CN111840513A - 一种负载促肿瘤凋亡蛋白和抗癌小分子的复合外泌体及其制备方法和应用 - Google Patents
一种负载促肿瘤凋亡蛋白和抗癌小分子的复合外泌体及其制备方法和应用 Download PDFInfo
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Abstract
本发明属纳米材料与肿瘤学技术领域,公开了一种负载促肿瘤凋亡蛋白和抗癌小分子的复合外泌体及其制备方法和应用,复合外泌体为EV‑T&抗癌小分子,其表面携带促肿瘤凋亡蛋白TRAIL,内部包裹抗癌小分子;先以表达人TRAIL基因的病毒载体转染人的外泌体的细胞资源,获得稳定高表达TRAIL的基因工程化间充质干细胞,标记为MSC‑flT;然后从MSC‑flT培养液中提取细胞分泌的外泌体,得到高纯度表达携带TRAIL的外泌体,标记为EV‑T;再将抗癌小分子包裹进EV‑T中制得。本发明的制备工艺简单、高效、成本低;复合外泌体用于抗肿瘤治疗效果显著,药物用量低且无毒副作用,为临床治疗癌症提供了纳米药物策略。
Description
技术领域
本发明属纳米材料与肿瘤学技术领域,更具体地,涉及一种负载促肿瘤凋亡蛋白和抗癌小分子的复合外泌体及其制备方法和应用。
背景技术
众所周知,癌症已成为人类健康的最大威胁之一。尽管人们对癌症研究已取得了许多重大突破,但是绝大多数癌症的临床治疗效果仍然不理想。因此,亟需新的有效且安全的治疗方法。
肿瘤坏死因子相关的凋亡诱导配体TRAIL(Tumour Necrosis Factor-relatedApoptosis-Inducing Ligand),是具有凋亡诱导活性的肿瘤坏死因子超家族新成员,能够快速诱导癌细胞大量凋亡,而对正常细胞无影响,在癌症治疗领域具有广泛的应用前景。TRAIL与细胞膜表面的死亡受体4(DR4)和5(DR5)结合可征募胞浆内接头蛋白Fas死亡结构域相关蛋白(FADD)和半胱天冬酶原8(Procaspase-8),形成一个死亡诱导信号复合体DISC(Death-inducing signaling complex),从而引发Procaspase-8激活进而诱导Procaspase3激活,然后引起细胞凋亡。文献表明,肿瘤细胞相对于正常细胞而言,其DR4/DR5表达水平较高,而正常组织除了DR4/DR5表达水平很低或不表达之外,往往还表达诱骗受体DcR1/2,从而躲避TRAIL对其产生的毒副作用。TRAIL高效特异性杀死癌细胞的特点吸引了人们广泛开展其临床应用研究。然而,迄今为止,重组可溶型TRAIL(rTRAIL)的临床试验效果并不理想。主要原因在于广泛存在肿瘤和癌症对rTRAIL的抗性。此外,rTRAIL在体内不稳定,半衰期只有30分钟,生物利用度低。这些缺点导致其临床效应不好,极大地限制了其临床应用。
间充质干细胞(MSC)是一类具有自我复制能力的多潜能成体干细胞,在体内或体外特定的诱导条件下,可分化为脂肪、骨、软骨、肌肉、神经等多种组织细胞。此外,干细胞还具有肿瘤归巢的靶向迁移能力。近年来,随着对间充质干细胞研究与应用的深入,已发现其大量分泌细胞外囊泡(外泌体和微囊泡),而外泌体在多种疾病模型中具有可与细胞匹配的活性和治疗潜力,并且可用作一种天然的生物纳米载体,具有细胞所没有的独特优势。包括;1)可携带膜结合的全长TRAIL(Full length TRAIL,FlT),这是已经发现的优于rTRAIL的活性更高的分子;2)免疫原性无或极低,进入体内后不会引起免疫反应;3)具有双层磷脂膜结构,可以保护所携带的分子,避免血液循环系统中酶的降解;4)30~200nm的纳米尺度,赋予其很好的组织渗透性,比如良好的透血脑屏障能力;5)良好的稳定性,可在-20℃或-80℃长期贮存;6)多重载药能力,可同时载送亲脂和亲水药物;7)一定的肿瘤靶向性,可提高药物在肿瘤部位的富集浓度。因此,用外泌体载药既能够克服rTRAIL体内不稳定性,又能增强其作用效率,还可通过联用致敏药物解决TRAIL的耐药性问题,有望创造一种高效且安全的新型生物纳米药物。
