Self-assembled micelles of well-defined pentaerythritol-centered amphiphilic A4B8 star-block copolymers based on PCL and PEG for hydrophobic drug delivery

Article abstract of DOI:10.1016/j.polymer.2011.04.054

Biodegradable star-shaped poly(ε-caprolactone) (PCL) with four arms were synthesized by ring-opening polymerization (ROP) from a symmetric pentaerythritol core via the ''core-first'' strategy. Subsequently, two samples of the amphiphilic A₄B₈ star-block copolymers with symmetrical topologies [4s(PCL-b-2sPEG)] were synthesized by a macromolecular coupling reaction between carboxyl-terminated poly(ethylene glycol) (PEG) and 4-arm star-shaped PCL macromers with eight -OH end groups. The latter was prepared by attaching 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoic acid (HHMPA) to 4sPCL using a simple two-step reaction sequence. The in vitro cytotoxicity test indicated no apparent cytotoxicity. The amphiphilic star-block copolymers are capable of self-assembling into spherical micelles in water at room temperature, and they possess low critical micelle concentrations (CMCs) of 2～8 mg/L in aqueous solution which was determined by fluorescence spectroscopy using pyrene as a probe. Transmission electron microscopy (TEM) measurement demonstrated that the micelles exhibit a spherical shape with a size range of 30～50 nm in diameter. In addition, the hydrophobic and anticancer drug, quercetin, is loaded effectively in the polymeric micelles, suggesting that these new materials are appropriate candidates as hydrophobic drug nanocarriers.

Full text of DOI:10.1016/j.polymer.2011.04.054

Polymer 52 (2011) 2799e2809
Contents lists available at ScienceDirect
Polymer
journal homepage: www.elsevier.com/locate/polymer
Self-assembled micelles of well-defined pentaerythritol-centered amphiphilic
A B star-block copolymers based on PCL and PEG for hydrophobic drug delivery
4
8
Mohammad Reza Nabid , Seyed Jamal Tabatabaei Rezaei , Roya Sedghi b, Hassan Niknejad c,
a
,
a
a,
*
d
b
b
Ali Akbar Entezami , Hossein Abdi Oskooie , Majid M. Heravi
Department of Chemistry, Shahid Beheshti University, G.C, 1983963113, Tehran, Iran
Department of Chemistry, School of Science, Azzahra University, Vanak, Tehran, Iran
Nanomedicine and Tissue Engineering Research Center, Shaheed Beheshti Medical University, Tehran, Iran
a
b
c
d
Lab of Polymer, Faculty of Chemistry, University of Tabriz, Tabriz, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 March 2011
Received in revised form
Biodegradable star-shaped poly(
3
-caprolactone) (PCL) with four arms were synthesized by ring-opening
polymerization (ROP) from a symmetric pentaerythritol core via the ‘‘core-first’’ strategy. Subsequently,
two samples of the amphiphilic A star-block copolymers with symmetrical topologies [4s(PCL-b-
sPEG)] were synthesized by a macromolecular coupling reaction between carboxyl-terminated poly
ethylene glycol) (PEG) and 4-arm star-shaped PCL macromers with eight eOH end groups. The latter
4 8
B
19 April 2011
2
(
Accepted 26 April 2011
Available online 5 May 2011
was prepared by attaching 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoic acid (HHMPA) to 4sPCL
using a simple two-step reaction sequence. The in vitro cytotoxicity test indicated no apparent cyto-
toxicity. The amphiphilic star-block copolymers are capable of self-assembling into spherical micelles in
water at room temperature, and they possess low critical micelle concentrations (CMCs) of 2w8 mg/L in
aqueous solution which was determined by fluorescence spectroscopy using pyrene as a probe. Trans-
mission electron microscopy (TEM) measurement demonstrated that the micelles exhibit a spherical
shape with a size range of 30w50 nm in diameter. In addition, the hydrophobic and anticancer drug,
quercetin, is loaded effectively in the polymeric micelles, suggesting that these new materials are
appropriate candidates as hydrophobic drug nanocarriers.
Keywords:
Star-block copolymers
Amphiphiles
Hydrophobic drug nanocarriers
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
permeability, biocompatibility and nontoxicity. The high olefin
content imparts polyolefin-like properties to PCL, while hydrolyti-
Over the past decades, polymeric micelles self-assembled from
amphiphilic block copolymers have received significant attention
as drug and gene nanocarriers due to their unique characteristics
such as core-shell structure, mesoscopic size range and prolonged
blood circulation [1e4]. Lipophilic molecules can be incorporated
into the hydrophobic core of polymeric micelles by physical
entrapment, while the hydrophilic shell composed of flexible
polymers provides steric protection and helps these nanoparticles
to escape from the reticuloendothelial system (RES) uptake after
intravenous administration [5,6]. The PCL is a U.S. Food and Drug
Administration (FDA) approved semi-crystalline, hydrophobic and
biodegradable polymer with an ester group and five methylene
groups in its repeating unit that might be used as a synthetic
biomaterial or a controlled drug release matrix due to its good drug
cally labile ester groups make PCL biodegradable under physio-
logical conditions [7]. The PEG is also a FDA approved hydrophilic
and nonionic polymer that has been widely used as a hydrophilic
polymer in conjunction with hydrophobic block [8] to inhibit the
uptake and clearance of colloidal particles by RES and increases the
blood circulation time of these carriers. This is due to steric
hindrance posed by PEG chains to complement activating system
toward the hydrophobic part of the delivery system. Poly(ethylene
glycol) is a nonbiodegradable polymer and longer chain length
could eventually lead to increased body load of the polymer.
