CN112641959A - 一种新型多功能纳米探针及其制备方法和应用 - Google Patents
一种新型多功能纳米探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种新型多功能纳米探针及其制备方法和应用,涉及纳米材料领域。本发明提供的制备新型多功能纳米探针方法制备的纳米探针包括金纳米颗粒核心、和搭载在所述金纳米颗粒核心表面的钆离子和新吲哚箐绿IR820,所述金纳米探针的粒径为50‑100nm,该纳米探针以可以进行酸响应的药物释放,提高了药物在肿瘤细胞部位的聚集,并在肿瘤部位的酸性环境下可以进行解离或者降解,***效率高,大大减小了纳米探针在肿瘤细胞内的滞留量以及对人体造成的毒性风险;并在双模式成像的指导下,进行更加精准的光热和光动力协同治疗,实现更灵敏的检测和更高效的治疗一体的多模式成像诊疗体系。
Description
技术领域
本发明属于纳米材料领域,特别涉及一种新型多功能纳米探针及其制备方法和应用。
背景技术
癌症已经成为威胁人类生命安全的首要原因和主要的公共健康问题之一。据报告,2015 年全球范围内新增癌症病例达1410万例,有820万人死于癌症,预计未来20年新发癌症病例数将增加70%。目前,肝癌的治疗主张根据不同阶段实施不同的方案,主要有早期的手术治疗,中晚期的化疗、放射治疗和放/化疗治疗,以及新兴的生物治疗。由于肝癌起病症状隐匿且目前临床早期诊断手段单一,许多肝癌患者难以在早期确诊,导致其较难获得预期的治疗效果。因此,建立一种新型的癌症早期精准诊断和治疗策略,提高癌症早期诊断和治疗的准确性和灵敏性,有效地降低癌症死亡率,是关系着国计民生的重大问题。
随着纳米技术与产业的发展,纳米材料在生物医学领域中的应用越来越广泛借助于其独特的性质,纳米材料已经在生物医学领域中的多模态成像、疾病早期诊断检测、药物递送以及肿瘤治疗方面发挥了重要作用[1-4]。
药物一般通过静电力吸附或者化学键偶联荷载到纳米颗粒上构建成纳米探针,纳米探针用于体内的癌症治疗时,除了通过材料自身物理化学特性进行的光动力治疗外,最多的是通过荷载上各种药物,利用纳米探针在体内的EPR(enhanced permeability andretention effect)效应对肿瘤组织靶向递送,进而进行靶向治疗。然而,这些纳米探针在经过静脉注射进入到生物体内后,若不能及时从体内代泄、过量滞留在体内将会对人体造成很大的毒性风险,从毒理学角度出发,纳米材料在体内可能引起的毒性不但与其元素组成、形貌尺寸以及表面配体的理化性质密切相关,并且也存在明显的量-效关系[5,6],要降低纳米材料在体内的毒性风险,最主要的解决办法就是降低纳米探针在体内的滞留量。
理想的纳米诊疗探针在体内最终可以通过主要的代泄途径(肝脏-粪便、肾脏-尿液)***到体外[7,8],从而最大地降低纳米材料在体内可能引起的毒性;影响纳米探针在体内分布及代泄的最大的因素就是尺寸,目前的研究中证明,尺寸在10-100纳米之间的纳米颗粒主要通过肝脏-粪便途径代泄,但是代泄率极低,大部分的纳米颗粒还是滞留在生物体内;而尺寸小于10纳米的纳米颗粒可以通过肾脏-尿液途径快速地从生物体内***,24小时后基本在生物体内清空[9-11]。但是这也造成一个矛盾,较大尺寸的纳米颗粒因为具有更长的血液循环时间,具有更好的肿瘤靶向能力,但是较难从体内代泄;小尺寸的纳米颗粒能快速地从体内代泄,但是肿瘤靶向能力较差,药物靶向释放可以减少或者避免药物的副作用,并且可以减小给药的剂量。
参考文献:
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发明内容
本发明的目的在于提供一种新型多功能纳米探针及其制备方法和应用,解决现有技术的纳米探针纳米颗粒在生物体内的过量滞留、对人体造成很大的毒性风险的问题,可用于肿瘤细胞的核磁共振成像、荧光成像,提供双模态成像模式,对肿瘤细胞进行精准诊疗。
本发明的技术方案:
第一方面,一种新型多功能纳米探针,所述新型多功能探针包括:金纳米颗粒核心、和搭载在所述金纳米颗粒核心表面的钆离子和新吲哚箐绿IR820,所述金纳米探针的粒径为 50-100nm。
