Microlabs, located in Bengaluru, India, provided a complimentary sample of levofloxacin (LFX). HPMC K4M, gelatin, PEG-400, sodium hydroxide, dihydrogen potassium orthophosphate, and buffer tablets were acquired by Fischer Scientific. The locally grown plants in Anantapur produced fresh Aloe barbadensis leaves. The remaining chemicals and reagents used in the study were of analytical reagent (AR) grade.

These were carried out during the preformulation phase, when in-depth compatibility tests were carried out to evaluate Levofloxacin's (LFX) compatibility with the selected excipients.To create the test samples, LFX and each excipient were dried in equal amounts. These mixes were examined using Fourier Transform Infrared (FTIR) spectroscopy to find any possible chemical or physical interactions.The FTIR analysis was a vital source of information on the compliance and maintainability of the creation of the ODDS with the selected formulation components (15).

Ocular films were made by means of solvent casting (16, 17). First, the polymer(s) of choice were dissolved in a pH 7.4 synthetic tear fluid to create the LFX reservoir, and the entire mixture was then stirred thoroughly on a magnetic stirrer to guarantee the full dissolution of the polymers, as well as their attainment of the desired concentrations. PEG-400 and other additives used to formulate the polymeric solution were added gradually to the solution under stirring to add film-forming properties and the general performance of the formulation with levofloxacin (0.5% w/v). The solution was then poured onto a mercury substrate and left to cast into films after obtaining a homogeneous mixture (18). There were 13 formulation batches that were optimized and designed through the Box-Behnken Design of Design Expert Software (version 11/ 13) (Table 1) to investigate the effects of the most important variables on film properties. Cast films were dried and then circular ocular inserts (8 mm in diameter) were neatly punched out with a cork borer and further in vitro analysis done (19).

Ocular films were measured through the digital caliper at three points of each film regarding thickness. The average thickness was then computed by averaging these values and uniformity of the film surface was measured using standard deviation.It is necessary to determine the mean and the standard deviation of the thickness to guarantee the uniformity of the ocular drug delivery system and its mechanical soundness as well as the quality of the system overall (20).

The homogeneity of the Levofloxacin (LFX) distribution in the cast films was measured with the help of a systematic analytical process. Three inserts of the different After carefully removing portions of the film, they were placed in 100 mL volumetric flasks that contained phosphate buffer (pH 7.4) to allow LFX to be completely extracted.After obtaining the solution, 1 mL of it was taken and appropriately diluted with the same buffer in order to bring the drug concentration to the range of detectable concentration. The absorbance of the diluted sample was then measured at 287 nm using the UV-visible spectrophotometer, with the blank serving as the standard (21).

To ensure analytical reliability, this was done three times for every batch of formulations. To ascertain the consistency of LFX loading in the films, the mean drug content and standard deviation were calculated. LFX concentrations in each sample were calculated among the dilution factor using the right formula (Eq. 1).

In this formula, GL stands for the concentration of LFX in the standard solution, and As stands for the absorbance of the sample solution. Gr stands for the standard LFX solution's absorbance (22).

A test on weight change was aimed to determine the consistency of the ocular films in each of the batches of the substance. The batch was chosen randomly with three films taken in different regions and weighed separately with precision. The average weight was obtained by taking an average of the three measurements and the standard deviation was obtained in order to consider the degree of weight variation across the films. A small value of standard deviation shows a greater degree of uniformity and a large value has a high degree of variation. With the dosing reliability, batch reliability, and the product quality in general, it is necessary to have a consistent film weight (23).

In order to ascertain the moisture absorption capacity of the ocular inserts, three samples each of the formulations batches were chosen as representative samples. The weight of every insert was measured separately and the weight of the insert taken to note (mg). After that the inserts were put in a desiccator at about 75% relative humidity (RH) during three days. The inserts were taken off after the exposure period and reweighed again to get the final weight of the inserts. Eq. 2 was used to calculate the percentage of moisture absorption.This analysis gives the stability and moisture uptake information of the ocular inserts in a high-humidity environment that is important in its storage, handling and general operation in an ocular drug delivery system (Eq. 2) (24).

