Alternating Bioactivity of Polymeric Layer-by-Layer Assemblies

Biomacromolecules 2002, 3, 724-731

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Alternating Bioactivity of Polymeric Layer-by-Layer Assemblies: Anticoagulation vs Procoagulation of Human Blood Takeshi Serizawa, Miyuki Yamaguchi, and Mitsuru Akashi* Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065, Japan Received January 3, 2002

The layer-by-layer assembly between cationic chitosan and anionic dextran sulfate was analyzed quantitatively by a quartz crystal microbalance technique in the absence and presence of 0.2, 0.5, and 1 M NaCl in the polymer solution. The apparent film thickness increased upon increasing the NaCl concentration. The antiversus procoagulant activity of these films against whole human blood was studied by the immersion of a substrate into blood for 30 min incubation time at 37 °C. The substrate was coated with films of varying NaCl concentrations and assembly step numbers. There was a critical concentration for the alternating activity; above a concentration of 0.5 M NaCl, both anti- and procoagulation could be observed on the dextran sulfate and chitosan surfaces, respectively. The underlying layer of the assembly was necessary for this alternating activity; after a five-step assembly, the activity was realized. The adsorption of a cationic dye (methylene blue) onto the films revealed that the anionic-charge density derived from dextran sulfate on the film surface was linearly increased with increased NaCl concentration. There was a critical charge density of the dextran sulfate for the anticoagulant activity. An assembly was also constructed from a combination of chitosan and heparin, but the activity was different from that of the former system; strong anticoagulant activity was observed even on the chitosan surface. We suggest that the polymer species and/or the assembly conditions are key factors for realizing the alternating bioactivities of films prepared by the layer-by-layer assembly. Introduction The surface modification of materials by organic or inorganic layers is significant for biomedical applications, because the intact materials can be engineered for specific bioaffinities. Coating a substrate surface with polymeric ultrathin films can maintain the original mechanical properties and/or fine structure of the substrate. If the polymers used are insoluble in an aqueous biological phase, then the coating is easily performed by methods such as dip- or spincoating. However, when the polymers are water-soluble, they should be immobilized by chemical bonds between the polymers and the substrate material. There are limitations in the latter case, and control of the chemical composition of the film surface is also necessary. Layer-by-layer assembly using water-soluble polyelectrolytes, in which polyion complexes are alternately formed on the substrate surface, promises to prepare polymeric ultrathin films.1 The assembly can be achieved by the alternate immersion of certain materials into oppositely charged, water-soluble polymers. The films obtained are very stable, even in aqueous phases, because of their strong electrostatic interactions. The importance of layer-by-layer assembly for biomedical applications is the control of the chemical composition of the surface, which affects the biological activity. Hubbell et al.2 prepared polyelectrolyte multilayers on biological surfaces to obtain bioinert surfaces, and cell

interactions with the underlying surface were suppressed by the coating. However, no alternating bioactivity of the multilayers was demonstrated. In our previous report,3 an alternating antivs procoagulant activity was observed on layer-by-layer assemblies prepared from anionic dextran sulfate (Dex)4 and cationic chitosan,5 which have anti- and procoagulant activities against whole blood, respectively, on a poly(ethylene terephthalate) substrate. The Dex and chitosan surfaces showed their anti- and procoagulant activities, respectively, when an adequate amount of NaCl was added into the aqueous polymer solutions for film preparation. In fact, the alternating activities were observed in the presence of 1 M NaCl in the polymer solutions, but not in its absence. Therefore, it is important to further analyze this system in detail for the suitable coating of biomedical materials. Creating anti- or procoagulant activity on the surfaces of materials such as polymeric or inorganic seats, tubes, and meshes is important in the biomedical field. Both activities are useful, depending on the application. Since layer-by-layer assembly includes a “wetting” immersion process of dipping the substrate into a polymer solution, variously shaped materials can be generated. Of course, it is easy to coat the surfaces inside or outside the tubes. In the present study, the antivs procoagulation activity against human whole blood of a layer-by-layer assembly prepared from Dex and chitosan was analyzed in terms of the NaCl concentration in the aqueous polymer solution and

