Micellar self-assemblies of gadolinium(iii)/europium(iii) amphiphilic complexes as model contrast agents for bimodal imaging

Article abstract of DOI:10.1039/c3dt52842a

The synthesis and characterization of two novel DTPA bisamide derivatives DTPA-BC₁₂PheA and DTPA-BC₁₄PheA functionalized with p-dodecylaniline and p-tetradecylaniline are described. The ligands were coordinated to Gd(iii) and Eu(iii), resulting in highly paramagnetic and luminescent complexes, respectively. Mixed micelles consisting of Gd/Eu-DTPA-BC₁₂PheA and DTPA-BC₁₄PheA with a homogeneous size distribution (33-40 nm) were prepared by the assembly of the amphiphilic complexes with phospholipid DPPC and a surfactant Tween 80. Taking into account the sensitivity difference between magnetic resonance and optical imaging techniques, the ratios of Gd and Eu complexes (Gd/Eu) 1:1, 2:1, 3:1, 20:1 and 50:1 were combined in one single micelle and their optical and relaxometric properties were characterized in detail. Upon excitation at 290 nm, the micelles display characteristic red emission bands due to the ⁵D₀→⁷F_J (J = 0-4) transitions of Eu(iii). The number of water molecules in the first coordination sphere of Eu(iii) (q_Eu = 0.1-0.2) was calculated from the lifetime measurements performed in H₂O and D₂O solutions. Micelles composed of exclusively europium complexes display quantum yields in the range of 1.0%, decreasing with the europium concentration when going from 1:1 to 50:1 Gd/Eu contents. The ligand-to-lanthanide sensitization efficiency for micelles consisting of Eu-DTPA-BC₁₂PheA and Eu-DTPA-BC₁₄PheA equals 3.8% and 4.1%, respectively. The relaxivity r₁ per Gd(iii) ion at 40 MHz and 310 K reaches a maximum value of 14.2 s^-1 mM^-1 for the Gd-DTPA-BC₁₂PheA assemblies and 16.0 s^-1 mM ^-1 for the micellar Gd-DTPA-BC₁₄PheA assemblies compared to a value of 3.5 s^-1 mM^-1 for Gd-DTPA (Magnevist). Theoretical fitting of the ¹H NMRD profiles results in τ_R values of 4.2 to 6.6 ns. The optimal concentration ratio of Gd/Eu compounds in the micelles in order to provide the required bimodal performance has been determined to be 20:1. In the search for other bimodal systems, this discovery can be used as a guideline concerning the load of paramagnetic agents with respect to luminescent probes.

Full text of DOI:10.1039/c3dt52842a

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Micellar self-assemblies of gadolinium(III)/
europium(III) amphiphilic complexes as model
contrast agents for bimodal imaging†
Cite this: Dalton Trans., 2014, 43,
3589
Elke Debroye,a Svetlana V. Eliseeva,b,c Sophie Laurent,d Luce Vander Elst,d
Robert N. Mullerd,e and Tatjana N. Parac-Vogt*a
The synthesis and characterization of two novel DTPA bisamide derivatives DTPA-BC12PheA and
DTPA-BC14PheA functionalized with p-dodecylaniline and p-tetradecylaniline are described. The ligands
were coordinated to Gd(III) and Eu(III), resulting in highly paramagnetic and luminescent complexes,
respectively. Mixed micelles consisting of Gd/Eu-DTPA-BC12PheA and DTPA-BC14PheA with a homo-
geneous size distribution (33–40 nm) were prepared by the assembly of the amphiphilic complexes with
phospholipid DPPC and a surfactant Tween 80®. Taking into account the sensitivity difference between
magnetic resonance and optical imaging techniques, the ratios of Gd and Eu complexes (Gd/Eu) 1 : 1,
2 : 1, 3 : 1, 20 : 1 and 50 : 1 were combined in one single micelle and their optical and relaxometric pro-
perties were characterized in detail. Upon excitation at 290 nm, the micelles display characteristic red
emission bands due to the 5D0→7FJ (J = 0–4) transitions of Eu(III). The number of water molecules in the
first coordination sphere of Eu(III) (qEu = 0.1–0.2) was calculated from the lifetime measurements per-
formed in H2O and D2O solutions. Micelles composed of exclusively europium complexes display
quantum yields in the range of 1.0%, decreasing with the europium concentration when going from 1 : 1
to 50 : 1 Gd/Eu contents. The ligand-to-lanthanide sensitization efficiency for micelles consisting of Eu-
DTPA-BC12PheA and Eu-DTPA-BC14PheA equals 3.8% and 4.1%, respectively. The relaxivity r1 per Gd(III)
ion at 40 MHz and 310 K reaches a maximum value of 14.2 s−1 mM−1 for the Gd-DTPA-BC12PheA assem-
blies and 16.0 s−1 mM−1 for the micellar Gd-DTPA-BC14PheA assemblies compared to a value of
3.5 s−1 mM−1 for Gd-DTPA (Magnevist®). Theoretical fitting of the 1H NMRD profiles results in τR values of
4.2 to 6.6 ns. The optimal concentration ratio of Gd/Eu compounds in the micelles in order to provide
the required bimodal performance has been determined to be 20 : 1. In the search for other bimodal
systems, this discovery can be used as a guideline concerning the load of paramagnetic agents with
respect to luminescent probes.
Received 9th October 2013,
Accepted 10th December 2013
DOI: 10.1039/c3dt52842a
www.rsc.org/dalton
which makes them suitable for magnetic resonance imaging
(MRI) applications. Compared to other imaging modalities,
Introduction
Paramagnetic gadolinium chelates are known to enhance MRI provides unique anatomical resolution, but the technique
remarkably the longitudinal relaxation rate of water protons, suffers from low sensitivity. To avoid the administration of
aKU Leuven, Department of Chemistry, Celestijnenlaan 200F, 3001 Heverlee,
(Fig. S3), Relative integral intensities of f–f-transitions for Eu(III) complexes
Belgium. E-mail: tatjana.vogt@chem.kuleuven.be; Fax: +32 16 327992;
Tel: +32 16 327612
bCentre de Biophysique Moléculaire CNRS UPR 4301, Rue Charles Sadron, 45071
Orléans Cedex 2, France
cLe STUDIUM® Institute for Advanced Studies, Orléans and Tours, France
dUniversity of Mons, Department of General, Organic and Biomedical Chemistry,
Place du Parc 23, 7000 Mons, Belgium
eCenter for Microscopy and Molecular Imaging (CMMI), 6041 Gosselies, Belgium
†Electronic supplementary information (ESI) available: ESI mass spectra of
the Eu(III) complexes (Fig. S1), 1H NMR spectra of ligand DTPA-BC12PheA and
Eu(III) complexes (Fig. S2), DLS measurements on the assembled micelles
(Table S1), Normalized excitation and absorption spectrum of micelles con-
sisting of Eu-DTPA-BC14PheA (Fig. S4), Relative integral intensities of f–f-tran-
sitions for micelles consisting of Eu(III) complexes (Table S2), Photophysical
data for micelles consisting of Eu-DTPA-BC12PheA and Eu-DTPA-BC14PheA in
water (Table S3), 1H NMRD profiles of micelles composed of Gd/Eu-
DTPA-BC12PheA or Gd/Eu-DTPA-BC14PheA complexes compared to Gd-DTPA
in water (Fig. S5, S6), fitted by using the Lipari–Szabo model (Fig. S7, S8),
Parameters obtained by theoretical fitting of the 1H NMRD data of micelles
consisting of Gd/Eu-DTPA-BC12PheA and Gd/Eu-DTPA-BC14PheA (Tables S4
and S5), using the Lipari–Szabo model (Tables S6 and S7). See DOI: 10.1039/
c3dt52842a
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high concentrations of the MRI agent (0.01–0.1 mM), signifi- antenna to capture the incident light and subsequently trans-
cant efforts have been made to increase the efficiency per fer the energy to the emissive state of the lanthanide ion.
