a modular and supramolecular approach to bioactive ... · a modular and supramolecular approach to...

Supplementary Information for Nature Materials

A modular and supramolecular approach to bioactive

scaffolds for tissue engineering

Patricia Y.W. Dankers1, Martin C. Harmsen2, Linda A. Brouwer2, Marja J.A. van

Luyn2 and E.W. Meijer1*

1. Laboratory of Macromolecular and Organic Chemistry, Eindhoven University of Technology,

PO Box 513, NL-5600 MB Eindhoven, The Netherlands

*e-mail: [email protected]

2. Department of Pathology and Laboratory Medicine, Medical Biology Section, University

Medical Center Groningen, Hanzeplein 1, NL-9713 GZ Groningen, The Netherlands

1. EXPERIMENTAL

1.1 General methods

General materials. Polycaprolactone diol (Mn = 2.1 kg/mole) was purchased from Acros. The Fmoc-

protected amino acids and the Wang resin for solid-phase peptide synthesis (SPPS) were obtained from

Bachem. Potassium hexafluoro phosphate was obtained from Acros. Commercial products were used

without further purification. All solvents purchased from Acros Chimica or Sigma-Aldrich were of p.a.

quality. Deuterated solvents were obtained from Cambridge Isotope Laboratories. Water was always

demineralized prior to use. Phosphate buffered saline (PBS) tablets were purchased from Sigma

(dissolution of the tablets in water resulted in a 0.01 M phosphate buffer with 0.0027 M potassiumchloride

and 0.137 M sodiumchloride, with pH = 7.4). Sodium azide was obtained from Acros. The trypsin-EDTA

(200 mg/L EDTA, 500 mg/L trypsin) and the EDTA (200 mg/L EDTA) solution was purchased from

BioWhittaker Cambrex Bio Science. The trypan blue was obtained from Biochrom AG.

© 2005 Nature Publishing Group

Cell culture medium. 3T3 mouse fibroblasts were cultured on a 1:1 mixture of Ham’s F-12 with L-

glutamine (BioWhittaker), supplemented with 1% penicillin, 1% streptomycin (Biochrom AG; 1000 u,

10000 µg/mL) and 10% Fetal Bovine Serum (FBS) (Biochrom AG) and of Dulbecco’s Modified Eagle’s

Medium (DMEM) with 1 g/L glucose without L-glutamine (BioWhittaker), supplemented with 1%

penicillin, 1% streptomycin (Biochrom AG; 1000 u / 10000 µg/mL), 1% glutamine (BioWhittaker, 200

mM in 0.85% sodiumchloride solution) and 10% Fetal Bovine Serum (FBS) (Biochrom AG).

Instrumentation. 1H NMR, 13C NMR, 19F NMR (with potassium hexafluoro phosphate as internal

standard) and 2D 1H,1H-COSY spectra were recorded on a Varian Mercury 400 MHz or Varian Inova 500

MHz spectrometer at 298 K. Chemical shifts are given in ppm (δ) values relative to tetramethylsilane

(TMS) or relative to the solvent residual peak. Infrared (IR) spectra were recorded on a Perkin Elmer

Spectrum One FT-IR spectrometer with a Universal ATR Sampling Accessory for solids. ES-QTOF-MS

experiments were recorded on a Q-tof Ultima GLOBAL mass spectrometer (Micromass). Analytical

reversed phase liquid chromatography (RPLC) was performed on a Shimadzu FCV-10 AL VP with a

Shimadzu SCL-10A VP system controller, Shimadzu LC-10AD VP liquid chromatography pumps (with an

Alltima C18 5u (150 mm x 3.2 mm) reversed phase column and gradients of water-acetonitrile,

supplemented with 0.2% trifluoro acetic acid), a Shimadzu DGU-14A degasser and a Shimadzu SPD-

M10A VP diode array detector. Preparative reversed phase liquid chromatography (prep-RPLC) was

performed on a system consisting of the following components: Shimadzu SCL-10A VP system controller

with Shimadzu LC-8A preparative liquid chromatography pumps (with an Alltima C18 5u (150 x 10 mm)

preparative reversed phase column and gradients of water-acetonitrile, supplemented with 0.2% trifluoro

acetic acid), a Shimadzu SIL-10AD VP auto injector, a Shimadzu FRC-10A fraction collector and a

Shimadzu SPD-10AV VP UV-Vis detector. Reversed phase liquid chromatograpy – mass spectroscopy

(RPLC-MS) was performed on a system consisting of the following components: Shimadzu SCL-10A VP

system controller with Shimadzu LC-10AD VP liquid chromatography pumps (with an Alltima C18 3u (50

mm x 2.1 mm) reversed phase column and gradients of water-acetonitrile supplemented with 0.1% formic

acid), a Shimadzu DGU-14A degasser, a Thermo Finnigan surveyor autosampler, a Thermo Finnigan

surveyor PDA detector and a Finnigan LCQ Deca XP Max. Gel permeation chromatography (GPC) was

performed on a Shimadzu FCV-10 AL VP with a Shimadzu SCL-10AL VP system controller, Shimadzu


