CN114487391A - 仿生免疫磁纳米颗粒、其制备方法及应用 - Google Patents
仿生免疫磁纳米颗粒、其制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种仿生免疫磁纳米颗粒、其制备方法及应用。该仿生免疫磁纳米颗粒包括:磁纳米颗粒,以及中性粒细胞膜囊泡,包覆在磁纳米颗粒上形成仿生磁纳米颗粒。该仿生磁纳米颗粒后续连接上靶向抗体,可用于生物成分的分离。该仿生免疫磁纳米颗粒至少还具有以下优势:Neu‑vesicles的脂质双层减少了非特异性蛋白质的吸附并保持了IMNs在血液中的靶向能力;CTCs和中性粒细胞之间的相互作用增强,提高了IMNs对CTC分离的效率;白细胞和中性粒细胞之间的相互作用减少,减少了WBCs在分离过程中的干扰,提高了分离CTC的纯度;Neu‑vesicles和CTC之间的软相互作用提高了分离的CTC的活力。
Description
技术领域
本发明涉及生物医药技术领域,具体而言,涉及一种仿生免疫磁纳米颗粒、其制备方法及应用。
背景技术
从临床外周血样本中分离循环肿瘤细胞(CTC)属于无创液体活检,在癌症早期检测和诊断方面具有广阔的前景。到目前为止,已经探索了多种新兴方法从外周血中分离CTC,例如基于物理性质的富集方法、基于生物特征的富集方法和有核细胞无差异的富集方法等。其中,最广泛使用的策略是基于免疫磁性纳米颗粒(IMN),其中磁性纳米颗粒被用作为分离介质,而特异性抗体被用作捕获配体。由于外周血中CTC的稀缺性(每毫升血液只有几个到几十个),如何高效率高纯度地分离具有天然生物活性的CTC用于后续细胞分析是一个重大挑战。
目前的分离循环肿瘤细胞方法仍存在一定的技术问题,例如:Qian-Fang Meng,etal.Biomimetic Immunomagnetic Nanoparticles with Minimal Non-SpecificBiomolecule Adsorption for Enhanced Isolation of Circulating Tumor Cells.ACSApplied Materials&Interfaces,2019,11,28732-28739.公开了一种红细胞膜囊泡包覆的磁性纳米颗粒MNs上,然后用靶向抗体对获得的磁性纳米颗粒进行修饰,用来分离CTC,可以一定程度提高分离CTC的效率,但是其分离CTC的纯度不高。又如:Lang Rao,etal.Platelet–Leukocyte Hybrid Membrane-Coated Immunomagnetic Beads for HighlyEfficient and Specific Isolation of Circulating Tumor Cells.AdvancedFunctional Materials.2018,28,1803531.公开了一种血小板和白细胞的混合膜包裹在MBs上,然后用靶向抗体对获得的磁性纳米颗粒进行修饰,用来分离CTC,可以一定程度提高CTC的分离纯度。但是其稳定性不好,程序复杂,包封率不高。
发明内容
本发明旨在提供一种仿生免疫磁纳米颗粒、其制备方法及应用,以提供一种新型的仿生免疫磁纳米颗粒,提高生物组分的分离效率和纯度。
为了实现上述目的,根据本发明的一个方面,提供了一种仿生免疫磁纳米颗粒。该仿生免疫磁纳米颗粒包括:磁纳米颗粒,以及中性粒细胞膜囊泡,包覆在磁纳米颗粒上形成仿生磁纳米颗粒。
进一步地,磁纳米颗粒为Fe3O4磁性纳米颗粒。
根据本发明的另一个方面,提供一种用于分离的仿生免疫磁纳米颗粒。该仿生免疫磁纳米颗粒包括:仿生免疫磁纳米颗粒,以及靶向抗体,靶向抗体与仿生磁纳米颗粒物理连接和/或共价连接。
进一步地,靶向抗体为CTC靶向抗体。
进一步地,CTC靶向抗体为抗上皮细胞黏附分子抗体。
进一步地,物理连接和/或共价连接为共轭连接或点击连接。
根据本发明的另一方面,提供了一种上述仿生免疫磁纳米颗粒的制备方法。该制备方法包括以下步骤:S1,制备中性粒细胞膜囊泡;以及S2,用中性粒细胞膜囊泡包覆磁纳米颗粒,获得仿生磁纳米颗粒。
进一步地,S1包括:从外周血中分离中性粒细胞制备中性粒细胞膜囊泡。
进一步地,采用梯度密度离心方法从外周血中分离中性粒细胞。
进一步地,分离的中性粒细胞通过低渗裂解缓冲液和反复冻融的方法破坏,超速离心收集细胞膜,然后通过微型推挤机挤出,制备得到中性粒细胞膜囊泡。
进一步地,微型推挤机通过800和400nm聚碳酸酯多孔膜挤出以得到中性粒细胞膜囊泡。
进一步地,S2包括:将中性粒细胞膜囊泡与磁纳米颗粒注入到微流控光穿孔芯片,中性粒细胞膜囊泡和磁纳米颗粒在光穿孔区域融合,使中性粒细胞膜囊泡包覆在磁纳米颗粒上形成仿生磁纳米颗粒。
进一步地,混合物以最优的2毫升/小时的流速通过光穿孔区域时,使用激光能量密度为0.12焦耳/平方厘米的脉冲激光进行照射,激光脉冲可以有效促进磁纳米颗粒进入中性粒细胞膜囊泡形成仿生磁纳米颗粒。
进一步地,中性粒细胞膜囊泡与磁纳米颗粒(1:1)~(1:10)。
