH 7.4). It was determined how much drug was in the microsponges[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of gelling capacity:\u003c/h2\u003e \u003cp\u003eThe gelling capacity was assessed by putting a drop of the in-situ gel in a test tube with 2 mL of freshly made simulated tear fluid (pH 7.4) that had been equilibrated at 35\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. The gel's gelling formation and dissolution times were visually observed, and the gelling capacity was calculated as follows:\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"1\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(-) No gelation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(+) The gel formed after a few minutes and dissolved rapidly\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(++) Immediately gel formation and remains for a few hours\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(+++) Immediate stiff gelation, which remains for a prolonged time.\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 \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eRheological studies:\u003c/h2\u003e \u003cp\u003eThe Brookfield viscometer is mainly employed to determine the viscosity of in situ eye gels. By increasing angular velocity gradually from 0.5 to 100 rpm, viscosity is both pre-gel and post-gelation[\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eIn vitro drug release and kinetic study of in situ gel containing AZM-loaded microsponges:\u003c/h2\u003e \u003cp\u003eAn in vitro release study based on dialysis was conducted at 50 rpm in 50 mL of simulated tear fluid (STF; pH 7.4, 35\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) that included 0.008% calcium chloride dihydrate, 0.2% sodium bicarbonate, and 0.67% sodium chloride[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The assembly was created by stretching a semipermeable standard egg membrane over the open end of a dialysis tube and agitating it at 50 rpm while keeping the temperature at 35\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. The dialysis tube's membrane was covered with either precisely weighted microsponges or prepared microsponge gels, each loaded with the necessary amount of medication. The membrane was suspended such that it stayed just below the surface of the buffered dialysis medium[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. At predefined intervals (0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, and 8 h), 2 mL aliquots were withdrawn from the release medium and analyzed spectrophotometrically at 265 nm against an identically processed STF blank. Sink conditions were ensured by replenishing withdrawn volumes with equivalent amounts of preheated STF (35\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C)[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The experiment was executed independently in triplicate. As detailed in the relevant section, kinetic analysis of the ocular in situ gel drug release was subsequently conducted[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eSterility testing:\u003c/h2\u003e \u003cp\u003eThe sterility test is performed on sterile drugs that meet the \"Indian Pharmacopoeia\" sterility benchmark. The contents of the vial were aseptically evaluated after the foil and outside were sterilised with alcohol, an antibacterial agent. For inoculation, 20 ml of sterile medium were directly applied aseptically to the in situ ocular gel formulations[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. 20 ml of fluid thioglycolate medium and 20 ml of soy casein digest medium were used as inoculation media, and they were incubated for 14 days at 30\u0026deg; to 35\u0026deg;C[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Using soybean-casein digest medium and aerobic incubation, microbial and fungal pollutants were investigated. Under aseptic conditions, the tests were carried out in triplicate to avoid accidental contamination. Using the positive and negative controls, respectively, growth promotion and sterility were investigated.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eEx vivo transcorneal permeation:\u003c/h2\u003e \u003cp\u003eAn optimised acetazolamide microsponge-based in situ gel formulation's ex vivo transcorneal permeability investigation uses freshly excised goat corneas, which are normally acquired from a slaughterhouse right after animal sacrifice to guarantee tissue viability[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. A Franz diffusion cell's donor and receptor chambers are separated by carefully dissecting the cornea, which is then mounted with the epithelial side toward the donor compartment[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Using phosphate-buffered saline (pH 7.4) or artificial tear fluid, the receptor compartment is kept at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C and constantly agitated to replicate physiological circumstances. An effective diffusion area (typically 2.0 cm\u0026sup2;) is covered by a measured volume (typically 2 mL) of the in-situ gel formulation applied to the ocular surface in the donor compartment[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In order to maintain sink conditions, samples are taken out of the receptor chamber at prearranged intervals and replaced with equal volumes of fresh buffer during the course of the permeation experiment, which is typically carried out for up to 8 hours[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. At each time point, the amount of acetazolamide that has penetrated the cornea is measured using an analytical technique that has been proven to work, like UV-visible spectrophotometry. Using a commercially available acetazolamide ophthalmic solution or traditional gel, the same process is carried out for comparison. The effectiveness of the microsponge-based in situ gel system is evaluated by plotting the cumulative drug permeation against time and calculating metrics like the permeability coefficient and steady-state flow[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Similar methods in the literature for various ophthalmic medications support the idea that this ex vivo model offers a reliable and repeatable way to assess and compare the transcorneal drug delivery effectiveness of novel ocular formulations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eIsotonicity test:\u003c/h2\u003e \u003cp\u003eThe ophthalmic formulation's safety, non-irritability, and non-toxicity with ocular secretions were confirmed by an isotonicity test. A few drops of blood were added, mixed with a few drops of the optimised formulation, and examined under a compound microscope with a 45X objective lens. RBCs were used in