血小板膜糖蛋白单克隆抗体洗脱血管内支架的实验研究
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摘要
经皮腔内血管成形术是治疗血管狭窄或闭塞性疾病的主要介入手段,但术后30%-50%的再狭窄率,严重地影响了其远期疗效。血管内支架置入术辅以抗凝、抗血小板治疗虽能有效地抑制血管急性弹性回缩和血管负性重塑,但因术后血管平滑肌细胞迁移、增殖所致的内膜过度增生仍使15%-25%的患者出现了支架再狭窄。许多抗细胞增殖药物虽在动物实验中取得了较好的效果,但在临床实践中却没有达到预期的疗效,一个重要的原因是全身给药很难在病变血管局部达到持续有效的药物浓度。药物洗脱支架通过包被于金属支架表面的聚合物携带药物,当支架置入病变部位后,药物自聚合物中缓慢释放而在损伤局部发挥持续高效的生物学效应,使再狭窄降至10%以下。
目前药物洗脱支架的研发主要集中在以下三个方面:药物载体材料的选择和制备、药物的筛选以及药物控释的实现。本文选用国内自主研发的抗血小板膜糖蛋白Ⅲa单克隆抗体为洗脱药物,聚乳酸为药物载体,利用超声雾化喷涂和被动吸附的方法制备单抗洗脱血管内支架。在制备并检测了聚合物涂层和单抗洗脱血管内支架体外特性的基础上,应用正常兔髂动脉置入过大支架的动物模型评价了单抗洗脱血管内支架的生物安全性和防治支架内再狭窄的有效性。论文的主要内容和结果如下:①根据喷涂方法的特点和超声雾化的原理,设计并实现了超声雾化血管支架
喷涂装置。该装置包括雾化模块、载气模块、载液模块(包括喷嘴)和载支架模块四个部分,其中雾化模块包括投入式雾化器和水槽;载气模块包括高压氮气瓶和气流表,实现对载气流量的控制和调节;载液模块和喷嘴为玻璃材质,便于雾化聚合物溶液和实验后仪器的清洗;载支架模块能够实现喷涂过程中支架的旋转往复运动,以利于制备出均匀、稳定的聚合物涂层。经后续的实验验证该装置操作简便,制备的聚合物涂层均匀、稳定,且与支架表面粘附牢固,易于实现多层涂层的喷涂制备。
②选择喷涂参数设计正交实验,对超声雾化喷涂装置制备聚乳酸涂层进行了工艺研究,通过不均匀区域记数法确定了适宜的喷涂条件,即喷涂时间为30s、聚乳酸浓度为1.0mg/ml、转头转速为50rpm。
③在适宜喷涂条件下制备聚乳酸涂层,采用荧光染色和扫描电镜确认并分析了聚乳酸涂层在支架表面的分布及其形貌特征,并结合计算机图像分析方法评价了涂层的均匀性、孔隙分布、表面粗糙度等性能。另外,对聚乳酸涂层支架进行体外扩张实验,比较了扩张前后支架表面聚乳酸涂层的变化情况,以考察涂层与支架的黏附和力学性能。实验结果证明聚乳酸涂层具有良好的孔隙度分布和表面形貌,扩张前后与支架的附着良好,涂层无龟裂、脱落或翘起等现象。
④应用免疫荧光的方法对吸附在聚乳酸涂层中的单抗进行染色,以确定单抗在聚乳酸涂层中的吸附;同时,对单抗、聚乳酸及其混合物进行差示扫描量热法检测,以确定单抗与聚乳酸是否结合及其结合方式,从而考察被动吸附法制备单抗洗脱血管内支架的可行性。结果表明单抗以被动吸附的方式结合到聚乳酸涂层上,且与聚乳酸涂层载体之间无共聚化反应发生,聚乳酸涂层对单抗的生物学活性没有明显影响。用扫描电镜观察与枸橼酸盐抗凝全血接触后的单抗洗脱支架发现几乎没有血小板的聚集,表明单抗洗脱支架在体外能显著抑制血小板的聚集。采用125I放射性标记单抗的方法对单抗在聚乳酸涂层上的吸附以及单抗在一定流量下的释放进行了定量的检测。从吸附实验的结果来看,单抗在聚乳酸涂层上的吸附量与单抗溶液的浓度和吸附时间有关。实验检测了支架丝在三种不同的单抗浓度中吸附120h的实验数据,最大的单抗吸附量是在2.0mg/ml的单抗溶液中吸附72h时得到的。支架上单抗的释放时间可达到2周以上,不同的制备方法和流量对药物的释放有一定影响。
⑤将单抗洗脱支架和对照316L不锈钢裸支架置入兔髂动脉,在1、4、12周后,对置入支架段血管及其两端的血管段、主要脏器组织和血清,运用硬组织切片、扫描电镜、透射电镜、免疫组化以及组织染色、生化检测等技术方法对此单抗洗脱支架的有效性和安全性进行检测评价。结果表明单抗洗脱支架在3个月的观察期内安全、可靠,同时能有效防止支架内再狭窄和血栓形成,其机理可能与单抗抑制血小板的粘附和聚集以及加速支架表面内皮化和抑制血管平滑肌细胞的迁移和增殖有关。
The percutaneous transluminal angioplasty (PTCA) as the main interventional therapies of stenosis and occlusion of blood vessel has induced restenosis (30%-50%), which influence it’s late curative effect. The introduction of stents in the clinical practice was a major breakthrough in the field of percutaneous coronary intervention. Its success assistant with the anticoagulant and antiplatelet therapies is thought to be due to its ability to reduce the acute elastic recoil and long-term negative remodelling associated with PTCA. The stents may also induce a more chronic and pronounced vascular response than angioplasty alone which has been defined as in-stent restenosis (ISR). There are still relatively high in-stent