CN111701031B - 靶向线粒体负载circRNA的纳米材料及其制备方法和应用 - Google Patents

靶向线粒体负载circRNA的纳米材料及其制备方法和应用 Download PDF

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CN111701031B
CN111701031B CN202010560444.6A CN202010560444A CN111701031B CN 111701031 B CN111701031 B CN 111701031B CN 202010560444 A CN202010560444 A CN 202010560444A CN 111701031 B CN111701031 B CN 111701031B
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苏士成
许小丁
刘嘉煜
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Sun Yat Sen Memorial Hospital Sun Yat Sen University
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Abstract

本发明涉及靶向线粒体负载circRNA的纳米材料，以相互电荷吸附的线粒体靶向肽和circRNA过表达载体为核心，以共价连接有pH响应聚合物的纳米颗粒为载体，通过如下步骤制备得到：（1）合成pH响应聚合物Meo‑PEG‑b‑PDPA和线粒体靶向肽；（2）合成circRNA，并将其克隆到circRNA的过表达载体pcD‑ciR中；（3）将合成的Meo‑PEG‑b‑PDPA溶解在二甲基甲酰胺中形成均相溶液；（4）将合成的circRNA过表达载体溶于去离子水中形成溶液；同时将合成的线粒体靶向肽溶于二甲基亚砜中形成溶液；（5）将circRNA过表达载体水溶液和线粒体靶向肽二甲亚砜溶液混合后，再与Meo‑PEG‑b‑PDPA均相溶液混合；混合液经强力搅拌后混合液滴加到去离子水中，形成靶向线粒体负载有circRNA纳米颗粒；（6）将靶向线粒体负载有circRNA纳米颗粒洗涤、除杂、定容。

Description

靶向线粒体负载circRNA的纳米材料及其制备方法和应用
技术领域
本发明涉及靶向线粒体负载circRNA的纳米材料及其制备方法和应用，属于纳米材料技术领域。
背景技术
线粒体是一种存在于大多数细胞中的由两层膜包被的细胞器，直径在在0.5到10微米左右。大多数真核细胞拥有线粒体，但它们各自拥有的线粒体在大小、数量及外观等方面上都有所不同。线粒体拥有自身的遗传物质和遗传体系，但因其基因组大小有限，所以线粒体是一种半自主细胞器，是细胞中制造能量的结构，细胞进行有氧呼吸的主要场所，为细胞的活动提供了能量。由于线粒体在维持生命稳态中发挥着重要作用，涉及细胞能量障碍及氧化损伤的多种疾病(如神经退行性疾病、癌症、衰老、心血管疾病等)都与线粒体的功能紊乱相关，因此，完善线粒体的研究，深入了解它的功能，将为疾病防治提供新策略。
环状RNA(circRNA)是一种特殊的内源性非编码RNA，是继微小RNA及长链非编码RNA后RNA家族的又一研究热点。近年来，随着RNA测序技术和生物信息学的迅速发展，人们通过分析大规模转录组数据发现circRNA大量存在，约占真核细胞转录本10％，相较于miRNA和长链非编码RNA，circRNA具有较为显著一些特性，如：具有环状闭合结构，无polyA尾巴，不易被核酸外切酶降解，能够更加稳定地存在真核细胞中，且序列多数高度保守，具有一定的组织结构特异性，使得circRNA在基因表达及其他生物学进程中占据重要地位。circRNA与多种疾病密切相关，在疾病的发生发展过程中起着至关重要的调控作用，因此，circRNA在未来可能会成为一种高效能的临床诊断标志物。
目前，关于靶向线粒体纳米材料负载的物质基本是药物或者探针，利用靶向线粒体纳米材料负载探针常常应用在线粒体成像或蛋白质标记中，线粒体成像能够实时监控细胞内线粒体的形态和数量变化，进而实现疾病的早期预判，蛋白质标记；利用靶向线粒体纳米材料负载药物能够有助于将药物递送到线粒体中，有效地治疗与线粒体有关的疾病；但是，关于负载circRNA等RNA表达载体的靶向线粒体纳米材料的研究鲜有，主要是因为普通的circRNA过表达载体难以导入线粒体且普通的circRNA表达载体导入线粒体的效率低。
