Development of polymeric drug delivery system for recognizing vascular endothelial dysfunction

Article abstract of DOI:10.1016/j.bmc.2008.01.006

The vascular endothelium plays an important role in regulating vascular homeostasis. Damage to the endothelium can lead to cardiovascular diseases such as arteriosclerosis. Therefore, early-stage detection and evaluation of vascular endothelium dysfunction would be very important for effective diagnosis and therapy. We synthesized a polymeric drug carrier bearing an Evans blue analogue as a probing unit for endothelium injury. The polymeric carrier spontaneously formed stable nanoparticles with micelle-like structure in aqueous media and could encapsulate hydrophobic doxorubicin (DOX). The encapsulated DOX showed a sustainable release profile over a period of 10-60 h depending on the loaded DOX concentration. The polymeric carrier specifically adsorbed against the endothelium-injured site in extracted porcine aorta. These properties of the polymeric drug carrier will be suitable for specific drug delivery to endothelium dysfunctional region.

Full text of DOI:10.1016/j.bmc.2008.01.006

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2811–2818
Development of polymeric drug delivery system for
recognizing vascular endothelial dysfunction
Kenjiro Ikuta,a Takeshi Mori,a,b,c Tatsurhiro Yamamoto,b Takuro Niidome,a,b,c
Hiroaki Shimokawae and Yoshiki Katayamaa,b,c,d,
*
aGraduate School of Systems Life Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan
bDepartment of Applied Chemistry, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan
cCenter for Future Chemistry, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan
dJapan Science and Technology Agency, CREST, 4-1-8 Honmachi, Kawaguchi 332-0012, Japan
eDepartment of Cardiovascular Medicine, Graduate School of Medicine, Tohoku University, 1-1 Seiryo-machi,
Aoba-ku, Sendai 980-8574, Japan
Received 8 November 2007; revised 3 January 2008; accepted 4 January 2008
Available online 10 January 2008
Abstract—The vascular endothelium plays an important role in regulating vascular homeostasis. Damage to the endothelium can
lead to cardiovascular diseases such as arteriosclerosis. Therefore, early-stage detection and evaluation of vascular endothelium dys-
function would be very important for effective diagnosis and therapy. We synthesized a polymeric drug carrier bearing an Evans
blue analogue as a probing unit for endothelium injury. The polymeric carrier spontaneously formed stable nanoparticles with
micelle-like structure in aqueous media and could encapsulate hydrophobic doxorubicin (DOX). The encapsulated DOX showed
a sustainable release profile over a period of 10–60 h depending on the loaded DOX concentration. The polymeric carrier specifically
adsorbed against the endothelium-injured site in extracted porcine aorta. These properties of the polymeric drug carrier will be suit-
able for specific drug delivery to endothelium dysfunctional region.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
For the diagnosis of and therapy for vascular disease,
many studies utilizing such tools as MRI, fluorescence
imaging, and drug and gene delivery have been re-
ported.5–10 In local drug delivery methods, the use of
drug- or gene-coated stents provides one of the most
promising approaches.9 These stents are coated with
polymers or gels including therapeutic agents. Elutions
of these therapeutic agents are controlled by the poly-
mer or gel. This method has primarily been used to pre-
vent restenosis after surgical treatment. Another
strategy is active targeting using drug carriers with spe-
cific ligands of marker proteins expressed on the cell sur-
face in vascular disease regions. ICAM-1, VCAM-1, and
E/P selectin are known to be expressed in dysfunctional
endothelium.11,12 Therefore, drug carriers such as lipo-
some and polymeric micelle modified with ligands13,14
or antibodies for these adhesion molecules14–18 and fib-
urin19–21 have been widely investigated as targeting tools
for vascular diseases.
The vascular endothelium is a cellular monolayer on the
inside blood vessel wall and plays an important role in
regulating vascular homeostasis.1–4 When the endothe-
lium is exposed to free radicals, hypertension, and oxi-
dized low-density lipoprotein (LDL), the endothelium
causes inflammation leading to the formation of athero-
sclerosis. Its earliest changes include endothelial dys-
function such as increasing endothelial permeability,
adherence of plasma constituents, and attraction of
monocytes.2 This endothelial dysfunction leads to for-
mation of a fatty streak in atherosclerosis and finally
brings about vascular disease such as the spasms in myo-
cardial infarction.2,4 Therefore, the detection of endo-
thelial dysfunction is important for early diagnosis of
and therapy for myocardial disease.
