CN111920957A - 一种线粒体靶向生育酚-齐墩果酸双药纳米粒的制备方法 - Google Patents

一种线粒体靶向生育酚-齐墩果酸双药纳米粒的制备方法 Download PDF

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CN111920957A
CN111920957A CN202010773355.XA CN202010773355A CN111920957A CN 111920957 A CN111920957 A CN 111920957A CN 202010773355 A CN202010773355 A CN 202010773355A CN 111920957 A CN111920957 A CN 111920957A
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oleanolic acid
tocopherol
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targeted
lecithin
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曾华辉
谭晓柯
朱鑫
张振强
武香香
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Henan University of Traditional Chinese Medicine HUTCM
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Abstract

本发明涉及线粒体靶向生育酚‑齐墩果酸双药纳米粒的制备方法，可有效提高药物的生物利用度及靶向性，其解决的技术方案是，将抗癌药物、靶向载体、卵磷脂及胆固醇分别溶解于有机溶剂中，至完全溶解，溶解的溶液混合在一起，旋转蒸发除去有机溶剂，得到在器壁形成的薄膜，放入真空干燥箱内干燥，彻底除去有机溶剂，加入水化溶剂，磁力搅拌水化后的所得物进行超声，依次通过不同孔径的聚碳酸酯膜，挤压破碎，即得线粒体靶向生育酚‑齐墩果酸双药纳米粒；本发明纳米粒是利用生育酚上连接的三苯基磷，其所带的正电荷易富集于线粒体，从而达到线粒体靶向给药的作用，是纳米粒的线粒体靶向性制剂上的创新。

Description

一种线粒体靶向生育酚-齐墩果酸双药纳米粒的制备方法
技术领域
本发明涉及医药领域，特别是一种线粒体靶向生育酚-齐墩果酸双药纳米粒的制备方法。
背景技术
α-生育酚琥珀酸酯（α-TOS）是维生素E 最主要成份α-生育酚（α-TOH）的一种酯化衍生物。作为天然抗氧化剂，维生素E 在体内的抗氧化作用已得到公认。维生素E 在储存和应用过程中易被氧化, 因而常将其酯化衍生物作为商业的供应形式，α-TOS 即是其中的一种。近年来研究发现，α-TOS 除作为维生素E 的供应前体外，具有广泛的抗肿瘤活性，可通过多种机制发挥抗癌作用，如诱导细胞凋亡、抑制细胞核转录因子KB功能、抑制血管生成等。此外，α-TOS能选择性杀死癌胞而对正常组织和细胞无不良反应。近些年来人们研制出多种α-TOS的新剂型，提高了α-TOS的溶解度，其中通过在其结构上连接具三苯基磷基团（TPP），其化学结构中含有3个苯基，使得整个分子具有很强的脂溶性，同时TPP中磷原子上的正电荷可以离域到3个苯环上，在更大空间分散正电荷，降低扩散渗透膜时的自由能，促使TPP穿越磷脂膜。