CN116036299A - 间皮素特异性外泌体载体、包含其的载药外泌体、其制备方法及其医药用途 - Google Patents
间皮素特异性外泌体载体、包含其的载药外泌体、其制备方法及其医药用途 Download PDFInfo
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Abstract
本发明提供了一种外泌体载体,其包含间皮素(MSLN)特异性嵌合抗原受体,其中,所述嵌合抗原受体包括跨膜部分和展示于外泌体膜表面的抗MSLN单链抗体,所述跨膜部分为人CD63。本发明还提供了包含该外泌体载体的载药外泌体、药物组合物及其医药用途。
Description
技术领域
本发明涉及生物医药领域,具体涉及一种间皮素特异性外泌体载体、包含其的载药外泌体、其制备方法及其医药用途。
背景技术
为解决常规给药药物利用率低的特点,近年来,纳米技术包纳药物的研究十分火热,并在药物缓释及递送方面已取得较好的研究成果。但由于纳米颗粒为外源性物质,具有免疫原性,无法逃避体内巨噬细胞的吞噬作用,因此体内循环稳定性差,利用度低,甚至可能造成机体排异损伤,影响其最终的治疗效果。外泌体最早发现于体外培养的绵羊红细胞上清液中,是细胞主动分泌的大小均一、直径在30-150nm之间的小囊泡。外泌体可以携带多种蛋白质、mRNA、miRNA,参与细胞之间的信号传导与交流。所有人体细胞都能分泌,其可携带不同的蛋白质和核酸,起着细胞间通讯的作用。外泌体作为自身细胞分泌物,生物稳定性好,渗透性强,是一种理想的药物递送载体。故可利用外泌体作为载药***,能最大限度地发挥其治疗作用。
实际上,探讨外泌体作为载体的研究已经越来越多。Dai,S.报道了通过基因工程使人结肠癌细胞LS-174T所分泌的外泌体内含有大量IL-18,可以有效的促进T细胞增殖以及IFN-γ释放,从而达到杀伤抑制肿瘤细胞的目的。当外泌体中高表达miR-122、miR-134、miR146b或是anti-miR-9等时,均可在抑制肿瘤细胞生长、促进肿瘤细胞凋亡或者是增强药物敏感性等方面发挥抗肿瘤的作用。2015年Yang,T报道了当用脑内皮细胞分泌的外泌体包裹化疗药物阿霉素和紫杉醇后,可以使药物通过血脑屏障,该发现对基于外泌体的治疗研究意义重大,提示外泌体作为药物运输载体具有非常大的应用潜能。2016年,Kim,M.S等人发表于nanomedicine上的文章显示应用巨噬细胞细胞系Raw264.7所分泌的外泌体包裹紫杉醇可以有效的杀伤多药耐药的肿瘤细胞,该研究证实了以外泌体作为化疗药物的运输载体的高效性。除此之外,Toffoli的研究证明当用外泌体包裹阿霉素的时候,可以有效的降低阿霉素的心脏毒性。但是单纯使用外泌体作为运药载体并无肿瘤细胞靶向性的作用,因此无法实现药物在肿瘤部位的富集。
卵巢癌是妇科癌症最为常见的一种恶性肿瘤,由于其发病隐匿,临床症状、体征不典型且缺乏有效的早期诊断方法,故80%的卵巢癌患者在确诊时已属晚期,晚期卵巢癌5年生存率不到30%。目前卵巢癌的主要治疗策略以减瘤术联合化疗为主,主要以紫杉醇联合卡铂进行化疗,但是传统化疗药物无法直接靶向肿瘤细胞,只能进行无选择性的细胞杀伤,在杀死肿瘤细胞的同时也会杀死正常的细胞,使身体的免疫力低下。同时化疗治疗的患者绝大部分会出现恶心、呕吐、便秘、腹泻等胃肠道反应症状。因此寻找一种可精准杀伤肿瘤细胞的同时能够降低患者胃肠道反应,提高患者生活质量的治疗方法就显得至关重要。
间皮素(MSLN)是一种在分离单克隆抗体K1(MabK)时发现的细胞表面表达的糖蛋白,其仅在体腔的间皮细胞中表达,在恶性间皮瘤、卵巢恶性肿瘤、胰腺癌中呈现高度表达。MSLN基因是卵巢癌患者体内表达上调最显著的基因之一。目前的研究显示,MSLN可作为卵巢癌的肿瘤标志物及其预后预测指标。
蛇床子素又名甲氧基欧芹酚或欧芹酚甲醚,是从伞形科植物中提取分离的纯天然香豆素类化合物。研究表明蛇床子素具有抗肿瘤、抗骨质疏松、抗肝炎、抗***反应、抑制血小板聚集,以及抗病毒、抗诱变等多种药理活性。
发明内容
本公开提供了一种外泌体载体,其包含间皮素(MSLN)特异性嵌合抗原受体,其中,所述嵌合抗原受体包括跨膜部分和展示于外泌体膜表面的抗MSLN单链抗体,所述跨膜部分为人CD63。
