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Surface Macromolecular Microarchitecture Design: Biocompatible Surfaces via Photo-Block-Graft-Copolymerization Using N,N-Diethyldithiocarbamate Y. Nakayama and T. Matsuda* Department of Bioengineering, National Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565, Japan Received November 10, 1998. In Final Form: April 27, 1999 The photo-block-graft-copolymerization method using an iniferter, benzyl N,N-diethyldithiocarbamate, was utilized to design a biomedically functional surface for fabricated devices. Ultraviolet light irradiation under sequential charge of vinyl monomers, such as acrylic acid (AA), n-butyl methacrylate (BMA), N,Ndimethylacrylamide (DMAAm), N-[3-(dimethylamino)propyl]acrylamide (DMAPAAm), or styrene (ST), produced block-grafted surfaces, in which different polymer blocks were sequentially formed on polyST (PST) partially derivatized with N,N-diethyldithiocarbamate groups. Examination of a cross-sectional view under a transmission electron microscope (TEM) revealed a bilayered structure of the PST-b-PDMAAm block-graft copolymer. Three different AB-type block-graft-copolymerized surfaces were prepared for biomedical applications, where block A was the bottom layer and block B the top layer: (1) For heparin immobilization, a polyPDMAPAAm (PDMAPAAm) (A)-polyDMAAm (PDMAAm) (B) block-graftcopolymerized surface was prepared, where the block A layer contained ionically immobilized heparin and the block B layer functioned as a protective layer to suppress protein adsorption and cell adhesion. (2) For protein immobilization, a PDMAAm (A)-polyAA (PAA) (B) block-graft-copolymerized surface was prepared, where the block B layer covalently fixed the protein and the block A layer functioned as a protective layer to prevent contact of the protein with the substrate. (3) For a drug-releasing surface, a PDMAAm (A)polyBMA (PBMA) (B) block-graft-copolymerized surface was prepared, where the block A layer contained the drug and the block B layer functioned as a barrier to regulate drug release. These macromolecularly designed surfaces were characterized by X-ray photoelectron spectroscopy, and the immobilization of heparin and protein was visualized by light microscopy, after staining with toluidine blue (for heparin), and fluorescence microscopy (for protein).
Introduction Surface design in biomedical devices is crucial to whether implanted devices would be accepted or rejected by the body defense mechanisms. Many reports have shown that the surface structure, composition, and properties of the blood- or tissue-contacting surfaces determine the nature and magnitude of the biological responses.1-6 Nonionic polymer graft surfaces prepared by graft polymerization have proven to minimize cell adhesion and protein adsorption.7-13 Graft-copolymeri* Corresponding author. Present address: Department of Biomedical Engineering, Kyushu University Graduate School of Medicine, 3-1-1 Maedashi, Higashi-ku, Fukuoka 812-8582, Japan. Tel.: (+81) 92-642-6210. Fax: (+81) 92-642-6212. E-mail: matsuda@ med.kyushu-u.ac.jp. (1) Curtis, A. S. G., Cell Adhesion. In The Cell Surface; Its Molecular Role in Morphogenesis; Academic Press: London, 1967. (2) Szycher, M. In Biocompatible Polymers, Metals and Composites; Technomic Publ. Co., Inc.; Lancaster, 1983. (3) Park, J. B. In Biomaterials Science and Bioengineering; Planum Press: New York, 1984. (4) Andrade, J. D. In Surface and Interfacial Aspects of Biomedical Polymers; Andrade, J. D., Ed.; Plenum Press: New York, 1985; Vol. 1, p 1. (5) Andrade, J. D. Clin. Mater. 1992, 11, 19. (6) Leonard, M. J. Chromatogr. B 1997, 699, 3. (7) Tazuke, S.; Kimura, H. J. Polym. Sci., Polym. Lett. Ed. 1978, 16, 497. (8) Ogiwara, Y.; Kanda, M.; Takumi, M.; Kubota, H. J. Polym. Sci., Polym. Lett. Ed. 1981, 19, 457. (9) Uchida, E.; Uyama, Y.; Ikada, Y. J. Polym. Sci., Polym. Chem. Ed. 1989, 27, 527. (10) Chapiro, A. Eur. Polym. J. 1983, 19, 859. (11) Suzuki, M.; Kishida, A.; Iwata, H.; Ikada, Y. Macromolecules 1986, 19, 1804.
