Self-assembled polymeric micelles based on THP and THF linkage for pH-responsive drug delivery

Article abstract of DOI:10.1016/j.polymer.2014.05.010

Developing smart nanocarriers for drug delivery system is advantageous for many kinds of successful biomedicinal therapy. In this study, we designed an amphiphilic block copolymers containing pH-responsive tetrahydropyran (THP) and tetrahydrofuran (THF) linkage. Their structures were confirmed by ¹H NMR and gel permeation chromatography (GPC). The release rate of encapsulated drugs depends upon the pH value and pH sensitive linkage in the backbone of copolymers. For PLA-THP-PEG micelles the cumulative release amount of doxorubicin (DOX) was 62% at pH 5.0, which is about four times higher than that at pH 7.4. Under the same conditions the release rate for PLA-THF-PEG micelles is a little faster than that of the PLA-THP-PEG micelles. Cellular uptake study demonstrates that DOX-loaded micelles can easily enter the cells and produce the desired pharmacological action and minimizing the side effect of free DOX. These findings indicate that THP and THF linked diblock copolymer micelles is a promising candidate for drug carrier.

Full text of DOI:10.1016/j.polymer.2014.05.010

Accepted Manuscript
Self-assembled Polymeric Micelles Based on THP and THF Linkage for pH-
Responsive Drug Delivery
Fangxia Zhu , Qinglai Yang , Yuan Zhuang , Yuanqing Zhang , Zhifeng Shao , Bing
Gong , Yu-Mei Shen
PII:
S0032-3861(14)00387-5
10.1016/j.polymer.2014.05.010
JPOL 16963
DOI:
Reference:
To appear in: Polymer
Received Date: 6 March 2014
Revised Date: 14 April 2014
Accepted Date: 6 May 2014
Please cite this article as: Zhu F, Yang Q, Zhuang Y, Zhang Y, Shao Z, Gong B, Shen Y-M, Self-
assembled Polymeric Micelles Based on THP and THF Linkage for pH-Responsive Drug Delivery,
Polymer (2014), doi: 10.1016/j.polymer.2014.05.010.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Self-assembled Polymeric Micelles Based on THP and THF
Linkage for pH-Responsive Drug Delivery
†
†
†
ǂ
‡
Fangxia Zhu , Qinglai Yang , Yuan Zhuang , Yuanqing Zhang , Zhifeng Shao , Bing
||,§,
†,
Gong * and Yu-Mei Shen *
†
Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine and
Bio-ID Center, Shanghai Jiao Tong University, Shanghai 200240, China
ǂShanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800,
China
‡
State Key Laboratory of Oncogenes and Bio-ID Center, School of Biomedical
Engineering, Shanghai Jiao Tong University, Shanghai 200240, 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
E-mail: yumeishen@sjtu.edu.cn; bgong@buffalo.edu

ACCEPTED MANUSCRIPT
ABSTRACT: Developing smart nanocarriers for drug delivery system is advantageous
for many kinds of successful biomedicinal therapy. In this study, we designed an
amphiphilic block copolymers containing pH-responsive tetrahydropyran (THP) and
1
tetrahydrofuran (THF) linkage. Their structures were confirmed by H NMR and gel
permeation chromatography (GPC). The release rate of encapsulated drugs depends upon
the pH value and pH sensitive linkage in the backbone of copolymers. For
PLA-THP-PEG micelles the cumulative release amount of doxorubicin (DOX) was 62%
at pH 5.0, which is about four times higher than that at pH 7.4. Under the same
conditions the release rate for PLA-THF-PEG micelles is a little faster than that of the
PLA-THP-PEG micelles. Cellular uptake study demonstrates that DOX-loaded micelles
can easily enter the cells and produce the desired pharmacological action and minimizing
the side effect of free DOX. These findings indicate that THP and THF linked diblock
copolymer micelles is a promising candidate for drug carrier.
KEYWORDS: Amphiphilic block copolymer; drug delivery; pH-responsive;
doxorubicin
1
. Introduction
A great challenge for hydrophobic anticancer drugs concerns limited availability of
1
-12
effective biocompatible delivery systems . Poor water solubility is the major problem
encountered with the anticancer drugs, so development of novel delivery systems
attracted significant attention in the past decades. Block copolymer micelles with
core-shell structures self-assembled from amphiphilic copolymers offer great potential

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1
3
and promising approach to deliver hydrophobic drugs into tumor site . The inner
hydrophobic core and outer hydrophilic shell of the micelles can be used to load and
protect hydrophobic drugs from inactivating under the biological environment. Recently
stimuli-responsive polymeric micelles have emerged as vehicles for smart drug delivery
based on the release of drugs can be readily modulated by exerting an appropriate
1
4-16
17-20
21-22
23-25
26-28
stimulus such as temperature
, pH
, glucose
, glutathione
, etc.
Stimuli-responsive release of drugs may result in significantly enhanced therapeutic
2
9-30
efficiency and minimal side effects
. Among available stimuli, pH responsiveness is
3
1-36
particularly appealing due to pH values in malignant tissue from about 5.8 to 7.6
.
Therefore, pH-sensitive polymeric micelles which rapidly respond to the mild acidic pH
trigger provide an opportunity for the achievement of programmable and controlled drug
delivery. Tumoral pH variation has been considered as an ideal trigger for drug delivery
based on the controlled release of anticancer drugs in tumor tissues and/or within tumor
cells.
Up to now, a number of acid-cleavable linkers have been successfully adopted in the
3
7-44
design and synthesis of pH-sensitive polymeric drug carriers
. While encouraging
progress has been made in this field, new strategies that lead to further improvement on
the responsive acuteness of the corresponding drug delivery systems are still in high
demand.
Tetrahydropyran (THP) and tetrahydrofuran (THF) as protecting group widely used
in organic synthesis have rarely been employed in constructing acid-responsive block
4
5-46
copolymers
. In our current work, THP and THF will be used as an acid-responsive