进一步地,S2还包括机械挤推和超声混合等其它常见方法,阐明了光穿孔方法相对于机械挤推和超声混合方法的优势。机械挤推法是将中性粒细胞膜囊泡与磁纳米颗粒混合,然后将得到混合物通过微型推挤机采用400nm孔多次反复挤出,使中性粒细胞膜囊泡包覆在磁纳米颗粒上形成仿生磁纳米颗粒。超声融合是将中性粒细胞膜囊泡与磁纳米颗粒混合,然后超声混合3分钟,使中性粒细胞膜囊泡包覆在所述磁纳米颗粒上形成仿生磁纳米颗粒。
根据本发明的再一方面,提供了一种用于分离的仿生免疫磁纳米颗粒的制备方法。该制备方法包括以下步骤:按照上述制备方法制备仿生免疫磁纳米颗粒,S3,将靶向抗体与仿生磁纳米颗粒通过物理连接和/或共价连接得到仿生免疫磁纳米颗粒。
进一步地,靶向抗体为CTC靶向抗体。
进一步地,CTC靶向抗体为抗上皮细胞黏附分子抗体。
进一步地,S3包括:将S2制备的仿生磁纳米颗粒用羧基-聚乙二醇-磷脂处理,然后加入NHS/EDC活化,接着交联上链霉亲和素,生物素化的CTC靶向抗体与之结合得到仿生免疫磁纳米颗粒。
根据本发明的又一方面,提供了一种用于分离的仿生免疫磁纳米颗粒在制备用于富集、分离或检测循环肿瘤细胞的产品中的应用。
根据本发明的再一方面,提供了一种用于分离的仿生免疫磁纳米颗粒在制备用于***转移相关疾病的药物中的应用。
进一步地,肿瘤转移相关疾病为上皮细胞粘附因子高表达相关的肿瘤,优选为结肠直肠癌、乳腺癌和胃癌。
应用本发明的技术方案,该仿生磁纳米颗粒后续连接上靶向抗体,可用于生物成分的分离,利用中性粒细胞膜衍生的囊泡(Neu-vesicles)包裹磁纳米颗粒得到仿生免疫磁纳米颗粒(Neu-IMNs)。该仿生免疫磁纳米颗粒至少具有以下优势:1)Neu-vesicles的脂质双层减少了非特异性蛋白质的吸附并保持了IMNs在血液中的靶向能力;2)CTCs和中性粒细胞之间的相互作用增强,提高了IMNs对CTC分离的效率;3)白细胞(WBCs)和中性粒细胞之间的相互作用减少,减少了WBCs在分离过程中的干扰,提高了分离CTC的纯度;4)Neu-vesicles和CTC之间的软相互作用提高了分离的CTC的活力,使后续的细胞分析成为可能。另外,本发明通过微流控光穿孔方法制备的中性粒细胞膜囊泡仿生磁纳米颗粒具备以下两点突出优势:1)应用的光穿孔制备方法相比于其它常见的制备方法(如机械挤推、超声混合)是最优的,包封率最高,需要的原材料最少,且CTC的捕获效率最高,因而最有应用转化前景;2)相比于其它杂化膜包裹的仿生磁纳米颗粒,本发明的中性粒细胞膜囊泡仿生磁纳米颗粒的包封率和稳定性都更好,能实现更高的CTC捕获效率。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。在附图中:
图1示出了利用微流控光穿孔芯片制备中性粒细胞膜囊泡仿生磁纳米颗粒的示意图。
图2示出了(a)制备Neu-IMNs的流程示意图以(b)增强CTC分离的示意图。
图3示出了微流控光穿孔芯片的实物图。
图4示出了Neu-MNs修饰anti-EpCAM抗体的路径示意图。
图5示出了实施例1中外周血样本分离的中性粒细胞的明场图。比例尺,100μm。
图6示出了实施例1中Fe3O4 MNs的XRD光谱。
图7示出了不同激光能量密度下的光穿孔包封效率,最优激光能量密度为0.12焦耳/平方厘米。
图8示出了微流控芯片中不同流速下的光穿孔包封效率,最优流速为2毫升/小时。
图9示出了应用不同制备方法(光穿孔、微型挤推器挤推、超声混合),在不同中性粒细胞膜囊泡和磁纳米颗粒比例(1:1,1:2,1:5和1:10)下的包封效率。
图10示出了单一中性粒细胞膜和杂化膜1(中性粒细胞和巨噬细胞杂化膜)、杂化膜2(白细胞和红细胞杂化膜)和磁纳米颗粒在不同比例(1:1,1:2,1:5和1:10)下的包封效率。
图11示出了单一中性粒细胞膜和杂化膜1(中性粒细胞和巨噬细胞杂化膜)、杂化膜2(白细胞和红细胞杂化膜)仿生纳米颗粒在2周内的粒径变化,单一中性粒细胞膜仿生纳米颗粒显示出更好的稳定性。
图12示出了单一中性粒细胞膜和杂化膜1(中性粒细胞和巨噬细胞杂化膜)、杂化膜2(白细胞和红细胞杂化膜)仿生纳米颗粒制备2周后的傅里叶红外表征结果,中性粒细胞膜仿生纳米颗粒展现了细胞膜的C-H键,显示出相比于杂化膜1(中性粒细胞和巨噬细胞杂化膜)、杂化膜2(白细胞和红细胞杂化膜)仿生纳米颗粒更好的包封稳定性。
图13示出了Neu-IMNs的表征:(a)MN和Neu-MN的平均直径和zeta电位。(b)MNs和(c)Neu-MNs的TEM图像。比例尺,100nm。(d)通过SDS-PAGE对Neu-vesicles和Neu-MNs进行蛋白质鉴定。(e)生物素-FITC标记的Neu-MNs的共聚焦图像。比例尺,20μm。(f)Neu-IMNs捕获的单个MCF-7细胞的SEM图像。比例尺,10μm和2.5μm(放大版)。箭头表示Neu-IMN。(g)Neu-MN或Neu-IMN对PBS中MCF-7和HeLa细胞的分离效率。所有数据表示为平均值±标准差(n=5)。
图14中的a示出了Neu-IMNs的磁滞曲线;b示出了Neu-IMNs水溶液经外磁场处理前后的照片。
