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