CN113318236B - 一种MSNAs-TPP多功能纳米颗粒及其制备方法、应用 - Google Patents
一种MSNAs-TPP多功能纳米颗粒及其制备方法、应用 Download PDFInfo
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Abstract
本发明公开了一种MSNAs‑TPP多功能纳米颗粒及其制备方法、应用,该制备方法包括:将SiO2纳米颗粒分散在水中,得到溶液A;将溶液A,水和无水乙醇混合,再添加高锰酸钾,然后将溶液转移至水热反应釜中反应;反应后冷却至室温,再离心收集MSNAs纳米颗粒;将MSNAs纳米颗粒添加到聚乙烯亚胺溶液中进行氨基化修饰,得到MSNAs‑PEI,作为溶液B;将三苯基溴化膦和1‑(3‑二甲氨基丙基)‑3‑乙基碳二亚胺盐酸盐溶解在二甲亚砜的水溶液中,得到溶液C;将n‑羟基琥珀酰亚胺添加至溶液C中,在室温下搅拌以形成TPP活化液D;将所述的溶液D添加到溶液B中,并在室温下搅拌,离心,洗涤,得到MSNAs‑TPP多功能纳米颗粒。
Description
技术领域
本发明涉及医药领域,尤其涉及一种MSNAs-TPP多功能纳米颗粒及其制备方法、应用。
背景技术
尽管肿瘤的治疗方式得到了长足的进步,但是癌症仍然是全球范围内导致死亡的主要原因之一。传统的治疗方式,例如手术、放疗和化疗等,只能发挥有限的治疗效果,并具有严重的副作用。在过去的几十年,已经开发出众多的功能性纳米材料用于肿瘤微环境响应的药物递送和特定的癌症治疗。但是,无论在患者之间还是在单个肿瘤内,肿瘤微环境的异质性都很高。因此,单纯依靠肿瘤微环境的靶向很难彻底消灭肿瘤。
线粒体是细胞中最重要的细胞器,负责细胞的能量供应和许多其他必要的细胞功能,包括生物合成、活性氧(ROS)生成、钙稳态调节等,并且是内源性凋亡途径的主要调节关口。因此,线粒体被认为是用于肿瘤治疗的更精确和有效的靶标。但是目前基于肿瘤线粒体的靶向治疗特异性、有效性仍然较低,治疗效果不理想。
另外,纳米药物经过全身循环进入到肿瘤组织需要经历一个漫长的过程。较大尺寸的纳米颗粒(50-200nm)具有较长的血液循环时间和较好的肿瘤靶向作用,但不利于在肿瘤组织内扩散。较小尺寸的纳米颗粒(小于20nm)可以大大提高肿瘤组织的扩散效率,但是在血液循环中容易被肾脏清除,半衰期较短。因此绝大部分的纳米药物肿瘤输运效率不高,治疗效果有限,很难彻底根治肿瘤。
发明内容
本申请实施例的目的是提供一种MSNAs-TPP多功能纳米颗粒及其制备方法、应用,以解决相关技术中存在的肿瘤线粒体特异性靶向治疗缺乏有效手段和纳米颗粒输运效率低的问题。
根据本申请实施例的第一方面,提供一种MSNAs-TPP多功能纳米颗粒的制备方法,包括:
将SiO2纳米颗粒分散在水中,得到溶液A;
将溶液A,水和无水乙醇混合,再添加高锰酸钾,然后将溶液转移至水热反应釜中反应;
反应后冷却至室温,再离心收集MSNAs纳米颗粒;
将MSNAs纳米颗粒添加到聚乙烯亚胺溶液中进行氨基化修饰,得到MSNAs-PEI,作为溶液B;
将三苯基溴化膦和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶解在二甲亚砜的水溶液中,得到溶液C;
将n-羟基琥珀酰亚胺添加至溶液C中,在室温下搅拌以形成TPP活化液D;
将所述的溶液D添加到溶液B中,并在室温下搅拌,离心,洗涤,得到MSNAs-TPP多功能纳米颗粒。
进一步地,所述溶液A中SiO2纳米颗粒的浓度为40-60mg/mL。
进一步地,所述溶液A、水和无水乙醇的体积比为1:(4~6.5):(5~2.5)。
进一步地,所述高锰酸钾与溶液A、水和无水乙醇的混合溶液的配比为(2.5~5):1(mg/mL)。
进一步地,所述水热反应釜中的反应温度为170-190℃,反应时间为18-30h。
进一步地,所述聚乙烯亚胺溶液的浓度为0.1~1mg/mL,MSNAs纳米颗粒和聚乙烯亚胺的质量比为(10~1):1。
进一步地,所述三苯基溴化膦在二甲亚砜水溶液中的浓度为2.5~5mg/mL,三苯基溴化膦和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的质量比为1:(1~2),二甲亚砜水溶液的体积分数为5%-15%。
进一步地,所述n-羟基琥珀酰亚胺与1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的质量比为1:(1~2)。
进一步地,所述溶液B和溶液D的体积比为(125~250):1。
