Synthesis and characterization of tailorable biodegradable thermoresponsive methacryloylamide polymers based on l-serine and l-threonine alkyl esters

Article abstract of DOI:10.1016/j.polymer.2010.04.010

A series of monomers based on the methyl, ethyl, and isopropyl esters of N^α-(methacryloyl)-serine and -threonine were synthesized, and used in an AIBN-initiated radical polymerization reaction to yield polymers with an M_n ranging between 6.6 and 23.8 kDa. The newly synthesized polymers showed LCST behavior in aqueous solution that could be tailored by subtle variations of the hydrophobicity of the monomers to obtain a broad range of cloud points between 1.5 and >100°C. According to HPLC, the hydrolytic t_1/2-values (pH 7.4 at 37°C) of the monomers were found to be 5, 12, and 40 days of the methyl, ethyl, and isopropyl esters, respectively, while the hydrolysis rate of poly[N^α-(methacryloyl)-Ser-OMe] and poly[N^α-(methacryloyl)-Thr-OMe] was found to be significantly lower compared to the corresponding monomers. In order to obtain thermoresponsive nanoparticles, N^α-(methacryloyl)-Thr-OEt was polymerized with (PEG monomethyl ether 5000)₂-ABCPA as macroinitiator to yield an amphiphilic block co-polymer, poly[N^α-(methacryloyl)-Thr-OEt]-b-(PEG monomethyl ether 5000), which forms particles of 300 nm at a temperature higher than its cloud point of 24°C. Incubation at physiological conditions induced ester hydrolysis resulting in a destabilization of the particles making these particles suitable for drug delivery purposes.

Full text of DOI:10.1016/j.polymer.2010.04.010

Polymer 51 (2010) 2479e2485
Contents lists available at ScienceDirect
Polymer
journal homepage: www.elsevier.com/locate/polymer
Synthesis and characterization of tailorable biodegradable thermoresponsive
methacryloylamide polymers based on
L-serine and
L-threonine alkyl esters
Maarten van Dijk a b, Tobias M. Postma , Dirk T.S. Rijkers , Rob M.J. Liskamp ,
,
a
,
b
b
b
a
a,
*
Cornelus F. van Nostrum , Wim E. Hennink
Division of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University,
PO Box 80082, 3508 TB Utrecht, The Netherlands
a
b
Division of Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Department of Pharmaceutical Sciences,
Faculty of Science, Utrecht University, PO Box 80082, 3508 TB Utrecht, The Netherlands
a r t i c l e i n f o
a b s t r a c t
a
Article history:
Received 20 November 2009
Received in revised form
A series of monomers based on the methyl, ethyl, and isopropyl esters of N -(methacryloyl)-serine and
threonine were synthesized, and used in an AIBN-initiated radical polymerization reaction to yield
polymers with an M ranging between 6.6 and 23.8 kDa. The newly synthesized polymers showed LCST
-
n
4
March 2010
behavior in aqueous solution that could be tailored by subtle variations of the hydrophobicity of the
Accepted 7 April 2010
Available online 20 April 2010
ꢀ
monomers to obtain a broad range of cloud points between 1.5 and >100 C. According to HPLC, the
ꢀ
hydrolytic t1/2-values (pH 7.4 at 37 C) of the monomers were found to be 5, 12, and 40 days of the methyl,
ethyl, and isopropyl esters, respectively, while the hydrolysis rate of poly[N -(methacryloyl)-Ser-OMe]
and poly[N -(methacryloyl)-Thr-OMe] was found to be significantly lower compared to the corre-
sponding monomers. In order to obtain thermoresponsive nanoparticles, N -(methacryloyl)-Thr-OEt was
polymerized with (PEG monomethyl ether 5000) -ABCPA as macroinitiator to yield an amphiphilic block
2
co-polymer, poly[N -(methacryloyl)-Thr-OEt]-b-(PEG monomethyl ether 5000), which forms particles of
00 nm at a temperature higher than its cloud point of 24 C. Incubation at physiological conditions
a
Keywords:
a
Biodegradable
Nanoparticles
Thermosensitivity
a
a
ꢀ
3
induced ester hydrolysis resulting in a destabilization of the particles making these particles suitable for
drug delivery purposes.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
drug. The incorporated biodegradability renders thermoresponsive
polymers suitable for clinical applications.
Thermoresponsive polymers with a lower critical solution
temperature (LCST) are soluble in aqueous solution below a certain
temperature referred to as the cloud point (CP), but precipitate
above this temperature due to dehydration of the polymer chains
One approach to induce biodegradability is the introduction of
ester moieties in the polymeric side chains. Studies by Neradovic
et al. [8,9] have shown that co-polymers consisting of N-isopropyl
acrylamide (NIPAAm) and 2-hydroxyethyl methacryloyl mono-
lactate (HEMA-monolactate) behave as thermoresponsive poly-
mers with a tunable CP. By increasing the HEMA-monolactate
content, the CP decreases, however, upon hydrolysis of the lactate
ester, the CP gradually increases during time [10e12]. Another
approach makes use of amino acid-based N-acryl amide-derived
polymers. The thermoresponsive behavior of such polymers
depends on the hydrophobic/hydrophilic balance of the polymer
and in case of amino acid esters (e.g. alanine [13,14] and proline
moieties [15e17]), hydrolysis of the ester results in partial
biodegradability as well as in a shift of the hydrophobic/hydro-
philic balance and thus in a variable CP during the time of
incubation.