gadolinium unit. For this, the commonly used diethylenetri-
In this report, we present a novel approach towards
aminepentaacetic acid (DTPA) or 1,4,7,10-tetraazacyclodo- bimodal contrast agents in which Gd- and Eu-DTPA bisamide
decane-1,4,7,10-tetraacetic acid (DOTA) scaffolds are complexes functionalized with p-dodecylaniline and p-tetra-
functionalized in order to obtain better structural or dynamic decylaniline self-assemble with a saturated phospholipid and
properties. Faster proton relaxivities can be achieved by a non-ionic surfactant, resulting into micelles with favorable
lengthening the rotational correlation time (τR) or shortening paramagnetic and luminescent properties. Although neutral
the residence time of the coordinated water molecules (τM). Gd-DTPA bisamides are generally characterized by a slow water
Already many studies have been performed to optimize this set exchange resulting in a larger τM value,1,36,37 a large increase
of parameters.1–3 The molecular tumbling rate of the contrast of relaxivity is observed due to the immobilization of the para-
agent can be lowered by conjugation of Gd(III) chelates to magnetic chelate into the micellar membrane.13,38 On the
macromolecules like linear polymers or dendrimers.2,4–7 Also a other hand, the integrated phenyl functions should ensure
non-covalent interaction of the paramagnetic entity with sensitization of the coordinated luminescent Eu(III) ions,
human serum albumin resulted in an amplified proton leading to a strong optical signal. Taking into account the
relaxation.8–11 Unfortunately, the theoretical maximum different sensitivities of the two imaging modalities, approxi-
efficiency has not yet been reached, since the local motions of mately a 100-fold higher concentration of the MRI probe with
the gadolinium complex accompany the slow molecular tum- respect to the optical probe should be administered. In the
bling of the carrier.12,13 Another approach to increase the sen- present case, several ratios of gadolinium versus europium
sitivity is the accumulation of a substantial amount of Gd(III) complexes are integrated into the micellar systems and the
agents in a small volume. Amphiphilic complexes can be ratio providing the optimal optical and magnetic combination
assembled in aqueous solutions forming supramolecular has been experimentally established. The composition of the
aggregates, like micelles or liposomes.1,13–16 Their structural ligands and of the corresponding Gd(III) and Eu(III) complexes
properties can easily be tuned by varying the charge of the has been confirmed by mass spectrometry, NMR, FT-IR and
hydrophilic head-group or the amount and length of hydro- elemental analysis. The size of the obtained supramolecular
phobic side chains.1 In micelles, the Gd(III) complexes are micelles has been determined by dynamic light scattering and
entirely exposed to the external aqueous surface, providing their luminescent and relaxometric properties have been inves-
easy access of the bulk water molecules to the paramagnetic tigated. It is generally known that the administration of non-
center.
ionic gadolinium based contrast agents in patients with
Among the bioimaging techniques, optical imaging is advanced renal impairment should be avoided.39 Nevertheless,
known to possess a low detection limit so that minute concen- during the performed experiments no release of free Gd(III)
trations can be perceived. In a recent effort to enhance the ions has been observed.
imaging performance of contrast agents, probes combining
MRI and luminescent activities have been created in order to
offer good resolution as well as high sensitivity.17,18 This
approach leads to the same biodistribution of the probe for
Results and discussion
Synthesis of ligands and complexes
both techniques, which is advantageous for in vivo biological
The synthetic pathway towards the ligands consists of two
investigations. Several organic dyes have been attached to para-
main steps. First, DTPA bisanhydride is formed starting from
magnetic complexes and their bimodal applications have been
exploited.19–22 In a different approach, transition metal com-
diethylene triamine pentaacetic acid and acetic anhydride.
Afterwards, two equivalents of the hydrophobic aromatic
plexes endowed with luminescent properties have been linked
to DTPA or DOTA.23–25 Bimodal agents based on magnetofluor-
amines p-dodecylaniline and p-tetradecylaniline are reacted
with DTPA bisanhydride leading to DTPA-bis-p-dodecylphenyl-
amide (DTPA-BC12PheA) and DTPA-bis-p-tetradecylphenyl-
escent liposomes26,27 and nanoparticles28–30 have also been
reported. Although significant improvements of MRI and
amide (DTPA-BC14PheA) respectively (Fig. 1). All ligands have
optical properties have been achieved, practical applications of
been characterized by nuclear magnetic resonance spec-
aforementioned bio-conjugates are limited by short lumines-
troscopy, mass spectrometry, elemental analysis and IR
cence lifetimes, a small Stokes shift and poor resistance to
spectroscopy.
photobleaching. During the last decade, lanthanide based
systems combining magnetic and optical properties are attract-
ing attention.31–35 Besides the excited state lifetimes in the
range of milliseconds, the lanthanide complexes exhibit very
sharp emission bands and an apparent shift between absorp-
tion and emission bands. Since the f–f transitions of Ln(III)
ions are Laporte forbidden and thus have low molar absorp-
tion coefficients, it is necessary to integrate an appropriate
chromophore into the coordinated ligand. This will serve as an (n = 10) or DTPA-bis-p-tetradecylphenyl-amide (n = 12).
