SURFACE MODIFICATION OF BLEND FILMS COMPOSED OF SILK FIBROIN AND POLY(ETHYLENE GLYCOL) MACROMER AND

Chinese Journal of Polymer Science Vol. 22, No. 4, (2004), 399 403Chinese Journal ofPolymer Science02004 Springer-VerlagNoteSURFACE MODIFICATION OF BLEND FILMS COMPOSED OF SILK FIBROINAND POLY(ETHYLENE GLYCOL) MACROMER AND THEIR IN VITROANTITHROMBOGENICITYHua-xin Sun, Song Wang" and He-sun ZhuResearch Center of Materials, Bejing Institute of Technology, Beijing 100081, ChinaAbstract In order to improve the blood compatibility of silk fbroin (SF), poly(ethylene glycol) macromer (PEGM) indifferent amounts was added to the SF flm to incorporate C=C group into the surface of blend films which were thenmodifed by SO2 gas plasma treatment. ATR-FITR and XPS were used to analyze the chemical change which had ocurtedon the fim's surface. When the content of sulfur on the surfce of blend films supasses 1.59%, the antithrombogncity ofplasma treated flms increases remarkably due to surface sulfonation. This result implies that SF with blend of PEGM aferSO2 plasma treatment have potential use for making blood-contacting biomaterials.Keywords sik fibroin, Poly(thylene glyco) macromer, Plasma, AntithrombogenicityINTRODUCTIONSilk fibroin (SF), as a natural biopolymer with outstanding physicomechanical properties, has been applied inbiological and biomedical fields such as enzyme immobilization, oxygen permeable membranes, drug permneablefilms and so on(!-3]. However, there also exist some disadvantages such as poor mechanical properties of SFflms in the aplication of biomaterials. Polylethyene glyco1) (PEG), as a widely used biomaterial, has beenchosen to change SF properties by many researchers. Wang et al. blended SF and PEG to prepare films toenhance mechanical properties in the dry and wet stel. Kawano et al. revealed that the PEG grafed on thesurface of silk fbroin could decrease platelet adhesion, fibrinogen adsorption and complement consumption ofsilk fbroinls. Many biomaterials must possess desirable blood compatibility, especially when they are consideredfor use as blod-contating matrials. The lowtemperature plasma glow technique is an efective method forsurface mdifcation. A previous report has shown that sulfur-containing groups can be incorporated onto thesubstrate of some polymer materals by sO2 plasma through surface sulfonation and thus reduce plateletdeposition and thereby improve antithormbogenectyos 7. In the present work, poly(ethylene glycol) macromer(PEGM), having an acrylated-terminated group, was chosen as the additive of SF. SO2 gas plasma was used toproduce sulfnic groups covalently atached to the surface of SF blend film. Then the sulfonated film was testedfor its antithrombogencity by in viro coagulation time tests, including the test of activated partial thromboplastintime (APTT). The resuts show that antithrombogenicity of SF/PEGM blend flms could be significantlyimproved by surface sulfonation.This work was supported by the National Basic Science Research and Development Grants (973) of China (No.91999064705) and the National High Technology Project (863) of China (No.2002AA326030).”Corresponding author: Song Wang (王松), E-mail: wangsong@bit.edu.cn中国煤化工Received October 9, 2003; Revised December 26, 2003; Accepted December|YHCNMHGH.S. Sun et al.400EXPERIMENTALMaterialsRaw silk was degummed twice with 0.5% weigh-ofiber (w.0.D sodium catbonate solution at 100^C for 30 minand then washed with distlle water. Degummed silk was dssolved in CaCl:H2O:ethanol = 1:8:2 mixed solvent.The solution was then dialyzed in a cllulose tube against running water. The fibroin concentration ofregenerated sik was nomally about 20 mgmL. PEG400 was purchased from Bejig Yili Chemicals Ltd.Acryloyl chloride was obtained from Acros Organics. Kits for in vitro coagulation time (APTT) test wereobtained from Huashan Hospital of Shanghai Medical University. Human blood plasma was obtained from theChina Red Cross Association. All other chemicals were extra pure reagent grade and the minimal water wasremoved with CaH2.Synthesis of PEG Macromer14 mL (36 mmo/L) PEG400 and 15 mL (107 mmolL) triethylamine were dissolved in 70 mL dichloromethanein a 250 mL round-bottomed flask. 