Comparison of MRI Properties between Multimeric DOTAGA and DO3A Gadolinium-Dendron Conjugates

Article abstract of DOI:10.1021/acs.inorgchem.9b01747

The inherent lack of sensitivity of MRI needs the development of new Gd contrast agents in order to extend the application of this technique to cellular imaging. For this purpose, two multimeric MR contrast agents obtained by peptidic coupling between an amido amine dendron and GdDOTAGA chelates (premetalation strategy, G1-4GdDOTAGA) or DO3A derivatives which then were postmetalated (G1-4GdDO3A) have been prepared. By comparison to the monomers, an increase of longitudinal relaxivity has been observed for both structures. Especially for G1-4GdDO3A, a marked increase is observed between 20 and 60 MHz. This structure differs from G1-4GdDOTAGA by an increased rigidity due to the aromatic linker between each chelate and the organic framework. This has the effect of limiting local rotational movements, which has a positive impact on relaxivity.

Full text of DOI:10.1021/acs.inorgchem.9b01747

Article
pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Comparison of MRI Properties between Multimeric DOTAGA and
DO3A Gadolinium-Dendron Conjugates
†
∥
Maleotane Ndiaye,†,⊥ Volodymyr Malytskyi,‡,⊥ Thomas Vangijzegem, Felix Sauvage, Mike Wels,∥
́
Cyril Cadiou,‡ Juliette Moreau, Celine Henoumont, Sebastien Boutry, Robert N. Muller,
‡
†
§
†,§
́
́
Dominique Harakat,‡ Stefaan De Smedt,∥ Sophie Laurent,*,†,§ and Francoise Chuburu*,‡
̧
†
‡
́
́
Laboratoire de RMN et d’Imagerie Moleculaire, Universite de Mons, B-7000 Mons, Belgium
́
́
Institut de Chimie Moleculaire de Reims, CNRS UMR 7312, Universite de Reims Champagne-Ardenne URCA, F-51685 Reims
Cedex 2, France
§Center for Microscopy and Molecular Imaging, Rue Adrienne Bolland 8, B-6041 Charleroi, Belgium
∥Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium
S
* Supporting Information
ABSTRACT: The inherent lack of sensitivity of MRI needs
the development of new Gd contrast agents in order to extend
the application of this technique to cellular imaging. For this
purpose, two multimeric MR contrast agents obtained by
peptidic coupling between an amido amine dendron and
GdDOTAGA chelates (premetalation strategy, G1-4GdDO-
TAGA) or DO3A derivatives which then were postmetalated
(G1-4GdDO3A) have been prepared. By comparison to the
monomers, an increase of longitudinal relaxivity has been
observed for both structures. Especially for G1-4GdDO3A, a
marked increase is observed between 20 and 60 MHz. This
structure differs from G1-4GdDOTAGA by an increased
rigidity due to the aromatic linker between each chelate and
the organic framework. This has the effect of limiting local rotational movements, which has a positive impact on relaxivity.
the inverse of T1 per millimolar metal ion. The GBCAs efficacy
to affect the relaxation of protons in tissues is related to several
parameters such as its molecular rotational correlation time τR,
the number of inner-sphere water molecules q, or the exchange
rate of these inner-sphere water molecules (kex).2,3 Low
molecular weight GBCAs are the active substances of the
commercially and clinically available MRI contrast agents.4
Their low molecular weight favors their rapid elimination from
the body through the renal pathway, which effectively avoids
deleterious side effects associated with gadolinium (Gd) body
accumulation (e.g. nephrogenic systemic fibrosis (NSF)).5
Moreover, the relaxivity of small GBCAs is limited by their
rapid tumbling motions which results in a fast rotational
correlation time τR (∼10−10 s) and then a low relaxivity
between 20 and 60 MHz (3−5 mM−1 s−1).
INTRODUCTION
■
Due to its high spatial and temporal resolutions, magnetic
resonance imaging (MRI) is a mainstream method for clinical
diagnosis and therapeutic decision-making. The MRI signals
1
arise from the H abundancy in the body. When the body is
1
exposed to a strong magnetic field, the H nuclei, which are
magnetically active, orient themselves within this magnetic
field. When the body is then submitted to radiofrequency
pulses, the energy as well as the orientation of the nuclei
change and as they relax to their initial states, a resonance
radio-wave is emitted. The recovery of this MRI signal and its
spatial encoding leads to image production.1 In MRI, the main
contrast parameters are 1H density, longitudinal T1, and
transversal T2 proton relaxation times. Although T1 and T2 are
generally reduced in injured tissues, there is considerable
overlap of relaxation times between healthy and injured
damaged tissues on native MRI. This stresses the need for MRI
contrast agent injection to enhance the contrast.
Paramagnetic contrast agents are known to exert a strong
stimulation to T1 relaxation, and gadolinium-based contrast
agents (GBCAs) are ideal candidates for producing MRI
contrastophores that affect T1 of water protons in tissues.2 The
MRI effectiveness of GBCAs is evaluated from their
longitudinal r1 relaxivity value, which refers to the increase in
In order to amplify the MRI efficacy, the conjugation of
small GBCAs with macromolecular carriers has been
investigated.2,3,6 Among them, dendrimers and particularly
polyamidoamines (PAMAM) have been explored because of
their more rigid structure,7 and GBCA conjugation with
PAMAM dendrimers of different generations was carried out.8
Received: June 12, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.9b01747
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Scheme 1. Structure of the Multimeric Systems Investigated Herein
As the result of the reduced rotational tumbling rate (1/τR),
the proton relaxivity was improved at intermediate fields, but
the efficiency of dendrimeric GBCAs did not show a linear
sensitivity as well as their kinetic inertness. Finally, their
harmlessness toward fibroblasts was investigated.
