制备及<em>体外</em>磁共振成像基于树状大分子造影剂的表征
Summary
本协议描述携带CYCLEN基于大环螯合物协调顺钆离子树枝状磁共振成像（MRI）造影剂的制备和表征。在一系列的体外 MRI实验中，相对于可商购的单体类似物时此代理产生的放大的磁共振信号。
Abstract
钆（III）与无环或大环螯合物的顺磁性配合物是用于磁共振成像（MRI）中最常用的造影剂（CA）的。他们的目的是提高在组织的水质子的弛豫率，从而增加了MR图像对比度和磁共振测量的特异性。当前临床批准的造影剂是正在迅速从体内清除的低分子量分子。作为顺磁性螯合剂的载体的使用树枝状聚合物可以在更高效的MRI造影剂的未来发展中发挥重要作用。具体地，增加在一个较高的信号对比度顺磁物质的结果的局部浓度。此外，此CA提供由于其高分子量和尺寸较长的组织的保留时间。这里，我们证明了基于聚（酰氨基胺）大分子MRI造影剂（PAMAM）与monomacro树枝状聚合物的制备便利过程环状DOTA型螯合剂（DOTA – 1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸）。螯合单元通过利用朝向PAMAM树枝状聚合物的胺基表面基团的异硫氰酸酯（NCS）基团的反应性，以形成硫脲桥追加。树枝状产物纯化并通过核磁共振谱，质谱和元素分析法分析。最后，高清晰度的MR图像记录和从制备树枝状和市售单体剂获得的信号反差进行比较。
Introduction
磁共振成像（MRI）是广泛用于生物医学研究和临床诊断的有力和非电离成像技术，由于其非侵入性和优良特性的软组织的对比。最常用的MRI方法利用从水的质子获得的信号的基础上，在水信号的密度差异的组织中提供高清晰度的图像和详细的信息。的信号强度和在MRI实验的特异性可以使用造影剂（CA）的进一步提高。这些都是影响的纵向（T 1）和横向分别1,2-顺磁性或超顺磁性物质（T 2）弛豫时间。
用聚氨基聚羧酸配体的镧系元素离子钆配合物是最常用的Ť1的CA。钆（III）缩短的T 1松弛因此水的质子的时间，增加的MRI实验3中的信号对比度。然而，钆离子是有毒的;它的大小近似于钙（Ⅱ），它严重影响钙辅助细胞信号传导。因此，无环和大环螯合物被用来抵消这种毒性。各种多齿配体迄今已开发的，导致具有高的热力学稳定性和动力学惰性1钆（Ⅲ）配合物。那些基于所述12元azamacrocycle CYCLEN，特别是其四衍生物DOTA（1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸）是此CA类的最研究和应用的复合物。
尽管如此，GdDOTA型CA是低分子量的系统，其中显示某些缺点，例如低对比度效率和快速的肾排泄。大分子和多价的CA可能是一个很好的解决了这些问题，4。由于CA biodistribu化主要是由它们的大小决定的，大分子的CA显示组织中更长的保留时间。同样重要的是，在单体的MR 探测器 （ 如 GdDOTA复合），基本上改善所获取的MR信号和测量质量的增加的局部浓度这些试剂的结果的多价。
树枝状聚合物之间的编写多价的CA用于MRI 4,5最好支架。这些具有良好定义的尺寸的高度支化的大分子易于在其表面上的不同的偶合反应。在这项工作中，我们报道的制备，纯化和树枝状的CA用于MRI由第4代（G4）聚（酰氨基胺），耦合到GdDOTA状螯合物（DCA）（PAMAM）树枝状聚合物的表征。我们描述了反应性的DOTA衍生物的合成及其与PAMAM树枝状耦合。在与钆（III），标准的理化性质络合procedu再DCA的进行。最后，进行核磁共振实验证明DCA的以产生具有比从低分子量的CA获得的那些较强的对比度的MR图像的能力。
Protocol
1. DCA的制备 单体单元4 6的合成。 4-（4-硝基苯基）-2-（4,7,10-三- 叔丁氧基羰基环十二烷-1,4,7,10-四氮杂-1-基）丁酸叔丁基酯（2）。 溶解（4,7-双- 叔丁氧基羰基- 1,4,7,10-四氮杂cyclododec -1-基）在N -乙酸叔丁基酯1（1.00克，1.94毫摩尔），N-二甲基甲（ DMF，5ml）中，加入碳酸钾（0.67克，…
Representative Results
DCA的制备包括两个阶段：1）的单体的DOTA型螯合剂的合成（ 图1）和2）与G4 PAMAM树枝状聚合物和树枝状钆（III）络合物的后续制备螯合剂的耦合（ 图2） 。在第一阶段，制备含有四个羧酸的基于CYCLEN-DOTA型螯合剂和适合于进一步合成修饰的正交基。从1（DO3A- 叔丁基酯）7开始制备，这是烷基化叔丁基2-?…
Discussion
所述树枝状MRI造影剂的制备需要的单体单元的适当选择（ 即，螯合剂为钆（III））。它们减少这种顺磁性离子的毒性，迄今为止，各种各样的无环的和大环螯合剂用于此目的1-3。在这些中，大环DOTA型螯合剂具有最高热力学稳定性和动力学惰性的，因此，是惰性MRI造影剂1,18的制剂最优选的选择。此外，它们易发生各种合成转化，这导致双官能螯合剂，能够链接到各种功能分…
