New Multiscale Characterization Methodology for Effective Determination of Isolation–Structure–Function Relationship of Extracellular Vesicles

Abstract

Extracellular vesicles (EVs) have been lauded as next-generation medicines, but very few EV-based therapeutics have progressed to clinical use. Limited clinical translation is largely due to technical barriers that hamper our ability to mass produce EVs, i.e., to isolate, purify, and characterize them effectively. Technical limitations in comprehensive characterization of EVs lead to unpredicted biological effects of EVs. Here, using a range of optical and non-optical techniques, we showed that the differences in molecular composition of EVs isolated using two isolation methods correlated with the differences in their biological function. Our results demonstrated that the isolation method determines the composition of isolated EVs at single and sub-population levels. Besides the composition, we measured for the first time the dry mass and predicted sedimentation of EVs. These parameters were likely to contribute to the biological and functional effects of EVs on single cell and cell cultures. We anticipate that our new multiscale characterization approach, which goes beyond traditional experimental methodology, will support fundamental understanding of EVs as well as elucidate the functional effects of EVs in in vitro and in vivo studies. Our findings and methodology will be pivotal for developing optimal isolation methods and establishing EVs as mainstream therapeutics and diagnostics. This innovative approach is applicable to a wide range of sectors including biopharma and biotechnology as well as to regulatory agencies.

Squid Skin Cell-Inspired Refractive Index Mapping of Cells, Vesicles, and Nanostructures

“…We furthermore interrogated the optical characteristics of the RfA2-based structures found within the EVs released by our engineered cells. To achieve this goal, we collected the vesicles from the cell culture media using established methodologies , and again probed them with a commercially available holotomography microscope (see Methods for full details). ,− The representative phase microscopy images collected for EVs from RfA2-expressing cells confirmed the presence of internal RfA2-based light-scattering structures, which featured a substantial contrast relative to their immediate surroundings (Figure A, left, and see also Figure S11). The representative three-dimensional refractive index maps formulated from multiple images of our RfA2-containing EVs revealed average global refractive indices of ∼1.41 ± 0.01, which were similar to those quantified for the engineered cells (Figure A, right, and see also Figure S11 and Video S2).…”

“…27,28,56 By We furthermore interrogated the optical characteristics of the RfA2-based structures found within the EVs released by our engineered cells. To achieve this goal, we collected the vesicles from the cell culture media using established methodologies 57,58 and again probed them with a commer-cially available holotomography microscope (see Methods for full details). 37,50−52 The representative phase microscopy images collected for EVs from RfA2-expressing cells confirmed the presence of internal RfA2-based light-scattering structures, which featured a substantial contrast relative to their immediate surroundings (Figure 3A, left, and see also Figure S11).…”

Advances in the Field of Micro- and Nanotechnologies Applied to Extracellular Vesicle Research: Take-Home Message from ISEV2021

“…(b) Schematic diagrams of the APTES-GA and PPB-avidin/streptavidin functionalization method where a capillary surface is coated with a self-assembled monolayer of APTES or a layer of a PLL-PEG copolymer conjugated with biotin, which are then linked to the capture antibody. This experimental setup was used in electrokinetic measurements [30] (adapted with permission from [30]. Copyright: American Chemical Society, 2021).…”

“…Streptavidin and avidin were used as linkers, followed by the immobilization of biotinylated antibodies or affinity capture probes against CD9, CD63 and EGFR. The objective was to optimize the experimental setup in order to reach the sensitivity required for the successful application in tumor detection by liquid biopsies still using a limited sample volume (PS05.03) [30] (Figure 2b). Similarly, another electrokinetic sensor was proposed by Dr. Cavallaro (KTH Royal Institute of Technology, Stockholm, Sweden), who described a multiplexed platform to detect and compare tumor markers such as EGFR and PD-L1 in small EVs from pleural effusions of non-small-cell lung cancer patients, demonstrating that the platform may be used for the monitoring of EVs' alterations during treatments (PS 05.13) [35].…”

Challenges in the Development of Drug Delivery Systems Based on Small Extracellular Vesicles for Therapy of Brain Diseases

“…Their cargo usually reflects both the intracellular origin and the cell type from which they are derived, and may contain cytoskeletal proteins, heat shock proteins, integrins, nucleic acids, bioactive lipids, and other active components expressed by the cells of origin ( Lv et al, 2019 ). Some markers used in their characterization are included in Figure 1 , though their specific characterization is not trivial ( Phan et al, 2021 ) and some of the markers despite being more abundant in lEVs can be also found in sEVs and vice-versa ( Théry et al, 2018 ; Saludas et al, 2022 ).…”