A new ditopic GdIII complex functionalized with an adamantyl moiety as a versatile building block for the preparation of supramolecular assemblies Topical Issue on Metal-Based MRI Contrast Agents. Guest editor: Valerie C. Pierre

Article abstract of DOI:10.1007/s00775-013-1050-0

A dimeric GdAAZTA-like complex (AAZTA is 6-amino-6-methylperhydro-1,4- diazepinetetraacetic acid) bearing an adamantyl group (Gd₂ L1) able to form strong supramolecular adducts with specific hosts such as β-cyclodextrin (β-CD), poly-β-CD, and human serum albumin (HSA) is reported. The relaxometric properties of Gd₂ L1 were investigated in aqueous solution by measuring the ¹H relaxivity as a function of pH, temperature, and magnetic field strength. The relaxivity of Gd₂ L1 (per Gd atom) at 40 MHz and 298 K is 17.6 mM^-1 s^-1, a value that remains almost constant at higher fields owing to the great compactness and rigidity of the bimetallic chelate, resulting in an ideal value for the rotational correlation time for high-field MRI applications (1.5-3.0 T). The noncovalent interaction of Gd₂ L1 with β-CD, poly-β-CD, and HSA and the relaxometric properties of the resulting host-guest adducts were investigated using ¹H relaxometric methods. Relaxivity enhancements of 29 and 108 % were found for Gd₂ L1-β-CD and Gd₂ L1-poly-β-CD, respectively. Binding of Gd₂ L1 to HSA (K _A = 1.2 × 10⁴ M ^-1) results in a remarkable relaxivity of 41.4 mM^-1 s ^-1 for the bound form (+248 %). The relaxivity is only limited by the local rotation of the complex within the binding site, which decreases on passing from Gd₂ L1-β-CD to Gd₂ L1-HSA. Finally, the applicability of Gd₂ L1 as tumor-targeting agent through passive accumulation of the HSA-bound adduct was evaluated via acquisition of magnetic resonance images at 1 T of B16-tumor-bearing mice. These experiments indicate a considerable signal enhancement (+160 %) in tumor after 60 min from the injection and a very low hepatic accumulation.

Full text of DOI:10.1007/s00775-013-1050-0

J Biol Inorg Chem (2014) 19:133–143
DOI 10.1007/s00775-013-1050-0
ORIGINAL PAPER
A new ditopic GdIII complex functionalized with an adamantyl
moiety as a versatile building block for the preparation
of supramolecular assemblies
•
Giuseppe Gambino Sara De Pinto
•
•
Lorenzo Tei Claudio Cassino Francesca Arena
•
•
•
Eliana Gianolio Mauro Botta
Received: 3 July 2013 / Accepted: 24 September 2013 / Published online: 8 October 2013
Ó SBIC 2013
Abstract A dimeric GdAAZTA-like complex (AAZTA
is 6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid)
bearing an adamantyl group (Gd2L1) able to form strong
supramolecular adducts with specific hosts such as
b-cyclodextrin (b-CD), poly-b-CD, and human serum
albumin (HSA) is reported. The relaxometric properties of
Gd2L1 were investigated in aqueous solution by measuring
M-1) results in a remarkable relaxivity of 41.4 mM-1 s-1
for the bound form (?248 %). The relaxivity is only lim-
ited by the local rotation of the complex within the binding
site, which decreases on passing from Gd2L1–b-CD to
Gd2L1–HSA. Finally, the applicability of Gd2L1 as tumor-
targeting agent through passive accumulation of the HSA-
bound adduct was evaluated via acquisition of magnetic
resonance images at 1 T of B16-tumor-bearing mice. These
experiments indicate a considerable signal enhancement
(?160 %) in tumor after 60 min from the injection and a
very low hepatic accumulation.
1
the H relaxivity as a function of pH, temperature, and
magnetic field strength. The relaxivity of Gd2L1 (per Gd
atom) at 40 MHz and 298 K is 17.6 mM-1 s-1, a value
that remains almost constant at higher fields owing to the
great compactness and rigidity of the bimetallic chelate,
resulting in an ideal value for the rotational correlation
time for high-field MRI applications (1.5–3.0 T). The
noncovalent interaction of Gd2L1 with b-CD, poly-b-CD,
and HSA and the relaxometric properties of the resulting
Keywords Gadolinium ꢀ Relaxivity ꢀ Contrast
agents ꢀ Magnetic resonance imaging ꢀ
Supramolecular chemistry
1
host–guest adducts were investigated using H relaxomet-
ric methods. Relaxivity enhancements of 29 and 108 %
were found for Gd2L1–b-CD and Gd2L1–poly-b-CD,
respectively. Binding of Gd2L1 to HSA (KA = 1.2 9 104
Introduction
Magnetic resonance imaging (MRI) allows the acquisition
of high resolution, three-dimensional images of the distri-
bution of tissue water in vivo. The quality and diagnostic
potential of these images are enhanced by the use of con-
trast agents that catalytically decrease the relaxation time
of protons of water coordinated to a paramagnetic metal
center. GdIII and MnII are ideally suited for such agents.
