Perparation Of DNA Nano-micelle Modified By Difunctional RGD-TAT And Its Intracellular Transpotr Mechanism

Gene therapy provides a promising tool to eradicate cancer by treating it at its source.The key to gene therapy is finding an ideal gene delivery vector. Delivery of nucleic acidsinto cells using cationic polymers has recently gained a remarkable interest in the field ofnon-viral gene therapy due to their structural diversity, easy production,non-immunogenicity and safety. One of the most effective and widely studied syntheticnon-viral gene delivery vectors is the polycation polyethylenimine (PEI). However,polyplexes of PEI/DNA have shown three outstanding problems. First of all, long PEIchains are highly effective in gene transfection, but more cytotoxic. Conversely, short PEIchains display lower cytotoxic, but lower efficient. And then, there is a contradictionbetween stability and cell penetrating. To increase the stability of PEI/DNA complexes,their hydrophilicity should be improved. But cell penetrating is affected accordingly andtransfection efficiency decreases. At last, the major drawback with gene delivery using PEIis the lack of satisfactory specificity towards tumor cells because there is no bindingselectivity between the positively charged polycation and the negatively charged bodycells.Above all, in this work at first we synthesized a kind of high molecular weight PEIderivate (P123-PEI) by cross-linking low molecular weight PEI with Pluronic P123. Theαvβ3receptors were highly expressed on tumor cells and tumor angiogenic blood vessels,whereas they were not detectable on quiescent blood vessels. Then we used αvβ3-targetingpeptide RGD, in conjunction with the cell-penetrating peptide TAT to yield a bifunctionalpeptide RGD-TAT named R13which can improve cell selection and increase cellularuptake, and at last adopted R13to modify P123-PEI so as to prepare a new polymeric genevector (P123-PEI-R13). Mechanisms of cellular uptake, subsequent intracellular traffickingand disassembly of this vector were also investigated. The purpose of the present studywas to solve the efficiency-versus-cytotoxicity and tumor-targeting problems of PEI usedas a non-viral gene delivery vector. The new non-viral gene vector P123-PEI-R13couldreduce cytotoxicity of PEI on the premise of ensuring higher transfection efficiency,improve its tumor targeting, increase cellular uptake of gene, and then enhance thetherapeutic effect of gene therapy on cancer. This study could find an available way togene therapy on cancer. The first part of this work was synthesis and characterizations of P123-PEI-R13. abifunctional peptide R13(RGDC-TAT) was prepared using the solid phase method atfirst. The sequence analysis, content assaying and binding assessment of R13to αvβ3positive cells (Hela cells and B16cells) were performed by ESI-MS, HPLC and HRPlabeling respectively. Then we activated Pluronic P123withbis-(trichloromethyl)-carbonate and solid N-hydroxysuccinimide and employed theactivated P123to crosslink PEI2KDa so as to prepare a high molecular weight PEIderivate of P123-PEI. At last R13was used to modify P123-PEI by SMCC andP123-PEI-R13was obtained. All reaction products were characterized by IR or1H-NMR.The results indicated that the bifunctional peptide R13was synthesized successfully withsequence of RGDCRKKRRQRRR, content of95.8677%and ability of binding to αvβ3positive cells in vitro. P123has been activated and P123-PEI has been also prepared bycross-linking PEI2KDa with the activated P123. The bifunctional peptide R13wascoupled with P123-PEI by SMCC successfully. Structural analysis of IR and1H-NMRrevealed the high modification degree and purity of the products, which showed that thesynthesis method had stability, controllability and repeatability.In the second part, the new synthesized gene vector P123-PEI-R13was characterized interms of its physico-chemical properties. Degradation of P123-PEI-R13was estimated bythe measurement of molecular weight. The sizes and zeta potential of polymer/DNAcomplexes in PBS buffer at room temperature were measured using an electrophoretic lightscattering spectrophotometer. The DNA condensation capacity, the resistance to DNase Idigestion and the resistance to FBS, sodium heparin disassembly of P123-PEI-R13wasdetermined by gel retardation experiments. The cytotoxicity of the polymers on the B16and Hela cells were measured using the MTT assay