The financial support of the Max-Planck Society, the Turkish Ministry of National Education (PhD fellowship to S. G.), and the German Exchange Academic Service (DAAD, PhD fellowship to T. S.) are gratefully acknowledged.

<ol>
	<li>Merbach, A. E., Helm, L., T&#243;th, &. #. 2. 0. 1. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The chemistry of contrast agents in medical magnetic resonance imaging. 2nd ed. , (2013).</li><li>Geraldes, C. F. G. C., Laurent, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classification+and+basic+properties+of+contrast+agents+for+magnetic+resonance+imaging.">Classification and basic properties of contrast agents for magnetic resonance imaging.</a> Contrast Media Mol. Imaging. 4 (1), 1-23 (2009).</li><li>Caravan, P., Ellison, J. J., McMurry, T. J., Lauffer, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gadolinium(III)+chelates+as+MRI+contrast+agents:+Structure,+dynamics,+and+applications.">Gadolinium(III) chelates as MRI contrast agents: Structure, dynamics, and applications.</a> Chem. Rev. 99 (9), 2293-2352 (1999).</li><li>Villaraza, A. J. L., Bumb, A., Brechbiel, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecules,+Dendrimers,+and+Nanomaterials+in+Magnetic+Resonance+Imaging:+The+Interplay+between+Size,+Function,+and+Pharmacokinetics.">Macromolecules, Dendrimers, and Nanomaterials in Magnetic Resonance Imaging: The Interplay between Size, Function, and Pharmacokinetics.</a> Chem. Rev. 110 (5), 2921-2959 (2010).</li><li>Langereis, S., Dirksen, A., Hackeng, T. M., van Genderen, M. H. P., Meijer, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendrimers+and+magnetic+resonance+imaging.">Dendrimers and magnetic resonance imaging.</a> New J. Chem. 31 (7), 1152-1160 (2007).</li><li>G&#252;nd&#252;z, S., Power, A., Maier, M. E., Logothetis, N. K., Angelovski, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+Characterization+of+a+Biotinylated+Multivalent+Targeted+Contrast+Agent.">Synthesis and Characterization of a Biotinylated Multivalent Targeted Contrast Agent.</a> ChemPlusChem. 80 (3), 612-622 (2015).</li><li>Pope, S. J. A., Kenwright, A. M., Heath, S. L., Faulkner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+luminescence+properties+of+a+kinetically+stable+dinuclear+ytterbium+complex+with+differentiated+binding+sites.">Synthesis and luminescence properties of a kinetically stable dinuclear ytterbium complex with differentiated binding sites.</a> Chem. Commun. (13), 1550-1551 (2003).</li><li>Vibhute, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+characterization+of+pH-sensitive,+biotinylated+MRI+contrast+agents+and+their+conjugates+with+avidin.">Synthesis and characterization of pH-sensitive, biotinylated MRI contrast agents and their conjugates with avidin.</a> Org. Biomol. Chem. 11 (8), 1294-1305 (2013).</li><li>Vogel, A. I., Furniss, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Vogel's textbook of practical organic chemistry. 5th ed. , (1989).</li><li>Lundanes, E., Reubsaet, L., Greibrokk, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chromatography : basic principles, sample preparations and related methods. , (2013).</li><li>Barge, A., Cravotto, G., Gianolio, E., Fedeli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+determine+free+Gd+and+free+ligand+in+solution+of+Gd+chelates.+A+technical+note.">How to determine free Gd and free ligand in solution of Gd chelates. A technical note.</a> Contrast Media Mol. Imaging. 1 (5), 184-188 (2006).</li><li>Keeler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Understanding NMR spectroscopy. 2nd ed. , (2010).</li><li>Hillenkamp, F., Peter-Katalini&#263;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> MALDI MS : a practical guide to instrumentation, methods and applications. , (2007).</li><li>Peters, J. A., Huskens, J., Raber, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lanthanide+induced+shifts+and+relaxation+rate+enhancements.">Lanthanide induced shifts and relaxation rate enhancements.</a> Prog. Nucl. Magn. Reson. Spectrosc. 28, 283-350 (1996).</li><li>Averill, D. J., Garcia, J., Siriwardena-Mahanama, B. N., Vithanarachchi, S. M., Allen, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation,+Purification,+and+Characterization+of+Lanthanide+Complexes+for+Use+as+Contrast+Agents+for+Magnetic+Resonance+Imaging.">Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging.</a> J. Vis. Exp. (53), e2844 (2011).