Tricine-modified chitosan as a strategy for enhancing hydrophilicity and gene delivery

In this study, we investigated the effect of chitosan modification with tricine on transfection efficiency by preserving its ability to form complexes with plasmid DNA (pDNA) and increasing its hydrophilicity. The inherent limitations of chitosan, such as poor solubility at physiological pH, insufficient cellular uptake, and strong ionic interactions with pDNA, typically result in low transfection efficiency. To overcome these challenges, Tricine, a hydrophilic molecule containing a secondary amine group, was conjugated to chitosan. Chitosan of three different molecular weights (low, medium, and high) was modified with tricine. Structural characterization of the modified chitosan was conducted using Fourier Transformed Infrared Spectroscopy (FTIR) and Nuclear Magnetic Resonance (NMR) analyses. The effects of tricine modification were assessed in terms of hydrophilicity/hydrophobicity, proton buffering capacity, particle size, PDI and zeta potential. Tricine modified low molecular weight chitosan nanoparticles (nLMWChi_Tri), which exhibit suitable properties for gene transfer studies, were evaluated regarding pDNA complexation ability, cytotoxicity and in vitro transfection efficiency. The results demonstrated that tricine modification enhanced the gene transfer potential of chitosan, making it competitive with the commercial transfection agent Lipofectamine™ 2000 and offering a promising strategy for non-viral gene therapy applications. Furthermore, the biocompatibility and biodegradability of chitosan, combined with the improved hydrophilicity provided by tricine, makes nLMWChi_Tri a safer and more sustainable option for repeated use in gene delivery, overcoming the major limitations associated with other synthetic vectors such as Lipofectamine™ 2000.