Keywords: Arteriosclerosis; Endothelium dysfunction; Drug delivery
systems; Evans blue analogue.
We have focused on a change of physical properties of
the endothelium surface in the early-stages of atheroscle-
*
Corresponding author. Tel./fax: +81 92 802 2850; e-mail:
ykatatcm@mbox.nc.kyushu-u.ac.jp
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.01.006

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K. Ikuta et al. / Bioorg. Med. Chem. 16 (2008) 2811–2818
rosis, and found recently that some organic dye mole-
cules can selectively adsorb to an injured endothelium
region. In these dyes, Evans blue (EB) shows the most
effective discriminating ability. Thus we have recently re-
ported specific MRI imaging of injured carotid artery in
rat with newly synthesized EB-based contrast agent, EB-
DTPA-Gd.22,23
namic light scattering and also investigated the
targeting ability of the nanoparticle using extracted por-
cine aorta. Our results showed that the nanoparticle
incorporating DOX demonstrates specific adsorption
to the injured aorta. Thus, our polymeric drug carrier
will potentially be suitable for specific drug delivery to
endothelium dysfunctional regions.
In this report, we describe the synthesis and evaluation
of endothelial dysfunction-specific drug carriers using
EB analogue (Fig. 1a and b). We synthesized a poly-
meric carrier, which consisted of a hydrophobic main
chain [poly(N-tert-butylacrylamide)], hydrophilic graft
chain [poly(ethylene glycol)], and EB analogue as a
probing unit for vascular disorder. This copolymer is ex-
pected to form a micelle-like nanoparticle24 that has a
hydrophobic core of poly(N-tert-butylacrylamide) and
a hydrophilic outer shell of poly(ethylene glycol). We
encapsulated doxorubicin (DOX) in the nanoparticle
as an inhibiting drug of smooth muscle cell prolifera-
tion.25 We then characterized the nanoparticle with dy-
2. Results and discussion
2.1. Loading of DOX in the polymeric carriers
We designed a graft-type polymeric carrier (Fig. 1a and
c) in which the hydrophobic main chain was grafted
with hydrophilic PEG chains and was modified with a
sensing unit for recognizing endothelium dysfunction.
This copolymer was synthesized by radical copolymeri-
zation with PEG macromonomer, NBAAm, and Boc-
AS as a precursor of the EB analogue (Fig. 1c). EB ana-
logue modification was achieved by diazo-coupling of
a
b
Nanoparticle
Inner blood Vessel
Drug
Endothelium
Dysfunction
Accumulation
Endothelium
Basal Membrane
Evans Blue Analogue
Smooth Muscle Cell
c
O
O
AIBN
HN
k
+
HN
+
m
l
O
n
O
HN
HN
O
NH
O
O
O
NH
O
O
k
NH
OH
SO3H
m
l
O
n
O
m
l
O
n
O
HCl, NaNO2
SO3H
TFA
HN
k
HN
HN
k
HN
NH2
N
N
O
O
OH
HO3S
NH
SO3H
Figure 1. (a) Schematic representation of spontaneous formation of drug-loaded polymeric carrier bearing Evans blue analogue, (b) targeting
concept of the dye-bearing polymeric carrier to endothelium dysfunction site, and (c) the scheme of synthesis for polymeric carriers.

K. Ikuta et al. / Bioorg. Med. Chem. 16 (2008) 2811–2818
Table 1. Fractions of each monomeric unit in copolymers
2813
Sample code
In feed NBAAm/Boc-AS/PEG (mol/mol/mol)
In copolymer
EB analogue (wt%)
Conversion (%)
PEG (mol%)
BEP10-1
BEP10-5
BEP20-5
BEP30-5
BEP0-5
89/10/1
85/10/5
75/20/5
65/20/5
95/0/5
3.8
4.2
8.8
5.3
—
0.3
7.0
4.7
3.5
2.4
4.8
75.4
54.0
66.7
68.3
71.8
42.0
BEP0-10
90/0/10
—
Table 2. Molecular weights of copolymers
2.2. Characterization of polymeric carriers
Sample code
Mn
Mw
Mw/Mn
The polymeric carrier was expected to form micelle-like
structure consisting of a hydrophilic PEG shell and a
hydrophobic polyNBAAm core in aqueous solution.
