Preparation Of Biodegradable Nano-gene Vector P407-PEI-K12 Modified By Bifunctional Peptide

Gene therapy is a new type of treatment in recent years,based on genetic engineering and molecular genetics.As the biological basis of tumor is a genetic mutation,gene therapy has greater potential to eradicate cancer,compared with the traditional therapeutic regimens.Gene therapy involves three aspects: target gene,gene vector and target cells.Firstly a kind of high molecular weight PEI derivate(P407-PEI)was synthesized by cross-linking low molecular weight PEI with Pluronic P407,then the tumor-targeting peptide tLyP-1 was used to connected with the nuclear loclization peptide NLS to yield a bifunctional peptide tLyP-1-NLS(K12),and lastly tLyP-1-NLS(K12)was adopted to modify the PEI derivates.Thus a new type of non-virus gene carrier(P407-PEI-K12)was prepared.The DNA nano-complex could self-assemble by mixing pGL3-Control solution and P407-PEI-K12 solution.The free P407 was also added to the complex solution to form a kind of sustained-release hydrogels.In Part one of this article,an overview was given on developing status of PEI,poloxamer,functional peptides as nonviral gene vectors.The main reason for limiting its clinical application was discussed and the strategies aiming at these problems were analyzed.Part two focused on synthesis and characterization of P407-PEI-K12.At first,the bifunctional peptide K12 was prepared using the solid phase Fmoc method.K12 was composed of the tumor-targeting peptide tLyP-1 and the nuclear loclization peptide NLS.The structure and purity of K12 were determined by MS and HPLC,respectively.Then P407 was used to crosslink with PEI 2 KDa to obtain a high molecular weight PEI derivate P407-PEI.Lastly P407-PEI was modified by bifunctional peptide tLyP-1-NLS(K12)to prepare P407-PEI-K12.P407-PEI and P407-PEI-K12 was characterized by 1H-NMR.The results indicated that thebifunctional peptide tLyP-1-NLS was synthesized successfully with the purity of98.75%.P407-PEI and P407-PEI-K12 has been also prepared successfully.The physical characterization of P407-PEI-K12 was in Part three.Firstly the complexes particle had good pH buffering and hydrolysis capabilities.Secondly the complexes particle sizes range was 200-500 nm,which were suitable for gene delivery.Thirdly the DNA binding ability of Polymer was measure though gel retardation experiments.DNA was condensed effectively by P407-PEI-K12-h and P407-PEI-K12-l.Fourthly the stability of the P407-PEI-K12/DNA was evaluated by serum,DNase I and sodium heparin.These results indicated that the P407-PEI-K12/DNA had advisable stability in the blood.And P407-PEI-K12 holds high potential as a safe gene vector.In part four,plasmid pEGFP-N2 and pGL3-Control were used as reporter genes to measure gene transfection efficiency of the vectors in vitro.The transfection efficiency of P407-PEI-K12 was demonstrated to be much higher than P407-10g-PEI and PEI in vivo.The addition of free P407 enhanced the transfection efficiency of P407-PEI-K12 and the greatest enhancing effect was achieved when the concentration of free P407 was 0.09%.At a certain temperature(37?)and the concentration,the P407 solution can form a homogeneous,delicate,transparent gel.At the same time,the free collagen had been added to the P407-PEI-K12/pGL3-Control solution to form a kind of sustained-release hydrogels for verify transfection capacity.The results indicated that the new sustained-release hydrogels can enhance the therapeutic effect of gene therapy on cancer.In this study,we developed a new gene vector by crosslinking LMW PEI with P407,then conjugated with bifunctional peptide K12,which composed of the tumor-targeting peptide t LyP-1 and the nuclear loclization peptide NLS.The new non-viral gene vector P407-PEI-K12 can reduce cytotoxicity on the premise of ensuring higher transfection efficiency and improve its tumor targeting.Then,a kind of sustained-release hydrogels had been made via free P407 had been added to the P407-PEI-K12/pGL3-Control solution for sustained expression.In the process of gel dissolution,the free P407 enhanced the transfection efficiency of P407-PEI-K12.