Single-Particle STORM Imaging Quantifies Coating Heterogeneity Among Cell Membrane-Coated Nanoparticles

Cell membrane-coated nanoparticles (CNPs) are a potentially transformative biomimetic targeted-delivery platform, which can engage therapeutic targets through source-cell membrane protein functions or homotypic interactions. However, their therapeutic development is limited by the current inability to accurately resolve membrane coating completeness, and CNP particle-to-particle variation. Here, we introduce a STochastic Optical Reconstruction Microscopy (STORM)-based approach that resolves population composition and quantifies membrane coating coverage at the single-particle level. Applying it to dendritic cell membrane- and HeLa cell membrane-coated nanoparticles (DCmPs and HeLamPs), we revealed substantial heterogeneity within both particle types and a cell-type dependence of coating outcomes, corroborated by confocal imaging and flow cytometry. We also quantified membrane protein composition, including peptide major histocompatibility complex class I (pMHC-I) among DCmPs, and confirmed that DCmPs preferentially engaged antigen-specific T cells in coculture. This single-particle framework establishes a quantitative standard for CNP characterization, providing a foundation for quality control and rational design.