CN115350287A - 一种具有pH/ROS双重响应性的纳米制剂及其制备方法与应用 - Google Patents
一种具有pH/ROS双重响应性的纳米制剂及其制备方法与应用 Download PDFInfo
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Abstract
本发明提供了一种pH/ROS双重响应性纳米药物载体,所述载体的结构为肉桂醛和4‑羟基苯硼酸频哪醇酯修饰的环糊精。并采用卵磷脂、磷脂‑聚乙二醇(DSPE‑PEG)和磷脂‑聚乙二醇‑RGD肽作为纳米药物的壳,核由糖皮质激素和制备的pH/ROS双重响应性药物载体构成纳米制剂。本发明设计了一种基于疾病部位的微环境而具有pH/ROS双重响应性药物载体,与单独的pH响应性或单独的ROS响应性药物载体相比,该载体微环境响应性更好,更能根据病变部位微环境进行可控的降解并释放出治疗分子。
Description
技术领域
本发明涉及纳米制剂技术领域,尤其涉及一种具有pH/ROS双重响应性的纳米制剂及其制备方法与应用。
背景技术
类风湿性关节炎是一种慢性全身性自身免疫性疾病。抗炎和降低免疫反应的药物治疗是除关节置换等外科治疗方式外类风湿性关节炎的主要治疗手段,其可以减轻患者痛苦和防止关节形态和功能的进一步损害。
治疗类风湿性关节炎的常用药物主要包括非甾体类抗炎药(如塞来昔布)、改善病情的抗风湿药物(如甲氨蝶呤、生物药物等)及糖皮质激素类药物(如地塞米松、甲基泼尼松龙等)。以糖皮质激素类药物为例,虽然糖皮质激素类药物能够在一定程度上控制病变部位炎症反应、缓解患者痛苦,但因其具有较严重的副作用而使用受限。传统口服给药方式能够引起骨质疏松、感染几率增加、甚至心肌梗死等严重不良反应。究其原因是为维持病变部位达到一定药物浓度而使血液循环中药物浓度维持在高水平,从而引起严重不良反应,导致糖皮质激素类药物使用严重受限,仅用于短期快速缓解症状的“过渡治疗”。而局部关节腔内给药方式由于给药不便、患者依从性差、给药间隔期太长(每次间隔不少于3-4个月)等原因,无法解决药物治疗在临床应用中面临的问题。
因此,开发能最大程度发挥现有类风湿性关节炎治疗药物的药效、而又同时能避免其不良反应的纳米药物对于类风湿性关节炎的药物治疗意义重大,且具有极大的临床应用前景。
发明内容
针对现有技术中所存在的不足,本发明提供了一种具有pH/ROS双重响应性的纳米制剂及其制备方法与应用,其解决了现有技术中存在的类风湿性关节炎治疗药物的药效不佳、不良反应多的问题。
本发明一方面,提供一种pH/ROS双重响应性纳米药物载体,所述载体为肉桂醛和4-羟基苯硼酸频哪醇酯修饰的环糊精,其具有下述通式:
其中,n=6时为α环糊精,n=7时为β环糊精,n=8时为γ环糊精。
本发明另一方面,提供一种pH/ROS双重响应性纳米药物载体的制备方法,包括以下步骤:
(1)将肉桂醛、环糊精和活化剂1溶解,加热,然后终止反应,过滤,洗涤,干燥得到肉桂醛修饰的环糊精;
(2)将肉桂醛修饰的环糊精溶解,加入活化剂2和N,N'-羰基二咪唑活化的4-羟基苯硼酸频哪醇酯,室温下反应,加水沉淀,过滤得到pH/ROS双重响应性纳米药物载体。
进一步地,步骤(1)中,肉桂醛、环糊精和活化剂1的摩尔比为2-4:1:2-4,所述活化剂1为对甲苯磺酰氯,加热温度为60-80℃,加热时间为1-2天。
