CN105477648B - 一种淋巴靶向的类普鲁士蓝纳米颗粒及其制备方法 - Google Patents
一种淋巴靶向的类普鲁士蓝纳米颗粒及其制备方法 Download PDFInfo
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- CN105477648B CN105477648B CN201510981682.3A CN201510981682A CN105477648B CN 105477648 B CN105477648 B CN 105477648B CN 201510981682 A CN201510981682 A CN 201510981682A CN 105477648 B CN105477648 B CN 105477648B
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Abstract
本发明提供了一种淋巴靶向的类普鲁士蓝纳米颗粒及其制备方法。该方法通过在二乙三胺五乙酸上交联上透明质酸并螯合在钆离子上,形成稳定的表面带有透明质酸的具有淋巴靶向的类普鲁士蓝纳米颗粒。该纳米颗粒表面包裹透明质酸,不仅具有保护类普鲁士蓝不受生理环境影响,而且具有淋巴靶向作用;在生理条件下非常稳定,提高了生物相容性和造影信号稳定性;合成反应在室温下进行,节能环保,操作简单易行;可以作为光吸收物质,光吸收效率较高,光稳定较好;其内核类普鲁士蓝纳米颗粒是利用钆取代三价铁的位置,具有更多的未配对电子,磁共振信号更强;其表面包裹物为透明质酸,透明质酸为人体组织成分之一,生物相容性非常好。
Description
技术领域
本发明属于生物医用材料领域,具体涉及一种淋巴靶向的类普鲁士蓝纳米颗粒及其制备方法。
背景技术
近年来,癌症的发病率呈明显的上升趋势,纳米材料由于其特殊性质在生物医学领域尤其是癌症诊断和治疗中的应用越来越备受关注,纳米材料有望解决传统生物医学方法的局限性。然而,当前纳米材料的研究仅限于体外试验,其在人体内的生物安全性得不到保障,严重限制了其进一步发展。解决纳米材料的生物安全性成为纳米材料在生物医学领域应用的关键问题。癌症的主要生物学特征是侵袭和转移,肿瘤转移主要通过血管和淋巴管途径实现,其中,淋巴管是其转移的主要通道。肿瘤一旦发生转移将会给治疗带来很大的困难,因此对淋巴管疾病和肿瘤的诊断具有重要意义。
透明质酸是由两个双糖单位D-葡萄糖醛酸及N-乙酰葡糖胺组成的高分子聚合物,存在于动物体大部分的软结缔组织中,其主要功能是润滑和保护细胞,调节细胞在粘弹性基质上移动,稳定胶原网状结构并使其免受机械性破坏。人体内的透明质酸受体包括毛细淋巴管内的淋巴内皮透明质酸受体LYVE-1(Lymphatic vascular endothelialhyaluronan receptor-1)和肿瘤细胞表面透明质酸受体CD44。因此,透明质酸受体可被视为肿瘤特异性靶点,不仅可以作为癌症治疗的靶点,还可以作为淋巴转移肿瘤影像学诊断的新靶点。
普鲁士蓝是一种临床用药,是医院的常规储备药物。普鲁士蓝长期的临床用药验证了其在人体内可靠的生物安全性和代谢途径。从结构上说,普鲁士蓝是一种二价铁和三价铁混合价态的六氰络铁酸盐,在普鲁士蓝纳米粒子结构中,二价铁与碳原子相连,是低自旋的,三价铁和氮原子相连,是高自旋的,在每个二价铁-碳-氮-三价铁的结构单元中,共有5个未配对的电子,由于普鲁士蓝的这种特殊结构,它可以缩短水质子的横向或纵向弛豫时间,从而用于磁共振成像。同时普鲁士蓝纳米粒子内的金属离子与氰基由于通过配位键连接,因此非常牢固,不易产生毒性的游离金属离子和氢氰酸,也不易产生氧自由基等有害物质,因此安全性很高。