Supporting Information for the paper entitled:
Carbohydrate-based crosslinking agents: Potential use in hydrogels
STEFAN M. PATERSON,1 JASPER CLARK,1 KEITH A. STUBBS,1 TRAIAN V. CHIRILA,2-5
MURRAY V. BAKER1,2
1
Chemistry M313, School of Biomedical, Biomolecular and Chemical Sciences, The University of
Western Australia, Crawley, W.A. 6009, Australia
2
Queensland Eye Institute, 41 Annerley Road, South Brisbane, Queensland 4101, Australia
3
Faculty of Science and Technology, Queensland University of Technology, Brisbane, Queensland
4001, Australia
4
Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St Lucia,
Queensland 4072, Australia
5
Faculty of Health Sciences, University of Queensland, Herston, Queensland 4006, Australia
Correspondence to: M. V. Baker (E-mail: murray.baker@uwa.edu.au) or K. A. Stubbs (E-mail:
keith.stubbs@uwa.edu.au).
Contents
1. Materials
S2
2. Synthesis of crosslinking agents
S3
3. Polymerization
S9
4. SEM
S9
5. References
S10
S1
1. Materials
Nuclear magnetic resonance spectra were recorded using Varian 400 (399.9 MHz for 1H and 100.5
MHz for 13C) Bruker 500 (500.1 MHz for 1H and 125 for 13C 150.15 MHz) or Bruker 600 (600.1 MHz
for 1H and 150.9 MHz for 13C) spectrometers at ambient temperatures. 1H and 13C spectral assignment
were made with the aid of DEPT, HMBC, and HSQC experiments. 1H and
13
C chemical shifts were
references to solvent signals. Optical rotation measurements were preformed using a Perkin Elmer 141
polarimeter. High resolution FAB-MS spectra were recorded using VG Auto Mass spectrometer. All
solvents were re-distilled prior to use. Anhydrous solvents were obtained by distillation from a suitable
drying agent under an inert atmosphere. Flash chromatography was preformed using Merck silica gel
60 (40-63µm). Size-exclusion chromatography was performed using LH-20 Sephadex resin (Aldrich).
All reactions were conducted under an inert atmosphere unless stated otherwise. 2-Hydroxyethyl
methacrylate (HEMA) (Bimax, Inc, USA > 99.0%) was distilled in batches (b.p. 38-39 °C /0.1 mm Hg)
and stored at -20 °C until use. 1,2,3,4,6-Penta-O-acetyl-β-D-glucose (Aldrich, 99%), 2-bromoethanol
(Aldrich, 95%), 2,2-dimethoxy-2-phenylacetophenone (DPAP) (Igracure 651, Aldrich, 97 %),
tetraethyleneglycol dimethacrylate (TEGDMA) (Fluka), NaN3 (Fluka), BF3.Et2O (Fluka), palladium on
carbon (Fluka, puriss 10 % Pd), (1-(3-dimethylaminopropyl)-3-ethyl)carbodiimide hydrochloride
(EDC.HCl, Alfa-Aeser), acetic anhydride (Ajax), triphenylmethyl chloride (Sigma), methanesulfonyl
chloride (Fluka), sodium methoxide (Sigma) and N-hydroxysuccinimide (Fluka) were all used as
received. N-Methacryloyl succinimide (MA-NHS) was prepared according to literature procedures.1
S2
2. Synthesis of crosslinking agent 1
2-Bromoethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside 3
Boron trifluoride diethyl etherate (7.1 ml, 50 mmol) was added dropwise to a solution of 2bromoethanol (1.6 g, 12 mmol) and 1,2,3,4,6 penta-O-acetyl-β-D-glucose 2 (4.0 g, 10 mmol) in dry
CH2Cl2 (30 mL) at 0 °C. The solution was then stirred at room temperature (12 h). The mixture was
then concentrated to yield a light brown residue which was subsequently extracted with EtOAc (3  20
