CN114377126A - 纳米钯片负载妥珠单抗的纳米复合体、制备方法及其在制备治疗炎症相关性贫血药物的应用 - Google Patents
纳米钯片负载妥珠单抗的纳米复合体、制备方法及其在制备治疗炎症相关性贫血药物的应用 Download PDFInfo
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Abstract
本发明公开了一种纳米钯片负载妥珠单抗的纳米复合体、制备方法及其在制备治疗炎症相关性贫血药物的应用。本发明通过表面修饰PdPL的方式提高其对TCZ的装载能力，由此构建得到PdPL‑PEG@TCZ纳米复合体。PdPL‑PEG@TCZ纳米复合体利用PdPL对肝脏组织被动靶向的特性，将TCZ高效的运输并富集在肝脏组织中，阻断肝脏细胞IL‑6/IL‑6R/JAK2/STAT3/Hepcidin信号通路，从而有效改善小鼠模型的贫血程度，同时可以减少TCZ在其它组织中的分布而诱发的副作用。本发明的PdPL‑PEG@TCZ纳米复合体为治疗AI提供了一种新的手段。
Description
技术领域
本发明涉及生物技术领域，特别涉及一种纳米钯片负载妥珠单抗的纳米复合体、制备方法及其在制备治疗炎症相关性贫血药物的应用。
背景技术
炎症相关性贫血(Anemia of inflammation，AI)的发病率位居全球贫血发病率的第二位。AI在感染性疾病、自身免疫性疾病、慢性肾病、充血性心力衰竭和癌症患者群体中普遍存在，该类疾病患者通常伴随免疫系统的异常激活。AI可对患者多个组织器官的生理机能产生不利影响，特别是对于正在接受化疗和(或)放疗的患者，AI可能加速其肿瘤进程。同时，有研究表明在老年患者(>60岁)群体中，AI的存在可显著降低患者的生活质量和生存率。然而，目前用于AI治疗的方法都具有局限性，治疗效果不够理想。因此，寻找有效治疗AI的方法是一个亟需攻克的科学问题。
研究表明，AI的发生与肝脏细胞分泌铁调素(Hepcidin)水平失调密切相关，而白细胞介素-6(Interleukin-6，IL-6)是AI患者中诱导肝脏细胞合成分泌Hepcidin的主要驱动因子，IL-6可通过Janus激酶2/信号转导和转录激活因子3(Janus kinase 2/signaltransducer and activator of transcription 3，JAK2/STAT3)信号通路促进肝脏细胞产生Hepcidin来抑制生物体内铁转运蛋白活性，从而减弱十二指肠上皮细胞对食物中铁的吸收，抑制巨噬细胞、肝脏和脾脏等铁储库内铁的释放，继而导致血清中的有效生物铁浓度降低，影响血红蛋白及红细胞的生成，最终导致贫血的发生。妥珠单抗(Tocilizumab，TCZ)是一种IL-6受体(Interleukin-6Receptor，IL-6R)单克隆抗体，因其可有效阻断IL-6/IL-6R/JAK2/STAT3/Hepcidin级联信号转导通路而被应用于类风湿性关节炎、系统性红斑狼疮等免疫相关性疾病的临床治疗中。但是，TCZ经血液进入体内后会分布至全身组织器官，降低了靶器官肝脏组织中的有效药物浓度，同时也会诱发全身其它组织器官的副作用。因此，需要寻求一种有效的方法提高TCZ在肝脏组织中的靶向富集浓度，以此来提高其治疗AI的效果，降低药物的副作用。
发明内容
本发明为了解决上述技术问题，提供了一种纳米钯片负载妥珠单抗的纳米复合体、制备方法及其在制备治疗炎症相关性贫血药物的应用。
第一方面，本发明提供了一种纳米钯片负载妥珠单抗的纳米复合体的制备方法，是采用以下技术方案实现的。
一种纳米钯片负载妥珠单抗的纳米复合体的制备方法，包括以下步骤：
S1.将纳米钯片与巯基丙酸混合，孵育3-3.5小时，巯基丙酸的加入量为200-300μL/mg纳米钯片；离心并洗涤沉淀后，将沉淀再次分散，然后加入1-(3-二甲基氨基丙基)-3-乙基碳二亚胺和N-羟基丁二酰亚胺，孵育1-1.5小时，1-(3-二甲基氨基丙基)-3-乙基碳二亚胺、N-羟基丁二酰亚胺和巯基丙酸的体积比为(2-3):(2-3):8；再加入NH2-PEG并孵育3-3.5小时，NH2-PEG与纳米钯片的质量比为(4-6):3；将离心得到的沉淀进行超滤、洗涤并重新分散得到PEG修饰的纳米钯片；
S2.将步骤S1得到的PEG修饰的纳米钯片与妥珠单抗混合，孵育过夜，PEG修饰的纳米钯片与妥珠单抗的质量比为(1-2):1；离心、洗涤并重悬沉淀，得到纳米钯片负载妥珠单抗的纳米复合体。
