Optimization of Antibiotic-Loaded Liposomes for Inhalation to Improve Lung Infection Treatment

Abstract

Cystic fibrosis (CF) and bronchiectasis (BE) are chronic respiratory disorders marked by airway inflammation, mucus obstruction, and recurrent bacterial infections that worsen disease progression. Oral antibiotics, the standard treatment for CF and BE infections, can lead to systemic side effects and antibiotic resistance. Pulmonary delivery of antibiotics offers a localized approach to minimize these issues. Also, inhaled antimicrobial liposomal formulations could offer several advantages over systemic administration, such as enhancing drug concentration directly in the airways, minimizing systemic side effects, and enabling prolonged drug release. This project focuses on manufacturing and optimization of formulations for the pulmonary delivery of apramycin-loaded liposomes in dry powder form with desirable physicochemical characteristics such as: size (< 150 nm), zeta potential (> 50 mV), polydispersity index (PDI) (≤ 0.3), encapsulation efficiency (EE) (>50% ), powder particle sizes (≤ 5 μm), glass transition (Tg) (>50 °C) and water content (≤ 3%) of the final dried powder apramycin products. Initially, Spray-drying parameters were optimized to produce liposomal formulations with small particle size (<150 nm), narrow size distribution (PDI ≤ 0.3), high EE > 50%, positive zeta potential (>30 mV), and respirable particle size (≤5 μm). Subsequently, the composition of the liposomes was optimized to develop an affordable and effective dry powder formulation for pulmonary delivery. The results show that formulation containing D-α tocopherol polyethylene glycol 1000 succinate (TPGS) consistently demonstrated better size and EE compared to those with poloxamer 407, which showed significant size increases post-spray drying. The formulations containing dimethyldioctadecylammonium bromide (DDAB) and TPGS exhibited desirable properties. Stability testing showed that the product remained stable for 24 weeks at 20°C but degraded at higher temperatures of 40 °C with 75 % humidity. Both formulations demonstrated comparable antibacterial efficacy against clinical isolates of Mycobacterium avium complex (MAC) and Pseudomonas aeruginosa (P. aeruginosa), maintaining consistent antimicrobial activity across the isolates. Additionally, the liposomal formulations showed minimal cytotoxicity, with over 80% cell viability at all tested concentrations, confirming their biocompatibility. Reducing the DDAB content to half its amount improved cost-effectiveness while maintaining liposome stability. A freeze-dried formulation was also developed to deliver the liposomes hydrated with apramycin for nebulization. Freeze-drying, with trehalose as a cryoprotectant, minimized water content, preserved a high Tg > 50°C, and enhanced long-term stability. Stability testing of freeze-dried formulations rehydrated into liquid form showed that those stored at 2°C exhibited a low degree of aggregation and performed better than those stored at 20°C. Transitioning from liquid to powder form further improved stability, reducing risks of aggregation and drug leakage. Powder formulations stored at both 2°C and 20°C demonstrated significantly enhanced stability, underscoring their suitability for pulmonary delivery. Finally, this work has demonstrated that inhalable liposomal antibiotic formulations could potentially serve as a novel therapeutic approach for treating lung infections associated with respiratory diseases.