CN114468295B - 一种基于藻蓝蛋白尺寸可控的叶黄素纳米粒子 - Google Patents
一种基于藻蓝蛋白尺寸可控的叶黄素纳米粒子 Download PDFInfo
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- CN114468295B CN114468295B CN202210141949.8A CN202210141949A CN114468295B CN 114468295 B CN114468295 B CN 114468295B CN 202210141949 A CN202210141949 A CN 202210141949A CN 114468295 B CN114468295 B CN 114468295B
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- phycocyanin
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- lutein
- procyanidine
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Abstract
本发明公开了一种基于藻蓝蛋白尺寸可控的叶黄素纳米粒子,属于纳米技术领域。本发明以食品级藻蓝蛋白、原花青素和香兰素为原料,基于曼尼希反应制备纳米载体,通过控制藻蓝蛋白、原花青素和香兰素单一浓度变量,获得具有不同粒径的纳米载体。该载体在清除游离自由基方面表现出强抗氧化性能;此外,该纳米载体安全无毒,对丙烯酰胺和过氧化氢引起的细胞氧化应激损伤具有改善作用;采用该载体负载叶黄素,制备叶黄素纳米粒子,装载率为74.42%,且能显著促进细胞增殖。本发明所使用原料为食品级,制备条件快速温和,易于规模化生产。
Description
技术领域
本发明涉及一种基于藻蓝蛋白尺寸可控的叶黄素纳米粒子,属于纳米技术领域。
背景技术
纳米载体的设计为克服功能因子的低溶解性、稳定性和生物利用度提供了可能。一个理想的纳米载体应具有良好的生物相容性,控制释放、靶向特性,以及与功能因子的协同增效功能。然而,多数纳米载体并未参与功能因子功效的发挥。
目前为止,多数纳米载体是采用不可再生的石化资源制备,基于天然可再生、安全、食品级的原料作为功能因子的纳米载体的策略正在出现。天然生物大分子如藻蓝蛋白是一种氨基酸组成完全的水溶性蓝色色素,具有抗氧化、抗炎等功效。原花青素,作为自然界中含量最丰富的类黄酮,可作为膳食补充剂和食物添加剂。它还具有两亲性,交联特性,抗氧化和抗炎效应,能与负载的功能因子发挥协同增效的作用。香兰素作为香草豆的主要成分之一,具备一定的药理特性。
发明内容
[技术问题]
本发明要解决的技术问题是利用藻蓝蛋白开发一种新型的纳米载体,并利用这种新型的纳米载体装载功能因子。
[技术方案]
本发明提供一种制备基于藻蓝蛋白的纳米载体的方法,包括如下步骤:
S1、将原花青素和香兰素分散在30mL水中,在室温条件下持续磁力搅拌1~3h得到混合溶液;混合溶液中所述原花青素的终浓度为1~7.5mg/mL,香兰素的终浓度为0.19~0.76mg/mL;
S2、向步骤S1所述的混合溶液中加入10mL藻蓝蛋白溶液触发纳米载体的自组装,在室温黑暗中搅拌12~24h;所述藻蓝蛋白的浓度为0.1~0.5mg/mL;
S3、将步骤S2所述的三元混合体系在10000rpm/min条件下离心20min,沉淀用蒸馏水洗2~3次,然后将沉淀用水重悬获得纳米载体溶液。
在某些实施方式中,步骤S1所述搅拌转速为800~1200rpm。
在某些实施方式中,步骤S2所述搅拌转速为800~1200rpm。