进一步地,步骤(2)中,肉桂醛修饰的环糊精、N,N'-羰基二咪唑活化的4-羟基苯硼酸频哪醇酯和活化剂2的摩尔比为1:1-3:1-3,所述活化剂2为4-二甲氨基吡啶,反应时间为2-3天。
本发明再一方面,提供一种含有上述pH/ROS双重响应性纳米药物载体的纳米制剂,所述pH/ROS双重响应性纳米药物载体包裹有疏水药物,所述pH/ROS双重响应性纳米药物载体外层覆盖有卵磷脂和二硬脂酰磷脂酰乙醇胺-聚乙二醇2000,并覆盖有二硬脂酰磷脂酰乙醇胺-聚乙二醇3400-RGD作为靶向单元。
进一步地,所述疏水药物包括糖皮质激素,优选地,所述糖皮质激素为甲基泼尼松龙。
进一步地,所述纳米制剂的粒径约为160-180纳米,聚合分散指数小于0.2,表面电位为-25ev~-30ev。
本发明又一方面,提供一种含有pH/ROS双重响应性纳米药物载体的纳米制剂的制备方法,包括以下步骤:
(1)将DSPE-PEG2000、DSPE-PEG3400-RGD和卵磷脂加入有机溶液中分散,然后加入水中超声分散,加热,得到溶液1;
(2)将pH/ROS双重响应性纳米药物载体和疏水药物用甲醇和二甲亚砜溶解,得到溶液2;
(3)将溶液2加入到溶液1中,搅拌,室温下自组装反应,得到纳米制剂。
进一步地,步骤(1)中,DSPE-PEG2000、DSPE-PEG3400-RGD和卵磷脂的质量比为1-2:1:1-2;步骤(2)中,pH/ROS双重响应性纳米药物载体和疏水药物的质量比为5-10:1;
优选地,步骤(1)中所述有机溶液为乙醇,加热温度为50-65℃,时间为30min;
优选地,步骤(3)中自组装反应时间为1.5-3小时。
本发明还提供一种pH/ROS双重响应性纳米药物载体、含有pH/ROS双重响应性纳米药物载体的纳米制剂在制备用于治疗风湿性关节炎药物中的应用。
本发明的技术原理为:发明人前期根据类风湿性关节炎的炎症部位有高浓度的活性氧(如H2O2,O2-,HO·等)和叶酸受体高表达的特点,制备了活性氧响应性地塞米松纳米制剂,对类风湿性关节炎的治疗取得了较好效果。在此基础上,申请人根据类风湿性关节炎病变部位具有低pH值和高活性氧浓度的特点,利用肉桂醛和4-羟基苯硼酸频哪醇酯对环糊精进行结构修饰,制备了pH/ROS双重响应性药物载体,发现其取得了更好的治疗效果,发明人进一步在药物载体外修饰靶向单元RGD肽,发现其取得了更好的靶向效果。
具体如下:肉桂醛的醛基和环糊精的羟基可以形成缩醛,缩醛在中性、碱性的条件下都保持较好的稳定性,而在酸性条件下可以断键。4-羟基苯硼酸频哪醇酯通过碳酸酯键与环糊精连接,在正常生理条件下能保持较好的稳定性,而在高H2O2条件下则可以快速断键。由于血液中几乎没有双氧水,pH值呈中性,所以纳米药物在血液循环中能保持较好的稳定性,直至聚集在关节炎部位。到达关节炎部位后,在关节炎部位的低pH值和高浓度的双氧水(H2O2)的刺激下,载体材料降解,纳米药物结构被破坏,从而可控的释放出抗炎药物糖皮质激素,达到靶向治疗和降低药物毒副作用的目的。另外,纳米药物外层有RGD肽修饰,其能与关节炎部位的αvβ3有较强的键合作用,故能主动靶向到关节炎部位,可以提高关节炎部位的药物浓度,降低药物在其它脏器的分布,从而减低药物的毒副作用。同时,环糊精的羟基被肉桂醛和4-羟基苯硼酸频哪醇酯修饰,水溶性降低,变为亲脂性。
相比于现有技术,本发明具有如下有益效果:
