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which readily reacts with carboxyl
groups on the polymer side chains of
random copolymers.
All copolymers 1–4 were dissolved
in aqueous solution and formed nano-
meter-sized self-assemblies (Table 1).
The molecular weights of copolymers
1–4 were about three times as large as
that of copolymer A, whereas the
diameters of the nanoparticles formed
by copolymers 1–4 were slightly larger
than that of A as observed by trans-
mission electron microscopy (TEM)
and dynamic light scattering (DLS).[16]
This slight difference in the diameters
might be caused by the high-density
cohesion of the hydrophobic cores in
the polymeric self-assemblies. Because
the critical aggregation concentrations
(cac) estimated by static light scattering
(SLS) were 7–40 times lower than that
of copolymer A, self-assemblies of
Figure 3. Amphiphilic copolymers A and 1–4. All segments inside bold brackets were polymer-
ized randomly; Mn(PMA)=5400 and Mn(PEG)=2000.
copolymers 1–4 were extremely stable
example for their application to tumor imaging in vivo. We
report here high-contrast tumor imaging probes consisting of
polymer backbones bearing poly(methacrylate) (PMA) and
poly(ethylene glycol) (PEG) polymer brushes, which are
obtained by ROMP.
under diluted conditions in aqueous solution.[17] The stronger
fluorescence of assemblies of copolymers 3 and 4 relative to
that of 1 and 2 indicates that hydrophobic ICG dye moieties
tethered by long side chains, which could enhance their
mobility, prefer to disperse in the hydrophobic core of the
assemblies and thereby display inherent fluorescence.
To examine the characteristics of the brush-like copoly-
mers 1–4 as tumor-specific probes in vivo, we performed a
series of optical imaging experiments (Figure 4). We intra-
venously injected polymer conjugates 1–4 into nude mice
bearing a subcutaneous tumor xenograft[18] in their right hind
leg and monitored their distribution using an optical in vivo
imaging device.[19] The probes gradually accumulated in
tumor tissues, and their fluorescence intensities exceeded
the threshold level within six hours after injection. As shown
in Figure 4a, the tumor site could be visualized through the
EPR effect of copolymer 1, despite the strong fluorescence
from the liver.[20] The accumulation of copolymer 2 in the liver
relative to that of copolymer 1 was slightly suppressed by the
effect of folate-receptor targeting (Figure 4b). In contrast,
optical in vivo imaging of copolymers 3 and 4 afforded high-
contrast images of clearly visualized tumor sites (Figure 4c,d).
We measured fluorescence intensities in two defined regions
of interest (ROIs): at a tumor site (ROI 1) and in the liver and
kidney (ROI 2, Figure 5a). The fluorescence intensities of
copolymers 3 and 4 in the tumor sites were four to six times as
strong as those of copolymers 1 and 2 (Figure 5b). The
contrast ratios (defined as the ratio of fluorescence intensities
of ROIs 1 and 2) show that copolymers 1 and 2 did not
accumulate efficiently in the tumor tissues (purple and green
lines in Figure 5c). However, copolymers 3 and 4 accumu-
lated in the tumor tissues and exhibited contrast ratios as high
as 1.5 after one day (red and blue lines in Figure 5c). In
ex vivo experiments, fluorescence is obviously stronger in the
tumor sites than in the kidneys, heart, and lungs (Figure 6),
although uptake of copolymers 3 and 4 was found in the
The synthesis of copolymers 1–4 bearing hydrophobic and
hydrophilic polymer brushes includes ROMP to introduce
PMA moieties as hydrophobic segments, copper-catalyzed
[2+3] cyclization[11] to graft PEG segments, and dihydroxy-
lation[12] of double bonds in the main chain of the polymer
(Figure 3). We prepared random copolymers of PEG, the
NIRF indocyanine green (ICG) dye, and targeting-agent
segments, followed by end-capping reactions of PMA macro-
monomers to obtain PMA-grafted copolymers.[13,14] The
random copolymerization enhanced the fluorescence inten-
sities of the amphiphilic copolymer assemblies because the
self-quenching of the dye moieties was restricted (see Fig-
ure S4 in the Supporting Information). The folate-containing
copolymer A, which forms self-assemblies without cross-
linked interface, was also prepared as control sample.[9b] For
its conjugation with cyclic RGD peptides (RGD) and glucos-
amine molecules (GA) as targeting agents, we synthesized a
new ICG derivative[15] bearing an amino functional group,
Table 1: Diameters, critical aggregation concentrations, and fluores-
cence intensities of the self-assembled copolymers 1–4 and A.
Polymer DTEM [nm][a] DDLS [nm][b] cac [gLÀ1 [c]
]
Relative FL intensity[d]
1
2
3
4
A
170Æ37
164Æ37
163Æ49
151Æ46
158Æ32
194
216
204
212
182
8.1ꢀ10À5
6.7ꢀ10À5
1.4ꢀ10À5
4.0ꢀ10À5
5.5ꢀ10À4
1.00
0.83–0.88
3.68–3.76
3.56–3.67
–
[a] Distribution of the diameters of nanoparticles determined by TEM.
[b] Hydrodynamic diameters of nanoparticles in aqueous solution
measured by DLS. [c] Measured by SLS. [d] Fluorescence intensities of
2–4 relative to that of 1 in aqueous solution (10 mgmLÀ1).
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2011, 50, 6567 –6570