CN110170055A - 一种新型双重脑缺血靶向纳米载体材料的制备方法 - Google Patents
一种新型双重脑缺血靶向纳米载体材料的制备方法 Download PDFInfo
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Abstract
本发明属于生物医药技术领域，具体涉及一种新型双重脑缺血靶向纳米载体材料的制备方法。本发明以聚赖氨酸树枝状大分子(PLLD)聚合物为药物载体，采用新型低密度脂蛋白受体亲和肽L57及中性粒细胞锚定肽PGP两种多肽作为脑靶向配体，利用双官能团修饰的聚乙二醇（MAL‑PEG‑NHS）将两种靶向多肽与PLLD连接，构建具有双重脑靶向性的纳米载体材料。本发明制备的脑缺血靶向纳米载体材料，采用了全新的PLLD聚赖氨酸树枝状大分子，其具有很高的生物相容性，能改善一些难溶性药物的生物利用度，利用L57与血脑屏障(BBB)表面LRP1受体的高亲和力，通过胞吞作用进入脑内，再通过PGP对CXCR2受体的高亲和力和特异性，使药物向缺血炎症病灶部位富集，提高脑缺血疾病的治疗效果。
Description
技术领域
本发明属于生物医药技术领域，涉及一种新型双重脑缺血靶向纳米载体材料的制备方法。
背景技术
聚赖氨酸树枝状大分子(Poly-L-lysine dendrimers,PLLD)是一种具有很好的生物相容性，并且表面存在大量的可修饰的氨基基团的纳米载体材料，是肽类树枝状分子中的一种，具有良好的生物相容性和生物可降解性，而赖氨酸也是人类必需的氨基酸。与聚酰胺-胺(PAMAM)和其他树状聚合物相比，肽类树枝状分子的免疫相容性和生物相容性更好，几乎无细胞毒性，被广泛用作新一代生物医用材料。其具有独特的树枝状结构，且粒径在纳米尺寸范围、单分散性、物理稳定性，其内部的空隙可以用来包封药物分子或小分子多肽，能够降低药物的毒性，促进药物的靶向输送，是一种很好的纳米递3药载体。
聚赖氨酸树枝状大分子(PLLD)经表面PEG修饰后，更加提高了树状分子的水溶性，增加药物溶解度和稳定性，减少多肽和蛋白质的免疫原性，抑制带电分子在修饰表面的非特异性结合，为后续多肽的连接提供了媒介，而同时，链接PEG后使得药物载体能够装载更多的药物，达到更高的治疗效果。
LRP1是一种低密度脂蛋白受体相关蛋白，可以通过受体介导的转运(Receptor-mediated transport,RMT)途径到达中枢神经系统(CNS)，L57(TWPKHFDKHTFYSILKLGKH-OH)是第一种具有血脑屏障(blood-brain barrier,BBB)穿透性的人工合成的LRP1结合肽，L57在小鼠血浆中稳定时长达20min，原位脑灌注实验显示L57的血脑屏障通透性明显增高。
CXCR2是一种典型的化学引诱物受体，因为它在吞噬细胞，特别是嗜中性粒细胞上呈现高表达，所以可为理想的炎性靶向受体，作为细胞外胶原的内源性降解产物，三肽激动剂Pro-Gly-Pro(PGP)对CXCR2受体具有高亲和力和特异性，并具有低免疫原性风险，PGP已被认为是炎症性疾病的关键趋化因子，并在炎症状态下的中性粒细胞(Neutrophils)内流中起重要作用。因此，PGP可以作为用于脑靶向递送的嗜中性粒细胞锚定肽。
发明内容
本发明的目的是为弥补脑缺血卒中的药物靶向治疗缺陷，提供一种新型双重脑缺血靶向纳米载体材料，采用四代聚赖氨酸树枝状大分子(PLLD)为药物载体，赖氨酸为人体必需氨基酸，可有效解决纳米载体生物相容性差、细胞毒性等问题，在此基础上，采用L57多肽为一级靶向因子，与LPR1受体的特异性结合，实现跨越血脑屏障转运；PGP中性粒细胞锚定肽为二级靶向因子，与CXCR2受体具有高度亲和性，在药物载体透过血脑屏障的同时，进一步锚定脑缺血炎症部位，实现精准靶向治疗，提高药物的利用度。
