Albumin and fibrinogen adsorption on PU–PHEMA surfaces

Biomaterials 24 (2003) 2067–2076

Albumin and fibrinogen adsorption on PU–PHEMA surfaces M.C.L. Martinsa,b,*, D. Wangc, J. Jia,c, L. Fengc,{, M.A. Barbosaa,b a

! INEB-Instituto de Engenharia Biom!edica, Laboratorio de Biomateriais, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal b ! Departamento de Engenharia Metalurgica e Materiais, Faculdade de Engenharia, Universidade do Porto, Porto, Portugal c Department of Polymer Science, Zhejiang University, Hangzhou, China Received 13 June 2002; accepted 17 December 2002

Abstract Materials that adsorb specific proteins may find a variety of applications in the biomedical field. The aim of this study was the preparation of a hydrophilic surface, with low protein adsorption, to be used in the future as a support for the immobilisation of several species, e.g. Cibacron Blue F3G-A, which has been described to induce specific albumin adsorption. Poly(hydroxyethylmethacrylate) (PHEMA) and poly(hydroxyethylacrylate) (PHEA) were chosen as the hydrophilic surface because they can be easily polymerised and possess hydroxyl groups that can be used for the immobilisation of different compounds. Thin films of PHEMA and PHEA were successfully graft polymerised onto the surface of a commercial poly(etherurethane) (PU) using ceric ion as initiator. Grafting polymerisations were followed by mass gain and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). Since stability tests demonstrated that only PU–PHEMA was stable in alkaline solutions, a necessary condition to future immobilisations, the investigation was focused on the coating of PU with PHEMA. PU–PHEMA films were characterised in detail using several techniques as mass gain, ATR-FTIR, contact angle measurements, X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). Protein adsorption was evaluated using radiolabelled albumin and fibrinogen from pure solutions and from mixtures of both proteins. PU surfaces modified with PHEMA have demonstrated low protein adsorption, showing their potential use as substrates. This opens the possibly of exploring the advantages of selective adsorption by appropriate immobilisation of specific molecules. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: PHEMA; Graft polymerisation; Pellethane; Protein adsorption; ATR-FTIR

1. Introduction Thrombus formation and microbial invasion associated infections are the two major complications affecting blood contacting medical devices. These processes are always initiated by protein adsorption [1–3]. A widely used strategy to increase haemocompatibility of the conventional polymers, is the creation of a protein-resistant surface by immobilisation of very hydrophilic polymers as poly(ethylene oxide) (PEO). Glow discharge plasma deposition (GDPD) of organic compounds called glymes has also been described as very effective in resisting protein adsorption and cell ! *Corresponding author. Laboratorio de Biomateriais, Instituto de Engenharia Biom!edica, Rua do Campo Algre 823, Porto 4150-180, Portugal. Tel.: +351-22-6074982; fax: +351-22-6094567. E-mail address: [email protected] (M.C.L. Martins). { Deceased.

adhesion [4,5] and preventing bacterial adhesion [5]. However, these materials also present problems in maintaining resistance to fouling for long-term biomedical applications. Another strategy to improve blood compatibility and decrease bacterial adhesion is the creation of an albumin-coated surface that resists adsorption of other proteins. A thin layer of albumin appears to minimise adhesion and aggregation of platelets, thus avoiding subsequent thrombus formation [6]. Surfaces pre-coated with albumin have also been associated with lower bacterial adhesion [7–9]. Several groups have developed different methods to modify polymeric surfaces in order to improve albumin binding to surfaces [9–15]. However, problems associated with denaturation of the blocking protein over time [16] or exchange of the albumin with other proteins in solution make this strategy only satisfactory for short-term use [17].

0142-9612/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0142-9612(03)00002-4

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Materials with resistance to non-specific protein adsorption can be obtained by the immobilisation of compounds with specificity for a certain protein. The immobilisation of these compounds requires an inert, hydrophilic support, which possesses non-fouling properties (low protein adsorption and cell adhesion). Synthetic polymers like poly(hydroxyethylmethacrylate) (PHEMA) and poly(hydroxyethylacrylate) (PHEA) can be easily polymerised and possess hydroxyl groups that can be used for the immobilisation of different compounds. Moreover, PHEMA has been widely used in many biomedical applications [18]. In the present work, we intend to evaluate if PHEMA and PHEA can be used as a support for the immobilisation of several species, e.g. Cibacron Blue F3G-A that has been described to adsorb albumin from human plasma in a selective and reversible way [9]. In this part of the work, we prepared the PHEMA and PHEA surfaces by graft polymerisation of their monomers onto the surface of a commercial poly(etherurethane) (PU). After surface characterisation, the surfaces were also evaluated in relation to protein adsorption. Characterisation of the films was carried out by mass gain, attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), contact angle measurements, X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). Protein adsorption was evaluated using radiolabelled albumin and fibrinogen from pure solutions and from mixtures of both proteins.

