CN105797171A - 具有靶向整合素αvβ3的复合碳纳米点及其在肿瘤新生血管成像和光热治疗中的应用 - Google Patents
具有靶向整合素αvβ3的复合碳纳米点及其在肿瘤新生血管成像和光热治疗中的应用 Download PDFInfo
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Abstract
本发明公开了具有靶向整合素αvβ3的复合碳纳米点及其在肿瘤新生血管成像和光热治疗中的应用，以及在光热治疗中的应用。其中构建基质为碳纳米点，包载组分为亚甲基蓝荧光分子，表面偶联为精氨酸‑甘氨酸‑天冬氨酸（RGD）多肽。该碳纳米点直径为2～30纳米，电位为‑25～‑10毫伏。本发明的制备步骤简单、条件温和、工艺绿色环保、能耗少、无三废及辐射与噪声等污染，分离与提纯工艺操作简便，所得体系稳定、易于保存。基于靶向复合碳纳米点良好的生物相容性、包载物质的荧光成像与光热治疗功能，有望在体内标记与示踪、生物医学影像和肿瘤的早期诊断与治疗等领域得到广泛应用，在生命健康与个性化医疗等方面产生良好的经济与社会效益。
Description
技术领域
本发明涉及一种具有靶向整合素αvβ3的新型碳纳米点复合材料，具体是复合碳纳米点的制备工艺及其特异性靶向肿瘤整合素αvβ3的荧光成像及作为潜在光热治疗剂的应用。
背景技术
荧光成像是一种常见的光学成像技术，以其直观、原位的可视化检测方式在生物检测和医学影像等领域应用广泛。荧光成像在实际应用中展现出其独特优势，如样品穿透性强、极高的灵敏度和选择性，但传统有机染料同时存在一些致命缺陷，如性质不稳定，容易被光漂白，不能长期使用等。因此，如何克服荧光染料在使用中的缺陷成为当前研究的重点。
随着纳米技术的不断发展，将纳米技术与高灵敏度的荧光分子相结合，发展制备包载荧光分子的分子影像探针，将为克服上述问题提供新的策略。近年来碳纳米点以其化学惰性、光闪烁缺乏、光漂白率低、毒性低、生物兼容性好等优点日益受到关注。研究发现，荧光小分子经过碳点包载后其荧光稳定性和结构稳定性都会有明显的提升。
当荧光探针表面与某些靶特异性分子（如蛋白质，DNA，RNA）结合时，会使探针与体内特定细胞所表达的抗原或受体进行结合，特异性作用于病变区域生物分子组成成分来突出显示病变部位，从而提高诊断的准确性与敏感性，这种靶向造影剂成为现今研究领域的热点。整合素αVβ3是肿瘤组织最常用的靶标之一，它由αV亚基（CD51，150000）和β3亚基（CD61，105000）组成，其中α链的胞膜外区能特异性识别含精氨酸-甘氨酸-天冬氨酸（RGD）序列的多肽，介导整合素与细胞外基质的黏附。整合素αV，β3在正常组织器官及成熟血管内皮细胞中不表达或低表达，在多种肿瘤（包括肺癌、成胶质细胞瘤、乳腺癌、骨肉瘤）细胞表面和新生血管内皮细胞中有高表达，在肿瘤的新生血管生成、侵袭和转移过程中起重要作用。特异性标记的外源性RGD多肽进入体内后可与整合素αVβ3位点高选择性结合，从而RGD多肽可以作为一种特异性配体靶向组织，并通过各种影像学方法进行示踪。
光热治疗剂是利用具有较高光热转换效率的材料，将其注射入活体内部，利用靶向性识别技术聚集在病灶附近，并在外部光源（一般是近红外光）的照射下将光致热效应杀死癌细胞的一种治疗方法。这种方法具有高效率、低痛苦、副作用小、作用时间短等优点，并且治愈效果明显，用于光热治疗的材料无毒无害，因此在肿瘤治疗方面具有非常广阔的应用前景。
发明内容
本发明的目的是为了克服现有技术存在的缺点和不足，而提供一种具有靶向整合素αvβ3的复合碳纳米点。
本发明的另一个目的是提供一种具有靶向整合素αvβ3的复合碳纳米点的制备工艺
本发明的另一个目的是提供具有靶向整合素αvβ3的复合碳纳米点在肿瘤新生血管成像的应用。
本发明的另一个目的提供基于具有靶向整合素αvβ3的复合碳纳米点的光热治疗剂。
为实现本发明的第一个目的，本发明的技术方案是以碳纳米点为构建基质，包载组分为荧光物质亚甲基蓝，表面偶联有特异性靶向多肽分子。
进一步设置是表面偶联的特异性靶向多肽分子为精氨酸-甘氨酸-天冬氨酸（RGD）三肽序列。