To find out the stability of ocular inserts at dry temperatures, the percent moisture loss was calculated. The insert was first weighed to get its original weight and then was transferred to the desiccator with anhydrous calcium chloride that offered a very dry environment. After three days of exposure, the implants were taken out and weighed again to determine their final weight. The loss in percent moisture was determined through the use of the equation. 3. This test is needed to determine the capacity of the inserts to maintain structural integrity and stability under low-moisture conditions which is vital in their storage, handling, and their appropriateness in ophthalmic use (Eq. 3) (25).

A bi-chambered donor was used for the in vitro permeation tests. receiver compartment system with a semipermeable, translucent regenerated cellulose membrane (Sigma Dialysis Membrane) to simulate the corneal epithelium

obstacle. After inserting this ocular insert into the donor compartment, 0.7 µL of To mimic the tear, phosphate buffer (pH 7.4) was added to the donor compartment. fluid surroundings. A magnetic stirrer was used in the receiver compartment to continually stir the phosphate buffer (pH 7.4) in this compartment to mimic the eyelid blinking system (26).

At prearranged intervals, the samples were removed from the receiver compartment and immediately replaced with an equal volume of new buffer. Using a UV-visible spectrophotometer (Shimadzu 1700, Japan) at a wavelength of 287 nm and phosphate buffer with a pH of 7.4 as a blank, the LFX content of the removed samples was ascertained.

To ascertain the release mechanism of LFX out of the inserts, the release data were modelled to different kinetic models which included Zero-order, First-order, Higuchi diffusion and Korsmeyer-Peppas models. The mechanism of drug release is described by the release exponent (n):n = 0.5: Fickian diffusion0.5 < n0.50.5 n = 1: Non-Fickian (anomalous) transportn = 0.5: Zero-order releasen0.5 > n > 1: Super case-II transportn = 1.

These studies give insights on the diffusional behavior and mechanism that governs the LFX permeation through the ocular films.

7 levofloxacin ocular films (LFX) were prepared successfully with different concentrations of Aloe barbadensis leaf mucilage (ABLM) gelatin, HPMC K4M. The polymers were chosen due to their biocompatibility, biodegradability, and good capabilities to form the films. PEG-400 was added to achieve plasticization to improve flexibility and handle ability of the films.

Design Expert software Box-Behnken Design (BBD) was used to formulate optimization because it allowed systematic analysis of the effect of each of the polymers on the desired film parameters. Solvent casting method was used to form smooth, even and soft films that may be used in the eye. The studies on FTIR compatibility also verified that no significant chemical interaction between LFX and the polymers took place, as evidenced by the fact that typical peaks were observed without any significant change, which proved the compatibility of the two components.

The thickness of the films was very uniform with values ranging between 0.15 +/- 0.01 and 0.19 +/- 0.01 mm and the mean film area was 0.502 cm 2 which shows that the films were very uniform. The fact that the thickness is uniform indicates how accurate the casting procedure is and the ability to reproduce the formulation process.

Mean weight per batch varied between 65.54 + 0.7 to 61.85 + 0.4 mg, the standard deviations were low, which indicated uniform distribution of materials in casting.

The LFX in the movies was between 88.00 + 3.99%-97.03 + 0.95% indicating a consistent drug loading. In all formulations, LF-6 had the greatest drug content. These results are the positive indication of the homogeneous distribution of LFX across the polymer matrix.

The moisture loss test which was conducted in a very dry environment and anhydrous calcium chloride was used as a dry weight revealed a percentage loss of 7.98 ± 0.5 to 9.55 ± 0.4%. The gelatin and PEG-400 characteristic of the loss of moisture to the atmosphere are found to contribute to the water-retention ability of the films.

On the contrary, the percentage absorption of moisture test showed that films with more gelatin absorbed more water because the substance is hydrophilic. The values of moisture absorption were within:

Highest: LF-6 → 18.05 ± 0.7%

Lowest: LF-7 → 12.65 ± 0.8%

Although the moisture uptake was different, all the films maintained their structural integrity with no evidence of deformation and stickiness, which implies great stability in the presence of high humidity conditions.

(Values expressed as mean ± SD; n = 3)

The results confirm that:

The chosen polymers (HPMC K4M, gelatin, ABLM) successfully produced consistent and flexible films.

LFX was uniformly distributed across all formulations.

Moisture loss and moisture absorption values indicate good environmental stability.

LF-6 emerged as the most promising formulation due to its optimal drug content, controlled thickness, and highest moisture absorption capacity suggesting good hydration and mucoadhesive potential.

