Redox-responsive flower-like micelles of poly(l-lactic acid)-b-poly(ethylene glycol)-b-poly(l-lactic acid) for intracellular drug delivery

Article abstract of DOI:10.1016/j.polymer.2016.03.030

Redox-responsive micelles self-assembled from triblock copolymers of poly(l-lactic acid)-poly(ethylene glycol)-poly(l-lactic acid) (PLA-PEG-PLA) with double-disulfide linkage in the backbone were synthesized and characterized by proton nuclear magnetic resonance (¹H NMR) and size exclusion chromatography (SEC), in which both PEG (M_n = 1000, 2000 and 4000 g mol^-1) and PLA (M_n = 1600 g mol^-1) have different molecular weights respectively. The triblock copolymers PLA₃₀₀₀-PEG₂₀₀₀-PLA₃₀₀₀ and PLA₃₀₀₀-PEG₄₀₀₀-PLA₃₀₀₀ can self-assemble into flower-like micelles in aqueous media with average diameters 110 nm and 43 nm and lower critical micelle concentrations (CMC) 0.017 and 0.014 mg mL^-1 respectively compared with that of diblock copolymers. Moreover, in vitro drug release analyses indicated that reductive environment can result in triggered drug release profiles. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl- 2-H-tetrazolium bromide (MTT) assay in vitro showed no significant cytotoxicity as NIH 3T3 cells incubated in the micelles even when the concentrations up to 1000 μg/mL. Additionally fluorescence microscopy measurements and MTT assay demonstrated that the micelles exhibited a faster drug release and higher cellular proliferation inhibition due to the effect of intracellular reduction responsiveness compared with that of diblock copolymers. The above results suggest that the reduction-responsive, biodegradable and biocompatibility micelles could provide a platform to construct potential drug delivery systems for cancer therapy.

Full text of DOI:10.1016/j.polymer.2016.03.030

Accepted Manuscript
Redox-responsive flower-like micelles of poly(L-lactic acid)-b-poly(ethylene glycol)-b-
poly(L-lactic acid) for intracellular drug delivery
Qinglai Yang, Changyu He, Zhen Zhang, Lianjiang Tan, Bingya Liu, Zhenggang Zhu,
Zhifeng Shao, Bing Gong, Yu-Mei Shen
PII:
S0032-3861(16)30170-7
10.1016/j.polymer.2016.03.030
JPOL 18522
DOI:
Reference:
To appear in: Polymer
Received Date: 27 October 2015
Revised Date: 8 March 2016
Accepted Date: 10 March 2016
Please cite this article as: Yang Q, He C, Zhang Z, Tan L, Liu B, Zhu Z, Shao Z, Gong B, Shen Y-M,
Redox-responsive flower-like micelles of poly(L-lactic acid)-b-poly(ethylene glycol)-b-poly(L-lactic acid)
for intracellular drug delivery, Polymer (2016), doi: 10.1016/j.polymer.2016.03.030.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Redox-responsive flower-like micelles of poly(L-lactic
acid)-b-poly(ethylene glycol)-b-poly(L-lactic acid) for
intracellular drug delivery
Qinglai Yang,† Changyu He,# Zhen Zhang,† Lianjiang Tan,† Bingya Liu #, Zhenggang Zhu#,
Zhifeng Shao¶, Bing Gong||,§ and Yu-Mei Shen†,*
¶
†Key Laboratory of Systems Biomedicine (Ministry of Education) and Bio-ID Center, School of
#
Biomedical Engineering, Shanghai Key Laboratory of Gastric Neoplasms, Department of
Surgery, Shanghai Institute of Digestive Surgery, Ruijin Hospital, Shanghai Jiao Tong University
School of Medicine, Shanghai 200025, China. ||College of Chemistry, Beijing Normal University,
Beijing 100875, China; §Department of Chemistry, University at Buffalo, State University of New
York, Buffalo, NY 14260, United States.
*Corresponding author. E-mail: yumeishen@sjtu.edu.cn
ABSTRACT: Redox-responsive micelles self-assembled from triblock copolymers of
poly(L-lactic acid)-poly(ethylene glycol)-poly(L-lactic acid) (PLA-PEG-PLA) with
double-disulfide linkage in the backbone were synthesized and characterized by
proton nuclear magnetic resonance (1H-NMR) and size exclusion chromatography
(SEC), in which both PEG (Mn = 1000, 2000 and 4000 g.mol-1) and PLA (Mn = 1600
g.mol-1) have different molecular weights respectively. The triblock copolymers
PLA3000-PEG2000-PLA3000 and PLA3000-PEG4000-PLA3000 can self-assemble into
flower-like micelles in aqueous media with average diameters 110 nm and 43 nm
and lower critical micelle concentrations (CMC) 0.017 and 0.014 mg/mL
respectivelycompared with that of diblock copolymers. Moreover, in vitro drug
release analyses indicated that reductive environment can result in triggered drug
release profiles. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl- 2-H-tetrazolium bromide
(MTT) assay in vitro showed no significant cytotoxicity as NIH 3T3 cells incubated

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in the micelles even when the concentrations up to 1000 µg/mL. Additionally
fluorescence microscopy measurements and MTT assay demonstrated that the
micelles exhibited a faster drug release and higher cellular proliferation inhibition due
to the effect of intracellular reduction responsiveness compared with that of diblock
copolymers. The above results suggest that the reduction-responsive, biodegradable
and biocompatibility micelles could provide a platform to construct potential drug
delivery systems for cancer therapy.
Keywords: amphiphilic triblock copolymer, PLA-PEG-PLA, double-disulfide
linkage, drug delivery carriers, redox-responsiveness
Introduction
In the past decades，there has been considerable interest in using amphiphilic
copolymers as drug delivery system for cancer therapy.[1-4] Amphiphilic copolymers
can self-assemble into core-shell structured micelles in aqueous solution with
hydrophobic interior and hydrophilic corona.[5-9] Such biodegradable copolymer
micelles encapsulate anticancer drugs for targeted delivery and controlled release in
high efficacy and low side effects.[10-13] Structure of the linking units in amphiphilic
copolymer can be sensitive to external or internal stimuli to control the encapsulated
drug release. These incentives include pH,[14-17] glutathione (GSH),[18-24]
30]
temperature,[25-28] glucose,[29,
and others.[31-35] Deserved to more attention,
disulfide-linkage in amphiphilic copolymers responsive to reductive GSH are
interesting to construct drug delivery systems[36-42] because of the presence of redox
potential gradient between the extra- and intracellular space.[43-45] Over the past years,
some efforts have been made to construct supramolecular block copolymers and
specific chemical reactions associated by H-bonding duplex with specific amide
sequencing.[46-50] Recently we reported amphiphilic diblock poly(ethylene
glycol)-poly(L-lactic
acid)
copolymers
(PEG-PLA)
and
chitosan
oligosaccharide-poly(L-lactic acid) copolymers (CSO-PLA) with double disulfide
linkage with H-bonding association units in the backbone.[51-53]