进一步的，步骤S1中，离心条件为12000-13000g离心8-10分钟。
进一步的，步骤S1中，1-(3-二甲基氨基丙基)-3-乙基碳二亚胺、N-羟基丁二酰亚胺和巯基丙酸的体积比为5:5:16。
进一步的，步骤S2中，离心条件为14000-15000rpm离心60-70分钟。
第二方面，本发明提供了一种纳米钯片负载妥珠单抗的纳米复合体，是采用以下技术方案实现的。
一种上述制备方法制备得到的纳米钯片负载妥珠单抗的纳米复合体。
第三方面，本发明提供了一种纳米钯片负载妥珠单抗的纳米复合体的应用，是采用以下技术方案实现的。
一种上述纳米钯片负载妥珠单抗的纳米复合体在制备治疗炎症相关性贫血药物的应用。
本申请具有以下有益效果。
本发明利用聚乙二醇(PEG)修饰纳米钯片(PdPL)提高其装载TCZ的效率和生物相容性，构建得到PdPL-PEG@TCZ纳米复合体。PdPL-PEG@TCZ纳米复合体利用PdPL对肝脏组织被动靶向的特性，将TCZ高效的富集到肝脏组织中，提高肝脏组织中的有效药物浓度。同时减少了TCZ在全身其它组织器官分布而引发的副作用。PdPL-PEG@TCZ纳米复合体中的TCZ可有效阻断肝脏细胞IL-6/IL-6R/JAK2/STAT3/Hepcidin信号通路，在AI动物模型中更高效的发挥改善AI作用。采用本发明方法构建的PdPL-PEG@TCZ纳米复合体可以为AI的治疗提供一种新的手段。
附图说明
图1是本发明PdPL、PdPL-PEG及PdPL-PEG@TCZ形貌结构表征图；
图2是本发明TCZ阻断IL-6/JAK2/STAT3/Hepcidin信号通路体外实验验证结果图；
图3是本发明BA诱导的AI模型建立及PdPL-PEG@TCZ纳米复合体改善其贫血效果图；
图4是本发明LLC肿瘤细胞诱导的AI模型建立结果图；
图5是本发明PdPL-PEG@TCZ纳米复合体改善LLC肿瘤细胞诱导的AI模型的贫血效果图。
具体实施方式
1.制备纳米钯片(PdPL)
将10mg乙酰丙酮钯(II)、30mg聚乙烯吡咯烷酮(Polyvinyl pyrrolidone，PVP)和10mg溴化钠加入玻璃压力反应器中，加入2mL N,N-二甲基甲酰胺(N,N-Dimethylformamide，DMP)混匀，待上述原料充分溶解后加入4mL双蒸水(Double distilledwater，ddH2O)充分混匀。随后采用CO气流排尽容器内空气，使容器内CO压力维持在1个标准大气压，将其置于油浴锅中持续搅拌，使其在30分钟内从室温加热至60℃，并在该温度下持续反应1.5小时后冷却至室温。再次向上述反应后容器中加入37.5mg乙酰丙酮钯(II)加入反应容器中并使其充分溶解，采用CO排空容器内空气并将容器内CO压力维持在1个标准大气压，在油浴锅中持续搅拌，使其在60分钟内从室温加热至60℃，并在该温度下持续反应1小时后冷却至室温。将所得产物转移至玻璃瓶中4℃保存，以备后续使用。
2.PdPL表面修饰
取1mL预合成的PdPL于离心管中，加入7mL丙酮沉积以去除多余的杂质，8,000g离心5分钟，去上清，于通风橱内风干数分钟。将沉淀物重新分散到3mL ddH2O中，超声后加入巯基丙酸(每1mgPdPL加入200μL)室温孵育3小时。随后将混合物12000g离心10分钟，用ddH2O轻柔洗涤沉淀物3次后，将沉淀物重新分散在3mL ddH2O中，超声充分分散后加入75μL1-(3-二甲基氨基丙基)-3-乙基碳二亚胺[1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride，EDC，200mM]和75μL N-羟基丁二酰亚胺(N-Hydroxysuccinimide，NHS，58mM)室温孵育1小时，随后加入2mg NH2-PEG并在室温下孵育3小时。离心去上清后ddH2O重悬，然后用超滤离心管(100K)过滤水溶液，用ddH2O洗涤沉淀物3次。最后，收集沉淀物并重新分散在无菌ddH2O或PBS中。
3.PdPL-PEG装载TCZ
量取1mL修饰后的PdPL溶液与30μL TCZ(20mg/mL)混合并于摇床上4℃孵育过夜，之后15,000rpm离心60分钟并收集沉淀物，随后无菌PBS洗涤3次并重悬于PBS中。PdPL、PdPL-PEG及PdPL-PEG@TCZ相关表征结果见图1。
