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A Core-Shell Structured Hydrogel Thin Layer on Surfaces by Lamination of a Poly(ethylene glycol)-b-poly(D,L-lactide) Micelle and Polyallylamine Kazunori Emoto,†,‡ Yukio Nagasaki,† and Kazunori Kataoka*,‡ Department of Materials Science and Technology, Science University of Tokyo, 2641 Yamazaki, Noda, Chiba 278-8510, Japan, and Department of Materials Science, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan Received November 4, 1999. In Final Form: March 22, 2000 A thin hydrogel possessing layer-by-layer structure was prepared on substrates from a stabilized reactive micelle from a poly(ethylene glycol)-poly(D,L-lactide) (PEG-PLA) bearing an acetal group at the PEG end and a methacryloyl group at the PLA end. The hydrogel layer was formed by coating the aminated surfaces with the micelle and polyallylamine (PAlAm) alternately in the presence of a reducing reagent. Each step of the alternate coating of the micelle and PAlAm was characterized with scanning probe microscopy and the ζ-potential measurement over a pH range of 2-11. When the micelle was the topmost layer, the ζ-potential exhibited a small absolute value, suggesting full masking of the electrostatic charge from the inner layer. The ζ-potential of the surface with a PAlAm top layer showed a large positive value of ∼50 mV up to pH 8 and declined to zero, attributed exclusively to the protonation and deprotonation of PAlAm. The alternation of ζ-potential with the coatings indicates the unmixing of the micelle and PAlAm. Scanning probe microscopy revealed that the surface coated with a monolayer of micelles consisted of granules on the order of the micelle size. With an increase in the number of coatings, the surface undulation was promoted and the nodular size increased. The thickness of the layer, estimated by tapping-mode scanning of the area scratched by the strong force contact mode, was found to increase with the number of coatings, and the increase corresponded approximately to the size of the micelle for each step of the coating. Of interest, the diminution of the well scratched by the probe was observed after a short period indicative of the reorganization of the elastic network to recover entropic reduction. Because of its unique structure, the thin hydrogel layer can be applied as a controlled release matrix of hydrophobic drugs.
Introduction Surface modification with hydrophilic polymers is paid remarkable attention for a variety of applications especially in the field of biomedicine.1-8 The coatings mask the surface charge, increase wettability and lubricity, and reduce the interaction of biomolecules. The coating method varies depending on the application: physical adsorption, chemical grafting of linear, branched, or star polymers, cross-linking to form hydrogels, etc.1-7 We recently reported a novel process of surface coating with polymeric micelles from amphiphilic block copolymers.9,10 In this * Corresponding author. E-mail:
[email protected]. Phone: +81-3-5841-7138. Fax: +81-3-5841-8653. † Science University of Tokyo. ‡ The University of Tokyo. (1) Bayer, E.; Rapp, W. In Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications; Harris, J. M., Ed.; Plenum Press: New York, 1992; p 325. (2) Andrade, J. D.; Hlady, V.; Jeon, S.-I. In Hydrophilic Polymers: performance with environmental acceptance; Glass, J. E., Ed.; American Chemical Society: Washington, DC, 1996; p 51. (3) Li, J.-T.; Carlsson, J.; Huang, S.-C.; Caldwell, K. D. In Hydrophilic Polymers: performance with environmental acceptance; Glass, J. E., Ed.; American Chemical Society: Washington, DC, 1996; p 61. (4) Preisler, J.; Yeung, E. S. Anal. Chem. 1996, 68, 2885. (5) Espadas-Torre, C.; Meyerhoff, M. E. Anal. Chem. 1995, 67, 3108. (6) Sofia, S. J.; Merrill, E. W. In Poly(ethylene glycol); Chemistry and Biological Applications; Harris, J. M., Zalipsky, S., Eds.; American Chemical Society: Washington, DC, 1997; p 342. (7) Sofia, S. J.; Premnath, V.; Merrill, E. W. Macromolecules 1998, 31, 5059. (8) Harris, J. M.; Zalipsky, S. In Poly(ethylene glycol): Chemistry and Biological Applications; Harris, J. M., Zalipsky, S., Eds.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997; p 1. (9) Emoto, K.; Nagasaki, Y.; Kataoka, K. Langmuir 1999, 15, 5212. (10) Emoto, K.; Iijima, M.; Nagasaki, Y.; Kato, M.; Kataoka, K. Colloid Surf., B, in press.
