Online Appendix for the following JACC article
TITLE: In Vivo Characterization of a New Abdominal Aortic Aneurysm Mouse Model
With Conventional and Molecular Magnetic Resonance Imaging
AUTHORS: Ahmed Klink, Joeri Heynens, Beatriz Herranz, Mark E. Lobatto, Teresa
Arias, Honorius M.H.F. Sanders, Gustav J. Strijkers, Maarten Merkx, Klaas Nicolay,
Valentin Fuster, Alain Tedgui, Ziad Mallat, Willem J.M. Mulder, Zahi A. Fayad
APPENDIX
Supplementary Methods
Synthesis of collagen-targeted multimodal nanoparticles
The paramagnetic micelles were prepared by the lipid film hydration method as
previously described1. Briefly, a lipid film was formed by rotary evaporation of GdDTPA-distearyl amide, DSPE-PEG2000, DSPE-PEG2000-maleimide, and Rhodamine-PE
or Cy5.5-PEG-DSPE-lipid in a molar ratio 50:39:10:1, dissolved in a mixture of
chloroform/methanol (4:1 v/v). After rotary evaporation to dryness, the lipid film was
hydrated with HEPES buffered saline (HBS, pH 6.7), containing 20 mM HEPES
(Sigma) and 135 mM NaCl (Sigma). The solution was slowly rotated at 65o C for 1 hour,
which resulted in a clear solution, indicative of micelle formation. CNA-35 was then
coupled to the micelles by a sulfhydrylmaleimide coupling method at a molar ratio of
1:50 (CNA-35: lipids). CNA-35 was reacted with N-succinimidyl S-acetylthioacetate
(SATA) at a molar ratio of 1:10 for 1 hour in a NaHCO3 buffer at pH 8.0. Uncoupled
SATA was removed using a centrifuge concentrator with a Molecular Weight Cut-off of
10 kDa (Sartorius). Subsequently, the thioacetate group is converted to a free thiol group
by deacetylation with a hydroxylamine solution (0.5M hydroxyl amine, 1M HEPES, 32
mM EDTA, pH 7.0) for 1 hour. Modified CNA-35 was then allowed to react with preformed micelles at 4 ºC overnight in HBS (pH 6.7). The maleimide moiety reacts with
free thiol groups of the protein to form a stable carbon-sulfur bond. Uncoupled CNA-35
was separated from CNA-35-functionalized micelles using centrifuge concentrators with
a MWCO of 100 kDa (Sartorius), during the process the pH was increased to 7.4.
Mutant-CNA-35 nanoparticles consisting micelles conjugated to a ‘nonbinding’ mutant
CNA-35 protein were synthesized following the same procedure.
Characterization and in-vitro binding experiment
The mean hydrodynamic diameter of the particles was determined by dynamic light
scattering, using a ZetaPALS (Brookhaven Instruments Corporation). The lipid
composition of the final particles was determined by phosphate determination according
to Rouser, and the amount of proteins was determined using a modified Lowry method.
In this method, equal lipid concentration in both the calibration curve and actual samples
were used, ensuring the same background signal of lipids.
1
H relaxivity measurements were performed to verify the expected relaxivity of the
micelles. The longitudinal relaxation time (T1) of a concentration series was determined
at 60 MHz and 37oC on a Bruker Minispec MQ60 (Bruker, Ettingen, Germany). The
relaxivity (r1) of the micelles was then calculated.
In order to check the binding of CNA-35 micelles to collagen, a fluorescence binding
experiment was performed. Wells of 8-well strip plate were incubated overnight at 4° C
with 50 μL of 55 μg/mL rat-tail collagen type I (C7661, Sigma-Aldrich) in HBS. Next,
wells were rinsed 4 times with 300 μL HBS. Wells were then blocked with 250 μL of 5%
(w/v) milk powder in HBS for 3 h at 20° C, aspirated and again rinsed 3 times with 300
μL HBS. 50 μL of a solution (concentration: 1 mM lipid) of targeted CNA micelles,
mutant CNA micelles and untargeted micelles in HBS was added to each well and
incubated for 3 h at 20 ° C. Wells were aspirated and washed 10 times with 300 μL HBS.
The fluorescence intensities of Rhodamine were measured using a BioTek Synergy 2
microplate reader with Gen5TM software using a 540±20 nm excitation filter and a
590±20 nm emission filter. In the case of Cy5.5 micelles, an IVIS Imaging System 200
(Xenogen, Alameda, CA) was used to measure the fluorescence intensities of Cy5.5
using a 615–665 nm excitation filter and a 695–770 nm emission filter.
