The Study On Gene Transfection And Therapy Through Non-lysosome Route

Gene therapy is one kind of treatment utilized endogenous or exogenous genetic materials to improve cell function, eliminate diseased lesions or enhance the body's own protection. There are three strategies for gene therapy:physical methods, viral vectors and non-viral vectors. Compared with the first two strategies, non-viral vectors can delivery gene with the advantages of no gene fragment limitation, low toxicity and immunogenicity, being easy to large-scale manipution and preservation. However, non-viral vectors generally achieve low transfection efficiency, mainly due to poor plasma stability, lysosomes retention and package and release contradiction. Especially, the problem of lysosomes retention leads to huge damage to genes in their acidic environment. Non-viral gene vectors usually utilize the'proton buffering' effect of cationic polymers to destroy lysosomes, which inevitably causees high cytotoxicity. Recently, by modifying polyethyleneimine with histone protein (H3) researchers prepared a novel vector which could reach the perinuclear region directly through non-lysosome route with high transfection efficiency and reduced toxicity. Such strategy based on the intracellular fate hints us how to design a novel non-viral vectors and shows high prospects. In this paper, two non-viral gene delivery systems through non-lysosome route were constructed:polyethyleneimine-modified carbon nanotubes and specific FAL peptide-modified cationic liposomes. The former vector possesses unique physicochemical properties and can be cellular internalized through the caveolin-mediated endocytosis mechanism, which can avoid the traditional lysosome fate and realize good gene transfection and therapy efficacy. The latter utilizes a cationic FAL peptide to modify ordinary cationic liposomes showing a safe and efficient intracellular fate.In addition to the problems mentioned above, non-viral gene vectors usually confront with contradiction of'package and release'. In order to prevent the nucleic acids from premature release and degradation by enzymes, non-viral vectors should bind tightly to nucleic acids to form stable particles. However, once the complex reaches the site of action, an overly packaged particle can't release the nucleic acids sufficiently. Photothermal gene transfection (PTT) is a new method to solve this contradiction. In this paper, we physically modified SWNTs which have photothermal conversion ability with three PEI-Chol (PEI25K-Chol, PEIioK-Chol ? PEI1.8K-Chol) to construct three 'PTT' agents (PEI-Chol/SWNTs, PCS25K> PCS10k> PCS1.8K). The infrared spectroscopy confirmed the success of the construction. The transmission electron microscopy, UV-Vis absorption spectroscopy and dynamic light scattering were used for characterizing the physiochemical properties of PCS systems. The agarose gel electrophoresis was applied to study the binding capability of PCS with DNA, which was related to the molecular weight of the polyethyleneimine, ie. PCS25K>PCS10K>PCS1.8K. The three complexes of PCS/DNA presented obvious in-vitro photothermal conversion ability. NIR light stimulation could lead to the detachment of DNA from the complex and the released DNA could be destroyed by DNasel enzyme. Without NIR stimulation, three PCS/DNA complexes presented good ability against DNasel. The cytotoxicity of PCS/DNA complexes to HEK293 cells and HeLa cells was detected by MTT assay. When the w/w ratio of vector to DNA remains unchanged, the cytotoxicity of PCS/DNA showed little lower than that of PEI/DNA complexes formed by corresponding MW of PEI and DNA. The introduction of NIR laser didn't significantly affect the cytotoxicity of PCS/DNA.The PCS labeled by fluorescein isothiocyanate (FITC) incubated with cyanine dye fluorescence (Cy3) modified DNA to form fluorescent complexes for cellular uptake mechanism study under different incubation conditions. The cell flow cytometer and confocal results founded that the PCS/DNA complexes entered into the cells mainly through caveolin. Further co-localization study proved that PCS/DNA can avoid the uptake of lysosomes which was the benefit for the stability of DNA. In addition, the NIR-induced detachment of PCS with DNA was observed, while such behavior failed to be founded in complexes formed by traditional transfection agent PEI25K with DNA. The plasmid of pEGFP-C1 was applied to conduct in vitro photothermal gene transfection assay. The efficiency of transfection without laser was: PCS25K>PCS10K>PCS1.8K and the laser irradiation can improve the expression of green fluorescence protein by PCS in HEK293 and HeLa cells. In order to discover the photothermal gene therapy potential of PCS, a tumor suppressor gene plasmid TP53 was chosen to combine with PCS10K to form PCS10K/pTP53 complexes. The cell cycle, apoptosis/mortality rate, mitochondrial membrane potential, nuclear debris, mitochondrial morphology and apoptosis-related proteins were detected to study the tumor inhibition effects and the apoptosis mechanism in HeLa cells. The breast xenograft tumor model was stablished for the evaluation the in-vivo anti-tumor effect of PCS10K/pTP53 complexes with the observation of tumor growth curve and histological section staining.A novel cationic liposome internalized through non-lysosome route was constructed by introducing a special peptide of FAL to an ordinary cationic liposome. Firstly, FAL peptide was utilized to modify DSPE-PEG-NH2 to form DSPE-PEG-FAL The product was detected by 1H-NMR and IR to prove the successful reaction. By mixing DOTAP, DOPE, DSPE-PEG-NH2 and DSPE-PEG-FAL, we prepared FAL peptide-modified liposome (Liposome-FAL) via film dispersion method. Utilizing the pEGFP-C1 as a reporter gene, the in-vitro transfection of Liposome-FAL in HEK293, A549 and MCF-7 cells was investigated by fluorescence inverted microscope. The results showed that Liposome-FAL containing 2.25% of DSPE-PEG-FAL was an efficient vector rather than Liposome-Non without DSPE-PEG-FAL. The in vivo transfection efficiency of Liposome-FAL was also better than that of commercial transfection reagent Lipofectamine, and the level of green fluorescent protein in Liposome-FAL group was also highly expressed. Utilizing plasmid pTP53 as a model gene drug, Liposome-FAL/pTP53 complex could inhibit the growth of breast cancer significantly and the p53 protein immunohistochemistry showed that the p53 protein level in Liposome-FAL group was also higher than Lipofectamine2000 group, while liposome-Non group showed poor anti-tumor effect. To sum up, FAL peptide modified cationic liposomes is a super excellent gene vector and the new strategy for designing non-viral gene vector via non-lysosome route presented great potential.