优选的,所述金纳米探针的粒径为70nm。
第二方面,本发明还提供了一种新型多功能纳米探针制备方法,包括以下步骤:
1)金种子溶液的制备:
在剧烈搅拌情况下,将1%柠檬酸三钠溶液加入到沸腾的1mmol/L的HAuCl4溶液中,持续搅拌,反应液由淡金黄色逐渐变为淡紫色,得到金种子溶液;
2)金纳米颗粒的制备
2.1)将上述步骤1)制得的金种子溶液冷却至室温后,用0.22μm硝酸纤维素膜过滤,将过滤后滤液在4℃温度下进行冷藏,在中度搅拌下,将冷藏后的滤液加入到含有1mol/L HCl和1.6mmol/L HAuCl4的混合溶液中,持续搅拌得到混合溶液;
2.2.)将新鲜制备的3mol/L AgNO3溶液和1mol/L抗坏血酸溶液分别快速地加入到上述步骤2.1)所得的混合溶液中,在剧烈搅拌情况下,反应溶液的颜色从浅红色迅速变为深红色时,迅速加入50mg/ml巯基-PEG-羧基溶液继续搅拌反应,得到金纳米颗粒悬液;
3)纳米探针的制备:
3.1)取上述步骤2)制备的金纳米颗粒悬液溶于去离子水中,在温度为37°水浴锅中搅拌,混匀后加入45-60mmol/L氯化钆溶液,反应过夜;
3.2)离心上述步骤3.1)所得溶液,弃去上清液,将沉淀洗涤后重悬于去离子水中,然后加入1mg/mL的新吲哚箐绿IR820溶液,继续搅拌反应24小时,然后再次离心,弃去上清液,将沉淀洗涤后重悬于去离子水中,获得纳米探针。
优选的,所述步骤1)中的柠檬酸三钠与HAuCl4的体积比为:15-20:100-150,进一步优选为15:100。
优选的,所述步骤2)中的金种子溶液、HCl溶液、HAuCl4溶液、AgNO3溶液、抗坏血酸溶液和巯基-PEG-羧基溶液的体积比为:1:1:100:1:0.5:0.02。
优选的,所述步骤3)中的金纳米颗粒悬液、新吲哚箐绿IR820溶液和氯化钆溶液的体积比:10:0.01-0.02:0.1-0.15,进一步优选为10:0.01:0.1。
优选的,所述步骤1)中所述的搅拌速率为500-1000rpm,搅拌时间为15min。
优选的,所述步骤2.1)中的搅拌速率为200-300rpm,搅拌时间为1-5min;步骤2.2)中的搅拌速率为500-1000rpm,搅拌时间为10-15min。
优选的,所述步骤3.1)中的搅拌速率为200-300rpm,水浴反应时间为12-18h;步骤3.2)中的两次离心均在室温下进行,第一次离心条件为:在离心力为8000-9000g的环境下离心10-15min,第二次离心条件为:在离心力为9000-1000g的环境下离心5-10min。
第三方面,本发明提供了一种新型多功能纳米探针的应用,其可应用于癌症靶向协同治疗、多模态成像引导的光热/光动力协同治疗。
本发明使用通网高分辨透射电镜(TEM)、紫外分光光度计(UV-Vis-NIR)、ZETA电位/粒度仪等手段表征制备的纳米探针;通过Hoechst即用型试剂盒检测和流式细胞术实验分别定量和定向的观察荧光来研究细胞对纳米探针的摄取情况;利用活性检测试剂盒(CCK-8)检测和流式细胞术实验评估纳米探针的安全性;通过激光共聚焦显微镜和流式细胞术分别定量和定向的观察荧光来评价纳米探针在肿瘤细胞内的分布;通过sosg单线态荧光光谱评估纳米探针的光动力特性;通过近红外热成像仪测定纳米探针的光热效应和光热衰减作用;通过ROS荧光探针和莱卡激光共聚焦显微镜观察经过纳米探针处理的细胞内单线态氧含量情况;通过体外光治疗实验后分别使用钙黄绿素/PI细胞双染法及细胞流式凋亡实验评估纳米探针对体外光动力治疗效果;最后通过对荷瘤鼠静脉给药评估纳米探针的体内毒性、抑瘤效果和体内光动力治疗效果。
有益效果:
1、本发明的纳米探针在肿瘤部位的酸性环境下可以进行解离或者降解,然后通过肝脏- 粪便途径和肾脏-尿液途径***,***效率高,大大减小了纳米探针在肿瘤细胞内的滞留量,避免了纳米颗粒在生物体内的过量滞留而可能引起的慢性/急性反应;
2、本发明的纳米探针是由Gd3+离子诱导形成的自组装体可以进行酸响应的药物释放,提高了药物在肿瘤细胞部位的聚集;
