CN111514316B - 一种炎症靶向及微环境响应性纳米系统及制备方法与应用 - Google Patents
一种炎症靶向及微环境响应性纳米系统及制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种炎症靶向及微环境响应性纳米系统及制备方法与应用,涉及分子影像学技术领域;纳米系统包括靶向功能分子、纳米载体、核心晶体;靶向功能分子为CD163靶向结合短肽,短肽通过共价连接的方式与纳米粒表面的聚乙二醇相连;核心晶体以生物矿化的方式包载在纳米载体内。该纳米系统静脉注射后,通过靶向CD163阳性巨噬细胞在癫痫灶富集,与H+和H2O2发生反应使癫痫灶的磁共振T1信号增强成像,准确、直观的显示特发性/隐源性癫痫灶,有助于临床提高治疗精确性、安全性;还可以催化清除癫痫灶的活性氧物质减轻具有氧化应激,产生氧气改善局部乏氧微环境,保护癫痫区的神经元活力。
Description
技术领域
本发明涉及分子影像学技术领域,涉及一种炎症靶向及微环境响应性纳米系统及制备方法与应用,尤其涉及一种针对脑癫痫灶的炎症靶向及微环境响应性纳米药物系统及其制备与应用。
背景技术
癫痫是最常见的慢性神经疾病之一,以脑神经元异常放电引起反复痫性发作为特征。全世界大约有6500万人患有癫痫,癫痫对患者的健康和生活质量以及整个社会的影响极其严重。目前没有药物能够治愈癫痫,疾病通常伴随患者终身。
现有的抗癫痫药多为对症治疗药,副作用多,并且在30-40%的患者中无效。手术切除癫痫灶是治愈癫痫的有效方法,而病灶的精确定位是手术的关键。
公告号为CN108113669A的中国发明专利提供一种癫痫病灶定位方法及系统,所述方法包括:步骤S1、基于预设的多个导联电极,采集每个导联电极对应的一维癫痫脑电信号;步骤S2、将所述一维癫痫脑电信号转换为二维癫痫脑电信号图像;步骤S3、对所述二维癫痫脑电信号图像进行图像处理和纹理特征提取,以确定所述多个导联电极中的癫痫病灶导联电极位置。
目前癫痫患者的术前评估主要依靠视频脑电图监测和磁共振检查。然而,在接受过外科治疗的癫痫患者中,有18%~43%患者的磁共振检查不能发现明确的癫痫病灶。而磁共振检查阴性的患者常常无法进行手术治疗,即使进行手术效果往往也不佳。因此,急需一种新的技术来实现对癫痫灶的准确定位。
发明内容
针对现有技术的缺陷,本发明的目的是提供一种炎症靶向及微环境响应性纳米粒及其制备与应用;具体涉及一种短肽修饰的纳米递药系统。本发明对比剂可通过靶向CD163+巨噬细胞而在癫痫灶富集,在微环境下响应性的磁共振T1信号增强而成像,提高癫痫的检出率和准确率。同时,纳米粒清除癫痫灶活性氧并改善乏氧微环境,起神经细胞保护作用。
本发明的目的是通过以下技术方案实现的:一种炎症靶向及微环境响应性纳米系统,所述纳米系统包括如下重量份数的各组分:
纳米载体40~80份;
二氧化锰(MnO2)6~10份;
聚乙二醇(PEG)28~32份;
靶向功能分子4~6份,以纳米载体60份、二氧化锰8份、聚乙二醇30份、靶向功能分子5份为优。
优选地,所述靶向功能分子为CD163靶向结合短肽(CTHRSSVVC),所述纳米载体为白蛋白(BSA),二氧化锰为核心晶体;所述聚乙二醇位于纳米载体的表面,所述靶向功能分子与纳米载体表面的聚乙二醇通过共价连接的方式相连接,所述核心晶体以生物矿化的方式包载在纳米载体内。
抗脑癫痫药物以包裹或共价连接的方式与纳米载体相结合,CD163靶向结合短肽通过共价连接的方式与纳米载体表面的聚乙二醇相连。
优选地,所述白蛋白包括牛血清白蛋白;所述白蛋白的活性基团包括羧基、硫醇基和氨基。
优选地,所述聚乙二醇的分子量为1000~10000;所述纳米系统的粒径为20~30nm。
一种炎症靶向及微环境响应性纳米系统的制备方法,包括如下步骤:
A、将白蛋白溶于超纯水中,加入MnCl2溶液,调节酸碱度至pH=10~12进行反应,获得BSA-MnO2纳米粒(BM);
B、取聚乙二醇加入BSA-MnO2纳米粒的水溶液中搅拌形成PEG化的BSA-MnO2纳米粒;
