Cell labeling via membrane-anchored lipophilic MR contrast agents

Article abstract of DOI:10.1021/bc500083t

Cell tracking in vivo with MR imaging requires the development of contrast agents with increased sensitivity that effectively label and are retained by cells. Most clinically approved Gd(III)-based contrast agents require high incubation concentrations and prolonged incubation times for cellular internalization. Strategies to increase contrast agent permeability have included conjugating Gd(III) complexes to cell penetrating peptides, nanoparticles, and small molecules which have greatly improved cell labeling but have not resulted in improved cellular retention. To overcome these challenges, we have synthesized a series of lipophilic Gd(III)-based MR contrast agents that label cell membranes in vitro. Two of the agents were synthesized with a multiplexing strategy to contain three Gd(III) chelates (1 and 2) while the third contains a single Gd(III) chelate (3). These new agents exhibit significantly enhanced labeling and retention in HeLa and MDA-MB-231-mcherry cells compared to agents that are internalized by cells (4 and Prohance).

Full text of DOI:10.1021/bc500083t

Article
pubs.acs.org/bc
Cell Labeling via Membrane-Anchored Lipophilic MR Contrast
Agents
Christiane E. Carney, Keith W. MacRenaris, Daniel J. Mastarone, David R. Kasjanski, Andy H. Hung,
and Thomas J. Meade*
Department of Chemistry, Molecular Biosciences, Neurobiology, Biomedical Engineering, and Radiology, Northwestern University,
2145 Sheridan Road, Evanston, Illinois 60208-3113, United States
S
* Supporting Information
ABSTRACT: Cell tracking in vivo with MR imaging requires
the development of contrast agents with increased sensitivity
that effectively label and are retained by cells. Most clinically
approved Gd(III)-based contrast agents require high incuba-
tion concentrations and prolonged incubation times for
cellular internalization. Strategies to increase contrast agent
permeability have included conjugating Gd(III) complexes to
cell penetrating peptides, nanoparticles, and small molecules
which have greatly improved cell labeling but have not resulted
in improved cellular retention. To overcome these challenges, we have synthesized a series of lipophilic Gd(III)-based MR
contrast agents that label cell membranes in vitro. Two of the agents were synthesized with a multiplexing strategy to contain
three Gd(III) chelates (1 and 2) while the third contains a single Gd(III) chelate (3). These new agents exhibit significantly
enhanced labeling and retention in HeLa and MDA-MB-231-mcherry cells compared to agents that are internalized by cells (4
and Prohance).
intracellular Gd(III) resulting in decreased MR image contrast,
thereby prohibiting long-term longitudinal imaging studies.9
Furthermore, internalized agents are typically compartmental-
ized in endosomes that can lead to relaxivity quenching and
contrast agent degradation.19,20
Our strategy to overcome these challenges is the develop-
ment of cell membrane-anchored contrast agents that label cells
without the need for cellular internalization. Li et al. reported a
membrane-anchored MR contrast agent that labels cell at low
incubation concentrations with long-term imaging potential.21
Their strategy (modeled after lipophilic fluorescent dyes such
as DiI and DiO) involves conjugating alkyl chains to a Gd(III)
complex to serve as anchors in the cell membrane. Several
alternative strategies for synthesizing lipophilic contrast agents
with alkyl chains have been developed; however, these agents
have only been used to investigate relaxometric properties and
have not been tested in vitro.22−27
We propose the further development of lipophilic MR
contrast agents using multimeric cyclen-based chelates to
increase Gd(III) payload and therefore amplify MR signal. We
have previously reported the synthesis of a multimeric MR
contrast agent that uses copper(I)-catalyzed azide alkyne
cycloaddition (CuAAC) “click” chemistry to attach azide
modified chelates to an alkyne modified scaffold.28 This
strategy generates rigid triazole linkages that have been
INTRODUCTION
■
The ability to track cells in vivo will facilitate applications such
as detection of tissue rejection,1 fate mapping of transplanted
stem cells,2−4 and early detection of cancer.5 MR imaging is
ideally suited for long-term cell tracking since it is noninvasive,
possesses high spatial resolution and unlimited penetration
depth, and provides tomographic information without the use
of ionizing radiation.6 Intrinsic MR image contrast is enhanced
using paramagnetic contrast agents, typically iron oxide
nanoparticles or Gd(III) complexes.7 Iron oxide nanoparticles
generate contrast by reducing the transverse relaxation time
(T2) of surrounding water protons thereby producing negative
(dark) image contrast. Gd(III) contrast agents decrease the
proton spin−lattice relaxation time (T1) of surrounding water
protons and generate positive (bright) image contrast.8 Few
examples of cell tracking with Gd(III) contrast agents exist in
the literature because these agents usually efflux rapidly from
cells prohibiting long-term imaging studies.2,9−11
For long-term imaging and tracking of cells using Gd(III)-
based contrast agents it is necessary to develop probes that
effectively label and are retained by cells. Clinically approved
Gd(III)-based MR agents (such as Magnevist and Prohance)
are largely confined to the extracellular space and require high
incubation concentrations and long incubation times for
cellular internalization. Attempts to increase agent permeability
have included conjugating Gd(III) complexes to cell penetrat-
ing peptides,12−15 nanoparticles,16−18 polymers,2 and small
molecules.9 While these labeling strategies improve agent
uptake, rapid cellular efflux leads to significant reduction of
Received: February 28, 2014
Revised: April 22, 2014
Published: April 30, 2014
© 2014 American Chemical Society
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Figure 1. Three lipophilic and two nonlipophilic contrast agents are described. 1 and 2 are multimeric MR contrast agents that contain three Gd(III)
chelates while 3, 4, and Prohance are monomeric complexes. 4 and Prohance were selected as controls because they accumulate in the cytosol
whereas the lipophilic agents label the cell membrane.
shown to hinder the local rotation of Gd(III) complexes and
enhance relaxivity.29 Herein, we modify this scaffold to include
lipophilic alkyl chains to generate a series of contrast agents
that label the cell membrane with increased labeling efficiency,
retention, and MR image contrast compared to nonlipophilic
monomeric Gd(III) contrast agents (Prohance and an azide-
modified Gd(III) complex, 4).
increase solubility 25-fold from 28 μM (in 10% DMSO) to 700
μM. Relaxivity was measured in 4 mM cholate at 1.41 and 7 T,
where the ionic relaxivities (r1) of 1 and 2 were 16.5
0.1
mM−1 s−1 and 17.4
1.3 mM−1 s−1 at 1.41 T, respectively
(Table 1), and decrease to 4.00 0.15 mM−1 s−1 for 1 and 4.86
0.19 mM−1 s−1 for 2 at 7 T (Table S1). These relaxivities are
consistent with those obtained from a previously reported
contrast agent that was synthesized from a similar scaffold (15.4
0.8 mM−1 s−1 at 1.41 T).28
RESULTS AND DISCUSSION
■
Synthesis and Characterization of Water-Soluble
Lipophilic Complex. To increase water solubility we prepared
a monomeric lipophilic contrast agent (Scheme 2). Specifically,
1-azidotetradecane was reacted with an alkyne modified chelate
(12) in 2:1 tBuOH:H2O with CuSO4 and sodium ascorbate to
afford the final product (3).
