A highly efficient charge-driven method to encapsulate bacteriophages in liposomes

Liposome-encapsulated bacteriophages offer promising potential for targeted antimicrobial therapy against multidrug-resistant infections, by enhancing phage stability and delivery. Current encapsulation methods face challenges due to low efficiency (≤50 %), phage inactivation, and limited scalability for industrial production. To overcome these challenges, this study presents an electrostatically driven encapsulation method to produce phage liposomal formulations using cationic lipids (DOTAP) in various scalable micromixing systems (AXF™mini, confined impinging jet (CIJ), microfluidic chip). Encapsulation was evaluated by mixing a lipid-carrying organic stream (ethanol) with a phage-containing aqueous stream at flow rate ratios (FRR) of 3:1, 2:1, and 3:2 in AXF™mini, total flow rates (TFR) of 5-35 mL/min in CIJ, and a fixed FRR of 2:1 with a TFR of 3 mL/min in the microfluidic chip. The electrostatic interactions between the positively charged lipids and negatively charged phages significantly improved the encapsulation efficiency to 90-91 % for podovirus PEV31 (short-tailed) and myovirus PEV1 (long-tailed) while maintaining phage viability with < 0.2 log₁₀ reduction in titre. This encapsulation approach suited both phage morphotypes, producing uniformly sized liposomal phages (< 1000 nm, PDI < 0.3) with a slightly positive zeta potential. Structural observation using transmission electron microscopy and atomic force microscopy infrared spectroscopy confirmed phage encapsulation within intact vesicles and lipid-phage colocalization. This versatile formulation strategy addresses key challenges in phage therapy by achieving high encapsulation efficiency, accommodating diverse phage morphotypes, and preserving viability while offering potential scalability for industrial applications.