EXPERIMENTAL SECTION
dependence with their molecular mass. Besides, the enhance-
■
ment clearly tends to diminish for larger systems. Moreover,
high-generation dendrimers are not easy to prepare and to
purify,8 and a latent cytotoxicity is observed due to a slow
degradation in vivo.9
In this report, we propose to take advantage of the
dendronization strategy to design multimeric GBCAs. The
All reagents were purchased from commercial suppliers and used
1
without further purification. H NMR and 13C NMR spectra were
recorded on a 600 MHz Bruker Avance III spectrometer equipped
with a TCI cryoprobe and on a Bruker Avance Neo 600 MHz. The
chemical shifts were reported in parts per million (δ scale). Traces of
residual solvents were used as internal standards for 1H and 13C
chemical shift referencing. Abbreviations: s: singlet, d: doublet, t:
triplet, q: quadruplet, m: multiplet, ws: wide signal.
FTIR absorption spectra were obtained on a Thermo Scientific
Nicolet iS5 spectrometer equipped with ATR iD5 accessory, and
wavenumbers are reported in cm−1.
All mass spectra were collected by using a SYNAPT G2-Si HDMS
(Waters Corp., Manchester, UK) equipped with an electrospray
ionization source. ESI mass spectra were acquired over the m/z 100−
4000 range. The desolvation gas was set to 800 L·h−1 at a temperature
of 200 °C, the cone gas was set to 40 L·h−1, and the source
temperature was set to 120 °C. The capillary voltage was set to 2500
V. Spectra were acquired in continuum and negative mode.
Column chromatographies were performed using silica gel (230−
400 mesh) (Macherey-Nagel GmbH, Germany). Inversed phase
chromatography was performed on C18 bonded silica (45−75 μm,
VersaFlash). The excess of Gd3+ ions was removed on Chelex 100
sodium form resin (50−100 mesh, purchased from Sigma).
Syntheses. The syntheses of starting dendrimer G1-4NH2,
DO3A-based ligand in the form of activated ester DO3A(tBu)3NHS,
as well as mononuclear complexes GdDOTAGA and GdDO3A-
COOH were performed as described in the Supporting Information
(SI 1, 2, Supporting Information).
dendritic structures prepared in this work (Scheme 1) rely on a
common bifunctional amido amine core (G1-4NH2). The
installation of MRI contrastophores will herein be carried out
by peptidic coupling with the amino groups of the G1-4NH2
core. On the resulting G1-4GdDOTAGA and G1-4GdDO3A
structures, there will remain an activatable group (benzyl ester
group), which may be released in the future, in order to
introduce either a targeting ligand or a second imaging probe.
Indeed, in order to reduce ambiguities in the MRI signal
analysis, it will be interesting to develop contrast agents able to
multiplex an MRI probe to a more sensitive one (for instance
an optical imaging probe),10 provided that differences in
concentration imposed by differences in sensitivity are taken
into account in the system design.
Two different approaches were used to obtain the desired
multimeric structures. For the first one, G1-4GdDOTAGA,
four premetalated GdDOTAGA chelates were grafted on the
central G1-4NH2 core. The second one G1-4GdDO3A, was
obtained by covalent linking of four DO3A macrocyclic units
on the same central G1-4NH2 core, followed by Gd
metalation. In order to ensure a good thermodynamic stability
of the corresponding metal complexes, these two structures
were designed on the basis of macrocyclic GBCAs, and their
synthesis is described in the paper. Before considering any
additional functionalization of these multimeric GBCAs, we
have evaluated their respective efficacy to enhance MRI
G1-4GdDOTAGA Synthesis. The G1-4NH2 dendrimer (200 mg,
0.20 mmol, 1 equiv) and Gd-chelate Na2GdDOTAGA 5 (84% purity
(SI 2, Supporting Information) 955 mg, 1.19 mmol, 6 equiv) were
loaded into a flask flashed with argon. Anhydrous DMSO (50 mL)
and triethylamine (166 μL, 1.19 mmol, 6 equiv) were added, and the
mixture was homogenized in ultrasonic bath.
After addition of 1-[bis(dimethylamino)methylene]-1H-1,2,3-
triazolo[4,5-b]pyridinium3-oxidhexafluorophosphate (HATU, 452
B
DOI: 10.1021/acs.inorgchem.9b01747
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mg, 1.19 mmol, 6 equiv), the mixture was stirred at room temperature
for 1 day. Addition of ethyl acetate (450 mL) provoked the formation
of a precipitate that was recovered and washed two times with ethyl
acetate (30 mL). The precipitate was taken up with 10 mL of distilled
water; the resulting solution was split into two, diluted to 12 mL with
water and loaded into two Vivaspin 15R tubes (MWCO 2000 g/mol)
for ultrafiltration (6000 g for 45 min). Three ultrafiltration cycles were
performed afterward; the remaining solutions were transferred into a
round-bottom flask and evaporated under reduced pressure to afford
G1-4GdDOTAGA (499 mg, Gd-purity found by relaxometric study
89%, 0.14 mmol, yield 73%) as a crystalline solid. The absence of free
gadolinium ions was checked by a colorimetric test with xylenol
orange as a dye.11 FTIR: 1595 cm−1. HR-MS (ESI): m/z calculated
for [M − H]−: 3071.8726/found: 3071.8738; calculated for [M −
2H]2−: 1535.4324/found: 1535.4336 (values are given for the highest
component of isotopic profile; MC107H167Gd4N25O40). Detailed
HRMS spectra with simulated isotopic distribution profiles are given
in SI 3, Supporting Information.