发明内容
本发明提供靶向线粒体负载circRNA的纳米材料及其制备方法，提高了circRNA过表达载体导入线粒体的效率，将靶向线粒体负载circRNA的纳米材料应用在细胞实验或动物实验中能够进入到特定器官中特定细胞的线粒体内，从而实现线粒体内特定circRNA的高表达，在细胞实验中circRNA的高表达方便探究该circRNA对线粒体功能的影响和在线粒体相关疾病的细胞模型中所起的治疗作用，在动物实验中circRNA的高表达中方便探究该circRNA对组织器官的影响，还可以提高对线粒体相关疾病诊断和治疗的准确性。
本发明采用的技术方案如下：
第一方面，本发明提供了靶向线粒体负载circRNA的纳米材料，所述纳米材料为线粒体靶向肽和circRNA过表达载体通过电荷吸附后成为核心的纳米颗粒，纳米颗粒表面共价连接有pH响应聚合物。
第二方面，本发明提供靶向线粒体负载circRNA的纳米材料的制备方法，包括如下步骤：
(1)合成pH响应聚合物Meo-PEG-b-PDPA和线粒体靶向肽；
(2)合成circRNA，并将其克隆到circRNA的过表达载体pcD-ciR中合成circRNA过表达载体；
(3)将经过步骤(1)合成的Meo-PEG-b-PDPA溶解在二甲基甲酰胺中，形成均相溶液；
(4)将经步骤(2)合成的circRNA过表达载体溶于去离子水中形成circRNA过表达载体水溶液；同时将经过步骤(1)合成的线粒体靶向肽溶于二甲基亚砜中形成线粒体靶向肽二甲亚砜溶液；
(5)将经过步骤(4)制得的circRNA过表达载体水溶液和线粒体靶向肽二甲亚砜溶液混合后，再与Meo-PEG-b-PDPA均相溶液混合；混合液经强力搅拌均匀后，将混合液滴加到去离子水中，最终形成负载有circRNA的靶向线粒体纳米颗粒；
(6)将经过步骤(5)形成的负载有circRNA的靶向线粒体纳米颗粒转移至超滤装置中，离心去除有机溶剂和游离化合物，然后用超纯水洗涤，最后用超纯水定容至1mL。
进一步地，所述步骤(3)中均相溶液的浓度为10mg/mL。
进一步地，所述步骤(4)中circRNA过表达载体水溶液的浓度为1mg/mL；所述线粒体靶向肽二甲亚砜溶液的浓度为5mg/mL。
第三方面，本发明还提供根据靶向线粒体负载circRNA的纳米材料的制备方法制得的靶向线粒体负载circRNA的纳米材料应用在动物实验或细胞实验中进而对线粒体相关疾病进行诊断和治疗。
本发明与现有技术相比，具有如下有益效果：
1、本发明提供了靶向线粒体负载circRNA的纳米材料及其制备方法，是对靶向线粒体的负载运用功能进行了进一步的扩展，在现有的靶向线粒体负载药物或探针的技术上进行了延伸，弥补了靶向线粒体负载circRNA的技术空白。
2、本发明提供的靶向线粒体负载circRNA纳米材料将其应用在动物实验或细胞实验中，能够将负载的circRNA过表达载体高效运送至特定细胞的线粒体内，从而在线粒体内富集特定的circRNA，实现线粒体内特定circRNA的高表达，为探究circRNA对线粒体功能的影响、对组织器官的影响以及研究circRNA在线粒体相关疾病的细胞模型中所起的治疗作用提供了新方向，同时方便线粒体相关疾病的治疗，提高线粒体相关疾病诊断和治疗的效率和准确性。
3、本发明提供的靶向线粒体负载circRNA的纳米材料的制备方法克服了现有技术中普通circRNA过表达载体难以导入线粒体以及普通circRNA表达载体导入线粒体的效率低的问题，在制备过程中利用带负电的circRNA过表达载体与带正电的线粒体靶向肽相互吸引吸附后在高速漩涡的作用下形成纳米颗粒，同时在纳米颗粒上共价连接pH响应聚合物，使之能够根据细胞内生理环境的变换根据进行pH调节响应，使得靶向线粒体负载circRNA的纳米材料能够顺利通过细胞的内吞作用输送到线粒体中，pH响应聚合物被质子化，从内体中释放出由线粒体靶向肽和circRNA过表达载体组成的复合物，复合物靶向运送至线粒体，在线粒体内实现特定circRNA的高表达。
附图说明
图1为本发明的制备流程以及靶向线粒体负载环状RNA的纳米材料在细胞内的反应顺序示意图；
