NUID UCD

Name of the infrastructure: UCD-TA

Location (town, country): Dublin, Ireland

Web site address: www.cbni.eu, www.ucd.ie

Legal name of organisation operating the infrastructure: National University of Ireland, Dublin / University College Dublin

 

Description of the Infrastructure MATERIALS AND EXPERTISE:

The key component of the UCD offer is the state of the art purpose built facility for nanobiology (nanotechnology in contact with living systems) which will include Class III facilities for nanoparticles synthesis and for tissue culture (primary cells and cell lines) for nanobiology, both of which will be run according to the principles of Good Laboratory Practice (GLP) and the technical and scientific support of a variety of core facilities which have been adapted to the needs of nanobiology, such as BionanoImaging, Nano-“omics” facilities, and protein engineering and expression. While many of the core facilities are already running in the Conway Institute for Biomolecular and Biomedical Engineering with the appropriate adaptions for nanobiology, the dedicated NanoBiology building will be operational from late 2010. UCD has unique expertise in understanding the behaviour (aggregation, dispersion, protein conformation, surface epitope expression, engagement with cellular machinery etc.) of nanoparticles in a biological milieu, and has developed the concept of the nanoparticle-biomolecule entity that is presented to living systems, for which it won the US National Academy of Sciences Cozzarelli prize for 2007. On this basis, UCD has developed a collection of materials and protocols specifically designed for nanobiology and nanosafety assessment. A unique accumulation of expertise is available through physical and biophysical scientists, material scientists, chemists, physicists, molecular biologists, cell biologists, medics and clinicians, all specialising in different aspects of the mechanisms of nanoparticles interactions with living systems, and co-located in the UCD Science District, a 32,000 m2 (rising to 67,000 m2 based on approved construction projects) constellation of science disciplines and facilities including, UCD Conway Institute of Biomolecular and Biomedical Research, UCD Health Sciences, UCD Centre for Agriculture and Food Science, UCD Veterinary Sciences Centre and UCD Computer Science and Informatics Centre. Fifteen dedicated core-funded staff support these facilities, and each core facility has a dedicated nanobiology-liaison who is working to translate the core technologies for nanobiology. Such a combination of expertise and facilities is unique in Europe. UCD has an internationally renowned research base of more than 1890 full time researchers, supported by some 400 technical staff. The latter will provide key expertise to support Users when accessing UCD’s state-of-the-art analytical facilities. All Users will be provided with individually-tailored training in the use of specific analytical facilities as a minor component of the researcher’s visit, ensuring Health and Safety regulations are adhered to. A significant challenge that exists for nanobiology is that many established techniques have had to be developed quite differently because of the sensitivity levels required (e.g. transcriptomics has had to be applied to levels not normally required in other research because of the subtle nature of nanoparticle cell responses), or other details of the science. Since the field is new, these are still being worked out in some arenas, and as such all of UCD’s core facilities are supported by a nano-liaison officer who has the required technical expertise but also a deep appreciation of the challenges of working with nanoparticles and nanomaterials.

 

SUPPORTING FACILITIES AND SERVICES:

 

UCD will be offering access to all four components listed in the project, as follows:

 

Particle Synthesis:

Class III Nanoparticle synthesis & handling: A dedicated GLP facility for handling, synthesis and characterisation of (potentially) harmful nanomaterials will be available from late 2010. The facility will be equipped with glove boxes, laminar flow hoods and fume cupboards, and is designed to ensure no contamination of nanoparticles synthesis. The facility will be supported by 1 technical staff member. Currently, synthesis facilities are available in a standard chemistry lab, equipped with fume cupboards and a glove box. UCD will focus on providing polystyrene and polymeric nanoparticles.

 

Particle Labelling:

Protein Expression Factory: State-of-the-art fully robotic protein (and other biopolymer) expression / synthesis / labelling, spotting and manipulation lab, capable of producing in modest-scale high purity proteins and peptides, RNA, DNA and others biopolymers as well as fluorescent and isotope and radio-labelled proteins, point-mutations and phage display capabilities, spotting onto arrays, surfaces and related techniques. The Expression Factory allows for automated primer design and sub-cloning of 384 constructs, their ligation into a matrix of vectors and transformation into E. coli or transfection into insect cells, followed by monitoring of cell growth and induction of protein expression. This facility is supported by 2 technical support personnel and a nano-liaison. In the present context, the interpretation of nanomaterials labelling is extended to include labelling with biological signalling markers, such as proteins.

 

Particle characterisation:

Nanoparticle dispersion & characterisation suite: Nanoparticle characterisation techniques available in CBNI include NanoSight, NanoSizer, Spinning Disc Centrifuge, and a Covaris Disperser. Transmission Electron Microscopy and Scanning, Electron Microscopy are available through the UCD Core Facilities. Other techniques such as rheology (viscosity), NMR (1H-FTPGSE-NMR probe with gradient and software and cold probe for the 600 MHz NMR spectrometer, and solid state NMR) are also integrated. Other techniques such as diffusing wave scattering, 2-colour scattering, fluorescence correlation spectroscopy, SAXS, and upstream and downstream processing for SANS are being implemented currently. This facility is supported by 2 technical support staff and 2 nano-liaisons.

