Magnetofluorescent Nanoaggregates Incorporating Terbium(III) Complexes as Potential Bimodal Agents for Magnetic Resonance and Optical Imaging

Article abstract of DOI:10.1002/ejic.201500310

Terbium(III) ions were coordinated to two diethylenetriaminepentaacetic acid (DTPA) amphiphilic bisamide ligands, and the complexes were assembled into micellar nanoaggregates. The magnetic and optical properties of the resulting nanoaggregates were examined in detail. Upon excitation into the ligand levels at 265 nm, the complexes show characteristic Tb^III emission at 546 nm with quantum yields of up to 7.6 %. Nuclear magnetic relaxation dispersion (NMRD) measurements have shown that the transverse relaxivity r₂ at 500 MHz and 310 K reaches a maximum value of 9.4 s^-1 mM^-1. The efficient T₂ relaxivity at high magnetic field strengths is sustained by the increased rotational correlation time of the nanoaggregates and high magnetic moment of the terbium ion.

Full text of DOI:10.1002/ejic.201500310

FULL PAPER
DOI:10.1002/ejic.201500310
Magnetofluorescent Nanoaggregates Incorporating
Terbium(III) Complexes as Potential Bimodal Agents for
Magnetic Resonance and Optical Imaging
CLUSTER
ISSUE
Michael Harris,[a] Sophie Carron,[a] Luce Vander Elst,[b,c]
Sophie Laurent,[b,c] and Tatjana N. Parac-Vogt*[a]
Keywords: Nanostructures / Nanoaggregates / Micelles / Imaging agents / Bimodal imaging / Lanthanides / Carboxylate
ligands
Terbium(III) ions were coordinated to two diethylenetri-
aminepentaacetic acid (DTPA) amphiphilic bisamide ligands,
7.6%. Nuclear magnetic relaxation dispersion (NMRD)
measurements have shown that the transverse relaxivity r2
and the complexes were assembled into micellar nanoaggre- at 500 MHz and 310 K reaches
a
maximum value of
gates. The magnetic and optical properties of the resulting
nanoaggregates were examined in detail. Upon excitation
into the ligand levels at 265 nm, the complexes show charac-
teristic TbIII emission at 546 nm with quantum yields of up to
9.4 s–1 –1. The efficient T2 relaxivity at high magnetic field
mM
strengths is sustained by the increased rotational correlation
time of the nanoaggregates and high magnetic moment of
the terbium ion.
(DOTA), which shorten the longitudinal relaxation time
(T1) of water protons and produce a positive contrast but
suffer from dramatic loss of efficiency at high magnetic
fields.[3]
Introduction
Powerful in vivo techniques such as magnetic resonance
imaging (MRI), positron emission tomography (PET),
fluorescence imaging and bioluminescence are indispens-
able in clinical diagnostics. As each imaging technique has
its own strengths and weaknesses, approaches that combine
different, complementary techniques could overcome the
inherent limitations associated with one individual tech-
nique. Although MRI is ideal for whole-body images owing
Alternatively, iron oxide nanoparticles can accelerate the
transverse relaxation time of water protons (T2) to produce
a negative contrast.[4] Decreasing the molecular tumbling
rate of the GdIII complex through the conjugation of
chelates to polymers or dendrimers,[5] noncovalent inter-
actions with human serum albumin[6] or incorporation into
supramolecular micelles or liposomes[7] has been frequently
used to increase the relaxation performance of contrast
agents, especially for high-magnetic-field applications.
In a recent effort to enhance the imaging performance of
CAs, probes combining MRI and luminescence properties
have been synthesized to combine good resolution with
high sensitivity and, thereby, allow the investigation of
samples in exquisite detail.[8] The attachment of DTPA and
DOTA to several organic dyes[9] and transition metal com-
plexes[10] have been investigated, and their bimodal applica-
tions have been exploited. The design and characterization
of fluorescent liposomes[11] and nanoparticles to enhance
the longitudinal[12] or transverse relaxation times[13] have
also been reported. The use of bioconjugates led to signifi-
cant improvements of MRI and optical properties; however,
practical applications are limited by the short luminescence
lifetimes (100–300 ns), small Stokes shifts and poor
resistance to photobleaching of the organic fluorophores.
In a different approach, transition metal complexes have
been linked to DTPA or DOTA to produce luminescent
to its good spatial resolution, large concentrations of GdIII
-
based contrast agents are required owing to its rather low
sensitivity.[1] On the other hand, luminescence-based im-
aging can provide high-resolution images, but this tech-
nique is only suitable for thin tissue samples because of the
low optical transparency of biological tissue.[2]
The current contrast agents (CAs) used in magnetic reso-
nance imaging (MRI) are based on gadolinium(III) com-
plexes with diethylenetriaminepentaacetic acid (DTPA) or
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid
[a] Department of Chemistry, KU Leuven,
Celestijnenlaan 200F, 3001 Leuven, Belgium
E-mail: tatjana.vogt@chem.kuleuven.be
http://chem.kuleuven.be/lbc/
[b] Department of General, Organic and Biomedical Chemistry,
NMR and Molecular Imaging Laboratory, University of Mons,
7000 Mons, Belgium
E-mail: luce.vanderelst@umons.be
[c] CMMI – Center for Microscopy and Molecular Imaging,
6041 Gosselies, Belgium
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejic.201500310.
