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Acc presentation lipid coated(2)
1. Lipid Coated SPIO;
Introducing a Novel Tracer for MR
Imaging of Macrophage Infiltration in
Vulnerable Atherosclerotic Plaque
Center for Vulnerable Plaque Research
University of Texas-Houston and
Texas Heart Institute, Houston, Texas
3. SPIOSPIO
Super ParamagneticSuper Paramagnetic
Iron OxideIron Oxide
lBlood pool magnetic resonance (MR) imaging
contrast media with a central core of iron
oxide generally coated by a polysaccharide
layer
lShortening MR relaxation time
lEngulfed by and accumulated inside cells
with phagocytic activity
8. Histopathologic studies of Thoracic aorta in Watanabe
Hereditary Hypercholesterolemic rabbit after SPIO
injection
H&E staining
Macrophage staining Iron staining
9. MR Angiography 3D with Gadolinium-DTPA in
Watanabe Rabbit
3D-TOF
TR=59ms
TE=7.0ms
Flip=30
3D-TOF
TR=59ms
TE=7.0ms
Flip=30
After SPIO injectionBefore SPIO injection
Baseline Day 5
10. Ex-vivo MR Study of Thoracic Aorta in SPIO-injected
Atherosclerotic and Normal Rabbits after Compared to Non-
injected Controls.
Watanabe rabbit
post-SPIO
Watanabe rabbit
without SPIO
NZW rabbit
17. Home Made SPIO
• 1. SPIO coated with chondroitin sulfate
• 2 SPIO coated with standard dextran (6K)
• 3 SPIO coated with Amino-silane
• 4 SPIO coated with lipids
• 5 Lipid encapsulated SPIO coated with
amino-silane
18. Uptake of SPIO by murine peritoneal
macrophages and monocytes
• Normal mice were treated with mineral oil
by ip injection.
• 24 hours later, equal amount of various
SPIO were injected intraperitoneally.
• 24 hours later, peritoneal macrophages and
monocytes were isolated.
• Uptakes of various SPIO were examined by
light microscopy.
19. Uptake by Mouse Macrophages
SPIO coated with standard Dextran
(6 K)
41. Principle
• Any event of phagocytosis is immediately
followed by a transient release of super oxide due
to the assembly of the NADPH oxidase against the
plasma membrane. Subsequently the oxidase
translocates onto the phagosomes containing the
SPIO to produce intracellular ROS.
• Thus an early extra cellular secretion of super
oxide is detectable (using luminol) soon after
phagocytosis and a later event of intracellular
secretion is measurable using DCFDA dye .
42. Detection of macrophage
viability after ROS production
• Viable macrophages convert the Alamar
blue dye into an orange colored product
measurable in a Elisa reader. AB is a widely
used method to measure viability.
• The hypothesis we are testing is that cells
that make ROS may die and lose viability.
Thus if SPIO stimulates ROS within the
cells does it lead to cell death over time ??
43. Method
∀ • The suspension of SPIO (1.25-10 micromol/mL)
was added to macrophages (1x104
/well in 96 well
plates). Cells were incubated for 1 h or 24 h and
washed to remove extracellular SPIO. For each
dose three wells were tested.
∀ • AB dye at 20% volume was added to the
macrophage plate and incubated for 4 h at 37o
C
and 5% Co2. They were read for viability using
an Elisa at 570 nm
44. Results
• Both SPIO treated as well as untreated
macrophages had same level of viability as
shown in the following slide at 24 h post
SPIO treatment.
47. Inhibition of the Uptake of SPIO Using Mannan and
Dextran ( Competitive Inhibitor of Mannose Receptor )
SPIO SPIO+Dex SPIO+Man Control
0
25
50
75
SPIO alone
SPIO+Dextran
SPIO+Mannan
Control
*
*
Combination
AFU/104Macrophages(SPIOUptake)
