Multivalent, high-relaxivity MRI contrast agents using rigid cysteine-reactive gadolinium complexes

Article abstract of DOI:10.1021/ja204516p

MRI contrast agents providing very high relaxivity values can be obtained through the attachment of multiple gadolinium(III) complexes to the interior surfaces of genome-free viral capsids. In previous studies, the contrast enhancement was predicted to de

Full text of DOI:10.1021/ja204516p

ARTICLE
pubs.acs.org/JACS
Multivalent, High-Relaxivity MRI Contrast Agents Using Rigid
Cysteine-Reactive Gadolinium Complexes
Praveena D. Garimella,†,‡ Ankona Datta,† Dante W. Romanini,†,‡ Kenneth N. Raymond,*,† and
Matthew B. Francis*,†,‡
†Department of Chemistry, University of California, Berkeley, California 94720-1460, United States
‡Materials Sciences Division, Lawrence Berkeley National Laboratories, Berkeley, California 94720-1460, United States
S Supporting Information
b
ABSTRACT: MRI contrast agents providing very high relax-
ivity values can be obtained through the attachment of multiple
gadolinium(III) complexes to the interior surfaces of genome-
free viral capsids. In previous studies, the contrast enhancement
was predicted to depend on the rigidity of the linker attach-
ing the MRI agents to the protein surface. To test this hypoth-
esis, a new set of Gd-hydroxypyridonate based MRI agents
was prepared and attached to genetically introduced cysteine
residues through flexible and rigid linkers. Greater contrast
enhancements were seen for MRI agents that were attached via
rigid linkers, validating the design concept and outlining a path
for future improvements of nanoscale MRI contrast agents.
’ INTRODUCTION
greater relaxivity values must be combined with signal amplifica-
tion strategies to generate a sufficient amount of signal difference
to allow their detection.3,4
Magnetic resonance imaging (MRI) is a powerful medical
diagnostic technique due to its ability to provide images of the
human body with great anatomical detail.1,2 However, its ability
to distinguish between two adjacent regions is often limited
by the inherently narrow range of relaxation rates for water
protons. As a result, a great deal of effort has been directed toward
increasing tissue contrast through the administration of contrast
enhancement agents prior to scanning. The most effective small
molecules for this purpose have contained paramagnetic metal
ions, which are capable of improving contrast by shortening the
relaxation time of nearby protons.1ꢀ6
Gadolinium(III) is most commonly used in MRI contrast
agents due to its large magnetic moment and long electronic
relaxation time.2 However, due to the high toxicity of its free form,
the metal must be complexed tightly by a coordinating agent prior
to injection into humans. Current commercially available Gd-
(III)-based contrast agents are based on poly(aminocarboxylate)
chelators (e.g., DOTA and DTPA), which have fairly low
relaxivity values (4ꢀ5 mMꢀ1sꢀ1 at 60 MHz and 25 °C). As a
result, gram quantities are typically injected to obtain the desired
level of contrast enhancement.7ꢀ9 A further limitation of these
agents is their low tissue retention and blood circulation times
(less than 30 min), which restrict their use in MRI applications
with larger data collection times.10 As a result, these agents have
only been successfully used to target sites where they can
accumulate in large concentrations. For the selective imaging of
biological targets bearing low (e.g., micromolar to nanomolar)
concentrations of specific markers, contrast agents with much
According to the Solomon-Bloembergen-Morgan (SBM) the-
ory of paramagnetic relaxation,11ꢀ15 the relaxivity of a Gd-based
contrast agent can be enhanced1 in three ways: by increasing the
number of water molecules (represented by q) coordinated to the
Gd center, by reducing the tumbling rate (1/τR) of the contrast
agent, and by keeping the exchange rate of the inner sphere water
molecules on Gd (1/τM) at an optimum value (e.g., τM ≈ 10 ns).