LC-10AD VP liquid chromatography pumps (with a PL gel 3 µm mixed-E column and chloroform with 5%

methanol as eluent), a Shimadzu DGU-14A degasser, a Shimadzu SPD-10AV UV-Vis detector and a

Polymer Laboratories PL-ELS 1000 detector. Thermal properties were investigated with differental

scanning calorimetry (DSC) on a Perkin Elmer Differential Scanning Calorimeter Pyris 1 with Pyris 1 DSC

Autosampler and Perkin Elmer CCA7 cooling element under a nitrogen atmosphere with heating and

cooling rates of 20ºC/min (samples of 8-12 mg were measured). Material properties were tested with stress-

strain measurements performed on a Zwick Z010 Universal Tensile Tester at an elongation rate of 0.1 min-1

(= 10% strain/min) with a preload of 0.1 N and a load cell of 20 N. Tensile bars with a test section or length

between the clamp region of 22 mm or 13 mm and a cross section of 2-3.5 mm2 or 0.7-0.8 mm2 were

obtained by punching them out of sheets of materials that were prepared by drop casting out of chloroform.

Water contact angle measurements were performed with a Krüss drop shape analysis system DSA 10 with

drop shape analysis 1.10 software. Optical microscopy pictures were taken on a Zeiss Axiovert 25

microscope with a Sony (Cybershot, 3.3 Megapixels) digital still camera DSC-S75 (Carl Zeis ACC

Terminal).

Materials processing. Films were made by solvent casting from chloroform solution or via compression

moulding (at approximately 20ºC above the melting temperature). Melt spinning (at a temperature of 90ºC)

and electrospinning (from chloroform solution) were used to make fibres and meshes. The electrospun

meshes were studied with scanning electron microscopy (SEM) on a Philips XL30 FEG E-SEM under high

vacuum. The grids consisting of filaments with a width down to approximately 220 µm were produced via

Fused Deposition Modelling (FDM)1 at temperatures just below 80ºC.

1.2 Synthesis and characterization of the building blocks

PCLdiUPy polymer. Hydroxy terminated polycaprolactone diol (Mn = 2.1 kg/mole; obtained via ring-

opening polymerization initiated by diethylene glycol) was reacted with the UPy-isocyanate-synthon, 2(6-

isocyanatohexylaminocarbonyl-amino)-6-methyl-4[1H]-pyrimidinone2, making use of a similar procedure

as described before2, resulting in 34 g (12.6 mmole) PCLdiUPy as a white fluffy material with a yield of

86%. PCLdiUPy was characterized with 1H NMR, 13C NMR, IR, DSC and tensile testing.


Mn = 2.7 kg/mole (calculated from 1H NMR). 1H NMR (CDCl3): δ = 13.13 (s, 2H, C-NH-C=N, UPy),

11.86 (s, 2H, CH2NH-(C=O)-NH, UPy), 10.14 (s, 2H, CH2NH-(C=O)-NH, UPy), 5.85 (s, 2H, C=CH,

UPy), 4.90 (s, 2H, CH2NH-(C=O)-OCH2), 4.23 (t, 4H, CH2-(C=O)-OCH2CH2O), 4.06 (t, 2nH, CH2-(C=O)-

OCH2), 3.69 (t, 4H, CH2-(C=O)-OCH2CH2O), 3.24 (m, 4H, CH2NH-(C=O)-NH), 3.16 (m, 4H, CH2NH-

(C=O)-OCH2), 2.31 (m, 2nH, CH2-(C=O)-OCH2), 2.23 (s, 6H, CH3, UPy), 1.64 (m, 4nH, CH2-(C=O)-

OCH2CH2CH2CH2CH2), 1.50 (m, 16H, NH-(C=O)-NH-CH2CH2CH2CH2CH2CH2-NH-(C=O)-O), 1.39 (m,

2nH, CH2-(C=O)-OCH2CH2CH2CH2CH2) ppm. 13C NMR (CDCl3): δ = 173.5, 173.1, 156.7, 156.5, 154.6,

148.2, 106.6, 69.0, 64.4, 64.1, 63.2, 40.6, 39.6, 34.0, 33.9, 29.7, 29.3, 28.7, 28.3, 26.2, 26.1, 25.5, 24.6,

24.5, 24.4, 18.9 ppm. IR (ATR): ν = 2941, 2865, 1729 (C=O stretch), 1699, 1669 (C=O stretch), 1587,

1527 (C=O stretch), 1461, 1418, 1359, 1251, 1162 (C-O stretch), 1105 cm-1. DSC: Tg = –59ºC, Tm = 41ºC

(∆H = 15 J/g), Tm = 64ºC (∆H = 5 J/g). Tensile testing: E = 49 ± 2 MPa, εbreak = 26.7 ± 1.8%, σbreak = 3.3 ±

0.1 MPa.

Peptides and UPy-peptides. The GRGDS (the loading of the Wang resin with Fmoc-Ser(tBu)-OH was

0.63 mmole/g) and PHSRN (the loading of the Wang resin with Fmoc-Asn(Trt)-OH was 0.43 mmole/g)

peptides were synthesized according to conventional SPPS techniques using standard Fmoc-coupling

chemistry3 on a Wang resin. The Fmoc-protection groups were deprotected with 20% piperidine in DMF.

