CN116327726B - 一种血小板膜包被的仿生纳米颗粒及其制备方法和应用 - Google Patents
一种血小板膜包被的仿生纳米颗粒及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种血小板膜包被的仿生纳米颗粒及其制备方法和应用,属于生物医药技术领域。一种血小板膜包被的仿生纳米颗粒,由聚吡咯和利伐沙班结合而成的单核纳米粒,该单核纳米粒以聚吡咯颗粒为核的中心,利伐沙班分子与聚吡咯颗粒之间以Π‑Π堆积和分子间疏水作用力相连接。本发明将抗凝药物利伐沙班与具有光热性能的聚吡咯共组装得到具有良好光热性能的载药纳米粒,并用血小板膜对纳米粒进行伪装,通过血小板膜功能性受体的黏附作用,可将载有利伐沙班的纳米粒富集到血栓部位从而达到药物精准靶向治疗的目的,同时可以显著降低治疗过程中的全身出血等风险。
Description
技术领域
本发明涉及一种血小板膜包被的仿生纳米颗粒及其制备方法和应用,属于生物医药技术领域。
背景技术
目前,心血管疾病成为世界范围内死亡率和发病率最高的疾病,其发病率远高于癌症。其中血栓性疾病严重危害身体健康,可以诱发缺缺血性脑卒中、心肌梗死和肺栓塞,引起人们广泛的关注。目前血栓治疗的方法主要包括药物治疗和手术干预。因高昂的手术费用及侵入性,使得药物治疗仍然是临床上血栓治疗的主要选择。传统药物治疗包括抗凝治疗、抗血小板治疗、溶栓治疗。但在药物治疗的时候面临着因药物脱靶导致全身出血等风险,因此,为了减少全身出血等副作用,我们提出药物的精准靶向达到快速溶栓的目的。
凝血因子Xa作为内源性和外源性凝血途径的交汇点,一分子的FXa可催化凝血酶原产生1000多个凝血酶分子,利伐沙班是直接FXa抑制剂,可以阻止凝血酶的产生,减少凝血酶介导的凝血和血小板的活化。同时作为上市新药,已经在临床预防和治疗血栓栓塞性疾病取得一定的进展。但在ROCKET AF试验中,最常见的不良反应为出血事件,发生率较高于华法林,因此在整个治疗期间时刻密切观察。
聚吡咯(PPy)是一种由五元杂环组成的聚合物,因其优异的光热性能和光声成像在生物医学领域广泛应用。目前,研究人员在药物新剂型的发现都使用单一聚吡咯纳米粒作为诊疗元素,很少有文献报道将聚吡咯与药物共组装成新型纳米粒用于疾病的治疗。
近年来提出的自上而下的纳米仿生策略在抗肿瘤、抗血栓、抗炎等方面取得良好的治疗效果。不同于自下而上的策略(对纳米粒表面进行化学靶向修饰)的低效、耗时、耗力、免疫原性引起的过敏反应等问题。我们将细胞膜涂覆在纳米粒表面可弥补传统药物递送的缺陷。最近仿生血小板纳米药物递送系统基于血小板自身特异性功能和可以储存药物的内腔的优势,在各种疾病治疗和诊断中表现出突出的优势,包括吞噬逃逸、免疫系统的激活,对受损血管及肿瘤组织的选择性粘附,使在药物主动靶向递送、是血小板膜在疾病治疗和诊断成像方面成为强大的治疗工具。同时血小板膜上的功能性受体只负责血小板的黏附,没有任何聚集作用。
发明内容
针对上述所述的问题,本发明提供一种具有抗血栓功能的仿生纳米粒及其制备方法及应用,利用利伐沙班和吡咯的Π-Π堆积、疏水相互作用共组装成具有光热作用的内核载药纳米粒,将从大鼠全血中提纯的血小板膜包裹在其外部,以实现血栓靶向的精准治疗。
