Pyridine-based lanthanide complexes: Towards bimodal agents operating as near infrared luminescent and MRI reporters

Article abstract of DOI:10.1039/b817343e

We report two prototype Ln³⁺ complexes that address requirements for both MRI and luminescence imaging and we demonstrate that the presence of two H₂O molecules bound to the Ln³⁺, beneficial for MRI applications of the Gd³⁺ analogue, is not a major limitation for the development of NIR luminescent agents. The Royal Society of Chemistry 2008.

Full text of DOI:10.1039/b817343e

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Pyridine-based lanthanide complexes: towards bimodal agents operating
as near infrared luminescent and MRI reportersw
ab
c
a
a
b
Laurent Pellegatti, Jian Zhang, Bohuslav Drahos, Sandrine Villette, Franck Suzenet,
b
ac
a
´
Gerald Guillaumet, Stephane Petoud* and Eva Toth*
´ ´ ´
Received (in Cambridge, UK) 2nd October 2008, Accepted 23rd October 2008
First published as an Advance Article on the web 12th November 2008
DOI: 10.1039/b817343e
3
+
We report two prototype Ln complexes that address require-
ments for both MRI and luminescence imaging and we demon-
3
+
strate that the presence of two H O molecules bound to the Ln
2
,
3
+
beneficial for MRI applications of the Gd analogue, is not a
major limitation for the development of NIR luminescent agents.
1
2
Scheme 1 Structure of the ligands L and L .
Among the state-of-the-art bioimaging modalities, some are
characterized by high resolution but low sensitivity (magnetic
resonance imaging, MRI), others by high sensitivity but low
resolution (optical imaging). Luminescent/MRI bimodal ima-
ging offers the advantage of coupling the high sensitivity of
1
providing a powerful way to validate in vivo experiments.
3
+
The majority of bimodal agents reported so far are Gd
complexes or iron-oxide nanoparticles conjugated to fluores-
1
1
luminescence with the high resolution of MRI. Lanthanide
cent organic dyes.
We report prototypes of a versatile scaffold for Ln
3
+
complexes are well suited for the design of bimodal imaging
probes: they combine optimized magnetic and optical proper-
ties, while the similar chemical reactivities allow facile sub-
complexation where MRI and luminescence requirements
are both satisfied using the same ligand. Through the appro-
3
+
3+
1
priate choice of a pyridine-based backbone, L and L ensure
2
stitution of one Ln
efficient MRI contrast agents. Several Ln
by another. Gd
,3
complexes are
3+
cations emit
2
3+
(i) high MRI efficacy when complexed to Gd , due to
4
5
in the visible or in the near infrared (NIR). NIR imaging is
more compatible with biological applications: (i) the lack of
native NIR fluorescence in biological systems makes the
detection highly sensitive, (ii) the low scattering of NIR
bishydration of the chelate, (ii) efficient sensitization of a
3+
3
+
NIR emitting Ln
when complexed to Nd
(Scheme 1).
They also fulfil the requirements of thermodynamic stability
and kinetic inertness, prerequisite for biological applications
of metal complexes. We demonstrate for the first time that the
6
photons allows for a better image resolution, (iii) NIR
photons can cross significant tissue depths for non-invasive
3
+
2
presence of two H O molecules bound to the Ln , beneficial
3+
7
imaging. In addition, lanthanides provide complementary
for MRI applications of the Gd analogue, is not an absolute
limitation for the development of NIR luminescent probes.
properties over organic fluorophores: resistance to photo-
bleaching, long luminescence lifetimes, no reabsorption, sharp
3
+
This work opens promising routes towards Ln -based
bimodal contrast agents for MRI and NIR optical imaging.
So far, only one bimodal MRI/luminescence agent with a NIR
4
,5
emission bands.
chelators combining MRI and luminescence activities has been
So far, the development of lanthanide
8
–12
3+
10
modest. The combined requirements for both imaging
techniques were considered to be non-compatible: the inner
emitting Ln
lanthanide sensitization was obtained through a Re complex
has been described. In that approach, the
1
and not an organic sensitizer. L was previously proposed for
sphere H O required for MRI is expected to induce dramatic
2
3
+
quenching of the Ln
1
luminescence through non-radiative
the development of bimodal imaging probes, however, only
11,14
3
3+
visible luminescent Ln cations were considered.
