Fusogenic Liposomes for the Intracellular Delivery of Phosphocreatine

Background/Objective: Maintaining intracellular adenosine triphosphate (ATP) levels is essential for numerous cellular functions, including energy metabolism, muscle contraction, and nerve impulse transmission. ATP is primarily synthesized in mitochondria through oxidative phosphorylation. It is also generated in the cytosol under anaerobic conditions using phosphocreatine (PCr) as a phosphate donor to adenosine diphosphate. However, the intracellular delivery of exogenous PCr is challenging as it does not readily cross the plasma membrane. This complicates the use of PCr as a therapeutic agent to maintain energy homeostasis or to treat conditions like cerebral creatine deficiency syndrome (CDS), which results from defective creatine transporters. Methods: This study employs the use of fusogenic liposomes to deliver PCr directly into the cytosol, bypassing membrane impermeability issues. We engineered various 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-based fusogenic liposomes, incorporating phospholipids such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in combination with phospholipid-aromatic dye components to facilitate membrane fusion and to enhance the delivery of the PCr cargo. Liposomal formulations were co-loaded with membrane-impermeable chromophores and PCr and studied on live cells using confocal microscopy. Conclusions: We demonstrated the successful intracellular delivery of these agents and observed a 23% increase in intracellular ATP levels in cells treated with PCr-loaded liposomes. This increase was not observed with free PCr, confirming the effectiveness of the liposome-based delivery system. Additionally, cell viability assays showed minimal toxicity from the liposomes. Our results indicate that fusogenic liposomes are a promising method for the delivery of PCr (and potentially other cell-impermeable therapeutic agents) to the cellular cytosol. The approach demonstrated here could be advantageous for treating energy-related disorders and improving cellular energy homeostasis.