This file was created by the Typo3 extension sevenpack version 0.7.14 --- Timezone: CEST Creation date: 2013-05-21 Creation time: 15-14-50 --- Number of references 69 article VibhuteEVMLA2012 Organic & Biomolecular Chemistry 2013 2 11 8 1294-1305 Responsive or smart contrast agents (SCAs) provide new opportunities in magnetic resonance imaging (MRI) to examine a number of physiological and pathological events. However, their application in vivo remains challenging. Therefore, much research is focused on the optimization of their properties, to enable their use in additional imaging modalities, pre-targeted delivery, or to increase the local concentration of the agent. The key feature in the SCA synthetic modification is the retention of their physicochemical properties related to the specific MR response. Here, we report the preparation and characterization of pH sensitive SCAs appended with a phosphonate pendant arm and either an aliphatic (GdL1) or aromatic linker (GdL2). The longitudinal relaxivity of GdL1 and GdL2 increases by 146% and 31%, respectively, while the pH decreases from 9 to 5. These two SCAs were converted to the biotinylated systems GdL3 and GdL4 and their interaction with avidin was investigated. The binding affinity with avidin was assessed with a fluorescence displacement assay and with MRI phantom experiments in a 3T MRI scanner. The fluorometric assay and MRI E-titrations revealed a 3 : 1 binding mode of GdL3–4 to avidin with the binding affinity as high as that of the parent avidin–biotin complex. The high binding affinity was confirmed with MRI by a competitive assay. The avidin–GdL3–4 complexes thus obtained exhibit changes in both r1 and r2 that are pH dependent. The results reveal a new pathway for the modification and improvement of SCAs to make them more suitable for in vivo application. http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Logothetis Department Scheffler http://pubs.rsc.org/en/content/articlepdf/2013/ob/c2ob26555a 10.1039/C2OB26555A svibhuteSMVibhute joernJEngelmann TVerbić MEMaier nikosNKLogothetis goranGAngelovski article JoshiFKDGMSE2013 Biological Chemistry 2013 1 394 1 125–135 The surface of spherical, nonporous silica nanoparticles (SiO2-NPs) was modified with gadolinium (Gd) complexes, fluorophores, and cell-penetrating peptides to achieve multifunctionality on a single particle. The Gd surface concentrations were 9–16 μmol/g resulting in nanomaterials with high local longitudinal and transversal relaxivities (~1×105 and ~5×105 /mm/s/NP, respectively). Rapid cellular uptake was observed in vitro; however, larger extracellular agglomerates were also formed. In vivo administration revealed a fast distribution throughout the body followed by a nearly complete disappearance of fluorescence in all organs except the lungs, liver, and spleen after 24 h. Such NPs have the potential to serve as efficient multimodal probes in molecular imaging. http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Scheffler http://www.degruyter.com/dg/viewarticle.fullcontentlink:pdfeventlink/$002fj$002fbchm.2013.394.issue-1$002fhsz-2012-0251$002fhsz-2012-0251.xml?t:ac=j$002fbchm.2013.394.issue-1$002fhsz-2012-0251$002fhsz-2012-0251.xml rajuRJoshi VFeldmann WKoestner CDetje sgottSGottschalk HAMayer MGSauer joernJEngelmann article KelirisMHLSE2012 Contrast Media and Molecular Imaging 2012 9 7 5 478–483 Here we report on a dual-modal 19F and 1H MRI paramagnetic probe with a self-immolative linker, Gd–DOMF–Gal. The enzymatic conversion of this probe by β-galactosidase resulted in a simultaneous turning on of the fluorine signal and changed ability of the Gd3+ complex to modulate the 1H MR signal intensity of the surrounding water molecules. A versatile imaging platform for monitoring a variety of enzymes by 19F and 1H MRI using this molecular design is proposed. http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Logothetis Department Scheffler http://onlinelibrary.wiley.com/doi/10.1002/cmmi.1470/pdf 10.1002/cmmi.1470 abrudAKeliris ilgarIMamedov ghagbergGEHagberg nikosNKLogothetis schefflerKScheffler joernJEngelmann article DhingraVermaMEBML2012 ChemPlusChem 2012 9 77 9 758–769 One major challenge in noninvasive mapping of various molecular targets is their inherently low in vivo concentration coupled with the insensitivity of imaging modalities, such as the widely used magnetic resonance imaging (MRI). Development of agents with high sensitivity and specificity is of paramount importance for structural and functional noninvasive imaging. The design, synthesis, and physiochemical characterization of two gadolinium-based contrast agents (CAs) for MRI, the sensitivity of which was optimized by exploiting the well-established biotin–avidin amplification strategies, are reported. The relaxivity of these agents showed a large increase if bound to avidin; specifically, the first compound showed an approximately 1000 % increase in transverse proton relaxivity (r2p), whereas the second compound had an approximately 250 % r2p increase. The increase in r2p was magnetic field independent in the range of 1.5–16.4 T whereas the longitudinal proton relaxivity (r1p) showed strong field dependence. The CAs were further characterized by measuring luminescence lifetimes and emission spectral changes upon addition of avidin to their Eu3+ analogues. The difference in relaxation rate behavior of both complexes was explained on the basis of hydration number modulation and the “global/internal motion concept”. The association constant of these CAs with avidin was found to be in the range of approximately 1015 M−1, which shows that the coupling of biotin to Gd-DO3A did not affect its affinity for binding to avidin (DO3A=1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid). http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Logothetis Department Scheffler http://onlinelibrary.wiley.com/doi/10.1002/cplu.201200064/pdf 10.1002/cplu.201200064 kirtiKDhingra Verma anuragrkAMishra joernJEngelmann bayoMBeyerlein MEMaier nikosNKLogothetis article MishraJEL2012 ACS Chemical Neuroscience 2012 4 3 4 268–273 Herein we report the design, synthesis, and in vitro evaluation of a gadolinium-containing biotinylated dextran-derived molecular imaging probe as a prospective neuroanatomical tracer by means of magnetic resonance imaging (MRI). The probe was effectively taken up by cultured differentiated murine neuroblastoma cells and significantly enhanced the contrast in T1- and T2-weighted MR images of labeled cells under physiological conditions. A significant longitudinal relaxation rate enhancement in the presence of avidin was observed allowing the verification of the results in the end of noninvasive longitudinal MRI connectivity studies by post-mortem histology. The in vitro results indicate that the probe has the potential to be used in vivo to identify the organization of global neuronal networks in the brain with MRI. http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Logothetis Department Scheffler http://pubs.acs.org/doi/pdf/10.1021/cn200112v 10.1021/cn200112v anuragrkAMishra rajuRJoshi joernJEngelmann nikosNKLogothetis article MamedovEEBL2011 Chemical Communications 2012 2 48 22 2755-2757 A Gd3+ based paramagnetic dextran conjugate has been developed, which enables the tracking of neuroanatomical connectivity in the brain by both MR and optical imaging. Cell studies and subsequent in vivo experiments in rodents demonstrate efficient internalisation and transport properties of the new tracer molecule. http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Logothetis Department Scheffler http://pubs.rsc.org/en/Content/ArticleLanding/2012/CC/c1cc15991g 10.1039/C1CC15991G ilgarIMamedov joernJEngelmann oeschenkoOEschenko bayoMBeyerlein nikosNKLogothetis article MishraGEP2011 Chemical Science 2012 1 3 1 131-135 The design, synthesis and evaluation of eight contrast agents for metabotropic glutamate receptors is reported. Each of the contrast agents contains a selective mGluR5 binding moiety linked to a ‘DOTA’-derived gadolinium complex. The potential of these systems was evaluated in vitro for application as responsive MR imaging probes. The targeting moieties mGluR5 antagonists based on aromatic alkyne and dipyridyl/heterobiaryl amide derivatives integrated in a modular fashion, involving linkage to the macrocyclic DOTA ligand to allow specific binding to the mGluR5 receptors. Signal intensity enhancements of up to 27% were observed by MRI in primary astrocyte suspensions and the reversibility of probe binding to the receptor sites, induced by added glutamate, was demonstrated using optical emission and the antagonistic activity of complexes was defined by calcium binding assays. http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Scheffler Department Logothetis http://pubs.rsc.org/en/content/articlepdf/2011/sc/c1sc00418b 10.1039/C1SC00418B anuragrkAMishra sgottSGottschalk joernJEngelmann DParker article FeldmannEGM2011 Journal of Colloid and Interface Science 2012 1 366 1 70-79 Spherical, nonporous and monodisperse silica nanoparticles (NPs) with a diameter of about 100 nm were synthesized and covalently functionalized with lanthanoid(III) (Ln = Gd or Y) chelate complexes, which serve as contrast agents (CAs) for magnetic resonance imaging (MRI). The materials were fully characterized after each synthetic step by different analytical methods, such as dynamic light scattering, scanning electron microscopy, DRIFT and NMR spectroscopy, thermogravimetry and elemental analysis, as well as zetapotential measurements. High surface concentrations of Gd(III) complexes (up to 50 μmol g−1) were determined by ICP-AES and T1-measurements, respectively. MRI experiments show the typical concentration-dependent increase of the longitudinal relaxation rate. T1-weighted images of samples with more than 25 μg NPs per 100 μL agar display a clear contrast enhancement in the agar layer. The transverse relaxivities r2 of the materials are significantly higher than r2 of the corresponding free Gd(III) complexes in water and medium, whereas the longitudinal relaxivities r1 are slightly increased. Due to the high loading of Gd(III) complexes, the relaxivities per particle are remarkably high (up to 2.78 × 105 mM−1 s−1 for r1). Thus, new hybrid materials, based on nonporous silica NPs with high local relaxivity values were synthesized, which can serve as very effective CAs for MRI. http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Scheffler http://www.sciencedirect.com/science?_ob=MiamiImageURL&_cid=272564&_user=29041&_pii=S0021979711011921&_check=y&_origin=&_coverDate=15-Jan-2012&view=c&wchp=dGLbVBA-zSkzS&md5=b5aad1284d2f3bfdc23e873b22b08b68/1-s2.0-S0021979711011921-main.pdf 10.1016/j.jcis.2011.09.053 VFeldmann joernJEngelmann sgottSGottschalk HAMayer article MishraSEBLC2011 ACS Chemical Neuroscience 2011 10 2 10 578–587 To investigate the connectivity of brain networks noninvasively and dynamically, we have developed a new strategy to functionalize neuronal tracers and designed a biocompatible probe that can be visualized in vivo using magnetic resonance imaging (MRI). Furthermore, the multimodal design used allows combined ex vivo studies with microscopic spatial resolution by conventional histochemical techniques. We present data on the functionalization of biocytin, a well-known neuronal tract tracer, and demonstrate the validity of the approach by showing brain networks of cortical connectivity in live rats under MRI, together with the corresponding microscopic details, such as fibers and neuronal morphology under light microscopy. We further demonstrate that the developed molecule is the first MRI-visible probe to preferentially trace retrograde connections. Our study offers a new platform for the development of multimodal molecular imaging tools of broad interest in neuroscience, that capture in vivo the dynamics of large scale neural networks together with their microscopic characteristics, thereby spanning several organizational levels. http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Logothetis Department Scheffler http://pubs.acs.org/doi/pdf/10.1021/cn200022m 10.1021/cn200022m anuragrkAMishra schuezASchüz joernJEngelmann bayoMBeyerlein nikosNKLogothetis canalsSCanals article SweidanEME2011 Letters in Organic Chemistry 2011 10 8 8 603-605 The reaction of 1,3-dimethyl-5-bis(thioethyl)methylene barbituric acid (2) with various vic-diamines leads to the formation of the respective 1,3-diazamethylene barbiturate derivatives 3 (a-f) in high yields. Solid-state 13C-NMR, MS and IR spectral data were employed