A multimeric MR-optical contrast agent for multimodal imaging

Article abstract of DOI:10.1039/c4cc05651e

We describe the design, synthesis and in vitro evaluation of a multimodal and multimeric contrast agent. The agent consists of three macrocyclic Gd(iii) chelates conjugated to a fluorophore and possesses high relaxivity, water solubility, and is nontoxic.

Full text of DOI:10.1039/c4cc05651e

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A multimeric MR-optical contrast agent for
multimodal imaging†
Cite this: Chem. Commun., 2014,
50, 11469
Victoria S. R. Harrison, Christiane E. Carney, Keith W. Macrenaris and
Thomas J. Meade*
Received 21st July 2014,
Accepted 14th August 2014
DOI: 10.1039/c4cc05651e
www.rsc.org/chemcomm
We describe the design, synthesis and in vitro evaluation of a multi- detection of cancer,10 tracking gene expression,11 and impor-
modal and multimeric contrast agent. The agent consists of three tantly, histological validation of MR signal.
macrocyclic Gd(III) chelates conjugated to a fluorophore and possesses
There have been an increasing number of reports of multi-
high relaxivity, water solubility, and is nontoxic. The modular synthesis modal MR-optical contrast agents using a wide variety of
is amenable for the incorporation of a variety of fluorophores to nanoconjugates and fluorophores.12,13 However, the intrinsic
generate molecular constructs for a number of applications.
variability of nanoparticles can be an obstacle for some appli-
cations. In order to address this issue, researchers have devel-
The advent of a number of highly sensitive, tomographic imaging oped small molecule MR-optical contrast agents consisting
modalities has enabled scientists and clinicians to acquire in vivo of a Gd(III)-based chelate conjugated to near-IR dyes,14 rhoda-
images of animals without the need for sacrifice.1–3 Each modality mine,15,16 cyanine7,17 and fluorescein.17 Typically, these agents
possesses unique strengths where the combination of two or have been shown to have relatively low relaxivities and limited
more imaging modalities continues to impact our understanding water solubility. Further, conjugating agents to dendrimer
of complex biological processes and drug development.4 Techni- scaffolds has been shown to increase relaxivity, but the resul-
ques for in vivo imaging include positron emission tomography tant agents are polydisperse and difficult to characterize.18,19
(PET), single-photon emission computed tomography (SPECT),
To overcome these limitations we report the synthesis, charac-
and magnetic resonance (MR).5 Although each modality provides terization and in vitro evaluation of a high relaxivity, multimeric and
high resolution in vivo tomographic information, MR imaging is multimodal MR-optical contrast agent. The design is based upon
well suited for longitudinal assessment of in vivo processes our previously reported agent where three Gd(III) chelates are
because it does not require ionizing radiation (CT) or the use of conjugated to a phenolic core.20 We modified this design to allow
radioactive tracers (PET and SPECT). For example, MRI has been conjugation of fluorescein to generate a highly water-soluble
used to fate map cells in developing embryos where the descen- MR-optical agent which labels cells with high efficiency and
dants of individual precursors were labeled with a stable, generates significant MR contrast enhancement at clinical
nontoxic, lineage tracer (MRI contrast agent) which allowed (1.4 T) and research (7 T) magnetic field strengths.
researchers to determine the cell location and migration responsible
To facilitate conjugation of 2 to fluorescein isothiocyanate, an
for embryonic development.6 However, the low probe sensitivity amine-functionalized linker was introduced onto the phenolic
and limited spatial resolution of MRI preclude the observation of core of the complex (see Fig. S1–S3, ESI† for further details). 1 was
molecular events.
synthesized via the direct reaction of complex 2 with commercially
Optical imaging is a modality that provides high resolution available fluorescein isothiocyanate in water at pH 9.0 using
and probe sensitivity to detect subcellular localization and potassium carbonate (Scheme 1). The reaction was performed in
molecular interactions. The integration of optical and MR the dark due to the photo-instability of fluorescein.
imaging is therefore an appealing approach to facilitate appli-
The relaxivity of 1 and 2 were determined to be 17.0 Æ
cations such as fate mapping transplanted stem cells,7–9 early 0.5 mMÀ1 sÀ1 and 14.9 Æ mMÀ1 sÀ1 respectively at 1.41 T
(Table 1). The observed ionic relaxivities decrease to 4.7 Æ
0.3 mMÀ1 sÀ1 for 1 and 5.2 Æ 0.3 mMÀ1 sÀ1 for 2 at 7 T and
Department of Chemistry, Molecular Biosciences, Neurobiology, Biomedical
Engineering, and Radiology, Northwestern University, 2145 Sheridan Road,
are consistent with values obtained from agents generated from
Evanston, Illinois 60208-3113, USA. E-mail: tmeade@u.northwestern.edu
similar scaffolds.20,21
† Electronic supplementary information (ESI) available: Experimental proce-
