Synthesis of OH-group-containing, biodegradable polyurethane and protein fixation on its surface

Article abstract of DOI:10.1021/bm2003658

A series of biodegradable polyurethanes containing free side hydroxyl groups (PUOH) were synthesized successfully in two steps: (1) PLA diol as soft segment, hexamethylene diisocyanate (HDI) as hard segment, and benzalpentaerythritol (BPO) as a chain extender were used to synthesize PUs with protected OH groups; (2) CF₃COOH was used as a deprotection agent to remove the benzal groups on PU to prepare PUOH. The properties of PU and PUOH were characterized by Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), water contact angle measurement, and gel permeation chromatography (GPC). The benzal groups were removed completely in 15 min without detrimental effect on PU main chains to obtain PUOHs. 4-Azidobenzoic acid was conjugated to PUOH through its esterification with the free OH groups on PUOH. The results of immunofluorescence assay showed that the phenyl azide groups formed were capable of binding mouse IgG under UV (254 nm) irradiation in 3 min; the bound mouse IgG retained its own biological activity and could further bind the FITC-labeled anti(mouse IgG). Therefore, this material has a potential in immunofluorescence assay and related fields.

Full text of DOI:10.1021/bm2003658

ARTICLE
pubs.acs.org/Biomac
Synthesis of OH-Group-Containing, Biodegradable Polyurethane and
Protein Fixation on Its Surface
Lixin Yang,†,‡ Jizheng Wei,†,‡ Lesan Yan,†,‡ Yubin Huang,† and Xiabin Jing*,†
†State Key Laboratory of Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences,
Changchun 130022, People’s Republic of China
‡Graduate School of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China
ABSTRACT: A series of biodegradable polyurethanes contain-
ing free side hydroxyl groups (PUOH) were synthesized
successfully in two steps: (1) PLA diol as soft segment,
hexamethylene diisocyanate (HDI) as hard segment, and
benzalpentaerythritol (BPO) as a chain extender were used to
synthesize PUs with protected OH groups; (2) CF3COOH was
used as a deprotection agent to remove the benzal groups on PU to prepare PUOH. The properties of PU and PUOH were
characterized by Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), differential scanning
calorimetry (DSC), water contact angle measurement, and gel permeation chromatography (GPC). The benzal groups were
removed completely in 15 min without detrimental effect on PU main chains to obtain PUOHs. 4-Azidobenzoic acid was conjugated
to PUOH through its esterification with the free OH groups on PUOH. The results of immunofluorescence assay showed that the
phenyl azide groups formed were capable of binding mouse IgG under UV (254 nm) irradiation in 3 min; the bound mouse IgG
retained its own biological activity and could further bind the FITC-labeled anti(mouse IgG). Therefore, this material has a potential
in immunofluorescence assay and related fields.
’ INTRODUCTION
surface hydrophilicity.8 A series of PUs prepared from L-lysine-
diisocyanate (LDI) are further transformed to cationic PUs with
noncytotoxicity and high transfection activity.9 Two novel segmen-
ted polyurethanes synthesized from poly(ε-caprolactone) diol,
HDI, and diester-diphenol or diurea-diol chain extenders have
potential in soft tissue engineering.10 An isobutyl-functionalized
polyhedral oligosilsesquioxanes (POSS) diol is used to synthesize
multiblock thermoplastic polyurethane hydrogels with higher shear
modulus, stiffness, and water swelling ratio.11 In short, design of
novel chain extenders or diisocyanates containing functional groups
is an effective strategy for the functionalization of PUs.
Many works focus on the introduction of hydroxyl groups to
polymers to improve the biocompatibility and hydrophilicity.12
To prepare such polymers, protection and subsequent deprotec-
tion of hydroxyl groups are usually necessary. Our lab introduced
hydroxyl groups to the main chains of poly(ester carbonates)
based on pentaerythritol13 and the free hydroxyl groups obtained
proved active for further reactions. We expect that the similar
synthetic strategy is applicable to the preparation of hydroxyl-
containing polyurethanes, that is, synthesizing benzal pentaery-
thritol (BPO) and reacting it with proper diisocyanates. To the
best of our knowledge, there is little work on the introduction of
free hydroxyl groups to PUs by this strategy. These free hydroxyl
groups are expected to further improve the biocompatibility and
Biodegradable polyurethanes (PUs) as medical materials have
been extensively investigated due to their good biocompatibility
and mechanical properties1 and have been applied as drug
carriers,2 tissue-engineering scaffolds,3 shape memory fibers
and implants,4 and so on.
