靶向上转换纳米光敏剂构建及其医学应用研究

	靶向上转换纳米光敏剂构建及其医学应用研究
	夏露
学位类型	博士
导师	孔祥贵
	2014-07
学位授予单位	中国科学院大学
学位专业	凝聚态物理
摘要	癌症是危害人类健康的重大疾病之一，据统计，仅我国每年因患癌症导致死亡的人数就达到180万之多，成为危害国民健康的头号杀手。癌症之所以成为一直危害人类健康的头号疾病，这主要是因为在现有的人类医学水平范围内，癌症的很难在早期被精确的诊断出来，目前临床医学中癌症患者在发病确诊时通常已经处在晚期，甚至已经在体内发生大面积转移，从而错过了最佳的治疗阶段，治疗起来难度极大。近几十年来，人们一直致力于对肿瘤的早期诊断和治疗不懈的深入研究，在传统的手术、放疗、化疗的基础上发展了许多新兴的抗肿瘤疗法，如基因疗法、光动力疗法等。随着纳米科学与纳米技术不断进步和发展，人们也在研究将纳米材料用于癌症治疗的新方法。纳米技术不仅对肿瘤的诊断和治疗更为精确，而且使患者的治疗痛苦大大降低，这不仅延长了患者的生存时间，也提高了患者的生存质量，是目前研究的热点。人们将化学、生物、纳米技术有机的结合，发展了一系列可以用于癌症早期诊断和治疗的纳米材料，如半导体量子点、聚合物纳米材料、介孔硅纳米粒子以及上转换发光纳米粒子等等。这些材料不仅具有对肿瘤靶向标记的作用，而且在肿瘤的早期诊治过程中成为新的研究热点。近几年来，随着纳米技术和分子生物学研究的不断发展，两个学科有机结合发展衍生出一个新兴的领域：纳米生物技术。稀土掺杂上转换纳米粒子由于其诸多的优点已经发展成为一类新兴的荧光生物探针和光动力治疗纳米材料。稀土掺杂上转换发光纳米粒子（UCNPs）可以将长波辐射转换为短波辐射。和传统的通过下转换过程发光的有机染料、半导体量子点等相比，上转换纳米粒子通过近红外光激发，因此在生物医学应用中有其不可比拟的优点：对生物组织影响小、组织穿透深、成像灵敏度高以及发光性质稳定、不易光漂白等优点。除此之外，上转换纳米粒子本身可以进行功能化修饰、生物毒性低等优点，因此在生物医学研究领域受到了越来越多的关注。目前人们对稀土掺杂的上转换发光纳米粒子的研究也越来越深入，对其研究从基本的合成与制备到化学修饰，再到生物医学中的光动力治疗、多模成像等等。但是这些研究中还存在着诸多问题，如上转换纳米粒子的发光效率低、表面生物功能化后的稳定性差、肿瘤靶向性差、光动力治疗单线态氧产量低等等。本论文中，我们通过改进合成条件和设计不同核壳及掺杂比例，制备了大小均一、结晶性好的稀土上转换发光纳米粒子；并采用胺基聚合物修饰的方法对纳米粒子的表面进行修饰，使纳米粒子表面带有亲水官能团的同时，还可用于生物功能化修饰。在此基础上，围绕提高单线态氧产量、降低辐照光功率密度、避免980nm激光热效应的目的，我们分别通过非共价吸附和共价偶联两种方式对纳米粒子进行光敏剂的组装，构建了同时具有诊断和治疗功能的上转换发光复合纳米粒子，极大的提高上转换光敏纳米粒子的单线态氧产量，降低了辐照光功率密度，并实现了对体外肿瘤细胞的靶向标记和荷瘤鼠动物模型的在体光动力治疗。本文针对如何提高上转换光敏纳米粒子的单线态氧产量、降低辐照剂量及增强近红外光动力治疗安全性进行了一系列研究，主要研究成果概括如下： (1) 制备了生物相容性的高效上转换纳米粒子：利用不同的表面配体，通过水热法、溶剂热法和高温热分解方法制备了粒径均一，结晶性良好，上转换发光效率高的NaYF4:Yb3+,Er3+上转换纳米粒子，通过改变稀土掺杂浓度及纳米粒子核壳结构，调控了NaYF4:Yb3+,Er3+上转换纳米粒子540nm和650nm发光强度。并利用聚丙烯胺（PAAM）作为配体将纳米粒子从油相成功的转移到水相。这为上转换纳米粒子在接下来的生物应用提供了材料基础。 (2) 物理吸附法实现可用于光动力治疗的上转换光敏剂纳米平台的组装:在合成胺基聚合物修饰的上转换纳米粒子的基础上，我们通过一步法将两种光敏剂包覆在纳米粒子表面。通过调控光敏剂的装载量，我们实现了上转换光敏剂纳米复合材料单线态氧产量的最大化。并在体外细胞水平证明了其可同时用于靶向细胞标记和光动力治疗。 (3) 构建了具有生物安全性和靶向性的发光上转换纳米光敏剂：利用PAAM表面修饰的红光增强NaYF4:Yb,Er上转换纳米粒子，通过共价偶联的方法将光敏剂分子锌酞菁（ZnPc）、靶向分子叶酸（FA）以及聚乙二醇分子（PEG）修饰到纳米粒子表面，使复合材料同时具备肿瘤靶向标记和光动力治疗的功能，共价偶联的方式不仅使得光敏剂的装载量达到最佳、而且提高了上转换到光敏剂的能量传递效率，是增加单态氧的产率的有效方法。在此基础上我们还研究了对不同叶酸受体表达程度的细胞的靶向性，及C57荷瘤鼠动物模型的光动力治疗作用。 (4) 构建了同时共价偶联双光敏剂的高效上转换光敏剂复合靶向材料：通过在核壳结构NaYF4:Yb,Er上转换纳米粒子表面同时共价不同吸收波长的双光敏剂，实现了上转换发光谱带能量的充分利用，进一步提高了上转换光敏剂复合材料的单线态氧产量。并且通过体外细胞实验证明其靶向性，在鼠动物模型上证明其对鼠肝癌治疗的安全性和有效性。
其他摘要	Cancer is one of the major diseases threatening human health. According to statistics, only in our country the deaths caused by cancer are more than 1.8 million every year. Why is cancer the top one killer among all the diseases threatening human health? The most important reason is that it is difficult to diagnose cancer precisely in early stages under the current medical level. The patients in the clinical medicine are usually diagnosed in terminal stages, and the cancer cells even have a widespread shift in the body then. Thus the best treatment phase has been missed, and the treatment becomes very difficult. In recent years, people have devoted themselves to the research of early diagnosis and treatment of tumor. Beside of the traditional surgery, radiotherapy and chemotherapy, other anti-tumor therapy approaches are emerging, such as the gene therapy, the photodynamic therapy, etc. People are studying new methods in the treatment of cancer using nanomaterial along with the development of the nanotechnology. Compared with traditional cancer treatments, the nanotechnology for tumor diagnosis and treatment has many advantages. It is more precise, and