CN116421740A - 一种主动靶向性和氧化还原敏感型纳米递送载体及其制备方法与应用 - Google Patents

一种主动靶向性和氧化还原敏感型纳米递送载体及其制备方法与应用 Download PDF

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CN116421740A
CN116421740A CN202310216612.3A CN202310216612A CN116421740A CN 116421740 A CN116421740 A CN 116421740A CN 202310216612 A CN202310216612 A CN 202310216612A CN 116421740 A CN116421740 A CN 116421740A
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崔兰
徐晴晴
杨硕晔
张鹏帅
屈凌波
娄卫爽
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Henan University of Technology
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Abstract

本发明公开了一种主动靶向性和氧化还原敏感型纳米递送载体，所述纳米递送载体为采用苯硼酸和γ‑聚谷氨酸修饰壳寡糖所得，制备方法包括以下具体步骤：(1)在苯硼酸PBA和壳寡糖COS中加入引发剂后反应，得到PBA‑COS；(2)在所述PBA‑COS和γ‑聚谷氨酸γ‑PGA中加入交联剂后反应，得到一种主动靶向性和氧化还原敏感型纳米递送载体PBA‑COS/γ‑PGA。本发明纳米载体制备方法简单，粒径分布均匀、理化性质稳定，载体对DOX的包封率、装载量高，并能实现在模拟肿瘤微环境中的响应性释药，实现对肝癌细胞的靶向作用，降低了潜在毒性，提高了抗肿瘤效果。

Description

一种主动靶向性和氧化还原敏感型纳米递送载体及其制备方 法与应用
技术领域
本发明属于药物制剂技术领域，具体涉及一种主动靶向性和氧化还原敏感型纳米递送载体及其制备方法与应用。
背景技术
肝细胞癌是世界上最常见的恶性肿瘤之一，其发病率呈逐年上升趋势。化疗是对不能切除的晚期肝内疾病的主要治疗方法，而化疗药物通常会引起全身效应、不可逆毒副作用、体内多药耐药风险等问题。肿瘤微环境(tumor microenvironment，TME)是肿瘤细胞赖以生存的场所，肿瘤微环境响应型纳米载药体系则是根据TME特点设计，在TME敏感性环境中结构、性质等发生响应性变化，从而提高靶向性并实现药物的集中定位释放。
近年来，纳米载体显示出对肿瘤的有效选择性和高载药率的巨大潜力，但是这些纳米载体被内化到肿瘤组织中时，它们会遇到如何将药物快速释放到细胞质的问题。为了应对这一挑战，技术人员对肿瘤微环境如(pH、氧化还原、酶敏感等)的纳米颗粒进行了广泛研究，以促进药物传递到肿瘤靶向位置，提高治疗效果。在这些微环境信号中，低pH值(6.5)和ROS浓度(50～100μM)是对纳米载体明显的内源性刺激，这种刺激可以以特定的控制方式导致纳米载体的不稳定性，进一步导致封装药物的快速释放；但是目前的药物递送系统在载药量、生物相容性和主动靶向性等方面性能并不能很好的满足需求。
因此，能够提供一种载药量高、生物相容性好、微环境敏感及主动靶向型的氧化还原及主动靶向性的纳米药物递送系统及其制备方法是本领域技术人员亟需解决的问题。
发明内容
有鉴于此，本发明提供了一种主动靶向性和氧化还原敏感型纳米递送载体及其制备方法，本发明以PBA、γ-PGA作为修饰剂对COS进行修饰，用作递送载体，该制备方法简单，所得的PBA-COS/γ-PGA、DOX@PBA-COS/γ-PGA样品粒径分布均匀、理化性质稳定，载体对DOX的包封率、装载量高，并能实现在模拟肿瘤微环境中的响应性释药，对HepG2细胞的毒性作用显著优于HeLa细胞和MCF-7细胞，实现对肝癌细胞的靶向作用。
