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MEDITRANS Final Meeting 2010

Held at Hotel Eden Roc, Sant Feliu de Guixols, Spain

MEDITRANS Annual Meeting 19-22 March 2010

Held at Saarland University, Saarbrücken, Germany

EURONANOMEDICINE 2009
The ‘EURONANOMEDICINE 2009 ’ conference was held on 28-30 September 2009 to disseminate the results of three EC-funded FP6 NMP Integrated Projects, MEDITRANS, NANOEAR and NANOBIOPHARMACEUTICS.

MEDITRANS Annual Meeting 26-29 March 2009

Held at Weizmann Institute of Science, Rehovot, Israel. See: http://www.weizmann.ac.il

Work Package 1: Naocarrier design

WP1 aims to identify materials suitable for serving as drug delivery devices. Hereto, it on the one hand deals with the synthesis and characterization of Emerging Materials, like fullerenes and nanotubes, as well as, on the other hand, with the evaluation and optimisation of already existing Candidate Materials, like polyplexes and polymeric micelles.

Regarding the Emerging materials, CEA, BRACCO and UU have in the past three years synthesized and tested several batches of surface-functionalized and surface-modified fullerenes and nanotubes, and have obtained a significant amount of data on the in vitro cytotoxicity of these systems. In addition to this, some initial proof-of-principle has been obtained with regard to drug loading, demonstrating that PEGylated carbon nanotubes can be reproducibly functionalized with budesonide (see Figure 1), and that certain cationic nanotubes can complex siRNA.

Concerning the Candidate materials, except for the Task dealing with molecularly imprinted particles (which was halted in May 2009), significant progress was made in all six other project lines, focusing on I) DNA- and II) siRNA-containing polyplexes, on III) polymeric micelles, on IV) iron oxide nanoparticles, on V) amino acid-based nanoparticles and on VI) stimuli-sensitive liposomes. All of these Tasks have been successfully completed. Those candidate materials for which particularly promising results have been obtained, i.e. siRNA-based nanoparticles, polymeric micelles, iron oxide nanoparticles and stimuli-sensitive liposomes, are now being evaluated in more detail in other WPs.

Figure 1. Preparation of budesonide-loaded PEGylated carbon nanotubes. A: Synthesis of the pyrene-PEG-budesonide conjugate. B: Surface adsorbtion of the pyrene-PEG-budesonide conjugate to ultrasound-shortened carbon nanotubes

Work Package 2: Development of highly sensitive imaging probes for guided drug delivery processes

Task 2.1 – Highly sensitive Gd(III)-based agents

UNITO, in collaboration with BRACCO and the University of Eastern Piedmont (Prof. M. Botta) has synthesized novel amphiphilic Gd(III) complexes properly designed to have an optimal reorientational correlation time, ?R, upon incorporation in nanocarriers (see, as a representative example, the complex depicted below).

The novelty of these systems is that the two aliphatic chains are linked to the 1,4 position of the DOTA skeleton and not on the same coordinating arm as done so far for amphiphilic macrocyclic chelates. In addition, the shorter C12 chains could shorten the extremely slow body excretion usually observed for probes with C16 or C18 chains. Actually, the relaxivity of the complex reported above when incorporated in conventional stealth liposomes (40 s-1mM-1 at 25°C and 0.5 T) is the highest so far reported for a liposome incorporated amphiphilic Gd(III) complex with a single metal coordinated water molecule.

Task 2.2 - Highly sensitive Chemical Exchange Saturation Transfer (CEST) agents
PHILIPS and UNITO prepared block copolymer vesicles containing paramagnetic lanthanide complexes that represent a versatile class of MRI contrast agents. By exploiting the water flux across the membrane, these nanovesicles can be employed as T1- and CEST MRI contrast agents. The incorporation of paramagnetic amphiphilic lipids (Gd-1 for T1 MRI CAs agents and Tm-2 for CEST MRI CAs) into the polymeric bilayer markedly improves the properties of the MRI contrast agents with respect to the longitudinal relaxivity (22 mM-1s-1 at 20 MHz) and the chemical shift of the intrapolymersomal water protons.

