Experimental

Materials and methods

Poly(vinyl alcohol) and low-molecular-weight chitosan (<100 kDa) were procured from Central Drug House (P) Ltd., Mumbai, India, while glutaraldehyde was purchased from NICE Chemicals (P) Ltd., Kochi, India. Nutrient agar and gum ghatti were purchased from Himedia (P) Ltd., Mumbai, India. Moxifloxacin was provided as a gift by Alkem Laboratories, and double-distilled water was used throughout the experiment.

Film-forming solutions and their preparation

10 % poly(vinyl alcohol) (10 g PVA in 90 g water) was prepared using an overhead stirrer (Q-19 A, Remi Elektrotechnik Limited, Kolkata, India). A CS solution (5 wt.%) was prepared by blending acetic acid and water (1:1) and stirring at room temperature for 8 hours. Different concentrations of GG solution (1, 2.5, 5 and 10 wt.%) were prepared by dissolving GG in distilled water and stirring until a clear solution formed. All solutions were then filtered through nylon cloth. All polymeric solutions were mixed as specified in Table 1. PVA/CS/GG solutions were prepared using an overhead mechanical stirrer (400 rpm, 20 min). Meanwhile, drug-containing films were made by mixing moxifloxacin into the film-forming solution until complete dissolution. In the final step, the crosslinker (glutaraldehyde solution: distilled water, 0.5:9.5) was added and the mixture was homogenized for 2 minutes. Then, the solution (20 mL) was poured into a 90-mm Petri dish and placed in a hot-air oven at 40 °C. After drying, the composite was packed in a sealed zippered container for subsequent use.

Formulation code | Content, g | Amount of moxifloxacin HCl, mg cm-2
PVA solution (10 wt.%) | CS solution (5 wt.%) | GG solution
PCG0 | 14 | 6 | 0 | -
PCG1 | 12 | 6 | 2a | -
PCG2 | 12 | 6 | 2b | -
PCG3 | 12 | 6 | 2c | -
PCG4 | 12 | 6 | 2d | -
PCG0D | 14 | 6 | 0 | 5
PCG1D | 12 | 6 | 2a | 5
PCG2D | 12 | 6 | 2b | 5
PCG3D | 12 | 6 | 2c | 5
PCG4D | 12 | 6 | 2d | 5

Physicochemical properties of the fabricated composites

Various physicochemical properties, including weight uniformity, ocular composite thickness, surface pH, opacity (Equation 1), folding endurance, and blood biocompatibility (Equation 2), were measured.

where x / nm represents the thickness of the films in mm and Abs600 represents the absorbance at 600 nm.

where ODtest is the absorbance of the tested film sample at 545 nm, ODnegative refers to the absorbance of the negative control at 545 nm and ODpositive is the absorbance of the positive control at 545 nm.

Transparency

Transparency of the ocular composites was measured both using a ruler and spectrophotometrically. Gently, the ruler was placed under the fabricated films, and photographs were taken. Visually, the number on the ruler was visible through the fabricated films. To assess the radiation effect and UV rays transmittance, the films were cut into 40×50 mm pieces, and transmittance was recorded from 200 to 800 nm.

Microscopy

Optical images were captured using a compound microscope equipped with a CCD camera (Magnus MX21i LED, Japan). A small piece of film was cut and placed on the slide, and microarchitecture was observed. The surface morphology of the prepared composite was examined using field-emission scanning electron microscopy (JEOL JSM-6510, Japan) at higher magnification [20].

Colour analysis

The effects of CS and GG on the colour coordinates were observed visually. To analyse the hue system in the prepared ocular film, the colour variation of the fabricated films was measured using a colourimeter (Premier Colorscan, SS 5100H, Mumbai, India). The measurement was performed in direct transmittance mode using the CIE 1976 L* (lightness), a* (+a redness or -a greenness) and b* (+b yellowness or -b blueness) colour space. Using the L*, a* and b* coordinates, colour difference (Δ_E_), whiteness (WI) and yellowness index (YI) were calculated using Equations (3), (4) and (5), respectively [21].

LC, aC, b*C are the values of the control film parameters (ocular film without gum ghatti), whereas the numerical values obtained for different GG-films were substituted with symbols having an ‘s’ tag.

Swelling profile

The swelling profile of the PVA/CS/GG was evaluated by cutting it into 20×20 mm squares and weighing the squares. Pre-weighted films were immersed in artificial tear fluid (ATF; pH 7.4) at room temperature. Briefly, the pre-weighted ocular film was submerged in a petri dish containing 2 mL of ATF. At predetermined time intervals, the film was carefully removed, and excess ATF was wiped off with tissue paper. The film weight was recorded, and the process was repeated until three consecutive measurements were identical. The swelling ratio was calculated using Equation (6).

