用于肿瘤诊断治疗的透明质酸基多功能纳米粒子的研究

发布时间：2018-01-20 06:47

本文关键词： 癌症诊断治疗透明质酸形貌调控协同治疗抗药性　出处：《浙江大学》2017年硕士论文　论文类型：学位论文

【摘要】：构建用于癌症快速诊断和治疗的多功能纳米粒子是肿瘤治疗领域的研究热点之一。透明质酸(HA)是细胞外基质的重要组成成分,具有非常好的生物相容性和体内可降解特性。此外,透明质酸可与多种癌细胞表面过度表达的CD44受体进行特异性结合,从而增加针对癌细胞的靶向作用,所以HA是一种非常好的表面修饰和药物载体材料。因此,本文选用HA作为主要原料,构建用于癌症诊断治疗的多功能纳米粒子,研究内容主要分为以下两个部分:1.多功能纳米粒子在癌症的诊断治疗方面引起越来越多的关注。除了尺寸和表面化学特性之外,形貌是影响纳米粒子体内循环时间、生物分布、靶向性以及细胞内吞的重要因素。由于金纳米粒子具有无毒、可以简单地大量合成、表面易于修饰、特殊的光学特性等优点,能满足癌症诊断治疗的不同需求。本文首先合成金纳米杆(AuNRs)和金纳米球(AuNSs)来构建两种不同形貌的金纳米探针,其中AuNRs的长约50nm,截面直径约为12mn,AuNSs直径约为20nm。通过近红外光荧光染料尼罗蓝(NB)标记的巯基化透明质酸(HS-HA-NB)来包裹两种不同形貌的金纳米粒子,构建稳定的纳米探针AuNR@HS-HA-NB和AuNS@HS-HA-NB。由于透明质酸优异的生物相容性和体内可降解特性,纳米探针在不同pH磷酸缓冲液(pH=4-8)和80%浓度的血清中均表现出很好的稳定性,同时乳腺癌(MCF-7)细胞毒性实验表明在纳米探针表面包裹HS-HA-NB后,毒性显著降低。对比在MCF-7细胞内的成像结果发现,相对于球形探针,杆形探针可以更加快速地被细胞内吞并产生荧光,这可能是由于其长轴与细胞膜表面接触时能够提供更大的接触面积,因此可以快速有效地被细胞内吞。探针的HA靶向性也通过竞争抑制实验得到了验证。而且,在近红外光照后,加入金纳米杆探针的实验组细胞观测到了 MCF-7细胞的凋亡。以上实验结果证明AuNR@HS-HA-NB纳米探针能够实现靶向快速准确成像和光热治疗。2.化疗已经被广泛地应用于多种类型的癌症治疗当中,抗药性和缺乏癌细胞的靶向性是导致癌症化疗失败的主要因素。采用光热治疗产生的过高热来解除癌细胞的抗药性受到了广泛的关注。本文采用胱胺(Cys)交联的HA包裹AuNRs形成HA-Cys@AuNR纳米凝胶来负载阿霉素(DOX),其载药量为0.5±0.017%,包封率为51.9±3.5%。并合成了不含有AuNRs的纳米凝胶HA-Cys,负载DOX后其载药量为0.5±0.014%,包封率为56.8±3.4%。由于Cys上存在二硫键,因此在GSH条件下,载药的HA-Cys@AuNR@DOX及HA-Cys@DOX纳米凝胶均能够有效的响应性释放DOX,4 h药物释放量分别为69.9%和72.24%。细胞实验表明HA-Cys@AuNR@DOX及HA-Cys@DOX纳米凝胶均能对有效杀伤药物敏感的MCF-7细胞,但是对抗药性的MCF-7 ADR细胞却无明显效果。在HA-Cys @AuNR@D OX纳米凝胶对癌细胞治疗的过程中加入近红外光照射,AuNRs的光热作用能有效解除癌细胞的抗药性,造成MCF-7ADR凋亡。通过荧光显微镜进一步研究发现,增加近红外光照后,细胞内部的DOX排出过程被抑制,使细胞的抗药性消失,这可能是由于抗药细胞在光热作用产生过高热的条件下,细胞膜通透性改变。在靶向性实验中,通过竞争抑制实验验证了纳米凝胶对MCF-7细胞的靶向性作用。采用HA成功制备了一种针对抗药性细胞的靶向性光热-药物协同治疗的多功能纳米凝胶。本文采用的基于HA设计的纳米探针及纳米药物载体,为癌症诊断治疗多功能纳米粒子的研究提供了新的思路。
[Abstract]:For the construction of multifunctional nanoparticles rapid diagnosis and treatment of cancer is one of the hot research field of cancer therapy. Hyaluronic acid (HA) is an important component of the extracellular matrix, has very good biocompatibility and in vivo biological degradable properties. In addition, hyaluronic acid can be overexpressed and surface multiple cancer cell CD44 receptor the specificity of binding, thereby increasing in cancer cell targeting, so HA is a very good surface modification and drug carrier material. Therefore, this paper chooses HA as the main raw material, for the construction of multifunctional nanoparticles for cancer diagnosis and treatment, the main content is divided into the following two parts: 1. multifunctional nanoparticles caused more and more attention in the diagnosis and treatment of cancer. In addition to the size and surface chemical properties, morphology is the influence of nanoparticle circulation time in vivo, biodistribution, targeted to Important factors and endocytosis. The gold nanoparticles are nontoxic, simple in large quantities, easy surface modification, special optical properties and other advantages, to meet the different needs of cancer diagnosis and treatment. This paper firstly synthesized gold nanorods (AuNRs) and gold nanoparticles (AuNSs) to construct two different morphologies of gold nano probe, which AuNRs is about 50nm, the diameter is about 12mn, the diameter of AuNSs is about 20nm. by near infrared fluorescent dye Nile Blue (NB) thiolated hyaluronic acid (HS-HA-NB) to mark the