Besifloxacin liposomes with positively charged additives for an improved topical ocular delivery

Bacterial conjunctivitis and keratitis are common ocular infections treated primarily with fluoroquinolone antibiotics such as besifloxacin. Despite available topical therapies (e.g., besifloxacin 0.6% suspension, Besivance, employing a viscous mucoadhesive system), ocular anatomy and physiology rapidly clear instilled drugs, resulting in poor bioavailability and potential discomfort from viscous vehicles. The study explores two complementary strategies to enhance besifloxacin corneal delivery: (i) iontophoresis as a rapid, clinic-applied burst delivery to quickly achieve therapeutic levels at the infection site; and (ii) passive delivery using liposomes engineered with a positive surface charge to increase mucoadhesion and residence time without increasing viscosity. The central hypotheses are that adding positive charge to liposomes may improve electromigration during iontophoresis, and that positive charge may prolong precorneal residence to enhance passive penetration, particularly under conditions simulating lacrimal flow.

Prior work highlights limitations of topical ocular delivery due to tear turnover and ocular barriers; polymeric systems like DuraSite can prolong contact but at the expense of high viscosity, discomfort, blurred vision, and dose variability. Iontophoresis can enhance ocular delivery via electromigration and electroosmosis, with the former being most relevant for charged molecules; however, besifloxacin is near neutral at physiological pH. Imparting charge to nanocarriers to exploit electromigration has shown mixed results in cutaneous models and had not been applied for ocular delivery. Liposomes are widely studied ocular carriers that can improve drug interaction with the corneal surface and penetration without high viscosity. Positive surface charge (via cationic polymers or coatings) can confer mucoadhesion, improving resistance to tear clearance, but often increases viscosity. Small cationic molecules (e.g., polyamines like spermine) offer a way to impart positive charge while maintaining low viscosity; their ocular application and biocompatibility have been reported, though this strategy had not been explored for ocular drug delivery of besifloxacin.

Materials included besifloxacin, phosphatidylcholine (PC), cholesterol, spermine (SPM), stearylamine (SA), buffers, and analytical reagents. Ex vivo porcine eyes were obtained post-mortem for permeation studies. - Drug quantification: HPLC-DAD at 340 nm using a C8 column, isocratic 0.01% phosphoric acid:methanol (60:40 v/v), 1.2 mL/min, 45 °C, 10 µL injection. Linear range 6.0–60.0 µg/mL (r=0.9999); LOD 1 µg/mL; LOQ 2 µg/mL; precision CV ≤5%; validated against ocular matrices with recoveries: cornea 90.6±5.9%, vitreous 85.6±11.2%, aqueous humor 93.9±3.1%. - Liposome preparation (lipid film hydration and extrusion through 200 nm membranes): Systematically varied (i) drug solubilization phase (lipid vs aqueous), (ii) solubilization pH (acidic pH 3 vs neutral), (iii) drug loading dose (1–4 mg), (iv) PC concentration and cholesterol ratios, and (v) cationic amines (SPM or SA) at PC:amine molar ratios 3:1 and 5:3 with drug solubilized in basic pH (11.6). Two optimized formulations selected: LP PC (PC only; drug solubilized in acidic phase; final pH 6.79) and LP PC:SPM (PC with spermine; drug solubilized in basic phase; final pH 9.70). - Characterization: Dynamic light scattering for hydrodynamic diameter and PdI; electrophoretic mobility for zeta potential. Encapsulation efficiency (EE%) determined by centrifugal filtration (10 kDa MWCO) to quantify free drug (FD) and total drug (TD) after liposome disruption with Triton X-100; EE%=(TD−FD)/TD×100; drug recovery (DR%)=TD/AD×100. TEM imaging assessed morphology. Mucoadhesion tested by measuring size shifts upon mixing with mucin (1% w/v) dispersion. In vitro release across cellulose membranes in Franz cells (donor 400 µL, acceptor 15 mL HEPES pH 7.4, 600 rpm) sampled up to 120 min. - Stability: Stored at 6 °C and monitored at days 1, 7, 14, 30 for size, PdI, zeta potential, and EE. Electrical stability: exposure to 2 mA for 30 min; measured size, PdI, zeta, DR, EE before/after; evaluated drug stability in diluted Besivance as well. EPR spectroscopy (Bruker EMX, X-band) with TEMPO and 5-DSA spin probes assessed membrane dynamics and effects of current (0–4 mA). DSC analyzed thermal behavior of components and mixtures. - Ocular tolerance (HET-CAM): CAM exposure to formulations (300 µL, 30 s), rinsed, observed 5 min; PBS negative control, 1 M NaOH positive control. - In vitro iontophoresis (ex vivo cornea): Porcine corneas in modified Franz cells (1.32 cm²). Donor 400 µL of LP PC, LP PC:SPM, or Besivance diluted to 500 µg/mL; acceptor 15 mL HEPES pH 7.4, 600 rpm. Anodal iontophoresis at 2 mA/cm² for 10 or 30 min via salt bridge to silver anode; cathode in acceptor. Passive counterparts run without current. After 30 min, acceptor sampled; corneas extracted for drug quantification. - Passive permeation with simulated lacrimal flow: Whole porcine globes mounted in a custom setup with donor cell and peristaltic pump delivering PBS at 20 µL/min over the corneal surface to mimic tear flow. Donor 400 µL of each formulation (500 µg/mL). After 30 min, cornea, aqueous, and vitreous collected for drug analysis. - Antimicrobial testing: MIC and MBC against Staphylococcus epidermidis ATCC 14990 and Pseudomonas aeruginosa ATCC 2327 by broth microdilution (final inoculum 5×10⁵ CFU/mL; treatment range 2.0×10¹ to 9.0×10⁻³ µg/mL). Treatments: free besifloxacin (pH 11 HEPES), LP PC, LP PC:SPM, Besivance, vehicle controls. Resazurin used for growth assessment; MBC by subculture of clear wells. - Statistics: Mean±SD, n≥3; one-way ANOVA with Tukey’s post hoc or Student’s t-test; α=0.05.