the method, and by comparing the optimised formulation of RBC with commercial eye drops, the RBCs were checked for any cell bulging or shrinkage[\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eHET-CAM Ocular Irritation Assessment\u003c/h2\u003e \u003cp\u003eFertilized hen eggs (\u0026lt;\u0026thinsp;7 days after laying) were incubated horizontally for 7 days at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C and 60\u0026ndash;70% humidity. On day 8, viable embryos with well-developed vascular networks were chosen by candling under intense light[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Post-candling, the air sac borders were carefully marked, and then sterile instruments were used to remove the aseptic shell at the blunt end. In order to reveal the intact chorioallantoic membrane (CAM) without vascular damage, the inner shell membrane was moistened with a 0.9% NaCl solution, carefully aspirated, and gently peeled under magnification[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Using a precision micropipette, the CAM was given 0.3 mL of the optimised formulation, which was matched by administrations of the positive control (1 N NaOH, a severe irritant) and negative control (0.9% w/v NaCl, a non-irritant). Post-application vascular endpoints hemorrhage (bleeding), lysis (vessel dissolution), and coagulation (protein precipitation) were timed within the 5-minute observation period under stereomicroscopy and scored per validated HET-CAM protocols[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIrritation score (IS) was calculated as IS = [((301 - hemorrhage time in s)/300) \u0026times; 5] + [((301 - lysis time in s)/300) \u0026times; 7] + [((301 - coagulation time in s)/300) \u0026times; 9], categorizing irritancy potential[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eHistological studies:\u003c/h2\u003e \u003cp\u003eCorneal biocompatibility of the optimized acetazolamide-loaded microsponge in situ gel was assessed using ex vivo goat cornea models following established ophthalmic safety protocols. Excised corneas were exposed to the formulation for 1 and 6 hours alongside negative (normal saline) and positive (0.1% SDS) controls, then fixed in 10% formalin, processed through graded alcohol dehydration, paraffin-embedded, sectioned (5 \u0026micro;m), and stained with hematoxylin-eosin[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Microscopic evaluation revealed no epithelial disruption, stromal edema, necrosis, or structural abnormalities in formulation-treated corneas, maintaining intact epithelial layering and stromal organization comparable to negative controls[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Positive controls exhibited marked epithelial desquamation and stromal separation, confirming assay sensitivity. These findings, consistent with physiological corneal hydration levels (76\u0026ndash;79%), demonstrate the formulation's excellent biocompatibility and absence of ocular irritancy, supporting its safety profile for glaucoma therapy and aligning with similar reports for mucoadhesive ophthalmic in situ gels[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eStability studies:\u003c/h2\u003e \u003cp\u003eStability studies were performed to assess the physicochemical and microbiological stability of acetazolamide (AZM)-loaded microsponges and in situ ocular gel containing AZM-loaded microsponges, in compliance with the International Conference on Harmonisation (ICH) recommendations Q1A(R2). In order to imitate accelerated storage conditions, the formulations were packaged in the proper containers (glass vials for gels and aluminum foil for inserts) and kept in a stability chamber that was kept at 40\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 75\u0026thinsp;\u0026plusmn;\u0026thinsp;5% relative humidity. The samples were extracted and evaluated for important criteria, such as physical appearance, drug content, pH (for gels), gelling capability, in vitro drug release, and sterility, at predetermined intervals of 15 days, 1 month, 2 months, and 3 months[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. All formulations showed no discernible changes in appearance, medication potency, or performance characteristics over the course of the three months[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. The in-situ gels maintained their consistency in gelling behavior, acceptable pH range (near physiological), and clarity. Sterility was maintained, and drug release patterns demonstrated sustained release with no discernible deviation. These results demonstrate the physical and chemical stability of the AZM-loaded microsponges and their ocular delivery systems under accelerated settings, guaranteeing their effectiveness and safety over the course of their shelf life[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Result and Discussion","content":"\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003ePreparation and characterization of microsponge formulation\u003c/h2\u003e \u003cp\u003eAcetazolamide-loaded microsponges were fabricated using ethyl cellulose via quasi-emulsion solvent diffusion, wherein drug and polymer were dissolved in dichloromethane (internal phase) and emulsified into polyvinyl alcohol aqueous solution (external phase) under stirring[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Rapid DCM diffusion induced polymer precipitation at droplet interfaces, forming porous matrices that crystallized acetazolamide within the core while counter-diffusion of water enhanced internal porosity. This simple, reproducible technique yielded discrete microsponges with high entrapment efficiency, minimized solvent toxicity, sustained release profiles, and suitability for ocular delivery by reducing burst effect and prolonging therapeutic residence[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of Production Yield\u003c/h2\u003e \u003cp\u003eAcetazolamide-loaded microsponges had a production yield ranging from 36.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.66% to 91.11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69%. Formulations F1 through F4 showed comparatively good manufacturing yields despite having lower amounts of the polymer (ethyl cellulose). On the other hand, formulations F6 through F9 showed a discernible decrease in yield as the polymer ratio rose. This pattern suggests that the manufacturing yield decreases with increasing polymer concentration, perhaps as a result of increased viscosity impeding the creation and recovery of microsponges. These findings imply that a key factor influencing the effectiveness of microsponge formation is polymer concentration[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003eDetermination of Drug Content\u003c/h2\u003e \u003cp\u003eThe drug concentration of the microsponges loaded with acetazolamide varied among the different formulations, ranging from 78.31\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5% to 7.