Nucleic acids-based next generation biopharmaceuticals (i.e., pDNA, oligonucleotides, short interfering RNA) are potential pioneering materials to cope with various incurable diseases. However, several biological barriers present a challenge for efficient gene delivery. On the other hand, developments in nanotechnology now offer numerous non-viral vectors that have been fabricated and found capable of transmitting the biopharmaceuticals into the cell and even into specific subcellular compartments like mitochondria. This overview illustrates cellular barriers and current status of non-viral gene vectors, i.e., lipoplexes, liposomes, polyplexes, and nanoparticles, to relocate therapeutic DNA-based nanomedicine into the target cell. Despite the awesome impact of physical methods (i.e., ultrasound, electroporation), chemical methods have been shown to accomplish high-level and safe transgene expression. Further comprehension of barriers and the mechanism of cellular uptake will facilitate development of nucleic acids-based nanotherapy for alleviation of various disorders.