CN115554992B - 一种聚合物修饰的磁性纳米材料、其制备方法及应用 - Google Patents
一种聚合物修饰的磁性纳米材料、其制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种聚合物修饰的磁性纳米材料、其制备方法及应用。本发明提供了一种聚合物修饰的磁性纳米材料的制备方法,其包括如下步骤:在惰性气氛存在下,在等离子体辉光存在下,将所述的聚合物与溶剂的混合物加热得到雾化形态,与所述的磁性纳米材料进行改性修饰;得到所述的聚合物修饰的磁性纳米材料即可。本发明提供的聚合物修饰的磁性纳米材料的聚合物修饰量高、稳定性好,可在糖基化蛋白、多肽类物质、核酸、循环肿瘤细胞、外泌体等的富集分离中应用,响应时间快;如可用作制备捕获外周血/尿液等体液中循环肿瘤细胞的药物或试剂。
Description
技术领域
本发明涉及一种聚合物修饰的磁性纳米材料、其制备方法及应用。
背景技术
随着人口数量的增长和老龄化以及环境问题等原因,我国癌症的发病率和死亡率一直在不断提高,癌症在中国乃至世界范围内都是致死的主要疾病之一,长期以来难以取得有效的治疗效果,因此对癌症的研究长期以来都是全球各地科学技术研究人员所热衷的领域。早期诊断和治疗对于挽救患者生命具有极其重要的意义,众多研究致力于癌症的早期诊断,通过早期诊断和治疗可以延长患者生存期、增加生存率和挽救患者的生命。
循环肿瘤细胞(CTC)是被广泛认为是从实体瘤患者肿瘤部位处脱落并进入患者血液循环系统中的各类肿瘤细胞的统称,也被普遍认为是导致癌症发生转移的主要因素,而癌症的转移是引发病患死亡的最根本原因,也是术后复发的重要因素,很多研究证明手术、化疗等治疗手段也是导致肿瘤细胞从病灶脱落入血从而形成循环肿瘤细胞的重要因素。循环肿瘤细胞被认为是最有潜力的多功能生物标志物,在乳腺癌、肺癌、结直肠癌乃至前列腺癌等众多不同类型的癌症中都有循环肿瘤细胞被发现,循环肿瘤细胞的检出、计数及相关分析对于患者病情判断有很重要的意义,有望应用于肿瘤早期检测、辅助治疗、疗效评估以及预后判断。
目前,已报道的CTC富集的方法主要有物理法和生物法。如细胞过滤器富集CTC,让小的血细胞通过截留大的肿瘤细胞;基于抗体识别细胞表面蛋白,如利用上皮细胞黏附分子(EpCAM)将癌变的上皮癌细胞捕获。但这些方法都不是基于肿瘤细胞独有的性质进行捕获的,检测存在假阳性或假阴性,且不具有广谱性,无法广泛应用。我们前期研究发现,由于肿瘤细胞的糖酵解产生大量乳酸,使其表面带大量负电荷,而正常细胞呈现电中性或带少量正电荷。因此,基于肿瘤细胞与正常细胞之间独特的电荷差异,可实现CTC的高效高选择性富集,且具有广谱性。构建高性能的正电荷纳米材料是捕获负电荷CTC的关键。
其中,四氧化三铁(Fe3O4)磁性纳米颗粒由于具有特殊的结构和优异的性能,在生物技术与医学相关领域受到广泛关注和研究。这类材料通常以已经相当成熟的方法制备得到的磁性纳米粒子为基础,表面包覆二氧化硅等无机材料或其它有机高分子材料,然后进一步进行反应或表面修饰,制备得到具有铁磁性的多功能复合纳米微粒。这种纳米材料通常在材料粒径大小、磁强度等特征易于控制,生物相容性以及稳定性都很好,且易于修饰,极大的拓展了其应用范围,在药物靶向运输、基因载体、生物磁分离、磁热疗、磁共振成像等诸多领域均有研究应用。基于Fe3O4纳米颗粒优良的物理化学性质,开发简单、可大量制备、生物相容性好、表面带正电荷的修饰方法,可为CTC富集、检测以及指导治疗提供高质量的纳米探针。
但是,目前现有技术报道的纳米材料存在聚合物量不足、稳定性差,响应时间长等不足。
发明内容
本发明所要解决的技术问题在于克服现有技术中磁性材料(带正电荷的磁性纳米粒子(positively charged magnetic nanoparticles,PCMNs))聚合物量不足、稳定性差,响应时间长等缺陷,而提供了一种聚合物修饰的磁性纳米材料、其制备方法及应用。本发明的聚合物修饰的磁性纳米材料稳定性好,响应时间快,能够实现从复杂样品中对糖基化蛋白、多肽类物质、核酸、循环肿瘤细胞、外泌体的高选择性、高重复性和高通量地富集;可应用于制备活体荧光和磁共振双模态成像显影剂或用于治疗癌症的光热治疗剂。
本发明是通过下述技术方案来解决上述技术问题的。
本发明提供了一种聚合物修饰的磁性纳米材料,其包括如下结构:
所述的聚合物为阳离子型聚合物;所述的聚合物包覆于磁性纳米粒子表面(即,位于所述壳的外层,简称涂层),形成带正电荷的所述聚合物修饰的磁性纳米材料;
所述的磁性纳米材料为核-壳结构,所述的核为磁性纳米粒子(核),所述的壳为改性层;所述的改性层附着或包覆于所述磁性纳米粒子的表面,形成改性层复合的磁性纳米粒子;
其中,所述的聚合物与所述的磁性纳米材料的质量比为1:10~20:1。
在本发明的某一方案中,所述的聚合物与所述的磁性纳米材料的质量比可为1:5至3:1;例如1:3。
在本发明的某一方案中,所述的聚合物修饰的磁性纳米材料的电位可为+5至+60mV,例如+10至+50mV,优选+20至+40mV(又例如+35mV)。
在本发明的某一方案中,所述的磁性纳米材料为带负电荷的磁性纳米材料,例如其电位可为-10至-60mV;例如-20至-40mV。
在本发明的某一方案中,所述的聚合物修饰的磁性纳米材料的粒径可为10nm至600nm;例如粒径为300nm至500nm,又例如350nm至400nm。
在本发明的某一方案中,所述的磁性纳米材料的粒径可为5nm至500nm;例如,300nm至350nm。
在本发明的某一方案中,所述的壳的厚度可为1nm至100nm,例如40nm至60nm。
在本发明的某一方案中,所述的磁性纳米粒子的粒径可为5nm至500nm;例如250nm至300nm。
在本发明的某一方案中,所述的聚合物为(树)支状聚合物。
在本发明的某一方案中,所述的聚合物可为重均分子量MW在2000至300000之间。
在本发明的某一方案中,所述的聚合物为本领域常规的阳离子型聚合物;例如,聚乙烯亚胺(PEI,Polyethylenimine)、壳聚糖(β-壳聚糖)和聚吡咯中的一种或多种。
在本发明的某一方案中,所述的聚合物可为:重均分子量可为2000-100000的聚乙烯亚胺,例如为MW=10000,99%纯度;重均分子量可为50000-300000的β-壳聚糖,例如MW=50000;重均分子量可为5000的聚吡咯。
在本发明中,所述的磁性纳米粒子可为本领域中常规的磁性纳米粒子,例如氧化物磁性纳米粒子(又例如Fe3O4、γ-Fe2O3)、磁性金属纳米粒子、磁性硫化物纳米粒子、磁性复合粒子中的一种或几种;再例如磁性Fe3O4纳米粒子(以下简称Fe3O4)。所述磁性纳米粒子可采用本领域常规的方法制备得到,例如溶剂热法、共沉淀法等。
所述磁性纳米粒子使得所述聚合物修饰的磁性纳米材料能够具有磁性,进而能够在磁铁的作用下进行移动,可作为探针。
在本发明中,所述的磁性纳米材料可为本领域中常规的磁性纳米材料,其中,所述的改性层包裹于所述的磁性纳米粒子的表面形成核-壳结构复合的磁性纳米材料;所述的改性层形成的壳(层)既可以阻止其团聚,又能防止其被破坏,还可以对其进行表面功能化。
