CN108354912B - 一种具Aβ蛋白抑制活性的EGCG-Fe/PVP纳米球及其制备方法和应用 - Google Patents
一种具Aβ蛋白抑制活性的EGCG-Fe/PVP纳米球及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种具Aβ蛋白抑制活性的EGCG‑Fe/PVP纳米球及其制备方法和应用，属于纳米材料制备和生物医学领域。所述EGCG‑Fe/PVP纳米球由表没食子儿茶素没食子酸酯（EGCG）和Fe3+及表面活性剂PVP按一定配合比自组装组成，其粒径为2‑6nm，电位为‑10mV。本发明制备的EGCG‑Fe/PVP纳米球具有粒径小、易清除、热稳定性高、生物相亲性好等优点，且在低浓度时即对Aβ蛋白有显著抑制效果，可作为治疗阿尔兹海默病的一种潜在药物。
Description
技术领域
本发明属于纳米材料制备和生物医学领域，具体涉及一种具Aβ蛋白抑制活性的EGCG-Fe/PVP纳米球及其制备方法与其在抑制淀粉样蛋白聚集中的应用。
背景技术
阿尔茨海默病（Alzheimer disease，AD），又叫老年性痴呆，是一种中枢神经系统变性病，起病隐袭，病程呈慢性进行性，是老年期痴呆最常见的一种类型，主要表现为渐进性记忆障碍、认知功能障碍、人格改变及语言障碍等神经精神症状，严重影响社交、职业与生活功能。AD的病因及发病机制尚未阐明，特征性病理改变为：β淀粉样蛋白沉积形成的细胞外老年斑和tau蛋白过度磷酸化形成的神经细胞内神经原纤维缠结，以及神经元丢失伴胶质细胞增生等。
大量研究表明Aβ在AD发病中起着主导作用。目前针对Aβ的神经毒作用的防治措施主要有以下几方面：减少Aβ的前体即β淀粉样前体蛋白的量，调节APP的裂解，减少Aβ的产生；促进Aβ的降解和清除；防止Aβ的聚集和沉积；阻断Aβ作用机制的关键环节等。
表没食子儿茶素没食子酸酯（EGCG）具有抗氧化性、清除体内自由基、抗辐射和紫外线、预防心脑血管疾病、调整脂糖代谢、治疗糖尿病、降血脂、抗癌、抗炎、抗突变、抗衰老及改善肝功能等生物活性。此外，EGCG还常被用来防止神经退行性疾病，如AD、PD、HD以及肌萎缩性侧索硬化症(amyotrophic lateral sclerosis，ALS)等。
虽然EGCG具有抗氧化、抗衰老、预防神经退行性疾病等特性，但是自由的EGCG分子易被氧化、热稳定性差，导致其生物利用度很低。本发明利用EGCG易与金属离子配位的特性，对其进行修饰，发展出一种粒径小、易被体内清除、热稳定性高且对Aβ蛋白的抑制治疗效果显著的EGCG-Fe/PVP纳米球，该项目获福建省自然基金（2017J01040）、国家自然基金（21271044）资助支持。
发明内容
本发明的目的在于提供一种具Aβ蛋白抑制活性的EGCG-Fe/PVP纳米球及其制备方法和应用，该EGCG-Fe-PVP纳米球在低浓度时对淀粉样蛋白Aβ的抑制率高达百分之三十，且其具有粒径小、易被体内清除、热稳定性高等优点，可作为一种治疗阿尔兹海默病的潜在药物。
为实现上述目的，本发明采用如下技术方案：
一种具Aβ蛋白抑制活性的EGCG-Fe/PVP纳米球，其是由表没食子儿茶素没食子酸酯（EGCG）、Fe3+及表面活性剂PVP自组装而成，其制备方法包括以下步骤：
1）将FeCl3分散至水中，然后加入PVP，在室温、450 rpm条件下混合搅拌1小时；
2）将EGCG溶解在乙醇中，再将其加入到步骤1）所得混合溶液中，搅拌反应过夜，产物经透析处理，得到EGCG-Fe/PVP纳米球。
所用PVP的分子量为10KD。
所用FeCl3与PVP的投料质量比为1:3.3~1:6，优选为1:5。
所用FeCl3与EGCG的投料质量比为1:0.13~1:0.2，优选为1:0.17。
所得具Aβ蛋白抑制活性的EGCG-Fe/PVP纳米球的粒径为2-6 nm，在最优质量比条件下，其粒径为3.2±1.2 nm，电位为-10 mV，其可作为淀粉样蛋白抑制剂，用于抑制淀粉样蛋白Aβ的聚集。
EGCG存在较多的酚羟基，可以与金属离子Fe3+通过强配位作用得到金属-多酚纳米球EGCG-Fe/PVP。
