The Construction Of Graphene Oxide-based Composite Nano-drug System And Its Anti-tumor Effect

In recent years,graphene oxide?Graphene oxide,GO?has been used as a kind of nanocarrier for drug delivery,which has become the research hotspot.GO exhibits unusual physical and chemical properties,such as high specific surface area,lower biotoxicity and better hydrophilicity,and can be used to load drugs via its unique aromatic conjugated structure.However,unmodified GO is prone to agglomeration in physiological solutions?such as PBS buffer and cell medium?,which affects its drug loading properties.Therefore,the selection of suitable materials and methods to modify GO to enhance its properties has become the focus of the current research.In this project,a preparation method of the novel anticancer drug nanocarrier was designed,for the first time,biological materials of chitosan oligosaccharide?Chitosan oligosaccharide,CO?and?-polyglutamic acid??-Polyglutamic acid,?-PGA?covalently modified GO,loaded anti-cancer drug doxorubicin hydrochloride?Doxorubicin,DOX?to obtain nano drug delivery system of graphene oxide based composites?GO-CO-?-PGA-DOX?,and its cytotoxicity and anti-tumor mechanism were studied.On this basis,in order to further improve the targeting of the system,the nucleic acid aptamer NH₂-AS1411?Aptamer NH₂-AS1411,APT?of targeted nucleolin?Nucleolin,also known as C23?was used to modify GO-CO-?-PGA to obtain the targeted nano drug delivery system of APT-GO-CO-?-PGA-DOX,and its anti-tumor effect was studied.?1?First and foremost,a simple and economical method was used to activate GO which binded with CO through amide bond.Afterthat,the pretreated?-PGA was connected to the CO on GO-CO via amidation to complete the fabrication of GO-CO-?-PGA.Characterization methods of fourier transform infrared spectroscopy?FTIR?,X-ray photoelectron spectroscopy?XPS?,ultraviolet-visible absorption spectra?UV-Vis?,atomic force microscope?AFM?,transmission electron microscopy?TEM?and the determination of Zeta potential were used to analyze its structure,composition,morphology,dispersion and other properties,and the results showed that GO-CO-?-PGA was successfully connected,presented layer structure with diameter of 200-300 nm,thickness of 7-8 nm,and exhibited excellent dispersibility in physiological environments.?2?The profiles of GO-CO-?-PGA loading and releasing DOX in vitro were investigated,the experimental results showed that the maximum drug loading capacity of GO-CO-?-PGA was1.1883 mg/mg,and GO-CO-?-PGA-DOX could release DOX under acidic conditions?pH 5.0?with a cumulative release rate of 52.58%,indicating a pH responsive release behavior.The results of cell experiments indicated that GO-CO-?-PGA had good biological safety,and could load DOX into Hela cells,release drugs into the nucleus by sensing the pH of the tumor microenvironment,and play an excellent anticancer effect,its IC₅₀ value was 3.50±0.01?g/mL.Cell experiments and Western Blot results confirmed that GO-CO-?-PGA-DOX caused the cell cycle arrest of Hela cells at G₂/M phase,up-regulated the expression of Bcl-2 protein and down-regulated the expression of Bax protein.The abnormal expression of the proteins led to mitochondrial depolarization and promoted the release of cytochrome C?Cytochrome C,Cytc?.Cytc in the cytoplasm was combined with apoptosis protease-activating factor-1?Apoptosis protease-activating factor-1,Apaf-1?,which recruited procaspase-9 to form apoptotic corpuscles,thereby activating Caspase-9.Caspase-9 further activated Caspase-3,cleaving poly ADP-ribose polymerase?Poly ADP-ribose polymerase,PARP?,which leaded to apoptosis.The induction of Hela cell apoptosis by GO-CO-?-PGA-DOX depended on the endogenous mitochondrial apoptosis pathway and Caspase activation.?3?In order to endow the nanocancer with the targeting property and enhance the anticancer effect of the drugs,GO-CO-?-PGA was further functionalized with NH₂-AS1411,and its structure,composition and dispersion were analyzed.It was shown that the maximum drug loading capacity of APT-GO-CO-?-PGA was 1.2135 mg/mg and higher than GO-CO-?-PGA,and showed the ability of controlled and sustained release of drugs through the study on the drug loading capacity of APT-GO-CO-?-PGA.The cell experiment results indicated that because nucleolins were overexpressed on the surface of Hela cells but not on the surface of Beas-2B cells,APT-GO-CO-?-PGA-DOX could specially bind with Hela cells,making more toxic to Hela cells than Beas-2B cell,and the IC₅₀ value of APT-GO-CO-?-PGA-DOX to Hela cells was 3.23±0.04?g/mL.All of the results demonstrated that APT-GO-CO-?-PGA could deliver anticancer drugs in a targeted manner,and achieve the effect of increasing effect and reducing poison,which has a broad application prospect in the field of biomedicine.