A Novel Approach to Design a Ofloxacin Nanoemulsion Based Formulation Development and Evaluation for the Management of Bacterial Keratitis

Bacterial keratitis (BK) is a severe ocular infection that can lead to significant visual impairment if not promptly and effectively treated. Ofloxacin (OFX), a second-generation fluoroquinolone, is widely utilized for treating BK due to its broad-spectrum antibacterial activity and favorable pharmacokinetic properties.

Conventional OFX ophthalmic solutions often suffer from rapid precorneal elimination and poor ocular bioavailability, necessitating frequent administration and potentially leading to patient non-compliance. To address these limitations, nanoemulsion-based drug delivery systems have emerged as promising alternatives. These systems offer advantages such as enhanced ocular retention, improved corneal permeability, and sustained drug release, thereby increasing therapeutic efficacy and patient adherence.

Nanoemulsions are colloidal dispersions comprising nanoscale droplets of oil and water stabilized by surfactants. Their small droplet size facilitates deeper penetration into ocular tissues, while their tunable properties allow for optimization of drug release profiles. The incorporation of OFX into nanoemulsion formulations can potentially overcome the drawbacks associated with traditional ophthalmic preparations.

This study aims to develop and evaluate an OFX-loaded nanoemulsion formulation for the management of bacterial keratitis, focusing on enhancing ocular bioavailability and therapeutic outcomes.

MATERIALS AND METHODS

Drug Profile

Ofloxacin (OFX) is a second-generation fluoroquinolone with the molecular formula C??H??FN?O? and molecular weight 361.37 g/mol. It functions by inhibiting bacterial DNA gyrase and topoisomerase IV, thereby preventing DNA replication. OFX is highly soluble in water and ethanol, with a plasma half-life of 4–5 hours and oral bioavailability of approximately 98%. The drug is widely used to treat ocular infections, including bacterial keratitis, and is generally well tolerated, with mild side effects such as burning, stinging, or transient photophobia. In the current study, OFX was incorporated into a chitosan-coated nanoemulsion to enhance ocular bioavailability, mucoadhesion, and therapeutic efficacy while reducing dosing frequency.

Materials
The study utilized Ofloxacin (Spectrum Lab), acetic acid (SD Fine), castor oil, olive oil, Tween 80, soya lecithin, chitosan, and other analytical-grade reagents. Instruments used included a bath sonicator, centrifuge, digital balance, FT-IR spectrometer, Franz diffusion cells, HPLC, and TEM.

Preformulation and Drug Authentication

Purity and identity of OFX were verified using UV spectrophotometry, FT-IR, melting point determination, solubility testing, and octanol–water partition coefficient assessment. UV spectra were recorded in methanol to determine λ_max, while FT-IR analysis employed the KBr pellet technique. Melting point was determined via the capillary method. Solubility studies in various solvents confirmed the drug’s suitability for nanoemulsion development.

Formulation Development

OFX nanoemulsions were formulated using ethyl oleate as the oil phase, Tween 80 as surfactant, and soya lecithin as cosurfactant. Pseudoternary phase diagrams were constructed to identify nanoemulsion regions and optimize Smix ratios. The drug was dissolved in the oil phase, mixed with Smix, and emulsified into the aqueous phase using vortexing followed by high-energy homogenization. Benzalkonium chloride was added as a preservative.

Optimization (Design of Experiments)

A Box–Behnken Design was implemented to optimize oil concentration (X?), Smix ratio (X?), and sonication time (X?). Responses measured included globule size (R?) and polydispersity index (R?). Response surface plots, contour plots, and desirability functions guided the identification of the optimal formulation. Validation was performed through checkpoint analysis comparing predicted and experimental outcomes.

Chitosan Coating

Chitosan-coated nanoemulsions were prepared by dropwise addition of 0.5% chitosan solution in acetate buffer (pH 5) to uncoated nanoemulsions under gentle stirring to ensure uniform coating.

In Vitro Characterization

Nanoemulsions were evaluated for particle size, polydispersity index, zeta potential, pH, viscosity, osmolarity, refractive index, drug content, and stability. Morphology was assessed via TEM. In vitro drug release was conducted using the dialysis bag method in PBS (pH 5.5) and analyzed using mathematical kinetic models. Mucoadhesion was evaluated viscometrically with mucin dispersions, and antimicrobial activity against E. coli and S. aureus was measured using the agar diffusion method, comparing results to commercial eye drops.

