脑靶向复方纳米胶束治疗HER-2阳性乳腺癌脑转移的实验研究
发布时间：2018-08-22 20:22
【摘要】：第一部分脑靶向紫杉醇-拉帕替尼复方载药纳米胶束的制备及表征目的:近二十年来,乳腺癌在全球女性中的发病率居高不下,尤其是在很多发达国家和地区,长居发病率首位,在欠发达国家和地区,乳腺癌的发病率也有不断增长的趋势。随着乳腺癌分子分型治疗模式的广泛应用,以及靶向治疗药物的不断问世,乳腺癌的总体预后得到了明显的改善。HER-2阳性型乳腺癌的总生存也随着抗HER-2靶向药物的加入而显著延长。然而,脑转移的发生率却随着伴随着生存的延长而增加。目前脑转移主要的治疗方法仍然是手术或脑立体定向放疗为主,尚缺乏一种有效的针对乳腺癌脑转移病灶的治疗药物。本部分研究旨在研发一种具有脑靶向功能的复方纳米载药系统,使载有紫杉醇和拉帕替尼的纳米胶束能较高浓度地通过血脑屏障,治疗乳腺癌脑转移。方法:以聚乙二醇-聚乳酸(PEG-PLA)为载体,采用薄膜水化法(溶剂挥发法)制备胶束,包裹水溶性差的拉帕替尼和紫杉醇,制成载药纳米胶束(以下简称胶束)。首先制备单药胶束,检测胶束各项理化性质,证实该种方法制备的胶束能够满足进一步实验要求。在此基础上制备复方胶束。分别测定复方胶束对紫杉醇和拉帕替尼的载药率包封率和稳定性。将具有亲大脑内皮细胞功能的靶向短肽分子Angiopep-2以氨基与聚合物外壳的羧基连接。在体外实验中,验证接有靶向分子的胶束能够穿透血脑屏障。结果:薄膜水化法制备的复方载药胶束(MIC-LPTN+PTX)粒径为16.23±0.75nm,接有靶向分子的胶束(ANG-MIC-LPTN+PTX)粒径为18.80±0.97 nm。紫杉醇的载药率是2.9±0.8%,包封率95.97±3.17%,对拉帕替尼的载药率是6.82±1.16%,包封率91.21±2.83%,并且能够在4°C时具有良好的稳定性。接有靶向分子的胶束在体外血脑屏障模型中,在相同条件相同时间下,与不接靶向分子的胶束相比,具有更明显的脑靶向功能优势。结论:载有紫杉醇和拉帕替尼的复方纳米胶束具有良好的物理特性。胶束粒径为20nm,能够少量穿透体外血脑屏障模型。连接Angiopep-2脑靶向分子后的复合胶束,具有良好的穿透血脑屏障的功能。第二部分Ang化复方胶束的脑靶向性研究及抗肿瘤效应的实验研究目的:目前针对乳腺癌脑转移,临床可用药物屈指可数,且效果欠佳。论文的第一部分已经证实,在体外实验中,我们制备的复方纳米载药胶束具有良好的脑靶向功能。本部分的研究旨在通过体外细胞毒实验及活体动物体内,验证复方靶向纳米载药胶束具有有效的抗HER-2阳性乳腺癌脑转移作用,能够延长荷瘤鼠的生存期。方法:体外细胞毒实验,使用两种复方胶束分别作用于HER-2阳性与阴性细胞株,分别比较两种胶束对同一细胞株的抑制能力,并且比较双极靶向复方胶束对两种细胞的抑制能力。使用脑定位立体定位仪给裸鼠定向接种乳腺癌细胞Skbr-3(HER-2过表达),若干天后观察裸鼠行为状态,随机选取两只裸鼠,取脑组织,肉眼观察证实脑部已有乳腺癌肿瘤组织。将裸鼠随机分组,每八只一组,三组分别为生理盐水组,未接靶向分子胶束组,接有靶向分子胶束组。每三天给药,共十次,给药量为7.5 mg/kg/3 d,拉帕替尼:紫杉醇=2:1。记录每组荷瘤鼠死亡日期,从第一次给药开始共观察60天。绘制生存曲线,得出中位生存期。结果:两种胶束比较,从对两种细胞的抑制能力而言,双极靶向复方纳米胶束对细胞的抑制能力更强。而双极靶向复方纳米胶束对HER-2阳性细胞株的抑制能力更强。在脑转移瘤模型鼠体内,接有靶向分子的胶束能够在更短的时间内穿透血脑屏障,到达病变组织。在三组荷瘤鼠中,接有靶向分子的胶束组,中位生存期为56天。未接靶向分子的胶束组,中位生存期为47天。生理盐水组,中位生存期为35天。注射连接Angiopep-2脑靶向分子的胶束组的荷瘤鼠生存明显延长。P=0.0465。结论:接有靶向分子的复方胶束对HER-2阳性的乳腺癌细胞株更有效,能够有效延长HER-2阳性乳腺癌脑转移模型鼠的生存期。
[Abstract]:Part I Preparation and Characterization of Brain Targeted Paclitaxel-Lapatinib Compound Drug-loaded Nanomicelles Objective: In the past 20 years, the incidence of breast cancer in women has been high in the world, especially in many developed countries and regions, the incidence of breast cancer has been the highest. In less developed countries and regions, the incidence of breast cancer has been increasing. The overall prognosis of breast cancer has been significantly improved with the widespread use of molecular typing and targeted therapies for breast cancer. The overall survival of HER-2-positive breast cancer has been significantly prolonged with the addition of anti-HER-2 targeted drugs. However, the incidence of brain metastasis has been associated with the prolongation of survival. The main treatment for brain metastasis is still surgery or stereotactic radiotherapy, but there is no effective drug for brain metastasis of breast cancer. This study aims to develop a compound nano-drug delivery system with brain-targeting function, so that the nano-micelles containing paclitaxel and rapatenil can be higher. Methods: Using PEG-PLA as carrier, the micelles were prepared by membrane hydration method (solvent evaporation method), and the drug-loaded nanomicelles were prepared by wrapping the water-soluble Lapatinib and Paclitaxel. The encapsulation efficiency and stability of the compound micelles to paclitaxel and rapatenil were determined respectively. Angiopep-2, a short peptide molecule with the function of pro-brain endothelial cells, was linked to the carboxyl group of the polymer shell with amino group. Results: Micelles with targeted molecules (MIC-LPTN+PTX) prepared by membrane hydration method had a particle size of 