CN114410293B - 一种高灵敏度硫化氢响应型纳米探针及其制备方法和应用 - Google Patents
一种高灵敏度硫化氢响应型纳米探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种高灵敏度硫化氢响应型纳米探针及其制备方法。并且对近红外二区纳米荧光探针的应用进行了初步尝试,与普通探针比较,具有深的组织穿透能力,几乎不受自体荧光干扰,EPR效应使得其在肿瘤细胞中具有更长的滞留时间,可以实现长时间高时空分辨的活体近红外二区荧光及光声成像,肿瘤的光热治疗效果显著,实现了对肿瘤的诊疗一体化。
Description
技术领域
本发明属于近红外及光声纳米探针生物成像技术领域,具体涉及新型高灵敏度H2S定量纳米探针ZNNPs和ZNNPs@FA的制备方法,以及ZNNPs活体H2S可视化定量和ZNNPs@FA结肠癌光动力治疗。
背景技术
细胞内硫化氢(H2S)是由L-半胱氨酸(L-半胱氨酸)相关的酶促生物合成衍生而来,已被充分证明是一种重要的气体传递素,与各种生理和病理过程密切相关。体内H2S的异常与许多疾病的发生有本质的联系,如阿尔茨海默病,肝硬化,炎症,癌症。因此,准确监测生命系统内内源性H2S,对H2S相关疾病的早期诊断和治疗具有重要意义。目前最常用的H2S检测方法有高压液相色谱法、气相色谱法、电化学分析法、亚甲蓝法等。这些方法表现出很高的检测灵敏度,但由于成本高、操作繁琐、不适用于活体检测,严重阻碍了H2S的活体检测。为此,近年来人们致力于探索智能H2S响应探针,用于H2S相关疾病的诊断和治疗。许多基于不同的机理的H2S响应的荧光探针已经被开发,这些探针在生物系统中检测H2S方面具有显著优势,响应快、无创、灵敏度高,为H2S的实时成像提供了强有力的工具。然而,由于组织穿透较浅、空间分辨率较差和严重的光子散射,大多数探针在活体硫化氢成像中依旧面临挑战。因此,开发准确定量评价深部组织H2S水平的新策略,对H2S相关疾病的诊断和治疗评价具有重要意义。
发明内容
为了克服上述现有材料及技术中存在的问题,本发明构建两种新型高灵敏度H2S响应型纳米探针,利用ZNNPs特异性H2S成像的优势,进行活体中硫化氢浓度的定量,同时利用ZNNPs@FA进行内源性H2S激活的光动力治疗癌症,比如结肠癌。
本发明采用以下技术方案:
一种高灵敏度硫化氢响应型纳米探针,其具有核壳结构;所述壳为两亲性聚合物,核为如下化学结构式的化合物(ZM1068-NB):
该核化合物称为化合物ZM1068-NB。
本发明中,作为壳的两亲性聚合物为聚乙二醇-聚酯(PEG-PCL)或者所述两亲性聚合物为聚乙二醇-聚酯(PEG-PCL)与聚乙二醇-聚酯-叶酸(PEG-PCL-FA);按数均分子量,优选聚乙二醇的分子量为2000~10000,聚酯的分子量为2000~5000。作为最佳示例,聚乙二醇-聚酯为PEG5000-PCL3000,聚乙二醇-聚酯-叶酸为PEG5000-PCL3000@FA;PEG-PCL、PEG-PCL-FA的化学结构式分别如下:
本发明中,聚乙二醇-聚酯可以单独作为探针的壳,也可以利用聚乙二醇-聚酯与聚乙二醇-聚酯-叶酸共同作为探针的壳,当两亲性聚合物为聚乙二醇-聚酯与聚乙二醇-聚酯-叶酸时,聚乙二醇-聚酯-叶酸的质量为两亲性聚合物质量的15%~25%。
本发明公开了上述高灵敏度硫化氢响应型纳米探针在H2S浓度定量,或者制备H2S浓度定量试剂中的应用,或者在制备活体H2S浓度定量试剂中的应用。优选的,本发明公开了高灵敏度硫化氢响应型纳米探针在体外H2S浓度定量中的应用;或者高灵敏度硫化氢响应型纳米探针在制备体外H2S浓度定量试剂中的应用;或者高灵敏度硫化氢响应型纳米探针在活体H2S浓度定量中的应用;或者高灵敏度硫化氢响应型纳米探针在制备活体H2S浓度定量试剂中的应用;或者高灵敏度硫化氢响应型纳米探针在肿瘤治疗中的应用;或者高灵敏度硫化氢响应型纳米探针在制备肿瘤治疗试剂中的应用。
本发明将化合物ZM1068-NB与两亲性聚合物在超声条件下自组装反应,得到高灵敏度硫化氢响应型纳米探针,化合物与两亲性聚合物的质量比为1∶(3.5~5.5),自组装为室温反应10~30min;进一步的,ZM1068-ketone与对硝基苯甲酰氯反应,得到化合物ZM1068-NB,ZM1068-ketone与对硝基苯甲酰氯的摩尔比为1∶(3~5);反应在氮气保护下进行,反应为常温反应5~20h。具体的,作为示例,本发明所述高灵敏度硫化氢响应型纳米探针的制备方法如下步骤:
(1)萘二甲酰亚胺与3-溴丙酸甲酯反应,得到化合物1;
(2)化合物1与甲基氯化镁反应,得到化合物2;
(3)化合物2与2-氯-3-(羟亚甲基)-1-环己烯-1-羧醛反应,得到化合物ZM1068;
(4)化合物ZM1068与N-羟基琥珀酰亚胺反应,得到化合物ZM1068-ketone;
(5)化合物ZM1068-ketone与对硝基苯甲酰氯,得到化合物ZM1068-NB;
(6)化合物ZM1068-NB与PEG5000-PCL3000在超声条件下自组装反应,得到高灵敏度硫化氢响应型纳米探针,称为ZNNPs;
或者
