Water-soluble poly(ε-caprolactone)-Paclitaxel prodrugs toward an efficient drug delivery system

Article abstract of DOI:10.1071/CH14517

In this paper, poly(ε-caprolactone)-graft-carboxylic acid (P(α-C2CL)) was prepared via a thio-bromo click reaction between mercaptosuccinic acid and poly(α-bromo-ε-caprolactone). It is readily soluble in aqueous solutions (pH 5.5-9.8) due to the presence

Full text of DOI:10.1071/CH14517

CSIRO PUBLISHING
Aust. J. Chem. 2015, 68, 1136–1143
Full Paper
http://dx.doi.org/10.1071/CH14517
Water-Soluble Poly(e-caprolactone)-Paclitaxel Prodrugs
Toward an Efficient Drug Delivery System
A
A
A
A
A B C
, ,
Ji Wang, Huanjiao Sun, Deshan Li, Jie Yuan, Xuefei Zhang,
A B C
, ,
and Haoyu Tang
A
College of Chemistry, Xiangtan University, Xiangtan 411105, Hunan Province, China.
Key Laboratory of Environmentally Friendly Chemistry and Applications of Ministry
of Education, Key Laboratory of Polymeric Materials and Application Technology
of Hunan Province, and Universities of Hunan Province, Xiangtan 411105, China.
B
C
Corresponding authors. Email: zxf7515@163.com; htang@xtu.edu.cn
In this paper, poly(e-caprolactone)-graft-carboxylic acid (P(a-C2CL)) was prepared via a thio-bromo click reaction
between mercaptosuccinic acid and poly(a-bromo-e-caprolactone). It is readily soluble in aqueous solutions (pH 5.5–9.8)
due to the presence of carboxylic groups in each repeating unit. A series of water-soluble P(a-C2CL)-paclitaxel prodrugs
with high drug contents (up to 41.4 wt-%) was prepared by esterification. Meanwhile, methyl tetrazolium (MTT) assays
showed that P(a-C2CL)-paclitaxel prodrugs exhibited a high antitumour effect on A549 and MCF-7 cells. These prodrugs
have appeared as a highly versatile and potent platform for cancer therapy.
Manuscript received: 23 August 2014.
Manuscript accepted: 8 December 2014.
Published online: 18 February 2015.
Introduction
received tremendous interest for targeted cancer therapy. Yet,
PHPMA and PEG are non-biodegradable and those polymers
with a high molecular weight (Mw . 45 kDa) are not readily
excreted from the body. Polymeric drugs with low molecular
weights, however, exhibit relatively short circulation time and
poor accumulation in tumours in vivo.[23] Thus, steadily increas-
ing attention has been paid to polymeric prodrugs based on fully
biodegradable and water-soluble polymers in the past few years.
For example, polypeptide materials (i.e. poly(glutamic acid),
poly(L-g-glutamylglutamine), poly(hydroxyethyl-L-asparagine),
poly(hydroxyethyl-L-glutamine)) are able to degrade in vivo
into their respective amino acids that can be metabolized by
physiological pathways.[24–26] Aliphatic polyesters (i.e. poly(e-
caprolactone), poly(D- or L-lactic acid), and polyglycolic) exhib-
it promising biocompatibility and are able to degrade through
hydrolyzation and/or enzymatic degradation. Poly(e-caprolactone)
(PCL) is a type of aliphatic polyester possessing good bio-
degradability, biocompatibility, non-toxicity, and high perme-
ability to drugs. PCL is hydrophobic and highly crystalline and
it is more resistant to hydrolytic degradation compared
with polylactide and polyglycolide.[27–30] However, PCL based
prodrugs with water solubility have been rarely studied due to
their hydrophobicity and absence of functional moieties, which
allow for introducing bioactive moieties. To address this issue,
reactive hydrophilic groups (i.e. hydroxy,[31] amino,[32] and
carboxylic acid[33]) have been incorporated into PCLs to
improve their water solubility and biocompatibility for the
preparation of novel fully biodegradable and water-soluble
polymeric prodrugs.
The efficacy of many potent and promising anticancer drug
molecules is limited by their poor water solubility, and highly
cytotoxic side effects,[1,2] thus necessitating an efficient way
of systemic transportation. Self-assembled nanovehicles
(e.g. micelles,[3,4] liposomes,[5] nanoparticles,[6,7] and polymer-
somes[8]) encapsulating hydrophobic drugs have been docu-
mented as a possible strategy to give improved efficacy of
chemotherapeutic drugs delivery to solid tumours and reducing
drug release to non-cancerous tissue. However, these nanove-
hicles tend to dissociate and release encapsulated drug upon
intravenous administration.[9,10] In addition, dosage of the drug
in nanoparticles or liposomes are not allowed to exceed
10 %[11,12] to minimize the initial drug release in the blood
compartments. In contrast, the conjugation of a drug with a
polymer forms a so-called ‘polymeric prodrug’ that reduces the
potential for gradual leakage of the drug from the delivery
system because of the covalent linkages between the polymers
and drug molecules; this has gained tremendous attention for
cancer therapy.[13–16] Polymeric prodrugs showed an increased
water solubility of hydrophobic drugs, thus, enhancement of
drug bioavailability. On one hand they preserved the drugs
activity during circulation, and transported it to the target organ
or tissue; on the other, they reduced the antigenic activity of the
drug and exhibited a less pronounced immunological body
response. They enabled passive or active targeting of drugs to
tumour tissues and cells.[17–19] So far, numerous polymeric
prodrugs have been successfully approved for different phases
of clinical trials during the past decades.[13]
Polymeric prodrugs based on biocompatible water-soluble
polymers such as poly(N-(2-hydroxypropyl)methacrylamide)
(PHPMA)[20,21] and poly(ethylene glycol) (PEG)[22] have
In this contribution, we report a series of poly(e-caprolactone)-
graft-carboxylic/paclitaxel (P(a-C2CL)/PTX) prodrugs which
were prepared from water soluble and biodegradable PCL bearing
Journal compilation Ó CSIRO 2015
www.publish.csiro.au/journals/ajc

Water-Soluble PCL-Paclitaxel Prodrugs
1137
a-carboxyl groups. Poly(e-caprolactone)-graft-carboxylic (P(a-
C2CL)) was synthesized by ring-opening polymerization of
a-bromo-e-caprolactone and subsequent postpolymerization
by a thio-bromo click reaction. It is water soluble, non-toxic,
and amendable to conjugate with paclitaxel by esterification,
which makes P(a-C2CL) a unique candidate towards an efficient
drug delivery system.