(1)本发明设计了一种基于疾病部位的微环境而具有pH/ROS双重响应性药物载体,与单独的pH响应性或单独的ROS响应性药物载体相比,该载体微环境响应性更好,更能根据病变部位微环境进行可控的降解并释放出治疗分子。
(2)该载体具有疏水性质,且能通过疏水-疏水作用对疏水药物如糖皮质激素进行包裹,在卵磷脂和磷脂-聚乙二醇-配体的稳定下形成核壳结构的纳米药物。
(3)该纳米药物具有较高分散性和较小尺寸,因此可以静脉注射。通过静脉注射后,由于纳米药物外层有聚乙二醇修饰,能在体内进行长循环,因此可以减少给药次数。
(4)该载体制备方法简单,两步完成,可以实现大规模的制备。利用该载体制备的纳米药物具有分散性好、载药量较好、靶向性强、微环境响应性好、体内抗炎效果好的优点。
附图说明
图1为本发明实施例1中制备pH/ROS双重响应性药物载体路线图。
图2为本发明实施例1中制备pH/ROS双重响应性药物载体的核磁共振氢谱图。
图3为本发明实施例1中制备纳米制剂的透射电镜图。
图4为本发明实施例1中制备纳米制剂的路线图。
图5为本发明实施例5中纳米制剂在关节炎模型小鼠的体内分布活体成像图。
图6为本发明实施例6中游离药物和纳米制剂治疗小鼠类风湿关节炎体内药效。
具体实施方式
下面结合附图及实施例对本发明中的技术方案进一步说明。本发明中所用试剂均可商业购买得到。
实施例1纳米制剂的制备方法
1、制备pH/ROS双重响应性药物载体
(1)将3.34毫摩尔的肉桂醛、1.67毫摩尔α环糊精和3.34毫摩尔对甲苯磺酰氯溶于14毫升二甲亚砜溶液中,60℃搅拌反应2天,然后停止反应。向反应液中加入140毫升丙酮,冰箱放置一夜后,过滤,50毫升丙酮洗涤,烘干,得肉桂醛修饰的环糊精白色固体。
(2)将肉桂醛修饰的环糊0.6毫摩尔溶于干燥的二甲亚砜溶液中,再加入0.6毫摩尔4-二甲氨基吡啶和0.6毫摩尔CDI-活化的4-羟基苯硼酸频哪醇酯,室温下搅拌反应3天,然后向反应瓶中加入40毫升超纯水,有固体析出,离心,固体转移至透析袋中,蒸馏水透析2天,冻干,得到pH/ROS双重响应性药物载体。
制备pH/ROS双重响应性药物载体路线图如图1所示。化合物的1HNMR如图2所示,从图中可以发现肉桂醛苯环和4-羟基苯硼酸频哪醇酯苯环的特征峰,表明肉桂醛和4-羟基苯硼酸频哪醇酯均已连接到环糊精上,载体制备成功。
2、纳米制剂的制备方法
将6毫克DSPE-PEG2000、6毫克DSPE-PEG3400-RGD和6毫克卵磷脂用0.4毫升无水乙醇超声分散,然后再加入7毫升的水继续超声分散,并置于65℃搅拌30分钟。另外称取50毫克步骤1制备的pH/ROS双重响应性药物载体,10毫克的甲基泼尼松龙,溶于0.4毫升甲醇和0.2毫升二甲亚砜溶液中,将该溶液滴加到上述分散相中,800rpm快速搅拌3min,然后在室温自组装2小时,离心,洗涤,得纳米制剂。并设立非靶向纳米制剂为对照组,非靶向纳米制剂没有RGD修饰,不能主动靶向到关节炎部位。纳米制剂的形状采用透射电镜观察。如图3所示,非靶向纳米制剂和靶向纳米制剂均为球形,粒径约为160-180纳米,聚合分散指数低于0.2,表面电位-25到-30ev,适合体内应用。纳米制剂的合成路线如图4所示。
实施例2纳米制剂的制备方法
1、制备pH/ROS双重响应性药物载体