根据本申请实施例的第二方面,提供一种第一方面所述的基于MSNAs-TPP多功能纳米颗粒在线粒体靶向纳米治疗剂诱导线粒体氧化应激的智能纳米平台药物中的应用。
本申请的实施例提供的技术方案可以包括以下有益效果:
由上述实施例可知,本发明实施例的MSNAs是以二氧化硅,高锰酸钾和乙醇作为前驱体,通过水热法制备。在反应中,乙醇可在高温高压的水热反应条件下生成二氧化碳等纳米气泡,从而充当材料制备中的软模板,而二氧化硅和高锰酸钾反应会在纳米气泡的气相表面生成硅酸锰,从而形成硅酸锰的小囊泡,这些硅酸锰小囊泡在原本的二氧化硅表面组装和堆积,随着二氧化硅和高锰酸钾的完全反应,将会在中间形成大的囊腔,从而形成两级中空的硅酸锰纳米材料,得到的MSNAs的尺寸约为100nm,组成MSNAs的硅酸锰小囊泡尺寸为6-7nm。
本申请与现有技术相比,本发明实施例的三苯基溴化膦(TPP)修饰的分级硅酸锰组装体(MSNAs-TPP),可用于线粒体靶向的癌症治疗。合成后的MSNAs-TPP因为是多级中空结构的纳米组装体,并可以响应肿瘤酸性微环境解体,因而解决了较大尺寸的纳米颗粒难以在肿瘤组织深度扩散的难题。图1为本发明实施例中的MSNAs-TPP纳米颗粒荷瘤小鼠静脉注射后肿瘤治疗机制示意图。本发明实施例的MSNAs-TPP可以在血液循环过程中保持结构和大小的稳定性(约100nm),通过肿瘤的高渗透长滞留(EPR)效应改善肿瘤的积累,并在肿瘤酸性微环境中迅速解体为超小型硅酸锰纳米囊泡(约6nm),有利于肿瘤组织渗透和线粒体靶向。
上述超小型硅酸锰纳米囊泡由于TPP的修饰,使之靶向肿瘤细胞的线粒体,迅速消耗高表达的线粒体谷胱甘肽,同时在生物降解过程中释放催化性的Mn2+离子,利用肿瘤细胞线粒体大量产生的CO2和H2O2诱导肿瘤细胞氧化应激的放大,和线粒体损伤,并导致肿瘤细胞凋亡。因此本发明实施例克服了以往线粒体靶向治疗特异性差的难题,提高了肿瘤治疗效率。所述MSNAs-TPP在小鼠肿瘤模型中显示出显著的抗肿瘤作用,而没有明显的副作用。
本发明实施例的分级硅酸锰组装体(MSNAs-TPP)具有肿瘤核磁共振成像和治疗的双重功能,可以用于肿瘤的早期诊断和对治疗过程进行实时监测,实现高效的肿瘤诊治一体。
迄今为止,尚未开发出一种高效的基于MSNAs-TPP纳米颗粒的肿瘤线粒体精准靶向治疗策略。而本发明则填补了这一空白。本发明的制备方法简单,所述MSNAs-TPP分散性稳定性良好,生物安全性高,成本低廉,适合大规模生产。
应当理解的是,以上的一般描述和后文的细节描述仅是示例性和解释性的,并不能限制本申请。
附图说明
此处的附图被并入说明书中并构成本说明书的一部分,示出了符合本申请的实施例,并与说明书一起用于解释本申请的原理。
图1为本发明实施例中的MSNAs-TPP纳米颗粒荷瘤小鼠静脉注射后肿瘤治疗机制示意图;
图2为本发明实施例中MSNAs纳米颗粒的电镜图,其中(a)为扫描电镜图,(b)为透射电镜图片,(c)为高分辨透射电镜图;
图3为本发明实施例中MSNAs纳米颗粒的Si、O、Mn元素的能谱图;
图4为本发明实施例中MSNAs纳米颗粒的XRD图谱;
图5为本发明实施例中MSNAs纳米颗粒的XPS图谱;
图6为本发明实施例中MSNAs纳米颗粒的氮气吸附脱附曲线;
图7为本发明实施例中TPP,MSNAs和MSNAs-TPP的FTIR图谱;
图8为本发明实施例中MSNAs纳米颗粒在弱酸性溶液中的解体性能,其中(a)为透射电镜图片,(b)为动态光散射粒径分布图;
图9为本发明实施例中MSNAs纳米颗粒溶液加入谷胱甘肽前后的紫外可见吸收曲线和溶液的光学照片;
图10为本发明实施例中MSNAs和对照材料HMSNs对谷胱甘肽的消耗曲线;
图11为本发明实施例中MSNAs在不同条件下催化亚甲基蓝(MB)降解的动力学曲线,其中(a)为不同浓度谷胱甘肽下的MB降解曲线,(b)为不同浓度H2O2中的MB降解曲线,(c)为不同浓度NaHCO3条件下的MB降解曲线;
图12为本发明实施例中MSNAs在不同溶液条件下Mn2+离子释放规律图;
图13为本发明实施例中MSNAs在不同溶液条件下的核磁共振造影成像弛豫率r1测试;
图14为本发明实施例中MSNAs和MSNAs-TPP的人脐静脉血管内皮细胞(HUVEC)相容性测试;
图15为本发明实施例中不同浓度MSNAs和MSNAs-TPP对4T1小鼠乳腺癌细胞的细胞活性抑制实验;
图16为本发明实施例中MSNAs和MSNAs-TPP处理4T1小鼠乳腺癌细胞后胞内活性氧含量检测实验;
图17为本发明实施例中MSNAs在不同细胞培养环境下对4T1小鼠乳腺癌细胞的细胞活性抑制实验;