resin was separated by centrifugation and the solution was
concentrated under reduced pressure. The resulting crude product
was dissolved in water (10 mL) and purified by dialysis (membrane
with MWCO 500−1000 Da, VWR) against 1 L of distilled water
(which was changed after 2.5, 6.5, 11.5, and 18 h). The dialyzed
solution was freeze-dried leading to G1-4GdDO3A (0.12 g, 0.038
mmol, yield 25%). FTIR: 1600 cm−1. HR-MS (ESI): m/z calculated
for [M − H]−: 3259.9368/found: 3259.9368; calculated for [M −
2H]2−: 1629.4645/found: 1629.4651 (values are given for the highest
component of isotopic profile; MC123H171Gd4N29O36). Detailed
HRMS spectra with simulated isotopic distribution profiles are given
in SI 5, Supporting Information.
Determination of Gd Concentration for Relaxometric
Measurements. To determine Gd3+ concentration, either ICP-
OES or relaxometric measurements were performed. Previously, the
absence of free Gd3+ ion in the analyzed samples was checked by
complexometry with xylenol orange as a dye.11 Furthermore, each
sample (200 μL) was submitted to an acidic digestion (H2O/HNO3
1:1 v/v, 200 μL) to release Gd3+. For ICP-OES measurements,
volumetric dilutions were carried out to achieve an appropriate Gd
concentration within the working range of the method. Samples were
analyzed using a Varian Liberty Series II spectrometer. Counts of Gd
were correlated to a Gd calibration curve generated by mixing
Gd(NO3)3 standard prepared under the same acidic conditions. For
relaxometry measurements, the longitudinal relaxation rate of the
digested acidic solution was then determined after subtraction of the
diamagnetic contribution (0.35 s−1 at 37 °C for the H2O/HNO3 1:1
v/v solution). This value was compared to the one of a 1 mM GdCl3
solution (11.76 s−1), and after taking into account the dilution factor
(2), the Gd3+ concentration was obtained for the sample of interest.
Both methods gave similar results since for a solution of G1-
4GdDOTAGA, a Gd concentration of 1.68 mM was found by ICP-
OES while the same solution analyzed by relaxometry gave a
concentration of 1.63 mM. Similar results were obtained for a G1-
4GdDO3A solution, i.e. 1.51 mM and 1.54 mM in Gd by ICP-OES
and relaxometry respectively (between 2 and 3% error between the
two methods). For determination of r1 relaxivities Gd concentration
determined by relaxometry was used.
G1-4GdDO3A Synthesis. G1-4[DO3A(tBu)3]. To a solution of
G1-4NH2 dendrimer (0.411 g, 0.42 mmol, 1 equiv) in DMF (5 mL)
and DIPEA (0.99 mL, 5.83 mmol, 14 equiv) was added the solution
of the activated ester 6 (2.112 g, 2.67 mmol, 6.3 equiv, prepared as
described in SI 4, Supporting Information) in dichloromethane (5
mL), and the mixture was stirred for 48 h under a nitrogen
atmosphere at room temperature. The solution was diluted with ethyl
acetate (15 mL) and extracted three times with distilled water (3 × 30
mL) and brine (3 × 30 mL). Organic phase was dried over sodium
sulfate, filtered, and concentrated under reduced pressure. A white
solid was isolated (0.48 g) containing the desired compound as
revealed by mass spectrometry. It was used in the further synthetic
step without purification.
HR-MS (ESI): m/z calculated for [C171H279N29O36+Cl]−:
3351.0620/found: 3351.0625.
G1-4DO3A. The compound G1-4[DO3A(tBu)3] (0.48 g) was
dissolved in a mixture of dichloromethane and trifluoroacetic acid (20
mL, 1:1 v/v), and this solution was stirred at room temperature for 48
h. The reaction mixture was concentrated under reduced pressure,
and the residue precipitated in diethyl ether and washed three times
with this solvent (3 × 30 mL). The obtained product was solubilized
in distilled water (10 mL) and purified by dialysis (dialysis membrane
MWCO 500−1000 Da, VWR) against 1 L of distilled water (which
was changed four times, after 2.5, 6.5, 11.5, and 18 h). After dialysis,
the resulting solution was freeze-dried and the desired dendron G1-
4DO3A obtained (0.40 g, 0.15 mmol, yield 36% over two steps from
G1-4NH2).
Relaxometric Measurements. T1 Measurements at 20 MHz
(0.47 T). T1 measurements were performed in water on a Bruker
mq20 minispec relaxometer (0.47 T) using an inversion recovery
pulse sequence. The measurements were performed at nine different
temperatures between 5 and 45 °C. The temperature was equilibrated
before each scan. The inverse of the paramagnetic longitudinal
relaxation time (1/T1para, s−1) of each sample was calculated according
to
1H NMR (600.16 MHz, DMSO-d6, 25 °C): δ 1.81 (ws, 4H,
-NHCH2CH2CH2N<), 1.90 (ws, NHCH2CH2CH2N<), 2.8−5.4 (ws,
dendrimer CH2 and cyclen CH2), 3.60 (s, 2H, >NCH2COOBn), 5.21
(1/T1para) = (1/T1observed) − (1/T
)
1dia
3
1/T1para values were divided by Gd3+ concentration (mM) to
determine G1-4GdDOTAGA and G1-4GdDO3A relaxivities r1 (s−1
mM−1).