(1)将5.01毫摩尔的肉桂醛、1.67毫摩尔β环糊精和5.01毫摩尔对甲苯磺酰氯溶于20毫升二甲亚砜溶液中,65℃搅拌反应1.5天,然后停止反应。向反应液中加入200毫升丙酮,冰箱放置一夜后,过滤,70毫升丙酮洗涤,烘干,得肉桂醛修饰的环糊精白色固体。
(2)将肉桂醛修饰的环糊0.6毫摩尔溶于干燥的二甲亚砜溶液中,再加入1.2毫摩尔4-二甲氨基吡啶和1.2毫摩尔CDI-活化的4-羟基苯硼酸频哪醇酯,室温下搅拌反应2.5天,然后向反应瓶中加入50毫升超纯水,有固体析出,离心,固体转移至透析袋中,蒸馏水透析2天,冻干,得到pH/ROS双重响应性药物载体。
2、纳米制剂的制备方法
将12毫克DSPE-PEG2000、6毫克DSPE-PEG3400-RGD和12毫克卵磷脂用0.4毫升无水乙醇超声分散,然后再加入7毫升的水继续超声分散,并置于65℃搅拌30分钟。另外称取50毫克步骤1制备的pH/ROS双重响应性药物载体,10毫克的甲基泼尼松龙,溶于0.4毫升甲醇和0.2毫升二甲亚砜溶液中,将该溶液滴加到上述分散相中,800rpm快速搅拌3min,然后在室温自组装2小时,离心,洗涤,得纳米制剂。
实施例3纳米制剂的制备方法
1、制备pH/ROS双重响应性药物载体
(1)将6.68毫摩尔的肉桂醛、1.67毫摩尔γ环糊精和6.68毫摩尔对甲苯磺酰氯溶于26毫升二甲亚砜溶液中,80℃搅拌反应1天,然后停止反应。向反应液中加入250毫升丙酮,冰箱放置一夜后,过滤,80毫升丙酮洗涤,烘干,得肉桂醛修饰的环糊精白色固体。
(2)将肉桂醛修饰的环糊0.6毫摩尔溶于干燥的二甲亚砜溶液中,再加入1.8毫摩尔4-二甲氨基吡啶和1.8毫摩尔CDI-活化的4-羟基苯硼酸频哪醇酯,室温下搅拌反应2天,然后向反应瓶中加入70毫升超纯水,有固体析出,离心,固体转移至透析袋中,蒸馏水透析2天,冻干,得到pH/ROS双重响应性药物载体。
2、纳米制剂的制备方法
将6毫克DSPE-PEG2000、6毫克DSPE-PEG3400-RGD和6毫克卵磷脂用0.4毫升无水乙醇超声分散,然后再加入7毫升的水继续超声分散,并置于60℃搅拌30分钟。另外称取50毫克步骤1制备的pH/ROS双重响应性药物载体,5毫克的甲基泼尼松龙,溶于0.4毫升甲醇和0.2毫升二甲亚砜溶液中,将该溶液滴加到上述分散相中,800rpm快速搅拌3min,然后在室温自组装3小时,离心,洗涤,得纳米制剂。
实施例4纳米制剂的制备方法
1、制备pH/ROS双重响应性药物载体
(1)将4.175毫摩尔的肉桂醛、1.67毫摩尔α环糊精和4.175毫摩尔对甲苯磺酰氯溶于17毫升二甲亚砜溶液中,70℃搅拌反应2天,然后停止反应。向反应液中加入160毫升丙酮,冰箱放置一夜后,过滤,60毫升丙酮洗涤,烘干,得肉桂醛修饰的环糊精白色固体。
(2)将肉桂醛修饰的环糊0.6毫摩尔溶于干燥的二甲亚砜溶液中,再加入0.9毫摩尔4-二甲氨基吡啶和0.9毫摩尔CDI-活化的4-羟基苯硼酸频哪醇酯,室温下搅拌反应0.5天,然后向反应瓶中加入45毫升超纯水,有固体析出,离心,固体转移至透析袋中,蒸馏水透析2天,冻干,得到pH/ROS双重响应性药物载体。
2、纳米制剂的制备方法
将9毫克DSPE-PEG2000、9毫克DSPE-PEG3400-RGD和9毫克卵磷脂用0.6毫升无水乙醇超声分散,然后再加入7毫升的水继续超声分散,并置于50℃搅拌30分钟。另外称取50毫克步骤1制备的pH/ROS双重响应性药物载体,10毫克的甲基泼尼松龙,溶于0.4毫升甲醇和0.2毫升二甲亚砜溶液中,将该溶液滴加到上述分散相中,800rpm快速搅拌3min,然后在室温自组装1.5小时,离心,洗涤,得纳米制剂。
