Synthesis and characterization of a new polymer-drug conjugate with pH-induced activity

Article abstract of DOI:10.1016/j.polymer.2012.06.006

A well-defined, stimuli-responsive tetrapolymer with pH-responsive characteristics and targeting specificity has been synthesized by radical copolymerization of methacrylic acid, N-(2-hydroxypropyl)methacrylamide, methacryloyl glycylglycyl sulfamethoxazol

Full text of DOI:10.1016/j.polymer.2012.06.006

Polymer 53 (2012) 3498e3507
Contents lists available at SciVerse ScienceDirect
Polymer
journal homepage: www.elsevier.com/locate/polymer
Synthesis and characterization of a new polymeredrug conjugate with
pH-induced activity
1
*
Hui-Ping Chang , Jian-Ying Chen, Pei-Shan Zhong, Yung-Hsien Chang, Mong Liang
Department of Applied Chemistry, National Chiayi University, Chiayi 60004, Taiwan, Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A well-defined, stimuli-responsive tetrapolymer with pH-responsive characteristics and targeting speci-
ficity has been synthesized by radical copolymerization of methacrylic acid, N-(2-hydroxypropyl)meth-
acrylamide, methacryloyl glycylglycyl sulfamethoxazole, and N-(methacryloyl)glycylglycine 4-nitrophenyl
ester. The structure and properties of tetrapolymer were investigated by NMR, FT-IR, UVevisible
absorption, TEM and gel permeation chromatography. Incorporation of maleimide linker into tetra-
polymer facilitates its conjugation with antibody fragments, as demonstrated by the solid-phase immu-
noassayexperiments. The TEM image shows that tetrapolymer had self-assembled a spherical micelle with
a diameter ranging from 50 to 150 nm. Altering the pH of the solution leads to a different extent of
aggregation at pH 6.5e3.5, responding in accordance with the properties associated with the extracellular
environment of solid tumors and endocytosis. Furthermore, fluorescence spectroscopy indicated a critical
micelle concentration (CMC) of 1 mg/mL. Because of the solvation and ionization effects, the tetrapolymer
showed considerably enhanced antibacterial activities against Escherichia coli in the presence of DMSO and
the antibacterial activity increased with decreasing pH value.
Received 17 April 2012
Received in revised form
26 May 2012
Accepted 4 June 2012
Available online 13 June 2012
Keywords:
Polymeredrug
Tumor-specific targeting
Stimuli-responsive
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
several site-specific drug delivery systems have been designed.
Here, the antibody acts as a homing device by attaching it to the
The application of water-soluble polymeredrug conjugate for
anti-cancer drug delivery represents a promising approach to
cancer therapy. The main benefits of using these macromolecular
therapeutics include: (1) improved drug accumulation in tumor
cells because of the enhanced permeability and retention (EPR)
effect [1]; (2) lower drug doses, thereby reducing non-specific
toxicity to healthy organs [2]; and (3) contributing to the devel-
opment of a site-specific drug that targets the tumor site to
maximize the efficiency and allows the creation of a programmed
profile of drug release [3]. Polymeredrug conjugate generally
consists of a water-soluble polymer containing drug molecules and
bioactive moieties in its side chains to ensure not only the site-
specific targeting of the pathological area, but also the modula-
tion of pharmacokinetics of the drug carrier. Significant progress
has been made in the design and synthesis of various
polymeredrug conjugates [4e10]. Some studies have focused on
designing macromolecular drug delivery systems by using the EPR
effect for tumor-seeking anti-cancer agents [11e14]. However, in
cases where the EPR effect is not operative (e.g., smaller tumors),
reactive polymer precursors [15e18]. Alternatively, it can be
modified with polymerizable groups, and then undergoes copo-
lymerization with water-soluble comonomers [19,20]. The use of
such targeting moiety not only reduces adverse side effects by
allowing the drug to be delivered to the specific site of action, but
also facilitates cellular uptake of the drug by receptor-mediated
endocytosis. Active targeting ligands such as monoclonal anti-
bodies [21,22], folates [23,24], transferrin [25,26], and luteinizing
hormone-releasing hormone [27] have shown to be effective in
delivering drugs to tumor cells. However, because of the absence
of a substantial improvement in clinical applications, a more effi-
cient strategy for designing anti-cancer drugs that exhibit a high
selectivity to tumor tissues must be developed. It is thought that
a combination of both active and passive targeting ligands may
produce synergistic effects and has higher efficiency than each
targeting ligands separately. By using a local stimuli characteristic
of the pathological site, an additional selectivity and drug accu-
mulation could be gained if these functional moieties were to be
incorporated into the drug carrier. Temperature and pH conditions
are 2 most commonly used stimuli for this purpose. Compared
with the normal physiological pH level of 7.4, the extracellular
environment of solid tumors is slightly acidic, with a pH level of
6.8 dropping between 5 and 6 in endosomes or between 4 and 5 in
* Corresponding author. Fax: þ886 52717901.
E-mail address: mliang@mail.ncyu.edu.tw (M. Liang).
Current address: St. Martin De Porres Hospital, Chiayi, Taiwan.
1
0032-3861/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.polymer.2012.06.006

H.-P. Chang et al. / Polymer 53 (2012) 3498e3507
3499
lysosomes after endocytosis [28,29]. TAT peptide-mediated poly-
meric micelles, which target tumor areas and are able to inter-
nalize into cells, were applied in the studies on the design of a pH-
sensitive drug carrier in response to the change of external pH
[30e34]. It has also been reported that drugs bound to a thiolated
protein carrier or serum albumin through acid-sensitive linkers
exhibited greater anti-cancer activity in animal tumor models than
in free drugs [35e38]. Despite extensive research efforts, linear,
drug-loaded, antibody-bearing and pH-responsive polymer
conjugates remain a great challenge to synthetic chemists and may
have potential applications in biomedicine.
dropped onto the carbon-coated copper grids and then allowed to
dry in air at room temperature before observation. Fluorescence
measurements were performed on a Shimatsu F-7000 FL Spec-
trophotometer. Ultraviolet (UV) spectroscopy and optical trans-
mittance of the aqueous polymer solutions (1 mg/mL) were
recorded by JASCO V-630 spectrophotometer. The pH of the test
solutions was controlled by adding tris(hydroxymethyl)amino-
methane or phosphoric acid from 9 to 3.