can reduce the pain of patients, which means it prolongs the patient's survival time, and improves the patient's quality of life at the same time. Therefore it is a hotspot of current research. People developed a series of materials for diagnosis and treatment of cancer by combining chemical, biological, and nanotechnology, for example: semiconductor quantum dots , polymer, mesoporous silica nanoparticles , up-conversion luminescence nanoparticles and so on. These nanomaterials have the abilities to target tumor and transport mark functions to achieve early diagnosis and treatment of the cancer, which brings new hope. In recent years, the combination of nanotechnology and molecular biology has developed into a new field: nano biotechnology. Upconversion nanoparticles doped by rare earth elements have become a kind of nanometer materials used as new fluorescent probes and optical power treatment due to its various advantages. Upconversion nanoparticles (UCNPs) doped by rare earth elements is a kind of materials which can transform the long wavelength radiation to the short wavelength radiation, compared with the traditional down conversion materials such as organic dyes or semiconductor quantum dots. Upconversion nanoparticles were excited by the near-infrared light, therefore it presents a series of unique advantages in biomedical applications: less damage to the biological tissue, deeper tissue penetration, high sensitivity imaging, stable luminescence properties, and hard to be bleached by light, etc. In addition, the upconversion nanoparticles themselves are easy to be prepared by chemical modification, and they are low in toxicity. So it has attracted a great deal of interest in the biomedical field. At present a series of researchs and explorations have been done in the synthesis and preparation, surface modification, photodynamic therapy, multimodal imaging of the up-conversion nanoparticles, and the imaging guided treatment of cancer. There are still many problems in these studies, such as the low efficiency of upconversion nanoparticles , the poor stability of the surface biological functionalization, targeting ability of tumor, the low efficiency of singlet oxygen generation in photodynamic therapy and so on. In this paper, we have prepared NaYF4: Yb3 +, Er3 + UCNPs in hexagonal phase by optimizing synthetic conditions and designing core-shell structure with different doping ratio, and the nanoparticles have good performance in uniform size distribution, crystallization and luminous efficiency; we have realized the surface modification of nanoparticles by the amino polymer obtaining the nanoparticles with an excellent water solubility and biocompatibility. On this basis, on purpose of raising the yield of singlet oxygen, reducing the irradiation light power density and avoiding the heat effect of the 980 nm laser, we also realized the assembly of the photosensitizer and the nanoparticles through covalent coupling and physical adsorption, building a kind of light-emitting composite nanoparticles which have the function of diagnosis and treatment at the same time. It greatly increases the yield of singlet oxygen of UCNPs and reduces the light power density of the irradiation. At last we accomplished targeted marker on tumor cells in vitro and the