(7)萘二甲酰亚胺与3-溴丙酸甲酯反应,得到化合物1;
(8)化合物1与甲基氯化镁反应,得到化合物2;
(9)化合物2与2-氯-3-(羟亚甲基)-1-环己烯-1-羧醛反应,得到化合物ZM1068;
(10)化合物ZM1068与N-羟基琥珀酰亚胺反应,得到化合物ZM1068-ketone;
(11)化合物ZM1068-ketone与对硝基苯甲酰氯,得到化合物ZM1068-NB;
(12)化合物ZM1068-NB与PEG5000-PCL3000& PEG5000-PCL3000@FA在超声条件下自组装反应,得到高灵敏度硫化氢响应型纳米探针,称为ZNNPs@FA;
上述技术方案中,萘二甲酰亚胺与3-溴丙酸甲酯的反应在第一有机溶剂中进行,化合物1与甲基氯化镁的反应在第二有机溶剂中进行;化合物2与2-氯-3-(羟亚甲基)-1-环己烯-1-羧醛的反应在第三有机溶剂中进行;化合物ZM1068与N-羟基琥珀酰亚胺的反应在第四有机溶剂中进行;化合物ZM1068-ketone与对硝基苯甲酰氯的反应在第五有机溶剂中进行;化合物ZM1068-NB与PEG5000-PCL3000或者化合物ZM1068-NB与PEG5000-PCL3000& PEG5000-PCL3000@FA在超声条件下自组装反应在第一无机溶剂中进行。优选的,所述第一有机溶剂为含有碳酸钾的N,N-二甲基甲酰胺;所述第二有机溶剂为四氢呋喃;所述第三有机溶剂为含有乙酸钠的乙酸酐,2-氯-3-(羟亚甲基)-1-环己烯-1-羧醛、乙酸钠的摩尔比为1∶2.2;所述第四有机溶剂为N,N-二甲基甲酰胺;所述第五有机溶剂为无水二氯甲烷;所述第一无机溶剂为去离子水或者超纯水。
上述技术方案中,萘二甲酰亚胺与3-溴丙酸甲酯的摩尔比为1∶3;化合物1和甲基氯化镁的摩尔比为1∶8;化合物2与2-氯-3-(羟亚甲基)-1-环己烯-1-羧醛的摩尔比为2.2∶1;化合物ZM1068与N-羟基琥珀酰亚胺的摩尔比为1∶3;化合物ZM1068-ketone与对硝基苯甲酰氯的摩尔比为1∶4;化合物ZM1068-NB与PEG5000-PCL3000的质量比为1∶4;化合物ZM1068-NB与PEG5000-PCL3000与PEG5000-PCL3000@FA的质量比为1∶3.8∶0.2。
根据本发明技术方案,其中:
步骤(1)中,萘二甲酰亚胺与3-溴丙酸甲酯的反应在N,N-二甲基甲酰胺溶剂中进行,萘二甲酰亚胺与3-溴丙酸甲酯的摩尔比为1∶3;优选的,反应在氮气保护下进行,反应为回流反应6 h。
步骤(2)中,化合物1与甲基氯化镁的反应在四氢呋喃中进行,化合物1和甲基氯化镁的摩尔比为1∶8;优选的,格式反应在氮气保护下进行,所述反应为回流反应2 h。
步骤(3)中,化合物2与2-氯-3-(羟亚甲基)-1-环己烯-1-羧醛的反应在乙酸钠存在的乙酸酐溶剂中进行,化合物2、2-氯-3-(羟亚甲基)-1-环己烯-1-羧醛和乙酸钠的摩尔比为2.2∶1∶2.2;优选的,反应在氮气保护下进行,反应为常温反应4 h。
步骤(4)中,化合物ZM1068与N-羟基琥珀酰亚胺的反应在三乙胺存在的无水DMF中进行,化合物ZM1068、N-羟基琥珀酰亚胺和三乙胺的摩尔比为2.9∶1∶3.6;优选的,反应在氮气保护下进行,反应为常温反应3 h。
步骤(5)中,化合物ZM1068-ketone与对硝基苯甲酰氯的反应在无水二氯甲烷中进行,化合物ZM1068-ketone与对硝基苯甲酰氯的摩尔比为1∶3;优选的,反应在氮气保护下进行,反应为常温反应12 h。
步骤(6)中,化合物ZM1068-NB与PEG5000-PCL3000在超声条件下自组装在水相溶剂中进行,化合物ZM1068-NB与PEG5000-PCL3000的质量比为1∶4;优选的,自组装为室温超声反应20min。
步骤(7)中,萘二甲酰亚胺与3-溴丙酸甲酯的反应在N,N-二甲基甲酰胺溶剂中进行,萘二甲酰亚胺与3-溴丙酸甲酯的摩尔比为1∶3;优选的,反应在氮气保护下进行,反应为回流反应6 h。
步骤(8)中,化合物1与甲基氯化镁的反应在四氢呋喃中进行,化合物1和甲基氯化镁的摩尔比为1∶8;优选的,格式反应在氮气保护下进行,所述反应为回流反应2 h。
步骤(9)中,化合物2与2-氯-3-(羟亚甲基)-1-环己烯-1-羧醛的反应在乙酸钠存在的乙酸酐溶剂中进行,化合物2、2-氯-3-(羟亚甲基)-1-环己烯-1-羧醛和乙酸钠的摩尔比为2.2∶1∶2.2;优选的,反应在氮气保护下进行,反应为常温反应4 h。
步骤(10)中,化合物ZM1068与N-羟基琥珀酰亚胺的反应在三乙胺存在的无水DMF中进行,化合物ZM1068、N-羟基琥珀酰亚胺和三乙胺的摩尔比为2.9∶1∶3.6;优选的,反应在氮气保护下进行,反应为常温反应3 h。
步骤(11)中,化合物ZM1068-ketone与对硝基苯甲酰氯的反应在无水二氯甲烷中进行,化合物ZM1068-ketone与对硝基苯甲酰氯的摩尔比为1∶3;优选的,反应在氮气保护下进行,反应为常温反应12 h。
