- Email: [email protected]
Immobilization of poly(ethylene glycol) or its sulfonate onto polymer surfaces by ozone oxidation
Biomaterials 22 (2001) 2115}2123
Immobilization of poly(ethylene glycol) or its sulfonate onto polymer surfaces by ozone oxidation Young Gun Ko , Young Ha Kim *, Ki Dong Park , Hee Jung Lee , Won Kyu Lee , Hyung Dal Park , Soo Hyun Kim , Gil Sun Lee, Dong June Ahn Biomaterials Research Center, Korea Institute of Science and Technology, P.O. Box 131, Cheongryang, Seoul 130-650, South Korea Department of Chemical Engineering, Korea University, Anamdong 5ga-1, Seoul 136-701, South Korea Received 7 September 2000; accepted 12 November 2000
Abstract A novel surface modi"cation method has been developed to improve biocompatibility of polymeric biomaterials. This approach involves ozonation and then followed by graft polymerization with acrylates containing PEG, sulfonated PEG or by coupling of PEG derivatives. All the reactions were con"rmed by ATR FT-IR and ESCA. The degree of ozonation measured by the iodide method was dependent on the ozone permeability of the polymers used. Surface hydrophilicity was investigated by measuring the contact angles. Ozonation itself yielded a slight increase in hydrophilicity and a decrease in platelet adhesion, but PEG immobilization showed a signi"cant e!ect on surface hydrophilicity and platelet adhesion to con"rm well-known PEG's passivity which minimize the adhesion of blood components on polymer surfaces. Both graft polymerization and coupling were e!ective for PU. In contrast, only grafting gave enough yields for PMMA and silicone. Platelet adhesion results demonstrated that all PEG modi"ed surfaces adsorbed lower platelet adhesion than untreated or ozonated ones. Polymers coupled with sulfonated PEG exhibited the lowest platelet adhesion when compared with control and PEG coupled ones by virtue of the synergistic e!ect of non-adhesive PEG and negatively charged SO groups. This PEG or sulfonated PEG immobilization technology using ozonation is relatively simple for introducing uniform surface modi"cation and therefore very useful for practical application of blood contacting medical devices. 2001 Elsevier Science Ltd. All rights reserved. Keywords: PEG/PEG}SO immobilization; Ozone treatment; Wettability; Platelet adhesion
1. Introduction A variety of approaches has been undertaken to improve the blood compatibility and to minimize cell adhesion on biomaterials surfaces [1}3]. One approach involves surface modi"cation by grafting a hydrophilic component, such as poly(ethyleneglycol) (PEG). PEG has unique solution properties and molecular conformation in aqueous solution. PEG-grafted surfaces exhibit speci"c non-adhesive property to proteins, blood components and cells mainly due to high surface mobility and steric stabilization e!ects [4}8]. In addition, sulfonated PEG, (PEG}SO )-grafted polymers, improved anti thrombogenicity, biostability, and anticalci"cation in-vitro,
* Corresponding author. Tel.: #82-2-958-5340; fax: #82-2-9585308. E-mail address: [email protected] (Y.H. Kim).
ex-vivo, and in-vivo by the synergistic e!ect of the #exible hydrated PEG chains and negatively charged sulfonate anticoagulant groups [9}12]. Surface ozone oxidation is widely applied in polymer areas because it has an advantage of uniformly introducing peroxides on the polymer surface and o!ers an easy-to-handle, inexpensive technique [13}16]. When polymer is exposed to ozone gas, peroxides are formed in addition to carbonyl and carboxyl groups [17]. The generated polymeric peroxides are capable of initiating polymerization of vinyl monomers, resulting in surface grafting onto the ozonated polymeric materials [13]. This article describes PEG or PEG}SO immobiliz ation onto polymethylmethacrylate (PMMA), polyethylene (PE), silicone, and polyurethane (PU) by ozonation (Fig. 1). The surface structures and properties of modi"ed polymers were investigated using attenuated total re#ectance Fourier transform infrared (ATR FT-IR), electron spectroscopy for chemical analysis (ESCA), atomic force
0142-9612/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 0 ) 0 0 4 0 0 - 2
2116
Y.G. Ko et al. / Biomaterials 22 (2001) 2115}2123
Fig. 1. Schematic illustration of surface modi"cation.
microscopy (AFM) and dynamic contact angle (DCA). In addition, the blood compatibility of PEG-modi"ed polymers was evaluated by in-vitro platelet adhesion study.
2. Materials and methods 2.1. Materials Polyurethane (PU, Pellethane, Dow Chemical Co., Midland, MI, USA) was extracted with methanol for 3 days to remove low molecular weight components, and dried under vacuum. The extracted PU was dissolved in N,N-dimethylacetamide, cast into "lms and dried for 1 week at 453C. Polymethylmethacrylate (PMMA), commercial sheets (LG Chemical Co., Korea), was washed by ethanol for 3 days. Low-density polyethylene (PE) "lms were purchased from Hanwha Petrochemical Co., Korea. Silicone sheets were supplied from Yushin Medical Co., Korea. All the sheets (size 1;3 cm, thickness 0.5}2 mm) were further sonicated for 30 min before ozonations. PEG mono-acrylate and diamino-terminated PEG (molecular weight of PEG 1000, NOF Co., Japan) were dissolved in chloroform for puri"cation and precipitated into diethyl ether, and then dried under vacuum. Sulfonation of PEG mono-acrylate(PEGA) was performed using Na metal and propane sultone. Na metal was put into PEGA to be reacted at 503C for 12 h. The resultant product was precipitated into diethyl ether,
"ltered, and puri"ed by dissolving in chloroform and then it was precipitated into diethyl ether and "nally dried overnight at room temperature. Diamino-terminated PEG (H N}PEG}NH ) was also sulfonated by propane sultone as described previously [11]. Brie#y, 10% (w/v) propane sultone in tetrahydrofuran was added by drops to 10% (w/v) H N}PEG}NH solution in tetrahydrofuran and reacted at 503C for 6 h. The resultant product was precipitated from medium as the reaction proceeded. The "ltered product was washed with cold tetrahydrofuran and dried overnight at room temperature. The structures of obtained sulfonated PEGs (PEGA}SO and H N}PEG}SO ) were con"rmed by a nuclear magnetic resonance spectrometer (H-NMR, C-NMR, Varian Gemini 200 MHz). 2.2. Ozonation and surface grafting or coupling of PEG derivatives Ozone was generated with dried oxygen gas passed through an ozone generator (Labor-Ozonizator 301, Sander Co., USA). The operating condition was set at 6 kV, 1 bar O pressure and 50 l/h O #ow rate at room temperature. After the ozonation, it was degassed by purging O to remove ozone adsorbed in the specimen. Polymeric peroxides introduced onto the treated "lms were determined by the iodide method spectrophotometrically [13,18]. This method utilizes an oxidation of sodium iodide by peroxides in the presence of ferric
Y.G. Ko et al. / Biomaterials 22 (2001) 2115}2123
chloride. The treated "lms were kept at 603C for 10 min in benzene}isopropyl alcohol solution (1 : 6 by vol) containing saturated sodium iodide and 1 ppm ferric chloride. After addition of water to stop the reaction, the oxidized iodine was measured as triiodide anion from the absorbance of the solution at 360 nm with the molar absorptivity of 2.3;10 l/mol cm. After ozonation, the "lms were immersed immediately into 20 wt% aqueous solutions of PEGA, PEGA}SO , H N}PEG}NH , or H N}PEG}SO in each glass am pule, respectively. After vigorous degassing by purging N , the ampules were sealed and kept at 603C for given periods of time. The PEG-immobilized "lms were washed by tridistilled water for 3 days to remove the unreacted PEG derivatives. 2.3. Surface characterization Dynamic contact angles (DCA) were measured at room temperature with a Wilhelmy plate equipment (DCA-322, Cahn Instruments Inc., USA). Tridistilled water was used for the measurement of each of four specimen. The velocity of the translation stage was 150.7 m/s. DCA by Wilhelmy plate method gives two angles, advancing angle when immersing into water and receding angle when withdrawing from water. It was generally known that advancing angles indicate hydrophobicity while receding ones express hydrophilicity of surfaces. All the specimens were hydrated for 1 day before measurements to be close to in-vivo conditions, although the values before and after hydration were not much di!erent from each other. Attenuated total re#ectance Fourier transform infrared (ATR FT-IR) was used to analyze the surfaces using a Bruker FT-IR (IFS 66, Germany) with KRS-5 crystal. ESCA spectra for each sample were obtained on an ESCA 2803-S (SSI, USA) spectrometer with Al K X-ray. In order to determine O/C, N/C, S/C, and Na/C stoichiometries, collecting factors of 2.50, 1.68, 1.80, and 8.5 were used for O , N , S , and Na , respectively. Binding energies were referenced to the C}H group of the C peak components at 285.0 eV. With the C spectra smoothed, the subpeaks were deconvoluted using a curve-"tting method from a series of Gauss}Lorentzian curves. The surface morphology of modi"ed polymers was examined with an AFM manufactured by Park Scienti"c Instruments (CP, USA). 2.4. In vitro platelet studies Human whole blood, which was treated with citric acid, was centrifuged at 800 g for 10 min at 253C to prepare platelet-rich plasma (PRP). The residue of the
2117
blood was centrifuged at 3000 rpm for 10 min to obtain platelet-poor plasma (PPP). The number of platelets was adjusted to 430,000 mm\ by adding PPP to PRP. Polymer "lms were introduced into the specially designed syringe and hydrated in phosphate-bu!ered saline (PBS) for 1 day. Prior to adhesion studies, the bu!er was removed from the syringe and 2 ml of PRP was introduced into it. The syringe was then tapped to remove air bubbles, sealed, and rotated in a shaking incubator at 373C. By this method, the polymer "lms were constantly exposed to PRP. Therefore, only surface/platelet interactive in#uences were observed. A set of syringes was prepared for platelet adhesion test along the time of 15, 30, 60 and 120 min with incubation in PRP. At each time, the syringes were quickly removed from the shaking incubator, and immediately depleted platelets were counted in the PRP with the Coulter counter or hematocytometer. The amount of platelets that adhered upon the specimen was calculated by subtracting the number of unadhered platelets from the number of diluted platelets that were initially incubated. The morphology of platelets adhered on "lms were examined by SEM (Hitachi, S-2460N, Japan). Polymer samples were withdrawn from PRP and rinsed in PBS with gentle agitation to remove weakly adhered platelets. The "lms were "xed with 2% glutaraldehyde solutions in PBS for 2 h at 43C, dehydrated with 50, 60, 70, 80, 90, and 100% dilutions of ethanol and water, and freeze dried for 1 day. Samples were coated with a gold layer and observed with SEM.
