Online Appendix for the following JACC article
TITLE: Targeted Iron Oxide Particles For In Vivo Magnetic Resonance Detection of
Atherosclerotic Lesions With Antibodies Directed to Oxidation-Specific Epitopes
AUTHORS: Karen C. Briley-Saebo, PHD, Young Seok Cho, MD, Peter X. Shaw,
PHD, Sung Kee Ryu, MD, PHD, Venkatesh Mani, PHD, Stephen Dickson, MS, Ehsan
Izadmehr, Simone Green, BS, Zahi A. Fayad, PHD, Sotirios Tsimikas, MD
APPENDIX
METHODS
In the current study the following two iron oxide particles were evaluated: oxidationspecific epitope targeted lipid coated superparamagnetic iron oxide particles
(LUSPIO) and the larger targeted lipid coated superparamagnetic iron oxide particles
(LSPIO). The rationale for comparing LUSPIOs and LSPIOs is related to the effect of
the core size on the magnetization (and resultant MR signal) as well as the effect of
hydrated particle size on the ability to penetrate the arterial wall (1-5). Whereas the
LUSPIOs are expected to exhibit better wall penetration, the LSPIOs are expected to
generate greater MR signal loss. It is currently unclear if it is more advantageous to
have a greater number of less effective particles within the wall or a fewer number of
more effective particles within the arterial wall.
Antibodies
Two murine (MDA2 and E06) and one fully human single-chain (IK17) antibody
fragment targeted to oxidation-specific epitopes were covalently attached to the
surface of the LUSPIO and LSPIO particles. The immunological characteristics of
these antibodies have been previously described in detail (13). Briefly, MDA2, is a
murine IgG monoclonal antibody that binds to malondialdehyde(MDA)-lysine
epitopes present on modified LDL or other MDA-modified proteins. E06 is a natural
murine IgM monoclonal antibody that binds to the phosphocholine head group of
oxidized phospholipids. IK17 is a fully human single chain Fv fragment that binds to
an MDA-like epitope present on MDA-LDL and copper-oxidized LDL. All three
antibodies were >99% pure and the binding specificity confirmed using in vitro
chemiluminescent enzyme-linked immunosorbent assay (ELISA), as described (12).
Synthesis of iron oxides particles
The lipid coated LUSPIOs were prepared by first synthesizing the iron core, as
previously reported for NC100150 (6). This method of synthesis was chosen since the
resultant USPIOs are mono-crystalline and mono-disperse (6). Additionally, PEG has
been conjugated to the surface of these particles thereby resulting in formation of
NC100150 Injection (Clariscan, Amersham/GE Health Care). In this method Fe(III)
chloride hexahydrate and Fe(II) chloride tetrahydrate were added to potato starch at
ratio of 0.18g:0.066g per gram starch. 28% ammonium hydroxide was then added
under vigorous stirring and the solution heated to 90°C for 3 hours. Excess
ammonium hydroxide was removed and the sample stored at 4°C overnight to allow
for gel formation. The gel was then washed with de-ionized water, slowly stirred then
allowed to settle. The supernatant was separated from the gel layer and the process of
gel agitation repeated. To release the particles from the washed gel, 12% hypochlorite
was added to the gel (at 45°C) and the sample slowly heated to 55°C for 30 minutes.
The temperature was then increased to 70°C and maintained for 45 minutes. The final
solution was then filtered through a Millipore 0.2 micron filter.
In order to incorporate the antibodies to the particle surface, the iron particles
were dried and diluted in chloroform. PEG-DSPE, PEG-malamide-DSPE, and Liss
rhodamine are added to the chloroform at a ratio of 17.3 mg:1.3 mg:0.12 mg per
milligram iron. The iron solution was then added drop wise into boiling HEPES
buffer (pH 7) under vigorous stirring until a clear brown solution was formed. Any
iron deficient micelles formed were removed via centrifugation with KBr
(density=1.25 g/ml, 14,000 rpm, 20 minutes, room temperature). The resultant
particles were then washed (via ultracentrifugation using filters with a MW cutoff of
