Synthesis and Relaxometric Characterization of New Poly[N,N-bis(3-aminopropyl)glycine] (PAPGly) Dendrons Gd-Based Contrast Agents and Their in Vivo Study by Using the Dynamic Contrast-Enhanced MRI Technique at Low Field (1 T)

Article abstract of DOI:10.1002/cbdv.201900322

The synthesis of poly[N,N-bis(3-aminopropyl)glycine] (PAPGly) dendrons Gd-based contrast agents (GdCAs) via an orthogonal protection of the different functional groups and an activation/coupling strategy wherein a specific number of synthetic steps add a generation to the existing dendron has been described. The aim of this protocol is to build up two different generations of dendrons (G-0 or dendron's core, and G-1) with peripheral NH₂ groups to conjugate a 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) derivative and afterwards to chelate with Gd³⁺ paramagnetic ions. These complexes, which have a well-defined molecular weight, are of relevance to MRI as an attempt to gain higher ¹H relaxivity by slowing down the rotation of molecule compared to monomeric Gd(III) complexes used as contrast agents and to increase the number of paramagnetic centers present in one molecular structure. From the study of their water ¹H longitudinal relaxation rate at different magnetic fields (NMRD, Nuclear Magnetic Relaxation Dispersion) and by evaluating the variable temperature ¹⁷O-NMR data we determined the parameters characterizing the water exchange rate and the rotational correlation time of each complex, both affecting ¹H relaxivity. Furthermore, these two novel PAPGly GdCAs were objects of i) an in vivo study to determine their biodistributions in healthy C57 mice at several time points, and ii) the Dynamic Contrast-Enhanced MRI (DCE-MRI) approach to assess their contrast efficiency measured in the tumor region of C57BL/6 mice transplanted subcutaneously with B16-F10 melanoma cells. The aim of the comparison of these two dendrons GdCAs, having different molecular weights (MW), is to understand how MW and relaxivity may influence the contrast enhancement capabilities in vivo at low magnetic field (1 T). Significant contrast enhancement was observed in several organs (vessel, spleen and liver), already at 5 min post-injection, for the investigated CAs. Moreover, these CAs induced a marked contrast enhancement in the tumor region, thanks to the enhanced permeability retention effect of those macromolecular structures.

Full text of DOI:10.1002/cbdv.201900322

Title: Synthesis and relaxometric characterization of new Poly[N,N-
bis(3-AminoPropyl)Glycine] (PAPGly) dendrons Gd-based
contrast agents and their in-vivo study by using the Dynamic
Contrast-Enhanced MRI technique at Low field (1 T)
Authors: Sophie Laurent, Luigi Granato, Dario Longo, L. Vander Elst,
C. Henoumont, S. Aime, and R.N. Muller
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Biodiversity 10.1002/cbdv.201900322
Link to VoR: http://dx.doi.org/10.1002/cbdv.201900322

10.1002/cbdv.201900322
Chemistry & Biodiversity
Synthesis and relaxometric characterization of new Poly[N,N-bis(3-
AminoPropyl)Glycine] (PAPGly) dendrons Gd-based contrast agents and
their in-vivo study by using the Dynamic Contrast-Enhanced MRI
technique at Low field (1 T)
L. Granato,1 D. Longo,4 S. Boutry,3 L. Vander Elst,1 C. Henoumont,1 S. Aime,2 R.N. Muller,1,3 S.
Laurent1,3
1
General, Organic and Biomedical Chemistry Unit, NMR and Molecular Imaging Laboratory,
University of Mons, 19 avenue Maistriau B-7000 Mons, Belgium
2 Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino Italy
3 Center for Microscopy and Molecular Imaging, 8 rue Adrienne Bolland, B-6041 Charleroi, Belgium
4 Institute of Biostructures and Bioimaging (IBB); Italian National Research Council (CNR); Torino,
Italy
Abstract
The synthesis of poly[N,N-bis(3-aminopropyl)glycine] (PAPGly) dendrons Gd-based contrast agents (GdCAs) via an
orthogonal protection of the different functional groups and an activation/coupling strategy wherein a specific
number of synthetic steps add a generation to the existing dendron has been described. The aim of this protocol is
to build up two different generations of dendrons (G-0 or dendron’s core, and G-1) with peripheral NH2-groups to
conjugate a 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) derivative and after to chelate with Gd3+
paramagnetic ions. These complexes, which have a well-defined molecular weight, are of relevance to MRI as an
1
attempt to gain higher H relaxivity by slowing down the rotation of molecule compared to monomeric Gd(III)
complexes used as contrast agents and to increase the number of paramagnetic centers present in one molecular
1
structure. From the study of their water H longitudinal relaxation rate at different magnetic fields (NMRD,
Nuclear Magnetic Relaxation Dispersion) and by evaluating the variable temperature 17O NMR data we
determined the parameters characterizing the water exchange rate and the rotational correlation time of each
complex, both affecting 1H relaxivity.
Furthermore, these two novel PAPGly GdCAs were objects of i) an in-vivo study to determine their biodistributions
in healthy C57 mice at several time points, and ii) the Dynamic Contrast-Enhanced MRI (DCE-MRI) approach
to assess their contrast efficiency measured in the tumour region of C57BL/6 mice transplanted subcutaneously
1
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10.1002/cbdv.201900322
Chemistry & Biodiversity
with B16-F10 melanoma cells. The aim of the comparison of these two dendrons GdCAs, having different
molecular weights (MW), is to understand how MW and relaxivity may influence the contrast enhancement
capabilities in vivo at low magnetic field (1 T). Significant contrast enhancement was observed in several organs
(vessel, spleen and liver), already at 5 min post-injection, for the investigated CAs. Moreover, these CAs induced a
marked contrast enhancement in the tumour region, thanks to the enhanced permeability retention effect of those
macromolecular structures.
Keywords : MRI, gadolinium complexes, dendrimers
Introduction
Magnetic resonance imaging (MRI) has become a powerful non-invasive diagnostic imaging modality
that provides high-quality anatomic images and other physiologic data. Its advantages include a high
spatial resolution, a non-ionizing radiation source, and the ability to extract, simultaneously,
[1, 2]
physiological and anatomical information of soft tissues.