Diverse biomedical applications of PUs need corresponding
different functionalities. To incorporate functionalities into PUs,
modifications may be made before, during, or after polymerization.
Surface modifications are often done on PUs such as surface grafting
of water-soluble polymers to improve their surface hydrophilicity,5
but simple surface modification is difficult to realize specific
functionalization such as that for selective protein fixation. For
modifications before or during polymerization, some new chain
extenders or diisocyanates containing functional groups such as
carboxyl and amino groups, and so on, are designed. One example is
2,2-bis(hydroxymethyl) propionic acid (DMPA). It is used in the
synthesis of water-soluble PUs.6 Zhang et al. utilized DMPA to
prepare anionic poly(lactic acid)-polyurethane (PULA) micelles.
The degradation rate of PULA and the drug-release rate of PULA
micelles were dependent on the content of DMPA.2a Du Prez et al.7
utilized two kinds of extenders, 3,5-bis(hydroxymethyl)-1-propar-
gyloxybenzene (PBM) and 2,2-di(prop-2-ynyl) propane-1,3-diol
(DPPD), to synthesize PUs containing alkyne groups, and further
modification was performed by virtue of click reactions of these
alkyne groups with azido-containing moieties under mild condi-
tions. A novel lysine-derivative chain extender containing gemini
quaternary ammonium side groups is used to synthesize poly(ε-
caprolactone) urethanes with rapid degradation rate and better
Received: November 23, 2010
Revised:
April 13, 2011
Published: April 14, 2011
r
2011 American Chemical Society
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|

Biomacromolecules
ARTICLE
biodegradability of PUs and to broaden their applications in
biomedical fields.
respectively. After 2 h, a solution of BPO (1.68 g, 7.5 mmol) in THF
(30 mL) was added, and the reaction solution was stirred at 90 °C for 6 h.
The products were precipitated in ethanol and dried to constant weight
under vacuum.
Preparation of Polyurethanes Containing Hydroxyl Groups
(PUOHs). A certain quantity of PU (0.3 g) was dissolved in CH2Cl2
(3 mL) in a 50 mL flask immersed in ice bath, and CF3COOH (3 mL) was
added. The mixture was stirred for a predetermined time period and
precipitated in ether. The precipitates were collected and dried under
vacuum until constant weight. Polyurethanes containing hydroxyl groups
were abbreviated as PUOHs.
In this work, therefore, BPO was used as a chain extender to
prepare a series of biodegradable PUs, and their protective benzal
groups were removed under acidic conditions to obtain PUOHs. To
demonstrate possible applications of PUOHs, the side hydroxyl
groups were converted to phenyl azide groups. By virtue of the
photoaffinity labeling ability of the phenyl azide, that is, reacting with
XꢀH (X = C, N, O) groups rapidly under UV irradiation,14 an
immunofluorescent protein was attached to PUOH and a model
immunofluorescence assay was performed. It was demonstrated
that the target proteins were effectively fixed on the polymer surface
and retained their own bioactivities. Therefore, the hydroxyl-
functionalized PU can serve as a biomedical material.
Azido-Labeled PUOHs. 4-Azidobenzoic acid was synthesized
according to literature.17 PUOH8K132 (2.1 g) was dissolved in dried
THF (20 mL) in a round-bottom flask, and 4-azidobenzoic acid (0.20 g),
DCC (0.26 g), and DMAP (0.13 g) were then added. The mixture was
stirred at room temperature for 12 h and then precipitated in ethanol.
The precipitate was collected and dried to constant weight under
vacuum. The product was coded as PUOH8K132ꢀN3.
’ EXPERIMENTAL SECTION
Electrospinning of Polymers. PUOH8K132ꢀN3 (2.0 g) and
PUOH8K132 (2.0 g) were dissolved in chloroform (10 mL), respec-
tively, for electrospinning. The electrospinning was performed accord-
ing the literature.18 The voltage was 20 kV, and the electrospun fibers
were collected onto glass slides.
