新型抗肿瘤纳米载药体系在肺癌治疗中的应用

发布时间：2018-11-16 12:25

【摘要】：背景:全球每年肺癌的发病率居首位,同时也是第一位常见的肿瘤死亡原因。迄今为止,手术切除被认为是治疗肺癌的主要手段,然而,大部分的肺癌病人就诊时已属于进展期,单纯手术疗效有限,因此化疗在肺癌治疗中占有极其重要的地位。化疗已被证实可以提高治疗效果、延长生存时间。紫杉醇(Paclitaxel,Ptx),作为化疗领域中里程碑式的药物,在肺癌治疗中也作为一线方案的重要组成部分被列入NCCN肺癌临床实践指南中。尽管如此,但其单药或者联合化疗的有效率均小于50%。此外,中药单体小分子化合物汉防己甲素(Tetrandrine,Tet)也在我们前期研究中被证实具有潜在抗肿瘤效应。因此如何减少化疗药物的毒副作用且能够发现新型的潜在药物来增加化疗效果成为抗肿瘤药物研发中的一个非常重要的问题。纳米载药体系不仅具有改善水溶性较差药物的水相溶解度,增加其稳定性,从而获得控制释放等传统药物输送体系所具有的作用外,由于应用了不同的设计理念,在抗肿瘤药物输送中又具有许多独特的应用优势。目的:本研究构建了两种不同的纳米载药体系。首先采用自行合成的聚吡咯烷酮-聚己内脂(PVP-PCL)两亲嵌段共聚物,用纳米沉淀法制备了汉防己甲素载药纳米微球(Tet-NPs)。同时,我们基于Carriar fre的概念合成了Ptx-SA小分子化合物,通过其自组装构建了负载紫杉醇的Ptx-SA载药纳米纤维。在随后的研究中考察了两种载药纳米体系的稳定性、缓释效应以及体外与体内协同抑瘤活性来考察载药纳米体系在肺癌治疗中的优势,为这两种新型的纳米给药体系的继续研发提供实践基础。方法:用化学合成法合成了 PVP-PCL二嵌段共聚物以及Ptx-SA小分子化合物。用原子力显微镜和透射电子显微镜观察两种载药体系的形貌,并用动态光散射仪和Zeta电位测定仪检测载药纳米体系的粒径大小和表面电位。同时采用高效液相色谱(HPLC)测定载药纳米体系的载药效率,并且考察两种载药体系的体外缓释特性。在体外细胞及体内动物模型上研究载药纳米微球的抗肿瘤效果及其可能的机制。结果:第一,本文中所构建的Tet-NPs在扫描电子显微镜和透射电子显微镜的观察下呈现为较规则的表面光滑的球形,粒径比较均一,在120nm左右。其载药量为18.2%左右。Tet-NPs具有良好的稳定性以及体外缓释特征。且带有荧光示踪的Tet-NPs能够在与细胞共孵育的短时间内有效的进入细胞。Tet-NPs的诱导凋亡的效果显著强于Tet裸药。同等剂量下Tet-NPs比Tet裸药能够更为有效的调控凋亡相关蛋白的表达。此外,Tet-NPs比同等剂量下的裸药能够更为显著抑制肺癌细胞的侵袭和转移,其机制可能为通过调控MMP相关蛋白的表达。第二,Ptx-SA在扫描电子显微镜和透射电子显微镜的观察下呈现为直径为30-40nm,长约1-3um的纤维状结构。随后的体外释放实验证实了该载药纳米纤维良好的体外缓释性。细胞摄取实验发现Ptx-SA载药纤维具有良好的亲细胞性。Ptx-SA与Ptx裸药所显示的细胞毒效应的效果都具有浓度依赖性,且Ptx-SA的效果强于Ptx裸药。Western blot结果显示同等剂量下Ptx-SA比Ptx裸药能够更为有效的调控Akt蛋白及凋亡相关蛋白的表达。在体内肺癌模型上也证实了Ptx-SA比Ptx裸药更为有效的抑瘤效果。结论:本文中所构建的两种载药纳米体系在体外以及体内均显示出了良好的抗肿瘤效果,并且在体内外的疗效评价中证实了相比于裸药的优越性。可见,制备新型的纳米载药体系,是抗肿瘤药物发展的新方向,具有良好的实用价值和发展前景。
[Abstract]:Background: The incidence of lung cancer in the world is the first and the first common cause of tumor death. So far, surgical resection is thought to be the main means to treat lung cancer. However, most of the patients with lung cancer have been in the stage of progression and the simple operation is limited, so the chemotherapy plays a very important role in the treatment of lung cancer. The chemotherapy has been proved to improve the treatment effect and prolong the survival time. Paclitaxel (Ptx), as a landmark drug in the field of chemotherapy, is also included in the NCCN lung cancer clinical practice guide as an important part of the first-line protocol in the treatment of lung cancer. Nevertheless, the effective rate of either single or combination chemotherapy is less than 50%. In addition, Tetrandrine (Tet), a small-molecule compound of the traditional Chinese medicine, is also proved to have a potential anti-tumor effect in our earlier studies. Therefore, how to reduce the toxic and side effect of the chemotherapy medicine and to find the new potential medicine to increase the effect of the chemotherapy becomes a very important problem in the research and development of the anti-tumor medicine. The nano-carrier system not only has the functions of improving the water phase solubility of the water-soluble poor drug, increasing the stability of the water phase, and thus obtaining the traditional drug delivery system such as controlled release, and has a plurality of unique application advantages in the anti-tumor drug delivery. Objective: In this study, two different nano-carrier systems were constructed. The nano-microball (Tet-NPs) of tetrandrine (Tetrandrine) was prepared by using the self-synthetic polydioxanone-polycaprolactone (PVP-PCL). At the same time, we synthesized the Ptx-SA small molecule compound based on the