Poly-β-cyclodextrin based platform for pH mapping via a ratiometric 19F/1H MRI method

Article abstract of DOI:10.1039/b914540k

The in vitro validation of a new pH mapping MRI method based on a supramolecular poly-β-cyclodextrin-¹⁹F-Gd adduct is reported.

Full text of DOI:10.1039/b914540k

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Poly-b-cyclodextrin based platform for pH mapping via a ratiometric
19F/1H MRI methodw
Eliana Gianolio, Roberta Napolitano, Franco Fedeli, Francesca Arena and Silvio Aime*
Received (in Cambridge, UK) 20th July 2009, Accepted 26th August 2009
First published as an Advance Article on the web 21st September 2009
DOI: 10.1039/b914540k
The in vitro validation of a new pH mapping MRI method based
on a supramolecular poly-b-cyclodextrin–19F–Gd adduct is
reported.
to the poly-b-CD substrate has been ensured by the
functionalization of both molecules with an adamantane
moiety that is known to strongly bind14 the b-CD cavity
(Fig. 1).
The pH responsiveness is provided by a Gd(III)-chelate
whose coordination cage consists of a tetraazamacrocycle
bearing three acetate and one substituted sulfonamide arms.
The protonation–deprotonation step of the latter functionality
takes place at pH = 6.7. Upon deprotonation, the sulfonamide
enters into the coordination cage of the Gd(III) ion by
replacing the two inner sphere water molecules (Scheme 1).
This change in hydration of the paramagnetic centre results in
a dramatic effect on the observed relaxivity of the complex that
becomes strongly pH-dependent at values close to neutrality
(Fig. 2a). Upon formation of the supramolecular adduct with
the poly-b-CD substrate, the lengthening of the molecular
reorientation time yields an increase in relaxivity that is
particularly pronounced at pH o 6 and at a magnetic field
of ca. 1 T (Fig. 2a). This gain in relaxivity enhancement
observed at acidic pHs and at fields around 1 T is clearly
depicted in Fig. 2b, where the NMRD profiles of the supra-
molecular adduct at pH 5.8 and 8.4 are reported. In acidic
conditions, a strong relaxivity peak centred around 1 T is
observed for the Gd-complex endowed with two inner sphere
water molecules, whereas in basic conditions a lower enhance-
ment is obtained in the case of the outer sphere system.
The binding affinity of the adamantane functionalized Gd-
complex towards the poly-b-CD substrate has been assessed
by the proton relaxation enhancement (PRE) method15 by
titrating a 0.1 mM solution of the paramagnetic complex with
Mapping pH is an important task in medical imaging as
changes in pH usually accompany the development of various
pathologies such as tumours, strokes and infections.
Several attempts to map pH by MRI have been
reported.1–10 One method relies on the use of paramagnetic
Gd(III) complexes whose structures have been designed in
order to make their relaxivities (r1p) responsive to the pHs of
the microenvironments in which they are distributed. In order
to be usable, the method requires an independent knowledge
of the concentration of the paramagnetic complex; otherwise
the observed changes in water protons T1 may be ascribed
either to changes in the relaxivity (r1p) or to changes in the
concentration.11 Sherry, Gillies et al.12,13 tackled this issue in
in vivo pH mapping experiments by assuming that the local
concentration of the pH-responsive agent was the same as that
of a related non-responsive paramagnetic complex. Clearly
this approach may be misleading as minor structural
changes in the complex may result in dramatic effects on its
biodistribution.
In principle, one may recover the concentration data by
introducing on the surface of the pH-responsive agent a
concentration reporting moiety consisting, for instance, of
an ensemble of highly sensitive NMR heteronuclei (e.g. 19F,
31P, 13C). The major drawback of this approach is related to
the broadening of the heteronuclear resonance caused by its
proximity to the fast relaxing Gd(III) ion. It is evident that the
occurrence of broadened resonances invariably leads to
inaccuracies in the determination of the concentration
parameter.