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unit in the formation of new pH-responsive block copolymers. We aim to obtain a novel
and smart drug delivery system with THP and THF as pH-responsive linkage.
Herein we report the synthesis of amphiphilic block copolymers in which the
hydrophilic poly(ethylene glycol) (PEG) and hydrophobic polylactic acid (PLA) are
connected through THP or THF linkage and their self assembled micelles as potential
acid sensitive nanocarriers. The micelles based on PLA-THP-PEG and PLA-THF-PEG
copolymers were developed for intracellular delivery of doxorubicin (DOX), an effective
anticancer drug. Their physical characteristics and pH-responsive properties of blank and
drug-loaded micelles were observed by transmission electron microscopy (TEM),
dynamic light scattering (DLS), in vitro drug release and confocal laser scanning
microscopy (CLSM) measurements. Under physical conditions the micelles as potential
drug delivery carriers remained stable up to 35 days, however under acidic conditions the
pH sensitive linkage was cleaved and resulted in the release of encapsulated drugs.
Therefore, the introduced THP and THF linkage in the backbone of copolymer drug
carriers is the key structure which determines the release of encapsulated drugs.
2
2
. Experimental section
.1. Materials
All chemicals were purchased from Aldrich or Aladdin and were used as received
unless otherwise mentioned. All reactions were monitored by thin-layer chromatography
precoated 0.25 mm silica gel plates from Aldrich). Flash column chromatography was
(
carried out with silica gel 60 (mesh 200-400). N, N-dimethylformamide (DMF) was dried
over calcium hydride and distilled under vacuum. Triethylamine (TEA) was refluxed

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with phthalic anhydride, potassium hydroxide, and calcium hydride successively and then
distilled before use. Monomethyl ether of PEG (M = 1900, 5000, determined by GPC)
n
was purchased from Shanghai Yarebio Co. Ltd. PLA (Mv = 1000, 3000, 5000,
determined by viscometry) was purchased from Jinan Daigang Biomaterial Co. Ltd. and
used as received.
2
.2. Methods
1
13
H and C NMR analyses were recorded on a Bruker Avance III 400 MHz
spectrometer with CDCl as solvents. Tetramethylsilane (TMS) was used as the internal
3
reference. Fourier transform infrared (FTIR) spectra were recorded by KBr sample holder
−
1
method on a Perkin-Elmer Paragon 1000 instrument in the range of 4000−400 cm . Ultra
Performance Liquid Chromatography with Tandem Level Four Pole Time of Flight Mass
Spectrometry (UPLC-TOF-MS) was recorded on a Waters UMS Q-Tof Premier, UPLC
conditions: Waters UPLC BEH C18 1.0 × 100 mm column, 0.4 mL/min, methanol-water
gradient elution; methanol 20% in 1 min followed by methanol 20% to 40% in 1 min,
methanol 40% to 60% in 6 min, finally, methanol 60-95% in 2 min. The identity of each
peak was confirmed by collecting the corresponding fraction that was then subjected to
analysis by mass spectrometry. Number-average molecular weight (M ) and
n
polydispersity (PDI = M /M ) were determined by GPC on an Agilent 1260 infinity
w
n
GPC/SEC system (10 ꢀm PLgel 600 × 7.5 mm column, linear polystyrene calibration)
equipped with a refractive index (RI) detector. The samples were estimated by GPC with
THF as eluent, which contained 0.01 mol/L lithium bromide at a flow rate of 1 mL/min at
o
3
5 C. Transmission Electron Microscopy (TEM) studies were performed with a JEOL
JEM-100CX-II instrument at a voltage of 200 kV. Samples were prepared by

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drop-casting micellar solution onto carbon-coated copper grids and then air-drying at
room temperature overnight without staining before measurement. Dynamic Light
Scattering (DLS) measurements were performed with a Malvern Zetasizer Nano S
apparatus (Malvern Instruments Ltd.) equipped with a 4.0 mW He-Ne laser operating at =
6
33 nm. All samples (1 mg/mL) were measured in aqueous solution at room temperature
o
(
25 C) and at a scattering angle of 173°.
2
.3. Synthesis of PLA-THP-PEG copolymers
To an ice-cold solution of (3, 4-dihydro-2H-pyran-2-yl) methanol (925 mg, 8.1
mmol) in CH Cl (40 mL), TEA (1.15 mL) was added, followed by 4-nitrobenzoyl
2
2
o
chloride (2.02 g, 8.3 mmol). The reaction mixture was stirred at 0 C for 1 h and then
continued at room temperature for 4 h. The resulting mixture was washed with saturated
sodium bicarbonate solution and extracted with ether
.
The combined organic extracts
),
were washed with water and brine, dried over anhydrous magnesium sulfate (MgSO
4
filtered, followed by removing solvent. Purification was accomplished by column
chromatography on silica gel by using dichloromethane/petroleum ether as eluent, which
1
gave the desired intermediate 1 as a yellowish green liquid. Yield: 1.6 g, 75%. H NMR
(
400 MHz, CDCl ): δ 8.27-8.19 (m, 4H), 6.34 (d, J = 6.4 Hz, 1H), 4.75-4.65 (m, 1H),
3
4
.50-4.38 (m, 2H), 4.20-4.12 (m, 1H), 2.20-2.08 (m, 1H), 2.05-1.95 (m, 1H), 1.95-1.85
1
3
(
m, 1H) , 1.80-1.70 (m, 1H); C NMR (100 MHz, CDCl
3
): δ 164.6, 150.6, 143.3, 135.4,
1
2
1
31.0, 123.6, 100.7, 72.6, 67.5, 24.3, 19.2; IR (KBr): 3357, 3112, 3060, 2954, 2923,
851, 1727, 1649, 1608, 1528, 1450, 1410, 1346, 1320, 1274, 1239, 1188, 1170, 1118,
103, 1070, 1014, 968, 950, 888, 845, 788, 762, 718.