process, micelles from poly(ethylene glycol)-poly(D,Llactide) (PEG-PLA) block copolymer bearing an acetal group at the PEG end and a methacryloyl group at the PLA end were prepared.11,12 After the formation of the micelle, the methacryloyl group was polymerized. The micelle with a polymerized core is stable and withstands the destructive environment such as the addition of surfactant. The acetal group at the PEG end was converted into an aldehyde under an acidic condition. The resultant micelle is reactive and can attach a variety of ligands on its outermost layer.13,14 The reactivity of the stabilized micelle allows its tethering to a variety of surfaces.9,10 It can be tethered onto soft polymer surfaces as well as rigid inorganic surfaces effectively as long as the surface can be activated for the chemical reaction. The protein rejection of the micelle-coated surface was found to be comparable to that of the dense coating of PEG although the micelle coating was performed under moderate conditions, i.e., from low concentration and at ambient temperature.10 The ligands and a variety of biomolecules can be tethered on the surface via the micelle as a linker. The micelle tethered on a surface may also hold hydrophobic drugs in its core and release them in a controlled manner. In the case of the latter application, the amount of the drug on the surface and the release rate should be controlled. In this regard, the surface density of the tethered micelle (11) Iijima, M.; Okada, T.; Nagasaki, Y.; Kato, M.; Kataoka, K. Macromolecules 1999, 32, 1140. (12) Kim, J.-H.; Emoto, K.; Iijima, M.; Nagasaki, Y.; Aoyagi, T.; Okano, T.; Sakurai, Y.; Kataoka, K. Polym. Adv. Technol. 1999, 10, 647. (13) Yamamoto, Y.; Nagasaki, Y.; Kato, M.; Kataoka, K. Colloid Surf., B 1999, 16, 135. (14) Nagasaki, Y.; Hori, Y.; Kato, M.; Kataoka, K. Manuscript in preparation.
10.1021/la9914514 CCC: $19.00 © 2000 American Chemical Society Published on Web 06/02/2000
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needs to be high to control the loading capacity and release rate of the drug. Although the density can be slightly increased to some extent by coating at high temperature, addition of salt, and so on, the increment is limited and the micelle may undergo distortion and aggregation under such conditions. The alternative method is the buildup of micelle multilayers using a mediating reagent. Since micelle particles repel each other, the multilayer structure cannot be formed without a mediator such as spacer molecules and polymers with reactive groups. By coating the aldehyde-bearing micelle and polyamine alternately in the presence of a reducing reagent, the multilayer of micelles may be constructed in a controlled manner without destroying the structure of the micelle. Unlike typical solid particles, the micelle consists of a hydrophobic hard core and hydrophilic soft shell. Consequently, the alternate coating of the core-shell-type micelle and polyamine will result in the formation of a thin hydrophilic layer of hydrogel on a surface. The thin surface hydrogel was found to prevent adsorption of protein and release hydrophobic reagents in a controlled manner.15 In this paper we present the characterization of the laminated micellar hydrogel on substrates. The growth of the layer has often been characterized by ellipsometry, electron spectroscopy for chemical analysis (ESCA), scanning probe microscopy (SPM), and quartz crystal microbalance (QCM).16-20 Since the coating involves a charged substrate and a polyion, an electrokinetic characterization was effective and allows one to semiquantitatively determine the density of the ionizable groups on the surface.21-25 SPM not only presents topographical mapping of surfaces but also allows one to estimate the thickness of the adsorbed layer by a destructive method.26-28 By combining the SPM with ζ-potential measurement, each of the multilayer formations was analyzed, and the topography and the growth of the layer were discussed. Experimental Section Multiple Coating of the Micelle via Polyallylamine (PAlAm). Synthesis of