9 mL (110 mmol/L) acryloyl chloride dssolved in 100 mL ofdichloromethane was added to the flask through a dropping funnel. The reaction mixture was stired for 24 h at0C, and then kept for 48 h at room temperature (R7). The reaction mixture was filtered to rermove trethylaminehydrochloride and then the filtrate was poured into an eXcess amount of petroleum ether to obtain the macromer.Finally, the macromer was dried at RT under reduced pressure for 24 h and then stored at a lower temperature.Preparation of Blend FilmsThe 2% SF solution and PEGM were mixed in the ratio of l:1, 2:1, 3:1, 4:1, 6:1 (WIW). The mixture was castonto a polystyrene plate and incubated at 50C for 24 h.Surface Sulfonation ofSF Blend Films by Using SOz PlasmaThe plasma reactor system was designed by ourselves. The chemical grating of sulfonyl group onto SF blendfilms was performed as described by Gu et al?!. The processing parameters of SO2 plasma treatment were set asfllows: base gas pressure, less than 3 Pa; gas flow rate, 20 mL/min; working pressure, 20 Pa; net plasma power,30 W; time, 10 min; temperature , RT.MeasurementsA FTIR spectometer (Bruker) was used to confirm the structure of PEG macromer. ATR spectra were obtainedusing a Tensor spectrometer (Bruker) to identif the existence of C=C group and sulfonated structure on thesurface of the blend films after plasma treatment.XPS analysis of SF flms was performed by a PHI 5300 X-ray spectrometer (Perkin-Elmer) equipped withan MgKa non-monochromatic X-ray source. The surface elemental compositins of C1s 013, NIs and S2p weremeasured and a detailed scan of S2p was made in the present experiments.In vitro Coagulation Time Tests for SF FilmsThe antithrombogenicity of materials can be reflected by an in viro coagulation time test (APTT) The mainprocedure was to put the testing flm into a transparent plastic tube with healty blood plasma and then thereagents were added for each coagulation time test immediatly. The cotting timne was measured by using aphoto-optical clot detection istrument Coag-A-Mate*-XM (Organon Teknika, USA).RESULTS AND DISCUSSIONATR-FTIR SpectraFigure 1 shows the FTIR spectra of (a) PEG and (b) the PEGM. The PEG macromer prepared by the reactionbetween PEG and acryloyl chloride shows new peaks at 1722 cm I and 1409 cm，which are atributed to thestretching vibration band of carbony1 and deformation vibation band of the C=C group, respectivly, indicaingthe formation of PEG diacrylael9. The ATR-FTIR spectra of (a) SF film, (b) SF/PBGM (1:1) blend flm and (c)SO2 plasma-treated SF/PEGM (1:1) blend film are shov中国煤化工bsorption bands atYHCNMHGSurface Modification of Blend Films of SF/PEGM and Their Antithrombogenicity4011637 (amide D), 1514 (amide II) and 1232 cm . (amide I), which are attributed to the B-sheet conformation onthe SF flm surface. On the other hand, the spectrum of the polymer blend composed of SF and PEGM is no morethan an overlapping spectrum of SF and PEGM. In spectra b and C there is a peak at 1409 cm^ ; however, itspeak intensity in spectra C is weaker than that in spectra b (Fig. 2). That means the c=C groups existed on thesurface of SF/PEGM blend flm and the concentration of C=C group was lowered after SOz plasma-treatment.This means the C=C group takes part in the chemical reaction when SO2 plasma-treated. The stretchingvibration of S=0 of sulfonate group should appear at 1120-1180 cm~", but in spectra C there is only one strongand broad peak at 990 -1180 cm , atributed to the C -0- -C group. So the stretching vibration absorption ofS=0 group may be shielded by the absorption of the C- -0 - C group.1723.77MYyV11409.46hirip\p"1725.2840003500300025002000150010004000 3500 3000 2500 2000 1500 1000Wavenumber (cm )Wavenumber (cm)Fig. 1 FTIR spectra of(a) PEG and (b) PEGMFig. 