MRI-visible polymer based on poly(methyl methacrylate) for imaging applications

Article abstract of DOI:10.1039/c5ra23646k

Macromolecular contrast agents are very attractive to afford efficient magnetic resonance imaging (MRI) visualization of implantable medical devices. In this work, we report on the grafting of a Gd-based DTPA contrast agent onto a poly(methyl methacrylate) derivative backbone by combining free radical polymerization and copper-catalyzed azide-alkyne cycloaddition (CuAAC). Using free radical polymerization, poly(methyl methacrylate-co-propargyl methacrylate) copolymers were prepared with a control of the ratio in propargyl methacrylate monomer units. The synthesis of a new azido mono-functionalized DTPA ligand was also reported and characterized by ¹H NMR and mass spectroscopy. After complexation with gadolinium, this ligand has been grafted on the polymer backbone by click chemistry reaction. The obtained macromolecular contrast agent was then coated on a polypropylene mesh using the airbrushing technique and the mesh was assessed for MRI visualization at 7 teslas. The polymeric contrast agent was also tested for cytocompatibility and stability to assess its suitability for biomedical applications.

Full text of DOI:10.1039/c5ra23646k

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MRI-visible polymer based on poly(methyl methacrylate) for
imaging applications
Mira Younis,a,b Vincent Darcos,a* Cédric Paniagua,a Pauline Ronjat,a Laurent Lemaire,c Benjamin
Nottelet,a Xavier Garric,a Youssef Bakkour,d John Hanna El Nakat,b Jean Coudanea
Macromolecular contrast agents are very attractive to afford efficient magnetic resonance imaging (MRI) visualization of
implantable medical devices. In this work, we report on the grafting of a Gd-based DTPA contrast agent onto a poly(methyl
methacrylate) derivative backbone by combining free radical polymerization and copper-catalyzed azide-alkyne
cycloaddition (CuAAC). Using free radical polymerization, poly(methyl methacrylate-co-propargyl methacrylate)
copolymers were prepared with a control of the ratio in propargyl methacrylate monomer units. The synthesis of a new
azido mono-functionalized DTPA ligand was also reported and characterized by 1H NMR and mass spectroscopy. After
complexation with gadolinium, this ligand has been grafted on the polymer backbone by click chemistry reaction. The
obtained macromolecular contrast agent was then coated on a polypropylene mesh using the airbrushing technique and
the mesh was assessed for MRI visualization at 7 Teslas. The polymeric contrast agent was also tested for cytocompatibility
and stability to assess its suitability for biomedical applications.
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
reduce its toxicity, GdIII chelates have been developed. 3Among
these GdIII complexes, hydrophilic and low molecular weight
complexes such as Magnevist®, Dotarem®, Omniscan® have been
1. Introduction
Magnetic resonance imaging (MRI) has become one of the most
popular imaging techniques in the last few decades, with nearly 60
million MRI scans performed yearly around the world.1 This
technique’s popularity and routine use by clinicians for diagnosis
arises from the fact that it is a non-invasive imaging technique with
high spatial resolution.2 In order to enhance the signal and thus
improve the diagnosis accuracy, various MRI contrast agents (CAs)
have been developed. CAs containing paramagnetic ions such as
Gadolinium (Gd) are currently the most employed owing to the
ion’s unique magnetic properties3,4 and high coordination number.5
However, free gadolinium (Gd3+) ion is highly toxic. In order to
extensively used for clinical uses in the last decades. However, large
amount of those low molar masses Gd chelates must be injected as
they are rapidly cleared from the body6 thus allowing MRI
visualization for only short periods of time. To circumvent this
clearance shortcoming and also to significantly increase the
relaxivity properties as relaxivity enhancement time,7 CAs have
been linked to macromolecular structures such as polymer,8
dendrimer,9 carbon nanotube,10 fullerenes.11 As a consequence,
new functional Gd-based CAs were recently prepared to improve
the ligation between the macromolecular structure and the CA.
