Fenretinide-loaded Mesenchymal Stromal Cell-derived Extracellular Vesicles as a Potential Drug Carrier to Treat Human Neuroblastoma

Neuroblastoma (NB) is the most common and aggressive extracranial solid tumor of pediatric age. Despite advances, about 50% of high-risk NB patients relapse, necessitating the development of new therapies. Fenretinide (4-HPR), a synthetic retinoid, has shown cytotoxic activity against cancer cells but its clinical effectiveness is hampered by its poor bioavailability. Here, extracellular vesicles (EVs) derived from umbilical cord Mesenchymal Stromal Cells (UC-MSCs) were proposed as a novel drug delivery system for 4-HPR. MSC-EVs loaded with 4-HPR (4-HPR-MSC-EVs) were obtained via passive loading, and isolated from the conditioned medium by ultracentrifugation. Nanoparticle tracking analysis revealed that 4-HPR-MSC-EVs had a concentration of about 1.53x10 11 particles/mL with sizes below 200 nm. The identity of MSC-EVs was confirmed by the presence of tetraspanin markers and specific MSC-EVs markers (CD29, CD44, CD49e, CD105), whereas markers of CD14, CD19, CD45, MHC class I and II were absent. High-performance liquid chromatography detected a 4-HPR concentration of 5.90 ± 0.21 μM. The binding of MSC-EVs to NB cells was demonstrated after their labelling with CFSE. 4-HPR-MSC-EVs induced a greater time-dependent reduction of NB cell viability both in 2D and 3D cell cultures. In response to 4-HPR-MSC-EVs NB cell lines underwent apoptotic cell death, as determined by Annexin-V assay, to a greater extent if compared to free 4-HPR. Our findings demonstrate that 4-HPR-MSC-EVs are a suitable and effective system to efficiently deliver the drug to NB cells, thus opening the door for further pre-clinical investigations to support a future potential clinical application. • Neuroblastoma is the most aggressive extracranial solid tumor of pediatric age • Fenretinide is a retinoid with poor bioavailability but with anticancer activity • Extracellular vesicles from Mesenchymal Stromal Cells as drug delivery system • Extracellular vesicles loaded with fenretinide