CN114558132A - 一种羟基磷灰石负载的四氧化三铁纳米材料及其制备方法和应用 - Google Patents

一种羟基磷灰石负载的四氧化三铁纳米材料及其制备方法和应用 Download PDF

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CN114558132A
CN114558132A CN202210162490.XA CN202210162490A CN114558132A CN 114558132 A CN114558132 A CN 114558132A CN 202210162490 A CN202210162490 A CN 202210162490A CN 114558132 A CN114558132 A CN 114558132A
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hydroxyapatite
ferroferric oxide
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CN114558132B (zh
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孔祥东
阿塞姆·穆斯塔
祖柏尔
侯亦可
唐喆
王世博
赵瑞波
罗丹丹
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Zhejiang Sci Tech University ZSTU
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Abstract

本发明公开了一种羟基磷灰石负载四氧化三铁纳米材料及其制备方法和应用。属于材料制备技术领域。复合材料形状规则，平均粒径为25～30nm，具有良好的分散性，该复合材料有多晶性质，为含铁的磁性纳米复合材料。制备方法如下：将硬脂酸钙和四丁基磷酸铵分别溶于油酸，混合之后加入油胺和制备好的四氧化三铁纳米粒子，最终得到的混合物转移至具有合适乙醇和水比例的内衬有聚四氟乙烯的高压釜中。将高压釜放入烘箱中加热。反应结束后冷却，用无水乙醇和丙酮洗涤，离心，分散在环己烷中。此外，用
Figure DDA0003515390230000011
F‑127对复合材料进行表面改性，可赋予该纳米复合材料良好的生物相容性。该方法制备的材料具有核磁共振增强效果和作为光热转换剂的潜力，并且具有抗菌协同作用。

Description

一种羟基磷灰石负载的四氧化三铁纳米材料及其制备方法和 应用
技术领域
本发明涉及一种纳米复合材料的制备方法，具体涉及一种羟基磷灰石负载的四氧化三铁(Fe3O4-HAp)纳米复合材料的制备方法，该方法简单稳定，可广泛应用于生物、医学领域。
背景技术
癌症早已成为地球上对人类生命威胁最大的疾病之一，在20世纪导致了数百万人的死亡。为了应对癌症对人类的威胁，近些年来科学家们进行了无数次的尝试，开创了许多种治疗方法。但是由于肿瘤的异质性和一部分并发症，导致患者的存活率并不高。为了解决这些问题，科学家们引入了纳米药物。
近年来，将纳米药物与癌症疗法相结合成为了肿瘤诊疗发展的一大趋势，而且已经有一部分纳米药物通过批准进入市场。纳米级治疗体系成为一种替代性诊疗肿瘤的模型，它有良好的靶向性和较小的副作用，克服了传统疗法的局限性。
分子成像技术经发明以来，作为可以准确诊断和检测肿瘤的技术已经被广泛应用，包括核磁共振成像(MRI)，超声成像(US)，计算机断层扫描(CT)等。其中核磁共振成像技术(MRI)由于其非电离辐射，拥有优异的软组织识别能力和高分辨率，一跃成为肿瘤早期诊断检测应用最广的技术方法。核磁共振造影剂(对比剂)能提高成像分辨率，增加正常与病变组织的成像对比度，从而提高核磁共振诊断肿瘤的敏感性和特异性。基于钆(Gd)的造影剂早已被美国食品和药物监督管理局(FDA)和欧洲药品管理局批准应用，只是近些年来有部分临床数据和研究表明，已经广泛应用的钆造影剂具有一定的肾毒性，可造成肾源性系统性纤维化疾病。为了克服上述缺点，学者们开发了基于铁(Fe)的核磁共振造影剂，虽然其可以作为优异的补铁剂，但是成像对比度没有钆造影剂优秀。
新型癌症治疗方法中，光动力疗法无创且疗效良好，获得了临床认可。与传统光敏剂相比，负载了Anti-trop 2的纳米复合材料具有高靶向性，低毒性和优秀的稳定性。同时还有一定的抗菌协同作用。
