CN108348599A - 包含金属纳米粒子、连接物及抗体的复合物 - Google Patents

包含金属纳米粒子、连接物及抗体的复合物 Download PDF

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CN108348599A
CN108348599A CN201680042509.XA CN201680042509A CN108348599A CN 108348599 A CN108348599 A CN 108348599A CN 201680042509 A CN201680042509 A CN 201680042509A CN 108348599 A CN108348599 A CN 108348599A
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noble metal
compound
metal nano
nano particles
attachment
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陈嘉南
陈嘉雄
赵月秀
吴奕宏
纪俐伶
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GNT BIOTECH & MEDICALS Corp
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Abstract

一种包含金属纳米粒子、多数个连接物结合物及多数个PEG的复合物，其中所述连接物结合物包含pH敏感性连接物及抗体。所述pH敏感性连接物具有下式(I)。所述连接物通过1,2‑二硫杂环戊烷基的硫原子直接连接至所述金属纳米粒子。

Description

包含金属纳米粒子、连接物及抗体的复合物
技术领域
本发明涉及一种金属纳米粒子、连接物及抗体或融合蛋白质的复合物。特定言之，本发明提供一种包含金属纳米粒子、连接物、聚乙二醇(PEG)及抗体或融合蛋白质的复合物。
背景技术
将治疗及诊断化合物特定、有效地传递至细胞(尤其至细胞溶质)为许多医药公司的主要目标。已使用多种不同方法以提升特定性及吸收。举例而言，纳米技术已广泛地用于药物传递及癌症疗法的新策略的研发中。已研发出pH敏感性纳米系统，其中具体通过酸性肿瘤环境来触发药物释放，且此等系统可改善癌症治疗的功效。Feng Wang等人通过经由酸不稳定键将小红莓(doxorubicin)系链于具有聚(乙二醇)位置的AuNP的表面上来揭示药物传递系统(American Chemical Society,2011,第5卷,第5期,第3679-3692页)。Tian-MengSun等人揭示使用经由腙键结合至金纳米粒子的小红莓的癌症干细胞疗法(Biomaterials35,2014,第836-845页)。US 2013/0331764涉及用于将抗癌药物传递至癌细胞的方法，其通过使抗癌药物与pH敏感性金属纳米粒子结合，以使其与癌细胞分开。WO 2013/139942提供包含金属纳米粒子及至少一种连接物的纳米粒子。
依那西普(Etanercept，)为TNF-α的有效抑制剂，已显示其在类风湿性关节炎(RA)及幼年型类风湿性关节炎(JRA)中提供快速及持久改善(Ann Rheum Dis 2000,59(增刊I):146-149)。存在改善向靶细胞传递依那西普且由此提升依那西普的治疗作用的需要。依那西普为由重组DNA产生的融合蛋白质。其将肿瘤坏死因子(TNF)受体融合至IgG1抗体的定端。其由将两种天然存在的可溶性人类75kDa TNF受体连接物与IgG1的Fc部分组合来制成。其为人工设计的二聚体融合蛋白质。
发明内容
本发明提供一种包含贵金属纳米粒子、多数个连接物结合物及多数个PEG的复合物，其中连接物具有下式：
其中m为1至4；
其中连接物结合物包含连接物及抗TNFα人工融合蛋白质或抗TNFα抗体，其中融合蛋白质及抗体添加有醛基，且连接物的NH2基结合至融合蛋白质或抗体的醛基上；
其中抗TNFα抗体为赛妥珠单抗(certolizumab)、英利昔单抗(infliximab)、利妥昔单抗(rituximab)、阿达木单抗(adalimumab)或托珠单抗(tocilizumab)或其生物类似物，且抗TNFα人工融合蛋白质为依那西普(etanercept)或其生物类似物；且
