From Colloids to Hydrogels: Concentration-Dependent Cytocompatibility of Carboxylated Cellulose Nanocrystals Prepared...

Cellulose nanocrystals (CNCs) are promising sustainable nanomaterials, yet their application in biointerfaces is often limited by fixed surface chemistries and limited insight into their concentration-dependent behavior. Here, we report a green and scalable strategy for CNC surface reprogramming using an oxalic acid-choline chloride deep eutectic solvent (ChCl-OAD DES), enabling controlled substitution of sulfate groups with carboxyl functionalities under mild conditions. The resulting carboxylated CNCs (CNC-COOH) preserve crystallinity and morphology while achieving tunable surface charge densities (up to ∼0.19 mequiv g^-1) and improved thermal stability. We systematically investigate two distinct concentration regimes: dilute dispersions below 0.4 wt %, where CNC-COOH behaves as stable colloids, and concentrated systems at 2 wt %, where percolated hydrogel networks are formed. This transition is governed by hydrogen bonding and ionic screening, leading to pronounced changes in nanoscale organization and viscoelastic behavior. In biologically relevant media, CNC-COOH forms soft, elastic hydrogels (G' ≈ 10² Pa) capable of supporting three-dimensional (3D) cell encapsulation. Importantly, cytocompatibility is strongly dependent on material state. In the colloidal regime (<0.4 wt %), CNC-COOH exhibits negligible cytotoxicity toward 3T3-L1 fibroblasts and weak to mild cytotoxic effects toward HT-29 epithelial cells. In contrast, hydrogel networks (2 wt %) promote high cell viability (>85-100%) and enable 3D cellular organization. Protein adsorption appears to be limited at the surface of the CNC-COOH hydrogel, as indicated by BSA studies, suggesting that ionic strength-mediated interactions play an important role in network formation under cell culture conditions. These findings establish direct correlations among sustainable surface modification, concentration-dependent assembly, and biological response, providing design principles for CNC-based nanomaterials in biointerfaces, 3D cell culture, and nanomedicine.