The relaxivity of the resultant agent was measured in water
(17.6 0.7 mM−1 s−1 at 1.41 T, 4.32 0.10 mM−1 s−1 at 7 T)
and 4 mM cholate (16.4 2.9 7 mM−1 s−1 at 1.41 T, 5.57
0.12 mM−1 s−1 at 7 T) (Table 1, Table S1). These values are
approximately 6-fold higher at 1.41 T and 2-fold higher at 7 T
than the relaxivity of 12 which can be attributed to contrast
agent aggregation.29
Previously, we have described a strategy to generate high-
relaxivity, multimeric MR contrast agents with “click” chemistry
using a phenol-based scaffold functionalized with three alkyne
groups.28 The resultant agents are synthesized from this
scaffold by attaching an azide modified Gd(III) complex (4)
where the observed relaxivity increases 5-fold when attached to
the scaffold. Here, we describe conjugating alky chains to this
scaffold to generate cell membrane-anchored MR contrast
agents (Figure 1).
Synthesis and Characterization of Multimeric Com-
plexes. Multimeric lipophilic agents were synthesized
according to Scheme 1. Alkyl chains were attached to 2,4,6-
tribromophenol via a Mitsunobu reaction followed by a
Sonogashira reaction to introduce the TMS-protected alkyne
groups. TMS deprotection was carried out using potassium
fluoride and the final product was obtained by conjugating 4
onto the scaffold via “click” chemistry in 1:1 DCM:MeOH with
CuI and TBTA. Two complexes were synthesized using this
procedure: the first with a 14 carbon single alkyl chain (1) and
the second with two alkyl chains of 10 and 14 carbon units (2).
The resultant agents have limited water solubility prohibiting
accurate relaxivity measurements. Therefore, 1 and 2 were
solubilized in the detergent sodium cholate hydrate (4 mM) to
Lipophilic Contrast Agents Form Micelles and Lip-
osomes in Solution. The extent of agent aggregation and the
effect of cholate on micelle formation were determined using
dynamic light scattering (DLS). The data show that under the
conditions measured complexes 1, 2, and 3 form micelles with a
size distribution of 6
3 nm. Additionally, 1 and 2 form a
marginal population (≪0.1%) of liposomes 50−200 nm in size
(Table 2). Conversely, the control solutions of water, 4 mM
cholate, 4 in water and cholate, and Prohance in water and
cholate do not form light-scattering structures (Figure S22B).
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a
Scheme 1. Synthesis of Multimeric Lipophilic Contrast Agents That Contain a Single or Double Alkyl Chain
a
Agents were synthesized with “click” chemistry and contain three Gd(III) chelates. The resultant complexes have limited water solubility and were
solubilized in 4 mM cholate.
Table 1. Ionic Relaxivities of 1−4 and Prohance at 1.41 T and 37 °C in Water and 4 mM Cholate Show That Use of Cholate as a
Solubilizing Agent Does Not Significantly Impact Relaxivity
a
a
r1 in water (mM−1
r2 in water (mM−1
r2/r1 in
cholate
s−1
)
r1 in 4 mM cholate (mM−1 s−1
)
s−1
)
r2 in 4 mM cholate (mM−1 s−1
)
r2/r1 in water
,
,
1
ND
ND
16.5 0.1
17.4 1.3
16.4 2.9
2.97 0.31
2.61 0.16
ND
ND
26.5 0.3
30.4 1.4
18.4 2.7
3.50 0.61
2.98 0.25
ND
ND
1.24
1.19
0.96
1.61
1.75
1.21
1.18
1.00
2
3
17.6 0.7
2.98 0.07
2.60 0.08
21.8 0.7
3.56 0.05
2.86 0.08
4
Prohance
a
Complexes 1 and 2 are not water-soluble. ND= Not Determined.
These data suggest that the use of cholate (required for
solubilization of 1 and 2) does not significantly alter micelle
formation or size distribution.
Cell Labeling and Toxicity of Lipophilic Contrast
Agents. In order to optimize cell labeling, the incubation
conditions of 1−4 and Prohance in HeLa cells were determined
by quantifying toxicity (Figure S30, Table S2, Figure S31,
Figure S32), nonspecific binding (Figure S33), and the effects
of cholate (Figure S34, Figure S35) and incubation time on cell
labeling (Figure S36). Based on these data, all incubations were
prepared in 0.4 mM cholate for 24 h at concentrations of 1−4
and Prohance that maintain ≥90% cell viability. Concentration-
dependent cell labeling was determined by two approaches:
normalizing contrast agent or Gd(III) incubation concen-
trations. Data from studies where agent incubation concen-
tration was normalized show that multiplexed agents (1 and 2)
have increased cell labeling compared to the monomeric
contrast agent (3) (Figure 2A).
Specifically, HeLa cells incubated with 1 and 2 exhibited a
1.5-fold and 2.7-fold increase in cell labeling, respectively,
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a
Scheme 2. Synthesis of Monomeric Lipophilic Contrast Agent 3 Prepared with “Click”Chemistry
a
The agent is soluble in water and 4 mM cholate.
a
compared to 3. The labeling of lipophilic 1, 2, and 3 is 48-, 88-,
and 32-fold higher, respectively, than nonlipophilic 4 and
Prohance. Interestingly, data from studies where Gd(III)
incubation concentration is normalized (i.e., 3, 4, and Prohance
are present at 3× the agent concentration of 1 and 2) show that
at high incubation concentrations ([Gd(III)] > 75 μM)
complexes 2 and 3 label cells approximately 2-fold more
effectively than 1 (∼6 fmol Gd(III)/cell versus ∼3 fmol
Gd(III)/cell, Figure 2B). This is likely due to the two alkyl
chains present on 2 increasing cell membrane affinity
(compared to one alkyl chain for 1), whereas the increased
labeling of 3 is likely due to higher packing density in the cell
membrane since 3 has less steric bulk than multimeric 1 and 2.
To ensure that cell labeling is independent of cell line
selection, agent labeling was determined using MDA-MB-231-
mcherry cells at both normalized agent and Gd(III) incubation
concentrations (Figure 2C and D). Multimeric 1 and 2 showed
Table 2. Summary of DLS Size Measurements
1 (cholate)
2 (cholate)
3 (cholate)
3 (water)
Cumulant Analysis
b
d
d
Z-average
(nm)
49.9 1.5
82.3 2.6
5.8 0.3
7.1 0.2
PDI
0.67 0.02
0.41 0.07
0.25 0.03
0.29 0.01
Distribution Analysis
6.0 1.6
Peak 1
c
5.9 2.4
117 69
6.1 2.5
-
6.7 2.0
-
(nm)
Peak 2
133 66
c
(nm)
a
Data was analyzed by autocorrelation as fitted by cumulant and
distribution analysis for 1−3 (see Supporting Information for a more
b
detailed description of the fitting).
represents standard deviation
c
of z-average and reflects reproducibility of the fit (N = 3).
represents standard deviation of size distribution based on intensity
and reflects variance in size (N = 3). Low expected accuracy due to
d
poor fitting.
Figure 2. Concentration-dependent cell labeling for 1−4 and Prohance. HeLa cells incubated with normalized contrast agent (A) or normalized
Gd(III) concentrations (B) for 24 h. MDA-MB-231-mcherry cells incubated with normalized contrast agent (C) or normalized Gd(III)
concentrations (D) for 24 h. Error bars represent standard deviation of the mean of triplicate samples.
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a 2.3- and 2.4-fold reduction in Gd(III) per cell, respectively,
compared to the values measured in HeLa cells. 3 showed the
same Gd(III) per cell in both cell lines suggesting it may be a
more universal cell labeling agent.
