Amphiphilic brush-like copolymers involving hydrophobic amino acid- and oligopeptide-side chains for optical tumor imaging in vivo

Article abstract of DOI:10.1246/bcsj.20120220

Amphiphilic brush-like copolymers having simple amino acids or oligopeptides and poly(ethylene glycol) as side chains were synthesized via ring-opening metathesis polymerization and further transformations. All of the synthesized copolymers formed spherical self-assemblies in water and their hydrodynamic diameters were 100250 nm, as measured by dynamic light scattering. Amphiphilic polymers bearing leucine and valine can form stable spherical self-assemblies in water because of the synergistic effects of intermolecular hydrogen bonding and hydrophobic interaction. The oligomerization of leucine moieties and capping with the proper length of fatty acyl groups at an N-terminus could significantly affect both the stability and fluorescence intensity of near-infrared fluorescent (NIRF) dye-containing polymeric self-assemblies. All of the self-assemblies consisting of leucine- and oligoleucine-grafted copolymers with a NIRF indocyanine green dye and hydrophilic targeting agents showed low cytotoxicity in cell viability tests. Furthermore, self-assemblies consisting of N-acetylleucine-grafted copolymers with strong fluorescence could visualize a tumor site with high-contrast in vivo, while low contrast images were obtained by utilizing oligoleucine-grafted self-assemblies because of their weak fluorescence. These results indicate that the precise control of both the number of leucine moieties and the length of the fatty acyl groups is important to produce highly functionalized tumor-targeting vehicles delivering a NIRF dye.

Full text of DOI:10.1246/bcsj.20120220

©
2012 The Chemical Society of Japan
Bull. Chem. Soc. Jpn. Vol. 85, No. 12, 1277­1286 (2012) 1277
Selected Papers
Amphiphilic Brush-Like Copolymers Involving Hydrophobic Amino
Acid- and Oligopeptide-Side Chains for Optical Tumor Imaging In Vivo
1
1
2
3
Koji Miki,* Katsuya Nakano, Hideki Matsuoka, Chan Joo Yeom,
3
4
1
Hiroshi Harada, Masahiro Hiraoka, and Kouichi Ohe*
1Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto University,
Nishikyo-ku, Kyoto 615-8510
2Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University,
Nishikyo-ku, Kyoto 615-8510
3Group of Radiation and Tumor Biology, Career-Path Promotion Unit for Young Life Scientists, Kyoto University,
Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501
4Department of Radiation Oncology and Image-applied Therapy, Graduate School of Medicine, Kyoto University,
54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507
Received August 14, 2012; E-mail: kojimiki@scl.kyoto-u.ac.jp, ohe@scl.kyoto-u.ac.jp
Amphiphilic brush-like copolymers having simple amino acids or oligopeptides and poly(ethylene glycol) as side
chains were synthesized via ring-opening metathesis polymerization and further transformations. All of the synthesized
copolymers formed spherical self-assemblies in water and their hydrodynamic diameters were 100­250 nm, as measured
by dynamic light scattering. Amphiphilic polymers bearing leucine and valine can form stable spherical self-assemblies
in water because of the synergistic effects of intermolecular hydrogen bonding and hydrophobic interaction. The
oligomerization of leucine moieties and capping with the proper length of fatty acyl groups at an N-terminus could
significantly affect both the stability and fluorescence intensity of near-infrared fluorescent (NIRF) dye-containing
polymeric self-assemblies. All of the self-assemblies consisting of leucine- and oligoleucine-grafted copolymers with a
NIRF indocyanine green dye and hydrophilic targeting agents showed low cytotoxicity in cell viability tests. Furthermore,
self-assemblies consisting of N-acetylleucine-grafted copolymers with strong fluorescence could visualize a tumor site
with high-contrast in vivo, while low contrast images were obtained by utilizing oligoleucine-grafted self-assemblies
because of their weak fluorescence. These results indicate that the precise control of both the number of leucine moieties
and the length of the fatty acyl groups is important to produce highly functionalized tumor-targeting vehicles delivering
a NIRF dye.
The development of high-performance tumor-specific car-
role in stabilizing their self-assemblies in dilute aqueous
solution compared with alkyl-grafted copolymers, thereby
specifically accumulating in tumor tissues. However, from a
practical application standpoint, hydrophobic PMA moieties
should be replaced by other simple and naturally occurring
materials.
riers is one of the most important topics in the fields of
chemical biology, noninvasive diagnosis of disease, and
chemotherapy utilizing drug delivery systems.1 Polymeric
self-assemblies have been intensively investigated as intelligent
tumor-specific carriers with an expectation that they passively
accumulate in solid tumors because of the enhanced perme-
Amino acids and their oligomers are naturally biosynthesized
molecules, and therefore have been utilized as a component
of various types of polymeric nanoparticles that have been
successfully applied as tumor-specific carriers, but selected as
2
ability and retention (EPR) effect. For ideal passive tumor
targeting by the EPR effect, it is known that the disassembly
of amphiphiles under dilute conditions, especially in blood
vessels, is a problem that remains to be solved. Recently, we
developed amphiphilic block copolymers having hydrophobic
alkyl groups or poly(methacrylate) (PMA) and poly(ethyl-
ene glycol) (PEG) brushes by using ring-opening metathesis
polymerization (ROMP) of norbornadienes, for high-contrast
1
f,2b,2f,4
a biocompatible material in most cases.
Because inter-
molecular hydrogen bonding between amide moieties can
form two- and three-dimensional networks, hydrogen bonding
of amino acids and oligopeptides have been utilized to tailor
the nanostructures of self-assemblies, such as nanofibers and
3
5
optical tumor imaging probes in vivo (Figures 1a and 1b). In
nanoparticles. Based on this background, to obtain stable
the previous study, we proposed that polymeric hydrophobic
side chains in brush-like copolymers would play an important
polymeric self-assemblies for efficient passive tumor targeting,
we have investigated the effect of intermolecular hydrogen
Published on the web December 1, 2012; doi:10.1246/bcsj.20120220

1
278 Bull. Chem. Soc. Jpn. Vol. 85, No. 12 (2012)
Amino Acid-Grafted Polymers for Tumor Imaging
hydrophobic interaction
between alkyl groups
Ph
(
(
(
a)
N
N
1) 1a (30 equiv)
2) 1b (n equiv)
Mes
Mes
n
30
Cl
self-
assembled
py* Ru
Cl
3) ethyl vinyl ether
5
5
py* Ph
HN
Br
Grubbs' 3rd catalyst
O
R
alkyl-grafted
NHAc
amphiphilic copolymers
radially arranged polymer backbone
2
partially cross-linked
interface
b)
OMe
m
O
HO
OH
HO
HO
OH
HO
HO
Ph
cat. K2OsO4
NMO
self-
assembled
cat. CuSO4
n
30
OH
NaN3 sodium ascorbate
OH
5
OH
5
monomers
HN
PEG
O
PMA-grafted
R
bent polymer backbone
Br
5
amphiphilic copolymers
NHAc
intermolecular
hydrogen bonding
c)
1a
Ac-Leu-3
(R = CH2CH(CH3)2, n = 3)
(R = CH CH(CH ) , n = 15)
(R = CH2CH(CH3)2, n = 30)
(R = CH(CH3)2, n = 15)
Ac-Leu-15
O
2
3 2
self-
assembled
Ac-Leu-30
Ac-Val-15
Ac-Ala-15
AcHN
N
H
5
R
(R = CH3, n = 15)
1
1
b-Leu (R = CH CH(CH ) )
b-Val (R = CH(CH3)2)
Ac-Phe-15
Ac-Trp-15
(R = phenylmethyl, n = 15)
2
3 2
(R = 3-indolylmethyl, n = 15)
amino acid-grafted
amphiphilic copolymers
radially arranged polymer backbone
1b-Ala (R = CH3)
1
1
b-Phe (R = phenylmethyl)
b-Trp (R = 3-indolylmethyl)
OMe
N
O
m
hydrophobic
:
hydrophobic
PMA chain
hydrophilic
PEG chain
hydrophobic
amino acid
PEG =
:
:
:
alkyl chain
N
N
Figure 2. Synthesis of amino acid-grafted amphiphilic
copolymers via ROMP. Mes: 2,4,6-trimethylphenyl. py*:
Figure 1. Spherical self-assemblies consisting of amphi-
philic brush-like copolymers conjugated with (a) alkyl
groups (previous work), (b) poly(methacrylate)s (previous
work), and (c) amino acids (this work) as hydrophobic side
chains.