图18为本发明实施例中MSNAs-TPP小鼠体内核磁共振成像照片,其中(a)为瘤内注射后肿瘤核磁共振成像照片,(b)为静脉注射后肿瘤核磁共振成像照片;
图19为本发明实施例中不同处理方式下小鼠肿瘤体积变化曲线;
图20为MSNAs-TPP静脉注射后小鼠体内各主要脏器和肿瘤的生物分布检查图;
图21为本发明实施例中小鼠治疗后不同时间点血液生化检查结果,其中(a)为谷丙转氨酶,(b)为直接胆红素,(c)为总胆红素,(d)为胆碱酯酶,(e)为谷草转氨酶,(f)为白蛋白,(g)为肌酐,(h)为总蛋白;
图22为治疗后小鼠体内主要脏器的H&E切片检查结果图。
具体实施方式
这里将详细地对示例性实施例进行说明,其示例表示在附图中。下面的描述涉及附图时,除非另有表示,不同附图中的相同数字表示相同或相似的要素。以下示例性实施例中所描述的实施方式并不代表与本申请相一致的所有实施方式。相反,它们仅是与如所附权利要求书中所详述的、本申请的一些方面相一致的装置和方法的例子。
在本申请使用的术语是仅仅出于描述特定实施例的目的,而非旨在限制本申请。在本申请和所附权利要求书中所使用的单数形式的“一种”、“所述”和“该”也旨在包括多数形式,除非上下文清楚地表示其他含义。还应当理解,本文中使用的术语“和/或”是指并包含一个或多个相关联的列出项目的任何或所有可能组合。
下面结合附图和具体实例对本发明作进一步的说明。
本发明实施例提供一种MSNAs-TPP多功能纳米颗粒的制备方法,包括:将SiO2纳米颗粒分散在水中,得到溶液A;将溶液A,水和无水乙醇混合,再添加高锰酸钾,然后将溶液转移至水热反应釜中反应;反应后冷却至室温,再离心收集MSNAs纳米颗粒;将MSNAs纳米颗粒添加到聚乙烯亚胺溶液中进行氨基化修饰,得到MSNAs-PEI,作为溶液B;将三苯基溴化膦和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶解在二甲亚砜的水溶液中,得到溶液C;将n-羟基琥珀酰亚胺添加至溶液C中,在室温下搅拌以形成TPP活化液D;将所述的溶液D添加到溶液B中,并在室温下搅拌,离心,洗涤,得到MSNAs-TPP多功能纳米颗粒。
由以上技术方案可知,MSNAs是以二氧化硅,高锰酸钾和乙醇作为前驱体,通过水热法制备。在反应中,乙醇可在高温高压的水热反应条件下生成二氧化碳等纳米气泡,从而充当材料制备中的软模板,而二氧化硅和高锰酸钾反应会在纳米气泡的气相表面生成硅酸锰,从而形成硅酸锰的小囊泡,这些硅酸锰小囊泡在原本的二氧化硅表面组装和堆积,随着二氧化硅和高锰酸钾的完全反应,将会在中间形成大的囊腔,从而形成两级中空的硅酸锰纳米材料,得到的MSNAs的尺寸约为100nm,组成MSNAs的硅酸锰小囊泡尺寸为6-7nm。
由于MSNAs是由硅酸锰小囊泡组装而成,因此在弱酸性环境,MSNAs可以与酸反应导致微弱的硅酸锰解离,从而弱化了硅酸锰小囊泡之间的连接而解体,形成大量分散的6-7nm小囊泡。该性能是MSNAs在静脉注射时保持结构和尺寸的稳定性从而提高血液中的循环时间,并通过肿瘤的高渗透和滞留效应高量聚集在肿瘤部位,并在进入弱酸性肿瘤微环境后响应性解体为小的硅酸锰囊泡,增加了材料在肿瘤组织部位的渗透和扩散。
由于在MSNAs合成过程中引入了高价的锰离子,因此MSNAs具有十分优异的谷胱甘肽消除能力,谷胱甘肽是细胞中的重要抗氧化剂和解毒剂,也是肿瘤多药耐药的重要因子,因此MSNAs的谷胱甘肽消除能力将有利于肿瘤的治疗。
可通过亚甲基蓝(MB)的降解实验来考查MSNAs的催化氧化能力。MSNAs的催化氧化效率依赖于谷胱甘肽,碳酸氢钠(二氧化碳)和过氧化氢的浓度。谷胱甘肽具有开关作用,只有在谷胱甘肽的存在下才能引发MSNAs的催化氧化作用,并且该催化氧化作用随谷胱甘肽,碳酸氢钠(二氧化碳)和过氧化氢的浓度的增加而提高。
线粒体是细胞的能量供应中心,通过三羧酸循环和电子传递链产生ATP,以满足细胞的主要能量需求。CO2是三羧酸循环过程的主要副产物,而且线粒体是唯一的细胞内CO2来源。当电子从电子传递链泄漏,会与O2反应形成超氧自由基(O2-·),然后通过线粒体中的超氧化物歧化酶2(SOD2)将其转化为过氧化氢(H2O2)。H2O2主要通过线粒体电子传递链生成,并参与各种信号传导途径。大多数恶性肿瘤与线粒体中CO2和H2O2产生的增加有关,这与众所周知的肿瘤微环境特征,如低pH和H2O2水平升高密切相关。同时线粒体中富集了更多的抗氧化剂谷胱甘肽(GSH)以消除ROS。因此,高水平的CO2、H2O2和GSH是肿瘤细胞线粒体的重要化学微环境特征。而这一特征与MSNAs的催化氧化规律高度匹配。有鉴于此,将常用的线粒体靶向分子三苯基溴化膦修饰在MSNAs上,得到MSNAs-TPP。
