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.

One of the greatest challenges in the manufacturing and development of nanotechnologies is the requirement for robust, reliable, and accurate characterization data. Presented here are the results of an interlaboratory comparison (ILC) brought about through multiple rounds of engagement with NanoSight Malvern and ten pan-European research facilities. Following refinement of the nanoparticle tracking analysis (NTA) technique, the size and concentration characterization of nanoparticles in liquid suspension was proven to be robust and reproducible for multiple sample types in monomodal, binary, or multimodal mixtures. The limits of measurement were shown to exceed the 30–600 nm range (with all system models), with percentage coefficients of variation (% CV) being calculated as sub 5% for monodisperse samples. Particle size distributions were also improved through the incorporation of the finite track length adjustment (FTLA) algorithm, which most noticeably acts to improve the resolution of multimodal sample mixtures. The addition of a software correction to account for variations between instruments also dramatically increased the accuracy and reproducibility of concentration measurements. When combined, the advances brought about during the interlaboratory comparisons allow for the simultaneous determination of accurate and precise nanoparticle sizing and concentration data in one measurement.

Fluorescence nanoparticle tracking analysis (fl-NTA) allows for accurate sizing, counting, and phenotyping of extracellular vesicles (EV). Here, we present two protocols for the analysis of EVs using fl-NTA, highlighting the potential pitfalls and challenges. The first protocol utilizes CellMask Orange™ (CMO) as a general membrane marker to label EVs derived from plasma. The second protocol describes the use of a Qdot-conjugated antibody to identify syncytiotrophoblast (STB)-derived EVs. "Standard" preparations of STB-derived EVs enriched for either microvesicles (STBMV) or exosomes (STBEX), containing a known amount of EV positive for the STB specific antigen placental alkaline phosphatase (PLAP), were also used to optimize fl-NTA camera settings.

Functionalised nanomaterials are gaining popularity for use as drug delivery vehicles and, in particular, mucus penetrating nanoparticles may improve drug bioavailability via the oral route. To date, few polymers have been investigated for their muco-penetration, and the effects of systematic structural changes to polymer architectures on the penetration and diffusion of functionalised nanomaterials through mucosal tissue have not been reported. We investigated the influence of poly(2-oxazoline) alkyl side chain length on nanoparticle diffusion; poly(2-methyl-2-oxazoline), poly(2-ethyl-2-oxazoline), and poly (2-n-propyl-2-oxazoline) were grafted onto the surface of thiolated silica nanoparticles and characterised by FT-IR, Raman and NMR spectroscopy, thermogravimetric analysis, and small angle neutron scattering. Diffusion coefficients were determined in water and in a mucin dispersion (using Nanoparticle Tracking Analysis), and penetration through a mucosal barrier was assessed using an ex vivo fluorescence technique. The addition of a single methylene group in the side chain significantly altered the penetration and diffusion of the materials in both mucin dispersions and mucosal tissue. Nanoparticles functionalised with poly(2-methyl-2-oxazoline) were significantly more diffusive than particles with poly(2-ethyl-2-oxazoline) while particles with poly(2-n-propyl-2-oxazoline) showed no significant increase compared to the unfunctionalised particles. These data show that variations in the polymer structure can radically alter their diffusive properties with clear implications for the future design of mucus penetrating systems.

The increasing use of nanoparticles in the pharmaceutical industry is generating concomitant interest in developing nanomaterials that can rapidly penetrate into, and permeate through, biological membranes to facilitate drug delivery and improve the bioavailability of active pharmaceutical ingredients. Here, we demonstrate that the permeation of thiolated silica nanoparticles through porcine gastric mucosa can be significantly enhanced by their functionalization with either 5 kDa poly(2-ethyl-2-oxazoline) or poly(ethylene glycol). Nanoparticle diffusion was assessed using two independent techniques; Nanoparticle Tracking Analysis, and fluorescence microscopy. Our results show that poly(2-ethyl-2-oxazoline) and poly(ethylene glycol) have comparable abilities to enhance diffusion of silica nanoparticles in mucin dispersions and through the gastric mucosa. These findings provide a new strategy in the design of nanomedicines, by surface modification or nanoparticle core construction, for enhanced transmucosal drug delivery.