Therefore, in place of increasing PEG chain length for increasing the
surface coverage, the use of short-chain PEG at higher density on
nanomicelles is a preferable strategy. For this objective, PEG is
coated physically on the surface of the nanoparticles, or multiple
PEG chains are covalently linked to the hydrophobic block of the
polymer. The last strategy is apparently more effective whereas
physical coating may fail to serve the objective because of the good
aqueous solubility of PEG.
*
Corresponding author. Tel.: þ98 21 29902800; fax: þ98 21 22431586.
E-mail address: m-nabid@sbu.ac.ir (M.R. Nabid).
0
032-3861/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.polymer.2011.04.054

2800
M.R. Nabid et al. / Polymer 52 (2011) 2799e2809
Although micelles self-assembled from PEG and PCL based
anhydrous toluene. Maleic anhydride (MAh) (Aldrich, 98%) was
recrystallized from toluene and then dried under vacuum
copolymers have been widely investigated as drug carriers due to
their biocompatibility, biodegradability and nonimmunogenicity,
most of works were focused on utilizing linear block copolymers
(0.1 mmHg) at room temperature for 24 h
3-Caprolactone (CL) was
purchased from Sigma and purified with CaH
2
by vacuum distillation.
[
9e18]. Recently several studies have revealed that the architecture of
Pentaerythritol, 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoic
acid (HHMPA), diethyl ether, methanol, and dichloromethane were
amphiphilic block copolymers has great influence on the
a
morphology, stability and dimensions of the micelles [19e22] which
are very important parameters to determine the drug loading effi-
ciency, releasing behavior, biodistribution and circulation in vivo
2
purchased from Merck Chemical Co. Stannous octoate (Sn(Oct) ),
4-(dimethylamino) pyridine (DMAP) and dicyclohexylcarbodiimide
(DCC) were purchased from Aldrich and used as received. Quercetin
dihydride was obtained from Fluka Chemical Co. All other chemicals
were of analytical grade and were used as received. For in vitro
cytotoxicity test, amniotic epithelial cells were obtained from elective
Cesarean. Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 and
fetal calf serum (FCS) were obtained from GIBCO Invitrogen Corpo-
ration. 3-(4,5 Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT), dimethyl sulfoxide (DMSO) and epidermal growth
factor (EGF) were from Sigma.
[23e38].
Star-shaped block polymers are the simplest examples of
branched macromolecules with all branches (arms) extending from
a single point. Compared to linear polymers, the special unique
properties of amphiphilic star-block copolymers originating from
their unique shape have been observed in both bulk state and in
solution. It has been proved that star-shaped polymers exhibit
a smaller hydrodynamic radius, lower solution viscosity and
improvement of micelle stability when compared to linear block
polymers of the same molecular weight and composition. More-
over, several recent reports also demonstrated that star-shaped
polymers have some advantages in drug delivery compared to
linear polymers [39e43]. From these reports it is expected that
developing new types of star-shaped block copolymers based on
PCL and PEG with unique architectures may provide novel insights
for fabricating superior drug carriers for clinical applications in
drug delivery systems (DDS).
2.2. Synthesis of amphiphilic star-shaped 4s(PCL-b-2sPEG)
copolymer
The total synthetic scheme for the star-shaped block polymer is
shown in Scheme 1, and the 4s(PCL-b-2sPEG) star copolymer was
prepared through five steps, which are detailed as following.
2.2.1. Synthesis of 4-arm star-Shaped PCL (4sPCL)
In this study, according to the “arm-first” and “core-first”
synthetic methods, a versatile strategy to prepare star-shaped n
2
The 4-arm star-Shaped PCL was synthesized by Sn(Oct) -cata-
lyzed ring-opening polymerization of CL at different monomer/
initiator feed ratios. Typical polymerization procedure was as
following: A certain amount of pentaerythritol (0.13 g, 1 mmol),
[
Poly(
3
-caprolactone)-b-mPoly(ethylene glycol)] (n ¼ 4 and m ¼ 2)
copolymers is successfully introduced, as shown in Scheme 1. In the
core-first method, the star-PCL polymer with four arms having
terminal primary hydroxyl groups were synthesized by ring-
opening polymerization (ROP) from a symmetric pentaerythritol
core as a initiating agent. In the arm-first strategy, two samples of
the amphiphilic star-block copolymers with symmetrical topolo-
gies [4s(PCL-b-2sPEG)] and different PCL chain length were
synthesized by a macromolecular coupling reaction between
carboxyl-terminated PEG and 4-arm star-shaped PCL macromers
with eight eOH end groups. The latter was prepared by attaching
2
Sn(Oct) (0.1 wt.% of 3-caprolactone) and 3-caprolactone (9.12 g,
80 mmol) were placed in a three-neck round-bottom flask equip-
ped with a reflux condenser under a nitrogen atmosphere. Then,
ꢀ
the reaction vessel was put in oil bath at 120 C, for 24 h with
stirring. After the reaction flask was cooled to room temperature,
the resulting product was dissolved in methylene chloride and then
poured into excess methanol to precipitate the polymerized
product. The 4-arm star-shaped PCL with a hydroxyl group at each
chain end was obtained after filtering and drying in a vacuum at
room temperature for 48 h. Yield: 8.38 g (90%). Here as shown in
Table 1, two products of 4sPCL with different PCL lengths are
labeled as 4sPCL-a (monomer to initiator ratio; 80/1) and 4sPCL-
b (monomer to initiator ratio; 132/1).
