PROPERTIES OF SILK FIBROIN/POLY(ETHYLENE GLYCOL)400 BLEND FILMS PROPERTIES OF SILK FIBROIN/POLY(ETHYLENE GLYCOL)400 BLEND FILMS

PROPERTIES OF SILK FIBROIN/POLY(ETHYLENE GLYCOL)400 BLEND FILMS

  • 期刊名字:高分子科学(英文版)
  • 文件大小:370kb
  • 论文作者:Song Wang,Qing-hua Wang,Xin-li
  • 作者单位:Research Center of Materials
  • 更新时间:2020-12-22
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论文简介

Chinese Journal of Polymer Science Vol. 21. No. 1, (2003), 87 -91Chinese Journal ofPolymer ScienceC2003 Springer-VerlagNotePROPERTIES OF SILK FIBROIN/POLY(ETHYLENE GLYCOL)400BLEND FILMSSong Wang, Qing-hua Wang, Xin-lin Yang, Liyong Wang and He-sun ZhuResearch Center of Materials, Bejing Institute of Technology, Beiing 100081, ChinaAbstract The blend flm of silk fbroin (SF) and poly(ethylene glyco)400 (PEG400) with a blend ratio of 2/1 (wt/wt) wasprepared simply by dropping a litle PEG400 into the SF solution and then casting the mixed aqueous solution at 50C. Theresuling film exhibited much better mechanical properties in the dry and wet state than SF itelf, owing to theconformational change of SF in the blends from the random coil to the -sheet structure and intermolecular hydrogen bondformation between SF and PEG400. Thermogravimetric analysis showed that the initial thermal decomposition temperatureofthe blend flm was 170C, which was 80^C lower than that of SF (250C) and 20C higher than that of PEG400 (150C),and indicated a strong interaction between two components of the blend, No crytlline peaks were observed in the X-raydiffaction curve of the blend film. Cel culture test showed that SF/PEG400 was a suitable substrate for the growth of humanumbilical vein endothelial cells (HUVEC).Keywords Silk fibroin, Poly(ethylene glycol)400, Blend filmn, Cell cultureINTRODUCTIONSilk fibroin (SF) has proven to be an invaluable biomaterial due to its inherent biocompatibility, good oxygenand water vapor permeability and biodegradability. In recent years, the application of SF flms has progressed toyield, e.g. the immobilization matrices for of enzymes, drug delivery systems, bum wound dressing1-3.However, pure SF films are very britle. To improve the mechanical properties, blend SF films have beenprepared by mixing SF with other synthetic or natural polymers, for example, poly(vinyl alcohol) (PVA), sodiumalginate, chitosan, and cellulosel4-7!.Poly(ethylene glycol) (PEG) is widely used as a biomaterial. The chemical mdifcation of SF with 2,4-bis[o-methoxypoly(ethyleneglycol)]-6-chloro-s-triazine (actPEG2, Mw = 104)8) and PEG diacrylate macromer(Mw. PEc = 8000)9 was reported. It was found that the tensile strength and elongation of SF/PEG were higher thanthose of SF itself. But the cell (L929) attachment and growth decreased significantly after PEG was introducedinto SF. In addition, the modifiers and initiators used in chemical reactions are usually toxic, and it is not easy toremove them thoroughly from the products. In this study, a very low molecular weight PEG400 was chosen as amodifier of SF. Since it is a liquid, we can obtain the blend flms simply by dropping PEG400 into the aqueoussolution of SF and then casting the mixed solution. The resulting flm of SF/PEG400 (2/1) shows a goodmechanical properties in the dry and wet state, and it is also a suitable substrate for the growh of humanumbilical vein endothelial cels (HUVEC).'This work was supported by the National Basic Science Rese中国煤化工(973) of China (No.G1999064705)." Coresponding author: Song Wang (王松), E-mail: songwl@95777.;YHCNMHGReceived April 6, 2002; Revised May 16, 2002; Accepted May 20, 200288S. Wang et al.EXPERIMENTALMaterialsDegunmed silk was dissolved in a mixed solvent of CaCl, H2O, and ethanol in a molar ratio of 1:8:2. Thesolution was then dialyzed in a cellulose tube against running water. The fibroin concentration of regenerated silkwas normally about 15 mg/mL. Analytical grade PEG400 from Bejing Yili Chemicals Ltd. was used withoutfurther purification.Preparartion of FilmsThe aqueous SF solution of 1% (g/mL) and PEG400 were carefully mixed in the ratio of 1000:5 (V/V). The filmsof SF and SF/PEG were obtained by casting the solutions onto tissue culture polystyrene (TCPS) dishes anddrying at 50C.MeasurementsThe tensile strength and elongation at break were measured by using a TOM 500J model tensile testing machineat ambient relative humidity at room temperature. In the case of measurements in the wet state the