Improving physiological solubility and gene transfer efficiency of chitosan via 3-nitrobenzaldehyde and amino acid conjugation

In this study, chitosan was chemically modified with 3-nitrobenzaldehyde (3NBA) and three amino acids (arginine, cysteine, and histidine) to enhance its gene delivery performance. 3-NBA was selected for its known DNA binding properties, while the amino acids were chosen based on their functional groups, which can improve solubility, facilitate cellular uptake, and contribute to endosomal escape. The modified chitosan polymers were characterized using Fourier Transform Infrared Spectroscopy (FTIR) and Nuclear Magnetic Resonance Spectroscopy (NMR). Nanoparticles were prepared using the ionotropic gelation method, and their particle size, polydispersity index (PDI), zeta potential were analyzed by dynamic light scattering (DLS). The particle sizes ranged from 105.07 ± 3.45 to 206.15 ± 10.39 nm, with PDI values between 0.29 ± 0.01 and 0.39 ± 0.02. Zeta potentials were measured between 32.05 ± 0.49 mV and 51.95 ± 0.35 mV. The cysteine-modified chitosan (Chi-3NBACys) exhibited approximately 8.4-fold higher solubility than unmodified chitosan. In vitro studies demonstrated that the modified chitosan nanoparticles exhibited low cytotoxicity in HEK293T cells. Among the tested formulations, Chi-3NBACys showed the highest transfection efficiency, comparable to commercial agent Lipofectamine™ 2000. These findings suggest that chitosan nanoparticles modified with 3-NBA and amino acids can be safe and efficient non-viral gene delivery vectors.