进一步设置是所述具有靶向整合素αvβ3的碳纳米点的直径为2～30纳米，电位为-25～-10毫伏。
实现本发明的第二个发明目的是技术方案是包括以下步骤：
a.取碳源和0.5～5.0毫克/毫升亚甲基蓝溶液，按体积比1:1～10:1混合，制得前体溶液；
b.将上述前体溶液放置微波反应仪中，仪器参数设置为：温度160℃、时间5h；
c.反应体系静置30～60分钟，离心分离出上清液，得复合碳纳米点粗产物；
d.取上述复合碳点粗产物，按比例依次加入精氨酸-甘氨酸-天冬氨酸（RGD），N-羟基丁二酰亚胺（NHS）和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐（EDC）；
f.将上反应体系置于25～45摄氏度水浴中搅拌5～48小时；
g.将上述反应液转移至离心管中分离提纯，取上清液；
h.将上清液透析30～180分钟即得具有靶向整合素αvβ3的复合碳纳米点。
实现本发明的第三个发明目的技术方案是一种靶向复合纳米荧光探针，其为具有靶向整合素αvβ3的复合碳纳米点，并用于肿瘤新血管成像。
实现本发明的第四个发明目的，用于光热治疗的治疗剂，其为所述的具有靶向整合素αvβ3的复合碳纳米点。
本发明所述新型靶向整合素αvβ3的纳米荧光探针及选择性构建方法对发展新型生物探针、拓展荧光造影剂的制备工艺、提高荧光探针的生物利用率、实现肿瘤新生血管的早期诊断与光热治疗具有重要意义。
本发明得到新型靶向整合素αvβ3的纳米荧光探针，其通用工艺与结构如图1所示。表面电位表征及多分布指数可由激光粒度仪测量得到（如图2）。荧光探针的形貌和粒径大小可透射电镜测得（如图3）。制成的靶向复合纳米荧光造影材料具有良好的生物相容性和安全性，荧光成像灵敏度高，靶向整合素αvβ3效果强，在荧光场中，经近红外光激发产生荧光信号响应经重建图像如图4所示。与非靶向对照组相比，靶向整合素αvβ3的复合碳纳米点可显着提高在肿瘤新生血管的荧光成像的对比度与分辨率。此外制成的靶向复合碳纳米点在热成像仪拍摄下，经808nm激光照射5钟后温度明显升高（图5），材料成像效果明显，从而确认该靶向复合纳米荧光探针在荧光成像，肿瘤细胞的早期诊断与光热治疗、荧光治疗药物的靶向富集等方面的广阔应用前景。
下面结合说明书附图和具体实施方式对本发明做进一步介绍。
附图说明
图1为本发明所涉及的靶向复合纳米荧光探针的通用结构与工艺（左）及结构（右）；
图2为新型靶向复合纳米荧光探针的电位表征图；
图3为新型靶向复合纳米荧光探针的原子力显微镜表征图；
图4为新型靶向复合纳米荧光探针的透射电镜表征图
图5a为靶向复合碳纳米点不同浓度下的荧光成像效果图；
图5b为小鼠尾静脉注射靶向复合碳纳米点注射后24小时内荧光成像效果图
图6a为靶向复合碳纳米点可见光下及808纳米激光照射5分钟前后的红外热成像效果
图6b为同功率808纳米激光照射下5分钟内靶向复合碳纳米点与碳点、同浓度亚甲基蓝溶液和水的升温比较
图6c不同浓度靶向复合碳纳米点在同功率808纳米激光照射下5分钟内升温比较（CMB’的亚甲基蓝浓度为CBM的一半）
图6d不同浓度靶向复合碳纳米点在不同功率的808纳米激光照射5分钟内升温情况。
具体实施方式
下面通过实施例对本发明进行具体的描述，只用于对本发明进行进一步说明，不能理解为对本发明保护范围的限定，该领域的技术工程师可根据上述发明的内容对本发明作出一些非本质的改进和调整。
本发明所采用的制备原料均为商品获得。