在一些实施方案中,所述人CD63的氨基酸序列如SEQ ID NO:1所示。
MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEFNNNFRQQMENYPKNNHT
在一些实施方案中,所述抗MSLN单链抗体的氨基酸序列如SEQ ID NO:2所示。
QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSGGGGSGGGGSSGGGSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTYGAGTKLEIK
在一些实施方案中,所述嵌合抗原受体包含信号肽和/或连接所述抗MSLN单链抗体和所述跨膜部分的连接肽。
在一些实施方案中,所述嵌合抗原受体的氨基酸序列如SEQ ID NO:3所示。
MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEFNNNFRQQMENYPKNNHTQVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSGGGGSGGGGSSGGGSDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTYGAGTKLEIK
在一些实施方案中,所述嵌合抗原受体由SEQ ID NO:4所示的核酸序列编码。
atggcggtggaaggaggaatgaaatgtgtgaagttcttgctctacgtcctcctgctggccttttgcgcctgtgcagtgggactgattgccgtgggtgtcggggcacagcttgtcctgagtcagaccataatccagggggctacccctggctctctgttgccagtggtcatcatcgcagtgggtgtcttcctcttcctggtggcttttgtgggctgctgcggggcctgcaaggagaactattgtcttatgatcacgtttgccatctttctgtctcttatcatgttggtggaggtggccgcagccattgctggctatgtgtttagagataaggtgatgtcagagtttaataacaacttccggcagcagatggagaattacccgaaaaacaaccacactgccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgggatcccaggtacaactgcagcagtctgggcctgagctggagaagcctggcgcttcagtgaagatatcctgcaaggcttctggttactcattcactggctacaccatgaactgggtgaagcagagccatggaaagagccttgagtggattggacttattactccttacaatggtgcttctagctacaaccagaagttcaggggcaaggccacattaactgtagacaagtcatccagcacagcctacatggacctcctcagtctgacatctgaagactctgcagtctatttctgtgcaagggggggttacgacgggaggggttttgactactggggccaagggaccacggtcaccgtctcctcaggtggaggcggttcaggcggcggtggctctagcggtggcggatcggacatcgagctcactcagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagtgccagctcaagtgtaagttacatgcactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaactggcttctggagtcccaggtcgcttcagtggcagtgggtctggaaactcttactctctcacaatcagcagcgtggaggctgaagatgat