利用高效液相色谱法制作甲基泼尼松龙的标准工作曲线,通过实施例1-4中离心分离后的纳米制剂再分散于200微升离子水中,取20微升分散的纳米制剂低温冷冻干燥后称重,并用甲醇和乙腈体积比1:1的混合溶液1毫升溶解,采用高效液相色谱法测定甲基泼尼松龙的药物含量,计算其包封率和载药量,计算结果如表1所示。其中:
表1甲基泼尼松龙纳米制剂的包封率和载药量
实施例1 | 实施例2 | 实施例3 | 实施例4 | |
包封率(%) | 86.1 | 81.5 | 89.6 | 83.8 |
载药量(%) | 9.2 | 8.9 | 6.5 | 8.3 |
实施例5纳米制剂的体内靶向效果评价
建立了小鼠类风湿性关节炎模型,实施例1制备的纳米制剂和非靶向纳米制剂用荧光探针Cy5进行了标记,游离药物与Cy5进行共价键连接,模型小鼠分别给予Cy5标记的游离药物,Cy5标记的非靶向纳米制剂,Cy5标记的靶向纳米制剂,对照组用正常小鼠,给予等计量的生理盐水。如图5所示,非靶向纳米制剂明显比游离药物在关节炎部位聚集的多,其原因是非靶向纳米制剂可以通过增强与滞留效应被动靶向聚集在关节炎部位,因此比游离药物在关节炎部位聚集的多。另外,靶向纳米制剂比非靶向纳米制剂明显的在关节炎部位聚集的多,其原因是靶向纳米制剂有RGD肽修饰,其能与关节炎部位高表达的αvβ3特异性结合,因此能主动靶向到关节炎部位,在关节炎部位有较多聚集。该结果说明本发明制备的pH/ROS双重响应性纳米制剂能主动靶向到关节炎部位。
实施例6体内治疗类风湿性关节炎活性验证
建立了小鼠类风湿性关节炎模型用于评价纳米制剂的体内抗炎活性。如图6所示,模型小鼠组的脚趾与正常小鼠相比,出现明显的肿胀,说明模型成功建立。采用空白纳米制剂(不含甲基泼尼松龙)治疗小鼠后,小鼠的脚趾肿胀减轻不是很明显,证明空白纳米制剂的抗炎效果欠佳。游离的甲基泼尼松龙组和非靶向纳米制剂组治疗后,小鼠的脚趾肿胀程度有一定缓解,证明游离的甲基泼尼松龙和非靶向纳米制剂(实施例1)有一定的治疗效果。然而,小鼠经靶向纳米制剂(实施例1)治疗后,脚趾的肿胀程度明显降低,基本接近于正常组,说明靶向纳米制剂治疗效果最好。小动物CT结果表明,模型小鼠的脚趾由于有炎症反应,侵蚀很严重,而经过游离药物和非靶向纳米制剂治疗后,小鼠骨侵蚀有所缓解。在靶向纳米制剂治疗后,小鼠骨关节变得光滑,基本接近正常组,说明靶向纳米制剂有很好的抗炎效果。
对比例1纳米制剂的制备方法
与实施例1类似,不同之处在于:步骤1中,只包括步骤(1),不包括步骤(2),即得到肉桂醛修饰的环糊精。同时,按照步骤2中的方法制备纳米制剂。
对比例2纳米制剂的制备方法
与实施例1类似,不同之处在于:步骤1中,不包括步骤(1),只包括步骤(2),即得到CDI-活化的4-羟基苯硼酸频哪醇酯修饰的环糊精。同时,按照步骤2中的方法制备纳米制剂。
通过实施例6的实验方法进行效果检测,结果显示:对比例1制备的纳米制剂的抗炎效果和对比例2制备的纳米制剂的效果明显不如实施例1制备的纳米制剂的效果。这可能是由于肉桂醛具有一定抗关节炎效果,在抗炎效果上两者具有相互促进和协同作用。