下面结合实施例以及附图来进一步对本发明的方案做详细的说明。
实施例1
本实例提供一种MSNAs-TPP多功能纳米颗粒的制备方法,该方法可以包括以下步骤:
步骤(1),将SiO2纳米颗粒分散在水中,得到溶液A;
具体地,所述SiO2纳米颗粒可以使用传统的法制备得到。进一步地,称取1.0g的氨水溶液,加入至包含30mL的无水乙醇和500μL的水的混合溶液中,后将1.5mL的TEOS滴加到上述溶液中。室温搅拌4h后,以12000rpm的转速离心10min收集SiO2纳米颗粒,用乙醇和水洗涤数次,然后再分散在10mL水中,得到60mg/mL溶液A。
步骤(2),将溶液A,水和无水乙醇混合,再添加高锰酸钾,然后将溶液转移至水热反应釜中反应;
具体地,将步骤(1)得到的2mL的60mg/mL的溶液A(即SiO2水溶液),13mL水和5mL无水乙醇混合,将100mg的高锰酸钾添加到混合物中并充分混合。然后将溶液转移至水热反应釜中,反应温度为190℃,反应时间为18h。
步骤(3),反应后冷却至室温,再离心收集MSNAs纳米颗粒;
具体地,将步骤(2)的溶液冷却至室温后,以12000rpm的转速离心10min收集MSNAs纳米颗粒,并用水洗涤数次以去除多余的残留物。
步骤(4),将MSNAs纳米颗粒添加到聚乙烯亚胺溶液中进行氨基化修饰,得到MSNAs-PEI,作为溶液B;
具体地,将10mg的MSNAs纳米颗粒添加到10mL聚乙烯亚胺溶液(PEI)中进行氨基化修饰,其中聚乙烯亚胺溶液的浓度为0.1mg/mL,将溶液在室温下搅拌3h,将得到的溶液离心并用水充分洗涤后,得到PEI修饰的MSNAs(即MSNAs-PEI)。
步骤(5),将三苯基溴化膦和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶解在二甲亚砜的水溶液中,得到溶液C;
具体地,本实例所述三苯基溴化膦为带有羧基的三苯基溴化膦,本实例以(4-羧丁基)三苯基溴化膦为例。将50mg的(4-羧丁基)三苯基溴化膦(TPP)和50mg的(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)(EDC)溶解在10mL的体积分数为10%的二甲亚砜(DMSO)的水溶液中,并在室温下避光搅拌15h,得到溶液C。
步骤(6),将n-羟基琥珀酰亚胺添加至溶液C中,在室温下搅拌以形成TPP活化液D;
具体地,将步骤(5)得到的溶液C中加入25mg的n-羟基琥珀酰亚胺(NHS),继续在室温下再搅拌5h以形成活化的TPP反应中间体溶液。
步骤(7),将所述的溶液D添加到溶液B中,并在室温下搅拌,离心,洗涤,得到MSNAs-TPP多功能纳米颗粒。
具体地,将80μL上述的TPP反应中间体溶液添加到10mL的1mg/mL的MSNAs-PEI溶液中,并在室温下搅拌3h,就得到了TPP修饰的MSNAs(MSNAs-TPP多功能纳米颗粒),进一步将得到的MSNAs-TPP多功能纳米颗粒离心并用水洗涤数次来纯化。
实施例2
本实例提供一种MSNAs-TPP多功能纳米颗粒的制备方法,该方法可以包括以下步骤:
步骤(1),将SiO2纳米颗粒分散在水中,得到溶液A;
具体地,所述SiO2纳米颗粒可以使用传统的法制备得到。进一步地,称取1.5g的氨水溶液,加入至包含30mL的无水乙醇和500μL的水的混合溶液中,后将1.0mL的TEOS滴加到上述溶液中。室温搅拌4h后,以12000rpm的转速离心10min收集SiO2纳米颗粒,用乙醇和水洗涤数次,然后再分散在10mL水中,得到40mg/mL的溶液A。
步骤(2),将溶液A,水和无水乙醇混合,再添加高锰酸钾,然后将溶液转移至水热反应釜中反应;
具体地,将步骤(1)得到的2mL的40mg/mL的溶液A(即SiO2水溶液),8mL水和10mL无水乙醇混合,将50mg的高锰酸钾添加到混合物中并充分混合。然后将溶液转移至水热反应釜中,反应温度为170℃,反应时间为30h。
步骤(3),反应后冷却至室温,再离心收集MSNAs纳米颗粒;
具体地,将步骤(2)的溶液冷却至室温后,以12000rpm的转速离心10min收集MSNAs纳米颗粒,并用水洗涤数次以去除多余的残留物。
步骤(4),将MSNAs纳米颗粒添加到聚乙烯亚胺溶液中进行氨基化修饰,得到MSNAs-PEI,作为溶液B;
