CN117482229A - 一种酶响应性多功能纳米点、制备方法及其在增强***癌成像和治疗方面的应用 - Google Patents
一种酶响应性多功能纳米点、制备方法及其在增强***癌成像和治疗方面的应用 Download PDFInfo
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Abstract
本发明提供了一种酶响应性多功能纳米点、制备方法及其在增强***癌成像和治疗方面的应用,属于纳米材料技术领域。该种酶响应性多功能纳米点AMNDs‑FTp在***癌微环境过表达的ALP发生去磷酸化反应后,在肿瘤部位显著地聚集,对于荧光、CT、核磁等多种模式成像效果均显著增强。增强的CT/核磁成像可以实现术前肿瘤的准确诊断,增强的荧光成像效果可以实现“术中视觉导航”,同时还对近红外光吸收并转换成热量的性能显著增强,提升了光热治疗***癌肿瘤的效果。本发明的酶响应性多功能纳米点材料提供一种有效的策略来提高***癌的诊断准确性和治疗效果,具有广阔的应用前景。
Description
技术领域
本发明属于高分子材料技术领域,具体涉及一种碱性磷酸酶响应的多功能纳米点的制备方法,及其在增强***癌成像和治疗方面的应用。
背景技术
***癌是男性泌尿***的恶性肿瘤之一,其发病率呈逐年提高的趋势。目前,针对***癌的诊断和治疗仍面临许多临床挑战。设计一种能够实现准确诊断和治疗***癌的纳米诊疗平台具有重要的临床意义。
近年来,金纳米粒子(AuNPs)由于其优异的荧光特性、生物相容性和易于表面功能化等特性,受到研究人员的广泛关注。锰(Mn)是维持人体生理功能必需的微量元素,而Mn2+是一种顺磁性金属离子,具有很好的核磁成像效果,在生物医学中有着广泛的应用。基于AuNPs和Mn2+的优势,开发一种有效且简单的方法将AuNPs和Mn2+组装成单一的多功能球形纳米颗粒,可以更好地发挥它们在肿瘤诊断和治疗中的优势。值得注意的是,金基纳米材料的抗肿瘤效果与其尺寸密切相关。较小的尺寸(小于20纳米)具有较好的荧光特性,穿透组织更深,在肿瘤组织内扩散更均匀。不幸的是,它们很容易被人体排出体外,导致在肿瘤处蓄积不足,限制了其诊断和治疗的效果。较大的尺寸(大于100纳米)可以发挥实体瘤的高通透性和滞留(EPR)效应,并且在近红外区域具有较强的吸收,因此适用于光热治疗。然而,它们很容易被网状内皮***捕获,导致治疗效果不理想。为了解决上述矛盾,通过外界刺激诱导纳米粒子的聚集可能提供一种解决方案,进而充分利用纳米颗粒的优势。
研究人员发现借助肿瘤微环境中过表达的酶可以实现纳米颗粒的自组装。据报道,碱性磷酸酶(ALP)在某些肿瘤的膜结构上过表达,如肝癌、***、骨肉瘤,尤其是***癌。它已成为相关肿瘤靶向成像、诊断和治疗的重要生物标志物。ALP的特点是对底物进行去磷酸化作用,产生更多的疏水产物,并伴有产物的自组装(或聚集)。因此,开发一种由ALP触发的金基纳米材料在肿瘤部位的自组装有望提高***癌的诊断准确性和治疗效果。
发明内容
本发明的目的是提供一种碱性磷酸酶响应性多功能纳米点的制备方法,及其在增强***癌成像和治疗方面的应用。