2. Materials and methods 2.1. Materials 2-Hydroxyethylmethacrylate (HEMA; 97%) and 2hydroxyethylacrylate (HEA; 96%) were obtained from Aldrich and purified using low-pressure distillation. 2.2. PU film preparation PU pellets (Pellethane 2363-80 AE, Dow Chemical) were extracted with absolute ethanol (Merck) for 3 days in a Soxhlet extractor. After drying for 2 days at 60
C in a vacuum oven, the pellets were dissolved in tetrahydrofuran (THF, Merck) to give a 12.5% (w/w) solution. 40 ml of PU solution were casted onto clean glass Petri dishes (140 mm diameter) to get the thin films. The petri dishes were covered with perforated aluminium foil and kept at room temperature to slowly vaporise the solvent. All the films were dried overnight in a vacuum oven at 60
C. Films were cut in plaques of 20  60 mm2.

2.3. HEMA and HEA graft polymerisation onto PU films PHEMA and PHEA films were produced by the graft polymerisation of the monomers HEMA and HEA onto PU films. The polymerisation was initiated using ceric ion [9,19]. The PU film with known weight and dimensions was introduced into a flask with 60 ml of monomer solution and stirred for 15 min under nitrogen. Then, 40 ml of fresh catalyst, prepared with ammonium cerium (IV) nitrate ((NH4)2Ce(NO3)6, Merck) in 400 mm nitric acid solution (HNO3, Merck), were added. The reaction was performed under nitrogen at room temperature using electromagnetic stirring. Graft polymerisations were carried out using different monomer concentrations and two ceric ion concentrations. The effect of the time of reaction was also evaluated. After reaction, the film was washed 4 times in 250 ml of water during 24 h with agitation at 250 rpm. Films were dried overnight in a vacuum oven at 60
C. After drying, they were cut with a borer in several discs with 6 mm diameter. All the samples were washed 3 times with hexane (5 min under ultrasonic agitation) followed by washing with methanol (5 min under ultrasonic agitation). 2.4. Surface characterisation 2.4.1. Grafting yield The surface grafting yield was obtained from the weight increase of the film according to the following equation: Grafted amount ðmg=cm2 Þ ¼

W 1  W0 ; A

where W0 is the initial dry weight of the PU film, W1 is the dry weight of PHEMA- or PHEA-grafted film and A is the surface area of the film. 2.4.2. ATR-FTIR measurements ATR-FTIR measurements were performed on a FTIR spectrophotometer from Perkin Elmer, model 2000, coupled with ATR accessory (split-pea). 100 scans were performed with a resolution of 4 cm1. 2.4.3. X-ray photoelectron spectroscopy XPS measurements were carried out on a VG Scientific ESCALAB 200A (UK) spectrometer using magnesium Ka (1253.6 eV) as radiation source. The photoelectrons were analysed at a take off angle of 0
. Survey spectra were collected over a range of 0–1150 eV with an analyser pass energy of 50 eV. High-resolution spectra of C1s, O1s and N1s were collected with an analyser pass energy of 20 eV. The binding energy (BE) scales were referenced by setting the C1s BE to 285.0 eV.

