CN112933251A - 四氧化三锰纳米颗粒制备方法及用途 - Google Patents

四氧化三锰纳米颗粒制备方法及用途 Download PDF

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CN112933251A
CN112933251A CN202110167362.XA CN202110167362A CN112933251A CN 112933251 A CN112933251 A CN 112933251A CN 202110167362 A CN202110167362 A CN 202110167362A CN 112933251 A CN112933251 A CN 112933251A
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陈春英
王亚玲
陈曦
赵宇亮
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National Center for Nanosccience and Technology China
GBA National Institute for Nanotechnology Innovation
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Abstract

本发明涉及化学、纳米材料及生物学的技术领域，特别涉及四氧化三锰纳米颗粒制备方法及用途。该制备方法包括：将交联剂溶液与生物分子缓冲溶液混合，得到第一溶液；将蛋白分子缓冲溶液与第一溶液混合，经交联反应后，透析纯化，冻干，得到修饰蛋白；将二价锰盐水溶液滴加入修饰蛋白溶液中，混匀，得到复合溶液；调节复合溶液pH值至10或10以上，搅拌，透析纯化，冻干，得到四氧化三锰纳米探针。本发明方法制备步骤简单，形貌结构及尺寸易控制。本发明的四氧化三锰纳米探针能够适用于核磁共振谱仪，具有较好的核磁共振成像能力。同时，其还可以作为生物纳米酶，有效催化过氧化氢。

Description

四氧化三锰纳米颗粒制备方法及用途
技术领域
本发明涉及化学、纳米材料及生物学的技术领域，特别涉及四氧化三锰纳米颗粒制备方法及用途。
背景技术
与其他体内成像技术相比，高分辨率和出色的软组织对比度是磁共振成像(MRI)成像的主要优势。通过开发更加高效和针对性的造影剂，MRI已发展成为具有多种功能的通用技术，并已成为生物医学设备中功能最强大的无创成像工具之一。与常规显像剂相比纳米显影剂有几个优点：第一是可装载性，在合成过程中可以控制每个纳米颗粒内显像剂浓度；另一个优点是纳米颗粒表面的可修饰性，通过修饰特定的分子，可以帮助纳米显影剂有效地延长在血液中的循环时间或特异性在体内的特定位置高度聚集。最后，纳米显影剂还可以结合自身的物理化学特性，实现更多的功能。
而由于其结构和形态在一定程度上的可调节性、独特的物理及化学特性以及出色的性能，锰氧化物纳米材料(MONs)及其衍生物在生物成像、生物传感、药物/基因传递和肿瘤治疗中的应用引起了越来越多的关注。早在二十年前，就有通过热分解法得到了锰氧化物纳米颗粒，随后陆续有多种锰氧化物用于核磁共振成像的研究报道。但是常规的锰氧化物纳米颗粒制备完成后用于生物体研究时，往往需要进行表面修饰处理，以提升其亲水性和生物适应性。制备步骤繁琐，形貌结构及尺寸不易控制，阻碍了锰氧化物纳米材料在生物学上的实际应用。
居晓玲于2017年发表一篇名为《四氧化三锰白蛋白纳米粒的制备与表征》硕士论文，在该四氧化三锰白蛋白纳米粒制备方法中，所使用的锰元素来源为高锰酸钾，锰氧化物纳米颗粒制备时，利用蛋白质的还原作用，将七价锰还原得到二价锰与三价锰共存的化合物Mn3O4；而且仅提及白蛋白的使用，但是若无功能性基团如细胞特定蛋白的靶向肽或透明质酸等修饰，是无法用于细胞特定蛋白的特异性识别和成像。