该方法首先通过电化学还原法制备金锰纳米点(AMNDs),然后通过化学键将一种ALP响应的多肽(Phe-Phe-Tyr(H2PO3)-OH,简写为FTp)与AMNDs相连,合成得到具有酶响应性的多功能纳米点(AMNDs-FTp)。该多功能纳米点能与在***癌微环境过表达的ALP发生去磷酸化反应,获得的产物显现出强疏水性和π-π堆积等分子间相互作用能进行自组装,表现出在肿瘤部位显著地聚集,尺寸增大和密度增加,增强了***癌成像和治疗效果。我们通过体内/外实验验证了该多功能纳米点材料用于***癌的成像和治疗的应用。多功能纳米点AMNDs-FTp具有一定的荧光/CT/核磁多种模式成像功能,当其与ALP反应并聚集后,对于荧光、CT、核磁等每种模式成像效果均显著提高。增强的CT/核磁成像可以实现术前肿瘤的准确诊断,增强的荧光成像效果可以实现“术中视觉导航”,在临床肿瘤检测和手术指导中显示出潜在的应用价值。此外,AMNDs-FTp由于发生聚集,对近红外光吸收并转换成热量的性能显著增强,提升了光热***的效果。总之,这种酶响应性的多功能纳米点AMNDs-FTp提供一种有效的策略来提高***癌的诊断准确性和治疗效果。
本发明所述的酶响应性多功能纳米点的制备方法,其具体步骤如下:
(1)制备金锰纳米点AMNDs:在圆底烧瓶中依次加入10mLH2O,1mL模版剂/还原剂银纳米点,不同摩尔比例的HAuCl4和MnO2,室温条件下持续搅拌30分钟。接下来,向体系中缓慢加入NaOH溶液,调节体系的pH为7~9。混合物搅拌均匀后,在80℃油浴中反应4h,降至室温后即得到产物溶液。将产物溶液以8800rpm离心15分钟后,在上清液中加入过量异丙醇。混合均匀后获得有固体颗粒的悬浮溶液,再以6600rpm离心8分钟,得到的沉淀进行烘干,即为产物金锰纳米点AMNDs。称取10mgAMNDs溶解于5mL去离子水中,即得到浓度为2mg/mL的AMNDs溶液,常温避光保存。
(2)制备酶响应性多功能纳米点AMNDs-FTp:依次将3mL浓度为2mg/mL的AMNDs溶液,7.2mg 1-乙基-(3-二甲基氨基乙基)碳二亚胺盐酸盐,3.6mgN-羟基琥珀酰亚胺加入到圆底烧瓶中,室温搅拌15min。向混合物中加入75μLPhe-Phe-Tyr(H2PO3)-OH(简写为FTp)溶液,在室温条件下反应8h。反应结束后,将得到的产物在去离子水中渗析12h,期间每3h更换一次水,以除去未反应的物质及杂质。最终所得到的溶液冻干得到固体样品,称取10mg固体溶解在5mL去离子水中,即获得浓度为2mg/mL的AMNDs-FTp溶液,常温避光保存备用。
本发明优点如下:1.酶响应性多功能纳米点AMNDs-FTp与***癌微环境过表达的ALP发生去磷酸化反应后,在肿瘤部位获得纳米聚集体,显著提高多模式成像效果(荧光/CT/核磁),实现精准诊断;2.增强的CT/核磁成像可以实现术前肿瘤的准确诊断,增强的荧光成像效果可以实现“术中视觉导航”,在临床肿瘤检测和手术指导中显示出潜在的应用价值;3.AMNDs-FTp聚集后在近红外区有较强的吸收,进一步提高了光热治疗效果;4.聚集后增加了纳米点在肿瘤部位的浓度和停留时间;5.多功能纳米点AMNDs-FTp具有良好的生物相容性,可作为一种安全的制剂用于体内肿瘤成像和治疗;6.制备操作过程简便,绿色无污染。7.多功能纳米点AMNDs-FTp实现了***癌肿瘤部位的原位聚集、成像诊断和治疗等多种功能,应用前景广阔。
附图说明
图1:为实施例1所制备的酶响应性多功能纳米点体外模拟聚集前(a)、后(b)的透射电镜图。聚集前纳米点的尺寸约为4.6纳米。在与ALP反应后,纳米点表现出明显的聚集,尺寸增加到约165纳米。
图2:图a为实施例1所制备的酶响应性多功能纳米点体外模拟聚集前、后的荧光光谱图。图b为体外荧光成像图。如图所示,纳米点聚集后,其荧光成像效果明显增强。图c,d分别为多功能纳米点体外CT、核磁成像图。不同浓度(50、100、150、200微克/毫升)的纳米点与ALP反应后表现出明显的聚集,CT和核磁信号逐渐增强,均高于相同浓度的未聚集时纳米点的成像效果。
