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CN116712556B - 一种靶向中枢神经系统的药物递送载体复合物及其制备方法和应用 - Google Patents

一种靶向中枢神经系统的药物递送载体复合物及其制备方法和应用 Download PDF

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CN116712556B
CN116712556B CN202310892804.6A CN202310892804A CN116712556B CN 116712556 B CN116712556 B CN 116712556B CN 202310892804 A CN202310892804 A CN 202310892804A CN 116712556 B CN116712556 B CN 116712556B
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施炜
刘倩倩
朱星佳
周友浪
游波
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Affiliated Hospital of Nantong University
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Abstract

本发明公开了一种靶向中枢神经系统的药物递送载体复合物及其制备方法和应用，涉及生物医药技术领域。该制备方法包括以下步骤：将载体细胞进行叠氮修饰，得到叠氮修饰载体细胞；将巯基和胆固醇分别修饰至治疗性miRNA的两端，再将修饰后治疗性miRNA与丙烯基复合物进行连接反应，形成miRNA复合物；将所述叠氮修饰载体细胞和所述miRNA复合物共同孵育，通过点击化学反应形成所述药物递送载体复合物。本发明制备得到的药物递送载体复合物对中枢神经系统具有良好的靶向性，可用于制备治疗中枢神经系统疾病的药物。

