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portion of the probe signal could be attributed to MMP9-specific activity.
MMP-sensitive probes have also been used to detect inflammatory responses
occurring after myocardial infarction in mice.
82
Smaller fluorescence molec-
ular probes have also been developed to image inflammation and enzyme-
related vascular pathologies.
86,129-131
PGC-based NIRF probes designed to target inflammation-specific mol-
ecules are also well suited for detecting osteo- and rheumatoid arthritis, as
the disease is characterized by early inflammatory responses in which release
of MMPs, cysteine proteases, and cathepsin B into the synovial fluid causes
the destruction of arthritic joints due to the degeneration of proteoglycan
and type II collagen. Because these events occur before morphological
changes in joint structure can be detected, there is a strong impetus for find-
ing noninvasive imaging methods to assess treatment options that may pre-
vent further joint damage. In two separate studies, a cathepsin B-activated
NIR probe (PGC-Cy5.5) injected 24 h prior to imaging resulted in a three-
fold increased fluorescence signal intensity in the affected joints of animals
with experimentally induced arthritis as compared to control animals.
78,80
In arthritic animals treated with the anti-inflammatory drug methotrexate
(35 mg of MTX/kg 48 h prior to probe injection), a significantly lower
fluorescence signal was observed as compared with untreated arthritic
animals.
78
In a more recent study, an altered form of the probe was used
in which poly-
D
-lysine replaced poly-
L
-lysine and a cathepsin K-sensitive
peptide linker (GHPG-GPQGKC) was used for attachment of Cy5.5.
This configuration resulted in a probe that was activatable only by
cathepsin K
132
and was subsequently shown to detect osteoclast-derived
cathepsin K-mediated bone degradation, which is largely responsible for
bone resorption.
The use of macromolecular enzyme-sensing probes has limitations in
certain cases where the extent of probe extravasation from the bloodstream
into the extravascular space of the affected organ is very limited. For exam-
ple, in the heart, which is located near the lungs and liver (the latter have
shown high levels of protease-sensing probe activation (see
Section 8
)
57
),
the resultant levels of fluorescent signal (TBR) can be rather small. Small-
molecular-weight probes such as qNIRF-ABPs can potentially accumulate
in the heart rapidly. However, high levels of accumulation can result in
cardiotoxicity. Therefore, we tested the approach that used the effect of
cyanine dye FL change in response to probe cleavage by proteases.
Although the far-red cyanine dye Cy5.5 gives the most pronounced increase
in lifetime after PGC proteolysis (approximately 5
, see
Table 9.2
), we used
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