CN108704143A - 一种负载肾脏纤维化治疗药物的聚合物纳米粒子及其制备方法 - Google Patents

一种负载肾脏纤维化治疗药物的聚合物纳米粒子及其制备方法 Download PDF

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CN108704143A
CN108704143A CN201810635548.1A CN201810635548A CN108704143A CN 108704143 A CN108704143 A CN 108704143A CN 201810635548 A CN201810635548 A CN 201810635548A CN 108704143 A CN108704143 A CN 108704143A
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renal fibrosis
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耿鑫然
胡建强
刘建宇
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South China University of Technology SCUT
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Abstract

本发明属于医药材料技术领域，公开了一种负载肾脏纤维化治疗药物的聚合物纳米粒子及其制备方法。将肾脏纤维化治疗药物、乳化剂、水溶性聚合物加入到含有醋酸的水相中搅拌混合均匀，得到前驱体溶液；将油溶性聚合物溶于有机溶剂中，超声混合均匀，搅拌条件下加入到前驱体溶液中，去除有机溶剂，离心，固相产物经洗涤，得到负载肾脏纤维化治疗药物的聚合物纳米粒子。本发明利用药物、油溶性聚合物和阳离子型的聚合物之间的静电相互作用，以及油溶性聚合物在水相中的自组装得到。所得的聚合物纳米粒子能够保护药物避免被酶降解，实现对肾脏纤维化的高效治疗，降低肾脏纤维化治疗药物的生物毒性。

Description

一种负载肾脏纤维化治疗药物的聚合物纳米粒子及其制备 方法
技术领域
本发明属于医药材料技术领域，具体涉及一种负载肾脏纤维化治疗药物的聚合物纳米粒子及其制备方法。
背景技术
近年来，慢性肾脏病已经成为全球公共健康的沉重负担之一。糖尿病、慢性肾炎和高血压等多种常见疾病都可导致慢性肾脏病。而慢性肾脏病在发展过程中会不断产生细胞外基质的累积，引起肾脏纤维化的发生，进而导致肾脏器官功能障碍甚至引起死亡。因此，肾脏纤维化的治疗在慢性肾脏病治疗中具有十分关键的地位。然而，目前治疗肾脏纤维化的药物普遍存在着细胞摄取效率低、体内稳定性差、肾脏浓度不足和副作用大的问题。因此，提高细胞对药物摄取效率，增强药物在体内的稳定性和实现药物的特异性的肾脏靶向是实现有效治疗肾脏纤维化并降低副作用的关键。但是，要实现药物的高效运载和特异性的肾脏靶向，进而有效的治疗肾脏纤维化仍然面临着巨大的挑战。
发明内容
针对以上现有技术存在的缺点和不足之处，本发明的首要目的在于提供一种负载肾脏纤维化治疗药物的聚合物纳米粒子的制备方法。
本发明的另一目的在于提供一种通过上述方法制备得到的负载肾脏纤维化治疗药物的聚合物纳米粒子。
本发明目的通过以下技术方案实现：
一种负载肾脏纤维化治疗药物的聚合物纳米粒子的制备方法，包括如下制备步骤：
(1)将肾脏纤维化治疗药物、乳化剂、水溶性聚合物加入到含有醋酸的水相中搅拌混合均匀，得到前驱体溶液；
(2)将油溶性聚合物溶于有机溶剂中，超声混合均匀，搅拌条件下加入到步骤(1)所得前驱体溶液中，去除有机溶剂，离心，固相产物经洗涤，得到负载肾脏纤维化治疗药物的聚合物纳米粒子。
