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

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.

Electrostatic Surface Properties of Blood and Semen Extracellular Vesicles: Implications of Sialylation and HIV-Induced Changes on EV Internalization

“…Additional studies with increased sample size are needed to examine the relationship between EV surface charge and microbial products contents in people living with HIV (PLWH). Furthermore, what is warranted is the use of purer EV preparations such as those isolated by particle purification liquid chromatography (PPLC) [96], since the EVs described in this study were isolated using the precipitation method. Other questions that remain unanswered include (i) why the ζ-potential of BEV differ from SEV?…”

Human Placenta Exosomes: Biogenesis, Isolation, Composition, and Prospects for Use in Diagnostics

“…Still, many impurity proteins co-isolated with exosomes during ultracentrifugation were found in the second and third peaks after size-exclusion chromatography [ 62 ]. Thus, gel filtration is an advantageous approach for the additional effective purification of exosomes from impurities, but there is still room for improvement [ 63 ].…”

The Past, the Present, and the Future of the Size Exclusion Chromatography in Extracellular Vesicles Separation

“…The group investigated the effects of HIV-1 infection and illicit drug use on the structure and function of SEVs, and concluded that HIV-SEV, Drug-SEV, and HIV-Drug-SEV may mediate pathological processes by differentially inducing monocytes' actin-cytoskeletal re-organization, adhesion, ECM-modifying metalloprotease secretion, and chemotactic migration toward HIV secretome [108]. Lessons from study [108] led the group to develop a gradient SEC (gSEC) column in which all available Sephadex beads (Table 1) were layered in a 100 x 1 cm column [109]. The gSEC column yielded four distinct peaks, the first two of which correspond to two (large and small) EV populations, the third peak was enriched in membraneless condensates, comprising small RNAs and a unique protein signature, but clearly lacking a membrane whereas the fourth contained small molecules and salts [109].…”

“…Lessons from study [108] led the group to develop a gradient SEC (gSEC) column in which all available Sephadex beads (Table 1) were layered in a 100 x 1 cm column [109]. The gSEC column yielded four distinct peaks, the first two of which correspond to two (large and small) EV populations, the third peak was enriched in membraneless condensates, comprising small RNAs and a unique protein signature, but clearly lacking a membrane whereas the fourth contained small molecules and salts [109]. Thus far, seminal plasma is one of the highest EV-containing body fluids, with diverse cargos and functions [61,110-113].…”

“…Noteworthy is that the authors' protocol involved multiple ultracentrifugation steps and protein reduction by DTT to remove soluble Tamm-Horsfall protein prior to SEC [117]. Interestingly, Kaddour et al [109] obtained better resolved chromatogram of healthy urine using ultrafiltration followed by chromatography on a 100 cm x 1 cm Sephadex gSEC column, without ultracentrifugation or protein reduction. On the other hand, Lozano-Ramos [118] used a 10 ml Sepharose-CL-2B packed column to show clear EV separation from bulk urine proteins, as evidenced by NTA, flowcytometry, cryo-TEM, immunoblot, and silver stain.…”