deactivation. Evidently, bimodal probes have to account for
the differences in sensitivity of the imaging modality. An MRI
1
The absorption spectrum of NdL shows a unique absorption
3
+
2
reporter (Gd
complex) has to be administered in at least
band (Fig. 1; for NdL see ESIw). Upon excitation of the
absorption band, the NIR emission spectra reveal three narrow
B100 fold higher concentration than the luminescent optical
probe. Being aware of this, bimodal agents are still attractive
as they enable the colocalization of the acquired images
3+
emission bands that correspond to Nd transitions (895, 1051
4
and 1323 nm assigned to transitions from the F3/2 level to the
4
I
4
4
9/2, I11/2 and I13/2 sublevels). These experiments demonstrate
a
1
2
that the electronic structures of L and L can sensitize Nd
3+
.
Centre de Biophysique Mole´culaire, CNRS, rue Charles Sadron,
4
5071 Orle´ans, France. E-mail: eva.jakabtoth@cnrs-orleans.fr;
Fax: +33 2 38 63 15 17; Tel: +33 2 38 25 76 25
Institut de Chimie Organique et Analytique, UMR 6005 CNRS/
Universite´ d’Orle´ans, Rue de Chartres, 45067 Orle´ans, France
Dept. of Chemistry, The University of Pittsburgh, 219 Parkman
Avenue, 15260 Pittsburgh, USA. E-mail: spetoud@pitt.edu
Importantly, emission signals were observed with a good
detection sensitivity using a commercial fluorimeter.
The luminescence lifetimes recorded upon ligand excitation
b
c
2 2 2
in H O and D O allow the presence of two H O coordinated
3+
1
2
13
to Ln in EuL and EuL to be concluded (Table 1). The
quantum yields, characterizing the efficiency of the sensitiza-
w Electronic supplementary information (ESI) available: Synthesis of
the ligands, details of luminescence, potentiometric and kinetic
measurements. See DOI: 10.1039/b817343e
1
tion and protection of lanthanide cations by L and L , are
2
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ab
Table 2 Complex stability constants and pM values
1
L
2
L
d
DTPA
c
c
log KGdL
log KNdL
log KZnL
log KZnHL
log KZnH2L
log KZn2L
log KCuL
log KCuHL
log KCaL
18.60 Æ 0.02
18.76 Æ 0.01
15.84 Æ 0.02
3.81 Æ 0.02
—
18.1
—
15.3
—
—
—
18.76 Æ 0.01
18.28 Æ 0.01
16.93 Æ 0.01
3.70 Æ 0.01
—
22.46
—
18.29
5.6
3.1
4.48
21.5
4.8
—
—
15.69 Æ 0.02
3.45 Æ 0.02
9.43 Æ 0.06
15.53 Æ 0.02
3.37 Æ 0.02
9.81 Æ 0.01
—
9.2
c
10.1
19.1
11.2
e
f
f
f
pGde
pGd
17.5
9.8
—
—
17.6
9.9
g
g
g
a
+
b
K
ML = [ML]/[M][L]; KMHL = [MHL]/[ML][H ]. The protona-
tion constants, log KHi, are 8.95, 7.85, 3.38, 2.48 and 8.91, 7.85, 3.01,
e
1
2
c
d
2
.42 for L and L , respectively. Ref. 18. Ref. 19. pGd = Àlog
Fig. 1 Absorption (a), normalized emission (lex = 267 nm) and
f
À6
À5
À5
g
À6
L
cGd = 10 M, c =
À4
1
[Gd]free
.
cGd = 10 M, c
L
= 10 M, pH 7.4.
excitation (lem = 1065 nm) spectra (b) of 5.1 Â 10 M NdL . 0.01 M
À5
1
0
M, cZn = 10 M, pH 7.4.
HEPES, pH 7.02, I = 0.01 M.