Nowadays, clinically approved metal-based commercial
agents are low molecular weight and aspecific chelates
characterized by low relaxivity. This translates into a
reduced sensitivity which can be overcome by developing
contrast agents with enhanced relaxivity. One of the main
goals for an effective signal amplification strategy is the
simultaneous optimization of the molecular parameters
governing the relaxometric behavior of the paramagnetic
probe (based on GdIII or MnII complexes). According to
Responsible Editor: Valerie C. Pierre.
Electronic supplementary material The online version of this
article (doi:10.1007/s00775-013-1050-0) contains supplementary
material, which is available to authorized users.
G. Gambino ꢀ S. De Pinto ꢀ L. Tei ꢀ C. Cassino ꢀ M. Botta (&)
Dipartimento di Scienze e Innovazione Tecnologica,
`
Universita del Piemonte Orientale ‘‘Amedeo Avogadro’’,
Viale T. Michel 11, 15121 Alessandria, Italy
e-mail: mauro.botta@unipmn.it
F. Arena ꢀ E. Gianolio
Department of Molecular Biotechnology and Health Sciences,
Molecular Imaging Center,
`
Universita di Torino,
Via Nizza 52, 10126 Turin, Italy
123

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the established theory of paramagnetic relaxation, large
longitudinal relaxivity (r1) enhancements can be mainly
pursued by (1) increasing the hydration number (q), (2)
optimizing the mean residence lifetime (sM) of the coor-
dinated water molecule(s), and (3) lengthening the rota-
tional molecular correlation time (sR) [1–3]. It was
recognized very early that the rotational dynamics is the
main factor controlling the efficiency of the low molecular
weight GdIII chelates. Therefore, over the last 20 years
different approaches have been developed to slow down
the rotation of the paramagnetic complex by conjugating
the complex to a large substrate [2–5]. The reversible
formation of adducts with human serum albumin (HSA)
has been one of the major routes to achieve high relaxation
enhancement for GdIII complexes as a result of the reduced
rotational tumbling rate (1/sR) of the macromolecular
adduct [4, 5]. Slow-tumbling systems with large sR can be
obtained through different approaches: (1) contrast agents
covalently bound to dendrimers, polymers, proteins, or
virus capsids [1, 6–9]; (2) aggregated systems, such as
micelles or liposomes [1, 10–12]; (3) suitably functional-
ized probes that can interact noncovalently with proteins or
other types of macromolecules [1, 4, 5]. The large array of
molecular recognition pathways provided by supramolecular
chemistry allows one to exploit several systems for attaining
large aggregates with high binding constants. For example,
HSA has been largely used for the formation of adducts with
GdIII chelates bearing hydrophobic functionalities, taking
advantage not only of the relaxivity enhancement generated
by the noncovalent interaction, but also of the lengthening of
the lifetime of the contrast agent in the bloodstream [1, 4, 5,
13, 14]. The latter issue plays a relevant role in the devel-
opment of magnetic resonance angiography probes.
In the case of GdIII-based contrast agents, a well-
established and attractive approach to attain large sR values
relies on the reversible formation of adducts between
suitably functionalized complexes and macromolecular
substrates [4, 5]. An optimized GdIII probe bearing a
hydrophobic moiety, able to form supramolecular adducts
with a given substrate, would be a promising system to
achieve high relaxivities. For this purpose we chose
GdAAZTA (AAZTA is 6-amino-6-methylperhydro-1,4-
diazepinetetraacetic acid) [15, 16] as a complex that
combines high thermodynamic and kinetic stabilities with
the presence of two water molecules in the inner coordi-
nation sphere (q = 2) in fast exchange with the bulk.
Moreover, the use of a recently reported [17] dimeric
bifunctional chelating agent bearing two AAZTA units and
an isothiocyanate group for conjugation allows further
optimization owing to the higher relaxivity (per Gd atom)
of the dimeric complex with respect to the monomer as a
consequence of its higher molecular weight, which trans-
lates into slower rotational dynamics.
A host–guest type of interaction widely explored to
obtain large supramolecular structures involves cyclo-
dextrins (CDs) and poly(b-CD)s (poly-b-CDs). CDs are
naturally occurring, water-soluble, toroidally shaped oli-
gosaccharides with a truncated cone-shaped hydrophobic
cavity that can host a variety of organic compounds,
especially those carrying a lipophilic group. GdIII com-
plexes bearing hydrophobic substituents such as adaman-
tyl, cyclohexyl, or phenyl moieties in their structure have
been shown to be able to form inclusion adducts with b-CD
(b-CD is a CD made up of seven sugar units) with high
binding constants and remarkable relaxivity enhancement
[18–21].
In this article, a new dimeric derivative based on
AAZTA and bearing an adamantyl moiety is described.
