Abstract: Background: Nanoparticle tracking analysis (NTA) and tunable resistive pulse sensing (TRPS) enable measurement of extracellular vesicles (EVs) in blood plasma but also measure other particles present in plasma. Complete isolation of EVs from similarly sized particles with full EV recovery is currently not possible due to limitations in existing isolation techniques.Aim: This study aimed to evaluate preanalytical, analytical, and biological variation of particle measurements with NTA and TRPS on blood plasma.Methods: Blood from 20 healthy subjects was sampled in the fasting and postprandial state. Platelet free plasma (PFP) was analyzed immediately and after a freeze-thaw cycle. Additionally, the effect of prandial state and a freeze-thaw cycle on EV-enriched particle fractions obtained via size-exclusion chromatography (SEC) was examined.Results: We observed analytical linearity in the range of 1.0–10.0 × 108 particles/mL for NTA and 1.0 × 108–1.8 × 109 particles/mL for TRPS. The analytical variat...

Abstract: Accurate analysis of specific microvesicles (MVs) from biofluids is critical and challenging. Here we described novel methods to purify and detect MVs shed from endothelial cells (ECs) and endothelial progenitor cells (EPCs) by combining microbeads with fluorescence quantum dots (Q-dots) coupled nanoparticle tracking analysis (NTA). In the in vitro screening systems, we demonstrated that 1) anti-CD105 (EC marker) and anti-CD34 (EPC marker) conjugated-microbeads had the highest sensitivity and specificity for isolating respective MVs, which were confirmed with negative controls, CD41 and CD235a; 2) anti-CD144 (EC marker) and anti-KDR (EPC marker) conjugated-Q-dots exhibited the best sensitivity and specificity for their respective MV NTA detection, which were confirmed with positive control, anti-Annexin V (MV universal marker). The methods were further validated by their ability to efficiently recover the known amount of EC-MVs and EPC-MVs from particle-depleted plasma, and to detect the dynamical changes of plasma MVs in ischemic stroke patients, as compared with traditional flow cytometry. These novel methods provide ideal approaches for functional analysis and biomarker discovery of ECs- and EPCs- derived MVs.

Abstract: Comprehensive characterization of nanomaterials for medical applications is a challenging and complex task due to the multitude of parameters which need to be taken into consideration in a broad range of conditions. Routine methods such as dynamic light scattering or nanoparticle tracking analysis provide some insight into the physicochemical properties of particle dispersions. For nanomedicine applications the information they supply can be of limited use. For this reason, there is a need for new methodologies and instruments that can provide additional data on nanoparticle properties such as their interactions with surfaces. Nanophotonic force microscopy has been shown as a viable method for measuring the force between surfaces and individual particles in the nano-size range. Here we outline a further application of this technique to measure the size of single particles and based on these measurement build the distribution of a sample. We demonstrate its efficacy by comparing the size distribution obtained with nanophotonic force microscopy to established instruments, such as dynamic light scattering and differential centrifugal sedimentation. Our results were in good agreement to those observed with all other instruments. Furthermore, we demonstrate that the methodology developed in this work can be used to study complex particle mixtures and the surface alteration of materials. For all cases studied, we were able to obtain both the size and the interaction potential of the particles with a surface in a single measurement.

Abstract: Diabetic nephropathy (DN), a leading cause of end‐stage renal disease, occurs in 25%–40% of patients with diabetes, highlighting the importance of early identification and management of DN. Urinary exosomes are 40–100 nm vesicles released by epithelial cells along the nephron and they may serve as biomarkers of renal dysfunction and structural injury. Kidney injury molecule‐1 (KIM‐1) is expressed on the apical membrane of dedifferentiated proximal tubule cells and its ectodomain can be cleaved and released into the urine in both rodents and humans with proteinuric kidney diseases. In this study, we investigated urinary exosomal KIM‐1 in type 2 diabetic patients to confirm its role as a non‐invasive biomarker for predicting kidney injury and dysfunction. Exosomes were prepared from spot urine samples of healthy (n=11), prediabetic (n=12) and type 2 diabetic patients (n=10) using serial ultracentrifugation. Nanoparticle tracking analysis (NTA) was applied to quantify the size and concentration of urinary exosomes. The majority of the captured particles had a size ranging from 20 nm to 100 nm with a peak size of 78±14 nm in healthy subjects, 67±10 nm in prediabetic, and 49±7 nm in diabetic patients with or without albuminuria. Urinary exosomes were significantly increased in diabetic patients (16.2±2.7 ×10 10 particles/mg creatinine) compared to healthy donors (7.4±0.9 ×10 10 particles/mg creatinine) or prediabetic patients (8.8±1.5 ×10 10 particles/mg creatinine). As expected, soluble KIM‐1 was significantly increased in the urine of diabetic patients with microalbuminuria. Interestingly, Western blot also detected an increase in urinary exosomal KIM‐1 in the diabetic patients without microalbuminuria. In conclusion, type 2 diabetes is associated with an increase in urinary exosomes and exosomal KIM‐1 may represent a novel candidate biomarker in diabetic renal complication. Support or Funding Information This work was supported by the NIH SDK096441, 8G12MD007602 and 8U54MD007588.

Abstract: The interest in extracellular vesicles (EVs) has grown exponentially over the last decade. Evolving evidence is demonstrating that these EVs are playing an important role in health and disease. They are involved in intercellular communication and have been shown to transfer proteins, lipids, and nucleic acids. This review focuses on the most commonly used techniques for detection of EVs, to include microparticles, 100-1,000 nm in size, and exosomes, 50-100 nm in size. Conventional flow cytometry is the most prevalent technique, but nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), and resistive pulse sensing have also been used to detect EVs. The accurate measurement of these vesicles is challenged by size heterogeneity, low refractive index, and the lack of dynamic measurement range for most of the available technologies. Sample handling during the preanalytical phase can also affect the accuracy of measurements. Currently, there is not one single method which allows phenotyping, sizing, and enumerating the whole range of EVs and, therefore, providing all the necessary information to truly understand the biology of these particles. A combination of methods is probably needed which might also include electron and atomic force microscopy and full RNA, lipid, and protein profiling.

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.