Description

一种靶向中枢神经系统的药物递送载体复合物及其制备方法 和应用
技术领域
本发明涉及生物医药技术领域，特别是涉及一种靶向中枢神经系统的药物递送载体复合物及其制备方法和应用。
背景技术
血脑屏障(blood-brainbarrier，BBB)，是脑毛细血管壁与神经胶质细胞形成的血浆与脑细胞之间的屏障和由脉络丛形成的血浆和脑脊液之间的屏障。它能严格控制特定营养物质(如氨基酸、葡萄糖、核苷和脂肪酸)从血管系统进入中枢神经系统的细胞外液，以及限制有害的外源性分子(如神经毒素)的通过，来维持中枢神经系统稳态。与此同时，血脑屏障也会阻止某些药物和生物制药等大分子治疗药物进入大脑，低水平的药物浓度会影响药物的治疗效率。所以急需一项有效的非侵入技术来将生物药物高效地输送到中枢神经系统。
目前，关于中枢神经系统靶向的技术大致分为以下几种：(1)受体介导的胞吞作用(RMT)；(2)噬神经病毒的应用；(3)纳米颗粒的递送系统；(4)外泌体介导的递送系统；(5)其他递送系统。
对于RMT在中枢神经系统递送技术的开发，理想的受体应该只在血脑屏障细胞上表达，并具有由选择性配体介导的高转运能力，这些配体不会干扰其自然功能，不会引发溶酶正常途径降解。尽管目前还没有发现这样的“理想”受体，但关于靶向转铁蛋白受体(TfR)、胰岛素受体和LDL受体相关蛋白1(LRP1)的药物正在进行的临床试验，这些结果可以证实基于RMT的技术是否可以安全有效地将治疗相关药物剂量递送到中枢神经系统，同时将外周和/或脱靶毒性作用降到最低。
嗜神经病毒，作为一类对神经组织有亲和力的病毒，也被尝试用作进入中枢神经系统的载体，最主要的包括腺相关病毒(AAV)和慢病毒，其他包括单纯疱疹病毒(HSV)和类病毒颗粒(VLPs)。关于这些病毒载体的应用目前仅限于基因治疗，并且通常依赖于需要对患者进行额外监测的侵入性给药方法，例如鞘内或肾腔内注射。开发出一种新型跨越BBB的病毒载体，如某些AAV变体，可以减少神经外科治疗过程中对侵入性治疗方式的依赖。噬神经病毒作为载体在使用过程中还应考虑的问题包括载体纯度、调节元件和转基因序列、非整合或自失活载体的使用、包装DNA的性质、给药途径，配方以及输注体积和速率。还需要更多的研究来进行强有力的安全性评估和选择最佳病毒载体。
纳米颗粒系统在载体的探索中应用广泛，如脂质体、聚合纳米颗粒和固体脂质纳米颗粒(SLNPs)。有研究表明非靶向脂质体更多的是被动摄取，这可能是由于血脑屏障完整性受损，而不是主动血脑屏障运输机制。而靶向性脂质体的应用，如谷胱甘肽(GSH)-聚乙二醇脂质体，其高效的脑靶向性已在一些临床前期研究中得到证实。以聚乳酸-羟基乙酸聚乙二醇共聚物(PLGA)为代表的纳米颗粒聚合物在神经退行性疾病以及脑肿瘤中也得到应用。总的来说，虽然研究表明以纳米颗粒为载体的药物递送在中枢神经系统疾病的治疗中有一定的临床价值，但是不得不承认大多数案例的成功是在脑癌的背景下，确切的说是BBB的完整性受到损害的前提下实现的。而纳米颗粒本身的固有属性(如粒径、表面电荷等)也会影响药物递送。纳米颗粒的内在稳定性也很重要，载体需要保持完整直到抵达大脑才能产生疗效。降解是许多纳米颗粒载体需要面对的问题。总之，目前尚未找到有效的办法将纳米颗粒靶向大脑并提高脑内的血药浓度，有待需要进一步研究。
外泌体是一种细胞来源的小囊泡，直径40-100nm，大多数类型的细胞都能够分泌。外泌体可以包含各种物质，包括蛋白质和核酸。近年来，越来越多的研究证明，外泌体有能力作为一种载体实现对中枢神经系统的药物递送，但要使其适用于中枢神经疾病的治疗，还有一些问题需要解决。首先是外泌体的获取，其含量取决于其产生的细胞，来自不同细胞的外泌体可以引起不同的效果，识别合适的外泌体来源可以帮助避免潜在的副作用。其次如果没有进行大脑靶向的修饰，只有一小部分注射的外泌体能够顺利到达大脑。而外泌体穿过BBB的机制仍不清楚，外泌体和细胞之间相互作用的主要机制是膜融合，随后将其内容送入细胞。另一个重要问题是外泌体的装载效率，现有的外泌体装载策略效率都较低，这会限制装载物的最大适用剂量，使其达到治疗上的低效率水平。要想使外泌体作为中枢神经系统治疗药物的输送工具，就必须解决这些问题。
细胞介导的脑传递是一种相对较新的方法，可以被视为细胞治疗更广泛背景下的一种特殊应用。这些细胞也可以被改造成产生与治疗相关的肽、蛋白质或RNA分子，或携带包裹药物的脂质体，既可以用于自体的、患者特异性的治疗，也可以作为输注到体循环中的异体通用细胞治疗。免疫细胞如巨噬细胞和单核细胞是神经炎症性疾病或具有神经炎症成分的神经退行性疾病的脑递送的合适候选者，因为这些细胞在脑炎症期间被招募穿过血脑屏障。这些免疫细胞依靠其自身的“浸润”过程，改变自身形状以穿越血脑屏障。
目前，关于细胞介导的中枢神经系统的递送载体的探索仍处于早期阶段，有许多悬而未决的问题。例如，目前尚不清楚能够穿过血脑屏障的细胞数量是否足以进行有效治疗，或者细胞是否到达与治疗相关的大脑区域。由于可能只有一小部分载体细胞会通过外周静脉给药进入大脑，而大多数细胞仍滞留在其他器官；因此，外周其他主要器官的载体细胞分布也是一个必须考虑的主要问题。