These findings collectively demonstrate the suitability of the prepared films for further evaluation as an ocular drug delivery system for levofloxacin.

In microbiology, precise media preparation is crucial for reliable experimental outcomes. For the current study, nutrient agar (NA) served as the foundational solid culture medium for all plate preparations. This general-purpose, rich medium is well-suited for supporting the expansion of several non-fastidious bacterial species, providing essential nutrients such as peptone and beef extract, along with agar to give it a solid consistency. The NA medium was sterilized, cooled, and then aseptically poured into Petri dishes to create a sterile surface conducive to bacterial colony development and isolation. Simultaneously, for the initial cultivation and robust growth of the bacterial strains before their use on plates, nutrient broth (NB) was employed as the liquid culture medium. NB shares the same nutritive components as NA but lacks the solidifying agent, making it ideal for preparing homogeneous bacterial suspensions. This liquid culturing step ensures a standardized and viable inoculum, which is essential for accurate and consistent results in downstream applications like antimicrobial susceptibility testing or viability assays (27).

In the case of microbiological assays, Muller-Hinton Agar (MHA) was carefully prepared. An accurate weight of 33.9 g of MHA powder was weighed with accuracy and fully dissolved in 1000 mL of distilled water. This solution was then autoclaved under normal sterilization conditions to make a totally sterile solution, with no microbial contaminants. After the sterilization, the molten sterilized mixture was mixed thoroughly to achieve homogeneity and aseptically transferred to 10 cm Petri plates (c. 30 ml of the mixture). This measured volume of pouring agar provided a uniform level of agar depth to all the plates which is essential in equal diffusion in antimicrobial susceptibility tests (28).

To render it sterile, 1,000 milliliters of pH-adjusted nutrient agar (NA) were made and autoclaved. After the molten medium cooled, it was aseptically transferred to pre-sterilized Petri plates in a chamber of Laminar Flow Hood at 42–45 o C. The plates were then solidified and allowed to dry and then, a 24 h sterility check incubation period was incubated (30).

Standard Levofloxacin discs that were commercially available were used as the reference antibiotic in order to compare the antimicrobial activity of all the bacterial strains used during the study (31).

In the case of antimicrobial susceptibility testing, 20 mL of Muller-Hinton medium with bacterial seeds was inoculated and then poured into sterilized Petri dishes and allowed to dry. These plates were left to incubate after one day so that initial bacteria growth could occur. Wells were then formed in agar with the help of sterile die and the formulations under study were measured and carefully put in the formed wells. Another day was then allowed to incubate the plates at 37 o C to ensure diffusion of the drug and inhibition of bacterial growth. The antimicrobial activity of the formulations was determined by the measurement of the diameter of the zone of inhibition formed around each well where the larger the zone formed, the greater the antimicrobial effect (32)

The estimation of antimicrobial activity was done through the use of paper disc diffusion technique. In order to make the bacterial inoculum, a small portion of slant culture was suspended in 5–6 drops of sterile water in a test tube. A portion of this activated bacterial suspension was in turn used on the surface of the agar plates with a sterile swab stick so that a continuous layer of microbes was formed on the culture medium. Plates were dried after use (15–30 minutes) to dry up any excess moisture. Then, paper discs with the test samples were positioned on such prepared plates in a certain distance to each other. After that, plates were left to incubate at 37 o C to one day which enabled the bacteria to grow and the antimicrobial agents to diffuse out of the discs to cause the development of a zone of inhibition (33).

The optimization of the batch of ocular inserts was done by subjecting it to UV radiation to sterilize it. The batches were subjected to UV rays over a period of 10 minutes keeping an exact distance of 250 mm between the UV lamp and the batch. This will be done to minimize surface microbial contamination of the inserts, which will make them qualified to be used in the eyes (34)