本发明提供所述基于藻蓝蛋白的纳米载体在装载功能因子中的应用,所述功能因子包括叶黄素、虾青素、姜黄素和白藜芦醇等疏水性物质。
本发明提供一种利用所述基于藻蓝蛋白的纳米载体制备尺寸可控的叶黄素纳米粒子的方法,包括如下步骤:
S1、将原花青素和香兰素分散在30mL水中,在室温条件下持续磁力搅拌1~3h得到混合溶液,混合溶液中所述原花青素的终浓度为1~7.5mg/mL,香兰素的终浓度为0.19~0.76mg/mL;
S2、向步骤S1所述的混合溶液中加入10mL藻蓝蛋白溶液触发纳米载体的自组装,在室温黑暗中搅拌12~24h;所述藻蓝蛋白的浓度为0.1~0.5mg/mL;
S3、将步骤S2所述的三元混合体系在10000rpm/min条件下离心20min,沉淀用蒸馏水洗2~3次,然后将沉淀用水重悬获得纳米载体溶液;
S4、将获得的纳米载体溶液与叶黄素的乙醇溶液等浓度(2mg/mL)等体积混合,将混合物在室温下搅拌24h,10000rpm/min条件下离心20min,沉淀用水重悬获得装载叶黄素的纳米载体溶液。
在某些实施方式中,步骤S1中,所述搅拌转速为800~1200rpm。
在某些实施方式中,步骤S2中,所述搅拌转速为800~1200rpm。
在某些实施方式中,随着原花青素浓度在1~7.5mg/mL范围内增加,纳米载体的粒径呈现先降低后上升的趋势;随着香兰素浓度在0.19~0.76mg/mL范围内增加,纳米载体的粒径呈现先降低后上升的趋势;藻蓝蛋白的浓度变化对纳米载体的粒径影响呈相反的趋势,随着藻蓝蛋白浓度在0.1~0.5mg/mL范围内增加,粒径则呈下降趋势。因此,通过改变原花青素、香兰素和藻蓝蛋白的浓度,可实现纳米载体的尺寸的控制,调控范围在138.32-744.87nm之间,例如,在原花青素、香兰素和藻蓝蛋白浓度分别为5mg/mL、0.57mg/mL和0.5mg/mL条件下,制备的纳米载体尺寸最小,仅为138.32nm。
本发明提供所述基于藻蓝蛋白的纳米载体或应用所述基于藻蓝蛋白的纳米载体装载功能因子得到的纳米粒子在制备食品、保健品、化妆品、药品中的应用。
[有益效果]
本发明采用“一锅法”提供一种基于藻蓝蛋白尺寸可控的叶黄素纳米粒子。本发明以藻蓝蛋白、原花青素和香兰素作为食品级原料,基于曼尼希反应合成具有功能特性的叶黄素纳米载体。所设计的纳米载体具有尺寸可控、强抗氧化性和良好的生物相容性,并对丙烯酰胺和过氧化氢引起的细胞氧化应激损伤具有抑制作用。
本发明方法制备食品级尺寸可控和强抗氧化性的纳米粒子,制备方法简单温和,所制备的纳米载体为球形,通过改变原花青素、香兰素和藻蓝蛋白的浓度可实现纳米载体尺寸的控制。抗氧化实验表明纳米载体具有清除DPPH和ABTS游离自由基的能力,细胞毒性实验表明所制备的纳米载体安全无毒,具有良好的生物相容性。此外,采用丙烯酰胺和过氧化氢诱导细胞氧化应激模型,藻蓝蛋白纳米载体对细胞氧化应激具有抑制效应。本发明所使用的原料为食品级,价格低廉,合成条件温和,易于规模化生产;本发明制备的纳米载体可用于营养物质叶黄素的递送,应用在护眼保健品及功能性食品领域。
本发明以原花青素、香兰素和藻蓝蛋白为原料,制备的叶黄素纳米粒子具有尺寸可控、强抗氧化性、安全无毒以及对细胞氧化应激模型的抑制效应。
本发明以食品级原花青素、香兰素和藻蓝蛋白为原料,基于曼尼希反应合成具有尺寸可控和强抗氧化性的藻蓝蛋白纳米载体,该体系可用于功能因子叶黄素的递送,应用在护眼等食品、医药领域。
附图说明
图1是本发明实施例1制备的纳米载体的扫描电镜图;
图2是本发明实施例2制备的纳米载体的扫描电镜图;
图3是本发明实施例3制备的纳米载体的扫描电镜图;
图4是本发明实施例4制备的纳米载体的扫描电镜图;
图5是本发明实施例5制备的纳米载体的扫描电镜图;
图6是本发明实施例6制备的纳米载体的扫描电镜图;
图7是本发明实施例7制备的纳米载体的扫描电镜图;
