CN113559247A - 一种多酚类纳米反应器及其制备方法和应用 - Google Patents
一种多酚类纳米反应器及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种多酚类纳米反应器及其制备方法和应用,所述的多酚类纳米反应器由MOF结构包载酶形成纳米颗粒后在纳米颗粒的表面形成TA‑PEI外壳,再对MOF结构进行刻蚀,最后在TA‑PEI外壳表面吸附盐酸阿霉素后得到。本发明所述的多酚类纳米反应器通过对MOF模板的刻蚀,形成中空结构,有利于酶活性的保持;利用TA‑PEI交联外壳对酶的保护,能够对抗胰酶等复杂环境;实现了饥饿治疗与化疗的协同治疗。
Description
技术领域
本发明属于生物医药领域,尤其是涉及一种多酚类纳米反应器及其制备方法和应用。
背景技术
葡萄糖氧化酶(GOx)是一种外源性氧化酶,由于其固有的生物相容性、无毒性和对β-D-葡萄糖高效、专一的催化特性,已被广泛应用于生物医学领域。GOx能够高效催化葡萄糖氧化产生葡萄糖酸和过氧化氢(H2O2),从而有效消耗肿瘤区的葡萄糖。然而,葡萄糖和氧气作为GOx催化反应的底物,在人体内无处不在。因此,基于GOx的纳米载体在体内循环时,负载于纳米载体中的GOx会与人体内的葡萄糖和氧气反应产生H2O2,H2O2的产生会引起严重的全身不良反应。过氧化氢酶(CAT)可以将H2O2分解为水和氧气,因此,将GOx与CAT共包载是解决上述问题的有效手段,可以在此外,其在消耗H2O2的同时产生氧气,改善肿瘤内乏氧环境。此外,目前针对肿瘤治疗的的多种治疗多种策略,例如癌症饥饿治疗,氧化治疗和多模式协同治疗等已有大量报道,其中饥饿治疗和化药的协同治疗是杀伤肿瘤的有力手段。
然而,由于葡萄糖氧化酶、过氧化氢酶等自身的不稳定性,易受环境中pH值与蛋白酶等多种因素的影响而失活,因此如何在体内保持酶的活性是亟待解决的问题。
通过研究发现,纳米反应器可以在一定程度上保护酶免受蛋白质水解消化或免疫清除,是解决该问题的一种有效策略。目前常用的纳米反应器材料有聚合物囊泡、纳米金属有机框架材料(MOFs)和介孔二氧化硅等。脂质体和聚合物囊泡是目前最主要的两种人工囊泡,这两种囊泡通过两亲性脂质或聚合物的自组装获得水性空腔来包载酶。然而囊泡的封装效率有限,并且脂质体和聚合物囊泡的疏水外壳会限制亲水底物从外层空间扩散到水腔,从而影响催化性能。金属有机框架材料是一类利用有机配体和金属离子或团簇通过配位作用制备的介孔材料,由于其优异的分子吸附性能,近年来作为酶固定载体被广泛探索。然而,在宿主材料中紧密包封的酶通常缺乏构象自由,从而影响酶的活性。此外,介孔二氧化硅材料由于在体内具有降解缓慢,生物相容性差等特点,也非酶固定化的理想载体。
因此,迫切需要探索新的纳米反应器平台来克服这些挑战,以求在有限的空间中不仅可以保护酶,还可以最大程度的发挥出酶活性。
发明内容
有鉴于此,本发明旨在克服现有技术中的缺陷,提出一种多酚类纳米反应器及其制备方法和应用。
为达到上述目的,本发明的技术方案是这样实现的:
一种多酚类纳米反应器的制备方法,包括如下步骤:
(1)将葡萄糖氧化酶、过氧化氢酶和2-甲基咪唑于水中混合均匀,然后迅速加入六水合硝酸锌溶液,低温条件下搅拌,反应后将所得反应液进行超滤离心,取浓缩液冻干后得GOx/CAT@ZIF纳米颗粒;
(2)将所得的GOx/CAT@ZIF纳米颗粒分散于含单宁酸(TA)的Tris-HCl溶液中,搅拌反应,离心、洗涤、取沉淀;
(3)将所得的沉淀分散于含聚乙烯亚胺(PEI)的Tris-HCl溶液中,搅拌反应,离心、洗涤、取沉淀得GOx/CAT@ZIF@TA-PEI;
(4)将所得的GOx/CAT@ZIF@TA-PEI加入柠檬酸-磷酸缓冲液中,室温孵育,离心、洗涤、取沉淀得GOx/CAT@TA-PEI;
(5)将所得的GOx/CAT@TA-PEI加入Tris-HCl溶液中,加入盐酸阿霉素,低温条件下搅拌,离心、洗涤、取沉淀得GOx/CAT@TA-PEI@DOX。