Therefore, a copolymer with insufficient PEG content
would not disperse stably in deionized water after the
dialysis. Indeed, BEP10-1, which has the smallest PEG
content (0.3 mol%) in the synthesized copolymers,
formed large aggregates (average diameter = 1.5 lm)
due to the low dispersion stability. In contrast, BEP10-
5, which contains a large number of PEG chains
(7.0 mol%), formed stable small particles with an aver-
age diameter of 152 nm. This particle is considered to
have consisted of about 25–30 polymer molecules, which
were estimated with static light scattering experiment
using Zetasizer Nano ZS.
BEP10-1
BEP10-5
BEP20-5
BEP30-5
BEP0-5
18,000
20,400
11,800
16,500
19,600
69,900
26,800
1.59
1.76
3.76
3.27
3.05
5.84
36,000
44,400
54,000
59,800
408,000
BEP0-10
the copolymer with 1-amino-8-naphthol-2,4-disulfonic
acid monosodium salt. The conditions of polymeriza-
tion and the molecular weights of the obtained polymers
are shown in Tables 1 and 2, respectively. When the
mole fraction of Boc-AS was increased, the molecular
weight distribution of the copolymer became wider
(BEP10-5, BEP20-5, BEP30-5). Because of the wide
molecular weight distribution, we did not use BEP20-5
and BEP30-5 for DOX loading experiment.
The average size and f-potential of DOX-loaded poly-
meric carriers (BEP10-5) are shown in Figure 2a–c.
When DOX was encapsulated, the average diameter of
the polymeric carrier became smaller than those of
non-loaded carriers (Fig. 2a and b). This change was
probably due to the increase in the compaction of the
hydrophobic core with the hydrophobic interaction be-
tween DOX and the EB analogue, because BEP0-5
and BEP0-10 that did not contain EB analogue could
not incorporate DOX. The f-potential of non-loaded
polymeric carrier was ꢀ20 mV (Fig. 2c). This result is
most likely due to the localization of the anionic EB
analogue on the surface of the particle. In contrast,
the f-potential approached to neutral with the DOX
loading, indicating that anionic EB core of the poly-
meric carrier was neutralized by the loading of cationic
DOX. This interaction between DOX and the EB ana-
logue would be based on the electrostatic as well as
the hydrophobic interaction.
Loading of DOX into the polymeric carrier was
achieved by changing the solvent from DMF to deion-
ized water with dialysis, and non-loaded DOX was re-
moved by ultrafiltration. The quantities of loaded
DOX in the polymeric carrier (BEP10-5) under various
loading conditions are summarized in Table 3. The
quantity of loaded DOX in the polymeric carrier in-
creased with increases in the DOX concentrations in
the preparation solutions (Run 1–6 and 7–9). In addi-
tion, the efficiency of DOX loading (loaded DOX/feed
DOX) also improved with increases in the carrier con-
centration (Run 4/7, 6/9). The maximum loading was
15.9 wt% in Run 9, and the loading efficiency was over
50%. These results indicate that these carriers can load
DOX with comparable efficiency to other reported poly-
meric carriers.26,27
Then, the stability of the polymeric carrier was evalu-
ated. The time course of the photon count rate per sec-
ond and the average size of DOX-loaded polymeric
carrier (Table 3, Run 9) in PBS(ꢀ) remained constant
up to 200 min (Fig. 2d). These results demonstrated
the high stability of the polymeric carrier under the
physical saline condition due to the hydrophilic PEG
shell.