图15示出了IMNs经与10%血浆共孵育前后的尺寸变化。
图16示出了Neu-IMNs显示出蛋白质吸附减少和与癌细胞的相互作用增强的特性。(a)通过与10%血浆共孵育后IMN上的蛋白质成分。(b)在10%血浆中孵育的Neu-IMN的大小变化。(c)在10%血浆中孵育后,热图展示Neu-IMN表面上一些最常见蛋白质的丰度变化。(d)ICP-AES测试了MCF-7细胞与各种MN之间的相互作用。(e)各种MN对不同癌细胞系的捕获效率。所有数据表示为平均值±标准差(n=5)。
图17示出了ICP-AES测定MCF-7细胞和浓度为100μg/ml不同纳米颗粒共培养不同时间的相互作用。
图18示出了不同制备方法(光穿孔、微型挤推器挤推、超声混合)制备的中性粒细胞仿生免疫磁纳米颗粒的MCF-7细胞捕获效率。
图19示出了单一中性粒细胞膜和杂化膜1(中性粒细胞和巨噬细胞杂化膜)、杂化膜2(白细胞和红细胞杂化膜)仿生免疫磁纳米颗粒的MCF-7细胞捕获效率。
图20示出了PBS中不同纳米颗粒的细胞捕获效率。
图21示出了Neu-IMN显示减少的WBC干扰并增强分离细胞的活力:(a)通过ICP-AES测量WBC与各种MN之间的相互作用。(b)在全血中添加不同数量MCF-7细胞的不同MN的捕获效率。(c)各种MN对MCF-7细胞的分离纯度。(d)IMNs和Neu-IMNs分离后的MCF-7细胞活力。所有数据表示为平均值±标准差(n=5)。
图22示出了ICP-AES测定白细胞和浓度为100μg/ml的不同纳米颗粒共培养不同时间的相互作用。
图23示出了不同制备方法(光穿孔、微型挤推器挤推、超声混合)制备的中性粒细胞仿生免疫磁纳米颗粒在每毫升血液样本中CTC的捕获数量。
图24示出了单一中性粒细胞膜和杂化膜1(中性粒细胞和巨噬细胞杂化膜)、杂化膜2(白细胞和红细胞杂化膜)仿生免疫磁纳米颗粒在每毫升血液样本中CTC的捕获数量。
图25示出了CTC的鉴定、计数和分析。(a)Neu-IMNs分离的CTC和WBC的共聚焦图像。比例尺,15μm。(b)从健康或乳腺癌患者血液样本中分离的CTC的计数和纯度。(c)Neu-IMNs分离和两轮培养后单个CTC的SEM图像。比例尺,5μm。(d)Neu-IMNs分离后CTCs的Sanger测序结果。
具体实施方式
需要说明的是,在不冲突的情况下,本申请中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本发明。
名词解释
CTC:循环肿瘤细胞。
MNs:磁性纳米粒子。
IMN:免疫磁性纳米颗粒。
Neu-vesicles:中性粒细胞膜衍生的囊泡,或称为中性粒细胞膜囊泡。
Neu-IMNs:利用中性粒细胞膜衍生的囊泡包裹磁纳米颗粒的仿生免疫磁纳米颗粒,其上连接有靶向抗体,在本申请中连接有CTC靶向抗体。
Neu-MNs:利用中性粒细胞膜衍生的囊泡包裹磁纳米颗粒的仿生免疫磁纳米颗粒。
WBCs:白细胞。
anti-EpCAM:抗上皮细胞黏附分子。
PBS:phosphate buffered saline,磷酸缓冲盐溶液。
用免疫磁纳米粒子(IMNs)检测和分析稀有循环肿瘤细胞(CTC)在无创癌症诊断中展现出有希望的前景。然而,本申请的发明人发现IMNs进入生物体液后会吸附非特异性蛋白质,形成的蛋白冠覆盖表面靶向配体,限制了IMNs的检测效率。此外,表面靶向配体与血液中的白细胞(WBC)有很强的相互作用,限制了IMN分离的CTC的纯度。针对此,本申请提出用中性粒细胞膜对IMNs进行表面功能化修饰,这样可以显著减少非特异性蛋白质吸附,增强与CTC的相互作用,减少对WBC的干扰,并提高分离的CTC的活性。
根据本申请一种典型的实施方式,提供一种用于分离CTC的仿生免疫磁纳米颗粒。参考图2中a所示,该用于分离CTC的仿生免疫磁纳米颗粒包括:磁纳米颗粒,中性粒细胞膜囊泡,包覆在磁纳米颗粒上形成仿生磁纳米颗粒。
仿生磁纳米颗粒与靶向抗体连接后,可以用于生物组分的分离,例如,靶向抗体可以是CTC靶向抗体,当然根据具体分离目标的需要,也可以连接其他的靶向抗体。
参考图2中b所示,该仿生免疫磁纳米颗粒至少还具有以下优势:1)Neu-vesicles的脂质双层减少了非特异性蛋白质的吸附并保持了IMNs在血液中的靶向能力;2)CTCs和中性粒细胞之间的相互作用增强,提高了IMNs对CTC分离的效率;3)白细胞(WBCs)和中性粒细胞之间的相互作用减少,减少了WBCs在分离过程中的干扰,提高了分离CTC的纯度;4)Neu-vesicles和CTC之间的软相互作用提高了分离的CTC的活力,使后续的细胞分析成为可能。
在本申请一种典型的实施方式中,CTC靶向抗体为抗上皮细胞黏附分子抗体。
在本申请一种典型的实施方式中,磁纳米颗粒为Fe3O4磁性纳米粒子。
根据本申请一种典型的实施方式,提供一种上述仿生免疫磁纳米颗粒的制备方法。该制备方法包括以下步骤:S1,制备中性粒细胞膜囊泡;S2,用中性粒细胞膜囊泡包覆磁纳米颗粒,获得仿生磁纳米颗粒。对应的,用于分离的仿生免疫磁纳米颗粒的制备方法包括上述仿生免疫磁纳米颗粒的制备方法以及S3,将CTC靶向抗体与仿生磁纳米颗粒物理连接和/或共价连接,该物理连接和/或共价连接为共轭连接或化学点击连接。