在本发明的某一方案中,所述的改性层的材料可为本领域常规的为有机物和/或无机物的改性层材料;例如二氧化硅或标记荧光和/或表面活性剂修饰的二氧化硅;例如二氧化硅或标记荧光的二氧化硅。即,所述的磁性纳米材料可为二氧化硅(SiO2)复合的磁性纳米粒子、或、标记荧光和/或表面活性剂修饰的二氧化硅复合的磁性纳米粒子。
在本发明的某一方案中,当所述的磁性纳米材料为二氧化硅改性层复合的磁性纳米粒子时,所述的二氧化硅改性层复合的磁性纳米粒子为二氧化硅改性层复合的磁性Fe3O4纳米粒子(以下简称Fe3O4@SiO2,四氧化三铁/二氧化硅复合微球)。
在本发明的某一方案中,所述的改性层(例如所述二氧化硅改性层)的表面含有修饰得到的氨基,进而使得其具有能够与进一步修饰(改性)的物质进行反应的基础。所述的修饰可采用本领域常规的表面修饰剂;例如,通过在二氧化硅的表面修饰氨基,进而使得其具有能够与具有羧基的荧光染料进行酰胺反应的基础。所述的标记荧光的二氧化硅复合的磁性纳米粒子中,荧光染料例如通过酰胺反应与二氧化硅改性层键合。在本发明的某一方案中,所述的二氧化硅复合的磁性纳米粒子中,所述二氧化硅层进行修饰是通过表面化学修饰剂在二氧化硅层的表面修饰上氨基。所述表面化学修饰剂可为本领域常规的能够对二氧化硅复合的磁性纳米粒子表面进行氨基修饰的表面修饰剂;例如氨水和/或APTES(3-氨丙基三乙氧基硅烷);又例如氨水。
在本发明的某一方案中,所述的改性层与所述的磁性纳米粒子的质量比可为50:1~1:10;例如10:1。
在本发明的某一方案中,所述聚合物修饰的磁性纳米材料的稳定时长为2年。
在本发明的某一方案中,所述聚合物修饰的磁性纳米材料的响应时间为3S至2min。
在本发明的某一方案中,所述的磁性纳米材料为标记荧光的二氧化硅复合的磁性纳米粒子,所述的标记荧光的二氧化硅复合的磁性纳米粒子中的荧光染料(或荧光标记物)可为本领域该类材料中常规的荧光染料,例如具有羧基或能够与氨基发生酰胺反应的荧光染料,例如异硫氰酸荧光素(fluorescenceisothiocyanate,FITC)和/或罗丹明类染料,和/或其被修饰物;所述荧光染料可为异硫氰酸荧光素、罗丹明B、罗丹明B异硫氰酸酯(Rhodamine B 5-isothiocyanate,RBITC)和四甲基异硫氰酸罗丹明(tetramethylrhodamineisothiocyanate,TRITC)中的一种或多种。所述的修饰物可为APS修饰的异硫氰酸荧光素和/或APS修饰的罗丹明类染料。所述APS可为3-氨基丙基三乙氧基硅烷(APTES)和/或3-氨基丙基三甲基硅烷(APTMS))。例如,所述的FITC被修饰物可为APS-FITC(或称FITC-APS/APS修饰FITC),又例如,所述的荧光染料为APS-FITC。
所述的标记荧光的二氧化硅复合的磁性纳米粒子中的荧光染料(或荧光标记物)采用该领域常规的方式修饰于所述的二氧化硅复合的纳米粒子的表面,形成标记荧光的二氧化硅复合的磁性纳米粒子;例如,通过连接键(例如如上所述的酰胺键)连接于二氧化硅复合的磁性纳米粒子的表面。例如,所述的标记荧光的二氧化硅复合的磁性纳米粒子为APS-FITC标记的Fe3O4@SiO2。所述的二氧化硅改性层复合的磁性纳米粒子为二氧化硅改性层复合的磁性Fe3O4纳米粒子(以下简称Fe3O4@SiO2,四氧化三铁/二氧化硅复合微球)
在本发明的某一方案中,所述的磁性纳米材料可为表面活性剂修饰的二氧化硅复合的磁性纳米粒子。所述的表面活性剂可包括乙酸钠、柠檬酸三钠、壳聚糖、聚乙烯吡咯烷酮、聚对苯二甲酸乙二醇酯、硬脂酸、阿拉伯树胶、羟丙基甲基纤维素、海藻酸钠、十二烷基硫酸钠、十二烷基苯磺酸钠、聚乙烯醇、长链脂肪酸、淀粉和十二硫醇中的一种或两种以上的组合。通过上述表面活性剂修饰,例如能够避免形成的纳米粒子进行团聚,从而达到对聚合物修饰的磁性纳米材料的粒径的控制。
在本发明的某一方案中,当所述的磁性纳米材料为标记荧光的二氧化硅改性层复合的磁性纳米粒子(例如APS-FITC标记的Fe3O4@SiO2)时,所述的二氧化硅复合的磁性纳米粒子与所述的荧光染料(例如APS-FITC)的质量比值可为20。
在本发明的某一方案中,所述的聚合物修饰的磁性纳米材料为聚乙烯亚胺(PEI,Polyethylenimine)修饰的APS-FITC荧光标记的Fe3O4@SiO2磁性纳米材料,其中,所述的PEI重均分子量MW=10000,99%(纯度);所述聚乙烯亚胺与所述磁性纳米材料的质量比可为1:3;所述的聚合物修饰的磁性纳米材料的粒径可为20nm~500nm;其电位可为+10mV~+60mV。
在本发明的某一方案中,所述的聚合物修饰的磁性纳米材料为β-壳聚糖修饰的APS-FITC荧光标记的Fe3O4@SiO2磁性纳米材料;其中,所述的β-壳聚糖的重均分子量MW=50000;所述β-壳聚糖与所述磁性纳米材料的质量比可为1:3;所述的聚合物修饰的磁性纳米材料的粒径可为20nm~500nm;其电位可为+10~+60mV。
在本发明的某一方案中,所述的聚合物修饰的磁性纳米材料为聚吡咯修饰的APS-FITC荧光标记的Fe3O4@SiO2磁性纳米材料;其中,所述的聚吡咯的重均分子量可为5000;所述聚吡咯与所述磁性纳米材料的质量比可为1:3;所述的聚合物修饰的磁性纳米材料的粒径可为20nm~500nm;其电位可为+10~+60mV。
本发明还提供了一种聚合物修饰的磁性纳米材料的制备方法,其包括如下步骤:
将聚合物与溶剂的混合物与磁性纳米材料进行改性修饰,得到所述的聚合物修饰的磁性纳米材料即可;其中,所述的聚合物与溶剂的混合物为雾化形态;
其中,所述的聚合物、所述的磁性纳米材料的定义如上所述的聚合物修饰的磁性纳米材料中任一方案所示。
其中,
在本发明的某一方案中,所述的聚合物与溶剂的混合物中,所述的溶剂可为该领域常规的溶剂,例如醇类溶剂,所述的醇类溶剂可为甲醇。所述的聚合物在所述的与溶剂的混合物中的质量体积比可为本领域常规的质量体积比,例如5mg/mL。
所述的聚合物与溶剂的混合物的雾化形态可为本领域常规的方法得到,例如通过加热所述的聚合物与溶剂的混合物得到,较佳地,将所述的聚合物与溶剂的混合物通过等离子体法加热得到所述的雾化形态。
所述的聚合物与溶剂的混合物的加入可为控制通入所述的雾化气体的体积流量在3-5sccm。(sccm为体积流量单位,又称质流单位(Mass flow),表示标准毫升/分钟:mL/min)。
所述的改性修饰的温度可为100至300℃;例如200℃。
较佳地,所述的改性修饰在惰性气氛存在下进行。所述的惰性气氛可为氮气和/或氩气。
本发明中,所述的改性修饰,较佳地,例如采用等离子体法进行改性修饰,所述的等离子体法的条件和操作可为本领域常规的等离子体法的条件和操作,本发明中优选如下步骤,在惰性气氛存在下,在等离子体辉光存在下,将所述的聚合物与溶剂的混合物加热得到雾化形态,与所述的磁性纳米材料进行改性修饰;得到所述的聚合物修饰的磁性纳米材料即可。
在本发明的某一方案中,所述的等离子体辉光可为如下步骤得到,在所述的惰性气氛下,调节射频功率,使等离子反应腔内产生等离子体辉光;所述惰性气氛的压力可为在300-400Pa之间;所述射频的功率可为10W±5W;较佳地,在真空下,射频电源预热,再向等离子体反应腔通所述的惰性气氛;所述的真空可为200Pa以下,例如150-200Pa。
在本发明的某一方案中,所述的反应的时间可为1-2小时。
