CN115317626A - 一种仿生纳米探针及其监测血脑屏障损伤的应用 - Google Patents
一种仿生纳米探针及其监测血脑屏障损伤的应用 Download PDFInfo
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Abstract
本发明公开了一种监测血脑屏障损伤的仿生纳米探针及其制备方法。所述仿生纳米探针是以负载发夹探针H1和H2的磷酸钙矿化的金属‑有机框架载体为核心,以脑微血管内皮细胞膜作为外壳。该仿生纳米探针在血液循环过程中,可以靶向血脑屏障,被脑内皮细胞摄取,在细胞内释放发夹探针H1和H2,可与血脑屏障损伤相关RNA杂交,触发杂交链式反应,荧光强度提高,可通过测定相应RNA的表达来检测血脑屏障损伤情况。本发明所述的纳米探针具有灵敏度高的优点,可检测血脑屏障的早期损伤,解决了传统探针检测血脑屏障损伤灵敏度低的问题。
Description
技术领域
本发明属于生物传感技术领域,具体涉及一种仿生纳米探针及其监测血脑屏障损伤应用。
背景技术
血脑屏障(BBB)是由紧密连接的脑微血管内皮细胞(BMEC)、内皮细胞外连续的基底膜、周细胞和星形胶质细胞所组成的复杂、动态的三维结构,可以保护大脑免受血液中有害物质的侵害。血脑屏障功能障碍的主要特征是细胞旁通透性增加,即内皮细胞间间隔增大,导致血浆成分外渗到脑实质。研究证实,在阿尔茨海默症(AD)、多发性硬化症(MS)、卒中、及一些神经炎症性疾病等许多脑部疾病的进程中,BBB通透性增加,会进一步加重疾病。血脑屏障损伤也发生在人类大脑老化的早期阶段,并可能导致认知障碍。事实上,BBB功能障碍最近被认为是神经系统疾病的早期标志。因此,发展监测血脑屏障损伤的有效方法,对于预测脑疾病进展和制定针对性治疗策略具有重要意义。
目前,测量外源性示踪剂脑外渗是评价血脑屏障通透性的主要方法。已经开发了许多血脑屏障渗透性示踪剂,如放射性标记蔗糖、荧光素钠、台潘蓝、辣根过氧化物酶和伊文思蓝等。但该方法往往需要采集脑组织切片来研究血脑屏障的破坏情况,无法监测体内血脑屏障通透性的动态变化。近年来,多种体内成像方法的发展使得实时监测血脑屏障损伤成为可能,例如核成像技术和磁共振成像技术。然而,核成像具有较高的电离辐射始终存在一定的安全隐患,磁共振成像需要较长的采集时间导致检测灵敏度较低。相比之下,荧光成像因具有灵敏度高、采集速度快、无电离辐射风险和实时检测等优点日益受到关注,因此有可能以更高的灵敏度实现体内血脑屏障通透性的动态评估。
近来,一些长波长发射的荧光显像剂已经被开发用于在体内评估血脑屏障的通透性。然而,这些荧光探针往往表现为“always-on”型,且不能特异性识别任何与血脑屏障通透性相关的靶点,其工作原理与上述传统外源性示踪剂类似。在这种情况下,只有血脑屏障被严重破坏,有足够数量的探针进入脑实质,才能检测到明显的荧光信号。因此,这些显像剂仍然缺乏灵敏度,只能检测到血脑屏障的严重损伤,而不能检测到血脑屏障通透性的细微变化。
发明内容
本发明所要解决的技术问题是针对上述现有技术存在的不足而提供一种仿生纳米探针及其监测血脑屏障损伤应用,可以高效靶向血脑屏障,通过测定脑内皮细胞中miRNA-155等RNA的表达来监测血脑屏障损伤。
本发明为解决上述提出的问题所采用的技术方案为:
一种监测血脑屏障损伤的仿生纳米探针,其特征在于所述仿生纳米探针是以负载发夹探针H1和H2的磷酸钙矿化的金属-有机框架载体为核心,以细胞膜作为外壳;所述发夹探针H1和H2能够与血脑屏障损伤的RNA杂交触发杂交链式反应。
按上述方案,所述的细胞膜为脑微血管内皮细胞膜。
按上述方案,所述的金属-有机框架载体主要是以锆(Zr)为基础的MOF材料,即Zr-MOFs,包括Uio-66,PCN-222,PCN-223和PCN-224等。这些载体需带正电荷,可通过电荷作用结合带负电荷的发夹探针;并且需以Zr为中心,利用配位作用高效结合发夹探针的磷酸根,提高发夹探针的负载量,同时可实现在酸性环境下释放发夹探针。