在本申请一种典型的实施方式中,CTC靶向抗体为抗上皮细胞黏附分子抗体。
在本申请一实施例中,S1包括:从外周血中分离中性粒细胞制备中性粒细胞膜囊泡;优选的,采用梯度密度离心方法从外周血中分离中性粒细胞;当然也可以从组织中进行中性粒细胞的分离。
优选的,分离的中性粒细胞通过低渗裂解缓冲液和反复冻融的方法破坏,离心收集细胞膜,然后通过微型推挤机挤出,制备得到中性粒细胞膜囊泡;在本申请一种典型的实施方式中,微型推挤机通过800nm和400nm聚碳酸酯多孔膜挤出以得到中性粒细胞膜囊泡,当然可以根据实际需要,选择其他孔径的多孔膜挤出。
常见的细胞膜囊泡包覆纳米颗粒的方法有机械挤推和超声混合,这些方法都存在包封效率不高,包封不稳定等缺陷。为了改进这些不足,本申请采用光穿孔的方法制备仿生磁纳米颗粒,参见图1,将中性粒细胞膜囊泡与磁纳米颗粒注入到微流控光穿孔芯片,中性粒细胞膜囊泡和磁纳米颗粒进入光穿孔区域时,激光脉冲促进磁纳米颗粒进入中性粒细胞膜囊泡形成仿生磁纳米颗粒。该方法可以有效提高包封效率和稳定性,且能提高后续CTC的捕获效率,如图9,图18和图23所示。
本申请采用单一中性粒细胞膜囊泡包覆磁纳米颗粒制备仿生磁纳米颗粒,相比于其它杂化膜囊泡包覆的磁纳米颗粒,包封率和稳定性都更好,能实现更高的CTC捕获效率,如图10、图11、图12,图19和图24所示。
在本申请一实施例中,S2包括:将中性粒细胞膜囊泡与磁纳米颗粒注入到微流控光穿孔芯片,中性粒细胞膜囊泡和磁纳米颗粒在光穿孔区域融合,使中性粒细胞膜囊泡包覆在磁纳米颗粒上形成仿生磁纳米颗粒;在本申请一实施例中,混合物在通过光穿孔区域时激光脉冲可以有效促进磁纳米颗粒进入中性粒细胞膜囊泡形成仿生磁纳米颗粒;优选的,中性粒细胞膜囊泡与磁纳米颗粒的摩尔比为(1:1)~(1:10),例如,1:1,1:2,1:5或1:10,可以进行很好的包覆。
S2还包括机械挤推和超声混合等其它常见方法,阐明了光穿孔方法相对于机械挤推和超声混合方法的优势。机械挤推法是将所述中性粒细胞膜囊泡与磁纳米颗粒混合,然后将得到混合物通过微型推挤机采用400nm孔多次反复挤出,使所述中性粒细胞膜囊泡包覆在所述磁纳米颗粒上形成仿生磁纳米颗粒。超声融合是将中性粒细胞膜囊泡与所述磁纳米颗粒混合,然后超声混合3分钟,使所述中性粒细胞膜囊泡包覆在所述磁纳米颗粒上形成仿生磁纳米颗粒。
根据本申请一种典型的实施方式,S3包括:将S2制备的仿生磁纳米颗粒用羧基-聚乙二醇-磷脂处理,然后加入NHS/EDC活化,接着交联上链霉亲和素,生物素化的CTC靶向抗体与之结合得到仿生免疫磁纳米颗粒。
在本申请另一典型的实施方式中,还包括S4,采用液相色谱串联质谱法验证Neu-IMNs的蛋白冠吸附减少以及桑格测序鉴定Neu-IMNs分离的循环肿瘤细胞。
根据本申请一种典型的实施方式,提供一种用于分离CTC的仿生免疫磁纳米颗粒在制备用于富集、分离或检测循环肿瘤细胞的产品中的应用。
根据本申请一种典型的实施方式,提供一种用于分离CTC的仿生免疫磁纳米颗粒在制备用于***转移相关疾病的药物中的应用。其中,肿瘤转移相关疾病为上皮细胞粘附因子高表达相关的肿瘤,优选为结肠直肠癌、乳腺癌和胃癌。
下面将结合试验数据进一步说明本发明的有益效果。在下述实施例中如果没有详细描述的步骤或试剂均可采用本领域的常规方法或试剂完成。
实施例1
中性粒细胞膜囊泡包覆的免疫磁纳米颗粒(Neu-IMNs)的制备和应用步骤如下:(1)制备微流控光穿孔芯片;(2)从临床外周血中分离中性粒细胞制备Neu-vesicles;(3)用制备好的Neu-vesicles包覆磁纳米颗粒(MNs)获得仿生磁纳米颗粒(Neu-MNs);以及(4)将抗上皮细胞黏附分子(anti-EpCAM)抗体与Neu-MNs进行生物共轭连接。(5)液相色谱串联质谱法验证Neu-IMNs的蛋白冠吸附减少以及桑格测序鉴定Neu-IMNs分离的循环肿瘤细胞。
具体步骤如下:
(1)微流控光穿孔芯片的制备:
匀胶:将清洗干净的硅片放在匀胶机上,旋涂一层SU-8-2050光刻胶,前转600rpm,运行30s后,将转速调为1000rpm,运行40s。前烘:65℃烘台上加热5min,再转移到95摄氏度烘台上加热12min,然后冷却10min。曝光:将硅片放在光刻机上,盖上掩模板,曝光35s。后烘:将曝光后的硅片放在65℃烘台上加热2.5min,在转95℃烘台加热8.5min,然后冷却10min。显影、坚膜:将硅片浸入显影液中,显影9min30s,拿镊子取出,洗去多余的显影液,用氮***吹干;然后放在120℃烘台上加热30min。倒PDMS:按A、B胶30:3的比例配制33gPDMS;将PDMS倒入硅片上,放置30min,用吹气球吹掉大的气泡;再用真空机抽真空两次,每次三分钟;然后放入烘箱在80℃下烘一个小时。打孔、键合:从硅片上取下PDMS,打孔并切好,和清洗干净的玻璃一起放入离子清洗机3min,取出来把玻璃和PDMS粘合,完成芯片通道的封闭,放在烘箱上烘一天。至此,微流控光穿孔芯片制作完毕(图3)。
