Meditrans News

Latest project news

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Would you like to learn more about the results from the MEDITRANS project? If so, we would like to hear from you. Please do not hesitate to get in touch with us via the Contact Us page of this website and we will be delighted to invite you to MEDITRANS dissemination events in 2009 and 2010.

MEDITRANS Annual Meeting 2010

The next MEDITRANS Annual Meeting is provisionally scheduled for 20-21 March 2010 in Saarbrücken, Germany. It is kindly being hosted by Saarland University’s Prof. Dr. Claus-Michael Lehr.

MEDITRANS Final Meeting 2010

The final MEDITRANS Meeting is scheduled for 28-30 October 2010 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

One of the main work packages of MediTrans, termed Nanocarrier Design, focuses on emerging materials, like fullerenes and nanotubes, and on candidate materials, like cationic polymers for DNA and siRNA delivery, polymeric micelles and stimuli-sensitive liposomes.

Regarding the emerging materials, as demonstrated in Figure 1A-C, CEA has recently managed to shorten multi-walled carbon nanotubes (MWCNT) to sizes well below 500 nm. In addition, as depicted schematically in Figure 1D, methods have been developed for funtionalizing nanotubes with carboxyl, hydroxyl and amine groups. These functional groups can (and have) been used to surface-modify the nanotubes, e.g. with hydrophilic polymers like PEG. For toxicity testing, CEA has provided BRACCO with several surface-functionalized and surface-modified fullerenes and nanotubes, and interestingly, it was observed that at concentrations up to 1 mg/ml (i.e. the maximal concentration that can be reproducibly dispersed), hardly any cytotoxicity was observed, neither for PEGylated nanotubes, nor for PEGylated fullerenes. Also for control formulations, i.e. for PEGylated liposomes and HPMA copolymers, hardly any toxicity was observed. Both the MTT and the LDH assay, and six different cell lines, were used in these analyses.

An additional interesting observation reported by BRACCO relates to the fact that the PEGylated nanomaterials were taken up very well by the cells, and apparently without harming them. This is illustrated in Figure 1E-F, which shows uptake of PEGylated MWCNT by J774 macrophages and by human umbilical vein endothelial cells (HUVEC), respectively. Uptake (i.e. phagocytosis) by macrophages is not very surprising, as one of the physiological functions of macrophages is to remove (nanosized) foreign materials. Uptake by endothelial cells, on the other hand, is quite remarkable, especially because very high amounts of the nanotubes were found to be internalized (Figure 1F). In future experiments, attempts will be made to confirm and to quantify these findings, and to evaluate the kinetics of this process.

Regarding the candidate materials, except for Task 1.3.3. (i.e. molecularly imprinted particles), all Tasks are on track for achievement. Significant progress has been made with regard to siRNA-based nanoparticles, polymeric micelles and stimuli-sensitive liposomes. Some of these systems have already been forwarded to other Work Packages, and they are currently being subjected to in vitro and in vivo evaluation.

Work Package 2: Development of highly sensitive imaging probes for guided drug delivery processes
A novel pH responsive Gd-based agent was synthesized and preliminarily characterized in vitro. The compound is based on a macrocyclic DOTA-like co-ordination cage bearing three acetate and one sulfonamido group. The pH dependence of the relaxivity is guaranteed by the protonation/deprotonation switch of the sulfonamido group that has a pKa value of about 6. Interestingly, the change in relaxivity does not seem to involve a variation of the hydration number of the metal ion, but rather a change in the tumbling motion of the complex, likely caused by a rearrangement of the solution structure of the complex.
As far as the progresses in the development of CEST agents is concerned, the attention has been primarily focused on the in vivo visualization of liposome-based CEST (LIPOCEST) probes. To this end, LIPOCEST were intratumorally injected in mice bearing xenografted B16 melanoma tumour and their contrast was followed over time.
Interestingly, the CEST contrast disappeared quite rapidly (Figure 1), because of the avid macrophage uptake of the liposomes as confirmed by confocal fluorescence microscopy.
In addition, LIPOCEST can also act as efficient T2 agents and the R2 values of the tumour after the liposome injection revealed a very different kinetic (Figure 1). However, the observed behaviour is still consistent with the cell internalization of the liposomes, owing to the long spatial range property of the T2 susceptibility effect generated by the vesicles.

Work Package 3: Formulation of drugs and imaging agents into carriers / physicochemical characterisation
Current formulations have focussed on non-propriatory drugs, such as mesopram (Crohn’s disease therapy), fludarabine phosphate (Anti-cancer drug) and corticosteroids.
Monodisperse PLGA nanoparticles of a size small enough to be retained in the inflamed intestinal tissue (<200 nm) have been prepared using a solvent-evaporation method. Nanoparticles have been shown to be stable during short term storage and have mesopram encapsulation efficiencies of >70%. However, no considerable sustained release of the anti-inflammatory drug has been demonstrated. The current formulations require further optimisation prior to in vivo testing in a rat colitis model.

Work Package 4: Recognition of targets: cells, tissues, organs
Development of a novel 3D model of the inflamed intestinal mucosa
Lipopolysaccharides (LPS) from both E. coli and S. typhimurium were previously shown to stimulate inflammatory activity in Caco-2 cells at the mRNA level (Figure 3).

