Multimeric Near IR-MR Contrast Agent for Multimodal in Vivo Imaging

Article abstract of DOI:10.1021/jacs.5b04509

Multiple imaging modalities are often required for in vivo imaging applications that require both high probe sensitivity and excellent spatial and temporal resolution. In particular, MR and optical imaging are an attractive combination that can be used to determine both molecular and anatomical information. Herein, we describe the synthesis and in vivo testing of two multimeric NIR-MR contrast agents that contain three Gd(III) chelates and an IR-783 dye moiety. One agent contains a PEG linker and the other a short alkyl linker. These agents label cells with extraordinary efficacy and can be detected in vivo using both imaging modalities. Biodistribution of the PEGylated agent shows observable fluorescence in xenograft MCF7 tumors and renal clearance by MR imaging. (Figure Presented).

Full text of DOI:10.1021/jacs.5b04509

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Article
A Multimeric Near IR-MR Contrast Agent for Multimodal In Vivo Imaging
Victoria S.R. Harrison, Christiane E Carney, Keith W. MacRenaris, Emily A. Waters, and Thomas J. Meade
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b04509 • Publication Date (Web): 17 Jun 2015
Downloaded from http://pubs.acs.org on June 22, 2015
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A Multimeric Near IR-MR Contrast Agent for MultimodalIn Vivo Imaging
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Victoria S. R. Harrison ,Christiane E. Carney , Keith W. MacRenaris , Emily A. Waters ,
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Thomas J. Meade
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Department of Chemistry, Molecular Biosciences, Neurobiology, Biomedical Engineering, and
Radiology, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208ꢀ3113,
United States
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Center for Advanced Molecular Imaging, Northwestern University, 2145 Sheridan Road,
Evanston, Illinois 60208ꢀ3113, United States
Email: tmeade@northwestern.edu
Author Contributions: V.S.R.H. and C.E.C. contributed equally to this work.
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Abstract
Multiple imaging modalities are often required for in vivo imaging applications that
require both high probe sensitivity and excellent spatial and temporal resolution.In particular,
MR and optical imaging are an attractive combination that can be used to determine both
molecular and anatomical information. Herein, we describe thesynthesis and in vivo testing of
two multimeric NIRꢀMR contrast agents thatcontain three Gd(III) chelates and an IRꢀ783 dye
moiety. One agent contains a PEG linker and the other a short alkyl linker. These agents label
cells with extraordinaryefficacy and can be detected in vivousing both imaging modalities.
Biodistribution of the PEGylated agent showsobservable fluorescence in xenograft MCF7 tumors
and renal clearance by MR imaging.
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Introduction
Magnetic resonance imaging (MRI) is a powerful diagnostic tool used in both clinical
and researchsettings due to its capacity to render images with high spatial and temporal
resolution. Unlike other imaging modalities, MR does not require the use of ionizing radiation or
suffer from limited depth penetration, making it well suited fornonꢀinvasive longitudinal studies.
As a result, MRI has become a major focus of translational imaging research, with the ultraꢀhigh
resolution achievable with highꢀfield magnets making it particularly well suited to applications
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1ꢀ3
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such as the fate mapping transplanted stem cells, detecting cancer, and tracking gene
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expression.
Contrast agents are typically used to enhance intrinsic MR image contrast. These agents
selectively shorten the longitudinal (T ) or transverse (T ) relaxation times of water protons in the
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region of interest thereby allowing the visualization of a wide range of otherwise undetectable
biomarkers. Gadolinium (III) complexesare the most commonly T contrast agents due to the
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metal’s seven unpaired electrons(S= 7/2) and high magnetic moment. The efficacy with
whichGd(III) shortens T is termed its relaxivity (r ); agents with higher relaxivities are more
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sensitive and are detectable at lower concentrations.
A significant limitation of T contrast agents is the low observed relaxivities,
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whichultimatelytranslate into signal ambiguity. One strategy to address this shortcoming
involves multiplexing an MR contrast agent with a more sensitive imaging modality, such as
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optical imaging.
In this combination, MR offers detailed anatomic imaging, while optical
imaging offers high probe sensitivity that can be used to image molecular targets at low
concentrations. In order to take advantage of this high sensitivity in vivo, it is necessary to use
fluorophoresthat excite in the Nearꢀinfrared (NIR) imaging window of 700 nm to 1000 nm as the
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biological matrix exhibits high absorption and autofluorescence background at shorter
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wavelengths.
