Abstract Synergistic effect of combined antibodies targeting distinct epitopes of a particular tumour antigen has encouraged some clinical trial studies and is now considered as an effective platform for cancer therapy. Providing several advantages over conventional antibodies, variable domain of heavy chain of heavy chain antibodies (VHH) is now major tools in diagnostic and therapeutic applications. Active targeting of liposomal drugs is a promising strategy, resulting in enhanced binding and improved cytotoxicity of tumour cells. In the present study, we produced four anti-HER2 recombinant VHHs and purified them via native and refolding method. ELISA and flow cytometry analysis confirmed almost identical function of VHHs in refolded and native states. Using a mixture of four purified VHHs, PEGylated liposomal doxorubicin was targeted against HER2-overexpressing cells. The drug release was analyzed at pH 7.4, 6.4 and 5.5 and dynamic light-scattering detector and TEM micrograph was applied to characterize the produced nanoparticles. The binding efficiency of these nanoparticles to BT474 and SKBR3 as HER2-positive and MCF10A as HER2-negative cell line was examined by flow cytometry. Our results indicated effective encapsulation of about 94% of the total drug in immunoliposomes. Flow cytometry results verified receptor-specific binding of targeted liposomes to SKBR3 and BT474 cell lines and more efficient binding was observed for liposomes conjugated with oligoclonal VHHs mixture compared with monoclonal VHH-targeted liposomes. Oligoclonal nanoparticles also showed more cytotoxicity compared with non-targeted liposomes against HER2-positive tumour cells. Oligoclonal targeting of liposomes was represented as a promising strategy for the treatment of HER2-overexpressing breast cancers.

中文翻译：

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通过寡克隆HER2靶向递送脂质体阿霉素有效抑制肿瘤细胞

摘要 针对特定肿瘤抗原的不同表位的联合抗体的协同效应鼓励了一些临床试验研究，现在被认为是癌症治疗的有效平台。重链抗体重链可变结构域 (VHH) 提供优于传统抗体的几个优势，现在是诊断和治疗应用中的主要工具。脂质体药物的主动靶向是一种很有前景的策略，可增强肿瘤细胞的结合并提高细胞毒性。在本研究中，我们生产了四种抗 HER2 重组 VHH，并通过天然和重折叠方法对其进行了纯化。ELISA 和流式细胞术分析证实了 VHH 在重折叠和天然状态下的功能几乎相同。使用四种纯化 VHH 的混合物，聚乙二醇化脂质体阿霉素靶向过度表达 HER2 的细胞。在 pH 7.4、6.4 和 5.5 下分析药物释放，并应用动态光散射检测器和 TEM 显微照片来表征产生的纳米颗粒。通过流式细胞术检查这些纳米颗粒与 BT474 和 SKBR3 作为 HER2 阳性细胞系和 MCF10A 作为 HER2 阴性细胞系的结合效率。我们的结果表明免疫脂质体中约 94% 的总药物有效包封。流式细胞术结果证实了靶向脂质体与 SKBR3 和 BT474 细胞系的受体特异性结合，与单克隆 VHH 靶向脂质体相比，与寡克隆 VHH 混合物缀合的脂质体观察到更有效的结合。与非靶向脂质体相比，寡克隆纳米粒子对 HER2 阳性肿瘤细胞也表现出更高的细胞毒性。脂质体的寡克隆靶向被认为是治疗 HER2 过表达乳腺癌的一种有前途的策略。