ONLINE SUPPLEMENT TO:
Collagen-specific peptide conjugated high-density lipoprotein nanoparticles as MR
contrast agents to evaluate compositional changes in murine atherosclerotic plaques
under regression conditions.
Wei Chen1, David P. Cormode1, Yuliya Vengrenyuk2, Beatriz Herranz1,3, Jonathan E
Feig2, Ahmed Klink1,4, Willem J. M. Mulder1,5, Edward A. Fisher2*, and Zahi A. Fayad1*
1. Translational and Molecular Imaging Institute, Departments of Radiology and
Medicine, Mount Sinai School of Medicine, New York, New York USA.
2. Department of Medicine, Leon H. Charney Division of Cardiology and the Marc
and Ruti Bell Program in Vascular Biology, New York University School of
Medicine, New York, New York, USA.
3. Department of Epidemiology, Atherothrombosis and Imaging. Fundacion Centro
Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), 28029 Madrid,
Spain
4. Paris Cardiovascular Research Center, INSERM Assistance Publique-Hopitaux de
Paris, Hopital Europeen Georges Pompidou, Paris, France
5. Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New
York, New York, USA.
* Corresponding Authors:
Zahi A. Fayad, PhD, FAHA, FACC
Mount Sinai School of Medicine
Translational and Molecular Imaging Institute
One Gustave L. Levy Place, New York, NY 10029, USA
Tel: 212-241-6858
Fax: 212-534-2683
Email: zahi.fayad@mssm.edu
Edward A. Fisher, MD, PhD
New York University School of Medicine, Smilow 8
522 First Ave. New York, NY 10016
Tel: 212-263-6631
1
Fax: 212-263-6632
Email: edward.fisher@nyumc.org
Methods
Materials
1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl -sn-glycero-3phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rhodamine-PE), 1,2distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene
glycol)-2000]
ammonium salt (DSPE-PEG-COOH) were purchased from Avanti Polar Lipids
(Alabaster, AL). Gadolinium diethylenetriaminepentaacetate-bis(stearylamide) (GdDTPA-BSA) was synthesized by IQSynthesis (St. Louis, MO). 1-ethyl-3-[3dimethylaminopropyl]carbodiimide hydrochloride (EDC) and N-Hydroxysuccinimide
(NHS) were obtained from Fisher Scientific. The collagen-specific peptide EP-3533 with
the sequence GKWH[CTTKFPHHYC]LYBip-CONH2, and the non-specific peptide EP3612 containing non-natural D-Cys as shown in Figure 1 were synthesized by Peptide
International. All other chemicals were of analytical grade or the best grade available.
Preparation of HDL nanoparticles: Gd-DTPA-BSA, Rhodamine-PE, DPPC, and DSPEPEG-COOH were dissolved in chloroform/ethanol (3:1 mol/mol, chloroform: ethanol),
then dried initially under airflow at room temperature, then under vacuum overnight. The
dried lipid film was dissolved in a sodium cholate buffer (0.15 M NaCl, 1 mmol EDTA,
pH 7.5) at 55 °C so that the lipid film was fully hydrated. Apolipoprotein A-I (apo A-I,
purified from human HDL) solution in PBS was then added to the cooled lipid solution
on ice. The molar ratio was 1:120:25:2.5:2:200 for apoAI:DPPC:Gd-DPTA-BSA:DSPEPEG-COOH:Rhodamine-PE:sodium cholate. After incubation at 0-5°C in an ice water
bath for 2 h, the mixture was dialyzed against buffer (0.15 M NaCl, 1 mmol EDTA, pH
7.5) to remove the sodium cholate at 4°C. The dialysate was then filtered through 0.22
concentrated and the
buffer exchanged to PBS with a Vivaspin 6 Centrifugal Filter Device (membrane cutoff
range: 10,000 kDa; Sartorius Corporation, Edgewood, NY).
The mean size of the different nanoparticles was determined by photon correlation
spectroscopy performed with a BIC ZetaPALS (Brookhaven Instruments Corporation,
New York, US). All samples were analyzed at 25 °C in filtered water (30 nm size cut-off).
