CN116602935A - 一种巨噬细胞靶向聚多巴胺纳米载药系统及其制备方法和应用 - Google Patents

一种巨噬细胞靶向聚多巴胺纳米载药系统及其制备方法和应用 Download PDF

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CN116602935A
CN116602935A CN202310574753.2A CN202310574753A CN116602935A CN 116602935 A CN116602935 A CN 116602935A CN 202310574753 A CN202310574753 A CN 202310574753A CN 116602935 A CN116602935 A CN 116602935A
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polydopamine
macrophage
mpda
nps
solution
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皮江
范书豪
徐军发
杨芬
金花
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Abstract

本发明涉及生物医学技术领域，具体涉及一种巨噬细胞靶向聚多巴胺纳米载药系统及其制备方法和应用，所述纳米载药系统以聚多巴胺为载体，Pluronic F‑127为乳化剂，TMB为稳定剂，甘露糖为表面修饰剂，具有良好的生物相容性和巨噬细胞靶向性，能高效地装载药物。所述纳米载药系统的制备方法包括：制备介孔聚多巴胺、甘露糖的修饰和装载药物，多巴胺在弱碱性条件下发生自聚合反应，反应后得到介孔聚多巴胺，将甘露糖修饰到介孔聚多巴胺上，抗结核药物可通过氢键和静电作用力，结合到介孔聚多巴胺的孔径中，得到具有巨噬细胞靶向性且装载抗结核药物的纳米载药系统。本发明中的纳米载药系统能够结合光热疗法和抗结核药物，其应用发挥重要作用。