Fig. 1 Chemical structure of
DTPA-bis-p-dodecylphenyl-amide
3590 | Dalton Trans., 2014, 43, 3589–3600
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The obtained ligands are coordinated to paramagnetic
Gd(III) and to luminescent Eu(III) in pyridine according to a
known procedure.40 The absence of free lanthanide ions is
verified by the addition of an arsenazo indicator solution.41
Molecular peaks [M + Na]+ appear with positive-mode electro-
spray ionization mass spectrometry (ESI-MS) indicating a 1 : 1
stoichiometry in solution (Fig. S1 in ESI† and Experimental
section). Proton NMR spectra of Eu(III)-DTPA-BC12PheA and
Eu(III)-DTPA-BC14PheA display broadening of the signals over a
large ppm scale (−13 to +32 ppm) due to the paramagnetic
nature of europium and an increase of proton resonances in
the aliphatic region which is consistent with the occurrence of
several interconverting isomers due to the coordination of a
lanthanide to a DTPA derivative42 (Fig. S2 in ESI†). IR spectra
of the ligands show strong absorptions in the region around
1600 cm−1, corresponding to the CvO carboxylic acid stretch-
ing modes. After complexation, the band shifts by 30 cm−1 to
lower wavenumbers, which suggests acetate oxygen’s coordi-
nation to the lanthanide(III) ions. The coordination sphere of
the lanthanide is further completed by two carbonyl oxygen
atoms of the amide groups and three nitrogen atoms. As a
result the complex consists of a hydrophilic center where the
DTPA unit implements an eight-fold coordination to the
lanthanide ion,36,37 flanked by two hydrophobic tails which
comprise a phenyl group and an aliphatic chain of 12 or 14
carbon atoms. X-ray structures of related DTPA bisamides
point out the formation of U-shaped complexes.43 The lantha-
nide–water vector is shown to be aligned parallel to the phenyl
rings which induces a hydrophobic environment. The long
alkyl chains in the para-positions create an even more defined
hydrophobic area around the lanthanide, hindering water
access especially in case of aggregation.
Table 1 Mean diameters of micelles consisting of DPPC, Tween 80®
and Ln-DTPA-BC12PheA or Ln-DTPA-BC14PheA (Ln = Gd/Eu)
Ln(III)-DTPA-
BC12PheA
z-Average
d (nm)
Ln(III)-DTPA-
BC14PheA
z-Average
d (nm)
Gd/Eu (1 : 1)
Gd/Eu (2 : 1)
Gd/Eu (3 : 1)
Gd/Eu (20 : 1)
Gd/Eu (50 : 1)
37.9
39.7
38.3
35.4
39.1
Gd/Eu (1 : 1)
Gd/Eu (2 : 1)
Gd/Eu (3 : 1)
Gd/Eu (20 : 1)
Gd/Eu (50 : 1)
33.1
39.2
34.3
37.1
36.6
Photophysical properties
Ln(III) complexes. The excitation spectra of Eu-DTPA-bis-p-
dodecylphenyl-amide and Eu-DTPA-bis-p-tetradecylphenyl-
amide in a 1 : 1 CHCl3–MeOH mixture are similar and reveal a
broad band in the range 240–300 nm with a maximum at
≈265 nm corresponding to the ligand electronic transitions
(Fig. 2). In addition, sharp features are seen in the range
360–410 nm, which can be assigned to f–f-transitions of the
Eu(III) ion.
Upon excitation into the ligand levels at 290 nm, Eu-DTPA-
bis-p-dodecylphenyl-amide
and
Eu-DTPA-bis-p-tetradecyl-
phenyl-amide exhibit characteristic red luminescence due to
5D0→7FJ (J = 0–4) transitions. No ligand-centered emission at
lower wavelengths is observed indicating that the ligands can
efficiently sensitize Eu(III) (Fig. 3). The integral intensities of
5
the hypersensitive D0→7F2 transition, relative to the magnetic
dipole 5D0→7F1 transition, equal 2.3 and 2.4, respectively for
Eu-DTPA-BC12PheA and Eu-DTPA-BC14PheA. For each complex,
5
5
the hypersensitive D0→7F2 transition and D0→7F4 transition
have almost equal integral intensities (Table S1 ESI†). The
highly forbidden 5D0→7F0 transition has a quite high intensity,
i.e. 14–16% relative to the magnetic dipole 5D0→7F1 transition,
which is typical for coordination geometry with Cs, Cn or
Cnv symmetry.44
Mixed micelle formation
Luminescence decays of both Eu complexes in a 1 : 1
CHCl3–MeOH mixture have been fitted by mono-exponential
equations confirming the presence of only one luminescent
lanthanide species in solutions. Luminescence lifetimes are
very similar, i.e. 0.83-(1) ms and 0.86-(1) ms for Eu-DTPA-
BC12PheA and Eu-DTPA-BC14PheA, respectively.
Due to their amphiphilic nature, the gadolinium and euro-
pium complexes of DTPA-BC12PheA and DTPA-BC14PheA
could be incorporated into mixed micelles. In this way, the
paramagnetic or luminescent agents become an integral part
of a supramolecular structure with a decreased rotational
motion, targeting a positive contribution to the relaxometric
properties. Micelles are formed by mixing 78 mol% phospho-
lipid (DPPC), 15.5 mol% surfactant (Tween 80®) and 6.5 mol%
of different ratios of Gd and Eu complexes of each ligand
(Gd/Eu = 1 : 1, 2 : 1, 3 : 1, 20 : 1, 50 : 1). The Gd and Eu com-
plexes are combined into one single micelle in order to find a
composition with an optimal bimodal performance consider-
ing magnetic and optical imaging requirements. The micelle
size has been determined by photon correlation spectroscopy
at room temperature. Since only one main kind of particle
appears in the size distribution profile, the solutions are
considered as monodisperse (Fig. S3 in ESI†). As can be seen
in Table 1, the mean diameters of the micelles are all within
the same range (33–40 nm), which implies that the phospho-
lipid DPPC is the most important factor determining the
micelle size.
Fig. 2 Normalized excitation spectrum of Eu-DTPA-BC12PheA (dashed
trace) and Eu-DTPA-BC14PheA (solid trace) measured while monitoring
Eu(III) emission at 614 nm (1 : 1 CHCl3–MeOH, 10−4 M, 298 K).
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Fig. 5 Corrected and normalized luminescence spectra of Eu-DTPA-
BC14PheA (1 : 1 CHCl3–MeOH mixture, solid trace) and micelles consist-
Fig. 3 Corrected and normalized luminescence spectra of Eu-DTPA-
BC12PheA (dashed trace) and Eu-DTPA-BC14PheA (solid trace) (1 : 1
CHCl3–MeOH mixture, 10−4 M, λexc = 290 nm, 298 K).
ing of Gd/Eu-DTPA-BC14PheA (H2O, dashed trace) (10−4 M, λexc
=
290 nm, 298 K).
Micelles. After assembly of the Ln(III) complexes with the
phospholipid DPPC and surfactant Tween 80®, the excitation
spectrum of the micelles exhibit a very similar ligand-centered
band in the range 240–300 nm with a maximum at 264 nm.
On the other hand, no sharp bands of f–f-transitions of the
Eu(III) ion are observed in contrast to the free complexes (Fig. 4).
Upon excitation at 290 nm, the emission spectra display the
unique sharp emission bands due to 5D0→7FJ (J = 0–4) tran-
sitions of the Eu(III) ion. The efficient energy transfer from
ligand to lanthanide is maintained since no ligand-centered
emission is detected. Relative integral intensities of 5D0→7FJ
(J = 0–4) transitions for both Eu(III) micelles (Table S2 ESI†) are
the same within experimental errors confirming similarity of
the local microenvironment around the lanthanide ions.