2.3. Synthesis
The aim of this study is to synthesize a novel polymeredrug
conjugate that would have both pH-responsive and bio-selective
sensors for drug delivery systems. For this purpose, we conju-
gated sulfamethoxazole (SMX) as a model drug to the polymer side
chain, testing the antimicrobial activity against Escherichia coli
(E. coli). The drug was chosen not only for its nucleophilic reactivity
toward the monomer containing 4-nitrophenoxy reactive groups,
but also for its characteristic absorption peaks of oxazole in 1H NMR
spectroscopy, which allowed a precise determination of molar
composition of the drug. It is assumed that a number of potential,
primary amine-containing antitumor drugs (e.g., doxorubicin, cla-
dribine, idarubicin, epirubicin, and gemcitabine) could also be
attached to the polymer backbone in a similar manner, increasing
tumor-targeting capabilities when combined with relevant cancer
cells.
2.3.1. Synthesis of N-methacryloyl glycylglycyl sulfamethoxazole
(MA-GG-SMX)
N-methacryloylglycylglycine 4-nitrophenyl ester (MA-GG-ONp)
was synthesized according to a literature protocol by the reaction of
methacryloyl chloride with glycylglycine followed by esterification
with p-nitrophenol in the presence of DCC [42]. Then, to a stirred
solution of MA-GG-ONp (0.64 g, 2.0 mmol) in anhydrous DMF (4 mL)
under nitrogen, sulfamethoxazole (SMX) (0.56 g, 2.2 mmol) was
added. The reaction mixture was heated to 110 ꢁC for 24 h. The
solvent was removed under high vacuum and the resulting residue
was purified by dissolving in a minimum amount of acetone and re-
precipitated in ethyl ether solution. The product was collected as
a brown powder and dried under vacuum for overnight (0.52 g,
yield: 60%). 1H NMR (DMSO-d6)
d: 1.86 (s, 3H, CH2]CH(CH3)e), 2.28
(s, 3H, eCH3 of oxazole unit), 3.76 (d, 2H, eNHeCH2eCONHRe,
J ¼ 6.0 Hz), 3.92 (d, 2H, eCONHeCH2eCONHAre, J ¼ 5.7 Hz), 5.36
(s, 1H, trans CH2]C(CH3)e), 5.74 (s, 1H, cis CH2]C(CH3)e), 6.09 (s,
1H, eH of oxazole ring), 7.78 (s, 4H, AreH), 8.27 (m, eNH), 10.19 (s,
2. Experimental
2.1. Materials
eNH). 13C NMR (DMSO-d6)
d
: 11.82,18.47, 42.41, 43.00, 94.99,119.13,
120.11, 127.93, 133.21, 139.46, 142.78, 157.25, 167.90, 168.49, 169.47,
170.25, 171.22. IR (KBr)
(cmꢀ1): 3358 (NeH), 3287 (NeH), 3115
2,20-Azobis-isobutyronitrile (AIBN, Showa) was recrystallized in
ethanol before use. Methacrylic acid (MAA, Showa) was purified by
distillation and stored under N2 prior to use. Tetrahydrofuran
(THF), ethyl ether (Et2O) were purchased from J. T. Baker. N-(2-
Hydroxypropyl)methacrylamide (HPMA) was prepared according
to a known procedure [39]. Methacryloyl chloride was obtained
from TCI. Glycylglycine and di-tert-butyl dicarbonate ((Boc)2O)
were purchased from Alfa Aesar. p-Nitrophenol, 1,4-
diaminobutane and trifluroacetic acid (TFA) were purchased
from Acros. N,N0-Dicyclohexylcarbodiimide (DCC) and sulfame-
thoxazole (SMX) were purchased from Fluka. 3,30,5,50-Tetrame-
thylbenzidine (TMB), albumin from bovine serum (BSA) were
purchased from Sigma. Luria-Bertani (LB) broth was obtained from
Lab M Limited. Pan CEA (H-8): sc-48364 mouse monoclonal IgG1
and goat anti-mouse IgG1-HRP (sc-2060) was obtained from Santa
Cruz Biotechnology. E. coli (DH5) was purchased from GeneMark,
Taicuung, Taiwan.
n
(CeH), 3071 (CeH), 2984 (CeH), 2925 (CeH),1718 (C]O),1674 (C]
O), 1648 (C]O), 1529 (C]C), 1324 (S]O asym), 1160 (S]O sym).
Elemental analysis: calcd for C18H21N5O6S: C 49.60, H 4.80, N 16.00, S
7.30; found: C 49.07, H 5.00, N 15.43, S 7.08.
2.3.2. Synthesis of poly(MAA-co-HPMA-co-MA-GG-SMX-co-MA-
GG-ONp) (1)
To a mixture of 0.045 g of HPMA (0.32 mmol), 0.05 g of MA-GG-
ONp (0.16 mmol), 0.068 g of MA-GG-SMX (0.16 mmol) and 0.014 g
of AIBN (0.085 mmol) under nitrogen was added a solution of MAA
(0.22 g, 2.56 mmol) in anhydrous acetone (3 mL) via cannula. The
solution was heated under nitrogen at 50 ꢁC with UV irradiation
(mercury lamp, 100 W) for 24 h. After polymerization, the polymer
was filtered and purified by dissolving in a minimum amount of
methanol and re-precipitated in a 10-fold excess of Et2O solution.
