Nanoparticle Tracking of Adenovirus by Light Scattering and Fluorescence Detection.

The phospholipid bilayer is a highly dynamic structure. Approximately 2% is recycled every 5–10 min, so the whole membrane is recycled every 1–2 h. The process involves constant formation of endosomes that bud off by endocytosis from the plasma membrane and become internalised into the cytoplasm. These endosomes, together with their associated intra-membranous proteins, represent a snapshot of that cell's plasma membrane composition. Further rounds of endocytosis within the endosomes themselves generate intracellular multivesicular bodies. Upon fusing with the plasma membrane, these endosomes release their contents into the circulation (they were first identified in the maturing mammalian reticulocyte) or, in the case of renal tubular epithelial cells, into the urine. The resultant urinary ‘exosomes’ may be characterised by their size (generally 20–100 nm) and density (1.10-1.19 mg ml−1). They are representative of the plasma membrane from which they originated and therefore offer a potential window into the pathophysiology of the kidney, providing information about changes in membrane or cytosolic composition from specific segments of the nephron. Chronic kidney disease (CKD) is highly prevalent and is expected to increase further in the next 5–10 years because of the rising prevalence of obesity and diabetes. Acute kidney injury (AKI), the loss of kidney function over hours to days, is also very common, being seen in up to 20% of acute hospital admissions. There are often delays in detection of both AKI and CKD, which can lead to worse clinical outcomes. The use of urinary biomarkers is key to the early detection of kidney disease (and also to some systemic conditions that might lead to changes in renal epithelial composition). Urine is an excellent fluid for biomarker discovery and development, having sufficient quantities of measureable peptides and/or proteins and being relatively easy to obtain non-invasively in reasonable quantities (assuming the patient is not oliguric). Hence, some urinary biomarkers such as albumin/creatinine ratio (ACR) are already used in routine clinical practice. Detection of increased ACR might, for example, be the first sign of diabetic nephropathy. AKI is currently defined by an increase in serum creatinine or a fall in urine output, but these changes can occur relatively late with respect to the renal injury, potentially leading to delays in treatment. Urinary biomarkers such as neutrophil gelatinase associated lipocalin (NGAL) and kidney injury molecule 1 (KIM-1) have shown promise as novel early biomarkers of AKI. Biomarker discovery and development is an active field of basic and clinical research. More detailed examination of the urinary proteome, including analysis of exosomes, creates further opportunity for discovery of clinically useful early biomarkers of disease. Putative exosomal biomarkers of AKI have been reported, such as the Na+/H+ exchanger isoform 3 (NHE3) (du Cheyron et al. 2003) and Fetuin-A (Zhou et al. 2006) but with improved technology for exosome analysis, more sensitive and specific biomarkers might be discovered. To date, the difficulty with determining and quantifying urinary exosomes has been their lability, small size and particular density. Accurate and reproducible identification has been labour intensive and expensive, and has required specific laboratory equipment and skills (e.g. Western blotting). A new study published in the current issue of the Journal of Physiology (Oosthuyzen et al. 2013) suggests a novel approach that may change this situation. Using nanoparticle tracking analysis (NTA) Oosthuyzen et al. successfully identified a range of particle sizes in urine, including those classified typically as exosomes. They validated the technique by fluorescently tagging known exosomal proteins such as CD24 (a cell surface marker) and aquaporin 2 (AQP2) and co-localising their fluorescent read-out in the range of particle sizes typically defining exosomes (20–100 nm). They prospectively identified an increase in the output of urinary exosomes tagged with AQP2 under known stimulatory conditions (treatment with the arginine vasopressin analogue, desmopressin). The authors conducted their studies in a cell line, then in an animal model, and finally in five healthy volunteers and a patient with central diabetes insipidus treated with desmopressin. The authors also established optimal conditions for urine storage for potential use in biomarker discovery studies using NTA. So what exactly is NTA? NTA was invented in the UK by Dr Bob Carr who subsequently founded Nanosight Ltd (http://www.nanosight.com/) in 2003. NTA is used to observe (in conjunction with a high-powered microscope) and analyse (using specialised software) particle movement within a solution. The rate of movement of these particles (Brownian motion) is determined by a number of factors including particle size, viscosity and temperature of the liquid but is not affected by particle density or refractive index. Thus, using NTA, a size distribution profile of small (10–1000 nm) particles in solution (e.g. urine) can be produced with minimal sample preparation and hence time associated with the procedure. With further development, refinements and validation then it may be possible for the analysis to be done in real time with little to no preparation. However, given the complexity of the equipment required, a simple point of care (‘bedside’) test would appear to be some time off. Nevertheless, by describing optimal handling of samples for NTA of urinary exosomes, the authors have contributed to bringing this exciting technique a step closer to routine clinical use. No doubt researchers in acute and chronic kidney disease, interested in identification of disease biomarkers, will take note.

