CN110393805A - 纳米酶修饰的聚合物载体及其制备方法、抗肿瘤纳米粒子 - Google Patents
纳米酶修饰的聚合物载体及其制备方法、抗肿瘤纳米粒子 Download PDFInfo
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Abstract
本发明涉及一种纳米酶修饰的聚合物载体及其制备方法、抗肿瘤纳米粒子。所述纳米酶修饰的聚合物载体的制备方法包括以下步骤:于二甲基亚砜中溶解4‑羧基苯硼酸频那醇酯,加入活化剂,得活化液;于水中溶解聚乙烯亚胺,加入所述活化液,第一次搅拌,得第一中间产物;于多糖上负载纳米酶,得第二中间产物;向所述第二中间产物中加入所述第一中间产物,第二次搅拌,得纳米酶修饰的聚合物载体。上述方法制得的聚合物载体可吸附承载负电荷的功能分子自组装形成稳定的纳米粒子,能在肿瘤组织较高H2O2含量和酸性环境条件下,实现药物功能分子在靶部位的特异性释放,同时,还能在较高H2O2含量和酸性环境条件下产生氧气,改善组织缺氧,并降低耐药性。
Description
技术领域
本发明涉及高分子化学、纳米医学领域,特别是涉及纳米酶修饰的聚合物载体及其制备方法、抗肿瘤纳米粒子。
背景技术
在现阶段,肿瘤诊断与治疗是纳米技术研究的重要方向,有利于改善肿瘤微环境的药物响应,涉及肿瘤药物的靶向释放,肿瘤微环境内的免疫应答。肿瘤化疗药物的高靶向性可使药物在肿瘤部位富集,从而减少副作用,故肿瘤化疗药物的靶向性研究是目前研究的热点问题。而且,由于细胞膜的生理特性,带负电荷的药物难以进入到细胞,发挥其对细胞的杀伤作用。因此,目前大多通过设计构建药物递送载体来实现药物入胞的目的。
但是,虽然已有的靶向递药体系能在肿瘤部位富集,却很难实现药物利用肿瘤微环境的特异释放。而且,肿瘤部位与正常组织相比具有偏酸和富集H2O2的特点,传统的递送体系旨在改善药物理化性质上的缺点,完成特异部位的靶向递药功能,受限于独特的微环境,往往药效不如预期,仍具有高的耐药性。
发明内容
基于此,本发明提供一种纳米酶修饰的聚合物载体的制备方法,所制得的聚合物载体可吸附承载负电荷的功能分子自组装形成稳定的纳米粒子,能在肿瘤组织较高H2O2含量和酸性环境条件下,实现药物功能分子在靶部位的特异性释放,同时,还能在较高H2O2含量和酸性环境条件下产生氧气,改善组织缺氧,并降低耐药性。
具体技术方案为:
一种纳米酶修饰的聚合物载体的制备方法,包括以下步骤:
于二甲基亚砜中溶解4-羧基苯硼酸频那醇酯,加入活化剂,得活化液;
于水中溶解聚乙烯亚胺,加入所述活化液,第一次搅拌,得第一中间产物;
于多糖上负载纳米酶,得第二中间产物;
向所述第二中间产物中加入所述第一中间产物,第二次搅拌,得纳米酶修饰的聚合物载体。
在其中一个实施例中,所述多糖选自透明质酸、果糖、羧基化壳聚糖和葡聚糖中的一种。
在其中一个实施例中,所述纳米酶选自纳米二氧化铈、纳米氧化铁和纳米氧化锰中的一种。
在其中一个实施例中,所述多糖为透明质酸,所述纳米酶为纳米二氧化铈,所述于多糖上负载纳米酶的具体方法为:
于水中溶解所述透明质酸,加入三价铈盐,反应20min-40min后,再加入碱性试剂,搅拌至溶液呈淡黄色。
在其中一个实施例中,所述第一中间产物与所述第二中间产物的摩尔比为1:(0.02-1)。
在其中一个实施例中,所述透明质酸的分子量为700Da-400000Da。
在其中一个实施例中,所述第一次搅拌的时间为24h-48h。
在其中一个实施例中,所述第二次搅拌的时间为20min-40min。
