CN112021558B - 一种自组装Cu/Zn-SOD纳米颗粒及其应用 - Google Patents
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Abstract
本发明公开了一种自组装Cu/Zn‑SOD纳米颗粒及其应用,通过单纯Cu/Zn‑SOD的受热自组装,并通过微滤、超滤分离得到的这种纳米颗粒,其比活力高于天然Cu/Zn‑SOD,含有Cu/Zn‑SOD受热变性聚集形成的纳米颗粒载体、以及结合在载体上未变性的Cu/Zn‑SOD分子。本发明涉及的Cu/Zn‑SOD纳米颗粒的制备与分离过程通过加热、微滤、超滤的方式,不涉及其他任何物质,因此安全性高,是绿色、无污染的纳米颗粒制备方案;分离制备得到的Cu/Zn‑SOD纳米颗粒的抗氧化性优于天然Cu/Zn‑SOD。
Description
技术领域
本发明涉及保健食品领域中的蛋白质剂型和制剂技术,具体涉及到一种自组装Cu/Zn-SOD纳米颗粒及其应用。
背景技术
超氧化物歧化酶(Superoxide dismutase,SOD)是生物抗氧化剂系统的第一道防线,能够特异性地清除机体内的超氧阴离子自由基。SOD在氧化应激引起的各种疾病的治疗中发挥着重要的作用,如糖尿病及其并发症、炎症性疾病、癌症以及缺血再灌注等。天然的SOD由于半衰期短、分子量大导致不易透过细胞膜、易被酶解失活等缺点,导致其在应用中受到限制。
将SOD与纳米技术结合能够改善天然SOD的应用局限性,提高其在机体内的生物利用度,这是由于纳米颗粒具有较大的比表面积、渗透率高以及具有缓释作用的优点。蛋白纳米颗粒的制备主要有四种:超声化学法、乳化法、超临界流体法以及自组装法。目前,研究人员主要将有机溶剂或交联剂与SOD混合,或将SOD与脂质体结合制备纳米SOD。但上述方法大多用到乙醚等有机试剂,对机体不可避免会有轻微毒害作用。此外,上述方法制备纳米SOD的制备工艺较复杂,以及需要制备后纯化的工序。
自组装法制备纳米SOD的研究目前较少,大多通过壳聚糖等糖类物质对SOD修饰或逐层自组装。本文采用的纳米颗粒是通过加热使得单纯Cu/Zn-SOD蛋白发生自组装形成纳米颗粒初制品;初制品通过0.45 μm的PES滤膜的微滤后,滤液再加入到100KDa超滤膜中,在5000 g的条件下超滤离心15 min,然后从截留液中得到Cu/Zn-SOD纳米颗粒,其比活力较天然的Cu/Zn-SOD高76.7%。
发明内容
本发明的目的在于提供一种具有比活力高于天然Cu/Zn-SOD分子的Cu/Zn-SOD纳米颗粒及其应用。本发明涉及的Cu/Zn-SOD纳米颗粒的制备与分离过程通过加热、微滤、超滤的方式,不涉及其他任何物质;它包括变性的Cu/Zn-SOD聚集形成的纳米载体,以及结合在载体上的活性Cu/Zn-SOD分子,这种结构的Cu/Zn-SOD纳米颗粒未见报道;而且这种纳米颗粒虽然经过加热、超滤等过程,但是其比活力没有降低、反而高于天然SOD。这是基于酶学专业知识预测不到的。另外,这种纳米颗粒是通过特殊的超滤处理才能得到,它的抗氧化性优于天然Cu/Zn-SOD。
为实现上述目的,本发明可以采用如下技术方案:
所述的Cu/Zn-SOD纳米颗粒比活力高于天然Cu/Zn-SOD,含有Cu/Zn-SOD受热变性聚集形成的纳米颗粒载体、以及结合在载体上未变性的Cu/Zn-SOD分子。
Cu/Zn-SOD纳米颗粒的制备方法如下:
纳米颗粒的初制品的制备方法为取Cu/Zn-SOD粉剂于锥形瓶中,加入蒸馏水使之浓度为1 mg/mL,完全溶解后置于60℃水浴预热60 min,再置于75℃水浴保温60 min,即得到Cu/Zn-SOD纳米颗粒的初制品。所述颗粒粒径范围为141.8—458.7 nm,平均粒径为247.3nm;Zeta电位为-19.5 mV。
初制品通过0.45 μm的PES滤膜滤掉其中的絮凝蛋白后,加入到100KDa超滤膜中,在5000 g的条件下离心15 min,所得截留液即为纯Cu/Zn-SOD纳米颗粒。所得纳米颗粒的比活力为66225.7 U/mg,较天然SOD高76.7%。所述颗粒平均粒径为275.2±10.3 nm,PdI为0.226,Zeta电位为-9.27±1.53 mV。
