Reaction kinetics of chitosan nanogels crosslinked by genipin

Crosslinking of chitosan chains in dilute solution by natural crosslinker genipin leads to biocompatible nanogels. Here we investigated the reaction kinetics between chitosan and genipin in a 200 mM acetate buffer at 37 °C, and the structural and conformational evolutions of the nanogels during the crosslinking reaction by multi detection asymmetric flow field-flow fractionation (AF4). Upon crosslinking by genipin, the z-average hydrodynamic radius R_hz of the chitosan chains increased from 26 nm to 130 nm, while the weight average molar mass M_w increased from 2.0 × 10⁵ g/mol to 1.8 × 10⁷ g/mol. The crosslinking reaction appeared to be first-order and size-dependent. In particular, the intrachain crosslinking reaction was preferentially for nanogels having the larger size, leading to formation of branched chains/nanogels having a wide range of molar masses between 10⁶ and 10⁸ g/mol but a similar radius of gyration R_g ∼ 40 nm. For the largest nanogel fractions with M > 2.0 × 10⁸ g/mol, both R_g and R_h showed a scaling relation with exponent 1/3 and a structure parameter R_g/R_h = 0.74, as expected for the hard sphere particle. The reaction was accompanied by a reduction of charge density and an increase in hydrophobicity of chitosan nanogels, which plays a key role in the formation of uniform size nanogels with chain density ρ(R_h) up to 0.45 g/cm³.