Materials. L-Lactide was prepared in our laboratory and recrystal-
lized two times in ethyl acetate just before use.15 1,4-Butanediol (BDO)
and hexamethylene diisocyanate (HDI) (from Tokyo Kasei Kogyo Co.)
were dried over 4 Å molecular sieves and distilled under reduced
pressure. Stannous(II) octoate (Sn(Oct)2; from Sigma Chemical Co.)
was used as catalyst. Dicyclohexylcarbodiimide (DCC) and 4-dimethy-
laminopyridine (DMAP) were obtained from Acros. Trifluoroacetic
acid and pentaerythritol (from Aladdin) were used as received. Tetra-
hydrofuran (THF) was dried over CaH2 and distilled over sodium just
before use. The mouse IgG powder and the fluorescein isothiocyanate
(FITC)-labeled goat anti(mouse IgG) (1.5 mg/mL) were obtained from
Beijing Dingguo Biotechnology Co. Ltd., China.
Synthesis of Chain Extender Benzal Pentaerythritol (BPO).
The chain extender BPO was synthesized according to the literature.13
Pentaerythritol (81.6 g, 0.6 mol) was dissolved in 500 mL of boiling
deionized water in a 1000 mL, three-neck, round-bottom flask equipped
with a mechanical stirrer, and then 3 mL of concentrated hydrochloric acid
was added and benzaldehyde (63.6 g, 0.6 mol) was dropped. The mixture
was stirred for 24 h. The white crude products were collected, washed with
deionized water, and recrystallized in a sodium carbonate solution. During
the whole polymerization of polyurethane, attention must be paid to two
aspects. One is the equal molar ratio of [OH]/[NCO], which determines
the molecular weight of the polyurethanes formed. The other is the purity of
BPO, because the impurity in crude BPO was mainly pentaerythritol, which
would cause cross-linking of the PUs on one hand and lower the molecular
weight of PU due to the unmatched [OH] and [NCO] on the other hand.
Therefore, complete purification of BPO is critical for successful PU
preparation. After recrystallization from dried toluene solution (2ꢁ), a
high purity of BPO (81.5 g, yield: 60%) was obtained. 1H NMR (CDCl3,
ppm vs TMS): 3.22 (d, 2H), 3.64 (d, 2H), 3.90 (d, 2H), 4.57 (t, 1H), 5.37
(s, 1H), 7.31ꢀ7.38 (m, 5H). Elem. Anal. Calcd for C12H16O4: C, 64.27; H,
7.19; O, 28.54. Found: C, 64.09; H, 7.21; O, 28.70.
Immunofluorescence Assay of the Electrospun Fibers. The
polymer fiber slides electrospun from PUOH8K132ꢀN3 and
PUOH8K132 were further divided into two groups. One was for
irradiation and the other was for control reaction under darkness. The
slides were immersed into 15 mL of mouse IgG solution (0.02 mg/mL in
PBS buffer, pH 7.4). The slides for irradiation were then placed under
UV light (254 nm, the distance of the irradiation was 10 cm) for 3 min,
while the control samples were kept in darkness. After that, all slides
were rinsed with PBS-T buffer (0.1% Tween 80 in PBS buffer, pH 7.4)
five times to remove free antigens. FITC-labeled goat anti(mouse IgG)
(1.5 mg/mL) was diluted in PBS at 1:100 (v/v), and then the slides were
immersed in it for 30 min under darkness, followed by rinsing five times.
A cover glass was adhered onto each slide with a drop of glycerin for
confocal laser scanning microscopic (CLSM) observation.
Molecular Weight Measurements. The numberꢀaverage molar
mass (Mn) and polydispersity index (PDI) of the polymer were measured
by means of size exclusion chromatography of TOSOH HLC-8220 GPC
(Column: Super HZM-H 33) at 40 °C using THF as eluent at a flow rate
of 0.35 mL/min. The Mn was calibrated against polystyrene standards.
Infrared Measurement. A Fourier-transform infrared (FT-IR)
spectrometer (Nicolet 6700, USA) was used to obtain the FT-IR spectra.
Each spectrum was recorded with a total of 32 scans. Film specimens were
used. They were solution-cast on a KBr plate from chloroform solution.
