CN113244409A - 一种具有清除活性氧及抗炎作用协同增强效果的纳米粒子 - Google Patents

一种具有清除活性氧及抗炎作用协同增强效果的纳米粒子 Download PDF

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CN113244409A
CN113244409A CN202110584984.2A CN202110584984A CN113244409A CN 113244409 A CN113244409 A CN 113244409A CN 202110584984 A CN202110584984 A CN 202110584984A CN 113244409 A CN113244409 A CN 113244409A
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仝维鋆
邱慧强
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Zhejiang University ZJU
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Abstract

本发明公开了一种具有清除活性氧及抗炎作用协同增强效果的纳米粒子，是中空二氧化锰纳米粒子经聚电解质修饰处理后，作为药物载体负载有抗炎药。所述的聚电解质修饰是先将中空二氧化锰纳米粒子加入聚阳离子溶液，得到聚阳离子修饰的粒子，随后加入聚阴离子溶液尤其是硫酸葡聚糖钠盐溶液中，得到聚电解质修饰处理后的粒子。所述纳米粒子可靶向炎症部位的促炎巨噬细胞，清除细胞内高水平的活性氧，降低炎症因子的表达，同时可以减轻免疫细胞富集，降低活性氧水平，阻断炎症反应的级联放大效应，实现协同增强的抗炎效果。本发明制备方法简单，材料来源广泛，得到的载体粒子可以靶向炎症部位、清除活性氧和负载抗炎药物等功能，具有良好应用前景。