The development of such new functional Gd-based CA that can
be grafted on polymeric structures opens up a new era in MR
imaging. Indeed, polymeric based implants as used in gynecology or
hernia repair are not visible using conventional MR imaging
examination12,13 even though the presence of collagen coating on
some of them allow a transitional visualization of the implant.14
However, a long term vizualisation of the prosthesis is required
to obtain information concerning their post-operation fate and
fixation inside the body.15,16 Polymeric gadolinium macromolecular
contrast agents are one of the very promising contrast agents for
long term visualization7,17 even if there are not so many examples
a Institut des Biomolécules Max Mousseron (IBMM), UMR 5247, CNRS, Université
Montpellier, ENSCM, Bâtiment I, Faculté de Pharmacie, 15 avenue Charles Flahault,
BP14491, 34093 Montpellier cedex 5, France
E-mail: vincent.darcos@univ-montp1.fr
b Department of Chemistry, Faculty of Sciences, University of Balamand, Al_Kurah, P.O.
Box 100, Lebanon
c Micro et Nanomédecines Biomimétiques-MINT, INSERM UMR-S1066, Université
Angers, 4 rue Larrey, 49933 Angers Cedex 9, France
d Laboratory of Applied Chemistry, Faculty of Science III, Lebanese University, P.O.Box
826, Tripoli, Lebanon
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concerning the use of hydrophobic polymers. Among the first
FT-IR: FT-IR spectra were obtained on a Perkin Elmer spectrum
examples of the use of hydrophobic polymers are MRI-visible PCL 100 FT-IR spectrophotometer with an attenuated total reflectance
and MRI-visible PMMA obtained by post-polymerization (ATR) method.
modification.18,19 However, there are some disadvantages
LC-MS and MALDI analyses: A Q-TOF (Waters) spectrometer
concerning the synthesis of these polymeric contrast agents, such with an electrospray interface was used to obtain LC-MS spectra. An
as lack of control over the substitution degree of the polymer and Ultra-FlexIII spectrometer (Bruker) with dithranol as matrix was
the necessity to use several protection/deprotection steps on the used to obtain MALDI-TOF spectra. All solvents were HPLC grade.
DTPA ligand before the complexation with gadolinium. In order to
Preparative HPLC: HPLC separation was performed at SYNBIO3,
control the substitution degree of CA, in a second approach the Montpellier on HPLC (Waters) with HD-C18 column using HPLC
Huisgen click reaction had been used in the synthesis of MRI poly(ε- acetonitrile in 0.1% trifluoroacetic acid as eluent.
caprolactone). 20
Size Exclusion Chromatography: Size exclusion chromatography
In this paper, we aim at designing and characterizing Gd3+- (SEC) was performed at room temperature using
Viscotek
a
functionalized poly(methyl methacrylate) using copper catalyzed GPCmax system equipped with a Viscotek guard column (10×4.6
[3+2] cycloaddition (scheme 1). The synthesis of new azide mono- mm) and two Viscotek columns LT 5000L mixed medium (300×7.8
functionalized DTPA and its corresponding gadolinium(III) complex mm), with a Viscotek VE 3580 refractometric detector, and a
is first reported. Then, the click reaction of a gadolinium DTPA Viscotek VE 3210 UV/Vis detector. Calibration was established with
complex onto an alkyne-functionalized poly(methyl methacrylate) is polystyrene standards from Polymer Laboratories. THF was used as
described. The obtained macromolecular contrast agent is then solvent with a flow rate of 1 mL.min-1.
coated on a commercial polypropylene mesh using the airbrushing
ICP-MS Analyses: Quantification of Gd3+ and Cu+ was performed
technique.21 The effect of the amount of gadolinium present in the on an Element XR sector field ICP-MS (inductively coupled plasma)
contrast agent on the MRI signal will be assessed, in addition to with an indium enriched ultrapure solution as an internal standard.
stability and cytocompatibility studies.
The analyses were performed in the GeoSciences laboratory at the
University of Montpellier.