进一步,所述的步骤(1)中的葡萄糖氧化酶、过氧化氢酶、2-甲基咪唑和六水合硝酸锌的质量比为1:1:460:23.76;所述的2-甲基咪唑的浓度为38.33mg/mL;所述的步骤(1)中的六水合硝酸锌水溶液的浓度为2.376mg/mL;所述的步骤(1)中的搅拌步骤的温度为4℃,时间为30min。
进一步,所述的步骤(2)中的Tris-HCl溶液的浓度为50mM,pH为8.0,TA的浓度为0.1mg/mL;所述的步骤(2)中的GOx/CAT@ZIF纳米颗粒与所述的Tris-HCl溶液的料液比为2-3:20;所述的步骤(2)中的搅拌步骤的时间为30min,速度为150-200rpm,离心力为4000g。
进一步,所述的步骤(3)中的Tris-HCl溶液的浓度为50mM,pH为8.0,PEI的分子量为600-800Da,浓度为0.04mg/mL;所述的步骤(3)中的Tris-HCl溶液的用量为步骤(2)中Tris-HCl溶液用量的1/2。
进一步,所述的步骤(3)中的搅拌步骤的时间为30min,速度为150-200rpm,离心力为4000g。
进一步,所述的步骤(4)中孵育步骤的时间为20min,离心力为4000g;所述的步骤(3)中的柠檬酸-磷酸缓冲液的pH为5.0。
进一步,所述的步骤(5)中的GOx/CAT@TA-PEI纳米颗粒与盐酸阿霉素的质量比为2:1;所述的步骤(5)中的盐酸阿霉素的终浓度为0.75mg/mL,搅拌的时间为2h,温度为4℃,离心力为4000g。
一种多酚类纳米反应器,所述的多酚类纳米反应器由MOF结构包载酶形成纳米颗粒后在纳米颗粒的表面形成TA-PEI外壳,再对MOF结构进行刻蚀,最后在TA-PEI外壳表面吸附盐酸阿霉素后得到。所述的多酚类纳米反应器,其既可以保护纳米反应器内封装的酶不被降解,又不影响各种底物分子的透过,并且具有良好的生物相容性,可以延长生物大分子在体内的循环时间。首先,生物大分子原位封装在可降解的MOFs中,形成GOx/CAT@ZIF,并作为后续GOx/CAT@TA-PEI@DOX核-壳结构的生长模板。之后TA-PEI聚合物在GOx/CAT@ZIF表面生长聚合,形成纳米反应器的外壳。而后,去除MOF核,释放空心反应器内的生物大分子。最后,通过π-π作用在TA-PEI外壳表面吸附阿霉素,形成多功能的纳米反应器。
所述的多酚类纳米反应器的应用,所述的多酚类纳米反应器在葡萄糖氧化酶、过氧化氢酶和化疗药物的递送体系中的应用。
相对于现有技术,本发明具有以下优势:
本发明所述的多酚类纳米反应器通过对MOF模板的刻蚀,形成中空结构,有利于酶活性的保持;利用TA-PEI交联外壳对酶的保护,能够对抗胰酶等复杂环境;实现了饥饿治疗与化疗的协同治疗。
本发明所述的多酚类纳米反应器的制备方法在温和的条件下完成,原料绿色环保,制备方法简单,反应易控制,重复性高,利于实现大规模生产。
附图说明
图1为本发明实施例1所述的GOx/CAT@ZIF纳米颗粒的TEM图;
图2为本发明实施例1所述的GOx/CAT@TA-PEI@DOX纳米反应器的TEM图;
图3为本发明实施例2所述的纳米反应器在葡萄糖溶液中的时间-浓度曲线图;
图4为本发明实施例3所述的纳米反应器在与胰酶溶液孵育后的残存活性图;
图5为本发明实施例3所述的纳米反应器对Pan02细胞的细胞毒性。
具体实施方式
除有定义外,以下实施例中所用的技术术语具有与本发明所属领域技术人员普遍理解的相同含义。以下实施例中所用的试验试剂,如无特殊说明,均为常规生化试剂;所述实验方法,如无特殊说明,均为常规方法。
下面结合实施例来详细说明本发明。
实施例1
一种多酚类纳米反应器的制备方法,包括如下步骤:
(1)GOx/CAT@ZIF纳米颗粒的制备:
将0.25mg GOx,0.25mg CAT与115mg 2-甲基咪唑溶于3mL去离子水中并混合均匀,再迅速加入2.5mL 4℃预冷的2.376mg/mL的六水合硝酸锌,并将上述溶液置于4℃搅拌反应30min,待溶液呈乳白色后,使用超滤管对溶液进行浓缩,取上层浓缩液并用去离子水反复清洗去除多余的GOx和未反应的前驱体,-20℃冷冻干燥,得GOx/CAT@ZIF纳米颗粒。应用透射电子显微镜对纳米颗粒进行形貌表征,如图1所示,GOx/CAT@ZIF纳米粒形貌规则,粒径均一,粒径约为90nm;
(2)GOx/CAT@TA-PEI纳米反应器的制备:
称取TA粉末60mg,加入纯水3ml,充分涡旋,超声,配置成20mg/mL的TA母液,取100μL加入20mL Tris-HCl溶液(50mM,pH 8.0)中,然后取步骤(1)制备得到的GOx/CAT@ZIF纳米颗粒2.5mg,分散于上述Tris-HCl溶液中,室温搅拌30min后,4000g离心,取沉淀用去离子水清洗2次去除多余的反应液,得GOx/CAT@ZIF@TA纳米颗粒沉淀;
(3)配制10mg/mL的PEI水溶液,取40μL加入10mL Tris-HCl溶液(50mM,pH 8.0)中,而后加入GOx/CAT@ZIF@TA纳米颗粒沉淀,搅拌反应30min,4000g离心,取沉淀用去离子水清洗2次去除多余的反应液,得GOx/CAT@ZIF@TA-PEI纳米颗粒沉淀;
(4)将上述GOx/CAT@ZIF@TA-PEI纳米颗粒加入2mL柠檬酸-磷酸缓冲液中,室温振荡20min,4000g离心取沉淀,用去离子水清洗沉淀2次,得GOx/CAT@TA-PEI纳米颗粒;
(5)GOx/CAT@TA-PEI@DOX纳米反应器的制备:
将上述GOx/CAT@TA-PEI纳米颗粒分散于1mL Tris-HCl溶液中,加入盐酸阿霉素0.75mg,4℃搅拌反应2h,4000g离心,去离子水清洗沉淀2次,取沉淀得GOx/CAT@TA-PEI@DOX。通过透射电子显微镜对纳米颗粒进行形貌表征,如图2所示,GOx/CAT@TA-PEI@DOX纳米粒形貌规则,可以明显的看到TA-PEI@DOX外壳,外壳厚度为15-20nm。
实施例2
一种多酚类纳米反应器的制备方法,包括如下步骤:
(1)GOx@ZIF纳米颗粒的制备:
将1mg GOx与460mg 2-甲基咪唑加入12mL去离子水中混合均匀,再迅速加入10mL4℃预冷的2.376mg/mL的六水合硝酸锌,并将上述溶液置于4℃搅拌反应30min,待溶液呈乳白色后,使用超滤管对溶液进行浓缩,取上层浓缩液并用去离子水反复清洗去除多余的GOx和未反应的前驱体,冷冻干燥,得GOx@ZIF纳米颗粒;
(2)GOx@TA-PEI纳米反应器的制备:
称取TA粉末60mg,加入纯水3ml,充分涡旋,超声,配置成20mg/mL的TA母液,取400μL加入80mL Tris-HCl溶液(50mM,pH 8.0)中,然后取步骤(1)制备的GOx@ZIF纳米颗粒12mg,分散于上述Tris-HCl溶液中,室温搅拌30min后,4000g离心,取沉淀用去离子水清洗2次去除多余的反应液,得GOx@ZIF@TA纳米颗粒沉淀;
(3)配制10mg/mL的PEI水溶液,取160μL加入40mL Tris-HCl溶液(50mM,pH 8.0)中,而后加入上述GOx@ZIF@TA纳米颗粒沉淀,室温搅拌反应30min,4000g离心,取沉淀用去离子水清洗2次去除多余的反应液,得GOx@ZIF@TA-PEI纳米颗粒沉淀;
(4)将GOx@ZIF@TA-PEI纳米颗粒加入8mL柠檬酸-磷酸缓冲液中,室温振荡20min,4000g离心取沉淀,用去离子水清洗沉淀2次,得GOx@TA-PEI纳米颗粒;
(5)GOx@TA-PEI@DOX纳米反应器的制备:将上述GOx@TA-PEI纳米颗粒分散于4mLTris-HCl溶液中,加入盐酸阿霉素3mg,4℃搅拌反应2h,4000g离心,去离子水清洗沉淀2次,取沉淀得GOx@TA-PEI@DOX;