samples werefirst equilibrated with ditlled water for a few minutes. The experimental conditions were 5 kg initial load cell,20 mm/min tensile speed, 60 x 8 x 0.03 mm sample dimensions.To measure the solubility in water, seven preweighed dry samples (190- 220 mg) were immersed in ditilledwater (10 mL) at various time intervals (3, 6,9, 12, 15, 18, and 21 days), respectively. The water was changedonce every three days. Finally the removed samples were dried and weighed again. Dissolved fibroin wasdetermined by spectrophotometry. The weight loss and the fibroin loss of the film were calculated from thefollowing equations:Weight loss = (Wo- W)/WoFibroin loss = (E10C; )/Wowhere Wo and W are the weights of the films before and after the immersion, 10 is the volume of the water inwhich the samples were immersed, and C; is the concentration of fbroin in the water after the immersion for3 days.FTIR spectra were obtained using a Perkin-Elmer 2000 spectrometer in the spectral region of 2000-400 cm^'. X-ray diffaction patterns were obtained with a D/max -2400 Diffractometer, and CuKa radiation with awavelength of 0.154 nm. Thermogravimetric analysis (TGA) was run under a flow of nitrogen gas at a scanningspeed of 10 K/min using a DuPont 2000 thermogravimeter.Cell Culture Test ProcedureThe films of SF and SF/PEG for the cell test were made by pouring each aqueous solution (0.5 mL) onto four-well tissue culture plates and drying at 50°C. The pure SF flms were immersed in alcohol for 30 min to makethem water- insoluble. All sample films were sterilized by UV irradiation for3 h.Human umbilical vein endothelial cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium),supplemented with 20% fetal bovine serum, 100 U/mL penicilin and 100 ug/mL streptomycin. Aliquot (0.5 mL)suspensions of HUVECs at 1x 10 cells/mL were plated, and maintained at 37C in water saturated atmosphere(5% CO - 95% air) for 72 h. Non-attached cells were removed by discarding the culture medium and then thefilms were carefully washed twice with phosphate buffered saline solution. The number of viable cells wasmeasured by MTT (3(4,5-dimethylthiazol-zy)2,5-dipbheay1 tetrazoliuim bromide)-based colorimetric assaylol andhe absorbance at 550 nm {ODsoonm) that was proportional to the number of cells on the sample flms wasrecorded by a MB-1II enzyme immune analyzer. The same procedure was repeated with the cells without films asa control. All data were analyzed using the means of duplicated? were subjected to中国煤化工5analysis by Student's t test. Differences were considered significTYHCNMHGProperties of Silk Fibroin/PEG400 Blended Film89RESULTS AND DISCUSSIONMechanical PropertiesThere were significant diferences in the mechanical behavior between SP and SF/PEG400 films. Pure silk filmswere extremely britte in the dry state and soluble in water, so they were unsuitable for measurements. However,films became soft and water-insoluble after PEG400 was introduced into SF. In particular, the breaking tensilestrength of SF/PEG with a blend ratio of 2/1 (wtwt) in the dry and wet state was 16.5 and 2.9 MPa, theelongation at break was 10% and 50%, respectively. These strong and soft blend films were used in subsequentmeasurements.It was found that the effect of the blend ratio on the mechanical behavior of the blend was remarkable; it willbe discussed in a following paper.SolubilityTable 1 shows the weight loss and fibroin loss of polymer blends composed of SF and PEG400 afer immersingthem in dstild water at37C for 3, 6, 9, 12, 15, 18 and 21 days, respectively. It was found that the blend flmscontain 33% and 23% PEG400 before and after the immersion and no more PEG diffused into water after 3 days.The total fibroin loss of the blend was 1.8% after 21 days' immersion, where the 3~day fibroin loss decreased asthe immersion time increased. The first 3-day loss was the most significant at 0.55%, but it was still less than onetenth of that of pure silk flm under the same immersion conditions (7%). This means that SF became nearlywater-insoluble after PEG400 was introduced.Table 1. Weight loss and fibroin loss of SF/PEG400 blend fims after immersion in waterImmersion/d39121518_2Weight loss (%)10.3410.3910.5110.4511.7210.70Total fibroin loss (%)0.55.0.901.191.451.591.731.80Last 3-day0.550.350.290.260.140.065fibroin loss (%)SPEE wm拿hw~SP4000 3500 3000 2500 2000 1500 1000 50020v (cmr)20()fig, 1 FTIR spectra of PEG400 (a), SF(b),Fig. 