3
-bromopyridine. NMO: 4-methylmorpholine-N-oxide.
Mn(PEG) = 2000.
bonding of amino acid- and oligopeptide-brushes in a hydro-
phobic core of self-assemblies (Figure 1c). Here, we report
the synthesis and evaluation of self-assemblies consisting of
various types of amino acid- and oligopeptide-grafted amphi-
philic copolymers and their application to high-contrast optical
tumor imaging in vivo.
an anhydrous and degassed CH Cl solution in the dark, and
2
2
used within two weeks after preparation. The abbreviation
MW(theor) means a theoretical molecular weight of copoly-
mers assuming that the transformation proceeded completely.
Number-average molecular weight (Mn,GPC) and PDI were
determined by GPC (Shodex GPC-104, Showa Denko K.K.,
Japan) with polystyrene standards.
Experimental
Synthesis of Polymers. Amphiphilic copolymers having
amino acids moieties were synthesized via ROMP, side chain
graft, and dihydroxylation (Figure 2). The representative syn-
thetic procedure of amino acid-grafted copolymers is shown
below. The synthetic procedures of oligoleucine-grafted copoly-
mers and tumor-targeting copolymers are summarized in the SI.
Grubbs’ 3rd catalyst (4.6 mg, 5.0 ¯mol) was placed in a
flame-dried Schlenk flask and dissolved in anhydrous and
degassed CH Cl (1.2 mL) at room temperature. To this solution
Materials. Grubbs’ 3rd catalyst was prepared from Grubbs’
2
nd catalyst with 3-bromopyridine according to the reported
6
procedure. Grubbs’ 2nd catalyst and potassium osmate
dihydrate were purchased from Sigma-Aldrich (St. Louis,
MO). Ethyl vinyl ether, sodium azide (NaN ), L-(+)-ascorbic
3
acid sodium salt, copper(II) sulfate pentahydrate (CuSO4¢
5
H O), N,N-dimethylformamide (DMF), chloroform (CHCl ),
2 3
acetone, and methanol (MeOH) were purchased from Nacalai
Tesque (Japan). Nile Red was purchased from Tokyo Chemical
Industry (TCI) Co., Ltd. (Japan). 4-Methylmorpholine N-oxide
2
2
was added a 0.10 M solution of 1a in anhydrous and degassed
CH2Cl2 (1.5 mL, 0.15 mmol) at room temperature. After stirring
for 10 min at room temperature, to the brown solution was added
a 0.10 M solution of 1b-Leu in anhydrous and degassed CH Cl
(
NMO), tetrahydrofuran (THF), and dichloromethane (CH Cl )
2 2
were purchased from Wako Pure Chemicals Inc. (Japan).
Dialysis membrane, Spectra/Por 6 (molecular weight cutoff
2
2
(0.75 mL, 0.075 mmol) at room temperature. The resulting
pale brown reaction mixture was stirred for 8 min at room
temperature and the reaction was quenched with ethyl vinyl
ether (2.0 mL, 20 mmol). After additional stirring for 5 min, the
organic solvent and excess amounts of ethyl vinyl ether were
(
MWCO): 3500 or 25000) was purchased from Spectrum
Laboratories Inc. (Rancho Dominguez, CA, USA).
Methods. Monomer 1a was synthesized according to
7
the reported procedures. The synthetic procedures of amino
acid-grafted monomers 1b and related oligoleucine-grafted
monomers are shown in the Supporting Information (SI). All
monomers were purified by gel permeation chromatography
removed under reduced pressure. The crude copolymer 2-Leu-
4
15 (64 mg, 4.9 ¯mol, 98%, MW(theor) = 1.2 © 10 , M
=
n,GPC
3
8.0 © 10 , PDI = 1.42) was obtained as a dark brown solid and
used for the next oxidation reaction without further purifica-
tion. The PDI values of obtained polymers are 1.10­1.50.
(GPC, recycling preparative HPLC LC-908, Japan Analytical
Industry Co., Ltd., Japan) with CHCl as an eluent, stocked as
3

K. Miki et al.
Bull. Chem. Soc. Jpn. Vol. 85, No. 12 (2012) 1279
1
1
2
-Leu-3: H NMR (400 MHz, CDCl ): ¤ 0.86­0.95 (br m,
Ac-Val-15: H NMR (300 MHz, CDCl ): ¤ 0.89­0.99 (m,
3
3
2
5H), 1.18­1.32 (br m, 74H), 1.81­2.60 (m, 322H), 3.31­3.66
735H), 1.25­1.62 (br m, 144H), 3.36­3.41 (m, 300H), 3.51­
4.15 (br m, 13398H), 4.13­4.25 (br m, 81H), 4.63­4.80 (br m,
(m, 353H), 5.12­5.36 (br m, 138H).
1
2
-Leu-15: H NMR (300 MHz, CDCl ): ¤ 0.91 (br m, 90H),
60H).
3
1
1
.25­1.62 (br m, 364H), 1.83­2.15 (m, 267H), 3.41 (br m,
Ac-Ala-15: H NMR (300 MHz, CDCl ): ¤ 1.20­1.37 (br m,
3
1
50H), 4.59 (br m, 15H), 5.24 (br m, 138H).
461H), 1.95­2.03 (m, 65H), 3.36­3.40 (br m, 290H), 3.47­3.90
(br m, 34210H), 4.19­4.24 (br m, 205H), 4.65­4.72 (br m,
1
2-Leu-30: H NMR (300 MHz, CDCl3): ¤ 0.87­1.00 (br m,
2
3
23H), 1.16­1.64 (br m, 1124H), 1.78­2.18 (m, 835H), 3.16­
60H).
1
.53 (br m, 212H), 5.06­5.53 (br m, 138H).
Ac-Phe-15: H NMR (300 MHz, CDCl ): ¤ 1.20­1.25 (m,
15H), 1.90­1.99 (m, 61H), 3.36­3.41 (br m, 162H), 3.50­3.85
3
1
2
-Val-15: H NMR (400 MHz, CDCl ): ¤ 0.81­0.97 (br m,
3
1
3
82H), 1.25­1.62 (br m, 3291H), 1.79­2.05 (m, 515H), 3.27­
(br m, 14026H), 4.19­4.21 (m, 60H), 4.50­4.60 (br m, 62H).
1
.65 (br m, 240H), 5.07­5.40 (br m, 138H).
Ac-Trp-15: H NMR (400 MHz, CDCl ): ¤ 3.37­3.39
3
1
2
-Ala-15: H NMR (300 MHz, CDCl ): ¤ 1.16­1.41 (br m,
(m, 416H), 3.41­3.90 (br m, 19792H), 4.20 (m, 56H), 4.69
(m, 60H).
Myr-Leu-30: 1H NMR (400 MHz, CDCl3): ¤ 0.85­1.06
(br m, 400H), 1.25­1.62 (br m, 620H), 3.36­3.38 (br, 174H),
3.40­3.87 (br m, 19433H), 4.15­4.35 (br m, 110H), 4.56­4.73
3
4
48H), 1.85­2.17 (br m, 314H), 3.15­3.63 (br m, 240H), 4.47­
4
.59 (br m, 10H), 5.05­5.49 (br m, 138H).
1
2
-Phe-15: H NMR (300 MHz, CDCl ): ¤ 1.05­2.16 (m,
3
4
423H), 2.88­3.07 (m, 20H), 3.36­3.68 (m, 167H), 5.06­5.45
(
br m, 138H). The chemical shifts of aromatic protons were
(br m, 60H).
1
difficult to determine because of their broad shapes.