for the characterization of these compounds. Antimicrobial screening revealed that none of the newly synthesized compounds showed appreciable antibacterial activity. http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Scheffler http://www.ingentaconnect.com/content/ben/loc/2011/00000008/00000008/art00018 10.2174/157017811797249353 sweidanKSweidan joernJEngelmann rajuRJoshi MSMubarak MMEl-Abadelah article PlacidiENLA2011 Chemical Communications 2011 9 47 41 11534-11536 Four ligand systems have been prepared whose characteristics are well suited to the design of bimodal MRI and luminescence probes. The lanthanide complexes display high relaxivities and luminescence quantum yields. These properties are retained at higher magnetic fields and in a range of competitive environments including model extracellular medium and cultured cells. http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Logothetis Department Scheffler http://pubs.rsc.org/en/content/articlepdf/2011/cc/c1cc14437e 10.1039/C1CC14437E matteoMPPlacidi joernJEngelmann LSNatrajan nikosNKLogothetis goranGAngelovski article KelirisZMPSUE2011 Biorganic & Medicinal Chemistry 2011 4 19 8 2529-2540 Noninvasive monitoring of intracellular targets such as enzymes, receptors, or mRNA by means of magnetic resonance imaging (MRI) is increasingly gaining relevance in various research areas. A vital prerequisite for their visualization is the development of cell-permeable imaging probes, which can specifically interact with the target that characterizes the cellular or molecular process of interest. Here, we describe a dual-labeled probe, Gd-DOTA-k(FR)-Gal-CPP, designed to report the presence of intracellular β-galactosidase (β-gal) enzyme by MRI. This conjugate consists of a galactose based core serving as cleavable spacer, incorporated between the cell penetrating peptide D-Tat49-57 and reporter moieties (Gd-DOTA, fluorescein (FR)). We employed a facile building block approach to obtain our bimodal probe, Gd-DOTA-k(FR)-Gal-CPP. This strategy involved the preparation of the building blocks and their subsequent assembly using Fmoc mediated solid phase synthesis, followed by the complexation of ligand 14 with GdCl3. Gd-DOTA-k(FR)-Gal-CPP showed a considerably higher relaxivity enhancement (16.8±0.6 mM-1s-1, 123 MHz) relative to the commercial Gd-DOTA (4.0±0.12 mM-1s-1, 123 MHz). The activation of Gd-DOTA-k(FR)-Gal-CPP was based on a cellular retention strategy that required enzymatic cleavage of the delivery vector from galactose moiety following the cell internalization to achieve a prolonged accumulation of the reporter components (Gd-DOTA/FR) in the β-gal expressing cells. Cellular uptake of Gd-DOTA-k(FR)-Gal-CPP in β-gal expressing C6/LacZ and enzyme-deficient parental C6 rat glioma cells was confirmed by fluorescence spectroscopy, MR imaging and ICP-AES measurements. All methods showed higher accumulation of measured reporters in C6/LacZ cells compared to enzyme deficient parental C6 cells. Fluorescence microscopy of cells labeled with Gd-DOTA-k(FR)-Gal-CPP indicated a predominantly vesicular localization of the green fluorescent conjugate around cell nuclei. This cellular distribution was most likely responsible for the observed nonspecific background signal in the enzyme deficient C6 cells. Even though, the specific accumulation of our bimodal probe has to be further improved, it could be already used for cell imaging by MRI and optical modalities. http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Scheffler http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6TF8-52CG6N8-3-D&_cdi=5220&_user=29041&_pii=S0968089611002045&_origin=gateway&_coverDate=04%2F15%2F2011&_sk=999809991&view=c&wchp=dGLbVlb-zSkzS&md5=00f8202c5dd25114e366170324c9576d&ie=/sdarticle.pdf 10.1016/j.bmc.2011.03.023 abrudAKeliris TZiegler rituRMishra rolfRPohmann MSauer KUgurbil joernJEngelmann article JhaMGWUME2011 Bioconjugate Chemistry 2011 3 22 3 319-328 Cell-penetrating peptides (CPPs) may have impli-cations in biomedical sciences by improving the delivery of a wide variety of drugs through the membrane barrier. CPPs are generally taken up by endocytotic pathways, and vesicular encapsulation is a limiting factor in the area of intracellular targeting. A novel, cationic cysteine-rich CPP, CyLoP-1, has been developed exhibiting distinguished diffused cytosolic distribution along with endosomal uptake at low micromolar concentrations. Comparative uptake analysis with known CPPs showed CyLoP-1 as a promising delivery vector to access the cytosol in a variety of cell types. In addition to the positively charged residues, the presence of cysteines and tryptophans proved to be essential to maintain its functionality. Also, the oxidation status of the cysteines played an important role for the uptake efficiency of CyLoP-1, with the disulfide-containing form being more effective. The distinct feature of CyLoP-1 to enter the cytosol was further explored by the covalent attachment of cargoes of different nature and sizes. In particular, induction of caspase-3 activity (indicating apoptosis) by a CyLoP-1-SmacN7 conjugate proved successful delivery of the pro-apoptotic cargo to its site of action in the cytosol. Efficient intracellular delivery into the entire cytosol already at low micromolar concentrations makes CyLoP-1 a promising candidate for cytosolic delivery of cargoes of small sizes. Thus, this peptide might prove to be useful for efficient transmembrane delivery of agents directed to cytosolic targets. http://www.kyb.tuebingen.mpg.dehttp://pubs.acs.org/doi/pdf/10.1021/bc100045s http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://pubs.acs.org/doi/pdf/10.1021/bc100045s 10.1021/bc100045s djhaDJha rituRMishra sgottSGottschalk KHWiesmüller KUgurbil MEMaier joernJEngelmann article 6943 Journal of Peptide Science 2011 1 17 1 8-13 Peptide nucleic acids (PNAs) are DNA mimics with a neutral peptide backbone instead of the negatively charged sugar phosphates. PNAs exhibit several attractive features such as high chemical and thermal stability, resistance to enzymatic degradation, and stable binding to their RNA or DNA targets in a sequence-specific manner. Therefore, they are widely used in molecular diagnosis of antisense-targeted therapeutic drugs or probes and in pharmaceutical applications. However, the main hindrance to the effective use of PNAs is their poor uptake by cells as well as the difficult and laborious chemical synthesis. In order to achieve an efficient delivery of PNAs into cells, there are already many published reports of peptides being used for transport across the cell membrane. In this protocol, we describe the automated as well as cost-effective semi-automated synthesis of PNAs and PNA-peptide constructs on an automated peptide synthesizer. The facile synthesis of PNAs will be helpful in generating PNA libraries us able, e.g. for high-throughput screening in biomolecular studies. Efficient synthetic schemes, the automated procedure, the reduced consumption of costly reagents, and the high purity of the products are attractive features of the reported procedure. http://www.kyb.tuebingen.mpg.de/fileadmin/user_upload/files/publications/JoshiR2011_6943[0].pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://onlinelibrary.wiley.com/doi/10.1002/psc.1305/pdf Biologische Kybernetik Max-Planck-Gesellschaft en 10.1002/psc.1305 rajuRJoshi djhaDJha wusuWSu joernJEngelmann article 6613 Inorganic Chemistry 2010 6 49 13 6124-6138 http://www.kyb.tuebingen.mpg.de/fileadmin/user_upload/files/publications/HenigJ2010_6613[0].pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://pubs.acs.org/doi/pdfplus/10.1021/ic1007395 Biologische Kybernetik Max-Planck-Gesellschaft en 10.1021/ic1007395 JHenig ÉTóth joernJEngelmann sgottSGottschalk HAMayer article 6384 Bioorganic and Medicinal Chemistry Letters 2010 4 20 7 2238-2241 A new mRNA targeting contrast agent consisting of three main functional domains, (i) gadolinium based magnetic resonance reporter part, (ii) antisense peptide nucleic acids targeted to mRNA, and (iii) cholesterol as the delivery vector, was developed and synthesized. The new contrast agent showed efficient cellular uptake and significant contrast enhancement at very low labeling concentrations (0.5 μM). However, after uptake into cells the agent was located predominantly in endosomes like a similar cell penetrating peptide conjugated probe. Our results indicate that this newly developed contrast agent could be used for the labeling of cells for optical as well as magnetic resonance imaging. http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6TF9-4YBKG37-9-D&_cdi=5221&_user=29041&_pii=S0960894X1000209X&_orig=search&_coverDate=02%2F08%2F2010&_sk=999999999&view=c&wchp=dGLbVlb-zSkzV&md5=f96c525963630 Biologische Kybernetik Max-Planck-Gesellschaft en 10.1016/j.bmcl.2010.02.019 rajuRJoshi rituRMishra rolfRPohmann joernJEngelmann article 6119 Bioconjugate Chemistry 2009 9 20 10 1860-1868 Molecular imaging of cells and cellular processes can be achieved by tagging intracellular targets such as receptors, enzymes, or mRNA. Seeking to visualize the presence of specific mRNAs by magnetic resonance (MR) imaging, we coupled peptide nucleic acids (PNA) with gadolinium-based MR contrast agents using cell-penetrating peptides for intracellular delivery. Antisense to mRNA of DsRed2 protein was used as proof of principle. The conjugates were produced by continuous solid-phase synthesis followed by chelation with gadolinium. Their cellular uptake was confirmed by fluorescence microscopy and spectroscopy as well as by MR imaging of labeled cells. The cell-penetrating peptide D-Tat57−49 was selected over two other derivatives of HIV-1 Tat peptide, based on its superior intracellular delivery of the gadolinium-based contrast agents. Further improved delivery of conjugates was achieved upon coupling peptide nucleic acids (antisense to mRNA of DsRed2 protein and nonsense with no natural counterpart). Significant enhancement in MR contrast was obtained in cells labeled with concentrations as low as 2.5 μM of these agents. Specific binding of the targeting PNA containing conjugate to its complementary oligonucleotide sequence was proven by in vitro cell-free assay. In contrast, a lack of specific enrichment was observed in transgenic cells containing the target due to nonspecific vesicular entrapment of contrast agents. Preliminary biodistribution studies showed conjugate-related fluorescence in several organs, especially the liver and bladder, indicating high mobility of the agent in spite of its high molecular weight. No conjugate related toxicity was observed. These results are encouraging, as they warrant further molecular optimization and consecutive specificity studies in vivo of this new generation of contrast agents. http://www.kyb.tuebingen.mpg.de/fileadmin/user_upload/files/publications/MishraR2009_6119[0].pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Logothetis Department MRZ http://pubs.acs.org/doi/pdf/10.1021/bc9000454 Biologische Kybernetik Max-Planck-Gesellschaft en 10.1021/bc9000454 rituRMishra wusuWSu rolfRPohmann josefJPfeuffer MGSauer KUgurbil joernJEngelmann article 4366 Contrast Media and Molecular Imaging 2007 2 2 1 42-49 In order to image mRNA transcription by in vivo magnetic resonance imaging (MRI), two intracellular MR contrast agents were developed, which are composed of a Gd-DOTA complex, a peptide nucleic acid (PNA) sequence and a cell-penetrating peptide. One was designed to bind to mRNA of dsRed (red fluorescent protein originating from Discosoma coral) by its PNA sequence, whereas the second one contains a nonsense sequence with no natural counterpart. The conjugates were synthesized using a continuous solid-phase synthesis scheme and characterized by ESI-MS. Fluorescence studies showed that both contrast agents could enter efficiently into 3T3 cells in a concentration-dependent manner from 0.5 to 9.0 µM. The contrast agent was located predominantly in vesicles around the nucleus, whereas no uptake into the nucleus was observed. The results of in vitro MR studies showed a statistically significant increase of the intracellular relaxation rate R 1,cell at a labeling concentration of only 0.5 µM, thus contrast enhanc ement was detectable too. These results suggest that the synthesized contrast agents could label cells for optical as well as MR imaging and in future might be useful to prove specific accumulation in cells containing target mRNA. http://www.kyb.tuebingen.mpg.de/fileadmin/user_upload/files/publications/CMMI126_Supplementary%20Information_[0].pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://www3.interscience.wiley.com/cgi-bin/fulltext/114128669/PDFSTART Biologische Kybernetik Max-Planck-Gesellschaft en 10.1002/cmmi.126 wusuWSu rituRMishra josefJPfeuffer KHWiesmüller KUgurbil joernJEngelmann article 3633 Bioconjugate Chemistry 2006 4 17 3 773-780 http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Logothetis Department MRZ http://pubs.acs.org/cgi-bin/article.cgi/bcches/2006/17/i03/pdf/bc050295b.pdf Biologische Kybernetik Max-Planck-Gesellschaft en 10.1021/bc050295b anuragrkAMishra josefJPfeuffer rituRMishra joernJEngelmann akmishraAMishra KUgurbil nikosNKLogothetis inproceedings 6760 Peptides 2008, Proceedings of 30th European Peptide Symposium 2008 9 550-551  http://www.kyb.tuebingen.mpg.de/fileadmin/user_upload/files/publications/JoshiR2009_[0].pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://www.eurpepsoc.com/proceedings-of-the-30th-european-peptide-symposium/ Lankinen, H. , J. Vallivirta, T. Strandin, J. Hepojoki FIPS