优选地，步骤(1)所述肾脏纤维化治疗药物为番茄红素、miR-29抑制剂、miR-21抑制剂、miR-192抑制剂、氯化钴、大黄素中的至少一种。
优选地，步骤(1)中所述乳化剂为聚乙烯吡咯烷酮、泊洛沙姆188、聚乙烯醇、十六烷基三甲基氯化铵、普朗尼克F127、聚乙二醇、脂肪酸聚氧乙烯酯、嵌段聚氧乙烯-聚氧丙烯醚中的至少一种。
优选地，步骤(1)中所述水溶性聚合物为阳离子型低分子量聚合物，包括分子量Mn为3000～10000Da的低分子量壳聚糖、分子量Mn为600～10000Da的低分子量聚醚酰亚胺、多聚赖氨酸、十六烷基三甲基氯化铵和聚环糊精中的至少一种。
优选地，步骤(1)中所述前驱体溶液中，醋酸的体积浓度为0.5％～1％；乳化剂的质量浓度为0.3％～2％；水溶性聚合物的浓度为0.05～1mg/mL。
优选地，步骤(2)中所述油溶性聚合物为聚乳酸(PLA)、聚乳酸-羟基乙酸共聚物(PLGA)、聚N-异丙基丙烯酰胺、聚酰胺-胺(PAMAM)中的至少一种。
优选地，步骤(2)中所述有机溶剂为丙酮、乙腈、二氯甲烷、四氢呋喃、二甲基甲酰胺、二甲基亚枫中的一种或两种以上的混合。
优选地，步骤(2)中所述的有机溶剂与步骤(1)中水相的体积比为(3～4):5。
优选地，步骤(1)中肾脏纤维化治疗药物与步骤(2)中油溶性聚合物的质量比为(0.3～1):10。
一种负载肾脏纤维化治疗药物的聚合物纳米粒子，通过上述方法制备得到。
本发明的制备方法及所得到的产物具有如下优点及有益效果：
(1)本发明利用纳米沉淀法制得的负载肾脏纤维化治疗药物的聚合物纳米粒子是利用药物、油溶性聚合物和阳离子型的聚合物之间的静电相互作用，以及油溶性聚合物在水相中的自组装得到。所得的聚合物纳米粒子尺寸在30nm左右，且能够保护药物避免被酶降解。
(2)本发明制备的纳米粒子具有暴露在外面的阳离子型靶向聚合物，能够特异性靶向到肾脏部位，并且高效的将药物转染进入细胞，实现对肾脏纤维化的高效治疗，降低肾脏纤维化治疗药物的生物毒性。
附图说明
图1为实施例1中所得负载肾脏纤维化治疗药物的聚合物纳粒子的扫描电镜图。
图2为实施例1中所得负载肾脏纤维化治疗药物的聚合物纳米粒子与核糖核苷酸酶共培养后的琼脂糖凝胶电泳图。
图3为实施例1中所得的负载肾脏纤维化治疗药物的聚合物纳米粒子与细胞共培养后的细胞存活率图。
图4为实施例1中所得的负载肾脏纤维化治疗药物的聚合物纳米粒子将药物转染进入细胞的效率图。
图5为实施例1中所得的负载肾脏纤维化治疗药物的聚合物纳米粒子静脉注射进入小鼠24h后的主要器官荧光成像图。
图6为实施例1中所得的负载肾脏纤维化治疗药物的聚合物纳米粒子静脉注射进入小鼠后的主要器官的H&E染色图。
图7为实施例1中所得的小鼠病理组和负载肾脏纤维化治疗药物的聚合物纳米粒子干预组的肾脏H&E染色图。
图8为实施例1中所得的小鼠病理组和负载肾脏纤维化治疗药物的聚合物纳米粒子干预组的肾脏Masson染色图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
实施例1
(1)在烧杯中加入5ml去离子水、25μL冰醋酸、15mg泊洛沙姆188、0.3mg低分子量壳聚糖(Mn 4261Da)、300μg miR-21抑制剂，并持续搅拌，混合均匀，得到前驱体溶液。
(2)将5mg PLGA溶于含有3mL丙酮的塑料离心管之中，超声30s后，在搅拌速度为1500rpm/min的条件下，将其快速倾倒进入步骤(1)中所得的前驱体溶液中。连续搅拌6h后，丙酮挥发完全，固体产物离心洗涤三次，即得到负载肾脏纤维化治疗药物miR-21抑制剂的聚合物纳米粒子。
实施例2
(1)在烧杯中加入5ml去离子水、25μL冰醋酸、20mg普朗尼克F127、0.3mg低分子量聚醚酰亚胺(Mn 2500Da)、300μg miR-29抑制剂，并持续搅拌，混合均匀，得到前驱体溶液。