2 ATR-FTIR spectra of (a) SF, (b) SF/PEGM(Mw, PEG = 400)(1:1) blend, (c) SO2 plasma-treated SF/PEGM(1:1) blend filmsXPS Analysis of FilmsXPS analysis reveals the existence of carbon (C), nitrogen (N), oxygen (O), and sulfur (S) atoms on the surfaceof untreated and plasma-treated SF blend films. The surface atomic relative percentages in different SF films arelisted in Table 1. The surface percentages ofC, O, N, and S of SF/PEGM blend film are 72.80%, 24.27%, 2.82%and 0.10%, respectively. The 0/C atomic ratios of the SO2 plasma-treated SF blend films are all higher than thatof untreated blend film, which indicates the strong oxidizing capability of SO2 plasmaln. Compared withuntreated sample, SF blend films treated by SO2 plasma have more abundant surface sulfur as shown by theintroduction of some sulfur-containing groups onto their surfaces. The percentages of S on the surface of filmsrange from 1.59% to 3.% (Table 1). First with the increase of PEGM content, the percentage of S increases andwhen the ratio of SF and PEGM increases to 3:1, the percentage of S reaches its maximum. It is presumed thatwhen the PEGM content is low, the surface C=C groups may react mainly with SO2. In contrast, when the ratioof SF and PEGM is above 3:1, the percentage of S decreases gradually. It is believed that the surface c=Cgroups may mainly be involved in crosslinking reactions because they have strong chemical activities'. In Fig. 3,spectum C shows the XPS analysis results of the surface S2p spectra of SF blend flms treated by SO2 plasma.There are two distinctive peaks near 169.1 eV and 164.2 eV in the S2p spectrum of SO2 plasma moified SF.Table 1. Surface atomic relative ratios of Cip O1 Na and S2 measured by XPS analysis in diferent modified SF flmsSampleRatioCig(%)03(%)N;(%) S2p (%)O/CS/C(SF/PEGM)(Atomic ratio)Silk fibroin78.3013.417.820.470.1710.006SF/PEGM blend film1:72.8024.272.820.100.3330.001SO2 plasma treated flm72.5525.490.381.590.3510.022SO2 plasma-treated flm2:69.1825.103.392.30.3630.0343:62.05 .29.684.373.900.4780.0634:167.2224.555.900.365 ;0.0356:65.2724.01中国煤化工-YHCNMHGH.S. Sun et al.402The higher energy peak may represent the sulfur atom at a higher oxidation state, assigned to sufur atomsbonded to two or three oxygen atoms such as sulfone, sulfonate or sulfonic acid. In contrast, the lower energypeak may be atributed to sulfur atoms linked to carbon or to one oxygen atom!". Interestingly, with theincreasing PEGM content on the surface of SO2 plasma-treated blend flms, the relative contents of sulfur witharound 170 eV binding energy gradually decrease while those of sulfur with around 165 eV binding energyincrease gradually (Fig.3).de178 174 170 166 162 158Binding energy (eV)Fig. 3 XPS analysis of the S relative contents ofthe plasma treated films (SF/PEGM)a) 6:1; b) 4:1; c) 3:1; d)2:1;e)1:1Table 2. In vitro coagulation timnes (APTT) of human blood plasma incubated withSF and SO2 plasma-treated SF blend fImsSO2 plasma-SO2 plasma- SO2 plasma-Blood plasmaUntreated SFtreated filmtreated flm(U:1)(2:1)(3:)(4:1)APTTS)32.631.3>150> 150'The data are the mean value of three repeated determinations.In vitro Coagulation Time Tests of SF FilmsIn vitro coagulation time tests (APTT) have widely been used for the clinical detetion of abnormnality of bloodplasma, the primary screning of anticogulative chemicalst10l and the evaluation of antithrombogenicity ofbiomaterials. As the mean value of APTT time for a blood plasma is 32.6 s (Table 2), the normal range of APTTtime is taken as 32.6土10s)I. It can be seen that when the t中国煤化工th untreated SF film,YHCNMHGSurface Modification of Blend Films of SF/PEGM and Their Anithrombogenicitythe APTT is 31.3 s, indicating that SF shows neither antithrombogenicity nor thrombogenicity towards the bloodplasma. 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