细胞外囊泡（EV）是细胞分泌的颗粒，被认为是细胞间通讯的天然载体。通过 EV 功能化捕获异质分子货物并靶向特定细胞群的能力有望在生物医学应用中取得进步。然而，获得的 EV 的效率、细胞暴露受体对 EV 相互作用的贡献，以及功能性货物释放与高分子量重组 mRNA 潜在共享的可预测性，对于在靶向治疗应用中推进异源 EV 至关重要。 ，我们选择流行的 EV 标记 CD81 作为融合蛋白的跨膜引导，其 C 端 GFP 报告基因包含或不包含针对 HER2 受体的曲妥珠单抗轻链。我们进行了高内涵成像分析来追踪 EV 细胞相互作用，包括具有受控 HER2 表达的同基因乳腺癌细胞。我们验证了携带阿霉素的重组 EV 在 EV 供体细胞处理后的功能性货物递送。然后，我们使用通常用作 HER2 难治性、曲妥珠单抗耐药模型的 JIMT-1 细胞进行了一项体内研究，以检测移植肿瘤中长度超过 2000 nt 的重组 mRNA。融合蛋白参与囊泡运输动力学并在分泌的 EV 上积累根据它们在 HEK293T 细胞中的表达水平。尽管存在 GFP，分泌的 EV 群体保留了 HER2 受体结合能力，并用于追踪 EV 细胞相互作用。在 HER2 阳性 (SK-BR-3) 或阴性 (MDA-MB-231) 乳腺癌细胞系之间整体 EV 分布没有变化的时间范围内，同基因细胞中的 HER2 暴露显着影响异源细胞的向性EV，展示了约 20% 的分泌性大囊泡的抗 HER2 EV 的特异性。这种特异性相互作用与异位表达 HER2 的 MDA-MB-231 中阿霉素-EV 细胞杀伤活性的提高以及敲除 HER2 受体的 SK-BR-3 中的毒性降低、克服游离药物的影响密切相关。有趣的是，重组 EV 中以全长 mRNA 形式呈现的融合蛋白相应转录物可以到达 JIMT-1 异种移植小鼠的原位乳腺肿瘤，从而提高我们在组织活检中检测渗透性货物的灵敏度。这项研究强调了创建背后的定量方面分泌型异源 EV 平台的研究，并显示了 EV 细胞接合机制背后的单一受体-配体相互作用的局限性，该机制现已成为预测功能向性和设计新一代基于 EV 的纳米车辆的关键步骤。© 2024。作者（ s）。

Extracellular vesicles (EVs) are cell-secreted particles conceived as natural vehicles for intercellular communication. The capacity to entrap heterogeneous molecular cargoes and target specific cell populations through EV functionalization promises advancements in biomedical applications. However, the efficiency of the obtained EVs, the contribution of cell-exposed receptors to EV interactions, and the predictability of functional cargo release with potential sharing of high molecular weight recombinant mRNAs are crucial for advancing heterologous EVs in targeted therapy applications.In this work, we selected the popular EV marker CD81 as a transmembrane guide for fusion proteins with a C-terminal GFP reporter encompassing or not Trastuzumab light chains targeting the HER2 receptor. We performed high-content imaging analyses to track EV-cell interactions, including isogenic breast cancer cells with manipulated HER2 expression. We validated the functional cargo delivery of recombinant EVs carrying doxorubicin upon EV-donor cell treatment. Then, we performed an in vivo study using JIMT-1 cells commonly used as HER2-refractory, trastuzumab-resistant model to detect a more than 2000 nt length recombinant mRNA in engrafted tumors.Fusion proteins participated in vesicular trafficking dynamics and accumulated on secreted EVs according to their expression levels in HEK293T cells. Despite the presence of GFP, secreted EV populations retained a HER2 receptor-binding capacity and were used to track EV-cell interactions. In time-frames where the global EV distribution did not change between HER2-positive (SK-BR-3) or -negative (MDA-MB-231) breast cancer cell lines, the HER2 exposure in isogenic cells remarkably affected the tropism of heterologous EVs, demonstrating the specificity of antiHER2 EVs representing about 20% of secreted bulk vesicles. The specific interaction strongly correlated with improved cell-killing activity of doxorubicin-EVs in MDA-MB-231 ectopically expressing HER2 and reduced toxicity in SK-BR-3 with a knocked-out HER2 receptor, overcoming the effects of the free drug. Interestingly, the fusion protein-corresponding transcripts present as full-length mRNAs in recombinant EVs could reach orthotopic breast tumors in JIMT-1-xenografted mice, improving our sensitivity in detecting penetrant cargoes in tissue biopsies.This study highlights the quantitative aspects underlying the creation of a platform for secreted heterologous EVs and shows the limits of single receptor-ligand interactions behind EV-cell engagement mechanisms, which now become the pivotal step to predict functional tropism and design new generations of EV-based nanovehicles.© 2024. The Author(s).