CN114191539B - 一种复合共载送小分子核酸和活性蛋白的外泌体纳米粒子及其制备方法和应用 - Google Patents
一种复合共载送小分子核酸和活性蛋白的外泌体纳米粒子及其制备方法和应用 Download PDFInfo
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- CN114191539B CN114191539B CN202111410482.4A CN202111410482A CN114191539B CN 114191539 B CN114191539 B CN 114191539B CN 202111410482 A CN202111410482 A CN 202111410482A CN 114191539 B CN114191539 B CN 114191539B
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Abstract
本发明公开了一种复合共载送小分子核酸和活性蛋白的外泌体纳米粒子及其制备方法和应用。所述纳米粒子包括来自细胞并载入了活性蛋白的复合外泌体，以及装载在复合外泌体内的小分子核酸。本发明先经过基因工程修饰构建慢病毒载体，通过慢病毒转染将活性蛋白的目的基因整合到细胞上，得到载入了活性蛋白的复合外泌体，再载入治疗核酸siRNA，实现外泌体共载送siRNA和活性蛋白。本发明表明，靶向凋亡抑制基因的siRNA和活性蛋白具有复合协同作用，能高效特异地杀死耐药性癌细胞，并且与抗肿瘤药物组合使用，能进一步提高治疗效果，可实现药物基于外泌体对耐药性癌症的多靶点联合精准治疗，为癌症治疗提供更多更高效的药物。
Description
技术领域
本发明属于纳米载药与肿瘤学技术领域，更具体地，涉及一种复合共载送小分子核酸和活性蛋白的外泌体纳米粒子及其制备方法和应用。
背景技术
肿瘤的耐药性是造成肿瘤难以治愈的主要原因，研究表明，肿瘤的复发与转移与耐药性肿瘤干细胞(cancer stem cells，CSCs)具有密切的联系。而CSCs的耐药性主要包括以下两个方面：获得性耐药性和天然耐药性。获得性耐药是指长期暴露于辐射或致癌因子后，CSCs和相近的子代细胞可通过与正常干细胞积累突变的同样机制(点突变、基因激活、基因扩增)等出现新的耐药性；天然耐药性指处于静止期的CSCs具有DNA自我修复能力和APC转运蛋白而获得的与生俱来的耐药性。而细胞周期蛋白依赖性激酶9(cyclindependent kinase9，CDK9)是一种DNA转录调节因子，与细胞凋亡调控有密切的关系。有研究表明，抑制CDK9可以重新激活在表观遗传上被癌症沉默的基因，同时调节凋亡抑制基因的表达，解除癌细胞的凋亡耐药性，致敏其对治疗药物的应答。因此，CDK9是一种潜在的癌症治疗分子靶位。
外泌体是细胞分泌的粒径在30-150nm的细胞外囊泡，外被磷脂双分子层结构，能够透过体内的天然屏障(如血脑屏障)，具有良好的肿瘤靶向性。此外，外泌体在机体内部还具有良好的生物稳定性和生物相容性、低免疫原性和低毒性。外泌体不仅具有细胞间信息传递的作用，还作为一种天然的生物纳米载体，被开发运用于各类分子(包括siRNA)的细胞间传输。与传统的药物载体相比，外泌体作为一种新型的，天然药物载体，显示诸如生物相容性好、免疫原性低，良好的肿瘤靶向性等优点。
小分子干扰核酸(small interfering RNA，siRNA)是一类通常长度为20-25个核苷酸的双链RNA，它进入体内后通过干扰mRNA影响翻译过程，从而沉默该基因的蛋白合成。目前已经有研究表明小干扰核酸疗法Inclisiran作为首创的降低低密度脂蛋白胆固醇(low density lipoprotein cholesterol，LDL-C)的siRNA疗法在欧洲获批用于治疗成人高胆固醇血症及混合性血脂异常，这将是引领慢性疾病治疗的一个全新的革命。但由于siRNA自身结构的不稳定性，在体内实验中通常需要对其进行化学修饰，这些化学修饰可能会改变siRNA的性质甚至影响其基因沉默的效果，且siRNA是无法自主进入细胞的，因此寻求一种行之有效的递载siRNA的方法就成为必要。