而在线粒体内膜上的质子泵造成了线粒体膜内的负电位，因此可以吸引正电荷分子。三苯基磷基生育酚琥珀酸酯（α-TOS-TPP）分子具有一定的亲脂性，将更容易透过细胞膜而富集于线粒体。然而癌细胞中的线粒体具有更高的膜电位，使得α-TOS-TPP这种离域型亲脂阳离子（DLC）更容易选择性富集癌细胞线粒体。α-TOS-TPP在高浓度下将表现出线粒体毒性，导致肿瘤细胞死亡，因此一定程度上使其具有了线粒体靶向性。
齐墩果酸（Oleanolic acid，OA）是一种五环三萜类天然产物，广泛存在于多种中药材中，如三七、怀牛膝、甘草、石斛夹竹桃和女贞等。研究发现，OA具有多种有益的药理学特性，比如抗氧化作用、抗炎作用、降糖作用、抗病毒作用、肝功能保护作用、胃黏膜保护作用、抗微生物作用、抗肿瘤学效应，等等。其分子式为C30H48O3，分子量为456.71，结构式如下：
Figure 253185DEST_PATH_IMAGE001
虽然OA具有一定抗癌活性，能够抑制多种肿瘤细胞的增殖。但该药物的水溶性较差，在水中的溶解度仅为 1 μg/m L，肠黏膜透过性较差具有较重的首过效应，致使其生物利用度低且个体间差异性较大，这些性质限制了其临床应用与开发。目前，齐墩果酸在市场上销售的药物制剂只有片剂和胶囊剂，国内外有关学者进行了齐墩果酸新剂型的研究，大多数新型制剂制备工艺较复杂，载药量低，对设备要求较高。而且存在靶向性较的低问题，使药物疗效不佳以及易产生副作用。因此，我们需要开发基于纳米粒的线粒体靶向性制剂来提高治疗效果，减少癌症患者的毒副作用。
发明内容
针对上述情况，为解决现有技术之缺陷，本发明之目的就是提供一种线粒体靶向生育酚-齐墩果酸双药纳米粒的制备方法，可有效提高药物的生物利用度及靶向性。
本发明解决的技术方案是，该纳米粒是由抗癌药物、靶向载体、卵磷脂、胆固醇及去离子无菌水制成；包括以下步骤：
1）将抗癌药物、靶向载体、卵磷脂及胆固醇分别溶解于有机溶剂中，在超声功率100~500w下，至完全溶解；
2）将步骤1）中溶解的溶液混合在一起，旋转蒸发除去有机溶剂，得到在器壁形成的薄膜；
3）放入真空干燥箱内干燥，彻底除去有机溶剂，加入水化溶剂2~10ml，水化温度在30~80 ℃下，磁力搅拌水化1~5 h；
4）将步骤3）中所得物进行超声，超声功率100~500w、超声时间1~10 min，超声后，在40~70 ℃下依次通过孔径为1.0~0.1μm的聚碳酸酯膜，挤压破碎，即得线粒体靶向生育酚-齐墩果酸双药纳米粒。
所述的抗癌药物为齐墩果酸，但不局限于此，药物还可以是用于治疗癌症的其他药物。
所述的靶向载体为三苯基磷修饰的生育酚琥珀酸酯（α-TOS-TPP），既为治疗药物，也为线粒体靶向载体，具有线粒体靶向性及表面活性的抗癌药物。
所述的有机溶剂为甲醇、乙醇、二氯甲烷、三氯甲烷、丙酮、异丙醇、四氢呋喃、二甲基亚砜、乙酸乙酯之一或以上任意组合的有机溶剂。
所述的水化溶剂为去离子无菌水、0.9%氯化钠水溶液或者磷酸盐缓冲液中的任意一种。
所述的卵磷脂与三苯基磷修饰的生育酚琥珀酸酯的质量比为1:1~1:5；卵磷脂与胆固醇的质量比为1:1~1:10；卵磷脂与齐墩果酸的质量比为1:1~1:10；抗癌药物与有机溶剂的质量体积比为1:20~1: 30。