此外,与传统纳米材料相比,如金纳米棒,碳纳米管等,普鲁士蓝纳米粒子的光热转换效率较高,且光热稳定性强,在光声成像应用中具有很明显的优势。已有研究证实了普鲁士蓝作为MRI造影剂的成像应用(J.Mater.Chem.,2010,20,5251-5259;Inorganic Chemistry Communications,2010,13,58-61;)和光声造影剂的成像应用(Chem.Commun.,2013,49,11029-11031;Biomaterials,2014,35,9844-9852.)。
由于在普鲁士蓝晶胞中,过渡金属离子占据两个不同的位置,而这两个位置上都可以用不同的其它过渡金属来替代,导致类普鲁士蓝粒子极大的品种多样性。因此类普鲁士蓝纳米颗粒将有潜力解决其它多数纳米材料生物安全性得不到保障且价格昂贵的医学难题,其研究和应用价值巨大。
然而,目前的普鲁士蓝纳米颗粒的磁共振成像信号不足,对于普鲁士蓝纳米的颗粒的保护修饰研究目前大部分利用非水溶性的有机物,而水溶性的有机物包裹的普鲁士蓝纳米颗粒采用的是静电吸附的方法,得到的产物在体内生理条件下,有机层容易被破坏,形成裸露的普鲁士蓝纳米颗粒,裸露的普鲁士蓝纳米颗粒不溶于水环境,易被机体清除,同时生理盐中的金属离子与裸露的普鲁士蓝容易发生置换反应,对生物体有潜在的毒性作用。
发明内容
为了解决上述技术问题,本发明的目的在于提供一种淋巴靶向的类普鲁士蓝纳米颗粒及其制备方法,通过在二乙三胺五乙酸上交联上透明质酸并螯合在钆离子上,形成稳定的表面带有透明质酸的具有淋巴靶向的类普鲁士蓝纳米颗粒。
本发明的目的通过以下技术方案得以实现:
一种淋巴靶向的类普鲁士蓝纳米颗粒的制备方法,包括以下步骤:
(1)将透明质酸(HA)溶于缓冲溶液中,混合搅拌均匀后,配制成A溶液,其中,每毫升A溶液中含有1-20mg的透明质酸,再在搅拌条件下缓慢加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)、N-羟基琥珀酰亚胺(NHS),反应5-30min,用PBS缓冲溶液调节pH值到7,然后再加入二胺类连接剂,反应6-48h后用去离子水透析处理,得到产物E;其中,透明质酸、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、N-羟基琥珀酰亚胺和二胺类连接剂的质量比为1:(1-10):(2-20):(10-100);
(2)将二乙三胺五乙酸溶于缓冲溶液中,混合搅拌均匀后,配制成B溶液,其中,每毫升B溶液中含有1-20mg的二乙三胺五乙酸,再在搅拌的条件下缓慢加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)、N-羟基琥珀酰亚胺(NHS),反应5-30min,用PBS缓冲溶液调节pH值到7,然后再加入产物E,反应6-48h后用去离子水透析处理,得到产物F;其中,二乙三胺五乙酸、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、N-羟基琥珀酰亚胺和产物E的质量比为1:(1-10):(2-20):(0.2-1);
(3)将六氰络铁酸盐溶于溶剂中,混合搅拌均匀后,配制成C溶液,其中,每毫升C溶液中含有1-20mg的六氰络铁酸盐;
(4)将钆盐溶于溶剂中,混合搅拌均匀后,配制成D溶液,其中,每毫升D溶液中含有1-20mg的钆盐;
(5)将C溶液磁力搅拌均匀,然后再逐滴加入D溶液,磁力搅拌反应20-120min,离心分离得到产物G,将产物G溶于水中,形成产物G的溶液;其中,钆盐与六氰络铁酸盐的质量比1:0.2-1;
(6)将产物F加入到产物G的溶液中,混合搅拌均匀,反应4-24h后用去离子水透析处理,得到表面带有透明质酸的淋巴靶向的类普鲁士蓝纳米颗粒;其中,产物G与产物F的质量比为1:0.2-2。