However, a major limitation of MRI
remains its inherent low sensitivity. To increase the sensitivity of MRI, scientists have developed non-
toxic contrast agents (CAs) over the last few decades, which are administered to the patient to enhance
image contrast between normal and diseased tissues or to indicate the status of organ function or blood
flow. [3, 4] The development of new MRI contrast agents offers intriguing challenges for investigators in
the chemical, physical, and biological sciences, including the design and synthesis of stable, non-toxic,
and tissue-specific metal complexes and the understanding of their effect on relaxation behaviour in
solution and in tissues. These biocompatible contrast agents alter the longitudinal (T1) and transverse
(T2) relaxation times of the surrounding water protons, therefore enhancing image contrast in tissues
of interest. For this reason, MRI CAs are generally categorized as T1 and T2 CAs based on their magnetic
properties and relaxation mechanisms. Gadolinium chelates are effective for increasing T1 relaxation
rate (1/T1) and commonly used as T1 CAs, generating a positive image contrast. [5] Superparamagnetic
iron oxide nanoparticles are more effective for increasing T2 relaxation rate (1/T2) and commonly used
as T2 CAs, producing negative image contrast. [6-7]
Clinical MRI contrast agents are low molecular weight complexes that have transient tissue retention
due to their short half-life. These agents are nonspecific, extravasate rapidly from blood circulation and
distribute throughout the extracellular space of normal and diseased tissues. Their unfavourable
pharmacokinetic profile makes these agents unsuitable for MR angiography and for the early detection
of cancer tissues. In addition, these low molecular weight complexes show some restrictions to reach
the ideal (or maximum attainable) relaxivity. [8]
2
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10.1002/cbdv.201900322
Chemistry & Biodiversity
In fact, the rapid extravasation of low MW contrast agents causes a fast decrease of their concentration
in the bloodstream after administration. As a consequence, this not only results in a lower signal
enhancement in the normal blood vessels, but also in a higher background signal from the surrounding
tissues. An increase in the background signal could reduce the spatial resolution such that blood vessels
can no longer be discriminated from their surroundings. Contrary to low MW contrast agents, high
MW contrast agents remain in high concentrations in the bloodstream. Due to the fact that high MW
contrast agents show little extravasation and long intravascular retention, they are commonly referred
to as blood pool agents, while low MW contrast agents are referred to as extravascular agents. [8] First
attempts to improve r1, hence, contrast efficiency, were pursued by non-covalent binding of low MW
contrast agents to the human serum albumin. [9-11]
Secondly, low MW contrast agents rapidly diffuse from the bloodstream into the interstitial space, even
in the case of healthy vessels. This relatively high permeability prevents their effective use in
differentiating between benign lesions (normal vessels) and malignant tumours (hyperpermeable
vessels) with contrast-enhanced MR imaging. On the other hand a better differentiation between these
two types of vessels is possible with high MW contrast agents. [12]
Thirdly, low MW contrast agents, such as Gd(III) complexes of 1,4,7,10-tetraazacyclododecane-1,4,7,10-
tetraacetic acid (DOTA), contain typically one Gd3+ ion per molecule. Consequently, the relatively low
signal enhancing effect (or longitudinal relaxivity r1, s-1 mM-1) requires that high concentrations (i.e. high
doses) of MRI contrast agent have to be administered to eventually obtain sufficient contrast between
the blood vessels and its surrounding tissues. A straightforward approach to increase the loading of
Gd3+ per molecule is to attach several Gd(III) chelates to a macromolecular scaffold. [13-14] An additional
advantage of (covalently) attaching multiple Gd(III)-based complexes to high MW structures is the
strong increase in r1 per Gd3+ as a result of the strong reduction in the molecular tumbling rate of the
Gd(III) complex. [8, 15]
Inulin-Gd-DO3A complexes, [16] several natural and synthetic polymers, such as starch polysaccharide,
[17-18] dextran, [19] or polylysine, [20] have been modified and conjugated to contrast agents, and showed
good efficiency in imaging. [21-23] However, their use was limited because of synthetic and purification
difficulties (polydispersity), low level of characterisation and non-reproducibility of the final chemical
entities.
Dendrimers can offer several advantages as contrast agents compared to the traditional linear polymers.
The dendritic architecture has a highly branched, three dimensionnal nanoscale structure with very low
polydispersity, high functionality and inherent rigidity higher than the macromolecular linear polymers.
Actually dendrimer-based contrast agents provide numerous benefits over low-molecular-weight
3
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10.1002/cbdv.201900322
Chemistry & Biodiversity
gadolinium chelates for MRI, including an enhanced r1 relaxivity due to slow rotational dynamic, [24,25]
[26,27]
a tunable pharmacokinetic that can be adapted for blood pool,
liver, kidney, and lymphatic
imaging, the ability to be a drug carrier, and a flexibility for labeling due to their inherent multivalency.
[28]
An important aspect for the in vivo application of these MRI contrast agents is that the relaxivity of
Gd(III) complexes is field-dependent and, in the case of the paramagnetic macromolecular systems, it
shows a maximum efficiency at ca. 1 T. [29-31] The theory of paramagnetic relaxation describes well this
experimental observation, since contrast agents with optimized water exchange rate (τM) show highest
overall relaxivity at around 1 T, which is mainly controlled by the long rotational correlation time (τR)
[32-35]
characteristic of the paramagnetic macromolecules.
On the other hand, high fields provide an
overall increase of the signal intensity (SI) that allows the rapid acquisition of highly resolved images.
The first dendrimeric MRI contrast agents were reported by Adam et al. [36] and Wiener et al. [37] Since
then, many dendrimeric MRI CAs have been reported using various dendrimers, including
[38]
[38, 39]
[40]
poly(amidoamine) (PAMAM),
poly(propylene imine) (PPI),
and poly-L-lysine (PLL).
Dendrimeric Gd(III) complexes have shown higher relaxivity due to the more rigid structure than their
[8, 41-43]
[39]
linear polymeric counterparts.
Langereis et al.
reported that a Gd-DTPA conjugated to
poly(propylene imine) dendrimer (the fifth generation G-5, Mw = 51 kDa) had r1 relaxivity as high as
19.7 s-1 mM-1 per Gd(III) (1.5 T, 20 °C), which was 4.7 times that of Gd-DTPA. Dendrimer-based Gd(III)
chelates were effective for contrast enhanced MRI of the kidneys, vasculature, liver, or tumours. [44-46]
Here we report a “convergent synthesis” of poly[N,N-bis(3-aminopropyl)glycine] (PAPGly), as new
dendrons Gd-based contrast agents. We describe the parameters governing the relaxivity of these new
materials comparing them with those reported previously. We report also an in-vivo study in healthy
mice and in mice with transplanted subcutaneously melanoma cells of those new PAPGly dendrons
Gd-based contrast agents, having generation G-0 and G-1, in order to determine and compare their
biodistributions and contrast efficiency by Dynamic Contrast-Enhanced MRI (DCE-MRI) at low
magnetic field (1 T).
Results and Discussion
Synthesis of poly[N,N-bis(3-aminopropyl)glycine] dendrons Gd-based contrast agents
The following approach described for the synthesis of these poly-amino-propyl-glycine (PAPGly)
dendrons (Fig. 1) was based on the suitably convergent method of multibranched dendrimers synthesis.
4
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10.1002/cbdv.201900322
Chemistry & Biodiversity
We synthesised two different generations of dendrons G-0 (or dendron’s core) and G-1, via an
orthogonal protection of the different functional groups and an activation/coupling strategy, wherein
each synthetic step adds a generation to the existing dendron.
NH2
O
NH2
NH2
N
NH
NH
NH2
NH2
N
N
OH
OH
O
N
O
O
NH2
Dendron G-0 (or core)
Dendron G-1
O
O
OH
HO
N
N
N
HO
N
O
O
HN
S
L1
HN
Figure 1: Chemical structures of ligand L1 and the PAPGly dendrons with two different generations (G-0, G-1).