To take advantage of the strengths of optical and MR imaging, our approach was to
design a multimodal agent that excites in the NIR range and offers a singlepharmacological
behavior for both imaging acquisitions.There have been an increasing number of reports of NIRꢀ
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MR contrast agents which incorporate a wide variety of nanoconjugates,
however; the
intrinsic variability and fast clearance of nanoparticles can make longꢀterm longitudinal studies
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impractical. Additionally, while researchers have developed a number of small molecule MRꢀ
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optical contrast agents conjugated to fluorophores such as rhodamine,
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fluorescein,
napthalimide,
BODIPY, and luminescent lanthanides,
these agents excite
predominately in the visible light spectrum and suffer from low relaxivities, thereby limiting in
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vivo applications. Small molecule MRꢀoptical agents that excite in the NIR range exist,
however they suffer from reduced chelate stability and a discrepancy in sensitivity between the
MR and optical component due to the 1:1 ratio of the fluorophore and Gd (III) chelate.
Previously, we have reported an agent that consisted of three macrocyclic Gd(III)
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chelates conjugated to a fluorescein moiety. This agent possessed high relaxivity, water
solubility, and excellent cell labeling capabilities but itsin vivo application was limited by the
excitation wavelength of fluorescein. To overcome this limitation wehave prepared a multimodal
agent using IRꢀ783, chosen due to its high extinction coefficient, excellent emission
wavelengths, and high photochemical and photophysical stability as compared to other
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commercially available NIR dyes.
Two agents were synthesized and evaluated forcell
labeling in vitro. Additionally, biodistribution of 1was evaluated in vivo using a xenograft MCF7
tumor model. Though further work is needed to increase the sensitivity of the MR component,
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here we show that these agents possess outstanding cell labeling capability and are detectable in
vivousing both MR and optical imaging.
Results and Discussion
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Synthesis and Characterization
Two multimericMR contrast agents conjugated to IRꢀ783 dye were synthesized and
characterized (Figure 1). The linker between IRꢀ783 and the phenolic core was varied to
investigate the effect on water solubility and the photophysical properties of the complexes. The
synthesis of these agents begins with the preparation of an amineꢀfunctionalized core. Complex
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was synthesized according to literature procedure, whereas 3 was synthesized from 1,11ꢀ
dichloroꢀ3,6,9ꢀtrioxaꢀundecane (See Scheme S1 for synthetic details). In order to incorporate IRꢀ
83 onto these scaffolds, an isothiocyanate functional handle was introduced onto the
commercially available dye (See Scheme S2for synthetic details).
and 2 were synthesized via the direct reaction of 3 or 4 with 5 in a mixture of
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bicarbonate buffer (pH 9.4), acetonitrile and dimethylꢀsulfoxide (Scheme 1). The addition of the
dimethylꢀsulfoxide and acetonitrile inhibited the aggregation of the dye and allowed the reaction
to proceed to completion. 1 and 2 were purified by semiꢀpreparative reverseꢀphase HPLC and
characterized by analytical reverseꢀphase HPLC and MALDIꢀTOF.
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Figure 1. Structure of agentsinvestigated in vitro. The presence of the IRꢀ783 dye moiety in
complexes1 and 2 increases cellular uptake and introduces the capacity to image usingoptical
imaging. Complex1 utilizes a PEG linker to increase water solubility and increase the distance
between the Gd(III) and NIR moieties.
O
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SO3ꢀ
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OH
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, 5:4:1
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OH
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Bicarbonate Buffer:MECN:DMSO
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OH
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Bicarbonate Buffer:MECN:DMSO
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H
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O
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Scheme 1.Synthesis of IRꢀ783 conjugated contrast agents (1 and 2). Complexes 3 and 4 were
designed for orthogonal modification through isothiocyanate conjugation to the primary amine.
The reaction was performed in the dark due to the photoꢀinstability of the IRꢀ783 derivative.
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The octanolꢀwater partition coefficients (log P) of 1-4 were measured to determine
hydrophobicity of the agents (Table 1). The negative log P values are characteristic of high
water solubility, indicating that the introduction of the IRꢀ783 moiety did not significantly
impact solubility. As a result, the relaxation and photophysical properties of these complexes
could be measured in aqueous solutions.
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The relaxivities of 1ꢀ3 were measured at pH 7.4 in 10 mM MOPS buffer. The ionic
ꢀ1 ꢀ1
ꢀ1 ꢀ1
relaxivities of 1 and 2 were determined to be 16.7 + 0.7 mM s and 17.5 + 0.4 mM s ,
ꢀ1 ꢀ1
respectively at 1.41 T (Table 1). The observed ionic relaxivities decreased to 4.8 + 0.4 mM s
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for 1 and 4.1 + 0.3 mM s for 2 at 7 T and are consistent with values obtained from agents
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generated from similar scaffolds.