目前，已有研究采用外泌体运载siRNA。如现有技术公开了一种复合siRNA纳米载体，是一种以自体肿瘤细胞来源的外泌体脂质膜包裹载送siRNA和血清白蛋白的复合纳米，应用于抗肿瘤。但是，该技术使用外泌体载送的siRNA和血清白蛋白两种分子只是复合载送，两者并无协同治疗效应。为了更好地应用外泌体作为天然药物载体提高治疗效果，寻找一种有效的装载siRNA并联合其他药物用于癌症协同治疗的新策略，对于开发高效的癌症精准治疗纳米药物有巨大的实际应用价值。
发明内容
本发明的目的在于提供一种新的复合载送小分子核酸和协同活性蛋白的外泌体纳米粒子用于癌症的治疗，能复合协同，高效特异地杀死具有耐药性的癌细胞，为癌症治疗提供了基于外泌体的更高效的药物联合治疗策略。
本发明的第二个目的在于提供所述制备方法及其应用。
本发明的第三个目的在于提供一种抗肿瘤药物组合物。
本发明的上述目的是通过以下技术方案给予实现的：
一种复合载送小分子核酸和协同活性蛋白的外泌体纳米粒子，包括来自细胞并载入了活性蛋白的复合外泌体，以及装载在复合外泌体内的小分子核酸。
本发明对细胞进行基因工程修饰后，得到载入了活性蛋白的复合外泌体，利用该复合外泌体装载小分子核酸纳米粒子可以对癌症进行联合治疗，还可以利用该复合外泌体装载小分子核酸纳米粒子联合其他药物用于癌症的治疗。
优选地，所述细胞包括间充质干细胞、骨髓干细胞、胚胎干细胞、脐带干细胞、肿瘤细胞、肿瘤干细胞、免疫细胞或成纤维细胞中的一种。
优选地，所述活性蛋白为TRAIL蛋白。
优选地，所述小分子核酸为siRNA-CDK9和/或siRNA-eif5B。
特别地，本发明采用的复合外泌体运载的小分子核酸，其siRNA可为癌症沉默基因，也可为其他的siRNA，是能根据需求不同可以变动的，并且不止是针对癌症治疗，在原则上是采用外泌体进行运载，所以是可以采用其他siRNA。
上述复合载送小分子核酸和协同活性蛋白的外泌体纳米粒子的制备方法，包括以下步骤：
S1.基因工程修饰构建慢病毒载体，通过慢病毒转染将活性蛋白的目的基因整合到细胞上，在培养箱中培养，提取外泌体，即得到载入了活性蛋白的复合外泌体；
S2.将步骤S1中得到的载入了活性蛋白的复合外泌体，进行重悬后与小分子核酸混合，冰上超声处理，摇床孵育，得到复合载送小分子核酸和协同活性蛋白的外泌体纳米粒子。
优选地，所述步骤S1中基因工程修饰包括慢病毒转染、逆转录病毒转染或腺病毒转染中的一种。
优选地，所述步骤S2中载入了活性蛋白的复合外泌体与小分子核酸混合的体积比为3～8:1。
更优选地，所述步骤S2中载入了活性蛋白的复合外泌体与小分子核酸混合的体积比为5:1。
优选地，所述步骤S2中超声处理的条件为：超声10～60s，暂停10～60s，功率10～30％，温度20～35℃，循环次数1～10次。
更优选地，超声处理的条件为：超声30s，暂停30s，功率25％，温度25℃，循环次数6次。
优选地，所述步骤S2中摇床孵育的条件为：在30～40℃孵育0.5～2h。
更优选地，摇床孵育的条件为：在37℃孵育1h。
优选地，所述步骤S1中培养箱的培养条件为：25～40℃，5～10％CO2，10～20h。
更优选地，培养箱的培养条件为：37℃，5％CO2，16h。
本发明提供所述复合载送小分子核酸和协同活性蛋白的外泌体纳米粒子在制备抗肿瘤药物中的应用。
本发明还提供一种抗肿瘤药物组合物，含有上述复合载送小分子核酸和协同活性蛋白的外泌体纳米粒子和抗肿瘤药物。
优选地，所述抗肿瘤药物包括化疗药物、免疫治疗药物、重构肿瘤微环境的药物中的至少一种。
