Sheddable micelles based on disulfide-linked hybrid PEG-polypeptide copolymer for intracellular drug delivery

Article abstract of DOI:10.1016/j.polymer.2011.06.013

A novel reduction-sensitive sheddable micelle based on disulfide-linked hybrid PEG-polypeptide mPEG-SS-Pleu was demonstrated for intracellular drug delivery. These micelles are composed of an mPEG shell and polypeptide core, characterized by FT-IR, ¹H NMR, fluorescence techniques, TEM, and DLS. Interestingly, they would undergo a fast sheddable process when encounter the reduction sensitive condition, indicated by the aggregation phenomena in the presence of DTT, a reduction agent, which could cleave the disulfide bond between the micellar core and shell and consequently leading to the aggregation of hydrophobic core. Cytotoxicity study revealed that copolymers in this system have good biocompatibility and their self-assembled micelles showed a high drug loading efficiency for DOX, a hydrophobic drug model, and released DOX quantitatively in response to the intracellular level of reducing potential. Cellular uptake experiments demonstrated that the fluorescently labeled micelles could be successfully internalized into human liver carcinoma HepG2 cells, evidenced by confocal laser scanning microscopy. Above results indicate that the copolymers may have great potential in drug delivery to achieve improved cancer therapy.

Full text of DOI:10.1016/j.polymer.2011.06.013

Polymer 52 (2011) 3580e3586
Contents lists available at ScienceDirect
Polymer
journal homepage: www.elsevier.com/locate/polymer
Sheddable micelles based on disulfide-linked hybrid PEG-polypeptide copolymer
for intracellular drug delivery
Tian-Bin Ren , Wen-Juan Xia, Hai-Qing Dong, Yong-Yong Li*
*
The Institute for Advanced Materials & Nano Biomedicine, School of Material Science and Engineering, Tongji University, Shanghai 200092, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 February 2011
Received in revised form
A novel reduction-sensitive sheddable micelle based on disulfide-linked hybrid PEG-polypeptide mPEG-
SS-Pleu was demonstrated for intracellular drug delivery. These micelles are composed of an mPEG shell
and polypeptide core, characterized by FT-IR, 1H NMR, fluorescence techniques, TEM, and DLS. Inter-
estingly, they would undergo a fast sheddable process when encounter the reduction sensitive condition,
indicated by the aggregation phenomena in the presence of DTT, a reduction agent, which could cleave
the disulfide bond between the micellar core and shell and consequently leading to the aggregation of
hydrophobic core. Cytotoxicity study revealed that copolymers in this system have good biocompatibility
and their self-assembled micelles showed a high drug loading efficiency for DOX, a hydrophobic drug
model, and released DOX quantitatively in response to the intracellular level of reducing potential.
Cellular uptake experiments demonstrated that the fluorescently labeled micelles could be successfully
internalized into human liver carcinoma HepG2 cells, evidenced by confocal laser scanning microscopy.
Above results indicate that the copolymers may have great potential in drug delivery to achieve
improved cancer therapy.
3
June 2011
Accepted 8 June 2011
Available online 15 June 2011
Keywords:
Reduction-sensitive
Micelle
Polypeptide
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
and interact with them, causing a long-term toxicity. So degradable
polymeric carriers are optimal to ensure elimination of adverse
Amphiphilic block copolymers have the capability of self-
assembly in dilute solution. One of the most intriguing formula-
tions includes nanoscaled micelles which compose a typical core-
shell structure and could effectively entrap hydrophobic drugs
within the hydrophobic cores in aqueous solution. These micellar
drug-delivery systems enjoy several advantages, such as less
susceptibility to uptake by the reticuloendothelial system (RES) [1],
resistance to dilution arising from the low critical micelle concen-
tration (CMC) to make them stable after in vivo administration [2],
good water solubility to improve bioavailability, and the separated
functionality due to the core-shell structure to protect transporting
substances from the outer environment [3]. More importantly, the
nanoparticles were reported to be capable of improving the drug
internalization into the cells while minimizing the recognition by
drug resistance system [4]. Thus, in the past decades, polymeric
micelles have been extensively investigated for the controlled drug
delivery applications [5e10]
effects [11]. Over the past decade, new synthetic tools have facili-
tated fabrication of well-controlled polymeric structures. Never-
theless, synthetic biopolymers are still far from matching the
structural and functional diversity of proteins and peptides [12].
Proteins are unique by combining three-dimensional architecture
with intricate, precise, and diverse patterns of surface chemistry.