C、将CD163短肽添加到PEG化的BM溶液中反应即得BSA-MNO2-CD163短肽纳米粒(BMC)。
优选地,所述步骤A具体包括如下步骤:将白蛋白溶于超纯水得到白蛋白溶液,所述白蛋白溶液的浓度为5~40mg/ml,最优为10mg/ml;
在剧烈搅拌的同时向白蛋白溶液中缓慢加入MnCl2溶液,述白蛋白与MnCl2的重量比为1:5~1:40,最优为1:10;所述剧烈搅拌的转速为100~200rad/min,以120rad/min为优,剧烈搅拌的时间大于或等于5min,以10min为优;
使用NaOH调节溶液酸碱度至pH=10~12,以PH=11为优,PH超过上限将导致纳米粒粒径明显增大,PH低于下限将导致MnO2生成量明显减少;在25~40℃,最优为37℃,搅拌下反应1~4小时,以2小时为优,反应时间超过上限将导致纳米粒粒径明显增大,反应时间小于下限将导致MnO2生成量明显不足,获得BSA-MnO2纳米粒溶液(BM);
所述步骤A与步骤B之间还包括如下步骤:使用透析袋将获得BSA-MnO2纳米粒溶液在超纯水中透析,所述透析袋为10kDa分子量透析袋,透析时间大于或等于48小时,以48小时为优。
优选地,所述步骤B具体包括如下步骤:取聚乙二醇加入所述BSA-MnO2纳米粒的水溶液,所述聚乙二醇与BSA-MnO2纳米粒的水溶液的摩尔比为10:1,在室温下搅拌1小时及以上,以1~2小时为优,即得PEG化的BSA-MnO2纳米粒溶液;所述聚乙二醇包括NHS-PEG-MAL;
所述步骤B与步骤C之间还包括如下步骤:使用超滤管将PEG化的BSA-MnO2纳米粒溶液超滤,除去未连接的PEG;所述超滤管为10kDa分子量(MWCO)的超滤管;所述超滤中离心的转速控制在3000~5000rad/min,以5000rad/min为优;所述超滤时间10~20min,以15min为优;次数为2~4次,以3次为优。
优选地,所述步骤C具体包括如下步骤:将CD163短肽添加到PEG化的BSA-MnO2纳米粒溶液中,所述CD163短肽与PEG化的BSA-MnO2纳米粒的水溶液的摩尔比为5:1,使CD163短肽的巯基与PEG化的BSA-MnO2纳米粒溶液的马来酰胺基(MAL)在室温下反应1小时,即得BSA-MNO2-CD163短肽纳米粒;
所述步骤C之后还包括如下步骤:使用超滤管将BSA-MNO2-CD163短肽纳米粒(BMC)溶液超滤,除去过量的短肽,所述超滤管为10kDa分子量(MWCO)的超滤管;所述超滤中离心的转速控制在3000~5000rad/min,以5000rad/min为优;所述超滤时间10~20min,以15min为优;所述次数2~4次,以3次为优。
一种炎症靶向及微环境响应性纳米系统的应用,将抗脑癫痫药物与纳米载体以包裹或共价连接的方式相连接,用于制造癫痫的磁共振成像和神经保护用的组合物的应用。
一种炎症靶向及微环境响应性纳米系统的应用,将抗脑癫痫药物与纳米载体以包裹或共价连接的方式相连接,用于制造以CD163+巨噬细胞为炎症靶点实现对癫痫灶精准成像和靶向递药的制剂的应用。
过去癫痫病被认为是神经元疾病,而最近越来越多的证据表明炎症在癫痫的病理生理中起着重要的作用。申请人研究发现,癫痫与外周免疫系统的活化有关,癫痫灶可见大量浸润的外周单核巨噬细胞。CD163是在M2型巨噬细胞表面特异性表达的受体,仅表达于活化单核巨噬细胞系细胞的细胞膜。在癫痫患者或癫痫模型动物的脑组织中,癫痫灶内CD163+巨噬细胞显著增加,而正常脑组织的CD163+巨噬细胞量极少。CD163作为癫痫灶的特异性诊疗靶点具有如下优势:第一、CD163+巨噬细胞存在于脑毛细血管内皮细胞管腔侧,药物无需通过血脑屏障即可被其特异性结合;第二、CD163+巨噬细胞可以通过趋化游走穿透血脑屏障,在癫痫灶聚集,有利于药物的靶向递送;第三、CD163+巨噬细胞可用于反映癫痫灶炎症的严重程度,与血脑屏障破坏、癫痫的发作次数和持续时间正相关,有助于评估疾病的病情、疗效和预后。先前已有研究以CD163为靶点成功实现血管粥样硬化和帕金森病的诊疗,然而靶向CD163的癫痫药物尚未见报道。因此,通过靶向CD163+巨噬细胞而实现癫痫灶的精确定位和靶向递药具有极大的临床应用价值。