Helsinki, Finland

Peptides 2008: Chemistry of Peptides in Life Science Technology and Medicine Biologische Kybernetik Max-Planck-Gesellschaft Helsinki, Finland 30th European Peptide Symposium (30 EPS) en 978-952-92-7697-4 rajuRJoshi rituRMishra wusuWSu joernJEngelmann inbook MishraDMSEBCL2011 2012 2 1 181-204 One of the most striking characteristic of the brain is its profuse neuronal connectivity. Not surprisingly, the function of the nervous system critically depends on the spatiotemporal pattern of intercommunication between different regions of the brain. Both macro- and microscopic aspects of the wiring diagrams of brain circuits are relevant and need to be understood in order to cope with the complexity of the brain function. In this way, for instance, the long-range connections that carry the functional specification of cortical territories need to be studied together with the detailed microcircuits inside a cortical column. Moreover, the temporal dimension of these wiring diagrams must be investigated since neuronal networks are dynamic structures exhibiting context-dependent changes in synaptic weights (Canals et al., 2009) and numbers (Chklovskii et al., 2004). Investigations over the last decades strongly suggest that stimulus or task related neural activity is distributed over large parts of the brain, covering different cortical and sub-cortical areas. For a detailed understanding of brain function, it is of prime importance to understand the organization of the neuronal connections. To chart the anatomical connections between the various components of brain networks, the neuronal tract tracing technique has been proved to be very useful. Thus, experimental tools that allow the exploration of brain circuits at diverse organizational levels are mandatory for the understanding of brain intercommunication and information processing. http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Logothetis Department Scheffler http://www.intechopen.com/articles/show/title/biocytin-based-contrast-agents-for-molecular-imaging-an-approach-to-developing-new-in-vivo-neuroanat Bright, P. InTech

Rijeka, Croatia

Neuroimaging - Methods 978-953-51-0097-3 10.5772/23806 anuragrkAMishra rituRMishra canalsSCanals nikosNKLogothetis bayoMBeyerlein joernJEngelmann schuezASchüz kirtiKDhingra poster MamedovEHEL2012 2012 5 20 1632 A Gd3+ based paramagnetic dextran conjugate has been developed, which enables the tracking of neuroanatomical connectivity in the brain by both MR and optical imaging. Cell studies demonstrated that the synthesized tracer was efficiently internalized into neuronal cells and transported toward the axons. Furthermore, our preliminary in vivo experiments revealed efficient transportation of the conjugate, thereby proving its applicability for neuroanatomical studies by T1-weighted MRI. Initial in vivo experiments in rodents demonstrated the significant potential of this method. http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Logothetis Department Scheffler http://www.ismrm.org/12/ Melbourne, Australia 20th Annual Meeting and Exhibition of the International Society for Magnetic Resonance in Medicine (ISMRM 2012) ilgarIMamedov joernJEngelmann ghagbergGHagberg oeschenkoOEschenko nikosNKLogothetis poster BrudMESZU2011 2011 P30 http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://www.cost.eu/domains_actions/cmst/Actions/D38 Tübingen, Germany CMST COST Action D38: Metal-Based Systems for Molecular Imaging Applications abrudABrud rituRMishra joernJEngelmann wusuWSu TZiegler KUgurbil poster GottschalkBPE2010_2 2010 9 2010 0926B Despite of decade-long research currently no method exists that could either accurately or non-invasively determine beta-cell mass in vivo. However, quantification of beta-cells would allow to understand the pathophysiology of diabetes, to identify pre-diabetic patients and to follow up cellular therapies (e.g. islet transplantations). Here, we present in vivo and ex vivo MRI data of the murine pancreas at ultra high fields (16.4T) and the first attempt to visualize pancreatic islets with a newly developed targeted contrast agent (CA) based on a single chain antibody fragment (SCA1, kindly provided by S. Schneider, Bochum, Germany). METHODS: Beta-cell specific SCA1 was covalently coupled to superparamagnetic cobalt nanoparticles (NPs). C57BL/6J-mice were injected intravenously with PBS (control), unlabeled NPs or SCA1-NPs. Five hours later the animals were anaesthetized with isoflurane, a constant breathing rate was maintained and T2*-weighted MR-Images were recorded. Then, the mice were sacrificed, organs were taken out and MR-images were recorded overnight (fig. 1). EX VIVO: As expected, punctuate loss of signal intensity (sizes are consistent to average diameters of islets) in the excised pancreas of SCA1-NPs treated mice was seen. Binding of the prospective CA to the islets was also verified by immunofluorescence (data not shown). IN VIVO: All organs can be easily identified and accumulation of unlabeled NPs as well as SCA1-NPs was clearly visible in liver and spleen. However, the anticipated punctuate signal-loss in pancreatic tissue was not yet detectable in vivo (data not shown). CONCLUSIONS: For the first time we have demonstrated the feasibility of in vivo MRI of the mouse abdomen at 16.4T. This allowed MR-microscopic sensitivity for structures <100µm and anatomical details of the pancreas were identified. Despite of the high spatial resolution at this field strength the cellular architecture of the pancreas, i.e. the location or amount of islets of Langerhans remains difficult to assess. Furthermore, using a novel targeted CA in vivo, beta-cell containing islets of Langerhans were identified in excised pancreas. Financial support of the Max-Planck Society and German Ministry for Education and Research (BMBF, FKZ: 01EZ0813) is gratefully acknowledged. http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Scheffler http://www.wmicmeeting.org/2010/Abstracts/forSystemUse/papers/P0926B.html Kyoto, Japan 2010 World Molecular Imaging Congress (WMIC) sgottSGottschalk balladDZBalla rolfRPohmann joernJEngelmann poster GottschalkJE2010 2010 9 2010 0949A Intracellular and especially cytosolic delivery with cell penetrating peptides (CPPs) can be severely limited by endocytotic uptake, leading to unwanted vesicular entrapment. A correlation between cell-surface thiols and uptake efficiency of disulfilde-containing CPPs was discussed and it was shown that these CPPs possess much better cytosolic targeting capability [1].Our newly developed, cysteine-rich CPP, CyLoP-1 (Cytosol Localizing Peptide 1, CRWRWKCCKK) showed pronounced cytosolic delivery already in its non-oxidized form. Aim of the present study was to evaluate if a controlled oxidation of the cysteines, generating disulfide linkages in CyLoP-1, further increases its cytosolic targeting. To test this hypothesis the pro-apoptotic peptide AVPIAQK (SmacN7) was attached to CyLoP-1. SmacN7 alone can not pass cellular membranes and needs to bind to its cytosolic target to exert a pro-apoptotic action and thus can be used to prove cytosolic delivery of CPP-conjugates. CyLoP-1 (covalently bound to lysine-FITC) was subjected to oxidizing conditions. SmacN7 was covalently conjugated to the N-terminus, yielding the construct SmacN7-K(FITC)-CyLoP-1, which was also oxidized. Internalization of all 4 compounds was evaluated on 3T3 fibroblasts (2.5µM, 18 hours, for method details see [2]). Caspase-3 activity as parameter for apoptosis-induction by the reduced and oxidized SmacN7-K(FITC)-CyLoP-1 was measured in HeLa cells. Intracellular uptake (measured by FITC-fluorescence) was significantly higher for the oxidized versions of both the peptide itself and SmacN7-CyLoP-1. This was confirmed by fluorescence microscopy of the same cells, displaying higher cytosolic fluorescence for the oxidized versions of the compounds. However, cargo-attachment reduced the internalization efficacy of CyLoP-1 in its reduced as well as oxidized form. SmacN7-CyLoP-1 was capable of inducing apoptosis in HeLa cells, while SmacN7 alone had no effect, proving efficient delivery of the bioactive cargo into the cytosol. Oxidation of SmacN7-CyLoP-1 further enhanced the amount of apoptotic cells (i.e. increased Caspase-3 activity). Thereby demonstrating that oxidative modification of cysteine-residues in CyLoP-1 enhances its cytosolic targeting capability. Conclusion: Our results suggest an important role of disulfides in the uptake mechanisms of cysteine-containing CPPs and also in their capability to deliver to the cytosol. The use of disulfide-motifs in CPPs might also be applicable for other types of cargos, like imaging agents. [1]Aubry,FASEB J.23(2009)2956 [2]Mishra,Bioconjug.Chem.2009 http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Scheffler http://www.wmicmeeting.org/2010/Abstracts/forSystemUse/papers/P0949A.html Kyoto, Japan 2010 World Molecular Imaging Congress (WMIC) sgottSGottschalk djhaDJha joernJEngelmann poster AngelovskiMEGBPL2010 2010 9 2010 0945B Magnetic resonance imaging (MRI) is a powerful tool in clinical diagnostics and is also used for the understanding of developmental and biological processes. It visualizes the differences in tissues and organs, as well as between normal and pathological states. Due to its noninvasive nature, excellent spatial resolution and tissue penetration, MRI became one of the preferential methods for molecular and cellular imaging. The specificity and sensitivity of MRI can be further enhanced by the introduction of contrast agents. Many of the currently existing contrast agents are restricted to the extracellular space, though novel approaches enforce generations of intracellular contrast agents that can be developed for targeted labeling of cells for the visualization of a specific biological process. However, the lower sensitivity of MRI as compared to other imaging techniques demands certain quantitative considerations for the rational design of these intracellular agents. Gadolinium complexes are the most frequent choice for T1-weigthed MR-imaging. Besides having a high longitudinal relaxivity (r1), these agents should also be delivered in a sufficient amount to target structures on the cell membrane or inside the cells. We have performed a study in an attempt to determine the minimum number of gadolinium ions needed for the efficient labeling of cells. The concentration dependent contrast enhancement of Gd-DOTA, Gd-DO3A or Gd-AAZTA in T1-weighted MR-images of phantoms with water only, cell culture medium containing serum, or in the presence of cells was followed at different, ultra-high magnetic fields (3T, 7T, 16.4T) and with the spatial resolution commonly used for in vivo measurements. The results suggest MRI detectability at low micromolar concentrations for all applied contrast agents, which correspond to the previously predicted number of metal atoms per cell [1]. However, this number is dependent on the r1 of the contrast agent and the magnetic field of the imaging scanner. Financial support of the Max-Planck Society and German Ministry for Education and Research (BMBF, FKZ: 01EZ0813) is gratefully acknowledged. http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Logothetis Department Scheffler http://www.wmicmeeting.org/2010/Abstracts/forSystemUse/papers/P0945B.html Kyoto, Japan 2010 World Molecular Imaging Congress (WMIC) goranGAngelovski ilgarIMamdeov joernJEngelmann sgottSGottschalk balladDZBalla rolfRPohmann nikosNKLogothetis poster EngelmannHGM2010 2010 5 2010 1897 