Methoxy-poly(ethylene glycol)-block-poly(ε-caprolactone) bearing pendant aldehyde groups as pH-responsive drug delivery carrier

Article abstract of DOI:10.1071/CH13297

A biodegradable amphiphilic block copolymer of methoxy poly(ethylene glycol)-block-poly(ε-caprolactone) bearing pendant aldehyde groups was synthesised by a combination of ring-opening polymerisation and thio-bromo 'click' chemistry. The free aldehyde gro

Full text of DOI:10.1071/CH13297

CSIRO PUBLISHING
Aust. J. Chem. 2013, 66, 1576–1583
Full Paper
http://dx.doi.org/10.1071/CH13297
Methoxy-Poly(ethylene glycol)-block-Poly(e-caprolactone)
Bearing Pendant Aldehyde Groups as pH-Responsive
Drug Delivery Carrier
A
A
A
A B C
, ,
Gejun Ma, Deshan Li, Ji Wang, 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; cranetang@gmail.com
A biodegradable amphiphilic block copolymer of methoxy poly(ethylene glycol)-block-poly(e-caprolactone) bearing
pendant aldehyde groups was synthesised by a combination of ring-opening polymerisation and thio-bromo ‘click’
chemistry. The free aldehyde groups on the copolymer were reacted with hydrophobic payloads (p-methoxylaniline as a
model drug) by a benzoic–imine linker, which was responsive to pH change. NMR, FTIR, and gel permeation
chromatography analysis confirmed the copolymer structures. In vitro release studies revealed that under acid stimulus,
hydrolysis of the benzoic–imine bond resulted in a rapid drug release. This new amphiphilic block copolymer is expected
to have promising applications in biodegradable controlled drug delivery systems.
Manuscript received: 10 June 2013.
Manuscript accepted: 20 August 2013.
Published online: 16 September 2013.
Introduction
normal extracellular matrices and blood is around 7.4 while the
pH value in the endocytic compartments of cells generally ranges
from 4.5 to 6.5.[6] Therefore, much effort has been made to
design and fabricate pH-responsive polymeric micelles to
achieve pH-triggered drug release.[7–9] For example, Yang’
group[10] developed a type of amphiphilic block copolymer
(i.e. methoxy poly(ethylene glycol)-block-poly(2,2-dimethyl-
1,3-dioxolane-4-yl)methyl acrylate, MPEG-b-PDMDMA) con-
taining pH-sensitive ketal groups attached to the hydrophobic
block. Such copolymers are able to form uniform micelles in
aqueous solutions that can rapidly release encapsulated pyrene in
a mildly acidic environment. Zhong and co-workers[11] reported
a type of acid activatable doxorubicin (DOX) pro-drug nanogel
that was prepared by conjugating DOX to poly(ethylene glycol)-
block-poly(2-hydroxyethyl methacrylate-co-glycine methacryl-
amide-hydrazide) (PEG-b-P(HEMA-co-GMA-hydrazide)) by
pH-sensitive hydrazone. Zhong and co-workers[12] demonstrated
the preparation of endosomal pH-activatable paclitaxel (PTX)
prodrug micellar nanoparticles by conjugating PTX to poly
(ethylene glycol)-block-poly(acrylic acid) (PEG-PAA) copoly-
mers by an acid-labile acetal bond to the PAA block. While this
drug delivery system showed triggered release of the payload in a
pH-dependent manner, it could not fully degrade into small
molecules and left polymeric residues, which limits its applica-
tion for practical use.
