acc presentation lipid coated(2)
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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 andTexas Heart Institute, Houston, Texas
Rupture Prone Inflamed Plaque
SPIOSPIOSuper Paramagnetic Super 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
FL-labeled SPIO Incubated Macrophages 24hr
Double DAPI Staining with Fluorescence-labeled SPIO Macrophages after 24hr Incubation
Hypercholesterolemic Rabbit, Aorta, 10 days after SPIO injection
Perls’ Staining H&E Staining X10 X10
Hypercholesterolemic Rabbit, Aorta, 4 days after SPIO injection
Perls’ Staining H&E Staining X40 X40
Histopathologic studies of Thoracic aorta in Watanabe Hereditary Hypercholesterolemic rabbit after SPIO
injection
H&E staining
Macrophage staining Iron staining
MR Angiography 3D with Gadolinium-DTPA in Watanabe Rabbit
3D-TOFTR=59msTE=7.0msFlip=30
3D-TOF
TR=59ms
TE=7.0ms
Flip=30
After SPIO injectionBefore SPIO injection
Baseline Day 5
Ex-vivo MR Study of Thoracic Aorta in SPIO-injected Atherosclerotic and Normal Rabbits after Compared to Non-
injected Controls.
Watanabe rabbitpost-SPIO
Watanabe rabbitwithout SPIO
NZW rabbit
Control
SPIO Injected
Schmitz et al J. Inv. Radiol. 2000
Goals:
• Increase contrast enhancement by increasing macrophage uptake of SPIO
• Decrease SPIO induced macrophage activity and free radical formation
SPIO Characteristics
• SIZE• Surface Charge• Coating Composition
Particle Core Size Particle Size Blood
(nm) (nm) Half-life
Combidex 5-6 20-30 8h
Feridex 4-6 35-50 2.4±0.2h
DDM 43/34/102 6.4 20-30 6h
Clariscan
MION 4-6 17 varies
Feruglose
--- --- --- ---
Examples of commercially available SPIOs
SPIO Coating√ Macrophage Uptake
Macrophage Activity
• Dextran Coated• Condrotin Sulfate Coated• Liposomal Coated• Combined lipid and protein Coated
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
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.
Uptake by Mouse Macrophages
SPIO coated with standard Dextran (6 K)
Uptake by Mouse Macrophages
SPIO coated with amino-silane
Uptake by Mouse Macrophages
SPIO coated with lipids
Uptake by Mouse macrophages
SPIO coated with CS
Uptake by Mouse Macrophages
Lipid encapsulated SPIO coated with amino-silane
SPIO Coating Macrophage Uptake
√ Macrophage Activity
• Dextran Coated• Condrotin Sulfate Coated• Liposomal Coated• Combined lipid and protein Coated
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 .
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 ??
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 37oC and 5% Co2. They were read for viability using an Elisa at 570 nm
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.
Viability Values with Alamar Blue
0.5850.59
0.5950.6
0.6050.61
0.6150.62
0.6250.63
1.25 /mL 2.5 /ml 5 /mL 10 /mL Untreated
Sample1Sample2Sample3
Studies of Macrophage Receptor Pathways for SPIO:
1) Uptake √2) Activity
Inhibition of the Uptake of SPIO Using Mannan andDextran ( Competitive Inhibitor of Mannose Receptor )
SPIO SPIO+Dex SPIO+Man Control0
25
50
75
SPIO aloneSPIO+DextranSPIO+Mannan
Control
*
*
Combination
AFU
/104
Mac
roph
ages
( SP
IO U
ptak
e)
Studies of Macrophage Receptor Pathways for SPIO:
1) Uptake 2) Activity √
Induction of Nitric Oxide by SPIO and LPIO-particles inMacrophages -24 h (Experiment-1)
DEX-1 DEX-2 LIP-1 LIP-2 LIP-3 LIP-4 Feridex Control0.0
0.1
0.2
0.3
DEX-1DEX-2
LIP-1LIP-2LIP-3LIP-4
FeridexControl
SPIOLPIO
SPIO or LPIO particles
OD
550
(Nitr
ite le
vel)
Conclusion
• Macrophage uptake of SPIO varies by SPIO size, surface charge and coating material.
• SPIO uptake does not affect macrophage viability.
• SPIO induces transient increased macrophage activity.
• Liposomal coated SPIOs combined with certain aminoglycans offer highest macrophage uptake with lowest oxidative stress.
Center for Vulnerable Plaque Research Houston, Texas
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