Stabilization of erythrocyte membrane coating on nanoparticle via strong substrate interaction confers enhanced anti-α-hemolysin immunity

The biomedical performance of cell membrane-coated nanoparticles is dependent on membrane coating stability. The canonical sonication-based coating method uses weak non-covalent substrate interactions to stabilize membrane coating, a mechanism that fails under strong physical/biological perturbations, leading to membrane detachment and loss of function. Here, we developed an "equilibrated incubation" method that creates stable erythrocyte membrane coatings on gold nanoparticles (AuNP@RBCM) via strong covalent membrane-substrate interactions. Acetylcholinesterase assays, glycocalyx characterization, and lipidomics indicated that the new method achieves intact and functional membrane coating. Linear stability analysis revealed that the increased stiffness physically counters negative membrane tension generated by shear stress and α-hemolysin (α-HL) insertion, preventing membrane detachment and nanoparticle aggregation, which was observed for the sonication-derived control. As an anti-α-HL vaccine, the α-HL-loaded AuNP@RBCM prepared via equilibrated incubation enhanced α-HL antigen delivery to antigen-presenting cells, yielding higher antibody titers and a higher lesion-free rate than the sonication-prepared controls in α-HL-challenged mice.