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CAs.[10a,10b,14] However, in terms of their luminescence ylamides, which can be excited at a wavelength of 265 nm
properties, lanthanides have several advantages as they can and upon energy transfer to the TbIII centre result in ob-
exhibit excited-state lifetimes in the range of milliseconds servable luminescence. A new procedure to create smaller
and, thereby, remove the problem of short-lifetime auto- micelles of approximately 10 nm has been developed to pro-
fluorescence. In addition, they have sharply spiked emission duce CAs that are potentially more biocompatible owing to
spectra,
a
large energy difference between their their possible easier excretion. The relaxation and lumines-
emission bands, and absorption bands of coordinated sensi- cence efficiency of the resulting nanoaggregates have been
tizers, which often results in impressive luminescence. How- investigated in detail, and their potential as CAs for MRI
ever, as gadolinium(III) ions are not luminescent, the and optical imaging has been discussed.
creation of a bimodal CA containing the gadolinium(III)
centre needed to produce an MRI signal and another
Results and Discussion
luminescent lanthanide centre for the optical signal is
challenging owing to the very similar coordination proper-
ties across the lanthanide series. We recently reported a
successful strategy for the creation of a heteropolymetallic
lanthanide complex[14a] with selectively incorporated
gadolinium(III) and europium(III) ions. The multistep syn-
thetic procedure resulted in a metallostar complex with
favourable luminescence and relaxometric properties. In an-
other approach, the incorporation of amphiphilic gadolini-
um(III) complexes with europium(III) complexes into mi-
celles resulted in magnetofluorescent nanoaggregates, in
which the relaxivity and luminescence intensity could be
tuned by changing the ratio of the amphiphilic gadolini-
um(III) and europium(III) complexes.[15]
Synthesis of Ligands, Complexes and Aggregates
Two DTPA bisamide derivatives with long hydrophobic
side chains were synthesized according a literature pro-
cedure.[18] The synthetic pathway and the obtained ligands
Tb-DTPA-BC12PheA and Tb-DTPA-BC14PheA are pre-
sented in Scheme 1. All of the ligands were characterized
by nuclear magnetic resonance spectroscopy, mass spec-
trometry, and IR spectroscopy.
However, the field of bimodal imaging with one single
chelated LnIII ion has been very scarcely explored. We re-
cently reported a new approach towards bimodal contrast
agents based on a single LnIII ion by exploiting the unique
properties of DyIII and TbIII ions.[16] DyIII and TbIII com-
plexes show strong luminescence owing to the effective
shielding of their valence electrons and are, therefore, char-
acterized by sharp emission lines, which make them appro-
priate for optical imaging (OI) applications. In addition,
owing to the large magnetic moments of DyIII and TbIII
ions, their complexes exhibit large T2 relaxivities at higher
magnetic fields. This is a very interesting property as
modern MRI instruments are progressively using higher
magnetic field strengths at which traditional GdIII T1 posi-
tive contrast agents such as Gd-DTPA and Gd-DOTA suf-
fer from dramatic decreases in performance.[17] Further-
more, a significant increase of T2 relaxivity is predicted if
the molecular volume and rotational correlation time (τC)
Scheme 1. (i) Acetic anhydride (2 equiv.), pyridine (4 equiv.), 65 °C,
of CAs increase. A convenient approach to increase the size 1 h. (ii) N,N-dimethylformamide (DMF), CHCl3, 50 °C, 8 h. (iii)
Pyridine, 70 °C, 3 h. 1: Tb-DTPA-BC12PhenA or Tb-DTPA-
of the CAs is to incorporate them into biocompatible
BC14PhenA with coordinated water molecule omitted; R = C12 or
nanoaggregates. The incorporation of amphiphilic DyIII
C14 alkyl chain.
and TbIII complexes into mixed micelles indeed resulted in
assemblies that showed large transverse relaxivities at mag-
The two ligands were coordinated to TbIII ions in pyr-
netic fields of 300–500 MHz.[16] A comparison between idine by a slightly modified procedure.[17] The DTPA chela-
DyIII and TbIII nanoaggregates revealed that the TbIII tor was dissolved in pyridine, and a solution of hydrated
nanoaggregates hold more potential as bimodal probes be- TbCl3 (1.1 equiv.) in water was added. The solution was
cause of their larger quantum yields and comparably ef- mixed for 3 h, and the solvents were evaporated. The solid
ficient transverse relaxivity at high fields. Therefore, in this product was suspended in acetone, collected by filtration,
study, we further explore the potential of nanoaggregates washed with acetone, acetone/water (50:50) and again with
based on TbIII complexes as potential CAs for MRI and acetone, and then dried in vacuo. The absence of free
optical imaging and report two new TbIII complexes based lanthanide ions was verified by the addition of an
on amphiphilic DTPA ligands, which were synthesized by a arsenazo(III) indicator solution.[19] The complex formation
facile route. The ligands were derivatized with p-alkylphen- was established by mass spectrometry (Figures S1 and S2
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in the Supporting Information), IR spectroscopy, and total
reflection X-ray fluorescence (TXRF) analysis.
