CN116270728A - 一种具有内质网靶向的抗氧化功能纳米酶及其制备方法和应用 - Google Patents
一种具有内质网靶向的抗氧化功能纳米酶及其制备方法和应用 Download PDFInfo
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Abstract
本发明属于生物制药领域,涉及具有内质网靶向的新药,特别是指一种具有内质网靶向的抗氧化功能纳米酶及其制备方法和应用。本申请的新型纳米药物ER‑Ru MOF为以对十二烷基苯磺酰作为内质网靶向功能基团,并以Ru纳米酶作为内部包载药物,能有效消除活性氧的积累,抑制脑出血后损伤部位ROS和MMP2/9的激活,发挥了减轻疼痛超敏反应的作用。相比于天然酶而言,纳米酶具有更强的修饰性,本发明将构建一种内质网精准抗氧化纳米酶将实现氧化应激的精准调控。
Description
技术领域
本发明属于生物制药领域,涉及具有内质网靶向的新药,特别是指一种具有内质网靶向的抗氧化功能纳米酶及其制备方法和应用。
背景技术
中枢性卒中后疼痛(CPSP)发生在卒中后,其患病率估计为56%,当丘脑出血或缺血时,其患病率更高。全身性痛觉过敏、痛觉超敏和/或各种形式的疼痛(颈痛、肩痛、手痛和脚痛)是最常见的症状,可用的药物治疗通常是无用的。因此,迫切需要新的治疗靶点来开发更有效的止痛药。脑出血(ICH)后,神经炎症的炎症级联反应被释放。作为回应,活化的小胶质细胞释放因子,如活性氧(ROS,例如超氧物)、趋化因子和细胞因子,以及基质金属蛋白酶(MMPs)。新的证据表明ROS和MMPs在疼痛传导中发挥重要作用。活性氧包括过氧化氢(H2O2)、超氧阴离子(•O2 −)和羟基自由基(•OH)等,其过度产生引起继发性损伤,并清除O2•−及H2O2(不是•OH)以模拟超氧化物歧化酶(SOD)和过氧化氢酶。然而,其生物利用度低、半衰期短、穿透血脑屏障效率低以及对肾和肝功能的副作用限制了其临床应用。因此,迫切需要设计和开发具有强大活性氧清除活性和理想理化性质的药物来治疗脑出血。
天然抗氧化剂比如过氧化氢酶、超氧化物歧化酶在炎症治疗以及组织工程材料领域广泛应用,酶固定化技术越来越受到人们越来越多的关注。近些年发展的天然抗氧化抗炎中仍然存在问题,如:1.价格昂贵;2.体内易降解;3.酶催化剂醇耐受性较差。
相比于天然酶而言,纳米酶的研发成本更低、稳定性更强,循环利用性优异,同时还可催化一些非自然发生的生物过程,已被应用于疾病诊疗、生物传感、环境治理以及抗菌防污等领域。专利202110706467.8公开了一种过渡金属单原子纳米酶及其制备方法和用途,该专利通过仿POD的活性实现高催化性能,用于抗菌、废水处理和免疫印迹;而本申请期望开发一种具备内质网靶向、用于缓解中枢性卒中后疼痛的新型纳米酶。
发明内容
为解决上述技术问题,本发明提出一种具有内质网靶向的抗氧化功能纳米酶及其制备方法和应用。
本发明的技术方案是这样实现的:
一种具有内质网靶向的抗氧化功能纳米酶的制备方法,具体包括以下步骤:
(1)称量2-甲基咪唑,十六烷基三甲基溴化铵溶于去离子水中,转速400 rpm搅拌混匀,再向混合溶液中依次滴入溶有1 mg/mL牛血清白蛋白的去离子水溶液和溶有29.06mg/mL Zn(NO3)2•6H2O的去离子水溶液,室温持续搅拌20 min,反应结束后,将反应液以转速5000 rpm离心5 min,收集沉淀物,使用溶有RuCl3的去离子水重悬沉淀物后进行超声分散,持续搅拌12 h加入硼氢化钠反应10 min,再次收集沉淀物,将沉淀物洗涤2次、冷冻、干燥后即得RuMOF,其中2-甲基咪唑,十六烷基三甲基溴化铵、牛血清白蛋白、Zn(NO3)2•6H2O、RuCl3和硼氢化钠的质量比为100-400:1:1:10-30。