</li><li>Hagberg, G. E., Scheffler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+r1+and+r2+relaxivity+of+gadolinium-based+contrast+agents+on+the+T1-weighted+MR+signal+at+increasing+magnetic+field+strengths.">Effect of r1 and r2 relaxivity of gadolinium-based contrast agents on the T1-weighted MR signal at increasing magnetic field strengths.</a> Contrast Media Mol. Imaging. 8 (6), 456-465 (2013).</li><li>Boswell, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis,+characterization,+and+biological+evaluation+of+integrin+alpha(v)beta(3)-targeted+PAMAM+dendrimers.">Synthesis, characterization, and biological evaluation of integrin alpha(v)beta(3)-targeted PAMAM dendrimers.</a> Mol. Pharmaceut. 5 (4), 527-539 (2008).</li><li>Sherry, A. D., Caravan, P., Lenkinski, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer+on+Gadolinium+Chemistry.">Primer on Gadolinium Chemistry.</a> J. Magn. Reson. Imaging. 30 (6), 1240-1248 (2009).</li><li>Caki&#263;, N., G&#252;nd&#252;z, S., Rengarasu, R., Angelovski, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+strategies+for+preparation+of+cyclen-based+MRI+contrast+agents.">Synthetic strategies for preparation of cyclen-based MRI contrast agents.</a> Tetrahedron Lett. 56 (6), 759-765 (2015).</li><li>Polasek, M., Hermann, P., Peters, J. A., Geraldes, C. F. G. C., Lukes, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAMAM+Dendrimers+Conjugated+with+an+Uncharged+Gadolinium(III)+Chelate+with+a+Fast+Water+Exchange:+The+Influence+of+Chelate+Charge+on+Rotational+Dynamics.">PAMAM Dendrimers Conjugated with an Uncharged Gadolinium(III) Chelate with a Fast Water Exchange: The Influence of Chelate Charge on Rotational Dynamics.</a> Bioconjugate Chem. 20 (11), 2142-2153 (2009).</li><li>Ali, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+relaxometric+studies+of+a+dendrimer-based+pH-responsive+MRI+contrast+agent.">Synthesis and relaxometric studies of a dendrimer-based pH-responsive MRI contrast agent.</a> Chem. Eur. J. 14 (24), 7250-7258 (2008).</li><li>Jackson, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+dendrimer+molecules+by+transmission+electron+microscopy+(TEM):+Staining+methods+and+Cryo-TEM+of+vitrified+solutions.">Visualization of dendrimer molecules by transmission electron microscopy (TEM): Staining methods and Cryo-TEM of vitrified solutions.</a> Macromolecules. 31 (18), 6259-6265 (1998).</li><li>Jain, K., Kesharwani, P., Gupta, U., Jain, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendrimer+toxicity:+Let's+meet+the+challenge.">Dendrimer toxicity: Let's meet the challenge.</a> Int. J. Pharm. 394 (1-2), 122-142 (2010).</li><li>Rudovsky, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAMAM+dendrimeric+conjugates+with+a+Gd-DOTA+phosphinate+derivative+and+their+adducts+with+polyaminoacids:+The+interplay+of+global+motion,+internal+rotation,+and+fast+water+exchange.">PAMAM dendrimeric conjugates with a Gd-DOTA phosphinate derivative and their adducts with polyaminoacids: The interplay of global motion, internal rotation, and fast water exchange.</a> Bioconjugate Chem. 17 (4), 975-987 (2006).</li><li>Xu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+improved+syntheses+of+dendrimer-based+magnetic+resonance+imaging+contrast+agents:+New+bifunctional+diethylenetriaminepentaacetic+acid+ligands+and+nonaqueous+conjugation+chemistry.">Toward improved syntheses of dendrimer-based magnetic resonance imaging contrast agents: New bifunctional diethylenetriaminepentaacetic acid ligands and nonaqueous conjugation chemistry.</a> J. Med. Chem. 50 (14), 3185-3193 (2007).</li><li>Nwe, K., Bryant, L. H., Brechbiel, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(amidoamine)+Dendrimer+Based+MRI+Contrast+Agents+Exhibiting+Enhanced+Relaxivities+Derived+via+Metal+Preligation+Techniques.">Poly(amidoamine) Dendrimer Based MRI Contrast Agents Exhibiting Enhanced Relaxivities Derived via Metal Preligation Techniques.</a> Bioconjugate Chem. 21 (6), 1014-1017 (2010).</li><li>Livramento, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+in+vivo+MRI+assessment+of+a+self-assembled+metallostar+compound+endowed+with+a+remarkable+high+field+relaxivity.">First in vivo MRI assessment of a self-assembled metallostar compound endowed with a remarkable high field relaxivity.</a> Contrast Media Mol. Imaging. 1 (1), 30-39 (2006).</li><li>Norek, M., Kampert, E., Zeitler, U., Peters, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+of+the+Size+of+Dy2O3+Nanoparticles+for+Optimal+Performance+as+an+MRI+Contrast+Agent.">Tuning of the Size of Dy2O3 Nanoparticles for Optimal Performance as an MRI Contrast Agent.</a> J. Am. Chem. Soc. 130 (15), 5335-5340 (2008).</li></ol>