上述制备方法中,主要采用的是四步法合成具有淋巴靶向的类普鲁士蓝纳米颗粒。其中,第一步是通过二胺类连接改变透明质酸反应官能团羧基,使之变为氨基;第二步是使带有氨基官能团的透明质酸通过EDC、NHS反应交联上二乙三胺五乙酸,得到连接有透明质酸的二乙三胺五乙酸产物;第三步是使钆盐与六氰络铁酸盐反应,形成类普鲁士蓝纳米颗粒;第四步是使带有透明质酸的二乙三胺五乙酸螯合在钆离子上,形成稳定的表面带有透明质酸的淋巴靶向的类普鲁士蓝纳米颗粒产物。
上述制备方法中,二乙三胺五乙酸是一种具有二乙烯三胺骨架的多元羧酸,对金属离子具有很强的螯合作用,其螯合能力比EDTA强100倍左右。由于二乙三胺五乙酸能形成八个配位键,而过渡金属的配位键一般少于八个,因此,二乙三胺五乙酸可以交联上其它的分子,形成衍生物后再与金属螯合,其稳定性和生物相容性较好。
上述制备方法中,优选地,所述透明质酸的分子量为6千-7万。透明质酸是由两个双糖单位D-葡萄糖醛酸及N-乙酰葡糖胺组成的高分子聚合物,存在于动物体大部分的软结缔组织中,其主要功能是润滑和保护细胞,调节细胞在粘弹性基质上移动,稳定胶原网状结构并使其免受机械性破坏。透明质酸受体可被视为肿瘤特异性靶点,不仅可以作为癌症治疗的靶点,还可以作为淋巴转移肿瘤影像学诊断的新靶点。
上述制备方法中,优选地,所述缓冲溶液可以为MES缓冲溶液、PBS缓冲溶液、Tris缓冲溶液、HEPES缓冲溶液、TES缓冲溶液、MOPS缓冲溶液和PIPES缓冲溶液等中的一种。
上述制备方法中,优选地,所述二胺类连接剂可以为脂肪族二胺或饱和环二胺;优选地,所述脂肪族二胺可以为乙二胺、1,3-丙二胺和2,2-二甲基-1,3-丙二胺等中的一种;更加优选地,所述饱和环二胺可以为环己二胺。
上述制备方法种,优选地,所述六氰络铁酸盐可以为亚铁氰化钾、亚铁氰化钠和亚铁氰化铵等中的一种。
上述制备方法中,优选地,所述溶剂可以为水、乙醇和乙二醇等中的一种。
上述制备方法中,优选地,所述钆盐可以为氯化钆、硝酸钆和硫酸钆等中的一种。由于在普鲁士蓝晶胞中,过渡金属离子占据两个不同的位置,而这两个位置上都可以用不同的其它过渡金属来替代,当用钆离子取代三价铁的位置时,能够增加结构单元中未配对的电子数,从而有效的增强磁共振成像效果。
本发明还提供了一种利用上述制备方法制备得到的表面带有透明质酸的淋巴靶向的类普鲁士蓝纳米颗粒。该纳米颗粒内核层为类普鲁士蓝纳米颗粒可产生磁共振信号和光声信号,外层为透明质酸,外层与内核层通过二乙三胺五乙酸与钆强配位交联,有效的保护类普鲁士蓝纳米颗粒不被破坏,能够增加普鲁士蓝纳米颗粒的水溶性和生物相容性,同时具有淋巴靶向作用。该纳米颗粒可作为磁共振和光声双功能成像造影剂,结合两种成像优势,为医学诊断提供更全面有效的信息。
本发明的有益效果:
本发明表面带有透明质酸的淋巴靶向的类普鲁士蓝纳米颗粒具有以下优点:
(1)表面包裹透明质酸,不仅具有保护类普鲁士蓝不受生理环境影响,而且具有淋巴靶向作用;
(2)在生理条件下非常稳定,能够提高生物相容性和造影信号稳定性;
(3)合成反应在室温下进行,节能环保,操作简单易行;
(4)可以作为光吸收物质,光吸收效率较高,光稳定较好;
(5)其内核类普鲁士蓝纳米颗粒是利用钆取代三价铁的位置,具有更多的未配对电子,磁共振信号更强;
(6)其表面包裹物为透明质酸,透明质酸为人体组织成分之一,生物相容性非常好。
附图说明
图1为实施例7的淋巴靶向的类普鲁士蓝纳米颗粒的扫描电镜图。
具体实施方式
为了对本发明的技术特征、目的和有益效果有更加清楚的理解,现对本发明的技术方案进行以下详细说明,但不能理解为对本发明的可实施范围的限定。
实施例1
