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

Robert Vogel,Gabriele Vella,Otmar Geiss,Marianne Marchioni,Adriele Prina‐Mello,John Savage,Dimitri Aubert,Julien Muzard,Luigi Calzolai,Fanny Caputo,Alice Law,Dora Mehn,Ben Peacock,Giacomo Della Camera

doi:10.1002/jev2.12052

Abstract

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.

Highlights

Extracellular vesicles (EVs) are small lipid membrane enclosed bodies of size greater than 30 nm in diameter, to be differentiated from intermediate-density lipoprotein, low-density lipoprotein, and high-density lipoprotein that are present in plasma
Performance of various platforms can be evaluated in a quantifiable way, while liposomes are chosen in step 3 as the synthetic lipid-based vesicles closer in nature to EVs
To exclude any subjective biases, measurements of monomodal and multimodal polystyrene samples were done in a blind fashion, with the instrument operators not knowing the content of the sample

Summary

Introduction

Extracellular vesicles (EVs) are small lipid membrane enclosed bodies of size greater than 30 nm in diameter, to be differentiated from intermediate-density lipoprotein, low-density lipoprotein, and high-density lipoprotein that are present in plasma. EVs have been considered a recent avenue for biomedical investigation in both regenerative and pathogenic contexts, with potential in diagnostic and therapeutic applications (Savage et al, 2018). Their potential use in manifold applications, including drug delivery, particle loading and other theragnostic applications have been reviewed recently (Piffoux et al, 2018). Physicochemical properties of EVs, such as particle size distribution (PSD), stability, particle concentration (quantification of particle number per unit of volume), and phenotype are critical quality attributes (CQAs) that must be measured in a standardized way to assure quality, potency, stability and batch to batch consistency. There are characterization infrastructures, such as the European Union Nanomedicine Characterisation Laboratory (EUNCL) and the NCI-Nanotechnology Characterization Laboratory (NCI-NCL), that endeavour to produce standard operating procedures for the characterization of medically relevant nanoparticles, and that are working for developing regulatory standards in collaboration with regulators and standardization bodies

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Results

Conclusion