The authors have nothing to disclose.

Preparation of the dendrimeric MRI contrast agent requires appropriate selection of the monomeric unit (i.e., the chelator for Gd(III)). They reduce the toxicity of this paramagnetic ion and, to date, a wide variety of acyclic and macrocyclic chelators serve this purpose1-3. Among these, macrocyclic DOTA-type chelators possess the highest thermodynamic stability and kinetic inertness and, hence, are the most preferred choice for the preparation of inert MRI contrast agents1,18. Furthermore,...

Magnetic resonance imaging (MRI) is a powerful and non-ionizing imaging technique widely used in biomedical research and clinical diagnostics due to its noninvasive nature and excellent intrinsic soft-tissue contrast. The most commonly used MRI methods utilize the signal obtained from water protons, providing high-resolution images and detailed information within the tissues based on differences in the density of the water signals. The signal intensity and the specificity of the MRI experiments can be further improved using contrast agents (CAs). These are paramagnetic or superparamagnetic species that affect the longitudinal (T1) and transverse (T2) relaxation times, respectively1,2.
Complexes of the lanthanide ion gadolinium with polyamino polycarboxylic acid ligands are the most commonly used T1 CAs. Gadolinium(III) shortens the T1 relaxation time of water protons, thus increasing the signal contrast in MRI experiments3. However, ionic gadolinium is toxic; its size approximates that of calcium(II), and it seriously affects calcium-assisted signaling in cells. Therefore, acyclic and macrocyclic chelates are employed to neutralize this toxicity. Various multidentate ligands have been developed so far, resulting in gadolinium(III) complexes with high thermodynamic stability and kinetic inertness1. Those based on the 12-membered azamacrocycle cyclen, in particular its tetracarboxylic derivative DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate) are the most investigated and applied complexes of this CA class.
Nevertheless, GdDOTA-type CAs are low molecular weight systems, displaying certain disadvantages such as low contrast efficiency and fast renal excretion. Macromolecular and multivalent CAs may be a good solution to these problems4. Since CA biodistribution is mainly determined by their size, macromolecular CAs display much longer retention times within tissues. Equally important, the multivalency of these agents results in an increased local concentration of the monomeric MR probe (e.g., GdDOTA complex), substantially improving the acquired MR signal and the measurement quality.
Dendrimers are amongst the most preferred scaffolds for the preparation of multivalent CAs for MRI4,5. These highly branched macromolecules with well-defined sizes are prone to various coupling reactions on their surface. In this work, we report the preparation, purification, and characterization of a dendrimeric CA for MRI consisting of a generation 4 (G4) poly(amidoamine) (PAMAM) dendrimer coupled to GdDOTA-like chelates (DCA). We describe the synthesis of the reactive DOTA derivative and its coupling to the PAMAM dendrimer. Upon complexation with Gd(III), the standard physicochemical characterization procedure of DCA was performed. Finally, MRI experiments were performed to demonstrate the ability of DCA to produce MR images with a stronger contrast than those obtained from low molecular weight CAs.

