Development of Novel High-Resolution Size-Guided Turbidimetry-Enabled Particle Purification Liquid Chromatography (PPLC): Extracellular Vesicles and Membraneless Condensates in Focus

International Journal of Molecular Sciences

Nadia Shouman¹  Yuan Lyu¹  ChiomaM. Okeoma¹  Hussein Kaddour¹  Mahesh Mohan² 

[1] Department of Pharmacology, Stony Brook University Renaissance School of Medicine, Stony Brook, NY 11794-8651, USA;Host Pathogen Interaction Program Southwest National Primate Research Center, Texas Biomedical Research Institute, San Antonio, TX 78227, USA;

关键词: extracellular vesicles (EVs);    exosomes;    membraneless condensates (MCs);    gradient size exclusion chromatography (gSEC);    UV-Vis spectroscopy;   

DOI  :  10.3390/ijms21155361

来源: DOAJ

【 摘 要 】

Acellular particles (extracellular vesicles and membraneless condensates) have important research, drug discovery, and therapeutic implications. However, their isolation and retrieval have faced enormous challenges, impeding their use. Here, a novel size-guided particle purification liquid chromatography (PPLC) is integrated into a turbidimetry-enabled system for dye-free isolation, online characterization, and retrieval of intact acellular particles from biofluids. The chromatographic separation of particles from different biofluids—semen, blood, urine, milk, and cell culture supernatants—is achieved using a first-in-class gradient size exclusion column (gSEC). Purified particles are collected using a fraction collector. Online UV–Vis monitoring reveals biofluid-dependent particle spectral differences, with semen being the most complex. Turbidimetry provides the accurate physical characterization of seminal particle (Sp) lipid contents, sizes, and concentrations, validated by a nanoparticle tracking analysis, transmission electron microscopy, and naphthopyrene assay. Furthermore, different fractions of purified Sps contain distinct DNA, RNA species, and protein compositions. The integration of Sp physical and compositional properties identifies two archetypal membrane-encased seminal extracellular vesicles (SEV)—notably SEV large (SEV_L), SEV small (SEV_S), and a novel nonarchetypalμμembraneless Sps, herein named membraneless condensates (MCs). This study demonstrates a comprehensive yet affordable platform for isolating, collecting, and analyzing acellular particles to facilitate extracellular particle research and applications in drug delivery and therapeutics. Ongoing efforts focus on increased resolution by tailoring bead/column chemistry for each biofluid type.

【 授权许可】

Unknown