The protected (if necessary) amino acids (3 eq.; (Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pmc)-

OH and Fmoc-Gly-OH for GRGDS) and (Fmoc-Arg(Pmc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-His(Trt)-OH

and Fmoc-Pro-OH for PHSRN)) were dissolved in DMF. As coupling reagents 1-hydroxybenzotriazole

(3.6 eq.) and diisopropylcarbodiimide (3.3 eq.) in DMF were used. The peptides were deprotected and

cleaved from the support with 95% trifluoro acetic acid (TFA) and 5% water. They were precipitated in

(cold) diethylether, spun down and washed three times with diethylether. Subsequently, they were freeze-

dried three times from water with 10-20% acetonitrile which resulted in white fluffy powders. For the

coupling of the UPy-units to the free amine of the last amino acid of the peptides, a method was developed

to perform these reactions on the solid support. The reaction of the free amine of the protected GRGDS

peptide with a 1,1’-carbonyldiimidazole activated4 methyl-isocytosine (5 eq.) was performed in dry DMF

(molsieves) under an argon atmosphere for 16 hours at 50ºC resulting in the protected UPy-GRGDS on the

resin. The excess of 1,1’-carbonyldiimidazole activated4 methyl-isocytosine was washed away with acidic


water. The protected UPy-PHSRN peptide was synthesized on the resin with the last step consisting of the

reaction of the free amine with 2(6-isocyanatohexylaminocarbonyl-amino)-6-methyl-4[1H]-pyrimidinone

(the UPy-isocyanate-synthon)2 (8 eq.) in dry chloroform (molsieves) for 16 hours at 21ºC resulting in the

protected UPy-PHSRN on the resin. The excess of UPy-isocyanate-synthon2 was washed away with

chloroform. The last steps of the work-up procedure of the UPy-GRGDS and UPy-PHSRN peptides after

deprotection and cleavage from the solid support with 95% trifluoro acetic acid with 5% water were the

same as for the GRGDS and PHSRN peptides. The UPy-peptides were purified using preparative reversed

phase liquid chromatography (RPLC). The compounds were characterized with NMR techniques, IR,

RPLC and mass spectrometry.

GRGDS. 1H NMR (D2O/ACN-d3): δ = 4.77 (t, 1H, NH-CH-CH2-COOH, Asp), 4.48 (t, 1H, NH-CH-CH2-

OH, Ser), 4.31 (t, 1H, NH-CH-CH2CH2CH2-NH-(C=NH)-NH2, Arg), 3.93-3.84 (s, 2H, NH-CH2, Gly(1); s,

2H, NH-CH2, Gly(2); m, 2H, NH-CH-CH2-OH, Ser), 3.17 (t, 2H, NH-CH-CH2CH2CH2-NH-(C=NH)-NH2,

Arg), 2.93-2.84 (m, 2H, NH-CH-CH2-COOH, Asp), 1.83-1.63 (3xm, 4H, NH-CH-CH2CH2CH2-NH-

(C=NH)-NH2, Arg) ppm. The assignment of the 1H NMR spectrum is confirmed by 2D 1H,1H-COSY

spectroscopy. 13C NMR (D2O/ACN-d3): δ = 173.2 (2x), 172.9, 171.3, 170.2, 166.4, 163.8, 60.2, 54.3, 52.9,

49.1, 41.6, 39.6, 39.5, 34.7, 27.1, 23.4 ppm. 19F NMR (D2O/ACN-d3), with potassium hexafluoro

phosphate as internal standard) showed that the sample contained less than 1.5 weight% TFA. IR (ATR): ν

= 3196, 3068, 2943, 2630, 1706, 1646 (C=O stretch), 1532, 1408, 1232, 1200, 1050 cm-1. RPLC-MS: one

peak in chromatogram with m/z: Calcd. 490.2 g/mole. Obsd. [M + H]+ = 491.3 g/mole and [M + H]2+ =

246.1 g/mole.