The lanthanide complexes of DTPA-BC12PhenA and
DTPA-BC14PhenA consist of a hydrophilic centre and two
hydrophobic tails with a phenyl ring and a chain of 12 or
14 carbon atoms. The amphiphilic nature permits them to
be incorporated into mixed micelles to create slowly
tumbling supramolecular structures with limited local
motion of the lanthanide complexes. Variation of the ratio
of surfactant to phospholipid and TbIII complex was inves-
tigated to gain control over the nanoaggregate formation.
We observed that increasing the surfactant to 50 mol-% or
greater resulted in small nanoaggregates. The nanoaggre-
gates were formed by mixing 1 equiv. of the Ln complex of
interest with 12 equiv. of phospholipid (DPPC) and 6.5
equiv. of surfactant (Tween 80®). Photon correlation spec-
troscopy measurements showed two different size distri-
bution profiles for the C12 and C14 alkyl chain systems (Fig-
ures S3 and S4). The C12 system had a narrower size distri-
bution than the C14 system, possibly because of the disrup-
tion of the DPPC packing in the micelles with the C14 sys-
tem. This has allowed completely transparent nanoaggreg-
ate solutions with distribution maxima at 9 nm for Tb-
DTPA-BC12PheA and 11 nm for Tb-DTPA-BC14PheA.
Furthermore, in the C12 distribution, a small peak at ca.
1 nm was observed and identified to most likely originate
from micelles formed by surfactant Tween 80® alone.
Figure 1. Normalized absorbance spectra of TbIII complexes in
water (pH 7.4, 1 wt.-%, 298 K).
Photophysical Properties
Owing to the πǞπ* transitions of the ligands, the ter-
bium complexes display well-defined absorption bands
(Figure 1). As shown in Figure 1, a ligand-centred band in
the range λ = 220–300 nm with a maximum at λ ≈ 240–260
is observed in the absorption spectrum of the supramolec-
ular structures corresponding to the ligand electronic tran-
sitions. The excitation spectrum of Tb-DTPA-BC12PhenA
(Figure 2) shows a small shoulder at λ ≈ 300 nm, which
could be caused by the varied incorporation of the complex
into the DPPC micelles. For both complexes, a wavelength
of 265 nm remains the most efficient for energy transfer to
the TbIII ions.
Figure 2. Corrected and normalized excitation spectra of TbIII
complexes in water (pH 7.4, 1 wt.-%, 298 K). Emission wavelength
546 nm.
The emission spectra of the micelles display sharp emis-
5
sion bands attributed to the D4Ǟ7FJ (J = 9–3) transitions
of the TbIII ions upon excitation at 265 nm (Figure 3). The
efficient energy transfer from the ligand to lanthanide ion
is maintained as little ligand-centred emission is detected.
The luminescence decays were measured in both H2O
and D2O, and the monoexponential fit indicates the pres-
ence of only one luminescent lanthanide species in the mi-
cellar structures. The luminescence lifetimes in H2O are 1.8
and 2.1 ms for the micelles consisting of Tb-DTPA-
BC12PheA and Tb-DTPA-BC14PheA, respectively. In D2O,
luminescence lifetimes of up to 2.6 and 2.7 ms were ob-
Figure 3. Corrected and normalized emission spectra of TbIII com-
plexes in water (pH 7.4, 1 wt.-%, 298 K). Excitation wavelength
265 nm.
tained for Tb-DTPA-BC12PheA and Tb-DTPA-BC14PheA, aminocarboxylate systems[20] was employed to determine
respectively. Within the uncertainty of the luminescence the number of coordinated water molecules q with an
method, the phenomenological Equation (1) for TbIII poly- accuracy of Ϯ0.2–0.3:
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qTb (H2O) = 5.0(Δkobs – 0.06)
(1)
Δkobs = 1/τH2O – 1/τD2O is expressed in ms–1. According to
Equation (1), q values of 0.4 and 0.3 were obtained for Tb-
DTPA-BC12PheA and Tb-DTPA-BC14PheA, respectively.
According to Scheme 1, the TbIII complex should have one
water ligand in its first coordination sphere. However, the
nonionic surfactant Tween 80® at the periphery of the
assembled structures is able to form hydrogen bonds with
H2O and act as a competitor in lanthanide coordination,
which results in a value of less than 1. As a consequence,
less nonradiative deactivation due to O–H vibrations will
lead to longer luminescence lifetimes and, consequently, to
a reduced q number in the phenomenological Equa-
tion (1).[21]
Figure 4. Proton longitudinal relaxivity of the micelles versus
proton Larmor frequency at 310 K.
The TbIII luminescence quantum yields were determined
by excitation at 265 nm into the ligand levels. The values
were calculated to be 7.6% for both complexes (Table 1).
lated by the rotational correlation time of the compound τR
and the translational correlation time τD; τD equals a2/D, in
which a is the distance of the closest approach between the
water protons and the paramagnetic centre, and D is the
relative diffusion constant of the water molecules. Thus, the
proton longitudinal relaxivity will increase slightly at higher
fields.
Table 1. Photophysical data for the TbIII complexes in water (pH
7.4, 1 wt.-%) at 298 K.
[a]
τ H2O [ms] τ D2O [ms]
q
QLTb [%]
DTPA-BC12PhenA 1.8(1)
DTPA-BC14PhenA 2.1(1)
2.4(1)
2.7(1)
0.4 7.6
0.3 7.6
[a] Estimated relative errors QLTb Ϯ10%. Quantum yields relative to
rhodamine 101 in EtOH.
