λ_max Determination and Calibration in Distilled Water: A working graph of Moxifloxacin HCl in distilled water is plotted. First, 10 mg of Moxifloxacin HCl was dissolved in 100 ml of distilled water to prepare a 100 μg/ml solution. Then, 10 ml was diluted to 100 ml to obtain a concentration of 10 μg/ml. Further dilutions were performed to achieve concentrations of 2, 4, 6, 8, and 10 μg/ml. λ_max was determined by scanning the 10 μg/ml solution in a Shimadzu double-beam UV spectrophotometer between 200 and 400 nm. Absorbance was measured accordingly.

λ_max Determination and Calibration in Simulated tear fluid (STF): In the STF, 10 mg of Moxifloxacin HCl was dissolved in 100 ml of medium. Ten millilitres of this solution were pipetted and further diluted to prepare 2–10 μg/ml solutions. The UV scan was performed between 200 and 400 nm using a 10 μg/ml solution. Absorbance was measured in triplicate. STF was prepared with NaCl (0.670 g), sodium bicarbonate (0.200 g), and calcium chloride (0.008 g) in 100 ml of purified water 8.

Melting Point Determination: Melting point of Moxifloxacin HCl was determined by the open capillary method using an Analab digital melting point apparatus.

Drug-Excipient Compatibility Study: Fourier Transform Infrared (FTIR) spectroscopy was employed to study the drug-excipient interactions. IR spectra were scanned in the region 400–4000 cm⁻¹ employing 0.1 mm thickness KBr pellets. Spectra of the pure drug and blends with excipients were compared to ensure absence of interaction 9, 10.

Dose Calculation: Moxifloxacin HCl eye drops at 0.5% w/v concentration release 0.25 mg per drop, with a standard treatment regimen of three drops daily for five days, equating to 3.75 mg. Considering the drainage of tears (approximately 75% loss of drug), the therapeutically active dose was only 0.938 mg. Hence, ocular inserts were constructed to release 1.1 mg of Moxifloxacin HCl each for five days, considering minimal loss of drug during delivery.

Drug Quantity for Petri Dish: For an ocular insert surface area of 0.65 cm², and the internal area of the petri dish being around 15.896 cm², around 27 mg of drug was needed per petri dish. Therefore, 54 mg of Moxifloxacin HCl was utilised for the 10 mL casting solution.

Various polymers and plasticisers have been evaluated for ocular inserts based on their folding endurance, flexibility, and texture. Chitosan was tacky, PVP K-30 brittle, and alginate blends unstable. PVA with PEG-400 was chosen as the reservoir film and EC with PVP-K30 and DBP as the rate-controlling membranes. The films were cast, dried overnight at 40°C, and assessed for their suitability.

Drug Reservoir Preparation: Films of the reservoir were prepared by casting PVA and PEG-400 in distilled water. A dose of 54 mg Moxifloxacin HCl was mixed with 10 ml of this solution. Sonication for 30–40 min was followed by casting 4 ml into glass petri dishes of 4.5 cm diameter and drying at 30°C for 24 h. Dried films were elliptically cut into inserts and stored in desiccators 9.

Rate-Controlling Membrane Preparation: Ethyl cellulose (EC), either alone or with PVP-K30, was employed to cast membranes in proportions of 6%, 4%, and EC:PVP ratios of 8:1, 4:1, 2:1, and 1:1. The polymers were dissolved in acetone with DBP, stirred for 40 min, and cast on petri dishes. Solvent evaporation was regulated using an inverted funnel. Films were dried overnight, cut, and stored 11.

Sealing of Films: Reservoir films were placed between the two rate-controlling membranes. These assemblies were kept in a beaker containing acetone:ethanol vapours (60:40) for 1–2 min to properly seal them. The inserts were kept in air-tight containers 9.

Uniformity of Thickness: Insert thickness was determined using a digital Vernier caliper at five points. Three inserts per batch were evaluated 10.

Uniformity of Weight: Three inserts from each batch were individually weighed by a digital balance and mean weight was reported 10.

Drug Content: Individual inserts were dissolved in 10 ml phosphate buffer (pH 7.4), filtered into a 25 ml flask, and diluted. The absorbance was read at 288 nm 10.