PEG-PLA possessing an acetal group at the PEG end and a methacryloyl group at the PLA end, preparation of the stabilized reactive micelle from the block copolymer, and the amination of a Si wafer and glass surfaces with (3-aminopropyl)triethoxysilane (Aldrich, Milwaukee, WI) are carried out following the previous publications.9-12,21-23 The (15) Emoto, K.; Iijima, M.; Nagasaki, Y.; Kataoka, K. J. Am. Chem. Soc., in press. (16) Lvov, Y.; Ariga, K.; Onda, M.; Ichinose, I.; Kunitake, T. Langmuir 1997, 13, 6195. (17) Sun, Y.; Hao, E.; Zhang, X.; Yang, B.; Shen, J.; Chi, L.; Fuchs, H. Langmuir 1997, 13, 5168. (18) Serizawa, T.; Takeshita, H.; Akashi, M. Langmuir 1998, 14, 4088. (19) Pfau, A.; Schrepp, W.; Horn, D. Langmuir 1999, 15, 3219. (20) Phuvanartnurks, V.; McCarthy, T. J. Macromolecules 1998, 31, 1906. (21) Emoto, K. Ph.D. Thesis, University of Alabama, Huntsville, AL, 1997. (22) Burns, N. L.; Van Alstine, J. M.; Harris, J. M. Langmuir 1995, 11, 2768. (23) Emoto, K.; Harris, J. M.; Van Alstine, J. M. Anal. Chem. 1996, 68, 3751. (24) Uchida, E.; Uyama, Y.; Ikada, Y. Langmuir 1994, 10, 1193. (25) Nagasaki, Y.; Kobayashi, J.; Tsujimoto, H.; Kato, M.; Kataoka, K.; Tsuruta, T. Nanobiology 1996, 4, 63. (26) Ge, S.; Kojio, K.; Takahara, A.; Kajiyama, T. J. Biomater. Sci. Polym. Ed. 1998, 9, 131. (27) Wadu-Mesthrige, K.; Xu, S.; Amro, N. A.; Liu, C. Langmuir 1999, 15, 8580. (28) Boland, T.; Johnston, E. E.; Hubber, A.; Ratner, B. D. In Scanning Probe Microscopy of Polymers; Ratner, B. D., Tsukruk, V. V., Eds.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998; p 342.
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Figure 1. Change in ζ-potential vs pH profile of aminated glass with the coatings of (9) micelle-1, (O) polyallylamine-1, (b) micelle-2, (4) polyallylamine-2, and (2) micelle-3. The number denotes the cycle of coatings. molecular weight of the block copolymer determined by gel permeation chromatography and neutron magnetic resonance spectrum was ca. 12 000 (PEG 7500 and PLA 4500) (Mw/Mn ≈ 1.1). The size of the micelle determined by dynamic light scattering was ∼30 nm and unchanged before and after the polymerization of the methacryloyl group. The aminated Si wafer and glass were coated with the micelle and polyallylamine (PAlAm; MW 10 000) (Nittobo, Tokyo, Japan) alternately. The substrate was immersed in ∼1.2 mg/mL micelle solution in 0.04 M HEPES (pH 6.7) containing 3.2 × 10-3 % (w/v) NaCNBH3 at 22 °C for 2 h. After light rinsing with Milli-Q water, the micellecoated substrate was immersed into 0.6% (w/v) PAlAm in 0.04 M HEPES (pH 6.7) containing 0.25% (w/v) NaCNBH3 at 22 °C for 2 h. The above procedure was repeated until the desired number of coatings was obtained. The final micelle coating was carried out in the same manner but at a higher concentration of NaCNBH3 (0.25%). Although NaCNBH3 is a mild reducing reagent, the micelle solution used for the coating with 0.25% reagent lost the aldehyde group.10 This is desirable for the PAlAm and the final micelle coatings, while it is not for the intermediate coating of the micelle. Whether the aldehyde is intact whereas the Schiff base is reduced was expected to depend on the concentration of the reagent. The above concentration variation was chosen for this reason. The sample was rinsed with Milli-Q water repeatedly and stored in water until use. ζ-Potential Measurement. The aminated glass surfaces coated with the micelle and PAlAm alternately were characterized by the ζ-potential measurement at 25 °C over a pH range of 2-11 by LEZA-600 (Otsuka Electronics, Osaka, Japan). The polyacrylamide-coated quartz cell with a 10 × 25 × 2 mm groove was covered with the glass slide sample of approximately 15 × 35 × 1 mm size. The space between the sample and the cell was then filled with 7.5 mM NaCl containing 1 µm polystyrene beads coated with neutral hydrophilic polysaccharide (Otsuka Electronics, Osaka, Japan). The pH of the medium was altered by adding the 7.5 mM HCl or NaOH into the above NaCl solution. The electric field of approximately 20 V/cm was applied perpendicular to the sample-cell