[1]. Thermoresponsive polymers with a CP around physiological
temperature are presently under investigation for biomedical and
pharmaceutical applications and can be used for the design of
thermosensitive drug delivery systems such as polymeric micelles
[2e5] and hydrogels [6,7]. In an ideal polymeric micellar delivery
system, the drug retains entrapped during transport through the
bloodstream, and will be released only after reaching the site of
action. These principally conflicting requirements can be
approached by developing polymeric micelles that gradually
become more hydrophilic resulting into a destabilization of the
micelles and therefore in a controlled release of the entrapped
In the present study it was our aim, to design and synthesize
a
a series of novel thermoresponsive polymers based on N -meth-
*
Corresponding author. Tel.: þ31 30 253 6964; fax: þ31 30 251 7839.
E-mail address: W.E.Hennik@uu.nl (W.E. Hennink).
acryloyl serine and threonine alkyl esters with tailorable CPs and
0
032-3861/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.polymer.2010.04.010

2480
M. van Dijk et al. / Polymer 51 (2010) 2479e2485
ꢀ
degradation kinetics. Subtle variations of the hydrophobicity, either
in the amino acid side chain (serine/threonine) or ester moiety
above 40 C, and as a consequence of this, polymers with a CP
ꢀ
>40 C (as determined in plain water) could not be analyzed.
(
methyl/ethyl/isopropyl), lead to tailorable thermosensitive
behavior. Since ester hydrolysis is strongly dependent on the alkyl
chain, differences in hydrolysis rate of the methyl/ethyl/isopropyl
ester will be used to control the degradation kinetics of the
polymers.
2.2.3. Dynamic light scattering (DLS)
DLS was performed on a Malvern CG-3 multi-angle goniometer
(Malvern Ltd., Malvern, U.K.) equipped with a He-Ne JDS Uniphase
laser (
l
¼ 632.8 nm, 22 mW output power), an optical fiber based
detector, a digital LV/LSE-5003 correlator and a temperature
controller (Julabo waterbath). Time correlation functions were
analyzed using the ALV-60X0 Software V.3.X provided by Malvern.
2
. Experimental
2
.1. Materials and general procedures
Scattering of the micellar dispersions was measured at an angle of
ꢀ
90
in an optical quality 8 mL borosilicate cell. A cell with
0
2
,2 -Azobis(isobutyronitrile) (AIBN) and serine (H-Ser-OH)
approximately 1 mL micellar dispersion (1 mg/mL) was incubated
in the DLS apparatus and measured at regular time intervals
were purchased from Acros Organics. Thionyl chloride, threonine
H-Thr-OH) and methacryloyl chloride were purchased from
ꢀ
(
between 17 and 25 C.
Fluka. Peptide synthesis grade N,N-dimethylformamide (DMF),
MeOH, EtOH, 2-propanol and dioxane were obtained from Bio-
solve. Triethylamine was purchased from Merck. Solvents used for
extractions and column chromatography were distilled prior to
use. Thin layer chromatography (TLC) was performed on Merck
silica gel 60 F-254 plates. Spots were visualized by UV-quenching,
2.2.4. Differential scanning calorimetry (DSC)
DSC was carried out on a Q1000 differential scanning calorim-
eter (TA Instruments). For the DSC measurement, the samples were
ꢀ
ꢀ
heated (10 C/min) from room temperature to 150 C. Then, these
ꢀ
ꢀ
samples were cooled to 10 C and subsequently heated (10 C/min)
0
0
0
ꢀ
ninhydrin, N,N,N ,N -tetramethyl-4,4 -diaminodiphenylmethane
for a second time to 150 C.
ꢀ
1
(
m
TDM)/Cl
m) was used for column chromatography. H NMR spectra were
recorded on a Varian Gemini 300 MHz spectrometer and chemical
shifts are given in ppm ( ) relative to the internal reference tet-
ramethylsilane (TMS) (0.00 ppm). C NMR spectra were recorded
at 75.5 MHz and chemical shifts are given in ppm ( ) relative to
either CDCl (77.0 ppm) or DMSO-d (39.5 ppm). Analytical RP-
HPLC runs were carried out on a Shimadzu automated HPLC
system equipped with a UV/VIS detector operating at
2 4
[18], KMnO . Silicycle silica 60 A (particle size 41e63
2.3. Synthesis
d
2.3.1. Monomer synthesis
2.3.1.1. HCl.H-Ser-OMe (3a). In a typical experiment, MeOH (200
mL) was cooled on ice and SOCl (18 mL, 250 mmol) was added
2
dropwise. After the addition was complete, H-Ser-OH (1) (5.25 g, 50
13
d
3
6
mmol) was added as a single portion and the reaction mixture was
ꢀ
l
¼ 214 and
stirred 1 h at 0 C followed by 16 h at room temperature. Then, the
2
54 nm and using an Alltech Prosphere C18 column (250 ꢁ 4.6
reaction mixture was concentrated to an oil, which was triturated
with ice-cold diethyl ether to give methyl ester 3a as a white solid
ꢀ
mm, particle size: 5
m
m, pore size: 300 A) at a flow rate of 1 mL/
O/
O 95:5 v/
min using a linear gradient of 100% buffer A (0.1% TFA in H
CH CN 95:5 v/v) to 100% buffer B (0.1% TFA in CH CN/H
v) in 20 min.
2
in 84% yield (6.6 g). R
v). H NMR (300 MHz, DMSO-d
f
¼ 0.87 (CHCl
3
d
/MeOH/25% NH
: 3.74 (s, 3H, OCH
CH), 5.64 (broad s,1H, OH), 8.60 (br s, 3H, NH
C NMR (75 MHz, DMSO-d : 53.1, 54.3, 59.8, 168.8.
4
OH 8:4:1.5 v/v/
), 3.83 (s, 2H,
).