2 X-ray diffaction curves of SF andSF/PEG400 blend (c).SF/PEG400 blend中国煤化工FTIR and WAXD AnalysisThe FTIR spectra of PEG400 (a), SF (b) and SF/PEG400 (c) al.MHC NMH G.ed asoption bandsat 1652 (amide D), 1541 (amide I) and 1237 cm:' (amide II), atributed to the random coil conformation of SF.90S. Wang et al.PEG400 exhibited absorption bands at 3437 and 1108 cm" , atributed to the 0 H and C-0- C strething,respectively. On the other hand, the spectrum of the blend filtm revealed that the amide II band of SF at 1541 cm'lshifted to 1518 cmi ! with increasing intensity, while the amide Il band of SF at 1237 cm' became sharp andshifted to 1246 cm^. In the case of absorption bands of PEG400 in the blend, the 0-H and C -0-C stretchingbands shifted from 3437 and 1108 cm*' to 3300 and 1105 cm,respectively. These results suggested that the .conformation change of SF occurred from a randorm coil to a B-sheet structure due to the formation ofintermolecular hydrogen bonds between SF and PEG in the blend filros.Figure 2 shows the X-ray diffraction curves of SF and SF/PEG400. Both samples showed amorphousdiffraction patterns.Thermal PropertiesFigure 3 shows the thermogravimetric curves of SF, SF/PEG400 and PEG400. The initial weight loss atapproximately 100C was due to the evaporation of water from SF. Then, the temperature increased and a sharpdrop of the TGA curves appeared at 250, 170 and 150C for SF, SF/PEG400 and PEG400, which indicated thebeginning of the thermal decomposition accompanied by a greater weight loss. The temperatures at which SF,SF/PEG400 and PEG400 reached a 20% weight loss were 307, 275 and 275C, respectively. These valuesindicated that the thermal stability of the blend film was intermediate between two pure components.Figure 4 shows the differential thermogravimetric (DTG) curves of SF, SF/PEG400 and PEG400. Themaximum decomposition temperatures of SF, SF/PEG400 and PEG400 were 305, 307 and 345C, respectively.The blend film composed of SF and PEG400 showed one maximum, indicating that the molecular interactionsbetween the two components were strong in the blend.8 801.5p一SF..+ SF/PEG40060-0叶中PEG400g40-。~ 0.5室2. SF/PEG40020F PEG4000.0-100 200300 400 s00500T(C)T(°C)Fig, 3 Thermogravimetric curves of SF, SF/PEG400Fig.4 DTG curves of SF, SF/PEG400 blendblend and PEG400and PEG4000.80.6).2 F0.CTCPS SF SF/PEG SF TCPS SF/PEGFig. 5 Proliferation of HUVECs on SF, SF/PEG400 blend and TCPSValues of OD are the average of eight independent measurements.Statistically significant differences between SF and SF/PEG400, TCPS:*p <0.05 and ** p<0.01Interaction Betweent Cultured Cells and SF/PEG Blend Flulm中国煤化工Figure 5 shows the proliferation of HUVECs on SF, SF/PEC les of DODsom wereproportional to the number of the viable cells growth on the fi.CNM H CsF gave de hgetProperties of Silk Fibroin/PEG400 Blended Film)1prolifertion of cells, whereas SF/PEG gave the same proliferation of cells as the TCPS. The result suggested thatSF/PEG400 is just as good a substrate for cell incubation as TCPS.Minoura et al.'U have reported that SF-PEG (Mw = 10000) conjugates exhibited very low cell (L-929)atachment and growth, where the content of the PEG component in SF-PEG was about 67% by weight. Theresult has been explained in terms of the high excluded volume effect of PEG. In this paper, the content of thePEG400 in the blend was about 33% by weight and its molecular weight was one twentieth of that of thePEG10000 used in Ref. [8]. Therefore, the excluded volume effect of PEG on the cell incubation was small.REFERENCES[ Demura, M, Asakura, T. and Kuroo; T, Biosensors, 1989, 4: 3612 Hanawa, T, Watanabe, A, Tsuchiya T, lkoma, R, Hidaka, M. and Sugihara, M.. Chem. Pharm. Bull, 1995, 43: 8723 Minoura, N, Tsukada, M. and Nagura, M, Biomalerials, 1990, 11: 4304 Yamaura, K, Kuranuki, N, Suzuki, M, Tanigami, T. and Matsuzawa, S.,J. Appl. Polym. Sci, 1990, 41: 24095 Liang, C.X. and Hirabayashi, K, J. Appl. Polym. Sci, 1992, 45: 19376 Park, S.J, Lee, KY, Ha, W.S. and Park, S.Y, J. Appl. Polym, Sci, 1999, 74: 25717 Freddi, G., Romarino, M, Massafra, M. R. and Tsukada, M, J. Appl. Polym. Sci, 1995, 56: 15378 Yohko, G, Masuhiro, T, Norihiko, M. and Imai, Y., Biomaterials, 1997, 18: 2679 Kweon, HY, Park, S.H, Yeo, J.H. Lee, Y.W. and Cho, C.S, J. Appl. Polym. Sci, 2001, 80: 184810 Mosmann, T, J. Immunol. Methods, 1983, 65: 55中国煤化工MYHCNMHG

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