Tumor-targeted CD40 ligand-functionalized extracellular vesicle mimetics enable chemoimmunotherapy for neuroblastoma.

Abstract

Neuroblastoma remains one of the most lethal pediatric solid tumors, and durable control of high-risk disease is hindered not only by inefficient tumor-selective drug delivery but also by a profoundly immunosuppressive tumor microenvironment. To address these dual barriers, we developed a chemoimmunotherapeutic vesicle-mimetic platform, DAS/CD40L-EM@DOX, by engineering CD40L-overexpressing HEK-293 T donor cells, generating extracellular vesicle mimetics through extrusion, decorating the membrane with a neuroblastoma-targeting DAS peptide, and post-loading doxorubicin. The resulting formulation preserved a nanoscale vesicular morphology, displayed CD40L on the membrane, and showed favorable particle size and zeta potential characteristics. In neuroblastoma cells, DAS decoration enhanced uptake in an α7 nicotinic acetylcholine receptor-associated manner and increased the cytotoxic and immunogenic effects of doxorubicin, as evidenced by augmented apoptosis, calreticulin exposure, and HMGB1 release. In macrophage assays, CD40L-containing vesicles shifted M2-like cells toward an M1-like phenotype, increasing CD86, TNF-α and IL-6 while decreasing CD206, IL-10 and TGF-β. Functionally, this repolarization enhanced tumor-cell phagocytosis and promoted CD8+ T-cell effector responses, including increased granzyme B, IFN-γ and IL-2 production. In tumor-bearing mice, DAS modification improved tumor accumulation and reduced off-target sequestration relative to unmodified vesicles. Therapeutically, DAS/CD40L-EM@DOX exerted the strongest inhibition of tumor growth, reduced tumor burden, prolonged survival, increased intratumoural M1-like macrophages and CD8+ T cells, decreased M2-like macrophages and regulatory T cells, and enhanced immunogenic cell-death markers in situ. Together, these findings support DAS/CD40L-EM@DOX as a dual-function vesicle-mimetic nanomedicine that couples neuroblastoma-targeted chemotherapy with macrophage reprogramming to remodel the tumor immune microenvironment and improve antitumour efficacy.