Description

一种巨噬细胞靶向聚多巴胺纳米载药系统及其制备方法和 应用
技术领域
本发明涉及生物医学技术领域，具体涉及一种巨噬细胞靶向聚多巴胺纳米载药系统及其制备方法和应用。
背景技术
目前的药物传递系统(drug delivery system)，如纳米给药系统，通常使用各种稳定剂制备稳定的纳米系统，装载药物后，可以主动或被动地聚集到靶器官、细胞或细胞器，增强药物的靶向性；利用纳米颗粒装载药物还可以提高药物的稳定性和可溶性，促进药物的跨膜转运，并延长药物的循环时间以提高药物药效，并降低游离药物对非靶器官/细胞的毒性。
多巴胺(DA)是大脑中最丰富的儿茶酚胺神经递质，其作为神经递质调节中枢神经系统的各种生理功能。通过DA合成的聚多巴胺纳米颗粒(PDANPs)具有制备简单、生物相容性高、易于功能化、粘附性好等优点，可以在不同配体修饰后作为靶向递送的药物载体。
光热疗法是近年来新兴的治疗方法，其利用具有较高的光热转换效率的材料，将其注射入人体内部，利用靶向性识别技术聚集在病灶处，并在近红外光的照射下将光能转化为热能来治疗的一种方法。但是目前仅有几种光热转换材料通过FDA的批准被用于临床，因此开发一种生物毒性低，光热转换效率高，易于修饰装载材料，能作为光热治疗的载体显得尤为重要。
结核病是由结核分枝杆菌引起的慢性传染性细菌性疾病，结核病具有高“传染性”、“高致病性”，导致全世界发病率和死亡率居高不下。中国一直是结核重灾区，结核病患者、耐药结核患者、结核病致死患者数量均居全球前列。在结核病例中，约20％为肺外结核，皮肤结核约占肺外结核的1-2％，皮肤结核是结核分枝杆菌直接侵犯皮肤或者由其它脏器结核灶内的结核分枝杆菌播散到皮肤组织所致的皮肤损害，属全身性感染的一部分。由于皮肤结核病灶的杆菌量常小于肺结核量，目前临床推荐皮肤结核的治疗方案，包括多种药物治疗和肺结核的治疗方案，及长周期、全身性地用药，尚未发现任何已讨论感染的局部治疗，但它们并非都完全有效，其属于结核病发展的晚期症状，强调尽早治疗，但皮肤科医生对其了解程度较低，故临床筛查率低且无法及时治疗。综上，目前临床治疗方案为全身性的广泛治疗、预后不佳，且对皮肤结核的针对性治疗方案的研究甚少，故加大对皮肤结核病的基础研究，并将研究成果向皮肤结核病防控转化，对尽快遏制皮肤结核病的蔓延以及控制我国结核病的流行具有重要意义。
发明内容
本发明的目的是为了解决上述现有技术的不足而提供一种巨噬细胞靶向聚多巴胺纳米载药系统及其制备方法和应用。
本发明的目的通过下述技术方案予以实现：本发明提供了一种巨噬细胞靶向聚多巴胺纳米载药系统，以聚多巴胺为载体，Pluronic F-127为乳化剂，TMB为稳定剂，甘露糖为修饰剂。
优选的，所述聚多巴胺载体为介孔聚多巴胺。
本发明提供了一种巨噬细胞靶向聚多巴胺纳米载药系统的制备方法，包括如下步骤：
(1)制备介孔聚多巴胺：称取0.1-0.5g盐酸多巴胺和0.1-0.5gPluronic F-127，溶于体积比为1-1.5:1的水和无水乙醇的混合溶液中，加入占体系总体积1-2％的TMB，超声混匀后，逐滴加入占体系总体积3.5-4％的氨水，在室温条件下搅拌，离心所得到的混合溶液，收集沉淀物，用体积比为1.5-2:1的乙醇和水的混合溶液洗涤沉淀物4-5次，得到mPDANPs；
(2)甘露糖的修饰：量取50-500μL溶解于乙酸钠缓冲液的d-甘露糖溶液和50-100mL的mPDANPs溶液，混合，孵育，离心孵育溶液，收集沉淀物，用1x PBS洗涤沉淀物2-3次，得到Man-mPDA NPs；
(3)装载药物：称取0.1-1mg的Man-mPDA NPs，分散到1-2mL的Rif溶液中，混匀，孵育，离心孵育液，用1x PBS洗涤2-3次，除去未装载的利福平，得到Rif@Man-mPDA NPs。
优选的，步骤(1)中的离心转速为10000-12000rpm，离心时间为5-10min。
优选的，步骤(2)中的孵育为在3-5℃下摇动孵育24-26h，离心转速为10000-12000rpm，离心时间为5-10min。
优选的，步骤(2)中的乙酸钠缓冲液的pH为3.5-4.5，体积摩尔浓度为0.05-0.15M，d-甘露糖溶液的体积摩尔浓度为90-110mM，mPDANPs溶液的质量浓度为0.2-0.5mg/mL。
优选的，步骤(3)中的孵育为在3-5℃下摇动孵育8-10h，离心转速为10000-12000rpm，离心时间为5-10min。
优选的，步骤(3)中的Rif溶液的质量浓度为0.2-0.5mg/mL。
本发明提供了上述巨噬细胞靶向聚多巴胺纳米载药系统在抗结核药物的应用。
本发明还提供了上述巨噬细胞靶向聚多巴胺纳米载药系统的制备方法在制备抗结核药物的应用。
本发明的有益效果在于：
1、本发明的巨噬细胞靶向聚多巴胺纳米载药系统具有良好的生物相容性，能高效地装载药物，并通过甘露糖修饰，具有巨噬细胞靶向性；
2、本发明的一种巨噬细胞靶向聚多巴胺纳米载药系统的制备方法，利用多巴胺能在弱碱性的条件下，发生自聚合反应，多巴胺发生自聚合后洗脱其聚合时包裹的PluronicF-127后得到具有孔径结构的介孔聚多巴胺，甘露糖在酸性条件下开环，暴露出活性羟基，与介孔聚多巴胺上的氨基反应，将甘露糖修饰到介孔聚多巴胺上，利福平通过氢键和静电作用力，结合到介孔聚多巴胺的孔径中，得到具有巨噬细胞靶向性且装载抗结核药物的纳米载药系统；