Where _S_0 and _S_1 represent the sample weight before and after absorption of ATF, respectively.

Molecular and mechanical evaluation

FTIR, DSC and XRD analyses of the prepared composite film should be performed using the methods described by the respective authors [22].

Mucoadhesion of the PVA/CS/GG ocular film was tested with a CTX texture analyser (Brookfield, Ametek, United States). A 35 mm cylindrical probe equipped with a 5 kg load cell was used to assess adhesive properties and was applied to the gelatine substrate (6.67 wt.%). 1 mL of ATF was evenly spread over the gelatine surface to mimic the ocular mucosa. The film was cut into a circular disc and attached to the probe tip. The probe was pressed onto the gelatine gel in a glass beaker with a force of 0.5 N for 120 seconds. Various mucoadhesion parameters, such as peak adhesion force (PAF), total work of adhesion (TWA), and cohesiveness, were measured and recorded using the built-in Texture Pro software.

To evaluate the breaking force of the ocular composite, prepared films were cut into 20×20 mm pieces, placed between two rings, and secured. A 4 mm-diameter cylindrical sensor (TA44) was used in the experiment. Throughout the experiment, the probe velocity was fixed at 2.00 mm s-1.

In vitro drug diffusion study

Drug diffusion analysis was performed using the method described by the various authors [23]. A modified Franz diffusion cell was used for the study. A film was cut into 10×10 mm and placed on the pre-hydrated dialysis membrane (soaked overnight in artificial tear fluid, ATF), which served as a semipermeable barrier separating the donor and receptor compartments. The temperature of the receptor compartment was maintained at 37±1 °C with continuous stirring at 100 rpm throughout the experiment. At a predetermined time interval, 1 mL of the sample was withdrawn and replaced with fresh ATF solution. The concentration of moxifloxacin in the withdrawn solution was measured using a UV-vis spectrophotometer (UV-1900i, Shimadzu Corporation, Japan) at 289 nm.

MTT-assay of the fabricated formulations

The MTT Assay was performed on a 96-well plate. Human corneal epithelial cells (HCECs) were used to evaluate the toxicity of the formulations. The test was conducted by the method described elsewhere [24]. In brief, HECE was incubated in a healthy environment (37 °C) for 24 hours. Inoculated HECE having 3000 to 4000 cells per well in 100 μL is taken as standard cell growth, and test samples were treated in 96-well plates after incubation. Test and reference samples were measured for absorbance at 570 nm in DMSO. Cell viability was determined as the percentage relative to the control value (set to 100%).

Antimicrobial activities

The antimicrobial activity of the composite film was evaluated against both Gram-positive (S. aureus; ATCC 29213) and Gram-negative (P. aeruginosa; ATCC 27853) bacteria. Prepared films were placed on a freshly prepared agar well plate using the disc diffusion method. The plates were incubated for 24 h at 37 ± 1 °C. The zone of inhibition (ZOI) was measured with a ruler.

Ocular irritation study

An ocular irritation test was conducted after obtaining approval from the Institutional Animal Ethics Committee, with protocol clearance number (CPCSEA-IAEC-IPT/04/23), dated 03 October 2023. During the experiment, ocular abnormality-free rabbits were used, and the study was conducted strictly in accordance with the ARRIVE guidelines and the Draize test protocol. A small piece of film was placed in the conjunctival sac of the left eye and considered the test group. The right eye was injected with 0.1 mL of normal saline and referred to as the control. After inserting the film at different time intervals, both eyes were examined for ocular abnormalities (redness, swelling, injury, reaction, or inflammation).

In vivo ocular inflammation potential of the composite

Healthy rabbits were used to assess the in vivo anti-inflammatory activity of the prepared film. For this, ocular abnormalities and disease-free rabbits were selected and divided into three groups of three rabbits each. Group I served as the negative control and was treated with normal saline. Group II was treated with carrageenan (200 μL; 2 % w/v) and designated as the test group, and Group III was used as the standard group (treated with the fabricated film formulation after injecting carrageenan). Inflammation was induced by injecting carrageenan, and a sterilized composite film (UV-irradiated at 25 cm for 10 min) was placed in the cul-de-sac region of the eyes 1 hour after inflammation. After the conclusion of the experiment, fluorescein staining of the cornea was done to evaluate the damage to the epithelium. Moxifloxacin eye drops (MOXI TOR; 0.5%) were used to prevent infection and facilitate recovery.

Statistical analysis

All experiments were conducted in triplicate, and results are presented as mean ± standard deviation (SD).