Description

一种具有清除活性氧及抗炎作用协同增强效果的纳米粒子
技术领域
本发明属于多功能抗炎药物制备领域，涉及一种具有清除活性氧及抗炎协同增强作用的纳米粒子。
背景介绍
活性氧(Reactive oxygen species，ROS)是一类含氧的化学活性分子，主要包括超氧阴离子，羟基自由基、过氧化氢等。ROS产生和代谢的动态平衡对于维持细胞和组织的正常功能是非常重要的，但是ROS的异常增高会导致氧化应激并产生一系列的细胞和组织损伤。越来越多的证据表明ROS诱导的氧化应激是炎症发生发展中的重要因素。ROS可以通过直接损伤和加重炎性反应发挥作用，也可直接刺激促炎细胞因子的产生，或者通过激活炎性小体引起炎性级联放大效应，从而加重炎症的症状。
炎症部位促炎巨噬细胞富集，通过靶向促炎巨噬细胞，可以实现炎症部位靶向。然而炎症部位的高表达的炎症因子会使免疫细胞富集和提高活性氧水平，限制抗炎效果。因此，抑制炎症因子的产生对抗炎治疗同样具有重要意义。糖皮质激素类抗炎药可以有效的抑制炎症因子产生，但由于其水溶性差，进入体内会迅速被代谢掉，使用载体可以很好的解决这一问题。
基于此本发明提供一种可以协同增强活性氧清除功能和抗炎药抗炎作用的纳米粒子，一方面可以清除炎症微环境活性氧，另一方面可以在炎症部位释放出抗炎药，阻断炎症反应的级联放大效应，从而实现协同增强的炎症性疾病治疗。
发明内容
为解决上述技术问题，本发明提供一种具有清除活性氧及抗炎协同增强作用的纳米粒子。本发明通过表面修饰纳米粒子可以促进靶向促炎巨噬细胞，制得的整个纳米粒子可以有效清除炎症微环境中的过量活性氧，可以阻断炎症反应的级联放大效应，起到协同增强的治疗炎症性疾病的效果。
本发明采用以下技术方案实现：
一种具有清除活性氧及抗炎协同增强作用的纳米粒子，为中空二氧化锰纳米粒子经聚电解质修饰处理后，作为药物载体负载有抗炎药。
所述的聚电解质修饰具体包括如下：先将中空二氧化锰纳米粒子加入聚阳离子溶液，震荡使粒子吸附聚阳离子，离心、洗涤后，得到聚阳离子修饰的粒子，随后加入聚阴离子溶液中，震荡使粒子吸附聚阴离子，离心、洗涤得到聚电解质修饰处理后的粒子。
所述的聚阴离子溶液采用硫酸葡聚糖钠盐溶液。
所述的聚阴离子溶液的浓度为1-2mg/ml。
所述的聚阳离子溶液为聚丙烯基胺盐酸盐溶液、聚二烯丙基二甲基季铵盐溶液、壳聚糖溶液、聚赖氨酸溶液或聚乙烯亚胺溶液，浓度可以为1-5mg/mL。
所述的中空二氧化锰纳米粒子为中空的花生状或椭球状的二氧化锰粒子，其粒径为500-600nm。
所述的中空二氧化锰纳米粒子采用如下方法制得：
在搅拌条件下，将碳酸钠溶液加入水溶性二价锰溶液中，室温下反应30s-8h，反应结束后，洗涤，得到碳酸锰模板纳米粒子；将所得的粒子分散到水中,再加入高锰酸钾溶液，反应0.25h-8h，洗涤后将粒子分散在水中，加入盐酸溶液，震荡反应0.25h-8h，洗涤得到中空二氧化锰纳米粒子。
所述的高锰酸钾溶液的浓度为1-100mg/mL；碳酸钠溶液加入水溶性二价锰溶液中，水溶性二价锰盐可以是氯化锰、硫酸锰、或硝酸锰等，混合后碳酸钠、二价锰的浓度可以均为0.01-0.1g/mL；所述盐酸溶液的质量分数可以为0.5％-10％。
中空二氧化锰纳米粒子经聚电解质修饰后，分散于溶解有抗炎药的溶剂中，进行载药。所述的抗炎药为地塞米松、布地奈德、姜黄素、泼尼松、甲泼尼松、倍他米松、丙酸倍氯米松、泼尼松龙或氢化可的松，所述的溶剂可以为乙醇、四氢呋喃、乙酸乙酯、甲醇、丙酮、二甲基亚砜、二氯甲烷等。
本发明通过二价锰离子和碳酸根离子之间的沉淀作用，形成纳米尺度的碳酸锰模板，用高锰酸钾氧化表面碳酸锰，再通过酸与碳酸盐的反应去除内部未反应的碳酸锰，得到分散良好的中空的二氧化锰纳米粒子。再利用二氧化锰纳米粒子静电吸附带正电的聚阳离子，使表面带上正电荷，随后再吸附带负电的聚阴离子，采用特定种类的聚阴离子使得制得的纳米粒子可以靶向促炎巨噬细胞表面的清道夫受体，同时利用该粒子的中空介孔结构，吸附装载大量的抗炎药物。在高水平活性氧的炎症微环境中，清除大量的活性氧，起到治疗炎症的效果，但高水平的炎症因子会导致免疫细胞聚集、产生更多活性氧，限制抗炎效果。与此同时释放的抗炎药，可以抑制炎症因子的产生，阻断上述级联放大效应，从而实现协同抗炎治疗。
本发明的有益效果在于：
本发明所用原料来源广泛，制备过程简单可控，可以放大规模；纳米粒子的表面修饰有清道夫受体靶向的聚电解质，可以靶向促炎细胞，将抗炎载体精准的带到炎症部位，并可以使清除活性氧和抑制炎症因子这两种作用协同增强，达到最佳效果。
附图说明
图1中a)是中空二氧化锰纳米粒子的透射电镜照片，b)是聚电解质修饰的二氧化锰纳米粒子的透射电镜照片。c)是抗炎药负载的聚电解质修饰的二氧化锰纳米粒子的透射电镜照片。
图2是载体的药物负载量。
图3中a)是抗炎药负载的聚电解质修饰的二氧化锰纳米粒子清除超氧阴离子活性。b)是抗炎药负载的聚电解质修饰的二氧化锰纳米粒子清除双氧水活性。
图4是抗炎药负载的聚电解质修饰的二氧化锰纳米粒子的生物相容性。
图5是荧光标记的抗炎药负载的聚电解质修饰的二氧化锰纳米粒子靶向促炎巨噬细胞荧光显微镜照片。
图6是抗炎药负载的聚电解质修饰的二氧化锰纳米粒子清除细胞内活性氧的照片。
图7中a)是小鼠结肠炎治疗期间体重变化，b)是结肠炎治疗期间疾病活动系数变化，c)是结肠炎治疗末期结肠长度。
图8是结肠组织切片染色。
图9是结肠中炎症因子水平。