Using peptides as building blocks, several attempts successfully
resulted in the synthesis of functional nanostructures [13]. Poly-
peptide hybrid block copolymers were first reported by Gallot and
co-workers in the mid 1970s [14]. At present, polypeptides based
on natural or non-natural amino acids have been exploited to
prepare polymeric carriers since they are associated with several
merits such as biodegradability, biocompatibility, and bio-
functionality due to their component and structure analogous with
natural biomolecules [15]
There are some reports about the works on the preparation of
polypeptide hybrid block copolymers, which could self-assemble
into micelles in aqueous systems [16e25]. In these reports, some
kinds of stimuli-sensitive micelles have been synthesized to release
drugs quickly in response to an appropriate stimulus including pH
While entering into the body, the micelles based on nonde-
gradable polymers would accumulate in the host cells and tissues
[
16e20] and temperature [21,22] to achieve an enhanced therapy
*
Corresponding authors. Tel.: þ86 21 6598 3706x815; fax: þ86 27 21 6598
706x0.
E-mail addresses: rtb002@163.com (T.-B.Ren),yongyong_li@tongji.edu.cn(Y.-Y. Li).
effect. With the aim to extend the stimuli-sensitive drug delivery
system, recently reduction sensitive micelles have received
3
0
032-3861/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.polymer.2011.06.013

T.-B. Ren et al. / Polymer 52 (2011) 3580e3586
3581
a tremendous amount of interest for intracellular drug delivery
10,26,27], due to the large difference in reducing potential
sulfoxide (DMSO). The solution was kept under magnetic stirring at
room temperature for 2 h. Chloroactic acid (0.5 g, 5.291 mmol) was
then added and the reaction mixture was heated to 60 C. The
reaction proceeded for an additional 24 h before adjusting its pH to
7.0 using HCl solution (37 wt %). The water was evaporated with
a rotary evaporator and the DMSO was removed by dialysis. The
residue was taken up in THF, filtered, precipitated in cold ethyl
ether twice and dried in vacuum overnight. Then white powder
mPEG-COOH was obtained.
[
ꢀ
between the tumor tissues and normal tissues, with at least 4-fold
higher concentrations of GSH in the tumor tissues over normal
tissues [28]. To the best knowledge of us, there was no report on
reduction sensitive self-assembly based on polypeptide hybrid.
In this paper, we developed a type of sheddable micelles which-
can detach their shells upon reduction stimuli, based on poly(-
ethylene glycol) monomethyl ether-SS-poly(D-leucine-L-leucine)
(
mPEG-SS-Pleu) copolymer for controlled drug delivery (Scheme 1).
The mPEG-SS-Pleu was synthesized by N-carboxyanhydride (NCA)
ring-openingpolymerization(ROP). Themicelles showed lowcritical
micelle concentration (CMC) and a high drug loading efficiency for
doxorubicin (DOX) and released DOX quantitatively in response to
dithiothreitol (DTT) analogous to the intracellular level of reducing
potential (Scheme 1). Moreover, the copolymers in this system are
non-toxicity to HepG2 cells due to the good biocompatibility of
mPEG and polyleucine. The micelles are highly promising for drug
delivery to achieve improved cancer therapy.
2.3. Synthesis of mPEG-cystamine
Cystamine dihydrochloride (2.0 g, 22.2 mmol) was dissolved in
NaOH solution (148 mM, 30 ml). The reaction was kept under
magnetic stirring at room temperature for 0.5 h. The solvent was
evaporated with a rotary evaporator and the residue was taken up
in CH Cl and filtered. The CH Cl was removed with a rotary
2
2
2
2
evaporator. After drying in vacuum overnight, cystamine was
obtained.
A mixture of mPEG-COOH (2.0 g, 0.3954 mmol), DCC (0.10 g,
0.47 mmol) and NHS (0.055 g, 0.47 mmol) was dissolved into 20 ml
2
. Experimental part
CH
2
Cl
2
and kept under magnetic stirring at room temperature for
Cl was
2.1. Materials
5 h. A solution of cystamine (0.95 g, 6.25 mmol) in 20 ml CH
2
2
then added dropwise. After completion of addition, the reaction
proceeded for an additional 24 h. The suspension was filtered and
the solvent was evaporated with a rotary evaporator. The residue
was precipitated in cold ethyl ether twice and then taken up in THF.
The solution was dialyzed (MWCO ¼ 3500Da) against distilled
water and the water was refreshed five times everyday to remove
the excess cystamine. The resulting product was collected by
freeze-drying.
Poly (ethylene glycol) methyl ether (mPEG, M
n
¼ 5000, Aladdin)
was dried in vacuum for 24 h before use. L-leucine (H-Leu-OH, GL
Biochem, shanghai), D-leucine (H-D-Leu-OH, GL Biochem,
shanghai), chloroactic acid (99%, Aladdin), potassium hydroxide
(
KOH, Aladdin), cystamine dihydrochloride (Cystamine$2HCl, Sig-
’
maeAldrich), N,N -dicyclohexylcarbodiimide (DCC, GL Biochem,
Shanghai), N-Hydroxysuccinimide (NHS, Aladdin) and dithio-
threitol (DTT, 99%, Merck) were used as received. Doxorubicin
hydrochloride (DOX$HCl, Zhejiang Hisun Pharmaceutical Co., Ltd,
China) was desalinated before use. Triphosgene (99%, Aladdin) was
2.4. Synthesis of leucine-N-carboxyanhydride (L-NCA)
’
recrystallized before use. Tetrahydrofuran (THF, SCRC), N,N -
dimethyl formamide (DMF, SCRC) and n-hexane (SCRC) were dried
The following synthesis was adapted from the literature [30].
