Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging
Summary
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.
Abstract
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 – 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.
Introduction
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.
Protocol
Representative Results
Discussion
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,…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Cyclen | CheMatech | C002 | |
| tert-Butyl bromoacetate | Alfa Aesar | A14917 | |
| N,N-Dimethylformamide | Fluka | 40248 | |
| Potassium carbonate | Sigma-Aldrich | 209619 | |
| 4-(4-Nitrophenyl)butryic acid | Aldrich | 335339 | |
| Thionyl chloride | Acros Organics | 382662500 | Note: Corrosive substance; toxic if inhaled |
| Bromine | Acros Organics | 402841000 | Note: causes severe skin burns, fatal if inhaled |
| Diethyl ether | any source | ||
| Sodium sulphate | Acros Organics | 196640010 | |
| Chloroform | VWR Chemicals | 22711.29 | |
| tert-Butyl 2,2,2-trichloroacetimidate | Aldrich | 364789 | Note: flammable substance; irritrant to skin and eyes |
| Boron trifluoride etherate | Acros Organics | 174560250 | 48 % BF3. Note: Flammable substance; causes skin burns, fatal if inhaled |
| Sodium bicarbonate | Acros Organics | 424270010 | |
| Ethyl-acetate | any source | For column chromatography | |
| n-Hexane | any source | For column chromatography | |
| Bulb-to-bulb (Kugelrohr) distillation apparatus | Büchi | Model type: Glass oven B-585 | |
| Silicagel | Carl Roth GmbH | P090.2 | |
| Methanol | any source | For column chromatography | |
| Dichloromethane | any source | For column chromatography | |
| Ethanol | VWR Chemicals | 20821.296 | |
| Ammonia | Acros Organics | 428381000 | 7N Solution in Methanol |
| Palladium | Aldrich | 643181 | 15 % wet |
| Hydrogenation apparatus PARR | PARR Instrument Company | ||
| Celite 503 | Aldrich | 22151 | |
| Sintered glass funnel | any source | ||
| Thiophosgen | Aldrich | 115150 | Note: irritrant to skin; toxic if inhaled |
| Triethylamine | Alfa Aesar | A12646 | |
| Dichloromethane | Acros Organics | 348460010 | Extra dry |
| Magnetic stirrer | any source | ||
| PAMAM G4 Dendrimer | Andrews ChemService | AuCS – 297 | 10 % wt. solution in MeOH |
| Lipophylic Sephadex LH-20 | Sigma | LH20100 | |
| Thin-layer chromatography plates | Merck Millipore | 1.05554.0001 | |
| Formic acid | VWR Chemicals | 20318.297 | |
| Lophylizer | any source | ||
| Gadollinium(III) chloride hexahydrate | Aldrich | G7532 | |
| Sodium hydroxide | Acros Organics | 134070010 | |
| pH meter | any source | ||
| Ethylenediaminetetraacetic acid disodium salt dihydrate | Aldrich | E5134 | |
| Mass spectrometer (ESI) | Agilent | Ion trap SL 1100 | |
| Acetate buffer | any source | pH 5.8 | |
| Xylenol orange | Aldrich | 52097 | 20 μM in acetate buffer |
| Hydrophylic Sephadex G-15 | GE Healthcare | 17-0020-01 | |
| Amicon Ultra-15 Centrifugal Filter Unit | Merck Millipore | UFC900324 | Ultracel-3 membrane (MWCO 3000) |
| Centrifuge | any source | ||
| NMR spectrometer | Bruker | Avance III 300 MHz | |
| Topspin | Bruker | version 2.1 | |
| Combustion analysis instrument | EuroVector SpA | EuroEA 3000 Elemental Analyser | |
| MALDI-ToF MS instrument | Applied Biosystems | Voyager-STR | |
| Deuteriumoxid | Carl Roth GmbH | 6672.3 | |
| tert-Butyl alcohol | Carl Roth GmbH | AE16.1 | |
| Vortex mixer | any source | ||
| Norell NMR tubes | Deutero GmbH | 507-HP-7 | |
| NMR coaxial tube | Deutero GmbH | coaxialb-5-7 | |
| DLS instrument | Malvern | Zetasizer Nano ZS | |
| 0.20 μm PTFE filter | Carl Roth GmbH | KC94.1 | |
| HEPES | Fisher BioReagents | BP310 | |
| Plastic tube vials | any source | ||
| Dotarem | Guerbet | NDC 67684-2000-1 | |
| MRI scanner | Bruker | BioSpec 70/30 USR magnet (7 T). Note: potential hazards related to high magnetic fields | |
| RF coil | Bruker | dual frequency volume coil (RF RES 300 1H/19F 075/040 LIN/LIN TR) | |
| Paravision (software) | Bruker | Version 5.1 |
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