CN115554272B - 一种纳米颗粒的制备方法及其应用 - Google Patents

一种纳米颗粒的制备方法及其应用 Download PDF

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CN115554272B
CN115554272B CN202211194704.8A CN202211194704A CN115554272B CN 115554272 B CN115554272 B CN 115554272B CN 202211194704 A CN202211194704 A CN 202211194704A CN 115554272 B CN115554272 B CN 115554272B
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王承潇
王丽云
崔秀明
于颖
崔颢
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Kunming University of Science and Technology
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Abstract

本发明公开一种纳米颗粒的制备方法及其应用，将牛血清白蛋白共价连接到羧基化的2‑甲基丙烯酰氧基乙基磷酰胆碱上，2‑甲基丙烯酰氧乙基磷酰胆碱为脑靶向的靶头，牛血清白蛋白可作为药物载体装载药物，功能化修饰白蛋白穿透血脑屏障，达到脑靶向的效果；本发明靶向修饰牛血清白蛋白跨血脑屏障脑靶向纳米制剂，在跨血脑屏障脑靶向和高生物相容性纳米材料、中枢神经系统疾病治疗方面具有潜在的应用前景。

Description

一种纳米颗粒的制备方法及其应用
技术领域
本发明属于生物医药技术领域，具体涉及一种纳米颗粒的制备方法及其应用。
背景技术
血脑屏障是脑靶向传输中最主要的障碍，由于血脑屏障(BBB)限制药物进入大脑，大分子药物和超过98％的小分子候选药物无法进入大脑，中枢神经系统疾病的诊断和治疗还远远不够。BBB主要由脑毛细血管内皮细胞及其紧密连接、嵌入毛细血管基底膜的周细胞和围绕毛细血管的星形胶质细胞末端组成。要把药物靶向大脑，血脑屏障是第一个障碍。现阶段克服或绕过血脑屏障的策略，包括通过细胞内化穿透血脑屏障、打开血脑屏障和鼻腔传递等。
MPC(2-甲基丙烯酰氧乙基磷酰胆碱)一种含有胆碱和乙酰胆碱类似物的分子，可与烟碱乙酰胆碱受体(nAChRs)和胆碱转运体(ChT s)以类似于乙酰胆碱和胆碱的方式相互作用。在尼古丁乙酰胆碱受体和胆碱转运体的介导下，胆碱部分通过静电作用，乙酰基部分通过氢键受体，可以有效地穿透血脑屏障并将蛋白质等传递到中枢神经系统。
蛋白质是一类天然的生物大分子，在新陈代谢、DNA复制中起着重要的作用。牛血清白蛋白(BSA)，是牛血清中的一种球蛋白，包含607个氨基酸残基，分子量为66.446KDa，等电点为4.7，原材料来源广，可从机体中获取，易于产生和纯化。
但是现在还没有很好的载体或方法可以实现跨血脑屏障，使药物很好的靶向大脑。
发明内容
本发明提供一种纳米颗粒的制备方法及其应用，将牛血清白蛋白共价连接到羧基化2-甲基丙烯酰氧基乙基磷酰胆碱MPC-COOH上，2-甲基丙烯酰氧乙基磷酰胆碱(MPC)为脑靶向的靶头，牛血清白蛋白(BSA)可作为药物载体装载药物，功能化修饰白蛋白穿透血脑屏障，达到脑靶向的效果。
本发明具体技术方案如下：
本发明提供一种纳米颗粒的制备方法，具体步骤如下：
称取100mgMPC-COOH溶解于5mL水中，加入60mgEDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)搅拌反应一个小时，再加入44mgNHS(N-羟基硫代琥珀酰亚胺)搅拌反应一个小时，得溶液B，称取100mgBSA溶解于2mL水中，加入溶液B中，反应24h，得溶液C，利用1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺的羧基活化作用，将溶液C装入3500Da分子量得透析袋中，以纯化水做接收相，充分透析，冻干，即得纳米颗粒。
所述MPC-COOH的制备方法如下：
(1)将400mg 2-甲基丙烯酰氧基乙基磷酰胆碱装入50mL反应瓶里，加入5mL醇并充分搅拌，使其溶解，再加入170mg巯基乙酸钠和14μL催化剂，反应瓶上迅速接一个磨塞三通阀，通入惰性气体，室温下搅拌反应6h，得溶液A；