本发明采用过膜挤压法制得线粒体靶向纳米粒，该纳米粒是利用生育酚上连接的三苯基磷，其所带的正电荷易富集于线粒体，从而达到线粒体靶向给药的作用，是纳米粒的线粒体靶向性制剂上的创新。
附图说明
图1为本发明实施例1制备的线粒体靶向生育酚-齐墩果酸双药纳米粒的电镜照片。
图2为本发明实施例3制备的线粒体靶向生育酚-齐墩果酸双药纳米粒的粒径分布图。
图3为本发明实施例2制备的线粒体靶向生育酚-齐墩果酸双药纳米粒的表面电荷图。
图4为本发明三苯基磷修饰的生育酚琥珀酸酯的标准曲线。
图5为本发明齐墩果酸的标准曲线。
图6为本发明实施例1制备的线粒体靶向生育酚-齐墩果酸双药纳米粒在4℃水中7天的粒径变化趋势图。
图7为本发明实施例1制备的线粒体靶向生育酚-齐墩果酸双药纳米粒在37℃水中3天粒径变化趋势图。
图8为本发明实施例1制备的线粒体靶向生育酚-齐墩果酸双药纳米粒对胰腺癌细胞凋亡的影响。
具体实施方式
以下结合附图和实施例对本发明的具体实施方式作进一步详细说明。
实施例1
本发明在具体实施时，该纳米粒是由抗癌药物、靶向载体、卵磷脂、胆固醇及去离子无菌水制成；具体是，精密称取5.0mg卵磷脂，10.0 mg α-TOS-TPP，5.0 mg齐墩果酸，5.0 mg胆固醇（1:2:1:1）分别溶于100ml二氯甲烷中，混合上述溶液，20 r/min，40 ℃下旋转蒸发除去二氯甲烷，得到在器壁形成的薄膜，40 ℃真空干燥2h，加入去离子无菌水5ml ，65 ℃下磁力搅拌水化2 h，250w超声5 min，在60 ℃下分别挤压过0.4μm、0.2μm、0.1μm聚碳酸酯膜，即得线粒体靶向生育酚-齐墩果酸双药纳米粒。粒径大小为137.72 nm，多分散系数0.228，表面电荷为+43.70 mV。
实施例2
本发明在具体实施时，该纳米粒是由抗癌药物、靶向载体、卵磷脂、胆固醇及去离子无菌水制成；具体是，精密称取1.0 mg卵磷脂，5.0 mg α-TOS-TPP，1.0 mg齐墩果酸，1.0 mg胆固醇（1:5:1:1）分别溶于25ml乙醇中，混合上述溶液，20 r/min，40 ℃下旋转蒸发除去乙醇，得到在器壁形成的薄膜，40 ℃真空干燥2h，加入去离子无菌水6ml ，65 ℃下磁力搅拌水化2h，250w超声5 min，在60 ℃下分别挤压过0.4μm、0.2μm、0.1μm聚碳酸酯膜，即得线粒体靶向生育酚-齐墩果酸双药纳米粒。粒径大小为110.12nm，多分散系数为0.218，表面电荷为+26.26 mV。
实施例3
本发明在具体实施时，该纳米粒是由抗癌药物、靶向载体、卵磷脂、胆固醇及去离子无菌水制成；具体是，精密称取2.0mg卵磷脂，10.0 mg α-TOS-TPP，4.0 mg齐墩果酸，6.0 mg胆固醇（1:5:2:3）分别溶于100ml二甲基亚砜中，混合上述溶液，20 r/min，40 ℃下旋转蒸发除去二甲基亚砜，得到在器壁形成的薄膜，40 ℃真空干燥2 h，加入去离子无菌水4 ml ，65℃下磁力搅拌水化2 h，250w超声5 min，在60 ℃下分别挤压过0.4μm、0.2μm、0.1μm聚碳酸酯膜，即得线粒体靶向生育酚-齐墩果酸双药纳米粒。粒径大小为149.74 nm，多分散系数为0.183，表面电荷为+27.41 mV。
实施例4