The polymer was collected by filtration, washed with Et2O and
dried under vacuum for overnight (0.29 g, yield: 77%). 1H NMR
2.2. Characterization
(DMSO-d6) d: 0.53e1.33 (m, CH3e of polymer backbone and
HPMA), 1.70 (bs, eCH2e of polymer backbone), 2.25 (s, CH3- of
oxazole), 2.88 (m, eNHeCH2e of HPMA), 3.61 (m,
eCH2eCH(CH)3OH of HPMA and eNHeCH2eCONHCH2e of MA-
GG-SMX), 3.90 (bs, eNHeCH2eCONHeAr of MA-GG-SMX and
eNHeCH2eCONHCH2e of MA-GG-ONp), 4.07 (bs, eNH-
CH2eCOOAr of MA-GG-ONp group), 4.69 (bs, OH of HPMA), 6.08
(s, eH of oxazole ring), 7.40 (d, ONpeH, J ¼ 6.9 Hz), 7.78 (s, AreH of
MA-GG-SMX), 8.28 (d, ONpeH, J ¼ 6.9 Hz), 8.39 (m, NHe), 10.19 (bs,
1H spectra were recorded on a 300 MHz VarianeMercuryþ300
spectrometer using deuterated solvent. FT-IR spectra were
measured using
a Shimadzu 8400 spectrophotometer. Gel
permeation chromatography (GPC) was carried out using
a DMF eluent that contained 0.05 mol Lꢀ1 LiBr at 80 ꢁC at a flow
rate of 0.8 mL/minꢀ1
.
Narrowly distributed poly(methyl
methacrylate) standards in the molecular weight range of
2500e520,000 g molꢀ1 (Polymer Standards Service, USA) were
utilized for calibration. Prior to GPC analysis, the polymers were
modified by methylation of the carboxylic acid groups using tri-
methylsilyldiazomethane in DMF [40,41]. TEM measurements
were carried out on a JEOL JEM-2100 electron microscope oper-
ated at an acceleration voltage of 100 kV. Polymer 1 solutions
(1 mg/mL) containing 0.05 wt.% phosphotungstic acid were
eNH), 11.33 (bs, eNH), 12.34 (bs, eCOOH). 13C NMR (DMSO-d6)
d:
11.82, 15.73e18.66, 21.99, 42.31, 42.41, 44.07, 47.40, 50.33e55.61,
65.02, 95.68, 119.04, 123.23, 125.23, 127.83, 133.40, 180.02, 142.88,
145.22, 155.21, 157.84, 170.05, 171.23, 177.29. IR (KBr) n
(cmꢀ1): 3410
(NeH), 2993, 2936 (CeH), 1713 (C]O), 1644 (C]O), 1532 (C]C),
1498 (N]O asym), 1382 (S]O asym), 1342 (N]O sym), 1257
(CeO), 1166 (CeO).

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H.-P. Chang et al. / Polymer 53 (2012) 3498e3507
2.3.3. Synthesis of N-(4-tert-butoxycarbonyl-aminobutyl)
maleimide (6)
(NeH), 2998, 2979 (CeH), 1708 (C]O), 1646 (C]O), 1542 (C]C),
1382 (S]O), 1254 (CeO), 1175 (CeO).
N-Boc protection of 1,4-diaminobutane was carried out with
(Boc)2O according to the literature [43]. N-Boc-protected 1,4-
diaminobutane (4) (2.37 g, 12 mmol) was added to a solution of
maleic anhydride (1.38 g, 14 mmol) in acetone (20 mL) and the
reaction mixture was stirred at 5 ꢁC for 1 h. The mixture was dried
under reduced pressure and re-dissolved in 20 mL of ethyl acetate.
The organic layer was extracted with three portions of water
(20 mL), dried over anhydrous magnesium sulfate and concen-
trated under reduced pressure to give crude N-(4-tert-butox-
ycarbonyl-aminobutyl)maleamic acid (5) (2.99 g, yield: 82%). The
resulting maleamic acid product (1.23 g, 4.3 mmol) was then heated
with sodium acetate (0.37 g, 4 mmol) in acetic anhydride (3.7 mL,
36 mmol) at 100 ꢁC for 1 h. The mixture was cooled to room
temperature, washed with excess water to remove most acetic
anhydride and the resulting brown oil residue was dissolved in
30 mL of ethyl acetate. The solution was extracted twice with equal
volumes of saturated NaHCO3 solution and water until the solution
was neutral. The organic layer was dried over anhydrous magne-
sium sulfate and concentrated under reduced pressure to leave the
residue, which was further purified by flash chromatography
(silica gel, 90:10 CH2Cl2/EtOAc) to afford the white product 6
2.4. Determination of critical micelle concentration (CMC) of
Polymer 1
4 mL of pyrene stock solution in acetone (1.26 mM) was added
to each of a series of 10-mL volumetric flasks and the acetone
evaporated. To each flask was then added 10 mL of Polymer 1
solution with various concentration (0.001 mg/mLe2.0 mg/mL).
Fluorescence spectra were obtained from 360 to 460 nm
following excitation at wavelength of 339 nm. The intensities
of excitation peaks at 373 nm and 393 nm were recorded
and the ratios were plotted as the function of polymer
concentrations.
2.5. Antimicrobial activity test
The antimicrobial activity of Polymer 1 was evaluated against
E. coil (DH5) using LB broth solutions. Prior to the test, Polymer 1
(0.2 mg) was dissolved with 200
mL of LB broth either in the exis-
tence or absence of 4 L DMSO at different starting pH values
m
(4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 and 8) to reach a final concentration of
(0.42 g, yield: 37%). 1H NMR (CDCl3)
d
: 1.43 (s, 9H, eCOOC(CH3)3),
0.1% (w/v).