为了实现上述目的，本发明采用如下技术方案：
一种主动靶向性和氧化还原敏感型纳米递送载体，所述纳米递送载体为采用苯硼酸和γ-聚谷氨酸修饰壳寡糖所得。
本发明以PBA、γ-PGA作为修饰剂对COS进行修饰，其中，天然阳离子碱性低聚糖-壳聚糖低聚糖(COS)因其易吸收、可生物降解、无毒等特点，被广泛应用于纳米药物载体，并可进行功能修饰进行靶向给药，特别是COS脱乙酰后带正电，可与带负电的细胞膜相互作用，在抗肿瘤治疗中具有良好的细胞亲和力；糖抗原-唾液酸(SA)是一种碳水化合物抗原，在人宫颈癌细胞系(HeLa)和人肝细胞细胞系(HepG2)等肿瘤细胞表面过表达，而在人乳腺癌细胞系(MCF-7)中低表达，而无毒、廉价、非免疫原性的苯硼酸(PBA)可与SA通过可逆共价作用形成共价键，对SA具有较高的亲和力和选择性，除了主动靶向外，PBA对活性氧的敏感性使其具有氧化还原特性；γ-谷氨酰基转移酶(GGT)在肝癌和病毒性肝炎中过表达，它是γ-谷氨酰基循环中重要的活性酶，有助于肝细胞癌的早期分析，聚(γ-谷氨酸)(γ-PGA)是一种免疫原性、生物相容性、生物降解性和无毒的天然聚合物，可减少与生理成分的非特异性相互作用，并通过静电相互作用部分屏蔽纳米颗粒表面的过多正电荷。
靶向纳米载体识别肿瘤细胞的效果受多个因素的影响，如配体与受体的结合亲和力、受体的分布和密度、内吞率和肿瘤分期等，这些都取决于受体的表达水平，为了解决上述问题，本发明纳米递送载体通过利用肿瘤微环境氧化还原特性将药物引导至精确的位置，从而提高药物的治疗效果，通过将主动靶向和氧化还原结合到药物递送系统中，可以将治疗剂高效递送至靶位，从而实现癌症治疗的高效率。
优选的，所述壳寡糖和所述苯硼酸通过聚合法连接后，再与所述γ-聚谷氨酸进行离子交联即可。
本发明将COS和PBA原子转移自由基聚合法连接生成PBA-COS，PBA的引入赋予纳米载体主动靶向和氧化特性；其次，γ-PGA、PBA-COS、TPP通过离子交联生成纳米载体PBA-COS/γ-PGA，并与DOX共交联可形成纳米药物递送系统DOX@PBA-COS/γ-PGA，γ-PGA的引入赋予纳米粒子酶响应特性，且交联剂TPP将DOX与纳米载体紧密连接，提高了药物的包封率和载药率，包封率高达93.74％，载药率高达28.84％。
优选的，所述纳米递送载体的粒径为140-170nm。
优选的，所述氧化响应性基团为对ROS敏感的载体。
优选的，所述ROS响应性载体为苯硼酸/酯、芳基草酸酯、脯氨酸中的任意一种。
优选的，所述主动靶向响应性基团为对GGT敏感的载体。
优选的，所述GGT敏感性载体为γ-PGA。
上述所述的一种主动靶向性和氧化还原敏感型纳米递送载体的制备方法，具体包括以下步骤：
(1)在苯硼酸PBA和壳寡糖COS中加入引发剂后反应，得到PBA-COS；
(2)在所述PBA-COS和γ-聚谷氨酸γ-PGA中加入交联剂后反应，得到一种主动靶向性和氧化还原敏感型纳米递送载体PBA-COS/γ-PGA。
优选的，步骤(1)所述苯硼酸PBA、所述壳寡糖COS和所述引发剂的摩尔比为1:1:1。
优选的，步骤(1)所述引发剂为硝酸铈铵。
优选的，步骤(2)所述PBA-COS、所述γ-聚谷氨酸γ-PGA和所述交联剂的质量比为1:3:2。
优选的，步骤(2)所述交联剂为三聚磷酸钠。
优选的，步骤(2)所述反应的条件为：反应速率为600rpm，反应时间为3h。
如上述所述一种主动靶向性和氧化还原敏感型纳米递送载体或者如上述所述制备方法得到的一种主动靶向性和氧化还原敏感型纳米递送载体在制备抗肿瘤药物中的应用。
优选的，所述PBA-COS和所述药物的质量比为1:2。