一种血小板膜包被的仿生纳米颗粒,所述仿生纳米颗粒是由血小板膜外壳包被内核纳米粒构成,其中,所述内核纳米粒为由聚吡咯和利伐沙班结合而成的单核纳米粒,该单核纳米粒以聚吡咯颗粒为核的中心,利伐沙班分子与聚吡咯颗粒之间以Π-Π堆积和分子间疏水作用力相连接。
优选地,所述聚吡咯的聚合度为2~20;进一步优选,聚吡咯的聚合度为10。
优选地,所的内核纳米粒的粒径为10~1000nm,进一步优选为100~250nm。
优选地,所述内核纳米粒是由利伐沙班和吡咯在过硫酸铵氧化聚合作用下组装成的内核载药纳米粒。
优选地,所述内核纳米粒按下述方法制得:常温下,将吡咯单体和利伐沙班同时溶解在十二烷基硫酸钠水溶液中,随后缓慢滴加氧化剂过硫酸铵置于冰浴中进行化学氧化反应,获得SDS-PPy/Riv NPs混合溶液,将其在15000rpm,10min的离心条件下进行离心洗涤除去游离的吡咯、利伐沙班、过硫酸铵及SDS,最终得到粒径均一的SDS-PPy/Riv NPs内核纳米粒,其中,
进一步地,所述十二烷基硫酸钠水溶液的浓度为60~100mg/ml;所述吡咯单体与过硫酸铵的摩尔比为1:1~5:1;所述的利伐沙班与吡咯单体的质量比为0.028:1~0.083:1;吡咯单体与十二烷基硫酸钠水溶液的比例为0.73~1.21g:1mL。
进一步地,所述缓慢滴加过硫酸铵的速度控制为每滴间隔20秒,边滴加边搅拌;所述化学氧化反应时间为2h,冰浴控制温度为4℃。
优选地,所述血小板膜为提取的天然血小板膜,在提取过程进行低温控制和添加蛋白酶抑制剂PMSF。
进一步地,所述血小板膜按下述方法获得:毛细管取大鼠眼眶的全血置于含有EDTA的抗凝管中,采用梯度离心法将提取到的血小板重悬于含蛋白酶抑制剂的PBS溶液中;然后经过-80℃~4℃的反复冻融循环制的血小板膜。
一种血小板膜包被的仿生纳米颗粒,所述仿生纳米颗粒是由血小板膜外壳包被内核纳米粒构成,其中,所述内核纳米粒是由利伐沙班和吡咯共组装成的内核载药纳米粒。
本发明所述血小板膜包被的仿生纳米颗粒中,所述内核载药纳米粒具有光热性能。
本发明所述血小板膜包被的仿生纳米颗粒中,所述内核纳米粒按下述方法制得:常温下,将吡咯单体和利伐沙班同时溶解在十二烷基硫酸钠水溶液中,随后缓慢滴加氧化剂过硫酸铵置于冰浴中进行化学氧化反应,获得SDS-PPy/Riv NPs内核纳米粒。
进一步地,所述十二烷基硫酸钠水溶液的浓度为60~100mg/ml;所述吡咯单体与过硫酸铵的摩尔比为1:1~5:1;所述的利伐沙班与吡咯单体的质量比为0.028:1~0.083:1;吡咯单体与十二烷基硫酸钠水溶液的比例为0.73~1.21g:1mL。
进一步地,所述缓慢滴加过硫酸铵的速度控制为每滴间隔20秒,边滴加边搅拌;所述化学氧化反应时间为2h,冰浴控制温度为4℃。
进一步地,所述将得到的SDS-PPy/Riv NPs混合溶液经过离心洗涤除去未参与反应的吡咯、利伐沙班、过硫酸铵及SDS,得到粒径均一的SDS-PPy/Riv NPs内核纳米粒。
本发明所述血小板膜包被的仿生纳米颗粒中,所述血小板膜为提取的天然血小板膜。
进一步地,所述血小板膜按下述方法获得:毛细管取大鼠眼眶的全血置于含有EDTA的抗凝管中,采用梯度离心法将提取到的血小板重悬于含蛋白酶抑制剂的PBS溶液中;然后经过-80℃~4℃的反复冻融循环制的血小板膜。