XPS Measurement. PU4K121 (10 mg) was dissolved in chloro-
form (10 mL). The solution was dropped on the surface of a piece of
glass slide and dried under vacuum at 80 °C for 5 h. The other samples
(PU4K132, PU4K143, PUOH4K121, PUOH4K132, PUOH4K143)
were prepared by the same procedure. The dried films were used for
XPS measurement. The surface elemental composition of the polymer
film was analyzed on an Escalab-MKII X-ray photoelectron spectro-
meter (VG Scientific Ltd., U.K.) using Mg KR radiation (1253.6 eV) as
the X-ray source for excitation. The typical operating pressure in the
analytical chamber was in the range of 10ꢀ9ꢀ10ꢀ10 Torr. The binding
energy of the experimental spectra was calibrated on the basis of the
most intense peak of C1s at 284.5 eV.
Synthesis of Polyurethanes (PUs). A series of PUs were
synthesized from PLA-diol, HDI, and BPO. Three different number
average molecular weights (4000, 6000, 8000 g molꢀ1) of PLA-diol and
3
three molar ratios of PLA-diol/HDI/BPO (1:2:1, 1:3:2, 1:4:3) for each
PLA-diol were designed, thus 9 PUs were prepared, abbreviated as
PU4k121, PU4K132, PU4K143, PU6K121, PU6K132, PU6K143,
PU8K121, PU8K132, and PU8K143. The three PLA-diol samples were
prepared according to the literature.16 A typical synthetic procedure for
PU4K143 was as follows: dried PLA-diol (Mn = 4 K, 10.15 g, 2.5 mmol)
was dissolved in THF (20 mL) in a dried polymerization bottle equipped
with a magnetic stirring bar. The reaction bottle was immersed in oil bath
at 90 °C for 30 min to make the PLA-diol dissolved completely. A
solution of HDI (1.76 g, 10.5 mmol) in THF (20 mL) and Sn(Oct)2
(1 mL) in a toluene solution (2.0 ꢁ 10ꢀ4 mol/mL) were added,
Water Contact Angle. The filmsofPUs and PUOHs were prepared
by the same method as in XPS measurements. The contact angles of the
films were measured by a DSA-10 drop shape analyzer (Germany)
equipped with a charge-coupled device (CCD) camera. The fixed volume
of every water droplet was 2 μL. When the water droplet attached the film
surface, the contact angle was calculated by the DSA software.
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ARTICLE
Scheme 1. Synthetic Routes of PLA-Diol, PU, and PUOH
Table 1. Synthesis Conditions of PU Samples
Mn of PLA-diol
feed molar ratios
product molar ratios
PLA-diol/HDI/BPO
Mn of PU
entry
(104 g molꢀ1
)
PLA-diol/HDI/BPO
(104 g molꢀ1
)
PU code
3
3
1
2
3
4
5
6
7
8
9
0.406
0.406
0.406
0.606
0.606
0.606
0.795
0.795
0.795
1/2.0/1.0
1/3.0/2.0
1/4.0/3.0
1/2.0/1.0
1/3.0/2.0
1/4.0/3.0
1/2.0/1.0
1/3.0/2.0
1/4.0/3.0
1/1.95/0.98
1/2.90/1.94
1/3.96/2.99
1/1.90/0.98
1/2.87/1.94
1/3.82/2.96
1/1.86/0.94
1/2.96/1.99
1/3.90/2.98
6.2
2.9
2.2
5.1
4.2
4.6
3.4
4.9
3.5
PU4K121
PU4K132
PU4K143
PU6K121
PU6K132
PU6K143
PU8K121
PU8K132
PU8K143
Thermal Analysis. Differential scanning calorimetry (DSC) was
carried out using a TA Instrument Q100. Each scan was performed at a
heating and cooling rate of 10 K/min under a nitrogen atmosphere, and the
scan temperature ranged from 25 to 180 °C. During the test, each sample was
heated to 180 °C, kept at 180 °C for 5 min to eliminate thermal history, cooled
to 25 °C, and then heated to 180 °C again. Glass transition temperature (Tg)
was taken as the inflection of the DSC curve and melting temperature (Tm)
was taken as the peak temperature of the endothermic peak.
measured by 1H NMR. Itis seen that the two sets of data are
close to each other. Because the PU products have their
molecular weights as high as 2.2ꢀ6.2 ꢁ 104 g molꢀ1, the
3
equimolarity of NCO and OH was reached and the activity
of BPO is high enough to serve as a chain extender. Careful
comparison shows that the molar amounts of HDI and
BPO in the PUs are less than the designed values with
respect to PLA-diol andthe totals of[PLA-diol] and[BPO]
are always more than [HDI], probably due to existence of
impurities in PLA-diol, such as monohydroxyl PLA.