A microbial challenge test was done to confirm the sterility of the anticipated batch of ocular inserts. The inserts were put in solidified agar media that were previously prepared with negative and positive control. S. aureus is a common strain of bacteria that has been used to test the ability of a media to promote growth and ensure the viability of the challenge organism, so specifically S. aureus was inoculated onto the positive control plates. All plates, the one with the predicted batch and the ones with the controls were then incubated at 37C during 24h. The existence or lack of microbial growth on the inserts and the formation of microbes of inhibition around them were noted and studied after incubation period. This test allowed the direct detection of sterility of the prepared ocular formulations, and it was clear whether it contained living microorganisms or not and therefore safe to use. A microbial challenge test was done to confirm that the expected batch of ocular inserts was sterile. The inserts were spotted on solid media of agar which had been prepared with negative and positive controls. S. aureus, a common bacterial strain in sterility tests, was specifically inoculated in the positive control plates to ensure that the media was growth-promoting and that the organism is alive. Plates containing the anticipated batch and controls were then incubated at 37 o C after which incubation was done on all the plates with 24 hours. Following incubation, the occurrence or absence of microbial growth on the inserts and the appearance of zones of inhibition thereof were keenly observed and studied. This test allowed the direct determination of sterility of the prepared ocular formulations such that it was or was not contaminated with viable microorganisms and hence safe to use (35, 36).

Two conventional microbiological methods, agar-well diffusion method and paper disc diffusion technique was used to determine the in vitro antibacterial activity of the formulations. In both approaches effectiveness of the formulations were evaluated by the measuring of the of zone diameter of inhibition that was formed in relation to certain bacterial strains that are E. coli and S. aureus. This value directly shows the level of inhibition of the microbial growth by the formulations (37).

The highest zone of inhibition of batch LF-6 was 19 mm to produce antibacterial activity against E. coli. Comparing batch LF-6 and S. aureus, batch LF-6 showed maximum zone of inhibition of 21 mm 2. (38) Bench marking of these results was done against a Levofloxacin disc that acted as a positive control as presented in Table 3.

The study's findings, as presented in Table 5.98, indicated that batch LF-6 exhibited the most significant antibacterial inhibition. Specifically, it produced an 18 mm zone of inhibition against E. coli and a 22 mm2 zone of inhibition against S. aureus (39).

The eye irritation test revealed that the tested formulas did not exhibit any visible signs of irritation (redness, inflammation, or an increase in tear production) over the 2.5-hour research period. Therefore, it might be determined that the formulas were safe and did not cause eye irritation is represents in figure no.1.

Figure 1. (A) Normal eye; (B) Carrageenan-induced ocular inflammation with full redness; (C) Film (EMA3) placed in the cul-de-sac; (D) Redness and inflammation have remarkably been disappeared after 2.5 h of film administration

The zone of inhibition in the treated group was also observed in this test which gave information on the drug that was released in the tear fluid. Sterile nutrient agar Petri dishes were prepared in advance and inoculated with a culture of S. aureus after which the dish was incubated at 37C in 24 hours to produce a homogeneous bacterial lawn. Sterile swabs were used in collecting tear fluid samples of the treated rabbit eyes after every 2hours up to 12 hours. The swabs that had been impregnated with the drug that was released out of the ocular inserts were then applied to the prepared Petri dishes alongside the test organism. A zone of inhibition was further incubated after 24 hours around each swab with Image-J software whereby the quantitative evaluation of antimicrobial activity of the released drug over time could be measured.

The conjunctivae of each animal were periodically evaluated (day 1 to day 5) in both eyes to evaluate the redness. This was done by comparing the treated eyes which were treated with the predicted sterile formulation and the control eyes. To compare the means of various groups, One-way ANOVA was applied and then it was followed by the Tukey-Kramer multiple comparison test to perform statistical analysis. The findings showed that the amount of redness scores had a significant reduction in the treated eyes relative to the positive control group (P < 0.01 and P < 0.001). As discussed in Table 5.100 and Fig. 5.45, ocular film, batch LF-6, the optimized film, was capable of reducing conjunctival redness by the 4th day of treatment. Redness scoring was determined in the following way: zero: no redness, one: mild redness, two: moderate redness, three: complete redness of the conjunctiva.

Monitoring of bacterial load in the treated eyes of each animal was periodically done (2 to 12 hours) following the administration of optimized sterile formulation, as compared to control eyes. The statistical analysis of the comparison between the means of the various groups was done using one-way ANOVA with a follow up multiple comparison test, the Tukey-Kramer test. There was a significant difference in the diameter of the zone of inhibition realized on the comparison to the positive control group (P less than 0.001). Results of samples obtained in the treated eyes, treated with the optimized batch of ocular film, show an amazing decrease in the bacterial load, where the largest possible inhibition zone was 23 mm 2 until 12 hours (Table. 6, Figure.2).