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Heparin-induced thrombocytopenia (HIT) is a thrombocytopenia caused by heparin and mediated by an atypical immune mechanism leading to a paradoxical high thrombotic risk, associated with severe morbidity or death. The diagnosis of HIT combines a clinical scoring of pretest probability and laboratory testing. First-line routine tests are antigen binding assays detecting specific antibodies. The most sensitive of these tests have a high HIT-negative predictive value enabling HIT diagnosis to be ruled out when negative. However, HIT-positive predictive value is low, and a functional assay evaluating the pathogenicity of the antibodies should be performed to exclude false-positive results. In contrast to screening assays, functional assays are highly specific but technically challenging, and are thus performed in referral laboratories, where platelet activation is detected using radioactive serotonin (serotonin release assay, SRA) or visually (heparin-induced platelet activation, HIPA). Flow cytometry is a possible alternative. It is, however, currently not widely used, mostly because of the lack of standardization of the published assays. This article describes and discusses the standardization of a HIT flow cytometry assay (HIT-FCA) method, which subsequently led to the development and commercialization of a CE-marked assay (HIT Confirm®, Emosis, France) as a suitable rapid HIT functional test.

Efficient and safe delivery of nanoparticles (NPs) into the cytosol of living cells constitutes a major methodological challenge in bio-nanotechnology. Electroporation allows direct transfer of NPs into the cytosol by forming transient pores in the cell membrane, but it is criticized for invasiveness, and the applicable particle sizes are not well defined. Here, in order to establish principles for efficient delivery of NPs into the cytosol with minimal cytotoxicity, the influence of the size of NPs on their electroporation and intracellular behavior is investigated. For this study, fluorescent dye-loaded polymer NPs with core sizes between 10 and 40 nm are prepared. Optimizing the electroporation protocol allows minimizing contributions of endocytosis and to study directly the effect of NP size on electroporation. NPs of20 nm hydrodynamic size are efficiently delivered into the cytosol, whereas this is not the case for NPs of30 nm. Moreover, only particles of core size15 nm diffuse freely throughout the cytosol. While electroporation at excessive electric fields induces cytotoxicity, the use of small NPs20 nm allows efficient delivery at mild electroporation conditions. These results give clear methodological and design guidelines for the safe delivery of NPs for intracellular applications.

International audience; Controlling emission of bright luminescent nanoparticles by a single molecular recognition event remains an ultimate challenge in the design of ultrasensitive probes for biomolecules. It would require an efficient Förster resonance energy transfer (FRET) from the nanoparticle to a single acceptor at its surface, which is not realized to date. Here, we developed 20-nm light-harvesting nanoantenna particles, built of a tailor-made hydrophobic charged polymer poly(ethyl methacrylate-co-methacrylic acid), encapsulating ~1000 strongly coupled and highly emissive rhodamine dyes with their bulky counterion. Being 87-fold brighter than quantum dots QDots605 in single-particle microscopy (with 550-nm excitation), these DNA-functionalized nanoparticles exhibit >50% total FRET efficiency to a single hybridized FRET acceptor, a highly photostable carbopyronine dye (ATTO665). The obtained FRET nanoprobes enable single-molecule detection of short DNA and RNA, encoding a cancer marker, and imaging single hybridization events by an epi-fluorescence microscope with ultralow excitation irradiance close to ambient sunlight.

Fluorescent nanoparticles (NPs) help to increase spatial and temporal resolution in bioimaging. Advanced microscopy techniques require very bright NPs that exhibit either stable emission for single-particle tracking or complete on/off switching (blinking) for super-resolution imaging. Here, ultrabright dye-loaded polymer NPs with controlled switching properties are developed. To this aim, the salt of a dye (rhodamine B octadecyl ester) with a hydrophobic counterion (fluorinated tetraphenylborate) is encapsulated at very high concentrations up to 30 wt % in NPs made of poly(lactic-co-glycolic acid) (PLGA), poly(methyl methacrylate) (PMMA), and polycaprolactone (PCL) through nanoprecipitation. The obtained 35 nm NPs are nearly 100 times brighter than quantum dots. The nature of the polymer is found to define the collective behavior of the encapsulated dyes so that NPs containing thousands of dyes exhibit either whole particle blinking, for PLGA, or stable emission, for PMMA and PCL. Fluorescence anisotropy measurements together with small-angle X-ray scattering experiments suggest that in less hydrophobic PLGA, dyes tend to cluster, whereas in more hydrophobic PMMA and PCL, dyes are dispersed within the matrix, thus altering the switching behavior of NPs. Experiments using a perylene diimide derivative show a similar effect of the polymer nature. The resulting fluorescent NPs are suitable for a wide range of imaging applications from tracking to super-resolution imaging. The findings on the organization of the load innside NPs will have impact on the development of materials for applications ranging from photovoltaics to drug delivery. journal article 2017 Dec 13 2017 11 29 imported "Supporting information" disponible sur le site de l'éditeur à l'adresse suivante : http://pubs.acs.org/doi/suppl/10.1021/acsami.7b12292

