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Experimental supplementary information

Synthesis of modified contrast agent

The starting material, named P717 in the manuscript, was provided by Guerbet Company. P717 is a carboxymethylated dextran substituted with a chelating moiety 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) via a diaminoethyl linker and filled with a gadolinium (III) ion. The average degrees of substitution (DS) in the different chemical groups are: DS(carboxymethyl) = 0.32 ± 0.02 (1.14 ± 0.05 meq COO-/g), DS(GdDOTA) = 0.23 (%N = 4.43 ± 0.30, %Gd = 8.0 ± 0.4 – w/w). The molecular weights determined by SEC-MALLS (see § Physicochemical characterizations) are 44 700 ± 2 200 g/mol (average number weight) and 74 000 ± 3 700 g/mol (average weight weight) with a hydrodynamic radius (Rh) of 6.0 ± 0.3 nm).

Others chemicals were purchased from Sigma-Aldrich, France.

Periodate oxidation was performed on P717 to create aldehyde function on the polysaccharidic chain. Aldehyde reacted with amino groups to form a Schiff’s base which was further reduced into an amine. Briefly, one gram of P717 (20 µmol) was incubated with one gram of NaIO4 (47.10-4 mol) in 50 mM acetate buffer pH = 5.5, overnight in the dark and at room temperature. The final solution was neutralized and dialyzed (MWCO = 1 000 Da) against bidistilled water. The solid product was obtained after freeze-drying as a fluffy powder in 70% yield.

Oxidized P717, Pox, was then conjugated with trityrosine. 50 mg of Pox was incubated with 25 mg of Tyr-Tyr-Tyr in a phosphate buffer 0.15 M (pH = 7.4). pH was carefully monitored during the 3 h of the reaction at 60°C and kept between 6 and 7.4. The solution was cooled down to room temperature and 248 mg of NaBH3CN (4 mmol) were added. The reaction was prolonged for 3 h at 60°C. The final solution was dialyzed against 1 M NaCl and water and freeze-dried to get PTriTyr in 90 % yield.

PoxR was obtained from the oxidation of P717 followed with the reduction in the same conditions as PTriTyr without the addition of Tyr-Tyr-Tyr.

Synthesis of compounds for NMR studies

Gadolinium free P717 was provided by Guerbet Company. The chelation with lanthane (III) ions was carried out by the addition of 1 La(III) (LaCl3, 7 H2O) per DOTA on the dextran backbone. After adjusting the pH to 6.5 with NaOH, the solution was maintained at room temperature overnight followed by heating at 60°C for 4h more. Chloride ions were removed by dialysis and a white product was obtained after freeze-drying. This product named P717(La) underwent the same chemical modifications as P717 leading to PoxR(La) and PTriTyr(La) devoid of paramagnetic properties.

NMR studies

For nuclear magnetic resonance spectroscopy analysis (400.16 MHz 1H NMR), 30-50 mg of dried samples were dissolved in 1 mL D2O (99.8%), lyophilized using a rotary concentrator and exchanged several times with 0.5 mL 99.8% D2O before final dissolution in 0.7 mL 99.99% D2O. One dimensional (1D) 1H NMR spectra of solutions were recorded at 333 K on a Bruker

Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2011

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ADVANCE III 400 spectrometer (Bruker, Germany), equipped with a 5 mm diameter tunable probe, with BRUKER software. Chemical shifts are given relative to HOD. Spectra were reprocessed for presentation using the software Topspin (Bruker, Germany).