direction, and the mobility of the particle was measured to obtain the ζ-potential of the sample. Characterization of Micelle-Coated Surfaces with Scanning Probe Microscopy (SPM). The APTS-Si wafers alternately coated with the micelle and PAlAm were further characterized by SPM in an aqueous solution. A sample was immobilized on a polystyrene Petri dish and soaked in 10 mM NaCl solution to reduce the capillary effect.29 The dish with the attached sample in NaCl was set on the unit of the Bioscope (Olympus Co., Tokyo, Japan) equipped with a controller by Digital Instruments (Santa Barbara, CA). The pyramidal cantilever made of Si3N4 (spring constant ∼0.09 N/m; Olympus Co., Tokyo, Japan) was used throughout the experiment. The scanning of the samples was carried out by the tapping mode (drive frequency 5-7 kHz) at 0.5 Hz. The drive amplitude was set so that the cantilever response (RMS) was ∼1.5 V. To obtain the thickness of the layer, the indentation was also carried out with the same cantilever by the contact mode. The area of 1 × 1 µm2 was scraped (29) Bemis, J. E.; Akhremitchev, B. B.; Walker, G. C. Langmuir 1999, 15, 2799.
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Figure 3. Scanning probe image of the APTS-Si wafer with a triple coating of the nonpolymerized micelle using PAlAm as the binder layer.
Figure 2. Scanning force images of the APTS-Si wafers coated with (a, top) a monolayer, (b, middle) double layers, and (c, bottom) triple layers of the micelle. with the same cantilever at a rate of 0.75 Hz with strong force for 30 min. Immediately after the indentation, the cantilever was retuned and the area of 4 × 4 µm2 was scanned by the tapping mode in water.
Results and Discussion ζ-potential vs pH Profile of Micelle Coated Surfaces. The characterization of the micelle and PAlAmcoated APTS-glass slides was carried out by measuring the ζ-potential. This method allows one to semiquantitatively determine the surface charge density and the
thickness of the polymer layer. The APTS-coated glass exhibited a positive ζ-potential at low pH and a negative potential at high pH on the basis of the protonation and deprotonation of amino and silanol groups on the surface (data not shown; see ref 9). Figure 1 shows the plot of the ζ-potential of the alternate micelle and PAlAm coatings on the aminated glass surface. After coating with the micelle, the variance in the ζ-potential with pH decreased due to the extended distance between the charged surface and the plane of shear. When the PAlAm was coated on the micelle layer, the ζ-potential was highly positive in the whole pH range. Since the ζ-potential is little varied below pH 8 and no negative potential was observed at higher pH, the effect of the silanol group on the substrate was fully masked, and the profile only reflected the protonation and deprotonation of the amino group of PAlAm on top of the micelle layer. When the second layer of micelles was coated on top of the PAlAm, the ζ-potential was decreased to the same level as the monolayer coating. A slightly negative potential is seen at high pH. The positive ζ-potential originated from the PAlAm layer under the top micelle layer. The micelle coatings screened the electrostatic potential from the underlaid surface charge,9,10 resulting in a slightly positive potential. Since the negative charge of the substrate silanol was completely screened, the slight but appreciable negative ζ-potential at high pH was probably due to the micelle itself. Note that a micelle from PEG-PLA block copolymer exhibited a slightly negative ζ-potential in aqueous solution above neutral pH.13 Although it did not have ionizable groups, the hydrophobic polymer surface including PLA shows a strongly negative ζ-potential over the moderate to high pH range, presumably due to the adsorption of anion.24,30 Such a property of the micelle reflected the ζ-potential at the high pH region. Further coatings of PAlAm and subsequent micelle also showed identical ζ-potential profiles to the previous coatings of PAlAm and micelle, respectively. SPM Imaging of Surfaces. Figure 2 shows the SPM height images of the aminated Si wafer coated with the micelle and polyallylamine alternately. Single micelle coating resulted in the surfaces covered with granules of approximately 30-50 nm diameter. As the micelle coating was multiplied, the undulated structure was pronounced. (30) Otsuka, H.; Nagasaki, Y.; Kataoka, K. Biomacromolecules 2000, 1, 39.