UPy-GRGDS. 1H NMR (D2O/ACN-d3): δ = 5.98 (s, 1H, C=CH, UPy), 4.78 (t, 1H, NH-CH-CH2-COOH,

Asp), 4.48 (t, 1H, NH-CH-CH2-OH, Ser), 4.32 (t, 1H, NH-CH-CH2CH2CH2-NH-(C=NH)-NH2, Arg), 3.98-

3.84 (s, 2H, NH-CH2, Gly(1); s, 2H, NH-CH2, Gly(2); m, 2H, NH-CH-CH2-OH, Ser), 3.15 (t, 2H, NH-CH-

CH2CH2CH2-NH-(C=NH)-NH2, Arg), 2.90-2.78 (m, 2H, NH-CH-CH2-COOH, Asp), 2.23 (s, 3H, CH3,

UPy), 1.85-1.61 (3xm, 4H, NH-CH-CH2CH2CH2-NH-(C=NH)-NH2, Arg) ppm. The assignment of the 1H

NMR spectrum is confirmed by 2D 1H,1H-COSY spectroscopy. 13C NMR (D2O/ACN-d3): δ = 173.3 (2x),

172.9, 172.0, 171.1 (2x), 170.2, 159.8, 155.9, 155.4, 150.2, 104.4, 60.3, 54.2, 52.7, 49.0, 41.9, 41.7, 39.7,

34.6, 27.0, 23.5, 19.7 ppm. 19F NMR (D2O/ACN-d3), with potassium hexafluoro phosphate as internal


standard) showed that the sample contained less than 0.1 weight% TFA. IR (ATR): ν = 3280, 3182, 3073,

2948, 2542, 1701, 1642 (C=O stretch), 1528, 1413, 1224, 1180, 1135, 1076, 1046 cm-1. RPLC-MS: one

peak in chromatogram with m/z: Calcd. 641.3 g/mole. Obsd. [M + H]+ = 642.2 g/mole and [M + H]2+ =

321.7 g/mole.

PHRSN. 1H NMR (D2O/ACN-d3): δ = 8.59 (s, 1H, NH-CH-N, His), 7.32 (s, 1H, CH-NH-CH-N, His), 4.73

(t, 1H, NH-CH-CH2, His), 4.66 (t, 1H, NH-CH-CH2-(C=O)-NH2, Asn), 4.35 (t, 1H, NH-CH-CH2-OH, Ser;

t, 1H, NH-CH-CH2-CH2-CH2, Pro; t, 1H, NH-CH-CH2CH2CH2-NH-(C=NH)-NH2, Arg), 3.83 (m, 2H, NH-

CH-CH2-OH, Ser), 3.36-3.16 (m, 2H, NH-CH-CH2, His; m, 2H, NH-CH-CH2-CH2-CH2, Pro; t, 2H, NH-

CH-CH2CH2CH2-NH-(C=NH)-NH2, Arg), 2.79-2.75 (m, 2H, NH-CH-CH2-(C=O)-NH2, Asn), 2.43 (m, 1H,

NH-CH-CH2-CH2-CH2, Pro), 2.02-1.99 (m, 1H, NH-CH-CH2-CH2-CH2, Pro; m, 2H, NH-CH-CH2-CH2-

CH2, Pro), 1.86-1.63 (3xm, 4H, NH-CH-CH2CH2CH2-NH-(C=NH)-NH2, Arg) ppm. The assignment of the

1H NMR spectrum is confirmed by 2D 1H,1H-COSY spectroscopy. 13C NMR (D2O/ACN-d3): δ = 173.8,

173.3, 172.1, 170.8, 170.6, 168.9, 133.2, 127.9, 117.1, 60.6, 59.1, 55.2, 52.8, 52.4, 49.1, 46.0, 40.1, 36.0,

29.2, 27.8, 26.0, 23.9, 23.3 ppm. 19F NMR (D2O/ACN-d3), with potassium hexafluoro phosphate as

internal standard) showed that the sample contained less than 1.5 weight% TFA. IR (ATR): ν = 3354, 3185,

2870, 2453, 1647 (C=O stretch), 1552, 1438, 1260, 1200, 1125, 1059 cm-1. RPLC-MS: one peak in

chromatogram with m/z: Calcd. 609.3 g/mole. Obsd. [M + H]+ = 610.4 g/mole and [M + H]2+ = 305.7

g/mole.

UPy-PHSRN. 1H NMR (D2O/ACN-d3): δ = 8.58 (s, 1H, NH-CH-N, His), 7.27 (s, 1H, CH-NH-CH-N,

His), 5.92 (s, 1H, C=CH, UPy), 4.73 (t, 1H, NH-CH-CH2, His), 4.66 (t, 1H, NH-CH-CH2-(C=O)-NH2,

Asn), 4.36 (t, 1H, NH-CH-CH2-OH, Ser; t, 1H, NH-CH-CH2-CH2-CH2, Pro), 4.15 (t, 1H, NH-CH-

CH2CH2CH2-NH-(C=NH)-NH2, Arg), 3.86 (m, 2H, NH-CH-CH2-OH, Ser), 3.40-3.05 (m, 2H, NH-CH-

CH2, His; m, 2H, NH-CH-CH2-CH2-CH2, Pro; t, 2H, NH-CH-CH2CH2CH2-NH-(C=NH)-NH2, Arg), 2.82-

2.73 (m, 2H, NH-CH-CH2-(C=O)-NH2, Asn), 2.21 (s, CH3, UPy), 2.15 (m, 1H, NH-CH-CH2-CH2-CH2,

Pro), 2.03-2.00 (m, 1H, NH-CH-CH2-CH2-CH2, Pro; m, 2H, NH-CH-CH2-CH2-CH2, Pro), 1.94-1.62 (3xm,

