多西紫杉醇靶向纳米药物对小细胞肺癌治疗作用的研究
发布时间：2018-08-23 14:41
【摘要】：流行病学统计结果显示,每年因癌症而死亡的病例中约有26%的人死于肺癌,而约15%的新增肺癌病例是小细胞肺癌(Small Cell Lung Cancer, SCLC)。小细胞肺癌是肺癌中恶性程度最高的肿瘤之一,临床治疗多以化疗为主,初治有效率高,但治疗过程中极易发生继发性耐药,而且治疗后易复发,患者的两年生存率极低。因此,人们迫切需要找到针对小细胞肺癌治疗的有效化疗药物。随着纳米技术的发展,越来越多的纳米药物被应用到肿瘤的靶向治疗中,如脂质体、树枝状分子、聚合物胶束、超顺磁氧化铁晶体和胶体金药物。已知纳米药物递送涉及到被动靶向和主动靶向。被动靶向是基于肿瘤周围脉管系统增强的渗透和滞留效应进行药物递送,无法从根本上克服常规化疗药物的非选择性杀伤作用。主动靶向则主要依赖配体与肿瘤细胞表面的受体结合,从而“引导”纳米粒子进入肿瘤细胞。现在已经开发的配体主要有叶酸、肽、蛋白、抗体、适配子和寡核苷酸。目前已有靶向纳米药物输送系统应用于多种肿瘤的治疗报道,有些药物甚至已经进入临床试验,但小细胞肺癌靶向递药系统的研究却鲜有报道。本研究旨在构建多西紫杉醇靶向纳米药物并评价其治疗效应。我们以SCLC细胞系H446为靶分子,采用噬菌体展示技术获得了与SCLC细胞系特异性结合的七肽。在采用乳化溶剂挥发法构建包载多西紫杉醇纳米粒子的基础上,将其与靶向肽相连接制备靶向纳米制剂。最后,通过体内外实验探究其抗肿瘤作用及其作用机制。主要研究结果如下：1. 以SCLC细胞系H446为靶分子,利用噬菌体展示技术筛选出能与之特异性结合的七肽(AP和AF)。并采用免疫组织化学技术评价其特异性。结果显示,筛选出的七肽具有与人小细胞肺癌病理组织特异性结合的能力。2.采用可生物降解的高分子材料聚乳酸(PLA)包载多西紫杉醇制备纳米药物,然后利用EDC缩合反应将其与靶向肽连接构建靶向纳米药物。理化性质分析结果显示,制备的纳米制剂平均粒径为195.4±3.9 nm,PDI平均值为0.19±0.03,平均Zeta电位值为-30.5±0.9 mV,而靶向纳米制剂的平均粒径为285.9±4.4 nm,PDI平均值为0.148±0.036,平均Zeta电位值为-30.1±0.6 mV. TEM电镜结果显示,靶向纳米粒子表面光滑,粒径均一。体外释放结果表明纳米药物能够有效释放。3.成功制备靶向纳米药物后进行体外抗肿瘤作用研究。细胞毒性实验结果显示,相同浓度的靶向纳米药物对小细胞肺癌细胞系H446的抑制作用显著强于常规药物多西紫杉醇,并具有一定的浓度依赖性。4. 细胞摄取结果表明,小细胞肺癌细胞系H446对靶向纳米制剂有较好的摄取能力,从而使靶向纳米制剂具有更强的杀伤作用。5.在体实验结果表明,与多西紫杉醇相比,多西紫杉醇靶向纳米制剂能够显著地抑制肿瘤生长,且动物对药物的耐受性良好。6.为探讨靶向纳米制剂的作用机制,我们对其体内分布情况进行了分析。实验结果显示,靶向纳米药物在肿瘤组织分布量是常规药物的4～7倍,而多西紫杉醇在动物肝脏及心脏的浓度均显著高于靶向纳米制剂。7. 各组动物的心、肝、脾、肺、肾、脑组织HE染色结果显示,各剂型药物均能抑制肺癌肝转移,但多西紫杉醇组和纳米药物组动物肝脏均有不同程度的肝细胞颗粒样、脂肪样变形,而靶向纳米药物组动物肝脏状态良好。综上所述,我们基于筛选得到的靶向肽构建了靶向纳米制剂,其在体内和体外实验均表现出明显优于常规药物的肿瘤细胞杀伤作用。靶向纳米药物在肿瘤组织具有较高的药物分布,此外还能有效地抑制小细胞肺癌的肝转移。
[Abstract]:Epidemiological statistics show that about 26% of cancer deaths each year are caused by lung cancer, and about 15% of new lung cancer cases are small cell lung cancer (SCLC). With the development of nanotechnology, more and more nano-drugs have been applied to targeted therapy of cancer, such as liposome, dendrimer, polymerization. Biomicelles, superparamagnetic iron oxide crystals and colloidal gold drugs. It is known that nanodrug delivery involves passive targeting and active targeting. Passive targeting is based on enhanced penetration and retention in the peripheral vascular system of the tumor, which can not fundamentally overcome the non-selective killing effect of conventional chemotherapeutic drugs. Active targeting is predominant. Ligands that have been developed are folic acid, peptide, protein, antibody, aptamer and oligonucleotide. Targeted nanodrug delivery systems have been reported to be used in the treatment of various tumors, and some drugs have even entered clinical practice. The aim of this study was to construct docetaxel-targeted nano-drug and evaluate its therapeutic effect. We used the SCLC cell line H446 as the target molecule and obtained the heptapeptides specifically binding to SCLC cell line by phage display technique. The heptapeptides were constructed by emulsion solvent evaporation method. Finally, the antitumor effect and mechanism of docetaxel nanoparticles were investigated by in vitro and in vivo experiments. The main results were as follows: 1. Using SCLC cell line H446 as the target molecule, seven specific binding compounds were screened by phage display technique. Peptides (AP and AF) were evaluated by immunohistochemistry. The results showed that the heptapeptides had the ability to bind specifically to the pathological tissues of human small cell lung cancer (SCLC). 2. Nano-drugs were prepared by biodegradable polymeric material polylactic acid (PLA) encapsulated with docetaxel and then targeted by EDC condensation reaction. The results of physicochemical properties analysis showed that the average particle size, PDI, Zeta potential and PDI were 195.4 (+ 3.9) nm, 0.19 (+ 0.03) and - 30.5 (+ 0.9) mV, respectively, while the average particle size, PDI and Zeta potential were 285.9 (+ 4.4) nm, 0.148 (+ 0.036) and - 30.1 (+ 0.6) mV.TEM. The results of electron microscopy showed that the surface of targeted nanoparticles was smooth and the particle size was uniform. The results of in vitro release showed that the nanoparticles could be effectively released. 3. The antitumor effect of targeted nanoparticles was studied in vitro after the successful preparation of targeted nanoparticles. The results of cell uptake showed that small cell lung cancer cell line H446 had a better uptake of targeted nanoparticles, which made targeted nanoparticles have a stronger killing effect. 5. In vivo results showed that compared with docetaxel, docetaxel had a stronger killing effect. Targeted nanoparticles can significantly inhibit tumor growth and are well tolerated by animals. 