1
3
3
2
6
)
3
b
CH
2
), 4.09 (m,1H,
a
3
1
3
6
) d
2.2. Characterization methods
2
.3.1.2. HCl.H-Ser-OEt (3b). R
f
¼ 0.87 (CHCl
3
/MeOH/25% NH
: 1.24 (t (J 7.0 Hz), 3H,
), 4.07 (t (J 3.4 Hz), 1H, CH), 4.20 (m,
), 5.62 (broad s, 1H, OH), 8.55 (broad s, 3H, NH
NMR (75 MHz, DMSO-d : 13.9, 54.3, 59.5, 61.7, 168.0.
4
OH
1
2.2.1. Gel permeation chromatography (GPC)
8:4:1.5 v/v/v). H NMR (300 MHz, DMSO-d
OCH CH ), 3.83 (s, 2H, CH
2H, OCH CH
6
) d
GPC was performed on a Waters 2695 Controller equipped with
a refractive index detector (model 2414). The analyses were run on
2
3
b
2
a
1
3
2
3
3
).
C
a PLgel MIXED-D column (particle size: 5
m
m) (Polymer Laborato-
6
) d
ꢀ
ries) at 40 C using 10 mM LiCl in DMF as the mobile phase at a flow
rate of 0.7 mL/min. The polymers were dissolved in DMF (con-
taining 10 mM LiCl) at a concentration of 5 mg/mL for at least 24 h
2.3.1.3. HCl.H-Ser-OiPr (3c). R
f
¼ 0.89 (CHCl
8:4:1.5 v/v/v). H NMR (300 MHz, DMSO-d : 1.24 (d (J 2.8 Hz), 6H,
OCH(CH ), 3.81 (s, 2H, CH ), 4.02 (m, 1H, CH), 4.99 (m, 1H, OCH
(CH ), 5.61 (broad s, 1H, OH), 8.52 (broad s, 3H, NH
MHz, DMSO-d : 21.4, 54.4, 59.5, 69.5, 167.5.
3 4
/MeOH/25% NH OH
1
6
) d
a
before filtering them through a 0.45
mm filter prior analysis. The
3
)
2
b
2
13
samples were analyzed and calibration was done using PEG as
standards (M: 194-439,600 g/mol; Polymer Laboratories). Peak
areas were determined with Empower Software Version 1154
3
)
2
3
). C NMR (75
6
) d
(
Waters Associates Inc.).
2.3.1.4. HCl.H-Thr-OMe (4a). R
f
¼ 0.92 (CHCl
:4:1.5 v/v/v). H NMR (300 MHz, DMSO-d : 1.20 (d (J 6.3 Hz), 3H,
CH ), 3.75 (s, 3H, OCH ), 3.93 (d (J 3.9 Hz), 1H, CH), 4.13 (m, 1H,
CH), 5.65 (broad d (J 5.0 Hz), 1H, OH), 8.40 (broad s, 3H, NH
: 20.0, 52.7, 57.9, 65.0, 168.7.
3 4
/MeOH/25% NH OH
1
8
g
a
6
) d
2
.2.2. Cloud point (CP) determination
Cloud point measurements were preformed on a Shimadzu UV-
450 UV/VIS spectrophotometer. Polymer sample solutions were
3
3
b
13
3
).
C
2
NMR (75 MHz, DMSO-d
6
)
d
2
prepared by dissolving 1 mg of polymer per mL H O. The samples
ꢀ
ꢀ
were placed in a cuvette, heated from 0 C to 95 C and then cooled
2.3.1.5. HCl.H-Thr-OEt (4b). R
f
¼ 0.88 (CHCl
8:4:1.5 v/v/v). H NMR (300 MHz, DMSO-d : 1.21 (d (J 6.6 Hz), 3H,
CH ), 1.24 (t (J 7.0 Hz), 3H, OCH CH ), 3.90 (m, 1H, CH), 4.13 (m,
1H, CH), 4.21 (q (J 7.2 Hz), 2H, OCH CH ), 8.39 (broad s, 3H, NH ).
: 13.9, 20.0, 57.8, 61.7, 65.0, 168.1.
3 4
/MeOH/25% NH OH
ꢀ
1
to 0 C while measuring the absorption at 450 nm at a heating/
cooling rate of 1 C/min. The cloud point was determined by
extrapolating the slope of the absorption increase to zero absorp-
tion. Another series (compounds 10a, 11, and 22) was measured in
6
) d
ꢀ
g
3
2
3
b
a
2
3
3
13
C NMR (75 MHz, DMSO-d
6
)
d
PBS-buffer (containing 0.15 M NaCl, 0.01 M Na
M NaH PO .2H O at pH 7.4) and PBS-buffer containing 10% foetal
calf serum. CP determinations in PBS-buffer were measured from
2 4 2
HPO .12H O, 0.002
2
4
2
2.3.1.6. HCl.H-Thr-OiPr (4c). R
8:4:1.5 v/v/v). H NMR (300 MHz, DMSO-d
f
¼ 0.92 (CHCl
3
/MeOH/25% NH
: 1.26 (m, 9H, OCH
CH), 4.11 (br s, 1H,
4
OH
1
6
) d
a
ꢀ
ꢀ
0
C to 40 C, as described above. PBS buffer alone became turbid
(CH (6H)/ CH (3H)), 3.83 (d (J 4.13), 1H,
3
)
2
g
3

M. van Dijk et al. / Polymer 51 (2010) 2479e2485
2481
b
8
CH), 5.01 (m, 1H, OCH(CH
3
)
2
), 5.64 (broad d (J 4.4 Hz), 1H, OH),
Hz), 1H,
a
CH), 4.64 (m, 1H,
b
CH), 5.41 (s, 1H, H
2
C](1H)), 5.80 (s, 1H,
13
13
.35 (broad s, 3H, NH
3
). C NMR (75 MHz, DMSO-d : 20.1, 21.3,
6
)
d
2
H C](1H)), 6.57 (broad d (J 7.7 Hz), 1H, NH). C NMR (75 MHz,
21.5, 57.9, 65.2, 69.5, 167.6.