4H, NH-CH-CH2CH2CH2-NH-(C=NH)-NH2, Arg), 1.54-1.46 (2xt, 4H, (C=O)-NH-CH2, hexyl spacer),

1.34-1.27 (m, 8H, (C=O)-NH-CH2CH2CH2CH2CH2CH2-NH-(C=O), hexyl spacer) ppm. The assignment of

the 1H NMR spectrum is confirmed by 2D 1H,1H-COSY spectroscopy. 13C NMR (D2O/ACN-d3): δ =


175.5, 173.9, 173.3 (2x), 172.3, 171.4, 171.1, 158.3, 156.6, 155.3, 151.4, 133.5, 129.0, 117.1, 104.9, 61.1,

60.7, 56.0, 52.9, 51.9, 49.2, 46.2, 40.4, 40.1, 39.3, 36.2, 29.6, 29.5, 28.7, 28.1 (2x), 25.8 (3x), 24.2 (2x)

ppm. 19F NMR (D2O/ACN-d3), with potassium hexafluoro phosphate as internal standard) showed that the

sample contained less than 1 weight% TFA. IR (ATR): ν = 3263, 2943, 1657 (C=O stretch), 1542, 1441,

1361, 1317, 1252, 1201, 1133, 1078 cm-1. RPLC-MS: one peak in chromatogram with m/z: Calcd. 902.4

g/mole. Obsd. [M + H]+ = 903.3 g/mole, [M + H]2+ = 452.3 g/mole and [M + H]3+ = 301.9 g/mole.

1.3 Toxixity tests in vitro

The toxicity of the UPy-unit was tested with two different viability tests, the MTT toxicity5 and the LDH

leakage test6, with rat NR8383 macrophages (passage 11) on two water-soluble UPy-molecules (kindly

provided by Gaby van Gemert and Henk Janssen); Tris-UPy and PEG-UPy (Mn = 5,6 kg/mole). The

toxicity of different concentrations of the molecules (1, 10–1, 10–2, 10–3 and 10–4 mM) was investigated. The

tests were performed in a similar manner as described in the reported references.5,6 The tetrazolium-based

colorimetric assay, or MTT test, is based on the fact that the yellow 3-[4,5-dimethylthiazol-2-yl]-2,4-

diphenyl tetrazolium bromide (MTT) is converted to purple formazan crystals by metabolically active

cells.5 The LDH (lactate dehydrogenase) leakage test is based on the leakage of NADH (nicotinamide

adenine dinucleotide) and LDH oxidases out of cells when their cell membranes have become permeable.6

1.4 Degradation studies in vitro

The degradation behaviour of PCLdiUPy polymer films was studied in duplicate in buffer and in buffer in

the presence of lipase enzymes, via mass measurements (the dry mass of the samples was measured on a

Sartorius microbalance), differential scanning calorimetry (DSC) and gel permeation chromatography

(GPC) after rinsing the samples three times with water and drying them at 40ºC for 1.5 hours.

Degradation in buffer. Films were made by compression moulding at approximately 20ºC above the

melting temperature (HMW polycaprolactone; Mn = 80 kg/mole) or drop casting from chloroform solution

(PCLdiUPy). The produced films were dried in vacuo at 35-40ºC for 2-3 days if necessary. Samples were

shaken in phosphate buffered saline (PBS) solution supplemented with sodiumazide (0.05%) at 37ºC for


130 days. The buffer was changed monthly. The mentioned techniques (stated above) and stress-strain

measurements were used to study the samples.

Enzymatic degradation. PCLdiUPy films were made via drop casting from chloroform solution

(PCLdiUPy) and dried in vacuo at 35-40ºC for 2-3 days prior to use. Samples were shaken in a lipase (from

Thermomyces lanuginosus, Aldrich) containing solution which was diluted 1000 times with PBS solution

supplemented with sodiumazide (0.05%) at 37ºC for 23 days. The mentioned techniques (stated above)

were used to study the samples.

1.5 Extraction experiments

The extraction of UPy-GRGDS and GRGDS out of the second set of PCLdiUPy films was investigated

with LC-MS measurements. The amount of peptide that was mixed in the PCLdiUPy films was 4 mole%.

Calibration was performed by quantification of one fragment of the parent ion (MS2) of the peptides using

different concentrations of the peptides. The surface area of the corresponding peak (in the total ion count)

was calculated with the ICIS algorithm. The extraction experiment was performed by incubation of the

second set of PCLdiUPy films with UPy-GRGDS or with GRGDS in a total volume of 5 mL water, added

in portions of 1 mL, during 80 minutes of incubation at 37ºC. So the procedure for one film was the

following; the film was incubated at 37ºC for 5 minutes in 1 mL water. Then the water was removed and

the concentration peptide was measured with the described LC-MS procedure (time-point: 5 minutes).

