Abstract: Exosomes are nanometer-scale, membrane-enclosed vesicles that can potentially be used to detect nephrotoxicity, and reveal the subsequent response of the kidney. Epithelial cells of every nephron segment can contribute to the urinary exosome population, which is rich in potential biomarkers, including membrane proteins such as transporters and receptors, transcription factors, and microRNAs. These exosomal biomarkers may be up- or downregulated upon nephrotoxicant exposure. Exosome isolation is an area of ongoing research. Although faster and simpler methods have been developed, ultracentrifugation remains a mainstay for purification. A single ultracentrifugation step provides an enriched preparation suitable for biomarker discovery, and a second ultracentrifugation on a sucrose/D2O cushion provides the purest exosome preparation currently available and may be preferred for bioactivity assays. The concentration of exosomes can be determined using Nanosight Nanoparticle Tracking Analysis and their contents studied with a variety of approaches including western blots for proteins and RT-qPCR for microRNAs.

Abstract: Introduction: Nanoparticle tracking analysis (NTA) enables measurement of extracellular vesicles (EVs) but lacks the ability to distinct between EVs and lipoproteins which are abundantly present in blood plasma. Limitations in ultracentrifugation and size exclusion chromatography applied for EV isolation may result in inadequate EV purification and preservation. In this proof of concept study, we aimed to evaluate the potential of antibody-mediated removal of lipoproteins from plasma prior to extracellular vesicle (EV) analysis by nanoparticle tracking analysis (NTA).Methods: Ten platelet-free plasma (PFP) samples from healthy fasting subjects were incubated with magnetic beads coated with antibodies against apolipoprotein B-48 and B-100 (ApoB). Plasma samples were analysed with NTA before and after application of the bead procedure. Four fasting PFP samples were analysed with an ELISA specific for human ApoB to estimate the degree of removal of lipoproteins and EV array analysis was used for iden...

Abstract: Nanoparticle tracking analysis (NTA) can be used to quantitate extracellular vesicles (EVs) in biological samples and is widely considered a useful diagnostic tool to detect disease. However, accurately profiling EVs can be challenging due to their small size and heterogeneity. Here, we aimed to provide a protocol to facilitate high-precision particle quantitation by NTA in plasma, the supernatant of activated purified platelets (the platelet releasate) and in serum, to increase confidence in NTA particle enumeration. The overall variance and the precision of NTA measurements were quantified by root mean square error and relative standard error. Using a bootstrapping approach, we found that increasing video replicates from 5 x 60 sec to 25 x 60 sec captures led to a reduction in overall variance and a reproducible increase in the precision of NTA particle-concentration quantitation for all three biofluids. We then validated our approach in an extended cohort of 32 healthy donors. Our results indicate that for vesicles sized between 50-120nm, the precision of routine NTA measurements in serum, plasma, and platelet releasate can be significantly improved by increasing the number of video replicates captured. Our protocol provides a common platform to statistical compare particle size distribution profiles in the exosomal-vesicle size range across a variety of biofluids and in both healthy donor and patient groups.

Abstract: Dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) are two orthogonal and complementary methods of measuring size of particles in a sample. These technologies use the theory of Brownian motion by analyzing the random changes of light intensity scattered by particles in solution. Both techniques can be used to characterize particle size distribution of proteins and formulations in the nanometer to low micron range.Each method has benefits over the other. DLS is a quick and simple measurement that is ideal for monodisperse particles and can also analyze a distribution of particles over a wide range of sizes. NTA provides a size distribution that is less susceptible to the influence of a few large particles, and has the added benefit of being able to measure particle concentration. Here we describe methods for measuring the particle size and concentration of an oil-in-water nanoemulsion.

Abstract: Characterization of submicron protein particles continues to be challenging despite active developments in the field. NTA is a submicron particle enumeration technique, which optically tracks the light scattering signal from suspended particles undergoing Brownian motion. The submicron particle size range NTA can monitor in common protein formulations is not well established. We conducted a comprehensive investigation with several protein formulations along with corresponding placebos using NTA to determine submicron particle size distributions and shed light on potential non-particle origin of size distribution in the range of approximately 50–300 nm. NTA and DLS are performed on polystyrene size standards as well as protein and placebo formulations. Protein formulations filtered through a 20 nm filter, with and without polysorbate-80, show NTA particle counts. As such, particle counts above 20 nm are not expected in these solutions. Several other systems including positive and negative controls were studied using NTA and DLS. These apparent particles measured by NTA are not observed in DLS measurements and may not correspond to real particles. The intent of this article is to raise awareness about the need to interpret particle counts and size distribution from NTA with caution.

