Abstract: The refractive index (RI) dictates interaction between light and nanoparticles and therefore is important to health, environmental, and materials sciences. Using nanoparticle tracking analysis, we have determined the RI of heterogeneous particles <500 nm in suspension. We demonstrate feasibility of distinguishing silica and polystyrene beads based on their RI. The hitherto unknown RI of extracellular vesicles from human urine was determined at 1.37 (mean). This method enables differentiation of single nanoparticles based on their RI.

Abstract: Dark field microscopy is a widely unknown method to measure the particle size distribution of diffusing nanoparticles by particle tracking. Here we demonstrate that by using the surface plasmonic resonance of Au nanoparticles, size differences of ca. 20 nm can be identified within the particle size distribution. For that purpose, we developed a software tool which helps to analyze color videos of diffusing nanoparticles retrieved from CCD or CMOS cameras. Polystyrene beads with a diameter of 100 and 200 nm were used to compare the results to those obtained with a well-established laser-based particle tracking system. The methodology will be discussed in the light of recent developments in the emerging field of optical nanoparticle tracking.

Abstract: Exosomes are vesicles that are released from the kidney into urine. They contain protein and RNA from the glomerulus and all sections of the nephron and represent a reservoir for biomarker discovery. Current methods for the identification and quantification of urinary exosomes are time consuming and only semi-quantitative. Nanoparticle tracking analysis (NTA) counts and sizes particles by measuring their Brownian motion in solution. In this study, we applied NTA to human urine and identified particles with a range of sizes. Using antibodies against the exosomal proteins CD24 and aquaporin 2 (AQP2), conjugated to a fluorophore, we could identify a subpopulation of CD24- and AQP2-positive particles of characteristic exosomal size. Extensive pre-NTA processing of urine was not necessary. However, the intra-assay variability in the measurement of exosome concentration was significantly reduced when an ultracentrifugation step preceded NTA. Without any sample processing, NTA tracked exosomal AQP2 upregulation induced by desmopressin stimulation of kidney collecting duct cells. Nanoparticle tracking analysis was also able to track changes in exosomal AQP2 concentration that followed desmopressin treatment of mice and a patient with central diabetes insipidus. When urine was stored at room temperature, 4°C or frozen, nanoparticle concentration was reduced; freezing at -80°C with the addition of protease inhibitors produced the least reduction. In conclusion, with appropriate sample storage, NTA has potential as a tool for the characterization and quantification of extracellular vesicles in human urine.

Abstract: Background : Extracellular vesicles (EVs) have clinical importance due to their roles in a wide range of biological processes. The detection and characterization of EVs are challenging because of their small size, low refractive index, and heterogeneity. Methods : In this manuscript, the size distribution of an erythrocyte-derived EV sample is determined using state-of-the-art techniques such as nanoparticle tracking analysis, resistive pulse sensing, and electron microscopy, and novel techniques in the field, such as small-angle X-ray scattering (SAXS) and size exclusion chromatography coupled with dynamic light scattering detection. Results : The mode values of the size distributions of the studied erythrocyte EVs reported by the different methods show only small deviations around 130 nm, but there are differences in the widths of the size distributions. Conclusion : SAXS is a promising technique with respect to traceability, as this technique was already applied for traceable size determination of solid nanoparticles in suspension. To reach the traceable measurement of EVs, monodisperse and highly concentrated samples are required. Keywords: exosome; microvesicle; erythrocyte; extracellular vesicles; freeze-fracture transmission electron microscopy; nanoparticle tracking analysis; resistive pulse sensing; small-angle X-ray scattering; dynamic light scattering; size exclusion chromatography (Published: 4 February 2014) Citation: Journal of Extracellular Vesicles 2014, 3 : 23298 - http://dx.doi.org/10.3402/jev.v3.23298 To access the supplementary material to this article, please see Supplementary files under Article Tools online.

Abstract: Silica nanoparticles could be promising delivery vehicles for drug targeting or gene therapy. However, few studies have been undertaken to determine the biological behavior effects of silica nanoparticles on primary endothelial cells. Here we investigated uptake, cytotoxicity and angiogenic properties of silica nanoparticle with positive and negative surface charge and sizes ranging from 25 to 115 nm in primary human umbilical vein endothelial cells. Dynamic light scattering measurements and nanoparticle tracking analysis were used to estimate the dispersion status of nanoparticles in cell culture media, which was a key aspect to understand the results of the in vitro cellular uptake experiments. Nanoparticles were taken up by primary endothelial cells in a size-dependent manner according to their degree of agglomeration occurring after transfer in cell culture media. Functionalization of the particle surface with positively charged groups enhanced the in vitro cellular uptake, compared to negatively charged nanoparticles. However, this effect was contrasted by the tendency of particles to form agglomerates, leading to lower internalization efficiency. Silica nanoparticle uptake did not affect cell viability and cell membrane integrity. More interestingly, positively and negatively charged 25 nm nanoparticles did not influence capillary-like tube formation and angiogenic sprouting, compared to controls. Considering the increasing interest in nanomaterials for several biomedical applications, a careful study of nanoparticle-endothelial cells interactions is of high relevance to assess possible risks associated to silica nanoparticle exposure and their possible applications in nanomedicine as safe and effective nanocarriers for vascular transport of therapeutic agents.

Abstract: Extracellular vesicles (EV) form a heterogeneous population of mostly spherical membrane structures released by almost all cells, including tumour cells, both in vivo and in vitro. Their size varies from 30 nm to 1 μm, and size is one of the main criteria of the selection of two categories of EV: small (30-100 nm), more homogeneous exosomes and larger fragments (0.1-1 μm) called membrane microvesicles or ectosomes. The presence of EV has already been detected in many human body fluids: blood, urine, saliva, semen and amniotic fluid. Formation of EV is tightly controlled, and their function and biochemical composition depend on the cell type they originate from. EV are the “vehicles” of bioactive molecules, such as proteins, mRNA and microRNA, and may play an important role in intercellular communication and modulation of e.g. immune system cell activity. In addition, on the surface of tumour-derived microvesicles (TMV), called oncosomes, several markers specific for cancer cells were identified, which indicates a role of TMV in tumour growth and cancer development. On the other hand, TMV may be an important source of tumour-associated antigens (TAA) which can be potentially useful as biomarkers with prognostic value, as well as in development of new forms of targeted immunotherapy of cancer.

Abstract: Amyloidogenesis is the process of formation of protein aggregates with fibrillar morphology. Because amyloidogenesis is linked to neurodegenerative disease, there is interest in understanding the mechanism of fibril growth. Kinetic models of amyloidogenesis require data on the number concentration and size distribution of aggregates, but this information is difficult to obtain using conventional methods. Nanoparticle tracking analysis (NTA) is a relatively new technique that may be uniquely suited for obtaining these data. In NTA, the two-dimensional (2-D) trajectory of individual particles is tracked, from which the diffusion coefficient, and, hence, hydrodynamic radius is obtained. Here we examine the validity of NTA in tracking number concentration and size of DNA, as a model of a fibrillar macromolecule. We use NTA to examine three amyloidogenic materials: beta-amyloid, transthyretin, and polyglutamine-containing peptides. Our results are instructive in demonstrating the advantages and some limitations of single-particle diffusion measurements for investigating aggregation in protein systems.