优选的，所述药物为疏水性药物或多肽、蛋白质、核酸类生物、大分子多醣或单糖、维生素、谷胱甘肽和二十八醇中的任意一种。
优选的，所述药物为阿霉素、紫杉醇、自由基清除剂和低聚糖中的任意一种。
阿霉素(DOX)是临床一线蒽环类广谱非特异性抗肿瘤药物，也是目前临床上使用较为广泛的抗肿瘤药物之一，但具有靶向性差、不良反应严重、治疗指数低等缺点。将其与本发明制备的响应性纳米载体结合，纳米药物通过血液循环到达肝癌细胞组织，γ-PGA对肝癌细胞表面的γ-谷氨酰基转移酶GGT迅速作出反应，使得γ-PGA被GGT迅速水解脱落，失去γ-PGA的纳米复合物增加了与HepG2细胞的亲和性；随后，PBA与细胞表面的SA形成共价键促使纳米药物通过内吞作用进入细胞；最后，DOX成功从溶酶体逸出至细胞核，促使细胞发生凋亡，本发明系统可显著提高药物的治疗作用，降低毒副作用，促进药物的靶向能力，促进其在肿瘤部位的聚集，并延长体内循环时间。
与现有技术相比，本发明具有如下有益效果：
(1)本发明选用生物相容性高、低细胞毒性、高细胞亲和性的壳寡糖为纳米载体，药物可通过离子交联被牢固装载到载体表面，载药量高；
(2)本发明选用的PBA能特异性识别肝癌细胞表面的糖抗原-唾液酸(SA)，形成稳定的共价键；且PBA对ROS敏感，利用PBA修饰COS可有助于提高COS纳米载体的ROS敏感性和对肝癌细胞的选择性；
(3)本发明选用的γ-PGA能被GGT特异性识别使其发生水解或转肽反应，且跨膜蛋白GGT在肝癌等上皮细胞膜表面高表达，利用γ-PGA修饰COS可有助于提高COS纳米载体对肝癌细胞的主动靶向特性。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1为本发明实施例1PBA-COS中CAN与COS的摩尔比例优化红外光谱图；
图2为本发明实施例1PBA-COS的核磁共振氢谱；其中，A：COS，B：PBA，C：PBA-COS；
图3为本发明实施例2纳米粒子粒径电位各影响因素粒径电位图，其中，A：PBA-COS浓度(Cγ-PGA:1mg/mL；CTPP:1mg/mL；转速:600rpm；反应时间:3h)，B：γ-PGA浓度(CPBA-COS:1mg/mL；CTPP:1mg/mL；转速:600rpm；反应时间:3h)，C：TPP浓度(CPBA-COS:1mg/mL；Cγ-PGA:1mg/mL；转速:600rpm；反应时间:3h)，D：搅拌速率(CPBA-COS:1mg/mL；Cγ-PGA:1mg/mL；CTPP:1mg/mL；反应时间:3h)，E：反应时间(CPBA-COS:1mg/mL；Cγ-PGA:1mg/mL；CTPP:1mg/mL；转速:600rpm)；
图4为本发明实施例2纳米粒子正交试验所得的优化组合粒径电位分析图，其中，A：以粒径最佳组合各条件反应测得的粒径电位图；B：以电位最佳组合各条件反应测得的粒径电位图；
图5为本发明实施例3DOX浓度对载药纳米粒子粒径电位及包封率、载药率影响分析，其中，A：不同DOX浓度下载药纳米粒子的粒径电位，B：不同DOX浓度下纳米粒子对DOX的包封率和载药率；
图6为本发明实施例3纳米粒子及载药纳米粒子的红外光谱图；
图7为本发明实施例3纳米粒子和载药纳米粒子的粒径电位图；
图8为本发明实施例3纳米粒子和载药纳米粒子的透射电镜图，其中，A：PBA-COS/γ-PGA，B：DOX@PBA-COS/γ-PGA；
图9为本发明应用例1载药纳米粒子在模拟肿瘤微环境条件下的药物释放图，其中，A：DOX@PBA-COS/γ-PGA pH响应的药物释放图，B：DOX@PBA-COS/γ-PGA和DOX@COS/γ-PGA的ROS响应的药物释放图，C：DOX@PBA-COS/γ-PGA GGT响应的药物释放图，D：DOX@COS和DOX@PBA-COS ROS响应的药物释放图，E：DOX@PBA-COS/γ-PGA和DOX@PBA-COS ROS/GGT响应的药物释放图；