Vaccine thermostability is key to successful global immunization programs as it may have a significant impact on the continuous cold-chain maintenance logistics, as well as affect vaccine potency. Modern biological and biophysical techniques were combined to in-depth characterize the thermostability of a formulated rabies virus (RABV) in terms of antigenic and genomic titer, virus particle count and aggregation state. Tunable resistive pulse sensing (TRPS) and nanoparticle tracking analysis (NTA) were used to count virus particles while simultaneously determining their size distribution. RABV antigenicity was assessed by NTA using a monoclonal antibody that recognize a rabies glycoprotein (G protein) conformational epitope, enabling to specifically count antigenic rabies viruses. Agreement between antigenicity results from NTA and conventional method, as ELISA, was demonstrated. Additionally, NTA and ELISA showed mirrored loss of RABV antigenicity during forced degradation studies performed between 5 °C and 45 °C temperature exposure for one month. Concomitant with decreased antigenicity, emergence of RABV particle populations larger than those expected for rabies family viruses was observed, suggesting RABV aggregation induced by thermal stress. Finally, using a kinetic-based modeling approach to explore forced degradation antigenicity data (NTA, ELISA), a two-step model accurately describing antigenicity loss was identified. This model predicted a RABV shelf-life of more than 3 years at 5 °C; significant loss of antigenicity was predicted for samples maintained several months at ambient temperature. This thorough characterization of RABV forced degradation study originally provided a time-temperature mapping of RABV stability.

Microparticles (MP) are shed extracellular vesicles that express the prothrombotic tissue factor (TF). Aerobic exercise may reduce MP count and TF expression. This study investigated the impact of acute running or rest followed by standardized meal consumption on MP phenotypes and TF expression. Fifteen males (age, 22.9 ± 3.3 yr; body mass, 81.9 ± 11.4 kg; V˙O2max, 54.9 ± 6.5 mL·kg·min; mean ± SD) completed 1 h of running (70% V˙O2max) or rest at 9:00 AM and consumed a standardized meal (1170 kcal, 43% CHO, 17% PRO, 40% fat) at 10:45 AM. Venous blood samples were taken at 9:00 AM, 10:00 AM, and 11:30 AM. The MP concentration, diameter, phenotypes, and TF expression were assessed using nanoparticle tracking analysis and flow cytometry. Nanoparticle tracking analysis identified no changes in MP concentration or diameter in response to time or trial. Flow cytometry revealed total MP count increased from 9:00 AM to 10:00 AM (1.62 ± 2.28 to 1.74 ± 2.61 × 10 L, P = 0.016, effect size (η) = 0.105), but was unaffected by trial. TF platelet-derived MP % reduced from 9:00 AM to 10:00 AM (44.0% ± 21.2% to 21.5% ± 9.3%, P = 0.001, η = 0.582) after exercise only (control, 36.8% ± 18.2% to 34.9% ± 11.9%; P = 0.972). TF neutrophil-derived MP percentage reduced from 9:00 AM to 11:30 AM (42.3% ± 17.2% to 25.1% ± 14.9%; P = 0.048, η = 0.801) in the exercise trial only (control, 28.5% ± 15.7% to 32.2% ± 9.6%; P = 0.508). Running induced a significant reduction in %TF platelet and neutrophil MP, suggesting a transient reduction in cardiovascular risk via reduced TF-stimulated thrombosis. This requires further investigation over longer periods in cardiovascular disease populations.