L-leucine (3.0 g, 22.9 mmol) was suspended in 100 ml THF and
ꢀ
by refluxing over CaH
2
and distilled prior to use. Dimethyl sulfoxide
heated to 70 C in a nitrogen atmosphere under stirring. A solution
(
DMSO, SCRC) was used as received. Dulbecco’s modified Eagle’s
of triphosgene (3.5 g, 11.7 mmol) in 40 ml THF was then added
dropwise to the mixture. After 3 h, the solution was poured into
300 ml n-hexane to obtain crude crystals of L-leucine-N-carbox-
yanhydride (L-L-NCA). L-L-NCA was recrystallized from n-hexane
twice. The D-leucine-N-carboxyanhydride (D-L-NCA) monomers
were also synthesized using the similar protocol.
medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin,
trypsin, Dubelcco’s phosphate buffered saline (DPBS) and 3-(4, 5-
dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT)
were obtained from Gibco Invitrogen Corp. 4% paraformaldehyde
was purchased from DingGuo Chang Sheng Biotech. Co., Ltd.
2.2. Synthesis of mPEG-COOH
2.5. Synthesis of mPEG-SS-Pleu
mPEG-COOH was synthesized according to the following
procedure [29]: an aqueous solution of KOH (40wt%, 48 ml) was
added to a solution of mPEG (5 g, 1 mmol) in 72 ml dimethyl
mPEG-SS-Pleu diblock copolymer was synthesized by ring-
opening polymerization of racemic leucine-N-carboxyanhydride
(rac-L-NCA) initiated by the mPEG-cystamine in N,N-
Scheme 1. Illustration of reduction-sensitive sheddable micelle based on disulfide-linked hybrid PEG-polypeptide mPEG-SS-Pleu for efficient release of doxorubicin (DOX) triggered
by dithiothreitol (DTT).

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T.-B. Ren et al. / Polymer 52 (2011) 3580e3586
dimethylformamide (DMF). The different feed reactant weight
ratios were showed in Table 1. The reaction was stirred at room
temperature in a nitrogen atmosphere for 72 h and then the
polymer was dialyzed against distilled water. The water was
refreshed five times everyday to remove the excess L-NCA. The
resulting product was collected by freeze-drying.
Table 1
Feed composition and some characteristic properties of mPEG-SS-Pleu copolymers.
Disulfide-linked
copolymer
mPEG-SS-NH /rac-L-NCA
(w/w)
2
Mna
Mnb
PDIb
mPEG-SS-Pleu
6
1/4
1/2
5850
6420
6230
6840
1.14
1.15
mPEG-SS-Pleu12
a
Determined by 1H NMR.
Determined by GPC.
b
2.6. Synthesis of FITC-labeled mPEG-SS-Pleu
For fluorescent microscopy evaluation fluorescein iso-
thiocyanate (FITC) was conjugated to mPEG-SS-Pleu following
a previously published procedure [31]. A solution of mPEG-SS-Pleu
2
.10. Reduction-triggered destabilization of mPEG-SS-Pleu micelles
The size change of micelles in response to 10 mM DTT in PBS
(
50 mg) in anhydrous DMF (5 mL) was combined with FITC (50 mg)
buffer (pH ¼ 7.4, 10 mM) was followed by DLS measurement [10].
in the presence of triethylamine (Et N). The reaction was allowed
3
10 mM DTT was added in the solution of mPEG-SS-Pleu micelles in
proceed at r.t. for 48 h before the desired mPEG-SS-Pleu-FITC
conjugate was isolated by dialysis and freeze-dried until further
use.
PBS buffer. At different time points, the size of micelles was
measured by DLS.
2.7. Characterization
2.11. Encapsulation and in vitro release of DOX
The structure of the terminal-modified mPEG was characterized
The diblock copolymer mPEG-SS-Pleu was amphiphilic and self-
1
by FT-IR and H NMR spectroscopy which used DMSO-d6 and D
2
O
assembled into micelles in aqueous solution. Using the dialysis
method, anticancer doxorubicin (DOX) was encapsulated into the
core of the micelles. Briefly, mPEG-SS-Pleu (1 mg) was dissolved in
DMF (4 ml). DOX$HCl (0.1 mg) was dissolved in DMSO (0.1 ml) and
as the solvent. FT-IR spectra were recorded on a Bruker Tensor 27
1
spectrometer. Samples were directly measured as powder. H NMR
spectra were recorded on a Bruker DMX-500 NMR spectrometer.
