CN113041358A - 一种超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂及其制备方法 - Google Patents
一种超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂及其制备方法 Download PDFInfo
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Abstract
本发明提供了一种超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂及其制备方法，属于工程化纳米囊泡载药系统技术领域。本发明所述制备方法制备得到的超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂，作用于肿瘤细胞株，发现超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂能够特异性靶向肿瘤细胞，诱导肿瘤细胞凋亡，抑制肿瘤生长，显著延长小鼠生存时间，改善药物对癌症的治疗效率。
Description
技术领域
本发明属于工程化纳米囊泡载药系统技术领域，具体涉及一种超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂及其制备方法。
背景技术
癌症是严重威胁人类健康的疾病，大多数癌症的诊断都处于中晚期，导致患者预后不佳。目前，化疗是治疗癌症的常用手段。然而，大多数药物对正常细胞有毒性作用，缺乏靶向性。因此，细胞外囊泡的出现为肿瘤的靶向治疗开辟了新前景。从天然细胞来源的纳米载体来看，细胞外囊泡具有较低的生物毒性，可以作为优良的纳米载体被设计成具有靶向抗肿瘤作用的复合物。迄今为止，已有诸多研究通过细胞外囊泡输送蛋白、RNA或其他小分子药物以达到治疗疾病的目的，但是仿生囊泡未曾在肿瘤领域进行过相关研究。
发明内容
有鉴于此，本发明的目的在于提供一种超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂及其制备方法，所述超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂可特异性靶向癌细胞，从而抑制癌细胞增殖、延缓癌发生发展。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂的制备方法，包括以下步骤：1)将外周血中性粒细胞与抗肿瘤药物共同培养，得摄取药物的中性粒细胞；
2)对所述摄取药物的中性粒细胞进行离心，重悬底部缓冲垫，得细胞悬液；
3)对所述细胞悬液进行连续物理挤压和梯度过滤，得中性粒细胞外泌体仿生囊泡包裹药物悬液；所述梯度过滤包括依次通过1μm、400nm和200nm聚碳酸酯过滤膜；
4)对所述中性粒细胞外泌体仿生囊泡包裹药物悬液离心后，重悬底部NNV-药物缓冲垫，得NNV-药物悬液；
5)将所述NNV-药物悬液与超顺磁材料混合后共孵育，对共孵育后的混合液进行磁分离，得超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂。
优选的，步骤1)每1×106个所述外周血中性粒细胞与50μg的抗肿瘤药物共同培养。
优选的，步骤1)所述共同培养的温度为37℃，时间为12～24h。
优选的，步骤2)所述离心包括差速离心，所述差速离心的离心力为800g，离心时间为5min。
优选的，步骤3)所述物理挤压包括使用mini-extrader挤压器对所述细胞悬液进行11次连续的物理挤压。
优选的，步骤4)所述离心包括超速离心，所述超速离心的离心力为10000g，离心时间为80min。
优选的，步骤5)所述超顺磁材料包括SPION-Tf，所述SPION-Tf在所述NNV-药物悬液中的浓度为0.5mg/ml。
优选的，步骤5)所述共孵育的温度为4℃，时间为4h。
优选的，步骤5)所述磁分离包括利用钕磁铁进行磁分离，MF＝1T。
本发明还提供了利用上述制备方法制备得到的超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂。
本发明提供了一种超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂的制备方法，利用mini-extrader挤压器将吞噬DOX的中性粒细胞通过1μm、400nm和200nm聚碳酸酯过滤膜挤压后，形成不同粒径的中性粒细胞外泌体仿生囊泡药物递送生物制剂。
