There is a need of an efficient and safe nonviral gene delivery carrier due to promising future of nucleic acid-based therapeutics in the treatment of intractable diseases. Cytotoxicity and cost are the major concerns with current quaternary ammonium-based cationic liposomes. The major aim of current research work was development and in vitro evaluation of arginine-anchored nanoliposomes for gene delivery. LArginine-fatty acid conjugate was synthesized and characterized using IR, NMR, and mass spectroscopy. Synthesized conjugate-lauroyl arginine ethyl ester (LAE) was successfully incorporated into liposomes. Effect of nanocarrier composition on DNA binding was evaluated by preparing solid lipid nanoparticle (SLN) and self nanoemulsifying system (SNES) using same LAE concentration. Effect of cationic head on DNA binding was also evaluated. Arginine-anchored nanoliposomes-arginoplexes (APX) showed superior DNA-binding affinity. Surface PEG was expected to cause hindrance in DNA binding in SLNs and SNES. Guanidino group was found to be a better cationic head for DNA binding compared to primary amine or quaternary amine. Gel retardation assay was performed to optimize the ratio of DNA to LAE in nanocarrier. Serum stability, haemolysis, cytotoxicity, and transfection studies were carried out to evaluate APX. Binding ofDNAtoAPXwas found to be stable in the presence of serum, and no degradation of DNA was observed. APX containing 2 mg/ml LAE which exhibited particle size of ∼72 nm with zeta potential of +57.5 mV, showed lower cytotoxicity and better transfection. APXcan be a promising carrier for gene delivery. © Springer Science+Business Media Dordrecht 2014.