CN113480992A - 羧基荧光素修饰的肾清除型银纳米颗粒及其制备方法与应用 - Google Patents
羧基荧光素修饰的肾清除型银纳米颗粒及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了羧基荧光素修饰的肾清除型银纳米颗粒及其制备方法与应用。本发明制备的超稳定银纳米颗粒FG‑AgNPs是将荧光染料(NHS‑FAM)共轭到制备好的GS‑AgNPs上，生成一种银‑染料共轭物。本发明合成的FG‑AgNPs，合成产率高，形貌较均一，并且具有优越的稳定性和良好的生物相容性，同时本发明的制备方法简单方便，原料来源广泛。通过对FG‑AgNPs和NHS‑FAM在pH从5‑10范围内的荧光强度研究表明与AgNPs的结合提高了NHS‑FAM的pH响应范围和灵敏度。基于此发现，这种荧光纳米传感器可以作为一种强有力的检测酸碱度的新型探针，有望在化学、医学和生物学领域得到广泛的应用。
Description
技术领域
本发明属于化学纳米材料领域，具体涉及羧基荧光素修饰的肾清除型银纳米颗粒及其制备方法与应用。
背景技术
纳米粒子由于其独特的光学和电子性质而受到了广泛的关注，并在纳米医学领域得到了广泛的应用。银纳米粒子由于其独特的物理、化学和生物特性，近年来引起了人们的极大兴趣。它在毒性、电阻、化学稳定性、催化活性和表面等离子共振方面也具有独特的物理化学性质，这些特性使得银纳米颗粒具有广泛的应用，包括药物输送、生物成像、医学诊断和治疗学、化学和生物化学传感、光学传感器、化妆品以及食品和制药等行业。此外，银纳米颗粒具有表面等离子体共振(SPR)、高稳定性和良好的生物相容性，可以结合一些配体如谷胱甘肽(GSH)，使其进入生物体后能够快速被肾脏清除，从而降低其生物毒性。例如：
等人在壳聚糖生物聚合物(BP/AgP)上制备了超小且稳定的银纳米粒子改善了生物聚合物膜的性能[Int.J.Mol.Sci.2020,21(24),9388]；M Shariq Ahmed等人通过内生细菌合成生物AgNPs，合成的AgNPs通过改变MCF-7细胞中凋亡蛋白(包括Bax，Bcl-2和炎性标记COX-2)的表达对MCF-7细胞表现出剂量依赖性的细胞毒性，从而诱导人乳腺癌MCF-7细胞凋亡[Environ Sci Pollut Res 26,26939–26946(2019)]。这些研究成果为贵金属银纳米颗粒的进一步发展和实际生物应用展开了一个很好的前景。
纯单体荧光有机染料常常有吸收系数低、毒性大、光漂白和发射寿命短等缺点，为了克服单体荧光有机染料的应用缺陷，需要对荧光材料进行改进处理，以便提高其在生物标记分析、免疫测定和诊断成像等领域的应用。
发明内容
针对现有技术的不足，本发明提供了一种羧基荧光素修饰的肾清除型银纳米颗粒及其制备方法与应用，本发明将荧光有机染料羧基荧光素NHS-FAM通过共价键连接到GS-AgNPs上，改善了单体荧光有机染料的缺点，并且AgNPs的结合提高了NHS-FAM对pH响应的范围和灵敏度，可用来检测酸碱度。制备合成的FG-AgNPs具有良好的荧光性质，且具有合成产率高，形貌较均一，稳定性高等特点。银纳米粒子与有机染料结合对于生物标记分析、免疫测定和诊断成像(包括癌症诊断)中的光学诊断非常有益，并且可应用于肿瘤成像及治疗方面。
将荧光团结合到纳米粒子上的优点之一是，荧光小分子的水溶性往往比较差，将其接在水溶性好的纳米颗粒表面，可以大大提高其水溶性。此外，纳米粒子大的比表面积使得其可以作为载体容纳多个荧光团分子，增加荧光团的光稳定性。但是需要考虑到荧光团-纳米粒子通过共价键或非共价键连接的效率。因此，设计适当的荧光团连接策略和荧光团-纳米粒子的详细表征很重要。
为解决现有技术问题，本发明采取的技术方案为：
羧基荧光素修饰的肾清除型银纳米颗粒，通过5(6)-羧基荧光素琥珀酰亚胺酯(NHS-FAM)和谷胱甘肽(GSH)为配体合成的GS-AgNPs之间发生反应获得银纳米粒子FG-AgNPs；所述银纳米粒子FG-AgNPs为分散性好、且直径为1.5-2.5nm的球形颗粒，在522nm波长处有强的荧光发射峰。