3
CDCl )
d: 14.0, 18.5, 20.0, 57.7, 61.5, 67.7, 120.5, 139.4, 169.2, 170.9.
a
a
2
.3.1.7. N -(Methacryloyl)-serine methyl ester (MA-Ser-OMe, 5a). In
a typical experiment, to a vigorously stirred and ice-cold solution of
HCl.H-Ser-OMe (3a) (6.7 g, 43 mmol) in dioxane/H O 1:1 v/v (300
2.3.1.12. N -(Methacryloyl)-threonine isopropyl ester (MA-Thr-OiPr,
1
6c). R
CDCl
CH ), 2.12 (broad s, 1H, OH), 4.36 (m, 1H,
5.10 (septet (J 6.2 Hz), 1H, OCH(CH ), 5.41 (s, 1H, H
(s, 1H, H C](1H)), 6.55 (broad d (J 8.0 Hz), 1H, NH,). C NMR (75
f
¼ 0.63 (Et
2
O). R
t
¼ 19.33 min (C18). H NMR (300 MHz,
(6H)/ CH (3H)), 2.01 (m, 3H,
CH), 4.62 (m, 1H, CH),
C](1H)), 5.80
2
3
)
d
: 1.26 (m, 9H, OCH(CH
3
)
2
g
a
3
mL), methacryloyl chloride (4.2 mL, 43 mmol) was added dropwise
and the pH of the reaction mixture was kept between 8 and 9 by the
addition of Et
3
b
3
)
2
2
13
3
N (18 mL, 129 mmol). After the addition was
2
complete, the reaction mixture was stirred for 16 h at room
temperature. Then, the solvents were removed by evaporation and
MHz, CDCl
170.6.
3
) d: 18.7, 20.2, 21.8, 57.8, 68.1, 69.5, 120.5, 139.6, 169.2,
the residue was dissolved in Et
removed by filtration and washed with Et
was concentrated in vacuo and the residue was purified by column
chromatography (hexane/Et O 1:1 v/v / Et O) to give MA-Ser-
OMe (5a) as a colorless oil in 33% yield (2.5 g). R O). R
¼ 0.19 (Et
: 1.99 (s, 3H, CH ), 3.05
), 3.96 (m, 2H, CH ), 4.71 (m,
C](1H)), 5.81 (s, 1H, H C](1H)), 6.82
broad d (J 6.3 Hz), 1H, NH). C NMR (75 MHz, CDCl : 18.7, 52.9,
2
O. The remaining solids were
2
O. The combined filtrate
2.3.2. Polymerization reactions
2.3.2.1. Homopolymer synthesis. The monomer (0.5 g) and AIBN
(monomer/initiator (M/I) molar ratio was 100/1) were dissolved in
dry DMF (5 mL) and the solution was degassed by three freeze-
pump-thaw cycles. The reaction mixture was stirred for 48 h at
2
2
f
2
t
¼
1
1
(
1
(
2.98 min (C18). H NMR (300 MHz, CDCl
broad s, 1H, OH), 3.80 (s, 3H, OCH
H, CH), 5.42 (s, 1H, H
3
)
d
3
ꢀ
3
b
2
70 C after which the solvent was removed in vacuo and the residue
a
2
2
2
was dissolved in MeOH (5 mL) and precipitated with ice-cold Et O
13
3
)
d
(40 mL). The precipitate was collected by centrifugation (3000 rpm,
5 min) and the solvent was decanted. After drying in vacuo, the
polymers were obtained as white solids in a yield of 82e96%.
5
5.1, 62.8, 121.3, 139.4, 169.3, 171.4.
a
2
0
(
(
5
(
.3.1.8. N -(Methacryloyl)-serine ethyl ester (MA-Ser-OEt, 5b). R
f
¼
1
.31 (Et
2
O). R
t
¼ 15.37 min (C18). H NMR (300 MHz, CDCl
CH ), 1.99 (s, 3H, CH ), 3.98 (m, 2H, CH
CH ), 4.69 (m, 1H, CH), 5.42 (s, 1H, H
C](1H)), 6.84 (broad d (J 6.1 Hz), 1H, NH). C NMR
75 MHz, CDCl : 13.7, 18.0, 54.5, 61.4, 62.4, 120.5, 138.8, 168.4,
3
)
d
: 1.31
), 4.26 (q
C](1H)),
2.3.2.2. Co-polymer synthesis. To an equimolar mixture of co-
monomer and co-monomer (1.5 mmol), AIBN (5 mg, 30 mmol)
1 2
was added and the solid compounds were dissolved in dry DMF (5
mL) and the obtained solution was degassed by three freeze-pump-
thaw cycles. The reaction mixture was stirred for 48 h at 70 C after
t (J 7.0), 3H, OCH
J 6.5), 2H, OCH
.82 (s, 1H, H
2
3
3
b
2
2
3
a
2
1
3
2
ꢀ
3
)
d
170.2.
which the solvent was removed in vacuo and the residue was dis-
3
solved in CHCl (5 mL) and precipitated with ice-cold hexane (40
a
2
R
1
3
a
(
.3.1.9. N -(Methacryloyl)-serine isopropyl ester (MA-Ser-OiPr, 5c).
mL). The precipitate was collected by centrifugation (3000 rpm, 5
min) and the solvent was decanted. After drying in vacuo, the co-
polymers were obtained as white solids in a yield of 32e95%.