发明内容
本发明的目的在于克服现有技术的不足，提供一种具有核磁共振增强效果和增强光动力疗效的羟基磷灰石负载的四氧化三铁纳米复合材料及其制备方法。
为了解决技术问题，本发明首先提供了一种羟基磷灰石负载的四氧化三铁纳米材料(Fe3O4-HAp纳米粒子)的制备方法，其包括如下步骤：
1)将Fe3O4纳米粒子分散在环己烷中：
2)将硬脂酸钙溶解在油酸中制成A溶液，将四丁基磷酸铵溶解在油酸中制备B溶液；在容器中加入一定体积的油酸、油胺，倒入溶液A和B，加入步骤1)所得的Fe3O4纳米粒子分散液；混合液转移到水热釜中加入乙醇与水，在水热釜中反应，反应结束后，离心洗涤，得到Fe3O4-HAP纳米管。
作为本发明的可选方案，所述的Fe3O4纳米粒子可以选自市售产品或采用现有技术中已经报道的方法制备得到，本发明对Fe3O4纳米粒子不做限定。优选的Fe3O4纳米粒子的制备方法可参照文献(M.Z.Iqbal,X.Ma,T.Chen,L.Zhang,W.Ren,L.Xiang,A.Wu,Silica-coated super-paramagnetic iron oxide nanoparticles(SPIONPs):a new typecontrast agent of T1 magnetic resonance imaging(MRI),J.Mater.Chem.B.3(2015)5172–5181)中的方法。
作为本发明的可选方案，所述步骤2)的A液中硬脂酸钙和油酸的摩尔比例为33:1～37：1，B液中四丁基磷酸铵和油酸的摩尔比例为30：1～33:1。
作为本发明的可选方案，混合液的pH值保持在9～10，水热釜中乙醇与水的体积比为3:1；反应温度为150℃～200℃，反应时间为4～8h。
作为本发明的可选方案，所述步骤2)的混合液中，油胺与油酸的体积比为1:1；所述步骤2)中Fe3O4纳米粒子分散液与混合液总体积的比例为3:70～3:90。
本发明还提供了一种
Figure BDA0003515390210000021
F-127包裹的Fe3O4-HAp纳米粒子，其制备方法包括如下步骤：
1)将权利要求1-4任一项所述方法制得的Fe3O4-HAp纳米粒子分散在环己烷中，得到Fe3O4-HAp纳米粒子分散液；
2)将
Figure BDA0003515390210000022
F-127溶解在氯仿中，将所得Fe3O4-HAp纳米粒子分散液加入其中，室温下搅拌；加入蒸馏水进行旋蒸，蒸发出氯仿，得到
Figure BDA0003515390210000031
F-127包裹的Fe3O4-HAp纳米粒子。
作为本发明的可选方案，所述步骤2)中
Figure BDA0003515390210000032
F-127和氯仿的质量比为1:13～1:16。
本发明还提供了一种羟基磷灰石负载的四氧化三铁纳米复合材料，其制备方法包括如下步骤：
3)将Anti-trop2抗体溶液与权利要求5所得的
Figure BDA0003515390210000033
F-127包裹的Fe3O4-HAp纳米粒子分散液搅拌混合，得到含有Anti-trop 2抗体的Fe3O4-HAp纳米粒子分散液；
4)在黑暗条件下，将ICG溶液与步骤3)含有Anti-trop 2抗体的Fe3O4-HAp纳米粒子分散液搅拌混合，将所得混合物离心，最终得到产物；或进一步将产物分散在PBS，保存。
优选的，所述步骤3)中，Anti-trop 2抗体溶液的浓度为1mg/ml，Anti-trop2抗体溶液与步骤2)的
Figure BDA0003515390210000034
F-127包裹的Fe3O4-HAp纳米粒子分散液以1：150～1:200的体积比混合，在4℃的条件下搅拌8h～18h，得到含Anti-trop 2抗体的Fe3O4-HAp纳米粒子分散液。
进一步的，所述步骤4)中将1mg/ml的ICG溶液与步骤3)含有Anti-trop2抗体的Fe3O4-HAp纳米粒子分散液以1:1300～1:1400的体积比混合，在4℃的条件下搅拌8h～18h，全部过程需要在黑暗下进行；然后，将混合物在10000转/分下离心20min得到最终产物，或进一步将产物重新分散到PBS中，并在4℃保存以备进一步使用。
本发明还提供了上述的羟基磷灰石负载的四氧化三铁纳米复合材料在制备核磁共振成像造影剂和肿瘤光动力疗法药物中的应用。