本发明所述血小板膜包被的仿生纳米颗粒中,所述仿生纳米颗粒按下述方法获得:将内核纳米粒与的血小板膜混合超声一段时间后,通过含有200nm的碳酸脂膜的挤出机反复挤压得到均一的仿生纳米粒。
进一步地,所述血小板膜包载内核纳米粒时的超声功率为100w,超声频率为42KHz,超声时间为20min。
本发明进一步提供血小板膜包被的仿生纳米颗粒在制备治疗心血管疾病中的应用。
本发明的有益效果为:受血小板膜的天然特性,将血小板膜包覆在纳米粒表面可以有效的将药物富集在血栓部位。血栓形成的原理是指血小板黏附在受损血管的表面,然后激活内部信号通路,释放更多促进血栓形成的细胞因子,加速形成纤维蛋白网状结构,进而促成血栓的形成。研究表明,血小板膜上的功能性受体只负责血小板的黏附,不参与血小板的激活与聚集作用,受此启发,血小板包覆的纳米粒将延长被负载药物的半衰期,有效将药物富集靶向到血栓部位。
本发明将抗凝药物利伐沙班与具有光热性能的聚吡咯共组装得到具有良好光热性能的载药纳米粒,并用血小板膜对纳米粒进行伪装,通过血小板膜功能性受体的黏附作用,可将载有利伐沙班的纳米粒富集到血栓部位从而达到药物精准靶向治疗的目的,同时可以显著降低治疗过程中的全身出血等风险。
本发明不同于纳米化学涂层的方法,制得的仿生纳米粒可以有效减缓药物的清除,显著延长药物的半衰期,提高生物利用率。同时,在血液循环过程中,仿生纳米粒缓慢释放利伐沙班,抑制凝血FXa,中断内源性外源性凝血途径,达到抑制血栓形成的目的。
本发明借助利伐沙班和吡咯的Π-Π堆积、疏水间相互作用,实现具有良好光热性能的载药纳米粒,在光热的辅助作用下,达到血栓的快速溶解。
附图说明
图1为本发明具有抗血栓功能的仿生纳米粒的制备流程图。
图2(a)和(b)分别为本发明中制备不同纳米粒的粒径分布图及PLT-PPy/Riv NPs的透射电镜照片。
图3(a)和(b)分别为本发明中利伐沙班和吡咯分子模拟和分子对接结果图。
图4为本发明中PLT-PPy/Riv NPs在含10%胎牛血清的PBS中利伐沙班泄露的情况。
图5为本发明中PLT-PPy/Riv NPs的在近红外激光照射下利伐沙班的体外释放图。
图6为本发明中不同纳米制剂在808nm激光照射下的体外温度升高曲线图。
图7(a)和(b)分别为本发明中PLT-PPy/Riv NPs对活化血小板黏附的流式细胞图及共聚焦显微镜图。
图8为本发明中PLT-PPy/Riv NPs对人工血块体外靶向的荧光强度数据图。
图9为本发明中不同制剂的体外溶栓结果图。
图10为本发明中股静脉血栓形成构建图。
图11(a)和(b)分别为本发明中PLT-PPy/Riv NPs的体内靶向荧光图及给药后激光照射时间的筛选。
图12(a)和(b)分别为本发明中不同制剂在808nm激光照射下的大鼠体内热成像图及体内温度升高图。
图13(a)和(b)分别为本发明中血栓治疗后H&E染色的组织切片图及血栓治疗率。
图14(a)和(b)分别为本发明中小鼠治疗后的小鼠断尾试验示意图和出血时间。
图15为本发明中对正常组织毒性的H&E染色图。
具体实施方式
下述非限制性实施例可以使本领域的普通技术人员更全面地理解本发明,但不以任何方式限制本发明。
下述实施例中所述试验方法,如无特殊说明,均为常规方法;所述试剂和材料,如无特殊说明,均可从商业途径获得。