图10为本发明应用例2纳米载体对乳腺癌细胞(MCF-7)、宫颈癌细胞(HeLa)和肝癌细胞(HepG2)的MTT测定结果图，其中，A：MCF-7，B：HeLa，C：HepG2；
图11为本发明应用例2载药纳米粒子在模拟肿瘤微环境条件下对乳腺癌细胞(MCF-7)、宫颈癌细胞(HeLa)和肝癌细胞(HepG2)的MTT测定结果图，其中，A、D：MCF-7，B、E：HeLa，C、F：HepG2。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
一种主动靶向性和氧化还原敏感型纳米递送载体，主要步骤包括：
(1)PBA-COS的制备：
将3.00g COS溶于50mL 2％(V/V)醋酸溶液，0.82g CAN溶于2mL 1M硝酸溶液，0.32g PBA与0.26mL DEAEA分别溶于2mL DMSO中，其中COS、CAN、PBA与DEAEA的摩尔比为1:1:1:1；将COS的醋酸溶液用注射器注入通氮气的三口瓶中，30℃，600rpm磁力搅拌0.5h，随后加入CAN，待温度升至60℃后，磁力搅拌1h，然后加入DEAEA和PBA，继续60℃磁力搅拌反应24h；最后将反应液用截留分子量为500Da的透析袋透析一天，冷冻干燥得到淡黄色絮状产品苯硼酸-壳寡糖PBA-COS；
为获得最优结构的PBA-COS，将引发剂CAN与COS的摩尔比按0.25-2:1的比例重复上述实验步骤；
将得到的PBA-COS分别进行红外光谱分析表征，结果见图1，扫描波数范围为500-4000cm-1，X轴表示透光率(％)，Y轴表示波数，单位为cm-1，由图可知，在3370cm-1处-OH的伸缩振动峰；由于引入烷基链，CH3和CH2的C-H的伸缩振动峰为2928和2879cm-1；1739cm-1处是羰基的伸缩振动峰，产物中含有羰基，说明DEAEA成功连接PBA和COS；1554cm-1处是N-H键的弯曲振动峰，1072和1031cm-1处是C-O键的伸缩振动峰；由此可见，PBA-COS成功合成，且当COS与CAN的摩尔比为1时，PBA-COS的主要特征峰最为明显；
图2是PBA-COS(COS与CAN的摩尔比为1)的核磁共振氢谱图，7.26和4.79ppm处的特征峰分别是氧化氘和氯化氘的化学位移特征峰，0.9-1.2ppm为PBA-COS上甲基和亚甲基(-CH3、-CH2)的特征峰，1.81ppm是乙酰基团上的甲基质子峰，2.45ppm处出现的丙烯酸N,N-二乙基氨基乙酯中亚甲基的化学位移，3.36ppm出现苯硼酸中亚甲基的化学位移，说明COS和PBA经DEAEA成功连接。
实施例2
PBA-COS/γ-PGA的制备：
将PBA-COS(实施例1中COS与CAN的摩尔比为1)、TPP和γ-PGA溶于超纯水中，分别配制不同浓度的原料液备用；
将10mL PBA-COS溶液与5mL TPP溶液混合，转速为600rpm下磁力搅拌15min，之后再逐滴加入10mLγ-PGA溶液，继续磁力搅拌3h，即得到苯硼酸-壳寡糖/γ-聚谷氨酸纳米粒子PBA-COS/γ-PGANPs；
用单因素实验考察了原料浓度、反应时间和磁力搅拌速率对纳米粒子的粒径和Zeta电位的影响，用正交试验进一步确定各原料的最佳浓度以及反应时间和转速，结果见表1-3和图3-4；