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The intermediate 1 (1.689 g, 6.4 mmol) was dissolved in 45 mL of tetrahydrofuran
(
THF), to which a solution of NH Cl (1.368 g, 25.6 mmol) in water (30 mL) was added.
4
o
Upon the mixture stirred at 70 C for 30 min, Fe powder (1.428 g, 25.6 mmol) was added.
o
The reaction mixture was stirred at 75 C for another 50 h, cooled to room temperature,
and then filtered through a celite pad. The filtrate was extracted with ethyl acetate. After
being washed with saturated sodium bicarbonate solution and brine, and dried over
anhydrous sodium sulfate, the organic extract was evaporated under reduced pressure.
The residue was subjected to column chromatography (silica gel, petroleum ether/ethyl
acetate) to give the desired intermediate 2 as a cream-coloured solid. Yield: 1.16 g,
1
7
6
2
7.7%. H NMR (400 MHz, CDCl ): δ 7.86 (d, J = 8.8 Hz, 2H), 6.61 (d, J = 8.8 Hz, 2H),
3
.39 (d, J = 6.0 Hz, 1H), 4.75-4.65 (m, 1H), 4.38-4.34 (m, 2H), 4.18-4.11 (m, 1H),
1
3
.18-2.07 (m, 1H), 2.06-1.96 (m, 1H), 1.95-1.87 (m, 1H), 1.82-1.71 (m, 1H); C NMR
(
100 MHz, CDCl ): δ 166.6, 151.2, 143.5, 131.9, 119.4, 113.8, 100.6, 73.1, 66.1, 24.5,
3
1
1
9.3; IR (KBr): 3466, 3368, 3231, 3060, 2951, 2923, 2850, 1694, 1648, 1626, 1602, 1518,
439, 1310, 1273, 1239, 1171, 1112, 1067, 1013, 973, 843, 770, 729, 699.
Under nitrogen atmosphere, PLA (1 mmol) and 2-(7-aza-1H-benzotriazole-1-yl)
-1,1,3,3- tetramethyluronium hexafluorophosphate (HATU) (2 mmol) were dissolved in
3
0 mL DMF, then N, N- diisopropylethylamine (DIPEA) (4 mmol) was added and the
o
reaction was stirred at 0 C for 0.5 h. After adding the intermediate 2 (2 mmol, dissolved
o
in 4 mL DMF) the reaction mixture was stirred at 30 C for an additional 45 h. The
resulting mixture was poured into a few drops of concentrated hydrochloric acid and
2 2
extracted with CH Cl . The combined organic extracts were washed with water and brine,
dried over anhydrous magnesium sulfate, filtered, and the filtrate was evaporated.

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Purification was accomplished by chromatography to give the desired intermediate 3 as
yellow semi-solid product.
1
3
a, yield: 81%. H NMR (400 MHz, CD OD): δ 8.04-7.96 (m, 3H), 7.63-7.55 (m,
3
2
4
1
1
2
1
H), 6.38 (d, J = 6.4 Hz, 1H), 5.20-5.05 (m, 53H), 4.72-4.66 (m, 1H), 4.40-4.35 (m, 2H),
.20-4.10 (m, 12H), 2.85-2.61 (m, 13H), 2.20-2.10 (m, 1H), 2.09-1.99 (m, 2H) ,
1
3
3
.98-1.85 (m, 1H), 1.61-1.38 (m, 168H); C NMR (100 MHz, CDCl ): δ 172.4, 170.4,
69.6, 169.4, 166.1, 143.3, 142.5, 130.9, 125.1, 118.9, 100.6, 72.8, 69.3, 66.5, 31.9, 29.1,
4.3, 19.2, 16.6; IR (KBr): 3371, 2993, 2944, 1755, 1649, 1598, 1530, 1453, 1409, 1383,
364, 1308, 1273, 1189, 1130, 1094, 1051, 860, 770, 741, 698, 665.
1
3
7
4
b, yield: 68%. H NMR (400 MHz, CD OD): δ 8.29 (s, 1H), 8.02-7.94 (m, 2H),
3
.63-7.55 (m, 2H), 6.36 (d, J = 6.0 Hz, 1H), 5.20-5.04 (m, 15H), 4.72-4.66 (m, 1H),
.39-4.35 (m, 2H), 4.20-4.12 (m, 5H), 2.84-2.76 (m, 2H), 2.76-2.60 (m, 3H), 2.20-2.06
1
3
(
m, 1H), 1.98-1.94 (m, 2H) , 1.80-1.70 (m, 1H), 1.62-1.42 (m, 47H); C NMR (100
MHz, CDCl ): δ 174.5, 171.2, 169.7, 169.2, 165.4, 143.1, 130.5, 124.5, 118.4, 100.2,
3
7
1
7
2.5, 69.0, 66.3, 38.3, 36.2, 31.1, 24.7, 20.1, 16.3; IR (KBr): 3502, 2994, 2944, 1756,
648, 1599, 1530, 1455, 1409, 1383, 1364, 1305, 1270, 1186, 1131, 1093, 1046, 955, 868,
56, 701.
1
3
6
4
c, yield: 64%. H NMR (400 MHz, CDCl
3
): δ 8.05-7.98 (m, 2H), 7.82-7.56 (m, 2H),
.39 (d, J = 6.4 Hz, 1H), 5.26-5.08 (m, 68H), 4.77-4.68 (m, 1H), 4.42-4.38 (m, 2H),
.20-4.16 (m, 2H), 2.86-2.78 (m, 7H), 2.78-2.66 (m, 2H), 2.20-2.10 (m, 1H), 2.08-2.01
1
3
(
m, 12H) , 2.00-1.90 (m, 1H) , 1.68-1.46 (m, 223H); C NMR (100 MHz, CDCl ): δ
3
1
74.8, 171.0, 169.4, 169.3, 169.1, 143.4, 130.8, 126.8, 125.6, 119.3, 118.7, 100.5, 72.7,

