Novel Graphene Oxide Particles As A Plasmid-based Stat3Sirna Carrier For Hepatoma Targeted Therapy

Background:The death proportion of hepatocellular carcinoma (Hepatocellularcarcinoma HCC), which is the primary liver cancer incidence of a diseasein a supreme, would be up to more than90%. There is the reason of thehigh proportion that occult and found late already, disease progressionquick and poor prognosis. Main means of treatment for advancedhepatocellular carcinoma is surgical resection or even for livertransplantation. However，it is too many complications, high cost andpoor prognosis. With the progress of science and technology, people learnto the HCC understanding from macro to micro at point of Molecularbiology view of HCC gene. The progress in HCC incidence and disease iscomposed of oncogene and anti-oncogene in different interaction andpromote the development of the disease. So to inhibit of HCCdevelopment, we can cut the line between oncogenes and anti-oncogeneby down regulation active oncogene related to the realization.Signal transducer and activator of transcription3gene is as regard asoncogenes. According to the report, Stat3in HCC is over expression andinvolve in cell proliferation, differentiation and apoptosis process; Stat3can be activated by the stimulation signal receiving the signal pathway ofcells such as growth factor and so on. Stat3can regulate the transcriptionprocess in nucleus specific DNA fragment, thus sustaining activation stateand leading to the malignant transformation of cells. Recently, Stat3wasinhibited the expression in tumors so as to achieve the anti tumor effect by the RNAi strategy. In another words, Stat3was regard as target genefor the treatment of cancer. Our laboratory in previous work, successfullyconstructed recombinant plasmid Stat3specific siRNA, which can inhibitthe expression of Stat3in hepatocellular carcinoma and prostate tumors.However, it cannot completely block the expression of Stat3.In order to realize to carry Stat3specific siRNA vector (si-Stat3) intotumor cells, it is important to synthetize safe and efficient gene carrier. Inrecent years, graphene oxide, a kind of polymer material, has attractedmuch attention in the reason of its unique electrical, mechanical andthermal properties. The graphene oxide surface was modified to carrygene and enhance the gene transfection efficiency. Polythyleneimine is akind of cationic gene transfer vector with strong binding plasmid capacity,protecting gene escape from nuclease degradation and endosomes.Polyethylene, Glycol can improve the solubility and stability of GO-PEIand prolong the complexes circulation time in vivo. In order to targetinhibit the growth of tumor, folic acid molecules as targeting carrier wasloaded onto GO-PEI-PEG. Meanwhile, the capacity of complexes bindinggene is up to the content of PEI. It means that the higher the content ofPEI and carrier binding stronger capacity. However, PEI has a certaintoxicity to the cell. So it is hanging over head to study the best proportionof GO and PEI as the Stat3specific siRNAgene carrier.Our experiment made use of the GO-PEI-PEG-FA complexes asStat3specific siRNA gene vector to loading the Stat3expression plasmidtargeting to tumor cells. Meanwhile, the infrared photothermal therapyhas no complication as well as damage tumor cells, we take advantage ofthis to use into inhibit tumor development. Objective:Novel graphene oxide particles as a Plasmid-based Stat3siRNAcarrier targeted to inhibit the growth of HCC by folic acid by relatedexperiment in vivo and in vitro.Methods:It is successful implementation of the chemical synthesis ofGO-PEI-PEG-FA complex according to the different of proportion of GOand PEI (GO: PEI=1:10,1:20,1:40,1:60,1:80, and1:100) by UVspectrometer (UV) and atomic force microscopy (AFM) to characteristicthe compound synthesis and surface potential. GO-PEI-PEG-FA (GO:PEI=1:10,1:20,1:40,1:60,1:80, and1:100)complexes respectively were pullinto physiological saline, PBS, stability in culture fluid and serum toobserve the ability of GO-PEI-PEG-FA complex.1%agarose gelelectrophoresis was to detect the ability of the complex of compound withsi-Stat3at different proportion. Cytotoxicity was detectedGO-PEI-PEG-FA on human hepatocellular carcinoma cell lineSMMC-7721by MTT at different proportion of GO-PEI-PEG-FA. Flowcytometry was used to detect transfection efficiency of GO-PEI-PEG-FAcomplexes carrying si-Stat3in H22cells. Observation and detection ofapoptosis for GO-PEI-PEG-FA carried si-Stat3on liver cancer by flowcytometry and inverted fluorescence microscope. Western blot andRT-PCR were used to detect the expression level of protein and gene invivo and in vitro; Immunohistochemical method was to observe theexpression of PCNA, which is responded of cell proliferation ability.Results:GO-PEI-PEG-FA complex was synthesized successfully withdifferent weight ratio of GO and PEI (GO:PEI=1:10,1:20,1:40,1:60,1:80and1:100),surface potential fluctuations of six kinds of complex was in the range of+40.2mV-+52.1mV, dynamic lightscattering can response to the GO-PEI-PEG-FA particle size in therange of442nm-223nm. MTT and transfection efficiency resultsshowed that the most suitable GO and PEI ratio was1:80, correspondingGO-PEI-PEG-FA complexes in PBS, physiological saline, culturemedium and serum possessed good dispersion ability and stability. Invitro experimental results show that GO-PEI-PEG-FA composite can beused as a gene vector delivered plasmid si-Stat3transfected intoSMMC-7721cells and H22cells. Meanwhile gene and protein expressionof stat3in GO-PEI-PEG-FA-si-Stat3(GO-FA-si-Stat3group) group wassignificantly lower than that GO-PEI-PEG-si-Stat3group (GO-si-Stat3group) and the other group. In vivo, GO-FA/si-Stat3targeting therapygroup were significantly prolonged survival time.Tumor volume andweight in GO-FA/si-Stat3group were smaller than the other groups.Compared with the other groups, the expression level of Stat3mRNA andprotein in GO-FA-si-Stat3group was significantly lower. The results ofimmunohistochemistry showed the expression of related proliferationmarkers PCNA in GO-FA-si-Stat3group can significantly inhibited. HEstaining was not morphology abnormality in the heart, liver, spleen,kidney and lung.Conclusion:Novel graphene oxide particles is effective and safe for targeted genedelivery vehicles, meanwhile playing the important role of targetinginhibition of tumor development to enter the tumor cells carrying si-Stat3.By the properties of graphene oxide can absorb infrared, GO-PEI-PEG-FA can further improve the inhibition effect of mouse liver carcinoma insitu with infrared irradiation.