前期已发现细胞周期蛋白依赖性激酶(CDK)抑制剂具有致敏癌细胞TRAIL应答的效应,因此可联用CDK抑制剂与TRAIL来增强治疗效果。CDK在细胞周期和基因转录调控中发挥重要作用,作为癌症和其它疾病的重要靶点受到越来越多的关注。Dinaciclib(SCH727965)(Dina)是一种新型的纳摩尔浓度的CDK抑制剂,能够高效特异性地抑制CDK1、CDK2、CDK5和CDK9活性,IC50分别是3,1,1,4nmol/L;Dina通过调控细胞周期停滞以及短寿命抗凋亡蛋白的转录,能够广泛抑制肿瘤细胞增殖以及诱导肿瘤细胞凋亡。Dinaciclib表现出对慢性白血病较好的治疗效果,已进入临床Ⅲ期治疗研究。
外泌体同时递送TRAIL和抗癌小分子(如Dina)既能够克服重组TRAIL的耐药性,体内不稳定性和生物利用率低的问题,同时通过两者联用产生协同效应,可大幅度降低药物使用浓度,增强癌细胞杀死效率和特异性,降低抗癌小分子的毒副作用,因而具有极大的临床应用潜力。目前尚无同时负载促肿瘤凋亡蛋白TRAIL和抗癌小分子(如Dina)的外泌体的报道。
发明内容
为了解决上述现有促凋亡蛋白TRAIL用于癌症治疗技术存在的不足和缺点,本发明提供一种负载促肿瘤凋亡蛋白和抗癌小分子的复合外泌体。该复合外泌体为双重载药外泌体,其所携带的抗癌化疗小分子药物能够协同增强促凋亡蛋白对肿瘤细胞的杀伤而对正常组织无显著毒性,为以后癌症的临床治疗提供可靠的实验数据及理论基础。
本发明的另一目的在于提供上述负载促肿瘤凋亡蛋白和抗癌小分子的复合外泌体的制备方法。
本发明的再一目的是提供上述负载促肿瘤凋亡蛋白和抗癌小分子的复合外泌体的应用。
本发明的目的通过下述技术方案来实现:
一种负载促肿瘤凋亡蛋白和抗癌小分子的复合外泌体,所述复合外泌体为EV-T&抗癌小分子,其表面携带促肿瘤凋亡蛋白TRAIL,内部包裹抗癌小分子;该复合外泌体是先以表达人TRAIL基因的病毒载体转染人的外泌体的细胞资源,获得稳定高表达TRAIL的基因工程化间充质干细胞,标记为MSC-flT;然后采用外泌体制备金标准-差速结合超速离心法,从MSC-flT培养液中提取细胞分泌的外泌体,得到高纯度表达携带TRAIL的外泌体,标记为EV-T;再将抗癌小分子包裹进EV-T中制得。
优选地,所述复合外泌体中促肿瘤凋亡蛋白TRAIL的浓度为0.5~4ng/mL;所述抗癌小分子的浓度为10~80nM;所述复合外泌体储存在-70~-85℃中。
优选地,所述人的外泌体的细胞资源为脐带间充质干细胞、骨髓间充质干细胞、脂肪间充质干细胞、牙髓间充质干细胞、胎盘、羊水、羊膜间充质干细胞、人胚肾细胞、心肌细胞、巨噬细胞、T细胞或肿瘤相关成纤维细胞中的一种以上。
优选地,所述抗癌小分子为化疗药物、天然活性分子、免疫治疗药物或核酸类药物中的一种以上。
更为优选地,所述的化学合成药物为Dinaciclib,SNS-032、Flavopiridol或阿霉素中的一种以上;所述的免疫治疗药物为DR4/5单克隆激动抗体或/和PD-1/PD-L1单克隆抗体;所述的核酸类药物为mRNA、siRNA、shRNA、lncRNA或micro RNA;所述天然活性分子为番茄红素或/和紫杉醇。
优选地,所述的病毒载体为慢病毒、逆转录病毒或腺病毒。
优选地,所述EV-T的粒径为30~200nm。
所述的负载促肿瘤凋亡蛋白和抗癌小分子的复合外泌体的制备方法,具体包括如下具体步骤:
S1.以表达人TRAIL基因的病毒载体转染人的外泌体的细胞资源,获得稳定高表达TRAIL的基因工程化间充质干细胞,标记为MSC-flT;
S2.采用外泌体制备金标准-差速结合超速离心法,从MSC-flT培养液中提取细胞分泌的外泌体,得到高纯度表达携带TRAIL的外泌体,标记为EV-T;
S3.将抗癌小分子包裹进EV-T中,得到载有促肿瘤凋亡蛋白和抗癌小分子的复合外泌体,即EV-T&抗癌小分子;所述复合外泌体的表面携带促肿瘤凋亡蛋白TRAIL,内部包裹抗癌小分子。
优选地,步骤S3中所述包裹的载药方法为超声法、膜穿孔法、电穿孔法或共孵育法。
所述的负载促肿瘤凋亡蛋白和抗癌小分子的复合外泌体在制备抗肿瘤治疗药物中的应用。优选地,所述的肿瘤为包括但不限于在人肺癌、胸膜间皮瘤、肾癌、神经胶质瘤、乳腺癌、肝癌、胃癌、结直肠癌、宫颈癌或黑色素瘤中对TRAIL敏感和高抗性的细胞。
与现有技术相比,本发明具有以下有益效果:
1.与现有的单纯负载TRAIL外泌体或单纯不经过纳米载体递送抗癌小分子(如Dina)治疗相比,本发明用外泌体同时递送TRAIL和抗癌小分子,一方面增强了TRAIL和抗癌小分子在体内的半衰期,提高了生物利用度,同时也大大降低了抗癌小分子的使用浓度,拓宽了其治疗窗口,可以大幅度降低其对正常组织的毒副作用。
2.本发明抗癌小分子联用的协同致敏效应显著增强了TRAIL对肿瘤细胞杀伤效果,尤其是对耐药性肿瘤细胞的杀死效果被急剧增强,联合用药后短时间内即杀死80%以上肿瘤细胞。肿瘤动物模型结果表明,EV-T&抗癌小分子联合治疗能够彻底清除体内肿瘤,同时对机体没有不利影响。
3.本发明中抗癌小分子(如Dina)能够通过重编程显著致敏高耐药性肿瘤细胞对TRAIL的应答;体内外实验表明此EV-T&抗癌小分子能够显著抑制肿瘤细胞增殖并强烈诱导耐药性肿瘤细胞凋亡。更重要的是,EV-T&抗癌小分子的体内活性高且无毒副作用。瘤内注射EV-T&抗癌小分子后,裸鼠皮下异种移植瘤完全消失,且未观察到对正常组织的任何细胞毒性。抗癌小分子协同增强促凋亡蛋白对肿瘤细胞杀伤而对正常组织无细胞毒性。
4.本发明阐述了EV-T&抗癌小分子(如Dina)高效杀死耐药性肿瘤细胞的分子机制,为其未来的临床应用铺垫了坚实的理论基础。
附图说明
图1为实施例1制得的表达TRAIL干细胞的共聚焦显微镜图(a)和流式分析图(b);
图2为实施例2制得的EV-T的粒径分布、形貌和蛋白免疫印迹(WB);
图3为Dina标准曲线图;
图4为实施例3制得的EV-T&Dina对对肿瘤细胞和正常细胞增殖抑制活性检测;
图5为实施例3制得的EV-T&Dina体内抑瘤生长曲线;
图6为实施例3制得的EV-T&Dina治疗肿瘤后,对肿瘤组织进行免疫组化分析;
图7为实施例3制得的EV-T&Dina治疗肿瘤后,对心、肝、脾、肺、肾、小肠HE肠染色分析。
图8为实施例3制得的EV-T&Dina抗肿瘤治疗的分子机制示意图。
具体实施方式
下面结合具体实施例进一步说明本发明的内容,但不应理解为对本发明的限制。若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
实施例1表达TRAIL干细胞的制备
1.制备:5-50μL(最优为30μL)包含TRAIL基因的慢病毒与5-20μg/mL(最优为8μg/mL)聚凝胺(polybrene)混匀后加入到1×106UC-MSCs中,37℃,5%CO2细胞培养箱中孵育6-24h(最优为10h),用新鲜的含有10%FBS(胎牛血清)DMEM/F12培养基取代旧的培养基继续培养2天。待细胞长满后,按照细胞接种成3瓶或4瓶为1:(3~4)进行细胞传代,得到表达TRAIL的干细胞(MSC-flT)。
