CN115368579B - 一种以锰卟啉为金属有机骨架纳米酶的制备方法和应用 - Google Patents
一种以锰卟啉为金属有机骨架纳米酶的制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种以锰卟啉为金属有机骨架纳米酶的制备方法，仅需要用三水合硝酸铜、中‑四（4‑羧基苯基）卟吩和氯化锰，通过两步即可合成一种具有类SOD和CAT活性的纳米材料，同时能够在体内外实验中成功缓解急性心肌梗死及缺血‑再灌注损伤。本发明的方法合成步骤简单、成本低、且具有较好生物相容性，具有较好的应用前景。
Description
技术领域
本发明涉及金属-纳米材料技术领域，具体涉及一种以锰卟啉为金属有机骨架纳米酶的制备方法和应用。
背景技术
急性心肌梗死是导致全球每年数百万人死亡的一大原因，也是慢性心力衰竭的主要原因。尽管介入治疗和非手术治疗取得了重大进展，但相当大比例的患者会出现术后心功能受损的情况，因此心梗及随后的心力衰竭仍然有待探寻更好的治疗方法以改善预后。值得注意的是，无论是在急性心肌梗死的缺血再灌注(IR)过程还是在心梗后心室重塑过程中，氧化应激都是心肌损伤的一个重要因素【1】。这类氧化应激损伤包括过量活性氧(ROS)的产生，细胞内pH的快速回升、细胞内钙离子超载等，而过量ROS的产生为核心因素。ROS主要包括超氧阴离子(O2·-)、过氧化氢(H2O2)和羟基自由基(OH·)。正常人体内产生的活性氧自由基，如超氧阴离子，可以被线粒体中的超氧化物歧化酶(SOD)催化为过氧化氢(H2O2)，过氧化氢可进一步被胞质中的过氧化氢酶(CAT)或谷胱甘肽过氧化物酶(GPx)等抗氧化酶分解为水和氧气，从而完成体内活性氧的清除。但发生缺血再灌注损伤后，ROS水平超过了自身天然酶的清除能力，发生氧化还原失衡。过量ROS可导致不可逆的线粒体破坏、DNA链断裂及蛋白和膜的氧化，从而诱导细胞死亡。同时，过量ROS可引起心脏炎症，炎症又反过来诱导ROS的进一步产生加重心肌损伤。因此，抗氧化治疗和减轻心脏炎症被认为是改善心肌缺血再灌注损伤的有效方法。传统的抗氧化治疗，如抗氧化剂、炎症调节剂、心脏保护因子等，存在药物生物利用度低，全身副作用大等限制，在临床上并不能取得很好的效果。
纳米酶(nanozymes)是受天然酶和传统人工酶启发而开发的一类具有类似酶催化活性的纳米材料，它通过模拟天然酶的催化位点或具备多价元素而能够进行氧化还原反应，为酶工程领域的一大突破【2】。它能够具备类似超氧化物歧化酶、过氧化氢酶等氧化还原酶的特性，从外界干预，有效地清除活性氧自由基，减轻氧化应激状态，达到抗氧化、抗炎治疗的作用。与天然酶相比，纳米酶不仅具有成本低、稳定性高、易于大规模生产等优势，而且纳米材料独特的物理化学性质也赋予了纳米酶多种功能，为纳米酶的设计开发和未来应用提供了更多可能。目前，纳米酶治疗在生物医学领域中，尤其是在肿瘤、自身免疫病、急慢性炎症性疾病等中得到了广泛研究。作者Wan S【3】在《ACS-nano》上发表了一种基于三价锰离子与卟啉一步合成的封闭金属有机骨架纳米体系Mn(III)-TCPP MOF，它能响应性地与谷胱甘肽反应而被分解为游离的Mn(II)和TCPP，在光动力学的作用下游离TCPP能够可控得调节ROS产生，从而杀伤肿瘤细胞。Zhang L【4】等报道了一种Cu-TCPP MOF纳米点(CTMDs)的材料，该材料由二维片层状MOF结构的Cu-TCPP经超声打碎，从而形成只有十几纳米的量子点。它不仅具有类SOD及GPx活性，能够满足天然SOD的模拟要求，并且纳米级的尺寸赋予其充分的扩散能力，更容易接近体内细胞代谢产物，从而发挥作用，在结构和功能上都模拟了超氧化物歧化酶的活性位点，具有仿生大小，在体内外都显示出极高的SOD活性及GPx活性，在内毒素血症的动物模型中证实了CTMDs能够清除活性氧自由基，有效减轻氧化应激，降低全身炎症反应。但是，Cu-TCPP纳米点因只掺入Cu2+，合成后活性位点单一，因此仅具有SOD活性，而不具备CAT活性，并没有达到O2·-→H2O2→H2O+O2的级联ROS清除反应的功效。