发明内容
有鉴于此，本发明提供了四氧化三锰纳米颗粒制备方法及用途。该方法制备步骤简单，形貌结构及尺寸易控制。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种四氧化三锰纳米探针的制备方法，包括如下步骤：
步骤1)将交联剂溶于溶剂中，得到交联剂溶液；将交联剂溶液与生物分子缓冲溶液混合，得到第一溶液；生物分子为肿瘤组织细胞高表达的蛋白质的靶向序列或透明质酸；
步骤2)将蛋白分子缓冲溶液与第一溶液混合，经交联反应后，透析纯化，冻干，得到修饰蛋白；蛋白分子为牛血清白蛋白、人血清白蛋白、铁红蛋白、纤连蛋白、蛋清蛋白中的一种或几种；
步骤3)将二价锰盐水溶液滴加入修饰蛋白溶液中，混匀，得到复合溶液；调节复合溶液pH值至10或10以上，搅拌，透析纯化，冻干，得到四氧化三锰纳米探针。
作为优选，步骤1)中，交联剂为4-(N-马来酰亚胺甲基)环己烷-1-羧酸磺酸基琥珀酰亚胺酯钠盐(Sulfo-SMCC)、4-(N-马来酰亚胺甲基)环己烷羧酸-N-琥珀酰亚胺酯(SMCC)、琥珀酰亚胺基-4-[N-马来酰亚胺甲基]-环己烷-1-羧基-[6-氨基己酸盐](LC-SMCC)、1-乙基-3-[3-二甲基氨基丙基]碳化二亚胺盐酸化物(EDC)中的一种或几种；所述溶剂为DMSO、DMF、pH为5～6的磷酸盐缓冲溶液(PBS)中的一种或几种。
作为优选，步骤1)中，交联剂溶液的浓度为2～10g/L，生物分子缓冲溶液的浓度为5～20mmol/L，生物分子与交联剂的摩尔比为1∶(1～2)。
作为优选，步骤1)中，肿瘤组织细胞高表达的蛋白质包含但不限于整合素蛋白、人表皮生长因子受体-2蛋白、纤连蛋白中的一种或几种。
作为优选，步骤1)中，生物分子缓冲溶液或蛋白分子缓冲溶液中的缓冲液为PBS缓冲液、醋酸盐缓冲液中的一种或两种。
作为优选，步骤1)中，交联反应的条件为室温搅拌20～60min。
作为优选，步骤2)中，蛋白分子缓冲溶液的浓度为5～50g/L，蛋白分子与生物分子的摩尔比为1：(1～20)。
作为优选，步骤2)中，透析纯化的条件为：截留分子量3000～15000Da，透析时间5～20小时；
作为优选，步骤3)中，透析纯化的条件为：截留分子量3000～15000Da，透析时间5～20小时。
作为优选，步骤3)中，二价锰盐选自氯化锰、硝酸锰、乙酸锰、硫酸锰中的一种或几种。
作为优选，步骤3)中，修饰蛋白与锰元素的摩尔比为1：(100～500)。
作为优选，步骤3)中，调节复合溶液pH值的试剂为碱性试剂，碱性试剂为氢氧化钠、氢氧化钾、氢氧化钙、氨水、三乙胺、吡啶、N-甲基吗啉、四甲基乙二胺中的一种或多种。
作为优选，步骤3)中，搅拌的温度为30～37℃，搅拌的时间为1～10小时，搅拌的转速为200～1000r/min。
本发明还提供了由上述制备方法制得的四氧化三锰纳米探针。
在本发明中，四氧化三锰纳米探针包括四氧化三锰纳米颗粒以及在复合在纳米颗粒外层的包覆物质，包覆物质为修饰蛋白，修饰蛋白为修饰有生物分子的蛋白分子。
作为优选，四氧化三锰纳米探针的粒径为1～100纳米。
本发明还提供了上述四氧化三锰纳米探针作为核磁共振成像增敏剂和/或生物酶中的用途。
本发明具有的技术效果为：
(1)本发明方法制备步骤简单，形貌结构及尺寸易控制。
(2)本发明的四氧化三锰纳米探针包括四氧化三锰纳米颗粒以及复合在纳米颗粒外层的包覆物质，所述包覆物质包括生物蛋白质分子和生物分子。其能够适用于临床核磁共振谱仪，具有较好的核磁共振成像能力。同时，其可以作为一种生物纳米酶，具有过氧化物酶活性，能够有效催化过氧化氢。