in comparison with PEI25kDa. Theresults indicated that P123-PEI-R13was degraded slowly and the degradation was nearlycompleted after about60h. The degradation profile of P123-PEI-R13could be welldescribed by first-order model. The particle size of P123-PEI-R13/DNA complexes wasaround100-250nm, with proper zeta potential. P123-PEI-R13was able to condense DNAeffectively and neutralized its charge at w/w ratio of2. The nanoparticles can protectplasmid DNA from being digested by DNase I at a concentration of6U DNase I/μg DNA.The nanoparticles were resistant to dissociation induced by50%fetal bovine serum and600μg/mL sodium heparin. P123-PEI-R13showed significantly higher cell viability ascompared with PEI25kDa. In the third part, gene transfection of this vector was investigated in vitro and in vivo.We examined the ability of P123-PEI-R13to transfect Hela cells, B16cells and HepG2cells using the plasmid pEGFP-N2and pGL3-Control. Percentage of GFP transfection andluciferase activity were respectively determined using flow cytometry and a luminometerso that the quantitative and qualitative study on in vitro transfection ofP123-PEI-R13/DNA complexes could be carried out. In addition, we established animaltumor model and investigated distribution and transfection efficiency ofP123-PEI-R13/DNA complexes using the plasmid pGL3-Control in vivo. The transfectionexperiments in vitro showed that transfection efficiency of P123-PEI-R13/DNA complexesincreased in correlation with the w/w ratio and decreased with R13modification degree.On a whole, all synthesized P123-PEI-R13showed much higher gene transfer abilitycompared with PEI2KDa, also higher than P123-PEI and similar to PEI2KDa, evenhigher. The transfection experiment in vivo showed that transfection of P123-PEI-R13inthe HepG2liver tumor model was similar to that in the B16melanoma tumor model.P123-PEI-R13showed higher gene transfer ability compared with P123-PEI and PEI25KDa, ameliorated the distribution of the complexes and improved the transfectionefficiency in tumor tissues significantly due to the produce of R13. This showed thepolymer P123-PEI-R13had tumor targeting in vivo. However, the transfection efficiencyof pGL3-Control in viv was lower than that in vitro.The last part of this work was the mechanisms of cellular uptake, subsequentintracellular trafficking and disassembly of this vector. The internalization pathways ofP123-PEI-R13/DNA complexes were investigated based on the effect of specific endocyticinhibitors on transfection efficiency. The mechanism of intracellular trafficking wasinvestigated based on the effect of endosome-lysosome acidification inhibitors,cytoskeleton and dynein inhibitors on transfection efficiency. The intracellular disassemblyof P123-PEI-R13/DNA complexes was also investigated based on the effect of cytokineand cellular environment on stability of polyplexes. Intracellular localization ofFITC-labeled P123-PEI-R13polyplexes in Hela cells was researched in order to furtherclarify the transport process in the cells. The results indicated that the modification ofP123-PEI-R13with R13made it display new property of internalization.P123-PEI-R13/DNA complexes were endocyzed by clathrin-mediated endocytosis,caveolin-mediated endocytosis, macropinocytosis and possible energy-independent route.After internalization, P123-PEI-R13/DNA complexes could escape from the endosome-lysosome system because of its acidification and further took microtubule as thetrack, dynein as the dynamic source to transport towards the microtubule (+) end, to witnucleus, under the action of microfilament and with the aid of intermediate filament.P123-PEI-R13had a higher affinity for RNA. It was RNA that resulted in the disassemblyof P123-PEI-R13/DNA complexes in the nucleus.We developed a new non-viral gene vector, PEI-P123-R13, by cross-linking lowmolecular weight PEI with P123and further coupling a bifunctional peptide R13to thepolymer for targeting tumor and increasing cellular uptake. This new polymer might be apotential candidate in gene delivery with low cytotoxicity and high transfection efficiency.Also, the mechanisms of cellular uptake, subsequent intracellular trafficking and disassemblyof this vector were investigated. This study can form the base of problems solving andpractical applications of PEI as a non-viral gene delivery vector. The contents of this workdisplay theoretical as well as practical values.