(2) As described above, the purity of BPO is another key factor
to influence PU preparation. Unpurified BPO always led
to cross-linking and gel formation. As shown in Table 3 the
PDIs of all samples were lower than 2 and no gelation was
observed during PU preparations so that there was little
pentaerythritol impurity in the BPO prepared.
’ RESULTS AND DISCUSSION
Synthesis of PUs. A series of PUs were prepared from PLA-
diol, HDI, and BPO (Scheme1). The PLA-diols were prepared in
our own laboratory by ring-opening polymerization of L-lactide
initiated by BDO, as published earlier.19 By adjusting the molar
ratio of L-lactide to BDO, three PLA-diol samples were obtained.
1
Their molecular weights determined by H NMR were 4060,
(3) Furthermore, solvent also plays a key role in PU synthesis.
In our earlier works, toluene was used for PU preparation
due to its high boiling point and its capability of dissolving
polyols. However, BPO cannot dissolve in toluene even at
120 °C. Several solvents were tried and THF was finally
chosen as a reaction solvent.
(4) The final factor is the reaction temperature. Considering
the lower reaction rate in the THF system than in toluene,
90 °C was finally chosen to realize rapid enough polym-
erization and to avoid possible cross-linking.
6060, and 7950 g molꢀ1, respectively. HDI was chosen because
3
of its aliphatic structure and lower toxicity compared to the
aromatic counterparts. BPO was used as a chain extender because
its two protected hydroxyl groups can be released to endow the
PU with OH-functionality. To prepare the desired PUs, four
factors were controlled:
(1) Equimolar feed ratio of [NCO]/[OH] is critical to ensure
high molecular weight of PUs. Table 1 lists molar ratios of
PLA-diol/HDI/BPO in reaction feeds and in PU products
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Table 2. Reaction of PU4K143 with CF3COOH Followed by
GPC and 1H NMR
b
time
deprotection ratio
(mol %)a
Mn
(104 g molꢀ1
)
Mw/Mn
b
entry
(min)
3
1
2
3
4
5
0
0
2.22
1.81
1.85
2.00
2.03
1.53
1.43
1.66
1.40
1.30
5
81
85
94
96
10
15
20
a Determined by 1H NMR. b Determined by GPC.
Figure 1. FT-IR spectra of PLA4K (A), PU4K143 (B), and
PUOH4K143 (C).
Figure 3. GPC curves of PU4K143 at different time points during the
deprotection.
NMR peaks of the benzal groups at 7.44, 7.33, and 5.40
ppm were weakened during the reaction. By measuring the
relative intensities of these peaks, the deprotection ratio can be
calculated. Table 2 lists the deprotection ratio at different time
intervals. It is seen that 81% of the benzal groups were removed
in the first 5 min and the deprotection ratio reaches 96% in 15
min for PU4K143, which contains the highest amount of BPO
among all samples prepared. Figure 3 shows the GPC traces of
the reaction products at different time intervals and their Mn and
distribution index are listed in Table 2. In the first 5 min, the Mn
decreased due to the leaving of the benzal groups. After 5 min, the
Mn increased gradually from 1.81 ꢁ 104 to 2.03 ꢁ 104, although
the benzal groups kept losing, evidenced by the 1H NMR data,
probably due to cross-linking or H-bond formation between the
new-born OH groups and the urethane units on the PU chains.20
In fact, as shown in Figure 1C, after the deprotection, a new peak
at 1701 cmꢀ1 appears, the width of the peak at 1755 cmꢀ1 (CdO
stretching) is wider, and the peak at 1537 cmꢀ1 (amide II) shifts
toward a higher wavenumber. These are the evidence of H-bond-
ing. Similarly, the deprotection reactions of other samples were
examined. The results are listed in Table 3. It is seen that 15 min
is enough to remove the benzal groups. All samples can be
divided into two groups according to their Mn changes during the
deprotection. Molecular weights of PU4K132, PU6K121,
PU6K132, PU6K143, and PU8K143 increase after deprotection,
while those of others decrease. The reasons for this difference are
not clear. It is safe to say that there are two trends in the system:
benzal leaving plus degradation of PU chains under acidic
conditions, which lead to a Mn decrease; and cross-linking plus
H-bond formation, which lead to Mn increase. The above results
imply that one of the two trends prevails over the other in a
certain sample.
Figure 2. 1H NMR spectra of PU4K143 at different time intervals
during the deprotection reaction.