Another 1 mL water was added to this sample and the film was incubated again for 5 minutes at 37ºC. Then

the water was removed and the concentration peptide was detected (time-point: 10 minutes). Another 1 mL

water was added and the sample was incubated for 10 minutes at 37ºC. Removal of the water and

measuring of the peptide concentration yielded the amount of extracted peptide at 20 minutes. Incubation at

37ºC in another 1 mL water for 20 minutes, removal of the water and measurement of the concentration of

the peptide showed the amount of peptide extracted after 40 minutes. The last time-point (80 minutes) was

obtained by incubation of the film at 37ºC for 40 minutes.

Another extraction experiment was performed by incubation of the PCLdiUPy films containing UPy-

GRGDS or GRGDS in 1 mL water at 37ºC for 2 hours. After this incubation step the concentration of

extracted peptide was measured with that same LC-MS procedure.


1.6 Quantification of extent and morphology of cell adhesion

The cells cultured on the bioactive UPy-films were quantified both on extent and on morphology by

counting the cells at 400 times magnification. This quantification was performed on seven films of each

material; PCldiUPy with UPy-GRGDS (1), PCLdiUPy with UPy-PHSRN (2), PCLdiUPy with both UPy-

GRGDS and UPy-PHSRN (3) and on the bare PCLdiUPy (4).

1.7 Stability test on bioactive UPy-films

To test the stability of the bioactive UPy-films in medium the whole cell adhesion and cell spreading

experiment was repeated, but this time the samples were incubated in medium without FBS (1 mL) in a

humidified incubator at 37ºC and 5% CO2 for 3 hours, prior to seeding of the cells on the polymers. After

this incubation step, the samples were washed two times with PBS solution and the cells were cultured in

medium without FBS on these films in a humidified incubator at 37ºC and 5% CO2 for 1 day. The cells

were studied with optical microscopy.

2. RESULTS

2.1 Toxicity tests in vitro on water-soluble UPy-molecules

The toxicity of the UPy-unit was tested with two different viability tests, the MTT toxicity5 and the LDH

leakage test6, with rat macrophages on two water-soluble UPy-molecules; Tris-UPy and PEG-UPy. The

toxicity of different concentrations of the molecules (1, 10–1, 10–2, 10–3 and 10–4 mM) was investigated. The

tetrazolium-based colorimetric assay (MTT test) showed that the viability of the cells stayed above 80% for

every concentration of Tris-UPy and stayed at least above 65% for every concentration of PEG-UPy (Fig.

2.1). The LDH leakage test displayed similar results (Fig. 2.1), although the viability of the cells incubated

with 1 mM of the two water-soluble UPy-molecules was somewhat lower when determined with this LDH

leakage test. These tests strongly indicate that the UPy-moiety is not toxic and thus biocompatible.


Figure 2.1 Toxicity studies on the UPy-moiety. MTT and LDH viability tests on two water-

soluble UPy-molecules, Tris-UPy and PEG-UPy (Mn = 5,6 kg/mole), with NR8383 rat

macrophages showed that the UPy-unit can be assumed to be not toxic.

2.2 Degradation studies in vitro

The supramolecular PCLdiUPy material was compared with high molecular weight PCL with respect to its

biodegradability in vitro. Polymeric films of PCLdiUPy and high molecular weight PCL were shaken in a

phosphate buffered saline solution. The high molecular weight PCL samples showed even after 130 days

almost no mass loss; 0.5% was observed. Moreover, no visible macroscopic changes occurred and the

materials remained as plastic films. The PCLdiUPy films showed somewhat different degradation

behaviour compared to PCL. Although after 130 days only 2% of the material was degraded, the films

became less clear and more brittle, indicating an increase of crystallinity upon degradation. This was

confirmed by differential scanning calorimetry (DSC) measurements; the melting enthalpy became higher

and the melting peaks slightly shifted to higher temperatures. The rearrangement is supposed to be a result

of the reversible binding of the repeating units in the supramolecular materials. Tensile testing showed that

the material became stiffer; an increase in the Young’s modulus and a decrease in strain at break upon

degradation were observed. During enzymatic degradation of PCLdiUPy with the lipase from

Thermomyces lanuginosus chain scission was demonstrated with gel permeation chromatography

0

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Tris-UPy PEG-UPy

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Tris-UPy PEG-UPy

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techniques. After 15 days already 90% mass loss was found. These findings show that our new

supramolecular polymers can be enzymatically degraded in vitro.