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Bioactivity of Polymeric Assemblies

the number of assembly steps. Furthermore, heparin, which is an anionic polymer and is well-known to show anticoagulant activity, was also used in a layer-by-layer assembly with chitosan, and its bioactivity was analyzed. The charge density onto the assembly surface was analyzed by the adsorption experiment of suitable dyes, to chemically follow the alternating bioactivity. The assembly of the polymer combinations was analyzed quantitatively by a quartz crystal microbalance (QCM),6 which has been previously used for analyzing the assembly of naturally occurring polymers.7 Experimental Section Materials. Dex (Mw 500 000) and chitosan (Mw 1 200 000) were purchased from Wako (Japan) and were used without further purification. Heparin (Mw ca. 20 000) and formic acid were purchased from Nacalai Tesque (Japan) and were used without further purification. The chitosan contained 10-25% N-acetylglucosamine units (as chitin) according to the data sheet. X-ray photoelectron spectral (XPS) analysis of the nitrogen peaks of the Dex-chitosan assembly showed that it contained 20% chitin units based on the peak ratio between the amide nitrogen (400.2 eV) and the amine one (402.2 eV).3 Sodium chloride (NaCl) (guaranteed reagent) was purchased from Nacalai Tesque (Japan) and was used without further purification. A cationic dye, methylene blue, was purchased from Aldrich and used without further purification. Anionic dyes, methyl orange (Aldrich), trypan blue (Wako), brilliant blue (Wako), and bromophenol blue (Ncalai Tesque) were also used without further purification. Ultrapure distilled water was provided by the MILLI-Q labo system. QCM Study. The quantitative analysis of the layer-bylayer assembly using a QCM was performed essentially as reported in previous studies.3,7 An AT-cut quartz crystal with a parent frequency of 9 MHz was obtained from USI (Japan). The crystal (9 mm in diameter) was coated on both sides with gold electrodes 4.5 mm in diameter. The frequency was then monitored by an Iwatsu frequency counter (model SC7201). The leads of the QCM were sealed and protected with a silicone-rubber gel in order to prevent degradation during immersion in the aqueous salt solutions. The amount of polymer adsorbed, ∆m, was calculated by measuring the frequency decrease in the QCM, ∆F, using Sauerbrey’s equation6 as follows -∆F )

2F02 A(Fqµq)1/2

∆m

where F0 is the parent frequency of the QCM (9 × 106 Hz), A is the electrode area (0.159 cm2), Fq is the density of the quartz (2.65 g cm-3), and µq is the shear modulus (2.95 × 1011 dyn cm-2). This equation was reliable when the measurements were performed in air as described in this study. The mass of the solvents was never detected as a frequency shift, and thus the effects of the viscosity of the absorbent can be ignored. Assembly. Before the assembly measurements, the QCM electrodes were treated three times with a piranha solution [concentrated H2SO4/H2O2 (30 wt % in water) ) 3/1, v/v]

for 1 min each time, followed by rinsing with pure water and drying with N2 gas in order to clean the surface. The cleaned QCM was then immersed in an aqueous chitosan solution (1 mg mL-1) containing 25% HCOOH for 10 min at ambient temperature, rinsed thoroughly with pure water, and then dried under N2 gas. The frequency decrease was then measured. The QCM was immersed again into an aqueous Dex (or heparin) solution (1 mg ml-1), and the same procedure was repeated. This alternate cycling was repeated for film assembly. NaCl was added at the given concentration into the polymer solutions. The apparent film thickness of the resulting assemblies was estimated from the frequency shifts, assuming the film density to be 1.2 g cm-3 and the film surface to be flat.8 In the present study, the assembly started from the chitosan step. The starting polymer does not affect the assembly, because the first step of the assembly is caused by nonspecific physisorption onto the silver surface. Coagulation Assay. The antiversus procoagulant activity assay was performed essentially as reported in previous studies.3,9 A sterilized cell disk of 13.5 mm diameter (normally used as a cell-culture plastic disk prepared by poly(ethylene terephthalate)) (Sumilon, Japan) was trimmed down to 10.5 mm diameter and was used as the substrate after rinsing with pure water. The ultrathin films were assembled onto the substrate by a method similar to the QCM study. The film-coated substrate was then immersed in whole human blood (1 mL), which was supplied by Mr. T. Matsuyama and Dr. K. Yamamoto (Kagoshima University), in a polypropylene tube (13.5 mm diameter) (Maruemu, Japan) at 37 °C. After a given incubation time, the substrate was removed, gently rinsed in PBS, and then photographed. Dye Adsorption onto Assembly. The dye adsorption experiment was basically followed by a previous study.10 The films were similarly prepared on a transparent quartz substrate (45 × 12.5 × 1 mm) by every 10 min immersion into both solutions. The film-coated substrate was immersed into dye solutions at a suitable concentration for 10 min at ambient temperature, rinsed with pure water, and then dried with nitrogen gas. The dye amount adsorbed was analyzed by ultraviolet-visible (UV-vis) spectrometry, Jasco model V-550, at ambient temperature at 600 nm for methylene blue. Others. Reflection absorption spectra (RAS) were obtained with a Herschel FT/IR-610 (Jasco, Japan) instrument in a nitrogen atmosphere at ambient temperature. One side of the poly(ethylene terephthalate) film, which was used as the substrate for the RAS measurements, was coated with gold in order to obtain a reflective surface. The ultrathin film was assembled onto the substrate by a method similar to the QCM study. The interferograms were coadded 50 times and Fourier transformed at a resolution of 4 cm-1. Atomic force microscopy (AFM) images were obtained with a Digital Instruments NanoScope III operating in a tapping mode in air at ambient temperature. We did not perform any image processing other than flat leveling. The mean roughness (Ra) in a given observed area was estimated from the following equation Ra )