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63%. Drug content and polymer ratio were shown to be inversely correlated; as polymer concentration rose, drug content steadily fell. This could be because a larger percentage of polymer forms a thicker matrix, diluting the amount of drug per unit weight and possibly decreasing the effectiveness of drug incorporation during microsponge generation[\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003eDetermination of Entrapment efficiency (%)\u003c/h2\u003e \u003cp\u003eThe microsponges loaded with acetazolamide showed a notable decrease in entrapment efficiency (EE%) from batch F1's 85.97% \u0026plusmn; 1.2 to batch F9's 20.83% \u0026plusmn; 0.3. Because of the increased viscosity of the polymer solution, which creates a denser and more rigid polymeric network, the EE% decreases as the polymer concentration rises. This structure limits the drug's diffusion into the microsponge matrix during the manufacturing phase by acting as a physical barrier. According to Morsi et al. (2016), who obtained similar results, higher polymer concentrations hinder drug encapsulation within the polymeric framework, hence reducing entrapment efficiency[\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e].\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\u003eEvaluation data of Acetazolamide-loaded microsponge (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/div\u003e \u003c/caption\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=\"char\" char=\"\u0026plusmn;\" 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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBatch\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDrug: Polymer ratio\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eProduction Yield (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eActual drug content (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEntrapment Efficiency (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e91.11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e78.31\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e85.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2:1.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e88.50\u0026thinsp;\u0026plusmn;\u0026thinsp;2.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e68.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e77.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2:1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e85.09\u0026thinsp;\u0026plusmn;\u0026thinsp;2.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e59.32\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e69.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2:1.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e77.62\u0026thinsp;\u0026plusmn;\u0026thinsp;2.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e47.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e61.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2:2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e68.03\u0026thinsp;\u0026plusmn;\u0026thinsp;2.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e36.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e53.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.2.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e56.19\u0026thinsp;\u0026plusmn;\u0026thinsp;2.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e25.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e45.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2:2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e50.86\u0026thinsp;\u0026plusmn;\u0026thinsp;2.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e18.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e37.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2:2.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e43.95\u0026thinsp;\u0026plusmn;\u0026thinsp;2.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e12.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e28.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2:3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e36.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e7.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e20.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\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 \u003c/div\u003e\n\u003ch3\u003eParticle size\u003c/h3\u003e\n\u003cp\u003eFor the F1 formulation, the average particle size of the acetazolamide-loaded microsponges was 7.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 \u0026micro;m, and it gradually rose as the polymer content increased. Larger emulsion globules are more likely to form during the quasi-emulsion solvent diffusion process due to the increased viscosity of the dispersed phase brought on by higher polymer concentrations. The overall size of the microsponge increases as a result of these larger globules being less likely to fragment into smaller particles[\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eZeta Potential and Colloidal Stability Assessment:\u003c/h3\u003e\n\u003cp\u003eZeta potential, a primary determinant of colloidal stability, quantifies electrostatic repulsion at the shear plane; values exceeding\u0026thinsp;\u0026plusmn;\u0026thinsp;30 mV confers optimal dispersion stability through interparticle charge repulsion. The optimized F1 microsponge batch demonstrated a zeta potential of -20.7 mV within the moderate stability range\u0026mdash;coupled with the smallest particle size amenable to ophthalmic administration. This synergistic profile facilitated superior mucoadhesion to corneal/conjunctival epithelia and extended precorneal residence via tear mucin interactions. Suboptimal batches exhibiting inferior zeta potentials and larger particle dimensions were excluded due to compromised colloidal stability and diminished ocular retention potential[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec37\" class=\"Section2\"\u003e \u003ch2\u003eScanning Electron Microscopy (SEM) Analysis\u003c/h2\u003e \u003cp\u003eSEM micrographs (1000\u0026times;, 2000\u0026times;, 4000\u0026times;) of optimized Batch F1 acetazolamide-loaded microsponges revealed discrete, spherical particles with smooth surfaces and characteristic porosity indicative of successful quasi-emulsion solvent diffusion fabrication. Higher magnifications disclosed well-developed micro-voids and channels essential for high drug loading and sustained release, with