Divulgations
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 |
References
- Merbach, A. E., Helm, L., Tóth, &. #. 2. 0. 1. ;. . The chemistry of contrast agents in medical magnetic resonance imaging. 2nd ed. , (2013).
- Geraldes, C. F. G. C., Laurent, S. Classification and basic properties of contrast agents for magnetic resonance imaging. Contrast Media Mol. Imaging. 4 (1), 1-23 (2009).
- Caravan, P., Ellison, J. J., McMurry, T. J., Lauffer, R. B. Gadolinium(III) chelates as MRI contrast agents: Structure, dynamics, and applications. Chem. Rev. 99 (9), 2293-2352 (1999).
- Villaraza, A. J. L., Bumb, A., Brechbiel, M. W. Macromolecules, Dendrimers, and Nanomaterials in Magnetic Resonance Imaging: The Interplay between Size, Function, and Pharmacokinetics. Chem. Rev. 110 (5), 2921-2959 (2010).
- Langereis, S., Dirksen, A., Hackeng, T. M., van Genderen, M. H. P., Meijer, E. W. Dendrimers and magnetic resonance imaging. New J. Chem. 31 (7), 1152-1160 (2007).
- Gündüz, S., Power, A., Maier, M. E., Logothetis, N. K., Angelovski, G. Synthesis and Characterization of a Biotinylated Multivalent Targeted Contrast Agent. ChemPlusChem. 80 (3), 612-622 (2015).
- Pope, S. J. A., Kenwright, A. M., Heath, S. L., Faulkner, S. Synthesis and luminescence properties of a kinetically stable dinuclear ytterbium complex with differentiated binding sites. Chem. Commun. (13), 1550-1551 (2003).
- Vibhute, S. M., et al. Synthesis and characterization of pH-sensitive, biotinylated MRI contrast agents and their conjugates with avidin. Org. Biomol. Chem. 11 (8), 1294-1305 (2013).
- Vogel, A. I., Furniss, B. S. . Vogel’s textbook of practical organic chemistry. 5th ed. , (1989).
- Lundanes, E., Reubsaet, L., Greibrokk, T. . Chromatography : basic principles, sample preparations and related methods. , (2013).
- Barge, A., Cravotto, G., Gianolio, E., Fedeli, F. How to determine free Gd and free ligand in solution of Gd chelates. A technical note. Contrast Media Mol. Imaging. 1 (5), 184-188 (2006).