3、本发明的纳米探针可以通过EPR效应对肝癌肿瘤部位进行主动靶向聚集,极大地提高药物的递送效率,有良好的生物相容性和靶向性;
4、本发明的纳米探针中的钆离子能形成核磁共振成像,IR820能形成荧光成像,从而提供双模态成像模式,在双模式成像的指导下,进行更加精准的光热和光动力协同治疗,从而为后续能够实现更灵敏的检测和更高效的治疗一体的多模式成像诊疗体系,提供了重要的前提条件,具有良好的应用前景和重要的现实意义。
附图说明
图1为实施例1制备得到的TEM电子显微图;
图2为实施例1制备得到金纳米颗粒、纳米探针、和IR820的紫外吸收光谱;
图3为实施例1制备得到的纳米探针的水合半径分析图;
图4为在激光共聚焦显微镜下的肝癌细胞内部的纳米探针的荧光强度及分布;
图5为纳米探针和癌细胞孵育不同时间后,流式定量定量分析细胞对纳米探针的摄取情况;
图6为不同浓度的纳米探针在肝癌细胞内的细胞凋亡图;
图7为分别不同浓度的Gd-AuNPS@IR820、PBS、Au和IR820培育下,肝癌细胞内的细胞相对活性曲线图;
图8为不同时间点的SOSG在荧光光度计上的荧光光谱图;
图9为PBS、Gd-AuNPS@IR820和IR820分别在808nm激光照射下的近红外热成像图;
图10为PBS、Gd-AuNPS@IR820和IR820分别在808nm激光照射下的照射时间与温度上升情况的曲线;
图11为PBS、Gd-AuNPS@IR820和IR820分别在808纳米的激光照射下进行5个循环的照射的温度的变化曲线图;
图12为分别加入PBS、IR820、Gd-AuNPS、Gd-AuNPS@IR820悬液培养的肝癌细胞在激光共聚焦显微镜下的观察图;
图13为分别加入PBS、IR820、Gd-AuNPS、Gd-AuNPS@IR820悬液培养,并进行激光(808 nm,6分钟,1W CM-2)介导的联合治疗的肝癌细胞,经过钙黄绿素/PI双染试剂盒染色后,在激光共聚焦显微镜下观察的细胞双染色荧光图;
图14为分别加入PBS、IR820、Gd-AuNPS、Gd-AuNPS@IR820悬液培养,并进行激光(808 nm,6分钟,1W CM-2)介导的联合治疗的肝癌细胞,进行流式细胞分析后的分析图;
图15为荷瘤鼠体静脉注射Gd-AuNPS@IR820悬液后各个时间分别在核磁成像仪上观察和拍摄成像图片;
图16为荷瘤鼠体静脉注射Gd-AuNPS@IR820悬液后各个时间分别在活体成像仪上观察和拍摄成像图片;
图17为荷瘤鼠体分别静脉注射同体积PBS、AuNPS溶液、IR820溶液、Gd-Au@IR820溶液、Gd-AuNPS@IR820悬液,然后进行808纳米激光照射介导的联合治疗完成后,第14 天取出的肿瘤组织KI-67和HE染色观察图;
图18为荷瘤鼠体分别静脉注射同体积PBS、AuNPS溶液、IR820溶液、Gd-Au@IR820溶液、Gd-AuNPS@IR820悬液,然后进行808纳米激光照射介导的联合治疗完成后,第14 天取出各治疗组的主要脏器,进行HE染色观察图;
图19为治疗后荷瘤鼠体重变化曲线图;
图20为治疗后荷瘤鼠体生存曲线图;
图21为治疗后荷瘤鼠体肿瘤体积变化曲线图。
具体实施方式
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解,在阅读了本发明讲授的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
本申请中,“AuNPS”指代金纳米颗粒;
“Gd-AuNPS@IR820”指代纳米探针;
“Gd-AuNPS”指代搭载钆离子的金纳米颗粒;
“Saline”指代生理盐水;
“PBS”指代磷酸缓冲盐溶液;
“IR820”指代新吲哚菁绿IR820;
实施例中所采用主要原料来源说明:
实施例中所采用主要仪器来源说明:
实施例1
1)金种子溶液的制备:
在搅拌速率为500rpm的情况下,将15mL的1%柠檬酸三钠溶液加入到100mL沸腾的1.0mM的HAuCl4溶液中,持续搅拌15min后,反应液由淡金黄色逐渐变为淡紫色,得到金种子溶液;
2)金纳米颗粒的制备:
2.1)将步骤1)制得的Au种子溶液冷却至室温后,用0.22μm硝酸纤维素膜过滤,将过滤后滤液在4℃温度下进行冷藏,取1mL冷藏后的滤液加入到由1ml(1mol/L)的HCl 溶液和100ml(1.6mmol/L)HAuCl4溶液组成的混合溶液中,在搅拌速率为200rpm的情况下,持续搅拌1min,得到混合溶液;
2.1)将新鲜制备的1ml(3mol/L)AgNO3溶液和0.5ml(1mol/L)抗坏血酸溶液分别快速地加入到上述步骤2.1)的混合溶液中,并将搅拌器的搅拌速率调整为为500rpm,反应溶液的颜色从浅红色迅速变为深红色时,迅速加入50mg/ml巯基-PEG-羧基溶液继续搅拌反应10min,得到金纳米颗粒悬液;
3)纳米探针的制备:
3.1)取10ml上述步骤2)制备的金纳米颗粒悬液溶于10mL去离子水中,在温度为37°水浴锅中搅拌,混匀后加入的10μL(45-60mmol/)L的氯化钆溶液,反应12h;
3.2)将上述3.2)所得溶液在离心力为8000g的环境下离心10min,弃去上清液,将沉淀洗涤后重悬于5mL去离子水中,然后再加入100μL(1mg/mL)的新吲哚箐绿IR820 溶液,在室温下搅拌反应24h,然后在离心力为9000g的环境下离心5min,弃去上清液,将沉淀洗涤后重悬于5mL去离子水中,获得新型多功能纳米探针。
通过通网高分辨透射电镜(TEM)、紫外分光光度计(UV-Vis-NIR)、ZETA电位/粒度仪等手段对本实施例制备得到的纳米探针(Gd-AuNPS@IR820)进行表征说明:
(1)通网高分辨透射电镜(TEM)
将实施例制备得到的纳米探针滴在碳支持膜铜网上,在室温下自然风干,在放置在通网高分辨透射电镜(TEM)上观察,表征结果见图1的TEM电子显微图,图1表示在100nm的标尺下,超小尺寸的金纳米颗粒粒径均匀,分散性好。
(2)紫外分光光度计(UV-Vis-NIR)
分别测量本实施例所用的IR820,和本实施例制备得到AuNPS、Gd-AuNPS、 Gd-AuNPS@IR820的紫外吸收光谱,表征结果见图2,如图2所示,超小尺寸金纳米颗粒在520纳米处呈现出金纳米颗粒的特征峰,IR820在695纳米和820纳米处呈现出主峰和肩峰,Gd-AuNPS@IR820的肩峰出现了约30nm的红移,在808nm处出现了更强的吸收峰,进一步表示该纳米探针的成功制备。
(3)ZETA电位/粒度仪
以ZETA电位/粒度仪的动态光散射对金纳米探针的粒径以及粒径分布进行表征,表征结果见图3,从图3可知金纳米探针的平均粒径为72.4,粒径分布系数为0.304,表明探针具有良好的粒径大小和分布,有利于后期的功能验证。
实施例2
1)金种子溶液的制备:
在搅拌速率为1000rpm的情况下,将20mL的1%柠檬酸三钠溶液加入到150mL沸腾的1.0mM的HAuCl4溶液中,持续搅拌15min后,反应液由淡金黄色逐渐变为淡紫色,得到金种子溶液;
2)金纳米颗粒的制备:
2.1)将步骤1)制得的Au种子溶液冷却至室温后,用0.22μm硝酸纤维素膜过滤,将过滤后滤液在4℃温度下进行冷藏,取1mL冷藏后的滤液加入到由1ml(1mol/L)的HCl 溶液和100ml(1.6mmol/L)HAuCl4溶液组成的混合溶液中,在搅拌速率为300rpm的情况下,持续搅拌5min,得到混合溶液;
2.1)将新鲜制备的1ml(3mol/L)AgNO3溶液和0.5ml(1mol/L)抗坏血酸溶液分别快速地加入到上述步骤2.1)的混合溶液中,并将搅拌器的搅拌速率调整为为500rpm,反应溶液的颜色从浅红色迅速变为深红色时,迅速加入60mg/ml巯基-PEG-羧基溶液继续搅拌反应15min,得到金纳米颗粒悬液;
3)纳米探针的制备:
3.1)取10ml上述步骤2)制备的金纳米颗粒悬液溶于10mL去离子水中,在温度为37°水浴锅中搅拌,混匀后加入的20μL(45-60mmol/)L的氯化钆溶液,反应18h;
3.2)将上述3.2)所得溶液在离心力为9000g的环境下离心15min,弃去上清液,将沉淀洗涤后重悬于5mL去离子水中,然后再加入150μL(1mg/mL)的新吲哚箐绿IR820溶液,在室温下搅拌反应24h,然后在离心力为10000g的环境下离心10min,弃去上清液,将沉淀洗涤后重悬于5mL去离子水中,获得新型多功能纳米探针。