package of two different morphologies of gold nanoparticles, build stable nano probe AuNR@HS-HA-NB and AuNS@HS-HA-NB. due to excellent hyaluronic acid the in vivo biocompatibility and biodegradable properties, nano probe in different pH phosphate buffer (pH=4-8) and 80% serum concentration showed good stability, and breast cancer (MCF-7) cell toxicity The experiment showed that the coated nano probe after HS-HA-NB toxicity was significantly reduced. Contrast imaging in MCF-7 cells and found that compared to the spherical probe, the rod probe can more quickly by intracellular annexation of fluorescence, which may be due to the long axis of the cell membrane surface contact can provide larger contact area, so you can quickly and efficiently internalized by cells. HA target probe to the competitive inhibition experiment was verified. Moreover, in the near infrared light, the cells in the experimental group with observation of gold nanorods probe to MCF-7 cell apoptosis. These results demonstrated that AuNR@HS-HA-NB nano probe can achieve fast and accurate imaging and targeting photothermal therapy.2. chemotherapy has been widely used in various types of cancer treatment, drug resistance and lack of cancer cell targeting is caused mainly due to the failure of cancer chemotherapy . high heat generated by photothermal therapy to relieve cancer resistance has been widespread concern. This paper used cysteamine (Cys) HA coated AuNRs crosslinked gel to form HA-Cys@AuNR nanoparticles loaded with doxorubicin (DOX), the drug loading was 0.5 + 0.017%, the encapsulation rate was 51.9 + 3.5%. and nano HA-Cys gel not containing AuNRs was synthesized, load DOX after its drug loading was 0.5 + 0.014%, the encapsulation rate was 56.8 + 3.4%. due to the presence of the two disulfide bonds in Cys, so under the condition of GSH, HA-Cys@AuNR@DOX and HA-Cys@DOX nano gel of the drug were able to respond effectively to release DOX, 4 h drug release amount was 69.9% experimental results show that HA-Cys@AuNR@DOX and 72.24%. cells and HA-Cys@DOX nano gel can effectively kill MCF-7 cells in drug sensitive and drug-resistant ADR cells but MCF-7 had no obvious effect. The treatment of cancer cells in HA-Cys @AuNR@D OX nanogel too With near infrared light in the process of resistance AuNRs photothermal effect can effectively relieve cancer cells, resulting in apoptosis of MCF-7ADR. Further study by fluorescent microscope, increase near infrared illumination, the cell's internal DOX discharge process is inhibited, the cell resistance disappeared, this may be due to the excessive heat generated in the resistant cells the photothermal effect under the condition of cell membrane permeability changes. To experiment in the target, the competitive inhibition experiment of nano gel on MCF-7 cell targeting preparation. A needle resistant cell using HA successfully targeted multifunctional nano gel of photothermal therapy. Drug co nano probe and the nano drug carriers based on HA design of this paper provides a new idea for the treatment of multifunctional nanoparticles for cancer diagnosis.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.4;TB383.1

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