2.表征:将1×106所得MSC-flT接种于共聚焦培养皿或6孔板中,待其贴壁生长12h后,PBS洗涤2次,4%多聚甲醛室温固定15min后,用含0.1%tween-20PBS溶液破膜10min,以PE偶联鼠来源的抗TRAIL抗体在常温下,避光染色30min,经PBS洗涤细胞3次,每次5min,清洗掉没有结合到细胞膜上的游离的抗体。以异硫氰酸酯(FICT)偶联的鬼笔环肽染色细胞骨架10min,PBS洗涤细胞3次,共聚焦显微镜下观察TRAIL表达。用于流式细胞仪检测的细胞处理如下:以PBS洗涤6孔板培养的细胞2次后,胰酶消化洗脱细胞,1000rpm/min离心收集细胞,PBS重悬后,加入PE偶联鼠来源的抗TRAIL抗体,常温避光染色30min,1000r/min离心,弃上清,PBS洗涤,离心,PBS重悬后以流式细胞仪检测TRAIL表达水平。
图1为实施例1制得的表达TRAIL干细胞的共聚焦显微镜图(a)和流式分析图(b)。从图1中可知,所制备的慢性毒高效率转染293T细胞后,流式细胞仪对TRAIL表达进行检测,所转染细胞TRAIL阳性率高达95%,进一步的免疫荧光染色肯定了TRAIL在293T中的高表达。
实施例2 EV-T的制备
1.制备:外泌体提取、纯化:收集MSC-flT细胞上清液500mL,按如下步骤进行差速和超速离心:300g×10min→2000g×10min→0.22μm亲水性滤膜过滤,除去大的细胞外囊泡→100KD超滤离心管浓缩5×→100000g×70min;终沉淀物用PBS轻柔洗涤两次后,加入5mLPBS重悬,制得EV-T,然后用0.22μm亲水性滤膜过滤除菌后分装于-80℃保存备用。
2.EV-T的表征:取10μL EV-T用PBS稀释后,滴加到镍网吸附10min,未吸附到镍网上的EV-T用PBS清洗;染后加入4%多聚甲醛常温固定10min,0.3%醋酸铀负染后自然晾干,以透射电子显微镜观察EV-T粒径分布和形貌;用于纳米激光粒度检测的样品处理如下:取10μL EV-T用PBS稀释,以福流生物公司纳米流式仪nanoFCM对其粒径分布进行检测。
图2为实施例2制得的EV-T的粒径分布、形貌和蛋白免疫印迹(WB)。其中,(a)TEM形貌图,(b)粒径分布图,(c)Elisa法对TRAIL定量检测,(d)蛋白免疫印迹。从图2中可知,超速离心分离得到的细胞外囊泡呈典型的双层囊泡结构,平均粒径为75nm,符合2018年国际细胞外囊泡所界定的小细胞外囊泡范畴;ELISA结合蛋白免疫印迹对细胞外囊泡携带的TRAIL以及外泌体四跨膜蛋白标志物CD63进行了检测,同样可以发现,制备得到的细胞外囊泡不仅可以用于TRAI递送,且绝大部分是以外泌体形式存在。
实施例3 EV-T&Dina的制备
1.制备:取300μL蛋白浓度为1mg/mL的EV-T加入1-10μL 10nmol/L的Dina,混匀后,将含有EV-T和Dina溶液超声处理30s后,停30s,继续超声30s停30s处理,总共循环6次。超声处理结束后,将含有EV-T和Dina混合液置于37℃,5%CO2培养箱中修复外泌体膜1h;向混合液中加入100μL外泌体沉淀试剂聚乙二醇溶液,冰浴下沉淀1h,12000g/15min沉淀外泌体后,PBS洗涤2次,加入300μL PBS重悬,制得EV-T&Dina,-80℃备用。
2.测试:先准确配制0.125,0.25,0.5,1.0,2.0,4.0,8.0μg/mL Dina标准液,以紫外分光光度计在255nm处检测对应浓度吸光值;再取10μL EV-T&Dina加入490μLPBS稀释后,紫外分光光度计检测其OD值,根据Dina标准曲线(图3)即可计算EV-T&Dina中Dina浓度。