此外，Liu Y【5】等人于2020年发表在《Science Advances》的纳米酶Pt@PCN222-Mn是一个兼具SOD和CAT活性的纳米材料。通过三步，由TCPP-Mn到PCN222-Mn，最后包裹铂纳米微粒，合成了一个以锆基金属有机骨架，封装有金属铂的均匀梭形透镜状材料。其中，连接在TCPP卟啉中心位点的Mn2+具有类SOD活性，Pt纳米微粒部分则发挥了类CAT活性，其高效的级联催化反应和较好的生物相容性，成功地在体内外缓解了炎症性肠病的疾病模型。但是，Pt@PCN222-Mn材料合成步骤比较繁琐，并且需要二氯氧化锆和铂纳米微粒等一些成本相对较高的纳米原材料；此外，TCPP存在一定的细胞毒副作用(在其细胞毒性实验中，超过100ug/ml的材料毒性明显增加)。该文献中，仅研究了炎症性肠病的疾病模型，并没有在急性心肌梗死及心肌缺血-再灌注损伤中做出相关验证。
但是，金属有机骨架相关纳米酶目前在急性心肌梗死及缺血-再灌注损伤中的研究还很少。针对这种氧化应激的机制，纳米酶对于急性心肌梗死及缺血再灌注损伤的治疗，具有非常好的前景。
【1】K Raedschelders,D.M.Ansley,D.Chen.The cellular and molecularorigin of reactive oxygen species generation during myocardial ischemia andreperfusion.Pharmacology&Therapeutics.
【2】Wu J,Wang X,Wang Q,et al.Nanomaterials with enzyme-likecharacteristics(nanozymes):next-generation artificial enzymes(II).Chem SocRev.
【3】Wan S,Cheng Q,Zeng X,et al.A Mn(III)-Sealed Metal-OrganicFramework Nanosystem for Redox-Unlocked Tumor Theranostics.ACS nano.2019.
【4】Zhang L,Zhang Y,Wang Z,et al.Constructing metal–organic frameworknanodots as bio-inspired artificial superoxide dismutase for alleviatingendotoxemia.Materials Horizons.
【5】Liu Y,Cheng Y,Zhang H,et al.Integrated cascade nanozyme catalyzesin vivo ROS scavenging for anti-inflammatory therapy.Science Advances.2020。
发明内容
发明目的：为解决现有技术中存在的技术问题，本发明提供了一种合成步骤简单、成本低、且具有较好生物相容性、同时还兼具较高SOD和CAT活性的级联纳米酶系统，能够在体内外实验中成功缓解急性心肌梗死及缺血-再灌注损伤。
为实现上述目的，本发明提出一种以锰卟啉为金属有机骨架纳米酶的制备方法，其特征在于，包括如下步骤：
(1)Cu-TCPP纳米片的制备：将二价铜盐溶于水，中-四(4-羧基苯基)卟吩溶于N-N-二甲基甲酰胺(DMF)或二甲基亚砜(DMSO)有机溶剂，再将两者混合后可加入苯甲酸，苯甲酸有助于卟吩的羧基与铜离子配位，充分超声至颗粒溶解，将溶液置于90℃油浴中反应3-5小时，反应结束后，将其高速离心，用有机溶剂洗涤三遍，浓缩得到纳米片溶液；
(2)Cu-TCPP-Mn纳米量子点的制备：将合成好的Cu-TCPP纳米片与MnCl2在反应器中，90℃油浴搅拌过夜，后用水洗涤2-3次，高速离心，收集浓缩溶液，再经超声2-6小时，100-300W打碎，5000-9000转，离心收集上清液，得到小于200nm尺寸的纳米量子点。
优选地，步骤(1)中，二价铜盐、中-四(4-羧基苯基)卟吩和苯甲酸的用量摩尔比范围为8-10：1-2：1-2，优选地，范围为10：1：1.5。
