Name of the infrastructure: FUNDP-TA
Location (town, country): Namur, Belgium
Web site address: http://www.nanotoxico.eu/
Legal name of organisation operating the infrastructure: Facultés Universitaires Notre-Dame de la Paix /University of Namur
Location of organisation (town, country): Namur, Belgium
Description of the Infrastructure
MATERIALS AND EXPERTISE: The two biggest Research Units of the faculty of sciences are conducting research on nanotechnology in the Dept. of Physics (LARN and LISE laboratories) and on molecular and cellular biology and toxicology in the Research Unit of Cell Biology (URBC) within the Dept. of Biology. The Dept. Physics has deep knowledge in the synthesis, manipulation and modification of nanomaterials. It has produced and functionalised several types of carbon nanotubes (CNTs) by the chemical vapour deposition (CVD) process. Fruit of that knowledge, the spin-off “Nanocyl” was founded in 2002 together with the U. of Liège and with the support of private investors. In 2009, Nanocyl is among the top 3 world producer of CNTs. URBC has acquired strong knowledge in in vitro toxicology, oxidative stress, reconstructed tissues like epidermis and intestinal epithelium, transcriptomics and proteomics. Fruit of that knowledge, the spin-off “Straticell” was founded in 2005 by private investors. In 2009, Straticell’s operating statement will be roughly one million euros, with an operating statement doubling each year since the creation of the company. Straticell (http://www.straticell.com) specializes in in vitro toxicological testing namely applying OECD tests within the REACH regulation.
These Research Units have 15 years of extensive experience in coordinating collaborative research projects under the EU’s framework programmes. More recently, FUNDP purchased Applied Biosystems 7900 HT realtime RT-PCR equipment for studying gene expression for instance linked with inflammation, immunity, etc. from 48 genes (8 samples) up to 384 genes at a time (1 sample). Microfluidics allows this system to work with minute amounts of RNA (500 ng) per sample and to increase sharply reproducibility. This system was used to establish an in vitro model of intestinal inflammation based on the differential expression of genes linked with inflammation (Piront et al., in preparation) or to study how reconstruction of skin tissues changes gene expression compared to monolayers of cells (Dedessus, submitted). The conjunction of the expertises of these two Research Units was a perfect cradle for the Nanotoxico project of excellence (10x106 €, 5 years) financed by the Wallonia Region and devoted to nanotoxicology. The expertise, interdisciplinary scientific thinking and equipment acquired thanks to Nanotoxico are the bases of the intervention of the University of Namur in this Infrastructure project.
SUPPORTING FACILITIES AND SERVICES
University of Namur will be offering access to two components in the project, as follows:
Particle Characterisation in situ & ex situ (including media standardisation, dispersion etc):
Cell biology facility: The total area of URBC is 1,120 m2 and includes 2 laboratories levels and 2 office levels devoted to research for up to 60 researchers (53 currently). Facilities are available for most of the methods related to cell biology including 4 cell culture rooms. The lab owns a confocal microscope with a dedicated technician and several visible and UV microscopes with digital image processing. The lab also contains the necessary instrument for other assays such as ELISA plate readers in colorimetry and fluorescence, spectrophotometers, microfluidic PCR and Real Time PCR, gene transfection, western blotting, transcriptional factors assays, reporter genes, animal facilities and radioactivity lab. Standard Operating Procedures are followed. Safety of the operators and respect of ethical rules are considered as basic rules in research. The lab has all the computer power and software required to perform the complex bioinformatics analyses required for the confocal microscope, 2D gel analysis, mass spectrometry and transcriptomic data analysis. Characterisation of nanomaterials at all conditions (powder, dispersion, modified, etc), must allow the tracking of surface and chemical composition changes, before and after manipulation. Two components are necessary in this respect: cell biologists for offering knowledge in culture media compatible with optimal cell development and physico-chemists offering knowledge on characterisation. FUNDP's specific expertise, concerning the physicochemical properties and the role of nanomaterials surfaces and its interactions with the dispersion media (Nanotoxico project), comes from joint expertise from URBC, Analysis by Nuclear Reactions Laboratory (LARN) and Electronic Spectroscopy Interdisciplinary Laboratory (LISE) which capitalizes 30 year experience with electron spectroscopies (XPS-ESCA, HREELS, AES, UPS, and synchrotron radiation) and electron diffraction. The LISE projects cover the preparation, the modification and the characterization of surfaces and interfaces.