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Atomic concentrations were quantified using tabulated sensitivity factors. 2.4.4. Contact angle measurements Contact angle measurements were performed with a contact angle measuring system from Data Physics, model OCA 15, equipped with a video CCD-camera and SCA 20 software. The equipment incorporates an electronic gas tight 500 ml dosing syringe (Hamilton). Films were stored in deionised water during a week. Before contact angle measurements, the films were dried overnight in a vacuum oven at 40
C. Samples were placed in a temperature controlled chamber saturated with the liquid, in order to prevent evaporation of the drop. Measurements were carried out using the sessile drop method with distilled and deionised water drops at 25
C. After drop deposition, images were taken every 2 s over 600 s. Digital images of the drop were acquired by the CCD-camera and used for the calculation of the contact angle. Droplet profiles were fitted using Young– Laplace mathematical function in order to calculate the contact angle. The water contact angle for each surface was calculated by extrapolating the time dependent curve to zero. Five replicates were used. 2.4.5. Scanning electron microscopy Samples were coated with a thin layer of gold by sputtering in an Ion Sputter JEOL JFC 1100. Observation was performed in a JEOL JSM-6301F SEM using an accelerating voltage of 10 kV. 2.4.6. Stability tests in alkaline solutions As already explained, it is our intention to use PU– PHEMA and PU–PHEA surfaces to immobilise several species, e.g. Cibacron Blue. This part of the research will be reported in another paper. However, since the immobilisation of Cibacron Blue has been performed by covalent coupling through the hydroxyl groups of PHEMA or PHEA using alkaline solutions, stability tests in these media were performed and followed by ATR-FTIR. 2.5. Protein adsorption measurements 2.5.1. Protein solutions Human serum albumin (HSA, Sigma, ref. A1653) and human fibrinogen (HFG, Sigma, ref. F4129) were obtained as lyophilised powders. The buffer used in all protein adsorption experiments was an isotonic citratephosphate buffered saline solution [4,20]. This buffer was composed of 0.01 m sodium citrate, 0.01 m sodium phosphate, 0.12 m sodium chloride, 0.01 m sodium iodide and 0.02% sodium azide, pH 7.4. All salts used in the buffer preparation were of reagent grade.

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2.5.2. Quantification of adsorbed albumin Quantification of adsorbed proteins on the polymer surfaces was performed using 125I-labelled proteins. HSA and HFG were labelled using the iodo-gen method [21,22]. Purification of the labelled protein was performed using Sephadex G-25 m columns (PD-10, Amersham Pharmacia biotech). The yield of iodination reaction was 87% for HSA and 93% for HFG determined by precipitating the 125I-labelled protein with 20% trichloro-acetic acid (TCA, Merck). 125Ilabelled protein was added to unlabelled protein solution in order to obtained a final activity of B107 cpm/mg. The samples for protein adsorption quantification were hydrated overnight in a degassed isotonic citrate– phosphate buffered saline solution at 37
C [4,20]. A buffer with iodide was used in order to inhibit adsorption of free radioactive 125I. Adsorption tests were carried out at 37
C during 2 h. After protein adsorption, samples were rinsed four times with 2 ml of buffer. The gamma activities were counted with the samples placed in radio-immunoassay tubes. Three replicates were used. The counts from each sample were averaged and the surface concentration was calculated by the equation Counts ðcpmÞCsolution ðng=mlÞ HSA ðng=sampleÞ ¼ ; Asolution ðcpm=mlÞ where the counts measure the radioactivity of the samples, the Csolution and Asolution are the concentration and the specific activity of the protein solution, respectively. Elution tests were carried out by immersing the labelled samples over 24 h in an unlabelled HSA pure solution (1 mg/ml). The samples were then washed with the buffer and their residual radioactivity counted.

3. Results 3.1. Surface characterisation 3.1.1. Grafting yield Table 1 shows the graft amount of the PU film after HEMA and HEA graft polymerisation using different concentrations of the monomers and two concentrations of ceric ion during 110 min. There is an increase of the amount of grafted PHEA with the increase of monomer concentration until a certain critical concentration is reached. Monomer concentrations higher than 1 m induce homopolymerisation. Higher yields of grafting were obtained when 0.66 m of HEA and 40 mm of ceric ion were used. Regarding HEMA polymerisation, for 40 mm of ceric ion the grafted amounts are similar for all the monomer concentrations, except for 3.3 m that induced homopolymerisation. This was also observed

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when 5 mm Ce4+ was used. Fig. 1 shows the grafting yield of the PHEMA and PHEA onto the PU surface versus reaction time, using 0.66 m of monomer and

40 mm Ce4+. Results demonstrate an increase of the amount of grafted PHEMA with increasing reaction time. Grafting of PHEMA on the surface of PU is always higher than for PHEA.