Task 2.3 - Novel iron-oxide based probes
The carbon-coated iron oxide particles (C@OxFe) produced by laser pyrolysis at CEA were obtained as heavy aggregates. C@OxFe have thus to be optimised for biological applications as imaging probes by reducing the nanoparticles size and increasing their colloidal stability. A post C@OxFe synthesis process has been developed to afford small particles clusters of ca 20-100 nm in diameter. The latter were coated with polymerizable amphiphilic polyethylene glycols (PEG-5000) yielding water dispersible nanoparticles.

The dispersed C@OxFe nanoparticles exhibit strong positive magnetic signals and are stable for days. Assessment of their relaxivity properties (r1 and r2) and iron amount is currently in progress by GUERBET.

Task 2.4 – Optical Imaging Probes
Work is ongoing with the aim of developing optical imaging protocols for the visualization of the release of fluorescent dyes from nanocarriers on living cells or in vivo.

Work Package 3: Formulation of drugs and imaging agents into carriers / physicochemical characterisation

Established Nanocarrier Systems
Significant progress has been made on the formulation and characterisation of established nanocarrier systems. In particular long-circulating liposomes for passive targeting, loaded with corticosteroids, have demonstrated favourable size and sufficient encapsulation. MOLPROF are currently performing more detailed physicochemical characterisation of these long circulating liposomes that have been selected for Industrial Exploitation. A whole host of techniques are currently being employed such as AFM, TEM, PCS, Nanosight, XRD, QCM-D.

Liposomal nanomedicines able to release their payload under the action of specific triggering stimuli, e.g. pH sensitive liposomes, have been prepared and characterised. The corticosteroid pro-drug prednisolone phosphate (PLP, provided by UU) has been co-incorporated in paramagnetic liposomes loaded with the amphiphilic paramagnetic Gd(III) complex.

Proteoliposomes have been successfully developed. Formulations with siRNA as the payload have been produced. In order to follow siRNA encapsulation/release from MscL-Liposomes a new method was optimised in order to obtain giant unilamellar MscL-liposomes.

Budesonide PLGA nanoparticles have been formulated and demonstrate high encapsulation rates (~80%) and a delayed drug release for at least 48 h. Long term release studies over the course of several weeks will further clarify the budesonide release profile.

Candidate Nanocarrier Systems
si-RNA loaded PLGA nanoparticles (<300nm) with a narrow size range and encapsulation of up to 58% have been produced. The particles demonstrate good colloidal stability and a sustained release profile. MOLPROF have been undertaking physicochemical characterisation of PLGA nanoparticles produced at CU for siRNA delivery. Early efforts have focused on atomic force microscopy (AFM) and surface chemical characterisation by time of flight secondary ion mass spectrometry (ToF-SIMS).

The chemistry and preparation methods to make dextran-HEMA nanogels is understood. A high amount of siRNA can be complexed and the relationship between charge, aggregation and concentration has been studied.

Folic acid decorated nanoparticles have been formulated for the delivery of DNA.

Iron-oxide particles with a stealth coating have been developed where PEG and aminosilanes are coupled to the iron oxide particles. MOLPROF have undertaken a program of characterisation on coated and uncoated iron oxide nanoparticles produced by MAGFORCE. Dynamic light scattering and single particle tracking (Nanosight™) have been used to study the particle size distributions at different pH levels and to investigate the influence of protein binding on particle size. The surface chemistry of the nanoparticles has been investigated using time of flight secondary ion mass spectrometry and X-ray photoelectron spectrometry and confirm coating integrity. X-ray powder diffraction has been used to investigate the crystal structure of the iron oxide cores and to estimate the size of crystalline domains. In addition, although pushing the resolution limits of the technique, AFM imaging can visualise individual particles.

Emerging Nanocarrier Systems

Significant progress has been made over the last 12 months with regard to the formulation of drug loaded carbon based nanocarriers (e.g. fullerenes and carbon nanotubes). These nanocarrier types have been successfully produced with favourable drug delivery characteristics. In addition, carbon nanotubes have been surface modified with various functionalities, as well as coating the nanotubes with a stealth coating of PEG based amphiphiles. Furthermore, carbon nanotube-drug complexes have been developed.

Work Package 4: Recognition of targets: cells, tissues, organs

Active targeting in cancer/ Recognition of target in cancer
Liposomal nanocarriers were evaluated for active and passive targeting of anticancer drugs. Two ligands with anti-angiogenic activity, Anginex (Anx) targeting galectin 1, and RGD targeting αvβ3 integrin, were conjugated to paramagnetic fluorescent liposomes. Co-application of the two single targeted formulations was compared to a dual targeting strategy. In addition, two different ligand concentrations were compared (heavy (H) and light (L)). Results show that simultaneous targeting of both ligands improves the uptake of liposomes by activated endothelial cells compared to single-targeting and that dual targeting provides synergistic targeting effects, with the high ligand concentration being more effective.

