CN112569253A - 一种用于急性肾损伤的纳米酶药物及其制备方法与应用 - Google Patents
一种用于急性肾损伤的纳米酶药物及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种用于急性肾损伤的纳米酶药物及其制备方法与应用，所述纳米酶药物包括：铈氧化物纳米颗粒，结合在所述铈氧化物纳米颗粒表面的表面配体。本发明纳米酶药物包括表面配体以及由所述表面配体保护的铈氧化物纳米颗粒(ceria NPs)。本发明的纳米酶药物具有超小的尺寸，能够有效的富集于小鼠肾脏，清除肾小管内大量的活性氧以缓解和治疗甘油诱导的急性肾损伤。这些纳米酶药物具有良好的治疗效果，同时具有优异的生物相容性和生物安全性。
Description
技术领域
本发明涉及生物医学材料技术领域，尤其涉及一种用于急性肾损伤的纳米酶药物及其制备方法与应用。
背景技术
急性肾损伤是人类重要的健康问题。由于其高发病率和死亡率,据估计全球每年有170万人死亡。目前,辅助治疗和肾移植是最常见的治疗方法。最近的研究表明,急性肾损伤的发病机理与细胞内过量的活性氧和活性氮物种相关。此前,一些小分子药物,例如,氨磷汀和乙酰半胱氨酸,已经被证明可以作为抗氧化剂,消除活性氧,以此来缓解急性肾损伤。然而，小分子药物具有较低的利用率，较大的毒副作用以及有限的疗效。这些都阻碍了他们的临床应用。但是，抗氧化剂的成功发展为急性肾损伤未来的治疗提供了充分的基础。
发明内容
发明人研究发现，相较于传统蛋白酶，纳米酶具有成本低、催化性质可调、可大规模制备等明显优势。同时，纳米酶，尤其是二氧化铈等材料具有广谱的活性氧的清除能力，同时具有良好的生物安全性。更重要的是，超小的纳米颗粒可以通过肾脏进行代谢，这就为急性肾损伤的治疗提供了可能。
基于此，本发明开发了利用纳米酶用于急性肾损伤的治疗。
具体地，本发明提供一种用于急性肾损伤的纳米酶药物及其制备方法与应用，旨在解决现有的小分子药物利用率低、副作用大，难以用于急性肾损伤治疗的技术问题。
本发明第一方面，提供一种用于急性肾损伤的纳米酶药物，其中，包括：铈氧化物纳米颗粒，结合在所述铈氧化物纳米颗粒表面的表面配体。这些表面配体能够有效的稳定铈氧化物纳米颗粒，控制铈氧化物纳米颗粒具有很小的尺寸。并且它们都具有良好的水溶性和生物安全性，不易与血清内蛋白发生作用，有利于纳米酶药物在血液中的循环。
可选地，所述铈氧化物纳米颗粒选自二氧化铈纳米颗粒、过氧化铈纳米颗粒中的一种或多种。
可选地，所述表面配体选自聚乙烯吡咯烷酮、壳聚糖、柠檬酸、聚乙二醇、聚氧乙烯聚氧丙烯醚嵌段共聚物等中的一种或多种，但不限于此。
可选地，所述表面配体为柠檬酸。
可选地，所述铈氧化物纳米颗粒和所述表面配体的质量比为1:(1-10)。
可选地，所述纳米酶药物为直径小于6nm的球形颗粒。
本发明第二方面，提供一种如上所述的纳米酶药物的制备方法，其中，包括步骤：将铈盐和表面配体混合于水中，搅拌溶解，得到混合溶液；将所述混合溶液加入氨水中，在搅拌下进行反应，分离洗涤，即得到所述纳米酶药物。
可选地，所述铈盐与氨水的摩尔比为1:(100-500)。
可选地，所述反应的时间为12-48小时，所述反应的温度为20-30摄氏度。
本发明第三方面，提供一种如上所述的纳米酶药物在制备治疗急性肾损伤制剂中的应用。