按上述方案,所述血脑屏障损伤相关的RNA包括miRNA-155,miRNA-15a/16-1,miRNA-501-3p,miRNA-182,miRNA-107,miRNA-143,miRNA-27a,miRNA-101,miRNA-132和miRNA-210等。
优选地,发夹探针H1的序列为:5’-TAA TCG TGA TAG GGG TAC AGG TCACCC CTATCA CGA TTA GCA TTA A-3’;发夹探针H2的序列为:5’-GAC CTG TAC CCC TAT-Cy3(TexasRed)-CAC GAT TAT TAA TGC TAA TCG-BHQ1(BHQ2)-GAT AGG GGT-3’;所述RNA为miRNA-155。
按上述方案,金属-有机框架载体中发夹探针H1和H2的总负载量在1×10-3~2.3×10-3nmol/ug。
本发明还提供一种上述监测血脑屏障损伤的仿生纳米探针的制备方法,主要步骤如下:
1)将金属-有机框架MOF载体加入发夹探针H1和H2的水溶液中,置于摇床中震荡,然后离心,得到负载发夹探针的MOF-H1/H2;
2)将负载发夹探针的MOF-H1/H2分散到DMEM中,加入CaCl2进行矿化,得到负载发夹探针H1和H2的磷酸钙矿化的金属-有机框架载体MOF-H1/H2-CaP;
2)将MOF-H1/H2-CaP与细胞膜在去离子水中混合,冰水浴超声,得到监测血脑屏障损伤的仿生纳米探针。
按上述方案,步骤1)中,金属-有机框架MOF载体在发夹探针H1和H2的水溶液的分散浓度为0.05~0.15mg/mL,发夹探针H1和H2的水溶液的浓度范围都是在50~200nM(两者比例是1:1),摇床的温度在27~37℃范围内,震荡的时间2~8h。
按上述方案,步骤2)中,负载发夹探针的MOF-H1/H2在DMEM中的分散浓度为0.05~0.15mg/mL,MOF-H1/H2与CaCl2之间的质量比为1:1~1:6,CaCl2在DMEM中的浓度在0.2~0.6mg/mL;矿化的时间4~12h,温度在27~37℃范围内。
按上述方案,步骤3)中,MOF-H1/H2-CaP在去离子水中浓度0.5~1mg/mL,MOF-H1/H2-CaP与细胞膜之间的质量比为1:1~1:2,细胞膜在去离子水中浓度在0.5~1mg/mL;冰水浴超声5~15min。
本发明还要求保护上述仿生纳米探针在靶向血脑屏障和/或监测血脑屏障损伤方面的用途以及其在检测血脑屏障损伤相关的RNA方面的应用。
本发明的原理以特异性检测血脑屏障相关miRNA-155为例,具体说明如下:首先设计了DNA发夹探针H1和H2,其可以通过触发杂交链式反应(HCR)特异性检测血脑屏障相关miRNA-155。为了提高发夹探针在脑内皮细胞中的摄取,将H1和H2负载于磷酸钙(CaP)矿化的MOF载体上,并在其表面包覆上一层脑微血管内皮细胞膜用于靶向脑内皮细胞,从而得到仿生纳米探针。该仿生纳米探针在进入血液循环后,利用内皮细胞膜表面丰富的具有相同黏附域的表面黏附分子(如Claudin 5和VE-cadherin)同源黏附脑内皮细胞,有效提高细胞摄取;进入细胞后表面矿化的CaP层在溶酶体酸性条件下降解,原位提供高浓度的磷酸根离子,促进MOF载体的降解,从而释放H1和H2同时促进其溶酶体逃逸;H1和H2通过触发HCR反应对目标miRNA-155进行原位放大检测,有效提高检测灵敏度,从而实现对血脑屏障早期损伤的灵敏检测。
与现有技术相比,本发明的有益效果是:该仿生纳米探针在血液循环过程中,可以靶向血脑屏障,被脑内皮细胞摄取,在细胞内释放发夹探针H1和H2,可与血脑屏障损伤相关RNA杂交,触发杂交链式反应,荧光强度提高,可通过测定相应RNA的表达来检测血脑屏障损伤情况,以及检测血脑屏障通透性轻微变化。本发明所述的纳米探针具有灵敏度高的优点,可检测血脑屏障的早期损伤,解决了传统探针检测血脑屏障损伤灵敏度低的问题。