Stability Studies

Formulations were stored under refrigerated (5 ± 3?°C) and room temperature (25 ± 2?°C / 60 ± 5% RH) conditions for three months. Physicochemical properties and drug release profiles were periodically evaluated. Statistical analysis was performed using ANOVA, and results were reported as mean ± standard deviation (SD).

RESULTS AND DISCUSSION

Pre-formulation Studies

The UV absorption spectrum of Ofloxacin showed a sharp λmax at 294 nm, confirming the drug’s purity and suitability for quantitative analysis (Figure 5.1). This wavelength was used for further spectroscopic estimations.

FT-IR analysis revealed distinct peaks corresponding to functional groups present in Ofloxacin, including –OH/–NH stretching (~3430 cm?¹), quinolone ketone C=O (~1720 cm?¹), aromatic C=C (~1610 cm?¹), and C–F (~650 cm?¹). These observations confirmed the structural integrity of the drug.

Melting point determination showed a range of 223–228 °C, which was consistent with the reported standard (225–227 °C), further confirming drug identity. Solubility profiling revealed high solubility in methanol, DMSO, and water, while ethanol showed limited solubility. The experimentally determined partition coefficient (log P = –0.34 ± 0.05) closely matched the reported value (–0.39), indicating hydrophilic characteristics and suitability for ocular formulations.

Calibration curves constructed in methanol and simulated tear fluid (STF, pH 7.2) exhibited linearity within the concentration range of 2–12 µg/mL (R² > 0.99), ensuring reliability for drug quantification.

Screening of Excipients

Ethyl oleate was identified as the most suitable oil phase due to its solubilizing capacity for Ofloxacin and ocular safety profile. Tween 80 was chosen as the surfactant for its stabilizing efficiency, while soya lecithin was used as co-surfactant to enhance interfacial stabilization and impart negative charge before cationic chitosan coating.

Phase Behavior and Optimization

Pseudo-ternary phase diagrams were constructed to evaluate different Smix ratios. The surfactant-to-co-surfactant ratio of 2:1 exhibited the widest nanoemulsion region with excellent clarity and stability (Figure 6.8). This ratio was selected as the optimized composition for subsequent formulation development.

Formulation Development

Ofloxacin nanoemulsion was prepared by high-energy emulsification. Ethyl oleate was selected as the oil phase, Tween 80 as the surfactant, and soya lecithin as the co-surfactant. Pseudoternary phase diagrams confirmed a 2:1 Smix ratio produced the most stable nanoemulsion region.

The drug was dissolved in the oil phase, mixed with Smix, and dispersed into the aqueous phase under stirring. The coarse emulsion was reduced to nanoscale using homogenization and probe sonication. Benzalkonium chloride was added as a preservative.

For improved ocular retention, a chitosan coating (0.5% solution) was applied to the optimized formulation, imparting positive charge and mucoadhesive properties. The final nanoemulsion was stable, transparent, and suitable for ocular delivery.

Optimization Using Design of Experiments (DoE)

The Box–Behnken Design (BBD) was applied to optimize Ofloxacin nanoemulsion variables. Three factors—oil concentration, Smix ratio, and sonication time—were studied at three levels. The responses considered were globule size and polydispersity index (PDI), with the aim of producing stable nano-sized globules (<200 nm).

Seventeen experimental runs were generated, and response surface analysis confirmed a quadratic model fit for both responses. High R² values (>0.96) indicated strong predictive accuracy. Oil concentration increased droplet size, while Smix ratio reduced it due to interfacial stabilization. Sonication time further improved particle size uniformity.

For PDI, oil content decreased variability, whereas higher Smix levels slightly increased it. Sonication helped achieve narrower distribution. ANOVA confirmed the significance of interaction terms, with oil concentration emerging as the most influential factor.

Checkpoint validation showed good agreement between predicted and observed values, with bias <6%. The optimized formulation achieved a mean globule size of ~104 nm, a PDI of 0.16, and a desirability index of 0.88, meeting the set quality criteria.

This DoE-based optimization ensured precise control of droplet size and uniformity, enhancing the stability and therapeutic potential of the ocular nanoemulsion.

