Gadolinium-doped LipoCEST agents: A potential novel class of dual 1H-MRI probes

Article abstract of DOI:10.1039/c1cc10172b

A novel class of paramagnetic liposome-based systems acting as dual T ₁ and CEST ¹H-MRI contrast agents is described. The vesicles contain a shift reagent in the aqueous core and a Gd-complex on the external surface conjugated throug

Full text of DOI:10.1039/c1cc10172b

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COMMUNICATION
Gadolinium-doped LipoCEST agents: a potential novel class of dual
1H-MRI probesw
Enzo Terreno,*a Cinzia Boffa,a Valeria Menchise,b Franco Fedeli,c Carla Carrera,c
Daniela Delli Castelli,a Giuseppe Digiliod and Silvio Aimea
Received 10th January 2011, Accepted 22nd February 2011
DOI: 10.1039/c1cc10172b
A novel class of paramagnetic liposome-based systems acting
as dual T1 and CEST 1H-MRI contrast agents is described.
The vesicles contain a shift reagent in the aqueous core and a
Gd-complex on the external surface conjugated through a bio-
degradable linker. As such, the probe can generate T1 contrast
only, but after the cleavage and removal of the Gd-coating, the
CEST contrast is switched on.
agents. A recently published example, still involving a dual
1H–19F agent, is represented by thermosensitive liposomes
co-encapsulating a paramagnetic shift reagent (SR) and a
19F-containing molecule.8 The former label enables the vesicles
to act as highly-sensitive liposomal CEST (dubbed LipoCEST,
CEST: Chemical Exchange Saturation Transfer) agents,9 thus
allowing the MRI detection of intact vesicles, whereas the
19F signal, silent for intact liposomes (due to the severe line
broadening effects operated by the SR), is generated following
the release of the liposomal content when the tempera-
ture exceeds the gel-to-liquid transition point of the vesicle
membrane.
Despite the good performance displayed by dual 1H–19F
agents, it is worth reminding that they require specific coils/
hardware for allowing the double signal detection necessary to
optimize the images overlay without changing the image
geometry.
Magnetic Resonance Imaging (MRI) agents able to generate
different contrast modalities have received recently a growing
interest in virtue of their potential to improve the already wide
application range of these xenobiotics. Most of the work reported
so far in this field has dealt with heteronuclear 1H–19F agents. In
some examples, the presence of paramagnetic Gd(III) centers is
exploited in virtue of the well known ability of such chemicals to
shorten the T1 relaxation of 19F spins through dipolar coupling,
thus reducing the total acquisition time of the MR images.1–3
Another explored possibility involves the use of 19F-containing
ligands in which the complexation with lanthanide ions acting as
shift reagents facilitates the 19F detection and offers intriguing
biomedical applications.4,5 In other cases, in addition to the
19F signal, also the proton T1 contrast generated by the Gd(III)
complexes was exploited to design smart tracers able to report
about drug delivery and release processes6 or to monitor pH in a
concentration-independent manner.7
Hence, the development of MRI homonuclear, possibly
1H, multicontrast agents appears to be still an important task,
and nanovesicular systems like liposomes are well suited
for achieving this scope. Interesting results on this class of
agents have been recently reported, where the capability of
paramagnetically labeled liposomes to simultaneously act as
dual T1/T2 (if encapsulating a Gd(III) complex) or T2/CEST
(if encapsulating a lanthanide-based shift reagent) 1H MR
agents has been exploited for investigating the in vivo intratumor
trafficking of the vesicular nanocarriers.10
On this basis, we deemed that the design of a dual 1H probe
in which one contrast is detectable only after the disappearance
of the other one could open new interesting applications,
especially in the field of smart tracers and reporters of drug
delivery/release processes.
More recently, the increased demand for developing MRI
protocols aimed at aiding and monitoring therapies has stimulated
research on dual, and more in general, on multicontrast
a Department of Chemistry IFM and Molecular & Preclinical Imaging
Centres, University of Torino, via Nizza 52, 10126, Torino, Italy.