每孔1×104接种神经胶质瘤细胞系Tet21/N,SH-SY5Y,肺癌细胞系A549,H727细胞,乳腺癌细胞系M231,宫颈癌细胞系Hela,人肾癌细胞系RCC10,人永生化角质形成细胞HaCaT和每孔0.5×104接种间充质干细胞MSC于96孔板中,37℃,5%CO2培养箱孵育12h后,换上新鲜的含有0.5,1.0,2.0ng/mL的EV-T,;10,15,20,40nM Dina以及EV-T&Dina,作用细胞24h,随后以CCK-8检测细胞存活率。
图4为实施例3制得的EV-T&Dina对对肿瘤细胞和正常细胞增殖抑制活性检测。其中,A-I分别表示单独的EV-T、Dina以及联合联合对Tet21/N、H727、A549、Hela、M231、RCC10、SH-SY5Y、HaCaT和MSC细胞增殖抑制活性检测。从图4中可知,Dina联用EV-T后能够显著增强EV-T对肿瘤细胞杀伤(除了SH-SY5Y肿瘤细胞外),而这一联合对正常细胞无显著细胞毒性。
实施例4 EV-T&Dina体内抑瘤实验
1.A549异种移植瘤皮下模型建立:将购于北京斯贝福3~5周龄雌性Balb/c裸鼠饲养于SPF级别的动物培养房中,以1×107个A549细胞接种于裸鼠右前侧,建立皮下实体瘤模型,待其肿瘤长至100mm3大小,分为4组,每组5只,分别为对照组,EV-T组,Dina组和EV-T&Dina组。
2.瘤内注射:将100μL分别含有50μg EV-T(4.5ng TRAIL),160μg Dina,同时含有50μg EV-T和160μg Dina以及100μLPBS作为对照,用胰岛素针注射到肿瘤内部,每48h注射一次,共注射3次;注射结束后,每两天对肿瘤体积及老鼠体重进行测量并记录,待治疗结束后继续饲养观察1个月,最后处死裸鼠,取出肿瘤对其体积和大小进行测量,并绘制肿瘤抑制生长曲线图。图5为实施例3制得的EV-T&Dina体内抑瘤生长曲线;从图5中可知,低浓度EV-T作用后,对肿瘤生长几乎无任何影响;而单独的Dina作用后,也仅是抑制了肿瘤生长;Dina联用EV-T即为EV-T&Dina(也表示为Combi)后,肿瘤完全消失。
实施例5免疫组化
1.组织切片及封闭:在最后一次药物(50μg EV-T(4.5ng TRAIL),160μg Dina,同时含有50μg EV-T和160μg Dina以及100μLPBS)注射24h后,处死小鼠,取出小鼠的肿瘤组织和其它器官(心、肝、脾、肺、肾、小肠)并做好标记。用石蜡包埋后切成3~5μm厚连续切片,对组织切片进行脱蜡、水化处理。组织切片置于盛满柠檬酸抗原修复缓冲液(pH=6)的修复盒中于微波炉内进行抗原修复,中火8min至沸,停火8min保温再转中低火7min,此过程中应防止缓冲液过度蒸发,切勿干片。自然冷却后将玻片置于PBS(pH=7.4)中在脱色摇床上晃动洗涤3次,每次5min,用3%的过氧化氢在室温封闭20min,以封闭内源性过氧化物酶;加入3%BSA均匀覆盖组织,室温封闭30min;
2.抗体染色:轻轻甩掉封闭液后,在切片上滴加以PBS按一定比例配好的一抗染液(抗Ki-67,抗cleaved caspase-3),切片平放于湿盒内,4℃孵育过夜。(湿盒内加少量水,防止抗体蒸发),玻片置于PBS(pH=7.4)中在脱色摇床上晃动洗涤3次,每次5min。切片稍甩干后在圈内滴加与一抗相应种属匹配的二抗(HRP标记)染液覆盖组织,室温孵育50min。
3.Tunel染色:按片子数量和组织大小取tunel试剂盒内适量试剂1(TdT)和试剂2(dUTP)按体积比1:9混合,加到圈内覆盖组织,切片平放于湿盒内,37℃水浴锅孵育2h,湿盒内加少量水保持湿度。
4.DAB显色:玻片置于PBS(pH=7.4)中,在脱色摇床上晃动洗涤3次,每次5min。切片稍甩干后在圈内滴加新鲜配制的DAB显色液,显微镜下控制显色时间,阳性为棕黄色。以自来水冲洗切片来终止显色。
