A new end group structure of poly(ethylene glycol) for hydrolysis-resistant biomaterials

Article abstract of DOI:10.1002/pola.24562

A new PEG end-group structure (α-PEG-MA) was designed and synthesized. When compared with the conventionally used PEG-acrylate structure, the vinyl group in the new structure showed comparable reactivity in thiol-ene reactions, but the formed materials were hydrolysis resistant because there is no ester linkage in the backbone chains. Copyright

Full text of DOI:10.1002/pola.24562

A New End Group Structure of Poly(ethylene glycol) for
Hydrolysis-Resistant Biomaterials
XINMING TONG, JINGJING LAI, BAO-HUA GUO, YANBIN HUANG
Department of Chemical Engineering, Tsinghua University, Beijing 100084, China
Received 9 November 2010; accepted 27 December 2010
DOI: 10.1002/pola.24562
Published online 20 January 2011 in Wiley Online Library (wileyonlinelibrary.com).
KEYWORDS: crosslinking; degradation; hydrogels; hydrolysis-resistant hydrogel; modification; PEG end group; thiol-ene reaction
INTRODUCTION Because of their excellent biocompatibility,
poly(ethylene glycol) (PEG) and its derivatives have been
widely used in biomedical applications, especially in drug
PEGylation, biomaterial surface modification, and hydrogel-
based implants and tissue scaffolds.1–8 To be incorporated
into a hydrogel structure, the hydroxyl end group of PEG is
usually and conveniently reacted with acryloyl chloride to
form an acrylate structure (PEG-A, Scheme 1),9–11 which
subsequently undergoes either a free radical polymerization
or the Michael-type addition reaction with thiol-containing
molecules.11–14 Consequently, the formed hydrogel structure
contains ester linkages at the crosslink points, and their
hydrolysis (half time of the ester bonds in the water-rich
environment is on the order of days at pH 7.4 and 37 ꢀC)
will result in disintegration of the gel,15–17 which is an
advantage for applications requiring gel degradation but a
disadvantage for longer term applications such as vitreous,
cartilage, and nucleus pulposus replacement.
with high yield using commercially available reagents. The
reactivity of the new structure in the Michael-type addition
was found comparable to that of the conventional acrylate
end group, and the hydrolysis-resistant property of the
formed hydrogel was demonstrated in this study.
EXPERIMENTAL
Materials and Methods
PEG (MW 2000, PEG2K), sodium hydride (NaH), 4-dimethy-
laminopyridine (DMAP), triethylamine (TEA), L-cysteine
hydrochloride, and 5,50-dithiobis-(2-nitrobenzoic acid)
(DTNB) were purchased from Alfa Aesar. Acryloyl chloride
was purchased from TCI Chemical. 4Arm-PEG-SH (MW
10,000) was purchased from Jen Kem Technology. Ethyl 2-
(hydroxymethyl) acrylate (EHMA) was purchased from
Jiachen Chemical (China). Phosphorus tribromide (PBr3) was
purchased from Sinopharm Chemical (China). All reagents
were used as received without further purification.
NMR spectra were recorded on a 300-MHz JOEL spectrometer.
UV tests were carried out on a PGeneral TU-1810 UV–Vis spec-
trophotometer. Matrix assisted laser desorption ionization
(MALDI)-time-of-flight mass spectrometer (TOF MS) was con-
ducted using an Applied Biosystems 4800 Plus TOF-MS and a-
cyano-4-hydroxycinnamic acid (CHCA) was used as matrix.
For such applications requiring hydrolysis-resistant PEG-
based biomaterials, PEG with vinyl sulfone end groups (PEG-
VS) seems the only structure that can be obtained through
direct reaction with hydroxyl-ended PEG.18,19 However, the
synthesis of PEG-VS either involves PEG reaction with divinyl
sulfone, which is highly toxic, or a lengthy multiple-step reac-
tion route. Therefore, alternative PEG end group structures
should be developed for hydrolysis-resistant biomaterials.
Synthesis of Ethyl 2-(Bromomethyl) Acrylate (EBrMA)20
EBrMA is commercially available; but in this study, we syn-
thesized it in-house following the protocol in literature.
EHMA (7.8 g, 60 mmol) was dissolved in 60 mL diethyl
ether, and PBr3 (2.0 mL, 21 mmol) was slowly added while
the vessel was cooled with an ice bath. Afterward, the mix-
ture was warmed to room temperature and stirred for 3 h.