优选地，步骤(1)中，浓缩为0.8-1mmol/L的Cu-TCPP纳米片溶液。
优选地，步骤(1)中，二价铜盐为Cu(NO3)2·3H2O、硫酸铜或氯化铜中的任意一种。更优选地，二价铜盐为Cu(NO3)2·3H2O。
步骤(2)中，MnCl2的用量与步骤(1)中的中-四(4-羧基苯基)卟吩的摩尔比为1：1。
其中，步骤(1)和步骤(2)中高速离心的条件为不小于11000转，离心时间为10-20min。
本发明进一步提出了上述制备方法得到的纳米酶在制备用于治疗或缓解急性心肌梗死及缺血再灌注损伤的药物中的应用。
有益效果：与现有技术相比，本发明具有如下优点：
(1)Cu-TCPP-Mn纳米量子点与现有技术相比，具有原材料成本低廉，合成步骤简便的优势，仅需要3种材料：三水合硝酸铜，中-四(4-羧基苯基)卟吩和氯化锰，通过两步即可合成一种具有类SOD和CAT活性的纳米材料；
(2)在体外实验中，该材料在较低浓度(0.1ug/ml)与黄嘌呤和黄嘌呤氧化酶共孵育时，通过二氢乙锭和NBT等活性氧自由基探针均可以检测到显著的活性氧减少，证实了其具备高SOD催化活性；同时，当材料与H2O2共孵育时，通过Ru(dpp)3+Cl检测到O2的生成，从而有力说明了材料具备较高的类CAT活性；
(3)在细胞水平实验中，MC38细胞在受t-BOOH刺激24h后，发生氧化应激产生过量活性氧。接着，该材料与氧化应激的MC38细胞共孵育24h后，通过DCFH-DA探针检测到代表细胞内活性氧的绿色荧光明显减少，证实了材料能够在细胞水平有效地清除细胞内活性氧；
(4)在急性心肌梗死的小鼠动物模型中，通过静脉给药的方式注射材料(0.5mg/kg剂量)，与对照组(仅注射PBS，仅注射Cu-TCPP组)相比，Cu-TCPP-Mn治疗组在超声心动图上心功能(左室射血分散、短轴收缩率)有显著的统计学差异；观察终点取小鼠心脏做Masson切片染色，治疗组梗死部位纤维化明显减少，均证实了材料治疗后小鼠心功能有明显的恢复。
附图说明
图1为Cu-TCPP纳米片的透射电镜显微镜图；
图2为Cu-TCPP-Mn纳米量子点的透射电镜显微镜图；
图3为Cu-TCPP纳米片的扫描电镜显微镜图；
图4为Cu-TCPP-Mn纳米量子点的扫描电镜显微镜图；
图5为Cu-TCPP和Cu-TCPP-Mn的XPS图；
图6为Cu-TCPP和Cu-TCPP-Mn的FT-IR图；
图7为Cu-TCPP-Mn的动态光散射图；
图8为Cu-TCPP和Cu-TCPP-Mn的UV-Vis图；
图9和图10为Cu-TCPP-Mn的体外实验结果；
图11为Cu-TCPP和Cu-TCPP-Mn与内皮细胞共孵育的细胞毒性实验；
图12和图13为Cu-TCPP-Mn在小鼠急性心肌梗死模型的超声心动图治疗结果；
图14为Cu-TCPP-Mn在小鼠急性心肌梗死模型心脏Masson切片染色的治疗结果；
图15为Cu-TCPP和Cu-TCPP-Mn在小鼠体内治疗后各脏器组织形态的HE染色图。
具体实施方式
下面结合具体实施例对本发明做进一步详细说明，实施例将有助于理解本发明，但是本发明的保护范围不限于下述的实施例。
其中，下面实施例子中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或按照产品说明书进行。下述实施例子中涉及的材料、试剂等，如无特殊说明，均可从商业途径购买得到。
如无特殊说明，以下实施例中的定量实验，均设置三次重复实验，取平均值。
实施例中使用的材料：中-四(4-羧基苯基)卟吩(MW 774.2):罗恩试剂，CAS号14609-54-2。三水合硝酸铜(Cu(NO3)2·3H2O)：Sigma-Aldrich公司，CAS号10031-43-3。氯化锰(MnCl2):Sigma-Aldrich公司，CAS号7773-01-5。苯甲酸(Benzoic acid):Sigma-Aldrich公司，CAS号65-85-0。N-N-二甲基甲酰胺(DMF)：罗恩试剂，CAS号172361-60-3。
RAW264.7细胞：小鼠单核巨噬细胞白血病细胞。MC38细胞：小鼠结肠癌细胞。BEND.3细胞：小鼠脑微血管内皮细胞。
