CN113262312B - 靶向肾脏的近红外荧光探针及其制备方法和应用 - Google Patents
靶向肾脏的近红外荧光探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明属于生物材料技术领域，具体为一种靶向肾脏的近红外发光探针及其制备方法和应用。本发明的近红外发光探针由肾脏靶向多肽与发光材料共价连接组成；多肽氨基酸侧链的残基（包括氨基、羧基或巯基）与荧光分子形成化学键合；多肽用于增强荧光分子的水溶性以及靶向肾脏中的肾小管结构；荧光分子用于荧光成像。本发明的肾脏靶向探针对肾脏靶向特异性高，70%以上注射剂量的探针在肾脏富集，所述肾脏靶向探针在肾脏中靶向停留时间大于48 h，可作为特异性肾脏靶向荧光成像剂，用于可用于对早期肾损伤状态进行诊断以及肾疾病治疗药物的靶向递送。
Description
技术领域
本发明属于生物材料技术领域，具体涉及一种基于靶向肾脏的荧光探针及其制备方法和应用。
背景技术
肾脏疾病在早期通常是没有临床症状的，但随着肾脏疾病的发展最终可能导致严重的肾功能衰竭。全世界近10%的成年人患有早期无症状的肾脏疾病，这些疾病每年导致约200万人死于后期的肾衰竭。目前临床对于肾脏疾病的诊断方法依赖于测定血尿素氮(BUN)和血肌酐(sCr)，然而，这些标志物往往在早期肾损伤时没有明显变化，在约50%的肾小球滤过率(GFR)丧失之前，这些临床检查标志物仍保持在正常范围内，因此不能用于早期肾损伤检测。非侵入性活体成像技术，如单光子发射计算机断层扫描(SPECT)，磁共振成像(MRI)，正电子发射断层扫描/计算机断层扫描(PET/CT)能被用于评估不同阶段的肾脏功能障碍，然而这些战略受到其高成本、辐射风险和低可及性的限制。
基于荧光的成像技术具有快速反馈、非电离辐射、高时空分辨率和灵敏度高等优点，可以直接显示生物体内的动态变化，目前已广泛应用于生物成像和生物传感领域。尤其是利用近红外第二窗口成像穿透更深，能降低生物组织的自身荧光，提高信噪比，更适合在活体深层组织成像。基于肾脏对小颗粒的快速滤过与清除能力，一些直径低于肾脏滤过阈值(5.5 nm)的超小荧光纳米材料包括金纳米颗粒(GNP)、量子点(QD)、康奈尔点和分子探针等可以用来对肾功能进行荧光成像。但是荧光探针的靶向成像通常需要分子具有特定的结构或特性，例如，肾探针分子需要能够通过肾脏清除代谢。而基于肾清除通路的肾脏荧光成像，探针在肾脏内停留时间短，且荧光信号会随排尿而波动，造成成像诊断或靶向药物递送的不稳定性。因此开发能够长时间靶向肾脏的探针从而进行肾脏荧光成像仍是一项挑战。在本发明中，我们设计了一种能用于长期靶向肾脏的荧光探针，通过将有机小分子或纳米颗粒与高极性的多肽偶联，从而实现在肾脏中高效长期的积累。
发明内容
本发明的目的在于提供一种制备工艺简单、水溶性好、生物相容性好，多肽肾脏靶向效率高且靶向停留时间长的荧光探针及其制备方法及其应用。
本发明提供的多肽靶向肾脏的荧光探针，由肾脏靶向多肽与发光材料共价连接组成；多肽氨基酸侧链的残基（包括氨基、羧基或巯基）与荧光分子形成化学键合；其中，多肽用于增强荧光分子的水溶性以及靶向肾脏中的肾小管结构；荧光分子用于荧光成像。本发明通过筛选多肽序列，选取不同荧光分子调控探针在肾脏中的靶向时间，最终得到可以高效长期靶向于肾脏的荧光探针。
本发明中，所述肾靶向多肽（记为KTPs）序列为：
SHSNTQTLAKAPEHTGC(SEQ. ID.NO 1) （记为KTP1）；
GHGNTQTLAKAPEHTGC(SEQ. ID.NO 2)（记为KTP2）；
SHSSTARDLWPHGKEGC(SEQ. ID.NO 3)（记为KTP3）；
优选KTP1。
本发明中，所述荧光分子包括有机小分子染料与无机纳米颗粒，所述有机小分子选自：cy3、cy5、FITC、AlexaFluor、DyLight、ICG、CX2、FD1080、LZ105；无机纳米颗粒选自金纳米颗粒GNP、量子点QD、康奈尔点CD、稀土纳米颗粒RENP。
本发明所提出的靶向肾脏的荧光探针制备方法，包括多肽-有机小分子荧光探针的制备和多肽-无机纳米荧光探针的制备两个部分；其中：
（1）多肽-有机小分子荧光探针制备的具体步骤为：