所述的磁性纳米材料可为本领域常规的制备方法制备得到。本发明中优选如下:
在本发明的某一方案中,当所述的磁性纳米材料为二氧化硅(SiO2)复合的磁性纳米粒子或标记荧光的二氧化硅复合的磁性纳米粒子、所述的二氧化硅复合的磁性纳米粒子为Fe3O4@SiO2时,本发明中优选采用如下步骤制备得到:
步骤(a)、在碱性试剂存在下,将硅试剂加入到Fe3O4磁性纳米微粒与溶剂的体系中进行改性反应,得到所述的Fe3O4@SiO2即可;和/或,
步骤(b)、在溶剂和碱性试剂中,将步骤(a)得到所述的Fe3O4@SiO2与荧光染料进行荧光标记反应,得到所述的标记荧光的二氧化硅复合的磁性纳米粒子即可。
步骤(a)中,所述的溶剂可为水,或水和醇类溶剂,所述的醇类溶剂可为乙醇。
所述的碱性试剂可为氨水。
所述的硅试剂可为正硅酸乙酯(TEOS)或正硅酸甲酯;例如TEOS。
所述的Fe3O4磁性纳米微粒与所述的二氧化硅试剂质量体积比可为1500g/L。
所述的二氧化硅试剂可以与所述的溶剂的混合物形式使用;例如2mL乙醇溶解100μl TEOS。
所述的碱性试剂的用量可为使所述的Fe3O4磁性纳米微粒与溶剂的体系的pH为9.5±0.5即可。
所述的改性反应,较佳地,在超声和/或机械搅拌条件下进行。
步骤(a)中,其还可包括后处理步骤,所述的后处理可为如下步骤,所述的反应结束后,洗涤经磁分离辅助条件获得的所述的Fe3O4@SiO2,即可;所述的洗涤可为分别使用乙醇和去离子水洗涤,例如洗涤三次;较佳地,洗涤后,将得到的Fe3O4@SiO2分散在去离子水中,配制成所需浓度的溶液待用即可,例如浓度为100mg/mL的溶液。
步骤(b)中,所述的溶剂可为醇类溶剂和水的混合物。所述的水可为去离子水。所述的醇类溶剂可为乙醇。所述的醇类溶剂和水的体积比可为9:1~10:1(例如9.7:1)。
所述的二氧化硅复合的磁性纳米粒子(例如Fe3O4@SiO2)与所述的溶剂的质量体积比可为0.56至0.6g/L。
所述的碱性试剂可为氨水。所述的二氧化硅复合的磁性纳米粒子与所述的氨水的质量体积比可为42至45g/L。
所述的荧光染料可为与所述的溶剂的混合物形式(例如溶液)使用,所述的溶液中的溶剂可为醇类溶剂,例如乙醇。所述的荧光染料与溶剂的混合物中,所述的溶剂与所述的荧光染料的体积质量可为1.7mL/mg。例如,当所述的荧光染料为APS-FITC时,所述的APS-FITC可为溶液形式,例如APS-FITC的乙醇溶液形式,又例如2.5mL乙醇中含1.5mg FITC。
所述的荧光标记反应可在超声和机械搅拌条件下进行。
所述的荧光标记反应可在避光条件下进行。
在本发明的某一方案中,所述的荧光标记反应中,还可加入如上所述的硅试剂(即,在荧光标记反应的同时,进行包被反应),即同时进行二氧化硅进一步包被。其中,所述的二氧化硅复合的磁性纳米粒子与所述的硅试剂的质量体积比可为1000g/L。所述的硅试剂可为与所述的溶剂的混合物形式;例如,正硅酸乙酯的乙醇溶液,又例如1mL乙醇中含30μl正硅酸乙酯。
在本发明的某一方案中,当所述的磁性纳米材料为标记荧光的二氧化硅复合的磁性纳米粒子,所述的标记荧光的二氧化硅复合的磁性纳米粒子为含APS-FITC荧光标记的二氧化硅复合的磁性纳米粒子(例如Fe3O4@SiO2)时,采用如下步骤制备得到,将TEOS和APS-FITC依次加入到Fe3O4@SiO2与溶剂和氨水的混合体系中进行反应,得到所述的标记荧光的二氧化硅复合的磁性纳米粒子即可。较佳地,在避光条件下,在超声和机械搅拌条件下,在TEOS缓慢地逐滴加入Fe3O4@SiO2与乙醇和氨的混合体系中后,快速加入APS-FITC的溶液,进行所述的荧光标记反应。
在本发明的某一方案中,所述的标记荧光的二氧化硅复合的磁性纳米粒子的制备方法中,其还可包括后处理步骤,所述的后处理的操作和条件可为本领域常规的操作和条件,本发明中所述的后处理可为如下步骤,所述的反应结束后,洗涤经磁分离辅助条件获得的所述的磁性纳米粒子,即可;所述的洗涤可为分别使用乙醇和去离子水洗涤,例如洗涤三次。
在本发明的某一方案中,所述的聚合物修饰的磁性纳米材料中,当所述的纳米粒子为被荧光染料标记、所述的荧光染料为APS-FITC时,所述的APS-FITC可为如下步骤制备得到:将APS加入到FITC的乙醇溶液中反应,得到所述的APS-FITC即可。其中,所述的反应较佳地在避光条件下进行;所述的反应以得到澄清的溶液即可,例如混合过夜,例如8-24小时。FITC与APS的质量体积比可为300g/L。
在本发明的某一方案中,所述的Fe3O4磁性纳米微粒与溶剂的体系采用如下步骤制备得到,在超声和机械搅拌条件下,在溶剂中,将Fe3O4纳米磁珠依次用盐酸、去离子水洗涤至上清液pH中性,即可。所述的盐酸可为3.6%~36%盐酸。
在本发明的某一方案中,当所述的磁性纳米材料中的磁性纳米粒子为Fe3O4时,本发明中优选如下步骤制备得到,将FeCl3·6H2O和碱金属盐的乙二醇溶液进行反应,得到所述的Fe3O4纳米微粒即可。
其中,所述的碱金属盐可选自柠檬酸三钠和/或NaAc。
所述的FeCl3·6H2O与NaAc的摩尔比可为1:10。
所述的溶剂与FeCl3·6H2O的体积摩尔比可为10L/mol。
所述的反应的温度可为200℃。
所述的反应的时间可为8小时。
所述的Fe3O4磁性纳米微粒的制备方法中,还可包括后处理步骤,所述的后处理可为如下步骤,所述的反应结束后,洗涤经磁分离辅助条件获得的所述的Fe3O4磁性纳米微粒,即可;所述的洗涤可为分别使用乙醇和去离子水洗涤,例如洗涤三次;较佳地,洗涤后,将得到的Fe3O4磁性纳米微粒分散在去离子水中,配制成所需浓度的溶液待用即可,例如浓度为100mg/mL的溶液。
本发明还提供了一种聚合物修饰的磁性纳米材料,其采用如上所述的制备方法中任一方案制备得到;
较佳的,所述的聚合物修饰的磁性纳米材料为如上所述的聚合物修饰的磁性纳米材料中任一方案所示。
本发明还提供了一种等离子体法在制备聚合物修饰的磁性纳米材料中的应用;所述的应用可为如下步骤:在等离子体辉光存在下,将聚合物与溶剂的混合物与纳米材料进行改性修饰反应。
其中,所述的操作和条件可如上所述的聚合物修饰的磁性纳米材料中任一方案所述的条件和操作所示。所述的聚合物修饰的磁性纳米材料以及所述的聚合物和所述的磁性纳米材料的定义可为如上所述的聚合物修饰的磁性纳米材料中任一方案所述。
本发明还提供了一种如上所述的聚合物修饰的磁性纳米材料在糖基化蛋白、多肽类物质、核酸、循环肿瘤细胞、外泌体的富集分离中的应用。
在本发明的某一方案中,所述的应用可为所述的聚合物修饰的磁性纳米材料在制备荧光和磁共振MRI双模态成像显影剂、电化学细胞传感器、用于循环肿瘤细胞捕获的药物和/或医疗产品(试剂)、或用于治疗癌症的光热治疗剂中的应用。例如,用于细胞示踪、肿瘤示踪成像、磁热疗成像或血管成像。
所述(循环)肿瘤细胞例如可为叶酸受体阳性的肿瘤细胞;优选地,所述肿瘤细胞选自以下的一种或多种:卵巢癌肿瘤细胞、宫颈癌肿瘤细胞、非小细胞肺癌肿瘤细胞、结肠癌细胞、肺癌细胞、直肠癌细胞、胃癌细胞、乳腺癌细胞(三阴性乳腺癌肿瘤细胞)、食管癌细胞、肝癌细胞、白血病;例如卵巢癌肿瘤细胞、宫颈癌肿瘤细胞、三阴性乳腺癌肿瘤细胞、结肠癌肿瘤细胞、非小细胞肺癌肿瘤细胞、白血病。