步骤(12)中,化合物ZM1068-NB与PEG5000-PCL3000& PEG5000-PCL3000-FA在超声条件下自组装在水相溶剂中进行,化合物ZM1068-NB与PEG5000-PCL3000与PEG5000-PCL3000-FA的质量比为1∶3.8∶0.2;优选的,自组装为室温超声反应20 min。
本发明中,化合物1、化合物2、化合物ZM1068、化合物ZM1068-ketone、化合物ZM1068-NB、纳米颗粒ZNNPs与纳米颗粒ZNNPs@FA(新型高灵敏度硫化氢响应型纳米探针)的化学结构式分别如下:
利用上述高灵敏度硫化氢响应型纳米探针ZNNPs进行活体硫化氢可视化定量的方法,包括以下步骤,将所述高灵敏度硫化氢响应型纳米探针的水溶液尾静脉注射入被检测小鼠的体内,90 min后在多光谱光声断层扫描仪上获得小鼠被检测部位680 nm和900 nm处的光声图像,用iThera MSOT成像软件测量小鼠被检测部位PA680和PA900光声信号强度平均值。将PA680/PA900代入定量方程y =-0.90573x2+5.30757x-2.23108,得到小鼠被检测部位H2S浓度(单位:μmol/g protein)。
利用上述高灵敏度H2S响应型纳米探针ZNNPs@FA进行结肠癌肿瘤成像的方法包括以下步骤,将所述高灵敏度硫化氢响应型纳米探针ZNNPs@FA的水溶液尾静脉注射入荷瘤小鼠的体内,麻醉状态下观察不同时间点近红外成像以及光声成像效果;完成活体结肠癌肿瘤成像成像。
利用上述高灵敏度H2S响应型纳米探针ZNNPs@FA进行结肠癌肿瘤光动力治疗的方法包括以下步骤,将所述高灵敏度H2S定量纳米探针ZNNPs@FA (1 mg/mL, 200 μL)的水溶液尾静脉注射入HCT116荷瘤小鼠的体内,16 h后用660 nm激光(50 mW/cm2)照射肿瘤3min,记录肿瘤体积及小鼠体重变化情,观察肿瘤治疗效果。
由于上述技术方案的运用,本发明与现有技术相比具有如下优点:
(1)本发明中设计合成了一种高灵敏度H2S响应型纳米探针ZNNPs,可以实现活体内源性硫化氢可视化定量;
(2)本发明中目标探针ZNNPs@FA对结肠癌肿瘤组织具有良好的被动靶向性,光动力治疗效果显著,可以实现对肿瘤的诊疗一体化。
附图说明
图1为两种新型高灵敏度硫化氢响应型纳米探针的制备示意图。
图2为化合物ZM1068-NB的核磁氢谱表征。
图3为化合物ZM1068-NB的核磁碳谱表征。
图4为化合物ZM1068-NB的高分辨质谱表征。
图5为(a)实验组探针ZNNPs在水溶液中的水合粒径,(b)实验组探针ZNNPs的TEM图像以及粒径统计结果,(c)实验组探针ZNNPs在含有100 μM NaHS PBS溶液中1-10 min紫外吸收光谱,(d)实验组探针ZNNPs在含有0-500 μM NaHS PBS溶液中10 min时的近红外荧光光谱变化;(e)实验组探针ZNNPs在含有0-500 μM NaHS PBS溶液中10 min时的近红外荧光图像及荧光强度量化,(f)实验组探针ZNNPs在含有0-500 μM NaHS PBS溶液中10 min时的光声图像(680 nm-900 nm),(g)实验组探针ZNNPs在含有0-500 μM NaHS PBS溶液中10 min时的比率光声信号PAS680/PAS900变化,(h)实验组探针ZNNPs与不同生物因子育后的荧光图像,(i)实验组探针ZNNPs与不同生物因子育后的比率光声信号PAS680/PAS900。
图6为(a)实验组探针ZNNPs@FA在水溶液中的水合粒径,(b)实验组探针ZNNPs@FA的TEM图像以及粒径统计结果,(c)实验组探针Q3-NPs尾静脉注射入小鼠体内不同时间点的近红外二区活体荧光成像,(b)对应(a)中荧光强度随时间变化,(c)实验组探针ZNNPs@FA的红外图谱,(d)实验组探针ZNNPs和ZNNPs@FA的Zeta电位。
图7为ZNNPs@FA通过细胞内H2S消耗抑制HCT116细胞增殖,(a)ZNNPs@FA(20μg/mL)处理HCT116细胞4小时,或ZnCl2(300μM)处理HCT116细胞10分钟后的细胞划痕实验,(b)图(a)中划痕实验结果量化,(c)不同浓度ZNNPs或ZNNPs@FA条件下培养HCT116 24 h后细胞毒性试验,(d)ZNNPs@FA(20μg/mL)与HCT116细胞孵育不同的时间点的线粒体与ZNNPs@FA的共定位实验,成像前细胞用PBS洗涤三次,然后用Mito Tracker(Green,100 nM,1 mL)培养30分钟,并用PBS洗涤以进行荧光显微镜成像。
图8为(a)用不同浓度L-Cys对小鼠实施腹腔注射30 min后尾静脉注射实验组探针ZNNPs 90 min后小鼠肝脏在680 nm和900 nm处的光声图像,(b)a中小鼠肝脏光声信号强度随腹腔注射L-Cys浓度的变化曲线,(c)a中小鼠肝脏光声比率信号PAS680/PAS900随腹腔注射L-Cys浓度的变化曲线,(d)a中小鼠腹腔注射不同浓度L-Cys 120 min后肝脏中硫化氢浓度变化曲线,(e)a中小鼠肝脏光声比率信号PAS680/PAS900和肝脏H2S浓度的拟合曲线,(f)不同浓度二甲双胍对小鼠连续实施腹腔注射7天后对小鼠尾静脉注射实验组探针90 min后的光声图像,(g)f中小鼠肝脏光声比率信号PAS680/PAS900量化,(h)f中小鼠肝脏拟合硫化氢浓度以及实际硫化氢浓度,(i)为ICH模型构建和内源性H2S检测的示意图说明,(j)PA图像、(k)在680和900 nm处的 PA强度、(l)PAS680/PAS900信号比率,(m)为小鼠脑切片的荧光图像。