图2为本发明实施例细胞实验中未经处理、负载空载体的纳米颗粒处理以及负载circRNA过表达载体的纳米颗粒处理的非酒精性脂肪性肝炎患者肝成纤维细胞内胞浆和线粒体中circRNA的相对表达量示意图；
图3为本发明实施例动物实验中注射靶向线粒体负载circRNA纳米材料以及注射裸Cy5标记载体的小鼠体内5种器官的荧光信号强度对比图；
图4为本发明实施例动物实验中注射靶向线粒体负载circRNA纳米材料和注射裸Cy5标记载体的小鼠体内Cy5阳性肝成纤维细胞的数量对比示意图；
图5为本发明实施例动物实验中靶向线粒体负载circRNA纳米材料在线粒体中和胞浆中的浓度对比示意图。
具体实施方式
下面结合附图和具体实施例来对发明进行详细的说明。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
本发明提供了靶向线粒体负载circRNA的纳米材料，所述纳米材料为线粒体靶向肽和circRNA过表达载体通过电荷吸附后成为核心的纳米颗粒，其中，circRNA过表达载体带负电，线粒体靶向肽带正电；纳米颗粒表面共价连接有pH响应聚合物。
如图1所示，本发明提供了靶向线粒体负载circRNA的纳米材料的制备方法，包括如下步骤：
(1)合成pH响应聚合物Meo-PEG-b-PDPA和线粒体靶向肽；pH响应聚合物Meo-PEG-b-PDPA和线粒体靶向肽的合成方法均为现有技术；
其中，pH响应聚合物Meo-PEG-b-PDPA的合成采用原子转移自由基法，具体的合成方法参见如下文献：
Xu,X.,Wu,J.,Liu,Y.,Saw,P.E.,Tao,W.,Yu,M.,Zope,H.,Si,M.,Victorious,A.,Rasmussen,J.,et al.(2017).Multifunctional Envelope-Type siRNA DeliveryNanoparticle Platform for Prostate Cancer Therapy.ACS nano 11,2618-2627；
Xu,X.,Wu,J.,Liu,Y.,Yu,M.,Zhao,L.,Zhu,X.,Bhasin,S.,Li,Q.,Ha,E.,Shi,J.,et al.(2016).Ultra-pH-Responsive and Tumor-Penetrating Nanoplatform forTargeted siRNADelivery with Robust Anti-Cancer Efficacy.Angewandte Chemie 55,7091-7094；
线粒体靶向肽的合成方法采用标准Fmoc化学方法，通过固相多肽合成线粒体靶向肽，具体的合成方法参见如下文献：
Chouchani,E.T.,Methner,C.,Nadtochiy,S.M.,Logan,A.,Pell,V.R.,Ding,S.,James,A.M.,Cocheme,H.M.,Reinhold,J.,Lilley,K.S.,et al.(2013).
Cardioprotection by S-nitrosation of a cysteine switch onmitochondrial complex I.Nat Med 19,753-759；
Zielonka,J.,Joseph,J.,Sikora,A.,Hardy,M.,Ouari,O.,Vasquez-Vivar,J.,Cheng,G.,Lopez,M.,and Kalyanaraman,B.(2017).Mitochondria-TargetedTriphenylphosphonium-Based Compounds:Syntheses,Mechanisms of Action,andTherapeutic and Diagnostic Applications.Chem Rev 117,10043-10120；
本发明对现有技术的改进不在于pH响应聚合物和线粒体靶向肽的合成，且本领域技术人员依据现有技术可知晓关于pH响应聚合物和线粒体靶向肽合成方法，故本发明对此不另做限定；
(2)合成circRNA，并将其克隆到circRNA的过表达载体pcD-ciR中合成circRNA过表达载体；circRNA过表达载体用化学合成法首先合成circRNA基因序列，以KpnI和BamHI对pcd-ciR载体双酶切去除载体中的stuffer序列，然后与经双酶切的circRNA基因序列片段进行连接克隆，circRNA过表达载体的具体合成方法参见如下文献：