Herein an alternative route to developing a ratiometric
Gd/19F method for pH mapping is reported. Instead of being
covalently bound, the Gd(III) and 19F containing moieties are
‘‘hosted’’ by the same carrier, which is a poly-b-cyclodextrin
(poly-b-CD) substrate consisting of 8–10 b-CD units. This
ensures that the 19F nuclei are sufficiently far from the Gd(III)
centre so as to avoid drawbacks in the signal acquisition. The
binding of the Gd-complexz and the 19F-containing reportery
Dept. of Chemistry IFM and Molecular Imaging Center,
Via Nizza 52, Torino, Italy. E-mail: silvio.aime@unito.it;
Fax: +39 0116706487; Tel: +39 0116706451
w Electronic supplementary information (ESI) available: Full details
on the synthesis of the Gd(III)- and F-containing probes, the
determination of their binding affinities toward poly-b-CD and the
calibration lines for the determination of Gd concentration are
provided. See DOI: 10.1039/b914540k
Fig. 1 Schematic representation of the supramolecular adduct
between poly-b-CD, the Gd(III)-complex and the 19F-reporting
molecule designed for pH mapping.
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F-reporter 7.5 mM, Gd-complex 1.5 mM at pH = 7.6;
(5) poly-CD 20 mM, F-reporter 5 mM, Gd-complex 1 mM
at pH = 8.2.
1
Upon acquiring a T1-weighted H-MR image (7.1 T),z the
obtained signal intensities (Fig. 3a) do not reflect in any way
the order of the pH values of the solutions contained in the
capillaries numbered 2–5. Actually tube 4 displays a markedly
higher intensity as a consequence of the highest concentration
of the paramagnetic probe. In Fig. 3b the 19F image of the five
capillaries recorded at 7.1 T has been reported; the 19F signal
intensity in each capillary is obtained on the basis of the ratio
of the signal generated by that capillary to the signal generated
by the reference 25 mM NaPF6 solution in capillary 1. Clearly,
the obtained 19F signal intensities correctly reflect the amounts
of fluorine in the specimens numbered 2–5 and therefore they
can be used to normalize the Gd(III) concentrations in the
T1-weighted 1H-MR images (calibration curves in ESIw). This
operation leads to the transformation of the relaxation
data (R1obs) into relaxivity (r1p) values, thus establishing the
Scheme
1
Mechanism of action of the pH-responsive Gd(III)-
complex: the protonation–deprotonation of the sulfonamide group
produces changes in the complex hydration.
Fig. 2 (a) Profiles of water proton relaxivity as a function of pH
measured for the supramolecular adduct poly-CD–19F-reporter–
Gd-complex (20 mM/5 mM/1 mM) at 298 K and 1 T (’) and 7.1 T
(&) and for the free Gd-complex at 298 K and 1 T (m); (b) NMRD
profiles of the supramolecular adduct poly-CD–19F-reporter–Gd-complex
(20 mM/5 mM/1 mM) at 298 K and pH = 5.8 (’) and 8.4 (&).
poly-b-CD (ESIw). Fitting of the titration curve yielded a KA
value of 1300 Mꢁ1 independent of the pH of the solution.
Analogously, the interaction of the 19F-containing reportery
with poly-b-CD has been assessed by the measurement of the
1H-NMR chemical shift changes upon formation of the
inclusion complex (ESIw). The resulting KA value (14 000 Mꢁ1
)
is one order of magnitude higher than that of the corres-
ponding Gd-complex. The observed behaviour may be
related to the overall enhanced hydrophobicity of the latter
compound as well as to the possible role of the OH group in
forming additional H-bonds with the outer OH groups of the
poly-b-CD substrate.
The proof of concept of this approach has been achieved
in vitro by acquiring the 1H and 19F-MR images of a phantom
consisting of four tubes filled with solutions of poly-
b-CD–19F-reporter–Gd-complex at different values of
concentration and pH. In order to guarantee that both the
Gd-complex and the fluorine reporter are fully bound to the
poly-b-CD substrate, the observed KA values suggested that
the supramolecular adduct components have to be present in
Fig. 3 MR Images of a phantom containing the following solutions:
(1) NaPF6 25 mM; (2) poly-CD 10 mM, F-reporter 2.5 mM,
Gd-complex 0.5 mM at pH = 6.8; (3) poly-CD 13 mM, F-reporter
3.25 mM, Gd-complex 0.65 mM at pH = 7.2; (4) poly-CD 30 mM,
F-reporter 7.5 mM, Gd-complex 1.5 mM at pH = 7.6; (5) poly-CD
20 mM, F-reporter 5 mM, Gd-complex 1 mM at pH = 8.2.