图1(a)为PdPL-PEG@TCZ纳米复合体合成流程示意图。首先合成PdPL，随后进行PEG修饰，得到PdPL-PEG，最后通过装载TCZ得到PdPL-PEG@TCZ纳米复合体。图1(b)为透射电子显微镜结果，从图中可以看出，PdPL呈现出较为均匀的二维片层状结构，在NH2-PEG对其表面修饰后，PdPL发生了一定程度的形态变化，主要表现为纳米材料边缘区域发生卷曲。在图1(c)和1(d)中，原子力显微镜(AFM)2D和3D成像结果显示NH2-PEG修饰可增加PdPL的厚度和尺寸，而TCZ装载后可进一步增加其尺寸，且形貌结构也发生进一步改变。在图1(e)中，水合粒径结构显示PdPL、PdPL-PEG和PdPL-PEG@TCZ的水合粒径分别为31.37nm、75.72nm和135.72nm，这表明PdPL在进行PEG修饰和进一步TCZ装载后其尺寸发生显著变化。图1(f)中，通过对SDS-PAGE凝胶考马斯亮蓝染色测定PdPL-PEG表面TCZ装载量，其结果显示即使在非常低的浓度(10μg/mL)下，TCZ也能有效地装载到PdPL-PEG上，并且随着浓度的增加，TCZ的装载量逐渐增加，显示出一定的剂量依赖性。
4.TCZ阻断IL-6/IL-6R/JAK2/STAT3/Hepcidin信号通路实验验证
人肝脏癌症HepG2细胞系在DMEM培养基(Dulbecco’s modified Eagle’s medium，美国Gbico BRL生命技术有限公司)中生长至细胞密度为50％-60％时，使用人类IL-6重组蛋白(10ng/mL)暴露细胞。30分钟后，弃去培养基，分别暴露TCZ(5μg/mL)、PdPL-PEG(10μg/mL)和PdPL-PEG@TCZ(PdPL-PEG浓度为10μg/mL)。24h后提取HepG2细胞总蛋白并采用Western Blotting实验方法检测磷酸化STAT3(P-STAT3，IL-6R/JAK2/STAT3信号通路激活标志)表达水平。同时，在HepG2细胞中构建Hepcidin荧光素酶报告系统，将处理过的HepG2细胞在无血清的DMEM培养基中培养24h。用人类IL-6重组蛋白(10ng/mL)暴露细胞30分钟，随后用TCZ(5μg/mL)暴露细胞2h。暴露完成后，将细胞继续培养6h。最后进行荧光素酶报告实验检测。TCZ阻断IL-6R/STAT3信号通路相关实验结果见图2。
图2(a)为IL-6分别与TCZ、PdPL-PEG、PdPL-PEG@TCZ联合暴露HepG2细胞后细胞内P-STAT3的表达水平变化情况。与对照组相比，单一IL-6暴露组P-STAT3表达水平显著增高；与单一IL-6暴露组相比，TCZ以及PdPL-PEG@TCZ联合暴露组P-STAT3表达水平显著降低，而PdPL-PEG联合暴露组P-STAT3表达水平无明显变化。这表明，IL-6可以诱导HepG2细胞内P-STAT3高表达，而TCZ可以有效逆转IL-6介导的HepG2细胞P-STAT3高表达。图2(b)为HepG2细胞中Hepcidin荧光素酶报告实验结果。与对照组相比，单一IL-6暴露组中Hepcidin表达显著增高；而当IL-6联合TCZ暴露时，HepG2细胞内Hepcidin表达量回到基线水平。这表明，IL-6可以诱导HepG2细胞内Hepcidin高表达，而TCZ可以有效逆转IL-6介导的HepG2细胞Hepcidin高表达。综上可知，外源性IL-6可以通过激活IL-6/IL-6R/JAK2/STAT3/Hepcidin信号通路提高HepG2细胞内P-STAT3、Hepcidin表达水平，而TCZ可以有效阻断IL-6/IL-6R/JAK2/STAT3/Hepcidin信号通路，最终逆转HepG2细胞内P-STAT3、Hepcidin高表达现象。
5.构建AI动物模型及PdPL-PEG@TCZ纳米复合体改善其贫血效果评估
(一)热灭活布鲁氏菌(BA)诱导的AI模型建立及PdPL-PEG@TCZ纳米复合体改善模型鼠贫血效果评估：采用体重约20g(6-8周龄，购自Vital River实验室(中国北京))的雄性C57BL/6小鼠，将5×108个热灭活布鲁氏菌注射于小鼠腹膜腔内，构建AI模型。于不同时间点经鼠尾静脉注射PdPL-PEG@TCZ纳米复合体至雄性C57BL/6小鼠体内进行实验干预。具体实验方法为：在实验周期的第1、7天采用鼠尾静脉注射的方式将PdPL-PEG@TCZ纳米复合体注射到雄性C57BL/6小鼠体内。于实验周期第3天按上述方法建立BA诱导的AI模型。在实验周期第17天收集模型鼠外周血，进行血细胞分析等相关指标检测。BA诱导的AI模型及PdPL-PEG@TCZ纳米复合体在该AI模型中改善贫血的相关实验结果见图3。