Ste-Leu-30: H NMR (400 MHz, CDCl ): ¤ 0.85­1.06 (br
3
1
2
-Trp-15: H NMR (400 MHz, CDCl ): ¤ 1.07­1.57 (br m,
m, 24H), 1.25­1.62 (br m, 154H), 3.36­3.40 (br m, 151H),
3.43­4.01 (br m, 65215H), 4.13­4.31 (br m, 89H), 4.60­4.83
(br m, 60H).
3
2
5
846H), 1.75­2.20 (br m, 570H), 3.32­3.44 (br m, 250H),
.07­5.48 (br m, 138H). The chemical shifts of aromatic
protons were difficult to be determined because of their
broadened shapes.
To crude copolymer 2-Leu-15 (64 mg, 4.9 ¯mol) in DMF
Ac-(Leu)3-10: 1H NMR (300 MHz, CDCl3): ¤ 0.82­0.95
(br m, 142H), 1.17­1.62 (br m, 364H), 1.86 (m, 51H), 3.32­
3.37 (br m, 167H), 3.54 (br m, 15041H), 4.12­4.22 (m, 54H),
4.61­4.83 (br, 60H), 7.48­7.59 (br, 40H).
(
5.0 mL) was added NaN (19 mg, 0.30 mmol) at room tem-
3
1
perature. The reaction mixture was stirred at 50 °C overnight.
The organic solvent was removed under reduced pressure.
The crude polymer was dissolved in small amounts of CHCl3.
The mixture was filtered. The organic solvent was removed
under reduced pressure to afford a crude polymer. To a solu-
tion of the obtained polymer in THF (5.0 mL) was added a
Myr-(Leu) -10: H NMR (400 MHz, CDCl ): ¤ 0.85­1.06
3
3
(br m, 6H), 1.22­1.33 (br m, 25H), 1.57­1.94 (br m, 482H),
3.35­3.39 (m, 123H), 3.42­4.15 (m, 3294H), 4.16­4.23 (m,
68H), 4.56­4.73 (br m, 60H).
Myr-(Leu)6-5: 1H NMR (300 MHz, CDCl3): ¤ 0.79­0.99
(br m, 150H), 1.21­1.36 (br m, 337H), 3.36­3.40 (br m, 127H),
3.45­4.11 (br m, 8549H), 4.17­4.31 (br m, 53H), 4.61­4.83
(br m, 60H), 7.61 (br, 44H).
4
solution of propargyl ether (HC¸C­CH ­(OCH CH ) ­OMe,
2
2
2 n
(
n = ca. 45), 0.90 g, 0.45 mmol), CuSO ¢5H O (8.0 mg, 0.030
4 2
1
mmol), and L-(+)-ascorbic acid sodium salt (12 mg, 0.060
Ste-(Leu) -10: H NMR (300 MHz, CDCl ): ¤ 0.85­1.06
3
3
mmol) in H O (5.0 mL) at room temperature. After stirring the
(br m, 46H), 1.22­1.33 (br m, 130H), 1.89­2.69 (br m, 727H),
3.35­3.39 (m, 421H), 3.42­4.15 (m, 45707H), 4.16­4.23
(m, 98H), 4.56­4.73 (br m, 60H), 7.51­7.76 (br m, 44H).
2
reaction mixture overnight at room temperature, the organic
solvent was removed under reduced pressure. The PEGylated
copolymer was dissolved in acetone (5.0 mL) and MeOH (5.0
mL), and to this solution were added NMO (53 mg, 0.45 mmol)
and potassium osmate dihydrate (1.0 mg, 3.0 ¯mol) at room
temperature. After stirring at room temperature overnight, the
1
Ste-(Leu)6-5: H NMR (300 MHz, CDCl3): ¤ 1.10­1.40
(br m, 46H), 1.80­2.29 (br m, 2265H), 3.35­3.39 (m, 1908H),
3.42­4.15 (m, 76160H), 4.16­4.23 (m, 283H), 4.42­4.75 (br m,
134H), 7.49­7.72 (br m, 30H).
1
mixture was dialysed against H O for 2 days by using Spectra/
Myr-(Leu) -30: H NMR (400 MHz, CDCl ): ¤ 0.85­0.99
2
3
3
Por 6 (MWCO = 25000). Dihydroxylated copolymer Leu-15
(br m, 286H), 1.20­1.32 (br m, 54H), 3.39­3.42 (br m, 131H),
3.45­4.01 (br m, 20850H), 4.15­4.23 (br m, 64H), 4.60­4.72
(br m, 60H).
4
(54 mg, 0.75 ¯mol, 15% for 3 steps, MW(theor) = 7.6 © 10 )
was obtained as a white solid after lyophilisation. As observed
1
1
from H NMR spectra, olefin moieties were converted almost
Myr-(Leu)6-30: H NMR (300 MHz, CDCl3): ¤ 0.87­1.04
quantitatively. Some of the proton signals both of alkyl and
aromatic protons could not be detected because of the large
(br m, 231H), 1.20­1.31 (m, 397H), 2.41­2.46 (m, 51H), 3.34­
3.40 (m, 872H), 3.54­4.00 (br m, 35257H), 4.18­4.22 (m,
94H), 4.66­4.72 (br m, 60H).
number of PEG methylene protons.
1
1
Ac-Leu-3: H NMR (400 MHz, CDCl ): ¤ 3.36­3.40 (m,
Myr-Me-(Leu) -10: H NMR (300 MHz, CDCl ): ¤ 0.86­
3
3
3
4
050H), 3.40­3.95 (br m, 247700H), 4.62­4.71 (m, 60H).
1.00 (br m, 169H), 1.25­1.62 (m, 381H), 3.34­3.40 (m, 166H),
3.54­4.00 (br m, 45829H), 4.18­4.22 (m, 54H), 4.66­4.72
(br m, 60H).
1
Ac-Leu-15: H NMR (400 MHz, CDCl ): ¤ 0.88 (br m,
3
6
4
0H), 1.26 (br m, 46H), 3.38 (m, 90H), 3.64 (br m, 16063H),
.20 (br m, 99H), 4.67 (br m, 60H).
Preparation of Nanoparticles. Amphiphilic copolymers
1
¹1
Ac-Leu-30: H NMR (300 MHz, CDCl ): ¤ 0.85­0.95 (br m,
were dissolved in pure water (5 mg mL ) and stored for 30 min
3
2
86H), 1.25­1.62 (br m, 364H), 1.83­2.15 (m, 63H), 3.35­3.42
in the dark. The resulting solution was filtered by syringe filter
(0.45 ¯m pore size, PVDF filter membrane, Whatman, Florham
Park, NJ). The aqueous solution was stored at ambient tem-
(br m, 219H), 3.47­3.90 (br m, 29670H), 4.20­4.31 (br m,
1
08H), 4.65­4.73 (br m, 60H).

1
280 Bull. Chem. Soc. Jpn. Vol. 85, No. 12 (2012)
Amino Acid-Grafted Polymers for Tumor Imaging
perature in the dark for at least one day and used for tumor
imaging within two weeks after preparation. The diameters and
critical aggregation concentration of nanoparticles were also
measured within two weeks after preparation.
CA, USA, irradiation wavelength: ­exc = 745 nm, detected
wavelength: ­em = 840 nm). The NIRF images of polymer
solutions are summarized in Figure S5.
Cell Viability Test.
Mouse embryonic fibroblast cells
Characterization of Nanoparticles by TEM Image
Analysis. Transmission electron microscopy (TEM, Model
H-9000NAR, Power Supply 200 V, 60 Hz, 15 kVA, Single
Phase, Hitachi, Ltd., Tokyo, Japan) was used to visualize the
morphology of dried self-assemblies. Samples were dropped
onto a TEM copper grid covered with a carbon film (200 mesh,
Nisshin EM, Japan) and dried for 3 h. The distribution of
diameters was determined by using TEM images of at least fifty
self-assemblies. The TEM images are summarized in the SI.