Previous studies in our laboratories have found that Gd(III)-
hydroxypyridonate (Gd-HOPO) based contrast agents have a
higher number of inner-sphere water molecules (q = 2 or even 3)
compared with other commercial agents.9,16,17 Furthermore, HOPO
complexes also have optimum water exchange rates, with τM
values ranging between 10 and 20 ns.18 By reducing the tumbling
rates of these contrast agents, it should be possible to obtain even
higher relaxivity values.
The tumbling rates of contrast agents can be reduced via
their covalent or noncovalent attachment to macromolecules
such as modified proteins,19 dendrimers,20,21 liposomes,22 carbo-
hydrates,23 or synthetic and micellar nanoparticles.4,24 In each
case, enhancements in the relaxivity values have been observed.
These approaches have the added benefit that they can allow the
attachment of multiple contrast agents to each carrier, further
increasing the total relaxivity.4
Received: May 17, 2011
Published: July 29, 2011
r
2011 American Chemical Society
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Figure 1. Design of highly efficient MRI contrast enhancement agents. (a) A crystal structure rendering of an MS2 coat protein dimer indicates the
positions of the introduced cysteine residues (Cys 87) in red. (b) A rendering of the full 27 nm MS2 capsid shows the interior and exterior surfaces, with
the 180 internal N87C positions highlighted in red. (c) The plan for increasing the overall relaxivity involved the use of more efficient cysteine
modification chemistry and the rigidification of several rotable bonds (indicated in blue).
As an alternative strategy, the construction of nanoscale
MRI contrast agents from viral capsids has been explored by
various laboratories in recent years because of the inherent
multivalency of these particles and the slow solution tumbling
rates resulting from their large size. In the first example, very
high relaxivity values (T1 = 202 mMꢀ1sꢀ1 at 61 MHz) were
obtained upon the chelation of gadolinium ions to the protein
coat of the cowpea chlorotic mottle virus (CCMV, 28.5 nm
diameter).25 However, the dissociation constant for gadoli-
nium was only 31 μM, and clinical applications require much
stronger chelation to prevent the release of toxic Gd3+ ions. In
another example, the attachment of Magnevist (Gd-DTPA) to
the exterior surface of MS2 viral capsids via lysine residue
modification26 has resulted in a 3-fold increase in relaxivity
(14ꢀ16.9 mMꢀ1sꢀ1 at 64 MHz) compared to free Magnevist.
Copper-catalyzed click chemistry has been used for the cova-
lent attachment of Gd(DOTA) to the exterior lysine residues
of cowpea mosaic virus(CPMV) while simultaneously exploit-
ing the natural affinity of the genomic RNA for Gd3+ ions in
order to increase the total metal loading. This resulted in
relaxivities of 11ꢀ15 mMꢀ1sꢀ1 per complex at 64 MHz.27 All
of these examples demonstrate the exceptional potential of
viral capsids for building highly efficient nanoscale contrast
agents.
Additional studies by our laboratories have explored the use
of bacteriophage MS2 to construct high-relaxivity MRI agents.
The capsid of this virus is icosahedral in symmetry and is
composed of 180 sequence identical protein subunits.28 The
resulting spherical shells are 27 nm in diameter and can be
prepared in genome-free assembled form through direct pro-
tein expression in E. coli.29 Access to the interior surface of the
capsid is afforded by 32 pores that are 2 nm in diameter,
permitting modification of the internal surface residues,
Figure 1a,b.30 The interior and exterior surfaces of the MS2
capsid can therefore be modified differentially by targeting a
variety of side chain groups, including lysine, tyrosine, and
artificial amino acids.29ꢀ31
We have previously reported the site-specific covalent mod-
ification of the exterior and the interior surfaces of MS2 viral
capsids with Gd-HOPO-based complexes.32,33 The Gd-com-
plexes were attached to tyrosine residues on the internal surface
and to lysineresidues on the external surface. Using this approach,
we demonstrated the advantage of housing the Gd-HOPO
complexes within MS2 capsids to improve their solubility at
high levels of modification. We also found that water transport
through the capsid shell was fast on the NMR time scale and that
the internally modified capsids yielded high relaxivity values
(31 mMꢀ1sꢀ1, 25 °C, pH 7, at an external field of 60 MHz). The
externally modified capsids gave lower relaxivity values, which
was attributed to the increased flexibility of the lysine modifica-
tion products relative to those formed with the internal tyrosine
residues.