E-mail: enzo.terreno@unito.it; Fax: +39 011-6706487;
Tel: +39 011-6706452
This communication reports about the first dual T1-CEST
liposomal agent, in which the CEST contrast can be detected
only after the removal of the Gd(III) units responsible for the
generation of T1 contrast. The mechanism of action of
the agent relies on the observation that CEST contrast arises
from the chemical exchange-mediated transfer of saturated
magnetization from a pool of protons of the agent, properly
irradiated by a radiofrequency pulse centered at their resonance
frequency, to the bulk site.11 Thus, if the longitudinal relaxa-
tion time of the proton at the bulk site is shorter than its
residence lifetime at that site, then the magnetization associated
b Institute for Biostructures and Bioimages (CNR),
c/o Molecular Biotechnology Center, University of Torino,
via Nizza 52, 10126, Torino, Italy
c Laboratory of Integrated and Advanced Methodologies,
Bioindustry Park of Canavese, via Ribes, 5, 10100,
Colleretto Giacosa (TO), Italy
d Department of Life and Environmental Sciences,
University of Eastern Piedmont ‘‘A. Avogadro’’, Alessandria, Italy
w Electronic supplementary information (ESI) available: Synthesis of
(C18)2–PDP compound, liposomes preparation and basic characteriza-
tion, relaxometric determination of the amount of Gd(III) and Tm(III)
complexes loaded in the liposomes, and Nuclear Magnetic Resonance
Dispersion (NMRD) profiles. See DOI: 10.1039/c1cc10172b
c
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Scheme 1 Schematic representation of the dual T1-CEST 1H-MRI
agent. The CEST contrast is activated following the cleavage, triggered
by a specific variable, and rapid wash-out of the Gd-units from the
biological target (SR: paramagnetic Shift Reagent).
with the spin will be no longer saturated, thus reducing or even
nullifying the CEST effect.
Scheme 2 Preparation of dual T1-CEST 1H-MRI liposomes. The
dashed arrow represents the transformation due to occur under a
reducing microenvironment.
The desired T1 shortening can be successfully achieved
by conjugating paramagnetic Gd(III) complexes on the outer
surface of a conventional LipoCEST agent. In such a way, the
system can generate T1 contrast, whereas the CEST contrast
will be silenced (Scheme 1). As long as the Gd(III)-coating is
removed and cleared from the biological microenvironment
hosting the nanoprobe, T1 contrast will disappear, whereas the
CEST contrast is restored. If the removal of the T1 agent is
triggered by a variable acting as a biomarker of a given
pathology (e.g. pH, enzymes, . . .), this dual probe can provide
information on the accumulation of the agent (via T1) and
signal the presence of the triggering variable (via CEST).
The in vitro demonstration of the performance of this new agent
was gained by preparing a conventional (i.e. spherical) LipoCEST
agent containing 75% (in moles) of DPPC (di-palmitoyl-
glycero-phosphatidylcholine), 5% of the negatively charged PI
(L-a-phosphatidylinositol, necessary for stabilizing the vesicles),
and 20% of an amphiphilic molecule, (C18)2–PDP, bearing a
3-(2-pyridyl)-dithio-propionate moiety at the terminal end of
the polar head (Chart 1, see ESIw for the synthetic details).
The necessary chemical shift separation between intra- and
extra-liposomal water protons was obtained by exploiting
the lanthanide-induced shift generated by the encapsulation
of a sufficient amount of the shift reagent (Tm-DOTMA)À
(Chart 1, synthesized according to the procedure reported in
ref. 12) in the liposome. A Dintralipo offset of 3.5 ppm, typical
for conventional LipoCEST,9 was observed at 37 1C. The
choice of exposing the reactive PDP moiety on the outer
surface of the LipoCEST was done in order to conjugate a
certain number of Gd(III) complexes via a disulfide linkage.
This is a typical ‘‘labile’’ bond prone to be reduced in bio-
logical environments, and the presence of a long spacer between
PDP and the aliphatic chains was designed to facilitate the
coupling reaction. Scheme 2 reports the three liposomal prepara-
tions tested in the work. As proof of concept, the Gd–DO3A–PDP
complex (Chart 1, synthesized according to the procedure reported
in ref. 13) was selected for the coupling with the LipoCEST. First,
the PDP groups exposed on the vesicle surface were transformed
to free thiols using a 5 : 1 molar excess of the reducing agent
TCEP (tris(2-carboxyethyl)-phosphine).
The reaction was carried out at room temperature and its
completeness was reached after 1 h as determined by measuring
the absorbance at 343 nm of the reaction product pyridine-
2-thione. The obtained LipoCEST–SH agent displayed good
CEST properties (Fig. 1, sample 2 right), thus indicating that
the reduction reaction did not affect any of the determinants of
the CEST contrast. After removing the unreacted TCEP by
exhaustive dialysis, the LipoCEST–SH agent was conjugated
to the PDP-containing Gd(III) complex. Again, the pyridine-
2-thione absorbance was used to monitor the reaction and, after its
completeness, the unreacted Gd-agent was removed from the
LipoCEST–S–S–Gd suspension by dialysis. The effective coupling
of the T1 agent to the vesicles surface was confirmed by measuring
the relaxivity of the liposomes suspension.
A value of 10 sÀ1 mMÀ1, about 20% times larger than
the relaxivity of the non-conjugated complex, was obtained at
0.5 T and 25 1C. The small relaxivity enhancement is likely the
result of a relatively fast rotational tumbling of the conjugated
complex due to the presence of the very long and flexible
linker. However, the acquisition of the magnetic field dependence
of the relaxivity confirmed the effective linkage of the complex,
because the profile displayed the typical broad hump indicative
Chart 1 Non-commercially available compounds used for the formula-
tion of the herein proposed dual agent.
c
4668 Chem. Commun., 2011, 47, 4667–4669
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detection can be still exploited using LipoCEST agents with
different saturation frequencies and conjugated with Gd(III)
complexes through linkers sensitive to different stimuli.