FEG-SEM (Field Emission Gun Scanning Electron Microscope): The Electron Microscopy Unit (EMU) counts along with others, with a Field Emission Gun Scanning Electron Microscope model JSM-7500f Jeol (operating since middle 2007). The FEG-SEM is coupled to an EDX detector (JED-2300) for measuring the elemental chemical composition of nanomaterials. Particle size distributions are determined with the SmileView software.
Analytical Centrifugation (Disc centrifuge CPS 24000): The Disc centrifuge belongs to both LARN and URBC, and is located next to the accelerator and the XPS spectrometer. The analytical centrifugation was chosen, among several other tested instruments, because of its simplicity and performance. FUNDP is included in the list of validated suppliers by IRMM (Institute for Reference Materials and Measurements, JRC) following our participation in an interlaboratory comparison using the disc centrifuge. Industrial demand is increasing. X-Ray Photoelectron Spectroscopy (SSI-100): The spectrometer SSX-100 (Surface Science Instruments) has a 0.65 eV energy resolution, lateral resolution (analysis spot: 150 μ), angular resolution: 6 deg.
Particle Exposure Assessment:
PIXE (Particle Induced X-ray Emission): LARN is equipped with a particle accelerator (Linear Tandetron ALTAÏS: "Accélérateur Linéaire Tandetron pour l'Analyse et l'Implantation des Solides", tandem type). The accelerator is used to assess the biodosimetry response of cells stressed with different types of radiation (from X-Rays to heavy ions). In addition, the particle accelerator is base equipment for analytical purposes. FUNDP scientists/technicians setup the accelerator (choice of the incident particles, current, beam size), the irradiation station (rotation for large volume investigation, biasing, cooling) and all the particles/X-Rays detection system (type of detector, one or several detectors, geometry, solid angle measurements, efficiency measurements, calibration setup), and instructs the user on how to change/manipulate the samples during the measurements. State of the art computer software (e.g. Gupix, Simnra) for data analysis and assistance from lab senior scientists are also included.
Gene expression profiling: The Applied Biosystems 7900 HT real time RT-PCR equipment described above will be available for the present project. It is possible to use both formatted cards and on-demand cards with genes of interest. Practically, samples of total RNA are shipped to FUNDP, and the quality and integrity (Nanodropand Agilent Bioanalyser) of RNA and checked. If RNA is usable, real-time RT-PCR is performed with the visiting scientist, the principle of data analysis is explained using pre-existing written procedures and advice, and the visitor scientist is followed throughout the analysis of the results.
Protein identification with mass spectrometry: A full proteomic facility is handled by a full time technician for the 2D gels and their analysis with a confocal scanner. After spot picking, an engineer identifies the proteins with 2mass spectrometers. In 2009, URBC possesses a high resolution Q-TOF mass spectrometer (maXis, BruckerDaltonics) coupled to a two dimension liquid chromatography system (2DLC – MS/MS, Ultimate 3000, Dionex).This system is completed with a second mass spectrometer, an ion-trap MS/MS with ETD capacity (HCT ultra, ETD II from Brucker) coupled to a nanoLC-MSMS (Dionex). We have a bioinformatic platform (ProteinScape,Brucker; Mascot, Matrix system). Thus this platform has a high throughput.
Research supported by the infrastructure
Past and current researches related to nanomaterials are: new materials such as HTC superconductors, C60, heterofullerenes, metallofullerenes, CNTs. Some fundamental goals are the determination of elemental composition and crystallographic structure of the surface, the identification of the chemical concentration profiles of elements at an interface, and the recording composition maps at a surface (with a lateral resolution of 10 μm). Radiation-matter interaction. Ion or electron beams and plasma discharge used to induce chemical reactions and structure modifications at the surface of polymers. Surface modifications by proton and ion beams.