- Keeler, J. . Understanding NMR spectroscopy. 2nd ed. , (2010).
- Hillenkamp, F., Peter-Katalinić, J. . MALDI MS : a practical guide to instrumentation, methods and applications. , (2007).
- Peters, J. A., Huskens, J., Raber, D. J. Lanthanide induced shifts and relaxation rate enhancements. Prog. Nucl. Magn. Reson. Spectrosc. 28, 283-350 (1996).
- Averill, D. J., Garcia, J., Siriwardena-Mahanama, B. N., Vithanarachchi, S. M., Allen, M. J. Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging. J. Vis. Exp. (53), e2844 (2011).
- Hagberg, G. E., Scheffler, K. Effect of r1 and r2 relaxivity of gadolinium-based contrast agents on the T1-weighted MR signal at increasing magnetic field strengths. Contrast Media Mol. Imaging. 8 (6), 456-465 (2013).
- Boswell, C. A., et al. Synthesis, characterization, and biological evaluation of integrin alpha(v)beta(3)-targeted PAMAM dendrimers. Mol. Pharmaceut. 5 (4), 527-539 (2008).
- Sherry, A. D., Caravan, P., Lenkinski, R. E. Primer on Gadolinium Chemistry. J. Magn. Reson. Imaging. 30 (6), 1240-1248 (2009).
- Cakić, N., Gündüz, S., Rengarasu, R., Angelovski, G. Synthetic strategies for preparation of cyclen-based MRI contrast agents. Tetrahedron Lett. 56 (6), 759-765 (2015).
- Polasek, M., Hermann, P., Peters, J. A., Geraldes, C. F. G. C., Lukes, I. PAMAM Dendrimers Conjugated with an Uncharged Gadolinium(III) Chelate with a Fast Water Exchange: The Influence of Chelate Charge on Rotational Dynamics. Bioconjugate Chem. 20 (11), 2142-2153 (2009).
- Ali, M. M., et al. Synthesis and relaxometric studies of a dendrimer-based pH-responsive MRI contrast agent. Chem. Eur. J. 14 (24), 7250-7258 (2008).
- Jackson, C. L., et al. Visualization of dendrimer molecules by transmission electron microscopy (TEM): Staining methods and Cryo-TEM of vitrified solutions. Macromolecules. 31 (18), 6259-6265 (1998).
- Jain, K., Kesharwani, P., Gupta, U., Jain, N. K. Dendrimer toxicity: Let’s meet the challenge. Int. J. Pharm. 394 (1-2), 122-142 (2010).
- Rudovsky, J., et al. 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. Bioconjugate Chem. 17 (4), 975-987 (2006).
- Xu, H., et al. Toward improved syntheses of dendrimer-based magnetic resonance imaging contrast agents: New bifunctional diethylenetriaminepentaacetic acid ligands and nonaqueous conjugation chemistry. J. Med. Chem. 50 (14), 3185-3193 (2007).
- Nwe, K., Bryant, L. H., Brechbiel, M. W. Poly(amidoamine) Dendrimer Based MRI Contrast Agents Exhibiting Enhanced Relaxivities Derived via Metal Preligation Techniques. Bioconjugate Chem. 21 (6), 1014-1017 (2010).
- Livramento, J. B., et al. First in vivo MRI assessment of a self-assembled metallostar compound endowed with a remarkable high field relaxivity. Contrast Media Mol. Imaging. 1 (1), 30-39 (2006).
- Norek, M., Kampert, E., Zeitler, U., Peters, J. A. Tuning of the Size of Dy2O3 Nanoparticles for Optimal Performance as an MRI Contrast Agent. J. Am. Chem. Soc. 130 (15), 5335-5340 (2008).
Citer Cet Article
Gündüz, S., Savić, T., Toljić, Đ., Angelovski, G. Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging. J. Vis. Exp. (118), e54776, doi:10.3791/54776 (2016).