NanoParticle Tracking and Analysis (NTA), has been commercially developed with over 600 systems installed, and is now considered a key characterisation technique in nanomedecine, studying environmental effects on nanoparticles in colloidal suspension and nanometrology. This technique gives significant advantage in sizing over DLS as the individual tracking of particles results in a better ability to measure polydispersed suspension and a better assessment of degree of aggregation. NTA also generates a number-based concentration measurement directly, a crucial parameter in the assessment of dosimetry for nanoparticles where weight-based concentration measurements are less relevant. The sample can have an electric field applied to allow the measurement of zeta potential - an important indicator of likelihood of future aggregation. Finally the technique can also be integrated with fluorescence filters to allow fluorescently labelled/loaded particles to be selectively analysed. This can be of particular import when analysing the sample in complex biological media, such as cell culture media or in the presence of protein suspensions. The technique is used for characterisation of exosomes shed from cells for e.g. cell signalling. In this case using NTA in conjunction with fluorescently labelled antibodies may enable speciating the exosomes. Another key application is in measuring a wide range of engineered nanoparticles in more complex media, including river and sea water, cell culture media and buffer solutions. The technique, novel developments and its application to the above fields will be described, explaining the importance of obtaining as complete characterisation as is possible, in as relevant media as possible.

Abstract Cellular microvesicles (100 nm-1µm) are shed into the circulation from many different cell types including; platelets, erythrocytes and endothelial cells. They have major potential as novel biomarkers as they have been shown to be implicated in a multitude of pathological conditions. However, developments in this area are constrained by limitations in the technology available for their measurement. Nanoparticle Tracking Analysis (NTA) (NanoSight Ltd, Amesbury) offers the potential to both enumerate and speciate (through fluorescent markers) these microparticles in a rapid manner. In this method a laser beam passes through a suspension at a low angle. The particles scatter light which is collected onto a CCD (or sCMOS) camera by a microscope-type configuration. Particles between 10-2000 nm are tracked individually and their diffusion coefficient, and therefore size, calculated directly from their speed. Therefore, this characterization method gives a direct measurement of the concentration and size distribution of the particles in the field of view. If the particles are fluorescently labelled, then the concentration and size distribution are those of the labelled particles only, allowing a proportion of labelled particles to be calculated. However, this requires a combination of aspects to be identified and optimised during the process. This talk will look at some of the challenges and considerations that need to be taken during the protocol development and measurement. It will also review the application of the technique in this field, compare and contrast it to other applicable technologies such as various forms of electron microscopy (EM) and to flow cytometry. Citation Format: Sonja Capracotta, Pauline Carnell, Andrew Malloy, Patrick Hole, Bob Carr. Using Nanoparticle Tracking Analysis (NTA) to characterize cellular microvesicles. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1869. doi:10.1158/1538-7445.AM2014-1869

INTRODUCTION:Optical techniques are routinely used to size and count extracellular vesicles (EV). For comparison of data from different methods and laboratories, suitable calibrators are essential. A suitable calibrator must have a refractive index (RI) as close to that of EV as possible but the RI of EV is currently unknown. To measure EV, RI requires accurate knowledge of size and light scattering. These are difficult to measure as most EVs cannot be resolved by light microscopy and their diameter is smaller than the wavelength of visible light. However, nanoparticle tracking analysis (NTA) provides both size and relative light scattering intensity (rLSI) values. We therefore sought to determine whether it was possible to use NTA to measure the RI of individual EVs. METHODS:NTA was used to measure the rLSI and size of polystyrene and silica microspheres of known size and RI (1.470 and 1.633, respectively) and of EV isolated from a wide range of cells. We developed software, based on Mie scattering code, to calculate particle RI from the rLSI data. This modelled theoretical scattering intensities for polystyrene and silica microspheres of known size (100 and 200 nm) and RI. The model was verified using data from the polystyrene and silica microspheres. Size and rLSI data for each vesicle were processed by the software to generate RI values. RESULTS:The following modal RI measurements were obtained: fresh urinary EV 1.374, lyophilised urinary EV 1.367, neuroblastoma EV 1.393, blood EV 1.398, EV from activated platelets 1.390, small placental EV 1.364-1.375 and 1.398-1.414 for large placental EV (>200 nm). Large placental EV had a significantly higher RI than small placental EV (p<0.0001). The spread of RI values was narrower for small EV than for the more heterogeneous large EV. DISCUSSION:Using NTA and Mie scattering theory, we have demonstrated that it is possible to estimate the RI of sub-micron EV using NTA data. EV typically had a modal RI of 1.37-1.39, whereas values of >1.40 were observed for some large (>200 nm) microvesicles. CONCLUSION:This method for measuring EV RI will be useful for developing appropriate calibrators for EV measurement.