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6
1
9.1, 66.5, 38.5, 29.6, 24.3, 20.9, 16.7; IR (KBr): 3502, 2995, 2945, 1755, 1648, 1599,
530, 1455, 1382, 1365, 1319, 1270, 1188, 1131, 1092, 1051, 956, 865, 739, 701.
PEG (0. 5 mmol) was dissolved in 1, 2-dichloroethane (1, 2-DCE) (90 mL) and
pyridinium p-toluenesulfonate (PPTS) solution (0.5 mmol). After stirring at room
temperature for 15 min, the intermediate 3 (0. 5 mmol) was added. The reaction was
o
refluxed at 95 C for 24 h. Then the solvent was evaporated and purified by
chromatography to give the desired terminal product block copolymer 4 as solid. An
inseparable mixture of trans/cis diastereomers was observed for the terminal product 4 in
some cases.
1
4
a (PLA1000-THP-PEG1900). Yield: 50.9%. H NMR (400 MHz, CDCl ) δ 8.00-7.94
3
(
m, 2H), 7.78-7.58 (m, 2H), 5.24-5.08 (m, 14H), 4.88 (s, 1H), 4.40-4.26 (m, 2H),
.26-4.22 (m, 2H), 3.90-3.42 (m, 206H), 3.36 (s, 3H), 2.84-2.75 (m, 2H), 2.75-2.60 (m,
4
2
1
6
1
5
1
3
3
H), 1.96-1.70 (m, 6H), 1.70-1.40 (m, 49H); C NMR (100 MHz, CDCl ): δ 172.0,
70.1, 169.2, 169.1, 165.8, 143.2, 130.4, 125.5, 119.2, 118.5, 100.3, 97.0, 77.4, 72.3, 70.3,
8.8, 67.6, 58.8, 31.7, 29.4, 24.1, 18.9, 16.0; IR (KBr): 3502, 2868, 1755, 1713, 1599,
536, 1454, 1410, 1383, 1350, 1272, 1255, 1186, 1103, 1040, 991, 952, 860, 771, 700,
77.
1
4
b (PLA3000-THP-PEG1900). Yield: 18.6%. H NMR (400 MHz, CDCl
3
) δ 8.02-7.92
(m, 2H), 7.68-7.58 (m, 2H), 5.22-5.08 (m, 20H), 4.87 (s, 1H), 4.42-4.20 (m, 2H),
4
6
1
.20-4.10 (m, 6H), 3.85-3.41 (m, 191H), 3.35 (s, 3H), 2.90-2.77 (m, 2H), 2.77-2.60 (m,
13
H), 1.96-1.60 (m, 10H), 1.60-1.46 (m, 63H); C NMR (100 MHz, CDCl ): δ 174.8,
3
72.1, 170.1, 169.4, 165.8, 142.9, 130.5, 124.6, 118.5, 102.2, 97.1, 71.7, 70.4, 69.1, 68.3,

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6
1
7
4.6, 58.8, 31.7, 29.1, 24.8, 20.3, 16.9; IR (KBr): 3501, 2921, 2871, 1755, 1716, 1598,
534, 1455, 1409, 1384, 1354, 1271, 1257, 1212, 1185, 1129, 1095, 1043, 994, 952, 862,
71, 756, 700.
1
4
c (PLA3000-THP-PEG5000). Yield: 12.7%. H NMR (400 MHz, CDCl ) δ 7.98-7.92
3
(m, 2H), 7.66-7.60 (m, 2H), 5.21-5.04 (m, 20H), 4.86 (s, 1H), 4.37-4.20 (m, 2H),
4
4
1
6
1
7
.20-4.14 (m, 5H), 3.81-3.41 (m, 501H), 3.35 (s, 3H), 2.88-2.73 (m, 3H), 2.73-2.62 (m,
1
3
3
H), 2.02-1.66 (m, 6H), 1.60-1.46 (m, 64H); C NMR (100 MHz, CDCl ): δ 174.6,
71.9, 171.2, 169.7, 169.2, 165.7, 142.8, 130.3, 124.4, 118.4, 102.1, 96.9, 77.4, 71.6, 70.2,
8.7, 64.4, 58.7, 31.1, 29.3, 24.7, 20.2, 16.3; IR (KBr): 3502, 2869, 1758, 1715, 1647,
560, 1534, 1455, 1409, 1383, 1350, 1298, 1255, 1184, 1100, 1042, 994, 950, 861, 771,
01.
1
4
d (PLA5000-THP-PEG5000). Yield: 12.7%. H NMR (400 MHz, CDCl
3
) δ 8.02-7.96
(m, 2H), 7.78-7.60 (m, 2H), 5.25-5.10 (m, 76H), 4.89 (s, 1H), 4.41-4.30 (m, 2H),
4
2
1
.28-4.24 (m, 1H), 3.84-3.42 (m, 427H), 3.36 (s, 3H), 2.88-2.75 (m, 2H), 2.75-2.60 (m,
H), 1.94-1.66 (m, 6H), 1.64-1.42 (m, 243H); IR (KBr): 3504, 2902, 2863, 1755, 1715,
705, 1454, 1384, 1362, 1269, 1186, 1128, 1092, 1046, 952, 862, 798, 739, 706.
Similarly the diblock copolymers PLA-THF-PEG 18 were synthesized and characterized
accordingly, the detailed procedure was in supporting information.
2
.4. Preparation of blank and DOX-loaded micelles
In brief, one of the copolymers PLA-THP-PEG (4a, 4b, 4c, 4d) and PLA-THF-PEG
(18a, 18b, 18c) (10 mg) was dissolved in 1 mL of DMF and stirred at room temperature.
The solution was then added slowly to 8 mL of deionized water and stirred for another 1