The molecular weight of the copolymers was determined by gel
permeation chromatograph (GPC) which used THF as the eluent at
then TEA (0.12 mL) was added to remove the HCl of DOX$HCl. These
two kinds of solution were mixed and then transferred into a dial-
ysis tube (MWCO ¼ 8000e12000 Da) to remove the unloaded DOX
and the solvent by dialysis against 500 mL of PBS buffer (pH ¼ 7.4,
a flow rate of 1.0 ml/min. GPC system was equipped with a Waters
ꢀ
1
50 C separations module and a Waters differential refractometer.
The molecular weight and molecular weight distributions were
calibrated against polystyrene standards, with THF as the eluent at
a flow rate of 1 ml/min. The size and size distribution of the
micelles were measured by dynamic light scattering (DLS, Malvern
Instruments Ltd., Worcestershire, UK). The micellar morphology
was determined by transmission electron microscopy (TEM, Tec-
nai-12 Bio-Twin, FEI, Netherlands). The samples of the TEM were
1
0 mM) for 24 h at room temperature. The PBS buffer was refreshed
every 12 h. The whole procedure was performed in the dark. The
amount of unloaded drug was analyzed by using fluorescence
measurement (excitation at 470 nm). Drug loading (DL) and
entrapment efficiency (EE) were calculated according to the
following formula:
prepared by dropping 10 mL of 0.125 mg/mL micellar solution on the
copper grid followed by staining with phosphotungstic acid and
dried at room temperature for 24 h [10].
DL (wt.%) ¼ (mass of drug loaded in micelles/mass of drug loaded
micelles) ꢁ 100%
EE (wt.%) ¼ (mass of drug loaded in micelles/mass of drug fed
initially) ꢁ 100%
2.8. Micelle formation
Membrane-dialysis method was used to prepare the mPEG-SS-
Pleu micelle [11]. The copolymer mPEG-SS-Pleu (1 mg) was dis-
solved in DMF (8 ml). Then the solution was dialyzed
After dialysis, the dialysis tube was directly immersed into
2
00 mL PBS buffer (pH ¼ 7.4, 10 mM). The release of the DOX from
mPEG-SS-Pleu micelles was studied in three different media. PBS
pH ¼ 7.4, 10 mM) with 1 mM DTT, PBS (pH ¼ 7.4, 10 mM) with
0 mM DTT and PBS (pH ¼ 7.4, 10 mM) without DTT. The solution
(
MWCO ¼ 8000e12000 Da) against distilled water to remove the
(
1
organic solvent and form micelles. The water was refreshed three
times a day. During the dialysis, the transparent solution changed
into translucent gradually, indicating the formation of micelles.
2.9. Critical micelle concentration of the copolymer
The critical micelle concentration (CMC) of the copolymer was
estimated by fluorescence spectroscopy using pyrene, a hydro-
phobic florescence probe, which was partitioned preferably in the
micelle core, causing changes in the photophysical properties of
the nanoparticles under investigation [32]. The concentration of
ꢂ6
pyrene in the aqueous solution was 7 ꢁ 10 mol/L. The concen-
ꢂ3
tration of the copolymer was varied from 15 ꢁ 10 to 250 mg/L.
The solution was kept at room temperature and equilibrated for
2
4 h before measurements. The fluorescence spectra were recor-
ded with the excitation wavelength of 310 nm. The emission
fluorescence at 373 nm was monitored. Fluorescence spectra were
recorded on a Hitachi F2500 luminescence spectrometer (Hitachi,
Ltd, Hong Kong).
Scheme 2. Synthesis of the mPEG-SS-Pleu copolymer via disulfide bond conjugation.

T.-B. Ren et al. / Polymer 52 (2011) 3580e3586
3583
6
Fig. 3. CMC of mPEG-SS-Pleu micelles measured by fluorescence spectroscopy.
Fig. 1. FT-IR spectra of: (A) mPEG; (B) mPEG-COOH; (C) mPEG-cystamine; (D) mPEG-
SS-Pleu copolymer.
ꢂ1
mL of fresh media and 20 mL of 5 mg mL sterile filtered
2
3
00
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(
MTT) solution prepared in PBS. The plate was incubated for addi-
ꢀ
ꢀ
was placed in a shaking bed at a speed of 150 rpm at 37 C. At
tional 4 h at 37 C, allowing viable cells to metabolically reduce MTT
desired time intervals, samples (2 mL) were withdrawn and
replaced with an equal volume of fresh media. The DOX concen-
tration was estimated by using fluorescence measurement (exci-
tation at 470 nm) based on the standard calibration curve obtained
from free DOX in DMSO.
into purple formazan. After addition of 150 mL of dimethyl sulfoxide
(DMSO) to each well, the plate was incubated at r.t. for 10 min on
a shaking platform before optical density (OD) was measured at
l
¼ 492 nm using a Multiscan MK3 plate reader (Thermo Fisher
Scientific, Waltham, MA, USA). Cell viability was calculated
according to the following equation:
2.12. Cell cytotoxicity
Cell viability (%) ¼ (ODtreated/ODcontrol) ꢁ 100%
Human liver carcinoma HepG2 cells were generously provided
where ODcontrol was obtained in the absence of polymer and
ODtreated was obtained in the presence of polymer.
by Cell Center of the Tumor Hospital at Fudan University. Routinely,
ꢀ
this cell line was maintained at 37 C in a humidified 5% CO
2
atmosphere using DMEM supplemented with 10% fetal bovine
serum and 0.1% penicillin-streptomycin. For experiments, HepG2
cells were seeded into 96-well plate at a density of 5000 cells/well.