After adding 5 mL water, the mixture was extracted with
hexane for three times. The organic solutions were com-
bined, washed with brine, and dried with MgSO4. The solvent
In this study, we designed and synthesized such a structure
in which the PEG chain is linked to the a-carbon atom of a
methacrylate group and forms an a-PEG-MA structure
(Scheme 1). In this structure, the electron-withdrawing ester
group is still connected to the vinyl group, making the latter
active for Michael-type addition reaction and possible free
radical polymerization. However, unlike in the PEG-A system,
ester hydrolysis will no longer affect the linkage of PEG to
the gel structure (Scheme 1). As shown below (Scheme 2),
the synthesis of the new structure is a one-step reaction
ꢀ
was removed in a rotary evaporator at 60 C to give the final
product (7.2 g, 90%).
Additional Supporting Information may be found in the online version of this article. Correspondence to: Y. Huang (E-mail: yanbin@tsinghua.edu.cn)
C
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Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 49, 1513–1516 (2011) 2011 Wiley Periodicals, Inc.
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JOURNAL OF POLYMER SCIENCE: PART A: POLYMER CHEMISTRY DOI 10.1002/POLA
SCHEME 1 Schematic illustration of hydrolysis effect on biomaterials based on traditional acrylate PEG and the new a-PEG-MA.
[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
1H NMR (CDCl3): 1.3 ppm (t, 3H, ACH3), 4.16 ppm (s, 2H,
ACH2ABr), 4.25 ppm (q, 2H, ACH2AOA), 5.9 and 6.3 (s, 1H,
¼¼CH2).
MW (MALDI-TOF MS, Fig. 2 and Supporting Information S1):
average Mn ¼ 2188, Mw/Mn ¼ 1.01. Melting point (differen-
tial scanning calorimetry (DSC), Supporting Information Fig.
S6): 43.8 ꢀC; degradation temperature (thermal graviꢀmetric
analysis (TGA), Supporting Information Fig. S5): 191.9 C.
Synthesis of PEG-A
PEG2K (10 g, 5 mmol) was dissolved in 200 mL dry tetra-
hydrofuran, and added with DMAP (305 mg, 2.5 mmol) and
TEA (4.34 mL, 30 mmol). Then acryloyl chloride (2.45 mL,
30 mmol) was added dropwise, and the reaction was carried
out overnight at room temperature. The resulting solution
was filtered, condensed by rotary evaporation, and precipi-
tated with diethyl ether.
Reactivity Comparison of a-PEG-MA and
PEG-A with Thiol
The reactivity of a-PEG-MA and PEG-A with thiol was evaluated
using cysteine as the model thiol compound and monitoring
thiol concentration with Ellman’s assay (DTNB).17 Briefly, 1
mM solution of cysteine and 0.5 mM PEG derivative (contain-
ing 1 mM double bond), prepared in phosphate buffer solution
(PBS) buffer (0.2 M, pH 8.0) were mixed at volume ratio of 1:1.
At scheduled times, 1 mL of the reaction mixture was mixed
with 9 mL DTNB solution (0.1 mM), and the absorbance at
412 nm was measured as an index of thiol concentration.
1H NMR (D2O): 3.6 ppm (200H, PEG backbone), 4.25 ppm
(t, 4H, CH2AOAC (O)A), 5.9 ppm (d, 2H, AC(O)ACH¼¼), 6.1
and 6.4 (q, d, 2H, ACH¼¼CH2).
Synthesis of a-PEG-MA
PEG2K (10 g, 5 mmol) was dissolved in 200 mL dry
dichloromethane, and NaH was added to the solution, at
threefold molar excess over OH groups. After that, EBrMA
(2.8 mL, 20 mmol) was added. The reaction was carried out
at room temperature overnight. The mixture was then
neutralized with acetic acid and filtered, and the solvent
was removed by rotary evaporation. After redissolving, the
residual in tetrahydrofuran and filtration, the solution was
condensed and precipitated with diethyl ether. The product
was washed with diethyl ether two times and vacuum dried
(yield > 90%).
Gel Preparation and Swelling Experiments
a-PEG-MA (or PEG-A) and 4arm-PEG-SH were first dissolved
in PBS (0.2M, pH 7.4) at a concentration of 10% (w/v) sepa-
rately. The two solutions were then mixed at a 5:2 volume
ratio (thiol:vinyl group molar ratio ¼ 1:1) and placed into
disc-shaped vessels (1 mL each). After being left overnight
to ensure complete reaction, the gels were taken out and
immersed in PBS (pH 7.4, 20 mM) at room temperature
1H NMR (300 MHz, D2O, Fig. 1): d (ppm) 1.23 (t, 6H, CH3A),
3.62 (s, 200H, PEG backbone), 4.17–4.21 (t, s, 8H,
ACH2AC(O)AOA, AOACH2AC(¼¼CH2)A), 5.90 (s, 1H,
AC¼¼CH2), 6.31 (s, 1H, AC¼¼CH2). 13C NMR (300 MHz,
CDCl3, Supporting Information Fig. S4): d (ppm) 14.21
(CH3A), 60.62 (CH3ACH2A), 69.24 (AC(¼¼CHA2)ACH2AOA),
70.56 (PEG backbone), 125.50 (AC¼¼CH2), 137.27
(AC¼¼CH2), 165.80 (AC¼¼O). FT-IR (KBr, Supporting Informa-
tion Fig. S7): m (cmꢁ1) 2887, 1718, 1643, 1470, 1344, 1281,
1424, 1147, 1113, 1063, 960, 843.