其中连接物经由连接物中的1,2-二硫杂环戊烷基的硫原子连接至贵金属纳米粒子，且PEG直接连接至贵金属纳米粒子；且
其中负载于贵金属纳米粒子上的融合蛋白质或抗体的比率为约1.0％至约6.0％(w/w)，且负载于贵金属纳米粒子上的PEG的比率为约0.5％(w/w)至约12％(w/w)。
在一个实施例中，贵金属纳米粒子为Au纳米粒子、Ag纳米粒子、Pd纳米粒子及Pt纳米粒子。优选地，贵金属纳米粒子为Au纳米粒子(AuNP)。在一个实施例中，贵金属纳米粒子为Au纳米粒子、Ag纳米粒子、Pd纳米粒子及Pt纳米粒子。优选地，贵金属纳米粒子为Au纳米粒子。在一个实施例中，本发明中所使用的PEG的分子量在约1000至约20,000Da的范围内。优选地，PEG的分子量为约2000Da。在一个实施例中，连接物为连接物I-1(LK)。
在一个实施例中，负载于贵金属纳米粒子上的抗TNFα融合粒子或抗体的比率为约1.0％至约5.0％(w/w)或约1％至约4％(w/w)。
在一些实施例中，对于尺寸为约20nm至约30nm的贵金属纳米粒子，负载于贵金属纳米粒子上的抗TNFα融合粒子或抗体的比率为约3.0％(w/w)至约6.0％(w/w)、约3.0％(w/w)至约5.0％(w/w)或约3.0％(w/w)至约4.0％(w/w)；更佳地，约3.5％(w/w)。对于尺寸为约40nm的贵金属纳米粒子，负载于贵金属纳米粒子上的抗TNFα抗体的比率为约2.0％(w/w)至约3％(w/w)；优选地，约2.5％(w/w)至约3％(w/w)；更佳地，约2.63％(w/w)。
在一个实施例中，负载于贵金属纳米粒子上的PEG的比率为约0.75％(w/w)至约10％(w/w)。
本发明亦提供一种医药组合物，其包含本发明的复合物及医药学上可接受的载剂。本发明亦提供用于治疗自体免疫及免疫调节障碍疾病的方法，其包含向个体投与有效量的本发明的复合物。
图式简单说明
图1(A)至(F)显示连接至不同粒度的AuNP的ETA/LK的饱和量。(A)及(B)占AuNP(20-30nm)的ETA/LK的饱和量为3.5％(w/w)。低于0.35μg的ETA/LK量导致AuNP(20-30nm)凝聚，呈现灰色-紫色；(C)及(D)占AuNP(40nm)的ETA/LK的饱和量在1.75％(w/w)至3.5％(w/w)的范围内，且平均值为2.625％(w/w)；及(E)及(F)占AuNP(80nm)的ETA/LK的饱和量在0.875％(w/w)至1.75％(w/w)的范围内，且平均值为1.31％(w/w)。
图2显示LK成功地连接至ETA。OD536值自1.8391降低至背景。
图3显示AuNP/LK/ETA复合物(与ETA结合的20-30nm AuNP)的电浆子峰(λmax)中的2nm红移。
图4显示在表面上结合有ETA融合蛋白质的各金纳米粒子。
图5显示AuNP/LK/ETA复合物的TEM图像。观察到1-5nm以金标记的二级抗人类IgG抗体与位于AuNP表面上的ETA结合。
图6显示AuNP/LK/ETA/PEG复合物的实例的示意图。
图7显示AuNP/ETA/LK及AuNP/ETA/2KLK中的不同连接物长度不会影响ETA的活性。各自封端有或不含PEG的AuNP/ETA/LK及AuNP/ETA/2KLK不会影响ETA的活性。
具体实施方式
本发明是基于至少一种金属纳米粒子的发现，其可同时结合pH敏感性连接物、融合蛋白或抗体及PEG以形成复合物。本发明的复合物可将融合蛋白质或抗体传递至疾病过程中所涉及的靶细胞或接近细胞，以使融合蛋白质或抗体可选择性地靶向及作用于细胞。
除非另外定义，否则本文所用的所有技术及科学术语均具有如一般技术者通常理解相同的含义。除非另外说明，否则本文中所提及的所有专利、申请案、公开的申请案及其他公开案均以全文引用的方式并入。除非另外说明，否则在本文术语存在多数个定义的情况下，以此部分中的定义为准。
从最广的意义上使用术语“抗体”，且其包含单株抗体(例如全长或完好单株抗体)、多株抗体、单价、多价抗体、多特异性抗体(例如双特异性抗体，只要其呈现所要的生物活性即可)，且亦可包括某些抗体片段(如本文中更详细所描述)。抗体可为嵌合、人类、人类化及/或亲和力成熟的。