Recently, significant progress has been made in the design and
fabrication of amphiphilic diblock copolymers used as carriers
for hydrophobic anticancer drugs, dyes, proteins, enzymes,
DNA, and so on.[1] Amphiphilic block copolymers can self-
assemble into micelles in aqueous solution, where the hydro-
phobic blocks form the hydrophobic micelle core with the
ability to incorporate various hydrophobic compounds while the
hydrophilic corona serves as a stabilising interface between
the hydrophobic core and external medium. The diameters of the
polymeric micelles can be tuned by adjusting the main chain
length of the hydrophilic/hydrophobic block according to the
demand. For example, polymeric micelles with a size between
10 and 100 nm can efficiently escape renal rapid excretion and
significantly increase the possibility of accumulation of the
micelle carriers in tumours with leaky vasculature by the well
accepted enhanced permeability and retention (EPR) effect.[2,3]
For a systematic application of the drug-loaded polymeric
micelles, a challenging step is to release the drug in a sustained
and tunable fashion at the target site.[4,5] That is to say, in terms
of controlled drug release, ideally, a drug should not be
released from the carriers during circulation in the bloodstream;
however, once the pro-drugs (i.e. the carriers with drugs) are
internalised by the cancer cells, a sufficient amount of anticancer
drug should be released in a short period of time to effectivelykill
the cancer cells. To achieve this goal, much attention has been
paid to develop pH-responsive polymeric micelles as drug
carriers since there are pH value differences between normal
and pathological states. It is well known that the pH value of
In this contribution, we report a facile synthetic method to
access biodegradable amphiphilic block copolymers based on
poly(ethylene glycol) (PEG) and poly(e-caprolactone) (PCL)
containing aldehyde side groups. Water soluble PEG has been
Journal compilation Ó CSIRO 2013
www.publish.csiro.au/journals/ajc

Amphiphilic Block Copolymer Bearing Pendant Aldehyde Groups
1577
chosen as the hydrophilic block due to its biocompatibility and
lower immunogenicity.[13] It constructs the corona in core–shell
micelles, which can minimise the clearance of the polymeric
micelles by the renal and reticuloendothelial systems (RES) and
prolong the circulation time.[14–16] PCL is a type of aliphatic
polyester possessing good biodegradability, biocompatibility,
high permeability to drugs, and less acidic degradation products
as compared with polylactide and polyglycolide,[17–20] and acts
as the hydrophobic block in the amphiphilic copolymers. Non-
modified PCL as a hydrophobic block is able to conjugate
hydrophobic cargos, such as drugs, peptides, and proteins, by
hydrophobic interactions, yet the resulting pro-drugs are char-
acterised by a lack of stability.[21] While many e-caprolactone
(CL) derivatives bearing functional groups, such as hydroxy,[22]
bromide,[23,24] amino,[25] and carboxylic acid,[26] have been
synthesised and used to prepare PCLs with corresponding
functional pendant groups, some functional groups are not
compatible with the propagating species, such as tin alkoxides,
which resulted in tedious protection and deprotection reactions.
In order to conquer these difficulties, we designed a new type of
amphiphilic block copolymer comprised of PEG and PCL
blocks in which the aldehyde groups can be formed directly
by the thio-bromo ‘click’ chemistry[27] without additional
protection and deprotection reactions. Hydrophobic drugs,
i.e. p-methoxylaniline (p-MA) as a model drug, were conjugated
to the aldehyde groups by benzoic–imine linker bond formation.
When compared with other widely used pH responsive linkers
(e.g. imine linkers including hydrazone or ketoxime
bonds),[28–32] the benzoic–imine linker is more stable at neutral
and basic pH due to the proper p–p conjugation extent and can
be hydrolysed under mild acidic conditions.[33] The pH-respon-
sive behaviour of this biodegradable amphiphilic block
copolymer under physiological conditions has been carefully
characterised. With the advantages of good biodegradability and
pH-responsive behaviour, this amphiphilic biodegradable block
copolymer may become a potential candidate for the delivery of
hydrophobic drugs in the human body.
Result and Discussion
Synthesis of the Monomer
The a-bromo-e-caprolactone (aBrCL) was prepared according
to the literature procedure[34] as described in Scheme 1A.
Cyclohexanone was first converted into a-bromocyclohexanone
through a substitution reaction with N-bromosuccinimide
(NBS) at room temperature with a good yield of 85.0 %. The
ketone was subsequently oxidised to yield aBrCL (44.9 % yield)
by the Baeyer–Villiger reaction using 3-chloroperbenzoic acid
O
O
O
Br
Br
(A)
O
(b)
OH
(a)
(c)
αBrCl
O
O
SH
OH
O
O
(d)
MPHEB
H
O
H
O
H
O
O
OH
(e)
O
H
O
O
O
O
(B)
O
m
nϪ 1
nϪ1
4
Br
O
O
(f)
O
H
O
O
O
nϪ 1
O
m
4
H
m
O
O
O
S
4
nϪ1
S
(g)
O
O
O
O
O
pH labile
covalent linker
O
H
N
O
CH3
H
O
Scheme 1. (A) The synthetic route of a-bromo-e-caprolactone (aBrCL) and 2-(4-formylphenoxy)ethyl-
3-mercaptopropanoate (MPHEB). Reaction conditions: (a) N-bromosuccinimide, NH4OAc, room temp.;
(b) m-chloroperoxybenzoic acid, CH2Cl2, room temp.; (c) 2-bromoethanol, K2CO3, tetrabutylammonium
bromide, 708C; (d) 3-mercaptopropanoic acid dicyclohexylcarbodiimide, 4-(N,N-dimethylamino)-pyridine,
CH2Cl2, 08C. (B) Synthesis of methoxy poly(ethylene glycol)-block-poly(a-bromo-e-caprolactone)-aldehyde/
p-methoxyaniline (p-MA) prodrug. Reaction conditions: (e) SnOct2, 1208C, 48 h; (f) triethylamine, room temp.,
2 h; (g) p-MA, 408C, 4 h.

