Engineered ATP-Loaded Extracellular Vesicles Derived from Mesenchymal Stromal Cells: A Novel Strategy to Counteract Cell ATP Depletion in an In Vitro Model

The use of adenosine triphosphate (ATP) has shown promising effects in alleviating ischemic damage across various tissues. However, the penetration of ATP into kidney tubular cells presents a challenge due to their unique anatomical and physiological properties. In this study, we introduce a novel bioinspired drug delivery system utilizing extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) and engineered to carry ATP. ATP-loaded liposomes (ATP-LPs) and ATP-loaded EVs (ATP-EVs) were prepared using microfluidic technology, followed by characterization of their morphology (DLS, NTA, SEM, TEM), ATP content, and release rate at 37 °C (pH 7.4). Additionally, the delivery efficacy of ATP-LPs and ATP-EVs was evaluated in vitro on renal cells (HK2 cells) under chemically induced ischemia. The results indicated successful ATP enrichment in EVs, with ATP-EVs showing no significant changes in morphology or size compared to naïve EVs. Notably, ATP-EVs demonstrated superior ATP retention compared to ATP-LPs, protecting the ATP from degradation in the extracellular environment. In an ATP-depleted HK2 cell model, only ATP-EVs effectively restored ATP levels, preserving cell viability and reducing apoptotic gene expression (BCL2-BAX). This study is the first to successfully demonstrate the direct delivery of ATP into renal tubular cells in vitro using EVs as carriers.