Self-assembled CM-PLH/PbAE/DNA Ternary Complexes Non-viral System For Gene Delivery

Efficient, safe and stable gene delivery vector for gene transfer remains to be a major challenge to successful gene therapy. Compared with viral gene delivery vectors, non-viral gene delivery vectors are most attractive due to their low toxicity, easy to synthesis, low immune response and ease to be modified and so on. As a result, the development of novel efficient non-viral gene delivery vectors has attracted much attention although the efficiency is not quite satisfactory. Cationic polymers are far to enter the clinical application and there are still many problems to be solved. For example, it will lead to low transfection efficiency after administered intravenously because the poor stability in the circulation, which hints us to develop efficiency, feasible and low toxicity in vivo non-viral cationic gene delivery system.The contents of the study involved pharmaceutical, polymer materials science and molecular biology techniques. The characteristics of the two materials the poly (β-amino ester)(PbAE) and carboxymethyl poly (L-histidine acid)(CM-PLH) were combined to construct CM-PLH/PbAE/DNA ternary complex vector system for gene delivery based on assembly electrostatic coating. The cationic polymer PbAE with positively charged which can effectively self-assembly condensed negatively charged plasmid DNA to form PbAE/DNA binary complex nanoparticles. And then the nanoparticles were modified with CM-PLH to form core-shell CM-PLH/PbAE/DNA ternary complex nanoparticles. The characteristics of the vector are showed as following:(1) CM-PLH can reduce the positive charge of the surface of PbAE/DNA complex, which can reduce the cell toxicity of cationic polymer;(2) it will extend the circulation time of the vector system in vivo, and maintain stability in vivo;(3) CM-PLH contains a large number of pH-sensitive imidazole groups, which can lead "proton sponge effect" to break through the lysosomal barrier and protect plasmids from enzymatic degradation and improve the efficiency of transfection.(4) to avoid reducing the efficiency of transfection after functional groups react with the carrier material.This study includes the synthesis, characterization and screening of PbAE; the preparation, characterization and optimization of PbAE/DNA binary complex; Synthesis and Characterization of the CM-PLH; CM-PLH/PbAE/DNA ternary complex carrier system selection of human embryonic kidney HEK293cells and melanoma cells B16F10and filter the prescription, and will ultimately determine the optimal prescription; the optimal prescription of the ternary complex vector system were used to transfer the genes pEGFP-N2and therapeutic genes pTNFSFlO-EGFP; to study the complex intracellular endocytic pathway and the transport mechanism by adding specific inhibitors; the confocal microscopy was used to observe the process of the complexes into the cell and intracellular translocation; subcutaneous tumor model and lung metastasis tumor model were used to study the gene transfection of ternary complex vector system in vivo.Different end groups capped PbAE was synthesized and characterized, and the safety evaluation and the transfection efficiency were determined in vitro. Using the static condensation method (coacervation) to prepared PbAE/DNA binary complex aand the mass ratio of50:1can be prepared by the average particle size of the complex at150nm nanoparticles, and the transfection efficiency in vitro was considerable to Lipofectamine2000.After the synthesis of the CM-PLH, CM-PLH/PbAE/DNA ternary complexes based on electrostatic coating were prepared. The ternary complexes were characterized with particle size and zeta potential, condensed ability, anti-DNase I enzymatic hydrolysis, anti-heparin sodium dissociation and serum dissociation. The results showed that PbAE could condense plasmid DNA within the experimental range. CM-PLH does not affect the stability of the complexes, and can completely prevent a concentration of0.4U/μg of DNA, DNase I degradation; and protect plasmid DNA from serum dissociation, even if the serum concentration to50%; complexes whether the anti-heparin sodium dissociation is closely related to the concentration of heparin. In selected heparin sodium concentration range, the complex is basically no dissociation. After electrostatic coating, with the increase of the amount of the CM-PLH, the cytotoxicity of CM-PLH/PbAE/DNA complex decreased. The cellular uptake behavior was time-dependent manner. The number of positive cells expressing green fluorescent protein was determined by Fluorescence microscopy and Flow cytometry. The