2
,2-bis(hydroxymethyl)propionic acid (BHPA) (this linker was used
to increase PEG density on PCL chain) to 4sPCL using a simple two-
step reaction sequence. The star structure of the block copolymers
was confirmed by several physicochemical methods. To establish
this system as a suitable drug carrier the micellar properties of the
star amphiphilic polymer in aqueous media were studied by fluo-
rescence techniques and dynamic light scattering. The anticancer
drug quercetin was then chosen as a model lipophilic drug to
investigate the drug entrapment efficiency and in vitro drug release
profile of drug-loaded 4s(PCL-b-2sPEG) micelles.
2.2.2. Synthesis of 2,2-Bis[(2,2-propyl)dioxymethyl]propionic acid
(BPMPA)
2,2-Bis[(2,2-propyl)dioxymethyl]propionic acid was synthe-
sized through the reaction of HHMPA, 2,2-dimethoxypropane, and
p-TSA in acetone [45].
Quercetin is an anticancer drug and is abundantly found in
citrus fruits, vegetables, herbs and related products. However, its
administration has been heavily hindered by its extremely poor
water-solubility. Many serious problems are associated with the
therapeutic use of poorly water-soluble drugs. This includes poor
absorption and bioavailability upon oral administration, emboli-
zation of blood vessels from intravenous injection of the water-
insoluble drug because of drug precipitation, and local tissue
toxicity and low systemic drug bioavailability [44].
2.2.3. Synthesis of 4-arm star-shaped PCL macromers with eight
eOH end groups [4s(PCL-(OH)
2
)]
In 50 ml flask with
a
a magnetic stirring bar, 4sPCL
(M
n
¼ 9500 g/mol, 3.8 g, 0.4 mmol) was dissolved in 10 ml of dry
methylene chloride. 4-dimethylaminopyridine (DMAP, 0.29 g,
2.4 mmol) and BPMPA (0.348 g, 2 mmol) were added subse-
quently. After the flask was cooled to 0 C, a diluted solution of
ꢀ
dicyclohexyl carbodiimide (DCC, 0.41 g, 2 mmol) was added
dropwise over 2 h. The reaction mixture was warmed to room
temperature and stirred for 24 h. After filtration, the crude product
was precipitated into excess methanol and collected by vacuum
2
. Experimental
2
.1. Materials
filtration. After redissolving the solid in CH
cycle was repeated twice. The solid product was dried in a vacuum
oven at room temperature for 24 h. Yield: 3.6 (90%).
Then, functionalized 4-arm star-shaped PCL (1 g, 0.1 mmol) was
2 2
Cl , the precipitation
Monomethoxy poly (ethylene glycol) (MPEG, M
n
¼ 2000 g/mol)
g
purchased from Fluka and was dried by azeotropic distillation using

M.R. Nabid et al. / Polymer 52 (2011) 2799e2809
2801
4 8
Scheme 1. Synthesis of pentaerythritol-centered amphiphilic A B star-block copolymers based on PCL and PEG.

2802
M.R. Nabid et al. / Polymer 52 (2011) 2799e2809
Table 1
Molecular weight and molecular weight distribution of carboxylated PEG, 4sPCL-a, 4sPCL-b, 4s(PCL-b-2sPEG)-a and 4s(PCL-b-2sPEG)-b
(g mol )a
ꢂ1
M
n
(g mol )b
ꢂ1
b
Sample
M
n
w n
M /M
PEG block
PCL block
Total
3
3
3
3
3
3
3
Carboxylated PEG
2.2 ꢁ 10
e
2.2 ꢁ 10
1.6 ꢁ 10
3.8 ꢁ 1 0
1.13
1.15
1.14
1.16
1.15
3
3
4
4
4
4
sPCL-a
sPCL-b
s(PCL-b-2sPEG)-a
s(PCL-b-2sPEG)-b
e
e
9.5 ꢁ 10
9.5 ꢁ 10
15.4 ꢁ 10
26.2 ꢁ 10
32.1 ꢁ 10
3
3
3
15.4 ꢁ 10
5.5 ꢁ 10
13.9 ꢁ 10
16.3 ꢁ 10
3
3
3
16.7 ꢁ 10
16.7 ꢁ 10
9.5 ꢁ 10
3
3
15.4 ꢁ 10
a
Determined by 1H NMR.
Determined by GPC.
b
dissolved in a mixture of 25 mL of THF and 10 mL of 0.1 M HCl
aqueous). The reaction mixture was stirred at room temperature
n
the number-average molecular weights (M ) of 4sPCL and 4s(PCL-
(
b-2sPEG) copolymers. The GPC system was equipped with Waters
2690D separations module, Waters 2410 refractive index detector.
THF was introduced as the eluent at a flow rate of 0.3 ml/min.
Waters millennium module software was used to calculate
molecular weight on the basis of a universal calibration curve
generated by polystyrene standard with narrow molecular weight
distribution. The morphology of the self-assembled micelles was
performed on a ZEISS EM-900 transmission electron microscopy
operating at an acceleration voltage of 80 kV. TEM sample was
prepared by dipping a copper grid with Formvar film into the
freshly prepared micelles solution. A few minutes after the depo-
sition, the aqueous solution was blotted away with a strip of filter
paper and stained with phosphotungstic acid aqueous solution,
then dried in the air. The mean particle size in aqueous solution at
room temperature and size distribution of self-assembled micelles
were determined by Nano-ZS ZEN3600. Each sample was diluted to
appropriate concentration with distilled water. The polymeric
for 4 h. Subsequently, the solvent was removed under reduced
pressure and the resulting bifunctional 4-arm star-shaped PCL was
dissolved in methylene chloride and then poured into excess
methanol to precipitate the deprotected product. The 4-arm star-
shaped PCL macromer with eight eOH end groups was obtained
after filtering and drying in a vacuum at room temperature after
4
8 h. Yield: 0.96 g (96%).