上述银纳米粒子FG-AgNPs与GS-AgNPs相比，形貌和尺寸并未发生明显改变。
上述羧基荧光素修饰的肾清除型银纳米颗粒的制备方法，包括如下步骤：
步骤1，分别取2-4mL浓度为100-200mmol/L的硝酸银溶液和1-2mL浓度为200-400mmol/L的谷胱甘肽溶液于100-200mL去离子水中，加5mol/L氢氧化钠溶液调节pH至4.9-5.1，再向混合溶液中加入200-400μL浓度为0.5mol/L硼氢化钠溶液，室温下以搅拌速度不低于900r/min的速度剧烈搅拌2天，待反应停止后，取反应溶液透析，并将溶液冷冻干燥，得谷胱甘肽封端银纳米颗粒GS-AgNPs；
步骤2，将谷胱甘肽封端银纳米颗粒GS-AgNPs粉末重新溶解在PBS中配制成0.05mmol/L的溶液，调pH为8-9，将荧光染料5(6)-羧基荧光素N-琥珀酰亚胺酯溶解在DMSO中配制成20mmol/L的溶液，二者在室温下振荡反应，反应完成得混合液，按V混合液：V乙醇为1:2的比例加入乙醇，在10000r/min的速度下离心2-3min后去掉上清液，并将沉淀重新溶解在PBS中，整个离心过程重复4次，最后一次离心后，将沉淀在PBS缓冲液中溶解完全，并过Sephadex LH-20柱，收集第一洗脱相溶液，即NHS-FAM与纳米颗粒AgNPs合成的FG-AgNPs。
作为改进的是，步骤2中所述振荡反应为室温下反应24小时。
其中，步骤2所述加乙醇高速离心后的沉淀重新溶解并通过Sephadex LH-20柱，去除未反应的NHS-FAM。
上述NHS-FAM包覆银纳米颗粒AgNPs合成的FG-AgNPs在制备荧光纳米传感器、制备诊断或治疗肿瘤的产品上的应用。
本发明利用pH敏感的荧光染料与谷胱甘肽为配体合成的GS-AgNPs，在室温下进行振荡反应，制备了稳定性良好的FG-AgNPs。合成的球形纳米颗粒FG-AgNPs相较于GS-AgNPs，在水中或PBS缓冲溶液中的稳定性得到大幅提高，具有高分散性，并且形貌尺寸未发生明显改变，依旧保留着在生物体内能够通过肾脏被快速清除的特性，因此可用于生物医学领域，尤其是肿瘤诊断和治疗。
有益效果：
与现有技术相比，本发明羧基荧光素修饰的肾清除型银纳米颗粒及其制备方法与应用，具有如下优势：
本发明制备的具有pH响应的银纳米颗粒FG-AgNPs是一种全新的纳米荧光材料，具有以下几个优点：纳米颗粒大的比表面积提高了荧光基团的负载率；此外，荧光团在大分子结构中的包封可以提高有机染料的稳定性，减少光漂白；与小分子相比，荧光标记的纳米粒子的另一个优点是它们能够将成像和药物输送结合起来作为治疗剂；当共价连接时，有机染料分子与纳米粒子的结合比单体荧光的物理吸收更有效且化学计量更精确；这种共价键连接的荧光团-纳米粒子可以减少染料从纳米粒子中逸出；NHS-FAM修饰的银纳米颗粒的荧光强度具有良好的pH响应性质，这可以作为一种检测细胞内酸碱度的新型探针；同时本发明的制备方法易于操控，原料易获得，可加工性强。本发明被NHS-FAM包覆的银纳米粒FG-AgNPs由于其超高稳定性和较小的生物毒性使其成为生物体内肿瘤诊断和治疗及微环境酸碱度检测的理想材料。
附图说明
图1为本发明制备的荧光染料分子NHS-FAM包覆银纳米颗粒GS-AgNPs合成的FG-AgNPs的理化图，其中(a)为HR-TEM图，(b)为HR-TEM图中的粒径分布；
图2为本发明制备的GS-AgNPs、FG-AgNPs和NHS-FAM的吸收光谱图和在468nm处激发的发射光谱图，其中(a)为吸收光谱，(b)为发射光谱；
图3为本发明制备的GS-AgNPs、FG-AgNPs和NHS-FAM的凝胶-电泳图，泳道1为GS-AgNPs，泳道2为FG-AgNPs，泳道3为游离NHS-FAM染料；
图4为本发明制备的FG-AgNPs和NHS-FAM在不同pH下的荧光强度，其中(a)为NHS-FAM，(b)为FG-AgNPs；
图5为本发明制备的FG-AgNPs在pH为6-10时的可逆性研究；
图6为本发明制备的FG-AgNPs和NHS-FAM的pH与荧光强度线性相关图,其中(a)为FG-AgNPs，(b)为NHS-FAM。
具体实施方式
以下结合附图和实施例对本发明作进一步说明。