2.3 Synthesis
2.3.1 Synthesis of 3-azido-1-propylamine: 3-azido-1-
propylamine was synthesized according to a known procedure.22 3-
chloropropylamine hydrochloride (4 g, 30.8 mmol), sodium azide (6
g, 92.3 mmol), and water (30 mL) were added in the round bottom
flask. The mixture solution was heated overnight at 80˚C. After
cooling in an ice bath, KOH (4 g) and diethyl ether (50 mL) were
added. After separation of the organic phase, the aqueous phase
was further extracted with diethyl ether (3×50 mL). The organic
phase was then dried over magnesium sulfate and evaporated
under reduced pressure. Purification of the crude product by
distillation yielded 2.46 g of the pure product as coloress oil with a
1
yield of 80 %. H NMR (300MHz, CDCl3): δ(ppm)= 3.3 (t,2H,CH2N3);
2.8 (t,2H, CH2NH2), 1.6 (m,2H, CH2CH2CH2), 1.4 (s, NH2). FT-IR (ATR,
cm-1): 2100(N3)
Scheme 1: Synthetic pathway for the preparation of PMMA MRI
contrast agent 4
2.3.2 Synthesis of azido-functionalized DTPA (monoN3-
2. Experimental Section
DTPA): DTPA bis-anhydride (1.9 g, 5.3 mmol,
imidazole (3.04 g, 44.2 mmol) were solubilized in DMF at 80 ˚C. The
mixture was then cooled to 50 ˚C, and mixture of
1 equiv.) and
2.1 Chemicals and Materials
a
azidopropylamine (0.53g, 5.3mmol, 1equiv.) and pyridine (0.22 mL)
was added dropwise. The mixture was left at 50 ˚C for 5 hours, then
Dimethylfornamide (DMF, Aldrich) and toluene (Aldrich) were
dried over calcium hydride overnight and distilled under reduced precipitated in acetone, filtered and lyophilized. The
pressure. Pyridine (Aldrich) was dried over KOH and distilled.
Tetrahydrofuran (THF, Aldrich) was dried by refluxing over a separation (0.555 g, yield 30 %). 1H NMR (400MHz, D2O) δ ppm: 1.8
monofunctional DTPA (1) was purified by preparative HPLC
benzophenone-sodium mixture until a deep blue colour appeared
(2H, m, CH2CH2N3 ), 3.4 (12H, t, N(CH2)2N and CH2N3), 3.7(2H, s,
and distilled. Methyl methacrylate (MMA, Aldrich) was purified NCH2CO2H), 4 (8H, s, NCH2CO2H). FT-IR (ATR, cm-1): 2100 (N3). LC-
through a basic aluminum oxide column in order to remove the MS (ES+, m/z): 476.2 Da [M+H+]
inhibitor. AIBN was recrystallized in ethanol. All other materials
2.3.3 Synthesis of Gd3+ complex (2): Pyridine (2 mmol, 0.16
were obtained from Aldrich and were used without any further
mL) was added to azido-functionalized DTPA 1 (100 mg, 0.2 mmol)
dissolved in few mL of H2O and stirred for 10 minutes at room
temperature. GdCl3.6H2O (0.15g, 0.4 mmol) was then added, and
the mixture was left at 40 ˚C for 24 hours. Solvents were removed
under vacuum, and an aqueous solution of the complex was stirred
purification.
2.2 Characterization
NMR Spectroscopy: AMX300 Bruker spectrophotometer
operating at 300MHz was used to record the NMR spectra. AMX400
Bruker spectrophotometer was used to obtain the 1H and 13C
spectra of the azido functionalized DTPA ligand. Deuterated
chloroform and water were used as solvent, and chemical shifts
were expressed in ppm with respect to tetramethylsilane (TMS).
in Chelex resin 100 overnight to remove free Gd3+ 23
. The product
was then filtered, lyophilized, and obtained as white solid. The
absence of free gadolinium was verified by methyl thymol blue
(MTB) calorimetric test24 and the amount of complexed Gd3+was
2 | J. Name., 2012, 00, 1-3
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calculated by ICP-MS, revealing 10% Gd by weight (64 mg; yield: circuit of the NMR probe was
ARTICLE
a
35-mm diameter birdcage
50%). MALDI-TOF (dithranol, m/z): [M+K+]=685.8 Da versus 685.78 resonator. Meshes (around 1 cm2) were embedded in degassed 1%
calculated. FT-IR (ATR, cm-1): 2100 (N3).
by weight agar gel prior to imaging. Gadolinium-free samples
corresponded to a polypropylene mesh without Gd contrast agent.
