In recent years, various approaches have been undertaken to eliminate lipoproteins co‐isolated with extracellular vesicles, as they were initially regarded as contaminating entities. However, novel discoveries are reshaping our perspective. In body fluids, these distinct particles not only co‐exist, but also interactions between them are likely to occur. Extracellular vesicles and lipoproteins can associate with each other, share cargo, influence each other's functions, and jointly have a role in the pathomechanisms of diseases. Additionally, their association carries important implications for therapeutic and pharmacological aspects of lipid‐lowering strategies. Extracellular vesicles and lipoprotein particles may have roles in the elimination of each other from the circulation. The objective of this minireview is to delve into these aspects. Here, we show that under certain physiological and pathological conditions, extracellular vesicles and lipoproteins are ‘partners’ rather than ‘strangers’ or ‘rivals’.

Extracellular vesicles (EVs) are lipid membrane-enclosed particles released by all cells and can be isolated from various sources, even from solid tissues. This study focuses on isolating and characterizing EVs from mouse lymph nodes (LNs). Male C57BL/6 mice were injected with complete Freund’s adjuvant, with or without ovalbumin. Inguinal and popliteal LNs were incised 9 days after immunization, and EV isolation was carried out using a combination of differential centrifugation and size-exclusion chromatography. The characteristic morphology of small and large EVs was confirmed by transmission electron microscopy. Particle size distribution and concentration were determined by nanoparticle tracking analysis, while protein and lipid contents were measured by bicinchoninic acid assay, and sulfo-phospho-vanillin assays, respectively, to calculate the protein-to-lipid ratio. Immune and EV markers were analyzed by using flow cytometry and Western blot assay, revealing significant changes between immunized mice compared to controls. This study establishes a novel protocol for isolating and characterizing EVs from LNs and highlights the impact of immunization on EV properties, offering insights into their roles in immune processes.

Exosomes, a major subpopulation of small extracellular vesicles (sEV), are conserved mediators of intercellular communication, yet the properties of their endosomal precursors, intraluminal vesicles (ILV), have not been systematically quantified across species or imaging modalities. This study systematically evaluates ILV sizes across diverse eukaryotic species and modalities while assessing their relationship to secreted sEV sizes. We carried out two complementary meta-analyses of ILV sizes based on transmission electron microscopy (TEM) and cryogenic electron microscopy (cryo-EM) data across species. This was followed by in situ assessment of sEVs secreted by HEK293T cells with TEM, nanoparticle tracking analysis and super-resolution microscopy characterization. Across species, imaging modalities, and cellular contexts, ILV sizes were under approximately 200 nm, with a mean diameter of 100.5 nm, overlapping with the size range of sEVs. This study addresses an existing knowledge gap by systematically evaluating ILV size across species and revealing an upper size limit of approximately 200 nm.

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 Statement: Extracellular 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.

Background: Mechanisms governing non-vesicular extracellular particle (NVEP) biogenesis and compositional diversity remain incompletely characterized. Method: This study integrates multiparametric profiling to define NVEPs. NVEPs were isolated by differential ultracentrifugation and characterized using complementary biochemical, lipidomic, and imaging approaches, including protein-to-lipid ratio analysis, lipid profiling, and advanced microscopy. Result: Comprehensive characterization demonstrated that NVEPs exhibit significantly elevated protein-to-lipid ratios compared to extracellular vesicles (EVs) subtypes. Lipidomic profiling revealed pronounced enrichment in cholesteryl esters and triacylglycerols, with distinct carbon chain length signatures differentiating NVEPs from small EVs. Super-resolution microscopy confirmed marker heterogeneity across populations, with NVEPs showing specific enrichment of Arf6 and CD63. Conclusion: These findings provide a robust compositional framework to distinguish non-vesicular extracellular particles from canonical EVs and underscore their emerging significance as mediators of intercellular communication and potential vehicles for targeted delivery.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has had a profound global impact on individuals with diabetes mellitus (DM). This review examines the interplay between COVID-19, cholesterol metabolism, and diabetes, focusing on the differences in lipid regulation between Type I (T1DM) and Type II diabetes (T2DM). Diabetes, characterized by impaired glucose regulation and lipid homeostasis, has been identified as a significant risk factor for severe COVID-19 outcomes, including increased rates of hospitalization, ICU admission, and mortality. Dysregulated cholesterol metabolism is often present in diabetic patients and exacerbates the severity of COVID-19. We explore the mechanisms by which SARS-CoV-2 infection affects cholesterol pathways, highlighting the role of cholesterol-rich lipid rafts in viral entry and replication. The review also discusses the potential therapeutic implications of targeting cholesterol metabolism in managing COVID-19 in diabetic populations. Understanding these complex interactions may provide insights into better clinical management strategies and improve outcomes for COVID-19 patients with diabetes.