(2)将十二烷基苯磺酸、卵磷脂和胆固醇溶于氯仿/甲醇(v/v=2:1),然后在旋转蒸发器下干燥,之后,用Ru MOF溶液(1 mg/mL)对干燥的脂质膜进行水合,接着探头超声5分钟,得到ER-Ru MOF;其中十二烷基苯磺酸、卵磷脂和胆固醇的质量比为1:1:5。
上述的方法制备的具有内质网靶向的抗氧化功能纳米酶,其以对十二烷基苯磺酰作为内质网靶向功能基团,并以Ru纳米酶作为内部包载药物。
上述的抗氧化功能纳米酶在制备抗氧化药物中的应用。
上述的抗氧化功能纳米酶在制备抑制脑出血后损伤部位ROS和MMP2/9的激活的药物中的应用。
上述的抗氧化功能纳米酶在制备缓解中枢性卒中后疼痛的药物中的应用。
本发明具有以下有益效果:
1、本申请提供了一种对丘脑出血疼痛有缓解作用的新型纳米药物,在微量注射后第1天出现,在微量注射后第3-7天达到峰值,在对侧微量注射后至少持续28天。系统性纳米给药消除了活性氧的积累,使MMP-2和MMP-9在CPSP小鼠中的激活受到快速有效的抑制,减轻了疼痛超敏反应。在适当的时间窗口内使用纳米不仅可以减少进一步的ICH损伤,而且可以产生镇痛作用。总之,本研究使用ZIF作为生物活性表面装饰,钌(Ru)纳米颗粒作为功能核心,不仅提供了一种新的原位合成协同纳米疗法的方法,还揭示了CPSP在对抗氧化应激诱导的出血性卒中损伤中的应用机制。
2、本申请的新型纳米药物ER-Ru MOF为以对十二烷基苯磺酰作为内质网靶向功能基团,并以Ru纳米酶作为内部包载药物,能有效消除活性氧的积累,抑制脑出血后损伤部位ROS和MMP2/9的激活,发挥了减轻疼痛超敏反应的作用。相比于天然酶而言,纳米酶具有更强的修饰性,本发明将构建一种内质网精准抗氧化纳米酶将实现氧化应激的精准调控。
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一些实施例,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,还可以根据这些附图获得其他的附图。
图1为实施例1和对比例1合成的Ru MOF和ER-Ru MOF的透射电子显微镜镜图。
图2为实施例1和对比例1合成的ER-Ru MOF类SOD/CAT酶酶活性测定数据结果图。
图3为实施例1合成的ER-Ru MOF内质网靶向性验证。
图4为CPSP小鼠出现疼痛超敏且损伤区域内ROS和炎症因子表达增高;其中A:动物模型制备及行为测试时间线设计方案,B:Ⅶ型胶原酶注射后第三天CPSP组小鼠脑出血示意图,C:CPSP组小鼠神经系统评分,D:CPSP组小鼠丘脑出血后对侧肢体产生了长期痛觉过敏现象,E:丘脑出血后不会影响两组小鼠同侧肢体痛觉,图F、G、H为丘脑出血导致ROS(8-OHdG作为氧自由基的标记)(F)及炎症因子TNFa(G)和IL-6(H)的表达增加,I为CPSP组小鼠丘脑损伤区域尼氏体数量明显减少。
图5 QT-PCR (A)、West Bloting(B)、免疫荧光(C)均提示CPSP小鼠丘脑损伤区域内疼痛关键蛋白MMP2/9表达增高。