图8是本发明实施例8制备的纳米载体的扫描电镜图;
图9是本发明对比例1制备的纳米载体的扫描电镜图;
图10是本发明对比例2制备的纳米载体的扫描电镜图;
图11是本发明对比例3制备的纳米载体的扫描电镜图;
图12是本发明对比例4制备的纳米载体的扫描电镜图;
图13是本发明原花青素、Vc、实施例8制备的纳米载体的DPPH清除率变化;
图14是本发明实施例8制备的纳米载体的细胞活力图;
图15是本发明未经处理的Caco-2细胞;
图16是本发明经丙烯酰胺处理的Caco-2细胞;
图17是本发明经原花青素预处理后经丙烯酰胺损伤的Caco-2细胞;
图18是本发明经藻蓝蛋白预处理后经丙烯酰胺损伤的Caco-2细胞;
图19是本发明经实施例8制备的纳米载体预处理后经丙烯酰胺损伤的Caco-2细胞;
图20是本发明未经处理、丙烯酰胺处理、经原花青素、藻蓝蛋白和实施例8制备的纳米载体预处理后经丙烯酰胺损伤的Caco-2细胞的荧光强度分析图;
图21是本发明未经处理的Caco-2细胞;
图22是本发明经过氧化氢处理的Caco-2细胞;
图23是本发明经原花青素预处理后经过氧化氢损伤的Caco-2细胞;
图24是本发明经藻蓝蛋白预处理后经过氧化氢损伤的Caco-2细胞;
图25是本发明经实施例8制备的纳米载体预处理后经过氧化氢损伤的Caco-2细胞;
图26是本发明未经处理、过氧化氢处理、经原花青素、藻蓝蛋白和实施例8制备的纳米载体预处理后经过氧化氢损伤的Caco-2细胞的荧光强度分析图;
图27是本发明实施例9制备的叶黄素纳米载体的扫描电镜图;
图28是本发明实施例9制备的叶黄素纳米载体的包埋率;
图29是叶黄素的细胞活力图;
图30是市售叶黄素咀嚼片的细胞活力图;
图31是市售叶黄素胶囊的细胞活力图;
图32是本发明实施例9制备的叶黄素纳米载体的细胞活力图。
具体实施方式
下面通过具体实施例对本发明作进一步说明。
下述各例中,所述原花青素的生产厂家为天津尖峰天然产物研究开发有限公司,Item#:002。
实施例1:纳米载体的制备
S1、将原花青素和香兰素分散在30mL水中,在室温条件下以1000rpm/min速度持续磁力搅拌2h。其中,所述原花青素的终浓度为1mg/mL,所述香兰素的终浓度为0.57mg/mL。
S2、向步骤S1所述的混合溶液中加入10mL浓度为0.2mg/mL藻蓝蛋白溶液,触发纳米载体的自组装,在室温黑暗中以1000rpm/min搅拌24h。
S3、将步骤S2所述的三元混合体系在10000rpm/min条件下离心20min,沉淀用蒸馏水洗3次,然后将沉淀用水重悬获得纳米载体溶液。
实施例2:纳米载体的制备
S1、将原花青素和香兰素分散在30mL水中,在室温条件下以1000rpm/min速度持续磁力搅拌2h。其中,所述原花青素的终浓度为5mg/mL,所述香兰素的终浓度为0.57mg/mL。
S2、向步骤S1所述的混合溶液中加入10mL浓度为0.2mg/mL藻蓝蛋白溶液,触发纳米载体的自组装,在室温黑暗中以1000rpm/min搅拌24h。
S3、将步骤S2所述的三元混合体系在10000rpm/min条件下离心20min,沉淀用蒸馏水洗3次,然后将沉淀用水重悬获得纳米载体溶液。
实施例3:纳米载体的制备
S1、将原花青素和香兰素分散在30mL水中,在室温条件下以1000rpm/min速度持续磁力搅拌2h。其中,所述原花青素的终浓度为7.5mg/mL,所述香兰素的终浓度为0.57mg/mL。
S2、向步骤S1所述的混合溶液中加入10mL浓度为0.2mg/mL藻蓝蛋白溶液,触发纳米载体的自组装,在室温黑暗中以1000rpm/min搅拌24h。
S3、将步骤S2所述的三元混合体系在10000rpm/min条件下离心20min,沉淀用蒸馏水洗3次,然后将沉淀用水重悬获得纳米载体溶液。