restenosis rates (15%-25%) after stenting, caused by the excess neointimal hyperplasia because of the smooth muscle cell migration and proliferation. Although many anti-proliferation drugs have good effects in animal experiments the clinical uses of them have depressed us. An important reason is that they could not reach the sufficient concentration at local vascular vessel by systemic therapy. Drug-eluting stents (DES) are coated with a bioresorbable polymer capable of releasing single or multiple bioactive agents into the bloodstream and surrounding tissues. Local deliveries to the vasculature achieves high regional drug concentrations and decrease the ISR to below 10%, with prolonged retention at lower doses and reduces systemic toxicity.
The issues in the preparation process of DES are generally focused on three chief aspects, including coating material and its preparation methods, the selection of biological agents, the control release of drugs. In this article the biological agent is domestic-made platelet glycoproteinⅢa receptor monoclonal antibody (mAb) and the coating material is L-polylactic acid (PLLA). The spray method based on ultrasonic atomization is used to prepare PLLA coated stents, which is immersed into the mAb solution passively absorbed by coating. After evaluating the qualities of polymer coating and mAb eluting stents, we have examined the biosecurity and validity of prevention and cure ISR by implanted exceeding mAb eluting stents into rabbit iliac arteries. The major contents and results of the research are as follows.
①The spray equipment for experiment was designed and produced according to the character of spray process and the principle of ultrasonic atomization. The equipment were composed of four parties, including atomization module, gas-carrier module, solution-container module, and stent-carrier module. The atomization module could atomize the polymer solution through a drop-in ultrasonic atomizer located in the cylindrical water container. The gas-carrier module could control and adjust the outflow flux of N2 in the high-pressure air tank through gas flowmeter. The polymer-solution container and nozzle were made of glass because they were easy to atomize polymer solution and clear. The stent-carrier module helped to prepare uniform polymer coating by the means of making the stent move back-and-forth in front of nozzle at the time of maintaining circumrotation. This equipment could prepare the ideal polymer coating on the stent surface and easy to spary multi-coating.
