CN114192117B - 一种磁性复合材料及其制备方法和应用 - Google Patents

一种磁性复合材料及其制备方法和应用 Download PDF

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CN114192117B
CN114192117B CN202111346568.5A CN202111346568A CN114192117B CN 114192117 B CN114192117 B CN 114192117B CN 202111346568 A CN202111346568 A CN 202111346568A CN 114192117 B CN114192117 B CN 114192117B
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陈立娜
张宏娟
丁莹莹
彭涛
张宇
王淑祯
高艳坤
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Nanjing Medical University
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Abstract

一种磁性复合材料及其制备方法和应用，包括：(1)Fe3O4纳米粒子的制备，(2)磁性金属有机骨架材料的制备，(3)磁性MOF@nNOS的制备，(4)磁性MOF@nNOS‑PSD95复合材料的制备。本发明制备的基于金属有机骨架材料的MOF@nNOS‑PSD95磁性纳米荧光材料，结合了磁性分离的便捷性、MOF材料的高亲和力和荧光检测的高灵敏性，可以快速、高效地发现PSD95‑nNOS解偶联剂类活性物质，为天然药物中抗缺血性脑卒中候选药物的筛选提供了一种新思路和新方法。

Description

一种磁性复合材料及其制备方法和应用
技术领域
本发明设计属于天然药物学和材料学领域，具体涉及一种磁性MOF@nNOS-PSD95复合材料及其制备方法，以及在筛选天然药物潜在抗脑卒中活性成分方面的应用。
背景技术
缺血性脑卒中是局部脑组织缺血、缺氧而引发脑组织软化、坏死的一种疾病，目前已经成为危害民众健康的主要疾病之一。在脑缺血过程中，N-甲基-D-天门冬氨酸受体(NMDARs)被激活，从而导致神经元型一氧化氮合酶(nNOS)过度表达，释放过量的一氧化氮，诱发神经损伤和死亡。通过阻断突触后密度蛋白-95(PSD95)与nNOS的偶联，可以在不影响nNOS和NMDAR的生理功能的前提下，有效减少缺血性脑卒中造成的损害。小分子ZL006和IC87201是根据该机制设计合成的有效的nNOS-PSD95偶联抑制剂。但是，它们生物相容性差、亲水性大、脑组织分布不理想，临床效果并不理想。因此，迫切需要寻找临床疗效更好的解偶联剂。
天然药物是指经现代医药体系证明具有一定药理活性的动物药、植物药和矿物药等。早有研究表明，麝香、牛黄、姜黄素、葛根素等天然药物对于脑卒中的预防和治疗具有良好的效果，而且药理作用强、副作用小。在此基础上，已有研究捕获到潜在的nNOS-PSD95解偶联剂如黄岑素、小檗碱等，以期用于缺血性脑卒中的治疗。然而，目前的研究需借助已知的具有解偶联效果的模板分子，难以实现广泛捕获潜在的结构多样的解偶联剂，因此需要寻找一种无需借助模板分子，能直接从天然药物中捕获解偶联剂的方法。
金属有机骨架(MOFs)，是近十年来发展迅速的一种配位聚合物，具有三维的孔结构，具有比表面积大、孔隙率高、孔道规则、孔径可调以及拓扑结构多样性等优点。已有研究表明，MOFs上的配位不饱和金属位点可以与His标签特异性结合，从而达到固定带有His标签的酶的作用。此外，磁性分离技术可以简化操作，加快样品的分离速度。目前，应用磁性MOF@nNOS-PSD95复合材料发现天然药物中的nNOS-PSD95解偶联剂尚未见报道。
发明内容
解决的技术问题：本发明提供一种磁性MOF@nNOS-PSD95复合材料及其制备方法，具体来说是从天然药物中快速、高效筛选nNOS-PSD95解偶联剂的方法，为找到活性显著的抗脑卒中候选药物奠定基础，同时为天然药物解偶联类活性成分的筛选提供了一种新策略。
技术方案：一种磁性复合材料的制备方法，步骤如下：(1)Fe3O4纳米粒子的制备：按比例，将60～100mL超纯水加到反应容器中，氮气保护下加入5～20mmol FeCl2·4H2O和10～40mmol FeCl3·6H2O，在油浴升温至60～120℃时逐滴加入氨水5～20mL，持续反应0.5～2h，然后在搅拌下加入聚乙二醇200 