Bacterial conjunctivitis and keratitis are common eye infections treated with fluoroquinolones like besifloxacin (Besivance). However, topical ophthalmic therapies face challenges due to low bioavailability resulting from the eye's anatomy and physiology, which quickly remove formulations from the ocular surface, reducing drug residence time and absorption. Current commercial formulations like Besivance, while employing mucoadhesive systems to prolong contact time, can cause discomfort (high viscosity), blurred vision, and dose variability. To overcome these limitations, this research explored iontophoresis as a potential "emergency burst delivery" method, and liposomes as a delivery system. Iontophoresis can enhance drug permeation through passive diffusion, electromigration (movement of charged molecules in an electric field), and electroosmosis (solvent flow driven by an electric potential). Since besifloxacin has a near-neutral charge at physiological pH, this study hypothesized that imparting a positive charge to liposomes as nanocarriers would improve electromigration during iontophoresis, resulting in faster drug delivery. Furthermore, the study investigated whether a positive charge on liposomes would prolong formulation residence time, enhancing passive drug penetration. The use of liposomes themselves offers advantages over viscous gels, facilitating corneal transport and improving drug deposition in the ocular region without discomfort. A mucoadhesive characteristic, often achieved with positively charged polymers, is desirable to improve the formulation's resistance to tear flow, which usually results in increased viscosity and discomfort. In contrast, the addition of small positive molecules, such as spermine, may offer an alternative by imparting positive charge without significantly increasing viscosity. This study aimed to incorporate besifloxacin into liposomes containing amines as positively charged additives to evaluate their influence on ocular iontophoresis (as a burst delivery approach) and passive drug delivery, using a novel in vitro model simulating lacrimal flow to evaluate passive delivery.

The literature extensively covers the challenges of topical ophthalmic drug delivery due to rapid clearance mechanisms of the eye. Studies highlight the use of mucoadhesive polymers and iontophoresis to improve drug bioavailability. While many studies propose liposomes for ocular delivery, the use of positively charged additives in liposomes for enhancing both iontophoretic and passive ocular drug delivery, particularly for a molecule like besifloxacin with near-neutral charge at physiological pH, was relatively unexplored prior to this study. Existing literature on iontophoresis for cutaneous drug delivery shows ambiguous results regarding the advantages of charged nanocarriers. The incorporation of positively charged polymers into the formulation or coating nanosystems is a commonly used strategy in passive drug delivery to confer mucoadhesive characteristics. However, these polymers might increase the viscosity of the formulation. This research aimed to address this gap by investigating the effect of small positive molecules as an alternative for imparting a positive charge to liposomes without increasing viscosity.

The study involved preparing liposomal formulations of besifloxacin using the lipid film hydration technique. Various parameters were optimized to achieve high drug recovery (DR) and encapsulation efficiency (EE). These parameters included the drug solubilization medium (lipid or aqueous phase), the pH of the solubilization medium, the drug loading dose, the phospholipid concentration, and the addition of cationic amines (spermine and stearylamine). Two formulations, LP PC (phosphatidylcholine liposomes) and LP PC:SPM (phosphatidylcholine liposomes with spermine), exhibited the best DR and EE and were selected for further analysis. The formulations were characterized using dynamic light scattering for hydrodynamic diameter and polydispersity index (PdI), electrophoretic mobility for zeta potential, and transmission electron microscopy (TEM) for morphology. Mucoadhesiveness was assessed by measuring hydrodynamic diameter changes after mixing with mucin. In vitro drug release profiles were determined using Franz-type diffusion cells. Formulation stability was evaluated over 30 days at 6 °C by assessing visual aspects, hydrodynamic diameter, PdI, zeta potential, and EE. Electrical stability was evaluated by subjecting formulations to a 2 mA electric current for 30 min. Ocular tolerance was assessed using the hen's egg test-chorioallantoic membrane (HET-CAM) test. Differential scanning calorimetry (DSC) and electron paramagnetic resonance (EPR) spectroscopy were employed to investigate the interactions between liposome components and the effect of electric current on membrane fluidity. In vitro iontophoretic drug permeation experiments were performed using excised porcine corneas in Franz-modified diffusion cells with a 2 mA/cm² current density for 10 or 30 min. Passive permeation experiments were also conducted under static conditions. A novel in vitro model simulating lacrimal flow (20 µL/min PBS flow) was used to evaluate passive permeation through the entire porcine ocular globe. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using the microdilution method against *Staphylococcus epidermidis* and *Pseudomonas aeruginosa*. Statistical analysis included one-way ANOVA with Tukey's post hoc test or Student's t-test.