In order to convert the MS2 capsids to imaging agents for
targeted imaging, we sought to improve the relaxivity further by
both increasing the bioconjugation efficiency of the Gd com-
plexes (resulting in the attachment of more contrast agents to
each capsid) and by rigidifying the linker that attaches them to
the protein scaffold. We recently reported the use of a muta-
genically introduced cysteine at position 87 (the N87C mutant
shown in Figure 1a,b) and targeted this residue with maleimide
reagents to introduce drug molecules34,35 or imaging agents36
to the interior surface of the capsids with high efficiency
(approaching 100%). On the basis of this success, this site was
also chosen for modification with Gd-HOPO complexes through
the use of linking groups that possessed a minimal number of
rotatable bonds. It was found that these design concepts were
indeed successful, providing capsids with effective relaxivities as
high as 7416 mMꢀ1sꢀ1 at 60 MHz while maintaining the high
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Scheme 1. Synthesis of Cysteine Reactive HOPO-Based Contrast Agentsa
a Reagents and conditions: (i) PFP-trifluoroacetate, DIPEA, dry THF, 77%; (ii) 3a, 3b, or 3c, DIPEA, DCM, slow addition, rt, 24 h. 4a = 69%, 4b = 66%,
4c = 67%; (iii) 5, DIPEA, DCM, rt, 3 h; (iv) TFA:DCM (1:1), 10% thioanisole, rt, 3 h, followed by ether precipitation. 6a = 50%, 6b = 55%, 6c = 56%
(after two steps). DIPEA = N,N-diisopropylethyl amine.
affinity for the gadolinium ions that is conferred by the HOPO
ligands.
the HOPO-ligand attached to the inside surface of each capsid.
The extent of attachment was determined using LC/ESI-MS
following disassembly of the capsids (see Figure 2 and Support-
ing Information). This level of modification therefore provided a
2-fold increase in bioconjugation efficiency over the previous
tyrosine-based strategy. There was no detectable protein pre-
cipitation and the capsids remained soluble despite the high
loading of the hydrophobic ligand. Capsid assembly (both before
and after the introduction of Gd(III) ions) was verified using
TEM, size-exclusion chromatography (SEC), and dynamic light
scattering (DLS) measurements (see Supporting Information).
Next, the conjugates were metalated with a solution contain-
ing 0.9ꢀ1.0 equiv of GdCl3 to afford the corresponding Gd
complexes. Initial control experiments in which the targeted
cysteine residues were capped with N-ethyl maleimide before
exposure to GdCl3 resulted in a significant amount of background
metal binding, Table 1. Dialysis against a citrate solution and size
exclusion chromatography did not remove this nonspecifically
bound Gd, with ∼49% of the amount added remaining. This
indicated that the N87C capsids have a non-negligible affinity for
gadolinium ions without a strong affinity ligand like HOPO being
present in solution.
However, it was found that background binding did not occur
in the presence of one equivalent of free bis-HOPO-TREN-TAM-
ethyl amine ligand17—conditions that more accurately repre-
sented the metalation reactions with conjugates 7aꢀc. This ligand
is similar to the HOPO-maleimide ligands that were synthesized,
but lacks the bioconjugation handle. In this case, we observed
that only a minimal amount (<5%) of the Gd3+ added remained
nonspecifically bound to MS2.
