The heterogeneity of exosomal populations has hindered our understanding of their biogenesis, molecular composition, biodistribution, and functions. By employing asymmetric-flow field-flow fractionation (AF4), we identified two exosome subpopulations (large exosome vesicles, Exo-L, 90-120 nm; small exosome vesicles, Exo-S, 60-80 nm) and discovered an abundant population of non-membranous nanoparticles termed “exomeres” (~35 nm). Exomere proteomic profiling revealed an enrichment in metabolic enzymes and hypoxia, microtubule and coagulation proteins and specific pathways, such as glycolysis and mTOR signaling. Exo-S and Exo-L contained proteins involved in endosomal function and secretion pathways, and mitotic spindle and IL-2/STAT5 signaling pathways, respectively. Exo-S, Exo-L, and exomeres each had unique N-glycosylation, protein, lipid, and DNA and RNA profiles and biophysical properties. These three nanoparticle subsets demonstrated diverse organ biodistribution patterns, suggesting distinct biological functions. This study demonstrates that AF4 can serve as an improved analytical tool for isolating and addressing the complexities of heterogeneous nanoparticle subpopulations.

In nature, biological nanomaterials are synthesized under ambient conditions in a natural microscopic‐sized laboratory, such as a cell. Biological molecules, such as peptides and proteins, undergo self‐assembly processes in vivo and in vitro, and these monomers are assembled into various nanometer‐scale structures at room temperature and atmospheric pressure. The self‐assembled peptide nanostructures can be further organized to form nanowires, nanotubes, and nanoparticles via their molecular‐recognition functions. The application of molecular self‐assemblies of synthetic peptides as nanometer‐scale building blocks in devices is robust, practical, and affordable due to their advantages of reproducibility, large‐scale production ability, monodispersity, and simpler experimental methods. It is also beneficial that smart functionalities can be added at desired positions in peptide nanotubes through well‐established chemical and peptide syntheses. These features of peptide‐based nanotubes are the driving force for investigating and developing peptide nanotube assemblies for biological and non‐biological applications.