(2)将5mg PLGA溶于含有3mL丙酮的塑料离心管之中，超声30s后，在搅拌速度为1500rpm/min的条件下，将其快速倾倒进入步骤(1)中所得的前驱体溶液中。连续搅拌6h后，丙酮挥发完全，固体产物离心洗涤三次，即得到负载肾脏纤维化治疗药物miR-29抑制剂的聚合物纳米粒子。
以上实施例1所得的负载肾脏纤维化治疗药物miR-21抑制剂的聚合物纳粒子的扫描电镜图如图1所示，所得的纳米粒子直径在30nm左右，单分散性好，粒子尺寸分布均一。
以上实施例1所得的负载肾脏纤维化治疗药物miR-21抑制剂的聚合物纳粒子的性能及效果测试：
(1)对纤维化治疗药物的保护能力效果测试：
分别向负载0.4μg肾脏纤维化治疗药物miR-21抑制剂的聚合物纳粒子和0.4μgmiR-21抑制剂的去离子水分散液中加入1μL的核糖核苷酸酶，在37℃下分别共培养4h、8h、12h、24h和48h，然后将所有的样品和5μL浓度为0.25M的EDTA培养10min以使核糖核苷酸酶失活，然后加入到浸泡在TAE缓冲溶液中的2％(w/v)的琼脂糖凝胶中，打开电源，控制电压为120V，10min后关闭电源，取出琼脂糖凝胶，在紫外灯下照射，拍照。
本实施例所得的聚合物纳米粒子对miR-21抑制剂保护能力结果(凝胶电泳图)如图2所示，相比于游离的miR-21抑制剂，负载在纳米粒子中的miR-21抑制剂在与核糖核苷酸酸酶培养24h仍然具有很高的活性，说明聚合物纳米粒子可以有效的保护miR-21抑制剂避免被降解，这为实现miR-21抑制剂在体内的治疗效果提供了一个不可或缺的保障。
(2)生物相容性测试：
将正常大鼠肾近端肾小管上皮细胞系(NRK-52E)和大鼠肾间质成纤维细胞株(NRK-49F)注入含有3mL完全培养基的塑料离心管中，在500rpm/min的转速下离心5min后弃去上清液，然后加入新鲜的培养基，接种至25mL的培养瓶当中，用含有10％的胎牛血清的DMEM培养基在37℃和5％CO2的条件下培养，每隔24h更换培养基一次，直至80～90％的细胞融合，弃去培养液。用10％的PBS缓冲溶液洗涤2次，加入0.7ml的胰蛋白酶-EDTA溶液，在37℃下孵育1min，再加入3mL的10％胎牛血清培养液，500rpm/min离心5min，分散至10％的胎牛血清的DMEM培养基，接种至96孔细胞培养板，培养24h后，再更换无血清培养基培养12h，分别加入含有0、50、100、150、200、300nM的本实施例所得负载miR-21抑制剂的聚合物纳米粒子，在37℃下分别培养24h、48h和72h。用PBS缓冲液洗涤2次，每孔加入10μL CCK-8，37℃下培养2h，用酶标仪测定450nm下的吸光度，计算细胞的存活率。
本实施例所得负载miR-21抑制剂的聚合物纳米粒子与细胞共培养后的细胞存活率如图3所示，在miR-21抑制剂浓度达到300nM，培养72h后，仍然具有和对照组相似的存活率，说明本发明所得负载miR-21抑制剂的聚合物纳米粒子具有很好的生物相容性。
(3)细胞转染效率测试：
1)、将NRK-52E细胞接种至细胞培养皿上，培养24h使细胞融合80～90％。
2)、将生理盐水、含有25pM的负载miR-21抑制剂的聚合物纳米粒子(miRi-PCNPs)和脂质体2000分别加入到DMEM培养基中，使培养基中miR-21抑制剂的最终浓度为25nM。
3)、将步骤2)中所得的液体加入至1)中所得的液体中，避光培养24h，然后用PBS缓冲溶液洗涤3次，用胰酶消化，离心处理，最后用200μL PBS缓冲溶液转入流式管，使用流式细胞仪检测，并用FlowJo软件进行分析。
以和生理盐水共培养的细胞为对照组，本实施例所得的聚合物纳米粒子将miR-21抑制剂转染进细胞的效率统计结果如图4所示，与细胞培养24h后，聚合物纳米粒子将miR-21抑制剂转染进细胞的效率达到88.3％，接近经典的miRNA转染载体脂质体2000的效率(76.8％)。这为提高miR-21抑制剂在细胞层面的治疗纤维化的效果提供了基础。