<table><tbody><tr><td>Cyclen</td><td>CheMatech</td><td>C002</td><td></td></tr><tr><td>&lt;em&gt;tert&lt;/em&gt;-Butyl bromoacetate&amp;nbsp;</td><td>Alfa Aesar</td><td>A14917</td><td></td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-Dimethylformamide</td><td>Fluka</td><td>40248</td><td></td></tr><tr><td>Potassium carbonate</td><td>Sigma-Aldrich</td><td>209619</td><td></td></tr><tr><td>4-(4-Nitrophenyl)butryic acid</td><td>Aldrich</td><td>335339</td><td></td></tr><tr><td>Thionyl chloride&amp;nbsp;</td><td>Acros Organics</td><td>382662500</td><td>Note: Corrosive substance; toxic if inhaled</td></tr><tr><td>Bromine</td><td>Acros Organics</td><td>402841000</td><td>Note: causes severe skin burns, fatal if inhaled&amp;nbsp;</td></tr><tr><td>Diethyl ether</td><td>any source</td><td></td><td></td></tr><tr><td>Sodium sulphate</td><td>Acros Organics</td><td>196640010</td><td></td></tr><tr><td>Chloroform&amp;nbsp;</td><td>VWR Chemicals</td><td>22711.29</td><td></td></tr><tr><td>&lt;em&gt;tert&lt;/em&gt;-Butyl 2,2,2-trichloroacetimidate</td><td>Aldrich</td><td>364789</td><td>Note: flammable substance; irritrant to skin and eyes</td></tr><tr><td>Boron trifluoride etherate</td><td>Acros Organics</td><td>174560250</td><td>48% BF&lt;sub&gt;3&lt;/sub&gt;. Note: Flammable substance; causes skin burns, fatal if inhaled</td></tr><tr><td>Sodium bicarbonate</td><td>Acros Organics</td><td>424270010</td><td></td></tr><tr><td>Ethyl-acetate</td><td>any source</td><td></td><td>For column chromatography</td></tr><tr><td>&lt;em&gt;n&lt;/em&gt;-Hexane</td><td>any source</td><td></td><td>For column chromatography</td></tr><tr><td>Bulb-to-bulb (Kugelrohr) distillation apparatus</td><td>B&amp;uuml;chi</td><td></td><td>Model type: Glass oven B-585</td></tr><tr><td>Silicagel</td><td>Carl Roth GmbH</td><td>P090.2</td><td></td></tr><tr><td>Methanol</td><td>any source</td><td></td><td>For column chromatography</td></tr><tr><td>Dichloromethane&amp;nbsp;</td><td>any source</td><td></td><td>For column chromatography</td></tr><tr><td>Ethanol</td><td>VWR Chemicals</td><td>20821.296</td><td></td></tr><tr><td>Ammonia</td><td>Acros Organics</td><td>428381000</td><td>7 N Solution in Methanol</td></tr><tr><td>Palladium</td><td>Aldrich</td><td>643181</td><td>15% wet</td></tr><tr><td>Hydrogenation apparatus PARR</td><td>PARR Instrument Company</td><td></td><td></td></tr><tr><td>Celite 503</td><td>Aldrich</td><td>22151</td><td></td></tr><tr><td>Sintered glass funnel</td><td>any source</td><td></td><td></td></tr><tr><td>Thiophosgen</td><td>Aldrich</td><td>115150</td><td>Note: irritrant to skin; toxic if inhaled</td></tr><tr><td>Triethylamine</td><td>Alfa Aesar</td><td>A12646</td><td></td></tr><tr><td>Dichloromethane&amp;nbsp;</td><td>Acros Organics</td><td>348460010</td><td>Extra dry&amp;nbsp;</td></tr><tr><td>Magnetic stirrer</td><td>any source</td><td></td><td></td></tr><tr><td>PAMAM G4 Dendrimer</td><td>Andrews