图6为纳米酶给药可减轻CPSP且未见明显毒副作用的效果图;其中图A:不同剂量纳米酶给药均可减轻CPSP组小鼠丘脑出血后遗留对侧肢体痛觉过敏现象且呈剂量依赖效应,B:不同剂量纳米酶给药不影响两组小鼠同侧肢体感觉 ,C:纳米酶(8mg/kg)不影响两组小鼠肝肾功能,未表现出体内毒性,D:试验期间未发现两组小鼠体重异常改变 。
图7为纳米酶给药可减轻损伤区域内脑损伤和ROS的表达图;较给予安慰剂组,纳米酶给药组小鼠丘脑病变区域ROS(8-OHdG作为氧自由基的标记)(A)、炎症因子TNFa(B)和IL-6(C)的表达明显减少,脑出血量(D)改善,尼氏体数量未见变化(E)。
图8为纳米酶给药可减轻损伤区域内MMP2/9的表达图;与既往文献报导的EMMPRIN非特异性抑制剂米诺环素相比,纳米酶给药后,疼痛行为学测试(A)显示CPSP模型小鼠疼痛过敏现象改善, QT-PCR (B)、West Bloting(C)均提示CPSP小鼠丘脑损伤区域脑组织疼痛关键蛋白MMP2/9表达降低。
具体实施方式
下面将结合本发明实施例,对本发明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付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
本实施例的具有内质网靶向的抗氧化功能纳米酶的制备方法,步骤如下:
称量331.65 mg 2-甲基咪唑,1 mg十六烷基三甲基溴化铵溶于5 mL去离子水中,转速400 rpm搅拌混匀,再向混合溶液中依次滴入1 mL溶有1 mg牛血清白蛋白的去离子水溶液和1 mL溶有29.06 mgZn(NO3)2•6H2O的去离子水溶液,室温持续搅拌20 min,反应结束后,将反应液以转速5000 rpm离心5 min,收集沉淀物,使用溶有1 mgRuCl3去离子水重悬沉淀物后进行超声分散,持续搅拌12 h加入1 mg硼氢化钠反应10 min,再次收集沉淀物,将沉淀物洗涤2次、冷冻、干燥后即得Ru MOF。
2 mg对十二烷基苯磺酰,2 mg胆固醇和10 mg卵磷脂溶于氯仿/甲醇(v/v=2:1),然后在旋转蒸发器下干燥。之后,用Ru MOF溶液(1 mg/mL,2 mL)对干燥的脂质膜进行水合,接着探头超声5分钟,得到ER-Ru MOF。
对产物Ru MOF和ER-Ru MOF进行透射电子显微镜分析,结果如图1所示,由图1可知ER-Ru MOF具有均一粒径,尺寸在100 nm左右,说明本发明有潜力有效穿透血脑屏障。
实施例2
本实施例的具有内质网靶向的抗氧化功能纳米酶的制备方法,步骤如下:
称量100 mg 2-甲基咪唑,1 mg十六烷基三甲基溴化铵溶于5 mL去离子水中,转速400 rpm搅拌混匀,再向混合溶液中依次滴入1 mL溶有1 mg牛血清白蛋白的去离子水溶液和1 mL溶有20 mgZn(NO3)2•6H2O的去离子水溶液,室温持续搅拌20 min,反应结束后,将反应液以转速5000 rpm离心5 min,收集沉淀物,使用溶有1 mgRuCl3去离子水重悬沉淀物后进行超声分散,持续搅拌12 h加入1 mg硼氢化钠反应10 min,再次收集沉淀物,将沉淀物洗涤2次、冷冻、干燥后即得Ru MOF。
1 mg对十二烷基苯磺酰,2 mg胆固醇和10 mg卵磷脂溶于氯仿/甲醇(v/v=2:1),然后在旋转蒸发器下干燥。之后,用Ru MOF溶液(1 mg/mL,2 mL)对干燥的脂质膜进行水合,接着探头超声5分钟,得到ER-Ru MOF。