the catalyst via a thio-bromo click reaction to yield P(a-C2CL).
Poly(a-bromo-e-caprolactone) with different number average
molecular weights and molecular weight distributions were
synthesised by controlling the initial monomer to initiator ratio
(Table 1). In the 1H NMR spectrum (Fig. 1), the integration ratio
of proton resonances at 4.16 and 3.8 ppm (Ha) was used to
calculate the degree of polymerization (DP) of poly(a-bromo-
e-caprolactone). The successful preparation of P(a-C2CL) was
verified by 1H NMR, 13C NMR, and FTIR spectroscopy and gel
permeation chromatography (GPC). As shown in Fig. 2, the
chemical shift at 3.75 ppm (Hg) was assigned to the character-
istic proton of the CH group of thiomalic acid adjacent to the
thioether. Integration of these characteristic protons (Hg, Hf)
demonstrated the quantitative reaction between thiomalic acid
and bromo-substituted PCL, indicating high fidelity of the click
reaction. In the FTIR spectra (Fig. 3b), a large broad peak from
2500 to 3500 cmꢀ1, corresponding to the carboxy absorption,
emerged after the click reaction, which further confirmed
the successful preparation of P(a-C2CL). In addition, the suc-
cessful preparations of the polymer was also confirmed by the
GPC analyses, as shown in Fig. 4. Unimodal peaks with a
low-molecular-weight distribution were clearly observed and
the molecular weight of the carboxyl-substituted PCL increased
appreciably compared with that of poly(a-bromo-e-capro-
lactone), suggesting that the carboxy groups were successfully
grafted to the PCL backbone and the polymer did not degrade
after the click reaction.
Results and Discussion
Polymer Synthesis
Poly(a-bromo-e-caprolactone) and P(a-C2CL) were synthe-
sized by following the procedure as shown in Scheme 1. Poly(a-
bromo-e-caprolactone) was prepared in a multistep route by
following a reported procedure.[34] Cyclohexanone was first
converted into a-bromocyclohexanone through a substitution
reaction with N-bromosuccinimide (NBS). Subsequently, the
a-bromo-e-caprolactone monomer was prepared by the Baeyer–
Villiger oxidation of a-bromocyclohexanone in the presence of
m-chloroperoxy benzoic acid. The monomer underwent a ring-
opening polymerization using Sn(Oct)2 as the catalyst and
methanol as the initiator, yielding a bromo-substituted PCL
(Poly(a-bromo-e-caprolactone)), which was then reacted with
thiomalic acid in acetonitrile in the presence of triethylamine as
O
O
O
Br
Br
NBS, CH3COONH4
mCPBA
DCM
O
Et2O, 25ꢀC
O
Br
e
O
Br
c
a
b
fꢂ
O
CH3OH
Sn(Oct)2, 110ꢀC
O
O
OH
n
f
d
O
OH
Br
n
O
O
Br
bꢁf
HO
O
dꢁe
CDCl3
SH
O
O
HO
OH
c
OH
O
S
O
O
a
OH
O
CH3CN, NEt3
n
fꢂ
S
OH
O
8
7
6
5
4
3
2
1
0
O
HO
O
P(α-C2CL)
[ppm]
Fig. 1. 1H NMR spectrum of poly(a-bromo-e-caprolactone) (P(a-BrCL))
in CDCl3.
Scheme 1. The synthetic route of poly(e-caprolactone)-graft-carboxylic
acid (P(a-C2CL)).
Table 1. Sn(Oct)2 mediated ring-opening polymerization of a-bromo-e-caprolactone
Polymerization
M0/
I0A
DPBn
MCn,theor
[g molꢀ1
MDn,GPC
[g molꢀ1
Mw/
MnD
Yield
[%]
]
]
1
2
3
4
30
50
28
47
5800
9700
7300
15300
23600
34500
1.24
1.15
1.27
1.31
94
95
96
95
80
77
15400
23200
120
115
ARatio of monomer to initiator.
BDegree of polymerization determined by 1H NMR spectroscopy.
CTheoretical number-average molecular weight.
DMn,GPC was determined by GPC (with polystyrene standards).