本公开的外泌体载体是间皮素特异性的,对卵巢癌细胞具有靶向性作用,并且可用于装载卵巢癌治疗剂,例如华蟾素、蛇床子素等,从而实现药物在卵巢癌病灶的富集和精准治疗。
本公开还提供了一种载药外泌体,其包含前述外泌体载体和装载于所述外泌体载体内的至少一种药物。
在一些实施方案中,所述药物为抗肿瘤剂。
在一些实施方案中,所述药物为卵巢癌治疗剂。卵巢癌治疗剂的实例包括但不限于:阿霉素、紫杉醇、多西紫杉醇、顺铂、卡铂、奥沙利铂、环磷酰胺、异环磷酰胺、吉西他滨、奥拉帕利、鲁卡帕尼、尼拉帕尼、氟唑帕利、帕米帕利、维利帕利、他拉唑帕利、帕唑帕尼、西地尼布、尼达尼布、舒尼替尼、卡博替尼、索拉非尼、厄洛替尼、布帕尼西、替西罗莫司、哌立福新。
在一些实施方案中,卵巢癌治疗剂是中药提取物,例如华蟾素、蛇床子素等。
本公开还提供了一种负载阿霉素的间皮素特异性外泌体,所述间皮素特异性外泌体如上述外泌体载体所定义。
本公开还提供了一种负载华蟾素的间皮素特异性外泌体,所述间皮素特异性外泌体如上述外泌体载体所定义。
本公开还提供了一种药物组合物,其包含前述载药外泌体,以及药学上可接受的载体或稀释剂。载体或稀释剂的实例包括但不限于水、盐水、林格氏溶液、右旋糖溶液和5%人血清白蛋白。此类介质和化合物用于药物活性物质的用途在本领域中众所周知。除非任何常规介质或化合物与本文所述的外泌体不相容,否则考虑其在组合物中的使用。本公开的药物组合物还可以包含第二治疗剂。通常,将药物组合物配制成可与其预期施用途径相容。
本公开还提供了上述外泌体载体用于装载或递送药物的用途。药物的定义如上所述。
本公开还提供了上述外泌体载体、载药外泌体或药物组合物在制备用于治疗疾病的药物中的用途。
在一些实施方案中,所述疾病为肿瘤或癌症。在一些实施方案中,所述疾病为实体瘤。
在一些实施方案中,所述肿瘤或癌症包括但不限于:卵巢癌、胰腺癌、肺癌、间皮瘤、非小细胞肺癌、胸腺瘤、胃癌、胆管肿瘤、腺癌、输卵管癌、腹膜恶性肿瘤、胰腺腺泡癌、卵巢上皮癌。
在一些实施方案中,所述疾病为卵巢癌。
本公开还提供了一种试剂盒,其包含上述外泌体载体、载药外泌体和药物组合物中的任一种。
本公开还提供了一种制备上述外泌体载体的方法,其包括:
(1)用包含编码间皮素(MSLN)特异性嵌合抗原受体的核酸的表达质粒转染生产细胞;
(2)培养后收集外泌体。
本公开还提供了一种制备上述载药外泌体的方法,其包括:
(1)用包含编码间皮素(MSLN)特异性嵌合抗原受体的核酸的表达质粒转染生产细胞;
(2)培养后分离和富集外泌体;
(3)将步骤(2)收集的外泌体进行药物装载,得到所述载药外泌体。
在一些实施方案中,所述生产细胞包括在合适条件下(例如在悬浮培养或提笔培养或任何其他类型的培养***中)能够产生外泌体的任何类型的细胞。
在一些实施方案中,所述生产细胞可以选自广泛范围的细胞和细胞系,例如基质细胞、成纤维细胞、羊膜细胞、骨髓抑制细胞、脂肪细胞、内皮细胞、成纤维细胞、人胚肾(HEK)细胞、中国仓鼠卵巢(CHO)细胞、红细胞、红系祖细胞、软骨细胞、干细胞、免疫细胞。
在一些实施方案中,所述生产细胞是干细胞,例如胚胎干细胞、体干细胞、间充质干细胞(MSC)、诱导多能干细胞(iPSC)和通过任何方法衍生的其它干细胞。对于待治疗的患者而言,所述细胞本质上可以是同种异体的、自体的、甚至异种的,即源细胞可以来自患者本人或者来自无关的、匹配的或不匹配的供体。
在一些实施方案中,所述生产细胞是免疫细胞,诸如抗原呈递细胞、T细胞、B细胞、自然杀伤细胞(NK细胞)、巨噬细胞、M2极化巨噬细胞、单核细胞、树突细胞、T辅助细胞或调控性T细胞(Treg细胞)。在一些实施方案中,生产细胞不是抗原呈递细胞(例如,树突细胞、巨噬细胞、B细胞、肥大细胞、嗜中性粒细胞、Kupffer-Browicz细胞或源自任何此类细胞的细胞)。
在一些实施方案中,所述生产细胞不是免疫细胞。
在一些实施方案中,外泌体载体的药物装载通过以下方式进行:直接共孵育、超声处理、电穿孔、冻融、挤压或透化作用。