(2)中性粒细胞膜囊泡(Neu-vesicles)的制备:
首先从新鲜临床外周血中用梯度密度离心方法分离中性粒细胞(图5,外周血样本分离的中性粒细胞的明场图。比例尺,100μm)。为了获得中性粒细胞膜囊泡,分离的中性粒细胞通过低渗裂解缓冲液和反复的冻融方法破坏。收集的细胞用冷PBS洗涤,然后悬浮在低渗溶液(Gibico品牌的PBS和去离子水按照1:4体积混合得到)中。之后,使用反复冻融法将中性粒细胞破碎3次,然后将溶液在4℃下以2000g离心30分钟,然后将富集的上清液进一步以100000g超速离心40分钟以收集细胞膜。将收集到的细胞膜重新悬浮在PBS中,并通过Avanti mini挤出机通过800和400nm聚碳酸酯多孔膜逐步挤出以产生中性粒细胞膜囊泡。
(3)磁纳米颗粒合成以及用中性粒细胞膜囊泡包覆磁纳米颗粒:
水热法合成了Fe3O4磁性纳米粒子(MNs)。先将1.35g FeCl3·6H2O加入40ml乙二醇中充分溶解,再加入3.6g NaAc。将混合溶液剧烈搅拌1h,然后密封在50ml内衬特氟隆的不锈钢高压釜中,在200℃下反应8h。反应产物用乙醇和去离子水洗涤数次。然后,通过X射线衍射仪(XRD;D8 Advance,AXS Instruments,Germany)对获得的MNs进行表征,结果见图6(Fe3O4 MNs的XRD光谱)。为了获得包覆细胞囊泡的磁纳米颗粒(Neu-MNs),将50μg磁纳米颗粒和中性粒细胞膜囊泡注入到微流控光穿孔芯片,中性粒细胞膜囊泡和磁纳米颗粒混合物在以2毫升/小时的最优流速通过光穿孔区域时,使用最优激光能量密度为0.12焦耳/平方厘米的脉冲激光进行照射,激光脉冲促进磁纳米颗粒进入中性粒细胞膜囊泡形成仿生磁纳米颗粒(图7,图8)。机械挤推法是将中性粒细胞膜囊泡与所述磁纳米颗粒混合,然后将得到混合物通过微型推挤机采用400nm孔多次反复挤出,使所述中性粒细胞膜囊泡包覆在所述磁纳米颗粒上形成仿生磁纳米颗粒。超声融合是将中性粒细胞膜囊泡与所述磁纳米颗粒混合,然后超声混合3分钟,使所述中性粒细胞膜囊泡包覆在所述磁纳米颗粒上形成仿生磁纳米颗粒。实验数据表明,在不同中性粒细胞膜囊泡和磁纳米颗粒比例(1:1,1:2,1:5和1:10)下,光穿孔均表现出相比于机械挤推和超声混合更高的包封效率(图9)。用动态光散射(DLS)对MNs和Neu-MNs的动态直径和zeta电势进行测量,用透射电子显微镜对MNs和Neu-MNs的形貌进行表征。Neu-MNs的磁性通过物理性质测量***(PPMS-9Quantum Design)测量。SDS-PAGE用于测试和分析Neu-vesicles和Neu-MNs的蛋白质成分。将Neu-vesicles和Neu-MNs的蛋白质在100℃下变性10分钟,并将具有相同蛋白质含量5μg的每个样品加载到10%SDS-聚丙烯酰胺凝胶(EpiZyme Biotechnology,China)的平行孔中。随后,电泳槽在110V下运行2h,得到的聚丙烯酰胺凝胶用考马斯亮蓝溶液染色30min,脱色过夜。然后用化学发光凝胶成像***曝光成像。
相比于其它杂化膜囊泡包覆的磁纳米颗粒,本申请采用单一中性粒细胞膜囊泡包覆磁纳米颗粒制备仿生磁纳米颗粒,包封率更高(图10)。通过动态光散射测定,Neu-MNs粒径相比于杂化膜仿生纳米颗粒的粒径在2周时间范围内更稳定(图11)。傅里叶红外光谱图证明Neu-MNs在放置2周后仍然具有细胞膜的C-H键,表面官能团更稳定(图12)。DLS结果表明,Neu-MNs的平均直径单纯的MN大约19nm(图13中的a)。通过TEM观测和记录MNs和Neu-MNs的形貌特征,图像表明,膜包覆的纳米颗粒比裸核厚约9nm(图13中的b,c),Neu-MNs展现出明显的核壳结构。随后,SDS-PAGE用于鉴定Neu-MNs中的膜蛋白成分,显示出Neu-MNs完全继承了Neu-vesicles膜蛋白成分(图13的d)。
(4)抗上皮细胞黏附分子(anti-EpCAM)抗体与Neu-MNs生物共轭连接:
将制备的Neu-MNs用DSPE-PEG-COOH(羧基-聚乙二醇-磷脂)处理,然后加入NHS/EDC(N-羟基琥珀酰亚胺/(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐))活化1小时,接着交联上链霉亲和素(Streptavidin),用biotin-FITC(生物素偶联的荧光标记物)标记链霉亲和素连接的Neu-MNs,然后用共聚焦成像验证Neu-MNs是否能够成功和抗上皮细胞黏附分子(anti-EpCAM)生物偶联。验证成功后,将anti-EpCAM抗体与Neu-MNs混合进行生物共轭连接。Neu-MNs修饰anti-EpCAM抗体的路径如图4所示。链霉亲和素连接的Neu-MNs被biotin-FITC标记,共聚焦图像显示在纳米颗粒上有明显的FITC荧光(图13的e),表明Neu-MNs能够和anti-EpCAM生物偶联。此外,发明人还证实Neu-IMN具有出色的顺磁性(图14),这保证了在复杂生物介质中能够成功分离CTC。
