Synthesis of multimeric MR contrast agents for cellular imaging

Article abstract of DOI:10.1021/ja0777990

We have prepared a series of molecular multimeric MR contrast agents for cell labeling that are easy to synthesize, relatively low molecular weight, and biocompatible. The relaxivities of the agents range from 17 to 85 mM^-1s^-1. Cellu

Full text of DOI:10.1021/ja0777990

Published on Web 05/02/2008
Synthesis of Multimeric MR Contrast Agents for Cellular Imaging
Ying Song, Ellen K. Kohlmeir, and Thomas J. Meade*
Departments of Chemistry, Biochemistry and Molecular and Cell Biology, Neurobiology and Physiology, Radiology,
Northwestern UniVersity, 2145 Sheridan Road,
EVanston, Illinois 60208-3113
Received October 10, 2007; E-mail: tmeade@northwestern.edu
Advances in magnetic resonance imaging (MRI) have extended
the application of this technique from routine clinical diagnosis to
1–3
in vivo cellular imaging. Gd(III) complexes are commonly used
in MR imaging to enhance local contrast. Gd(III) ions reduce the
1
longitudinal relaxation time (T ) of surrounding water protons,
rendering an increase in signal intensity in an appropriately weighted
4
,5
MR image.
The inherent lack of sensitivity of Gd(III) contrast agents limits
the application of MR for cellular imaging when compared to
6
agents used for positron emissive tomography (PET) and light
7
microscopy. A number of Gd(III) complexes (low and high
molecular weights) have been reported for in vitro and in vivo
8
–12
applications.
been optimized with regard to ease of synthesis, biocompatibility,
cellular translocation, and molecular rotational correlation time (τ ),
lifetime (τ ), and the number of coordinated water molecules (q).
However, the majority of these agents have not
R
m
Here, we describe a facile and inexpensive approach to prepare
three new low molecular weight, monodispersed MR contrast agents
with multiple Gd(III) complexes. Click chemistry is employed to
1
3
generate three pretemplated macromolecular architectures. Ac-
cording to the Solomon-Bloembergen-Morgan theory, elongation
4
of τ
R
will result in an increase in relaxivity. Moreover, the robust
Figure 1. Synthetic scheme of multimeric MR contrast agents via click
chemistry.
reaction between the azide substrate and the DOTA-Gd-alkyne
derivative forms a triazole ring linkage (Figure 1). The rigid nature
of the triazole linker hinders the local rotation of the Gd(III)
Table 1. Gd(III) Ionic and Molecular Relaxivities of the New MRI
Contrast Agents 4, 5, and 6 at 60 MHz, 37 °C
1
4,15
complexes to further enhance the relaxivity.
The complexes
possess from three to seven Gd(III) chelates/molecule and exhibit
excellent biocompatibility and high molecular relaxivities (Table
MW
(g/mol)
Gd ionic
s
no.
of Gd
molecular
(mM
-1 -1
-1 -1
compounds
r
1
(mM
)
r
1
s )
1
).
ꢀ
Gd-DOTA-alkyne derivative 595.70 3.21
1
3
6
7
3.21
-Cyclodextrin (ꢀ-CD) has been shown to associate with DOTA-
4
5
6
2030.34 5.90 ( 0.15
3982.57 10.97 ( 0.13
5480.01 12.20 ( 0.54
17.7 ( 0.47
65.8 ( 0.76
85.4 ( 3.74
Gd and DTPA-Gd derivatives in a noncovalent fashion and form
inclusion complexes. Each cyclodextrin molecule can host only
one Gd(III) complex with an association constant between 10 and
1
6
3
4
17
10 M. Covalent attachment of Gd(III) complexes to ꢀ-CD ensures
the formation of the products (see Table 1 and Supporting
Information).
the stability of the assembly, which is crucial in conducting long-
term cell tracking.
To evaluate the in vitro efficiency of these agents, T relaxivity
1
The DOTA-Gd-alkyne derivative was synthesized by modifica-
was measured on a Bruker Minispec 60 MHz relaxometer at 37
°C in Millipore water using a standard inversion-recovery pulse
1
8,19
tion of existing procedures.
ligand was performed before the cycloaddition reaction because
Metalation of the DOTA-alkyne
sequence. The relaxivity of the Gd-DOTA-alkyne derivative was
20
-1 -1
DOTA has a high affinity for Cu(II). The trismethylazido benzene,
, hexakis(azidomethyl) benzene, 2, and heptakis-6-azido-6-deoxy-
-cyclodextrin, 3, were synthesized from corresponding com-
measured to be 3.21 mM
s
at 60 MHz, which is comparable
-
1
1
ꢀ
1
to the reported value of DOTA-Gd (Dotarem, r ) 2.7-3.1 mM
-1
22
s
at 60 MHz, 37 °C).
mercially available halides in one step. These azide compounds
are known to be stable at room temperature; however, they should
be handled with extreme care due to the explosive nature of organic
azides. The click chemistry was carried out in a water/dimeth-
ylformamide mixture with heating or under microwave irradiation
After conjugation with 1, 2, and 3, the measured relaxivities of
the complexes increased dramatically and are summarized in Table
1. With an increase of the molecular weight and restricted rotation
of the Gd(III) chelates, the relaxivity per Gd(III) increased 2-4-fold.
The molecular relaxivities of the three new agents are significantly
higher than those of commercially available agents.
2
1
(
Figure 1). HPLC and Sephadex size exclusion chromatography
were used to purify these complexes. High resolution MS and
MALDI-MS characterization of complexes 4, 5, and 6 confirmed
MR phantoms were imaged using a 4.7 T Bruker Biospec MR
Imager with increasing molecular concentrations of DOTA-Gd, 4,
6
662 9 J. AM. CHEM. SOC. 2008, 130, 6662–6663
10.1021/ja0777990 CCC: $40.75  2008 American Chemical Society

C O M M U N I C A T I O N S
be carried out to determine the longevity of the label and any long-
term effects on cellular function.
In summary, we have prepared three new MR contrast agents
with varying numbers of Gd(III) complexes via click chemistry.
This inexpensive and facile synthesis results in contrast agents with
increased relaxivity and good biocompatibility. The agents are
monodispersed and have relatively low molecular weights. We have
demonstrated that these agents do not affect cellular viability in
culture and are able to effectively label cells as observed in acquired
MR images.
Acknowledgment. This research was supported by Center of
Excellence in Translational Human Stem Cell Research, 1P50
NS054287-01 and NIH grant 1RO1 EB005866-01. MR Imaging was
performed on instruments supported by NIH/NCRR Grant S10
RR15685-01.
Figure 2. (A) T1 weighted spin-echo MR phantom images of DOTA-
Gd, 4, 5, and 6 in Millipore water at equal molar concentration of each
contrast agent. Images were acquired at 4.7 T at 25 °C (TR/TE ) 300/18.4
ms). Concentrations (from left to right): 62, 51, 37, 21, 7, 0 µM. (B) T1
weighted spin-echo images of pelleted NIH-3T3 cells incubated with
DOTA-Gd, 5, and 6 at approximately equal molar concentrations of Gd(III)
Supporting Information Available: Experimental details. This
material is available free of charge via the Internet at http://pubs.acs.org.
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JA0777990
J. AM. CHEM. SOC. 9 VOL. 130, NO. 21, 2008 6663