no surface drug crystals confirming internal encapsulation within the ethyl cellulose matrix. The uniform size distribution, absence of aggregation, and mechanical integrity validate F1's suitability for incorporation into ocular in situ gels, consistent with established microsponge morphology literature[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec38\" class=\"Section3\"\u003e \u003ch2\u003eFTIR analysis\u003c/h2\u003e \u003cp\u003eThe structural integrity of acetazolamide (AZM) was validated by FTIR analysis, which showed distinctive peaks at 3271.71 cm⁻\u0026sup1; (N\u0026ndash;H sulfonamide), 1674.21 cm⁻\u0026sup1; (C\u0026thinsp;=\u0026thinsp;O amide), 1541.12 cm⁻\u0026sup1; (N\u0026ndash;H bending), 1427/1361 cm⁻\u0026sup1; (C\u0026ndash;N/SO₂), and 1244\u0026ndash;1083 cm⁻\u0026sup1; (S\u0026thinsp;=\u0026thinsp;O/C\u0026ndash;N). With slight changes due to polymer interactions, the physical mixture of AZM and ethyl cellulose showed similar peaks at 3440 cm⁻\u0026sup1; (N\u0026ndash;H/O\u0026ndash;H overlap), 1650 cm⁻\u0026sup1; (C\u0026thinsp;=\u0026thinsp;O), 1545 cm⁻\u0026sup1; (amide II), 1363 cm⁻\u0026sup1; (S\u0026thinsp;=\u0026thinsp;O), and 1160\u0026ndash;1190 cm⁻\u0026sup1; (S\u0026thinsp;=\u0026thinsp;O/C\u0026ndash;O). AZM and ethyl cellulose do not chemically interact, as confirmed by the lack of new peaks or a notable peak disappearance, confirming drug-polymer compatibility for microsponge formulation[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec39\" class=\"Section2\"\u003e \u003ch2\u003eDifferential Scanning Calorimetry (DSC) analysis of AZM:\u003c/h2\u003e \u003cp\u003eThe DSC analysis was performed to confirm the thermogram for pure acetazolamide (AZM); it revealed a prominent endothermic peak at 274.97\u0026deg;C, with an onset temperature of 273.66\u0026deg;C. The enthalpy of fusion was measured at 192.99 J/g. The recommended standard range of 274\u0026ndash;276\u0026deg;C, as stated in pharmacopeial references such as the United States Pharmacopoeia (USP) and the Merck Index, 15th Edition, is extremely close to these values. The narrow and univocal character of the endothermic peak reflects the crystalline state of the drug and lack of polymorphic transition or degradation[\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. The large value of enthalpy also confirms the presence of a stable crystalline form of AZM. No other thermal phenomenon was observed, indicating that the drug remains stable up to its melting point and is amenable to formulation development. These results verify that the sample meets the pharmaceutical specification for pure acetazolamide, validating its suitability for subsequent formulation development[\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec40\" class=\"Section3\"\u003e \u003ch2\u003eIn vitro drug release from microsponges and in vitro drug release kinetics study of microsponges:\u003c/h2\u003e \u003cp\u003eThe in vitro drug release profile of selected microsponge formulations loaded with acetazolamide (F1, F2, F3, and F4) was assessed because of their moderate to high entrapment efficiency and favorable particle size. These formulations released between 10.23% and 17.15% of the drug within the first hour, according to the cumulative drug release statistics shown in the table and visually depicted in the figure. As seen in formulation F4, the results unequivocally show that the ethyl cellulose (EC) polymer efficiently delays drug release, which becomes increasingly noticeable as the polymer concentration rises[\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. Acetazolamide's absorption into the microsponges' porous interior structure, which functions as a micro-reservoir and promotes a continuous release pattern, is responsible for this slower release rate. Higher EC concentrations also produced microsponges with larger particle sizes and thicker polymer walls, which effectively decreased the surface area accessible for drug diffusion and aided in the extended release[\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. The floating properties of microsponges and the hydrophobic nature of ethyl cellulose may also play a role in the delayed drug diffusion, leading to a slow and prolonged drug release over time. These results highlight the possibility of using microsponge systems to deliver acetazolamide to the eyes in a regulated manner[\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e% CDR of selected AZM-loaded microsponge (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/div\u003e \u003c/caption\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\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eAcetazolamide loaded microsponge formulation (% CDR)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF4\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\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0.5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.15\u0026thinsp;\u0026plusmn;\u0026thinsp;1.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29.08\u0026thinsp;\u0026plusmn;\u0026thinsp;1.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33.35\u0026thinsp;\u0026plusmn;\u0026thinsp;2.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e44.25\u0026thinsp;\u0026plusmn;\u0026thinsp;2.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25.53\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e58.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51.11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e51.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e55.66\u0026thinsp;\u0026plusmn;\u0026thinsp;3.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e39.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e59.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60.16\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e43.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e73.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e69.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e65.