Silsesquioxane-based macromolecular MRI-contrast agents have recently been reported to have a compact globular structure resulting in significantly higher relaxivities when compared to smaller MRI-probes. The stability of the core under physiological conditions is unknown so far. We therefore studied the stability of Gadoxane, a new silsesquioxane-based contrast agent connected to eight DOTAGA-gadolinium chelates. The silsesquioxane core was almost completely hydrolyzed under physiological conditions while the integrity of the gadolinium chelate was maintained. In conclusion, these probes cannot be considered as stable anymore. However, their degradability might improve their in vivo-applicability due to a faster renal excretion of the smaller fragments. http://www.kyb.tuebingen.mpg.defileadmin/user_upload/files/publications/ISMRM-ESMRMB-2010-Engelmann.pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Scheffler http://www.ismrm.org/10/ Stockholm, Sweden ISMRM-ESMRMB Joint Annual Meeting 2010 joernJEngelmann JHenig sgottSGottschalk HAMayer poster 7018 2010 5 2010 1879 Despite of decade-long research, the quantification of insulin producing beta-cells in the pancreas is still not ready for clinical application. Currently, no method exists that could either accurately or non-invasively determine the betacell mass in humans [1]. The quantification of beta-cells not only would allow to understand the pathophysiology of both type 1 and 2 diabetes in more detail, but also to identify pre-diabetic patients and to follow up cellular therapies (e.g. islettransplantations). Several in vivo imaging approaches are being developed (e.g. fluorescence, positron emission tomography). But these methods have some inherent limitations (low depth in tissue penetration, ionizing radiation, low resolution). Magnetic Resonance Imaging (MRI) on the other hand offers the advantages of using non-ionizing radiation and having a high spatial resolution. Here we present in vivo MRI of the mouse abdomen and pancreas at ultra high fields (16.4T) and the first attempt to visualize pancreatic islets with a newly developed beta-cell specific superparamagnetic contrast agent based on a single chain antibody (kindly provided by PD Dr. S. Schneider, Bochum, Germany). http://www.kyb.tuebingen.mpg.defileadmin/user_upload/files/publications/ISMRM-ESMRMB-2010-5511.pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://www.ismrm.org/10/ Biologische Kybernetik Max-Planck-Gesellschaft Stockholm, Sweden ISMRM-ESMRMB Joint Annual Meeting 2010 en sgottSGottschalk balladDZBalla rolfRPohmann joernJEngelmann poster JoshiME2009 2009 9 2009 0647 Magnetic Resonance Imaging (MRI) is meanwhile one of the most important medical diagnostic tools. Its specificity and sensitivity can be further extended by contrast agents (CAs). As many clinically valuable targets like DNA, mRNA or protein/enzymes reside inside the cell membrane, development of efficient intracellular targeted MR CA is required. However, prerequisite for intracellular targeting is not only the efficient delivery inside the cell but also the co-localization with the target. Recently, cell penetrating peptides (CPP) are used to achieve an efficient uptake of cargo molecules. However, it has been shown that these conjugates were predominantly taken up by an endosomal mechanism preventing a proper interaction with targets located in the cytosol. Cholesterol coupling has been reported to facilitate cellular import of siRNAs for effective silencing of protein expression [1]. We developed a contrast agent based on a lipid mediated delivery system by using cholesterol. Uptake and MR contrast enhancement ability was compared with a CPP based CA previously reported by our group [2]. To image the presence of specific mRNAs the probes composed of Gd-DOTA, FITC, a sequence to bind to target mRNA (DsRed), and CPP (D-Tat) or cholesterol for cellular delivery. Fmoc continuous solid phase chemistry was used for synthesis. Fluorescence and MR studies were performed using a mouse fibrosarcoma cell line expressing DsRed protein and its parent cell line deficient of the target sequence. Fluorescence spectroscopy showed that the CPP based CA (CPP-CA) could enter efficiently in both cell types without observable toxicity up to a concentration of 5μM. The cholesterol based CA (Chol-CA) was even more efficient. However, this conjugate was not soluble in aqueous solution at concentrations > 3µM. Both probes were able to enhance MRI contrast in labeled target containing as well as non-targeted parent cells. Intracellular relaxation rate increased already at a labeling concentration of 1µM for CPP-CA and 0.5µM for Chol-CA. However, fluorescence microscopy demonstrated that Chol-CA was also predominantly localized inside endosomes. Coupling of cholesterol further improved uptake and contrast enhancement. However, the reduced solubility in physiological aqueous media is restricting the applicability for MR imaging purposes. In addition, endosomal entrapment poses a still unsolved problem. Modifications to circumvent both drawbacks have to be implemented to achieve a sufficient cytosolic distribution of CA. http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ Department Logothetis http://www.wmicmeeting.org/abstracts/data/papers/0647.html Montréal, Canada 2009 World Molecular Imaging Congress (WMIC) rajuRJoshi rituRMishra joernJEngelmann poster JhaMWUE2009 2009 9 2009 0683 Cell Penetrating Peptides (CPPs) have gained attention in biomedical field because of their ability to deliver a variety of hydrophobic moieties through the membrane barrier. CPPs are generally taken up by endocytotic pathways and vesicular encapsulation limits the interaction of the attached cargo with targets present in the cytosol. Here we present the systematic development of a cysteine rich CPP (derived from polypeptide Crotamine [1]) by Structure Activity Relationship (SAR) studies. Various peptides were synthesized by Fmoc/tBu strategy and labeled with fluorescein isothiocyanate at the N-terminus. After purification by RP-HPLC, the products were lyophilized and characterized by ESI-MS. Uptake of fluorescently labeled peptides was assessed in NIH-3T3 mouse fibroblasts by fluorescence spectroscopy and microscopy. Our studies identified a novel CPP, CyLoP-1, which is efficiently delivered into the entire cytoplasm of cells besides being encapsulated in vesicles at low concentrations of 2.5 µM in a non-cytotoxic and temperature independent fashion. It proved to be most efficient in its natural L-form as SAR studies indicated a clear negative effect of chiral inversion of the residues on cellular uptake and distribution of CyLoP-1. The additional feature of cytosolic gain was further explored by the covalent attachment of different cargoes to CyLoP-1. Payload consisted of fluorophore, a Magnetic Resonance Imaging agent (Gd)-DOTA, a bio-active peptide (SmacN7), the larger peptide Penetratin, or a PNA molecule. The regular decrease in the uptake and localization pattern concomitant with the increase in the cargo size was observed. This indicates the dependence of internalization and distribution profile of CyLoP-1 constructs on the nature, charge as well as size of the cargo. Efficient intracellular delivery into the entire cytosol and a low cytotoxicity makes CyLoP-1 a promising candidate for cytosolic delivery of cargoes of small sizes like intracellular targeted probes. [1] Kerkis A. et al, FASEB J. 2004, 18, 1407. http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Scheffler http://www.wmicmeeting.org/abstracts/data/papers/0683.html Montréal, Canada 2009 World Molecular Imaging Congress (WMIC) djhaDJha rituRMishra sgottSGottschalk K-HWiesmüller KUgurbil joernJEngelmann poster MishraJGUE2009 2009 9 2009 0776 Endocytosis is a fundamental process through which essential biomolecules otherwise unable to permeate the plasma membrane are taken up by cells. This process is mainly used to fulfill the nutritional requirements of cells. The idea of exploiting this uptake mechanism to transfer exogenous macromolecules into cells is widely used. After crossing the cellular membrane via endocytosis most macromolecules enter the so-called endo-Iysosomal degradation pathway. This still is a severe hindrance for the development of therapeutics and diagnostic tools based on intracellular targets like. DNA or oligonucleotides. Cell penetrating peptides (CPPs) seemed to be promising tools for improving intracellular delivery of cargos. However, recent studies questioned the maintenance of biological activity of specific molecules following CPP-assisted uptake. For most CPPs it has now been established that the mechanism of internalization mainly involves endocytosis/macropinocytosis, by this limiting access to targets located in the cytosol. Inspired by Crotamine, a toxin in snake venom, we developed the novel CPP CyLoP-1 that was able to pass through the cellular membrane barrier. Recently, we have shown its capability of delivering a MRI contrast agent into the cytosol. Herein we present the characterization of CyLoP-1 in comparison to other well-studied CPPs. The fluorescently labeled CPPs were assessed for cellular and cytosolic localization in 3T3 fibroblasts by fluorescence spectroscopy and microscopy. We also studied the distribution in vivo by injecting CyLoP-1 into mice followed by ex vivo observation of fluorescence in organs. CyLoP-1 required the presence of serum for solubility in medium and for cytosolic delivery. Without serum CyLoP-1 stuck to the outer membrane. In contrast, arginine-rich CPPs showed diffuse labeling in serum-free medium. Cytosolic delivery of CyLoP-1 was maintained at 4°C indicating the uptake to be independent or complementary to endocytosis. In vivo studies showed that the peptide was readily dispersed into the blood stream after ip injection and it excretion via the kidneys after 4h. Apart from the kidneys and the bladder, many other organs (liver, spleen, lungs) were fluorescent. Interestingly, CyLoP-1 showed faint labeling in the brain as well. Our study reveals that CyLoP-1, a cysteine and arginine-rich peptide, is remarkably different in comparison to other available CPPs. Its peculiar cytosolic localization makes it a likely candidate for targeted delivery of drugs and agents for molecular imaging. http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Scheffler http://www.wmicmeeting.org/abstracts/data/papers/0776.html Montréal, Canada 2009 World Molecular Imaging Congress (WMIC) rituRMishra djhaDJha sgottSGottschalk KUgurbil joernJEngelmann poster DhingraMESCPBML2009 2009 9 2009 0617 Magnetic resonance imaging (MRI) is one of the powerful imaging modality. To circumvent its low sensitivity, there has been a substantial interest on the development of the contrast agents. In the present scenario, there is a need to develop contrast agents which are target specific and can report the changes in the physiological environment around them. On the similar lines we are reporting here a novel precursor (tris-tert-Bu-(Z)-Ser-DO3A (Figure 1)). This precursor contains an amine and a carboxylate groups in an orthogonally protected condition, which allows their selective de-protection and coupling to different moieties. Out of the various possibilities, we explored two strategies of coupling that lead to a potential targeted CA and another CA with potential of tracing neuroanatomy in the brain. The special design of these agents not only provides the stability against their enzymatic degradation which is important for their in vivo applicability but also has the possibility to amplify its signal once recognized by the target site. This could be done by exploiting the biotin/strept(avidin) high affinity and the pretargeting strategy, which is well established in nuclear medicine. The CA when bound to avidin showed an enhancement in the relaxivity (r1 and r2) at 1.5T. A substantial increase of ≥1000% in r2 was observed at all magnetic fields studied (1.5T, 3T, 7T, 9.4T) while r1 showed an increase of 260% at 1.5T and an expected decrease with further increase of field strength. The relaxivity changes at 1.5T suggest the structural requirement of a CA to fit in to avidin and optimize the parameters determining relaxivity of the complex matches well with our synthesized agent. Using the same precursor, we have also synthesized a CA which can potentially be used for tracing the neuronal tracks in the brain. Biocytin was used as the basic tracer. Coupling an MR detectable moiety to a well known neuroanatomical tracer would open up new possibilities to