MR Imaging of Cells Labeled with Lipophilic Contrast
Agents. To evaluate contrast enhancement of HeLa cells
labeled with lipophilic contrast agents 1−3, MR images of cell
pellets were acquired at 7 T. HeLa cells were incubated with
either 20 μM contrast agent (Figure 3A) or 90 μM normalized
Figure 4. Localization of contrast agents determined by cell
fractionation. The internalized (cytosol + endosomes) and membrane
fraction were analyzed by ICP-MS for Gd(III) content. All lipophilic
complexes show higher membrane accumulation than Prohance with 2
having the greatest membrane localization. Error bars represent
standard deviation of the mean of triplicate samples.
1 and 3, with single alkyl chains, contained 5.4- and 3.2-fold
more Gd(III) in the membrane, respectively. The only complex
internalized into cells was Prohance. Since the lipophilic
complexes preferentially label cell membranes, we tested
whether this membrane anchoring translated to long-term
Gd(III) retention compared to internalized contrast agents.
Cellular Proliferation and Retention of Lipophilic
Agents. Cellular proliferation and retention of the agents
was investigated by incubating HeLa, MDA, MB-213-mcherry,
and NIH/3T3 cells with varying concentrations of 1−3 and
Prohance (normalizing Gd(III) per cell). Following incubation,
labeled cells were replaced in contrast agent free media (time =
0) and allowed to proliferate for 72 h (media was replaced
every 24 h). Cell count and Gd(III) content was determined at
time = 0 and 72 h. Cells incubated with the lipophilic contrast
agents showed similar proliferative ability compared to cells
incubated with Prohance (Figure 5A).
Cellular retention of the contrast agents was determined by
calculating the fold change in cellular Gd(III) content between
time = 0 and 72 h. Complexes 1 and 2 showed similar retention
in all cell lines with 10−20-fold of the label diluted after 72 h
(Figure 5B). These retention values represent a 10-, 6-, and 3.2-
fold improvement over the retention of Prohance in HeLa,
MDA-MB-231-mcherry, and NIH/3T3 cells, respectively. The
cellular retention of 3 was highly cell line dependent with 36-
fold of the label diluted in HeLa cells, 105-fold in MDA-MB-
231-mcherry cells, and 1000-fold in NIH/3T3 cells. In all cases,
these values represent lower cellular retention compared to
multimeric 1 and 2 indicating that the increased water solubility
of 3 may reduce contrast agent retention.
Figure 3. T1-weighted cell pellet images of HeLa cells incubated with
1−4 and Prohance acquired at 7 T. A: Probe concentration was
equalized at 20 μM. B: Gd(III) concentration was equalized at 90 μM.
For both images TE = 11 ms, TR = 500 ms, MTX = 256 × 256, and
slice thickness is 1.0 mm. Scale bars represent 1 mm. Error represents
1 standard deviation of the mean of four 1-mm slices. These images
show that 3 produces the most significant contrast enhancement
compared to untreated cells.
Gd(III) (Figure 3B). For both studies, lipophilic complexes 1−
3 reduced the T1 relaxation time (and thereby increased MR
image contrast) compared to untreated cells more effectively
than 4 or Prohance.
Interestingly, complex 3 produced the greatest reduction in
T1 relaxation time and therefore the highest image contrast for
both studies (Figure 3, T2 data is in Figure S32). Since the
Gd(III) concentration per cell is lower for 3 in both
experiments, the enhanced image contrast observed is likely
due to increased relaxivity when embedded in the cell
membrane or 1 and 2 being limited as T1 contrast agents by
their higher r2/r1 ratios (approximately 2.8-fold higher than 3 at
7 T, Table S1).
Lipophilic Contrast Agents Localized in the Cell
Membrane. To examine the cellular distribution of lipophilic
1−3 and Prohance, HeLa cells were incubated with varying
concentrations of each agent (concentrations were chosen to
normalize Gd(III) labeling) and were subjected to a cell
fractionation kit. The membrane and internalized (cytosol +
endosomes) fractions were collected and analyzed for Gd(III)
content by ICP-MS. 2 (possessing two alkyl chains) displayed
the highest membrane affinity with 18-fold increased Gd(III)
present in the membrane compared to the internalized fraction
(Figure 4).
Surprisingly, the retention of 3 in MDA-MB-231-mcherry
and NIH/3T3 cells is lower than that of Prohance suggesting
that 3 may not be ideal for long-term cell tracking studies.
These results demonstrate the cell line dependence of cellular
retention and show that this property must be measured in any
new cell line before long-term cell tracking experiments are
performed.
CONCLUSIONS
■
We have developed a series of lipophilic MRI contrast agents
that efficiently label cell membranes in vitro. Two multimeric
agents were synthesized to increase Gd(III) payload per
complex (1 and 2). These agents were found to be insoluble in
water, which necessitated the use of cholate (a detergent) for
solubilizaion to allow biological testing. In order to overcome
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Figure 5. Cellular proliferation and retention of contrast agents 1−3 and Prohance after 72 h of leaching in HeLa, MDA-MB-231-mcherry, and
NIH/3T3 cell lines. A: Cellular proliferation was determined by calculating the fold difference in cell count at t = 0 and 72 h. The data show that
proliferation is not affected by labeling with lipophilic contrast agents. B: Cellular retention was determined by calculating the fold change in Gd(III)
per cell at t = 72 and 0 h. The data show that, while retention is influenced by cell line selection, 1 and 2 have consistently enhanced retention
compared to 3 and Prohance.
solubility constraints, a water-soluble lipophilic contrast agent
was synthesized using a single Gd(III) chelate (3).
Standard grade 60 Å 230−400 mesh silica gel (Sorbent
Technologies, Norcross, GA, USA) was used for flash
1
chromatography. H and 13C NMR spectra were obtained on
All three lipophilic contrast agents showed increased labeling
in HeLa and MDA-MB-231-mcherry cells compared to contrast
agents that are internalized by cells (4 and Prohance) without
significantly affecting cell viability or proliferation. In studies
where contrast agent incubation concentrations were normal-
ized, 1 and 2 effectively delivered the most Gd(III) to cells;
however, in studies where Gd(III) incubation concentrations
were normalized 2 and 3 were the most effective agents.
Additionally, at 7 T cells incubated with 3 show the greatest
reduction in T1 and therefore the most significant contrast
enhancement at both normalized contrast agent and Gd(III)
incubation concentrations. The surprising efficacy of 3 is
attributed to higher packing in the cell membrane since it has
less steric bulk than 1 and 2. Multimeric 1 and 2 had
significantly enhanced cellular retention compared to Prohance
in HeLa, MDA-MB-231-mcherry, and NIH/3T3 cells. Com-
plex 3 showed decreased cellular retention compared to
Prohance in MDA-MB-231 and NIH/3T3 cells, which will
likely minimize its utility as a long-term cell tracking agent.
Future work on this design will include modifying the aliphatic
chain length and branching to increase complex hydrophobicity
which should translate to improved cellular retention without
sacrificing the significant contrast enhancement produced at
high field strengths.
a Bruker 500 MHz Avance III NMR spectrometer (Billerica,
MA, USA). Electrospray ionization mass spectroscopy (ESI-
MS) spectra were taken on a Varian 1200 L single quadrupole
mass spectrometer Agilent Technologies, Santa Clara, CA,
USA). Matrix assisted laser desorption ionization mass
spectroscopy (MALDI-MS) were aquired on a Bruker Autoflex
III MALDI. Analytical reverse-phase HPLC-MS was performed
on an Agilent 1200 series system (Agilent Technologies, Santa
Clara, CA, USA) using a Phenomenex (Torrance, CA, USA)
Luna C8 column (4.6 × 50, 5 μm). This system is equipped
with an Agilent G1315C DAD detector and an Agilent 6130
quadrupole MS detector (Agilent Technologies, Santa Clara,
CA, USA). Preparative runs were performed on a Phenomenex
(Torrance, CA, USA) Luna C8 column (21.20 × 150, 5 μm)
with a mobile phase of water (A) and HPLC-grade acetonitrile
(B).