Han,D.,Li,J.,Wang,H.,Su,X.,Hou,J.,Gu,Y.,Qian,C.,Lin,Y.,Liu,X.,Huang,M.,et al.(2017).Circular RNAcircMTO1 acts as the sponge of microRNA-9tosuppress hepatocellular carcinoma progression.Hepatology 66,1151-1164；
本发明对现有技术的改进不在于circRNA过表达载体的合成，且本领域技术人员依据现有技术可知晓关于circRNA过表达载体的合成方法，故本发明对此不另做限定；
(3)将经过步骤(1)合成的Meo-PEG-b-PDPA溶解在二甲基甲酰胺中，形成10mg/mL的均相溶液；
(4)将经步骤(2)合成的circRNA过表达载体溶于去离子水中形成1mg/mL的circRNA过表达载体水溶液；同时将经过步骤(1)合成的线粒体靶向肽溶于二甲基亚砜中形成5mg/mL的线粒体靶向肽二甲亚砜溶液；
(5)将经过步骤(4)制得的circRNA过表达载体水溶液和线粒体靶向肽二甲亚砜溶液混合后，再与200μL的Meo-PEG-b-PDPA均相溶液混合；混合液经1000rpm强力搅拌均匀，混合液在高速旋涡的作用下最终形成所需要的纳米颗粒，将混合液滴加到去离子水中，最终形成负载有circRNA的靶向线粒体纳米颗粒；
(6)将经过步骤(5)形成的负载有circRNA的靶向线粒体纳米颗粒转移至超滤装置中，离心去除有机溶剂和游离化合物，然后用超纯水洗涤，最后用超纯水定容至1mL。
将根据本发明提供的制备方法制得的靶向线粒体负载circRNA纳米材料进行性能测试，测试结果如下：
1、粒径和zeta电位测试：
对制得的靶向线粒体负载circRNA纳材料通过动态光散射测定纳米颗粒的尺寸和Zeta电位，结果显示，靶向线粒体负载circRNA纳米材料的粒径为98.60±2.28nm，Zeta电位为2.96±0.15mv；
2、circRNA过表达载体的包载率：
实验组：使用Label
Figure GDA0004149630770000071
TrackerTM胞内核酸定位试剂盒，在步骤(5)前用0.5μL的Cy5荧光染料标记10μg的circRNA过表达载体，然后再负载形成纳米颗粒，取用5μL的制得的纳米材料溶液与20倍的二甲基亚砜混合；
对照组：将5μL未负载到纳米颗粒中的裸Cy5标记载体(10μg/μL)与20倍的二甲基亚砜混合制成标准品(Standard)；
用多功能微孔板读数仪测量荧光强度(FI)，计算circRNA过表达载体的包载率：EE％＝(FI-NPs/FI-Standard)×100％，结果显示，包载率为90.86±3.24％。
3、靶向线粒体负载circRNA纳米材料的释放和表达
将成纤维细胞接种于24孔板中，在含10％胎牛血清的DMEM培养基中培养，24h后，取浓度为200ng/mL的根据本发明提供的制备方法制得的靶向线粒体负载circRNA纳米颗粒与成纤维细胞共同孵育48～72h，成纤维细胞摄取纳米颗粒后，pH响应聚合物被质子化，从内体中释放出靶向线粒体负载circRNA纳米颗粒，并靶向运送至线粒体，在线粒体内高表达circRNA；靶向线粒体负载circRNA纳米材料的释放和表达如图1所示。
本发明还提供根据靶向线粒体负载circRNA的纳米材料的制备方法制得的靶向线粒体负载circRNA的纳米材料应用在动物实验或细胞实验中进而对线粒体相关疾病进行诊断和治疗。
本发明制得靶向线粒体负载circRNA的纳米材料在细胞实验中应用的方法如下：
分别将空载体和circRNA过表达载体按照本发明提供的方法负载进纳米颗粒中，然后将非酒精性脂肪性肝炎患者的肝成纤维细胞与纳米颗粒共同孵育48～72h，通过逆转录定量PCR分别检测未经处理、负载空载体的纳米颗粒处理以及负载circRNA过表达载体的纳米颗粒处理的非酒精性脂肪性肝炎患者肝成纤维细胞内胞浆和线粒体中circRNA的表达水平；结果如图2所示，负载circRNA过表达载体的纳米颗粒处理的成纤维细胞内线粒体中circRNA的表达量显著高于未经处理组以及负载空载体的纳米颗粒处理组；负载circRNA过表达载体的纳米颗粒处理的成纤维细胞内circRNA在线粒体中的表达量显著高于胞浆。