ChemService</td><td>AuCS - 297</td><td>10% wt. solution in MeOH</td></tr><tr><td>Lipophylic Sephadex LH-20</td><td>Sigma</td><td>LH20100</td><td></td></tr><tr><td>Thin-layer chromatography plates</td><td>Merck Millipore</td><td>1.05554.0001</td><td></td></tr><tr><td>Formic acid</td><td>VWR Chemicals</td><td>20318.297</td><td></td></tr><tr><td>Lophylizer&amp;nbsp;</td><td>any source</td><td></td><td></td></tr><tr><td>Gadollinium(III) chloride hexahydrate</td><td>Aldrich</td><td>G7532</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Acros Organics</td><td>134070010</td><td></td></tr><tr><td>pH meter</td><td>any source</td><td></td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid disodium salt dihydrate</td><td>Aldrich</td><td>E5134</td><td></td></tr><tr><td>Mass spectrometer (ESI)</td><td>Agilent</td><td></td><td>Ion trap SL 1100&amp;nbsp;</td></tr><tr><td>Acetate buffer</td><td>any source</td><td></td><td>pH 5.8</td></tr><tr><td>Xylenol orange</td><td>Aldrich</td><td>52097</td><td>20 &amp;mu;M in acetate buffer</td></tr><tr><td>Hydrophylic Sephadex G-15</td><td>GE Healthcare</td><td>17-0020-01</td><td></td></tr><tr><td>Amicon Ultra-15 Centrifugal Filter Unit</td><td>Merck Millipore</td><td>UFC900324</td><td>Ultracel-3 membrane (MWCO 3000)</td></tr><tr><td>Centrifuge</td><td>any source</td><td></td><td></td></tr><tr><td>NMR spectrometer&amp;nbsp;</td><td>Bruker</td><td></td><td>Avance III 300 MHz</td></tr><tr><td>Topspin</td><td>Bruker</td><td></td><td>Version 2.1</td></tr><tr><td>Combustion analysis instrument</td><td>EuroVector SpA</td><td></td><td>EuroEA 3000 Elemental Analyser&amp;nbsp;</td></tr><tr><td>MALDI-ToF MS instrument</td><td>Applied Biosystems</td><td></td><td>Voyager-STR</td></tr><tr><td>Deuteriumoxid</td><td>Carl Roth GmbH</td><td>6672.3</td><td></td></tr><tr><td>&lt;em&gt;tert&lt;/em&gt;-Butyl alcohol</td><td>Carl Roth GmbH</td><td>AE16.1</td><td></td></tr><tr><td>Vortex mixer</td><td>any source</td><td></td><td></td></tr><tr><td>Norell NMR tubes</td><td>Deutero GmbH</td><td>507-HP-7</td><td></td></tr><tr><td>NMR coaxial tube</td><td>Deutero GmbH</td><td>coaxialb-5-7</td><td></td></tr><tr><td>DLS instrument</td><td>Malvern</td><td></td><td>Zetasizer Nano ZS</td></tr><tr><td>0.20 &amp;mu;m PTFE filter&amp;nbsp;</td><td>Carl Roth GmbH</td><td>KC94.1</td><td></td></tr><tr><td>HEPES</td><td>Fisher BioReagents</td><td>BP310</td><td></td></tr><tr><td>Plastic tube vials</td><td>any source</td><td></td><td></td></tr><tr><td>Dotarem</td><td>Guerbet</td><td></td><td>NDC 67684-2000-1</td></tr><tr><td>MRI scanner</td><td>Bruker</td><td></td><td>BioSpec 70/30 USR magnet (7 T). Note: potential hazards related to high magnetic fields</td></tr><tr><td>RF coil</td><td>Bruker</td><td></td><td>Dual frequency volume coil (RF RES 300 1H/19F 075/040 LIN/LIN TR)</td></tr><tr><td>Paravision (software)</td><td>Bruker</td><td></td><td>Version 5.1</td></tr></tbody></table>