(2)45℃下减压旋蒸溶液A，旋蒸干燥后，再加入11mL丙酮以去除未反应的巯基乙酸钠和副反应产物，得白色沉淀物，上清再用11mL丙酮沉淀一次，合并沉淀，45℃旋蒸除去残留有机溶剂，干燥后用水溶解沉淀，冻干即得MPC-COOH。
所述醇为甲醇或乙醇。
所述催化剂为二异丙胺。
所述惰性气体为氮气。
所述纳米颗粒作为药物载体在制备跨血脑屏障脑靶向药物中的应用，本发明通过化学偶联或者物理包裹将药物载入白蛋白中，可提高难溶性药物的溶解度，提高生物利用度，避免有机溶剂和表面活性剂的使用以及一些毒副作用的发生；白蛋白纳米粒中的药物，随着白蛋白的缓慢降解释放，具有缓释性能，可避免药物突释效应，此外，白蛋白纳米载体可以保护药物以防止被降解酶降解，延长药物半衰期，白蛋白易于修饰，其表面的氨基及羧基容易与靶向配体连接，从而实现药物的主动运输，降低药物对正常组织的毒副作用。
本发明的有益效果：
本发明利用MPC与血脑屏障(BBB)上的烟碱乙酰胆碱受体(nAChRs)和胆碱转运体(ChT s)相互作用，胆碱部分通过静电作用，乙酰基部分通过氢键受体，可以有效地穿透血脑屏障并将蛋白质等传递到中枢神经系统(CNS)。本发明提供一种靶向修饰牛血清白蛋白跨血脑屏障脑靶向纳米制剂的制备方法，在跨血脑屏障脑靶向和高生物相容性纳米材料，中枢神经系统疾病治疗方面具有潜在的应用前景。
附图说明
图1为MPC的1H-NMR谱图；
图2为MPC的13C-NMR谱图；
图3为MPC-COOH的1H-NMR谱图；
图4为MPC-COOH的13C-NMR谱图；
图5为MPC和DIPA物理混合、MPC-COOH的傅立叶变换-红外光谱图；
图6为BSA、MPC-BSA的傅立叶变换-红外光谱图；
图7为Balb/C裸鼠尾静脉注射BSA和BSA-MPC 2h后滞留活体图片；
图8为细胞存活率柱状图。
具体实施方式
下面通过实施例和附图进一步阐述本发明的实质性特点和显著的进步，但本发明的保护范围绝非仅局限于实施例。实施例中使用的原材料均可市场购买得到，如未特殊说明，其操作方法及使用原料均为公知常识。
实施例1
MPC-COOH的制备方法，具体步骤如下：
(1)将400mg 2-甲基丙烯酰氧基乙基磷酰胆碱装入50mL反应瓶里，加入5mL甲醇并充分搅拌，使其溶解，再加入170mg巯基乙酸钠和14μL二异丙胺，反应瓶上迅速接一个磨塞三通阀，通入氮气，室温下搅拌反应6h，得溶液A；
(2)45℃下减压旋蒸溶液A，旋蒸干燥后，再加入11mL丙酮以去除未反应的巯基乙酸钠和副反应产物，得白色沉淀物，上清再用11mL丙酮沉淀一次，合并沉淀，45℃旋蒸除去残留有机溶剂，干燥后用水溶解沉淀，冻干即得MPC-COOH，其反应方程式如下所示：
图1为MPC的1H-NMR谱图，根据图1的1H-NMR结果，1H-NMR(600MHz,DeuteriumOxide)δ6.10(s,1H),5.67(s,1H),4.31(t,J＝6.1,2.7,1.1Hz,2H),4.21(t,J＝9.2,5.1,2.4Hz,2H),4.08(t,2H),3.58(t,2H),3.13(s,9H),1.86(t,J＝1.3Hz,3H)。
图2为MPC的13C-NMR谱图，13C-NMR(151MHz,Deuterium Oxide)δ169.43,135.63,126.96,65.77(dt,J＝7.1,3.2Hz),64.28(d,J＝7.3Hz),63.79(d,J＝5.3Hz),59.32(d,J＝5.0Hz),53.94–53.61(m),17.26。
图3为MPC-COOH的1H-NMR谱图，根据图3的1H-NMR结果，1H-NMR(600MHz,DeuteriumOxide)δ4.28(s,1H),4.28-4.21(m,4H),4.05(dt,J＝6.7,4.5Hz,3H),3.62(t,J＝4.6Hz,3H),3.42(s,1H),.22-3.14(m,14H),3.13(s,1H),2.86-2.73(m,2H),2.68(dd,J＝13.0,5.4Hz,1H),1.23(d,J＝6.6Hz,1H),1.18(d,J＝6.8Hz,3H)。