在本发明的某一方案中,所述的应用可为所述的聚合物修饰的磁性纳米材料在制备捕获循环肿瘤细胞的药物或试剂中的应用。
优选的,所述药物或试剂的检测对象为外周血/体液样本;所述的体液可为尿液、胸腔液、腹水、脑脊液等。
如上所述循环肿瘤细胞包括卵巢癌肿瘤细胞、宫颈癌肿瘤细胞、非小细胞肺癌肿瘤细胞、结肠癌细胞、肺癌细胞、直肠癌细胞、胃癌细胞、乳腺癌细胞(三阴性乳腺癌肿瘤细胞)、食管癌细胞、肝癌细胞、白血病。
优选的,所述药物或试剂捕获外周血样本中循环肿瘤细胞的方法具体包括以下步骤:
S1、取外周血样本,用密度梯度液进行密度梯度离心,取中间段的白细胞层,去掉血浆和红细胞;
S2、将白细胞层的细胞稀释离心,重悬细胞获得细胞悬浮液,去掉蛋白及杂质;
S3、超声活化所述药物或试剂,并将活化后的药物或试剂与S2获得的细胞悬浮液以体积比3:100混合,进行吸附反应;
S4、磁场分离S3中吸附细胞悬浮液后的药物或试剂,富集循环肿瘤细胞,然后将细胞重悬、甩片、迪夫快速染色;
S5、显微镜下阅片,并根据肿瘤形态学进行鉴定和计数。
优选地,S1中密度梯度离心前外周血样本采用PBS稀释3-4倍。
优选地,S3中的吸附反应、S4中的磁场分离及富集反应均是在4℃条件下进行。
优选的,所述聚合物修饰的磁性纳米材料包括:磁性纳米粒子核,改性层的壳,和阳离子型聚合物的涂层;所述的聚合物附着或包覆于磁性纳米材料表面,形成带正电荷的所述聚合物修饰的磁性纳米材料;所述的磁性纳米材料为核-壳结构,所述的核为磁性纳米粒子,所述的壳为改性层;所述的改性层附着或包覆于所述磁性纳米粒子的表面,形成改性层复合的磁性纳米粒子。其中,所述的聚合物修饰的磁性纳米材料中,所述的聚合物与所述的磁性纳米材料的质量比为1:10至20:1。
术语
“去离子水”是指是指除去了呈离子形式杂质后的纯水。国际标准化组织ISO/TC147规定的“去离子”定义为:“去离子水完全或不完全地去除离子物质。
在不违背本领域常识的基础上,上述各优选条件,可任意组合,即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于:(1)传统的表面改性方法(如表面涂覆、表面氧化、高能射线处理、表面接枝改性等)存在如表面结构破坏,形态和厚度无法控制,材料表面原性能消失,后处理繁杂等缺陷,而气相自由基聚合法进行表面改性可规避这些不足,但也有聚合物浓度稀薄,需要真空条件,聚合时间过长等缺点。作为气相聚合的改良方法,本发明提出了雾聚合修饰的概念,即将聚合物溶于有机溶剂后,雾化该聚合物溶液,形成雾状聚合物液滴凝结于等离子体处理表面发生反应,实现对高分子材料的表面改性。主要通过等离子体处理表面引发雾聚合修饰,制备具有特殊形态表面的高分子材料。分别以聚乙烯亚胺(PEI)、壳聚糖、聚吡咯为基质,使用等离子体处理后,引发雾化聚合物在基质表面的聚合反应,改善材料的表面性能。
(2)本发明制得的聚合物修饰的磁性纳米材料可在循环肿瘤细胞检测中的应用,具体是用作制备捕获外周血中循环肿瘤细胞的药物或试剂,且检测对象为外周血样本。与现有技术相比,本发明具有以下一种或多种优点:(1)本发明所述聚合物修饰的磁性纳米材料的新用途具有敏感度高、检出率高、特异性好的优势,且捕获的CTC具有活性,可用于后续研究;(2)所述新用途与现有的CTC检测方法相比,所用的样本血少、检测快速、操作简便;(3)所述新用途单次检测成本低,仅需配备显微镜和磁分离器即可,从而降低医疗负担;(4)所述新用途适用于肿瘤病人的疗效评估、复发预警及预后值达到等多种场景,为医生提供用药和治疗的参考。
附图说明
图1为实施例1-7中各种纳米材料的电位表征和荧光光谱图;其中,A为电位表征;依次分别为Fe3O4@SiO2,PEI正电磁珠,等离子体聚合法PEI正电磁珠,壳聚糖正电磁珠,等离子体聚合法壳聚糖正电磁珠,聚吡咯正电磁珠,等离子体聚合法聚吡咯正电磁珠;B为荧光光谱图。
图2为实施例3中等离子体聚合法PEI正电磁珠电位与pH的关系。
图3为实施例3中磁分离前后等离子体聚合法正电磁珠的图片,(A)磁分离前;(B)磁分离后。
图4为实施例3中等离子体聚合法修饰PEI的磁性颗粒TEM图。
图5为实施例2和3材料的稳定性比较,(A)电位比较、(B)粒径比较。
图6为实施例2和3材料的响应性能-不同时间捕获CTC的回收率比较。
图7为实施例2和3材料的聚合物的接枝修饰百分量比较。
图8为应用实施例1中循环肿瘤细胞检测流程示意图;
图9为应用实施例2中正常人和恶性肿瘤患者的检出率比较图;
图10应用实施例1中肿瘤细胞光学显微镜图;
图11是应用实施例1中捕获的CTC培养10天、20天、30天的结果图。
具体实施方式
下面通过实施例的方式进一步说明本发明,但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。
流量单位sccm(Standard Cubic Centimeter per Minute)是表示每分钟标准毫升。
实验试剂
氯化高铁(FeCl3·6H2O)、氨水(NH3·H2O)、浓盐酸(HCl,37%)、无水乙醇等购自于国药集团;正硅酸乙酯(TEOS)、乙酸钠(NaAc)、乙二醇(EG)、3-氨聚乙烯亚胺(PEI)(MW=10000)、β-壳聚糖(MW=50000)、聚吡咯(MW=5000)、3-氨丙基三乙氧基硅烷((3-Aminopropyl)triethoxysilane,APTES)和异硫氰酸荧光素(Fluoresceinisothiocyanate,FITC)等购自Sigma公司;实验过程中的去离子水(Deionized water,DIW,18.2MΩ·cm)由实验室Thermo Easypure II UF纯水制备系统自制。
实验主要仪器设备
表1实验仪器设备
实施例1多功能磁性纳米材料的制备
①超顺磁性四氧化三铁纳米颗粒的制备
四氧化三铁纳米粒子的溶剂热法制备:准确称取FeCl3·6H2O 0.81g(六水合三氯化铁,0.003mol)和NaAc 2.56g(无水乙酸钠,0.03mol)磁力搅拌30min使之完全溶解于30mLPEG(乙二醇)中,得到棕黄色混合溶液,将上述溶液转移入耐高温高压的不锈钢反应釜中,放入高温烘箱,温度调整至200℃,恒温反应8h;反应结束后,取出反应釜,使用流动水快速冷却至室温;通过磁铁吸附将产物从反应液中分离出来,去掉反应液,然后在磁铁辅助分离条件下,分别使用乙醇和去离子水各洗涤三次,最后得到黑色产物,将洗涤后的产物重新在去离水中稀释分散,根据粗估配制成100mg/mL的粗浓度,采用固含量测定方法测定相对准确浓度,标记,统一存放。
②四氧化三铁/二氧化硅复合微球的制备
采用HCl处理制备好的四氧化三铁纳米微粒:将1mL 36%浓盐酸加入9mL上述分散于去离子水的四氧化三铁溶液,于圆底烧瓶中超声(温度30-40℃,功率80-120W)搅拌处理10-15min,磁分离去掉水溶液,去离子水洗6-7次,至上清液pH中性停止;称取乙醇83.8g、去离子水25.7g于三口烧瓶中,加入经盐酸洗涤并用去离子水清洗的四氧化三铁纳米磁珠150mg,超声辅助条件(温度30-40℃,功率80-120W)下机械搅拌15分钟左右,加入氨水调节pH至9.5左右,然后用2mL乙醇溶解100μl TEOS并加入用于上述反应中,继续机械搅拌12小时,然后通过磁分离获得产物并在磁分离辅助下使用无水乙醇和去离子水各洗三次,将洗涤后的产物分散在去离子水中,根据粗估配制成100mg/mL的粗浓度,采用固含量测定方法测定相对准确浓度,标记室温保存。
