Biology Reference
In-Depth Information
The use of polylysine backbones with “encoded” cathepsin-sensing
specificity within the backbone has proved feasible in numerous
in vivo
ap-
plications (see
Table 9.1
and
Sections 7 and 8
). However, there are
numerous hydrolases,
including cathepsins,
that can cleave backbones
with either free or acylated N-
-amino group-conjugated PGC sensors.
These sensors were not initially designed with the intent of providing
high selectivity and specificity for given hydrolases. Such specificity was
eventually provided by using iodoacetylated PGC as a “template” for
linking synthetic peptides encoding specific peptide sequences and for
linking cysteines as well as free amino groups
99
(
Fig. 9.5
). The latter were
modified with NIR dyes (e.g., Cy5.5). This design resulted in probes that
rendered large fluorescence intensity increase (350-fold) after reacting
with hydrolases that were capable of cleaving the linker. The addition of
specific linkers may assist in solving the problem of potential lack of
accessibility to NIR fluorophores if the latter are linked directly to the
backbone N-
e
-amino groups of lysine. However, the caveat is that even
the above more sophisticated design of PGC sensor is susceptible to an
attack of multiple extracellular proteases, including the secreted cathepsin
B (reviewed in Ref.
100
) which is still capable of hydrolyzing both the
backbone and the linkers. One potential solution is to use a poly-
D
-lysine
backbone, which is not cleavable by naturally occurring proteases. This
approach has been used to manufacture a true noncleavable long-
circulating imaging agent
e
(Angiosense)
that
is useful
for monitoring
tumoral blood volume using NIR imaging.
101
6. PHARMACOKINETICS OF PGC AND IMAGING
OF ACTIVATION
Macromolecular sensors based on long-circulating PGC graft copol-
ymers with built-in enzyme-mediated fluorophore activation are large mol-
ecules that as a rule have low uptake in the organs of the reticuloendothelial
system (liver, spleen). Only PGC based on MPEG-bPLL with low degree of
PEGylation or a very low degree of backbone modification of the backbone
(10% of amino groups) is usually removed from the circulation rapidly (half-
life in blood
1 h). The biodistribution of fluorophore-linked PGC in an-
imals (rats, rabbits, and nonhuman primates) was found to approximate that
of GdDTPA-labeled PGC, suggesting that blood half-life of PEGylated
enzyme-sensitive macromolecular sensors ranges in various species between
24 (e.g., in rats) and up to 32 h (in rabbits). Long circulation times in the
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