3、本发明的巨噬细胞靶向聚多巴胺纳米载药系统在抗结核药物的应用：甘露糖作为天然配体靶向结合CD206，CD206是一类细胞膜受体，在大多数类型的巨噬细胞和DC细胞中高表达，因此可以作为巨噬细胞和DC细胞的靶向分子，经过甘露糖修饰并装载有抗结核药物的介孔聚多巴胺能够很好地靶向巨噬细胞，以更好地激活相关机制抑制Mtb免疫逃避。
4、本发明的巨噬细胞靶向聚多巴胺纳米载药系统的制备方法在制备抗结核药物的应用：巨噬细胞靶向聚多巴胺纳米载药系统的光热效应具有浓度依赖性和功率依赖性，随着浓度升高温度变化或激光功率升高温度变化，光热效应更为显著，且热效应主要依赖于激光的照射，巨噬细胞靶向聚多巴胺纳米载药系统能够结合光热疗法和抗结核药物，对结核分枝杆菌产生抑制作用，有效杀伤和清除宿主细胞内的结核分枝杆菌。
附图说明
图1是mPDANPs,Man-mPDANPs和Rif@Man-mPDA NPs的水合粒径图；
图2是mPDA NPs,Man-mPDA NPs和Rif-@Man-mPDA NPs的Zeta表面电位图；
图3是Rif@Man-mPDA NPs装载利福平的质量浓度图；
图4是mPDANPs,Man-mPDANPs和Rif@Man-mPDA NPs的透射电镜图；
图5是PBS、mPDA NPs、Man-mPDA NPs、Rif@mPDA NPs和Rif@man-mPDA NPs溶液的升温统计图；
图6是PBS、mPDA NPs和Rif@Man-mPDA NPs溶液的热像图；
图7是不同浓度Rif@man-mPDA NPs近红外激光照射下的温升曲线图；
图8是不同近红外激光照射下Rif@man-mPDA NPs的温升曲线图；
图9是Man-mPDA NPs的胞外抑菌图；
图10是Man-mPDA NPs的胞外菌落计数图；
图11是Rif@Man-mPDA NPs介导光热疗法的胞内抑菌图；
图12是Rif@Man-mPDA NPs介导光热疗法的胞内菌落计数图。
具体实施方式
为了便于本领域技术人员的理解，下面结合实施例1-3和附图1-12对本发明作进一步的说明，实施方式提及的内容并非对本发明的限定。
实施例1
本实施例提供了一种巨噬细胞靶向聚多巴胺纳米载药系统的制备方法，包括如下步骤：
(1)称取0.15g盐酸多巴胺和0.1gPluronic F-127，溶于体积比为1:1的水和无水乙醇的混合溶液中，加入占体系总体积1％的TMB，超声混匀后，逐滴加入占体系总体积3.5％的氨水，并使用磁力搅拌器在室温条件下搅拌，将得到的溶液在10000rpm下离心5min，收集沉淀物，用体积比为2:1的乙醇和水的混合溶液清洗5次，得到mPDA NPs。
(2)量取20μL溶解于pH为4.2、体积摩尔浓度为0.1M乙酸钠缓冲液的体积摩尔浓度为100mM的d-甘露糖溶液和10mL质量浓度为0.2mg/mL的mPDA NPs溶液，混合，在3℃条件下摇动孵育24h，孵育后，将孵育液在10000rpm转速下离心5min，收集沉淀物，用1x PBS洗涤沉淀物3次，得到Man-mPDA NPs。
(3)称取0.5mg的Man-mPDA NPs，分散到1mL质量浓度为0.2mg/mL的Rif溶液中，混匀，在3℃条件下摇动孵育8h，孵育后，将孵育液在10000rpm转速下离心5min，用1x PBS3次洗涤，除去未装载的利福平，得到Rif@Man-mPDA NPs。
将上述制备得到的mPDA NPs、Man-mPDA NPs和Rif@Man-mPDA NPs溶液进行激光粒度仪和透射电镜分析，参阅图1-4，说明mPDA NPs、Man-mPDA NPs和Rif@Man-mPDA NPs的水合粒径主要为200-400nm，透射电镜显示mPDA NPs、Man-mPDA NPs和Rif@Man-mPDA NPs均为200nm左右的球形纳米材料，mPDA NPs的平均电位为-45mV、Man-mPDA NPs的平均电位为-32mV、Rif@Man-mPDA NPs的平均电位为-37mV，均具有较稳定的结构，每1mg材料装载了0.13μg的利福平。
实施例2
(1)称取0.15g盐酸多巴胺和0.1gPluronic F-127，溶于体积比为1:1的水和无水乙醇的混合溶液中，加入占体系总体积1％的TMB，超声混匀后，逐滴加入占体系总体积3.5％的氨水，并使用磁力搅拌器在室温条件下搅拌，将得到的溶液在10000rpm下离心5min，收集沉淀物，用体积比为2:1的乙醇和水的混合溶液清洗5次，得到mPDA NPs。
(2)量取20μL溶解于pH为4.2、体积摩尔浓度为0.1M乙酸钠缓冲液的体积摩尔浓度为100mM的d-甘露糖溶液和10mL质量浓度为0.2mg/mL的mPDA NPs溶液，混合，在3℃条件下摇动孵育24h，孵育后，将孵育液在10000rpm转速下离心5min，收集沉淀物，用1x PBS洗涤沉淀物3次，得到Man-mPDA NPs。
