Application Of Polyamidoamine Dendrimers Gene Delivery System To Gene Delivery In Vitro And In Vivo And Gene Therapy Of Leukemia

【The research status of nanoparticles】Gene therapy is an efficient and economical approach to disease prevention and therapy. In order to succeed in gene therapy however, efficient delivery and expression of exogenous genes into target cells are of critical importance. To date, about three quarters of gene therapy trials undertaken are for cancer, mostly based on recombinant viral vectors to carry the gene of interest. Although recombinant viral vectors have high transfection efficiency, they are associated with immunogenicity, toxicity, lack of tissue specificity, unknown long-term effects et al. Non-viral vectors have become attractive alternatives, but most non-viral vectors are limited for their low efficiency of transfection, especially in vivo. The recent topic has been focused on new kinds of efficient, safe and targeting non-viral vectors.With the development of nanotechnology, recent interest has been focused on a new kind of non-viral vector, nanoparticles, which have the characteristics of surface effect, small size effect and macroscopic quantum tunneling effect. Nanoparticles have low cytotoxicity, delivering the interesting DNA to cells efficiently, attaching gene therapy molecular, such as DNA, ODN, PNA, et al., in their surface and are taken up by endocytosis, then nanoparticles are degraded by lysosome and the targeting genes are released and enter the nucleus to play a role of gene therapy. Among these nanoparticles, polyamidoamine (PAMAM) dendrimers have significant advantages over others. PAMAM dendrimers are new class of highly branched spherical polymers that are highly soluble in aqueous solution and have unique surface of positively charged primary amine groups, which make them form stable complexes with plasmid DNA, oligonucleotides, antibodies or drugs. The molecular weight of the dendrimer doubles, the number of surface amine groups exactly doubles. So PAMAM dendrimers are effective in gene transfer or applications in different areas of biology and medMne with high transfection efficiencies and minimal cytotoxicity.【The ability of PAMAM binding and protecting DNA】This study detailed some biological characteristics of G5 PAMAM dendfimers, their distribution in vivo and gene therapy of chronic myelogeneous leukemia. Atomic force microscopy indicated that the diameter of PAMAM is about 8 nm±0.5nm. As a gene vector, the DNA binding ability is essential requirement. DNA binding ability of PAMAM has been determined by observing whether DNA mobility was retarded on gel analysis and co-sedimentation assay. Results showed that from pH 5 to pH 9, PAMAM can bind DNA, but at pH 3, PAMAM couldn't bind DNA, which means that PAMAM dendrimers could bind DNA and guarantee its integrity at a certain wide range ofpH values. The binding is achieved by interaction between the negatively charged DNA phosphate groups and positively charged gene vectors. DNase I digestion assay showed that PAMAM dendrimer/DNA complexes could protect DNA from digestion at different pH values and in serum. Whether PAMAM and PAMAM/DNA complexes influenced cell viability was investigated in several cell lines, including COS7, SMF, K562 and Jurkat cells through MTT assays, which showed that PAMAM/DNA complexes showed no obvious cytotoxicity from ratio of 2:1 to 4:1, but at ratio beyond 6:1, these complexes had some cytotoxicity.【Study of PAMAM and PAMAM/DNA complexes in vitro and in vivo】Besides the requirements for gene delivery mentioned above such as high affinity for nucleic acids and small size, the ability to cross the cell membrane is necessary too. In this study, PAMAM was shown to internalize and accumulate at the cytoplasm and nucleus of the treated cells by transmission electron microscopy. To test the ability of PAMAM to transfer gene in vitro, assays of delivery of reporter plasmid DNA into cells were performed, pEGFP-C2, encoding green fluorescent protein, reporter gene system was chosed to evaluate the efficiency of transfection. PAMAM transfer exogenous gene into COS7 cells. Maximal transfection and expression of GFP were obtained when the gram ratio of PAMAM to DNA was 4:1.To define the tissue distribution of PAMAM/DNA in vivo, tissue frozen section assay, western blotting and transmission electron microscopy ass were analyzed in many organs after the intravenous injection of PAMAM/DNA to BALB/c mice. PAMAM mediated reporter plasmid EGFP-C2 encoding green fluorescent protein in vivo was analyzed after intravenous injection of PAMAM/DNA complexes to BALB/c mice by fluorescent microscopy. Fluorescent micrographs showed fluorescent signal apperared in cells of liver, kidney, lung and spleen compared with the control, but in heart, brain, stomach and intestine, there were only very weak or no GFP expression. It was further verified by Western blot that GFP could expressed in heart, brain, lung, liver, kidney, spleen, stomach and intestine tissues, and the expression was stronger in liver, kidney, lung and spleen. With electron micrographs, it could be seen that PAMAM and PAMAM/DNA complexes can be distributed in the liver, kidney, lung, spleen, pancreas, marrow, lymphonode and brain. After intravenous injection, PAMAM was distributed in the renal tubule, indicating that PAMAM can be excreted from the kidney, not resulting in cumulation.【Effect of PAMAM/DNA complexes on gene therapy of chronic myelogeneous leukemia】Leukemia is one of the most threatening hematological malignant cancers currently, and has distinct advantage in gene therapy field, so many researchers pay more attention to the gene therapy of hematological system disease, p16 gene is an antioncogene, and deleted in 25～64 % leukemia cell lines. LRRC4 gene is cloned by our laboratory, which involves in signal pathway as a receptor, receptor kinase, or adhesion molecule.To determine the possibility of the anti-tumor effect of p16 gene and LRRC4 gene, and the application of PAMAM as a vector for gene therapy, a series of experiments had been carried out 24 hrs after mice injected cyclophosphamide (2 mg) for two days, K562 cells were inoculated i.v. into the SCID mice. Treatment was given once a week from the 2nd day to the 28th day. Results indicated that PAMAM/pl6＋LRRC4 could decrease the percentage of immature granular leudocytes and monocytes among the peripheral blood, alleviate the infiltration of tumor cells into the liver, spleen and marrow. The average survival time of PAMAM/pl6＋LRRC4 treated groups were prolonged for 30 % compared with the PAMAM treated group. The effect of PAMAM/pl 6＋LRRC4 treated group was more effective than that of PAMAM/p16 and PAMAM/LRRC4 treated group. These results suggest that p16 and LRRC4 have in vivo anti-tumor activity in the human leukemia xenograft models when used together and PAMAM was an efficient vector for in vivo application.In conclusion, PAMAM can bind and protection DNA effectively, be uptaken by cells, and distributed in cytoplasm and nuclear of cells in vitro and in vivo. Using reporter gene system, PAMAM was demonstrated to transfer exogenous genes to cells and express encoded protein in vitro. PAMAM can transfer exogenous genes to organs, such as liver, kdney, lung, and spleen, and express encoded protein. In this study, PAMAM was firstly used as non-viral gene vector for gene therapy of leukemia. Our studies also demonstrate potent antitumor activity of p 16 and LRRC4 used together in K562 xenograft models, p16 and LRRC4 used together could decrease the percentage of immature granular leudocytes and monocytes among the peripheral blood, alleviate the infiltration of tumor cells into the liver, spleen and marrow. The average survival time of p16 and LRRC4 co-treated groups were longer than that of the PAMAM treated group. A novel non-viral gene vector which is somewhat characteristic of high efficiency, safety, targetting was developed using the advantage of nanoparticles. This study has also provided foundation for gene therapy, espacially for gene therapy of CML disease. Thus, our findings provide new insights into the therapy of leukemia, and polyamidoamine dendrimers, high-efficiency, low-cytotoxicity gene vector, appear to have potential for fundamental research and gene therapy in vitro and in vivo.