2.2.4. Synthesis of carboxyl-terminated monomethoxy
poly(ethylene glycol) (MPEG-CO
Carboxyl-terminated MPEG was synthesized as follows: MPEG
5.0 g, M
2
H)
(
(
n
¼ 2000 g/mol, 2.5 mmol) was dissolved in dry toluene
25 mL). In order to removing trace water in MPEG azeotropic
ꢀ
distillation was carried out at 125 C. Then, maleic anhydride
ꢀ
(
2.45 g, 25 mmol) was added. The mixture was stirred at 75 C for
2
4 h, and then added into diethyl ether at room temperature to
precipitate the reaction product. The precipitate was filtered, and
dried at 30 C in a vacuum oven for 24 h. The carboxyl-terminated
solutions (mg/L) were passed through a 0.2 mm pore size filter
ꢀ
before measurement. The thermal transitions of the polymers were
determined using a PerkineElmer DSC7 differential scanning
calorimeter under nitrogen purge. The samples were heated at
2
poly(ethylene glycol) (MPEG-CO H) (4.8 g) was obtained in 96%
yield.
ꢀ
a rate of 10 C/min.
2
.2.5. The coupling reaction of 4s(PCL-(OH)
The amphiphilic star-shaped 4s(PCL-b-2sPEG) copolymers were
synthesized using a molar feed ratio [4s(PCL-(OH) ) (4.75 g,
¼ 9500 g/mol)]/[MPEG-CO H (10.56 g, M ¼ 2200 g/mol)]/[DCC
1.64 g)]/[DMAP (0.97 g)] of 1:8.8:16:16. The calculated amounts of
2 2
) and MPEG-CO H
2.4. Micelle formation
2
M
(
n
2
n
We used dialysis bag method to fabricate the micelles. Briefly,
s(PCL-b-2sPEG) copolymer (10 mg) was dissolved in 3 ml DMF.
The solution was put into a dialysis tube and subjected to dialysis
against 1000 ml distilled water, which was renewed every 3 h, then
lyophilized before further examinations.
4
reactants were added into a three-necked round-bottom flask.
Then, anhydrous dichloromethane was poured into the mixture
under a nitrogen atmosphere. The reaction was performed at room
temperature for 24 h under vigorous stirring [33]. After that the
mixture was evaporated to dryness, the obtained product was
dissolved in distilled water. The solution was put into a dialysis bag
2
.5. Fluorescence measurements
(
MWCO ¼ 3500) and dialyzed against 2000 mL distilled water,
which was renewed every 12 h at room temperature for 5 days
before freeze-drying for 48 h.
Fluorescence spectra were recorded on a Varian Cary Eclipse
spectrofluorophotometer. Pyrene was used as a hydrophobic
fluorescent probe to determine the critical micelle concentration
ꢂ6
2.3. Characterization of copolymers and micelles
(CMC). Aliquots of pyrene solutions (6 ꢁ 10 M in acetone, 1 ml)
were added to containers, and acetone was allowed to evaporate
at room temperature. 10 ml aqueous copolymer solutions at
different concentrations were then added to the containers con-
taining the pyrene residue. The solutions were kept at room
temperature for 24 h to reach the solubilization equilibrium of
pyrene in the aqueous phase. Excitation was carried out at
340 nm, and emission spectra were recorded ranging from 350 to
470 nm. The excitation and emission bandwidths were 10 nm and
5 nm, respectively. A CMC value was determined from the ratios of
FT-IR spectra were recorded on a 102 MB BOMEM apparatus.
The samples of freeze-dried micelles were pressed into potassium
bromide (KBr) pellets. Additionally, other samples for FT-IR
measurements were prepared by dissolving them in 1.5 wt% chlo-
roform solution on the surface of a silicon wafer and the solvent
1
was completely evaporated before measurements. H NMR spectra
were recorded on a BRUKER DRX-300 AVANCE spectrometer at
3
00.13 MHz using CDCl
measurement was performed on lyophilized micelles using an
X-ray powder Diffractometer (XD-3A) with Cu K radiation. The gel
permeation chromatographic (GPC) system was used to determine
3
as the solvent. Crystallographic assay
pyrene intensities at 381 (I
the intersection of two tangent plots of I
3 1
3
) and 373 (I
1
) nm and calculated from
/I versus log concen-
a
trations of polymers.

M.R. Nabid et al. / Polymer 52 (2011) 2799e2809
2803
2.6. Cell cultures
3. Results and discussion
Human amniotic membrane was prepared using fresh human
3.1. Synthesis and characterization of the star-shaped poly
placenta as described previously [46]. Briefly, the amnion was
mechanically peeled of the chorion and washed several times with
PBS. To release amniotic epithelial cells, the amniotic membrane
was incubated at 37 C with 0.15% trypsin-EDTA. Trypsin was
inactivated with FCS and the solution centrifuged at 2500 rpm for
(
3
-caprolactone)
The star-shaped poly(
3
-caprolactone) homopolymer was
ꢀ
ꢀ
synthesized by ring-opening polymerization of CL at 120 C. The
pentaerythritol with four hydroxyl groups was chose as the initi-
ator in order to produce the hydroxyl-terminated star-shaped PCL.
By changing the feed ratio of monomer to initiator, two star-shaped
polymers of 4sPCL-a and 4sPCL-b with different lengths of PCL
chain were easily obtained.The IR spectrum of the 4sPCL is shown
in Fig. 1A. The IR spectrum of the star-shaped PCL had a band
1
2 min. Cells were washed with PBS and cultured in DMED/F12
containing 10% FCS, 10 mg/ml EGF, 2 mM -glutamine, 1% nones-
sential amino acid, 55 M 2-mercaptoethanol, 1 mM sodium
pyruvate and 100 U/ml penicillin/streptomycin solution.
