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Bcr-Abl and Lyn tyrosine kinase activity in cell extracts. 128 The combination of
probes that provide a readout of different enzymatic activities offers a means of
assessing the differential behavior of distinct targets in a given signaling pathway.
Moreover, from a therapeutic perspective, it can provide information on the
efficacy of drug cocktails and dosing regimens prior to therapy and during
therapy, depending on disease progression and emergence of resistance.
5.2. In vivo imaging of protein kinase activities
Despite thecomplexityof enzymatic reactions in living systems,major advances
have been made in probe design and application for in vivo imaging. 164,165
Efforts have been made in developing fluorescent proteins for imaging in
living cells and tissues, 94 and several synthetic near-infrared fluorescence
(NIRF) probes have been developed by chemists for in vivo application. 166
Moreover, imaging technologies have improved significantly 167 and
strategies have been designed for in vivo imaging through engineering of
activatable or targeted probes. 168-173 Nevertheless, in vivo imaging of
fluorescent biosensors remains extremely challenging for several reasons.
With respect to genetically encoded biosensors, the challenge resides
essentially in the heterogeneity and lack of control associated with ectopic
expression. This said, recent developments are contributing to establish
stable expression of FRET biosensors in cell lines and expression in
transgenic mouse models. 174,175 With respect to nongenetic biosensors, the
challenge lies in the choice of synthetic probes that are compatible with
in vivo imaging and in delivering peptide or polypeptide scaffolds in vivo
in an efficient fashion. As mentioned above, the development of NIRF
probes which are appropriate for in vivo applications 166 solves part of the
problem. Moreover, the development of nanoparticle strategies for delivery
of peptides and proteins into cells 107,108 will be of considerable help in
promoting the successful application of nongenetic biosensors in vivo .
5.3. Delivery, targeting, and activation strategies
A major bottleneck for in vivo applications concerns the delivery of fluores-
cent biosensors into cells, tissues, and organs. In practice, this involves
engineering biosensor formulations with nanocarriers which are stable
in bodily fluids and which can be delivered to their target passively, through
prolonged circulation and enhanced permeability retention in the tumor, for
instance, or actively, thanks to specific targeting sequences. One of the fu-
ture priorities for biosensor application in vivo therefore concerns the
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