1138
J. Wang et al.
Water Solubility of P(a-C2CL)
Synthesis of P(a-C2CL)-PTX
P(a-C2CL)s (10 mg) with different degrees of polymerization
(DP ¼ 28, 47, 77, 115) were dissolved in NaOH aqueous solu-
tion (2 mL, pH 9.8) by ultrasonicating for 10 min and standing
for 24 h at room temperature (Fig. 5, A1–A4). When regulating
the pH value to 7.8 by addition of HCl, all polymer solutions
became turbid (Fig. 5, B1–B4). P(a-C2CL) gradually precipi-
tated when further decreasing the pH value below 4.4 (Fig. 5,
C1–C4). Meanwhile, the GPC traces showed unimodal peaks
with low-molecular-weight distributions and a similar retention
time of P(a-C2CL) under basic and neutral conditions, indicat-
ing that the polymers are relatively stable under basic conditions
(Fig. 4).[35] The water solubility of P(a-C2CL) with different
main-chain length and at various pH values are summarised in
Table 2. These results indicate that P(a-C2CL)s are readily
dissolved in aqueous solutions when pH . 8.5 because of the
deprotonation of carboxylic groups.[36] The effect of the DP on
their water solubility is not noticeable.
P(a-C2CL)-paclitaxel prodrugs (P(a-C2CL)-PTX) were pre-
pared by esterification between the carboxylic acid side chains
of P(a-C2CL) and paclitaxel (PTX) in the presence of dicyclo-
hexylcarbodiimide (DCC) (Scheme 2). Successful preparation
of P(a-C2CL)-PTX was verified by FTIR, 1H NMR, and UV-vis
spectroscopy. As shown in Fig. 3c, P(a-C2CL)-PTX exhibits
several new characteristic absorptions attributed to the benzene
rings of PTX, such as the skeleton vibration at 1600, 1585, 1500,
and 1450 cmꢀ1, and the bending vibration at 730 cmꢀ1. In the
1H NMR spectrum (Fig. 6), P(a-C2CL)-PTX shows character-
istic resonances of paclitaxel moieties at 7.0–8.2 ppm. Notably,
the GPC curves of P(a-C2CL)-PTX exhibited unimodal peaks,
these peaks did not overlap with the signal of the free PTX
(Fig. S4, Supplementary Material). The results revealed that
PTX was successfully grafted to the P(a-C2CL) backbone and
the polymer-PTX prodrug did not degrade after esterification.
In addition, unreacted PTX was removed completely. In the
UV-vis spectra (Fig. 7), P(a-C2CL)-PTX shows the same
absorption band at 274 nm as that of PTX in a 1/2 (v/v)
methanol/DMF solution.
OH
OH
O
O
O
O
PTX Loading Efficiency and Water Solubility
of P(a-C2CL)-PTX
S
e
c
a
b
fꢂ
O
O
OH
n
d
f
S
g
HO
The concentration of PTX was linearly correlated with the
absorbance at 274 nm in the range of 0.5 ꢁ 10ꢀ5 to 3.25 ꢁ
10ꢀ5 g mLꢀ1 (the linear regression equation was A ¼ 0.70563C þ
0.09698, r ¼ 0.9982). Therefore, the PTX contents in the P(a-
C2CL)-PTX prodrugs were determined by UV-vis spectroscopy
and summarised in Table 3. A single narrow size distribution is
observed and the average diameter is 20 nm by dynamic light
scattering (DLS) (Fig. S3, Supplementary Material). The
average diameter determined was attributed to the pH of the
solution of P(a-C2CL)-PTX being slightly lower than neutral,
which resulted in spontaneous self-assembly of P(a-C2CL)-
PTX. In order to demonstrate whether the P(a-C2CL)-PTX
aggregated in aqueous solution at a lower pH value, we
measured the solution properties of P(a-C2CL)-PTX at dif-
ferent pH values. When regulating the pH value from 8.0–7.0
to 7.0–6.0, the transmittance of P(a-C2CL)-PTX solutions
O
h
O
OH
DMSO
H2O
b
aꢁfꢂ
cꢁdꢁe
g
f
h
5
4
3
2
1
[ppm]
Fig. 2. 1H NMR spectrum of poly(e-caprolactone)-graft-carboxylic acid
(P(a-C2CL)) in DMSO-d6.
(a)
(b)
a
b
c
(c)
730
3000
2500
2000
1500
1000
500
12
13
14
15
16
17
18
Wavenumber [cmꢃ1
]
Elution time [min]
Fig. 3. FTIR spectra of (a) poly(a-bromo-e-caprolactone) (P(a-BrCL)),
(b) poly(e-caprolactone)-graft-carboxylic acid (P(a-C2CL)), and (c) poly
(e-caprolactone)-graft-carboxylic acid–paclitaxel prodrugs (P(a-C2CL-
PTX)).
Fig. 4. Gel permeation chromatography traces of (a) poly(a-bromo-
e-caprolactone) (P(a-BrCL)), (b) poly(e-caprolactone)-graft-carboxylic
acid (P(a-C2CL)), and (c) P(a-C2CL) under basic condition standing
for 24 h.

Water-Soluble PCL-Paclitaxel Prodrugs
1139
A1
B1
C1
A2
B2
C2
A3
B3
C3
A4
B4
C4
Fig. 5. Digital photos for the aqueous solution of P(a-C2CL)28 (1), P(a-C2CL)47 (2), P(a-C2CL)77 (3) and P(a-C2CL)115 (4). P(a-C2CL):
poly(e-caprolactone)-graft-carboxylic acid. (A: at pH 8.5–9.8; B: at pH 5.5–7.8; C: at , pH 4.4).
