Fluorescent Macromolecular Sensors of Enzymatic Activity for In Vivo Imaging - Progress in Molecular Biology and Translational Science - page 375

xenografts were imaged using fluorescence-mediated tomography (FMT).
The comparison of the overall FMT-reconstructed fluorophore
concentration in the subcutaneous (ectopic) mammary tumors showed
that, in the case of qNIRF-ABPs, the probe concentration was higher.
However, high-molecular-mass commercial PGC analogs (ProSense TM )
showed much better tumor/lung ratios and overall better target volume/
background ratios, with a much brighter signal achieved in tumors at late
time points (8 h, see Fig. 9.1 C). 57 In all likelihood, the observed
differences between qNIRF-ABPs and PGC-based sensors were due to
differences in both the kinetics and retention of the respective dye
molecules. ABPs are small and have rapid distribution in extravascular
space and clearance, whereas ProSense shows slow diffusion into the
extravascular space and slow uptake by cells, where the liberated
fluorophores have much longer resident times than qNIRF-ABPs. The
latter undergo rapid degradation and exocytosis or compaction in
lysosomes after covalent or noncovalent interaction with the target enzymes.
Other non-PGC-based macromolecular NIRF probes have also been
developed. Scherer and colleagues showed detection of intestinal neoplasia
using a PEGylated polyamidoamine (PAMAM)-Generation 4 dendrimer
core covalently coupled to a Cy5.5-labeled peptide which acted as a selec-
tive substrate for MMP7. The probe is “self-quenched” (optically silent) in
the absence of MMP7 but leads to a 2.2-fold increase in fluorescence in
MMP7-expressing tumors 93 at 3 h postinjection. Ex vivo imaging yielded
even higher TBR (sixfold) and allowed identification of tumors as small
as 0.01 cm. 2 However, unlike polyamino acid-based PGCs, PAMAM den-
drimers are not biodegradable.
A well-appreciated difficulty in imaging enzyme activity, especially for
in vivo applications, is the somewhat broad specificity of most biological en-
zymes (especially proteolytic enzymes). Thus, the resulting fluorescence sig-
nal will lack strict specificity for a single enzyme and, in addition, may lack
strict specificity for diseased tissue, as even healthy tissues express enzyme
activity. One approach that has been implemented to overcome this limita-
tion is to use higher specificity disease targets, such as membrane receptors, as
the targeted moiety (reviewed in Ref. 119 ). Kobayashi et al. have described
the use of a fluorescent sensor (Av-3ROX) that is composed of a self-
quenching avidin-rhodamine X conjugate, which has affinity for lectin
on certain cancer cells (such as ovarian, colon, gastric, and pancreatic cancer,
which have high metastatic potential). Upon binding to the lectin receptor,
the probe is internalized and fluorescence is activated after the probe is