CN117100876B - 一种用于脑部递送系统的纳米胶囊、其制备方法及应用 - Google Patents
一种用于脑部递送系统的纳米胶囊、其制备方法及应用 Download PDFInfo
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Abstract
本发明涉及一种用于脑部递送系统的纳米胶囊、其制备方法及应用，属于纳米药物技术领域。本发明以2‑甲基丙烯酰氧乙基磷酸胆碱、N‑（3‑氨基丙基）甲基丙烯酰胺为单体，N,N’‑亚甲基双丙烯酰胺为交联剂，利用自由基聚合形成的纳米材料，以牛血清白蛋白为模型蛋白包裹在纳米材料内部，形成纳米胶囊，并在其表面修饰纳洛酮分子，利用其与脑部阿片受体特异性结合，形成以受体介导的高效跨越血脑屏障的新型蛋白药物纳米载体。
Description
技术领域
本发明涉及一种用于脑部递送系统的纳米胶囊、其制备方法及应用，属于纳米药物技术领域。
背景技术
血脑屏障(BBB)是保护大脑免受血液毒素和传染性物质影响的生理屏障。 其主要由内皮细胞、周细胞和星形胶质细胞组成。 如非主动运输，血脑屏障可阻碍98%的分子通过。因此血脑屏障成为脑部药物递送难以逾越的障碍。
近年来，科学家们提出了各种策略来规避血脑屏障。例如通过吸附介导的脑靶向，因为带负电荷的 BBB 可以在静电效应的基础上与带正电荷的药物输送系统相互作用，从而促进药物通过血脑屏障。然而，选择性差是吸附介导靶向的固有缺点，因为大多数生物膜带负电荷。由于这个原因，吸附介导的靶向很少用于全身给药。此外，可通过暂时打开 BBB让化合物或纳米颗粒直接扩散到大脑中。目前虽然可以通过几种物理或化学学方法暂时打开 BBB，然而这些方法均会对BBB产生一定损伤性。
发明内容
本发明的目的是针对现有技术存在的缺陷，提出一种用于脑部递送系统的纳米胶囊、及制备方法及应用。
本发明通过以下技术方案解决技术问题：本发明首先提供一种用于脑部递送系统的纳米材料，由盐酸纳洛酮衍生物修饰的纳米胶囊组成。所述盐酸纳洛酮衍生物具有游离的羧基。所述纳米胶囊由2-甲基丙烯酰氧乙基磷酸胆碱、N-（3-氨基丙基）甲基丙烯酰胺、N,N’-亚甲基双丙烯酰胺、过硫酸铵、四甲基乙二胺，以牛血清白蛋白为模型蛋白形成。
内源性阿片肽主要是β-内啡肽(β-EP)，较多分布于下丘 脑、垂体、大脑皮层、脑干、膈肌及脑脊液中。血液中β-EP主要由垂体释放，它在镇痛、内分泌活动、神经精神及呼吸循环系统起着重要调节作用。盐酸纳洛酮，商品名Narcan，为羟二氢吗啡酮的衍生物，是阿片受体的纯拮抗剂无激动活性，对阿片受体的亲和力大于吗啡和脑啡肽，能竞争性的阻断并取代阿片样物质与受体的结合。纳洛酮的脂溶性较高，可迅速的分布于全身，尤以脑、心、肺、肾脏中分布较高，给药后5 min在脑内的分布高于血浆4-6倍。
因此，基于纳洛酮的易于穿过BBB的特性，将其与可提高药物稳定性的纳米胶囊相结合以形成以阿片受体介导的高效跨越BBB的新型蛋白药物纳米载体，这种新型纳米载体的应用将为跨BBB脑部药物递送提供新的解决方案。
本发明进一步提供该材料的制备方法，具体是用于脑部递送系统的纳米材料的制备方法，包括以下步骤：
第一步、将纳洛酮分子苯环上的羟基修饰出一个游离的羧基；
第二步、以2-甲基丙烯酰氧乙基磷酸胆碱和N-（3-氨基丙基）甲基丙烯酰胺作为单体，N,N’-亚甲基双丙烯酰胺为交联剂，过硫酸铵和四甲基乙二胺作为引发剂，通过自由基聚合制备纳米材料，以牛血清白蛋白为模型蛋白，形成纳米胶囊；
第三步、将第一步的化合物和第二步的纳米胶囊偶联，得到以纳洛酮作为表面修饰的纳米胶囊。