Characterization of Size Distribution Using DLS. The
dynamic light scattering (DLS) was measured on an Otsuka
Photal SLS-6000HL equipped with a correlator (Photal GC-
derived from ICR were seeded onto 96-well plate (1000 cells/
well) and cultured in DMEM cell culture medium containing
the indicated concentration of imaging probe for 24 h in a cell
culture incubator (95% air and 5% CO , 37 °C, well-humidified
2
conditions). Then the cells were washed with phosphate
buffered saline (PBS, pH 7.4) twice and subjected to survival
fraction assay so as to quantify viable cells according to the
manufacturer’s instructions (Nacalai Tesque, Kyoto, Japan).
Cell viability was calculated as the percentage of the absorb-
ance value in each condition against that in medium without
any probes.
Imaging Experiments In Vivo. For the current study, the
human cervical epithelial adenocarcinoma cell line (HeLa) was
purchased from American Type Culture Collection. Cells were
cultured in 10% FBS-Dulbecco’s modified Eagle’s medium
1
000) and He-Ne laser (wavelength, 632.8 nm). The sample
solutions were diluted in pure water and filtered prior to analy-
sis (syringe filter, 0.45 ¯m pore size, PVDF filter membrane,
Whatman, Florham Park, NJ). The concentrations of the
and cultured in a well-humidified incubator with 5% CO and
2
¹
1
6
analyzed sample solutions were 0.05 g L (all copolymers
formed self-assemblies in this concentration). The measure-
ments were performed at 25 °C. The time correlation functions
were analyzed by single-exponential fitting and double-expo-
nential fitting. The measurements were performed at scattering
angles of 60, 75, 90, and 105° (30 min © 4). The diffusion
coefficient was calculated from the slope of the straight line
95% air at 37 °C. The suspension of HeLa cells (2 © 10 ) was
subcutaneously inoculated into the right hind leg of 6-week-
old nude mice (BALB/c nu/nu mice, SHIMIZU Laboratory
Supplies Co., Ltd., Kyoto, Japan). Polymers, Ac-Leu-15-
RGD, Ac-Leu-15-GA, Myr-(Leu) -10-GA, Myr-(Leu) -10-
3
3
RGD, Myr-(Leu) -5-GA, and Myr-(Leu) -5-RGD, in pure
6
6
¹
1
water (5.0 mg mL ) as fluorescent probes were prepared and
kept in the dark at room temperature for at least 24 h before
injection. The solutions of fluorescent probes (500 ¯L) were
intravenously injected in each experiment. Optical imaging
experiments to detect the distribution of the fluorescent probes
in the tumor-bearing mice were carried out with an IVIS-200 in
vivo imaging device (Xenogen, Alameda, CA, USA, irradi-
2
in the decay rate ¥ vs. q plot, where q is the scattering vector
(
q = 4³n sin ª/­, where n is the refractive index of solvent,
2
ª is the scattering angle, and ­ is the wavelength).
Measurement of Critical Aggregation Concentration
(cac). The cac values of copolymers were determined using
static light scattering (SLS, SLS-7000DL, Otsuka Electronics
Co., Ltd., Japan). The aqueous sample solutions of copolymers
in several concentrations were filtered prior to analysis (syringe
filter, 0.45 ¯m pore size, PVDF filter membrane, Whatman,
Florham Park, NJ). A He-Ne laser (15 mW, wavelength: 632.8
nm) was used for the measurement. SLS measurements (3 min
each) were carried out in a concentration range from 1.0 to
ation wavelength: ­exc = 745 nm, detected wavelength: ­em
=
840 nm). During the imaging, the mice were anesthetized with
¹
1
2.5% isoflurane gas in the oxygen flow (1.5 L min ). Images
were analyzed using Living Image 2.50-Igor Pro 4.09 software
(Xenogen), according to the manufacturer’s instructions. In
all imaging experiments, the fluorescent probe in the same
batch was injected to at least two mice and one set of experi-
ment was repeated at least twice to confirm reproducibility,
and representative images are shown. In order to emphasize
the areas where imaging probes specifically accumulated, we
set a threshold and represent intense fluorescence above it.
More details are shown in the SI. All of our animal experi-
ments were approved by the Animal Research Committee
of Kyoto University and carried out in accordance with its
guidelines.
¹
6
¹1
5
.0 © 10 g L at the detection angle, 90°. The measurements
were performed at 25 °C. Two separate measurements of static
light intensity were taken at each concentration, and the
reported cac values were determined for the average results.
More details are shown in the SI.
Dye-Releasing Test. To a polymer solution in degassed
¹
1
H O (2.0 mL, 1.0 mg mL ) was added a solution of Nile Red
2
¹
1
in degassed THF (2.0 ¯L, 1.0 ¯g mL ). The resulting solution
was immingled and then incubated in the dark at 25 °C for
1
5 min. The dye releasing behavior was determined by time-
Ex Vivo Experiments. One day after injection, mice were
sacrificed and the tumors as well as the major organs were
harvested for ex vivo imaging. Before imaging, the organs were
washed with PBS. Optical imaging experiments to detect
the distribution of the fluorescent probes in the major organs
dependent decrement of fluorescence intensities. The fluo-
rescence spectra were measured with a spectrofluorometer
(HORIBA JOBIN YVON, FluoroMax-3 integrating sphere).
The baseline fluorescence intensity was determined by measur-
ing the fluorescence intensity of a mixture of H O (2.0 mL)
were carried out with IVIS-200 (irradiation wavelength: ­exc =
2
¹
1
with a THF solution of Nile Red (2.0 ¯L, 1.0 ¯g mL ).
Measurement of Fluorescence Intensity of Polymer Solu-
tions. Optical imaging experiments to determine the fluores-
cence intensity of polymer solution in water were carried out
with an IVIS-200 in vivo imaging device (Xenogen, Alameda,
745 nm, detected wavelength: ­em = 840 nm). In all imaging
experiments, the fluorescent probe in the same batch was
injected to at least two mice and one set of experiments was
repeated at least twice to confirm reproducibility, and repre-
sentative images are shown. More details are shown in the SI.

K. Miki et al.
Bull. Chem. Soc. Jpn. Vol. 85, No. 12 (2012) 1281
(
a)
(b)
HO
Table 1. Properties of Self-Assemblies Consisting of Amino
Acid-Grafted Polymers
OH
HO
OH
HO OH
HO
Ph
O
O
N
NEt2
15
OH
30
OH
0.4 OH
HO
a)
DDLSb)
/nm
DTEMc)
/nm
OH
cac
Entry
Copolymer
HO
5
HO
HO
NHNs
/
©10 g L¹1
¹
4
C H11O
5
PEG
5
5
Nile Red
A
1
2
3
4
5
6
7
Ac-Leu-3
Ac-Leu-15
Ac-Leu-30
Ac-Val-15
Ac-Ala-15
Ac-Phe-15
Ac-Trp-15
2.8
0.61
1.5
0.71
11
206
204
190
234
252
218
220
115 « 32
170 « 45
146 « 31
159 « 42
69 « 17
(
c)
(d)
1
1
0.97
2.4
106 « 34
87 « 10
a) Measured by static light scattering. b) Measured by dy-
namic light scattering. c) Measured by transmission electron
microscopy.
0
5
0
60
750
560
750
wavelength/nm
wavelength/nm
All of our animal experiments were approved by the Animal
Research Committee of Kyoto University and carried out in
accordance with its guidelines.
Figure 3. Structures of (a) Nile Red and (b) alkyl-grafted
copolymer A. Emission spectra of Nile Red encapsulated
in self-assemblies of (c) Ac-Leu-15 and (d) alkyl-grafted
copolymer A measured after 0, 0.25, 0.5, 1, 2.75, 6, 10,
and 23 h. Excited wavelength: ­ex = 530 nm. Ns: 2-nitro-
benzenesulfonyl.
