Eco-Friendly Synthesis of Dicyandiamide-Coupled Chitosan for Superior Gene Delivery in Cancer Cells

Chitosan (CS) is valued in biomedical applications for its biocompatibility and cationic nature but is limited by poor solubility in water and organic solvents. To address this, we synthesized a highly water-soluble chitosan derivative, dicyandiamide-coupled chitosan (CSDCDA), via a novel, eco-friendly pathway and evaluated its efficacy as a gene delivery agent. CSDCDA was prepared by grafting dicyandiamide onto low molecular weight CS using cyanuric chloride as a green hydrochloric acid precursor at 60°C, eliminating the need for microwave irradiation or high-temperature reflux. The successful modification, confirmed by FT-IR, ¹ H NMR, XRD, SEM, and EDX analyses, introduced guanidinium groups mimicking cell-penetrating peptides, enhancing solubility, DNA binding, and biocompatibility. CSDCDA exhibited compact polyplexes (reduced size and zeta potential) and excellent cytocompatibility, outperforming native CS and Lipofectamine®. Notably, at a 1:5 pDNA/CSDCDA ratio, transfection efficiency in A549 cells was 18.2 times higher than pDNA/Lipofectamine and 45.3 times higher than pDNA/CS, with improved performance in serum. Superior buffering capacity, assessed via acid–base titration and bafilomycin A1 assays, facilitated rapid endosomal escape. These findings position CSDCDA as a promising gene delivery vector, warranting further mechanistic and in vivo studies.