1.51e1.62 (m, 4H, eNHCH2e(CH2)2eCH2NHe), 3.11 (m, 2H,
eCH2eCH2eNHCOOBu), 3.54 (t, 2H, eCH2eCH2emaleimide,
J ¼ 7.05 Hz), 4.50 (bs, eNH), 6.66 (s, 2H, eCH]CHe). 13C NMR
The bacteria suspension (5
m
L, 4 ꢂ 104 CFU/mL of E. coli) was
then added in triplicate into each corresponding media in 96-
well culture plates and incubated at 37 ꢁC, 160 rpm for 18 h.
Microbial growth was measured as an increase of optical density
at 630 nm (OD630) by a microplate reader after incubation and
the results of antimicrobial effects were expressed by optical
density ratio (treated sample/control sample). The low value of
the OD630 ratio represents the high level of the antimicrobial
efficiency.
(CDCl3)
170.87.
d: 25.75, 26.98, 28.32, 37.22, 39.60, 79.34, 134.32, 155.83,
2.3.4. Synthesis of N-(4-aminobutyl)maleimide(AMI) (7)
Trifluoroacetic acid (1.0 mL, 13 mmol) was added to a stirred
solution of compound 6 (0.44 g, 1.6 mmol) in anisole (0.2 mL) at
room temperature. After stirring for 1 h, the solvent was evaporated
under reduced pressure and the resulting residue was dissolved in
a minimum amount of ethyl acetate. The solution was added to
a 10-fold excess of n-hexanes and the product was isolated as
a white powder by filtration, rinsed with n-hexanes and vacuum-
2.6. Preparation of Fab0 and F(ab0)2 fragments
The antibody Fab0 and F(ab0)2 fragments were prepared freshly
using the Pierce Mouse IgG1 Fab0 and F(ab0)2 Micro Preparation
Kit (Thermo Fisher Scientific Inc., Rockford, IL, USA) following the
instructions provided with the Kit. Pan CEA (H-8): sc-48364
mouse monoclonal IgG1 antibody in digestion buffer was mixed
with immobilized ficin for 24 h at 37 ꢁC to give F(ab0)2 and the
undigested IgG was removed on an immobilized Protein A
column. The F(ab0)2 was further reduced to Fab0 with 20 mM
cysteine (Sigma) in 20 mM TriseHCl buffer (pH 8.5) for 1 h at
37 ꢁC. The collected fractions containing Fab0 fragments were
passed through the column (Amicon Ultra-4) using PBS buffer and
stored at 4 ꢁC.
dried for overnight (0.26 g, yield: 96%). 1H NMR (D2O)
d: 1.48 (m,
4H, NH2CH2e(CH2)2eCH2eimide), 2.84 (t, 2H, NH2eCH2eCH2e,
J ¼ 7.05 Hz), 3.38 (t, 2H, eCH2eCH2eimide, J ¼ 7.05 Hz), 6.68 (s,
2H, eCH]CHe). 13C NMR (DMSO-d6)
d: 24.33, 24.92, 36.65, 38.40,
134.09, 171.03.
2.3.5. Synthesis of poly(MAA-co-HPMA-co-ma-GG-SMX-co-MA-
GG-AMI) (2)
The AMI (0.01 g, 0.06 mmol) and Polymer 1 (0.10 g) were dis-
solved in 1.0 mL of anhydrous DMSO and heated to 80 ꢁC under
nitrogen for 24 h. The reaction mixture was added to a 10 mL
portion of acetone and the polymer was filtered, purified by dis-
solving in a minimum amount of methanol and re-precipitated into
a 10-fold excess of Et2O solution. The product was isolated as
a light-yellow powder by filtration, rinsed with Et2O and vacuum-
2.7. Solid-phase immunoassay
Nunc F96 MicroWell Plates (Thermo Fisher Scientific Inc.,
Rockford, IL, USA) were coated with 100
m
L Polymer 2 (3 wt % in
dried for overnight (0.06 g, 60%). 1H NMR (DMSO-d6):
d: 0.53e1.31
phosphate-buffered saline, PBS) for 4 days at 25 ꢁC. The arrays were
(m, CH3e of polymer backbone and HPMA),1.33e1.70 (m, eCH2e of
polymer backbone and AMI linker), 2.25 (s, CH3e of oxazole),
2.88e2.89 (m, eNHeCH2e of HPMA and eCONHeCH2eCH2e of
AMI linker), 3.61e3.90 (m, eCH2eCH(CH)3OH of HPMA and
eNHeCH2eCONHCH2e of MA-GG-SMX and AMI groups), 4.69 (bs,
OH of HPMA), 6.08 (s, eH of oxazole ring), 6.96 (s, eCH]CHe), 7.36
(m, eNH), 7.78 (s, AreH of MA-GG-SMX), 8.39 (m, eNH), 10.19 (bs,
rinsed with PBS and then, 50 m
L of Fab, F(ab0)2 and BSA were added
in triplicate to each well and incubated for 2 h in the dark at 25 ꢁC.