可以通过本领域已知的方法从生产细胞中分离和富集外泌体。在一些实施方案中,外泌体由生产细胞释放至细胞培养基中。预期分离外泌体的所有已知方式均视为适用于本文中。举例而言,可采用外泌体的物理性质来将它们与介质或其它源材料分离,所述分离包括基于电荷的分离(例如电泳分离)、基于尺寸的分离(例如过滤、分子筛分等)、基于密度的分离(例如常规离心或梯度离心)、基于Svedberg常数的分离(例如在有或无外力情况下的沉降等)。可替代地或除此之外,分离可基于一种或多种生物特性,并且包括可采用表面标记物的方法(例如用于沉淀、与固相的可逆结合、FACS分离、特异性配体结合、非特异性配体结合、亲和纯化等)。分离和富集可按通常包括连续离心的一般且非选择性方式进行。可替代地,分离和富集可按诸如使用外泌体或生产细胞特异性表面标记物的更具特异性和选择性的方式进行。举例而言,特异性表面标记物可用于免疫沉淀、FACS分选、亲和纯化和与珠粒结合配体的磁力分离。在一些实施方案中,尺寸排阻色谱法可用于分离外泌体。尺寸排阻色谱技术是本领域已知的。本文提供了示例性非限制性技术。在一些方面中,空隙体积级分经分离并且包含目标外泌体。此外,在一些方面中,可在如本领域中公知的通过离心技术(对一种或多种色谱法级分)进行色谱分离之后进一步分离外泌体。在一些方面中,举例而言,可利用密度梯度离心以进一步分离细胞外囊泡。在某些方面中,进一步分离生产细胞来源的外泌体与其它起源的外泌体可以是合乎需要的。举例而言,可通过使用对生产细胞具有特异性的抗原抗体进行免疫吸附捕获来使生产细胞来源的外泌体与非生产细胞来源的外泌体分离。在一些实施方案中,外泌体的分离可涉及多种方法的组合,所述方法包括但不限于差速离心、基于尺寸的膜过滤、免疫沉淀、FACS分选和磁力分离。
在一些实施方案中,通过将步骤(2)收集的外泌体与药物直接共孵育进行药物装载。
本公开的间皮素特异性外泌体载体及载药外泌体具有更好的靶向性,更低的毒副作用,更高效的抗肿瘤效果,具体地:
(1)本公开的外泌体载体靶向间皮素(MSLN),能特异性识别并结合MSLN阳性肿瘤细胞表面的MSLN,然后内吞入肿瘤细胞,使药物(例如华蟾素、蛇床子素等)在肿瘤细胞内大量富集,可以大大提高药物的有效使用率,起到增加药物抗肿瘤疗效的作用;
(2)显著改善了传统化疗药物由于其非特异性杀伤作用造成的正常组织严重损伤、患者免疫力降低、严重胃肠道反应等副作用;
(3)MSLN基因是卵巢癌患者体内表达上调最显著的基因之一,是一种极好的卵巢癌治疗的靶标,因此本公开的MSLN特异性外泌体载体特别适合在卵巢癌的治疗中用作载药载体;例如本公开的MSLN特异性外泌体载体运载中药提取物(例如华蟾素、蛇床子素等)后在杀死卵巢癌细胞的同时可显著调节患者免疫***,降低肠道反应,提高患者生活质量;因此提供了一种全新的基于精准干预和***调节的中西医结合治疗卵巢癌的新策略;
(4)用本公开的外泌体载体递送的药物具有良好的缓释和长效效果,外泌体的膜结构对药物储存具有保护作用,负载药物的靶向外泌体具有更好的免疫调节作用和更好的肠道微生态调节作用。
附图说明
本发明的上述和其他方面、特征和优点将从以下结合附图的实施例的详细描述中变得显而易见:
图1显示了荧光显微镜检测的抗MSLN外泌体的靶向效率;
图2显示了HEK297T细胞(A)和DC细胞来源(B)的抗MSLN外泌体的流式细胞术检测结果;
图3显示了蛇床子素负载的anti-MSLN外泌体载药效率结果;
图4显示了蛇床子素负载的anti-MSLN外泌体处理SKOV3细胞增殖测定-CCK8的结果;
图5显示了负载蛇床子素的anti-MSLN外泌体处理SKOV3细胞增殖测定显微镜检结果。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
如本文使用的术语“嵌合抗原受体”或“CAR”指的是被工程化以在外泌体上表达并特异性地结合抗原的工程化外泌体。CAR还可以包括细胞外结构域跨膜结构域和任选的细胞内活化结构域,该细胞外结构域包括肿瘤相关抗原结合区。在一些方面,CAR包括融合单链可变片段(scFv)衍生的单克隆抗体,其被融合至外泌体特异性的CD63跨膜结构域。CAR设计的特异性可以源自受体的配体(例如,肽)。在一些实施方式中,通过重定向表达特异于肿瘤相关抗原的CAR的外泌体的特异性,例如CAR可以靶向MSLN阳性癌症。