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In this paper, amphiphilic triblock copolymers poly(L-lactic acid)-poly(ethylene
glycol)-poly(L-lactic acid) (PLA-PEG-PLA) relying on H-bonding instructed
association units with double disulfide linkages were synthesized and self-assembled
into micelles for intracellular drug delivery (Scheme 1). Similar to AB diblock
copolymers PEG-PLA, ABA triblock copolymers PLA-PEG-PLA also consisting of
hydrophilic B (PEG) block and a hydrophobic block A (PLA), with a proper
suppressing of the interchain bridging in a microphase inversion, most of the water
insoluble PLA blocks can undergo an intrachain association prior to the interchain
assembly into a flower-like micelles with a collapsed PLA core and a swollen shell
made of PEG loops.[54-56] We found that PLA-PEG-PLA flower-like micelles have
lower CMC compared with that of diblock copolymer micelles,[51-53] while
maintaining redox-responsive properties with efficient intracellular drug release
triggered by intracellular reductants GSH and therefore enhance inhibition of cell
proliferation. Such redox-responsive micelles are desirable for constructing a
promising platform for anticancer drug delivery.
Scheme 1. Illustration of triblock copolymer micelles for controlled release of encapsulated drugs
Experimental section
Materials. All chemicals were purchased from Aldrich or Aladdin and were used as
received unless otherwise indicated. All reactions were followed by thin-layer

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chromatography (TLC) (precoated 0.25 mm silica gel plates from Aldrich), and silica
gel column chromatography was carried out with silica gel 60 (mesh 200-400).
Dichloromethane, N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO)
were dried over calcium hydride and then purified by vacuum distillation.
Amine-terminated poly(ethylene glycol) (NH2-PEG-NH2) was purchased from
Shanghai Yare Bio. Co. Ltd. (number average molar mass (Mn) determined by size
exclusion chromatography (SEC), Mn = 1000, 2000 and 4000 g.mol-1, Mw/Mn = 1.07,
1.05 and 1.05) and used as received. PLA3000-PEG4000-PLA3000 (without disulfide
linkage in the backbone) and polylactic acid (PLA) were purchased from Jinan
Daigang Biology Co. Ltd. (for PLA viscosity-average molar mass (Mv) determined by
viscometer, Mv = 3000 g.mol-1; Mn = 1600 g.mol-1) and used as received.
Measurements. Nuclear magnetic resonance (NMR) analyses were recorded on
Bruker Avance Ⅲ 400 MHz spectrometer with deuterated methanol (CD3OD) or
chloroform (CDCl3) as solvent. The number average molar mass (Mn), weight
average molar mass (Mw) and polydispersity index (PDI) were measured by size
exclusion chromatography (SEC). SEC was performed on Agilent SEC series 1260
infinty, 10 µm PLgel 600 × 7.5 mm column, THF used as the mobile phase at a flow
rate of 1.0 mL/min at 35 °C, linear polystyrene calibration, equipped with a refractive
index (RI) detector. Spectrometer Dynamic Light Scattering (DLS) measurements
were performed in aqueous solution using a Malvern Zetasizer Nano S apparatus
equipped with a 4.0 mW laser operating at λ = 633 nm. All samples of 1 mg/mL were
measured at 25 °C and at a scattering angle of 173°. Transmission electron
microscopy (TEM) studies were performed with a Tecnai G2 Spirit Biotwin
instrument operated at 120 kV. The samples were prepared by directly dropping the
solution of micelles onto carbon-coated copper grids and dried at room temperature
overnight without staining before measurement.

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Synthesis and Characterization
Synthesis of strand A, strand B and hydrophobic block PLA-B51-53
O
O
O
1.Thirourea,EtOH
Ph3CCl, DMF
Ph3CS
HS
Br
OH
O
OH
OH
2.
NaOH,H2O
5
4
3
O
O
COOCH3
COOH
5
H2SO4/CH3OH
O
Ph3CS
SCPh3
H2N
NH2
EDC, HOBt
H2N
NH2
N
N
H
H
7
6
1
O
OH
O
O
1. 1M NaOH
2. 1M HCl
Ph3CS
SCPh3
N
N
H
H
A
Ph3COH/TFA
SH
SCPh3
H2N
H2N
9
8
NH2
NH2
PLA
H
N
H
N
9/DMF
Ph3CS
SCPh3
EDC, HOBt
HOOC
COOH
O
O
B
10
O
PLA
HN
H
H
N
N
Ph3CS
SCPh3
O
O
PLA-B
Scheme 2. Synthesis of the strands A, B and intermediate PLA-B
4-(Tritylthio)butanoic acid (5)49. 4-Bromo-butyric acid 3 (16.48 g, 98 mmol) was
refluxed for 3 hrs with thiourea (8.5 g, 107 mmol) in 30 mL ethanol. Then 50 mL of
aqueous NaOH (4 M, 200 mmol) was added and refluxing was continued for another
2 hrs. The solution was concentrated in vacuo, acidified with aqueous HCl (6 M), and
extracted with ether, the extract dried over anhydrous sodium sulfate (Na2SO4),
evaporated, and the residue distilled in vacuo to give compound 4 (10.45 g, 87.1
mmol, 87%) as a light pink oil. Then, mixture of compound 4 (3 g, 25 mmol) and
trityl chloride (10 g, 38 mmol) were stirred in 40 mL DMF for 5 days at room
temperature. A 10% sodium acetate solution (350 mL) was then added, and the