(5)液相色谱串联质谱法(LC-MS/MS)验证Neu-IMNs的蛋白冠吸附减少以及桑格测序鉴定Neu-IMNs分离的循环肿瘤细胞:
蛋白质冠的蛋白质成分通过BCA试剂盒(KeyGEN BioTECH,中国)进行LC-MS/MS分析。将所有样品加入400μl摩尔浓度为1M尿素和25mM碳酸氢铵的溶液中,然后与5μl摩尔浓度为1M的二硫苏糖醇混合。然后,混合溶液在200℃下反应40分钟,随后冷却降至室温。混合物之后加入最终摩尔浓度为15mM的碘乙酰胺溶液。然后,将二硫苏糖醇水溶液加入混合物中以淬灭碘乙酰胺并在室温下振荡10分钟。随后使用蛋白质组学级胰蛋白酶在37℃下消化上述样品16小时。在使用LC-MS/MS(Thermo Scientific,USA)分析之前,用Speedvac(Thermo Scientific,USA)除去多余的溶剂。通过LC-MS/MS分析具有相同蛋白质含量的样品,并辅以连接到超高性能LC的质谱仪。质谱仪检测到最丰富的峰,并使用BioworksBrowser3.3.1SP1软件搜索光谱。使用以下等式实现光谱计数的归一化:
用桑格测序鉴定分离的循环肿瘤细胞,首先使用显微操作器来获取分离的细胞。使用全基因组扩增(WGA)试剂盒分别提取和扩增每个CTC的gDNA。之后,使用外显子9和外显子20特异性引物进行PCR扩增PIK3CA基因。gDNA在PIK3CA基因的外显子9和外显子20上扩增。最后,在青科生物科技有限公司使用上述引物进行Sanger测序以检测PIK3CA突变。此外,WBCs也被测序作为对照。
其中,PIK3CA-外显子9正向引物(SEQ ID NO:1):5’-GGGAAAAATATGACAAAGAAAGC-3’;
PIK3CA-外显子9反向引物(SEQ ID NO:2):5’-CTGAGATCAGCCAAATTCAGTT-3’;
PIK3CA-外显子9序列(SEQ ID NO:3):5’-TAGCTAGAGACAATGAATTAAGGGAAA-3’;
PIK3CA-外显子20正向引物(SEQ ID NO:4):5’-CTCAATGATGCTTGGCTCTG-3’;
PIK3CA-外显子20-反向引物(SEQ ID NO:5):5’-TGGAATCCAGAGTGAGCTTTC-3’;
PIK3CA-外显子20序列(SEQ ID NO:6):5’-TTGATGACATTGCATACATTCG-3’。
将Neu-IMNs与EpCAM阳性的MCF-7细胞共孵育来验证癌细胞的捕获性能。SEM图像显示大量Neu-IMNs附着在MCF-7细胞表面(图13的f),表明Neu-IMNs与癌细胞之间存在强结合。此外,发明人发现在最佳孵育浓度和时间条件下(100μg/ml,60min),Neu-IMNs对MCF-7细胞的捕获效率约为95%(图13的g),而Neu-MNs的这个值低于20%,有效地证明了经anti-EpCAM修饰后捕获效率大大提高。
实施例2
纳米粒子一旦进入生物环境(生理液中),很快就会在其表面形成蛋白冠,这将改变纳米粒子的界面结构和功能,并对纳米粒子在生物体液中命运产生至关重要的影响。在确认中性粒细胞膜包覆成功后,发明人发现该中性粒细胞膜能够有效减少蛋白冠的形成:
将IMNs(IMNs的制备步骤与Neu-MNs偶联上抗体的步骤相同)和Neu-IMNs分别与10%的人血浆进行了共孵育(100μg/ml,60min),随后将它们离心并用PBS洗涤以去除过量的生物分子。处理后,IMNs的尺寸明显增加(图15),这是由于IMNs表面产生蛋白冠所致。LC-MS/MS结果表明某些生物分子在孵育一段时间后已经聚集到IMN上(图16中的a)。与之形成鲜明对比的是,Neu-IMNs的大小没有明显改变(图16中的b),表明中性粒细胞膜以有效地抑制非特异性生物分子的吸附。处理前后蛋白质成分基本保持不变(图16中的c和表1),表明中性粒细胞膜涂层可以有效减少非特异性生物分子在Neu-IMN表面的聚集。
表1.在10%血浆中孵育4小时后,通过液相色谱串联质谱鉴定Neu-IMN上的每种蛋白质前后变化的标准化百分比的综合列表。NSpC:归一化光谱计数。
实施例3
在肿瘤转移过程中,中性粒细胞能够区分CTCs并与之交流,在本申请中发明人发现Neu-IMNs可以加强与CTC的相互作用:
将多种MNs与MCF-7细胞共培养(浓度100μg/ml,时间60min),并通过ICP-AES测试结合能力,结果表明中性粒细胞膜涂层可以有效增强MNs与癌细胞之间的相互作用(图16中的d和图17)。应用光穿孔方法制备的Neu-IMNs表现出相比于机械挤推和超声混合方法更高的MCF-7细胞捕获效率(图18)。Neu-IMNs在PBS中表现出比杂化膜仿生纳米颗粒更高的MCF-7细胞捕获效率(图19)。Neu-IMNs在PBS中显示出比IMNs显着提高的分离效率(图20),这得益于Neu-IMNs与癌细胞之间增强的相互作用。此外,发明人将MCF-7、PC-3和HeLa细胞加入10%的血浆中,并测试了细胞分离效率。与其他IMN相比,Neu-IMN表现出优异的EpCAM阳性细胞(即MCF-7和PC-3细胞)捕获效率(图16中的e)。对于EpCAM阴性细胞(即HeLa细胞),Neu-IMNs的捕获效率几乎是IMNs的2倍,表明单独的中性粒细胞膜涂层可以显着提高细胞分离效率,这得益于中性粒细胞-CTC的相互作用。此外,与PBS中的约90%的捕获效率相比,IMN在10%血浆中的细胞捕获效率急剧下降至约60%(图16中的e和图20),这可能是由蛋白冠的形成引起的。与此形成鲜明对比的是,Neu-IMNs在复杂的生物环境中保持了MCF-7细胞的优异分离效率,进一步证实了细胞膜涂层可以有效地减少anti-EpCAM的阻断。