有益效果：本发明纳米酶药物包括表面配体以及由所述表面配体保护的铈氧化物纳米颗粒，这些表面配体能够有效的稳定铈氧化物纳米颗粒，控制铈氧化物纳米颗粒具有很小的尺寸，使得最终获得的纳米酶药物具有超小的尺寸。本发明纳米酶药物具有超小的尺寸，能够有效的富集于小鼠肾脏，清除肾小管内大量的活性氧以缓解和治疗甘油诱导的急性肾损伤。另外，这些纳米酶药物具有良好的治疗效果，同时具有优异的生物相容性和生物安全性。
附图说明
图1为本发明具体的实施例中纳米酶药物的合成路线图；
图2为本发明具体的实施例中纳米酶药物的TEM图；
图3为本发明具体的实施例中纳米酶药物的XRD图；
图4为本发明具体的实施例中纳米酶药物过氧化氢清除率图；
图5为本发明具体的实施例中纳米酶药物羟基自由基清除率图；
图6为本发明具体的实施例中纳米酶药物超氧阴离子清除率图；
图7为本发明具体的实施例中纳米酶药物自由基清除率图；
图8为本发明具体的实施例中纳米酶药物处理肾小管细胞存活率图；
图9为本发明具体的实施例中纳米酶药物清除过氧化氢刺激下肾小管细胞中活性氧图；
图10为本发明具体的实施例中纳米酶药物提高过氧化氢刺激下肾小管细胞存活率图；
图11为本发明具体的实施例中纳米酶药物在不同时间小鼠主要器官分布变化图；
图12为本发明具体的实施例中纳米酶药物不同治疗组小鼠血清中血尿素氮含量图；
图13为本发明具体的实施例中纳米酶药物不同治疗组小鼠血清中血肌酐含量图；
图14为本发明具体的实施例中注射纳米酶药物和磷酸缓冲液(对照)老鼠的体重随时间的变化图。
具体实施方式
本发明提供一种用于急性肾损伤的纳米酶药物及其制备方法与应用，为使本发明的目的、技术方案及效果更加清楚、明确，以下对本发明进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
本发明实施例提供一种用于急性肾损伤的纳米酶药物，其中，包括：铈氧化物纳米颗粒，结合在所述铈氧化物纳米颗粒表面的表面配体。
本发明实施例纳米酶药物包括表面配体以及由所述表面配体保护的铈氧化物纳米颗粒，这些表面配体能够有效的稳定铈氧化物纳米颗粒，控制铈氧化物纳米颗粒具有很小的尺寸，使得最终获得的纳米酶药物具有超小的尺寸。该纳米酶药物具有超小的尺寸，能够有效到达小鼠肾脏，通过清除肾小管内大量的活性氧以缓解和治疗急性肾损伤。
在一种实施方式中，所述铈氧化物纳米颗粒选自二氧化铈纳米颗粒、过氧纳米颗粒化铈等中的一种或多种，但不限于此。
在一种实施方式中，所述表面配体选自聚乙烯吡咯烷酮、壳聚糖、柠檬酸、聚乙二醇、聚氧乙烯聚氧丙烯醚嵌段共聚物(F127等)等中的一种或多种，但不限于此。这些表面配体能够有效的稳定铈氧化物纳米颗粒，控制铈氧化物纳米颗粒具有很小的尺寸。并且它们都具有良好的水溶性和生物安全性，不易与血清内蛋白发生作用，有利于铈氧化物纳米颗粒在血液中的循环。
在一种实施方式中，所述铈氧化物纳米颗粒和所述表面配体的质量比为1:(1-10)，如1：1。该比例范围内得到的纳米酶药物具有良好的分散性和稳定性，并且具有很小的尺寸。
在一种实施方式中，所述纳米酶药物为直径小于6nm的球形颗粒。超小的纳米颗粒有利于到达小鼠肾脏，且超小的纳米颗粒有利于通过肾脏进行代谢。
