LAUSR

&NA; The present study aimed to investigate in vitro DNA transfection efficiency of three novel chitosan derivatives: thiolated trimethyl chitosan (TMC‐Cys), methylated 4‐N,N dimethyl aminobenzyl N,O carboxymethyl chitosan(MABCC) and thiolated trimethyl aminobenzyl chitosan(MABC‐Cys). After polymer synthesis and characterization, nanoparticles were prepared using these polymers and their size, zeta potential and DNA condensing ability were measured. After that, cytotoxicity and transfection efficiency of nanocomplexes were carried out in three different cells. The results showed that all polymers could condense DNA plasmid strongly from N/P 2 and nanocomplexes had eligible sizes and zeta potentials. Moreover, the nanocomplexes had negligible cytotoxicity and MABC‐Cys was the most effective vehicle for gene delivery in HEK‐293T cells. In the two other cell lines, SKOV‐3 and MCF‐7, TMC‐Cys exhibited the highest transfection efficiency. This study indicated that chemical structure of these novel chitosan derivatives in the interaction with the cell type can lead to successful gene delivery. Graphical abstract Figure. No caption available.