上述方法的所述第一步中，修饰的步骤为纳洛酮图1（A）与溴乙酸叔丁酯反应得到中间化合物纳洛酮-乙酸叔丁酯即化合物1图1 (B)；用三氟乙酸将化合物1的叔丁基脱去，得到游离的羧基即化合物2图1 (C)。反应如下：
所述第二步中， 2-甲基丙烯酰氧乙基磷酸胆碱:N-（3-氨基丙基）甲基丙烯酰胺:N,N’-亚甲基双丙烯酰胺:过硫酸铵:四甲基乙二胺:牛血清白蛋白=10000：10：5000：300：10：1，按2-甲基丙烯酰氧乙基磷酸胆碱、N-（3-氨基丙基）甲基丙烯酰胺、N,N’-亚甲基双丙烯酰胺、四甲基乙二胺、牛血清白蛋白、过硫酸铵的顺序依次按比例加入离心管,在摇床上反应过夜，通过疏水柱纯化除去游离蛋白，得到纳米胶囊。
所述第三步中，首先将纳洛酮修饰的化合物2溶于1×PBS，利用稀HCL溶液调整pH至5-6，加入1.2倍当量的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐，约1min后利用稀NaOH溶液调整PH至8-9左右加入1.2倍当量的N-羟基琥珀酰亚胺， 2-3min，以纳米胶囊:化合物2=1:100的比例加入纳米胶囊。所述纳米胶囊的浓度以其包裹的蛋白浓度为准。
本发明再进一步提供用于脑部递送系统的纳米材料的应用，包括纳米材料在制备脑部靶向药物中的应用。
该应用还包括纳米材料在制备递送大分子治疗药物到脑部疾病部位的药物中的应用。
本发明以2-甲基丙烯酰氧乙基磷酸胆碱（MPC）、N-（3-氨基丙基）甲基丙烯酰胺（APM）为单体N,N’-亚甲基双丙烯酰胺（BIS）为交联剂，过硫酸铵（APS）、四甲基乙二胺（TEMED）为引发剂，利用自由基聚合形成的纳米材料，以牛血清白蛋白（BSA）为模型蛋白包裹在纳米材料内部，形成纳米胶囊，并在其表面修饰纳洛酮分子，利用其与脑部阿片受体特异性结合，形成以受体介导的高效跨越血脑屏障的新型蛋白药物纳米载体。
通过研究发现其可以有效的将大分子蛋白转运到脑部。与天然的BSA相比在装入修饰过纳洛酮的纳米胶囊后，BSA在脑部聚集量高出8倍之多。同时与此前研究的具有脑靶向功能的nBSA-PMPC纳米载体相比，无论是尾静脉、滴眼睛、滴鼻子给药，修饰了纳洛酮的nBSA-PMPC-Naloxone纳米颗粒具有更好的脑靶向效果。
附图说明
图1 纳诺酮修饰步骤。
图2化合物B的H-NMR谱图。
图3化合物C的H-NMR谱图。
图4BSA的MALDI-TOF MS谱图。
图5BSA-Naloxone的MALDI-TOF MS谱图。
图6nBSA-PMPC-Naloxone及BSA的DLS测定粒径结果。
图7nBSA-PMPC-Naloxone的TEM。
图8为nBSA-PMPC-Naloxone及BSA的Zeta电位
图9通过尾静脉给药nBSA-PMPC与nBSA-PMPC-Naloxone的小鼠脑组织荧光成像图。
图10通过尾静脉给药nBSA-PMPC与nBSA-PMPC-Naloxone的小鼠脑组织荧光成像ROI定量。
图11通过尾静脉给药nBSA-PMPC与nBSA-PMPC-Naloxone的小鼠脑组织研磨液荧光强度。
图12通过眼部给药nBSA-PMPC与nBSA-PMPC-Naloxone的小鼠脑组织荧光成像图。
图13通过眼部给药nBSA-PMPC与nBSA-PMPC-Naloxone的小鼠脑组织荧光成像ROI定量。
图14通过眼部给药nBSA-PMPC与nBSA-PMPC-Naloxone的小鼠脑组织研磨液荧光强度。
图15通过鼻腔给药nBSA-PMPC与nBSA-PMPC-Naloxone的小鼠脑组织荧光成像图。
图16通过鼻腔给药nBSA-PMPC与nBSA-PMPC-Naloxone的小鼠脑组织荧光成像ROI定量。