实施例4:纳米载体的制备
S1、将原花青素和香兰素分散在30mL水中,在室温条件下以1000rpm/min速度持续磁力搅拌2h。其中,所述原花青素的终浓度为5mg/mL,所述香兰素的终浓度为0.19mg/mL。
S2、向步骤S1所述的混合溶液中加入10mL浓度为0.2mg/mL藻蓝蛋白溶液,触发纳米载体的自组装,在室温黑暗中以1000rpm/min搅拌24h。
S3、将步骤S2所述的三元混合体系在10000rpm/min条件下离心20min,沉淀用蒸馏水洗3次,然后将沉淀用水重悬获得纳米载体溶液。
实施例5:纳米载体的制备
S1、将原花青素和香兰素分散在30mL水中,在室温条件下以1000rpm/min速度持续磁力搅拌2h。其中,所述原花青素的终浓度为5mg/mL,所述香兰素的终浓度为0.29mg/mL。
S2、向步骤S1所述的混合溶液中加入10mL浓度为0.2mg/mL藻蓝蛋白溶液,触发纳米载体的自组装,在室温黑暗中以1000rpm/min搅拌24h。
S3、将步骤S2所述的三元混合体系在10000rpm/min条件下离心20min,沉淀用蒸馏水洗3次,然后将沉淀用水重悬获得纳米载体溶液。
实施例6:纳米载体的制备
S1、将原花青素和香兰素分散在30mL水中,在室温条件下以1000rpm/min速度持续磁力搅拌2h。其中,所述原花青素的终浓度为5mg/mL,所述香兰素的终浓度为0.76mg/mL。
S2、向步骤S1所述的混合溶液中加入10mL浓度为0.2mg/mL藻蓝蛋白溶液,触发纳米载体的自组装,在室温黑暗中以1000rpm/min搅拌24h。
S3、将步骤S2所述的三元混合体系在10000rpm/min条件下离心20min,沉淀用蒸馏水洗3次,然后将沉淀用水重悬获得纳米载体溶液。
实施例7:纳米载体的制备
S1、将原花青素和香兰素分散在30mL水中,在室温条件下以1000rpm/min速度持续磁力搅拌2h。其中,所述原花青素的终浓度为5mg/mL,所述香兰素的终浓度为0.57mg/mL。
S2、向步骤S1所述的混合溶液中加入10mL浓度为0.1mg/mL藻蓝蛋白溶液,触发纳米载体的自组装,在室温黑暗中以1000rpm/min搅拌24h。
S3、将步骤S2所述的三元混合体系在10000rpm/min条件下离心20min,沉淀用蒸馏水洗3次,然后将沉淀用水重悬获得纳米载体溶液。
实施例8:纳米载体的制备
S1、将原花青素和香兰素分散在30mL水中,在室温条件下以1000rpm/min速度持续磁力搅拌2h。其中,所述原花青素的终浓度为5mg/mL,所述香兰素的终浓度为0.57mg/mL。
S2、向步骤S1所述的混合溶液中加入10mL浓度为0.5mg/mL藻蓝蛋白溶液,触发纳米载体的自组装,在室温黑暗中以1000rpm/min搅拌24h。
S3、将步骤S2所述的三元混合体系在10000rpm/min条件下离心20min,沉淀用蒸馏水洗3次,然后将沉淀用水重悬获得纳米载体溶液。
实施例9:制备装载叶黄素的纳米粒子
S1、将原花青素和香兰素分散在30mL水中,在室温条件下以1000rpm/min速度持续磁力搅拌2h。其中,所述原花青素的终浓度为5mg/mL,所述香兰素的终浓度为0.57mg/mL。
S2、向步骤S1所述的混合溶液中加入10mL浓度为0.5mg/mL藻蓝蛋白溶液,触发纳米载体的自组装,在室温黑暗中以1000rpm/min搅拌24h。
S3、将步骤S2所述的三元混合体系在10000rpm/min条件下离心20min,沉淀用蒸馏水洗3次,然后将沉淀用水重悬获得纳米载体溶液。
S4、将获得的纳米载体溶液与叶黄素的乙醇溶液等浓度(2mg/mL)等体积混合,将混合物在室温下搅拌24h,在温度为45℃条件下旋蒸除去乙醇,用水重悬获得装载叶黄素的纳米载体溶液。
对比例1:
S1、将原花青素和香兰素分散在30mL水中,在室温条件下以1000rpm/min速度持续磁力搅拌2h。其中,所述原花青素的浓度为5mg/mL,所述香兰素的浓度为0.57mg/mL。
S2、向步骤S1所述的混合溶液中加入10mL浓度为0.5mg/mL牡蛎蛋白溶液,触发纳米载体的自组装,在室温黑暗中以1000rpm/min搅拌24h。
S3、将步骤S2所述的三元混合体系在10000rpm/min条件下离心20min,沉淀用蒸馏水洗3次,然后将沉淀用水重悬获得纳米载体溶液。
对比例2:
S1、将原花青素和紫苏醛分散在30mL水中,在室温条件下以1000rpm/min速度持续磁力搅拌2h。其中,所述原花青素的浓度为5mg/mL,所述紫苏醛的浓度为0.57mg/mL。
S2、向步骤S1所述的混合溶液中加入10mL浓度为0.5mg/mL半胱氨酸溶液,触发纳米载体的自组装,在室温黑暗中以1000rpm/min搅拌24h。
S3、将步骤S2所述的三元混合体系在10000rpm/min条件下离心20min,沉淀用蒸馏水洗3次,然后将沉淀用水重悬获得纳米载体溶液。
对比例3:
S1、将原花青素和甲醛分散在30mL水中,在室温条件下以1000rpm/min速度持续磁力搅拌2h。其中,所述原花青素的浓度为4.3mg/mL,所述甲醛添加量为60μL。
S2、向步骤S1所述的混合溶液中加入10mL浓度为0.25mg/mL甘氨酸溶液,触发纳米载体的自组装,在室温黑暗中以1000rpm/min搅拌24h。
S3、将步骤S2所述的三元混合体系在10000rpm/min条件下离心20min,沉淀用蒸馏水洗3次,然后将沉淀用水重悬获得纳米载体溶液。
对比例4:
S1、将原花青素和甲醛分散在30mL水中,在室温条件下以1000rpm/min速度持续磁力搅拌2h。其中,所述原花青素的浓度为4.3mg/mL,所述甲醛添加量为60μL。
S2、向步骤S1所述的混合溶液中加入10mL浓度为0.25mg/mL精氨酸溶液,触发纳米载体的自组装,在室温黑暗中以1000rpm/min搅拌24h。
S3、将步骤S2所述的三元混合体系在10000rpm/min条件下离心20min,沉淀用蒸馏水洗3次,然后将沉淀用水重悬获得纳米载体溶液。
对上述各例制备的纳米载体的微观结构、抗氧化性、细胞活力和线粒体膜电位等进行分析。
表1、实施例1~8制备的纳米载体的粒径和多分散指数
表1和图1~8为不同原花青素、香兰素和藻蓝蛋白条件下制备的纳米载体,从图中可以看出制备的实施例1~8的纳米载体均为球形。随着原花青素浓度的增加,纳米载体的粒径呈现先降低后上升的趋势,尺寸分别为254.90nm(实施例1),207.95nm(实施例2)和479.00nm(实施例3)。不同香兰素浓度下,纳米载体的粒径也呈现类似的趋势,尺寸分别为404.90nm(实施例4),283.27nm(实施例5)和744.87nm(实施例6)。藻蓝蛋白的浓度变化对纳米载体的粒径影响呈相反的趋势,随着藻蓝蛋白浓度的增加,粒径则呈下降趋势,分别为664.56nm(实施例7),207.95nm(实施例2)和138.32nm(实施例8)。因此,经上述分析可知通过改变原花青素、香兰素和藻蓝蛋白的浓度,可实现纳米载体的尺寸的控制,调控范围在138.32-744.87nm之间,且在原花青素,香兰素和藻蓝蛋白浓度分别为5.00,0.57和0.50mg/mL条件下,制备的实施例8纳米载体尺寸最小,仅为138.32nm。
图9为对比例1采用牡蛎蛋白代替藻蓝蛋白,在相同的反应机理条件下,未形成均匀的球形。
图10为对比例2采用紫苏醛代替香兰素,半胱氨酸代替藻蓝蛋白,在相同的反应机理条件下,也未形成均匀的球形。
图11为对比例3采用甲醛代替香兰素,甘氨酸代替藻蓝蛋白,在相同的反应机理条件下,形成均匀的球形。