The mean background reactions were evaluated by the BSA test
samples. After washing three times with PBS, each well was
blocked with 50
blocking solution was removed and the plate was washed succes-
sively with PBS and subsequently, 50 L of HRP-conjugated anti-
mL of BSA for 1 h at room temperature. The
m
eNH), 12.34 (bs, eCOOH). 13C NMR (DMSO-d6)
d
:
11.82,
mouse IgG1 (diluted 1:1000 in PBS) was added. After incubation for
1 h at room temperature, the plate was washed three times with
15.73e18.66, 21.99, 25.80, 26.38, 36.84, 38.22, 42.31, 42.41, 44.07,
47.40, 50.33e55.61, 65.02, 95.68, 119.04, 127.83, 133.40, 142.88,
157.84, 170.05, 171.23, 177.29, 180.02. IR (KBr) e (cmꢀ1): 3406
PBS buffer and 50
mL/well TMB substrate was added. Then 2 N HCl
(50 L) was added to each well and the chromogenic reactions were
m

H.-P. Chang et al. / Polymer 53 (2012) 3498e3507
3501
measured on a microplate reader (PowerWave 340, BioTek) at
450 nm.
protons of oxazole, respectively. The 13C NMR spectrum also
confirmed all carbon resonances of each monomer. Four aromatic
protons of MA-GG-SMX appeared as a singlet at 7.78 ppm
(denoted as i and j in Fig. 1), whereas 2 doublet peaks of
3. Results and discussion
p-nitrophenol (
d
¼ 7.42 and 8.28) were observed on MA-GG-ONp.
These differences, in addition to the characteristic resonances of
oxazole, were employed to estimate the relative molar concen-
tration of each component.
3.1. Preparation and characterization
3.1.1. Monomers and Polymer 1
Fig. 2 shows the representative 1H NMR spectra and the
assignment of the resonances of Polymer 1. The broad resonance
peak at 2.7e3.0 ppm (denoted as g) was attributed to the methy-
lene protons of HPMA and applied in the molar composition
analysis. Subsequently, the molar ratios of each component
(denoted as m, n, o, and p) were calculated by comparing the
The tetrapolymer poly(MAA-co-HPMA-co-MA-GG-SMX-co-
MA-GG-ONp) (1) was synthesized in good yield by radical
copolymerization of MAA and HPMA with a polymerizable drug
derivative MA-GG-SMX and a reactive monomer MA-GG-ONp,
as shown in Scheme 1. The MAA segment in the drug carrier
was selected for its ability to respond to pathologic changes
of pH and to precipitate in the acidic environment. The HPMA
segment was chosen for its non-toxic, biocompatible and
hydrophilic nature that can enhance the water solubility of
hydrophobic drugs.
average integrals at
(denoted as g, v and c0, respectively), with the peak area of methyl
protons at
d
¼ 2.70e3.10 ppm, 2.25 ppm, and 7.42 ppm
d
¼ 0.53e1.33 ppm (denoted as a, d, i, k and w, respec-
tively). The equation is displayed as follows:
MA-GG-ONp was prepared by the reaction of glycylglycine
with methacylchloride in an aqueous alkaline solution, followed
by esterification with p-nitrophenol in the presence of DCC.
Scheme 2 shows how the treatment of the ONp ester with SMX in
DMF provided the methacryloylglycylglycine derived drug
monomer MA-GG-SMX in a 60% yield. The formation of MA-GG-
ONp and MA-GG-SMX was confirmed by NMR spectroscopy, as
shown in Fig. 1. The 1H NMR spectrum of MA-GG-SMX showed 2
n ¼ ðg=2Þ=f½ða þ d þ i þ k þ wÞ ꢀ ððg=2Þ*3Þꢃ=3g
o ¼ ðv=3Þ=f½ða þ d þ i þ k þ wÞ ꢀ ððg=2Þ*3Þꢃ=3g
p ¼ ðc0=2Þ=f½ða þ d þ i þ k þ wÞ ꢀ ððg=2Þ*3Þꢃ=3g
sharp singlet signals at
d
¼ 6.09 ppm and
d
¼ 2.28 ppm (denoted
as l and m in Fig. 1) corresponding to the vinyl and methyl
Scheme 1. Synthetic route for the preparation of polymeredrug conjugate.

3502
H.-P. Chang et al. / Polymer 53 (2012) 3498e3507
the eONp group. A representative 1H NMR spectrum of Polymer 2
m ¼ 1 ꢀ n ꢀ o ꢀ p
is shown in Fig. 3. For Polymer 2, the 1H NMR spectrum exhibits
a singlet at
d
¼ 6.96 ppm (vinyl protons of maleimide, denoted as h0
in Fig. 3). This, in addition to the concomitant disappearance of the
nitrophenol peaks (denoted as c0 and d0 in Fig. 2), indicated
a successful aminolysis reaction. Based on GPC chromatography
results, Mn was approximately 18,000, with a polydispersity of 2.9.
Presumably, the antibody fragments are capable of conjugating to
the polymer by the Michael addition reaction between a sulfhydryl-
containing molecule, such as cysteine, and maleimide, thereby
forming a thioether bond [44]. In this case, the antibody-mediated
targeting of tumor cells could increase the tumor-specific accu-
mulation of drugs. Furthermore, the subsequent precipitation
caused by pH changes in the endosome or tumor surface could
assist drug accumulation within the tumor cell.
3.2. Stimulus-responsive transition of Polymer 1
The pH-responsive behavior of Polymer 1 can be described as an
ionization level of the polymer in an aqueous solution, resulting in
transmittance changes of the solution. Fig. 4 shows the pH-
dependent turbidity of the micelle solution. When the solution
pH level decreased, the relative transparency of the solution was
reduced by 75% at pH levels of 3.5. The cloud point of the polymer
solution occurred at pH 6.5e3.5, responding in accordance with the
properties associated with the extracellular environment of solid
tumors and endocytosis. At normal physiological or higher pH
values, the carboxylic acid groups of MAA were ionized and
solvated in water. This resulted in the hydrophobic sulfonamide
units being embedded within the microspheres, with a diameter
ranging from 50 to 150 nm, as shown in the TEM image (Fig. 5a).
