Biocompatible multifunctional nanomedicines (NMs) are known to be an attractive platform for targeted anticancer theranosis. However, these nanomedicines are of interest only if they efficiently target diseased cells and accumulate in tumors. Here we report the synthesis of a new generation of immunotargeted nanomedicines composed of a superparamagnetic iron oxide nanoparticle (SPION) core, polyethylene glycol coating and the anti-HER2 single chain fragment variable (scFv) of Trastuzumab antibody. We developed two novel bioengineered scFv carrying two cysteines located (i) at the end (4D5.1-cys₂) or (ii) at the beginning (4D5.2-cys₂) of its hexahistidine tag. The scFv bioconjugation was controlled via heterobifunctional linkers including a second generation maleimide (SGM). Our data indicated that the insertion of cysteines at the beginning of the hexahistidine tag was allowed to obtain nearly 2-fold conjugation efficiency (13 scFv/NP) compared to NMs using classical maleimide. As a result, the NMs-4D5.2 built using the optimal 4D5-cys₂ and linkers equipped with SGM showed the enhanced recognition of HER2 in an ELISA format and on the surface of SK-BR-3 breast cancer cells in vitro. Their stability in serum was also significantly improved compared to the NMs-4D5. Our results showed the fundamental importance of the controlled ligand conjugation in the perspective of rational design of NMs with tailored physicochemical and biological properties.

中文翻译：

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第二代顺丁烯二酰亚胺对ScFv特定位点缀合至多功能纳米药物的影响

已知生物相容性多功能纳米药物（NMs）是靶向抗癌治疗的有吸引力的平台。然而，仅当这些纳米药物有效地靶向病变细胞并在肿瘤中积累时，它们才是令人关注的。在这里，我们报告了由超顺磁性氧化铁纳米粒子（SPION）核心，聚乙二醇涂层和曲妥珠单抗的抗HER2单链片段变量（scFv）组成的新一代免疫靶向纳米药物的合成。我们开发了两种新型的生物工程化的scFv，它们携带了两个位于（i）末端（4D5.1-cys ₂）或（ii）开头（4D5.2-cys _2）的半胱氨酸。）的六组氨酸标签。通过包括第二代马来酰亚胺（SGM）的异双功能接头控制scFv生物缀合。我们的数据表明，与使用经典马来酰亚胺的NM相比，半胱氨酸在六组氨酸标签开始处的插入允许获得接近2倍的缀合效率（13 scFv / NP）。结果，使用最佳4D5-cys ₂构建的NMs-4D5.2带有SGM的接头在ELISA形式和体外SK-BR-3乳腺癌细胞表面显示出对HER2的增强识别。与NMs-4D5相比，它们在血清中的稳定性也显着提高。我们的结果表明，从合理设计具有定制理化和生物学特性的NMs的角度来看，受控配体结合的根本重要性。