results showed that the percentage of positive cells after adding CM-PLH is far more than the binary complexes. Further study showed that the transfection efficiency of the ternary complex with cell-dependent, time-dependent and serum tolerance manner.We successfully constructed the ternary complex vector system which was used to study delivery of therapeutic genes plasmid pTNFSF10-EGFP. With EGFP as an index, indirect quantitative treatment gene pTNFSF10-EGFP transfection efficiency by flow cytometry. PCR quantitative analysis was used to study the TRAIL therapeutic gene expression. The results show that the therapeutic gene transfection of ternary complex vector system in Hela and MCF7two kinds of tumor cells is superior to the positive control, Lipofectamine2000. TRAIL protein expression level is1.5times than the positive control. Western blot analysis showed that of TRAIL activation of the apoptotic signaling pathway protein caspase-8expression were up-regulated and anti-apoptotic factor BCL-2expression decreased. This proved that TRAIL is to promote apoptosis of tumor cells through the extracellular or cytoplasmic pathway and two kinds of intracellular or mitochondrial pathway were involved.Select the optimal prescription of the complexes to investigate the internalization pathways and whether the certain pathway could result in effective transfection by using of specific endocytic inhibitors. Ways to inhibit clathrin mediated endocytosis (CME) include chloropromazine (CPZ), hypertonic glucose solution. For inhibition of caveolae mediated endocytois (CvME), the inhibitors are filipin III and genistein. The inhibitors against macropinocytosis is5-(N,N-Dimethyl)amiloride hydrochloride (DMA). After inhibition of certain internalization pathway, the uptake of complexes and EGFP was determined. The results showed that hypertonic glucose and chloropromazine and Filipin Ⅲ, genistein against CvME decreased the uptake and transfection seperately. DMA had no significant effect on the uptake and the transfection efficiency. So, we can infer that the ternary complexes was internalized by the cells through both CME and CvME pathways and both of them lead to effective transfection. Macropinocytosis was not involved in the uptake of the complexes. To further clarify the internalization routes, we labelled complexes, CME and CvME endocytic markers and colocalized their relative position in the cells afer internalization. The colocalization experiments proved that the complexes are endocyzed by the cells with both CME and CvME pathways.Endosome-lysosome system, cell skeleton and motor proteins play very important roles in the transport of membrane vesicles. We investigated the effects of endosome-lysosome acidification inhibitors NH4Cl and monensin, actin depolymerizing agent (Cytochalasin D), microtubule-depolymerizing agent (nocodazole, NCZ), microtubule-stablizing agent (paclitaxel, PTX), dynein inhibitor sodium orthovanadate (SOV) and kinesin Eg5inhibitor (monastrol) on the transfection efficiency by determination of EGFP. The results indicated that nocodazole decreased the transfection efficiency significantly whereas paclitaxel had little effect. Cytochalasin D decreased the transfection efficiency by about50%. Sodium orthovanadate decreased the transfection efficiency by35%and monastrol had no significant effect on the gene expression. These results demonstrated that endosome-lysosome system, cell skeleton and motor proteins had pronounced influnce on the transport of the complexes.To further prove that endosome-lysosome system were involved in the transport of the complexes, their relative position was investigated with colocalization experiments. The results suggested that the complexes was transported through endosome-lysosome to nucleus after internalization.B16F10subcutaneous tumor models and lung metastasis tumor model were involved to study the transfection efficiency of ternary complex in vivo. For subcutaneous tumor model, the expression results in vivo showed that the CM-PLH can significantly increase the expression of the luciferase in the tumor. The expression of the ternary system is4times than the binary system. Transfection efficiency of ternary vector system in lung of lung metastasis model was also4times than the binary vector system; the luciferase expression of ternary complex system in lung of tumor-bearing of rat is2.8times than the lung of healthy rat. The above experiments show that CM-PLH/PbAE/DNA ternary complex vector system is a safe, effective and stable non-viral gene delivery system which provides a new method for the rational design of carrier systems, but also for further studies in vivo.