本发明经过基因工程修饰了间充质干细胞然后分离提纯的外泌体，在外泌体上能同时载运siRNA和活性蛋白，使siRNA和活性蛋白能协同表达，结果表明靶向凋亡抑制基因的siRNA和活性蛋白(TRAIL蛋白)的复合协同能高效杀死耐药性的癌细胞。
与现有技术相比，本发明具有以下有益效果：
本发明利用复合外泌体装载小分子核酸，能够大大提高小分子核酸在体内的稳定性，同时也能使其借助外泌体的天然肿瘤趋向性向病灶部位聚集。本发明可解决CSCs耐药性和药物靶向性差的问题，该复合外泌体载入小分子核酸并联合其他药物后通过静脉给药进入血液后，能够高效的蓄积于肿瘤组织、深部穿透进入肿瘤病灶深部并且显著抑制肺癌的生长，明显降低CSCs的分化。且靶向凋亡抑制基因的siRNA和活性蛋白具有复合协同作用，能高效特异地杀死耐药性癌细胞，并且与抗肿瘤药物组合使用，能进一步提高治疗效果，可实现药物基于外泌体对耐药性癌症的多靶点联合精准治疗，为癌症治疗提供更高效的药创新物。
附图说明
图1为EV-T透射电子显微镜照片；
图2为流式细胞仪分析细胞的TRAIL阳性转染率；
图3为TRAIL ELISA试剂盒测出EV和EV-T携带TRAIL的量；
图4为MSCs及EVs中TRAIL表达分析；
图5为HPLC检测siRNA含量结果；
图6为EV-T超声装载siRNA-eif5B后作用Huh7细胞不同时间下的蛋白表达情况；
图7为不同作用条件下A549细胞的存活率；
图8为小鼠皮下肿瘤模型。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。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。
除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1TRAIL基因工程化MSC干细胞及其外泌体的制备与表征
传代培养P2-P4脐带来源间充质干细胞(UC-MSC)(此细胞为某干细胞公司惠赠，也可从新鲜脐带分离培养而来)，将状态良好的干细胞MSC以1×106个/孔的密度接种于细胞培养六孔板，在37℃，5％CO2条件下，细胞培养箱过夜。
对上述培养的MSC细胞进行基因工程化修饰，使用携带TRAIL表达框的慢病毒，以MOI＝3的病毒浓度，采用8μg/mL聚凝胺(polybrene)增强转染，孵育10h；将培养基换成含有10％FBS(胎牛血清)DMEM/F12的新鲜培养基，继续培养2-3天；待细胞长满培养基后，再将细胞转移到培养瓶进行增殖传代培养，得到表达TRAIL的干细胞(MSCflT)。
将六孔板转染后的表达TRAIL的干细胞(MSCflT)消化，离心后取沉淀，再加入250μl裂解液(99％ripa，1％蛋白酶抑制剂)，冰上裂解1h，离心(4℃，10min，12000g)后取上清液，加入5×loading buffer进行加热蛋白(100℃，10min)，再采用BCA试剂盒测试其蛋白浓度后定量。
将上述转染后的MSCflT细胞传代培养后收集其上清液，先低速离心(4℃，10min，1000g)去除死细胞和细胞碎片；然后以0.22μm滤膜过滤除去大于220nm的沉淀外泌体EV-T或其它细胞残片和颗粒等物质；接着可以用100kD超滤离心(4℃，10min，3000g)将外泌体沉淀EV-T溶液浓缩5倍；最后进行超速离心(4℃，2h，120000g)，采用经0.22μm滤膜过滤过的PBS溶液重悬外泌体沉淀EV-T，分装并冻存于-80℃保存备用。
EV-T的透射电子显微镜图如图1所示，经过流式细胞仪分析细胞的TRAIL阳性转染率，结果如图2所示。采用TRAIL ELISA试剂盒测出EV和EV-T携带TRAIL的量结果如图3所示。