如本文所使用，术语“肿瘤”是指所有瘤性细胞生长及增殖(无论恶性或良性)，及所有癌前及癌性细胞及组织。术语“癌症”、“癌性”、“细胞增生性病症”、“增生性病症”及“肿瘤”在本文中提及时不相互排斥。
如本文所使用，术语“癌症”是指哺乳动物中的生理病况，其典型特征在于失调的细胞生长/增殖。癌症的实例包括(但不限于)癌瘤、淋巴瘤(例如霍奇金氏(Hodgkin's)及非霍奇金氏淋巴瘤(non-Hodgkin's lymphoma))、胚细胞瘤、肉瘤及白血病、鳞状细胞癌、小细胞肺癌、非小细胞肺癌、肺腺癌、肺鳞状癌、腹膜癌、肝细胞癌、肠胃癌、胰脏癌、神经胶母细胞瘤、子宫颈癌、卵巢癌、肝癌(liver cancer)、膀胱癌、肝癌(hepatoma)、乳癌、结肠癌、结肠直肠癌、子宫内膜或子宫癌、唾液腺癌、肾脏癌、肝癌(liver cancer)、前列腺癌、外阴癌、甲状腺癌、肝癌(hepatic carcinoma)、白血病及其他淋巴组织增生病症及各种类型的头颈癌。
如本文所使用，术语“治疗(treating)”、“处理(treatment)”、“医疗(therapeutic)”或“疗法(therapy)”未必意谓完全治愈或消除疾病或病况。疾病或病况的任何不期望的征候或症状达到任何程度上的任何缓解均可视为治疗及/或疗法。此外，治疗可能包括可能使患者的总体健康或外观恶化的作用。
“有效量”是指在所需剂量及时段下，可有效达成所需治疗或预防结果的量。
本发明的物质/分子的“治疗有效量”可根据诸如个体的疾病病况、年龄、性别、及重量、及物质/分子在个体中引起所需反应的能力等因素而变化。治疗有效量亦为治疗有益效应超过物质/分子的任何毒性或有害效应的量。“预防有效量”是指在所需剂量及时段下，可有效达成所需预防结果的量。通常但并非必需，因为预防剂量是用在个体疾病之前或在疾病早期，所以预防有效量将低于治疗有效量。
术语“医药学上可接受的盐”是指对其所投与的生物体不产生显着刺激且不消除化合物的生物活性及特性的化合物的盐。
术语“生物类似物”(亦称为后续生物制品或后入生物制品)是指近似为原始产物的相同复制物的生物医学产物。
在一个态样中，本发明提供包含贵金属纳米粒子、多数个连接物结合物及多数个PEG的复合物，其中连接物具有下式：
其中m为1至4；
其中连接物结合物包含连接物及抗TNFα人工融合蛋白质或抗体，其中融合蛋白质及抗体添加有醛基，且连接物的NH2基结合至融合蛋白质或抗体或生物类似物的醛基上；
其中抗TNFα抗体为赛妥珠单抗(certolizumab)、英利昔单抗(infliximab)、利妥昔单抗(rituximab)、阿达木单抗(adalimumab)或托珠单抗(tocilizumab)或其生物类似物，且抗TNFα人工融合蛋白质为依那西普(etanercept)或其生物类似物；且
其中连接物经由连接物中的1,2-二硫杂环戊烷基的硫原子连接至贵金属纳米粒子，且PEG直接连接至贵金属纳米粒子；且
其中负载于贵金属纳米粒子上的融合蛋白质或抗体的比率为约1.0％至约6.0％(w/w)，且负载于贵金属纳米粒子上的PEG的比率为约0.5％(w/w)至约12％(w/w)。
在一个实施例中，贵金属纳米粒子为Au纳米粒子、Ag纳米粒子、Pd纳米粒子及Pt纳米粒子。优选地，贵金属纳米粒子为Au纳米粒子。
在一个实施例中，贵金属纳米粒子的尺寸在约20nm至约100nm的范围内。在一些实施例中，尺寸为约20nm至约80nm、约20nm至约50nm、约20nm至约40nm或约20nm至约30nm。
在一个实施例中，连接物具有下式：
在一个实施例中，本发明中所使用的PEG的分子量在约1,000至约20,000Da的范围内。优选地，PEG的分子量为约1,000至约5,000Da或约2,000Da。
在一个实施例中，负载于贵金属纳米粒子上的抗TNFα融合蛋白质或抗体的比率为约1.0％至约5％(w/w)、约1.0％至约4％(w/w)、约1％至约3.5％(w/w)、约1.3％至约3.5％(w/w)、约1.3％至约3％(w/w)或2.5％至3.5％。在一些实施例中，对于尺寸为约20nm至约30nm的贵金属纳米粒子，负载于贵金属纳米粒子上的抗TNFα融合蛋白质或抗体为约3.0％(w/w)至约6％(w/w)、约3.0％(w/w)至约5％(w/w)或约3.0％(w/w)至约4％(w/w)；更佳地，约3.5％(w/w)。对于尺寸为约40nm的贵金属纳米粒子，负载于贵金属纳米粒子上的抗TNFα融合蛋白质或抗体为约2.0％(w/w)至约3％(w/w)；优选地，约2.5％(w/w)至约3％(w/w)；更佳地，约2.63％(w/w)。对于尺寸为约80nm的贵金属纳米粒子，负载于贵金属纳米粒子上的抗TNFα融合蛋白质或抗体为约1.0％(w/w)至约2％(w/w)；优选地，约1.0％(w/w)至约1.5％(w/w)；更佳地，约1.31％(w/w)。
