The Preparation Of Functionalized Graphene Oxide Nano Carrier And The Effects On The Proliferation Of The Carrier Loading HTERT SiRNA On Cervical Cancer Hela Cells

Partl The preparation of functionalized grapheneoxide nano carrier and structural characterizationObjectivePrepare functional oxide graphene nano-carrier which is characterized ofthe carrier structure, and detected any cytotoxicity on Hela cell targeting andtransfection efficiencyMethods1、Prepare two kinds of different functions of graphene oxidation nano-carrier: GO-PEG-FA-PyNH₂and GO-PEG-PyNH₂.2、Prove the vectors were successfully constructed or not by characterizingthe carrier structure with Fourier transform infrared spectrometer （FTIR） andUV-visible absorption spectroscopy （UV）; observe the morphological changes inthe functional graphene oxide with atomic force microscopy （AFM）.3、Culture cervical cancer Hela cells,then observe the prepared carrier thathave cytotoxicity or not by WST assay and DAPI staining.4、Observe target and transfective efficiency of tumor cells with laserscanning confocal microscope （LSCM） by transfecting the two differentnanocarrier loading DNA-FAM into Hela cells. Results1、The functionalized graphene oxide nano-carrier, GO-PEG-FA-PyNH₂and GO-PEG-PyNH₂, were successfully prepared.Characteristic absorptionpeak of PEG-FA and PyNH₂were tested with UV which confirmed that PEG-FAand PyNH₂were conjugated to GO, The conjugation of PEG with FA andPEG-FA on GO were further characterized by FTIR. After GO was modified,theheight of the GO was increased and many protuberances were observed on thesurface of GO by AFM, showing that the GO were prepared successfully.2、WST results showed that the PEG modified GO were not cytotoxic.There were no differences between the two groups in morphology of nuclearand cell after DAPI staining, The cell morphology and the nuclear werecompleted in GO-PEG-FA-PyNH₂group. All above indicated that the carrierhad no cytotoxicity.3、Observed target and transfective efficiency of Hela cells which weretransfected into two carrier load DNA-FAM with LSCM. Green fluorescence onthe surface of and inside the cells in GO-PEG-FA-PyNH₂group was strongerthan the GO-PEG-PyNH₂group at the point of1h later,which indicated that theformer FA modification had a better target to tumor, but transfection efficiencyhad no significant differences between two carrier at the point of5h later whichindicated that both were efficient to transfect gene into cervical cancer cells.ConclusionsTwo kinds of nanocarrier:GO-PEG-FA-PyNH₂andGO-PEG-PyNH₂,wereSuccessfully prepared and had no cytotoxicity to Hela cells; transfectionefficiency of the two kinds of carrier was not significantly different, despiteGO-PEG-FA-PyNH₂had better tumor target. Part11The effects on the proliferation of functionalizedgraphene oxide loading hTERTsiRNA on cervical cancerHela cells.ObjectiveTo observe the effects of functionalized graphene oxide loadinghTERTsiRNA on cervical cancer Hela cells,and whether hTERTsiRNA anddoxorubicin contained in the carrier have synergistic inhibition of tumor cellsornot.Methods1、Design siRNA to target hTERT, and prepare functional oxide grapheneloading hTERTsiRNA gene complexes to transfect Hela cells. Observe theactivity of cell proliferation with inverted microscope.2、 Analyze the expression of hTERT mRNA and protein byWestern blot and RT-PCR.3、 Transfect functionalized graphene oxide which contains bothhTERTsiRNA and doxorubicin into Hela cells. Detect the activity of cellproliferation inhibition to discover whether there is synergistic effect or not withthe MTT assay.Results1、The carrier gene complexes can inhibit cell proliferation and promote cellapoptosis after observing transfected cell morphology and number12h,24h,48h,72h later.2、The carrier gene complex could inhibit the expression of hTERT mRNAand protein of cervical cancer cells by RT-PCR and Western blot assays, andthe differences among groups were statistically significant （P＜0.05）.The pairwise comparison results showed that there was statistical significanceexcept the blank cell group and the carrier group （P＞0.05）, such indicated thatthe carrier had none cell cytotoxicity；the nano carrier had higher efficiencythan liposomes；carrier connected folic acid had higher efficiency than nonefolic acid and the higher concentration was, the higher transfection efficiency ofcarrier was which showed there was a certain dose-dependent effect.3、The functionalized graphene oxide which contained both hTERTsiRNAand doxorubicin were successfully transfected into Hela cells. the differencesamong GO-PEG-FA-PyNH₂contained siRNA and Dox and other groups werestatistically significant by measuring absorbance values of cells in each groupwith MTT assay （P＜0.01）, indicating that they can play a synergisticinhibitioned role during cell proliferation, which had time-dose dependence.ConclusionsThe functionalized graphene oxide which contained hTERTsiRNA wassuccessfully transfected into Hela cells, the complexes could inhibit the activityof cell proliferation as well as the expression of hTERT mRNA and the proteinof cervical cancer cells; functionalized graphene oxide armed hTERTsiRNAcoupled with Dox performed synergistic inhibitioned function on cellproliferation.