图9为小鼠肝脏中H2S的活体荧光和光声成像,Balbc-Nu小鼠腹腔注射生理盐水(100μL)、L-Cys(6mm,100μL)或PAG(2mg/mL,100μL),以构建肝脏中H2S水平不同的小鼠;(a)通过光声断层扫描(PAT)可视化小鼠肝脏中硫化氢浓度的示意图,(b)各组小鼠尾静脉注射ZNNPs(10 mg/kg)后肝脏区域720和1070 nm处的平均荧光强度(n=5),(c)b图中荧光强度量化,(d)各组小鼠尾静脉注射ZNNPs(10 mg/kg)后肝脏区域680和900 nm处的平均荧光强度(n=5),(e)d图中比率光声信号(PAS680/PAS900)量化(n=5)。
图10为(a)实验组探针ZNNPs@FA (100 mg/mL, 200 μL)尾静脉注射的的HCT116皮下瘤小鼠在720和1070 nm处的实时荧光图像,(b) a中肿瘤荧光强度定量,(c) 实验组探针ZNNPs@FA (100 mg/mL, 200 μL)尾静脉注射的的HCT116皮下瘤小鼠在680和900 nm处的实时光声图像,(d) c中肿瘤中光声信号强度定量。
图11为(a)分别用 PBS缓冲液, ZnCl2 (40 μg/mL, 10 min), NaHS (1 mM, 1h), L-Cys (24 μg/mL, 1 h), PAG (50 μg/mL, 0.5 h)+L-Cys (24 μg/mL, 1 h), LPS(1 μg/mL, 6 h)+L-Cys (24 μg/mL, 1 h), or PAG (50 μg/mL, 0.5 h)+LPS (1 μg/mL,6 h)+L-Cys (24 μg/mL, 1 h)处理各组细胞,然后用ZNNPs@FA (12 h, 50 μg/mL)+660 nm(50 mW/cm2, 3 min)处理各组细胞,并且用DCF-DA 试剂盒检测细胞中1O2的生成,(b)a图中各组细胞中DCF荧光亮度量化,(c)实验组两组细胞分别用ZNNPs@FA (12 h, 50 μg/mL),ZNNPs@FA (12 h, 50 μg/mL)+660 nm (50 mW/cm2, 3 min) 处理后,用 JC-1 试剂盒检测细胞中线粒体膜电位变化,(d)c中各组细胞存活率变化。
图12为(a)静脉注射ZNNPs(10 mg/kg)和(b)ZNNPs@FA(10 mg/kg)后3、6、12、24和48 h各器官中ZM1068-ketone的定量分析。
图13为(a) HCT116皮下瘤模型构建和PDT治疗示意图,(b) 对HCT116皮下瘤模型尾静脉注射200 mL PBS缓冲液或者ZNNPs@FA (10 mg/kg) 16 h后,660 nm激光(50 mW/cm2, 3 min)照射小鼠肿瘤后肿瘤体积随时间的变化曲线,(c)各组治疗结束时肿瘤图像及(d)肿瘤重量,(e)治疗后各组小鼠的体重变化,(f)治疗两天后肿瘤切片的H&E、Tunnel和ki67染色图像。
图14为小鼠尾静脉注射(a)ZNNPs(10 mg/kg)或(b)ZNNPs@FA(10 mg/kg)后3、6、12、24和48 h后脏器旳离体NIR-I荧光图像(Em = 640/720 nm)和NIR-II荧光图像(Em =808/1070 nm)。(c)a图中720 nm荧光强度量化。(d)a图中1070 nm荧光强度量化。(e)b图中720 nm荧光强度量化。(f)b图中1070 nm荧光强度量化。
具体实施方式
下文将结合附图和具体实施例来进一步阐述本发明。应当理解的是,这些实施例仅用于解释和说明本发明中的技术方案,而并非旨在限制本发明的范围。此外,除非另有说明,下列实施例中所使用的材料、试剂、仪器等均可通过商业手段获得。除非另有说明,所有化学品均从萨安化学技术(上海)有限公司购买,且未经进一步净化即可使用。m PEG5000-PCL3000和m PEG5000-PCL3000-FA购自上海东永生物科技有限公司。N-羟基琥珀酰亚胺、DL-炔丙基甘氨酸(PAG)、L-半胱氨酸、二甲双胍、IV型胶原酶、GAPDH活性测定试剂盒和三乙胺购自Sigma-Aldrich。1,3-二苯基异苯并呋喃和对硝基苯甲酰氯购自TCI(上海)。硫化氢(H2S)比色分析试剂盒购自Elabscience Biotechnology。Mito Tracker Green和MTT购自Beyotime Biotechnology。安捷伦海马XF细胞有丝分裂应激测试试剂盒和安捷伦海马XF糖酵解应激测试试剂盒购自安捷伦科技有限公司。DCFH-DA购自Solarbio(北京)。荷瘤(HCT116结肠癌)裸鼠的建模以及其他实验方法,都为常规技术。
本发明构建、合成新型高灵敏度硫化氢响应型纳米探针ZNNPs和ZNNPs@FA的步骤如下:
萘二甲酰亚胺与3-溴丙酸甲酯发生亲核取代反应,得到化合物1;化合物1与甲基氯化镁反应,得到化合物2;化合物2与2-氯-3-(羟亚甲基)-1-环己烯-1-羧醛反应,得到化合物ZM1068;化合物ZM1068与N-羟基琥珀酰亚胺反应,得到化合物ZM1068-ketone;化合物ZM1068-ketone与对硝基苯甲酰氯,得到化合物ZM1068-NB;化合物ZM1068-NB与聚合物(比如PEG5000-PCL3000或PEG5000-PCL3000-FA)在超声条件下自组装反应,得到高灵敏度硫化氢响应型纳米探针ZNNPs和ZNNPs@FA。
(1)新型高灵敏度硫化氢响应型纳米探针ZNNPs的肝脏H2S可视化定量:
将L-Cys (1、2、3、4、5 mM, 100 μL)注射到Balbc-Nu小鼠腹腔,调控小鼠肝脏H2S表达水平;30 min后,小鼠尾静脉注射将所述高灵敏度H2S定量纳米探针ZNNPs@FA (1 mg/mL, 200 μL);90 min后,用光谱光声断层扫描器(MSOT, iThera medical, Germany)采集小鼠肝脏光声图像(680 nm & 900 nm),应用iThera MSOT成像软件测量小鼠肝脏PA680和PA900光声信号强度平均值,使用H2S比色试剂盒测定肝脏中H2S的实际浓度,然后将PA680/PA900与肝脏中H2S的实际浓度进行拟合,得到小鼠内源性H2S光声比率信号定量方程y = -0.90573x2 + 5.30757x - 2.23108。Balbc-Nu小鼠(18 g、0.5 g) 连续7天腹腔注射二甲双胍(0、1、3、5 mg/100 μL PBS缓冲液);将所述高灵敏度H2S定量纳米探针ZNNPs@FA (1 mg/mL, 200 μL)经尾静脉注射小鼠;90 min,在多光谱光声断层扫描仪上获得小鼠肝脏680 nm和900 nm处的光声图像,用iThera MSOT成像软件测量小鼠肝脏PA680和PA900光声信号强度平均值。将PA680/PA900代入定量方程y = - 0.90573x2 + 5.30757x - 2.23108,得到小鼠肝脏H2S浓度。
(2)新型高灵敏度硫化氢响应型纳米探针ZNNPs@FA的活体荧光成像:
将上述获得的实验组探针ZNNPs@FA溶于PBS溶液中(浓度:1 mg/mL,体积:200 μL),以尾静脉注射的方式将探针注入荷瘤(HCT116结肠癌)裸鼠体内,随后分别置于小动物Serious II 900-1700 nm近红外二区活体成像系统中(激发波长:808 nm)和IVIS小动物成像系统(激发波长:640 nm,发射波长:720 nm),实时观察成像效果,最终通活体成像分析软件计算裸鼠的肿瘤部位在不同时间点的荧光强度。
(3)新型高灵敏度硫化氢响应型纳米探针ZNNPs@FA的活体光声成像:
将上述获得的实验组探针ZNNPs@FA溶于PBS溶液中(浓度:1 mg/mL,体积:200 μL),以尾静脉注射的方式将探针注入荷瘤(HCT116结肠癌)裸鼠体内,同时打开小动物光声断层扫描成像系统,待光声成像仪水浴池中的水温达37℃时,放入麻醉好的小鼠,扫描小鼠的肿瘤部位图像。之后将获得的光声成像数据使用MSOT InSight/inVision分析软件进行重建分析。
(4)新型高灵敏度硫化氢响应型纳米探针ZNNPs@FA的光动力治疗:
将荷瘤(HCT116结肠癌)裸鼠(肿瘤体积约20 mm3)随机分成4组(n=5):仅尾静脉注射PBS(10 mM, 200μL)的小鼠(第1组,简写Control);尾静脉注射PBS(10 mM, 200μL)16h后用660 nm激光(50 mW/cm2)照射肿瘤3 min(第2组,简写Control+660 nm);仅尾静脉注射ZNNPs@FA(1 mg/mL, 200μL)的小鼠(第3组,简写ZNNPs@FA);尾静脉注射ZNNPs@FA(1 mg/mL, 200μL)16 h后用660 nm激光(50 mW/cm2)照射肿瘤3 min(第4组,简写ZNNPs@FA+660nm)。记录肿瘤体积及小鼠体重变化情,观察肿瘤治疗效果。
实施例1:新型高灵敏度硫化氢响应型纳米探针ZNNPs和ZNNPs@FA的合成。反应示意图以及涉及各步产物的化学结构式如图1中所示。
(1)氮气保护下,150 mL圆底烧瓶中加入萘二甲酰亚胺(1.69 g, 10mmol)、3-溴丙酸甲酯(2.38 g, 20 mmol)以及10 mL无水N,N-二甲基甲酰胺作为溶剂,混合液加热到100℃磁力搅拌并回流5 h。反应完成,冷却至室温,混合物倒入500 mL冰水混合液中,并用1mol/L盐酸水溶液调节pH至中性,减压抽滤,粗产物使用硅胶柱层析纯化(石油醚:乙酸乙酯= 9:1,v/v),产物为黄色固体化合物1(1.56 g,产率:75 %)。
