Beyond Exosomes: Biological Properties, Isolation Challenges, and Functional Redefinition of Non-Vesicular Extracellular Nanoparticles (NVEPs).

Extracellular vesicles (EVs) have dominated research on intercellular communication for decades. However, the discovery of exomeres in 2018 revealed that conventional exosome preparations contain large quantities of long-overlooked non-membranous nanoparticles. Additional non-vesicular extracellular nanoparticles (NVEPs), including supermeres and SECmeres, have since been reported. Most of these particles are smaller than 50 nm in diameter, yet they carry protein and RNA cargo that was previously thought to be delivered exclusively by exosomes. Consequently, certain functions once attributed to exosomes now require reassignment. This review summarizes the classification, cargo composition, and structural features of NVEPs. It then compares the respective limitations of differential ultracentrifugation, density gradient centrifugation, size-exclusion chromatography, and flow field-flow fractionation for NVEP isolation, together with strategies for their combined use. Furthermore, the functional mechanisms that distinguish NVEPs from vesicular EVs are discussed at three levels: the extracellular stability of non-vesicular extracellular RNA (nv-RNA), cellular uptake pathways, and downstream effects. Current evidence indicates that NVEPs are enriched for disease-associated cargo in cancer and neurodegenerative diseases and show potential for liquid biopsy and drug delivery. Nevertheless, ambiguous definitional boundaries, the absence of reference materials, and insufficient in vivo evidence jointly hinder quantification and clinical translation in this field. Because these limitations arise largely from the absence of unified technical criteria, establishing internationally recognized standards for isolation and characterization is a prerequisite for the maturation of NVEP research.