实施例2PEI正电磁珠的制备
(1)APS-FITC的制备
称量1.5mg的FITC染料在1.5mL离心管中,用0.5mL无水乙醇溶解FITC,然后转入小玻璃反应瓶中,使用2mL无水乙醇稀释,磁力搅拌1分钟使其混合均匀。然后加入5μL APS,体系颜色立刻变成橘黄色,继续避光磁力搅拌过夜,直至产物状态变为为澄清溶液,得到APS-FITC溶液,实验过程中注意避光。
(2)荧光标记和TEOS包被制备负电荧光磁珠
量取无水乙醇45mL,去离子水5mL,加入氨水0.7mL,机械搅拌混匀,加入30mg实施例1制备所得Fe3O4@SiO2继续超声搅拌30min左右至分散均匀;将30μl TEOS溶于1mL无水乙醇中,在超声和机械搅拌条件下缓慢地逐滴加入上述溶液,继续超声搅拌15min后将APS-FITC溶液快速加入上述反应体系,在避光条件下继续超声和机械搅拌4小时,然后停止超声,机械搅拌18小时,磁分离辅助条件获得产物并分别使用乙醇和去离子水洗涤三次。至此反应得到了荧光负电磁珠,标记为“荧光负电磁珠-生产日期”,测算和标记浓度,配制成10mg/mL分散液,分类存放,冰箱4度下避光保存。
(3)PEI修饰制备正电荧光磁珠
称取甲醇25mL,加入上一步反应得到的负电荧光磁珠18mg,避光条件下边超声边机械搅拌10min至混合均匀,然后称量PEI(MW=10000,99%,购买于阿拉丁)10mg用2mL甲醇溶解,加入上述反应溶液,继续超声搅拌2小时,磁分离获得产物并用甲醇洗涤一次水洗三册。样品处理:水分散液标记“荧光正电磁珠-生产日期”,测算浓度,配制成10mg/mL分散液;分类存放,冰箱4度下避光保存。
实施例3等离子体聚合法PEI正电磁珠的制备
(1)APS-FITC的制备
称量1.5mg的FITC染料在1.5mL离心管中,用0.5mL无水乙醇溶解FITC,然后转入小玻璃反应瓶中,使用2mL无水乙醇稀释,磁力搅拌1分钟使其混合均匀。然后加入5μL APS,体系颜色立刻变成橘黄色,继续避光磁力搅拌过夜,直至产物状态变为为澄清溶液,得到APS-FITC溶液,实验过程中注意避光。
(2)荧光标记和TEOS包被制备荧光负电磁珠
量取无水乙醇45mL,去离子水5mL,加入氨水0.7mL,机械搅拌混匀,加入30mg实施例1制备所得Fe3O4@SiO2继续超声搅拌30min左右至分散均匀;将30μl TEOS溶于1mL无水乙醇中,在超声和机械搅拌条件下缓慢地逐滴加入上述溶液,继续超声搅拌15min后将APS-FITC溶液快速加入上述反应体系,在避光条件下继续超声和机械搅拌4小时,然后停止超声,机械搅拌18小时,磁分离辅助条件获得产物并分别使用乙醇和去离子束洗涤三次。至此反应得到了荧光负电磁珠,标记为“荧光负电磁珠-生产日期”,测算和标记浓度,配制成10mg/mL分散液,分类存放,冰箱4度下避光保存。
(3)PEI修饰制备正电荧光磁珠
具体实验操作步骤如下:将荧光负电磁珠粉末18mg放入等离子体反应腔,检测整体气密性——开启机械泵抽真空至200Pa以下,同时打开射频电源预热15-20分钟——打开氮气阀门,在机械泵运转的同时通氮气,使氮气压力稳定在300-400Pa之间——打开射频设备,调节射频电流和电压,使反应腔体内产生等离子体辉光,同时调节射频功率稳定在10W左右;加热反应使PEI(10mg溶于2mL甲醇,通入等离子反应腔中)挥发,通过流量计调节单体流量至3-5sccm——保持各反应条件稳定,反应1-2小时。
实施例4壳聚糖正电磁珠的制备
(1)APS-FITC的制备
称量1.5mg的FITC染料在1.5mL离心管中,用0.5mL无水乙醇溶解FITC,然后转入小玻璃反应瓶中,使用2mL无水乙醇稀释,磁力搅拌1分钟使其混合均匀。然后加入5μL APS,体系颜色立刻变成橘黄色,继续避光磁力搅拌过夜,直至产物状态变为澄清溶液,得到APS-FITC溶液,实验过程中注意避光。
(2)荧光标记和TEOS包被制备负电荧光磁珠
量取无水乙醇45mL,去离子水5mL,加入氨水0.7mL,机械搅拌混匀,加入30mg实施例1制备所得Fe3O4@SiO2继续超声搅拌30min左右至分散均匀;将30μl TEOS溶于1mL无水乙醇中,在超声和机械搅拌条件下缓慢地逐滴加入上述溶液,继续超声搅拌15min后将APS-FITC溶液快速加入上述反应体系,在避光条件下继续超声和机械搅拌4小时,然后停止超声,机械搅拌18小时,磁分离辅助条件获得产物并分别使用乙醇和去离子束洗涤三次。至此反应得到了荧光负电磁珠,标记为“荧光负电磁珠-生产日期”,测算和标记浓度,配制成10mg/mL分散液,分类存放,冰箱4度下避光保存。
(3)壳聚糖修饰制备正电荧光磁珠
称取甲醇25mL,加入上一步反应得到的负点荧光磁珠18mg,避光条件下边超声边机械搅拌10min至混合均匀,然后称量β-壳聚糖10mg用2mL甲醇溶解,加入上述反应溶液,继续超声搅拌2小时,磁分离获得产物并用甲醇洗涤一次水洗三次。样品处理:水分散液标记“荧光正电磁珠-生产日期”,测算浓度,配制成10mg/mL分散液;分类存放,冰箱4度下避光保存。
实施例5等离子体聚合法壳聚糖正电磁珠的制备
(1)APS-FITC的制备
称量1.5mg的FITC染料在1.5mL离心管中,用0.5mL无水乙醇溶解FITC,然后转入小玻璃反应瓶中,使用2mL无水乙醇稀释,磁力搅拌1分钟使其混合均匀。然后加入5μL APS,体系颜色立刻变成橘黄色,继续避光磁力搅拌过夜,直至产物状态变为为澄清溶液,得到APS-FITC溶液,实验过程中注意避光。
(2)荧光标记和TEOS包被制备负电荧光磁珠
量取无水乙醇45mL,去离子水5mL,加入氨水0.7mL,机械搅拌混匀,加入30mg实施例1制备所得Fe3O4@SiO2继续超声搅拌30min左右至分散均匀;将30μl TEOS溶于1mL无水乙醇中,在超声和机械搅拌条件下缓慢地逐滴加入上述溶液,继续超声搅拌15min后将APS-FITC溶液快速加入上述反应体系,在避光条件下继续超声和机械搅拌4小时,然后停止超声,机械搅拌18小时,磁分离辅助条件获得产物并分别使用乙醇和去离子水洗涤三次。至此反应得到了荧光负电磁珠,标记为“荧光负电磁珠-生产日期”,测算和标记浓度,配制成10mg/mL分散液,分类存放,冰箱4度下避光保存。
(3)壳聚糖修饰制备正电荧光磁珠
具体实验操作步骤如下:将荧光负电磁珠粉末18mg放入等离子体反应腔,检测整体气密性——开启机械泵抽真空至200Pa以下,同时打开射频电源预热15-20分钟——打开氮气阀门,在机械泵运转的同时通氮气,使氮气压力稳定在300-400Pa之间——打开射频设备,调节射频电流和电压,使反应腔体内产生等离子体辉光,同时调节射频功率稳定在10W左右——加热反应使β-壳聚糖(10mg用2mL甲醇溶解,通入等离子反应腔中)挥发,通过流量计调节单体流量至3-5sccm——保持各反应条件稳定,反应1-2小时。
最后得到的样品处理:水分散液标记“等离子体聚合法壳聚糖-正电磁珠-生产日期”,测算浓度,配制成10mg/mL分散液;分类存放,冰箱4度下避光保存。