羧基荧光素修饰的肾清除型银纳米颗粒，通过5(6)-羧基荧光素N-琥珀酰亚胺酯(NHS-FAM)和谷胱甘肽(GSH)修饰的GS-AgNPs之间发生反应获得银纳米粒子FG-AgNPs；所述银纳米粒子FG-AgNPs为分散性好、直径为1.5-2.5nm的球形状颗粒，在522nm波长处具有强的荧光发射峰。
上述羧基荧光素修饰的肾清除型银纳米颗粒的制备方法，包括以下步骤：
(1)将97mL去离子水，2mL、100mmol/L的硝酸银溶液，1mL、200mmol/L的谷胱甘肽溶液混合，加5mol/L氢氧化钠溶液调节pH至4.9-5.1，再向混合溶液中加200μL浓度为0.5mol/L硼氢化钠溶液，室温下以搅拌速度不低于900r/min的速度剧烈搅拌2天；
(2)将上述溶液反应2天后，停止反应，取出反应溶液转移至透析袋，透析3天，最后冷冻干燥24h，得到GS-AgNPs粉末；
(3)将上述GS-AgNPs粉末重新溶解在PBS中配制成0.05mmol/L的溶液，调pH为8-9，将pH敏感的荧光染料5(6)-羧基荧光素N-琥珀酰亚胺酯溶解在DMSO中配制成20mmol/L的溶液，二者在室温下振荡反应；
(4)将上述溶液反应24h后停止反应，将溶液倒入离心管，加2倍体积的乙醇在10000r/min离心2-3min，除去上清液，将沉淀在PBS缓冲液中重新溶解，整个离心过程重复4次，最后一次离心后，将沉淀在PBS缓冲液中完全溶解，并通过Sephadex LH-20柱去除未反应的NHS-FAM，收集第一洗脱相溶液，即NHS-FAM与纳米颗粒AgNPs合成的FG-AgNPs。
实施例1
对本发明制备的一种超稳定银纳米粒FG-AgNPs进行荧光、紫外吸收、HR-TEM、电泳等测试。
将荧光染料NHS-FAM包覆银纳米颗粒GS-AgNPs，采用上述实施案例的制备方法合成FG-AgNPs。图1为合成的FG-AgNPs的HR-TEM图，可以看出颗粒为球形结构，分散性好，直径小于5.5nm的尺寸满足在生物体中肾清除的要求。
如本发明制备的FG-AgNPs，图2(a)给出了GS-AgNPs、FG-AgNPs和NHS-FAM的吸收光谱，如下图所示，NHS-FAM在490nm处有一个强吸收峰。GS-AgNPs没有表现出任何显著的吸收。在图2(b)中，NHS-FAM的荧光光谱在550nm处显示最大波长，而FG-AgNPs在522nm处显示发射峰。发射波长的蓝移是由于染料分子(NHS-FAM)在银纳米颗粒表面形成了二聚体。从图中可以清楚地看出，在468nm激发下，银纳米粒子没有任何荧光。
实施例2
取10μL本发明制备的FG-AgNPs加入胶板的样品小槽内，接导线进行电泳，当移到距前沿1-2cm时，停止电泳，取出后在紫外灯下观察。图3(a)为本发明制备的FG-AgNPs与GS-AgNPs以及染料分子NHS-FAM的凝胶-电泳图。泳道1代表GS-AgNPs，泳道2中的浅黄色显示NHS-FAM与GS-AgNPs共轭形成的FG-AgNPs，泳道3中的深黄色代表游离NHS-FAM染料。尽管所有3条泳道都在同一个方向上移动，但从游离的NHS-FAM染料来看，GS-AgNPs和FG-AgNPs以相同的速度和相同的时间移动。此外，比较泳道2和3中的条带，显示sephadex技术成功地去除了未反应的染料。通过在紫外光照下显示的琼脂糖凝胶荧光图像来进一步确认结合，如图3(b)所示。
实施例3
测试实施例制备的FG-AgNPs在pH 5-10范围内的荧光强度变化并与NHS-FAM对比。图4(a)显示了NHS-FAM在不同酸碱度下的荧光强度，在468nm激发下，随着pH值从5-7的增加，NHS-FAM非常灵敏，550nm处的发射强度增加到两倍。然而，相比pH 5-7，从pH 8-10没有明显的荧光强度增加，说明NHS-FAM在pH较小范围内具有敏感的响应性。图4(b)显示了FG-AgNPs在不同酸碱度下的荧光强度，在468nm激发下，随着pH值从5-10的增加，FG-AgNPs在522nm处的荧光强度增加到五倍。比较不同pH下的NHS-FAM和FG-AgNPs的荧光强度，可以看到，从pH值5-7开始，两者的荧光强度都随着pH值的增加而大幅度增加，但从pH 7-10开始，FG-AgNPs显示出比NHS-FAM更显著的强度增加。这一结论表明，FG-AgNPs可以在更大的范围内对pH响应。通过在pH 6-10范围内对FG-AgNPs进行可逆性研究，进一步证实了这一点，如图5所示，在pH 6-10的范围内荧光强度可以可逆性地降低或恢复。