To test signal enhancement, T1 weighting was introduced into the
MR images using an inversion pulse in rapid three-dimensional (3-D)
acquisition with relaxation enhancement (RARE) sequence (TR =
3000 ms; mean echo time (TEm) = 8 ms; RARE factor = 8; FOV = 3 ×
3 × 1.5 cm; matrix 128 × 128 × 64). Inversion time was set at
1100 ms, sufficient to allow canceling of the embedding gel.
2.3.4 Synthesis of propargyl methacrylate: Methacryloyl
chloride (13.95 g, 133 mmol) in 50 mL DCM was added dropwise at
0 ˚C during 30 minutes to propargyl alcohol (5 g, 89 mmol) and Et3N
(13.51 g, 133 mmol) in 50 mL DCM. The reaction was then left
overnight at room temperature. After removal of ammonium salts
by filtration over celite, the product was washed with 3×50mL of
saturated NaHCO3 and 3×50mL H2O until pH = 7. The product was
dried over MgSO4 and solvents were removed under reduced
2.5 Stability and Cytocompatibility Studies
pressure. The final product was obtained as colorless oil by vacuum
2.5.1 Film Formation: Films containing different w/w % of
1
distillation. (4g, yield=36%) H NMR (300 MHz, CDCl3) δ (ppm): 1.97 Gd3+ have been prepared by diluting the MRI-visible polymer with
(3H, s, CH3-C=CH2), 2.48(1H, s, C≡CH), 4.76(2H, s, OCH2), 5.63(1H, s, commercial PMMA and dissolving in DCM. After solvent
C=CHH), 6.18(1H,s,C=CHH).
2.3.5 Synthesis of poly(methyl methacrylate-co-propargyl
evaporation, films containing various amounts of Gd3+ were then
obtained.
2.5.2 Stability Studies: Films containing 0.10% and 0.43% by
weight of Gd3+ were placed in PBS solution at 37˚C under stirring at
130 rpm. At scheduled time points (1, 7, 30 and 90 days) 1 mL of
PBS buffer was withdrawn (then replaced by 1 mL fresh buffer) and
analyzed by ICP-MS for Gd3+ release study.
2.5.3: In Vitro Cytotoxicity Evaluation: Cytocompatibility
tests were performed on L-929 murine fibroblast cells. Cells were
cultured in MEM culture media by adding horse serum (10% by
volume), penicillin streptomycin and 1% GLUTAMAX. Cells were
grown in 5% CO2 incubator at 37 ˚C for around 24 hours till a
confluence of around 80%. At this point, the culture medium was
sucked out and thrown. Different films were then gently placed on
these cells. The films contained 0.23, 0.5 and 1.34% Gd3+ by weight.
Films of gadolinium-free poly(methyl methacrylate-co-propargyl
methacrylate) were also placed in addition to blank samples,
negative control (the cells with cell lysis) and positive control (the
cells). To each of the wells 1000 μL of LDH reaction mixture was
added and samples were incubated for 24 hours. Each sample was
repeated 3 times. After 24 hours, 10 μL of each sample were
transferred to UV wells to which 100 μL of LDH were
added.Sample was tested for UV absorbance at 490 nm by Victor V3
Perkin Elmer UV spectrophotometer.
methacrylate): Polymerization was carried out in toluene solution
using a standard Schlenk technique under inert atmosphere of
argon. Typically, methyl methacrylate (5 g, 49.9 mmol, 97.5 equiv.)
and propargyl methacrylate (0.158 g, 1.279 mmol, 2.5 equiv.), AIBN
(0.05 g, 0.3 mmol, 1% w/w), and toluene (20 mL) were placed in an
oven-dried Schlenk tube. The tube was fitted with a rubber septum.