Ex vivo near-infrared fluorescence imaging of AAA
AAAs were induced in C57BL/6 mice according to the model described above. After
aneurismal development, the animals were injected with Cy5.5 labeled CNA-35 micelles
in order to visualize the spatial distribution of the nanoparticles throughout the
aneurismal region. Animals with AAA that were not injected with micelles as well as
healthy animals injected with Cy5.5 CNA-35 micelles served as controls. The animals
were sacrificed 32 hours post injection and the aortas excised were imaged with the IVIS200 optical imaging system (Xenogen, Alameda, CA, USA) using a 615-667nm
excitation filter and a 695-770nm emission filter. The photon count was quantified and
compared between the two groups.
MR imaging parameters for multi-contrast high-resolution in vivo MRI
Repetition time/echo time [TR/TE] 800ms/8.6ms, 2500ms/30ms, 2500ms/10ms for T1Weighted (T1W), T2-Weighted (T2W) and Proton Density-Weighted (PDW)
respectively; field of view 3.0 cm and 2.5 cm respectively for T1W and T2W/PDW;
matrix size 256x256 and 192x256 respectively for T1W and T2W/PDW; voxel resolution
117μm and 130x97μm respectively for T1W and T2W/PDW; n=6 and n=8 excitations
respectively for T1W and T2W/PDW; slice thickness 1mm for all the different
sequences.
Supplementary Figure 1
Figure 1 – (A) Illustration of CNA-35 or mutant CNA-35 micelles. Micelles are composed of
gadolinium-DTPA (Gd-DTPA) and fluorescent lipids (rhodamine B or Cy5.5). Targeting
proteins CNA-35 or the non-binding mutant CNA-35 are coupled to PEG-DSPE chains. (B)
Results of the in vitro binding experiment performed with Gd-DTPA and rhodamine labeled
CNA-35 micelles. CNA-35 micelles revealed a strong binding to collagen after extensive
washing, while minor binding occurred for mutant CNA-35 or unconjugated micelles. (C)
Results of the in vitro experiment performed with Gd-DTPA and Cy5.5 labeled CNA-35
micelles. Similarly to rhodamine labeled micelles, CNA-35 micelles showed a strong binding to
collagen compared to minor binding for mutant CNA-35 and unconjugated micelles.
Supplementary Figure 2
Figure 2 - Near infrared images of Ex-vivo aortas injected with Gd-DTPA/Cy5.5 CNA-35
micelles in healthy and aneurismal aortas. (A) A non-injected aneurismal aorta was used as a
control and showed no near infrared fluorescent signal. (B) Aneurismal aorta injected with GdDTPA/Cy5.5 CNA-35 micelles. Cy5.5 labeled CNA-35 micelles were clearly appreciated in the
AAA region compared to both the control aorta (A) and healthy animals injected with Cy5.5
CNA-35 micelles (C).
Supplementary Figure 3
Figure 3 – Representative images obtained pre and post injection of CNA-35 micelles in healthy
animals. No significant signal enhancement was observed.
Supplementary Figure 4
Figure 4 – Diagram representing the region of interest traced around the aneurysms – excluding
the lumen – in order to quantify the normalized signal enhancement percentage after the injection
of CNA-35 micelles.
Supplementary Figure 5
Figure 5 – Images from the 3 separate fluorescent channels of a representative AAA section
injected with Gd-DTPA/rhodamine CNA-35 micelles, shown on a fluorescent confocal
microscope. The section was stained with DAPI (A) and collagen-I (B). The fluorescent signal
originating from the micelles is shown on the rhodamine channel (C). The overlay of the 3
channels (D) shows the precise colocalization of CNA-35 micelles and collagen-I therefore
proving the specificity of the micelles for collagen.
Supplementary Figure 6
Figure 6 - Images from the 3 separate fluorescent channels of a representative AAA section
injected with Gd-DTPA/rhodamine mutant CNA-35 micelles, shown on a fluorescent confocal
microscope. The section was stained with DAPI (A) and collagen-I (B). The fluorescent signal
originating from the mutant CNA-35 micelles is shown on the rhodamine channel (C) and proves
to be minor compared to CNA-35 micelles. (D) The overlay of the 3 channels showcases the
absence of rhodamine signal in areas of collagen-I staining.
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Nicolay K, Griffioen AW. Quantum dots with a paramagnetic coating as a bimodal molecular
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