Uptake levels of liposomal contrast agent achieved with different active targeting strategies (Kluza et al., Nano Letters, 2010, 10(1):52-8)

Passive targeting in Crohn’s disease/ Recognition of targets in Crohn’s disease
Previously, an in vitro model of the inflamed intestinal mucosa for testing of drug formulations targeted to inflammatory bowel disease was established at UDS (Leonard et al., Molecular Pharmaceutics 2010, in reply). The model was now used to evaluate three different budesonide formulations - FA-PLGA budesonide nanoparticles (UDS), liposomal budesonide (UU) and free budesonide solution monitoring the anti-inflammatory effect as well as the particle distribution in the inflamed model. Both the budesonide solution and FA-PLGA budesonide nanoparticles reduced the IL-8 release to the level of the non-inflamed control and fully restored epithelial barrier function, while budesonide loaded liposomes further increased IL-8 release compared to the non-treated inflamed model. PLGA showed a prolonged effect in decreasing IL-8 release, surpassing the free budesonide.

Work Package 5: Target Cell Uptake and Intracellular Trafficking

5.1. Quantification of cellular uptake.
Quantification of cellular uptake has been completed for siRNA containing nanogels, showing efficient intracellular uptake in about 90% of the cells. Also, PEGylation had no significant inhibiting effect on the internalization of the nanoparticles. The cellular uptake of siRNA drug loaded nanocarriers, based on branched polyesters (supplied by MARBURG) is ongoing.

5.6. Quantification of the biological activity.
The biological activity of PEGylated and non-PEGylated nanogels has been monitored, resulting in a good downregulation efficiency, comparable with that of a commercially available lipid-based carrier (LipofectaminTM RNAiMAX). In both cases PCI had a positive effect on the biological activity obtained, by enhancing the endosomal escape of the nanogels (Figure 1 and Figure 2).

Figure 1. Application of PCI 6 days post-transfection on cells transfected with non-PEGylated nanogels (?) and Lipofectamine™ RNAiMAX (?). The (light and dark) grey colored zones indicate the time window during which more than 50 % knockdown of EGFP was observed.

Figure 2. Transfection efficiency of PEGylated nanogels with different PEGylation degree withouth (black bars) and with (gray bars) PCI. LF equals Lipofectamine™ RNAiMAX

Work Package 6: Stimulus induced release / activation

A liposome with a remote controlled valve, a mechanosensitive channel of large conductance (MscL), has been engineered (RUG). Opening and closing of the channel could be controlled on command by both light and pH at the same time. Channels modified with light-pH label do not open at any pH in the dark. After deprotection through irradiation for 10 min at 365 nm (Spectroline longlife™ filter, 365 mW/cm2), channels become activated and released the liposomal content depending on the ambient pH.

An optimized magnetic resonance-guided high intensity focused ultrasound (MRgHIFU) system was constructed. The existing MRI system was adapted such that MR images could be acquired without image distortion during sonications. Furthermore, an MR compatible HIFU system was designed and constructed (see Figure), taking into account all requirements for performing ablation as well as hyperthermia experiments in small animals. Communication software for the MRgHIFU system was purchased from the laboratory of molecular and functional imaging in Bordeaux and will be further developed by UMC UTRECHT.

Figure: CAD design of ultrasound transducer housing with hydraulic system and mono-element transducer inside (a). Photo of the constructed housing with hydraulic system. The transducer still has to be added in this stadium

An in vivo cerebral tumour model for the evaluation of extracellular pH effects in the delivery of chemotherapeutic drugs from pH sensitive liposomes/particles was completed by CSIC and is now available for the MEDITRANS researchers to test their pH sensitive particles.

HIFU-sensitive liposomes were developed (UMC UTRECHT) and tested on a magnetic resonance-guided HIFU system (MRgHIFU; PHILIPS). The Figure (a) shows a schematic drawing of the treatment planning positioned on the MR image of an agarose phantom containing HIFU-sensitive liposomes. Figure b and c show the temperature distribution during MRgHIFU-mediated heat treatment (42°C) of the liposomes. The release profile of the encapsulated fluorescein dye from the liposomes was similar compared to the release profile as observed in studies with a warm water bath.

