| The malignant tumor is one of the three kinds of diseases which do harm to human's health,and is characterized by out-of-control growth of abnormal cells,which scatter from primary position to other positions,infiltrate critical organs,and at last cause the exhaustion of normal function and death.The main therapeutics of malignant tumor at present are operation,radiotherapy,chemotherapy,immunotherapy,thermotherapy et al.Chemotherapy is still the most important therapy method of the malignant tumor,though chemotherapy drugs have many side effects and the therapeutic index is very low.Targeting chemotherapy is the development direction.The key of the chemotherapy is to improve the targeting efficacy to tumor tissue and reduce the side effect to the normal healthy tissue.To make this come true,smart drug delivery system is demanded.Thanks to the environment-reponsive drug delivery system,enhanced anti-tumor efficacy and reduced side effect are obtained.Doxorubicin(DOX)is one of the most prescribed conventional chemotherapeutic agents in clinical application presently due to its excellent anti-tumor efficiency against various solid tumors.DOX is an anthracycline antibiotic,with intricate features of binding to DNAs and inhibiting nucleic acid synthesis.However,clinical application of DOX has been severely hindered because of its side effects,such as the dose-dependent cardiotoxicity,myelosuppression,nephrotoxicity,and development of multi drug resistance.To solve the above problems,the environment-reponsive drug delivery system is the key.Graphene possesses a two-demensional honeycomb structure and it is the thinnest and strongest material in the world.Owing to its excellent physical-chemical properties,various filed is focusing on it.In my research,graphite was the original material and it was oxided via modified Hummers' method,and thus GO was obtained.Heprin(Hep)was selected to modify GO via a linker ADH.The final product(GO-ADH-Hep)was prepared.It was characterized by many methods such as Atom force microscopy(AFM)?Raman spectrum?Infrared spectroscopy(IR)?Ultraviolet spectrum(UV)?~1H Nuclear Magnetic Resonance(~1H NMR)and Elemental analysis.According to the AFM,the thickness of GO was about 1nm,while the thickness of GO-ADH-Hep was about 3nm.The thickness increase of GO-ADH-Hep was due to the Hep modification.All characterization methods confirmed the successful synthesis.Based on the results of the thermogravimetric analysis(TGA),the content of Hep was about 25%.To make this nanocarrier into clinical use,safety problem was a vital issue.According to the results of hemolytic experiment,the prepared nanocarrier was blood biocompatible and could be used via intravenous injection.In addition,the photothermal conversion efficacy was also studied in my research.GO-ADH-Hep possessed photothermal conversion efficacy and could be a potential photothermal therapy agent.After the successful synthesis of GO-ADH-Hep,drug loading was carried out.Through the simple physical mixing in darkness,the nanocomplex(GO-ADH-Hep/DOX)was finally achieved and its drug loading capacity was reached to 64%.The drug loading process was related to pH value and the ratio between DOX and GO-ADH-Hep.The drug loading and encapsulation efficiency were better at pH 7.4 than that of 5.0.In addition,when the ratio of DOX and GO-ADH-Hep was 2:1,the drug loading and encapsulation efficiency were higher than those of 1:1 and 1:2.So 2:1 was chosen for further experiment.Based on the fluorescence quenching property of GO,the loading of DOX onto GO-ADH-Hep nanosheets was confirmed by fluorescence spectroscopy.The fluorescent intensity of DOX was substantially quenched upon loading onto GO-ADH-Hep nanosheets.The drug release profile was sustained pH-sensitive.The DOX release rate was faster at pH 5.0 than pH 7.4.Compared to the DOX solution,the drug release rate of GO-ADH-Hep/DOX was slower.This drug release profile was beneficial to reduce DOX leakage and improve its therapy efficacy.In my research,the in vitro anti-tumor efficacy of the GO-ADH-Hep/DOX was also investigated.DOX has excellent anti-tumor efficiency against various solid tumors,so in this study,HepG2 and MCF-7 cells were selected to study the anti-tumor efficiency of GO-ADH-Hep/DOX in vitro.Firstly,the cytotoxicity of blank nanocarrier was carried out and the results were that the prepared GO-ADH-Hep was nontoxic to both cells.And then,the anti-cancer efficiency of DOX solution and GO-ADH-Hep/DOX was studied.Based on the results,both the DOX solution and the GO-ADH-Hep/DOX nanocomplexes induced cytotoxicity to MCF-7 and HepG2 cells in a dose-and time-dependent manner.However,compared to DOX solution,at equivalent DOX concentration,the cell inhibition ratio of GO-ADH-Hep/DOX was lower.This could be explained by the truth that owing to the strong ?-? interactions between the loaded DOX and GO-ADH-Hep,GO-ADH-Hep/DOX nanocomplexes experienced a slowly sustained DOX release and thus leading to the lower cytotoxicity.In order to find out the cell uptake profile,cellular uptake test was carried out.The results were that in cytoplasm,the fluorescence intensity of GO-ADH-Hep/DOX was stronger than that of the DOX solution.The DOX solution might be untaken by cells via the inefficient passive diffusion while the GO-ADH-Hep/DOX might be through efficient non-specific endocytosis.So the fluorescence intensity of GO-ADH-Hep/DOX was stronger.Apart from the above investigations of the in vitro anti-tumor activity,the in vivo behaviors of the GO-ADH-Hep/DOX was also studied by the method of fluorescence spectrophotometry including pharmacokinetic and biodistribution studies.Firstly,the pharmacokinetic studies of DOX solution and GO-ADH-Hep/DOX were studied in wistar rats.Compared to the DOX solution,the peak of maximum concentration(Cmax),half life(t1/2),the mean residence time(MRT0-t)and the area under the concentration-time curve(AUC0-t)of GO-ADH-Hep/DOX was increased.While the total body clearance(CL)of the GO-ADH-Hep/DOX was decreased.All these results indicated that the DOX behavior in wistar rats was changed after loading onto the surface of GO-ADH-Hep.Its bioavailability was improved and adverse effects were reduced.In addition,the GO-ADH-Hep/DOX possessed long circulating action.What's more,we also studied the biodistribution of the GO-ADH-Hep/DOX.After a single intravenous injection via tail vein,the concentration of DOX(GO-ADH-Hep/DOX group)in heart,lung and kidney was lower than that of free DOX solution group,which was beneficial for reducing its cardiotoxicity,pulmonary toxicity and renal toxicity.The current research proposed a way for the modifications of GO so as to enhance its biocompatibility and hence offered a promising platform for the study of the biological applications of GO-based derivatives.In a nutshell,Hep was first used to modify GO and its safety profile was greatly improved.The novel nanocomplex was pH-sensitive and had a sustained drug release behavior.The in vivo study confirmed that the GO-ADH-Hep/DOX possessed long circulating action and reduced side effects.The potential advantages were worthwhile for further research. |
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