Multivalent bacteriophage nanoemulsions enhance T4 phage gastrointestinal stability and retention for bactericidal...

Bacteriophages are promising antibacterial agents across the farm-to-fork continuum. Oral phage efficacy against infections, however, is limited by poor digestion tolerance. To enhance oral phage delivery, we synthesized T4 phage-chitosan conjugates and self-assembled them with phospholipids into protective nanoemulsions. We evaluated chitosan concentration effects on grafting efficiency, phage viability in simulated gastrointestinal (GI) conditions, and antibacterial activity. Nanoemulsions with moderate chitosan concentrations (50-100 μg/mL) optimally protected phages during gastric transit, rescuing ∼5 log₁₀ PFU/mL viability versus free phages. Higher concentrations impaired phage viability and release, while lower concentrations provided inadequate assembly. The optimized formulation (Phage-CL-50) showed superior efficacy against invasive bacteria and biofilms in intestinal epithelia, enabling recoverable transepithelial transport. In mice, Phage-CL-50 significantly enhanced GI phage retention and antibacterial performance. This nanoemulsion strategy offers a rational approach for developing robust oral phage formulations in food industry applications through targeted formulation optimization.