(6)纳米反应器酶活性测定:为了评估葡萄糖浓度,使用3,5-二硝基水杨酸(DNS)试剂检测葡萄糖浓度。在pH 7.4葡萄糖最终浓度为0.8mg/mL的条件下,分别加入GOx、GOx@TA-PEI@DOX和实施例1制备的GOx/CAT@TA-PEI@DOX(GOx等量浓度为2U/mL),分别于0.5h,1h,1.5h,2h,3h,4h和5h取出0.2mL溶液至EP管中,10000rpm离心5min,取上清0.1mL并与0.2mL DNS试剂混合,之后在100℃下加热5min,取出,自来水冷却至室温,加入去离子水0.9mL,540nm处测定各管的吸光度,依据标准曲线计算葡萄糖浓度。结果如图3所示,各组葡萄糖消耗速率为入GOx>GOx/CAT@TA-PEI@DOX>GOx@TA-PEI@DOX,因GOx/CAT的级联催化体系将产生的有毒产物H2O2同时催化为O2和H2O,生成的O2又可以进一步促进GOx的催化反应,因此GOx/CAT@TA-PEI@DOX的葡萄糖消耗率快于GOx@TA-PEI@DOX。
实施例3
一种多酚类纳米反应器的制备方法,包括如下步骤:
(1)GOx/CAT@ZIF纳米颗粒的制备:
将0.5mg GOx,0.5mg CAT与230mg 2-甲基咪唑加入6mL去离子水中混合均匀,再迅速加入5mL 4℃预冷的2.376mg/mL的六水合硝酸锌,并将上述溶液置于4℃搅拌反应30min,待溶液呈乳白色后,使用超滤管对溶液进行浓缩,取上层浓缩液并用去离子水反复清洗去除多余的GOx和未反应的前驱体,得GOx/CAT@ZIF纳米颗粒;
(2)GOx/CAT@TA-PEI纳米反应器的制备:
称取TA粉末60mg,加入纯水6ml,充分涡旋,超声,配置成10mg/mL的TA母液,取400μL加入40mL Tris-HCl溶液(50mM,pH 8.0)中,然后取步骤(1)制备的GOx/CAT@ZIF纳米颗粒,分散于上述Tris-HCl溶液中,室温搅拌30min后,4000g离心,取沉淀用去离子水清洗2次去除多余的反应液,得GOx/CAT@ZIF@TA纳米颗粒沉淀;
(3)配制20mg/mL的PEI水溶液,取40μL加入20mL Tris-HCl溶液(50mM,pH 8.0)中,而后加入GOx/CAT@ZIF@TA纳米颗粒沉淀,搅拌反应30min,4000g离心,取沉淀用去离子水清洗2次去除多余的反应液,得GOx/CAT@ZIF@TA-PEI纳米颗粒沉淀;
(4)将GOx/CAT@ZIF@TA-PEI纳米颗粒加入5mL柠檬酸-磷酸缓冲液中,室温振荡20min,4000g离心取沉淀,用去离子水清洗沉淀2次,得GOx/CAT@TA-PEI纳米颗粒;
(5)GOx/CAT@TA-PEI@DOX纳米反应器的制备:
将上述GOx/CAT@TA-PEI纳米颗粒分散于2mL Tris-HCl溶液中,加入盐酸阿霉素1.5mg,4℃搅拌反应2h,4000g离心,去离子水清洗沉淀2次,取沉淀得GOx/CAT@TA-PEI@DOX;
(6)GOx/CAT@TA-PEI@DOX纳米反应器与胰酶孵育后的活性测定:
取GOx/CAT@TA-PEI@DOX 2mg溶于5mL的胰酶溶液中(PBS,pH 7.4),胰酶终浓度为1mg/mL,37℃孵育。在预定的时间点,取1mL混合溶液,4000g离心10min弃去上清液,将沉淀分散于6.25mL葡萄糖溶液中(PBS,pH7.4),葡萄糖浓度为0.8mg/mL,室温下孵育1.5h,取上清测定葡萄糖浓度,与未与胰酶孵育的制剂消耗葡萄糖量对比,计算GOx/CAT@TA-PEI@DOX残存活性。结果如图4所示,纳米反应器与1mg/mL胰酶孵育6h后,仍保持较高活性,表明该纳米反应器在对抗体内复杂环境方面有很大优势;