以上结果说明，根据本发明制得的靶向线粒体负载circRNA的纳米材料在细胞实验中可以高效、特异性地将负载的circRNA过表达载体运送到线粒体中，从而实现线粒体内特定circRNA的高表达。
本发明制得靶向线粒体负载circRNA的纳米材料在动物实验中应用的方法如下：
使用Label
Figure GDA0004149630770000081
TrackerTM胞内核酸定位试剂盒，用Cy5荧光染料标记circRNA过表达载体，将未负载到纳米颗粒中的裸Cy5标记载体、负载Cy5标记circRNA过表达载体的靶向线粒体纳米颗粒和磷酸缓冲盐溶液PBS分别通过尾静脉注射到10周龄的健康雄性C57BL/6J小鼠中，注射24h后处死小鼠，采集小鼠的肺、心、脾、肝和肾，用小动物活体成像系统对上述器官进行荧光成像，并通过ImageJ软件量化每个器官的荧光强度；
如图3所示，注射靶向线粒体负载circRNA纳米颗粒的小鼠体内5种器官的荧光信号强度均显著高于注射裸Cy5标记载体的小鼠，且在检测的5种器官中，肝的荧光信号强度最高，即靶向线粒体纳米颗粒的累积量最多；从小鼠肝脏分离出肝成纤维细胞，通过流式细胞仪检测分离的成纤维细胞的荧光强度，如图4所示，注射靶向线粒体负载circRNA纳米颗粒的小鼠体内Cy5阳性肝成纤维细胞的数量和比例均显著高于注射裸Cy5标记载体的小鼠；
分离肝成纤维细胞的线粒体和胞浆部分，并在裂解缓冲液中均质化，随后将裂解物转移到96孔板中，用多功能微孔板读数仪测量样品的荧光强度。将负载Cy5标记circRNA过表达载体的靶向线粒体纳米颗粒进行梯度稀释，生成标准曲线，再根据标准曲线计算每个样品中Cy5标记circRNA过表达载体的浓度，结果如图5所示，靶向线粒体负载circRNA纳米颗粒在线粒体中的浓度显著高于胞浆。
以上结果均说明，本发明在动物实验中可以将负载的circRNA过表达载体高效地运送至特定器官中特定细胞的线粒体内，从而实现线粒体内特定circRNA的高表达。
以上所述仅为本发明的实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书及附图内容所作的等效结构或等效流程变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。

Claims (3)

1.靶向线粒体负载circRNA的纳米材料，其特征在于：所述纳米材料为线粒体靶向肽和circRNA过表达载体通过电荷吸附后成为核心的纳米颗粒，纳米颗粒表面共价连接有pH响应聚合物；
其中，线粒体靶向肽的化学结构式为：
Figure FDA0004152306480000011
所述靶向线粒体负载circRNA的纳米材料的制备方法，包括如下步骤：
(1)合成pH响应聚合物Meo-PEG-b-PDPA和线粒体靶向肽；
(2)合成circRNA，并将其克隆到circRNA的过表达载体pcD-ciR中合成circRNA过表达载体；
(3)将经过步骤(1)合成的Meo-PEG-b-PDPA溶解在二甲基甲酰胺中，形成均相溶液；
(4)将经步骤(2)合成的circRNA过表达载体溶于去离子水中形成circRNA过表达载体水溶液；同时将经过步骤(1)合成的线粒体靶向肽溶于二甲基亚砜中形成线粒体靶向肽二甲基亚砜溶液；
(5)将经过步骤(4)制得的circRNA过表达载体水溶液和线粒体靶向肽二甲基亚砜溶液混合后，再与Meo-PEG-b-PDPA均相溶液混合；混合液经强力搅拌均匀后，将混合液滴加到去离子水中，最终形成负载有circRNA的靶向线粒体纳米颗粒；
(6)将经过步骤(5)形成的负载有circRNA的靶向线粒体纳米颗粒转移至超滤装置中，离心去除有机溶剂和游离化合物，然后用超纯水洗涤，最后用超纯水定容至1mL。
2.如权利要求1所述的靶向线粒体负载circRNA的纳米材料，其特征在于：所述步骤(3)中均相溶液的浓度为10mg/mL。
3.如权利要求1所述的靶向线粒体负载circRNA的纳米材料，其特征在于：所述步骤(4)中circRNA过表达载体水溶液的浓度为1mg/mL；所述线粒体靶向肽二甲基亚砜溶液的浓度为5mg/mL。
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