CN108175861A - 一种抗肿瘤小核酸药物的递送系统及其应用 - Google Patents
一种抗肿瘤小核酸药物的递送系统及其应用 Download PDFInfo
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Abstract
本发明公开一种抗肿瘤小核酸药物的递送系统及其应用，属于生物技术领域。该系统由小核酸药物、包裹小核苷酸药物的脂膜囊状物组成，特别提供了脂膜囊状物为外泌体exosome来递送抑癌小核酸let‑7的给药系统。本发明还提供了该递送系统exosome‑Let‑7来抑制肿瘤细胞的应用：细胞水平实验结果说明采用本发明提供的递送系统则可将let‑7有效的递送到细胞内发挥功能来抑制乳腺癌细胞的增殖和迁移；在体小鼠水平实验结果说明exosome‑let‑7与其他对照组相比在小鼠体内可明显抑制乳腺癌肿瘤增殖，可将let‑7更有效的递送到肿瘤组织中发挥抑制肿瘤增殖和迁移功能。
Description
技术领域
本发明属于生物技术领域，涉及一种药物递送系统，具体涉及一种抗肿瘤小核酸药物的递送系统及其应用。
背景技术
近年来，核酸药物在人类重大疾病尤其是肿瘤治疗的研究中显示出了巨大潜力，为现代生物医药产业开辟了一个崭新的方向。但是裸露的核酸药物存在以下缺点：(1)核酸药物在组织或细胞中易被核酶降解；(2)核酸药物的细胞靶向能力较差；(3)核酸药物自身带有负电荷，与带有负电荷的细胞膜之间存在排斥作用，导致其细胞内吞和逃逸内涵体的能力较差。以上的缺点导致核酸药物的治疗效果不佳。因此，需要寻求合适的载体负载核酸药物，将其递送到靶细胞，有效逃离内涵体吞噬，释放基因治疗药物进入细胞质，以实现该药物的高效表达。所以目前核酸药物治疗领域中最大的瓶颈就是核酸药物载体。
传统的基因药物主要依靠病毒载体运输，但是病毒作为药物载体，可能引起免疫反应、整合入基因组的隐患及病毒具有复活毒性的可能等所造成的系列安全问题一直饱受争议。另有许多科学家利用纳米粒子(LPNs)作为基因药物载体，如阳离子脂质及脂质类似物或阳离子多聚体，例如多聚赖氨酸、多聚精氨酸、组蛋白、脱乙酰壳多糖、聚乙烯亚胺(PEI)等。但是脂质体给药系统上存在缺乏靶向性、转染率低和全身给药的毒性等问题。因此其毒性和递送药物的脱靶效应等问题，限制了脂质体在体内的应用。此外，研究发现利用聚合物载体作为基因传递系统效率非常低，大多被陷在了细胞回收利用器—胞内体中，只有极少数核酸药物可以被运到细胞质中发挥作用。Gaurav Sahay对纳米颗粒运输siRNA进入细胞质的效率进行了深入研究，发现大多数的siRNAs陷入到了细胞的胞吞回收通路中，一些随后移动到溶酶体(lysosome)中被降解；而另一些则基本困在了内吞膜泡(endocyticvesicles)中，只有极少数的siRNA能够达到细胞质发挥生物功能。因此，选择一种具有高效性、靶向性和安全性非病毒基因药物载体是基因治疗领域中最主要、最关键的问题之一。
Exosome是由细胞内多胞体(multivesicular body，MVB)与细胞膜融合后释放到细胞外基质的一种直径约为30～120nm的小囊泡。大多数生物细胞都可以分泌这种小囊泡，并且它们天然存在于体液中，如血液、唾液、尿液和母乳等。无论是培养基中的exosome，还是各种器官分泌到体液中的exosome，都像货车(cargo)一样运载货物然后穿梭于各种细胞参与细胞间通信、免疫反应的促进、抗原呈递、程序性细胞死亡、血管生成、炎症和凝血等。Exosome的蛋白质组学分析结果显示，不同类型的细胞和体液(血液、乳汁和尿液)中，exosome膜上或泡内含有相同的标记蛋白分子，这些分子分别来自细胞质、质膜及高尔基体和内质网的生物膜。典型的exosome标记蛋白有：(1)四跨膜蛋白超家族，如CD9、CD63和CD81蛋白；(2)细胞质蛋白，如肌动蛋白(actin)、钙磷脂结合蛋白(annexins)、Rab蛋白；(3)参与生物合成的分子，如凋亡连接基因2相互作用蛋白X(apoptosis-linked gene2-interacting protein X，ALIX)、肿瘤易感基因101的蛋白质(tumor susceptibilitygene101，TSG101)、热休克蛋白(heat shock proteins，HSPs)70和热休克蛋白90。