初制品通过0.45 μm的PES滤膜滤掉其中的絮凝蛋白后,用Sephacryl S-100凝胶过滤柱分离探讨Cu/Zn-SOD纳米颗粒的组成形式,得出Cu/Zn-SOD纳米颗粒由无酶活力的SOD纳米聚集体和有酶活力的SOD分子组成。
本发明的优点在于:本发明涉及的Cu/Zn-SOD纳米颗粒的制备与分离过程通过加热、超滤的方式,不涉及其他任何添加剂或交联剂,因此安全性高,是绿色、无污染的纳米颗粒制备方法;分离制备得到的Cu/Zn-SOD纳米颗粒的抗氧化性优于天然SOD。因此可望用于由氧化应激引起的各种疾病的治疗。
附图说明
图1 Cu/Zn-SOD纳米颗粒及其初制品的粒径分布图。
图2 Cu/Zn-SOD纳米颗粒初制品在不同pH体系中的酶活力变化图。
图3 Cu/Zn-SOD纳米颗粒初制品在不同pH体系中的粒径变化图。
图4 Cu/Zn-SOD纳米颗粒初制品在不同pH体系中的电位变化图。
图5 Cu/Zn-SOD纳米颗粒初制品的Sephacryl S-100凝胶过滤层析图。
图6 凝胶层析峰值所在样品的粒径分布图。
图7 Cu/Zn-SOD纳米颗粒及相关样品的全波长扫描图。
图8 Cu/Zn-SOD纳米颗粒对百草枯刺激的L929细胞存活率的影响(**为各组与空白组相比,p<0.01;#为同浓度间组间的比较,p<0.05;n=4。)。
图9 Cu/Zn-SOD纳米颗粒初制品对百草枯刺激的L929细胞存活率的影响(*为各组与空白组比较,p<0.05;#为同浓度之间的比较,p<0.05;n=4。)。
图10 Cu/Zn-SOD纳米颗粒初制品凝胶层析样品对百草枯刺激的L929细胞存活率的影响(*为各组与空白组比较,p<0.05;#为同浓度之间的比较,p<0.05;n=4。)。
具体实施方式
实施例1:Cu/Zn-SOD纳米颗粒的制备
称取Cu/Zn-SOD(以下简称SOD)粉末16 mg于锥形瓶中,加入16 mL蒸馏水,制备成1mg/mL的SOD溶液,溶解后于60℃水浴预热60 min,再置于75℃水浴保温60 min,制备得到初制品溶液。用马尔文激光粒度仪测定其粒径和Zeta电位,测得其平均粒径为247.3±1.9nm,聚合物分散性指数(Polymer dispersity index ,PdI)为0.019,Zeta电位为-19.5±0.3。
将上述初制品通过0.45 μm的PES滤膜滤掉其中的絮凝蛋白后,加入到100 KDa超滤膜中,在5000 g的条件下离心15 min,所得截留液即为Cu/Zn-SOD纳米颗粒。所得纳米颗粒的比活力为66225.7 U/mg,较天然SOD高76.7%,平均粒径为275.2±10.3 nm,PdI为0.226,Zeta电位为-9.27±1.53 mV。
Cu/Zn-SOD纳米颗粒及其初制品的粒径分布图见附图1。
实施例2:Cu/Zn-SOD纳米颗粒的pH稳定性
分别用0.1 M的HCl与0.02 M的NaOH将Cu/Zn-SOD纳米颗粒初制品的pH调至1-12,静置60 min后,过0.45 μm滤膜。通过马尔文激光粒度仪测定粒径的变化,再将纳米颗粒溶液pH调至7,用盐酸羟胺法测定酶活。Cu/Zn-SOD纳米颗粒的酶活力变化图见附图2,粒径变化图见附图3,Zeta电位变化见附图4。
结果显示Cu/Zn-SOD纳米颗粒在pH 4-11的范围内,酶活力没有损失,粒径也无变化,在强酸环境中,酶活力保留率较低,但是仍保持纳米结构,在强碱环境中,酶活力保留率低且纳米结构遭到破坏。
实施例3:Cu/Zn-SOD纳米颗粒的结构分析
上述制备得到的初制品通过0.45 μm的PES滤膜滤掉其中的絮凝蛋白后,用Sephacryl S-100凝胶过滤柱(1×60cm)分离。以0.2 mL/min流速分离Cu/Zn-SOD纳米颗粒,每5 min收集一管样品。所得收集液测定OD258,凝胶过滤色谱图见附图5;两个色谱峰的馏分粒径分布图见附图6,全波长扫描结果如附图7。