The ditopic GdIII complex was specifically designed to
form host–guest adducts with b-CDs and poly-b-CDs, as
the adamantyl moiety is known to interact strongly with the
b-CD cavity [22, 23]. To the best of our knowledge, only a
few GdIII chelates bearing an adamantyl moiety have been
reported [18, 24]. A detailed relaxometric characterization
of the complex was performed in aqueous solution by
investigating the 1H relaxivity dependence on pH, tem-
perature, and magnetic field strength. The noncovalent
interaction with both b-CD and poly-b-CD was evaluated
by using the proton relaxation enhancement (PRE) method,
and a detailed relaxometric study on the resulting host–
guest adducts was conducted. Furthermore, to assess the
possible application of such a system as an MRI contrast
agent, and thus its stability in the presence of endogenous
competitive species, the interaction with HSA was also
investigated. Moreover, an in vivo test on B16-tumor-
bearing mice was performed by acquiring magnetic reso-
nance images at 1 T over a period of 24 h.
Results and discussion
We have recently reported an isothiocyanate functionalized
AAZTA-like dimer (I) that acts as a bifunctional chelating
agent for the development of larger assemblies [17]. The
synthetic route towards the desired ditopic Gd2L1 complex
(Scheme 1) starts from the reaction between I and (1-ad-
amantyl)methylamine in CH2Cl2 under mild basic condi-
tions to afford II. The intermediate II was then deprotected
by using a 1:1 mixture of trifluoroacetic acid (TFA) and
CH2Cl2, yielding the final ligand L1. Both L1 and its
protected form II were fully characterized by mass spec-
trometry and NMR spectroscopy. Although ditopic and/or
multimeric GdIII complexes are widely investigated
[25–27], not many of them are functionalized with a
reactive or a target-specific moiety [17, 28–32]. For
example, we have recently reported a series of ditopic GdIII
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J Biol Inorg Chem (2014) 19:133–143
135
COOR
ROOC
straight line obtained corresponds to the relaxivity (r1) of
the complex, i.e., the R1 enhancement per millimolar
concentration of paramagnetic metal at a given temperature
and magnetic field strength (typically 298 K/310 K and
20 MHz). The r1 value of the ditopic complex at 20 MHz
and 298 K was found to be 16.7 mM-1 s-1 (per Gd atom),
over two times greater than that of the corresponding
monomeric GdAAZTA complex [15, 16]. This corresponds
to an enhancement of 135 %, which can be largely attrib-
uted to the increase in molecular size, which translates into
a corresponding increase of sR. In fact, the short linkers
connecting the two metal chelates and the adamantyl
moiety to the central aromatic group are expected to
minimize the flexibility of the system and favor long
rotational correlation times in both the free form and the
bound form.
ROOC
N
COOR
COOR
N
N
O
NH
COOR
COOR
N
O
ROOC
O
N
N
H
N
O
SCN
I
i
HN
S
NH
ROOC
ROOC
O
N
N
NH
O
COOR
COOR
O
N
N
N
To get more insight, the 1H nuclear magnetic relaxation
dispersion (NMRD) profiles of Gd2L1 were recorded in
aqueous solution at pH 7 and at 283, 298, and 310 K (Fig. 1,
left). The relaxivity per gadolinium atom is comparable to or
even higher than that of other GdIII dimers [25–27], and is
almost unchanged over a large range of maꢁg1netic fields
HN
ROOC
O
N
COOR
COOR
COOR
II R= t-Bu
R= H
ii
L1
iii
(r140 MHz ¼ 17:6 mMꢁ1 sꢁ1; r170MHz ¼ 17:4 mM
s
ꢁ1), thus
being interesting for applications in high-field MRI studies.
Moreover, the r1 value (20 MHz, 298 K) remains constant
over a large pH range (4–11; Fig. S1), indicating its stability
towards acid-catalyzed and base-catalyzed dissociation.
The experimental data were then analyzed by fitting
the data to the established theory for paramagnetic
relaxation, using the well-known Solomon–Bloembergen–
Morgan (SBM) equations and Freed’s model for inner-
sphere and outer-sphere contributions to the proton re-
laxivity, respectively [2, 3]. There are several parameters
that enter in the description of the magnetic field depen-
dence of r1, but some of these can be set at standard
values. The number of coordinated water molecules
(q) was fixed to two, the distance of closest approach of
the outer-sphere water molecules to Gd3? was set to
HN
S
NH
O
N
O
O
O
N
O
O
O
O
HN
NH
Gd3+
Gd3+
O
O
O
O
O
O
N
N
N N
O
O
O
O
O
O
Gd2L1
Scheme 1 Synthesis of Gd2L1: i
CH2Cl2, Et3N; ii trifluoroacetic acid, CH2Cl2; iii GdCl3, H2O, pH 6.5
(1-adamantyl)methylamine,
˚
4.0 A, a typical value for small metal chelates, and for the
complexes bearing hydrophobic moieties or a functional
group obtained through a Ugi four-component reaction,
including a study of the interaction between the octadecyl
derivative and HSA [32]. However, in that case, the use of
the less efficient GdDOTA-monoamide complexes (DOTA
is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid)
led to results hampered by the presence of only one coor-
dinated water molecule in slow exchange with the bulk.
The GdIII complex was prepared by adding small volumes
of a stock solution of Gd(NO3)3 to a solution of L1 while
maintaining the pH at 6–7 with dilute NaOH. The com-
plexation process was monitored by measuring the change
in the longitudinal water proton relaxation rate (R1) as a
function of the amount of Gd3? added. The slope of the
relative diffusion coefficient (D) the standard value of
2.24 9 10-5 cm2 s-1 (298 K) was used. The Gd–water
proton distance (rGd–H) and sR are parameters that are
strongly correlated, and then the former is generally fixed.