②The technics process was introduced in this text. The optimal parameters, which were determined by counting the un-uniform district, were spray time 30s, PLLA concentration 1.0 mg/ml, rotate speed 50 rpm.
③The distribution and morphologies of PLLA coatings on the stent surface prepared under the optimal parameters were visualized through fluorescence staining and scanning electron microscopy (SEM), combined with computer image analysis method to estimate the uniformity, hole distribution and surface roughness of coating. In addition the morphologies of PLLA coating were examined both pre- and post-expansion in vitro. The results suggest the PLLA coating has favourable hole distribution and surface morphology, and no cracks, break-off, or turnup both pre- and post-expansion.
④The presence of the PLLA coating over the entire surface of the stent was evidenced by means of a fluorescence staining method. Whether if the PLLA combine with the mAb or not was confirmed by the DSC. After these two experiments we could make certain the feasibility of passive absorption of mAb in the PLLA coating on the stent surface. The convergence degree of fluorescence intensity distribution indicated the uniformity of PLLA coating pore distribution, which was closely related to the distribution of mAb in PLLA coating. The results indicated there are no chemistry reaction between the PLLA and mAb, and PLLA has no influence on the biology activity of mAb.
Stents eluting the mAb were tested for their adsorption characteristics by radioisotope technique with 125I labelled mAb. The elution rates were then measured in looped circuits at different velocities and durations. The amount of mAb adsorbed onto the PLLA coating was dependent on the concentration and duration of immersion in the solution. We have tested three different concentrations for 120h. Maximal mAb binding was therefore defined as the amount of agent bound to stent wires after 72 hours immersion in a 2 mg/ml solution. The method of preparation the mAb eluting stent and flow velocity significantly influenced the elution characteristics for a continuous perfusion of more than 2 weeks.
⑤We implant the mAb eluting stents and 316L stainless steel stents the control in rabbit iliac arteries to study the bio-security validity of prevention and cure ISR by sliding microtome, SEM, transmission electron microscope (TEM), immunohistochemi- stry, tissue stain and biochemistry methods. The blood vessel and serum samples were got from the rabbits after stents implantation for 1week, 4weeks and 12weeks. The results suggests the mAb eluting stents are biocompatible and can inhibit the thrombosis and neointimal hyperplasia by accelerating the endothelialization of the stent surface and inhibit the smooth muscle cell migration and proliferation.