发明内容
本发明的目的是提供一种靶向中枢神经系统的药物递送载体复合物及其制备方法和应用，以解决上述现有技术存在的问题，该药物递送载体复合物对中枢神经系统具有良好的靶向性，可用于制备治疗中枢神经系统疾病的药物。
为实现上述目的，本发明提供了如下方案：
本发明提供一种靶向中枢神经系统的药物递送载体复合物的制备方法，包括以下步骤：
将载体细胞进行叠氮修饰，得到叠氮修饰载体细胞；
将巯基和胆固醇分别修饰至治疗性miRNA的两端，再将修饰后治疗性miRNA与丙烯基复合物进行连接反应，形成miRNA复合物；
将所述叠氮修饰载体细胞和所述miRNA复合物共同孵育，通过点击化学反应形成所述药物递送载体复合物。
进一步地，所述载体细胞包括小胶质细胞或中性粒细胞。
进一步地，所述载体细胞为小胶质细胞。
进一步地，所述治疗性miRNA为miR-138-5pmimic。
进一步地，所述丙烯基复合物为二苯基环辛炔-聚乙二醇-丙烯酸酯。
进一步地，所述将载体细胞进行叠氮修饰的方法，包括将所述载体细胞与叠氮修饰的甘露糖共同培养。
本发明还提供一种根据上述的制备方法制备得到的靶向中枢神经系统的药物递送载体复合物。
本发明还提供上述的药物递送载体复合物在制药过程中提高中枢神经系统靶向性中的应用。
本发明还提供一种治疗中枢神经系统疾病的药物，包括上述的药物递送载体复合物。
本发明公开了以下技术效果：
本发明通过基于代谢糖工程和生物正交点击化学的生物正交标记策略构建得到了一种新的原创性的靶向中枢神经系统的药物递送载体复合物。该药物递送载体复合物能够顺利穿过血脑屏障，并对中枢神经系统具有良好的靶向性。本发明为制备治疗中枢神经系统疾病的药物开发了新的载药系统，从而可用通过外周静脉给药实现对中枢神经系统疾病的治疗，避免了侧脑室原位注射给药带来的感染风险。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明载体复合物的构建示意图；
图2为载体复合物搭载治疗性miRNA的体外、体内效果验证实验的结果；其中，A为荧光验证miR-138-5pmimic在体外附着在细胞表面的情况；红色表示CY5，蓝色表示细胞核；B为体外CY5的荧光值的定量统计结果；C为两种载体复合物(mimic-NE和mimic-HAPI)在MCAO大鼠的主要器官的分布情况；D为载体复合物携带miR-138-5pmimic到大鼠的中枢神经系统；红色表示CY5，蓝色表示细胞核，箭头表示用CY5标记的代表性miR-138-5pmimic；E为体内CY5的定量分析结果；F为通过共聚焦显微镜观察大鼠神经元对miR-138-5pmimic的摄取情况；红色表示CY5，绿色表示神经元，蓝色表示细胞核，箭头表示用CY5标记的代表性miR-138-5pmimic；G为定量分析神经元中的CY5的结果；H为CES2在MCAO大鼠损伤区的表达的Westernblot分析结果；I为CES2的Westernblot的灰度值分析结果；J为miR-138-5p的下游靶基因的Westernblot分析结果；K为miR-138-5p的下游靶基因的Westernblot的灰度值分析结果；所有数据代表三个独立实验的平均值±SEM；P值采用two-wayANOVAorStudentttests；n.s.P≥0.05，*P＜0.05，**P＜0.01，***P＜0.001，****P＜0.0001。
具体实施方式
现详细说明本发明的多种示例性实施方式，该详细说明不应认为是对本发明的限制，而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式，并非用于限制本发明。另外，对于本发明中的数值范围，应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值，以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明，否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料，但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入，用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时，以本说明书的内容为准。
在不背离本发明的范围或精神的情况下，可对本发明说明书的具体实施方式做多种改进和变化，这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本发明说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等，均为开放性的用语，即意指包含但不限于。
术语说明：
本发明所指的miR-138-5pmimic是一种化学合成的miRNA模拟物，能模拟内源性miRNA，上调miRNA活性，经苏州吉玛基因股份有限公司构建并修饰
本发明所指的二苯环辛炔-聚乙二醇-丙烯基(DBCO-PEG-AC)复合物是指二苯基环辛炔-聚乙二醇-丙烯酸酯，购自上海拓旸生物公司
