The Construction And Study Of Nanometer Delivery System For Hepatic Targeting Therapy Using Graphene Oxide As A Carrier

Cancer,malignant tumor,is a disease caused by the disorder of cell growth and proliferation mechanism.Cancer,cardiovascular disease and diabetes,are the three killers that endanger human health.Liver cancer is one of the most common cancers in the worldwide.According to the American Cancer Society,the death rate of liver cancer is increasing year by year with a survival rate of only 18%.The current treatments include surgical resection,chemotherapy,radiotherapy,gene therapy,immunotherapy,physical therapy,traditional Chinese medicine treatment and so on.And surgical resection,chemotherapy,and radiotherapy are the main approaches,and the other treatments have also been developing rapidly.Drug therapy is an important approach for cancer theray.Gene drugs have the merits of strong targeting,low toxicity,low dosage and automatic degradation,showing great potential in disease treatment.However,its shortcomings such as poor stability,easy degradation,short half-life and low transfection efficiency severely limit its further development.In order to solve these problems mentioned above,it is necessary to construct an effective targeting carrier to transport the gene to the target organ or cells to achieve targeted gene therapy successfully.In recent years,the rapid development of graphene-based nanomaterials has provided new strategies for our work.The distinct physicochemical properties of graphene-based nanomaterials have highlighted their promise as novel nano-carriers for biomedicine.Gene therapy,using nuclei acids to regulate,replace,repair,add or delete a fraction of responsible gene sequence,has made significant progress.In recent years,incorporation of gene with graphene-based nanomaterials to construct safe and effective delivery systems has witnessed rapid advance for treating disease at the genetic roots.SiRNA has great potential in sequence-specific degradation and mRNA transcription inhibition,and has been widely studied and applied,so we employed anti-VEGFa siRNA as a tool drug for liver cancer therapy.In order to solve the shortcomings of poor stability,easy degradation,short half-life,and low transfection efficiency of siRNA,we have designed glycyrrhetinic acid(GA),polyethylene glycol(PEG)and PAMAM Dendrimer(Dendrimer)modified graphene oxide nanocomplexes GA-PEG-NGO-Dendrimer/anti-VEGFa siRNA(GPND/anti-VEGFa siRNA).We characterized its structure in detail,deeply studied its physicochemical properties,and evaluated the in vitro and in vivo activity of the nanocomplexes.We employed a modified Gabriel synthesis method to prepare bis-amino polyethylene glycol PEG-NH2.GA-PEG and PAMAM Dendrimer were linked to the surface of graphene oxide via covalent bond to obtain GPND.The structure of GPND was confirmed by nuclear magnetic resonance spectroscopy,infrared spectroscopy;MALDI-TOF/TOF mass spectrometry and elemental analysis.Then,we studied the physicochemical properties of GPND.The results showedthat comparing with NGO,the particle size and thickness of GPND increased,and the zeta potential changed from negative to positive electricity.In addition,the stability of GPND under physiological conditions was greatly improved.Compared with the rapid precipitation of NGO,GPND could keep stable for one week in physiological saline,phosphate buffer and medium containing 10%fetal bovine serum.Furthermore,GPND had excellent biocompatibility,and even at a high concentration of 250 ?g/mL,the hemolysis rate of GPND was less than 2%.We further studied the in vitro activity of GPND/anti-VEGFa siRNA.The result of MTT assay indicated that the toxicity of GPND was greatly reduced after modification.Even at a high concentration(100 mg/L),the cell survival rate could reach 80%,demonstrating the high security of the prepared nanocarrier GPND.Besides,cell imaging and flow cytometry experiments performed qualitative and quantitative analysis of cellular uptake of GPND/siRNA.The results showed that GPND/siRNA could overcome the shortcomings of siRNA to penetrate the cell membrane to achieve obviously enhanced cell uptake,and the cell transfection efficiency was much higher than that of Lipofectamine2000/siRNA.In addition,we found that GPND could protect siRNA against degradation by RNase.Moreover,we used QPCR and western blot experiments to evaluate the influence of GPND/anti-VEGFa siRNA nanocomplexes on the expression of VEGFa in both mRNA and protein levels in HepG2 cells.The results suggested that after treatment with GPND/anti-VEGFa siRNA nanocomplexes,the mRNA and protein expression of VEGFa in HepG2 cells were both significantly reduced,indicating that GPND/siRNA nanocomplexes could escape from lysosome and then realize effective release of siRNA.Therefore,in vitro,GPND can overcome multiple biological barriers and the disadvantages of siRNA,and effectively deliver siRNA to hepatocellular carcinoma cells,and eventually achieve the gene silencing of VEGFa.Next,we further validated the in vivo bio-evaluation of GPND/anti-VEGFa siRNA nanocomplexes,using naked mice as animal models.Firstly,the targeting of GPND was investigated.It has been reported that the bioactive molecule glycyrrhetinic acid,owing a variety of clinically applicable diseases,has a high affinity for hepatocytes and can specifically recognize liver cancer cells.Studies indicated that GPND could protect siRNA from degradation by RNase in vivo.It had strong targeting ability,and could efficiently deliver siRNA to liver tumor tissues of nude mice,and eventually achieved growth inhibition of liver tumor tissues,improving the shortcomings of poor stability,easy degradation and short half-life of siRNA.Finally,histopathologic analysis was employed to evaluate the pathological changes of tissues and organs of naked mice in different groups,H&E staining results proved that no obvious pathological changes were found of the mice treated with normal saline,siRNA and GPND/siRNA nanocomplexes,preliminary proving the high in vivo safety of GPND.Taken together,GPND/anti-VEGFa siRNA nanocomplexes have a great promise to achieve targeted gene therapy for liver cancer therapy in vivo,expected for further clinical application.It provides the experimental basis for gene therapy of non-viral vectors,and has profounded significance.Notably,this is a multiple delivery system,which could achieve different targeting by the modification of suitable ligands.Moreover,the constructed nanocarrier could deliver not only gene,but also drug,photosensitizers and other biomolecules,beneficial for combined therapy.