However, compared to the emission profiles of the complexes
which are not incorporated into the micellar structure, the
Tween 80® contains hard donor atoms with affinity for the
europium ion and is able to form hydrogen bonds with the co-
ordinated water molecule.45–47
As can be seen in Fig. 6, the luminescence drops with
increasing Gd(III) and decreasing Eu(III) content. A linear
relationship between the intensity decreases and the Eu(III)
concentration is observed. In Table 2, total integrated intensi-
5
ties of D0→7FJ (J = 0–4) transitions for micelles with decreas-
ing europium contents, relative to micelles composed of
exclusively Eu(III) complexes, are represented. The emission of
the micelles consisting of Gd/Eu 20 : 1 is still clearly visible,
whereas the characteristic red luminescence gradually fades
for micelles with less than 1 Eu(III) ion per 20 Gd(III) ions.
5
intensity of the D0→7F4 transition decreases by 26–33% while
the intensity of the hypersensitive 5D0→7F2 transition
increases by 14–22%. In addition, slight changes in the crystal-
field splitting of the f–f transitions are detected (Fig. 5).
All these observations reflect variations in the coordination
environment around the Eu(III) ion when going from com-
plexes to micelles. These are induced by a change of solvent
(CHCl3–MeOH vs. H2O) and also the influence of the micellar
components should not be excluded. The non-ionic surfactant
Fig. 4 Normalized excitation spectra of micelles consisting of Eu-
DTPA-BC12PheA (dashed trace) and Eu-DTPA-BC14PheA (solid trace)
(H2O, 10−4 M, λem = 614 nm, 298 K).
Fig. 6 Corrected and normalized luminescence spectra of micelles
consisting of Gd/Eu-DTPA-BC12PheA (top) and Gd/Eu-DTPA-BC14PheA
(bottom) (H2O, 10−4 M, λexc = 290 nm, 298 K).
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of 0.98% and 1.10% respectively were obtained. Emission
intensity of the micelles (Fig. 6) decreases linearly with increas-
ing Gd/Eu ratio reflecting constancy of quantum yield. Direct
excitation of the Eu(III) ion is very inefficient as the relevant f–f
Table 2 Eu(III) contents relative to micelles composed of exclusively
Eu(III) complexes and their corresponding relative total emission intensi-
ties (H2O, 10−4 M, λexc = 290 nm, 298 K)
Eu(III) content
DTPA-BC12PheA
DTPA-BC14PheA
transitions are Laporte forbidden, so intrinsic quantum yields
Eu
Eu
Gd/Eu (0 : 1)
Gd/Eu (1 : 1)
Gd/Eu (2 : 1)
Gd/Eu (3 : 1)
Gd/Eu (20 : 1)
Gd/Eu (50 : 1)
1.00
0.50
0.33
0.25
0.05
0.02
1.00
0.53
0.38
0.27
0.08
0.04
1.00
0.56
0.38
0.29
0.09
0.04
(Q
) have been estimated according to the following
equations comprising the ratio between the observed (τobs
and radiative (τrad) lifetimes:
)
ꢀ
ꢁ
1
τrad
Itot
IMD
¼ AMD;0n3
ð2aÞ
ð2bÞ
τobs
Eu
Eu
Luminescence lifetimes in both H2O and D2O have been
obtained after fitting luminescence decays by mono-exponen-
tial equations, which confirm the presence of only one lumi-
nescent lanthanide species in the micelles. Luminescence
lifetimes in H2O are equal to 1.36–1.39 ( 0.01) ms for both
Q
¼
τrad
The Einstein coefficient AMD,0 equals 14.65 s−1, n is the
refractive index set equal to that of the neat solvent, nH O
=
2
1.34, and (Itot/IMD) represents the ratio of the total integrated
5D0→7FJ (J = 0–4) emission area to the intensity of the 5D0→7F1
micelles. These τH O values are remarkably high compared to
2
other lanthanide complexes with DTPA bisamide deriva-
tives.48,49 In D2O, luminescence lifetimes are prolonged up to
2.19–2.21 ( 0.04) ms. Two phenomenological equations develo-
ped for cyclen50 and aminocarboxylate derivatives51 allow us to
determine the number of coordinated water molecules q with
an accuracy of 0.2–0.3 for eqn (1) and 0.1 for eqn (2):
magnetic dipole (MD) transition. The side chain length of the
Eu
complexes hardly influences the Q value of the micelles and
Eu
rather high rates of 26–27% are obtained. This result is in
agreement with the former determined elimination of water
molecules from the first coordination sphere of the lumines-
cent lanthanide ion. At last, the ratio between the acquired
Eu(III) quantum yield under ligand excitation (QELu) and Eu(III)
qEuðH2OÞ ¼ 1:2ðΔkobs ꢀ 0:25 ꢀ 1:2qNH ꢀ 0:075qCONH
qEuðH2OÞ ¼ 1:11ðΔkobs ꢀ 0:31 ꢀ 0:44qOH ꢀ 0:99qNH
Þ
ð1Þ
Eu
Eu
intrinsic quantum yield (Q
efficiency (ηsens) of the ligand:
) defines the sensitization
QLEu
ꢀ 0:075qCONH
Þ
ð2Þ
ηsens
¼
ð3Þ
Eu
Q
Eu
Δkobs represents the difference of the decay rate constants
kH O(1/τH O) and kD O(1/τD O) and qX stands for the number of
OH, NH or CONH groups participating in lanthanide coordi-
nation. In the present case, only amide groups have been con-
sidered, qCONH = 2. After calculation, qEu values obtained using
resulting in 3.8% for micelles consisting of Eu-DTPA-BC12PheA
and 4.1% for micelles consisting of Eu-DTPA-BC14PheA.
2
2
2
2
Relaxometric studies
both equations are in agreement and lie in the range of Proton NMRD profiles give an idea about the efficiency of a
0.1–0.2 water molecules. Additionally, luminescence lifetimes 1 mM solution of Gd(III) agent to shorten the longitudinal
upon direct excitation into the europium ion (393 nm, relaxation time (T1) by measuring the water proton relaxivity
7F0,1←5L6) have been measured and found to be 1.27–1.28 (r1) as a function of the magnetic field strength. The relaxation
( 0.03) ms in H2O and 2.02–2.04 ( 0.02) ms in D2O. The rate is enhanced by dipolar interactions between the gadoli-
corresponding number of coordinated water molecules is cal- nium ion and the proximate water molecules exchanging with
culated to be 0.2. These outcomes are not in agreement with bulk water. Besides the inner sphere contribution54,55 compris-
other studied DTPA bisamide complexes in aqueous solu- ing the interactions between Gd(III) and directly coordinated
tions52,53 in which the lanthanide ion is eight-fold coordinated water molecules, also the longer distance interactions with
while one water molecule is capable of occupying the ninth second56 and outer sphere57 water molecules, should not be
coordination site. As mentioned before, the non-ionic surfac- neglected. Inner sphere interactions are defined by several
tant at the periphery of the assembled micelles is able to form parameters such as the number of water molecules in the first
hydrogen bonds with H2O and acts as a competitor in lantha- coordination shell (q), the distance between Gd(III) and the
nide coordination. Moreover, the complexes possess a defined water proton nuclei (r), the water residence time (τM), the
hydrophobic area in the lanthanide region, further inhibiting rotational correlation time of the paramagnetic compound
water access. Less non-radiative deactivation due to O–H (τR), the electronic relaxation time of Gd(III) at zero field (τS0)
vibrations will take part, leading to longer luminescence life- and the correlation time modulating the electronic relaxation
times and as a consequence to a reduced q number found by (τV). Concerning outer sphere interactions, the distance of
the phenomenological eqn (1) and (2).