noninvasively study the neuronal networks by MRI. http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Logothetis Department MRZ http://www.wmicmeeting.org/abstracts/data/papers/0617.html Montréal, Canada 2009 World Molecular Imaging Congress (WMIC) kirtiKDhingra anuragrkAMishra joernJEngelmann schuezASchüz canalsSCanals rolfRPohmann bayoMBeyerlein MEMaier nikosNKLogothetis poster MishraDMSELC2009 2009 9 2009 0081 Gaining insights into brain function by identifying neuronal circuits connecting different regions of the brain is of prime interest in cognitive neuroscience. For visualization of anatomical connections, the tract-tracing technique has been proved to be an extremely useful tool which provides valuable information on afferent and efferent connectivity of the brain. A variety of neuroanatomical tracers exists, and numerous investigations using classical tracers have contributed valuable descriptions of connectivity in the mammalian brain (1). These studies, however, require fixed, histologically processed tissue for data analysis and therefore cannot be applied for noninvasive/longitudinal investigations. Manganese-enhanced Magnetic Resonance Imaging is a recently introduced non-invasive technique that represents the first effort in the direction of studying neuronal connectivity in vivo by means of MRI (2). It is based on the paramagnetic properties of Mn2+ ion and on the fact that, once taken up by neurons, Mn2+ is transported anterogradely along the axon. However, the technique presents several drawbacks that can challenge its applicability, the most important being the potential toxicity of the ion in the tissue. In this study, we have developed an entirely new tracing technique where we have synthesized stable and bifunctional neuronal tracers that allow both, in vivo brain connectivity studies by means of MRI and postmortem microscopic investigation in fixed tissue, in the same experimental animal. Existing tracer molecules with excellent tracing properties could be covalently connected to organic macrocyclic moiety caging gadolinium as MR reporter. We started with biocytin and biotinylated-dextranamine (BDA) as model molecules which are well known anterograde and also retrograde (as opposed to Mn2+) neuronal tracer. Due to high interaction of biotin-avidin, both tracers can be visualized in postmortem tissue by using a host of avidin conjugated markers at the light- and electron microscope level. We have performed in vitro cell studies in neuroblastoma cell lines to prove their uptake competence and in vivo MR/histological visualization to prove their in vivo tracing efficiency as potential neuroanatomical tract-tracers. Our results reveal that these new tracers allow for a new strategy of neuronal tract-tracing, combining the powerful spatial resolution of the conventional microscopic techniques and in vivo visualization by MRI. References. (1) Prog Neurobiol (2000), 62, 327 and (2) Neuroimage (2008), 40, 458. http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Logothetis Department MRZ http://www.wmicmeeting.org/abstracts/data/papers/0081.html Montréal, Canada 2009 World Molecular Imaging Congress (WMIC) anuragrkAMishra kirtiKDhingra rituRMishra schuezASchüz joernJEngelmann nikosNKLogothetis canalsSCanals poster 6261 2009 4 17 3123 A novel cell penetrating peptide (CyLoP-1) was obtained by structure activity relationship studies. It exhibited efficient transport across the cell membrane and distribution in the entire cytosol as well. These distinctive properties were only slightly influenced by the coupling of Gd-DOTA. This conjugate was slightly less internalized into 3T3 fibroblasts, but the cellular distribution was retained. MR studies on labeled cells revealed an exceptional high contrast enhancement in T1-weighted images. These results demonstrate its potential to be used for efficient transmembrane delivery of imaging agents and as vector for probes specifically targeted to cytosolic constituent. http://www.kyb.tuebingen.mpg.defileadmin/user_upload/files/publications/ISMRM-2009-03123.pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://www.ismrm.org/09/ Biologische Kybernetik Max-Planck-Gesellschaft Honolulu, HI, USA 17th Annual Meeting of the International Society for Magnetic Resonance in Medicine (ISMRM 2009) en joernJEngelmann djhaDJha rituRMishra K-HWiesmüller KUgurbil poster 5286 Journal of Peptide Science 2008 9 14 S1 154-155 Cell penetrating peptides (CPP) are a special class of peptides that possess the property to traverse the formidable barrier of the plasma membrane and deliver cargos into cells. Using CPP as vectors and DNA, mRNA or proteins/enzymes as potential intracellular targets, a new generation of intracellular contrast agents (CAs) can be developed. These agents have prospective use for molecular imaging (both optical and magnetic resonance imaging) by targeted labeling of cells. Aiming to image the presence of specifi c mRNAs or enzymes, two mRNA targeting (contains a PNA sequence antisense or non-sense to the target mRNA of DsRed) and one enzyme targeted (contains a unit cleavable by -galactosidase) CAs were tested for their activity in the presence and absence of respective targets. The antisense targeting CA, their nonsense derivative and the enzyme targeted CA were taken up effi ciently into cells by an exclusively endosomal mechanism as observed by fl uorescence microscopy. Cell free binding assays proved a specifi c interaction with a synthetic target for the antisense but not for non-sense CA. Magnetic Resonance studies showed a higher uptake in transgenic DsRed expressing cells than the parent cells. However, no difference was observable for antisense versus non-sense CA in DsRed cells, due to the vesicular entrapment which is preventing the specifi c interaction between CA and cytosolic target. Since a comparable cellular distribution was visible for the enzyme targeted agent, a specifi c accumulation in -galactosidase containing cells is also unlikely. The results show that even though the designed CAs were effi ciently taken up into cells, they can interact specifi cally with the target only if colocalization is achieved. However, a lack of specifi city is caused by the endosomal entrapment. Further modifi cations are required to achieve the release from endosomes or a direct uptake into the cytosol. http://www.kyb.tuebingen.mpg.de/fileadmin/user_upload/files/publications/Ritu%20Poster_EPS2008_5286[0].pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://www.eurpepsoc.com/proceedings-of-the-30th-european-peptide-symposium/ Biologische Kybernetik Max-Planck-Gesellschaft Helsinki, Finland 30th European Peptide Symposium (30 EPS) en 10.1002/psc.1090 rituRMishra wusuWSu abrudABrud MGSauer josefJPfeuffer KUgurbil joernJEngelmann poster 5373 Journal of Peptide Science 2008 9 14 S1 157 Peptide nucleic acid (PNA) is a DNA mimic consisting of the four common bases of DNA on a pseudopeptide backbone that makes it extremely stable in biological fl uids. Antisense PNA is targeted against mRNA in cytoplasm in a sequence specifi c manner. However, the main hindrance to the effective use of PNAs has been their relatively poor uptake by cells. Endosomal release or direct uptake into cytosol of agents is mandatory for attaining mRNA based targeting. There are reports on the cell penetrating peptide (CPP) based delivery system. It has also been reported that conjugates of cholesterol and siRNAs facilitate cellular import (1). The aim of this study was to synthesize different sequences of cholesterol coupled antisense PNA and to compare its uptake characteristics with a CPP-PNA conjugate. The synthesis of PNA (anti-dsRed PNA (agcgcctgtacc), specifi cally targeted to mRNA of dsRed, a red fl uorescent protein) conjugated to CPP (d-Tat) or cholesterol was performed in fully automated synthesizer (Prelude, Protein Technologies, Inc.) using continuous solid phase chemistry. To increase the solubility in water, linkers (AEEA) and additional charged amino acids were coupled or the sequence of peptide, PNA and cholesterol was changed. All compounds were labelled with FITC to confi rm the cellular uptake by fl uorescence microscopy and spectroscopy. Cell uptake studies showed that the CPP bound PNA was located predominantly in vesicles indicating an endosomal uptake mechanism and subsequent entrapment in vesicles. Cholesterol bound PNA was also effi ciently internalized. However, it was also located inside vesicles without detectable cytosolic distribution. PNA-Cholesterol has fewer synthetic steps than PNA-CPP. However, it was also located inside vesicles restricting its applicability for mRNA targeting. The effi cient uptake might make it a promising cellular delivery agent after further improvements. http://www.kyb.tuebingen.mpg.de/fileadmin/user_upload/files/publications/EPS30-2008-Joshi_[0].pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://www3.interscience.wiley.com/cgi-bin/fulltext/121431165/PDFSTART Biologische Kybernetik Max-Planck-Gesellschaft Helsinki, Finland 30th European Peptide Symposium (30 EPS) en 10.1002/psc.1090 rajuRJoshi rituRMishra wusuWSu joernJEngelmann poster 5795 2008 9 2008 0706 Molecular imaging of cells or cellular processes can be obtained by targeting e.g. specific enzymes, receptors or mRNA. Aiming to track specific enzymes present in the cytosol an intracellular MR contrast agents (CA) targeting the enzyme b-galactosidase (b-gal) was synthesized as a model system. The conjugate was composed of four domains: a gadolinium loaded ligand 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-acetate (DOTA), fluorescent dye FITC for optical imaging, an enzymatically cleavable “sensor unit” based on the enzyme targeting sugar moiety (b-D-galactopyranose), and a cell penetrating peptide (d-Tat57-49) as delivery vector. In the presented approach the MR detectable part should be selectively accumulated in the target cells containing b-gal, due to the enzymatic cleavage of internalization vector (CPP) from the enzyme targeting sugar moiety. Cellular uptake of CA was confirmed by the fluorescence microscopy and spectroscopy in C6 glioma cells as well as in the transgenic C6/LacZ7 cell line containing the targeted b-gal. Fluorescence studies have shown that the obtained CA was efficiently internalized into both cell lines in a concentration dependent manner from 5µM to 30µM without inducing significant toxicity. However, no selective accumulation of CA was observed in the b-gal expressing cells. The vesicular localization of CA around the nucleus indicates to a predominantly endosomal uptake mechanism without any detectable release into the cytosol (Fig.1). Thus, this endosomal entrapment of CA might explain, that there is no selective accumulation of CA in targeted cells, because of the inability of the contrast agent inside the vesicles to interact with the targeted enzyme located in the cytosol. Nonetheless, the synthesized CA showed an excellent ability for intracellular delivery and might prove to be useful in the cellular labeling and tracking studies. http://www.kyb.tuebingen.mpg.defileadmin/user_upload/files/publications/WMIC-2008-Brud.pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://www.abstractsonline.com/viewer/viewAbstract.asp?CKey={4FFED844-5A88-4CB8-A900-F9A994034DE9}&MKey={B47BAE74-CCA9-4C27-80FB-0005AFC9E5C0}&AKey={A4C6DD8F-4BF2-400D-97ED-20C14381CDBB}&SKey={31FBC176-CC46-4787-8098-ABA5FA33266C} Biologische Kybernetik Max-Planck-Gesellschaft Nice, France 2008 World Molecular Imaging Congress (WMIC) en abrudAJBrud rituRMishra KUgurbil TZiegler joernJEngelmann poster 5353 2008 9 2008 1006 The success of intracellular targeted delivery of imaging probes or drugs depends on the efficient transmembrane delivery of the compound as well as interaction of the payload with the desired targets. Cell penetrating peptides (CPPs) are extensively used as the delivery tools. However, confinement of biomolecules into endosomes limits their use for intracellular targeting. Herein, we focus on the development of a novel cysteine rich CPP (derived from polypeptide Crotamine [1]) by Structure Activity Relationship (SAR) studies optimizing cellular uptake efficacy as well as distribution. Series of peptides were synthesized by Fmoc strategy and were N-terminally labeled with fluorescein isothiocyanate (via an additional lysine residue) for optical imaging. The optimized peptide (DJ23) is 10 amino acids long and contains aside of cationic amino acids (common for various CPPs) cysteine and tryptophane residues. It is markedly distinct showing an efficient