CN114767852A - 一种光控释放型中空金银纳米探针在用于制备肿瘤诊疗一体化制剂中的应用 - Google Patents
一种光控释放型中空金银纳米探针在用于制备肿瘤诊疗一体化制剂中的应用 Download PDFInfo
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Abstract
本发明公开一种光控释放型中空金银纳米探针在用于制备肿瘤诊疗一体化制剂中的应用。主要是以氯金酸为金源、硝酸银为银源，依次加入还原剂、拉曼信号分子和稳定剂，采用种子生长法制备而成。该方法简单温和、绿色环保，所制备的中空金银纳米颗粒具有表面增强拉曼散射(SERS)成像、光声(PAT)成像和荧光(FL)成像等多模态成像功能，可以实现肿瘤手术切除的精准可视化。该纳米颗粒在808nm近红外光照射下，具有较高的光热转换效率，可作为肿瘤光热治疗(PTT)的治疗剂。此外，该纳米探针在近红外激光的照射下释放具有抑菌杀菌效果的银离子，可实现手术切口细菌感染的协同治疗。
Description
技术领域
本发明涉及一种光控释放型中空金银纳米探针在用于制备肿瘤诊疗一体化制剂中的应用，即在肿瘤多模态成像介导手术切除以及术后协同光热治疗和切口抗感染中的应用，在作为光热试剂产生光热效应的同时还可以实现多模态成像，为外科手术提供影像指导；而且中空金银纳米颗粒在被激光照射后还会释放出银离子，银离子具有杀菌效果，可以解决术后的伤口细菌感染问题。
背景技术
恶性肿瘤严重危害人类健康。目前，有多种干预措施可用于肿瘤治疗，如化疗、放射治疗、手术切除等，这些方法的协同应用在很大程度上改善了肿瘤的治疗效果。但是这些治疗方法会在一定程度上给患者生理上带来很大的痛苦，尤其是手术切除，还会出现诊断不够精准以及术后伤口感染等问题。纳米材料由于具有独特的物理化学性质而被用于肿瘤的诊断与治疗(纳米诊疗)。通过对纳米材料进行合理设计，开发出一种集多模态成像和治疗于一体的多功能纳米平台，可实现肿瘤的诊断与治疗的时空统一。
表面增强拉曼散射(Surface-Enhanced Raman Scattering，SERS)成像是一种不断发展中的振动光谱学技术，当一些分子被吸附到某些粗糙的金属，如金、银或铜的表面时，它们的拉曼谱线强度会得到极大地增强，这在肿瘤组织的识别和成像中展现出巨大的研究和使用价值。传统的SERS基底主要以Au、Ag、Cu为基础制备而成，这三种金属在大部分可见及近红外波长范围内具有表面等离子体基元共振(Localized Surface PlasmonResonance，LSPR)效应，而Cu在空气中稳定性较差，因此Au和Ag是最常用的SERS基底。金纳米颗粒的尺寸易于控制、稳定性高、生物相容性好；银纳米颗粒被用作SERS基底时，其SERS效应通常发生在可见光区域，从而限制了其在生物医学领域的应用，但是银离子的抗菌谱广、不易产生耐药性、创面愈合快，对于慢性伤口及术后切口的感染具有良好的治疗效果。因此，本发明以金、银两种常用的拉曼增强金属元素为原料构建结构中空的金银纳米颗粒(Hollow gold-silver nanoparticle，HGS)，并将HGS作为拉曼增强基底，通过层层组装(layerby layer)的方式在双金属纳米颗粒表面包载拉曼信号分子和稳定剂，从而构建一种光控释放型诊疗一体化纳米探针。
基于近红外激光照射的光热治疗(Photothermal therapy，PTT)是一种安全有效的肿瘤治疗方法。目前主要采用近红外Ⅰ区(NIR-Ⅰ，650～950nm)的激光用于肿瘤光热治疗，利用具有较高光热转换效率的纳米材料，将其注射入生物体内部后，纳米材料在被动靶向作用下会聚集在肿瘤组织内，在外部近红外光源的照射下将光能转换为热能来杀死癌细胞。光热治疗区别于传统治疗手段(外科手术、放疗、化疗)，它是一种非侵入性的治疗方法，且具有治疗时间短、疼痛少、对人体副作用小、治疗特异性好等优点。中空金银纳米颗粒是一种具有较高光热转换效率的材料，它在近红外光下迅速将光能转化为热能，将癌细胞“烫”死，并且对正常组织无多大伤害。