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Preparation of EP3533-HDL and EP3612-HDL nanoparticles: The conjugation of EP3533 peptides to HDL nanoparticles is schematically illustrated in Figure 1. First, the
HDL nanoparticles were transferred into HEPES buffer (pH=5.0) and diluted to 1 mg/ml
apo A-I, into which 10 mg/ml EDC and 10 mg/ml NHS in HEPES (pH=5.0) was added
in a 1:5:10 DSPE-PEG-COOH:EDC:NHS molar ratio. After incubation at room
temperature for 2 h, the reaction solution was thoroughly washed with a HEPES buffer
(pH=5.0) using a Vivaspin 6 Centrifugal Filter Device (membrane cutoff range: 10,000
kDa). Then the pH of the nanoparticle solution was adjusted to 7.0-8.0, to which 1 mg/ml
EP-3533 peptides in dimethyl sulfoxide was added in a 1:1.1 DSPE-PEG-COOH:EP3533 molar ratio. This preparation was kept at room temperature for 4 h, stored at 4 °C
overnight, and then purified using a Vivaspin 6 Centrifugal Filter Device (membrane
cutoff range: 10,000 kDa) by exchanging the HEPES buffer to PBS. The final
nanoparticles in solution were named EP3533-HDL.
Non-specific control nanoparticles (EP3612-HDL) were prepared by the same procedure
using non-specific control EP-3612 peptides.
Gadolinium content and relaxivity determinations: The Gd concentrations of
nanoparticles were analyzed by inductively coupled plasma mass spectrometry (ICP-MS).
The relaxivity measurements were done on a Minispec (Bruker Medical BmbH, Ettingen)
operating at 60 MHz and 40 °C.
In vitro binding assay: Black, flat-bottom, Costar 96-well protein binding plates (Corning,
US) were incubated overnight on a rotating table at 4 °C with 50 μl of 30 μg/ml protein in
PBS. Proteins were aspirated from the wells, and the wells were rinsed three times with
250 μl PBS at room temperature. Wells were blocked with 100 μl of 50mg/ml BSA in
PBS for 3 h at 37 °C, aspirated, and rinsed three times with 250 μl PBS. Then 50 μl of a
solution of nanoparticles in PBS was added to each well and incubated 3 h at 37°C. After
cooling to room temperature, the total fluorescence I0 from Rhodamine-PE on
nanoparticles was recorded with a Bio-Rad microplate reader (excitation: 540 ± 25 nm;
emission: 590 ± 35 nm). Then the nanoparticle solutions were aspirated and washed three
times with 250 μl PBS, after which the binding fluorescence Ibind was recorded with BioRad microplate reader again (excitation: 540 ± 25 nm; emission: 590 ± 35 nm). Each
binding experiment was done in triplicate. The average fluorescence reading of empty
wells Iempty was subtracted from the raw fluorescence data. In order to correct the loss of
fluorescence labeling of Rhodamine-PE from HDL nanoparticles during peptide
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conjugation, the binding of nanoparticles was normalized as Normalized Binding = (Ibind
– Iempty)/(I0 – Iempty)*[Gd].
Animal use: The Institutional Animal Care and Use Committee of Mount Sinai School of
Medicine approved the animal protocols and procedures. Reversa mice were homozygous
on 4 alleles (Ldlr-/-ApoB100/100Mttpfl/flMx1-Cre+/+). At 4-weeks of age, Reversa mice were
weaned and then fed on Western diet containing a 0.15% cholesterol and 21% fat
(Research Diets) for 16 weeks. Mice were then divided into “regression” and control
groups as shown in Figure 1. The regression group was fed on a chow diet and Cre was
induced by i.p. injection of polyinosinic-polycytidylic ribonucleic acid (pIpC) (Sigma)
(250 µg every other day for a total of 4 injections). The baseline was defined at Day 0
(the 9th day after 1st pIpC injection). The mice were scanned by MR imaging at Day 0
(n=5) and Day 28 (n=5) and then sacrificed for histological staining. The control group
was fed the Western diet for the duration of the experiment and injected with saline.
