Fluorescent Sensors of Protein Kinases: From Basics to Biomedical Applications - Progress in Molecular Biology and Translational Science - page 264

refinement of noninvasive strategies for the delivery of fluorescent

biosensors into living organisms.
With respect to targeting strategies, there are two different levels that
may have a major impact on the signal reported by the biosensor. The first
consists in targeting a specific intracellular compartment, thanks to subcel-
lular targeting sequences. Indeed, several kinase biosensors have been spe-
cifically targeted to the nucleus, the Golgi, the endoplasmic reticulum,
the mitochondria, or the plasma membrane, revealing differences in kinase
activities and dynamics, as well as differences attributable to differential lo-
calization of discrete subpopulations or specific kinase isoforms. Hence, sub-
cellular targeting allows monitoring the differential activity associated with
subcellular localization, whereas it may not be as clear-cut if the biosensor is
simply distributed randomly within the cellular cytoplasm. The second level
of targeting consists in targeting specific cell types, which involves a more
complex approach, requiring conjugation or complexation of the biosensors
with formulations that direct the biosensor to cell-surface receptors.
Aside from targeting strategies, “smart” strategies based on specific activa-
tion of probes are being developed, notably for imaging cancer and metastasis,
and some of these have been successfully applied to image-guided sur-
gery. 168,176 These activatable probes make use of properties inherent to
cancer cells for specific penetration and/or activation within tumor
vicinity. For instance, metalloproteases secreted in the vicinity of tumors
cleave target sequences which normally prevent the probe from penetrating
cells in a nonspecific fashion, thereby releasing a cell-penetrating sequence
that allows for endosomal uptake of the probe. 169 Other systems are
activated by the acidic pH that characterizes the tumor environment, such
as pH-activatable fluorescent moieties coupled to cancer-targeting
antibodies 172 and pH-activatable CPPs conjugated to a fluorescent probes
(pHLIP technology). 173 Yet another strategy consists in silencing a
fluorescent probe through molecular quenching until specific enzymes
release the molecular cage. This strategy has been applied to generate a
fluorescent probe activated by tumor-specific g -glutamyl transferases
(GGT), 177 which can be applied topically to probe fluorescence at local
tumor sites and which allows for rapid visualization of surface lesions upon
intraoperative detection of tumors and metastases.
Other examples include targeting of NIRF probes to specific cell-surface
antigens and receptors, such as integrins and Glutamate Transporter recep-
tors (GLUT), through conjugation with Cyclic Arginine-Glycine-Aspartate
(RGD) and 2-deoxyglucose, respectively, to image tumors in vivo. 178,179