图4为MPC-COOH的13C-NMR谱图，13C-NMR(151MHz,Deuterium Oxide)δ177.44(d,J＝28.3Hz),176.93,65.81(dq,J＝7.0,3.4Hz),64.37(q,J＝8.8Hz),63.70(td,J＝10.0,5.0Hz),59.39(dp,J＝11.4,6.6,5.8Hz),53.86(td,J＝6.8,6.3,3.4Hz),47.03,42.59,39.85–39.23(m),36.78–36.32(m),34.84(t,J＝6.7Hz),18.23(q,J＝3.3Hz),16.19(d,J＝2.6Hz)。
从氢谱和碳谱图中可以得到本实施例得到的产物的化学式与方程式的结果一致。
图5为MPC和DIPA物理混合、MPC-COOH的傅立叶变换-红外光谱图，与反应物的物理混合红外图相比，MPC-COOH在2555cm-1和1640cm-1无红外吸收，表明产物中无-SH和-C＝C-，反应物通过碳碳双键和巯基的迈克尔加成反应连接起来，表明MPC-COOH的成功合成。
实施例2
BSA-MPC的制备方法，具体步骤如下：
称取100mgMPC-COOH溶解于5mL水中，加入60mgEDC搅拌反应一个小时，再加入44mgNHS搅拌反应一个小时，得溶液B，称取100mgBSA溶解于2mL水中，加入溶液B中，反应24h，得溶液C，将溶液C装入3500Da分子量得透析袋中，以纯化水做接收相，充分透析(3-4天)，除去没有反应的MPC，袋内剩余物冻干，即得MPC接枝的BSA(MPC-BSA)，其反应方程式如下所示：
图6为BSA、BSA-MPC的傅立叶变换-红外光谱图；从图中可知，BSA-MPC的曲线在1673cm-1，1591cm-1附近出现新的吸收峰，为MPC-COOH特征吸收峰，表明MPC接枝到介孔上。
实施例3
应用试验，具体步骤如下：
实验动物准备：雄性Balb/C裸鼠，6～8周龄，体重(20±2)g，购自昆明医科大学实验动物中心，饲养于昆明理工大学实验动物房SPF级中，动物在室温(22±2)℃、相对湿度50％条件下自由摄取食水，两周后开始试验；
制备BSA-Cy5.5：将1mgCy5.5-NHS溶于10μLDMSO中，制成Cy5.5-NHS溶液，将BSA溶于碳酸钠-碳酸氢钠缓冲液(pH9.3，0.1M，碳酸盐缓冲液配置：1L，NaHCO37.5524g，Na2CO31.0969g)中，制成BSA溶液(0.5mg/mL)，在常温避光条件下，BSA溶液和Cy5.5-NHS溶液于摇床以100rpm的速度反应24h，3500Da的透析袋中充分透析，冷冻干燥即得Cy5.5接枝标记的牛血清白蛋白BSA-Cy5.5，将BSA-Cy5.5作为酰胺反应的反应物，即将BSA-Cy5.5替换实施例2中的BSA按照实施例2的方法制备得到Cy5.5标记的MPC-BSA；
为了揭示所制备的MPC-BSA纳米体系血脑屏障穿透能力，通过IVIS Lumina LTSeriesⅢ光学分子成像系统对健康Balb/C裸鼠进行小动物活体成像，获取脑区的药物积累情况，成像系统使用673nm激发和695nm发射波长进行成像。
分别尾静脉注射Cy5.5标记的BSA(即BSA-Cy5.5)0.5mg/mL和同样浓度的MPC-BSA溶液，每只200.0μL，注射后使用健康的裸鼠进行小动物成像，注射后2h将小鼠用异氟烷气麻全身麻醉，从背部成像，MPC-BSA组的小鼠在大脑区域看到强烈的近红外(NIR)荧光信号，如图7所示，对于没有MPC修饰的纳米颗粒BSA组，则未到达脑部在动物体内的脑靶向效应。
使用体外MTT实验来评估纳米体系的细胞毒性：
将对数生长期的小鼠脑微血管内皮细胞bEnd.3，用胰蛋白酶消化细胞制成单细胞悬液，以每孔5×103的密度100μL铺在96孔板中，培养体系为10％FBS+1％双抗+89％DMEM高糖培养基，之后将不同浓度的BSA、MPC-BSA分别加入相应的孔板中，以不含样品的孔作为对照组，不含细胞以及样品的孔作为调零组，每个样品3个复孔，孵育24h，将每个孔中的培养基吸出，并小心用灭菌的PBS洗3次，然后再加入新鲜培养基，每孔100μL，再向每个孔中加入20μL，浓度为5.0mg/mL的MTT溶液，并在37℃孵育4h，之后小心将每孔溶液吸出，再向其中加入150μLDMSO以溶解形成的甲瓒，并置于摇床上震荡10-15min使其均匀充分溶解，用酶标仪测定其在490nm波长处的吸光度(OD值)。
细胞存活率通过以下公式进行计算：
细胞存活率＝(实验孔OD值-调零OD孔值)/(对照组OD值-背景组OD值)×100％
结果发现，BSA组和MPC-BSA组均未观察到明显的细胞毒性，其细胞存活率均大于80％(图8)，说明BSA和MPC-BSA组均具有良好的生物相容性。