Structural Characterization of PUs. Structural characteriza-
tion of PUs was performed by FT-IR and 1H NMR. To illustrate the
transformation from PLA-diol to PU, FT-IR spectra of PLA-diol and
PU4K143 are shown in Figure 1. PLA-diol displays two character-
istic peaks at 3544 (OH stretching) and 1755 cmꢀ1 (CdO
stretching). After the polymerization, the 3544 cmꢀ1 peak disap-
pears, while two new peaks appear at 3401 (NH stretching) and
1526 cmꢀ1 (amide II) in PU4K143, indicating formation of PU.
This is in agreement with the literature.19 The 1H NMR spectrum of
PU4K143 is shown in Figure 2. The peaks i at 7.44 and 7.33
ppm and h at 5.40 ppm are assigned to the protons of benzal
protective groups. The peaks marked with a (1.6 ppm) and b (5.1
ppm) can be assigned to the protons in PLA repeat units. The peaks
c(3.13ppm), e(1.55ꢀ1.48 ppm), and d (1.30 ppm) are assigned to
protons of the hexamethylene group adjacent to the urethane
linkages. The peaks marked with f (4.44ꢀ4.13 ppm) and g (3.86
ppm) are assigned to the derived structure of pentaerythritol. In
short, all the 1H NMR signals of the PLA-diol, HDI and BPO units
can be found in the PU synthesized.
Preparation of PUOHs. CF3COOH as an effective deprotec-
tion reagent is widely used to realize the transformation from
acetal groups to vicinal-diols. In this work, the benzal groups in
the PU were removed with CF3COOH to get free OH groups
at ice bath temperature. A large excess of CF3COOH was
used to ensure the complete removal of the protective
1
groups. The reaction was followed by H NMR and GPC
1
measurements. As shown in Figure 2, the characteristic H
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Table 3. GPC Data of PUs before and after Deprotection for 15 Min
before deprotection
after deprotection
Mna (104 g molꢀ1
)
Mw/Mn
Mna (104 g molꢀ1
)
Mw/Mn
deprotection ratio in 15 min (mol %)b
a
a
3
3
PU4K121
PU4K132
PU4K143
PU6K121
PU6K132
PU6K143
PU8K121
PU8K132
PU8K143
6.22
2.86
2.22
5.11
4.23
4.65
3.41
4.90
3.51
1.67
1.53
1.53
1.52
1.75
1.74
1.71
1.45
1.72
5.06
3.77
2.00
5.28
4.77
4.89
3.20
3.90
3.80
1.68
1.40
1.40
1.39
1.64
1.24
1.64
1.42
1.50
100
87
91
100
84
88
100
100
100
a Determined by GPC. b Measured by 1H NMR.
Table 4. DSC Results (Second Heating Scan) of PUs before
and after Deprotection
before deprotection
after deprotection (i.e., PUOH)
Tg
Tm ΔHm
(°C) (J/g)
Tg
Tm
ΔHm
sample
(°C)
(°C)
(°C)
(J/g)
PLA4K
42.1
50.0
54.0
53.8
45.7
51.9
52.9
54.0
48.1
57.9
57.2
56.3
143 31.8
PU4K121
PU4K132
PU4K143
PLA6K
0
0
54.0
55.7
55.5
141
142
136
3.15
14.3
14.6
0
152 32.3
149 1.5
146 0.4
0
PU6K121
PU6K132
PU6K143
PLA8K
57.8
56.0
57.1
149
146
149
5.4
18.2
4.1
Figure 4. DSC results of PUs (A: PU4K121, B: PU6K121, C:
PU8K121) and PUOHs (a: PUOH4K121, b: PUOH6K121, c:
PUOH8K121).
156 34.8
152 0.8
150 0.3
0
PU8K121
PU8K132
PU8K143
58.6
57.8
57.6
150
149
155
15.2
13.9
16.2
Thermal Properties. The major change in PUs after deprote-
cion is the formation of hydroxyl groups, which strengthen the
H-bonding interaction between molecular chains, both within
the hard phase and between the hard and soft phases and, thus,
affect the microphase separation in PUs and crystallization of
PLA chains. Therefore, the thermal properties of PLA-diols and
those of PUs before and after deprotection were investigated by
DSC. Figure 4 collects all DSC curves of the samples with molar
ratio of PLA-diol/HDI/BPO of 1:2:1. It is seen that the Tg
increases with increasing Mn of PLA-diols in PUs; deprotection
leads to Tg increase; the endothermic Tm peak increases with
increasing Mn of PLA-diols in PUs; and deprotection leads to
enhancement of both cold-crystallization peak and melting peak.