目前药物洗脱支架的研发主要集中在以下三个方面:药物载体材料的选择和制备、药物的筛选以及药物控释的实现。本文选用国内自主研发的抗血小板膜糖蛋白Ⅲa单克隆抗体为洗脱药物,聚乳酸为药物载体,利用超声雾化喷涂和被动吸附的方法制备单抗洗脱血管内支架。在制备并检测了聚合物涂层和单抗洗脱血管内支架体外特性的基础上,应用正常兔髂动脉置入过大支架的动物模型评价了单抗洗脱血管内支架的生物安全性和防治支架内再狭窄的有效性。论文的主要内容和结果如下:①根据喷涂方法的特点和超声雾化的原理,设计并实现了超声雾化血管支架
喷涂装置。该装置包括雾化模块、载气模块、载液模块(包括喷嘴)和载支架模块四个部分,其中雾化模块包括投入式雾化器和水槽;载气模块包括高压氮气瓶和气流表,实现对载气流量的控制和调节;载液模块和喷嘴为玻璃材质,便于雾化聚合物溶液和实验后仪器的清洗;载支架模块能够实现喷涂过程中支架的旋转往复运动,以利于制备出均匀、稳定的聚合物涂层。经后续的实验验证该装置操作简便,制备的聚合物涂层均匀、稳定,且与支架表面粘附牢固,易于实现多层涂层的喷涂制备。
②选择喷涂参数设计正交实验,对超声雾化喷涂装置制备聚乳酸涂层进行了工艺研究,通过不均匀区域记数法确定了适宜的喷涂条件,即喷涂时间为30s、聚乳酸浓度为1.0mg/ml、转头转速为50rpm。
③在适宜喷涂条件下制备聚乳酸涂层,采用荧光染色和扫描电镜确认并分析了聚乳酸涂层在支架表面的分布及其形貌特征,并结合计算机图像分析方法评价了涂层的均匀性、孔隙分布、表面粗糙度等性能。另外,对聚乳酸涂层支架进行体外扩张实验,比较了扩张前后支架表面聚乳酸涂层的变化情况,以考察涂层与支架的黏附和力学性能。实验结果证明聚乳酸涂层具有良好的孔隙度分布和表面形貌,扩张前后与支架的附着良好,涂层无龟裂、脱落或翘起等现象。
④应用免疫荧光的方法对吸附在聚乳酸涂层中的单抗进行染色,以确定单抗在聚乳酸涂层中的吸附;同时,对单抗、聚乳酸及其混合物进行差示扫描量热法检测,以确定单抗与聚乳酸是否结合及其结合方式,从而考察被动吸附法制备单抗洗脱血管内支架的可行性。结果表明单抗以被动吸附的方式结合到聚乳酸涂层上,且与聚乳酸涂层载体之间无共聚化反应发生,聚乳酸涂层对单抗的生物学活性没有明显影响。用扫描电镜观察与枸橼酸盐抗凝全血接触后的单抗洗脱支架发现几乎没有血小板的聚集,表明单抗洗脱支架在体外能显著抑制血小板的聚集。采用125I放射性标记单抗的方法对单抗在聚乳酸涂层上的吸附以及单抗在一定流量下的释放进行了定量的检测。从吸附实验的结果来看,单抗在聚乳酸涂层上的吸附量与单抗溶液的浓度和吸附时间有关。实验检测了支架丝在三种不同的单抗浓度中吸附120h的实验数据,最大的单抗吸附量是在2.0mg/ml的单抗溶液中吸附72h时得到的。支架上单抗的释放时间可达到2周以上,不同的制备方法和流量对药物的释放有一定影响。
⑤将单抗洗脱支架和对照316L不锈钢裸支架置入兔髂动脉,在1、4、12周后,对置入支架段血管及其两端的血管段、主要脏器组织和血清,运用硬组织切片、扫描电镜、透射电镜、免疫组化以及组织染色、生化检测等技术方法对此单抗洗脱支架的有效性和安全性进行检测评价。结果表明单抗洗脱支架在3个月的观察期内安全、可靠,同时能有效防止支架内再狭窄和血栓形成,其机理可能与单抗抑制血小板的粘附和聚集以及加速支架表面内皮化和抑制血管平滑肌细胞的迁移和增殖有关。
The percutaneous transluminal angioplasty (PTCA) as the main interventional therapies of stenosis and occlusion of blood vessel has induced restenosis (30%-50%), which influence it’s late curative effect. The introduction of stents in the clinical practice was a major breakthrough in the field of percutaneous coronary intervention. Its success assistant with the anticoagulant and antiplatelet therapies is thought to be due to its ability to reduce the acute elastic recoil and long-term negative remodelling associated with PTCA. The stents may also induce a more chronic and pronounced vascular response than angioplasty alone which has been defined as in-stent restenosis (ISR). There are still relatively high in-stent restenosis rates (15%-25%) after stenting, caused by the excess neointimal hyperplasia because of the smooth muscle cell migration and proliferation. Although many anti-proliferation drugs have good effects in animal experiments the clinical uses of them have depressed us. An important reason is that they could not reach the sufficient concentration at local vascular vessel by systemic therapy. Drug-eluting stents (DES) are coated with a bioresorbable polymer capable of releasing single or multiple bioactive agents into the bloodstream and surrounding tissues. Local deliveries to the vasculature achieves high regional drug concentrations and decrease the ISR to below 10%, with prolonged retention at lower doses and reduces systemic toxicity.