(2)氮气保护的冰浴条件下,100 mL圆底烧瓶中加入化合物1(1.03 g,5.0 mmol)以及20 mL无水四氢呋喃作为溶剂,加入甲基氯化镁的四氢呋喃溶液(13.4 mL,3 mol/L),随后撤去冰浴,混合液回流搅拌反应2 h,然后倒入酸性(pH1,盐酸调节)冰水浴中,搅拌条件下加入六氟磷酸钾(0.92 g,5.0 mmol),继续室温搅拌6 h。反应完成,析出浅绿色固体后,抽滤,得绿色固体化合物2,产物室温保存,无需进一步处理,进行下一步反应。
(3)氮气保护下,50 mL圆底烧瓶中加入化合物2(0.77 g,2.19 mmol)、2-氯-3-(羟亚甲基)-1-环己烯-1-羧醛(0.17 g,0.98 mmol)、无水醋酸钠(0.16 g,2.0 mmol)以及20mL无水乙酸酐作为溶剂,混合液室温搅拌反应4 h。反应完成,混合液减压浓缩,硅胶柱层析纯化(二氯甲烷:甲醇 = 50:1,v/v),产物为深绿色固体化合物ZM1068(0.44 g,产率:63%)。
(4)将N-羟基琥珀酰亚胺(334 mg,2.9 mmol)和三乙胺(0.5 mL)混合在10 mL无水DMF中,然后在搅拌下加入ZM1068(644 mg,1 mmol)溶解于6 mL无水DMF中的溶液,随后在氮气气氛下室温搅拌3 h,最后在HPLC纯化后获得化合物ZM1068-ketone(488 mg,78%)。
(5)将ZM1068-ketone(100 mg,0.16 mmol)溶解于10 mL无水二氯甲烷中,然后在搅拌下加入溶解于5 mL无水二氯甲烷中的对硝基苯甲酰氯(89 mg,0.48 mmol),室温搅拌12h后,经HPLC纯化得到ZM1068-NB(68mg),产率为54.9%。
图2为化合物ZM1068-NB的核磁氢谱表征,图3为化合物ZM1068-NB的核磁碳谱表征,图4为化合物ZM1068-NB的高分辨质谱表征。
(6)ZM1068-NB (1 mg)和PEG5000-PCL3000 (4mg)分别溶于100 µL二甲基亚砜中。将ZM1068-NB的二甲基亚砜溶液加入到聚合物的二甲基亚砜溶液中,超声10分钟得到混合溶液。在超声条件下将混合物滴加到4mL超纯水中,再超声处理20分钟。所得溶液用蒸馏水透析2天,在分子量截止(MWCO)为14 kDa的透析袋中去除DMSO,得到ZNNPs。
(5)ZM1068-NB (1 mg) 溶于100 µL二甲基亚砜中,P PEG5000-PCL3000(3.8 mg)+PEG5000-PCL3000-FA (0.2 mg)溶于100 µL二甲基亚砜中。将ZM1068-NB的二甲基亚砜溶液加入到聚合物的二甲基亚砜溶液中,超声10分钟得到混合溶液。在超声条件下将混合物滴加到4mL超纯水中,再超声处理20分钟。所得溶液用蒸馏水透析2天,在分子量截止(MWCO)为14kDa的透析袋中去除DMSO,得到ZNNPs@FA。
实施例2:ZNNPs和ZNNPs@FA的化学表征。
首先通过动态光散射(DLS)测定的平均水动力尺寸分别为70和75 nm,表明ZNNPs和ZNNPs@FA在水溶液中呈单分散纳米颗粒(图5a和图6a)。同理,图5b和图6b的透射电镜(TEM)结果显示,它们的平均直径分别为70±3.5 nm和77±5 nm。然后通过傅里叶变换红外光谱(IR)、zeta电位分析和紫外-可见吸收光谱对纳米粒子的结构进行了表征。图6c的红外光谱结果显示,在3200-3300 m-1和1637 cm-1处的特征吸收峰分别对应于氨基和酯基。此外,ZNNPs和ZNNPs@FA在1000 nm处记录的另一个最大吸收峰应该是醚键,这与ZM1068-NB的红外吸收曲线非常一致。Zeta电位分析显示mPEG5000-PCL3000, ZNNPs和ZNNPs@FA的电势分别为-21±0.63 mV, 29.3±0.93, 24.6±0.6mV(图6d)。此外,还通过DLS监测纳米探针的流体力学尺寸变化来评价纳米探针的稳定性,图6c和图6d显示的结果表明这两种纳米探针在37℃的磷酸盐缓冲盐水(PBS, pH=7.4)中储存7天时具有良好的稳定性。
为了研究ZNNPs对H2S的特异性反应,将1.4 mg ZNNPs溶解在1 mL PBS缓冲液中,得到1.4 mg/mL的备用溶液。采用紫外-可见光谱法(UV-vis)检测了ZNNPs原液(20 μL, 1.4mg/mL)在1.98 mL硫氢化钠(100 μM)溶液中的吸收变化(图5c)。此外,配制不同浓度的硫氢化钠溶液(0~500 μM),分别添加20μL ZNNPs备用液到1.98 mL不同浓度硫氢化钠溶液并37℃孵育10 min,然后测试每个样品的荧光光谱和600~900nm光声图像(图5d和图5f)。
为考察ZNNPs对H2S的特异性和选择性,将30 μL (1.4 mg/mL) PBS缓冲液(pH7.4)加入1.97 mL不同生物相关因子(100 µM NaHS, 1 mM SCN-, 1 mM NO3 -, 1 mM SO4 2-,1 mM K+, 1 mM Ca2+, 1 mM Na+, 1 mM Mg2+, 1 mM NO2 -, 1.25 mM VC, 10 mM GSH, 1 mMH2O2, 1 mM CO3 2-, 1 mM S2O3 2-, 1 mM SO3 2-, 1 mM AC-)中并在37 ℃下孵育10 min,分别用IVIS光谱成像系统记录荧光图像(λex/λem = 640/720 nm),同时在多光谱光声断层扫描仪上分别获得各样品680 nm和900 nm的PA图像并量化分析得到光声比率信号PAS680/PAS900(图5i)。