Following an overnight attachment period, cells were preincubated
with and without 10 mM GSH. The preincubation medium was
removed after 2 h and cells were washed with prewarmed PBS. As
a negative control, mPEG-SS-Pleu micelles without DOX dispersed
2
.13. Confocal laser scanning microscopy observation of mPEG-SS-
Pleu micelle treated HepG2 cells
5
HepG2 cells were seeded in a 6-well plate at a density of 1 ꢁ10
cells/well with complete medium. The cells were incubated for 24 h
in a humidified atmosphere with 5% CO
washed by PBS and incubated at 37 C for 24 h with mPEG-SS-Pleu
micelles in complete DMEM. The cells were then washed with PBS
twice and fixed with 4% paraformaldehyde. The slides were
mounted and observed with a confocal laser scanning microscope
ꢀ
2
at 37 C. Cells were
ꢀ
in complete DMEM (100
mL) were added to cells preincubated
without GSH. Treatment groups with DOX-loaded mPEG-SS-Pleu
micelles included both cells with and without preexposure to
1
0 mM GSH. mPEG-SS-Pleu copolymers were tested at a concen-
(Olympus, FV300, IX71, Tokyo, Japan) equipment.
ꢂ3
ꢂ1
trations ranging from 7.8 ꢁ 10 to 1 mg mL . All suspensions
were prepared in complete cell culture media. After a 24 h
incubation period, the volume in each well was replaced with
3
. Results and discussion
3.1. Synthesis of mPEG-SS-Pleu diblock copolymer
The mPEG-SS-Pleu diblock copolymers with different molecular
weights were obtained by varying the mPEG-cystamine/L-NCA feed
weight ratios (Table 1). The synthetic approach was shown in
Scheme 2. The mPEG-SS-Pleu was prepared by ring-opening
polymerization of racemic leucine-N-carboxyanhydride (rac-L-
NCA), initiated by the primary amine end-group on the mPEG
chain. Deming recorded that use of rac-leucine would prevent
a-helix formation and increase chain flexibility. Disordered racemic
polypeptides also presented exposed amide groups capable of
facilitating H-bonding interactions between hydrophobic chains in
the assemblies [33]. Furthermore, use of disordered racemic
hydrophobic segments was found to favor formation of spherical
micelles in water that can entrap cargos and possess a nonionic,
PEG-like corona potentially useful for drug delivery applications
[34]. The synthesis procedure included three steps: (1) the prepa-
ration of mPEG-COOH, which was obtained by the nucleophilic
Fig. 2. 1H NMR spectra of: (A) mPEG-SS-Pleu copolymer in DMSO-d6; (B) mPEG-SS-
Pleu micelles in D O.
2

3584
T.-B. Ren et al. / Polymer 52 (2011) 3580e3586
6 6
Fig. 4. (A) Size distribution of mPEG-SS-Pleu micelles determined by DLS; (B) TEM image of mPEG-SS-Pleu micelles.
substitution reaction between chloroactic acid and mPEG. The
mPEG-SS-Pleu calculated by GPC is consistent with the results of
the H NMR spectrum (Table 1).
1
polymer was purified by exhausted dialysis to remove the residual
ꢂ1
chloroactic acid. The typical C]O band of the polymer at 1741 cm
arising from the introduced eCOOH, in the FT-IR of the polymer
3.2. Micelle formation and reduction-responsive destabilization
(Fig. 1B), proved the successfully conjugation of carboxyl group
onto the terminal mPEG; (2) the preparation of mPEG-cystamine,
which was synthesized by condensation reaction between the
excess cystamine and mPEG-COOH in the presence of DCC and NHS
Micelles of mPEG-SS-Pleu copolymers were prepared by dialysis
method. With increasing concentration of polymer solution,
micelle formation occurs at a certain concentration, which is
defined as the critical micelle concentration (CMC). The CMC was
determined using pyrene as a probe. The plot of fluorescence
intensity versus copolymer concentration was shown in Fig. 3. As
the copolymer concentration increased, an abrupt increase of
intensity was observed at a certain concentration, which indicated
the formation of micelles and the transfer of pyrene into the
in CH
2
Cl
2
. The FT-IR of mPEG-cystamine was shown in Fig. 1C. The
ꢂ1
typical amide I and II bands at 1687 and 1533 cm were observed
from the FT-IR spectroscopy, which revealed the successful
synthesis of the polymer; (3) the preparation of mPEG-SS-Pleu. The
rac-L-NCA reacted with mPEG-cystamine to obtain mPEG-SS-Pleu
copolymer, whose structure was characterized by both FT-IR and
1
H NMR. As shown in the FT-IR in Fig. 1D, the higher stretching
6
hydrophobic core of the micelles. mPEG-SS-Pleu showed a CMC of
ꢂ1
variation absorbance of NeH at 1687 and 1533 cm compared
2
.8 mg/L, while the CMC of mPEG-SS-Pleu12 could not be obtained
with that of mPEG-cystamine were observed from the spectros-
copy, which revealed the successful synthesis of the copolymer.