Figure 3: Induction of IL-8 and TNF-Alpha mRNA by S. typhimurium LPS

However, further testing of the inflammatory potential, revealed only a very weak stimulation of actual IL-8 production at the protein level (Figure 4) and no effect on tight junctional function and expression, as transepithelial electrical resistance (TEER) of LPS treated Caco-2 monolayers was not significantly different to the control.

Figure 4: IL-8 concentration in Caco-2 cell supernatant 48 h after induction of inflammation

Therefore IL-1 beta was introduced as a co-stimulator. In combination with IL-1 beta, LPS from S. typhimurium induced a 150-200 stronger release of IL-8 from Caco-2 cells compared to LPS individually, and reduced TEER values of Caco-2 monolayers to 85% of the starting values.

Work Package 5: Target Cell Uptake and Intracellular Trafficking
Confidential.

Work Package 6: Stimulus induced release / activation
Confidential.

Work Package 7: Application to Rheumatoid Arthritis and Crohn’s Disease
A high relaxivity Gd(III)DOTA-DSPE-based liposomal contrast agent for magnetic resonance imaging
The field of molecular imaging aims to visualize (patho)physiological processes at the cellular and molecular level and vitally depends on sensitive and site-targeted contrast agents. Hence, we report on recent achievement of the TUE team to develop a lipid-based magnetic resonance imaging (MRI) detectable liposome with high relaxivity and stability. To this end, Gd(III)DOTA-DSPE was synthesized and incorporated in a liposomal formulation. The resulting liposomes were extensively characterized in vitro in terms of contrast agent efficiency and structural properties. The liposomes were shown to have a high longitudinal relaxivity (r1 ~ 4 mM-1s-1), which is beneficial for the detection of low concentration molecular markers in molecular imaging studies. We also demonstrated that liposomal Gd(III)DOTA-DSPE exhibits no detectable transmetallation upon incubation with Zn(II) in the presence of phosphate. This is important as it significantly contributes to the biocompatibility of the contrast agent. The present liposome preparation will serve as a versatile, and well characterized, platform for molecular imaging and targeted drug delivery studies.
This work was recently submitted for publication. When published, the reference will be listed on the MEDITRANS publications webpage.

Work Package 8: Application to Multiple Sclerosis
The gadolinium-based pro-contrast agent sensitive to a family of matrix metalloproteinase (MMP) active enzymes is currently being tested in vitro: DOTA-gadolinium has been covalently linked to a peptidic sequence that is a substrate for some MMPs, and to an insolubilizing moiety. This assembly constitutes a prodrug form of the CA (i.e. the proCA). The peptide part in the proCA is released from the carrier upon cleavage by MMPs at the site of action, thus activating an MRI signal in T1-weighted images. The insolubilising moiety in the proCA, which suppresses any T1-enhancement, makes these particles T2-relaxation agents in T2-weighted image acquisitions. This proCA has been tested in biochemical assays as well as in ex-vivo blood samples from disease animal models mimicking aspects of Multiple Sclerosis, the experimental autoimmune encephalomyelitis (EAE). In biochemical assays, the proCA showed a dose-response increase in R1 values in the presence of increasing concentrations of MMP-1, -9 or -12. In ex-vivo blood samples from EAE mice, the R1 values were significantly increased above healthy control values at time of inflammatory peaks of the disease when MMPs are highly enriched in blood stream. Nanosizing the particle will allow testing the pro-CA in in vivo situations.
In rat EAE in vivo MRI modalities have been applied to visualize not only blood brain barrier lesions in the central nervous system (CNS) lesions but also to assess the degree of macrophage migration. Indeed EAE models are characterized by lymphocytic / macrophagic inflammatory responses leading into demyelination and axonal loss within spinal cord and brain regions. Imaging the trafficking of some inflammatory cells such as macrophages is key to understand MMP expression and activity and thus will be very valuable for the next steps of the program. Thus, to characterize the imaging signature of inflammatory lesions in rat acute EAE, longitudinal T2 weighted, T1-Gadolinium and USPIO uptake signals have been measured over the disease at the level of the pontine-cerebellar interface. Assessment of various USPIO nanoparticles and doses is currently ongoing to highlight potential differential pathogenic mechanisms and imaging approaches. This characterization will allow testing in vivo nanosized MMP-proCA during the next phases of the work package.

Work Package 9: Application to Cancer
UNITO have investigated the in vivo properties of highly sensitive Gd-based stealth liposomes encapsulating the antitumoral drug doxorubicin. Two kinds of liposome formulations were studied differing for the presence on the external surface of a peptide targeting vector (C3d) recognizing NCAM receptors. Both liposomes contained amphiphilic Gd-based imaging probes in their bilayer and Doxorubicin in their aqueous cavity. SCID mice bearing human KAPOSI xenograft tumours were i.v. injected with the liposomes every week for three weeks (Doxorubicin dose 5 mg/kg b.w., Gd dose 0.01 mmole/kg b.w.). The animals were subjected to the first MRI investigation 1 day before the first administration in order to evaluate the tumour size. Interestingly, non targeting liposomes showed brighter contrast than targeting ones, either 5 h or 24 h post injection and this observation was confirmed by a biodistribution study that emphasized the different blood circulation times of the two liposomes. Actually, targeting liposomes were removed from circulation much faster than the control, thus explaining the different contrast enhancement in the tumour. Nevertheless, the therapeutic response (evaluated by measuring the tumour size) observed for targeting liposomes was higher than the control.  This result suggests the need to design new MRI protocols to improve intratumoral localization (extra- vs intra-cellular) of the drug.

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.

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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