Effect of Sample Preprocessing and Size-Based Extraction Methods on the Physical and Molecular Profiles of Extracellular Vesicles.

Extracellular vesicles (EVs) are nanometric lipid vesicles that shuttle cargo between cells. Their analysis could shed light on health and disease conditions, but EVs must first be preserved, extracted, and often preconcentrated. Here we first compare plasma preservation agents, and second, using both plasma and cell supernatant, four EV extraction methods, including (i) ultracentrifugation (UC), (ii) size-exclusion chromatography (SEC), (iii) centrifugal filtration (LoDF), and (iv) accousto-sorting (AcS). We benchmarked them by characterizing the integrity, size distribution, concentration, purity, and expression profiles for nine proteins of EVs, as well as the overall throughput, time-to-result, and cost. We found that the difference between ethylenediaminetetraacetic acid (EDTA) and citrate anticoagulants varies with the extraction method. In our hands, ultracentrifugation produced a high yield of EVs with low contamination; SEC is low-cost, fast, and easy to implement, but the purity of EVs is lower; LoDF and AcS are both compatible with process automation, small volume requirement, and rapid processing times. When using plasma, LoDF was susceptible to clogging and sample contamination, while AcS featured high purity but a lower yield of extraction. Analysis of protein profiles suggests that the extraction methods extract different subpopulations of EVs. Our study highlights the strengths and weaknesses of sample preprocessing methods, and the variability in concentration, purity, and EV expression profiles of the extracted EVs. Preanalytical parameters such as collection or preprocessing protocols must be considered as part of the entire process in order to address EV diversity and their use as clinically actionable indicators.

Extracellular Vesicle Antibody Microarray for Multiplexed Inner and Outer Protein Analysis.

Proteins are found both outside and inside of extracellular vesicles (EVs) and govern the properties and functions of EVs, while also constituting a signature of the cell of origin and of biological function and disease. Outer proteins on EVs can be directly bound by antibodies to either enrich EVs, or probe the expression of a protein on EVs, including in a combinatorial manner. However, co-profiling of inner proteins remains challenging. Here, we present the high-throughput, multiplexed analysis of EV inner and outer proteins (EVPio). We describe the optimization of fixation and heat-induced protein epitope retrieval for EVs, along with oligo-barcoded antibodies and branched DNA signal amplification for sensitive, multiplexed, and high-throughput assays. We captured four subpopulations of EVs from colorectal cancer (CRC) cell lines HT29 and SW403 based on EpCAM, CD9, CD63, and CD81 expression, and quantified the co-expression of eight outer [integrins (ITGs) and tetraspanins] and four inner (heat shock, endosomal, and inner leaflet) proteins. The differences in co-expression patterns were consistent with the literature and known biological function. In conclusion, EVPio analysis can simultaneously detect multiple inner and outer proteins in EVs immobilized on a surface, opening the way to extensive combinatorial protein profiles for both discovery and clinical translation.

Cell-specific optoporation with near-infrared ultrafast laser and functionalized gold nanoparticles.

Selective targeting of diseased cells can increase therapeutic efficacy and limit off-target adverse effects. We developed a new tool to selectively perforate living cells with functionalized gold nanoparticles (AuNPs) and near-infrared (NIR) femtosecond (fs) laser. The receptor CD44 strongly expressed by cancer stem cells was used as a model for selective targeting. Citrate-capped AuNPs (100 nm in diameter) functionalized with 0.01 orthopyridyl-disulfide-poly(ethylene glycol) (5 kDa)-N-hydroxysuccinimide (OPSS-PEG-NHS) conjugated to monoclonal antibodies per nm(2) and 5 µM HS-PEG (5 kDa) were colloidally stable in cell culture medium containing serum proteins. These AuNPs attached mostly as single particles 115 times more to targeted CD44(+) MDA-MB-231 and CD44(+) ARPE-19 cells than to non-targeted CD44(-) 661W cells. Optimally functionalized AuNPs enhanced the fs laser (800 nm, 80-100 mJ cm(-2) at 250 Hz or 60-80 mJ cm(-2) at 500 Hz) to selectively perforate targeted cells without affecting surrounding non-targeted cells in co-culture. This novel highly versatile treatment paradigm can be adapted to target and perforate other cell populations by adapting to desired biomarkers. Since living biological tissues absorb energy very weakly in the NIR range, the developed non-invasive tool may provide a safe, cost-effective clinically relevant approach to ablate pathologically deregulated cells and limit complications associated with surgical interventions.