To prepare the MS2-HOPO-Gd(III) conjugates under analo-
gous conditions, capsids bearing internal HOPO ligands (MS2-
HOPO-Lin, MS2-HOPO-R,R and MS2-HOPO-S,S) were pre-
pared and concentrated using ultrafiltration. The complexes were
next metalated using 0.95 equiv. of GdCl3 relative to the estimated
’ RESULTS AND DISCUSSION
To test the hypothesis that increasing the linker rigidity would
increase the relaxivity, we designed HOPO-based contrast agents
bearing maleimide groups through either a flexible linear linker
or a rigid cyclohexyl linker. The synthesis of the newly designed
cysteine reactive HOPO-contrast agents involved the attach-
ment of a linker containing a terminal maleimide group to the
heteropodal trisaminoethylamine(TREN)-bis(HOPO)-tereph-
thalimide(TAM) moiety (Scheme 1). The TAM starting materi-
al was activated as the pentafluorophenyl (PFP) ester (2). This
molecule was then treated with linear maleimide-amine 3a37 to
give TAM-PFP-ethyl-maleimide 4a. This compound was next
coupled to TREN-bis-HOPO-(OBn)2 (5) to yield the benzyl
protected ligand. Benzyl deprotection with concentrated hydro-
chloric and acetic acid was not possible, as it led to chloride addition
to the maleimide double bond. Optimization of this reaction using a
mixture of 50% trifluoroacetic acid in dichloromethane with 10%
thioanisole as a scavenger afforded fully deprotected ligand 6a with
minimal amounts of side products. The material was purified via
ether precipitation and used without further purification.
For the synthesis of the rigid linker, the two enantiomers of
trans-1,2-cyclohexyldiamine were converted to corresponding
maleimide-amines 3b and 3c in three steps (see the Supporting
Information for details). Both enantiomers were used in order to
determine if there would be a difference in relaxivity due to their
interaction with the chiral capsid protein. Activated PFP ester 2
was reacted with 3b and 3c and then deprotected to yield rigid
ligands 6b(S,S) and 6c(R,R) in a sequence similar to that used to
access the linear linker.
Exposure of assembled N87C MS2 capsids to 20 equiv of ligands
6aꢀc (based on protein monomer concentration) for 2ꢀ3 h led
to virtually quantitative modification, resulting in 180 copies of
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Figure 2. Bioconjugation of HOPO-maleimide 6a to N87C MS2 and mass spectral characterization of the MS2-conjugates (See Supporting
Information for additional spectra). (a) Conditions: 10 mM phosphate, TRIS buffer pH 8, 6a (20 equiv), 3 h. (b) ESI-MS reconstruction of MS2 N87C
monomers after capsid dissociation. Expected mass: [M+H+] = 13719 m/z. (c) ESI-reconstruct of linear linker protein conjugate 7a, showing virtually
complete conversion (corresponding to 180 copies per capsid). Expected mass of HOPO-linear-MS2: [M+H+] = 14469 m/z. All mass values agreed to
within 0.03% of those expected. Full charge ladders appear in Figure S1 of the Supporting Information.
The relaxivity values of the bioconjugates were measured
to quantify the effect of linker rigidity on contrast enhancement.
T1 relaxation times of the protein conjugate solutions (appro-
ximately 200 μM in [Gd3+]) were measured at 60 MHz field
strengths using an inversion recovery pulse sequence. The
relaxivity values for the capsids conjugated to the linear and rigid
linkers were calculated using the following equation:
Table 1. Quantitation of Gd(III) Binding to HOPO-
Modified Viral Capsids
entry
sample
MS2-NEMc
MS2+HOPO-EAd
[protein] (μM)a [Gd] (μM)b % Gd bound
1
2
3
4
5
413
413
41
201
49
5
20
MS2-HOPO-Lin(7a)
MS2-HOPO-S,S(7b)
MS2-HOPO-R,R(7c)
41.1 ( 0.4
33.0 ( 0.2
20.6 ( 0.4
>95%
>95%
89
33
ꢀ
ꢁ
1
1
23
ꢀ
a Protein concentrations were determined using UV/vis, and are taken to be
within 10% of the actual values. b Gadolinium concentrations were deter-
mined using ICP-AES. Control reactions were conducted to check for
nonspecific binding. c N-ethyl maleimide capped MS2 treated with 0.95ꢀ1
equiv. of GdCl3 (entry 1). d N-ethyl maleimide capped MS2 treated with
0.95ꢀ1 equiv. of GdCl3 and 1 equiv. of free HOPO-ethyl amine (entry 2).