如本文使用的术语“转染”指的是如下过程:通过该过程外源性核酸被转移或引入宿主细胞。“转染”的细胞是已经用外源性核酸转染的细胞。细胞包括原代对象细胞和其子代。
下面通过具体实施例的对比对本发明方法做更加详细的说明,以便于本领域技术人员的理解。如无特殊说明,本发明所用的试剂和仪器为本领域的市售产品,本发明采用的实验方法为本领域常规方法。
实施例1 anti-MSLN-外泌体载体的制备与鉴定
按照以下步骤制备anti-MSLN-外泌体载体:
1、细胞准备:将HEK293T细胞铺六孔板,每孔8.5×105个细胞,于37℃、5%CO2细胞培养箱中培养,次日进行质粒转染;
2、质粒准备:质粒终浓度(MSLN-scFv质粒:210ng/μl;空载体:206ng/μl);载体为pIRES2-EGFP,目的基因序列如下:
atggcggtggaaggaggaatgaaatgtgtgaagttcttgctctacgtcctcctgctggccttttgcgcctgtgcagtgggactgattgccgtgggtgtcggggcacagcttgtcctgagtcagaccataatccagggggctacccctggctctctgttgccagtggtcatcatcgcagtgggtgtcttcctcttcctggtggcttttgtgggctgctgcggggcctgcaaggagaactattgtcttatgatcacgtttgccatctttctgtctcttatcatgttggtggaggtggccgcagccattgctggctatgtgtttagagataaggtgatgtcagagtttaataacaacttccggcagcagatggagaattacccgaaaaacaaccacactgccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgggatcccaggtacaactgcagcagtctgggcctgagctggagaagcctggcgcttcagtgaagatatcctgcaaggcttctggttactcattcactggctacaccatgaactgggtgaagcagagccatggaaagagccttgagtggattggacttattactccttacaatggtgcttctagctacaaccagaagttcaggggcaaggccacattaactgtagacaagtcatccagcacagcctacatggacctcctcagtctgacatctgaagactctgcagtctatttctgtgcaagggggggttacgacgggaggggttttgactactggggccaagggaccacggtcaccgtctcctcaggtggaggcggttcaggcggcggtggctctagcggtggcggatcggacatcgagctcactcagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagtgccagctcaagtgtaagttacatgcactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaactggcttctggagtcccaggtcgcttcagtggcagtgggtctggaaactcttactctctcacaatcagcagcgtggaggctgaagatgat(SEQ ID NO:4)
3、质粒转染:
1)将六孔板中的培养基弃去,更换为Opti-MemTM培养基,每孔1.5ml,然后放入37℃、5%CO2细胞培养箱;
2)配制B液:MSLN-scFv质粒(10μl Lipo 2000+250ml Opti-MemTM)*3;空载体(10μlLipo 2000+250ml Opti-MemTM)*3
用枪头轻轻缓慢吹吸混匀,在室温下孵育5min;
3)配制A液:MSLN-scFv质粒(10μl MSLN-scFv质粒+250ml Opti-MemTM)*3;空载体(10μl空载体+250ml Opti-MemTM)*3
将孵育完成的B液分别加入至A液中,用枪头轻轻缓慢吹吸混匀,然后在室温下孵育20min,混合液体分别记作a、b;