短肽CTHRSSVVC是通过噬菌体短肽库筛选出的可以与CD163蛋白特异性结合的短肽,其具有分子量小、空间结构稳定、亲和力高等特点(Silva R A.CTHRSSVVC Peptide asa Possible Early Molecular Imaging Target for Atherosclerosis[J].Int J MolSci,2016,17(9))。因此,通过短肽CTHRSSVVC构建靶向性纳米探针及递药系统,有望改善现有癫痫诊疗手段的不足,提高疾病诊断的特异性与灵敏度,并为癫痫的药物靶向递送带来了新的契机。目前国内外未见报道。
纳米载体是靶向性纳米探针和药物递送系统的基础。基于蛋白质的纳米载体具有无免疫原性、生物相容性好、生物可降解等特点。而生物矿化法制备简单绿色,并且纳米粒径可控、载药量高,可以有效地提高材料成像和治疗的效果,具有良好的生物应用前景。
二氧化锰由于其优异的生物相容性、微环境响应的降解途径以及高效的催化性能在肿瘤诊疗一体化的应用中已备受青睐,但目前尚未见二氧化锰在癫痫诊疗中应用的研究。癫痫灶神经元反复异常放电,耗氧、耗能明显增加,细胞发生无氧酵解产生乳酸,局部形成乏氧的酸性微环境。同时,癫痫灶存在明显的炎症反应,胶质细胞的活化和巨噬细胞的浸润产生了大量的活性氧物质(ROS)。乏氧的微环境和活性氧物质将共同导致神经元的变性和坏死。二氧化锰具有良好的酸和H2O2响应性,在癫痫微环境下可以产生Mn2+离子,缩短磁共振T1弛豫时间,放大磁共振成像信号。另外,二氧化锰可以作为催化剂清除超氧阴离子和H2O2,并产生氧气,具有清除活性氧,改善组织乏氧的价值。
根据上述背景,本申请发明人拟构建基于白蛋白的纳米探针及药物递送系统,修饰CD163靶向性结合短肽作为靶向功能基团,并以MnO2微环境响应晶体作为纳米核心,为癫痫疾病的诊疗一体化提供新的技术和方法。
综上所述,与现有技术相比,本发明具有如下的有益效果:
(1)本发明公开了一种炎症靶向及微环境响应性纳米系统及制备方法与应用,用于癫痫的磁共振成像和神经保护的一体化诊疗,所述纳米系统包括靶向功能分子,纳米载体和核心晶体;所述的靶向功能分子为针对癫痫灶区聚集的CD163阳性细胞具有高亲和力的短肽,短肽通过共价连接的方式与纳米粒表面的聚乙二醇相连;所述核心晶体以生物矿化的方式包载在纳米载体内;
(2)该纳米系统静脉注射后,以CD163+巨噬细胞为炎症靶点便于实现对癫痫灶的精准成像和靶向递药,通过靶向CD163阳性巨噬细胞而在癫痫灶富集,与癫痫微环境中的H+和H2O2发生反应使癫痫灶的磁共振T1信号增强而成像,从而能够准确、直观显示特发性/隐源性癫痫灶,有助于临床提高治疗的精确性和安全性;便于研发基于锰基的磁共振T1WI对比剂进行癫痫灶的微环境响应性成像;
(3)该纳米系统可以催化清除癫痫灶的活性氧物质减轻具有氧化应激,产生氧气改善局部乏氧微环境,从而保护癫痫区的神经元活力,建立了基于MnO2的纳米酶在改善癫痫疾病氧化应激和乏氧环境中的应用;
(4)从炎症显像的角度解决现有技术无法早期准确显示癫痫灶,能直观显示特发性/隐源性癫痫;
(5)该纳米系统与CD163+细胞特异性结合,通过量化间接反映CD163细胞数量,有助于早期评估癫痫灶的炎症细胞数量,反应神经炎症的严重程度。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述,本发明的其它特征、目的和优点将会变得更明显:
图1为实施例1一种炎症靶向及微环境响应性纳米系统的制备方法示意图;
图2为实施例1一种炎症靶向及微环境响应性纳米系统的表征图;
图3为实施例1一种炎症靶向及微环境响应性纳米系统的MR-T1加权成像图;
图4为实施例1一种炎症靶向及微环境响应性纳米系统的H2O2清除和氧气产生曲线图;
图5为实施例2一种炎症靶向及微环境响应性纳米系统的应用中建立体外CD163+巨噬细胞模型,比较有无地塞米松诱导下J774A.1细胞的CD163蛋白表达结果图;