实施例6聚吡咯正电磁珠的制备
(1)APS-FITC的制备
称量1.5mg的FITC染料在1.5mL离心管中,用0.5mL无水乙醇溶解FITC,然后转入小玻璃反应瓶中,使用2mL无水乙醇稀释,磁力搅拌1分钟使其混合均匀。然后加入5μL APS,体系颜色立刻变成橘黄色,继续避光磁力搅拌过夜,直至产物状态变为为澄清溶液,得到APS-FITC溶液,实验过程中注意避光。
(2)荧光标记和TEOS包被制备负电荧光磁珠
量取无水乙醇45mL,去离子水5mL,加入氨水0.7mL,机械搅拌混匀,加入30mg实施例1制备所得Fe3O4@SiO2继续超声搅拌30min左右至分散均匀;将30μl TEOS溶于1mL无水乙醇中,在超声和机械搅拌条件下缓慢地逐滴加入上述溶液,继续超声搅拌15min后将APS-FITC溶液快速加入上述反应体系,在避光条件下继续超声和机械搅拌4小时,然后停止超声,机械搅拌18小时,磁分离辅助条件获得产物并分别使用乙醇和去离子水洗涤三次。至此反应得到了荧光负电磁珠,标记为“荧光负电磁珠-生产日期”,测算和标记浓度,配制成10mg/mL分散液,分类存放,冰箱4度下避光保存。
(3)聚吡咯修饰制备正电荧光磁珠
称取甲醇25mL,加入上一步反应得到的负点荧光磁珠18mg,避光条件下边超声边机械搅拌10min至混合均匀,然后称量聚吡咯10mg,用2mL甲醇溶解,加入上述反应溶液,继续超声搅拌2小时,磁分离获得产物并用甲醇洗涤一次水洗三次。样品处理:水分散液标记“荧光正电磁珠-生产日期”,测算浓度,配制成10mg/mL分散液;分类存放,冰箱4度下避光保存。
实施例7等离子体聚合法聚吡咯正电磁珠的制备
(1)APS-FITC的制备
称量1.5mg的FITC染料在1.5mL离心管中,用0.5mL无水乙醇溶解FITC,然后转入小玻璃反应瓶中,使用2mL无水乙醇稀释,磁力搅拌1分钟使其混合均匀。然后加入5μL APS,体系颜色立刻变成橘黄色,继续避光磁力搅拌过夜,直至产物状态变为为澄清溶液,得到APS-FITC溶液,实验过程中注意避光。
(2)荧光标记和TEOS包被制备负点荧光磁珠
量取无水乙醇45mL,去离子水5mL,加入氨水0.7mL,机械搅拌混匀,加入30mg实施例1制备所得Fe3O4@SiO2继续超声搅拌30min左右至分散均匀;将30μl TEOS溶于1mL无水乙醇中,在超声和机械搅拌条件下缓慢地逐滴加入上述溶液,继续超声搅拌15min后将APS-FITC溶液快速加入上述反应体系,在避光条件下继续超声和机械搅拌4小时,然后停止超声,机械搅拌18小时,磁分离辅助条件获得产物并分别使用乙醇和去离子束洗涤三次。至此反应得到了荧光负电磁珠,标记为“荧光负电磁珠-生产日期”,测算和标记浓度,配制成10mg/mL分散液,分类存放,冰箱4度下避光保存。
(3)聚吡咯修饰制备正电荧光磁珠
具体实验操作步骤如下:将荧光负电磁珠粉末18mg放入等离子体反应腔,检测整体气密性——开启机械泵抽真空至200Pa以下,同时打开射频电源预热15-20分钟——打开氮气阀门,在机械泵运转的同时通氮气,使氮气压力稳定在300-400Pa之间——打开射频设备,调节射频电流和电压,使反应腔体内产生等离子体辉光,同时调节射频功率稳定在10W左右——加热反应使聚吡咯(10mg溶于2mL甲醇,通入等离子反应腔中)其挥发,通过流量计调节单体流量至3-5sccm——保持各反应条件稳定,反应1-2小时。
效果实施例1多功能磁性纳米材料的性能
通过将实施例1-7制备所得多功能磁性纳米材料分散在水溶液中观察材料的分散性,将磁铁放置与装有制备的磁性纳米材料的样品瓶一侧,通过磁场对材料的吸引大致确定材料的磁力学性能;材料水合粒径大小和表面电位的测定采用激光粒度仪ZetasizerNano-ZS(Malvern,UK),使用动态光散射法测定纳米颗粒粒径大小分布情况及纳米颗粒表面电位情况;通过透射电子显微镜观察分析纳米颗粒的形貌;用荧光分光光度仪测制备所得纳米材料的荧光发射情况。
(1)多功能磁性纳米材料Zeta电位及荧光分析
实施例1-7中各种Fe3O4纳米材料的电位表征和荧光光谱图,如图1所示。
(2)溶液pH对纳米材料Zeta电位的影响
实施例3中等离子体聚合法PEI正电磁珠电位与pH的关系,如图2所示。
(3)磁性纳米材料在溶液中的分散性和磁性能观察
实施例3中磁分离前后等离子体聚合法正电磁珠,如图3所示,(A)磁分离前;(B)磁分离后
(4)纳米材料的电镜
实施例3中等离子体聚合法修饰PEI的磁性颗粒TEM图,如图4所示。
效果实施例2非等离子体聚合法与等离子体聚合的比较
(1)材料稳定性:电位(A)、粒径(B)比较
实施例2与实施例3材料的稳定性比较,如图5所示,(A)电位比较显示,实施例2中电位随着时间显著衰退,在200天时,从40衰退至15左右。实施例3中得到的材料的电位可保持在2年没有明显变化,显著优于实施例2。(B)粒径比较中,实施例3中得到的材料的水合粒径可保持在2年没有明显变化,显著优于实施例2。
(2)响应性能:不同时间捕获CTC的回收率比较
实施例2与实施例3材料的不同时间捕获CTC的回收率比较,如图6所示。
采用本发明的等离子体聚合发的实施例3中的材料的响应时间可达3S,显著快于采用常规的非等离子体聚合法得到的材料。
(3)聚合物的接枝百分量比较
实施例2与实施例3材料的聚合物的接枝百分量比较,如图7所示。
实施例3所得聚合物修饰的磁性纳米材料中聚合物的质量与投料量的比例可达60%以上。
实施例2所得聚合物修饰的磁性纳米材料中聚合物的质量与投料量的比例仅在15%左右。
实施例5和7与实施例4和6相比,可得类似效果。
应用实施例1
聚合物修饰的磁性纳米材料在制备捕获循环肿瘤细胞的药物或试剂中的应用。如图8所示,具体应用步骤如下:
(1)取外周血样本4mL;
(2)将密度梯度分离液(Percoll细胞分离液)按顺序分层缓慢加入15mL离心管中;
(3)将外周血样本混匀,取1mL外周血样本用PBS稀释3-4倍;
(4)将稀释后的外周血样本缓慢加入上述装有梯度分离液的离心管中,离心1600r×30min;
(5)离心后,取白细胞层于15mL离心管中,加4-6mL PBS重悬细胞,离心1600r×7min;
(6)离心后,去上清,用1mL PBS重悬细胞,并同步转移至1.5mL离心管中;
(7)超声活化药物或试剂(实施例3,PEI涂层的磁性纳米颗粒),加30μL药物或试剂至上述1.5mL离心管中,并置于迷你旋转培养器上,4℃条件下4rpm/min孵育10分钟;
(8)孵育后,将离心管插入多功能磁分离器上,4℃磁吸附10分钟;
(9)去上清,加入1mL PBS混匀,再将离心管插入多功能磁分离器上,4℃磁吸附10分钟;
(10)去上清,加入200μL PBS,重悬混匀,甩片1-2张;
(11)用迪夫快速染色液(Diff-Quik Stain)进行染色;
(12)光学显微镜下判定计数。
如图10所示:细胞体积大;核质比高;核形态不一,可出现巨核、双核或多核现象;核深染且染色不均;细胞质常见脂肪粒;细胞膜表面褶皱或边界清楚。以上为肿瘤细胞的形态特征,满足上述4个及4个以上特征即判定为肿瘤细胞。
本实施例与市售强生CellSearch产品相比,本实施例仅需4mL外周血,且检测时间在2小时内完成;而强生CellSearch技术需要7.5mL外周血,检测至少需要6小时。