本发明采用聚赖氨酸树枝状大分子(PLLD)为基础药物载体，以两种小分子多肽L57及PGP作为脑靶向配体，采用琥珀酰亚胺及马来酰亚胺双修饰的聚乙二醇(NHS-PEG-MAL)对PLLD进行表面修饰，既有利于后续靶向多肽L57及PGP两种多肽的修饰，也可以避免聚赖氨酸树枝状大分子(PLLD)被网状内皮系统吞噬，有利于提高体内循环时间，同时降低载体的细胞毒性。
为了达到以上目的，本发明采用如下技术方案：
本发明提供的一种新型双重脑缺血靶向纳米载体，主要是由靶向多肽、双功能聚乙二醇NHS-PEG-MAL以及聚赖氨酸树枝状大分子(PLLD)。其中所述的聚赖氨酸树枝状大分子(PLLD)与聚乙二醇(NHS-PEG-MAL)的摩尔比为1:5～1:16；PLLD与多肽的摩尔比为1:2～1:5。
本发明中所述的高分子聚合物材料为聚赖氨酸树枝状大分子(PLLD)；所述的聚乙二醇PEG分子链两端分别用马来酰亚胺(MAL)和琥珀酰亚胺(NHS)基团修饰，分子量为2000～3400；所述的两种靶向多肽分别为L57及PGP多肽。
本发明所提供的一种新型双重脑缺血靶向纳米载体材料，其包括以下步骤：
(1)将四代聚赖氨酸树状大分子(PLLD)与NHS-PEG-MAL按摩比1:5～1:16溶于PH＝6.0～8.5的磷酸盐缓冲溶液(PBS)中，室温下避光搅拌反应1～6小时，将PEG连接到PLLD载体上；
(2)将反应后的溶液转移到超滤管(MW10kDa)，转数8000～12000r/min离心10～15min，每次用双蒸水洗涤，用薄层色谱法检测，一直到反应液中无游离的PEG，然后冷冻干燥得到PEG-PLLD；
(3)将冻干后的PEG-PLLD与L57及PGP两种多肽按摩尔比为1:2～1:5溶于PH＝7.0的PBS溶液中，室温下遮光反应24小时，得到粗产物L57&PGP-PEG-PLLD；
(4)将反应后的混合溶液转移到超滤管(MW30kDa)，转数8000～12000r/min离心10～15min，离心5～8次每次用双蒸水洗涤除去未反应的多肽，然后冷冻干燥，得到纯化的L57&PGP-PEG-PLLD。
其中，所述步骤(1)中利用NHS-PEG-MAL的琥珀酰亚胺基团与PLLD表面氨基反应形成酰胺键，PEG的分子量为2000～3400。
其中，所述步骤(3)中利用MAL-PEG-PLLD中未参与反应的马来酰亚胺基团MAL与多肽末端巯基发生反应形成靶向纳米载体L57&PGP-PEG-PLLD。
本发明构建的一种新型双重脑缺血靶向纳米载体材料，适用于脑缺血组织细胞的靶向富集。
本发明的优点是：
1.本发明设计合理，工艺简单，节能环保。采用聚赖氨酸树枝状大分子(PLLD)作为药物载体，并利用亲水性强的双功能聚乙二醇(NHS-PEG-MAL)对其修饰合成PEG-PLLD药物载体，同时利用L57&PGP-PEG-PLLD载体表面的L57与PGP两种多肽，首先与血脑屏障表面的LRP1受体通过胞吞作用，提高纳米载体跨越血脑屏障的效率，再次通过PGP与CXCR2受体的特异性结合，锚定缺血炎症部位，达到靶向富集效果；
2.本发明所制备脑缺血靶向纳米递药系统，载体材料在体内可生物降解，不会形成药物栓塞，可用于静脉注射。可使药物选择性的向脑缺血病灶部位富集，达到更优越的药效。
附图说明
图1中的A图和B图分别是制备的纳米载体PEG-PLLD和L57&PGP-PEG-PLLD的核磁共振氢谱图。
图2是PLLD，PEG，PEG-PLLD三种纳米颗粒的红外谱图。
图3是制备的纳米载体L57&PGP-PEG-PLLD的扫描电镜图。
图4是制备的纳米载体L57&PGP-PEG-PLLD的纳米粒径分布图。