Results and Discussion
Synthesis of Amino Acid-Grafted Amphiphilic Copoly-
mers and Formation of Their Self-Assemblies. To check
the stability of polymeric self-assemblies in aqueous solution,
we initially synthesized amphiphilic block copolymers having
hydrophobic amino acid moieties and hydrophilic PEG moie-
The critical aggregation concentration (cac) values of
amphiphilic polymers were measured by SLS. Compared with
the cac values of Ac-Leu-3, Ac-Leu-15, and Ac-Leu-30,
copolymer Ac-Leu-15, with a 1:2 ratio of hydrophobic and
hydrophilic segments, formed the most stable self-assemblies
in water (Entries 1­3). The more hydrophobic the amino acid
moieties conjugated with a polymer main chain are, the more
stable the polymeric self-assemblies become (Entries 2, 4, and
5). Intermolecular hydrogen bonding between amide moieties
and hydrophobic interactions between alkyl moieties would
synergistically enhance the stability of self-assemblies of Ac-
Leu-15. Hydrogen bonding as well as ³­³ interaction between
aromatic rings in a hydrophobic core of Ac-Phe-15 and Ac-
Trp-15 would stabilize their self-assemblies.
8
ties by the reported procedure (Figure 2). ROMP of norbor-
nadiene monomers 1a having a bromo group and 1b having
various types of N-acetyl-capped hydrophobic amino acids
with a variety of mole ratios afforded diblock copolymers
2
in good yields. Bromide moieties on the side chains were
converted to PEG moieties via nucleophilic azidation followed
by copper-catalyzed [2 + 3] Huisgen cyclization.9 Finally,
double bonds on the main chain were transformed to vic-diols
under osmium-catalyzed dihydroxylation conditions.1
0,11
All polymers formed spherical self-assemblies in water.
Their hydrodynamic diameters (DDLS) determined by DLS
and diameters (DTEM) estimated by TEM are summarized in
Table 1. The theoretical molecular lengths of copolymers in
the extended form are approximately 30­40 nm, which might
indicate that the PEGylated copolymers formed self-assemblies
like vesicles or multimicellar aggregates.3 In our previous
work, the observed DTEM of dried ROMP-based polymeric self-
The fact that the cac values of Ac-Leu-15 and Ac-Val-15 are
smaller than those of amphiphilic copolymers having simple
alkyl chains instead of amino acid moieties (cac of alkyl-
¹4
¹1 3b,8a
grafted copolymers are larger than 4.0 © 10 g L )
would
support the presence of intermolecular hydrogen bonding in a
hydrophobic core. To check the existence of a polar hydro-
phobic core in self-assemblies of Ac-Leu-15, we performed
a dye-releasing test. In general, hydrophobic dyes, such as
Nile Red, are used as a model payload to estimate the effec-
tiveness of drug release because of the difference in fluores-
cence intensities of dyes between the inside and the outside
assemblies were slightly smaller than the D
of swollen
DLS
8
self-assemblies in water. Ac-Leu-15, Ac-Leu-30, and Ac-Val-
5 having bulky alkyl groups showed a similar tendency, while
the DTEM of Ac-Leu-3 was half the length of its DDLS (Entries
­4). Because Ac-Leu-3 has only a small number of hydro-
1
1
1
3
phobic segments, we suppose that Ac-Leu-3 might form
spherical nanogel-like self-assemblies involving a large num-
of self-assemblies. The dye-retention ability was determined
by a time-dependent decrease in the fluorescence intensities
(Figure 3). The fluorescence intensity of Nile Red in self-
assemblies of Ac-Leu-15 decreased more slowly than those
1
2
ber of water molecules in their supramolecular networks.
Indeed, Ac-Ala-15 having alanine as a less hydrophobic amino
acid formed fairly swollen self-assemblies such as nanogel-
like particles involving a large number of water molecules
3
b,8a
of alkyl-grafted copolymer A.
Seventy-five percent of dye
molecules were released from self-assemblies of A after 1 h,
while fifty percent of dye molecules still remained in self-
assemblies of Ac-Leu-15 even after 6 h. This indicates that
the self-assembly of Ac-Leu-15 has a polar hydrophobic core
in which polar hydrophobic Nile Red can be entrapped by
hydrophobic interaction as well as intermolecular hydrogen
(Entry 5). Ac-Phe-15 and Ac-Trp-15 having hydrophobic
amino acid moieties with bulky aromatic rings could not form
a tight hydrophobic core and therefore might provide some
room for water molecules, resulting in relatively large dif-
ferences between DDLS and DTEM (Entries 6 and 7).

1
282 Bull. Chem. Soc. Jpn. Vol. 85, No. 12 (2012)
Amino Acid-Grafted Polymers for Tumor Imaging
HO OH
HO
OH
HO
Ph HO
OH
OH
HO OH
HO
Ph
Table 2. Properties of Self-Assemblies Consisting of Leu-
cine- and Oligoleucine-Grafted Polymers
n
30
n
OH
30
OH
OH
OH
OH
a)
DDLSb)
/nm
HO
5
HO
5
HO
5
HO
5
cac
Entry
Copolymer
R¤
HN
PEG
HN
PEG
©10¹4 g L¹1
/
Leu
Leu
Leu
Leu
Leu
Leu
Leu
1
2
3
Ac-Leu-30
CH3
1.5
0.92
®
190
142
®
O
R'
Ac-(Leu) -10
3
Myr-Leu-30
Ste-Leu-30
(R' = C13H27, n = 30)
(R' = C17H35, n = 30)
Ac-(Leu) -5
6
4
Myr-Leu-30
C H
1
3
27
1.2
98
5
6
7
8
9
10
Myr-(Leu)3-10
Myr-(Leu)6-5
Ste-Leu-30
0.26
0.11
16
4.1
2.4
120
174
111
184
102
96
HO
OH
HO OH
HO
Ph
O
R'
n
30
OH
17
C H
35
OH
OH
Ac-(Leu)6-5
(R' = CH3, n = 5)
Ste-(Leu)3-10
HO
HO
Myr-(Leu) -5 (R' = C13H27, n = 5)
5
5
6
HN
PEG
Ste-(Leu) -5
Myr-(Leu) -30 (R' = C13H27, n = 30)
6
6
Leu
Leu
Leu
Ste-(Leu) -5 (R' = C17H35, n = 5)
Myr-(Leu)3-30
C13H27
4.6
6
11
Myr-(Leu)6-30
3.2
179
O
a) Measured by static light scattering. b) Measured by dynamic
light scattering.
R'
Ac-(Leu) -10 (R' = CH , n = 10)
3
3
Myr-(Leu) -10 (R' = C13H27, n = 10)
3
Myr-(Leu) -30 (R' = C13H27, n = 30)
3
assemblies, probably because of the additional stabilization
effect derived from a hydrophobic interaction between long
alkyl chains. In the case of N-stearoyl-protected polymers,
larger hydrophobic aggregates inside the self-assemblies might
disrupt intermolecular hydrogen bonding. From the compari-
son of copolymers having the same number of hydrophobic
segments, it is obvious that the proper number of leucine
moieties in a single polymer molecule is important for the
stability of self-assemblies (Entries 4, 10, and 11).
Ste-(Leu) -10 (R' = C17H35, n = 10)
3
Figure 4. Amphiphilic copolymers bearing leucine units as
hydrophobic chains. Leu: ­COCH(CH2CH(CH3)2)NH­.
bonding. It is known that the ­max of fluorescence emission
spectra of Nile Red bathochromically shifts in a more polar
1
4
solvent. The ­max of emission spectra of Nile Red in self-
assemblies of Ac-Leu-15 and copolymer A were observed at
6
54 and 640 nm, respectively. This also supports the existence
As mentioned above, we assumed that the high stability
of a polar hydrophobic core of self-assemblies results from
the intermolecular hydrogen bonding between amide N­H
groups and C=O groups. To confirm this, we prepared the N-
of a polar hydrophobic core in self-assemblies of amino acid-
grafted copolymers.