本实施例提供了一种淋巴靶向的类普鲁士蓝纳米颗粒的制备方法,包括以下步骤:
(1)将分子量为1万的透明质酸溶于MES缓冲溶液中,混合搅拌均匀后,配制成A溶液,其中,每毫升A溶液中含有1mg的透明质酸,取50mL的A溶液,在搅拌条件下缓慢加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐200mg、N-羟基琥珀酰亚胺300mg,反应15min,用PBS缓冲溶液调节pH值到7,然后再加入乙二胺3mL,反应24h后用去离子水透析处理,得到产物E。
(2)将二乙三胺五乙酸溶于缓冲溶液中,混合搅拌均匀后,配制成B溶液,其中,每毫升B溶液中含有1mg的二乙三胺五乙酸,取50mL的B溶液,在搅拌的条件下缓慢加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐200mg、N-羟基琥珀酰亚胺300mg,反应15min,用PBS缓冲溶液调节pH值到7,然后再加入产物E,反应24h后用去离子水透析处理,得到产物F。
(3)将亚铁氰化钾溶于乙醇中,混合搅拌均匀后,配制成C溶液,其中,每毫升C溶液中含有1mg的亚铁氰化钾;
(4)将硝酸钆溶于乙醇中,混合搅拌均匀后,配制成D溶液,其中,每毫升D溶液中含有1mg的硝酸钆;
(5)将C溶液磁力搅拌均匀,然后再逐滴加入D溶液,磁力搅拌反应30min,离心分离得到产物G,将产物G溶于水中,形成产物G的溶液;其中,硝酸钆与亚铁氰化钾的质量比1:0.5;
(6)将产物F加入到产物G的溶液中,混合搅拌均匀,反应12h后用去离子水透析处理,得到表面带有透明质酸的淋巴靶向的类普鲁士蓝纳米颗粒;其中,产物G与产物F的质量比为1:1。
实施例2
本实施例提供了一种淋巴靶向的类普鲁士蓝纳米颗粒的制备方法,包括以下步骤:
(1)将分子量为6800的透明质酸溶于TES缓冲溶液中,混合搅拌均匀后,配制成A溶液,其中,每毫升A溶液中含有5mg的透明质酸,取50mL的A溶液,在搅拌条件下缓慢加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐1g、N-羟基琥珀酰亚胺1.5g,反应10min,用PBS缓冲溶液调节pH值到7,然后再加入乙二胺5mL,反应12h后用去离子水透析处理,得到产物E。
(2)将二乙三胺五乙酸溶于TES缓冲溶液中,混合搅拌均匀后,配制成B溶液,其中,每毫升B溶液中含有5mg的二乙三胺五乙酸,取50mL的B溶液,在搅拌的条件下缓慢加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐1g、N-羟基琥珀酰亚胺1.5g,反应10min,用PBS缓冲溶液调节pH值到7,然后再加入产物E,反应12h后用去离子水透析处理,得到产物F。
(3)将亚铁氰化钠溶于水中,混合搅拌均匀后,配制成C溶液,其中,每毫升C溶液中含有5mg的亚铁氰化钠;
(4)将氯化钆溶于水中,混合搅拌均匀后,配制成D溶液,其中,每毫升D溶液中含有5mg的氯化钆;
(5)将C溶液磁力搅拌均匀,然后再逐滴加入D溶液,磁力搅拌反应120min,离心分离得到产物G,将产物G溶于水中,形成产物G的溶液;其中,氯化钆与亚铁氰化钠的质量比1:0.2;
(6)将产物F加入到产物G的溶液中,混合搅拌均匀,反应12h后用去离子水透析处理,得到表面带有透明质酸的淋巴靶向的类普鲁士蓝纳米颗粒;其中,产物G与产物F的质量比为1:0.2。
实施例3
本实施例提供了一种淋巴靶向的类普鲁士蓝纳米颗粒的制备方法,包括以下步骤:
(1)将分子量为3万的透明质酸溶于Tris缓冲溶液中,混合搅拌均匀后,配制成A溶液,其中,每毫升A溶液中含有10mg的透明质酸,取50mL的A溶液,在搅拌条件下缓慢加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐2g、N-羟基琥珀酰亚胺3g,反应30min,用PBS缓冲溶液调节pH值到7,然后再加入1,3-丙二胺3mL,反应48h后用去离子水透析处理,得到产物E。
(2)将二乙三胺五乙酸溶于Tris缓冲溶液中,混合搅拌均匀后,配制成B溶液,其中,每毫升B溶液中含有10mg的二乙三胺五乙酸,取50mL的B溶液,在搅拌的条件下缓慢加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐2g、N-羟基琥珀酰亚胺3g,反应30min,用PBS缓冲溶液调节pH值到7,然后再加入产物E,反应24h后用去离子水透析处理,得到产物F。
(3)将亚铁氰化铵溶于乙二醇中,混合搅拌均匀后,配制成C溶液,其中,每毫升C溶液中含有20mg的亚铁氰化铵;
(4)将硫酸钆溶于乙二醇中,混合搅拌均匀后,配制成D溶液,其中,每毫升D溶液中含有20mg的硫酸钆;
(5)将C溶液磁力搅拌均匀,然后再逐滴加入D溶液,磁力搅拌反应20min,离心分离得到产物G,将产物G溶于水中,形成产物G的溶液;其中,硫酸钆与亚铁氰化铵的质量比1:0.5;
(6)将产物F加入到产物G的溶液中,混合搅拌均匀,反应6h后用去离子水透析处理,得到表面带有透明质酸的淋巴靶向的类普鲁士蓝纳米颗粒;其中,产物G与产物F的质量比为1:2。
实施例4
本实施例提供了一种淋巴靶向的类普鲁士蓝纳米颗粒的制备方法,包括以下步骤:
(1)将分子量为5万的透明质酸溶于MOPS缓冲溶液中,混合搅拌均匀后,配制成A溶液,其中,每毫升A溶液中含有20mg的透明质酸,取50mL的A溶液,在搅拌条件下缓慢加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐4g、N-羟基琥珀酰亚胺6g,反应30min,用PBS缓冲溶液调节pH值到7,然后再加入2,2-二甲基-1,3-丙二胺5mL,反应6h后用去离子水透析处理,得到产物E。
(2)将二乙三胺五乙酸溶于MOPS缓冲溶液中,混合搅拌均匀后,配制成B溶液,其中,每毫升B溶液中含有20mg的二乙三胺五乙酸,取50mL的B溶液,在搅拌的条件下缓慢加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐4g、N-羟基琥珀酰亚胺6g,反应30min,用PBS缓冲溶液调节pH值到7,然后再加入产物E,反应48h后用去离子水透析处理,得到产物F。
(3)将亚铁氰化钾溶于水中,混合搅拌均匀后,配制成C溶液,其中,每毫升C溶液中含有10mg的亚铁氰化钾;
(4)将硝酸钆溶于水中,混合搅拌均匀后,配制成D溶液,其中,每毫升D溶液中含有10mg的硝酸钆;
(5)将C溶液磁力搅拌均匀,然后再逐滴加入D溶液,磁力搅拌反应20min,离心分离得到产物G,将产物G溶于水中,形成产物G的溶液;其中,硝酸钆与亚铁氰化钾的质量比1:1;
(6)将产物F加入到产物G的溶液中,混合搅拌均匀,反应6h后用去离子水透析处理,得到表面带有透明质酸的淋巴靶向的类普鲁士蓝纳米颗粒;其中,产物G与产物F的质量比为1:1。
实施例5
本实施例提供了一种淋巴靶向的类普鲁士蓝纳米颗粒的制备方法,包括以下步骤:
(1)将分子量为7万的透明质酸溶于PIPES缓冲溶液中,混合搅拌均匀后,配制成A溶液,其中,每毫升A溶液中含有20mg的透明质酸,取50mL的A溶液,在搅拌条件下缓慢加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐4g、N-羟基琥珀酰亚胺6g,反应30min,用PBS缓冲溶液调节pH值到7,然后再加入乙二胺5mL,反应6h后用去离子水透析处理,得到产物E。