图17通过鼻腔给药nBSA-PMPC与nBSA-PMPC-Naloxone的小鼠脑组织研磨液荧光强度。
具体实施方式
下面将结合本发明实施例的技术方案进行清楚、完整地描述。
实施例
1. 纳洛酮的修饰
实验步骤：
(1)将图1A（800 mg，2.198 nmol）和5 eq的碳酸钾（1518.8 mg）加入10 ml乙腈在50度下搅拌30 min，后加入2 eq的溴乙酸叔丁酯升温至90度，TLC监控，反应时间约为2 h。将反应液过滤，得到的液相经过旋转蒸发，得到黄色的油状液体通过硅胶柱层析纯化（流动相二氯甲烷：甲醇=20:1）。化合物B的H-NMR见图2。
(2)将图1B（100 mg\0.226 nmol）中加入2 ml三氟乙酸室温下搅拌1 h，TLC检测反应结束后，分三次每次加入5 ml氯仿，旋转蒸发。后分两次每次加入5 ml乙醚旋转蒸发将得到的化合物中加入1 ml左右乙醇完全溶解，后加入约为乙醇体积50倍的乙醚，有白色固体析出。抽滤得到的白色固体即为图1化合物C。化合物C的H-NMR见图3。
2. 纳米胶囊的合成
BSA溶于1×PBS中，随后将MPC、APM、BIS（10000：10：5000：300：10：1，n/n, MPC:APM: BIS: BSA）加到溶液中，然后加入APS (500:1, n/n, APS: BSA)和TEMED（1000:1, n/n, TEMED: BSA，pH=7.0）引发自由基聚合，在常温下反应2 h。得到nBSA-PMPC。通过疏水柱纯化分离
3. 纳洛酮与纳米胶囊的偶联
化合物C溶于1×PBS中后，用稀HCL溶液调pH至5-6左右。并在加入相应量的EDC后，用稀NaOH调pH至8-9左右后，加入NHS。最后加入nBSA-PMPC（100:1.2:1.2:1，n/n, 化合物C：EDC：NHS：nBSA-PMPC）后，摇床反应12 h，透析除去得到nBSA-PMPC-Naloxone。
4. 纳米胶囊的表征
取在PB溶液中透析过的纳米胶囊1 mL，用Malvern Zetasizer Nano ZSE仪器进行动态光散射测试，获取纳米胶囊的zeta电位。
5. Cy7荧光标记
(1)避光条件下,将1 mg sulfo-Cy7-NHS溶于200 μl DMSO，终浓度5 mg/ml，用铝箔纸包好，置于-20℃冰箱避光冷冻备用。
(2)将nBSA-PMPC与用NaOH溶液调至pH=8，以摩尔比1:3的比例加入ulfo-Cy7-NHS加入体系后，室温条件下避光震荡反应4 h。
(3)室温下避光透析每4h换一次透析液，透析12h。
一、具体实施方式
6. 组织成像
(1)尾静脉注射给药：取8只小鼠，分为两组每组4只分别为实验组（nBSA-PMPC-Naloxone）和对照组（nBSA-PMPC)，每只给药25.5 mg/kg，10 h后对小鼠进行心脏灌注后，取脑组织进行荧光成像。
(2)鼻腔给药：取8只小鼠，分为两组每组4只分别为实验组（nBSA-PMPC-Naloxone）和对照组（nBSA-PMPC)，通过移液器向小鼠鼻腔滴加样品50 μl，每只给药6.4 mg/kg，10 h后对小鼠进行心脏灌注后，取脑组织进行荧光成像。
(3)眼部给药：取8只小鼠，分为两组每组4只分别为实验组（nBSA-PMPC-Naloxone）和对照组（nBSA-PMPC），过移液器向小鼠两只眼睛共滴加样品50 μl，每只给药1.28mg/kg，10 h后对小鼠进行心脏灌注后，取脑组织进行荧光成像。
7. 荧光含量测定
