Abstract: Exosomes are vesicles that are released from the kidney into urine. They contain protein and RNA from the glomerulus and all sections of the nephron and represent a reservoir for biomarker discovery. Current methods for the identification and quantification of urinary exosomes are time consuming and only semi-quantitative. Nanoparticle tracking analysis (NTA) counts and sizes particles by measuring their Brownian motion in solution. In this study, we applied NTA to human urine and identified particles with a range of sizes. Using antibodies against the exosomal proteins CD24 and aquaporin 2 (AQP2), conjugated to a fluorophore, we could identify a subpopulation of CD24- and AQP2-positive particles of characteristic exosomal size. Extensive pre-NTA processing of urine was not necessary. However, the intra-assay variability in the measurement of exosome concentration was significantly reduced when an ultracentrifugation step preceded NTA. Without any sample processing, NTA tracked exosomal AQP2 upregulation induced by desmopressin stimulation of kidney collecting duct cells. Nanoparticle tracking analysis was also able to track changes in exosomal AQP2 concentration that followed desmopressin treatment of mice and a patient with central diabetes insipidus. When urine was stored at room temperature, 4°C or frozen, nanoparticle concentration was reduced; freezing at -80°C with the addition of protease inhibitors produced the least reduction. In conclusion, with appropriate sample storage, NTA has potential as a tool for the characterization and quantification of extracellular vesicles in human urine.

Abstract: 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.

Abstract: Nanoparticle Tracking Analysis (NTA), developed by NanoSight, has been proved to be a highly useful, simple and efficient characterisation tool to differentiate between the capping efficiencies of various biomass-derived stabilising agents (e.g. starch, alginic acid and waste-derived hemicellulosic syrup) of aqueous colloidal silver suspensions. The results indicated that the use of a complex biorefinery-derived hemicellulosic syrup containing a mixture of C5 and C6 sugars, as well as oligomers, provided comparable capping and stabilisation properties to those of the most efficient pure polysaccharides including alginic acid. These findings illustrate the potential of waste-derived feedstocks for the stabilisation of nanoparticles in solution.

Abstract: Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10. A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

Abstract: 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.

Abstract: The primary objective of my thesis work is to establish a set of design criteria for nanoparticles whose purpose is to safely and efficiently access the brain after systemic injection. Nanoparticles that can access the brain may be able to deliver therapeutic molecules to the brain that otherwise would be excluded by the blood-brain barrier. E. coli glycoprotein 96 (Ecgp96) is explored as a candidate receptor on the blood-brain barrier that could potentially facilitate nanoparticle-receptor mediated transcytosis into the brain. Results from studies utilizing PET/CT, SPECT/CT, MRI, Xenogen fluorescence imaging, and confocal microscopy conclude that Ecgp96 is observed in the blood-brain barrier endothelial cells, but is not accessible from the blood of adult or neonatal mice under normal, non-pathological conditions. Transferrin receptor is a well-characterized receptor on the blood-brain barrier that is accessible from the blood and known to transcytose transferrin. I focused on this receptor and on synthesizing and characterizing a well-defined set of transferrin containing gold nanoparticles of various sizes and transferrin compositions that would be investigated during in-vivo studies. Nanoparticle sizes were measured by DLS and nanoparticle tracking analysis. Zeta potentials were also measured. Nanoparticle transferrin content was directly measured by labeling transferrin with 64Cu and measuring the nanoparticle associated gamma activity. The nanoparticle binding avidities to mouse transferrin receptors were ranked by a silver enhancement fluorescence-based method using the mouse Neruo2A cell line. Each nanoparticle formulation was systemically injected into mice, and localization in the mouse brain was observed by silver enhancement light microscopy, and TEM. The quantitation of the gold was determined by ICP-MS. Nanoparticles with large amounts of transferrin remain strongly attached to brain endothelial cells, while nanoparticles with less transferrin are capable of both interacting with transferrin receptor on the luminal side of the blood-brain barrier and detaching from transferrin receptor on the brain side of the blood-brain barrier. These results highlight the fact that the nanoparticle avidity must be tuned to maximize the number of nanoparticles exiting the endothelial cells and entering the brain tissue. Lanthanum nitrate perfusion-fixation studies demonstrate that the nanoparticle formulations investigated do not degrade the blood-brain barrier integrity and enter the brain by transferrin receptor-mediated transcytosis. The results from these studies provide initial design criteria for creating nanoparticle therapeutics for delivery to the brain from systemic administrations.

Abstract: Nanoparticle tracking analysis (NTA) was first applied to biologically active nanocomplexes to obtain concurrent information on their size, state of aggregation, concentration, and antigenic specificity in liquid. The subject of the NTA was an immunogenic complex (a candidate nanovaccine) comprised of spherical particles (SPs) generated by thermal remodeling of the tobacco mosaic virus and Rubella virus tetraepitopes exposed on the surface of SP.