细胞划痕试验。将6孔板上的HCT116细胞用ZNNPs@FA (20 μg/mL)处理4 h或用ZnCl2 (300 μM)处理10min。用200 μL的移液管尖划伤。然后将细胞与不含胎牛血清的培养基孵育。用Olympus IX73倒置显微镜在0和24小时捕捉划痕图像。利用image J软件对划痕宽度进行分析。ZNNPs与ZNNPs@FA的细胞毒性试验。HCT116细胞被接种到96孔板24 h。然后用不同浓度的ZNNPs或ZNNPs@FA (0、0.1、1、10、25、50、100μg/mL)孵育细胞24 h。最后采用标准MTT法测定细胞活力。ZNNPs@FA与线粒体的共定位实验。将HCT116细胞(~ 5×104)接种于共聚焦培养皿中,24 h后贴壁。将各组细胞与ZNNPs@FA (20 μg/mL)分别孵育不同时间点,然后PBS缓冲液洗涤3次。最后,细胞与Mito-Tracker Green (100 nM, 1 mL)孵育30分钟,PBS缓冲液冲洗,最后用荧光显微镜对各组细胞进行成像。以上结果参见图7。
实施例3:新型高灵敏度硫化氢响应型纳米探针ZNNPs的活体硫化氢定量方程推导及验证
为了建立体内内源性H2S的定量方法,Balbc-Nu小鼠(18 ± 0.5 g)腹腔注射100μL的L-Cys溶液(0、1、2、3、4、5 mM),构建肝脏中H2S浓度不同的小鼠模型。30min后,将实施例1中制得的实验组探针ZNNPs (1 mg/mL, 200 μL)通过尾静脉注射。采集90 min时间点680 nm 和900 nm PA图像(图8a),用iThera MSOT成像软件测量90 min时肝脏ROI区PA信号的平均强度并绘制曲线(图8b)。最后,将各组(n=3)的肝脏解剖匀浆,使用H2S比色试剂盒测定肝脏中H2S的实际浓度(图8d)。然后将PA680/PA900(图8c)与肝脏中H2S的实际浓度进行拟合,得到活鼠内源性H2S的定量方程(图8e)。
为验证活鼠内源性H2S的定量方程,Balbc-Nu小鼠(18 g 0.5 g)腹腔注射二甲双胍(0、1、3、5 mg / 100 μL PBS缓冲液),连续7天。每只小鼠尾静脉注射ZNNPs (1 mg/mL,200 μL)。注射后90 min在多光谱光声断层扫描仪上分别采集680 nm和900 nm处小鼠肝脏的PA图像(图8f),并用iThera MSOT成像软件测量肝脏ROI区域光声信号强度平均值。将PA680/PA900代入定量方程y = - 0.90573x2 + 5.30757x - 2.23108,得到小鼠肝脏中H2S的浓度。最后将各组(n = 2)肝脏解剖匀浆,采用H2S比色测定试剂盒测定肝脏中H2S的实际浓度(图8g和8h),图8i为ICH模型构建和内源性H2S检测的示意图说明,静脉注射ZNNPs(10mg/kg小鼠)的PA图像、在680和900 nm处的 PA强度、PAS680/PAS900信号比率见图8j、k、l(n=3),图8m为小鼠脑切片的荧光图像。
小鼠肝脏内源性H2S的近红外/PA成像。为评价ZNNPs对肝脏H2S的反应,Balbc-Nu小鼠腹腔注射生理盐水(100 μL)、L-Cys (6 mM, 100 μL)或PAG (2 mg/mL, 100 μL),从而调控小鼠肝脏H2S表达水平。30 min后,静脉注射ZNNPs (1 mg/mL, 200 μL),在IVIS (Ex:640 nm, Em: 720 nm)和NIR-II成像系统(Ex: 808 nm, Em: 1070 nm)上获取指定时间点的图像。PA图像(680 nm &用多光谱光声层析成像扫描仪在指定时间点(900 nm)采集。结果见图9。
实施例4:新型高灵敏度硫化氢响应型纳米探针ZNNPs@FA的活体结肠癌皮下瘤成像。
HCT116荷瘤小鼠尾静脉注射ZNNPs@FA (1 mg/mL, 200 μL)。3%异氟醚混合空气麻醉小鼠,用IVIS小动物成像系统、NIR-II荧光成像系统和MSOT采集不同时间点小鼠图像(图10)。之后将获得的数据进行重建分析,结果表明,实验组随时间的变化肿瘤部位的光声及荧光强度呈现先增后减的变化趋势,且在8 h时附近信号强度达到最大值。