图中，M为二氧化锰，MPD为聚电解质修饰的二氧化锰，MPDB为布地奈德负载的聚电解质修饰二氧化锰，LPS为脂多糖，DCFH-DA为二氯二氢荧光素-乙酰乙酸酯，DSS为硫酸葡聚糖钠盐，BF为眀场。
具体实施方式
以下结合实例进一步说明本发明，但这些实例并不用来限制本发明。
实施例1
1)在剧烈磁力搅拌条件下，将碳酸钠溶液加入四水合氯化锰溶液中，使其浓度分别为25mg/mL、45mg/mL，室温反应1min。反应结束后离心去除上清液，加入去离子水洗涤粒子得到碳酸锰模板纳米粒子。
2)将步骤1)所得的粒子分散到水中，浓度为5mg/mL，再加入高锰酸钾溶液，使其浓度为1mg/mL，反应1h。加入去离子水洗涤粒子，将粒子分散在水中，往粒子中加入水。超声分散后加入盐酸溶液，浓度为0.5％，震荡0.5h，重复一次。反应结束后离心去除上清液，加入去离子水洗涤粒子，离心去除上清液从而得到中空二氧化锰纳米粒子。
3)将步骤2)所得的粒子分散到水中，加入1mg/mL的聚丙烯基胺盐酸盐溶液，震荡0.5h，反应结束后离心去除上清液，加入去离子水洗涤粒子。将粒子重新分散到水中,加入1mg/mL的硫酸葡聚糖钠盐溶液，震荡0.5h，反应结束后离心去除上清液，加入去离子水洗涤粒子，离心去除上清液从而得到具有炎症部位靶向功能的活性氧清除抗炎药载体。
4)取10mg步骤3)制备的粒子，离心弃去上清，分散在四氢呋喃中，搅拌下加入含10mg布地奈德的四氢呋喃溶液,搅拌24小时，反应结束后离心去除上清液，加入去离子水洗涤粒子，离心去除上清液从而得到具有炎症部位靶向功能的活性氧清除和抗炎药负载纳米粒子。
实施例2
步骤同实施例1，但在步骤1)中用硫酸锰代替氯化锰，该步成功制得碳酸锰模板纳米粒子。
实施例3
步骤同实施例1，但在步骤3)中用壳聚糖代替聚丙烯基胺盐酸盐；最终成功制得了具有炎症部位靶向功能的活性氧清除抗炎药载体。
实施例4
步骤同实施例1，但在步骤3)中用透明质酸代替硫酸葡聚糖钠盐；最终成功制得了具有炎症部位靶向功能的活性氧清除抗炎药载体。
实施例5
步骤同实施例1，但在步骤4)中用姜黄素代替布地奈德；最终成功制得了具有炎症部位靶向功能的活性氧清除和抗炎药负载纳米粒子。
本发明方法制备的具有炎症部位靶向功能的活性氧清除和抗炎药负载纳米粒子的电镜照片见图1中c)。图1中a)为中空二氧化锰纳米粒子透射电镜照片。图1中b)聚电解质修饰的二氧化锰纳米粒子的透射电镜照片。从图中可知，粒子为中空花生状或椭球形，约为500～600纳米，壁厚约70纳米，表面有比较多的针刺结构，组装和载药后尺寸不变。
图2表明载体的药物负载量约为15％，且重复性好。
图3中a)是抗炎药负载的聚电解质修饰的二氧化锰纳米粒子清除超氧阴离子活性。图3中b)是抗炎药负载的聚电解质修饰的二氧化锰纳米粒子清除双氧水活性。从图中可以看出，双氧水和超氧阴离子的清除能力都随粒子浓度增大而提高。
图4是抗炎药负载的聚电解质修饰的二氧化锰纳米粒子的生物相容性。表明材料在12.5ug/ml以下具有良好的生物相容性。
图5利用一种荧光探针罗丹明B来标记抗炎药负载的聚电解质修饰的二氧化锰纳米粒子，红色越强代表进入细胞内的粒子越多。LPS刺激后，将小鼠腹腔巨噬细胞直接与粒子共孵育，内吞程度显著提高。说明粒子具有激活巨噬细胞靶向能力。但用游离硫酸葡聚糖钠盐占据激活的巨噬细胞表面的清道夫受体之后，再与粒子孵育，内吞提高程度降低。证明这种靶向作用是粒子表面的硫酸葡聚糖带来的。
图6是抗炎药负载的聚电解质修饰的二氧化锰纳米粒子清除细胞内活性氧的照片。利用DCFH-DA探针来表征细胞内ROS水平，它本身无荧光，可以自由穿过细胞膜，进入细胞内后，可以被细胞内的酯酶水解生成DCFH。而DCFH不能通透细胞膜，从而使探针很容易被装载到细胞内。细胞内的活性氧可以氧化无荧光的DCFH生成有荧光的DCF。检测DCF的绿色荧光就可以知道细胞内活性氧的水平。载药与不载药的聚电解质修饰二氧化锰纳米粒子与巨噬细胞共孵育，不会提高细胞内ROS水平。LPS刺激后，细胞内ROS水平提高，不载药的聚电解质修饰二氧化锰纳米粒子预处理组提高程度降低，载药的聚电解质修饰二氧化锰纳米粒子预处理组进一步降低。表明具有抗氧化和药物协同治疗的抗炎药负载的聚电解质修饰二氧化锰纳米粒子，比不载药的聚电解质修饰二氧化锰载体具有更好的清除和抑制ROS的产生的效果。这是由于高水平炎症因子会募集大量免疫细胞，进而产生大量的ROS。抗炎药可以抑制炎症因子的产生，从而降低ROS水平。
图7中a)是小鼠结肠炎治疗期间体重变化，在治疗的第六天，健康组小鼠体重略微降低，DSS灌喂的小鼠体重明显下降，表明模型建立成功。自由药组体重降低更为严重，这与文献报道的结果一致，表明自由药具有一定的毒性。MPD组体重降低程度有所改善，证明载体具有治疗结肠炎的效果。图7中b)是结肠炎治疗期间疾病活动系数变化，疾病活动系数越高，炎症越严重。MPDB治疗组疾病活动系数比单独使用自由药或载体治疗组都更低。图7中c)是结肠炎治疗末期结肠长度，肠长度越短代表结肠炎更严重，MPDB组结肠比单独使用自由药和载体的都更长。这些结果都证明了药物与载体的协同治疗作用。
图8是结肠组织切片染色。MPDB组在粘膜完整性、线体和隐窝数量、免疫细胞浸润等方面，都表现出比载体或自由药组更好的治疗效果，证明了药物与载体的协同治疗作用。
图9是结肠中炎症因子水平。MPDB组的炎症因子比单独使用载体或自药组都更低，证明了药物与载体的协同治疗作用。