在本发明实施例中，利用本发明所述制备方法制备得到的超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂，作用于肿瘤细胞株，发现超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂具有特异性靶向肿瘤细胞，诱导肿瘤细胞凋亡，抑制肿瘤生长，显著延长小鼠生存时间，改善药物对癌症的治疗效率。
附图说明
图1为实施例1中超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂的制备方法流程图；
图2为实施例1中人外周血中性粒细胞对阿霉素脂质体的摄取情况；
图3为实施例1中人外周血中性粒细胞外泌体仿生囊泡包裹药物的生物制剂的制备和透射电镜图；
图4为实施例1中人外周血中性粒细胞外泌体仿生囊泡包裹药物的生物制剂的粒径大小和zeta电位；
图5为实施例1中超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂对肿瘤细胞株的细胞毒性作用，其中a为HGC-27细胞与SPION-NNV-DOX/MF体外共培养模式图；b为CCK8细胞增殖实验检测不同处理下HGC-27细胞增殖能情况。
具体实施方式
本发明提供了一种超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂的制备方法，包括以下步骤：1)将外周血中性粒细胞与抗肿瘤药物共同培养，得摄取药物的中性粒细胞；
2)对所述摄取药物的中性粒细胞进行离心，重悬底部缓冲垫，得细胞悬液；
3)对所述细胞悬液进行连续物理挤压和梯度过滤，得中性粒细胞外泌体仿生囊泡包裹药物悬液；所述梯度过滤包括依次通过1μm、400nm和200nm聚碳酸酯过滤膜；
4)对所述中性粒细胞外泌体仿生囊泡包裹药物悬液离心后，重悬底部NNV-药物缓冲垫，得NNV-药物悬液；
5)将所述NNV-药物悬液与超顺磁材料混合后共孵育，对共孵育后的混合液进行磁分离，得超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂。
本发明将外周血中性粒细胞与抗肿瘤药物共同培养，得摄取药物的中性粒细胞。本发明优选每1×106个所述外周血中性粒细胞与50μg的抗肿瘤药物共同培养。本发明对所述外周血中性粒细胞和抗肿瘤药物的来源并没有特殊限定，利用本领域的常规提取方法提取或市售产品即可。本发明所述抗肿瘤药物优选包括阿霉素脂质体(DOX-CL)。本发明优选用含10％胎牛血清的培养基RPMI1640培养人外周血中性粒细胞6h，然后添加抗肿瘤药物的溶液至培养液中共同培养24h，所述共同培养的温度优选为37℃，时间优选为12～24h。经过所述共同培养后，中性粒细胞胞质内含有大量带有红色荧光的抗肿瘤药物(DOX)。
得摄取药物的中性粒细胞后，本发明对所述摄取药物的中性粒细胞进行离心，重悬底部缓冲垫，得细胞悬液。本发明所述离心优选包括差速离心，所述差速离心的离心力优选为800g，离心时间优选为5min。本发明经所述离心后，优选收集底部的缓冲垫，用PBS再次冲悬沉淀制备中性粒细胞包裹药物的细胞悬液。
得细胞悬液后，本发明对所述细胞悬液进行连续物理挤压和梯度过滤，得中性粒细胞外泌体仿生囊泡包裹药物悬液；所述梯度过滤包括依次通过1μm、400nm和200nm聚碳酸酯过滤膜。本发明所述物理挤压优选包括使用mini-extrader挤压器(Avanti PolarLipids,USA)对所述细胞悬液进行11次连续的物理挤压，所述物理挤压的参数优选包括：5mL细胞悬液挤压参数为：300psig，气密注射器温度为10℃以上。本发明通过物理挤压可以产生不同粒径大小的中性粒细胞外泌体仿生囊泡，可以简单快速制备出大量中性粒细胞外泌体仿生囊泡。本发明使经过所述物理挤压的细胞悬液依次通过1μm、400nm、200nm聚碳酸酯过滤膜，收集挤压后的滤过液，制备得到中性粒细胞外泌体仿生囊泡包裹药物悬液。
得中性粒细胞外泌体仿生囊泡包裹药物悬液后，本发明对所述中性粒细胞外泌体仿生囊泡包裹药物悬液离心后，重悬底部NNV-药物缓冲垫，得NNV-药物悬液。本发明所，述离心优选包括超速离心，所述超速离心的离心力优选为10000g，离心时间优选为80min。本发明所述重悬优选包括收集经所述超速离心后底部的NNV-药物缓冲垫，将NNV-药物沉淀再分散于PBS缓冲液中进一步纯化。