在其中一个实施例中,所述聚乙烯亚胺的分子量为800Da-25000Da。
在其中一个实施例中,所述活化剂选自二环己基碳二亚胺和N-羟基琥珀酰亚胺。
本发明还提供上述制备方法制得的纳米酶修饰的聚合物载体。
本发明还提供一种抗肿瘤纳米粒子。
具体技术方案为:
一种抗肿瘤纳米粒子,其原料包括上述纳米酶修饰的聚合物载体以及阴离子功能分子。
在其中一个实施例中,所述阴离子功能分子选自带负电的荧光染料、带负电的药物。
在其中一个实施例中,所述阴离子功能分子为吲哚菁绿和带负电荷的肿瘤抗原分子中的一种。
与现有技术相比,本发明具有以下有益效果:
本发明发明人结合长期对金属氧化物抗氧化和过氧化氢催化的研究,设计合成了以多糖为母链的纳米酶复合物,尤其是以透明质酸为母链的氧化铈复合物。之后通过苯硼酸介导的特异性点击修饰,将上述复合物修饰与丙聚乙烯亚胺-苯硼酸(PEI-PBA)纳米结构表面,制备了聚乙烯亚胺-苯硼酸-多糖/纳米酶的多功能聚合物载体。所述聚合物载药体系兼具对肿瘤组织的靶向和靶部位的特异释放,其中,在多糖上引入纳米酶,将长效的纳米功能材料引入肿瘤局部微环境,改善药物的治疗效率,并通过纳米酶的生物学研究,提高肿瘤微环境中的免疫应答。更重要的是,以多糖为母链的纳米酶复合物能够将肿瘤部位过表达的过氧化氢转化为氧气分子,改善肿瘤乏氧,有利于负载的功能分子进一步发挥作用,降低耐药性。同时,利用肿瘤组织富含H2O2和酸性微环境的特点,引起聚合物载体分子内共价键的断裂,使苯硼酸的硼酸基脱落,破坏纳米结构,使负载的功能分子特异性释放。所述聚合物载体既可以携带药物,在肿瘤部位特异性释放,又能携带荧光染料,对肿瘤组织成像,实现诊疗一体。
可利用上述聚合物载体分子自身结构的两亲性,完成温和条件下的纳米结构的自组装。即通过聚合物载体所带的正电荷,吸附阴离子功能分子,自组装形成稳定性、分散性良好的纳米结构。具体为:第一步,活化后的4-羧基苯硼酸频那醇酯(PBA)上的羧基与PEI(聚乙烯亚胺)的伯胺基团反应形成酰胺键,合成PEI-PBA;第二步,在多糖引入纳米酶形成多糖/纳米酶复合体系,优选为透明质酸/氧化铈复合体系(HA-CeNPs);第三步,利用PEI-PBA的苯硼酸上的羟基与具有肿瘤靶向的多糖上的顺二醇反应形成的分子间引力,结合载体上所带的亲疏水基团和正电荷,完成对阴离子功能分子的吸附和组装,得到稳定的球状纳米粒子。实现阴离子功能分子与纳米酶协同作用、增强肿瘤治疗效率。所述阴离子功能分子选自带负电荷的荧光染料、药物等。上述聚乙烯亚胺-苯硼酸-透明质酸/纳米酶载体可以克服这些阴离子功能分子到达肿瘤组织前在生物体液中的降解,保护功能分子通过恶劣的人体内环境和免疫系统的攻击,还有利于实现肿瘤多功能化治疗体系构建,且具有很高的靶细胞给药潜力。
相比于传统递药体系,本发明的纳米粒表面化学性质可控,并且在温和条件下就能实现阴离子药物在聚合物载体上的固定,实现载体的多功能化。
附图说明
图1为第一中间产物PEI-PBA的透射电镜图
图2为第二中间产物HA/CeO2的透射电镜图;
图3为纳米酶修饰的聚合物载体PEI-PBA-HA/CeO2的透射电镜图;
图4为纳米酶修饰的聚合物载体PEI-PBA-HA/CeO2与阴离子功能分子的自组装过程示意图;
图5为不同浓度的PEI-PBA-HA/CeO2与ICG反应后离心情况示意图;
图6为不同浓度的PEI-PBA-HA/CeO2吸附带负电蛋白质的SDS-PAGE电泳凝胶条带示意图;
图7为纳米酶修饰的聚合物载体与H2O2反应产物的透射电镜图;