在图3(a)中，与对照组相比，BA腹腔注射组小鼠的肺脏和肝脏组织经过HE染色后可以检测到炎症细胞聚集和浸润(箭头处)。如图3(b)-3(e)所示，腹腔注射BA后，小鼠血清中IL-6和白细胞水平升高，红细胞计数和血红蛋白含量明显降低。以上数据表明BA小鼠腹腔注射可以诱导AI。与未经药物干预的AI小鼠相比，经过PdPL-PEG@TCZ处理后的AI小鼠血清中IL-6和白细胞水平明显下降，而红细胞计数和血红蛋白含量明显升高，其他治疗组的AI小鼠各项指标均未出现明显变化。这些数据表明PdPL-PEG@TCZ纳米复合体可以有效改善BA诱导的AI。
(二)小鼠肺癌LLC细胞诱导的肿瘤相关AI模型建立及PdPL-PEG@TCZ纳米复合体改善模型鼠贫血效果评估。向体重约20g(6-8周龄，购自Vital River实验室(中国北京))的雄性C57BL/6小鼠腹膜腔内注射3×106个LLC细胞建立肿瘤相关AI模型。于不同时间点经鼠尾静脉注射PdPL-PEG@TCZ纳米复合体至雄性C57BL/6小鼠体内进行实验干预。具体实验方法为：在实验周期的第1、7、14天采用鼠尾静脉注射的方式将PdPL-PEG@TCZ纳米复合体注射至雄性C57BL/6小鼠体内。于实验周期第3天通过腹腔注射的方式建立肺癌LLC细胞诱导的肿瘤相关AI模型。在实验周期第21天经收集模型鼠外周血用于血细胞分析等相关指标的检测。LLC细胞诱导的肿瘤相关AI模型及PdPL-PEG@TCZ纳米复合体在该AI模型中改善贫血的相关实验结果见图4-5。
图4(a)-(f)为腹腔注射LLC细胞后，小鼠血液中血红蛋白含量、红细胞计数、白细胞计数、血清IL-6、Hepcidin表达水平以及铁浓度随时间的变化关系。如图4(a)、4(b)所示，血红蛋白含量和红细胞计数随时间推移逐渐减少，与未处理组相比，第4周血红蛋白含量和红细胞计数减少近60％。这表明腹腔注射LLC细胞可以诱导小鼠出现贫血表现。在图4(c)、4(d)中，小鼠血液中白细胞计数逐渐增加，在第4周时白细胞计数较对照组增加2倍以上；在第3周时，小鼠血清中IL-6水平达到高峰，其表达水平较对照组增加近1倍。这些数据表明，腹腔注射LLC细胞可以引起小鼠的全身炎症反应。如图4(e)、4(f)，腹腔注射LLC细胞后，小鼠血清中Hepcidin表达水平在第2-3周上升，第4周回到基线水平；而血清铁含量在第3-4周出现明显下降。这可能与高表达的Hepcidin调节体内铁再分配有关。综合以上数据可知，腹腔注射LLC肿瘤细胞可以诱导小鼠出现AI表现，该表现可能与血清中Hepcidin上调从而促进体内铁再分配相关。
如图5(a)-5(d)所示，腹腔注射LLC细胞后，小鼠血清中IL-6和白细胞水平显著升高，红细胞计数和血红蛋白含量显著降低，这些数据表明LLC细胞腹腔注射可以成功诱导肿瘤相关性AI。与未经药物干预的AI小鼠相比，经过PdPL-PEG@TCZ处理后的AI小鼠血清中IL-6和白细胞水平显著下降，而红细胞计数和血红蛋白含量显著升高，其他治疗组的AI小鼠各项指标均未出现明显变化。这些数据表明PdPL-PEG@TCZ纳米复合体可以有效的改善肿瘤相关性AI。
本具体实施方式的实施例均为本发明的较佳实施例，并非依此限制本发明的保护范围，故：凡依本发明的结构、形状、原理所做的等效变化，均应涵盖于本发明的保护范围之内。
Claims (6)
1.一种纳米钯片负载妥珠单抗的纳米复合体的制备方法，其特征在于：包括以下步骤：
S1.将纳米钯片与巯基丙酸混合，孵育3-3.5小时，巯基丙酸的加入量为200-300μL/mg纳米钯片；离心并洗涤沉淀后，将沉淀再次分散，然后加入1-(3-二甲基氨基丙基)-3-乙基碳二亚胺和N-羟基丁二酰亚胺，孵育1-1.5小时，1-(3-二甲基氨基丙基)-3-乙基碳二亚胺、N-羟基丁二酰亚胺和巯基丙酸的体积比为(2-3):(2-3):8；再加入NH2-PEG并孵育3-3.5小时，NH2-PEG与纳米钯片的质量比为(4-6):3；将离心得到的沉淀进行超滤、洗涤并重新分散得到PEG修饰的纳米钯片；
S2.将步骤S1得到的PEG修饰的纳米钯片与妥珠单抗混合，孵育过夜，PEG修饰的纳米钯片与妥珠单抗的质量比为(1-2):1；离心、洗涤并重悬沉淀，得到纳米钯片负载妥珠单抗的纳米复合体。