2007年Valadi等发现，MC/9和人肥大细胞系分泌的exosome包含了约1300个基因的mRNA以及small RNA，其中包括121个microRNA。这项研究让人们意识到miRNA功能可能不仅仅在细胞内调控基因表达，还可以利用exosome这个运载工具通过各种体液在细胞间进行信息的传递。
与其它转运膜泡和载体相比，exosome作为药物载体有诸多优越性：如在血液中能够稳定存在；以及用病人自身的exosome会有较低的免疫原性等；且组织特异性的exosome可以携带miRNA、miRNA的拮抗物和小的效应分子靶向穿梭于特定组织中。
2011年英国科学家Alvarez-Erviti.L等人报道了从实验鼠的树突状细胞内提取出来的exosome，让其与从实验鼠的病毒中提取出来的蛋白质靶头---脑特异性识别多肽—RVG结合在一起。随后他们将GAPDH siRNA通过电转方式装载到exosome中，再将此exosome复合体从体外注射到小鼠体内。利用exosome的内源性、通透性特点突破血脑屏障，将siRNA递送到大脑细胞并敲除基因BACE1(该基因与与阿尔茨海默病相关)。结果显示，BACE1基因的表达降低了60％。这是科学家首次使用天然系统向大脑内递送药物。一年后，瑞典的Wahlgren J团队通过电转染的方式将siRNA装载到正常人类血清来源的exosome中，证明装载siRNA的exosome可以有效地传递MAKP1-siRNA到作为受体的外周血单核细胞，体外敲除了特定基因。T.A.Shtam等也用类似的方法递送RAD51-siRNA和RAD52-siRNA来诱导两个基因的敲除并降低纤维素肉瘤细胞的生存和增殖。L.Alvarez-Erviti等在研究中发现，在靶向递送富含siRNA的exosome后，无论管家基因mRNA如GAPDH表达的下调还是阿尔茨海默病相关的基因BACE1的下调，都能在被靶向递送的特定神经细胞中被观察到。还有一些关于短发夹RNAs(shRNA)的使用和所谓的自我递送RNAs被包含在exosome中作为治疗药物的研究。例如，Q.Pan等研究发现，一种抑制病毒进入受体和丙型肝炎病毒(HCV)复制的shRNAs，通过exosome介导可以使肝细胞的HCV的感染率降低。因为exosome天生携带miRNAs，这一特征的治疗应用也是符合逻辑的，正如一些将这一方法应用于不同疾病模型的研究所阐述的那样。研究发现Exosome包裹的miR-150能降低内皮细胞的迁移，介导效应T细胞的抑制；当293T细胞培养在含有exosome(来自miR-122转导的293T细胞)的条件培养基时，细胞内的miR-122的表达提高数倍。在体外，富含miR-133b的MSC exosome提高了神经突触的生长速率，提示这种MSC exosome成为大脑缺血的潜在治疗药物。另外，miR-214可以通过exosome穿梭到肝星状细胞，造成CCN2表达的降低，而CCN2基因在调控肝脏纤维化中起到很重要的作用。正如近期Heidi G等对多形性成胶质母细胞瘤(GBM)的评论中表述的那样，大多数肿瘤类型都有miRNAs的异常表达。MSC衍生的exosome抑制miRNA--miR-146b的高表达，在GBM的异植瘤模型中可抑制肿瘤的生长，显示了exosome-based药物递送和miRNA-based治疗两者之间存在的相关性。在外GBM细胞体研究中，MSC分泌的exosome递送anti-miRs来敲除致癌的miR-9从而提高了GBM细胞对替莫唑胺化学治疗的敏感性。exosome递送miR-143和let-7a，在体外可分别抑制前列腺癌和乳腺癌的生长。但在正常的前列腺上皮细胞中用装载miR-143的exosome处理后没有观察到类似的效果。