The issues in the preparation process of DES are generally focused on three chief aspects, including coating material and its preparation methods, the selection of biological agents, the control release of drugs. In this article the biological agent is domestic-made platelet glycoproteinⅢa receptor monoclonal antibody (mAb) and the coating material is L-polylactic acid (PLLA). The spray method based on ultrasonic atomization is used to prepare PLLA coated stents, which is immersed into the mAb solution passively absorbed by coating. After evaluating the qualities of polymer coating and mAb eluting stents, we have examined the biosecurity and validity of prevention and cure ISR by implanted exceeding mAb eluting stents into rabbit iliac arteries. The major contents and results of the research are as follows.
①The spray equipment for experiment was designed and produced according to the character of spray process and the principle of ultrasonic atomization. The equipment were composed of four parties, including atomization module, gas-carrier module, solution-container module, and stent-carrier module. The atomization module could atomize the polymer solution through a drop-in ultrasonic atomizer located in the cylindrical water container. The gas-carrier module could control and adjust the outflow flux of N2 in the high-pressure air tank through gas flowmeter. The polymer-solution container and nozzle were made of glass because they were easy to atomize polymer solution and clear. The stent-carrier module helped to prepare uniform polymer coating by the means of making the stent move back-and-forth in front of nozzle at the time of maintaining circumrotation. This equipment could prepare the ideal polymer coating on the stent surface and easy to spary multi-coating.
②The technics process was introduced in this text. The optimal parameters, which were determined by counting the un-uniform district, were spray time 30s, PLLA concentration 1.0 mg/ml, rotate speed 50 rpm.
③The distribution and morphologies of PLLA coatings on the stent surface prepared under the optimal parameters were visualized through fluorescence staining and scanning electron microscopy (SEM), combined with computer image analysis method to estimate the uniformity, hole distribution and surface roughness of coating. In addition the morphologies of PLLA coating were examined both pre- and post-expansion in vitro. The results suggest the PLLA coating has favourable hole distribution and surface morphology, and no cracks, break-off, or turnup both pre- and post-expansion.
④The presence of the PLLA coating over the entire surface of the stent was evidenced by means of a fluorescence staining method. Whether if the PLLA combine with the mAb or not was confirmed by the DSC. After these two experiments we could make certain the feasibility of passive absorption of mAb in the PLLA coating on the stent surface. The convergence degree of fluorescence intensity distribution indicated the uniformity of PLLA coating pore distribution, which was closely related to the distribution of mAb in PLLA coating. The results indicated there are no chemistry reaction between the PLLA and mAb, and PLLA has no influence on the biology activity of mAb.
Stents eluting the mAb were tested for their adsorption characteristics by radioisotope technique with 125I labelled mAb. The elution rates were then measured in looped circuits at different velocities and durations. The amount of mAb adsorbed onto the PLLA coating was dependent on the concentration and duration of immersion in the solution. We have tested three different concentrations for 120h. Maximal mAb binding was therefore defined as the amount of agent bound to stent wires after 72 hours immersion in a 2 mg/ml solution. The method of preparation the mAb eluting stent and flow velocity significantly influenced the elution characteristics for a continuous perfusion of more than 2 weeks.
⑤We implant the mAb eluting stents and 316L stainless steel stents the control in rabbit iliac arteries to study the bio-security validity of prevention and cure ISR by sliding microtome, SEM, transmission electron microscope (TEM), immunohistochemi- stry, tissue stain and biochemistry methods. The blood vessel and serum samples were got from the rabbits after stents implantation for 1week, 4weeks and 12weeks. The results suggests the mAb eluting stents are biocompatible and can inhibit the thrombosis and neointimal hyperplasia by accelerating the endothelialization of the stent surface and inhibit the smooth muscle cell migration and proliferation.
引文
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