1～4mL反应5～20min，反应全程用氮气保护，产物磁性分离，并用去离子水洗至中性得Fe3O4纳米粒子，50℃真空干燥后备用；(2)磁性金属有机骨架材料的制备：取上述Fe3O4纳米粒子、金属源和有机配体分散于溶剂中，在反应装置中23℃～120℃反应1～24h，降至室温，产物磁性分离，用洗涤剂洗涤数次，最后在50℃下真空干燥24h，得到磁性金属有机骨架材料；其中金属源为铁、锆或铜，有机配体为氨基对苯二甲酸、均苯三甲酸、富马酸或4，4'-联吡啶，溶剂为N,N-二甲基甲酰胺、乙醇、超纯水或三者的混合溶液，Fe3O4纳米粒子和金属源的有机配体的摩尔比为1:1～1:4，金属源和有机配体的摩尔比为1:1～1:20，金属源的摩尔量和溶剂的体积用量的比例为1:2～1:180，反应装置为磁力搅拌器或反应釜，洗涤剂为超纯水、N,N-二甲基甲酰胺或无水乙醇；(3)磁性MOF@nNOS的制备，将上述磁性金属有机骨架材料加到His-nNOS溶液中，4℃下170rpm振摇混匀，磁性分离，即得；其中磁性金属有机骨架材料和His-nNOS的质量比为20:1～4:1，振摇时间为15～90min；(4)磁性MOF@nNOS-PSD95的制备：将磁性MOF@nNOS加到GFP-PSD95溶液中，4℃下165rpm振摇过夜，磁性分离，即得；其中nNOS和PSD95的质量比为0.8:1～2:1。
上述制备方法制得的磁性复合材料。
上述磁性复合材料在筛选天然药物潜在抗脑卒中活性成分中的应用。
上述磁性复合材料在制备筛选天然药物潜在抗脑卒中活性成分产品中的应用。
应用具体方法为，将磁性复合材料加到不同天然药物活性成分的溶液中，4℃下125rpm振摇5h，磁性分离，测定磁性复合材料的荧光强度，即可比较筛选出效果较好的天然药物活性成分。
有益效果：本发明将纳米材料、磁性分离、荧光检测和金属有机骨架材料固定蛋白技术相结合，设计合成了一种表面固定大量活性蛋白的磁性荧光纳米材料，从而实现对天然药物中潜在抗脑卒中活性成分的高效筛选。除此之外，本发明的优良效果还表现在：1、本发明制备得到的磁性MOF@nNOS-PSD95复合材料方法简单、成本低、条件易于控制；2、本发明所得的磁性MOF@nNOS-PSD95复合材料磁力强劲，其饱和磁化强度可达60emu/g～70emu/g，可以实现对固定酶的简单快速分离；3、本发明制备得到的磁性金属有机骨架材料对带有His标签的神经元型一氧化氮合酶的固定效率高，其负载率可达90％以上；4、本发明所得的磁性MOF@nNOS-PSD95复合材料无需借助模板分子，利用荧光检测的灵敏性，能直接从天然药物中发现潜在的解偶联剂，快捷高效。
附图说明
图1是实施例1制备的磁性金属有机骨架材料的扫描电镜图；
图2是实施例1制备的磁性MOF@nNOS-PSD95复合材料的磁滞回线(磁化强度由高到低依次为Fe3O4；磁性金属有机骨架材料；磁性MOF@nNOS；磁性MOF@nNOS-PSD95)；
图3是实施例1制备的磁性金属有机骨架材料固定His-nNOS和Flag-nNOS负载率对比；
图4是各实施例制备的不同金属有机骨架材料固定His-nNOS的负载量对比。
具体实施方式
以下是本发明内容的具体实施例，用于阐述本申请文件中所要解决技术问题的技术方案，有助于本领域技术人员理解本发明内容，但本发明技术方案的实现并不限于这些实施例。
实施例1:
材料的制备：(1)Fe3O4纳米粒子的制备：将80mL超纯水加到250mL的三颈瓶中，氮气保护下加入10mmol FeCl2·4H2O和20mmol FeCl3·6H2O，在油浴升温至80℃时逐滴加入氨水10mL，持续反应1h，然后在剧烈搅拌下加入聚乙二醇200 2mL反应10min，反应全程用氮气保护。产物磁性分离，并用去离子水洗至中性，50℃真空干燥后备用；(2)磁性金属有机骨架材料的制备：取上述Fe3O4纳米粒子2.78mmol和氨基对苯二甲酸2.78mmol分散于60mL DMF，磁力搅拌器中120℃油浴4h，然后加入800μL醋酸(事先加热15min)，放冷后分别用N,N-二甲基甲酰胺和乙醇洗涤，最后在50℃下真空干燥24h，得到产物；(3)磁性MOF@nNOS的制备：将3mg磁性MOF@nNOS加到250μL His-nNOS(1mg/mL)溶液中，4℃下170rpm振摇60min，磁性分离，即得；(4)磁性MOF@nNOS-PSD95的制备：将上述磁性MOF@nNOS加到250μL GFP-PSD95(1.25mg/mL)溶液中，4℃下165rpm振摇过夜，磁性分离，即得。
应用：将磁性MOF@nNOS-PSD95加到不同天然药物活性成分的溶液中，4℃下125rpm振摇5h，磁性分离，测定磁性MOF@nNOS-PSD95的荧光强度，即可比较筛选出效果较好的天然药物活性成分。
实施例2:
材料的制备：(1)MOF的制备：取ZrCl4 2mmol溶解于18mL混合溶液(HCl：DMF，1:5v/v)，氨基对苯二甲酸2.8mmol溶解于20mL DMF，混合均匀，反应釜80℃反应过夜，放冷后分别用N,N-二甲基甲酰胺和乙醇洗涤，最后在50℃下真空干燥24h，得到产物；(2)MOF@nNOS的制备：将1mg MOF@nNOS加到250μL His-nNOS(1mg/mL)溶液中，4℃下170rpm振摇15min，离心分离，即得；(3)MOF@nNOS-PSD95的制备：将上述MOF@nNOS加到250μL GFP-PSD95(1mg/mL)溶液中，4℃下165rpm振摇过夜，离心分离，即得。