图6为实施例2一种炎症靶向及微环境响应性纳米系统的应用中建立体外CD163+巨噬细胞模型,分析靶向纳米粒对CD163蛋白的特异性结合的结果示意图;
图7为实施例3一种炎症靶向及微环境响应性纳米系统的应用中建立癫痫动物模型,比较癫痫组与假手术组海马区CD163+细胞示意图;
图8为实施例4一种炎症靶向及微环境响应性纳米系统的应用中建立癫痫动物模型,分析靶向纳米粒对CD163蛋白的特异性结合的结果示意图;
图9为实施例5一种炎症靶向及微环境响应性纳米系统的应用中建立J774A.1细胞和PC12细胞氧化应激模型,分析纳米粒清除细胞内活性氧的免疫荧光图;
图10为实施例5一种炎症靶向及微环境响应性纳米系统的应用中建立J774A.1细胞和PC12细胞氧化应激模型,分析纳米粒对细胞凋亡的影响;
图11为实施例5一种炎症靶向及微环境响应性纳米系统的应用中建立J774A.1细胞和PC12细胞氧化应激模型,分析纳米粒对细胞活力的影响;
图12为实施例6一种炎症靶向及微环境响应性纳米系统的应用中建立癫痫动物模型,分析纳米粒对改善癫痫灶乏氧、细胞凋亡和神经元损伤的影响。
具体实施方式
以下实施例将有助于本领域的技术人员进一步理解本发明,但不以任何形式限制本发明。对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,还可以做出若干变化和改进,这些都属于本发明的保护范围。在本文中所披露的范围的端点和任何值都不限于该精确的范围或值,这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说,各个范围的端点值之间、各个范围的端点值和单独的点值之间,以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围,这些数值范围应被视为在本文中具体公开,下面结合具体实施例对本发明进行详细说明:
一种炎症靶向及微环境响应性纳米系统,包括如下重量份数的各组分:40~80份纳米载体;6~10份二氧化锰;28~32份聚乙二醇;4~6份靶向功能分子。其中靶向功能分子为CD163靶向结合短肽,纳米载体为白蛋白,白蛋白为牛血清白蛋白;白蛋白的活性基团包括羧基、硫醇基和氨基。二氧化锰为核心晶体;聚乙二醇位于纳米载体的表面,靶向功能分子与纳米载体表面的聚乙二醇通过共价连接的方式相连接,核心晶体以生物矿化的方式包载在纳米载体内。聚乙二醇的分子量为1000~10000;纳米系统的粒径为20~30nm。
其制备方法包括如下步骤:
A、BSA-MnO2纳米粒的制备:配置白蛋白BSA水溶液(5~40mg/ml),(100~200rad/min)剧烈搅拌下缓慢加入MnCl2溶液,(白蛋白与MnCl2的重量比为1:5~1:40),搅拌的时间为5min。使用NaOH(0.1uM)调节溶液酸碱度至pH=(10~12),在(25~40℃)剧烈搅拌下反应(1~4小时)。
使用10kDa分子量(MWCO)透析袋在超纯水中透析48小时,除去过量的前体即获得BSA-MnO2纳米粒(BM)。
B、BSA-MnO2-CD163短肽纳米粒的制备:取28~32份NHS-PEG-MAL加入BM的水溶液中中,在环境温度下搅拌1小时以形成PEG化的BSA-MnO2。
使用10kDa分子量(MWCO)的超滤管除去过量未连接的PEG。
C、将4~6份的CD163短肽以5:1摩尔比例添加到PEG化的BM溶液中,使CD163短肽的巯基与PEG化的BM的马来酰胺(MAL)基在室温下反应1小时。
使用10kDa分子量(MWCO)的超滤管除去过量的短肽即获得靶向纳米粒,制备过程如图1。
实施例1
一种炎症靶向及微环境响应性纳米系统,包括如下重量份数的各组分:60份纳米载体;8份二氧化锰;30份聚乙二醇;5份靶向功能分子。其中靶向功能分子为CD163靶向结合短肽,纳米载体为白蛋白,白蛋白为牛血清白蛋白;白蛋白的活性基团包括羧基、硫醇基和氨基。二氧化锰为核心晶体;聚乙二醇位于纳米载体的表面,靶向功能分子与纳米载体表面的聚乙二醇通过共价连接的方式相连接,核心晶体以生物矿化的方式包载在纳米载体内。聚乙二醇的分子量为3400;纳米系统的粒径为25nm。