(4)药物靶向性测试：
将200uL的10％PBS缓冲溶液、负载miR-21抑制剂的聚合物纳米粒子分别通过尾静脉注射进入单侧输尿管结扎(UUO)的Balb/c小鼠模型当中，24h后牺牲小鼠，取心、肝、脾、肺、肾、胸腺、小肠、肌肉和大脑等主要器官，用生理盐水清洗后在动物活体荧光成像仪下成像。
负载miR-21抑制剂的聚合物纳米粒子打入小鼠体内24h后的成像结果如图5所示，聚合物纳米粒子主要富集于肾脏部位，这说明负载miR-21抑制剂的聚合物纳米粒子具有肾脏靶向性，这为提高miR-21抑制剂在肾脏病灶部位的浓度，进而提高治疗效率提供了一个保证。
(5)生物毒性测试：
将Balb/c小鼠尾静脉分别注射200μL生理盐水和负载miR-21抑制剂的聚合物纳米粒子溶液，每两天给药一次，给药量为miR-21抑制剂20mg/kg，第七天牺牲小鼠。收集小鼠的心、肝、脾、肺、肾，并置于10％的中性甲醛当中，在4℃下固定24h后，再进行脱水和石蜡包埋。最后，切片并用苏木素-伊红进行染色。具体脱水包埋步骤如下：
1)、75％乙醇脱水3次，然后80～100％梯度乙醇脱水各一次，每次10min。然后二甲苯清洗两次，每次10min。
2)、用石蜡包埋处理两次，每次50min。
3)、石蜡包埋冷冻2h后，切片(厚2μm)，并在65℃下烘烤1h。二甲苯脱蜡两次，每次30min，梯度乙醇清洗，每次5min。
4)、苏木素染色20min，纯水冲洗，1％盐酸乙醇分化后，氨水反蓝。纯水和95％乙醇分别冲洗一次。伊红染色5min，95％乙醇和无水乙醇脱水，二甲苯透明2次后，中性树脂封片。
注射负载miR-21抑制剂的聚合物纳米粒子的小鼠主要器官H&E染色结果如图6，生理盐水组和聚合物纳米粒子干预组的老鼠的心、肝、脾、肺、肾等主要器官组织形态并未存在明显差异，说明负载miR-21抑制剂的聚合物纳米粒子对主要器官不存在明显的生物毒性，是一种安全的纳米载药系统。
(6)药物治疗效果测试：
将Balb/c小鼠随机分为4组：假手术组(正常喂食)、病理组(UUO，尾静脉注射生理盐水)、病理干预组(UUO，尾静脉注射游离的miR-21抑制剂)、病理干预组(UUO，尾静脉注射负载miR-21抑制剂的聚合物纳米粒子)。UUO模型为左侧单侧输尿管结扎手术，手术后的第1、3、5天分别给药20mg/kg(miR-21抑制剂)。手术后第七天处死老鼠，收集小鼠肾脏，进行H&E染色，观察肾脏形态。小鼠肾脏的H&E染色结果如图7，病理组的肾小管细胞萎缩、肾小管扩张、间质纤维化、炎性细胞浸润等病理性状明显。给了游离的miR-21抑制剂的病理组，其病理特征并未出现明显改变，而尾静脉注射miR-21抑制剂负载的聚合物纳米粒子组的小鼠的肾小管细胞形态出现明显改善，间质纤维化和炎性细胞浸润等明显减弱。这表明聚合物纳米载药体系比游离的药物具有更高的治疗肾小管损伤和炎症的能力。
(7)肾小管细胞间质纤维化改善效果测试：
按照(6)中药物治疗效果测试分组的实验，收集小鼠肾脏，进行肾脏行胶原纤维三色(Masson)染色，其具体步骤如下：
1)、苏木素染色15min，去离子水洗，然后1％盐酸乙醇分化后，1％的氨水反蓝；
2)、去离子水冲洗，立春红染色20min，2％的冰醋酸水溶液浸泡1min；
3)、1％磷钼酸分化4min；
4)、苯胺蓝染色5min，2％的冰醋酸水溶液浸泡1min；
5)、95％乙醇、无水乙醇分别脱水，各5min；
5)、二甲苯透明2次后，中性树脂封片。
小鼠肾脏的Masson染色结果如图8所示，病例组的肾小管细胞间质的纤维化面积显著增加，而静脉注射游离miR-21抑制剂的病理组的间质纤维化出现了一定程度的改善，但并不明显，而静脉注射负载miR-21抑制剂的聚合物纳米材料后，纤维化面积出现了明显的减少。