Table 1 Graft amount (mg/cm2) versus monomer and two different ceric ion concentrations. Reaction time was 110 min

3.1.2. Attenuated total reflection Fourier transform infrared spectroscopy Fig. 2 shows the ATR-FTIR spectra for the original PU film, the PU film after HEMA graft polymerisation (PU–PHEMA) and the homopolymerised PHEMA. These spectra demonstrate that the graft polymerisation of HEMA onto the PU surface was successful, since the ATR-FTIR spectra of the PU–PHEMA film shows the presence of the characteristic PHEMA absorption bands. These are located at B3400 cm1 from the hydroxyl groups (–OH), B1720 cm1 from the ester stretching band of the carboxyl group (C=O), 1075 cm1 from the stretching band of the alcohol group (C–O), presenting also the absorption bands characteristic of the carboxylic acid esters at B1150 cm1 [23]. The decrease of the intensity of the characteristic peaks of PU, namely the absorption bands at 1595 and at 1413 cm1, from the benzene ring, and at 1528 cm1, from the N–H bending and C–N stretching mode of the urethane group [24,25], are indicative of the coverage of the surface. The peak at 1636 cm1, observed for the spectrum of homopolymerised PHEMA, is characteristic of the stretching vibration of the C=C double bond [23]. The presence of the double bond vibration absorption band indicates that some of the monomer used was not polymerised. After graft polymerisation the disappearance of this peak indicates that all the monomer was polymerised.

Monomer (m)

0.33 0.50 0.66 0.80 1.0 1.5 3.3

PHEA (mg/cm2)

PHEMA (mg/cm2)

40 mm Ce4+

5 mm Ce4+

40 mmCe4+

5 mmCe4+

98 128 141 — HP HP HP

20 66 HP — HP HP HP

— — 240 212 — 242 HP

— — HP — — — HP

HP—Homopolymerisation.

2

Polymer grafted ( µ g/cm )

500 400 300

PHEMA PHEA

200 100 0 30

60

90

120

180

210

300

Reaction time (min)

Fig. 1. Grafting yield of the PHEMA and PHEA graft polymerisation onto the PU surface using different reaction time.

PU 3322

1728 1413

1595 1700

1308 1528 1078

%T

PU-PHEMA

1221 1103

3384 1451 1025

1722

1075 1157

PHEMA 3419 1636 1451 1024 1076 1715

4000

3000

2000

1161

1500

1000

750

cm-1

Fig. 2. ATR-FTIR spectra of the Pellethane film (PU), the PU film after HEMA graft polymerisation (PU–PHEMA) and the PHEMA homopolymerised using the same procedure for graft polymerisation onto the PU surface.

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1701

PU

1366

1596

1730

1413

2071

1309

1530 1222

1106 1079

PU-PHEMA (60 min)

%T

PU-PHEMA (90 min)

PU-PHEMA (120 min)

PU-PHEMA (180 min)

1800

1600

1200

1400 cm

1000

-1

Fig. 3. ATR-FTIR spectra of the PU films after HEMA graft polymerisation with different reaction times.

Fig. 3 shows the ATR-FTIR spectra of the PU films after HEMA graft polymerisation using 0.66 m of HEMA and 40 mm Ce4+ with different reaction times. It shows that 180 min of reaction time is necessary to give a good surface coverage, since the typical absorption bands of PU disappear. Similar results were obtained for HEA graft polymerisation (data not shown). Fig. 4 shows the ATR-FTIR spectra of PU–PHEA surface during immersion in solutions with different concentrations of NaOH. For solutions with 1 m NaOH, it is possible to detect the characteristic peaks of the PU substrate, indicating that the PHEA was removed from the surface. When the polymer was immersed in solutions with low NaOH concentrations, the PU– PHEA retains the typical absorption bands of PHEA. PU–PHEMA maintains its characteristic peaks in all the NaOH concentrations used (data not shown). These results demonstrate that PU–PHEMA was very stable in alkaline solutions, contrary to PU–PHEA. Therefore, it was decided to use only the former substrate for further studies, including protein adsorption. 3.1.3. X-ray photoelectron spectroscopy XPS survey spectra of PU and PU–PHEMA films have demonstrated that no elements other than those expected were found, indicating that no contamination took place.