closest approach (d) and the relative diffusion coefficient (D)
The micelles composed of exclusively europium complexes of neighbouring water molecules are also taken into account.
of DTPA-BC12PheA or DTPA-BC14PheA show a slight difference For the second sphere contribution, three additional para-
in Eu(III) quantum yields under ligand excitation (QELu). Values meters have to be considered: the number of water molecules
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in the second hydration sphere (qss), the distance between the
protons of these molecules and the Gd(III) ion (rss) as well as
the correlation time modulating the interaction (τss). Although
this theoretical model certainly gives a rough description of
such complex systems, the proton NMRD profiles of the
micelles have been theoretically fitted considering inner
sphere, second sphere and outer sphere contributions (Fig. 7).
The parameters r, d and rss are fixed to 0.31, 0.36 and 0.36 nm
respectively, D is set to 3.0 × 10−9 m2 s−1 and τM is adjusted to
500 ns consistent with other Gd-DTPA bisamide com-
plexes.35,48,58,59 Since the photophysical study points out that
hardly any water molecule is directly bound to the gadolinium
ion, the hydration number q is allowed to vary between 0.0 and
Table 3 Averaged values of the parameters obtained by theoretical
fitting of the 1H NMRD profiles of the micelles in water at 37 °C. The
fittings and parameters of each kind of micelle are shown in Fig. S5, S6
and in Tables S4, S5
Micelles DTPA-
BC12PheA
Micelles DTPA-
BC14PheA
τR (ns)
q
qss
4.2 1.4
0.40 0.04
2.4 0.5
6.6 2.4
0.58 0.04
1.9 0.9
τss (ps)
τSO (ps)
τV (ps)
33.4 11.5
179 18
31.0 4.0
25.5 11.0
121 21
24.9 3.4
0.6, while the number of water molecules in the second coordi- supramolecular structures. At 30–40 MHz, r1-values of 14.2
and 16.0 s−1 mM−1 per Gd(III) ion have been obtained for the
nation shell qss is allowed to vary between 0.0 and 10.0. The
aggregated Gd-DTPA-BC12PheA and Gd-DTPA-BC14PheA com-
Gd- and Eu-DTPA-BC12PheA in water at 37 °C, all follow the plexes respectively. τR values of 4.2 to 6.6 ns are obtained,
NMRD profiles of the micelles consisting of different ratios of
which is in good agreement with the averaged diameter
same trend. For clarity, only those with Gd/Eu ratios of 1 : 1
and 50 : 1 are depicted in Fig. 7 in comparison with the data of
(33–40 nm) of the micelles.37 The relatively high τR values are
the clinically administered Gd-DTPA. In a similar way, the caused by the anchoring of two aliphatic chains of the para-
magnetic chelate into the micellar membrane, leading to a
strong reduction of the local motions.13,38 Although eqn (1)
NMRD results of micelles consisting of Gd- and Eu-DTPA-
BC14PheA are shown. For both assemblies, the different pro-
files are very similar resulting in comparable parameters suggests the absence of water molecules in the first coordi-
nation sphere of the lanthanide ion, a qGd of 0.4 to 0.6 is
achieved by fitting the proton NMRD profiles. Despite the
obtained by theoretical fitting. An overview of the averaged par-
ameter values for each kind of micelle is displayed in Table 3.
The characteristic maximum for slowly tumbling systems at minimization of the luminescence quenching effect induced
by Tween 80®, still a certain amount of coordinated water
molecules can be monitored in MR measurements. The
modest number of water molecules capable of coordinating to
Gd(III) is important for a more efficient relaxation enhance-
ment. Nevertheless, second and outer sphere interactions
between water molecules and the paramagnetic center cannot
be neglected. The fitting indicates the presence of 2.0–2.5
water molecules in the second coordination shell, which also
contributes to the observed relaxivity.
frequencies between 20 and 60 MHz provides a clear indi-
cation for the assembly of the amphiphilic components into
Taken into account the formation of supramolecular assem-
blies, the Lipari–Szabo model has also been used in the analy-
sis of the relaxometric data.60 In this approach, the hydration
number has been fixed to q = 1, neglecting second sphere con-
tributions, r and d are fixed to 0.31 and 0.36 nm respectively, D
is set to 3.0 × 10−9 m2 s−1, τM is adjusted to 500 ns and two
additional parameters have been introduced. In this model, the
global rotational correlation time τRG, as well as the correlation
time governing the local motions τRL, are considered. Further-
more, the order parameter S2 describes the internal flexibility
where S2 = 1 stands for a completely rigid system and S2 = 0 rep-
resents a completely flexible system. Fig. 8 depicts the fitted
proton NMRD profiles of both kinds of micelles and the corres-
ponding averaged parameter values are displayed in Table 4.
According to the Lipari–Szabo model, the average global
correlation time τRG equals 2.6 and 4.1 ns, while the corre-
lation time governing the local motions τRL is about 150 and
204 ps, for the BC12 and BC14 assemblies respectively. More-
over, values of S2 = 0.15–0.23 for the Gd/Eu-DPTA-BC12PheA
micelles and S2 = 0.30–0.41 for the Gd/Eu-DPTA-BC14PheA
micelles have been obtained. The Gd/Eu-DPTA-BC14PheA
Fig. 7 Proton NMRD profiles of micelles composed of 1 : 1 and 50 : 1
Gd/Eu-DTPA-BC12PheA (top) and of 1 : 1 and 50 : 1 Gd/Eu-DTPA-
BC14PheA (bottom) compared to Gd-DTPA in water at 37 °C. The dashed
traces represent the fitted data.
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Fig. 9 Relaxivity per Gd for micelles based on Gd-DTPA-BC16, 50 : 1
Gd/Eu-DTPA-BC12PheA and -DTPA-BC14PheA at 20 and 40 MHz.
Fig. 8 Proton NMRD profiles of micelles composed of 1 : 1 and 50 : 1
Gd/Eu-DTPA-BC12PheA (top) and of 1 : 1 and 50 : 1 Gd/Eu-DTPA-
BC14PheA (bottom) compared to Gd-DTPA in water at 37 °C. The dashed
traces represent the fitted data using the Lipari–Szabo model.
Fig. 10 Proton NMRD profiles of micelles composed of 20 : 1 Gd/Eu-
DTPA-BC14PheA in water at 25 °C, 50 °C and 37 °C. The dashed traces
represent the fitted data.
Table 4 Averaged values of the parameters obtained by theoretical
fitting of the 1H NMRD profiles of the micelles in water at 37 °C using
the Lipari–Szabo model. The fittings and parameters of each kind of
micelle are shown in Fig. S7, S8 and in Tables S6, S7
analogs. On the other hand, the incorporation of Gd-
DTPA-BC14PheA enhances the proton longitudinal relaxation
in a more efficient way.