Table 1. Characterization of IRꢀ783 conjugated agents (1 and 2) and precursors (3 and 4)
including log P, and relativity at low and high fields.
a
b
Log P
Relaxivity 1.41 T (60 MHz)
Relaxivity 7 T (300 MHz)
Ionic
mM s )
Molecular
(mM s )
Ionic
(mM s )
4.8 + 0.4
Molecular
(mM s )
ꢀ1 ꢀ1
ꢀ1 ꢀ1
ꢀ1 ꢀ1
ꢀ1 ꢀ1
(
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ꢀ3.0 + 0.1
ꢀ1.9 + 0.1
16.7 + 0.7
17.5 + 0.4
13.0 +0.1
14.9 + 0.5
50.1+2.1
52.5 + 1.2
39.0 + 0.3
45.0 + 1.5
14.4 + 1.2
12.4 + 1.2
15.4 + 0.6
15.9 + 0.9
4.1 + 0.3
5.1 + 0.2
5.2 + 0.3
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7° C, pH 7.4 10 mM MOPS buffer
5° C, pH 7.4 10 mM MOPS buffer
b
The photophysical properties of 1 and 2 were measured at pH 7.4 in 10 mM MOPS
buffer and in DMSO (Table 2). The maximum absorption and emission wavelengths were in the
range of 767 to 803 nm (typical of IRꢀ783 derivatives) and well within the NIR in vivo imaging
window. The quantum yields of 1 and 2 in MOPS buffer are typical of heptamethine dye
derivatives, which tend to form higher order aggregates in aqueous solutionsand selfꢀ
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quench.
1 and 2 have large extinction coefficients in both DMSO and aqueous solution.
Unlike clinically used indocyanine green, the fluorescence of 1 and 2 has a linear relationship
with concentration indicating that observed fluorescence has a direct relationship with the
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amount of dye present.
Table 2.Photophysical Propertiesof 1 and 2
ꢀ1
ꢀ1
λexcitation(nm)
λemission(nm)
ɸfl
ε (M cm )
MOPS
DMSO
786
MOPS
DMSO
803
MOPS
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0.02
DMSO
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MOPS
DMSO
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2
769
767
787
785
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1.36E+05
2.45E+05
1.93E+05
786
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CellularUptake
Concentrationꢀdependent cellular uptakewas determined by incubating MCF7 cells with
concentrations of 1ꢀ3 that maintain ≥ 90% cell viability (Figure S7 and Figure S8) for 24 hours
to maximize labeling (Figure S9). Both 1 and 2 showed significantly enhanced cell uptake with
an approximately 910ꢀfold and 430ꢀfold increase in labeling, respectively compared to 3(Figure
2
).Complex 1 attains the highest cellular uptake with a maximum of 230 ± 10fmolGd(III) per cell
with only a 60 ꢁM incubation concentration.
This labeling is surprising because although some Gd(III)ꢀbased nanoparticle contrast
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agents have attained over 100 fmol Gd(III) per cellwith low incubation concentrations,
small
molecule agents typically require incubations in the tens to hundreds of millimolar to achieve
comparable cell labeling. Furthermore, the cellular uptake of 1 and 2 represent a significant
increase in labeling compared to our previous fluoresceinꢀconjugated agent that uses a similar
chelate scaffold indicating that the high cell labeling can be attributed to the IRꢀ783
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derivative. Confocal micrographs of 1 and 2 showed intracellular accumulation of contrast
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agent indicating that the labeling is unlikely to be a result of nonꢀspecific binding to the cell
membrane(Figure 3). Additionally, the high labeling achieved with 1 allowed the agent to be
detected with transmitted laser light (seen as dark spots on the image). This phenomena is likely
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attributable to light scattering and is frequently observed with nanoparticles.
Figure 2. Concentrationꢀdependent cell labeling in MCF7 cells incubated with 0ꢀ100 ꢁM1ꢀ3.
Complexes 1 and 2 with the IRꢀ783 derivative achieve significantly higher cell labeling than 3.
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Figure 3.Confocal micrographs of 1 and 2 showing intracellular accumulation of contrast agent.
The high labeling of 1 enables detection with transmitted laser light.Scale bar = 25 ꢁm. Red =
NIR.