concept of Carriar fre, and the Ptx-SA drug-loaded nano-fiber loaded with paclitaxel was constructed by self-assembly. In the following study, the stability, sustained-release effect of the two drug-containing nano-systems, and the in vitro and in vivo co-inhibition of the tumor-inhibiting activity were investigated in order to study the advantages of the drug-containing nano-system in the treatment of lung cancer, and provide the practical basis for the continuation and development of the two new drug delivery systems. Methods: PVP-PCL diblock copolymer and Ptx-SA small molecular compound were synthesized by chemical synthesis. The morphology of two drug-carrying systems was observed by atomic force microscope and transmission electron microscope, and the particle size and surface potential of the drug-containing nano-system were detected by dynamic light-scattering instrument and Zeta potential tester. The drug-carrying efficiency of the drug-containing nano-system was determined by high performance liquid chromatography (HPLC), and the in vitro release characteristics of the two drug-carrying systems were investigated. The anti-tumor effect and possible mechanism of the drug-loaded nanospheres were studied in vitro and in vivo animal models. Results: First, the Tet-NPs constructed in this paper showed a smooth spherical surface with a uniform particle size of about 120nm under the observation of a scanning electron microscope and a transmission electron microscope. The drug loading is about 18. 2%. Tet-NPs have good stability and in vitro slow-release characteristics. and a Tet-NPs with a fluorescent trace can effectively enter the cells within a short period of time with the cell co-incubation. The effect of Tet-NPs on the induction of apoptosis was stronger than that of Tet. Tet-NPs at the same dose can be used to control the expression of apoptosis-related protein more effectively than the Tet naked drug. In addition, Tet-NPs can significantly inhibit the invasion and metastasis of lung cancer cells than those of the same dose, and the mechanism may be to regulate the expression of MMP-related proteins. Second, Ptx-SA is present in a fibrous structure having a diameter of 30-40nm and a length of about 1-3um under the observation of a scanning electron microscope and a transmission electron microscope. The subsequent in vitro release experiment confirmed the good in vitro release of the drug-containing nanofibres. The cell uptake experiments showed that the Ptx-SA drug-containing fiber had good cell affinity. The effect of Ptx-SA with the cytotoxic effect shown by the Ptx naked drug has a concentration dependence, and the effect of the Ptx-SA is stronger than that of the Ptx naked drug. Western blot showed that Ptx-SA could more effectively control the expression of Akt protein and apoptosis-related protein at the same dose. In vivo lung cancer model, a more effective tumor-inhibiting effect of Ptx-SA than Ptx bare drug was also confirmed. Conclusion: The two drug-loading nano-systems constructed in this paper show good anti-tumor effect in vitro and in vivo. It can be seen that the preparation of a novel nano-carrier system is a new direction for the development of anti-tumor drugs, and has good practical value and development prospect.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R734.2

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