CN113866073A - 用于高效捕获癌细胞的酶敏感纳米材料及制备方法和应用 - Google Patents
用于高效捕获癌细胞的酶敏感纳米材料及制备方法和应用 Download PDFInfo
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Abstract
本发明公开了用于高效捕获癌细胞的酶敏感纳米材料及制备方法和应用，所述纳米材料为具有三层结构且带正电的磁性纳米颗粒，三层结构依次是：最外层为识别并捕获癌细胞的效应高分子层，中间层为酶敏感高分子层，内核为四氧化三铁。与现有技术相比，本发明具有以下优点：(1)本发明所述酶敏感纳米材料能够高效捕获离体血样中的循环肿瘤细胞，并实现无损、低损释放，且无需考虑其体内生物安全性及纳米材料尺寸的限制；(2)仅通过淀粉酶水解即可实现肿瘤细胞的释放，因此释放的循环肿瘤细胞可直接用于后续研究。
Description
技术领域
本发明属于纳米材料技术领域，涉及一种经表面修饰的纳米材料，具体为用于高效捕获癌细胞的酶敏感纳米材料及制备方法和应用。
背景技术
循环肿瘤细胞在癌症治疗中，因其检测方式的非介入性、采样的便捷和及时以及其与原发病灶部位的同源性，具有非常高的潜在研究价值。磁性纳米颗粒在通过表面修饰抗体或带电高分子材料后，能够通过抗原抗体反应或异性电荷相吸的作用与血液中的癌细胞紧密结合。研究者们期望能够实现对捕获到的循环肿瘤细胞的释放，从而能够对患者来源的循环肿瘤细胞进行深入的后续研究。因此，发明一种高效捕获癌细胞的酶敏感纳米材料具有重要意义。
现有技术中关于生物医药用的纳米材料种类繁多，其中针对肿瘤细胞检测的多为体内应用。如中国专利申请CN1822254A公开了一种制备亲水性有机高分子物质包裹的磁性纳米颗粒，用于细胞分离、肿瘤热疗、磁共振造影和靶向给药等生物医学领域，它包括磁流体的制备和表面醛基化，通过将铁盐溶液加入淀粉溶液中在氮气保护下搅拌，调节溶液的pH至10～11后60～80℃搅拌1～2h，除杂后得到表面富含羟基的磁性淀粉复合纳米颗粒，均匀分散于水相后得到该磁流体；而后在磁流体样品中加入高碘酸钠，在氮气保护下避光发生氧化反应，得到表面富含醛基的磁性淀粉复合纳米颗粒。中国专利申请CN1993469A公开了一种由磁性氧化铁内核和天然高分子材料外壳组成的纳米颗粒，它包括将高分子材料在酸性溶液中溶解，加入γ-Fe2O3粒子作为水相，石蜡、表面活性剂、助表面活性剂混合为油相，二者混合得到油包水型反向微乳液，而后加入交联剂搅拌后洗涤除杂即可得到产物。以上两个现有技术均是考虑体内应用，极大地限制了磁性纳米颗粒表面修饰的可能性。例如，若要在表面修饰带正电性的高分子，就很难应用于体内。
发明内容
解决的技术问题：为了克服现有技术的不足，获得用于捕获循环肿瘤细胞的磁性纳米颗粒，且专门针对离体血样检测，本发明提供了用于高效捕获癌细胞的酶敏感纳米材料及制备方法和应用。
技术方案：用于高效捕获癌细胞的酶敏感纳米材料，所述纳米材料为具有三层结构且带正电的磁性纳米颗粒，三层结构依次是：最外层为识别并捕获癌细胞的效应高分子层，中间层为酶敏感高分子层，内核为四氧化三铁。
优选的，最外层为抗体或正电性高分子，中间层为直链淀粉。
优选的，所述正电性高分子为聚乙烯亚胺，壳聚糖或氨基聚乙二醇。
以上任一所述用于高效捕获癌细胞的酶敏感纳米材料的制备方法，所述方法首先将直链淀粉包裹于四氧化三铁磁颗粒表面使其带有负电荷，然后通过正负电荷吸附的原理在直链淀粉层外进行抗体或正电性高分子的表面修饰，从而制备获得表面带正电荷的酶敏感纳米材料。