by the same method. This phenomena might be explained by
Deming’s findings on that the long hydrophobic segments of Pleu
would lead to fast aggregation into precipitations [33]. Although
1
Otherwise, in the H NMR spectrum of the copolymer, shown in
Fig. 2A, the signals assigned to both mPEG (
leucine ( 0.87, 1.24 and 1.45) were detectable. The resonances at
.42 ppm attributed to the methylene protons neighboring to the
disulfide bond (-CH -SS-CH -) were also detected. These results
d 3.54 and 3.24) and
6
without long hydrophobic segments, the mPEG-SS-Pleu showed
d
very low CMC down to 2.8 mg/L, providing the possibility of
keeping stabilization of micelles and allowing their use in very
dilute aqueous milieu such as blood and body fluids. This feature
was important for drug delivery uses.
3
2
2
supported successful synthesis of the copolymer.
The structure of the copolymer was further verified by GPC. The
Another evidence for the micelle formation of mPEG-SS-Pleu
6
1
1
results of GPC analysis and H NMR estimation of the molecular
was obtained by the H NMR spectroscopy in D
2
O. The signals of the
n
weight of copolymers were summarized in Table 1. The M value of
hydrogen atoms of methylene group in mPEG units and the solvent
Fig. 5. The size change of mPEG-SS-Pleu
.4) determined by DLS measurement.
6
micelle in response to DTT in PBS buffer (pH
Fig. 6. In vitro reduction-triggered release profiles of DOX from mPEG-SS-Pleu
micelle in PBS buffer (pH 7.4) at 37 C.
6
ꢀ
7

T.-B. Ren et al. / Polymer 52 (2011) 3580e3586
3585
structure of aggregates [35] and the dehydration of the micelles
and the shrinkage of the PEG shell induced by water evaporation
under the high vacuum conditions before TEM observation [10,36].
The size change of micelles in response to 10 mM DTT in PBS
buffer (pH 7.4, 10 mM) was studied using DLS. Following addition of
10 mM DTT, the micelle size increased from 160 nm to about
300 nm in 6 h (Fig. 5). Large aggregates with sizes of over 4000 nm
were also observed. The aggregates were formed most probably
due to the cleavage of the intermediate disulfide bonds, which
resulted in shedding of the PEG shells. In contrast, no change in
micelle sizes was discerned after 24 h in absence of DTT under
otherwise the same conditions. This phenomenon was best suitable
to our expectation and revealed that the copolymer mPEG-SS-Pleu
possessed the reduction sensitive property.
6
Fig. 7. Cell proliferation of HepG2 cancer cells after 24 h incubation with mPEG-SS-
3.3. Loading and triggered release of DOX
Pleu
6
nanomicelles alone; DOX-loaded mPEG-SS-Pleu
6
nanomicelles with GSH
treated or untreated.
The drug release behavior of micelles was studied. DOX was
chosen as the model drug. DOX is one of the most potent and
clinically useful anticancer drugs, which have the capability of
inhibiting the synthesis of DNA and RNA and used widely in the
treatment of different types of solid malignant tumors [37e39]. It is
crucial, therefore, to deliver and release DOX in the cytoplasm and/
or right into the cell nucleus. The aim of this study was to develop
triggered intracellular delivery systems for DOX, which may lead to
enhanced cancer chemotherapy. The intrinsic fluorescent proper-
ties of DOX allow us to follow the internalization mechanism and
localization during in vitro experiments.
DOX was loaded into micelles by dialysis of a polymer/DOX
solution in DMSO against PBS buffer (PH 7.4, 10 mM) and the drug
in excess was removed. The drug loading content and encapsula-
tion efficiency were determined by fluorescence measurement. The
theoretical drug loading content was set at 10 wt%. The result
1
2
peak of D O were detectable at 3.61 ppm and 4.68 ppm in the H
NMR spectrum, shown in Fig. 2 B. The signals of the hydrogen
atoms in the leucine segment almost disappeared in D O as
compared with that of the copolymer in DMSO shown in Fig. 2 A,
which indicated that the copolymer formed a core-shell micellar
structure with an isolated hydrophobic inner core and a hydrophilic
outer shell.
The micellar structure was further confirmed by dynamic light
scattering (DLS) and transmission electron microscopy (TEM). DLS
2
6
measurements showed that mPEG-SS-Pleu formed micelles with
sizes of 160 nm (Fig. 4A). TEM micrograph revealed that these
micelles had a spherical morphology with an average size of 70 nm
(Fig. 4B). The larger diameter obtained from DLS measurement may
be due to the existence of compound micelles with an internal
6
Fig. 8. Confocal laser scanning images of HepG2 cells cultured with FITC labeled mPEG-SS-Pleu copolymers. (A) shows the bright-field image; (B) shows the FITC fluorescence; (C)
the blue fluorescent was derived from the nuclei stained with DAPI and the green fluorescence was from FITC. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article).