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\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\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation and characterization of ocular in situ gel containing AZM-loaded microsponges\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOptimized F1 acetazolamide-loaded ethyl cellulose microsponges were incorporated into a pH/thermo-responsive in situ gel comprising Carbopol 940 (0.5\u0026ndash;0.7% w/v) and HPMC E50LV (0.2% w/v). Carbopol 940, a high molecular weight crosslinked polyacrylic acid, undergoes rapid sol-gel transition at physiological tear pH (~\u0026thinsp;7.4) via carboxylic group ionization, forming mucoadhesive networks that prolong precorneal residence. HPMC E50LV, a low-viscosity non-ionic cellulose ether, synergistically enhances viscosity, elasticity, Spreadability, and thermosensitive gelation at ocular temperature (~\u0026thinsp;35\u0026deg;C) through hydrophobic methoxy interactions, enabling lower Carbopol concentrations to minimize irritation while strengthening the gel matrix[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. This dual-polymer system provides sustained drug release via multi-layered diffusion control, superior ocular comfort, and enhanced therapeutic performance compared to single-polymer formulations[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eDetermination of pH:\u003c/h3\u003e\n\u003cp\u003eOcular in situ gel compositions' pH is crucial for maintaining patient comfort and therapeutic effectiveness. The pH of the formulation should ideally be around 7.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2, which is the same as that of natural tear fluid, to reduce irritation and prevent excessive tear formation, which can lead to premature medication clearance and decreased effectiveness[\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. A number around physiological pH is ideal, even though the eye can withstand pH levels in the wider range of 4.5 to 11.5. The pH values for the acetazolamide-loaded microsponge in situ gel formulations ranged from 6.81 to 7.02, indicating good ocular compatibility and a little chance of irritation when administered. Crucially, even after autoclaving, the pH values did not change, indicating the formulation's stability in sterile settings.\u003c/p\u003e\n\u003ch3\u003eDetermination of Drug Content:\u003c/h3\u003e\n\u003cp\u003eAcetazolamide was distributed uniformly and consistently across all gel formulations, according to drug content analysis, with content ranging from 81.67% to 89.27%. This demonstrated the preparation method's dependability and reproducibility[\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eDetermination of Gelling Capacity:\u003c/h3\u003e\n\u003cp\u003eGelling behavior at physiological pH: formulations including Carbopol 940 and HPMC E50 LV as gelling agents demonstrated a rapid sol-to-gel conversion, producing strong, stable gels suitable for use in the eyes. In the relatively alkaline environment of the eye, Carbopol 940, which is renowned for its pH-sensitive properties, produces a viscous gel, while HPMC E50 LV aids by boosting the gel's viscosity and mechanical strength[\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. Higher concentrations of these polymers led to lower gelation temperatures and higher gel strengths, which reinforced a sustained drug release profile and enhanced gel integrity over time. A concentration of 0.1% W/V to 0.4% W/V of Carbopol shows less gelation than a concentration of 0.5% W/V to 0.8% W/V, which shows good gelling capacity for the stiff gel. However, a concentration of 0.8% to 1% W/V of Carbopol causes stiff gel formation and ocular irritation. Overall, formulations optimized with appropriate ratios of HPMC E50 LV and Carbopol 940 showed good physicochemical characteristics, such as optimal pH, rapid gelation, and stable gel structure, which made them extremely promising for acetazolamide ocular administration[\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEvaluation of ocular in situ gel containing AZM-loaded microsponge (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/div\u003e \u003c/caption\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=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eFormulation code\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eISG-1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eISG-2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eISG-3\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eDrug content\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e89.27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.64\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e81.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.87\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGelling capacity\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e+++\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e+++\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e+++\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eViscosity\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eAt non-physiological conditions\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e168\u0026thinsp;\u0026plusmn;\u0026thinsp;102\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e324\u0026thinsp;\u0026plusmn;\u0026thinsp;102\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e636\u0026thinsp;\u0026plusmn;\u0026thinsp;110\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eAt physiological conditions\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e2256\u0026thinsp;\u0026plusmn;\u0026thinsp;108\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e2586\u0026thinsp;\u0026plusmn;\u0026thinsp;136\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e2706\u0026thinsp;\u0026plusmn;\u0026thinsp;178\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eIn vitro drug release and kinetic study of in situ gel containing AZM-loaded microsponges:\u003c/h3\u003e\n\u003cp\u003eIn the ocular cul-de-sac, the migration of simulated tear fluid (STF) into the in-situ gel formulation leads to fast gelation, producing a hard gel matrix. Through the porous microsponge structure, acetazolamide diffuses into the gel matrix once the STF hydrates the gel and penetrates the microsponge particles. The drug then keeps seeping into the STF (diffusion medium) surrounding it. One of the two simultaneous mechanisms in this release technique is the diffusion of microsponges into the gel matrix. diffusion into the surrounding media of the gel. The following factors are responsible for the sustained release profile, which ranges from 85.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43% to 73.