Proton Transverse Relaxation Tate
The T2 enhancements at 20, 60, 300 and 500 MHz by the
complexes are depicted in Figure 5. At the proton Larmor
frequency of 20 MHz, the transverse relaxivities for the
complexes are slightly higher in comparison to the longitu-
dinal values (r2 = 0.17 mm–1 s–1). This increase is due to the
relative large values of τM and ΔωM. At higher magnetic
fields (νo Ͼ 100 MHz), r2 increases. Although the transverse
Relaxometric Studies
Proton Longitudinal Relaxation Rate
The enhancement of the relaxation rate by 1 mm of the relaxivity depends on the square of the magnetic field, r2
TbIII compound determines the proton longitudinal relaxi- shows a strong reliance on the τM value as the external mag-
vity (r1). In Figure 4, the proton longitudinal relaxivities of netic field increases. In that particular case, it is important
the two compounds at 20, 60, 300, and 500 MHz are de- that the chemical shift difference between coordinated and
picted. The profiles of the two compounds follow the same bulk water (ΔωM) remains low relative to the water ex-
trend. At low magnetic fields (20–60 MHz), low r1 values of change to avoid limitation by τM.[22a] The chemical shift
0.10–0.11 mm–1 s–1 were obtained. At higher magnetic fields of the coordinated water molecule is proportional to the
(300–500 MHz), a slight increase of r1 to 0.12–0.15 mm–1 s–1 magnetic field and is the sum of contact and pseudocontact
was observed. The variation between the complexes corre- terms.
sponds to the variation of the q values, as has been pre-
The fitting of the data was performed by using the equa-
tions defining the inner- and outer-sphere contributions as
viously observed with Tb-DOTA complexes.[16b]
Both inner- (1/T1is) and outer-sphere (1/T1os) contri- described by Vander Elst et al.[22a] The inner-sphere contri-
butions to the proton longitudinal relaxation rate are de- butions depend on the ratio [Tb complex]/[water], the value
fined by the sum of dipolar (1/T1DD) and Curie (1/T1C) con- of q, the water residence time τM and the transverse relax-
tributions. At low magnetic fields, the longitudinal relax- ation rate of the coordinated water molecule 1/T2M. The
ation rate is mainly modulated by the dipolar interactions latter factor results from dipolar, dipolar Curie, and Curie
between the water protons and the static magnetic moments contact contributions. The correlation time τC modulates
of the electrons of the TbIII centres and is highly dependent the dipolar interaction and is related to τR, τS and τM
on the electronic relaxation time τS.[22] Those findings ex- through τC–1 = τR–1 + τS–1 + τM–1, whereas the Curie contri-
–1
plain the low r1 values in this region, as TbIII ions are char- bution is modulated by τCC, which is defined as τCC
=
acterized by very short τS values (ca. 0.5 ps).[22a,23] At high τR–1 + τM–1. During the fitting procedure, the parameters q
magnetic fields, the Curie inner- and outer-sphere contri- = bound water molecules(s) as determined by luminescence
butions become more significant. These terms are modu- lifetimes, r = 0.31 nm, a = 0.36 nm, D = 3.3ϫ10–9 m2 s–1
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Figure 6. Ratios of proton transverse versus longitudinal relaxivity
at 20, 60, 300 and 500 MHz for the TbIII complexes at 310 K.
Conclusions
The amphiphilic terbium(III) complexes built into mixed
micelles reported in this study show magnetic and optical
properties that make them potential candidates for mag-
netic resonance and optical imaging. The micelles showed
long luminescence lifetimes in H2O at the emission wave-
length of 546 nm and high quantum yields of up to 7.6%.
The complexes also exhibit transverse relaxivities r2 of close
to 10 s–1 mm–1 at 500 MHz and 310 K. In comparison with
previously reported DyIII-DTPA micelle complexes, the
TbIII complexes provides a large increase in luminescence
quantum yields. Additionally, in comparison with the
values for previously reported DyIII-DTPA micelle com-
plexes,[16a] a decrease in r2 has been observed, which could
mainly result from a significant decrease in the nanoaggreg-
ate sizes for the TbIII complexes. Smaller micelles are ad-
vantageous for biocompatibility and elimination from the
body, and in this work we created monodisperse nanoaggre-
gates; the C12 alkyl system showed the narrowest distri-
bution in the 9 nm range. In comparison with our recently
reported Tb-DOTA complexes,[16b] the DTPA complexes
show slightly less efficient T2 relaxivity. This is most likely
a consequence of decreased water exchange kinetics and a
lower number of bound water molecules in the DTPA com-
plexes. However, the advantage of the complexes reported
in this work is their more facile synthetic route. The exci-
tation at 265 nm could be overcome by further optimization
of the complexes by using different chromophores. This
may lead to further improvements to the optical properties
and establish TbIII complexes as a class of potential candi-
dates for magnetic resonance and optical imaging.
Figure 5. Proton transverse relaxivity of the micelles versus proton
Larmor frequency at 310 K. The lines represent the fitted data.
The concentration of DTPA-BC12Phen A was 6.38 mm, and that
of DTPA-BC14Phen A C14 was 6.03 mm.
and τR = 1 ns were fixed. The parameters τS, τM and ΔωM
were extracted from the fit and are listed in Table 2 but
should only be considered as estimates as the fit is only over
four points.