(s, 2H, −COOCH2Ph), 7.32−7.44 (m, 5H, CH), 7.66 (d, 8H, J =
3
8.4 Hz, CH), 7.83 (d, 8H, J = 8.4 Hz, CH), 8.63 (s, 4H, NH), 8.86
(s, 2H, NH) 10.78 (ws, 4H, NH).
13C NMR (150.9 MHz, DMSO-d6, 25 °C): δ 24.03, 26.81, 28.27,
36.35, 36.53, 51.40, 51.51, 51.74, 52.35, 53.11, 53.47, 54.91, 55.05,
113.54, 115.51, 117.47, 118.58, 119.00, 119.43, 128.19, 128.45,
128.54, 129.15, 130.82, 141.09, 157.87, 158.09, 158.32, 158.54,
163.66, 164.60, 165.93. FTIR: 1733 cm−1, 1669 cm−1.
1
NMRD Profiles. H NMRD profiles were measured on a Stelar
Spinmaster FFC fast field cycling NMR relaxometer (Stelar, Mede,
Pavia, Italy) over a range of magnetic fields extending from 0.24 mT
1
to 0.7 T (which corresponds to H Larmor frequencies from 0.01 to
30 MHz) using 0.5 mL samples in 7.5 mm o.d. tubes. The
temperature was kept constant at 37 °C. Additional relaxation rates at
60 and 300 MHz were obtained with Bruker Minispec mq60 and
Bruker Avance-300 MHz spectrometers, respectively (Bruker,
Karlsruhe, Germany). The least-squares fitting of the 1H NMRD
data was performed using a homemade program (Fitting2000) and
equations from the Solomon and Bloembergen (SBM) theory for the
small gadolinium complexes or from the Lipari-Szabo theory for the
slow rotating macromolecular contrast agents. In the fitting
procedure, the rGdH distance was fixed to 3.10 Å and the closest
approach of the bulk water protons to the Gd3+ ion, dGdH, to 3.6 Å.12
The diffusion constant D has been fixed to 3.0 × 10−9 m2 s−1,13 and
the hydration number q was 1, as determined by luminescence
lifetime measurements on the Eu3+ analogues (SI 6, Supporting
Information). The water exchange rate kex = 1/τm where τm is the
HR-MS (ESI): m/z calculated for [C123H183N29O36−H]−:
2642.3333/found: 2642.3303; calculated for [C123H183N29O36-
2H]2−: 1320.6627/found: 1320.6633.
G1-4GdDO3A. The dendron G1-4DO3A (0.40g, 0.15 mmol, 1
equiv) was dissolved in distilled water (2.5 mL), and the pH of the
corresponding solution was adjusted to 5.5 with 1 M solution of
sodium hydroxide. Gadolinium chloride (GdCl3·6H2O, 0.34 g, 0.91
mmol, 6 equiv) was dissolved in distilled water (10 mL) and added to
the solution of dendron G1-4DO3A. The pH of the mixture was
adjusted again to 5.5, and the reaction mixture was stirred at room
temperature for 1 day. The solution was treated for 1 h with Chelex
100 resin to remove the excess of gadolinium ions (in G1-4GdDO3A
sample, the absence of free gadolinium ions was checked as
previously, by complexometry with xylenol orange as a dye).11 The
C
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1
Table 1. Best-Fit Parameters Obtained from the Fitting of the H NMRD Profiles to the SBM Theory, Including the Lipari−
a
Szabo Approach for Internal Flexibility
b
b
c
b
d (nm)
r (nm)
τM (ns)
q
τSO (ps)
τV (ps)
τRG (ps)
τRL (ps)
S2
G1-4GdDOTAGA
G1-4GdDO3A
0.36
0.36
0.31
0.31
97
505
1
1
332 11
31.1 4.3
37.4 1.8
700 112
800 94
150.0 0.9
167 13
0.051 0.015
0.20 0.02
126
3
a
b
c
The definition of the parameters is given in the Experimental Section. Fixed parameters. Value obtained from 17O experiments.
Scheme 2. Synthesis of G1-4NH2 Dendron
residence time of the coordinated water molecule in the inner sphere,
was fixed to the value obtained by independent 17O measurements of
the transverse relaxation time of water (1/T2) according to the
temperature. The fitting procedure allowed the determination of τSO,
the gadolinium electronic relaxation time at zero field, τV, the
correlation time that modulates the electronic relaxation, and τR, the
rotational correlation time (if the SBM theory is used), or τRG and τRL,
i.e. the rotational correlation times describing the global and local
motions, respectively, and S2 that reflect the degree of spatial
restriction of the local motion with respect to the global motion (0 ≤
S2 ≤ 1,S2 = 0 for isotropic internal motions, S2 = 1 for completely
restricted internal motions), if the Lipari−Szabo approach is used.
The best-fit parameters obtained from the analysis of 1H NMRD data
are summarized in Table 1.
activation energy related to τv; B, related to the mean-square of the
zero field splitting energy Δ (B = 2.4Δ2); and ΔH‡ and ΔS‡ the
enthalpy and entropy of activation, respectively, of the water exchange
process. The number of coordinated water molecules was set to 1.