zation is usually achieved by the formation of highly reactive radical-generating species such as trapped polymer radicals and peroxide groups, via γ-ray irradiation,10 ultraviolet (UV) light irradiation,7-9 plasma, glow discharge, or ozone gas treatments,11 followed subsequently by radical polymerization at an elevated temperature in solution. Due to the inherent nature of radical polymerization, where highly reactive radicals undergo various adverse reactions such as coupling and/or disproportionation, control of the reaction is very difficult. Therefore, a well-controlled graft-copolymerization method is needed for precise surface design in biomedical applications. Otsu et al. developed a quasi-living polymerization method based on iniferter, dithiocarbamte.14-16 Based on the characteristic features of polymerization, diblock and triblock with well-controlled block lengths were prepared under appropriate conditions.17-19 That is, a benzyl radical, which was produced by photolysis of benzyl N,N-diethyldithiocarbamate under ultraviolet light (UV) irradiation (reaction I in Scheme 1), initiates radical polymerization in the presence of monomer A to produce a homopolymer (12) Nakayama, Y.; Matsuda, T. J. Polym. Sci., Polym. Chem. 1993, 31, 3299. (13) Nakayama, Y.; Matsuda, T. J. Appl. Phys. 1996, 80, 505. (14) Otsu, T.; Yoshida, M. Macromol. Chem., Rapid Commun. 1982, 3, 127. (15) Otsu, T.; Yoshida, M.; Tazaki, T. Macromol. Chem., Rapid Commun. 1982, 3, 133. (16) Otsu, T.; Matsunaga, T.; Doi, T.; Matsumoto, A. Eur. Polym. J. 1995, 31, 67. (17) Otsu, T.; Yoshida, M. Polym. Bull. (Berlin) 1982, 7, 197. (18) Otsu, T.; Kuriyama, A. J. Macromol. Sci. Chem. 1984, A21, 961. (19) Turner, S. R.; Blevins, R. W. Polym. Prepr. 1988, 29, 6.
10.1021/la981581x CCC: $18.00 © 1999 American Chemical Society Published on Web 06/26/1999
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Scheme 1. Reaction Mechanism of Block Copolymerization Using Benzyl N,N-Diethyldithiocarbamate
A whose propagating end is coupled with dithiocarbamyl radical (reactions II, III, and IV). When the monomer A solution was changed to monomer B solution and polymer A was subsequently UV-irradiated, the copolymerization of monomer B is initiated from the chain end of polymer A, resulting in the formation of an AB-type diblock copolymer (reactions V, VI, and VII). An ABC-type triblock copolymer can be prepared by UV irradiation of an ABtype block copolymer in a monomer C solution (reactions VIII and IX). We utilized this unique photograft copolymerization method to precisely control the surface design of biomedical devices,20-22 since polymerization proceeds only during photoirradiation and at photoirradiated regions. The following types of surfaces were prepared in our previous studies: (1) a regionally precise, microprocessed surface onto which different polymers were regionally grafted,20-22 (2) a gradient surface in which the thickness of the graft layer was gradually increased in one direction,20,21 and (3) surface block-graft copolymers in which different polymer blocks20 were sequentially formed. As a continuation of a series of our studies, we prepare four types of block-graft-copolymerized surfaces composed of block A (bottom layer) and block B (top layer) for functional biomedical surface design. These are (1) poly(N,N-dimethylacrylamide) (PDMAAm; block A)-b-PST (block B) to examine whether the A and B layers are segregated or phase-separated by transmission electron microscopy (TEM), (2) poly(N-[3-(dimethylamino)propyl]acrylamide) (PDMAPAAm)-b-PDMAAm for heparin immobilization in block A from which slow release of heparin takes place through a nonionic top layer, (3) PDMAAmb-poly(acrylic acid) (PAA) for protein immobilization in block B through a nonionic spacer polymer, and (4) (20) Nakayama, Y.; Matsuda, T. Macromolecules 1996, 29, 8622. (21) Higashi, J.; Nakayama, Y.; Marchant, R. E.; Matsuda, T. Langmuir 1999, 15, 2080. (22) DeFife, K. M.; Colton, E.; Nakayama, Y.; Matsuda, T.; Anderson, J. M. J. Biomed. Mater. Res. 1999, 45, 148.