1 LxLy

∫0Ly ∫0Lx |F(x,y)| dx dy

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Figure 1. Frequency shifts of the QCM plotted against the assembly step for a Dex-chitosan assembly prepared in the presence of the following concentrations of NaCl: (a) 0 M; (b) 0.2 M; (c) 0.5 M; (d) 1 M. The open and closed symbols represent the chitosan and Dex steps, respectively.

where F(x,y) is the surface relative to the center plane that is a flat plane parallel to the mean plane, and Lx and Ly are the dimensions of the surface. Results and Discussion Layer-by-Layer Assembly of Dex and Chitosan. The total thickness of the ultrathin polymer films prepared by layer-by-layer assembly increased with increasing salt concentration in the polymer solution, possibly because the salt reduced the electrostatic repulsion of the coiled polymers and released the hydrated water. A similar salt effect has been observed in previous studies.7a,11 To analyze the effects of the apparent thickness of the outermost layer on the antior procoagulant activity, the layer-by-layer assembly between Dex and chitosan was performed in the presence of NaCl at various concentrations. Figure 1 shows the frequency shifts plotted against the assembly step of the layer-by-layer assemblies in chitosan and Dex solutions, in the absence and presence of NaCl at ambient temperature. The frequencies shifted with an increasing assembly step, indicating stepwise polymer deposition. The amount (corresponding to -∆F) of polymer assembled gradually increased upon the addition of NaCl into the solution because the coiled polymers seemed to adsorb in the presence of NaCl (as mentioned above). At all NaCl concentrations, a larger amount of polymer was assembled after a five-step assembly. The layer thickness of the outermost surfaces, as well as the total thickness, could be controlled by the addition of NaCl to the polymer solution at suitable concentrations. The total thickness of the films prepared in the presence of 0, 0.2, 0.5, and 1 M NaCl at a six-step assembly were estimated to 1.8, 13, 30, and 64 nm, respectively. In the following sections, five- and six-step assemblies were generally used. In our previous study,3 X-ray photoelectron spectra were obtained for assemblies prepared in the absence of NaCl, on a cell disk that was utilized as the substrate for the antivs procoagulant activity, to analyze the presence of each polymer in the assembly. Peaks corresponding to nitrogen and sulfur, which were assigned to chitosan and Dex, respectively, could be observed and their ratios were reason-

Serizawa et al.

Figure 2. RAS of a Dex-chitosan assembly prepared in the presence 1 M NaCl for a six-step assembly.

able, thus demonstrating the presence of each polymer. However, it was difficult to analyze the presence of each polymer in films prepared in the presence of NaCl, because the analyzing depth (approximately 10 nm) for XPS was insufficient for the thicker films. Therefore, in the present study, a reflection absorption spectrum of an assembly prepared in the presence of 1 M NaCl at a six-step assembly was obtained, as shown in Figure 2. The peaks were assigned to the amine and amide groups and to the sulfate groups, which are typical functional groups in chitosan-containing chitin units and Dex, respectively, thus demonstrating the presence of each polymer. The surface topologies of six-step assemblies prepared in the presence of various NaCl concentrations were analyzed by AFM in tapping mode, as shown in Figure 3. The QCM substrate showed a gold-sputtered surface with a mean roughness of 1.5 nm. On the other hand, after polymer assembly, the surface still appeared to be molecularly smooth. The roughness of the assemblies prepared in the presence of 0, 0.2, 0.5, and 1 M NaCl were estimated to 0.97, 1.4, 3.2, and 3.2 nm, respectively. It would have been extremely difficult to consider the confounded effects of the above nanometer-ordered roughness on the anti- procoagulant activities. Although we ignored them in the present study, a delicate analysis is necessary soon. In fact, the total thickness, which corresponds to the increased roughness, did not affect the activities.3 Blood Anti- vs Procoagulant Activity on a DexChitosan Assembly. In our previous study,3,9 the anti- or procoagulant activities of various films to which certain functional polymers had been grafted or assembled were studied by simple immersion into human blood for an adequate period of time. The Dex-chitosan films prepared by the layer-by-layer assembly were similarly analyzed.3 Films prepared in the presence of 1 M NaCl showed alternate antivs procoagulant activity for an outermost surface of Dex versus chitosan, respectively, thus demonstrating the intact characteristics of the polymers on these films. The activities were clearly observed after 30 min of immersion in blood. In fact, the anticoagulant activity was observed after only 15 min of immersion. However, the alternate activities were not observed in the absence of NaCl. In the present



