腦靶向復(fù)方納米膠束治療HER-2陽(yáng)性乳腺癌腦轉(zhuǎn)移的實(shí)驗(yàn)研究
發(fā)布時(shí)間：2018-08-22 20:22
【摘要】：第一部分腦靶向紫杉醇-拉帕替尼復(fù)方載藥納米膠束的制備及表征目的:近二十年來(lái),乳腺癌在全球女性中的發(fā)病率居高不下,尤其是在很多發(fā)達(dá)國(guó)家和地區(qū),長(zhǎng)居發(fā)病率首位,在欠發(fā)達(dá)國(guó)家和地區(qū),乳腺癌的發(fā)病率也有不斷增長(zhǎng)的趨勢(shì)。隨著乳腺癌分子分型治療模式的廣泛應(yīng)用,以及靶向治療藥物的不斷問(wèn)世,乳腺癌的總體預(yù)后得到了明顯的改善。HER-2陽(yáng)性型乳腺癌的總生存也隨著抗HER-2靶向藥物的加入而顯著延長(zhǎng)。然而,腦轉(zhuǎn)移的發(fā)生率卻隨著伴隨著生存的延長(zhǎng)而增加。目前腦轉(zhuǎn)移主要的治療方法仍然是手術(shù)或腦立體定向放療為主,尚缺乏一種有效的針對(duì)乳腺癌腦轉(zhuǎn)移病灶的治療藥物。本部分研究旨在研發(fā)一種具有腦靶向功能的復(fù)方納米載藥系統(tǒng),使載有紫杉醇和拉帕替尼的納米膠束能較高濃度地通過(guò)血腦屏障,治療乳腺癌腦轉(zhuǎn)移。方法:以聚乙二醇-聚乳酸(PEG-PLA)為載體,采用薄膜水化法(溶劑揮發(fā)法)制備膠束,包裹水溶性差的拉帕替尼和紫杉醇,制成載藥納米膠束(以下簡(jiǎn)稱(chēng)膠束)。首先制備單藥膠束,檢測(cè)膠束各項(xiàng)理化性質(zhì),證實(shí)該種方法制備的膠束能夠滿足進(jìn)一步實(shí)驗(yàn)要求。在此基礎(chǔ)上制備復(fù)方膠束。分別測(cè)定復(fù)方膠束對(duì)紫杉醇和拉帕替尼的載藥率包封率和穩(wěn)定性。將具有親大腦內(nèi)皮細(xì)胞功能的靶向短肽分子Angiopep-2以氨基與聚合物外殼的羧基連接。在體外實(shí)驗(yàn)中,驗(yàn)證接有靶向分子的膠束能夠穿透血腦屏障。結(jié)果:薄膜水化法制備的復(fù)方載藥膠束(MIC-LPTN+PTX)粒徑為16.23±0.75nm,接有靶向分子的膠束(ANG-MIC-LPTN+PTX)粒徑為18.80±0.97 nm。紫杉醇的載藥率是2.9±0.8%,包封率95.97±3.17%,對(duì)拉帕替尼的載藥率是6.82±1.16%,包封率91.21±2.83%,并且能夠在4°C時(shí)具有良好的穩(wěn)定性。接有靶向分子的膠束在體外血腦屏障模型中,在相同條件相同時(shí)間下,與不接靶向分子的膠束相比,具有更明顯的腦靶向功能優(yōu)勢(shì)。結(jié)論:載有紫杉醇和拉帕替尼的復(fù)方納米膠束具有良好的物理特性。膠束粒徑為20nm,能夠少量穿透體外血腦屏障模型。連接Angiopep-2腦靶向分子后的復(fù)合膠束,具有良好的穿透血腦屏障的功能。第二部分Ang化復(fù)方膠束的腦靶向性研究及抗腫瘤效應(yīng)的實(shí)驗(yàn)研究目的:目前針對(duì)乳腺癌腦轉(zhuǎn)移,臨床可用藥物屈指可數(shù),且效果欠佳。論文的第一部分已經(jīng)證實(shí),在體外實(shí)驗(yàn)中,我們制備的復(fù)方納米載藥膠束具有良好的腦靶向功能。本部分的研究旨在通過(guò)體外細(xì)胞毒實(shí)驗(yàn)及活體動(dòng)物體內(nèi),驗(yàn)證復(fù)方靶向納米載藥膠束具有有效的抗HER-2陽(yáng)性乳腺癌腦轉(zhuǎn)移作用,能夠延長(zhǎng)荷瘤鼠的生存期。方法:體外細(xì)胞毒實(shí)驗(yàn),使用兩種復(fù)方膠束分別作用于HER-2陽(yáng)性與陰性細(xì)胞株,分別比較兩種膠束對(duì)同一細(xì)胞株的抑制能力,并且比較雙極靶向復(fù)方膠束對(duì)兩種細(xì)胞的抑制能力。使用腦定位立體定位儀給裸鼠定向接種乳腺癌細(xì)胞Skbr-3(HER-2過(guò)表達(dá)),若干天后觀察裸鼠行為狀態(tài),隨機(jī)選取兩只裸鼠,取腦組織,肉眼觀察證實(shí)腦部已有乳腺癌腫瘤組織。將裸鼠隨機(jī)分組,每八只一組,三組分別為生理鹽水組,未接靶向分子膠束組,接有靶向分子膠束組。每三天給藥,共十次,給藥量為7.5 mg/kg/3 d,拉帕替尼:紫杉醇=2:1。記錄每組荷瘤鼠死亡日期,從第一次給藥開(kāi)始共觀察60天。繪制生存曲線,得出中位生存期。結(jié)果:兩種膠束比較,從對(duì)兩種細(xì)胞的抑制能力而言,雙極靶向復(fù)方納米膠束對(duì)細(xì)胞的抑制能力更強(qiáng)。而雙極靶向復(fù)方納米膠束對(duì)HER-2陽(yáng)性細(xì)胞株的抑制能力更強(qiáng)。在腦轉(zhuǎn)移瘤模型鼠體內(nèi),接有靶向分子的膠束能夠在更短的時(shí)間內(nèi)穿透血腦屏障,到達(dá)病變組織。在三組荷瘤鼠中,接有靶向分子的膠束組,中位生存期為56天。未接靶向分子的膠束組,中位生存期為47天。生理鹽水組,中位生存期為35天。注射連接Angiopep-2腦靶向分子的膠束組的荷瘤鼠生存明顯延長(zhǎng)。P=0.0465。結(jié)論:接有靶向分子的復(fù)方膠束對(duì)HER-2陽(yáng)性的乳腺癌細(xì)胞株更有效,能夠有效延長(zhǎng)HER-2陽(yáng)性乳腺癌腦轉(zhuǎn)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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R737.9
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相關(guān)期刊論文 前6條
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