L
m
ꢂ1
characteristic for the ester carbonyl at 1727 cm and two bands for
hydroxyl at 3540 and 3439 cm . These bands could be associated
2.7. In vitro cytotoxicity studies
ꢂ1
to different hydrogen bonding interactions. There is only one
hydroxyl group per PCL chain so the intensity of the hydroxyl band
was low; reflecting the low content of eOH in the sample. H NMR
Amniotic epithelial cells (AEC) were seeded at density of
4
7
.5 ꢁ 10 per well in gelatin-coated 24-well plate and incubated
1
ꢀ
2
overnight in incubator (37 C, 5% CO ). The next day, AEC was
spectrum of the star-shaped PCL is shown in Fig. 2A. The typical
signal of the methylene (a) protons of the initiator pentaerythritol
was clearly detected at 4.37 ppm. The major resonance peaks (bee)
were attributed to PCL. The peak of the methylene (e) protons was
detected at 4.05 ppm when the peak of the protons of the terminal
methylene (e’) was observed at 3.65 ppm indicating that PCL is
terminated by hydroxyl groups. The average degrees of polymeri-
zation for the PCL arms were calculated from the integration ratio
between the methylene protons in the repeat units (e) and those in
treated with 4s(PCL-b-2sPEG)-a copolymer with concentration
ꢀ
1
mg/ml and incubated at 37 C for 24 h. The cells without treat-
ment were used as control group. For cytotoxicity assay, 250 mL of
MTT solution (5 mg/mL) was added to each cell and the plates were
incubated for 4 h. The MTT formazan crystals were then dissolved
with 1 ml DMSO at room temperature. The optical density (OD) was
measured at 570 nm with a spectrophotometer (CE7500, Cecil, UK).
The blank well containing only medium material was used for zero
adjustment. The viable rate was calculated by the following
equation:
1
the terminal unit (e’) based on H NMR spectrum. The molecular
1
weights of 4sPCL-a and 4sPCL-b was determined by H NMR
spectrum and GPC analysis were listed in Table 1.
Viable rate ¼ ðODtreated=ODcontrolÞ ꢁ 100%
Where ODcontrol was obtained in the absence of polymers and
ODtreated was obtained in the presence of polymers.
3.2. Synthesis and characterization of the 4-arm star-shaped PCL
macromers with eight eOH end groups
The star-shaped 4s(PCL-(OH)
reaction of 4sPCL with the BPMPA. The molecular weight of 4s(PCL-
OH) ) increased slightly and the molecular weight distribution was
similar to that of 4sPCL. The IR spectrum of the star-shaped 4s(PCL-
2
) macromer was obtained by the
2
.8. General procedure for the preparation of drug-loaded micelles
(
2
4
s(PCL-b-2sPEG)-a (10 mg) and quercetin (0.5 mg) were dis-
solved in 3 mL DMF, then the solution was put into a dialysis tube
and subjected to dialysis against 1000 ml distilled water. The whole
dialysis process lasted for 24 h and the water was renewed every
(
OH)
2
) macromer (Fig. 1B) had a band characteristic for the ester
ꢂ1
carbonyl at 1728 cm and two bands for hydroxyl at 3544 and
3
ꢂ1 1
441 cm
H NMR spectrum was shown in Fig. 2B. The peak
2
h during the initial 12 h to remove the unloaded drug. To deter-
assigned to the methylene protons of the PCL block at 3.65 ppm
disappeared, while novel signals corresponding to methyl (g)
protons and ester methylene (f) protons of terminal BHPAs
appeared at 1.16 and 3.72 ppm, indicating complete reaction of all
the terminal hydroxyl groups.
mine the entrapment efficiency (EE), the drug-loaded micelle
solution was lyophilized, and then dissolved in DMF and analyzed
by UV absorbance at 373 nm, using a standard calibration curve
experimentally obtained with quercetin/DMF solutions. The EE was
calculated based on the following formula:
EEðwt:%Þ ¼ ðWeight of the drug in micelles=Weight of the
feeding drugsÞ ꢁ 100
2.9. In vitro drug release
The drug-loaded micelle solution (2 mL, [Quercetin]
¼
0.008e0.011 g/L) was placed in a dialysis tube (MWCO ¼ 12 kDa).
The dialysis tube was sealed and immersed in 30 mL of phosphate-
buffered saline (PBS) (pH 7.4). In vitro drug release study of drug-
ꢀ
loaded micelle was carried out in a shaking water bath at 25 C. A
3
mL aliquot of solution was taken out and the same volume of PBS
solution was added after each sampling at predetermined time
intervals. Also, as a control experiment, quercetin solution was used
with 0.01 g/L in a solvent mixture of ethanol-water-PEG400 (4:3:3 v/
v). The concentration of the drug in the release samples was deter-
mined by UV spectra.
2
Fig. 1. FT-IR spectra of 4sPCL, 4s(PCL-(OH) ), and 4s(PCL-b-2sPEG).

2804
M.R. Nabid et al. / Polymer 52 (2011) 2799e2809
Fig. 2. Chemical structures and 1H NMR spectra of copolymers: (A) 4sPCL, 4s(PCL-(OH)
) (B), carboxylated PEG (C), 4s(PCL-b-2sPEG) (D).