Abstract: Due to their size, small extracellular vesicles such as exosomes have been difficult to identify and to quantify. As the roles that exosomes play in intercellular signalling become clearer, so does their potential utility as both diagnostic biomarkers for disease and as therapeutic vectors. Accurate assessment of exosomes, both their number and their cargo, is important for continued advancement in the field of vesicle research. To that end, several technologies, including nanoparticle tracking analysis, have been developed to define the physical characteristics of vesicle preparations and determine their concentration. This chapter describes a method for identifying the size and concentration of a subpopulation of vesicles in biological samples, using nanoparticle tracking analysis. Characterization of distinct exosomes is enabled by specific marker antibodies, coupled to fluorescent quantum dots.

Abstract: Aim Extracellular vesicles, such as exosomes, are present in urine with reports of roles in intercellular signalling and diagnostic utility. However, the extent to which the concentration and characteristics of urinary vesicles are altered in albuminuric renal disease has not been well characterized. In this study, we examined the number and characteristics of extracellular vesicles in albuminuric urine. Methods Vesicles were isolated from the urine of 32 patients with varying levels of albuminuria using ultracentrifugation and density gradient purification and were examined using nanoparticle tracking analysis, immunoblotting and transmission electron microscopy. The size profile of particles in these urine preparations was compared with albumin-containing solutions. Results Overall, there were no substantial differences in the number, or characteristics, of vesicles released into proteinuric urine. Analysis of albumin-containing solutions showed particles of exosome-like size, suggesting that such particles can mimic exosomes in standard nanoparticle tracking analysis. Albumin and IgG depletion of proteinuric urine resulted in a substantial reduction in the concentration of particles detected by nanoparticle tracking analysis. Conclusion There was no increase in urinary vesicle concentration in patients with albuminuria. Furthermore, these results demonstrate the need for cautious interpretation of nanoparticle tracking analysis of vesicle concentration in biological fluids containing protein and for sophisticated preparative methods in vesicle purification from urine.

Abstract: Nanoparticle tracking analysis (NTA) is a recently developed nanoparticle characterization technique that offers certain advantages over dynamic light scattering for characterizing polyplex nanoparticles in particular. Dynamic light scattering results in intensity-weighted average measurements of nanoparticle characteristics. In contrast, NTA directly tracks individual particles, enabling concentration measurements as well as the direct determination of number-weighted particle size and zeta-potential. A direct number-weighted assessment of nanoparticle characteristics is particularly useful for polydisperse samples of particles, including many varieties of gene delivery particles that can be prone to aggregation. Here, we describe the synthesis of poly(beta-amino ester)/deoxyribonucleic acid (PBAE/DNA) polyplex nanoparticles and their characterization using NTA to determine hydrodynamic diameter, zeta-potential, and concentration. Additionally, we detail methods of labeling nucleic acids with fluorophores to assess only those polyplex nanoparticles containing plasmids via NTA. Polymeric gene delivery of exogenous plasmid DNA has great potential for treating a wide variety of diseases by inducing cells to express a gene of interest.

Abstract: Exosomes are small extracellular vesicles (EVs) of endocytic origin and get released by a multitude of cell types. These vesicles have been found in a variety of body fluids and more recently an important role in exchanging genetic information between cells was postulated for exosomes. The aims of this thesis were to investigate the npcRNA content of exosomes and analyse their putative role in cell-cell communication. I established three different exosome isolation techniques yielding EVs in the appropriate size range determined by nanoparticle tracking analysis (NTA) and detected three exosomal marker proteins, i.e. Alix, TSG101 and CD9, by immunoblot. Furthermore, the identity of exosomes was confirmed by electron microscopy. Stable cell lines were generated to investigate the effect of the individual marker proteins on exosome production and characteristics. The tetraspanin CD9 positively influenced exosome and microvesicle production, while overexpression of Alix or TSG reduced the amount of EVs. In the context of lentiviral vector production a role in membrane fusion was identified for CD9, indicating a direct participation in fusion of exosomes with target cell membranes. However, extracellular CD9-enriched and WT vesicles comprise similar npcRNA content, suggesting no active role of CD9 in sorting of npcRNAs to exosomes. The extracellular npcRNA content of four human cell lines was successfully examined via Next Generation Sequencing. Data analysis was performed using the DESeq package in R with self-made, question-adjusted, scripts. NGS data analysis revealed secretion (exosome) and retention (cell) motifs for miRNAs, an important finding for further cell-cell communication studies. Furthermore, I identified differentially expressed miRNAs after Jurkat exosome treatment in Raji cells by NGS and in silico analysis revealed two target genes (DNMT1 and Bcl-2) for these candidates. Extracellular vesicles derived from T-lymphocytes transferred sufficient amounts of functional miRNAs to induce a significant knockdown of DNMT1 as was confirmed by qPCR analysis. We generated a reporter gene assay that enabled detection of the horizontal transfer of synthetic siRNA (siGL-2) in exosomes. The results indicated a direct and functional communication via extracellular vesicles. In contact-inhibiting co-cultures we were able to confirm an EV mediated transfer of fluorescent labelled siRNAs between human B-lymphocytes and colon carcinoma cells, reflecting the cellular communication in the tumor microenvironment. Modification of cells with npcRNAs to study EV-functions is difficult in some cell types and hard to adapt for in vivo research. We utilized a gene delivery technology based on virus-like particles (VLP) that facilitates modification of cells in vivo. Feasibility was confirmed in a rat animal model by systemic delivery of functional siRNAs. The directed in vivo manipulation of cells opens new prospects for the exosome research and has to be expanded in the future.