(2)将二乙三胺五乙酸溶于PIPES缓冲溶液中,混合搅拌均匀后,配制成B溶液,其中,每毫升B溶液中含有20mg的二乙三胺五乙酸,取50mL的B溶液,在搅拌的条件下缓慢加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐4g、N-羟基琥珀酰亚胺6g,反应30min,用PBS缓冲溶液调节pH值到7,然后再加入产物E,反应48h后用去离子水透析处理,得到产物F。
(3)将亚铁氰化钾溶于水中,混合搅拌均匀后,配制成C溶液,其中,每毫升C溶液中含有10mg的亚铁氰化钾;
(4)将硝酸钆溶于水中,混合搅拌均匀后,配制成D溶液,其中,每毫升D溶液中含有10mg的硝酸钆;
(5)将C溶液磁力搅拌均匀,然后再逐滴加入D溶液,磁力搅拌反应20min,离心分离得到产物G,将产物G溶于水中,形成产物G的溶液;其中,硝酸钆与亚铁氰化钾的质量比1:1;
(6)将产物F加入到产物G的溶液中,混合搅拌均匀,反应6h后用去离子水透析处理,得到表面带有透明质酸的淋巴靶向的类普鲁士蓝纳米颗粒;其中,产物G与产物F的质量比为1:1。
实施例6
本实施例提供了一种淋巴靶向的类普鲁士蓝纳米颗粒的制备方法,包括以下步骤:
(1)将分子量为1万的透明质酸溶于PBS缓冲溶液中,混合搅拌均匀后,配制成A溶液,其中,每毫升A溶液中含有1mg的透明质酸,取50mL的A溶液,在搅拌条件下缓慢加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐200mg、N-羟基琥珀酰亚胺300mg,反应15min,用PBS缓冲溶液调节pH值到7,然后再加入环乙二胺3mL,反应24h后用去离子水透析处理,得到产物E。
(2)将二乙三胺五乙酸溶于PIPES缓冲溶液中,混合搅拌均匀后,配制成B溶液,其中,每毫升B溶液中含有1mg的二乙三胺五乙酸,取50mL的B溶液,在搅拌的条件下缓慢加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐200mg、N-羟基琥珀酰亚胺300mg,反应15min,用PBS缓冲溶液调节pH值到7,然后再加入产物E,反应24h后用去离子水透析处理,得到产物F。
(3)将亚铁氰化钾溶于水中,混合搅拌均匀后,配制成C溶液,其中,每毫升C溶液中含有1mg的亚铁氰化钾;
(4)将硝酸钆溶于水中,混合搅拌均匀后,配制成D溶液,其中,每毫升D溶液中含有1mg的硝酸钆;
(5)将C溶液磁力搅拌均匀,然后再逐滴加入D溶液,磁力搅拌反应30min,离心分离得到产物G,将产物G溶于水中,形成产物G的溶液;其中,硝酸钆与亚铁氰化钾的质量比1:0.5;
(6)将产物F加入到产物G的溶液中,混合搅拌均匀,反应12h后用去离子水透析处理,得到表面带有透明质酸的淋巴靶向的类普鲁士蓝纳米颗粒;其中,产物G与产物F的质量比为1:1。
实施例7淋巴靶向的类普鲁士蓝纳米颗粒扫描电镜实验
采用实施例1制备得到的淋巴靶向的类普鲁士蓝纳米颗粒,对其进行扫描电镜实验,采用的扫描电镜为Nova NanoSEM,扫描电镜参数为:mag 50000;det TLD;WD 5.2mm;HV10kV;实验结果如图1所示。
实验结果表明:该透明质酸包裹的淋巴靶向的类普鲁士蓝纳米颗粒粒径范围为40-100nm,表面具有有机层,说明透明质酸的有效包裹,粒径范围非常适合肿瘤成像的粒径范围(肿瘤成像的纳米粒径范围20-200nm)。