Micelles DTPA-
BC12PheA
Micelles DTPA-
BC14PheA
qa
1.0
2.57 0.65
162 28
44.5 5.0
150.0 4.9
0.20 0.03
1.0
4.12 1.69
114 13
30.7 2.0
203.5 131
0.33 0.04
In order to gain more information about the influence of τR
on the relaxivity enhancement, the NMRD profiles of the 20 : 1
Gd/Eu-DTPA-BC14PheA micelles have also been measured at
different temperatures (25 °C and 50 °C) (Fig. 10).
τRG (ns)
τSO (ps)
τV (ps)
τRL (ps)
S2
The relaxivity at 40 MHz and 25 °C equals 18.9 s−1 mM−1
,
which is 14 and 52% higher than the relaxivity at 37 and 50 °C
respectively. Consequently, we can presume that in the present
case the τR parameter plays a significant role. Increasing temp-
erature leads to higher molecular motion, resulting in a
decrease of proton relaxation efficiency. The profiles have been
fitted taking into account inner, second and outer sphere con-
tributions to the paramagnetic relaxation rate. The parameters
r, d and rss are fixed to 0.31, 0.36 and 0.36 nm respectively, D is
set to 2.2 × 10−9 m2 s−1 at 25 °C, 3.0 × 10−9 m2 s−1 at 37 °C and
4.0 × 10−9 m2 s−1 at 50 °C. Likewise, τM is adjusted to 700 ns at
25 °C, 500 ns at 37 °C and 200 ns at 50 °C. Considering the
previous fitting, the hydration number q is fixed to 0.6 and the
number of water molecules in the second coordination shell
qss is allowed to vary between 0.0 and 10.0. As can be seen in
Table 5, the τR value reaches 11.1 ns at 25 °C, while a 3- and
16-fold decrease at 37 °C and 50 °C respectively are observed.
Less important, but noteworthy, is the number of water
a Fixed value.
complexes clearly display less local flexibility in the supramole-
cular assemblies due to better insertion into the micellar
monolayer leading to a stronger proton relaxation enhance-
ment at frequencies between 20 and 60 MHz.
Some years ago, Kimpe et al.37 prepared mixed micelles
consisting of Gd-DTPA-bisamide derivatives with alkyl chains
containing 14, 16 and 18 carbon atoms. The micellar com-
pounds with C16 chains displayed the best relaxation pro-
perties. In Fig. 9, the relaxivities at 20 MHz (0.47 T) and
40 MHz (0.94 T) of the micelles containing Gd-DTPA-BC16,
50 : 1 Gd/Eu-DTPA-BC12PheA and -BC14PheA are compared to
the corresponding values of Gd-DTPA.
The relaxivity of the micelles with DTPA-BC12PheA com-
plexes is only slightly higher than that of the DTPA-BC16
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Table 5 Parameter values obtained by theoretical fitting of the 1H
NMRD profiles of micelles consisting of 20 : 1 Gd/Eu-DTPA-BC14PheA in
water at 25, 37 and 50 °C
Table 6 Parameter values obtained by theoretical fitting of the 1H
NMRD profiles of micelles consisting of 20 : 1 Gd/Eu-DTPA-BC14PheA in
water at 25, 37 and 50 °C using the Lipari–Szabo model
25 °C
37 °C
50 °C
25 °C
37 °C
50 °C
τR (ns)
q
qss
τss (ps)
τSO (ps)
τV (ps)
τM (ns)
11.1 2.8
0.6
3.8 0.2
100.0 1.0
3.9 0.6
0.6
3.1 0.1
44.0 0.8
0.7 0.1
0.6
2.6 0.5
τRG (ns)
τSO (ps)
τV (ps)
3.07 0.56
2.07 0.44
1.42 0.33
165
4
127
4
104
2
29.9 1.0
667 377
0.64 0.11
700
33.2 1.0
471 129
0.34 0.09
500
37.7 1.0
367 14
0.10 0.04
200
49.1 5.6 τRL (ps)
70
3
92
1
81
5
S2
32.0 1.0
700
28.6 0.8
500
30.0 2.0 τMa (ns)
200
a Fixed value.
molecules in the second coordination sphere gradually drops
from 3.8 to 2.6, promoting an attenuation of relaxation
efficiency. In addition, these observations give an indication
about the formation of micelles instead of liposomes. If lipo-
somes had been formed, the increase of the temperature
would probably induce an increase of the relaxivity since the
exchange of water through the membrane would be faster and
the contribution from the complexes in the inner cavity would
be increased.61,62 However, a decrease of the relaxivity is
observed when temperature is increased, so rather micellar
structures have been obtained.
ratio and even 55.3 s−1 mM−1 for the 50 : 1 Gd/Eu ratio are
obtained (Fig. 12). Similarly, the relaxation was enhanced up
to 30.0 s−1 mM−1 and 62.7 s−1 mM−1 per mmol micelle con-
sisting of Gd/Eu-DTPA-BC14PheA complexes.
Conclusions
In this study, mixed micelles with a homogeneous size distri-
bution based on amphiphilic DTPA complexes with Gd(III) and
Fitting by the Lipari–Szabo model for which q has been
fixed to 1 also suggests the importance of the τR parameter
(Fig. 11). At the lower temperature of 25 °C, τRG is 1.5 to 2.2
times higher compared to the values at 37 and 50 °C, respect-
ively, and the local motions are more restricted. The corres-
ponding fitted parameter values are displayed in Table 6.
Regarding the sizes of the micelles, the DPPC phospho-
lipids and the Gd(III) and Eu(III) complexes, each assembly
comprises at least 50 compounds.1 Since micelles are formed
starting from 1 lanthanide complex per 12 DPPC molecules,
we can presume the theoretical presence of at least four
lanthanide complexes per particle. This leads to the assump-
tion of two Gd(III) complexes per micelle in the 1 : 1 Gd/Eu-
DTPA-BCnPheA assemblies, three Gd(III) complexes per micelle
in the 3 : 1 Gd/Eu assemblies and approximately four Gd(III)
complexes in the 50 : 1 Gd/Eu composition. Per mmol micelle
consisting of Gd/Eu-DTPA-BC12PheA complexes, relaxation
enhancements of maximum 24.8 s−1 mM−1 for the 1 : 1 Gd/Eu
Fig. 11 Proton NMRD profiles of micelles composed of 20 : 1 Gd/Eu-
DTPA-BC14PheA in water at 25, 37 and 50 °C. The dashed traces rep-
resent the fitted data using the Lipari–Szabo approach.
Fig. 12 Relaxation enhancement per mmol micelle based on Gd/Eu-
DTPA-BC12PheA (top) and Gd/Eu-DTPA-BC14PheA (bottom) at 20 and
40 MHz.
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Eu(III) were synthesized and evaluated for their magnetic and obtained from Alfa Aesar (Ward Hill, USA) and europium(III)
optical properties. Different ratios of Gd and Eu complexes chloride hexahydrate was obtained from Acros Organics (Geel,
were combined into one single micelle in order to find the Belgium).
composition with optimal magnetic and optical performance.