如图1所示;其制备方法包括如下步骤:
A、BSA-MnO2纳米粒的制备:将120mg白蛋白BSA溶于12mL超纯水(10mg/ml),120rad/min剧烈搅拌下缓慢加入0.8mL MnCl2溶液(1.02mg),搅拌的时间为5min。使用NaOH(0.1uM)调节溶液酸碱度至pH=11,在37℃剧烈搅拌下反应2小时。
使用10kDa分子量(MWCO)透析袋在超纯水中透析48小时,除去过量的前体即获得BSA-MnO2纳米粒(BM)。
B、BSA-MnO2-CD163短肽纳米粒的制备:取NHS-PEG-MAL 50mg,以10:1的摩尔比例加入BM的水溶液中(100mg,10mg/ml)中,在环境温度下搅拌1小时以形成PEG化的BSA-MnO2。
使用10kDa分子量(MWCO)的超滤管除去过量未连接的PEG。
C、将CD163短肽(0.828mg)以5:1摩尔比例添加到PEG化的BM溶液中,使CD163短肽的巯基与PEG化的BM的马来酰胺(MAL)基在室温下反应1小时。
使用10kDa分子量(MWCO)的超滤管除去过量的短肽即获得靶向纳米粒,制备过程如图1。
采用粒径分析仪测定纳米粒的粒径及Zeta电位,经过1%(w/v,pH 7.0)磷钨酸负染色后,透射电镜观察粒子形态,结果如图2所示:BMC纳米粒外观圆整,大小均一,水合粒径约为22nm。
采用MRI分析纳米粒的磁敏感特征及T1信号强度,结果如图3所示:纳米粒在酸性和H2O2环境下纵向弛豫性能显著增加,在PH5.0和H2O2环境下的r1值为11.38mM-1S-1)是其在中性溶液的7.8倍,为Gd-DTPA的2.3倍。
使用紫外分光光度仪评估纳米粒清除H2O2的效能,采用氧溶解测定仪检测BMC在酸性和H2O2环境下的产氧能力,结果如图4所示:其中,A为免疫荧光共聚焦定性分析图,地塞米松诱导组(右)J774A.1细胞的CD163蛋白表达明显高于无地塞米松诱导组(左);B为流式细胞仪PE荧光定量分析,地塞米松诱导(右)RBCECs的P-糖蛋白增高4.4倍;BMC可以体外高效地清除H2O2,消耗率达85.07%;BMC在体外酸性和H2O2的环境中具有显著产氧能力。
实施例2
一种炎症靶向及微环境响应性纳米系统的应用:
采用250nM地塞米松刺激J774A.1细胞24小时诱导J774A.1巨噬细胞构建CD163+细胞模型,结果如图5所示:图4所示地塞米松可诱导J774A.1细胞高表达CD163蛋白。
将香豆素-6标记的BMC/BMP(200ug/mL)分别与地塞米松诱导组及无地塞米松诱导组的J774A.1细胞共孵育15分钟。再取地塞米松诱导的J774A.1细胞另设游离CD163短肽组,即在加入BSA-MNO2-CD163短肽纳米粒(BMC)孵育前,加入CD163短肽(200ug/mL)提前孵育15分钟。通过采用免疫荧光共聚焦成像、流式细胞仪分析在体外验证BMC对CD163的靶向结合能力,结果如图6所示:其中,A为免疫荧光共聚焦定性分析图,CD163+细胞对靶向纳米粒靶向摄取较非靶向纳米粒明显增高;B为流式细胞仪荧光定量分析,CD163+细胞对靶向纳米粒靶向摄取是非靶向纳米粒的2.1倍;说明靶向功能分子CD163短肽可以结合CD163蛋白的结合位点,有助于纳米粒转运与显影。
实施例3
一种炎症靶向及微环境响应性纳米系统的应用:
立体定位SD大鼠右侧海马,微针注射3μL红藻氨酸(KA,0.5mg/mL),建立癫痫动物模型;注射3μL PBS(磷酸缓冲液)为对照组;4%多聚甲醛心脏灌流,取脑,通过免疫组化成像观察癫痫组、假手术组海马区CD163+细胞的情况,结果如图7所示:其中,A为CD163+免疫组化图;B为CD163+细胞计数图,癫痫组CD163+细胞明显增高;图7显示癫痫组海马区CD163+细胞数明显高于对照组,说明可以提示癫痫灶CD163+细胞增多。
实施例4