photodynamic therapy of a tumor-beared mice animal model. This paper mainly discusses several questions on how to improve the production of singlet oxygen of UCNPs, reduce the radiation dose and enhance the security at the near-infrared light power treatment. The main results of the study are summarized as follows: (1) The preparation of efficient UCNPs with good biocompatibility: we have prepared the NaYF4: Yb3 +, Er3 + UCNPs which have good performance in uniform size distribution, crystallization by thermal decomposition and the hydro(solvo)thermal method. By changing the concentration of the doped rare earth and the core-shell structure of the UCNPs, we have regulated the luminous intensity at the wavelength of 540 nm and 650 nm. We realized the phase transfer from hydrophobic to hydrophilic solution by using Poly(allylamine) (PAAM) as the ligand of the UCNPs. The nanoparticles have very good water solubility and biocompatibility, which lay a solid foundation for the multifunction of biological and the application in the future. (2) The physical adsorption method realizes assembly of the upconversion nano photosensitizer platform which is used as the photodynamic therapy: basing on the synthesis of amino modified polymer nanoparticles, we have loaded two kinds of photosensitizers on the surface of the nanoparticles by one-step. By regulating the loading capacity of the photosensitizer, we maximized the yield of singlet oxygen from the nanocomposite of the up-conversion photosensitizer. We also proved the feasibility of the targeted imaging and the photodynamic therapy in vitro at the same time. (3) The preparation of the upconversion nano photosensitizer with high biological safety, high efficiency and good performance in targeting cancer cell: based on red light-enhanced NaYF4: Yb3 +, Er3 + UCNPs with the surface modification of PAAM, photosensitizer zinc phthalocyanine (ZnPc), targeted molecular folic acid (FA) and polyethylene glycol (PEG) were modified to the surface of UCNPs by the covalent method, and we built a composite platform which had the function of targeting imaging and photodynamic therapy, greatly improving the drug loading capacity of the photosensitizer, the efficiency of the energy transition from UCNPs to photosensitizer, and the yield of the singlet oxygen. We also studied the ability of the targeting to the cells at different levels of the expression of folate receptor, and the photodynamic therapy of the C57 tumor-beared mice animal model (4)The preparation of composite targeting materials which are covalent double photosensitizers on the efficient upconversion nanopaticles: the double photosensitizers with different absorption wavelength are covalent on the surface of the core-shell structure NaYF4: Yb, Er up-conversion nanoparticles at the same time, and it made full use of the energy of the upconversion emission band, further improving the production of the singlet oxygen of the photosensitizer composite materials. And it shows its targeting in vitro cell, proving its safety and efficacy in the treatment of the mice animal model.
语种	中文
文献类型	学位论文
条目标识符	http://ir.ciomp.ac.cn/handle/181722/41486
专题	中科院长春光机所知识产出
推荐引用方式 GB/T 7714	夏露. 靶向上转换纳米光敏剂构建及其医学应用研究[D]. 中国科学院大学,2014.

条目包含的文件
文件名称/大小	文献类型	版本类型	开放类型	使用许可
夏露.pdf（8466KB）			开放获取	CC BY-NC-SA	请求全文