Abstract: Exosomes are 50- to 150-nm-diameter extracellular vesicles secreted by all mammalian cells except mature red blood cells and contribute to diverse physiological and pathological functions within the body. Many methods have been used to isolate and analyze exosomes, resulting in inconsistencies across experiments and raising questions about how to compare results obtained using different approaches. Questions have also been raised regarding the purity of the various preparations with regard to the sizes and types of vesicles and to the presence of lipoproteins. Thus, investigators often find it challenging to identify the optimal exosome isolation protocol for their experimental needs. Our laboratories have compared ultracentrifugation and commercial precipitation- and column-based exosome isolation kits for exosome preparation. Here, we present protocols for exosome isolation using two of the most commonly used methods, ultracentrifugation and precipitation, followed by downstream analyses. We use NanoSight nanoparticle tracking analysis and flow cytometry (Cytek® ) to determine exosome concentrations and sizes. Imaging flow cytometry can be utilized to both size exosomes and immunophenotype surface markers on exosomes (ImageStream® ). High-performance liquid chromatography followed by nano-flow liquid chromatography-mass spectrometry (LCMS) of the exosome fractions can be used to determine the presence of lipoproteins, with LCMS able to provide a proteomic profile of the exosome preparations. We found that the precipitation method was six times faster and resulted in a ∼2.5-fold higher concentration of exosomes per milliliter compared to ultracentrifugation. Both methods yielded extracellular vesicles in the size range of exosomes, and both preparations included apoproteins. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Pre-analytic fluid collection and processing Basic Protocol 2: Exosome isolation by ultracentrifugation Alternate Protocol 1: Exosome isolation by precipitation Basic Protocol 3: Analysis of exosomes by NanoSight nanoparticle tracking analysis Alternate Protocol 2: Analysis of exosomes by flow cytometry and imaging flow cytometry Basic Protocol 4: Downstream analysis of exosomes using high-performance liquid chromatography Basic Protocol 5: Downstream analysis of the exosome proteome using nano-flow liquid chromatography-mass spectrometry.

Abstract: Extracellular vesicles (EVs) are membrane vesicles secreted by cells and can modulate biological activities by transferring their content following uptake into recipient cells. Labelling of EVs is a commonly used technique for understanding their cellular targeting and biodistribution. A reliable fluorescent technique needs to preserve the size of EVs since changes in size may alter their uptake and biodistribution. Lipophilic fluorescent dye molecules such as the PKH family have been widely used for EV labelling. Here, the effect of PKH labelling on the size of EVs was systematically evaluated using nanoparticle tracking analysis (NTA), which is a widely used technique for determining the size and concentration of nanoparticles. NTA analysis showed a size increase in all the PKH labelling conditions tested. As opposed to lipophilic dye molecules, no significant shift in the size of labelled EVs was detected with luminal binding dye molecules such as 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE, hereinafter CFSE). This finding suggests that PKH labelling may not be a reliable technique for the tracking of EVs.

Abstract: Human plasma is a complex fluid, increasingly used for extracellular vesicle (EV) biomarker studies. Our aim was to find a simple EV-enrichment method for reliable quantification of EVs in plasma to be used as biomarker of disease. Plasma of ten healthy subjects was processed using sedimentation rate- (sucrose cushion ultracentrifugation-sUC) and size- (size exclusion chromatography-SEC) based methods. According to nanoparticle tracking analysis (NTA), asymmetrical flow field-flow fractionation coupled to detectors (AF4-UV-MALS), miRNA quantification, transmission electron microscopy and enzyme-linked immunosorbent assay, enrichment of EVs from plasma with sUC method lead to high purity of EVs in the samples. High nanoparticle concentrations after SEC resulted from substantial contamination with lipoproteins and other aggregates of EV-like sizes that importantly affect downstream EV quantification. Additionally, sUC EV-enrichment method linked to quantification with NTA or AF4-UV-MALS is repeatable, as the relative standard deviation of EV size measured in independently processed samples from the same plasma source was 5.4% and 2.1% when analyzed by NTA or AF4-UV-MALS, respectively. In conclusion, the sUC EV-enrichment method is compatible with reliable measurement of concentration and size of EVs from plasma and should in the future be tested on larger cohorts in relation to different diseases. This is one of the first studies using AF4-UV-MALS to quantify EVs in blood plasma, which opens new possible clinical utility for the technique.