通过荧光酶标仪对脑组织荧光强度进一步定量研究，探明nBSA-PMPC-Naloxone-Cy7在脑部的聚集程度。
(1)将取出的脑组织每100 g/1ml，1×PBS,放入4 ml离心管中。
(2)高通量组织研磨机，50 Hz,10 s,经三次研磨后高速离心机离心后取其上清溶液。
(3)加入黑色96孔板中，每孔100 μl。
(4)荧光酶标仪测定。
8. 实验结果
经过图1步骤的反应，通过核磁结果（图2-图3）证明了Naloxone的修饰成功。通过TOF（图4-图5）证明了Naloxone与BSA通过羧基与氨基的缩合反应成功偶联。DLS（图6）的粒径结果发现nBSA-PMPC的粒径为30 nm左右，TEM（图7）成像也进一步证实了这一点，这一粒径显然大于天然BSA（＜10 nm），结合蛋白表面zeta电位的变化（图8）这均说明了nBSA-PMPC的成功合成。
为了验证nBSA-PMPC-Naloxone可跨越BBB对脑部的靶向递送能力，我们首先通过大鼠尾静脉给药来观察nBSA-PMPC-Naloxone在大鼠脑部的递送情况。通过对荧光标记的样品分布观察（图9-图10）结果我们发现nBSA-PMPC给药的小鼠蛋白在脑部聚集明显低于nBSA-PMPC-Naloxone给药的小鼠，脑组织匀浆荧光强度（图11）也进一步证明了这一结论。进一步的，在滴眼睛给药（图12-图14）和小鼠鼻腔给药（图15-图17）后，我们也观察到类似的结果，nBSA-PMPC给药的小鼠蛋白在脑部聚集明显低于nBSA-PMPC-Naloxone给药的小鼠。这些结果表明，在多种给药途径中纳诺酮修饰过的纳米胶囊nBSA-PMPC-Naloxone均可显著提高蛋白跨域BBB在脑部蓄积的能力，其与可提高药物稳定性的纳米胶囊相结合以形成以阿片受体介导的高效跨越BBB的新型蛋白药物纳米载体。其的应用将为跨BBB脑部药物递送提供新的解决方案。
除上述实施外，本发明还可以有其他实施方式。凡采用等同替换或等效变换形成的技术方案，均落在本发明要求的保护范围。
Claims (4)
1.一种用于脑部递送系统的纳米材料，由盐酸纳洛酮修饰的纳米胶囊组成，其制备方法包括以下步骤：第一步、将纳洛酮分子苯环上的羟基修饰出一个游离的羧基；第二步、以2-甲基丙烯酰氧乙基磷酸胆碱和N-（3-氨基丙基）甲基丙烯酰胺作为单体，N,N’-亚甲基双丙烯酰胺为交联剂，过硫酸铵和四甲基乙二胺作为引发剂，通过自由基聚合制备纳米材料，以牛血清白蛋白为模型蛋白，形成纳米胶囊；第三步、将第一步的化合物和第二步的纳米胶囊偶联，得到以纳洛酮作为表面修饰的纳米胶囊；所述第一步中，修饰的步骤为纳洛酮与溴乙酸叔丁酯反应得到中间化合物纳洛酮-乙酸叔丁酯即化合物B；用三氟乙酸将化合物B的叔丁基脱去，得到化合物C；所述第三步中，首先将纳洛酮修饰的化合物C溶于1×PBS，利用稀HCL溶液调整pH至5-6，加入1.2倍当量的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐，1min后利用稀NaOH溶液调整pH至8-9左右加入1.2倍当量的N-羟基琥珀酰亚胺，2-3min，以纳米胶囊:化合物C=1:100的比例加入纳米胶囊。
2.根据权利要求1所述用于脑部递送系统的纳米材料，其特征在于：所述纳米胶囊的浓度以其包裹的蛋白浓度为准。
3.根据权利要求1所述脑部递送系统的纳米材料在制备脑部靶向药物中的应用。
4.根据权利要求3所述用于脑部递送系统的纳米材料的应用，其特征在于：包括纳米材料在制备递送大分子治疗药物到脑部疾病部位的药物中的应用。
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