This research was supported by funding from Regione
Piemonte (PIIMDMT and Nano IGT projects), EU-FP6
project Meditrans (NMP4-CT-2006-026668) and ESF COST
Action D38 (metal-based systems for molecular imaging applica-
tions). Bracco Imaging S.p.A. is gratefully acknowledged for
providing the DOTMA ligand.
Fig. 1 MR images at 7 T and 37 1C of: (1) LipoCEST–PDP,
(2) LipoCEST–SH, (3) LipoCEST–S–S–Gd, and (4) sample 3 after
reduction with TCEP and removal of the detached Gd–DO3A–SH
complex. Left: T1w image. Right: CEST map upon irradiation at
3.5 ppm overlaid to a T2w-image of the phantom.
Notes and references
1 A. M. Neubauer, J. Myerson, S. D. Caruthers, F. D. Hockett,
P. M. Winter, J. Chen, P. J. Gaffney, J. D. Robertson,
G. M. Lanza and S. A. Wickline, Magn. Reson. Med., 2008, 60,
1066.
of the occurrence of a slower rotational mobility of the Gd(III)
complex (see ESIw).
2 E. Terreno, M. Botta, W. Dastru and S. Aime, Contrast Med.
Mol. Imaging, 2006, 1, 101.
3 H. Lee, R. R. Price, G. E. Holburn, C. L. Partain, M. D. Adams
and W. P. Cacheris, J. Magn. Reson. Imaging, 1994, 4, 609.
4 A. M. Kenwright, I. Kuprov, E. De Luca, D. Parker, S. U.
Pan-dya, P. K. Senanayake and D. G. Smith, Chem. Commun.,
2008, 2514.
5 K. H. Chalmers, H. Kirsten, E. De Luca, N. H. M. Hogg,
A. M. Kenwright, I. Kuprov, D. Parker, M. Botta, J. L. Wilson
and A. M. Blamire, Chem.–Eur. J., 2010, 16, 134.
6 R. E. Port, C. Schuster, C. R. Port and P. Bachert,
Cancer Chemother. Pharmacol., 2006, 58, 607.
Importantly, the LipoCEST–S–S–Gd sample did generate
a good T1 contrast at 7 T, whereas the CEST effect was
significantly quenched (Fig. 1, sample 3). The performance
of the LipoCEST–S–S–Gd agent as a dual MRI reporter of
reducing environment was tested in vitro by adding TCEP and
removing the detached Gd(III) chelate in the reduced form
(Gd–DO3A–SH). The obtained suspension, containing the
LipoCEST–SH probe, was silent in the T1w image, but fully
recovered the CEST contrast (Fig. 1, sample 4).
7 E. Gianolio, R. Napolitano, F. Fedeli, F. Arena and S. Aime,
Chem. Commun., 2009, 6044.
In summary, a novel dual 1H T1-CEST liposomal MR agent
has been proposed. The probe has the peculiarity to be
detected in the T1w image as long as a triggering stimulus
cleaves the Gd(III)-complex from the vesicle, thus silencing the
T1 contrast and switching on the CEST effect that in such a
way becomes responsive to the triggering factor. It is impor-
tant to remind that the efficacy of this probe is based on the
integrity of the vesicle. Work is in progress to use these agents
in cell-containing alginate capsules where T1 contrast may
report about cell localization and the CEST effect may provide
information about the functional status of the cells. In addition,
the peculiar property of CEST agents to allow multiple probe
8 S. Langereis, J. Keupp, J. L. J. van Velthoven, I. H. C. de Roos,
D. Burdinski, J. A. Pikkemaat and H. J. Gruell, J. Am. Chem. Soc.,
2009, 131, 1380.
9 S. Aime, D. Delli Castelli and E. Terreno, Angew. Chem., Int. Ed.,
2005, 44, 5513.
10 D. Delli Castelli, W. Dastru, E. Terreno, E. Cittadino, F. Mainini,
E. Torres, M. Spadaro and S. Aime, J. Controlled Release, 2010,
144, 271.
11 E. Terreno, D. Delli Castelli and S. Aime, Contrast Med. Mol.
Imaging, 2010, 5, 78.
12 S. K. Hekmatyar, P. Hopewell, S. K. Pakin, A. Babsky and
N. Bansal, Magn. Reson. Med., 2005, 53, 294.
13 G. Digilio, V. Menchise, E. Gianolio, V. Catanzaro, C. Carrera,
R. Napolitano, F. Fedeli and S. Aime, J. Med. Chem., 2010, 53,
4877–4890.
c
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Chem. Commun., 2011, 47, 4667–4669 4669