实施例1
1.方法
1.1载体复合物的构建
如图1所示的构建策略进行如下操作：
(1)载体细胞的准备和预处理
从8-12周龄的SD大鼠的胫骨和股骨中制备原代中性粒细胞(NE)，并在含有10％胎牛血清的RPMI1640培养基(Gibco)中培养。HAPI细胞(大鼠小胶质细胞)购自上海ATCC细胞库。HAPI细胞在DMEM培养基(Gibco)中培养，辅以10％FBS和100U/mL青霉素-链霉素。
(2)治疗性miRNA的预修饰
将步骤(1)培养后的HAPI细胞和NE细胞与叠氮修饰的甘露糖(CAS号：361154-30-5)共同培养1.5小时，在生物正交标记前进行共孵育预处理以分别形成细胞复合物，即叠氮修饰HAPI细胞和叠氮修饰NE细胞。
miR-138-5pmimic的两端事先经过化学修饰，mimic的反义链的5’端修饰巯基，3’端修饰胆固醇，为方便荧光观察，3’端再添加荧光染料Cyanine5(CY5)标记。修饰过的miR-138-5pmimic与二苯环辛炔-聚乙二醇-丙烯基(DBCO-PEG-AC)复合物在25℃下孵育0.5h，这两种模拟化合物通过巯基和丙烯基之间形成酯键连接，形成mimic复合物。
(3)将载体细胞和mimic复合物通过点击化学反应连接
将叠氮修饰HAPI细胞和叠氮修饰NE细胞分别与mimic复合物在25℃共孵育，通过点击化学反应分别形成HAPI细胞载体复合物(mimic-HAPI)和NE细胞载体复合物(mimic-NE)。
1.2载体复合物搭载治疗性miRNA，通过体外荧光观察、体内活体成像、荧光观察、下游蛋白水平的改变验证实验效果。
2.结果
在载体复合物的构建完成后，荧光显微镜观察到miR-138-5pmimic被加载到细胞表面(图2中A和B)。利用炎症细胞靶向受损炎症区域的特性，本发明选择了不同的炎症细胞(中性粒细胞NE和小胶质细胞HAPI)作为载体，分别探讨了它们靶向传递miRNA到受损区域的效率。本发明将miR-138-5pmimic加载到NE细胞或HAPI细胞的表面。通过观察用Cyanine5(CY5)标记的miR-138-5pmimic在MCAO大鼠器官中的分布，发现mimic-NE和mimic-HAPI都能到达缺血损伤区，且HAPI细胞的传递能力优于NE细胞，而miRNA复合物在其他器官的分布主要集中在肺和肾脏(图2中C)。染色结果还显示，CY5标记的miR-138-5pmimic可以到达缺血受损区域(图2中D和E)并被神经元摄取(图2中F和G)。因此，本发明发现HAPI细胞和NE细胞都可以作为载体用于中枢神经系统靶向，并且实验证实HAPI细胞具有更好的靶向性，因此，本发明在随后的实验中使用HAPI细胞作为载体。
当miRNA被HAPI细胞运送到受损区域后，必须从装载的细胞中分离出来才能发挥作用。为此，本发明用巯基修饰miRNA，通过在细胞表面酯化丙烯基形成酯键。因为受损区域细胞破碎释放酯酶，比如羧酸酯酶，这些酯酶可以切断酯键，进而将miRNA从细胞表面分离出来。在过去的几十年里，羧酸酯酶(包括CES1和CES2)的分子特性及其在体内和外源性代谢中的生物作用得到了广泛的研究。最近的研究表明，CES参与了大量内源性酯类的水解。用CES2细胞与mimic-HAPI共培养8小时的体外实验显示mimic与HAPI分离(图2中A和B)。体内实验证实，CES2的表达在MCAO大鼠的缺血损伤区明显升高(图2中H和I)，表明体内存在酯键解离的内部环境。
最后，检测治疗后MCAO大鼠损伤区的DNMT3A、Rheb和Rhebl1的表达。结果显示，miR-138-5pmimic在到达受损区域后可以发挥类似的功能，减少DNMT3A的表达，从而提高Rhebl1的水平。与之前的结果相似，HAPI细胞比NE细胞具有更强的靶向能力(图2中J和K)。
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。

Claims (3)

1.一种靶向中枢神经系统的药物递送载体复合物的制备方法，其特征在于，包括以下步骤：
将载体细胞进行叠氮修饰，得到叠氮修饰载体细胞；
将巯基和胆固醇分别修饰至治疗性miRNA的两端，再将修饰后治疗性miRNA与丙烯基复合物进行连接反应，形成miRNA复合物；
将所述叠氮修饰载体细胞和所述miRNA复合物共同孵育，通过点击化学反应形成所述药物递送载体复合物；
所述载体细胞为小胶质细胞或中性粒细胞；
所述丙烯基复合物为二苯基环辛炔-聚乙二醇-丙烯酸酯；
所述将载体细胞进行叠氮修饰的方法，包括将所述载体细胞与叠氮修饰的甘露糖共同培养；所述叠氮修饰的甘露糖的CAS号为361154-30-5；
所述治疗性miRNA为miR-138-5p mimic。
2.一种根据权利要求1所述的制备方法制备得到的靶向中枢神经系统的药物递送载体复合物。
3.一种治疗中枢神经系统疾病的药物，其特征在于，包括权利要求2所述的药物递送载体复合物。
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