Response surface optimization of thermosensitive chitosan-polyvinyl alcohol hybrid hydrogels with enhanced betel leaf extract release for localized anti-inflammatory therapy.

Thermoresponsive biopolymer hydrogels offer a versatile platform for localized delivery of bioactive compounds; however, their performance is often limited by the competing requirements of network integrity, mechanical compliance, and stimulus-regulated release. Herein, thermosensitive chitosan/β-glycerophosphate/polyvinyl alcohol (CS/GP/PVA) hybrid hydrogels were optimized for the delivery of anti-inflammatory betel leaf extract (BLE) under in vitro conditions simulating an acute inflammatory microenvironment (pH 6.5 and 39 °C). Non-optimized hydrogels previously released only 61% of the loaded BLE, with negligible lysozyme-triggered release attributed to dense chitosan cross-linking that constrained enzyme-accessible transport pathways. Response surface methodology was applied to balance cumulative BLE release and elastic modulus. The optimized formulation (0.88% CS, 4.76% GP, 0.5% PVA, and 4000 µg mL-1 BLE) achieved a cumulative BLE release of 73.41%, corresponding to a 20% relative increase over the non-optimized hybrid hydrogel, and an elastic modulus of 1.54 kPa. The optimized hydrogel also displayed pronounced microenvironment-responsive release, including an eightfold higher BLE release at pH 6.5 than at pH 8.0 and lysozyme-triggered release within 24 hours, indicating that it restored pH- and enzyme-regulated transport. In cell-free assays, the hydrogel extract inhibited hyaluronidase activity, scavenged DPPH radicals, and suppressed Staphylococcus aureus growth. The hydrogel was cytocompatible and significantly suppressed nitric oxide production in lipopolysaccharide-stimulated macrophages, confirming that anti-inflammatory activity is retained in a cellular model. These findings establish the optimized hybrid hydrogel as a bio-functional, thermo-responsive network capable of coupling elasticity with bioactive extract release behavior, offering a promising candidate towards localized anti-inflammatory therapy.