FIGURE 1 1H NMR spectrum of a-PEG-MA (D2O, 300 MHz).
[Color figure can be viewed in the online issue, which is
available at wileyonlinelibrary.com.]
SCHEME 2 Synthesis of a-PEG-MA.
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NOTE
FIGURE 2 MALDI-TOF MS spectrum of a-PEG-MA.
for swelling studies. The swelling ratio, Q, was calculated as:
Q ¼ Wt/W0, where Wt is weight at time t and W0 is the ini-
tial weight of the gel.
and not added to the C¼¼C double bond, which may result in
polymer chain coupling. This high conversion was also veri-
fied by the MALDI-TOF MS spectrum (Fig. 2 and Supporting
Information Fig. S2), which showed only the targeted a-PEG-
MA peaks, ionized with sodium and potassium.
RESULTS AND DISCUSSION
The reactivity of this new PEG end group with thiol was eval-
uated and compared with that of the usually used PEG-A
structure. As shown in Figure 3, the reaction of cysteine with
both a-PEG-MA and PEG-A was complete within 45 min at pH
8.0, and the two PEG structures showed comparable reactivity.
The a-PEG-MA synthesis was convenient, and the structure
was confirmed by 1H NMR (in D2O) and MALDI-TOF MS
analysis. The 1H NMR spectrum was shown in Figure 1. All
the resonance peaks were identified and their peak area
ratios were found to be consistent with the target structure.
The degree of vinyl end group, calculated from the integra-
tion area ratio of the proton resonance peak of C¼¼CH2 to
that of PEG backbone was >97%. This high conversion
means that the sodium alkoxide, formed by hydroxyl group
of PEG reacting with NaH, selectively reacted with Br-group
To verify the hydrolysis stability of the hydrogel formed by the
new PEG structure and 4arm-PEG-SH through the Michael-type
addition reaction, swelling studies were carried out and com-
pared with those of PEG-A and 4arm-PEG-SH. The swelling
FIGURE 4 Swelling ratio of gels prepared by a-PEG-MA (n) and
PEG-A (~) with 4arm-PEG-SH. The study was made with six
samples in each group, and the error bars were too small to
be seen in most of the data points (X means that the gels were
too weak to be weighed.)
FIGURE 3 Reaction of cysteine with a-PEG-MA (~) and PEG-A
(l), carried out in PBS at pH 8.0 and room temperature. [Color
figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
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JOURNAL OF POLYMER SCIENCE: PART A: POLYMER CHEMISTRY DOI 10.1002/POLA
3 Knop, K.; Hoogenboom, R.; Fischer, D.; Schubert, U. S.
behavior of the gels was illustrated in Figure 4. The PEG-
acrylate-based gels continued to swell until the point of ulti-
mate gel disintegration, due to the hydrolysis of its ester bond
and breakdown of the crosslinking points. In contrast, the a-
PEG-MA-based gels reached the equilibrium swelling after
about 3 days and no significant further swelling was observed.
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To further compare the hydrolysis stability, accelerated
hydrolysis experiments (with 0.2 M NaOH solution) were
done with gels formed between 4arm-PEG-SH and PEG-dia-
crylate (PEG-A), PEG-dimethacrylate (PEG-MA), and our pro-
posed new structure a-PEG-MA. The results (Supporting
Information Fig. S3) showed that PEG-MA gel was more sta-
ble than the PEG-A gel, but our new PEG systems are much
more resistant to hydrolysis than both of the other two.
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An a-PEG-MA structure was proposed and synthesized
through a one-step reaction with high yield (>90%). This
new PEG derivative reacts with thiols in the Michael-type
addition reaction with reactivities comparable with the con-
ventionally-used acrylated PEG. More importantly, the link-
ages of the addition product are hydrolysis-stable and, hence,
suitable for long-term applications such as vitreous replace-
ment and implanted device surface coating. It should be
pointed out that this newly proposed structure is not only
applicable to double- and mono-functionalized PEG, but also
to other polymers with hydroxyl groups such as poly(vinyl
alcohols) and polysaccharides.
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This work was supported by the Beijing Municipal Science and
Technology Commission (grant number: Z08000303220801).
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