3
+
Table 1 Luminescence quantum yields and lifetimes of Nd
Eu complexes of L and L (10 M, 0.01 M HEPES, I = 0.01 M,
pH = 7.02)
and
2+
2+
or Cu , in contrast to GdDTPA under identical
16
Zn
3+
1
2
À4
conditions. The rigid pyridinic skeleton also contributes to
the slowness of the acid-catalyzed dissociation. We studied the
1
L
2
L
1
proton catalyzed dissociation of GdL in the exchange reaction
1
3+
1
3+
3+
Nd
À5a
À4a
À5b
À4b
GdL + Eu
conditions (pH 3.6–5.2; cGdL = 0.5 mM, cEu = 5–30 mM). The
observed rate constants show quadratic dependency
- EuL + Gd , under pseudo-first order
F
H O
9.7(4) Â 10
0.059(1)
5.5(2) Â 10
0.310(1)
0.403(1)
1.85(2)
7.5(7) Â 10
0.072(1)
4.0(3) Â 10
0.314(2)
0.35(4)
2
c
c
t
H O (ms)
2
F
D O
2
a
t
t
t
D O (ms)
H O (ms)c
D O (ms)
+
+
+ 2
2
3+
Eu
c
on [H ] (kobs = k
characterizing the dissociation via mono- and diprotonated com-
0 1 2
+ k [H ] + k [H ] ). The constants
2
1.79(6)
2.4
2
À1 À1
À2 À1
À1 À1
q
2.1
plexes are k s
lower values than those for GdDTPA, k
= 0.17 Æ 0.08 M
and k
= 520 Æ 30 M
s
and
,
1
2
a
b
c
lex = 266 nm.
lex = 250 nm.
lex = 266 nm.
1
= 0.58 M
s
4
À2 À1 16
k
2
= 9.7 Â 10 M
s . Since the dissociation pathway via
transmetallation, which represents 98% of
2+
Zn
2+
or Cu
the overall dissociation for GdDTPA under physiological
3
+
remarkable for a bishydrated Nd complex. They are in the
same range as those of the non-hydrated Nd-T2soxMe
16
1
conditions, is not important for GdL , and the proton catalyzed
1
dissociation is also slower, GdL has a remarkable inertness for a
(
0.027%), a system optimized for fully protecting the NIR
3
+
15
emitting Ln for aqueous application. This result demon-
strates for the first time that the non-radiative deactivation of
NIR emitting lanthanides, even resulting from two water
molecules, can be compensated by an efficient ligand to
lanthanide energy transfer.
bishydrated chelate.
At this proof of principle stage of the project, we demonstrated
3+
that even bishydrated Nd
complexes can have a quantum
yield in the same range as those of the most optimized chelates so
1
far reported. The LnL chelates are remarkably inert, and do not
17
Ternary complex formation with endogenous donors can be an
3+
important limitation to the MRI use of bishydrated Gd
form ternary complexes, in contrast to DO3A-type chelates.
These properties make the pyridinic synthon a prime candidate
for the development of bimodal MRI/NIR imaging probes.
In the perspective of biological applications, we are modifying
the ligand structure by attaching different substituents to the
pyridine to shift the excitation wavelength towards higher values
in order to prevent damages to biological samples and to allow
deeper tissue penetration of the excitation photons. These struc-
tural modifications, however, will not influence the bishydrated
nature and kinetic inertness of the complexes. The pyridine-
based platform provides high synthetic versatility. It offers easy
routes for coupling the probes to biological vectors and to
increase the molecular size in order to optimize proton relaxivity.
This work was financially supported by the Institut
National du Cancer, La Ligue contre le Cancer, France, the
National Science Foundation (award DBI0352346) and was
carried out in the frame of the COST Action D38. S.P.
gratefully acknowledges Le Studium (Agency for research
and international hosting associate researchers in ‘‘Region
1
À1 À1
chelates. The relaxivity of GdL , r
00 MHz), remains unaffected in bovine serum, indicating that the
inner sphere H O molecules are not replaced by potential donor
1
= 6.21 mM
s
(25 1C,
5
2
1
groups. The emission spectra of EuL recorded in pure water and
in the presence of 600 equivalents of carbonate or phosphate (pH
7
.4) are identical, excluding the formation of ternary complex.
3+
The toxicity of Ln complexes depends on the release of free
3+
Ln , controlled by the thermodynamic stability and kinetic
2+
inertness. Zn and Cu were found to be particularly active
2+
3+
16
in transmetallation of acyclic Ln chelates. Stability constants
determined by pH-potentiometry demonstrate a significant selec-
2+
tivity of L and L for Gd vs. Ca , Zn and Cu (Table 2).
1
2
3+
2+
2+
It has been also shown that the formation of the dinuclear
Zn
2 5
DTPA (H DTPA = diethylenetriamine-pentaacetic acid) is
16
an important driving force in the transmetallation of GdDTPA.
1
Such a dinuclear complex is not detectable with L and L , which
can likely be explained by their rigidity. No transmetallation of
2
1,2
LnL is observed in the presence of a 100-fold excess of
Centre’’), Orle
´
ans, France, for financial support.
6
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