In the first step (Scheme 1), the primary amine functions in the commercially available polyamine 3, 3'-
diaminodipropylamine 1 were protected using t-Boc groups producing compound 2 in 75% yield. The
t-Boc group was used since it is stable towards hydrogenation and basic reaction conditions. In the next
step, benzyl bromo acetate was used as alkylation reagent in the presence of DIEA using DMF as solvent.
Purification of 3 by silica gel column chromatography using EtOAc/Heptane (70:30) as eluent
produced white crystals with 90% yield. Removal of the benzyl protecting group of compound 3 was
accomplished by hydrogenation at 60 psi using MeOH as solvent giving the polyamine derivative 4.
Subsequently the Boc groups were removed by using TFA in DCM, to give the dendron G-0.
[47]
The ligand L1 (Fig. 1) was prepared via the synthetic route reported recently.
The intermediate
isocyanate-activated DO3A derivative (DO3A-NCSPP) of the chelate L1, freshly prepared, was used for
the reaction with the dendron G-0 in order to obtain the product G-0(L1)2.
5
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10.1002/cbdv.201900322
Chemistry & Biodiversity
BocHN
N
NHBoc
i
ii
iii
O
H2N
N
H
NH2
BocHN
N
NHBoc
H
O
1
2
3
O
O
O
O
OH
N
N
N
N
N
N
HO
OH
HO
N
N
O
S
O
OH
HO
O
NCS
O
N
H
S
N
BocHN
N
NHBoc
H2N
N
NH2
DO3A-NCSPP
v
H
O
O
O
O
HO
O
HO
H
N
N
iv
OH
OH
H
N
N
N
OH
O
N
4
G-0
N
HO
O
G-0(L1)2
O
O
O-
N
N
-O
vi
Gd3+
N
N
S
-O
O
O
N
H
HN
O
HO
N
O
H
-O
O
NH
N
N
Gd3+
N
S
O-
N
N
O
-O
O
G-0(GdL1)2
Scheme 1: Synthesis of dendron generation G-0 (or core) and its gadolinium complex G-0(GdL1)2: (i) tert-butyl phenyl
carbonate, DMF, room temperature (rt); (ii) benzylbromo acetate, DIPEA, DMF; (iii) Pd/C 10 %, MeOH, H2 at 60 psi; (iv)
CF3COOH/CH2Cl2 50%, rt; (v) CSCl2, CCl4/water, rt; (vi) GdCl3·6H2O, pH 6.5-7.0.
The generation one of the dendron (G-1) was efficiently carried out in solution (CH3CN) through the
activation of the intermediate 5 (2.8 eq.) by using the reagent HATU, 1-[bis(dimethylamino)methylene]-
1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate, to generate an active ester from the
carboxylic acid, and subsequent N-acylation with the primary amines of intermediate 4, in the presence
of DIPEA as organic base (Scheme 2). This typical peptide coupling leads the product 6, which was
purified by flash chromatography on silica gel using DCM/CH3OH as mobile phase, obtaining a white
crystal compound after evaporation of the solvent. The orthogonal deprotection of the carboxylic acid
and the primary amine groups of compound 6 provides G-1 dendron, the conjugation of which with
the intermediate DO3A-NCSPP derivative of ligand L1 gives the product G-1(L1)4 (Scheme 2).
6
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10.1002/cbdv.201900322
Chemistry & Biodiversity
BocHN
N
NHBoc
BocHN
N
NHBoc
H2N
N
NH2
O
O
O
i
ii
OH
O
O
4
3
5
NH2
NHBoc
O
O
O
NH
N
O
NH
NH
N
OH
O
NH2
NHBoc
N
N
iii
i, ii
4 + 5
NHBoc
NH2
NH
N
N
O
O
6
G-1
NHBoc
O
O
NH2
O
O-
N
N
O
O
-O
O
Gd3+
N
N
N
HO
N
N
N
N
OH
HO
OH
N
N
S
N
O
O-
O
S
O
N
O
OH
O
O
OH
O
O
H
O
NCS
HN
N
H
O
O-
N
N
-O
HN
Gd3+
N
N
N
OH
HO
DO3A-NCSPP
iv
N
N
N
H
N
N
H
O-
H
N
O
O
N
O
N
H
OH
v
O
O
N
N
H
O
O
O
N
H
O
O
S
O-
N
N
S
-O
N
N
N
Gd3+
HO
OH
N
N
S
S
N
N
OH
O
N
OH
O
N
H
O
N
H
N
H
O
N
H
O
O-
O
O
OH
O
NH
NH
N
N
O
O
O-
OH
HN
HN
N
N
Gd3+
H
H
G-1(GdL1)4
G-1(L1)4
O
O
N
N
N
N
N
N
O-
OH
N
N
S
S
O
O
OH
O-
O
O
Scheme 2: Synthesis of dendron generation G-1 and its gadolinium complex G-1(GdL1)4: (i) Pd/C 10 %, MeOH, H2 at 60
psi; (ii) CF3COOH/CH2Cl2 50%, rt; (iii) CH3CN, HATU, DIPEA; (iv) CSCl2, CCl4/water, rt; (v) GdCl3·6H2O, pH 6.5-
7.0.
The Gd3+ complexes of both dendrons were prepared by adding stoichiometric amount of Gd3+ chloride
to an aqueous solution of the ligands G-0(L1)2 and G-1(L1)4 (Schemes 1 and 2) while maintaining the
pH of the solution between 6.5-7.0 by addition of 1 M NaOH aqueous solution. After 3-4 days the
reaction was stopped, and the excess of free lanthanide of dendron complex G-0 was removed by
centrifugation as Gd(OH)3 precipitate, which appeared at pH 10 after addition of 1 M NaOH, whereas
for G-1 complex the free gadolinium was removed by membrane ultra-filtration. The absence of free
Gd3+ ions was confirmed in both cases by a xylenol orange test. The NMR characterization before the
complexation with gadolinium as well as the mass spectra after the complexation are available as a
supporting information (figures S1 – S4).
The hydrodynamic diameter of the higher generation dendrimer G-1(GdL1)4 was evaluated by Dynamic
Light Scattering (DLS), and a Z-ave diameter of 3.2 nm was obtained (figure S5).
7
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10.1002/cbdv.201900322
Chemistry & Biodiversity
Evaluation of the relaxivity and its parameters
The relaxivity depends on various factors including the external field, the number of coordinated water
molecules, water exchange rate, rotational diffusion, first and second coordination hydration sphere,
310
the gadolinium electronic relaxation times (described by the electronic relaxation time at zero field τS0
and by the correlation time that modulates the electronic relaxation τV) and the ion to water proton
distance. [15] DOTA-like chelates, such as the DO3A-p-aminophenyl-propan-1-one, [16] have a single water
molecule coordinated to the Gd3+ ion. Parameters such as the residence time of the coordinated water
(τM) and the rotational dynamics of the molecule (τR) are most affected when attaching contrast agents
to a polymer. Ideally for a small organic contrast agent, τM should be at maximum 10 ns and τR should
be higher than 10 ns at a magnetic field of 1.5 T (most commonly used in clinical MRI scanners). [48-49]
The results of relaxometric studies by fitting the 17O NMR and 1H NMRD data of the G-0(GdL1)2 and
G-1(GdL1)4 dendrons complexes (Table 1) indicated an overall increase in relaxivity compared to the r1
of the monomeric GdL1 complex previously studied, [47] and of the commercial Gd-DOTA complex. As
expected, the increase is the highest for the dendron agent of high generation (G-1) (Fig. 2b), with its
relaxivity per gadolinium being 4 times higher than the monomeric macrocyclic Gd3+poly(amino
carboxylate) complex (Gd-DOTA and GdL1, Table 1).