Azide modified Gd(III)-chelate (4),28 1-azidotetradecane
(11),30 and alkyne modified Gd(III)-chelate (12)29 were
synthesized according to literature procedures.
(5) 1,3,5-Tribromo-2-(tetradecyloxy)benzene. To a
solution of tribromophenol (0.8980 g, 2.721 mmol) in DCM
(18 mL) was added 1-tetradecanol (0.5953 g, 2.777 mmol) and
PPh3 (1.0781 g, 4.115 mmol). The reaction was placed under a
nitrogen atmosphere and cooled to 0 °C. DIAD (0.80 mL,
4.067 mmol) was added dropwise over 2 min. The solution
became yellow during the addition and a yellow suspension
formed while the reaction was stirred at 0 °C over 15 min. The
reaction was removed from the ice-bath and allowed to warm to
room temperature. The reaction was stirred for 12 h and
concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel (hexanes) and the product was
isolated as a white solid (1.1611 g, 81%). 1H NMR (500 MHz,
CDCl3) δ 7.64 (s, 2H, -CHar−CBr−CHar-), 3.97 (t, J = 6.6 Hz,
2H, -OCH2-), 1.85 (dt, J = 14.9, 6.7 Hz, 2H, -OCHH2-CH2-),
1.51 (p, J = 7.3 Hz, 2H, -OCHH2−CH2−CH2-), 1.40−1.22 (m,
20H, -CH2−(CH2)10−CH3), 0.88 (t, J = 6.9 Hz, 3H, -(CH2)13-
CH3). 13C NMR (126 MHz, CDCl3) δ 153.06 (CarH−
OCHH2), 134.98 (CarH−CBr−CarH), 119.13 (CarBr−Car−
CarBr), 117.11 (CarH−CarBr−CarH-), 73.77 (-OCH2-), 31.96
(-CH2−CH2−CH3), 30.00 (CH2−(CH2)2−CH3), 29.70
The high cell labeling and retention of complex 2 may be
well suited for in vivo applications such as fate mapping and
stem cell tracking that will require highly efficient membrane-
anchored contrast agents to combat the decrease in MR image
contrast that arises from agent efflux and endosomal entrap-
ment of intracellular contrast agents.
MATERIALS AND METHODS
■
Synthetic Methods. Unless noted, materials and solvents
were purchased from Sigma-Aldrich Chemical Co. (St. Louis,
MO, USA) and used without further purification. All reactions
were performed under an inert nitrogen atmosphere.
Acetonitrile, triethylamine, and dichloromethane were purified
using a Glass Contour solvent system. Deionized water was
obtained from a Millipore Q-Guard System equipped with a
quantum Ex cartridge (Billerica, MA, USA). Thin layer
chromatography (TLC) was performed on EMD 60F 254
silica plates and stained either with iodine or with UV light.
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((CH2)7-(CH2)3-CH3), 29.41 (-OCH2−CH2), 25.86 (-OCH2−
CH2−CH2-), 22.74 (CH2−CH3), 14.18 (CH2−CH3).
through the reaction for 5 min. Trimethylsilyl acetylene (3.5
mL, 26.27 mmol) was added and nitrogen gas was bubbled
through the reaction for 5 min. PdCl2(PPh3)2 (0.1803 g, 0.2569
mmol) was added to the reaction and it was left under nitrogen
at 70 °C for 18 h. The mixture was concentrated in vacuo and
filtered with hexanes. The resulting orange residue was purified
by flash-column chromatography on silica gel (35:1 hexane-
s:ethyl acetate) and the product was isolated as an orange oil
(6) 1,3,5-Tribromo-2-((2-decyltetradecyl)oxy)benzene.
To a solution of tribromophenol (1.0239 g, 3.102 mmol) in
DCM (18 mL) was added 2-decyltetradecan-1-ol (1.3 mL,
3.085 mmol) and PPh3 (1.2194 g, 4.654 mmol). The reaction
was placed under an inert nitrogen atmosphere and cooled to 0
°C. DIAD (0.90 mL, 4.576 mmol) was added dropwise over 2
min. The solution became yellow during the addition and a
yellow suspension formed while the reaction was stirred at 0 °C
over 15 min. The reaction was removed from the ice-bath and
allowed to warm to room temperature. The reaction was stirred
for 12 h and concentrated in vacuo. The residue was purified by
flash-column chromatography on silica gel (hexanes) and the
1
(1.6686 g, 92%). H NMR (CDCl3, 500 MHz) δ 7.28 (s, 2H,
-CHar−Car−CHar-), 4.00 (d, 2H, J = 5.7 Hz, -OCHH2-), 1.31−
1.39 (m, 5H, CH2-CH−CH2-), 1.15−1.23 (m, 6H, CH2−
CH2−CH−CH2−(CH2)2-)), 1.06 (s, 30H, -(CH2)9-CH3,
-(CH2)6-CH3), 0.69 (t, 6H, J = 6.9 Hz, 2 CH3), 0.04 (s,
27H, 3 -Si-(CH3)3). 13C NMR (125 MHz, CdCl3) δ 162.21
(-CarOCH2-), 137.84 (-CarH-Car-CarH-), 117.76 (-C-Si-
(CH3)3), 116.19 (-C-Si-(CH3)3), 103.18 (-Car-CarO,Car-),
100.42 (CarH-Car-CarH-), 99.64 (CalkyneC-Si-(CH3)3), 94.30
(-C-Si-(CH3)3), 88.10 64 (-CalkyneC-Si-(CH3)3), 86.08
(-CalkyneC-Si-(CH3)3), 39.37 (−-CH2−CH2‑), 32.10 (2 -CH2−
CH2−CH3), 31.18 (-CH2−CH−CH2-), 30.28 (2 -CH-
(CH2)2CH2-), 29.87 ((CH2)3-(CH2)3-CH-(CH2)3-(CH2)5-),
29.54 (2 -CH2-(CH2)2-CH3), 27.16 (2 -CH-(CH2)2-CH2-),
22.87 (2 -CH2−CH3) 0.00 (3 -Si-(CH3)3). ESI-MS m/z [M +
Na]+ calcd. for C45H78OSi3Na 741.5; observed 741.6.
1
product was isolated as a clear oil (1.6878 g, 82%). H NMR
(500 MHz, CDCl3) δ 7.63 (s, 2H, -CHar−Car−CHar-), 3.86 (d,
2H, J = 5.7 Hz, -OCHH2-), 1.83−1.89 (m, 1H, OCH2-CH-),
1.52−1.56 (m, 4H, CH2−CH−CH2-), 1.34−1.46 (m, 6H,
CH2−CH2−CH−CH2−(CH2)2-), 1.22−1.45 (m, 30H, CH3−
(CH2)6−(CH2)3−CH−(CH2)2−(CH2)9-), 0.89 (t, 6H, J = 6.9
Hz, 2 CH3). 13C NMR (126 MHz, CDCl3) δ 153.20
(-CarOCH2-), 135.16 (-CarH−CBr−CarH-), 119.26 (-CarBr−
Car−CarBr-), 117.16 (-CarH−CarBr−CarH-), 76.42 (-OCH2-),
39.14 (-OCH2−CH-), 32.12 (2 CH2−CH2−CH3), 31.16 (2
-CH−CH2-), 30.20 (2 -CH−(CH2)2−CH2-), 29.87 ((CH2)5-
(CH2)3-CH3 and ((CH2)3-(CH2)3-CH3), 29.56 (2 CH2-
(CH2)2-CH3), 27.10 (2 CH−CH2−CH2-), 22.89 (2 CH2−
CH3), 14.32 (2 -CH3).