Abstract: Renal cell carcinoma is a lethal disease that is often discovered incidentally. New non-invasive biomarkers are needed to aid diagnosis and treatment. Extracellular vesicles (EVs), membranous vesicles secreted by all cells, are a promising potential source for cancer biomarkers, but new methods are required that are both sensitive and specific for cancer identification. We have developed an EV isolation protocol optimized for kidney tumor and normal kidney tissue that yields a high vesicle concentration, confirmed by nanoparticle tracking analysis (NanoSight) and by nanoscale flow cytometry (NanoFCM). Using Western blot, we confirmed presence of EV markers CD81, CD63, flotillin-1, and absence of cellular debris, calnexin. Transmission electron microscopy images demonstrate intact membranous EVs. This new method improves existing protocols with additional steps to reduce contaminants in the EV product. Characterization of our isolation product confirms successful isolation of EVs with minimal contamination. The particle yields of our protocol are consistent and high as assessed by both standard and novel methods. This optimized protocol will contribute to biomarker discovery and biological studies of EVs in renal cancer.

Abstract: Accurate physico-chemical characterization of exosomes and liposomes in biological media is challenging due to the inherent complexity of the sample matrix. An appropriate purification step can significantly reduce matrix interferences, and thus facilitate analysis of such demanding samples. Electrical Asymmetrical Flow Field-Flow Fractionation (EAF4) provides online sample purification while simultaneously enabling access to size and Zeta potential of sample constituents in the size range of approx. 1-1000 nm. Hyphenation of EAF4 with Multi-Angle Light Scattering (MALS) and Nanoparticle Tracking Analysis (NTA) detection adds high resolution size and number concentration information turning this setup into a powerful analytical platform for the comprehensive physico-chemical characterization of such challenging samples. We here present EAF4-MALS hyphenated with NTA for the analysis of liposomes and exosomes in complex, biological media. Coupling of the two systems was realized using a flow splitter to deliver the sample at an appropriate flow speed for the NTA measurement. After a proof-of-concept study using polystyrene nanoparticles, the combined setup was successfully applied to analyze liposomes and exosomes spiked into cell culture medium and rabbit serum, respectively. Obtained results highlight the benefits of the EAF4-MALS-NTA platform to study the behavior of these promising drug delivery vesicles under in vivo like conditions.

Abstract: There are concerns that effectiveness and consistency of biopharmaceutical formulations, including vaccines, may be compromised by differences in size, concentration and shape of particles in suspension. Thus, a simple method that can help monitor and characterize these features is needed. Here, nanoparticle tracking analysis (NTA) was used to characterize particle concentration and size distribution of a highly-purified rabies vaccine (RABV), produced in Vero cells without raw materials of animal origin (RMAO). The NTA technique was qualified for characterization of RABV particles by assessing the stability profile of vaccine particles over 5-55 °C. Antigenicity of the viral particle was also monitored with the enzyme-linked immunosorbent assay (ELISA) and NTA. RABV particle size diameters were 100-250 nm (mean:150 nm), similar to sizes obtained when labelled with rabies anti-G D1-25 monoclonal antibody, suggesting mainly antigenic virus-like particles, also confirmed by transmission electron microscopy. Thermal stress at 55 °C decreased the concentration of anti-G D1-25-labelled particles from 144 hours, coherent with conformational changes leading to loss of G protein antigenicity without impacting aggregation. Results from RABV antigenicity assessment during the 24 months monitoring of stability showed good correlation between NTA and ELISA. NTA is a suitable approach for the characterization of biopharmaceutical suspensions.