一种炎症靶向及微环境响应性纳米系统的应用:
建立SD大鼠癫痫动物模型,按靶向纳米粒BMC、非靶向纳米粒分为两组,以5mg Mn/kg剂量尾静脉给药,2h后行3.0T MRI颅脑T1WI扫描,如图8所示:其中,A为MRI直观显示靶向纳米粒(左)在癫痫灶的分布明显高于非靶向纳米粒(右);B为脑组织冰冻切片免疫荧光成像示癫痫灶内荧光标记的靶向纳米粒高度聚集。
如图8A结果所示,靶向纳米粒BMC在脑癫痫部位的分布明显多于非靶向纳米粒,其T1信号强度明显增高。
以0.25mg/kg DiR的剂量分别对SD癫痫大鼠尾静脉注射DiR标记的靶向纳米粒BMC和非靶向纳米粒。于给药后2小时采用免疫荧光染色及激光共聚焦成像观察海马区纳米粒与CD163的共定位情况,如图8B结果所示,CD163短肽修饰的靶向纳米粒在脑海马区的分布明显增多,在动物水平证实了CD163短肽可与CD163蛋白的特异性结合,靶向功能分子CD163短肽的修饰有助于纳米粒分布到癫痫部位。
实施例5
一种炎症靶向及微环境响应性纳米系统的应用:
采用活性氧诱导剂Rosup诱导J774A.1细胞和PC12细胞,使细胞内活性氧水平增高,并与纳米粒共孵育,通过DCFH-DA荧光探针考察纳米粒对细胞内活性氧的影响;活性氧诱导剂Rosup诱导J774A.1细胞和PC12细胞,使细胞内活性氧水平增高,并与纳米粒共孵育,通过DCFH-DA荧光探针考察纳米粒对细胞内活性氧的影响,结果如图9所示:其中,A为纳米粒治疗后J774A.1细胞后细胞内活性氧荧光明显减低,B为纳米粒治疗PC12细胞后活性氧荧光明显减低,加入Rosup后,J774A.1细胞和PC12细胞的细胞内出现明显的活性氧荧光,加入纳米粒共孵育后,细胞内荧光明显下降;表明纳米粒可以有效清除细胞内活性氧。
如图10所示:其中,A为纳米粒治疗后J774A.1细胞细胞凋亡明显减低,B为纳米粒治疗PC12细胞细胞凋亡明显减低,表明纳米粒可以有效减轻氧化应激引起的细胞凋亡;即加入纳米粒孵育后,H2O2处理的J774A.1细胞和PC12细胞的凋亡率显著下降。
如图11所示:其中,A为纳米粒治疗后J774A.1细胞的细胞活力明显增高,B为纳米粒治疗PC12细胞细胞活力明显增高,表明纳米粒可以有减轻氧化应激引起的细胞活力减低;即加入纳米粒孵育后,H2O2处理的J774A.1细胞和PC12细胞的细胞活力显著上升。
实施例6
一种炎症靶向及微环境响应性纳米系统的应用:
将KA癫痫大鼠分为2组,以5mg Mn/kg的剂量尾静脉注射BMC纳米粒或等量PBS。于给药2小时,制作大鼠海马冰冻切片,采用HIF-1α免疫荧光染色观察大鼠海马组织缺氧的情况;给药后24小时,制作大鼠海马切片,采用TUNEL免疫荧光染色观察大鼠海马的细胞凋亡情况,采用尼氏神经元染色评估癫痫大鼠海马的神经元细胞的情况。
如图12所示,A为纳米粒治疗后大鼠癫痫灶乏氧荧光明显减弱,表明纳米粒可改善癫痫灶的乏氧;B为纳米粒治疗后大鼠癫痫灶细胞凋亡信号明显减少,表明纳米粒可减轻癫痫灶的细胞凋亡;C为纳米粒治疗后大鼠癫痫灶神经元细胞明显增多,排列规则,表明纳米粒可以有效保护神经元细胞。
即如图12A结果所示,KA+PBS组大鼠海马区可见明显的HIF-1α绿色信号,KA+BMC组药物治疗后大鼠海马区绿色荧光明显减弱,提示纳米粒可改善癫痫灶的乏氧环境。图12B结果所示,KA+PBS组大鼠海马区可见明显的细胞凋亡红色信号,KA+BMC组在药物治疗后大鼠海马区仅见少量红色荧光,表明纳米粒可减轻海马组织细胞凋亡。图12C结果所示,KA+PBS组大鼠海马CA3区神经元丢失明显,细胞排列紊乱,细胞核固缩、尼氏体数量减少;KA+BMC组药物治疗后大鼠海马区神经元边缘清晰,神经元排列规则,尼氏体数量明显增加,神经元丢失明显改善,说明纳米粒对癫痫灶神经元细胞具有保护作用。
实施例7