因此,本发明制备的具有pH/ROS双重响应性药物载体的效果明显优于单独的pH响应性或单独的ROS响应性的药物载体的效果。
最后说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的宗旨和范围,其均应涵盖在本发明的权利要求范围当中。
Claims (10)
2.权利要求1所述一种pH/ROS双重响应性纳米药物载体的制备方法,包括以下步骤:
(1)将肉桂醛、环糊精和活化剂1溶解,加热,然后终止反应,过滤,洗涤,干燥得到肉桂醛修饰的环糊精;
(2)将肉桂醛修饰的环糊精溶解,加入活化剂2和N,N'-羰基二咪唑活化的4-羟基苯硼酸频哪醇酯,室温下反应,加水沉淀,过滤得到pH/ROS双重响应性纳米药物载体。
3.如权利要求2所述的一种pH/ROS双重响应性纳米药物载体的制备方法,其特征在于,步骤(1)中,肉桂醛、环糊精和活化剂1的摩尔比为2-4:1:2-4,所述活化剂1为对甲苯磺酰氯,加热温度为60-80℃,加热时间为1-2天。
4.如权利要求2所述的一种pH/ROS双重响应性纳米药物载体的制备方法,其特征在于,步骤(2)中,肉桂醛修饰的环糊精、N,N'-羰基二咪唑活化的4-羟基苯硼酸频哪醇酯和活化剂2的摩尔比为1:1-3:1-3,所述活化剂2为4-二甲氨基吡啶,反应时间为2-3天。
5.一种含有权利要求1所述的pH/ROS双重响应性纳米药物载体的纳米制剂,其特征在于,所述pH/ROS双重响应性纳米药物载体包裹有疏水药物,所述pH/ROS双重响应性纳米药物载体外层覆盖有卵磷脂和二硬脂酰磷脂酰乙醇胺-聚乙二醇2000,并覆盖有二硬脂酰磷脂酰乙醇胺-聚乙二醇3400-RGD作为靶向单元。
6.如权利要求5所述的含有pH/ROS双重响应性纳米药物载体的纳米制剂,其特征在于,所述疏水药物包括糖皮质激素,优选地,所述糖皮质激素为甲基泼尼松龙。
7.如权利要求5所述的含有pH/ROS双重响应性纳米药物载体的纳米制剂,其特征在于,所述纳米制剂的粒径约为160-180纳米,聚合分散指数小于0.2,表面电位为-25ev~-30ev。
8.一种含有pH/ROS双重响应性纳米药物载体的纳米制剂的制备方法,其特征在于,包括以下步骤:
(1)将DSPE-PEG2000、DSPE-PEG3400-RGD和卵磷脂加入有机溶液中分散,然后加入水中超声分散,加热,得到溶液1;
(2)将pH/ROS双重响应性纳米药物载体和疏水药物用甲醇和二甲亚砜溶解,得到溶液2;
(3)将溶液2加入到溶液1中,搅拌,室温下自组装反应,得到纳米制剂。
9.如权利要求8所述的一种制备含有pH/ROS双重响应性纳米药物载体的纳米制剂的方法,其特征在于,步骤(1)中,DSPE-PEG2000、DSPE-PEG3400-RGD和卵磷脂的质量比为1-2:1:1-2;步骤(2)中,pH/ROS双重响应性纳米药物载体和疏水药物的质量比为5-10:1;
优选地,步骤(1)中所述有机溶液为乙醇,加热温度为50-65℃,时间为30min;
优选地,步骤(3)中自组装反应时间为1.5-3小时。
10.权利要求1所述的pH/ROS双重响应性纳米药物载体、权利要求5-7所述的含有pH/ROS双重响应性纳米药物载体的纳米制剂在制备用于治疗类风湿性关节炎药物中的应用。
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