本发明实施例提供一种如上所述纳米酶药物的制备方法，其中，包括步骤：将铈盐和表面配体混合于水中，搅拌溶解，得到混合溶液；将所述混合溶液加入氨水中，在搅拌下进行反应，分离洗涤，即得到所述纳米酶药物。
在一种实施方式中，所述铈盐与氨水的摩尔比为1:(100-500)。
在一种实施方式中，所述表面配体为柠檬酸。以摩尔比计，铈盐：柠檬酸：氨水＝1：1：200。柠檬酸可以起到稳定合成的氧化铈纳米颗粒，使它得到超小的尺寸，而过量的氨水可以与铈盐反应生成氢氧化物，随后发生水解得到二氧化铈纳米颗粒。
在一种实施方式中，所述反应的时间为12-48小时(如12小时)。
在一种实施方式中，所述反应的温度为20-30摄氏度。
在一种实施方式中，所述铈盐为硝酸铈等，但不限于此。
一种本发明实施例所述的纳米酶药物在制备治疗急性肾损伤制剂中的应用。
下面通过具体的实施例对本发明的技术方案作进一步地说明。
实施例1：合成纳米酶药物
纳米酶药物合成：如图1所示，将0.5克柠檬酸和0.5克硝酸铈五水合物加入到30毫升水中，然后搅拌将其溶解。25℃条件下搅拌加入100毫升氨水(3摩尔/升)，25℃下反应12小时。随后12000rpm离心并用水洗涤数次，得到的溶液通过冻干得到最终产品。
图1为合成纳米酶药物的路线图，其中Ce(NO3)3·5H2O代表硝酸铈五水合物。所述纳米酶药物中的柠檬酸表面配体能够很好地稳定二氧化铈纳米颗粒。
图2是合成的纳米酶药物的TEM图；图3是合成的纳米酶药物的XRD图；图2和图3表明纳米酶药物具有超小的尺寸。
实施例2：纳米酶药物清除各种活性氧的能力
不同浓度纳米酶药物(0-400μg/mL)清除过氧化氢的效率是通过过氧化氢酶检测试剂盒(碧云天公司)测定的。测试是按照制造商提供的方案进行的。
如图4所示，纳米酶药物能够有效的清除过氧化氢，并且具有浓度依赖的特性。
不同浓度纳米酶药物(0-200μg/mL)清除羟基自由基的效率是通过羟基自由基抗氧化能力(HORAC)试剂盒(Cell Biolabs,Inc.,USA)测定的。测试是按照制造商提供的方案进行的。
如图5所示，纳米酶药物能够有效的清除羟基自由基，并且具有浓度依赖的特性。
不同浓度纳米酶药物(0-200μg/mL)清除超氧阴离子的效率是通过SOD检测试剂盒(Sigma-Aldrich,USA)测定的。测试是按照制造商提供的方案进行的。
如图6所示，纳米酶药物能够有效的清除超氧阴离子，并且具有浓度依赖的特性。
纳米酶药物清除ABTS(2,2'-联氮双(3-乙基苯并噻唑啉-6-磺酸)二铵盐)自由基的测试
用ABTS自由基阳离子脱色法测定了纳米酶药物的自由基清除能力。ABTS(7mM)溶于水，加入2.45mM过硫酸钾反应12小时，可产生ABTS自由基阳离子(·ABTS+)。然后在734nm处测定纯·ABTS+溶液(AB)和不同浓度(0-50μg/mL)纳米酶药物与·ABTS+混合溶液的吸光度值。ABTS清除效率的计算公式为[(AB-AP)/AB]*100。所有的测量都是一式三次。
如图7所示，纳米酶药物能够有效的清除自由基，并且具有浓度依赖的特性。
实施例3：纳米酶药物细胞毒性和通过清除各种活性氧保护肾细胞采用标准的MTT法，评价纳米酶药物对293T肾胚胎细胞存活率的影响。
