Doxorubicin (DOX) is widely used in chemotherapy, yet it significantly contributes to heart failure-associated death. Acetylcholine (ACh) is cardioprotective by enhancing heart rate variability and reducing mitochondrial dysfunction and inflammation. Nonetheless, the protective role of ACh in countering DOX-induced cardiotoxicity (DIC) remains underexplored as current approaches to increasing ACh levels are invasive and unsafe for patients. In this study, we explore the protective effects of ACh against DIC through three distinct ACh administration strategies: i) freely-suspended 100µM ACh
ii) ACh-producing cholinergic neurons (CNs)
or iii) ACh-loaded nanoparticles (ACh-NPs). These are tested in