100 KD, 4000 rpm, 10 minutes) and suspended in HEPES buffer.
The LSPIOs were prepared according to previously reported techniques (7). In
short, a colloidal suspension of oleic acid coated magnetite (Fe3O4, 10−15 nm) in
toluene carrier liquid (7). These particles were chosen since they exhibit large iron
cores while maintaining a relatively small particle size. The particles were dried and
diluted in chloroform and PEG-DSPE, PEG-malamide-DSPE, and Liss rhodamine
added at a ratio of 17.3:1.3mg:0.12 mg per mg Fe. The lipid mixture was heated to
65 C and added drop wise into boiling HEPES buffer (pH 7). All micelles formed
during synthesis were removed via centrifugation with KBr (density=1.25 g/ml,
14,000 rpm, 20 minutes, room temperature) and the resultant LSPIOs washed and
suspended in HEPES buffer.
Antibodies (0.35 mg/mg Fe) were attached the LUSPIO and LSPIO surface
via S-acetylthioglycolic acid N-hydroxysuccinimide ester (SATA) modification, as
shown in Fig. 1. In order to remove any excess SATA and/or hydroxylamine used in
antibody attachment, the iron oxide particles are purified via dialysis (membrane
cutoff 100 KD, 4° C, 24 hours).
Characterization of iron oxides particles
All samples were concentrated to 1 mg Fe/ml, sterile filtered, and characterized with
respect to: iron content, hydrated particle size, particle structure, relaxation properties
and stability (with respect to storage).
Total Iron Content: The total iron content of all formulations was determined using
inductively coupled plasma mass spectrometry (ICP-MS, Cantest Ltd, Burnaby,
British Columbia).
Particle size and structure: Dynamic light scattering was used to determine the mean
hydrated particle diameter at 25 C in HEPES buffer (Malvern Instruments, Malvern,
UK). All particle diameters were reported as the number weighted average. Negative
stain transmission electron microscopy (TEM, model CX-100; JEOL, Toyko, Japan)
was preformed to evaluate the iron oxide core and particle structure.
Relaxation Properties: The longitudinal (r1) and transverse (r2) relaxivities were
determined in HEPES buffer at 60 MHz and 40° C (Bruker Minispec). The
longitudinal (R1) and transverse (R2) relaxation rates were determined at six different
concentration levels (0-1 mmol/l Fe) using an inversion recovery sequence (15
different inversion times) or CPMG spin echo sequence (echo time of 1 ms),
respectively. All relaxivity values were obtained as the slope associated with a linear
fit of iron oxide concentration (mmol/l Fe) versus R1 (s-1) or R2 (s-1).
Proton R1 and R2 nuclear magnetic resonance dispersion (NMRD) profiles
were measured over an applied magnetic field strength ranging from 0.00024 to 0.47
T (corresponds to a proton frequency of 0.01 to 20 MHz) using a Stelar field-cycling
relaxometer operating at 37 C (by Dr. Silvio Aime at the Universita del Piemonte
Orientale, Italy). The error associated with the R1 and R2 measurements has been
reported to be less than 2% (8). Data points obtained over a frequency range of 20 to
70 MHz were collected using a Stelar Spinmaster spectrometer operated at variable
field. Inversion recovery sequences with at least 8 inversion times were used to obtain
all R1 values. R2 values were obtained using a CPMG spin echo sequence with an
echo time of 1 millisecond.
Stability Testing: Three batches of each untargeted formulation were prepared and the
batch variability, with respect to size and r2/r1 determined. The formulations were
then stored at 4 C (dark, ambient humidity) and the particle size and r2/r1 values
determined over a 30 day time interval post storage.