本发明的显著优点在于：
（1）本发明所得EGCG-Fe/PVP纳米球粒径小、稳定性高、生物相容性好，并易被体内清除；
（2）本发明首次将多酚与金属离子组装形成EGCG-Fe/PVP纳米球，并将其应用于淀粉样蛋白聚集的抑制。本发明所得EGCG-Fe/PVP纳米球在低浓度（2μg/mL）时，对Aβ蛋白的抑制率高达30%，可有效降低因淀粉样蛋白聚集所引起的毒性。
附图说明
图1为实施例1所制备EGCG-Fe/PVP纳米球的Zeta电位仪粒径图。
图2为实施例1所制备EGCG-Fe/PVP纳米球的TEM图（a）及粒径分布图（b）。
图3为实施例1所制备EGCG-Fe/PVP纳米球放置不同时间的光学照片。
图4为实施例1所制备EGCG-Fe/PVP纳米球在不同浓度下的生物相亲性情况图。
图5为淀粉样蛋白Aβ与不同浓度EGCG-Fe/PVP纳米球一起孵育不同时间所测得的荧光结果图。
图6为淀粉样蛋白Aβ与不同浓度EGCG-Fe/PVP一起孵育48h后的TEM图，其中a为Aβ（20μM）+0μg/mL EGCG-Fe/PVP，b为Aβ（20μM）+2μg/mL EGCG-Fe/PVP，c为Aβ（20μM）+50μg/mLEGCG-Fe/PVP；
图7为淀粉样蛋白Aβ与不同浓度EGCG-Fe/PVP一起孵育48h后的AFM图，其中a为Aβ（20μM）+0μg/mL EGCG-Fe/PVP，b为Aβ（20μM）+2μg/mL EGCG-Fe/PVP，c为Aβ（20μM）+10μg/mLEGCG-Fe/PVP，d为Aβ（20μM）+50μg/mL EGCG-Fe/PVP。
具体实施方式
为了使本发明所述的内容更加便于理解，下面结合具体实施方式对本发明所述的技术方案做进一步的说明，但是本发明不仅限于此。
实施例1
1）将FeCl3分散到去离子水中，配成浓度为100 mg/mL的FeCl3溶液，将PVP（Mw=10kD）分散在去离子水中，配成浓度为10 mg/mL的PVP溶液；
2）在搅拌条件下，在200μL FeCl3溶液中加入10 mL PVP溶液，然后将此混合液在室温、450 rpm转速条件下搅拌1小时；
3）向上述混合液中逐滴加入1 mL含3.36 mg EGCG的乙醇溶液，搅拌反应过夜，经过24 h透析，即得EGCG-Fe/PVP纳米球。
1. 将所得EGCG-Fe/PVP纳米球加入到去离子中，配制成浓度为1 mg/mL的溶液，测其粒径，结果如图1所示。由图1可见，EGCG-Fe/PVP纳米球的光学粒径约为10 nm。
2. 将所得EGCG-Fe/PVP纳米球加入到去离子中，配制成浓度为1 mg/mL的溶液，然后滴加到铜网上，待其干燥后，进行电镜扫描，结果如图2所示。由图2可见，透射电镜下EGCG-Fe/PVP纳米球的粒径为3.2±1.2 nm。
3. 将所得EGCG-Fe/PVP加入到去离子中，配制成浓度为1 mg/mL的溶液，混匀，然后分别加入到7个1.5mL的EP管中，分别放置1天、3天、5天、7天、15天、20天、30天，观察EGCG-Fe/PVP的稳定性，结果如图3所示。由图3可见，所得EGCG-Fe/PVP纳米球的稳定性良好，30天内没发生团聚现象；
4. 为了考察EGCG-Fe/PVP纳米球的生物相亲性情况，以EGCG-Fe/PVP纳米球对HeLa细胞的毒性实验为根据。
待HeLa细胞生长至90%左右进行细胞传代与接种操作，其步骤如下：先将旧培养基去掉，然后加入2mL PBS溶液轻柔清洗，之后加入1mL胰蛋白酶消化细胞1min左右，去除胰酶，加入2mL RPMI-1640培养基停止消化；轻轻吹打细胞制备成细胞原液，取1/4的细胞原液分装入新的培养瓶中，置于37℃培养箱中继续培养。