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Figure 4. Estimation of the thickness of the micelle/PAlAm layer by scratching the surface of (a) a monolayer, (b) double layers, and (c) triple layers of the micelle with the SPM cantilever.
The nodular size was more than 50 nm and varied. It is important to note that although the APTS-Si was exposed to the micelle solution for an extended time up to 16 h, the topography of the surface was little different from that in Figure 2a. The kinetics of the micelle coating to aminated surface observed by OWLS and FTIR-ATR indicated that the increase in the amount of the micelle stopped within less than 2 h.31 Under the present coating condition, the surface-attached micelle prevented the (31) Huang, N.; Csucs, G.; Emoto, K.; Kataoka, K.; Textor, M.; Spencer, N. D. Proceedings of the World Biomaterials Conference, Hawaii, 2000.
further attachment of the micelle in the bulk solution, and the multilayer or aggregation formation may have not taken place. The undulation may have resulted from the low surface coverage and the conformation of PAlAm on the micelle-covered surface. On the basis of the ζ-potential of the silica surface with ∼5 silanol/nm2 of -80-90 mV at the fully deprotonated state22 and that of the APTS-coated glass with 0.4 amine/nm2 of 40 mV at the fully protonated state,23 the estimated surface amine density of the PAlAm top layer was ∼1 amine/nm2, or the occupational area of PAlAm was 175 nm2 (r ) 7.5 nm) provided the amino group of PAlAm was fully protonated.
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Although the polyamine and the method for the determination of density were different, the density of polyamine is lower than the result by Serizawa et al. and Pfau et al. of 5-11 amine/nm2.18,19 The PAlAm did not seem to be abundant to fully cover the micelle-coated surface due to the weak attractive force between the PAlAm and the micelles on the surface. The polymer molecule attached on the surface preferentially takes a coiled structure rather than a completely stretched structure. When the micelle is attached to the PAlAm, the loop structure of the PAlAm is more pronounced, leading to the undulation of the surface. We previously showed that the nonpolymerized micelle disrupted upon attachment to the surface.9 Multilayered coating of the nonpolymerized micelle was carried out and observed by SPM. Figure 3 shows the image of the coating of the nonpolymerized micelle on an aminated surface. Ditches are seen on the surface as in Figure 3. The depth of the ditch is about 100 nm, and the length and width vary. Temporary dehydration of the sample during mounting to the SPM stage or impact by the probe at the engagement32 may have formed such ditches although it could not be determined here. On the other hand, such a ditch was not seen in the polymerized micelle coatings. The network formed between the polymerized micelle particles via the PAlAm linker prevented the ditch formation, while the nonpolymerized micelle did not have a strong driving force to form a stable network on the surface. The thickness of the layer was also measured by the SPM. The area of 1 × 1 µm2 scraped by strong force was scanned by the tapping mode. As Figure 4a shows, the height of the single micelle layer is ca. 25 nm. This is somewhat smaller than the size determined by dynamic light scattering. It is either due to the artifact by the SPM imaging or the flattening of the particle upon attachment to the surface. After the second coating of the micelle, the thickness of the layer was increased to about 45 nm as in Figure 4b. The cross section in Figure 4b shows serrated nodes, and the size of the serrated grain seems to be comparable