- Optimized liposomes: LP PC (neutral/slightly negative) and LP PC:SPM (positively charged) showed high drug recovery and encapsulation. LP PC: DR 93.0±0.5%, EE 51.8±1.9%, final pH 6.79; LP PC:SPM: DR 68.0±2.5%, EE 63.3±1.8%, final pH 9.70. - Physicochemical properties: LP PC size 177.2±2.7 nm, zeta −5.7±0.3 mV, PdI 0.02±0.01; LP PC:SPM size 175.4±1.9 nm, zeta +19.5±1.0 mV, PdI 0.071±0.032. TEM showed unilamellar vesicles ≈200 nm. - Mucoadhesion: LP PC:SPM exhibited significant size increase upon mixing with mucin, indicating mucoadhesive interactions; LP PC did not. - Release: Similar controlled release from both liposomes; e.g., at 30 min ≈9–10% released (LP PC 9.10±1.29%; LP PC:SPM 8.49±1.06%). - Stability: At 6 °C for 30 days, no significant changes in size, PdI, EE for both formulations; zeta potential shift observed for LP PC:SPM. Under 2 mA for 30 min, sizes/PdI unchanged; small EE decrease only for LP PC; DR unchanged, indicating drug and carrier electrical stability. - EPR/DSC: DSC showed SPM melting shifted from 33.1 °C to 57.0 °C in mixtures, evidencing interaction with lipids. EPR with 5-DSA showed no significant changes in bilayer fluidity (2A||) upon adding besifloxacin or SPM or applying current up to 4 mA. TEMPO partitioning into membranes increased with current intensity, more pronounced for SPM-containing systems, indicating interfacial reorganization without compromising bilayer order. - Ocular tolerance: HET-CAM classified LP PC, LP PC:SPM, and Besivance as non-irritant (similar to PBS; positive control NaOH caused hyperemia/hemorrhage). - Iontophoresis (ex vivo cornea): Current at 2 mA/cm² enhanced corneal drug retention for all formulations vs passive. After 30 min, absolute retained amounts: LP PC 6.95±0.83 µg/cm²; LP PC:SPM 6.94±0.83 µg/cm²; Besivance 4.02±0.25 µg/cm². This represented ≈65% increase for liposomes vs ≈23% for Besivance relative to passive. As % of applied dose: passive LP PC 2.99±0.17% vs LP PC:SPM 4.12±0.43%; iontophoresis 30 min LP PC 4.90±0.67% vs LP PC:SPM 6.82±0.96% (≈40% higher for SPM liposomes), mirroring passive differences; therefore, positive charge did not confer additional iontophoretic advantage beyond baseline formulation differences. - Passive permeation with simulated lacrimal flow (20 µL/min): Only LP PC:SPM significantly outperformed control and LP PC: corneal retention 4.26±0.74% vs control 2.29±0.15% and LP PC 2.65±0.34% (p<0.002 and p<0.008, respectively). No drug detected in aqueous or vitreous at 30 min. - Antimicrobial activity: For S. epidermidis, MIC=MBC=0.156 µg/mL for all besifloxacin treatments (free, liposomal, Besivance). For P. aeruginosa, MIC=MBC=0.625 µg/mL for free besifloxacin, LP PC, and LP PC:SPM; Besivance had higher MIC=MBC=1.25 µg/mL. Vehicle controls showed no antibacterial effect.

