CN111773394A - 一种β-半乳糖苷酶荧光探针纳米微球及其制备方法和用途 - Google Patents
一种β-半乳糖苷酶荧光探针纳米微球及其制备方法和用途 Download PDFInfo
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Abstract
本发明属于生物检测领域，涉及一种β‑半乳糖苷酶荧光探针纳米微球及其制备方法和用途。尤其涉及一种用于活体检测动脉粥样硬化斑块的β‑半乳糖苷酶荧光探针纳米微球及其制备方法。本发明中，将新型β‑半乳糖苷酶荧光探针包裹在可生物降解材料PLGA中，通过乳剂扩散法的方法制成纳米微球；利用新型β‑半乳糖苷酶荧光探针对β‑半乳糖苷酶的识别作用，实现对动脉粥样硬化斑块中衰老VSMCs的识别，进而实现对动脉粥样硬化的活体检测，本发明的β‑半乳糖苷酶荧光探针微球用于进行动脉粥样硬化活体无毒无创成像检测，具有非常积极的临床意义。
Description
技术领域
本发明属于生物检测领域，涉及一种β-半乳糖苷酶荧光探针纳米微球及其制备方法和用途。尤其涉及一种用于活体检测动脉粥样硬化斑块的β-半乳糖苷酶荧光探针纳米微球及其制备方法。
背景技术
文献公开了心脑血管疾病仍然是人类健康的重要威胁之一，动脉粥样硬化是引发心脑血管疾病的主要诱因，临床实践对动脉粥样硬化的诊断和治疗已有了较成型的手段，但是在早期诊疗中还存在一定的不足，如何能够更早地发现血管内病变并在病患处准确的释放药物是目前研究的热点之一。
研究显示血管平滑肌细胞(VSMCs)是动脉粥样硬化(AS)斑块的重要组成部分，VSMCs细胞衰老促进了AS的发展和动脉粥样硬化斑块的不稳定性，动脉粥样硬化斑块是由脂质和衰老的VSMCs细胞沉积而成。
衰老细胞是指细胞在执行生命活动过程中，随着时间的推移，细胞增殖与分化能力和生理功能逐渐发生衰退的变化过程，研究表明，衰老细胞与多种老年性疾病如动脉粥样硬化、血管瘤、炎症疾病、肿瘤等有关；细胞在衰老状态下β-半乳糖苷酶会过表达，β-半乳糖苷酶可作为衡量细胞衰老状态的重要指标，因此对β-半乳糖苷酶的检测可在一定程度上反映生物体系的衰老状态，尤其对动脉硬化斑块中的衰老VSMCs细胞中过表达的β-半乳糖苷酶进行检测，可在一定程度上实现对动脉粥样硬化的检测。
近年来。荧光分析法具有高灵敏度、高选择性、高时空分辨率、操作简单并可实现对生物活性分子原位无损伤、实时快速的可视化成像等优点已成为本领域研究热点，然而，已报道的β-半乳糖苷酶荧光探针尚存在存在背景信号高，发射波长短，荧光穿透能力弱，易代谢无法到达作用部位，毒性大等缺点，以至于无法应用于活体检测动脉粥样硬化。
具有良好的生物相容性的可降解聚乳酸/乙醇酸
(polylactide-co-glycolide，PLGA)纳米微粒用于制备生物降解型缓释或定向给药体系已经研究了近30年，是国内外研究的热点。PLGA(聚乳酸/乙醇酸)目前已获美国FDA批准用作手术缝合线，心血管支架控释药物涂层，以及注射用微囊、微球、埋植剂等的材料。根据药物的性质、给药途径和释药时间，选用不同分子量、不同光学活性的乳酸共聚，不同种丙交酯和乙交酯的聚合比例，采用相应的制备工艺控制药物达到不同的释放模型；还可以通过PLGA(聚乳酸/乙醇酸)优选药物的输送系统，从而获得药物的新剂型。纳米粒能够使给药局部的药物浓度长时间维持在治疗水平，减少给药次数和用药量，从而在多个层面上防止或减轻毒副作用。聚乳酸水解的最终产物是水和二氧化碳，中问产物乳酸也是体内正常糖代谢产物，故该聚合物无毒、无刺激性，并具有很好的生物相溶性。