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74% over 6 hours: Acetazolamide and the microsponge polymers (such as ethyl cellulose) form a hydrogen bond, which slows the release of drugs. entrapment inside the gel matrix, which delays nasolacrimal discharge and increases retention. Kinetics of Release; Dominance of the Higuchi model suggests diffusion-controlled release. For complicated systems such as microsponge-gel hybrids, non-Fickian diffusion (anomalous transport) combines diffusion and polymer relaxation[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003e%CDR of AZM-loaded microsponge-based ocular in situ gel (mean\u003c/b\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;\u003cb\u003eSD, n\u0026thinsp;=\u0026thinsp;3)\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e%CDR of AZM-MCP loaded ocular in-situ gel\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eISG-1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eISG-2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eISG-3\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1hrs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2hrs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.027\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3hrs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4hrs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38.37\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5hrs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e62.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e52.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6hrs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e73.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e62.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e68.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7hrs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e79.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e74.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e73.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8hrs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e85.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e82.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e77.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\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\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRelease kinetics data of AZM-loaded microsponge-based in situ gel\u003c/p\u003e \u003c/div\u003e \u003c/caption\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\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eRelease kinetics of optimized formulation\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\u003eBatch\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eZero order\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eFirst order\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eHiguchi model\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eKorsmeyer-peppas (n)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eISG-1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0.9795\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.9914\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.9942\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.89\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eEx vivo transcorneal permeation study:\u003c/h3\u003e\n\u003cp\u003eThe ex vivo transcorneal permeation of the optimized formulation was carried out through goat cornea. The marketed eye drop formulation showed 91.36% drug permeation within 2 hours, whereas the AZM-loaded microsponge incorporated in situ gel exhibited 85.25% drug permeation over 8 hours. Hence, the prepared formulation demonstrated a sustained release profile. With a steady-state flux (Jₛₛ) of 1373.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40 \u0026micro;g/cm\u0026sup2;/h and an apparent permeability coefficient (Pₐₚₚ) of 6.87 \u0026times; 10⁻\u0026sup2; cm/h, the ex vivo transcorneal permeation study showed superior controlled release from acetazolamide-loaded microsponge in situ gel (ISG-1). This is roughly three times lower than the commercial Dorzox 2% solution (Jₛₛ: 4118.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 \u0026micro;g/cm\u0026sup2;/h; Pₐₚₚ: 20.59 \u0026times; 10⁻\u0026sup2; 10⁻\u0026sup2; cm/h). The \"reservoir-in-matrix\" architecture, in which the drug first partitions from porous microsponges and then diffuses through the Carbopol\u0026ndash;HPMC network, is reflected in this change from rapid burst permeation to sustained diffusion. This creates kinetic resistance that prolongs precorneal residence and lowers tear turnover loss. Consistent therapeutic levels were ensured by ISG-1's near-linear permeation over 8 hours (R2\u0026thinsp;=\u0026thinsp;0.9798), in contrast to the high-flux commercial solution that is susceptible to systemic drainage and bioavailability variations. By maintaining the concentration gradient for hydrophilic acetazolamide (Log P\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.26) across the lipophilic cornea, mucoadhesive characteristics further improved epithelial contact, increasing bioavailability, lowering dosage frequency, and optimising glaucoma therapy[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSterility study\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSterility Testing for AZM-Loaded Microsponge-Based in Situ Gel as per Indian Pharmacopoeia (I.P.) The sterility test for acetazolamide (AZM)-loaded microsponge-based in situ gel formulations follows the direct inoculation method as per Indian Pharmacopoeia (I.P.) guidelines. The inoculated media are incubated under the following conditions:\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eObservation: There was no microorganism growth observed in the optimized ISG-1 formulation at the end of the 28 days, indicating the prepared ISG-1 formulation passed the preservative/sterility test.\u003c/p\u003e\n\u003ch3\u003eIsotonicity study:\u003c/h3\u003e\n\u003cp\u003eIn this method, a normal blood cell combines with a hypertonic solution (NaCl 2%), causing the shrinkage of the cells. In the second step, the addition of the hypotonic solution (NaCl 0.02%) causes the swelling of the biconcave structure of the blood cells.\u003c/p\u003e \u003cp\u003eThe optimized preparations had normal blood cells, just like the isotonic control solution, according to the results of a 45x microscope view. The observations were conducted using an isotonic control solution (NaCl 0.9%), a hypertonic control solution (NaCl 2%), and a hypotonic control solution (NaCl 0.2%), as shown in Figs.\u0026nbsp;13 and 14. Consequently, the test verifies the isotonicity of the developed ISG-1 formulation[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eHET-CAM: Precision Ocular Irritancy Profiling\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eGiven the eye's exquisite sensitivity, ocular formulations mandate rigorous tolerance evaluation; the HET-CAM assay serves as a validated alternative to the Draize rabbit test, leveraging vascular responses in the developing chorioallantoic membrane (CAM) that closely correlate with conjunctival irritancy. Irritation scores were determined for acetazolamide-loaded microsponge-incorporated in situ gel (AZM-loaded microsponge-ISG F-1), alongside negative control (0.9% NaCl) and positive control (1 N NaOH). The optimized formulation yielded a mean irritation score of 0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08, indicative of non-irritant potential (IS 0-0.9), with no observable hemorrhage, vascular lysis, or coagulation within the 5-minute assessment period. The negative control scored 0 (non-irritant), while the positive control registered 13 (severely irritant), confirming assay validity (Table\u0026nbsp;8). AZM-loaded microsponge-ISG F-1 scores aligned closely with saline control, demonstrating excellent ocular biocompatibility. Representative images of treated CAM [Figs.