Single‐Particle Analysis Reveals Heterogeneity in Membrane Coating

Coating nanoparticles with cell membranes has emerged as a powerful strategy for developing biomimetic nanocarriers that replicate essential cellular features such as immune evasion, prolonged circulation, and site-selective distribution. Despite substantial efforts to optimize ligand presentation on cell membranes, achieving efficient, uniform, and scalable membrane coating remains challenging. Moreover, precise quantification of coating degree, particularly at the single-particle level to reveal inter-particle heterogeneity, has been lacking. Here, we establish Single-Particle Analysis of CEll MembrANe (SPACEMAN), a quantitative platform that enables accurate evaluation of coating integrity and heterogeneity with single-nanoparticle resolution. Using SPACEMAN, we demonstrate that a nano-vesiculation strategy based on ultrasonication achieves a significantly higher overall coating rate (82.2% of particles) compared with extrusion (68.0%) and sonication (61.0%), and yields a greater proportion of particles with high surface coverage (50%-75% and 75%-100% of surface area). Nanoparticles coated via this method exhibited superior immune evasion and prolonged circulation in both in vitro and in vivo models. Indeed, SPACEMAN offers a generalizable single-particle analytical framework for quantitatively probing heterogeneity in physicochemical properties, drug loading, and surface modifications of nanoparticle formulations.