O
HO
S
O
Table 2. Water solubility of poly(e-caprolactone)-graft-carboxylic
acid (P(a-C2CL)) with various degree of polymerization at different
pH values
O
OH
OH
PTX
O
O
n
OH
S
O
O
Polymers
Solubility
O
pH 2.5, 3.2, 4.4
pH 5.5, 6.7, 7.8
pH 8.5, 9.3, 9.8
O
HO
O
O
O
insolubleA
insoluble
insoluble
insoluble
slightly solubleB
slightly soluble
slightly soluble
slightly soluble
solubleC
soluble
soluble
soluble
O
P(a-C2CL)28
P(a-C2CL)47
P(a-C2CL)77
P(a-C2CL)115
OH
O
DCC, DMAP
PTX, DCM
C2ꢂ
NH
O
O
HO
O
O
O
O
NaHCO3
ꢁNaꢃO
AInsoluble (,1 mg mLꢀ1).
BSlightly soluble (1–5 mg mLꢀ1).
CSoluble (.5 mg mLꢀ1).
O
O
PTX
O
O
S
O
O
O
H
O
O
x
y
z
decreased sharply at low pH values (Fig. S5, Supplementary
Material). Furthermore, the water solubility of P(a-C2CL)-
PTX decreased as the PTX contents increased (Table 3). In
order to ensure relatively high drug loading and good water-
solubility of the produgs, the drug-loading efficacy was
relatively suitable in the range of 10 to 40 %.
OꢃNaꢁ
OꢃNaꢁ
S
S
O
O
O
PTX
ꢁNaꢃO
O
O
O
Scheme 2. The synthetic route of P(a-C2CL)-PTX conjugate.
In Vitro Cytotoxicity
The in vitro antitumour activity of the P(a-C2CL)-PTX
conjugate was also studied in MCF-7 and A549 cells. The cell
viabilities were evaluated after 24, 48, or 72 h incubation with
the P(a-C2CL)-PTX conjugate, and free PTX was used as a
control. As shown in Fig. 9, the P(a-C2CL)-PTX conjugate
exhibited dose and time dependent cell proliferation inhibition
for both MCF-7 and A549 cells. The viabilities of MCF-7 and
A549 cells treated with the P(a-C2CL)-PTX conjugate were
The in vitro cytotoxicity of P(a-C2CL) was evaluated by methyl
tetrazolium (MTT) assay. Two cell lines (i.e. MCF-7 and A549
cells) were applied. As shown in Fig. 8, the viabilities of MCF-7
and A549 cells treated with P(a-C2CL) were in the range of 80
to 110 % at all test concentrations up to 10 mg mLꢀ1 after 72 h
incubation, revealing the low toxicity and good compatibility of
the polymer to the cells.

1140
J. Wang et al.
HO
S
O
O
PTX
O
O
O
c
e
a
b
f
O
d
O
H
O
O
m
n
k
g
OH
S
O
OH
S
O
h
O
O
O
HO
O
PTX
O
i
O
l
O
m
PTX
O
n
O
i
OH
C2ꢂ
j
NH
O
O
HO
O
O
O
i
k
O
O
l
j
b
g
d
f
i
nꢁc
m
h
k
e
8
7
6
5
4
3
2
1
[ppm]
Fig. 6. 1H NMR spectrum of poly(e-caprolactone)-graft-carboxylic acid–paclitaxel prodrugs (P(a-C2CL-PTX)) in
DMSO-d6.
(a)
(b)
1.2
1.0
0.8
0.6
0.4
0.2
0.5 ꢄ 10ꢃ5
0.75 ꢄ 10ꢃ5
1.0
1.0 ꢄ 10ꢃ5
1.5 ꢄ 10ꢃ5
1.75 ꢄ 10ꢃ5
2.0 ꢄ 10ꢃ5
2.5 ꢄ 10ꢃ5
3.25 ꢄ 10ꢃ5
0.9
Solvent
P(α-BrCL)
P(α-C2CL)
P(α-C2CL-PTX)
0.8
280
300
320
340
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Concentration (ꢄ 10ꢃ5) [g mLꢃ1
]
Wavelength [nm]
Fig. 7. (a) UV-vis spectra of PTX in DMF/methanol at different concentrations and poly(a-bromo-e-caprolactone) (P(a-BrCL)), poly(e-caprolactone)-graft-
carboxylic acid (P(a-C2CL)), and poly(e-caprolactone)-graft-carboxylic acid–paclitaxel prodrugs (P(a-C2CL-PTX)) in DMF/methanol. (b) Absorption
intensity of P(a-C2CL)-PTX at 274 nm in their DMF/methanol solutions of different concentrations.
Table 3. Water solubility of poly(e-caprolactone)-graft-carboxylic
acid–paclitaxel (P(a-C2CL-PTX)77) prodrugs with different paclitaxel
(PTX) contents
conjugate displayed a slightly higher drug efficacy than that of
free PTX in MCF-7 cells at a concentration of 4 mg PTX equiv.
mLꢀ1 and lower. When drug concentrations were higher than
4 mg PTX equiv. mLꢀ1, the antitumour activity of the PTX
prodrug became lower than that of free PTX. The maximal half
inhibitory concentrations (IC50) were 3.45 and 3.89 mg PTX
equiv. mLꢀ1 for the P(a-C2CL)-PTX conjugate and free PTX,
respectively. After 72 h incubation, the P(a-C2CL)-PTX conju-
gate displayed a slightly higher drug efficacy then free PTX in
MCF-7 cells between a concentration of 0.31 and 5.76 mg PTX
equiv. mLꢀ1. The IC50 of the P(a-C2CL)-PTX conjugate and
free PTX were 0.694 and 0.95mg PTX equiv. mLꢀ1, respectively.