1
f
¼ 0.47 (Et
2
O). R
t
¼ 17.70 min (C18). H NMR (300 MHz, CDCl
), 2.00 (m, 3H, CH
.04 (broad s, 1H, OH), 3.97 (d (J 3.9 Hz), 2H, CH ), 4.64 (m, 1H,
CH), 5.10 (septet (J 6.3 Hz), 1H, OCH(CH ), 5.41 (s, 1H, H C]
C](1H)), 6.82 (broad d (J 6.1 Hz), 1H, NH).
NMR (75 MHz, CDCl : 18.7, 21.9, 55.4, 63.9, 70.1, 121.0, 139.4,
68.9, 170.2.
3
) d:
.89 (dd (Jax 2.2, Jbx 6.3 Hz), 6H, OCH(CH
3
)
2
3
),
b
2
3
)
2
2
2.4. Degradation studies
13
1H)), 5.82 (s, 1H, H
2
C
3
)
d
2.4.1. Degradation of the monomers
1
The monomers 5aec and 6aec (15 mg) were dissolved in 100
ꢀ
mM sodium phosphate buffer pH 7.4 (5 mL) and incubated at 37 C;
a
2
6
CDCl
2
(
(
.3.1.10. N -(Methacryloyl)-threonine methyl ester (MA-Thr-OMe,
during the experiment the pH was checked to ensure the same
1
a). R
f
¼ 0.28 (Et
: 1.24 (d (J 6.6 Hz), 3H,
.29 (broad s, 1H, OH), 3.79 (s, 3H, OCH
m, 1H, CH), 5.42 (s, 1H, H C](1H)), 5.81 (s, 1H, H
broad d, 1H, NH). C NMR (75 MHz, CDCl : 18.8, 20.2, 52.9, 57.5,
2
O). R
t
¼ 14.92 min (C18). H NMR (300 MHz,
CH ), 2.01 (t (J 1.2 Hz), 3H, CH ),
), 4.39 (m, 1H, CH), 4.66
C](1H)), 6.60
start- and end-value. At regular time intervals a sample (100
mL)
3
)
d
g
3
3
was drawn and immediately quenched with ice-cold 1 M sodium
ꢀ
acetate buffer pH 3.8 (200 mL) and stored at 4 C prior to HPLC
3
b
a
2
2
analysis. The t1/2-values were determined by plotting the ln(AUC)
versus time (AUC: area under the curve).
1
3
3
)
d
6
8.3, 120.8, 139.7, 169.0, 171.8.
2
.4.2. Degradation of the polymers
The homopolymers 7 and 8 (5 mg) were dissolved in a D O/
a
2
R
(
(
.3.1.11. N -(Methacryloyl)-threonine ethyl ester (MA-Thr-OEt, 6b).
2
1
f
¼ 0.47 (Et
m, 6H, OCH
broad s,1H, OH), 4.24 (q (J 7.2 Hz), 2H, OCH
2
O).R
t
¼ 17.10 min (C18). H NMR (300 MHz, CDCl
3
)
d
: 1.26
), 2.24
), 4.37 (broad d (J 3.0
sodium phosphate buffer (100 mM, pH 7.4, 5 mL) and incubated at
37 C. The degradation of the polymer was followed by H NMR
spectroscopy by monitoring the decrease of the COOCH3 peak (d:
ꢀ
1
2
CH
3
(3H)/ CH (3H)), 2.01 (t (J 0.8 Hz), 3H, CH
g
3
3
2
CH
3
methacryloyl chloride/Et3N
in dioxane/H2O 1:1 v/v
0°C to rt, 16h
R1 OH
OH
2
R1 OH
OR2
R1 OH
SOCl2 in R OH
0
O
°C to rt, 16h
OR2
H2N
HCl.H N
N
H
2
O
O
O
1
1
R1 = H; R = Me: 3a (84%)
2
1
1
1
1
1
1
2
R
R
= H: Ser (1)
= CH3: Thr (2)
R
R
R
R
R
R
= H; R = Me: 5a (33%)
1
1
1
1
1
2
2
R
R
R
R
R
= H; R = Et: 3b (98%)
= H; R = Et: 5b (47%)
2
2
= H; R = iPr: 3c (80%)
= H; R = iPr: 5c (75%)
2
2
= CH3; R = Me: 4a (99%)
= CH3; R = Et: 4b (92%)
= CH3; R = iPr: 4c (99%)
= CH3; R = Me: 6a (88%)
= CH3; R = Et: 6b (96%)
= CH3; R = iPr: 6c (79%)
2
2
2
2
Scheme 1. Synthesis of the monomers 5aec (serine-based) and 6aec (threonine-based).

2482
M. van Dijk et al. / Polymer 51 (2010) 2479e2485
A
O
HN
O
AIBN (M/I = 100/1) in DMF
n
48h, 70°C (82%)
OH
HN
O
O
O
HO
O
5
a
7
B
O
O
HN
O
HN
AIBN (M1/M2/I = 50/50/1) in DMF
8h, 70°C (80%)
m
n
4
OH
+
O
OH
HN
O
O
HN
O
O
O
O
HO
HO
O
O
5
a
6c
17
Scheme 2. Synthesis of the polymers. A. Homopolymer based on 5a (entry 1, Table 1). B. Co-polymer based on 5a and 6c (entry 13, Table 1).
3
.64 ppm) and the increase of the CH
3
OD peak (
d: 3.20 ppm) during
3
CHCl (5 mL) and the polymer was precipitated with ice-cold
time. The HDO peak ((
reference.
d
: 4.64 ppm) was used as an internal
hexane (40 mL). The precipitate was collected by centrifugation
(3000 rpm, 5 min) and the solvent was decanted. After drying in
vacuo, the amphiphilic co-polymer was obtained as a white solid
in 63% yield (0.46 g).