本发明在具体实施时，该纳米粒是由抗癌药物、靶向载体、卵磷脂、胆固醇及去离子无菌水制成；具体是，精密称取5.0mg卵磷脂，10.0 mg α-TOS-TPP，5.0 mg齐墩果酸，10.0 mg胆固醇（1:2:1:2）分别溶于110ml丙酮中，混合上述溶液，20 r/min，40 ℃下旋转蒸发除去丙酮，得到在器壁形成的薄膜，40 ℃真空干燥3 h，加入去离子无菌水9ml ，65 ℃下磁力搅拌水化2 h，200w超声8 min，在60 ℃下分别挤压过0.4μm、0.2μm、0.1μm聚碳酸酯膜，即得线粒体靶向生育酚-齐墩果酸双药纳米粒。粒径大小为101.30 nm，多分散系数为0.245，表面电荷为33.46 mV。
一、以线粒体靶向生育酚-齐墩果酸双药纳米粒为例做以下性能测试
线粒体靶向生育酚-齐墩果酸双药纳米粒制备及表征。本发明经过多次反复试验，制得的线粒体靶向生育酚-齐墩果酸双药纳米粒有合适的粒径、表面电荷和载药量及较好的体外稳定性。对胰腺癌细胞的凋亡作用明显优于游离药物组合，相关实验资料如下：
（1）大小、形状和表面电荷的确定
采用动态光散射(DLS)和透射电子显微镜(TEM) 对α-TOP-TPP-OA-NPs的形状、大小及表面电荷进行评价。粒径大小为137.72 nm，多分散系数0.228，表面电荷为+43.70 mV。评价结果见图1、图2及图3所示。
（2）包封率及载药量
通过超滤法分离纳米粒和游离药物，通过紫外可见光分光光度法和高效液相色谱法分别测定分离前的含药物的量、纳米粒中药物的量及游离药物的量，得到α-TOS-TPP包封率为72.74 %，载药量为42.79 %，OA包封率为81.10 %，载药量为4.77%。α-TOS-TPP和OA的标准曲线如图4、图5所示。
（3）体外稳定性
为了临床转化的成功，α-TOP-TPP-OA-NPs在4 ℃和37 ℃时应具有储存稳定性。取适量实施例1制备的纳米粒，分别放入4 ℃冰箱和37 ℃恒温培养箱中一定时间。一定时间记录粒径变化。稳定性数据清楚地说明了α-TOP-TPP-OA-NPs在贮存条件下一周和在血液循环模拟2天是稳定的。结果如图6、图7所示。
（4）MTT法检测细胞凋亡
用游离的α-TOS和OA混合溶液、α-TOP-TPP-OA-NPs分别作用于胰腺癌细胞，48h后检测胰腺癌细胞活性。α-TOS和OA混合溶液的IC50=27.22μM、α-TOP-TPP-OA-NPs的IC50=9.032μM，与相同α-TOS/ OA比例的游离药物组合相比，α-TOS-TPP-OA-NPs诱导更多的癌细胞死亡，细胞活力数据清楚地表明，与游离的单药治疗以及药物组合相比，双药加载的α-TOS-TPP-OA-NPs的效果要优，各浓度下胰腺癌细胞活性如图8所示。
本发明采用过膜挤压法制得线粒体靶向纳米粒，粒径为100~150 nm，多分散系数为0.23±0.01，表面电荷+26.26mV~+43.09 mV，α-TOS-TPP包封率为72.74 %，载药量为42.79 %，OA包封率为81.10 %，载药量为4.77%，该纳米粒是利用生育酚上连接的三苯基磷，其所带的正电荷易富集于线粒体，从而达到线粒体靶向给药的作用，是纳米粒的线粒体靶向性制剂上的创新，具有实际的临床意义和推广价值。

Claims (5)

1.一种线粒体靶向生育酚-齐墩果酸双药纳米粒的制备方法，其特征在于，该纳米粒是由抗癌药物、靶向载体、卵磷脂、胆固醇及去离子无菌水制成；包括以下步骤：
1）将抗癌药物齐墩果酸、靶向载体三苯基磷修饰的生育酚琥珀酸酯、卵磷脂及胆固醇分别溶解于有机溶剂中，在超声功率100~500w下，至完全溶解；
2）将步骤1）中溶解的溶液混合在一起，旋转蒸发除去有机溶剂，得到在器壁形成的薄膜；