经测试,所得纳米探针的表征结果与实施例1中相近。
实施例3
1)金种子溶液的制备:
在搅拌速率为750rpm的情况下,将18mL的1%柠檬酸三钠溶液加入到125mL沸腾的1.0mM的HAuCl4溶液中,持续搅拌15min后,反应液由淡金黄色逐渐变为淡紫色,得到金种子溶液;
2)金纳米颗粒的制备:
2.1)将步骤1)制得的Au种子溶液冷却至室温后,用0.22μm硝酸纤维素膜过滤,将过滤后滤液在4℃温度下进行冷藏,取1mL冷藏后的滤液加入到由1ml(1mol/L)的HCl 溶液和100ml(1.6mmol/L)HAuCl4溶液组成的混合溶液中,在搅拌速率为250rpm的情况下,持续搅拌3min,得到混合溶液;
2.1)将新鲜制备的1ml(3mol/L)AgNO3溶液和0.5ml(1mol/L)抗坏血酸溶液分别快速地加入到上述步骤2.1)的混合溶液中,并将搅拌器的搅拌速率调整为为750rpm,反应溶液的颜色从浅红色迅速变为深红色时,迅速加入45mg/ml巯基-PEG-羧基溶液继续搅拌反应13min,得到金纳米颗粒悬液;
3)纳米探针的制备:
3.1)取10ml上述步骤2)制备的金纳米颗粒悬液溶于10mL去离子水中,在温度为37°水浴锅中搅拌,混匀后加入的15μL(45-60mmol/)L的氯化钆溶液,反应15h;
3.2)将上述3.2)所得溶液在离心力为9000g的环境下离心13min,弃去上清液,将沉淀洗涤后重悬于5mL去离子水中,然后再加入125μL(1mg/mL)的新吲哚箐绿IR820溶液,在室温下搅拌反应24h,然后在离心力为10000g的环境下离心8min,弃去上清液,将沉淀洗涤后重悬于5mL去离子水中,获得新型多功能纳米探针。
经测试,所得纳米探针的表征结果与实施例1中相近。
实施例4
本实施例通过Hoechst即用型试剂盒检测和流式细胞术实验分别定量和定向的观察荧光来研究细胞对纳米探针的摄取情况;
1)细胞培养:
将肝癌HCC-LM3细胞分别种植在四室皿和24孔板中,过夜培育后,将培养基换成分别加入PBS、IR820、Gd-AuNPS@IR820(来自实施例1)新鲜的DMEM细胞培养基,继续孵育HCC-LM3细胞2小时和8个小时;
2)采用多聚甲醛固定四室皿内细胞后,采用激光共焦进行观察,在激光共聚焦显微镜下观察肝癌细胞内部的荧光强度及分布,结果见图4,孵育2小时后,IR820和 Gd-AuNPS@IR820组出现荧光信号,但Gd-AuNPS@IR820组的荧光信号明显强于IR820 组;共孵育8小时后,Gd-AuNPS@IR820组的荧光信号仍然强于IR820处理组;说明细胞对纳米探针有良好的内吞能力,且纳米探针进入细胞的能力强于IR820。
3)24孔中的细胞收集后,按照Hoechst即用型试剂盒操作步骤操作,再通过流式细胞仪定量分析肝癌细胞对IR820的摄取量,结果见图5,结果表明,时间越长细胞荧光强度越高,证明IR820和Gd-AuNPS@IR820进入细胞越多,且Gd-AuNPS@IR820进入细胞的能力强于IR820,与共聚焦的结果相符。
本实施例表明肝癌细胞可有效的摄取本发明制备的纳米探针。
实施例5
本实施例利用活性检测试剂盒(CCK-8)检测和BD细胞凋亡试剂盒检测评估纳米探针的安全性。
1)细胞培养
将肝癌HCC-LM3细胞种植在24孔板中,过夜培育后,将细胞分为Au对照组、PBS 对照组、IR820对照组、和Gd-AuNPS@IR820悬液实验组,将Gd-AuNPS@IR820悬液实验组的细胞培养基换成新鲜配置的分别加入含浓度为:0μg/mL、1μg/mL、2μg/mL、4μg/mL、 8μg/mL、16μg/mL的纳米探针悬液(来自是实施例1)的DMEM细胞培养基,PBS对照组的细胞培养基中加入相应体积PBS溶液,Au对照组和IR820对照组分别相应浓度(0-16 μg/mL)金纳米颗粒溶液和IR820溶液,加入细胞继续培养8个小时后,收集细胞;
2)BD细胞凋亡试剂盒检测