具体实施方案之一:
1)提取血小板膜,备用:大鼠眼眶毛细管取一定量的全血置于含有EDTA的抗凝管中,采用梯度离心法将提取到的血小板重悬于含蛋白酶抑制剂的PBS溶液中;然后经过-80℃~4℃的反复冻融循环制的血小板膜。
2)内核纳米粒的制备:取SDS置于去离子水中搅拌至全部溶解后,同时加入一定量的吡咯单体和利伐沙班常温搅拌至全部溶解,随后缓慢滴加氧化剂过硫酸铵置于冰浴反应,随后获得具有光热性能的SDS-PPy/Riv NPs内核纳米粒。
3)仿生纳米颗粒的制备:将提取到的内核纳米粒与提取到的血小板膜混合超声一段时间后,通过含有200nm的碳酸脂膜的挤出机反复挤压得到均一的仿生纳米粒。
作为优选的技术方案的,步骤2)中,所述的SDS水溶液的浓度为60mg/ml;
作为优选的技术方案的,步骤2)中,所述的吡咯单体与过硫酸铵的摩尔比为5:1;
作为优选的技术方案的,步骤2)中,所述的利伐沙班和吡咯的质量比为0.055:1;
作为优选的技术方案的,步骤2)中,所述吡咯单体与十二烷基硫酸钠水溶液的比例为1.21g:1mL;
作为优选的技术方案的,步骤1)中的梯度离心法是第一次离心是以200g离心力离心10min,第二次离心是以800g离心力离心10min;
作为优选的技术方案的,步骤1)中的反复冻融循环的次数为3次;
作为优选的技术方案的,步骤2)中的缓慢滴加过硫酸铵的速度控制为每滴间隔20秒,边滴加边搅拌;
作为优选的技术方案的,步骤2)中化学氧化反应时间为2h,冰浴控制温度为4℃;
作为优选的技术方案的,步骤3)中血小板膜包载内核纳米粒时的超声功率为100w,超声频率为42KHz,超声时间为20min;
其次,本发明提供一种具有抗血栓功能的仿生纳米粒,该仿生纳米粒可通过上述的制备方法制备而成。
再次,本发明还提供一种前述的具有抗血栓功能的仿生纳米粒的应用,具体为在制备和研发抗血栓功能药物的应用。
实施例1
本实施例是制备一种功能性血小板膜包被的纳米粒,该血小板包被纳米粒的制备技术路线如图1所示,具体操作如下:
提取血小板膜:采用梯度离心法从Sprague-Dawley大鼠中取全血分离提纯血小板。操作步骤如下,从毛细管取大鼠眼眶的全血置于EDTA抗凝管中,200g离心10min取上层富血小板血浆层,等体积加入含有1μM PGE1的ACD缓冲溶液,800g离心10min将得到的白色沉淀即血小板重悬于含蛋白酶抑制剂的PBS缓冲溶液中。经过3次的-80℃-4℃的反复冻融循环,使血小板破裂经离心洗涤去除其细胞器得到血小板膜,备用。
内核纳米粒SDS-PPy/Riv NPs:取0.03g的SDS于500μL去离子水中常温搅拌至溶解,同时加入7.5μL的Py单体和0.4mg的利伐沙班常温搅拌4h使其全部溶解后置于准备好的4℃水浴锅中搅拌,随后缓慢滴加0.00492g的过硫酸铵,维持4℃冰浴,氧化聚合2h,溶液从透明色过渡到黄棕褐色最后变为黑色溶液。期间借助利伐沙班与吡咯分子间Π-Π堆积相互作用将利伐沙班成功封装,实现载药聚吡咯纳米粒在静脉血栓治疗中的应用。
仿生纳米粒PLT-PPy/Riv NPs的制备:将步骤SDS-PPy/Riv NPs与4×108的血小板混匀,100w,42Khz连续超声20min,得到具有抗血栓功能的仿生纳米粒。
对比例1