mL). The organic fractions were then combined, washed with H2O (20 mL), brine (10 mL) and dried
(MgSO4). Concentration, followed by flash chromatography of the residue (EtOAc/hexane, 2:3)
yielded 3 as a light brown solid (4.1 g 87%). The 1H and 13C NMR spectra were consistent with those
previously reported.2
2-Azidoethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside 4
Sodium azide (2.9 g, 45 mmol) was added to a solution of 3 (4.1 g, 9.0 mmol) in DMF (100 mL) and
stirred (12 h) at 70 °C. The solution was then concentrated to yield a white solid which was
subsequently extracted with EtOAc (3  20 mL). The organic fractions were then combined, washed
with H2O (20 ml), brine (20 mL) and dried (MgSO4). Concentration followed by followed by flash
chromatography of the residue (EtOAc/hexane, 2:3) yielded 4 as a yellow solid (3.4 g, 90%). Rf: 0.57
(SiO2, hexanes/EtOAc, 1:1). Mp: 88-90oC (EtOAc/hexane), [α]D -25.7° (CHCl3). max (KBr)/cm-1
2109 (N3), 1759, 1742 (OCOCH3). 1H NMR (600 MHz, CDCl3) δ 5.21 (dd, J = 9.4, 9.6 Hz 1H), 5.09
(dd, J1 = 9.6 Hz, J2 = 10.0 Hz, 1H), 5.02 (dd, J1 = 7.9 Hz, J2 = 9.4 Hz, 1H), 4.95 (d, J = 7.9 Hz, 1H),
4.24 (dd, J1 = 4.7 Hz, J2 = 12.0 Hz, 1H), 4.15 (dd, J1 = 2.4 Hz, J2 = 12.0 Hz, 1H), 4.02 (ddd, J1 = 1.8
Hz, J2 = 4.0 Hz, J3 = 12.0 Hz, 1H), 3.75-3.65 (m, 2H), 3.49 (ddd, J1 = 3.4 Hz, J2 = 9.0 Hz, J3 = 12.0
Hz, 1H), 3.28 (ddd, J1 = 4.6 Hz, J2 = 5.0 Hz, J3 = 14.0 Hz 1H), 2.08 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H),
S3
1.98 (s, 3H).
13
C HMR (150 MHz, CDCl3) δ 170.6, 170.2, 169.3 (C=O) 100.6 (C1) 72.7, 71.9, 71.4,
68.2, 68.5, 61.8 (C2,3,4,5,6,CH2), 50.5 (CH2N3), 20.7, 20.6, 20.5, 20.5 (CH3). HRMS (m/z): calcd for
C16H23N3O10, 417.1337; found, 417.1383 (M)+.
2-Azidoethyl β-D-glucopyranoside 5
Sodium methoxide (20 mg, 0.37 mmol) was added to a solution of 4 (5.9 g, 140 mmol) in methanol (30
mL) and stirred (1 h). The solution was neutralised with resin (Amberlite IR-120[H+]) and then filtered
and concentrated yielding the title compound 5 as a colourless oil (3.5 g, 98%). Rf: 0.35 (SiO2,
EtOAc/MeOH, 9:1). [α]D -15.7° (MeOH) 1H-NMR (600 MHz, D2O) δ 4.31 (d, J = 7.8 Hz, 1H), 4.02
(ddd, J1 = 3.3 Hz, J2 = 3.3 Hz, J3 = 6.6 Hz, 1H), 3.87 (dd, J1 = 1.6 Hz, J2 = 12.0 Hz, 1H), 3.79-3.70 (m,
3H), 3.67 (dd, J1 = 2.0 Hz, J2 = 4.0 Hz, 1H), 3.50-3.44 (m, 2H), 3.27 (dd, J1 = 2.0 Hz, J2 = 9.0 Hz, 1H),
3.21-3.10 (br s, 4H) 3.18 (dd, J1 = 7.8 Hz, J2 = 9.0 Hz, 1H). 13C-NMR (150 MHz, CDCl3) δ 104.5 (C1)
78.0, 75.1, 71.6, 68.8, 69.3, 61.8 (C2,3,4,5,6,CH2), 49.4 (CH2N3). HRMS (m/z): calcd for C8H15N3O6,
249.0961; found, 249.0963 (M)+.
2-Azidoethyl 6-O-(triphenylmethyl)-β-D-glucopyranoside 6
Triphenylmethyl chloride (4.1 g, 150 mmol) was added to a solution of 5 (3.5 g, 133 mmol),
triethylamine (3.3 mL), DMAP (81 mg, 0.70 mmol) in dry CH2Cl2 (30 mL) and the mixture stirred (12
h). The mixture was then quenched (MeOH) and concentrated to yield a yellow solid. This was
subsequently extracted with EtOAc (3  30 mL) and the organic fractions were then combined, washed
with H2O (20 mL), brine (20 mL) and dried (MgSO4). Concentration followed by followed by flash
chromatography of the residue (EtOAc) yielded 6 as a yellow oil (4.7 g, 74%). Rf: 0.70 (SiO2, EtOAc).