Table 2. Values of τS, τM and ΔωM obtained by fitting the 1H r2
data of the TbIII complexes at 310 K.
τs [ps]
τM [ns]
ΔωM
[105 rads–1 T–1]
DTPA-BC12PhenA
DTPA-BC14PhenA
0.25
0.25
500
500
2.3
2.1
The τS values were 0.25 ps. The fit of the proton nuclear
magnetic relaxation dispersion (NMRD) profiles of the mi-
celles indicates that there is little difference between the C12
and C14 complexes. The slow molecular motion leads to
transverse relaxivities of 9.4 and 6.6 s–1 mm–1 at 500 MHz
and 310 K for Tb-DTPA-BC12PheA and Tb-DTPA-
BC14PheA, respectively. The slight variation of the number
of coordinated water molecules (0.4 to 0.3) shows that the
Tb-DTPA-BC12PheA micelles with the greatest q value are
the most efficient negative contrast agent. The perform-
ances of the compounds as efficient r2 agents are enforced
by the high magnetic moment of the terbium ion (μ =
9.81 μB), the presence of water molecules in the first coordi-
nation sphere and long rotational correlation time.[22b]
Furthermore, DPPC forms micelles of 50 phospholipid
molecules[24] in which the TbIII load would most likely be
four molecules per micelle.
Experimental Section
Materials: Reagents and solvents were obtained from Sigma–
Aldrich (Bornem, Belgium), Acros Organics (Geel, Belgium),
ChemLab (Zedelgem, Belgium), Matrix Scientific (Columbia,
USA) and BDH Prolabo (Leuven, Belgium) and were used without
further purification. Terbium(III) chloride hexahydrate was ob-
tained from Sigma–Aldrich (Bornem, Belgium).
As tissues display a shorter T2 than T1, contrast agents
with significant r2/r1 ratios can be beneficial through the
use of appropriate pulse sequences in clinical applications.
In Figure 6, the r2/r1 ratios are depicted. The ratio increases
for both complexes with increasing magnetic field strength.
The largest ratio of r2/r1 is observed for Tb-DTPA-
BC12PheA; however, the two complexes show very similar
behaviour.
Instrumentation: 1H NMR spectra were recorded by using a Bruker
Avance 300 spectrometer (Bruker, Karlsruhe, Germany) operating
at 300 MHz.
IR spectra were recorded by using a Bruker Vertex 70 FTIR spec-
trometer (Bruker, Ettlingen, Germany).
Mass spectra were obtained by using a Thermo Finnigan LCQ
Advantage mass spectrometer. Samples for the mass spectrometry
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were prepared by dissolving the product (2 mg) in methanol
(1 mL), and then adding this solution (200 μL) to a water/methanol
mixture (50:50, 800 μL). The resulting solution was injected at a
flow rate of 5 μLmin–1.
DTPA-BC14PhenA: Yield 1.2 g, 91%. 1H NMR (300 MHz, [D5]-
pyridine, 25 °C, TMS): δ = 0.88 [t, 6 H, CH3(CH2)11CH2CH2Ar],
1.26 [m, 44 H, CH3(CH2)11CH2CH2Ar], 1.59 [m, 4 H, CH3(CH2)9-
CH2CH2Ar], 2.56 [t, 4 H, CH3(CH2)9CH2CH2Ar], 3.19 (t, 8 H,
NCH2CH2N), 3.82, 3.84, 3.86 [t, 10 H, NCH2C(O)], 7.22, 7.26 ppm
(d, 4 H, under solvent signal, phenyl CH), 8.14, 8.17 (d, 4 H, phenyl
CH), 10.68 ppm (s, 2 H, amide NH). ESI-MS (+): m/z = 937.8 [M
+ H]+, 958.9 [M + Na]+.
The TXRF measurements were performed with a Bruker S2 Pico-
fox spectrometer (Bruker, Berlin, Germany) with a molybdenum
source. Terbium(III) solutions of approximately 1000 ppm in milli-
Q water were prepared and these solutions (500 μL) were mixed
with a 1000 ppm Chem-Lab gallium standard solution (500 μL,
1000 μg/mL, 2–5% HNO3). This mixture with similar TbIII and
GaIII concentrations was placed on a Bruker AXS quartz glass
sample plate for measurement.
Terbium(III) DTPA-BC12PhenA and DTPA-BC14PhenA Complexes:
The ligand (0.1 g, Ϯ0.1 mmol, 1 equiv.) was dissolved in pyridine
(5 mL), and a solution of hydrated TbCl3 hexahydrate salt
(0.11 mmol, 1.1 equiv.) in H2O (0.2 mL) was added. The mixture
was heated to 70 °C for 3 h, after which the solvents were evapo-
rated. The crude product was suspended in acetone (10 mL) and
filtered through a Büchner funnel. The solid was washed with an
acetone/water 50:50 mixture (2ϫ 5 mL) to remove any free TbIII
ions, rinsed again with acetone (2ϫ 10 mL) and dried in vacuo.
The absence of free lanthanide ions was checked with an arsenazo
indicator.
The solutions were dispersed with a 180-W Bandelin Sonorex RK
510 H sonicator equipped with a thermostatic heating bath.
The absorption spectra were recorded with a Varian Cary 5000
spectrophotometer with freshly prepared aqueous solutions in
quartz Suprasil cells (115F-QS) with an optical pathlength of 1 cm.