In order to determine the water exchange rate, a variable temperature 17O NMR study was performed
on the dendritic Gd3+-complexes of the two different generations and on the monomeric complex.
From the Table 1 and the Figure 2a, it appears that there is no dramatic difference between the
monomer and the G-0(GdL1)2 complex, whereas for G-1(GdL1)4 complex we can observe a somewhat
longer water residence time. This is probably due to a steric constraint around the second coordination
sphere of the Gd3+-complex which limits the access of the bulk water to the paramagnetic centre. The
study of 1H NMRD profiles of these complexes (Fig. 2b), which takes into account an eventual second-
sphere contribution, showed that the number of water molecules in the second-sphere (qss) is zero for
the G-1(GdL1)4 dendron complex comparing to the G-0(GdL1)2, where qss ~3 (Table 1). This is in line
with the previous observation, confirming the steric hindrance around the paramagnetic center which
limits the water access. As one of the most important outcomes of this study, we can thus confirm that
the attachment of the macrocyclic unit to a large dendritic polymer molecule does not dramatically
influence the kinetics of water exchange of the complex. These results are in line with most of the
general conclusions reported in literature concerning macrocyclic units attached to the well definite
dendritic architecture with its higher degree of symmetry and rigidity than the hyperbranched and
linear polymers macromolecular systems. [8, 38, 43, 46]
8
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10.1002/cbdv.201900322
Chemistry & Biodiversity
This finding allows to predict water exchange rates for Gd(III) complexes attached to
dendrons/dendrimers by determining this parameter for the monomeric chelate, before the tedious
synthesis of the whole molecule.
Figure 2 (a) Temperature dependence of the reduced 17O transverse relaxation rate at 11.75 T; (b) comparison between 1H
NMRD profiles registered at 37°C of GdL1 1.78 mM (spheres), G-0(GdL1)2 1.59 mM (squares), G-1(GdL1)4 0.90 mM
(triangles) and Gd-DOTA 1 mM (stars). The lines represent simulation using the best-fit parameters (Table 1).
The most important expectation for the dendritic contrast agents was that the rotational correlation
time, τR, which limits the proton relaxivity of current contrast agents at imaging fields, would be
increased. The longer the rotational correlation time, the greater is the potential for a higher relaxivity
of the agent. In our present study, we address the problem of the paramagnetic enhancement by
increasing the molecular weight of the contrast agent. As expected for large molecules previously
reported, and for these systems described in the present work, the values of the τR are higher than those
found for monomeric Gd3+ complexes (Table 1), which results in an increase of the relaxivity mainly at
high field where a hump can be observed on the NMRD profile of G-1(GdL1)4 (the relaxivities per
molecule are of 49.6 and 14.4 s-1 mM-1 for G-1(GdL1)4 and G-0(GdL1)2 respectively, at 20MHz (37 ° C)).
1
Table 1 Parameters of GdL1, G-0(GdL1)2 and G-1(GdL1)4 obtained from the analysis of 17O NMR and H NMRD data
compared with Gd-DOTA as a reference.
Reference
New Complexes
(41)
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10.1002/cbdv.201900322
Chemistry & Biodiversity
Parameters
Gd-DOTA
3.5
GdL1
G-0(GdL1)2
7.2
G-1(GdL1)4
12.4
1
1
r1(b) [mM− s− ]
4.7
r1(c) [mM− s− ]
τV 310(ps)
τM 310(ns)
τR310(ps)
τS0310(ps)
qSS
3.5
4.7
14.4
49.6
1
1
7 ± 1.0
122 ± 10
53 ± 1.3
404 ± 24
19.2 ± 1.1 (23.8)a
331 ± 44
93.1 ± 2.2
74.8 ± 1.2
22.6 ± 2.14 (22)a 47.1 ± 1.92 (6)a
364 ± 22
541 ± 60
152 ± 4.87
72.0 ± 5.43
3.15 ± 0.108
18.3 ± 2.89
473 ± 10.9
127 ± 1.63
τSS(ps)
(a) The first values were obtained from the fitting of the proton NMRD profile; the values in
parentheses were obtained from the fitting of the 17O NMRD data; (b) r1 corresponds to the relaxivity
per Gd-complex, (c) per molecule, both calculated at 20 MHz and 37 °C.
The relaxivity at 20 MHz and 37°C in the presence of human serum albumin (HSA) was also measured
in order to evaluate the binding of both dendrimers G-0(GdL1)2 and G-1(GdL1)4 to HSA. The obtained
per gadolinium relaxivities are slightly higher than the corresponding relaxivities in water (10.0 s-1 mM-
1 and 15.4 s-1 mM-1 for G-0(GdL1)2 and G-1(GdL1)4 respectively) but this increase can be explained by
the higher viscosity of the HSA solution compared to water and can not be attributed to a specific
binding of the dendrimers to HSA. This should thus allow a fast elimination of the compounds from
the body, principally via glomerular filtration.
In vivo evaluation of biodistribution and contrast enhancing properties in tumour bearing mice
Following the results obtained in the presence of HSA, an evaluation of the blood half-life and of the
elimination of both dendrimers through the kidneys was performed in healthy mice at 9.4T after an
injection of 100 μmoles Gd/kg (n = 2). Blood half-life are based on curves collected from signal intensity
measurements in left ventricle of mice on MR images (dynamic T1-weighted GE acquisitions until 16
min post-injection with 1 min time resolution). It has however to be noted that signal intensity data
also depend on the sensitivity of MR to detect Gd and are not a direct measurement of Gd plasma
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10.1002/cbdv.201900322
Chemistry & Biodiversity
concentration. Using the peak signal intensity as « Initial quantity N0 » and the signal intensity at a
time t of 15,5 min as « Quantity remains N(t) », we obtained blood half-life of 11.65 min and 8.11 min
for G-0(GdL1)2 and G-1(GdL1)4 respectively (https://www.miniwebtool.com/half-life-calculator). The
same experiment performed on Gd-DOTA gave a blood half-life of 7 min, meaning that the grafting to
a macromolecular entity such as a dendrimer does not increase dramatically the blood half life of the
complexes, which could be expected considering the absence of binding to human serum albumin.
The kidneys biodistribution was also evaluated on those mice and it was observed that kidney remained
contrasted on MR images at day 6 after injection (T1-weighted spin echo collected at day 0 and day 6).
Subsequent experiments with G1 compound only were performed and images were collected also at
day one and at one month after injection. We present here below (figure 3) measurements performed
in cortical and medullar region of the kidney, suggesting that this organ recovers its normal MR signal
intensities at one month.