附图说明
图1为实施例1中,MOF载体、MOF-H1/H2、MOF-H1/H2-CaP、MOF-H1/H2-CaP@M的透射电镜图。
图2中,左图为MOF-H1,MOF-H1-CaP和MOF-H1-CaP@M在pH为7.4或pH为5.0溶液中的释放效率;右图为MOF-H1/H2,MOF-H1/H2-CaP和MOF-H1/H2-CaP@M在pH为7.4或pH为5.0溶液中在目标ssDNA存在时的荧光强度比值。
图3为仿生纳米探针在炎症刺激脑内皮细胞中检测miRNA-155的共聚焦成像图。其中control代表脑内皮细胞不经过任何处理,lps(-)代表脑内皮细胞未经过LPS刺激,但与仿生纳米探针共孵育8h,lps(+)代表脑内皮细胞经过LPS刺激并与仿生纳米探针共孵育8h。仿生纳米探针与目标物结合后在细胞中的发出红光;细胞核用Hoechst 33342染色,发出蓝光;Merge代表蓝光和红光重叠。
图4为MOF-H1/H2-CaP@M和MOF-H1/H2-CaP随着时间变化在小鼠体内的成像图。
图5为仿生纳米探针MOF-H1/H2-CaP@M可以在不同程度脑部炎症小鼠体内的成像图。
图6为仿生纳米探针MOF-H1/H2-CaP@M在不同年龄AD小鼠和野生型小鼠体内的成像图。
具体实施方式
为了更好地理解本发明,下面结合实施例进一步阐明本发明的内容,但本发明不仅仅局限于下面的实施例。
下述实施例中,所述的细胞膜来源于鼠源脑微血管内皮细胞(bEnd.3细胞)。
实施例1仿生纳米探针的制备
(1)称取0.0747g 2-氨基对苯二甲酸将其溶于8mL DMF中,随后再量取45μL三乙胺加入上述溶液中。将0.1g四氯化锆和2.07mL冰醋酸分别加入到8mL DMF中。随后将上述溶液混合,转移至50mL反应釜中,于85℃反应24h。冷却后离心,随后用按顺序用DMF、甲醇、去离子水洗涤三次,得到MOF载体(UIO-66-NH2),在去离子水中分散备用。
(2)将0.1mg的UIO-66-NH2纳米载体加入发夹探针H1和H2(H1和H2分别是100nM,比例1:1)的水溶液(1mL)中,置于37℃摇床震荡4h,随后取出离心15min得到负载发夹探针的MOF-H1/H2。
其中,发夹探针H1的序列为:5’-TAA TCG TGA TAG GGG TAC AGG TCACCC CTA TCACGA TTA GCA TTA A-3’;发夹探针H2的序列为:5’-GAC CTG TAC CCC TAT-Cy3(TexasRed)-CAC GAT TAT TAA TGC TAA TCG-BHQ1(BHQ2)-GAT AGG GGT-3’。
为了测定MOF-H1/H2中发夹探针负载量和包封率,由于发夹探针H1/H2在没有目标物存在的条件下无法通过荧光分析测定负载量和包封率,所以通过在MOF载体(1mL,100μg/mL)溶液中加入相同体积的带有Cy5荧光团的发夹探针H1-Cy5,测定带有Cy5荧光团的发夹探针H1-Cy5的负载量和包封率,来间接得到MOF-H1/H2中发夹探针负载量和包封率。具体方法如下:
将0.1mg UIO-66-NH2纳米载体加入探针H1-Cy5的水溶液中(1mL,H1-Cy5是200nM)置于37℃摇床震荡4h,随后取出离心15min,得到负载H1-Cy5的MOF-H1-Cy5。
将离心收集的上清液进行荧光分析,采用标准曲线法测定上清液中的探针H1-Cy5的浓度。上清液的荧光强度是1969,标曲曲线方程为y=852.72x+1884.55,测得上清液中探针H1-Cy5浓度为0.1nM。其中,H1-Cy5的标准曲线测定方法如下:用去离子水制备一系列不同浓度的H1-Cy5溶液(400nM,200nM,100nM,50nM,25nM,12.5nM,6.25nM),测定其荧光强度,激发波长650nm,发射波长670nm。