Previously, IRꢀ783 derivatives have been shown to targetorganicꢀanion transporting
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polypeptides (OATPs).
cell labeling achieved with 1 and 2, MCF7 cells were incubated with bromosulfopthalein (BSP)
a competitive inhibitor of OATPs) for 30 minutes prior to agent labeling(Figure 4).Labeling
To investigate whether OATPs may be responsible for the excellent
(
decreased by 3.2ꢀfold for 1 and 2.1ꢀfold for 2 compared to cells that did not receive BSP
treatment suggesting that uptake is at least partially mediated by OATPs.An active transport
mechanism of cell labeling was further confirmed by incubating cells at 4 °C which resulted in a
59ꢀfold and 14ꢀfold decrease in labeling for 1 and 2, respectively compared to controls incubated
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at 37 °C. Overall, these results suggest that the uptake of 1 and 2 is facilitated by an energyꢀ
dependent active transport mechanism and is likely at least partially mediated by OATPs.
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Figure 4. The mechanism of cell uptake was investigated by incubating cells with BSP (an
inhibitor of OATPs) or 4 °C for 30 min prior to contrast agent labeling. To determine the
statistical significance from controls, labeling was compared using an unpaired tꢀtest where * p<
0.05, ** p< 0.01, *** p< 0.001.
In Vivo Fluorescence Imaging
The biodistribution of 1 was evaluated using a xenograft MCF7 tumor model in athymic
nude mice. Complex 1 was dissolved in DMSOand injected into the intraperitoneal cavity (n =
12) at a dose of 10 mg/kg. The mice were imaged with nearꢀIR fluorescence imaging 2, 4, 24,
and 48 hours postꢀinjection. Tumors were visibly distinct by fluorescence imaging with the
maximum intensity attained 4 hours postꢀinjection (Figure 5A and 5B).Fluorescence signal
significantly decreased at the 24 and 48ꢀhour time points suggesting that the complex is cleared
efficiently. This tumor uptake profile differs from other IRꢀ783 derivatives that report maximum
fluorescence intensity in subcutaneous tumor models 48 hours postꢀinjection and persistence of
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signal for up to 20 days.
This suggests that conjugation of IRꢀ783 to the Gd(III) chelate
scaffold increases clearance from tumors.
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Figure 5. NearꢀIR fluorescence images of MCF7 xenograft nude mice were acquired 2, 4, 24,
and 48 hours after i.p. injection of 1. A: Representative images of mice show accumulation of 1
into the tumor. The maximum fluorescence signal is observed 4 hours postꢀinjection. B:
Quantitative representation of background subtracted radiant efficiency in the tumors over time.
C: Organs were harvested after each time point and imaged ex vivo. These representative images
taken 4 hours postꢀinjection show the greatest fluorescence intensity in the uterus, kidney,
intestine, and tumor. Taken together, these images suggest clearance of the complex over time.
Biodistibution of Complex 1
To further investigate the biodistribution of 1, organs were collected 2, 4, 24, and 48
hours postꢀinjection and imaged ex vivo. Representative images of organs harvested at 4 hours
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show significant fluorescence intensity in the uterus, kidneys, intestines, and tumor (Figure
5C).The fluorescence intensity of organs collected at allꢀtime points was plotted and showed that
1
has the greatest accumulation in the liver and intestines followed by the kidneys, tumor, and
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uterus (Figure S10). Accumulation in the uterus is attributed to the i.p. injection method as the
uterine tubes open into the peritoneal cavity. While fluorescence imaging can provide some
preliminary information regarding biodistribution, this technique is limited by differences in light
scattering and absorption between tissue types and the potential for dye quenching. In particular,
studies that comparebiodistributionby fluorescence and radiolabeling methods have reported
attenuated fluorescence signal in the liver and spleen that can lead to inaccurate conclusions
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regarding biodistribution.
To obtain a quantitative assessment of biodistribution of 1, organs were digested and
analyzed for Gd(III) content by ICPꢀMS (Figure 6). Unlike the fluorescence analysis, Gd(III)
content was normalized to the total mass of the tissue and analysis of blood and urine samples
was performed. These data show that complex 1 has the highest accumulation in the intestines,
urine, and spleen followed by the liver, kidneys, ovaries, and uterus. Accumulation in the spleen
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and liver is associated with uptake by the reticuloendothelial system (RES) system,
whereas
accumulation in the intestines, urine, and kidneys likely indicates clearance of the complex.
Uptake of 1 in the tumor was not significantly different than accumulation in the muscle at any
time point. All organs except the liver show significantly decreased accumulation of 1after 48
hours compared to the early time points further suggesting clearance of the complex. Taken
together, these data suggest that complex 1 is taken up by the RES and eliminated over time
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possibly by renal, fecal, or hepatic clearance.
