Ligands Conjugated Cationic Polymers As Non-viral Gene Vector

The use of various synthetic lipids and polymers to deliver DNA for gene therapy applications has been the subject of intense examination for the last 20 years. Our understanding of the processes involved in the delivery of DNA, although still limited, can be described in terms of specific physical and chemical barriers encountered along the delivery pathway. Successful engagement of this pathway involves avoiding inactivation in the extracellular compartment and initial favorable interactions with the cell surface. Internalization of the delivery system by endocytosis results in a poorly defined endosomal trafficking process which, if not escaped, leads to degradation of the therapeutic DNA in lysosomes. For the small fraction of material that is able to escape this vesicular trafficking pathway, the cytosol provides additional physical and metabolic barriers to further trafficking to the nucleus. Finally, nuclear uptake has been demonstrated to be a significant barrier to gene delivery. In this work, the daul-ligand cationic polymeric nanovectors with target ability and transmembrane function were desigined for highly efficient gene delivery to tumor cells.First, cationic polysaccharides have been of interest and importance for safe and efficient gene delivery systems. Coixan polysaccharide (CP), derived from a traditional Chinese medicine, possesses varieties of pharmacological activities. Herein, coixan polysaccharide-graft-polyethylenimine (Mw～1200 Da)-folate (CP-PEI-FA) was prepared as an effective vector for in vitro and in vivo tumor-targeted gene delivery. The structures of the polymers were characterized, and the DNA condensation capability, particle sizes, zeta potentials, cytotoxicity and in vitro (and in vivo) transfection of CP-PEI-FA were examined. The cytotoxicity of CP-PEI-FA was significantly less than that of high molecular weight PEI 25 kDa and close to that of PEI 1200. The in vitro transfection of CP-PEI-FA was tested in C6 and HeLa cells (folate-receptor (FR) positive cells) and A549 cells (FR-negative cells). CP-PEI-FA demonstrated high levels of targeting specificity and good gene transfection efficiency in FR positive cells. Importantly, in vivo transfaction assay indicated that CP-PEI-FA is also quite effective in tissue tumors. These results clearly show that CP-PEI-FA is a safe and effective polyplex-forming agent for both in vitro and in vivo transfection of plasmid DNA.In order to overcome the barrers of cell membrance, for the first time, target ligands (folic acid, FA) and cell-penetrating peptides (octaarginine, R8) were coupled with the gene vectors (PEI600-CyD, PC) composed ofβ-cyclodextrin (β-CyD) and low-molecular-weight polyethylenimine (PEI, Mw 600) to form the daul-ligand nanovectors for highly efficient gene delivery to tumor cells. The resultant ternary nanocomplexes of FA-PC/R8-PC/pDNA produced excellent gene transfaction abilities in vitro and in vivo. This study provides a promising means to produce novel daul-ligand gene nanovectors for in vivo applications.PC-CC9 were designed and synthesized with different ratios of peptide CC9 and The structures of the polymers were characterized, and the DNA condensation capability, particle sizes, zeta potentials, cytotoxicity and in vitro (and in vivo) transfection of PC-CC9 were examined. The high gene transfection efficiency has been proved when PC-CC9 condensed with pEGFP-N1 and pGL-3 plasmid. The transfection efficiency of PC-CC9/DNA (CC9 conjugated) was also comparied with PC/DNA+CC9 (CC9 mixed). And the cells apoptosis and cell cycle were determined by flow cytometer after the cells transfection by PC-CC9/miRNA-34a.eGFP and PC/ miRNA-34a.eGFP+CC9. Western blot and qRT-PCR experiments proved that PC-CC9/miRNA-34a.eGFP and PC/miRNA-34a.eGFP+CC9 complexes could inhibitor the miRNA and protein expression of BCL-2,E2F3,CyclinE2和c-myc. After the Balb/c mice were injected with PC-CC9/miRNA-34a.eGFP and PC/miRNA-34a.eGFP+CC9 complexes solution by tail intravenous injection with the optimum ratio, tumor volume and tumor weigh of mice were investigated for 21 days. The results showed that the conplexes could inhibit the pancreatic cancer growth to great extent.The research above indicated that the couple used of target and cell penetrating properties couled overcome the barrers of extracellular and mambrance of cell, improve the targedted and transfection efficiently of backbone in vitro and in vivo. Therefore, our study has a great significance for cancer clinic therapy.