2.根据权利要求1所述的一种纳米钯片负载妥珠单抗的纳米复合体的制备方法，其特征在于：步骤S1中，离心条件为12000-13000g离心8-10分钟。
3.根据权利要求1所述的一种纳米钯片负载妥珠单抗的纳米复合体的制备方法，其特征在于：步骤S1中，1-(3-二甲基氨基丙基)-3-乙基碳二亚胺、N-羟基丁二酰亚胺和巯基丙酸的体积比为5:5:16。
4.根据权利要求1所述的一种纳米钯片负载妥珠单抗的纳米复合体的制备方法，其特征在于：步骤S2中，离心条件为14000-15000rpm离心60-70分钟。
5.一种权利要求1-4任一所述制备方法制备得到的纳米钯片负载妥珠单抗的纳米复合体。
6.一种权利要求5所述纳米钯片负载妥珠单抗的纳米复合体在制备治疗炎症相关性贫血药物的应用。
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| US20170216218A1 (en) * | 2014-10-14 | 2017-08-03 | The Brigham And Women S Hospital, Inc. | Nanoparticles and Methods of Use |
| CN108348599A (zh) * | 2015-07-22 | 2018-07-31 | 华上生技医药股份有限公司 | 包含金属纳米粒子、连接物及抗体的复合物 |
| US20180250420A1 (en) * | 2014-11-13 | 2018-09-06 | The Curators Of The University Of Missouri | Multiple human antibody-nanoparticle conjugates and methods of formation |
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2021
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170216218A1 (en) * | 2014-10-14 | 2017-08-03 | The Brigham And Women S Hospital, Inc. | Nanoparticles and Methods of Use |
| US20180250420A1 (en) * | 2014-11-13 | 2018-09-06 | The Curators Of The University Of Missouri | Multiple human antibody-nanoparticle conjugates and methods of formation |
| CN108348599A (zh) * | 2015-07-22 | 2018-07-31 | 华上生技医药股份有限公司 | 包含金属纳米粒子、连接物及抗体的复合物 |
Non-Patent Citations (2)
| Title |
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| WEI ZHUANG等: "Efficient nanobiocatalytic systems of nuclease P1 immobilized on PEG-NH2 modified graphene oxide: effects of interface property heterogeneity", COLLOIDS AND SURFACES B: BIOINTERFACES, vol. 145, pages 2 * |
| ZHU J.等: "A Palladium Nanosheet-Based Tocilizumab Delivery System Inhibits Prostate Cancer Progression By Correcting Cancer Related Anemia In Mice", EUR UROL OPEN SCI, vol. 19, no. 2, pages 1841 * |
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