For all compositions, excitation into the ligand levels at
290 nm resulted in the typical red emission bands due to the
5D0→7FJ (J = 0–4) transitions of Eu(III). Extremely low qEu
values obtained from the luminescence studies indicate com-
petition between the micelle’s non-ionic surfactant and H2O
for coordination to the lanthanide ion. Moreover, the hydro-
phobic character of the DTPA bisamides prevents easy water
access to the coordinated lanthanide ions. The micelles are
characterized by a high relaxivity r1 per Gd(III) ion, reaching a
maximum value of 14.2 s−1 mM−1 for the Gd-DTPA-BC12PheA
assemblies and 16.0 s−1 mM−1 for the micellar Gd-DTPA-
Apparatus and methods
Elemental analysis was performed by using a CE Instruments
EA-1110 elemental analyzer. 1H and 13C NMR spectra were
recorded using a Bruker Avance 300 spectrometer (Bruker,
Karlsruhe, Germany), operating at 300 MHz for 1H and
75 MHz for 13C, or on a Bruker Avance 400 spectrometer, oper-
1
ating at 400 MHz for H and 100 MHz for 13C. IR spectra were
measured using
a Bruker Vertex 70 FT-IR spectrometer
(Bruker, Ettlingen, Germany). Mass spectra were obtained
using a Thermo Finnigan LCQ Advantage mass spectrometer.
Samples for the mass spectrometry were prepared by dissol-
ving the product (2 mg) in methanol (1 mL), then adding
200 µL of this solution to a water–methanol mixture (50 : 50,
800 µL). The resulting solution was injected at a flow rate of
5 µL min−1. The metal contents were detected on a Varian
720-ES ICP optical emission spectrometer with reference to a
Chem-Lab gadolinium standard solution (1000 µg mL−1, 2–5%
HNO3). Solutions were dispersed in a 180 W Bandelin Sonorex
RK 510 H sonicator equipped with a thermostatic heating
bath. Absorption spectra were measured on a Varian Cary 5000
spectrophotometer on freshly prepared aqua solutions in
quartz Suprasil® cells (115F-QS) with an optical path-length of
0.2 cm. Emission spectra and luminescence decays of Eu(III)
complexes were recorded on an Edinburgh Instruments FS920
steady state spectrofluorimeter. This instrument is equipped
with a 450 W xenon arc lamp, a high energy microsecond
flashlamp μF900H and an extended red-sensitive photomulti-
plier (185–1010 nm, Hamamatsu R 2658P). All spectra are cor-
rected for the instrumental functions. Luminescence decays
were determined under ligand excitation (290 nm) monitoring
emission of the 5D0→7F2 transition for Eu(III) complexes. Lumi-
nescence decays were analyzed using Edinburgh software; life-
times are averages of at least three measurements. Quantum
yields were determined by a comparative method with quinine
sulfate (Fluka) in 1 N sulfuric acid (Q = 54.6%) as a standard
reference;63 estimated experimental errors for quantum yield
determination 10%. Solutions with a concentration of about
10−5 M were prepared in order to obtain an optical density
lower than 0.05 at the excitation wavelength.
1
BC14PheA at 40 MHz and 310 K. Theoretical fitting of the H
NMRD profiles indicates the presence of 0.4 to 0.6 water mole-
cules in the first and 2.0 to 2.5 water molecules in the second
coordination sphere. The slow tumbling rate of the micellar
systems, the few water molecules capable of coordinating to
Gd(III) and the second and outer sphere interactions between
water molecules and the paramagnetic center are important
for a very efficient relaxation enhancement. The higher relaxi-
vity values for the micelles containing DTPA-BC14PheA com-
plexes is due to restricted local motions because of a more
efficient insertion into the lipid monolayer as could be found
by fitting the profiles according to the Lipari–Szabo model,
fixing q to 1. Temperature dependent relaxivity measurements
reveal that τR plays a significant role concerning the relaxo-
metric properties of the micelles. Lower temperatures promote
the proton relaxation efficiency since the molecular motion of
the compound is reduced.
Considering the luminescence spectra and the NMRD pro-
files of all prepared Gd/Eu micelles and taking into account
the sensitivity of the imaging techniques, the optimal ratio of MR
versus optical reporter in the discussed mixed micelles is deter-
mined to be 20 : 1. In these particular systems, the combination
of magnetic resonance and optical imaging modalities in one
single probe are perfectly compatible. Due to the lack of an inte-
gral coordinated inner sphere water molecule, the Eu(III) lumines-
cence suffers less from non-radiative deactivation. Meanwhile,
very high relaxivity values are obtained which can be attributed to
second and outer sphere interactions and to relatively high τR
values. Since the sensitivity difference between optical and mag-
netic resonance imaging is of great importance, these findings
can contribute to the development of other model bimodal
systems with an optimal load of both imaging probes.
Proton NMRD
Proton nuclear magnetic relaxation dispersion (NMRD) pro-
files were measured on a Stelar Spinmaster FFC, fast field
cycling NMR relaxometer (Stelar, Mede (PV), Italy) over a mag-
netic field strength range extending from 0.24 mT to
0.7 T. Measurements were performed on 0.6 mL samples con-
tained in 10 mm o.d. pyrex tubes. Additional relaxation rates
Experimental
Chemicals
Reagents and solvents were obtained from Acros Organics at 20, 60 and 300 MHz were respectively obtained on a Minis-
(Geel, Belgium), Sigma-Aldrich (Bornem, Belgium) and BDH pec mq20, a Minispec mq60, and a Bruker Avance 300 spectro-
Prolabo (Leuven, Belgium), and were used without further meter (Bruker, Karlsruhe, Germany). The proton NMRD curves
purification. Gadolinium(III) chloride hexahydrate was were fitted using data-processing software,64,65 including
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different theoretical models describing the nuclear relaxation and dried in vacuo at 50 °C. The absence of free lanthanide
phenomena (Minuit, CERN Library).54,55,57
ions was checked with an arsenazo indicator.41
157Gd(III)-DTPA-BC12PheA. Yield: 68%. ESI-MS in MeOH: m/z
calcd 1057.4 [M + Na]+, found 1057.7 [M + Na]+. Anal. calcd for
DLS measurements
Photon correlation spectroscopy was performed at room temp- C50H78GdN5O8·H2O: C, 57.06; H, 7.66; N, 6.65; found: C, 56.77;
erature with a BIC multiangle laser light scattering system with H, 7.85; N, 6.72. FT-IR: ν (cm−1) = 1595 (COO− asym. stretch),
a 90° scattering angle (Brookhaven Instruments Corporation, 1514 (amide II), 1390 (COO− sym. stretch).
Holtsville, USA). The intensity weighted micellar diameter was
measured on 1 × 10−4 M diluted suspensions and calculated calcd 1052.2 [M + Na]+, found 1052.0 [M + Na]+. FT-IR: ν (cm−1
by a non-negatively constrained least-squares (multiple pass) = 1595 (COO− asym. stretch), 1514 (amide II), 1390 (COO− sym.