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Figure 6. Biodistribution of 1 was determined 2, 4, 24, and 48 hours postꢀinjection in MCF7
xenograft nude mice (n = 3 per time point). Briefly, organs, blood, and urine were harvested and
acid digested for analysis of Gd(III) content by ICPꢀMS. These data show the most significant
accumulation of 1 occurs in the intestines and urine. Accumulation of 1decreases over time for
all organs except the liver. These data suggest clearance of the complex through the renal
system. A: all organs, B: all organs except the spleen, intestines, and urine.
In Vivo MR Imaging
Based on the biodistribution and fluorescence imaging data, MR images of xenografted
nude mice were acquired 0, 2, 4, and 24 hours postꢀinjection of complex 1(n = 2). Images show
significant contrast enhancement in the bladder at allꢀtime points (Figure 7). Contrastꢀtoꢀnoise
ratios (CNR) were determined by subtracting the mean intensity of the muscle from the bladder
and dividing by the standard deviation of the noise. The highest CNR was obtained 2 hours postꢀ
injection with a 2.7ꢀfold increase compared to the preꢀscan image. The CNR at 4 and 24 hours
postꢀinjection was the same with a 1.7ꢀfold increase compared to the preꢀscan. No significant
contrast enhancement was observed in the tumors. These images agree with the ICPꢀMS
biodistribution data that shows high accumulation of 1 in the urine and no significant difference
in accumulation between the muscle and tumors.
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Figure 7. T ꢀweighted MR images at 9.4 T of MCF7 xenograft nude mice were acquired 0, 2, 4,
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and 24 hours after i.p. injection of 1 (n = 2 per time point). Images show contrast enhancement in
the bladder with a 2.7ꢀfold increase in CNR compared to the preꢀscan after 2 hours and a 1.7ꢀfold
increase after 4 and 24 hours.
Conclusions
We have developed two multimeric and multimodal contrast agents containing three
Gd(III) chelates conjugated to an IRꢀ783 derivative. One agent was synthesized with a PEG
linker (1) while the other contained a short alkyl linker (2). Both agents achievedhigh cell
labeling in cell culture with a maximum of 228 fmolGd(III) per for1 and 108 fmol Gd(III) per
cell for 2. This result is significantbecause the majority of small molecule agents require
incubations in the tens to hundreds of millimolar to achieve labeling of 100+ fmolGd(III) per cell
whereas our agents were incubated at only 60 ꢁM. Additionally, we found that the labeling
occurred via an active transport mechanism likely mediated by OATPs. The biodistribution of
complex 1 was investigated in MCF7 xenograft nude mice and showed renal clearance of the
agent. Accumulation in the tumors was detected with fluorescence imaging but not MR imaging.
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To address this issue, synthetic modifications that improve solubility and allow for a higher
injection dose or alternate route of administration (i.v. instead of i.p. injection) could be
investigated in future work.
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Materials and Methods
Synthetic Methods Unless otherwise noted, materials and solvents were obtained from
commercial suppliers and used without further purification. All reactions were performed under
an inert nitrogen atmosphere. EMD 60F 254 silica gel plates were used for thin layer
chromatography and visualized using UV light or ninhydrin stain. Column chromatography was
1
performed using standard grade 60 Å 230 – 400 mesh silica gel (Sorbent Technologies). H
1
3
NMR and C NMR spectra were obtained at room temperature on a Bruker Avance III 500 MHz
NMR spectrometer. An Agilent 6210 LCꢀTOF spectrometer was used to acquire electrospray
ionization mass spectra (ESIꢀMS). MatrixꢀAssisted Laser Desorption Ionization TimeꢀOfꢀFlight
(MALDIꢀTOF) mass spectrometry was carried out using a Bruker Autoflex III MALDI. Semiꢀ
preparative HPLC was performed on a Waters 19 x 250 mm Xbridge C18 Column. Analytical
HPLC was performed using a Waters 4.6 X 250 mm 5 ꢁMXbridge C18 column using the Varian
Prostar 500 system equipped with a Varian 363 fluorescence detector, and a Varian 335 UV/Vis
Detector.
The amine functionalized Gd(III) scaffold (4) was synthesized according to literature
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0
procedure. For a detailed synthetic procedureof the peg functionalized scaffold (3) and the
isothiocyanate functionalized dye (5) see the supporting information.