对产物Ru MOF和ER-Ru MOF进行透射电子显微镜分析,结果如图1所示 ,由图1可知ER-Ru MOF具有均一粒径,尺寸在100 nm左右,说明本发明有潜力有效穿透血脑屏障。
实施例3
本实施例的具有内质网靶向的抗氧化功能纳米酶的制备方法,步骤如下:
称量400 mg 2-甲基咪唑,1 mg十六烷基三甲基溴化铵溶于5 mL去离子水中,转速400 rpm搅拌混匀,再向混合溶液中依次滴入1 mL溶有1 mg牛血清白蛋白的去离子水溶液和1 mL溶有10 mgZn(NO3)2•6H2O的去离子水溶液,室温持续搅拌20 min,反应结束后,将反应液以转速5000 rpm离心5 min,收集沉淀物,使用溶有1 mgRuCl3去离子水重悬沉淀物后进行超声分散,持续搅拌12 h加入1 mg硼氢化钠反应10 min,再次收集沉淀物,将沉淀物洗涤2次、冷冻、干燥后即得Ru MOF。
2 mg对十二烷基苯磺酰,2 mg胆固醇和10 mg卵磷脂溶于氯仿/甲醇(v/v=2:1),然后在旋转蒸发器下干燥。之后,用Ru MOF溶液(1 mg/mL,4 mL)对干燥的脂质膜进行水合,接着探头超声5分钟,得到ER-Ru MOF。
对产物Ru MOF和ER-Ru MOF进行透射电子显微镜分析,结果如图1所示 ,由图1可知ER-Ru MOF具有均一粒径,尺寸在100 nm左右,说明本发明有潜力有效穿透血脑屏障。
对比例
本实施例的具有内质网靶向的抗氧化功能纳米酶的制备方法,步骤如下:
称量331.65 mg 2-甲基咪唑,1 mg十六烷基三甲基溴化铵溶于5 mL去离子水中,转速400 rpm搅拌混匀,再向混合溶液中依次滴入1 mL溶有1 mg牛血清白蛋白的去离子水溶液和1 mL溶有29.06 mgZn(NO3)2•6H2O的去离子水溶液,室温持续搅拌20 min,反应结束后,将反应液以转速5000 rpm离心5 min,收集沉淀物,使用溶有1 mg RuCl3去离子水重悬沉淀物后进行超声分散,持续搅拌12 h加入1 mg硼氢化钠反应10 min,再次收集沉淀物,将沉淀物洗涤2次、冷冻、干燥后即得Ru MOF。
2 mg胆固醇和10 mg卵磷脂溶于氯仿/甲醇(v/v=2:1),然后在旋转蒸发器下干燥。之后,用Ru MOF溶液(1 mg/mL,2 mL)对干燥的脂质膜进行水合,接着探头超声5分钟,得到ER-RuMOF。
对产物Ru MOF和ER-Ru MOF进行透射电子显微镜分析,结果如图1所示 ,由图1可知ER-Ru MOF具有均一粒径,尺寸在100 nm左右,说明本发明有潜力有效穿透血脑屏障。
实施效果例
1、实验动物和流程:
我们采用的C57雄性小鼠(7-8周,20-25g)购买并饲养于郑州大学动物实验中心,每个标准化笼位5只小鼠,接受标准的12小时光照/黑暗循环中饲养,可随意获取清洁的水源和食物。动物实验得到了郑州大学第二附属医院医学伦理委员会的批准(伦理批号:2021056)。为了最小化行为学测试的个体差异,动物在进行行为测试之前均接受1-2天的环境适应。在行为测试过程中,为保证客观真实,实验者对小鼠的治疗条件不知情。
2、CPSP模型建立