与现有技术相比，本发明具有的有益效果包括：
(1)本发明制备的羟基磷灰石负载的四氧化三铁(Fe3O4-HAp纳米粒子)纳米复合材料，r1弛豫率为2.1mM-1s-1，在核磁共振成像造影剂方面有潜在的应用；
(2)负载了ICG的Fe3O4-Hap纳米粒子还在体外表现出有效的光协同作用，对肿瘤细胞有一定的光动力杀伤作用；
(3)本发明制备的羟基磷灰石负载的四氧化三铁(Fe3O4-HAp)纳米复合材料，还表现出一定的抗菌性能；
(4)本发明对实验仪器的要求低，方法简单易于操作，得到的纳米颗粒形状尺寸均一，分散性良好。
附图说明
图1为本发明实施例1所得产物的透射电镜图片。
图2为本发明实施例1所得产物的透射电镜图片。
图3为本发明实施例1所得产物的X射线能谱分析(EDS)图片。
图4为本发明实施例1所得产物的核磁共振成像图片和T1弛豫率比例。
图5为本发明实施例1所得产物在4T1细胞中摄取的激光共聚焦图片，图中比例尺为2微米。
图6为本发明实施例1所得产物对4T1细胞的活死实验结果，图中比例尺为100微米。
图7为本发明实施例1所得产物对金黄色葡萄球菌和大肠杆菌的抗菌效果。
图8为本发明反向实施例3所得产物的透射电镜图片。
具体实施方式
以下实施例向本领域普通技术人员提供如何制造和评价本发明，所述实施例仅是本公开内容的示范且不圈定限制范围。尽管已经尽力确保关于数值(例如，量、温度等)的准确性，但是应当考虑一些误差和偏差。除非另外说明，否则温度是以℃为单位或者在环境温度下。
实施例一
将Fe3O4 NPs分散在10mL环己烷中。将60mg硬脂酸钙溶解于1mL油酸中以制备溶液A。同样，将34mg磷酸四丁胺(TBA)溶解于1mL油酸中制备溶液B。然后，在烧瓶中取2mL油酸，倒入溶液A和B，然后添加300μL Fe3O4NCs和4mL油胺。混合物的pH值为8.7。将溶液搅拌10分钟，倒入50mL聚四氟乙烯管中，并转移到另一个100mL聚四氟乙烯管中，该管内衬不锈钢高压釜，其中含有体积比为3:1的乙醇-水。在高压釜反应200℃，6h。反应后，通过在11000rpm下离心10min并使用乙醇和丙酮的混合物重复洗涤来沉淀MHNCs。最后，将MHNCs分散在环己烷中以供进一步使用。
800mg
Figure BDA0003515390210000051
F-127PF-127溶解在80mL氯仿中。超声处理10分钟后，将1mL合成的MHNCs溶液轻轻添加到混合物中，并保持搅拌4h。将蒸馏水添加到混合物中，并使用旋转蒸发器蒸发CHCl3。此外，将水中的MHNCs混合物离心并重新分散在蒸馏水中，以消除多余的PF-127。
30μL的Anti-trop 2(1mg/mL)和5mL的PF-127包被的MHNCs在100rpm、4℃的条件下搅拌8h，将Anti-trop 2与PF-127包被的MHNCs偶联。然后，将混合物以10000转/分的速度离心，分散在5mL的PBS中，在4℃保存7天内使用。
将1mg/mL的ICG溶液与5mL含抗体的MHNCs溶液混合，在100rpm和4℃下搅拌过夜。然后，将混合物在10000转/分下离心20min，重新分散到5mL的PBS中，并在4℃保存以备进一步使用。
实施例1所得产物的透射电镜图片如图1和2所示，所得产物的X射线能谱分析(EDS)图片如图3所示，所得产物的核磁共振成像图片和T1弛豫率比例如图4所示。从图1和2中可见，所得的羟基磷灰石负载的四氧化三铁(Fe3O4-HAp纳米粒子)纳米复合材料具有良好的分散性，且粒径均一稳定；从图3中可见，所得的羟基磷灰石负载的四氧化三铁(Fe3O4-HAp纳米粒子)纳米复合材料确实含有Ca和Fe等元素；从图4中可见，羟基磷灰石负载的四氧化三铁(Fe3O4-HAp纳米粒子)纳米复合材料具有优异的核磁共振增强效果。
图5是将所得的羟基磷灰石负载的四氧化三铁(Fe3O4-HAp纳米粒子)纳米复合材料与4T1(小鼠乳腺癌细胞)细胞共培养12h，然后用DiI、DAPI染料分别对细胞膜、核进行染色，发现经过一段时间的迁移，羟基磷灰石负载的四氧化三铁(Fe3O4-HAp纳米粒子)纳米复合材料可以进入细胞质内，且分散均匀。
图6是将不同浓度的羟基磷灰石负载的四氧化三铁(Fe3O4-HAp纳米粒子)纳米复合材料和4T1(小鼠乳腺癌细胞)细胞共培养，再通过808nm波长的激光照射不同时间之后，经过细胞活死染色，发现大部分细胞死亡，从而证明此纳米复合材料的光动力治疗效果良好。