2.5. Nanoparticles of a thermoresponsive amphiphilic block co-
polymer
2.5.2. Particle preparation and particle destabilization studies of
poly(MA-Thr-OEt)-b-(PEG monomethyl ether 5000)
A sample of poly(MA-Thr-OEt)-b-(PEG monomethyl ether 5000)
co-polymer (3 mg) was dissolved in ice-cold sodium phosphate
2
5
.5.1. Synthesis of poly(MA-Thr-OEt)-b-(PEG monomethyl ether
000) (26)
a
N -(methacryloyl)-Thr-OEt 6b (493 mg, 2.30 mmol) and (PEG
3
buffer (100 mM, pH 7.4, 3 mL) containing 0.02% NaN . The solution
2
monomethyl ether 5000) -ABCPA 25 [19] (238 mg 0.023 mmol)
were dissolved in dry DMF (5 mL) and the solution was subjected
to three freeze-pump-thaw cycles for degassing and the reaction
mixture was stirred at 70 C for 72 h. Then, the solvent was
removed under reduced pressure and the residue was dissolved in
was stirred and rapidly heated until the temperature was above CP.
ꢀ
Then, the polymer solution was incubated at 37 C (pH 7.4) and the
ꢀ
particle size and mean count rate was determined at regular time
intervals by dynamic light scattering.
Table 1
a
Characterization of the polymers.
Polymer b
Yield %
M
n
ckDa
M
ckDa
PDI d
CP e
ꢀ
C
CPf
ꢀ
C
calcd CP g
ꢀ
C
ꢀ
g
T C
Entry
Compound
w
1
2
3
4
5
6
7
8
9
7
8
9
poly(MA-Ser-OMe)
poly(MA-Thr-OMe)
poly(MA-Ser-OEt)
poly(MA-Thr-OEt)
82
90
96
84
81
78
90
88
88
50
83
65
80
91
78
89
95
75
88
32
15.7
12.3
14.4
12.6
6.6
23.8
13.8
9.8
16.2
13.0
14.6
23.4
13.2
13.3
13.7
14.0
18.2
16.3
17.8
15.2
29.7
22.4
25.0
20.2
12.1
46.7
26.9
16.7
26.2
22.6
26.3
45.3
23.6
25.0
25.3
22.7
33.4
30.0
29.8
24.8
1.9
1.8
1.7
1.7
1.8
2.0
1.9
1.7
1.6
1.7
1.8
1.9
1.8
1.9
1.8
1.6
1.8
1.7
1.7
1.6
>100
64.5
49.5
24.0
24.5
19.5
6.5
e
e
e
e
>150
83
101
89
83
92
91
92
113
80
94
100
92
88
88
83
92
93
89
80
e
e
10a
10b
10c
11
12
13
14
15
16
17
18
19
20
21
22
23
24
18.5 (18.5)
e
e
h
poly(MA-Thr-OEt)
poly(MA-Thr-OEt)
e
i
e
e
poly(MA-Ser-OiPr)
poly(MA-Thr-OiPr)
3.5 (2.5)
e
e
1.5
e
poly(MA-Ser-OMe-co-MA-Thr-OMe)
poly(MA-Ser-OMe-co-MA-Ser-OEt)
poly(MA-Ser-OMe-co-MA-Thr-OEt)
poly(MA-Ser-OMe-co-MA-Ser-OiPr)
poly(MA-Ser-OMe-co-MA-Thr-OiPr)
poly(MA-Ser-OiPr-co-MA-Thr-OMe)
poly(MA-Ser-OiPr-co-MA-Ser-OEt)
poly(MA-Thr-OiPr-co-MA-Ser-OEt)
poly(MA-Thr-OiPr-co-MA-Thr-OMe)
poly(MA-Ser-OiPr-co-MA-Thr-OEt)
poly(MA-Thr-OiPr-co-MA-Thr-OEt)
poly(MA-Thr-OiPr-co-MA-Ser-OiPr)
83.0
81.0
46.0
32.0
26.0
25.0
24.8
17.0
14.0
14.0
10.5
6.0
e
>82
>75
>62
>53
>50
35
28
33
25
14
13
4
10
11
12
13
14
15
16
17
18
19
20
e
e
e
e
e
e
e
e
10 (10)
e
e
a
b
c
d
e
f
The polymers are listed based on the CP-value.
Polymerization reactions were performed at a molar M/I ratio of 100/1. The co-polymer ratios (1:1 w/w) could not be verified by 1H NMR due to overlapping signals.
and M were determined by GPC with 10 mM LiCl in DMF as eluent and PEG as standards for calibration.
PDI: polydispersity index (M /M ).
CP: cloud point, measured in H O.
M
n
w
w
n
2
CP measured in PBS-buffer (PBS-buffer with 10% foetal calf serum).
g
h
i
calcd CP: calculated cloud point: (CPhomopolymer 1 þ CP homopolymer 2)/2.
M/I ¼ 50/1.
M/I ¼ 200/1.

M. van Dijk et al. / Polymer 51 (2010) 2479e2485
2483
Table 2
buffer and PBS-buffer containing 10% foetal calf serum, and the CP-
value was determined (Table 1). It was found that PBS had
a measurable, however, a rather small influence on the CP which
was in accordance with the data from Soga et al. [12]
Half-life times (t1/2) of monomers 5aec and 6aec.a
Entry
Monomer
t
1/2 (days)b
1
2
3
4
5
6
MA-Ser-OMe 5a
MA-Ser-OEt 5b
MA-Ser-OiPr 5c
MA-Thr-OMe 6a
MA-Thr-OEt 6b
MA-Thr-OiPr 6c
5.0 ꢃ 0.6
11.0 ꢃ 0.1
40.5 ꢃ 0.5
5.4 ꢃ 0.4
The newly synthesized polymers were also characterized by
g
determination of their glass transition temperature (T , Table 1).