在一个实施例中，负载于贵金属纳米粒子上的PEG的比率为约0.75％(w/w)至约10％(w/w)；优选地，0.75％(w/w)至约9％(w/w)、0.75％(w/w)至约8％(w/w)、0.75％(w/w)至约7％(w/w)、0.75％(w/w)至约6％(w/w)、0.75％(w/w)至约5％(w/w)、0.75％(w/w)至约4％(w/w)、约1％(w/w)至约10％(w/w)、约1％(w/w)至约9％(w/w)、约1％(w/w)至约8％(w/w)、约1％(w/w)至约7％(w/w)、约1％(w/w)至约6％(w/w)、约1％(w/w)至约5％(w/w)、约1％(w/w)至约4％(w/w)、约2％(w/w)至约6％(w/w)、约2％(w/w)至约10％(w/w)、约2％(w/w)至约8％(w/w)、约2％(w/w)至约5％(w/w)或约2％(w/w)至约4％(w/w)。
如本文所描述，抗TNFα抗体或融合蛋白质经氧化以具有醛基。可通过首先在连接物末尾的NH2基处使连接物与抗TNFα融合蛋白质或抗体结合，且将连接物结合物及PEG连接至金属纳米粒子以形成复合物来制备本发明的复合物。连接物经由连接物中的1,2-二硫杂环戊烷基的硫原子连接至贵金属纳米粒子，且PEG直接连接至贵金属纳米粒子。抗TNFα融合蛋白质或抗体以饱和状态负载于贵金属纳米粒子上。用于连接物与抗TNFα融合蛋白质或抗体的结合及连接物结合物及PEG与金属纳米粒子的连接的方法为此项技术中已知的。
如本文所使用，抗TNFα抗体为赛妥珠单抗、英利昔单抗、利妥昔单抗、阿达木单抗或托珠单抗或其生物类似物。抗TNFα融合蛋白质为依那西普或其生物类似物。
鉴于本发明，可使用熟习此项技术者已知的方法制备本发明的连接物I-酰胺连接物。举例而言，可如以下方案中显示制备本发明的优选连接物。
在一些情况下，亦可使用聚乙二醇化以减小连接物或纳米粒子及生物部分之间的电荷相互作用，例如通过在连接物或纳米粒子的表面上产生可避免聚合物与生物部分相互作用的亲水层。在一些情况下，添加聚(乙二醇)重复单元可延长聚合性结合物的电浆半衰期，例如通过减少由吞噬细胞系统导致的聚合性结合物的吸收同时减少由细胞导致的转染/吸收功效。本领域普通技术人员将知晓用于聚乙二醇化聚合物的方法及技术，举例而言，如下文实例中所论述，通过开环聚合技术(ROMP)或类似者，通过使用EDC(1-乙基-3-(3-二甲胺基丙基)氢氯化碳化二亚胺)及NHS(N-羟基丁二酰亚胺)以使聚合物与以胺收端的PEG基反应。
在一个态样中，本发明提供一种医药组合物，其包含本发明的复合物及医药学上可接受的载剂。
在另一态样中，本发明提供用于治疗自体免疫及免疫调节障碍疾病的方法，其包含向个体投与有效量的本发明的复合物。在一些实施例中，疾病为发炎性肠病、牛皮癣、化脓性汗腺炎及顽固性哮喘类风湿性关节炎、幼年型类风湿性关节炎及牛皮癣性关节炎、斑状牛皮癣或僵直性脊椎炎。
本文所描述的一些实施例涉及一种组合物，其可包含连接本发明的抗TNFα融合蛋白质或抗体的金属纳米粒子复合物及医药学上可接受的载剂。医药组合物有助于向生物体投与连接抗TNFα融合蛋白质或抗体的金属纳米粒子复合物。用于经口、经直肠、局部或非经肠(包括皮下、腹膜内、肌内及静脉内)投与的合适医药学上可接受的载剂为熟习此项技术者所已知的。载剂必须在与调配物的其他成分兼容且不对其受体有害的意义上为医药学上可接受的。存在于此项技术中的投与化合物的多种技术包括(但不限于)经口、注射、喷雾、非经肠及局部投与。
举例而言，适于非经肠投与的组合物宜包括活性化合物的无菌水性制剂，其优选与接受者的血液呈等张性。适用调配物亦包括含有本发明的复合物的浓缩溶液或固体，其一旦用适当溶剂稀释，即产生适于上述非经肠投与的溶液。
举例而言，对于经口投与，本发明的复合物可并入至惰性载剂中，呈离散单位形式，诸如胶囊、扁胶剂、锭剂或口含剂，各自含有预定量的抗TNFα抗体；呈粉末或颗粒状；或呈悬浮液或水性液态溶液或非水性液体状，例如糖浆、酏剂、乳液或饮剂。合适载剂可为淀粉或糖且包括崩解剂、润滑剂、调味剂、黏合剂及其他相同性质的物质。