In the A549 cell line, the P(a-C2CL)-PTX conjugate exhibited a
lower antitumour effect than free PTX. After 48 h incubation,
the IC50 of the P(a-C2CL)-PTX conjugate and free PTX were
3.15 and 1.81 mg PTX equiv. mLꢀ1, respectively. However, the
IC50 of the P(a-C2CL)-PTX conjugate was 1.01 mg PTX equiv.
Prodrugs
PTX content [wt-%]
Calculated Observed
Water solubility
[mg mLꢀ1
]
P(a-C2CL-PTX)77–1
P(a-C2CL-PTX)77–2
P(a-C2CL-PTX)77–3
P(a-C2CL-PTX)77–4
P(a-C2CL-PTX)77–5
13.8
12.1
.250
.200
.150
.100
,50
24.2
39.4
44.8
53.2
20.3
31.5
35.4
41.4
in the range of 74 to 100 % at all test concentrations up to
10 mg PTX equiv. mLꢀ1 after 24 h incubation, indicating that the
P(a-C2CL)-PTX conjugate displayed almost no antitumour
activity within 24 h. After 48 h incubation, the P(a-C2CL)-PTX

Water-Soluble PCL-Paclitaxel Prodrugs
1141
120
100
80
60
40
20
0
mLꢀ1 after 72 h incubation, which was lower than that of free
PTX (1.33 mg mLꢀ1). The results show that the MCF-7 cells
were much more sensitive to the PTX prodrug and free PTX than
A549 cells after 72 h incubation. Meanwhile, the prodrug
exhibited a lower antitumour efficacy as compared with the
free PTX.[37,38] These results suggest that the PTX-polymers
release PTX in a pristine state and it retains its potent therapeutic
activity, as such P(a-C2CL)-PTX prodrugs possess great anti-
tumour ability towards an efficient drug delivery system.
MCF-7
A549
Conclusions
A new family of poly(e-caprolactone)s bearing carboxylic
pedants (P(a-C2CL)) was synthesized via a combination of ring-
opening polymerization and thio-bromo click reaction. The
anticancer drug (i.e. paclitaxel) was successfully conjugated
to P(a-C2CL) via esterification with a high PTX content (up to
40 wt-%). P(a-C2CL)-PTX are readily dissolved in aqueous
solutions (. 50 mg mLꢀ1) when the carboxylic side-chains are
deprotonated. P(a-C2CL)-PTX prodrugs showed potent cellular
growth inhibition abilities in MCF-7 (IC50 ¼ 0.694 mg PTX
equiv. mLꢀ1) and A549 (IC50 ¼ 1.01 mg PTX equiv. mLꢀ1) cell
2.5
P(α-C2CL) concentration [mg mLꢃ1
10
5
0.625
0.15625 0.3125
1.25
]
Fig. 8. In vitro cell cytotoxicity of poly(e-caprolactone)-graft-carboxylic
acid (P(a-C2CL)) against A549 and MCF-7 cells at different concentrations
after 72 h.
24h
110
100
90
80
70
60
50
40
A549
MCF-7
110
100
90
80
70
60
50
40
30
20
10
0
30
PTX to A549
P(α-C2CL)-PTX to A549
PTX to MCF-7
20
P(α-C2CL)-PTX to MCF-7
10
0
0.1
1
10
0.1
1
10
PTX concentration [μg mLꢃ1
]
48h
110
100
90
80
70
60
50
40
30
20
10
110
100
90
80
70
60
50
40
30
20
10
0
PTX to A549
PTX to MCF-7
P(α-C2CL)-PTX to A549
P(α-C2CL)-PTX to MCF-7
0
0.1
1
10
0.1
1
10
PTX concentration [μg mLꢃ1
]
72h
110
100
90
80
70
60
50
40
30
20
10
0
110
100
90
80
70
60
50
40
30
20
10
0
PTX to MCF-7
P(α-C2CL)-PTX to MCF-7
PTX to A549
P(α-C2CL)-PTX to A549
0.1
1
10
0.1
1
10
PTX concentration [μg mLꢃ1
]
Fig. 9. In vitro cell viability of A549 and MCF-7 cells against free paclitaxel (PTX) and the poly(e-caprolactone)-graft-carboxylic acid–paclitaxel prodrug
(P(a-C2CL-PTX)) at different concentrations and incubation time.

1142
J. Wang et al.
lines after 72 h incubation. Our work indicates that the
P(a-C2CL)-PTX will be a great potential drug delivery system
for cancer therapy.
(m, 2H, –CH2COOH–), 3.6 (s, 3H, –CH3), 3.7 (m, 2H, –CH2OH),
3.75 (m, 1H, –CHCOOH–). dC (400 MHz, d6-DMSO) 173.4,
173.1, 172.5, 64.8, 46.6, 43.1, 37.5, 31.6, 31.1, 23.4
Experimental
Synthesis of P(a-C2CL)-PTX
Instruments and Measurements
Under a N2 atmosphere, a mixture of P(a-C2CL) (0.1 g,
0.45 mmol), DCC (0.024 g, 0.085 mmol), and catalytic amounts
of dimethylaminopyridine (DMAP) in DMF (2 mL) was stirred
at 08C for 0.5 h. PTX (0.065 g, 0.08 mmol) was then added and
the mixture kept stirring at room temperature for 24 h. The
consumption of PTX was monitored by thin-layer chromato-
graphy. The solvent was removed at reduced pressure and the
residue was dissolved in deionized water (50 mL) by regulating
the pH value to 8.5–9.0 with 2 M NaOH aqueous solution. The
polymer aqueous solution was extracted with CH2Cl2 and ethyl
acetate three times to remove the impurities. The polymer was
precipitated by regulating the pH to 3.5 with 6 M HCl(aq),
filtered off, and freeze-dried to yield a white sticky solid
(obtained: 0.12 g, yield: 73 %). dH (400MHz, d6-DMSO) 1.23–
1.75(m, 6H, –CH2CH2CH2–), 2.67 (m,2H, –CH2COOH–), 3.6(s,
3H, –CH3), 3.7 (m, 2H, –CH2OH), 3.75 (m, 1H, –CH(COOH)–),
4.0 (m, 3H, –COOCH2– and –COCH–), 7.3–8.4 (m, 15H, Ar-H).