Synthesis of Oligoleucine-Grafted Amphiphilic Copoly-
mers and Formation of Their Self-Assemblies. It is easy to
exchange amino acid brushes for oligopeptides and to function-
alize an N-terminus with long alkyl fatty acyl groups. We
next examined the effects of the conjugation of oligoleucine
as hydrophobic brushes and their N-capping agents on the
stability of self-assemblies (Figure 4). We prepared amphi-
philic copolymers having trileucine and hexaleucine moieties
as hydrophobic brushes in a similar synthetic manner (see the
SI). To check the effect of the length of alkyl groups, myristoyl
and stearoyl groups were introduced into N-termini of oligo-
leucine moieties. All of the synthesized copolymers formed
spherical self-assemblies in water and their hydrodynamic
diameters were 100­190 nm, as measured by DLS. The cac
values of self-assemblies measured by SLS are summarized
in Table 2. We checked the effect of repeated leucine moieties
on the stability of self-assemblies by fixing the number of
leucine moieties in a single polymer molecule to 30. Although
methylated derivative of Myr-(Leu) -10 from the correspond-
ing monomer (Figure 5). Because N-methylated amide moie-
ties cannot form intermolecular hydrogen bonding, it was
3
supposed that N-methylated polymer Myr-Me-(Leu) -10 forms
3
unstable self-assemblies in water. Indeed, Myr-Me-(Leu) -10
3
formed spherical self-assemblies in water but their hydro-
dynamic diameter measured by DLS was 213 nm, much larger
than that of Myr-(Leu) -10 (D
of this polymer, determined by SLS, was 1.5 © 10 g L , six
= 120 nm). The cac value
3
DLS
¹4
¹1
¹
5
times higher than that of Myr-(Leu) -10 (cac = 2.6 © 10
3
¹
1
g L ). These data clearly show the existence of intermolecular
hydrogen bonding between leucine moieties in a hydrophobic
core.
Synthesis and Properties of Leucine- and Oligoleucine-
Grafted Copolymers Having Tumor-Targeting Agents.
Because the cac values of Ac-Leu-15 as well as polymers
having leucine or oligoleucine with an N-myristoyl-capping
group at an N-terminus were comparable to those of previ-
Ac-(Leu) -5 could not be synthesized because of the low
6
¹5
¹4
solubility of the corresponding monomer in common organic
solvents, we successfully synthesized Ac-Leu-30 and Ac-
ously reported PMA-grafted polymers (1.0 © 10 ­1.0 © 10
¹
1 3
g L ), we decided to synthesize a NIRF dye and targeting
agent-grafted Ac-Leu- and Myr-Leu-based polymers as tumor-
targeting probes. The main backbone of copolymers having
leucine moieties with N-acetyl groups and copolymers having
oligoleucine with N-myristoyl groups was prepared according
to the synthesis of the corresponding Ac-Leu and Myr-Leu
copolymers (Figure 6 and see also the SI). To monitor the
tumor-targeting behavior of self-assemblies in vivo, a NIRF
(
Leu) -10 and measured their cac values (Entries 1­3). The
3
cac data indicate that amphiphilic copolymers having oligo-
leucines rather than those having leucine form more stable self-
assemblies in water. In both copolymers having myristoyl and
stearoyl caps, the same tendencies were observed (Entries 4­9).
As to N-capping moieties, the introduction of an appropriate
length of an alkyl chain at the N-termini can stabilize self-

K. Miki et al.
Bull. Chem. Soc. Jpn. Vol. 85, No. 12 (2012) 1283
N-methylated trileucine
(no hydrogen bonding)
HO OH
HO
OH
HO
HO Ph
10
30
OH
OH
self-
assembled
OH
HO
5
5
MeN
PEG
O
(
H C) HCH C
3
2
2
radially arranged
polymer backbone
NMe
3
cac = 1.5 x 10-4 g L-1
Myr
DDLS = 213 nm
Figure 5. Self-assembly of N-methylated derivative of Myr-(Leu)3-10.
HO OH
HO
HO
OH
HO
HO
OH
HO
OH
HO
Ph
HO OH
HO
OH
HO
OH
HO
OH
HO
HO
Ph
1
5
30
0.4
1
X
30
0.4
1
OH
OH
OH
5
OH
5
OH
5
OH
5
OH
5
OH
5
OH
5
OH
5
HO
HO
HO
HO
HO
HN
PEG
ICG
TA
HN
PEG
ICG
TA
O
O
Myr-(Leu) -10-GA (X = 10, Y = 3, TA = GA)
3
Ac-Leu-15-GA (TA = GA)
NHAc Ac-Leu-15-RGD (TA = RGD)
Myr-(Leu) -10-RGD (X = 10, Y = 3, TA = RGD)
3
NH
Myr-(Leu)6-5-GA
(X = 5, Y = 6, TA = GA)
Y
Myr
Myr-(Leu)6-5-RGD (X = 5, Y = 6, TA = RGD)
O
HN
HO
H2N
O
O
NH HN
HO
NH
COOH
OH
ICG =
GA =
HO
RGD =
O
N
N
3
Cl-
H
NH HN
O
O
NH
N
H
N
H
Et
H
3
N
N
N
H
N
6
3
O
3
O
O
4
H
O
O
O
Ph
Figure 6. Amphiphilic copolymers having hydrophilic targeting molecules. All segments in bold brackets were polymerized
randomly.
indocyanine green (ICG) dye derivative was conjugated with
side-chain termini using a condensation reaction. ICG moieties
Table 3. Properties of Self-Assemblies
a)
DDLSb)
/nm
cac
were randomly grafted in hydrophilic segments to avoid
Entry
Copolymer
/©10 g L¹1
¹
4
fluorescence self-quenching of dye moieties.3,15 Tumor-target-
1
6
1
2
3
4
5
6
Ac-Leu-15-GA
16
19
136
129
119
133
128
134
ing agents (TA), such as glucosamine molecule (GA) and
1
7
Ac-Leu-15-RGD
Myr-(Leu)3-10-GA
Myr-(Leu)3-10-RGD
Myr-(Leu)6-5-GA
Myr-(Leu)6-5-RGD
cyclic RGD peptide (arginine­glycine­aspartic acid, RGD),
3.5
4.6
1.5
2.7
were also introduced for the purpose of active targeting of
tumor-specific receptors.
All of the copolymers also form spherical self-assemblies
in water, although their stabilities slightly decreased, probably
because of the disruption of hydrogen bonding as well as
hydrophobic interactions by random localization of hydro-
phobic planar dye moieties (Table 3). Although the diameters
of all copolymers are similar (around 120­130 nm), the cac
values of Ac-Leu-based copolymers are higher than those of
oligoleucine-grafted copolymers. Because the number of amide
moieties is smaller and the lengths of alkyl cappings are
shorter than those of self-assemblies having N-myristoyl-
capped oligoleucines, both the intermolecular hydrogen bond-
ing and hydrophobic interactions in Ac-Leu-based self-assem-
blies are assumed to be weaker. Therefore, Ac-Leu-based
copolymers formed less stable self-assemblies in water. Inter-
estingly, these less stable self-assemblies consisting of Ac-Leu-
based copolymers emit strongly in water, compared with Myr-
Leu-based polymers (Figure 7a). In general, hydrophobic dyes
emit strongly in hydrophobic media, such as nonpolar organic
a) Measured by static light scattering. b) Measured by dynamic
light scattering.
solvents, rather than protic solvents.15 The strong fluorescence
of Ac-Leu-based self-assemblies indicates that hydrophobic
ICG dye could localize deep inside the hydrophobic core
because of the weak interaction between hydrophobic side
chains. In contrast, planar ICG dye moieties could not disperse
in the tight hydrophobic core of self-assemblies of copolymers
having N-myristoyl-capped oligoleucines, resulting in lower
fluorescence in water. The cac values of GA-containing poly-
mers are slightly smaller than those having RGD moieties. We
assume that the cyclic RGD oligopeptide might be localized
inside a polar hydrophobic core, probably because of inter-
molecular hydrogen bonding between amino acid moieties,
thereby reducing the stability of self-assemblies. The cell

1
284 Bull. Chem. Soc. Jpn. Vol. 85, No. 12 (2012)
Amino Acid-Grafted Polymers for Tumor Imaging
(
a)
We also measured fluorescence intensities in two defined
regions of interest (ROIs): a tumor site (ROI 1) and the liver
and kidney (ROI 2) (Figure 8g). The contrast ratios, defined as
the ratio of fluorescence intensities of ROIs 1 and 2, showed
that all of the copolymers accumulated in tumor tissues
5
4
3
2
efficiently within 6 to 9 h, except for Myr-(Leu) -10-RGD
3
(
Figures 8h and 8i). The better tumor specificity of Ac-Leu-
based copolymers might support the reduced interaction
between tumor-targeting agents and leucine moieties in a polar
hydrophobic core.