PDMAAm-b-poly(butyl methacrylate) (PBMA) for creating a drug reservoir in block A from which a drug is released through the hydrophobic top layer. The potential biomedical applications of these designed surfaces are discussed. Experimental Section Materials. ST was purchased from Ohken Co., Ltd. (Tokyo, Japan). DMAPAAm was obtained from Kohjin Co., Ltd. (Tokyo, Japan). 1-Ethyl-3,3-dimethylaminopropylcarbodiimide hydrochloride (WSC) was obtained from Dojindo (Kumamoto, Japan). Solvents and other reagents, all of which were of special reagent grade, were obtained from Wako Pure Chem. Ind. Ltd. (Osaka, Japan) and used after standard purification. PST film was obtained from Asahi Chemical Industry Co. Ltd. (Tokyo, Japan). Cross-linking of PST film was carried out by irradiation with γ-rays from a 60Co source under argon atmosphere. The detailed irradiation conditions have been reported previously.20 Preparation of N,N-Diethyldithiocarbamate-Derivatized PST Film. Dithiocarbamate-derivatized PST films were prepared according to a previously reported procedure.20 Briefly, a cross-linked PST film was chloromethylated and then dithiocarbamated using potassium N,N-diethyldithiocarbamate trihydrate. The treated PST film was stored in the dark until use. The content of N,N-diethyldithiocarbamyl groups in the outermost layers of the PST film was 0.5 mol %, which was calculated from the elemental ratio of nitrogen to carbon as measured by X-ray photoelectron spectroscopy (XPS). Surface Block-Graft-Copolymerization. Surface graftpolymerization was performed on the dithiocarbamate-derivatized PST film. The PST film was vertically mounted in a 30-mL quartz cell with 20 mL of a monomer-containing solution (methanol or toluene; concentration of monomer, 5.0 mol/dm3). A stream of dry nitrogen was introduced through a gas inlet to sweep the cell for at least 5 min. The film was then UV-irradiated from a locus of approximately 5 cm with the light of an ultrahighpressure mercury-vapor lamp (Spot Cure 250, Ushio, Tokyo, Japan) through a quartz optical fiber (diameter, 5 mm; length, 1 m), while nitrogen was bubbled through the solution. The light intensity which was measured using a photometer (UVR-1, Topcon, Tokyo, Japan) was adjusted to 5 mW/cm2. The temperature of the polymerization samples was maintained at 20-25
5562 Langmuir, Vol. 15, No. 17, 1999 °C. The treated film was thoroughly rinsed to remove unreacted monomers and homopolymers and then dried in air. Surface blockgraft-copolymerization was performed on the graft-copolymerized surface by repeating UV irradiation in a sequential monomer charge under the same conditions as described above. Physical Measurements. XPS spectra were measured with a Shimadzu ESCA 750 (Kyoto, Japan) using a magnesium anode (Mg KR radiation) connected to a data processor ESCAPAC-760 at room temperature and 2 × 10-7 Torr (8 kV, 20 mA) at a takeoff angle of 90°, the takeoff angle being defined as the angle between the sample surface and the electron optics of the energy analyzer. As the elemental analysis data determined by XPS measurement were reproducible (n ) 3, s.d. < 10%), only the average values were reported. Transmission electron microscopy (TEM) was conducted using with a Philips/CM-120 TEM (Eindhoven, The Netherlands) operated at 120 kV, using a side-entry goniometer stage. Samples were prepared by embedding then in Spurr’s low-viscosity resin (TAAB Laboratories, Berks, CA) for 3 days at 60 °C and cut into approximately 800-Å sections using a diamond knife at room temperature. All TEM samples were collected on copper mesh grids and stained in iodine vapor for 12 h. Immobilized heparin was visualized by light microscopy with a Nikon Optiphoto system (Tokyo, Japan) after staining with toluidine blue (1.0 w/v % aqueous solution).23 Images of the distribution of the fluorescence intensity (excitation wavelength, 490 nm) were recorded with an Argus20 system (Hamamatsu Photonics, Shizuoka, Japan) equipped with a CCD (charge-coupled device) video camera (C-2400-77i, Hamamatsu Photonics) controlling the image acquisition and display. The concentration of propranolol hydrochloride released into a saline solution was determined spectrophotometrically at 290 nm (JASCO, Ubest-30 UV/VIS spectrophotometer, Tokyo, Japan). The molar absorption coefficient of propranolol hydrochloride was ) 6500 at 290 nm in water.