According to a pulsed electron–nuclear double resonance
study by Caravan et al. [33], the rGd–H values for selected
diethylenetriaminepentaacetic acid (DTPA)- and DOTA-
like GdIII complexes fall in the range 3.0–3.2 A. We
˚
˚
obtained the best results by fixing rGd–H at 3.0 A. As a
function of temperature, r1 follows an exponential
dependence, as typical for gadolinium-based systems
under a fast-water exchange regime (Fig. 1, right). We
then used for the exchange lifetime the same value
reported for [Gd(AAZTA)(H2O)2]-, i.e., 298sM = 90 ns
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Fig. 1 Left: 1H nuclear magnetic relaxation dispersion (NMRD)
profiles of Gd2L1 (see Scheme 1 for the structure) at 283, 298, and
310 K. Right: r1 as a function of temperature for Gd2L1 (20 MHz,
pH 7). The dashed line represents a simple fit to a monoexponential
function to confirm the fast-exchange condition
1
Table 1 Best-fit parameters obtained by analysis of the H nuclear magnetic relaxation dispersion profiles for Gd2L1 (see Scheme 1 for the
structure) and its adducts with b-cyclodextrin (b-CD), poly-b-CD, and human serum albumin (HSA)
Parameter
Gd2L1
Gd2L1–b-CD
Gd2L1–poly-b-CD
Gd2L1–HSA
GdAAZTAa
298
r
(mM-1 s-1 b
)
16.7
2.5
21.6
1.2
33.9
1.3
41.4
1.4
7.1
2.2
31
1p
D2 (1019 s-2
298sV (ps)
)
31
44
37
11.1c
21
11.1c
298
k
s
s
(106 s-1
)
11.1c
0.29
107
0.66
2
11.1c
0.62
227
0.29
2
11.1
0.074
–
ex
298
298
(ns)
2.3
28
RG
RL
(ps)
410
0.17
2
479
0.10
2
S2
qc
–
2
c
˚
rGdH (A)
3.0
3.0
3.0
3.0
3.1
4.0
2.24
c
˚
a (A)
298D (10-5 cm2 s-1 c
4.0
4.0
4.0
4.0
)
2.24
2.24
2.24
2.24
AAZTA 6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid
a
From [6, 7]
b
At 20 MHz
c
Fixed in the fitting procedure
[15, 16]. Nevertheless, the relaxivity is not affected by
this parameter, and the result of the fit does not change
for values of sM in the range 50–150 ns. We then used as
adjustable parameters D2, sV, and sR. The electronic
relaxation parameters, D2 and sV, are rather similar to
those of the monomer, indicating that the typical coor-
dination cage of AAZTA is retained in the ditopic com-
plex. The rotational correlation time is of the order of
220 ps (at 298 K), about three times longer.
overall tumbling motion [34, 35]. These two types of
motion, a relatively fast local rotation of the coordination
cage about the linker to the central aromatic scaffold
superimposed on the global reorientation of the complex,
are characterized by different correlation times: sRL and
s
RG, respectively. The degree of correlation between global
and local rotations is given by the parameter S2, which
takes values between 0 (completely independent motions)
and 1 (entirely correlated motions). A five-parameter (D2,
sV, sRL, sRG, and S2) least-squares fit of the data was per-
formed. The best-fit parameters for Gd2L1 are listed in
Table 1 and are compared with those for the parent com-
plex [Gd(AAZTA)(H2O)2]-. The correlation times for the
local and global rotations differ by only a factor of 3, hence
confirming the low level of rotational flexibility of the
Although the fit can be considered rather satisfactory, it
is not of very high quality, especially for the data at 283 K.
Much better results are obtained using the Lipari–Szabo
approach for the description of the rotational dynamics.
This model allows one to take into account the presence of
a certain degree of internal rotation superimposed on the
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J Biol Inorg Chem (2014) 19:133–143
137
Fig. 2 Left: Water proton relaxation rate of a 0.08 mM aqueous solution of Gd2L1 as a function of increasing amounts of b-cyclodextrin (b-CD)
1
(20 MHz, 298 K). Right: H NMRD profiles of Gd2L1 b-CD at 283, 298, and 310 K
ditopic complex. This is also evidenced by the large value
of the order parameter S2.
enabled us to calculate the affinity constant [KA =
(1.05 0.09) 9 104 M-1] and the relaxivity of the adduct
(r1b = 21.4 0.1 mM-1 s-1). The formation of the inclu-
sion compound was then accompanied by an increase of r1
of 29 % relative to the isolated complex, which reflects the
slowing down of molecular tumbling. The analysis of the
NMRD profiles indicates that both the local rotational
correlation time and the global rotational correlation time
(Table 1) are more than doubled as compared with the free
dimer, whereas the S2 value is sensibly reduced (from 0.66
to 0.23), suggesting great flexibility of the adamantyl
moiety in the b-CD cavity.
