白蛋白融合或结合是一种已经建立起来的肿瘤递送技术，主要是通过白蛋白诱导的洞穴内吞作用来介导的。我们报告说，由人血清白蛋白（HSA）激活的洞穴依赖内吞信号不足以诱导细胞摄取，主要是由于白蛋白与带负电荷的细胞表面硫酸化糖胺聚糖（GAGs）之间的静电相互斥作用。而将细胞表面保留的蛋白与HSA融合是一种有效的策略，可以激活HSA诱导的内吞信号，从而提高其细胞内摄取。在这项研究中，我们选择了人乳铁蛋白（hLF），这是一种与GAGs一起积累在细胞表面的蛋白质，用于递送到人肺腺癌PC-14细胞中。当外源添加hLF融合的HSA（hLF-HSA）时，成功地发生内吞，而同时添加HSA和hLF则无法引发内吞，这表明仅通过HSA激活内吞信号的效率较低，并且说明其融合对于肿瘤递送的重要性。重要的是，已知的GAG硫酸化抑制剂氯酸盐处理细胞显著抑制了hLF-HSA的内吞，因为hLF-GAG相互作用的丧失。因此，融合与细胞表面保留蛋白质的细胞表面定位增强了其与相关受体的结合，这提高了作为白蛋白融合平台的细胞内递送。opyright © 2023. Published by Elsevier B.V.

Albumin fusion or conjugation is a well-established technique for tumor delivery and is mainly mediated by albumin-induced caveolae-dependent endocytosis. We report that caveolae-dependent endocytic signaling activated by human serum albumin (HSA) is not sufficiently strong to induce cellular uptake, mainly due to its electrostatic repulsion from the negatively charged cell surface sulfated glycosaminoglycans (GAGs), and fusion of the cell-surface-retained protein with HSA is an effective strategy to activate the HSA-induced endocytic signal, thereby improving its intracellular uptake. In this study, human lactoferrin (hLF), a protein that accumulates on the cell surface along with GAGs, was selected for delivery into human lung adenocarcinoma PC-14 cells. When added exogenously, hLF-fused HSA (hLF-HSA) was successfully endocytosed, whereas the simultaneous addition of HSA and hLF did not result in endocytosis, indicating less efficient activation of endocytic signaling by HSA alone and the importance of its fusion. Importantly, the treatment of cells with chlorate, a known inhibitor of GAG sulfation, dramatically suppressed the endocytosis of hLF-HSA owing to the loss of the hLF-GAG interaction. Therefore, the cell-surface localization of HSA imposed by fusion with the cell-surface-retained protein enhances its binding to the relevant receptor, which improves intracellular delivery as an albumin-fusion platform.Copyright © 2023. Published by Elsevier B.V.