Gene carriers made from synthetic materials are of interest in relation to gene therapy but suffer from lack of transfection efficiency upon systemic delivery. To address this problem, a novel lipo-peptide-PEG conjugate constituted by a lipid-anchor, a peptide sensitive to proteases and a poly (ethylene glycol) (PEG) chain is investigated. Utilizing ethanol-mediated nucleic acid encapsulation to prepare lipo-nanoparticles (LNPs), LNPs that are stable in serum are obtained. The LNPs constitute a highly effective gene delivery systems in vitro and possess the right features for further investigation in vivo including a PEG layer and a net negative charge that should ensure long-circulating properties before being activated by proteases in diseased tissue. Protease activation leads to detachment of PEG and a charge switching where the LNPs become positive due to the presence of glutamates in the cleaved peptide moiety. The cationic lipid DOTAP is used mainly to complex DNA and proton titratable DODAP is used to increase endosomal escape and enhance transfection efficiency. The idea of using a mixture of permanently charged and titratable cationic lipids shielded by a protease sensitive negatively charged lipo-peptide-PEG coat appears to be a highly efficient solution for achieving effective non-viral gene delivery and the results warrant further investigations.