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precipitate was filtered and washed with water. The product was distilled in vacuo to
give compound 5 (8.1 g, 22.5 mmol, 90%) as white powder. 1H NMR (400 MHz,
CDCl3) δ7.43-7.37 (m, 6H, ArH), 7.29-7.23 (m, 6H, ArH), 7.22-7.15 (m, 3H, ArH),
2.38 (t, J = 7.4 Hz, 2H, -CH2COO-), 2.30 (t, J = 7.2 Hz, 2H, -CH2S-), 1.76-1.73 (m,
2H, -CH2-).
Methyl 3,5-diaminobenzoate (7). 3,5-Diaminobenzoic acid 6 (1.52 g, 10 mmol) was
dissolved in 20 mL 10% sulfuric acid-methanol solution under an ice water bath, then
refluxed at 90 °C for 6 hrs. After cooling, the solution was concentrated in vacuo,
extracted with ethyl acetate, neutralized with saturated sodium bicarbonate solution,
the extract was dried over anhydrous Na2SO4, ethyl acetate evaporated, and the
residue was distilled in vacuo to give compound 7 (1.48 g, 8.9 mmol, 89%). 1H NMR
(400 MHz, CD3OD) δ 6.76-6.73 (m, 2H, ArH), 6.32-6.31 (m, 1H, ArH), 3.82 (s, 3H,
CH3O-).
Methyl 3,5-bis(4-(tritylthio)butanamido)benzoate (1). To a stirred solution of
compound 5 (1.2 g, 3.3 mmol) and 7 (0.2 g, 1.2 mmol) in 30 mL methylene chloride
were added 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDCI)
(1.0 g, 5.2 mmol) at 0 °C. Stirring was continued for 12 hrs upon the mixture was
warmed up to room temperature. Solvent was evaporated, and the residue was
dissolved in methanol at 0 °C about 1 hr. Recrystallized from methanol afforded
compound 1 (0.63 g, 0.73 mmol, yield 61%). 1H NMR (400 MHz, DMSO-d6) δ 10.07
(s, 2H, -NH(C=O)-), 8.10 (s, 1H, ArH), 7.91 (s, 2H, ArH), 7.43-7.12 (m, 30H, ArH),
3.84 (s, 3H, -OCH3), 2.29 (t, J = 7.2 Hz, 4H, -CH2CO-), 2.15 (t, J = 7.1 Hz, 4H,
-CH2S-), 1.73-1.55 (m, 4H, -CH2CH2CH2-); 13C NMR (100 MHz, DMSO-d6) δ171.1,
166.7, 145.0, 140.2, 130.8, 129.5, 128.4, 127.1, 114.9, 114.2, 66.5, 55.2, 52.7, 35.8,
31.4, 24.4; HRMS (ESI) calcd for C54H50N2O4S2Na 877.3110 (M + Na+), found
877.3132.
3,5-Bis(4-(tritylthio)butanamido)benzoic acid (Strand A). Compound 1 (0.3 g,
0.35 mmol) was dissolved in 6 mL DMSO then heated to 120 °C. To the solution, a

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solution of 0.3 mL NaOH (1.0 M) in water was added in several portions. The
reaction was monitored by TLC. The solution was poured into 30 mL of HCl (1.0 M)
solution, and the precipitate was filtered, washed with water and distilled in vacuo to
give the strand A (0.25 g, 0.3 mmol, 86%). 1H NMR (400 MHz, DMSO-d6) δ 9.99 (s,
2H, -NH(C=O)-), 8.06 (s, 1H, ArH), 7.84 (m, 2H, ArH), 7.31-7.23 (m, 24H, ArH),
7.20-7.15 (m, 6H, ArH), 2.26 (t, J = 7.2 Hz, 4H, -CH2S-), 2.10 (t, J = 7.1 Hz, 4H,
-CH2CO-), 1.65-1.54 (m, 4H, -CH2CH2CH2-); 13C NMR (100 MHz, DMSO-d6)
δ171.0, 167.6, 148.3, 145.0, 140.0, 131.8, 129.5, 128.5, 128.3, 127.8., 127.1, 115.2,
66.5, 35.8, 31.4, 24.4; HRMS (ESI) calcd for C53H48N2O4S2Na 863.2953 (M + Na+),
found 863.2990.
2-(Tritylthio)ethanamine (9). Triphenylmethanol (2.29 g, 8.8 mmol) was added to a
solution of 2-aminoethanethiol hydrochloride 8 (1.0 g, 8.8 mmol) in 4 mL
trifluroacetic acid (TFA). The resulted solution was stirred at room temperature for
about 40 min. TFA was removed in vacuo. The residue was triturated with ethyl ether.
The formed white precipitate was filtered, partitioned with aqueous NaOH (25 mL, 1
M) and extracted with ethyl acetate. The organic layers were dried over anhydrous
Na2SO4. After evaporation, the compound 9 was obtained as white solid (2.35 g, 7.4
mmol, 84%). 1H NMR (400 MHz, CD3OD) δ 7.46-7.41 (m, 6H, ArH), 7.34-7.27 (m,
6H, ArH), 7.26-7.21 (m, 3H, ArH), 2.54-2.44 (m, 4H, -NCH2-, -SCH2-).
5-Amino-N1,N3-bis(2-(tritylthio)ethyl)isophthalamide (Strand B). To a stirred
solution of 5-aminoisophthalic acid 10 (0.36 g, 2.0 mmol) and compound 9 (1.91 g,
6.0 mmol) in 20 mL N,N-dimethylformamide (DMF) were added
1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDCI) (0.95g, 4.8
mmol), and N-hydroxybenzotriazole (HOBt) (0.65 g, 4.8 mmol) at 0 °C. Stirring was
continued for 4 hrs while the mixture was warmed up to room temperature (RT). The
solution was poured into 30 mL ice-water solution, the precipitate was filtered and
1
distilled in vacuo to give strand B (0.97g，1.24 mmol, 62%). H NMR (CDCl3, 400
MHz) δ 7.43-7.34 (m, 12H, ArH), 7.28-7.14 (m, 19H, ArH), 7.06 (s, 2H, ArH), 6.34

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(t, J = 5.6Hz，2H，-NHCH2-), 3.27-3.18 (m, 4H, -NCH2-), 2.48 (t, J = 6.4Hz, 4H,
-SCH2-); 13C NMR (100 MHz, CDCl3) δ166.9, 147.1, 144.6, 135.9, 129.5, 128.0,
126.9, 116.2, 114.6, 67.9, 38.8, 32.0; HRMS (ESI) calcd for C50H45N3O2S2Na
806.2851 (M+Na+), found 806.2824.
PLA-B. Common procedure: Under nitrogen atmosphere, PLA (1.0 mmol),
N-methylmorpholine (NMM) (1.0 mmol), 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HATU) (1.5 mmol) and the intermediate
strand B (1.5 mmol) in anhydrous DMF (15 mL) were cooled in ice-water bath with
stirring by a magnetic. After stirring for 1 hr, reaction was continued at room
temperature for additional 24 hrs. The resulting mixture was poured into water and
extracted with CH2Cl2, the combined organic extracts were washed with water and
brine, dried over anhydrous MgSO4 and evaporated in vacuo. The hydrophobic block
PLA-B was further purified by chromatography on silica gel by using
CH2Cl2/CH3OH (100/1 to 50/1) as eluent.
PLA3000-B. Purified isolated yield: 76%; 1H NMR (400 MHz, CDCl3) δ 8.19-8.10 (m,
2H, ArH), 7.80 (s, 1H, ArH), 7.45-7.38 (m, 12H, ArH), 7.31-7.23 (m, 12H, ArH),
7.22-7.16 (m, 6H, ArH), 6.34 (brs, 2H ,-NH-), 5.40-5.02 (m, 21H, 1H per repeating
unit, PLA-CH-), 4.39-4.31 (m, 1H, PLA-CH-), 3.30-3.22 (m, 4H, -NCH2-), 2.51 (t, J
= 6.4Hz, 4H, -SCH2-), 1.65-1.44 (m, 66H,3H per repeating unit, PLA-CH3-); GPC
retention time: 6.40 min, GPC: Mn = 2400, Mw = 2900, PDI = 1.21; IR (KBr, cm-1)
3385, 2994, 2941, 1757, 1657, 599, 1536, 1449, 1379, 1359, 1266, 1189, 1129, 1092,
1047, 951, 866, 803, 746, 702, 622, 507.
Synthesis of hydrophilic block A-PEG-A. In a typical procedure: under nitrogen
atmosphere, strand A (1.0 mmol), NMM (2.0 mmol), HATU (2.0 mmol) and
NH2-PEG-NH2 (0.4 mmol) in anhydrous DMF (15 mL) were cooled in ice-water bath
with stirring by a magnetic. After stirring for 1 h, the reaction was continued at room
temperature for additional 14 h. The resulting mixture was poured into water and