图5显示,Cu/Zn-SOD纳米颗粒初制品中包括峰1和峰2两种成分;将两种成分分别收集、测定粒径分布。如图6的结果显示,峰1为纳米形态的颗粒,峰2为与天然SOD粒径相同的物质,因此确定峰1为纳米颗粒,峰2为SOD;测定峰1(纳米颗粒)的比活力为353.2 U/mg,峰2(SOD分子)的比活力为34619.3 U/mg(与天然SOD比活力相似)。因此确定自组装Cu/Zn-SOD纳米颗粒中含有SOD受热变性聚集形成的纳米颗粒载体、以及结合在载体上未变性的SOD分子。
实施例4:超滤法分离纯化Cu/Zn-SOD纳米颗粒
采用100KDa的超滤管,首先用一级水进行清洗;样品在超滤前需要过0.45 µm的滤膜,除去制备过程中变性絮凝的蛋白;按照说明书,上样量为5mL;5000xg下离心15 min;取出滤液和截留液;出于对滤膜上可能粘附蛋白的考虑,用800 µL pH7.8 酶活力测定用的PBS溶液冲洗滤膜,最后得到滤液与截留液。滤液与截留液的二级结构、酶活、粒径、电位和蛋白含量进行了测定。
本实验经过筛选,排除了截留分子量为300KDa等的超滤管,最后采用截留分子量为100KDa的超滤管进行超滤。超滤的滤液中存在的天然Cu/Zn-SOD(分子量为32KDa),截留液中得到了Cu/Zn-SOD纳米颗粒。超滤的过程中蛋白质基本无损失,酶活力损失5.4%,Cu/Zn-SOD纳米颗粒的产率为21.3%。截留液与滤液的比活分别为66225.7 U/mg与53443.5 U/mg,即截留液(含Cu/Zn-SOD纳米颗粒)的比活力比滤液高12782.2U/mg(23.9%),比天然的SOD溶液高28747.2 U/mg(76.7%)。经过超滤的分离后,截留液与滤液的PdI都有略微增大,但两者体系都处于均一状态;二者的平均粒径分别为275.2nm和5.1nm。
实施例5:Cu/Zn-SOD纳米颗粒在氧化应激损伤修复中的作用
以L929成纤维细胞为模型,通过6 mmol/L的百草枯溶液刺激细胞20 h后用天然Cu/Zn-SOD与Cu/Zn-SOD纳米颗粒初制品、超滤分离得到的Cu/Zn-SOD纳米颗粒以及凝胶层析收集样品进行氧化应激损伤修复,通过CCK-8测定细胞的存活率,探究不同样品在不同浓度下对L929细胞氧化应激的修复作用。Cu/Zn-SOD纳米颗粒对细胞氧化应激的修复作用见附图8-10。
结果显示,在酶浓度为200 U/mL内,Cu/Zn-SOD纳米颗粒及其初制品均对百草枯刺激引起的氧化应激的修复作用优于Cu/Zn-SOD(p<0.05)。凝胶层析收集样品中,纳米载体基本无作用,而纳米载体与SOD混合后具有比天然SOD更好的修复作用(p<0.05)。
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 (2)
1.一种自组装Cu/Zn-SOD纳米颗粒,其特征在于:其是取Cu/Zn-SOD粉剂溶解于蒸馏水中,然后通过加热使得单纯Cu/Zn-SOD蛋白发生自组装形成纳米颗粒;再通过0.45μm PES滤膜的微滤,得到的滤液再加入到100KDa超滤膜中,在5000g的条件下超滤离心15min后,从截留液中得到Cu/Zn-SOD纳米颗粒;所述颗粒平均粒径为275.2±10.3 nm,PdI为0.226,Zeta电位为-9.27±1.53mV;
所述加热的工艺条件为:先置于60℃水浴预热60min,再置于75℃水浴保温60min;
所述自组装Cu/Zn-SOD纳米颗粒,其部分组成物质是Cu/Zn-SOD受热变性聚集形成无活性的纳米颗粒载体,其余部分是结合在载体上未变性的Cu/Zn-SOD分子,从而形成具有高比活力的Cu/Zn-SOD纳米颗粒。
2.权利要求1所述的自组装Cu/Zn-SOD纳米颗粒在制备去除超氧阴离子自由基的功能食品或者药剂中的应用。
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