将收集的细胞,按照BD细胞凋亡试剂盒操作步骤操作,最后采用BD流式细胞仪进行分析,结果见图6,在孵育浓度范围内(0-16μg/mL),HCC-LM3细胞未发生明显凋亡。
3)活性检测试剂盒(CCK-8)检测
将收集的细胞,按照活性检测试剂盒(CCK-8)检测,结果见图7,对照组和 Gd-AuNPS@IR820实验组孵育浓度范围内,HCC-LM3细胞的活性没有出现明显下降;
本实施例表明本发明制备的纳米探针在正常情况下,与肝癌细胞有良好的生物相容性,且不具备毒性。
实施例6
本实施利用SOSG作为单线态捕获剂,检测纳米探针的单线态氧生成能力,用于说明纳米探针的光动力特性;
实验方法:取实施例1制备得到的纳米探针悬液,和与其浓度相同的新吲哚菁绿IR820 溶液,然后再加入SOSG单线态氧探针(10-12M),在808nm激光照射下进行测量,对不同时间点的单线态氧的含量进行记录;
实验结果:见图8所示,在808nm的激光照射下,纳米探针产生的单重态粒子数量迅速增加,10分钟后,单线态氧的量达到0.5分钟辐照的5.6倍,表明该纳米探针具有增强光动力治疗的巨大潜力。
实施例7
本实施例用于说明纳米探针的光热效应和光热衰减作用;
(1)光热效应实验
取1ml实施例1制备的Gd-AuNPS@IR820悬液、1ml IR820溶液、和1ml PBS作为空白对照组,其中IR820溶液和Gd-AuNPS@IR820悬液中的IR820的含量相同,分别置于1.5ml 离心管中,然后在808nm激光照射下进行温度监控(1W CM-2),通过近红外热成像仪监控,结果见图9和图10,图9为近红外热成像图,图10为以数据结果绘制照射时间与温度上升情况的曲线,通过图9可以看出该纳米探针在近红外热像仪下,IR820和 Gd-AuNPS@IR820悬液在808nm的激光照射下持续120秒,温度上升很快,但由图10可以看出,在相同的IR820含量下,Gd-AuNPS@IR820的温度明显升高,而PBS组在整个辐照期间温度没有明显升高,因此排除了激光辐照功率过大导致的温度升高的可能,表明本发明制备的纳米探针具有很显著的光热效应。
(2)光热衰减作用实验
取1ml实施例1制备的Gd-AuNPS@IR820悬液、1ml IR820溶液、和1ml PBS作为空白对照组,其中IR820溶液和Gd-AuNPS@IR820悬液中的IR820的含量相同,分别在808nm 的激光照射下进行5个循环的照射,记录温度的变化,结果见图11,由图11可见后期IR820 的热衰减作用很明显,所以相比于IR820,合成的纳米探针有更持续性的产热作用,表明本发明制备得到的纳米探针热衰减的速度更慢,拥有有更长久的产热作用,且在光热条件下对IR820有保护作用。
实施例8
本实施例用于说明经过纳米探针处理的细胞内单线态氧含量情况;
1)细胞培养和治疗
将肝癌细胞分别种植在四室皿中,过夜培育后,将培养基换成分别加入分别加入相同体积的PBS、IR820、Gd-AuNPS、Gd-AuNPS@IR820悬液的新鲜DMEM细胞培养基,继续培养8个小时后,用808nm激光(1W CM-2)照射细胞6min进行激光介导的联合治疗,治疗完成后,继续培养4小时。
2)实验方法与结果
四室皿细胞经过ROS荧光探针染色后,再通过激光共聚焦显微镜观察,结果见图12:在808nm的激光照射下,Gd-AuNPS@IR820悬液组的细胞产生了最多的单线态氧,由于肿瘤细胞的代谢是无氧呼吸,单线态氧越多就说明肿瘤细胞内产生的毒性越大,进而说明纳米探针在光热/光动力联合治疗的环境下对体外肝癌细胞明显的细胞毒性。
实施例9
本实施例用于说明纳米探针对体外肝癌细胞的光热/光动力治疗效果
1)细胞培养和治疗:
将肝癌细胞分别种植在四室皿中,过夜培育后,换分别加入相同体积的PBS、IR820、 Gd-AuNPS、Gd-AuNPS@IR820悬液的新鲜细胞培养液,其中Gd-AuNPS、Gd-AuNPS@IR820 悬液均由实施例1制备得到,继续培养8个小时后,用808nm激光(1W CM-2)照射细胞6min进行激光介导的联合治疗,治疗完成后,继续培养4小时。
2)实验方法与结果
