Proximity Proteomics Maps Candidate Cellular Uptake Pathways for Cell-Derived Nanovesicles.

Although extracellular vesicles (EVs) facilitate selective molecular exchange between cells, their low yields and inherent heterogeneity limit translational applications. Cell-derived nanovesicles (CDNs), produced by mechanical extrusion of donor cells, offer a scalable alternative while retaining key membrane features of EVs. Yet how fabrication reshapes vesicle-cell communication remains poorly understood. Here, vesicle-side proteomics is integrated with TurboID proximity labeling of recipient-cell proteins to construct quantitative, dual-sided maps of CDN and small extracellular vesicle (sEV) interactions. Despite similar size and surface charge, CDNs displayed a substantially more diverse proteome and a broader repertoire of predicted uptake-associated signatures. Recipient-cell proximity proteomics further resolved distinct molecular cohorts. The CDN-associated cohort contained recipient-derived CALR and NCL, which are literature-linked to LRP1/CD91-associated efferocytic recognition and NCL-associated macropinocytic processes, respectively, whereas the sEV-associated cohort contained signatures consistent with HSPG-assisted docking and clathrin-mediated or CLIC/GEEC-related uptake. Together, these complementary datasets reveal molecular interfaces for extrusion-generated CDNs that are distinct from those of naturally secreted sEVs. Receptor dependence and the contributions of surface association and internalization require direct testing; however, the identified associations define specific mechanistic targets for further investigation. This dual-sided proteomic strategy establishes a quantitative framework for dissecting vesicle-cell communication and engineering membrane-based nanocarriers.