得NNV-药物悬液后，本发明将所述NNV-药物悬液与超顺磁材料混合后共孵育，对共孵育后的混合液进行磁分离，得超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂。本发明所述超顺磁材料优选包括SPION-Tf，所述SPION-Tf在所述NNV-药物悬液中的浓度为0.5mg/ml。本发明所述共孵育的温度优选为4℃，时间优选为4h。本发明对共孵育后的混合液进行磁分离，所述磁分离优选包括利用钕磁铁进行磁分离，MF＝1T。
本发明还提供了利用上述制备方法制备得到的超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂。本发明实施例中，所述超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂是包裹阿霉素脂质体(DOX-CL)的中性粒细胞挤压囊泡与SPION-Tf结合后磁分离得到的粒径在200nm的杯状膜形囊泡；所述超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂的活性成份包括中性粒细胞相关的毒性蛋白(FasL,GranzymeA/B,Perforin)；所述的超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂通过运载毒性蛋白和化疗药物DOX在胃癌细胞中发挥双重杀伤作用，显著抑制肿瘤生(载药量为15％)。
下面结合实施例对本发明提供的一种超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂及其制备方法进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂提取与鉴定
人外周血中性粒细胞培养试剂：多形核白细胞分离液(PolymorphPrep分离液，Norway)、RPMI1640(Bioind，USA)、胎牛血清(Gibco，USA)、胰蛋白酶(Sigma，USA)、二氧化碳培养箱(Forma公司)、无血清培养基(Excell，China)；
倒置显微镜(Nikon，Japan)、超净工作台，台式离心机(Eppendorf，Germany)，超速离心机(Beckman，USA)。
SPION-NNV-DOX生物制剂的提取试剂：SPION材料和阿霉素脂质体(Xi an ruixiBiological Technology，China)、Mini-extrader挤压器(Avanti Polar Lipids，USA)、1μm，400nm，200nm不同孔径的聚碳酸酯过滤膜(Xi an ruixi Biological Technology，China)、透射电子显微镜(FEI Tecnai 12，Philips公司)、超速离心机(Beckman，USA)、全景式流式细胞仪(Flow sight，USA)、NanoSight LM10 system(nanosight trackinganalysis，UK)。
按照图1所示流程制备超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂：
1.将人外周血中性粒细胞(Neu，Neutrophils，中性粒细胞)与阿霉素脂质体(DOX-CL)在孵箱内37℃共培养24h(图2)；
2.800g，5min(Eppendorf Centrifuge 5804/5804离心机)离心，收集底部的缓冲垫，用PBS冲悬制备细胞悬液；
3.使用mini-extrader挤压器对细胞悬液进行连续挤压11次，依次通过1μm，400nm，200nm聚碳酸酯过滤膜，制备中性粒细胞外泌体仿生囊泡包裹的DOX(NNV-DOX，NNV：Neutrophil Nano Vesicle，中性粒细胞挤压囊泡)；
4.通过超速离心法：10000g，80min，(Beckman超速离心机)离心沉淀收集NNV-DOX，将NNV-DOX沉淀再分散于PBS中；
5.NNV-DOX悬液与SPION-Tf(0.5mg/ml)超顺磁材料共同孵育，4℃，4h；
6.通过外加钕磁铁(MF＝1T)对混合液进行磁分离获得SPION-NNV-DOX复合物。
透射电镜和Nanoparticle Tracking Analysis观察(NNV-DOX)形态特征：阿霉素脂质体、中性粒细胞挤压囊泡和NNV-DOX溶液各20μL，混匀后滴加于直径2mm的载样铜网上，静置5min后，用滤纸吸去残余液体，将铜网倒扣于30g/L磷钨酸(pH6.8)液滴上，25℃条件下负染5min，白炽灯下烘干，透射电镜下观察拍照，图3所示阿霉素脂质体的直径约为50nm，中性粒细胞挤压囊泡为大小在180nm左右的囊泡状结构；NNV-DOX的粒径约为220nm左右；图4显示NNV-DOX的粒径大小为200nm±20左右，电位为-29.04±0.45mV。