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extracted with CH2Cl2. The combined organic extracts were washed with water and
brine, dried over anhydrous MgSO4 and evaporated in vacuo. The residue was
purified by silica gel column chromatography using an eluent gradient
CH3OH/CH2Cl2 (1/20-1/10) to give the desired intermediate A-PEG-A.
1
A-PEG1000-A, isolated yield 76%, H NMR (400 MHz, CDCl3) δ 8.05 (s, 4H, ArH),
7.65 (s, 2H, ArH), 7.43-7.31 (m, 24H, ArH), 7.28-7.22 (m, 24H, ArH), 7.20-7.14 (m,
12H, ArH), 3.88-3.41 (m, 88H, -OCH2CH2O-), 2.32-2.22 (m, 16H, -COCH2-, -SCH2-),
1.80-1.72 (m, 8H, -CH2CH2CH2-); IR（KBr）2921, 1756, 1650, 1603, 1541, 1452,
1352, 1252, 1248, 1193, 1099, 952, 845, 668, 558.
1
A-PEG2000-A, isolated yield 68%, H NMR (400 MHz, CDCl3) δ 8.08 (s, 4H, ArH),
7.67 (s, 2H, ArH), 7.41-7.37 (m, 24H, ArH), 7.29-7.23 (m, 24H, ArH), 7.25-7.16 (m,
12H, ArH), 3.84-3.40 (m, 164H, -OCH2CH2O-), 2.32-2.23 (m, 16H, -COCH2-,
-SCH2-), 1.80-1.70 (m, 8H, -CH2CH2CH2-); IR（KBr）2916, 1755, 1650, 1602, 1544,
1451, 1352, 1252, 1249, 1192, 1099, 951, 845, 668, 557.
1
A-PEG4000-A, isolated yield 83%, H NMR (400 MHz, CDCl3) δ 8.01 (s, 4H, ArH),
7.63 (s, 2H, ArH), 7.46-7.39 (m, 24H, ArH), 7.34-7.26 (m, 24H, ArH), 7.25-7.14 (m,
12H, ArH), 3.83-3.42 (m, 368H, -OCH2CH2O-), 2.36-2.25 (m, 16H, -COCH2-,
-SCH2-), 1.83-1.61 (m, 8H, -CH2CH2CH2-); IR（KBr）2915, 1756, 1653, 1601, 1548,
1451, 1363, 1252, 1248, 1190, 1099, 951, 846, 665, 555.
Synthesis of triblock copolymers PLA-PEG-PLA. In a typical procedure:
PLA3000-B (30 mL) and A-PEG-A (10 mL) were taken from CH2Cl2 stock solutions
(1.0 mM) separately and mixed in a 250 mL round bottom flask. Solvent was
evaporated in vacco and the residue was dissolved with 60 mL of iodine solution (6
mM) in CH2Cl2. The resulting mixture was stirred at room temperature without inert
gas protection. After 1 h, the reaction mixture was cooled in an ice-water bath and a
sodium thiosulfate aqueous solution (3 mM) was added until the color of iodine
disappeared. The mixture was then extracted with CH2Cl2. The organic layer was

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washed with water and brine and dried over anhydrous Na2SO4. Purification was
accomplished by thin-layer chromatography on silica gel by using CH2Cl2/CH3OH
(10/1) as eluent to give the desired triblock copolymer PLA-PEG-PLA.
1
PLA3000-PEG1000-PLA3000, isolated yield: 48%, H NMR (400 MHz, CDCl3) δ
8.33-7.32 (m, 12H, ArH), 5.26-5.12 (m, 42H, 1H per repeating unit, PLA-CH-),
4.35-4.31 (m, 1H, PLA-CH-), 3.76-3.41 (m, 94H, 2H per repeating unit,
-OCH2CH2O-), 2.94-2.73 (m, 16H, -NCH2-, -SCH2-), 2.53-2.05 (m, 24H, -(C=O)
CH2-, -SCH2-, -CH2-), 1.74-1.21 (m, 129H, 3H per repeating unit, PLA-CH3-); IR
(KBr) 3467, 2883, 1755, 1651, 1601, 1544, 1451, 1380, 1350, 1256, 1183, 1092, 956,
863, 763, 704, 667, 645.
1
PLA3000-PEG2000-PLA3000, isolated yield: 37%, H NMR (400 MHz, CDCl3) δ
8.30-7.35 (m, 12H, ArH), 5.27-5.11 (m, 40H, 1H per repeating unit, PLA-CH-),
4.38-4.32 (m, 1H, PLA-CH-), 3.80-3.45 (m, 174H, 2H per repeating unit,
-OCH2CH2O-), 2.98-2.76 (m, 16H, -NCH2-, -SCH2-), 2.50-2.00 (m, 24H, -(C=O)
CH2-, -SCH2-, -CH2-), 1.75-1.24 (m, 123H, 3H per repeating unit, PLA-CH3-); IR
(KBr) 3455, 2878, 1755, 1650, 1602, 1544, 1450, 1380, 1350, 1256, 1180, 1098, 956,
863, 759, 704, 667, 648.
1
PLA3000-PEG4000-PLA3000, isolated yield: 53%, H NMR (400 MHz, CDCl3) δ
8.31-7.33 (m, 12H, ArH), 5.26-5.13 (m, 44H, 1H per repeating unit, PLA-CH-),
4.45-4.31 (m, 1H, PLA-CH-), 3.80-3.45 (m, 364H, 2H per repeating unit,
-OCH2CH2O-), 2.97-2.73 (m, 16H, -NCH2-, -SCH2-), 2.56-2.08 (m, 24H, -(C=O)
CH2-, -SCH2-, -CH2-), 1.78-1.21 (m, 135H, 3H per repeating unit, PLA-CH3-); IR
(KBr) 3459, 2883, 1755, 1655, 1600, 1544, 1451, 1380, 1352, 1256, 1183, 1098, 956,
865, 759, 705, 667, 645.
Preparation and Characterization of the Micelles. In brief, 10 mg PLA-PEG-PLA
was dissolved in 1 mL of DMSO and stirred at room temperature for 30 min. Then,
the solution was added slowly to 8 mL of deionized water within 10 min and stirred