1578
G. Ma et al.
(a)
b
(m-CPBA) as the oxidative reagent. 1H NMR spectroscopy
confirmed the successful synthesis of the monomer, which
agreed with previous literature.[34]
O
a
f
h
b
d
O
e
O
c
113
mH
O
O
g
i
b
Synthesis of 2-(4-Formylphenoxy)ethyl-3-
mercaptopropanoate
Br
The general strategy applied for the synthesis of 2-(4-
formylphenoxy)ethyl-3-mercaptopropanoate (MPHEB) is
shown in Scheme 1A. First, 4-hydroxybenzaldehyde reacted
with 2-bromoethanol in the presence of absolute potassium
carbonate to extend the hydroxy group to obtain 4-(2-
hydroxyethoxy)benzaldehyde, and the hydroxy group was then
converted into a mercapto functional group by using 3-
mercapto-propanoic acid and dicyclohexylcarbodiimide (DCC)
in CH2Cl2 at room temperature. The products were purified by
silica gel chromatography with an overall yield of 75.1 %. The
molecular structure of MPHEB was verified by 1H NMR
spectroscopy (Fig. 1).
H2O
eϩ i
h
g
c
a
f
d
7
b
6
5
4
3
2
1
0
[ppm]
(b)
Synthesis of Amphiphilic Diblock Copolymer MPEG114
-
b-PaBrCL19
O
The synthesis of the methoxy poly(ethylene glycol)-block-poly
(a-bromo-e-caprolactone) (MPEG-b-PaBrCL) copolymer is
outlined in Scheme 1B. The amphiphilic diblock copolymer was
prepared by the ring-opening polymerisation (ROP) of a-BrCL
using methoxy poly(ethylene glycol) (MPEG) as the macro-
initiator and SnOct2 as the catalyst. Typically, the ROP was
carried at 1208C for 48 h with a monomer-to-initiator ratio of 25.
After the polymerisation, the monomer conversion was deter-
mined by gravimetric analysis and it is 76 % after 48 h. MPEG-
b-PaBrCL was obtained by precipitating from diethyl ether after
concentration and dried under vacuum. The molecular structure
a
d
f
c
O
O
mH
k
O
H
O
O
e
g
113
b
i
S
O
O
j
h
m
l
O
n
H2O
c
a
k
j
fϩe
l
m
n
h
i
g
1
d
of the resulting polymer was characterised by H NMR spec-
troscopy (Fig. 2a). The degree of polymerisation (DP) of
PaBrCL was calculated from the integration ratio of the proton
resonance at 3.38 ppm (methyl in the MPEG block) and the
proton resonance at 1.49 ppm (Hf). The DP of PaBrCL was
calculated to be 19, which was consistent with the theoretical
result at this conversion. Therefore, we coded this amphiphilic
diblock copolymer as MPEG114-b-PaBrCL19. From the IR
spectrum of MPEG114-b-PaBrCL19 (Fig. 3), we observed the
11
10
9
8
7
6
5
4
3
2
1
0
[ppm]
Fig. 2. (a) 1H NMR spectra of methoxy poly(ethylene glycol)-block-poly
(a-bromo-e-caprolactone) (MPEG-b-PaBrCL) and (b) MPEG-b-PaBrCL-
aldehyde in CDCl3.
O
d
a
f
b
O
60
g
HS
O
e
c
h
H
(c)
d
50
e
h
f
g
O
b
1685
40
(b)
c
839
H2O
30
(a)
a
20
1732
10
4000
3500
3000
2500
2000
1500
1000
500
Wave number [cmϪ1
]
11
10
9
8
7
6
5
4
3
2
1
0
[ppm]
Fig. 3. FTIR spectra of (a) methoxy poly(ethylene glycol)-block-poly(a-
bromo-e-caprolactone) (MPEG-b-PaBrCL), (b) MPEG-b-PaBrCL-
aldehyde, and (c) MPEG-b-PaBrCL-aldehyde/p-methoxyaniline prodrug.
Fig. 1. 1H NMR spectrum of 2-(4-formylphenoxy)ethyl-3-mercapto-
propanoate (MPHEB) in CDCl3.