内核纳米粒SDS-PPy NPs:制备方法同实施例1的步骤2),不同在于:实施例1在步骤2)中“同时加入7.5μL的Py单体和0.4mg的利伐沙班”,在该对比例改为加入7.5μL的Py单体。
纳米粒PLT-PPy NPs的制备:SDS-PPy NPs与4×108的血小板混匀,100w,42Khz连续超声20min,得到具有抗血栓功能的纳米粒。
对比例2
SDS-PPy/Riv NPs:取0.03g的SDS于500μL去离子水中常温搅拌至溶解,同时加入7.5μL的Py单体和0.4mg的利伐沙班常温搅拌4h使其全部溶解后置于准备好的4℃水浴锅中搅拌,随后缓慢滴加0.00492g的过硫酸铵,维持4℃冰浴,氧化聚合2h,溶液从透明色过渡到黄棕褐色最后变为黑色溶液。期间借助利伐沙班与吡咯分子间Π-Π堆积相互作用将利伐沙班成功封装,实现载药聚吡咯纳米粒在静脉血栓治疗中的应用。
对比例3
Riv Sol:取4mg的利伐沙班溶于含有60mg/mL的SDS水溶液中常温搅拌,使得利伐沙班全部溶解。
性能考察:
一、对实施例1中制备的具有抗血栓功能的仿生纳米粒和对比例1、2制备的纳米粒的形貌表征及体外释药性能、光热性能的考察。
1)表征:将实施例1和对比例1、2制得的SDS-PPy/Riv NPs、PLT-PPy NPs与PLT-PPy/Riv NPs分别制成的溶液,使用Zetasizer仪器分别检测粒径分布、电位。结果如图2所示,从图中可以看到PLT-PPy/Riv NPs的粒径比内核纳米粒SDS-PPy/Riv NPs高15-20nm,且电位升高到-19V,均表示血小板膜的成功包覆,PLT-PPy/Riv NPs的透射电镜图也验证该结果。
2)分子间作用力的考察:我们将在chemdraw中绘制的吡咯单体和利伐沙班的化学结构导入Materials studio中,吡咯单体聚合的程度(从n=1~20)分别与利伐沙班进行结合能的计算,结果如表1,发现吡咯自聚合到10时与利伐沙班的结合能最大。如图3中的分子对接结果显示,聚吡咯与利伐沙班存在的作用力有Π-Π分子堆积和分子间疏水作用力。图3中分子模拟展示的是PLT-PPy/Riv NPs构建的三维晶胞图。
表1.不同聚合程度的聚吡咯与利伐沙班的结合能(kcal/mol)
3)生理环境中稳定性的考察:我们将实施例1和对比例2的血小板膜包被的纳米粒置于含10%的胎牛血清的PBS溶液中12h,分别于0h、2h、4h、6h、8h、10h、12h取出置于30Kda的超滤管中4000rpm,10min离心,采用高效液相色谱法计算分析得到的上清液的利伐沙班含量进而得到PLT-PPy/Riv NPs的包封率。结果如图4所示,在12h内,利伐沙班的包封率稳定在90%左右,不存在药物泄露的情况。
4)体外释药性能的考察:我们将实施例1和对比例2中的血小板膜包被的纳米粒置于透析袋中,以0.2%的SDS的PBS(pH=7.4)为释放介质。光照组需要事先给与纳米粒一定的光照(808nm,2.22W/cm2,10min)。在预先设定的时间点取2ml的样品进高效液相色谱测定利伐沙班的含量并计算其累计释放率。结果如图5所示,激光治疗组中利伐沙班在48h内的累计释放量可达60%,且一定的光照可促进利伐沙班的释放。
