【作者】 徐成；

【导师】 王太宏；

【作者基本信息】 湖南大学 ， 化学， 2016， 博士

【摘要】 石墨烯(graphene)纳米材料是一种单原子层厚,sp2杂化的新型碳纳米材料。由于其独特的物理、化学性质近年来吸引了全世界各个领域科学家的广泛关注。在生物、医学领域,石墨烯的衍生物氧化石墨烯(graphene oxide,GO)由于生物相容性好、比表面积大、表面易修饰等特点,在药物递送、生物成像、生物传感器、抗菌材料、癌症的诊断与治疗等方面都取得了较大的应用。本论文围绕氧化石墨烯纳米材料的生物应用为核心,致力设计和构建基于氧化石墨烯复合材料的纳米载体,高效地将药物、DNA分子或造影剂负载,并成功递送到了特定部位,同时利用纳米载体本身的光、热、磁特性,实现了对肿瘤的成像与治疗。主要内容如下:(1)结合并改良文献报道的方法,采用一种较为便捷、高效的方法,以天然石墨为原材料,合成了一种适用于生物医学应用的纳米级氧化石墨烯材料,其纳米片的平均直径在100–200 nm。同时生物相容性好、细胞毒性小、氧化程度高,表面经过羧基(-COOH)化处理,利于进一步功能化修饰连接其他生物分子。这种纳米级氧化石墨烯的制备,为后续基于氧化石墨烯的复合材料的构建及其生物医学应用打下了基础。(2)采用一种快速、简便、廉价的方法,将氧化石墨烯纳米片高效地包裹在金纳米颗粒和金纳米棒材料的表面上。包裹后,一方面增加了金纳米材料的亲水性与表面可利用的官能团,为进一步功能化修饰提供了位点;另一方面,减小了金纳米材料由于表面活性剂CTAB(十六烷基三甲基溴化铵)带来的细胞毒性。接着在氧化石墨烯/金纳米材料的表面通过共价键连接有机高分子PEI(聚乙烯亚胺),将其构建为一种阳离子基因载体。递送含GFP(绿色荧光蛋白)的外源质粒DNA进入Hela细胞,取得了高效(>65%转染率)、低毒(>90%细胞存活率)的转染结果。并且此载体构建的方法具有通用性,可拓展应用到其他一些无机纳米颗粒的表面修饰和药物\基因载体的构建。(3)构建了表面共价连接透明质酸(hyaluronic acid,HA)分子的氧化石墨烯/金纳米棒核壳结构纳米材料。透明质酸分子的修饰既提高了金纳米棒的亲水性、生物相容性,又给予了复合材料靶向表达受体CD44肿瘤细胞的能力。同时此载体还可以通过π-π堆积作用负载抗癌药物阿霉素(Doxorubicin,DOX)。并且利用金纳米棒材料本身的光热效应,本章将氧化石墨烯-透明质酸/金纳米棒复合材料构建为一种结合化学治疗和光热治疗的多功能肿瘤治疗载体。化疗-光热治疗(chemophotothermal therapy)相结合的协同治疗方法,比之只采用化疗或者只采用光热治疗分别提升了1.5倍和4倍的疗效。同时该载体对表达CD44受体的肿瘤细胞具有靶向性,对其他非靶向细胞有低的毒性与副作用。(4)采用一步溶剂热法合成了还原型氧化石墨烯/四氧化三铁纳米颗粒复合材料(reduced graphene oxide(RGO)/Fe3O4),并创新性地先后使用了两种不同类型的聚乙二醇(PEG)分子进行表面修饰。第一段聚乙二醇分子(C18PMH-PEG5000-NH2)通过疏水作用与还原型氧化石墨烯相连接;第二段聚乙二醇(MAL-PEG5000-SCM)分子共价连接在第一段PEG末端的氨基上。两段PEG分子的修饰能极大的增加RGO/Fe3O4纳米材料的水溶性和生物相容性,使其在体内的血液循环时间大大增加。标记同位素64Cu后将RGO/Fe3O4纳米材料通过尾静脉注射入老鼠体内,进行活体正电子发射扫描成像(positron emission tomography,PET)。由于其超长的血液循环时间和增强的肿瘤通透性与滞留性效应(enhanced permeability and retention,EPR effect)。在肿瘤部位取得了非常高的信号富集量(15.5%ID/g)。其被动肿瘤靶向效果几乎等同于或超过现行的一些被美国FDA批准的纳米抗肿瘤药物(如Doxil)。并且利用此载体本身的磁性和光学特性,同时进行了肿瘤的磁共振成像(magnetic resonance imaging,MRI)与光声成像(photoacoustic imaging),成功验证了PET中结果的准确性。多种模态的生物成像方法提供了更多维度和更详细的肿瘤信息。(5)使用表面活性剂十六烷基三甲基氯化铵(CTAC)和油酸合成了产率极高的金纳米棒材料。利用合成后表面残存的CTAC分子,直接在其表面生长一层孔径较大的(5–10 nm)介孔二氧化硅层。接着通过介孔二氧化硅层表面及孔内的硅氧键,实现了在无需螯合剂的情况下,高效地(>95%)地共价连接同位素89Zr。在表面修饰上聚乙二醇后,本工作成功地将介孔二氧化硅/金纳米棒复合材料构建为一种能进行活体肿瘤成像与光热治疗的多功能纳米载体,并取得了有较好的光热治疗的疗效与较高的活体肿瘤信号富集量(9.5%ID/g)更多还原