Abstract: As extracellular vesicles (EVs) have become a prominent topic in life sciences, a growing number of studies are published on a regular basis addressing their biological relevance and possible applications. Nevertheless, the fundamental question of the true vesicular nature as well as possible influences on the EV secretion behavior have often been not adequately addressed. Furthermore, research regarding endothelial cell-derived EVs (EndoEVs) often focused on the large vesicular fractions comprising of microvesicles (MV) and apoptotic bodies. In this study we aimed to further extend the current knowledge of the influence of pre-isolation conditions, such as cell density and conditioning time, on EndoEV release from human umbilical vein endothelial cells (HUVECs). We combined fluorescence nanoparticle tracking analysis (NTA) and the established fluorescence-triggered flow cytometry (FT-FC) protocol to allow vesicle-specific detection and characterization of size and surface markers. We found significant effects of cell density and conditioning time on both abundance and size distribution of EndoEVs. Additionally, we present detailed information regarding the surface marker display on EVs from different fractions and size ranges. Our data provide crucial relevance for future projects aiming to elucidate EV secretion behavior of endothelial cells. Moreover, we show that the influence of different conditioning parameters on the nature of EndoEVs has to be considered.

Abstract: Due to their extremely small size, nanoparticles cannot be analyses by conventional approaches such as light microscopy. To visualise particles in the nanoscale range, a combination of an ultra-microscope and a laser illumination unit has to be applied. This combinatory technique is called Nanoparticle Tracking Anlysis (NTA) and can be used of thr nalysis of particles in a size range of approximately 10 nm up to 1 μm in liquid suspension.

Abstract: This chapter describes the technology of particle tracking analysis, PTA (also called nanoparticle tracking analysis, NTA), which analyses the Brownian motion of particles to determine size and size distribution and counts the number of particles present within a volume to measure particle concentration (in particles/millilitre). It also discusses the advantages and disadvantages of PTA and describes particular applications where the technology has found most significant benefit.

Abstract: Advancements in the field of characterization techniques have broadened the opportunities to deepen into nanoparticle production bioprocesses. Gag-based virus-like particles (VLPs) have shown their potential as candidates for recombinant vaccine development. However, comprehensive characterization of the production process is still a requirement to meet the desired critical quality attributes. In this work, the production process of Gag VLPs by baculovirus (BV) infection in the reference High Five and Sf9 insect cell lines is characterized in detail. To this end, the Gag polyprotein was fused in frame to the enhanced green fluorescent protein (eGFP) to favor process evaluation with multiple analytical tools. Tracking of the infection process using confocal microscopy and flow cytometry revealed a pronounced increase in the complexity of High Five over Sf9 cells. Cryogenic transmission electron microscopy (cryo-TEM) characterization determined that changes in cell complexity could be attributed to the presence of occlusion-derived BV in High Five cells, whereas Sf9 cells evidenced a larger proportion of the budded virus phenotype (23-fold). Initial evaluation of the VLP production process using spectrofluorometry showed that higher levels of the Gag-eGFP polyprotein were obtained in High Five cells (3.6-fold). However, comparative analysis based on nanoparticle quantification by flow virometry and nanoparticle tracking analysis (NTA) proved that Sf9 cells were 1.7- and 1.5-fold more productive in terms of assembled VLPs, respectively. Finally, analytical ultracentrifugation coupled to flow virometry evidenced a larger sedimentation coefficient of High Five-derived VLPs, indicating a possible interaction with other cellular compounds. Taken together, these results highlight the combined use of microscopy and flow cytometry techniques to improve vaccine development processes using the insect cell/BV expression vector system. © 2020 International Society for Advancement of Cytometry.