\u0026nbsp;15 (A), (B), \u0026amp; (C)] visually corroborate the absence of vascular disruption, affirming the formulation's safety for ophthalmic administration[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab8\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eScore obtained in the HET-CAM test for optimized formulation\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" 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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSr. No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHemorrhage time (sec)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLysis time (sec)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCoagulation time (sec)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHET-CAM score\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePositive control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e47.8\u0026thinsp;\u0026plusmn;\u0026thinsp;16.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e51.2\u0026thinsp;\u0026plusmn;\u0026thinsp;8.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e74.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e17.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOptimized formulation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e291.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e297.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e300.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNegative control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e289.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e294.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e299.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\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\u003e \u003c/p\u003e\n\u003ch3\u003eAssessment of local irritation in goat cornea:\u003c/h3\u003e\n\u003cp\u003eThe present study found that after treatment with the acetazolamide-loaded microsponge-based in situ gel formulation, corneal hydration levels ranged from 76% to 79%, indicating that the formulation did not cause any dehydration or structural damage to the corneal tissue. Histological evaluation further confirmed the formulation's non-toxic nature. The normal cornea maintains a hydration level of approximately 75\u0026ndash;80%, which is essential for its transparency and physiological function. As a positive control, Fig.\u0026nbsp;19 (C) shows that corneal tissue was severely damaged by 0.1% (w/w) sodium dodecyl sulphate (SDS) in phosphate-buffered saline (PBS). However, as shown in Fig.\u0026nbsp;19(A), corneas treated with normal saline (negative control) showed no evidence of injury and remained structurally intact. Significantly, ocular tissues exposed to the optimised acetazolamide-loaded in situ gel formulation (ISG-1) retained normal epithelial and stromal integrity and displayed no histological indications of toxicity or irritation (Fig.\u0026nbsp;19(B)). These results validate the new formulation's ocular safety and biocompatibility, bolstering its potential for long-term, safe use in the treatment of glaucoma[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eA (6hr) B(6hr)\u003c/h3\u003e\n\u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eC(6hr)\u003c/h3\u003e\n\u003cp\u003e \u003cb\u003eFigure\u0026nbsp;16 Histological study of goat cornea (A) Normal saline (Negative control) B. Acetazolamide-loaded microsponge in situ gel formulation, C. sodium dodecyl sulphate (SDS) in phosphate buffer saline (PBS) (positive control)\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eStability study\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eStability analysis of an acetazolamide-loaded microsponge-based in situ gel, developed in accordance with ICH Q1A(R2) recommendations for rapid stability testing. According to these standards, tests should be conducted at 40\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 75\u0026thinsp;\u0026plusmn;\u0026thinsp;5% RH every three months. Data on pH, drug concentration, gelling capacity, and viscosity at both physiological and non-physiological temperatures are included in the optimised acetazolamide-loaded microsponge-based in situ gel (IGF-1)[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab9\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 9\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStability studies of ocular in situ gel containing AZM-loaded microsponge at 40\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C with 75% RH\u0026thinsp;\u0026plusmn;\u0026thinsp;5% (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, n\u0026thinsp;=\u0026thinsp;3)\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=\"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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFormulation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTime Interval\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003epH (\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDrug Content (%) (\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGelling Capacity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eViscosity (cP)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAt Non-Physiological Temp (25\u0026deg;C) (\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAt Physiological Temp (37\u0026deg;C) (\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eAZM microsponge loaded in situ gel (ISG-1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInitial (0 month)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e89.27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e168\u0026thinsp;\u0026plusmn;\u0026thinsp;10.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2256\u0026thinsp;\u0026plusmn;\u0026thinsp;108\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15 Days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e88.95\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e165\u0026thinsp;\u0026plusmn;\u0026thinsp;9.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2239\u0026thinsp;\u0026plusmn;\u0026thinsp;97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 Month\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e88.