光热效应除了能杀死癌细胞外，还能产生声波，被探测并转化为成像信号，即光声(Photoacoustic，PAT)成像。PAT提供了肿瘤诊断的另一种成像方式。将中空金银纳米颗粒注入小鼠体内后，可利用PAT影像对肿瘤组织进行定位、利用SERS成像对肿瘤边界进行精准识别，为手术切除肿瘤提供高分辨率的影像依据，从而实现诊疗一体化。
对癌症患者进行手术切除肿瘤后，由于患者的自身免疫力低下，患者的术后切口易出现细菌感染。中空金银纳米颗粒在接受近红外光照射时，释放出银离子，因为银离子带正电荷，细菌内含巯基(-SH)带负电荷，正负电荷相互吸引，使活性银离子释放出来后可以快速地穿透细菌的细胞壁，导致细菌迅速失去活性。银离子不仅杀菌能力特别强，还不伤害正常细胞。因此本发明中制备的光控释放型诊疗一体化纳米探针不仅可以基于多模态成像实现肿瘤的精准手术导航以及术后光热治疗，还可以实现术后残存肿瘤的根除及切口细菌感染的治疗。
目前还没有发现基于中空金银纳米颗粒的SERS/PAT/FL多模态成像用于肿瘤手术导航及术后光热辅助治疗功能于一体的多功能纳米颗粒的制备方法的报道。
发明内容
本发明的第一个目的在于针对现有技术的不足，提供一种简单温和、绿色环保的中空金银双金属纳米基的表面增强拉曼散射基底的合成方法。
本发明一种中空金银双金属纳米基的表面增强拉曼散射基底的合成方法，具体包括如下步骤：
步骤(1)、避光条件下，向沸腾的还原剂中加入银源溶液，使溶液在沸腾条件下反应10～20min，停止加热，继续搅拌10～20min，即得到银纳米颗粒；
作为优选，银纳米颗粒的直径为20～30nm；
作为优选，银源采用硝酸银；
步骤(2)、避光条件下，以步骤(1)银纳米颗粒为牺牲模板，向银纳米颗粒悬浮液中加入金源溶液，利用Ag和HAuCl4之间的置换反应，制备得到中空金银双金属纳米颗粒HGS悬浮液；所述中空金银双金属纳米颗粒为中空球形结构并通过控制金源用量来实现对LSPR的精密调控；
作为优选，中空金银双金属纳米颗粒HGS的直径为30～40nm；内部空腔为15～30nm；
作为优选，金源采用氯金酸；
作为优选，步骤(1)中所述的还原剂为硼氢化钠、柠檬酸钠、抗坏血酸、或盐酸羟胺。
本发明的第二个目的是提供一种中空金银双金属纳米基的表面增强拉曼散射基底，采用以上方法制备得到。
本发明的第三个目的是提供一种以中空金银双金属纳米颗粒为核、拉曼信号分子和稳定剂为壳的纳米探针的制备方法，具体是：
避光条件下，在中空金银双金属纳米基的表面增强拉曼散射基底中加入拉曼信号分子溶液，轻微摇晃过夜后再加入稳定剂，搅拌，得到表面包覆拉曼信号分子和稳定剂的中空金银纳米核壳材料。
作为优选，稳定剂的厚度为2～5nm。
作为优选，所述的拉曼信号分子为3，3’-二乙基硫代三碳菁碘化物(DTTC)、吲哚菁绿(ICG)、IR-780碘化物或IR-782碘化物。
作为优选，所述的稳定剂为聚乙烯吡咯烷酮、聚乙二醇、壳聚糖、透明质酸、牛血清白蛋白、人血清白蛋白或无机盐。
本发明的第四个目的是提供一种以中空金银双金属纳米颗粒为核、拉曼信号分子和稳定剂为壳的纳米探针，采用以上方法制备得到。
本发明的第五个目的是将上述表面包覆拉曼信号分子和稳定剂的中空金银纳米颗粒用于制备肿瘤诊疗一体化制剂中的应用，在肿瘤光热治疗中作为光热试剂产生光热效应的同时还可以作为多模态造影剂实现多模态成像，为外科手术提供影像指导；而且中空金银纳米颗粒作为细菌抑制剂，在被激光照射后还会释放出银离子，银离子具有杀菌效果，可以解决术后的伤口细菌感染问题。
所述的中空金纳米颗粒具备金银双金属核壳结构，由于双金属的协同作用及两种颗粒间电磁场的耦合使该材料在近红外区具有较强的吸收，因而具有表面增强拉曼散射成像和光声成像的功能，与此同时拉曼信号分子还具有荧光成像的功能。