【Abstract】 Graphene is a two-dimensional crystal of sp2-hybridized carbon atom arranged in six-membered rings. Grephene has sparked tremendous interest on the filed of materials science and nanodevice due to their unique magnetic, optical and chemical properties. Graphene oxide(GO), a derivative of graphene, prepared from natural graphite has attracted great attentions in biomedical applications such as drug and gene delivery, bioimaging, cancer diagnosis and therapy because of its good biocompatibility, large surface area, nanoscale size and abundant surface functional groups. There are a lot of groups reported that GO can effectively delivery drug for tumor chemotherapy and radiolabeld GO can be a platform for in vivo tumor imaging and therapy.In this thesis we construct a few nanocomposites that based on GO nanomaterials for drug or gene delivery to cancer cells and in vivo tumor imaging. The main contents are as follows:(1) Using a simple, efficient and convenient method to prepare graphene oxide nanosheets(NGO) with good biocompatibility, large surface area and abundant functional groups(-COOH, OH) on the surface. And almost all the nanocaomposites in the following chapters are based on this NGO.(2) Based on NGO have been synthesized, we constructed a highly biocompatible and hydrophilic shell on individual gold nanoparticle and nanorod surface by encapsulating nanoparitcles or nanorods in GO nanosheets and used these core/shell hybrids for the first time as gene vectors to delivery p DNA into He La cells. It exhibited good DNAbinding capacity and condensed plasmid DNA into nanoscale particles(150 nm). In vitro gene transfection tests demonstrated that GOpolyethylenimine/gold nanoparticles(GOPEI-Au NPs) presented much lower cytotoxicity and comparable transfection efficiency(65% efficiency and 9 0% viability) with commercial PEI 25 k Da in He La cells, which can be attributed to the small size and spherical structure facilitating cellular uptake. Considering the excellent dispersibility, biocompatibility and high transfection efficiency of GOPEI-Au NPs, their applications can be extended to si RNA delivery and photothermal therapy.(3) A novel nanostructure with gold nanorods(Au NRs) encapsulated in NGO shells is developed to be an ultraefficient chemophotothermal cancer therapy agent. The NGO shells decrease the toxicity of surfactant-coated Au NRs and provide anchor points for the conjugation of hyaluronic acid(HA). The HA-conjugated NGO-enwrapped Au NR nanocomposites(NGOHA-Au NRs) perform higher photothermal efficiency than Au NRs and have the capabili ty of targeting hepatoma Huh-7 cells. NGOHA-Au NR is applied to load doxorubicin(DOX), and it exhibits p H-responsive and near-infrared light-triggered drug-release properties. Chemophotothermal combined therapy by NGOHA-Au NRs-DOX performs 1.5-fold and 4-fold higher targeting cell death rates than single chemotherapy and photothermal therapy, respectively, with biosafety to nontargeting cells simultaneously.(4) We developed double-PEGylated biocompatible reduced graphene oxide nanosheets anchored with iron oxide nanoparticles(RGO-IONP-1stPEG-2ndPEG). The nanoconjugates exhibited prolonged blood circulation half-life(~27.7 h) and remarkable tumor accumulation(15.5 %ID/g) via enhanced permeability and retention(EPR) effect. Due to strong near-infrared absorbance and superparamagnetism of RGO-IONP-1st PEG-2nd PEG, multimodality imaging combining positron emission tomography imaging(PET) with magnetic resonance imaging(MRI) and photoacoustic(PA) imaging was successfully achieved. The promising results suggest great potential of these nanoconjugates for multi-dimensional and more accurate tumor diagnosis and therapy in the future.(5) By using surfactant cetyltrimethylammonium chloride(CTAC) and oleic acid we synthesize Au NRs with high yields. And we coated a biodegradable mesoporous silica layer on the surface of Au NRs by take advantage of residual CTAC molecules on the surface. Biodegradable mesoporous silica layer coated Au NRs(Au NRs@b MSN) can chelator-free label isotope 89 Zr due to the deprotonated silanol groups. After conjugated PEG to surface, Au NRs@b MSN-PEG has been used as a nonoplarform for in vivo tumor imaging and photothermal therapy, and got a remarkable PET imaging signal(9.5 %ID/g) in tumor area.更多还原