以下结合实施例对本发明作进一步说明，但本发明并不局限于这些实施例。
具体实施方式
实施例1
分别称取5.0mg PLLD树状大分子和25.08mg的MAL-PEG-NHS，两者的摩尔比为1:5，溶于10ml pH 8.38的PBS溶液中，室温下N2保护遮光搅拌反应6h，双功能聚乙二醇(MAL-PEG-NHS)中的NHS基团与PLLD表面的氨基特异性的反应，将反应后的溶液转移至离心超滤管(MW10kDa)中，转速6000rpm/min，离心15min，离心8次，每次用去离子水替换缓冲溶液，除去未反应的MAL-PEG-NHS，冷冻干燥即制得PEG-PLLD，并进行核磁共振氢谱(H1-NMR)表征，如图1中的A所示。
实施例2
分别称取5.0mg PLLD树状大分子和25.08mg的MAL-PEG-NHS，两者的摩尔比为1:5，溶于10ml pH 8.38的PBS溶液中，室温下N2保护遮光搅拌反应6h，双功能聚乙二醇(MAL-PEG-NHS)中的NHS基团与PLLD表面的氨基特异性的反应，将反应后的溶液转移至离心超滤管(MW10kDa)中，转速8000rpm/min，离心10min，离心6次，每次用去离子水替换缓冲溶液，除去未反应的MAL-PEG-NHS，冷冻干燥即制得PEG-PLLD，按PLLD与L57及PGP多肽摩尔比为1：5的比例，分别取3mg L57及0.5mg PGP与20mg PEG-PLLD溶于2ml pH 7.00的PBS溶液中，室温下N2保护遮光搅拌反应24h，将反应后的溶液转移至离心超滤管(MW10kDa)，转速10000rpm/min，离心10min，离心6次，每次用去离子水替换缓冲溶液，除去未反应L57及PGP肽，即制得L57&PGP-PEG-PLLD纳米复合物，用重水作为H1-NMR谱的测试溶剂，进行核磁共振氢谱的表征，如图1中的B所示。
实施例3
分别称取5.0mg PLLD树状大分子和25.08mg的MAL-PEG-NHS，两者的摩尔比为1:5，溶于10ml pH 8.38的PBS溶液中，室温下N2保护遮光搅拌反应6h，双功能聚乙二醇(MAL-PEG-NHS)中的NHS基团与PLLD表面的氨基特异性的反应，将反应后的溶液转移至离心超滤管(MW10kDa)中，转速6000～8000rpm/min，离心10～15min，离心5～8次，每次用去离子水替换缓冲溶液，除去未反应的MAL-PEG-NHS，冷冻干燥即制得PEG-PLLD，按PLLD与L57及PGP多肽摩尔比为1：2的比例，分别取1mg L57及0.2mg PGP与20mg PEG-PLLD溶于2ml pH 7.00的PBS溶液中，室温下N2保护遮光搅拌反应24h，将反应后的溶液转移至离心超滤管(MW10kDa)，转速8000rpm/min，离心15min，离心8次，每次用去离子水替换缓冲溶液，除去未反应的L57及PGP肽，即制得L57&PGP-PEG-PLLD纳米复合物。
实施例4
分别称取6.25mg PLLD树状大分子和55.75mg的MAL-PEG-NHS，两者的摩尔比为1:16，溶于10ml pH 8.38的PBS溶液中，室温下N2保护遮光搅拌反应6h，双功能聚乙二醇(MAL-PEG-NHS)中的NHS基团与PLLD表面的氨基特异性的反应，将反应后的溶液转移至离心超滤管(MW10kDa)中，转速8000rpm/min，离心12min，离心7次，每次用去离子水替换缓冲溶液，除去未反应的MAL-PEG-NHS，冷冻干燥即制得PEG-PLLD，按PLLD与L57及PGP多肽摩尔比为1：5的比例，分别取3mg L57及0.5mg PGP与20mg PEG-PLLD溶于2ml pH 7.00的PBS溶液中，室温下N2保护遮光搅拌反应24h，将反应后的溶液转移至离心超滤管(MW10kDa)，转速10000rpm/min，离心12min，离心7次，每次用去离子水替换缓冲溶液，除去未反应L57及PGP，即制得L57&PGP-PEG-PLLD纳米复合物。