3586
T.-B. Ren et al. / Polymer 52 (2011) 3580e3586
showed that the drug loading efficiency was approximately 70% for
mPEG-SS-Pleu micelles.
The release of DOX from mPEG-SS-Pleu
potential. The cell internalization experiment exhibited that fluo-
rescent micelles could be internalized into the cells. The cytotox-
icity study showed that the copolymer exhibited good
biocompatibility and it was evident that these micelles hold great
promise to be utilized as a potential hydrophilic drug carrier to
enhance the efficiency of cancer chemotherapy.
6
6
micelles was investi-
gated using a dialysis tube (MWCO 12000) in PBS buffer (pH 7.4,
ꢀ
1
0 mM) at 37 C in presence or absence of 10 mM DTT. The drug
release data was shown in Fig. 6. Remarkably, the results showed
that in presence of 10 mM DTT, a reductive environment analogous
to that of the intracellular compartments such as cytosol and the
cell nucleus, the drug loaded micelles exhibited a much faster drug
release rate and 40% of the encapsulated DOX was released within
Acknowledgement
This work was financially supported by National Natural Science
Foundation of China (21004045&30800230), and Shanghai Natural
Science Foundation (10ZR1432100).
12 h, while it took nearly 100 h to release 25% of loaded DOX in
absence of DTT. There are many factors affecting the drug release
rate, such as polymer degradation, molecular weight, the strength
of the interactions between the drug and the core-forming block of
polymer, and so on [40]. In this study, drug release rates from
mPEG-SS-Pleu micelles increased in parallel with increasing DTT
concentrations (i.e., DOX release: 10 mM DTT > 1 mM DTT > 0 mM
DTT). Previous reports suggested that a hydrophilic PEG shell may
act as a significant diffusion barrier, thus negatively affecting
release of encapsulated payload [41,42]. However, the results from
this and previous studies [27,41] experimentally underline the
feasibility to modulate release kinetics of micelle-encapsulated
drug by chemically cleaving the PEG shell from the nanomicelle
architecture. Therefore, stimulus-induced shedding of the PEG shell
References
[1] Kataoka K, Matsumoto T, Yokoyama M, Okano T, Sakurai Y, Fukushima S, et al.
J Control Release 2000;64(1e3):143e53.
[2] Li YY, Zhang XZ, Cheng H, Zhu JL, Li UN, Cheng SX, et al. Nanotechnology 2007;
18:505101 (8pp).
[3] Kim SY, Shin IG, Lee YM, Cho CS, Sung YK. J Control Release 1998;51(1):13e22.
[4] Elamanchili P, McEachern C, Burt H. J Pharm Sci 2009;98(3):945e58.
[
[
5] Kataoka K, Harada A, Nagasaki Y. Adv Drug Deliver Rev 2001;47(1):113e31.
6] Qiu LY, Bae YH. Pharm Res 2006;23(1):1e30.
[7] Ganta S, Devalapally H, Shahiwala A, Amiji M. J Control Release 2008;126(3):
187e204.
[
[
8] Peer D, Karp JM, Hong S, FaroKhzad OC, Margalit R, Langer R. Nat Nanotechnol
007;2(12):751e60.
9] Rijcken CJF, Soga O, Hennink WE, Nostrum CFv. J Control Release 2007;120(3):
6
from biodegradable mPEG-SS-Pleu micelles via reductive cleavage
seems a promising approach to accelerate payload release under
tumor-relevant conditions.
2
131e48.
[
10] Sun HL, Guo BN, Cheng R, Meng FH, Liu HY, Zhong ZY. Biomaterials 2009;
0(31):6358e66.
11] Fischer D, Bieber T, Li Y, Elsasser HP, Kissel T. Pharm Res 1999;16(8):
273e9.
3
3.4. Cell cytotoxicity and internalization
[
1
[
[
[
12] Dehn S, Chapman R, Jolliffe KA, Perrier S. Polym Rev 2011;51(2):214e34.
13] Versluis F, Marsden HR, Kros A. Chem Soc Rev 2010;39(9):3434e44.
14] Perly B, Douy A, Gallot B. CR Acad Sci Paris 1974;279C:1109.
A cytotoxicity study was carried out to investigate whether
reductive cleavage of the mPEG-SS-Pleu copolymer affects cell
6
proliferation of the HepG2 human liver carcinoma cell line. Results
are summarized in Fig. 7. As an important control experiment, it
[15] Wang K, Dong HQ, Wen HY, Xu M, Li C, Li YY, et al. Macromol Biosci 2011;
1(1):65e71.
1
[
[
16] Rao JY, Luo ZF, Ge ZS, Liu H, Liu SY. Biomacromolecules 2007;8(12):3871e8.
