Design and evaluation of ciprofloxacin hydrophobic ion pairing liposomal nanosuspension for ocular drug delivery system

다국어 초록 (Multilingual Abstract)

The aim of this study was to design a novel liposome-based ocular drug delivery system containing ciprofloxacin (CIP) for the treatment of bacterial ocular infections such as keratitis. To enhance encapsulation efficiency within the liposomes, Ciprofloxacin-pamoate (CP) hydrophobic ion pair (HIP) was first prepared. Based on foundational research focused on the characterization of the CP HIP complex, the optimal molar ratio of CP ion pair binding was determined. This study investigates the development of a new formulation of pegylated liposomes encapsulating the CP HIP complex for ocular drug delivery. The CP HIP-loaded pegylated liposome (CPPL) appeared as spherical particles under TEM, with a uniform particle size of 72.46 ± 1.29 nm, a PDI of 19.50 ± 2.12%, and a zeta potential of -21.23 ± 0.76 mV. The encapsulation efficiency (EE) of CPPL was significantly enhanced to 73.43 ± 0.99%, more than three times higher than that of ciprofloxacin-loaded pegylated liposomes (CL). This improvement was attributed to the increased hydrophobicity due to the use of DSPE-PEG 2000 and the HIP, resulting in enhanced encapsulation of the drug (CP) within the liposomal bilayer. CPPL demonstrated stable physicochemical properties over 4 weeks at 4°C, 25°C, and 40°C. In vitro release studies showed a sustained release profile, with less than 80% of the drug released over 72 hours, indicating controlled release characteristics. CPPL also exhibited excellent penetration and antibiofilm activity against Pseudomonas aeruginosa biofilms, demonstrating superior antimicrobial effects in various microbial assays. These results suggest that CPPL has the potential to be a promising formulation for the treatment of bacterial ocular infections, overcoming the limitations of current liposomal drug formulations.

국문 초록 (Abstract)

이 연구의 목적은 각막염과 같은 세균성 안구 질환 감염 치료를 위한 ciprofloxacin (CIP)을 함유한 새로운 리포좀 기반 안과 약물 전달 시스템을 설계하는 것이었다. 리포좀 내에서의 봉입 효율을 향상시키기 위해, 먼저 Ciprofloxaicn-pamoate (CP) 소수성 이온쌍(HIP)을 준비하였다. CP HIP의 최적 복합화 효율을 나타내는 결합 몰 비율을 선정하고 특징화에 중점을 둔 기초 연구를 바탕으로, CP HIP를 봉입한 페길화된 리포좀의 새로운 제형 개발 응용연구를 진행하였다. CP HIP를 함유한 페길화된 리포좀 (CPPL)은 TEM 분석에서 구형 입자로 났으며, PDI 19.50 ± 2.12 %으로, 72.46 ± 1.29 nm의 균일한 입자크기와 제타전위 -21.23 ± 0.76 mV를 보였다. CPPL의 봉입 효율(EE)은 73.43 ± 0.99%로, CIP을 함유한 페길화 리포좀(CL)보다 3배 이상 증가했다. 이러한 개선은 DSPE-PEG 2000과 HIP 사용으로 인한 소수성 증가로, 리포좀 이중층 내 약물(CP) 봉입 향상의 결과로 해석된다. CPPL은 4 25 40 에서 4주 동안 안정적인 물리화학적 성질을 유지하였다. In vitro 방출 연구 결과, 72시간 동안 약 80 % 미만의 약물이 방출되어 지속적인 방출 프로파일 보였으며, 이는 조절 방출 특성을 나타낸다. CPPL은 또한 녹농균 바이오필름에 대해 우수한 침투력과 항바이오필름 활성을 보였으며, 다양한 미생물 시험에서 탁월한 향균 효과를 나타냈다. 이러한 결과는 CPPL이 세균성 안구 질환 치료를 위한 유망한 제형이 될 가능성을 시사하며, 현재의 리포좀 약물 제형의 한계를 극복할 수 있는 잠재력을 지니고 있음을 보여준다.

목차 (Table of Contents)

• 1. Introduction 1
• 2. Materials and Methods 5
• 2.1. Materials 5
• 2.2. High-Performance Liquid Chromatography (HPLC) Method for Ciprofloxacin Analysis 6
• 2.3. Preparation of Ciprofloxacin-Pamoate (CP) Hydrophobic Ion Pair (HIP) complex 8
• 2.3.1. Solubility of CP HIP complex 9
• 2.4. Preparation of Liposomal HIP: Ciprofloxacin-Pamoate Hydrophobic Ion Pair complex-loaded PEGylated Liposome (CPPL) 10
• 2.5. Physicochemical Characterization of Liposomal HIP 13
• 2.5.1. Measurement of Particle Size (PS), Polydispersity Index (PDI) and Zeta Potential (ZP) 13
• 2.5.2. Encapsulation Efficiency (EE) 13
• 2.5.3. Morphology 15
• 2.5.4. In Vitro Drug Release from Liposome 15
• 2.6. Study of Antibacterial Activity 16
• 2.6.1. Bacterial Strain and Culture Conditions 16
• 2.6.2. In Vitro Disk Diffusion 17
• 2.6.3. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Assay 20
• 2.6.4. In Vitro Antibiofilm Activity Assay 21
• 2.7. Stability of CPPL 22
• 2.8. Statistical Analysis 23
• 3. Results and Discussion 24
• 3.1. Solubility of CP HIP complex 24
• 3.2. Physicochemical Characterization of Liposomal HIP 26
• 3.2.1. Characterization of CL, CPL and CPPL 26
• 3.2.2. Evaluation of Encapsulation Efficiency 29
• 3.2.3. Morphology 32
• 3.2.4. In Vitro CIP Release from Liposome 34
• 3.3. Determination of Antibacterial Activity 37
• 3.3.1. Confirmation of Bacterial Susceptibility 37
• 3.3.2. Determination of MIC and MBC Assay 39
• 3.3.3. Assessment of Antibiofilm Activity Assay 46
• 3.4. Assessment of CPPL Stability 50
• 4. Conclusion 53
• 5. Reference 54
• 6. Abstract in Korean 58