T1-Weighted Images. MR imaging of G1-4GdDOTAGA and G1-
4GdDO3A were performed using a 1.0 T MRI device (ICON,
Bruker) using a T1-weighted spin−echo sequence (TR = 300 ms, TE
= 12 ms, resolution = 208 × 195 μm, slice thickness = 1.25 mm, 4
averages). Solutions of G1-4GdDOTAGA and G1-4GdDO3A were
tested in the 0.1−1 mM range.
Transmetalation Experiments. The reactions of G1-4GdDOTA-
GA and G1-4GdDO3A with Zn2+ ions were monitored by measuring
the longitudinal relaxation rates R1 (1/T1) of water protons on a
Bruker mq20 minispec relaxometer (0.47 T), by the inversion
recovery method. 12 μL of a 2.5 × 10−1 mol L−1 solution of ZnCl2
was added to 300 μL of a phosphate buffered solution (pH 7) of the
paramagnetic complex (2.5 × 10−3 mol L−1). The temperature was
equilibrated and maintained at 37 °C during the experiments. The
mixture was stirred, and the measurements were started immediately
Temperature Dependent 17O NMR Measurements. The trans-
verse 17O relaxation rates (1/T2) were measured in aqueous solutions
of G1-4GdDOTAGA and G1-4GdDO3A (12 mM) in the temper-
ature range 7−77 °C, on a Bruker Avance 500 (11.7 T, 67.8 MHz)
spectrometer. The temperature was calculated according to a previous
calibration with ethylene glycol and methanol. Proton decoupling was
applied during all the acquisitions. Transverse relaxation times (T2)
were obtained by the measurement of the signal width at midheight.
p
and followed during 5 days. The R1 (t) relaxation rate was obtained
after subtraction of the diamagnetic contribution of the proton water
relaxation (0.2826 s−1) from the observed relaxation rate R1(t).
In Vitro Cytotoxicity Studies. NIH/3T3 (ATCC CRL-1658) cell
viability was evaluated with an MTT assay (Sigma-Aldrich) in the
presence of G1-4GdDOTAGA, G1-4GdDO3A, Na2GdDOTAGA,
and GdDO3ACOOH. NIH/3T3 cells were chosen as they are
commonly used to screen the toxicity of chemicals15 and nanoma-
terials.16 They are well characterized, uniform, and known to provide
reproducible results.17 NIH/3T3 cells were cultured in Dulbecco’s
Modified Eagle Medium GlutaMax supplemented with 2 mM L-
glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10%
fetal bovine serum and maintained at 37 °C under a humidified
R
The data are presented as the reduced transverse relaxation rate 1/T2
P
= 55.55/([Gdcomplex]·q·T2 ), where [Gdcomplex] is the molar concen-
tration of the complex, q is the number of coordinated water
molecules, and T2 is the paramagnetic transverse relaxation rate
P
obtained after subtraction of the diamagnetic contribution from the
observed relaxation rate. The treatment of the experimental data was
performed as described elsewhere.14 The following parameters were
determined: A/ℏ, the hyperfine coupling constant between the
oxygen nucleus of the bound water molecule and the Gd3+ ion; τv, the
correlation time modulating the electronic relaxation of Gd3+; Ev, the
D
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Scheme 3. Synthesis of G1-4GdDOTAGA
Figure 1. ESI-HRMS spectrum of G1-4GdDOTAGA with [M − H]− and [M − 2H]2−/2 signals.
atmosphere containing 5% CO2. The cells were seeded in a 96 well
plate (15.000 cells per well) and were allowed to settle overnight. The
next day, G1-4GdDOTAGA, G1-4GdDO3A, Na2GdDOTAGA, and
GdDO3ACOOH were added on cells in the following concentrations
0.1, 0.3, 0.5, 0.75, and 1.05 mM of Gd and incubated for 24 h at 37
°C. The medium containing the dendrimers was then removed, and
the MTT reagent (100 μL, 1.5 mM in cell medium) was added to
each well for 3 h at 37 °C. After taking off the reagents, DMSO was
added under shaking for 1 h to dissolve the formazan crystals. The
absorbance of each plate was measured at 595 nm with a plate
spectrophotometer (PerkinElmer VICTOR3 1420 Multilabel Coun-
ter).
G0 gave the bis-amine 4 (92% yield). Acid 3 and amine 4 were
then coupled using 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetrame-
thyluronium hexafluorophosphate (TBTU) to give the
pentavalent protected dendrimer G1 (28% yield after silica
chromatography). Finally, deprotection of tert-butoxycarbonyl
groups under acidic conditions afforded dendron G1-4NH2
(35% yield).
G1-4GdDOTAGA Synthesis. G1-4GdDOTAGA synthesis
was achieved from synthon G1-4NH2 in the presence of Gd-
DOTAGA 5 (Scheme 3). DOTAGA complexation with
Gd2O3 afforded complex 5, and the excess of Gd3+ was
removed by precipitation after NaOH addition. The peptidic
coupling between 5 and G1-4NH2 was then performed in the
presence of 1-[bis(dimethylamino)methylene]-1H-1,2,3-
triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate
(HATU). In this approach, the chelate was then metalated
before being grafted to the dendron (premetalation strategy).
G1-4GdDOTAGA synthesis was confirmed by means of
ESI-MS spectroscopy. The ESI-MS spectrum of G1-
4GdDOTAGA clearly showed intense signals for molecular
ions at [M − H]− and [M − 2H]2−/2, with an isotopically
resolved profile matching well with the corresponding
simulated signals (Figure 1 and SI 3, Supporting Information).
RESULTS AND DISCUSSION
■
Preparation of G1-4GdDOTAGA and G1-4GdDO3A.
