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

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.

Keywords: extracellular vesicles; liposomes; multimodal samples; nanomedicine; orthogonal techniques; particle concentration; particle size distribution; polystyrene.

FIGURE 1

NTA, TRPS, nFCM, CLS, AF4‐MALS and MADLS measurements of monomodal CPN100, CPN150, CPN200 and CPN240. The nominal standard deviation of the NIST‐traceable CPN150 standards as given by the particle provider are indicated for all six techniques. NTA, TRPS, nFCM and MADLS measurements were averaged over 3 runs and CLS over 2. AF4‐MALS was only performed once. AF4‐MALS measurement of sample H was scaled down by a factor of 5, to make it comparable with the other sample measurements

FIGURE 2

NTA, TRPS, CLS, nFCM and AF4‐MALS measurements of trimodal sample J (CPN60/CPN100/CPN150 at 1/1/1). NTA, TRPS and nFCM measurements were averaged over 3 runs, and CLS over two runs. AF4‐MALS was only performed once. The NTA inset shows the multi‐Gaussian fit of its PSD. The CLS inset shows the baseline‐correction of the mass‐weighted PSD, whilst the AF4‐MALS inset shows the respective number density‐based fractogram and sample specific particle peak positions

FIGURE 3

NTA, TRPS, CLS, nFCM, AF4‐MALS and MADLS measurements of quadrimodal sample C (CPN100/CPN150/CPN200/CPN240 at 25/25/25/25). NTA, TRPS, nFCM and MADLS measurements were averaged over 3 runs and CLS over 2. AF4‐MALS was only performed once. The NTA inset shows the multi‐Gaussian fit of its PSD. The CLS inset shows the base‐line correction of the respective mass‐weighted PSD, whilst the AF4‐MALS inset shows the number density‐based fractogram and sample specific particle peak positions. The MADLS inset shows the intensity‐weighted PSD

FIGURE 4

NTA, TRPS, nFCM, CLS, AF4‐MALS and MADLS measurements of quadrimodal sample I (CPN100/CPN150/CPN200/CPN240 at 10/50/30/10). NTA, TRPS, nFCM and MALDS measurements were averaged over 3 runs and CLS over 2. AF4‐MALS was only performed once. The NTA inset shows the multi‐Gaussian fit of its PSD. The CLS inset shows the base‐line correction of the respective mass‐weighted PSD, whilst the AF4‐MALS inset shows the number density‐based fractogram and sample specific particle peak positions. The MADLS inset shows the intensity‐weighted PSD

FIGURE 5

Number‐weighted PSDs of PEGylated liposome sample, as measured with NTA, TRPS, nFCM and AF4‐MALS. All results are averaged over 3 repeat runs each

FIGURE 6

Number‐weighted PSDs of EV containing plasma sample, as measured with NTA, TRPS, nFCM and CLS. NTA and TRPS results are averaged over 3 repeat runs each. For CLS the two repeat runs are shown in order to demonstrate the discrepancy between repeat runs. Respective mass‐weighted PSDs are shown In the CLS inset. nFCM was only performed once