T1 T1d
relaxivity ¼
ð1Þ
½Gdꢁ
where [Gd] represents the metal concentration measured by
ICP-AES and T1d is the intrinsic diamagnetic solvent relaxation
time. The relaxivity values were measured in triplicate at pH 7
in 12.5 mM HEPES solution at 25 °C as well as the more
physiologically relevant temperature of 37 °C (Table 2).
concentration of protein after the ultrafiltration step (which
could vary by about 10%). Following complex formation, any
remaining Gd3+ was removed by gel filtration and dialysis against
a solution of ammonium citrate (as a low-affinity competitive
ligand), followed by overnight dialysis against 12.5 mM HEPES
buffer at pH 7. To ensure accurate relaxivity measurements, the
Gd-content of the resulting capsid samples was measured using
inductively coupled plasma-atomic emission spectroscopy (ICP-
AES), and the protein concentrations were independently
determined using UVꢀvis spectroscopy, Table 1. The compar-
ison of these values indicated a high degree of Gd incorpora-
tion (>95% for MS2-HOPO-Lin (7a) and MS2-HOPO-S,S (7b)
and 89% for MS2-HOPO-R,R (7c)). DLS and SEC analyses
confirmed that the capsids remained stable and intact after
metalation (see the Supporting Information for full characteriza-
tion of the capsid conjugates).
The measured relaxivity values indicated that increasing the
linker rigidity had a positive effect on contrast enhancement
in some cases. The (S,S)-linked rigid conjugate gave a higher
relaxivity value (41 mMꢀ1sꢀ1 at 60 MHz) compared with the
(R,R) linker (30 mMꢀ1sꢀ1, 60 MHz) and the linear linker
(33 mMꢀ1sꢀ1, 60 MHz). Also, due to the nearly quantitative
conversion achieved in the maleimide coupling reaction, the total
relaxivity per capsid was as high as 7416 mMꢀ1sꢀ1 for MS2-
HOPO-S,S (7b). The lysine and tyrosine bioconjugates from our
previous work gave relaxivity values of 23.2 and 30.9 mMꢀ1sꢀ1
,
respectively, at 60 MHz.32,33 Since each capsid contained
only ∼95 complexes, total relaxivities of up to 2900 mMꢀ1sꢀ1
were observed. The current MS2-Gd-HOPO conjugates therefore
possess up to 2.5-fold higher overall relaxivity through the
combination of rigidification effects and higher loading.
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Table 2. Comparison of Relaxivity Values at 60 MHza
relaxivity at 60 MHz
(mM-1s-1)b
relaxivity/capsid at 60 MHz
(mM-1s-1)c
sample
25 °C
37 °C
25 °C
37 °C
Lin (Gd-7a) 32.6 ( 0.1 29.7 ( 0.1
SS (Gd-7b) 41.2 ( 0.2 38.2 ( 0.6
RR (Gd-7c) 29.6 ( 0.1 25.4 ( 0.3
5868
7416
4736
5346
6876
4064
a Relaxivity values were measured in 12.5 mM HEPES (pH 7) at 25 and
37 °C for the MS2-Gd-HOPO conjugates. b Reported per Gd(III)
complex. c The total relaxivities of the full capsids were calculated
assuming 180 Gd(III) complexes for 7a, 180 Gd(III) complexes for
7b, and 160 Gd(III) complexes for 7c. These estimates are taken to be
within 5ꢀ10% of the actual values, based on the errors associated with
protein concentration determination.
A marked difference in relaxivity was observed for the two
enantiomeric ligands when attached to the protein. This dis-
crepancy is reasonable considering the intrinsic chirality of the
protein, which will experience complex and different interactions
with the two enantiomeric linkers. Previous studies38ꢀ42 have
reported differences in water exchange rates for diastereomeric
small molecule contrast agents that include Gd3+. In particular,
Burai et al.38 have observed differential water-exchange rates for
the diastereomers of [Gd(EPTPA-bz-NH2)H2O)]2- and [Gd-
(DTPA-bz-NH2)H2O)]2-. They predicted that upon slowing the
tumbling rates of these diastereomers, the differences in water-
exchange rates would be reflected in the relaxivity values.
