ORIGINAL PAPER

A new ditopic GdIII complex functionalized with an adamantylmoiety as a versatile building block for the preparationof 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

� 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

the 1H 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

host–guest adducts were investigated using 1H 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

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.

Keywords Gadolinium � Relaxivity � Contrast

agents � Magnetic resonance imaging �Supramolecular chemistry

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

the established theory of paramagnetic relaxation, large

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00775-013-1050-0) contains supplementarymaterial, 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

J Biol Inorg Chem

DOI 10.1007/s00775-013-1050-0

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 supramolec-

ular 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 function-

alities, 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 development 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

J Biol Inorg Chem

123

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

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.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 magnetic fields

(r40 MHz1 ¼ 17:6 mM�1 s�1; r70MHz

1 ¼ 17:4 mM�1 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

4.0 A, a typical value for small metal chelates, and for 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

N

N

NROOC

ROOC

ROOC COOR

ONH

O

N

N

NCOOR

COOR

COORCOOR

O

HN

O

SCN

N

N

NCOOR

COOR

COORCOOR

OHN

O

N

N

NROOC

ROOC

ROOCCOOR

ONH

O

NH

HN

S

ii R= t -Bu

L1 R= HII

i

iii

I

NH

HN

S

HN NH

O O O ON

NNO

OO

Gd3+

O

OO OO

N

N NO

OO

Gd3+

O

O OOO

Gd2L1

Scheme 1 Synthesis of Gd2L1: i (1-adamantyl)methylamine,

CH2Cl2, Et3N; ii trifluoroacetic acid, CH2Cl2; iii GdCl3, H2O, pH 6.5

J Biol Inorg Chem

123

[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.

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

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

sRG, 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

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

Table 1 Best-fit parameters obtained by analysis of the 1H 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

298r1p (mM-1 s-1)b 16.7 21.6 33.9 41.4 7.1

D2 (1019 s-2) 2.5 1.2 1.3 1.4 2.2298sV (ps) 31 44 37 21 31298kex (106 s-1) 11.1c 11.1c 11.1c 11.1c 11.1298sRG (ns) 0.29 0.62 2.3 28 0.074298sRL (ps) 107 227 410 479 –

S2 0.66 0.29 0.17 0.10 –

qc 2 2 2 2 2

rGdH (A)c 3.0 3.0 3.0 3.0 3.1

a (A)c 4.0 4.0 4.0 4.0 4.0298D (10-5 cm2 s-1)c 2.24 2.24 2.24 2.24 2.24

AAZTA 6-amino-6-methylperhydro-1,4-diazepinetetraacetic acida From [6, 7]b At 20 MHzc Fixed in the fitting procedure

J Biol Inorg Chem

123

ditopic complex. This is also evidenced by the large value

of the order parameter S2.

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

through the well-established 1H 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 rb1 (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)

enabled us to calculate the affinity constant [KA =

(1.05 ± 0.09) 9 104 M-1] and the relaxivity of the adduct

(rb1 = 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.

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

(rb1 = 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 rb1 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

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)

(20 MHz, 298 K). Right: 1H NMRD profiles of Gd2L1 b-CD at 283, 298, and 310 K

J Biol Inorg Chem

123

(including the Lipari–Szabo model) affords the parameters

listed in Table 1.

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 rb1 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

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.

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. 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

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

J Biol Inorg Chem

123

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 (rb1 = 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 rb1 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

(rb1 = 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 rb1 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 ProHance� [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

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

J Biol Inorg Chem

123

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 ProHance� 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 ProHance� 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].

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

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-

Hance� 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

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

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)

J Biol Inorg Chem

123

conducted using silica gel (Merck silica gel 60, 230 ± 400

mesh) presoaked in the starting eluent. 1H 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 Atlantis�

reversed-phase C18 column (150 mm 9 4.6 mm, 5 lm).

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,

0.109 mmol, 71 % yield). 1H NMR (400 MHz, CD3Cl): d7.58 (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, CHCy2 ), 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 (CArq ), 108.2 (CHAr), 105.9

(CHAr), 81.0, 80.9 (CCH3), 67.9 (CH2O), 63.3 (CCyq ), 62.3,

62.1 (CH2CO), 59.0, 51.6 (CHCy2 ), 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

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

(CH2C=O), 63.0 (CCyq ), 60.6, 56.6 (CH

Cy2 ), 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.

1H NMR relaxation measurements

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

J Biol Inorg Chem

123

Larmor frequencies of 0.01–10 MHz). The relaxometer

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.

Animal models

Six C57Bl/6 female mice 6–7 weeks old weighing about

15 g were housed in a ventilated, temperature-controlled

room, with a 12-h light/dark cycle. Subcutaneous injection

of 0.2 ml of a single suspension containing approximately

1 9 106 B16 murine melanoma cells into their flanks was

performed. Tumor growth was monitored, and the mice

underwent MRI analysis 10–12 days after inoculation of

cells. All tumors were approximately the same size at the

time of image acquisition (approximately 200 mm3). The

investigation complied with national legislation about the

care and use of laboratory animals.

MRI acquisition

Mice were anesthetized by injecting them with tiletamine/

zolazepam (Zoletil 100), 20 mg/kg, and xylazine (Rom-

pum), 5 mg/kg, and were placed in the prone position inside

a 3.5 cm inner diameter transmitter–receiver birdcage coil.

Images were acquired using an Aspect M2 system (Aspect

Imaging, Shoam, Israel) operating at 1 T equipped with a

horizontal bore MRI magnet. Coronal spin echo and trans-

verse multislice, fast spin T2-weighted images (rapid

acquisition with relaxation enhancement , effective echo

time 44 ms) were acquired for tumor localization and good

visualization of extratumoral tissues. T1-weighted spin echo

sequences (repetition time 250 ms, echo time 8.9 ms, num-

ber of excitations 5, flip angle 90�) were acquired before and

after administration of Gd2L1 (three mice) and ProHance�

(three mice). Mice were examined by MRI, and a phantom

containing 0.5 mM ProHance solution was inserted in the

field of view and used as an external reference standard.

Acknowledgments The authors thank Regione Piemonte (PI-

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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