1H NMR and 13C NMR spectra were recorded on a Bruker AV
400 M. CDCl3 and DMSO-d6 were used as the solvents. Average
molecular weights (Mn) and polydispersities (PDI) were mea-
sured on a PL-GPC120 setup equipped with a column set con-
sisting of two PL gel 5 mm MIXED-D columns (7.5 ꢁ 300 mm,
effective molecular weight range of 0.2–400.0 kg molꢀ1) using
N,N-dimethylformamide that contained 0.01 M LiBr as the
eluent at 808C at a flow rate of 1.0 mL minꢀ1. Narrowly dis-
tributed polystyrene standards in the molecular weight range of
0.5–7500.0 kg molꢀ1 (PSS, Mainz, Germany) were utilised for
calibration. FTIR spectra were recorded on a Perkin–Elmer
Spectrum one FTIR spectrophotometer, five scans were signal-
averaged with a resolution of 10 cmꢀ1 at room temperature.
Samples were prepared by dispersing the complexes in KBr and
compressing the mixtures to form disks. UV-vis spectra were
measured using a PE Lambda 20 spectrophotometer. UV irra-
diation was carried out with a 300 W high-pressure mercury
lamp coupled with UV filters (,360 nm). The size of particles
was measured by DLS with a vertically polarized He–Ne laser
(DAWNEOS, Wyatt Technology, USA) at a fixed scattering
angle of 908 and at a constant temperature of 258C. The sample
was filtered through Millipore membranes with pore sizes of
0.45 mm before measurement.
Water Solubility of P(a-C2CL)
The effect of degree of polymerization (DP ¼ 28, 47, 77, 115)
and pH (pH ¼ 2.5, 3.2, 4.4, 5.5, 6.7, 7.8, 8.5, 9.3 and 9.8) on the
water-solubility of P(a-C2CL) was determined by sonicating
P(a-C2CL) (10 mg) in a 10 mL aqueous solution for 10 min at
room temperature. The pH value was adjusted using NaOH or
HCl aqueous solutions.
Methods
PTX Loading Efficiency and Water Solubility
of P(a-C2CL)-PTX
Synthesis of Poly(a-bromo-e-caprolactone)
by Ring-Opening Polymerization
Loading Efficiency
a-Bromo-e-caprolactone (1.00 g, 0.005 mol), toluene
(5 mL), methanol (1.7 mg, 0.52 mmol), and Sn(Oct)2 (21 mg,
0.5 mmol) were added into a H2O-free polymerization flask
under nitrogen, followed by stirring at 1108C for 48 h. The
solvents were then removed by rotary evaporation and the
residue was dissolved in CH2Cl2. The polymer/CH2Cl2 solution
was precipitated from cold methanol to yield a colourless,
transparent, and sticky solid product and dried under vacuum
overnight at room temperature (obtained: 0.95 g, yield: 95 %).
dH (400 MHz, CDCl3) 1.48–2.14 (m, 6H, –CH2CH2CH2–), 3.7
(m, 2H, –CH2OH), 3.8 (s, 3H, –CH3), 4.16 (m, 3H, –CHBr–,
–COOCH2–). dC (400 MHz, CDCl3) 169.65, 65.44, 45.64,
45.62, 34.26, 27.70, 23.72.
The PTX-loading efficiency of the P(a-C2CL)-PTX prodrug
was determined by UV-vis spectroscopy. Briefly, PTX was
dissolved in a methanol/DMF solution (1/2, v/v) to yield a serꢀie5s
of standard solutions at certain concentrations (i.e. 0.5 ꢁ 10
,
,
0.75 ꢁ 10ꢀ5, 1.0 ꢁ 10ꢀ5, 1.5 ꢁ 10ꢀ5, 1.75 ꢁ 10ꢀ5, 2.0 ꢁ 10ꢀ5
2.5 ꢁ 10ꢀ5, 3.25 ꢁ 10ꢀ5 g mLꢀ1). A calibration curve that cor-
relates the absorbance at 274 nm with the concentration of PTX
was constructed. The concentration of PTX in the P(a-C2CL)-
PTX) aqueous solution was obtained using the calibration curve.
Thus, the estimated PTX-loading efficiency was calculated
using the following equation:
Percentage drug loading ¼ ðconcentration of PTX in Pða-C2CLÞ-
PTX=concentration of Pða-C2CLÞ-PTXÞ ꢁ 100 %
Synthesis of P(a-C2CL) by a Thio-Bromo ‘Click’
Reaction
ð1Þ
Triethylamine (6 mL) was added dropwise to an acetonitrile
solution (10 mL) containing thiomalic acid (1.00 g, 0.006 mol)
under N2, followed by slow addition of acetonitrile solution
containing poly(a-bromo-e-caprolactone) (0.50 g, 0.041 mol).
