Intense cardiac-targeted small extracellular vesicles-mediated delivery of RAGE siRNA attenuates inflammation in rat myocarditis model

Abstract

Abstract Background Despite advances in the field and new therapeutics being developed, heart disease remains the leading cause of mortality worldwide. Small extracellular vesicles (sEV) are 30–150 nm in diameter and ferry RNA and proteins to cells. sEV are a natural carrier of many signaling molecules and play an important role in heart disease. However, the high probability of off-target effects associated with these carriers is a major barrier to translation into clinical application. Purpose Our aim was to evaluate whether sEVs engineered to express potent cardiac targeting peptides (CTPs) could deliver siRNA to the heart and exert a therapeutic effect. Methods We use vectors encoding LAMP2B (sEV) or CTP-LAMP2B (PsEV) into HEK 293 cells expressing. sEVs were purified from culture media of HEK 293 cells by serial centrifugation followed by tangential flow filtration (TFF) system. sEV and PsEV were loaded with siRNAs by Exo-Fect™ exosome transfection reagent, and were treated into H9C2 rat cardiomyocyte. TNF-alpha were then added to the cells to induce inflammation. And sEVs were intravenously injected into myocarditis rat. Inflammation factors of in vitro and in vivo inflammation model were identified by western blot. Echocardiographic examination was also performed in rat. Results The successful development of PsEVs was analyzed by Western blot and TEM. We observed a 4 fold increase from that of the previously developed sEVs in H9C2 cells and a 200% increase in cardiac-specific delivery without toxicity in rat model. AGE is involved in proinflammatory/pro-apoptotic processes. To block RAGE, we loaded RAGE siRNA (siRAGE) in sEV that had high expression of PsEV. Characteristics of sEVs were maintained despite siRNA load.In inflammatory cell models and rat disease models, PsEV-treated groups significantly reduced molecular levels associated with inflammatory responses such as RAGE, IL-6, TNF-alpha, COX2, HMGB1, and p-p65 / p65. Conclusions Our results suggest that PsEV can potentially serve as a treatment delivery vehicle for heart disease. Scheme Funding Acknowledgement Type of funding source: None