Minimizing phage stress in poly lactic-co-glycolic acid (PLGA) encapsulation and simplifying production with three-fluid nozzle spray drying

Bacteriophages (phages) have emerged as an alternative to combat multidrug resistant (MDR) infections. Formulating phages with polymers such as poly lactic-co-glycolic acid (PLGA) were investigated to improve retention in the body, thereby enabling prophylactic use of phage or potentially improving therapeutic outcome. A key challenge lies in phage sensitivity to organic solvents and agitation during emulsion-based processing. To investigate the possibility of employing a three-fluid nozzle spray drying technique, which separates the aqueous phage suspension and polymeric organic phase until mixing at the nozzle tip—minimizing phage exposure to organic solvent. Two anti- Pseudomonas aeruginosa phages (PEV2 or PEV61) were formulated with PLGA, using trehalose as a phage stabilizer. We investigated formulations with various trehalose: PLGA mass ratios (5:1, 1:1, 1:5) in terms of phage viability and stability, formulation characterizations, release, and bacterial killing efficacy. All powder formulations achieved ≤ 1.5 log-PFU/mg titer loss in phage viability (phage loads ranging from 3.42 × 10 6 to 4.49 × 10 7 PFU/mg) after three-fluid nozzle spray drying. During 3-month storage, titer loss ranged from 0.24–1.16 log-PFU/mg at 4°C and 3.45–5.59 log-PFU/mg at 25°C, with higher trehalose ratios providing better stability. The release for all PEV61 formulations reached around 10 7 PFU/mg after 2 hours, with a higher initial burst associated with lower PLGA mass ratio. Phage-PLGA particles significantly inhibited bacterial growth over 24 hours. Three-fluid nozzle spray drying offers a viable and simplified strategy for producing stable phage-PLGA powders. The improved bacterial killing indicated a promising potential of using phage-PLGA powder for treating MDR bacterial infections.