In vitro macrophage uptake: Previous in vitro studies have shown that oxidationspecific epitope targeted micelles exhibit increased macrophage uptake, particularly
when pre-incubated with the MDA-LDL(5). Since iron oxide particles are known to
also passively target macrophages, in vitro cell studies were performed to determine
the extent of passive macrophage uptake (5,9-14). It was also of interest to establish
whether the size of the particles influenced passive uptake. For this study 8th
generation J774A.1 macrophages were plated into 12-well plates in Dulbecco’s
modified Eagle’s medium (DMEM) containing fetal calf serum. Macrophages were
activated by incubating (37 C) the cells with MDA-LDL (5 µg/ml) for 24 hours. The
wells were then washed with fresh DMEM (3x) and the following materials added to
the macrophages: (1) 1 mmol/l Fe of untargeted LUSPIO (n=2), (2) 1 mmol/l Fe of
MDA2 labeled LUSPIO (n=2), (3) 1 mmol/l Fe of untargeted LSPIO (n=2), and (4) 1
mmol/l Fe of MDA2 labeled LSPIO (n=2). Feridex (1 mmol/l Fe) and saline (0.15ml) were used as controls. The rationale for using Feridex as a positive control is
relate to published studies that show strong macrophage uptake of dextran coated iron
oxide particles, such as Feridex, fractionated Feridex, and Combidex (5,15). All cells
were then incubated for an additional 24 hours at 37 C. The macrophages were then
gently scraped off the plates, washed in PBS (3x) and split into 2 fractions. For the
first fraction, cells were counted using a bright line counting chamber (Hausser
Scientific, USA) and iron content determined using inductively coupled plasma mass
spectrometry (ICP-MS). For the second fraction, the cells were fixed on glass slides
(4% paraformaldehyde, 10 minutes) and either fluorescently labeled with
VectaShield® containing 1.5µg/ml DAPI (VectorLaboratories, Burlingame, CA) for
laser scanning confocal microscopy or processed for Perl’s Prussian Blue staining (for
determination of intracellular iron deposition), using standard methods (5). All
confocal-scanning microscopy (Zeiss LSM 510 META microscope, Carl, Zeiss AG,
Oberkochen, Germany) was performed in an inverted configuration. The system was
equipped with 4 lasers and 3 confocal detectors and data was captured and analyzed
using Zeiss LSM 510 Meta and Image Browser software (Carl, Zeiss AG,
Oberkochen, Germany). The spatial relationship of iron particles (rhodamine) to
macrophage nuclei (DAPI) or iron (Perl’s) to cell nuclei (H&E) were then visually
assessed.
Animal Models
Eight to ten month old apoE-/- mice on a C57BL/6 background were used for all studies. All
apoE-/- mice were placed on a high-cholesterol diet (0.2% total cholesterol, Harlan Teklad,
Madison, Wisconsin) ad libitum beginning at 6 weeks until 32-40 weeks of age. The ethics
committee at Mount Sinai approved all of the animal experiments. For imaging studies, a total
of 51 ApoE-/- mice were used: n=5 untargeted LUSPIOs and LSPIOs, n=9 MDA2 labeled
LUSPIOs and LSPIOs, n=6 E06 labeled LUSPIOs and LSPIOs, and n=5 IK17 labeled
LUSPIOs and LSPIOs, and n=3 for competitive inhibition (MDA2 labeled LUSPIOs only).
The number of mice chosen was based upon power testing of pilot data (for MDA2 LUSPIO)
that showed a critical sample size of 4 (mean group 1 = 213, mean group 2 = 407, sigma =10,
delta =10, critical = 2.92, actual power = 0.9999, using GPower version 2, Faul & Erdfelder)
was required to evaluate significant changes in the R2* values of the arterial wall. All mice
were randomized into each of the groups upon arrival from the vendor.
Age matched C57BL/6 wild type (WT) mice were used as a control in the
study. The mice were maintained on normal chow until 32-40 weeks of age. For
imaging, a total of 20 WT mice were used (n=5 untargeted LUSPIOs and LSPIOs,
and n=5 MDA2 labeled LUSPIOs and LSPIOs). Due to limitations on antibody
availability only the untargeted and MDA2 labeled iron particles were tested in WT
mice.
Pharmacokinetics and Reticuloendothelial System (RES) uptake
The blood half-lives were determined in age matched apoE-/- and WT mice. Animals
were administered 3.9 mg Fe/Kg LSPIO or LSPIO via retro-orbital injection and
blood was drawn (into heparinized tubes) over a 24 hour time period post injection
(n=5 time points, n=3 mice/time point/formulation). Standard relaxometry methods
were used to determine the amount LUSPIO or LSPIO present in the blood as
function of time post injection (5,15). The blood half-life values were calculated from
the resultant iron oxide concentration versus time curves using standard noncompartmental bi-exponential pharmacokinetic analysis (16).