图3为纳米粒子粒径电位各影响因素粒径电位图，由图3中A-E可知，随着PBA-COS浓度的增加，PBA-COS与γ-PGA的结合先紧密后变得疏松，而表面电荷逐渐减少，这是因为PBA-COS表面电荷呈正值，因此当其浓度超过一定限度时，只能疏松的吸附在PBA-COS/γ-PGA表面；而随着TPP浓度增加，纳米粒子更加紧密连接，表面电荷逐渐增加，同理，这是因为TPP是反应的交联剂，它会使得PBA-COS与γ-PGA的连接更为紧致，但由于TPP是阴离子交联剂，过量会使体系负离子浓度增加，影响PBA-COS/γ-PGA的表面电荷；随着转速的增加，纳米粒子同样先紧密后疏松，而对电位影响较小，适当的转速会促进反应的进行，使纳米粒子紧密结合，但是转速过大会破坏原本形成的稳定体系导致纳米粒子变得疏松甚至解离，但转速的变化并没有影响反应体系的电荷分布，因此对表面电荷影响很小；在试验浓度范围内γ-PGA浓度对粒径电位并无显著影响，说明γ-PGA增加的浓度并不会影响体系的电荷分布，只是更充分的与PBA-COS交联；反应时间对纳米粒子粒径电位大小变化可忽略不计，因为在适当的转速条件下，形成的纳米体系相对稳定，时间对其粒径电位无明显影响，说明形成的纳米体系稳定性较好，不会随着时间的变化而改变自身的表面性质；综合上述研究结果，PBA-COS和TPP浓度对PBA-COS/γ-PGA NPs的粒径和Zeta电位影响较大，时间和γ-PGA浓度对纳米粒子的Zeta电位影响相对较小，而转速对纳米粒子的粒径影响较明显；
表1纳米粒子粒径电位正交试验分析表
Figure SMS_1
表2粒径条件优化方差分析表
Figure SMS_2
表3电位条件优化方差分析表
Figure SMS_3
表1、2和3为本发明实施例2纳米粒子粒径电位正交试验分析表，根据直观分析表1所示，各因素粒径的极差大小为：CPBA-COS＞Cγ-PGA＞时间＞CTPP＞转速；电位的极差大小为：Cγ-PGA＞CPBA-COS＞转速＞CTPP＞时间；则PBA-COS浓度、γ-PGA浓度对纳米粒子粒径和电位的影响最显著，转速、TPP和时间对粒径和电位的影响较小；
在方差分析表2和3中，各因素的粒径F比的大小为：CPBA-COS＞Cγ-PGA＞CTPP＞时间＞转速，各因素的电位F比的大小为：Cγ-PGA＞CPBA-COS＞转速＞CTPP＞时间，结合实验需要，粒径的最佳组合为组合一：CPBA-COS＝0.5mg/mL，Cγ-PGA＝1.5mg/mL，CTPP＝1mg/mL，时间为3h，转速为800rpm；电位的最佳组合为组合二：CPBA-COS＝0.5mg/mL，Cγ-PGA＝1.5mg/mL，CTPP＝2mg/mL，时间为3h，转速为600rpm。
图4为纳米粒子正交试验所得的优化组合粒径电位分析图，A为组合一的粒径电位分布图，粒径为190.1nm，PDI值为0.910，电位值为-9.56mV，纳米粒子分散度较差；B为组合二的粒径电位分布图，平均粒径为141.6nm，PDI为0.421，电位值为-19.6mV，纳米粒子分布性良好；纳米粒子的粒径在150nm左右，Zeta电位在-15mV以上时，可被细胞有效摄取和吸收，故选组二：CPBA-COS＝0.5mg/mL，Cγ-PGA＝1.5mg/mL，CTPP＝2mg/mL，时间为3h，转速为600rpm，来合成PBA-COS/γ-PGA纳米粒子。
实施例3
DOX@PBA-COS/γ-PGA的制备：
将盐酸阿霉素溶于超纯水中，配成1mg/mL的DOX溶液；
将10mL 0.5mg/mL的PBA-COS(实施例1中COS与CAN的摩尔比为1)溶液与5mL 2mg/mL的TPP溶液混合，转速为600rpm室温下磁力搅拌15min，随后加入10mL 1.5mg/mL的γ-PGA溶液和10mL 1mg/mL的DOX溶液，磁力搅拌3h后通过离心、洗涤和冷冻干燥收集载药纳米粒子，即得到载药苯硼酸-壳寡糖/γ-聚谷氨酸纳米粒子DOX@PBA-COS/γ-PGA；DOX@COS、DOX@PBA-COS载药纳米粒子的合成方法同上。
配制不同浓度的DOX溶液，合成DOX@PBA-COS/γ-PGA，方法同上，DOX的浓度分别为0.2mg/mL、0.5mg/mL、0.75mg/mL、1mg/mL和2mg/mL，考察DOX浓度对载药纳米粒DOX@PBA-COS/γ-PGA粒径电位以及包封率和载药率的影响，结果见图5-8；