Claims (10)

1.一种具有清除活性氧及抗炎协同增强作用的纳米粒子，其特征在于，所述纳米粒子为中空二氧化锰纳米粒子经聚电解质修饰处理后，作为药物载体负载有抗炎药。
2.根据权利要求1所述的具有清除活性氧及抗炎协同增强作用的纳米粒子，其特征在于，所述的聚电解质修饰具体包括如下：先将中空二氧化锰纳米粒子加入聚阳离子溶液，震荡使粒子吸附聚阳离子，离心、洗涤后，得到聚阳离子修饰的粒子，随后加入聚阴离子溶液中，震荡使粒子吸附聚阴离子，离心、洗涤得到聚电解质修饰处理后的粒子。
3.根据权利要求2所述的具有清除活性氧及抗炎协同增强作用的纳米粒子，其特征在于，所述的聚阴离子溶液采用硫酸葡聚糖钠盐溶液。
4.根据权利要求2所述的具有清除活性氧及抗炎协同增强作用的纳米粒子，其特征在于，所述的聚阴离子溶液的浓度为1-2mg/ml。
5.根据权利要求2所述的具有清除活性氧及抗炎协同增强作用的纳米粒子，其特征在于，所述的聚阳离子溶液为聚丙烯基胺盐酸盐溶液、聚二烯丙基二甲基季铵盐溶液、壳聚糖溶液、聚赖氨酸溶液或聚乙烯亚胺溶液。
6.根据权利要求1所述的具有清除活性氧及抗炎协同增强作用的纳米粒子，其特征在于，所述的中空二氧化锰纳米粒子为中空的花生状或椭球状的二氧化锰粒子，其粒径为500-600nm。
7.根据权利要求1所述的具有清除活性氧及抗炎协同增强作用的纳米粒子，其特征在于，所述的中空二氧化锰纳米粒子采用如下方法制得：
在搅拌条件下，将碳酸钠溶液加入水溶性二价锰溶液中，室温下反应30s-8h，反应结束后，洗涤，得到碳酸锰模板纳米粒子；将所得的粒子分散到水中,再加入高锰酸钾溶液，反应0.25h-8h，洗涤后将粒子分散在水中，加入盐酸溶液，震荡反应0.25h-8h，洗涤得到中空二氧化锰纳米粒子。
8.根据权利要求7所述的具有清除活性氧及抗炎协同增强作用的纳米粒子，其特征在于，所述的高锰酸钾溶液的浓度为1-100mg/mL。
9.根据权利要求1所述的具有清除活性氧及抗炎协同增强作用的纳米粒子，其特征在于，中空二氧化锰纳米粒子经聚电解质修饰后，分散于溶解有抗炎药的溶剂中，进行载药。
10.根据权利要求9所述的具有清除活性氧及抗炎协同增强作用的纳米粒子，其特征在于，所述的抗炎药为地塞米松、布地奈德、姜黄素、泼尼松、甲泼尼松、倍他米松、丙酸倍氯米松、泼尼松龙或氢化可的松。
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