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cleaved into fragments (dissociation of avidin tetramers to monomers) by
lysosomal proteases. This scheme takes advantage of both the broad specificity
of lysosomal enzymes and the relative selectivity of the lectin membrane re-
ceptor. In addition, because the fluorescent fragments are sequestered inside
the cells, there may be greater opportunity for buildup of signal as the non-
reacted probe is eliminated from the tissue.
89
Using intravital imaging in
murine models of peritoneal ovarian metastases, Av-3ROX allowed imag-
ing of submillimeter cancer nodules (sensitivity of 92% and specificity of
98%) with minimal contamination by background signal. Higher resolution
is likely if the probe is detected using current state-of-the-art animal imaging
techniques. In a subsequent study, a nonimmunogenic alternative of avidin,
galactosamine-conjugated serum albumin (GmSA), was used with similar
results.
90
9. MACROMOLECULAR SENSORS IN FLUORESCENT
IMAGING OF INFLAMMATION
AND VASCULAR DISEASE
Macromolecular fluorescence-based enzyme sensors have also shown
utility in detecting pathological changes occurring in cardiovascular disease
(reviewed in Ref.
120
). Vascular wall inflammation precedes atherosclerosis,
and one of the early pathological signs of the disease, the fatty streak, is char-
acterized by the accumulation of monocyte-derived macrophages and T lym-
phocytes and their associated proteases.
121
Proteolytic enzymes from highly
activated macrophages also hasten plaque erosion and rupture, and their levels
are correlated with clinical outcomes in patients with cardiovascular dis-
ease.
101,122-124
Elevated levels of MMP9 have been found in the brain
tissue of stroke patients and also correlate with clinical outcome.
125-128
Experimentally induced atherosclerotic lesions in mice were successfully
imaged using FMT after the injection of PGC-based NIRF probes for ca-
thepsin B
71
and MMPs (MMP2 and 9).
85
Histological analysis showed
colocalization of the probes with the targeted enzymes and with activated
macrophages. In addition, the probe-mediated signal increase could be di-
minished after treatment with protease inhibitors.
85
PGC-based MMP-
sensitive probes (MMPSense
TM
) have also been used to image increased
MMP levels occurring after middle cerebral artery occlusion (MCAO) in
mice.
95
In this study, TBRs were 1.3 times higher in the affected hemi-
sphere, and both the fluorescent signal and lesion volume could be dimin-
ished by MMP inhibitors. When repeated in MMP9-deficient mice, a major
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