(M1430-03-18) Developing a Liposomal Ciprofloxacin Formulation Using Passive and Active Loading Strategies for Enhanced Ocular Delivery in Bacterial Endophthalmitis

Purpose: Bacterial endophthalmitis (BE) is a bacterial infection of the eye that results in inflammatory reaction of the intraocular fluids or tissues. Despite treatment, it often results in vision loss. FDA-approved ciprofloxacin (CIP) eye drops are commonly used as a preventive measure for endogenous bacterial endophthalmitis.[1] CIP ophthalmic solution has been utilized to treat various ocular bacterial infections, including allergic and bacterial conjunctivitis, endophthalmitis, corneal keratitis, and corneal ulcers.[2] CIP is a fluoroquinolone antibiotic with broad-spectrum activity against aerobic Gram-positive and Gram-negative bacteria. CIP has low toxicity and slow resistance development, but its poor ocular bioavailability limits efficacy, requiring frequent dosing, reducing compliance.[3] CIP is water-soluble (35 mg/mL), but its solubility drops at tear fluid pH, causing precipitation and reduced ocular bioavailability, which limits therapeutic efficacy. There is a critical need for novel delivery system to enhance the bioavailability of commercial CIP ophthalmic solutions and reduce frequent dosing.[4] This study aims to develop and characterize CIP-loaded liposomal nanoparticles to enhance therapeutic efficacy by increasing ocular retention and enabling sustained drug release over four days. Encapsulation is expected to protect CIP from enzymatic degradation at the corneal surface and ensure gradual release via lipid bilayer erosion. Different CIP forms were evaluated to identify the optimal candidate, addressing bioavailability limitations of conventional eye drops like Ciloxan®.

Methods: UPLC Method Development and Validation for Ciprofloxacin Quantification: A reverse-phase UPLC method was developed and validated according to USP and ICH guidelines for quantifying ciprofloxacin and its HCl salt, supporting applications such as encapsulation efficiency and in vitro release. Chromatographic separation was achieved using a Phenomenex® C18 column (with a column temperature 40 °C) and a mobile phase consisted of phosphate buffer: acetonitrile (87:13, apparent pH 3) at 1 mL/min, with an UV detection at 278 nm. Validation included specificity, linearity, intra- and inter-precision. Liposome Formulation and Physicochemical Characterization: Liposomes composed of DPPC and DC-Cholesterol (1:1 molar ratio) were prepared using the thin-film hydration method, followed by hydration with phosphate buffer containing EDTA and trehalose. The suspension was probe-sonicated to obtain uniform nanosized vesicles. Stability was assessed in liquid form, aligning with the intended ocular use despite exploring lyophilization. Particle size, polydispersity, and zeta potential were evaluated using DLS to ensure colloidal stability for ophthalmic delivery. Drug Loading Strategies for Ciprofloxacin Encapsulation: Passive loading was performed by incorporating ciprofloxacin in separate phases based on solubility but yielded limited encapsulation efficiency as reported in literature. To enhance loading, an active method was employed wherein liposomes were hydrated with ammonium sulphate buffer (pH 6.5), followed by sonication, homogenization, and pH gradient formation via dialysis against PBS (pH 7.4). This enabled protonated CIP HCl to diffuse into vesicles and precipitate via ion trapping. Active loading was carried out in an incubator shaker at 60 °C for 1 hour. Physical Stability Assessment and Lipid Distribution Analysis: Formulations were stored at 4 °C, and physical stability was evaluated by monitoring particle size, PDI, and zeta potential. For encapsulation efficiency and lipid distribution, liposomes were ultracentrifuged at 100,000×g for 1 h at 4 °C. Supernatant and redispersed pellets were separately analysed. Lipids were extracted, saponified, and quantified using GC-MS. Higher supernatant signals indicated greater phospholipid presence. In Vitro Cytotoxicity

Assessment: Cytotoxicity of blank liposomes with varying DPPC: DC-Chol ratios was evaluated on ARPE-19 cells using the resazurin assay at 48 and 72 hours