Fig. 5 shows the C1s high resolution spectrum of the PU surface before and after HEMA graft polymerisation. For the PU surface, the C1s spectrum shows the characteristic peaks of this material [26]. The dominant peaks at 285.0 eV (C–C/C–H bonds) and 286.5 eV (C–O bonds) are originated from the polyether soft segment and the peak at 289.5 eV is due to the carbamate carbon (O=C–NH) [27]. The increase of the ester (O–C=O) peak at 289.3 eV in the C1s high-resolution spectrum of the PU–PHEMA surface relative to the PU surface, also demonstrates that graft polymerisation occurred. These XPS results are similar to those found by Hsiue et al. [28] for PHEMA. Table 2 gives the atomic percentages of carbon, oxygen and nitrogen, and the O–C=O/C–C and N/C ratios of the PU and PU–PHEMA surfaces. The atomic composition found for the PU film was similar to the one reported by other authors [29]. The increase of the O–C=O/C–C ratio and the decrease of N/C ratio after surface modification indicates the presence of the PHEMA. The atomic composition of the surface is similar to the theoretical values for PHEMA (66.7% of carbon and 33.3% of oxygen), in spite of some nitrogen from the PU being found. 3.1.4. Contact angle measurements Water contact angle data of the PU and PU–PHEMA surfaces, using the sessile drop technique, are reported in Table 3. The contact angles are close to those reported

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2072

PU-PHEA

1726

1158

1075

%T

PU-PHEA NaOH (0.001M)

PU-PHEA NaOH (0.01M)

PU-PHEA NaOH (1M)

1221 1730

1595

1700

2000

1800

1413

PU 1080 1018

1309

1530

1600

1400

1200

1000

cm-1

Fig. 4. ATR-FTIR spectra of the degradation of the PHEA from the surface of PU–PHEA after immersion of the films during 24 h in solutions with different concentrations of NaOH.

Fig. 5. XPS C1s high resolution spectra of the PU and PU–PHEMA surfaces.

Table 2 XPS atomic composition of the PU and PU–PHEMA surface Atomic (%) Sample

C1s

O1s

N1s

N/C

PU PU–PHEMA

74.7 66.4

22.1 32.6

3.2 1.0

0.043 0.015

O  C ¼ O=C  C % % 0.03 0.18

Table 3 Water contact angle data of the PU and PU–PHEMA surfaces Sample

Contact angle (
)

PU PU–PHEMA

7976 5979

for advancing water contact angles described in the literature (85
73
for Pellethane [24], and 69
[30] and 61
72
[31] for PHEMA).

3.1.5. Scanning electron microscopy The surface morphology of PU and PU–PHEMA was studied by SEM (Fig. 6). The modified surface presents a much rougher morphology than the cast PU. The roughness of the modified surface is considerably homogeneous. 3.1.6. Protein adsorption measurements Fig. 7 shows the adsorption of human serum albumin (HSA) and fibrinogen (HFG) onto PU and PU– PHEMA surfaces from pure solutions (0.1 mg/ml). These results demonstrate that graft polymerisation of HEMA onto PU surface decreases albumin and fibrinogen adsorption. The decrease of adsorption after surface modification is higher for albumin (50%) than for fibrinogen (18%). However, there is always more albumin adsorbing on both materials. Fig. 8 shows the effect of the presence of unlabelled HFG on the adsorption of HSA onto PU and PU–PHEMA surfaces (competition studies). The

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PU

2073

PU-PHEMA

Fig. 6. Surface morphologies of PU and PU–PHEMA surfaces determined by SEM. Bar represents 10 mm.

600

120 HFG (0.1mg/mL)

400 300 200

110

HSA adsorption (%)

Protein adsorption (ng/sample)

HSA (0.1mg/mL)

500




100




90

PU PU-PHEMA

80




70

100

60 0

0.05

0.1

Fibrinogen concentration (mg/mL)

0 PU-PHEMA

Fig. 7. Human serum albumin (HSA) and Fibrinogen (HFG) adsorption onto PU and PU–PHEMA surfaces from pure solutions.

concentration of 125I-labelled HSA was kept at 0.1 mg/ml and unlabelled HFG was added to the solution in concentrations of 0.01 and 0.1 mg/ml. Calculations were performed considering the concentration of adsorbed HSA from a pure solution as 100% adsorption. The presence of low amounts of fibrinogen (0.01 mg/ml) increases the adsorption of HSA onto PU– PHEMA surface. Using solutions with equal concentrations of albumin and fibrinogen (0.1 mg/ml), the decrease of albumin adsorption was similar for both surfaces. The reversibility of adsorption to different surfaces was evaluated by the exchange of the preadsorbed 125I-labelled HSA and HFG by unlabelled HSA in solution. Fig. 9 shows the percentage of HSA and HFG retention onto PU and PU–PHEMA after soaking in a HSA solution (1 mg/ml) for 24 h. The retention of the 125I-HSA and 125I-HFG after soaking in unlabelled HSA is higher on the PU surface than on PU–PHEMA.