Nanotoxico couples physico-chemical characterisation, molecular & cellular biology, tissue engineering and in vitro toxicology, in vivo toxicology, and chemistry of nanoparticles. http://www.nanotoxico.eu/. FUNDP brought its expertise in in vivo and in vitro toxicology to test silica-derived nanoparticles in the Silicalloy project (1.5x106 €, 2009-12). FUNDP scientists have developed expertise in the dispersion of nanoparticles in life-compatible media such as culture media (Piret et al., J. Nanoparticle Res., in press). FUNDP scientists have also developed a model of reconstructed skin which allows testing nanoparticles in more ‘physiological” conditions (Vankoningsloo et al., Nanotoxicology, in press) and adapted in vitro tests to the possible artifacts generated by nanoparticles and to tridimensional conditions, which is quite unique. Tests comprise transepithelial electrical resistance, MTS, LDH, incorporation of tritiated thymidine into DNA, etc. Differentiated intestinal and lung epithelium are used for estimating the effects of nanoparticles on the gut, in addition to using the most common in vitro model of hepatotoxicology: monolayers of HepG2 cells. Electronic (TEM) and confocal microscopy is also available for biological localisation of nanoparticles. FUNDP performs acute and subchronic oral exposure with rodents (blood chemistry, hematocompatibility, anatomopathology). With the help of mathematical turbulence models, the FUNDP team have developed the concepts a new system of respiratory exposure with several isolated single animals (with collars to avoid self-licking). Biopersistence studies apply here as well. http://www.youtube.com/watch?v=jf-drcTPjEw.
FUNDP synthesize radioactive nanoparticles and study the cellular response to their exposure (TARGAN project in the field on cancerology). The amount deposited is measured, but also estimated using codes and software developed at FUNDP.
Services currently on offer and scientific highlights
Particle Characterisation in situ & ex situ (including media standardisation, dispersion etc):
FEG-SEM (Field Emission Gun Scanning Electron Microscope): The science behind CNTs synthesis had permitted FUNDP scientists to get deeper into the nanomaterials field. A fruitful cooperation between FUNDP chemists and physicists has generated knowledge in the field of nanomaterials surfaces, alloys and medical microdevices (Stents). All these competences, together with an important knowledge in the characterisation of several other nanoparticles (CuO, Cu2O, CeO2, SiO2, SiC, TiC, Au, ZnO, C60, etc) within several regional, national and European projects, has strengthened the skills of the EMU.
Analytical Centrifugation (Disc centrifuge CPS 24000): FUNDP's recent expertise concerns the MWCNT dispersions, aqueous and biocompatible types, as well as other manufactured nanomaterials (CuO, Cu2O,CeO2, SiO2, SiC, TiC, Au, ZnO, C60, etc).
Particle Exposure Assessment:
What makes the LARN’s accelerator unique, is that particle induced “nuclear” reactions techniques (PIXE, PIGE, RBS), allows on one hand, to measure the biodistribution of inorganic elements in all type of organs down to parts per million ranging from aluminium to uranium. The highly energetic proton beam penetrates deeper in the samples (50 vs 5 μm by EDX) and a specific rotating target has been designed to analyse a larger surface of sample (ex-vivo measurements). On the other hand, the same technique is used to characterise the bulk elemental concentration of raw nanoparticles, to which animals or humans are exposed. Concerning our expertise with nanoparticles, very recently we obtained results regarding the biodistribution study of SiC (Silicon Carbide) and TiC (Titanium Carbide) nanoparticles in rat lungs (Lozano et al, 2011). Results show a very different behaviour between the biodistribution of the two nanoparticles: while SiC concentration decreases with time as expected, TiC concentration remains about constant; this indicates clearly a TiC biopersistant behaviour. The infrastructure provides all items requested to perform elemental analysis: dryer, ball mill (sample reduction to powder), press (pellet preparation), and vacuum plasma deposition (to depose an internal reference, e.g. 80 nmCr layer).
Mass spectrometry analysis.
FUNDP is part of the steering committee of the EC integrated project ‘Proteomage’ dealing with proteomics. The mass spectrometry facility collaborates with pharmaceutical companies for more than 15 years.
Gene expression profiling.
In the framework of the Plan Marshall project ‘Walnut-20’, interested namely in nanoparticles in foods, FUNDP scientists perform gene expression profiling on a model of intestinal epithelium where they provide a service for testing the effects of protective compounds produced by five different companies, on intestinal inflammation.