An electron microscopy based method for the detection and quantification of nanomaterial number concentration in environmentally relevant media.

Nanoparticle tracking analysis

Chronic Helicobacter pylori (H. pylori) infection is considered the main risk factor for the development of gastric cancer. Pathophysiological changes in the gastric mucosa initiated by this bacterium can persist even after pharmacological eradication and are likely attributable also to changes induced in non-infected cells as a consequence of intercellular communication via extracellular vesicles (EVs). To better understand what such changes might entail, we isolated EVs from immortalized normal gastric GES-1 cells infected (EVHp+) or not with H. pylori (EVHp-) by ultracentrifugation and characterized them. Infection of GES-1 cells with H. pylori significantly increased the release of EVs and slightly decreased the EV mean size. Incubation with EVHp+ for 24 h decreased the viability of GES-1 cells, but increased the levels of IL-23 in GES-1 cells, as well as the migration of GES-1 and gastric cancer AGS cells. Furthermore, incubation of GES-1 and AGS cells with EVHp+, but not with EVHp-, promoted cell invasion and trans-endothelial migration in vitro. Moreover, stimulation of endothelial EA.hy926 cells for 16 h with EVHp+ promoted the formation of linked networks. Finally, analysis by mass spectrometry identified proteins uniquely present and others enriched in EVHp+ compared to EVHp-, several of which are known targets of hypoxia induced factor-1α (HIF-1α) that may promote the acquisition of traits important for the genesis/progression of gastric pre-neoplastic changes associated with H. pylori infection. In conclusion, the harmful effects of H. pylori infection associated with the development of gastric malignancies may spread via EVs to non-infected areas in the early and later stages of gastric carcinogenesis.

/pdf/extracellular-vesicles-from-gastric-epithelial-ges-1-cells-1z7gxuys.pdf

Extracellular vesicles from gastric epithelial GES-1 cells infected with Helicobacter pylori promote changes in recipient cells associated with malignancy

The loading of therapeutic actives into polymeric nanoparticles represents one of the approaches towards drug transport through the blood-brain barrier – the main obstacle to drug delivery into the central nervous system. The non-toxic, biocompatible and biodegradable polysaccharides chitosan and dextran were modified with permeation-enhancing alkylglyceryl pendant chains through reaction with epoxide precursors. The modified polysaccharides were characterised by spectroscopic methods (1H-, 13C-NMR and FT-IR). These polysaccharides were further formulated into nanoparticles using three methods, namely: nanoprecipitation, solvent displacement via dialysis and electrospraying. The resultant colloidal systems formed were characterised using Dynamic Light Scattering, Nanoparticle Tracking Analysis and Electrophoretic Mobility Measurements. Dried nanoparticles were further characterised by Scanning Electron Microscopy and Atomic Force Microscopy. Formulations of alkylglyceryldextran derivatives were found to be stable at the physiologically relevant pH of 7.4. Over the same range of pH values, formulations of alkyglyceryl-chitosans formed aggregates. Respectively dependent upon the method of formulation and the pH, nanoparticles from poly(lactic acid)-graft-butylglyceryl-modified dextran exhibited diameters in the range 100-400 nm and zeta potentials of between -15 and -30 mV. The preparation of nanoparticulate congeners that incorporated a fluorescent marker molecule (Doxorubicin, Rhodamine B or Fluorescein) allowed the studies of the capabilities of nanoparticles to accommodate and release a model therapeutic load. Rhodamine B-loaded nanoparticles further allowed the study of the uptake of nanoformulations by mouse (bEnd3) brain endothelial cells. The interactions of nanoparticles with modelled blood-brain barriers (mouse bEnd3 and human hCMEC/D3) were studied by Electric Cell Substrate Impedance Sensing and also by means of the Transwell model. Data from MTT and Presto Blue assays were consistent with the absence of nanoparticle-induced cytotoxic effects. An in ovo study that used 3-day chicken embryos indicated the absence of whole-organism acute toxicity effects but failed to unmask the biodistribution profile of nanoparticles. The results have shown that poly(lactic)-graftalkylglyceryl- modified dextran nanoparticles possess some promising features (size, stability, loading capacity, and toxicity) that render them candidates for further evaluation as biocompatible nanocarriers for drug delivery to the brain.

Nanoparticles from alkylglyceryl-modified polysaccharides for drug delivery to the brain

One of the main challenges of sizing methods for nanoparticle (NP) suspensions is to distinguish between particles and particle populations with very small size differences. This would be especially important to follow various surface functionalization processes of nanoparticles resulting in small alterations of their size. In this respect, methods involving the detection of single particles, such as resistive pulse sensing (RPS) or nanoparticle tracking analysis, are generally considered superior to ensemble measuring methods such as dynamic light scattering. However, to compare the exact capabilities of these methodologies require systematic investigations in optimized conditions for each method. Here, such a study is presented for a narrow size range of spherical latex nanoparticles (60–200 nm). It is concluded that the RPS methodology based on quartz nanopipets as single nanopore counters, is the only sizing method among those studied capable to fully resolve a ternary mixture of 70, 110, and 140 nm average diameter NPs. The practical usefulness of this size resolution is demonstrated by following the increase in diameter of latex nanoparticles after their surface modification with antibodies.

Resistive Pulse Sensing as a High‐Resolution Nanoparticle Sizing Method: A Comparative Study

The assessment of the complete distribution of nanoparticle sizes within a suspension is notoriously difficult to carry out. We demonstrate the Nanoparticle Tracking Analysis (NTA) technique that sizes nanoparticles in suspension, based on their Brownian motion. This technique has found significant use in the field of nano- and eco-toxicology, in several research groups showing of the technique to assess a range of engineered nanoparticles including gold, SiO2, TiO2 and polystyrene. This capability shares many features in common with conventional flow cytometry but is unique in this deeply sub-micron size range. NTA is a direct and fast technique by which nanoparticles in their natural solvated state in a liquid can be rapidly detected, sized and counted. The technique can be used to complement existing techniques for the sizing of nanoparticles (e.g., DLS, PCS) allowing data obtained from these methods to be validated by direct microscopical observation of the sample.

Characterisation of nanoparticle size and concentration for toxicological studies.

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Characterisation of nanoparticle size and concentration for toxicological studies.