As shown in Table 4, Tg of PLA-diols increases with increasing
molecular weight of PLA-diols and is lower than Tg of PUs.
These changes can be explained as follows. Tg is Mn-dependent
melting peaks implies reduced constraint exerted by the hard
segments. In other words, the H-bonds formed from the free OH
groups within the hard microphases are predominant.
Similar DSC measurements were performed for PUs and
PUOHs of other compositions. For a given composition, for
example, 1:3:2 or 1:4:3, similar results (PLA length dependence
of Tg and crystallinity and deprotection-caused enhancement of
crystallinity) are observed as in the case of PLA-diol/HDI/BPO
= 1:2:1, as shown in Table 4. Comparison between the three
compositions show that higher hard segment content corre-
sponds to higher Tg, lower Tm and ΔHm, for all PU samples.
Especially in the case of PLA-diol/HDI/BPO = 1:4:3, no melting
endotherms are observed at all. This can be ascribed to the
contribution of the higher constraints from the hard micro-
phases. After deprotection, all PUOH samples exhibit melting
peaks, indicating less constraint applied to the crystallization of
PLA segments, possibly due to more thorough microphase
separation between the hard and soft segments in the systems.
Hydrophilicity of PUOHs. To investigate the hydrophilicity of
PUs after deprotection, water contact angles of PU4K121,
PU4K132, and PU4K143 were measured before and after deprotec-
tion. The sample films were prepared by solution-casting on glass
slides and by annealing at 80 °C for 5 h to make free hydroxyl groups
move outside. As shown in Table 5, the water contact angles of
PUOH4K series are about 5 °C lower than those of the PU4K
series. To explain the change of hydrophilicity of PUs after
deprotection, XPS was adopted to determine the oxygen content
over the Mn range below 10000 g molꢀ1 for pure PLAs;21 due to
3
the microphase separation and the interphase interaction, PLA
segment mobility is further depressed by the hard phases. That is
why the Tg of PUs increases with PLA length. After deprotection,
free OH groups formed enhance H-bond formation between
hard and soft segments, and therefore bring about more hin-
drance to PLA chain movements, leading to higher Tg. In a PU
system, the PLA segments crystallize under a constraint condi-
tion as a result of microphase separation. Longer PLA segment
means less hard segment ratio and less constraint. Therefore,
the areas of the melting peak increase with PLA-diol for both
PUs and PUOHs. Enlargement of both cold-crystallization and
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Table 5. Water Contact Angles (°) of PU and PUOH Samples
sample
before deprotection
after deprotection (i.e., PUOH)
PU4K121
PU4K132
PU4K143
88.3
88.4
88.5
84.0
84.2
83.4
Table 6. Surface Element Contents of PU and PUOH Films
Based on XPS Analysis
surface element contents (at %)
samples
C
O
PU4K121
69.84
65.95
67.34
65.37
63.90
66.76
30.14
34.05
32.66
34.63
36.10
33.24
PU4K132
PU4K143
PUOH4K121
PUOH4K132
PUOH4K143
Figure 6. Fluorescent (left) and bright field (right) images of
PUOH8K132ꢀN3 under UV (A), PUOH8K132 under UV (B),
PUOH8K132ꢀN3 under darkness (C), and PUOH8K132 under dark-
ness (D) after the immunofluorescent assay procedure described in
the text.
Figure 5. FT-IR spectra of 4-azidobenzoic acid (a), PUOH8K132 (b),
and PUOH8K132ꢀN3 (c).
on the film surface. As shown in Table 6, the oxygen contents on
the surface of PUOH series are higher than those of PU4K series.
Therefore, the enhanced hydrophilicity is attributed to the
existence of the free OH groups on the sample surface as well
as in the hard microphases. The enhanced hydrophilicity would
help improve the biocompatibility of the PU materials.
PUOH8K132ꢀN3 (c). The new absorption peak at 2125 cmꢀ1 in
spectrum (c) indicates successful incorporation of the N3
functionality onto the PUOH8K132 chains.
