载药纳米粒子心血管内局部传递用于血管再狭窄的防治

【作者】 梅林；

【导师】 宋存先； 孙洪范；

【作者基本信息】 中国协和医科大学 ， 生物医学工程， 2007， 博士

【摘要】 经皮腔内冠状动脉成形术(Percutaneous Transluminal CoronaryAngioplasty，P7CA)是冠状动脉硬化性心脏病的主要治疗手段，但PTCA术后血管再狭窄的发生率高达15—60％，迄今仍是临床亟待解决的难题。如今，局部给药治疗血管再狭窄的方法主要有药物洗脱支架(Drug-eluting stent，DES)和基于球囊导管的药物传递系统。虽然已经有DES进入了临床应用，但是DES仍然存在一些有效性和安全性的问题。DES重要的缺陷就是在支架撑杆上药物浓度最高，抗增殖药物可抑制内皮的愈合，而此部位的内皮愈合是最重要的。另一方面，支架边缘和撑杆之间的部位却不能完全抑制新生内膜的形成，从而影响了DES的疗效。此外，DES不能应用在小血管和分支血管的病变部位。一些重要的安全问题也没有完全明确，如血栓、晚期支架贴壁不良、形成动脉瘤、边缘效应、用于结合药物所选用的聚合物引起的晚期炎症反应、释放毒素。因此，有必要开发其它局部药物传递系统作为DES的补充。这些系统可以将药物输送到没有被支架直接覆盖的血管部位，特别是小血管和多支血管。然而局部灌注药物溶液的传递效率和在血管腔内驻留的时间都相当低，影响了药物疗效充分发挥。因此，局部灌注纳米粒悬液也存在动脉壁内驻留效率低的问题。使用阳离子表面活性剂溴化双十二烷基二甲基铵(didodecyldimethylammonium bromide，DMAB)修饰纳米粒可增加其在动脉壁的驻留，从而增加其防治血管再狭窄的效果。紫杉醇能抑制兔颈动脉剥脱损伤模型的新生内膜的增生，达到此效果所需的血药浓度比治疗肿瘤所需的浓度低100到1000倍，由于很小的剂量即能抑制新生内膜的增生，因此被选用为本实验的治疗药物。本文第一章综述了PTCA术后再狭窄的发病机理及局部药物传递治疗血管再狭窄的现状。本文具体研究内容如下：1．制备了载尿激酶和紫杉醇PLGA纳米微球，用物理吸附法对载紫杉醇纳米微球进行DMAB表面修饰。首先对纳米微球进行表征，使用高效液相色谱仪对包封率和体外蓟物释放进行分析，测定了纳米微球的粒径和表面Zeta电位。建立兔颈动脉损伤模型，在血管局部灌注不同浓度的修饰纳米粒。28天后，取出局部给药的颈动脉血管，进行HE染色和弹力纤维染色。结果显示，尿激酶纳米微球呈两相的体外释放特征，48小时后，其在释放液中释放的尿激酶的活性急剧下降。尿激酶不适合作为纳米粒子缓释制剂应用。同时制备成粒径300纳米左右、包封率80％以上且表面带正电荷的载紫杉醇纳米微球。体外药物释放呈两相释放。28天后，血管内局部灌注紫杉醇纳米悬液可有效抑制血管内皮增生，抑制效果随纳米粒悬液浓度的增加而提高。浓度达到30mg／ml时，可完全抑制血管内膜增生。相比于未修饰纳米粒，DMAB修饰纳米粒能显著抑制新生内膜的增生(P＜0.05)。(第二章)2．合成PCL／F68材料，并利用溶剂替代法制备了载紫杉醇PCL／F68纳米微球，用物理吸附法对载紫杉醇纳米微球进行DMAB表面修饰。首先对纳米微球进行表征，载药量、包封率和体外药物释放使用高效液相色谱仪进行分析，测定了纳米粒子的粒径和表面Zeta电位。建立兔颈动脉损伤模型，在血管局部灌注不同浓度的修饰纳米粒。90天后，取出局部给药的颈动脉血管，进行HE染色和弹力纤维染色。结果合成了粘均分子量为44，000的材料，制备成包封效率75％以上，载药量3.5％左右，平均粒径在300nm左右，粒径范围窄，体外释放药物呈两相特征的表面带正电荷的载紫杉醇PCL／F68纳米粒。90天后，血管内局部灌注载紫杉醇PCL／F68纳米悬液可有效抑制血管内皮增生，其抑制效果比载紫杉醇PLGA纳米悬液要好(P＜0.05)。(第三章)综上所述，DMAB修饰纳米粒能显著抑制新生内膜的增生，其效果比未修饰纳米粒更好。纳米制剂与介入治疗相结合将提供一种极具前途的血管再狭窄的治疗方法。更多还原