% Moisture Absorption and Loss: Films were stored in desiccators in humid (aluminum chloride) or dry (calcium chloride) conditions for three days, weighed initially and finally, and % change determined 10.

Folding Endurance: Each strip of film was folded over and over at the same location until it ruptured. The fold number was counted 10.

Surface pH: Swollen inserts were measured using a digital pH meter after 30 min in distilled water.

Release studies were conducted using a Franz diffusion cell and a dialysis membrane. Inserts were incubated with 40 ml STF at pH 7.4 and maintained at 37±0.5°C. The mixture was then stirred at 20 rpm. Aliquots were removed at intervals, supplemented with fresh STF, and analyzed spectrophotometrically at 288.5 nm 11, 12.

Release Kinetics: Data were analysed by fitting to the zero-order, first-order, Higuchi, and Korsmeyer–Peppas models. Best-fit model was identified from regression coefficients (R² values) 13.

Sterilization: Optimized inserts were sterilized under UV radiation for 15 minutes at 0.305 m height under aseptic conditions and packaged in pre-sterilized amber vials 14.

Sterility Testing: Sterility was determined by using the direct inoculation method following Indian Pharmacopoeia guidelines, utilizing five inserts from FM6 formulation 15.

In vitro antimicrobial activity tests: In vitro antimicrobial activity tests were conducted to evaluate the biological activity of the synthesized ocuserts against certain microorganisms. The test was performed using the agar diffusion method with the cup-plate technique. A layer of nutrient agar seeded with Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) was first poured into sterile Petri plates and left to solidify. A sterile 4 mm borer was used to create wells in agar seeded with microbes. Standard Moxifloxacin drops and test formulations were added, diffused for two hours, and incubated at 37°C for 24 h. Zones of inhibition were measured and compared; tests were done in triplicate to ensure reliability and reproducibility 16.

The best ocular insert was microbiologically tested for controlled drug release over a period of five days. S. aureus and P. aeruginosa were used as test microorganisms in the investigation. Ten millilitres of seeded agar were poured into plates, solidified, and an ocular insert was placed. After 24-hour incubation, the inserts were transferred daily to fresh plates for five days. Zones of inhibition were measured daily to assess drug release using standard calibration curves 9.

Ocular irritation studies were conducted using the Draize test on albino rabbits, with the right eye treated and left eye as the control. A sterilised ocular insert was placed in the conjunctival sac and the eyes were monitored at intervals of up to one week for signs of irritation. Observed changes in the cornea, iris, and conjunctiva at 1, 24, 48, and 72 hours, and then one week post-administration were scored using a standard grading system to evaluate the degree of irritation 12, 17.

The in vivo drug release was studied in five healthy rabbits following IAEC approval. Sterile inserts and blanks were placed in the right and left eyes, respectively. The inserts were removed at intervals and analysed by UV spectrophotometry at 288 nm. The drug release was calculated by subtracting the residual content from the initial drug load. Drug release data were analysed using one-way ANOVA and Dunnett’s test to assess the membrane effects. Results are expressed as the mean ± SD (n=3), with significance set at p < 0.05 15.

The in vitro - in vivo drug release correlation was determined by comparing the in vitro and in vivo percentages of the drug released at corresponding time periods. The release data obtained by in vivo studies were compared against the in vitro data, and a correlation coefficient was determined by plotting the result to find whether the in vitro model is predictive and linear with respect to in vivo environment 18.

Accelerated three-month short-term stability studies were performed for ocular insert formulations. The samples were stored under two conditions in amber-coloured glass vials: room temperature and refrigeration (2–8°C). Samples were withdrawn monthly and examined for visual appearance changes, pH, and drug content. This evaluation was used to determine the physical and chemical stability of the formulations under different storage conditions 18, 19.

Determination of λmax and Construction of Calibration Curve in Distilled Water: The λmax of Moxifloxacin HCl in distilled water was determined to be 288.5 nm. A calibration curve was plotted using the absorbance values at this wavelength, which increased linearly with concentration, showing compliance with Beer's Law. The absorbance at concentrations from 0 to 10 μg/ml was measured, and the maximum absorbance was found to be 0.918 ± 0.0025 at 10 μg/ml.

Determination of λ_max and Construction of Calibration Curve in STF: In STF at pH 7.4, the λ_max of Moxifloxacin HCl did not change at 288.5 nm. The standard plot shows a linear correlation between the concentration and absorbance. The highest absorbance recorded was 0.810 ± 0.0025 at 10 μg/ml.

