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

Authors

Khaitan, S., Agrawal, T., Gulati, P., Akansha, A., Natasha, N., Rakshit, T., Pal, S.

Abstract

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 beta-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 {beta}-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 {beta}ketoenamine chemistry into a general platform for robust, stimuli-responsive protein biomaterials.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 11 Sep 2026.

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