【Abstract】 PTCA （Percutaneous Transluminal Coronary Angioplasty） has become one of the most important strategies to treat coronary artery diseases. However, restenosis has been observed in pathological and clinical studies, and poses a formidable problem （15-60%）. Currently, there are mainly two local drug delivery syetems are employed to prevent restensis after balloon angioplasty including drug-eluting stent/radioactive stent and catheter-based drug delivery system. The proliferation of VSMC, the cause of restenosis development, could be inhibited by the application of radioactive and drug eluting stents. However, their efficacy and safety have not been confirmed in all clinical settings. It should be also noted that 30-40% of critical lesions cannot be stented, largely because they occur at branch sites or in small arteries. Important safety issues such as thrombosis, late stent malapposition, aneurysm formation, edge effect, late inflammation due to choice of polymer used to bind the drug, the release of toxins, and potential interaction with brachytherapy have not been completely addressed. Thus, non-stent-based local delivery of antiproliferative drugs may offer additional flexibility and efficacy in the entire range of applications. It may also deliver drugs to vessel areas not directly covered by the stent, which could be of special interest for small and tortuous vessels. However, the delivery efficiency and intramural retention time of infused drug solution remains rather low. Nanoparticles modified with a cationic surfactant, didodecyldimethylammonium bromide （DMAB） can greatly enhance arterial retention in animal angioplasty models. Paclitaxel can inhibit neointimal formation in vivo with plasma paclitaxel levels about 100 to 1000 times lower than the concentrations to treat neoplasms. Thus, paclitaxel was selected as a better pharmacologic component to be formulated into nanoparticles.In chapter 1, the recent progress of the research topic was reviewed.The major contents of this paper are shown as follows:Chapter 2. This study tested the possibility of localized intravascular infusion of positive charged paclitaxel-loaded nanoparticles （NPs） to better prevent neointimal formation in a rabbit carotid artery injury model. Paclitaxel-loaded NPs were prepared by oil/water emulsion/solvent evaporation technique using biodegradable poly （lactide-co-glycolide） （PLGA）. Urokinase-loaded PLGA NPs were prepared by w/o/w emulsion/solvent evaporation technique. A cationic surfactant, didodecyldimethylammonium bromide （DMAB）, was absorbed on the NP surface by electrostatic attraction between positive and negative charges. NPs were characterized in such aspects as size, surface morphology, surface charges as well as in vitro drug release profile. Balloon-injured rabbit carotid arteries were treated with single infusion of paclitaxel-loaded NP suspension and observed for 28 days. The inhibitory effects of NPs on neointima formation were evaluated as end-point. NPs showed spherical shape with a diameter ranging from 200 to 500 nm. Negatively charged PLGA NPs shifted to positive after the DMAB modification. The in vitro drug release profile showed a biphasic release pattern. After 48 hours, the specific activity of released urokinase droped dramatically, indicating that urokinase cann’t be used to prepare nanoparticles for a prolonged release. Morphometric analyses on the retrieved artery samples revealed that the inhibitory effect of intima proliferation was dose-dependent. At a concentration of 30 mg ml^-1, NP infusion completely inhibited intima proliferation in a rabbit vascular injury model. Paclitaxel-loaded NPs with DMAB modification were proven an effective means of inhibiting proliferative response to vascular injury in a rabbit model.Chapter 3. PCL/F68 was synthesized by bulk polymerization as described previously. Paclitaxel-loaded nanoparticles（NPs） were prepared by solvent displacement method using biodegradable PCL/F68. A cationic surfactant, didodecyldimethylammonium bromide （DMAB）, was absorbed on the NP surface by electrostatic attraction between positive and negative charges. NPs were characterized in such aspects as size, surface morphology, surface charges as well as in vitro drug release profile. Balloon-injured rabbit carotid arteries were treated with single infusion of paclitaxel-loaded NP suspension and observed for 90 days. The inhibitory effects of NPs on neointima formation were evaluated as end-point. The obtained PCL/F68 compound has a viscosity average molecular weight of 44,000. PCL/F68 NPs showed porous surface and spherical shape with a mean diameter around 300nm. Negatively charged PLGA NPs shifted to positive after the DMAB modification. The in vitro drug release profile showed a biphasic release pattern. Morphometric analyses on the retrieved artery samples revealed that PCL/F68 NP infusion completely inhibited intima proliferation in a rabbit vascular injury model. Compared with PLGA NPs, the paclitaxel-loaded PCL/F68 has a better effect of inhibiting intima proliferation in animal model （P＜0.05）. Paclitaxel-loaded PCL/F68 NPs with DMAB modification represents a longer delay and an effective means of inhibiting proliferative response to vascular injury in a rabbit model.In conclusion, the modified paclitaxel-loaded nanoparticles as local delivery system provide an effective means of inhibting proliferative response to vascular injury in the rabbits.更多还原