图7是将不同浓度的羟基磷灰石负载的四氧化三铁(Fe3O4-HAp纳米粒子)纳米复合材料和大肠杆菌和金黄色葡萄球菌共培养，再通过808nm波长的激光照射不同时间之后，发现大部分细菌死亡，从而证明此纳米复合材料具有良好的抗菌效果。
实施例二
将Fe3O4 NPs其分散在10mL环己烷中。将60mg硬脂酸钙溶解于1mL油酸中以制备溶液A。同样，将36mg磷酸四丁胺(TBA)溶解于1mL油酸中制备溶液B。然后，在烧瓶中取2mL油酸，倒入溶液A和B，然后添加400μL Fe3O4NCs和4mL油胺。混合物的pH值为9-10。将溶液搅拌10分钟，倒入50mL聚四氟乙烯管中，并转移到另一个100mL聚四氟乙烯管中，该管内衬不锈钢高压釜，其中含有体积比为3:1的乙醇-水。在高压釜反应200℃，6h。反应后，通过在11000rpm下离心10min并使用乙醇和丙酮的混合物重复洗涤来沉淀MHNCs。最后，将MHNCs分散在环己烷中以供进一步使用。
700mg
Figure BDA0003515390210000061
F-127PF-127溶解在70mL氯仿中。超声处理10分钟后，将1mL合成的MHNCs溶液轻轻添加到混合物中，并保持搅拌4h。将蒸馏水添加到混合物中，并使用旋转蒸发器蒸发CHCl3。此外，将水中的MHNCs混合物离心并重新分散在蒸馏水中，以消除多余的PF-127。
50μL的Anti-trop 2(1mg/mL)和5mL的PF-127包被的MHNCs在100rpm、4℃的条件下搅拌8h，将Anti-trop 2与PF-127包被的MHNCs偶联。然后，将混合物以10000转/分的速度离心，分散在5mL的PBS中，在4℃保存7天内使用。
将20μL的ICG溶液(1mg/mL)与5mL含抗体的MHNCs溶液混合，在100rpm和4℃下搅拌过夜，全部过程在黑暗下进行。然后，将混合物在10000转/分下离心20min，重新分散到5mL的PBS中，并在4℃保存以备进一步使用。
对比例
将Fe3O4 NPs其分散在10mL环己烷中。将60mg硬脂酸钙溶解于1mL油酸中以制备溶液A。同样，将36mg磷酸四丁胺(TBA)溶解于1mL油酸中制备溶液B。然后，在烧瓶中取2mL油酸，倒入溶液A和B，然后添加400μL Fe3O4NCs和4mL油胺。混合物的pH值为9-10。将溶液搅拌10分钟，倒入50mL三颈烧瓶中在200℃下反应6h。反应后，通过在11000rpm下离心10min并使用乙醇和丙酮的混合物重复洗涤来沉淀MHNCs。最后，将MHNCs分散在环己烷中以供进一步使用。
700mg
Figure BDA0003515390210000071
F-127PF-127溶解在70mL氯仿中。超声处理10分钟后，将1mL合成的MHNCs溶液轻轻添加到混合物中，并保持搅拌4h。将蒸馏水添加到混合物中，并使用旋转蒸发器蒸发CHCl3。此外，将水中的MHNCs混合物离心并重新分散在蒸馏水中，以消除多余的PF-127。
50μL的Anti-trop 2(1mg/mL)和5mL的PF-127包被的MHNCs在100rpm、4℃的条件下搅拌8h，将Anti-trop 2与PF-127包被的MHNCs偶联。然后，将混合物以10000转/分的速度离心，分散在5mL的PBS中，在4℃保存7天内使用。
将20μL的ICG溶液(1mg/mL)与5mL含抗体的MHNCs溶液混合，在100rpm和4℃下搅拌过夜，全部过程在黑暗下进行。然后，将混合物在10000转/分下离心20min，重新分散到5mL的PBS中，并在4℃保存以备进一步使用。
从图8中可见，通过此对比例得的羟基磷灰石负载的四氧化三铁(Fe3O4-HAp纳米粒子)纳米复合材料粒径不均一，且分散效果不好。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。

Claims (10)

1.一种羟基磷灰石负载的四氧化三铁纳米材料的制备方法，其特征在于，包括如下步骤：
1)将Fe3O4纳米粒子分散在环己烷中：
2)将硬脂酸钙溶解在油酸中制成A溶液，将四丁基磷酸铵溶解在油酸中制备B溶液；在容器中加入一定体积的油酸、油胺，倒入溶液A和B，加入步骤1)所得的Fe3O4纳米粒子分散液；混合液转移到水热釜中加入乙醇与水，在水热釜中反应，反应结束后，离心洗涤，得到Fe3O4-HAp纳米粒子，即所述的羟基磷灰石负载的四氧化三铁纳米材料。