[α]D - 37.9° (CHCl3). 1H NMR (600 MHz, CDCl3) δ 7.47-7.42 (m, 6H), 7.34-7.28 (m, 6H), 7.28-7.22
(m, 3H), 4.36 (d, J = 7.7 Hz, 1H), 4.07 (ddd, J1 = 3.0 Hz, J2 = 5.4 Hz, J3 = 10.0 Hz, 1H), 3.75 (ddd, J1
= 3.0 Hz, J2 = 7.7 Hz, J3 = 10.0 Hz, 1H), 3.60-3.50 (m, 3H), 3.47-3.34 (m, 5H), 3.15 (s, 3H). 13C HMR
S4
(150 MHz, CDCl3) δ 143.5, 128.6, 127.9, 127.1 (Ar), 102.7 (C1), 77.3, 76.1, 74.2, 73.5, 61.9, 68.5,
64.0 (CPh3,C2,3,4,5,6,CH2), 50.7 (CH2N3). HRMS (m/z): calcd for C29H30N3O6, 492.2135; found,
492.2164 (M+H)+.
2-Azidoethyl 2,3,4-tri-O-acetyl-6-O-(triphenylmethyl)-β-D-glucopyranoside 7
Acetic anhydride (2.0 g, 20 mmol) was added to a solution of the alcohol 6 (2.4 g, 4.9 mmol) in
pyridine (20 mL) and the mixture stirred (12 h). The mixture was then quenched (MeOH) and
concentration of the solution yielded a colourless residue that was subsequently extracted with EtOAc
(3  20 mL). The organic fractions were then combined, washed with H2O (30 ml), brine (20 ml) and
dried (MgSO4). Concentration followed by flash chromatography of the residue (EtOAc/hexane, 2:3)
yielded the title compound 27 as a white solid (2.8 g, 87%). Rf: 0.61 (SiO2, hexanes/EtOAc, 4:1). Mp:
154-156 °C (EtOAc/hexane) [α]D +1.0° (CHCl3). 1H NMR (400 MHz, CDCl3) δ 7.45-7.40 (m, 5H),
7.30-7.18 (m, 10H), 5.15 (dd, J1 = 6.5 Hz, J2 = 6.5 Hz, 1H), 5.15 (dd, J1 = 12.1 Hz, J2 = 18.0 Hz, 1H),
5.06 (dd, J1 = 6.4 Hz, J2 = 7.6 Hz, 1H), 4.58 (d, J = 7.6 Hz, 1H), 4.08 (ddd, J1 = 3.6 Hz, J2 = 4.5 Hz, J3
= 10.0 Hz, 1H), 3.75 (ddd, J1 = 3.1 Hz, J2 = 7.3 Hz, J3 = 10.0 Hz, 1H), 3.58-3.50 (m, 2H), 3.32 (ddd, J1
= 3.1 Hz, J2 = 4.2 Hz, J3 = 12.6 Hz, 1H), 3.25 (dd, J1 = 4.2 Hz, J2 = 18.0 Hz, 1H), 3.09 (dd, J1 = 4.7 Hz,
J2 = 18.0 Hz, 1H), 2.04 (s, 3H), 1.97 (s, 3H), 1.71 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 170.6, 169.5,
168.9 (C=O) 143.5, 128.6, 127.7, 127.0 (Ar) 100.6 (C1), 76.6, 73.4, 73.5, 71.2, 68.2, 68.6, 61.8
(CPh3,C2,3,4,5,6,CH2), 50.5 (CH2N3), 20.7, 20.6, 20.3 (CH3). HRMS (m/z): calcd for C33H36N3O9,
617.2373; found, 617.2342 (M)+.