图3:为实施例1所制备的酶响应性多功能纳米点体内多模式成像图(荧光/CT/核磁)。如图所示,在***癌小鼠模型中,聚集组表现出增强的荧光、CT和核磁成像效果,表明在***癌微环境过表达的ALP催化去磷酸化后,纳米点在肿瘤部位表现出显著的聚集,增强了***癌成像效果。
图4:为实施例1所制备的酶响应性多功能纳米点的体外光热升温曲线图(a)和细胞光热杀伤图(b)。如图所示,与非聚集组相比,纳米点在聚集后表现出更好的升温效果。在相同的光照射条件下,聚集后的纳米点表现出更强的肿瘤细胞杀伤作用,增强了***癌治疗效果。
具体实施方式
实施例1:
(1)制备金锰纳米点AMNDs:在圆底烧瓶中依次加入10mLH2O,1mL模版剂/还原剂银纳米点,摩尔比例为1:0.3的HAuCl4和MnO2,室温条件下持续搅拌30分钟。接下来,向体系中缓慢加入NaOH溶液,调节体系的pH为7。混合物搅拌均匀后,在80℃油浴中反应4h,降至室温后即得到产物溶液。将产物溶液以8800rpm离心15分钟后,在上清液中加入过量异丙醇。混合均匀后获得有固体颗粒的悬浮溶液,再以6600rpm离心8分钟,得到的沉淀进行烘干,即为产物金锰纳米点AMNDs。称取10mgAMNDs溶解于5mL去离子水中,即得到浓度为2mg/mL的AMNDs溶液,常温避光保存。
(2)制备酶响应性多功能纳米点AMNDs-FTp:依次将3mL浓度为2mg/mLAMNDs溶液,7.2mg 1-乙基-(3-二甲基氨基乙基)碳二亚胺盐酸盐,3.6mgN-羟基琥珀酰亚胺加入到圆底烧瓶中,室温搅拌15min。向混合物中加入75μLPhe-Phe-Tyr(H2PO3)-OH(简写为FTp)溶液,在室温条件下反应8h。反应结束后,将得到的产物在去离子水中渗析12h,期间每3h更换一次水,以除去未反应的物质及杂质。最终所得到的溶液冻干得到固体样品,称取10mg固体溶解在5mL去离子水中,即获得浓度为2mg/mL的AMNDs-FTp溶液,常温避光保存备用。
实施例2:
(1)制备金锰纳米点AMNDs:在圆底烧瓶中依次加入10mLH2O,1mL模版剂/还原剂银纳米点,摩尔比例为1:0.4的HAuCl4和MnO2,室温条件下持续搅拌30分钟。接下来,向体系中缓慢加入NaOH溶液,调节体系的pH为8。混合物搅拌均匀后,在80℃油浴中反应4h,降至室温后即得到产物溶液。将产物溶液以8800rpm离心15分钟后,在上清液中加入过量异丙醇。混合均匀后获得有固体颗粒的悬浮溶液,再以6600rpm离心8分钟,得到的沉淀进行烘干,即为产物金锰纳米点AMNDs。称取10mgAMNDs溶解于5mL去离子水中,即得到浓度为2mg/mL的AMNDs溶液,常温避光保存。
(2)制备酶响应性多功能纳米点AMNDs-FTp:依次将3mL浓度为2mg/mLAMNDs溶液,7.2mg 1-乙基-(3-二甲基氨基乙基)碳二亚胺盐酸盐,3.6mgN-羟基琥珀酰亚胺加入到圆底烧瓶中,室温搅拌15min。向混合物中加入75μLPhe-Phe-Tyr(H2PO3)-OH(简写为FTp)溶液,在室温条件下反应8h。反应结束后,将得到的产物在去离子水中渗析12h,期间每3h更换一次水,以除去未反应的物质及杂质。最终所得到的溶液冻干得到固体样品,称取10mg固体溶解在5mL去离子水中,即获得浓度为2mg/mL的AMNDs-FTp溶液,常温避光保存备用。
实施例3:
(1)制备金锰纳米点AMNDs:在圆底烧瓶中依次加入10mLH2O,1mL模版剂/还原剂银纳米点,摩尔比例为1:0.5的HAuCl4和MnO2,室温条件下持续搅拌30分钟。接下来,向体系中缓慢加入NaOH溶液,调节体系的pH为9。混合物搅拌均匀后,在80℃油浴中反应4h,降至室温后即得到产物溶液。将产物溶液以8800rpm离心15分钟后,在上清液中加入过量异丙醇。混合均匀后获得有固体颗粒的悬浮溶液,再以6600rpm离心8分钟,得到的沉淀进行烘干,即为产物金锰纳米点AMNDs。称取10mgAMNDs溶解于5mL去离子水中,即得到浓度为2mg/mL的AMNDs溶液,常温避光保存。
