Simultaneous Targeting of Tumor Cells and Tumor-Associated Macrophages To Reprogram Glioblastoma Using Trypsinized Extracellular Vesicles Carrying Tumor Suppressive MicroRNA.

Glioblastoma (GBM) remains difficult to treat due to poor drug delivery across the blood-brain barrier and an immunosuppressive tumor microenvironment (TME). Tumor-suppressive microRNAs (miRNAs) offer a promising strategy to reprogram both tumor cells and the TME, but inefficient delivery systems limit their clinical application. We previously reported that tumor-suppressive miR-138 regresses tumor growth in preclinical GBM models. Here, we demonstrate that trypsin digestion of extracellular vesicles (EVs) enhances labeling efficiency with folate (FA), enhancing selective targeting of folate receptor (FR)-positive GBM cells and enabling simultaneous targeting of tumor-associated macrophages (TAMs). FA-labeled trypsinized EVs (tEVs) loaded with miR-138 inhibit tumor growth, depolarize TAMs, and enhance antitumor immunity. This study represents the first preclinical attempt to modulate tumor cells and innate immunity via miRNA-loaded tEVs, offering a novel and more effective therapeutic approach to GBM treatment.