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Figure 1. Schematic diagram of the preparation of a photoblock-graft-copolymerized surface on the dithiocarbamatederivatized PST film.
Results Cross-Sectional Structure Of Block-Grafted Surface. Surface photo-graft-copolymerization was carried out on N,N-diethyldithiocarbamated PST films (content of dithiocarbamyl group, 30.5 mol %), as described previously.20 Surface block-graft-copolymerization with DMAAm and ST was carried out as follows. DMAAm (monomer A) was first surface-graft-copolymerized on the dithiocarbamate-derivatized PST film by UV irradiation for 10 min in a methanol solution (step I in Figure 1). After cessation of UV irradiation and subsequent thorough rinsing with methanol, the PDMAAm-graft-copolymerized film was immersed into a methanol solution of ST (monomer B) and UV-irradiated for 10 min (step II). After thorough rinsing with methanol, a cross-section of the sample was examined by TEM. Upon staining with iodine, two horizontal bands were observed, consisting of a relatively light band and a dark band on the dithiocarbamated PST film (Figure 2). The bands were approximately 30 nm (bright band) and approximately 120 nm (dark band) thick. Since iodine reacts preferentially with polar functional groups, it renders the iodine-binding microdomains dark in the image of the electron beam. Thus, the dark regions were ascribed to the PDMAAm layer, and light regions to PST. This result suggests that ST (B) was surface-graft-copolymerized on the PDMAAmgraft-copolymerized (A) surface, and a PST layer (b in Figure 2) was apparently segregated from the PDMAAm layer (c in Figure 2). Thus, the sequential formation of different polymer blocks of grafted chains on the surface was visually confirmed. (23) Nakayama, Y.; Matsuda, T. J. Polym. Sci., Polym. Chem. 1993, 31, 977.
Figure 2. TEM micrograph of the cross-section of a PDMAAmb-PST block-graft-copolymerized surface stained in iodine vapor. (a) Spurr’s resin, (b) PST layer; (c) PDMAAm layer; and (d) dithiocarbamate-derivatized PST film.
Heparin-Immobilized Surface. Heparin immobilization has been achieved via ion complex formation of heparin with cationically charged surfaces.24,25 Thrombus formation after the complete release of heparin is a potential drawback of these surfaces because the noncomplexed cationically charged surface may induce platelet adhesion and aggregation probably due to the electrostatic interactions between the negatively charged (24) Miyama, H.; Harumiya, N.; Mori, Y.; Tanzawa, H. J. Biomed. Mater. Res. 1977, 11, 251. (25) Mori, Y.; Nagaoka, S.; Masubuchi, Y.; Itoga, M.; Tanzawa, H.; Kikuchi, T.; Yamada, Y.; Yonaha, T.; Watanabe, H.; Idezuki, Y. Trans. Am. Soc. Artif. Intern. Organs 1978, 24, 736.
