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Chitosan nanoparticles have shown considerable promise as gene vectors but do not mediate transfection with satisfactory efficiency. To improve upon the transfection efficiency of chitosan, we approached the development of alginate–chitosan nanoparticles with the goals of maintaining low toxicity and biocompatibility. Through ionic gelation, particles were formed with a mean Z-average diameter of 157 nm and a zeta potential of +32 mV. Competition binding assays indicated that the presence of alginate reduces the strength of interaction between chitosan and DNA, contributing to improved transfection. Cell viability assays indicated that nanoparticles exhibit the same low toxicity as chitosan, and significantly reduced toxicity compared to a commercial liposome formulation. As well, complexation with nanoparticles maintained DNA integrity and protected it from nuclease degradation better than chitosan alone. Alginate–chitosan nanoparticles were able to mediate transfection of 293T cells four times that achieved by chitosan nanoparticles; at 48 h, the transfection efficiency was as high as with Lipofectamine™, with significantly reduced cytotoxicity. Overall, alginate inclusion improved the vector properties of chitosan-based nanoparticles, demonstrating superior transfection ability while maintaining biocompatibility and low toxicity.