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hr. Subsequently, the solution was dialyzed against deionized water for 24 hrs using
dialysis tubing having molecular weight cut off (MWCO) of 2000, with the deionized
water being changed every 5 hrs. The appearance of turbidity in the aqueous solution
indicated the formation of aggregations. DOX-loaded micelles were prepared as above.
Besides, adding 1 mg of DOX·HCl and 1.5 equivalents of triethylamine to the solution.
In order to confirm that no more free DOX could be removed by dialysis, the change of
the fluorescent emission intensity of dialysate with time was monitored. To determine the
drug loading content (DLC) and drug loading efficiency (DLE), the DOX-loaded micelle
solution was lyophilized and dissolved in DMSO. The fluorescence excitation spectra at
4
85 nm was measured to determine the DOX concentration based on the standard curve
of the fluorescence excitation spectra of DOX in DMSO solution. DLC and DLE were
calculated according to the following formula:
DLC (wt %) = (weight of loaded drug/weight of polymer) × 100%
DLE (%) = (weight of loaded drug/weight of drug in feed) × 100%
2
.5. CMC measurement
The change of fluorescence emission of pyrene was plotted against the
corresponding concentration of the block copolymer, leading to the determination of
critical micelle concentration (CMC). Pyrene was dissolved in acetone and added to 5 mL
-3
volumetric flasks to the concentration of 6.4 × 10 mM in the final solutions. Acetone
was then evaporated and replaced with aqueous polymeric micelle solution with
-5
-1
concentrations ranging from 5.0 × 10 to 5.0 × 10 mg/mL. Samples were stirred at room
temperature for 24 hrs. The emission wavelength and excitation bandwidth were set at

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3
34 and 5 nm respectively. The intensity ratio of peaks at 373 nm to those at 384 nm was
plotted against the logarithm of copolymer concentration to measure CMC.
2
.6. Evaluation of Drug Release
A solution (2.5 mL) of DOX-loaded PLA-THP-PEG and PLA-THF-PEG micelles,
prepared as described above, was transferred into a membrane bag with a MWCO of
2
5
000. The membrane bag was immersed in 200 mL of phosphate buffer (pH 7.4) or pH
o
.0 solutions in a shaking water bath at 37 C to acquire sink conditions. At
predetermined time intervals, 2.5 mL of the external buffer was withdrawn and
replenished with an equal volume of fresh media for analysis. Drug release study was
performed for 100 hrs. The amount of released DOX was analyzed with fluorescence
measurement (BioTek Synergy 2, excitation wavelength at 485 nm, emission wavelength
at 490 nm). All DOX-release experiments were performed in triplicate, and the results
were the average data with standard deviations.
2
.7. Confocal laser scanning microscope (CLSM) studies
CLSM was utilized to visualize the cellular internalization and investigate the
intracellular distribution of DOX-loaded micelles and free DOX. For the CLSM study,
6
HeLa cells were seeded in 6-well plates at 2× 10 cells per well in 1 mL complete
DMEM and cultured for 24 hrs, followed by removing culture medium and adding
DMEM solution of free DOX and DOX-loaded micelles at finally the DOX concentration
o
of 9 ꢀg/mL, respectively. The cells were incubated at 37 C for the predetermined
interval 60 min and then washed with PBS and fixed with 4% paraformaldehyde at room
temperature for 30 min before the cell nucleus were stained by DAPI

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(4',6-diamidino-2-phenylindole) (excitation/emission: 486 nm/596 nm) for 10 min.
Thereafter, the slides were rinsed with PBS for three times and cover slips were placed
onto glass microscope slides. Finally the slides were installed and observed with CLSM.
2
.8. In vitro cell assay
The cytotoxicity of DOX-loaded PLA-THP-PEG, PLA-THF-PEG micelles and free
DOX against HeLa cancer cells was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2,
-diphenyl tetrazolium bromide (MTT) assay. The cells were seeded in 96-well plates
5
4
o
with a density of 1×10 cell per well and were cultured in 200 ꢀL DMEM at 37 C under
atmosphere containing 5% CO for 24 hrs to allow cell attachment. Then, the medium
2
was replaced with 200 ꢀL fresh medium containing the indicated concentration of
DOX-loaded PLA-THP-PEG, PLA-THF-PEG micelles and free DOX. After incubation
for 48 h, the medium was aspirated and replaced by 100 mL of fresh medium containing
-1
2
0 µL 5 mg mL of MTT. After 4 hrs, the medium was replaced by 200 µL DMSO.
Afterward, the absorbance at a wavelength of 490 nm of each well was measured using a
microplate reader. The relative cell viability (in percentage) was determined by the
following equation:
Cell viability (%) = (Isample — Iblank) / (Icontrol — Iblank
)
Where Isample, Icontrol, and Iblank represents the absorbance intensity at 490 nm
determined for cells treated with sample containing different amounts of DOX, for
control cells (nontreated), for blank well without cells, respectively.

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3
3
. Results and discussion
.1. Synthesis and Characterization
To design block copolymers containing THP or THF linkages in the backbone, the
intermediate 3 with the dihydropyran (DHP) terminals was prepared by condensation of
carboxyl group in PLA with the amine in modified DHP moiety. The synthetic route of
PLA-THP-PEG was shown in Scheme 1. First, the carboxyl group of macromolecular
PLA was reacted with the amine terminals of the DHP with the feeding molar ratio of 1:2,
affording the intermediate product 3. The molecular weight of DHP modified PLA was
determined directly by the starting material PLA. Subsequently, the intermediate 3 was
further coupled to different molecular weight of PEG by DHP group reacted with
hydroxyl group of PEG, resulted in the formation of terminal product PLA-THP-PEG (4)
as diblock copolymer.