17] Wang L, Zeng R, Li C, Qiao RZ. Colloid Surf B 2009;74(1):284e92.
was demonstrated that micellar aggregates of mPEG-SS-Pleu
6
copolymer without DOX do not significantly affect cell viability and
proliferation of this cell line at concentrations ꢃ1000 mg/L. Inclu-
sion of DOX into these nanomicelles effectively reduced cell
viability in a dose-dependent fashion. Most importantly, however,
[18] Huang CJ, Chang FC. Macromolecules 2008;41(19):7041e52.
[19] Lin JP, Zhu JQ, Chen T, Lin SL, Cai CH, Zhang LS, et al. Biomaterials 2009;30(1):
108e17.
[
[
20] Guo XD, Zhang LJ, Chen Y, Qian Y. AIChE J 2010;56(7):1922e31.
21] Ghoorchian A, Cole JT, Holland NB. Macromolecules 2010;43(9):4340e5.
the cytotoxic efficacy of DOX-loaded mPEG-SS-Pleu
6
nanomicelles
[22] Liu XM, Yang YY, Leong KW. J Colloid Interf Sci 2003;266(2):295e303.
[23] Atmaja B, Cha JN, Marshall A, Frank CW. Langmuir 2009;25(2):707e15.
[24] Li T, Lin JP, Chen T, Zhang SN. Polymer 2006;47(13):4485e9.
[25] Osada K, Kataoka K. Adv Polym Sci 2006;202:113e53.
was dramatically enhanced following pretreatment of HepG2 cells
with 10 mM GSH. These data support our hypothesis that reductive
cleavage of the PEG shell accelerates payload release from this
novel nanomicelle design.
To examine whether the micelles could enter HepG2 cells for
potential antitumor therapy, confocal laser scanning microscopy
[26] Tang LY, Wang YC, Li Y, Du JZ, Wang J. Bioconjug Chem 2009;20(6):1095e9.
[
27] Sun HL, Guo BN, Li XQ, Cheng R, Meng FH, Liu HY, et al. Biomacromolecules
010;11(4):848e54.
2
[28] Kuppusamy P, Li H, Ilangovan G, Cardounel AJ, Zweier JL, Yamada K, et al.
Cancer Res 2002;62(1):307e12.
[
[
29] Zhu J, Zhu XL, Kang ET, Neoh KG. Polymer 2007;48(24):6992e9.
30] Koo AN, Lee HJ, Kim SE, Chang JH, Park C, Kim C, et al. Chem Commun 2008;
(
CLSM) was used to observe the internalization of the micelles into
HepG2 cells. The hybrid polypeptide mPEG-SS-Pleu was labeled by
6
48:6570e2.
FITC. After 24 h of incubation with the micelles, the CLSM images of
HepG2 cell were demonstrated in Fig. 8. It was found that the
micelles could be internalized into the HepG2 cells because the
green fluorescence of FITC was mainly localized in internal cell. The
CLSM results demonstrated that the micelle was an efficient carrier
to deliver DOX into the human liver carcinoma HepG2 cells.
[31] Riddles PW, Blakeley RL, Zerner B. Methods Enzymol 1983;91:49e60.
[
32] Bae JW, Lee E, Park KM, Park KD. Macromolecules 2009;42(10):3437e42.
[33] Hanson JA, Li ZB, Deming TJ. Macromolecules 2010;43(15):6268e9.
[34] Kataoka K, Kwon GS, Yokoyama M, Okano T, Sakurai Y. J Control Release 1993;
24(1e3):119e32.
[35] Zhang LF, Eisenberg A. J Am Chem Soc 1996;118(13):3168e81.
[
36] Hu Y, Zhang L, Cao Y, Ge H, Jiang X, Yang C. Biomacromolecules 2004;5(5):
756e62.
1
[37] Akinc A, Anderson DG, Lynn DM, Langer R. Bioconjug Chem 2003;14(5):
979e88.
4
. Conclusion
[
[
[
38] Adams ML, Lavasanifar A, Kwon GS. J Pharm Sci 2003;92(7):1343e55.
39] Jimenez C, Tramontano A. Tetrahedron Lett 2001;42(44):7819e22.
40] Hua SH, Li YY, Liu Y, Xiao W, Li C, Huang FW, et al. Macromol Rapid Comm
2010;31(1):81e6.
In summary, a polypeptide hybrid copolymer mPEG-SS-Pleu has
been prepared. The micelles were nontoxic, showed a high drug
loading efficiency for DOX and displayed low drug release under
a non-reductive environment while released drug rapidly and
quantitatively in response to the intracellular level of reducing
[
[
41] Wen HY, Dong HQ, Xie WJ, Li YY, Wang K, Pauletti GM, et al. Chem Commun
2011;47(12):3550e2.
42] Masuda T, Akita H, Niikura K, Nishio T, Ukawa M, Enoto K, et al. Biomaterials
2009;30(27):4806e14.