2-Azidoethyl 2,3,4-tri-O-acetyl-β-D-glucopyranoside 8
The triacetate 7 (2.7 g, 4.5 mmol) was dissolved in AcOH:H2O (4:1, 50 mL) and the mixture stirred
overnight at 80 °C. The solution was concentrated to yield a yellow residue that was subsequently
S5
extracted with EtOAc (3  30 mL). The organic fractions were combined, washed with H2O (30 mL),
brine (20 mL) and dried (MgSO4). Concentration followed by flash chromatography of the residue
(EtOAc/hexane, 3:7) yielded 8 as a white solid (1.4 g, 81%). Rf: 0.48 (SiO2, hexanes/EtOAc, 3:2). Mp:
91-94°C (EtOAc/hexane) [α]D -45.6° (CHCl3). 1H NMR (400 MHz, CDCl3) δ 5.25 (dd, J1 = 8.8 Hz, J2
= 9.4 Hz, 1H), 5.04 (dd, J1 = 8.8 Hz, J2 = 9.4 Hz, 1H), 4.99 (dd, J1 = 7.6 Hz, J2 = 9.4 Hz, 1H), 4.61 (d,
J = 7.6 Hz, 1H), 4.03 (ddd, J1 = 2.9 Hz, J2 = 5.2 Hz, J3 = 10.4 Hz, 1H), 3.75 (dd, J1 = 1.4 Hz, J2 = 11.8
Hz, 1H), 3.70 (ddd, J1 = 2.9 Hz, J2 = 7.4 Hz, J1 = 10.4 Hz, 1H), 3.60 (dd, J1 = 4.4 Hz, J2 = 11.8 Hz,
1H), 3.53 (ddd, J1 = 1.4 Hz, J2 = 4.4 Hz, J3 = 8.8 Hz, 1H), 3.47 (ddd, J1 = 2.9 Hz, J1 = 7.4 Hz, J3 = 11.8
Hz, 1H), 3.30 (ddd, J1 = 2.9 Hz, J2 = 5.2 Hz, J3 = 11.8 Hz, 1H), 2.05 (s, 3H), 2.04 (s, 3H), 2.00 (s, 3H),
2.03 (br s, 1H). 13C NMR (100 MHz, CDCl3) δ 170.2, 170.1, 169.4 (C=O) 100.5 (C1), 74.1, 73.6, 71.2,
68.4, 68.5, 61.3 (C2,3,4,5,6,CH2), 50.5 (CH2N3), 20.7, 20.6, 20.3 (CH3). HRMS (m/z): calcd for
C14H21N3O9, 375.1278; found, 375.1253 (M)+.
2-Azidoethyl 2,3,4-tri-O-acetyl-6-O-(methanesulfonyl)-β-D-glucopyranoside 9
Methanesulfonyl chloride (468 µL, 6.05 mmol) was added to a solution of the alcohol 8 (1.4 g, 4.0
mmol) and triethylamine (1.1 mL) in dry CH2Cl2 (20 mL) at -30 °C. The solution was then stirred and
allowed to warm to room temperature (2 h). Concentration of the solution yielded a yellow residue that
was subsequently extracted with Et2O (3 x 30 mL). The organic fractions were then combined, washed
with H2O (20 ml), brine (10 ml) and dried (MgSO4). Concentration followed by flash chromatography
of the residue (EtOAc/hexane, 1:3) yielded the 9 as white needles (1.3 g, 72%). Rf: 0.62 (SiO2,
hexanes/EtOAc, 3:2). Mp: 83-86°C (EtOAc/hexane). [α]D -21.7° (CHCl3) max (KBr)/cm-1 2109 (N3),
1237 (O=S=O). 1H NMR (400 MHz, CDCl3) δ 5.21 (dd, J1 = 7.5 Hz, J2 = 9.1 Hz, 1H), 5.04 (dd, J1 =
9.1 Hz, J2 = 9.6 Hz, 1H), 5.00 (dd, J1 = 7.5 Hz, J2 = 8.0 Hz, 1H), 4.61 (d, J = 8.0 Hz, 1H), 4.29 (d, J =
2.9 Hz, 2H), 4.02 (ddd, J1 = 2.9 Hz, J2 = 4.4 Hz, J3 = 10.3 Hz, 1H), 3.80 (ddd, J1 = 2.9 Hz, J2 = 2.9 Hz,
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J3 = 9.6 Hz, 1H), 3.70 (ddd, J1 = 2.9 Hz, J2 = 7.4 Hz, J3 = 10.3 Hz, 1H), 3.47 (ddd, J1 = 2.9 Hz, J2 = 7.4
Hz, J3 = 13.3 Hz, 1H), 3.30 (ddd, J1 = 2.9 Hz, J2 = 4.4 Hz, J3 = 13.3 Hz, 1H), 3.05 (s, 3H), 2.05 (s, 3H),
2.04 (s, 3H), 1.99 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 170.2, 169.5, 169.3 (C=O) 100.5 (C1) 72.4,
71.7, 70.9, 68.1, 68.6, 66.5 (C2,3,4,5,6,CH2), 50.5 (CH2N3), 20.7, 20.6, 20.3 (CH3). HRMS (m/z): calcd
for C15H23N3O11S, 453.1053; found, 453.1053 (M)+.