Biological compounds

LDL and HDL were isolated from healthy volunteers’ plasma sampled on lithium heparin (Vacuntainer®, BD, France) by ultracentrifugation. In brief, plasma density was adjusted to 1.35 with KBr and overlaid with KBr solutions (d = 1.063 and d = 1.21). Ultracentrifugation was performed at 100 000 g for 20 h at 10°C. The upper lipoprotein fraction containing LDL was adjusted to a density of 1.25 with KBr and then overlaid with a KBr solution (d = 1.006) before another ultracentrifugation at 100 000 g for 20 h at 10°C. The LDL fraction was recovered as a single band, and was then extensively rinsed with saline-EDTA and concentrated using a centrifugation concentrating device (Cut-off 5 kDa, Vivascience, Stonehouse, UK). The density of the bottom fraction resulting from the first ultracentrifugation and containing HDL was adjusted to 1.25 with KBr and overlaid with KBr saline solution (d = 1.21). The second ultracentrifugation and subsequent washing steps were similar to those for LDL, the HDL fractions representing the top layer of the tube. All fractions were desalted either by dialysis against PBS or by centrifugation and three washes with PBS, and kept under nitrogen.

Physical and chemistry characterizations:

Molecular weight and Rh: The weight average molecular weight (Mw) and the number average molecular weight (Mn) were determined by size-exclusion chromatography (SEC) coupled on-line with multi angles laser light scattering (MALLS) and viscometry.

SEC was performed on a TSK gel G3000 PWXL (Interchim Sa, France) with LC10Ai pump (Shimadzu, France) and PBS, LiNO3 150 mM pH = 7.4 as the eluent at a flow rate of 0.5 ml/min at 25°C. The MALLS apparatus was the EOS (18 detectors) from Wyatt Technology (Ca, USA) filled with a K5 cell and a Ga–As laser (λ = 690 nm). The other on-line detectors were a viscometer (Vicostar II, Wyatt Technology Corp. Santa Barbara, Ca., USA) and a differential refractometer RID 10 A (Shimadzu, France). dn/dC of 0.15 mL.g-1 was used for all samples. Data have been exploited thanks to the Astra 5.3.4.14 software (Zimm order 1 with angles between 52° and 142°).

Gadolinium content and relaxivity: The free Gd(III) and the free La(III) contents were measured by the Arzenazo III method at pH = 8.5. The total Gd content was measured by inductively coupled plasma atomic spectrometry (ICP-AES) on a Beckman Spectraspan VII instrument (Beckman, Germany).

The longitudinal relaxivity (r1) was determined, on a clinical 1.5 T MR Imager (GE, France) from a linear fit of Gd concentration (mM Gd) vs. 1/T1 (1/T1 = R1, s–1), as described by

R1= r1x + R1m where R1 is the relaxation rate of the contrast agent containing Gd (1/s), x is the concentration of Gd present (in mM Gd), r1 is the slope of the linear correlation (equal to the relaxivity in s–1mM–1), and R1m is the relaxation rate of the matrix. Coupling :

The amount of tri-tyrosine residues (TriTyr) was determined from UV spectra (UVmc2 SAFAS, Monaco) with a calibration curve at 274 nm.


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Infrared spectrometry:

IR was performed on a FT-IR Avatar (Thermo Nicolet, France) with KBr pellets.

The green circle depicts the vibration from C=C stretching coming from the aromatic ring of trityrosine residues.

Figure S1: IR spectra of PoxR and PTriTyr on KBr pellets.

LogP determination:

LogP of PTriTyr (= Log([PTriTyr]oct/[PTriTyr]water) was obtained by the shake-flask method. Briefly, water and octanol were premixed in 50/50 V/V and equilibrium was reached before experiment. Then a known amount of PTriTyr was dissolved in the octanol/water mixture which was vigorously shaken and then allowed to diphase. PTriTyr concentration was measured by UV spectrophotometry at 274 nm (n=3).


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In vitro affinity

Surface Plasmon resonance measurements were performed using a BIAcore® X100 instrument (GE Healthcare Bio-sciences AB, Uppsala, Sweden). The running buffer for lipoprotein immobilization was 145 mM NaCl, 5 mM EDTA, 10 mM HEPES, pH = 7.4. Lipoproteins were diluted to 20-50 μg/ml in 10 mM sodium acetate buffer, pH = 5 and immobilized onto a carboxymethylated dextran surface of a CM5 sensor chip (GE Healthcare Bio-sciences AB, Uppsala, Sweden) using the amine coupling chemistry (BIAcore AB amine coupling kit) according to manufacturer’s instruction.