The solution was further degassed by three freeze-thaw-pump
cycles. The resulting mixture was placed in a thermostatically
controlled oil bath at 70˚C for 2 hours. The reaction was stopped
with liquid nitrogen. The polymer (3) was precipitated in cold
methanol, collected by filtration, and dried in vacum. 1H NMR
(300MHz, CDCl3) δ(ppm): 3.5(s,3H, OCH3) and 4.2 (s,2H,CH2- C≡CH ).
Mn,SEC = 40000 g.mol-1, Ð=1.8.
2.3.6 Synthesis of MRI visible poly(methyl methacrylate):
In
a Schlenk tube were placed poly(methyl methacrylate-co-
propargyl methacrylate) (50 mg, 2.2% mol propargyl unit), Gd DTPA
complex (7 mg, 10 µmol, 1.2 equiv.), CuBr (1.5 mg, 10 µmol, 1.2
equiv.), and the least amount of DMF to solubilize the components.
The tube was fitted with a rubber septum. The solution was further
degassed by three freeze-pump-thaw cycles. The mixture was
stirred under argon, and PMDETA (3.7 mg, 22 µmol, 2.4 equiv.) was
added. The reaction was left at 40 ˚C for 48 hours. The crude
material was purified by dialysis (dialyzing tubing 6000-8000
MWCO) against acetone which was renewed regularly. After 3 Days,
acetone was evaporated to yield pure gadolinated PMMA (4) (40
mg, yield 80%).
3. Results and Discussion
Azido mono-functionalized diethylenetriaminepentaacetic acid
(compound 1) was synthesized in one step using DTPA
bis(anhydride) according to the procedure outlined in Scheme 1. In
order to synthesize this DTPA ligand, a versatile method is the
reaction of an amino precursor, namely the azidopropylamine, with
DTPA bis(anhydride). However, the reaction of DTPA bisanhydride
with amine groups usually yields a mixture of mono-amide and bis-
amide products,25,26,27 and the separation can be difficult and
fastidious depending on the type of amine used (Scheme 2).
2.4 MRI Visualization
2.4.1 Mesh Preparation for MRI Visualization: Meshes
were sprayed with PMMA MRI contrast agent having different Gd3+
percentages prepared as follows: the synthesized polymeric
contrast agent has been diluted by commercial PMMA and
dissolved in DCM to obtain 0.14, 0.23 and 0.79% w/w of Gd3+. On
commercial polypropylene mesh (3x3cm2) each polymeric solution
was sprayed using Infinity Airbrush system supplied by Harder &
Steenbeck (Osteinbeck, Germany) under a pressure of argon of 3
bars and at a distance of 5 cm. Meshes were dried overnight under
vacuum to obtain a constant weight.
Scheme2. Chemical structure of mono-amide DTPA and bis-amide-
DTPA
Moreover, the synthesis of bis-amide-DTPA is relatively easy
using an excess of amine without the need for tedious purification
steps. Indeed, many publications reported the synthesis in which an
2.4.2 MR Imaging protocol: MR imaging experiments were
performed on a Bruker Biospec 70/20 system operating at a
magnetic field of 7T (Bruker, Wissembourg, France). The resonant
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excess of amine is used to ensure the formation of bis-amide.28,29
MRI-visible poly(methyl methacrylate) (4) was obtained by
Perez-Baena et al. reported the synthesis of alkyne functionalized combination of free radical polymerization and Huisgen click
bis-amide DTPA in almost quantitative yield.30 Azido-functionalized chemistry.
