Fluorescent Macromolecular Sensors of Enzymatic Activity for In Vivo Imaging - Progress in Molecular Biology and Translational Science - page 365

complement activation and activation of immune response. During the
course of optimization experiments, we established that only MPEG with
an average mass of 5000 was sufficient for providing single-exponential
blood elimination kinetics and no antibody formation to acylated poly- L -
lysine and attached small molecular weight molecules was observed after re-
peated i.v. injections of PGC.
The resultant PGC copolymers used in initial in vivo experiments (which
in the fluorophore-free state are composed of MPEG-grafted PLL) have an
approximate molecular mass of 350-450 kD and carry approximately 50-55
amino groups per molecule 99 which can be readily covalently modified. In
the presence of an excess of free activated amino-reactive ester of cyanine
dyes, because of steric constraints, not all of the available amino groups
can be efficiently modified in the water milieu: it is usually feasible to link
approximately 30-45 molecules of large cyanine dyes per molecule of PGC,
depending on the structure of the dye, and produce a macromolecule in
which most of the fluorophores end up in a quenched nonfluorescent state.
The background fluorescence of dye-conjugated PGC protease sensors is
usually low, while the activation efficiency of the sensors depends on several
factors: for example, (1) the ability of the dyes to maintain a quenched state,
that is, the strength of dye dimerization; (2) quantum yield of fluorescence in
the nonquenched state; and (3) the ease of backbone fragmentation by
protease. All the above parameters vary, though not very widely, among var-
ious NIR fluorochromes. Spectral properties of the dye-conjugated PGC
depend on loading density. If the PGC carrier is linked to less than 12
dyes/molecule, their absorbance spectra indicate very little close interaction
between individual dyes (blue-shifted absorbance peak is very small;
Fig. 9.4 ). If the loading is high, that is,

35 dyes/molecule, there is a pro-
nounced blue-shifted absorbance peak which is characteristic of H-dimer
formation. This peak is much less prominent in samples after the proteolytic
cleavage of the probes. Fluorescent properties change dramatically after the
cleavage with multifold increase of fluorescence intensity (see Fig. 9.4 and
Table 9.2 ).
At this time, the backbone-linked heptamethine IRDye 800CW Ò has
provided the best performance in terms of the robustness of fluorescence in-
tensity increase as a function of proteolytic activation (fragmentation) of
dye-linked PGC ( Table 9.2 ). The cleavage of PGC probes by model pro-
tease depends on whether the cyanine dyes are linked at high or low density
to the backbone and whether MPEG protective chains are linked to the
backbone via ester (semistable) linkers or not. Semistable linkers are

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