Physical Characterization and Stabilization of a Lentiviral Vector Against Adsorption and Freeze-Thaw

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.

Nanoparticle Tracking Analysis (NTA) is an emerging analytical technique developed for detection, sizing, and counting of sub-micron particles in liquid media. Its feasibility for use in biopharmaceutical development was evaluated with particle standards and recombinant protein solutions. Measurements of aqueous suspensions of NIST-traceable polystyrene particle standards showed accurate particle concentration detection between 2 × 10(7) and 5 × 10(9) particles/mL. Sizing was accurate for particle standards up to 200 nm. Smaller than nominal value sizes were detected by NTA for the 300-900 nm particles. Measurements of protein solutions showed that NTA performance is solution-specific. Reduced sensitivity, especially in opalescent solutions, was observed. Measurements in such solutions may require sample dilution; however, common sample manipulations, such as dilution and filtration, may result in particle formation. Dilution and filtration case studies are presented to further illustrate such behavior. To benchmark general performance, NTA was compared against asymmetric flow field flow fractionation coupled with multi-angle light scattering (aF4-MALS) and dynamic light scattering, which are other techniques for sub-micron particles. Data shows that all three methods have limitations and may not work equally well under certain conditions. Nevertheless, the ability of NTA to directly detect and count sub-micron particles is a feature not matched by aF4-MALS or dynamic light scattering. Thorough characterization of particulate matter present in protein therapeutics is limited by the lack of analytical methods for particles in the sub-micron size range. Emerging techniques are being developed to bridge this analytical gap. In this study, Nanoparticle Tracking Analysis is evaluated as a potential tool for biologics development. Our results indicate that method performance is molecule-specific and may not work as well under all solution conditions, especially when testing opalescent solutions. Advantages and disadvantages of Nanoparticle Tracking Analysis are discussed in comparison to other analytical techniques for particles in the sub-micron size range.

Growing interest in extracellular vesicles (EV) has necessitated development of protocols to improve EV characterization as a precursor for myriad downstream investigations. Identifying expression of EV surface epitopes can aid in determining EV enrichment and allow for comparisons of sample phenotypes. This study was designed to test a rigorous method of indirect fluorescent immunolabeling of single EV with subsequent evaluation using nanoparticle tracking analysis (NTA) to simultaneously determine EV concentration, particle size distribution, and surface immunophenotype. In this study, EV were isolated from canine and human cell cultures for immunolabeling and characterized using NTA, transmission electron microscopy, and Western blotting. Indirect fluorescent immunolabeling utilizing quantum dots (Qd) resulted in reproducible detection of individual fluorescently labeled EV using NTA. Methods were proposed to evaluate the success of immunolabeling based on paired particle detection in NTA light scatter and fluorescent modes. Bead-assisted depletion and size-exclusion chromatography improved specificity of Qd labeling. The described method for indirect immunolabeling of EV and single vesicle detection using NTA offers an improved method for estimating the fraction of EV that express a specific epitope, while approximating population size distribution and concentration.

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 50-300nm. NTA and DLS are performed on polystyrene size standards as well as protein and placebo formulations. Protein formulations filtered through a 20nm filter, with and without polysorbate-80, show NTA particle counts. As such, particle counts above 20nm 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.

PurposeTo evaluate the nanoparticle tracking analysis (NTA) technique, compare it with dynamic light scattering (DLS) and test its performance in characterizing drug delivery nanoparticles and protein aggregates.MethodsStandard polystyrene beads of sizes ranging from 60 to 1,000 nm and physical mixtures thereof were analyzed with NTA and DLS. The influence of different ratios of particle populations was tested. Drug delivery nanoparticles and protein aggregates were analyzed by NTA and DLS. Live monitoring of heat-induced protein aggregation was performed with NTA.ResultsNTA was shown to accurately analyze the size distribution of monodisperse and polydisperse samples. Sample visualization and individual particle tracking are features that enable a thorough size distribution analysis. The presence of small amounts of large (1,000 nm) particles generally does not compromise the accuracy of NTA measurements, and a broad range of population ratios can easily be detected and accurately sized. NTA proved to be suitable to characterize drug delivery nanoparticles and protein aggregates, complementing DLS. Live monitoring of heat-induced protein aggregation provides information about aggregation kinetics and size of submicron aggregates.ConclusionNTA is a powerful characterization technique that complements DLS and is particularly valuable for analyzing polydisperse nanosized particles and protein aggregates.

