Background

Extracellular vesicles (EVs) are attractive candidate drug delivery systems due to their ability to functionally transport biological cargo to recipient cells. However, the apparent lack of target cell specificity of exogenously administered EVs limits their therapeutic applicability. In this study, we propose a novel method to equip EVs with targeting properties, in order to improve their interaction with tumour cells.

Methods

EV producing cells were transfected with vectors encoding for anti-epidermal growth factor receptor (EGFR) nanobodies, which served as targeting ligands for tumour cells, fused to glycosylphosphatidylinositol (GPI) anchor signal peptides derived from decay-accelerating factor (DAF). EVs were isolated using ultrafiltration/size-exclusion liquid chromatography and characterized using western blotting, Nanoparticle Tracking Analysis, and electron microscopy. EV–tumour cell interactions were analyzed under static conditions using flow cytometry and under flow conditions using a live-cell fluorescence microscopy-coupled perfusion system.

Results

V analysis showed that GPI-linked nanobodies were successfully displayed on EV surfaces and were highly enriched in EVs compared with parent cells. Display of GPI-linked nanobodies on EVs did not alter general EV characteristics (i.e. morphology, size distribution and protein marker expression), but greatly improved EV binding to tumour cells dependent on EGFR density under static conditions. Moreover, nanobody-displaying EVs showed a significantly improved cell association to EGFR-expressing tumour cells under flow conditions.

Conclusions

We show that nanobodies can be anchored on the surface of EVs via GPI, which alters their cell targeting behaviour. Furthermore, this study highlights GPI-anchoring as a new tool in the EV toolbox, which may be applied for EV display of a variety of proteins, such as antibodies, reporter proteins and signaling molecules.

中文翻译：

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在细胞外囊泡上展示 GPI 锚定的抗 EGFR 纳米抗体可促进肿瘤细胞靶向。

背景

细胞外囊泡 (EVs) 是有吸引力的候选药物递送系统，因为它们能够将生物货物功能性地运输到受体细胞。然而，外源性 EV 明显缺乏靶细胞特异性限制了它们的治疗适用性。在这项研究中，我们提出了一种为 EV 配备靶向特性的新方法，以改善它们与肿瘤细胞的相互作用。

方法

用编码抗表皮生长因子受体 (EGFR) 纳米体的载体转染产生 EV 的细胞，该载体用作肿瘤细胞的靶向配体，与源自衰变加速因子 (DAF) 的糖基磷脂酰肌醇 (GPI) 锚定信号肽融合。使用超滤/尺寸排阻液相色谱分离 EV，并使用蛋白质印迹、纳米粒子追踪分析和电子显微镜对其进行表征。使用流式细胞仪在静态条件下和使用活细胞荧光显微镜耦合灌注系统在流动条件下分析 EV-肿瘤细胞相互作用。

结果

V 分析表明，与亲代细胞相比，GPI 连接的纳米抗体成功地展示在 EV 表面，并且在 EV 中高度富集。在 EV 上显示 GPI 连接的纳米抗体并未改变一般 EV 特征（即形态、大小分布和蛋白质标记表达），但在静态条件下极大地改善了 EV 与依赖于 EGFR 密度的肿瘤细胞的结合。此外，在流动条件下，显示纳米体的 EV 与表达 EGFR 的肿瘤细胞的细胞结合显着改善。

结论

我们表明，纳米抗体可以通过 GPI 锚定在 EV 的表面，这会改变它们的细胞靶向行为。此外，本研究强调 GPI 锚定作为 EV 工具箱中的一种新工具，可用于 EV 展示各种蛋白质，如抗体、报告蛋白和信号分子。