ꢀ
These values fall in the range between 80 and 113 C, with one
11.8 ꢃ 0.8
33.7 ꢃ 4.5
exception: poly(MA-Ser-OMe) (compound 7, entry 1), which had
ꢀ
a T
g
of >150 C. We did not observe a clear correlation between
a
ꢀ
Reaction conditions: pH 7.4 at 37 C.
Average ꢃ SD.
b
structure of the polymers or their physicochemical properties (e.g.
hydrophobicity or CP-values) with the measured T
g
.
As described in the literature for poly(N-isopropyl acrylamide)
PNIPAAM) [21] and poly(2-n-propyl-2-oxazoline) (PnPropOx)
22], the CP decreased with an increasing molecular weight.
3
. Results and discussion
(
[
3.1. Synthesis of the monomers
Therefore, this effect was also studied with poly(MA-Thr-OEt) since
the effect of different M/I ratios (50/1, 100/1, and 200/1) on M in
relation to CP was investigated. As can be seen from Table 1 (entries
The monomers 5aec and 6aec were synthesized via a two-step
approach as shown in Scheme 1. In the first step, the aimed esters
3, 4) were synthesized by reacting -serine (1) or -threonine (2)
n
4
e6), the influence of M
n
on CP was rather small in case of M/I 50/1
(
L
L
ꢀ
and M/I 100/1, respectively (24.5 and 24 C), however, in case of M/I
with the corresponding alcohol in the presence of thionyl chloride
in good to excellent yield (80e99%) [20]. In the second step, the
amino acid esters were N-terminally acylated by treatment with
methacryloyl chloride under SchotteneBaumann conditions in the
ꢀ
200/1 the CP was significantly lower (19.5 C).
3.3. Degradation studies
3
presence of Et N as base. After purification by column chroma-
tography, the monomers 5aec and 6aec were characterized by
3.3.1. Degradation of the monomers
NMR and analyzed by HPLC (purity >98%) and were obtained in
To obtain insight into the chemical stability of the polymers, the
degradation of the monomers under physiological conditions (pH
7.4, 37 C) was studied and expressed in half-life time (t1/2) values
ꢀ
3
3e88% yield. The monomers 5aec and 6aec were stored at ꢂ20 C
ꢀ
prior to use.
(Table 2). In this series, the hydrolysis of the esters followed the
order: methyl > ethyl > isopropyl, irrespective of the amino acid
residue. Apparently, the hydrolysis rate is independent of the
overall hydrophobicity, in contrast to the cloud points of the cor-
responding monomers (Table 1), and followed the leaving group
character of the alkyl moiety.
3
.2. Synthesis of the polymers
The monomers were polymerized via a free radical polymeriza-
0
tion, mediated with 2,2 -azobis(isobutyronitrile) (AIBN) as radical
initiator. The polymerization reactions were run at 70 C during 48 h
ꢀ
in dry, O
00/1. A series of homopolymers (7e12) and a set of co-polymers
13e24) were synthesized in good to very high yield (82e96%), as
shown in Scheme 2 and Table 1. The polymers were obtained as
white solids and characterized by GPC, which gave M -values
ranging between 6.6 and 23.8 kDa with a PDI between 1.6 and 2.0.
2
-free, DMF with a molar monomer/initiator (M/I) ratio of
3
.3.2. Degradation of the polymers
1
(
The rate of ester hydrolysis was studied with polymers 7 and 8
1
by means of H NMR spectroscopy. For this purpose the relative
intensity of the COOCH
two weeks. The intensity of this peak was correlated to
and CH Thr in case of 7 and 8, respectively. It was found that the
3
peak (
d: 3.64 ppm) was monitored during
n
b
CH Ser
2
g
3
hydrolysis of both polymers was rather slow compared to the
individual monomers, since the conversion after 14 days was w25
and w12% for 7 and 8, respectively. This reduction in hydrolysis rate
may be explained by the hydrophobic nature of the polymer main-
chain, in which solvation of the side chains is reduced and thereby
slowing down hydrolysis of the ester bond [19].
3
.2.1. Cloud point determinations
The homopolymers 7e12 (entries 1e8, Table 1) were found to
ꢀ
have cloud points (CP) ranging from 1.5 to >100 C, which was in
direct correlation with the overall hydrophobicity (methyl versus
ethyl versus isopropyl, and serine versus threonine) of the poly-
mers, since a lower CP was found with an increased hydrophobicity
of the polymer. This property offered us an opportunity for tailoring
the CP by synthesizing a series of co-polymers 13e24 (entries
3.4. Thermoresponsive polymeric micelles
9
e20, Table 1) with a 1:1 co-monomer molar ratio to arrive at a CP
between those of the homopolymers.
Since it is known that PBS may have an influence on the CP-
value, three compounds (10a, 11, and 22) were measured in PBS-
To study the applicability of the serine- and threonine-based
methacryloyl polymers as thermoresponsive polymeric micelles,
a thermoresponsive amphiphilic block co-polymer was synthesized
reference 19
DMF
2h, 70°C (63%)
O
CN
CN
7
O
N
O
+
O
O
O
n
N
O
O
HN
m
CN
O
n
O
n
O
OH
HN
O
O
2
5
26
O
HO
6
b
O
a
Scheme 3. Synthesis of poly[N -(methacryloyl)-Thr-OEt]-b-(PEG monomethyl ether 5000) 26 as a thermoresponsive amphiphilic block co-polymer.

