Magnetic Resonance Imaging (MRI) of Atherosclerosis Using Molecularly Targeted Nano-scale Gadolinium

Immunomicelles.Vardan Amirbekian, M.D., 2004-06 Sarnoff Fellow

Department of Radiology

Brigham and Women’s Hospital – Harvard Medical School

Current Contrast-Enhanced MRI Limited Plaque Activity Information

Gd-chelate enhanced-non specific

Targeted enhanced contrast agent - specific interaction with

plaque

Sirol M; Fayad ZA et al.

Free cholesterol

Cholesteryl ester

NC

EH

AC

AT

-1

MSR-A

SR-B1

Ox-LDL

VCAM-1ICAM-1

ABCA-1

macrophage foam cell

LDL

VLA-4

monocyte

MCP-1

apoAI

HDL

endothelium

cholesterol disposal In liver

M-CSF

exit to lymphaticsystem

LCAT

Ch

ou

dh

ury

R e

t a

l. N

atu

re C

ard

iov

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lar

Me

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CD-36

Gd-Immunomicelles

Self-assembleAmphilic componentsHydrophobic lipid core with polar surface

Conjugation of Gd(III) to hydrophillic side chains (corona)

Incorporation of ligands that specifically target a molecule of interest (the ligand can be a Antibody, peptide and any other structure with specific affinity for a molecule of interest).

Conjugation of Gd(III) to hydrophillic side chains

Diameter~ 80-120 nm diameter

4,000 Gd/particle

r1=~20 s-1mM-1 (63 MHz in H20)

Hypothesis

Targeting Gadolinium-containing MRI contrast-agents, such as immunomicelles, to the Macrophage Scavenger Receptor (MSR) will facilitate detection of atherosclerosis in Apo E knockout mice and furthermore may provide information about macrophage plaque content and plaque activity. The goals of the current study were: (i) to evaluate the relationship between macrophage content of plaques and the changes seen in MRI signal intensity using immunomicelles targeted to MSR; (ii) to examine the effect of MSR inhibition on immunomicelles-mediated MRI enhancement of atherosclerosis.

Micelles, immunomicelles, and standard (Gd-DTPA) paramagnetic contrast agents were tested in ApoE KO mice.

Mice were imaged at baseline with a 9.4T MR system (Bruker) using a high-spatial resolution sequence (98µmx98µmx500µm).

Mice were then injected with either micelles, immunomicelles, or standard.

Mice were then imaged at 1-hour, 24-hrs, 48-hrs and 72-hrs and 1-week post-contrast-injection.

Mice were all sacrificed at the end of the imaging period. Afterwards various types of analyses were performed including standard pathology, histology, and immunohistopathology.

Experimental Design - Methods

Pre-contrast 60 min. post 24hr-post

ApoE-KO

WT

In Vivo Aortic Atherosclerotic Plaque in ApoE-KO Mouse Gd-Immunomicelles Targeted to MSR

Amirbekian, V. et al. PNAS 2007

Amirbekian, V. et al. PNAS 2007

CD68 Macrophage Immunostaining of the Plaques Imaged in vivo with Immunomicelles

Macrophages were counted per HPF and these values were plotted against the %NER observed by Immunomicelles-mediated

MRI at the same anatomic level.

Amirbekian, V. et al. PNAS 2007

Relationship Between MRI Enhancement and Macrophage Content of the Atherosclerotic Aortas

y = 0.1968x - 7.6692

R2 = 0.753

0

2

4

6

8

10

12

14

16

50 60 70 80 90 100 110

% NER of Aorta (MRI Enhancement of Aorta)

Ma

cro

ph

ag

es

pe

r H

PF

Amirbekian, V. et al. PNAS 2007

Macrophages and MSR-Targeted Immunomicelles in Atherosclerotic Plaque

DAPINuclei

CD68Macrophages

FluorescentImmunomicelles

Overlay

Amirbekian, V. et al. PNAS 2007

Conclusions The current in vivo study shows that, using MRI,

immunomicelles targeted to macrophages may aid in the non-invasive detection of atherosclerotic plaque.

There appears to be a direct relationship between macrophage content of plaques and the changes seen in MRI signal intensity using immunomicelles targeted to MSR.

Immunomicelles may prove useful in detection of high-macrophage density typical of high-risk plaques. They may also provide valuable information about plaque activity.

Hypothesis

Immunomicelles targeting the macrophage scavenger receptor-B (CD36) will specifically improve ex-vivo MR detection and characterization of human aortic atherosclerosis.

Gd-containing micelles, anti-CD36 immunomicelles and Fc-micelles were created.

Macrophages were incubated with fluorescent micelles and immunomicelles to determine uptake via confocal microscopy and inductively coupled plasma mass spectroscopy (ICP-MS) was performed to quantify Gd uptake.

Human aortic specimens with moderate to severe atherosclerosis were harvested at autopsy. Using a 1.5 T Siemens clinical scanner, T1, T2, and PDW 3-dimensional scans were performed and post-contrast scans were repeated after 24 h incubation.

T1 analysis and cluster analysis were performed comparing immunohistopathology with MR images.

P-values<0.05 were considered significant.

Experimental Design - Methods

Immunomicelles – macrophage uptake in vitro and human atherosclerotic uptake ex vivo

MRI Results in Human Atherosclerosis

Co-localization of Immunomicelles and Macrophages

I am Grateful to:Brigham and Women’s Hospital

Harvard Medical School Dept. of Radiology

Dr. Barbara N. Weissman Dr. Steven E. Seltzer Dr. Frank J. Rybicki Dr. Salvatore G. Viscomi Bruce Boynton Jenna Hastings

Mount Sinai School of Medicine Institute for Translational and Molecular Imaging

Dr. Zahi A. FayadSmbat Amirbekian, BS.Dr. Fabien HyafilDr. Esad VucicDr. Marc SirolDr. Juan G.S. AguinaldoDr. Karen C. Briley-SaeboDr. Edward FisherDepts. of Radiology &

Cardiologyat Mount Sinai

Johns Hopkins University

School of Medicine

Dr. Donna Magid

Dr. Charles Lowenstein