The uptake into the liver was also determined in apoE-/- mice
(n=5/formulation) 24 hours after the administration of a 3.9 mg Fe/Kg dose of
LUSPIO or LSPIO. Mice were sacrificed using compressed CO2, saline perfused, and
the liver excised. The tissue was then cleaned, weighed and homogenized for the
determination of iron content using relaxometry methods (5,15). The percent injected
dose was determined based upon the iron concentration per gram wet organ weight.
MR Imaging
All MR imaging was performed at 9.4 Tesla using a 89 mm bore system operating at
a proton frequency of 400 MHz (Bruker Instruments, Billerica, MA). In order to
obtain in vivo R2* maps, multiple echo GRE sequences with the following pulse
sequence parameters were applied: TR=29.1ms, TE=5.1ms to 10 ms (n=5), flip
angle=30, number of signal averages (NEX)=6, in-plane resolution=0.098mm2, and
100% z-rephasing gradient. Twenty slices were acquired from the level of the renal
arteries to the iliac bifurcation. Since the abdominal aorta was retroperitoneal, it was
stationary in the axial and longitudinal planes relative to the vertebrae, spinal cord,
and paraspinous muscles. As a result, no respiratory or cardiac gating was necessary.
Slices were matched pre and post iron oxide injection based upon the unique anatomy
associated with the vertebral and paraspinous muscular anatomy. R2*-maps were
generated for the matched pre and post images on a pixel-by-pixel basis using a
custom Matlab program (The Mathworks, R2007b). The signal intensity associated
with each pixel was normalized to the standard deviation of adjacent noise (placed
above the spine of the mouse) prior to linear fitting of the signal-to-noise ratio versus
echo time (TE). Unblinded readers then identified relative changes in the R2*-maps
pre and post injection. R2* values were then obtained using regions of interest (ROI)
drawn on the arterial wall on slices (n>5) exhibiting either R2* modulation post
contrast or arterial wall thickening, indicative of plaque deposition. Since the mouse
aorta is typically 4 pixels in thickness, only 10-20 pixels were used to make up any
given RIO. All areas associated with the ROIs were kept constant between the pre and
post image analysis. The relative percent change in the R2* values were determined
as % change = ((R2*post-R2*pre)/R2*post)*100.
Immediately following GRE acquisition, a GRASP sequence was applied
using 50% of the z-rephasing gradient. The reduction in the rephasing gradient from
100% to 50% caused a gradient imbalance to occur that effectively reduced the signal
in locations that are void of iron labeled cells (17,18). In locations where local
magnetic inhomogeneities were present, the gradient balance was restored thereby
inducing signal enhancement. GRASP is extremely useful when trying to determine if
MR signal loss is due to iron oxide deposition or other endogenous artifacts (motion,
partial voluming, and peri-vascular effects) that may also promote signal loss.
GRASP cannot be used alone, however, since this sequence does not provide
adequate anatomic information. In similarity to GRE sequences, GRASP signal may
be observed in lymphatic tissue that may also sequester the iron oxide particles.
However, this sequence is extremely useful to differentiate between iron oxide
deposition and artifacts that are often present when imaging the arterial wall.
Previously published studies at lower magnetic fields (1.5T to 3T) have shown that
the GRASP sequence is most effective when the rephrasing gradient is reduced from
100% to 25-30% (17,18). However, at 9.4T, preliminary studies indicate that a
reduction in the z-rephasing gradient from 100% to 50% allows for adequate
susceptibility matching and signal gain. As result, for the GRASP sequence all
parameters were held constant (relative to the GRE sequence) expect for the zrephasing gradient that was reduced to 50%. Since equivalent imaging geometry was
used for both GRE and GRASP, the imaging results obtained from these sequences
were directly matched and compared. Using the same ROI location/area used to
determine the R2* values, the signal intensity values associated with the GRASP
images were recorded. The standard deviation associated with the background was
also determined for each slice. The percent relative change in the signal-to-noise
ratios (SNR) pre and post injection were then determined for the GRASP sequence.