2.3 Extraction experiments

Extraction experiments using quantitative LC-MS measurements on the second set of PCLdiUPy films

containing 4 mole% UPy-GRGDS or 4 mole% GRGDS show that dissolution of GRGDS without a UPy-

moiety proceeds extremely fast in water at 37ºC; within 5 minutes almost all of the peptide is dissolved

(Fig. 2.2). After 80 minutes of incubation at 37ºC in a total volume of 5 mL water the whole quantity of

GRGDS peptide (105%) is dissolved. If a PCLdiUPy film with 4 mole% GRGDS is incubated for 2 hours

at 37ºC in 1 mL water also the whole amount of GRGDS peptide (101%) is dissolved. The extraction of

UPy-GRGDS with water from the PCLdiUPy film is a slower process. After incubation at 37ºC of the

PCLdiUPy film containing 4 mole% of UPy-GRGDS for 80 minutes in a total volume of 5 mL water

ultimately 76% of the UPy-peptide is dissolved (Fig. 2.2). However, if this PCLdiUPy film with 4 mole%

UPy-GRGDS is incubated for 2 hours in 1 mL water at 37ºC 64% of the UPy-GRGDS peptide is dissolved.

This indicates that the UPy-unit is important for the tuneable but dynamic binding of the peptide to the

polymer.

Figure 2.2 Extraction experiments on PCLdiUPy films with UPy-GRGDS or GRGDS. The

GRGDS peptide is extracted much faster in water than the UPy-GRGDS peptide.

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2.4 Cell adhesion after 3 hours

After seeding the mouse 3T3 fibroblasts on the second set of different supramolecular polymer-peptide

blends in the absence of FBS, the samples were followed in time by optical microscopy. Some aspecific

cell adhesion, but hardly any cell spreading, was already visible after 3 hours on all samples, even on the

controls (Fig. 2.3).

Figure 2.3 Cell adhesion in vitro after 3 hours. Fibroblast cell (5·104 cells/cm2) adhesion on

different films of mixtures of PCLdiUPy with UPy-GRGDS (1), PCLdiUPy with UPy-PHSRN (2),

PCLdiUPy with both UPy-GRGDS and UPy-PHSRN (3), PCLdiUPy alone (4) and on PS in the

absence of FBS and on PS in the presence of FBS (PS + FBS) after three hours of cell culturing.

In all cases 4 mole% of peptide was mixed with the PCLdiUPy. The cells were visualized on the

polymer films with optical microscopy; scale bars represent 100 µm.

2.5 Quantification of extent and morphology of cell adhesion

The cell adhesion and spreading experiments on the different films of mixtures of PCLdiUPy with UPy-

GRDGS (1), PCLdiUPy with UPy-PHSRN (2), PCLdiUPy with both UPy-GRGDS and UPy-PHSRN (3)

and PCLdiUPy alone (4) were quantified by counting the amount of adhered cells after 1 day of culturing

(Fig. 2.4). The amount of adhered cells was divided into two categories; the spread and the round cells.

PCLdiUPy

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PS PS + FBSPCLdiUPy

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PS PS + FBS


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Figure 2.4 Quantification of extent and morphology of cell adhesion. The amount of spread

and round fibroblast cells determined at 400 times magnification on the different films of mixtures

of PCLdiUPy with UPy-GRGDS (1), PCLdiUPy with UPy-PHSRN (2), PCLdiUPy with both UPy-

GRGDS and UPy-PHSRN (3), PCLdiUPy alone (4).

2.6 Cell adhesion controls

Fibroblast cell adhesion and spreading experiments were performed on the first set of different

supramolecular polymer-peptide blends for 2 days in the absence of FBS (Fig.3 in the paper). PCLdiUPy

polymers were mixed with UPy-GRGDS (1), with UPy-PHSRN (2) and with both UPy-GRGDS and UPy-

PHSRN (3). In all cases 4 mole% of peptide was mixed with the PCLdiUPy solution prior to preparing the

films. Not only the bare PCLdiUPy polymer (4) was used as a control (Fig. 3 in the paper), but also

peptides without a UPy-unit were mixed in. The controls showed that they were not able to induce cell

adhesion and spreading (Fig. 2.5).


Figure 2.5 Controls of the cell adhesion experiments in vitro. Fibroblast cell (5·104 cells/cm2)

adhesion on different drop cast films of mixtures of PCLdiUPy polymers and peptides without a

UPy-unit, after two days of cell culturing in the absence of FBS. In all cases 4 mole% of peptide

was mixed with the PCLdiUPy. As negative controls, PCLdiUPy with GRGDS, PCLdiUPy with

PHSRN, PCLdiUPy with GRGDS and PHSRN, glass and polystyrene (PS) are shown. The

positive control (PS + FBS) was polystyrene (PS) in the presence of FBS. The cells were

visualized on the polymer films with optical microscopy; scale bars represent 100 µm.