293T细胞以每孔1×104密度接种到96孔板中，并置于37度、5％CO2条件下培育12h。接着，吸出96孔板中的旧培养基，分别加入含有不同浓度纳米酶药物的培养基溶液。继续培养44h后，吸出96孔板中的旧培养基，在每个孔中加入100μLMTT的培养基溶液(0.8mg/mL，继续培养4h。吸出96孔板中的残余培养基，在每个孔中加入150μL DMSO溶液，轻轻摇晃后，在Synergy H1型酶标仪上检测每孔的OD值(检测波长为570nm)，用如下公式计算细胞存活率。细胞存活率(cell viability)(％)＝(样品的OD570值/空白OD570值)×100％。
如图8所示，合成的纳米酶药物对293T肾胚胎细胞的细胞存活率，在浓度达到最大使用浓度200μg/mL时，细胞依然保持90％以上的存活率。表明本实施例的纳米酶药物具有较低的细胞毒性。
以纳米酶药物为例，293T细胞提前4小时处理纳米酶药物(200μg/mL)后，加入含2mM过氧化氢的培养基。再使用活性氧(图9)探针染色，洗涤之后使用激光共聚焦显微镜进行成像。如图9所示，与过氧化氢刺激后的细胞相比，预处理纳米酶药物细胞中的荧光明显减弱，接近于对照组细胞。这说明纳米酶药物能够有效清除细胞中活性氧，进而保护细胞。同时，如图10所示，提前预处理不同浓度的二氧化铈纳米颗粒4小时，再接受0.5mM过氧化氢刺激后的细胞存活率会大幅提高，说明纳米颗粒能够保护肾脏细胞免受活性氧损伤。
实施例4：纳米酶药物肾脏蓄积和治疗所有的实验操作均按照临床中心动物保健和使用委员会通过的动物使用和保健制度。雌性无胸腺小白鼠(六周,20-25g)，在小白鼠后腿肌肉注射8mL/kg 50％的甘油溶液建立老鼠急性肾衰竭模型。2小时后，注射纳米酶药物。
在不同的时间点取出小鼠主要器官，使用电感耦合等离子体质谱仪对小鼠器官中铈元素进行检测。如图11所示，二氧化铈主要分布在小鼠的肾脏和肝脏，其中在4小时时含量最高，说明纳米酶药物能够快速的到达小鼠肾脏。
实施例5：纳米酶药物治疗急性肾损伤和生物安全性评价
所有的实验操作均按照临床中心动物保健和使用委员会通过的动物使用和保健制度。雌性无胸腺小白鼠(六周,20-25g)，在小白鼠后腿肌肉注射8mL/kg 50％的甘油溶液建立老鼠急性肾衰竭模型。2小时后，注射小分子药物氨磷汀或者纳米酶药物。
小鼠随机分为5组：(1)健康鼠注射磷酸缓冲液；(2)健康鼠注射纳米酶药物；(3)甘油诱导的急性肾衰竭鼠注射磷酸缓冲液；(4)甘油诱导的急性肾衰竭鼠注射纳米酶药物；(5)甘油诱导的急性肾衰竭鼠注射等量的氨磷汀。健康鼠和甘油诱导的急性肾衰竭鼠24小时后安乐死小鼠，取小鼠血液离心获得血清，测量肌酐和血尿素氮含量。注射使用磷酸缓冲液为150μL，纳米酶药物为2mg，氨磷汀为2mg。
如图12-13所示，健康鼠注射纳米酶药物的肌酐和血尿素氮含量没有明显变化。而注射纳米酶药物的急性肾衰竭小鼠肌酐和血尿素氮含量明显低于只注射磷酸缓冲液的小鼠，并接近健康鼠的水平。另一方面，同等剂量的氨磷汀并不能有效的降低两个指标。这说明四种纳米酶药物能够有效的缓解和治疗急性肾衰竭，并具有比小分子药物氨磷汀更好的治疗效果。
此外，使用健康鼠注射磷酸缓冲液和纳米酶药物，记录小鼠一个月内的体重变化情况。如图14所示，与对照组相比，注射纳米酶药物的小鼠体重没有明显差异。
