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peptide. These activatable CPPs will accumulate in the interstitial space but
cannot enter cells until the linker is cleaved by extracellular MMP which
then frees the CPP and allows it to enter the cell. In this way, the
enzyme-activated probe can be retained in the tumor cells, while
nonactivated probes are, over time, eliminated from the tissue/animal, thus
creating detectable contrast between anatomical regions with high versus
low protease activity. Tumor cells secreting MMPs (2 and 9) in mice and
biopsied human squamous cell carcinomas could be identified by a two-
to threefold increase in NIR fluorescence, compared to surrounding tissue.
Subsequent studies showed that the probe could detect several different can-
cer types (including breast cancer) and that probe accumulation was highest
at the tumor-stromal interface.
115
A similar strategy uses a protease-targeted reactive group (typically an
electrophile) to covalently attach a quenched fluorescent dye to the active
site of the enzyme. Upon covalent modification, the fluorophore is
unquenched and thus becomes activatable. In this way, the fluorescence sig-
nal can be directly linked to the activity of the enzyme. Such activity-based
probes (ABPs)
116,117
have been developed for
in vitro
detection of proteases,
kinases, and phosphatases.
116
When injected into tumor-bearing athymic
mice, quenched near-infrared fluorescent activity-based probes (qNIRF-
ABPs) targeting lysosomal cysteine proteases allowed
in vivo
detection of
cathepsin-producing tumors (SNR of tumor was roughly ninefold higher
than surrounding tissue)
118
and determination of enzyme activity
attenuation after therapeutic intervention. Covalent attachment to the
enzyme allows the dye to be sequestered in cathepsin-producing tissue,
thus permitting
ex vivo
microscopic analysis subsequent to
in vivo
imaging.
An important difference between ABP-based enzyme probes and PGC-
based sensors is that the former inactivates the target enzyme once it has
been covalently modified, whereas the latter leaves the enzyme active and
thus capable of cleaving and activating additional dye-linked molecules in
the tissue (amplification effect). Because of the difference in time required
in each case for generating optical signal of acceptable magnitude, direct
comparisons of the two methods (i.e., long-circulating “quenched”
macromolecular sensors and qNIRF-ABPs) are difficult. However, a
recent study suggests that the amplification effect does not invariably
result in an overall higher signal generation.
57
Low-molecular-mass
qNIRF-ABPs sensors of cathepsin B showed overall high reconstructed
volume-averaged fluorescent
intensity signals
in tumors
and low
backgrounds
in liver and spleen when animals bearing subcutaneous
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