5)体外光热性能的考察:PLT-PPy/Riv NPs的光热效应我们采用红外热成像仪进行测量。我们将PBS、SDS-PPy/Riv NPs、PLT-PPy NPs、PLT-PPy/Riv NPs置于功率密度为2.22W/cm2的808nm激光器下照射10min,记录照射过程中的温度变化及停止照射后的温度变化。结果如图6所示,SDS-PPy/Riv NPs、PLT-PPy NPs、PLT-PPy/Riv NPs随时间的照射温度在升高,PLT-PPy/Riv NPs在10min内最高温度可达56℃,说明血小板膜包裹纳米粒这一过程并不影响内核纳米粒的光热性能,同时Time-Inθ线性拟合图计算得到PLT-PPy/RivNPs的光热转换率达30.86%。
二、对实施例1中制备的具有抗血栓功能的仿生纳米粒和对比例1制备的纳米粒体内外靶向性的考察
1)体外靶向性考察:将DiD标记的PLT-PPy/Riv NPs分别与活化血小板和非活化血小板共孵育30min,随后用PBS连续洗涤数次除去游离的NPs,PBS重悬后进流式观察。结果分析图如图7中流式细胞分析图所示,从图中可以看到非活化组的荧光强度与对照组相近,而活化组荧光强度显著高于非活化组,验证所制得的PLT-PPy/Riv NPs对活化血小板的粘附性。图7中的共聚焦显微镜图进一步验证此猜想。
为了进一步评估所制得的仿生纳米制剂的靶向性,我们采用毛细管眼眶取血法收集大鼠新鲜全血于微量离心管中,管内装有终浓度为1U/mL凝血酶和2.5mM的CaCl2溶液,37℃放置1h得人工血块。将得到的人工血块与DiD标记的PLT-PPy/Riv NPs共孵育30min后用PBS洗涤除去多余的纳米粒。最后用采用IVIS检测其荧光强度,结果如图8所示,结果发现仿生纳米粒相比于内核纳米粒的荧光强度有显著性差异。
2)体内靶向性能考察:首先,在Sprague-Dawley大鼠左腿内测建立股静脉血栓模型。将大鼠麻醉,用胶带固定后,使用脱毛膏去除皮毛,在大腿内部做切口使血管暴露在外,然后用浸有10%的氯化铁溶液饱和滤纸(1cm×1cm)覆盖在血管表面5min,结果如图10所示,血管变成深红色即血栓造模成功,随后使用生理盐水除去残留的FeCl3。
给药后激光照射时间的筛选:随后我们分别尾静脉注射PBS、DiD标记的SDS-PPy/Riv NPs和PLT-PPy/Riv NPs后,采用小动物活体成像仪记录荧光信号,结果如图11所示,与PBS和SDS-PPy/Riv NPs相比,PLT-PPy/Riv NPs组在血管栓塞部位荧光信号最强,证明了PLT-PPy/Riv NPs的体内靶向性。同时小动物活体成像仪记录了PLT-PPy/Riv NPs在不同时间点的荧光强度并绘制荧光强度曲线,结果如图11所示,在75min时,PLT-PPy/Riv NPs在大鼠血栓处荧光强度最强,意味着PLT-PPy/Riv NPs在血栓处富集程度最多,我们将75min作为纳米给药后808nm激光照射的最佳时间。