Fig. 8. Competitive adsorption of HSA (0.1 mg/ml) and HFG to PU and PU–PHEMA surfaces. Calculations were performed considering as 100% the concentration of the adsorbed HSA obtained from a pure albumin solution. The concentrations of unlabelled HFG added to the HSA solution were 0.01 and 0.1 mg/ml.

100 HSA (0.1mg/mL)

80 Protein retention (%)

PU

HFG (0.1mg/mL)

75

68 58 56

60

40

20

0 PU

PU-PHEMA

Fig. 9. Human serum albumin (HSA) and fibrinogen (HFG) retention onto PU and PU–PHEMA surfaces after washing them with a HSA solution (1 mg/ml) during 24 h.

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4. Discussion The aim of this study was the preparation of a hydrophilic surface, with low protein adsorption, to be used as a support for the immobilisation of several species that may induce specific protein adsorption. PHEMA and PHEA were chosen as the hydrophilic surface because they can be easily polymerised and possess hydroxyl groups that can be used for the immobilisation of different compounds. In order to obtain a material with acceptable mechanical and physical properties, the monomers were graft polymerised onto a PU surface. Medical grade PU (Pellethane 2363–80AE), was chosen as a substrate, due to its well-known blood compatibility. Elan et al. [32] studied the adsorption of coagulation proteins from whole blood and platelet activation onto different polymers, such as PU (Pellethane 2363–80AE), polyvinylchloride (PVC), poly(tetrafluorethylene) (PTFE) and silicone rubber (SIL). They described PU as the polymer which presents the lowest protein adsorption and platelet activation. The present study has shown that thin films of PHEMA and PHEA can be obtained by graft polymerisation of their monomers onto the surface of PU using ceric ion as the initiator. Results from mass gain (Table 1) and ATR-FTIR spectra (Fig. 2) have demonstrated a good incorporation of the monomers when their concentration is 0.66 m and that of ceric ion is 40 mm. ATR-FTIR spectra (Fig. 3) demonstrate that a good coverage of the surface of PU was obtained when a reaction time of 180 min was used. Feng et al. [19] have studied the reactive site and mechanism of graft copolymerisation of acrylamide onto PU initiated by ceric ion. They concluded that the main reactive site for graft polymerisation would predominantly be the nitrogen atom of the phenyl carbamate group (1,4-C6H4NHCOO–) of the hard segment of PU. They also showed that the reactivity of the ceric ion with PU is higher than with acrylamide monomer (AAM), because graft polymerisation of AAM onto the PU film always took place before homopolymerisation. Our results have shown that the reactivity of the ceric ion with HEMA, HEA and PU is dependent upon monomer and ceric ion concentration (Table 1). SEM (Fig. 6), XPS (Fig. 5 and Table 2) and water contact angles measurements (Table 3) also demonstrate the success of PHEMA graft polymerisation onto the PU surface. The stability studies demonstrate that PU–PHEMA is the most stable under alkaline conditions. Since one of the objectives of our research programme is to use these surfaces to immobilise several species that require alkaline conditions, the investigation concentrated on the coating of PU with PHEMA.

