Post-Synthetic Surface Quaternization of Agarose Cryogels: Optimizing Structural Integrity and Broad-Spectrum Growth Inhibition

Abstract

Agarose is a promising biopolymer for wound healing due to its biocompatibility and ability to form stable macroporous cryogels. However, its bioinert nature limits its antibacterial functionality, while conventional chemical modification can disrupt its macroporous architecture and mechanical integrity. Here, we present a versatile post-synthetic heterogeneous surface quaternization strategy to transform prefabricated agarose cryogels into surface-modified agarose cryogels (SMACs) with contact-active antibacterial functionality while preserving their interconnected supermacroporous architecture. Using 3-chloro-2-hydroxypropyltrimethylammonium chloride (CHPTAC) in an alkaline environment, quaternary ammonium moieties were covalently immobilized onto the agarose surface. A multivariable optimization approach examining reaction time, temperature, and scaffold concentration yielded surface quaternization values (DSEA) of 0.05 and 0.11 for SMAC-4 and SMAC-24, respectively, with successful modification confirmed via SEM-EDX and FT-IR analyses. Quaternization altered the physicochemical and mechanical properties of the scaffolds; notably, the average porosity increased from 66.7% in AC-0 to 78.7% in SMAC-24 while maintaining the integrity of the porous structure. Biological evaluation against Escherichia coli and Staphylococcus aureus identified SMAC-24 as the most favorable formulation, exhibiting the strongest antibacterial activity and maintaining 88.1 ± 1.6% human umbilical vein endothelial cell (HUVEC) viability after 24 h of exposure to scaffold extracts. These findings demonstrate that post-synthetic surface quaternization provides agarose cryogels with contact-active antibacterial functionality and favorable cytocompatibility, highlighting their potential as non-leaching antibacterial agarose scaffolds for chronic wound care.