PEGylation Overcomes Pharmacological Barriers to Improve Systemic Pharmacokinetics and Therapeutic Efficacy of Phages against MDR <i>Escherichia coli</i>

Systemic bacteriophage therapy against multidrug-resistant (MDR) <i>Escherichia coli</i> is fundamentally limited by rapid immune-mediated clearance, complement activation, and phagocytic sequestration, collectively constituting pharmacological barriers that restrict systemic bioavailability, shorten circulation half-life, and attenuate therapeutic efficacy. We hypothesized that PEGylation, by sterically shielding phage capsids from host immune clearance mechanisms, would enhance systemic stability, improve pharmacokinetic (PK) behavior, and augment therapeutic efficacy <i>in vivo</i>. Four lytic <i>E. coli</i> phages were covalently conjugated with 5-kDa mPEG-S-NHS, achieving >60% surface amine modification as confirmed by fluorescamine assay. PEGylation resulted in a ~1.5-5 log₁₀ reduction in infectious titer and modestly slowed adsorption kinetics but preserved latent period and burst size, confirming intact replication competence. In serum, wild-type phages were undetectable within 24-48 h, whereas PEGylated phages retained ~2-3 log₁₀ PFU ml^-1 at 24 h and persisted longer within RAW264.7 macrophages and HT-29 epithelial cells. In mice, PEGylation markedly increased systemic exposure (AUC₀-∞ up to 50-fold), prolonged circulation, and reduced clearance >15-fold. In infected hosts, PEG-EC.W2-6 and PEG-EC. W15-4 achieved plasma titers up to 100-fold higher with >30-fold lower clearance, accelerating bacterial elimination (72 h vs 96 h). Despite partial IgG induction upon repeated dosing, PEGylated phages maintained superior PK and significantly suppressed infection-driven IL-6, IFN-γ, TNF-α, and IL-1β, normalizing cytokine profiles toward baseline. Overall, PEGylation markedly improves systemic persistence, intracellular stability, and immunomodulatory efficacy, representing a robust strategy to overcome PK barriers and optimize systemic phage therapy against MDR <i>E. coli</i>.