建立CPSP模型小鼠的方法参考先前的文献,在我们进行预实验的基础上做了一些修改,1%戊巴比妥钠溶液腹腔注射麻醉小鼠(50mg/kg),固定于脑立体定位装置。在立体定位装置引导下,用微量进样针抽取Ⅶ型胶原酶(Coll Ⅶ,C0773,Sigma-Aldrich Co., MO;0.025 U/0.25 ul,溶解于盐水),注入丘脑右侧VPM和VPL区域(前囟前0.82-后2.3mm,侧面至中线外侧1.30-1.95 mm,深度至颅骨表面3.01-4.25 mm)。假手术组注射0.25ul无菌生理盐水。给药后保持微量进样针10min,防止反流。随后将进样针缓慢旋出。微量注射后,用石蜡封闭针孔,缝合皮肤,碘伏擦拭手术部位皮肤。
3、 Bederson 评分
采用改良的Bederson评分法评价四组小鼠的神经功能情况。将小鼠尾巴捏住提起距台面10cm,观察其行为学。按以下评分系统:0分,无肢体异常表现;1分,前肢弯曲;2分,单侧肢体侧推阻力减小;3分,肢体单向盘旋;4分,肢体纵向旋转;5分,单侧肢体瘫痪。
4、行为学
参照先前的研究方法测量机械刺激下的小鼠缩足频率。小鼠单置于高网眼筛网上的有机玻璃室中,适应环境1小时。将两种校准的von frey丝(0.07g和0.4 g, StoeltingCo.)扎在小鼠后爪上约1秒,重复10次。双侧后爪测量间隔20分钟。小鼠迅速缩回爪子是一种积极的反应。对每10次刺激的缩足频率以百分比计算:[(缩足次数/10次试验)× 100% =反应频率]。
冷刺激:同上述小鼠机械痛静置方式,待小鼠安静适应环境时,0.3毫升注射器抽取适量丙酮(12.5uL)喷洒至小鼠后掌心,观察小鼠行为并按照下列标准进行评分:0分:无反应;1分:快速抬、甩脚;2分:长期、反复抬甩脚,舔脚背:3分:反复、交替舔脚心。连续测试三次,每次间隔5min,记录各处理组小鼠评分平均值。双侧后爪测量间隔20分钟。
5、血清生化指标监测
小鼠尾静脉注射PBS溶液或Nano溶液(溶于PBS,8mg/kg),三天后深度麻醉小鼠,针刺眼眶取血,置于1.5ml 离心管,4度冰箱静置过夜。次日于离心机3000转,离心10分钟,抽取上清。按生化试剂盒步骤(雷杜,深圳),检测血清相应指标。
6、脑出血量评估
异氟醚深度麻醉小鼠,经心脏灌注50-100 ml 4%多聚甲醛(0.1 M PBS, pH 7.4)。取出大脑,4°C固定24小时。将小鼠脑组织置于小鼠脑槽中,从前额部向枕部依次切片,厚度1mm。用imagine-pro 5.0软件分析小鼠脑血肿体积(立方毫米)。血肿体积按照文献报道的公式V=t x(A1+A2+…+An)计算,V表示体积,t表示切片厚度,A表示血肿面积。
7、尼氏染色
小鼠灌注取脑组织后,脱水,石蜡固定包埋,切片,厚度5um。使用二甲苯脱蜡,并使用不同浓度的无水乙醇洗涤,尼氏染色液(碧云天生物技术公司,C0117)染色30分钟,在进行脱水和透明处理后,使用中性树胶封片,病理切片扫描机器进行拍照。用Image J分析病变区域尼氏小体的数量。
8、Western blotting
小鼠深度麻醉后取脑,然后在冰上操作,剔除脑血肿,取出脑血肿周围组织进行蛋白提取。蛋白样品与上样缓冲液混合在在99°C加热5分钟,装入7.5%聚丙烯酰胺凝胶,电泳,随后转印至pvdf膜(密理博,美国)上,将其置于3%脱脂牛奶中,常温摇床封闭两小时。然后取出pvdf膜4度与一抗孵育过夜,包括EMMPRIN(santa cruze,美国sc-46700)、MMP9(武汉三鹰10375-2-AP)、MMP2(ABCAM,ab92536)、GAPDH(武汉三鹰10494-1-AP)。次日TBST清洗pvdf膜三次,每次10分钟,二抗共同孵育2小时,包括山羊抗兔二抗(武汉三鹰,SA00001-2)、山羊抗小鼠二抗(ABCAM,ab6789),随后TBST清洗3次,每次10分钟。使用ECL发光液(碧云天,P0018AFT)和ChemiDoc XRS系统进行显影。使用Image软件对印迹强度进行密度测定。