将有机小分子染料溶于有机溶剂中，使用EDC/NHS进行活化，随后将反应液旋转蒸发干燥，再用柱色谱分离，得到活化后的小分子染料；将上述小分子染料溶于溶剂中，使用三乙胺调节溶液pH值，并加入多肽进行偶联反应，得最终产物；其中，溶剂选自氯仿、二氯甲烷、甲醇、乙酸乙酯、二甲基甲酰胺，二甲基亚砜、去离子水中的一种或几种；小分子染料与EDC/NHS、多肽的反应摩尔比为(1-10) : (1-30)/(1-50) : 1；活化时间为5-40 h，活化温度为20-65摄氏度；调节溶液pH值范围为7-12；与多肽偶联时间为3-48 h，偶联反应温度为20-95摄氏度。
上述反应中，优选的技术方案为，有机染料分子的浓度为100 µM-5 mM，溶剂为二氯甲烷或甲醇，EDC/NHS的浓度为300 µM-5 mM/500 µM-10 mM，活化温度为20-40摄氏度，活化时间为12-24 h，所述的多肽的摩尔浓度为1 µM-2 mM，与多肽偶连反应时间为6-12 h，反应pH值为7-9。
本发明中，所得多肽-有机小分子荧光探针产物生成后，使用真空旋转干燥除去溶剂，并用甲醇或二氯甲烷重新分散干燥产物，弃上清溶液得沉淀产物，用甲醇或二氯甲烷重复清洗所得沉淀并干燥，得最终肾脏靶向探针。
（2）多肽-无机纳米荧光探针制备的具体步骤为：
将无机纳米颗粒溶于溶剂中，加入多肽进行配位反应，即得最终产物；其中，所述溶剂选自氯仿、二氯甲烷、甲醇、乙醇、二甲基甲酰胺，二甲基亚砜、去离子水中的一种或几种；无机纳米颗粒与多肽的反应质量浓度比为(1-10) : 1；反应时间为3-48 h，反应温度为20-95摄氏度。
上述反应中，为了保证肾脏靶向探针的合成，优选的技术方案为，所述的无机纳米材料的质量浓度为5-20 mg/mL，溶剂为去离子水或二甲基亚砜，所述多肽的质量浓度为2-10 mg/mL。反应时间为12-18 h。
本发明中，所得多肽-无机纳米荧光探针产物生成后，使用葡聚糖凝胶色谱柱分离并干燥，得最终肾脏靶向纳米探针（固体粉末）。
本发明中，所述肾脏靶向探针，对肾脏靶向特异性高，70 %以上注射剂量的探针在肾脏富集，所述肾脏靶向探针在肾脏中靶向停留时间大于48 h，可以用于制备特异性肾脏靶向荧光成像剂。
本发明中，所述肾脏靶向荧光成像剂，是将肾靶向探针（固体粉末）溶于去离子水或生理盐水中，浓度为0.5-5 mg/mL。
本发明中，所述肾脏靶向荧光成像剂，通过小鼠静脉注射进行肾脏活体成像；具体操作步骤为：使用胰岛素针取100-300 µL，通过尾静脉注射到小鼠体内；在荧光成像装置下，5-10 min后可观察到荧光探针在小鼠肾脏部位聚集，在肾脏中靶向停留时间超过48 h。
附图说明
图1为肾脏靶向探针ICG-KTP1的吸收和发射光谱图。
图2为金纳米颗粒GNP、量子点QD及稀土纳米颗粒RENP的透射电镜图。
图3为肾脏靶向探针GNP-KTP1注射后不同时间点在小鼠体内的生物分布。
图4肾脏靶向探针ICG-KYP1注射10 min后对小鼠肾脏成像图。
图5肾脏靶向探针ICG-KYP1注射48 h后对小鼠肾脏成像图。
图6为肾脏靶向探针GNP-KYP1注射48 h后对小鼠肾脏成像图。
具体实施方式
实施例1
称取ICG-COOH 20 mg置于圆底烧瓶，加入6 mL二氯甲烷溶解，随后加入15 mg EDC和15 mg NHS，上述反应液置于室温中避光搅拌24 h。随后使用真空旋转蒸发干燥出去反应溶剂，得活化后的ICG混合物。上述混合物使用硅胶柱色谱分离，得活化后的ICG纯产物。称取活化后的上述产物3 mg溶解于3 mL二甲基甲酰胺中，加入10 mg三乙胺调节溶液pH值为8-9，随后加入7 mg KTP1，上述反应避光置于室温搅拌12 h，得ICG-KTP1肾脏靶向探针混合物。使用真空旋转蒸发干燥去除溶剂二甲基甲酰胺，所得固体粉末用二氯甲烷重新分散，得悬浊液，弃上清取沉淀物继续用二氯甲烷清洗3次，干燥后的肾脏靶向探针ICG-KTP1产物粉末。
实施例2
