Study On The Nanoscale Transport Vector Of The Prodrug Of Podophyllotoxin And FCY1 Suicide Gene

At present,cancer is one of the most important causes of death in the world and a serious threat to human life.With the increasing number of cancer patients worldwide,traditional treatment methods,including surgical treatment,chemotherapy and radiotherapy,have certain curative effect on cancer,but their limited therapeutic effect and severe toxic and side effects make them still unable to meet the current clinical needs.With the development of nanotechnology and its application in imaging and tumor molecular biology,it provides more effective strategies for the early diagnosis and treatment of tumors.In this study,the combination of nanomaterials GO-PEG and DCM-S-PPT,a glutathione(GSH)responsive prodrug of podophyllotoxin(PPT),achieve good tumor inhibition effect.The combination of polyethyleneimine(PEI)functionalized embeded dual mesoporous silica nanospheres(EDMSNs)and gene therapy(CD/5-FC suicide gene system)resulted in the effective treatment of HCT116 transplanted tumor in vivo.The research content and main conclusions of this paper are summarized as follows:1,By combining 6-arm PEG-NH2 with carboxylated GO through covalent binding,the GO-PEG was obtained.Then,the GSH-responsive prodrug of podophyllotoxin(DCM-S-PPT)was loaded onto the surface of GO-PEG by means of accumulation and hydrophobic interaction to obtain GO-PEG/DCM-S-PPT.The cytotoxicity of GO-PEG/DCM-S-PPT on cells with different GSH content were investigated.In vivo,by tail vein injection,the inhibitory effect of GO-PEG/DCM-S-PPT on HeLa transplanted tumor in mice was studied.It was found that PEG-functionalized GO was a nanomaterial with good physiological solution stability and biocompatibility,which provides the possibility for future use as a drug delivery carrier.DCM-S-PPT could be loaded on GO-PEG with a high load rate 13 8%.GO-PEG/DCM-S-PPT could react with intracellular GSH and release active molecule PPT to induce cell apoptosis and affect cell viability.GO-PEG/DCM-S-PPT can specifically react with GSH in HeLa cells with high GSH content,release active molecule PPT and inhibit the proliferation of tumor cells,which is conducive to reducing the toxic and side effects of PPT on normal cells with low GSH content.Animal experiments have shown that GO-PEG/DCM-S-PPT has good targeting of tumor tissues,and a good effect of inhibiting tumor growth.2,By amplifying FCY1,the gene encoding cytosine deaminase(CD),from the yeast genome,the recombinant plasmid pEGFP-N1-FCY1 was established and the suicide gene system CD/5-FC was formed.Then,we established a new capillary zone electrophoresis method that can qualitatively and quantitatively analyze the trace 5-FC and 5-FU in cell extracts at the same time.Using this method,5-FU was detected and quantitatively analyzed in the extracts of 293T cells transfected with pEGFP-N1-FCY1 and HCT116-FCY1 cells expressing CD-GFP fusion protein cultured with 5-FC.The result further explained the cause of apoptosis of HCT116-FCY1 cells cultured with 5-FC.On the one hand,the detection results proved the success of the construction of the suicide gene system;on the other hand,it proved a capillary zone electrophoresis method for the detection of trace 5-FC and 5-FU in the cell extract,providing a quick and simple analysis method for the related research on the CD/5-FC suicide gene system.3,The key to carrying out suicide gene therapy is to obtain effective gene delivery vector.Therefore,based on the above research work,the research on PEI-functionalized GO as the nano transport carrier of CD/5-FC system was carried out.The 25 kDa branched PEI was used to bind to the GO surface by attraction of positive and negative charges to obtain the GO-PEI.Biocompatibility was evaluated by cell activity assay.In addition,its ability to bind plasmids,transfection efficiency of cells in vitro,and transfection effect in vivo were investigated to evaluate GO-PEI as a nano gene delivery vector.Studies have shown that the GO modified by PEI has physiological solution stability and stable positive charge on the surface,and can completely bind to plasmid DNA under low N/P conditions.Under a certain concentration range or N/P condition,GO-PEI has good biocompatibility and good transfection effect in vitro.However,by tail vein injection and intratumoral injection of tumor-bearing mice,fluorescence imaging and Western blot analysis of protein expression showed that the synthesized GO-PEI could not deliver genes to tumor tissues in vivo,and could not achieve the target protein expression by tail vein injection and intratumoral injection.4,In order to obtain an effective nanoscale gene delivery vector,the EDMSNs as a nanometer carrier for CD/5-FC suicide gene system was studied.First,EDMSNs were modified with different amounts of 25 kDa branched PEI to obtain EDMSNs-PEI complexes with different PEI loads.The transfection effect of EDMSNs-PEI as gene delivery vector was evaluated by investigating its effect on cell activity,binding ability with plasmids,transfection efficiency in vitro,and transfection results after tail vein injection and intratumoral injection of tumor tissue in vivo.The anti-tumor effect of EDMSNs-PEI with CD/5-FC suicide gene system was evaluated by investigating the tumor growth of HCT116 transplanted tumor in vivo.Agarose gel electrophoresis showed that EDMSNs-PEI had good plasmid binding ability.Cytotoxicity experiments showed that EDMSNs-PEI had good biocompatibility within a certain concentration range.In vitro cell transfection determined that the mass ratio of EDMSNs and PEI was 2,that is EDMSNs-PEI(2)transfected HCT116 cells had the best effect.Tail vein injection could not achieve transfection of tumor tissue,but intratuminal injection could achieve successful transfection of tumor tissue,indicating that PEI-modified EDMSNs had the ability of gene delivery to tumor tissue.Finally,in vivo inhibition of tumor growth was studied by intratumoral injection.EDMSNs-PEI/pEGFP-N1-FCY1/5-FC group could slow tumor growth,which had a good effect of inhibiting tumor growth in vivo,and had a low toxic and side effect on mice.In conclusion,our study shows that PEG-functionalized GO can be used as a nano-carrier drug delivery for DCM-S-PPT.According to the characteristics of tumor tissue and microenvironment,the passive targeting of nanomaterials is combined with the active targeting of prodrugs,making a beneficial attempt to achieve effective treatment of tumors.However,PEI-functionalized GO,as a gene transport vector for the CD/5-FC suicide gene system,only achieved good transfection effect at cellular level,but failed to achieve the expected transmission effect at in vivo level,so further optimization is needed in the future.Studies on EDMSNs as gene delivery vector showed that PEI-modified EDMSNs had the gene delivery capacity of tumor tissue,and could inhibit tumor growth by intratumoral injection.These studies provide experimental evidence for the development of safe and effective cancer treatment strategies by combining nanomaterials with chemotherapy and/or gene therapy.