Liposomal Encapsulation of Two Novel Lytic Bacteriophages targeting Klebsiella pneumoniae: Preliminary Ex Vivo Evaluation of a Protective Delivery Platform for Pulmonary Infections

Multidrug resistance, exacerbated by inappropriate antibiotic use and limited development of new antimicrobials, represents a critical global health challenge. Klebsiella pneumoniae, characterized by its protective capsule and strong colonization capacity, is a major cause of hospital-acquired pneumonia, emphasizing the urgency of alternative therapeutic strategies. Phage therapy has emerged as a promising option, and a novel “Trojan Horse” approach using liposome-encapsulated phages (LEP) may enhance treatment by enabling phage delivery while evading immune detection. This study investigated the encapsulation of a lytic bacteriophage cocktail within liposomes to target multidrug-resistant K. pneumoniae. LEP presented a negative Zeta potential similar to that of K. pneumoniae cells, suggesting potential electrostatic repulsion between LEP and bacterial surfaces. Antimicrobial susceptibility testing confirmed that the evaluated strain was sensitive only to amikacin, gentamicin, and tetracycline. Physicochemical characterization showed that LEP exhibited a 21% smaller diffusion coefficient than empty liposomes, consistent with the increased size of the loaded structures. In vitro, LEP demonstrated sustained phage release and effectively prevented bacterial regrowth after 9 hours of treatment. Ex vivo assays using artificially contaminated canine lung tissue revealed that LEP achieved a maximal bacterial reduction of 1.04 log CFU/mL after 12 hours, markedly outperforming free phages, which allowed bacterial resurgence after 9 hours. The limited phage diffusion within the solid lung tissue matrix likely reduced phage-bacterium interactions, indicating that higher multiplicity of infection may be required to enhance therapeutic efficacy in structured biological environments.