Supramolecular adducts with b-CD and poly-b-CD
As mentioned already, an alternative route for increasing
the relaxivity consists in the formation of host–guest non-
covalent interactions between suitably functionalized
complexes and slowly tumbling substrates. A major
advantage is that the structural integrity of the complex is
preserved, and then the unbound form can be easily sepa-
rated from the free from. In addition, the association
constant can be modulated as it depends on the physico-
chemical properties of the targeting group. The relaxivity
of the supramolecular structure depends on several prop-
erties of the paramagnetic guest: the number of bound
water molecules (q), their rate of exchange with the bulk,
and the degree of rotational mobility of the complex in the
bound form, which is also correlated with the compactness
and stereochemical rigidity of the free complex. Finally,
also the nature of the pendant group dictates the strength of
the interaction. The presence of an adamantyl moiety in the
ditopic Gd2L1 complex allows its strong interaction with
the hydrophobic cavity of b-CDs: for such adducts, high
affinity constants (KA = 103–104 M-1) have often been
reported [13]. In the context of this work, the properties of
the adducts between Gd2L1 and either monomeric b-CD or
high molecular weight poly-b-CD were investigated
An analogous binding interaction was exploited to form
larger supramolecular assemblies using as the host a polymer
of b-CD with a molecular mass of approximately 15 kDa. In
this case up to 13 Gd2L1 units can be loaded into the b-CD
cavities to form a final system (Gd2L1–poly-b-CD) with a
molecular mass of about 34 kDa. Each of the 13 b-CD units
of poly-b-CD can bind the adamantyl moiety of Gd2L1. This
linear polymer is characterized by nearly equivalent binding
sites and, as a consequence, will affect the rotational
dynamics of each bound chelate approximately equivalently.
Consequently, the interaction of Gd2L1 with poly-b-CD
reproduces, to a first approximation, a simple 1:1 binding
model (Fig. 3). The data were fitted to a simple model that
considers the presence of 13 equivalent and independent
binding sites, yielding the values of the association constant
[KA = (1.27 0.13) 9 104 M-1
]
and the relaxivity
1
(r1b = 33.9 0.3 mM-1 s-1) for the bound ditopic com-
plex. The relaxivity enhancement is then of the order of
100 % over the dimer and 61 % as compared with Gd2L1–b-
CD. It is worth stressing that the r1b value represents an
average value and not the absolute value of a well-identified
ditopic chelate. The NMRD profiles were recorded at two
temperatures under conditions ensuring that more than 98 %
of the chelate was bound to the macromolecular substrate
(Fig. 3, right). Fitting the profiles to the SBM theory
through the well-established H PRE technique. This con-
sists in measuring the variation in R1 for increasing con-
centrations of the host. With this method, the binding
parameters KA, n (the number of equivalent and indepen-
dent binding sites), and r1b (the relaxivity of the resulting
supramolecular adducts) can be assessed. To this end, a
dilute solution of Gd2L1 was titrated with b-CD, at
20 MHz and 298 K, and the least-squares fit of the R1
versus b-CD concentration binding isotherm (Fig. 2, left)
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Fig. 3 Left: Water proton relaxation rate of a 0.05 mM aqueous solution of Gd2L1 as a function of increasing amounts of poly-b-CD (20 MHz,
298 K). Right: NMRD profiles of Gd2L1–poly-b-CD at 298 and 310 K
(including the Lipari–Szabo model) affords the parameters
listed in Table 1.
be explained on the basis of an increased rigidity of the
systems (greater sRL) due to multisite interactions that
cannot occur in the case of the monoadamantyl-substituted
bimetallic chelate.
The marked decrease in molecular tumbling is reflected
in the formation of relaxivity peaks centered at about
40 MHz, distinctive of slowly rotating systems. However,
an increase of the local degree of rotation of the discrete
dimer at the binding site also occurs, which limits the re-
laxivity. This is shown by the decrease of S2 (from 0.29 for
Gd2L1–b-CD to 0.17 for Gd2L1–poly-b-CD) and by the
larger difference between sRG (2.3 ns) and sRL (410 ps).