Table 3. Amount of loaded DOX in BEP10-5 carrier
Run Polymer Feed DOX Loaded DOX/ Loaded DOX/
(mg/mL) (mg/mL)
polymer (wt%) feed DOX (wt%)
1
2
3
4
5
6
7
8
9
0.4
0.4
0.4
0.4
0.4
0.4
2.0
2.0
2.0
0.06
0.1
0.2
0.3
0.4
0.6
0.3
0.5
0.6
4.6
10.0
10.8
14.4
14.4
15.8
5.9
31.0
39.8
21.6
19.2
14.4
10.6
39.0
44.8
53.1
2.2.1. Release of DOX from the polymeric carriers.
Determination of the release rate of DOX from the
polymeric carriers was carried out by measuring the
fluorescence of DOX in the eluting solution during
11.2
15.9

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K. Ikuta et al. / Bioorg. Med. Chem. 16 (2008) 2811–2818
180
24
20
16
12
8
BEP10-5
a
c
b
160
DOX loaded BEP10-5
140
120
100
80
60
40
20
0
4
0
1
10
100
1000
10000
0
0.2
0.4
0.6
Diameter (nm)
Feed DOXConcentration (mg/mL)
0
180
160
140
120
100
80
d
400
300
200
100
0
-5
-10
-15
-20
-25
60
40
20
0
0.0
0.2
0.4
0.6
0.8
0
50
100
150
200
Feed DOX Concentration (mg/mL)
Time (min)
Figure 2. Size distribution and f-potential of the polymeric carriers (1.0 mg/mL) in the presence or absence of DOX. (a) Size distribution of non-
loaded and DOX-loaded BEP10-5 (Run 9), (b) average diameter, (c) f-potential of the polymeric carrier, and (d) time course of the average diameter
and the mean count rate of DOX-loaded BEP10-5 (Run 9) after the dissolution in PBS(ꢀ).
the dialysis of the carrier dispersion. When 0.6 mg/mL
of DOX was used for the loading (Table 3, Run 9),
26% of the loaded DOX was released over a period
of 10 h (Fig. 3a, •). The time course of the DOX re-
lease did not have a burst phase which was observed
in free DOX (Fig. 3a, ꢁ), suggesting a relatively strong
interaction between DOX and the carrier. When the
loading DOX concentration was 0.3 mg/mL (Table 3,
Run 7), the time course of the DOX release similarly
showed no burst phase (Fig. 3b, •). In both the
DOX concentrations, DOX was released gradually
over a period of 10–60 h. These sustainable release pro-
files will be sufficient for successful application as a
drug carrier.
2.2.2. Evaluation for the adsorption of polymeric carrier
to Matrigel-coated and cultured endothelium surfaces.
The basal membrane and extracellular matrix are ex-
posed in the injured endothelial area of vascular surface
due to a detachment of endothelium. Since Matrigel
consists of component proteins of basal membrane, we
used a Matrigel-coated surface as a model of injured
endothelial region. The adsorption of the polymeric car-
rier was evaluated from the decrease of the carrier con-
centration in supernatant solution after treatment with a
Matrigel-coated surface. Matrigel was diluted with med-
ium and was used to coat each well of a 96-well plate.
Microscopic images of the gel-coated and cell-coated
wells are shown in Figure 4a and b, respectively. The
120
120
b
a
100
100
80
60
40
20
0
80
60
40
20
0
0
50
100
150
0
20
40
60
80
Time (hour)
Time (hour)
Figure 3. Time course of DOX release from the carrier during dialysis. 0.6 mg/mL (a) or 0.3 mg/mL (b) of DOX was used in the nanoparticle
preparation. Closed and open circles indicate DOX-loaded carriers and free DOX.

K. Ikuta et al. / Bioorg. Med. Chem. 16 (2008) 2811–2818
2815
b
a
110.0
100.0
90.0
80.0
70.0
60.0
50.0
110.0
c
d
100.0
90.0
80.0
70.0
60.0
50.0
non
coated
0.98
1.96
3.37
4.90
0.0
0.5
1.0
2.0
3.0
5.0
Cell Concentration (x104 cell/well)
Matrigel Concentration (mg/mL)
Figure 4. Microscopic images of Matrigel-coated (a) or endothelium-coated (b) wells, and changes of the carrier concentrations in supernatant after
the treatment with Matrigel-coated (c) or endothelium-coated (d) wells.
reddish violet color resulting from the carrier adsorption
was observed clearly in the gel-coated well. The adsorp-
tion of the carrier increased with increases in the Matri-
gel concentration in the coating solution (Fig. 4c). On
the other hand, the adsorption of the carrier to endothe-
lial cells was not observed irrespective of the number of
cells in the well (5000–50,000 cell/well) (Fig. 4d). These
results indicate that the polymeric carrier tended not
to adsorb to areas of intact endothelium, but did adsorb
to an extracellular proteins in areas of dysfunctional
endothelium.