如图11所示,通过对捕获的CTC进行培养,可以看到培养10天、20天、30天后有明显的增殖,表明捕获的CTC是活细胞。
应用实施例2
采用应用实施例1的应用方法分别检测158例健康志愿者和853例恶性肿瘤志愿者,收集2020年1月至2021年12月于泉州第一医院确诊的215例结肠癌患者,年龄34-86周岁,男性149例,女性66例;188例肺癌患者,年龄32-85周岁,男性113例,女性75例;145例直肠癌患者,年龄42-78岁,男性104例,女性41例;94例胃癌患者,年龄30-87岁,男性61例,女性33例;86例乳腺癌患者,年龄33-74岁,男性1例,女性85例;74例食管癌患者,年龄51-82岁,男性55例,女性19例;51例肝癌患者,年龄43-76岁,男性40例,女性11例;并选择同时期该院的158例健康体检者为健康对照,年龄在22-75周岁,男性101例,女性57例。
本实施例中志愿者的选择标准如下:
1.纳入标准:
1.1恶性肿瘤志愿者
a、年龄在18岁以上(包括18岁),性别不限;
b、经影像学及病理学确认的结肠癌、肺癌、直肠癌、胃癌、乳腺癌、食管癌、肝癌的单一恶性肿瘤患者;
c、仍处于放化疗、免疫治疗或靶向治疗中。
1.2健康志愿者
a、年龄在18岁以上(包括18岁),性别不限;
b、血常规或尿常规结果正常。
2.排除标准
2.1恶性肿瘤志愿者
a、有其他并发症的恶性肿瘤患者;
b、结肠癌/肺癌/直肠癌/胃癌/乳腺癌/食管癌/肝癌外,既往有其他恶性肿瘤史。
2.2健康志愿者
a、长期服用药物;
b、有慢性病或肿瘤家族史;
c、体检发现结节或疑似肿瘤。
结果如图9所示,其中健康对照组的158例中仅有1例检出,假阳性率极低,仅为0.6%;215例结肠癌组中检出205例,检出率达95.3%;188例肺癌组检出183例,检出率达97.3%;145例直肠癌组检出139例,检出率达95.9%;94例胃癌组检出92例,检出率达97.9%;86例乳腺癌组检出76,检出率达88.4%;74食管癌组检出68,检出率达91.9%;51例肝癌组检出50例,检出率高达98%。
Claims (9)
1.一种聚合物修饰的磁性纳米材料,其特征在于,其包括如下结构:
所述的聚合物为阳离子型聚合物;所述的聚合物附着或包覆于磁性纳米材料表面,形成带正电荷的所述聚合物修饰的磁性纳米材料;
所述的磁性纳米材料为核-壳结构,所述的核为磁性纳米粒子,所述的壳为改性层;所述的改性层附着或包覆于所述磁性纳米粒子的表面,形成改性层复合的磁性纳米粒子;
所述聚合物修饰的磁性纳米材料满足下述条件:
(1)所述的聚合物与所述的磁性纳米材料的质量比为1:3至3:1;
(2)所述的聚合物修饰的磁性纳米材料的电位为+5~+60mV;
(3)所述的磁性纳米材料为带负电荷的磁性纳米材料;
(4)所述的聚合物修饰的磁性纳米材料的粒径为10nm至600nm;
(5)所述的磁性纳米材料的粒径为5nm至500nm;
(6)所述的壳的厚度为1nm至100nm;
(7)所述的磁性纳米粒子的粒径为5nm至500nm;
(8)所述的聚合物为聚乙烯亚胺、壳聚糖和聚吡咯中的一种或多种;
(9)所述的聚合物为支状聚合物;
(10)所述的聚合物为重均分子量MW在2000至300000之间;
(11)所述的磁性纳米粒子为氧化物磁性纳米粒子、磁性金属纳米粒子、磁性硫化物纳米粒子、磁性复合粒子中的一种或几种;所述氧化物磁性纳米粒子为Fe3O4或γ-Fe2O3;
(12)所述的改性层的材料为二氧化硅或标记荧光和/或表面活性剂修饰的二氧化硅;
(13)所述的改性层的表面含有修饰得到的氨基;
(14)所述的改性层与所述的磁性纳米粒子的质量比为50:1~1:10;
(15)所述聚合物修饰的磁性纳米材料的稳定时长为2年;
(16)所述聚合物修饰的磁性纳米材料的响应时间为3S;
所述聚合物修饰的磁性纳米材料满足下述条件:
(1)当所述的聚合物为聚乙烯亚胺时,所述的聚乙烯亚胺的重均分子量为 2000-100000;
(2)当所述的聚合物为β-壳聚糖时,所述的β-壳聚糖重均分子量为50000-300000;
(3)当所述的聚合物为聚吡咯时,所述的聚吡咯的重均分子量为5000;
(4)当所述的磁性纳米材料为标记荧光的二氧化硅复合的磁性纳米粒子时,所述的标记荧光的二氧化硅复合的磁性纳米粒子中的荧光染料为异硫氰酸荧光素和/或罗丹明类染料,和/或其被修饰物;所述的修饰物为APS修饰的异硫氰酸荧光素和/或APS修饰的罗丹明类染料;所述APS为3-氨基丙基三乙氧基硅烷和/或3-氨基丙基三甲基硅烷;
(5)所述的磁性纳米材料为表面活性剂修饰的二氧化硅复合的磁性纳米粒子;所述的表面活性剂包括乙酸钠、柠檬酸三钠、壳聚糖、聚乙烯吡咯烷酮、聚对苯二甲酸乙二醇酯、硬脂酸、阿拉伯树胶、羟丙基甲基纤维素、海藻酸钠、十二烷基硫酸钠、十二烷基苯磺酸钠、聚乙烯醇、长链脂肪酸、淀粉和十二硫醇中的一种或两种以上的组合;
(6)当所述的磁性纳米材料为标记荧光的二氧化硅复合的磁性纳米粒子时,所述的二氧化硅复合的磁性纳米粒子与所述的荧光染料的质量比值为20;
(7)当所述的磁性纳米材料为标记荧光的二氧化硅复合的磁性纳米粒子时,所述的聚合物修饰的磁性纳米材料的荧光强度为40-1200;
(8)当所述的磁性纳米材料为二氧化硅改性层复合的磁性纳米粒子时,所述的二氧化硅改性层复合的磁性纳米粒子为Fe3O4@SiO2;
(9)当所述的磁性纳米材料为标记荧光的二氧化硅改性层复合的磁性纳米粒子时,所述的标记荧光的二氧化硅改性层复合的磁性纳米粒子APS-FITC标记的Fe3O4@SiO2。
2.如权利要求1所述的聚合物修饰的磁性纳米材料,其特征在于,
所述的聚合物修饰的磁性纳米材料选自如下任一方案:
方案1、
所述的聚合物修饰的磁性纳米材料为聚乙烯亚胺修饰的APS-FITC荧光标记的Fe3O4@SiO2;其中,所述的聚乙烯亚胺重均分子量MW=10000,所述聚乙烯亚胺与所述磁性纳米材料的质量比为1:3;所述的聚合物修饰的磁性纳米材料的粒径为20nm~500nm;其电位为+10mV~+60mV;
方案2、
所述的聚合物修饰的磁性纳米材料为β-壳聚糖修饰的APS-FITC荧光标记的Fe3O4@SiO2;其中,所述的β-壳聚糖的重均分子量MW=50000;所述β-壳聚糖与所述磁性纳米材料的质量比为1:3;所述的聚合物修饰的磁性纳米材料的粒径为20nm~500nm;其电位为+10~+60mV;
方案3、
所述的聚合物修饰的磁性纳米材料为聚吡咯修饰的APS-FITC荧光标记的Fe3O4@SiO2;
其中,所述的聚吡咯的重均分子量为5000;所述聚吡咯与所述磁性纳米材料的质量比为1:3;所述的聚合物修饰的磁性纳米材料的粒径为20nm~500nm;其电位为+10~+60mV。
3.一种聚合物修饰的磁性纳米材料的制备方法,其特征在于,其包括如下步骤:
将聚合物与溶剂的混合物与磁性纳米材料进行改性修饰,得到聚合物修饰的磁性纳米材料即可;其中,所述的聚合物与溶剂的混合物为雾化形态;
所述聚合物、所述磁性纳米材料的定义如权利要求1-2中任一项所述。
4.如权利要求3所述的制备方法,其特征在于,所述制备方法满足下述条件:
(1)所述的混合物与所述的磁性纳米材料采用等离子体法进行改性修饰;