Holmium is an attractive element since it can be imaged by MRI, CT and if radioactive by nuclear imaging. Micelles of the biodegradable block copolymers of mPEG-b-p(HPMAm-Lac2) containing holmium nanoparticles (np) will be tested for their ultrasound sensitivity. At this moment Ho2(CO3)3 and Ho(C5H7O2)3-np will be characterized and tested.

Work Package 7: Application to Rheumatoid Arthritis and Crohn’s Disease

The in vivo imaging part of this project deals with the evaluation and optimisation of targeted nanoparticles, loaded with imaging probes and drugs, for application in MRI guided imaging and therapy of inflammation. In vivo RA studies (MRI and CT) are performed in mice at TUE, jointly with UU. The data in this study demonstrate the profound anti-inflammatory activity of Dexamethasone-PEG-Liposomes, where the reduction of paw inflammation was rapid and the therapeutic effect of a single dose lasted more than a week (Figure 1). This indicates that the encapsulation of the drug in the nanocarrier system can strongly enhance its beneficial effect in RA.

Figure 1: The graph shows paw inflammation clinical scores and total area of the MRI images of the paws of mice after single treatment. Score was set at 100 % at the day of treatment, indicated by arrow. In vivo MRI indices and clinical score correlate well with each other. Symbols: ( ) saline group; ( ) 10 mg/kg of free Dexamethasone and ( ) 10 mg/kg of Dexamethasone-PEG-Liposomes

Further work has been performed in establishing appropriate read outs of in vivo models for measuring TNF-α silencing. Comparative studies of MEDITRANS carriers (dendrimers (CU), PLGA nanoparticles (CU), polymers (UU) and nanogels (GHENT)) will be done in vivo on RA model at UU and will be followed up by TUE using MRI & CT scanning.

CSIC developed a DSS animal model for CD studies. The 11 week old C57Black6 male mice were divided in two groups. One group received normal drinking water. The other group received 2.5% (w/v) DSS in drinking water for 5 days, followed by two days on normal water. The treatment with DSS led to swelling of the ascending colon (Figure 2).

Figure 2: The left side panel shows the gastrointestinal tract of a control mouse and the right side panel shows the gastrointestinal tract of a DSS treated mouse, where the ascending colon (arrow) is swollen, due to inflammation

A new protocol for dynamic 3D MRI visualization of the progression of Gd (III) doped materials through the gastrointestinal tract in live mice was established. Isofluorane anesthetized adult C57/BL6 mice (1%) received an intragastric administration of 0.4 mL Gd(III)DTPA and the passage of the contrast agent through the GI tract was followed by MRI during the next four hours. Acquisition conditions were: echo time TE=10ms, repetition time TR=450ms, slice thickness = 1mm, matrix = 192x18x256 voxels.

Figure 3: Snapshots extracted from 3D reconstructed images acquired 36 (upper row) and 72 (lower row) minutes after Gd-DTPA administration. The middle and right images are rotated 90º and 270º respectively from the initial orientation (left). Note the excellent 3D visualization of the colonic cavities downstream from the stomach

3D images were reconstructed with a maximal intensity projection algorithm. Figure 3 shows representative results. It was possible to observe the residual amount of contrast agent remaining in the stomach after the injection and how the different colonic cavities were filled sequentially, including the duodenum, jejunum, ileum and caecum.

Work Package 8: Application to Multiple Sclerosis

Multiple sclerosis (MS) is characterized by over-expression of several types of matrix metalloproteinases (MMPs). Activity of MMP-2/3/7/9 is detected in the brain of MS patients. Treatment with MMP inhibitors is thought to be a promising approach to control neuro-inflammation. The assessment of the activity of MMPs in demyelinating regions before/during treatment with MMP inhibitors would be of great value for the typization of the pathological process and calibration of the therapy. We are developing new methodologies for the simultaneous targeted delivery of therapeutics and visualisation of drug delivery by DCE-MR molecular imaging. The strategy is based upon MRI contrast agents composed of a MMP cleavable peptide substrate, conjugated at the N-terminus with a Gd(III)DOTA chelate and at the C-terminus with a hydrophobic alkyl chain (Figure 1).

