Dual-functional bacterial extracellular vesicles with therapeutic oligonucleotides for synergistic treatment of ischemia/reperfusion injury in the brain

INTRODUCTION: Ischemia/reperfusion (I/R) injury refers to secondary damage that occurs following ischemic stroke when reperfusion is achieved using thrombolytic agents. The abrupt restoration of blood flow induces excessive production of reactive oxygen species (ROS), inflammatory cytokines, and infiltration of immune cells, resulting in additional tissue injury. Although the anti-inflammatory effects of LpEVs have been researched, their potential as gene delivery vehicles has not been investigated. OBJECTIVES: LpEV, which possesses anti-inflammatory properties and a lipid membrane structure, has the potential to be utilized as a gene delivery vehicle. Our study focuses on whether LpEVs act as delivery vehicles for anti-miRNA-181a oligonucleotides (AMO181a) and exert therapeutic effects on I/R injury. METHODS: LpEVs were isolated by PEG precipitation method. Physical characterization of LpEVs was performed by dynamic light scattering. In vitro cytokine assays were performed to evaluate the anti-inflammatory effect. For delivery of AMO181a, cholesterol-conjugated AMO181a (AMO181a-chol) was loaded into the LpEVs by hydrophobic interaction. Delivery efficiency of AMO181a was evaluated in Neuro2A cells by flow cytometry. The therapeutic effects of LpEVs and AMO181a were measured in the ischemia/reperfusion animal models. RESULTS: Anti-inflammatory effects of LpEVs were confirmed in the activated macrophages in vitro, reducing pro-inflammatory cytokines. Also, LpEVs decreased the infarct volume in an I/R animal model. Delivery efficiency of AMO181a-chol by LpEVs was higher than that of naked AMO181a-chol and comparable to the AMO181a-chol/polyethylenimine (25 kDa, PEI25k) complex in vitro and in vivo. Furthermore, LpEVs did not induce cytotoxicity. As a result, AMO181a-chol-loaded LpEVs had higher therapeutic effects than controls including LpEV alone, naked AMO181a-chol, and AMO181a-chol/PEI25k. CONCLUSION: LpEVs may be useful for the treatment of ischemia/reperfusion injury with dual functions of its own anti-inflammatory effects and for delivery of therapeutic oligonucleotides.