MR imaging: The animals underwent in vivo MR imaging of the abdominal aorta using a
9.4 T, 89 mm bore magnet system (Bruker Instruments, Billerica, MA). The animals were
anesthetized with a 4% isoflurane/O2 gas mixture (400 cc/min initial dose) and
maintained with a 1.5% isoflurane/O2 gas mixture (100 cc/min maintenance dose)
delivered through a nose cone and positioned in a 30 mm birdcage coil. A respiratory
sensor connected to a monitoring and gating system (SA Instruments, Inc, Stony Brook,
NY) was placed on the abdomen to monitor the depth and frequency of respiration. The
abdominal aorta was identified in a coronal section on a localizing sequence. Highresolution T1-weighted images were generated using a black blood spin echo (SE)
sequence (TR/TE=800 ms/8.6 ms, FOV=3.0×3.0 cm, matrix size=256×256, 22
contiguous 500 μm-thick axial slices, number of averages=16, total scan time=54 min).
After pre-contrast imaging the animals were administered a nanoparticle contrast agent
(50 μmol of Gd/kg) via the tail vein. Post-contrast imaging with the same parameters was
performed on each animal at 24 h after injection. In this study we performed postinjection imaging at 24 hours, as this was the time point we have previously used with the
HDL contrast agent platform (Chen W et al, Contrast Media Mol Imaging. 2008,3(6):233-242).
We observed strong and significant contrast at this time point. However, it may be the
case that the contrast could be stronger at other time points. The slices of the postcontrast scan were matched to the pre-contrast scan by using unique vertebral and
paraspinous muscular anatomy as landmarks. Since MR images were acquired
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contiguously from the aortic bifurcation to the level of the renal arteries, accurate
registration was achieved for all the slices during pre- and post- contrast imaging.
Immunohistochemistry: Abdominal aortas matching the MR imaging area from Reversa
mice were collected and embedded in TissueTek®. Eight-micrometer frozen sections
were fixed in 4% paraformaldehyde and washed in PBS twice. The tissue sections were
permeabilized in PBS with 0.5% Triton X-100 at room temperature for 30 min and
washed with PBS twice. The sections were blocked in 2% goat serum in PBS for 45 min
at room. After blocking, the sections were incubated in 2% goat serum in PBS with a
primary antibody: rat anti-mouse CD 68 (Serotec, Raleigh, NC) or rabbit anti-mouse
collagen I (COL1A2:M-80, Santa Cruz Biotechnology) for 120 min at room temperature,
and washed by PBS twice. Thereafter the sections were incubated with a secondary
antibody: Alexa Fluor 647 conjugated goat anti-rat Ab (Serotec, Raleigh, NC) or Alexa
Fluor 647 conjugated goat anti-rabbit Ab (Serotec, Raleigh NC) corresponding to the
primary antibody for 60 min at room temperature, and washed by PBS twice again.
Finally, the sections were mounted with DAPI-containing VectaShield mounting media
and sealed with cover slips, shielded from light, and kept at 4 °C until imaged with laserscanning confocal fluorescence microscopy imaging (performed within 48 h of slide
preparation). One section without any antibody and one section with secondary antibody
only were used as negative controls. Confocal imaging was performed using a Leica
SP5DM microscope. The system is equipped with four lasers: a 405-nm blue diode, an
Argon (488 nm), a green HeNe (543 nm) and a red HeNe (633 nm).
Frozen sections were stained by Sirius red to detect interstitial collagen (type I and III)
using birefringency illumination with polarized light. The sections were rinsed with
distilled water and then incubated with 0.1% sirius red F3BA (Polysciences Inc) in
saturated picric acid for 90 minutes. After rinsing with 0.01N HCl for 1 minute twice, the
sections were immersed in distilled water, dehydrated in a graded ethanol series and
xylene substitute (Fisher Scientific) mounted in xylene substitute Mountant (Fisher
Scientific), and finally coverslipped. The sections were imaged under polarized light with
the same exposure time for each section.
For immunohistochemical CD68 staining, the frozen sections were fixed in cold acetone
for 10 min and then rinsed in PBS. After blocking with diluted serum from
VECTASTAIN ABC-AP kit (Vector Laboratories, Inc; CA USA) for 10 min, the
sections were incubated with primary antibody: rat anti-mouse CD68 (Serotec, Raleigh,
NC) in diluted serum for 1 h. After rinsing in PBS three times, the sections were
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incubated with secondary antibody: biotinylated rabbit anti-rat antibody from
VECTASTAIN ABC-AP kit (Vector Laboratories, Inc; CA USA) for 10 min. The
sections were then rinsed with PBS three times, incubated with VECTASTAIN ABCalkaline phosphatase solution from VECTASTAIN ABC-AP kit (Vector Laboratories,
Inc; CA USA) for 5 min, rinsed with PBS three times again, and then incubated in Vector
Red solution for 10 min. The sections were rinsed in deionized water and counter stained
with Vector Hematoxylin QS (Vector Laboratories, Inc; CA USA). After rinsing, the
sections were dehydrated in a graded ethanol series and xylene substitutive (Fisher
Scientific), mounted in xylene substitute Mountant (Fisher Scientific), and finally
coverslipped. The sections were imaged under bright field microscope with the same
exposure time for each section.
