Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

Acc presentation lipid coated(2)

145 views

Published on

SHAPE Society

Published in: Health & Medicine
  • Login to see the comments

  • Be the first to like this

Acc presentation lipid coated(2)

  1. 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
  2. 2. Rupture Prone Inflamed Plaque
  3. 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
  4. 4. FL-labeled SPIO Incubated Macrophages 24hr
  5. 5. Double DAPI Staining with Fluorescence-labeled SPIO Macrophages after 24hr Incubation
  6. 6. Hypercholesterolemic Rabbit, Aorta, 10 days after SPIO injection Perls’ Staining H&E Staining X10 X10
  7. 7. Hypercholesterolemic Rabbit, Aorta, 4 days after SPIO injection Perls’ Staining H&E Staining X40 X40
  8. 8. Histopathologic studies of Thoracic aorta in Watanabe Hereditary Hypercholesterolemic rabbit after SPIO injection H&E staining Macrophage staining Iron staining
  9. 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. 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
  11. 11. Control SPIO Injected Schmitz et al J. Inv. Radiol. 2000
  12. 12. Goals: • Increase contrast enhancement by increasing macrophage uptake of SPIO • Decrease SPIO induced macrophage activity and free radical formation
  13. 13. SPIO Characteristics • SIZE • Surface Charge • Coating Composition
  14. 14. 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
  15. 15. SPIO Coating √ Macrophage Uptake Macrophage Activity • Dextran Coated • Condrotin Sulfate Coated • Liposomal Coated • Combined lipid and protein Coated
  16. 16. 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
  17. 17. 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.
  18. 18. Uptake by Mouse Macrophages SPIO coated with standard Dextran (6 K)
  19. 19. Uptake by Mouse Macrophages SPIO coated with amino-silane
  20. 20. Uptake by Mouse Macrophages SPIO coated with lipids
  21. 21. Uptake by Mouse macrophages SPIO coated with CS
  22. 22. Uptake by Mouse Macrophages Lipid encapsulated SPIO coated with amino-silane
  23. 23. SPIO Coating Macrophage Uptake √ Macrophage Activity • Dextran Coated • Condrotin Sulfate Coated • Liposomal Coated • Combined lipid and protein Coated
  24. 24. 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 .
  25. 25. 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 ??
  26. 26. 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
  27. 27. 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.
  28. 28. Viability Values with Alamar Blue 0.585 0.59 0.595 0.6 0.605 0.61 0.615 0.62 0.625 0.63 1.25 /mL 2.5 /ml 5 /mL 10 /mL Untreated Sample1 Sample2 Sample3
  29. 29. Studies of Macrophage Receptor Pathways for SPIO: 1) Uptake √ 2) Activity
  30. 30. 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)
  31. 31. Studies of Macrophage Receptor Pathways for SPIO: 1) Uptake 2) Activity √
  32. 32. Induction of Nitric Oxide by SPIO and LPIO-particles in Macrophages -24 h (Experiment-1) DEX-1 DEX-2 LIP-1 LIP-2 LIP-3 LIP-4 Feridex Control 0.0 0.1 0.2 0.3 DEX-1 DEX-2 LIP-1 LIP-2 LIP-3 LIP-4 Feridex Control SPIO LPIO SPIO or LPIO particles OD550(Nitritelevel)
  33. 33. 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.
  34. 34. Center for Vulnerable Plaque Research Houston, Texas

×