具体地,将10mg的MSNAs纳米颗粒添加到10mL聚乙烯亚胺溶液(PEI)中进行氨基化修饰,其中聚乙烯亚胺溶液的浓度为1mg/mL,将溶液在室温下搅拌3h,将得到的溶液离心并用水充分洗涤后,得到PEI修饰的MSNAs(即MSNAs-PEI)。
步骤(5),将三苯基溴化膦和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶解在二甲亚砜的水溶液中,得到溶液C;
具体地,本实例所述三苯基溴化膦为带有羧基的三苯基溴化膦,本实例以(4-羧丁基)三苯基溴化膦为例。将25mg的(4-羧丁基)三苯基溴化膦(TPP)和50mg的(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)(EDC)溶解在10mL的体积分数为15%的二甲亚砜(DMSO)的水溶液中,并在室温下避光搅拌15h,得到溶液C。
步骤(6),将n-羟基琥珀酰亚胺添加至溶液C中,在室温下搅拌以形成TPP活化液D;
具体地,将步骤(5)得到的溶液C中加入50mg的n-羟基琥珀酰亚胺(NHS),继续在室温下再搅拌5h以形成活化的TPP反应中间体溶液。
步骤(7),将所述的溶液D添加到溶液B中,并在室温下搅拌,离心,洗涤,得到MSNAs-TPP多功能纳米颗粒。
具体地,将40μL上述的TPP反应中间体溶液添加到10mL的1mg/mL的MSNAs-PEI溶液中,并在室温下搅拌3h,就得到了TPP修饰的MSNAs(MSNAs-TPP多功能纳米颗粒),进一步将得到的MSNAs-TPP多功能纳米颗粒离心并用水洗涤数次来纯化。
实施例3
本实例提供一种MSNAs-TPP多功能纳米颗粒的制备方法,该方法可以包括以下步骤:
步骤(1),将SiO2纳米颗粒分散在水中,得到溶液A;
具体地,所述SiO2纳米颗粒可以使用传统的法制备得到。进一步地,称取1.4g的氨水溶液,加入至包含30mL的无水乙醇和500μL的水的混合溶液中,后将1.15mL的TEOS滴加到上述溶液中。室温搅拌4h后,以12000rpm的转速离心10min收集SiO2纳米颗粒,用乙醇和水洗涤数次,然后再分散在10mL水中,得到50mg/mL的溶液A。
步骤(2),将溶液A,水和无水乙醇混合,再添加高锰酸钾,然后将溶液转移至水热反应釜中反应;
具体地,将步骤(1)得到的2mL的50mg/mL的溶液A(即SiO2水溶液),11mL水和7mL无水乙醇混合,将74mg的高锰酸钾添加到混合物中并充分混合。然后将溶液转移至水热反应釜中,反应温度为180℃,反应时间为24h。
步骤(3),反应后冷却至室温,再离心收集MSNAs纳米颗粒;
具体地,将步骤(2)的溶液冷却至室温后,以12000rpm的转速离心10min收集MSNAs纳米颗粒,并用水洗涤数次以去除多余的残留物。
步骤(4),将MSNAs纳米颗粒添加到聚乙烯亚胺溶液中进行氨基化修饰,得到MSNAs-PEI,作为溶液B;
具体地,将10mg的MSNAs纳米颗粒添加到10mL聚乙烯亚胺溶液(PEI)中进行氨基化修饰,其中聚乙烯亚胺溶液的浓度为0.5mg/mL,将溶液在室温下搅拌3h,将得到的溶液离心并用水充分洗涤后,得到PEI修饰的MSNAs(即MSNAs-PEI)。优选地,所述聚乙烯亚胺可通过静电吸附作用连接于所述MSNAs纳米颗粒上。
步骤(5),将三苯基溴化膦和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶解在二甲亚砜的水溶液中,得到溶液C;
具体地,本实例所述三苯基溴化膦为带有羧基的三苯基溴化膦,本实例以(4-羧丁基)三苯基溴化膦为例。将25mg的(4-羧丁基)三苯基溴化膦(TPP)和25mg的(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)(EDC)溶解在10mL的体积分数为5%的二甲亚砜(DMSO)的水溶液中,并在室温下避光搅拌15h,得到溶液C。优选地,所述(4-羧丁基)三苯基溴化膦(TPP)通过EDC/NHS交联反应与MSNAs-PEI上的氨基形成酰胺键连接于所述MSNAs-PEI上。
步骤(6),将n-羟基琥珀酰亚胺添加至溶液C中,在室温下搅拌以形成TPP活化液D;
具体地,将步骤(5)得到的溶液C中加入15mg的n-羟基琥珀酰亚胺(NHS),继续在室温下再搅拌5h以形成活化的TPP反应中间体溶液。
步骤(7),将所述的溶液D添加到溶液B中,并在室温下搅拌,离心,洗涤,得到MSNAs-TPP多功能纳米颗粒。