将HeLa细胞原液用培养液稀释并分散成HeLa细胞悬液，其密度为105个/mL，并以每孔104个细胞接种至96孔板中，置于37℃，5% CO2的培养箱中培养24h后，移去培养液，加入浓度依次为0、2、10、50、100、200μg/mL的EGCG-Fe/PVP纳米球培养液，每个浓度设置4个重复孔。培养6h后，清洗2次，加入100μL新鲜培养液继续培养19h后，每孔加入10μL浓度为5mg/mL的MTT，孵育4~6h，小心移去培养液，加入150μL DMSO，37℃培养15min；测490nm处的吸收值，计算存活率，以评价EGCG-Fe/PVP纳米球的生物相亲性，结果如图4所示。由图4可见，即使EGCG-Fe/PVP纳米球在浓度为200μg/mL时，细胞存活率也在80%以上，说明EGCG-Fe/PVP纳米球的生物相亲性好。
应用实施例1
a）将淀粉样蛋白Aβ进行预处理，即将蛋白先用高极性溶剂六氟异丙醇（HFIP）溶解，其终浓度为1mg/mL，然后在4℃、450rpm下搅拌2小时，然后经冷冻干燥后，用pH=7.410mM PBS和去离子水重分散，得到浓度为0.25mM的淀粉样蛋白储备液；
b）将实施例1制得的EGCG-Fe/PVP纳米球与步骤a）制备的淀粉样蛋白储备液按不同比例混合，使所得混合液中蛋白浓度为20μM，纳米球浓度分别为0μg/mL、2μg/mL、10μg/mL、50μg/mL；然后将其分别加入到96孔板中，每孔加入100μL，于振荡器中37 ℃、150 rpm进行振荡。
1. 于不同时间分别测定混合溶液中蛋白的荧光（测荧光前，加入100μL 100μMThT，10min后，将96孔板置于酶标仪中进行荧光测定），并记录数据；
根据所测得的荧光值计算出EGCG-Fe/PVP纳米球对蛋白的抑制率，结果如图5所示。
由图5可见，含有EGCG-Fe/PVP纳米球的蛋白组的荧光值比不含EGCG-Fe/PVP纳米球的蛋白组的荧光弱，说明EGCG-Fe/PVP纳米球对蛋白的聚集有一定的抑制效果；且随着EGCG-Fe/PVP纳米球浓度的增高，抑制效果更强；浓度为2μg/mL、10μg/mL、50μg/mL EGCG-Fe/PVP纳米球的对Aβ的抑制率分别为29.4%、33%、35.3%；
2. 两天后，将所得混合溶液滴加到铜网上，静置数分钟，然后用滤纸从铜网边缘吸去多余的液体，滴上负染色液，染色1~2分钟用滤纸吸去负染色液，再用蒸馏水滴在铜网上洗1~2次，待其干燥后进行电镜扫描，结果如图6所示。
由图6可见，不含EGCG-Fe/PVP纳米球的Aβ易聚集形成纤维蛋白，而含有EGCG-Fe/PVP纳米球的蛋白Aβ仅部分聚集形成纤维蛋白，说明EGCG-Fe/PVP纳米球对Aβ的聚集具有抑制效果。
3. 两天后，将所得混合溶液进行超滤处理，然后取超滤上清20μL滴加到云母片上，待其干燥后用去离子水清洗1~2次，干燥后进行原子力显微镜（AFM）测试，结果如图7所示。
由图7可见，不含EGCG-Fe/PVP纳米球的Aβ聚集形成大量纤维蛋白，而含有EGCG-Fe/PVP纳米球的蛋白Aβ仅有部分聚集形成纤维蛋白，且随着EGCG-Fe/PVP纳米球浓度的增加，大部分Aβ蛋白只形成小颗粒而不是纤维，这进一步说明EGCG-Fe/PVP纳米球对Aβ的聚集具有抑制效果。
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。
Claims (3)
1.一种EGCG-Fe/PVP纳米球在制备淀粉样蛋白抑制剂中的应用，其特征在于：所述EGCG-Fe/PVP纳米球的制备方法包括以下步骤：
1）将FeCl3分散至水中，然后加入PVP混合搅拌1小时；
2）将EGCG溶解在乙醇中，再将其加入到步骤1）所得混合溶液中，搅拌反应过夜，产物经透析处理，得到EGCG-Fe/PVP纳米球；
所得EGCG-Fe/PVP纳米球的粒径为2-6 nm，电位为-10mV。
2.根据权利要求1所述的EGCG-Fe/PVP纳米球在制备淀粉样蛋白抑制剂中的应用，其特征在于：步骤1）所用PVP的分子量为10KD，其与FeCl3的投料质量比为3.3:1~6:1。
3.根据权利要求1所述的EGCG-Fe/PVP纳米球在制备淀粉样蛋白抑制剂中的应用，其特征在于：步骤2）所用EGCG与FeCl3的投料质量比为0.13:1~0.20:1。
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