Hydrophobic Cationic Polymers PP80Delivery Of Anti-tumor Gene Therapy

Safety and efficacy of gene carriers were regarded as the basis for tumor gene therapy.Recently, liposomes, lipoid and polymer had been used as gene carries to substitute virusvector because of showing advantages such as lower adverse effect and easier obtaining.However, modification and alteration for the polymers to improve its transfection efficiencyand biocompatibility were primarily considered in material synthesis area. Branchedpolyethylenimine, especially PEI25kDa, has generally been accepted as gene carrier,howerver, its nonbiodegradability, high cytotoxicity from much charge density limited itsapplication in clinical trials. Through Modification of PEI to improve the biocompatibility andtransfection efficiency was regarded the foundation to further apply it. Some polymer graftedby hydrophilic segments showed favorable biocompatibility but its transfection efficiency wasreduced due to the increasing of particle size. Therefore, a series of hydrophobic modifiedpolymers were gradually pay noticed. The hydrophobic modified materials possessed betteraffinity to the lipid surface of cell membrane which promoted its entry into the cells. Studieshave shown that hydrophobic cationic polymers could effetively mediate the delivery of drug,siRNA and plasmid DNA with low cell toxicity. In our previous study, we synthesized PPhenby NCA ring opening polymerization and monomer grafting of phenylalanines (Phe) toPEI25K which possessed small particle size and good stability. In order to expore thepotential of PP80as a gene carrier for tumor treatment in vivo, it is necessary to assess thesafety and efficacy of gene carriers.Firstly, PP80was complexed with GFP gene and separately added into different kinds ofadherent tumor cells grown on six-well plate. As the results showed GFP was expressed inHeLa, MCF-7and HepG2cells. The transfected efficiency was up to60%in HeLa cells but30%and20%separately in MCF-7and HepG2celles. It indicated that PP80possessed goodaffinity for tumor cell, especially for HeLa cells. These results demonstrated that PP80was aneffective gene carrier for targeting tumor cells.In order to verify the safety of PP80as gene carrier, the biocompatibility wasinvestigated in vitro and in vivo. As shown in WST-1experiments, there were no obviousinfluences on NIH3T3cell growth after7days incubation with PP80-pDNA complex.Subsequently, the complex was added into fresh rabbit blood incubating for30min, theresults showed that only PP80strongly induced hemolysis but not PP80-pDNA complex. Inaddition, this complex was also shown no inhibition on the tumor growth when injected intoHeLa xenograft compared with nude pDNA or PBS. To investigate the possible inflammatory reaction induced by PP80, the levels of cytokines were measured. The results showed thatthere were no significant increases in either mIL-12p70or IFN-g in any groups. Meanwhile,CK, LDH and CK-MB, ALT and AST, as well as Cr and BUN, were quantitatively examinedto evaluate the functions of heart, liver and kidney, respectively. As shown in Table1, none ofthese enzymes were significantly changes in all mice. Moreover, pyrogenetic reaction fromrabbit treating with complex was also not observed. Finally, the complex was injected intomice peritoneal cavity to examine its genetoxic by micronucleus tests, from the resultsverdicted that there was no inhibition chromosomal aberration. Taken together, the aboveresults demonstrated that PP80was safe for the delivery of pDNA in vitro and in vivo.The above study exhibited that PP80was well biocompatible with animals and effectivelymediated gene expression in tumor. To verify the further application in gene therapy, thecomplexed ratio of PP80and pDNA was optimized by assessing the gene expression in HeLaxenograft. From the gel retardation and size assay, the luciferase was abundantly expressed inthe tumor tissue which was injected PP80/pDNA complex at the ratio of0.5. Subsequently,tumor growth rate was decreased significantly by injecting PP80-pKH3-rev-casp-3complex.In the process of therapy, poly (ADP-ribose) polymerase (PARP1) was cleaved into twofragments of85kDa and25kDa, which lost the repaired function for the damaged DNA due tothe overexpression of rev-casp-3, and at last induced cell or tumor apoptosis. Meanwhile, thestatus of cell proliferation in tumor tissue was also detected, discovering that the Ki-67positive cells were strongly decreasded in rev-casp-3overexpressing group, it is supposed thatthe slow growth of tumor was possibly due to cell apoptosis inducing by rev-casp-3. TUNELanalysis also provided evidence that PP880deliveried rev-casp-3can effectively inhibit thetumoral growth mainly though apoptotic pawthway. This apoptosic phenomenon was furtherconfirmed by overexpressing rev-casp-3in HeLa cells.To further verify the extensive applicability of PP80as a gene vector, we also combinedPP80with a single-chain antibody gene (scFv1C9) against fibroblast growth factor1(FGF-1)to cure tumor. In our study, it was demonstrated that scFv1C9resulted in the G0/G1areest byregulation of the expression of p21and CDK2. Futhermore, PP880deliveried scFv1C9caneffectively inhibit HepG2xenograft growth.In summary, these work mainly foucus on developing the safety and effectiveness ofhydrophobic cationic as gene carrier. First of all, through biological safety evaluation, wedemonstrated PP80was safety and can be applied in vitro and in vivo. In addition, PP80caneffectively deliver rev-casp-3and scFv1C9expression in tumor and inhibit xenogaraft growth.These works have provided that PP80was a potential gene carrier for cancer gene therapy.