Qinmei Wang,1,* Ying Chen,1,* Lichun Wang,1 Xinchun Zhang,2 Hongzhang Huang,2 Wei Teng2 1Key Laboratory on Assisted Circulation, Ministry of Health, Cardiovascular Division, First Affiliated Hospital, Sun Yat-sen University, Guangzhou, People’s Republic of China; 2Hospital of Stomatology, Institute of Stomatological Research, Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou, People’s Republic of China *These authors contributed equally to this work Abstract: Successful in vivo gene delivery mediated by nonviral vectors requires efficient extracellular and intracellular gene delivery, but few studies have given attention to the former. That is why numerous gene delivery systems have succeeded in vitro, while the expected clinical success has not come about. To realize efficient extracellular gene delivery, the stability of vectors and/or their complexes with genes in body fluids is first required, which prevents loaded genes from premature unloading and degradation. Furthermore, the storage stability of vectors under common conditions is important for their widespread applications. Lipopolysaccharide-amine nanopolymersomes (NPs), a gene vector developed by our group recently, have higher than 95% in vitro transfection efficiency in mesenchymal stem cells when delivering pEGFP, and induce significant angiogenesis in zebrafish when delivering plasmid encoding vascular endothelial growth factor deoxyribonucleic acid (pVEGF). To reveal their extracellular delivery ability and storage stability, in this study their stability in various simulant physiological environments and storage conditions was systematically studied by monitoring their changes in disassembly, size, zeta potential, and transfection efficiency. Additionally, damage to the mitochondria of mesenchymal stem cells was evaluated. Results show that NPs and plasmid deoxyribonucleic acid (pDNA)-loaded NPs (pNPs) have acceptable stability against dilution, anions, salts, pH, enzyme, and serum, presumably assuring their efficient extracellular delivery in vivo. Moreover, both the lyophilized NPs at room temperature and NP/pNP solution at 4°C have high storage stability, and pNPs show low damage to the mitochondria. The acceptable stability of NPs combined with compatibility and efficient gene transfection highlight their huge potential in the clinic as a gene delivery vector. Keywords: nanopolymersomes, extracellular delivery stability, storage stability, toxicity, polyethyleneimine, alginate, cholesterol, mesenchymal stem cells