优选的，所述方法具体包括以下步骤：
S1：量取二甲亚砜(DMSO)加入去离子水中，二甲亚砜(DMSO)与去离子水的体积比为9:1；
S2：将直链淀粉溶于S1制得的溶液中，直链淀粉质量与溶液的体积比为1-5:1，90℃水浴直至淀粉充分糊化，冷却至室温；
S3：向S2糊化后的淀粉中加入四氧化三铁纳米颗粒超声分散均匀，逐滴加入无水乙醇，继续超声分散均匀，然后进行磁分离、洗涤得到淀粉包裹的磁性纳米颗粒，其中四氧化三铁纳米颗粒与直链淀粉的质量比为1:25，无水乙醇与糊化后淀粉的体积比为1-2.5:1；
S4：将淀粉包裹的磁性纳米颗粒分散于无水甲醇中，并向其中滴加0.8-1mg/mL的PEI甲醇溶液；或向其中滴加0.5-1mg/mL的壳聚糖乙酸溶液，其中乙酸浓度为1％；或向其中滴加0.5-1mg/mL的PEG-NH2水溶液，超声搅拌均匀后分离即可获得酶敏感纳米材料。
优选的，S4中PEI与磁性纳米颗粒的质量比为1:1-1.5。
以上任一所述用于高效捕获癌细胞的酶敏感纳米材料在制备捕获离体血样中癌细胞试剂盒中的应用。
本发明所述酶敏感纳米材料高效捕获癌细胞的原理在于：本发明的酶敏感纳米材料为三层结构，具体为四氧化三铁磁性内核、酶敏感的高分子中间层、识别并捕获癌细胞的最外层，本发明具有三层核壳结构的纳米材料通过最外层快速、高效的识别并捕获离体血液中的循环肿瘤细胞，然后经淀粉酶水解实现癌细胞的无损、低损释放，从而将循环肿瘤细胞用于后续研究。
有益效果：(1)本发明所述酶敏感纳米材料能够高效捕获离体血样中的循环肿瘤细胞，并实现无损、低损释放，且无需考虑其体内生物安全性及纳米材料尺寸的限制；(2)仅通过淀粉酶水解即可实现肿瘤细胞的释放，因此释放的循环肿瘤细胞可直接用于后续研究。
附图说明
图1是本发明所述酶敏感纳米材料的三层结构模拟图；
图2是所述酶敏感纳米材料捕获循环肿瘤细胞并酶解释放的模拟图；
图3是淀粉修饰后的磁性纳米颗粒透射电镜图；
图4是修饰上正电性高分子层后纳米材料的电位图；
图5是捕获率与释放率图。
具体实施方式
以下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。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改和替换，均属于本发明的范围。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。
实施例1
本实施例所述酶敏感纳米材料由以下方法制得：
(1)量取45mL DMSO，加入5mL去离子水，使DMSO:水(体积比)＝9:1；
(2)称取100mg直链淀粉溶于上述溶液中，直链淀粉的质量与溶液体积的比例为5:1至1:1，90℃下水浴，使淀粉充分糊化；
(3)冷却至室温后，加入4mg四氧化三铁纳米颗粒，充分超声分散均匀；
(4)逐滴滴加无水乙醇，继续超声，无水乙醇的体积50-125mL；
(5)磁分离，洗涤可得淀粉包裹的磁性纳米颗粒；
(6)将上述产物溶于25mL无水甲醇中，超声搅拌15分钟使得纳米颗粒分散均匀；
(7)称取PEI(Mw＝10000)3-5mg溶于5ml无水甲醇中，逐滴加入到溶液中。超声搅拌2小时；
(8)磁分离，洗涤可得三层结构的带正电磁性纳米颗粒。
结果分析：图3淀粉修饰后可看出，淀粉修饰后的纳米颗粒间呈现出膜状粘连。且进一步修饰PEI，电荷从负电变成了正电。