a.将收集的四室皿细胞经过钙黄绿素/PI双染试剂盒染色,通过激光共聚焦显微镜观察,观察结果见图13所示,图13为细胞的双染色荧光图,当不进行激光照射处理时,这些处理组的细胞基本上没有出现死亡现象,在激光照射处理后,PBS组仍然没有出现明显的细胞死亡,而IR820处理组和Gd-AuNPs有明显的细胞死亡,而Gd-AuNPS@IR820组大部分细胞已经死亡,只有少数细胞存活;
b.将收集的四室皿细胞,按照BD细胞凋亡试剂盒操作步骤操作,最后采用BD流式细胞仪进行分析,结果见图14,激光照射后PBS组的凋亡率很低,而IR820处理组和 Gd-AuNPS则上升至15-20%之间,Gd-AuNPS@IR820组的凋亡率最高,是前面两组的两倍多,达到40%以上,其结果与双染的结果相符。
本实施例表明本发明制备的纳米探针在808nm激光介导的光热/光动力学联合治疗的环境下,能对体外肝癌细胞进行杀伤,具有明显的光热/光动力治疗效果,且效果明显优于单一的IR820或者Gd-AuNPs。
实施例10
本实施例用于说明荷瘤鼠静脉给药后的纳米探针荷瘤小鼠上的分布和代谢,尤其是肿瘤部位的积累;
实验方法:
取实施例1中制备得到的纳米探针悬液经尾静脉注射到提前异氟烷麻醉的荷瘤鼠体内,然后在各个时间分别在核磁成像仪和活体成像仪上观察和拍摄成像图片,每种成像模式下,在不同的时间点拍摄的参数一致,结果见图15和图16;
实验结果:图15为T1-MR成像观察图片,显示当Gd-AuNPS@IR820经尾静脉注射到荷瘤小鼠体内1小时后,Gd-AuNPS@IR820主要分布在肝脏,在肿瘤中也有明显的聚集;当到达第3小时观察时间点时,肿瘤部位Gd-AuNPS@IR820数量进一步增加,肾脏部位信号明显增强,这证明了纳米探针可以在肝脏分离后通过肾-尿代谢途径***;肿瘤部位 T1-MR信号结果同时也表明,随着观察时间的增加,纳米探针在肿瘤部位的聚集量逐渐增加,12小时后肿瘤部位聚集量达到峰值;
图16为活体荧光成像实时观察图片,根据纳米探针加载的IR820的荧光特性,实时荧光成像照片清晰显示Gd-AuNPS@IR820经尾静脉注射到荷瘤小鼠体内,聚焦于肝脏,肝脏内的荧光信号随着时间的推移逐渐减弱,证明纳米探针可以通过从肝脏中消散的游离,而肿瘤处的荧光信号逐渐增加,在注射后12h达到峰值;
上述结果表明纳米探针可通过肝脏-粪便途径和肾脏-尿液途径***,***效率高,减小了纳米探针在肿瘤细胞内的滞留量,肿瘤部位的荧光信号逐渐增加,说明纳米探针的肿瘤靶向能力强。
实施例11
本实施例用于说明荷瘤鼠静脉给药后纳米探针的体内毒性和抑瘤效果;
1)实验方法:
取相同体积PBS、AuNPS溶液、IR820溶液、Gd-Au@IR820溶液、Gd-AuNPS@IR820 悬液(来自实施例1)分别经尾静脉注射到提前异氟烷麻醉的荷瘤鼠体内,然后进行808nm 激光照射介导的联合治疗;其中Gd-Au@IR820指代的是靶向性更差的一种对照材料,这种相对于纳米探针的粒径更广泛,靶向性更差。
2)结果处理:
a.在治疗完成的第14天取出各组治疗鼠的肿瘤组织进行HE和KI-67染色和观察;并且在第14天取出各治疗组的主要脏器,进行HE染色和病理学观察,结果见图17和图18,图17为肿瘤组织KI-67和HE染色观察,从图中可以看出生理盐水组肿瘤组织未见明显的肿瘤细胞死亡,但AuNPS组、IR820组和Gd-Au@IR820组可以观察到肿瘤细胞空泡化和死亡,而Gd-AuNPS@IR820组肿瘤细胞死亡最多,这些体内治疗结果充分证明本发明制备的纳米探针在激光辐照介导的光热/光动力学联合治疗,能够有效的杀死肿瘤细胞,具有显著的治疗效果;图18为各不同处理组荷瘤鼠在注射Gd-AuNPS@IR820悬液14天后各脏器 HE染色,可以清楚地看出,各处理组的主要器官均未出现明显的病理损伤,证明在激光照射介导的联合治疗的环境下,纳米探针具有良好的体内生物相容性,对正常细胞以及组织毒副作用很小;