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Scheme 1 Synthesis and self-assembly of diblock copolymer PLA-THP-PEG
For diblock copolymers PLA-THF-PEG 18 (see supplementary material Scheme S1), the
synthetic route has a little difference with that of copolymers PLA-THP-PEG 4. First, the
hydroxyl group of PEG was condensed with hydroxyl group in modified tetrahydrofuran
(THF), affording THF modified PEG 16 with nitro functional ends. Followed by
reduction of the nitro group to amine catalyzed by Pd/C to give THF-modified PEG 17.
Accordingly, the amine group in the intermediate 17 was further reacted with carboxyl
group of PLA to afford the terminal product diblock copolymers PLA-THF-PEG 18.
1
Figure 1A and 1B provides the H NMR spectra of the intermediate 3c and the
terminal product diblock copolymer PLA-THP-PEG 4d. The signal around 6.39 and 4.72
ppm in Figure 1A corresponds to the characteristics of DHP. After the following coupled
reaction with PEG, the DHP signal disappeared as shown in Figure 1B. Meanwhile, the
signal at 4.87 ppm suggested the successful coupling of the intermediate 3c with the
hydroxyl terminal of PEG monomethyl ether to give the terminal product 4d. Similarly
1
from the H NMR spectra of the intermediate 17b (Figure 1C) and the terminal product
diblock copolymer PEG-THF-PLA 18c (Figure 1D), the signal of 5.33 corresponds to
the THF characteristics, the signals of 5.13, 2.68 and 1.50-1.60 ppm corresponds to PLA,

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and the signals of 3.35 and 3.45-3.75 ppm corresponds to PEG segment in Figure 1D.
1
Therefore, H NMR spectra in Figure 1 elucidate the successful synthesis of the
intermediates and the terminal products PLA-THP-PEG 4d and PLA-THF-PEG 18c.
1
Figure 1 H NMR spectra of (A) modified PLA 3c (m = 69) (B) the terminal product 4d
(m = 69, n = 114) (C) intermediate 17b (n = 114) (D) the terminal product 18c (m = 69, n
=
114) (400 MHz, CDCl , 298 K).
3
The proposed structure of the block copolymers PEG-THP-PLA and PEG-THF-PLA was
further confirmed by the gel permeation chromatography (GPC) traces (Figure S19).

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Table 1 lists the number-average molecular weights and polydispersity index (PDI) of
PEG, the intermediate 3, the terminal product 4, PLA, modified PEG 17 and the terminal
product diblock copolymers 18. All the GPC traces of PLA-THP-PEG and
PLA-THF-PEG showed monomodal symmetric distribution. The GPC profile of the
block copolymers PLA-THP-PEG and PLA-THF-PEG compared with their precursor
modified PLA or modified PEG affords an evident shift toward higher molecular weight,
suggesting successful coupling between PEG and PLA via THP or THF linkage. For
3
typical terminal product 4d the number-average molecular weight is 10x10 , with a PDI
value of 1.08. The other copolymers with different molecular weight gave similar results.
Moreover, the amphiphilic block copolymers display a good solubility in common
organic solvent, like THF, DMF, and DMSO. Besides, this polymeric amphiphile can
make itself spontaneously aggregate in aqueous media due to the common amphiphilic
nature, which is beneficial for further biomedical applications.
Table 1 GPC Data of PEG, the intermediate 3, terminal product 4, PLA, intermediaate 17 and
a
terminal product 18
3
3
Polymer
PEG1900
PEG5000
M (x10 )
M (x10 )
PDI
1.04
1.07
1.65
1.23
1.35
1.13
1.12
1.08
1.08
1.82
2.80
1.62
1.03
n
w
2.1
5.1
1.9
3.4
6.1
3.4
4.3
7.6
10
2.2
5.4
3.2
4.2
8.3
3.9
4.8
8.2
11
3
a
3
b
3c
4
a
4
b
4c
4
d
PLA1000
PLA3000
PLA5000
1.0
1.2
3.7
2.0
1.7
3.4
6.0
2.0
17a

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1
7b
8a
8b
8c
4.5
2.8
7.8
11
5.0
3.1
8.4
13
1.10
1.12
1.08
1.23
1
1
1
a
Estimated by GPC (THF, 1 mL/min) using polystyrene standards.
3
.2. Self-Assembly of Block Copolymers
Considering that the critical micelle concentration (CMC) is one of the most
important parameters for micelles, the CMC value of PLA-THP-PEG and
PLA-THF-PEG in water was investigated. The determination of CMC was further
verified the successful formation of PLA-THP-PEG and PLA-THF-PEG micelles in
aqueous media. CMC value was determined by using pyrene as a fluorescent probe. In
the process of aggregation, the absorbance of DHP is close to zero when the
concentration is below the CMC value, whereas absorbance intensity of DHP is enhanced
exponentially with the growing concentration when the hydrophobic probe is
encapsulated into the micelles. Therefore a graph with the intensity ratio I /I of two
3
73 384
linear segments having two different slopes was obtained. The intersection point of the
two segments gave the CMC value (Figure S20). As shown in Table 2, the CMC of each
4
7
-6
micelles being in the range of 10 M, indicating the high stability characteristics of
4
8
polymeric micelles . Further the CMC value was decreased when the molecular weight
of copolymers was increased (Table 2). These results confirmed that the molecular
4
9
weight of the copolymers are larger, the resultant micelles will be more stable .
To further study the properties of the polymeric micelles, both DLS and TEM
measurements were performed. Diameter and PDI of PLA-THP-PEG and
PLA-THF-PEG micelles were characterized by DLS. The results from DLS showed that

