Preparation And Anti-tumor Effect Of DNA Nano-micelle Modified By Low-molecular Weight Polyethylenimine And Difunctional Peptide TLyP-1-NLS

A tumor is a swelling caused by overgrowth tissue.Malignant tumours are charactherized by the ability to metastasize,that is,to invade anatomically distant normal tissues and to seed and growth there.Malignant cells can cause either regional lymphadenopathy which was hard to cure.The traditional treatments such as surgical operation,chemotherapy and radiotherapy have a number of issues,like physical injury,high risk and incidence.Gene therapy because of its unique breakthrough point brought hope for fighting the tumors.The main obstacle of gene therapy is a lake of a useful and controllable vector.Non-viral vector because of its low cytotoxicity,easy preparation,no infection and risk of deterioration and so on advantages favored by researchers.Recently,Cationic polymer polyethyleneimine(PEI)have attracting much attention due to its unique "proton sponge effect" and good transfection efficiency.However,if the gene delivery system relies on PEI completely,there are some problems,for instance,high cytotoxicity,low transfection and poor stability.To solve these problems,we trying to synthesizing a PEI derivative by cross-linking branched-PEI 2kDa with quaternization chitosan(named as OTMCS)and then conjugated with a bifunctional peptide t Lyp-1—NLS(named as K12)to pursue high transfection and low cytotoxicity.Chapter one introduced the application and major drawback of PEI and chitosan of being gene delivery vectors,and the necessary to modify chitosan first before connecting with PEI.Peptide t Lyp-1 can specifically bound to NRPs receptors which were highly expressed in tumor neovascularization.NLS is a specific signal aminoacid sequence mediating proteins entering cell nuclei and targeting to cell sub micro-structure.Therefore,we attempt to yield a new peptide by conjugating tLyp-1 with NLS to improve cell selection,promote cargo transport and enhance transfection efficiency.Chapter two introduced the specific methods of constructing a new gene delivery system.Chitosan was modified by Potassium borohydride and N-methyl-2-pyrrolidone to OTMCS first,and then connected to activated PEI 2kDa to structure OTMCS-PEI.Bifunctional peptide K12 was conjugated by solid state reaction and then crosslinking with polymer precursor OTMCS-PEI by SMCC.Through controlling the mole ratios of K12 to OTMCS-PEI,we can obtain OTMCS-PEI-K12-2,OTMCS-PEI-K12-5,OTMCS-PEI-K12-10.Finally,the peptide K12 and functional polymer OTMCS-PEI-K12 were characterized by MS,HPLC and 1H-NMR.The results indicated that K12 and OTMCS-PEI-K12 had been successfully synthesized with high purity and can be used in follow-up study.By in-vitro experiments,chapter three showed the physical and chemical properties of OTMCS-PEI-K12,the results were compared with OTMCS-PEI and PEI 2kDa.Through acid-base titration,OTMCS-PEI-K12 displayed appropriate buffering capacity which can promote DNA to escape from endosome.Agarose electrophoresis experiment was used to inspect the DNA condensation capacity.The results indicated that the polymer with different K12 feeding ratio all showed wonderful DNA condensation ability and can protect DNA against at least 50 U DNase I/?g DNA,200?g/mL sodium heparin degradation.Samples of SEM and Zeta potentiometer analysis showed that the particle sizes of OTMCS-PEI-K12/DNA complexes were about 100-600 nm and zeta potentials about 1—36mV,in addition,the surface topography of nanoparticles were regular near spherical.At room temperature,the complexes were fairly homogenous and could be slowly degraded to small molecules after 60 h.More importantly,compared with PEI 25 kDa,OTMCS-PEI-K12 showed lower cytotoxicity even at high concentration.Taken together,the polymer OTMCS-PEI-K12 has good biodegradability and biocompatibility.In chapter four,we used Green fluorescent protein pEGFP-N2 and Luciferase pGL3-Control as reporter genes,to measure gene transfection efficiency of the OTMCS-PEI-K12 in U87 and B16 cell lines,and to explode the complexes intracellular operation mechanism.PEI 25 kDa was used as positive control.The results showed that OTMCS-PEI-K12 developed the highest transfection efficiency than any other polymers with the same cell line and polymer-to-pDNA w/w ratios,demonstrating that the modification of PEI was necessary.In addition,the transfection efficiency of OTMCS-PEI-K12 was better in U87 than that in B16,due to the t Lyp-1 special NRPs receptors bounding ability.Polymers with different K12 feeding ratios revealed different transfection efficiency,OTMCS-PEI-K12-5 showed the best result.As the w/w ratios increasing,the transfection efficiency of polymers all offered upgrade firstly than descending latter tendency,probably due to the increasing cytotoxicity.Chemical inhibitors PTX,NCZ,SOV and CytoB were used to interfere with microtubule dynamic balance and restrain activity of dynein to investigate intracellular transport mechanism of OTMCS-PEI-K12/DNA.The results showed concentration-dependent effect of CytoB and SOV on the response alterations of transfection efficiency.Indicating that transfection of the nanoparticle complexes in cell interior were mediated by the specific receptor and suitable for undifferentiated cells.On the base of the previous study,OTMCS-PEI-K12-5 was selected as the subsequent experimental material for its well transfection efficiency.In chapter five,A U87 mouse tumor model has been successfully establish to test the targeting property of OTMCS-PEI-K12-5,the method of tail vein injection and intra-tumor injection were both taken.The result showed that OPK5/DNA nanoparticles with the w/w of 12 have the best biocompatibility and tumor targeting ability to induce the apoptosis of tumor cells.In conclusion,a new polymer OTMCS-PEI-K12 was successfully developed with high transfection efficiency,low cytotoxicity,well physicochemical property and tumor targeting,indicating that the new synthetic polymer is suitable for using as a safe and efficient gene deliver vector.