一种炎症靶向及微环境响应性纳米系统,包括如下重量份数的各组分:40份纳米载体;6份二氧化锰;28份聚乙二醇;4份靶向功能分子。其中靶向功能分子为CD163靶向结合短肽,纳米载体为白蛋白,白蛋白为牛血清白蛋白;白蛋白的活性基团包括羧基、硫醇基和氨基。二氧化锰为核心晶体;聚乙二醇位于纳米载体的表面,靶向功能分子与纳米载体表面的聚乙二醇通过共价连接的方式相连接,核心晶体以生物矿化的方式包载在纳米载体内。聚乙二醇的分子量为1000;纳米系统的粒径为20nm。
其制备方法包括如下步骤:
A、BSA-MnO2纳米粒的制备:配置白蛋白BSA水溶液(5mg/ml),100rad/min剧烈搅拌下缓慢加入MnCl2,(白蛋白溶液与MnCl2溶液的重量比为40:6),搅拌的时间为5min。使用NaOH(0.1uM)调节溶液酸碱度至pH=(10),在25℃剧烈搅拌下反应2小时(1小时)。
使用10kDa分子量(MWCO)透析袋在超纯水中透析48小时,除去过量的前体即获得BSA-MnO2纳米粒(BM)。
B、BSA-MnO2-CD163短肽纳米粒的制备:取NHS-PEG-MAL 28份加入BM的水溶液中,在环境温度下搅拌1小时以形成PEG化的BSA-MnO2。
使用10kDa分子量(MWCO)的超滤管除去过量未连接的PEG。
C、将CD163短肽(4份)添加到PEG化的BM溶液中,使CD163短肽的巯基与PEG化的BM的马来酰胺(MAL)基在室温下反应1小时。
使用10kDa分子量(MWCO)的超滤管除去过量的短肽即获得靶向纳米粒,制备过程如图1。
该纳米系统静脉注射后,以CD163+巨噬细胞为炎症靶点便于实现对癫痫灶的精准成像和靶向递药,通过靶向CD163阳性巨噬细胞而在癫痫灶富集,与癫痫微环境中的H+和H2O2发生反应使癫痫灶的磁共振T1信号增强而成像,能够准确、直观显示特发性/隐源性癫痫灶,提高治疗的精确性和安全性;还可以催化清除癫痫灶的活性氧物质减轻具有氧化应激,产生氧气改善局部乏氧微环境,从而保护癫痫区的神经元活力,建立了基于MnO2的纳米酶在改善癫痫疾病氧化应激和乏氧环境中的应用;从炎症显像的角度解决现有技术无法早期准确显示癫痫灶,能直观显示特发性/隐源性癫痫;对比剂与CD163+细胞特异性结合,通过量化间接反映CD163细胞数量,有助于早期评估癫痫灶的炎症细胞数量,反应神经炎症的严重程度。
实施例8
一种炎症靶向及微环境响应性纳米系统,包括如下重量份数的各组分:80份纳米载体;10份二氧化锰;32份聚乙二醇;6份靶向功能分子。其中靶向功能分子为CD163靶向结合短肽,纳米载体为白蛋白,白蛋白为牛血清白蛋白;白蛋白的活性基团包括羧基、硫醇基和氨基。二氧化锰为核心晶体;聚乙二醇位于纳米载体的表面,靶向功能分子与纳米载体表面的聚乙二醇通过共价连接的方式相连接,核心晶体以生物矿化的方式包载在纳米载体内。聚乙二醇的分子量为10000;纳米系统的粒径为30nm。
其制备方法包括如下步骤:
A、BSA-MnO2纳米粒的制备:配置白蛋白BSA水溶液40mg/ml),(200rad/min剧烈搅拌下缓慢加入10份MnCl2溶液,(白蛋白溶液与MnCl2溶液的重量比为80:40),搅拌的时间为5min。使用NaOH(0.1uM)调节溶液酸碱度至pH=12),在37℃(40℃)剧烈搅拌下反应4小时)。
使用10kDa分子量(MWCO)透析袋在超纯水中透析48小时,除去过量的前体即获得BSA-MnO2纳米粒(BM)。
B、BSA-MnO2-CD163短肽纳米粒的制备:取32份NHS-PEG-MAL加入BM的水溶液中中,在环境温度下搅拌1小时以形成PEG化的BSA-MnO2。
使用10kDa分子量(MWCO)的超滤管除去过量未连接的PEG。
C、将CD163短肽(6份)添加到PEG化的BM溶液中,使CD163短肽的巯基与PEG化的BM的马来酰胺(MAL)基在室温下反应1小时。
使用10kDa分子量(MWCO)的超滤管除去过量的短肽即获得靶向纳米粒,制备过程如图1。