First, poly(methyl methacrylate-co-propargyl
bis-amide DTPA ligands were also described20. Both ligands were methacrylate) with 2.5 % of propargyl methacrylate monomer units
used as MRI CAs and were grafted onto macromolecular was synthesized by free radical polymerization in toluene using
architectures via click chemistry reactions. To the best of our AIBN as initiator and without any protection of the alkyne bond
knowledge, only few publications are reporting on mono-amide (Table 1). Best reaction conditions were found by using 1% by
DTPA compounds. The main reason resides in the fact that these weight AIBN and a reaction time of 2 hours (entry 3). Experimental
compounds are usually obtained in low yields with multi-step molar mass (Mn,exp= 40000 g.mol-1) determined by SEC was in good
reactions. For example, a mono-maleimide derivative of DTPA agreement with the expected value. Indeed, the molar mass of the
bearing an amide link was prepared in a multi-steps reaction PMMA polymer has to be high enough to allow a perfect coating of
leading to a global low yield.31 Despite the challenging synthesis, materials. Copolymers having 2.3% propargyl units were obtained
mono-amide DTPA ligands are more interesting than bis-amide as shown by 1H NMR. Experimental molar ratios were in good
because the chelating power of DTPA in momo-amide DTPA is agreement with theoretical compositions, which is consistent with a
higher than in the corresponding bis-amide DTPA25.Another study good incorporation of propargyl methacrylate monomer during the
revealed that micelles containing gadolinium monoamide DTPA polymerization and a low loss of alkyne moiety. Nevertheless,
complexes showed higher relaxivity than micelles with bis-amide protective groups such as trialkylsilyl can be used to overcome this
DTPA complexes.32 In addition, the dissociation rate of linear problem. In our contribution, in order to limit the loss of alkyne
bisamide Gd-DTPA chelates is higher than in other chelates due to group during the free radical polymerization, polymerizations were
insufficient thermodynamic and kinetic stabilities.33 Moreover, carried at 70 °C for a short time period, typically 2 hours, for the
compared to DOTA ligand, DTPA ligand has been chosen because entry 3. As a consequence, conversion ratio of the polymerization
the chemistry to prepare functional ligand is probably more simple, remains low, below 50 %.
and DTPA is much cheaper than DOTA.
Therefore, the aim was to find a simple and versatile method to
Table1. Characteristics of the poly(methyl methacrylate-
synthesize azido mono-functionalized DTPA ligand under mild cpropargyl methacrylate) copolymers
conditions. DTPA ligand
1 was prepared in one step by
a
b
Entry
AIBN
Time
Conva
%
FPMA,exp
Mn,SEC
Ðb
condensation of the commercially available DTPA bis(anhydride)
with azidopropylamine in DMF at 80 °C in the presence of
imidazole. It was found that the optimal condition is an equimolar
ratio between DTPA bis(anhydride) and azidopropylamine.
Imidazole was used for its good promotion ability attributed to the
low melting point of imidazolium salt, and its ability to homogenize
the reaction mixture in dry media.34 The LC-MS spectrum of the
crude material revealed the presence of mono-functionalized DTPA
and di-functionalized DTPA (Figure S1). After preparative HPLC
separation, pure azido mono-functionalized DTPA 1 was obtained as
a white powder with a 30% yield. The LC-MS spectrum revealed the
presence of a unique peak at 476.2 Da [M+H+] as compared to
476.45 theoretical (Figure S2). The formation of the target
compound was further confirmed by 1H NMR (Figure S3) and FTIR.
FT-IR analysis revealed the presence of the azide group at 2100 cm-
%wt
hours
g.mol-1
1
2
2
3
0.5
0.5
1
2
4
2
4
30
50
40
52
1.8
1.6
2.3
2.0
45000
54000
40000
33000
1.6
2
1.8
2
1
a Determined by 1H NMR. bDetermined by size exclusion chromatography in
THF
1
.
To prepare MRI-visible poly(methyl methacrylate) polymer 4,
alkyne-functionalized PMMA 3 was coupled with azido mono-
The functionalized ligand
1 was then complexed with
functionalized Gd-DTPA
2 by copper-catalyzed azide-alkyne
GdCl3.6H2O resulting in a gadolinium complex 2 with a quantitative
yield. Purification on Chelex 100 resin was used to efficiently
remove the free non complexed Gd3+. The absence of free Gd3+ was
cycloaddition (CuAAC). Click reaction was performed in DMF at 40
°C using a CuBr/PMDETA complex as catalyst. The reaction was
stopped after 48 hours, and the macromolecular CA was then
purified by dialysis against acetone using dialysis membranes with
MWCO 6000-8000. The amount of Gd3+ in the polymeric CA was
then measured by ICP-MS analysis as presented in Table 2. The
calculated weight percentage of Gd3+ was 2.25, while the
experimental weight percentage was 1.34. The difference between
the calculated and the experimental values is due to the low
efficiency of the click reaction, i.e. 60 %. However, it is well-know
that the efficiency of Huisgen click reaction is not quantitative in
macromolecular chemistry due to the steric hindrance, the poor
solubility of the reactant, etc… Nevertheless, the amount of Gd3+
into the macromolecular contrast agent is high enough for the next
step, the coating of polypropylene mesh. ICP-MS measurements
showed the copper amount in the macromolecular CA is below 1
ppm.