随后,根据公式计算负载量和包封率。负载量=(加入H1-Cy5量(nmoL)-上清液中的H1-Cy5量(nmoL))/MOF载体质量(μg);包封率=(加入H1-Cy5量(nmoL)-上清液中的H1-Cy5量(nmoL))/加入H1-Cy5量(nmoL)×100。计算得到负载量为0.002nmol/μg,包封率为100%。
其中,H1-Cy5的序列为:5’-Cy5-TAA TCG TGA TAG GGG TAC AGG TCA CCC CTATCA CGA TTA GCA TTA A-3’。
(3)将制备好的MOF-H1/H2(0.1mg)用1mL DMEM(含有固有的1.8mM CaCl2和0.9mMNaH2PO4)分散,浓度0.1mg/mL。在溶液中加入1M CaCl2(1.2μL),使CaCl2的最终浓度达到3mM,MOF-H1/H2和CaCl2质量比1:3,在37℃恒温条件下矿化6h。经离心净化以去除残留的DMEM,得到MOF-H1/H2-CaP,并在4℃的去离子水中保存。
(4)提取bEnd.3细胞的细胞膜。提取方式如下:将消化后得到的bEnd.3细胞用PBS洗涤三次后,均匀分散于膜蛋白提取试剂中,在冰水浴中静置15min,于-80℃冷冻后解冻,随后反复冻融三次。接下来在4000rpm的转速下离心,得到细胞悬液在13300rpm的转速下离心,最终得到bEnd.3细胞的细胞膜,于去离子水中冷冻保存。
将MOF-H1/H2-CaP溶液(0.2mL,1mg/mL)和细胞膜溶液(0.1mL,2mg/mL)混合,冰水浴超声10min,离心后得到仿生纳米探针MOF-H1/H2-CaP@M。
结果如图1所示,MOF载体平均直径为74.0±4.7nm,将发夹探针H1和H2负载到MOF载体后,MOF-H1/H2纳米粒子的分散性有所改善,但形貌并未发生明显变化;在矿化磷酸钙后,可以观察到MOF-H1/H2-CaP纳米颗粒表面轮廓变得粗糙且富有颗粒感;包覆了细胞膜后,MOF-H1/H2-CaP@M纳米探针表面呈现出了清晰的细胞膜层,其厚度约为7nm。这些结果意味着细胞膜被成功地涂覆在了MOF-H1/H2-CaP纳米探针的表面。
实施例2仿生纳米探针在酸性环境下降解并高效响应目标miRNA-155
(1)为测定实施例1所制备的仿生纳米探针MOF-H1/H2-CaP@M在pH为7.4或pH为5.0溶液中的降解情况,由于发夹探针H1/H2在没有目标物存在的条件下无法在降解后通过荧光分析测定释放效率,所以将实施例1制备过程中的发夹探针H1和H2替换为H1-Cy5,相应制备得到MOF-H1,MOF-H1-CaP和MOF-H1-CaP@M,测定其释放效率,从而间接得到MOF-H1/H2-CaP@M的释放效率。具体方法如下:
将MOF-H1,MOF-H1-CaP和MOF-H1-CaP@M分别分散于pH为7.4或pH为5.0的Tris-HCl杂交缓冲溶液(10mM,含1mM MgCl2和100mM KCl)中,H1-Cy5的浓度为100nM,摇床震荡12h后,离心收集上清液进行荧光分析,结合实施例1中的H1-Cy5荧光标准曲线,得到上清液中的H1-Cy5的浓度,并计算H1-Cy5的释放效率。如图2中左图可知,矿化磷酸钙的载体(MOF-H1-CaP和MOF-H1-CaP@M)可以在酸性环境下降解,并释放出负载的带荧光的H1-Cy5,释放率(约80%),显著高于没有矿化磷酸钙的载体MOF-H1的释放率(约5%)。由此,可以说明本发明所述仿生纳米探针也可以在酸性环境下降解释放出发夹探针。
(2)将实施例1制备的MOF-H1/H2,MOF-H1/H2-CaP和MOF-H1/H2-CaP@M分别分散于pH为7.4或pH为5.0的Tris-HCl杂交缓冲溶液(10mM,含1mM MgCl2和100mM KCl)中,确保每组中H1和H2的终浓度均为100nM,随后加入目标ssDNA(可与发夹探针中基因片段互补配对的单链DNA,作为miRNA-155的替代物,浓度100nM),摇床震荡1h后,使用荧光光谱仪测得荧光信号。F0和F代表了探针在目标ssDNA不存在和存在时的荧光强度。