具体地,将60μL上述的TPP反应中间体溶液添加到10mL的1mg/mL的MSNAs-PEI溶液中,并在室温下搅拌3h,就得到了TPP修饰的MSNAs(MSNAs-TPP多功能纳米颗粒),进一步将得到的MSNAs-TPP多功能纳米颗粒离心并用水洗涤数次来纯化。
图2中的(a),(b),(c)分别是MSNAs多级中空纳米囊泡的的SEM,TEM和高分辨TEM图,可以看出,MSNAs为规则的中空球体,尺寸约为100nm,尺寸均匀,表面由大量的更小的中空囊泡组成,小囊泡尺寸为6-7nm。图3是通过透射电镜EDX元素分析考查MSNAs的化学组成,Si、O和Mn元素均匀分布在整个空心粒子中。图4是MSNAs的X射线粉末衍射(XRD)图,显示了硅酸盐相的几个宽峰(JCPDS 41-1367),表明MSNAs是非晶形结构。图5是通过XPS进一步分析MSNAs中Mn的价态。Mn2p3/2的XPS曲线在641.2ev、642.0ev和643.1ev处有三个特征峰,分别对应17.5%Mn(Ⅱ)、23.6%Mn(Ⅲ)和58.9%Mn(Ⅳ),证明了MSNAs中高量高价锰的存在。图6是MSNAs的N2吸附-解吸等温线,表明MSNAs的表面积高达416.7m2/g。图7为TPP,MSNAs和MSNAs-TPP的FTIR图谱,MSNAs-TPP在1656cm-1处有一条很强的谱带是由于酰胺的羰基伸缩振动引起的,表明酰胺键的形成。C-P键在1437cm-1处出现伸缩振动,P与三个苯基的键合出现在1111cm-1。这些证据证实了MSNAs-TPP纳米复合材料的成功制备。
实施例4
本实施例提供一种上述实施例1-3任一项的制备方法制备得到的基于MSNAs-TPP多功能纳米颗粒在线粒体靶向纳米治疗剂诱导线粒体氧化应激的智能纳米平台药物中的应用。
该应用包括:该纳米药物静脉注射后具有良好的血液循环稳定性和肿瘤靶向作用,实现肿瘤酸性微环境响应的解体和深度扩散,且通过小鼠的核磁共振成像监测了这一过程。通过肿瘤细胞线粒体的靶向实现了胞内活性氧的有效放大,有效诱导了肿瘤细胞的凋亡。制备得到的MSNAs-TPP是由中空硅酸锰小囊泡组成的多级中空纳米颗粒,合适的尺寸和良好的胶体稳定性使MSNAs-TPP具有较长的血液循环时间和肿瘤靶向作用。到达肿瘤部位后,在酸性微环境下,由于部分硅酸锰的解离,MSNAs-TPP裂解成硅酸锰小囊泡,从而能够在肿瘤组织中深度扩散。由于线粒体靶向分子TPP的修饰,上述小囊泡被肿瘤细胞胞吞后靶向聚集到线粒体部位,消耗线粒体谷胱甘肽的同时释放出锰离子,锰离子同线粒体呼吸作用产生的副产物CO2和H2O2一起通过催化氧化反应放大了胞内活性氧水平,并最终诱发了肿瘤细胞凋亡。
以下实施例对实施例3中的MSNAs-TPP纳米颗粒进行性能表征。
实施例5
将上述得到的MSNAs浸泡于pH=6.5的PBS缓冲溶液中考查MSNAs在弱酸性环境下的解体性能。图8中的(a)为MSNAs纳米颗粒在弱酸性溶液中解体后的透射电镜图片,图8中的(b)为解体前后动态光散射粒径分布分析。可以看出MSNAs可以在弱酸性溶液中解体,由尺寸较大的中空硅酸锰纳米颗粒变为尺寸较小的硅酸锰小囊泡。将MSNAs加入到10mM的谷胱甘肽溶液中,反应1h,考查其生物降解性能。图9为MSNAs纳米颗粒溶液加入谷胱甘肽前后的紫外可见吸收曲线和溶液的光学照片,MSNAs与谷胱甘肽反应后,紫外可见吸收几乎消失,溶液颜色由棕色变成无色透明,并伴随丁达尔效应的消失,由此可见,MSNAs可以在生理谷胱甘肽浓度下完全降解为离子。
实施例6
将1.3mg/mL的MSNAs加入到10mM的谷胱甘肽溶液中,在不同时间点取上清溶液检测剩余谷胱甘肽的量,并与其他硅酸锰材料(HMSNs)做对比,考查MSNAs的谷胱甘肽消除能力。图10为MSNAs和对照材料HMSNs对谷胱甘肽的消耗曲线,由此可见,MSNAs具有十分优异的谷胱甘肽消除性能。
实施例7
取2mL的MSNAs溶液,加入50μL亚甲基蓝,并分别加入不同浓度的谷胱甘肽溶液(0mM,1mM,2mM),过氧化氢溶液(4mM,8mM,16mM)和碳酸氢钠溶液(15mM,25mM,40mM),分别考查谷胱甘肽,过氧化氢和碳酸氢钠对MSNAs催化氧化的影响。图11为MSNAs在不同浓度(a)谷胱甘肽,(b)H2O2和(c)NaHCO3条件下催化亚甲基蓝(MB)动力学曲线。可以看出,MSNAs的催化氧化效率依赖于谷胱甘肽,碳酸氢钠(二氧化碳)和过氧化氢的浓度。谷胱甘肽具有开关作用,只有在谷胱甘肽的存在下才能引发MSNAs的催化氧化作用,并且该催化氧化作用随谷胱甘肽、碳酸氢钠和过氧化氢的浓度的增加而提高。
实施例8