Exosome除了可以装载干扰RNAs进行治疗外，其他类型的物质也可以被装载进行递送，例如载入细胞毒素的阿霉素的exosome或者是exosome类似物纳米膜泡，可体外抑制乳腺癌和结肠腺癌异植瘤增殖。另外，通过修饰未成熟树突细胞分泌的exosome的膜蛋白使该exosome靶向递送阿霉素到肿瘤组织能力，而且研究结果表明这种方式既显著提高了阿霉素的功效，又降低了药物的毒副作用。
此外，很多研究分析了exosome装载蛋白进行疾病治疗的效果。例如，exosome在包裹药物的稳定性或抗炎症影响的优点，学者们在GBM的小鼠模型中，将STAT3抑制剂JSI-124(葫芦素I)装载进入exosome后进行小鼠给药，结果显示该exosome可有效的抑制肿瘤增殖。也有研究通过转染包含胞嘧啶脱氨酶和尿嘧啶磷酸核糖转移酶的载体到HEK293T细胞中后，该细胞可分泌出胞内富集该蛋白酶的exosome，并且这类exosome与化疗药物前体--5-氟胞嘧啶联合使用，在一个神经鞘瘤模型中，能有效的促进活性5-氟尿嘧啶和5-氟代-脱氧尿苷-单磷酸盐之间的转换，这一联合处理导致了肿瘤细胞显著凋亡并达到抑制肿瘤增殖的效果。该研究结果也进一步强调了蛋白负载exosome在恶性肿瘤治疗中的潜力。
除此之外，越来越多的证据证明exosome可以在基因治疗中作为新的纳米传递系统以及作为疾病生物标记物用于分子诊断等功能。例如在利用MRI的癌症诊断治疗中，负载超顺磁性氧化铁纳米粒子(SPIONs)的exosome利用局部磁高热达到治疗的目的，显示出较大的应用潜力。
发明内容
为了克服现有技术的缺点与不足，本发明的首要目的在于提供一种抗肿瘤小核酸药物的递送系统。该系统以外泌体(exosome)为载体来介导抑癌miRNA、siRNA到体内肿瘤组织发挥抗肿瘤生物活性作用，是一套全新、高效、低副作用的药物递送系统。
本发明的另一目的在于提供上述抗肿瘤小核酸药物的递送系统的应用。
在细胞学水平和小鼠动物学水平上应用上述抗肿瘤小核酸药物的递送系统，提供抗肿瘤方面的一系列数据信息。
本发明的目的通过下述技术方案实现：
一种抗肿瘤小核酸药物的递送系统，由小核酸药物、包裹小核苷酸药物的脂膜囊状物组成。
作为本发明中运送小核酸药物的脂膜囊状物，既可以是来自细胞内源产生的外泌体或者内囊泡，也可以是外源人工合成的脂质体。
外泌体是本发明主要采用的一种载体，但基于外泌体内源性的直径约为30～120nm的小囊泡，因此有同样类似特征的内源性的内囊泡也可以作为本发明的载体。类似地，本发明的所采用的载体不限于外泌体和内囊泡，包括任何有类似脂膜结构和大小的外源性人工合成的脂质体，如阳离子脂质体、单层脂质体、多层脂质体和多囊脂质体等。
本发明提供的外泌体(exosome)，不限于本发明内容公开的来自于属于免疫细胞的DC细胞，还有T细胞、B细胞等，而且还可以来自间充质干细胞、间质细胞、上皮细胞等多种自体产生或者细胞株产生的外泌体；但不包括来自任何肿瘤细胞的外泌体。
本发明提供的外泌体，当实际应用时，应采用人源的外泌体；但限于小鼠在体动物实验的局限性，本研究的实施例采用的是小鼠来源的外泌体。
本发明所装载的小核酸药物是siRNA、miRNA、piRNA或者人工合成的30nt以内单链及双链RNA分子，不限于本发明提供的siRNA-VEGF和miRNA let-7，也可以是已知的具有明确抑制癌症功能的其他小核酸药物，比如miRNA-16、miRNA-15b、miRNA-20a、miRNA-20b、miRNA-221、miRNA-222、miRNA-486、miRNA-937等。
所述的抗肿瘤小核酸药物的递送系统在制备抗肿瘤药物方面的应用，特别是在制备抑制肿瘤生长和抑制肿瘤转移的治疗药物上的应用。
本发明相对于现有技术，具有如下的优点及效果：