图5为DOX浓度对载药纳米粒子粒径电位及包封率、载药率的影响分析从图A可看出，随着DOX浓度的增加，包封率逐渐增加，随后保持稳定；载药率逐渐增加，这说明DOX浓度在1mg/mL时，DOX、PBA-COS和γ-PGA能够与TPP充分结合，包封率达到最高；图B是载药纳米粒子的粒径电位随DOX浓度的变化，随着DOX浓度的增加，粒径先减小后增加，电位逐渐增加，在DOX浓度为1mg/mL时，DOX与TPP的交联程度达到最大，但随着DOX浓度的增加，DOX与TPP的交联变得疏松，导致粒径变大，而由于DOX带有正电荷，随着DOX浓度的增加，DOX@PBA-COS/γ-PGA的电位会逐渐变大；
图6为纳米粒子及载药纳米粒子的红外光谱图，如图所示，与PBA-COS/γ-PGA相比，DOX@PBA-COS/γ-PGA在3382cm-1处的羟基峰变宽，在2938和2883cm-1有C-H的伸缩振动吸收峰；1614cm-1处是DOX的双羰基伸缩振动峰，在805和1220cm-1(＝C-O-CH3)的特征吸收峰与DOX相关，在1285和1412cm-1代表的是DOX的C-OH和C-C特征峰，结果表明DOX成功交联到PBA-COS/γ-PGANPs上；
图7为纳米粒子和载药纳米粒子的粒径电位图，如图所示PBA-COS/γ-PGA的粒径为141.8nm，电位为-19.6mV；而载入DOX后，DOX@PBA-COS/γ-PGA的粒径和电位略有增加，粒径为164.2nm，电位为-11.4mV，纳米粒子的负电荷有利于进入细胞；
图8为纳米粒子和载药纳米粒子的透射电镜图，从图可看出，纳米粒子呈球形，且图A粒径比图B中的略小，可能是由于用马尔文粒度仪测定粒径时，水溶液使得纳米粒子膨胀的缘故。
综合以上各项表征结果可知，本发明成功制备了氧化还原及主动靶向性的苯硼酸-壳寡糖/γ-聚谷氨酸纳米系统，该纳米系统具有良好的物理稳定性，分散性好，具有作为抗肿瘤药物优良递送载体的可能；
对制备的DOX@PBA-COS/γ-PGA，测定DOX的负载效率，方法为：取DOX@PBA-COS/γ-PGA反应液，离心后量取上清液体积，用紫外分光光度计进行测定，将吸光度代入标准曲线求出上清液中游离DOX含量，UV法测定DOX：选择480nm作为DOX的检测波长测定其含量，建立DOX的标准曲线，为DOX的包封率和载药量的计算，以及体外释放实验中DOX含量的测定做准备；
根据公式计算DOX的包封率和载药量，计算公式为：
Figure SMS_4
Figure SMS_5
COS/γ-PGA对DOX的包封率和载药率分别为87.98％和20.70％；而PBA-COS/γ-PGA对DOX的包封率和载药率分别为93.74％和28.84％，均大于COS/γ-PGA对DOX的包封率和载药率。
应用例1
对载药纳米粒子样品的pH响应、ROS响应和GGT响应性释药进行验证实验
将实施例3制备的DOX@PBA-COS/γ-PGA载药纳米粒子5mg分别分散于5mL的pH5.5、6.8和7.4的PBS缓冲溶液；分别将5mg的DOX@COS/γ-PGA和DOX@PBA-COS/γ-PGA置于5mLpH5.5和7.4的含有100μM H2O2的PBS缓冲溶液中，将上述液体分别放入截留分子量500Da的透析袋中，然后置于对应的50mL PBS缓冲溶液中，在37℃的恒温振荡箱内持续振荡，每隔一定时间吸取3mL释放液，同时迅速补充3mL新鲜的不同条件的PBS缓冲液，通过紫外分光光度计测定收集的样品在480nm吸收波长处的吸光度，根据DOX·HCl在不同pH条件下的标准曲线和公式，计算相应的浓度及累计释放率Qt，绘制药物的累积释放率与释放时间的关系图，
Figure SMS_6
其中，Cn为补充新鲜PBS溶液前收集的释放液浓度，V0为振荡箱内用于药物释放的PBS缓冲液体积，Vi为每次取样体积，Ci为第i次置换时释放液中的药物浓度，m为载药纳米粒子的药物总量；