与居晓玲发表的制备方法相比，本发明还具有如下优势：
①本发明所使用的锰元素来源为二价锰化合物，在制备过程和存放时二价锰源安全性高于高锰酸钾，锰氧化物纳米颗粒制备时，利用溶解氧的弱氧化作用，将部分二价锰氧化制备二价锰与三价锰共存的化合物Mn3O4，避免了表面修饰还原性分子时，高锰酸钾强氧化性使分子还原性受到影响。
②本发明中除了白蛋白外，还验证了在结构和性能上与白蛋白有明显差异的卵清蛋白、铁红蛋白、纤连蛋白等在本发明所提及实验上能得到相近的结果。
③本发明利用功能性基团修饰，则赋予了所构建的Mn3O4纳米颗粒靶向结合细胞特定蛋白的能力，而功能性基团的修饰并不是所有方法可用，有一些修饰方法会造成纳米颗粒聚集或功能性基团靶向能力缺失等，本发明分享了可有效修饰并保留功能性基团靶向能力的实验方法。这些在居晓玲的方法中并未提及。
④本发明所构建的Mn3O4纳米颗粒用途中还包括了过氧化物酶活性用途，这在居晓玲的方法中并未提及。基于此，本发明无论纳米颗粒构建所依据的基本原理、所使用的反应底物种类、产品用途等均与居晓玲的方法有明显不同，有着自己特色的创新性和改进性所在。
附图说明
图1为本发明实施例1制备的纳米颗粒探针的TEM图像；
图2为本发明实施例2制备的纳米颗粒探针的TEM图谱；
图3为实施例1不同锰元素浓度下，纵向弛豫T1和横向弛豫T2成像强度曲线；
图4为实施例1不同锰元素浓度下，过氧化氢吸光度随时间变化速率的曲线；
图5为纳米探针制备示意图；
图6为以滴加方式制备的纳米探针的TEM图像；
图7为以倾倒方式制备的纳米探针的TEM图像；
图8为纳米探针的谷胱甘肽过氧化物酶样活性。
具体实施方式
本发明公开了四氧化三锰纳米颗粒探针的制备方法及用途，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明四氧化三锰纳米颗粒探针的制备方法及用途中所用原料或试剂均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1
本实施例制备纳米颗粒探针的步骤如下：
A)设计含有可特异性靶向人表皮生长因子受体-2蛋白(HER2)的多肽序列KCCYSL，记为肽序列HV，取10mg合成好的肽序列HV加入到1mL PBS中，随后加入溶解有3mg Sulfo-SMCC的0.5mL的DMSO溶液，室温搅拌使之混合均匀，得到第一溶液。
B)搅拌均匀后将卵清蛋白溶液加入到步骤A得到的第一溶液中，室温搅拌半小时，之后选取截留分子量8000的透析袋透析10小时，冻干保存，得到肽序列HV修饰的卵清蛋白分子(以下记为HV-OVA)。
C)于2mL以卵清蛋白含量测算浓度为0.1mmol/L的HV-OVA水溶液中，加入2mL25mmol/L的氯化锰水溶液，剧烈搅拌使之混合均匀。随后加入2mol/L的KOH水溶液，调节溶液pH至11，于34℃继续搅拌2小时，经过与步骤B)相同的透析纯化冻干过程后得到探针粉末，记为纳米颗粒探针。参见图1。图1为本发明实施例1制备的纳米颗粒探针的TEM图像。
由图1可知，本发明实施例1制备的纳米颗粒探针平均粒径是15.2纳米，且分散性好、尺寸均一。
实施例2
本实施例制备纳米颗粒探针的步骤如下：
A)取透明质酸20mg加入到2mLpH为5.5的磷酸盐缓冲液(简称PBS)中，随后加入溶解有3mg EDC的0.5mL的pH为5.5的PBS，室温搅拌使之混合均匀，得到第一溶液。
B)搅拌均匀后将含有30mg牛血清白蛋白(BSA)的3mL PBS加入到步骤A)得到的第一溶液中，室温搅拌半小时，之后选取截留分子量8000的透析袋透析10小时，冻干保存，得到透明质酸HA修饰的牛血清蛋白分子(以下记为HA-BSA)。