将HCT116细胞(~ 5×104)接种于共聚焦培养皿中,12 h后细胞贴壁。共设4组实验细胞和1组对照组。第二组用NaHS处理1 h,第三组用DMEM培养基中300 μM的ZnCl2处理10min;第4组用DMEM培养基中L-Cys (200 μM)处理1 h;第五小组用DMEM培养基中PAG(50 mg/mL),L-Cys(200μM)分别处理0.5 h和1 h。所有的细胞都洗净后与ZNNPs@FA(20μg/mL)孵育4h。细胞用PBS缓冲洗三遍,然后与DCFH-DA(10μM, 1毫升)孵育20分钟。最后使用无血清培养基洗涤细胞3次,进一步暴露在660 nm (50 mW/cm2)激光下3 min,然后在共聚焦显微镜下对细胞中DCF成像以评估单线态O2的产生。将HCT116细胞(~ 5×104)接种于共聚焦培养皿中,12 h后细胞贴壁。PBS缓冲液或ZNNPs@FA (12 h, 50 μg/mL)或ZNNPs@FA (12 h, 50 μg/mL)+660 nm (50 mW/cm2, 3 min)处理细胞。24 h后,用PBS缓冲液洗涤细胞。用JC-1 (1μM)测定线粒体膜电位。低温透射电镜观察细胞线粒体形态。HCT116细胞中H2S激活ZNNPs@FA的PDT。将HCT116或HEK293细胞以8×103个/孔的密度接种于96孔板中,培养24 h。第三组HCT116细胞先与氯化锌(300 μM)共培养10 min,然后各组分别与ZNNPs@FA (10 μg/mL)共培养24 h,PBS缓冲液洗涤细胞3次,660 nm (50 mW/cm2)激光照射3 min,最后采用标准MTT法测定细胞活力。结果见图11。
静脉注射ZNNPs(10 mg/kg)和ZNNPs@FA(10 mg/kg)后3、6、12、24和48 h各器官中ZM1068-ketone的定量分析。各个时间点小鼠脏器离体匀浆,每100 μL器官匀浆添加1 μL50 mM NaHS溶液进行预处理,并在37℃下孵育2 h后用酶标仪检测ZM1068-ketone的荧光强度,并带入标曲计算各器官药物浓度。结果见图12。
实施例5:新型高灵敏度硫化氢响应型纳米探针ZNNPs@FA的活体结肠癌皮下瘤治疗。
HCT116荷瘤小鼠随机分组(每组5只)。每只小鼠通过尾空静脉注射PBS缓冲液(200μL)或ZNNPs@FA (100 mg/mL, 200 μL)。16h后,用50mw /cm2的660 nm激光照射第二组和第四组肿瘤3 min,每2天测量一次肿瘤大小,第20天处死小鼠。对心、肺、脾、肝、肾、肿瘤组织切片进行H&E染色分析。对肿瘤切片进行TUNEL和Ki67免疫荧光染色。表明实验组探针ZNNPs@FA具有很好的生物安全性,且对结肠癌皮下瘤有很好的治疗效果(图13)。
参见图14,小鼠尾静脉注射(a)ZNNPs(10 mg/kg)或(b)ZNNPs@FA(10 mg/kg)后3、6、12、24和48 h后脏器旳离体NIR-I荧光图像(Em = 640/720 nm)和NIR-II荧光图像(Em =808/1070 nm)。(c)a图中720 nm荧光强度量化。(d)a图中1070 nm荧光强度量化。(e)b图中720 nm荧光强度量化。(f)b图中1070 nm荧光强度量化。
本发明克服了传统荧光成像受组织强光吸收和散射干扰的局限性,提供了一种具有深度组织穿透能力和高空间分辨率的成像方式。由于这些显著的优点,本发明探针可用于检测活体中的微量物质,如H2S。现有技术基于单个响应性光声信号的多种生理和病理过程的活体光声成像,往往受到探针浓度、环境条件、组织光散射等因素的严重影响。本发明成像能够克服这些缺点,利用两个分离波长进行自校准,提高光声成像的信噪比,因此,本发明开发具有诊断功能的H2S响应性光声成像探针被认为是实现H2S相关疾病准确诊断和有效治疗的一种有前景的策略。
Claims (8)
1.一种高灵敏度硫化氢响应型纳米探针,其特征在于,所述高灵敏度硫化氢响应型纳米探针具有核壳结构;所述壳为两亲性聚合物,核为如下化学结构式的化合物:
;
所述两亲性聚合物为聚乙二醇-聚酯或者所述两亲性聚合物为聚乙二醇-聚酯与聚乙二醇-聚酯-叶酸;聚乙二醇的分子量为2000~10000,聚酯的分子量为2000~5000。
2.权利要求1所述高灵敏度硫化氢响应型纳米探针的制备方法,其特征在于,将权利要求1所述化合物与两亲性聚合物在超声条件下自组装反应,得到高灵敏度硫化氢响应型纳米探针。
3.根据权利要求2所述高灵敏度硫化氢响应型纳米探针的制备方法,其特征在于,ZM1068-ketone与对硝基苯甲酰氯反应,得到所述化合物;所述ZM1068-ketone的化学结构式如下:
。
4.根据权利要求3所述高灵敏度硫化氢响应型纳米探针的制备方法,其特征在于,ZM1068-ketone与对硝基苯甲酰氯的摩尔比为1∶(3~5);反应在氮气保护下进行,反应为常温反应5~20h。
5.根据权利要求2所述高灵敏度硫化氢响应型纳米探针的制备方法,其特征在于,化合物与两亲性聚合物的质量比为1∶(3.5~5.5);自组装为室温反应10~30min。
6.根据权利要求5所述高灵敏度硫化氢响应型纳米探针的制备方法,其特征在于,两亲性聚合物为聚乙二醇-聚酯或者所述两亲性聚合物为聚乙二醇-聚酯与聚乙二醇-聚酯-叶酸;当两亲性聚合物为聚乙二醇-聚酯与聚乙二醇-聚酯-叶酸时,聚乙二醇-聚酯-叶酸的质量为两亲性聚合物质量的15%~25%。
7.权利要求1所述高灵敏度硫化氢响应型纳米探针在制备H2S浓度定量试剂中的应用,或者在制备活体H2S浓度定量试剂中的应用。
8.权利要求1所述高灵敏度硫化氢响应型纳米探针在制备结肠癌治疗试剂中的应用。
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