针对GGT响应性，分别将实施例3制备的DOX@PBA-COS/γ-PGA载药纳米粒子以及DOX@PBA-COS纳米粒子各5mg分散于5mL的pH7.4、100μM H2O2并加有10U/mL GGT以及pH6.5、100μM H2O2和加有10U/mL GGT的PBS缓冲溶液，将其放入截留分子量500Da的透析袋中，然后置于对应的50mL PBS缓冲溶液中，在37℃的恒温振荡箱内持续振荡，后续步骤同上；相关结果见图9。
由图9可知，DOX@PBA-COS/γ-PGA有明显的pH响应性，在pH5.5条件下，其在48h后的累积释放率为66.24％，是pH7.4时的1.62倍；与DOX@COS/γ-PGA和DOX@COS相比，DOX@PBA-COS/γ-PGA有明显的ROS响应性，当pH5.5，H2O2浓度为100μM时，DOX@PBA-COS/γ-PGA在48h后的累积释放率可达到79.62％，而DOX@COS/γ-PGA在此条件下的累积释放率为67.33％，DOX@COS在此条件下的累计释放率为51.23％；无论是DOX@PBA-COS/γ-PGA还是DOX@COS/γ-PGA，在pH5.5时的药物累积释放率均明显高于pH7.4时的累积释放率，如图9(E)所示，在pH7.4，100μM H2O2和10U/mL GGT条件下，DOX@PBA-COS/γ-PGA在48h后的累计释放率为65.45％，而DOX@PBA-COS的累计释放率为42.46％，说明DOX@PBA-COS/γ-PGA相比于DOX@PBA-COS具有显著的GGT响应性；且在pH6.5，100μM H2O2和10U/mL GGT条件下，DOX@PBA-COS/γ-PGA在48h后的累计释放率为90.36％，是pH7.4时的2.22倍，而且比其在pH5.5时的释放率还要高出23.7％；由上可知多重响应性载药纳米粒DOX@PBA-COS/γ-PGA不仅具有良好的pH响应性，而且PBA和γ-PGA的引入也使得载药纳米粒子具备了ROS和酶响应性。
应用例2
细胞毒性验证
以MTT法分别考察纳米载体及载药纳米粒子的细胞毒性，称取各纳米载体2mg溶于无血清DMEM培养基中配制成200μg/mL的溶液，利用细胞培养液配制成200、100、50、20和10μg/mL的浓度；游离DOX和载药纳米粒子以DOX浓度为定量依据，设置浓度梯度为2、1、0.5、0.2和0.1μg/mL；将其分别加入到96孔板中，每个浓度设置5个平行孔，24h后加入0.5mg/mL的MTT溶液，4h后吸取含有MTT的培养液，加入DMSO，用酶标仪在490nm波长处测定各孔吸光值；同理，设置pH6.5和pH6.5加10μM H2O2的培养基分别培养细胞，操作步骤同上；相关结果见图10和11；
其中，图10为实施例2(COS与CAN的摩尔比为1)纳米载体对乳腺癌细胞(MCF-7)、宫颈癌细胞(HeLa)和肝癌细胞(HepG2)的MTT测定结果图，如图所示，MCF-7细胞活性随着载体材料浓度的增加无明显变化；HeLa和HepG2细胞在高浓度的COS作用下，细胞活力会有所降低，但在纳米载体浓度提高到200μg/mL时，HeLa和HepG2细胞活性仍大于80％，因此该COS纳米载体可作为药物递送载体使用；
图11为实施例3制备载药纳米粒子在模拟肿瘤微环境条件下对乳腺癌细胞(MCF-7)、宫颈癌细胞(HeLa)和肝癌细胞(HepG2)的MTT测定结果图，如图所示是DOX、DOX@COS/γ-PGA和DOX@PBA-COS/γ-PGA分别与MCF-7、HeLa和HepG2培养24和48h后的细胞活性，从A-C可看出在24h后，DOX@PBA-COS/γ-PGA对MCF-7、HeLa和HepG2的细胞毒性明显高于游离DOX和DOX@COS/γ-PGA的细胞毒性，且pH6.5、10μM H2O2条件下的细胞毒性更强；DOX@PBA-COS/γ-PGA在pH6.5和10μM