The emission spectra and luminescence decays of the TbIII micellar
complexes were recorded with an Edinburgh Instruments FS920
steady-state spectrofluorimeter. This instrument was equipped with
a 450W xenon arc lamp, a high-energy microsecond flashlamp
(mF900H) and an extended red-sensitive photomultiplier (185–
1010 nm, Hamamatsu R 2658P). All spectra are corrected for the
instrumental functions. The luminescence decays were determined
under ligand excitation (265 nm) with the emission of the 5D4Ǟ7FJ
(J = 9–3) transition of the TbIII complexes monitored. The lumines-
cence decays were analyzed by using Edinburgh software; the life-
times are averages of at least three measurements. The quantum
yields were determined by a comparative method with a standard
reference; the estimated experimental errors for the quantum yield
determinations are Ϯ10%. Rhodamine 101 (Sigma) in ethanol (Q
= 100%) was used as a standard for the complexes. Solutions with
concentrations of ca. 10–5 m were prepared to obtain an optical
density lower than 0.05 at the excitation wavelength.
TbIII-DTPA-BC PhenA: Yield 80%. IR: νmax = 1596 (COO– asym.
˜
12
stretch), 1514 (amide II), 1391 cm–1 (COO– sym. stretch). ESI-MS
(+): m/z = 1036.8 [M + H]+, 1058.8 [M + Na]+.
TbIII-DTPA-BC PhenA: Yield 82%. IR: νmax = 1595 (COO– asym.
˜
14
stretch), 1514 (amide II), 1392 cm–1 (COO– sym. stretch). ESI-MS
(+): m/z = 1092.8[M + H]+, 1116.0[M + Na]+.
Preparation of Micelles: 1,2-Dipalmitoyl-sn-glycero-3-phospho-
choline (DPPC, 80 mg, 0.109 mmol, 12 equiv.) and the amphiphilic
complex (10 mg, Ϯ0.091 mmol, 1 equiv.) were dissolved in a 1:1
chloroform/methanol solution (2 mL). After the evaporation of the
solvents from a flask with a septum and a needle fitted in a vacuum
oven at 50 °C, the obtained thin film was rehydrated with hot water
(2 mL, 70 °C). To improve the solubility, the suspension was soni-
cated with a 180 W sonicator with a thermostatic bath at 65 °C for
15 min. Polyoxyethylene sorbitan monooleate or Tween 80®
(77 mg, 0.06 mmol, 6.5 equiv.) was added as a surfactant followed
by another 15 min of sonication to complete the process of micelle
formation. The water was evaporated in a flask with a septum and
needle fitted in a vacuum oven overnight at 50 °C to leave a thin
film. A small sample was removed for dynamic light scattering
(DLS) measurements. For the preparation of samples for relaxome-
try measurements, the thin film was rehydrated with Milli-Q water
(1 mL), sonicated for 15 min and passed through a 200 nm PTFE
filter. The terbium(III) concentrations were analyzed by TXRF be-
fore relaxometric measurements.
Relaxometry: 1H T1 and T2 measurements were performed at 310 K
and 0.47, 1.41, 7.05 and 11.75 T with Bruker Minispec mq-20, mq-
60, Avance-300 and Avance-500 instruments, respectively. The T1
values were measured by using the inversion-recovery sequence,
and the T2 values were obtained by using the CMPG sequence.
The echo time was set to 1 ms. The diamagnetic contribution was
the contribution of pure water.
Dynamic Light Scattering Measurements: Photon correlation
spectroscopy was performed at room temperature with a BIC
multiangle laser light-scattering system with a 90° scattering angle
(Brookhaven Instruments Corporation, Holtsville, USA). The Acknowledgments
intensity-weighted micellar diameter was measured for 0.1 wt.-%
L. V. E. and S. L. thank Fabian Rouffiange for his help, the French
diluted suspensions in Milli-Q water, sonicated for 15 min, passed
through a 200 nm polytetrafluoroethylene (PTFE) filter before
analysis and calculated by a non-negatively constrained least-
squares (multiple pass) routine.
Community of Belgium for support through ARC programs
(AUWB-2010-10/15-UMONS-5), the Fonds National de la Recher-
che Scientifique (FNRS), the Belgian Science Policy (UIAP VII
program), for support and sponsorship concerted by COST Ac-
tions (D38 and TD1004), the European Network of Excellence
EMIL (European Molecular Imaging Laboratories) program
(LSCH-2004-503569), and the Center for Microscopy and Molecu-
lar Imaging (CMMI), supported by the European Regional Devel-
Synthesis: DTPA bisanhydride and DTPA ligands were synthesized
as described previously.[16a]
1
DTPA-BC12PhenA: Yield 1.05 g, 62%. H NMR {300 MHz, [D5]-
pyridine, 25 °C, tetramethylsilane TMS): δ = 0.88 [t,
6 H,
CH3(CH2)9CH2CH2Ar], 1.26 [m, 36 H, CH3(CH2)9CH2CH2Ar], opment Fund and the Walloon Region.