图12为对比例4采用甲醛代替香兰素,精氨酸代替藻蓝蛋白,在相同的反应机理条件下,仅形成部分球形。因此,并不是所有含有氨基的物质都可基于曼尼希反应合成球形纳米载体。
图13为原花青素、实施例8制备的纳米载体与对照Vc的DPPH清除率变化情况。分别将原花青素浓度、实施例8制备的纳米载体中的原花青素浓度、和Vc的浓度梯度稀释为1μg/mL、2.5μg/mL、5μg/mL、10μg/mL、20μg/mL、40μg/mL,测定其清除DPPH能力。其中,实施例8制备的纳米载体中的原花青素浓度为3.75mg/mL,经过稀释得到原花青素浓度分别为1μg/mL、2.5μg/mL、5μg/mL、10μg/mL、20μg/mL、40μg/mL的纳米载体溶液。清除DPPH能力的测定方法参照文献李晓英,薛梅,樊汶樵.蓝莓花、茎、叶酚类物质含量及抗氧化活性比较[J].食品科学,2017,3,142-147。结果显示,在原花青素浓度为1~20μg/mL范围内,原花青素及纳米载体对DPPH自由基清除能力呈现明显的剂量依赖效应;且在原花青素浓度为40μg/mL时,DPPH清除率均高于80%。但是实施例8的纳米载体清除DPPH的能力均低于原花青素和Vc,这与原花青素和藻蓝蛋白之间的相互作用有关。
图14为实施例8制备的纳米载体对Caco-2细胞活性的影响。将Caco-2细胞以1×105个/孔的密度接种在96孔培养板上,细胞在培养箱内孵育24h后,暴露于含有实施例8制备的纳米载体的培养基内(原花青素的浓度为0,1,2,2.5,5,7.5和10μg/mL),经24h孵育后,加入20μL浓度为5μg/mL的MTT进一步孵育4h。除去上清液,然后加入150μL的二甲基亚砜,震荡均匀后使用酶标仪检测细胞在570nm处的吸光值。结果表明随着原花青素浓度的增加,Caco-2细胞的增殖能力呈上升趋势。在原花青素浓度为10μg/mL时,细胞活力最高为151.48%,说明制备的纳米载体无毒,具有良好的生物相容性。
图15~19为本发明未经处理、丙烯酰胺处理、经原花青素、藻蓝蛋白、实施例8制备的纳米载体预处理后经丙烯酰胺损伤的Caco-2细胞的线粒体膜电位荧光图。将Caco-2细胞以1×105个/孔的密度接种在12孔培养板上,细胞贴壁生长24h后,与原花青素(10μg/mL),藻蓝蛋白(1μg/mL)和实施例8制备的纳米载体(原花青素10μg/mL,藻蓝蛋白1μg/mL)预孵育24h,随后暴露于含有浓度800μM的丙烯酰胺培养基中4h。其中,分别以与单独MEM和丙烯酰胺孵育的Caco-2细胞作为阴性对照和阳性对照。随后,将Caco-2细胞与含有5μg/mL罗丹明123的培养基在37℃下孵育30min。采用荧光倒置显微镜观察细胞线粒体膜电位的变化情况。图16为丙烯酰胺对Caco-2细胞的损伤,线粒体膜的通透性增加,与图15未经处理的Caco-2细胞相比荧光强度降低,为41.07%。相反地,经原花青素、藻蓝蛋白和实施例8制备的纳米载体预干预后,由丙烯酰胺引起的线粒体膜电位下降得到改善,分别增加了26.11%、14.98%和44.06%。图20为采用Image J处理分析后,不同条件下细胞的相对荧光强度。罗丹明123是一种绿色荧光染料,能通过细胞膜聚集在活细胞的线粒体内。
图21~25为本发明未经处理、过氧化氢处理、经原花青素、藻蓝蛋白、实施例8制备的纳米载体预处理后经过氧化氢损伤的Caco-2细胞的线粒体膜电位荧光图。实验步骤与图15~19细胞处理方式一致,不同处在于采用浓度为800μM的过氧化氢代替丙烯酰胺诱导细胞损伤。图22为过氧化氢对Caco-2细胞的损伤,线粒体膜的通透性增加,与图21未经处理的Caco-2细胞相比荧光强度降低,为30.50%。经原花青素、藻蓝蛋白和实施例8制备的纳米载体预干预后,由过氧化氢引起的线粒体膜电位下降得到改善。图26为采用Image J处理分析后,不同条件下细胞的相对荧光强度。