(9) 1,3,5-Triethynyl-2-(tetradecyloxy)benzene. To a
solution of 7 (0.8214 g, 1.419 mmol) in DCM:methanol (50
mL DCM, 20 mL methanol) was added KF (1.0214 g, 17.58
mmol) and the mixture was stirred for 24 h at 30 °C. The
mixture was concentrated in vacuo, taken up in water, and
extracted with diethyl ether (30 mL × 3). The organic layer was
dried with MgSO4 and concentrated in vacuo. The mixture was
purified by flash-column chromatography on silica gel (19:1
hexanes:ethyl acetate) to give the product as an orange solid
(7) ((2-(Tetradecyloxy)benzene-1,3,5-triyl)tris(ethyne-
2,1-diyl))tris(trimethylsilane). To a flame-dried flask was
added PPh3 (0.1836 g, 0.7007 mmol) and CuI (0.0882 g,
0.4631 mmol). Triethylamine (30 mL) was used to dissolve 5
(1.1611 g, 2.203 mmol) and the resulting solution was added to
the reaction flask. Nitrogen gas was bubbled through the
reaction for 5 min. Trimethylsilyl acetylene (3.1 mL, 22.08
mmol) was added and nitrogen gas was again bubbled through
the reaction for 5 min. PdCl2(PPh3)2 (0.1567 g, 0.2232 mmol)
was added to the reaction and left under nitrogen at 70 °C for
18 h. The mixture was concentrated in vacuo and filtered with
hexanes. The resulting orange residue was purified by flash-
column chromatography on silica gel (50:1 hexanes:ethyl
acetate) and the product was isolated as an orange oil (0.8214
1
(0.4112 g, 80%). H NMR (CDCl3, 500 MHz,) δ 7.55 (s, 2H,
-CHar−Car−CHar-), 4.24 (t, J = 6.6 Hz, 2H, -OCH2-), 3.27 (s,
2H, 2 CHalkyne), 3.03 (s, 1H, CHalkyne), 1.80 (dt, J = 14.7, 6.7 Hz,
2H, -OCHH2−CH2-), 1.52−1.43 (m, 2H, -OCHH2−CH2−
CH2-), 1.40−1.21 (m, 20H, -OCHH2−(CH2)2−(CH2)10-),
0.88 (t, J = 6.9 Hz, 3H, −CH3). 13C NMR (126 MHz, CDCl3)
δ 162.59 (-CarOCH2-), 138.12 (-CarH-Car-CarH-), 117.30 (Car-
CCHalkyne), 116.95 (Car-CarO-Car-), 82.34 (2 -C-CHalkyne),
81.39 (-C-CHalkyne), 78.60 (2 -C-CHalkyne), 77.62 (-C−
CHalkyne), 74.70 (-OCH2-), 31.92 (-CH2−CH2−CH3), 30.20
(-CH2-(CH2)2-CH3), 29.69 (-(CH2)7-(CH2)3-CH3), 29.40
(-OCH2−CH2-), 25.82 (-OCH2−CH2−CH2-), 22.70 (-CH2−
CH3), 14.14 (CH3). ESI-MS m/z [M + H]+ calcd. for C26H36O
363.3; observed 363.3.
1
g, 64%). H NMR (500 MHz, CDCl3) δ 7.26 (s, 2H, -CHar−
CBr−CHar-), 4.01 (t, J = 6.5 Hz, 2H, -OCH2-), 1.63−1.52 (m,
2H, -OCHH2-CH2-) 1.33−1.24 (m, 2H, -OCHH2−CH2−CH2-
), 1.19−0.98 (m, 20H, -CH2−(CH2)10−CH3), 0.67 (t, J = 6.9
Hz, 3H, -(CH2)13-CH3), 0.02 (s, 27H, -Si(CH3)3). 13C NMR
(126 MHz, CDCl3) δ 162.09 (-CarOCH2-), 137.47 (-CarH−
Car−CarH-), 133.82 (-C-Si-(CH3)3), 128.63 (-C-Si-(CH3)3),
118.32 (-C-Si-(CH3)3), 117.78 (Car−CarO−Car-), 103.16
(CarH-Car-CarH-), 100.11 (CalkyneC−Si−(CH3)3), 99.69
(CalkyneC−Si−(CH3)3), 94.51 (CalkyneC−Si−(CH3)3), 74.52
(-OCH2-), 32.11 (−CH2−CH2−CH3), 30.58 (CH2−(CH2)2−
CH3), 29.86 ((CH2)7-(CH2)3-CH3), 29.55 (-OCH2−CH2-),
26.25 (-OCH2−CH2−CH2-), 22.88 (-CH2−CH3), 14.33
(CH3), 0.06 (3 -Si-(CH3)3). ESI-MS m/z [M + Na]+ calcd.
for C35H58OSi3Na 601.4; observed 601.4.
(10) 2-((2-Decyltetradecyl)oxy)-1,3,5-triethynylben-
zene. To a solution of 8 (1.6686 g, 2.323 mmol) in
DCM:methanol (50 mL DCM, 20 mL methanol) was added
KF (1.8041 g, 31.05 mmol) and the mixture was stirred 24 h at
30 °C. The mixture was concentrated in vacuo, brought up in
water, and extracted with diethyl ether (30 mL × 3). The
organic layer was dried with MgSO4 and concentrated in vacuo.
The mixture was purified by flash-column chromatography on
silica gel (19:1 hexanes:ethyl acetate) to give the product as an
1
orange oil (0.8928 g, 77%). H NMR (500 MHz, CDCl3) δ
(8) ((2-((2-Decyltetradecyl)oxy)benzene-1,3,5-triyl)-
tris(ethyne-2,1-diyl))tris(trimethylsilane). To a flame-
dried flask was added PPh3 (0.2091 g, 0.7981 mmol) and
CuI (0.1026 g, 0.5388 mmol). Triethylamine (30 mL) was used
to dissolve 6 (1.6878 g, 2.530 mmol) and the resulting solution
was added to the reaction flask. Nitrogen gas was bubbled
7.55 (s, 2H, -CHar-Car-CHar-), 4.15 (d, J = 5.4 Hz, 2H,
-OCHH2-), 3.25 (s, 2H, 2 CHalkyne), 3.03 (s, 1H, CHalkyne),
1.72−1.79 (m, 1H, -OCHH2−CH-), 1.50−1.58 (m, 6H, -CH2−
CH2−CH−CH2−(CH2)2-), 1.26−1.40 (m, 34H, -CH−CH2−
CH2−(CH2)9, -CH−CH2−(CH2)2−(CH2)6-), 0.88 (t, J = 6.9
Hz, 6H, 2 CH3). 13C NMR (126 MHz, CDCl3) δ 162.95
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(-CarOCH2-), 138.39 (-CarH-Car-CarH-), 117.24 (-Car-
CCHalkyne), 116.86 (Car-CarO-Car-), 82.51 (2 -C-CHalkyne),
81.58 (-C-CHalkyne), 78.92 (2 -C-CHalkyne), 77.70 (-C−
CHalkyne), 77.26 (-OCH2-), 39.25 (-OCH2−CH-), 32.09 (2
CH2−CH2−CH3), 31.20 (2 -CH-CH2-), 30.19 (2 -CH-(CH2)2-
CH2-), 29.86 (-(CH2)5-(CH2)3-CH3, -(CH2)3-(CH2)3-CH3),
29.53 (2 -CH2−(CH2)2−CH3), 27.09 (2 -CH−CH2−CH2-),
22.86 (2 -CH2−CH3), 14.30 (2 CH3). ESI-MS m/z [M + H]+
calcd. for C36H55O 503.4, observed 503.4.