1.8
1.6
1.4
1.2
1
Cortex
0.8
Medulla
0.6
0.4
0.2
0
Day 0
Day 1
Day 6
1 month
Figure 3 : Mean signal intensity values in cortex and medulla of mice kidney at different times after
injection of G1 compound (n=5 ; data were processed by dividing signal intensity values by the signal
intensity of an external Gd-based reference placed beside the mouse).
Considering the quite long elimination of the compounds through the kidneys, a more complete
biodistribution of the two investigated molecules was studied in healthy C57 mice at several time points
following intravenous injection at doses of 0.05 mmol Gd/kg (Fig. 4, left). All the molecules were
completely cleared from the vascular compartment 24h post their administration. A pronounced
accumulation within the spleen was observed for both compounds, even the day after the
administration, with similar contrast enhancements values 24h post injection, probably due to the large
11
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10.1002/cbdv.201900322
Chemistry & Biodiversity
size of these compounds. A different distribution was observed within the liver, with higher contrast
enhancement values for G1(GdL1)4 in comparison to G-0(GdL1)2.
Fig. 4 (right) shows the dynamic signal intensity enhancements in the tumour regions of C57BL/6 mice
transplanted subcutaneously with B16-F10 melanoma cells. Significant contrast enhancement was
observed, already at 5 min post-injection, for G-1(GdL1)4 and G-0(GdL1)2, of 42%, and 28%,
respectively. The contrast enhancement slowly reduced thereafter for G-1(GdL1)4, while for G-0(GdL1)2
it remained quite constant along time. Measurements of relative area under the curve (AUC) clearly
show a higher contrast enhancement inside the tumour region for G-1(GdL1)4 that becomes more
evident 30 min after the injection.
All the investigated CAs induced a good contrast enhancement in the tumour region, thanks to the
enhanced permeability retention effect, at the investigated doses of 0.05 mmol Gd/kg (Fig. 5). G-
0(GdL1)2 showed the lower contrast enhancement and accumulation inside tumour region, despite
almost no uptake in the liver. This can be explained by the small size of this system that results in faster
wash-in and wash-out properties, combined with the lowest relaxivity efficiency. Conversely, G-1(GdL1)4
showed a better contrast enhancement and accumulation within tumour.
DCE_Enh
vessel
60
40
60
40
20
0
30 min
1h
2h
4h
24h
G0(GdL1)2
G1(GdL1)4
20
0
0
20
40
Time (min)
60
4
2
)
)
1
1
L
L
d
d
G
G
(
(
1
0
G
G
AUC
spleen
25
100
80
60
40
20
0
30 min
1h
2h
4h
24h
20
15
10
5
G0(GdL1)2
G1(GdL1)4
0
0
0
0
0
5
1
2
3
UC
UC
UC
UC
4
2
)
)
E
E
E
E
r
r
r
r
1
1
L
L
d
d
G
G
(
(
1
0
G
G
12
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10.1002/cbdv.201900322
Chemistry & Biodiversity
liver
30
20
10
0
30 min
1h
2h
4h
24h
4
)
2
)
1
L
1
L
d
d
G
(
G
(
1
0
G
G
Figure 4: (Left): signal intensities contrast enhancements measured in the heart (top), spleen (middle) and liver (bottom) at
various time points post-injection (30 min, 1h, 2h, 4h and 24h) of G-1(GdL1)4 and G-0(GdL1)2 at the same dose of 0.05
mmol Gd/kg. Values are shown as mean ±SD (n=2 mice) for each contrast agent; (Right): time course of MRI signal intensity
enhancement (top) and calculated area under the curve (AUC, bottom) in tumour bearing mice after intravenous injection
of G-0(GdL1)2 (black squares), and G-1(GdL1)4 (red squares) at a dose of 0.05 mmol Gd/kg at several time points post
injection 10, 20, 30 and 50 min. Values are shown as mean ±SD (n=3 mice) for each contrast agent.
Figure 5: Representative tumour SI enhancement maps over imposed onto anatomical T2w images upon injection of G-
0(GdL1)2, and G-1(GdL1)4 at a dose of 0.05 mmol Gd/kg.
Conclusions
A convenient methodology has been presented for the synthesis of poly[N,N-bis(3-aminopropyl)glycine]
(PAPGly) dendrons Gd-based contrast agents for MR imaging. By orthogonal chemistries, a novel
synthetic methodology has been developed to obtain two generations of PAPGly dendrons conjugated
with a low molecular weight Gd-DO3A-based complex. The longitudinal (r1) relaxivities of these MRI
contrast agents increase along with their size, indicating a longer rotational correlation time for the
high dendron generation. Consequently, very high relaxivities were observed (r1 = 14.4 s-1 mM-1 and
13
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Chemistry & Biodiversity
49.6 s-1 mM-1 for the G-0(GdL1)2 and G-1(GdL1)4 per molecular complex, respectively). These values are
significantly higher in comparison with the parent monomeric GdL1 (r1 = 4.7 s-1 mM-1) complex and
with the commercial one (Gd-DOTA, r1 = 3.5 s-1 mM-1). Our MRI results support the hypotheses that
the Gd-based dendrons with high molecular weight, or macromolecular dendrimers Gd-complexes, can
improve contrast enhancement as a result of their higher relaxivities compared to the low molecular
weight contrast agents. Furthermore, high structural and chemical homogeneity of the dendritic
polymers are beneficial for their contrast agent properties as compared to linear polymers. For example,
biodistribution and pharmacokinetic properties can be tuned more easily by controlling dendrimer size
and conformation.
Consequently, when those dendritic contrast agents are applied for DCE-MRI in mice they provide an
opportunity to noninvasively extract important physiological and pathophysiological information.
Experimental Section
General methods and materials
All reagents and chemicals were obtained from commercial sources and used without further
purification. Solvents were distilled and/or dried prior to use by standard methodology except for
those, which were reagent grades. Water was demineralized prior to use. Unless stated otherwise, all
the reactions were carried out under a nitrogen atmosphere and the reaction flasks were pre-dried by
heat gun under vacuum. The pH values of the samples were measured at ambient temperature using a
Corning 125 pH meter with a calibrated micro-combination electrode purchased from Aldrich. The
pH values of the solution were adjusted using 1M NaOH, 1N pyridine or 1N HCl. Most of the reactions
were monitored by TLC on silica plates detecting the components by UV light or by applying the
Dragendorff reagent, while other product formations were monitored via analytical HPLC (Waters
W600, with a quaternary pump gradient module) with an UV detector coupled with a Waters
Micromass ZQ system (Waters, Belgium). For low molecular weight characterization, ESI-QTOF-MS
were obtained on a Q‐TOF Ultima GLOBAL mass spectrometer (Waters-Micromass, Manchester, UK),
whereas for high molecular weight products, MALDI spectra were obtained on MALDI-TOF
spectrometer (Waters-Micromass, Manchester, UK).
