A Covalent Organic Framework-Inspired β-Ketoenamine Crosslinking Strategy for Robust, Injectable Bovine Serum Albumin Hydrogels with pH-Triggered Drug Release

Globular proteins are difficult to convert into robust hydrogels, as their compact, folded structures bury reactive residues, forcing conventional strategies to rely on denaturation or synthetic-polymer reinforcement that compromise the native protein. Inspired by the β-ketoenamine bond-forming chemistry of covalent organic frameworks (COFs), we report the crosslinking of native bovine serum albumin (BSA) with 1,3,5-triformylphloroglucinol (TFP), a C3-symmetric trialdehyde, into a chemically defined hydrogel. TFP reacts with surface-exposed lysine residues through an irreversible enol-to-keto tautomerization, confirmed by FTIR and NMR spectroscopy, generating stable β-ketoenamine crosslinks under mild aqueous conditions without denaturing the protein, as verified by intrinsic tryptophan fluorescence. The resulting hydrogels are mechanically robust compared to a reversible-imine control, injectable and self-recovering, exhibit reversible shape memory and substantial load-bearing capacity, and remain stable across a broad pH range over extended periods. The network shows consistent swelling behavior at physiological and mildly acidic pH, with modest compaction under strongly basic conditions; scanning electron microscopy reveals a dense, nodular network for the TFP hydrogel versus an open, sheet-like lamellar morphology for the reversible-imine control. The hydrogel efficiently encapsulates doxorubicin and displays pH-triggered, acid-selective release, which comparative kinetic analysis attributes principally to pH-dependent weakening of DOX–BSA binding affinity (linked to the N-to-F conformational transition of BSA near its isoelectric point) rather than to bulk network swelling or degradation. Doxorubicin-loaded hydrogels show enhanced killing of MCF-7 breast cancer cells relative to the free drug while remaining cytocompatible toward normal mammalian cells, and a ciprofloxacin-loaded variant exhibits potent antibacterial activity against both Gram-positive (M. luteus) and Gram-negative (E. coli) bacteria. This work translates reticular β-ketoenamine chemistry into a general platform for robust, stimuli-responsive protein biomaterials.