3）放入真空干燥箱内干燥，彻底除去有机溶剂，加入水化溶剂2~10ml，水化温度在30~80 ℃下，磁力搅拌水化1~5 h；
4）将步骤3）中所得物进行超声，超声功率100~500w、超声时间1~10 min，超声后，在40~70 ℃下依次通过孔径为1.0~0.1μm的聚碳酸酯膜，挤压破碎，即得线粒体靶向生育酚-齐墩果酸双药纳米粒；
所述的有机溶剂为甲醇、乙醇、二氯甲烷、三氯甲烷、丙酮、异丙醇、四氢呋喃、二甲基亚砜、乙酸乙酯之一或以上任意组合的有机溶剂；
所述的水化溶剂为去离子无菌水、0.9%氯化钠水溶液或者磷酸盐缓冲液中的任意一种；
所述的卵磷脂与三苯基磷修饰的生育酚琥珀酸酯的质量比为1:1~1:5；卵磷脂与胆固醇的质量比为1:1~1:10；卵磷脂与齐墩果酸的质量比为1:1~1:10；抗癌药物与有机溶剂的质量体积比为1:20~1: 30。
2.根据权利要求1所述的线粒体靶向生育酚-齐墩果酸双药纳米粒的制备方法，其特征在于，精密称取5.0mg卵磷脂，10.0 mg α-TOS-TPP，5.0 mg齐墩果酸，5.0 mg胆固醇分别溶于100ml二氯甲烷中，混合上述溶液，20 r/min，40 ℃下旋转蒸发除去二氯甲烷，得到在器壁形成的薄膜，40 ℃真空干燥2h，加入去离子无菌水5ml ，65 ℃下磁力搅拌水化2 h，250w超声5 min，在60 ℃下分别挤压过0.4μm、0.2μm、0.1μm聚碳酸酯膜，即得线粒体靶向生育酚-齐墩果酸双药纳米粒。
3.根据权利要求1所述的线粒体靶向生育酚-齐墩果酸双药纳米粒的制备方法，其特征在于，精密称取1.0 mg卵磷脂，5.0 mg α-TOS-TPP，1.0 mg齐墩果酸，1.0 mg胆固醇分别溶于25ml乙醇中，混合上述溶液，20 r/min，40 ℃下旋转蒸发除去乙醇，得到在器壁形成的薄膜，40 ℃真空干燥2h，加入去离子无菌水6ml ，65 ℃下磁力搅拌水化2h，250w超声5 min，在60 ℃下分别挤压过0.4μm、0.2μm、0.1μm聚碳酸酯膜，即得线粒体靶向生育酚-齐墩果酸双药纳米粒。
4.根据权利要求1所述的线粒体靶向生育酚-齐墩果酸双药纳米粒的制备方法，其特征在于，精密称取2.0mg卵磷脂，10.0 mg α-TOS-TPP，4.0 mg齐墩果酸，6.0 mg胆固醇分别溶于100ml二甲基亚砜中，混合上述溶液，20 r/min，40 ℃下旋转蒸发除去二甲基亚砜，得到在器壁形成的薄膜，40 ℃真空干燥2h，加入去离子无菌水4 ml ，65 ℃下磁力搅拌水化2h，250w超声5 min，在60 ℃下分别挤压过0.4μm、0.2μm、0.1μm聚碳酸酯膜，即得线粒体靶向生育酚-齐墩果酸双药纳米粒。
5.根据权利要求1所述的线粒体靶向生育酚-齐墩果酸双药纳米粒的制备方法，其特征在于，精密称取5.0mg卵磷脂，10.0 mg α-TOS-TPP，5.0 mg齐墩果酸，10.0 mg胆固醇分别溶于110ml丙酮中，混合上述溶液，20 r/min，40 ℃下旋转蒸发除去丙酮，得到在器壁形成的薄膜，40 ℃真空干燥3 h，加入去离子无菌水9ml ，65 ℃下磁力搅拌水化2 h，200w超声8min，在60 ℃下分别挤压过0.4μm、0.2μm、0.1μm聚碳酸酯膜，即得线粒体靶向生育酚-齐墩果酸双药纳米粒。
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