(2)制备酶响应性多功能纳米点AMNDs-FTp:依次将3mL浓度为2mg/mLAMNDs溶液,7.2mg 1-乙基-(3-二甲基氨基乙基)碳二亚胺盐酸盐,3.6mgN-羟基琥珀酰亚胺加入到圆底烧瓶中,室温搅拌15min。向混合物中加入75μLPhe-Phe-Tyr(H2PO3)-OH(简写为FTp)溶液,在室温条件下反应8h。反应结束后,将得到的产物在去离子水中渗析12h,期间每3h更换一次水,以除去未反应的物质及杂质。最终所得到的溶液冻干得到固体样品,称取10mg固体溶解在5mL去离子水中,即获得浓度为2mg/mL的AMNDs-FTp溶液,常温避光保存备用。
Claims (7)
1.一种酶响应性多功能纳米点制备方法,其步骤如下:
(1)制备金锰纳米点AMNDs:在圆底烧瓶中依次加入10mLH2O,1mL模版剂/还原剂银纳米点,不同摩尔比例的HAuCl4和MnO2,室温条件下持续搅拌30分钟;接下来,向体系中缓慢加入NaOH溶液,调节体系的pH为7~9;混合物搅拌均匀后,在80℃油浴中反应4h,降至室温后即得到产物溶液;将产物溶液以8800rpm离心15分钟后,在上清液中加入过量异丙醇;混合均匀后获得有固体颗粒的悬浮溶液,再以6600rpm离心8分钟,得到的沉淀进行烘干,即为产物金锰纳米点AMNDs;称取10mgAMNDs溶解于5mL去离子水中,即得到浓度为2mg/mL的AMNDs溶液,常温避光保存;
(2)制备酶响应性多功能纳米点AMNDs-FTp:依次将3mL浓度为2mg/mL的AMNDs溶液,7.2mg 1-乙基-(3-二甲基氨基乙基)碳二亚胺盐酸盐,3.6mgN-羟基琥珀酰亚胺加入到圆底烧瓶中,室温搅拌15min;向混合物中加入75μLPhe-Phe-Tyr(H2PO3)-OH(简写为FTp)溶液,在室温条件下反应8h;反应结束后,将得到的产物在去离子水中渗析12h,期间每3h更换一次水,以除去未反应的物质及杂质;最终所得到的溶液冻干得到固体样品,称取10mg固体溶解在5mL去离子水中,即获得浓度为2mg/mL的酶响应性多功能纳米点AMNDs-FTp溶液,常温避光保存备用。
2.如权利要求1所述的一种酶响应性多功能纳米点制备方法,其特征在于:步骤(1)中所述的HAuCl4和MnO2的摩尔比例为1:0.3~0.5。
3.如权利要求1所述的一种酶响应性多功能纳米点制备方法,其特征在于:步骤(1)中所述的调节体系的pH为7~9。
4.根据权利要求1~3任一所述的酶响应性多功能纳米点制备方法所制得的多功能纳米点兼具有***癌肿瘤部位的原位聚集、多模式成像、光热治疗等多种功能。
5.根据权利要求4所述的一种酶响应性多功能纳米点在与***癌微环境过表达的碱性磷酸酶ALP发生去磷酸化反应后,在肿瘤部位显著地聚集,尺寸增大和密度增加,显著提升了多功能纳米点的多模式成像效果(荧光/CT/核磁),实现对***癌的精确诊断。
6.根据权利要求4所述的一种酶响应性多功能纳米点通过与***癌肿瘤部位ALP反应后发生聚集,获得增强的CT/核磁成像可以实现术前肿瘤的准确诊断,获得增强的荧光成像效果可以实现“术中视觉导航”,在***癌肿瘤检测和手术指导中显示出重要应用价值。
7.根据权利要求4所述的一种酶响应性多功能纳米点与***癌肿瘤部位ALP反应并发生聚集后,对近红外光吸收并转换成热量的性能增强,显著提升了光热治疗***癌肿瘤的效果。
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