Amphiphilic Block Copolymer Bearing Pendant Aldehyde Groups
1579
characteristic band of carbonyl groups at 1732 cmꢀ1, which
further confirmed the successful preparation of MPEG114-b-
PaBrCL19. The molecular weight and polydispersity (PDI) of
MPEG114-b-PaBrCL19 was determined by GPC (Fig. 4 and
Table 1), which showed a single peak with a PDI of 1.03.
backbone can be determined from the integration ratio of the
resonance at 2.96–2.79 ppm to that at 3.38 ppm. The value was
calculated to be 34/3, indicating that the average number of
pendant aldehyde groups on each polymer chain was 17. As
shown in Fig. 4, the GPC trace of MPEG114-b-PaBrCL19-
aldehyde17 was a unimodal peak with a shorter elution time than
that of MPEG114-b-PaBrCL19 due to the increase of molecular
weight after the thio-bromo click reaction.
Conversion of the Bromine Groups into Aldehyde Groups
by Thio-Bromo ‘Click’ Chemistry
The thio-bromo click reaction[27] between MPEG114-b-
PaBrCL19 and 2-(4-formylphenoxy) ethyl-3-mercapto-
propanoate was performed in dried acetonitrile at 258C in the
presence of triethylamine as shown in Scheme 1B. The alde-
hyde-modified MPEG114-b-PaBrCL19 was characterised by 1H
NMR, FTIR, and gel permeation chromatography (GPC) anal-
Synthesis of MPEG114-b-PaBrCL19-aldehyde17/
p-MA Prodrug
p-MA was conjugated to MPEG114-b-PaBrCL19-aldehyde17 by
an amine–aldehyde reaction. In the FTIR spectrum of the con-
jugated product, a new characteristic band appeared at
1685 cmꢀ1, indicating the formation of the benzoic imine link-
age in the side chains (Fig. 3). The molecular structure has also
been verified by 1H NMR spectroscopy, which showed that the
resonance of aldehyde groups at 9.90 ppm disappeared with
the appearance of a new peak at 8.39 ppm corresponding to the
CH¼N protons (Fig. 5). The molar fraction of the addition
product, Fp-MA, was calculated by integrating the peaks at
3.38 ppm (IM) (Fig. 1) (CH3O protons of MPEG) and at 8.4 ppm
(IB) (Fig. 5a) (CH¼N proton) (Eqn 1).
1
ysis. In the H NMR spectrum (Fig. 2b), the appearance of
typical resonances of aldehyde groups at 9.90 ppm indicates the
occurrence of the click reaction. In the FTIR spectrum, as shown
in Fig. 3, we observed the appearance of a new characteristic
band at 839 cmꢀ1 corresponding to the phenyl groups. The
average number of aldehyde groups conjugated onto the
(c)
(b)
(a)
(d)
IB
Fp-MA
¼
ð1Þ
IM=3
The molecular weight of the amphiphilic block copolymer-
conjugated p-MA (i.e. the prodrug) was investigated by GPC,
which showed a notable increase of molecular weight. This
observation confirmed that p-MA has been successfully conju-
gated to the copolymer.
Formation of the MPEG114-b-PaBrCL19-aldehyde17/
p-MA Prodrug Micelle
The MPEG114-b-PaBrCL19-aldehyde17/p-MA prodrug pro-
vides an opportunity to form micelles in water. The water-
soluble MPEG chains serve as the hydrophilic shell stabilising
the nanoparticle, and the PaBrCL19-aldehyde17/p-MA con-
stitutes the hydrophobic core. A complete reduction of char-
acteristics peaks of PaBrCL19-aldehyde17/p-MA was seen in the
micellar spectra in D2O while the PEG peak remained when
13.0
13.5
14.0
14.5
15.0
15.5
16.0
Elution time [min]
Fig. 4. GPC curves of (a) methoxy poly(ethylene glycol) (MPEG),
(b) MPEG-b-poly(a-bromo-e-caprolactone) (PaBrCL), (c) MPEG-b-
PaBrCL-aldehyde, and (d) MPEG-b-PaBrCL-aldehyde/p-methoxyaniline
prodrug.
1
compared with the H NMR spectra in CDCl3, indicating that
PaBrCL19-aldehyde17/p-MA was entrapped within the micelles
Table 1. Physical properties of methoxy poly(ethylene glycol)-block-poly(a-bromo-e-caprolactone) (MPEG-b-PaBrCL), MPEG-b-PaBrCL-
aldehyde, and MPEG-b-PaBrCL-aldehyde/p-methoxyaniline (p-MA) copolymers
Sample
Molar ratio in feed
Molar ratio in copolymerA
MnA
MnB
Mw/MBn
YieldC
[%]
(MPEG/aBrCL/MPHEB/p-MA)
(MPEG/aBrCL/MPHEB/p-MA)
(ꢁ104)
(ꢁ104)
[g molꢀ1
]
[g molꢀ1
]
MPEG114
–
–
–
0.51
1.29
1.49
1.01
1.03
1.02
–
MPEG114-b-PaBrCL
MPEG114-b-PaBrCL-
aldehyde
1 : 25 : 0 : 0
1 : 19 : 30 : 0
1 : 19 : 0 : 0
1 : 2 : 17 : 0
0.86
1.16
70.2
60.3
MPEG114-b-PaBrCL-
aldehyde/p-MA
1 : 19 : 17 : 50
1 : 2 : 3 : 14
1.31
1.53
1.05
81.0
ACalculated from 1H NMR spectra analysis.
