Nanoparticle Tracking Analysis Method Research Articles

Nanoparticle Tracking Analysis of Urinary Extracellular Vesicle Proteins as a New Challenge in Laboratory Medicine.

Urinary extracellular vesicle (uEV) proteins may be used as specific markers of kidney damage in various pathophysiological conditions. The nanoparticle-tracking analysis (NTA) appears to be the most useful method for the analysis of uEVs due to its ability to analyze particles below 300 nm. The NTA method has been used to measure the size and concentration of uEVs and also allows for a deeper analysis of uEVs based on their protein composition using fluorescence measurements. However, despite much interest in the clinical application of uEVs, their analysis using the NTA method is poorly described and requires meticulous sample preparation, experimental adjustment of instrument settings, and above all, an understanding of the limitations of the method. In the present work, we demonstrate the usefulness of an NTA. We also present problems encountered during analysis with possible solutions: the choice of sample dilution, the method of the presentation and comparison of results, photobleaching, and the adjustment of instrument settings for a specific analysis. We show that the NTA method appears to be a promising method for the determination of uEVs. However, it is important to be aware of potential problems that may affect the results.

Sonication is a suitable method for loading nanobody into glioblastoma small extracellular vesicles

Glioblastoma is one of the deadliest cancers, therefore novel efficient therapeutic approaches are urgently required. One of such are nanobodies, prospective nano-sized bio-drugs with advantageous characteristics. Nanobodies can target intracellular proteins, but to increase their efficiency, the delivery system should be applied. Here, we examined small extracellular vesicles as a delivery system for anti-vimentin nanobody Nb79. Nb79 was loaded in small extracellular vesicles either by incubation with glioblastoma cells, by passive loading into isolated small extracellular vesicles or by sonication of isolated small extracellular vesicles. Small extracellular vesicles secreted by glioblastoma cells were isolated by ultracentrifugation on sucrose cushion. The size distribution and average size of sonicated and non-sonicated small extracellular vesicles were determined by nanoparticle tracking analysis method. The loading of Nb79 into small extracellular vesicles by incubation with cells, passive loading or sonication was confirmed by Western blot and electron microscopy. The effect of small extracellular vesicles on cell survival was determined by WST-1 reagent. Loading of small extracellular vesicles by incubation of cells with Nb79 was unsuccessful and resulted in substantial cell death. On the other hand, as confirmed by Western blot and electron microscopy, sonication is a successful method for obtaining Nb79-loaded small extracellular vesicles. Small extracellular vesicles also had an effect on cell viability. Small extracellular vesicles without Nb79 increased survival of U251 and NCH644 cells for 20–25%, while the Nb79-loaded small extracellular vesicles decreased survival of NCH421k by 11%. We demonstrated that sonication is a suitable method to load nanobodies into exosome, and these small extracellular vesicles could in turn reduce cell survival. The method could be translated also to other applications, such as targeted delivery system of other protein-based drugs.

Effect of PEG Anchor and Serum on Lipid Nanoparticles: Development of a Nanoparticles Tracking Method.

Polyethylene glycol (PEG) is used in Lipid Nanoparticles (LNPs) formulations to confer stealth properties and is traditionally anchored in membranes by a lipid moiety whose length significantly impacts the LNPs fate in vivo. C18 acyl chains are efficiently anchored in the membrane, while shorter C14 lipids are quickly desorbed and replaced by a protein corona responsible for the completely different fate of LNPs. In this context, a method to predict the biological behavior of LNPs depending on the lipid-PEG dissociation was developed using the Nanoparticle Tracking Analysis (NTA) method in serum. Two formulations of siRNA-containing LNPs were prepared including CSL3 or SM-102 lipids and were grafted with different lipids-PEG (C18, C14 lipids-PEG, and Ceramide-PEG). The impact of the lipid-PEG on the interactions between LNPs and serum components was demonstrated by monitoring the mean particle size and the concentration over time. In vitro, these formulations demonstrated low toxicity and efficient gene knockdown on tumor MDA-MB-231 cells, but serum was found to significantly impact the efficiency of C18-PEG-based LNPs, while it did not impact the efficiency of C14-PEG-based LNPs. The NTA method demonstrated the ability to discriminate between the behaviors of LNPs according to serum proteins' interactions. CSL3 lipid and Cer-PEG were confirmed to have promise for LNP formulation.

Measurement and characterization of bulk nanobubbles by nanoparticle tracking analysis method

Measurement and characterization of bulk nanobubbles by nanoparticle tracking analysis method

Nanostructured motherwort extract and its use in ice-cream production

The paper presents data on the use of nanostructured motherwort extract in ice-cream production to be used as a functional food product for preventive purposes. Using the Nanoparticle Tracking Analysis method (NTA method), we determined nanostructured motherwort extract sizes, depending, to a large extent, on the membrane. Thus, the size of motherwort extract nanocapsules greatly depends on the carbohydrate membrane nature: 10% of nanocapsules are from 63 to 88 nm, only in sodium alginate particles are smaller 3 25 nm. The smallest average nanocapsule size is 192 nm in konjac gum, the average 227.00-249.80 nm in carrageenan and sodium alginate, respectively, the highest average size (322 nm) is in sodium carboxymethyl cellulose.