2484
M. van Dijk et al. / Polymer 51 (2010) 2479e2485
Table 3
Characterization of poly[N -(methacryloyl)-Thr-OEt]-b-(PEG monomethyl ether
000) 26 and its precursor 25.
a
5
Polymer Yield
%)
M
kDa
n
M
kDa
w
M
(PDI)
w
/M
n
M
b
n
b
Thr-
(kDa)
Z
nm
ave
PD c
a
c
(
2
2
5
6
46
94
11.1
13.0
12.2
22.7
1.1
1.7
e
e
e
6.4
216
0.06
a
n w
M and M were determined by GPC with 10 mM LiCl in DMF as eluent and PEG-
based standards for calibration.
b
c
M
n
of the N -(methacryloyl)-Thr-OEt block was determined by 1H NMR.
a
Z-average diameter and polydispersity (PD) as determined by DLS at 1 mg/mL
ꢀ
dispersion in H
2
O at 25 C.
(Scheme 3). Based on a literature procedure according to Neradovic
et al. [19], the macroinitiator (PEG monomethyl ether 5000)
2
-
a
ABCPA 25 was prepared and used in combination with N -(meth-
acryloyl)-Thr-OEt (6b) in a radical polymerization reaction to obtain
a
poly[N -(methacryloyl)-Thr-OEt]-b-(PEG monomethyl ether 5000)
a
Fig. 2. Stability of particles formed by poly[N -(methacryloyl)-Thr-OEt]-b-(PEG mon-
omethyl ether 5000) incubated at 37 C and pH 5.0 (dashed line) and 7.4 (solid line),
2
6 in 63% yield. At an M/I ratio of 100/1 the isolated polymer had an
ꢀ
a
average of 30 repeating units of N -(methacryloyl)-Thr-OEt as
judged by H NMR analysis (Table 3).
respectively. The data are shown as average ꢃ SD, based on three independent
1
determinations.
Based on Dynamic Light Scattering (DLS) measurements, parti-
a
cles were formed when a solution of poly[N -(methacryloyl)-Thr-
at pH 5.0, the mean count rate was almost constant over a period of
700 h, indicating that at this pH the particles were stable (Fig. 2).
Moreover, the diameter and PD of the particles remained constant
during the time of incubation. This was in sharp contrast with the
particles that were incubated at pH 7.4. At this pH, ester hydrolysis
induced an increase of the hydrophilic character of the polymer
which resulted in a destabilization of the particles and thus in
a gradual decrease of the mean count rate and an increase in the PD
from 0.1 at t ¼ 0 to 0.5 at t ¼ 700 h. Furthermore, the half-life time
(t ) of the particles could be estimated to be approximately 400 h
OEt]-b-(PEG monomethyl ether 5000) in H
slowly heated from 0 to 25 C, since the clear solution became
turbid at 22 C resulting in a high particle count rate (Fig. 1). The
measured particle size (Table 3) was rather high, which indicated
that polymer aggregates (larger aggregates consisting of smaller
polymeric aggregates) rather than core-shell micelles were formed,
most likely due to the slow heating of the polymer solution, as
previously described by Neradovic et al. [8,9] Cooling of the
2
O (1 mg/mL) was
ꢀ
ꢀ
ꢀ
dispersion resulted in a decrease of particle count rate, and at 20 C
1
/2
almost no particles could be detected, indicating that the amphi-
philic block co-polymer was completely soluble. The critical
(16.7 days) as the mean count rate was 50%of its starting value, while
the monomer N -(methacryloyl)-Thr-OEt had a similar half-life time
(t1/2) of 11.8 days (Table 2). Again, the slower degradation kinetics of
the polymer as compared to that of the monomer can be ascribed to
dehydration of the thermosensitive block above its CP.
a
ꢀ
aggregation temperature between 20 and 25 C corresponded very
ꢀ
a
well with the cloud point of 24 C of the homopolymer, poly[N -
(
methacryloyl)-Thr-OEt] 10a (Table 1).
The polymer poly[N -(methacryloyl)-Thr-OEt]-b-(PEG mono-
a
methyl ether 5000)wasdissolved in anaqueousbuffer (either pH 5.0
or 7.4) and the solution was incubated at 37 C after particle forma-
4. Conclusions
ꢀ
tion by heating the polymer solution. The stability of the particles
was analyzed by DLS as function of the incubation time. During each
transfer of the sample from the incubation bath to the DLS machine
at the measuring time points, the sample was allowed to cool down
and subsequently slowly heated in the DLS machine, which
explained, as already mentioned above, the rather high particle size
a
A series of N -methacryloyl amino acid alkyl esters was
synthesized and used for the preparation of a particular set of
homo- and co-polymers. It turned out that the cloud point of these
polymers could be tailored by variation of the hydrophobicity of the
monomer. Furthermore, the leaving group character of the ester
moiety was a major determinant of the half-life time of the
monomers in aqueous solution, while the polymers were hydro-
lyzed at least with a four-fold lower rate compared to the respective
monomer. Functionalization with a hydrophilic PEG-chain resulted
(Z
ave: 300 nm and PD: 0.1e0.15). Since ester hydrolysis is rather slow
a
in a co-polymer, poly[N -(methacryloyl)-Thr-OEt]-b-(PEG mono-
methyl ether 5000), which was soluble in aqueous buffer below its
ꢀ
cloud point, while nanoparticles were formed above 21 C. Incu-
bation of these microparticles at physiological conditions resulted
in a gradual decrease in particle amount, since ester hydrolysis
induced hydrophilicity and thus increasing the CP which resulted in
destabilization of the microparticles. The independently tunable
LCST and degradation behavior of the newly designed amino acid-
based polymers may find application in the controlled release of
bioactive molecules.
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