The mixture was stirred at room temperature for 24 h. The
solvent was then removed and the residue was dissolved in
deionized water by regulating the pH value to 8.5–9.0 with 2 M
NaOH aqueous solution. The polymer aqueous solution was
extracted with CH2Cl2 and ethyl acetate three times to remove
the impurities. The polymer was precipitated by regulating the
pH to 3.5 with 6 M HCl(aq), filtered off, and freeze-dried to
yield a white sticky solid (obtained: 0.51 g, yield: 77 %). dH
(400 MHz, d6-DMSO) 1.23–1.75 (m, 6H, –CH2CH2CH2–), 2.67
Water Solubility
P(a-C2CL)-PTX was first dissolved in a NaHCO3 aqueous
solution, followed by dialyzing against distilled water and
lyophilization to obtain a solid product for the water-solubility
measurement. Typically, 100 mg of P(a-C2CL)-PTX and deio-
nized water were mixed in a 10 mL flask by ultrasonicating for
10 min. The volume of the solution was measured to calculate
the water solubility. The size of P(a-C2CL)-PTX in aqueous
solution was measured by DLS. The influence of pH on the
water solubility of P(a-C2CL)-PTX was determined by sonicat-
ing P(a-C2CL)-PTX (100 mg) in a 10 mL aqueous solution for

Water-Soluble PCL-Paclitaxel Prodrugs
1143
10 min at room temperature. The pH value was adjusted using
HCl aqueous solutions.
[11] J. Callahan, P. Kopeckova´, J. Kopecek, Biomacromolecules 2009, 10,
1704. doi:10.1021/BM801514X
[12] H. M. Deutsch, J. A. Glinski, M. Hernandez, R. D. Haugwitz, V. L.
Narayanan, M. Suffness, L. H. Zalkow, J. Med. Chem. 1989, 32, 788.
doi:10.1021/JM00124A011
[13] S. Parveen, S. K. Sahoo, J. Drug Target. 2008, 16, 108. doi:10.1080/
10611860701794353
[14] Y. Shen, E. Jin, B. Zhang, C. J. Murphy, M. Sui, J. Zhao, J. Wang,
J. Tang, M. Fan, E. Van Kirk, W. J. Murdoch, J. Am. Chem. Soc. 2010,
132, 4259. doi:10.1021/JA909475M
Cell Culture
A549 (human pulmonary carcinoma) and MCF-7 cells were
cultured in Dulbecco’s modified Eagle’s medium (DMEM) with
high glucose levels containing 10 % fetal bovine serum,
supplemented with 50 U mLꢀ1 penicillin and 50 U mLꢀ1 strep-
tomycin, and incubated at 378C in 5 % CO2 atmosphere.
[15] R. Tong, J. Cheng, Angew. Chem. 2008, 120, 4908. doi:10.1002/
ANGE.200800491
[16] (a) H. S. Yoo, K. H. Lee, J. E. Oh, T. G. Park, J. Control. Release 2000,
68, 419. doi:10.1016/S0168-3659(00)00280-7
In Vitro Cytotoxicity Assay
The in vitro cytotoxicity was assessed with a MTT viability
assay against A549 and MCF-7 cells. A549 cells and MCF-7
cells were seeded in 96-well plates at 10000 cells per well in
100 mL of DMEM medium and incubated for 24 h, followed by
removing the culture medium and adding P(a-C2CL), PTX,
P(a-C2CL)-PTX (200 mL in complete DMEM medium) at
different concentrations. The cells were subjected to MTT
assay after being incubated for another 24, 48, or 72 h. At the
end of the experiments, 20 mL of MTT solution (5 mg mLꢀ1 in
phosphate buffered saline) was added to each well. The plate
was returned to the incubator. After 4 h, the MTT solution was
carefully removed from each well, and the MTT-formazan
generated by live cells was dissolved in 200 mL of DMSO. The
absorbance of the solution was measured on a Bio-Rad 680
microplate reader at 492 nm. Cell viability (%) was calculated
by (Asample/Acontrol) ꢁ 100, where Asample and Acontrol denote the
absorbance of the sample well and control well, respectively.
Data are presented as average ꢂ s.d. (n ¼ 3).
(b) V. R. Caiolfa, M. Zamai, A. Fiorino, E. Frigerio, C. Pellizzoni,
R. d’Argy, A. Ghiglieri, M. G. Castelli, M. Farao, E. Pesenti, M. Gigli,
F. Angelucci, A. Suarato, J. Control. Release 2000, 65, 105.
doi:10.1016/S0168-3659(99)00243-6
(c) E. S. Lee, K. Na, Y. H. Bae, J. Control. Release 2005, 103, 405.
doi:10.1016/J.JCONREL.2004.12.018
(d) O. M. Koo, I. Rubinstein, H. Onyuksel, Nanomedicine 2005, 1, 77.
doi:10.1016/J.NANO.2004.11.002
[17] K. Kataoka, A. Harada, Y. Nagasaki, Adv. Drug Deliv. Rev. 2001, 47,
113. doi:10.1016/S0169-409X(00)00124-1
[18] E. Fleige, M. A. Quadir, R. Haag, Adv. Drug Deliv. Rev. 2012, 64, 866.
doi:10.1016/J.ADDR.2012.01.020
[19] V. P. Torchilin, Nat. Rev. Drug Discov. 2005, 4, 145. doi:10.1038/
NRD1632
[20] M. E. Davis, D. M. Shin, Nat. Rev. Drug Discov. 2008, 7, 771.
doi:10.1038/NRD2614
[21] R. Cheng, F. Meng, C. Deng, H.-A. Klok, Z. Zhong, Biomaterials
2013, 34, 3647. doi:10.1016/J.BIOMATERIALS.2013.01.084
[22] F. Meng, Z. Zhong, J. Feijen, Biomacromolecules 2009, 10, 197.
doi:10.1021/BM801127D
[23] K. Knop, R. Hoogenboom, D. Fischer, U. S. Schubert, Angew. Chem.