称取FD1080 10 mg置于圆底烧瓶，加入4 mL二氯甲烷溶解，随后加入5 mg EDC和5mg NHS，上述反应液置于室温中避光搅拌12 h。随后使用真空旋转蒸发干燥出去反应溶剂，得活化后的FD1080混合物。上述混合物使用硅胶柱色谱分离，得活化后的FD1080纯产物。称取活化后的上述产物2 mg溶解于2 mL甲醇中，加入10 mg三乙胺调节溶液pH值为8-9，随后加入5 mg KTP2，上述反应避光置于室温搅拌12 h，得FD1080-KTP2肾脏靶向探针混合物。使用真空旋转蒸发干燥去除溶剂甲醇，所得固体粉末用乙酸乙酯重新分散，得悬浊液，弃上清取沉淀物继续用乙酸乙酯清洗3次，干燥后的肾脏靶向探针FD1080-KTP2产物粉末。
实施例3
称取金纳米颗粒GNP (粒径2.5 nm，3 mg) 置于玻璃瓶中，加入5mL去离子水溶解，随后加入KTP1多肽5 mg，将上述反应瓶置于45摄氏度水浴锅中，搅拌反应18 h，得肾脏靶向多肽偶联的金纳米颗粒。将上述产物置于旋转蒸发干燥器中除去水，得GNP-KTP1荧光探针粉末。使用葡聚糖凝胶G25色谱柱对上述产物进行分离，得纯化后的肾脏靶向探针GNP-KTP1。
应用例
1、使用肾脏靶向探针ICG-KTP1对小鼠肾脏进行近红外二区成像。具体步骤如下：
取实施例1中所得ICG-KTP1探针1 mg溶解于1 mL生理盐水中，通过静脉注射200 μL上述溶液于麻醉小鼠体内，用808 nm外置激光器照射小鼠背部，激光器功率密度为120mW/cm2，1200 nm长通滤光片下可进行小鼠肾脏成像(参见图4-5)；
2、使用肾脏靶向纳米探针GNP-KTP1对小鼠肾脏进行成像。具体步骤如下：
取实施例3中所得GNP-KTP1探针1 mg溶解于1 mL生理盐水中，通过静脉注射200 μL上述溶液于麻醉小鼠体内，用655 nm外置激光器照射小鼠背部，激光器功率密度为200mW/cm2，850 nm长通滤光片下可进行小鼠肾脏成像(参见图6)。
序列表
<110> 复旦大学
<120> 靶向肾脏的近红外荧光探针及其制备方法和应用
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Claims (3)
1.一种近红外发光探针在制备特异性肾脏靶向荧光成像剂中的应用，该探针由肾脏靶向多肽与发光材料共价连接组成，其中多肽氨基酸侧链的残基与荧光分子形成化学键合；所述肾脏靶向多肽序列为：SHSNTQTLAKAPEHTGC；其中，所述荧光分子包括有机小分子染料与无机纳米颗粒，所述有机小分子选自：cy3、cy5、FITC、AlexaFluor、DyLight、ICG、CX2、FD1080、LZ105；所述无机纳米颗粒选自金纳米颗粒GNP、量子点QD、康奈尔点CD、稀土纳米颗粒RENP；
所述近红外发光探针的制备方法包括多肽-有机小分子荧光探针的制备和多肽-无机纳米荧光探针的制备两个部分；其特征在于：
（1）多肽-有机小分子荧光探针制备的具体步骤如下：
将有机小分子染料溶于有机溶剂中，使用EDC/NHS进行活化，随后将反应液旋转蒸发干燥，再用柱色谱分离，得到活化后的有机小分子染料；
将上述活化后的有机小分子染料溶于溶剂中，使用三乙胺调节溶液pH值，并加入多肽，进行偶联反应，即得最终产物；其中，所述溶剂选自氯仿、二氯甲烷、甲醇、乙酸乙酯、二甲基甲酰胺，二甲基亚砜、去离子水中的一种或几种；所述有机小分子染料与EDC/NHS、多肽的反应摩尔比为(1-10) : (1-30)/(1-50) : 1；活化时间为5-40 h，活化温度为20-65摄氏度；调节溶液pH值范围为7-12；与多肽偶联时间为3-48 h，偶联反应温度为20-95摄氏度；
（2）多肽-无机纳米荧光探针制备的具体步骤为：
将无机纳米颗粒溶于溶剂中，加入多肽进行配位反应，即得最终产物；其中，所述溶剂选自氯仿、二氯甲烷、甲醇、乙醇、二甲基甲酰胺，二甲基亚砜、去离子水中的一种或几种；无机纳米颗粒与多肽的反应质量浓度比为(1-10) : 1；反应时间为3-48 h，反应温度为20-95摄氏度。
2. 根据权利要求1所述的应用，是将肾脏靶向荧光成像剂溶于去离子水或生理盐水中，浓度为0.5-5 mg/mL。
3.根据权利要求1所述的应用，所述肾脏靶向荧光成像剂，通过小鼠静脉注射进行肾脏活体成像。
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