举例而言，经直肠投与的调配物可使用公知载剂，例如可可豆油或Witepsol S55(Dynamite Nobel Chemical,Germany的商标)作为栓剂基质，形成栓剂。举例而言，本文所描述的某些实施例涉及一种药物传递系统，其包含连接一或多种相同或不同治疗剂或诊断剂的一或多种金属纳米粒子复合物。
组合物可宜呈单位剂型，且可通过药学技术中熟知的任何方法来制备。
任何组合物可视情况包括一或多种其他组分，诸如镇痛剂(诸如NSAID)、COX-2抑制剂及改善疾病的抗风湿药(DMARD)。DMARD的实例包括(但不限于)甲胺喋呤(methotrexate)、羟基氯喹(hydroxychloroquine)、柳氮磺胺吡啶(sulfasalazine)、来氟米特(leflunomide)、TNFα抑制剂(赛妥珠单抗、英利昔单抗及依那西普)、利妥昔单抗、托珠单抗、阿巴西普(abatacept)及阿那白滞素(anakinra)。NSAID的实例包括(但不限于)阿司匹林(aspirin)、二氟尼柳(diflunisal)、双水杨酸酯(salsalate)、布洛芬(ibuprofen)、右布洛芬(dexibuprofen)、非诺洛芬(fenoprofen)、酮基布洛芬(ketoprofen)、右酮洛芬(dexketoprofen)、氟比洛芬(flurbiprofen)、奥沙普嗪(oxaprozin)、洛索洛芬(Loxoprofen)萘普生(naproxen)。COX-2抑制剂的实例包括(但不限于)塞内昔布(celecoxib)。
投与连接抗TNFα融合蛋白质或抗体的金属纳米粒子复合物的多种技术存在于此项技术中，其包括(但不限于)经口、经直肠、局部、喷雾、注射及非经肠传递，包括肌内、皮下、静脉内、髓内注射、鞘内、直接室内、腹膜内、鼻内及眼内注射。
实例
实例1制备连接物I-1
步骤1将乙醇胺(1.28g，1.05当量)及类脂酸(4.13g，1.0当量)溶解于80mL DCM中，且缓慢添加EDC·HCl(4.98g，1.3当量)、羟基丁二酰亚胺(NHS)(2.30g，1.0当量)及三乙胺(6.4mL，2.3当量)。在室温下进行反应达至少5小时且反应之后为TLC。随后，通过添加ddH2O来淬灭混合物，且用DCM萃取2-3次。收集有机层，用无水硫酸镁干燥，用MgSO4过滤且随后移除DCM。通过使用DCM:MeOH＝9.8:0.2用管柱来纯化萃取物。产物为浅黄色液体。产量：4.04g；81.0％。
步骤2将氯甲酸4-硝基苯酯(3.35g，1.15当量)置于双颈瓶中。在真空下达1小时，将三通阀转向至氮气器件且用注射器添加60mL DCM。用10-15mLDCM溶解经称重的步骤1产物1，用注射器将其注入至反应烧瓶中，且随后缓慢添加三乙胺(4.5mL，2.3当量)。在冰浴中进行反应约1小时且随后回到室温下持续整夜。反应完成后，通过添加ddH2O来淬灭混合物，且用DCM萃取3次。收集有机层，用无水硫酸镁干燥，用MgSO4过滤且随后移除DCM。通过使用EtOAc:Hex:MeOH＝1:1:0.2用管柱来纯化萃取物。产物为浅黄色液体。纯化后产率：51.6％。
步骤3将经称重的步骤2产物2(3.5g，1.0当量)溶解于120mL DCM中，且随后缓慢添加水合肼(6.5mL，10.0当量)。在室温下进行反应约24小时。溶液颜色自浅黄色变为橙色。反应完成后，通过添加ddH2O来淬灭混合物，且用DCM萃取2-3次。收集有机层，用无水硫酸镁干燥，用MgSO4过滤且随后移除DCM。通过使用DCM:MeOH＝9.5:0.5用管柱来纯化萃取物。在此步骤中，产物阻塞于管柱中且可提高MeOH至5％来洗涤最终产物。产物为浅黄色黏性固体。纯化后产率：19.5％。
连接物I-1(连接物I-酰胺，m＝2)。1H(400MHz)δ1.23-1.31(2H,m),1.40-1.51(3H,m),1.57-1.64(1H,m),1.78-1.86(1H,m),2.05(2H,t),2.32-2.39(1H,m),3.01-3.18(4H,m),3.39(2H,t),3.52-3.57(3H,m),4.03-4.19(4H,m),7.94(1H,s),8.14(1H,s)。13C(100MHz)δ25.9,29.0,34.8,36.1,38.9,39.5,40.9,57.2,64.7,69.3,69.6,159.5,174.9ppm。H-质量(m/z):(M+Na)＝374.1171