9、QT-PCR
用异氟醚对小鼠实施安乐死后,取出血肿周围脑组织于1.5ml无菌无酶管中,放入液氮速冻,随后置于-80度冰箱保存。使用RNA柱式提取试剂盒进行RNA提取(艾科瑞)。反转录为cDNA。引物序列为EMMPRIN 上游5‘-GGTCGGAAAGAAATCAGAGCAT-3’,下游5‘-GCAGTGAGATGGTTTCCCGAG-3’,MMP9上游5‘- GCTGGCAGAGGCATACTTGTAC-3’下游5‘-GGTGTTCGAATGGCCTTTAGTG-3’,MMP2上游5‘-TGATAACCTGGATGCCGTCG-3’下游5‘-CCAGCCAGTCTGATTTGATGC-3’,GAPDH上游5‘- CCTCGTCCCGTAGACAAAATG-3’,下游5‘-TGAGGTCAATGAAGGGGTCGT-3’。随后进行QT-PCR,操作按照制造商的说明书进行。
10、 统计分析
使用GraphPad Prism 7软件进行统计分析,分析结果如图所示。采用单因素或双因素方差分析(ANOV A)对3个分类独立组的数据进行比较。两组间比较采用非配对t检验。在所有统计检验中P值小于0.05被认为是显著的。数据以平均值±SEM表示。
结论:
利用实施例1中制备的产物Ru MOF和ER-Ru MOF以及对比例合成的ER-RuMOF进行酶活性测定,具体检测方法如下:对照组吸光度ΔA1的测试条件:
酶标仪550 nm测1 min,调试黄嘌呤氧化酶浓度,使ΔA1稳定在0.0225左右;
实验组吸光度的ΔA2的测试条件:
酶标仪550 nm测1 min,梯度稀释样品,比如:1、1/5、1/5^2、1/5^3、1/5^4、1/5^5、1/5^6;紫外分光光度计550 nm测1min ΔA2。
结果如图2所示,由图2可知,Ru MOF和ER-Ru MOF具有良好超氧化物歧化酶(SOD)活性分析,内质网靶向脂质体包载并不会影响纳米酶的类酶活性,说明本发明能够有效清除自由基。
实施例的ER-Ru MOF内质网靶向性验证:
使用FITC对ER-Ru MOF进行染色,并使用商用ER-Tracker进行共定位,结果如图3所示,显示本发明中所制备的ER-Ru MOF能够较好的靶向到内质网,从而将精准调控内质网氧化应激。
CPSP小鼠出现疼痛超敏且损伤区域内MMP2/9和ROS表达增高:
如图4、图5所示,与单侧注射生理盐水组相比,单侧微注射Coll IV(图4A/B)导致了对0.07 g和0.4 g机械性痛觉超敏。这些疼痛过敏发生在病变后一天,对侧后肢的机械阈值和冷痛反应潜伏期(丙酮试验)显著增加,与假对照组相比持续至少28天(图4D),同侧肢体没有明显的疼痛相关变化(图4E),且损伤区域内ROS(图4F)及炎症因子TNF-a(图4G)、IL-6(图4H)以及疼痛关键蛋白MMP9/2(图5A-C)表达增高。
纳米酶给药可减轻CPSP且减少损伤区域内MMP2/9和ROS的表达:
纳米酶(3、5或8mg/kg)和载体(PBS)在微注射Ⅶ型胶原酶或生理盐水后30分钟通过尾静脉给药。在CollIV/生理盐水微注射前1天和CollIV/生理盐水微注射后第1天和第3天,分别在纳米/载体给药后30分钟进行行为测试。结果如图6-图8证实,不同剂量均可减轻CPSP组小鼠丘脑出血后遗留对侧肢体痛觉过敏现象且呈剂量依赖效应(图6),给予8 mg/kg纳米材料组CPSP小鼠疼痛超敏性显著减弱,且减少损伤区域内MMP2/9和ROS的表达。由图7可知纳米酶给药后,脑损伤区域内ROS(8-OHdG氧自由基标记物)(图7A)、炎症因子TNFa(图7B)和IL-6(图7C)的表达明显减少,脑出血量(图7D)改善。由图8可知,纳米酶给药后,疼痛行为学测试(图8A)显示纳米酶给药组小鼠疼痛过敏现象改善,QT-PCR (图8B)、WestBloting(图8C)均提示CPSP小鼠丘脑损伤区域脑组织疼痛关键蛋白MMP2/9表达降低,这也与既往文献报道的emmprin非特异性抑制剂米诺环素效果相一致。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (9)
1.一种具有内质网靶向的抗氧化功能纳米酶的制备方法,其特征在于,步骤如下:
(1)将2-甲基咪唑、十六烷基三甲基溴化铵溶于去离子水中,搅拌均匀后,再依次滴入牛血清白蛋白的去离子水溶液和Zn(NO3)2•6H2O的去离子水溶液,持续搅拌至发应结束,离心收集沉淀物;
(2)用溶有RuCl3的去离子水将沉淀物重悬,然后超声分散,再在搅拌条件下加入硼氢化钠反应,再次收集沉淀,经洗涤、冷冻、干燥得Ru MOF;
(3)将十二烷基苯磺酸、卵磷脂和胆固醇溶于氯仿-甲醇混合溶液中,经旋蒸干燥后得脂质膜,再用含有步骤(2)的Ru MOF的溶液进行水合、超声,得ER-Ru MOF,即抗氧化功能纳米酶。
2.根据权利要求1所述的具有内质网靶向的抗氧化功能纳米酶的制备方法,其特征在于:所述步骤(1)中2-甲基咪唑、十六烷基三甲基溴化铵、牛血清白蛋白和Zn(NO3)2•6H2O的质量比为100-400:1:1:10-30。
3. 根据权利要求1所述的具有内质网靶向的抗氧化功能纳米酶的制备方法,其特征在于:所述步骤(1)中牛血清白蛋白的去离子水溶液的浓度为1mg/mL,Zn(NO3)2•6H2O的去离子水溶液的浓度为29.06mg/mL。
4. 根据权利要求2或3所述的具有内质网靶向的抗氧化功能纳米酶的制备方法,其特征在于:所述步骤(2)中RuCl3、沉淀物和硼氢化钠的质量比为:1: 100 :1
根据权利要求4所述的具有内质网靶向的抗氧化功能纳米酶的制备方法,其特征在于:所述步骤(3)中十二烷基苯磺酸、卵磷脂和胆固醇的质量比为1:1:5,其中氯仿-甲醇混合溶液中氯仿和甲醇的体积比为2:1。
5.权利要求1-3、5任一项所述的方法制备的具有内质网靶向的抗氧化功能纳米酶。
6.根据权利要求6所述的抗氧化功能纳米酶,其特征在于:以对十二烷基苯磺酰作为内质网靶向功能基团,并以Ru纳米酶作为内部包载药物。
7.权利要求6或7所述的抗氧化功能纳米酶在制备抗氧化药物中的应用。
8.权利要求6或7所述的抗氧化功能纳米酶在制备抑制脑出血后损伤部位ROS和MMP2/9的激活的药物中的应用。
9.权利要求6或7所述的抗氧化功能纳米酶在制备缓解中枢性卒中后疼痛的药物中的应用。
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