152Eu(III)-DTPA-BC12PheA. Yield: 60%. ESI-MS in MeOH: m/z
)
routine.
stretch).
157Gd(III)-DTPA-BC14PheA. Yield: 79%. ESI-MS in MeOH: m/z
DTPA-BC12PheA and DTPA-BC14PheA according to a slightly
modified procedure37,66
calcd 1113.5 [M + Na]+, found 1112.8 [M + Na]+. Anal. calcd for
C54H86GdN5O8·H2O: C, 58.51; H, 8.00; N, 6.32; found: C, 58.08;
p-Dodecylaniline (0.52 g, 2 mmol, 2 eq.) or p-tetradecylaniline H, 8.34; N, 6.37. FT-IR: ν (cm−1) = 1595 (COO− asym. stretch),
(0.58 g, 2 mmol, 2 eq.) were dissolved in CHCl3 (30 mL). The 1514 (amide II), 1392 (COO− sym. stretch).
solution was brought to 40 °C and then added dropwise to a
152Eu(III)-DTPA-BC14PheA. Yield: 80%. ESI-MS in MeOH: m/z
solution of DTPA-bisanhydride (0.36 g, 1 mmol, 1 eq.) in dry calcd 1108.3 [M + Na]+, found 1108.4 [M + Na]+. FT-IR: ν (cm−1
)
DMF (40 mL, 40 °C). The reaction mixture was stirred for eight = 1595 (COO− asym. stretch), 1514 (amide II), 1392 (COO− sym.
hours at 50 °C after which the solvents were removed. To the stretch).
yellowish powder, acetone (20 mL) was added and the suspen-
sion was filtered over a Büchner. The pale compound was
Preparation of micelles37
washed with acetone (2 × 20 mL) and was dried overnight in
vacuo at 50 °C. The powder was stirred in water (150 mL) at
80 °C for three hours to dissolve excess of DTPA. After fil-
tration, the residue was washed with acetone (2 × 20 mL) again
and brought to reflux conditions in 150 mL CHCl3 for another
three hours. A creamy white powder was finally obtained after
filtration and rinsing with acetone (2 × 20 mL). The pure com-
pound was dried overnight in vacuo at 50 °C.
1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, 225 mg,
0.3 mmol, 12 eq.) and the amphiphilic complex (25 mg,
0.025 mmol, 1 eq.) were dissolved in 50 mL of a 1 : 1 chloro-
form–methanol solution. After evaporation of the solvents, a
thin film was obtained which was rehydrated with 5 mL of hot
water (70 °C). To improve the solubility, the suspension was
sonicated in a 180 W sonicator with a thermostatic bath at
65 °C for 15 min. Polyoxyethylene sorbitan monooleate or
Tween 80® (75 mg, 0.06 mmol, 2.4 eq.) was added as a surfac-
tant followed by another 15 minutes of sonication to fulfill the
process of micelle formation. Water was evaporated while
slowly reducing pressure.
1
DTPA-BC12PheA (0.55 g, 62%) H NMR (CD3OD, 400 MHz):
δ (ppm) 0.92 (t, 6H, CH3), 1.29 (m, 44H, alkyl CH2), 2.06, 2.19
(s, 10H, N–CH2–C(O)), 2.91, 2.98, 3.51 (t, 8H, N–CH2–CH2–N),
7.02 (d, 2H, phenyl CH), 7.38 (dd, 4H, phenyl CH), 7.54 (d, 2H,
phenyl CH). ESI-MS in MeOH: m/z calcd 926.2 [M + 2Na]+ and
949.2 [M + 3Na]+, found 925.6 [M + 2Na]+ and 947.0
[M + 3Na]+. Anal. calcd for C50H81N5O8·H2O: C, 66.86; H, 9.31;
N, 7.80; found: C, 66.65; H, 9.78; N, 7.98. FT-IR: ν (cm−1) =
1626 (CvO free acid), 1529 (CvO amide).
Micelles were obtained by assembling different ratios of
gadolinium and europium complexes of DTPA-BCnPheA with
n = 12 or 14. The ratios Gd/Eu equaled 0 : 1, 1 : 1, 2 : 1, 3 : 1,
10 : 1, 20 : 1, 50 : 1.
1
DTPA-BC14PheA (0.81 g, 86%) H NMR (CD3OD, 400 MHz):
δ (ppm) 0.91 (t, 6H, CH3), 1.28 (m, 52H, alkyl CH2), 2.36, 2.50
(s, 10H, N–CH2–C(O)), 2.91, 3.02, 3.56 (t, 8H, N–CH2–CH2–N),
Acknowledgements
7.11 (d, 4H, phenyl CH), 7.48 (d, 4H, phenyl CH). ESI-MS in E.D. and T.N.P.V. acknowledge the IWT Flanders (Belgium)
MeOH: m/z calcd 937.3 [M + H]+ and 959.3 [M + Na]+, found and the FWO Flanders (project G.0412.09) for financial
938.0 [M + H]+ and 959.4 [M + Na]+. Anal. calcd for support. S.V.E. was a visiting postdoctoral fellow of the FWO
C54H89N5O8: C, 69.27; H, 9.58; N, 7.48; found: C, 69.55; H, Flanders (project G.0412.09) and now works at the Centre de
9.65; N, 7.53. FT-IR: ν (cm−1) = 1626 (CvO free acid), 1529 Biophysique Moléculaire – CNRS in Orléans. S.V.E thanks the
(CvO amide).
French National Research Agency (Project ANR-10-BLAN-1513).
CHN microanalysis was performed by Mr Dirk Henot. ESI-MS
measurements were done by Mr Dirk Henot and Mr Bert
Ln(III)-DTPA-BC12PheA and Ln(III)-DTPA-BC14PheA37
The ligand (1 mmol) was dissolved in pyridine (30 mL) and a Demarsin and ICP-OES measurements were performed by Ms.
solution of hydrated LnCl3 salt (1.1 mmol) in H2O (1 mL) was Elvira Vassilieva (Department of Earth and Environmental
added. The mixture was brought to 70 °C for 3 hours after Sciences). Mr Karel Duerinckx is acknowledged for his help
which the solvents were evaporated. The crude product was with the NMR measurements. S.L., L.V.E. and R.N.M. thank
then heated at reflux in ethanol for one hour. The suspension the ARC Programs of the French Community of Belgium, the
was cooled to room temperature, the complex was filtered off FNRS (Fonds National de la Recherche Scientifique), the
3598 | Dalton Trans., 2014, 43, 3589–3600
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support and sponsorship concerted by COST Actions (D38 and 21 A. Keliris, T. Ziegler, R. Mishra, R. Pohmann, M. G. Sauer,
TD1004), the EMIL and ENCITE programs and the Center for
Microscopy and Molecular Imaging (CMMI, supported by the
K. Ugurbil and J. Engelmann, Bioorg. Med. Chem., 2011, 19,
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