2
3.3. Synthesis of carboxyl-terminated PEG
2 1 2
CO H. Two signals at 6.18 (f ) and 6.37 ppm (f ) are assigned to vinyl
protons in the terminal group; the signals at 3.65 (c) and 4.30 ppm
(e) belong to ether and ester methylene protons, respectively;
Carboxyl-terminated monomethoxy poly(ethylene glycol)
(
MPEG-CO
2
H) was prepared from the reaction of MPEG-OH with
terminal methyl and methylene (CH
3.32e3.40 ppm. The reaction extent is estimated by the integration
3
OCH
2
) peaks (a þ b) appear at
1
maleic anhydride. Fig. 2C exhibits the H NMR spectrum of MPEG-

M.R. Nabid et al. / Polymer 52 (2011) 2799e2809
2805
methyl (g) and ester methylene (f) protons in BHPA units. The peak
at 3.38 is attributed to the methyl (j) protons of eCH in MPEG
3
units. Two signals at 6.2 and 6.4 ppm are assigned to vinyl (h)
protons in the maleic anhydride units and the signal at 4.37 ppm
belongs to ester methylene (a) protons in pentaerythritol core. All
of these peaks indicate that the 4s(PCL-b-2sPEG) copolymer was
successfully synthesized.
Fig. 2D shows that the proton resonance of vinyl group between
the two ester groups is clearly observed at 6.2 and 6.4 ppm, indi-
cating that carboxylated PEGs are successfully coupled with the
4-arm star-shaped PCL. Since the proton resonance of the pen-
taerythritol moiety is observed at 4.37 ppm, the degree of coupling
between carboxylated PEG and 4-arm PCL can be estimated from
the ratio of the peak area at 6.2 and 6.4 ppm to the peak area at
4
9
2
.37 ppm; the degree of coupling for our system is estimated to be
5%. All the GPC traces of carboxylated PEG, 4sPCL and 4s(PCL-b-
sPEG) copolymer exhibit monomodal molecular weight distribu-
1
tions, as shown in Fig. 3. Therefore, both the results of H NMR and
GPC confirm the successful synthesis of the star-shaped copolymer.
The molecular weights and molecular weight distributions of
carboxylated PEG, 4-arm star-shaped PCL and star-shaped copoly-
mers are listed in Table 1. It has been demonstrated that star-
shaped polymers exhibit a smaller hydrodynamic radius when
compared to linear polymers of the same molecular weight and
composition. So, these results (Table 1) are predictable: the star
copolymers compared to linear polymers of the same molecular
Fig. 3. GPC traces of polymers: (A) carboxylated PEG, (B) 4sPCL-a, (C) 4sPCL-b, (D)
s(PCL-b-2sPEG)-a, (E) 4s(PCL-b-2sPEG)-b.
4
ratio of the peaks f
1
and f
2
to the peak a þ b, the value is close to 2:5,
indicating almost quantitative reaction of hydroxyl group of MPEG
with MAh. The MAh-terminated MPEG was successfully prepared.
weight can be detected in higher elution time. This lead to smaller
1
M
n
compared to actual M
n
or M
n
( H NMR).
3
2
.4. Synthesis and characterization of the star-shaped 4s(PCL-b-
sPEG) copolymers
3.5. Self-assembly amphiphilic star-shaped copolymers [4s(PCL-b-
sPEG)]
2
Finally, the pre-synthesized 4-arm star-shaped PCL macromer
[
4s(PCL-(OH)
star-shaped block copolymers of 4s(PCL-b-2sPEG)-a and 4s(PCL-b-
sPEG)-b with different lengths of PCL chain. In the IR spectrum of
2
] was coupled with the carboxylated PEG to yield
The amphiphilic star-block copolymer self-assembled into
micelles in aqueous solution using the dialysis method. The critical
micelle concentrations (CMC) of these star-shaped block copoly-
mers were analyzed by fluorescence spectra using pyrene as
a hydrophobic probe. Pyrene, initially dissolved in water, undergoes
partition in the hydrophobic core of forming micelles, where it is
more soluble. The variation of microenvironmental polarity results
in a red shift of pyrene excitation maximum. The excitation spectra
of pyrene at different copolymer concentrations in water were
2
star 4s(PCL-b-2sPEG) copolymer (Fig.1C), the band belonging to the
hydroxyl end groups of the 4-arm star-shaped PCL macromer
ꢂ1
[4s(PCL-(OH)
2
] at 3544 to 3441 cm disappeared, confirming the
conjugation of 4-arm star-shaped PCL macromer [4s(PCL-(OH)
and PEG blocks. A stronger band at 1112 cm for CeOeC appeared,
consistent with the addition of PEG ether units. The H NMR
spectrum of 4s(PCL-b-2sPEG) copolymer is shown in Fig. 2D. In H
2
]
ꢂ1
1
1
acquired and the intensities of absorptions at 381 (I
3 1
) and 373 (I )
NMR, peaks at 1.37, 1.62, 2.28, and 4.03 ppm are assigned to
nm are plotted as I /I ratio versus polymer concentration in solu-
3 1
methylene (b-e) protons of e(CH
in PCL units respectively. The sharp peak at 3.65 ppm is attributed
to methylene (i) protons of eCH CH Oe in MPEG units in block
copolymer. The very weak peaks at 1.18 and 4.17 are assigned to the
2
)
3
e, eCH
2
COOe, and eOCCH
2
e
tion (Fig. 4). It was found that the ratio is almost constant at rela-
tively low concentrations. After the concentration of the copolymer
got to a critical value, ratio starts to enhance dramatically with the
increasing concentration, then the creation of micelles as well as
2
2
3 1
Fig. 4. I /I plotted as a function of polymer concentrations of 4s(PCL-b-2sPEG)-a (A) and 4s(PCL-b-2sPEG)-b (B).