SUN-045 URINARY EXTRACELLULAR VESICLES AS BIOMARKER OF RENAL TUBULAR INJURY

Extracellular vesicles (EV) are microstructures that participate in cellular communication. They are released by all biological fluids and are carriers of proteins, lipids, DNA, RNA mainly miRNA, that reflect their cell of origin. Among their populations, we can mention microvesicles and exosomes. Exosomes are structures that are 40-150 nm and have preserved proteins, such tetraspanins CD9, CD63 and CD81. They are released after injury, including in acute renal injury. Urinary exosomes (uEV) are potential marker of renal tubular injury. However, few studies have confirmed the origin of exosomes excreted in the urine. This study aims to compare the clearance of fluorescent beads similar to exosomes with the clearance of inulin and creatinine, substances excreted in the urine only by glomerular filtration. This strategy aims to determine whether uEV come from the kidney itself or from systemic origin through glomerular filtration and/or tubular secretion. Wistar rats were anesthetized, submitted to an intrajugular injection of inulin (10%) or fluorescent beads (0.1%). Serum and urine samples were collected before and 2h after the beginning of the experiment. The clearance of inulin, creatinine and fluorescent beads were determined by respective quantifications in serum and urine samples. Creatinine and inulin concentrations were determined by spectrophotometry and beads concentrations were determined by fluorescence quantification by Nanoparticle Tracking Analysis (NTA). The location of beads in renal tissue was determined by immunofluorescence microscopy. According to the NTA method, the beads commercially obtained had a diameter of 97 nm, consistent with the exosomes. There was no significant difference between creatinine clearances in the group inulin (1.13 ± 0.31 mL/min) or beads (1.13 ± 0.17 mL/min) at the end of the experiment, suggesting that administration of the particles did not alter renal function. On the contrary, at the end of the experiment, the clearance of beads was significantly lower (0.26 ± 0.26 mL/min) than inulin clearance (2.02 ± 0.29 mL/min), suggesting that particles of similar diameter to exosomes are poorly filtered by the glomerulus. Additionally, we identified the beads in the glomerulus and peritubular space, demonstrating their presence in the kidney. However, we observed an increase in the clearance of non-fluorescent particles compared to fluorescent ones, suggesting that most of the uEVs come from the urinary system itself. This work suggests that uEVs released in the urine come from the urinary system itself, reinforcing its potential as a biomarker for kidney diseases, like acute kidney injury and renal tubular injury.

Validation of Nanoparticle Tracking Analysis in Characterizing Extracellular Vesicle Isolated from Polydisperse Biological Samples

Nanoparticle tracking analysis (NTA) has become a desired method of characterizing extracellular vesicles (EVs) in recent years. The NTA method is distinguished from other EV detection techniques by its ability to simultaneously analyze size and concentration measures in polydisperse EV samples. However, a recent study (Tian et al., 2016) suggested using a single parametric setting for sizing and counting of polydisperse particles in a broad size range is challenging. The OBJECTIVE of this study is to evaluate the accuracy of the NTA when analyzing polydisperse, biological EV samples isolated at different centrifugation speeds.METHODSHuman venous blood samples were collected in a 3.2% buffered sodium citrate‐containing tube. Plasma fraction was separated within 2 hours by a double centrifugation (2,500 g for 15 min each) and stored at −80 °C freezer until analyzed. Microvesicles were isolated from the plasma sample with an ultracentrifugation method using differing speeds (12,000 g, 16,000 g, 20,000 g, and 32,000 g) at 4 °C. Exosomes were isolated by double centrifugation (120,000 g for 70 min each) at 4°C from conditioned media from C2C12 myotubes. Samples of known size microsphere beads with 200, 500, and 800 nm diameters were evaluated in either monodisperse or polydisperse samples for comparison. The NanoSight NS300 was used to measure size distribution and concentration of the particles per manufacturer's procedures. All samples were analyzed using identical camera settings and detection parameters.RESULTSThe mean particle size was ranged from 140 nm to 222 nm across the EV samples from human blood. The mean particle size for exosomes from C2C12 cultured media was 204.0 ± 9.1 nm. There was no correlation of mean particle size with centrifugation speed. In all samples, higher concentrations of small particles (< 300 nm) and lower concentrations of larger particles (> 300 nm) were observed. In analyses of sized microsphere samples, monodisperse samples of 200 nm beads accurately measured expected size distribution with a high repeatability. Increases in concentrations by factors of 2 and 4 resulted in accurately measured increases for the 200 nm beads. In contrast, monodisperse samples of 500 and 800 nm beads measured lower concentrations when increased by the same factors. Results for polydisperse samples of 200, 500, and 800 nm beads, with equal concentration ratios (1:1:1) showed lower concentrations for 500 and 800 nm beads, compared to 200 nm (7.29×107, 3.78×107, vs, 10.3×107, respectively).CONCLUSIONRegardless of the actual composition of EV subpopulation, NTA measures an average particle size of ~200 nm in polydisperse samples. Analysis of synthetic size beads revealed that this may be because, at least in part, the NTA method underestimates the concentration for larger particles with a size greater than 200 nm. Our results suggest that caution should be used when analyzing polydisperse samples (i.e. serum/plasma) using a nanoparticle tracking method.Support or Funding InformationSupported by NIH Grant R01NS102157This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Use of ultrasound-assisted co-precipitation route to obtain CuMnO2 semiconductor nanomaterials