2.根据权利要求1所述的制备方法，其特征在于，所述步骤2)的A液中硬脂酸钙和油酸的摩尔比例为33:1～37：1，B液中四丁基磷酸铵和油酸的摩尔比例为30：1～33:1。
3.根据权利要求1所述的制备方法，其特征在于，混合液的pH值保持在9～10，水热釜中乙醇与水的体积比为3:1；反应温度为150℃～200℃，反应时间为4～8h。
4.根据权利要求1所述的制备方法，其特征在于，所述步骤2)的混合液中，油胺与油酸的体积比为1:1；所述步骤2)中Fe3O4纳米粒子分散液与混合液总体积的比例为3:70～3:90。
5.一种
Figure FDA0003515390200000011
F-127包裹的Fe3O4-HAp纳米粒子，其特征在于，其制备方法包括如下步骤：
1)将权利要求1-4任一项所述方法制得的Fe3O4-HAp纳米粒子分散在环己烷中，得到Fe3O4-HAp纳米粒子分散液；
2)将
Figure FDA0003515390200000013
F-127溶解在氯仿中，将所得Fe3O4-HAp纳米粒子分散液加入其中，室温下搅拌；加入蒸馏水进行旋蒸，蒸发出氯仿，得到
Figure FDA0003515390200000012
F-127包裹的Fe3O4-HAP纳米管。
6.根据权利要求5所述的
Figure FDA0003515390200000014
F-127包裹的Fe3O4-HAp纳米粒子，其特征在于，所述步骤2)中
Figure FDA0003515390200000015
F-127和氯仿的质量比为1:13～1:16。
7.一种羟基磷灰石负载的四氧化三铁纳米复合材料，其特征在于，其制备方法包括如下步骤：
1)将Anti-trop2抗体溶液与权利要求5所得的
Figure FDA0003515390200000016
F-127包裹的Fe3O4-HAp纳米粒子分散液搅拌混合，得到含有Anti-trop2抗体的Fe3O4-HAP纳米管分散液；
2)在黑暗条件下，将ICG溶液与步骤1)含有Anti-trop2抗体的Fe3O4-HAp纳米粒子分散液搅拌混合，将所得混合物离心，最终得到产物；或进一步将产物分散在PBS，保存。
8.根据权利要求7所述的羟基磷灰石负载的四氧化三铁纳米复合材料，其特征在于，所述步骤1)中，Anti-trop 2抗体溶液的浓度为1mg/ml，Anti-trop 2抗体溶液与权利要求5步骤2)的
Figure FDA0003515390200000021
F-127包裹的Fe3O4-HAp纳米粒子分散液以1：150～1:200的体积比混合，在4℃的条件下搅拌8h～18h，得到含Anti-trop 2抗体的Fe3O4-HAp纳米粒子分散液。
9.根据权利要求7所述的羟基磷灰石负载的四氧化三铁纳米复合材料，其特征在于，所述步骤2)中将1mg/ml的ICG溶液与步骤1)含有Anti-trop2抗体的Fe3O4-HAp纳米粒子分散液以1:1300～1:1400的体积比混合，在4℃的条件下搅拌8h～18h，全部过程需要在黑暗下进行；然后，将混合物在10000转/分下离心20min得到羟基磷灰石负载的四氧化三铁纳米复合材料，或进一步将产物重新分散到PBS中，并在4℃保存以备进一步使用。
10.权利要求7所述的羟基磷灰石负载的四氧化三铁纳米复合材料在制备核磁共振成像造影剂和肿瘤光动力疗法药物中的应用。
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CN116371434A (zh) * 2023-04-06 2023-07-04 中国科学院生态环境研究中心 一种新型铁基固体芬顿催化剂及其制备方法与应用
CN116395747A (zh) * 2023-02-22 2023-07-07 浙江理工大学 一种四氧化三锰-硫化银Janus结构纳米复合材料及其制备方法

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CN115282944A (zh) * 2022-08-12 2022-11-04 山东大学 硬脂酸改性鲍鱼壳磁性多孔羟基磷灰石微球及其制备方法
CN115282944B (zh) * 2022-08-12 2024-04-12 山东大学 硬脂酸改性鲍鱼壳磁性多孔羟基磷灰石微球及其制备方法
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