(a) T1-weighted spin-echo 1H-MR image acquired at 7.1
(TR/TE/NEX (80/4.4/32), FOV 1.6 cm, 1 slice 1 mm); (b) T1-weighted
spin-echo 19F-MR Image acquired at 7.1
(TR/TE/NEX
T
the following molar ratio: poly-b-CD
: :
19F-reporter
Gd-complex = 20 : 5 : 1. The phantom (Fig. 3) consists of
five capillaries containing the following solutions: (1) NaPF6
25 mM as a reference specimen for the quantitative assessment
of 19F present in the other four capillaries; (2) poly-CD
10 mM, F-reporter 2.5 mM, Gd-complex 0.5 mM at
pH = 6.8; (3) poly-CD 13 mM, F-reporter 3.25 mM,
Gd-complex 0.65 mM at pH = 7.2; (4) poly-CD 30 mM,
T
(2000/1.4/384), FOV 2 cm, 1 slice 3 mm); (c) T1-weighted spin-echo
1H-MR image acquired at 7.1 T and normalized to the concentrations
found from image (b); (d) T1-weighted spin-echo 1H-MR image
acquired at 1 T (TR/TE/NEX (80/7.2/32), FOV 2 cm, 1 slice, 2 mm)
and normalized to the concentrations found from image (b);
(e) pH-map image derived from image (d).
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relationship between the latter data and the pH values; this is
shown in Fig. 3c.
treatment with CF3COOH removed the tert-butyl protecting groups
and reaction with GdCl3 in water at pH 7 led to the isolation of the
neutral complex.
A further increase in the sensitivity of the pH assessment can
1
be obtained from acquiring the T1-weighted H-MR image at
y The 19F-containing reporter has been synthesized from the
alkylation of 1-adamantamine by 4[(2,3-epoxy)propyloxy]phenylacetic
acid phenyl methyl ester (CH3CN, DIPEA, 5 days at 50 1C), obtained
from the reaction of benzyl 4-hydroxybenzoate with epibromohydrin
(DMF, K2CO3, 6 hours at 70 1C). Cleavage of the benzyl group has
been achieved by hydrogenation with Pd/C at atmospheric pressure
to yield the carboxylic acid, that has been coupled to 2,20,200-trifluoro-
ethylamine (DMF, DIPEA, HBTU, 16 hours at RT). The final
product has been carefully purified to 4 98% by liquid chromato-
graphy on Amberchroms CG161 resin.
1 T where the relaxivity gap between q = 2 and q = 0
conditions is maximized (Fig. 3d).
In summary, the proof of concept reported here shows that
a novel, highly accurate pH mapping can be pursued by MRI
using the heteronuclear 19F signal to normalize the relaxation
enhancement values induced by a pH-responsive Gd-complex.
Moreover, the use of poly-b-CD as a macromolecular carrier
can be further exploited to host analogous adamantane
functionalized moieties to endow the supramolecular adduct
with targeting as well as multimodal capabilities.
z Magnetic resonance images have been acquired on a Bruker
Avance300 spectrometer operating at 7.1
T equipped with a
microimaging probe and on an Aspect scanner (Netanya, Israel)
operating at 1 T using standard T1-weighted multislice multiecho
sequences.
For an in vivo translation of the proposed method, it
appears necessary to improve the binding affinity of the
adamantane functionalized Gd-complex to the poly-b-CD
substrate. The system used here shows a good binding to
HSA (KA = 13 000 Mꢁ1). Thus, at the physiological protein
concentration of 0.6 mM and a 1 mM Gd-complex concentration
(and poly-b-CD and 19F-reporter in the above established
ratio), ca. 20% of the paramagnetic complex will be bound
to HSA.
1 M. P. Lowe, D. Parker, O. Reany, S. Aime, M. Botta,
G. Castellano, E. Gianolio and R. Pagliarin, J. Am. Chem. Soc.,
2001, 123, 7601.
2 S. R. Zhang, K. C. Wu and A. D. Sherry, Angew. Chem., Int. Ed.,
1999, 38, 3192.
3 S. Aime, M. Botta, S. Geninatti, G. B. Giovenzana, G. Palmisano
and M. Sisti, Chem. Commun., 1999, 1577.
4 S. Aime, A. Barge, M. Botta, J. A. K. Howard, R. Kataky,
M. P. Lowe, J. M. Moloney, D. Parker and A. De Sousa, Chem.
Commun., 1999, 1047.
5 R. Hovland, C. Glogard, A. J. Aasen and J. Klaveness, J. Chem.
Soc., Perkin Trans. 2, 2001, 929.