Figure 1. Structure of the MMP cleavable MRI probes

The activity of MMP in tissues can be detected on the basis of the differences between the wash-out kinetics of the intact (hydrophobic) probe and that of the more hydrophilic fragment, still bearing the Gd(III) chelate, formed after cleavage by MMPs. Compound K11N can be efficiently cleaved in vitro by MMP and interacts with serum albumin with a KD of 50 µM, thus having a relatively long plasma half-life. K11N has been tested on a rat model of peripheral nerve inflammation (nerve crush model) to assess wash-in/wash-out kinetics in the absence of specific targeting vectors. K11N was administered at 30 µmol/kg at week 2 post injury (when neuroinflammation reaches a maximum). The signal enhancement was only detected on the injured nerve (arrows) at 20min post injection and with a maximum uptake at 40min. No uptake was observed at week 3 post injury (at this time point, inflammation has mostly faded away). Once in the nerve the compound interacts with lipids and hydrophobic components of the ECM with an increase of the retention time in tissues, and wash out occurs after rescue of the Gd(III)DOTA peptide fragment by MMP dependent cleavage of the probe. Experiments are ongoing to assess whether treatment with MMP inhibitors can cause a decrease in the wash out kinetics, thus revealing at the same time drug delivery and a map of MMP activity.

Figure 2. Uptake of K11N by the injured sciatic nerve (arrows)

In parallel with the untargeted approach to the MR molecular imaging of MMPs, a targeted nanosized system is under development. This system is based upon a β-cyclodextrin vesicle that offers a wealth of binding sites for i) the MMP responsive MRI contrast agent (compound KADA) ii) a MMP-inhibitor; and iii) a vector targeting integrins based on the RGD peptide sequences conjugated with adamantane to form host/guest complexes with β-cyclodextrins.

Figure 3. Nanosized system for the MR molecular imaging of MMPs

Work Package 9: Application to Cancer

Liposomal channel proteins
An engineered channel protein MscL was reconstituted into stealth liposomes. Liposomes were loaded with corticosteroids and tested for release of the encapsulated corticosteroids on command. The facilitated encapsulation and release of siRNA by using MscL-liposomes is in progress.

Targeted siRNA delivery
In order to track nanoparticle delivery by in vivo bioimaging IDT synthesized EGFP DsiRNA covalently labelled with LI-COR IRdye 800CW on the 5´-end. Additionally, AlexaFluor 488 and AlexaFluor 647 tagged EGFP Evader DsiRNAs have been made available. Particles tested in vivo for suppression of reporter gene expression showed toxicity and non specific inhibition in vivo.

Targeted pDNA delivery
Hydrodynamic diameters of complexes with siRNA were measured. Over the investigation of more than one hour, no aggregation could be observed.The conjugates proved to be less toxic than PEI with IC50 values above 0.05 mg/ml. The conjugates were lyophilized and sent to UU for distribution between UU and WEIZMANN.

Folate receptor as a target in ovarian cancer
USPIO contrast media (p904 and p1133; GUERBET) were administered intravenously to CD-1 nude mice bearing subcutaneous MLS human ovarian carcinoma tumour xenografts.

The animals were monitored by MRI (4.7T). In addition, particles were incubated ex vivo with isolated macrophages and were then administered by adoptive transfer to tumour bearing mice. Delivery of the label to the tumour was followed by histology and by MRI.

Histological sections were stained with Folate-BSA-ROX or BSA-ROX (200 µg/ml). USPIO were detected with Prussian blue staining. Macrophages were detected using F4/80 (Serotec, Germany) and with LYVE1 antibodies, detecting also lymphatic vessels.

Two -photon intravital microscopy was done using dorsal skinfold window chambers. Tumours were initiated 48 hours after surgery by injection of MLS-EGFP cells to the centre of the chamber. When the tumour was established, P904-rhodamine was injected intravenously, followed 24h later by intravenous administration of DiD, labeling the blood vessels.

Hypoxia imaging (CSIC)
Hypoxia is known to play a central role in the development of oncologic diseases and their treatment. In this period CSIC has investigated a novel protocol for the non invasive detection of hypoxia in tumours in vivo using 1H Magnetic Resonance Spectroscopy using Misonidazol and 1H MRS or 1H MRSI.

