Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer, lacking targeted therapeutic options. Hydrogels, particularly gelatin methacrylate (GelMA), have emerged as promising materials for localized drug delivery due to their biocompatibility and tunable properties. This study investigates a dual-delivery system for enhancing the treatment efficacy of triple-negative breast cancer (TNBC) using a combination of extracellular vesicles (EVs) derived from Citrus limon L. and the chemotherapeutic drug doxorubicin (DOX). We fabricated 3D bioprinted GelMA scaffolds to achieve localized and controlled release of EVs and evaluated their synergistic effects with systemic DOX delivery on both primary and metastatic 3D TNBC models.

The GelMA scaffolds, especially those with 95 % methacrylation, exhibited higher stiffness, which enhanced their sustained release. Following 48-h incubation, the combination of EVs and DOX significantly increased cytotoxicity in the primary 3D TNBC model, reducing cell viability to approximately 30 % compared to controls. This was notably more effective than treatments with DOX or EVs alone. During the extended 7-day incubation period, the combination treatment continued to show superior efficacy, with persistently high levels of ROS generation and further reduction in cell viability. In a metastatic 3D TNBC model, a significant sensitivity to the combined treatment was observed, which notably inhibited aggregate formation and migration. Importantly, EVs-embedded scaffolds promoted the proliferation of human fibroblasts, highlighting their non-toxic nature, while concurrently inhibiting TNBC cell growth. This approach provides a promising strategy to improve the treatment outcomes of TNBC by exploiting the synergistic effects of local EVs release and systemic chemotherapy.