取上述转染后的MSC细胞裂解后的上清液及EV-T通过蛋白免疫印迹法检查细胞是否转染成功以及其外泌体的标志蛋白CD63以及其携带的TRAIL情况。结果如图4所示，为MSCs及EVs中TRAIL表达情况，其中图4A表示MSC细胞和表达TRAIL的干细胞(MSCflT)通过蛋白免疫印迹法检测细胞是否转染成功，由图可知MSCflT已经成功转染；图4B表示外泌体的标志蛋白CD63以及其携带的TRAILB情况，由图可知CD63已经成功携带EV-T。
实施例2外泌体装载小分子核酸siRNA-CDK9/siRNA-eif5B
使用的siRNA-CDK9/siRNA-eif5B由广州艾基生物科技有限公司设计合成，其序列为：
Hs-siRNA-CDK9-1 Sense:5’-GUGAUGAAGUUUCCAAAUAdTdT-3’；
Hs-siRNA-CDK9-1 Antisense:5’-UAUUUGGAAACUUUCAUCACdTdT-3’；
Hs-siRNA-CDK9-2 Sense:5’-GGCAGGCUGGAGAGAGAAAdTdT-3’；
Hs-siRNA-CDK9-2 Antisense:5’-UUUCUCUCUCCAGCCUGCCdTdT-3’；
Hseif5B-2012-s：CGGCGACUUGAACAUAGUAAA(dT)(dT)；
Hseif5B-2012-a：UUUACUAUGUUCAAGUCGCCG(dT)(dT)；
Hseif5B-3047-s：GAUGAAUUGAUCCAUGAGUUA(dT)(dT)；
Hseif5B-3047-a：UAACUCAUGGAUCAAUUCAUC(dT)(dT)。
将沉淀外泌体EV-T与siRNA按照质量比5:1进行混合，使用超声仪对其进行超声装载，设置参数为：超声30s，暂停30s，功率25％，温度25℃，循环次数6次。在超声结束后放置在37℃的摇床，孵育1h，帮助外泌体恢复膜结构；装载siRNA后的EV-T利用外泌体沉淀试剂盒沉淀(贝贝生物科技有限公司，郑州)，去掉上清中游离的siRNA，再用PBS溶解后加入ripa裂解液，使包裹siRNA的EV-T将siRNA全部释放出来，再利用HPLC检测siRNA含量结果如图5所示，计算对应的装载率。通过计算装载率得出超声的装载率约为18％。
实施例3Western blotting检测基因沉默后的eif5B蛋白表达情况
将A549和Huh7细胞(购于上海富衡生物科技有限公司)按1×106个/孔的密度种植于细胞培养六孔板，在37℃，5％CO2条件下进行培养箱培养，待融合率达到70％，加入装载了siRNA的EV-T，继续培养72h后，将六孔板转染后的MSC细胞消化，离心取沉淀，加入250μl裂解液(99％ripa，1％蛋白酶抑制剂)，冰上裂解1h，离心(4℃，10min，12000g)后取上清液，加入5×loading buffer加热蛋白(100℃，10min)，再采用BCA试剂盒测试其蛋白浓度后定量，通过蛋白免疫印迹法检测基因沉默情况，结果如图6所示，为eif5B不同时间段的基因沉默情况。
实施例4CCK8检测细胞毒性及对肿瘤细胞的杀伤情况
为了验证装载了siRNA的EV-T对正常细胞的毒性以及对肿瘤细胞的杀伤效果，我们选取了正常细胞MSC和NF，肿瘤细胞A549、Huh7和HepG2，使其在单独的EV-T(包含活性蛋白)、游离的siRNA和复合外泌体载送siRNA作用下，分别检验其细胞活性。
结果由图7可知，在单独的EV-T和游离的siRNA作用下，对正常的细胞和肿瘤细胞的活性基本没有影响；而复合外泌体载送siRNA对正常细胞基本无毒性，对肿瘤细胞有着较好的杀伤效果。
我们还进一步研究发现，在复合外泌体载送siRNA的使用上同时加入氯喹(chloroquine，CQ)一种自噬抑制剂，能进一步的提高复合外泌体载送siRNA对肿瘤细胞的杀伤效果。可能的机理在于氯喹抑制了细胞内溶酶体对进入细胞的外泌体的融合分解，从而促进了外泌体载送siRNA进入细胞核发挥抑制靶分子mRNA作用。
实施例5复合外泌体载送siRNA-CDK9后在A549肿瘤模型中的杀伤情况