As the pH decreased, the carboxyl groups of the MAA segments
became protonated and intrapolymer chainechain interactions
dominated via intramolecular hydrogen bonding. As a result, the
micelles started to collapse and partially precipitated into hydro-
phobic polymer particles which led to a reduced transparency of
the solution (Fig. 4) and certain degree of aggregation (Fig. 5b). This
pH-induced phase transition is reversible and may presumably
allow the temporary “shield” for drugs during delivery.
The tetrapolymer was isolated as a light-yellow solid in good
yield in acetone, wherein the approximate molar ratio of m, n, o and
p was 13:2.5:1:1.5 (entry 1, Table 1), respectively. The molar
composition, molecular weight and yield varied slightly when
polymerizations were carried out in DMF at 50 ꢁC (entry 2). The GPC
chromatographs of both polymers showed unimodal peaks of Mn
ranging from 8400 to 15 ,000, with a polydispersity of 1.9e2.5,
revealing
a smooth radical polymerization obtained under
different reaction conditions. Because of the higher yield and
molecular weight, the product from entry 1 was used for subse-
quent reactions.
3.1.2. Linker and Polymer 2
N-[4-aminobutyl]maleimide (AMI) is a thiol-reactive, non-
biodegradable spacer that connects polymers and target ligands,
making the antigen-binding site more approachable in pathological
areas. AMI synthesis is shown in Scheme 3, where the linker length
(Cn) can be modified, if necessary. Initially, the diaminobutane was
monoalkylated with (Boc)2O to yield 5, which was then reacted
with maleic anhydride followed by cyclization with Ac2O/NaOAc
yielding the N-Boc-protected aminomaleimide (6). Removal of the
N-Boc group was achieved by treating with TFA to provide the AMI
(7) in good yield. Then, the linker was incorporated into the side
chain by reacting Polymer 1 with AMI in DMSO with the removal of
The ability of Polymer 1 to form micelles in the aqueous solu-
tion was tested by fluorescence spectroscopy using pyrene as
a probe. The pyrene was mixed with the polymer solutions of
various concentrations, and the emission spectra were recorded.
Fig. 6 displays the intensity ratios of the first to the third vibra-
tional band at 373 nm and 393 nm (I373 and I393), measured as
Scheme 2. Preparation of N-methacryloyl glycylglycyl sulfamethoxazole.

H.-P. Chang et al. / Polymer 53 (2012) 3498e3507
3503
Fig. 1. 1H NMR spectra of MA-GG-ONp and MA-GG-SMX.
Fig. 2. A representative 1H NMR spectra of 1.

3504
H.-P. Chang et al. / Polymer 53 (2012) 3498e3507
Table 1
Reaction conditions and characterization data of Polymer 1.a
PDIb
[h]
Temp (ꢁC)
t (h)
Solvent
yield (%)
b
b
c
Entry
Composition
Mn
Mw
(m:n:o:p)
Mw/Mn
(dL/g)
1
2
13:2.5:1:1.5
17:4:1:1.65
15,085
8411
38,175
15,927
2.5
1.9
0.12
0.05
60
50
24
24
Acetone
DMF
77
50
a
Feed ratio (mol) m:n:o:p ¼ 16:2:1:1.
b
c
Prior to GPC analysis, the polymers were modified by methylation of the carboxylic acid groups using trimethylsilyldiazomethane in DMSO at room temperature.
Intrinsic viscosity ([
]) was measured at 26 ꢁC using Ubbelohde type viscometer in methanol.
h
a function of polymer concentrations. At low polymer concentra-
tions, I373 and I393 had a plateau value of approximately 1.3, indi-
cating the absence of polymeric micelles in the solution [45,46]. At
concentrations above 1 mg/mL, I373 and I393 ratios dropped
sharply, signaling micelle formation and partitioning of pyrene
inside the micelle hydrophobic cores. The critical micelle
concentration (CMC) of 1 mg/mL was estimated after the sharp
drop in I373 to I393 ratios.
The solution turned yellow as the pH value increased because of
the ionization of the sulfonamide group at a pH higher than its pKa
value [47], wherein the deprotonated sulfonamide groups pre-
sented intense charge-transfer absorptions at 400 nm, as shown in
Fig. 7. The UVevis spectra of Polymer 1 were recorded from 230 to
600 nm at different pH values in water. The absorptions relative to
the phenyl ring in SMX were observed at 270 nm at pH 5.5e6.5.
They were reduced with the concomitant appearance of new
absorption bands at 400 nm, when the pH of the solution was
increased or shifted to 312 nm when the pH level decreased. The
development of new bands in the absorption spectra, as well as
the change in color of the solution, indicate different charge-
transfer interactions between the protonated and deprotonated
forms of the sulfonamide group. These results are in accordance
with pKa1 and pKa2 values of sulfonamide derivatives, as reported
by Sanli et al. [48].
3.3. Conjugation of antibody fragments
The conjugation between the antibody fragments and the
polymers is evaluated by the solid-phase immunoassay. Antibody
fragments (such as Fab0 or (Fab0)2) are an attractive alternative to
intact mAbs for antibody attachment because of more efficient
antigen binding and a simpler structure that provides greater
control of the structures of polymer conjugates [49]. The anti-
body fragments Fab0 and (Fab0)2 were prepared from the pan CEA
(H-8) antibody: sc-48364 mouse monoclonal IgG1 antibody
according to supplier’s protocol. This carcinoembryonic antigen is
capable of examining various cancer diseases [50]. Polymers 1
and 2 were pre-coated onto Nunc F96 MicroWell plates. Prior to
use, each well was treated with BSA, and Fab0 and (Fab0)2 frag-
ments for conjugation. The plate was washed thoroughly with
PBS, and then the secondary antibody and chromogenic substrate
were added. The test samples were analyzed on the microplate
reader at 450 nm. For Polymer 2, the optical density values for
the BSA, Fab0 and (Fab0)2 were 0.152, 0.353, and 0.861, respec-
tively, with positive-to-negative (P/N) ratios of 2.32 for Fab0 and
5.37 for (Fab0)2, indicating successful attachment of both antibody
fragments. As a result, the thiol groups of cysteine and primary
amine groups of lysine in antibody fragments both could undergo
Michael addition, forming the polymer bioconjugate. Polymer 1
Scheme 3. Preparation of N-(4-aminobutyl)maleimide (AMI, 7).