5.复染细胞核:苏木素复染3min左右,以自来水洗;加苏木素分化液分化数秒,自来水冲洗;加苏木素返蓝液返蓝,流水冲洗。对组织进行脱水处理后,以树胶封片。采用Leica公司的Pathologic microtome进行图片和数据采集。
图6为实施例3制得的EV-T&Dina治疗肿瘤后,对肿瘤组织进行免疫组化分析;从图6中可知,H&E染色结果显示,Dina、EV-T联合Dina作用肿瘤后,肿瘤细胞核发生皱缩,细胞核数量相对于对照组和EV-T作用组明显减少,表明肿瘤细胞发生凋亡;单独Dina作用后,cleaved caspase-3相对于对照组表达水平显著上升,联合EV-T后,cleaved caspase-3表达水平进一步上升。cleaved caspase-3上调提示肿瘤细胞发生明显凋亡,与H&E染色结果相一致;Tunel染色结果进一步验证了这一结论。Ki-67是衡量细胞增殖快慢的一个特异性指标,其表达量越高,表明肿瘤细胞增殖越快,其恶性程度越高。Dina组作用肿瘤后,Ki-67相对于对照组和EV-T组,表达量显著降低,联合EV-T后,表达水平进一步下降,这也就很好的解释了Dina组对肿瘤抑制率达到72%,联合治疗组能够彻底根除肿瘤。
图7为实施例3制得的EV-T&Dina治疗肿瘤后,对心、肝、脾、肺、肾、小肠H&E染色分析。从图7中可知,EV-T&Dina相对与对照组,H&E染色并无显著差异,表明EV-T&Dina对各脏器无显著毒副作用。
实施例6 EV-T&Dina抗肿瘤治疗的分子机制阐述
图8为实施例3制得的EV-T&Dina抗肿瘤治疗的分子机制示意图。从图8中可知,Dina能致敏肿瘤细胞对EV-T杀伤应答,其机理具体为:细胞外囊泡上膜结合的TRAIL在溶液中锌离子作用和膜流动下形成三聚体,与死亡受体4(DR4)和死亡受体5(DR5)结合;TRAIL受体DR4/5的胞浆区含有死亡结构域(death domain,DD),可募集胞浆内接头蛋白FADD和半胱氨酸蛋白酶原8(procaspase8),形成一个死亡诱导信号复合体DISC(Death-InducingSignaling Complex),“DR4/5-FADD-procaspase8”,引发caspase8激活、酶解级联和凋亡。但对于TRAIL抗性的肿瘤细胞,可以通过高表达procaspase-8同源物抗肿瘤凋亡蛋白cFLIP与FADD竞争性结合,cFLIP因缺少能够酶解procaspase活性的关键氨基酸,从而抑制TRAIL对肿瘤细胞杀伤。此外,肿瘤细胞还可以通过表达Bcl-2,Mcl-1,XIAP等抗凋亡蛋白帮助肿瘤细胞逃避TRAIL的杀伤,而这些抗凋亡蛋白表达主要受到细胞周期蛋白依赖激酶(CD)9的调控。Dinaciclib作用A549细胞后,通过抑制CDK9表达,进而抑制抗凋亡蛋白cFLIP、Bcl-2,Mcl-1表达;一方面解除了cFLIP对DISC抑制,从而增强EV-T对肿瘤杀伤效果;另外,通过下调Bcl-2,Mcl-1和上调Bax蛋白,从而导致线粒体膜电位下降,激活caspase-9进一步增强caspase-3凋亡诱导作用。Dinaciclib除了抑制CDK9表达外,还有效抑制了肿瘤细胞CDK1表达,从而将肿瘤细胞增殖分裂阻滞在G2/M期,最终使得肿瘤细胞对EV-T杀伤更加敏感。
上述抗癌小分子为化疗药物同样适用其他化学合成药物如SNS-032、Flavopiridol或阿霉素等,还可适用于天然活性分子、免疫治疗药物或核酸类药物。其中,免疫治疗药物可选为DR4/5单克隆激动抗体或/和PD-1/PD-L1单克隆抗体;所述的核酸类药物课选为mRNA、siRNA、shRNA、lncRNA或micro RNA;所述天然活性分子可选为所述天然活性分子为番茄红素或/和紫杉醇。上述复合外泌体EV-T&抗癌小分子均可联合治疗能够彻底清除体内肿瘤,抗肿瘤治疗效果显著,药物用量低且无毒副作用,同时对机体没有不利影响,为临床治疗癌症提供了纳米药物策略。