WOS:000471728200021; International audience; Imaging single molecules and nanoparticles in complex biological media is highly challenging notably due to autofluorescence of cells and tissues. Lanthanides and lanthanide complexes, with their particularly long luminescence lifetimes, offer the possibility to perform time-gated imaging and thus to strongly reduce the autofluorescence background. However, their very low brightness and photon flux have limited their use in single-molecule imaging. Here, we encapsulate high amounts of Europium complexes into poly(methyl methacrylate)-based particles of 10, 20, and 30 nm size. The resulting particles contain up to 5000 copies of the complex with a quantum yield of \textgreater= 0.2, resulting in a per particle brightness of up to 4 x 10(7) M-1 cm(-1). They can be imaged at the single-particle level using low illumination intensities (0.24 W cm(-2)) and low acquisition times (300 ms) and internalize well into living cells, where they can be monitored through time-gated imaging at illumination conditions compatible with living specimen. These Eu-complex-loaded nanoparticles can thus be applied for highly sensitive and autofluorescence-free imaging and have the potential to become very performant probes for fast intracellular tracking of single biomolecules.

International audience; Intracellular applications of fluorescent nanoparticles (NPs) as probes and labels are currently limited by the significant molecular crowding and the high level of complexity encountered inside living cells. The solution is to develop very small, bright, and non-interacting (stealth) NPs. Combining these properties requires to implement the stealth behavior through the thinnest possible hydrophilic shell. Here, we propose a one-step process for preparing ultrasmall and bright stealth nanoparticles (NPs) based on a zwitterionic methacrylate based copolymer. Dye-loaded polymer nanoparticles are assembled through nanoprecipitation of the copolymer together with the salt of a rhodamine B derivative and a bulky hydrophobic counterion to achieve high particle brightness. We found that 10 mol% zwitterionic groups in the polymer yields NPs of less than 15 nm that are stable in physiological salt conditions and practically resistant to protein adsorption, as suggested by fluorescence correlation spectroscopy. The combination of the very small size with the non-fouling nature of these particles enables spreading of zwitterionic polymer NPs in the whole cytosol after their microinjection into living cells. In addition, single-particle tracking showed an up to 4 times faster diffusion of zwitterionic NPs in the cytosol compared to PEGylated nanoparticles. The obtained dye-loaded zwitterionic polymer NPs open the route to intracellular single-particle tracking and biosensing applications.

Nanoprecipitation is a straightforward yet powerful technique to synthesize polymer nanoparticles loaded with various biologically active compounds or contrast agents. Particle formation in this approach is kinetically controlled and various assembly parameters have been used to control the size distribution and properties of the formed nanoparticles. Here, the influence of the nature of the polymer on the formation of nanoparticles in nanoprecipitation is studied systematically by varying its hydrophobicity and charge over a broad range. For this, methacrylate copolymers with different of types and fractions of hydrophobic, hydrophilic, and charged side groups are synthesized. Nanoprecipitation of these polymers shows that particle size increases with increasing global hydrophobicity of the polymers. At the same time, both hydrophilic and charged groups reduce particle size. In this way, we achieve control over particle size from ~10 to 200 nm. Furthermore, the effect of the polymer nature on the photophysical properties of nanoparticles loaded with a fluorescent dye, a rhodamine B derivative with a bulky hydrophobic couterion (fluorinated tetraphenylborate), is studied. It is found that the hydrophobic/hydrophilic balance of the polymer modulates to a large extent the spectral properties and fluorescence quantum yield of the dye encapsulated at high concentration, which reflects changes in the dye aggregation within the polymer matrix. Thus, we show how polymer chemistry can tune kinetically controlled formation of nanoparticles and encapsulation of the load. The concepts introduced here should be valuable tools for the design of nanoparticles for imaging and drug-delivery applications.