uptake at low concentrations (&amp;#8804;2.5&amp;#956;M) and a cytosolic distribution along with vesicular uptake unlike known for other common CPPs (e.g. Tat or Antennapedia) at these concentrations [2]. Fluorescence microscopic results highlight the importance of cysteines and tryptophans in the peptide. Additional coupling of Gd-DOTA to this peptide showed proficient uptake maintaining the cytosolic localization of the conjugate (Fig.). Furthermore, fluorescence spectroscopic data showed that the internalization efficacy at 2.5 &amp;#956;M increased significantly by about 50% in comparison to the conjugate containing the d-form of Tat peptide, known to be an efficient CPP (Fig.). Thus, this novel peptide might prove useful for efficient transmembrane delivery of agents directed to cytosolic targets. http://www.kyb.tuebingen.mpg.de/fileadmin/user_upload/files/publications/DJ-SMI-2008_[0].pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://www.wmicmeeting.org/dev/ Biologische Kybernetik Max-Planck-Gesellschaft Nice, France 2008 World Molecular Imaging Congress (WMIC) en joernJEngelmann djhaDJha rituRMishra K-HWiesmüller KUgurbil poster 5351 Journal of Peptide Science 2008 9 30 S1 157 Introduction: Crossing the plasma membrane is a prerequisite for intracellular targeted drug delivery. Cell penetrating peptides are actively used as the delivery tool for intracellular delivery of various cargos. However, confinement of biomolecules into endosomes limits their use for intracellular targeting. Therefore, there is a need for vectors capable of transferring cargo molecules directly into the cytoplasm. Herein, we focus on the development of a novel CPP (derived from polypeptide Crotamine [1]) which shows an efficient uptake at low concentrations (&amp;amp;amp;amp;#8804;2.5 &amp;amp;amp;amp;#956;M) and cytosolic distribution along with vesicular uptake. Methods: Series of peptides were synthesized by Fmoc strategy, introducing mutations in Cro (27-39) (proposed CPP sequence in Crotamine). All were N-terminally labeled with fluorescein isothiocyanate for optical imaging. Structure Activity Relationship (SAR) studies were done by substitution and/or deletion of amino acid residues in the sequence observing the uptake behaviour by fluorescence spectroscopy and microscopy. Results: Amongst 60 synthesized peptides, one of shorter length showed the best intracellular delivery and cytosolic distribution at lower concentration (2.5 &amp;amp;amp;amp;#956;M) when compared to other CPP. Replacing or deleting cysteines had negative impact on internalization. Results also displayed the involvement of tryptophans in cellular uptake indicating, along with cationic amino acids, the importance of each residue in this optimized sequence. Conclusions: SAR studies identified a novel cell penetrating peptide showing, besides of endosomal uptake, also an efficient delivery into the cytoplasm at low concentrations. Thus, this peptide might prove useful for efficient transmembrane delivery of agents directed to cytosolic targets. http://www.kyb.tuebingen.mpg.de/fileadmin/user_upload/files/publications/DJ_EPS-2008_5351[0].pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://www.30eps.fi/ Biologische Kybernetik Max-Planck-Gesellschaft Helsinki, Finland 30th European Peptide Symposium (30 EPS) en 10.1002/psc.1090 djhaDJha rituRMishra KUgurbil joernJEngelmann K-HWiesmüller poster 5352 2008 6 2 A88 79 http://www.kyb.tuebingen.mpg.de/fileadmin/user_upload/files/publications/DJ_Cardiff-2008_[0].pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://www.cdtm2008.cardiff.ac.uk/CDTM_2008_symposium.html Biologische Kybernetik Max-Planck-Gesellschaft Cardiff, UK 2nd International Symposium "Cellular Delivery of Therapeutic Macromolecules 2008" (CDTM 2008) en djhaDJha K-HWiesmüller rituRMishra KUgurbil joernJEngelmann poster 5151 2008 6 2 A73 71 http://www.kyb.tuebingen.mpg.de/fileadmin/user_upload/files/publications/CDTM2008%20Abstract_Ritu_[0].pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://www.cdtm2008.cardiff.ac.uk/CDTM_2008_symposium.html Biologische Kybernetik Max-Planck-Gesellschaft Cardiff, UK 2nd International Symposium "Cellular Delivery of Therapeutic Macromolecules 2008" (CDTM 2008) en rituRMishra wusuWSu MSauer josefJPfeuffer KUgurbil joernJEngelmann poster 5349 FEBS Journal 2008 6 275 Supplement S1 221 Introduction: Cell Penetrating Peptides (CPPs) are potential tools for the intracellular delivery of wide range of cargos. Though the exact translocation mechanism is still unknown, endocytosis is the most prevalent uptake mechanism seen for highly cationic peptides. Release from endosomes for colocalization of cargo/drug and target in the cytoplasm is the major hurdle of targeting approaches. Therefore, there is a need for vectors capable for transferring cargo molecules directly into the cytoplasm. Herein, we focus on the development of a novel CPP derived from Crotamine (polypeptide in venom of rattle snake) which shows an efficient uptake at low concentrations (&amp;amp;amp;#8804;2.5 &amp;amp;amp;#956;M) and cytosolic distribution along with vesicular uptake. Methods: Series of peptides were synthesized by Fmoc strategy, introducing mutations in Cro(27-39) (proposed CPP sequence in Crotamine). All were labeled with fluorescein isothiocyanate at the N-terminal. SAR studies were done by substitution and/or deletion of amino acid residues in the sequence observing the uptake behaviour by fluorescence spectroscopy and microscopy. Results: Amongst 61 synthesized peptides one of shorter length was showed the best intracellular delivery and cytosolic distribution. Replacing or deleting cysteines had negative impact on internalization. Results also show the involvement of tryptophans in cellular uptake indicating along with cationic amino acids the importance of each residue in this optimized sequence along with cationic amino acids. Conclusions: SAR studies identified a peptide showing, besides of endosomal uptake, also an efficient delivery into the cytoplasm. Thus, this peptide might prove useful for efficient transmembrane delivery of agents directed to cytosolic targets. http://www.kyb.tuebingen.mpg.de/fileadmin/user_upload/files/publications/DJ_FEBS-2008_[0].pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://www3.interscience.wiley.com/cgi-bin/fulltext/119878150/PDFSTART Biologische Kybernetik Max-Planck-Gesellschaft Athens, Greece 33rd FEBS Congress and 11th IUBMB Conference: "Biochemistry of Cell Regulation" en 10.1111/j.1742-4658.2008.06448.x djhaDJha K-HWiesmüller rituRMishra KUgurbil joernJEngelmann poster 5372 2008 6 2 A82 76 http://www.kyb.tuebingen.mpg.de/fileadmin/user_upload/files/publications/CDTM2008-Joshi_[0].pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://www.cdtm2008.cardiff.ac.uk/CDTM_2008_symposium.html Biologische Kybernetik Max-Planck-Gesellschaft Cardiff, UK 2nd International Symposium "Cellular Delivery of Therapeutic Macromolecules 2008" (CDTM 2008) en rajuRJoshi wusuWSu rituRMishra joernJEngelmann poster 5152 FEBS Journal 2008 6 275 Supplement S1 172 Introduction: Crossing the plasma membrane is a prerequisite for intracellular targeted drug delivery. Cell penetrating peptides (CPPs) are known to transport cargo molecules attached to it into cells primarily by endocytosis. Nevertheless, confinement of biomolecules into endosomes limits their use for intracellular targeting. We developed a novel cysteine-rich peptide that has the ability to enter directly into the cytosolic compartment of the cell. The factors affecting cytosolic distribution of peptide are reported. Methods: Uptake of fluorescently labeled peptide was assessed in NIH-3T3 mouse fibroblasts. Effect of varying the labeling concentration, time course, changing incubation temperature from 37°C to 4°C, etc. on intracellular distribution was observed by fluorescence spectroscopy and microscopy. Results: Our novel peptide exhibited mainly cytosolic localization along with some vesicular uptake in cells at a concentration as low as 2.5 &amp;amp;amp;#956;M. Diffused appearance was visible earliest after 4h. A reduction in vesicular uptake was observed on incubation at 4°C, however cytosolic uptake was almost unaffected indicating for an additional non-endosomal pathway. Due to higher stability, d-form of most of the well studied cationic CPPs (like Tat, arginines, etc.) is reported to be taken up better than the l-form. Our peptide differs as the l-form shows the most efficient cytosolic uptake while the d-form and retro-inverso form exhibited especially reduced cytosolic diffusion. Conclusions: Thus, a novel cysteine-rich peptide has been found that is directly taken up into cytosol avoiding endosomal entrapment. Therefore, it has the potential to be used as CPP for efficient cytosolic drug delivery for intracellular targeting. http://www.kyb.tuebingen.mpg.de/fileadmin/user_upload/files/publications/FEBS2008Abstract_Ritu_[0].pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://www3.interscience.wiley.com/cgi-bin/fulltext/119878150/PDFSTART Biologische Kybernetik Max-Planck-Gesellschaft Athens, Greece 33rd FEBS Congress and 11th IUBMB Conference: "Biochemistry of Cell Regulation" en 10.1111/j.1742-4658.2008.06448.x rituRMishra djhaDJha K-HWiesmüller KUgurbil joernJEngelmann poster 5298 2008 P34 http://www.kyb.tuebingen.mpg.de/fileadmin/user_upload/files/publications/COSTD38_Mishra_[0].pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://www.cost.eu/domains_actions/cmst/Actions/D38 Biologische Kybernetik Max-Planck-Gesellschaft Tübingen, Germany CMST COST Action D38: Metal-Based Systems for Molecular Imaging Applications en rituRMishra wusuWSu josefJPfeuffer KUgurbil joernJEngelmann poster EngelmannSBMPU2008 2008 P30 http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://www.cost.eu/domains_actions/cmst/Actions/D38 Tübingen, Germany CMST COST Action D38: Metal-Based Systems for Molecular Imaging Applications joernJEngelmann wusuWSu abrudABrud rituRMishra JPfeuffer KUgurbil poster 5015 2007 9 1032 http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://www.ami-imaging.org/index.php?option=com_content&view=article&id=93&Itemid=111 Biologische Kybernetik Max-Planck-Gesellschaft Providence, RI, USA AMI/SMI Joint Molecular Imaging Conference 2007 en djhaDJha wusuWSu rituRMishra joernJEngelmann KHWiesmüller MEMaier josefJPfeuffer KUgurbil poster 5014 2007 9 1018 http://www.kyb.tuebingen.mpg.de/fileadmin/user_upload/files/publications/Ritu%20Poster_SMI2007_final_5014[0].pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://www.ami-imaging.org/index.php?option=com_content&view=article&id=93&Itemid=111 Biologische Kybernetik Max-Planck-Gesellschaft Providence, RI, USA AMI/SMI Joint Molecular Imaging Conference 2007 en rituRMishra wusuWSu joernJEngelmann MSauer josefJPfeuffer KUgurbil poster 5141 2007 5 2007 1161 251 An antisense MR contrast agent and its nonsense counterpart, conjugated to a peptide nucleic acid and a cell penetrating peptide were synthesized capable of targeting cellular mRNA. Intracellular uptake in non-targeted 3T3 fibroblasts was confirmed by fluorescence and MR studies. The intracellular relaxation rate R1,cell increased significantly already at a labeling concentration of 0.5 μM, thus contrast enhancement was also detectable. An in vitro binding assay provided first evidence of a higher specificity of the antisense contrast agent. These results demonstrate its potential to be used as a targeted contrast agent for tracking mRNA transcription in cells by MRI. http://www.kyb.tuebingen.mpg.de/fileadmin/user_upload/files/publications/CA_ISMRM2007_JE_[0].pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://www.ismrm.org/07/ Biologische Kybernetik Max-Planck-Gesellschaft Berlin, Germany 2007 Joint Annual Meeting ISMRM-ESMRMB en joernJEngelmann wusuWSu rituRMishra josefJPfeuffer KHWiesmüller KUgurbil poster 5355 2007 3 8 P53 http://www.kyb.tuebingen.mpg.de/fileadmin/user_upload/files/publications/DJ_GPS-2007_[0].pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ Biologische Kybernetik Max-Planck-Gesellschaft Heidelberg, Germany 8th German Peptide Symposium en djhaDJha SWu rituRMishra joernJEngelmann K-HWiesmüller MEMaier josefJPfeuffer KUgurbil poster 5297 2007 P30 http://www.kyb.tuebingen.mpg.de/fileadmin/user_upload/files/publications/RM_DJ_COST%202007final_[0].pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://www.cost.eu/domains_actions/cmst/Actions/D38 Biologische Kybernetik Max-Planck-Gesellschaft Tübingen, Germany CMST COST Action D38: Metal-Based Systems for Molecular Imaging Applications