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、组合和简化,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)
1.一种负载促肿瘤凋亡蛋白和抗癌小分子的复合外泌体,其特征在于,所述复合外泌体为EV-T&抗癌小分子,其表面携带促肿瘤凋亡蛋白TRAIL,内部包裹抗癌小分子;该复合外泌体是先以表达人TRAIL基因的病毒载体转染人的生产外泌体的细胞资源,获得稳定高表达TRAIL的基因工程化间充质干细胞,标记为MSC-flT;然后采用外泌体制备的金标准-差速结合超速离心法,从MSC-flT培养液中提取细胞分泌的外泌体,得到高纯度表达携带TRAIL的外泌体,标记为EV-T;再将抗癌小分子包裹进EV-T中制得。
2.根据权利要求1所述的负载促肿瘤凋亡蛋白和抗癌小分子的复合外泌体,其特征在于,所述复合外泌体中促肿瘤凋亡蛋白TRAIL的浓度为0.5~4ng/mL;所述抗癌小分子的浓度为10~80nM;所述复合外泌体储存在-70~-85℃中。
3.根据权利要求1所述的负载促肿瘤凋亡蛋白和抗癌小分子的复合外泌体,其特征在于,所述人的外泌体的细胞资源为脐带间充质干细胞、骨髓间充质干细胞、脂肪间充质干细胞、牙髓间充质干细胞、胎盘、羊水、羊膜间充质干细胞、人胚肾细胞、心肌细胞、巨噬细胞、T细胞或肿瘤相关成纤维细胞中的一种以上。
4.根据权利要求1所述的负载促肿瘤凋亡蛋白和抗癌小分子的复合外泌体,其特征在于,所述抗癌小分子为化学合成药物、免疫治疗药物、天然活性分子或核酸类药物中的一种以上。
5.根据权利4要求所述的负载促肿瘤凋亡蛋白和抗癌小分子的复合外泌体,其特征在于,所述的化学合成药物为Dinaciclib,SNS-032、Flavopiridol或阿霉素中的一种以上;所述的免疫治疗药物为DR4/5单克隆激动抗体或/和PD-1/PD-L1单克隆抗体;所述的核酸类药物为mRNA、siRNA、shRNA、lncRNA或micro RNA;所述天然活性分子为番茄红素或/和紫杉醇。
6.根据权利要求1所述的负载促肿瘤凋亡蛋白和抗癌小分子的复合外泌体,其特征在于,所述的病毒载体为慢病毒、逆转录病毒或腺病毒。
7.根据权利要求1所述的负载促肿瘤凋亡蛋白和抗癌小分子的复合外泌体,其特征在于,所述EV-T的粒径为30~200nm。
8.根据权利要求1-7任一项所述的负载促肿瘤凋亡蛋白和抗癌小分子的复合外泌体的制备方法,其特征在于,具体包括如下步骤:
S1.以表达人TRAIL基因的病毒载体转染人的外泌体的细胞资源,获得稳定高表达TRAIL的基因工程化间充质干细胞,标记为MSC-flT;
S2.采用外泌体制备金标准-差速结合超速离心法,从MSC-flT培养液中提取细胞分泌的外泌体,得到高纯度表达携带TRAIL的外泌体,标记为EV-T;
S3.将抗癌小分子包裹进EV-T中,得到载有促肿瘤凋亡蛋白和抗癌小分子的复合外泌体,即EV-T&抗癌小分子;所述复合外泌体的表面携带促肿瘤凋亡蛋白TRAIL,内部包裹抗癌小分子。
9.根据权利要求8所述的负载促肿瘤凋亡蛋白和抗癌小分子的复合外泌体的制备方法,其特征在于,步骤S3中所述包裹的载药方法为超声法、膜穿孔法、电穿孔法或共孵育法。
10.权利要求1-7任一项所述的负载促肿瘤凋亡蛋白和抗癌小分子的复合外泌体在制备抗肿瘤治疗药物中的应用,其特征在于,所述的肿瘤为在人肺癌、胸膜间皮瘤、肾癌、神经胶质瘤、乳腺癌、肝癌、胃癌、结直肠癌、宫颈癌或黑色素瘤中对TRAIL敏感和高抗性的细胞。
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