H.-P. Chang et al. / Polymer 53 (2012) 3498e3507
3505
Fig. 3. A representative 1H NMR spectra of 2.
was also found to be conjugated to Fab0 and (Fab0)2 with P/N
ratios of 3.12 and 7.23, respectively. This study argues that,
because of excellent reactivity of primary amine groups toward
a nucleophilic attack, antibody fragments could replace eONp
groups.
3.4. Antibacterial activities
The growth inhibiting effect of Polymer 1 and its structural
analog poly(MAA-co-HPMA-co-MA-GG-ONp) against E. coli was
evaluated by the survival ratio in the medium containing the
polymer and E. coli cells. The capability to inhibit the growth of the
tested microorganisms (denoted as OD) at different pH media is
shown in Fig. 8. Compared with the control sample, both polymers
reduced bacterial viability by approximately 10%e20% at a pH level
of 4e8 in LB broth media. Furthermore, both polymers had no
appreciable difference in antibacterial activity. Albeit adding
a small amount of DMSO (2 vol%) not having any major effect on the
activity of the tertpolymer (without drug units), it reduced viability
(50%e60%) of Polymer 1 significantly. These drastic changes in the
antimicrobial activity could be explained by greater solvation
ability, thereby the hydrophobic units and the sulfonamide groups
embedded inside became more exposed to the test bacteria under
culture conditions. Although further investigations are necessary to
elucidate the antibacterial mechanism against E. coli, the intrinsic
activity of the tertpolymer can be partially attributed to the glycine
units, which are known to exert antibacterial effect against E. coli
[51,52].
pH
Growth inhibition was also found to decrease as the pH value
increased. The decreased antimicrobial activity at higher pH levels
could be accounted for by the polymer’s ionization in the basic
Fig. 4. Transmittance changes of Polymer 1 aqueous solution as a function of pH
measured at 600 nm.

3506
H.-P. Chang et al. / Polymer 53 (2012) 3498e3507
Fig. 5. TEM images of Polymer 1 at a polymer concentration of 1 mg/mL in aqueous solution; (a) pH ¼ 7.5, and (b) pH ¼ 5.5.
solution, wherein fully ionized polymers interact less favorably
with the negatively charged E. coli outer membrane [53], thereby
exhibiting the least antimicrobial activity at pH 8. The in vitro
results are promising because they indicate that appropriate
polymer aggregation in the specific situation can increase the
effectiveness of the drug and facilitate its contact with acidic
surfaces. Further studies are in progress for replacing SMX with
other anti-cancer drugs (doxorubicin, epirubicin, and gemcitabine)
and its effects on antitumor activity.
3
2.5
2
1.5
1
0.5
0
Polymer concntration (mg/mL)
Fig. 6. Relation between intensity ratio (I373/I393
(
) of pyrene emission spectra
lex ¼ 339 nm) and Polymer 1 concentration at pH ¼ 7.5, 25 ꢁC.
Fig. 7. UVevis spectra of Polymer 1 at different pH levels.

H.-P. Chang et al. / Polymer 53 (2012) 3498e3507
3507
device. Such conjugates demonstrate the feasibility of combining
pH sensitivity and bio-selectivity that facilitate site-specific accu-
mulation and possess a high potential for designing new multi-
functional antitumor drug delivery systems.
a
Acknowledgment
Financial supports from National Science Council (NSC 99-2113-
M-415-010-MY3) of Taiwan, R.O.C. is gratefully acknowledged.
References
[1] Maeda H, Greish K, Fang J. Adv Polym Sci 2006;193:103e21.
[2] Greish K, Nagamitsu A, Fang J, Maeda H. Bioconjug Chem 2005;16:230e6.
[3] Gabizon AA. Cancer Invest 2001;19:424e36.
[4] Lutz J, Börner HG. Prog Polym Sci 2008;33:1e39.
[5] Minko T. Drug Discov Today Tech 2005;2:15e20.
[6] Li C, Wallace S. Adv Drug Deliv Rev 2008;60:886e98.
[7] Park JH, Lee S, Kim J, Park K, Kim K, Kwon IC. Prog Polym Sci 2008;33:113e37.
[8] Klok HA. J Polym Sci Part A Polym Chem 2005;43:1e17.
[9] Li C. Adv Drug Deliv Rev 2002;54:695e713.
[10] Larson N, Ghandehar H. Chem Mater 2012;24:840e53.
[11] Maeda H, Bharate GY, Daruwalla J. Eur J Pharm Biopharm 2009;71:409e19.
[12] Iyer AK, Khaled G, Fang J, Maeda H. Drug Discov Today 2006;11:812e8.
[13] Duncan R. Nat Rev Drug Discov 2003;2:347e60.
pH
[14] Duncan R. Nat Rev Cancer 2006;6:688e701.
ꢀ
[15] Putnam D, Kopecek J. Adv Polym Sci 1995;122:55e123.
b
ꢀ
ꢀ
[16] Kovár M, Strohalm J, Etrych T, Ulbrich K, Ríhová B. Bioconjug Chem 2002;13:
206e15.
ꢀ
[17] Omelyanenko V, Kopecková P, Gentry C, Shiah JG, Kopecek J. J Drug Target
1996;3:357e73.