图8为纳米酶修饰的聚合物载体与H2O2反应产生氧气情况示意图;
图9为氧化铈过氧化氢酶检测结果示意图;
图10为光敏剂在肿瘤靶向的递送结果示意图。
具体实施方式
以下结合具体实施例对本发明作进一步详细的说明。本发明可以以许多不同的形式来实现,并不限于本文所描述的实施方式。相反地,提供这些实施方式的目的是使对本发明公开内容理解更加透彻全面。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
以下具体实施方式中所采用的原料,若无特殊说明,均可来源于市售。
实施例1
本实施例提供一种纳米酶修饰的聚合物载体及其制备方法,步骤如下:
步骤1、于50mL的二甲基亚砜(DMSO)中溶解0.75g的4-羧基苯硼酸频那醇酯(PBA),然后加入0.94g的二环己基碳二亚胺(DCC)和0.52g的N-羟基琥珀酰亚胺(NHS),搅拌活化2h,得活化液。
步骤2、于50ml超纯水中溶解0.06g分子量为800Da的聚乙烯亚胺(PEI),得PEI水溶液,将活化液移至恒压漏斗,室温下,向所述PEI水溶液中边搅拌边缓慢滴加上述活化液,全部滴加完毕后,继续搅拌24h,使混合体变为带有乳光的均匀体系,用截留分子量为8000Da的透析袋透析两天后收样,得到第一中间产物PEI-PBA,所述第一中间产物PEI-PBA的透射电镜图如图1所示。
步骤3、于48ml水中溶解0.1g的分子量为700Da的透明质酸钠(HA),完全溶解后定容至50mL,再加入200μL 0.7M的CeCl3·7H2O,室温搅拌反应30min后,快速搅拌并加入100μL浓氨水,搅拌至溶液变为淡黄色,用截留分子量为14000Da的透析袋透析两天后收样,得第二中间产物HA/CeO2。所述第二中间产物HA/CeO2的透射电镜图如图2所示。
步骤4、向第二中间产物HA/CeO2中边搅拌边滴加完上述第一中间产物PEI-PBA[经过计算,控制中间产物PEI-PBA与HA/CeO2的摩尔比在1:(0.02-1)的范围内],室温搅拌30min,得聚合物载体PEI-PBA-HA/CeO2,所述聚合物载体PEI-PBA-HA/CeO2的透射电镜图如图3所示。
本实施例还提供一种抗肿瘤纳米粒子及其制备方法,步骤如下:
取上述聚合物载体PEI-PBA-HA/CeO2,加入ICG(吲哚菁绿)水溶液,涡旋5min混匀吸附,避光静置。另外,还可参见图4的顺序,制备上述抗肿瘤纳米粒子。
图5为不同浓度的PEI-PBA-HA/CeO2与ICG反应后离心情况示意图。从左到右,PEI-PBA-HA/CeO2的浓度分别为8mg/mL、4mg/mL、2mg/mL和0mg/mL,最右为等量ICG水溶液对照,ICG的浓度均为40μg/mL。
由图5可知,ICG吸附到载体,组装在载体内核,在高速离心条件下变为下沉液,而游离的ICG不受离心作用仍为均匀的溶液,颜色不发生改变;加载体组离心后出现沉淀,上清颜色变浅,并且所加载体量越多,上清残余游离ICG越少。说明ICG可以通过电荷吸附至聚合物载体PEI-PBA-HA/CeO2,并且可以组装在载体内核。
实施例2
本实施例提供一种纳米酶修饰的聚合物载体及其制备方法,步骤如下:
步骤1、于55mL的二甲基亚砜(DMSO)中溶解0.75g的4-羧基苯硼酸频那醇酯(PBA),然后加入0.94g的二环己基碳二亚胺(DCC)和0.52g的N-羟基琥珀酰亚胺(NHS),搅拌活化2h,得活化液。