C)于2mL以牛血清蛋白含量测算浓度为0.3mmol/L的HA-BSA水溶液中，加入5mL20mmol/L的氯化锰水溶液，剧烈搅拌使之混合均匀。随后加入1mol/L的NaOH水溶液，使得溶液pH为10.5，于37℃继续搅拌2小时，透析纯化冻干后得到探针粉末，记为纳米颗粒探针。参见图2。图2为本发明实施例2制备的纳米颗粒探针的TEM图像。
由图2可知，本发明实施例2制备的纳米颗粒探针平均粒径是8.3纳米，且分散性好、尺寸均一。
下面以实施例1得到的纳米颗粒探针(纳米探针)，实施核磁共振成像增敏能力和生物纳米酶用途。
实施例3纳米探针核磁共振成像增敏能力
A)将实施例1得到的纳米探针加入到磷酸盐缓冲液(简称PBS)中，配制成锰元素摩尔浓度为0、0.2、0.4、0.8、1.6、3.75、7.5、15、30mmol/L的PBS溶液。
B)使用7.0T核磁共振成像扫描仪对步骤A)得到的溶液进行核磁工作成像(仪器型号：Bruker公司BioSpec70/20USR型小动物核磁共振成像仪(BS-70))。结果参见图3，由图3可知，实施例1得到的纳米探针能有效增强核磁共振成像强度，可用于核磁共振成像增敏剂用途。
实施例4纳米探针纳米酶样活性催化过氧化氢
A)将实施例1得到的纳米探针以重量浓度为0～20ng/μL的浓度，加入到含有浓度为1mmol/L的过氧化氢的磷酸缓冲液中(磷酸盐浓度为50mmol/L，pH值为7.0)。
B)使用紫外可见分光光度计的动力学模式，采用分光光度法监测240纳米处H2O2吸光度值的降低来确定纳米探针酶样活性。在25℃测量步骤A)的溶液在2分钟的反应速率。结果参见图4，由图4可知，实施例1得到的纳米探针能有效催化过氧化氢，具有优异的纳米酶催化效果。
实施例5
(一)探究BSA:Mn不同摩尔比对合成影响
在6mL 10mg/mL BSA水溶液中分别滴加3mL不同浓度的氯化锰溶液，使得蛋白与氯化锰的摩尔比分别为1:340、1:220、1:500，反应5min后再加入250μL 0.25M的氢氧化钠溶液，用水将反应体积补到10mL，剧烈搅拌，反应5min后移入34℃继续反应2h，用10k透析袋透析8h，置于4℃保存。反应后pH和状态见表1。
表1.BSA:Mn不同摩尔比得到产物pH和状态
Figure BDA0002936365450000071
由上述实验结果可知，控制BSA的含量为10mg/mL，BSA：Mn的摩尔比分别为1:340、1:220、1:500。当Mn的摩尔比过高，溶液澄清度及稳定性降低。
(二)探究BSA:Mn相同摩尔比、Mn盐加入方式对合成影响
表2.不同加入方法获得产物的TEM结果
Figure BDA0002936365450000081
(三)BSA-Mn3O4纳米颗粒探针的谷胱甘肽过氧化物酶样活性
为了研究其谷胱甘肽过氧化物酶样活性，采用了谷胱甘肽还原酶(GR)测定，并使用UV-Vis分光光度计在340nm处监测了两分钟内NADPH浓度的降低。当改变纳米颗粒的浓度(0～20ngμL-1)并使其他反应物的浓度保持恒定时，在GSH(2mM)GR(1.7单位)，NADPH(400μM)和BSA-Mn3O4纳米颗粒(0～20ngμL-1)的情况下观察反应速率。反应在25℃的磷酸盐缓冲液(25mM，pH 7.4)中进行。结果见图8，图8示出了BSA-Mn3O4纳米颗粒探针具有优异的谷胱甘肽过氧化物酶样活性。
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视为本发明的保护范围。

Claims (10)

1.一种四氧化三锰纳米探针的制备方法，其特征在于，包括如下步骤：
步骤1)将交联剂溶于溶剂中，得到交联剂溶液；将交联剂溶液与生物分子缓冲溶液混合，得到第一溶液；所述生物分子为肿瘤组织细胞高表达的蛋白质的靶向序列或透明质酸；