实施例2
超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂对肿瘤细胞的体外抗肿瘤作用
96孔细胞培养板(JET Biofil公司)，RPMI1640(Bioind，USA)、胰蛋白酶(Sigma，USA)、CCK8检测试剂盒(Vazyme，China)、酶标仪(FLX800，United States)。
1.用胰蛋白酶消化HGC-27细胞，离心沉淀将HGC-27细胞接种于96孔细胞培养板中，待细胞贴壁以后分别添加不同的物质(DOX-CL(45μg/mL)，NNV(40μg/mL)，SPION-NNV-DOX(40μg/mL)，SPION-NNV-DOX/MF(在外加磁场下发挥抗肿瘤作用的超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂)(40μg/mL))处理HGC-27细胞；
2.按上述处理24h后，分别在96孔细胞培养板中添加CCK8检测试剂，将培养板置于CO2孵箱中继续培养；
3.利用酶标仪检测HGC-27细胞在450nm的吸光值。
通过CCK8实验观察SPION-NNV-DOX/MF对HGC-27增殖能力的影响。结果如图5所示，通过外加磁场可以促进SPION-NNV-DOX在HGC-27细胞的积累，磁铁作用时间越长，HGC-27细胞吞噬的SPION-NNV-DOX越多；SPION-NNV-DOX/MF显著抑制HGC-27细胞增殖。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂的制备方法，其特征在于，包括以下步骤：1)将外周血中性粒细胞与抗肿瘤药物共同培养，得摄取药物的中性粒细胞；
2)对所述摄取药物的中性粒细胞进行离心，重悬底部缓冲垫，得细胞悬液；
3)对所述细胞悬液进行连续物理挤压和梯度过滤，得中性粒细胞外泌体仿生囊泡包裹药物悬液；所述梯度过滤包括依次通过1μm、400nm和200nm聚碳酸酯过滤膜；
4)对所述中性粒细胞外泌体仿生囊泡包裹药物悬液离心后，重悬底部NNV-药物缓冲垫，得NNV-药物悬液；
5)将所述NNV-药物悬液与超顺磁材料混合后共孵育，对共孵育后的混合液进行磁分离，得超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂。
2.根据权利要求1所述制备方法，其特征在于，步骤1)每1×106个所述外周血中性粒细胞与50μg的抗肿瘤药物共同培养。
3.根据权利要求1所述制备方法，其特征在于，步骤1)所述共同培养的温度为37℃，时间为12～24h。
4.根据权利要求1所述制备方法，其特征在于，步骤2)所述离心包括差速离心，所述差速离心的离心力为800g，离心时间为5min。
5.根据权利要求1所述制备方法，其特征在于，步骤3)所述物理挤压包括使用mini-extrader挤压器对所述细胞悬液进行11次连续的物理挤压。
6.根据权利要求1所述制备方法，其特征在于，步骤4)所述离心包括超速离心，所述超速离心的离心力为10000g，离心时间为80min。
7.根据权利要求1所述制备方法，其特征在于，步骤5)所述超顺磁材料包括SPION-Tf，所述SPION-Tf在所述NNV-药物悬液中的浓度为0.5mg/ml。
8.根据权利要求1所述制备方法，其特征在于，步骤5)所述共孵育的温度为4℃，时间为4h。
9.根据权利要求1所述制备方法，其特征在于，步骤5)所述磁分离包括利用钕磁铁进行磁分离，MF＝1T。
10.利用权利要求1～9任一项所述制备方法制备得到的超顺磁修饰中性粒细胞外泌体仿生囊泡药物递送生物制剂。
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| CN110934851A (zh) * | 2019-11-21 | 2020-03-31 | 饶磊 | 靶向细胞膜的多肽药物外泌体纳米载药系统及其制备方法 |
| CN111035769A (zh) * | 2019-11-19 | 2020-04-21 | 饶磊 | 超顺磁纳米铁修饰的外泌体载药纳米系统及其制备和血糖响应方法 |
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