Azidoethyl 2,3,4-tri-O-acetyl-6-azido-6-deoxy-β-D-glucopyranoside 10
Sodium azide (950 mg, 14.0 mmol) was added to a solution of the mesylate 9 (1.3 g, 2.90 mmol) in
DMF (30 mL) at 0 °C. The solution was then stirred at 70 °C (12 h). Concentration yielded a yellow
residue that was subsequently extracted with EtOAc (3  30 mL). The organic fractions were
combined, washed with H2O (30 mL), brine (20 mL) and dried (MgSO4). Concentration followed by
flash chromatography of the residue (EtOAc/hexane, 1:4) yielded the title compound 10 as a white
solid (1.10 g, 92%). Rf: 0.55 (SiO2, hexanes/EtOAc, 3:2). Mp: 79-81oC (EtOAc/hexane). [α]D -65.4o
(CHCl3) max (KBr)/cm-1 2148, 2113 (N3). 1H NMR (400 MHz, CDCl3) δ 5.20 (dd, J1 = 9.3 Hz, J2 =
9.7 Hz, 1H), 5.01 (dd, J1 = 8.0 Hz, J2 = 9.3 Hz, 1H), 4.97 (dd, J1 = 9.7 Hz, J2 = 10.2 Hz, 1H), 4.62 (d, J
= 8.0 Hz, 1H), 4.05 (ddd, J1 = 2.6 Hz, J2 = 3.4 Hz, J3 = 10.4 Hz, 1H), 3.72-3.66 (m, 2H), 3.48 (ddd, J1
= 2.6 Hz, J2 = 10.4 Hz, J3 = 13.9 Hz, 1H), 3.40 (dd, J1 = 6.9 Hz, J2 = 13.0 Hz, 1H), 3.28 (ddd, J1 = 2.6
Hz, J2 = 3.4 Hz, J3 = 13.9 Hz, 1H), 3.20 (dd, J1 = 1.7 Hz, J2 = 13.0 Hz, 1H), 2.03 (s 3H), 2.02 (s 3H),
1.99 (s 3H).
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C NMR (100 MHz, CDCl3) δ 170.1, 169.5, 169.3 (C=O) 100.4 (C1), 73.7, 72.4, 71.0,
68.5, 69.5 (C2,3,4,5,CH2), 51.1, 50.4 (C6, CH2N3), 20.7, 20.6, 20.3 (CH3). HRMS (m/z): calcd for
C14H20N6O8, 400.1376; found, 400.1343 (M)+.
2-Azidoethyl 6-azido-6-deoxy-β-D-glucopyranoside 11
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Sodium methoxide (20 mg, 0.37 mmol) was added to a solution of 10 (1.10 g, 2.70 mmol) in methanol
(20 mL) and stirred (1 h). The solution was neutralised with resin (Amberlite IR-120[H+]) and then the
solution was then filtered and concentrated yielding the title compound as a white solid (650 mg, 84%).
Rf: 0.36 (SiO2, EtOAc). Mp: 71-74°C (MeOH) [α]D -44.3° (MeOH) max (KBr)/cm-1 2109, 2089 (N3).
1
H NMR (400 MHz, CDCl3) δ 3.15 (d, J = 5.7 Hz, 1H), 2.62 (ddd, J1 = 2.5 Hz, J2 = 4.4 Hz, J3 = 10.0
Hz, 1H), 2.44 (ddd, J1 = 2.5 Hz, J2 = 4.4 Hz, J3 = 13.3 Hz, 1H), 2.25- 2.10 (m, 8H), 2.06 (dd, J1 = 4.2
Hz, J2 = 7.1 Hz, 1H), 1.97 (dd, J1 = 5.7 Hz, J2 = 7.1 Hz, 1H), 1.88 (dd, J1 = 4.2 Hz, J2 = 5.7 Hz, 1H).
13
C NMR (100 MHz, CDCl3) δ 100.4 (C1), 75.3, 74.7, 72.9, 68.5, 70.3 (C2,3,4,5,CH2), 50.8, 50.4 (C6,
CH2N3). HRMS (m/z): calcd for C8H15N6O5, 275.1104; found, 275.0192 (M+H)+. Anal. calcd for
C8H14N6O5: C, 37.74; H, 5.70. Found: C, 37.83; H, 5.75%.