体内光热性能的考察:我们尾静脉注射不同制剂(PBS、SDS-PPy/Riv NPs、PLT-PPyNPs和PLT-PPy/Riv NPs)75min后使用808nm激光器在大鼠的血栓部位激光照射10min,期间记录在血栓部位的温度升高图,结果如图12所示,PLT-PPy/Riv NPs和PLT-PPyNPs经连续照射10min后血栓部位温度高达43℃,其体内光热转换率高于SDS-PPy/Riv NPs(约37℃)。研究发现纤维蛋白凝块是热敏感的,在加热后血块得到崩解(温度升高约42~50℃),同时较低温度在溶栓的同时减少对正常组织的损伤。因此,相比于PLT-PPy NPs和SDS-PPy/RivNPs,PLT-PPy/Riv NP自身具备的体内靶向性和载药性能成为最佳溶栓制剂,表现出最佳溶栓效果。
效果例1
本效果例是对实施例1中制备的具有抗血栓功能的仿生纳米粒(PLT-PPy/RivNPs)进行体外溶栓评估。具体如下:
我们采用Drabkin法评估不同纳米制剂的体外溶栓效果,首先,进行采血和凝血。采用毛细管眼眶取血将收集100μL的Sprague-Dawley大鼠的新鲜血液吸取到微量离心管中,并且管内装有终浓度为1U/mL凝血酶和2.5mM的CaCl2溶液。将试管37℃放置1h得到柔软的人工血凝块。
然后溶栓试验,我们将制备好的血凝块分为四组,分别进行溶栓试验。具体实验组分别为:Riv Sol处理组、SDS-PPy/Riv NPs处理组、PLT-PPy NPs处理组、PLT-PPy/Riv NPs处理组。结果如图所示,仿生纳米粒PLT-PPy/Riv NPs的加入具有良好的血栓治疗效果,并且结合激光治疗效果更好。
不同处理组经治疗后,经离心后得到上清液在540nm处吸光度如图10所示,可以看出该仿生纳米粒的治疗效果是单一药物治疗的1.8倍,是单一光热治疗的1.72倍,是未具有靶向性能的1.50倍。
效果例2
本效果例是为了探究实施例1中制备的具有仿生纳米粒的体内静脉溶栓效果。
首先,在Sprague-Dawley大鼠左腿内测建立股静脉血栓模型。将大鼠麻醉,用胶带固定后,使用脱毛膏去除皮毛,在大腿内部做切口使血管暴露在外,然后用浸有10%的氯化铁溶液饱和滤纸(1cm×1cm)覆盖在血管表面5min,血管变成深红色即血栓造模成功。
然后进行静脉血栓的治疗,将造模好的大鼠股静脉血栓模型分别在尾静脉注射不同制剂后进行光热治疗。具体试验及对照组如下:PBS处理组、Riv Sol、SDS-PPy/Riv NPs+激光照射处理组、PLT-PPy NPs+激光照射处理组及PLT-PPy/Riv NPs+激光照射处理组。静脉注射剂量为利伐沙班等剂量的0.9mg/Kg,在注射后75min后进行激光照射,治疗结束后进行伤口缝合。24h后对各组小鼠进行安乐死,将大鼠的血管、心、肝、脾、肺、肾对其解剖,放入4%的多聚甲醛溶液中,最后进行H&E染色,以评估后续的治疗效果。
大鼠股静脉血栓的H&E染色结果及溶栓比例如图13所示,从计算结果能够看出,游离的Riv有轻微的抗血栓能力和激光照射的具有靶向作用的PLT-PPy NPs具有一定的溶栓效果,但因Riv在体内的快速清除及脱靶表现出微弱的溶栓效果,同时表明激光照射在一定程度上增强抗血栓作用。同样激光照射没有靶向作用的SDS-PPy/Riv NPs增强了抗血栓作用,暗示光热治疗和药物治疗的协同作用。正如预期的那样,PLT-PPy/Riv NPs在表现出优异的溶栓效果,这归因于Riv在血栓部位的特异性累积及局部光热联合作用的结果。