Table 1: Variables in BBD for the formulation

Table 2: Optimization by box-Behnken Design for formulation of Ofloxacin loaded nanoemulsion

(All values are reported as mean ± S.D., n=3)

Table 3: Results of regression analysis for responses R1and R2 for fitting to models

Table 4: Regression equation of the fitted model as per above models

Influence on Globule Size

Oil concentration significantly increased droplet size, whereas Smix reduced it by lowering interfacial tension and enhancing droplet stability. Sonication time further minimized size variation. Response surface and contour plots confirmed this trend, with ANOVA showing that oil concentration (p < 0.0001) was the most critical factor, followed by Smix and sonication. Quadratic terms also contributed significantly, indicating complex factor interactions.

Influence on PDI

Oil concentration had a negative influence on PDI, indicating improved uniformity, while higher Smix and co-surfactant ratios produced a slight increase in heterogeneity. Sonication reduced variability and helped achieve narrower distributions. ANOVA results confirmed strong quadratic effects, with oil concentration and Smix emerging as dominant contributors.

Checkpoint Analysis

Predicted and observed results were closely aligned, with percentage prediction error <6%, confirming the robustness of the optimization model. The optimized formulation (checkpoint batch) showed a mean globule size of 103.9 ± 2.24 nm, PDI of 0.161 ± 0.01, and a desirability of 0.88, validating the DoE-based approach.

Development & Characterization of Chitosan-Coated Nanoemulsion

Ofloxacin nanoemulsion was coated with chitosan to impart a positive surface charge. The cationic coating enhances electrostatic interactions with the negatively charged ocular mucosa, thereby improving retention and drug absorption.

Physicochemical Properties
Coating increased globule size, PDI, viscosity, osmolarity, and refractive index compared to the uncoated system, confirming successful deposition of chitosan. The zeta potential shifted from –21.32 mV to +31.13 mV, reflecting strong cationic surface charge. Both systems remained translucent, near-neutral in pH, and exhibited high drug content, indicating stability and ocular safety.

In Vitro Release and Kinetics
Drug release studies showed rapid release from plain drug, while nanoemulsions provided controlled release over 24 h. The chitosan coating further slowed release (82% in 24 h), following the Higuchi model, demonstrating diffusion-controlled sustained release suitable for prolonged ocular therapy.

Table5: In Vitro Characterization of Chitosan-Coated Nanoemulsion

In Vitro drug release study and Release Kinetics

Mucoadhesion Strength

Mucoadhesion significantly improved upon coating. Chitosan-coated nanoemulsion exhibited a strength of 7.6 F compared to 1.4 F for the uncoated system, consistent with the positive surface charge promoting stronger interaction with mucin.

Microscopy Analysis

Optical and TEM images confirmed uniform droplets with smooth morphology for both coated and uncoated systems, with visible size enlargement after chitosan adsorption. with the negatively charged ocular surface, improving residence time and drug absorption.

Antimicrobial Efficacy

The antimicrobial activity of chitosan-coated Ofloxacin nanoemulsion was evaluated against Pseudomonas aeruginosa and Staphylococcus aureus. Results showed that the formulation produced inhibition zones comparable to, or slightly higher than, marketed Ofloxacin eye drops. At all tested concentrations, the coated nanoemulsion demonstrated strong antibacterial action. For P. aeruginosa, inhibition zones increased from 2.23 cm at 1 µg/mL to 5.32 cm at 100 µg/mL, which were consistently larger than those of the control. Similarly, for S. aureus, the nanoemulsion showed zones of 1.59 cm, 2.76 cm, and 3.67 cm at respective concentrations, slightly exceeding marketed drops.

Table8: The diameter of the zone of inhibitions generated by nanoemulsion formulation and control formulation against both test organisms

These findings confirm that chitosan coating did not compromise the inherent antimicrobial potential of Ofloxacin. Instead, the modified formulation retained broad-spectrum efficacy with enhanced performance, making it a promising candidate for ocular infections caused by resistant bacterial strains.

Stability Studies

The stability of Ofloxacin-loaded chitosan-coated nanoemulsion was evaluated at refrigerated (5 ± 3 °C) and room temperature (25 ± 2 °C/60 ± 5% RH) conditions over three months.

At refrigerated storage, the formulation showed negligible changes in globule size, zeta potential, and drug content, with the physical appearance remaining homogenous throughout. In contrast, samples kept at room temperature exhibited a marked increase in globule size and a reduction in both zeta potential and residual drug content over time.

*Refrigerated (5 ± 3 ºC) **Room Temp (25 ± 2 ºC / 60 ± 5% RH)