en rituRMishra djhaDJha wusuWSu joernJEngelmann K-HWiesmüller MEMaier josefJPfeuffer KUgurbil poster 5140 2006 5 14 1887 374 For the development of intracellular Magnetic Resonance contrast agents (CAs) it is a prerequisite that they cross the plasma membrane. Cell penetrating peptides (CPPs) are known to transport cargo molecules attached to it into cells. We synthesized intracellular CAs containing known CPPs or modified peptides. Gd-diethylenetriaminepenta-acetic acid (Gd-DTPA) was coupled to CPPs for MR visualization and fluorescence imaging agent fluorescein isothiocyanate (FITC) for tracking the agent inside cells by optical imaging. Compared on the basis of these measurements, modifications in CPPs enhanced the intracellular delivery ability of our CAs providing a tool for stable labeling of cells for MR imaging. http://www.kyb.tuebingen.mpg.de/fileadmin/user_upload/files/publications/AGKUISMRM06_final_5140[0].pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://www.ismrm.org/06/ Biologische Kybernetik Max-Planck-Gesellschaft Seattle, WA, USA 14th Scientific Meeting of the International Society of Magnetic Resonance in Medicine (ISMRM 2006) en joernJEngelmann wusuWSu djhaDJha rituRMishra josefJPfeuffer KUgurbil poster 5296 2006 4 P30 Advances in Magnetic Resonance Imaging (MRI) have revolutionized the way in which cellular and physiological processes are being investigated. The specificity and sensitivity of MR imaging can be further enhanced by the introduction of contrast agents (CA). Most of the available CA like Gd-DTPA, Gd-DOTA are non-specific and restricted to the extracellular space. A new generation of intracellular CA can be developed for labeling cells specifically by coupling with special peptides known as cell penetrating peptides (CPP) that convey cargo molecules attached to it across the cell membrane. Amongst the large variety of CPP available, Tat49-57 peptide (derived from HIV-1 Tat protein) is the most intensively studied and has high delivery ability for vast variety of cargos. It has been reported that better internalization is observed with retro-inverso isomer of Tat using d-form of the peptide [1] and also by introduction of modifications in sequence like replacement of glutamine (q) with ornithine (Orn, o) [2]. We present here synthesis of bimodal (magnetic and fluorescent) MR CA based on Gd-DTPA conjugated to a fluorescent dye fluorescein isothiocyanate (FITC) and CPP (Figure 1). The CPP fragments were synthesized on solid phase by Fmoc (9-fluorenylmethoxycarbonyl) mediated scheme. One lysine residue was coupled to the CPP fragment as a linker for DTPA dianhydride (via α-amino group) and FITC (via ε-amino group). The peptide conjugates were then cleaved from solid phase. After purification by reversed-phase HPLC, the conjugates were chelated with Gd3+. The products were again purified by HPLC and characterized by ESI-MS. The obtained bimodal CA were tested on cells for their internalization efficiency and cytotoxicity using fluorescence and MR imaging. Orn-d-Tat CA exhibited much better internalization compared to d-Tat CA but was also accompanied with increased toxicity. Also a difference in internalization mechanism was observed with orn-d-Tat CA. To conclude, a balance needs to be maintained between the internalization efficiency and toxicity in order to obtain a proficient intracellular MR CA that can be used for cellular tracking. http://www.kyb.tuebingen.mpg.de/fileadmin/user_upload/files/publications/Ritu_COST18abstract_2006_[0].pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://www.cost.eu/domains_actions/cmst/Actions/D18 Biologische Kybernetik Max-Planck-Gesellschaft Orléans, France COST Chemistry Action D18: Lanthanide Chemistry for Diagnosis and Therapy en rituRMishra joernJEngelmann wusuWSu josefJPfeuffer KUgurbil poster BrudEPZU2006 2006 4 P23 http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://www.cost.eu/domains_actions/cmst/Actions/D18 Orléans, France COST Chemistry Action D18: Lanthanide Chemistry for Diagnosis and Therapy abrudABrud joernJEngelmann JPfeuffer TZiegler KUgurbil poster 5295 2006 4 P23 http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://www.cost.eu/domains_actions/cmst/Actions/D18 Biologische Kybernetik Max-Planck-Gesellschaft Orléans, France COST Chemistry Action D18: Lanthanide Chemistry for Diagnosis and Therapy en djhaDJha rituRMishra joernJEngelmann josefJPfeuffer KUgurbil poster 5354 2005 9 4 545 In recent years, many contrast agents have been developed and are being used as non-specific extracellular MR contrast agents. Another class is intracellular contrast agents whose prerequisite is to cross the plasma membrane by different mechanisms like endocytosis. For therapeutic applications, internalization is an absolute precondition, as the desired moiety has to reach its target within the cell. Cell penetrating peptides (CPPs) have been used to facilitate the delivery of macromolecules into the cells both in vitro and in vivo. The transfection ability of D-octaarginines, L-octaarginines and stearyloctaarginines has already been studied; stearyl-D-octaarginines showed improved transfection efficiency (1). This versatile approach can be used for the internalization of contrast agents into the cell for bimodal optical and MR imaging. Synthesis was performed on polystyrene based Wang resin, containing Fmoc-protected arginine.The synthesized peptide was coupled with lysine linked to FITC by its ε amino group and further conjugated with DTPA. In case of lipid modified octaarginines stearyl-lysine was additionally introduced in between polyarginine and contrast agent. Finally complexation with Gd (III) was performed at pH 5-6 in room temperature. Comparative studies on the uptake mechanism were done by both optical (fluorescent microscopy and spectroscopy) and MR imaging techniques. The optimization of the cellular uptake can prove useful by coupling to the arginines and its derivative (stearyl) for the development of new intracellular targeted contrast agents. http://www.kyb.tuebingen.mpg.de/fileadmin/user_upload/files/publications/DJ_SMI-2005_[0].pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ Biologische Kybernetik Max-Planck-Gesellschaft Köln, Germany 4th Annual Meeting of the Society for Molecular Imaging (SMI 2005) en djhaDJha rituRMishra joernJEngelmann akmishraAKMishra josefJPfeuffer KUgurbil poster BrudEPZU2005 2005 9 4 543 Reporter genes are widely applied to study gene expression and regulation in biological systems. Bacterial LacZ gene, which encodes enzyme beta-galactosidase, represents one of the most commonly used reporter genes, among others such as green fluorescent protein and luciferase. Together with fluorogenic and chromogenic substrates of the enzyme beta-galactosidase, LacZ has been utilized as a standard method of assaying clonal insertion, transcriptional activation, protein expression and protein interaction as well. The use of MR detectable substrates provides a new non-invasive tool for detection of gene expression [1]. beta-Galactosidase catalyzes the hydrolysis of the glycosidic bond between the anomeric carbon at position C1 of the beta-D-galactopyranose and an aglycone part. The replacement of hydroxyl groups at positions C2 - C6 of galactopyranose may induce the loss of enzyme activity. However, it offers a possibility to develop new marker molecules for bi-modal detection by MR/optical imaging. Here we report the syntheses of a series of compounds containing a MR detectable part (Gd(III)-DO3A) attached to beta-galactopyranose moiety by different types of linkers, and a chromophore (p-nitrophenol or 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO) [2]) at the anomeric carbon for testing enzyme activity. The tests are based on colorimetric detection of released yellow p-nitrophenol or the absorbance/fluorescence shift of released DDAO upon beta-galactosidase hydrolysis. These compounds can prove helpful for the development of new gene expression markers offering the possibility of detection by MR and optical imaging. Coupling to molecules which facilitate internalization into cells can provide a new class of intracellular contrast agents. http://www.kyb.tuebingen.mpg.defileadmin/user_upload/files/publications/4th-Soc-Mol-Imag-Brud.pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ Köln, Germany 4th Annual Meeting of the Society for Molecular Imaging (SMI 2005) abrudABrud joernJEngelmann JPfeuffer TZiegler KUgurbil poster 5142 2005 9 4 379 Noninvasive imaging techniques like MRI possess the prospective to observe molecular-genetic and cellular processes. But the exogenously administered molecular imaging agents are often unable to reach their molecular and cellular targets as the lipid bilayer of the cell poses a formidable natural barrier. However, a unique class of peptides known as cell penetrating peptides (CPPs) has the ability to traverse this barrier and convey cargo molecules attached to it across the cell membrane. Amongst a variety of natural and chimeric CPPs, HIV-1 tat protein derived Tat peptide (Tat49-57) has received much attention mainly because of its high efficiency to deliver a large variety of cargo molecules across the membrane. Considering the potential of Tat as a molecular transporter, we coupled its derivatives with fluorescence imaging agent FITC as well as with MR agent Gd-DTPA, thus obtaining bimodal cell internalizing agents. We aimed at comparing the effect of chirality on the internalization efficiency and thus synthesized l-Tat49-57 and its retro-inverso isomer d-Tat57-49. The effect of the two isomers on the vitality of cells and induction of metabolic changes was also studied. Fluorescence microscopy and spectroscopy as well as MRI were used on cell cultures for these studies. Although both the peptides showed concentration and time dependent cellular delivery, the unnatural d-Tat coupled agent exhibited better cellular internalization compared to the natural form. This could be attributed to the increased stability of the d-form of the peptide to enzymatic cleavage. Also the d-form affected the cell vitality at concentrations above 9µM and induced slight changes in metabolic activities. http://www.kyb.tuebingen.mpg.defileadmin/user_upload/files/publications/SMI-2005-Mishra.pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ Biologische Kybernetik Max-Planck-Gesellschaft Köln, Germany 4th Annual Meeting of the Society for Molecular Imaging (SMI 2005) en rituRMishra joernJEngelmann wusuWSu josefJPfeuffer KUgurbil poster 5299 2005 9 However, a unique class of peptides known as cell penetrating peptides (CPPs) has the ability to traverse this barrier and convey cargo molecules attached to it across the cell membrane [1]. CPPs are short peptides (generally less than 30 residues) with net positive charge and acting in a receptor- and energy-independent manner. Amongst a variety of natural and chimeric CPPs, HIV-1 tat protein derived Tat peptide (Tat ) has received much attention mainly because of its high efficiency to deliver a 49-57 large variety of cargo molecules across the membrane. Noninvasive imaging techniques like MRI possess the prospective to observe molecular-genetic and cellular processes. The combination of these exogenously administered molecular imaging agents with CPPs may enhance their intracellular delivery, thus solving several queries at sub-cellular level. Improved cellular uptake of the unnatural retro-inverso isomer of Tat, d-Tat57-49 (rrrqrrkkr), has been reported in comparison to l-Tat (RKKRRQRRR) [2]. 