Results: The developed UPLC method showed high linearity (R² > 0.99) and specificity for both ciprofloxacin and CIP HCl, with retention times of 8.26 ± 0.10 min and 7.99 ± 0.09 min, respectively (Figure 1). Liposomal formulations exhibited an average particle size of ~150-200 nm, PDI < 0.3, and zeta potential values confirming good colloidal stability (Figure 2A). Passive loading resulted in moderate encapsulation efficiency, with CIP HCl demonstrating slightly higher entrapment and mass transfer than the free base (Figure 2B). No significant changes in particle size, PDI, or zeta potential were observed over 15 days of storage at 4 °C, indicating good physical stability (Figures 2C–E). Cytotoxicity assessment on ARPE-19 cells showed >85% viability at all tested lipid concentrations at 48 and 72 hours (Figure 3), confirming biocompatibility and guiding lipid level selection for active loading. These findings support continued development of the active loading approach for enhanced encapsulation.

Conclusion: The developed UPLC method reliably quantified ciprofloxacin and CIP HCl with excellent linearity and distinct retention times. Liposomal formulations exhibited desirable nanoscale size, low PDI, and stable zeta potential, with no significant changes during storage at 4 °C. Passive loading achieved moderate encapsulation efficiency, with CIP HCl performing slightly better than the free base. High cell viability ( >85%) across lipid concentrations confirmed biocompatibility and supported safe lipid levels for active loading. These results validate the formulation platform and underscore the need for active loading strategies to improve encapsulation efficiency and enable sustained drug release for ocular therapy.

References: [1] Bertino 2009 Impact of antibiotic resistance in the management of ocular infections: the role of current and future antibiotics OPTH 507
[2] Callegan M, Gilmore M, Gregory M, Ramadan R, Wiskur B, Moyer A, Hunt J and Novosad B 2007 Bacterial endophthalmitis: Therapeutic challenges and host–pathogen interactions Progress in Retinal and Eye Research 26 189–203
[3] Davis R, Markham A and Balfour J A 1996 Ciprofloxacin: An Updated Review of its Pharmacology, Therapeutic Efficacy and Tolerability Drugs 51 1019–74
[4] Hyndiuk R A, Eiferman R A, Caldwell D R, Rosenwasser G O, Santos C I, Katz H R, Badrinath S S, Reddy M K, Adenis J-P, Klauss V, Adenis J P, Badrinath S S, Caldwell D R, Cohen E, Marechal-Courtois C, Murta J, Darrell R W, Denis P, Eiferman R A, Heidemann D H, Hyndiuk R A, Insler M S, John T, Katz H R, Klauss V, Lass J H, Limberg M B, McCulley J P, Reddy M K, Rosenwasser G O D, Rowsey J J, Santos C, Sher N, Ullman S, Verin P, Wilhelmus K R, Wills R and Wolf T C 1996 Comparison of Ciprofloxacin Ophthalmic Solution 0.3% to Fortified Tobramycin-Cefazolin in Treating Bacterial Corneal Ulcers Ophthalmology 103 1854–63

Acknowledgements: The authors gratefully acknowledge Dr. Erik Haas, Associate Professor, Department of Chemistry and Biochemistry, College of Arts and Sciences, for his valuable support in lipid quantification using GC-MS. We also thank the School of Pharmacy and Health Professions at Creighton University for their continued collaboration.

Funding: This work was financially supported by funding from Health Science Strategic Investment Fund & the Department of Pharmacy Sciences, Creighton University. Conflict of Interest: The authors declare no conflicts of interest. No financial support or relationships influenced the design, execution, or outcomes of this study.

Figure 1: Specifications and parameters of the method developed.

Figure 2: A) Characterization of Liposomal Formulations (Blank, Passive Loading=PL & Active Loading= AL), B) Mass Transfer, Loading Capacity and Entrapment Efficiency of CIP free base and CIP HCl using Passive Loading (PL) method in 1:10 ratio of drug: lipid, C) D) & E) Physical Stability of Liquid Liposomal Formulations at 4°C, over 15 days