G1-4NH2 Synthesis. G0 (Scheme 2, SI 1, Supporting
Information) that will become the core of G1 dendrimers
was synthesized according to a procedure inspired from a
published approach,18,19 where the primary amino groups of
3,3′-iminobis(propylamine) 1 were regioselectively protected
by tert-butoxycarbonyl groups20 to give amine 2 (88% yield).
Amine 2 was further alkylated with benzylbromoacetate20
(63% yield) to provide the core structure G0 with Boc-
protected terminal amine functions and a central carboxylic
acid function protected by a benzyl (Bn) group. Hydro-
genolysis20 of G0 gave acid 3 (98% yield) while acidolysis18 of
E
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Scheme 4. Synthesis of G1-4GdDO3A
Figure 2. ESI-HRMS spectrum of G1-4GdDO3A with [M − H]− (inset: signal at 3260 m/z) and [M − 2H]2−/2 signals.
Therefore, G1-4GdDOTAGA, that exhibited 4 Gd3+
chelates/per molecule was obtained and readily isolated after
ultrafiltration in 73% yield.
dendron (9% yield over 3 steps). Excess of Gd3+ was removed
using a Chelex 100 resin followed by dialysis.
The ESI-MS spectrum of G1-4GdDO3A (Figure 2 and SI 5,
Supporting Information) clearly showed an intense signal for
bis-anionic molecular ion [M − 2H]2−/2, with an isotopically
resolved profile matching well with its corresponding simulated
signals.
To have an insight of G1-4GdDOTAGA and G1-
4GdDO3A hydrodynamic diameters, dynamic light scattering
(DLS) was used (SI 7, Supporting Information). DLS analysis
indicated Z-ave diameters of 1.70 and 2.50 nm for G1-
4GdDOTAGA and G1-4GdDO3A, respectively. In parallel,
DOSY experiments carried out on diamagnetic analogues (G1-
4LaDOTAGA and G1-4LaDO3A) revealed hydrodynamic
diameters of 2.08 and 1.75 nm, respectively (SI 7, Supporting
Information). Given the uncertainties affecting each of these
measurements, it can be concluded that the hydrodynamic
diameters of G1-4GdDOTAGA and G1-4GdDO3A were
inferior to 2.6 nm.
G1-4GdDO3A Synthesis. To obtain G1-4GdDO3A, a
different approach, based on DO3A ligand grafting to G1-
4NH2, followed by insertion of Gd3+ into DO3A chelators of
the corresponding dendrimeric ligand, was followed (post-
metalation strategy). In a preliminary step, the well-known
DO3A-tris-tBu-ester21 was alkylated with methyl 4-
[(bromoacetyl)amino]benzoate. Then the methyl ester group
was selectively saponified with lithium hydroxide22 and the
corresponding benzylic acid was treated with EDC and NHS
to afford the activated ester 6 (50% yield over 3 steps after a
flash chromatography) (SI 4, Supporting Information). The
ester 6 was then attached to G1-4NH2 to provide tBu-
protected G1-4[DO3A(tBu)3] (Scheme 4). Trifluoroacetol-
ysis of G1-4[DO3A(tBu)3] gave G1-4DO3A dendron that
was finally treated with GdCl3 to afford G1-4GdDO3A
F
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Figure 3. r1 relaxivity at different temperatures (20 MHz) for (a) G1-4GdDOTAGA and (b) G1-4GdDO3A.
Finally, to determine the hydration number of Gd3+ in G1-
4GdDOTAGA and G1-4GdDO3A, the luminescence lifetimes
of the corresponding Eu3+ complexes in H2O and D2O were
measured.23 τH2O lifetimes were 0.649 and 0.523 ms for
Na2EuDOTAGA and EuDO3ACOOH, respectively, while
τD2O lifetimes were 2.410 and 1.346 ms, giving a number of
water molecules directly coordinated to Eu3+ of 1.18 for
Na2EuDOTAGA and 1.23 for EuDO3ACOOH i.e. very close
to 1 (SI 6, Supporting Information). Assuming the fact that
luminescence lifetimes are similar for mononuclear and
multimeric complexes, one can conclude that each Gd3+ ion
in G1-4GdDOTAGA and G1-4GdDO3A structures exhibited
one coordinated water molecule in the inner sphere.
Relaxometric Properties of G1-4GdDOTAGA and G1-
4GdDO3A. The proton relaxivity, which describes the MRI
efficacy of chelates as contrast agents was first recorded at 20
MHz and 37 °C for both structures. On a per millimolar Gd
basis, G1-4GdDOTAGA and G1-4GdDO3A presented r1
relaxivities of 6.6 mM−1 s−1 and 8.1 mM−1 s−1, respectively.
These values are significantly higher than relaxivities found for
the corresponding mononuclear chelates Na2GdDOTAGA
and GdDO3ACO2H (3.9 and 4.6 mM−1 s−1, respectively), as
expected for a longer value of the rotational correlation time
associated with the increased molecular size of G1-
4GdDOTAGA and G1-4GdDO3A.8 In order to characterize
the parameters that govern proton relaxivity, r1 longitudinal
relaxivity evolution with the temperature was recorded at 20
MHz for both complexes (Figure 3).