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Figure 4. Photomicrograph and cross-sectional illustration of a patterned heparin-immobilized, PDMAPAAm-b-PDMAAmblock-grafted surface stained with toluidine blue. (a) Dithiocarbamate-derivatized PST film; (b) DMAPAAm-graft-copolymerized layer; (c) heparin; (d) DMAAm-graft-copolymerized layer; (e) PDMAPAAm-b-PDMAAm-grafted regions with immobilized heparin which was stained with toluidine blue; and (f) DMAAm-graft-copolymerized regions. Figure 3. Schematic diagram of the preparation of a heparinimmobilized, PDAPAAm-b-PDMAAm-block-graft-copolymerized surface. (a) Dithiocarbamate-derivatized PST film; (b) PDMAPAAm graft polymer; (c) heparin; and (d) PDMAAm graft polymer. Table 1. Chemical Compositional Changes of Dithiocarbamate-Derivatized PST Film Surface after Modification as Shown in Figure 3a N/C O/C S/C
I
II
III
IV
0.038 0.059 0.049
0.20 (0.22) 0.15 (0.11) 0.041
0.14 0.36 0.044
0.13 (0.2) 0.15 (0.2) 0.016
a I, II, III, and IV correspond to those in Figure 3. Parentheses enclose the theoretical values.
cellular membranes and cationically charged surface. However, if the cationically charged layer is completely shielded by a nonionic hydrophilic layer to avoid direct contact with blood components, protein adsorption and blood cellular adhesion would be minimal. For this purpose, the diblock-graft-copolymerized surface was designed as illustrated in Figure 3. First, the dithiocarbamated PST film was UV-irradiated for 10 min in a DMAPAAm methanol solution (step II in Figure 3). After through rinsing with methanol, the irradiated film was immersed in an aqueous heparin solution (10 w/v %; step III). Subsequently, the treated film was UV-irradiated for 10 min in a DMAAm methanol solution (step IV). XPS measurements revealed that after UV irradiation of the film in a DMAPAAm methanol solution and subsequent rinsing with methanol, significant XPS spectral changes occurred. Intensities of the nitrogen and oxygen peaks increased. Significantly, elemental ratios of N/C and O/C increased from 0.038 to 0.20 for N/C and from 0.059 to 0.15 for O/C (Table 1), which are close to the theoretical values for PDMAPAAm (N/C ) 0.22 and O/C ) 0.11). The relative content of two higher binding energy components in the C1s, assigned to C-N (286.0 eV) and CdO (287.6 eV), compared to the aliphatic hydrocarbonlike component (C-H; 285.0 eV), increased. The S/C ratio decreased slightly from 0.049 before irradiation to 0.041
after irradiation. These indicate that a considerable number of dithiocarbamyl groups was present at the outermost surface layer. After immersion in an aqueous heparin solution, the N/C elemental ratio decreased to 0.14 and the O/C ratio increased to 0.36. There was little appreciable change in the S/C ratio after treatment with heparin (before treatment, 0.041; after treatment, 0.044). The S2p signal contained two components of a new higher binding energy signal at 169 eV, which was derived from heparin, and lower binding energy components consisting of two signals at 162 and 164 eV, both of which were derived from dithiocarbamate groups. The signal intensity of the higher energy component was approximately four times higher than the sum of that of the lower energy components, indicating that heparin was immobilized onto the DMAPAAm graft layer. When the heparinized surface was UV-irradiated in a DMAAm methanol solution, a marked reduction in the O/C ratio (0.15) was observed. S2p signals derived from heparin were scarcely appreciable, suggesting that the newly formed PDMAAm layer completely covered the heparin-immobilized layer. To confirm the presence of immobilized heparin in the PDMAPAAm-b-PDMAAm diblock-graft-copolymerized film, a stripe-patterned PDMAPAAm layer was prepared by UV irradiation using a projection metal mask with a stripe pattern (width of lines ) 100 µm). The photomask was placed in contact with the substrate film following graft copolymerization with DMAPAAm and subsequent immersion in an aqueous heparin solution. The partially grafted surface was completely graft-copolymerized with DMAAm without using the photomask. When the resultant film was immersed into an aqueous solution of toluidine blue, a dye specific for acidic polysaccharides such as heparin and chondroitin sulfate, only the PDMAAm-b-PDMAPAAm diblock-grafted stripe regions were stained in blue (Figure 4), indicating that heparin was immobilized on the PDMAPAAm layer which was covered by the nonionic polymer layer. Protein-Immobilized Surface. Protein immobilization has been achieved on surfaces derivatized or grafted with functional groups such as carbonyl-, amine-, or hydroxyl groups. These groups were coupled with func-
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Nakayama and Matsuda Table 2. Chemical Compositional Changes of Dithiocarbamate-Derivatized PST Film Surface after Modification as Shown in Figure 5a N/C O/C S/C
I
II
III
0.038 0.059 0.049
0.13 (0.2) 0.16 (0.2) 0.052
0.024 (0) 0.43 (0.5) 0.039
a I, II, and III correspond to those in Figure 5. Parentheses enclose the theoretical values.