实施例4
循环中的同源白细胞不会形成细胞团簇,而中性粒细胞是白细胞的一种,本发明发现Neu-IMN继承WBC这一特性:
将大量MNs与从新鲜人外周血样本中分离的WBC共同孵育(浓度100μg/ml,时间60min),然后通过ICP-AES进行检测。结果表明,Neu-IMNs在WBCs中的积累较少(图21中的a和图22),表明中性粒细胞膜可以成功地限制Neu-IMNs和WBCs之间的相互作用。此外,为了更好地模拟体内复杂的生理环境,将MCF-7细胞掺入新鲜的健康血液样本中并进行相同的实验。Neu-IMNs对癌细胞的分离效率优于其他IMNs(图21中的b),进一步证实即使在全血中,Neu-IMNs也可以有效提高癌细胞的捕获效率。更重要的是,当存在大量WBC时,Neu-IMN显示出比其他IMN更好的分离纯度(图21中的c),因为减少了WBC的干扰。值得注意的是,人体血液样本中CTC的数量大大低于人造样本。因此,在分离过程中,背景白细胞的影响会被急剧放大。化学和材料科学的最新进展表明,生物膜界面和细胞之间的软相互作用使分离细胞具有更好的活力。发明人测试了Neu-IMNs对分离细胞活力的影响,发现Neu-IMNs确实比IMNs具有更好的生物相容性(图21中的d),保证了后续细胞分析的可行性。
实施例5
基于临床血液样本,发明人比较了Neu-IMN与IMN对CTC的捕获效率。分离的CTC通过免疫荧光染色进行鉴定,其中DAPI、PE-anti-CK和FITC-anti-CD45分别用于区分细胞核、CTC和WBC。通过共聚焦显微镜观察,荧光信息用于区分CTC,即DAPI+、CK+、CD45-,以及WBC,即DAPI+、CK-、CD45+(图25中的a)。对于来自健康供体的血液样本,IMN或Neu-IMN未检测到CTC信号(图25中的b);而对于乳腺癌血液样本,与IMN相比,Neu-IMN的分离效率和纯度明显更高(图25中的b)。在乳腺癌患者捐献的20份血液样本中,有19个样本通过Neu-IMN成功分离出CTCs。值得一提的是,Neu-IMNs捕获的CTCs纯度高于IMNs。此外,发明人还比较了不同方法制备的Neu-IMN对CTC的捕获效率,光穿孔方法制备的Neu-IMN具有最高的CTC捕获效率(图23)。同其它杂化膜仿生免疫磁纳米颗粒相比,Neu-IMN也表现出更高的CTC捕获效率(图24)。
利用Neu-IMNs优异的生物相容性,成功培养了Neu-IMNs分离的CTC,并在SEM下观察(图25中的c)。更重要的是,考虑到CTC的不同突变点,发明人测试了捕获的CTC的频繁突变的PIK3CA基因。聚合酶链式反应(PCR)和Sanger测序用于基因突变分析。结果显示癌症转移与3140A/G突变呈正相关(图25中的d)。相反,对于分离的WBC,PIK3CA没有突变。结果表明,Neu-IMNs捕获的CTCs具有高纯度和优异的生物活性,可以直接用于CTC测序。
从以上的描述中,可以看出,本发明上述的实施例实现了如下技术效果:
1)相比于Qian-Fang Meng,et al.Biomimetic Immunomagnetic Nanoparticleswith Minimal Non-Specific Biomolecule Adsorption for Enhanced Isolation ofCirculating Tumor Cells.ACS Applied Materials&Interfaces,2019,11,28732-28739.,本申请通过增强与CTC的相互作用以及减少对白细胞的干扰,提高CTC的分离效率和纯度,使分离的CTC具有良好的生物活性便于后续的分析;
2)相比于Lang Rao,et al.Platelet–Leukocyte Hybrid Membrane-CoatedImmunomagnetic Beads for Highly Efficient and Specific Isolation ofCirculating Tumor Cells.Advanced Functional Materials.2018,28,1803531.,本申请中性粒细胞膜囊泡的制备不需要复杂的流程,设计更为方便,由于是单一的细胞膜,其包封率的稳定性和包封率也会更好。基于以上的优点,该技术能以较小成本实现CTC的高效高纯分离,并使分离的CTC具有良好的生物活性便于后续的分析。