该纳米系统静脉注射后,以CD163+巨噬细胞为炎症靶点便于实现对癫痫灶的精准成像和靶向递药,通过靶向CD163阳性巨噬细胞而在癫痫灶富集,与癫痫微环境中的H+和H2O2发生反应使癫痫灶的磁共振T1信号增强而成像,能够准确、直观显示特发性/隐源性癫痫灶,提高治疗的精确性和安全性;还可以催化清除癫痫灶的活性氧物质减轻具有氧化应激,产生氧气改善局部乏氧微环境,从而保护癫痫区的神经元活力,建立了基于MnO2的纳米酶在改善癫痫疾病氧化应激和乏氧环境中的应用;从炎症显像的角度解决现有技术无法早期准确显示癫痫灶,能直观显示特发性/隐源性癫痫;对比剂与CD163+细胞特异性结合,通过量化间接反映CD163细胞数量,有助于早期评估癫痫灶的炎症细胞数量,反应神经炎症的严重程度。
以上对本发明的具体实施例进行了描述。需要理解的是,本发明并不局限于上述特定实施方式,本领域技术人员可以在权利要求的范围内做出各种变化或修改,这并不影响本发明的实质内容。在不冲突的情况下,本申请的实施例和实施例中的特征可以任意相互组合。
Claims (9)
2.根据权利要求1所述的炎症靶向及微环境响应性纳米系统,其特征在于,所述白蛋白包括牛血清白蛋白;所述白蛋白的活性基团为羧基、硫醇基或氨基。
3.根据权利要求1所述的炎症靶向及微环境响应性纳米系统,其特征在于,所述聚乙二醇的分子量为1000~10000;所述纳米系统的粒径为20~30nm。
4.一种根据权利要求1-3中任一炎症靶向及微环境响应性纳米系统的制备方法,其特征在于,包括如下步骤:
A、将白蛋白溶于超纯水中,加入MnCl2溶液,调节酸碱度至pH=10~12进行反应,获得BSA-MnO2纳米粒;
B、取聚乙二醇加入BSA-MnO2纳米粒的水溶液中搅拌形成PEG化的BSA-MnO2纳米粒;
C、将CD163短肽添加到PEG化的BM溶液中反应即得BSA-MnO2-CD163短肽纳米粒。
5.根据权利要求4所述的炎症靶向及微环境响应性纳米系统的制备方法,其特征在于,所述步骤A具体包括如下步骤:将白蛋白溶于超纯水得到白蛋白溶液,所述白蛋白溶液的浓度为5~40mg/ml;
在剧烈搅拌的同时向白蛋白溶液中加入MnCl2溶液,所述白蛋白与MnCl2的重量比为1:5~1:40;所述剧烈搅拌的转速为100~200rad/min,以120rad/min为优,剧烈搅拌的时间大于或等于5min;
使用NaOH调节溶液酸碱度至pH=10~12;在25~40℃,搅拌下反应1~4小时,获得BSA-MnO2纳米粒溶液;
所述步骤A与步骤B之间还包括如下步骤:使用透析袋将获得BSA-MnO2纳米粒溶液在超纯水中透析,所述透析袋为10kDa分子量透析袋,透析时间大于或等于48小时。
6.根据权利要求4所述的炎症靶向及微环境响应性纳米系统的制备方法,其特征在于,所述步骤B具体包括如下步骤:取聚乙二醇加入所述BSA-MnO2纳米粒的水溶液,所述聚乙二醇与BSA-MnO2纳米粒的水溶液的摩尔比为10:1,在室温下搅拌1小时及以上,即得PEG化的BSA-MnO2纳米粒溶液;所述聚乙二醇包括NHS-PEG-MAL;
所述步骤B与步骤C之间还包括如下步骤:使用超滤管将PEG化的BSA-MnO2纳米粒溶液超滤;所述超滤管为10kDa分子量的超滤管。
7.根据权利要求4所述的炎症靶向及微环境响应性纳米系统的制备方法,其特征在于,所述步骤C具体包括如下步骤:将CD163短肽添加到PEG化的BSA-MnO2纳米粒溶液中,所述CD163短肽与PEG化的BSA-MnO2纳米粒的水溶液的摩尔比为5:1,在室温下反应1小时,即得BSA-MNO2-CD163短肽纳米粒;
所述步骤C之后还包括如下步骤:使用超滤管将BSA-MNO2-CD163短肽纳米粒溶液超滤,所述超滤管为10kDa分子量的超滤管。
8.一种根据权利要求1-2中任一炎症靶向及微环境响应性纳米系统的应用,其特征在于,将抗脑癫痫药物与纳米载体以包裹或共价连接的方式相连接,用于制造癫痫的磁共振成像和神经保护用的组合物的应用。
9.一种根据权利要求1-2中任一炎症靶向及微环境响应性纳米系统的应用,其特征在于,将抗脑癫痫药物与纳米载体以包裹或共价连接的方式相连接,用于制造以CD163+巨噬细胞为炎症靶点实现对癫痫灶精准成像和靶向递药的制剂的应用。
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