【作者】 吴少玲；

【导师】 王欣；

【作者基本信息】 山东大学 ， 内科学（专业学位）， 2013， 博士

【摘要】 研究背景恶性肿瘤是严重危害人类健康的疾病,也是当今社会蔓延最快的疾病之一,全世界每年约有1000多万新增病例。根据2008年WHO统计,因肿瘤致死人数占世界总死亡人数的13%。在美国、新加坡等国家,肿瘤致死人数占到了总死亡人数的1/4-1/3,仅次于心脏病致死率。虽然近5年来,肿瘤生存率与过去几十年相比有了明显提高,但肿瘤相关死亡率没有显著的变化,生存率的提高归功于肿瘤的早期发现及治疗手段的提高。在西方国家,急性白血病、恶性淋巴瘤、多发性骨髓瘤等血液肿瘤均位于恶性肿瘤发病的前十位,在我国发病率也呈逐年升高的趋势。我国每年新增淋巴瘤患者约2.5万人,其中非霍奇金淋巴瘤的发病人数年递增率达到3%-5%,是所有恶性肿瘤中增长速度最快的。目前,恶性血液肿瘤的治疗主要有化疗、放疗、诱导分化治疗以及造血干细胞移植等方法。化疗是一种有效而经典的肿瘤治疗手段,但化疗药物通常在杀死肿瘤细胞的同时也杀死正常细胞如小肠内皮细胞和骨髓细胞,从而产生严重的不良反应和并发症,不但限制了化疗药物足量应用而达到最佳疗效,还直接导致原发肿瘤不能被控制,最终导致治疗效果不理想。因此,针对恶性血液肿瘤的靶向药物治疗已成为近年来的研究热点。纳米技术的发展使得纳米靶向药物在肿瘤治疗领域显示出巨大的应用潜能。纳米医学是一门新型学科,该学科是随着纳米生物医药发展起来的用一系列纳米技术解决医学问题的学科。纳米技术的发展将给生物医学领域产生深刻的影响,主要体现在诊断和治疗肿瘤方面。纳米药物因其科学价值和潜在应用前景日益受到关注。多柔比星是临床上常用治疗恶性血液肿瘤的蒽环类化疗药物之一,其对血液肿瘤的治疗效果显著。但由于多柔比星在实际应用中的副作用,如心脏毒性、肝脏毒性、骨髓抑制等,导致多柔比星在临床上的应用受到一定限制。为了达到较好的疗效而最大限度减少多柔比星的毒副作用,开发具有高吸附能力和快速吸附的新药物载体引起国内外同行的密切关注。氧化石墨烯(graphene oxide, GO)是碳家族的一种新型二维纳米材料,有学者在2004年成功制备出氧化石墨烯,由于成功制备了氧化石墨烯而在2010年获得诺贝尔奖。氧化石墨烯在很多方面的应用研究取得了很好的成果,但在药物载体方面潜在价值的研究还处在初级阶段,在药物载体方面的研究得到了本课题的密切关注。目的和方法本课题首先成功制备了纳米新碳材料-氧化石墨烯,应用相应仪器和技术对氧化石墨烯进行了一系列的表征,然后以斑马鱼为动物模型研究了氧化石墨烯的胚胎发育毒性。第二,研究了氧化石墨烯负载抗血液肿瘤药物(多柔比星)的载药量和氧化石墨烯负载多柔比星的影响因素,为制备抗血液肿瘤纳米药物提供理论基础。第三,利用细胞培养和细胞毒试验研究了氧化石墨烯负载多柔比星后对多发性骨髓瘤细胞的杀伤作用,为血液肿瘤靶向纳米药物的开发提供理论依据。研究结果1.通过Hummers方法制备了氧化石墨烯,利用透射电子显微镜(TEM)、傅里叶红外光谱仪、Zeta电位仪、元素分析等仪器或方法对氧化石墨烯的理化性进行了表征。氧化石墨烯(GO)的形态结构通过透射电子显微镜(TEM)进行表征,结果显示氧化石墨烯是单层纳米材料。元素分析显示氧化石墨烯的元素组成有碳(C)、氮(N)、氢(H)和氧(0),这些元素在氧化石墨烯中所占比例分别为碳(C),45.17%；氮(N),0.78%；氢(H),3.15%：氧(0),50.90%。用傅里叶红外光谱对氧化石墨烯所含的官能团进行分析,结果显示氧化石墨烯含有羟基(-OH)官能团、羰基(C=0)官能团和C-0官能团,这些官能团都是含氧官能团。Zeta电位仪检测zeta电位显示pH值在3.4～8.5之间,氧化石墨烯都是负电位,即氧化石墨烯所带的电荷为负电荷。2.以斑马鱼为动物模型,利用斑马鱼胚胎研究了氧化石墨烯的胚胎发育毒性,研究结果如下：在氧化石墨烯浓度为100mg/L的情况下,斑马鱼胚胎的孵化率达0.9446±0.01604,与正常对照组比较,p=0.079,差异没有统计学意义：在氧化石墨烯浓度为100mg/L的情况下,斑马鱼胚胎畸形率为0.2191±0.13236,与正常对照组比较,p=0.087,差异无统计学意义；在氧化石墨烯浓度为100mg/L的情况下,斑马鱼胚胎死亡率达0.2426±0.15717,与正常对照组比较,p=0.068,差异无统计学意义：在氧化石墨烯浓度为100mg/L的情况下,在60hpf时,斑马鱼心跳为35.1840±3.91240/15s,与正常对照组比较,p=0.342,差异无统计学意义；在氧化石墨烯浓度为100mg/L的情况下,在72hpf时,斑马鱼幼鱼体长为3.3835±0.18943,与正常对照组比较,p=0.374,差异无统计学意义。