For G1-4GdDOTAGA, the continuous decrease of the
relaxivity with increasing temperature is characteristic of a fast
exchange of the coordinated water molecule in the inner
sphere, which means in this case that water exchange will not
limit the relaxivity. On the contrary, the observed evolution for
the compound G1-4GdDO3A is typical of a slower exchange
of the coordinated water molecule, limiting the relaxivity. In
order to confirm these results and to have an insight into the
microscopic parameters governing the dynamics of the system,
variable-temperature 17O T2 measurements were performed to
have access to the water exchange rate kex (equal to the
reciprocal lifetime of the water molecule in the inner sphere of
the complex, 1/τm) for G1-4GdDO3A and G1-4GdDOTAGA
(Figure 4).
Figure 4. 17O NMR measurements for (a) G1-4GdDOTAGA and
(b) G1-4GdDO3A
nated water molecule. The experimental data were fitted with a
theoretical model as described previously,14 which allowed
extraction of τM values of 97 4 ns (kex = 1.03 × 107 s−1) and
505
17 ns (kex = 1.99 × 106 s−1) at 37 °C for G1-
4GdDOTAGA and G1-4GdDO3A, respectively, as well as the
thermodynamic parameters governing the exchange (SI 8,
Supporting Information). The lower value of kex obtained for
G1-4GdDO3A is consistent with data from the literature,24
where a similar Gd-complex was grafted on a polysaccharide
derived from inulin, giving a similar kex of 1.6 × 106 s−1. This
difference can be correlated with the structure of the two
complexes and more precisely to the functional groups directly
coordinated to the Gd center. It is indeed well-known that the
presence of an amide group, as in G1-4GdDO3A, slows down
the exchange of the coordinated water molecule.25
Nuclear magnetic relaxation dispersion (NMRD) profiles
were also recorded in the field range 0.01−300 MHz for all the
complexes.
The G1-4GdDOTAGA NMRD profile (Figure 5a) was
analyzed using the classical inner sphere and outer sphere
theories, which led to a rotational correlation time τR of 147.0
2.0 ps (SI 9, Supporting Information), i.e. almost two times
higher than τR of GdDOTAGA (65.1 1.9 ps). This NMRD
profile is nevertheless characteristic of a system in a fast
rotational motion, which is in relative contradiction with its
high molecular weight. When the Lipari−Szabo treatment was
incorporated in the fit (Table 1), the order parameter S2, which
describes the degree of spatial restriction of the local rotational
motion of the Gd3+-waterproton axis with respect to the global
system motion, was almost 0 (S2 = 0.05), i.e. typical of
isotropic internal motions. This explains the low value of τR
extracted from the fitting with the SBM theory.
The G1-4GdDO3A NMRD profile (Figure 5b) was
analyzed in a similar way, which led to a rotational correlation
time τR of 242.0 10.0 ps (SI 9, Supporting Information), i.e.
almost three times higher than τR of GdDO3ACO2H (71.2
0.8 ps). The profile exhibited a hump at medium field which is
typical of slowly rotating species. When the Lipari−Szabo
The observed behavior was completely different for the two
complexes and confirmed the results obtained by the evolution
of the relaxivity with the temperature. Indeed, a decrease of the
transverse relaxation rate was observed with increasing
temperature on most of the temperature domain for G1-
4GdDOTAGA, which is typical of a fast exchange of the
coordinated water molecule. The opposite was observed for
G1-4GdDO3A, indicating a slower exchange of the coordi-
G
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Figure 5. NMRD profiles at 37 °C for (a) G1-4GdDOTAGA and (b) G1-4GdDO3A (the line represents the least-square fit to the experimental
data point as described in the text).
Figure 6. T1-weighted images of G1-4GdDOTAGA and G1-4GdDO3A with DOTAREM and water as controls. Samples imaged at 1 T, 37 °C,
and with a standard spin echo (SE) sequence.
treatment was incorporated in the fit, the order parameter S2
Table 2. G1-4GdDOTAGA and G1-4GdDO3A (1 mM, 37
was 0.20, which indicated a higher rigidity of this system by
comparison to G1-4GdDOTAGA (Table 1). This could be
correlated with the presence in G1-4GdDO3A of the short and
rigid aromatic linker,8e which was a positive feature to limit the
rotation of the small Gd3+ chelates. This has thus a positive
impact on the relaxivity of this compound, especially at
medium field where a hump is observed on the NMRD profile.
To demonstrate how relaxivity enhancements were trans-
lated into contrast, T1-weighted images of phantoms
containing solutions of G1-4GdDOTAGA and G1-
4GdDO3A were acquired at 1T with DOTAREM as the
control (Figure 6).
For both systems, the bright signal enhancement pro-
gressively increased with Gd concentration. Furthermore, for
each Gd concentration, G1-4GdDO3A or G1-4GdDOTAGA
signals were brighter than those of the control, corroborating
the relaxometric results.
°C, 20 MHz) Relaxivities in the Presence of HSA (4%)
rH1 SA
/
r1 (s−1 mM−1, H2O) r1 (s−1 mM−1, HSA)
rH1 O
2
G1-4GdDOTAGA
G1-4GdDO3A
6.90
8.96
7.50
13.37
1.09
1.49
medium viscosity. For G1-4GdDO3A a more significant
difference (49%) was observed, but it is still not high enough
to conclude to a noncovalent association to HSA. This should
thus allow a fast elimination of the compounds from the body,
probably via glomerular filtration, given the estimated
hydrodynamic diameter of both structures (SI 7, Supporting
Information).