preparation and in vitro characterization of dendrimer-based contrast agents for magnetic resonance imaging

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance imaging (MRI). Their purpose is to enhance the relaxation rate of water protons in tissue, thus increasing the MR image contrast and the specificity of the MRI measurements. Current clinically approved contrast agents are low molecular weight molecules that are rapidly cleared from the body. The use of dendrimers as carriers of paramagnetic chelators can play an important role in the future development of more efficient MRI contrast agents. Specifically, the increase in local concentration of the paramagnetic species results in a higher signal contrast. Furthermore, this CA provides a longer tissue retention time due to its high molecular weight and size. Here, we demonstrate a convenient procedure for the preparation of macromolecular MRI contrast agents based on poly(amidoamine) (PAMAM) dendrimers with monomacrocyclic DOTA-type chelators (DOTA &#8211; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate). The chelating unit was appended by exploiting the reactivity of the isothiocyanate (NCS) group towards the amine surface groups of the PAMAM dendrimer to form thiourea bridges. Dendrimeric products were purified and analyzed by means of nuclear magnetic resonance spectroscopy, mass spectrometry, and elemental analysis. Finally, high resolution MR images were recorded and the signal contrasts obtained from the prepared dendrimeric and the commercially available monomeric agents were compared.

The preparation of DCA consisted of two stages: 1) synthesis of the monomeric DOTA-type chelator (Figure 1) and 2) coupling of the chelator with the G4 PAMAM dendrimer and subsequent preparation of the dendrimeric Gd(III) complex (Figure 2). In the first stage, a cyclen-based DOTA-type chelator containing four carboxylic acids and an orthogonal group suitable for further synthetic modifications was prepared. The preparation commenced from...

Watch this Scientific Journal Video about Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging at JoVE.com

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

This protocol describes the preparation and characterization of a dendrimeric magnetic resonance imaging (MRI) contrast agent that carries cyclen-based macrocyclic chelates coordinating paramagnetic gadolinium ions. In a series of MRI experiments in vitro, this agent produced an amplified MRI signal when compared to the commercially available monomeric analogue.

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance ...

preparation-vitro-characterization-dendrimer-based-contrast-agents

Research

JoVE Journal

Chemistry

9.8K Views.  Max Planck Institute for Biological Cybernetics. This protocol describes the preparation and characterization of a dendrimeric magnetic resonance imaging (MRI) contrast agent that carries cyclen-based macrocyclic chelates coordinating paramagnetic gadolinium ions. In a series of MRI experiments in vitro, this agent produced an amplified MRI signal when compared to the commercially available monomeric analogue.