图27为实施例9制备的叶黄素纳米粒子的微观形貌,所制备的颗粒均为球形,尺寸为370±50nm。采用3000rpm/min离心5min条件,对纳米颗粒进行离心,根据叶黄素的标准曲线y=0.5915x+0.0104(R2=0.9995)计算叶黄素的装载率为74.42%(图28)。图29~32为细胞毒性实验,将人角膜上皮细胞以1×105个/孔的密度接种在96孔培养板上,细胞贴壁生长24h后,与叶黄素,市售叶黄素咀嚼片,市售叶黄素胶囊和实施例9制备的装载叶黄素纳米载体孵育24h后(叶黄素浓度为0,10,20,40,60,80和100μg/mL),加入20μL浓度为5μg/mL的MTT进一步孵育4h。除去上清液,然后加入150μL的二甲基亚砜,震荡均匀后使用酶标仪检测细胞在570nm处的吸光值。图29为对照组叶黄素的细胞活力,叶黄素对人角膜上皮细胞活力有促进作用,但无浓度依赖性。图30为对照组市售叶黄素咀嚼片的细胞活力,对细胞活力无明显的促进作用。图31为对照组市售叶黄素胶囊的细胞活力,对细胞活力有促进作用,且存在浓度依赖性。而图32为实施例9装载叶黄素的纳米载体对细胞活力的影响,可以显著促进细胞增殖,且成浓度依赖特性,高于单独叶黄素,或市售叶黄素咀嚼片和胶囊的促增殖效果。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (9)
1.制备基于藻蓝蛋白的纳米载体的方法,其特征在于,包括如下步骤:
S1、将原花青素和香兰素分散在30mL水中,在室温条件下持续磁力搅拌1~3h得到混合溶液;混合溶液中所述原花青素的终浓度为1~7.5mg/mL,香兰素的终浓度为0.19~0.76mg/mL;
S2、向步骤S1所述的混合溶液中加入10mL藻蓝蛋白溶液触发纳米载体的自组装,在室温黑暗中搅拌12~24h;所述藻蓝蛋白的浓度为0.1~0.5mg/mL;
S3、将步骤S2获得的混合体系在10000rpm/min条件下离心20min,沉淀用蒸馏水洗2~3次,然后将沉淀用水重悬获得纳米载体溶液。
2.根据权利要求1所述的制备基于藻蓝蛋白的纳米载体的方法,其特征在于,步骤S1所述搅拌转速为800~1200rpm,步骤S2所述搅拌转速为800~1200rpm。
3.应用权利要求1或2所述方法制备得到的基于藻蓝蛋白的纳米载体。
4.权利要求3所述基于藻蓝蛋白的纳米载体在装载功能因子中的应用。
5.根据权利要求4所述的应用,其特征在于,所述功能因子包括叶黄素、虾青素、姜黄素、白藜芦醇或其他疏水性物质。
6.应用权利要求3所述基于藻蓝蛋白的纳米载体装载功能因子得到的纳米粒子。
7.一种利用基于藻蓝蛋白的纳米载体制备叶黄素纳米粒子的方法,其特征在于,包括如下步骤:
S1、将原花青素和香兰素分散在30mL水中,在室温条件下持续磁力搅拌1~3h得到混合溶液,混合溶液中所述原花青素的终浓度为1~7.5mg/mL,香兰素的终浓度为0.19~0.76mg/mL;
S2、向步骤S1所述的混合溶液中加入10mL藻蓝蛋白溶液触发纳米载体的自组装,在室温黑暗中搅拌12~24h;所述藻蓝蛋白的浓度为0.1~0.5mg/mL;
S3、将步骤S2获得的混合体系离心,离心所得沉淀用蒸馏水洗2~3次,然后将沉淀用水重悬获得纳米载体溶液;
S4、将获得的纳米载体溶液与叶黄素的乙醇溶液等浓度等体积混合,将混合物在室温下搅拌,然后旋蒸,随后用水重悬获得装载叶黄素的纳米粒子。
8.应用权利要求7所述方法制备得到的叶黄素纳米粒子。
9.权利要求3所述纳米载体或权利要求6所述纳米粒子或权利要求7所述方法制得的纳米粒子在制备食品、保健品、化妆品、药品中的应用。
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