3.研究了氧化石墨烯作为抗血液肿瘤药物载体的可行性。研究氧化石墨烯吸附多柔比星的过程,以及在此过程中,氧化石墨烯与多柔比星的接触时间、氧化石墨烯用量、反应温度和pH等参数对氧化石墨烯(GO)吸附多柔比星(DOX)的影响。结果显示在室温时(288K),氧化石墨烯吸附多柔比星的最大吸附值为1428.57mg/g,氧化石墨烯吸附多柔比星的吸附等温线数据符合Langmuir模型,吸附的动力学符合准二级动力学模型。对氧化石墨烯吸附多柔比星的热力学的研究表明氧化石墨烯吸附多柔比星是自发的、吸热的,即在加热的情况下有利于氧化石墨烯吸附多柔比星。同时,氧化石墨烯与药物之间的作用力与溶液的酸碱度相关。氧化石墨烯对多柔比星的高效负载是依靠氧化石墨烯巨大的比表面积以及与多柔比星之间的π-π堆积作用。4.以氧化石墨烯、多柔比星、氧化石墨烯装载多柔比星为研究对象,运用细胞培养技术和细胞毒检测技术,研究了氧化石墨烯、多柔比星、氧化石墨烯装载多柔比星对多发性骨髓瘤RPMI8226细胞的杀伤作用。4.1cck-8法检测纳米氧化石墨烯、多柔比星、氧化石墨烯装载多柔比星对RPMI8226细胞增殖的影响。氧化石墨烯(浓度为0,10,25,50,100mg/L)时对多发性骨髓瘤细胞的作用与正常对照组比较,细胞存活率(p均<0.05),差异有统计学意义,表明氧化石墨烯在0-100mg/L浓度下对多发性骨髓瘤细胞有低毒性。在氧化石墨烯浓度为50mg/L,多柔比星浓度为2m/L时,氧化石墨烯负载多柔比星(GO50mg/L+DOX2mg/L)时CCK-8检测结果如下：氧化石墨烯组细胞存活率为(84.6±0.4)%,多柔比星组细胞存活率为(71.7±1.0)%,氧化石墨烯装载多柔比星细胞存活率为(62.6+7.3)%。结果显示,氧化石墨烯装载多柔比星组与多柔比星组比较,1p=0.027,差异有统计学意义,但去除氧化石墨烯的影响比较,p>0.05,说明氧化石墨烯不降低多柔比星对多发性骨髓瘤细胞的细胞毒性。4.2流式细胞仪测定氧化石墨烯、多柔比星、氧化石墨烯装载多柔比星对RPMI8226细胞的细胞周期的变化。在多柔比星浓度为2m/L,氧化石墨烯浓度为50mg/L,氧化石墨烯负载多柔比星(GO50mg/L+DOX2mg/L)时,细胞周期检测结果显示,GO/Gl、S、G2/M期,实验组与正常对照组相比,P均>0.05,差异均无统计学意义。结果表明氧化石墨烯不影响多发性骨髓瘤细胞的细胞周期。4.3氧化石墨烯对多发性骨髓瘤细胞的凋亡检测结果显示正常对照(0mg/L)组与GO组(浓度为10,50,100mg/L)比较,p均>0.05,差异均无显著性。表明GO浓度在0-100m/L时不诱导细胞凋亡。在多柔比星浓度为2mg/L,氧化石墨烯浓度为50mg/L时,氧化石墨烯负载多柔比星(GO50mg/L+DOX2mg/L)时,细胞凋亡检测结果显示多柔比星组、氧化石墨烯负载多柔比星组与正常对照组比较,P<0.05,差异有统计学意义。氧化石墨烯负载多柔比星组与多柔比星组比较(P>0.05),无统计学差异。结果显示多柔比星可以诱导细胞凋亡,氧化石墨烯不降低多柔比星诱导细胞凋亡的能力。研究结论1、本课题成功制备了氧化石墨烯,并以斑马鱼为动物模型研究发现氧化石墨烯没有明显的胚胎发育毒性；2、研究氧化石墨烯作为药物载体时,发现氧化石墨烯具有很强的载药能力,研究影响氧化石墨烯对抗血液肿瘤药物(多柔比星)的载药能力的因素时,发现温度的变化、pH值的改变、氧化石墨烯的加入量能影响氧化石墨烯对抗血液肿瘤药物(多柔比星)的载药能力；3、氧化石墨烯负载抗血液肿瘤药物(多柔比星)后对多发性骨髓瘤的杀伤能力的研究发现,氧化石墨烯负载多柔比星对多发性骨髓瘤RPMI8226细胞有显著抑制增殖的作用,但氧化石墨烯不降低多柔比星对细胞的抑制作用。其抑制作用不是通过阻滞血液肿瘤细胞的细胞周期实现的,氧化石墨烯低浓度时不诱导细胞凋亡,不降低多柔比星诱导细胞凋亡的能力。可适合做抗血液肿瘤纳米药物的载体。更多还原

【Abstract】 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.更多还原