The study tested whether adding positive charge to besifloxacin-loaded liposomes enhances electromigration during iontophoresis and increases residence time for passive delivery. Under iontophoresis, all formulations benefited, but LP PC:SPM did not confer additional enhancement over LP PC beyond their baseline difference, indicating that carrier charge did not significantly augment electromigration-driven delivery for besifloxacin. This likely reflects concurrent mechanisms: electromigration of charged liposomal carriers vs opposing movement of differently charged drug species at the formulation pH, and significant contribution of the free drug fraction to passive diffusion. EPR and stability data corroborate that the electric current perturbs the interfacial environment without compromising bilayer order, supporting safe application of iontophoresis for liposomal systems. In contrast, for passive delivery, especially under simulated tear flow, the positively charged LP PC:SPM significantly improved corneal deposition compared to neutral liposomes and the commercial suspension. The gain is attributed to enhanced mucoadhesion and resistance to precorneal clearance, and potentially to membrane interactions from polyamines that can modestly facilitate tissue permeation. Tolerance testing confirmed non-irritancy, and antimicrobial assays showed liposomal besifloxacin maintained full activity; notably, for P. aeruginosa, liposomal formulations achieved lower MIC/MBC than the diluted commercial product, possibly due to differences in vehicle viscosity and drug release. Collectively, findings support positively charged liposomes as advantageous for passive ocular delivery while iontophoresis is effective as a burst-delivery strategy irrespective of carrier charge for this drug.

Besifloxacin was successfully loaded into stable, non-irritant liposomes with and without a positive charge additive (spermine). Positively charged LP PC:SPM exhibited superior passive corneal delivery, including under a simulated lacrimal flow, due to enhanced mucoadhesion, without increasing formulation viscosity. Iontophoresis at 2 mA/cm² significantly increased corneal deposition from all formulations, but adding positive charge to liposomes did not yield additional iontophoretic benefit for besifloxacin. Liposomal besifloxacin retained antimicrobial efficacy, with improved in vitro performance versus the commercial suspension against P. aeruginosa. Future work could assess in vivo performance and tolerability, optimize charge density and formulation pH, explore other cationic additives, and evaluate broader pathogen panels and dosing regimens.

Experiments were conducted ex vivo using porcine corneas and whole globes; in vivo ocular dynamics, blinking, and tear turnover may differ. The simulated lacrimal flow model used a scaled flow rate and a 30-minute window, which may not fully capture clinical conditions. Only one polyamine (spermine) and two bacteria species were tested. Iontophoresis parameters were limited to 2 mA/cm² and short durations. Long-term stability was assessed for 30 days at 6 °C; broader storage conditions were not evaluated. The need for trained personnel and specialized equipment for iontophoresis is a practical constraint.