H2O2条件下HepG2细胞的半数致死浓度(IC50)为0.26μg/mL，而MCF-7和HeLa在此条件下的IC50浓度分别为0.99μg/mL和0.56μg/mL，均大于HepG2细胞的IC50；且HepG2细胞在pH6.5、10μM H2O2条件下的IC50明显低于在pH 7.4和pH 6.5条件下的IC50；在作用48h后(D-F)，DOX@PBA-COS/γ-PGA对细胞的毒性更为显着，这是因为DOX@PBA-COS/γ-PGA外层的COS具有一定的黏附性，而且COS的引入，使得药物得到了缓释，从而在细胞中能够更长时间的发挥作用，引入的PBA和γ-PGA基团属于ROS和GGT响应性基团，使得DOX@PBA-COS/γ-PGA对HepG2细胞有更好的选择性，在模拟肿瘤微环境pH 6.5，H2O2以及GGT存在条件下，将更有利于DOX@PBA-COS/γ-PGA通过内吞作用进入癌细胞内部释放DOX。
综上，本发明构建了一种氧化还原及主动靶向性的苯硼酸-壳寡糖/γ-聚谷氨酸纳米药物递送系统，以COS为纳米载体，PBA和γ-PGA修饰赋予纳米载体ROS和GGT响应性能，该制备方法简单，所得的PBA-COS/γ-PGA和DOX@PBA-COS/γ-PGA样品粒径分布均匀、理化性质稳定，载体PBA-COS/γ-PGA对DOX的包封率和装载量高，并能实现在模拟肿瘤微环境条件下的响应释药，对HepG2细胞的毒性作用优于HeLa细胞和MCF-7细胞，因此，DOX@PBA-COS/γ-PGA是一种优良高效的纳米药物递送系统。
各个实施例采用递进的方式描述，每个实施例重点说明的都是与其他实施例的不同之处，各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (10)

1.一种主动靶向性和氧化还原敏感型纳米递送载体，其特征在于，所述纳米递送载体为采用苯硼酸和γ-聚谷氨酸修饰壳寡糖所得。
2.根据权利要求1所述的一种主动靶向性和氧化还原敏感型纳米递送载体，其特征在于，所述壳寡糖和所述苯硼酸通过聚合法连接后，再与所述γ-聚谷氨酸进行离子交联即可。
3.根据权利要求1所述的一种主动靶向性和氧化还原敏感型纳米递送载体，其特征在于，所述纳米递送载体的粒径为140-170nm。
4.根据权利要求1-3任一项所述的一种主动靶向性和氧化还原敏感型纳米递送载体的制备方法，其特征在于，具体包括以下步骤：
(1)在苯硼酸PBA和壳寡糖COS中加入引发剂后反应，得到PBA-COS；
(2)在所述PBA-COS和γ-聚谷氨酸γ-PGA中加入交联剂后反应，得到一种主动靶向性和氧化还原敏感型纳米递送载体PBA-COS/γ-PGA。
5.根据权利要求4所述的一种主动靶向性和氧化还原敏感型纳米递送载体的制备方法，其特征在于，步骤(1)所述苯硼酸PBA、所述壳寡糖COS和所述引发剂的摩尔比为1:1:1。
6.根据权利要求4所述的一种主动靶向性和氧化还原敏感型纳米递送载体的制备方法，其特征在于，步骤(1)所述引发剂为硝酸铈铵。
7.根据权利要求4所述的一种主动靶向性和氧化还原敏感型纳米递送载体的制备方法，其特征在于，步骤(2)所述PBA-COS、所述γ-聚谷氨酸γ-PGA和所述交联剂的质量比为1:3:2。
8.根据权利要求4所述的一种主动靶向性和氧化还原敏感型纳米递送载体的制备方法，其特征在于，步骤(2)所述交联剂为三聚磷酸钠。
9.如权利要求1-3任一项所述一种主动靶向性和氧化还原敏感型纳米递送载体或者如权利要求4-8任一项所述制备方法得到的一种主动靶向性和氧化还原敏感型纳米递送载体在制备抗肿瘤药物中的应用。
10.根据权利要求9所述的应用，其特征在于，所述PBA-COS和所述药物的质量比为1:2。
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