3)实验数据表明,应用光穿孔方法制备中性粒细胞膜囊泡包覆的免疫磁纳米颗粒(Neu-IMNs)相比于其它常见的制备方法(如机械挤推、超声混合)是最优的,包封率最高,需要的原材料最少,且CTC的捕获效率最高,因而最有应用转化前景。
4)实验数据表明,相比于其它杂化膜囊泡(中性粒细胞和巨噬细胞杂化膜,红细胞和白细胞杂化膜)包覆的仿生磁纳米颗粒,基于光穿孔方法开发的中性粒细胞膜囊泡仿生磁纳米颗粒的包封率和稳定性都更好,能实现更高的CTC捕获效率。
5)实验数据表明,中性粒细胞膜囊泡包覆的免疫磁纳米颗粒(Neu-IMNs)可以同时减少非特异性蛋白质吸附,增强与CTC的相互作用,减少WBC的干扰,提高分离的CTC的活性。与包覆红细胞膜囊泡的磁纳米颗粒相比,Neu-IMNs在提高CTC捕获效率的同时还能提高捕获CTC的纯度,一举两得。在外周血样本中分离CTC的临床实践中,本申请设计的Neu-IMNs表现出优异的性能,能够为Neu-IMNs在无创早期诊断中的广泛应用奠定良好的基础。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 深圳湾实验室
<120> 仿生免疫磁纳米颗粒、其制备方法及应用
<130> PN173729SZWSY
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Claims (21)
1.一种仿生免疫磁纳米颗粒,其特征在于,包括:
磁纳米颗粒,以及
中性粒细胞膜囊泡,包覆在所述磁纳米颗粒上形成仿生磁纳米颗粒。
2.根据权利要求1所述的仿生免疫磁纳米颗粒,其特征在于,所述磁纳米颗粒为Fe3O4磁性纳米颗粒。
3.一种用于分离的仿生免疫磁纳米颗粒,其特征在于,包括:
权利要求1或2中所述的仿生免疫磁纳米颗粒,以及
靶向抗体,所述靶向抗体与所述仿生磁纳米颗粒物理连接和/或共价连接。
4.根据权利要求3所述的仿生免疫磁纳米颗粒,其特征在于,所述靶向抗体为CTC靶向抗体。
5.根据权利要求4所述的仿生免疫磁纳米颗粒,其特征在于,所述CTC靶向抗体为抗上皮细胞黏附分子抗体。
6.根据权利要求3所述的仿生免疫磁纳米颗粒,其特征在于,所述物理连接和/或共价连接为共轭连接或点击连接。
7.一种如权利要求1或2所述的仿生免疫磁纳米颗粒的制备方法,其特征在于,包括以下步骤:
S1,制备中性粒细胞膜囊泡;以及
S2,用所述中性粒细胞膜囊泡包覆磁纳米颗粒,获得仿生磁纳米颗粒。
8.根据权利要求7所述的制备方法,其特征在于,所述S1包括:从外周血中分离中性粒细胞制备所述中性粒细胞膜囊泡。
9.根据权利要求8所述的制备方法,其特征在于,采用梯度密度离心方法从外周血中分离所述中性粒细胞。
10.根据权利要求8所述的制备方法,其特征在于,分离的所述中性粒细胞通过低渗裂解缓冲液和反复冻融的方法破坏,超速离心收集细胞膜,然后通过微型推挤机挤出,制备得到所述中性粒细胞膜囊泡。
11.根据权利要求10所述的制备方法,其特征在于,所述微型推挤机通过800nm和400nm聚碳酸酯多孔膜挤出以得到所述中性粒细胞膜囊泡。
12.根据权利要求7所述的制备方法,其特征在于,所述S2包括:将所述中性粒细胞膜囊泡与所述磁纳米颗粒注入到微流控光穿孔芯片,所述中性粒细胞膜囊泡和所述磁纳米颗粒在光穿孔区域融合,使所述中性粒细胞膜囊泡包覆在所述磁纳米颗粒上形成所述仿生磁纳米颗粒。
13.根据权利要求12所述的制备方法,其特征在于,所述中性粒细胞膜囊泡和所述磁纳米颗粒的混合物通过所述微流控光穿孔芯片的光穿孔区域时的流速为2毫升/小时,使用激光能量密度为0.12焦耳/平方厘米的脉冲激光进行照射,促进磁纳米颗粒进入中性粒细胞膜囊泡得到所仿生磁纳米颗粒。
14.根据权利要求12所述的制备方法,其特征在于,所述中性粒细胞膜囊泡与所述磁纳米颗粒的摩尔比为(1:1)~(1:10)。
15.一种如权利要求3至6中任一项所述的用于分离的仿生免疫磁纳米颗粒的制备方法,其特征在于,包括以下步骤:
按照如权利要求7至14中任一项所述的制备方法制备仿生免疫磁纳米颗粒,
S3,将靶向抗体与所述仿生磁纳米颗粒通过物理连接和/或共价连接得到仿生免疫磁纳米颗粒。
16.根据权利要求15所述的制备方法,其特征在于,所述靶向抗体为CTC靶向抗体。
17.根据权利要求16所述的制备方法,其特征在于,所述CTC靶向抗体为抗上皮细胞黏附分子抗体。
18.根据权利要求17所述的制备方法,其特征在于,所述S3包括:将所述S2制备的所述仿生磁纳米颗粒用羧基-聚乙二醇-磷脂处理,然后加入NHS/EDC活化,接着交联上链霉亲和素,生物素化的CTC靶向抗体与所述链霉亲和素的结合得到所述仿生免疫磁纳米颗粒。
19.一种权利要求3至6中任一项所述的用于分离的仿生免疫磁纳米颗粒在制备用于富集、分离或检测循环肿瘤细胞的产品中的应用。
20.一种权利要求3至6中任一项所述的用于分离的仿生免疫磁纳米颗粒在制备用于***转移相关疾病的药物中的应用。
21.根据权利要求20所述的应用,其特征在于,所述肿瘤转移相关疾病为上皮细胞粘附因子高表达相关的肿瘤,优选为结肠直肠癌、乳腺癌和胃癌。
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