1.58 [m, 4 H, CH3(CH2)9CH2CH2Ar], 2.55 [t, 4 H, CH3(CH2)9-
CH2CH2Ar], 3.19 (t, 8 H, NCH2CH2N), 3.82, 3.84, 3.86 [t, 10 H,
[1] E. Debroye, T. N. Parac-Vogt, Chem. Soc. Rev. 2014, 43, 8178–
NCH2C(O)], 7.22, 7.26 ppm (d, 4 H, under solvent signal, phenyl
CH), 8.14, 8.17 (d, 4 H, phenyl CH), 10.68 ppm (s, 2 H, amide
NH). ESI-MS (+): m/z = 881.2 [M + H]+, 903.0 [M + Na]+.
8192.
[2] J. C. Hebden, S. R. Arridge, D. T. Delpy, Phys. Med. Biol. 1997,
42, 825–840.
Eur. J. Inorg. Chem. 2015, 4572–4578
4577
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.eurjic.org
FULL PAPER
[3] P. Caravan, J. J. Ellison, T. J. McMurry, R. B. Lauffer, Chem.
Rev. 1999, 99, 2293–2352.
G. V. Velde, A. Ketkar-Atre, U. Himmelreich, M. D. Cuyper,
Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2011, 3, 197–
211.
[4] a) F. Hu, Nanoscale 2012, 4, 6235; b) S. L. Jeon, M. K. Chae,
E. J. Jang, C. Lee, Chem. Eur. J. 2013, 19, 4217–4222; c) F. Ye,
Contrast Media Mol. Imaging 2012, 7, 460–468; d) S. Soenen,
Contrast Media Mol. Imaging 2009, 4, 207–219; e) H. B. Na,
I. C. Song, T. Hyeon, Adv. Mater. 2009, 21, 2139–3148.
[5] a) A. J. L. Villaraza, A. Bumb, M. W. Briechbiel, Chem. Rev.
2010, 110, 2921–2959; b) P. Lebdusˇková, J. Kotek, P. Hermann,
L. Vander Elst, R. N. Muller, I. Lukesˇ, J. A. Peters, Bioconjug-
ate Chem. 2004, 15, 881–889; c) C.-H. Huang, K. Nwe, A. A.
Zaki, M. W. Brechbiel, A. Tsourkas, ACS Nano 2012, 6, 9419–
9424; d) Y. Li, M. Beija, S. Laurent, L. Vander Elst, R. N.
Muller, H. T. T. Duong, A. B. Lowe, T. P. Davis, C. Boyer,
Macromolecules 2012, 45, 4199–4204; e) K. Luo, G. Lui, W.
She, Q. Wang, G. Wang, B. He, H. Ai, Q. Gong, B. Song, Z.
Gu, Biomaterials 2011, 32, 7951–7960.
[6] a) M. Zhen, J. Zheng, L. Ye, S. Li, C. Jin, K. Li, D. Qiu, H.
Han, C. Shu, Y. Yang, C. Wang, ACS Appl. Mater. Interfaces
2012, 4, 3724–3729; b) T. N. Parac-Vogt, K. Kimpe, S. Laurent,
L. Vander Elst, C. Burtea, F. Chen, R. N. Muller, Y. C. Ni, A.
Verbruggen, K. Binnemans, Chem. Eur. J. 2005, 11, 3077–3086;
c) P. Caravan, Acc. Chem. Res. 2009, 42, 851–862; d) C. Henou-
mout, V. Henrotte, S. Laurent, L. Vander Elst, R. N. Muller,
K. Binnemans, J. Inorg. Biochem. 2008, 102, 721–730.
[12] W. J. Rieter, J. S. Kim, K. M. L. Taylor, H. An, W. Lin, T. Tarr-
ant, W. Lin, Angew. Chem. Int. Ed. 2007, 46, 3680–3682; An-
gew. Chem. 2007, 119, 3754.
[13] a) P. Howes, M. Green, A. Bowers, D. Parker, G. Varma, M.
Kallumadil, M. Hughes, A. Warley, A. Brain, R. Botnar, J.
Am. Chem. Soc. 2010, 132, 9833–9842; b) S. Ronchi, M. Co-
lombo, P. Verderio, S. Mazzucchelli, F. Corsi, C. D. Palma, R.
Allevi, E. Clementi, D. Prosperi, AIP Conf. Proc. 2010, 1275,
102–105; c) D. Bhattacharya, M. Das, D. Mishra, I. Banerjee,
S. K. Sahu, T. K. Maiti, P. Pramanik, Nanoscale 2011, 3, 1653–
1662; d) Y. Chen, H. Chen, S. Zhang, F. Chen, L. Zhang, J.
Zhang, M. Zhu, H. Wu, L. Guo, J. Feng, J. Shi, Adv. Funct.
Mater. 2011, 21, 270–278.
[14] a) E. Debroye, M. Ceulemans, L. Vander Elst, S. Laurent,
R. N. Muller, T. N. Parac-Vogt, Inorg. Chem. 2014, 53, 1257–
1259; b) A. Boulay, C. Deraeve, L. Vander Elst, N. Leygue, O.
Maury, S. Laurent, R. N. Muller, B. Mestre-Voegtlé, C. Picard,
Inorg. Chem. 2015, 54, 1414–1425.
[15] E. Debroye, S. V. Eliseeva, S. Laurent, L. Vander Elst, R. N.
Muller, T. N. Parac-Vogt, Dalton Trans. 2014, 43, 3589–3600.
