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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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The aim of this study is to synthesize new amphiphilic chitosan containing myristic acid as the hydrophobic tail and tertiary amine groups as the hydrophilic head and to evaluate the gene delivery efficiency. In this context, the primary amine groups of chitosan were first modified with myristic acid (Chi-M), followed by the modification of the methylol groups with 3-dimethylamino-1-propyl chloride hydrochloride. The chemical characterization of this chitosan formulation (Chi-MA) was determined using nuclear magnetic resonance (NMR), Fourier-transform infrared spectroscopy (FTIR) analysis and gel permeation chromatography-size exclusion chromatography. Chi-MA nanoparticles were prepared via ionic gelation, and particle size, polydispersity and zeta potential were determined. The nanoparticles were evaluated for their proton buffering capacity and gene complexing capacity. Additionally, the cytotoxicity of Chi-MA on HEK293T cells was determined via MTT assay, and the transfection efficiency of Chi-MA was analyzed by a flow cytometer. The results indicate a significant increase in gene complexing capacity (8-fold) and nanoparticle formation ability of Chi-MA compared to other chitosan formulations. Chi-MA nanoparticles showed no toxicity against HEK293T cells and exhibited the highest transfection efficiency with significantly lower nanoparticle: gene ratios compared to previous studies. These findings demonstrate the effective use of amphiphilic Chi-MA as a gene carrier., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)