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e162\u0026thinsp;\u0026plusmn;\u0026thinsp;8.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2213\u0026thinsp;\u0026plusmn;\u0026thinsp;102\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 Months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e87.88\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e160\u0026thinsp;\u0026plusmn;\u0026thinsp;10.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2194\u0026thinsp;\u0026plusmn;\u0026thinsp;110\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 Months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e87.21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e158\u0026thinsp;\u0026plusmn;\u0026thinsp;9.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2175\u0026thinsp;\u0026plusmn;\u0026thinsp;106\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 trial lasted three months, and all parameters stayed within reasonable bounds. There was no discernible change in the drug concentration, pH, or gelling behaviors, indicating both chemical and physical stability. Rheological consistency and performance integrity were guaranteed by the low variance in viscosity values. Sterility, clarity, and gelation behavior, all crucial for the safety and effectiveness of the eyes, were maintained in the formulation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAcetazolamide-loaded ethyl cellulose microsponges (optimized F1 formulation) were synthesized using the quasi-emulsion solvent diffusion method, yielding discrete, spherical, porous particulates (SEM verified) with a mean particle diameter of 7.43 \u0026micro;m, conducive to enhanced ocular mucoadhesion and bioavailability. These microsponges were incorporated into a pH- and thermosensitive in situ gelling system composed of Carbopol\u0026reg; 940 (0.5% w/v) and HPMC E50 LV (0.2% w/v), which exhibited shear-thinning (pseudoplastic) rheological behavior optimal for patient compliance and precorneal residence. Ex vivo release profiles followed Higuchi matrix diffusion kinetics, confirming controlled permeation. Rigorous biocompatibility evaluation affirmed ophthalmic safety through compliance with Indian Pharmacopoeia sterility criteria (no microbial proliferation over 14 days), confirmed isotonicity, preserved corneal hydration levels, showed unremarkable goat corneal histoarchitecture (intact stratified epithelium and stroma akin to saline negative control), and demonstrated HET-CAM non-irritancy, obviating concerns. Collectively, these attributes endorse the formulation's viability for sustained glaucoma pharmacotherapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are no conflicts of interest declared by the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNA\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, Keshav Shinde; Data collection and writing-original draft, Keshav Shinde; Editing, Keshav Shinde; Diagrams, Keshav Shinde; Revised the draft, Keshav Shinde, Rohan Barse; Supervision Rohan Barse; Vijay Jagtap read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNA\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval/declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNA\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNA\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNA\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNA\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNA\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBorawake PD, Kauslya A, Shinde JV, Chavan RS. 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Int J Adv Res (Indore). 2019;7:1218\u0026ndash;25. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.21474/IJAR01/9154\u003c/span\u003e\u003cspan address=\"10.21474/IJAR01/9154\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Glaucoma, in situ gel, microsponge, Acetazolamide, Polymer, Ocular delivery","lastPublishedDoi":"10.21203/rs.3.rs-9302048/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9302048/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe aim of this study was to reduce the systemic side effects of oral medication while increasing the antiglaucoma efficacy by creating an acetazolamide in situ gel based on ocular microsponges. Acetazolamide-loaded microsponges were created via quasi-emulsion solvent diffusion using ethyl cellulose at different drug-to-polymer ratios. They were then added to a pH-sensitive in situ gel made of 0.5% Carbopol 940 and HPMC E50LV. With a mean particle size of about 7 \u0026micro;m, a PDI of 0.287, and a good entrapment efficiency (85.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2%), the optimised formulation F1 (drug: polymer 2:1) is appropriate for ocular administration. When compared to a free-drug gel and commercial product, the resulting in situ gel (ISG-1) demonstrated suitable pH, quick gelation, favorable rheological behaviors, sustained in vitro drug release, and superior ex vivo trans corneal penetration, which translated into improved. It was demonstrated that the optimised acetazolamide-loaded microsponge in situ gel (ISG-1) was isotonic, non-hemolytic, and non-irritating, maintaining normal corneal hydration and epithelial-stromal integrity while delivering prolonged ocular drug release. It maintained adequate pH, drug concentration, viscosity, gelling capability, sterility, and clarity under three-month accelerated settings (40\u0026deg;C/75% RH), indicating strong physical, chemical, and microbiological stability for long-term glaucoma therapy. These findings demonstrate the potential of acetazolamide microsponge in situ gel for ophthalmic administration.\u003c/p\u003e","manuscriptTitle":"Engineered Acetazolamide Microsponges in pH‑Responsive In-Situ Gel: A Novel Platform for Long‑Acting Glaucoma Therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-23 10:17:14","doi":"10.21203/rs.3.rs-9302048/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":"b91829b9-18e9-4c86-9b68-78192bfc7a91","owner":[],"postedDate":"April 23rd, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-18T11:35:11+00:00","index":35,"fulltext":""},{"type":"reviewerAgreed","content":"144938073031604283374603054238450947065","date":"2026-05-13T12:22:17+00:00","index":34,"fulltext":""},{"type":"reviewerAgreed","content":"169481629954769324442908188220972543616","date":"2026-05-12T11:06:13+00:00","index":33,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-23T10:17:15+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-23 10:17:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9302048","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9302048","identity":"rs-9302048","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}