CuMnO2 nanoparticles were obtained for the first time by sonochemical synthesis in an aqueous solution of sodium hydroxide containing soluble copper and manganese nitrate reactants without any further thermal treatment. Experiments were performed at different lengths of time (20 min, 30 and 40 min) and ultrasound amplitude of 80%. The materials present the expected crednerite structure (C2/m space group), and the nanoparticles average size and morphology for each sample are studied by nanoparticle tracking analysis method, respectively, by transmission electron microscopy. The as-obtained materials display p-type semiconductivity behavior at room temperature and the value for direct optical band gap EG is estimated to be approximately ~ 3.6 eV. The study of the magnetic properties for the nanocrystalline crednerite samples reveals 2D magnetic behavior below 110 K and a weak ferromagnetic component at ~ 20 K.

304. Particle Titer Determination and Characterization of rAAV Molecules Using Nanoparticle Tracking Analysis

The development and commercialization of a biological pharmaceutical, such as recombinant adeno associated virus (rAAV), requires rigorous quantitative and qualitative testing. An accurate particle titer measurement is important for the development of purification processes. Current methods for titering rAAV include qPCR, for measuring genome copy titer, and ELISA, for measuring particle titer. Additionally, methods such as dynamic light scattering (DLS) have been considered for assessment of particle size and aggregation but with limited success for rAAV. Here, a novel method is described called Nanoparticle Tracking Analysis (NTA), to provide rAAV titer (concentration) within minutes, as opposed to hours or days required by traditional techniques. NTA characterization was enabled by a gold labeling technique to increase the light scattered from rAAVs. With a purified gold labelled rAAV sample, NTA results indicated a monodisperse size distribution and a measured rAAV particle titer equivalent to the concentration based on genome copies by qPCR. Particle titer determination can also be relevant for assessment of product stability. NTA was used to assess the stability of rAAV over a 3 month thermal stress test. The NTA method enabled the detection of a reduction of the primary rAAV particle peak within 1 month, as well as further reductions over time. These initial results demonstrate the potential uses of NTA for product characterization.

590 UVB generates microvesicle particles via Platelet-activating Factor-receptor signaling: a novel pathway by which a skin-specific stimulus exerts systemic effects

590 UVB generates microvesicle particles via Platelet-activating Factor-receptor signaling: a novel pathway by which a skin-specific stimulus exerts systemic effects

Properties of nano encapsulated L-arginine and resveratrol in water-soluble polymers

This article analyzes properties of nano encapsulated L-arginine and resveratrol using a Nanoparticle Tracking Analysis method, and studies their supramolecular properties using a self-organization method.

Positive dielectrophoresis used for selective trapping of nanoparticles from flue gas in a gradient field electrodes device

This paper investigates the possibility to use positive dielectrophoresis (pDEP) for selective trapping of nanoparticle dispersed in flue gas in a vertical pDEP-based microfluidic system. The experimental gradient field electrodes device contains as main part a vertical deposition plate with parallel planar electrodes in single connection on an insulating substrate, parallel to the reference electrode—a dielectric plate with a metalized side. The performances of the device were described and analyzed by numerical simulations and experimental tests in terms of two new specific parameters, called Retention rate and Filtration, related to the trapping of nanoparticles in suspension inside the device and the consequent purification of flue gas. It is outlined, both numerically and experimentally, that the concentration of particles trapped inside the device decreases as they are moving away from the inlet zone. The experimental results also highlight the nanoparticle size distribution of the particles collected from the deposition plate, using a nanoparticle tracking analysis method, and their selective capture on the deposition plate, depending on the amplitude and shape of the applied voltage, in a good agreement with the numerical simulations results.

Nanoparticle Tracking Analysis of Latex Standardized Beads

Abstract The most popular technique for particle size characterization is the dynamic light scattering (DLS). In recent years new advanced method based on counting each single particle movement was introduced giving perspective for measurement of each component of mixture. This study presents some advantages of nanoparticle tracking analysis (NTA) method in comparison to DLS. For tests standard polystyrene beds were chosen vary diameter of 22, 61 and 150 nm and its mixtures. Experiments showed that the particles size resolution allows to distinguish each population in two population suspension opposed to DLS. The NTA method permits to eliminate the negative effects i.e. dust or aggregates in sample during post processing, that permits to use it in a variety of studies.