4)取出六孔板,分别加入500μl步骤3)中的a或b液体,各3个孔,轻晃,然后放入37℃、5%CO2细胞培养箱孵育5小时;
5)5小时后,将六孔板中的培养基弃去,更换为含10%FBS、1%链霉青霉素的DMEM(GibcoTM)培养基,每孔2ml;
4、次日将培养基弃去,更换为DMEM(GibcoTM)培养基,每孔2ml;
5、三日后收集上清提取外泌体;
6、提取外泌体:取出六孔板,收集细胞上清(各约5ml),离心(2000g,4℃,30min),取上清(各4.5ml),加入上清1/2体积(2.25ml)的Total Exosome Isolation Reagent(Thermofisher/4478359),涡旋混匀,冰箱4℃过夜,次日取出,摇晃混匀,分装至1.5ml EP管中,离心(10000g,4℃,1h),彻底弃去上清,最后各用100μl PBS重悬,使得MSLN-scFv外泌体终浓度为75.9ng/μl,空载体外泌体终浓度为78.9ng/μl;
7、蛋白测定(BCA):将步骤6所得外泌体重悬液稀释10倍(MSLN-scFv外泌体:7.59ng/μl,空载体外泌体:7.89ng/μl);
8、单链抗体鉴定
1)向100μl MSLN-scFv外泌体和100μl空载体外泌体中各加入1μl DiL荧光染料(1mM),吹吸混匀;
2)于水浴锅(37℃,摇晃,避光)中孵育1小时;
3)用PBS将体积增至1ml;
4)按脱盐柱说明(thermofisher89892)处理步骤3)中的液体;
5)用过滤器(0.45μm)过滤:采用1ml注射器操作,各剩约700μl;
6)取出于前一天以每孔3×105个细胞铺板SKOV3细胞、细胞密度达80%左右的六孔板,将培养基换为每孔1ml,并且每孔各加入100μl步骤5)中所得的溶液,分三组——PBS组、MSLN-scFv组和空载体组,每组重复两个孔,轻晃混匀;
7)放入37℃、5%CO2细胞培养箱孵育过夜;
8)次日取出,弃去上清,用1ml PBS清洗三次;
9)各孔再加入1mL含10%FBS、1%链霉-青霉素的Gibco RPMI 1640培养基);
于倒置荧光显微镜下观察,结果见图1。抗MSLN外泌体和非特异性外泌体经膜染料标记后与MSLN+细胞系孵育,荧光显微镜检测靶向效率,证实靶向效率是由于MSLN-scFv与MSLN抗原的特异性结合。
本实施例还采用上述相同的方法制备了树突状细胞(DC)来源的MSLN特异性外泌体载体。
将所制备的两类外泌体载体与相应的外泌体特异性磁珠(Thermo Fisher:106-06D)共孵育,然后加入MSLN抗原((Sino Biological13128-H08H-50))孵育一段时间后,再加入anti-MSLN-PE孵育,再应用流式细胞仪进行检测,验证外泌体上的单链抗体特异性。流式细胞术检查结果显示上述制备方法可有效地将单链抗体表达于外泌体表面见图2中的A和2中的B。
实施例2负载蛇床子素的anti-MSLN-外泌体的制备
1)载药:anti-MSLN-外泌体载体和非特异性外泌体载体质量分别为7.5μg(100μl),蛇床子素195.2μg(5μl),吹吸混匀;载药方式:37℃水浴锅中共孵育4小时;透析:用注射器吸取载药后的液体,注射到含1×PBS透析卡中,4℃透析过夜;吸出透析卡中的液体,各约800μl;用0.45μm滤器过滤,各剩约650μl。
2)载药效率测定——HPLC
液相条件:色谱柱XdbC8,流动相为乙腈:水=7:3,检测波长303nm,流速1ml/min,进样量10μl,停止时间5min;
标准品配制:蛇床子素储液浓度160mM,用PBS将其稀释为五个浓度,分别为0.390864μg/ml,0.781728μg/ml,1.95432μg/ml,3.90864μg/ml,7.81728μg/ml,每个浓度1ml,标记为a、b、c、d、e;
样品准备:将4℃透析过夜后的蛇床子素负载外泌体,用5ml注射器从透析卡中吸出,四种载药条件下,共孵育2小时(co-2)、共孵育4小时(co-4)、电穿孔后恢复2小时(e-2)、电穿孔后恢复4小时(e-4),各液体体积为4.5ml、4.5ml、1ml、1ml,各取500μl移至新的1.5mlEP管中,分别标记为co-2、co-4、e-2、e-4,离心(4℃,7000rpm)5min,各取上清200μl至新的1.5ml EP管中;