综上所述,本发明的表面带有透明质酸的淋巴靶向的类普鲁士蓝纳米颗粒表面包裹透明质酸,不仅具有保护类普鲁士蓝不受生理环境影响,而且具有淋巴靶向作用;在生理条件下非常稳定,能够提高生物相容性和造影信号稳定性;合成反应在室温下进行,节能环保,操作简单易行;可以作为光吸收物质,光吸收效率较高,光稳定较好;其内核类普鲁士蓝纳米颗粒是利用钆取代三价铁的位置,具有更多的未配对电子,磁共振信号更强;其表面包裹物为透明质酸,透明质酸为人体组织成分之一,生物相容性非常好。
Claims (10)
1.一种淋巴靶向的类普鲁士蓝纳米颗粒的制备方法,其特征在于,包括以下步骤:
(1)将透明质酸溶于缓冲溶液中,混合搅拌均匀后,配制成A溶液,其中,每毫升A溶液中含有1-20mg的透明质酸,再在搅拌条件下缓慢加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、N-羟基琥珀酰亚胺,反应5-30min,用PBS缓冲溶液调节pH值到7,然后再加入二胺类连接剂,反应6-48h后用去离子水透析处理,得到产物E;其中,透明质酸、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、N-羟基琥珀酰亚胺和二胺类连接剂的质量比为1:(1-10):(2-20):(1-100);
(2)将二乙三胺五乙酸溶于缓冲溶液中,混合搅拌均匀后,配制成B溶液,其中,每毫升B溶液中含有1-20mg的二乙三胺五乙酸,再在搅拌的条件下缓慢加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、N-羟基琥珀酰亚胺,反应5-30min,用PBS缓冲溶液调节pH值到7,然后再加入产物E,反应6-48h后用去离子水透析处理,得到产物F;其中,二乙三胺五乙酸、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、N-羟基琥珀酰亚胺和产物E的质量比为1:(1-10):(2-20):(0.2-1);
(3)将六氰络铁酸盐溶于溶剂中,混合搅拌均匀后,配制成C溶液,其中,每毫升C溶液中含有1-20mg的六氰络铁酸盐;
(4)将钆盐溶于溶剂中,混合搅拌均匀后,配制成D溶液,其中,每毫升D溶液中含有1-20mg的钆盐;
(5)将C溶液磁力搅拌均匀,然后再逐滴加入D溶液,磁力搅拌反应20-120min,离心分离得到产物G,将产物G溶于水中,形成产物G的溶液;其中,钆盐与六氰络铁酸盐的质量比1:0.2-1;
(6)将产物F加入到产物G的溶液中,混合搅拌均匀,反应4-24h后用去离子水透析处理,得到表面带有透明质酸的淋巴靶向的类普鲁士蓝纳米颗粒;其中,产物G与产物F的质量比为1:0.2-2。
2.根据权利要求1所述的制备方法,其特征在于:所述透明质酸的分子量为6千-7万。
3.根据权利要求1所述的制备方法,其特征在于:所述缓冲溶液为MES缓冲溶液、PBS缓冲溶液、Tris缓冲溶液、HEPES缓冲溶液、TES缓冲溶液、MOPS缓冲溶液和PIPES缓冲溶液中的一种。
4.根据权利要求1所述的制备方法,其特征在于:所述二胺类连接剂为脂肪族二胺或饱和环二胺。
5.根据权利要求4所述的制备方法,其特征在于:所述脂肪族二胺为乙二胺、1,3-丙二胺和2,2-二甲基-1,3-丙二胺中的一种。
6.根据权利要求4所述的制备方法,其特征在于:所述饱和环二胺为环己二胺。
7.根据权利要求1所述的制备方法,其特征在于:所述六氰络铁酸盐为亚铁氰化钾、亚铁氰化钠和亚铁氰化铵中的一种。
8.根据权利要求1所述的制备方法,其特征在于:所述溶剂为水、乙醇和乙二醇中的一种。
9.根据权利要求1所述的制备方法,其特征在于:所述钆盐为氯化钆、硝酸钆和硫酸钆中的一种。
10.一种权利要求1-9任意一项所述的制备方法制备得到的表面带有透明质酸的淋巴靶向的类普鲁士蓝纳米颗粒。
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