The purification of the compounds was performed on KP-silica and KP-C18 Biotage cartridges over a
Biotage flash chromatography instrument, Uppsala, Sweden. 1H-NMR, 13C-NMR spectra were recorded
on either a Bruker Avance-300 MHz or a Bruker Avance II‐500 MHz spectrometer, at 25°C. Chemical
shifts are given in ppm (δ) values. Data are reported as follows: chemical shift (multiplicity: s = singlet,
14
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10.1002/cbdv.201900322
Chemistry & Biodiversity
d = doublet, t = triplet, q = quadruplet, m = multiplet, br = broad resonance), J = coupling constant
(Hz), integration, peak assignment in italic form.
Synthesis of bis-(3-tert-butoxycarbonyl-aminopropyl) amine (2)
To a solution of the bis-(3-aminopropyl) amine 1 (10 g, 0.0762 mol) in DMF (75 ml) was added tert-
butyl phenyl carbonate (32.63 g, 0.168 mol). The reaction mixture was stirred for 40h at rt. and poured
into a phosphate buffer (1L; 0.025 M K2HPO4 and 0.025 M NaH2PO4). The pH was adjusted to 3 with
aq. H2SO4 (2M) and mixture was extracted with CH2Cl2 (2x250 ml). The aq. phase was made strongly
alkaline with aq. NaOH (9M) and extracted with CH2Cl2 (3x250 ml). The organic phase was dried
(Na2SO4), filtered and concentrated in vacuo. The residual solvents (DMF and H2O) were removed by
drying the protected amine at less than 0.1 mmHg at 40 °C, to obtain a white solid compound 2 (18.9
g, 75% of yield). ESI MS: m/z 332 [M + H]+; 1H NMR (300 MHz, CDCl3, 25 °C): δ 1.35 (s, 18 H), 1.58
(4H, q, 2xCH2CH2CH2), 2.06 (2H, br s, 2xCONH), 2.57 (4H, t, 2xNHCH2CH2), 3.11 (4H, br t,
13
2xCONHCH2), 5.32 (1H, br s, CH2NHCH2). C NMR (300 MHz, CDCl3, 25 °C): δ 28.35, 29.60,
38.68, 47.16, 77.88, 156.16.
Synthesis of benzyl N,N-bis {3-[(tert-butoxycarbonyl) amino] propyl} glycinate (3)
Compound 2 (6.78 g, 0.02 mol) and 10 ml DMF were mixed in a three-necked flask under stirring.
The mixture was cooled by using an ice bath followed by addition of benzyl bromo-acetate (4 ml, 0.025
mol) and DIPEA (3.5 ml, 0.02 mol) over a time period of 30 min. The resulting reaction mixture was
stirred at 0 °C for additional 30 min, whereupon it was allowed to reach rt. The mixture was reacted
for additional 4h at rt. Ether (70 ml) was added and the formed white precipitate was collected by
filtration, while the mother liquid was evaporated to dryness. The produced oil was dissolved in 70 ml
DCM and carefully washed sequentially with 3 x 50 ml water, 2 x 50 ml 1M HCl, and 3 x 50 ml water.
The organic phase was dried over Na2SO4 followed by evaporation to dryness. The crude product was
purified by silica gel column chromatography using EtOAc/Heptane (70:30) as eluent, and the pure
1
product 3 was obtained in a yield of 90%, 8.63 g. ESI MS: m/z 480 [M + H]+; H NMR (300 MHz,
CDCl3, 25 °C): δ 1.45 [18H, s, 2xC(CH3)3], 1.65 (4H, br s, 2xCH2CH2CH2), 2.62 (4H, br s,
2xNCH2CH2), 3.18 (4H, br t, 2xCH2CH2NH), 3.37 (2H, br s, COCH2N), 5.17 (2H, s, COOCH2Ar),
5.24 (2H, br s, 2xCONH), 7.37 (5H, br s, ArH5). 13C NMR (300 MHz, CDCl3, 25 °C): δ 27.32, 28.45,
38.79, 52.11, 55.03, 66.30, 78.82, 128.37, 128.39, 128.59, 135.67, 156.10, 171.33.
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Synthesis of N,N-bis{3-[(tert-butoxycarbonyl) amino] propyl} glycine (4)
Compound 3 (2.1 g, 4.37 mmol), Pd/C 10% (324 mg) and 100 ml MeOH were mixed in a sealed tube,
whereupon hydrogen pressure of 60 psi was applied. The mixture was reacted for 22h at rt. The
resulting reaction mixture was filtered through a Celite pad and carefully washed with 150 ml MeOH.
The mother liquid was evaporated to dryness, and white product 4 was obtained in a yield of 1.6 g. ESI
MS: m/z 390 [M + H]+; 1H NMR (300 MHz, CD3OD, 25 °C): δ 1.45 [18H, s, 2xC(CH3)3], 1.91 (4H, br
s, 2xCH2CH2CH2), 3.17 (4H, br s, 2xNCH2CH2), 3.36 (4H, br t, 2xCH2CH2NH), 3.69 (2H, br s,
COCH2N). 13C NMR (300 MHz, CD3OD, 25 °C): δ 26.04, 28.84, 38.23, 54.54, 56.78, 80.33, 158.74,
170.07.
Synthesis of N,N-bis(3-aminopropyl) glycine (G-0)
In a round bottom flask, compound 4 (1.0 g, 2.1 mmol) was dissolved in 20 ml of 50% solution of TFA
in dry DCM, whereupon the mixture was stirred for 24h at rt. The reaction mixture was evaporated to
dryness under reduced pressure, and the crude product was washed 2 times with ethyl ether (in a sonic
bath) in order to remove further impurities, to give the compound G-0 (1.358 g, white colour, TFA-
salt). ESI MS: m/z 280 [M + H]+; 1H NMR (300 MHz, CDCl3, 25 °C): δ 1.69 (4H, q, 2xCH2CH2CH2),
2.56 (4H, t, 2xNCH2CH2), 2.71 (4H, t, 2xCH2CH2NH2), 3.86 (2H, s, COCH2N). 13C NMR (300 MHz,
CDCl3, 25 °C): δ 23.78, 38.28, 53.30, 55.55, 163.25, 170.75.
Synthesis of G-0(L1)2
Compound G-0 (190 mg, 0.456 mmol) was dissolved in water (25 ml), and the pH was adjusted to 9-
10 with diluted NaOH aqueous solution. DO3A-NCSPP (771 mg, 1.44 mmol) freshly prepared as
previously reported (39), was added, and the reaction mixture was stirred at room temperature for 48
h. The solution was freeze-dried to get a yellow powder (832 mg). A portion of crude compound (100
mg) was purified by using a semi-preparative HPLC condition: RP-C18 column; CH3CN/H2O (buffer
NH4HCO3, pH=7.5) as mobile phase; gradient 5-30% CH3CN in 30 min with a flow rate 1 ml/min;
to obtain 70 mg of pure pale-yellow compound G-0(L1)2. ESI MS: m/z 1261 [M + H]+; 1H NMR (500
MHz, D2O, 25 °C): δ 1.22 (6H, br s, 2xCH3CHCO), 1.97 (4H, br s, CH2CH2CH2), 2.82-3.91 (54H, 2
br m), 4.81 (2H, br s, 2xCH3CHCO), 7.31 and 7.70 (8H, 2 br d, ArH). 13C NMR (500 MHz, D2O, 25
°C): δ 13.10, 23.67, 41.09, 44.07, 45.24, 48.88, 50.89, 51.50, 52.71, 54.87, 55.68, 56.57, 56.93, 100.00,
123.37, 129.80, 132.21, 142.48, 169.56, 170.35, 170.54, 177.07, 179.97, 203.07.