进样序列:各取50μl放入进样瓶中,进样序列为PBS、a、b、c、d、e、co-2、co-4、e-2、e-4;
蛇床子素出峰时间为3min左右。
载药效率测定结果见表1和图3。
表1:各载药条件下的载药效率
实施例3负载蛇床子素的anti-MSLN外泌体对SKOV3细胞的增殖作用实验
1、细胞准备:将SKOV3以每孔约3×105个细胞铺六孔板,完全培养基(含10%FBS和1%青链霉素的1640培养基)培养,每孔2ml,次日加药;
2、加药:取出六孔板,弃去上清,用1ml PBS清洗3次,再每孔加入1ml完全培养基,各加入300μl PBS、300μl负载蛇床子素的anti-MSLN外泌体、300μl负载蛇床子素的非特异性外泌体,重复两个孔,轻晃,放入孵箱孵育48小时和72小时;
3、加CCK:48小时后,弃去上清,用1ml PBS清洗2次,再加1ml完全培养基,然后各孔加入100μl CCK溶液,轻晃混匀,放入孵箱孵育2小时;
4、放入孵箱孵育2小时后,将上清转移至96孔板中,且6孔板每孔移至96孔板6个孔,每孔100μl,然后弃去余下上清,用1ml PBS清洗2次,再每孔加入1ml完全培养基,继续培养24小时;
5、酶标仪测量:96孔板用酶标仪测量,单波长,450nm;
6、将步骤5中的六孔板取出,每孔加100μl CCK溶液,轻晃混匀,放入孵箱孵育2小时;
7、重复步骤4、5。
检测结果:48小时和72小时CCK试验结果见图4,24小时、48小时、72小时的荧光显微镜镜检细胞存活状态的结果见图5,均显示出负载蛇床子素的anti-MSLN外泌体对SKOV3细胞(MSLN+)有毒性,而且这种作用随着孵育时间的增加而增加。这些结果表明,负载蛇床子素的anti-MSLN外泌体应具有低副作用(相同毒性更小剂量)、缓慢释放、长作用时间和逐渐增强的毒性。
Claims (10)
1.一种外泌体载体,其包含间皮素(MSLN)特异性嵌合抗原受体,其中,所述嵌合抗原受体包括跨膜部分和展示于外泌体膜表面的抗MSLN单链抗体,所述跨膜部分为人CD63。
2.根据权利要求1所述的外泌体载体,其中,所述人CD63的氨基酸序列如SEQ ID NO:1所示。
3.根据权利要求1或2所述的外泌体载体,其中,所述抗MSLN单链抗体的氨基酸序列如SEQ ID NO:2所示。
4.根据权利要求3所述的外泌体载体,所述嵌合抗原受体还包含信号肽和/或连接所述抗MSLN单链抗体和所述跨膜部分的连接肽。
5.根据权利要求4所述的外泌体载体,其中,所述嵌合抗原受体的氨基酸序列如SEQ IDNO:3所示;优选的,所述嵌合抗原受体由SEQ ID NO:4所示的核酸序列编码。
6.一种载药外泌体,其包含权利要求1至5中任一项所述的外泌体载体和装载于所述外泌体载体内的至少一种药物,所述药物优选为抗肿瘤剂,更优选为卵巢癌治疗剂,最优选为蛇床子素。
7.一种药物组合物,其包含根据权利要求6所述的载药外泌体,以及药学上可接受的载体或稀释剂。
8.根据权利要求1至5中任一项所述的外泌体载体用于装载或递送药物的用途,所述药物优选为抗肿瘤剂,更优选为卵巢癌治疗剂,例如蛇床子素。
9.根据权利要求1至5中任一项所述的外泌体载体、权利要求6所述的载药外泌体或权利要求7所述的药物组合物在制备用于治疗疾病的药物中的用途,所述疾病优选为肿瘤或癌症,更优选为卵巢癌。
10.一种制备根据权利要求6所述的载药外泌体的方法,其包括以下步骤:
(1)用包含编码间皮素(MSLN)特异性嵌合抗原受体的核酸的表达质粒转染生产细胞;
(2)培养步骤(1)转染的生产细胞后,分离和富集外泌体;
(3)将步骤(2)所得的外泌体进行药物装载,得到所述载药外泌体;
所述生产细胞优选选自基质细胞、成纤维细胞、羊膜细胞、骨髓抑制细胞、脂肪细胞、内皮细胞、成纤维细胞、人胚肾(HEK)细胞、中国仓鼠卵巢(CHO)细胞、红细胞、红系祖细胞、软骨细胞、干细胞和免疫细胞,更优选为HEK293T细胞。
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