Study On Graphene Oxide Loaded With Doxorubicin And Effect Of Graphene Oxide Loaded With Doxorubicin On Multiple Myeloma Cell

BackgroundAt present, tumor is one of the serious diseases threatening human health, and is also one of the fastest spreading disease in the modern society. There are about10000000new cases of tumor each year all over the world, and the number of tumor patients is increasing year by year. According to the statistics of world health organization (WHO) in2008, the number of people died of tumor has taken up13%of all deaths in the world. The number of tumor deaths accounted for1/4-1/3of all deaths in some countries such as USA and Singapore, ranking behind the heart disease. In the past5years, cancer-related mortality has not decreased evidently though the survival rate of cancer has obviously increased compared with that in the past several decades. Early diagnosis and the improvement of treatment for the patients with tumors are the main reasons to increase the survival rate of tumors. In western countries, hematological malignant tumors including acute leukemia, malignant lymphoma and multiple myeloma are located at top10places in the incidence of malignant tumors, their incidence in China is increasing year by year. There are about25000new cases of lymphoma each year in China, the incidence of Non-hodgkin’s lymphoma has increased by3%-5%every year, non-Hodgkin’s lymphoma is the most rapid growth in all malignant tumors. At present, the treatment methods of malignant hematological tumors are chemotherapy, radiotherapy, targeted therapy, differentiation therapy and hematopoietic stem cell transplantation. Treatment of hematologic malignant tumors is one of the hotspot in medical research. Chemotherapy is an effective, classic treatment method for hematological malignant tumor, chemotherapy usually kill tumor cells, meanwhile chemotherapy also kill normal cells, such as intestinal endothelial cells and bone marrow cells. This leads to serious adverse reactions, the clinical application of chemotherapy drugs are limited, so treatment effect is not ideal. Therefore, the targeted therapy of hematologicl malignant tumor has become a hot topic in recent years. The nanodrugs show great potential applications in the treatment of tumor.Nanodrug is a new discipline, which is the medical discipline use a series of nanotechnology to solve the medical problems as development of nanomedical technology. The development of nanotechnology will bring unexpected changes in the field of biomedical applications, mainly reflected in the treatment and diagnosis of the tumor. Nanodrug is increasingly concerned because of its scientific value and potential prospects of application. As is known to all, doxorubicin (DOX) is one of chemotherapy drugs in clinical treatment of hematological malignant tumor, and has obvious therapeutic effect on hematological malignant tumor. However, DOX have some side effects, such as cardiac toxicity, liver toxicity, bone marrow depression in practical applications, these side effects led the limited of DOX in the clinical application. In order to achieve a better curative effect and minimize the side effects of DOX, to develop novel drug carriers with high and rapid adsorption capacity attracted domestic and foreign experts’ attention. Graphene oxide (GO) is new two-dimensional carbon nanomaterial, some scholars had prepared successfully graphene oxide in2004, and they won the Nobel Prize in2010. Research about application of GO in many aspects achieved some good results, but the research about the potential value in nanodrug carrier is still in its primary stage. This paper has been played close attention to research about GO in nanodrug carrier.Objective and MethodsFirst, GO (new carbon nanomaterial) was successfully prepared in the experiment, physico-chemical properties of GO was characterized by corresponding instruments and technology. To study the embryonic developmental toxicity of GO used zebrafish as animal model.Second, the GO loading with DOX and the influence factors of GO loading with DOX were studied, the study provided the theoretical foundation for the preparation of nanodrug in treatment of hematological malignancy tumor.Third, cell culture and toxicity assay were used to study the effect in killing cells of the GO loading with DOX on multiple myeloma cells. These studies ultimately provided theoretical basis for the development on hematological malignant tumor-targeting nanodrug.Results1. GO was prepared by Hummers’s method. The physico-chemical properties of GO were characterized by transmission electron microscope (TEM), Fourier infrared spectrometer, Zeta potentiometer, element analysis. The morphological structure of GO was characterized by the TEM, the results showed that GO was single nanomaterial. Elemental analysis showed composition of GO with carbon (C), nitrogen (N), hydrogen (H) and oxygen (O). These elements in the proportion of GO were carbon (C),45.17%; Nitrogen (N),0.78%; Hydrogen (H),3.15%; Oxygen (O),50.90%. Using Fourier transform infrared spectrum analysis of GO contained functional groups such as hydroxyl (OH), carbonyl (C=O) and C-O functional groups, these functional groups were oxygen containing functional groups. Examination of the Zeta potential through Zeta potentiometer showed when pH value was between3.4-8.5, GO was negative potential, namely GO with the charge was negative.2. Zebrafish was used as model animal to study the embryonic developmental toxicity of GO. The results were as follows, when GO concentration was100mg/L, zebrafish embryo hatching rate under the condition was0.9446±0.01604, compared with the control group, p=0.079, the difference was no statistically significant; when GO concentration was100mg/L, zebrafish embryo abnormality rate under the condition was0.2191±0.13236, compared with the control group, p=0.087, the difference was no statistically significant; when GO concentration was100mg/L, zebrafish embryo mortality rate under the condition was0.2426±0.15717, compared with the control group, p=0.068, the difference was no statistically significant; when GO concentration was100mg/L, at60hpf, zebrafish heart rate was35.1840±3.91240/15s, compared with the control group, p=0.342, the difference was no statistically significant; when GO concentration was100mg/L, at72hpf, zebrafish larvae’s body length was3.3835±0.18943mm, compared with the control group, p=0.374, the difference was no statistically significant.3. The feasibility of GO as hematological malignant tumor drug carrier was studied. When adsorption process of DOX onto GO were studies, influence factor of the GO loading with DOX such as the contact time, the dosage of graphene oxide, temperature and pH value were also studied. The results showed that GO had a maximum adsorption capacity of1428.57mg/g at room temperature (288k), and the adsorption isotherm data fitted the Langmuir model. Adsorption kinetics fits a pseudo-second-order model. The thermodynamic studies indicated that the adsorption of DOX onto GO was endothermic, namely heating may increase for GO loaded with DOX. At the same time, the reaction of drug onto GO associated with the pH value of solution. GO has high loading effciency due to its huge specific surface area and DOX adsorbed by GO through π-π stacking interaction.4. Grapheme oxide, graphene oxide loaded with DOX (GO/DOX), DOX were used as research objects, using cell culture technigues and cytotoxic testing technigue to study grapheme oxide, DOX, graphene oxide loaded with DOX on multiple myeloma RPMI8226cells in vitro.4.1The effect of GO, DOX, GO/DOX on cell viability was evaluated by measurment of CCK-8. The RPMI8226cells were treated with GO (0,10,25,50,100mg/L size<100nm). GO groups compared with the control group, the difference was statistically significant in cell viability (p<0.05). The results showed that GO in the0-100mg/L concentration on multiple myeloma cells had low cytotoxicity. The findings of GO (50mg/L), DOX (2mg/L), GO/DOX(GO50mg/L+DOX2mg/L) were as follows. Cell viability of GO (50mg/L) was (84.6±0.4)%, cell viability of DOX (2mg/L) was (71.7±1.0)%, cell viability of GO/DOX(GO50mg/L±DOX2mg/L) was (62.6±7.3)%. The results showed there was significant defference in cell viability between GO/DOX group and DOX group (p<0.05), but removing the impact of the cell viability of GO, there was no significant defference in cell viability between GO/DOX group and DOX group (p>0.05), indicating that GO did not decrease DOX cytotoxicity.4.2Cell cycle of GO, DOX, GO/DOX on RPMI8226cells were detected by flow cytometry. The G0/G1, S, G2/M phase of GO (50mg/L), DOX (2mg/L), GO/DOX (GO50mg/L+DOX2mg/L) on RPMI8226cells respectively compared with control group, there were no statistically significant difference (p>0.05). The results indicated GO did not change the cell cycle of RPMI8226cells.4.3There was no significant difference in cell apoptotic rate between GO (10,50,100mg/L) groups and the control groups. The results indicated that GO (0-100mg/L) did not induce cell apoptosis. There was significant difference between DOX (2mg/L) group and control group, and GO/DOX(GO50mg/L+DOX2mg/L) group and control group (P<0.05). There was no significant difference between the DOX/GO group and DOX group (P>0.05). The results indicated that DOX could induce cell apoptosis, and GO did not change the ability of DOX to induce apoptosis.Conclusions1.GO was prepared successfully, and GO had no obvious embryonic development toxicity on zebrafish.2. Study on GO as drug carrier, the test showed GO can have a strong force on drugs. Research on influence factors on GO loaded with drugs of hematologic malignancies (DOX), the test showed that the changes of temperature, pH value, dosage could affect the GO loaded with drugs of hematologic malignancies (DOX).3. The study on cell viability of GO, DOX, GO/DOX on multiple myeloma cells found that GO/DOX had significant effect on inhibiting the proliferation of multiple myeloma RPMI8226cells. Research showed that GO could not change toxic effect of DOX on hematologic malignancie tumor cells. GO did not change the cell cycle of RPMI8226cells. The results also indicated that GO did not change the ability of DOX to induce apoptosis. So GO could be suitable for antitumor nanodrug carrier.