N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-5-(3-(2,4,6)-Tris(1-2(hydroxyl-3-(1H-1,2,3-
triazol-1-yl(propyl)-3,7,10-tris(carboxymethyl)-
tetraazacyclododecylgadolinium(III)phenoxy)-
1,4,7,10-
4-(2-{4-[(4-
pentanamide-
isothiocyanatophenyl)oxy]-7-[3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene]-3,5-
propane-1,3-diyl)-1,3,5-heptatrien-1-yl}-3,3-dimethyl-3H-indolio)butanesulfonate (1): To a
(
solution of 3 (52.47 mg, 0.023 mmol) in 50 mL pH 9.4 Bicarbonate buffer and 40 mL
acetonitrile was added a solution of 5 (78.6 mg, 0.093 mmol) in 10 mL dimethyl sulfoxide. The
reaction was covered with tinfoil, and left to stir for 48 hours under nitrogen. The solvent was
removed by lyophilization, and the product was purified by reverse phase HPLC, using a C18
column, held at 13% for 5 minutes and eluting with a gradient of 13% ꢀ 22% acetonitrile in pH
1
0.38 buffered water over 20 minutes, t = 19.51 min. This gave 38 mg of the product as a green
r
solid (54% yield). The purity and identity of the product was confirmed using analytical HPLCꢀ
MS on a C18 column, held at 10% for 5 minutes, and eluting with a gradient of 10%ꢀ 62%
acetonitrile in pH 10.38 buffered water over 19 minutes, t = 13.55 min. MS (MALDIꢀTOF): m/z
r
+
+
observed= 3083.16 [M + 2H ] m/z calculated= 3082.86 [M + 2H ]
3
1
-(2,4,6)-Tris(1-2(hydroxyl-3-(1H-1,2,3-triazol-1-yl(propyl)-3,7,10-tris(carboxymethyl)-
,4,7,10-tetraazacyclododecylgadolinium(III)phenoxy)-propan-1-amino- 4-(2-{4-[(4-
isothiocyanatophenyl)oxy]-7-[3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene]-3,5-
(propane-1,3-diyl)-1,3,5-heptatrien-1-yl}-3,3-dimethyl-3H-indolio)butanesulfonate (2): To a
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solution of 4 (46.5 mg, 0.023 mmol) in 8 mL pH 9.4 Bicarbonate buffer and 6 mL acetonitrile
was added a solution of 5 (60 mg, 0.071mmol) in 2 mL dimethyl sulfoxide. The reaction was
covered with tinfoil, and allowed to stir for 48 hours under nitrogen. The solvent was removed
by lyophilization, and the product was purified by reverse phase HPLC (C18 column) held at
13% for 5 minutes and eluting with a gradient of 13% ꢀ 22% acetonitrile in pH 10.38 buffered
water over 20 minutes, t = 16.92 min. This gave 29 mg of the product as a green solid (46%
r
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yield). The purity and identity of the product was confirmed using analytical HPLCꢀMS on a
C18 column, held at 10% for 5 minutes, and eluting with a gradient of 10%ꢀ 48% acetonitrile in
pH 10.38 buffered water over 12.8 minutes, t = 13.12 min. MS (MALDIꢀTOF): m/z observed=
r
+
+
2862.165 [M + 2H ] m/z calculated= 2862.45 [M + 2H ]
Octanol Water Partition Coefficients Approximately 1 mg of compound was dissolved in 1
mL of a 1:1 mixture of water:octanol. After vortexing the sample tube for 30 s, the tube was
placed on a rotator for gentle mixing over 8 hours. The tube was removed from the rotator and
allowed to sit for 12 hours to ensure complete separation of the aqueous and organic phases. An
aliquot was removed from each layer and analyzed by ICPꢀMS to determine the Gd(III)
concentration in each layer. Partition coefficients were calculated from the equation logP =
log(C / C ), where logP is the logarithm of the partition coefficient, C is the concentration of
o
w
o
Gd in the 1ꢀoctanol layer, and C is the concentration of Gd in the water layer.
w
Relaxation Time Measurements at 1.4 TA 1 mM solution of each gadolinium complex was
made up in pH 7.4 10 mM MOPS buffer. These samples were serially diluted four times to give
500 ꢁL of five different sample concentrations. After 30 minutes of incubation at 37°C, the T1
and T relaxation times were measured on a Bruker mq60 NMR analyzer equipped with
2
Minispec V2.51 Rev.00/NT software (Billerica, MA, USA) operating at 1.41 T (60 Mhz) and
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37°C. Relaxation time determination and analysis was performed as previously described.