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Synthesis of benzyl N,N-bis(3-aminopropyl) glycinate (5)
In a round bottom flask, compound 3 (1.1 g, 2.3 mmol) was dissolved in 20 ml of 50% solution of TFA
in dry DCM, whereupon the mixture was stirred for 24h at rt. The reaction mixture was evaporated to
dryness under reduced pressure, and the crude product was washed 2 times with ethyl ether (in a sonic
bath) in order to remove further impurities, to give the compound 5 (1.58 g, white colour, TFA-salt).
ESI MS: m/z 280 [M + H]+; 1H NMR (300 MHz, CDCl3, 25 °C): δ 2.09 (4H, q, 2xCH2CH2CH2), 3.06
(4H, t, 2xNCH2CH2), 3.21 (4H, t, 2xCH2CH2NH2), 4.06 (2H, s, COCH2N), 5.27 (2H, s, COCH2Ar),
13
7.37-7.42 (5H, m, ArH5). C NMR (300 MHz, CDCl3, 25 °C): δ 23.94, 38.40, 53.15, 55.02, 69.02,
129.69, 129.71, 129.75, 136.43, 169.38.
Synthesis of benzyl N,N-bis{3-[(N,N-bis{3-[(tert-butoxycarbonyl) amino] propyl} glycyl) amino]
propyl} glycinate (6)
Into a two-necked round bottom flask, under dry condition (N2), a suspension of compound 4 (1.1 g,
2.82 mmol), 3 eq. of 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid
hexafluorophosphate (HATU) (1.289 g, 3.39 mmol), and 2.5 eq. of DIPEA (0.365 g, 2.83 mmol) in
CH3CN HPLC‐grade (70 ml) was stirred for 30 min at RT. Benzyl N,N-bis(3-aminopropyl) glycinate
(TFA-salt) (5) (0.7 g, 1.13 mmol) was first dissolved in 20 ml of CH3CN with 4 eq. of DIPEA, and added
dropwise to the reaction mixture over 30 min, [reaction monitored by TLC: CH2Cl2/CH3OH, 9:1,
with a ninhydrin test]. After 5 h, the reaction was stopped and poured into ethyl acetate (150 ml). The
organic layer was first washed three times with saturated NaHCO3 solution, and then three times with
H2O, dried over Na2SO4, and evaporated to dryness to give an oil (1.342 g). A 1.2 g aliquot of residue
product was purified by flash chromatography on silica gel using CH2Cl2/CH3OH (9.5:0.5) as mobile
phase. After evaporation of the solvent, 812 mg (0.77 mmol, with 75.4 % of total yield) of 6 was
obtained as a white crystal compound. ESI MS: m/z 1055 [M + H]+; 1H NMR (300 MHz, CDCl3, 25
°C): δ 1.42 [36H, s, 4xC(CH3)3], 1.62-1.84 (12H, br m, 6xCH2CH2CH2), 2.48 (8H, t, 4xNCH2CH2),
2.61 (4H, t, 2xNCH2CH2), 3.00 (4H, s, 2xNCH2COONH), 3.13 (8H, q, 4xCH2CH2NHCOOtert-But),
3.31 (4H, q, 2xCH2CH2NHCO), 3.37 (2H, s, NCH2COOCH2), 5.12 (2H, s, COCH2Ar), 7.32 (5H, br
13
m, ArH5). C NMR (300 MHz, CDCl3, 25 °C): δ 27.63, 27.68, 28.43, 37.14, 38.53, 51.85, 52.66,
54.91, 58.52, 66.16, 79.03, 100.00, 128.19, 128.27, 128.55, 135.75, 156.06, 171.31, 171.38.
Synthesis of N,N-bis(3-{[N,N-bis(3-aminopropyl)glycyl]amino}propyl)glycine (G-1)
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Chemistry & Biodiversity
The dendron G-1 (156 mg, 0.29 mmol) was obtained after a complete deprotection of compound 6
(325 mg, 0.31 mmol) by using the same reaction conditions as described for the synthesis of dendron
1
G-0. ESI MS: m/z 532 [M + H]+; H NMR (300 MHz, CDCl3, 25 °C): δ 1.99-2.05 ( 4H, br m,
2xCH2CH2CH2), 2.14-2.20 (8H, br m, 4xCH2CH2CH2) 3.06-3.09 (8H, t, 4x CH2CH2NH2), 3.32-3.38
(16H, br m, 6xNCH2CH2 and 2xCH2CH2NHCO), 4.08 (4H, s, 2xNCH2CONH), 4.12 (2H, s,
NCH2COOH). 13C NMR (300 MHz, CDCl3, 25 °C): δ 27.08, 30.43, 37.14, 38.53, 51.85, 52.66, 54.91,
58.52, 170.31, 173.38.
Synthesis of G-1(L1)4
Compound G-1 (156 mg, 0.29 mmol) was dissolved in water (20 ml), and the pH was adjusted to 9-10
with diluted NaOH aqueous solution. DO3A-NCSPP (770 mg, 1.43 mmol) freshly prepared as
previously reported (39), was added, and the reaction mixture was stirred at room temperature for 48
h. The crude product was purified at pH = 7, by ultrafiltration using a membrane with a molecular
1
weight cut-off of 1 kDa, to get a pale yellow solid of 0.2 g. MALDI Q-TOF: m/z 2673 [M + H]+; H
NMR (500 MHz, D2O, 25 °C): δ 1.31 (12H, br s, 4xCH3CHN), 1.78-1.95 (12H, br d, 6xCH2CH2CH2),
2.91-4.00 (122H, br m), 7.42-7.91 (16H, 2br m, 4xArH4). 13C NMR (500 MHz, D2O, 25 °C): δ 12.95,
13.30, 23.73, 24.05, 36.39, 41.50, 44.12, 45.28, 48.25, 50.91, 51.54, 52.45, 52.86, 55.45, 56.64, 56.96,
62.50, 72.06, 122.74, 129.74, 131.77, 142.84, 142.97, 169.53, 170.10, 170.50, 177.78, 179.71, 202.78.
