Acidic pH‐Triggered Membrane Fusion Enables Lyophilized Lipid Nanoparticles as Potent Cryoprotectants for Human Erythrocytes

Abstract The cryopreservation of human red blood cells (RBCs) is essential for maintaining a stable blood supply, but current glycerol‐based methods are hindered by complex and time‐consuming post‐thaw processing. While trehalose is a promising biocompatible cryoprotectant, its efficient intracellular delivery into non‐endocytic RBCs remains a major hurdle. Here, a lyophilized, ionizable lipid nanoparticle (LNP) platform is reported that overcomes this challenge via an acidic pH‐triggered membrane fusion mechanism. This approach, inspired by mRNA vaccine technology, enables rapid (<30 min) and efficient intracellular delivery of trehalose. Systematic optimization of the LNP composition and process parameters resulted in a remarkable post‐thaw RBC recovery rate of 85%, an efficacy comparable to the clinical glycerol standard but achieved with a simple, single‐wash protocol. The efficacy depends on the specific lipid synergy to enable pH‐responsive fusion. Critically, the lyophilized LNP powder retains full functionality, ensuring long‐term stability and simplifying storage and transportation logistics. This study establishes a clinically relevant, translation‐ready cryopreservation strategy that bridges nanotechnology and transfusion medicine, offering a next‐generation solution to longstanding challenges in cell preservation.