T) and 37 °C using an inversion recovery pulse sequence on a
Bruker mq60 minispec NMR spectrometer with 4 averages, 15
s repetition time, and 10 data points (Bruker Canada; Milton,
Ontario, Canada). The Gd(III) concentration of each solution
was determined using inductively coupled plasma-mass
spectrometer (ICP-MS). The inverse of the longitudinal
relaxation time (1/T1, s−1) was plotted against Gd(III)
concentration (mM) and fitted to a straight line with R2 >
0.99. The slope of the fitted line was recorded as the relaxivity,
r1.
(1) 5-(2,4,6-Tris(1-(2-hydroxy-3-(1H-1,2,3-triazol-1-yl)-
propyl)-4,7,10-tris(carboxymethyl)-1,4,7,10-
tetraazacyclododecylgadolinium(III))phenoxy) tetradec-
yl. To a solution of 9 (30.20 mg, 0.0637 mmol) in 1:1
DCM:MeOH (4 mL each) was added 4 (110.45 mg, 0.1841
mmol), CuI (3.15 mg, 0.1654 mmol), and TBTA (3.90 mg,
0.00735 mmol). The mixture was heated to 40 °C until the
reaction was shown to be complete by MALDI-TOF-MS
(typically 3−5 days). The mixture was concentrated in vacuo
and diluted with ice water. The resulting solution was
centrifuged and decanted. The solid was washed with MeCN
to afford the desired product as a white powder (69.1 mg,
50%). Purity was confirmed with analytical HPLC-MS using a
C8 column eluting with a gradient of 10−70% acetonitrile in
water over 30 min, tr = 20.5 min. MALDI-TOF-MS m/z [M +
H]+ calcd. for C77H119Gd3N21O22 2161.646; observed
2161.595. Anal. Calcd (%) for C77H130Gd3N21Na6O28: C,
38.41; H, 5.44; N, 12.22. Found: C, 38.42; H, 6.12; N, 11.60.
(2) 5-(2,4,6-Tris(1-(2-hydroxy-3-(1H-1,2,3-triazol-1-yl)-
propyl)-4,7,10-tris(carboxymethyl)-1,4,7,10-
tetraazacyclododecylgadolinium(III))phenoxy) 2-decyl-
tetradecyl. To a solution of 10 (13.69 mg, 0.0378 mmol) in
1:1 DCM:MeOH (4 mL each) was added 4 (75.52 mg, 0.1259
mmol), CuI (1.63 mg, 0.0086 mmol), and TBTA (2.34 mg,
0.0044 mmol). The mixture was heated to 40 °C until the
reaction was shown to be complete by MALDI-TOF-MS
(typically 3−5 days). The mixture was concentrated in vacuo
and diluted with ice water. The resulting solution was
centrifuged and decanted. The solid was washed with MeCN
to afford the desired product as a white powder (42.4 mg,
51%). Purity was confirmed with analytical HPLC-MS using a
C8 column eluting with a gradient of 10−70% acetonitrile in
water over 30 min, tr = 31.8 min. MALDI-TOF-MS m/z [M +
H]+ calcd. for C87H139Gd3N21O22 2302.912, observed 2303.322.
Anal. Calcd (%) for C87H150Cl2Gd3N21Na2O28: C, 41.35; H,
5.98; N, 11.64. Found: C, 41.54; H, 6.64; N, 11.17.
Dynamic Light Scattering (DLS). Samples for DLS were
prepared at 1.2 mM Gd(III) concentration and filtered by 0.2
μm filters. Data was acquired on a Malvern Instruments
Zetasizer Nano Series Nano-ZS equipped with Dispersion
Technology Software v 5.03 (Worcestershire, United King-
dom). Measurements were performed in triplicate at room
temperature in SARSTEDT clear polystyrene 10 × 10 × 45
mm3 cuvettes. Data analysis was performed on Zetasizer
Software v 7.02 (Worcestershire, United Kingdom).
General Cell Culture Methods. Dulbecco’s modified
phosphate buffered saline (DPBS), media, and dissociation
reagents were purchased from Life Technologies (Carlsbad,
CA). CorningBrand cell culture consumables (flasks, plates,
etc.) and sera were purchased from VWR Scientific (Radnor,
PA). HeLa cells (ATCC CCL-2) and NIH/3T3 cells (ATCC
CRL-1658) were purchased from the American Type Culture
Collection (Manassas, VA). MDA-MB-231-mcherry cells were
a gift of Northwestern’s Developmental Therapeutics Core.
Both HeLa and MDA-MB-231-mcherry cells were cultured in
phenol red free minimum essential media (MEM) supple-
mented with 10% fetal bovine serum (FBS). NIH/3T3 cells
were cultured in Phenol Red free Dulbecco’s modified eagle
medium (DMEM) supplemented with 10% calf bovine serum.
Prior to all experiments, cells were plated and allowed to
incubate for 24 h before dosing. Cells were harvested with
0.25% TrypLE for 10 min at 37 °C in a 5.0% CO2 incubator.
All doses were filtered with 0.2 μm sterile filters prior to
administration. Cells were grown in a humidified incubator
operating at 37 °C and 5.0% CO2 unless otherwise specified.
Cell Counting. Cell counting was accomplished using a
Guava EasyCyte Mini Personal Cell Analyzer (EMD Millipore,
Billerica, MA). After cell harvesting, an aliquot (50 μL) of the
cell suspensions was mixed with Guava ViaCount reagent (150
μL) and allowed to stain at room temperature for 5 min.
Stained samples were vortexed for 10 s and then cells were
counted using a Guava EasyCyte Mini Personal Cell Analyzer
(PCA) using the ViaCount software module. For each sample,
1000 events were acquired with dilution factors that were
determined based upon optimum machine performance (∼20−
70 cells/μL). Instrument reproducibility was assessed daily
using GuavaCheck Beads and following the manufacturer’s
suggested protocol using the Daily Check software module.
Cellular Labeling Studies. Cell labeling studies were
performed with either HeLa or MDA-MB-231-mcherry cells.
Specifically 20,000−25,000 cells were plated in each well of a
24-well plate. For concentration-dependent labeling studies,
complexes 1−4 and Prohance were dissolved in 0.4 mM
cholate and media at concentrations of 120, 60, 30, and 15 μM
of Gd(III) and incubated with cells for 24 h (180 μL dose). For
time-dependent labeling studies, cells were incubated with 50
μM of Gd(III) for 1, 2, 4, 8, and 24 h. Cells were rinsed twice
with 500 μL 0.4 mM cholate PBS and trypsinized following
contrast agent incubation and pelleted at 1000g for 5 min at 4
(3) Gd(III)-1,4,7-tris(carboxymethyl)-2,2′,2″-(10-(2-
oxo-2-(((1-tetradecyl-1H-1,2,3-triazol-4-yl)methyl)-
amino)ethyl)-1,4,7,10-tetraazacyclododecane. To a sol-
ution of 11 (40.8 mg, 0.1707 mmol) in 2:1 tBuOH:H2O (10
mL: 5 mL) was added 12 (109.7 mg, 0.1844 mmol), CuSO4
(4.90 mg, 0.0309 mmol) and sodium ascorbate (40.1 mg,
0.2024 mmol). The mixture was heated at 60 °C for 2 days.