(2)所述的溶剂为醇类溶剂,所述的醇类溶剂为甲醇;
(3)所述的聚合物在所述的混合物中的质量体积比为5mg/mL;
(4)所述的雾化形态通过加热所述的聚合物与溶剂的混合物得到;
(5)所述的聚合物与溶剂的混合物的加入为控制所述的混合物体积流量在3-5sccm;
(6)所述的改性修饰的温度为100至300℃
(7)所述的改性修饰在惰性气氛存在下进行;所述的惰性气氛为氮气和/或氩气;
(8)所述的改性修饰的反应的时间为1-2小时;
(9)当所述的磁性纳米材料为二氧化硅复合的磁性纳米粒子或标记荧光的二氧化硅复合的磁性纳米粒子、所述的二氧化硅复合的磁性纳米粒子为Fe3O4@SiO2时,所述的磁性纳米材料采用如下步骤制备得到:
步骤(a)在碱性试剂存在下,将硅试剂加入到Fe3O4磁性纳米微粒与溶剂的体系中进行改性反应,得到所述的Fe3O4@SiO2即可;和/或,步骤(b)将所述的Fe3O4@SiO2与荧光染料进行荧光标记反应,得到所述的标记荧光的二氧化硅复合的磁性纳米粒子即可。
5.如权利要求4所述的制备方法,其特征在于,所述的磁性纳米材料的制备中,满足下述条件:
(1)所述的聚合物修饰的磁性纳米材料的制备方法,其包括如下步骤:在惰性气氛存在下,在等离子体辉光存在下,将所述的聚合物与溶剂的混合物加热得到雾化形态,与所述的磁性纳米材料进行改性修饰;得到所述的聚合物修饰的磁性纳米材料即可;
其中,所述的等离子体辉光为如下步骤得到,在所述的惰性气氛下,调节射频功率,使等离子反应腔内产生等离子体辉光;所述惰性气氛的压力为在300-400Pa之间;所述射频的功率为10W±5W;(2)步骤(a)中,所述的溶剂为水,或水和醇类溶剂,所述的醇类溶剂为乙醇;
(3)所述的碱性试剂为氨水;
(4)所述的硅试剂为正硅酸乙酯或正硅酸甲酯;
(5)所述的Fe3O4磁性纳米微粒与所述的二氧化硅试剂质量体积比为1500g/L;
(6)所述的二氧化硅试剂以与所述的溶剂的混合物形式使用;
(7)所述的碱性试剂的用量为使所述的Fe3O4磁性纳米微粒与溶剂的体系的pH为9.5±0.5即可;
(8)所述的改性反应在超声和/或机械搅拌条件下进行;
(9)步骤(a)中,其还包括后处理步骤,所述的后处理为如下步骤,所述的反应结束后,洗涤经磁分离辅助条件获得的所述的Fe3O4@SiO2,即可;所述的洗涤为分别使用乙醇和去离子水洗涤;洗涤后,将得到的Fe3O4@SiO2分散在去离子水中,配制成所需浓度的溶液待用即可;
(10)步骤(b)中,所述的溶剂为醇类溶剂和水的混合物;所述的水为去离子水;所述的醇类溶剂为乙醇;所述的醇类溶剂和水的体积比为9:1~10:1;
(11)步骤(b)中,所述的二氧化硅复合的磁性纳米粒子与所述的溶剂的质量体积比为0.56至0.6g/L;
(12)步骤(b)中,所述的碱性试剂为氨水;所述的二氧化硅复合的磁性纳米粒子与所述的氨水的质量体积比为42至45g/L;
(13)步骤(b)中,所述的荧光染料为与所述的溶剂的混合物形式使用;所述的溶剂与所述的荧光染料的体积质量为1.7mL/mg;
(14)所述的荧光标记反应在超声和机械搅拌条件下进行;
(15)所述的荧光标记反应在避光条件下进行;
(16)步骤(b)中,所述的荧光标记反应中,还加入如步骤(a)所述的二氧化硅试剂,即同时进行二氧化硅进一步包被;所述的二氧化硅复合的磁性纳米粒子与所述的二氧化硅试剂的质量体积比为1000g/L;所述的硅试剂为与所述的溶剂的混合物形式;
(17)步骤(b)中,其还包括后处理步骤,所述的后处理为如下步骤,所述的反应结束后,洗涤经磁分离辅助条件获得的所述的磁性纳米粒子,即可;所述的洗涤为分别使用乙醇和去离子水洗涤;
(18)当所述的荧光染料为APS-FITC时,为如下步骤制备得到:将APS加入到FITC的乙醇溶液中反应,得到所述的APS-FITC即可;其中,所述的反应在避光条件下进行;所述的反应以得到澄清的溶液即可,混合过夜;FITC与APS的质量体积比为300g/L;
(19)步骤(a)中,所述的Fe3O4磁性纳米微粒与溶剂的体系采用如下步骤制备得到:在超声和机械搅拌条件下,在溶剂中,将Fe3O4纳米磁珠依次用盐酸、去离子水洗涤至上清液pH中性,即可;所述的盐酸为3.6%~36%盐酸;
(20)步骤(a)中,当所述的磁性纳米材料中的磁性纳米粒子为Fe3O4时,其由如下步骤制备得到:将FeCl3·6H2O和碱金属盐的乙二醇溶液进行反应,得到所述的Fe3O4纳米微粒即可;其中,所述的碱金属盐选自柠檬酸三钠和/或NaAc;所述的FeCl3·6H2O与NaAc的摩尔比为1:10;所述的溶剂与FeCl3·6H2O的体积摩尔比为10L/mol;所述的反应的温度为200℃;其还包括后处理步骤,所述的后处理为如下步骤,所述的反应结束后,洗涤经磁分离辅助条件获得的所述的Fe3O4磁性纳米微粒,即可;所述的洗涤为分别使用乙醇和去离子水洗涤;洗涤后,将得到的Fe3O4磁性纳米微粒分散在去离子水中,配制成所需浓度的溶液待用即可。
6.一种等离子体法在制备聚合物修饰的磁性纳米材料中的应用;所述的应用中,在等离子体辉光存在下,将聚合物与溶剂的混合物与纳米材料进行改性修饰反应;
所述的混合物、所述的磁性纳米材料及所述的制备方法的操作和条件如权利要求4或5所述的聚合物修饰的磁性纳米材料的制备方法中任一方案所述的混合物、所述的磁性纳米材料及所述的条件和操作所示;
和/或,相应的聚合物修饰的磁性纳米材料的定义如权利要求1-2中任一项所述的聚合物修饰的磁性纳米材料中任一方案所示。
7.一种聚合物修饰的磁性纳米材料在糖基化蛋白、多肽类物质、核酸、循环肿瘤细胞、外泌体的富集分离中的应用方法,其特征在于,所述的聚合物修饰的磁性纳米材料的定义如权利要求1-2中任一项所述的聚合物修饰的磁性纳米材料中任一方案所示;
所述肿瘤细胞选自以下一种或多种:卵巢癌肿瘤细胞、宫颈癌肿瘤细胞、非小细胞肺癌肿瘤细胞、结肠癌细胞、肺癌细胞、直肠癌细胞、胃癌细胞、乳腺癌细胞、食管癌细胞、肝癌细胞、白血病。
8.如权利要求7所述的应用方法,其特征在于,所述的应用为所述的聚合物修饰的磁性纳米材料在制备捕获循环肿瘤细胞的药物或试剂中的应用。
9.如权利要求7所述的应用方法,其特征在于,所述药物或试剂的检测对象为外周血/体液样本;和/或,所述循环肿瘤细胞包括卵巢癌肿瘤细胞、宫颈癌肿瘤细胞、非小细胞肺癌肿瘤细胞、结肠癌细胞、肺癌细胞、直肠癌细胞、胃癌细胞、乳腺癌细胞、食管癌细胞、肝癌细胞、白血病;
和/或,所述药物或试剂捕获外周血样本中循环肿瘤细胞的方法具体包括以下步骤:
S1、取外周血样本,用密度梯度液进行密度梯度离心,取中间段的白细胞层,去掉血浆和红细胞;
S2、将白细胞层的细胞稀释离心,重悬细胞获得细胞悬浮液,去掉蛋白及杂质;
S3、超声活化所述药物或试剂,并将活化后的药物或试剂与S2获得的细胞悬浮液以体积比3:100混合,进行吸附反应;
S4、磁场分离S3中吸附细胞悬浮液后的药物或试剂,富集循环肿瘤细胞,然后将细胞重悬、甩片、迪夫快速染色;
S5、显微镜下阅片,并根据肿瘤形态学进行鉴定和计数;
S1中密度梯度离心前外周血样本采用PBS稀释3-4倍;
S3中的吸附反应、S4中的磁场分离及富集反应均是在4℃条件下进行。
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