(3)称取0.5mg的Man-mPDA NPs，分散到1mL质量浓度为0.2mg/mL的Rif溶液中，混匀，在3℃条件下摇动孵育8h，孵育后，将孵育液在10000rpm转速下离心5min，用1x PBS洗涤3次，除去未装载的利福平，得到Rif@Man-mPDA NPs。
将上述制备得到的mPDA NPs、Man-mPDA NPs和Rif@Man-mPDA NPs溶液进行光热性能验证，用功率为0.8W/cm2的808nm波长的激光分别对PBS、mPDA NPs、Man-mPDA NPs、Rif@mPDA NPs和Rif@man-mPDA NPs溶液进行照射5min，并通过红外相机监测每分钟的温度变化，参阅图5-6，说明纳米系统具有良好的光热性能。
用功率为0.8W/cm2的808nm波长的激光分别对50μg/mL、100μg/mL、220μg/mL的Rif@Man-mPDA NPs溶液进行照射，参阅图7，说明巨噬细胞靶向聚多巴胺纳米载药系统的光热效应具有浓度依赖性，随着浓度升高温度变化，光热效应更为显著。
分别用功率为0.8W/cm2、1.2W/cm2、1.5W/cm2、2.0W/cm2的808nm波长的激光对200μg/mL的Rif@Man-mPDA NPs溶液进行照射，参阅图8，说明巨噬细胞靶向聚多巴胺纳米载药系统的光热效应具有功率依赖性，随着激光功率升高温度变化，光热效应更为显著。
实施例3
(1)称取0.15g盐酸多巴胺和0.1gPluronic F-127，溶于体积比为1:1的水和无水乙醇的混合溶液中，加入占体系总体积1％的TMB，超声混匀后，逐滴加入占体系总体积3.5％的氨水，并使用磁力搅拌器在室温条件下搅拌，将得到的溶液在10000rpm下离心5min，收集沉淀物，用体积比为2:1的乙醇和水的混合溶液清洗5次，得到mPDA NPs。
(2)量取20μL溶解于pH为4.2、体积摩尔浓度为0.1M乙酸钠缓冲液的体积摩尔浓度为100mM的d-甘露糖溶液和10mL质量浓度为0.2mg/mL的mPDA NPs溶液，混合，在3℃条件下摇动孵育24h，孵育后，将孵育液在10000rpm转速下离心5min，收集沉淀物，用1x PBS洗涤沉淀物3次，得到Man-mPDA NPs。
将上述制备得到的Man-mPDA NPs溶液进行抑菌功能验证，以H37Ra作为Mtb的模型菌，将H37Ra用PBS稀释成1×107CFU/mL的悬液，分别添加浓度10μg/mL、20μg/mL、50μg/mLl、100μg/mL和20μg/mL的Man-mPDA NPs溶液作为处理组，将100μL的Man-mPDA NPs溶液和100μL的H37Ra悬液加到48孔板中，充分混匀后，用功率为1.2W/cm2的808nm波长的激光照射5min后，孵育24h，孵育后用7H9培养基将H37Ra混合液稀释成不同倍数，涂布于7H11培养板，在37℃条件下孵育3周后，采用平板菌落计数法进行菌落计数，参阅图9-10，说明巨噬细胞靶向聚多巴胺纳米载药系统对H37Ra具有明显的杀伤作用，能够显著抑制H37Ra的生长。
实施例4
(1)称取0.15g盐酸多巴胺和0.1gPluronic F-127，溶于体积比为1:1的水和无水乙醇的混合溶液中，加入占体系总体积1％的TMB，超声混匀后，逐滴加入占体系总体积3.5％的氨水，并使用磁力搅拌器在室温条件下搅拌，将得到的溶液在10000rpm下离心5min，收集沉淀物，用体积比为2:1的乙醇和水的混合溶液清洗5次，得到mPDA NPs。
(2)量取20μL溶解于pH为4.2、体积摩尔浓度为0.1M乙酸钠缓冲液的体积摩尔浓度为100mM的d-甘露糖溶液和10mL质量浓度为0.2mg/mL的mPDA NPs溶液，混合，在3℃条件下摇动孵育24h，孵育后，将孵育液在10000rpm转速下离心5min，收集沉淀物，用1x PBS洗涤沉淀物3次，得到Man-mPDA NPs。
(3)称取0.5mg的Man-mPDA NPs，分散到1mL质量浓度为0.2mg/mL的Rif溶液中，混匀，在3℃条件下摇动孵育8h，孵育后，将孵育液在10000rpm转速下离心5min，用1x PBS洗涤3次，除去未装载的利福平，得到Rif@Man-mPDA NPs。
将上述制备得到的Rif@Man-mPDA NPs溶液进行抑菌功能验证，以H37Ra作为Mtb的模型菌，首先将生长状态良好的THP-1用PMA(100nM)刺激贴壁24h，然后用MOI＝1的H37Ra感染THP-1 24h后，分别加入培养基和10μg/ml的Rif@Man-mPDA NPs，处理4h后用功率为1.2W/cm2的808nm波长的激光照射Rif@Man-mPDA NPs处理组5min后，孵育24h，然后用0.1％的SDS裂解细胞后将悬液稀释涂布于7H11培养板，在37℃条件下孵育3周后，采用平板菌落计数法进行菌落计数，参阅图11-12，说明Rif@Man-mPDA NPs介导的光热疗法对胞内的H37Ra具有明显的杀伤作用，能够显著抑制H37Ra的生长。
上述的具体实施例是对本发明技术方案和有益效果的进一步说明，并非对实施方式的限定。对本领域技术人员来说，在不脱离本发明构思的前提下任何显而易见的替换均在本发明的保护范围之内。