Mn2+是良好的核磁共振成像T1造影剂,我们将MSNAs分别浸泡在pH=7.4、pH=6.5、pH=7.4/10mM谷胱甘肽和pH=6.5/10mM谷胱甘肽的PBS溶液中,考查其在不同溶液环境下的Mn2+释放性能。并将这四种溶液按照浓度梯度:0.0625mM,0.125mM,0.25mM,0.5mM和1mM在临床使用的3T磁共振成像系统上检测其T1磁共振造影性能。图12为MSNAs在不同溶液条件下Mn2+离子释放规律图,图13为MSNAs在不同溶液条件下的核磁共振造影成像弛豫率r1测试。由此可见,MSNAs可以灵敏的响应谷胱甘肽并释放Mn2+,并且是一种优异的谷胱甘肽响应型的磁共振造影剂。
实施例9
本实验通过人脐静脉血管内皮细胞(HUVEC)来评价MSNAs和MSNAs-TPP的细胞相容性。将HUVEC细胞种在96孔板中,等到细胞生长到对数生长期时,按照浓度梯度:5μg/mL、10μg/mL、15μg/mL、20μg/mL、25μg/mL、30μg/mL将MSNAs和MSNAs-TPP加入到细胞中,24小时后,用cck8法检测细胞活性。图14为MSNAs和MSNAs-TPP的人脐静脉血管内皮细胞(HUVEC)相容性测试,可以看出,在一定的浓度范围内(0-30μg/mL),MSNAs和MSNAs-TPP都具有良好的细胞相容性。随后,我们评价了MSNAs和MSNAs-TPP的体外抗肿瘤性能,使用的肿瘤细胞模型是4T1小鼠乳腺癌细胞。将4T1细胞种在96孔板中,等到细胞生长到对数生长期时,按照浓度梯度:5μg/mL、10μg/mL、15μg/mL、20μg/mL、25μg/mL、30μg/mL将MSNAs和MSNAs-TPP加入到细胞中,24小时后,用cck8法检测细胞活性。图15为不同浓度MSNAs和MSNAs-TPP对4T1小鼠乳腺癌细胞的细胞活性抑制实验结果,可以看出,4T1肿瘤细胞活性均随着材料浓度的升高而降低,并且MSNAs-TPP的抗肿瘤性能要显著优于MSNAs。为了揭示MSNAs和MSNAs-TPP的抗肿瘤机理,我们以DCFH-DA作为荧光探针检测了MSNAs和MSNAs-TPP处理的4T1细胞的胞内氧化应激水平。图16为MSNAs和MSNAs-TPP处理4T1小鼠乳腺癌细胞后胞内活性氧含量检测荧光照片结果。由此可见,MSNAs和MSNAs-TPP都能引起胞内氧化应激水平的上升,但是MSNAs-TPP的作用更加明显。图17为MSNAs在不同细胞培养环境下对4T1小鼠乳腺癌细胞的细胞活性抑制实验。实现结果表明当在细胞培养基中加入100μM的H2O2和15mM的NaHCO3时,MSNAs的4T1细胞杀伤作用都会增强,这与MSNAs催化氧化特性相吻合,并解释了MSNAs-TPP相比于MSNAs抗肿瘤效果提升的原因。
实施例10
本实验通过小鼠的核磁共振成像来评价MSNAs-TPP的肿瘤组织内扩散和肿瘤靶向性能。将MSNAs-TPP溶液直接瘤内注射于4T1荷瘤小鼠的肿瘤内,用临床3T核磁共振成像系统对小鼠进行磁共振扫描,扫描序列主要采用T1WI序列,参数如下:TR:520ms;TE:15ms;FOV:7×7cm;层厚:0.8mm;层间距:0.5mm;激励次数(Nex):2;矩阵:256×256;最大层数:21。图18中的(a)为MSNAs-TPP瘤内注射后肿瘤核磁共振成像照片,瘤内注射MSNAs-TPP溶液后,在肿瘤组织内注射区域周围清晰地观察到MRI信号增强。注射2h后,MRI信号分布于肿瘤各处,提示MSNAs-TPP由于其对肿瘤微环境的反应性解体而具有良好的肿瘤穿透性能。随后,我们将MSNAs-TPP溶液尾静脉注射到荷瘤小鼠体内,图18中的(b)为MSNAs-TPP尾静脉注射后肿瘤核磁共振成像照片,由此可见,肿瘤部位的磁共振信号随着时间而加强,说明MSNAs-TPP是一种良好的磁共振成像造影剂,同时也说明了MSNAs-TPP可通过肿瘤的高渗透和滞留效应高效靶向到肿瘤部位。
实施例11
本实验通过在动物(小鼠)层面的肿瘤杀伤来说明材料在肿瘤细胞杀伤方面的应用。随机将注射4T1细胞后的老鼠分为四组,组1注射PBS溶液,组2注射对照材料HMSNs纳米颗粒溶液(5mg/mL,150μL),组3注射MSNAs纳米颗粒溶液(5mg/mL,150μL),组4注射MSNA-TPPs纳米颗粒溶液(5mg/mL,150μL)。每隔一天测量老鼠的肿瘤尺寸。图19为不同处理方式下小鼠肿瘤体积变化曲线。可以看出,对照组(组1)的肿瘤体积大小随着时间变化不断增大,而MSNAs注射组和MSNAs-TPP注射组的肿瘤体积大小得到比较明显的遏制,并且MSNAs-TPP注射组的抑瘤效果最为显著。
实施例12