The crude mixture was purified using reverse phase semi-
preparative HPLC with a C8 column eluting with 35−100%
acetonitrile over 8 min, tr = 4.4 min. The desired product was
collected as a white powder (43.4 mg, 26%). ESI HRMS m/z
[M + H]+ calcd. for 836.3669, observed 836.3684. Anal. Calcd.
(%) for C33H61Cl2GdN8Na2O9: C, 41.35; H, 5.98; N, 11.64.
Found: C, 41.54; H, 6.64; N, 11.17.
Relaxivity (r1). Solutions of 1 and 2 were prepared in 500
μL of 4 mM cholate solution for T1 acquisition. Solutions of 3
were prepared in both 4 mM cholate and Millipore H2O. T1
(spin−lattice relaxation time) was determined at 60 MHz (1.41
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Article
°C. The media was aspirated off and the cell pellet was
resuspended in 200 μL of media. 50 μL of the cell suspension
was used for cell counting and 130 μL was used for Gd(III)
content analysis via ICP-MS.
Instrument performance is optimized daily through an autotune
followed by verification via a performance report (passing
manufacturer specifications). Instrument calibration was
accomplished by preparing individual-element Gd(III) stand-
ards (Inorganic Ventures, Christiansburg, VA, USA) using
concentrations of 0.78125, 1.5625, 3.125, 6.25, 12.50, 25.00,
50.00, 100.0, and 200.0 ng/mL containing 3.0% nitric acid (v/
v) and 5.0 ng/mL of multi-element internal standard.
Cell Pellet MR Imaging. MR imaging and T1/T2
measurements were performed on a Bruker Pharmscan 7 T
imaging spectrometer fitted with shielded gradient coils at 25
°C. For cell pellet images, ∼5 × 105 HeLa cells were incubated
in 25 cm2 T-flasks with complexes 1−4 and Prohance for 24 h,
rinsed with DPBS (2 × 1 mL/flask), and harvested with 500 μL
of trypsin. After addition of 500 μL of fresh complete media,
cells were transferred to 1.5 mL microcentrifuge tubes and
centrifuged at 1000 × g at 4.0 °C for 5 min. The supernatant
was removed; the cell pellets were resuspended in 1 mL of
complete media, added to 53/4″ flame-sealed Pasteur pipets,
and centrifuged at 100 × g at 4.0 °C for 5 min. The bottom
sections of the flame-sealed pipets were then scored with a glass
scribe, broken into small capillaries, and imaged using a RF RES
300 1H 089/023 quadrature transmit receive 23 mm volume
coil (Bruker BioSpin, Billerica, MA, USA). Solution phantoms
were prepared, imaged, and analyzed as previously described
using serially diluted solutions.
Spin−lattice relaxation times (T1) were measured using a
rapid-acquisition rapid-echo (RARE-VTR) T1-map pulse
sequence, with static TE (11 ms) and variable TR (150, 250,
500, 750, 1000, 2000, 4000, 6000, 8000, and 10000 ms) values.
Imaging parameters were as follows: field of view (FOV) = 25
× 25 mm2, matrix size (MTX) = 256 × 256, number of axial
slices = 4, slice thickness (SI) = 1.0 mm, and averages (NEX) =
3 (total scan time = 2 h 36 min). T1 analysis was carried out
using the image sequence analysis tool in Paravision 5.0 pl3
software (Bruker, Billerica, MA, USA) with monoexponential
curve-fitting of image intensities of selected regions of interest
(ROIs) for each axial slice.
Spin−spin relaxation times (T2) were measured using a
multislice multiecho (MSME) T2-map pulse sequence, with
static TR (5000 ms) and 32 fitted echoes in 11 ms intervals
(11, 22, ···, 352 ms). Imaging parameters were as follows: field
of view (FOV) = 25 × 25 mm2, matrix size (MTX) = 256 ×
256, number of axial slices = 4, slice thickness (SI) = 1.0 mm,
and averages (NEX) = 3 (total scan time = 48 min). T2 analysis
was carried out using the image sequence analysis tool in
Paravision 5.0 pl3 software (Bruker, Billerica, MA, USA) with
monoexponential curve-fitting of image intensities of selected
regions of interest (ROIs) for each axial slice.
Cell Leaching Studies. Leaching studies were performed
with HeLa, MDA-MB-231-mcherry, and NIH/3T3 cells.
Specifically, 50,000 cells were plated in each well of a 12-well
plate and incubated for 24 h. Cells were incubated with
complexes 1−3 and Prohance in 0.4 mM cholate in media (600
μL dose) for 24 h at various concentrations to equalize cell
labeling. Cells were washed with 0.5 mL 0.4 mM cholate DPBS
and trypsinized. The resulting solution was centrifuged at 1000g
for 5 min at 4 °C. The media was aspirated and cell pellets were
suspended in 500 μL of media. A small aliquot (50 μL) was
used for cell counting and 100 μL was used for analysis by ICP-
MS. The remainder was replated in 6 well plates. Every 24 h,
the media was collected, centrifuged, and 250 μL was used for
analysis by ICP-MS. The cells were washed with 1 mL 0.4 mM
cholate PBS and fresh media was added to the cells. After the
72 h time point, cells were trypsinized and centrifuged. The
media was aspirated and the cell pellet was resuspended in 200
μL media. A 20 μL aliquot was used for cell counting and the
remainder analyzed by ICP-MS.
Cell Fractionation. The BioVision FractionPREP Cell
Fractionation system (Milpitas, CA) was used to fractionate
HeLa cells labeled with complexes 1−3 and Prohance
(dissolved in 0.4 mM cholate in media) for 24 h at various
incubation concentrations chosen to equalize cell labeling. The
manufacturer’s protocol was followed to isolate cytosol and
membrane fractions. Each fraction was analyzed by ICP-MS
and the ratio between the two was used to determine
localization.
Cytotoxicity. The CellTiter 96 AQueous Non-Radioactive
Cell Proliferation Assay (Promega, Madison, WI) was used to
measure cell viability. HeLa cells were plated in 96-well plates
(5000 cells per well for 24 h incubations and 1000 cells per well
for 72 h incubations). Complexes 1−4 and Prohance were
dissolved in 0.4 mM cholate and media at various
concentrations and incubated with cells (50 μL doses) for 24
or 72 h. After incubation, the assay was performed according to
the manufacturer’s suggested protocol. Absorbance at 490 nm
was measured using a Biotek Synergy4 microplate reader
(Winooski, VT).
ICP-MS Sample Preparation and Instrument Parame-
ters. For relaxivity and cell studies Gd(III) content was
measured via ICP-MS. Specifically, samples were prepared by
adding ACS reagent grade nitric acid (70%) to solutions of
contrast agent or cell suspensions (1:1 v/v sample:nitric acid)
in 15 mL conical tubes. Samples were incubated at 70 °C for at
least 4.0 h to allow for complete sample digestion. Following
sample digestion, multi-element internal standard (containing
Bi, Ho, In, Li, Sc, Tb, and Y, Inorganic Ventures,
Christiansburg, VA) and filtered deionized H2O (18.2 MΩ·
cm) were added, producing a final ICP-MS sample of 3% (v/v)
nitric acid and 5 ng/mL internal standard.