Int. Ed. 2010, 49, 6288. doi:10.1002/ANIE.200902672
[24] C. Li, S. Wallace, Adv. Drug Deliv. Rev. 2008, 60, 886. doi:10.1016/
J.ADDR.2007.11.009
Supplementary Material
The 13C NMR spectrum and size distribution profiles of poly-
mers (Figs S1–S3), GPC curves of PTX-polymers and free PTX
(Fig. S4), and digital photos of the aqueous solution of PTX-
polymers (Fig. S5) are available on the Journal’s website.
[25] B. Romberg, J. M. Metselaar, L. Baranyi, C. Snel, R. Bunger,
W. Hennink, J. Szebeni, G. Storm, Int. J. Pharm. 2007, 331, 186.
doi:10.1016/J.IJPHARM.2006.11.018
Acknowledgements
[26] J. Pytela, V. Saudek, J. Drobnik, F. Rypa´cˇek, J. Control. Release 1989,
10, 17. doi:10.1016/0168-3659(89)90014-X
[27] G. L. Brode, J. V. Koleske, J. Macromol. Sci., Chem. 1972, 6, 1109.
doi:10.1080/10601327208056888
Financial support was provided by the National Natural Science Foundation
of China (51273169, 51473141).
[28] E. L. Prime, J. J. Cooper-White, G. G. Qiao, Aust. J. Chem. 2006, 59,
534. doi:10.1071/CH06144
References
[1] G. Pasut, F. M. Veronese, Prog. Polym. Sci. 2007, 32, 933.
doi:10.1016/J.PROGPOLYMSCI.2007.05.008
[2] R. Duncan, Nat. Rev. Cancer 2006, 6, 688. doi:10.1038/NRC1958
[3] H. Maeda, G. Y. Bharate, J. Daruwalla, Eur. J. Pharm. Biopharm.
2009, 71, 409. doi:10.1016/J.EJPB.2008.11.010
[4] Y. H. Bae, H. Yin, J. Control. Release 2008, 131, 2. doi:10.1016/
J.JCONREL.2008.06.015
[5] A. Rosler, G. W. M. Vandermeulen, H. A. Klok, Adv. Drug Deliv. Rev.
2001, 53, 95. doi:10.1016/S0169-409X(01)00222-8
[6] R. Duncan, Nat. Rev. Drug Discov. 2003, 2, 347. doi:10.1038/
NRD1088
[29] C. Allen, J. Han, Y. Yu, D. Maysinger, A. Eisenberg, J. Control.
Release 2000, 63, 275. doi:10.1016/S0168-3659(99)00200-X
[30] V. R. Sinha, K. Bansal, R. Kaushik, R. Kumria, A. Trehan, Int. J.
Pharm. 2004, 278, 1. doi:10.1016/J.IJPHARM.2004.01.044
[31] M. Liu, N. Vladimirov, J. M. J. Fre´chet, Macromolecules 1999, 32,
6881. doi:10.1021/MA990785X
[32] J. Yan, Y. Zhang, Y. Xiao, Y. Zhang, M. Lang, React. Funct. Polym.
2010, 70, 400. doi:10.1016/J.REACTFUNCTPOLYM.2010.03.008
[33] M. Trollsas, V. Y. Lee, D. Mecerreyes, P. Lo¨wenhielm, M. Mo¨ller,
R. D. Miller, J. L. Hedrick, Macromolecules 2000, 33, 4619.
doi:10.1021/MA992161X
[7] C. Li, S. Wallace, Adv. Drug Deliv. Rev. 2008, 60, 886. doi:10.1016/
J.ADDR.2007.11.009
[34] G. Ma, D. Li, J. Wang, X. Zhang, H. Tang, Aust. J. Chem. 2013, 66,
1576. doi:10.1071/CH13297
[8] J. Khandare, T. Minko, Prog. Polym. Sci. 2006, 31, 359. doi:10.1016/
J.PROGPOLYMSCI.2005.09.004
[35] Y. Hu, Z. Jiang, R. Chen, W. Wu, X. Jiang, Biomacromolecules 2010,
11, 481. doi:10.1021/BM901211R
[9] K. Hoste, K. De Winne, E. Schacht, Int. J. Pharm. 2004, 277, 119.
doi:10.1016/J.IJPHARM.2003.07.016
[10] J. Kopecˇek, P. Kopecˇkova´, T. Minko, Z.-R. Lu, Eur. J. Pharm.
Biopharm. 2000, 50, 61. doi:10.1016/S0939-6411(00)00075-8
[36] S. Q. Zhang, Biomed. Sci., Eng. Technol. 2012, 24, 588.
[37] R. Duncan, Nat. Rev. Cancer 2006, 6, 688. doi:10.1038/NRC1958
[38] Y. Gu, Y. Zhong, F. Meng, R. Cheng, C. Deng, Z. Zhong, Bioma-
cromolecules 2013, 14, 2772. doi:10.1021/BM400615N