Stabilisation, nano-spectroscopic analysis and charge-driven liposomal encapsulation of bacteriophages for advanced therapeutic delivery

Abstract

The global antibiotic-resistance crisis has renewed interest in bacteriophage (phage) therapy as a targeted treatment for multidrug-resistant respiratory infections. However, phage stability during formulation, storage, and aerosol delivery remains a critical challenge. Conventional characterization methods often cannot detect subtle nanoscale changes affecting phage stability. To address this challenge, advanced nanocharacterisation techniques—including scattering scanning near-field optical microscopy (s-SNOM) and atomic force microscopy–infrared spectroscopy (AFM-IR)—were employed to probe individual phage particles and reveal molecular-level alterations. Using these tools, phage chemical heterogeneity was mapped and the impact of external stressors (organic solvents, heat, and pH shifts) on two distinct phage morphotypes was assessed.

Results show that phage stability depends on phage type and environmental conditions. For instance, myoviruses tolerated moderate organic solvent levels better than podoviruses, whereas severe heat or acid stress caused capsid damage and genome release, especially in short-tailed phages. Building on these insights, an electrostatically driven liposomal encapsulation method was developed to improve formulation efficiency.

Mixing cationic lipids with phage suspensions in scalable microfluidic systems produced uniformly nanosized liposome–phage formulations with ~90% encapsulation efficiency and preserved infectivity. Encapsulation also protected phages during nebulization, minimizing titer loss and enabling efficient lung delivery. Collectively, this work provides a framework—from molecular characterization to formulation and aerosol delivery—for enhancing phage stability and efficacy, advancing inhalable phage therapeutics against multidrug-resistant bacterial infections.