Concerning protein adsorption, albumin and fibrinogen were used in these studies since they are the proteins with higher abundance in plasma, and they have different behaviour in relation to haemocompatibility of biomaterials surfaces. While albumin is considered a passivating protein [6], fibrinogen is an adhesive protein with a central role in coagulation and platelet activation and aggregation [33]. The surface modification with PHEMA decreases albumin and fibrinogen adsorption (Fig. 7). This figure also shows that there is always more albumin adsorbed than fibrinogen. Horbett [34] also demonstrated a higher affinity of albumin than fibrinogen for PHEMA surfaces using plasma solutions. The observed amounts of protein adsorption could be due to some hydrophobic interactions between the proteins and the non-polar parts of the PHEMA. However, due to low amounts taken up, it is probable that the interactions are weak, as suggested by Garrett et al. [35]. Adsorption of proteins to biomaterials usually occurs from complex mixtures and involves competition of all the proteins in the mixture for the available surface sites. However, because it is very difficult to control all the important variables using complex mixtures of proteins, competitive adsorption studies from mixtures of two proteins are usually carried out [20,36]. Competition studies, using mixtures of albumin and fibrinogen, have shown that the presence of a lower concentration of fibrinogen in relation to albumin (ratio between proteins similar to that existing in blood), increases the adsorption of albumin in modified surfaces (Fig. 8). These studies indicate that in the presence of a low concentration of fibrinogen, PU–PHEMA presents higher albumin affinity than the unmodified PU surface. However, using solutions with the same concentration of albumin and fibrinogen, the decrease of albumin adsorption due to the presence of fibrinogen was similar for both surfaces. This figure also shows that, for both surfaces, there is only a 20% decrease of albumin adsorption when equal concentrations of the two proteins were used. If two proteins were equally effective in competing for the adsorption sites, we would expect a decrease of albumin adsorption of 50% when the same concentration of the two proteins was used [20]. Thus, these results also suggest a higher affinity of albumin for PU and PU–PHEMA compared to fibrinogen. PU–PHEMA presents lower retention of the adsorbed albumin and fibrinogen when soaked in albumin during 24 h (Fig. 9), indicating that the surface favours reversible adsorption. The adsorption of proteins to polymers is usually irreversible. The loss of reversibility was related to changes in conformation of the surface bound proteins with time [37] and is dependent on the nature of the surface. More hydrophobic polymers exhibited greater retention of adsorbed fibrinogen [38]. The decrease of albumin and fibrinogen retention on

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PU–PHEMA in relation to the PU surface could be related with the hydrophilicity of the former. The water contact angle changes from 79
76 (PU) to 59
79 (PU– PHEMA). This explains why other authors have used PHEMA to avoid protein adsorption. Morra et al. [31] demonstrated a decrease of albumin and fibrinogen adsorption to non-woven poly(butyleneterephtalate) (PBT) after plasma deposition of a PHEMA film. However, the protein adsorption was affected by the deposition conditions of the PHEMA film. Tanaka et al. [39] studied albumin and fibrinogen adsorption and its relationship to platelet adhesion onto poly(2-methoxyethylacrylate) (PMEA) and other acrylic polymers, namely PHEMA and PHEA. They conclude that PMEA and PHEMA present similar and low protein adsorption. However, the conformation of the adsorbed protein was less affected by the PMEA surface. The adsorption of proteins to biomaterials from blood and plasma is very complex and cannot be simply predict from the behaviour of pure protein solutions and mixtures of two proteins [20]. The composition of the adsorbed protein layer from blood or plasma changes with time (usually in the first minutes adsorption). The high concentration proteins dominate the surface at short times due to the higher collision rates. As time passes various exchange processes may occur and protein with higher surface affinity dominate the surface, even if their bulk solution concentration is very low. This exchange process is generally referred as the Vroman effect [40–43]. Brash’s studies using plasma with different dilutions, demonstrated that the initially fibrinogen adsorbed to glass [44] and standard polymeric materials [45] can be replaced by other plasma proteins. Although the surface modification with PHEMA can decrease albumin and fibrinogen adsorption from pure solutions, an investigation is now been conducted by our team using proteins adsorbed from plasma.

5. Conclusions Thin films of PHEMA and PHEA were successfully graft polymerised onto the surface of PU. However, only PU–PHEMA was stable in alkaline solutions. Its properties were characterised in detail using several techniques. PU surfaces modified with PHEMA have demonstrated low protein adsorption, showing their potential use as substrates suitable for attempting to reduce the process even further. This opens the possibly of exploring the advantages of selective adsorption by appropriate immobilisation of specific molecules. Of potential interest is the observation that small concentrations of fibrinogen in the presence of albumin

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increase the adsorption of the latter, when their concentration ratio is similar to that found in blood.

Acknowledgements This research work was supported by the PortugalChina Scientific Agreement (Blood Contact Materials Project) and the PEDIP II (CARE Project). The authors would like to thank to Dow Chemical Company (Belgium) for the Pellethane 2363-80AE. Ma Cristina L. Martins is grateful to the Portuguese Foundation for Science and Technology (FCT) for awarding her a scholarship under the programme PRAXIS XXI.

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