结果如图2中右图所示,MOF-H1/H2-CaP和MOF-H1/H2-CaP@M)在酸性环境下降解,并释放出发夹探针,证实其能在酸性环境中高效响应目标miRNA-155。其中,加入目标物(ssDNA)后,MOF-H1/H2在不同pH条件下的荧光强度基本保持不变,表明pH并没有直接影响发夹探针的释放,MOF载体未发生降解。而在与目标ssDNA孵育后,MOF-H1/H2-CaP或MOF-H1/H2-CaP@M在酸性环境(pH 5.0)下的荧光信号强于中性环境(pH 7.4)。这可以归因于磷酸钙外层的存在,在酸性条件下降解生成大量磷酸根离子,促进MOF载体降解,释放出发夹探针H1和H2,并与目标物反应,荧光增强。
实施例3仿生纳米探针可以检测炎症刺激的内皮细胞中miRNA-155的表达。
将bEnd.3细胞接种于35mm的培养皿中,每皿的细胞密度为1×105个细胞,于37℃培养24h。取0.1mL MOF-H1/H2-CaP@M仿生纳米探针水溶液(实施例1制备的)(含H1和H2分别为2mM)加入到0.9mL新鲜培养基中,摇匀,用移液枪吸取全部溶液加入一皿bEnd.3细胞中,其中H1和H2分别为200nM。将细胞与MOF-H1/H2-CaP@M共孵育4h,随后加入LPS(终浓度为0.5μg/mL)再培养4h,用HEPES缓冲溶液仔细清洗细胞三次,加入细胞核染料Hoechst 33342染核10min,用HEPES缓冲溶液清洗细胞三次,用共聚焦显微镜进行细胞成像。
结果如图3所示,仿生纳米探针可以灵敏检测LPS刺激前后内皮细胞中miRNA-155表达的变化。
实施例4仿生纳米探针可以在正常老鼠中靶向完整的血脑屏障
将健康的BALB/c裸鼠(脑部有少量的内源性miRNA-155表达)(雌性,20g±2g)分为两组,每组五只。一组小鼠尾静脉注射MOF-H1/H2-CaP纳米探针(实施例1制备,5mg/mL,200μL),另一组小鼠尾静脉注射MOF-H1/H2-CaP@M仿生纳米探针(实施例1制备,5mg/mL,200μL)。使用3%异氟烷麻醉小鼠,随后用小动物活体成像仪进行活体荧光成像。
结果如图4所示,随着时间的延长,注射了MOF-H1/H2-CaP@M仿生纳米探针的小鼠脑部荧光信号强度明显高于注射MOF-H1/H2-CaP纳米探针的小鼠的脑部荧光信号强度,说明包裹了bEnd.3细胞膜的纳米探针在正常小鼠的脑部富集提高,并与脑内内源性的miRNA-155反应荧光增强,从而证明MOF-H1/H2-CaP@M仿生纳米探针可以在正常老鼠中靶向完整的血脑屏障。
实施例5仿生纳米探针可以在不同程度脑部炎症小鼠模型中检测血脑屏障通透性变化。
将健康的BALB/c裸鼠(雌性,20g±2g)分为三组。空白组腹腔注射生理盐水,剩下两组实验组小鼠分别腹腔注射不同剂量的含LPS的PBS缓冲溶液(LPS浓度分别为4mg/kg和8mg/kg,经LPS刺激后引起炎症,脑微血管内皮细胞中的miRNA-155的表达提高,血脑屏障损伤加剧)。6h后,每组小鼠尾静脉注射MOF-H1/H2-CaP@M仿生纳米探针(实施例1制备,5mg/mL,200μL),使用3%异氟烷麻醉小鼠,随后用小动物活体成像仪进行活体荧光成像。
结果如图5所示,与作为对照的空白组正常小鼠相比,实验组的炎症小鼠的大脑显示出明显增强的荧光信号,轻度炎症下荧光信号增强较弱,而在重度炎症下荧光信号增强更强,这表明MOF-H1/H2-CaP@M仿生纳米探针可以灵敏检测到不同炎症鼠脑中miRNA-155表达的上调,从而反映了不同炎症程度小鼠血脑屏障通透性的细微变化。
实施例6仿生纳米探针可以在不同年龄AD小鼠模型中检测血脑屏障通透性变化。
对健康的C57BL/6J小鼠(4月龄,野生型)和APP/PS1转基因AD小鼠(4月龄和10月龄)尾静脉注射MOF-H1/H2-CaP@M仿生纳米探针(实施例1制备,5mg/mL,200μL)。AD鼠比健康鼠的脑部表达更多的miRNA-155,并且年龄越大,miRNA-155表达水平越高,血脑屏障损伤越严重。随后,使用3%异氟烷麻醉小鼠,随后用小动物活体成像仪进行活体荧光成像。