to the micelle size. For the triple coating of the micelle, the thickness increased to over 80 nm, but the nodes were larger than the single and double coatings of the micelles as in Figures 2 and 4. Although the increase in thickness was not proportional to the number of coatings probably due to the conformation of the PAlAm between the micelle layers, the increase was stepwise with the number of coatings. The thickness of the polymer layer in aqueous solution is often determined by ellipsometry.33,34 Our result indicates that the present method is also applicable to the determination of the thickness of soft thin materials on the surface in water as well as rather rigid reagents in an ambient environment, although the surface-water and probe-water interactions, scratching of the silanated layer, and deposition of the removed polymer to the probe must be taken into account. The scraped topography and its width for the triple micelle coating in Figure 4c apparently differed from the other two. The scratched well appeared more like circular than square and smaller than the size of the scratch (1 × 1 µm). It took 30 min to obtain an image after the scratch due to the retuning of the drive frequency, optimization (32) Overney, R. M.; Tsukruk, V. V. In Scanning Probe Microscopy of Polymers; Ratner, B. D., Tsukuruk, V. V., Eds.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998; p 2. (33) Malmsten, M.; Lassen, B.; Van Alstine, J. M.; Nilsson, U. R. J. Colloid Interface Sci. 1996, 178, 123. (34) Elbert, D. L.; Herbert, C. B.; Hubbell, J. A. Langmuir 1999, 15, 5355.
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Figure 5. Change in the SPM image of the triple micelle coating 1.5 h after scratching.
of the parameters, slow scanning rate (0.5 Hz) in aqueous solution, and repetitive engagement and capture. The size of the scraping diminished, and the shape of the well changed with time. Figure 5 shows the image of triple coating 1.5 h after scratching. Within an hour the indentation disappeared and the scraped area was somewhat swollen. When the surface was scratched, the layers of the micelle and PAlAm were pushed toward the side of the formed well. Due to the cross-link between the micelle layers with the PAlAm mediator, the deformation of the gel resulted in the unfavorable elongation and shrinkage. Consequently the reorganization of the elastic network may have occurred to compensate for entropic reduction. Although the recovery of the gel structure took place, it could not be completed as the scratch may have destroyed the micelle structure in the gel. This resulted in the protrusion of the scratched area. Conclusion In this paper, we presented the formation of a new type of hydrogel thin layers from a stabilized reactive polymeric micelle on surfaces prepared by coating of the surface with the micelle and PAlAm alternately and its characterization by ζ-potential measurement and SPM. The PAlAm on the micelle-coated APTS surface completely masked the substrate surface charge and exhibited only a positive ζ-potential over the pH range of measurement. However, the ζ-potential suggested relatively low coverage. Combined with the conformation of PAlAm on the surface, the low density of the polymer resulted in the facilitated undulation after the further coating of the micelle. This may be overcome by the optimization of the coating condition for PAlAm as well as the micelle and/or the use of an alternative linker for micelle layers. We showed reduction of protein adsorption onto the surface coated with the micelle.10 Similar protein rejection is expected for the thin gel surface, which is important for the biomedical and bioanalytical applications. Since the hydrophobic core can incorporate drugs, the surface can release drugs in a controlled manner. These will be presented in another paper.15 Acknowledgment. This work was financially supported by the Japan Research Promotion Society for Cardiovascular Diseases. Advice from Prof. Atsushi Takahara of Kyushu University for the estimation of thickness by SPM is greatly acknowledged. LA9914514