应用：将MOF@nNOS-PSD95加到不同天然药物活性成分的溶液中，4℃下125rpm振摇5h，离心分离，测定MOF@nNOS-PSD95的荧光强度，即可比较筛选出效果较好的天然药物活性成分。
实施例3:
材料的制备：(1)MOF的制备：取ZrCl4 1.04mmol和氨基对苯二甲酸2.08mmol溶解于混合溶液(30mL水和1950μL甲酸的混合溶液，反应釜120℃反应24h，放冷后用水洗涤，最后在50℃下真空干燥24h，得到产物；(2)MOF@nNOS的制备：将2mg MOF@nNOS加到250μL His-nNOS(1mg/mL)溶液中，4℃下170rpm振摇30min，离心分离，即得；(3)MOF@nNOS-PSD95的制备：将上述MOF@nNOS加到250μL GFP-PSD95(0.5mg/mL)溶液中，4℃下165rpm振摇过夜，离心分离，即得。
应用：将MOF@nNOS-PSD95加到不同天然药物活性成分的溶液中，4℃下125rpm振摇5h，离心分离，测定MOF@nNOS-PSD95的荧光强度，即可比较筛选出效果较好的天然药物活性成分。
实施例4:
材料的制备：(1)MOF的制备：取Cu(NO3)2·2.5H2O 3mmol和均苯三甲酸6mmol溶解于6mL水中，磁力搅拌器室温反应60min，用乙醇洗涤，最后在50℃下真空干燥24h，得到产物；(2)MOF@nNOS的制备：将4mg MOF@nNOS加到250μL His-nNOS(1mg/mL)溶液中，4℃下170rpm振摇45min，离心分离，即得；(3)MOF@nNOS-PSD95的制备：将上述MOF@nNOS加到250μLGFP-PSD95(1mg/mL)溶液中，4℃下165rpm振摇过夜，离心分离，即得。
应用：将MOF@nNOS-PSD95加到不同天然药物活性成分的溶液中，4℃下125rpm振摇5h，离心分离，测定MOF@nNOS-PSD95的荧光强度，即可比较筛选出效果较好的天然药物活性成分。
实施例5:
材料的制备：(1)MOF的制备：取Cu(NO3)2·2.5H2O 0.5mmol溶解于1mL乙醇溶液中，4,4’-联吡啶10mmol溶解于90mL超纯水中，磁力搅拌器室温反应2h，用超纯水洗涤，最后在50℃下真空干燥24h，得到产物；(2)MOF@nNOS的制备：将5mg MOF@nNOS加到250μL His-nNOS(1mg/mL)溶液中，4℃下170rpm振摇75min，离心分离，即得；(3)MOF@nNOS-PSD95的制备：将上述MOF@nNOS加到250μL GFP-PSD95(1mg/mL)溶液中，4℃下165rpm振摇过夜，离心分离，即得。
应用：将MOF@nNOS-PSD95加到不同天然药物活性成分的溶液中，4℃下125rpm振摇5h，离心分离，测定MOF@nNOS-PSD95的荧光强度，即可比较筛选出效果较好的天然药物活性成分。
实施例6:
材料的制备：(1)Fe3O4-COOH纳米粒子的制备：取FeCl3·6H2O 3.2436g和柠檬酸钠0.5265g溶解于60mL乙二醇中，加入无水乙酸钠3.594g，持续搅拌30min，反应釜200℃反应12h，乙醇洗涤数次，真空干燥。；(2)磁性金属有机骨架材料的制备：取FeCl3·6H2O2.78mmol和氨基对苯二甲酸2.78mmol分散于60mL DMF，加入上述Fe3O4-COOH纳米粒子50mg，磁力搅拌器中120℃油浴4h，然后加入800μL醋酸(事先加热15min)，放冷后分别用N,N-二甲基甲酰胺和乙醇洗涤，最后在50℃下真空干燥24h，得到产物；(3)磁性MOF@nNOS的制备：将6mg磁性MOF@nNOS加到250μL His-nNOS(1mg/mL)溶液中，4℃下170rpm振摇90min，磁性分离，即得；(4)磁性MOF@nNOS-PSD95的制备：将上述磁性MOF@nNOS加到250μL GFP-PSD95(0.5mg/mL)溶液中，4℃下165rpm振摇过夜，磁性分离，即得。
应用：将磁性MOF@nNOS-PSD95加到不同天然药物活性成分的溶液中，4℃下125rpm振摇5h，磁性分离，测定磁性MOF@nNOS-PSD95的荧光强度，即可比较筛选出效果较好的天然药物活性成分。
实施例1的测试实施例：
测试实施例1.负载率测定实验
分别将1mg、2mg、3mg、4mg、5mg磁性金属有机骨架材料加到250μL His-nNOS(1mg/mL)溶液中，4℃下170rpm振摇60min，磁性分离，用Bradford法测定上清残留蛋白浓度，根据式(1)测定制备的磁性金属有机骨架材料的负载率:
loading efficiency％＝(C0-Ct)/C0×100％
式中：C0和Ct分别为His-nNOS的初始浓度和反应t时间后残留的上清浓度(mg/mL)。
从图3可以看出，不同量的磁性金属有机骨架材料固定带有His标签的nNOS的负载率均高于带有Flag标签的nNOS，这是因为本发明制备的磁性金属有机骨架上的配位不饱和金属位点可以与His标签上的咪唑基团特异性结合，实现高选择性和高效吸附。