实施例2
与实施例1的区别在于：步骤(7)中称取壳聚糖2.5-5mg溶于5ml的1％乙酸中，逐滴加入到溶液中，超声搅拌2小时；从而制备获得正电性高分子为壳聚糖的三层结构的带正电磁性纳米颗粒。
结果分析：如图4所示，修饰壳聚糖后，表面电荷从负电变成了正电。
实施例3
与实施例1的区别在于：步骤(7)中称取PEG-NH2 2.5-5mg溶于5ml去离子水中，逐滴加入到溶液中，超声搅拌2小时；从而制备获得正电性高分子为氨基聚乙二醇的三层结构的带正电磁性纳米颗粒。
结果分析：如图4所示，修饰PEG-HN2后，表面电荷从负电变成了正电。
实施例4
采用实施例1制得的酶敏感纳米材料进行癌细胞捕获：
(1)将已知量的癌细胞加入定量PBS中，可得到初始癌细胞浓度；
(2)将本发明的酶敏感纳米材料以40μg/mL的浓度加入(1)中，4℃下孵育10分钟
(3)用磁铁或磁力架对(2)进行磁分离，取上清计数用于计算脱靶率；
(4)取沉淀加入4mg/mL低温α-淀粉酶，调节pH值为6，37℃下共孵育10-30分钟
(5)用磁铁或磁力架对(4)进行磁分离，取上清计数用于计算释放率。
结果分析：如图5所示，经过脱靶率计算，捕获率可达90％以上；释放率可达80％。
Claims (7)
1.用于高效捕获癌细胞的酶敏感纳米材料，其特征在于，所述纳米材料为具有三层结构且带正电的磁性纳米颗粒，三层结构依次是：最外层为识别并捕获癌细胞的效应高分子层，中间层为酶敏感高分子层，内核为四氧化三铁。
2.根据权利要求1所述的用于高效捕获癌细胞的酶敏感纳米材料，其特征在于，最外层为抗体或正电性高分子，中间层为直链淀粉。
3.根据权利要求2所述的用于高效捕获癌细胞的酶敏感纳米材料，其特征在于，所述正电性高分子为聚乙烯亚胺、壳聚糖或氨基聚乙二醇。
4.权利要求1-3任一所述用于高效捕获癌细胞的酶敏感纳米材料的制备方法，其特征在于，所述方法首先将直链淀粉包裹于四氧化三铁磁颗粒表面使其带有负电荷，然后通过正负电荷吸附的原理在直链淀粉层外进行抗体或正电性高分子的表面修饰，从而制备获得表面带正电荷的酶敏感纳米材料。
5.根据权利要求4所述用于高效捕获癌细胞的酶敏感纳米材料的制备方法，其特征在于，所述方法具体包括以下步骤：
S1：量取DMSO加入去离子水中，DMSO与去离子水的体积比为9:1；
S2：将直链淀粉溶于S1制得的溶液中，直链淀粉质量与溶液的体积比为1-5:1，90℃水浴直至淀粉充分糊化，冷却至室温；
S3：向S2糊化后的淀粉中加入四氧化三铁纳米颗粒超声分散均匀，逐滴加入无水乙醇，继续超声分散均匀，然后进行磁分离、洗涤得到淀粉包裹的磁性纳米颗粒，其中四氧化三铁纳米颗粒与直链淀粉的质量比为1:25，无水乙醇与糊化后淀粉的体积比为1-2.5:1；
S4：将淀粉包裹的磁性纳米颗粒分散于无水甲醇中，并向其中滴加0.8-1mg/mL的PEI甲醇溶液；或向其中滴加0.5-1mg/mL的壳聚糖乙酸溶液，其中乙酸浓度为1％；或向其中滴加0.5-1mg/mL的PEG-NH2水溶液，超声搅拌均匀后分离即可获得酶敏感纳米材料。
6.根据权利要求5所述的用于高效捕获癌细胞的酶敏感纳米材料的制备方法，其特征在于，S4中PEI与磁性纳米颗粒的质量比为1:1-1.5。
7.权利要求1-3任一所述用于高效捕获癌细胞的酶敏感纳米材料在制备捕获离体血样中癌细胞试剂盒中的应用。
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