2-Aminoethyl 6-amino-6-deoxy-β-D-glucopyranoside 12
Palladium-on-charcoal (10%, 100 mg) was added to a solution of the diazide 11 (650 mg, 2.2 mmol) in
MeOH (20 mL) and the solution stirred under an atmosphere of hydrogen (1 atm., 4 h). The mixture
was then filtered through a celite pad and concentrated yielding the title compound as a colourless oil
(485 mg, 95%) which was used without further purification.
2-Methacryloylamidoethyl 6-deoxy-6-(2-methacrylamido)-β-D-glucopyranoside 1
To a solution of the diamine 12 (255 mg, 1.1 mmol) in a THF:50 mM NaHCO3 (aq) solution (1:1, 15mL)
with triethylamine ( mL) was added a solution of MA-NHS (613 mg, 3.45 mmol) in a THF/H2O
solution (1:1, 10 ml). The solution was stirred (12 h) then concentrated and the resultant aqueous
solution treated with resin (Amberlite IR-120[H+]) and the resin was eluted with MeOH and then
concentrated. Concentration gave a residue which was added to a column of Sephadex (LH-20) and the
column eluted with MeOH. Concentration followed by lyophilization gave a white solid. (120 mg,
31%). [α]D -14.7° (H2O). 1H NMR (500 MHz, d6-DMSO) δ 7.80 (br s, 2H), 6.12 (s, 1H), 5.82 (s, 1H),
S8
5.65 (s, 1H), 5.32 (s, 1H), 4.12 (d, J = 8.0 Hz, 1H), 3.74 (ddd, J1 = 7.5 Hz, J2 = 8.5 Hz, J3 = 11.0 Hz,
1H) 3.57-3.49 (m, 2H), 3.28-3.19 (m, 2H), 3.17-3.11 (m, 2H), 3.00-2.90 (m, 2H) 2.46-2.42 (m, 2H)
2.39-2.35 (m, 2H), 1.93 (s, 3H) 1.84 (s, 3H)
13
C NMR (125 MHz, d6-DMSO) δ 167.9, 167.6 (C=O),
139.8, 139.8 (C=CH2), 119.2, 119.1 (C=CH2), 103.2 (C1), 76.0, 74.2, 73.5, 72.0, 67.5 (C2,3,4,5,CH2),
41.1 39.8 (C6, CH2NH), 18.6, 18.5 (CH3). HRMS (m/z): calcd for C16H27N2O7, 359.1818; found,
359.1803 (M+H)+. Anal. calcd for C16H26N2O8: C, 53.62; H, 7.31. Found: C, 53.58; H, 7.40%
3. Polymerization studies
Polymer hydrogels were prepared as described previously3 by photoinitiated polymerization of HEMA.
Briefly, DPAP (0.1 mol% with respect to HEMA) was added to a 80:20 H2O:HEMA solution
containing 1 or TEGDMA (1 mol% with respect to HEMA) and the solution was irradiated under a UV
lamp (UVP Blak-Ray®, 365 nm, 120 W) for 30 min. After polymerization, the hydrogels were soaked
in water for one week to remove any unreacted monomers, with water being exchanged daily. After
soaking, all polymer samples were cut into 300-µm thick cross-sections (Vibratome 3000) and these
sections were further cut into disks using a 5-mm biopsy cutter. After sectioning, the samples were
carefully transferred with soft plastic tweezers into vials of deionized water where they were stored
until required.
4. SEM
Samples were dehydrated by freeze-drying (Dynavac FD2) and then were mounted on double-sided
carbon tabs and coated with a layer of carbon (approximately 30 nm thick) using a carbon evaporator
(Speedivac 12E6/1178, Edwards High Vacuum LTD). The samples were then imaged by SEM (Zeiss
1555 VF-FESEM) at 3 kV, using a working distance of 6 mm and an aperture of 10 µm. To acquire an
image, frame integration was used to prevent charging on the surface of the polymer.
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References
1. Monge, S, Haddleton, DM. Eur Polym J 2004, 40, 37–45
2. Dahmén J, Frejd T, Grönberg G, Lave T, Magnusson G, Noori G. Carbohydr Res
1983, 116, 303-307.
3. Baker MV, Brown DH, Casadio YS, Chirila TV. Polymer 2009, 50, 5918-5927.
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