All mice were anesthetized using 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. Imaging was performed prior to
and 24 hours after the administration of a 3.9 mg Fe/Kg dose. MR sections were
matched to histology based upon the distance from the renal arteries and/or the iliac
bifurcation as follows: immediately following the last MR scan, the mice were
sacrificed and the length of the intact aorta from the renal arteries to the iliac
bifurcation was measured. The mice were then saline perfused, and the aorta excised
(cleaned and weighed) and fixed with 4%PFA/sucrose. After the aorta was fixed it
was stretched and carefully pinned to a Styrofoam block. The aorta section from the
renal arteries to iliac bifurcation was then re-measured to account for any tissue
shrinkage caused by the fixation method.
Competitive Inhibition Studies
The specificity of the MDA2 labeled LUSPIOs for MDA-lysine epitopes within the
vessel wall of apoE-/- mice was evaluated using in vivo competitive inhibition. Age
matched apoE-/- mice (n=3) were co-administered 3 mg of free MDA2 antibody and
3.9 mg Fe/Kg MDA2 labeled LUSPIOs via tail vein injection. The MR enhancement
(as R2*) of arterial wall was determined and compared to the enhancement observed
after the administration of MDA2 labeled LUSPIOs only (n=9).
Immunohistochemistry
Cut sections and/or cells were fixed with 4% paraformaldehyde for 2 hours at room
temperature. The aortas were then embedded in paraffin and serial 8-mm-thick
sections were rehydrated and the following procedures performed: (1) Atherosclerotic
regions of wild type and apoE-/- mice aortas were embedded in paraffin, cut into serial
7-m sections, placed on microscope slides, deparaffinized in Histo-Clear (National
Diagnostics, Atlanta, Georgia), and rehydrated in ethanol gradient. Sections with
abundant atherosclerotic plaques were stained with Mac3 (BD Biosciences, San
Jose, California), a rat monoclonal antibody to mouse macrophage (1:20 dilution), and
MDA3, a guinea pig antiserum for MDA-lysine epitopes (1:250 dilution). Staining
was detected by biotinylated secondary antibodies (goat anti-rat IgG antibody for
Mac3 and goat anti-guinea pig IgG antibody for MDA3) and an avidin-biotinalkaline phosphatase system (Vector Laboratories, Burlingame, California). Negative
control sections were stained without primary antibody. All sections were
counterstained with Hematoxylin QS (Vector Laboratories, Burlingame, California),
dehydrated, covered with slides by Permount (Fisher scientific, Fair Lawn, New
Jersey), and examined under light microscope. (2) Perls Prussian Blue was performed
to stain for iron oxide deposition by incubating the slides (20 minutes) with 2%
potassium ferrocyanide in 2% hydrochloric acid. The slides were then rinsed with
distilled water and counterstained with nuclear fast red and dehydrated in ethyl
alcohol (90, 95, and 100%) prior to cover-slipping. Images were acquired with a
Nikon microscope using specialized software (SOFT, Diagnostic Instruments, MI),
and (3) aorta sections were blocked (PBS containing 0.5% BSA and 5% Horse
Serum) and macrophages were fluorescently stained using RPE-labeled anti-CD68
primary antibodies (Serotec, Inc.). The sections were then washed with hypertonic
PBS, rinsed, and mounted with VectaShield® containing 1.5µg/ml DAPI
(VectorLaboratories, Burlingame, CA) to stain cell nuclei. All slides were stored at
4°C for less than 48 hours prior to confocal microscopy. Co-localization of the
fluorescently labeled LUSPIOs (red) with macrophages with the arterial wall was then
performed.
Statistics
One-way ANOVA with Bonferroni post hoc multiple comparison tests were used to
compare the R2* values obtained as a function of time pre and post injection between
the iron oxide groups. One way ANOVA analysis was used to compare the mean
values obtained 24 hours post injection. For all statistical analysis, p<0.05 was
considered significant. The ANOVA analysis was performed using Number
Crunching Statistical System 2001 (NCSS, statistical and number crunching software,
Utah, USA).
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