A comparison between the relaxivity values of the
supramolecular adducts Gd2L1–b-CD and Gd2L1–poly-b-
CD and those measured for related paramagnetic systems
underlines the fact that the r1b values reported here are the
highest for chelates functionalized with a single hydro-
phobic group [18, 20]. On the other hand, GdIII complexes
of DTPA, DOTA, or 1,4,7,10-tetraazacyclododecane-1,4,7-
triacetic acid (DO3A) bearing three cyclohexyl or benzyl-
oxy groups were found to form inclusion compounds with
b-CD or poly-b-CD of even higher relaxivity [19]. This can
Interaction with HSA
The presence of a hydrophobic pendant arm on the GdIII
complex makes possible noncovalent interactions with
hydrophobic binding sites on HSA. The noncovalent
binding with plasma proteins is generally exploited to
increase the lifetime of the paramagnetic probe in the
vascular system, for angiographic MRI applications, and
to expand the time window for imaging. The interaction is
accompanied by a strong relaxivity enhancement associ-
ated with the reduced tumbling rate (lengthening of sR) of
the adduct [1, 4, 5, 13]. Like for the case of b-CD, the
binding parameters were determined according to the PRE
method by measuring R1 as a function of the concentra-
tion of added protein (Fig. 4, left). Fitting of the
Fig. 4 Left: Water proton relaxation rate of an aqueous solution of 0.07 mM Gd2L1 as a function of increasing amounts of human serum
albumin (HSA) (20 MHz, 298 K). Right: NMRD profiles of Gd2L1–HSA at 298 and 310 K
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J Biol Inorg Chem (2014) 19:133–143
139
Fig. 5 Top: Comparison of the signal enhancement in tumor, liver, and
kidneys, calculated from the mean signal in the region of interest with
respect to the precontrast images, after intravenous injection of
0.05 mmol/kg Gd2L1 (top left) and ProHance (top right). Bottom:
Axial T1-weighted magnetic resonance images recorded at 1 T on a
mouse before (proton relaxation enhancement, PRE) and after (30 min,
6 h, and 24 h) the administration of 0.05 mmol/kg Gd2L1 (A) and
ProHance (B). The tumor region is highlighted with a red circle
experimental data was performed assuming the presence
of one equivalent and independent binding site (n = 1),
even though the presence of multiple affinity sites on
HSA cannot be excluded. In this way, we could estimate
the value of the thermodynamic association constant,
which was rather similar to that with b-CD
[KA = (1.2 0.8) 9 104 M-1], and the relaxivity of
the resulting paramagnetic metalloprotein (r1b = 41.4
1.1 mM-1 s-1). The NMRD profiles of the Gd2L1–HSA
adduct were measured at 298 and 310 K (Fig. 4, right) for
a 0.07 mM solution of Gd2L1 in the presence of 1.0 mM
HSA. Under this condition, more than 99 % of the ditopic
complex is bound to the protein. The data were analyzed
only in the high field region because of the known limi-
tations of the SBM theory to completely account for the
behavior of slowly rotating systems at low magnetic field
strengths [13]. The r1b value represents an additional
increase of about 20 % compared with the relaxivity of
Gd2L1–poly-b-CD and is comparable to that reported
for the commercial blood pool contrast agent MS-325
(r1b = 49 mM-1 s-1) [36]. The best-fit parameters (Table 1)
indicate that the relaxivity of the adduct is markedly
limited by the poor correlation between global and local
rotational motions. A more pronounced degree of immo-
bilization by protein binding would make it possible to
attain a further, large relaxivity enhancement. It is worth
noting that even a nonoptimal rate of exchange could
hamper the achievement of higher relaxivity; however,
the decrease of r1b with increasing temperature suggests
that this parameter is not a dominant factor.
In vivo MRI analysis
In vivo tumor, liver, and kidney accumulation of Gd2L1
was investigated through a 24-h acquisition of magnetic
resonance images at 1 T of B16-tumor-bearing mice.
Control experiments were conducted by using the com-
mercial nonspecific contrast agent ProHanceÒ [gadolinium
10-(2-hydroxypropyl)-DO3A] at the same time intervals
and concentration (Fig. 5). We subcutaneously injected
approximately 1 9 106 B16 murine melanoma cells into
C57Bl/6 female mice (6–7 weeks old). Tumor growth was
123

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J Biol Inorg Chem (2014) 19:133–143
monitored, and the mice underwent MRI analysis
10–12 days after inoculation, when the tumor dimensions
were about a few hundred cubic millimeters. We noticed a
strong signal enhancement in the tumor region up to
50 min after the administration of Gd2L1 (0.05 mmol/kg),
which was not observed when ProHanceÒ was adminis-
tered. Moreover, the signal enhancement in the tumor
appears to be significantly high (approximately 40 %) even
24 h after the administration of the probe. The enhanced
tumor retention observed after the administration of Gd2L1
is likely related to its high affinity towards serum albumin.
The binding with serum proteins allows prolonged reten-
tion of the paramagnetic probe in the vascular system and
its gradual accumulation in the tumor region on the basis of
the well-known enhanced permeability and retention effect
[37]. Albumin-bound MRI contrast agents pass through the
highly permeable blood vessels in tumor, but not in normal
tissues, and do not diffuse back into blood capillaries or
end up in the lymphatic system. In contrast, the neutral and
nonspecific ProHanceÒ complex, which is not able to bind
to serum proteins, is rapidly eliminated from the vascular
system and does not accumulate in the tumor region. The
signal enhancements observed for liver and kidney suggest
a preferential renal elimination of Gd2L1, as expected for
low molecular weight gadolinium-based contrast agents.
The mean signal enhancement percentage in kidneys
reaches a peak during the first 30 min, with a subsequent
decrease after 6 and 24 h due to the renal elimination (see
the electronic supplementary material). Despite the lipo-
philic properties of Gd2L1 and its quite strong interaction
with serum albumin, a very low hepatic accumulation has
been observed [38].
Fig. 6 1H relaxivity values of GdAAZTA (AAZTA is 6-amino-6-
methylperhydro-1,4-diazepinetetraacetic acid) and GdL1 and its
supramolecular adducts with b-CD, poly-b-CD, and HSA (20 MHz,
298 K)
gadolinium atom show a constant increase from Gd2L1 to
Gd2L1–HSA, corresponding to an enhancement of up to
approximately 250 %, as illustrated in Fig. 6. In conclu-
sion, as the CD cavity may also host drug molecules,
properly functionalized targeting vectors, or optical dyes,
the optimized Gd2L1 complex has good potential to be
used in CD-based probes in association with a vector or a
dye for molecular imaging applications or in association
with a drug for theranostic or pharmaceutical uses.