2806
M.R. Nabid et al. / Polymer 52 (2011) 2799e2809
Fig. 5. TEM micrography of micelles from 4s(PCL-b-2sPEG)-a (A), 4s(PCL-b-2sPEG)-b (B) and size distribution detected by DLS of the micelles of 4s(PCL-b-2sPEG)-a (C), 4s(PCL-b-
sPEG)-b (D) in distilled water.
2
the simultaneous transfer of pyrene into the hydrophobic micellar
core are initiated. Thus, the transition concentration was defined as
the CMC of 7.75 and 2.83 mg/L for 4s(PCL-b-2sPEG)-a and 4s(PCL-b-
Furthermore, the diameters of the nanomicelles are around
30e50 nm. The information is more likely to support the opinion
that the micellization occur as a result of molecular association
rather than aggregation of smaller micelles. The PDI of corre-
sponding micelles, very low about 0.135 and 0.092 determined by
the DLS, also reinforced this opinion though the micelles exhibit
larger average diameter of 78 nm and 122 nm, respectively, deter-
mined by the DLS (Fig. 5C and D). This difference in micelles size
measured by TEM and DLS should be attributed to that the latter is
the hydrodynamic diameter of micelles in water, whereas the former
reveals the morphology size of the micelles in solid state.
2
sPEG)-b, respectively. The difference of the CMC values is probably
due to the relatively stronger hydrophobic interactions or
decreased solvency resulting from the more hydrophobic core in
the presence of longer PCL chains. Thus the CMC value exhibits
a decreasing tendency with the increased length of PCL chain and
might be tuned accordingly to a certain extent. Micelles with small
CMC values are expected to be more stable against dilution after the
IV injections [47].
To understand the micellization behavior of the obtained star-
block copolymers, the HLB values were estimated based on
composition of the copolymers according to equation [48,49]:
3.6. DSC characterization
The thermal behavior of the synthesized star-block copolymers
as well as 4sPCL-b and PEG-COOH samples was investigated using
differential scanning calorimetry (DSC). Results are summarized in
HLB ¼ ½W =ðWH þ W Þꢃ ꢁ 20
H
L
Here, W
H
and W
L
are the weight fraction of the hydrophilic
Table 2. Peak attribution was made taking into account the T
m
of
(
PEG) and hydrophobic (PCL) segment, respectively. The HLB values
of the copolymers are 12.7 and 10.4 for 4s(PCL-b-2sPEG)-a and
s(PCL-b-2sPEG)-b, respectively. These results demonstrate that
precursor PEG and PCL blocks. The melting enthalpy of PCL blocks
4
the HLB values decrease as the length of PCL chain increases, with
similar trends observed for the CMC values and this partly sustains
the conclusions concerning the CMC result.
To further evaluate the properties of micelles, both the
morphology and the mean size of these 4s(PCL-b-2sPEG) micelles
were studied by the TEM measurement and dynamic light scattering
Table 2
DSC result of polymers.
Samples
T
m
(◦C)
m
DH (J/g)
PEG
PCL
PEG
PCL
PEG-COOH
57
e
e
164
e
e
4
4
4
sPCL-b
s(PCL-b-2sPEG)-a
s(PCL-b-2sPEG)-b
55.7
62
61
80
81
80
(
DLS). It is seen from TEM pictures (Fig. 5A and B) that the self-
55.5
55
99
94
assembled micelles are well dispersed as individual nanoparticles
with regularly spherical shapes confirming the micellization.

M.R. Nabid et al. / Polymer 52 (2011) 2799e2809
2807
Table 3
result is due to the crystallization of PCL blocks which is related to
the higher molecular weight of PCL blocks (M
¼ 2.3e3.8 kDa) and
therefore disturbing the organization of PEG blocks (M
¼ 2.0 kDa).
Properties of blank and quercetin-loaded polymeric micelles.
n
Samples
CMCa
mg/L)
DLb
(%)
EEc
(%)
Quercetin Std
g/ml)
n
(
(m
As micelle stability is related also to the physical form of the core-
forming blocks, development of PCL crystallinity in the micelle core
contributes to improve their kinetic stability.
4
4
s(PCL-b-2sPEG)-a
s(PCL-b-2sPEG)-b
7.75
2.83
3.75
5
75
80
750
800
a
Critical micelle concentration of the micelles in water.
Percent drug loading ¼ weight of quercetin in micelles ꢁ 100/weight of micelles
b
3.7. Drug solubilization in aqueous solutions
tested.
c
Percent entrapment efficiency ¼ weight of quercetin in micelles ꢁ 100/weight
of quercetin used in micelles preparation.
One of the main aims of encapsulating quercetin into 4s(PCL-b-
sPEG) micelles was to increase its aqueous solubility. Quercetin is
d
Quercetin solubility in water.
2
a lipophilic molecule with very low aqueous solubility, <10
mg/mL
[
50]. Low bioavailability, low drug efficacy and limited treatment
in copolymers was almost equal to that of parent macromers, thus
suggesting that PCL crystallization is difficulty influenced by the
presence of PEG blocks. A decrease in DH for the PEG component
m
in star copolymers was observed compared with PEG-COOH. This
options of poorly soluble drugs are common problems in drug
development and can have negative consequences for patients
[51,52]. Table 3 presents quercetin aqueous solubility up to 800 mg/
mL observed for 4s(PCL-b-2sPEG) micelles at polymer concentra-
tion of 10 g/L; this corresponds to w80 folds increase in quercetin
Fig. 6. Light microscopic images of quercetin (A), empty micelles (B) and
Fig. 7. X-ray diffraction spectra of quercetin crystal, empty micelles and drug-loaded
quercetin-loaded micelles (C) added into cell culture medium.
micelles.

