Orange-Derived Extracellular Vesicles Labeled with MRI Gd-Containing Contrast Agents

Abstract Plant-derived extracellular vesicles (PEVs) are emerging as scalable, biocompatible nanocarriers for drug delivery; however, their biodistribution and in vivo behavior remain poorly understood. In this work, we describe an integrity-preserving MRI labeling approach for orange-derived extracellular vesicles (oEVs) employing macrocyclic Gd-based contrast agents Gd-CAs. A comparative analysis was performed using two probes: the clinically approved neutral complex Gd-HPDO3A and its amino-substituted derivative Gd-AHPDO3A. Passive loading under transient osmotic conditions enabled efficient encapsulation of both agents while preserving vesicle morphology and size, as confirmed by nanoparticle tracking analysis and TEM. Notably, Gd-AHPDO3A exhibited significantly higher loading efficiency, likely driven by electrostatic interactions with negatively charged membrane components, including phosphatidic acid. Relaxometric analysis revealed enhanced longitudinal relaxivity at low intravesicular Gd content, whereas higher loading levels led to relaxivity quenching due to restricted water exchange across the vesicle membrane. MRI phantom studies at 7 T demonstrated strong T1 contrast enhancement, with Gd-AHPDO3A-labeled oEVs achieving up to a 3-fold higher signal compared to the neutral analogue and a detection limit below ∼50 μM Gd. Importantly, labeled oEVs were efficiently internalized by HCT-116 colon cancer cells, producing a detectable MRI signal that correlated with intracellular Gd content.