连接物I-2(连接物I-酰胺，m＝1)。1H(300MHz)δ1.30-1.38(2H,m),1.45-1.57(3H,m),1.60-1.72(1H,m),1.80-1.92(1H,m),2.06(2H,s),2.36-2.46(1H,m),3.07-3.24(4H,m),3.56-3.65(1H,m),3.95(2H,t),4.02(2H,s),7.88(1H,t),8.11(1H,s)。13C(100MHz)δ25.6,28.8,34.6,35.9,38.8,39.4,40.7,56.9,63.7,163.8,174.3ppm。H-质量(m/z):(M+Na)＝330.0894。
连接物I-3(连接物I-酰胺，m＝3)。1H(300MHz)δ1.30-1.37(2H,m),1.45-1.57(3H,m),1.60-1.72(1H,m),1.80-1.91(1H,m),2.06(2H,t),2.35-2.46(1H,m),3.07-3.23(4H,m),3.39(2H,t),3.47-3.65(7H,m),4.02(2H,s),4.05-4.08(2H,m),7.84(1H,t),8.18(1H,s)。13C(100MHz)δ25.7,28.9,34.7,35.9,38.8,39.4,40.7,60.0,64.4,69.4,69.6,70.1,70.3,159.2,174.2ppm。H-质量(m/z):(M+Na)＝418.1466。
连接物I-4(连接物I-酰胺，m＝4)。1H(300MHz)δ1.30-1.38(2H,m),1.45-1.57(3H,m),1.60-1.72(1H,m),1.80-1.91(1H,m),2.06(2H,t),2.36-2.46(1H,m),3.07-3.23(4H,m),3.39(2H,t),3.49-3.65(11H,m),4.02(2H,s),4.05-4.08(2H,m),7.83(1H,t),8.18(1H,s)。13C(100MHz)δ25.7,28.9,34.7,35.9,38.8,39.4,40.7,56.9,64.4,69.4,69.6,70.2,70.3,70.4,159.2,174.1ppm。H-质量(m/z):(M+Na)＝440.1875。
实例2制备本发明的复合物
使用10kDa MWCO离心式过滤器(Millipore,UFC501024)浓缩依那西普(Etanercept，ETA)(Mycenax biotech,TuNEX)，且以1mg/mL将其溶解于100mM Na2HPO4(pH值为7.4的缓冲剂)中。随后，将20μL含有100mM NaIO4的水溶液添加至100μL依那西普溶液中，且在暗处保温混合物达30分钟。通过添加250μL 1×PBS来淬灭反应物。此时，氧化依那西普Fc部分上的碳水化合物部分以使其具有醛基。随后，将LK(连接物I-1)添加至依那西普溶液中。LK在分子相对位置上具有酰肼及二硫醇基。酰肼部分与改质依那西普分子的Fc部分的醛基相互作用。在室温下保温反应混合物达2小时，且随后使用离心式过滤器收集硫醇化依那西普，且在0.1M磷酸钠缓冲剂(pH值为7.4)中对其进行再悬浮。将硫醇化依那西普与0.5mL金纳米粒子混合，且在室温下保温悬浮液达1小时以获得AuNP/LK/ETA复合物。
与AuNP连接的ETA/LK的饱和度及药物负载量。在溶液中，将以不同量(0μg、0.0438μg、0.0875μg、0.175μg、0.35μg、0.7μg及1.4μg)存在于0.1M磷酸盐缓冲剂中的ETA/LK与10μg AuNP(20-30nm)混合，且随后在室温下保温达60分钟以用于饱和度测试。使用UV-Vis分光亮度计量测与AuNP连接的ETA/LK的饱和度。使用AuNP的颜色测定与AuNP连接的ETA/LK的饱和度。
将14μg ETA/LK添加至1mL的50ppm AuNP(其具有不同尺寸)中，且随后混合。在室温下保温所得混合物达60分钟。随后，以15000rpm离心所得混合物达20分钟。对所得上清液进行BCA分析以量测药物负载量。根据下式计算药物负载量：