为更准确说明本发明制备的FG-AgNPs对pH的高敏感性，比较FG-AgNPs与NHS-FAM的线性相关性，如图6(a)所示，从pH 5-10开始，FG-AgNPs有很好的线性相关性，其线性回归(R2)为0.9992，而NHS-FAM上的大多数强度点远离线性拟合。因此，在pH 5-10范围内，FG-AgNPs比NHS-FAM对酸碱度更敏感。
银纳米粒子也可以与对酸碱度不敏感的染料(NHS-TAMRA)结合，可用于生物体内成像并且具有良好的肾清除特性，但其荧光强度没有良好的pH响应关系，与对pH不敏感的NHS-TAMRA修饰的银纳米颗粒相比，NHS-FAM修饰的银纳米颗粒的荧光强度具有良好的pH响应性质，这种荧光纳米传感器可以作为一种检测细胞内酸碱度的新型探针。
以上所述，仅为本发明较佳的具体实施方式，本发明的保护范围不限于此，任何熟悉本技术领域的技术人员在本发明披露的技术范围内，可显而易见地得到的技术方案的简单变化或等效替换均落入本发明的保护范围内。
Claims (4)
1.羧基荧光素修饰的肾清除型银纳米颗粒，其特征在于，通过5(6)-羧基荧光素琥珀酰亚胺酯和谷胱甘肽为配体合成的GS-AgNPs之间发生反应获得银纳米粒子FG-AgNPs；所述银纳米粒子FG-AgNPs为分散性好、且直径为1.5-2.5nm的球形颗粒，在522nm波长处有强的荧光发射峰。
2.基于权利要求1所述的羧基荧光素修饰的肾清除型银纳米颗粒的制备方法，其特征在于，包括如下步骤：
步骤1，分别取2-4mL浓度为100-200mmol/L的硝酸银溶液和1-2mL浓度为200-400mmol/L的谷胱甘肽溶液于100-200mL去离子水中，加5mol/L氢氧化钠溶液调节pH至4.9-5.1，再向混合溶液中加入200-400μL浓度为0.5mol/L硼氢化钠溶液，室温下以搅拌速度不低于900r/min的速度剧烈搅拌2天，待反应停止后，取反应溶液透析，并将溶液冷冻干燥，得谷胱甘肽封端银纳米颗粒GS-AgNPs；
步骤2，将谷胱甘肽封端银纳米颗粒GS-AgNPs粉末重新溶解在PBS中配制成0.05mmol/L的溶液，调pH为8-9，将荧光染料5(6)-羧基荧光素N-琥珀酰亚胺酯溶解在DMSO中配制成20mmol/L的溶液，二者在室温下振荡反应，反应完成得混合液，按V混合液：V乙醇为1:2的比例加入乙醇，在10000r/min的速度下离心2-3min后去掉上清液，并将沉淀重新溶解在PBS中，整个离心过程重复4次，最后一次离心后，将沉淀在PBS缓冲液中溶解完全，并过Sephadex LH-20柱，收集第一洗脱相溶液，即NHS-FAM与纳米颗粒AgNPs合成的FG-AgNPs。
3.根据权利要求2所述的羧基荧光素修饰的肾清除型银纳米颗粒的制备方法，其特征在于，步骤2中所述振荡反应为室温下反应24小时。
4.基于权利要求1或权利要求2所述的NHS-FAM包覆银纳米颗粒AgNPs合成的FG-AgNPs在制备荧光纳米传感器、制备诊断或治疗肿瘤的产品上的应用。
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| CN111973573A (zh) * | 2020-08-04 | 2020-11-24 | 中山大学附属第七医院(深圳) | 一种磷酸钙纳米颗粒及其制备方法和应用 |
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| CN111973573A (zh) * | 2020-08-04 | 2020-11-24 | 中山大学附属第七医院(深圳) | 一种磷酸钙纳米颗粒及其制备方法和应用 |
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