PUOH8K132ꢀN3 is expected to show photoactivity as its
phenyl azide groups can react with XꢀH bonds (X = C, N, O)
under irradiation of 254 nm UV light.14 To demonstrate fixation
of protein molecules on the polymer surface and to check
harmful effect of the 254 nm UV irradiation on proteins,
PUOH8K132ꢀN3 was electrospun into nanofibers, and the
fibers were allowed to react with mouse IgG under UV light
for 3 min. After thorough rinse, the fibers were allowed to react
with FITC-labeled goat anti(mouse IgG) for 30 min in darkness,
followed by thorough rinsing and CLSM observation. For
comparison, the PUOH8K132ꢀN3 fibers were allowed to react
with mouse IgG under darkness and then with FITC-labeled goat
anti(mouse IgG) similarly, and the same test was carried out for
electrospun fibers of PUOH8K132 both under and without UV
irradiation. As is shown in Figure 6, after going through these test
procedures, only the first procedure resulted in fluorescent fiber
4-Azidobenzoic Acid Grafting and Protein Fixation. Many
works focus on the modification of PUs. The characteristics of
PUs can be improved by incorporating active groups onto PUs.
The active groups can be directly introduced onto the chain
extenders or diisocyanate units before polymerization of PUs or
by further reaction of these active groups on PU molecular chains
with desired molecules. In the present study, 4-azidobenzoic acid
was attached to the PUOH chains via esterification between the
free OH groups and the carboxyl group on 4-azidobenzoic acid in
the presence of DCC and under a mild condition. When molar
ratio of 4-azidobenzoic acid to free hydroxyl groups is 1.1, the
1
graft ratio of 4-azidobenzoic acid determined by H NMR is
35.7% (mol/mol). Figure 5 shows FTIR spectra of 4-azidoben-
zoic acid (a), PUOH8K132 (b), and their reaction product
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Biomacromolecules
ARTICLE
products (Figure 6A). Two conclusions can be drawn from
Figure 6: (1) For fixation of mouse IgG onto the electrospun
fibers, two conditions are necessary, that is, phenyl azide and UV
irradiation. Absence of either one or both of them leads to failure
as shown in Figure 6BꢀD; (2) Although the fibers and protein
solutions were irradiated with 254 nm light, the fixed mouse IgG
still retains its own bioactivity under the experimental conditions.
Otherwise, it cannot combine goat anti(mouse IgG) and the fiber
cannot show FITC fluorescence.
It is noticed that several surface modifications have been
reported in the literature,22 such as those via biotin/avidin
coupling or azide/triple bond coupling. Compared to these
techniques, the present procedure based on the photoactive
phenyl azide displays at least two advantages: (1) It is not
necessary to modify the protein molecules themselves before
coupling; (2) The reaction conditions are mild and no toxic
catalysts are involved.
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’ CONCLUSION
A novel strategy for the preparation of biodegradable poly-
urethane containing free hydroxyl groups (PUOH) was success-
fully developed, that is, to use a benzal-containg diol BPO as a
chain extender to prepare a benzal-containing PU and to
deprotect the OH groups to get the OH-containing PU
(PUOH) by using CF3COOH. CF3COOH was highly effective
for removal of benzal groups on the BPO residues under a mild
condition. The deprotection ratio could reach 100% in a few
minutes. The length of the PLA segments and the molar ratio of
BPO/PLA-diol determine the properties of PUs and PUOHs.
The OH groups on PUOH were further esterified by 4-azido-
benzoic acid. By using the phenyl azide groups formed as a
photoactive moiety, model protein mouse IgG was immobilized
on PUOH surface and the fixed mouse IgG retained its bioactiv-
ity to combine with goat anti(mouse IgG). Therefore, the
PUOHs synthesized may find biomedical applications in immu-
nofluorescence assay and related fields such as protein fixation
and identification, enzyme fixation and catalysis, and so on.
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Biomaterials 2008, 29, 1118–1126.
’ AUTHOR INFORMATION
Corresponding Author
*Tel. and Fax: þ86-431-85262775. E-mail: xbjing@ciac.jl.cn.
’ ACKNOWLEDGMENT
The financial support was provided by the National Nature
Science Foundation of China (Project No. 51003101), by “100
Talents Program” of the Chinese Academy of Sciences (No.
KGCX2-YW-802), and by the Ministry of Science and Technol-
ogy of China (“973 Project”, No. 2009CB930102; “863 Project”,
No. 2007AA03Z535).
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