BDetermined from gel permeation chromatography (eluent, DMF; flow rate, 1.0 mL minꢀ1; standards, polystyrene).
CCalculated from the diethyl ether-insoluble parts.

1580
G. Ma et al.
(Fig. 5). Fig. 6 presents the size distribution of the micelles
determined by dynamic light scattering (DLS). A single narrow
size distribution is observed and the average diameter is 150 nm.
In addition, the polymer–drug conjugate has a critical micelle
concentration (CMC) of 5.24 mg Lꢀ1 (Fig. 7).
hydrolysed to release the payload upon decreasing the pH. Our
results shed some light on a novel biodegradable amphiphilic
block copolymer bearing a pendant aldehyde group that may
have great potential applications as biodegradeable carriers and
controlled-release drug delivery systems.
Experimental
pH-Dependent Release of p-MA
General Procedures and Requirements
In this study, the pH-sensitive drug release behaviour of the
prodrug solutions was evaluated at a series of pH values. It has
been previously reported that PCL is only slightly degradable in
acid at 378C after 10 days,[35] which reveals that the pH-sensi-
tive drug release behaviour is mainly due to the hydrolysis of the
benzoic–imine bond instead of the effect of degradation of the
PCL core. As shown in Fig. 8, the release rate of the p-MA
increased consistently as the pH value decreased within the
range of 7.4 to 3.6, leading to the change of charge property in
the hydrophobic part. At pH 7.4, no parent drug release was
observed in 68 h, which is a desirable drug delivery character-
istic for drug nanocarriers. As shown in Fig. 8, the drug release
was accelerated within the pH range of 4.5 to 6.5, which is the
pH value of endocytic compartments. At pH 5.0, ,18 % of
p-MA was released after 68 h, suggesting that once the micelles
are internalised into the tumour cells, the conjugated drug can
be released effectively to improve the therapeutic effect of
the chemotherapy.
1H NMR spectra were recorded on a Bruker AV 400 M. Deu-
terium oxide (D2O) or deuterated chloroform (CDCl3) were
used as the solvent. Average molecular weights (Mn) and
polydispersity (PDI) were measured on a PL-GPC120 setup
100
80
60
40
20
0
Conclusion
We have demonstrated a simple and efficient method to prepare
a biodegradable amphiphilic block copolymer bearing aldehyde
side groups by ROP and thio-bromo ‘click chemistry’. Hydro-
phobic compounds such as p-MA can be conjugated to the
hydrophobic block by benzoic–imine linkages, which show a
pH-response in aqueous media. The polymeric solution is stable
at physiological pH (7.4), but the benzoic–imine linkages can be
0
50
100
150
200
250
300
Size [nm]
Fig. 6. Size distribution profiles of methoxy poly(ethylene glycol)-
block-poly(a-bromo-e-caprolactone)-aldehyde/p-methoxyaniline pro-
drug micelles determined by dynamic light scattering at a polymer
concentration of 0.1 mg mLꢀ1
.
f
a
b
d
g
c
e
i
O
113
O H
n
O
O
n
H
S
O
O
k
j
h
N
O
O
q
l
m
CH3
o
p
b
CDCI3
q
H2O
l
a
iϩh
fϩe
p
j
kϩg
o
n
m
d
(a)
(b)
D2O
10
8
6
4
2
0
[ppm]
Fig. 5. 1H NMR spectra of methoxy poly(ethylene glycol)-block-poly(a-bromo-e-caprolactone)-aldehyde/
p-methoxyaniline prodrug in (a) CDCl3 and (b) D2O.

Amphiphilic Block Copolymer Bearing Pendant Aldehyde Groups
1581
red shift
0.1(g LϪ1
0.05
0.01
0.005
0.0001
)
equipped with a column set consisting of two PL gel 5 mm
MIXED-D columns (7.5 ꢁ 300mm2, 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 distributed 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 spectro-
photometer, five scans were signal-averaged with a resolution of
10 cmꢀ1 at room temperature. Samples were prepared by dis-
persing the complexes in KBr and compressing the mixtures to
form disks. Ultraviolet–visible (UV–vis) spectra were measured
using a PE Lambda 20 spectrophotometer. UV irradiation was
carried out with a 300 W high-pressure mercury lamp coupled
with UV filters (,360nm). The size of the micelles was deter-
mined using DLS with a vertically polarised 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.