实施例5
取少量PLLD，PEG，PEG-PLLD纳米颗粒分别与KBr倒入研钵中，充分的研磨后压片，进行红外测试，检测的波长范围是4000～400cm-1，结果如图2所示。
实施例6
用导电胶固定到金属片上，真空下镀金，利用扫描电镜(SEM)观察纳米粒的外观形貌及分散性，可以看出L57&PGP-PEG-PLLD纳米颗粒，具有较规整的球形以及分散性良好，结果如图3所示。
实施例7
将制备的L57&PGP-PEG-PLLD纳米颗粒溶于蒸馏水中，制备成1mg/mL的溶液，经过0.22μm微孔滤膜过滤后，置于Zeta-sizer电位粒度分析仪中检测粒径分布，样品测试重复三次，取平均值，测得L57&PGP-PEG-PLLD纳米颗粒的粒径为215±0.263nm，结果如图4所示。
在上述实施例中，仅用于说明本发明但是不仅局限于此，应该理解在不脱离本发明的范围内还可以有多种变通或替换方案。
Claims (7)
1.一种新型双重脑缺血靶向纳米载体材料的制备方法，其特征在于，是由两种靶向多肽：新型低密度脂蛋白受体亲和肽L57 (TWPKHFDKHTFYSILKLGKH-OH)和中性粒细胞锚定肽PGP (proline-glycineproline- proline)，以及双功能聚乙二醇（NHS-PEG-MAL）、聚赖氨酸树状大分子(PLLD)所组成的具有脑靶向性能的纳米递药系统，所述的聚合物材料与聚乙二醇的摩尔比为1: 5~1: 16；所述的聚合物材料与两种多肽的摩尔比为1: 2~1: 5。
2.根据权利要求1所述的一种新型双重脑缺血靶向纳米载体材料，其特征在于步骤如下：
（1）将四代聚赖氨酸树状大分子(G4.0 PLLD)与NHS-PEG-MAL按摩比1: 5~1: 16溶于PH= 6.0~8.5的磷酸盐缓冲溶液(PBS)中，室温下避光搅拌反应1~6小时，将PEG连接到PLLD载体上；
（2）将反应后的溶液转移到超滤管（MW10kDa），转数8000~12000 r/min离心10 ~15min，每次用双蒸水洗涤，用薄层色谱法检测，一直到反应液中无游离的PEG，然后冷冻干燥得到PEG-PLLD；
（3）将冻干后的PEG-PLLD与L57及PGP两种多肽按摩尔比为1: 2～1: 5溶于PH = 7.0的PBS溶液中，室温下避光反应24小时，得到粗产物L57&PGP-PEG-PLLD；
（4）将反应后的混合溶液转移到超滤管（MW30kDa），转数8000～12000 r/min离心10～15 min，离心5～8次每次用双蒸水洗涤除去未反应的多肽，然后冷冻干燥，得到纯化的L57&PGP-PEG-PLLD聚合物纳米载体。
3.根据权利要求2所述的一种新型双重脑缺血靶向纳米载体材料，其特征在于,步骤（1）中的所述的PEG通过琥珀酰亚胺基团（NHS）接于PLLD树枝状大分子，形成PLLD-PEG-MAL。
4.根据权利要求6所述的一种新型双重脑缺血靶向纳米载体材料，其特征在于，步骤（3）所述多肽与NHS-PEG-MAL的链接方法优选多肽的末端疏基，与PEG链一端的马来酰亚胺基团反应连接制得。
5.根据权利要求1所述的一种新型双重脑缺血靶向纳米载体材料，其特征在于，所述的聚乙二醇分子链两端分别为马来酰亚胺（MAL）和琥珀酰亚胺（NHS），分子量为2000～3400。
6.根据权利要求1所述的一种新型双重脑缺血靶向纳米载体材料，其特征在于，所述的两种靶向多肽分别为L57 (TWPKHFDKHTFYSILKLGKH-OH)，PGP (Prolyl-Glycyl-Proline)。