ASSOCIATED CONTENT
■
S
* Supporting Information
Compound characterization, DLS fitting, 7 T relaxivity, toxicity
profiles, IC50 values, time-dependent labeling, nonspecific
binding, and cell pellet T2 analysis. This material is available
free of charge via the Internet at http://pubs.acs.org.
ICP-MS was performed on a computer-controlled (Plasma-
lab software) Thermo X series II ICP-MS (Thermo Fisher
Scientific, Waltham, MA, USA) operating in standard mode
equipped with an ESI SC-2 autosampler (Omaha, NE, USA).
Each sample was acquired using 1 survey run (10 sweeps) and
3 main (peak jumping) runs (100 sweeps). The isotopes
selected for analysis were 154,157,158Gd with 115In and 165Ho
isotopes selected as internal standards for data interpolation.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: tmeade@northwestern.edu.
Notes
The authors declare no competing financial interest.
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(19) Terreno, E., Geninatti Crich, S., Belfiore, S., Biancone, L.,
Cabella, C., Esposito, G., Manazza, A. D., and Aime, S. (2006) Magn.
Reson. Med. 55, 491.
(20) Di Gregorio, E., Gianolio, E., Stefania, R., Barutello, G., Digilio,
G., and Aime, S. (2013) Anal. Chem. 85, 5627.
ACKNOWLEDGMENTS
■
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.). A portion of this work was completed at the
Northwestern University Integrated Molecular Structure
Education and Research Center. 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.
MRI was performed on the 7 T Bruker Pharmscan system
purchased with the support of NCRR 1S10RR025624-01.
Metal analysis was performed at the Northwestern University
Quantitative Bioelemental Imaging Center generously sup-
ported by NASA Ames Research Center NNA06CB93G. Plate-
based assays were performed at the Northwestern High
Throughput Analysis Lab. Semipreparative HPLC was done
by ChemCore at the Center for Molecular Innovation and
Drug Discovery which is generously supported by the Chicago
Biomedical Consortium with support from The Searle Funds at
The Chicago Community Trust.
(21) Zheng, Q., Dai, H., Merritt, M. E., Malloy, C., Pan, C. Y., and Li,
W. H. (2005) J. Am. Chem. Soc. 127, 16178.
(22) Gianolio, E., Giovenzana, G. B., Longo, D., Longo, I.,
Menegotto, I., and Aime, S. (2007) Chemistry 13, 5785.
(23) Andre, J. P., Toth, E., Fischer, H., Seelig, A., Macke, H. R., and
Merbach, A. E. (1999) Chem.Eur. J. 5, 2977.
(24) Nicolle, G. M., Toth, E., Eisenwiener, K. P., Macke, H. R., and
Merbach, A. E. (2002) J. Biol. Inorg. Chem. 7, 757.
(25) Hovland, R., Glogard, C., Aasen, A. J., and Klaveness, J. (2003)
Org. Biomol Chem. 1, 644.
(26) Torres, S., Martins, J. A., Andre, J. P., Geraldes, C. F. G. C.,
Merbach, A. E., and Toth, E. (2006) Chem.Eur. J. 12, 940.
(27) Vaccaro, M., Accardo, A., Tesauro, D., Mangiapia, G., Lof, D.,
Schillen, K., Soderman, O., Morelli, G., and Paduano, L. (2006)
Langmuir 22, 6635.
(28) Mastarone, D. J., Harrison, V. S., Eckermann, A. L., Parigi, G.,
Luchinat, C., and Meade, T. J. (2011) J. Am. Chem. Soc. 133, 5329.
(29) Song, Y., Kohlmeir, E. K., and Meade, T. J. (2008) J. Am. Chem.
Soc. 130, 6662.
(30) Pajk, S., Garvas, M., Strancar, J., and Pecar, S. (2011) Org.
Biomol Chem. 9, 4150.
REFERENCES
■
(1) Zhang, Y. Q., Dodd, S. J., Hendrich, K. S., Williams, M., and Ho,
C. (2000) Kidney Int. 58, 1300.
(2) Modo, M., Cash, D., Mellodew, K., Williams, S. C., Fraser, S. E.,
Meade, T. J., Price, J., and Hodges, H. (2002) NeuroImage 17, 803.
(3) Modo, M., Mellodew, K., Cash, D., Fraser, S. E., Meade, T. J.,
Price, J., and Williams, S. C. (2004) NeuroImage 21, 311.
(4) Modo, M., Beech, J. S., Meade, T. J., Williams, S. C., and Price, J.
(2009) NeuroImage 47 (Suppl 2), T133.
(5) Anderson, S. A., Glod, J., Arbab, A. S., Noel, M., Ashari, P., Fine,
H. A., and Frank, J. A. (2005) Blood 105, 420.
(6) Modo, M., Hoehn, M., and Bulte, J. W. (2005) Mol. Imaging 4,
143.
(7) Caravan, P., Ellison, J. J., McMurry, T. J., and Lauffer, R. B.
(1999) Chem. Rev. 99, 2293.
(8) Merbach, A. E. T., Ed. The Chemistry of Contrast Agents in Medical
Magnetic Resonance Imaging; John Wiley & Sons, Ltd.: New York,
2001.
(9) Endres, P. J., MacRenaris, K. W., Vogt, S., and Meade, T. J.
(2008) Bioconjugate Chem. 19, 2049.
(10) Adler, E. D., Bystrup, A., Briley-Saebo, K. C., Mani, V., Young,
W., Giovanonne, S., Altman, P., Kattman, S. J., Frank, J. A., Weinmann,
H. J., Keller, G. M., and Fayad, Z. A. (2009) JACC: Cardiovasc. Imaging
2, 1114.
(11) Nolte, I. S., Gungor, S., Erber, R., Plaxina, E., Scharf, J.,
Misselwitz, B., Gerigk, L., Przybilla, H., Groden, C., and Brockmann,
M. A. (2008) Magn. Reson. Med. 59, 1014.
(12) Bhorade, R., Weissleder, R., Nakakoshi, T., Moore, A., and
Tung, C. H. (2000) Bioconjugate Chem. 11, 301.
(13) Wunderbaldinger, P., Josephson, L., and Weissleder, R. (2002)
Bioconjugate Chem. 13, 264.
(14) Allen, M. J., MacRenaris, K. W., Venkatasubramanian, P. N., and
Meade, T. J. (2004) Chem. Biol. 11, 301.
(15) Endres, P. J., Macrenaris, K. W., Vogt, S., Allen, M. J., and
Meade, T. J. (2006) Mol. Imaging 5, 485.
(16) Song, Y., Xu, X. Y., MacRenaris, K. W., Zhang, X. Q., Mirkin, C.
A., and Meade, T. J. (2009) Angew. Chem., Int. Ed. 48, 9143.
(17) Vuu, K., Xie, J., McDonald, M. A., Bernardo, M., Hunter, F.,
Zhang, Y., Li, K., Bednarski, M., and Guccione, S. (2005) Bioconjugate
Chem. 16, 995.
(18) Tseng, C. L., Shih, I. L., Stobinski, L., and Lin, F. H. (2010)
Biomaterials 31, 5427.
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