(a)
333 nm
338 nm
400
300
200
100
0
0.000001
300
310
320
330
340
350
360
Wavelength [nm]
1.25
1.20
1.15
1.10
1.05
1.00
0.95
0.90
0.85
0.80
0.75
0.70
(b)
Methods
Monomer Synthesis
The aBrCL was prepared according to a modified procedure
as follows (Scheme 1).[34]
Synthesis of a-Bromo-cyclohexanone
Cyclohexanone (20.00 g, 0.20 mol), NBS (38.14 g,
0.21 mol), ammonium acetate (1.54 g, 0.02 mol), and diethyl
ether (300 mL) were added to a 500 mL one-neck round bottom
flask fitted with a magnetic stirring bar and a reflux condenser
equipped with a nitrogen bag. After stirring at room temperature
for 0.5 h, the mixture was filtered. The filtrate was washed with
deionised H2O three times, separated, and dried over anhydrous
MgSO4. After filtration, the solution was concentrated and
the product was purified using silica gel chromatography
with 1/10 (v/v) ethyl acetate/petroleum ether as an eluent
(obtained: 30.09 g, yield: 85.0 %). dH (400 MHz, CDCl3) 4.39
(m, 1H, –CHBr–), 2.99 (m, 1H, –CH2CO–), 2.09–2.39 (m, 3H,
–CH2CO– and –CH2CHBr–), 1.88–2.05 (m, 2H, –CH2CH2CO–),
1.67–1.91 (m, 2H, –CH2CH2CHBr–).
Ϫ6
Ϫ5
Ϫ4
Ϫ3
Ϫ2
Ϫ1
0
logC [g LϪ1
]
Fig. 7. (a) Fluorescence excitation spectra of pyrene as a function
concentration (g Lꢀ1) in water at lem ¼ 390 nm. (b) Plot of the intensity
ratio from pyrene excitation of methoxy poly(ethylene glycol)-block-poly
(a-bromo-e-caprolactone)-aldehyde/p-methoxyaniline spectra v. log C
([pyrene] ¼ 5 ꢁ 10ꢀ7 mol Lꢀ1).
Synthesis of aBrCL
In a dry one-neck 500 mL flask equipped with a magnetic
stirring bar and a nitrogen bag, a-bromo-cyclohexanone
(17.70 g, 0.10 mol) and m-CPBA (20.64 g, 0.12 mol) were
dissolved in CH2Cl2 (300 mL). After stirring at 258C for
48 h, the mixture was filtered. The filtrate was cooled to
ꢀ258C and filtered again. The filtrate was washed with
Na2S2O3/H2O solution, saturated NaHCO3/H2O solution,
and deionised H2O sequentially. The organic layer was
separated and dried over MgSO4. After filtration, the solution
was concentrated and the product was purified using silica gel
chromatography with 1/10 (v/v) ethyl acetate/petroleum ether
as an eluent (obtained: 8.68 g, yield: 44.9 %). dH (400 MHz,
CDCl3) 4.85–4.83 (m, 1H, –CHBr–), 4.75–4.69 (m, 1H,
–CH2CO–), 4.33–4.26 (m, 1H, –CH2CO–), 2.15–2.06 (m,
2H, –CH2CHBr–), 2.06–1.92 (m, 2H, –CH2CH2O–), 1.92–
1.77 (m, 2H, –CH2CH2CH2O–).
35
pH 3.6
pH 5.0
pH 6.5
pH 7.4
30
25
20
15
10
5
0
0
10
20
30
40
50
60
70
Time [h]
Synthesis of 4-(2-Hydroxyethoxy)benzaldehyde
To a 250mL two-neck flask equipped with a magnetic stirring
bar and a reflux condenser was added 4-hydroxybenzaldehyde
Fig. 8. Time dependence of p-methoxyaniline released from the prodrug
solutions at different pH at 37 8C.

1582
G. Ma et al.
(10.00 g, 0.82 mol), 2-bromoethanol (12.30 g, 0.98 mol),
anhydrous potassium carbonate (17.00 g, 1.23 mol), 150 mL of
acetone, and a catalytic amount of tetrabutylammonium bro-
mide (TBAB) under nitrogen atmosphere. After stirring at 708C
for 48 h, the reaction mixture was filtered and concentrated. The
crude product was extracted with CH2Cl2 three times. After
drying over anhydrous MgSO4, the solution was concentrated
and the product was purified using silica gel chromatography
with 1/2 (v/v) ethyl acetate/petroleum ether as an eluent.