Abstract: Nanoparticle tracking analysis is an excellent tool for the characterization of mono- and polydisperse nanoparticle systems within the 10-2000 nm size range. The suitability of this technique relies on its ability to track all particles in solution simultaneously based on their Brownian motion giving an accurate size distribution. The tracked rate of particle movement is related to the particle's hydrodynamic radius using the Stokes-Einstein equation for determining the size distribution. Here we describe the characterization β-casein nanocarriers encapsulating a model hydrophobic compound, 8-anilino-1-naphthalenesulfonic acid, and the natural bioactive curcumin using the Malvern NanoSight NS300. Utilizing both normal light scattering and fluorescent modes of the NS300 enabled the differentiation of particles that had encapsulated the two fluorescent molecules and provided an accurate size distribution of the nanocarriers.

Abstract: Extracellular vesicles (EVs) are membranous structures that protect RNAs from damage when circulating in complex biological fluids, such as plasma. RNAs are extremely specific to health and disease, being powerful tools for diagnosis, treatment response monitoring, and development of new therapeutic strategies for several diseases. In this context, EVs are potential sources of disease biomarkers and promising delivery vehicles. However, standardized and reproducible EV isolation protocols easy to implement in clinical practice are missing. Here, a size exclusion chromatography-based protocol for EV-isolation from human plasma was optimized. We propose a workflow to isolate EVs for transcriptional research that allows concomitant analysis of particle number and size, total protein, and quantification of a major plasma contaminant. This protocol yields 7.54 × 109 ± 1.22 × 108 particles, quantified by nanoparticle tracking analysis, with a mean size of 115.7 ± 11.12 nm and a mode size of 83.13 ± 4.72 nm, in a ratio of 1.19 × 1010 ± 7.38 × 109 particles/μg of protein, determined by Micro Bicinchoninic Acid (BCA) Protein Assay, and 3.09 ± 0.7 ng RNA, assessed by fluorescence-based RNA-quantitation, from only 900 μL of plasma. The protocol is fast and easy to implement and has potential for application in biomarkers research, therapeutic strategies development, and clinical practice.

Abstract: Virus-like particles (VLPs) have emerged as a powerful scaffold for antigen presentation and delivery strategies. Compared to single protein-based therapeutics, quality assessment requires a higher degree of refinement due to the structure of VLPs and their similar properties to extracellular vesicles (EVs). Advances in the field of nanotechnology with single particle and high-resolution analysis techniques provide appealing approaches to VLP characterization. In this study, six different biophysical methods have been assessed for the characterization of HIV-1-based VLPs produced in mammalian and insect cell platforms. Sample preparation and equipment set-up were optimized for the six strategies evaluated. Electron Microscopy (EM) disclosed the presence of several types of EVs within VLP preparations and cryogenic transmission electron microscopy (cryo-TEM) resulted in the best technique to resolve the VLP ultrastructure. The use of super-resolution fluorescence microscopy (SRFM), nanoparticle tracking analysis (NTA) and flow virometry enabled the high throughput quantification of VLPs. Interestingly, differences in the determination of nanoparticle concentration were observed between techniques. Moreover, NTA and flow virometry allowed the quantification of both EVs and VLPs within the same experiment while analyzing particle size distribution (PSD), simultaneously. These results provide new insights into the use of different analytical tools to monitor the production of nanoparticle-based biologicals and their associated contaminants.

Abstract: Samples of water dispersion of polystyrene and gold nanoparticles were studied using dynamic light scattering and nanoparticle tracking analysis. It was shown the correlation between the width of the particle size distribution and the difference between mean particle sizes measured by both methods.

Abstract: Nanoparticles emerge to be powerful probes for the sensing of critical biomarkers because of their characteristic optical/electrochemical/magnetic properties. However, the versatility and prevalence of nanoparticle-based assays remain hampered because each kind of nanoprobe has designated properties, which requires different instruments for the readout of the signals. Herein, a versatile nanoparticle tracking analysis (NTA)-based strategy is innovatively proposed for the in vitro detection of critical biomarkers without demanding specific properties of the nanoparticles. Based on the sandwich-type immunoreaction and the duplex-specific nuclease-assisted cycling nucleic acid cleavage, the quantitative relationships are rationally built up between the level of both protein and microRNA biomarkers and the nanoparticles’ number variation. Under proper conditions, NTA will provide the absolute number count and accurate size distribution of the nanoparticles in the solution phase in a real-time manner to facile...