Claims (10)

1.一种巨噬细胞靶向聚多巴胺纳米载药系统，其特征在于：以聚多巴胺为载体，Pluronic F-127为乳化剂，TMB为稳定剂，甘露糖为修饰剂。
2.根据权利要求1所述的一种巨噬细胞靶向聚多巴胺纳米载药系统，其特征在于：所述聚多巴胺载体为介孔聚多巴胺。
3.根据权利要求1-2中任意一项所述的一种巨噬细胞靶向聚多巴胺纳米载药系统的制备方法，其特征在于：包括如下步骤：
(1)制备介孔聚多巴胺：称取0.1-0.5g盐酸多巴胺和0.1-0.5gPluronic F-127，溶于体积比为1-1.5:1的水和无水乙醇的混合溶液中，加入占体系总体积1-2％的TMB，超声混匀后，逐滴加入占体系总体积3.5-4％的氨水，在室温条件下搅拌，离心所得到的混合溶液，收集沉淀物，用体积比为1.5-2:1的乙醇和水的混合溶液洗涤沉淀物4-5次，得到mPDA NPs；
(2)甘露糖的修饰：量取50-500μL溶解于乙酸钠缓冲液的d-甘露糖溶液和50-100mL的mPDA NPs溶液，混合，孵育，离心孵育溶液，收集沉淀物，用1x PBS洗涤沉淀物2-3次，得到Man-mPDA NPs；
(3)装载药物：称取0.1-1mg的Man-mPDA NPs，分散到1-2mL的Rif溶液中，混匀，孵育，离心孵育液，用1x PBS洗涤2-3次，除去未装载的利福平，得到Rif@Man-mPDA NPs。
4.根据权利要求3所述的一种巨噬细胞靶向聚多巴胺纳米载药系统的制备方法，其特征在于：步骤(1)中的离心转速为10000-12000rpm，离心时间为5-10min。
5.根据权利要求3所述的一种巨噬细胞靶向聚多巴胺纳米载药系统的制备方法，其特征在于：步骤(2)中的孵育为在3-5℃下摇动孵育24-26h，离心转速为10000-12000rpm，离心时间为5-10min。
6.根据权利要求3所述的一种巨噬细胞靶向聚多巴胺纳米载药系统的制备方法，其特征在于：步骤(2)中的乙酸钠缓冲液的pH为3.5-4.5，体积摩尔浓度为0.05-0.15M，d-甘露糖溶液的体积摩尔浓度为90-110mM，mPDA NPs溶液的质量浓度为0.2-0.5mg/mL。
7.根据权利要求3所述的一种巨噬细胞靶向聚多巴胺纳米载药系统的制备方法，其特征在于：步骤(3)中的孵育为在3-5℃下摇动孵育8-10h，离心转速为10000-12000rpm，离心时间为5-10min。
8.根据权利要求3所述的一种巨噬细胞靶向聚多巴胺纳米载药系统的制备方法，其特征在于：步骤(3)中的Rif溶液的质量浓度为0.2-0.5mg/mL。
9.如权利要求1-2任意一项所述的巨噬细胞靶向聚多巴胺纳米载药系统在抗结核药物的应用。
10.如权利要求3所述的巨噬细胞靶向聚多巴胺纳米载药系统的制备方法在制备抗结核药物的应用。
CN202310574753.2A 2023-05-19 2023-05-19 一种巨噬细胞靶向聚多巴胺纳米载药系统及其制备方法和应用 Pending CN116602935A (zh)

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Cited By (1)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117323442A (zh) * 2023-08-30 2024-01-02 广东医科大学 一种巨噬细胞靶向二氧化锰纳米系统的制备方法及其应用
CN117323442B (zh) * 2023-08-30 2024-05-03 广东医科大学 一种巨噬细胞靶向二氧化锰纳米系统的制备方法及其应用

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