confirmed by
a methyl thymol blue test (MTB) using UV
spectroscopy. MALDI-TOF analysis using dithranol as a matrix
revealed one peak at [M+K+] = 685.8 Da as compared to 685.78
theoretical. The complexation ratio of Gd3+ in 1 was determined by
ICP-MS and was found to be 40 %. Further experiments were done
in order to improve the complexation ratio. Unfortunately, the
complexation ratio was not improved as it is not judicious to
increase the temperature due to the thermo-labile azide groups. In
fact, in most cases high temperature and long reaction time were
used to increase the complexation ratio. Moreover, it is well-known
that commercial DTPA CAs like Magnevist® contained non
complexed ligands. Finally it is interesting to have an excess of
ligand to reduce the proportion of the uncomplexed toxic free
gadolinium.
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imaging. Circular films were prepared with the PMMA contrast
agent containing 0.1 and 0.43% by weight of Gd3+ and placed in PBS
buffer at 37˚C up to 90 days. The amount of released Gd3+ was
measured by ICP-MS analysis. Films containing 0.1% Gd3+ w/w
showed no detectable release of Gd3+ in PBS over a period of 90
days. Films containing 0.43% Gd3+ by weight (79 μg of Gd3+) showed
a release of 0.4% Gd3+ (0.231 μg Gd3+) throughout the 90 days. In
both cases this represents a very low release of Gd3+ showing the
stability of the CA during this period of time. Moreover, µgram scale
of gadolinium use can be compare to gram scale of gadolinium use
for conventional MR scan where patients receive 1.0−2.5 g Gd3+ as a
bolus (posology of 0.1−0.2 mmol/kg of contrast agent for an
intravenous injection of Magnevist).
Table 2. Characteristics of the macromolecular contrast agent
Gd content
wt%
Gd contenta
wt %
Reaction efficiency yield
%
%
calculated
experimental
2.25
1.34
60
80
a Determined by ICP-MS
MRI-PMMA CAs with different amounts of Gd3+ (0.23, 0.5, and
1.34% Gd3+) were placed in contact with murine fibroblast cells in
order to determine their cytocompatibility.. Figure 2 showed that
MRI-PMMAs with 0.23 and 0.5% Gd3+ were cytocompatible and did
not have any effect on cell growth after 24 hours whereas those
containing 1.34% Gd3+ were detrimental for the cells. The LDH
direct contact cytotoxicity test used to assess the cytocompatibility
works as follows: LDH or lactate dehydrogenase is an enzyme
liberated in culture media once cells are dead. The presence of LDH
reduces tetrazolium salt in LDH mixture to formazan dye having a
red color and a maximum absorbance at 490 nm. Thus, the
presence of dead cells is indicated by both a red color and high UV
absorbance at this wavelength. This had been the case with the
TCPS negative control which contained the cell lysis, and in the case
of the direct contact of PMMA MRI films containing 1.34% Gd3+ as
revealed in Figure 2. The graph also reveals that MRI PMMA films
containing 0.23 and 0.5% Gd3+(w/w) had almost no UV absorbance,
and thus no cytotoxicity was observed. The result is not surprising
because a study performed by Porsio et al.36 revealed that MRI
visible PCL nanoparticles with 1% Gd3+(w/w) caused lower cell
proliferation as compared to the same nanoparticles with lower
Gd3+ amounts. Hence, polymeric contrast agents with the least
amounts of Gd3+ are recommended in order to assure
cytocompatibility by keeping a good MRI visibility.
MRI-visible PMMA CA was then sprayed onto commercial
polypropylene mesh using the airbrushing technique. The
airbrushing is a versatile method allowing the preparation of
homogenous and regular film without altering the mesh shape and
mesh mechanical properties.19 Moreover, it was found that Gd-
DTPA-PMMA using a PMMA with a molar mass of 40000 g.mol-1 had
suitable filmogenic properties on polypropylene mesh.