b.在治疗完成后的21天内,分别记录各治疗组荷瘤鼠肿瘤体积、体重及生存率,结果见图19、图20和图21,图19为治疗后荷瘤鼠体重变化,各组间体重变化无显著性差异;图20为治疗后荷瘤鼠体生存曲线,生存率有显著差异,在21天观察期结束时, Gd-AuNPS@IR820组的生存率最高;图21为治疗后各组荷瘤鼠肿瘤体积变化, Gd-AuNPS@IR820组肿瘤体积没有明显增大,而其他组肿瘤体积明显增大,说明该纳米探针抑瘤效果好。
综上需要说明的是,以上所述仅为本发明的优选实施例而已,并不用过于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案,在不脱离本发明构思的前提下,还可以做出若干简单推演、替换、或者对其中部分技术特征进行等同替换,凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (9)
1.一种新型多功能纳米探针,其特征在于:所述新型多功能探针包括:金纳米颗粒核心、和搭载在所述金纳米颗粒核心表面的钆离子和新吲哚箐绿IR820,所述金纳米探针的粒径为50-100nm。
2.一种如权利要求1所述的新型多功能纳米探针制备方法,其特征在于,包括以下步骤:
1)金种子溶液的制备:
在剧烈搅拌情况下,将1%柠檬酸三钠溶液加入到沸腾的1mmol/L的HAuCl4溶液中,持续搅拌,反应液由淡金黄色逐渐变为淡紫色,得到金种子溶液;
2)金纳米颗粒的制备
2.1)将上述步骤1)制得的金种子溶液冷却至室温后,用0.22μm硝酸纤维素膜过滤,将过滤后滤液在4℃温度下进行冷藏,在中度搅拌下,将冷藏后的滤液加入到含有1mol/L HCl和1.6mmol/L HAuCl4的混合溶液中,持续搅拌得到混合溶液;
2.2.)将新鲜制备的3mol/L AgNO3溶液和1mol/L抗坏血酸溶液分别快速地加入到上述步骤2.1)所得的混合溶液中,在剧烈搅拌情况下,反应溶液的颜色从浅红色迅速变为深红色时,迅速加入50mg/ml巯基-PEG-羧基溶液继续搅拌反应,得到金纳米颗粒悬液;
3)纳米探针的制备:
3.1)取上述步骤2)制备的金纳米颗粒悬液溶于去离子水中,在温度为37°水浴锅中搅拌,混匀后加入45-60mmol/L氯化钆溶液,反应过夜;
3.2)离心上述步骤3.1)所得溶液,弃去上清液,将沉淀洗涤后重悬于去离子水中,然后加入1mg/mL的新吲哚箐绿IR820溶液,继续搅拌反应24小时,然后再次离心,弃去上清液,将沉淀洗涤后重悬于去离子水中,获得纳米探针。
3.根据权利要求3所述的一种新型多功能纳米探针制备方法,其特征在于:所述步骤1)中的柠檬酸三钠与HAuCl4的体积比为:15-20:100-150。
4.根据权利要求3所述的一种新型多功能纳米探针制备方法,其特征在于:所述步骤2)中的金种子溶液、HCl溶液、HAuCl4溶液、AgNO3溶液、抗坏血酸溶液和巯基-PEG-羧基溶液的体积比为:1:1:100:1:0.5:0.02。
5.根据权利要求3所述的一种新型多功能纳米探针制备方法,其特征在于:所述步骤3)中的金纳米颗粒悬液、氯化钆溶液和新吲哚箐绿IR820溶液的体积比:10:0.01-0.02:0.1-0.15。
6.根据权利要求3所述的一种新型多功能纳米探针制备方法,其特征在于:所述步骤1)中所述的搅拌速率为500-1000rpm,搅拌时间为15min;
7.根据权利要求3所述的一种新型多功能纳米探针制备方法,其特征在于:所述步骤2.1)中的搅拌速率为200-300rpm,搅拌时间为1-5min;步骤2.2)中的搅拌速率为500-1000rpm,搅拌时间为10-15min。
8.根据权利要求3所述的一种新型多功能纳米探针制备方法,其特征在于:所述步骤3.1)中的搅拌速率为200-300rpm,水浴反应时间为12-18h;步骤3.2)中第一次离心条件为:在离心力为8000-9000g的环境下离心10-15min,第二次离心条件为:在离心力为9000-1000g的环境下离心5-10min。
9.一种如权利要求1所述的新型多功能纳米探针的应用,其特征在于:所述纳米探针可用于癌症靶向协同治疗、多模态成像引导的光热/光动力协同治疗。
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