Kinetic Inertness and Cytotoxicity Evaluations of G1-
4GdDOTAGA and G1-4GdDO3A. Before testing G1-
4GdDOTAGA and G1-4GdDO3A safety against cells, their
kinetic inertness was evaluated by relaxometry in the presence
of Zn2+ in a phosphate-buffered solution.26
Finally, relaxivity studies of G1-4GdDOTAGA and G1-
4GdDO3A in the presence of human serum albumin (HSA)
were performed (Table 2).
G1-4GdDOTAGA demonstrated a very small increase of
relaxivity (9%) which could be related to an increase of the
If the transmetalation reaction occurs, it will result in Gd3+
release which then will precipitate into GdPO4. Consequently,
it will lead to a subsequent decrease of the proton
26
p
paramagnetic relaxation rate (R1 ). G1-4GdDOTAGA and
H
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G1-4GdDO3AR1p evolutions gave therefore a good indication
70 and 76% over the monomer units). It should be emphasized
that the two multimeric systems were characterized by
exchange regimes of water molecule not limiting the relaxivity
for G1-4GdDOTAGA while limiting for G1-4GdDO3A, in
agreement with the presence of amide groups at the vicinity of
the lanthanide ion. Nevertheless, G1-4GdDOTAGA exhibited
an NMRD profile typical of a system in a fast rotational motion
while the G1-4GdDO3A NMRD profile was characteristic of a
system in a slower rotational motion. The analysis of NMRD
profiles by the Lipari−Szabo theory showed for G1-
4GdDOTAGA a significant limiting role of the local rotational
motions of the chelates which resulted in an effective value of
τR lower than that expected based on molecular dimensions.
On the other hand, the S2 value determined for G1-
4GdDO3A suggested a more restricted motion of the chelates
located at the periphery of the assembly. This local rigidity was
induced by the aromatic linker used to hook the chelate on the
organic framework. Therefore, G1-4GdDO3A that still
exhibits a derivatizable site is a good candidate for the design
of targeting paramagnetic probes for molecular imaging or
multimodal probes that combine at least two different imaging
techniques. Further works are in progress in these two
directions.
of potential transmetalation. Figure 7 showed the evolution of
p
p
their normalized paramagnetic relaxation rates R1 (t)/R1 (0).
p
p
Figure 7. Evolution of R1 (t)/R1 (0) vs time (at 20 MHz, 37 °C) for
(a) G1-4GdDOTAGA and (b) G1-4GdDO3A in the presence of
Zn2+ in phosphate buffer (pH = 7)
p
By comparison to the initial relaxation rate R1 (0), no
significant changes were observed over a 4 days period for both
dendrons, indicating that for G1-4GdDOTAGA and G1-
4GdDO3A no Gd3+ release occurred.
G1-4GdDOTAGA and G1-4GdDO3A in vitro safety was
then tested against NIH/3T3 cells (Figure 8).
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.9b01747.
Experimental details of new compounds’ synthesis and
analytical data (HR-ESI-MS mass spectra, luminescence
lifetime measurements, hydrodynamic diameters, and
fitted parameters of 17O and NMRD data) (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail: sophie.laurent@umons.ac.be.
*E-mail: francoise.chuburu@univ-reims.fr.
■
Figure 8. Metabolic activity of fibroblasts (NIH/3T3) incubated with
(a) G1-4GdDOTAGA and (b) G1-4GdDO3A for 24 h
(Na2GdDOTAGA and GdDO3ACOOH being used respectively as
controls).
ORCID
́
Felix Sauvage: 0000-0002-8065-4439
Cyril Cadiou: 0000-0002-2737-9976
For G1-4GdDO3A no significant decrease in metabolic
activity was observed up to a Gd concentration of 1.05 mM
(>90% viability for both), and this behavior is similar to that of
control. However, a slight and progressive decrease was
observed with increasing concentrations of G1-4GdDOTAGA
to reach 75% at a Gd concentration of 1.05 mM. This follows
the same trend as for Na2GdDOTAGA alone, which likely
means the decreasing in metabolic activity cannot be totally
attributed to the dendrimer. Na2GdDOTAGA is ionic while
GdDO3ACOOH is neutral which implies differences in
osmolarity that can likely explain these discrepancies in toxicity
between the two compounds.
Stefaan De Smedt: 0000-0002-8653-2598
Franco̧ ise Chuburu: 0000-0002-4937-173X
Author Contributions
⊥M.N. and V.M. contributed equally. The manuscript was
written through contributions of all authors. All authors have
given approval to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors would like to thank the “Programme de
cooperation transfrontaliere Interreg France-Wallonie-Vlaan-
CONCLUSION
̀
■
Two G1-4GdCA assemblies containing GdDOTAGA and
deren” for funding the “Nanocardio” project (http://
nanocardio.eu), the thesis of M. Ndiaye, T Vangijzegem
salary, and the postdoctoral fellowships of V. Malytskyi and F.
Sauvage. The bioprofiling platform and the Center for
Microscopy and Molecular Imaging (CMMI), both supported
by the European Regional Development Fund and the Region
Wallone, and the PlAneT platform, supported by the European
Regional Development Fund and the Region Champagne
GdDO3A units were prepared and subjected to a H and 17O
1
NMR relaxometric investigation. These compounds have
similar mass and molecular sizes but differ by the linkers
used to graft the chelates to the organic framework. The
multimeric nature of G1-4GdCA assemblies allowed an
increase in molecular size which was translated into a
corresponding increase of τR and hence of relaxivity (between
I
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DOI: 10.1021/acs.inorgchem.9b01747
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
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DOI: 10.1021/acs.inorgchem.9b01747
Inorg. Chem. XXXX, XXX, XXX−XXX