步骤2)将蛋白分子缓冲溶液与第一溶液混合，经交联反应后，透析纯化，冻干，得到修饰蛋白；所述蛋白分子为牛血清白蛋白、人血清白蛋白、铁红蛋白、纤连蛋白、蛋清蛋白中的一种或几种；
步骤3)将二价锰盐水溶液滴加入修饰蛋白溶液中，混匀，得到复合溶液；调节复合溶液pH值至10或10以上，搅拌，透析纯化，冻干，得到四氧化三锰纳米探针。
2.根据权利要求1所述的制备方法，其特征在于，步骤1)中，所述交联剂为4-(N-马来酰亚胺甲基)环己烷-1-羧酸磺酸基琥珀酰亚胺酯钠盐(Sulfo-SMCC)、4-(N-马来酰亚胺甲基)环己烷羧酸-N-琥珀酰亚胺酯(SMCC)、琥珀酰亚胺基-4-[N-马来酰亚胺甲基]-环己烷-1-羧基-[6-氨基己酸盐](LC-SMCC)、1-乙基-3-[3-二甲基氨基丙基]碳化二亚胺盐酸化物(EDC)中的一种或几种；所述溶剂为DMSO、DMF、pH为5～6的磷酸盐缓冲溶液(PBS)中的一种或几种。
3.根据权利要求1所述的制备方法，其特征在于，步骤1)中，所述交联剂溶液的浓度为2～10g/L，所述生物分子缓冲溶液的浓度为5～20mmol/L，所述生物分子与交联剂的摩尔比为1∶(1～2)。
4.根据权利要求1所述的制备方法，其特征在于，步骤1)中，所述肿瘤组织细胞高表达的蛋白质包含但不限于整合素蛋白、人表皮生长因子受体-2蛋白、纤连蛋白中的一种或几种。
5.根据权利要求1所述的制备方法，其特征在于，步骤2)中，所述蛋白分子缓冲溶液的浓度为5～50g/L，蛋白分子与生物分子的摩尔比为1：(1～20)。
6.根据权利要求1所述的制备方法，其特征在于，步骤2)中，所述透析纯化的条件为：截留分子量3000～15000Da，透析时间5～20小时；
步骤3)中，所述透析纯化的条件为：截留分子量3000～15000Da，透析时间5～20小时。
7.根据权利要求1所述的制备方法，其特征在于，步骤3)中，所述二价锰盐选自氯化锰、硝酸锰、乙酸锰、硫酸锰中的一种或几种，所述修饰蛋白与锰元素的摩尔比为1：(100～500)。
8.根据权利要求1所述的制备方法，其特征在于，步骤3)中，所述调节复合溶液pH值的试剂为碱性试剂，所述碱性试剂为氢氧化钠、氢氧化钾、氢氧化钙、氨水、三乙胺、吡啶、N-甲基吗啉、四甲基乙二胺中的一种或多种；所述搅拌的温度为30～37℃，搅拌的时间为1～10小时，搅拌的转速为200～1000r/min。
9.权利要求1至8中任一项所述制备方法制得的四氧化三锰纳米探针，其特征在于，所述四氧化三锰纳米探针的粒径为1～100纳米。
10.权利要求9所述四氧化三锰纳米探针在制备核磁共振成像增敏剂和/或生物酶中的用途。
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CN114793528A (zh) * 2022-03-25 2022-07-29 浙江大学 一种纳米酶在重金属胁迫下促进玉米生长中的应用
WO2024066191A1 (zh) * 2022-09-30 2024-04-04 广东邦普循环科技有限公司 磷掺杂纳米级四氧化三锰及其制备方法和电池

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999066964A1 (en) * 1998-06-23 1999-12-29 Surgical Sealants, Incorporated Carbodiimide cross-linked albumin for bioadhesives, surgical sealants and implantable devices
US20130017147A1 (en) * 2010-01-08 2013-01-17 Fujifilm Corporation Targeting agent for tumor site