Melting Point: The melting point of moxifloxacin hydrochloride was 242°C, which confirms its purity and thermal stability.

Interaction Studies: Compatibility studies were performed using FTIR spectroscopy. The IR spectra of the pure drug and polymers (PVA, PVP-K30, and EC) and the combined formulation were compared. (Figure 1) Spectral analysis demonstrated the existence of all characteristic peaks of Moxifloxacin HCl in the final formulation, indicating that there was no substantial interaction between the drug and polymers. The -NH group stretching appeared at 3528 cm^-1, and the carbonyl group C=O stretch appeared near 1708 cm^-1, which is consistent with the pure drug.

Physicochemical Evaluations: The Ocular inserts were tested for different physicochemical characteristics. The thickness varied from 0.289 ± 0.006 to 0.341 ± 0.004 mm. The weights were between 19.82 ± 0.24 mg and 20.29 ± 0.33 mg. The pH on the surface varied from 6.5 to 7.27, which is within the acceptable ocular pH. Drug content varied from 0.97 ± 0.04 mg to 0.991 ± 0.06 mg. Moisture uptake varied from 4.67 ± 0.003% to 12.45 ± 0.21%, whereas moisture loss varied from 7.2 ± 0.011% to 12.18 ± 0.012%. (Table 1) Folding endurance was between 75 ± 4.6 and 92 ± 3.5°, showing good flexibility of the inserts.

Release studies were initially performed on drug reservoirs (FR1–FR4) without rate-controlling membranes. FR1 was the highest at 98.36% at 10 h, which was closely followed by FR3 at 98.1% at 8 h (Figure 2). Based on their performance, FR1 and FR2 were chosen for the formulation.

Formulations FM1–FM5 were conducted for 48 h, and the highest release was found in FM5 (59.1%). FM6 to FM8 were screened for 5 days (120 hours), and FM6 indicated the highest drug release of 99.1% and was thus the optimized formulation (Figure 3).

Kinetic modelling was used for FM6, FM7, and FM8. For each formulation, the Higuchi model provided the best fit (R² values: 0.991 for FM6, 0.9703 for FM7, and 0.9831 for FM8), suggesting diffusion-controlled release. The "n" values from the Korsmeyer-Peppas model (between 0.60 and 0.61) supported non-Fickian (anomalous) transport behaviour (Table 2).

Sterility Studies: Sterility tests performed on FM6, FM7, and FM8 using Fluid Thioglycollate Medium as well as Soybean Casein Digest Medium validated that all three products were sterile throughout a period of 7 days' incubation period, with no microbial growth recorded.

Optimized formulation FM6 was assessed for drug release for five days by both in vitro and microbiological tests. The percentage of drug released increased progressively with time. On day one, the in vitro drug release was 42.85%, whereas S. aureus and P. aeruginosa released 37.52% and 25.43% of the drug, respectively. On day five, the drug release was 99.1% in vitro, whereas those of S. aureus and P. aeruginosa were 99.5% and 95.2%, respectively. These findings demonstrate sustained and consistent drug release from the ocular insert.

For in vivo testing, FM6 was inserted inside the cul-de-sac region of a rabbit eye, and drug release for five days was observed. On the first day, 39.5% of the drug was released. Every day, an increase in the percentage of drug release was recorded, reaching 97.67% by the fifth day. These results confirmed that the formulation ensured prolonged release of the drug in a biotic environment.

An in vivo-in vitro correlation between the drug release data of the in vivo and in vitro experiments of the formulation FM6 was found. The in vivo-in vitro correlation was strong, with an R² value of 0.9975. The drug percentages released in vivo were found to be in close agreement with the in vitro results on each day of the study, validating the predictive nature of in vitro testing for in vivo performance.

Three-month stability studies were performed at room temperature under refrigeration. At room temperature, no alterations in the visual appearance of the formulation were observed. Slight pH (from 7.17 to 6.81) and a decrease in drug content (from 98.72% to 97.12%) were observed. Similarly, under refrigerated conditions (4°C), no visual alterations occurred, with only slight pH (from 7.15 to 6.92) and drug content (from 98.15% to 97.11%). These results further confirm that FM6 was stable and active during the test.