从珠海百试通生物科技有限公司购入4-5周龄的BALB/c雌性裸鼠，在SPF级饲养环境下饲养其体重达到20g左右；选取状态良好的小鼠用于皮下肿瘤模型造膜。按照500万/只的细胞数量接种肿瘤细胞造膜，再分别注射Ctrl(空白对照水)，沉淀外泌体EV-T(包含活性蛋白)，siRNA-CDK9，复合外泌体载送siRNA-CDK9(siCDK9@EV-T)，通过皮下注射接种到小鼠皮下，接种28天以后随机将小鼠分为5组：Ctrl，EV-T，siRNA-CDK9，siCDK9@EV-T，按照100ugsiRNA/只，8ng EV-T/只的药物剂量通过皮下注射的方式给药，每次给药间隔2天，共给药7次，最后检测小鼠体内肿瘤细胞的抑制情况。
由结果可知，在治疗完成后发现单独使用的EV-T和siRNA-CDK9基本对高抗性A549肿瘤没有抑制作用，而在使用了复合外泌体载送siRNA-CDK9是可以提高对肿瘤的抑制作用，图8为小鼠皮下肿瘤模型图。
综上所述，先经过基因工程修饰间充质干细胞，使其分泌表达了治疗蛋白的外泌体，分离纯化此外泌体后，再载入治疗核酸siRNA，实现基于外泌体的共载递送siRNA和抗癌蛋白；而与单独的沉淀外泌体EV-T(包含活性蛋白)和siRNA-CDK9相比，采用复合外泌体载送siRNA-CDK9能提高对肿瘤的抑制作用还能高效杀死癌症细胞，这是在单独作用EV-T(包含活性蛋白)和siRNA-CDK9所达不到的效果，表明两种治疗分子的协同效应可显著增强对癌细胞的致死效果；结果表明靶向凋亡抑制基因的siRNA和抗癌蛋白的复合协同能高效特异性地杀死耐药性癌细胞，可实现基于外泌体的对耐药性癌症的多靶点联合精准治疗。
以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，对于本领域的普通技术人员来说，在上述说明及思路的基础上还可以做出其它不同形式的变化或变动，这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。
Claims (4)
1.一种复合载送小分子核酸和协同活性蛋白的外泌体纳米粒子，其特征在于，包括来自细胞并载入了活性蛋白的复合外泌体，以及装载在复合外泌体内的小分子核酸；
所述活性蛋白为TRAIL蛋白，所述细胞为脐带来源的间充质干细胞；
所述小分子核酸为siRNA-CDK9，siRNA-CDK9的序列为Sense:5’-GGCAGGCUGGAGAGAGAAAdTdT-3’；Antisense:5’-UUUCUCUCUCCAGCCUGCCdTdT-3’。
2.权利要求1所述纳米粒子的制备方法，其特征在于，包括以下步骤：
S1.基因工程修饰构建慢病毒载体，通过慢病毒转染将活性蛋白的目的基因整合到细胞上，在培养箱中培养，提取外泌体，即得到载入了活性蛋白的复合外泌体；所述活性蛋白为TRAIL蛋白，所述细胞为脐带来源的间充质干细胞；
S2.将步骤S1中得到的载入了活性蛋白的复合外泌体，进行重悬后与小分子核酸混合，冰上超声处理，摇床孵育，得到复合载送小分子核酸和活性蛋白的外泌体纳米粒子；所述载入了活性蛋白的复合外泌体与小分子核酸混合的体积比为3～8:1；所述小分子核酸为siRNA-CDK9，siRNA-CDK9的序列为Sense:5’-GGCAGGCUGGAGAGAGAAAdTdT-3’；Antisense:5’-UUUCUCUCUCCAGCCUGCCdTdT-3’。
3.权利要求1所述复合载送小分子核酸和协同活性蛋白的纳米粒子在制备抗肿瘤药物中的应用，其特征在于，所述肿瘤为肺癌。
4.一种抗肿瘤药物组合物，其特征在于，含有上述权利要求1所述复合载送小分子核酸和协同活性蛋白的纳米粒子和抗肿瘤药物；所述抗肿瘤药物选自化疗药物、免疫治疗药物、重构肿瘤微环境的药物中的至少一种，所述肿瘤为肺癌。
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