黄嘌呤(Xanthine):Sigma-Aldrich公司，CAS号69-89-6。黄嘌呤氧化酶(XanthineOxidase):Sigma-Aldrich公司，CAS号9002-17-9。二氢乙锭(Hydroethidine):Aladdin，CAS号104821-25-2。DCFH-DA(全称2’,7’-Dichlorofluorescein Diacetate):Aladdin,CAS号2044-85-1。NBT(全称nitrotetrazolium blue chloride)：Sigma-Aldrich公司，CAS号298-83-9。叔丁基过氧化氢(t-BOOH)：Sigma-Aldrich公司，CAS号75-91-2。
实施例1 Cu-TCPP-Mn纳米酶的制备与表征。
1.1.Cu-TCPP-Mn纳米量子点的制备：
(1)将Cu(NO3)2·3H2O(12.1mg)、去离子水(5ml)和中-四(4-羧基苯基)卟吩(7.9mg)加于50ml的N-N-二甲基甲酰胺(DMF)有机溶剂中混合，然后加入0.9mg的苯甲酸，充分超声。将溶液置于90℃油浴中反应4小时。反应结束后，将其用DMF溶剂，13000转，10分钟洗涤三遍，浓缩为1mmol/L的Cu-TCPP纳米片溶液。
(2)完成步骤(1)后，将Cu-TCPP纳米片与MnCl2以1:1摩尔比混合于500ml圆底烧瓶中，90℃油浴搅拌过夜，后用水洗涤3次，13000rmp,10min，收集浓缩溶液。再经超声3小时打碎，9000转收集上清液，得到小于200nm尺寸的纳米量子点。
1.2Cu-TCPP-Mn纳米量子点的表征；
利用透射电镜显微镜(TEM)观察步骤1制备的Cu-TCPP和Cu-TCPP-Mn纳米酶的形态和大小，结果如图1和2所示。利用扫描电镜显微镜(SEM)观察Cu-TCPP和Cu-TCPP-Mn的表面形态，结果如图3和4所示。利用动态光散射粒径仪(DLS)分析Cu-TCPP-Mn的水合粒径和分散性，结果如图7所示。可见Cu-TCPP纳米片为大于500nm的片层状结构，Cu-TCPP-Mn为尺寸均一的纳米量子点，分散性良好，粒径约80nm。
利用X射线光电子谱(XPS)、傅里叶红外光谱仪(FT-IR)、UV-Vis对Cu-TCPP和Cu-TCPP-Mn进行表征，结果如5、6、8所示。XPS检测产物的组成和化学键态，表明Cu-TCPP主要由Cu、C、O、N元素组成，Cu-TCPP-Mn主要由Mn、Cu、C、O、N元素组成，均无明显杂质，证实合成成功。FT-IR检测产物的官能团或化学键的变化，1474～1000cm-1区域特征峰的变化证实了材料的合成成功。UV-Vis中500～700nm波段中546nm和646nm处的峰证实了TCPP与Cu2+、Mn2+配位后的形成的Q带。
实施例2、Cu-TCPP-Mn纳米量子点的类酶活性体外实验验证。
(1)Cu-TCPP-Mn纳米量子点的类SOD活性体外实验
利用二氢乙锭作为·O2-探针评估Cu-TCPP和Cu-TCPP-Mn的体外SOD活性。将黄嘌呤(0.6mM)，黄嘌呤氧化酶(0.05U/ml)和Cu-TCPP，Cu-TCPP-Mn(0.1，0.5,1ug/ml)混合于tris-HCL缓冲液(0.1M，pH6.8)在37℃孵育20分钟。然后加入二氢乙锭(0.5mg/ml)再孵育15分钟。通过酶标仪记录吸收度，设置激发和发射波长分别为470nm和610nm。示例性的结果见图9。
(2)Cu-TCPP-Mn纳米量子点的类CAT活性体外实验
利用Ru(dpp)3Cl2评估Cu-TCPP和Cu-TCPP-Mn的体外CAT活性。将Cu-TCPP，Cu-TCPP-Mn(1ug/ml、2ug/ml)，Ru(dpp)3Cl2(1ug/ml)与H2O2(0.1M)于37℃共孵育30min，通过酶标仪记录吸收，设置激发和发射波长分别为463nm和620nm。示例性的结果见图10。
实施例3、Cu-TCPP-Mn纳米量子点的体内治疗效果实验。
(1)制备小鼠急性心肌梗死、缺血-再灌注模型