[16] a) E. Debroye, S. Laurent, L. Vander Elst, R. N. Muller, T. N.
Parac-Vogt, Chem. Eur. J. 2013, 19, 16019–16028; b) M. Harris,
S. Carron, L. Vander Elst, S. Laurent, R. N. Muller, T. N. Pa-
rac-Vogt, Chem. Commun. 2015, 51, 2984–2986.
[7] a) F. Kielar, L. Tei, E. Terreno, M. Botta, J. Am. Chem. Soc.
2010, 132, 7839–7837; b) T. N. Parac-Vogt, K. Kimpe, S. Laur-
ent, C. Piérart, L. Vander Elst, R. N. Muller, K. Binnemans, [17] S. Laurent, T. N. Parac-Vogt, K. Kimpe, C. Thirifays, K.
Eur. J. Inorg. Chem. 2004, 3538–3543; c) C. Vanasschen, N.
Bouslimani, D. Thonon, J. F. Desreux, Inorg. Chem. 2011, 50,
8949–8958.
Binnemans, R. N. Muller, L. Vander Elst, Eur. J. Inorg. Chem.
2007, 2061–2067.
[18] E. Debroye, S. Laurent, L. Vander Elst, R. N. Muller, T. N.
Parac-Vogt, Chem. Eur. J. 2013, 19, 16019–16028.
[19] H. Onishi, K. Sekine, Talanta 1972, 19, 473–478.
[20] A. Beeby, I. M. Clarkson, R. S. Dickins, S. Faulkner, D. Parker,
L. Royle, A. S. d. Sousa, J. A. G. Williams, M. Woods, J. Chem.
Soc. Perkin Trans. 2 1999, 493–504.
[8] a) A. Louie, Chem. Rev. 2010, 110, 3149–3195; b) L. Frullano,
T. J. Meade, J. Biol. Inorg. Chem. 2007, 12, 939–949; c) D. Jan´-
czewski, Y. Zhang, G. K. Das, D. K. Yi, P. Padmanabhan,
K. K. Bhakoo, T. T. Y. Tan, S. T. Selvan, Microsc. Res. Tech.
2011, 74, 568–576.
[9] a) C. Bernhard, C. Goze, Y. Rousselin, F. Denat, Chem. Com-
mun. 2010, 46, 8267–8269; b) A. Keliris, T. Ziegler, R. Mishra,
R. Pohmann, M. G. Sauer, K. Ugurbil, J. Engelmann, Bioorg.
Med. Chem. 2011, 19, 2529–2540.
[10] a) T. Koullourou, L. S. Natrajan, H. Bhavar, S. J. A. Pope, J. H.
Feng, J. Narvainen, R. Shaw, E. Scales, R. Kauppinen, A. M.
Kenwright, S. Faulkner, J. Am. Chem. Soc. 2008, 130, 2178–
2179; b) G. Dehaen, S. V. Eliseeva, K. Kimpe, S. Laurent, L.
Vander Elst, R. N. Muller, W. Dehaen, K. Binnemans, T. N.
Parac-Vogt, Chem. Eur. J. 2012, 18, 293–302; c) G. Dehaen,
[21] a) I. Hemmilä, S. Dakubu, V.-M. Mukkala, H. Siitari, T.
Lövgren, Anal. Biochem. 1984, 137, 335–343; b) I. Hemmilá,
V.-M. Mukkala, Crit. Rev. Clin. Lab. Sci. 2001, 38, 441–519;
c) J. A. Keelan, J. T. France, P. M. Barling, Clin. Chem. 1987,
33, 2292–2295; d) I. Hemmilä, V. Laitala, J. Fluoresc. 2005, 15,
529–542.
[22] a) L. Vander Elst, A. Roch, P. Gillis, S. Laurent, F. Botteman,
J. W. M. Bulte, R. N. Muller, Magn. Reson. Med. 2002, 47,
1121–1130; b) P. Caravan, M. T. Greenfield, J. W. M. Bulte,
Magn. Reson. Med. 2001, 46, 917–922.
S. V. Eliseeva, P. Verwilst, S. Laurent, L. Vander Elst, R. N. [23] a) S. Viswanathan, Z. Kovacs, K. N. Green, S. J. Ratnakar,
Muller, W. Deborggraeve, K. Binnemans, T. N. Parac-Vogt, In-
org. Chem. 2012, 51, 8775–8783; d) E. Debroye, G. Dehaen,
S. V. Eliseeva, S. Laurent, L. Vander Elst, R. N. Muller, K.
Binnemans, T. N. Parac-Vogt, Dalton Trans. 2012, 41, 10549– [24] A. Accardo, D. Tesauro, L. Aloj, C. Pedone, G. Morelli, Coord.
10556.
A. D. Sherry, Chem. Rev. 2010, 110, 2960–3018; b) J. A. Peters,
J. Huskens, D. J. Raber, Prog. Nucl. Magn. Reson. Spectrosc.
1996, 28, 283–350.
Chem. Rev. 2009, 253, 2193–2213.
[11] a) N. Kamaly, T. Kalber, G. Kenney, J. Bell, M. Jorgensen, A.
Miller, Org. Biomol. Chem. 2010, 8, 201–211; b) S. J. Soenen,
Received: March 23, 2015
Published Online: June 23, 2015
Eur. J. Inorg. Chem. 2015, 4572–4578
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