Claims (5)

1.一种磁性复合材料的制备方法，其特征在于，步骤如下： (1) Fe3O4纳米粒子的制备：按比例，将60~100 mL超纯水加到反应容器中，氮气保护下加入5~20 mmol FeCl2·4H2O和10~40 mmol FeCl3·6H2O，在油浴升温至60~120℃时逐滴加入氨水5~20 mL，持续反应0.5~2h，然后在搅拌下加入聚乙二醇200 1~4 mL反应5~20 min，反应全程用氮气保护，产物磁性分离，并用去离子水洗至中性得Fe3O4纳米粒子，50℃真空干燥后备用；(2) 磁性金属有机骨架材料的制备：取上述Fe3O4纳米粒子、金属源和有机配体分散于溶剂中，在反应装置中23℃~120℃反应1~24 h，降至室温，产物磁性分离，用洗涤剂洗涤数次，最后在50℃下真空干燥24 h，得到磁性金属有机骨架材料；其中金属源为铁、锆或铜，有机配体为氨基对苯二甲酸、均苯三甲酸、富马酸或4，4'-联吡啶，溶剂为N,N-二甲基甲酰胺、乙醇、超纯水或三者的混合溶液，Fe3O4纳米粒子和金属源的有机配体的摩尔比为1:1~1:4，金属源和有机配体的摩尔比为1:1~1:20，金属源的摩尔量和溶剂的体积用量的比例为1:2~1:180，反应装置为磁力搅拌器或反应釜，洗涤剂为超纯水、N,N-二甲基甲酰胺或无水乙醇；(3) 磁性MOF@nNOS的制备，将上述磁性金属有机骨架材料加到His-nNOS溶液中，4℃下170 rpm振摇混匀，磁性分离，即得；其中磁性金属有机骨架材料和His-nNOS的质量比为20:1~4:1，振摇时间为15~90 min；(4) 磁性MOF@nNOS-PSD95的制备：将磁性MOF@nNOS加到GFP-PSD95溶液中，4℃下165 rpm振摇过夜，磁性分离，即得；其中nNOS和PSD95的质量比为0.8:1~2:1。
2.权利要求1所述制备方法制得的磁性复合材料。
3.权利要求2所述磁性复合材料在筛选天然药物潜在抗脑卒中活性成分中的应用。
4.权利要求2所述磁性复合材料在制备筛选天然药物潜在抗脑卒中活性成分产品中的应用。
5.根据权利要求3所述的应用，其特征在于，将磁性复合材料加到不同天然药物活性成分的溶液中，4℃下125 rpm振摇5 h，磁性分离，测定磁性复合材料的荧光强度，即可比较筛选出效果较好的天然药物活性成分。
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