The optimized LP PC formulation achieved a DR of 93 ± 0.5% and an EE of >50%, while LP PC:SPM showed a DR of 68 ± 2.5% and an EE of 63 ± 1.86%. Both formulations had mean hydrodynamic diameters around 175 nm. LP PC had a negative zeta potential (-5.7 ± 0.3 mV), whereas LP PC:SPM was positively charged (+19.5 ± 1.0 mV). TEM images confirmed the unilamellar liposomal structure. LP PC:SPM demonstrated mucoadhesive characteristics. In vitro drug release was similar for both formulations. Both formulations remained stable at 6 °C for 30 days. Iontophoresis did not significantly alter liposome size or PdI, but it did affect zeta potential. The drug remained stable under iontophoresis. The HET-CAM test showed both formulations to be non-irritant. DSC analysis indicated interactions between liposome components. EPR spectroscopy showed that neither besifloxacin nor spermine significantly altered membrane fluidity. Iontophoresis increased besifloxacin permeation across porcine corneas for both formulations compared to passive permeation. However, the positively charged LP PC:SPM did not show a significant additional advantage over LP PC during iontophoresis. In the simulated lacrimal flow model, LP PC:SPM exhibited significantly higher passive corneal permeation than LP PC and the control (Besivance). MIC and MBC values for both liposomal formulations against *S. epidermidis* and *P. aeruginosa* were comparable or lower than those of Besivance.

The study successfully produced stable, non-irritant, and mucoadhesive liposomal formulations of besifloxacin. The positive charge imparted by spermine enhanced mucoadhesion, leading to improved passive drug delivery, particularly under simulated tear flow conditions. While iontophoresis increased drug permeation for both formulations, the added positive charge did not confer a further advantage in iontophoretic delivery, likely due to the complex interplay of factors influencing besifloxacin permeation (drug charge, liposome charge, passive diffusion). The superior performance of LP PC:SPM in passive delivery under simulated lacrimal flow highlights the importance of mucoadhesion in improving topical ophthalmic drug delivery. The comparable or superior antimicrobial activity of the liposomal formulations compared to Besivance suggests that controlled drug release from liposomes does not negatively impact efficacy. This enhanced efficacy might be linked to better interaction between the liposomal formulation and the bacteria, particularly for *P. aeruginosa* due to the interaction of the liposome with extracellular polymeric substances produced by the bacteria. This research demonstrates that positively charged liposomes, utilizing smaller positive molecules instead of polymers, provide a potential strategy for improving the efficacy and comfort of topical ophthalmic treatments.

This study demonstrated the successful incorporation of besifloxacin into stable, non-irritant, and mucoadhesive liposomes. While iontophoresis significantly enhanced drug delivery, the addition of positively charged spermine did not provide a further enhancement during iontophoretic delivery. However, the positively charged liposomes significantly improved passive drug delivery, especially under conditions mimicking the lacrimal flow. This approach offers a promising strategy for enhancing topical ophthalmic treatments by providing a stable, safe, and easily administrable formulation. Future research could focus on optimizing liposome composition for further enhancing delivery, investigating the mechanism of improved delivery, and exploring the potential of this approach for other ophthalmic drugs.

The study used an in vitro model, which may not perfectly replicate in vivo conditions. The in vitro model for passive permeation, while simulating lacrimal flow, still simplifies the complex dynamics of the ocular surface. The limited number of bacterial strains tested restricts the generalizability of the antimicrobial activity findings. While the HET-CAM test is a useful alternative to animal testing, it has limitations in terms of sensitivity and accuracy in predicting human eye irritation.