Caravan et al.39 have studied the diastereomers of the contrast
agent MS-325, which can bind noncovalently to human serum
albumin (hSA) and give different relaxivity enhancements for
the two diastereomers. The authors presented detailed NMRD
studies on the complexes and predicted that the relaxivity differ-
ences were also possibly due to differences in water-exchange rates
between the two diastereomers.
To clarify the way in which the chiral linkers alter the inter-
actions of the complexes with the protein surface, molecular
models were generated starting with the crystal structure of the
MS2 coat protein.28 The Gd(HOPO) complexes were based on
a reported crystal structure43 and altered to display the linkers
corresponding to 7b and 7c. Each complex was generated as a
set of three possible coordination isomers (defined in Support-
ing Information Figure S5). For the modeling studies, two
copies of each complex were attached to an adjacent pair of
C2-symmetric Cys 87 residues (green in Figure 3cꢀf). The
structures were minimized using MacroModel, using no con-
straints on the metal complexes or the amino acids within a 20 Å
radius.
Representative results for Gd-7b and Gd-7c complexes are
shown in Figure 3, with the full set appearing in Supporting
Information Figure S8. Little change was observed for the
protein backbone or the gadolinium coordination geometries,
but the orientations of the cysteine 87 side chains were altered
somewhat in response to the linker chirality, Figure 3a,b.
Interestingly, the chiral 1,2-diaminocyclohexane groups exhib-
ited striking effects on the display of the ligands attached to the
sulfur atoms, with the S,S-linker (Gd-7b) orienting the com-
plexes above the protein surface, Figure 3c,d. This likely leads to
unencumbered exchange of the bound water molecules with the
bulk solvent. In contrast, the R,R-linker (Gd-7c) flips the
complex toward the protein surface (Figure 3b). This geometry
Figure 3. Models of sterically hindered Gd-HOPO complexes at-
tached to the interior MS2 surface. Structures attached to two adjacent
Cys87 groups were minimized simultaneously using MacroModel, and
three different coordination isomers were considered for each. The Gd
coordination spheres were based on a previously reported crystal
structure.43 Representative structures are shown here, with the full set
appearing in Supporting Information Figures S5 through S8. Overlays
of S,S (Gd-7b, gray) and R,R (Gd-7c, brown) complexes are shown for
(a) a top view and (b) a side view. The rigid linker of S,S-complex
Gd-7b (c and e) forces an upright conformation that leaves the water
molecules (yellow) unobstructed. R,R-complex Gd-7c (d and f) places
the water molecules much closer to the capsid surface, likely restricting
their exchange. The Cys 87 attachment points are shown in green
in c-f.
places significant constraints on the water molecules and may
allow the protein side chains to compete with their binding.
Either of these effects could explain the lower overall relaxivity
of the R,R-complexes. The S,S-linkages also generated markedly
increased interactions between the complexes, which could
further restrict their conformational flexibilities. These obser-
vations were consistent throughout the full series of complexes
that were modeled. In ongoing studies, we are analyzing the
temperature dependence of relaxivity and the NMRD profiles
for these capsid-attached complexes, with the goal of gaining
further insight into the nature of these differences.
’ CONCLUSIONS
These studies demonstrate that increasing the rigidity of the
linker between Gd-containing contrast agents and a viral capsid
scaffold can lead to significant increases in relaxivity properties.
The obtained relaxivity values are some of the highest reported for
high-affinity Gd3+ complexes, paving the way for their future use
in applications that target nonabundant biological markers.
Current efforts are focused on the addition of cell binding ligands
on the external surfaces of the assemblies35 and the determination
of the detection limits associated with these complexes in
physiological environments.
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