Uncovering the release of micro/nanoplastics from disposable face masks at times of COVID-19.

A critical review on microplastics, interaction with organic and inorganic pollutants, impacts and effectiveness of advanced oxidation processes applied for their removal from aqueous matrices

We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single-particle interferometric reflectance imaging sensing (SP-IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized by transmission electron microscopy (TEM) and Western blot (WB). Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP-IRIS events could not be used to estimate particle concentrations. For sizing, SP-IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP-IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP-IRIS platform in fluorescence mode was able to detect at least two markers on the same particle, while NFCM detected either CD81 or CD63. Based on the results of this study, we can draw conclusions about existing single-particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies.

Characterization of extracellular vesicles and synthetic nanoparticles with four orthogonal single‐particle analysis platforms

Abstract Mounting evidence suggests that storage has an impact on extracellular vesicles (EVs) properties. While −80°C storage is a widespread approach, some authors proposed improved storage strategies with conflicting results. Here, we designed a systematic study to assess the impact of −80°C storage and freeze‐thaw cycles on EVs. We tested the differences among eight storage strategies and investigated the possible fusion phenomena occurring during storage. EVs were collected from human plasma and murine microglia culture by size exclusion chromatography and ultracentrifugation, respectively. The analysis included: concentration, size and zeta potential (tunable resistive pulse sensing), contaminant protein assessment; flow cytometry for the analysis of two single fluorescent‐tagged EVs populations (GFP and mCherry), mixed before preservation. We found that −80°C storage reduces EVs concentration and sample purity in a time‐dependent manner. Furthermore, it increases the particle size and size variability and modifies EVs zeta potential, with a shift of EVs in size‐charge plots. None of the tested conditions prevented the observed effects. Freeze‐thaw cycles lead to an EVs reduction after the first cycle and to a cycle‐dependent increase in particle size. With flow cytometry, after storage, we observed a significant population of double‐positive EVs (GFP+‐mCherry+). This observation may suggest the occurrence of fusion phenomena during storage. Our findings show a significant impact of storage on EVs samples in terms of particle loss, purity reduction and fusion phenomena leading to artefactual particles. Depending on downstream analyses and experimental settings, EVs should probably be processed from fresh, non‐archival, samples in majority of cases.

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/jev2.12162

The impact of storage on extracellular vesicles: A systematic study

Microplastics in seawater and zooplankton: A case study from Terengganu estuary and offshore waters, Malaysia.

Measuring particle size distribution and mass concentration of nanoplastics and microplastics: addressing some analytical challenges in the sub-micron size range

The measurement of physicochemical properties of polydisperse complex biological samples, for example, extracellular vesicles, is critical to assess their quality, for example, resulting from their production and isolation methods. The community is gradually becoming aware of the need to combine multiple orthogonal techniques to perform a robust characterization of complex biological samples. Three pillars of critical quality attribute characterization of EVs are sizing, concentration measurement and phenotyping. The repeatable measurement of vesicle concentration is one of the key-challenges that requires further efforts, in order to obtain comparable results by using different techniques and assure reproducibility. In this study, the performance of measuring the concentration of particles in the size range of 50-300 nm with complementary techniques is thoroughly investigated in a step-by step approach of incremental complexity. The six applied techniques include multi-angle dynamic light scattering (MADLS), asymmetric flow field flow fractionation coupled with multi-angle light scattering (AF4-MALS), centrifugal liquid sedimentation (CLS), nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (TRPS), and high-sensitivity nano flow cytometry (nFCM). To achieve comparability, monomodal samples and complex polystyrene mixtures were used as particles of metrological interest, in order to check the suitability of each technique in the size and concentration range of interest, and to develop reliable post-processing data protocols for the analysis. Subsequent complexity was introduced by testing liposomes as validation of the developed approaches with a known sample of physicochemical properties closer to EVs. Finally, the vesicles in EV containing plasma samples were analysed with all the tested techniques. The results presented here aim to shed some light into the requirements for the complex characterization of biological samples, as this is a critical need for quality assurance by the EV and regulatory community. Such efforts go with the view to contribute to both, set-up reproducible and reliable characterization protocols, and comply with the Minimal Information for Studies of Extracellular Vesicles (MISEV) requirements.

Measuring particle concentration of multimodal synthetic reference materials and extracellular vesicles with orthogonal techniques: Who is up to the challenge?

Extracellular vesicles (EVs) are of great interest in biological research, and though they are a relatively recent discovery, they have rapidly shown great potential for use in clinical applications. The various techniques used in EV isolation along with their respective strengths, weaknesses, and potential for downstream applications are outlined here. A brief description of the different approaches in exosome characterisation are subsequently described. It has been highlighted that despite the recent developments in these processes, there is still a great deal of refinement to be made. EVs are produced by almost all cell types, found in many biological samples, and are implicated in multiple biological processes including cargo trafficking, cell-cell communication, and signal transduction. The presence of these EVs and their varied cargo in a biological sample can be indicative in disease diagnosis, and guide precision medicine-based approaches to disease management. EVs have been reported to act in the benefit of the cell through moderating repair and regeneration, but they can also act to the detriment of the cell through increased tumorigenesis and metastasis. This duality is intriguing as it can allow for the use of EVs in distinct therapeutic approaches and displays their versatility in potential downstream applications. In this review, examples of the cellular roles of EVs and their applications in pathological and regenerative contexts are explored. In reviewing some of the developments made in recent times, EVs are shown to be very promising both in diagnostic and therapeutic approaches.

[READ ARTICLE](https://precisionnanomedicine.com/article/6474)

/pdf/origins-to-outcomes-a-role-for-extracellular-vesicles-in-4ly5pqyhe3.pdf

John Savage

Papers

Measuring particle size distribution and mass concentration of nanoplastics and microplastics: addressing some analytical challenges in the sub-micron size range

Measuring particle concentration of multimodal synthetic reference materials and extracellular vesicles with orthogonal techniques: Who is up to the challenge?

Origins to Outcomes: A Role for Extracellular Vesicles in Precision Medicine