Assessing Interactions with DNA, Gene Transfection Potential, and Cytotoxicity of Amide‐Bonded Cationic Gemini Surfactants: The Role of Chain Length

Abstract Gemini surfactants or simply Gemini with dual hydrophobic tails and hydrophilic head groups show promise in gene delivery applications due to their strong electrostatic interactions with negatively charged biomolecules. This study investigates the in‐vitro transfection efficiency of three amide‐bonded Gemini (AG‐12, AG‐14, AG‐16) at varying charge ratios (−/+). Optimal transfection efficiency was observed between 0.6 and 0.2, with AG‐16 (16‐carbon chain) demonstrating the highest efficiency, although its performance declined at higher concentrations due to strong DNA binding. All three Gemini outperformed the commercial standard, Turbofectamine, highlighting their potential in gene therapy. Ethidium bromide displacement assays revealed that the Gemini bind DNA through electrostatic and hydrophobic interactions, enhancing DNA compaction without significant conformational changes. Longer alkyl chains promoted DNA condensation and increased binding affinity. Dynamic light scattering studies revealed the stability of DNA‐Gemini complexes, which was influenced by charge ratio (−/+), providing insights into gene transfection outcomes. Cytotoxicity studies conducted on RAW 264.7 and HEK 293 cells revealed that the Gemini surfactants exhibited a greater toxic effect on the cancerous cells than on the healthy ones at higher concentrations. These findings underscore Gemini's potential for gene delivery, emphasizing the importance of optimizing charge ratio and chain length for efficient, low‐toxicity applications.