1
In ex vivo experiments, although the uptake of self-
assemblies was observed in the liver and kidneys, the fluores-
cence detected in tumor tissues was significantly more intense
than that in lungs and heart (Figure 9).
0
a
b
c
d
e
f
(
b)
1
20
00
The contrast ratios of GA-containing copolymers were better
1
8
than those containing RGD moieties (Figures 8h and 8i). This
difference might be caused by the stability and the structure
of the self-assemblies. More stable GA-grafted self-assemblies
could efficiently accumulate in the tumor site by the EPR
effect (Table 3). Cyclic RGD peptides localized inside a polar
hydrophobic core might not efficiently come in contact with
¡v¢3 receptors on the tumor surface, while GA moieties
localized on the surface of self-assemblies could efficiently
recognize glucose transporters on tumor cells. In the previous
study, we confirmed that there are receptors of both GA and
RGD moieties on the surface of HeLa cells and that the
GA- and RGD-targeting probes similarly accumulated in tumor
1
8
6
4
2
0
0
0
0
0
5
50
125
250
g mL−1
500
concentration/
µ
3
a
tissues. Although it is difficult to explain differences in
recognition on the tumor surface that depend on targeting
moieties, the present result indicates that glucosamine is a
better tumor-targeting ligand for tumor-specific polymeric
nanoparticles having amino acid- and oligopeptide-based polar
hydrophobic cores. Further experiments related to a binding
assay between targeting agents conjugated with self-assemblies
and receptors on the tumor surface both in vitro and in vivo
would be necessary to form any conclusion on the interaction
between targeting moieties and their receptors.
Figure 7. (a) Fluorescence intensity of copolymers (a: Ac-
Leu-15-GA, b: Ac-Leu-15-RGD, c: Myr-(Leu)3-10-GA,
d: Myr-(Leu)3-10-RGD, e: Myr-(Leu)6-5-GA, f: Myr-
¹1
(Leu)6-5-RGD) in water (5 mg mL ). The data are given
as mean « standard deviation (n = 3). (b) Cell viability of
copolymers (Myr-(Leu)3-10-GA (black) and Myr-(Leu)6-
5-GA (gray)) after incubation for 24 h. The data are given
as mean « standard deviation (n = 4).
viability tests showed low cell cytotoxicity (IC = ca. 300
5
0
¹
1
¯
g mL ) of GA- and RGD-containing polymers (Figure 7b
Conclusion
and see also the SI).
In Vivo and Ex Vivo Tumor Imaging Using Leucine- and
Oligoleucine-Grafted Copolymers. We performed the in
vivo optical tumor imaging of targeting agents-conjugated
amphiphilic copolymers having amino acid moieties to check
their ability as tumor-targeting probes (Figure 8). Tumor-
targeting polymers were intravenously injected into nude mice
bearing a subcutaneous tumor xenograft in their right hind legs,
and the distributions were monitored using an optical in vivo
imaging device. The probes gradually accumulated in tumor
tissues and their fluorescence intensities exceeded the threshold
level and became detectable within 3 to 6 h after injection
In summary, we have developed amino acid- and oligopep-
tide-grafted amphiphilic brush-like copolymers via ROMP,
click reaction, and dihydroxylation for optical tumor imaging
in vivo. Among the copolymers investigated, amphiphilic
polymers having amino acid brushes with long alkyl chains,
such as leucine and valine, could form the most stable self-
assemblies in water because of the synergistic effects of
intermolecular hydrogen bonding and hydrophobic interaction.
By the combination of intermolecular hydrogen bonding and
³­³ interaction, self-assemblies of amphiphilic polymers
having aromatic ring-containing amino acids, such as phenyl-
alanine and tryptophan, can also be stabilized. Because the
stabilities of self-assemblies having amino acids with long
alkyl chains as well as aromatic rings are comparable to those
of PMA-grafted polymers in aqueous media, the conjugation
of hydrophobic amino acid side chains is one of the most
facile and useful methods to make polymeric self-assemblies
more stable in water. We also showed that the introduction of
oligoleucine brushes as well as capping with the proper length
of fatty acyl groups at an N-terminus affects both stability and
(Figures 8a­8f). Fluorescence intensities of Ac-Leu-15-GA
and Ac-Leu-15-RGD were noticeably stronger than those of
oligoleucine-grafted copolymers; this tendency is similar to
that observed in the measurement of fluorescence intensities
in water (vide supra). This indicates that the fluorescence of
ICG dye moieties in a hydrophobic core of self-assemblies
is not significantly decreased or quenched in vivo, and self-
assemblies that have a flexible hydrophobic core would be
more suitable as optical tumor imaging probes in vivo.

K. Miki et al.
Bull. Chem. Soc. Jpn. Vol. 85, No. 12 (2012) 1285
(a)
(b)
(c)
6
(x106)
3 h
9 h
(x106)
20
6
h
9 h
(x10 )
3 h
9 h
70
20
50
10
10
(d)
(e)
(f)
(x106)
20
6
h
9 h
(x106)
3 h
9 h (x106)
20
3 h
9 h
7
0
5
0
10
(i)
9
(g)
(h)
2
.0
.5
2.0
1
1.5
1.0
0.5
0
*
ROI 2
ROI 1
1.0
0
.5
0
0
6
12
time/h
18
24
0
6
12
time/h
18
24
Figure 8. NIRF images (photons s¹1) of tumor-bearing mice after injection (3 or 6 h and 9 h) of 500 ¯L of polymeric self-assemblies
(
a) Ac-Leu-15-GA, (b) Myr-(Leu)3-10-GA, (c) Myr-(Leu)6-5-GA, (d) Ac-Leu-15-RGD, (e) Myr-(Leu)3-10-RGD, and (f) Myr-
¹1
(Leu)6-5-RGD (5.0 mg mL ). Black: low intensity, yellow: high intensity, and red dashed circle: tumor site. In order to clarify the
data, lower and upper limits were appropriately established. (g) ROIs (region of interests: two orange circles were same size, and
used for all experiments in Figure 8). ROI 1: tumor site and ROI 2: liver and kidney. Fluorescence intensity ratios (ROI 1/ROI 2) of
optical images of (h) Ac-Leu-based copolymers (Ac-Leu-15-GA (blue), and Ac-Leu-15-RGD (red)) and (i) Myr-(Leu)3- and Myr-
(
Leu)6-based copolymers (Myr-(Leu)3-10-GA (purple), Myr-(Leu)3-10-RGD (green), Myr-(Leu)6-5-GA (blue), and Myr-(Leu)6-
-RGD (red)), are summarized. In order to clarify the data, lower limits were appropriately established. The data are given as
mean « standard deviation (n = 3). *: p < 0.1.
5
(
a)
(b)
2.0
1.5
1.0
0.5
0
6
4
2
0
*
*
*
*
*
*
*
*
tumor lungs heart kidneys liver
tumor lungs heart kidneys liver
Figure 9. ROI analysis of the ex vivo NIRF imaging (photons s¹1 cm¹2) of dissected major organs of the tumor-bearing mice one
¹1
day after intravenous injection of (a) Ac-Leu-15-GA and (b) Myr-(Leu)6-5-GA (dose: 500 ¯L, 5 mg mL ). The data are given as
mean « standard deviation (n = 3). **: p < 0.01.