综上所述，本发明的纳米酶药物通过简单的合成方法，可制备出大量超小纳米颗粒，能够有效的清除各类活性氧物种，具有广谱的活性氧清除能力。并且对293T肾细胞的毒副作用较低，与细胞共培养48小时后细胞存活率均达到90％以上；同时它们可以通过清除细胞内多余的活性氧来保护细胞免受过氧化氢刺激。借助纳米酶药物自身特殊的性质，可以通过检测主要器官中铈元素来监测纳米酶药物在小鼠肾脏的有效累积。此外，纳米酶药物在甘油诱导的急性肾衰竭小鼠显示出良好的治疗效果。更重要的是，纳米酶药物具有良好的生物相容性和生物安全性。
应当理解的是，本发明的应用不限于上述的举例，对本领域普通技术人员来说，可以根据上述说明加以改进或变换，所有这些改进和变换都应属于本发明所附权利要求的保护范围。
Claims (10)
1.一种用于急性肾损伤的纳米酶药物，其特征在于，包括：铈氧化物纳米颗粒，结合在所述铈氧化物纳米颗粒表面的表面配体。
2.根据权利要求1所述的纳米酶药物，其特征在于，所述铈氧化物纳米颗粒选自二氧化铈纳米颗粒、过氧化铈纳米颗粒中的一种或多种。
3.根据权利要求1所述的纳米酶药物，其特征在于，所述表面配体选自聚乙烯吡咯烷酮、壳聚糖、柠檬酸、聚乙二醇、聚氧乙烯聚氧丙烯醚嵌段共聚物中的一种或多种。
4.根据权利要求3所述的纳米酶药物，其特征在于，所述表面配体为柠檬酸。
5.根据权利要求1所述的纳米酶药物，其特征在于，所述铈氧化物纳米颗粒和所述表面配体的质量比为1:(1-10)。
6.根据权利要求1所述的纳米酶药物，其特征在于，所述纳米酶药物为直径小于6nm的球形颗粒。
7.一种如权利要求1-6任一所述的纳米酶药物的制备方法，其特征在于，包括步骤：将铈盐和表面配体混合于水中，搅拌溶解，得到混合溶液；将所述混合溶液加入氨水中，在搅拌下进行反应，分离洗涤，得到所述纳米酶药物。
8.根据权利要求7所述的纳米酶药物的制备方法，其特征在于，所述铈盐与氨水的摩尔比为1:(100-500)。
9.根据权利要求7所述的纳米酶药物的制备方法，其特征在于，所述反应的时间为12-48小时，所述反应的温度为20-30摄氏度。
10.一种如权利要求1-6任一所述的纳米酶药物在制备治疗急性肾损伤制剂中的应用。
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| CN114681482A (zh) * | 2021-08-30 | 2022-07-01 | 深圳大学 | 一种纳米酶及其制备方法与应用 |
| CN114681482B (zh) * | 2021-08-30 | 2023-05-05 | 深圳大学 | 一种纳米酶及其制备方法与应用 |
| CN115317516A (zh) * | 2022-08-29 | 2022-11-11 | 中南大学 | 一种超小抗氧化纳米点及其在急性肾损伤中的应用 |
| CN115317516B (zh) * | 2022-08-29 | 2023-07-25 | 中南大学 | 一种超小抗氧化纳米点及其在急性肾损伤中的应用 |
| CN117695247A (zh) * | 2023-12-13 | 2024-03-15 | 南方医科大学珠江医院 | 一种掺锶二氧化铈纳米酶的制备方法及其应用 |
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