Claims (2)

1.一种纳米颗粒的制备方法，其特征在于，具体步骤如下：
称取 100mgMPC-COOH溶解于5mL水中，加入60mgEDC搅拌反应一个小时，再加入44mgNHS搅拌反应一个小时，得溶液B，称取100mgBSA溶解于2mL水中，加入溶液B中，反应24h，得溶液C，将溶液C装入3500Da分子量得透析袋中，以纯化水做接收相，充分透析后冻干，即得纳米颗粒，
所述MPC-COOH的制备方法如下：
（1）将400mg 2-甲基丙烯酰氧基乙基磷酰胆碱加入5mL醇并充分搅拌，再加入170mg巯基乙酸钠和14μL催化剂，通入惰性气体，室温下搅拌反应6h，得溶液A；
（2）45℃下减压蒸馏溶液A，旋蒸干燥后，加入11mL丙酮，沉淀白色沉淀物，上清再用11mL丙酮沉淀一次，合并沉淀，45℃旋蒸，用水溶解沉淀，冻干即得MPC-COOH；
所述醇为甲醇或乙醇；
所述催化剂为二异丙胺；
所述惰性气体为氮气；
制备得到的纳米颗粒的结构如下所示：
2.权利要求1所述制备方法制备得到的纳米颗粒作为药物载体在制备跨血脑屏障脑靶向药物中的应用。
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