Work Package 10: Preclinical toxicology

1) Liposomal dexamethasone for the treatment of cancer.
Liposomal corticosteroids designed to achieve inhibition of cancer-related inflammation have become one of the most successful targeted nanomedicines within MEDITRANS.

The toxicological profile of liposomal dexamethasone will be established through an extensive repeated dose toxicity study in rats. These studies will be done under GLP conditions using only certified GMP samples of the selected nanomedicine.

2) Polymeric micelles for the treatment of cancer.
Core-crosslinked micelles represent a novel, highly attractive platform technology suitable for the targeted delivery of small molecules and/or imaging agents.

Preclinical safety assessment of micellar paclitaxel will include the following studies: safety pharmacology, pharmacokinetics, biodistribution study and single dose toxicity.

3) Liposomal gadolinium for imaging-guided drug delivery.
The research work carried out in MEDITRANS has generated different types of gadolinium (Gd)-loaded liposomes that yield a marked contrast in the magnetic resonance (MR) images of the regions in which they distribute and are therefore an innovative diagnostic tool.

Safety pharmacology, pharmacokinetic and biodistribution studies will be performed as described for the previous micellar paclitaxel. Furthermore, the toxicological profile will in detail be investigated in an extended single dose toxicity study and a dose escalation study in rats.

4) Ultrasmall superparamagnetic iron-oxide (USPIO) nanoparticles for imaging-guided drug delivery.
P904 is a small iron-oxide nanoparticle with a low polydispersity and a relatively long half-life in the blood, thus allowing uptake of P904 mainly by activated macrophages that are involved in the physiopathological process of several diseases.

The toxicity of P904 will be estimated by using both in vitro and in vivo approaches. This includes studying of the haemocompatibility, activation of the complement system and the potential inflammatory effects. Next, the lethal dose (LD50) will be measured and the systemic toxicity will be evaluated through haematological and histopathological examinations in rats.

Work Package 11: Industrial exploitation

The following four selected nanomedicines have entered Industrial Exploitation (WP11):

1) Liposomal dexamethasone for treatment of cancer
2) Polymeric micelles for treatment of cancer
3) Liposomal gadolinium for imaging-guided drug delivery
4) Ultrasmall superparamagnetic iron-oxide (USPIO) nanoparticles for imaging-guided drug delivery

Recent important publications by MEDITRANS participants:

Scientists at WEIZMANN use MRI to detect transcriptional regulation of gene expression:

Cohen, B., Ziv, K., Plaks, V., Israely, T., Kalchenko, V., Harmelin, A., Benjamin, L.E., Neeman, M. (2007). MRI detection of transcriptional regulation of gene expression in transgenic mice. Nature Medicine 13, 498 – 503.

Scientists at UU provide new insights into lactadherin-dependent phagocytosis:

Fens, M.H.A.M., Mastrobattista, E., de Graaff, A.M., Flesch, F.M., Ultee, A., Rasmussen, J.T., Molema, G., Storm, G. and Schiffelers, R.M. (2008). Angiogenic endothelium shows lactadherin-dependent phagocytosis of aged erythrocytes and apoptotic cells. Blood Published on-line 21st Feb. 2008.

Scientists at UU review Tumour-targeted Nanomedicines:

Lammers, T., Hennink, W.E., and Storm, G. (2008). Tumour-targeted Nanomedicines: Principles and practice. Brit J Cancer 99, 392-397.

Drug targeting systems are nanometre-sized carrier materials designed for improving the biodistribution of systemically applied (chemo)therapeutics. Various different tumour-targeted nanomedicines have been evaluated over the years, and clear evidence is currently available for substantial improvement of the therapeutic index of anticancer agents. Here, we briefly summarise the most important targeting systems and strategies, and discuss recent advances and future directions in the development of tumour-targeted nanomedicines.

Scientists at GHENT use dextran microgels to deliver siRNA in a time-controlled manner:

Raemdonck, K. Van Thienen, T.G., Vandenbroucke, R.E., Sanders N.N., Demeester, J., and De Smedt, S.C. (2008). Dextran Microgels for Time-Controlled Delivery of siRNA. Advanced Functional Materials 18 (7), 993 – 1001.

An Integrated Project funded by the European Commission under the "nanotechnologies and nano-sciences, knowledge-based multifunctional materials and new production processes and devices" (NMP) thematic priority of the Sixth Framework Programme. Contract Number: NMP4-CT-2006-026668