The effect of the proportion of Gd3+ in the polymeric CA on MRI
visualization is shown on Figure 1. Polypropylene meshes covered
with CA were visualized using 7T MRI. In a first step, a 7T MRI
scanner was used for preliminary studies. Then, further
experiments will be done with a clinical scanner (1.5T or 3T). In the
absence of CA, the polypropylene mesh was not visible. The mesh
turned out to be visible by spraying it with the polymeric CA. For
this, polypropylene meshes were coated with PMMA samples at
different Gd3+ concentrations of 0.14 and 0.24 % by weight. The
amounts of Gd3+ present on each mesh were 1.4 and 2.3 μg per mg
of polymeric contrast agent sprayed on the mesh. The amounts
were determined depending on the amount of contrast agent
placed on the mesh usually around 10 mg. Regardless the amount
of Gd3+, meshes turned out to be visible even with concentrations
of Gd3+ as low as 1.4 μg per mg of polymeric contrast agent,
corresponding to 0.14% by weight Gd3+. The presence of 0.14% by
weight of Gd3+ in the polymeric CA was sufficient to make the mesh
visible. However, with the increase of Gd3+ content no signal
enhancement or magnetic susceptibility effect were observed for
MRI visualization.
Figure 2. Effect of amount of Gd3+ present in film on UV absorbance
Figure 1: 7T MR images of polypropylene meshes coated with
polymeric contrast agents containing (A) blank (B) 0.14%, and (C)
0.23% by weigh Gd3+
4. Conclusion
Gd-complexes are gaining more importance because of
nephrogenic systemic fibrosis (NSF). NSF mainly occurs in patients
with severe renal failure. These patients take longer time to clear
This work described the synthesis of a new azido mono-
functionalized DTPA ligand using a simple pathway for long term
visualization of implanted meshes. The complexation of this DTPA
ligand with Gd3+ lead to the synthesis of Gd-DTPA contrast agent.
Macromolecular contrast agents based on poly(methyl
methacrylate) were prepared by click chemistry of the Gd-DTPA
ligand bearing an azide group onto alkyne-functionalized PMMA.
This new MRI-visible polymer was characterized by ICP-MS analysis
Gd-complexes, which increases the risk of transmetallation
35
between Gd3+ and Zn2+ thus releasing free Gd3+ For this, stable
.
contrast agents are a necessity to eliminate any risks associated
with free Gd3+ and prevent the precipitation of gadolinium salts.
In vitro stability studies were carried out in PBS buffer to detect
any release of free toxic gadolinium and to evaluate long-term MR
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
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ARTICLE
Journal Name
20 S. El Habnouni, B. Nottelet, V. Darcos, B. Porsio, L. Lemaire,
F. Franconi, X. Garric and J. Coudane, Biomacromolecules,
2013, 14, 3626-3634.
21 A. Behrens, B. Casey, M. Sikorski, K. Wu, W. Tutak, A. Sandler
and P. Kofinas, ACS Macro Lett., 2014, 3, 249-254.
in order to determine the amount of Gadolinium. The results
revealed that an increase in the amount of Gd3+ did not improve
visualization of the mesh. Thus amounts of Gd3+ as low as 0.1% by
weight rendered polypropylene meshes visible on a 7T MRI. PMMA
MRI contrast agents with amounts of Gd3+ as low as 0.1% by weight
were also cytocompatible and stable. This allows their long-term
use in the body for biomedical applications after further in vivo
stability studies.
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25 M. Ardestani, A. Arabzadeh, Z. Heidari, A. Hosseinzadeh, H.
Ebrahimi, E. Hashemi, M. Mosayebnia, M. Shafiee-Alavidjeh,
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The authors thank the Balamand Internal Research Grant (BIRG)
and the SATT AxLR for funding. The authors also thank Synbio3
platform (IBMM, Montpellier) for the preparative HPLC
experiments and PRIMEx-IRM platform (Angers University) for the
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