Materials and methods
All chemicals were purchased from Sigma-Aldrich or Alfa
Aesar and were used without further purification. Pro-
HanceÒ was kindly provided by Bracco Imaging (Colle-
retto Giacosa, Italy). ‘‘Petroleum ether’’ refers to petroleum
ether with a boiling point in the range 313–333 K. Poly-b-
CD, consisting of a mixture of oligomers (molecular mass
3–15 kDa), was purchased from Cyclolab (Budapest,
Hungary). According to the producer, adjacent b-CD units
are linked through a 1-chloro-2,3-epoxypropane group. The
commercial product (1.5 g) was dissolved in water
(40 mL) and dialyzed for 20 h against water using cellu-
lose membrane dialysis tubing of size 25 mm 9 16 mm
(molecular mass cutoff 14 kDa, Sigma). The final com-
pound was freeze-dried, yielding 1.03 g of high molecular
weight poly-b-CD as a white solid, which was analyzed by
high-performance liquid chromatography (HPLC; Waters
Conclusions
A novel ditopic GdIII complex bearing a hydrophobic ad-
amantyl moiety, based on two stable and q = 2
GdAAZTA-like complexes, has been synthesized and
characterized. The 1H relaxometric properties and the
ability to form supramolecular adducts with CD, poly-b-
CD, and HSA have been investigated in detail. Gd2L1 has
several excellent properties which make it a promising
highly efficient MRI probe: (1) each GdIII ion possesses
two inner-sphere water molecules characterized by a suf-
ficiently fast rate of exchange; (2) it is stable over a wide
pH range; (3) it has a good degree of compactness and
limited rotational flexibility, which results in a sR value
nearly ideal for MRI applications at high field strengths
(1.5–3.0 T); (4) it is endowed with a strong ability to
interact noncovalently with a number of substrates of
increasing complexity (KA * 104 M-1), thus affording
large relaxivity enhancements. The relaxivity values per
˚
BioSuite size-exclusion chromatography column, 125 A,
4.6 mm 9 300 mm; see the electronic supplementary
material). Thin-layer chromatography was conducted on
silica plates (silica gel 60 F254, Merck 5554), with visual-
ization with a UV lamp (254 nm) or staining with basic
KMnO4 solution. Preparative column chromatography was
123

J Biol Inorg Chem (2014) 19:133–143
141
conducted using silica gel (Merck silica gel 60, 230 400
1
15.9 min. 1H NMR (500 MHz, D2O) d 7.37 (br s, 2H, CHAr),
7.19 (br s, 1H, CHAr), 4.18 (s, 4H, CH2O), 5.28 (s, 16H,
CH2C=O), 2.80–2.57 (m, 16H, cycle), 1.96 (s, 2H, CH2Ad),
1.93–1.51 (m, 13H,CH/CH2MeAd). 13C NMR (125 MHz,
D2O): d 181.0 (CS), 180.5, 179.3 (COOH), 155.3 (NCO),
137.8, 120.1 (CAr), 118.2, 115.4 (CHAr), 66.2 (CH2O), 64.3
mesh) presoaked in the starting eluent. H and 13C NMR
spectra were recorded with Bruker Avance III spectrome-
ters operating at 11.74 T. Chemical shifts are reported
relative to tetramethylsilane and were referenced using the
residual proton solvent resonances. Chemical shifts are
reported in parts per million and splitting patterns are
described as singlet (s), broad singlet (br s), doublet (d), or
multiplet (m). Electrospray ionization (ESI) mass spectra
were recorded with a SQD 3100 mass detector (Waters),
operating in positive or negative ion mode, with 1 % v/v
HCOOH in methanol as the carrier solvent. HPLC analyses
were performed with a Waters 1525 liquid chromatograph
equipped with a Waters 2489 UV/vis detector and a Waters
SQD 3100 mass spectrometer and using a Waters AtlantisÒ
reversed-phase C18 column (150 mm 9 4.6 mm, 5 lm).
(CH2C=O), 63.0 (CqCy), 60.6, 56.6 (CH2Cy), 57.9 (CH2Ad),
39.9, 39.7, 36.6, 36.5, 28.1 (CAd). ESI? MS: m/z 1,138
(M ? H?), 569 (M ? 2H?)/2 (calcd for C48H69N10O20S,
1,138.17).
Synthesis of Gd2L1
Gd2L1 was synthesized by complexometric titration mon-
itoring the change in the longitudinal water proton relax-
ation rate (R1) as a function of the amount of Gd3? added.
