Intracellular Mechanism Of Gene Delivery Of A Novel ER Targeted Cationic Liposome

As a major public health problem,cancer threats health and the mortality rate caused by caner are increasing year by year.Tumor gene therapy has shown a promising prospect in the field of tumor therapy by introducing exogenous tumor suppressor genes to inhibit tumor proliferation,invasion and metastasis.In order to achieve excellent tumor gene therapy effects,a safe and efficient gene delivery system needs to be developed.Currently,the delivery systems commonly used in gene therapy include viral vectors,virus-like vectors,and non-viral vectors,wherein the viral vectors have high gene transfection efficiency.But because of their random integration of viral genes into the host genome,instability and other shortcomings,viral vectors have limited development in clinical applications.Cationic liposome,a closed vesicle of phospholipid bilayer structure,is composed of cationic phospholipids and neutral phospholipids under appropriate conditions.With the advantages of no immunogenicity,biodegradability and convenient preparation,cationic liposome is commonly used as a nano-scale non-viral vector for tumor gene therapy,but limited transfection efficiency is the bottleneck for its further development and application.In order to improve the targeting and transfection efficiency of cationic liposomes,the strategy of PEG,glycosylation,polypeptide modification are always used to achieve gene delivery efficiency comparable to viral vectors.Gene delivery mechanisms have important reference significance for the development and preparation of novel cationic liposomes.By studying how liposome/genes complexes enter cells,complete intracellular transport,and delivery exogenous genes into the nucleus,as well as by understanding the key steps and key sites of intracellular gene delivery,it is more promising to discover new targets for gene delivery,and develop high-quality carriers with good specificity and safety.At the same time,the virus with high efficiency of gene delivery,like simian virus?SV40?,is of great significance for us to design and prepare highly efficient non-viral gene vectors.SV40 can penetrate into the cell through the caveolin-mediated endocytosis,break through the local cortical cytoskeleton barrier,and carry out intracellular transport along the microtubule,directly reach the peri-nuclear endoplasmic reticulum?ER?,and complete the shelling in the ER.The viral genome is released near the nuclear pore complexes,allowing it to enter the nucleus more efficiently.Based on these theories,this paper reports that an ER-targeted cationic liposome?Liposome-Padaxin,Lipo-Par?modified by cationic peptide Pardaxin?GE33?was prepared.Pardaxin,a cationic antibacterial peptide derived from the red sea flatfish,which amino acid sequence is NH2-GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE,has hydrophobicity and pore-forming properties.It can be inserted into the phospholipid bilayer and quickly localized to ER by non-lysosomal intracellular transport pathway,wherein ERs was induced by pardaxin.DSPE-PEG₂₀₀₀-Par,a material for synthesizing Lipo-Par,can be obtained by condensation reaction of the amino group of DSPE-PEG₂₀₀₀-NH₂ with the carboxyl group of Pardaxin polypeptide.Together with cationic phospholipid DOTAP,neutral phospholipid DOPE and DSPE-PEG,Lipo-Par can be prepared by the film dispersion,the particle size and zeta-potential of which is about 120 nm and 27 mV.It was found by agarose gel electrophoresis experiments that Lipo-Par and pDNA/ssDNA were tightly complexed when the N/P ratio was 6:1.In the in vitro transfection experiments of HEK 293T,MCF-7,MCF-7?PDX?,SKOV-3,Hela cell lines,Lipo-Par showed comparable transfection performance to commercial liposome Lipofectamine^TM 2000.Relevant experiments in vitro,including uptake,intracellular transport and nuclear delivery,were designed and performed to explore the intracellular mechanism of Lipo-Par for its efficient transfection.The uptake tests showed that the Lipo-Pars were internalized mainly through caveolin-mediated endocytosis,and were relied on the lipid raft,a region rich in cholesterol and sphingomyelin on the cell membrane.As for the study of the intracellular transport pathway mechanism,we found that Lipo-Pars targeted the ER through the cytoskeleton including microtubules and microfilaments,so that the DNA carried by Lipo-Pars could be protected from the acidic environment of lysosomes.Besides,compared to the non Pardaxin-modified liposome,Lipo-Nons,exogenous DNA carried by Lipo-Pars can be released ealier after internalization.In addition,mild ER stress?ERS?induced by the calcium ion chelating agents BAPTA-AM and brefeldin?BFA?can both enhance gene delivery efficiency of Lipo-Non and Lipo-Par.It is well known that cell mitosis has an important influence on the transfection efficiency of cationic liposomes.Compared to the cells in the interphase,Cells in the mitotic phase are more likely to uptake DNA.To test how mitosis affects the transfection performance of Lipo-Par,we used the mitotic chemical inhibitor paclitaxol?PTX?to inhibit cell mitosis.The results showed that Lipo-Par was still targeted to the ER when the cell mitosis was inhibited and the microtubule structure remained intact.What's more,compared with Lipofectamine ^TM 2000,mitotic inhibitor PTX has less effect on the transfection efficiency of Lipo-Par.