DOX-loaded control carrier poly(benzylmethacryla-
mide-co-PEG), which did not have EB analogue moi-
ety, the adsorption of the carrier to both the injured
and intact areas was not observed. These results indi-
cate that the EB analogue modified on the carrier is
essential for endothelium-injured sites’ recognition.
The introduced PEG side chains may suppress the
interaction between the EB analogue and endothe-
lium-injured site, due to the steric hindrance. However,
the negative value of f-potential of our particle indi-
cates that the PEG side chains cannot shield the nega-
tive charge of the EB analogue units and the particle
actually recognized the endothelium injury. Thus, we
expect that our polymer may form loose and dynamic
complex, which is different from common core-shell
type polymer micelle that is seen in block copolymer’s
micelle. The EB analogue units may be able to appear
sometimes on the surface of the particle due to the dy-
namic motion of the polymer chains. Although the tar-
get molecule of the EB analogue recognition has not
been clarified yet, the experiment using a Matrigel-
coated surface revealed that the EB analogue adsorbs
to extracellular matrix. The EB analogue of polymeric
carrier may bind to some proteins expressed specifically
in the inflammation site because a MRI contrast agent
having the same EB analogue unit was also adsorbed in
early-stage atherosclerosis in ApoE knockout mice.28
2.3. In vitro evaluation of the targeting ability of
polymeric carriers against endothelium-injured area
The targeting abilities of the polymeric carrier and
drug-loaded carrier were investigated using excised por-
cine aorta, in which endothelium of half area was in-
jured mechanically. Marked accumulations of the
polymeric carrier were observed only in the injured area
(Fig. 5a–f). The reddish color became deeper when
polymeric carrier concentrations were increased
(Fig. 5c and d). DOX concentrations on the aorta slip
were compared between injured and intact areas of
endothelium. Thus, the same area (1 · 1 cm) of injured
or normal regions was taken and lysed. The DOX was
then extracted with 2-propanol. As shown in Figure 5g,
when the aorta sample was treated with the saline solu-
tion of the DOX-loaded carrier, the amount of DOX in
the injured area was found to be fourfold greater than
that in the intact area. In the case of serum solution, the
accumulation of DOX to the injured area was still three
times larger. On the other hand, in the case of the
3. Conclusion
We report here a drug delivery system for vascular endo-
thelial dysfunction using EB analogue as a ligand. The

2816
K. Ikuta et al. / Bioorg. Med. Chem. 16 (2008) 2811–2818
a
d
b
c
e
f
3
g
Endothelium Injured
Endothelium Intact
2.5
2
1.5
1
0.5
0
DOX-loaded
Carrier in
Saline
DOX-loaded
Carrier in
Serum
DOX-loaded
Control Carrier
in Serum
Figure 5. Specific adsorption of the polymeric carriers against porcine aorta having injured (left area) and intact regions (right area). Photographs of
the opened aorta slips after treatment with the polymeric carriers (a–f). Photoimages after the treatment with saline (a), BEP10-1 (20 mg/mL) (b),
BEP10-5 (20 mg/mL) (c), BEP10-5 (40 mg/mL) (d), DOX-loaded BEP10-5 (Run 9) (20 mg/mL in saline) (e), or DOX-loaded BEP10-5 (Run 9)
(20 mg/mL in serum) (f). (g) Amounts of DOX in the injured (closed bar) and intact areas (open bar).
synthesized polymeric carrier was found to be capable of
encapsulating hydrophobic drug at sufficient concentra-
tions for delivery to an endothelium dysfunction region.
In addition, the polymeric carrier was observed to accu-
mulate specifically to the endothelium injured region of
the porcine aorta slip. These properties of the polymeric
carrier are potentially useful for the development of
medical treatment for vascular disease.
endothelial cell (HCAEC) was purchased from Cambrex
(USA). Matrigel was purchased from Becton–Dickinson
and Company (NJ, USA). Endothelium Growth Med-
ium was purchased from Takara (Tokyo, Japan).