取正常雄性BALB/c小鼠(8-10周，25g左右)，4％水合氯醛10mg/kg腹腔注射，深度麻醉，确认麻醉状态后，头部与四肢固定在手术台上，备皮，消毒局部皮肤，经口气管插管，连接呼吸机(呼吸频率110次/分，呼：吸为1:1，潮气量10ml/min)。剪开左侧三至四肋间皮肤，钝性分离肌肉、打开心包膜，根据左心耳与左心室连接位置暴露左冠状动脉前降支，用6-0无菌丝线快速结扎，打实结，看到心脏底部变白即为急性心肌梗死造成，用荷包缝合法快速关闭胸腔，挤压胸廓多余气体防止气胸，缝合严实完成后拔气管等待小鼠苏醒，即为急性心肌梗死模型造模完成。
缺血-再灌注模型步骤与急性心肌梗死模型类似，不同的是：在开胸结扎过程中，打活结，一端拉出留在肋间皮肤外，暂时关闭胸腔，继续通气30min，后解开丝线，用荷包缝合法快速关闭胸腔，挤压胸廓多余气体防止气胸，缝合严实完成后拔气管等待小鼠苏醒，即为缺血再灌注造模完成。
(2)给药及效果评价
将小鼠模型分成分成4组，每组至少3只。
空白对照组(假手术组)：对小鼠进行开胸手术，而不进行结扎。不进行治疗。
阴性对照组：(生理盐水治疗组)：对模型小鼠进行PBS静脉注射安慰治疗，术后第二天起连续三天给药。
试验1组(Cu-TCPP治疗组)：对模型小鼠进行Cu-TCPP以0.5mg/kg剂量静脉注射治疗，术后第二天起连续三天给药。
试验2组(Cu-TCPP-Mn治疗组)：对模型小鼠进行Cu-TCPP-Mn以0.5mg/kg剂量静脉注射治疗，术后第二天起连续三天给药。
术后4h进行超声心动图评估，作为基线。
此后每隔一周进行超声心动图扫描，持续4周，采集每只老鼠的左室射血分数、左室短轴收缩率、左室收缩期末容积、左室舒张期末容积等数据。第四周观察终点处死小鼠，取心脏做HE染色和Masson切片染色观察纤维化程度。
治疗效果结果如图12、13、14图所示，Cu-TCPP-Mn纳米酶缓解了小鼠的心梗炎症状态，对心功能恢复有明显促进作用，具有较好的治疗效果。并且药物对小鼠的各个脏器没有明显毒副作用，结果如图15所示。
本发明提供了一种以锰卟啉为金属有机骨架纳米酶的制备方法和应用的思路，具体实现该技术方案的方法和途径很多，以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。本实施例中未明确的各组成部分均可用现有技术加以实现。
Claims (8)
1.一种以锰卟啉为金属有机骨架纳米酶的制备方法，其特征在于，包括如下步骤：
（1）Cu-TCPP纳米片的制备：将二价铜盐溶于水，中-四（4-羧基苯基）卟吩溶于N-N-二甲基甲酰胺或二甲基亚砜有机溶剂，再将两者混合后加入苯甲酸，充分超声至颗粒溶解，将溶液置于80-90℃油浴中反应3-5小时，反应结束后，将其高速离心，用有机溶剂洗涤三遍，浓缩为约1mmol/L的Cu-TCPP纳米片溶液；
（2）Cu-TCPP-Mn纳米量子点的制备：将合成好的Cu-TCPP纳米片与MnCl2在反应器中，90℃油浴搅拌过夜，后用水洗涤2-3次，高速离心，收集浓缩溶液，再经超声2-6小时，100-300W打碎，5000-9000转离心收集上清液，得到小于200nm尺寸的纳米量子点。
2.根据权利要求1所述的制备方法，其特征在于，步骤（1）中，二价铜盐、中-四（4-羧基苯基）卟吩和苯甲酸的用量摩尔比为8-10：1-2：1-2。
3.根据权利要求1所述的制备方法，其特征在于，步骤（1）中，二价铜盐为Cu(NO3)2•3H2O、硫酸铜或氯化铜中的任意一种。
4.根据权利要求1所述的制备方法，其特征在于，步骤（1）中，浓缩为0.8-1mmol/L的Cu-TCPP纳米片溶液。
5.根据权利要求1所述的制备方法，其特征在于，步骤（2）中，MnCl2的用量与步骤（1）中的中-四（4-羧基苯基）卟吩的摩尔比为1：1。
6.根据权利要求1所述的制备方法，其特征在于，步骤（1）中高速离心的条件为不小于11000转，离心时间为10-20min。
7.根据权利要求1所述的制备方法，其特征在于，步骤（2）中高速离心的条件为不小于11000转，离心时间为10-20min。
8.权利要求1所述的制备方法得到的纳米酶在制备用于治疗或缓解急性心肌梗死及缺血再灌注损伤的药物中的应用。
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