ꢀ
ꢀ
ꢀ
ꢀ
[18] Ríhová B, Kopecková P, Strohalm J, Rossmann P, Vetvicka V, Kopecek J. Clin
Immunol Immunopathol 1988;46:100e14.
[19] Auditore-Hargreaves K, Houghton RL, Monji N, Priest JH, Hoffman AS,
Nowinski RC. Clin Chem 1987;33:1509e16.
[20] Monji N, Hoffman AS. Appl Biochem Biotechnol 1987;14:107e19.
[21] Brannon-Peppas L, Blanchette JO. Adv Drug Deliv Rev 2004;56:1649e59.
[22] Kohler G, Milstein C. Nature 1975;256:495e7.
[23] Sudimack J, Lee RJ. Adv Drug Deliv Rev 2000;41:147e62.
[24] Hilgenbrink AR, Low PS. J Pharm Sci 2005;94:2135e46.
[25] Bellocq NC, Pun SH, Jensen GS, Davis ME. Bioconjug Chem 2003;14:1122e32.
[26] Sahoo SK, Labhasetwar V. Mol Pharmacol 2005;2:373e83.
[27] Dharap SS, Qiu B, Williams GC, Sinko P, Stein S, Minko T. J Control Release
2003;91:61e73.
[28] Xu P, Van Kirk EA, Zhan Y, Murdoch WJ, Radosz M, Shen Y. Angew Chem Int
Ed 2007;46:4999e5002.
[29] Ulbrich K, Subr V. Adv Drug Deliv Rev 2004;56:1023e50.
[30] Sethuraman VA, Bae YH. J Control Release 2007;118:216e24.
[31] Lee ES, Na K, Bae YH. Nano Lett 2005;5:325e9.
[32] June RK, Gogoi K, Eguchi A, Cui XS, Dowdy SF. J Am Chem Soc 2010;132:
10680e2.
[33] Wadia JS, Dowdy SF. Adv Drug Deliv Rev 2005;57:579e96.
[34] Vivès E, Schmidt J, Pèlegrin A. Biochim Biophys Acta 2008;1786:126e38.
[35] Kratz F, Beyer U, Roth T, Tarasova N, Collery P, Lechenault F, et al. J Pharm Sci
1998;87:338e46.
pH
Fig. 8. Growth inhibition of E. coli control (A), Polymer 1 (:) and poly(MAA-co-
HPMA-co-MA-GG-ONp) (-) for 18 h at 37 ꢁC, 160 rpm at different pH levels in (a) in LB
broth media and (b) DMSO-mediated LB broth media.
[36] Beyer U, Roth T, Schumacher P, Maier G, Unold A, Frahm AW, et al. Med Chem
1998;41:2701e8.
4. Conclusions
[37] Drevs J, Hofmann I, Marme D, Unger C, Kratz F. Drug Deliv 1999;6:89e95.
[38] Tomlinson R, Heller J, Brocchini S, Duncan R. Bioconjug Chem 2003;14:
1096e106.
This research developed a water-soluble drug delivery system
capable of carrying tumor-specific targeting moieties and activating
drug precipitation processes under acidic conditions. The fluores-
cence spectra and TEM image showed that Polymer 1 self-
assembled to form a spherical micelle with a diameter ranging
from 50 to 150 nm at pH 7.5. Altering the pH of the solution leads to
a different extent of aggregation at pH 6.5e3.5, in accordance with
the properties associated with the extracellular environment of
solid tumors and endocytosis. Because of the solvation and
ꢀ
[39] Kopecek J, Bazilova H. Eur Polym J 1973;9:7e14.
[40] Hashimoto N, Aoyama T, Shioiri T. Chem Pharm Bull 1981;29:1475e8.
[41] Couvreur L, Lefay C, Belleney J, Charleux B, Guerret O, Magnet S. Macromol-
ecules 2003;36:8260e7.
ꢀ
[42] Rejmanova P, Labsky J, Kopecek J. Makromol Chem 1977;178:2159e68.
[43] Krapcho AP, Kuell CS. Synth Commun 1990;20:2559e64.
[44] Smyth DG, Blumenfeld OO, Konigsberg W. Biochem J 1964;91:589e95.
[45] Kalyanasundaram K, Thomas JK. J Am Chem Soc 1977;99:2039e44.
[46] Ringsdorf H, Venzmer J, Winnik FM. Macromolecules 1991;24:1678e86.
[47] Kang SI, Bae YH. J Control Release 2002;80:145e55.
[48] Sanli S, Altun Y, Sanli N, Alsancak G, Beltran JL. J Chem Eng Data 2009;54:
3014e21.
[49] Pedley RB, Boden JA, Boden R, Begent RHJ, Turner A, Haines AMR, et al. Br J
Cancer 1994;70:1126e30.
[50] Hance KW, Zeytin HE, Greiner JW. Mutat Res 2005;576:132e54.
[51] Baumann T, Kämpfer U, Schürch S, Schaller J, Largiadér C, Nentwig W, et al.
Cell Mol Life Sci 2010;67:2787e98.
ionization effects, Polymer
1 showed considerably enhanced
antibacterial activities against E. coli in the presence of DMSO,
and the growth inhibition decreased as the pH value increased.
The chemical structures of Polymers
characterized and both polymers successfully conjugated with
antibody fragments. Theoretically, number of antitumor
1 and 2 are well-
a
[52] Hammes W, Schleifer KH, Kandler OJ. Bacteriol 1973;116:1029e53.
[53] Sonohar R, Muramatsu N, Ohshima H, Kondo T. Biophys Chem 1995;55:
273e7.
polymeredrug conjugates could be altered with the replacement of
different drug molecules and conjugated to relevant homing