3. Results and discussion 3.1. Ewect of ozonation on polymer surfaces Fig. 2(a) shows the concentration of peroxides evolved on PU, PMMA, PE, and silicone "lms treated by ozone. In the case of PMMA, the concentration of peroxide increased gradually with ozonation time. But the peroxide concentrations on silicone, PE, and PU "lms were increased rapidly upto 1 h and leveled o! to a constant value. PU and silicone exhibited a high concentration of peroxide but PE showed low concentration. The change of contact angles (receding angles, ) of the ozonated "lms was compared on Fig. 2(b). All the angles which decreased after the ozonation indicate increase in hydrophilicity, but the extent of decrease varied depending on the "lms. PMMA exhibited no signi"cant change. The contact angles of PU and PE were decreased to a signi"cant extent; those of PU were decreased gradually as time went by but those of PE was decreased rapidly in the beginning and leveled o!. It is notable that the peroxides formation after ozonation and the increase in the hydrophilicity varied depending on the specimen. The ozonation is basically a surface
2118
Y.G. Ko et al. / Biomaterials 22 (2001) 2115}2123
centration of peroxides include those inside the material as well as on the surface. PMMA showed the gradually increased peroxide concentration as time in the large amount. However the receding contact angle did not decrease much and it indicated that surface concentration of peroxides may be low and most of them would be formed inside the polymer. PE was observed to show low concentration but rapid increase in peroxides while the contact angle was rapidly decreased to a substantial amount, which indicates most peroxides were formed on the surface. In the case of PU, both peroxide concentration and contact angles were changed to the greatest extent. Also, the decrease in the contact angle was not as fast as the increase in peroxides concentration. It means that peroxides were not generated only from the surface but throughout the whole specimen. Silicone exhibited similar increase in peroxide concentration to PU. However the decrease in the contact angle was very small but fast. This behavior of silicone would result from its speci"c reactivity to ozone and/or hydrophobic character. 3.2. PEGA or PEGA}SO3 grafting onto polymer surfaces
Fig. 2. (a) Concentration of peroxide generated and (b) receding contact angles, , of ozonated polymers at various ozonation time: (£) PU, (*) silicone, (䊏) PMMA, (䉱) PE.
reaction but ozone can penetrate into the inside of polymers. Moreover, the iodide method used here to measure the concentration of peroxide is a chemical method so that the values should include the peroxides not only on the surface but also in the inside [13]. Therefore in this case, the yield of ozone reaction should be "rst of all dependent on the permeability of ozone into polymers used. In Fig. 2(a), PU and silicone exhibited a high peroxide concentration and fast increase as they are soft materials having high ozone permeability. On the contrary, PE is hard and crystalline so that it yields low peroxide concentration due to the low ozone permeability. PMMA is also hard but it is an amorphous polymer. Thus the peroxides were increased gradually as time went by as gases can penetrate into the amorphous region much easier than in the crystalline domain. It is also interesting to "nd that the formation of peroxides as mentioned above does not seem to be directly re#ected to the contact angle values apparently. It should be recalled here that contact angles indicate the characteristics of the outermost surfaces while the con-
The scheme of PEGA or PEGA}SO grafting is depic ted in Fig. 1. Peroxides generated by ozonation were decomposed into radicals to initiate the polymerization so that the #exible PEGA or PEGA}SO chains were grafted onto polymer surfaces, resulting in a comb-type structure. The surface structures of modi"ed polymers were investigated using ESCA and contact angles. ESCA spectra of ozonated and PEGA-grafted silicone are shown in Fig. 3. The "lms were treated with ozone for
Fig. 3. C ESCA spectra for the modifed silicones: (a) untreated, 1 (b) ozonated, (c) PEGA grafted.
Y.G. Ko et al. / Biomaterials 22 (2001) 2115}2123 Table 1 Atomic percentage values from ESCA for modi"ed PMMA
Nontreated (measured) Nontreated (theoretical) O treated 1 h O /PEGA grafted O /PEGA}SO grafted
C (%)
O (%)
S (%)
Na (%)
71.09 71.43 70.90 68.71 64.59
28.91 28.57 29.10 31.29 31.39
1.98
2.04
Grafted with PEGA or PEGA}SO after ozonation for 1 h.
2119
percentage values of modi"ed PMMA are summarized in Table 1. Increased oxygen values and the presence of sulfur peak near 228 eV are ascribed to the surface grafting of PEGA and PEGA}SO , respectively. Fig. 4(a) represents receding contact angle data of PEGA-grafted PMMA, silicone, and PU, respectively which were ozonated for various periods and then grafted for 24 h. In the case of PMMA and PU, the receding angle decreased substantially with increased ozonation time and reached to complete wet state after 4 and 3 h treatment, respectively. Similar behavior was observed on silicone although plateau value was relatively high (around 303). Table 2 summarizes the contact angle data of modi"ed polymers. Those "lms were ozonated for 1 h and then grafted by PEGA or PEGA}SO for 24 h. After ozona tion, the contact angles decreased more or less as shown in Fig. 2(a). However they were further decreased due to the grafting of PEG polymers. The e!ect of grafted PEG polymers was more signi"cant than that of the ozonation, indicating the usefulness of PEG grafting. In the case of PEGA}SO -grafted PMMA, the contact angle was higher than that of PEGA-grafted one, although PEGA}SO contains strong hydrophilic ionic groups. This may be due to the low grafting yield of PEGA}SO . There are many reports to describe that monomers containing ionic salt groups polymerize slowly, especially in
Table 2 Contact angles data of modi"ed polymers
Fig. 4. (a) Receding contact angles of PEGA grafted polymers for 24 h after various ozonation time: (£) PU, (*) silicone, (䊏) PMMA, and (b) platelet adhesion on PEGA grafted ones: ( ) silicone, (k) PU.
