Bioorthogonal Catalytic Microvesicle-Mediated Prodrug Activation Against Liver Cancer

Objective Exploring the role and mechanism of a novel bioorthogonal system using transition metals as catalysts in the treatment of hepatocellular carcinoma (HCC). Methods Initially, a catalytic ruthenium (Ru) complex and the substrate alloc-RH 110 were synthesized, followed by the identification of their structures utilizing mass spectrometry and nuclear magnetic resonance (NMR) techniques. The catalytic efficacy of the Ru complex was then assessed using a fluorescence spectrophotometer. Subsequently, employing HepG2 cells as the cellular source, cell-derived vesicles encapsulating the Ru complexes, designated as EVs@Ru, were prepared. The EVs@Ru were characterized by measuring their particle size and Zeta potential, observing morphological features under transmission electron microscopy (TEM), and detecting specific protein expressions via Western blot analysis. Drug loading within the EVs@Ru was quantified using inductively coupled plasma mass spectrometry (ICP-MS), and their catalytic efficiency was evaluated. In vitro, the low-activity prodrug alloc-DOX was synthesized and its toxicity, along with the drug concentration in EVs@Ru, was determined. Further, the catalytic cytotoxicity of alloc-DOX against HepG2 cells encapsulated in EVs@Ru was analyzed through microscopic observation, CCK-8 assays, and apoptosis experiments. For in vivo studies, a tumor-bearing mouse model was established using human liver cancer HepG2 cells to observe the antitumor effects. Finally, the primary organs of each group of tumor-bearing mice were assessed for in vivo safety. Results ESI-MS and 1H NMR confirmed the accurate structure of Ru complexes and alloc-RH 110. The Ru complexes achieved full catalytic conversion of alloc-RH 110 within 24 hours. EVs and EVs@Ru exhibited particle sizes of ∼116.85 nm and ∼281.88 nm, respectively, with Zeta potentials of ∼-20.86 mV and ∼-25.89 mV, both appearing quasi-circular under TEM. WB analysis verified the presence of vesicle-specific marker proteins in both, confirming their cell-derived nature. ICP-MS determined a drug loading of 21.90 μg/mL for EVs@Ru, with an encapsulation efficiency of ∼24.86%. Fluorescence spectrophotometry demonstrated 100% catalytic efficiency for EVs@Ru. Synthetic alloc-DOX validated by 1H NMR and ESI-MS matched literature data. MTT and CCK-8 assays confirmed low toxicity for alloc-DOX and Ru complexes, setting the experimental drug concentration at 4μM. In vitro, the EVs@Ru+alloc-DOX group exhibited potent HepG2 cell killing and apoptosis. In vivo, this group significantly inhibited tumor growth in tumor-bearing mice, with no observed toxicity to vital organs, indicating good biosafety. Conclusion The integration of bio-derived microvesicles (MVs) with transition metal catalysts has resulted in a biologically orthogonal system for efficient Ru complex delivery to tumor sites. This system facilitates controlled release of the Ru complexes, enabling tumor cell elimination. This innovative strategy holds great promise for enhancing tumor immunity and targeted therapeutic approaches.