所述的中空金纳米颗粒具有优异的光热转换效率，在808nm激光的照射下，几分钟后便可将局部温度快速升温至70℃以上，使肿瘤细胞被杀灭，因此该材料可被当作光热转化剂以用于光热治疗。
所述的中空金银纳米颗粒在激光照射下能释放银离子，因为银离子带正电荷，细菌内含巯基(-SH)带负电荷，正负电荷相互吸引，使活性银离子释放出来后可以快速地穿透细菌的细胞壁，导致细菌迅速失去活性。银离子不仅杀菌能力特别强，还不伤害正常细胞。因此该材料可用作术后切口的抑菌剂。
本发明所述的中空金银纳米颗粒可通过任何已知的递送方式给药：全身递送(静脉注射)，动脉内，肿瘤内，胃肠外，肺腔内，局部、或局部给药的区域递送形式。如动脉内注射能被用来“局域效果”成像及特定区域的光热治疗。
本发明的有益效果是：本发明提出以金、银两种常用的拉曼增强金属元素为原料构建结构中空的金银纳米颗粒(Hollow gold-silver nanoparticle，HGS)，并将HGS作为拉曼增强基底，构建多功能的诊疗一体化纳米探针，赋予该探针高灵敏的肿瘤诊断性能、优良的光热治疗性能，以及抗菌物质的控制释放性能，实现了肿瘤手术的精准导航，术后残存肿瘤的彻底根除，手术切口细菌感染的协同抑制。
本发明将HGS作为拉曼增强基底，构建多功能的诊疗一体化纳米探针，赋予该探针高灵敏的肿瘤诊断性能、优良的光热治疗性能，以及抗菌物质的控制释放性能，实现了肿瘤手术的精准导航，术后残存肿瘤的彻底根除，手术切口细菌感染的协同抑制。
附图说明
图1为中空金银纳米粒制备流程；
图2为加入不同体积的氯金酸溶液的中空金银纳米颗粒的紫外可见吸收光谱图；
图3为银纳米颗粒的透射电镜图；
图4为HGS的透射电镜图；
图5为HGS@DTTC/BSA的透射电镜图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，下面结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的结构图及具体实施例仅用以解释本发明，并不用于限定本发明。
实施例1：
在装有47mL的超纯水的二口烧瓶中加入700μL，10mM的还原剂(此例中所使用的还原剂为抗坏血酸)，避光搅拌条件下煮沸。向1.25mL超纯水中加入一定量的还原剂(1mL，此例中采用柠檬酸钠)和0.25mL，1wt％的硝酸银溶液，室温下避光搅拌5min。将硝酸银溶液加到沸腾的抗坏血酸溶液中，继续加热15min后，停止加热，继续避光反应15min，制得粒径约为20～30nm的单分散银纳米颗粒。取一定量的Ag纳米颗粒悬浮液(本例中取用5mL)，煮沸后加入50～100μL，2.5mM的氯金酸溶液，纱布过滤，得到纯化的中空金银纳米颗粒的悬浮液，该中空金银纳米粒制备流程见图1。
向纯化的纳米悬浮液中加入浓度为1μg/mL～1mg/mL的拉曼探针(如DTDC、DTTC、ICG、CV、或IR-780)1～30mL，避光孵育12～48h，然后用去离子水漂洗2～3遍，去除游离的拉曼探针，得到装载拉曼探针的中空金银纳米颗粒备用；再向纳米颗粒悬浮液中加入稳定剂(本例中使用的稳定剂为牛血清白蛋白)，避光孵育12～48h，然后用去离子水漂洗2～3遍。
上述制备的包载了拉曼探针和稳定剂的银基的中空金银纳米颗粒，直径约35～45nm，并具备了拉曼增强成像、光声成像以及光热治疗的功能。
使用紫外可见分光光度计(UV-Vis)、透射电子显微镜(TEM)对合成的中空金银纳米颗粒进行表征：
(1)紫外可见分光光度计(UV-Vis)
紫外可见近红外光谱，表明该方法合成得到中空金银纳米颗粒的特征光谱。结果参照图2，通过调控氯金酸的加入量，可得到吸收峰在不同位置的具有不同光学性质的HGS纳米颗粒。由图2可见，银溶胶在可见光区域的LSPR峰位于401nm处，随着氯金酸的加入，401nm处的吸收峰逐渐降低，直至消失。同时在长波方向上出现了新的吸收峰，这是因为金-银合金颗粒之间产生了共振耦合。
