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Multiparametric Orthogonal Characterization of Extracellular Vesicles by Liquid Chromatography Combined with In-Line Light Scattering and Fluorescence Detection.

ABSTRACT: Extracellular vesicles (EVs) are membrane-enclosed biological nanoparticles with potential as diagnostic markers and carriers for therapeutics. Characterization of EVs poses severe challenges due to their complex structure and composition, requiring the combination of orthogonal analytical techniques. Here, we demonstrate how liquid chromatography combined with multi-angle light scattering (MALS) and fluorescence detection in one single apparatus can provide multiparametric characterization of EV samples, including concentration of particles, average diameter of the particles, protein amount to particle number ratio, presence of EV surface markers and lipids, EV shape, and sample purity. The method requires a small amount of sample of approximately 10⁷ EVs, limited handling of the sample and data analysis time in the order of minutes; it is fully automatable and can be applied to both crude and purified samples.

SUBMITTER: Normak K

PROVIDER: S-EPMC10448444 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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Multiparametric Orthogonal Characterization of Extracellular Vesicles by Liquid Chromatography Combined with In-Line Light Scattering and Fluorescence Detection.

Normak Karl K Papp Marcell M Ullmann Michael M Paganini Carolina C Manno Mauro M Bongiovanni Antonella A Bergese Paolo P Arosio Paolo P

Analytical chemistry 20230809 33

Extracellular vesicles (EVs) are membrane-enclosed biological nanoparticles with potential as diagnostic markers and carriers for therapeutics. Characterization of EVs poses severe challenges due to their complex structure and composition, requiring the combination of orthogonal analytical techniques. Here, we demonstrate how liquid chromatography combined with multi-angle light scattering (MALS) and fluorescence detection in one single apparatus can provide multiparametric characterization of E ...[more]

PMID: 37556360

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Project description:BackgroundPatients with HER2-positive breast cancer can significantly benefit from HER2-directed therapy - such as the monoclonal antibody trastuzumab. However, some patients can develop therapy resistance or change HER2 status. Thus, we urgently need new, noninvasive strategies to monitor patients frequently. Extracellular vesicles (EVs) secreted from tumor cells are emerging as potential biomarker candidates. These membrane-delimited nanoparticles harbor molecular signatures of their origin cells; report rapidly on changes to cellular status; and can be frequently sampled from accessible biofluids.ResultsUsing Single Extracellular VEsicle Nanoscopy (SEVEN) platform that combines affinity isolation of EVs with super-resolution microscopy, here we provide multiparametric characterization of EVs with ~ 8 nm precision and molecular sensitivity. We first interrogated cell culture EVs affinity-enriched in tetraspanins CD9, CD63, and CD81; these transmembrane proteins are commonly found on EV membranes. SEVEN robustly provided critical parameters of individual, tetraspanin-enriched EVs: concentration, size, shape, molecular cargo content, and heterogeneity. Trastuzumab-resistant cells (vs. trastuzumab-sensitive) secreted more EVs. Additionally, EVs from trastuzumab-resistant cells had lower tetraspanin density and higher HER2 density. We also evaluated EVs affinity-enriched in HER2; we found that these EVs (vs. tetraspanin-enriched) were larger and more elongated. We further optimized analytical sample processing to assess a rare population of HER2-enriched EVs from patient plasma. In breast cancer patients with elevated HER2 protein expression (vs. controls), HER2-enriched EVs had distinct characteristics including typically increased number of tetraspanin molecules and larger size. Importantly, these EVs were on average 25-fold more abundant compared to no cancer controls.ConclusionsSEVEN revealed unique characteristics of HER2-enriched EVs in cultured cells and complex biological fluid. In combination with current clinical approaches, this method is well poised to support precise therapeutic decisions.

Dataset Information

Multiparametric Orthogonal Characterization of Extracellular Vesicles by Liquid Chromatography Combined with In-Line Light Scattering and Fluorescence Detection.

Publications

Multiparametric Orthogonal Characterization of Extracellular Vesicles by Liquid Chromatography Combined with In-Line Light Scattering and Fluorescence Detection.

Similar Datasets

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