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for another 1 h. Subsequently, the solution was dialyzed against deionized water for
24 hrs (molecular weight cut off (MWCO) = 2000 g.mol-1), and the deionized water
was exchanged every 4 h for 2 days, and the dialysate through the 0.45 µm filter
membrane to give micelles solution. The critical micelle concentration (CMC) was
determined using 1,6-diphenylhexa-1,3,5-triene (DPH, purity 98% ) as UV probe by
monitoring the absorbance at 313 nm. The concentration of block copolymer varies
from 0.5 × 10-4 to 0.5 mg.mL-1 and the DPH concentration was fixed at 5 × 10-6
mol.L-1. The absorbance spectra of all solutions were recorded by using BioTek
Synergy 2.
Preparation of the DOX-Loaded Micelles. Typically, 10 mg of PLA-PEG-PLA
was dissolved in 1 mL of DMSO, followed by the addition of 1 mg DOX·HCl and
two molar equivalents of triethylamine (TEA, purity 99%), and stirred at room
temperature for 1 h. Then, the mixture was added slowly to 8 mL of deionized water
within 10 min and stirred for another 2 h. Subsequently, the solution was dialyzed
against deionized water for 24 h (MWCO = 2000 g.mol-1), and the deionized water
was exchanged every 5 h. To determine the total loading of drug, the DOX-loaded
micelle solution was lyophilized and then dissolved in DMSO again. The UV
absorbance of the solution at 485 nm was measured to determine the total loading of
DOX. Drug loading content (DLC) and drug loading efficiency (DLE) were
calculated according to the following formula.
DLC (wt %) = (weight of loaded drug/weight of polymer) × 100%
DLE (wt %) = (weight of loaded drug/weight in feed) × 100%
Redox-Triggered Destabilization. The size change of PLA-PEG-PLA micelles in
response to 10 mM of DL-Dithiothreitol (DTT) in deionized water was determined by
DLS measurement. In brief, DTT was added to 10 mL of micelle solution (1 mg/mL)
to achieve a final concentration at 10 mM, which was then degassed in advance with

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nitrogen for 15 min. The size at different time intervals was measured by DLS.
In Vitro Drug Release. Typically, DOX-loaded PLA-PEG-PLA micelle (1 mg.mL-1)
with treatment of DTT (0.1, 1.0 and 10 mM) was immediately measured by
fluorescence measurements (BioTek Synergy 2, excitation (EX) wavelength 485 nm,
emission (EM) wavelength 590 nm (DOX)) at different time intervals, and 2.0 mL of
the micelle solution was transferred into a membrane bag (molecular weight cut off
(MWCO) = 2000 g.mol-1). It was immersed in 100 mL of PBS (50 mM, pH 7.4) or
PBS containing 1 mM DTT in a shaking water bath at 37 °C to ensure sink conditions.
At predetermined time intervals, 10 mL of external buffer solution was replaced with
10 mL of fresh PBS or PBS with 1 mM of DTT. The amount of DOX released was
determined by using fluorescence measurement, and calculated by the standard curve
plotted in advance (BioTek Synergy 2, EX wavelength at 485 nm, EM wavelength at
490 nm (DOX)). All DOX-release experiments were conducted in triplicate, and the
results are the average data with standard deviations. The cumulative release Er was
calculated according to the following formula:
Er: The total cumulative release % of DOX; Ve: The replacement of PBS volume (10
mL); V0: The total amount of phosphate buffered saline (PBS) volume (100 mL); Ci:
DOX concentration of thei-th replacement liquid (µg/mL) (determined by
fluorescence measurement); Cn: DOX concentration of the last replacement liquid
(µg/mL); mdrug: The total amount of DOX in micelle (µg).
Cell Culture. NIH 3T3 cells (a mouse embryonic fibroblast cell line) and HeLa cells
(a human cervical carcinoma cell line) were cultivated in DMEM (Dulbecco’s
modified Eagle’s medium) containing 10 % FBS (fetal bovine serum) and antibiotics

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(50 units.mL-1 penicillin and 50 units.mL-1 streptomycin) at 37 °C in a humidified
atmosphere containing 5 % CO2.
Cytotoxicity Measurements. The relative cytotoxicity of micelles was estimated by
MTT Assay. NIH 3T3 cells were seeded into 96-well plates at 4 × 103 cells per well in
200 µL of DMEM. After 12 h of incubation, the culture medium was removed and
replaced with 200 µL of DMEM containing serial dilutions of PLA-PEG-PLA. The
cells were grown for another 48 h. Then, 15 µL of 5 mg.mL-1 MTT assays stock
solution in PBS was added to each well. After the cells were incubated for 4 h, the
medium containing unreacted MTT was carefully removed. The obtained blue
formazan crystals were dissolved in 200 µL.well-1 of DMSO, and the absorbance was
measured in a BioTek Synergy 2 at a wavelength of 490 nm.
Intracellular Drug Release. The experiments of intracellular drug release were
performed on fluorescence microscopy.
Fluorescence microscopy. HeLa cells were seeded in 6-well plates at 1.6 × 105 cells
per well in 1 mL of complete DMEM and cultured for 12 h and then treated with
GSH-OEt for 2 h. Cells were washed by PBS and incubated at 37 ℃ for an
additional 1 h with DOX-loaded PLA-PEG-PLA micelles at a final DOX
concentration of 6.8 µg.mL-1 in complete DMEM. Cells without GSH-OEt treatment
were used as the control. Then, the culture medium was removed and cells were
washed with PBS three times. Thereafter, the cells were fixed with 4% formaldehyde
for 15 min at room temperature, and the slides were rinsed with PBS solution three
times. Finally, the cells were stained with 2-(4-amidinophenyl)-6-indolecarbamidine
dihydrochloride (DAPI) for 10 min, and the slides were rinsed again with PBS three
times. The slides were mounted and observed by an Olympus BX51TF.