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all of the micelles exhibited unimodal size distribution, with mean diameters ranging
from 19 nm to 53 nm (Table 2). As shown in Table 2, the average diameter of the
micelles increases with the length of the hydrophobic PLA block, while the size of the
PEG block has little effect on the diameter of the micelles. The longer hydrophobic PLA
block leads to enhanced hydrophobicity and more convenient self-assembly process
driven by hydrophobic interaction. A typical TEM image of the blank micelles formed
from copolymer 4d and 18c was shown in Figure 2A and Figure 2B respectively which
appear approximately spherical. Both of the size of the micelles 4d (Figure 2C) and 18c
(
Figure 2D) determined by DLS was in accordance with the data from TEM.
Table 2 Properties of the PLA-THP-PEG 4 and PLA-THF-PEG 18 micelles
a
a
b
-6
Copolymer
Diameter (nm)
PDI
CMC (10 M)
4
a
28
31
41
40
30
32
53
0.274
0.245
0.201
0.124
0.156
0.157
0.200
10.5
6.2
4
b
4
c
2.8
4
d
1.7
1
8a
8b
8c
12.5
2.6
1
1
1.6
a
Diameter and polydispersity index (PDI) of micelles were determined by dynamic
light scattering (DLS); Critical micelle concentration (CMC) was determined by
UV/vis spectrometer.
b

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Figure 2 TEM image of (A) 4d and (B) 18c micelles; DLS measurement of the
hydrodynamic diameter of micelles (C) 4d and (D) 18c in aqueous solution.
3
.3. Size and morphology of DOX-loaded micelles 4 and 18
Doxorubicin (DOX) is one of the most widely used antitumor drugs, which acts by
inhibiting the synthesis of nucleic acids in tumor cells. The polymeric micelles described
above had a size distribution in the range of 19 to 53 nm, which affords the polymeric
micelles passive targeting function to tumor tissues via the enhanced permeability and
5
0
retention (EPR) effect . Furthermore, the micelles described here were characterized
with core-shell architecture. Thus, hydrophobic drugs could be entrapped into the core,
and the hydrophilic shell could effectively protect the core against the external biological
media, inhibit nonspecific protein absorption, and increase the plasma clearance of
half-life. In this paper, DOX as a model drug was encapsulated into the micelles, and the

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drug loading content of micelles 4d and 18c were 13.0% and 14.3%, respectively (Figure
S27). The morphology of the DOX-loaded micelles in aqueous solution was studied by
TEM technique. As shown in Figure 3A and 3B, spherical micelles with an average
diameter of about 80 nm were observed for both 4d and 18c. To further confirm the TEM
observation, DLS measurements were performed to analyze the hydrodynamic diameter
of the DOX-loaded micelles. Figure 3C and 3D showed the average size of the
DOX-loaded micelles 4d and 18c were 131 nm and 130 nm with a PDI value of 0.155
and 0.148 respectively, which is larger than that of blank micelles while still kept narrow
distribution in aqueous media. The size of the micelle is decided by the hydrophobic core
and DOX as hydrophobic drug which easily associated with the hydrophobic block of the
5
1
micelle. Therefore, the size of micelle increased upon DOX was loaded . Moreover, the
size of micelles observed with DLS was also larger than that of TEM. The smaller size
measured by TEM as compared to DLS was most probably due to shrinkage of the PEG
shell upon drying.

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C
D
Figure 3 TEM image of DOX-loaded micelle (A) 4d and (B) 18c; DLS measurement of
the hydrodynamic diameter of DOX-loaded micelle (C) 4d and (D) 18c in aqueous
solution.
3
.4. Stability and pH Responsiveness of Blank and DOX-Loaded Micelles 4 and 18
The stability of blank micelles 4 and 18 was studied at room temperature using DLS
(
Figure S24 and S25). As shown in Figure 4, the micelles 4d and 18c remained stable up
to 35 days (Figure 4A) and 32 days (Figure 4C) at room temperature in the absence of
acid, respectively. Furthermore, the DOX-loaded micelle 4d was examined under various
o
acidic conditions as shown in Figure 4B which remained stable for up to 4 hrs at 37 C
and pH 6.8, its average diameter increases from 100 nm to 1000 nm after 13 hrs.
Similarly as shown in Figure 4D, the DOX-loaded micelle 18c began to change at 6 hrs
and the size of micelles increases from 62 nm to 258 nm after 10 hrs. The driving force
for the change of micelle size at low pH value is attributed to the cleavage of THP or
THF linkage, which leads to the shedding of hydrophilic PEG shell from the micelles and
5
2
the aggregation of hydrophobic PLA core . Under acid conditions the micelles was
degraded and the hydrophobic PLA core aggregated to become larger. In contrast, no
obvious change in micelle size was observed after 35 days and 32 days at pH = 7.4 for
blank micelle 4d (Figure 4A) and 18c (Figure 4B), respectively. From these results it can
be concluded that the prepared micelles is highly stable and the release behavior is pH

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dependent. That is, significantly faster drug release at mildly acidic pH 6.8 compared to
that of physiological pH. These pH-responsive biodegradable micelles are promising as
smart nanovehicles for delivery of anticancer drugs.
A
B
C
D
o
Figure 4 DLS plots of size change of micelles (A) blank 4d (25 C, pH 7.4); (B)
o
o
DOX-loaded 4d (37 C, pH 6.8); (C) blank 18c (25 C, pH 7.4); (D) DOX-loaded 18c (37
C, pH 6.8).
o
3
.5. In Vitro Drug Release
As an initial test to verify the utility of prepared DOX-loaded PLA-THP-PEG and
PLA-THF-PEG micelles for drug release, the in vitro drug release profiles were
monitored at pH 7.4 and 5.0 by fluorescence spectrophotometry. As shown in Figure 5,
DOX release under physiological conditions (pH 7.4) was significantly lower than that
under acidic conditions (pH 5.0) for both DOX-loaded PLA-THP-PEG and
PLA-THF-PEG micelles. The accumulated drug release was 62% for PLA-THP-PEG
micelles and 69% for PLA-THF-PEG micelles at pH 5.0 within 100 hrs, respectively. In
contrast, the release was 16% for PLA-THP-PEG micelles and 20% for PLA-THF-PEG