步骤2、于55ml超纯水中溶解0.11g分子量为800Da的聚乙烯亚胺(PEI),得PEI水溶液,将活化液移至恒压漏斗,室温下,向所述PEI水溶液中边搅拌边缓慢滴加上述活化液,全部滴加完毕后,继续搅拌24h,使混合体变为带有乳光的均匀体系,用截留分子量为8000Da的透析袋透析两天后收样,得到第一中间产物PEI-PBA。
步骤3、于48ml水中溶解0.1g的分子量为700Da的透明质酸钠(HA),完全溶解后定容至50mL,再加入200μL 0.7M的CeCl3·7H2O,室温搅拌反应30min后,快速搅拌并加入100μL浓氨水,搅拌至溶液变为淡黄色,用截留分子量为14000Da的透析袋透析两天后收样,得第二中间产物HA/CeO2。
步骤4、向第二中间产物HA/CeO2中边搅拌边滴加完上述第一中间产物PEI-PBA[经过计算,控制中间产物PEI-PBA与HA/CeO2的摩尔比在1:(0.02-1)的范围内],室温搅拌30min,得聚合物载体PEI-PBA-HA/CeO2。
本实施例还提供一种抗肿瘤纳米粒子及其制备方法,步骤如下:
取上述聚合物载体PEI-PBA-HA/CeO2,加入带负电荷蛋白质水溶液,静置30min至蛋白质和载体吸附完全。
图6为浓度分别为100μg/mL、200μg/mL、400μg/mL和800μg/mL的PEI-PBA-HA/CeO2吸附带负电蛋白质的SDS-PAGE电泳凝胶条带示意图。1-4分别对应为上述浓度体系离心后上清游离蛋白质,5为等量蛋白质空白对照,6-9对应为离心后与上述浓度体系PEI-PBA-HA/CeO2吸附的蛋白质。其中蛋白质的用量均为25μg。
由图6可知,带负电荷的蛋白质吸附在聚合物载体上,通过离心作用,出现在沉淀中,而为吸附的游离蛋白质则仍在上清液中,通过分别将离心后的沉淀和上清液分离上样做SDS-PAGE凝胶电泳分析,以不加载体的游离蛋白质作为对照。加入载体量越多,上清液的游离蛋白质条带逐渐变浅,而沉淀的蛋白质条带逐渐加深。
实施例3
本实施例提供一种纳米酶修饰的聚合物载体及其制备方法,步骤如下:
步骤1、于60mL的二甲基亚砜(DMSO)中溶解0.75g的4-羧基苯硼酸频那醇酯(PBA),然后加入0.94g的二环己基碳二亚胺(DCC)和0.52g的N-羟基琥珀酰亚胺(NHS),搅拌活化2h,得活化液。
步骤2、于60ml超纯水中溶解0.33g分子量为800Da的聚乙烯亚胺(PEI),得PEI水溶液,将活化液移至恒压漏斗,室温下,向所述PEI水溶液中边搅拌边缓慢滴加上述活化液,全部滴加完毕后,继续搅拌24h,使混合体变为带有乳光的均匀体系,用截留分子量为8000Da的透析袋透析两天后收样,得到第一中间产物PEI-PBA。
步骤3、于48ml水中溶解0.1g的分子量为700Da的透明质酸钠(HA),完全溶解后定容至50mL,再加入200μL 0.7M的CeCl3·7H2O,室温搅拌反应30min后,快速搅拌并加入100μL浓氨水,搅拌至溶液变为淡黄色,用截留分子量为14000Da的透析袋透析两天后收样,得第二中间产物HA/CeO2。
步骤4、向第二中间产物HA/CeO2中边搅拌边滴加完上述第一中间产物PEI-PBA[经过计算,控制中间产物PEI-PBA与HA/CeO2的摩尔比在1:(0.02-1)的范围内],室温搅拌30min,得聚合物载体PEI-PBA-HA/CeO2。
实施例1-3可制备能够稳定保存的聚合物载体PEI-PBA-HA。另外,保证HA/CeO2过量,且浓度较低,可以得到稳定的PEI-PBA-HA/CeO2类球形纳米结构。
实施例4