Binding of polysaccharides was measured over 15 000 RU of immobilized LDL and 7 800 RU of immobilized HDL at a flow-rate of 10 μL/min. Regeneration of the surfaces was achieved by injection of 15 μL of NaOH 25 mM and 30 μL of NaCl 5 M.

Sensorgrams were analyzed using the BIAevaluation software (GE Healthcare Bio-sciences AB, Uppsala, Sweden). For each curve, blank flow cell was subtracted for non specific affinity before evaluation, see the following figure. Sensorgrams are representative of at least 3 independent experiments.

The association and dissociation constants were determined by steady-state affinity with Prism 5® software. Different non linear regressions were evaluated. The one site-specific binding with Hill slope gave the best results. This model takes into account the heterogeneity of both lipoproteins and polysaccharides and also the multiple binding sites and the cooperative interaction.


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Figure S2: BIAcore® sensorgrams. Association and dissociation curves of contrast agents, PoxR or PTriTyr, with lipoproteins, LDL or HDL, at different concentrations (0.025mg/ml to 5.0 mg/ml)

In vivo experiment and MRI evaluation

The procedure and the animal care complied with the “Principles of animal care” formulated by the European Union. Apoliprotein E KO (ApoE-/-) mice (Charles River, France) were fed a western diet with 21% fat and 0.15% cholesterol (SAFE, France) 6 months prior to the experiment. As a control wild-type (WT) mice (Charles River, France) were used on a control diet (SAFE, France). 7 ApoE -/- mice were injected with PTriTyr, 3 ApoE-/- mice were injected with PoxR and 3 WT mice were injected with PTriTyr.

Mice were injected in the retro orbital sinus. They were scanned with a 4.7 T small animal MRI (Bruker, Germany) before and after injection using a dedicated coil. Whole body scout images (coronal section) were recorded before and after injection to estimate the distribution of the contrast agents (figure S3).

Figure S3 : Coronal scout section (low resolution) before and 5 hours after the injection of the contrast agent. In post injection picture, contrast in the bladder is seen resulting for a renal excretion pathway.

For black-blood high resolution imaging of the aortic vessel wall, a RARE T1 sequence with cardiac gating (H. Alsaid, Invest Radiol, 2009) was used with the following parameter: minimum TR = 540.8 ms, TE = 10 ms, FOV= 2.5 cm and a matrix size of 256 x 256. Continuous slices (slice thickness = 0.8 cm) were made under the renal aorta bifurcation. The figure S4 presents a transversal section with the area focusing on the arterial wall.


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Figure S4 : Transversal section with the selected area focusing on the arterial wall. Red arrows indicate kidney enhancement.

Kinetics of arterial wall uptake were performed with PTriTyr. Mice were imaged every hour for the first eight hours after the injection, then at 24h, 25h and 48h after the injection (figure S5).

Figure S5 : Follow-up of contrast to noise ratio of PTriTyr contrast agent. Best contrast occurs 5 hours after the injection. No enhancement was seen after 8 hours.

At each time point, the slices were matched to the baseline pre-contrast scan by using the unique vertebral and paraspinous muscular anatomy as landmarks. Five slices per study were chosen and

CNR kinetics

0 5 10 15 20 25 30 35 40 45 50

0.5

1.0

1.5

PTriTyr

Time (h)

CNR


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the signal intensity of the atherosclerotic plaque and the paraspinous muscle were measured using ImageJ (Image J NIH, USA). Since the plaque was heterogeneous, only the enhancing areas of the aortic wall were used to quantify the signal. The standard deviation (SD) of noise from the background was also obtained.

For each slice the percent enhancement (% ENH) relative to the muscle, was calculated (according to the equation below) in order to evaluate the enhancement of the aortic wall.

%ENH=(CNRpost/CNRpre-1)x100 where CNR is the Contrast to Noise Ratio, defined as the ratio of the signal intensity measured in the arterial wall to the signal intensity in the muscle.


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