7.根据权利要求1所述的一种新型双重脑缺血靶向纳米载体材料，其特征在于，所述的聚合物材料具有15个赖氨酸的树状大分子。
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102614524A (zh) * | 2011-02-01 | 2012-08-01 | 复旦大学 | 低密度脂蛋白受体相关蛋白配体多肽修饰的脑肿瘤靶向基因递释复合物 |
| US20160015823A1 (en) * | 2012-08-14 | 2016-01-21 | Angiochem Inc. | Peptide-dendrimer conjugates and uses thereof |
| CN107789632A (zh) * | 2017-09-06 | 2018-03-13 | 哈尔滨理工大学 | 一种t7肽修饰的主动脑靶向纳米递药系统及其制备方法 |
| CN108771763A (zh) * | 2018-07-02 | 2018-11-09 | 哈尔滨理工大学 | 一种脑缺血靶向纳米递药系统的制备方法及用途 |
| CN109381706A (zh) * | 2017-08-11 | 2019-02-26 | 复旦大学 | 以pgp为活性中心的多肽在制备缺血性脑卒中靶向递药系统中的用途 |
-
2019
- 2019-06-04 CN CN201910478941.9A patent/CN110170055A/zh active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102614524A (zh) * | 2011-02-01 | 2012-08-01 | 复旦大学 | 低密度脂蛋白受体相关蛋白配体多肽修饰的脑肿瘤靶向基因递释复合物 |
| US20160015823A1 (en) * | 2012-08-14 | 2016-01-21 | Angiochem Inc. | Peptide-dendrimer conjugates and uses thereof |
| CN109381706A (zh) * | 2017-08-11 | 2019-02-26 | 复旦大学 | 以pgp为活性中心的多肽在制备缺血性脑卒中靶向递药系统中的用途 |
| CN107789632A (zh) * | 2017-09-06 | 2018-03-13 | 哈尔滨理工大学 | 一种t7肽修饰的主动脑靶向纳米递药系统及其制备方法 |
| CN108771763A (zh) * | 2018-07-02 | 2018-11-09 | 哈尔滨理工大学 | 一种脑缺血靶向纳米递药系统的制备方法及用途 |
Non-Patent Citations (2)
| Title |
|---|
| SAKAMOTO K等: "A novel LRP1-binding peptide L57 that crosses the blood brain barrier", 《BIOCHEMISTRY AND BIOPHYSICS REPORTS》 * |
| SAKAMOTO K等: "A novel LRP1-binding peptide L57 that crosses the blood brain barrier", 《BIOCHEMISTRY AND BIOPHYSICS REPORTS》, vol. 12, 12 August 2017 (2017-08-12), pages 135 - 139 * |
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