4.2. Synthesis of polymeric carriers
N-Boc-4-aminostyrene (Boc-AS) was synthesized from
4-aminostyrene and di-tert-butyl dicarbonate according
to the literature.29 N-Methacryloyl-PEG was prepared
from NH2-PEG (Mw 5000) and N-methacryloyloxysuc-
cinimide according to the previously reported method.30
Polymeric carriers were synthesized by radical polymer-
ization of N-methacryloyl-PEG macromonomer,
NBAAm, and Boc-AS in N,N-dimethylformamide
(DMF) (total monomer concentration 0.5 M) at 75 ꢁC
overnight under a nitrogen atmosphere using AIBN as
a radical initiator. The copolymers were purified with
GPC (Bio-Beads S-X-1, Bio-Rad Laboratories, Rich-
mond, CA) by using DMF as an eluent. The desired
product was dissolved in trifluoroacetic acid for 3 h to
deprotect Boc-AS. The reaction solution was neutralized
with sodium bicarbonate and desalted with sodium
bicarbonate. The solution was lyophilized to yield a pale
yellow solid. The deprotected polymer was then dis-
4. Experimental
4.1. Materials
N-tert-Butylacrylamide (NBAAm), di-tert-butyl dicar-
bonate, sodium nitrite, N-acryloxysuccinimide, benzyl-
amine, and DOX were purchased from Wako Pure
Chemical Industries, Ltd (Osaka, Japan). 4-Aminosty-
rene and 1-amino-8-naphthol-2,4-disulfonic acid mono-
sodium salt were from Tokyo Kasei Co., Ltd (Tokyo,
Japan). O-(2-Aminoethyl)-O0-methylpoly-ethylene gly-
col (NH2-PEG, Mw 5000) was purchased from Sigma–
Aldrich Japan K.K. (Tokyo, Japan). 2,20-Azobis-isobu-
tyronitrile (AIBN) was purchased from Nacalai Tesque,
Inc. (Kyoto, Japan). Normal human coronary artery

K. Ikuta et al. / Bioorg. Med. Chem. 16 (2008) 2811–2818
2817
solved in distilled water/THF [1:1 (v/v)] containing
hydrochloric acid on an ice bath. Sodium nitrite was
added in small portions to the polymer solution, fol-
lowed by stirring for 30 min to diazotize below 5 ꢁC.
The diazonium salt solution was added dropwise to 1-
amino-8-naphthol-2,4-disulfonic acid monosodium salt
aqueous solution containing sodium bicarbonate on an
ice bath and then stirred for 2 h. The reaction mixture
was purified with GPC (Sephadex G-50, Amersham Bio-
sciences, Uppsala, Sweden) in distilled water and then
lyophilized to yield a reddish violet solid.
ment Ltd, Worcestershire, UK) at 37 ꢁC using a He–Ne
laser (k = 633 nm) as a light source.
4.5. Quantification of loaded and released DOX
The amounts of loaded drug in polymeric carriers were
determined as follows. The polymeric carrier was dis-
solved in DMF for dissociation and was ultrafiltrated.
Fluorescence of the DOX in the filtrate was measured
with a fluorescence spectrometer (RF-5300PC, Shima-
dzu Co. Ltd, Kyoto, Japan) (kex = 485 nm, kem
586 nm).
=
A control polymeric carrier poly(benzylmethacrylamide-
co-PEG), which did not contain EB analogue units and
was used for the targeting experiment using extracted
porcine aorta, was synthesized as follows. The poly(N-
acryloxysuccinimide) (polyNAS) was synthesized by
radical polymerization of N-acryloxysuccinimide in
DMF (monomer concentration 1.0 M) at 75 ꢁC over-
night under a nitrogen atmosphere using AIBN as a rad-
ical initiator. The obtained polymer was purified by
reprecipitation with acetone. The polyNAS was reacted
with benzylamine and NH2-PEG in DMF for 12 h to
obtain a desired copolymer. The reaction mixture was
purified with ultrafiltration (quantitative molecular
weight limit 70 kDa, Apollo, Orbital Biosciences, LLC,
MA, USA) and then the copolymer was dissolved in
deionized water and lyophilized to yield a pale yellow
solid.
The amount of DOX released from the polymeric carri-
ers was determined by the fluorescence intensity at
586 nm in the eluent of dialysis against PBS(ꢀ) using
dialysis tubing (molecular weight cut-off 10,000 Spec-
tra/Por CE, Spectrum Laboratories Inc., CA, USA).