结果显示占AuNP(20-30nm)的ETA/LK的饱和量为3.5％(w/w)。低于0.35μg的ETA/LK量导致AuNP凝聚，呈现灰色-紫色(参见图1(A)及(B))。根据上文所提及的量及步骤，在饱和度测试中使用尺寸为40nm的AuNP。结果显示占AuNP(40nm)的ETA/LK的饱和量在1.75％(w/w)至3.5％(w/w)的范围内，且平均值为2.625％(w/w)(参见图1(C)及(D))。类似地，根据上文所提及的量及步骤，在饱和度测试中使用尺寸为80nm的AuNP。结果显示占AuNP(80nm)的ETA/LK的饱和量在0.875％(w/w)至1.75％(w/w)的范围内，且平均值为1.31％(w/w)(参见图1(E)及(F))。上文所提及的结果概括于下表中。
ETA连接至LK(连接物I-1)的证明。
用UV-Vis分光亮度计(Beckman,DU 800)分析LK与ETA的醛基的连接。在室温下，将ETA的醛基与10mg/mL purpald溶液混合达2小时。醛基的吸收是在536nm处。LK连接至ETA的醛基以成为ETA/LK。当在室温下将ETA/LK与10mg/mL purpald溶液混合达2小时时，无法在536nm处量测到吸收值。图2显示LK成功地连接至ETA。
ETA/LK连接至AuNP的证明。用UV-Vis分光亮度计(Beckman,DU 800)分析AuNP/LK/ETA复合物。所记录的表面电浆子共振谱显示AuNP/LK/ETA复合物的电浆子峰(λmax)中的2nm红移(图3)。使用荧光显微镜(Nikon,TE2000-U)量测以AuNP/LK/ETA复合物标记的AlexFluor 568二级抗体的红色荧光图像。各金纳米粒子显示红色荧光，其表明各金纳米粒子在表面上与ETA抗体结合(图4)。吾等通过与结合有1-5nm AuNP的二级抗体结合来进一步检测AuNP/LK/ETA复合物。在AuNP/LK/ETA复合物的TEM图像中，观察到1-5nm以金标记的二级抗人类IgG抗体结合至位于AuNP表面上的ETA(图5)。
连接至AuNP/LK/ETA复合物的AuNP的PEG。将具有2000Da分子量的PEG添加至AuNP/LK/ETA/PEG复合物溶液中。将所得混合物置放于室温下达20分钟，且随后以15000rpm离心20分钟以移除溶液。将所得沈淀物添加至水中以调配所需浓度的AuNP/LK/ETA/PEG复合物溶液。AuNP/LK/ETA/PEG复合物的实例的示意图显示于图6中。将AuNP/LK/ETA/PEG复合物保持在4℃来储存以供进一步使用。所得AuNP/LK/ETA/PEG复合物具有以下特征，其列于下表中。
实例3AuNP尺寸、连接物长度及封端有或不含PEG对阻断TNFα治疗的影响
AuNP尺寸。使用尺寸为20-90nm的AuNP连接至ETA/LK以测试AuNP尺寸对阻断TNF-α治疗的影响。用以下混合物治疗MCF-7细胞(5x105)：(A)仅50ng/ml TNF-α，(B)30μg/mL尺寸为20-50nm的AuNP，(C)50ng/ml TNF-α及30μg/mL尺寸为20-50nm的AuNP，(D)50ng/ml TNF-α及4.2μg/mL AuNP(20-50nm)/LK/ETA复合物，(E)50ng/ml TNF-α及与AuNP(20-50nm)/LK/ETA复合物的ETA相等的ETA。在5％CO2下在37℃下培养经治疗的细胞达48小时。结果显示将尺寸为20-50nm的AuNP连接至ETA/LK不会影响ETA的活性。类似地，亦使用尺寸为70-90nm的AuNP且显示相同结果，亦即将尺寸为20-50nm的AuNP连接至ETA/LK不会降低ETA的活性。
连接物长度及封端有或不含PEG。第一，使用AuNP连接至ETA/LK或ETA/2KLK(2000Da连接物)，且测试所得AuNP/ETA/LK及AuNP/ETA/2KLK以评估连接物长度对阻断TNF-α治疗的影响。第二，使用AuNP/LK/ETA复合物及AuNP/LK/ETA/PEG复合物测试封端有或不含PEG对阻断TNF-α治疗的影响。用2.1μg/mL AuNP/LK/ETA复合物、2.1μg/mL AuNP/LK/ETA/PEG复合物、2.1μg/mL AuNP/2KLK/ETA复合物及2.1μg/mL AuNP/2KLK/ETA/PEG复合物的混合物治疗MCF-7细胞。将上文所提及的复合物、30μg/mL尺寸为20-50nm的AuNP及50ng/mlTNF-α添加至6孔培养盘(各孔接种有5X105MCF-7细胞)中，且在5％CO2下在37℃下培养48小时。结果显示AuNP/ETA/LK及AuNP/ETA/2KLK中的不同连接物长度不会影响ETA的活性，以及各自封端有或不含PEG的AuNP/ETA/LK及AuNP/ETA/2KLK不会影响ETA的活性(参见图7)。