A yellowish oily liquid was obtained (8.61 g, yield: 64.9 %).
dH (400 MHz, CDCl3) 9.9 (s, 1H, –CH¼O), 7.86–7.84 (m, 2H,
C6H5), 7.05–7.01 (m, 2H, C6H5), 4.22–4.15 (m, 2H,
HOCH2CH2–), 4.03–3.94 (m, 2H, HOCH2CH2–).
C6H5), 7.05–7.01 (m, 2H, C6H5), 4.54–4.43 (m, 2H, –OCH2-
CH2O–), 4.31–4.21 (m, 2H, –OCH2CH2O–), 2.68–2.87 (m, 4H,
–SCH2CH2O–), 1.56–1.67 (m, 1H, –CHSCH2CH2O–), 4.17–
4.05 (m, 2H, –CH2CH2CH2O–), 3.76–3.53 (m, nH, OCH2CH2O
of MPEG), 3.38 (s, 3H, CH3O of MPEG), 2.96–2.79 (m, 2H,
–SCH2CH2COO–), 2.73–2.61 (m, 2H, –SCH2CH2COO–),
1.96–1.81 (m, 2H, –CH2CH2CH2O–), 1.57–1.34 (m, 4H,
–CH2CH2CH2O–).
Synthesis of MPEG114-b-PaBrCL19-aldehyde17/
p-MA Prodrug
The Schiff-base was prepared according to a reported proce-
dure.[36] Typically, in a 100 mL flask, MPEG114-b-PaBrCL19-
aldehyde17 (0.75 g, 0.065 mmol) and p-MA (0.40 g, 3.2 mmol)
were dissolved in 10 mL of DMSO. The reaction mixture was
heated to 408C and stirred at that temperature for 4 h. The crude
product was washed with diethyl ether and methanol, and dried
over anhydrous MgSO4. The solution was condensed and
precipitated from an ice-cooled diethyl ether. The product was
dried under vacuum (obtained: 0.65 g, yield: 81 %). dH
(400 MHz, CDCl3) 8.39 (s, 1H, –CH¼N), 8.01 (m, 2H, C6H5),
7.41 (m, 2H, C6H5), 6.79–6.73 (m, 2H, C6H5), 6.67–6.64 (m,
2H, C6H5), 4.48–4.43 (m, 2H, –OCH2CH2O–), 4.22–4.11 (m,
nH, –OCH2CH2O– and –CH2CH2CH2O–), 3.64 (s, 3H,
–OCH3), 3.55–3.46 (m, nH, OCH2CH2O of MPEG), 3.37 (s,
3H, CH3O of MPEG), 3.03–2.51 (m, 4H, –SCH2CH2COO–),
1.89–1.81 (m, 2H, –CH2CH2CH2O–), 1.51–1.21 (m, 4H,
–CH2CH2CH2O–).
Synthesis of 2-(4-Formylphenoxy)ethyl
3-Mercaptopropanoate
In a dry 50 mL one-neck round bottom flask, 4-(2-
hydroxyethoxy)benzaldehyde 2.00 g (0.01 mol) and 3-mercapto-
propanoic acid 1.40 g (0.013 mol) were dissolved in 20 mL of
CH2Cl2. A catalytic amount of 4-(N,N-dimethylamino)-pyridine
(DMAP) and DCC (2.99 g, 0.015 mol) were added under nitro-
gen atmosphere in an ice/H2O bath. The solution mixture was
stirred at room temperature and TLC was used to monitor the
reaction progress. The mixture was filtered at high reaction
conversion. The filtrate was concentrated and the product was
purified using silica gel chromatography with 1/2 (v/v) ethyl
acetate/petroleum ether as an eluent. A colourless oily liquid
was obtained (2.29 g, yield: 75.1 %). dH (400 MHz, CDCl3) 9.9
(s, 1H, –CH¼O), 7.86–7.84 (m, 2H, C6H5), 7.05–7.01 (m, 2H,
C6H5), 4.52–4.48 (m, 2H, –OCH2CH2O–), 4.28–4.26 (m, 2H,
–OCH2CH2O–), 2.68–2.87 (m, 4H, HSCH2CH2COO–),
1.56–1.67 (m, 1H, HSCH2CH2COO–).