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micelles at pH 7.4, respectively. The drug release from micelles PLA-THF-PEG is a little
faster than that from PLA-THP-PEG micelle under the same pH conditions due to the
linkage THF is more acid sensitive than THP. In contrast, the drug release profile of
DOX-loaded PLA-PEG micelles without pH-sensitive linkage (PLA-PEG) was also
carried out under the same conditions. Both of the release amounts were less than 7% at
pH 7.4 and pH 5.0, indicating the significant role of THP and THF linker in pH
responsiveness.
Although the mechanism of drug release from polymeric matrices is very complex
and is still not completely understood, it can be simplistically classified as either pure
5
3
diffusion, erosion controlled release or a combination of the two mechanisms . In this
study, we supposed that the release of DOX from PLA-THP-PEG and PLA-THF-PEG
copolymer micelles under acidic conditions may be determined by the cleavage of
pH-sensitive linkages and eventually lead to the micelle size increases as the copolymers
degrade at low pH.

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Figure 5 DOX release profiles of DOX-loaded PLA-THF-PEG 18c, PLA-THP-PEG 4d
o
and PLA-PEG micelles at 37 C in pH 5.0 and pH 7.4, respectively
3
.6. Confocal Microscopy Studies
CLSM was exploited to further investigate and compare the cell uptake behavior and
intracellular distribution of the DOX loaded micelles with that of free DOX by HeLa
o
cells. Prior to the incubation of HeLa cells at 37 C for predetermined interval 60 min,
both of DOX loaded PLA-THP-PEG and PLA-THF-PEG micelles were added to culture
medium with a DOX concentration of 9 ꢀg/mL. Stained with DAPI, the nuclei and
cytoplasm of pretreated cells were observed by CLSM. By comparison with the control in
Figure 6A, the observation reveals that free DOX is largely accumulated in the cell nuclei
of HeLa cells in Figure 6B, while DOX released from DOX loaded micelles is mainly
located in the cytoplasm in Figure 6C and 6D. The result also indicates that free DOX is

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taken up by diffusion through the cell membrane and the DOX loaded micelles are taken
up by the cells via the indocytosis process. CLSM analyses confirmed that the DOX
loaded micelles can be efficiently internalized by the HeLa cells.
Figure 6 CLSM images of HeLa cells incubated with (A) neither free DOX nor
DOX-loaded micelles (B) free DOX, (C) DOX-loaded PLA-THP-PEG 4d micelles and (D)
o
DOX-loaded PLA-THF-PEG 18c micelles at 37 C for 60 min; Cell nuclei were stained
with DAPI.
3
.7. Activity Analysis of the Drug-Loaded Micelles
The drug loaded self-assembled micelles were further investigated to evaluate the
potential therapeutic efficacy. The in vitro cytotoxicity of DOX-loaded PLA-THP-PEG

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and PLA-THF-PEG micelles compared with that of free DOX was determined by MTT
assay against HeLa cells. To analyze the activity of DOX loaded nanoparticles, HeLa
cells were cultured in the solutions of free DOX and DOX loaded micelles at different
DOX concentrations ranging from 0.5 to 10 ꢀg/mL for 48h, respectively. As shown in
Figure 7, the viability of HeLa cells depends on DOX concentration. It can be found that
DOX loaded micelles perform potent effect of inhibition to HeLa cell proliferation
similar to free DOX. The inhibition of cancer cell growth is attributed to the intracellular
DOX released from the DOX loaded micelles and final entry into the nuclei of HeLa cells.
Moreover, the DOX loaded PLA-THP-PEG and PLA-THF-PEG micelles and free DOX
doses required for 50% cellular growth inhibition (IC ) are 2.08, 2.14 and 0.83 mg/mL,
5
0
respectively. Both of the DOX loaded micelles show slightly lower cytotoxicity than free
DOX due to the time-consuming DOX release from DOX loaded micelles in comparison
to free DOX at the same DOX concentration, proved by the in vitro DOX release. The
result reveals that DOX released from the micelles could exploit a potent drug efficacy as
free DOX after entry into the HeLa cells, producing the desired pharmacological action
and minimizing the side effect of free DOX.

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Figure 7 In vitro cell viability of HeLa cells against free DOX and DOX-loaded THP and
THF micelles (4d and 18c) at different concentrations
4
. Conclusions
In conclusion, the paper constructed an acid sensitive drug delivery system based on
amphiphilic PLA-THP-PEG and PLA-THF-PEG diblock copolymers. The obtained
1
diblock copolymers were characterized by H NMR and GPC, their self assembled
micelles exhibited unimodal size distribution, with mean diameters ranging from 19 nm
to 53 nm. Their average size of DOX-loaded micelles 4d and 18c increased compared
-6
with that of blank micelles. The CMC of every diblock copolymers is in the range of 10
M, indicated the high stability of polymeric micelles. Further study confirmed that the
micelles kept stable up to 35 days under physiological conditions. Both of the
PLA-THP-PEG and PLA-THF-PEG polymeric micelles showed accelerated drug release
at pH 5.0 compared with that at pH 7.4. Under the same conditions the release rate of
micelles with THF linkage is faster than that owns THP linkage. Moreover the copolymer