CN104758956A (zh) * 2015-04-03 2015-07-08 国家纳米科学中心 一种肿瘤靶向的t1-t2双核磁共振成像造影剂及其制备方法和应用
CN105288666A (zh) * 2015-11-03 2016-02-03 中国科学院高能物理研究所 一种水溶性蛋白包覆的磁性纳米颗粒及其制备方法
CN105497923A (zh) * 2015-12-15 2016-04-20 上海纳米技术及应用国家工程研究中心有限公司 一种诊断与热化疗相结合的新型淋巴瘤靶向探针的制备方法
CN109771442A (zh) * 2019-03-15 2019-05-21 华中科技大学 一种增敏肿瘤放疗的复合纳米颗粒及其制备方法和应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999066964A1 (en) * 1998-06-23 1999-12-29 Surgical Sealants, Incorporated Carbodiimide cross-linked albumin for bioadhesives, surgical sealants and implantable devices
US20130017147A1 (en) * 2010-01-08 2013-01-17 Fujifilm Corporation Targeting agent for tumor site
CN104758956A (zh) * 2015-04-03 2015-07-08 国家纳米科学中心 一种肿瘤靶向的t1-t2双核磁共振成像造影剂及其制备方法和应用
CN105288666A (zh) * 2015-11-03 2016-02-03 中国科学院高能物理研究所 一种水溶性蛋白包覆的磁性纳米颗粒及其制备方法
CN105497923A (zh) * 2015-12-15 2016-04-20 上海纳米技术及应用国家工程研究中心有限公司 一种诊断与热化疗相结合的新型淋巴瘤靶向探针的制备方法
CN109771442A (zh) * 2019-03-15 2019-05-21 华中科技大学 一种增敏肿瘤放疗的复合纳米颗粒及其制备方法和应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHUN XIAN GUO等: "Ethylenediamine-mediated synthesis of Mn3O4 nano-octahedrons and their performance as electrocatalysts for the oxygen evolution reaction", 《NANOSCALE》 *
金刚: "《食品毒理学基础与实训教程》", 30 April 2010, 中国轻工业出版社 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113856768A (zh) * 2021-09-26 2021-12-31 沈阳医学院 一种纳米酶溶液及粉末的制备方法
CN114793528A (zh) * 2022-03-25 2022-07-29 浙江大学 一种纳米酶在重金属胁迫下促进玉米生长中的应用
CN114793528B (zh) * 2022-03-25 2023-02-14 浙江大学 一种纳米酶在重金属胁迫下促进玉米生长中的应用
WO2024066191A1 (zh) * 2022-09-30 2024-04-04 广东邦普循环科技有限公司 磷掺杂纳米级四氧化三锰及其制备方法和电池

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