1 h and subsequently reacted with PEGA for 24 h. The carbon binding energies of the following groups, }C}O} (286.6$0.2 eV), }C"O (287.8$0.2 eV), and }O}C"O (289.0$0.2 eV), are reported [19]. After the surface modi"cation, new peaks resulting from newly formed }C}O}, }C"O, and }O}C"O groups were observed on ESCA spectra of silicone (Fig. 3) to con"rm the PEGA grafting, although the concentration of peroxides, and therefore radicals, were presumably quite low. Atomic
PMMA Untreated O treated for 1 h O /PEGA grafted O /PEGA}SO grafted O /H N}PEG}NH coupled O /H N}PEG}SO coupled Silicone Untreated O treated for 1 h O /PEGA grafted O /H N}PEG}NH coupled O /H N}PEG}SO coupled PU Untreated O treated for 1 h O /PEGA grafted O /H N}PEG}NH coupled O /H N}PEG}SO coupled PE Untreated O treated for 1 h O /PEGA grafted O /H N}PEG}NH coupled O /H N}PEG}SO coupled
81.2$1.4 74.3$5.1 80.1$1.8 67.1$2.3 91.6$4.4 90.7$5.7
40.5$4.5 35.3$2.8 5.4$7.6 17.9$5.6 30.6$3.3 28.3$4.0
102.3$11.5 118.4$3.1 107.3$7.4 119.7$0.4 118.7$3.2
69.6$4.5 51.3$7.6 23.1$4.1 51.3$8.7 48.0$3.1
100.7$0.8 94.5$1.9 78.9$4.8 62.6$2.1 69.7$8.4
71.1$1.0 65.7$4.4 21.6$0.1 12.7$6.0 Wet
103.1$1.4 76.4$1.3 74.0$3.4 88.1$3.8 83.7$2.7
80.1$1.8 41.8$3.7 23.2$1.7 31.9$7.8 31.2$0.9
2120
Y.G. Ko et al. / Biomaterials 22 (2001) 2115}2123
the case of grafting, due to the salt e!ect [20]. This should be studied further in detail. 3.3. H2 N}PEG}NH2 or H2 N}PEG}SO3 coupling onto polymer surfaces The schematic representation of H N}PEG}NH or H N}PEG}SO coupling is also shown in Fig. 1. While PEGA or PEGA}SO , vinyl monomer, was graft-poly merized by polymeric radicals evolved from peroxides, H N}PEG}NH or H N}PEG}SO is expected to be coupled onto polymeric radicals via a two-step reaction (in this text the terminology of grafting or coupling was di!erently used). In addition, the polymeric radicals would attack H N}PEG}NH or H N}PEG}SO to yield radicals of the PEG derivatives, and then these radicals would react to the polymer surface. Although the yield of such a coupling method might not be so high as the graft polymerization reaction, H N}PEG}NH - or H N}PEG}SO -coupled surfaces do not have acrylic backbones like as PEGA or PEGA}SO grafting, and therefore it can yield di!erent surface characteristics. The specimens were treated with ozone for 1 h and were coupled to H N}PEG}NH or H N}PEG}SO at 603C for 24 h similarly to the graft-polymerization procedure. The coupling of H N}PEG}NH or H N}PEG}SO was con"rmed by ATR FT-IR spectra of modi"ed PE as shown in Fig. 5. After the coupling, an amino group newly appeared under 3500 cm\ and the a strong C}O peak was observed at 1103 cm\. In the H N}PEG}SO coupled PE, S}O group was assigned at 943 cm\ too. In
addition, H N}PEG}NH -coupled PU was con"rmed again by its ESCA spectra (not shown here) in which the intensity of atomic peak of N near 398 eV increased as compared with the untreated one. S peak was also observed near 228 eV in H N}PEG}SO -coupled PU. Dynamic contact angles of H N}PEG}NH - or H N}PEG}SO -coupled PE, PU, silicone, and PMMA were compared with untreated samples and PEGA or PEGA}SO grafted ones in Table 2. The e!ect of coup ling on receding angles varied depending on polymers. In the case of PU, the e!ect of coupling on receding angles was most signi"cant. It exhibited very low values down to complete wetting, which indicates higher hydrophilicity than the previous grafting method. On the contrary, there was no decrease in receding angle after the coupling for silicone, although it showed slight decrease in the case of grafting. PMMA revealed also no e!ect by coupling contrary to the large decrease in the case of grafting. PE also exhibited a small decrease. Fig. 6 shows the surface morphology of modi"ed PMMA examined by AFM. The untreated PMMA was relatively smooth and it has the root-mean-square (RMS) roughness of 5.21 As . After the ozonation, the smoothness of the treated samples was increased and their RMS is 2.68 As . However, RMS of PEG-modi"ed PMMA was increased to be 11.8 As for H N}PEG}NH and 10.8 As for H N}PEG}SO . Ozonation is a relatively simple and inexpensive method to modify polymers by introducing peroxides uniformly to the surfaces. Such polymeric peroxides can be produced also by irradiation with gamma-rays [21,22], electron beams [23], and ultraviolet radiations
Fig. 5. ATR FT-IR spectra of modi"ed PE; (a) untreated, (b) H N}PEG}NH coupled, (c) H N}PEG}SO coupled.
Y.G. Ko et al. / Biomaterials 22 (2001) 2115}2123
2121
Fig. 6. AFM images of modi"ed PMMA; (a) untreated, (b) ozonated for 1 h, (c) H N}PEG}NH coupled, (d) H N}PEG}SO coupled; RMS roughness; (a) 5.21, (b) 2.68, (c) 11.8, (d) 10.8 As .
[24] in addition to glow [25] or corona [26}29] discharge. However, ozone method has a superior advantage over those in terms of uniformity. It is well known that oxidation of polymeric "lms by ozonation occurs not only on the surface but also in the inside due to the di!usion of ozone into the "lm , and peroxides evolved by ozonation can be decomposed signi"cantly on storage at room temperature. In this study, several polymers were treated by ozone and graft-polymerized with PEGA/PEGA}SO -contain ing vinyl groups or coupled with H N}PEG}NH / H N}PEG}SO to investigate the e!ect of PEG chain on hydrophilicity in terms of contact angles. It was found that the extent of ozonation, the yield of graft polymerization or coupling, and the e!ect on hydrophilicity depend on the characteristics of the polymers. The extent of ozonation depended on ozone permeability of the polymer and the order is as follows; PU'silicone'PE' PMMA (after 1 h), but it increased gradually to a large extent for PMMA (Fig. 2a). The distribution of polymeric peroxides evaluated from receding contact angles demonstrated di!erent behaviors; they seemed to be concentrated on the surface on PE, but evenly located throughout the specimen in PU, silicone, and PMMA, while silicone and PMMA revealed only a small decrease in contact angles (Fig. 2b). The e$ciency of PEG compounds grafted or coupled onto polymeric surfaces was di!erent from polymers to polymers as evaluated from the change of receding contact angles (Table 2). For PU, both grafting and coupling were very e!ective to exhibit large decreases in contact angles. However, for silicone and PMMA only grafting was e!ective in decreasing the receding angles. The e$ciency of grafting evaluated from
the decreases in contact angles might be described in the order of PMMA'PE"PU"silicone, but that of coupling PU'PE
2122
Y.G. Ko et al. / Biomaterials 22 (2001) 2115}2123
fonated PEG (PEG}SO ) exhibited blood compatibility by a synergistic interaction of the non-adhesive property of PEG and anticoagulant activity of sulfonate group (negative cilia concept) [9}12]. It was also investigated that the interaction of PEG- or PEG}SO -grafted PU surfaces with platelets [30]. In the study, all the PEGgrafted PU surfaces adsorb less platelets than does control PU, and furthermore, PU}PEG}SO shows the lowest platelet adhesion and activation, con"rmed by measuring cytoplasmic-free calcium concentrations in platelets contacting modi"ed surfaces. The result from this study is well correlated with our previous reports to recon"rm the negative cilia concept of PEG}SO . 4. Conclusion
Fig. 7. Platelet adhesion on PEG-coupled polymers after ozonation for 1 h: (䉬) untreated, (*) ozonated, (䊐) H N}PEG}NH coupled, (䉱) H N}PEG}SO coupled.
found that the platelet adhesion onto the modi"ed surfaces was decreased with the period of ozonation and with the degree of decreased receding contact angle. Such an e!ect of PEG was also observed for H N}PEG}NH or H N}PEG}SO coupled polymers as shown in Fig. 7. PU, PE, and silicone were ozonated for 1 h and subsequently reacted with H N}PEG}NH or H N}PEG} SO for 24 h. It is to be pointed out that only the ozonation decreases the platelet adhesion due to hydrophilization except silicone. It is very interesting to "nd that all the H N}PEG}SO -coupled polymers re vealed less adhesion than those coupled with H N}PEG}NH . It suggests that the superior blood compatibility of sulfonated PEG to amino-terminated PEG. It is well accepted that PEG has a unique non-adhesive property to blood components and cells to be biocompatible [4}10]. This speci"c character of PEG is explained by the excluded volume e!ect and the dynamic motion of fully hydrated #exible chains. The result of this study is in full agreement with the previous reports. It was also previously reported by our group that sul-
PEG or sulfonated PEG was successfully immobilized onto polymeric surfaces by ozonation and then was followed by graft polymerization with acrylates containing PEG or sulfonated PEG or by coupling of PEG derivatives. All the reactions were con"rmed by ATR FT-IR and ESCA. The degree of ozonation measured by the iodide method was dependent on the ozone permeability of the polymers used. The measurement of a contact angle demonstrated improved hydrophilicity. Ozonation itself yielded an substantial increase of hydrophilicity and decrease in platelet adhesion, but PEG immobilization showed an additional e!ect on hydrophilicity and platelet adhesion due to the well-known characteristics of PEG. Both graft polymerization and coupling methods were e!ective for PU, but only grafting gave enough yields for PMMA and silicone. Platelet adhesion results demonstrated that all PEG modi"ed surfaces adsorbed less platelets than untreated or ozonated ones. Sulfonated-PEG-coupled polymers exhibited the lowest platelet adhesion when compared with untreated ones and PEG-coupled ones by virtue of the synergistic e!ect of non-adhesive property of PEG and negatively charged SO groups. Such a PEG or sulfonated-PEG immobiliz ation technology using ozonation was relatively simple for uniform surface modi"cation, and therefore very useful for practical application of blood-contacting medical devices. Acknowledgements This study was supported by KOREA MOST Grant N19020. References [1] Kim YH, Park KD, Han DK. Blood compatible polymers. In: Salamone JC, editor. Polymeric materials encyclopedia, vol. 1. Boca Raton, FL: CRC Press, 1996. p. 825}35.
Y.G. Ko et al. / Biomaterials 22 (2001) 2115}2123 [2] Ishihara K. Blood compatible polymers. In: Tsuruta T, editor. Biomedical applications of polymeric materials. Boca Raton, FL: CRC Press, 1993. p. 89}115. [3] Ikada Y. Surface modi"cation of polymers for medical applications. Biomaterials 1994;15(10):725}36. [4] Mori Y, Nagaoka S, Takiuchi H, Kikuchi T, Noguchi N, Tanzawa H, Noishiki Y. A new antithrombogenic material with long PEG chains. Trans Am Soc Artif Intern Organs 1982;28:459}63. [5] Merill EW, Salzman EW. Poly(ethylene oxide) as a biomaterial. Am Soc Artif Intern Organ 1983;6:60}4. [6] Lee JH, Kopecek J, Andrade JD. Protein-resistant surfaces prepared by PEO-containing block copolymer surfactants. J Biomed Mater Res 1989;23:351}68. [7] Desai NP, Hubbell A. Biological response to PEG modi"ed PET surfaces. J Biomed Mater Res 1991;25:829}43. [8] Park KD, Kim SW. PEO modi"ed surface-blood compatibility of in vitro and in vivo studies. In: Harris JM, editor. Biomedical applications of polyethyleneglycol chemistry. New York: Plenum Press, 1992. p. 283. [9] Han DK, Jeong SY, Kim YH, Min BG, Cho HI. Negative cilia concept for thromboresistance: Synergistic e!ect of PEO and sulfonate groups grafted onto polyurethanes. J Biomed Mater Res 1991;25:561}75. [10] Han DK, Park KD, Jeong SY, Kim YH, Kim UY, Min BG. In vivo biostability and calci"cation-resistance of surface-modi"ed PU}PEO}SO . J Biomed Mater Res 1993;27:1063}73. [11] Han DK, Lee NY, Park KD, Kim YH, Cho HI, Min BG. Heparin-like anticoagulant activity of sulphonated PEO and sulphonated PEO grafted PU. Biomaterials 1995;16:467}71. [12] Kim YH, Han DK, Park KD. Negative cilia concept for thromboresistance. In: Wise DL, editor. Encyclopedic handbook of biomaterials and bioengineering, Part B2. New York: Marcel Dekker, 1995. p. 1071}88. [13] Fujimoto K, Takebayashi Y, Inoue H, Ikada Y. Ozone-induced graft polymerization onto polymer surface. J Polym Sci A: Polym Chem 1993;31:1035}43. [14] Dasgupta S. Surface modi"cation of polyole"ns for hydrophilicity and bondability: ozonization and grafting hydrophilic monomers on ozonized polyole"ns. J Appl Polym Sci 1990;41:233}48. [15] Yamauchi J, Yamaoka A, Ikemoto K, Matsui T. Graft copolymerization of methyl-methacrylate onto polypropylene oxidized with ozone. J Polym Sci 1991;43:1197}203. [16] Karlsson JO, Gatenholm P. Preparation and characterization of cellulose-supported HEMA hydrogels. Polymer 1997;38(18): 4727}31.
2123
[17] Smets G, Weinano G, Deguchi S. Radial block polymerization of vinyl chloride: I. ozonized polypropylene oligomers as heterofunctional initiator. J Polym Sci: Polym Chem Ed 1978;16: 3077}90. [18] Frew JE, Jones P, Scholes G. Spectrophotometric determination of hydrogen peroxide and organic hydroperoxides at low concentration in aqueous solution. Anal Chim Acta 1983;155: 139}50. [19] Chtovrou H, Riedl B, Kokta BV, Adnot A, Kaliaguine S. Synthetic pulp "ber ozonation: An ESCA and FTIR study. J Appl Polym Sci 1993;49:361}73. [20] Mayhan KG, Janssen RA, Bertrand WJ. USP 4589964, 20 May 1986. [21] Suzuki M, Tamada Y, Iwata H, Ikada Y. Polymer surface modi"cation to attain blood compatibility of hydrophobic polymer. In: Mittel KL, editor. Physicochemical aspects of polymer surfaces, vol. 2. New York: Plenum Press, 1983. p. 923}41. [22] Ratner B, Weathersby PK, Ho!man AS, Kelly MA, Scharpen LH. Radiation-grafted hydrogels for biomaterial applications at studied by the ESCA technique. J Appl Polym Sci 1978;22: 643}64. [23] Poncin-Epaillard F, Chevet B, Brosse J-C. Modi"cation of isotactic PP by a cold plasma or an electron beam and grafting of the acrylic acid onto these activated polymers. J Appl Polym Sci 1994;53:1291}306. [24] Uyama Y, Ikada Y. Graft polymerization of acrylamide onto UV-irradiated "lms. J Appl Polym Sci 1988;36:1087}96. [25] Suzuki M, Kishida A, Iwata H, Ikada Y. Graft copolymerization of acrylamide onto a PE surface pretreated with a glow discharge. Macromolecules 1986;19:1804}8. [26] Iwata H, Kishida A, Suzuki M, Hata Y, Ikada Y. Oxidation of PE surface by corona discharge and the subsequent graft polymerization. J Polym Sci: Polym Chem Ed 1988;26:3309}22. [27] Okada T, Ikada Y. Modi"cation of silicone surface by graft polymerization of acrylamide with corona discharge. Makromol Chem 1991;192:1705}13. [28] Blythe AR, Briggs D, Kendall CR, Rance DG, Zichy VJI. Surface modi"cation of PE by electrical discharge treatment and the mechanism of autoadhesion. Polymer 1978;19:1273}8. [29] Dong S, Sapieha S, Schreiber HP. Mechanical properties of corona-modi"ed cellulose/PE composites. Polym Engng Sci 1993; 33(6):343}6. [30] Park KD, Suzuki K, Lee WK, Lee JE, Kim YH, Sakurai Y, Okano T. Platelet adhesion and activation on PEG modi"ed PU surfaces. ASAIO J 1996;42:M876}81.
Immobilization of poly(ethylene glycol) or its sulfonate onto polymer surfaces by ozone oxidation Young Gun Ko , Young Ha Kim *, Ki Dong Park , Hee Jung Lee , Won Kyu Lee , Hyung Dal Park , Soo Hyun Kim , Gil Sun Lee, Dong June Ahn Biomaterials Research Center, Korea Institute of Science and Technology, P.O. Box 131, Cheongryang, Seoul 130-650, South Korea Department of Chemical Engineering, Korea University, Anamdong 5ga-1, Seoul 136-701, South Korea Received 7 September 2000; accepted 12 November 2000
Abstract A novel surface modi"cation method has been developed to improve biocompatibility of polymeric biomaterials. This approach involves ozonation and then followed by graft polymerization with acrylates containing PEG, sulfonated PEG or by coupling of PEG derivatives. All the reactions were con"rmed by ATR FT-IR and ESCA. The degree of ozonation measured by the iodide method was dependent on the ozone permeability of the polymers used. Surface hydrophilicity was investigated by measuring the contact angles. Ozonation itself yielded a slight increase in hydrophilicity and a decrease in platelet adhesion, but PEG immobilization showed a signi"cant e!ect on surface hydrophilicity and platelet adhesion to con"rm well-known PEG's passivity which minimize the adhesion of blood components on polymer surfaces. Both graft polymerization and coupling were e!ective for PU. In contrast, only grafting gave enough yields for PMMA and silicone. Platelet adhesion results demonstrated that all PEG modi"ed surfaces adsorbed lower platelet adhesion than untreated or ozonated ones. Polymers coupled with sulfonated PEG exhibited the lowest platelet adhesion when compared with control and PEG coupled ones by virtue of the synergistic e!ect of non-adhesive PEG and negatively charged SO groups. This PEG or sulfonated PEG immobilization technology using ozonation is relatively simple for introducing uniform surface modi"cation and therefore very useful for practical application of blood contacting medical devices. 2001 Elsevier Science Ltd. All rights reserved. Keywords: PEG/PEG}SO immobilization; Ozone treatment; Wettability; Platelet adhesion
1. Introduction A variety of approaches has been undertaken to improve the blood compatibility and to minimize cell adhesion on biomaterials surfaces [1}3]. One approach involves surface modi"cation by grafting a hydrophilic component, such as poly(ethyleneglycol) (PEG). PEG has unique solution properties and molecular conformation in aqueous solution. PEG-grafted surfaces exhibit speci"c non-adhesive property to proteins, blood components and cells mainly due to high surface mobility and steric stabilization e!ects [4}8]. In addition, sulfonated PEG, (PEG}SO )-grafted polymers, improved anti thrombogenicity, biostability, and anticalci"cation in-vitro,
* Corresponding author. Tel.: #82-2-958-5340; fax: #82-2-9585308. E-mail address: [email protected] (Y.H. Kim).
ex-vivo, and in-vivo by the synergistic e!ect of the #exible hydrated PEG chains and negatively charged sulfonate anticoagulant groups [9}12]. Surface ozone oxidation is widely applied in polymer areas because it has an advantage of uniformly introducing peroxides on the polymer surface and o!ers an easy-to-handle, inexpensive technique [13}16]. When polymer is exposed to ozone gas, peroxides are formed in addition to carbonyl and carboxyl groups [17]. The generated polymeric peroxides are capable of initiating polymerization of vinyl monomers, resulting in surface grafting onto the ozonated polymeric materials [13]. This article describes PEG or PEG}SO immobiliz ation onto polymethylmethacrylate (PMMA), polyethylene (PE), silicone, and polyurethane (PU) by ozonation (Fig. 1). The surface structures and properties of modi"ed polymers were investigated using attenuated total re#ectance Fourier transform infrared (ATR FT-IR), electron spectroscopy for chemical analysis (ESCA), atomic force
0142-9612/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 0 ) 0 0 4 0 0 - 2
2116
Y.G. Ko et al. / Biomaterials 22 (2001) 2115}2123
Fig. 1. Schematic illustration of surface modi"cation.
microscopy (AFM) and dynamic contact angle (DCA). In addition, the blood compatibility of PEG-modi"ed polymers was evaluated by in-vitro platelet adhesion study.
2. Materials and methods 2.1. Materials Polyurethane (PU, Pellethane, Dow Chemical Co., Midland, MI, USA) was extracted with methanol for 3 days to remove low molecular weight components, and dried under vacuum. The extracted PU was dissolved in N,N-dimethylacetamide, cast into "lms and dried for 1 week at 453C. Polymethylmethacrylate (PMMA), commercial sheets (LG Chemical Co., Korea), was washed by ethanol for 3 days. Low-density polyethylene (PE) "lms were purchased from Hanwha Petrochemical Co., Korea. Silicone sheets were supplied from Yushin Medical Co., Korea. All the sheets (size 1;3 cm, thickness 0.5}2 mm) were further sonicated for 30 min before ozonations. PEG mono-acrylate and diamino-terminated PEG (molecular weight of PEG 1000, NOF Co., Japan) were dissolved in chloroform for puri"cation and precipitated into diethyl ether, and then dried under vacuum. Sulfonation of PEG mono-acrylate(PEGA) was performed using Na metal and propane sultone. Na metal was put into PEGA to be reacted at 503C for 12 h. The resultant product was precipitated into diethyl ether,
"ltered, and puri"ed by dissolving in chloroform and then it was precipitated into diethyl ether and "nally dried overnight at room temperature. Diamino-terminated PEG (H N}PEG}NH ) was also sulfonated by propane sultone as described previously [11]. Brie#y, 10% (w/v) propane sultone in tetrahydrofuran was added by drops to 10% (w/v) H N}PEG}NH solution in tetrahydrofuran and reacted at 503C for 6 h. The resultant product was precipitated from medium as the reaction proceeded. The "ltered product was washed with cold tetrahydrofuran and dried overnight at room temperature. The structures of obtained sulfonated PEGs (PEGA}SO and H N}PEG}SO ) were con"rmed by a nuclear magnetic resonance spectrometer (H-NMR, C-NMR, Varian Gemini 200 MHz). 2.2. Ozonation and surface grafting or coupling of PEG derivatives Ozone was generated with dried oxygen gas passed through an ozone generator (Labor-Ozonizator 301, Sander Co., USA). The operating condition was set at 6 kV, 1 bar O pressure and 50 l/h O #ow rate at room temperature. After the ozonation, it was degassed by purging O to remove ozone adsorbed in the specimen. Polymeric peroxides introduced onto the treated "lms were determined by the iodide method spectrophotometrically [13,18]. This method utilizes an oxidation of sodium iodide by peroxides in the presence of ferric
Y.G. Ko et al. / Biomaterials 22 (2001) 2115}2123
chloride. The treated "lms were kept at 603C for 10 min in benzene}isopropyl alcohol solution (1 : 6 by vol) containing saturated sodium iodide and 1 ppm ferric chloride. After addition of water to stop the reaction, the oxidized iodine was measured as triiodide anion from the absorbance of the solution at 360 nm with the molar absorptivity of 2.3;10 l/mol cm. After ozonation, the "lms were immersed immediately into 20 wt% aqueous solutions of PEGA, PEGA}SO , H N}PEG}NH , or H N}PEG}SO in each glass am pule, respectively. After vigorous degassing by purging N , the ampules were sealed and kept at 603C for given periods of time. The PEG-immobilized "lms were washed by tridistilled water for 3 days to remove the unreacted PEG derivatives. 2.3. Surface characterization Dynamic contact angles (DCA) were measured at room temperature with a Wilhelmy plate equipment (DCA-322, Cahn Instruments Inc., USA). Tridistilled water was used for the measurement of each of four specimen. The velocity of the translation stage was 150.7 m/s. DCA by Wilhelmy plate method gives two angles, advancing angle when immersing into water and receding angle when withdrawing from water. It was generally known that advancing angles indicate hydrophobicity while receding ones express hydrophilicity of surfaces. All the specimens were hydrated for 1 day before measurements to be close to in-vivo conditions, although the values before and after hydration were not much di!erent from each other. Attenuated total re#ectance Fourier transform infrared (ATR FT-IR) was used to analyze the surfaces using a Bruker FT-IR (IFS 66, Germany) with KRS-5 crystal. ESCA spectra for each sample were obtained on an ESCA 2803-S (SSI, USA) spectrometer with Al K X-ray. In order to determine O/C, N/C, S/C, and Na/C stoichiometries, collecting factors of 2.50, 1.68, 1.80, and 8.5 were used for O , N , S , and Na , respectively. Binding energies were referenced to the C}H group of the C peak components at 285.0 eV. With the C spectra smoothed, the subpeaks were deconvoluted using a curve-"tting method from a series of Gauss}Lorentzian curves. The surface morphology of modi"ed polymers was examined with an AFM manufactured by Park Scienti"c Instruments (CP, USA). 2.4. In vitro platelet studies Human whole blood, which was treated with citric acid, was centrifuged at 800 g for 10 min at 253C to prepare platelet-rich plasma (PRP). The residue of the
2117
blood was centrifuged at 3000 rpm for 10 min to obtain platelet-poor plasma (PPP). The number of platelets was adjusted to 430,000 mm\ by adding PPP to PRP. Polymer "lms were introduced into the specially designed syringe and hydrated in phosphate-bu!ered saline (PBS) for 1 day. Prior to adhesion studies, the bu!er was removed from the syringe and 2 ml of PRP was introduced into it. The syringe was then tapped to remove air bubbles, sealed, and rotated in a shaking incubator at 373C. By this method, the polymer "lms were constantly exposed to PRP. Therefore, only surface/platelet interactive in#uences were observed. A set of syringes was prepared for platelet adhesion test along the time of 15, 30, 60 and 120 min with incubation in PRP. At each time, the syringes were quickly removed from the shaking incubator, and immediately depleted platelets were counted in the PRP with the Coulter counter or hematocytometer. The amount of platelets that adhered upon the specimen was calculated by subtracting the number of unadhered platelets from the number of diluted platelets that were initially incubated. The morphology of platelets adhered on "lms were examined by SEM (Hitachi, S-2460N, Japan). Polymer samples were withdrawn from PRP and rinsed in PBS with gentle agitation to remove weakly adhered platelets. The "lms were "xed with 2% glutaraldehyde solutions in PBS for 2 h at 43C, dehydrated with 50, 60, 70, 80, 90, and 100% dilutions of ethanol and water, and freeze dried for 1 day. Samples were coated with a gold layer and observed with SEM.
3. Results and discussion 3.1. Ewect of ozonation on polymer surfaces Fig. 2(a) shows the concentration of peroxides evolved on PU, PMMA, PE, and silicone "lms treated by ozone. In the case of PMMA, the concentration of peroxide increased gradually with ozonation time. But the peroxide concentrations on silicone, PE, and PU "lms were increased rapidly upto 1 h and leveled o! to a constant value. PU and silicone exhibited a high concentration of peroxide but PE showed low concentration. The change of contact angles (receding angles, ) of the ozonated "lms was compared on Fig. 2(b). All the angles which decreased after the ozonation indicate increase in hydrophilicity, but the extent of decrease varied depending on the "lms. PMMA exhibited no signi"cant change. The contact angles of PU and PE were decreased to a signi"cant extent; those of PU were decreased gradually as time went by but those of PE was decreased rapidly in the beginning and leveled o!. It is notable that the peroxides formation after ozonation and the increase in the hydrophilicity varied depending on the specimen. The ozonation is basically a surface
2118
Y.G. Ko et al. / Biomaterials 22 (2001) 2115}2123
centration of peroxides include those inside the material as well as on the surface. PMMA showed the gradually increased peroxide concentration as time in the large amount. However the receding contact angle did not decrease much and it indicated that surface concentration of peroxides may be low and most of them would be formed inside the polymer. PE was observed to show low concentration but rapid increase in peroxides while the contact angle was rapidly decreased to a substantial amount, which indicates most peroxides were formed on the surface. In the case of PU, both peroxide concentration and contact angles were changed to the greatest extent. Also, the decrease in the contact angle was not as fast as the increase in peroxides concentration. It means that peroxides were not generated only from the surface but throughout the whole specimen. Silicone exhibited similar increase in peroxide concentration to PU. However the decrease in the contact angle was very small but fast. This behavior of silicone would result from its speci"c reactivity to ozone and/or hydrophobic character. 3.2. PEGA or PEGA}SO3 grafting onto polymer surfaces
Fig. 2. (a) Concentration of peroxide generated and (b) receding contact angles, , of ozonated polymers at various ozonation time: (£) PU, (*) silicone, (䊏) PMMA, (䉱) PE.
reaction but ozone can penetrate into the inside of polymers. Moreover, the iodide method used here to measure the concentration of peroxide is a chemical method so that the values should include the peroxides not only on the surface but also in the inside [13]. Therefore in this case, the yield of ozone reaction should be "rst of all dependent on the permeability of ozone into polymers used. In Fig. 2(a), PU and silicone exhibited a high peroxide concentration and fast increase as they are soft materials having high ozone permeability. On the contrary, PE is hard and crystalline so that it yields low peroxide concentration due to the low ozone permeability. PMMA is also hard but it is an amorphous polymer. Thus the peroxides were increased gradually as time went by as gases can penetrate into the amorphous region much easier than in the crystalline domain. It is also interesting to "nd that the formation of peroxides as mentioned above does not seem to be directly re#ected to the contact angle values apparently. It should be recalled here that contact angles indicate the characteristics of the outermost surfaces while the con-
The scheme of PEGA or PEGA}SO grafting is depic ted in Fig. 1. Peroxides generated by ozonation were decomposed into radicals to initiate the polymerization so that the #exible PEGA or PEGA}SO chains were grafted onto polymer surfaces, resulting in a comb-type structure. The surface structures of modi"ed polymers were investigated using ESCA and contact angles. ESCA spectra of ozonated and PEGA-grafted silicone are shown in Fig. 3. The "lms were treated with ozone for
Fig. 3. C ESCA spectra for the modifed silicones: (a) untreated, 1 (b) ozonated, (c) PEGA grafted.
Y.G. Ko et al. / Biomaterials 22 (2001) 2115}2123 Table 1 Atomic percentage values from ESCA for modi"ed PMMA
Nontreated (measured) Nontreated (theoretical) O treated 1 h O /PEGA grafted O /PEGA}SO grafted
C (%)
O (%)
S (%)
Na (%)
71.09 71.43 70.90 68.71 64.59
28.91 28.57 29.10 31.29 31.39
1.98
2.04
Grafted with PEGA or PEGA}SO after ozonation for 1 h.
2119
percentage values of modi"ed PMMA are summarized in Table 1. Increased oxygen values and the presence of sulfur peak near 228 eV are ascribed to the surface grafting of PEGA and PEGA}SO , respectively. Fig. 4(a) represents receding contact angle data of PEGA-grafted PMMA, silicone, and PU, respectively which were ozonated for various periods and then grafted for 24 h. In the case of PMMA and PU, the receding angle decreased substantially with increased ozonation time and reached to complete wet state after 4 and 3 h treatment, respectively. Similar behavior was observed on silicone although plateau value was relatively high (around 303). Table 2 summarizes the contact angle data of modi"ed polymers. Those "lms were ozonated for 1 h and then grafted by PEGA or PEGA}SO for 24 h. After ozona tion, the contact angles decreased more or less as shown in Fig. 2(a). However they were further decreased due to the grafting of PEG polymers. The e!ect of grafted PEG polymers was more signi"cant than that of the ozonation, indicating the usefulness of PEG grafting. In the case of PEGA}SO -grafted PMMA, the contact angle was higher than that of PEGA-grafted one, although PEGA}SO contains strong hydrophilic ionic groups. This may be due to the low grafting yield of PEGA}SO . There are many reports to describe that monomers containing ionic salt groups polymerize slowly, especially in
Table 2 Contact angles data of modi"ed polymers
Fig. 4. (a) Receding contact angles of PEGA grafted polymers for 24 h after various ozonation time: (£) PU, (*) silicone, (䊏) PMMA, and (b) platelet adhesion on PEGA grafted ones: ( ) silicone, (k) PU.
1 h and subsequently reacted with PEGA for 24 h. The carbon binding energies of the following groups, }C}O} (286.6$0.2 eV), }C"O (287.8$0.2 eV), and }O}C"O (289.0$0.2 eV), are reported [19]. After the surface modi"cation, new peaks resulting from newly formed }C}O}, }C"O, and }O}C"O groups were observed on ESCA spectra of silicone (Fig. 3) to con"rm the PEGA grafting, although the concentration of peroxides, and therefore radicals, were presumably quite low. Atomic
PMMA Untreated O treated for 1 h O /PEGA grafted O /PEGA}SO grafted O /H N}PEG}NH coupled O /H N}PEG}SO coupled Silicone Untreated O treated for 1 h O /PEGA grafted O /H N}PEG}NH coupled O /H N}PEG}SO coupled PU Untreated O treated for 1 h O /PEGA grafted O /H N}PEG}NH coupled O /H N}PEG}SO coupled PE Untreated O treated for 1 h O /PEGA grafted O /H N}PEG}NH coupled O /H N}PEG}SO coupled
81.2$1.4 74.3$5.1 80.1$1.8 67.1$2.3 91.6$4.4 90.7$5.7
40.5$4.5 35.3$2.8 5.4$7.6 17.9$5.6 30.6$3.3 28.3$4.0
102.3$11.5 118.4$3.1 107.3$7.4 119.7$0.4 118.7$3.2
69.6$4.5 51.3$7.6 23.1$4.1 51.3$8.7 48.0$3.1
100.7$0.8 94.5$1.9 78.9$4.8 62.6$2.1 69.7$8.4
71.1$1.0 65.7$4.4 21.6$0.1 12.7$6.0 Wet
103.1$1.4 76.4$1.3 74.0$3.4 88.1$3.8 83.7$2.7
80.1$1.8 41.8$3.7 23.2$1.7 31.9$7.8 31.2$0.9
2120
Y.G. Ko et al. / Biomaterials 22 (2001) 2115}2123
the case of grafting, due to the salt e!ect [20]. This should be studied further in detail. 3.3. H2 N}PEG}NH2 or H2 N}PEG}SO3 coupling onto polymer surfaces The schematic representation of H N}PEG}NH or H N}PEG}SO coupling is also shown in Fig. 1. While PEGA or PEGA}SO , vinyl monomer, was graft-poly merized by polymeric radicals evolved from peroxides, H N}PEG}NH or H N}PEG}SO is expected to be coupled onto polymeric radicals via a two-step reaction (in this text the terminology of grafting or coupling was di!erently used). In addition, the polymeric radicals would attack H N}PEG}NH or H N}PEG}SO to yield radicals of the PEG derivatives, and then these radicals would react to the polymer surface. Although the yield of such a coupling method might not be so high as the graft polymerization reaction, H N}PEG}NH - or H N}PEG}SO -coupled surfaces do not have acrylic backbones like as PEGA or PEGA}SO grafting, and therefore it can yield di!erent surface characteristics. The specimens were treated with ozone for 1 h and were coupled to H N}PEG}NH or H N}PEG}SO at 603C for 24 h similarly to the graft-polymerization procedure. The coupling of H N}PEG}NH or H N}PEG}SO was con"rmed by ATR FT-IR spectra of modi"ed PE as shown in Fig. 5. After the coupling, an amino group newly appeared under 3500 cm\ and the a strong C}O peak was observed at 1103 cm\. In the H N}PEG}SO coupled PE, S}O group was assigned at 943 cm\ too. In
addition, H N}PEG}NH -coupled PU was con"rmed again by its ESCA spectra (not shown here) in which the intensity of atomic peak of N near 398 eV increased as compared with the untreated one. S peak was also observed near 228 eV in H N}PEG}SO -coupled PU. Dynamic contact angles of H N}PEG}NH - or H N}PEG}SO -coupled PE, PU, silicone, and PMMA were compared with untreated samples and PEGA or PEGA}SO grafted ones in Table 2. The e!ect of coup ling on receding angles varied depending on polymers. In the case of PU, the e!ect of coupling on receding angles was most signi"cant. It exhibited very low values down to complete wetting, which indicates higher hydrophilicity than the previous grafting method. On the contrary, there was no decrease in receding angle after the coupling for silicone, although it showed slight decrease in the case of grafting. PMMA revealed also no e!ect by coupling contrary to the large decrease in the case of grafting. PE also exhibited a small decrease. Fig. 6 shows the surface morphology of modi"ed PMMA examined by AFM. The untreated PMMA was relatively smooth and it has the root-mean-square (RMS) roughness of 5.21 As . After the ozonation, the smoothness of the treated samples was increased and their RMS is 2.68 As . However, RMS of PEG-modi"ed PMMA was increased to be 11.8 As for H N}PEG}NH and 10.8 As for H N}PEG}SO . Ozonation is a relatively simple and inexpensive method to modify polymers by introducing peroxides uniformly to the surfaces. Such polymeric peroxides can be produced also by irradiation with gamma-rays [21,22], electron beams [23], and ultraviolet radiations
Fig. 5. ATR FT-IR spectra of modi"ed PE; (a) untreated, (b) H N}PEG}NH coupled, (c) H N}PEG}SO coupled.
Y.G. Ko et al. / Biomaterials 22 (2001) 2115}2123
2121
Fig. 6. AFM images of modi"ed PMMA; (a) untreated, (b) ozonated for 1 h, (c) H N}PEG}NH coupled, (d) H N}PEG}SO coupled; RMS roughness; (a) 5.21, (b) 2.68, (c) 11.8, (d) 10.8 As .
[24] in addition to glow [25] or corona [26}29] discharge. However, ozone method has a superior advantage over those in terms of uniformity. It is well known that oxidation of polymeric "lms by ozonation occurs not only on the surface but also in the inside due to the di!usion of ozone into the "lm , and peroxides evolved by ozonation can be decomposed signi"cantly on storage at room temperature. In this study, several polymers were treated by ozone and graft-polymerized with PEGA/PEGA}SO -contain ing vinyl groups or coupled with H N}PEG}NH / H N}PEG}SO to investigate the e!ect of PEG chain on hydrophilicity in terms of contact angles. It was found that the extent of ozonation, the yield of graft polymerization or coupling, and the e!ect on hydrophilicity depend on the characteristics of the polymers. The extent of ozonation depended on ozone permeability of the polymer and the order is as follows; PU'silicone'PE' PMMA (after 1 h), but it increased gradually to a large extent for PMMA (Fig. 2a). The distribution of polymeric peroxides evaluated from receding contact angles demonstrated di!erent behaviors; they seemed to be concentrated on the surface on PE, but evenly located throughout the specimen in PU, silicone, and PMMA, while silicone and PMMA revealed only a small decrease in contact angles (Fig. 2b). The e$ciency of PEG compounds grafted or coupled onto polymeric surfaces was di!erent from polymers to polymers as evaluated from the change of receding contact angles (Table 2). For PU, both grafting and coupling were very e!ective to exhibit large decreases in contact angles. However, for silicone and PMMA only grafting was e!ective in decreasing the receding angles. The e$ciency of grafting evaluated from
the decreases in contact angles might be described in the order of PMMA'PE"PU"silicone, but that of coupling PU'PE
2122
Y.G. Ko et al. / Biomaterials 22 (2001) 2115}2123
fonated PEG (PEG}SO ) exhibited blood compatibility by a synergistic interaction of the non-adhesive property of PEG and anticoagulant activity of sulfonate group (negative cilia concept) [9}12]. It was also investigated that the interaction of PEG- or PEG}SO -grafted PU surfaces with platelets [30]. In the study, all the PEGgrafted PU surfaces adsorb less platelets than does control PU, and furthermore, PU}PEG}SO shows the lowest platelet adhesion and activation, con"rmed by measuring cytoplasmic-free calcium concentrations in platelets contacting modi"ed surfaces. The result from this study is well correlated with our previous reports to recon"rm the negative cilia concept of PEG}SO . 4. Conclusion
Fig. 7. Platelet adhesion on PEG-coupled polymers after ozonation for 1 h: (䉬) untreated, (*) ozonated, (䊐) H N}PEG}NH coupled, (䉱) H N}PEG}SO coupled.
found that the platelet adhesion onto the modi"ed surfaces was decreased with the period of ozonation and with the degree of decreased receding contact angle. Such an e!ect of PEG was also observed for H N}PEG}NH or H N}PEG}SO coupled polymers as shown in Fig. 7. PU, PE, and silicone were ozonated for 1 h and subsequently reacted with H N}PEG}NH or H N}PEG} SO for 24 h. It is to be pointed out that only the ozonation decreases the platelet adhesion due to hydrophilization except silicone. It is very interesting to "nd that all the H N}PEG}SO -coupled polymers re vealed less adhesion than those coupled with H N}PEG}NH . It suggests that the superior blood compatibility of sulfonated PEG to amino-terminated PEG. It is well accepted that PEG has a unique non-adhesive property to blood components and cells to be biocompatible [4}10]. This speci"c character of PEG is explained by the excluded volume e!ect and the dynamic motion of fully hydrated #exible chains. The result of this study is in full agreement with the previous reports. It was also previously reported by our group that sul-
PEG or sulfonated PEG was successfully immobilized onto polymeric surfaces by ozonation and then was followed by graft polymerization with acrylates containing PEG or sulfonated PEG or by coupling of PEG derivatives. All the reactions were con"rmed by ATR FT-IR and ESCA. The degree of ozonation measured by the iodide method was dependent on the ozone permeability of the polymers used. The measurement of a contact angle demonstrated improved hydrophilicity. Ozonation itself yielded an substantial increase of hydrophilicity and decrease in platelet adhesion, but PEG immobilization showed an additional e!ect on hydrophilicity and platelet adhesion due to the well-known characteristics of PEG. Both graft polymerization and coupling methods were e!ective for PU, but only grafting gave enough yields for PMMA and silicone. Platelet adhesion results demonstrated that all PEG modi"ed surfaces adsorbed less platelets than untreated or ozonated ones. Sulfonated-PEG-coupled polymers exhibited the lowest platelet adhesion when compared with untreated ones and PEG-coupled ones by virtue of the synergistic e!ect of non-adhesive property of PEG and negatively charged SO groups. Such a PEG or sulfonated-PEG immobiliz ation technology using ozonation was relatively simple for uniform surface modi"cation, and therefore very useful for practical application of blood-contacting medical devices. Acknowledgements This study was supported by KOREA MOST Grant N19020. References [1] Kim YH, Park KD, Han DK. Blood compatible polymers. In: Salamone JC, editor. Polymeric materials encyclopedia, vol. 1. Boca Raton, FL: CRC Press, 1996. p. 825}35.
Y.G. Ko et al. / Biomaterials 22 (2001) 2115}2123 [2] Ishihara K. Blood compatible polymers. In: Tsuruta T, editor. Biomedical applications of polymeric materials. Boca Raton, FL: CRC Press, 1993. p. 89}115. [3] Ikada Y. Surface modi"cation of polymers for medical applications. Biomaterials 1994;15(10):725}36. [4] Mori Y, Nagaoka S, Takiuchi H, Kikuchi T, Noguchi N, Tanzawa H, Noishiki Y. A new antithrombogenic material with long PEG chains. Trans Am Soc Artif Intern Organs 1982;28:459}63. [5] Merill EW, Salzman EW. Poly(ethylene oxide) as a biomaterial. Am Soc Artif Intern Organ 1983;6:60}4. [6] Lee JH, Kopecek J, Andrade JD. Protein-resistant surfaces prepared by PEO-containing block copolymer surfactants. J Biomed Mater Res 1989;23:351}68. [7] Desai NP, Hubbell A. Biological response to PEG modi"ed PET surfaces. J Biomed Mater Res 1991;25:829}43. [8] Park KD, Kim SW. PEO modi"ed surface-blood compatibility of in vitro and in vivo studies. In: Harris JM, editor. Biomedical applications of polyethyleneglycol chemistry. New York: Plenum Press, 1992. p. 283. [9] Han DK, Jeong SY, Kim YH, Min BG, Cho HI. Negative cilia concept for thromboresistance: Synergistic e!ect of PEO and sulfonate groups grafted onto polyurethanes. J Biomed Mater Res 1991;25:561}75. [10] Han DK, Park KD, Jeong SY, Kim YH, Kim UY, Min BG. In vivo biostability and calci"cation-resistance of surface-modi"ed PU}PEO}SO . J Biomed Mater Res 1993;27:1063}73. [11] Han DK, Lee NY, Park KD, Kim YH, Cho HI, Min BG. Heparin-like anticoagulant activity of sulphonated PEO and sulphonated PEO grafted PU. Biomaterials 1995;16:467}71. [12] Kim YH, Han DK, Park KD. Negative cilia concept for thromboresistance. In: Wise DL, editor. Encyclopedic handbook of biomaterials and bioengineering, Part B2. New York: Marcel Dekker, 1995. p. 1071}88. [13] Fujimoto K, Takebayashi Y, Inoue H, Ikada Y. Ozone-induced graft polymerization onto polymer surface. J Polym Sci A: Polym Chem 1993;31:1035}43. [14] Dasgupta S. Surface modi"cation of polyole"ns for hydrophilicity and bondability: ozonization and grafting hydrophilic monomers on ozonized polyole"ns. J Appl Polym Sci 1990;41:233}48. [15] Yamauchi J, Yamaoka A, Ikemoto K, Matsui T. Graft copolymerization of methyl-methacrylate onto polypropylene oxidized with ozone. J Polym Sci 1991;43:1197}203. [16] Karlsson JO, Gatenholm P. Preparation and characterization of cellulose-supported HEMA hydrogels. Polymer 1997;38(18): 4727}31.
2123
[17] Smets G, Weinano G, Deguchi S. Radial block polymerization of vinyl chloride: I. ozonized polypropylene oligomers as heterofunctional initiator. J Polym Sci: Polym Chem Ed 1978;16: 3077}90. [18] Frew JE, Jones P, Scholes G. Spectrophotometric determination of hydrogen peroxide and organic hydroperoxides at low concentration in aqueous solution. Anal Chim Acta 1983;155: 139}50. [19] Chtovrou H, Riedl B, Kokta BV, Adnot A, Kaliaguine S. Synthetic pulp "ber ozonation: An ESCA and FTIR study. J Appl Polym Sci 1993;49:361}73. [20] Mayhan KG, Janssen RA, Bertrand WJ. USP 4589964, 20 May 1986. [21] Suzuki M, Tamada Y, Iwata H, Ikada Y. Polymer surface modi"cation to attain blood compatibility of hydrophobic polymer. In: Mittel KL, editor. Physicochemical aspects of polymer surfaces, vol. 2. New York: Plenum Press, 1983. p. 923}41. [22] Ratner B, Weathersby PK, Ho!man AS, Kelly MA, Scharpen LH. Radiation-grafted hydrogels for biomaterial applications at studied by the ESCA technique. J Appl Polym Sci 1978;22: 643}64. [23] Poncin-Epaillard F, Chevet B, Brosse J-C. Modi"cation of isotactic PP by a cold plasma or an electron beam and grafting of the acrylic acid onto these activated polymers. J Appl Polym Sci 1994;53:1291}306. [24] Uyama Y, Ikada Y. Graft polymerization of acrylamide onto UV-irradiated "lms. J Appl Polym Sci 1988;36:1087}96. [25] Suzuki M, Kishida A, Iwata H, Ikada Y. Graft copolymerization of acrylamide onto a PE surface pretreated with a glow discharge. Macromolecules 1986;19:1804}8. [26] Iwata H, Kishida A, Suzuki M, Hata Y, Ikada Y. Oxidation of PE surface by corona discharge and the subsequent graft polymerization. J Polym Sci: Polym Chem Ed 1988;26:3309}22. [27] Okada T, Ikada Y. Modi"cation of silicone surface by graft polymerization of acrylamide with corona discharge. Makromol Chem 1991;192:1705}13. [28] Blythe AR, Briggs D, Kendall CR, Rance DG, Zichy VJI. Surface modi"cation of PE by electrical discharge treatment and the mechanism of autoadhesion. Polymer 1978;19:1273}8. [29] Dong S, Sapieha S, Schreiber HP. Mechanical properties of corona-modi"ed cellulose/PE composites. Polym Engng Sci 1993; 33(6):343}6. [30] Park KD, Suzuki K, Lee WK, Lee JE, Kim YH, Sakurai Y, Okano T. Platelet adhesion and activation on PEG modi"ed PU surfaces. ASAIO J 1996;42:M876}81.