Chitosan Stabilizes Curcumin against Hydrolysis and Potentiates Antimicrobial Activity against Pseudomonas aeruginosa in a pH-Dependent Manner

Abstract

Curcumin exhibits antimicrobial activity against Gram-negative bacteria but is limited by rapid hydrolytic degradation at near-neutral pH. We hypothesized that chitosan, a cationic polymer that independently destabilizes the Gram-negative outer membrane, could simultaneously stabilize curcumin and potentiate its antibacterial activity. Curcumin degradation kinetics in cation-adjusted Mueller–Hinton broth (35–85 °C) revealed a concentration-dependent transition in chitosan-mediated stabilization: low chitosan ratios (1:0.5–1:1) reduced degradation rates without altering activation energy (∼33–35 kJ/mol), consistent with kinetic shielding, whereas higher ratios (1:2–1:3) increased activation energy to 39.0–45.8 kJ/mol, indicating formation of a protective curcumin–chitosan complex. Complementary microplate kinetic spectroscopy at 35 °C across pH 6.6, 7.2, and 7.4 confirmed that chitosan-mediated stabilization is itself pH-dependent, extending curcumin’s half-life by up to 10.8-fold at pH 7.2 (1:2 ratio) and 5.9-fold at pH 6.6 (1:3 ratio). Checkerboard synergy assays against six Pseudomonas aeruginosa isolates revealed pH-dependent potentiation consistent with synergy (Bliss Independence model), with mean Bliss Δ shifting from +0.112 at pH 6.6 to −0.019 at pH 7.4, tracking the ionization of chitosan’s amine groups (pKa 6.2–6.5). At a 24-h end point, the 1:2 combination produced greater nucleic acid and protein leakage than either agent alone; however, a complementary short-duration kinetic assay (0–120 min) across three pH values indicated that this enhancement reflects chitosan-mediated stabilization extending curcumin’s effective exposure duration rather than direct mechanistic synergy at the membrane. Together, these results identify a composite mechanism in which chitosan protects curcumin from hydrolysis while both agents contribute pH-dependent membrane disruption, with maximal antibacterial potentiation in the pH range characteristic of the cystic fibrosis airway.