Novel vaccine platforms for delivery of nucleic acids based on viral and non-viral vectors, such as recombinant adeno associated viruses (rAAV) and lipid-based nanoparticles (LNPs), hold great promise. However, they pose significant manufacturing and analytical challenges due to their intrinsic structural complexity. During product development and process control, their design, characterization, and quality control require the combination of fit-for-purpose complementary analytical tools. Moreover, an in-depth methodological expertise and holistic approach to data analysis are required for robust measurements and to enable an adequate interpretation of experimental findings. Here the combination of complementary label-free biophysical techniques, including dynamic light scattering (DLS), multiangle-DLS (MADLS), Electrophoretic Light Scattering (ELS), nanoparticle tracking analysis (NTA), multiple detection SEC and differential scanning calorimetry (DSC), have been successfully used for the characterization of physical and chemical attributes of rAAV and LNPs encapsulating mRNA. Methods’ performance, applicability, dynamic range of detection and method optimization are discussed for the measurements of multiple critical physical−chemical quality attributes, including particle size distribution, aggregation propensity, polydispersity, particle concentration, particle structural properties and nucleic acid payload.

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.

Protein-conjugated gold nanoparticles (AuNPs) have been extensively explored for the development of many novel protein assays. In this article, we demonstrate that nanoparticle tracking analysis (NTA) can be used as a rapid and simple analytical tool to monitor bioconjugation and to study protein-protein interactions. The adsorption of protein A onto gold nanoparticles was analyzed using NTA. The conjugation resulted in a measurable increase in hydrodynamic radius that correlated with protein A concentration, allowing conditions for complete conjugation to be elucidated. NTA was then used to investigate the binding of mouse IgG to protein A-conjugated AuNPs and the K(a) was measured as 2.00 × 10(7) M(-1). Furthermore, an assay for the detection of mouse IgG was developed using NTA to detect the binding to antibody-AuNP conjugates. This assay provided a detection limit of 3.2 ng mL(-1); however, the formation of aggregates resulting from the use of a polyclonal antibody and multiple binding sites on the antigen prevented the determination of binding affinity for this antibody-antigen system. To measure the binding affinity for this antibody-antigen system the IgG antigen was conjugated to the AuNPs and NTA was used to monitor the binding of the antibody. In this configuration aggregation of conjugates was not detected and a binding affinity constant of 2.80 × 10(8) M(-1) was measured. NTA results obtained in this work were validated by comparison to DLS. This work represents the first evaluation of NTA as an analytical tool for characterizing AuNP bioconjugates, investigating protein-protein binding, and detecting low levels of antigen in a bioassay.

Nanoparticle tracking analysis versus dynamic light scattering: Case study on the effect of Ca2+ and alginate on the aggregation of cerium oxide nanoparticles.

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. 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. 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. 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.

We show that the electrochemical particle-impact technique (or 'nano-impacts') complements light scattering techniques for sizing both mono- and poly-disperse nanoparticles. It is found that established techniques - Dynamic Light Scattering (DLS) and Nanoparticle Tracking Analysis (NTA) - can accurately measure the diameters of '30 nm' silver particles assuming spherical shapes, but are unable to accurately size a smaller '20 nm' sample. In contrast, nano-impacts have a high accuracy (<5% error in effective diameters) and are able to size both individual '20 nm' and '30 nm' silver NPs in terms of the number of constituent atoms. Further study of a '20 nm and 30 nm' bimodal sample shows that the electrochemical technique resolves the two very similar sizes well, demonstrating accurate sizing regardless of particle size polydispersity, whereas due to inherent limitations of light scattering measurements this is not possible for DLS and NTA. Electrochemical sizing is concluded to offer significant attractions over light scattering methods.