Abstract
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 (SEVL), SEV small (SEVS), and a novel non-archetypal-membraneless 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.
Highlights
Most mammalian cells produce and release small acellular particles or structures into biofluids, including semen, blood, urine, milk, saliva, and conditioned culture medium
Size exclusion separation is based on the principle of size discrimination, where larger molecules are excluded from the beads and flush-out directly, while smaller molecules are included in the beads and, travel a longer time through the column
The large-sized molecules elute in the void peak, while the small-sized molecules elute in the latter peak (Figure 1a)
Summary
Most mammalian cells produce and release small acellular particles or structures into biofluids, including semen, blood, urine, milk, saliva, and conditioned culture medium (supernatant). The most widely studied lipid bilayer membrane-encased particles are the nanosized extracellular vesicles (EVs) These membrane-enclosed nanoparticles are secreted by most cell types; are present in all body fluids, such as cerebrospinal fluid, urine, blood, saliva, breast milk, vaginal fluid, and semen, as well as cell culture supernatants; and these EVs facilitate distal and proximal intercellular communications [1,2]. The diversity of EV-mediated regulations of cellular functions has been attributed to (i) their bioactive cargo, including different RNA species (mRNA and miRNA), proteins, lipids, and double stranded DNA (dsDNA) [6], and (ii) the ability to protect the cargo against degradation These properties of EVs, as well as their endogenous natures, allow them to be considered as promising candidates of drug delivery and therapeutic agents [7]
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