Abstract: The use of nanomaterials is rapidly increasing, while little is known about their possible ecotoxicological effects. In addition to toxicity assessment the particles were characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). An Ag particle was within the small nanometer size range when dispersed in pure water. In media with higher ionic strength and DOC, particles tended to agglomerate. Cytotoxicity assessments showed that Ag nanoparticles caused a significant reduction in membrane integrity and cellular metabolic activity in a concentration dependent manner. The current study shows that size of particle, ionic strength and adverse effect on at low mg/l concentrations.

Abstract: Nanotechnology is a promising field of science and technology that deals with the effects of engineered or manufactured nanomaterial and their applications on living organisms. In addition to toxicity evaluation, these particles were characterized by different techniques like transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Titanium nanoparticle was within the small nanometer size range when dispersed in pure water. The particles were tended to agglomerate in media with higher ionic strength. The assessments of cytotoxicity showed that Ti nanoparticles caused a significant lessening in membrane integrity and cellular metabolic activity in a concentration dependent manner. The current study shows that size of Titanium nanoparticles and their ionic strength is adequate and also can be used at low concentrations to determine adverse effect in further studies.

Abstract: The invention belongs to the field of medicinal biology and particularly relates to a method of detecting distribution of nanoparticles in cells and tissues A simple and reliable qualitative and/or quantitative detection method is provided by using nanoparticle tracking analysis technology to evaluate nanoparticle intake by cells as well as distribution of the nanoparticles in bodily tissues By subjecting the nanoparticles to fluorescence labeling, the quantity and particle size of nanoparticles entering tissue organs are specifically analyzed by means of separation so as to investigate dynamic changes of the nanoparticles in a human body The method of the invention has the advantages that operating is simple, time is saved, and much effective data are provided

Abstract: Numerous nanometrology techniques have been developed in recent years to determine the size, concentration, and a number of other characteristics of engineered nanomaterials (ENM) in environmental matrices. Among the many available techniques, nanoparticle tracking analysis (NTA) can measure individual particles to create a size distribution and measure the particle number. Therefore, we explore the possibility to use these data to calculate the particle mass distribution. Additionally, we further developed the NTA methodology to explore its suitability for analysis of ENM in complex matrices by measuring ENM agglomeration and sedimentation in municipal solid waste incineration landfill leachates over time. 100 nm Au ENM were spiked into DI H₂O and synthetic and natural leachates. We present the possibility of measuring ENM in the presence of natural particles based on differences in particle refractivity indices, delineate the necessity of creating a calibration curve to adjust the given NTA particle number concentration, and determine the instruments linear range under different conditions. By measuring the particle size and the particle number distribution, we were able to calculate the ENM mass remaining in suspension. By combining these metrics together with transmission electron microscopy (TEM) analyses, we could assess the extent of both homo- and heteroagglomeration as well as particle sedimentation. Reporting both size and mass based metrics is common in atmospheric particle measurements, but now, the NTA can give us the possibility of applying the same approach also to aqueous samples.

Abstract: Outer membrane vesicles (OMVs) are spherical membrane nanoparticles released by Gram-negative bacteria. OMVs can be quantified in complex matrices by nanoparticle tracking analysis (NTA). NTA can be performed in static mode or with continuous sample flow that results in analysis of more particles in a smaller time-frame. Flow measurements must be performed manually despite the availability of a sample changer on the NanoSight system. Here we present a method for automated measurements in flow mode. OMV quantification in flow mode results in lower variance in particle quantification (coefficient of variation (CV) of 6%, CV static measurements of 14%). Sizing of OMVs was expected to be less favorable in flow mode due to the increased movement of the particles. However, we observed a CV of 3% in flow mode and a CV of 8% in static measurements. Flow rates of up to 5 µL/min displayed correct size and particle measurements, however, particle concentration was slightly lower than in static measurements. The automated method was used to assess OMV release of batch cultures of Neisseria meningitidis. The bacteria released more OMVs in stationary growth phase, while the size of the vesicles remained constant throughout the culture. Taken together, this study shows that automated measurements in flow mode can be established with advanced scripting to reduce the workload for the user.

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.