dures, 1H, 13C, and mass spectra of compounds, cellular toxicity, and cellular
The complexes were further characterized by quantum yield
labelling of 2. See DOI: 10.1039/c4cc05651e
and octanol–water partition coefficients (log P) measurements.
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Scheme 1 Synthetic route to fluorescein-conjugated contrast agent 1. 2 was designed for orthogonal modification through isothiocyanate conjugation
to the primary amine. For complete synthetic details, see Fig. S1–S3, ESI.†
Table 1 Relaxivities of 1 and 2 in 10 mM PBS buffer (pH 7.4) at 1.41 T
(37 1C) and 7 T (25 1C)
1.41 T (60 MHz)
Ionic
7 T (300 MHz)
Ionic
Molecular
(mMÀ1 sÀ1
Molecular
(mMÀ1 sÀ1
)
(mMÀ1 sÀ1
)
)
(mMÀ1 sÀ1
)
1
2
17.0 Æ 0.5
14.9 Æ 0.5
51.0 Æ 1.5
45.0 Æ 1.5
4.7 Æ 0.3
5.2 Æ 0.3
14.1 Æ 0.9
15.9 Æ 0.9
The quantum yield of 1 was determined to be 0.56. The log P
values of 1 and 2 were À2.0 and À1.9 respectively. These
negative log P values are characteristic of high water solubility,
indicating that conjugation of fluorescein to the contrast agent
scaffold did not significantly impact solubility. As a result,
incubation concentrations for in vitro studies can be made in
the mM range which is important for maximizing intracellular
agent concentration.
Efficient cell penetration is crucial for the use of imaging
probes to investigate biological mechanisms such as fate mapping
cells. Cellular localization of 1 was determined using confocal laser
scanning microscopy where micrographs showed intracellular
accumulation of the probe (Fig. 1, see ESI† for z-stacks and images
Fig. 1 Confocal fluorescence micrographs of HeLa cells incubated with
85 mM of 1 for 4 hours and 1 mM DAPI for 10 minutes. Images show
intracellular accumulation of 1. Images of unlabeled cells and z-stacks can
be found in the ESI.† Scale bar = 20 mm. Blue = DAPI, green = fluorescein.
of unlabeled cells). Larger cell populations were examined with achieved in HeLa cells (6.8 fmol Gd(III) per cell), followed by
analytical flow cytometry to determine the efficiency of cell label- B16-F10 cells (2.9 fmol Gd(III) per cell), and MDA-MB-213-
ing. Specifically, HeLa cells were incubated with 21 to 170 mM of 1 mcherry cells (2.2 fmol Gd(III) per cell). In all cell lines, uptake
and showed increasing accumulation of fluorescein with increasing plateaued indicating that cells were saturated with 1. These
incubation concentration (Fig. S25, ESI†). Labeling efficiency of values represent a 10-fold increase in cell uptake compared to
100% was attained at each concentration examined.
ProHances (Fig. S27, ESI†).
Cellular uptake of Gd(III) was investigated by incubating
To demonstrate that cell labeling of 1 is sufficient to produce
HeLa, B16-F10, or MDA-MB-231-mcherry cells with concentra- T1-weighted contrast enhancement of cell populations, MR images
tions of 1 ranging from 27 to 260 mM (Fig. 2). Cells were washed of cell pellets were acquired at 7 T at cell densities that approximate
and pelleted prior to analysis to reduce non-specific binding. tumor densities in vivo (Fig. 3). HeLa cells were incubated with
The cell lines were chosen to address variability in labeling 250 mM equalized Gd(III) of 1, 2, or ProHances (the concentration
arising from differences in tissue of origin (cervix, breast, and of ProHances is 3Â higher than 1 or 2). The most significant
skin), species of origin (human and mouse), cell size (12–20 mm contrast enhancement was observed in cells labeled with 1 with a
diameter),22,23 and cell growth rate (20–38 h doubling times).24–26 64% reduction in T1 compared to untreated cells, followed by
Cell uptake increased with cell volume with the highest labeling ProHances (20% reduction in T1), and 2 (8% reduction in T1).
11470 | Chem. Commun., 2014, 50, 11469--11471
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Graduate Research Fellowship (C.C.). Imaging was performed
at the Northwestern University Center for Advanced Molecular
Imaging generously supported by NCI CCSG P30 CA060553
awarded to the Robert H Lurie Comprehensive Cancer Center.
Notes and references
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Fig. 2 Concentration-dependent cell uptake was determined by incubating
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In conclusion, we have developed a new multimeric and
multimodal contrast agent that contains three Gd(III) chelates
conjugated to a fluorophore. The agent has high relaxivity at both
low (1.4 T) and high magnetic fields (7 T), is highly water soluble,
cell-permeable, and possesses excellent cell labeling capabilities.
The agent shows significantly increased labeling and image
contrast at 7 T compared to clinically available ProHances.
Further, the molecular architecture described can accommodate
a large variety of fluorophores to generate additional molecular
constructs for enhanced multimodal imaging.
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This work was supported by the National Institutes of Health
(NIH grant R01EB005866) and by a National Science Foundation
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 11469--11471 | 11471