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Langmuir 2005, 21, 9660-9665
Photoiniferter-Based Thermoresponsive Graft Architecture with Albumin Covalently Fixed at Growing Graft Chain End Takehisa Matsuda*,† and Shoji Ohya‡ Division of Biomedical Engineering, Graduate School of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan, and Department of Bioengineering, National Cardiovascular Center Research Institute 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan Received January 26, 2005. In Final Form: July 27, 2005 The aim of this study was to develop a novel surface graft architecture in which albumin is covalently fixed at the growing chain end of the hydrophilic polymers: poly(N, N-dimethylacylamide), PDMAM, and poly(N-isopropylacrylamide), PNIPAM. Photoiniferter-based surface-grafted polymers were prepared using either an albuminated iniferter or a nonalbuminated iniferter, both of which were derivatized on glass surfaces, and ultraviolet (UV)-light-irradiated in the presence of a DMAM or NIPAM monomer. Surface chemical composition analysis by X-ray photoelectron spectroscopy, contact angle measurement, immunostaining using fluorescence labeled antibody and the measurement of graft thickness, as determined from force-distance curves obtained in water at 25 °C and 37 °C by atomic force microscopy, evidenced that the thickness of graft layer increased with photoirradiation time and albumin molecules exist at growing chain ends. For PNIPAM-grafted surfaces, the interconversion between swollen and collapsed graft chains was observed below and above the lower critical solution temperature of PNIPAM. The potential application of a thermoresponsive graft with albumin covalently fixed at its growing chain end was discussed in terms of “active” nonfouling surface design based on the temperature-dependent switching of phase transition.
1. Introduction The blood- and tissue-contacting surfaces of medical implant devices have to be accepted by a body during service.1,2 Among the currently well-accepted surface design approaches to acquiring high blood compatibility in hostile living environments, various methods of surface design directing high nonprotein-adsorption and high noncell-adhesion potentials have been proposed and developed.3-6 Among them, nonspecific approach without using anticoagulant or antiplatelet agents involve either the formation of a highly swollen nonionic polymer graft layer,3-5 which suppresses protein adsorption and enhances desorption under flow due to very low physicochemical interaction force with biocolloids such as proteins, or surface albumin derivatization with or without a hydrophilic spacer.7,8 The latter approach is based on the fact that albumin, a major protein abundant in blood, is * To whom correspondence should be addressed. Tel: +81-92-642-6210. Fax: +81-92-642-6212. E-mail: matsuda@ med.kyushu-u.ac.jp. † Kyushu University. ‡ National Cardiovascular Center Research Institute. (1) Ratner, B. D.; Hoffman, A. F.; Schoen, F. J.; Lemons, J. E. Biomaterials Science: An Introduction to Materials in Medicine; Academic Press: New York, 1996; 193. (2) Kim, S. W.; Jacobs, H. Blood Purif. 1996, 14, 357. (3) Mori, Y.; Nagaoka, S.; Takiuchi, H.; Kikuchi, T.; Noguchi, N.; Tanzawa, H.; Noishiki, Y. Trans. Am. Soc. Artif. Intern. Organs 1982, 28, 459. (4) Ikada, Y. Biomaterials 1994, 15, 725. (5) Andrade, J. D. Polymer Surface Dynamics; New York: Plenum Press, 1985, Vol. 1, Chapter 2; 214. (6) Merrill, E. W.; Salzman, E. W. Am. Soc. Artif. Intern. Org. J. 1983, 6, 60. (7) Amiji, M.; Park, K. J. Biomater. Sci., Polymer ed. 1993, 4, 217. (8) Mcfarland, C. D.; Filippis, C.; Jenkins, M.; Tunstell, A.; Rhodes, N. P.; Williams, D. F.; Steele, J. G. J. Biomater. Sci., Polymer 1998, 9, 1227.
really bioinert,9 that is, it activates neither humoral nor cellular body defense mechanisms. In this article, a new surface graft architecture, in which albumin is bound at the growing chain end of a hydrophilic polymer graft, was developed using photoiniferter-based living radical polymerization. The photoiniferter used has a dithiocarbamate group, which produces a radical pair under ultraviolet (UV) light irradiation, and acts as an iniferter (initiator-transfer agent and terminator) during photopolymerization; its chemistry was first reported by Otsu et al.,10,11 almost a quarter century ago. Under appropriate reaction conditions, polymer chains steadily grow with photoirradiation time, indicating that living radical polymerization proceeds with time. The authors have extensively used this technique for generating precision graft-architectured surfaces in which graft chain length and the degree of branching are well controlled.12-15 The monomers used in this study are N,N-dimethylacrylamide (DMAM) and N-isopropylacrylamide (NIPAM). Poly(DMAM), PDMAM, is nonthermoresponsive watersoluble polymer, whereas poly(NIPAM), PNIPAM, exhibits thermoresponsive phase transition at the lower critical solution temperature (LCST: 32 °C) in water, and is soluble below LCST but insoluble above LCST.16 The (9) Peter, T., Jr. Serum Albumin. Advances in Protein Chemistry. Academic Press: New York, 1985; Vol 37, 161. (10) Otsu, T.; Yoshida, M.; Tazaki, T. Makromol. Chem., Rapid Commun. 1982, 3, 133. (11) Otsu, T.; Matsumoto, A. Adv. Polym. Sci. 1998, 136, 75. (12) Nakayama, Y.; Matsuda, T. Macromolecules 1996, 29, 8622. (13) Lee, H. J.; Nakayama, Y.; Matsuda, T. Macromolecules 1999, 32, 6968. (14) Kidoaki, S.; Ohya, S.; Nakayama, Y.; Matsuda, T. Langmuir 2001, 17, 2402. (15) Kidoaki, S.; Nakayama, Y.; Matsuda, T. Langumuir 2001, 17, 1980. (16) Heskins, M.; Guillet, J. E. J. Makromol. Sci. Chem. 1968, A2, 1441.
10.1021/la050221o CCC: $30.25 © 2005 American Chemical Society Published on Web 09/03/2005
Photoiniferter-Based Thermoresponsive Graft Architecture
Figure 1. Schematics of albumin-bound poly(N-isopropylacrylamide) (PNIPAM)-grafted surface via albuminated dithiocarbamate iniferter and hypothetical action of temperaturedependent switching of protein desorption by squeezing out and mechanical motion.
iniferters used include albuminated and nonalbuminated dithiocarbamates. Upon UV irradiation on an albuminated iniferter derivatized surface, a monomer is successively inserted between an albuminated iniferter and a substrate surface, resulting in the progressive growth of a graft chain. When the livingness is maintained during photoirradiation, such a graft chain should have an albumin molecule at its growing chain end (designated AlbPDMAM- or Alb-PNIPAM-graft). The schematics of a growing graft chain and plausible thermoresponsive nonfouling, temperature-driven selfcleaning surface mechanism are shown in Figure 1. The authors’ hypothetical strategy leading to the acquisition of a nonfouling potential on the designed Alb-PIPAMgraft architecture is a result of the combined effects of a weak physicochemical interaction force due to the hydrophilic polymer spacer and bioinertness of albumin. This architecture may exhibit high blood compatibility due to suppressed protein adsorption and reduced cell adhesion. For an Alb-PNIPAM-graft surface, even though protein adsorbs on albumin at the collapsed graft chain at 37 °C, the intermittently loaded temperature switching of phase transition above and below LCST, leading to the interconversion between the swollen and collapsed states of the PNIPAM graft chain, should cause the squeezing-out of proteins absorbed in the graft layer and the detachment of proteins adsorbed on the graft layer due to the mechanical action or mobility of the graft chain, thus possibly cleaning up the graft layer from protein adsorption and absorption, as well as cell adhesion. In this article, we report the preparation of an albuminbound surface graft architecture and a temperatureinduced structural change of a graft layer in water, and discuss its possible applications of temperature-induced self-cleaning nonfouling surfaces in biomedical engineering and biotechnology fields. 2. Experimental Section 2.1. Materials. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (WSC) and N-(dithiocarboxy)sarcosine, disodium salt, dihydrate (DTCS) were obtained from Dojindo Laboratories (Kumamoto, Japan). 4-Chloromethylphenylethyl trichlorosilane (CTS) was obtained from Shin-Etsu Chemical (Niigata, Japan). Albumin (bovine fraction V) was obtained from Sigma-Aldrich (MO). N-Hydroxysuccinimide (NHS) and FITC conjugated rabbit anti-bovine albumin (FITC-anti-Alb,Intercell Technologies Inc., FL) were obtained from Wako Pure Chemicals (Osaka, Japan). The solvents and other reagents used, all of which were of special reagent grade, were purchased from Wako Pure Chemicals and conventionally purified prior to use. 2.2. Preparation of Polymer Glass Surface. The preparation of iniferter-derivatized surfaces and photopolymerization were basically followed by our previous method.15 Cover glasses
Langmuir, Vol. 21, No. 21, 2005 9661 (f ) 5 and 15 mm, Matsunami Glass Co., Ltd., Saitama, Japan), washed with acetone and ethanol under sonification, was immersed in the piranha solution (composition, concentrated H2SO4: 30% H2O2 ) 7:3) at 80 °C for 1 h. After sequential thorough washing in water, ethanol and toluene, the clean cover glass was immersed in carboxyl group-bearing silane (CTS)-containing toluene solution (5%) with gentle shaking for 18 h. Subsequently, the cover glass was washed sequentially in toluene, ethanol and water, followed by heating at 115 °C for 10 min to produce chloromethyl group-derivatized glass (Cl-glass). The Cl-glass was immersed in methanol solution of N-(dithiocarboxy)sarcosine, disodium salt, dehydrate (DTCS) (5 w/v%) and shaken at room temperature for 18 h to produce carboxyl group-bearing dithiocarbamate-derivatized glass (DC-glass). DC-glass was immersed in NaH2PO4 aqueous solution (pH ) 4.6, 5 mL) containing WSC (30 mg/mL) and NHS dioxane solution (20 mg/mL) (mixing ratio ) 1:9) at 0 °C for 30 min. The glass was rinsed in water and subsequently immersed in albumin containing phosphate-buffered solution (PBS, 1 mg/mL, 5 mL). The glass was washed in water and dried to produce albumin-derivatized DC-glass (Alb-DC-glass). A drop of PBS solutions (30 mL) of DMAM and NIPAM (0.1 M), subjected to nitrogen gas purging for 5 min, were placed on the nontreated glass, and then covered with DC-glass or AlbDC-glass (thickness of solution: 1.5 mm). Ultraviolet light (UV) from a high-pressure mercury lamp (100 mW/cm2 at 325 nm, USHIO, Tokyo, Japan) was irradiated onto the DC-glass and Alb-DC-glass for 3-7 min (Figure 1). The treated glass was washed with water and subsequently air-dried. 2.3. Characterization of Glass Surfaces. The water contact angles of the surfaces prepared above were measured using a contact angle meter (CA-D, Kyowa Kaimenkagaku, Saitama, Japan) at 25 °C and 37 °C. The chemical compositions of the treated glasses were measured by ESCA (AXIS-His, Shimadzu, Kyoto, Japan). The force-distance (f-d) curves of the glass surfaces in PBS at 25 °C and 37 °C were measured by atomic force microscopy (AFM: Molecular Force Probe 3D, Asylum Research, Santa Barbara, CA) to examine the thickness of the graft layer using a Si probe tip (OMCL-TR400PSA-1, Olympus Optical, Co. Ltd., Tokyo). As the AFM probe tip approached the surface, the load gradually increased due to the repulsive interaction between the tip and the graft chains. When the tip hit on the hard surface, the tip cannot indent further. The distance from the onset of the gradual increase in load to the nondeformed point was defined as the repulsive distance obtained from forcedistance curve (f-d curve, see Figure 3). The glasses (Alb-DC-glass, DC-glass, Alb-PNIPAM-glass, and PNIPAM-glass) were immersed in PBS solution containing FITCanti-albumin antibody (1 mg/mL) at room temperature for 2 h in the dark. The glasses were vigorously washed with PBS. The samples were observed by confocal laser scanning microscopy (CLSM; Radiance 2000, Bio-Rad Laboratories, Hercules, CA) and the average fluorescence intensity were determined by scanning the surfaces at various parts.
3. Results 3.1. Preparation of Albuminated Polymer-Grafted Glass Surfaces. The preparation of an albuminated polymer-grafted glass surface, in which albumin was bound at the end of its graft chain, proceeded with four sequential steps as described in Figure 2. The glass was derivatized with a chloromethyl group-bearing silane coupling agent (CTS). Both advancing and receding contact angles of the treated surfaces were very high, that is, 96.3 ( 2.9 and 90.3 ( 3.9 deg, respectively. The surface elemental ratio of Cl/C, determined by ESCA, was 0.03, indicating that chloromethyl group was derivatized on the surface (designated Cl-glass). Subsequent treatment with sodium carboxylated dithiocarbamate (DTCS) resulted in reduced contact angles; the advancing and receding contact angles were 64.0 ( 1.9 and 52.8 ( 1.1 deg, respectively. The Cl atom disappeared, but both N and S atoms appeared: N/C, 0.06; and S/C, 0.04. Upon treatment of DC-glass with albumin in the presence a
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Matsuda and Ohya
Figure 2. Synthetic route of albumin-bound polymer-grafted glass.
Figure 3. Representative force-distance curves (A,B) and schematics of repulsion distance (C) of albumin-bound polymer-grafted glass at 25 °C and 37 °C. Monomer: DMAM (A), NIPAM (B). Graft layers of both polymer grafts swelled at 25 °C but PNIPAM graft collapsed at 37 °C.
water-soluble carbodiimide (WSC) and N-hydroxysuccinimide (NHS), the treated surface became quite wettable with water: the receding contact angle of the DC-glass derivatized with albumin was 20.4 ( 4.5 deg. Concomitantly, N/C increased to 0.34, while S/C remained small (0.03), indicating that albumin was chemically bound to carboxyl group of the iniferter of DC-glass (Alb-DC-glass). Upon UV irradiation in DMAM- or NIPAM-containing PBS solution on Alb-DC-glass or DC-glass, the contact angles of both surfaces at 25 °C decreased with irradiation time, irrespective of the type of monomer (Table 1), indicating that photopolymerization proceeded with time. The N/C elemental ratios of PDMAM-glass remained in the range of 0.06∼0.13, which is below the theoretical
value (0.20) of PDMAM, whereas those of PNIPAM-grafted surfaces were close to the theoretical value (0.17) of PNIPAM. On the other hand, the N/C elemental ratio increases: approximately 0.25∼0.31 for Alb-PDMAMsurface, and 0.13∼0.34 for Alb-PNIPAM-glass. These results indicate that polymerization proceeds on the surface, regardless of the type of monomer and the presence or absence of albumin at the terminus of the graft chain. The contact angle of PNIPAM-glass at 37 °C markedly increased, compared with that at 25 °C, indicating that PNIPAM grafts aggregate at 37 °C, thus exhibiting a more hydrophobic character. The receding contact angles of Alb-PNIPAM-glass were similar to those of Alb-DC, irrespective of irradiation time, suggesting that
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Table 1. Surface Characterization of Treated Glass
run
sample codea
1 2 3 4 5 6 7 8 9 10 11 12
Glass CL DC Alb-DC PDMAM PDMAM Alb-PDMAM Alb-PDMAM PNIPAM PNIPAM Alb-PNIPAM Alb-PNIPAM
irradiation time (min)
contact angle at 25 °C (degree) advancing receding 32.1 ( 4.4 96.3 ( 2.9 64.0 ( 1.9 52.9 ( 6.9 34.5 ( 2.1 28.1 ( 3.0 38.5 ( 3.3 27.7 ( 6.0 57.0 ( 2.8 35.7 ( 3.5 48.4 ( 6.3 50.9 ( 3.3
3 5 3 5 5 7 5 7
25.4 ( 3.0 90.3 ( 3.9 52.8 ( 1.1 20.4 ( 4.5 17.7 ( 0.5 18.6 ( 4.7 18.9 ( 1.7 14.4 ( 3.1 33.6 ( 6.1 16.1 ( 3.3 19.4 ( 3.7 19.1 ( 4.7
contact angle at 37 °C (degree advancing receding
N/C
59.9 ( 3.6 67.5 ( 5.2 45.7 ( 3.4 52.7 ( 2.3
0 0 0.06 0.34 0.13 0.06 0.25 0.31 0.21 0.16 0.13 0.34
40.7 ( 11.4 53.1 ( 4.9 26.3 ( 3.0 33.1 ( 8.6
elemental ratio S/C Cl/C 0 0 0.04 0.03 0.01 0.05 0.02 0.01 0 0.03 0.03 0.01
0 0.03 0 0 0 0 0 0 0 0 0 0
a CL: Chloromethyl group-derivatized glass, DC: Dithiocarbamate-derivatized glass, Alb-DC: Albumin-derivatized DC-glass, PDMAM: Poly(N,N-dimethylacrylamide)(PDMAM)-grafted glass, Alb-PDMAM: PDMAM-grafted glass with albumin, PNIPAM: Poly(N-isopropylacrylamide)(PNIPAM)-grafted glass, Alb-PNIPAM: PNIPAM-grafted glass with albumin. Elemental ratios were determined by ESCA.
Table 2. Repulsion Thickness of Graft Layer Measured by AFM run
sample code
irradiation time (min)
T (°C)
repulsion thicknessa (nm)
1 2 3 4 5 6 7 8 9 10 11 12
PDMAM PDMAM Alb-PDMAM Alb-PDMAM PNIPAM PNIPAM PNIPAM PNIPAM Alb-PNIPAM Alb-PNIPAM Alb-PNIPAM Alb-PNIPAM
3 5 3 5 5 5 7 7 5 5 7 7
25 25 25 25 25 37 25 37 25 37 25 37
92 ( 19 489 ( 62 111 ( 44 481 ( 49 106 ( 33 < 10 416 ( 87 < 10 73 ( 23