16.23.75 nm, and micelles with targeted molecules (ANG-MIC-LPTN+PTX) had a particle size of 18.80.97 nm. The drug loading rate and encapsulation efficiency of tinib were 6.82 6550 Tini composite nanomicelles have good physical properties. The micelle size is 20 nm and can penetrate a small amount of blood brain barrier model in vitro. The composite micelles linked with Angiopep-2 brain targeting molecules have good function of penetrating blood brain barrier. Part II Brain targeting study of Angiopep-2 compound micelles and experimental study of anti-tumor effect. The first part of this paper has confirmed that the compound nano-drug-loaded micelles prepared by us have good brain targeting function in vitro. The purpose of this part is to validate the compound targeting sodium by cytotoxicity test in vitro and in vivo in vivo. METHODS: In vitro cytotoxicity test, two kinds of compound micelles were used to treat HER-2 positive and HER-2 negative cell lines respectively, and the inhibitory effects of the two micelles on the same cell line were compared, and the bipolar targeted compound micelles on the same cell line were compared. Skbr-3 (HER-2 overexpression) was inoculated into nude mice by stereotactic brain localization system. The behavior of nude mice was observed several days later. Two nude mice were randomly selected and brain tissues were taken from the nude mice. Normal saline group, non-targeted micelles group, followed by targeted micelles group. Every three days, a total of 10 times, dosage of 7.5 mg/kg/3 days, Lapatinib: Paclitaxel = 2:1. Record the death date of each group of tumor-bearing mice, from the first administration to observe a total of 60 days. Bipolar targeting compound nanomicelles have stronger inhibitory effect on HER-2 positive cells than bipolar targeting compound nanomicelles. In brain metastasis model mice, micelles with targeted molecules can penetrate the blood-brain barrier and reach the lesion tissue in a shorter time. The median survival time was 56 days in micelles with targeted molecules. The median survival time was 47 days in micelles without targeted molecules. The median survival time was 35 days in normal saline group. The survival time of micelles with targeted molecules was significantly prolonged in micelles injected with Angiopep-2. It was more effective to HER-2 positive breast cancer cell lines and prolonged the survival time of HER-2 positive breast cancer brain metastasis model mice.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9
[Abstract]:Part I Preparation and Characterization of Brain Targeted Paclitaxel-Lapatinib Compound Drug-loaded Nanomicelles Objective: In the past 20 years, the incidence of breast cancer in women has been high in the world, especially in many developed countries and regions, the incidence of breast cancer has been the highest. In less developed countries and regions, the incidence of breast cancer has been increasing. The overall prognosis of breast cancer has been significantly improved with the widespread use of molecular typing and targeted therapies for breast cancer. The overall survival of HER-2-positive breast cancer has been significantly prolonged with the addition of anti-HER-2 targeted drugs. However, the incidence of brain metastasis has been associated with the prolongation of survival. The main treatment for brain metastasis is still surgery or stereotactic radiotherapy, but there is no effective drug for brain