Gd2L1 was also synthesized on a larger scale by adding
dropwise 1 mL of a water solution of GdCl3 (34.4 mg,
0.0926 mmol) at pH 6.4 to a solution of L1 (42 mg,
0.037 mmol) in H2O (10 mL) while keeping the pH
between 5.5 and 6.5. The mixture was further stirred for
1.5 h. The pH of the solution was then adjusted to 11 with
0.1 M NaOH to precipitate the excess of Gd3? as Gd(OH)3.
The suspension was centrifuged, filtered, and lyophilized to
obtain Gd2L1 as a white solid. HPLC analysis: H2O (sol-
vent A) and CH3OH (solvent B) starting from 100 % sol-
vent A for 5 min and then with a gradient to 100 % in
11 min at a flow rate of 1 mL min-1 over a total run of
20 min; retention time 9.86 min. ESI- MS: m/z: 721 (M-
2H?)/2 [calcd for (C48H60Gd2N10O20S)/2, 721.8]; isotopic
distribution consistent with GdIII complex.
Synthesis of II
A
solution of (1-adamantyl)methylamine (33 mg,
0.199 mmol) in dry CH2Cl2 (2 mL) was added to a solution
of I (243 mg, 0.153 mmol) and Et3N (97 lL, 0.701 mmol)
in dry CH2Cl2 (10 mL) under an N2 atmosphere. The
mixture was stirred overnight at room temperature, and
then it was evaporated in vacuo. The crude product was
purified by column chromatography (SiO2, 7:3 petroleum
ether/EtOAc), yielding II as a yellow oil (124 mg,
1
0.109 mmol, 71 % yield). H NMR (400 MHz, CD3Cl): d
7.58 (br s, 2H, CHAr), 7.29 (s, 1H, CHAr), 7.17 (br s, 2H,
NHCO), 6.47 (br s, 2H, NHCS), 4.22 (s, 4H, CH2O), 3.25
(s, 16H, CH2C=O), 3.10–2.64 (m, 16H, CHC2 y), 2.04–1.51
(m, 13H, CHAd), 2.02 (s, 2H, CH2Ad), 1.42 (br s, 72H,
CH3). 13C NMR (100 MHz, CDCl3): d 172.0, 170.8
(COOBut), 153.1 (NCO), 140.4 (CqAr), 108.2 (CHAr), 105.9
1H NMR relaxation measurements
(CHAr), 81.0, 80.9 (CCH3), 67.9 (CH2O), 63.3 (CCq y), 62.3,
The noncovalent interaction of Gd2L1 with b-CD, poly-b-
CD, and HSA was evaluated using the PRE method: R1 was
monitored as a function of the amount of b-CD, poly-b-CD,
and HSA added to solutions of 0.1–0.05 mM Gd2L1, at
298 K and 20 MHz, while maintaining the pH at 6–7. The
exact concentrations of gadolinium were determined by
measuring the bulk magnetic susceptibility shifts of the t-
BuOH 1H NMR signal with a Bruker Avance III spectrom-
eter (11.7 T). The 1H relaxation times (T1) were acquired by
the standard inversion recovery method with a typical 90°
pulse width of 3.5 ls, and 16 experiments of four scans. The
reproducibility of the T1 data was 5 %. The temperature
was controlled with a VTC-91 airflow heater (Stelar, Mede,
Italy) equipped with a calibrated copper–constantan ther-
mocouple (uncertainty of 0.1 K). The proton 1/T1 NMRD
profiles were measured with a fast field-cycling Stelar
SmarTracer relaxometer over a continuum of magnetic field
strengths from 0.00024 to 0.25 T (corresponding to proton
62.1 (CH2CO), 59.0, 51.6 (CHC2 y), 41.9 (CH2Ad), 37.1,
34.1, 21.1, (CAd), 28.3, 28.2 (CH3). ESI? mass spectrom-
etry (MS): m/z 1,587 (M ? H?), 794 (M ? 2H?)/2 (calcd
for C80H133N10O20S, 1,587.02).
Synthesis of L1
TFA (3 mL) was added dropwise to a solution of II (124 mg,
0.0782 mmol) in CH2Cl2 (3 mL). The mixture was stirred at
room temperature overnight and then evaporated in vacuo.
The solid residue was washed twice with Et2O (10 mL) and
isolated by centrifugation. Then it was dried in vacuo,
resulting in L1 as its trifluoroacetate salt. HPLC analysis:
H2O/0.1 % TFA (solvent A) and CH3OH/0.1 % TFA (sol-
vent B) starting from 100 % solvent A for 5 min and then
with a gradient to 100 % in 11 min at a flow rate of
1 mL min-1 over a total run of 20 min; retention time
123

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J Biol Inorg Chem (2014) 19:133–143
4. Aime S, Botta M, Fasano M, Terreno E (2001) In: Merbach AE,
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operated under computer control with an absolute uncer-
tainty in 1/T1 of 1 %. Additional data points in the range
15–70 MHz were obtained with a Bruker WP80 NMR
electromagnet adapted to variable-field measurements.
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a 3.5 cm inner diameter transmitter–receiver birdcage coil.
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after administration of Gd2L1 (three mice) and ProHanceÒ
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IMDMT and Nano-IGT projects) and Ministero dell’Universita e
della Ricerca (PRIN 2009) for support. This study was performed
under the auspices of the Consorzio Interuniversitario di Ricerca in
Chimica dei Metalli nei Sistemi Biologici and EU-COST Action
TD1004.
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