Fluorometric Analysis The photophysical properties of 1 and 2 were examined in an aqueous
solution buffered to pH 7.4 (10 mM MOPS buffer) and DMSO.UvꢀVisible spectra were recorded
on an Agilent 8453 spectrophotometer. Steadyꢀstate fluorescence emission and excitation spectra
were obtained using a Hitachi Fꢀ45000 Fluorescence Spectrophotometer. Theexcitation slit
width, emission slit width and photomultiplier voltages were 5 nm, 5 nm, and 700 V
respectively. Relative fluorescence quantum efficiencies of 1 and 2 were determined by
comparing the area under the emission of the sample with that of indocyanine green (ICG) in
DMSO (ɸ=0.13)
Cell Line & CultureMCF7 (ATCC® HTBꢀ22) cells were purchased from American Type
Culture Collection (Manassas, VA, USA) and cultured in phenol red free RPMIꢀ1640 media
supplemented with 10% FBS. Cells were grown in a humidified incubator operating at 37 °C and
5
.0% CO and harvested with 0.25% TrypLE unless otherwise indicated. Cells were allowed to
2
plate for 24 hours before all experiments.All agents were filtered with 0.2 ꢁm sterile filters prior
to incubation with cells.
Cellular ToxicityMCF7 cells were seeded at a density of 6,000 cells per well in a 96ꢀwell plate.
Cells were incubated with concentrations of 1 and 2 ranging from 0 – 80 ꢁM (50 ꢁL volume, 8
concentrations) for 24 hours. After incubation, 50 ꢁL of CellTiterꢀGlo 2.0® (Promega, Madison,
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WI, USA) was added to each well and the assay was run according to the manufacture’s
protocol. Luminescence was read on a Synergy 4 Microplate Reader (BioTek, Winooski, VT,
USA). The measured cell viability range was confirmed during each labeling experiment using a
Guava EasyCyte Mini Personal Cell Analyzer. An aliquot of cell suspension was mixed with
ViaCount solution to obtain a total volume of 200 ꢁL. Samples were counted and viability was
measured using Viacount software run according to the manufacture’s protocol.
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Confocal MicroscopyMCF7 cells were seeded at a density of 50,000 cells per plate on a 35 mm
FluoroDish (World Precision Instruments, Sarasota, FL, USA). Cells were incubated with 30
ꢁM1 and 2 (500 ꢁL) for 24 hours. Cells were washed with DPBS (2 x 1 mL) and 500 ꢁL of fresh
media was added. Cells were imaged using a Zeiss LSM 510 confocal laser scanning inverted
microscope equipped with a modeꢀlocked Mai Tai DeepSee® Ti:sapphire twoꢀphoton laser
(Spectra Physics, Mountain View, CA, USA) with an excitation wavelength of 780 nm. For all
images a PlanꢀAppochromat 40x/1.20NA water immersion Korr UVꢀVisꢀIR M27 objective lens
with a numerical aperture of 1.2 was used.
Cellular Uptake Concentrationꢀdependent uptake was determined in MCF7 cells plated at a
density of 30,000 cells per well of a 24ꢀwell plate. Complexes 1ꢀ3 were dissolved in media at
concentrations ranging from 0ꢀ100 ꢁM and incubated with cells for 24 hours. After incubation,
cells were washed twice with 0.5 mL DPBS and centrifuged at 1000xg for 5 min at 4 °C. The
media was aspirated and cells were reꢀsuspended in 200 ꢁL media. An aliquot of 50 ꢁL was used
for cell counting and 130 ꢁL was used for analysis of Gd(III) content by ICPꢀMS. For timeꢀ
dependent uptake, the same procedure was followed except cells were incubated with 20 ꢁM1ꢀ3
for1, 2, 4, 8, or 24 hours.
Mechanism of UptakeMCF7 cells were plated at a density of 45,000 cells per well of a 24ꢀwell
plate. Cells were incubated with 180 ꢁL of either blank media (4 °C or 37 °C) or 250
ꢁ
Mbromosulfophtalein (37 °C) for 30 minutes prior to the addition of 20 ꢁL of a 10X solution of
1
or 2. Cells were incubated for an additional 4 hoursand then harvested as described in the cell
uptake section.
ICP-MSQuantification of Gd(III) content in solutions, cell suspensions, and liquefied organs
was accomplished using ICPꢀMS. Samples were prepared and analyzed according to previously
30
published procedures.
MR Imaging of Solutions at 7 TA 300ꢁM solution of each gadolinium complex was made up
in pH 7.4 10 mM MOPS buffer. These samples were serially diluted three times to give 500 ꢁL
of four different sample concentrations. Solutions for determining relaxivity were imaged using a
BrukerPharmscan7 T imaging spectrometer. Image acquisition and analysis was performed as
30
previously described.