AuNP/LK/ETA/PEG复合物的例示性生物活性
将500μL、0.025ng/μL TNF-α及500μL、1ng/μL AuNP/LK/ETA/PEG复合物或游离ETA混合达2小时。将所得混合物添加至6孔板(各孔接种有5x105MCF-7细胞)中以分别在5％CO2下在37℃下进行培养。两天后，对细胞进行统计。比较AuNP/LK/ETA/PEG复合物及游离ETA的生物活性，发现AuNP/LK/ETA/PEG复合物中和TNF-α的阻断能力与游离ETA的阻断能力相同，因此AuNP、LK及PEG的结合不会影响ETA活性。游离ETA及在5％CO2下在37℃下的复合物的相对细胞存活率(％)显示于下表中。

Claims (15)

1.一种复合物，其包含贵金属纳米粒子、多数个连接物结合物及多数个PEG，其中所述连接物具有下式：
其中m为1至4；
其中连接物结合物包含连接物及抗TNFα人工融合蛋白质或抗体，其中所述融合蛋白质及所述抗体已添加醛基，且所述连接物的NH2基结合至所述融合蛋白质或所述抗体或生物类似物的醛基上；
其中所述抗TNFα抗体为赛妥珠单抗(certolizumab)、英利昔单抗(infliximab)、利妥昔单抗(rituximab)、阿达木单抗(adalimumab)或托珠单抗(tocilizumab)或其生物类似物，且所述抗TNFα人工融合蛋白质为依那西普(etanercept)或其生物类似物；且
其中所述连接物经由所述连接物中的1,2-二硫杂环戊烷基的硫原子连接至所述贵金属纳米粒子，且这些PEG直接连接至所述贵金属纳米粒子；且
其中负载于所述贵金属纳米粒子上的所述融合蛋白质或抗体的比率为约1.0％至约6.0％(w/w)，且负载于所述贵金属纳米粒子上的所述PEG的比率为约0.5％(w/w)至约12％(w/w)。
2.如权利要求1的复合物，其中所述贵金属纳米粒子为Au纳米粒子、Ag纳米粒子、Pd纳米粒子及Pt纳米粒子。
3.如权利要求1的复合物，其中所述贵金属纳米粒子为Au纳米粒子。
4.如权利要求1的复合物，其中所述贵金属纳米粒子的尺寸在约20nm至约100nm的范围内。
5.如权利要求1的复合物，其中所述连接物具有下式：
6.如权利要求1的复合物，其中用于本发明中的PEG的分子量在约1,000至约20,000Da的范围内。
7.如权利要求1的复合物，其中负载于所述贵金属纳米粒子上的所述抗TNFα融合蛋白质或抗体的比率为约1％至约4％(w/w)。
8.如权利要求1的复合物，其中对于尺寸为约20nm至约30nm的贵金属纳米粒子，负载于所述贵金属纳米粒子上的所述抗TNFα抗体或融合蛋白质为约3.0％(w/w)至约6.0％(w/w)；对于尺寸为约40nm的贵金属纳米粒子，负载于所述贵金属纳米粒子上的所述抗TNFα抗体为约2.0％(w/w)至约5％(w/w)；或对于尺寸为约80nm的贵金属纳米粒子，负载于所述贵金属纳米粒子上的所述抗TNFα抗体为约1.0％(w/w)至约3％(w/w)。
9.如权利要求1的复合物，其中负载于所述贵金属纳米粒子上的所述PEG的比率为约0.75％(w/w)至约10％(w/w)。
10.如权利要求1的复合物，其中负载于所述贵金属纳米粒子上的所述PEG的比率为约2％(w/w)至约5％(w/w)。
11.一种医药组合物，其包含如权利要求1的复合物及医药学上可接受的载剂。
12.如权利要求11的医药组合物，其可进一步包含一或多种其他组分。
13.如权利要求14的医药组合物，其中所述其他组分为镇痛剂(NSAID)、COX-2抑制剂或改善疾病的抗风湿药(DMARD)。
14.一种用于治疗自体免疫及免疫调节障碍疾病的方法，其包含向个体投与有效量的本发明的复合物。
15.如权利要求19的方法，其中所述疾病为发炎性肠病、牛皮癣、化脓性汗腺炎及顽固性哮喘类风湿性关节炎、幼年型类风湿性关节炎及牛皮癣性关节炎、斑状牛皮癣或僵直性脊椎炎。
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