Studies On Non-covalently Functionalized Modifed Graphene Oxide As Carrier For Anticancer Drug Delivery

With the deterioration of air quality and human lifestyle changes,the incidence of cancer and mortality increased year by year,effective cancer treatment without delay.Chemotherapy is an effective and classic treatment,but most of the chemotherapy drugs including doxorubicin have many disadvantages such as low selectivity,serious side effects especially cardiac toxicity,short half-life,multi-drug resistance and so on.Therefore,in order to improve the therapeutic effect and reduce the serious side effect of anticancer drugs,it is essential to develop the new and effective drug carrier.Since the discovery of graphene by some scholars in 2004,by virtue of its unique structural features,excellent mechanical,thermodynamic and optical characteristics,it has attracted great research enthusiasm in various fields.GO,which is the oxidized derivative of graphene,by virtue of its large specific surface area and excellent photo-thermal effect has become the research hotspot in the field of nanomedicine.GO is dispersible in water but it will aggregate in physiological solution,which will greatly limit its further application in drug delivery.Therefore,proper surface functionalization is critical to improve its stability and dispersion in physiological solution.Generally,the large ?-conjugated structure of GO and rich oxygen-containing on the surface and edges can be used to realize non-covalent and covalent modification.Due to the presence of chemical reaction and organic solvent,covalent modification usually destroys the conjugated structure and alters the physical properties of GO.Compared with covalent modification,non-covalent modification can avoid the cumbersome chemical reaction and better retain its super conjugated structure and nature properties.So non-covalent modification has been attracted more and more attention.The ideal drug delivery system should possess the following advantages.The preparations are stable in blood circulation and normal tissue.Through active or passive targeting,the preparations are concentrated in the tumor sites,and then into the cells through endocytosis,finally under the influence of cellular environment,the drug rapidly release.Only in this way,the anticancer drugs can achieve the best therapeutic effect and the lowest side effects.Therefore environmental stimuli-responsive drug delivery system has received extensive attention.We can take advantage of the environmental characteristics of tumor cells(such as low pH,high GSH concentration and so on)to design smart drug carrier.According to the change of tumor cellular environment,the drug delivery system will release drug to produce therapeutic effect,so as to achieve the purpose of improving the therapeutic effect and reducing the side effects.In order to allow full play to the functions and properties of GO,this paper employ two different biocompatible macromolecules to modify GO by non-covalent methods.The pH sensitive active targeted drug delivery system and redox sensitive drug delivery system are constructed and evaluated in vitro and in vivo.The contents of this paper are as follows:(1)The folate receptor-mediated active targeting drug delivery system was developed by using folate receptor that overexpressed on the surface of certain tumor cells.FA-BSA/GO was prepared by non-covalent modification of GO with FA-BSA,which binds active targeting molecule FA to biocompatible BSA via amide reaction.FA-BSA acts as both the stabilizer,dispersant,but also has the targeting function.The morphology,lamellar size and thickness were monitored by TEM,AFM and DLS.Zeta potential was employed to monitor the stability.The hemolysis assay was used to determine its good biocompatibility.(2)FA-BSA/GO as drug carrier was associated with anticancer drug DOX through ?-? and hydrogen-bonding interactions,resulting in forming the pH sensitive active targeting drug delivery system.When the mass ratio of DOX to FA-BSA/GO was 3:1,the drug loading can reach maximum and it was 437.43 ?g/mg FA-BSA/GO,which was much higher than traditional preparation.The in vitro release assay demonstrated the drug delivery system exhibited significant pH sensitivity and sustained release behavior in comparison with free DOX.The in vitro cell cytotoxicity and cellular uptake analysis suggested that FA can further improve the uptake of FA-BSA/GO/DOX into tumor cells,which can improve the therapeutic effect of DOX and reduce its toxicity to normal tissues.(3)In order to further solve the problems that the stabilizer of GO in physiological solution and incomplete,slow drug release behavior of based on GO drug delivery system,the project takes advantage of GSH concentration difference and employs redox sensitive prodrug molecule mPEG2K-PCL2K-SS-DOX as stabilizer and dispersant to decorate GO by non-covalent for the first time,finally the redox sensitive drug delivery system is developed and it's in vitro and in vivo are studied.(4)The presence of prodrug molecule make the prepared GOPN stable in physiological solution and prolong the time of GOPN in the body due to the long cycle of PEG,so as to avoid repeated administration.The lamellar morphology and size were observed by TEM and DLS.The plasma stability was monitored by DLS,and the results showed that the GOPN exhibited good stability.When the mass ratio of DOX to NGO was 3:1,the drug loading can reach maximum and it was 1.04 mg DOX/mg NGO.Compared with conventional preparation,the drug loading is significantly improved.(5)In vitro release results showed that GOPN exhibited excellent redox sensitivity and the drug release was more complete than NGO/DOX.In vitro cytotoxicity assay showed that the inhibition rate of GOPN was higher than that of free DOX and NGO/DOX,because the redox sensitive GOPN entered the cell via endocytosis,under the influence of high GSH concentration,the disulfide bond was broken and the drug released,finally it could kill cancer cells rapidly.Therefore our prepared GOPN possesses better therapeutic effect.In vitro cellular uptake result suggested that the uptake of GOPN in A549 cells was higher than that of free DOX;the high uptake of GOPN could assure the high concentration of drug in tumor site,which was an important reason to enhance the tumor lethality.(6)In vivo antitumor efficacy of GOPN was studied on B16 melanoma-bearing Kunming mice.From the relative tumor volume change,we can find that our prepared GOPN has a certain inhibitory effect on tumor growth.The safety of GOPN was evaluated by relative body weight change and histological sections of tumor-bearing mice,the results showed that GOPN could significantly reduce the cardio toxicity induced by free DOX,Therefore the GOPN has the effective in vivo antitumor effect and good biosafety.In conclusion,we successfully for the first time employed two biocompatible macromolecules(FA-BSA and mPEG2K-PCL2K-SS-DOX)to modify GO by non-covalent modification and construct pH sensitive active targeting drug delivery?system and redox sensitive drug delivery system,which not only successfully solve the problems of GO instability in physiological solution and incomplete,slow drug release behavior of based on GO drug delivery system,but also can improve the DOX therapeutic effect,reduce side effects,improve patient tolerance,importantly can broaden the application of GO in biomedical field.Therefore this topic has important significance.