我们对MSNAs-TPP在小鼠体内长期的生物分布及其毒性进行测试。将MSNAs-TPP溶液通过尾静脉注射到小鼠体内,将小鼠处死,取出主要的脏器和肿瘤进行称重,用硝酸:高氯酸=9:1的酸液在高温下消解组织,定容并用等离子体发射(ICP-OES)测定其中锰离子的含量,并计算每克组织中的锰含量。收集不同时间点(3天,7天和30天)的小鼠的血液进行小鼠的血生化和血常规指标的测定。另外取出小鼠主要的脏器(心,肝,脾,肺和肾)用4%甲醛固定,切片,并用苏木精-伊红染色法进行染色,在光学显微镜下观察MSNAs-TPP对主要的脏器有无损伤。图20为各主要脏器和肿瘤的生物分布检查,图21为小鼠的血液生化检查(包括肌酐,白蛋白,谷丙转氨酶,谷草转氨酶,直接胆红素,总胆红素,胆碱酯酶和总蛋白),图22为治疗后小鼠体内各主要脏器的苏木精-伊红(H&E)切片检查。由此可见,MSNAs-TPP静脉注射后可以在小鼠体内顺利代谢,体内各血液生化指标表现正常,也未对各主要脏器造成损伤,说明了MSNAs-TPP具有良好的生物安全性。
应理解为上述实施例只用于对本发明进行进一步说明,不能理解为对本发明保护范围的限制,本领域的技术人员根据本发明的上述内容作出的一些非本质的改进和调整均属于本发明的保护范围。上述实例具体的工艺参数等也仅是合适范围中的一个示例,即本领域技术人员可以通过本文的说明做合适的范围内选择,而并非要限定于下文示例的具体数值。
本领域技术人员在考虑说明书及实践这里公开的内容后,将容易想到本申请的其它实施方案。本申请旨在涵盖本申请的任何变型、用途或者适应性变化,这些变型、用途或者适应性变化遵循本申请的一般性原理并包括本申请未公开的本技术领域中的公知常识或惯用技术手段。说明书和实施例仅被视为示例性的,本申请的真正范围和精神由下面的权利要求指出。
应当理解的是,本申请并不局限于上面已经描述并在附图中示出的精确结构,并且可以在不脱离其范围进行各种修改和改变。本申请的范围仅由所附的权利要求来限制。
Claims (10)
1.一种MSNAs-TPP多功能纳米颗粒的制备方法,其特征在于,包括:
将SiO2纳米颗粒分散在水中,得到溶液A;
将溶液A,水和无水乙醇混合,再添加高锰酸钾,然后将溶液转移至水热反应釜中反应;
反应后冷却至室温,再离心收集MSNAs纳米颗粒;
将MSNAs纳米颗粒添加到聚乙烯亚胺溶液中进行氨基化修饰,得到MSNAs-PEI,作为溶液B;
将三苯基溴化膦和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶解在二甲亚砜的水溶液中,得到溶液C;
将n-羟基琥珀酰亚胺添加至溶液C中,在室温下搅拌以形成TPP活化液D;
将所述的溶液D添加到溶液B中,并在室温下搅拌,离心,洗涤,得到MSNAs-TPP多功能纳米颗粒。
2.根据权利要求1所述的制备方法,其特征在于,所述溶液A中SiO2纳米颗粒的浓度为40-60mg/mL。
3.根据权利要求1所述的制备方法,其特征在于,所述溶液A、水和无水乙醇的体积比为1:(4~6.5):(5~2.5)。
4.根据权利要求1所述的制备方法,其特征在于,所述高锰酸钾与溶液A、水和无水乙醇的混合溶液的配比为(2.5~5):1(mg/mL)。
5.根据权利要求1所述的制备方法,其特征在于,所述水热反应釜中的反应温度为170-190℃,反应时间为18-30h。
6.根据权利要求1所述的制备方法,其特征在于,所述聚乙烯亚胺溶液的浓度为0.1~1mg/mL,MSNAs纳米颗粒和聚乙烯亚胺的质量比为(10~1):1。
7.根据权利要求1所述的制备方法,其特征在于,所述三苯基溴化膦在二甲亚砜水溶液中的浓度为2.5~5 mg/mL,三苯基溴化膦和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的质量比为1:(1~2),二甲亚砜水溶液的体积分数为5%-15%。
8.根据权利要求1所述的制备方法,其特征在于,所述n-羟基琥珀酰亚胺与1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的质量比为1:(1~2)。
9.根据权利要求1所述的制备方法,其特征在于,所述溶液B和溶液D的体积比为(125~250):1。
10.一种权利要求1所述的MSNAs-TPP多功能纳米颗粒的制备方法所得到的多功能纳米颗粒在制备线粒体靶向纳米治疗剂诱导线粒体氧化应激的智能纳米平台药物中的应用。
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