1、向实施例1所制得纳米酶修饰聚合物载体中加入H2O2,反应产物的透射电镜图如图7所示,说明分子中硼酸基与苯环相连的键是H2O2敏感的,而聚合物载分子的HA通过顺式二醇与硼酸基上的两个羟基反应连接,在H2O2的作用下,可以达到缓慢释放所连的功能分子的作用。其中,硼酸基脱落时发生如下反应:
2、取两份实施例1所制得的纳米酶修饰聚合物载体,分别加超纯水稀释得到浓度为6.7mg/mL和2mg/mL的两组溶液,并设置等体积的超纯水作为空白对照组。
向上述三组溶液中加入H2O2,产生氧气的情况如图8所示,可见,本发明所制得的纳米酶修饰聚合物载体可将肿瘤部位过表达的过氧化氢转化为氧气分子,改善肿瘤乏氧,有利于负载的功能分子进一步发挥作用,降低耐药性。
3、使用过氧化氢酶活力检测试剂盒,检测氧化铈和实施例1的纳米酶修饰聚合物载体的过氧化氢酶活性,结果证实实施例1的纳米酶修饰聚合物载体能够完成过氧化氢酶的催化作用,如图9所示。
实施例5
使用热成像仪检测光敏剂在肿瘤靶向的递送。使用光敏剂吲哚菁绿作为模式药物,经实施例3的纳米酶修饰聚合物载体吸附装载后,进行荷瘤小鼠尾静脉注射,24小时后用808nm激光器照射肿瘤部位,评价肿瘤升温的能力,并设置游离药物组、生理盐水组和靶向载体组进行对照,由此判断上述纳米酶修饰聚合物载体靶向递送的能力强,能够增强光敏剂疗效。结果如图10所示,结果显示:实施例3的纳米酶修饰聚合物载体吸附装载光敏剂吲哚菁绿后,升温效率高于游离药物组和生理盐水对照组。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (10)
1.一种纳米酶修饰的聚合物载体的制备方法,其特征在于,包括以下步骤:
于二甲基亚砜中溶解4-羧基苯硼酸频那醇酯,加入活化剂,得活化液;
于水中溶解聚乙烯亚胺,加入所述活化液,第一次搅拌,得第一中间产物;
于多糖上负载纳米酶,得第二中间产物;
向所述第二中间产物中加入所述第一中间产物,第二次搅拌,得纳米酶修饰的聚合物载体。
2.根据权利要求1所述的纳米酶修饰的聚合物载体的制备方法,其特征在于,所述多糖选自透明质酸、果糖、羧基化壳聚糖和葡聚糖中的一种。
3.根据权利要求1所述的纳米酶修饰的聚合物载体的制备方法,其特征在于,所述纳米酶选自纳米二氧化铈、纳米氧化铁和纳米二氧化锰中的一种。
4.根据权利要求1所述的纳米酶修饰的聚合物载体的制备方法,其特征在于,所述多糖为透明质酸,所述纳米酶为纳米二氧化铈,所述于多糖上负载纳米酶的具体方法为:
于水中溶解所述透明质酸,加入三价铈盐,反应20min-40min后,再加入碱性试剂,搅拌至溶液呈淡黄色。
5.根据权利要求4所述的纳米酶修饰的聚合物载体的制备方法,其特征在于,所述第一中间产物与所述第二中间产物的摩尔比为1:(0.02-1)。
6.根据权利要求4所述的纳米酶修饰的聚合物载体的制备方法,其特征在于,所述透明质酸的分子量为700Da-400000Da;及/或,
所述聚乙烯亚胺的分子量为800Da-25000Da。
7.一种权利要求1-6任一项所述的制备方法制得的纳米酶修饰的聚合物载体。
8.一种抗肿瘤纳米粒子,其特征在于,其原料包括权利要求7所述的纳米酶修饰的聚合物载体以及阴离子功能分子。
9.根据权利要求8所述的抗肿瘤纳米粒子,其特征在于,所述阴离子功能分子选自带负电的荧光染料和带负电的药物中的一种。
10.根据权利要求8或9所述的抗肿瘤纳米粒子,其特征在于,所述阴离子功能分子为吲哚菁绿和带负电荷的肿瘤抗原分子中的一种。
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