(2)透射电子显微镜(TEM)
透射电子显微镜照片表明银纳米颗粒、HGS以及装载有拉曼探针和稳定剂的HGS纳米颗粒的形貌和尺寸。由图3可见，银纳米颗粒粒径分布均匀，粒径大小约为20～30nm。当加入的氯金酸的体积为100μL时，如图4所示，可清晰观察到纳米颗粒中心部位的中空结构。图5可以看到HGS外层较为均匀的包覆层，且包覆的BSA厚度约为2～5nm，得到的HGS@DTTC/BSA纳米颗粒的粒径约为35～45nm。
以上对本发明的描述是说明性的，而非限制性的，本专业技术人员理解，在权利要求限定的精神与范围之内可对其进行许多修改、变化或等效，但是它们都将落入本发明的保护范围内。
Claims (10)
1.一种光控释放型中空金银纳米探针在用于制备肿瘤诊疗一体化制剂中的应用，所述肿瘤诊疗一体化制剂为集光热试剂、多模态造影剂、细菌抑制剂的一体化制剂；其特征在于所述光控释放型中空金银纳米探针采用以下方法制备得到：
步骤(1)、中空金银双金属纳米基的表面增强拉曼散射基底的合成
1-1避光条件下，向沸腾的还原剂中加入银源溶液，使溶液在沸腾条件下反应10～20min，停止加热，继续搅拌10～20min，即得到银纳米颗粒；
1-2避光条件下，以步骤1-1银纳米颗粒为牺牲模板，向银纳米颗粒悬浮液中加入金源溶液，利用Ag和HAuCl4之间的置换反应，制备得到中空金银双金属纳米颗粒HGS悬浮液；所述中空金银双金属纳米颗粒为中空球形结构并通过控制金源用量来实现对LSPR的精密调控；
步骤(2)、避光条件下，在中空金银双金属纳米基的表面增强拉曼散射基底中加入拉曼信号分子溶液，摇晃过夜后再加入稳定剂，搅拌，得到表面包覆拉曼信号分子和稳定剂的中空金银纳米核壳材料。
2.如权利要求1所述的应用，其特征在于步骤1-1中，银纳米颗粒的直径为20～30nm。
3.如权利要求1所述的应用，其特征在于步骤1-1中，银源采用硝酸银。
4.如权利要求1所述的应用，其特征在于步骤1-2中，中空金银双金属纳米颗粒HGS的直径为30～40nm；内部空腔为15～30nm。
5.如权利要求1所述的应用，其特征在于步骤1-2中，金源采用氯金酸。
6.如权利要求1所述的应用，其特征在于步骤1-2中，还原剂为硼氢化钠、柠檬酸钠、抗坏血酸或盐酸羟胺。
7.如权利要求1所述的应用，其特征在于步骤(2)中，稳定剂的厚度为2～5nm。
8.如权利要求1所述的应用，其特征在于步骤(2)中，所述的拉曼信号分子为3，3’-二乙基硫代三碳菁碘化物(DTTC)、吲哚菁绿(ICG)、IR-780碘化物或IR-782碘化物。
9.如权利要求1所述的应用，其特征在于步骤(2)中，所述的稳定剂为聚乙烯吡咯烷酮、聚乙二醇、壳聚糖、透明质酸、牛血清白蛋白、人血清白蛋白或无机盐。
10.一种以中空金银双金属纳米颗粒为核、拉曼信号分子和稳定剂为壳的纳米探针，其特征在于采用以下方法制备得到：
步骤(1)、中空金银双金属纳米基的表面增强拉曼散射基底的合成
1-1避光条件下，向沸腾的还原剂中加入银源溶液，使溶液在沸腾条件下反应10～20min，停止加热，继续搅拌10～20min，即得到银纳米颗粒；
1-2避光条件下，以步骤1-1银纳米颗粒为牺牲模板，向银纳米颗粒悬浮液中加入金源溶液，利用Ag和HAuCl4之间的置换反应，制备得到中空金银双金属纳米颗粒HGS悬浮液；所述中空金银双金属纳米颗粒为中空球形结构并通过控制金源用量来实现对LSPR的精密调控；
步骤(2)、避光条件下，在中空金银双金属纳米基的表面增强拉曼散射基底中加入拉曼信号分子溶液，摇晃过夜后再加入稳定剂，搅拌即可。
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