实施例1：将2毫升现磨豆浆和20毫升亚甲基蓝溶液（0.5毫克/毫升）混合制得前体溶液，并取5毫升前体溶液投入5毫升微波反应器专用玻璃瓶，并将其放置于微波反应仪中，反应条件设置为160摄氏度、5小时。反应体系静置60分钟后离心，保留上层清液，得复合碳纳米点粗产物。取2毫升该复合碳纳米点粗产物，按比例依次加入精氨酸-甘氨酸-天冬氨酸（RGD）,N-羟基丁二酰亚胺（NHS）和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐（EDC），搅拌48小时后，离心，保留上清液，然后超滤3小时，即得靶向整合素αvβ3复合荧光碳纳米点。用紫外分光亮度计标准曲线法测得复合碳纳米点中亚甲基蓝的浓度为5微克/毫升。由动态光散射原理经激光粒度仪测量，表面电位为:-25.6±5.77毫伏（图2a）。其形貌及粒径由原子力显微镜检查而得（图3），粒径大小为4.6±2.5纳米。制成的靶向复合纳米荧光造影材料具有良好的生物相容性和安全性，荧光成像灵敏度高，靶向效果强，在荧光场中，靶向复合碳纳米点稀释不同倍数后材料成像的效果,经近红外光激发产生荧光信号响应经重建图像如图5a所示。眼眶静脉注射接种MDA-MB231细胞株的裸鼠24小时内小鼠体内成像的结果如图5b所示，靶向整合素αvβ3复合碳纳米点可显着提高在肿瘤新生血管的荧光成像的对比度与分辨率。并且制成的靶向复合碳纳米点在热成像仪拍摄下，经808nm激光照射5钟后温度明显升高（图5a），并且靶向复合碳纳米点的光热效应明显强于单纯碳纳米点和同浓度亚甲基蓝溶液（如图5b）.确认该靶向复合纳米荧光探针在荧光成像，肿瘤细胞的早期诊断与光热治疗、荧光治疗药物的靶向富集等方面的广阔应用前景。
实施例2：将2毫升纯牛奶和2毫升亚甲基蓝溶液（5毫克/毫升）混合制得前体溶液，并前体溶液投入5毫升微波反应器专用玻璃瓶，并将其放置于微波反应仪中，反应条件设置为160摄氏度、5小时。反应体系静置60分钟后离心，保留上层清液，得复合碳纳米点粗产物。取2毫升该复合碳纳米点粗产物，按比例依次加入精氨酸-甘氨酸-天冬氨酸（RGD）,N-羟基丁二酰亚胺（NHS）和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐（EDC），45℃搅拌5小时后，离心，保留上清液，然后超滤0.5小时，即得靶向整合素αvβ3复合荧光碳纳米点。用紫外分光亮度计标准曲线法测得复合碳纳米点中亚甲基蓝的浓度为15微克/毫升。由动态光散射原理经激光粒度仪测量，表面电位为:-11.8±6.27毫伏（图2b）。其形貌及粒径由透射电镜检测而得（图4），粒径大小为25.2±7.6纳米。并且制成的靶向复合碳纳米点在热成像仪拍摄下，经808nm激光照射5钟后,其光热效应存在浓度依赖和功率依赖（图6c,6d）。
Claims (6)
1.一种具有靶向整合素αvβ3的复合碳纳米点，其特征在于：以碳纳米点为构建基质，包载组分为荧光物质亚甲基蓝，表面偶联有特异性靶向多肽分子。
2.根据权利要求1所述的具有靶向整合素αvβ3的复合碳纳米点，其特征在于：表面偶联的特异性靶向多肽分子为精氨酸-甘氨酸-天冬氨酸三肽序列。
3.根据权利要求2所述的一种具有靶向整合素αvβ3的复合碳纳米点，其特征在于：所述具有靶向整合素αvβ3的碳纳米点的直径为2～30纳米，电位为-25～-10毫伏。
4.一种制备如权利要求1-3之一所述的复合碳纳米点的制备工艺，其特征在于包括以下步骤：
a.取碳源和0.5～5.0毫克/毫升亚甲基蓝溶液，按体积比1:1～10:1混合，制得前体溶液；
b.将上述前体溶液放置微波反应仪中，仪器参数设置为：温度160℃、时间5h；
c.反应体系静置30～60分钟，离心分离出上清液，得复合碳纳米点粗产物；
d.取上述复合碳点粗产物，按比例依次加入精氨酸-甘氨酸-天冬氨酸三肽序列，N-羟基丁二酰亚胺和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐；
f.将上反应体系置于25～45摄氏度水浴中搅拌5～48小时；
g.将上述反应液转移至离心管中分离提纯，取上清液；
h.将上清液透析30～180分钟即得具有靶向整合素αvβ3的复合碳纳米点。
5.一种如权利要求1-3之一所述的复合碳纳米点，其特征在于所述的具有靶向整合素αvβ3的复合碳纳米点运用于肿瘤新血管成像。
6.一种如权利要求1-3之一所述的复合碳纳米点的应用方法，其特征在于所述的具有靶向整合素αvβ3的复合碳纳米点作为光热治疗剂。
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