Preparation of the MPEG114-b-PaBrCL19-aldehyde17/
p-MA Prodrug Micelle
Polymicelles were prepared as follows: 0.1 g of polymer
conjugate was dissolved in 10 mL of THF in a 100 mL volumet-
ric flask and 40 mL of doubly distilled water was added with
gentle agitation, followed by removal of THF with a water
aspirator at 258C for 4 h. The size and size distribution were
determined by DLS measurements at a polymer concentration of
Synthesis of MPEG-b-PaBrCL
A typical synthetic procedure of MPEG-b-PaBrCL is
described as follows: To a flame-dried one-neck 25 mL flask
fitted with a magnetic stirring bar, 0.97 g (5 mmol) of dried
aBrCL (1.0 g, 0.2 mmol), dried MPEG (Mn 5000), and solution
of 25.5 mg of SnOct2 in 17 mL of dried toluene were added
under nitrogen atmosphere. After stirring at 1208C for 48 h, the
solution was concentrated under vacuum, followed by precipi-
tating from diethyl ether and dried under vacuum (obtained:
1.12 g, yield: 70.2 %). dH (400 MHz, CDCl3) 4.19–4.31 (m, 3H,
–CHBr– and –CH2CH2CH2O–), 3.76–3.53 (m, nH, OCH2CH2O
of MPEG), 3.38 (s, 3H, CH3O of MPEG), 2.19–1.97 (m, 2H,
–CH2CHBr–), 1.81–1.78 (m, 2H, –CH2CH2CH2O–), 1.46–1.51
(m, 2H, –CH2CH2CH2O–).
0.1 mg mLꢀ1
.
Formation of the micelles was confirmed using fluores-
cence technology, with pyrene as a probe.[37] A predeter-
mined amount of pyrene solution in acetone (0.1 mg Lꢀ1) was
added into a series of volumetric flasks, and the acetone was
evaporated completely. The solid pyrene left in each flask
was in such an amount that when the flask was filled with a
solution to the calibration mark, the pyrene concentration in
the final solution was 5 ꢁ 10ꢀ7 M. The micelle solution and
doubly distilled water were added consecutively into the
volumetric flasks containing the solid pyrene. A series of
copolymer concentrations from 10ꢀ6 to 1 g Lꢀ1 were achieved
by predetermining the volume of the copolymer solution. The
pyrene and polymer in the doubly distilled water were mixed
and incubated at room temperature for 24 h before measure-
ment. Excitation spectra were obtained using a fluorescence
spectrophotometer (Perkin LS 50B) with the detection wave-
length lem being set at 390 nm. The spectra were recorded
with a scan rate of 500 nm minꢀ1. The excitation spectra
showed a shift from 332.8 to 338 nm, indicating the incorpo-
ration of pyrene into the hydrophobic core of the micelle core.
Aldehyde-Functionalisation of MPEG-b-PaBrCL by a
Thio-Bromo ‘Click’ Reaction
In a dried and nitrogen-purged 25 mL flask, 1.00 g
(0.12 mmol) MPEG114-PaBrCL19 was dissolved in 5 mL of
acetonitrile, followed by adding 0.91 g (3.6 mmol) of 2-(4-
formylphenoxy) ethyl-3- mercaptopropanoate under a nitrogen
stream. Triethylamine (0.58 mL) was added dropwise to the
mixture using syringe over 2 min at room temperature under
nitrogen atmosphere. After stirring for 2 h, the reaction mixture
was filtered, concentrated, and dialysed against distilled water
for 48 h. The product was precipitated from diethyl ether and
dried under vacuum (obtained: 0.75 g, yield: 60.3 %). dH
(400 MHz, CDCl3) 9.9 (s, 1H, –CH¼O), 7.86–7.84 (m, 2H,
The intensity ratio at wavelengths of 338 and 333 nm (I338
I333) from the excitation spectra were analysed as a function
/
of the logarithm of polymer concentration.

Amphiphilic Block Copolymer Bearing Pendant Aldehyde Groups
1583
In Vitro Release Studies
[13] Y. Liu, J. Liu, J. Xu, S. Feng, T. P. Davis, Aust. J. Chem. 2010, 63,
1413. doi:10.1071/CH10168
The pH-dependent release behaviours of p-MA were studied
at five different pH values. Prodrug solutions (12.5 mL,
1 mg mLꢀ1 in double-distilled water) were divided into five
2.5 mL samples. Each sample was then transferred into dialysis
membrances (molecular weight cutoff of 5 kDa) and subse-
quently dialysed in four 100 mL flasks containing 50 mL of
phosphate buffer at pH 7.4, 6.5, 5.0 and 3.6 at 378C. At pre-
determined time points, 1 mL of solution was collected from
the flask and the amount of released drug was determined by
UV–vis spectra. The volume of the release medium in the flask
was kept constant by adding 1 mL of fresh medium after each
sampling. The results were expressed as the percentage of
p-MA released versus time.
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Financial support was provided by the National Natural Science Foundation
of China (NSFC51273169).
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