2.7 Two methods for the preparation of the bioactive films

Two methods were used to prepare bioactive supramolecular UPy-films. Figure 2.6 displays the cells on the

blends that are made by first drop casting the polymer solution and then drop casting the peptide solution

on the dried polymer film (method 2). Similar results were observed as shown before (Fig. 3 in the paper)

when a bioactive blend was produced by mixing the two solutions prior to drop casting (method 1).

glass PS PS + FBS

GRGDS & PHSRNGRGDS PHSRN

glass PS PS + FBS

GRGDS & PHSRNGRGDS PHSRN


Figure 2.6 Cell adhesion and spreading in vitro. Fibroblast cell (5·104 cells/cm2) adhesion and

spreading on different films of mixtures of PCLdiUPy polymers and peptides after 1 day of

culturing in the absence of FBS. The samples were prepared via method 2. In all cases 4 mole%

of peptide was put on the PCLdiUPy polymers. The cells were cultured on PCLdiUPy with UPy-

GRGDS (1), PCLdiUPy with UPy-PHSRN (2), PCLdiUPy with both UPy-GRGDS and UPy-

PHSRN (3). As controls PCLdiUPy with GRGDS, PCLdiUPy with PHSRN, PCLdiUPy with

GRGDS and PHSRN, PCLdiUPy alone (4), glass and polystyrene (PS) were used. The positive

control (PS + FBS) was polystyrene (PS) in the presence of FBS. All cells were visualized on the

polymer films with optical microscopy; scale bars represent 100 µm.

2.8 Different UPy-GRGDS concentrations

21 3

4GRGDS & PHSRNGRGDS PHSRN

PCLdiUPy


PS PS + FBSglass

21 3

4GRGDS & PHSRNGRGDS PHSRN

PCLdiUPy


PS PS + FBSglass


If blends of 1 with 1, 2 or 4 mole% UPy-peptide (prepared via method 2) were compared after 1 day of cell

culturing, hardly any difference was observed between cell adhesion and spreading between the three

different concentrations (Fig. 2.7).

Figure 2.7 Different UPy-GRGDS concentrations. Fibroblast cell (5·104 cells/cm2) adhesion and

spreading on films of mixtures of PCLdiUPy polymers with 1, 2 or 4 mole% UPy-GRGDS after 1

day of culturing in the absence of FBS. The cells were visualized on the polymer films with optical

microscopy; scale bars represent 100 µm.

2.9 Stability test on bioactive UPy-films

To test their stability, the bioactive UPy-films (prepared via method 2) were incubated in medium without

FBS for 3 hours before seeding of the cells on the films. After this incubation step, the samples were

washed two times with a PBS solution and cells were cultured on these films for 1 day again in the absence

of FBS. This resulted in similar adhesion and spreading patterns as shown before (Fig. 2.8).

1 1 1UPy-GRGDS

1 mole% 2 mole% 4 mole%

1 1 1UPy-GRGDS

1 mole% 2 mole% 4 mole%


Figure 2.8 The stability of the bioactive UPy-films. Fibroblast cell (5·104 cells/cm2) adhesion

and cell spreading on different films of mixtures of PCLdiUPy polymers and UPy-peptides, after

an incubation and two washing steps before culturing of the cells for 1 day in the absence of FBS.

In all cases 4 mole% of UPy-peptide was mixed with the PCLdiUPy. The cells were cultured on

PCLdiUPy with UPy-GRGDS (1), PCLdiUPy with UPy-PHSRN (2), PCLdiUPy with both UPy-

GRGDS and UPy-PHSRN (3). As controls PCLdiUPy alone and polystyrene (PS) were used. The

positive control (PS + FBS) was polystyrene (PS) in the presence of FBS. All cells were

visualized on the polymer films with optical microscopy; scale bars represent 100 µm.

3. REFERENCES

1. Hutmacher, D.W., Schantz, T., Zein, I., Ng, K.W., Teoh, S.W. & Tan, K.C. Mechanical properties

and cell cultural response of polycaprolactone scaffolds designed and fabricated via fused

deposition modeling. J. Biomed. Mater. Res. 55, 203-216 (2001).

2. Folmer, B.J.B., Sijbesma, R.P., Versteegen, R.M., van der Rijt, J.A.J. & Meijer, E.W.

Supramolecular polymer materials: chain extension of telechelic polymers using a reactive

hydrogen-bonding synthon. Adv. Mater. 12, 874-878 (2000).

21 3

4

PCLdiUPy


PS PS + FBS

21 3

4

PCLdiUPy


PS PS + FBS


3. Fields, G.B. & Nobel, R.L. Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl

amino acids. Int. J. Peptide Protein Res. 35, 161-214 (1990).

4. Keizer, H.M., Sijbesma, R.P. & Meijer, E.W. The convenient synthesis of hydrogen-bonded

ureidopyrimidinones. Eur. J. Org. Chem. 2004, 2553-2555 (2004).

5. Gerlier, D. & Thomasset, N. Use of MTT colorimetric assay to measure cel activation. J.

Immunol. Methods 94, 57-63 (1986).

6. Koh, J.Y. & Choi, D.W. Quantitative determination of glutamate mediated cortical neuronal injury

in cell culture by lactate dehydrogenase efflux assay. J. Neurosc. Methods 20, 83-90 (1987).


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