Preparation And Properties Of Long Circulating Spontaneous Escape Gene Vectors

Cancer is the main cause of death worldwide in the 21^st century.In cancer treatment,gene therapy is a revolutionary option by introducing therapeutic genes into target cells and restoring the normal function of organs through the expression of target genes.However,viral vectors have potential risks of immune response and insertional mutation.Non-viral vectors such as cationic liposomes have short circulating half-lives in blood and proteolytic degradation in cells,resulting in low transfection efficiency.In this paper,a series of PEG lipid molecules with long circulation and spontaneous endosomal escape properties were designed and synthesized,and neutral liposomes were prepared to explore the relationship between lipid structure and transfection efficiency,which provides the theoretical and experimental basis for neutral liposomes in cancer gene therapy.In this paper,four asymmetric double-hydrophobic end lipid molecules with different free hydrophobic ends were synthesized(CH₃-PEG_2k-DOAP,C₁₂H₂₅-PEG_2k-DOAP,C₁₆H₃₃-PEG_2k-DOAP,C₁₈H₃₅-PEG_2k-DOAP).Using tert-butyl N-（2,3-dihydroxypropyl）carbamate and oleic acid as starting materials,DOAP was obtained through esterification reaction,and different hydrophobic ends were connected at both ends of polyethylene glycol through amidation reaction.The post-processing operation of this route was convenient and the yield attained 90%.The intermediates and lipid molecular structures were correctly characterized by ¹H NMR and mass spectrometry.The relationship between PEG lipid structure and in vitro transfection efficiency was preliminarily explored through the study of self-assembly performance,encapsulation performance,biosafety performance,and in vitro transfection performance of PEG liposomes.The critical micelle concentrations of PEG lipids were 1.61×10^-4 mol/L,1.82×10^-5 mol/L,1.91×10^-4 mol/L,and 3.58×10^-4 mol/L,respectively.which were lower by one or two orders of magnitude compared with traditional surfactants such as SDBS or SDS,showing excellent aggregation or self-assemble performance of the synthesized PEG lipids.PEG liposomes were prepared by self-assembly with PEG lipid and neutral lipid DOPC,the hydrodynamic diameter of the liposomes was about 100 nm.With the increasing length of the free hydrophobic end,the hydrodynamic diameter of the liposomes was slightly increased.MTT test showed that the cytotoxicity of PEG liposomes was much lower than cationic liposomes.Neutral liposome complexes were prepared by co-inducing neutral PEG lipids with Ca²⁺and ethanol to encapsulate plasmids.When the concentration of ethanol was 50%and the concentration of Ca²⁺was 25 m M,the encapsulation efficiency of neutral lipid complexes was up to 50%.In He La cells,the transfection efficiency of PEG liposomes was enhanced with the increase of free hydrophobic end carbon chains,and the neutral liposome complexes(C₁₈H₃₅-PEG_2k-DOAP)were 1.5 times the transfection efficiency of the commonly used cationic liposome（DOTAP）and 0.9 times the transfection efficiency of the cationic polymer PEI_1.8k.