One of the key challenges in the field of nanoparticle (NP) analysis is in producing reliable and reproducible characterisation data for nanomaterials. This study looks at the reproducibility using a relatively new, but rapidly adopted, technique, Nanoparticle Tracking Analysis (NTA) on a range of particle sizes and materials in several different media. It describes the protocol development and presents both the data and analysis of results obtained from 12 laboratories, mostly based in Europe, who are primarily QualityNano members. QualityNano is an EU FP7 funded Research Infrastructure that integrates 28 European analytical and experimental facilities in nanotechnology, medicine and natural sciences with the goal of developing and implementing best practice and quality in all aspects of nanosafety assessment. This study looks at both the development of the protocol and how this leads to highly reproducible results amongst participants. In this study, the parameter being measured is the modal particle size.

Understanding nanoparticle diffusion within non-Newtonian biological and synthetic fluids is essential in designing novel formulations (e.g., nanomedicines for drug delivery, shampoos, lotions, coatings, paints, etc.), but is presently poorly defined. This study reports the diffusion of thiolated and PEGylated silica nanoparticles, characterized by small-angle neutron scattering, in solutions of various water-soluble polymers such as poly(acrylic acid) (PAA), poly(N-vinylpyrrolidone) (PVP), poly(ethylene oxide) (PEO), and hydroxyethylcellulose (HEC) probed using Nano Sight nanoparticle tracking analysis. Results show that the diffusivity of nanoparticles is affected by their dimensions, medium viscosity, and, in particular, the specific interactions between nanoparticles and the macromolecules in solution; strong attractive interactions such as hydrogen bonding hamper diffusion. The water-soluble polymers retarded the diffusion of thiolated particles in the order PEO > PVP > PAA > HEC whereas for PEGylated silica particles retardation followed the order PAA > PVP = HEC > PEO. In the absence of specific interactions with the medium, PEGylated nanoparticles exhibit enhanced mobility compared to their thiolated counterparts despite some increase in their dimensions.

Cellular microvesicles and nanovesicles (exosomes) are involved in many disease processes and have major potential as biomarkers. However, developments in this area are constrained by limitations in the technology available for their measurement. Here we report on the use of fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles. In this system vesicles are visualized by light scattering using a light microscope. A video is taken, and the NTA software tracks the brownian motion of individual vesicles and calculates their size and total concentration. Using human placental vesicles and plasma, we have demonstrated that NTA can measure cellular vesicles as small as similar to 50 nm and is far more sensitive than conventional flow cytometry (lower limit similar to 300 nm). By combining NTA with fluorescence measurement we have demonstrated that vesicles can be labeled with specific antibody-conjugated quantum dots, allowing their phenotype to be determined.From the Clinical Editor: The authors of this study utilized fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles, demonstrating that NTA is far more sensitive than conventional flow cytometry. (C) 2011 Elsevier Inc. All rights reserved.

A novel addition to a technique for the analysis of nanoparticles in a suspension is described. The Nanoparticle Tracking Analysis (NTA) technique sizes individual nanoparticles, based on their Brownian motion. NTA allows nanoparticles to be sized on a particle-byparticle basis, resulting in a higher resolution analysis and therefore a better understanding of polydispersity than ensemble methods (such as dynamic light scattering, DLS) and it also yields directly a count/concentration measurement. Analysis of scattering intensity is a recent development allowing sub-populations of nanoparticles with varying scattering characteristics to be resolved in a complex mixture. Now this technique has been extended to the analysis and differentiation of fluorescently labeled nanoparticles. With the appropriate wavelength lasers and optical filters, the technique has been shown to be able to differentiate between sub-populations in a heterogeneous mixture.