由以上结果所示，本发明所述的聚合物纳米粒子其尺寸在30nm左右，纳米粒子表面暴露着具有肾脏靶向能力的低分子量壳聚糖(Mn 4261Da)，并可以负载治疗肾脏纤维化的药物。一方面，聚合物纳米粒子可以保护负载的肾脏纤维化药物免受酶的降解。另一方面，低分子量壳聚糖赋予聚合物纳米粒子表面正电荷，使聚合物纳米粒子可以精准的靶向到肾脏病灶部位，并高效的转染进入肾脏细胞，实现对肾脏纤维化的显著治疗，降低药物的系统毒性。本发明制备的聚合物纳米载药粒子具有生物相容性好，药物保护高，肾脏靶向能力强的优点。动物实验模型表明，本发明制备的负载肾脏纤维化治疗药物的聚合物纳米粒子可以实现肾脏的特异性靶向富集，并在肾脏中具有可观的停留时间，可以有效的肾脏纤维化的高效治疗，是一种理想的负载肾脏纤维化治疗药物的载药系统，可以应用于临床实践。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其它的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

1.一种负载肾脏纤维化治疗药物的聚合物纳米粒子的制备方法，其特征在于包括如下制备步骤：
(1)将肾脏纤维化治疗药物、乳化剂、水溶性聚合物加入到含有醋酸的水相中搅拌混合均匀，得到前驱体溶液；
(2)将油溶性聚合物溶于有机溶剂中，超声混合均匀，搅拌条件下加入到步骤(1)所得前驱体溶液中，去除有机溶剂，离心，固相产物经洗涤，得到负载肾脏纤维化治疗药物的聚合物纳米粒子。
2.根据权利要求1所述的一种负载肾脏纤维化治疗药物的聚合物纳米粒子的制备方法，其特征在于：步骤(1)所述肾脏纤维化治疗药物为番茄红素、miR-29抑制剂、miR-21抑制剂、miR-192抑制剂、氯化钴中的至少一种。
3.根据权利要求1所述的一种负载肾脏纤维化治疗药物的聚合物纳米粒子的制备方法，其特征在于：步骤(1)中所述乳化剂为聚乙烯吡咯烷酮、泊洛沙姆188、聚乙烯醇、十六烷基三甲基氯化铵、普朗尼克F127、聚乙二醇、脂肪酸聚氧乙烯酯、嵌段聚氧乙烯-聚氧丙烯醚中的至少一种。
4.根据权利要求1所述的一种负载肾脏纤维化治疗药物的聚合物纳米粒子的制备方法，其特征在于：步骤(1)中所述水溶性聚合物为分子量Mn为3000～10000Da的低分子量壳聚糖、分子量Mn为600～10000Da的低分子量聚醚酰亚胺、多聚赖氨酸、十六烷基三甲基氯化铵和聚环糊精中的至少一种。
5.根据权利要求1所述的一种负载肾脏纤维化治疗药物的聚合物纳米粒子的制备方法，其特征在于：步骤(1)中所述前驱体溶液中，醋酸的体积浓度为0.5％～1％；乳化剂的质量浓度为0.3％～2％；水溶性聚合物的浓度为0.05～1mg/mL。
6.根据权利要求1所述的一种负载肾脏纤维化治疗药物的聚合物纳米粒子的制备方法，其特征在于：步骤(2)中所述油溶性聚合物为聚乳酸、聚乳酸-羟基乙酸共聚物、聚N-异丙基丙烯酰胺、聚酰胺-胺中的至少一种。
7.根据权利要求1所述的一种负载肾脏纤维化治疗药物的聚合物纳米粒子的制备方法，其特征在于：步骤(2)中所述有机溶剂为丙酮、乙腈、二氯甲烷、四氢呋喃、二甲基甲酰胺、二甲基亚枫中的一种或两种以上的混合。
8.根据权利要求1所述的一种负载肾脏纤维化治疗药物的聚合物纳米粒子的制备方法，其特征在于：步骤(2)中所述的有机溶剂与步骤(1)中水相的体积比为(3～4):5。
9.根据权利要求1所述的一种负载肾脏纤维化治疗药物的聚合物纳米粒子的制备方法，其特征在于：步骤(1)中肾脏纤维化治疗药物与步骤(2)中油溶性聚合物的质量比为(0.3～1):10。
10.一种负载肾脏纤维化治疗药物的聚合物纳米粒子，其特征在于：通过权利要求1～9任一项所述的方法制备得到。
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