Video: Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring 

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

Bioengineering

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

The development of fluorescent indicators represented a revolution for life sciences. Genetically encoded and synthetic fluorophores with sensing abilities allowed the visualization of biologically relevant species with high spatial and temporal resolution. Synthetic dyes are of particular interest thanks to their high tunability and the wide range of measureable analytes. However, these molecules suffer several limitations related to small molecule behavior (poor solubility, difficulties in targeting, often no ratiometric imaging allowed). In this work we introduce the development of dendrimer-based sensors and present a procedure for pH measurement in vitro, in living cells and in vivo. We choose dendrimers as ideal platform for our sensors for their many desirable properties (monodispersity, tunable properties, multivalency) that made them a widely used scaffold for several biomedical devices. The conjugation of fluorescent pH indicators to the dendrimer scaffold led to an enhancement of their sensing performances. In particular dendrimers exhibit reduced cell leakage, improved intracellular targeting and allow ratiometric measurements. These novel sensors were successfully employed to measure pH in living HeLa cells and in vivo in mouse brain.

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

Fluorescence sensors are powerful tools in life science. Here we describe a methodology to synthesize and use dendrimer-based fluorescent sensors to measure pH in living cells and in vivo. The dendritic scaffold enhances the properties of conjugated fluorescent dyes leading to improved sensing properties.

The development of  fluorescent indicators represented a revolution for life sciences. Genetically  encoded and synthetic fluorophores with sensing ...

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Biology

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Immunology and Infection

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.

To demonstrate MR cancer molecular imaging with a small peptide targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma in a mouse prostate cancer model.

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

Medicine

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Iron Oxide Nanoparticles for Image-Guided Therapy Delivery in Murine Glioblastoma

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a significant hurdle in the delivery of therapies to GBM. Reported here is an image-guided, iron oxide-based therapeutic delivery nano platform capable of bypassing this physiological barrier by virtue of size and accumulating in the tumor region, delivering its payload. This 25 nm nano platform consists of crosslinked dextran-coated iron oxide nanoparticles labeled with Cy5.5 fluorescent dye and containing antisense oligonucleotide as a payload. The magnetic iron oxide core enables tracking of the nanoparticles through in vivo magnetic resonance imaging, while Cy5.5 dye allows tracking by optical imaging. This report details the monitoring of the accumulation of this nanoparticle platform (termed MN-anti-miR10b) in orthotopically implanted glioblastoma tumors following intravenous injection. In addition, it provides insight into the in vivo delivery of RNA oligonucleotides, a problem that has hampered the translation of RNA therapeutics into the clinic.

Author Spotlight: Innovative Cancer Therapies with Iron Oxide Nanoparticles for Glioblastoma Treatment

The blood-brain barrier is a significant hurdle in the delivery of therapies for glioblastoma, a disease for which there is no cure. Here, we report an in vivo image-guided iron oxide therapeutic nano platform that can bypass this physiological barrier by virtue of size and accumulate in the tumor.

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a ...

Cancer Research

Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for magnetic resonance imaging (MRI). Many commercially available ligands are especially useful because they contain 
functional groups that allow for fast, high-purity, and high-yielding conjugation to macromolecules and biomolecules via amine-reactive 
activated esters and isothiocyanate groups or thiol-reactive maleimides. While metalation of these ligands is considered common 
knowledge in the field of bioconjugation chemistry, subtle differences in metalation procedures must be taken into account when 
selecting metal starting materials. Furthermore, multiple options for purification and characterization exist, and selection of the most 
effective procedure partially depends on the selection of starting materials. These subtle differences are often neglected in published 
protocols. Here, our goal is to demonstrate common methods for metalation, purification, and characterization of lanthanide complexes 
that can be used as contrast agents for MRI (Figure 1). We expect that this publication will enable biomedical scientists to incorporate 
lanthanide complexation reactions into their repertoire of commonly used reactions by easing the selection of starting materials and 
purification methods.

We demonstrate the metalation, purification, and characterization of lanthanide complexes. The complexes described here can be conjugated to macromolecules to enable tracking of these molecules using magnetic resonance imaging.

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

Summary

Explore More Videos

Chapters in this video

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Monitoring Dendritic Cell Migration using ¹⁹F / ¹H Magnetic Resonance Imaging

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

In vivo ¹⁹F MRI for Cell Tracking

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells