CN112458119A - 一种递送特定蛋白质的工程化仿生外泌体的制备方法及其应用 - Google Patents
一种递送特定蛋白质的工程化仿生外泌体的制备方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种递送特定蛋白质的工程化仿生外泌体的制备方法及其应用,包括如下步骤:S1.利用工程细胞包装病毒,制备特定的慢病毒颗粒;S2.用所得慢病毒侵染目的细胞,并加入抗性药物筛选,从而制备高纯度表达特定基因的稳定细胞系;S3.培养所得稳定细胞系,提取其细胞膜并将其制成膜纳米囊泡,最后离心提纯,即所得工程化仿生外泌体。本发明提供了一种大量制备特定蛋白质药物递送系统的合成方法,该法可以有效增强蛋白质类生物大分子药物在体内的稳定性、延长药效时长、实现药物的有效递送,为蛋白质类生物大分子药物的给药途径提供了一种新的思路,可用于治疗肿瘤的免疫治疗,安全性好、作用效果强,具有非常高的应用价值和临床转化前景。
Description
技术领域
本发明涉及生物医药材料技术领域,更具体地,涉及一种递送特定蛋白质的工程化仿生外泌体的制备方法及其应用。
背景技术
近年来,包括免疫检查点在内的免疫治疗给肿瘤的治疗方式带来了革命性的转变,为癌症的治愈带来了曙光。[NATURE REVIEWS CANCER,2015,12,4,252-264]。免疫检查点分为两类,一类是为人们所熟知的以PD-1/PDL1、CTLA4为代表的负向免疫检查点,会抑制机体的免疫系统,目前免疫检查点单抗药物绝大多数都是基于负向免疫检查点;另一类则是正向免疫检查点,会激活机体的免疫系统。[NATURE BIOMEDICAL ENGINEERING,2017,1,0011]。虽然免疫检查点单抗类药物获得了巨大的成功,然而有一部分患者并没有从中获益,表现出对于免疫检查点的抗性。此时,开发一种新的治疗途径尤为重要。OX40/OX40L是一条正向调节的免疫检查点通路,OX40L蛋白可有效激活T细胞,激活免疫,用于治疗肿瘤、微生物感染等疾病。[IMMUNOLOGICAL REVIEWS,229,173-191]。因此,OX40L蛋白是一种强力有效的候选药物。蛋白质类大分子药物具有高度的选择性、作用的高效性、可大量制备等优势,然而体内不稳定、易降解、生物半衰期短是其一直以来难以克服的问题。因此急需解决OX40L蛋白的递送方式问题。
细胞外囊泡,例如外泌体和微泡,是细胞以组成型或诱导型方式分泌的小膜颗粒。释放的EVs通过将核酸和蛋白质转运至下游细胞,充当细胞间的信使。最近的研究表明,EVs具备作为强大而可行的纳米载体进行药物递送的潜力。[SCIENCE,367,640]与现有的递送系统相比,EVs的独特优势是其天然来源,这使得它们能够逃避吞噬作用,延长治疗剂的半衰期并降低免疫原性。[SCIENCE ADVANCES,2020,6,33]尤其是巨噬细胞来源的EVs,基于其免疫细胞的特性,还具有较好的肿瘤靶向性。用巨噬细胞来源的外泌体类生物膜系统装载蛋白,可以极大程度地提高大分子药物的稳定性。[ADVANCED MATERIALS,2020,2002054]因此,基于巨噬细胞来源的EVs的药物递送系统可能是操纵OX40L蛋白治疗肿瘤的有效候选者。
然而,EVs的产量过低一直是其难以克服的缺点,极大的阻碍了其临床转化以及产业化的进程。[ACS NANO,2018,12,8977-8993]鉴于生物膜系统的相似性以及EVs的产生方式,本发明提出了用巨噬细胞膜来人工制备仿生外泌体,用来递送药物。
发明内容
本发明的目的在于克服现有蛋白类药物体内不稳定以及外泌体产量低的缺陷,提供一种递送特定蛋白质的工程化仿生外泌体的制备方法。
本发明的第二个目的在于提供所述递送特定蛋白质的工程化仿生外泌体的制备方法的应用。
本发明的上述目的是通过以下技术方案给予实现的:
一种递送特定蛋白质的工程化仿生外泌体的制备方法,包括如下步骤:
S1.利用工程细胞包装病毒,制备特定的慢病毒颗粒;
S2.用所得慢病毒侵染目的细胞,并加入抗性药物筛选,从而制备高纯度表达特定基因的稳定细胞系;
S3.培养所得稳定细胞系,提取其细胞膜并将其制成膜纳米囊泡,最后离心提纯,即所得工程化仿生外泌体。
进一步地,所述工程细胞为HEK-293T细胞。
进一步地,所述特定慢病毒颗粒为含有OX40L编码基因的慢病毒颗粒。
进一步地,所述目的细胞为巨噬细胞。
进一步地,所述抗性药物为嘌呤霉素。
进一步地,所述抗性药物的浓度为1.5~3.5μg/mL。
进一步地,所述稳定细胞系为OX40L巨噬细胞系。
本发明创造性的提出了一种改良的基因编辑法制备工程化仿生外泌体,通过特定慢病毒侵染目的细胞制备稳定细胞系,而后提取其细胞膜,通过挤出等方法得到膜纳米囊泡,而后通过密度梯度离心,得到高纯度的工程化仿生外泌体。
由上述方法制备得到的工程化仿生外泌体。所述工程化仿生外泌体上稳定表达OX40L蛋白。
本发明制备得到的工程化仿生外泌体具有较高的OX40L蛋白的表达,生物相容性好,靶向能力强,可用于肿瘤或病原微生物感染的免疫治疗中。因此,本发明还提供了上述工程化仿生外泌体在免疫治疗中的应用。所述制剂可应用于免疫疗法治疗肿瘤,具有很高的实用价值和临床前景。
与现有技术相比,本发明具有以下有益效果:
(1)本发明提供了一种递送特定蛋白质的工程化仿生外泌体的制备方法,率先制备出递送OX40L蛋白的生物膜体系,通过应用基因编辑的技术手段,将OX40L蛋白表达在细胞膜上,大大提高了OX40L蛋白在体内的稳定性、生物半衰期。
(2)本发明将基因编辑的巨噬细胞膜用于递送系统,基于其免疫细胞的特性,具有较好的生物安全性、较低的免疫原性以及具有较高的免疫逃逸效果,且具有很好的靶向性,能够更有效的靶向T细胞,生物安全性和生物利用度大大提高。
附图说明
图1为本发明实施例1中HEK-293T细胞生产慢病毒颗粒的荧光图;200μm。
图2为本发明实施例1中稳定细胞系的流式细胞仪检测图。
图3为本发明实施例1中稳定细胞系的共聚焦显微镜检测图;20μm。
图4为本发明实施例1中工程化仿生外泌体的透射电镜检测图;100nm。
图5为本发明实施例1中工程化仿生外泌体的粒径分布和Zeta电位图。
图6为本发明实施例1中工程化仿生外泌体的共聚焦显微镜检测图;10μm。
图7为本发明实施例1中工程化仿生外泌体的流式细胞仪检测图。
图8为本发明实施例1中工程化仿生外泌体与靶细胞结合的研究;10μm。
图9为本发明实施例1中不同浓度的工程化仿生外泌体体外毒性的研究。
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1
1.工程化仿生外泌体的制备
于6孔板培养HEK-293T细胞,至其密度为70%左右时,换用Opti-MEM培养。每孔加入混合好的1μg目的质粒、1.2μg包装质粒以及4μL的lipo2000,转染并孵育。6-8小时后换液,用含10%FBS的DMEM培养液培养。于特定时间荧光显微镜下观察处理后的HEK-293T细胞。其结果如图1所示,HEK-293T细胞可以有效的产生特定慢病毒。分别于24h、48h、72h时收集病毒上清,存放于4℃。而后将收集的病毒上清于500g下离心10min,并收集上清用0.45μm的滤器过滤,以除去上清中的细胞碎片。而后将滤液与病毒纯化试剂(LentiConcentrator)按照1:5的比例混合,冰上过夜。之后3500g离心30min,尽量去除上清,得到病毒颗粒,用微量(100μL每孔)PBS重悬慢病毒颗粒。
用所得慢病毒颗粒侵染巨噬细胞。将巨噬细胞培养于6孔板中,而后加入100μL上述所得慢病毒颗粒以侵染巨噬细胞。之后加入3μg/mL的嘌呤霉素筛选,1~2周后,用流式细胞仪检测。其结果如图2所示,GFP+细胞群比例高达95%以上。同时,用共聚焦显微镜观测GFP-OX40L的表达,其结果如图3所示,巨噬细胞上显著表达GFP-OX40L。检测结果一致表明得到高纯度的GFP-OX40L稳定细胞系。
之后收集稳定巨噬细胞系,PBS洗3次,于冰上用杜恩斯组织匀浆器破碎细胞200次左右。将所得裂解液放置于干净的玻璃容器内,于冰水里水浴超声(42kHZ,50W,5min)。之后离心(1000g、5min、4℃),以除去未裂解的细胞。收集上清,继续离心(3000g、5min、4℃),以除去较大的细胞碎片或细胞器。收集上清,通过0.8μm和0.45μm的滤器挤出,得到较为均一的膜囊泡。之后超速离心(10万g,90min,4℃),收集沉淀,用PBS重悬,洗涤1次(10万g,90min,4℃)。继续收集沉淀,用适量PBS重悬,即得工程化仿生外泌体。
将上述所得工程化仿生外泌体稀释一定的倍数,用透射电镜观察其形态。其结果如图4所示,具有典型的膜结构,与天然外泌体外貌上高度相似。布鲁克林粒度仪检测其粒径分布。其结果如图5所示,平均水动力粒径大致为90nm,Zeta电位大致为-20mV。用共聚焦显微镜观测其蛋白表达情况,其结果如图6所示,观测到明显的GFP荧光颗粒。用流式细胞仪检测,其结果如图7所示,GFP+颗粒的比例高达87%以上。这些结果表明稳定表达特定蛋白OX40L的工程化仿生外泌体(GFP-OX40L NVs)成功制备。
2.工程化仿生外泌体的生物学功能研究
将OFP-OX40质粒转染进3T3细胞内,共聚焦显微镜观测转染后的细胞。其结果如图8所示,可见OFP-OX40在3T3细胞上的明显表达。而后将OFP-OX40 3T3细胞分为3组,编号①、②、③,其中第③组提前用OX40抗体处理6小时,之后分别将DiO-free NVs、GFP-OX40L NVs、GFP-OX40L NVs加入至①、②、③中,共孵育2-4小时,之后于共聚焦显微镜下观察。其结果如图8所示,所制工程化外泌体与其配体具有很好的共定位,且可被抗体阻断。表明具有良好生物活性,且具有特异性靶向结合的优势,预测临床应用时在体内也具有较好的靶向性。
之后考察其体外毒性,用CCK-8法检测了不同浓度的工程化仿生外泌体对细胞存活率的影响。其结果如图9所示,对于所测细胞基本没有细胞毒性。表明本发明中的工程化仿生外泌体是一种安全性高、生物相容性好的生物材料。