2808
M.R. Nabid et al. / Polymer 52 (2011) 2799e2809
aqueous solubility. Also, we provide light microscopic images of
quercetin, empty micelles, and quercetin-loaded micelles added to
cell culture medium. As shown in Fig. 6AeC, the culture mediums
contain empty micelles and drug-loaded micelles which are quite
soluble whereas, quercetin in culture medium is partially insoluble;
these are in good agreement with above mentioned results.
3.8. Drug loading and encapsulation efficiency
The anticancer drug, quercetin-loaded micelles were prepared
by the dialysis method. The mood of the entrapped drug in the
nanomicelles matrix is one of the main factors in the release profile
of drug delivery systems. Fig. 7 shows the X-ray diffraction scans of
the pure quercetin, drug free micelles and drug-loaded core-shell
type micelles. It is observed that there is no diffraction peak of the
pure quercetin in the drug-loaded micelles and the diffraction
peaks of quercetin-loaded micelles are similar to those of
quercetin-free micelles. This means that no drug crystal is visual-
ized even on the surface of the nanoparticles demonstrating that
the quercetin is dispersed molecularly in the core of nanoparticles.
Indeed, similar results were reported for other drug-loaded poly-
meric micelles [53e55].
Fig. 9. In vitro cytotoxicity of the 4s(PCL-b-2sPEG)-a copolymer in two form of
nanoparticles (micelle) and synthesized polymer with concentration 1 mg/ml.
first stage. In the second stage, the release rate is much slowerand the
process may persist for several days. Forthe quercetin-loaded 4s(PCL-
b-2sPEG)-a and 4s(PCL-b-2sPEG)-b micelles, there were both
a similar burst release for initial 15 h, followed by a gradual release up
to 160 h. In contrast, the release rate of quercetin from micelles is
much slower than that of the free quercetin. After 160 h, 4s(PCL-b-
The drug loading contents of 4s(PCL-b-2sPEG)-a and 4s(PCL-b-
2
sPEG)-b were 3.75 and 5%, respectively, indicate that the quercetin
2sPEG)-a and 4s(PCL-b-2sPEG)-b micelles released w94 and 86% of
is effectively loaded into the micelles. Quercetin loading capacity
and encapsulation efficiency increase from 3.75 to 5.0 wt% and
from 75.0 to 80.0 wt%, respectively by increasing the PCL molecular
weight from 2.3 to 3.8 kDa. These results confirm that quercetin
loading capacity and encapsulation efficiency of the micelles
depend on the block length of the PCL arm. Thereby, hydrophobic
interactions of quercetin with PCL arm are an important factor for
the drug loading capacity and encapsulation efficiency of 4s(PCL-b-
their quercetin contents, respectively. These results demonstrate that
a strong interaction between the drug and core-forming block in
nanoparticles leads to a decreased rate of drug release from micelles.
3.9. In vitro cytotoxicity studies
Biocompatibility is a great concern for biomaterials and in this
2
sPEG) micelles (Table 3) [56].
study we performed preliminary evaluation on the cytotoxicity of
obtained materials by the MTT assay. The highest possible
concentration (1 mg/ml) of the copolymer was used to evaluate the
cytotoxicity. The results in Fig. 9 demonstrate that the resulted
copolymer does not exhibit apparent cytotoxicity.
The in vitro release behavior of quercetin-loaded 4s(PCL-b-2sPEG)
micelles and free quercetin in PBS (pH 7.4) at 25 C was studied and
the results are shownin Fig. 8. Quercetin solubilityin the PBS medium
ꢀ
was 150
the release experiments given the release volume (30 mL) and
quercetin amounts in the micelles (260e350 g). Free quercetin
mg/mL confirming the maintenance of sink conditions during
m
quickly diffused out of the dialysis tube when dissolved in the dialysis
medium. Almost complete release was achieved after 15 h. It was
observed that the release pattern of quercetin-loaded 4s(PCL-b-
4
. Conclusions
4 8
Well-defined pentaerythritol-centered amphiphilic A B star
2
sPEG)-a and 4s(PCL-b-2sPEG)-b micelles were a typical two phase
copolymers [4s(PCL-b-2sPEG)] have been successfully designed
and synthesized. These amphiphilic star-block copolymers self-
assemble into micelles in which hydrophobic arms of the copoly-
mers form a core while the hydrophilic arms form a corona. The
results demonstrate that such micelles provide an excellent nano-
carrier for hydrophobic drugs such as quercetin, in which aqueous
solubility of the drug is dramatically enhanced. Moreover, the
micelles can effectively load hydrophobic quercetin and encapsu-
lation efficiency of up to 80 wt% is achieved. The release behavior of
quercetin from loaded micelles is shown a sustained manner which
protects drug from precipitation in physiological medium. The star
copolymers exhibit no apparent cytotoxicity. With these enhanced
properties the star-block copolymer micelles composed of PEG and
PCL have potential applications as a versatile nanocarrier of various
liposolubility drugs.
release pattern which means that the release rate is very fast at the
Acknowledgment
Special thanks to Dr. Shant Shahbazian for his assistance in
preparing the manuscript. The financial support of Research
Council of Shahid Beheshti University is gratefully acknowledged.
Fig. 8. Cumulative drug release profiles of the 4s(PCL-b-2sPEG)-a and 4s(PCL-b-
ꢀ
2sPEG)-b in PBS pH 7.4 at 25 C .

M.R. Nabid et al. / Polymer 52 (2011) 2799e2809
2809
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