本实例还对PLT-PPy/Riv NPs进行出血性风险的评估。首先,分别将上述实验组尾静脉注射到昆明小鼠中,在注射后4h,用手术剪刀剪小鼠尾巴的远端(1cm),分别记录尾巴的出血时间和凝血时间以衡量PLT-PPy/Riv NPs的出血风险,结果如图14所示,表明相比于利伐沙班处理组,PLT-PPy/Riv NPs+激光照射处理组有很低的出血风险,表明PLT-PPy/RivNPs+激光照射处理组在有良好的治疗效果不会损坏血管,诱发全身出血等副作用,具有良好的使用安全性。
本实验还对PLT-PPy/Riv NPs进行体内安全性评估。我们经治疗后取得的心、肝、脾、肺、肾进行H&E染色,结果如图15所示,与PBS对照组相比,在PLT-PPy/Riv NPs处理的大鼠分离的器官中未观察到明显的病理变化,包括坏死、纤维化和水变性。以上结果证明,PLT-PPy/Riv NPs具有很高的治疗安全性。
本发明受血小板膜天然优势,将血小板膜包覆在纳米粒表面,血小板膜上的功能性受体代替外源性化学修饰分子,可降低蛋白酶的降解作用,减缓药物在体内的清除,显著延长负载药物的半衰期,使得利伐沙班大量富集在血栓部位,实现血栓的精准靶向治疗,具有良好的医疗应用前景和实用价值。
Claims (5)
1.一种血小板膜包被的仿生纳米颗粒,其特征在于:所述仿生纳米颗粒是由血小板膜外壳包被内核纳米粒构成,其中,所述内核纳米粒为由聚吡咯和利伐沙班结合而成的单核纳米粒,该单核纳米粒以聚吡咯颗粒为核的中心,利伐沙班分子与聚吡咯颗粒之间以Π-Π堆积和分子间疏水作用力相连接,
所述聚吡咯的聚合度为2~20;所的内核纳米粒的粒径为100~250nm;
所述内核纳米粒是由利伐沙班和吡咯在过硫酸铵氧化聚合作用下组装成的内核载药纳米粒,具体按下述方法制得:常温下,将吡咯单体和利伐沙班同时溶解在十二烷基硫酸钠水溶液中,随后缓慢滴加氧化剂过硫酸铵置于冰浴中进行化学氧化反应,获得SDS-PPy/RivNPs混合溶液,将其在15000rpm,10min的离心条件下进行离心洗涤除去游离的吡咯、利伐沙班、过硫酸铵及SDS,最终得到粒径均一的SDS-PPy/Riv NPs内核纳米粒,其中,
所述十二烷基硫酸钠水溶液的浓度为60~100mg/ml;所述吡咯单体与过硫酸铵的摩尔比为1:1~5:1;所述的利伐沙班与吡咯单体的质量比为0.028:1~0.083:1;吡咯单体与十二烷基硫酸钠水溶液的比例为0.73~1.21g:1mL。
所述缓慢滴加过硫酸铵的速度控制为每滴间隔20秒,边滴加边搅拌;所述化学氧化反应时间为2h,冰浴控制温度为4℃。
2.根据权利要求1所述的血小板膜包被的仿生纳米颗粒,其特征在于:所述血小板膜为提取的天然血小板膜,在提取过程进行低温控制和添加蛋白酶抑制剂PMSF。
3.根据权利要求1所述的血小板膜包被的仿生纳米颗粒,其特征在于:所述仿生纳米颗粒按下述方法获得,将内核纳米粒与的血小板膜混合超声一段时间后,通过含有200nm的碳酸脂膜的挤出机反复挤压得到均一的仿生纳米粒。
4.根据权利要求1所述的血小板膜包被的仿生纳米颗粒,其特征在于:所述血小板膜包载内核纳米粒时的超声功率为100w,超声频率为42KHz,超声时间为20min。
5.权利要求1~4任一项所述的血小板膜包被的仿生纳米颗粒的制备在治疗静脉血栓中的应用。
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