基于现有技术的基础，本申请的发明人拟利用PLGA(聚乳酸/乙醇酸)作为缓释材料的，提供一种用于活体检测动脉粥样硬化斑块的β-半乳糖苷酶荧光探针纳米微球及其制备方法，利用新型β-半乳糖苷酶荧光探针对动脉粥样硬化进行活体无毒无创成像的微球，具有非常积极的临床意义。
发明内容
基于现有技术的基础及存在的问题，本发明的第一个目的是提供一种基于β-半乳糖苷酶荧光探针的用以活体检测动脉粥样硬化斑块的微球。
本发明第二个目的提供所述微球的制备方法。
本发明第三个目的提供所述微球在用于制备对动脉粥样硬化，在体内，体外实现高选择性和高灵敏度的检测制剂中的用途。
本发明的目的是通过以下技术方案实现的：
将β-半乳糖苷酶荧光探针，包裹在可以生物降解的聚合物PLGA(聚乳酸/乙醇酸)内，并通过乳剂扩散法得到纳米微球。
本发明通过以下步骤制备可生物降解β-半乳糖苷酶荧光探针纳米微球：
第一步，将β-半乳糖苷酶荧光探针和PLGA(聚乳酸/乙醇酸)溶解在有机溶剂中得到探针-PLGA(聚乳酸/乙醇酸)有机溶液并对其进行超声乳化；
所述的β-半乳糖苷酶荧光探针，具有通式(1)结构：
第二步，将探针-PLGA(聚乳酸/乙醇酸)有机溶液加入到聚乙烯醇溶液中，经常温搅拌处理后，离心过滤水洗，获得可生物降解纳米微球；
所述的探针聚乳酸/乙醇酸PLGA的丙酮甲醇溶液中，探针浓度为0.005-0.03g/μL；
所述的PLGA的浓度为1-5g/μL，其中DL-LA∶GA的摩尔比为90∶10、80∶20、75∶25、60∶40或50∶50；
可生物降解纳米微球的制备方法中，所述的有机溶剂为：氯仿、甲醇、二氯甲烷、丙酮、乙醇或乙酸乙酯中的一种或其组合；
可生物降解纳米微球的制备方法中，所述超声乳化处理是指：设定超声功率4-100W进行超声分散1-5分钟后在700rpm/min条件下持续搅拌混合1小时；
所述的室温搅拌处理是指在室温环境下以300-1000rpm/min的转速搅拌4-16小时；
所述的离心过滤水洗是指离心速度在6000-24000rpm/min之间，离心时间5-30分钟，经过滤后采用去离子水洗涤3次；
第三步，将生物降解纳米微球冻干，获得微球冻干粉剂。
本发明提供了一种用于活体检测动脉粥样硬化斑块的β-半乳糖苷酶荧光探针纳米微球及其制备方法，其中，将新型β-半乳糖苷酶荧光探针包裹在可生物降解材料PLGA中，通过乳剂扩散法的方法制备成纳米微球；利用新型β-半乳糖苷酶荧光探针对β-半乳糖苷酶的识别作用，实现对动脉粥样硬化斑块中衰老VSMCs的识别，进而实现对动脉粥样硬化的活体检测，本发明中利用新型β-半乳糖苷酶荧光探针对动脉粥样硬化进行活体无毒无创成像的微球，具有非常积极的临床意义。
附图说明
图1限制了微球粒径图；
图2显示了微球电位图；
图3显示了微球投射电镜照片；
图4显示了微球尾静脉注射三天后，活体成像图；
图5显示了微球尾静脉注射一天，分离出主动脉器官荧光成像图。
具体实施方式
下面通过实施例和附图对本发明进行说明，但本发明的内容不受具体实施例的限制。
实施例1.