1
286 Bull. Chem. Soc. Jpn. Vol. 85, No. 12 (2012)
Amino Acid-Grafted Polymers for Tumor Imaging
fluorescence intensity of self-assemblies. The in vitro cell
viability tests showed low cytotoxicity of all self-assemblies of
leucine- and oligoleucine-grafted copolymers having targeting
agents, while self-assemblies with strong fluorescence could
visualize a tumor site with high contrast in in vivo optical
tumor imaging. The present study demonstrates the potential of
amino acid- and oligopeptide-grafted amphiphilic copolymer-
based self-assemblies for versatile in vivo applications includ-
ing optical imaging, and might provide important principles
about designing the structure of highly functionalized tumor-
targeting vehicles delivering imaging agents and antitumor
drugs.
4
a) A. Makino, S. Kimura, React. Funct. Polym. 2011, 71,
272. b) X. L. Wu, J. H. Kim, H. Koo, S. M. Bae, H. Shin, M. S.
Kim, B.-H. Lee, R.-W. Park, I.-S. Kim, K. Choi, I. C. Kwon, K.
Kim, D. S. Lee, Bioconjugate Chem. 2010, 21, 208. c) M. Oba, S.
Fukushima, N. Kanayama, K. Aoyagi, N. Nishiyama, H. Koyama,
K. Kataoka, Bioconjugate Chem. 2007, 18, 1415.
5
a) S. Zhang, Nat. Biotechnol. 2003, 21, 1171. b) M. Ehrbar,
S. C. Rizzi, R. G. Schoenmakers, B. S. Miguel, J. A. Hubbell, F. E.
Weber, M. P. Lutolf, Biomacromolecules 2007, 8, 3000. c) E. P.
Holowka, D. J. Pochan, T. J. Deming, J. Am. Chem. Soc. 2005,
1
27, 12423. d) R. Deng, S. Liu, J. Li, Y. Liao, J. Tao, J. Zhu, Adv.
Mater. 2012, 24, 1889. e) M. R. Molla, S. Ghosh, Chem.®Eur. J.
2
012, 18, 9860. f) S. Han, S. Cao, Y. Wang, J. Wang, D. Xia, H.
Xu, X. Zhao, J. R. Lu, Chem.®Eur. J. 2011, 17, 13095. g) S. E.
This work is supported by Modality Xn program of Kyoto
University/Canon joint research project­Innovative Techno-
Hub for Integrated Medical Bio-imaging from MEXT Budget
for the Promotion of Science and Technology, and Funding
Program for Next Generation World-Leading Researchers
Paramonov, H.-W. Jun, J. D. Hartgerink, J. Am. Chem. Soc. 2006,
1
28, 7291.
T.-L. Choi, R. H. Grubbs, Angew. Chem., Int. Ed. 2003, 42,
6
1743.
7
C. Yip, S. Handerson, G. K. Tranmer, W. Tam, J. Org.
(NEXT Program), Japan. We acknowledge Professor Takeshi
Chem. 2001, 66, 276.
Abe and Dr. Kohei Miyazaki at Kyoto University for TEM
measurements, and Ms. Akiyo Morinibu at Kyoto University
for technical assistance in in vivo imaging experiments.
8
a) K. Miki, K. Oride, S. Inoue, Y. Kuramochi, R. R. Nayak,
H. Matsuoka, H. Harada, M. Hiraoka, K. Ohe, Biomaterials 2010,
31, 934. b) K. Miki, Y. Kuramochi, K. Oride, S. Inoue, H. Harada,
M. Hiraoka, K. Ohe, Bioconjugate Chem. 2009, 20, 511.
Supporting Information
9
H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem.,
Int. Ed. 2001, 40, 2004.
a) H. C. Kolb, M. S. VanNieuwenhze, K. B. Sharpless,
Synthesis of monomers and polymers, TEM, DLS, and
SLS measurements, and details of in vivo and ex vivo tumor
imaging. This material is available free of charge on the web
at: http://www.csj.jp/journals/bcsj/.
1
0
Chem. Rev. 1994, 94, 2483. b) P. Dupau, R. Epple, A. A. Thomas,
V. V. Fokin, K. B. Sharpless, Adv. Synth. Catal. 2002, 344, 421.
11
In the previous study, we have found that the dihydroxyl-
ation of double bonds on the polymer backbone improved the
stability of self-assemblies compared with non-dihydroxylated
copolymers. See Ref. 8a.
References
1
a) A. Llevot, D. Astruc, Chem. Soc. Rev. 2012, 41, 242.
b) F. M. Kievit, M. Zhang, Adv. Mater. 2011, 23, H217. c) J. A.
Barreto, W. O’Malley, M. Kubeil, B. Graham, H. Stephan, L.
Spiccia, Adv. Mater. 2011, 23, H18. d) C. Minelli, S. B. Lowe,
M. M. Stevens, Small 2010, 6, 2336. e) G. Orive, O. A. Ali, E.
Anitua, J. L. Pedraz, D. F. Emerich, Biochim. Biophys. Acta, Rev.
Cancer 2010, 1806, 96. f) S. Aluri, S. M. Janib, J. A. Mackay, Adv.
Drug Delivery Rev. 2009, 61, 940.
12 a) Y. Sasaki, K. Akiyoshi, Chem. Lett. 2012, 41, 202. b) Z.
An, Q. Qiu, G. Li, Chem. Commun. 2011, 47, 12424. c) Y. Sasaki,
K. Akiyoshi, Chem. Rec. 2010, 10, 366.
13 a) E. R. Gillies, T. B. Jonsson, J. M. J. Fréchet, J. Am.
Chem. Soc. 2004, 126, 11936. b) A. Klaikherd, C. Nagamani, S.
Thayumanavan, J. Am. Chem. Soc. 2009, 131, 4830.
14 A. K. Dutta, K. Kamada, K. Ohta, J. Photochem. Photo-
biol., A 1996, 93, 57.
2
a) M. Yokoyama, J. Exp. Clin. Med. 2011, 3, 151. b) H.
Cabral, K. Kataoka, Sci. Technol. Adv. Mater. 2010, 11, 014109.
c) D. B. Pike, H. Ghandehari, Adv. Drug Delivery Rev. 2010, 62,
15 a) W. West, S. Pearce, J. Phys. Chem. 1965, 69, 1894.
b) A.-K. Kirchherr, A. Briel, K. Mäder, Mol. Pharmaceutics 2009,
6, 480.
16 Z. Cheng, J. Levi, Z. Xiong, O. Gheysens, S. Keren, X.
Chen, S. S. Gambhir, Bioconjugate Chem. 2006, 17, 662.
17 a) W. Arap, R. Pasqualini, E. Ruoslahti, Science 1998, 279,
377. b) M. Schottelius, B. Laufer, H. Kessler, H.-J. Wester, Acc.
Chem. Res. 2009, 42, 969.
18 We performed the in vivo imaging of an amphiphilic
copolymer bearing a tumor-targeting folate moiety as a control
experiment to check the tumor-targeting ability of GA and RGD
moieties. From this result, we confirmed that GA- and RGD-
bearing self-assemblies could accumulate in tumor tissues by both
passive and active targeting.
167. d) A. S. Mikhail, C. Allen, J. Controlled Release 2009, 138,
214. e) M. E. Fox, F. C. Szoka, J. M. J. Fréchet, Acc. Chem. Res.
2009, 42, 1141. f) E. Segal, R. Satchi-Fainaro, Adv. Drug Delivery
Rev. 2009, 61, 1159. g) Y. Matsumura, K. Kataoka, Cancer Sci.
009, 100, 572. h) A. K. Iyer, G. Khaled, J. Fang, H. Maeda, Drug
2
Discovery Today 2006, 11, 812. i) Y. Matsumura, H. Maeda,
Cancer Res. 1986, 46, 6387.
3
a) K. Miki, A. Kimura, K. Oride, Y. Kuramochi, H.
Matsuoka, H. Harada, M. Hiraoka, K. Ohe, Angew. Chem., Int. Ed.
011, 50, 6567. b) K. Miki, K. Oride, A. Kimura, Y. Kuramochi,
H. Matsuoka, H. Harada, M. Hiraoka, K. Ohe, Small 2011, 7,
536.
2
3