6. To investigate the mechanism of targeting nanoparticles, we analyzed the distribution in vivo. The results showed that the distribution of targeted nanoparticles in tumor tissue was 4-7 times as much as that of conventional drugs, while docetaxel in animal liver. HE staining of heart, liver, spleen, lung, kidney and brain tissues showed that all the dosage forms of drugs could inhibit liver metastasis of lung cancer, but the liver of both docetaxel group and nano-drug group had different degrees of hepatocyte granule-like and fatty deformation. In conclusion, we have constructed targeted nanoparticles based on the selected targeting peptides, which exhibit better cytotoxicity than conventional drugs in vivo and in vitro. Targeted nanoparticles have higher drug distribution in tumor tissues and can effectively inhibit small cell lung cancer. Liver metastasis.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R734.2
本文编号：2199376
[Abstract]:Epidemiological statistics show that about 26% of cancer deaths each year are caused by lung cancer, and about 15% of new lung cancer cases are small cell lung cancer (SCLC). With the development of nanotechnology, more and more nano-drugs have been applied to targeted therapy of cancer, such as liposome, dendrimer, polymerization. Biomicelles, superparamagnetic iron oxide crystals and colloidal gold drugs. It is known that nanodrug delivery involves passive targeting and active targeting. Passive targeting is based on enhanced penetration and retention in the peripheral vascular system of the tumor, which can not fundamentally overcome the non-selective killing effect of conventional chemotherapeutic drugs. Active targeting is predominant. Ligands that have been developed are folic acid, peptide, protein, antibody, aptamer and oligonucleotide. Targeted nanodrug delivery systems have been reported to be used in the treatment of various tumors, and some drugs have even entered clinical practice. The aim of this study was to construct docetaxel-targeted nano-drug and evaluate its therapeutic effect. We used the SCLC cell line H446 as the target molecule and obtained the heptapeptides specifically binding to SCLC cell line by phage display technique. The heptapeptides were constructed by emulsion solvent evaporation method. Finally, the antitumor effect and mechanism of docetaxel nanoparticles were investigated by in vitro and in vivo experiments. The main results were as follows: 1. Using SCLC cell line H446 as the target molecule, seven specific binding compounds were screened by phage display technique. Peptides (AP and AF) were evaluated by immunohistochemistry. The results showed that the heptapeptides had the ability to bind specifically to the pathological tissues of human small cell lung cancer (SCLC). 2. Nano-drugs were prepared by biodegradable polymeric material polylactic acid (PLA) encapsulated with docetaxel and then targeted by EDC condensation reaction. The results of physicochemical properties analysis showed that the average particle size, PDI, Zeta potential and PDI were 195.4 (+ 3.9) nm, 0.19 (+ 0.03) and - 30.5 (+ 0.9) mV, respectively, while the average particle size, PDI and Zeta potential were 285.9 (+ 4.4) nm, 0.148 (+ 0.036) and - 30.1 (+ 0.6) mV.TEM. The results of electron microscopy showed that the surface of targeted nanoparticles was smooth and the particle size was uniform. The results of in vitro release showed that the nanoparticles could be effectively released. 3. The antitumor effect of targeted nanoparticles was studied in vitro after the successful preparation of targeted nanoparticles. The results of cell uptake showed that small cell lung cancer cell line H446 had a better uptake of targeted nanoparticles, which made targeted nanoparticles have a stronger killing effect. 5. In vivo results showed that compared with docetaxel, docetaxel had a stronger killing effect. Targeted nanoparticles can significantly inhibit tumor growth and are well tolerated by animals. 6. To investigate the mechanism of targeting nanoparticles, we analyzed the distribution in vivo. The results showed that the distribution of targeted nanoparticles in tumor tissue was 4-7 times as much as that of conventional drugs, while docetaxel in animal liver. HE staining of heart, liver, spleen, lung, kidney and brain tissues showed that all the dosage forms of drugs could inhibit liver metastasis of lung cancer, but the liver of both docetaxel group and nano-drug group had different degrees of hepatocyte granule-like and fatty deformation. In conclusion, we have constructed targeted nanoparticles based on the selected targeting peptides, which exhibit better cytotoxicity than conventional drugs in vivo and in vitro. Targeted nanoparticles have higher drug distribution in tumor tissues and can effectively inhibit small cell lung cancer. Liver metastasis.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R734.2
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