本发明首先提供了在细胞水平采用该递送系统递送抑癌小核酸Let-7抑制细胞增殖和迁移的应用结果，结果说明裸露的let-7不能抑制乳腺癌细胞迁移，而采用本发明提供的递送系统则可将let-7有效的递送到细胞内发挥功能来抑制乳腺癌细胞的增殖和迁移。其次，本发明还提供了小鼠在体水平采用该系统递送Let-7抑制肿瘤增殖的应用，结果说明exosome-let-7与其他对照组相比在小鼠体内可明显抑制乳腺癌肿瘤增殖，可将let-7更有效的递送到肿瘤组织中发挥抑制肿瘤增殖和迁移功能(*P<0.05)。
附图说明
图1是实施例1获得exosome的透射电镜结果；其中，箭头指示的含有多个白色“环装”结构(Scare bare＝100nm)。
图2是实施例2中WST-8分析MDA-MB-231细胞活性。
图3是实施例2中转移小室法分析exosome-let-7对MDA-MB-231细胞迁移作用。
图4是实施例2中小鼠体内分析exosome-let-7对肿瘤的抑制作用。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
以下实施例中除特别说明外，均为本领域内的常规实验方法和操作步骤。
实施例1：Exosome的收集
收集培养小鼠来源的未成熟树突状细胞(immature dendritic cell，iDC)的培养上清，300×g离心10min，收集上清，经过0.22μm滤膜过滤去除细胞碎片和杂质，然后用100kDa超滤管浓缩上清(5000×g，30min)，得到浓缩液，再用PBS稀释到原来的体积，100kDa超滤管5000×g离心30min再次浓缩。接着按照浓缩液：exosome抽提试剂＝2:1的体积比来添加exosome抽提液，充分混匀，4℃孵育过夜。最后12000×g离心2h，去上清，收集exosome，用灭菌后的PBS重悬。用Nandrop 2000分光光度计测试蛋白浓度后，-20℃保存，一个月内使用可在4℃保存。
用约10μg纯化的exosome，加入等体积4％戊二醛，室温固定30min。然后将样品放到超声仪中超声2min，涡旋震荡仪震荡1min。取10μL滴在铜网上待液体风干。然后用2％醋酸双氧铀染色15min，再用滤纸将液体吸干。样品充分晾干后，常温储备以待透射电镜观察。在电镜下观察exosome的大小、形貌，并拍摄具有代表性的电镜照片。
如图1透射电镜结果显示，箭头指示的含有多个白色“环装”结构(Scare bare＝100nm)，这些“环装”结构直径大小在50nm左右，属于exosome粒径范围，说明成功抽取获得exosome。
实施例2：Exosome-Let-7抗肿瘤活性检测
Exosome-Let-7的制备：将150μg exosome与150μg miRNA let-7用OPTI-MEM无血清培养基混匀后加入0.4cm电击杯，冰浴，之后在0.7kV，350微秒、20次脉冲条件下用Eppendorf公司出品的multiporator点击装载。
(1)Exosome-Let-7体外细胞水平抗肿瘤活性检测
将乳腺癌细胞系MDA-MB-231接种于96空板中，每孔细胞约2,000个细胞。待细胞贴壁后加入15μg的exosome-let-7和等量的对照组样品。加10μL WST-8溶液到含有100μL培养液的细胞中，孵育1～4小时。用酶标仪在450nm波长条件下检测吸光度值，并根据吸光值计算细胞活率。结果如图2所示，发现，实验组exosome-let-7与对照组相比可明显抑制MDA-MB-231细胞活性。而裸露的let-7，exosome对MDA-MB-231细胞没有明显作用。结果说明裸露的let-7不能抑制乳腺癌细胞增殖，而exosome可将let-7有效的递送到细胞内发挥功能来抑制乳腺癌细胞增殖(*P<0.05)。