4.6. Evaluation of the polymeric carrier adsorption onto
Matrigel or cultured endothelium
Matrigel coating was carried out by the thin coat meth-
od described as follows. Matrigel (9.8 mg/mL, 100–
500 lL) was diluted with Dulbecco’s modified Eagle’s
medium (DMEM) (500–900 lL) without serum and
phenol red. The solution 50 lL was dropped into each
well of a 96-well microtiter plate and incubated at
37 ꢁC for 2 h. After aspiration of the solution, Matrigel
coating plate was washed by DMEM without serum and
phenol red. HVAEC were also seeded into each well of a
96-well microtiter plate and incubated with supplement
(5% FBS, 0.04% hydrocortisone, 0.4% hFGF-B, 0.1%
VEGF, 0.1% R3-IGF-1, 0.1% ascorbic acid, 0.1%
hEGF, and 0.1% GA-1000) at 37 ꢁC under a 5% CO2
atmosphere. After an overnight incubation, the culture
medium was exchanged to DMEM without serum and
phenol red.
The molecular weight of the copolymer was determined
by GPC using TSKa-gel 3000 and 4000 (TOSOH Co.
Ltd, Tokyo, Japan). GPC was performed using TriSEC
302TDA (Viscotek, TX, USA) with refractive-index, vis-
cosity, and light-scattering detectors at 40 ꢁC. DMF
containing 10 mM LiBr was used as an eluting solvent
with a flow rate of 0.6 mL/min. The mole fractions of
EB analogue and PEG were evaluated with the UV–
vis absorbance spectrum (UV-2550, Shimadzu Co.
Ltd, Kyoto, Japan) and 1H NMR (400 Hz, JNM-
ECP400, JEOL Ltd, Tokyo, Japan).
The carrier solution (1.0 mg/mL) was then added to the
DMEM without serum and phenol red in the Matrigel
coating plate or the HVAEC seeded plate and incubated
at 37 ꢁC under a 5% CO2 for 30 min. Absorbance at
536 nm of the supernatant was measured by UV–vis
spectrophotometer (Beckman Coulter. Inc, CA, USA).
The phase contrast image was obtained by BZ-8000
(Keyence, Osaka, Japan).
4.3. Loading of DOX into carriers
Loading of drug into the polymeric carrier was carried
out by dialysis. DOX and the polymeric carrier were dis-
solved in DMF. The solution was dialyzed against
deionized water using dialysis bag (molecular weight
cut-off 10,000 Spectra/Por 6, Spectrum Laboratories
Inc., CA, USA), and the carrier solution was ultrafiltrat-
ed to exclude non-loaded DOX (quantitative molecular
weight limit 10 kDa, Apollo, Orbital Biosciences, LLC,
MA, USA) and lyophilized to yield a reddish violet
solid.
4.7. Evaluation of the targeting ability of polymeric
carriers
An excised porcine aorta was opened to a flat sheet. Half
the area of the slip was then scratched softly to cause
endothelium damage. The aorta was dipped into an
aqueous solution of the polymeric carrier or serum solu-
tion for 60 s and then washed with saline. DOX-loaded
poly(benzylmethacrylamide-co-PEG) carrier that did
not contain EB analogue was used as a control carrier
(average hydrodynamic diameter: 83.6 nm). The aorta
was cut to 1 · 1 cm2 and homogenized in acidified 2-pro-
panol (9/1 = 2-propanol/1 M hydrochloric acid).31 The
lysate solutions were allowed to stand at 4 ꢁC for 12 h
to extract the drug and then centrifuged at 8000g for
4.4. Size distribution and f-potential measurements
The non-loaded carriers or DOX-loaded carriers were
dissolved in distilled water or PBS(ꢀ) (1.0 mg/mL),
and the samples were filtered with a 0.45-lm filter.
The hydrodynamic diameter distributions and the f-po-
tential of non-loaded carriers or DOX-loaded carriers
were determined by Zetasizer Nano ZS (Malvern Instru-

2818
K. Ikuta et al. / Bioorg. Med. Chem. 16 (2008) 2811–2818
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Acknowledgment
We thank the Uehara Memorial Foundation for their
financial support.
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