Brain endothelial cells (BECs) hinder macromolecules from reaching brain parenchyma, necessitating the evaluation and engineering of therapeutic immunoglobulin γ (IgG) for improved brain delivery. Emerging fluorescent-based approaches to assess IgG brain exposure can expedite and complement current methods; however, alterations in IgG pharmacokinetics following fluorophore conjugation, which remain unexplained, indicate that conjugation may confound analysis of native IgG processing. Here, changes in transcytosis and intracellular processing of IgG conjugates (with sulfonated cyanine 5) were examined using human induced pluripotent stem cell-derived BECs (iBECs). Above a critical degree of labeling, transcytosis rates increased significantly but could be attenuated by nonspecific protein competition. Concurrent increases in intracellular accumulation, which was not attributable to disrupted binding by the neonatal Fc receptor (FcRn), are indicative of indirect reduction of FcRn-mediated recycling that agrees with reported aberrations in the pharmacokinetics of certain unconjugated IgGs. Overall, these findings support the notion that certain fluorophore–IgG conjugates can engage in adsorptive interactions with cell surface moieties, reminiscent of phenomena exhibited by cationized IgG, and provide in vitro criteria to identify changes in IgG processing following fluorophore conjugation.

中文翻译：

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Sulfo-Cy5 标记的 IgG 的吸附介导的内吞作用导致脑内皮样细胞异常的 IgG 加工

脑内皮细胞 (BEC) 会阻碍大分子到达脑实质，因此需要对治疗性免疫球蛋白 γ (IgG) 进行评估和工程化，以改善大脑递送。新兴的基于荧光的评估 IgG 脑暴露的方法可以加速和补充当前的方法；然而，荧光团缀合后 IgG 药代动力学的改变，仍然无法解释，表明缀合可能会混淆对天然 IgG 加工的分析。在这里，使用人诱导多能干细胞衍生的 BEC (iBEC) 检查了 IgG 偶联物（含磺化花青 5）的转胞吞作用和细胞内加工的变化。超过标记的临界程度，转胞吞率显着增加，但可能会因非特异性蛋白质竞争而减弱。细胞内积累的同时增加，这不是由于新生儿 Fc 受体 (FcRn) 的结合中断，表明 FcRn 介导的再循环间接减少，这与报道的某些未结合 IgG 的药代动力学异常一致。总体而言，这些发现支持某些荧光团 - IgG 偶联物可以与细胞表面部分发生吸附相互作用的观点，让人联想到阳离子化 IgG 所表现出的现象，并提供用于识别荧光团缀合后 IgG 加工变化的体外标准。