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Activity Analysis. The cytotoxicity of DOX-loaded PLA-PEG-PLA micelles against
HeLa cells was evaluated in vitro by MTT assay. HeLa cells were seeded into 96-well
plates at 8 × 103 cells per well in 200 µL of medium and further incubated for 12 hrs.
The cells were then treated with glutathione reduced ethylester (GSH-OEt) for 2 hrs
or BSO for 12 hrs. Cells without pretreatment were used as the control. After
GSH-OEt or buthionine sulfoxide (BSO) was washed with PBS, DOX-loaded
PLA-PEG-PLA micelles diluted in complete DMEM (200 µL) obtained series of
DOX concentration were added to cells, and the cells were further incubated for 48 h.
After the incubation, the culture medium was removed and washed with PBS twice.
Then, 200 µL of DMEM and 20 µL of 5 mg.mL-1 MTT assays stock solution in PBS
were added. After the cells were incubated for 4 h, the medium containing unreacted
MTT was carefully removed. The obtained blue formazan crystals were dissolved in
300 µL.well-1 of DMSO, and the absorbance was measured in a wavelength of 490
nm (BioTek Synergy 2).
Results and discussion
Synthesis and characterization of triblock copolymers PLA-PEG-PLA. Triblock
copolymers PLA-PEG-PLA containing double disulfide linkages associated with
H-bonded specific sequence were synthesized according to a reported procedure[51]
(Scheme 3). The modified hydrophiphilic block A-PEG-A and hydrophobic block
PLA-B were synthesized from reaction of carboxyl group in strand A with amino
group in NH2-PEG-NH2 and reaction of amine group in strand B with carboxyl group
in PLA in the presence of HATU/NMM, respectively. The desired triblock copolymers
PLA-PEG-PLA were then obtained from A-PEG-A reacted with PLA-B under
iodine oxidation conditions at room temperature. The intermediates A-PEG-A,
1
PLA-B and the triblock copolymers PLA-PEG-PLA were characterized with H
1
NMR and SEC. In the H-NMR spectrum of PLA3000-PEG4000-PLA3000 (Figure 1),
there not only reveals signals of PEG block (3.86-3.40 ppm from the repeating
-OCH2CH2O- unit), PLA block (repeating LA residues 5.29-5.05 ppm from the -CH-

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groups, and CH3- groups of the LA units 1.76-1.24 ppm), but also those of the amide
linking units (3.00-1.98 ppm from -COCH2-, -SCH2-, -CH2-, -NCH2-, -SCH2-). The
dominant signals of the trityl groups in the spectra of A-PEG4000-A, PLA3000-B
disappeared completely in the spectrum of copolymers PLA3000-PEG4000-PLA3000
indicating that disulfide crosslinking units indeed happened. In comparison with the
corresponding signals of either A-PEG4000-A or PLA3000-B, the signals of protons
(3.00-1.98 ppm) of the amide association units show noticeable shifts. Figure S3 gives
the SEC traces of A-PEG-A, PLA-B and PLA-PEG-PLA. Obviously the Mn value of
PLA-PEG-PLA increased compared with that of A-PEG-A and PLA-B. Table 1 lists
Mn and PDI of polymers. The above results approve the structure of the amphiphilic
triblock copolymers and demonstrate that the desired cross coupling reaction between
the three blocks did happen. The same cross coupling reactions were also happened in
forming double disulfide linkage assciated with triblock copolymers
1
PLA3000-PEG1000-PLA3000 and PLA3000-PEG2000-PLA3000, their H-NMR spectra
were shown in Figure S1-S2.
COOH
O
O
O
NH2
Ph3CS
SCPh3
H2N
O
n
+
N
N
H
H
NH2-PEG-NH2
HATU
NMM
DMF
Ph3CS
SCPh3
O
O
O
NH
HN
HN
O
H
N
O
PLA-B
O
O
N
n
H
O
NH
O
Ph3CS
SCPh3
CH2Cl2
I2
HO
O
S
S
S
O
S
O
NH
O
O
HN
O
O
O
NH
O
O
HN
O
H
N
O
NH
O
HN
O
n
N
H
O
O
O
NH
HN
O
NH
O
HN
O
S
O
S
S
S
PLA-PEG-PLA
OH
Scheme 3. Synthesis of thetriblock copolymer PLA-PEG-PLA

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Figure 1. 1H-NMR (400 MHz, CDCl3) spectra of A-PEG4000-A, PLA3000-B and
PLA3000-PEG4000-PLA3000
Table 1. Characterization of the intermediates and copolymers
Mn (g.mol-1)
Mw (g.mol-1)
PDIa
Mn (g.mol-1)
a
a
b
Polymers
PLA3000-B
A-PEG1000-A
2400
2700
3700
5600
5500
6600
8400
2900
2900
3800
5800
6600
8000
10200
1.21
1.06
1.02
1.04
1.21
1.22
1.22
2368
2614
3450
5694
5432
6168
8546
A-PEG2000-A
A-PEG4000-A
PLA3000-PEG1000-PLA3000
PLA3000-PEG2000-PLA3000
PLA3000-PEG4000-PLA3000
aEstimated by SEC (THF, 1 mL/min) using polystyrene standards, bEstimated from 1H NMR
Characteristics of self-assembled micelles. Amphiphilic triblock copolymer
PLA-PEG-PLA can spontaneously form a core/shell structured micelles in
aqueous solution driven by the strong hydrophobic/hydrophilic interaction
between linear chains of PLA core and PEG shell. Self-assembled blank
micelles (or DOX-loaded micelles) from copolymer PLA-PEG-PLA were
prepared by dissolving copolymer (or DOX and copolymer together) in DMSO
and dialyzed against deionized water. The digital pictures of blank micelles or

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DOX-loaded micelles’ solution were shown in Figure S4. The
PLA3000-PEG1000-PLA3000 aggregate was undissolved in water and formed
suspension, the observation suggests it is difficult to form stable blank or
DOX-loaded micelles in water, where DOX precipitated. In contrast, the other
two triblock copolymers PLA3000-PEG2000-PLA3000 and PLA3000-PEG4000
-
PLA3000 can self-assemble stable blank and DOX-loaded micelles in water.
Using DPH as a hydrophobic probe[57], we investigated CMC of the micelles by
UV/vis spectra. The relationship of the absorbance intensity of DPH as a
function of the copolymer PLA-PEG-PLA concentration in aqueous solution
at room temperature is shown in Figure 2 and all of the CMC values are listed
in Table 2. CMC values were 0.014 (Figure 2) and 0.017 mg mL-1 (Figures S5)
for PLA3000-PEG4000-PLA3000 and PLA3000-PEG2000-PLA3000 micelles
respectively, which confirms the polymeric micelles were highly stable in
dilute solution.[58, 59]
0.4
0.36
0.32
CMC = 0.014 mg.mL-1
0.28
0.24
0.2
0.00001 0.0001
0.001
0.01
0.1
1
Concentration (mg mL-1)
Figure 2. Relationship of the absorbance intensity of DPH as a function of the copolymer
concentration of PLA3000-PEG4000-PLA3000 in aqueous solution at room temperature
The size of micelles is imperative for intracellular drug delivery as small
size (< 200 nm) of micelle is beneficial to maintaining low level of
reticulo-endothelial system (RES) uptake and minimal renal excretion.[60,61]
DLS results show that the micelles of PLA3000-PEG4000-PLA3000 and
PLA3000-PEG2000-PLA3000 exhibit unimodal size distribution with a mean