将transwell小室接种乳腺癌细胞MDA-MB-231细胞，每孔5,000个细胞。细胞贴壁后分别加入200μg的exosome-let-7，exosome、let-7和PBS培养48h。结晶紫染色：去掉培养基，PBS洗小室3次，用4％多聚甲醛室温静置固定20min；PBS洗3次，室温晾干；用0.1％结晶紫染色1～2min，PBS洗3次，用棉签轻轻刮掉小室内表面的细胞，室温晾干。显微镜下拍照，比较细胞迁移数目。转移小室法分析结果如图3所示，发现，实验组exosome-let-7与其他对照组相比可明显抑制细胞迁移作用，而裸露的let-7、exosome对MDA-MB-231细胞没有明显作用。结果说明裸露的let-7不能抑制乳腺癌细胞迁移，而exosome可将let-7有效的递送到细胞内发挥功能来抑制乳腺癌细胞迁移。
(2)Exosome-Let-7在体动物水平抗肿瘤活性检测
将传代获得的MDA-MB-231细胞，经过PBS清洗两遍后，用无血清的1640培养基重悬并在显微镜下观察并计数，再以无血清的1640培养液调节细胞浓度到2×107个/mL，并收集在15mL的离心管中备用。然后分别按每只小鼠0.2mL细胞混悬液的量给随机选取的雌性BABL/cL裸鼠注射于右上肢腹股沟处，随后每天观察成瘤情况，并记录，一周左右基本上全部成瘤。
根据瘤体大小，随机将负荷乳腺癌的裸鼠分成4组并编号，每组6只。每组名称分别为：PBS组、裸露let-7组、exosome组、exosome-let-7组。
待小鼠肿瘤增长到0.8cm3体积(体积＝长×宽×宽/2)后，尾静脉给药150μg的exosome-let-7，裸露let-7，exosome以及与实验组等量的PBS。每隔一天尾静脉方法注射一次，注射25天，共注射13次。每次给药前用游标卡尺测量小鼠的肿瘤长和宽来统计各组小鼠肿瘤生长情况。待最后一天将小鼠用脱颈发处死并解刨小鼠肿瘤进行拍照。
应用SPSS17软件进行统计分析，单组数据间使用t检验，多组数据间使用单因素方差分析，P＜0.05为有统计学意义。
结果如图4所示，显示，exosome-let-7与其他对照组相比在小鼠体内可明显抑制乳腺癌肿瘤增殖。这个结果也进一步说明exosome对乳腺癌肿瘤有更强的靶向性，可将let-7更有效的递送到肿瘤组织中发挥抑制肿瘤增殖和迁移功能(*P<0.05)。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (10)
1.一种抗肿瘤小核酸药物的递送系统，其特征在于由小核酸药物、包裹小核苷酸药物的脂膜囊状物组成。
2.根据权利要求1所述的抗肿瘤小核酸药物的递送系统，其特征在于：所述的脂膜囊状物，是来自细胞内源产生的外泌体或者内囊泡，或是外源人工合成的脂质体。
3.根据权利要求2所述的抗肿瘤小核酸药物的递送系统，其特征在于：所述的外源人工合成的脂质体为阳离子脂质体、单层脂质体、多层脂质体或多囊脂质体。
4.根据权利要求2所述的抗肿瘤小核酸药物的递送系统，其特征在于：所述的外泌体来自于免疫细胞、间充质干细胞、间质细胞或上皮细胞，但不包括任何肿瘤细胞。
5.根据权利要求4所述的抗肿瘤小核酸药物的递送系统，其特征在于：所述的免疫细胞为DC细胞、T细胞或B细胞。
6.根据权利要求1所述的抗肿瘤小核酸药物的递送系统，其特征在于：所述的小核酸药物是siRNA、miRNA、piRNA或者人工合成的30nt以内单链及双链RNA分子。
7.根据权利要求6所述的抗肿瘤小核酸药物的递送系统，其特征在于：所述的siRNA为siRNA-VEGF。
8.根据权利要求6所述的抗肿瘤小核酸药物的递送系统，其特征在于：
所述的miRNA为miRNA let-7、miRNA-16、miRNA-15b、miRNA-20a、miRNA-20b、miRNA-221、miRNA-222、miRNA-486或miRNA-937。
9.权利要求1～8任一项所述的抗肿瘤小核酸药物的递送系统在制备抗肿瘤药物方面的应用。
10.根据权利要求9所述的应用，其特征在于：所述的抗肿瘤小核酸药物的递送系统在制备靶向抑制肿瘤生长和抑制肿瘤转移的治疗药物上的应用。
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