Tumor Xenograft Model Female athymicNCrnude mice were purchased from Taconic
(Hudson, NY, USA). The mice were handled and processed according to a protocol approved by
Northwestern University Animal Care and Use Committee in accordance with current guidelines
from the National Institutes of Health Model Procedure of Animal Care and Use. A 17βꢀestradiol
pellet (Innovative Research of America, Sarasota, FL, 60 day release, 0.18 mg/pellet) was
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implanted into the nape of the neck of the mice due to their intrinsic low circulating estradiol
levels. This pellet ensures the growth of the estrogenꢀdependent MCF7 cells. Seven days later, 5
x 10 cells were suspended in 1:1 Matrigel:DPBS and injected into the right rear flank of each
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animal. Mice were monitored for tumor growth every day after inoculation until tumors reached
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50ꢀ250 mm . Tumor growth took approximately 2ꢀ3 weeks (n = 12, 100% uptake).
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In Vivo IVIS ImagingXenograftedathymicnude mice were injected I.P. with 10 mg/kg of
complex1 dissolved in 100% DMSO (40 ꢁL injection volume). Images were acquired 2 (n =12),
4 (n =9), 24 (n = 6), and 48 (n = 3) hours postꢀinjection on anIVIS Spectrum (Perkin Elmer,
Waltham, MA, USA) using an excitation wavelength of 745 nm and emission of 810 nm. During
imaging, mice were held under 1ꢀ3 % inhaled isoflurane anesthesia. Mice were allowed to
recover between imaging time points. Images were processed using Living Image software
where ROI’s corresponding to the tumor and background signal intensity were used to determine
background subtracted radiant efficiency in the tumors.
In Vivo MR ImagingA subset of mice that underwent IVIS imaging were also imaged with MRI
immediately after the corresponding IVIS imaging session. Images were acquired at baseline
and 2 (n=2), 4 (n=4), and 24 (n=2) hours post injection on a 9.4T Bruker Biospec (Bruker
Biospin, Billerica, MA, USA) using a 38mm quadrature mouse body volume coil. T weighted
1
rapid spin echo (RARE) images were acquired with TR/TE=1500 ms / 4.9 ms, field of view 3.5
cm x 3.5 cm, matrix 128x128, 0.7 mm slice thickness, 19 slices, and 1 average. During imaging,
mice were held under 1ꢀ2 % inhaled isoflurane anesthesia and respiration was monitored using
an SA Instruments MR compatible monitoring system (SA Instruments, Stonybrook, NY, USA).
Mice were allowed to recover between imaging time points. Images were processed using JIM 6
software (Xinapse Systems, Essex, United Kingdom). Contrast to noise ratios were measured by
placing signal regions of interest in the paraspinal skeletal muscle and the bladder, and a noise
region in the corner of the image, subtracting muscle signal from bladder signal, and dividing by
the standard deviation of the noise.
Biodistribution Mice were injected I. P. with 10 mg/kg of complex 1. Organs were harvested 2,
4, 24, and 48 hours postꢀinjection (n = 3 per time point). Organs were imaged on the IVIS using
excitation of 745 nm and emission of 810 nm. Living Image software was used to draw ROIs
around each organ and calculate the average radiant efficiency.
Following ex vivo imaging, organs were digested and analyzed for Gd(III) content by ICPꢀMS.
The heart, lungs, ovaries, uterus, kidneys, muscle, and tumors were placed into preꢀweighed
Teflon tubes, weighed, and dissolved in 9: 1 ACS reagent grade nitric acid: hydrogen peroxide(1
mL for kidneys, 500 ꢁL for remaining organs). The solutions were digested using a EthosEZ
microwave digestion system (Milestone, Shelton, CT, USA) with a 120 °C ramp for 30 min
followed by a 30 min hold and a 45 min exhaust cycle. The livers and intestines were placed into
preꢀweighed TFM vessels, weighed, and dissolved in 9:1 ACS reagent grade nitric acid:
hydrogen peroxide (10 mL). The resultant solutions were weighed and an aliquot was transferred
to a preꢀweighed 15 mL conical tube. The final ICPꢀMS sample was prepared as described above
in the ICPꢀMS procedure.
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Acknowledgements
This work was supported by the National Institutes of Health (NIH grants R01EB005866 and
P01HL108795) and by a National Science Foundation Graduate Research Fellowship (C.C.).
Animal studies were performed with the assistance of the Center for Developmental
Therapeutics at Northwestern University. 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. Metal analysis was performed at
the Northwestern University Quantitative Bioelemental Imaging Center generously supported by
NASA Ames Research Center NNA06CB93G.
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Supporting Information Available: Synthetic procedures, r , cell viability, timeꢀdependent
uptake, fluorescence biodistribution. This information is available free of charge via the Internet
at http://pubs.acs.org.
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References
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