Image analysis: For in vivo MR images, the signal-to-noise ratio (SNR) of the region of
interest (ROI) is defined by SNRROI= IROI/Inoise where IROI is the intensity of either aortic
vessel wall (for SNRw) or surrounding muscle (for SNRm); Inoise is the standard deviation
(S.D.) outside the animal. The contrast-to-noise ratio (CNRw) to muscle is defined as
CNRw=SNRw-SNRm. The difference of CNRw between pre- and post-contrast is defined
as ΔCNRw=(CNRw)post-(CNRw)pre. The normalized enhancement ratio of aortic wall
(NERw) to muscle is defined as NERw=[(SNRw/SNRm)post-(SNRw/SNRm)pre]/
(SNRw/SNRm)pre * 100%. Five aortic MR images of the matched (pre- and post-contrast)
slices for each mouse were used for analysis. The mean ± S.D. of NER w and ΔCNRw
were reported. For ex vivo microscopy images, the percentages of positive area for
macrophage and collagen contents were analyzed in ImageJ. The total plaque area (Stotal)
was calculated in pixels by selecting plaque area as a ROI. The positive area (S positive) of
CD68+ macrophages (red in immunohistochemical staining) or collagen (bright in
polarized light) was calculated using color threshold in ImageJ. The percentage of
positive area is defined as Spositive/Stotal * 100%. The data are presented as mean ± S.D.
Statistics: Data are presented as the mean ± S.D. For differences between groups of
regression and control, two-way ANOVA was used. For differences between each
nanoparticle contrast agent within the regression or control group, one-way ANOVA was
used. One-way ANOVA was also used to compare the positive percentages of
immunohistochemical staining of CD68 and Sirius Red staining of collagens. Multiple
comparisons with Bonferroni correction after ANOVA were used to distinguish the
groups of significant difference. A value of p < 0.05 was considered statistically
significant. A least squares method and a linear regression model are used to analyze the
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correlation between MR signals and histological data. The analysis was performed on
Matlab® R2007b.
Figure S1: Confocal microscopy of atherosclerotic plaques at Day 0 from aortic vessel
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walls of Reversa mice in the regression group with CD68 (green) or Collagen type I (Col
I, green) staining. The nuclei were stained with DAPI (blue) and nanoparticles were
labeled with Rhodamine-PE (red). The colocalization of green and red signal appears as
yellow/orange colors. Scale bar: 25μm
After in vivo MR imaging, the mice were sacrificed and the aortas were collected for
optical and histological analysis at 24 hrs post injection of contrast agent. All three types
of nanoparticles were labeled with Rhodamine-PE (displayed in red). The colocalizations
of nanoparticles with CD68 positive macrophages and collagen type I were investigated
by immunofluorescent staining and confocal microscopy. The HDL nanoparticles were
found to localize mainly with macrophages as shown by the co-localization of
fluorescence signals from CD68-stained macrophages with Rhodamine-PE at Day 0
(Figure S1A), but not colocalized with collagen type I (Figure S1B). The EP3533-HDL
nanoparticles colocalized with CD68-positive macrophages but not with collagen type I
at Day 0 (Figure S1C-D). Most of the non-specific EP3612-HDL nanoparticles were not
found to localize with CD68 positive macrophages or collagen type I at both Day 0,
though some of these nanoparticles were seen at the same positions of CD68 signals in
confocal images (Figure S1E-F).
Figure S2. Split channel images of Figure 5D. Collagen type I (Collagen I; green) was
stained by anti-collagen I antibody. The nuclei were stained with DAPI (blue) and
EP3533-HDL nanoparticles were labeled with Rhodamine-PE (red). The colocalization
of green and red signal appears as yellow/orange colors in merged channel image.
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