结果如图6所示,与健康4月龄的C57BL/6J小鼠相比,AD小鼠脑部荧光信号显著增强,且随着年龄的增加,10月龄AD小鼠脑部荧光比4月龄AD小鼠脑部荧光更强。这表明该仿生纳米探针能灵敏的检测到AD小鼠模型中miRNA155表达的上调,证明仿生纳米探针可以通过对AD小鼠脑部miRNA-155的即时成像反应血脑屏障的通透性变化从而预测AD的疾病进展。
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该视为本发明的保护范围。
Claims (10)
1.一种监测血脑屏障损伤的仿生纳米探针,其特征在于所述仿生纳米探针是以负载发夹探针H1和H2的磷酸钙矿化的金属-有机框架载体为核心,以细胞膜作为外壳;所述发夹探针H1和H2能够与血脑屏障损伤相关的RNA杂交触发杂交链式反应。
2.根据权利要求1所述的仿生纳米探针,其特征在于发夹探针H1的序列为:5’-TAA TCGTGA TAG GGG TAC AGG TCACCC CTA TCA CGA TTA GCA TTA A-3’;发夹探针H2的序列为:5’-GAC CTG TAC CCC TAT-Cy3(Texas Red)-CAC GAT TAT TAA TGC TAA TCG-BHQ1(BHQ2)-GAT AGG GGT-3’;所述RNA为miRNA-155。
3.根据权利要求1所述的仿生纳米探针,其特征在于所述的金属-有机框架载体主要是金属锆的有机骨架材料;所述的细胞膜为脑微血管内皮细胞膜。
4.根据权利要求1所述的仿生纳米探针,其特征在于金属-有机框架载体中发夹探针H1和H2的总负载量在1×10-3~2.3×10-3nmol/ug。
5.一种监测血脑屏障损伤的仿生纳米探针的制备方法,其特征在于主要步骤如下:
1)将金属-有机框架MOF载体加入发夹探针H1和H2的水溶液中,置于摇床中震荡,然后离心,得到负载发夹探针的MOF-H1/H2;
2)将负载发夹探针的MOF-H1/H2分散到DMEM中,加入CaCl2进行矿化,得到负载发夹探针H1和H2的磷酸钙矿化的金属-有机框架载体MOF-H1/H2-CaP;
3)将MOF-H1/H2-CaP与细胞膜在去离子水中混合,冰水浴超声,得到监测血脑屏障损伤的仿生纳米探针。
6.根据权利要求5所述的监测血脑屏障损伤的仿生纳米探针的制备方法,其特征在于步骤1)中,金属-有机框架载体在发夹探针H1和H2的水溶液的分散浓度为0.05~0.15mg/mL,发夹探针H1和H2的水溶液的浓度范围都是在50~200nM范围内,摇床的温度在27~37℃范围内,震荡的时间2~8h。
7.根据权利要求5所述的监测血脑屏障损伤的仿生纳米探针的制备方法,其特征在于步骤2)中,负载发夹探针的MOF-H1/H2在DMEM中的分散浓度为0.05~0.15mg/mL,MOF-H1/H2与CaCl2之间的质量比为1:(1~3),CaCl2在DMEM中的浓度在0.1~0.3mg/mL;矿化的时间4~12h,温度在27~37℃范围内。
8.根据权利要求5所述的监测血脑屏障损伤的仿生纳米探针的制备方法,其特征在于步骤3)中,MOF-H1/H2-CaP在去离子水中浓度0.5~1mg/mL,MOF-H1/H2-CaP与细胞膜之间的质量比为1:(1~2),细胞膜在去离子水中浓度在0.5~1mg/mL;冰水浴超声5~15min。
9.权利要求1所述的仿生纳米探针在靶向血脑屏障和/或监测血脑屏障损伤方面的应用。
10.根据权利要求9所述的应用,其特征在于应用方法为:所述仿生探针进入血液循环后,发夹探针H1和H2与血脑屏障损伤相关的RNA杂交触发杂交链式反应,根据反应前后荧光强度的变化检测血脑屏障损伤相关的RNA的浓度。
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