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diameter of 43 nm and 110 nm respectively (Figure 3a and Figure S6a). The
morphology of PLA3000-PEG4000-PLA3000 micelles was further visualized by
TEM (Figure 3c). The triblock copolymer PLA-PEG-PLA aggregates to form
flower-like micelles with PLA blocks at the core and PEG blocks on the shell in
aqueous solution and the micellar core sizes determined by TEM were in
accordance with that of DLS. As shown in Table 2, the average diameter of the
micelles increased with the proportion of hydrophobic PLA block. The longer
hydrophobic PLA block leads to enhanced hydrophobicity and will facilitate
the formation of self-assembled micelles driven by hydrophobic interaction.[17,
62]
Further, when both hydrophiphilic and hydrophobic blocks’ molecular
weights are similar (such as PLA3000-PEG4000-PLA3000 and PEG5000-PLA5000),
the triblock copolymer micelle has lower CMC than that of the diblock
copolymer (Table 2).
(a)
(b)
(d)
(c)
Figure 3. DLS plots and TEM images of blank (a), (c) and DOX-loaded (b), (d)
PLA3000-PEG4000-PLA3000 micelles
DOX, one of the most potent anticancer drugs,[64, 65] was selected as a model
to investigate drug loading property. The loading amount of DOX in micelles was
measured by UV analysis and the results show DLC of PLA3000-PEG2000-PLA3000
and PLA3000-PEG4000-PLA3000 micelles were 5.05% and 4.73% respectively (the

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theoretical DLC was set as 10%). With the proportion of hydrophobic PLA block
increases the formed self-assembled micelles become more stable.[65, 66] Therefore
triblock copolymer PLA3000-PEG4000-PLA3000 was selected as a model drug carrier
for further evaluations, DLS and TEM imaging of DOX-loaded
PLA3000-PEG4000-PLA3000 micelles (Figure 3b and 3d) show that the DOX-loaded
micelles exhibited bimodal distribution with a small amount of µm-level aggregates,
the mean diameter of DOX-loaded micelles is larger than that of the blank micelles.
The DLS results of blank and DOX-loaded PLA3000-PEG2000-PLA3000 micelles were
similar with that of PLA3000-PEG4000-PLA3000 (Figure S6). These findings indicate
that the triblock copolymers PLA-PEG-PLA can self-assemble into flower-like
micelles in water with an appropriate size for drug delivery.
Table 2. Properties of the triblock copolymer PLA-PEG-PLA micelles
Copolymers
CMCa
(mg mL-1) (nm)b(*/#)
Diameter
PDIb(*/#)
DLCc
(%)
PLA3000-PEG1000-PLA3000
PLA3000-PEG2000-PLA3000
PLA3000-PEG4000-PLA3000
PEG5000-PLA5000
Suspension
0.017
0.014
0.080
110/179
43/104
107*
0.118/0.281
0.187/0.274
0.205*
5.05
4.73
3.20
Determined by UV/vis spectrometera or DLSb, drug loading contentc (*blank
and #DOX-loaded micelles)
Redox responsiveness and stimuli triggered drug release. The disulfide bonds
incorporated into copolymer micelles are responsive to reducing agents.[68] Here we
investigate the redox responsiveness of PLA-PEG-PLA micelles under reductive
conditions of 10 mM DTT within a period of 8 h. The DLS curves of
PLA3000-PEG4000-PLA3000 micelles are shown in Figure 4, and for copolymer
PLA3000-PEG2000-PLA3000 it was shown in Figure S7. The results demonstrate that
the average diameter of the micelles after treatment with DTT became bimodal or
trimodal distributions, due to cleavage of disulfide linkage followed by detachment of
the hydrophilic shell, therefore the aggregation of hydrophobic PLA core enhances
hydrophobic interaction between the polyester inner cores[67]. The average diameter of
diblock copolymer PEG-PLA micelles has similar change after treatment with DTT

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(Figure S12). In contrast, the micelles without DTT treatment were stable in aqueous
solution within the same period. The release profile which similar with that of diblock
copolymer micelles (Figure S13) suggested the good stability of DOX-loaded
micelles under physiological conditions.
Figuer 4. DLS curves of PLA3000-PEG4000-PLA3000 micelle before and after treatment with DTT
(10 mM) for 8 h at 37 oC
Additionally, in vitro release of encapsulated DOX from PLA-PEG-PLA
micelles were observed under a reductive environment. The in vitro release behavior
was investigated by dialyzing the micelles in 0 mM and 1.0 mM DTT solution at 37
oC. It was found that in the presence of 1.0 mM DTT, the drug release from the
DOX-loaded micelles was accelerated compared with that of without DTT treatment.
The total amount of drug released from PLA3000-PEG2000-PLA3000 and
PLA3000-PEG4000-PLA3000 micelles were 30% and 22% respectively over 15 h in the
absence of DTT. In contrast, the total amount of DOX released from these micelles
increased to 66% and 59% respectively in the presence of 1.0 mM DTT solution over
the same period (Figure 5 and Figure S8). The released of DOX loaded
PLA3000-PEG4000-PLA3000 micelles (without disulfide linkage) has no difference in 0
mM and 1.0 mM DTT solution (Figure S9). The triggered drug release experiments in
vitro demonstrate that the disulfide linkages in these micelles can be broken, which
destabilizes the micelles’ structure and therefore accelerates the release of the
encapsulated DOX.