以上所述仅表达了本发明的优选实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形、改进及替代,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (9)
1.一种递送特定蛋白质的工程化仿生外泌体的制备方法,其特征在于,包括如下步骤:
S1.利用工程细胞包装病毒,制备特定的慢病毒颗粒;
S2.用所得慢病毒侵染目的细胞,并加入抗性药物筛选,从而制备高纯度表达特定基因的稳定细胞系;
S3.培养所得稳定细胞系,提取其细胞膜并将其制成膜纳米囊泡,最后离心提纯,即所得工程化仿生外泌体。
2.根据权利要求1所述的方法,其特征在于,所述工程细胞为HEK-293T细胞。
3.根据权利要求1所述的方法,其特征在于,所述特定慢病毒颗粒为含有OX40L编码基因的慢病毒颗粒。
4.根据权利要求1所述的方法,其特征在于,所述目的细胞为巨噬细胞。
5.根据权利要求1所述的方法,其特征在于,所述抗性药物为嘌呤霉素。
6.根据权利要求1或5所述的方法,其特征在于,所述抗性药物的浓度为1.5~3.5μg/mL。
7.根据权利要求1所述的方法,其特征在于,所述稳定细胞系为OX40L巨噬细胞系。
8.权利要求1~7任一所述的方法得到的工程化仿生外泌体。
9.权利要求8所述的工程化仿生外泌体在肿瘤的免疫治疗中的应用。
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114404571A (zh) * | 2022-01-20 | 2022-04-29 | 中山大学·深圳 | 一种装载化疗药物且tigit过表达的工程化载药细胞膜囊泡以及制备方法和应用 |
CN114657145A (zh) * | 2022-04-29 | 2022-06-24 | 中山大学·深圳 | 一种基因编辑t细胞的细胞外囊泡的制备方法与应用 |
CN115261410A (zh) * | 2022-07-20 | 2022-11-01 | 中山大学 | 一种基因编辑工程化m1型巨噬细胞外泌体的制备方法及其应用 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110215514A (zh) * | 2019-04-22 | 2019-09-10 | 上海瑞可新生物科技有限公司 | 一种基因工程化细胞膜纳米囊泡及其制备与应用 |
WO2019178113A1 (en) * | 2018-03-12 | 2019-09-19 | Board Of Regents, The University Of Texas System | Immuno-exosomes and methods of use thereof |
CN111787945A (zh) * | 2018-02-15 | 2020-10-16 | 北卡罗莱纳州立大学 | 作为癌症免疫治疗的检查点阻断剂的工程化纳米囊泡 |
-
2020
- 2020-11-10 CN CN202011251716.0A patent/CN112458119A/zh active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111787945A (zh) * | 2018-02-15 | 2020-10-16 | 北卡罗莱纳州立大学 | 作为癌症免疫治疗的检查点阻断剂的工程化纳米囊泡 |
WO2019178113A1 (en) * | 2018-03-12 | 2019-09-19 | Board Of Regents, The University Of Texas System | Immuno-exosomes and methods of use thereof |
CN110215514A (zh) * | 2019-04-22 | 2019-09-10 | 上海瑞可新生物科技有限公司 | 一种基因工程化细胞膜纳米囊泡及其制备与应用 |
Non-Patent Citations (1)
Title |
---|
张盈盈等: "外泌体作为药物递送载体的研究进展", 《药学学报》 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114404571A (zh) * | 2022-01-20 | 2022-04-29 | 中山大学·深圳 | 一种装载化疗药物且tigit过表达的工程化载药细胞膜囊泡以及制备方法和应用 |
CN114657145A (zh) * | 2022-04-29 | 2022-06-24 | 中山大学·深圳 | 一种基因编辑t细胞的细胞外囊泡的制备方法与应用 |
CN115261410A (zh) * | 2022-07-20 | 2022-11-01 | 中山大学 | 一种基因编辑工程化m1型巨噬细胞外泌体的制备方法及其应用 |
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