Abstract: The use of disease-specific signatures of microRNAs (miRNAs) in exosomes has become promising for clinical applications, either as biomarkers or direct therapeutic targets. However, a new approach for exosome enrichment and quantification of miRNAs is urgently needed for its clinical application, since the commercial techniques have shortcomings in quantity and quality. To overcome these deficiencies, we developed a new method for purification of exosomes with subsequent miRNA extraction, followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR), and compared our assays with commercial techniques. For the establishment of these methods, numerous reagents, parameters, and combinations thereof were examined. Our new technique for exosome extraction is based on a mannuronate-guluronate polymer (MGP) which avoids co-precipitating plasma proteins. Quality, concentration and biological activity of the isolated exosomes were examined by Western blot, Nanoparticle Tracking Analysis (NTA), and confocal microscopy. A combination of chaotropic and non-chaotropic salts was used to extract miRNAs from plasma, serum, and exosomes, allowing the exclusion of hazardous components, such as phenol/chloroform. The performance of the miRNAs extraction was verified by RT-qPCR. The chemistry and TaqMan probe were also optimized for RT-qPCR. Sensitivity, efficiency, and linearity of RT-qPCR were tested on serial dilutions of synthetic miR-16 and miR-142. Our established procedure covers all steps of miRNA analyses, and measures the levels of either cell-free and exosomal miRNAs in plasma, serum and other body fluids with high performance.

Abstract: We use single-particle tracking (SPT) to explore the role of nanoparticles/polymer interactions and polymer molecular weight on nanoparticle (NP) diffusion in unentangled polymer melts. The very dilute NP concentrations (∼10^–7 wt %) in SPT measurements enable tuning NP/polymer interactions so that the systems with unfavorable or neutral NP/polymer interactions in polymer melts can be studied without nanoparticle aggregation. Here, the diffusion coefficients of weakly interacting (methyl-capped, CH₃ QDs) and strongly interacting (carboxylic acid-capped, COOH QDs) nanoparticles (radius = 6.6 nm) in poly­(propylene glycol) (PPG) melts were measured via SPT. Mean-squared displacements and van Hove distributions of nanoparticle motion are consistent with Brownian motion of single nanoparticles in the long-time diffusion regime. The effective COOH QD size increases with PPG molecular weight as <i>M</i>_w^0.5, indicating a long-lived bound layer. However, for weakly interacting CH₃ QDs, the effective nanoparticle radius is independent of PPG <i>M</i>_w due to the absence of a bound layer. In contrast to ensemble average methods (i.e., X-ray photon correlation spectroscopy), SPT methods directly detect spatial and temporal diffusion behavior of individual nanoparticles and provide previously inaccessible information about nanoparticle diffusion in polymer melts.

Abstract: Nanoparticle Tracking Analysis (NTA) allows for the simultaneous determination of both size and concentration of nanoparticles in a sample. This study investigates the accuracy of particle size and concentration measurements performed on an LM10 device. For experiments, standard nanoparticles of different sizes composed of two materials with different refractive indices were used. Particle size measurements were found to have a decent degree of accuracy. This fact was verified by the manufacturer-reported particle size-determined by transmission electron microscopy (TEM)-as well as by performed scanning electron microscopy (SEM) measurements. On the other hand, concentration measurements resulted in overestimation of the particle concentration in majority of cases. Thus, our findings confirmed the accuracy of nanoparticle sizing performed by the LM10 instrument and highlighted the overestimation of particle concentration made by this device. In addition, an approach of swift correction of the results of concentration measurements received for samples is suggested in the presented study.