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90
80
70
60
50
40
30
20
10
0
0 mM DTT
1.0 mM DTT
-1
4
9
14 19
Time / h
Figure 5. DOX released from DOX loaded PLA3000-PEG4000-PLA3000 micelle against 1.0 mM
DTT as monitored with the fluorescence intensity of DOX (EX: 485 nm，EM: 590 nm)
120
100
80
60
40
20
0
Micelles concentration (µg.mL-1)
Figure 6. Viability of NIH 3T3 cells against PLA3000-PEG4000-PLA3000 blank micelles for 48 h by
MTT assay. Data are presented as the average ± standard deviation (n = 4).
Cytotoxicity of PLA-PEG-PLA micelles. As potential drug delivery materials,
biocompatibility or cytotoxicity is a key parameter for its applications.[69]
Cytotoxicity of PLA3000-PEG4000-PLA3000 micelles in vitro was evaluated by MTT
method against NIH3T3 cell lines. Figure 6 shows the cell viability after 48 h

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incubation with the micelles at the concentration varying from 0.5 to 1000 µg/mL.
The cell viability remained above 88% for PLA3000-PEG4000-PLA3000 micelles even
when the concentration up to 1000 µg/mL. Therefore, PLA-PEG-PLA micelles have
good biocompatibility and are suitable for drug delivery. The advantage of low
cytotoxicity of PLA-PEG-PLA allows high doses to be administered as required.
Intracellular reduction-sensitive drug release. It is important to study the redox
responsiveness of PLA-PEG-PLA micelles in living cells. The cellular uptake was
observed by fluorescence microscopy. It is documented that GSH-OEt can penetrate
cell membrane and rapidly reach a high intracellular concentration of GSH through
ethyl ester hydrolysis in cytoplasm, and no cytotoxicity to HeLa cells was observed at
this concentration.[19, 70,71] HeLa cells were first incubated with 10 mM GSH-OEt for 2
h to manipulate the intracellular concentration of GSH. The cells without GSH-OEt
pretreatment was used as a control. After incubated with PLA3000-PEG4000-PLA3000
micelles at the DOX concentration of 6.8 µg.mL-1, the cells were observed using a
fluorescence microscope. As shown in Figure 7, the control cells incubated for 30 min
emitted red fluorescence characteristic of DOX mainly in cytoplasm, whereas the
GSH-OEt pretreated cells showed the fluorescence in both cytoplasm and nucleus.
With the incubation time increased to 2 h, the control cells displayed red fluorescence
in both cytoplasm and nucleus. As expected, the intracellular fluorescence in the
pretreated cells was mainly located in the nucleus. It is suggested that high
intracellular GSH concentration accelerates the degradation of disulfide bonds. The
intracellular DOX release from the micelles is hence promoted, leading to rapid
localization of DOX in cell nucleus. In contrast, cellular uptake of HeLa cells
incubated with DOX-loaded PLA3000-PEG4000-PLA3000 (without disulfide linkage)
micelles was not influenced by the pretreatment of GSH-OEt (Figure S10). These
results confirm that the pretreated HeLa cells with GSH-OEt up-regulates the
intracellular concentration of GSH, promoting the cleavage of disulfide bond and
accelerates the intracellular release of DOX from the micelles.

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Figure 7. Representative fluorescence microscopy images of HeLa cells incubated with
DOX-loaded PLA3000-PEG4000-PLA3000 micelles: (a) 0.5 h, cells without pretreatment; (b) 0.5 h,
cells pretreated with 10 mM GSH-OEt; (c) 2.0 h, cells without pretreatment; (d) 2.0 h, cells
pretreated with 10 mM GSH-OEt. Cell nuclei were stained with DAPI.
In Vitro Cytotoxicity of DOX-Loaded Micelles. The proliferation inhibition of
drug-loaded micelles against tumor cells is a key parameter for drug delivery efficacy.
The smart redox responsiveness and good biocompatibility of PLA-PEG-PLA
micelles gave us a favorable chance to evaluate their potential as efficient drug
delivery vehicles. The inhibition of HeLa cell proliferation in vitro by the
DOX-loaded PLA3000-PEG4000-PLA3000 micelles was analyzed by MTT assay.
According to a reported process,[19, 20] the cells were treated with 10 mM GSH-OEt
for 2 h or 0.1 mM BSO for 12 h, respectively, and then incubated with the
DOX-loaded micelles. The cells without pretreatment were used as a control. It
proved that BSO is an inhibitor for the intracellular synthesis of GSH, and no
cytotoxicity to HeLa cells was observed at this concentration. As shown in Figure 8,
the HeLa cells pretreated by GSH-OEt or BSO exhibited higher or lower inhibition
efficiency compared with the control cells. All these results demonstrate that the
accelerated destabilization of PLA3000-PEG4000-PLA3000 micelles triggered by the

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higher intracellular GSH concentration leads to rapid DOX release. The rapid drug
release from the PLA3000-PEG4000-PLA3000 micelles in cells accelerates the nucleus
uptake of DOX, and thus enhances proliferation inhibition of HeLa cells. In contrast,
cellular uptake and proliferation of HeLa cells incubated with DOX-loaded
PLA3000-PEG4000-PLA3000 (without disulfide linkage) micelles have no effect by
pretreatment of GSH-OEt and BSO (Figure S11). These results demonstrated that the
DOX were successfully released by a change in intracellular GSH and were
pharmaceutical active to suppress the growth of HeLa cells.
120
GSH
Control
BSO
100
80
60
40
20
0
Concentration of load-DOX / µg mL -1
Figure 8. Viability of HeLa cells incubated with DOX-loaded PLA3000-PEG4000-PLA3000 micelles
at different drug concentrations for 48 hrs. The cells were pretreated with 10 mM GSH-OEt or 0.1
mM BSO. The nonpretreated cells were used as control. Data are presented as the average ±
standard deviation (n = 4).
Conclusions
Amphiphilic triblock copolymer PLA-PEG-PLA micelles containing double
disulfide linkages in the backbone have been developed successfully. The copolymer
micelles of PLA3000-PEG2000-PLA3000 and PLA3000-PEG4000-PLA3000 have a low
CMC with 0.017 and 0.014 mg mL-1 and DLC with 5.05% and 4.73% respectively.
Biodegradable triblock copolymers with effective sensitivity to environmental stimuli
are regarded as good candidates for drug delivery [72,73]. The copolymer can

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self-assemble into flower-like micelles in aqueous media with an average diameter of
110, 43 nm (blank micelles) and 179, 104 nm (DOX-loaded micelles) respectively.
Moreover, drug release from the copolymer micelles can be triggered by reductive
environment. The micelles exhibit a fast drug release behavior in GSH-OEt pretreated
HeLa cells. In vitro cell viability evaluation further confirms that destruction of
micelles by intracellular GSH accelerates DOX release and thus enhances the cell
proliferation inhibition. These results indicate a drug delivery system that reponds to
chemical signals in vivo would be important in the field of controlled release. Such
redox responsive amphiphilic triblock copolymeric micelles provide a platform for
constructing drug delivery systems for cancer therapy.
Appendix A. Supplementary data: Supplementary data related to this article can be
found at http://dx.doi.org.
Corresponding Author:
yumeishen@sjtu.edu.cn
Acknowledgment
This work was partially supported by Grants 11374207, 31370750, 81071250, and
91227109 from NSFC, Key Projects in the National Science & Technology Pillar
Program of China (No. 2014BAI09B03).
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