Preparation of G-0(GdL1)2 and G-1(GdL1)4 dendrons complexes
The dendrons G-0(GdL1)2 and G-1(GdL1)4 complexes were prepared by a stepwise addition of
GdCl3·6H2O to a solution of 83 mg (0.066 mmol) and 71 mg (0.026 mmol) of G-0(L1)2 and G-1(L1)4
respectively, in 5 ml of H2O, maintaining the pH between 6.5 and 7.0 by addition of 1 M NaOH
aqueous solution. After 4 days the reaction mixture was stopped, and the excess of free lanthanide for
the G-0(GdL1)2 complex was removed by centrifugation as Gd(OH)3 precipitate, which appeared at pH
10 after addition of 1 M NaOH, whereas for G-1(GdL1)4 complex the excess of free gadolinium was
removed by ultra-filtration using a membrane with a cut‐off of 3 kDa. The absence of free Gd3+ ions
was confirmed by a xylenol orange test, with a final pH of both solutions around 6.5-7.00, and the
purity of the solutions was verified by HPLC (RP-18 columns, elution time of 1.5 min with 70/30
methanol/water as elution solvents at a flow of 1ml/min, Agilent). The solutions were freeze-dried to
give a pale-yellow solid for both final complexes: G-0(GdL1)2 95 mg, ESI MS: m/z 1569 [M + H]+; and
18
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10.1002/cbdv.201900322
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75 mg G-1(GdL1)4, MALDI Q-TOF: m/z 3290 [M + H]+. Stock solutions of at least 80 mM can be
prepared and stored for a long time at 4°C.
NMRD profiles
1
1/T1 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 to 0.7 T and
1
corresponding to H Larmor frequencies from 0.01 to 30 MHz using 0.6 ml samples in 10 mm o.d.
tubes. The samples were prepared by dissolution of appropriate amounts of freeze-dried complexes of
GdL1, G-0(GdL1)2, and G-1(GdL1)4 in Milli-Q water with the pH in the range 6.5-7.0. The exact Gd3+
concentration was determined by proton relaxivity measurements at 20 MHz and 37 °C after complete
hydrolysis in concentrated HNO3. Additional relaxation rates at 20 and 60 MHz were obtained with
Bruker Minispec mq-20 and mq-60 spectrometers (Bruker, Karlsruhe, Germany), respectively. The 300
MHz data were obtained on a Bruker Avance-300 high resolution spectrometer.
1
Fitting of the H NMRD was performed with data processing software that uses different theoretical
models describing observed nuclear relaxation phenomena (Minuit, CERN Library). The theoretical
model takes into account the inner-sphere interaction, the outer‐sphere interaction and when necessary
the second sphere contribution. [8]
17O NMR studies
17O NMR relaxation rate measurements were performed on a Bruker Avance II-500 spectrometer using
5 mm diameter sample tubes containing 325 μl (pH 6.5-7.00) of G-0(GdL1)2 (25.5 mM) and G-1(GdL1)4
(15.9 mM) solutions. The temperature was regulated by air or N2 flow controlled by a BVT-3200 unit.
17O transverse relaxation times of pure water (diamagnetic contribution) were measured using a CPMG
sequence and a subsequent two-parameter fit of the data points. The 90 and 180° pulse lengths were
27.5 and 55 μs, respectively. Broadband proton decoupling was applied during the acquisition of all
17
17O NMR spectra. O T2 of water in complex solution was obtained from line width measurement.
R
The data are presented as the reduced transverse relaxation rate 1/T2 =55.55/([Gd complex]⋅q⋅T2P,
where [Gd complex] is the molar concentration of the complex, q is the number of coordinated water
molecules and T2P is the paramagnetic transverse relaxation rate obtained after subtraction of the
diamagnetic contribution from the observed relaxation rate. The number of water molecules in the first
coordination sphere (q) of the Gd3+ complex G-0(GdL1)2 and G-1(GdL1)4 was determined at 60 °C using
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10.1002/cbdv.201900322
Chemistry & Biodiversity
a Bruker Avance II‐500 spectrometer. The tubes with 5 mm external diameter containing 300 μl of the
sample with 50 μl of D2O for the lock were used for the measurements.
In vivo studies
Mice
6-old-week male C57BL/6 mice (n=3 for each molecule) were inoculated subcutaneously with 3×105
B16-F10 cell in 100 µL of PBS on both flanks. C57BL/6 mice (Charles River Laboratories Italia S.r.l.,
Calco Italia) were maintained under specific pathogen free conditions in the animal facility of the
Molecular Biotechnology Center, University of Torino, and treated in accordance with the EU
guidelines (EU2010/63). All in vivo studies were conducted according to approved procedures of the
Institutional Animal Care and Use Committee of the University of Torino. Before imaging, mice were
anaesthetized with a mixture of tiletamine/zolazepam (Zoletil 100; Vibac, Milan, Italy) 20mg/kg and
xylazine (Rompun; Bayer, Milan, Italy) 5mg/kg and during the acquisition their breath rate was
monitored throughout in vivo MRI experiments using a respiratory probe. Cannulation of the lateral
tail vein with a catheter was exploited for intravenous injection of the investigated molecules.
DCE-MRI experiments
Mice were placed supine in a solenoid Tx/Rx coil with an inner diameter of 3.5 cm. The breath rate
was monitored throughout in vivo MRI experiments using a respiratory probe (SAII Instruments, Stony
Brook, NY - USA). MR images were acquired with a 1 Tesla Aspect M2 MRI scanner (Aspect Magnet
Technologies Ltd., Israel). After the scout image acquisition, a T2-weighted (T2w) anatomical image was
acquired with a Fast Spin Echo sequence (TR 2500 ms; TE 44ms; number of slices 10; slice thickness
1.5 mm; FOV 40 mm; matrix 152 × 160; four averages; acquisition time 3 m 20 s). The DCE-MRI
dynamic protocol consisted of the application of an axial T1-weighted (T1w) 3D spoiled Gradient Echo
sequence with 3 initial pre-contrast images acquisition that were followed by the injection through the
catheter of one of the two investigated compounds at the same dose of 0.05 mmolGd/kg. After
injection, 47 dynamic post-contrast images were acquired with the following parameters: TR = 40 ms;
TE = 2.1 ms, flip angle = 60°, number of slices = 10, slice thickness = 1.5 mm, FOV = 40 mm, matrix
= 128 × 128, acquisition time 58 s.
All the DCE-MRI images were analysed using in-house developed software in MatLab (MathWorks,
Natick, MA).
Biodistribution
20
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10.1002/cbdv.201900322
Chemistry & Biodiversity
6-old-week male C57BL/6 mice (n=2 for each molecule) were administered with the two dendrons Gd-
based contrast agents at a dose of 0.05 mmol Gd/kg. T1w images were acquired before and after i.v.
injection at several time points: 30 min, 1h, 2h, 4h and 24h. Region of interests (ROIs) were manually
placed in several organs (liver, spleen, major vessel) and signal intensity (SI) values were measured and
contrast enhancement calculated as (SIpost-SIpre)/SIpre.
Acknowledgements
This work was performed with the financial support of the FNRS, FEDER, the ARC, the Walloon
Region (Gadolymph, Holocancer and Interreg projects), the Interuniversity Attraction Poles of the
Belgian Federal Science Policy Office and the COST actions. Authors thank the Center for Microscopy
and Molecular Imaging (CMMI, supported by European Regional Development Fund Wallonia).
Author Contribution Statement
LG and DL performed the experiments, analysed the data and wrote the paper. CH and LVE
contributed to the analysis of the data and to the writing of the paper. SA, RNM and SL contributed
samples/reagents/materials/analysis tools and conceived and designed the experiments. All authors
read and approved the final manuscript.
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Graphical abstract
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