第一步，将β-半乳糖苷酶荧光探针20mg和200mg PLGA(DL-LA∶GA＝75∶25)溶解在甲醇和丙酮中得到探针-PLGA(聚乳酸/乙醇酸)有机溶液，并在40W功率超声辅助乳化30分钟；
第二步，在700r/min搅拌条件下，将探针-PLGA(聚乳酸/乙醇酸)有机溶液加入到200mL 1％聚乙烯醇溶液中，在经室温搅拌3小时后，甲醇和丙酮会充分分散在水中，乳滴变硬成球，再于21000rpm下离心10分钟，过滤、水洗，获得可生物降解纳米微球；
第三步，将生物降解纳米微球冻干，可获得微球冻干粉剂，密封于4℃保存。
本实施例制得到的基于β-半乳糖苷酶荧光探针的用以活体检测动脉粥样硬化斑块的微球，微球是由PLGA(聚乳酸/乙醇酸)包裹β-半乳糖苷酶荧光探针而成的，经激光粒度仪测量以及投射电镜成像(如图3所示)，其平均粒径在80-150nm(如图1所示)，平均Zeta电位为-28.9mV(如图2所示)，β-半乳糖苷酶荧光探针包封率在50-80％之间。
实施例2.
使用Ang II诱导Apoe敲除小鼠产生动脉粥样硬化，以C57背景正常鼠为对照，将微球配置成33.3mg/ml的溶液，小鼠尾静脉注射300μl微球溶液；
体内反应三天后，使用VISQUE InVivo Smart小动物活体中的Cy5.5模式，拍摄活体荧光，可见衰老组老鼠其主动脉周围出现荧光信号(如图4所示)；在体内反应一天时，活体成像荧光信号最强，此时将老鼠主动脉解剖分离，用Cy5.5模式拍摄荧光，结果显示衰老组老鼠主动脉有明显荧光，对照组主动脉无荧光(如图5所示)。
Claims (5)
1.一种将β-半乳糖苷酶荧光探针，包裹在聚合物聚乳酸/乙醇酸PLGA内，通过乳剂扩散法制得到纳米微球；
所述的β-半乳糖苷酶荧光探针，具有通式(1)结构：
2.权利要求1的β-半乳糖苷酶荧光探针纳米微球的制备方法，其特征在于，其包括步骤：
第一步，将所述的β-半乳糖苷酶荧光探针与聚乳酸/乙醇酸PLGA溶解在有机溶剂中制得探针聚乳酸/乙醇酸PLGA有机溶液，对其进行超声乳化；
第二步，将探针聚乳酸/乙醇酸PLGA有机溶液加入到聚乙烯醇溶液中，经常温搅拌处理后，离心过滤水洗，获得可生物降解纳米微球；
第三步，将制得的生物降解纳米微球冻干，获得微球冻干粉剂。
3.按权利要求2所述的方法，其特征在于，步骤一中，
所述的探针聚乳酸/乙醇酸PLGA的丙酮甲醇溶液中，探针浓度为0.005-0.03g/μL；
所述的聚乳酸/乙醇酸PLGA有机溶液的浓度为1-5g/μL，其中DL-LA∶GA的摩尔比为90∶10、80∶20、75∶25、60∶40或50∶50；
所述的有机溶剂为：氯仿、甲醇、二氯甲烷、丙酮、乙醇或乙酸乙酯中的一种或其组合；
所述超声乳化处理是：设定超声功率4-100W进行超声分散1-5分钟后在700rpm/min条件下持续搅拌混合1小时。
4.按权利要求2所述的方法，其特征在于，步骤二中，
所述的常温搅拌处理是，室温环境下以300-1000rpm/min的转速搅拌4-16小时；
所述的离心过滤水洗是，离心速度在6000-24000rpm/min之间，离心时间5-30分钟，经滤后采用去离子水洗涤3次。
5.权利要求1的β-半乳糖苷酶荧光探针纳米微球在制备用于体内、外检测动脉粥样硬化的检测制剂中的用途。
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