49-57 Considering the potential of Tat as a molecular transporter, we coupled l-Tat and d- 49-57 Tat with fluorescence imaging agent FITC as well as with MR agent Gd-DTPA, 57-49 thus obtaining l-Tat-Lys(FITC)-(Gd)DTPA (l-Tat CA) and d-Tat-ys(FITC)-(Gd)DTPA (d-Tat CA), respectively. Based on optical imaging and relaxation time measurements we compared cellular internalization and contrast enhancement efficiencies of these two bimodal cell internalizing contrast agents. http://www.kyb.tuebingen.mpg.de/fileadmin/user_upload/files/publications/WuRituWorkshop-2005-poster_[0].pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ Biologische Kybernetik Max-Planck-Gesellschaft Köln, Germany COST Chemistry Action D18: Lanthanide Chemistry for Diagnosis and Therapy en wusuWSu rituRMishra joernJEngelmann josefJPfeuffer KUgurbil poster MishraESPU2005 2005 9 4 544 Noninvasive imaging techniques like MRI possess the prospective to observe molecular-genetic and cellular processes. But the exogenously administered molecular imaging agents are often unable to reach their molecular and cellular targets as the lipid bilayer of the cell poses a formidable natural barrier. However, a unique class of peptides known as cell penetrating peptides (CPPs) has the ability to traverse this barrier and convey cargo molecules attached to it across the cell membrane. Amongst a variety of natural and chimeric CPPs, HIV-1 tat protein derived Tat peptide (Tat49-57) has received much attention mainly because of its high efficiency to deliver a large variety of cargo molecules across the membrane. Considering the potential of Tat as a molecular transporter, we coupled its derivatives with fluorescence imaging agent FITC as well as with MR agent Gd-DTPA, thus obtaining bimodal cell internalizing agents. We aimed at comparing the effect of chirality on the internalization efficiency and thus synthesized l-Tat49-57 and its retro-inverso isomer d-Tat57-49. The effect of the two isomers on the vitality of cells and induction of metabolic changes was also studied. Fluorescence microscopy and spectroscopy as well as MRI were used on cell cultures for these studies. Although both the peptides showed concentration and time dependent cellular delivery, the unnatural d-Tat coupled agent exhibited better cellular internalization compared to the natural form. This could be attributed to the increased stability of the d-form of the peptide to enzymatic cleavage. Also the d-form affected the cell vitality at concentrations above 9μM and induced slight changes in metabolic activities. http://www.kyb.tuebingen.mpg.defileadmin/user_upload/files/publications/4th-Soc-Mol-Imag-Mishra2.pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ Köln, Germany 4th Annual Meeting of the Society for Molecular Imaging (SMI 2005) rituRMishra joernJEngelmann wusuWSu JPfeuffer KUgurbil poster SuMEPU2005 2005 9 4 542 Recent developments in MR imaging have enabled in vivo imaging at near microscopic resolution. In order to visualize and track cells by MR imaging, it is necessary to tag cells magnetically. Cell-penetrating peptides (CPPs) have been used as an efficient way of internalizing a number of marker proteins into cells. Here we describe the synthesis and testing of a series of bi-labeled (magnetic and fluorescent) Gd(III)-based MR contrast agents conjugated to fluorescent dye and CPPs, including l-Tat49-57, d-Tat57-49, PTD-4 and NLS (Fig. 1). The CPP fragments were synthesized by solid phase with the Fmoc (9-fluorenylmethoxycarbonyl) mediated scheme. FITC (fluorescein isothiocyanate) was coupled to Fmoc-lysine at first. Then FITC-Fmoc-lysine and diethylenetriaminepenta-acetic dianhydride (DTPA dianhydride) were coupled to CPPs, respectively. Finally the conjugates were chelated with Gd3+. The products were purified by reversed-phase HPLC and characterized by ESI-MS. Cellular uptake of these agents were confirmed by fluorescent microscopy and spectroscopy, as well as by T1 and T2 MR analysis of the Gd(III) agents in NIH 3T3 fibroblasts. Further optical and MR evaluation is under progress. The comparison of these different CPP conjugates can provide helpful data for the design of new intracellular MR contrast agents for in vivo tracking of cells. http://www.kyb.tuebingen.mpg.defileadmin/user_upload/files/publications/4th-Soc-Mol-Imag-Mishra.pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ Köln, Germany 4th Annual Meeting of the Society for Molecular Imaging (SMI 2005) wusuWSu rituRMishra joernJEngelmann JPfeuffer KUgurbil conference Engelmann2011 2011 11 http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Logothetis http://www.e-smi.eu/index.php?id=2024&tx_ttnews[tt_news]=1663&tx_ttnews[backPid]=1996&cHash=48bb25e2e12657c057314b0bc387b2b3 Tübingen, Germany Symposium on MRI Probes for Molecular Imaging: From Design to Application joernJEngelmann conference GottschalkMEP2011 2011 6 mediator of excitatory signals in the nervous system and is involved in nearly all aspects of normal brain functioning (cognition, memory, learning). Our idea was to develop glutamate ‘responsive’ magnetic resonance imaging (MRI) contrast agents (CAs) to image changes in specific brain regions upon neural activation. As CAs directly responsive to glutamate would not be feasible due to the very short half-life of glutamate in the extracellular space, we chose CAs that bind to glutamate receptors instead (to be specific metabotropic glutamate-receptor subtype 5 (mGluR5)), by this increasing image contrast. Ideally, upon glutamate-binding to the receptor (e.g. after glutamaterelease at the synapse) the CA will be released, hence leading to a reduction in image-contrast, followed by a restoration of equilibrium and re-binding of the CA to the receptor. These events are believed to occur over a period of seconds allowing data acquisition using modern FLASH pulse techniques[1]. Here, we present a proof-of-concept study for such ‘indirect’ glutamate-responsive MRI CAs. Methods: We have designed and synthesized different prospective CAs derived from various potent mGluR5-receptor antagonists (alkynes like MPEP, MTEP and dipyridyl/heterobiaryl amides) coupled to DOTA-derived macrocyclic lanthanidechelates. The CAs were evaluated in cultured primary cortical rat astrocytes, expressing mGluR5 (verified by immunofluorescence). MRI-measurements to examine the ability of the CAs for cellular labeling were done with a 3T human whole body scanner. Antagonistic potency of the CAs was assessed with a calcium fluorescence assay, by which glutamate induced intracellular calcium-transients mediated by mGluR5 were measured. Antagonistic activity of the CAs was calculated as changes in EC50 of glutamate. Receptor binding was measured for the dipyridyl derivaties, as these compounds have an inherent fluorescence that changes upon binding. Commercially available receptor membrane preparations containing human mGluR5A were used for these experiments. Results: Two of the gadolinium complexes retained significant antagonistic activity, one in each structural class. For the alkyne-derivative, about a threefold increase of the EC50(glutamate) (100μM CA, 15min, P<0.001) was found while under similar conditions the cellular relaxation rate R1,cell increased to 126% of control (100μM, 45 minutes incubation time, P<0.001). The CA derived from dipyridyl amides increased the EC50(glutamate) about fourfold (p<0.001) and the R1,cell to 115% (p<0.05). Fluorescence measurements of the latter CA showed enhanced emission upon binding to mGluR5-membrane preparations. This was reversed when increasing concentrations of glutamate were added, consistent with the a reversibility of CA-receptor binding Conclusions: Using primary rat astrocytes as cellular model system to investigate newly developed glutamate-responsive MRI contrast agents, we were able to identify two promising candidates. These CAs are based on the structures of antagonists to mGluR5 and our studies establish the validity of the concept, by which it might be possible to use MRI to image transient changes in the neurotransmitter glutamate. http://www.kyb.tuebingen.mpg.defileadmin/user_upload/files/publications/2011/EMIM-2011-Gottschalk.pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Scheffler Department Logothetis Abstract Talk http://www.e-smi.eu/index.php?id=2415 Max Planck Institute for Biological Cybernetics Leiden, Netherlands 6th European Molecular Imaging Meeting (EMIM 2011) sgottSGottschalk anuragrkAMishra joernJEngelmann DParker conference BallaGGPE2011 2011 5 19 665 Detection of single pancreatic islets has many applications in diabetes research, but can be achieved in vivo with proton MRI only after transplantation of ex vivo labeled islets. Recently, successful visualization of islets in the excised mouse pancreas at 16.4T following i.v. injection of a novel beta-cell specific paramagnetic contrast agent was reported. Here we present the methodical optimization for in vivo experiments. Signal efficient 2D acquisition methods were considered. Best results were obtained with a two slice navigator based sequence. Single islets are visualized for the first time in vivo in mice after i.v. administration of a labeling agent. http://www.kyb.tuebingen.mpg.defileadmin/user_upload/files/publications/ISMRM-2011-665.pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Scheffler Abstract Talk http://www.ismrm.org/11/ Montréal, Canada 19th Annual Meeting and Exhibition of the International Society for Magnetic Resonance in Medicine (ISMRM 2011) balladDBalla sgottSGottschalk shajangGShajan SUeberberg SSchneider rolfRPohmann joernJEngelmann conference GottschalkJMUE2010 2010 7 http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Scheffler Abstract Talk http://www.controlledreleasesociety.org/meetings/Archives/2010AnnualMeeting/Pages/default.aspx Max Planck Institute for Biological Cybernetics Portland, OR, USA 37th Annual Meeting and Exposition of the Controlled Release Society (CRS 2010) sgottSGottschalk djhaDJha rituRMishra KUgurbil joernJEngelmann conference GottschalkBPE2010 2010 5 http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department Scheffler Abstract Talk http://www.ismrm.org/10/ Max Planck Institute for Biological Cybernetics Stockholm, Sweden ISMRM-ESMRMB Joint Annual Meeting 2010 sgottSGottschalk balladDZBalla rolfRPohmann joernJEngelmann conference 5150 2008 7 http://www.kyb.tuebingen.mpg.de/fileadmin/user_upload/files/publications/JCR2008Abstract_Ritu_[0].pdf http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ Abstract Talk http://www.controlledrelease.org/meeting/default.cfm Biologische Kybernetik Max-Planck-Gesellschaft New York, NY, USA 35th Annual Meeting and Exposition of the Controlled Release Society 2008 en rituRMishra wusuWSu josefJPfeuffer KUgurbil joernJEngelmann conference 5292 2008 7 http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ Abstract Talk http://www.controlledreleasesociety.org/Pages/default.aspx Biologische Kybernetik Max-Planck-Gesellschaft New York, NY, USA 35th Annual Meeting and Exposition of the Controlled Release Society 2008 en abrudABrud rituRMishra KUgurbil wusuWSu TZiegler joernJEngelmann patent 5281 2008 2 EP2090584 A1 The present invention relates to a nucleic acid molecule encoding a peptide capable of being internalized into a cell, wherein said nucleic acid molecule comprises (a) a nucleic acid molecule encoding a peptide having the amino acid sequence of SEQ ID NO: 2; (b) a nucleic acid molecule having the DNA sequence of SEQ ID NO: 1, wherein T is U if the nucleic acid molecule is RNA; (c) a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of a nucleic acid molecule of (a) or (b), wherein the peptide encoded by said nucleic acid molecule has a cysteine at least at two positions selected from the group consisting of positions 1, 7 and 8 of SEQ ID NO: 2 and an arginine or a lysine at least at four positions selected from the groups consisting of position 2, 4, 6, 9 or 10 of SEQ ID NO: 2; (d) a nucleic acid molecule encoding a peptide having at least 70% sequence identity with that of SEQ ID NO: 2, wherein at least at two positions selected from the group consisting of positions 1, 7 and 8 of SEQ ID NO: 2 a cysteine is present and wherein at least at four positions selected from the groups consisting of position 2, 4, 6, 9 or 10 of SEQ ID NO: 2 an arginine or a lysine is present; or (e) a nucleic acid molecule degenerate with respect to the nucleic acid molecule of (c) or (d). The present invention also relates to a peptide encoded by the nucleic acid of the invention, a fusion molecule comprising the peptide of the invention and a composition comprising the peptide or the fusion molecule of the invention. Furthermore, the present invention relates to a method of detecting the internalization behaviour of a fusion molecule of the invention, the composition of the invention for treating and/or preventing a condition selected from cancer, enzyme deficiency diseases, infarcts, cerebral ischemia, diabetes, inflammatory diseases, infections such as bacterial, viral or fungal infections, autoimmune diseases such as systemic lupus erythematodes (SLE) or rheumatoid arthritis, diseases with amyloid-like fibrils such as Alzheimer's disease (AD) and Parkinson's disease (PD) or certain forms of myopathy. http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de http://www.kyb.tuebingen.mpg.de Department MRZ http://www.google.com/patents/EP2090584A1?cl=en Biologische Kybernetik Max-Planck-Gesellschaft Max-Planck Institute for Biological Cybernetics, Tübingen, Germany en djhaDJha KUgurbil joernJEngelmann rituRMishra K-HWiesmüller