Figure 5. Schematic diagram of the preparation of proteinimmobilized, PAA-b-PDMAAm block-graft-copolymerized surface. (a) Dithiocarbamate-derivatized PST film; (b) DMAAm graft polymer; (c) AA graft polymer; and (d) protein.
tional groups of their respective proteins to form an amide or ester linkage, resulting in the covalent immobilization of protein on the surface. A time-dependent inactivation of the biological activity of these immobilized proteins, often caused by contact due to surface attachment, has been reported.26,27 Therefore, immobilization of proteins on a surface was designed, in which contact between the proteins and the substrate was prevented. The preparative method is illustrated in Figure 5. First, the dithiocarbamated PST film was UV-irradiated in a DMAAm methanol solution (step II in Figure 5) and then in an acrylic acid (AA) methanol solution for 10 min each (step III). Subsequently, the treated film was coupled with a protein (FITC-IgG: fluorescence-labeled IgG) in the presence of 1-ethyl-3,3-dimethylaminopropylcarbodiimide hydrochloride (WSC) as a condensation reagent (step IV). After UV irradiation of the film in the DMAAm methanol solution, N/C and O/C elemental ratios increased from 0.038 to 0.13 (N/C) and from 0.059 to 0.16 (O/C; Table 2). Little appreciable change in S/C ratio was observed after irradiation (before irradiation; 0.049, after irradiation; 0.052). On the other hand, UV irradiation of the film treated in the AA methanol solution resulted in an increase of the O/C ratio (0.43), and decrease of the N/C and S/C ratios (0.024 and 0.039, respectively). The N/C and O/C ratios obtained after irradiation in each monomer solution were close to the theoretical values for the respective polymers (theoretical values of PDMAAm: N/C ) 0.2, O/C ) 0.2, those of PAA: N/C ) 0, O/C ) 0.5). These results indicate that the AA was graft-copolymerized on the (26) Kim, H. P.; Byun, S. M.; Yeom, Y. I.; Kim, S. W. J. Pharm. Sci. 1983, 72, 225. (27) Aldenhoff, Y. B.; Koole, L. H. J. Biomed. Mater. Res. 1995, 29, 917.
Figure 6. Fluorescence microphotograph and cross-sectional illustration of patterned FITC-IgG-immobilized surface. (a) Dithiocarbamate-derivatized PST film; (b) DMAAm-graftcopolymerized layer; (c) AA-graft-copolymerized layer; (d) protein; (e) PAA-b-PDMAAm-block-graft-copolymerized regions with surface-immobilized FITC-IgG; and (f) DMAAm-graftcopolymerized regions.
PDMAAm-grafted, dithiocarbamated PST film, suggesting that PDMAAm-b-PAA diblock-graft copolymerization had occurred. To verify protein immobilization, FITC-IgG was reacted with a diblock-grafted surface in which PAA was regionally grafted with the photomask used above. Fluorescence, derived from FITC bound to IgG, was observed only at the PAA grafted regions (Figure 6), indicating that the protein was covalently fixed onto the PAA graft-copolymerized regions. Controlled Drug-Release Surface. The surface blockgraft-copolymerization method was utilized to prepare controlled drug-release surfaces. DMAAm was first graftcopolymerized on a dithiocarbamated PST film with UV irradiation in DMAAm methanol solution for 10 min (step II in Figure 7). After rinsing, the DMAAm-graft-copolymerized surface was impregnated with propranolol hydrochloride, which is a β-blocker used for cardiovascular diseases as a drug in experimental model, by coating with an aqueous solution of the drug (10 w/v %) and then drying at room temperature (step III). Subsequently, the treated film was graft-copolymerized with n-butyl methacrylate (BMA) by UV irradiation in BMA-toluene solution (step IV). Three types of drug-impregnated films were prepared to evaluate the drug-releasing characteristics of the surface: (1) a DMAAm surface graft-copolymerized film without the PBMA layer (Figure 8): (2) a PDMAAm-graftcopolymerized surface with a micropatterned PBMA layer that was obtained by graft-copolymerization with BMA using a lattice-patterned projection mask (ratio of irradiated area/nonirradiated one ) 1); and (3) a PDMAAmgrafted surfaces with different thicknesses of the PBMA layer which were obtained by graft-copolymerization with
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Figure 9. Characteristics of release of the drug from four different types of microarchitectural, PBMA-b-PDMAAm blockgraft-copolymerized surfaces where the drug was entrapped in the PDMAAm block layer, (a) without PBMA layer; (b) with a micropatterned PBMA layer; and with a completed thin (c) and thick (d) PBMA layer.
variation of PBMA layer thickness could allow control of the rate of drug release. Figure 7. Schematic diagram of the preparation of a drugreleasing BMA-b-PDMAAm-block-graft-copolymerized surface. (a) Dithiocarbamate-derivatized PST film; (b) DMAAm graft polymer; (c) drug; and (d) BMA graft polymer.
Figure 8. Design of the graft copolymerization area of an upper PBMA block on a DMAAm-graft-copolymerized surface that was impregnated with drug. Areas a-d correspond to those in Figure 7.
PBMA at different irradiation times (5 and 10 min). The release of the drug from the grafted films in saline solution at 25 °C was monitored. As indicated in Figure 9, almost all of the loaded drug was released within 5 min for the non-PBMA-grafted sample. After regional grafting with PBMA, approximately 50% of the loaded drug was released within several minutes after immersion, followed by the continuous release of approximately 35% of the remaining drug over 10 h. Complete surface grafting with PBMA resulted in a marked reduction of drug release to 15-30% after 24 h of immersion. Increasing the irradiation time in the BMA solution (step IV in Figure 8), which should yield a thicker PBMA graft layer, resulted in a lower rate of drug release for a prolonged period. This indicates that
Discussion Since surface graft-copolymerization may provide appropriate composition and physicochemical properties at the surface different from those of the bulk, this method has been utilized for surface modification of biomedical devices. In particular, surfaces graft-copolymerized with nonionic water-soluble monomers such as acrylamide, poly(ethylene glycol) acrylate and phosphorylcholylethyl methacrylate on blood-contacting devices have proven to possess antithrombogenic potential, especially in acutephase implantation. This is attributed to minimal interaction with blood components. Surface graft copolymerization via iniferter-based quasi-living polymerization method enhanced to prepare well-controlled surface macromolecular architectures.14-18 The sequential steps of preparation of block-graft surfaces were followed by XPS measurements. An appreciable content of S atoms even after the graft copolymerization proceeded, which was determined by XPS measurements, implies that the growing chain ends preferentially recombine with dithiocarbamate radicals. In the resultant PDMAAm-b-PST-block-grafted surface, two blocks were well segregated, which was evidenced by TEM (Figure 2). Antithrombogenic surfaces are obtained by the immobilization of biologically active substances with antithrombogenic activity, such as heparin,28-30 urokinase,31-33 prostaglandin derivatives,34 and human thrombomodulin.35,36 Heparin immobilization has been achieved via ionic complex formation of heparin on cationically charged (28) Miura, Y.; Aoyagi, S.; Kusada, Y.; Miyamoto, K. J. Biomed. Mater. Res. 1980, 14, 619. (29) Park, K. D.; Okano, T.; Nojiri, C.; Kim, S. W. J. Biomed. Mater. Res. 1988, 22, 977. (30) Nojiri, C.; Park, K. D.; Grainger, D. W.; Jacobs, H. A.; Okano, T.; Koyanagi, H.; Kim, S. W. ASAIO Trans. 1990, 36, M168. (31) Ohshiro, T.; Kosaki, G. Artif. Organs 1980, 4, 58. (32) Kaetsu, I.; Kumakura, M.; Asano, M.; Yamada, A.; Sakurai, Y. J. Biomed. Mater. Res. 1980, 14, 199. (33) Forster, R. I.; Bernath, F. Am. J. Surg. 1988, 156, 130. (34) Chandy, T.; Sharma, C. P. J. Biomed. Mater. Res. 1984, 18, 1115. (35) Yagi, K.; Hirota, K.; Yamasaki, S.; Uwai, A.; Miura, Y. Chem. Pharm. Bull. (Tokyo) 1989, 37, 732.
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polymer substrates.24,25 Release of heparin from the surface with time into blood eventually occurs, exposing the cationic polymer surface to the blood, which may cause massive platelet adhesion and aggregation resulting from electrostatic interactions with the exposed cationic charged surface. If a cationic graft polymer complicated with heparin is completely shielded or overlayered by a nonionic water-soluble polymer layer, cell adhesion on the device surfaces may be passively suppressed even after the heparin is completely released. On the basis of the design concept above, we prepared PDMAPAAm-b-PDMAAmAB-type-block-graft-copolymerized surfaces using the iniferter-based copolymerization method. Heparin was ionically bound to a PDMAPAAm layer, which was confirmed by XPS measurements and dye-staining (Table 1 and Figure 4). The PDMAAm layer, formed as a top layer, should function as a protective layer against adhesion or adsorption of biocolloids such as cells, that minimizes occurrence of thrombus formation even after heparin is completely released. Surface covalent immobilization of proteins on polymer surfaces has been carried out extensively in order to provide bioactive functions.37,38 A major problem experienced with immobilization of proteins on surfaces is the significant reduction in bioactivity after the immobilization (surface induced denaturation).26,27 A spacer inserted between the substrate surface and the immobilized protein helped to avoid surface-induced denaturation.6,26,27 To meet this requirement, a PDMAAm-b-PAA-AB-type-blockcopolymerized surface was designed. Protein was covalently fixed onto the PAA with a condensation reagent, which was observed using a fluorescence microscope (Figure 6). The PDMAAm layer, formed as a bottom layer, should function as a protective layer preventing contact (36) Kishida, A.; Ueno, Y.; Maruyama, I.; Akashi, M. Biomaterials 1994, 15, 1170. (37) Jacobs, H.; Grainger, D.; Okano, T.; Kim, S. W. Artif. Organs 1988, 12, 506. (38) Matsuda, T.; Sugawara, T. Langmuir 1995, 11, 2272.
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between the substrate and the proteins fixed onto the PAA at an upper layer. Use of a projection mask during UV irradiation in BMA toluene solution resulted in regional control of a PBMAgrafted area formed on DMAAm-grafted surfaces impregnated with a water-soluble model drug, propranolol, which is used for treatment of hypertension and angina pectoris. The bottom water-dissolved PDMAAm layer functions as a drug holding layer, and the top hydrophobic PBMA layer functions as a barrier that regulates drug release. The release of the drug from these treated surfaces is fast during the first several minutes of immersion in water, followed by a relatively slow release rate, which is shown in Figure 9. The amount of drug that was burstleaked during the initial release period was reduced by completely covering the surface with the PBMA graft polymer on the PDMAAm surfaces. Increasing the thickness of the PBMA graft layers further decreased the drug release rate and thus prolonged the period of drug release. Moreover, because the PBMA surface is sticky, it probably functions as an adhesive layer on the skin as a device for percutaneous delivery of drugs. In conclusion, surface photo-block-graft-copolymerization, which was based on the photochemistry of N,Ndiethyldithiocarbamate, was applied to precisely design biocompatible and functional surfaces for biomedical applications. This method has proven to be a powerful tool in the design of biocompatible or functional surfaces, by which high reliability, high quality, high performance, and high regional precision are realized for fabricated devices. Acknowledgment. The authors thank the Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research (OPSR) for the financial support of this work under Grant No. 97-15. LA981581X