metastasis of breast cancer. This study aims to develop a compound nano-drug delivery system with brain-targeting function, so that the nano-micelles containing paclitaxel and rapatenil can be higher. Methods: Using PEG-PLA as carrier, the micelles were prepared by membrane hydration method (solvent evaporation method), and the drug-loaded nanomicelles were prepared by wrapping the water-soluble Lapatinib and Paclitaxel. The encapsulation efficiency and stability of the compound micelles to paclitaxel and rapatenil were determined respectively. Angiopep-2, a short peptide molecule with the function of pro-brain endothelial cells, was linked to the carboxyl group of the polymer shell with amino group. Results: Micelles with targeted molecules (MIC-LPTN+PTX) prepared by membrane hydration method had a particle size of 16.23.75 nm, and micelles with targeted molecules (ANG-MIC-LPTN+PTX) had a particle size of 18.80.97 nm. The drug loading rate and encapsulation efficiency of tinib were 6.82 6550 Tini composite nanomicelles have good physical properties. The micelle size is 20 nm and can penetrate a small amount of blood brain barrier model in vitro. The composite micelles linked with Angiopep-2 brain targeting molecules have good function of penetrating blood brain barrier. Part II Brain targeting study of Angiopep-2 compound micelles and experimental study of anti-tumor effect. The first part of this paper has confirmed that the compound nano-drug-loaded micelles prepared by us have good brain targeting function in vitro. The purpose of this part is to validate the compound targeting sodium by cytotoxicity test in vitro and in vivo in vivo. METHODS: In vitro cytotoxicity test, two kinds of compound micelles were used to treat HER-2 positive and HER-2 negative cell lines respectively, and the inhibitory effects of the two micelles on the same cell line were compared, and the bipolar targeted compound micelles on the same cell line were compared. Skbr-3 (HER-2 overexpression) was inoculated into nude mice by stereotactic brain localization system. The behavior of nude mice was observed several days later. Two nude mice were randomly selected and brain tissues were taken from the nude mice. Normal saline group, non-targeted micelles group, followed by targeted micelles group. Every three days, a total of 10 times, dosage of 7.5 mg/kg/3 days, Lapatinib: Paclitaxel = 2:1. Record the death date of each group of tumor-bearing mice, from the first administration to observe a total of 60 days. Bipolar targeting compound nanomicelles have stronger inhibitory effect on HER-2 positive cells than bipolar targeting compound nanomicelles. In brain metastasis model mice, micelles with targeted molecules can penetrate the blood-brain barrier and reach the lesion tissue in a shorter time. The median survival time was 56 days in micelles with targeted molecules. The median survival time was 47 days in micelles without targeted molecules. The median survival time was 35 days in normal saline group. The survival time of micelles with targeted molecules was significantly prolonged in micelles injected with Angiopep-2. It was more effective to HER-2 positive breast cancer cell lines and prolonged the survival time of HER-2 positive breast cancer brain metastasis model mice.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9
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相关期刊论文 前6条
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