6 M. Woods, S. Zhang, E. Von Howard and A. D. Sherry,
Chem.–Eur. J., 2003, 9, 4634.
Finally, as it is known that in Gd-DO3A derivatives,z the
two inner sphere water molecules can be replaced by the
coordination of endogenous oxoanions,1,16,17 an improvement
in the design of the pH-responsive coordination cage should be
pursued. It has already been shown that the introduction of
carboxyalkyl substituents on the acetate arms inhibits anion
binding while maintaining the pH responsiveness.1,18
Economic and scientific support from MIUR (FIRB
RBI P06293N_001 and PRIN 2007W7M4NF projects),
EC-FP6-projects DiMI (LSHB-CT- 2005-512146), EMIL
(LSHC-CT-2004-503569), MEDITRANS (Targeted Delivery
of Nanomedicine: NMP4-CT-2006-026668), ENCITE
(European Network for ‘‘Cell Imaging and Tracking
Expertise’’ 201842) and EU-COST D38 Action is gratefully
acknowledged.
7 S. Aime, A. Barge, D. Delli Castelli, F. Fedeli, A. Mortillaro,
F. U. Nielsen and E. Terreno, Magn. Reson. Med., 2002, 47, 639.
8 J. A. Pikkemaat, R. T. Wegh, R. Lamerichs, R. A. van de
Molengraaf, S. Langereis, D. Burdinski, A. Y. F. Raymond,
H. M. Janssen, B. M. F. de Waal, N. P. Willard, E. W. Meijer
and H. Grull, Contrast Media Mol. Imaging, 2007, 2, 229.
9 M. Oishi, S. Sumitani and Y. Nagasaki, Bioconjugate Chem., 2007,
18, 1379.
10 A. M. Kenwright, I. Kuprov, E. De Luca, D. Parker, S. U. Pandya,
P. K. Senanayake and D. G. Smith, Chem. Commun., 2008, 2514.
11 S. Aime, F. Fedeli, A. Sanino and E. Terreno, J. Am. Chem. Soc.,
2006, 128, 11326.
12 N. Raghunand, C. Howison, A. D. Sherry, S. R. Zhang and
R. J. Gillies, Magn. Reson. Med., 2003, 49, 249.
13 M. L. Garcia-Martin, G. V. Martinez, N. Raghunand,
A. D. Sherry, S. R. Zhang and R. J. Gillies, Magn. Reson. Med.,
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Notes and references
14 J. Carrazana, A. Jover, F. Meijide, V. H. Soto and J. Vasquez
Tato, J. Phys. Chem. B, 2005, 109, 9719.
z The synthesis of the ligand involved alkylation of the DO3A–
tris-tert-butylester with N-(benzyloxycarbonyl)-2-bromoethylamine
(CH3CN, K2CO3, 4 days at 50 1C). Following chromatographic
purification on silica, cleavage of the benzyloxycarbonyl group
has been achieved by hydrogenation with Pd/C at atmospheric
pressure to yield the free primary amino group. This group allowed
the subsequent coupling with 4-(methyloxycarbonylmethylenoxy)-
benzenesulfonyl chloride (CH3CN, triethylamine, 3 days at RT),
obtained by reaction of methyl phenoxyacetate with chlorosulfonic
acid in dichloromethane. After purification by chromatography on
silica and hydrolysis of the methyl ester (MeOH–H2O, pH 12, 1 hour),
the carboxylic acid was coupled with 1-adamantamine (DMF, HATU,
20 hours at RT). Following chromatographic purification on silica,
15 S. Aime, M. Botta, M. Fasano and E. Terreno, in The chemistry of
Contrast Agents, ed. A. Merbach and E. Toth, John Wiley & Sons,
Chichester, UK, 2001, pp. 193–241.
16 E. Terreno, M. Botta, P. Boniforte, C. Bracco, L. Milone,
B. Mondino, F. Uggeri and S. Aime, Chem.–Eur. J., 2005, 11,
5531.
17 S. Aime, E. Gianolio, E. Terreno, G. B. Giovenzana, R. Pagliarin,
M. Sisti, G. Palmisano, M. Botta, M. P. Lowe and D. Parker,
J. Biol. Inorg. Chem., 2000, 5, 488.
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S. Lopinski, M. P. Lowe, D. Parker, R. D. Peacock, J. J. B. Perry,
S. Aime and M. Botta, J. Am. Chem. Soc., 2000, 122, 9674.
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6046 | Chem. Commun., 2009, 6044–6046