 

Mass Spectrometry Resource, Transcriptomics, Metabolomics and Lipidomics facilities: Omics technologies are well established in biology, but their application to biomaterials and especially nanoparticles is less well established, and needs very careful handling to ensure that very small perturbations (rather than major events such as apoptosis and necrosis) are detected. Current techniques in protein and antibody microarrays, RT-PCR, transcriptomics, mass spectrometry, lipidomics and metabolomics in the Conway Institute are complemented by new equipment with much higher sensitivities which will be placed in the NanoBiology building (late 2010). Equipment envisaged for the new facility (Q4 2010) includes Waters Ion Wave Guide Mass Spectrometer, XEP3 Label free detection system, BioRobot Protein System, Protein ArrayWorkstation, Aj120 Inkjet Microarray Spotter and other robotic devices. The existing Mass Spectrometry, transcriptomics, lipidomics and metabolomics facilities are supported by several technical support sTAf, and by three nano-liaisons. Imaging suites (Electron, confocal, flow cytometry, high content analysis): This is an advanced facility for in situ spectroscopies and microscopies to track the spatio-temporal aspects of nanoparticle-cell interaction dynamics in real-time and also to study equilibrium and non-equilibrium cellular properties and functions perturbed by nanoparticles interactions. This core facility has expertise in in-cell spectroscopies, using techniques like Confocal-FRET, FRET-FLIM, multi-photon confocal spectroscopies of other kinds, Confocal-Raman, Fluorescence Correlation Spectroscopy, as well as optical imaging techniques including super-resolution fluorescence imaging and nano-IR imaging techniques, and surface plasmon resonance coupled to Raman, radio- and isotope imaging.

 

High Content Analysis (currently being modified with FRET) will allow high-throughput screening as well as connection to the nano-omics platform. Electron Microscopy facilities include TEM, and variable pressure SEM equipped with FIB, STEM, EDX, WDS, CL and EBSD. Each of the imaging and spectroscopy facilities have full technical support, and a dedicated nano-liaison.

 

Class III GLP suite for Tissue Culture: Separately ventilated TC facilities for primary cells and cell lines will be available from late 2010, which will be run under GLP conditions, allowing the widest range of cells to be evaluated, including genetically modified cells and organisms. Each lab will have a dedicated technical support and nano-liaison.

 

Particle exposure assessment:

Animal Facilities: a full animal house is available on-site for mice and rats, a controlled aquarium facility for zebra fish, xenopus and mussels, and a range of other biological model species, including daphnia, earthworms, algae etc., as well as the biological expertise in each species is available. Full technical support, and nano-liaisons available for each species. Exposure Facilities: The unique PEAC facility (Programme for Experimental Atmospheres and Climate) allows us to examine the response of organisms to environmental factors via an extensive suite of controlled and contained environments where the specific effects of environmental factors - light, temperature, photoperiod, atmospheric gas composition, can be examined both independently and in concert with other environmental factors such as water and nutrient availability. In vivo Imaging Facilities: Whole animal imaging via PET/SPECT and histology facilities. There are 2 dedicated supporting technical staff and a nano-liaison.

 

Research supported by the infrastructure UCD CBNI’s (Centre for BioNano Interactions)

Research is organised into a logical framework with 6 specific foci, as described below: 1. Synthesis (including labelling and surface functionalisation) of high quality, reproducible, pure nanoparticles suitable for biological applications; 2. Dispersion of nanoparticles in biological fluids such as cell culture media, standard OECD test media, plasma etc., and characterisation of the dispersion evolution; 3. Determination of the biological entity – the nanoparticles-biomolecules complex, its stability, evolution and exposed surface epitopes; 4. Visualisation of the spatiotemporal aspects of nanoparticles uptake and trafficking in vitro; 5. Assessment of the functional impacts of nanoparticles as a function of sub-cellular localisation by advanced “omics” and other technologies; and 6. Connection of the observed effects in vitro to effects in vivo and to disease scenarios. Quantitative reproducibility is at the heart of every aspect of the work, and understanding and exploiting the fundamental mechanisms of interactions between nanoparticles and living systems is the end-goal.

 

Specific projects can be grouped under the 4 TA components as follows:

 

Particle synthesis:

Synthesis of a range of nanoparticles (polymeric (PS, thermo- and pH responsive, biodegradable etc.), SiO2, TiO2, CeO, Au, calcium phosphates) of well defined properties (including mono-dispersity, range of sizes, structures, surface modifications); For QNano, the only particles that will be offered are polystyrene and labelled polystyrene.

 

Particle labelling & functionalisation:

High-throughput expression of large numbers and variants of proteins and peptides, attachment to nanoparticles surfaces via EDC and click chemistries; fluorescent labelling approaches using Quantum Dots, AlexaFluor dyes etc., radiolabelling via doping and synthesis from radioactive precursors.

 

Particle characterisation:

Dispersion of nanomaterials in biofluids including various cell culture media, OECD standard test media, plasma, cerebrospinal fluid, Natural Organic Matter from river water, sludge and waste materials, food and other complex matrices, etc.) and using biocompatible dispersants; characterisation of eluants from nanoparticles (dyes, catalysts, constituent components etc.) in biological milieu. Studies of the reactivity of nanoparticles (solubility, aggregation, behaviour in biological and environmental media) and biotransformation of nanoparticles in situ. Characterisation of the biomolecule corona (structure, conformation and identity) surrounding nanoparticles in situ in various media and its evolution as nanoparticles interact with living systems. Advanced “Omics” technologies to correlate uptake and localisation with biomolecule corona composition and functional impacts of the nanoparticles.

 

Particle exposure studies on labelled nanoparticles in vitro and in vivo in a range of model system:

Animal behavioural studies, and focus on nanoparticle interactions with the brain and passage through biological barriers such as the Blood brain Barrier.

 

Summary of Access UCD (CBNI) offers a dynamic interdisciplinary research environment, the particular strength of which is the close collaboration of scientists from a range of disciplines, specifically physical and biophysical scientists, material scientists, chemists, physicists, molecular biologists, cell biologists, medics and clinicians, as well as the availability of full technical support and a specialised nanobiology scientific liaison when accessing the core facilities and analytical equipment.