(7)GOx/CAT@TA-PEI@DOX纳米反应器的细胞毒性:选择对数生长期的Pan02小鼠胰腺癌细胞,调整细胞数为3×104个/mL,接种于96孔板中,每孔100μL,细胞贴壁生长24h后,加入由培养基稀释的不同浓度的GOx/CAT@TA-PEI@DOX纳米颗粒与Pan02细胞共同培养,继续培养48h后,弃去旧培养基,每孔加入含有10μL CCK8的新鲜培养基100uL后继续孵育1.5h,使用酶标仪测定各孔在450nm处的吸光度(OD值),计算细胞的活力。细胞存活率(Cellviability,%)=(ODsample-ODblank)/(ODcontrol-ODblank)×100。结果如图5所示,GOx/CAT@TA-PEI@DOX具有较强的细胞毒性,对Pan02细胞的IC50为410nM。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (9)
1.一种多酚类纳米反应器的制备方法,其特征在于:包括如下步骤:
(1)将葡萄糖氧化酶、过氧化氢酶和2-甲基咪唑于水中混合均匀,然后迅速加入六水合硝酸锌溶液,低温条件下搅拌,反应后将所得反应液进行超滤离心,取浓缩液冻干后得GOx/CAT@ZIF纳米颗粒;
(2)将所得的GOx/CAT@ZIF纳米颗粒分散于含TA的Tris-HCl溶液中,搅拌反应,离心、洗涤、取沉淀;
(3)将所得的沉淀分散于含PEI的Tris-HCl溶液中,搅拌反应,离心、洗涤、取沉淀得GOx/CAT@ZIF@TA-PEI;
(4)将所得的GOx/CAT@ZIF@TA-PEI加入柠檬酸-磷酸缓冲液中,室温孵育,离心、洗涤、取沉淀得GOx/CAT@TA-PEI;
(5)将所得的GOx/CAT@TA-PEI加入Tris-HCl溶液中,加入盐酸阿霉素,低温条件下搅拌,离心、洗涤、取沉淀得GOx/CAT@TA-PEI@DOX。
2.根据权利要求1所述的多酚类纳米反应器的制备方法,其特征在于:所述的步骤(1)中的葡萄糖氧化酶、过氧化氢酶、2-甲基咪唑和六水合硝酸锌的质量比为1:1:460:23.76;所述的2-甲基咪唑的浓度为38.33mg/mL;所述的步骤(1)中的六水合硝酸锌水溶液的浓度为2.376mg/mL;所述的步骤(1)中的搅拌步骤的温度为4℃,时间为30min。
3.根据权利要求1所述的多酚类纳米反应器的制备方法,其特征在于:所述的步骤(2)中的Tris-HCl溶液的浓度为50mM,pH为8.0,TA的浓度为0.1mg/mL;所述的步骤(2)中的GOx/CAT@ZIF纳米颗粒与所述的Tris-HCl溶液的料液比为2-3:20;所述的步骤(2)中的搅拌步骤的时间为30min,速度为150-200rpm,离心力为4000g。
4.根据权利要求1所述的多酚类纳米反应器的制备方法,其特征在于:所述的步骤(3)中的Tris-HCl溶液的浓度为50mM,pH为8.0,PEI的分子量为600-800Da,浓度为0.04mg/mL;所述的步骤(3)中的Tris-HCl溶液的用量为步骤(2)中Tris-HCl溶液用量的1/2。
5.根据权利要求1所述的多酚类纳米反应器的制备方法,其特征在于:所述的步骤(3)中的搅拌步骤的时间为30min,速度为150-200rpm,离心力为4000g。
6.根据权利要求1所述的多酚类纳米反应器的制备方法,其特征在于:所述的步骤(4)中孵育步骤的时间为20min,离心力为4000g;所述的步骤(3)中的柠檬酸-磷酸缓冲液的pH为5.0。
7.根据权利要求1所述的多酚类纳米反应器的制备方法,其特征在于:所述的步骤(5)中的GOx/CAT@TA-PEI纳米颗粒与盐酸阿霉素的质量比为2:1;所述的步骤(5)中的盐酸阿霉素的终浓度为0.75mg/mL,搅拌的时间为2h,温度为4℃,离心力为4000g。
8.一种多酚类纳米反应器,其特征在于:所述的多酚类纳米反应器多酚类纳米反应器由MOF结构包载酶形成纳米颗粒后在纳米颗粒的表面形成TA-PEI外壳,再对MOF结构进行刻蚀,最后在TA-PEI外壳表面吸附盐酸阿霉素后得到。
9.权利要求8所述的多酚类纳米反应器的应用,其特征在于:所述的多酚类纳米反应器在葡萄糖氧化酶、过氧化氢酶和化疗药物的递送体系中的应用。
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