Cholesteryl esters and high protein-to-lipid ratios distinguish Non-Vesicular Extracellular Particles from Extracellular Vesicles

Extracellular vesicles (EVs) are central to intercellular communication, yet the mechanisms underlying their biogenesis and diversity remain incompletely understood. Here, we integrate meta-analysis, advanced lipidomic, protein-to-lipid profiling, and super-resolution imaging to define the fundamental principles governing EV heterogeneity. Our meta-analysis of published transmission electron microcrographs across kingdoms reveals a highly conserved 110 nm average diameter and 200 nm upper size limit for intraluminal vesicles (ILVs), which are secreted as exosomes. Besides classical EV populations, we also characterize a distinct nanoparticle class: 167000 xg pellet of non-vesicular extracellular particles (167k-NVEPs), which exhibit a significantly higher protein-to-lipid ratio than 14000 xg pellet of large EVs (14k-lEVs) and 100000 xg pellet of small EVs (100k-sEVs), as measured by both biochemical assays and Raman spectroscopy. Lipid profiling demonstrates that 167k-NVEPs exhibit significant enrichment in cholesteryl esters and triacylglycerols, lipids typically associated with lipid droplets and the endosome/lysosome system. Analysis of lipid carbon-chain lengths reveals distinct signatures: 167k-NVEPs show pronounced enrichment at 16 and 18 carbons, while 100k-sEVs display enrichment at 32 and 34 carbons. This divergence indicates a potential connection to flexible biogenesis pathways. Marker heterogeneity across EV populations, confirmed by confocal and super-resolution microscopy, further underscores the limitations of relying on canonical tetraspanins for EV classification. Notably, 167k-NVEPs (likely exomeres) exhibit enrichment of Arf6 and CD63. Together, our findings provide compositional, biophysical, and molecular evidence supporting the formal recognition of 167k-NVEPs as a distinct class of extracellular particles and enabling exploring in disease biology and therapeutic delivery. Significance StatementExtracellular vesicles (EVs) are critical mediators of intercellular communication, yet their classification remains clouded by ambiguity in terms of their composition and biogenesis. This study resolves critical uncertainties through a cross-kingdom meta-analysis, establishing a conserved [~]110nm diameter and [~]200 nm upper size limit for intraluminal vesicles (ILVs), the precursors to exosomes. More significantly, we identify non-vesicular extracellular particles (167k-NVEPs) as a distinct class based on their unique sterol-rich lipidome, enrichment in lipids of 16 and 18 carbon chain length, elevated protein-to-lipid ratio, and functional cargo delivery. These features, alongside evidence of non-canonical origin and functional cargo delivery, establish NVEPs as a discrete class of extracellular particles.