Plant-derived extracellular vesicles as a natural drug delivery platform for glioblastoma therapy: A dual role in preserving endothelial integrity while modulating the tumor microenvironment

Glioblastoma (GBM) is the most aggressive primary brain tumor, with limited treatment options due to the restrictive blood-brain barrier (BBB) and the heterogeneity of the blood-tumor barrier (BTB). Temozolomide (TMZ), the standard chemotherapy, suffers from poor BBB permeability, rapid degradation, and systemic toxicity. Plant-derived extracellular vesicles (PDEVs) have emerged as promising natural nanocarriers, offering biocompatibility, stability, and the ability to cross biological barriers. This study investigates the use of extracellular vesicles from <i>Citrus limon</i> L. (LDEs) to encapsulate and deliver TMZ (EVs@TMZ) for GBM treatment. LDEs were isolated, characterized, and loaded with TMZ via ultrasonication. Encapsulation efficiency, stability, and physicochemical properties were assessed using UV-Vis and FTIR spectroscopy. A 3D BTB model was developed using bioprinted U87 glioblastoma cells in Matrigel, co-cultured with hCMEC/D3 endothelial cells to replicate the tumor microenvironment. Barrier integrity was evaluated through TEER and FITC-dextran assays. Uptake, cytotoxicity, and tumor invasion were assessed in this model, along with oxidative stress and VEGF-A secretion. LDEs effectively encapsulated TMZ, enhancing drug stability under physiological conditions. EVs@TMZ crossed the endothelial barrier while preserving barrier integrity and reducing TMZ-induced ROS production. In the 3D glioblastoma model, EVs@TMZ showed strong cytotoxic effects on tumor cells while minimizing endothelial toxicity and oxidative stress. Moreover, VEGF-A secretion was suppressed, disrupting pro-tumorigenic pathways. These findings highlight Citrus-derived EVs as biocompatible, efficient carriers for TMZ delivery, offering a promising approach to overcome current challenges in GBM therapy and supporting further development of PDEVs for brain tumor treatment.