Figure 3. AFM images of Dex-chitosan assemblies prepared in the presence of the following concentrations of NaCl for a six-step on the QCM: (a) QCM substrate; (b) 0 M; (c) 0.2 M; (d) 0.5 M; (e) 1 M.

section, the critical NaCl concentration as well as the assembly step to generate these alternate activities was analyzed by a similar method. We used a cell disk as a substrate and assembled the Dex-chitosan films in a manner similar to that described above.3 Figure 4 shows photographs of the antivs procoagulant activity against whole blood of the (chitosan-Dex)3 (six steps) and (chitosan-Dex)2-chitosan (five steps) assemblies (Dex and chitosan surfaces, respectively), which were prepared in various concentrations of NaCl for 30 min at 37 °C. The strong coagulation was observed for 30 min on a cell disk after slight coagulation at 15 min. On the chitosan surfaces, the blood was coagulated even though the NaCl concentration was altered from 0 to 1 M. Since chitosan has intrinsic procoagulant activity, this observation is reasonable. On the other hand, there was a critical NaCl concentration for the anticoagulant activity on the Dex surfaces. The blood was

coagulated at a NaCl concentration of less than 0.2 M, but anticoagulation was observed above 0.5 M. It is important to discuss why there is a critical concentration. Dex has intrinsic anticoagulant activity. As shown in the former section, the film thickness increased upon increasing the NaCl concentration, possibly due to the coiled polymers that were absorbed onto the film surface. This means that excess charges, which do not participate in the polyion complex formation with chitosan, are present on the outermost surface of the polymers. Since the anticoagulant activity of Dex can be contributed to the sulfate groups of the polymer, the excess charges on the Dex film surface prepared in the presence of suitable concentrations of NaCl seem to result in intact activity. In other words, the fabrication process can control the antivs procoagulant activity on the films prepared by layer-by-layer assembly. Other bioactivities on ultrathin films can also be controlled

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Figure 4. The antivs procoagulation activity of Dex-chitosan assemblies prepared in the presence of various NaCl concentrations against whole human blood.

Figure 5. The antivs procoagulation activity of Dex-chitosan assemblies prepared in the presence 1 M NaCl with the step number against whole human blood.

following the above concept. In fact, the total thickness did not affect the bioactivity. When the polymers were assembled in the absence of NaCl on the thick films prepared in the presence of NaCl, no antivs procoagulant activity was observed. Note that the alternating activity of the films prepared in the presence of 1 M NaCl was maintained for at least 3 h. Analyzing the effect of the step number on the antivs procoagulant activity is important in minimizing the fabrication process for the surface modification of certain substrates. In addition, it is also important to understand the step at

which the intact characteristics of the polymer surfaces emerge during the layer-by-layer assembly. Figure 5 shows the antivs procoagulation of whole blood on the outermost surface of Dex or chitosan, for one- to eight-step assemblies after a 30 min incubation at 37 °C. All of the films were assembled in the presence of 1 M NaCl in order to obtain the alternating activity. No alternating activity was observed until a step number of 4 and was observed consistently after step 5. It is difficult to reasonably account for these observations. Considering Figure 1d, the films might have incompletely covered the substrate or the underlying-polymer


Figure 6. The dependence of the absorbance at 600 nm against the NaCl concentration during the assembly, when methylene blue was adsorbed from its aqueous 1 mM solution for 10 min at ambient temperature onto the following assemblies: (a) (chitosan-Dex)3 (six steps); (b) (chitosan-Dex)2-chitosan (five steps); (c) (chitosan-Dex)2 (four steps) prepared in the presence of 1 M NaCl.

surface. Thus, at least four assembly steps were necessary to establish the antivs procoagulant activity. It is important to discuss the mechanism responsible for the antivs procoagulant activity. One possibility is that the coagulation was caused by the initial adsorption of plasma proteins such as fibronectin on the film surface and the subsequent platelet aggregation with fibrin polymer formation. The Dex surface prepared in the presence of more than 0.5 M NaCl seemed to suppress the above procoagulation pass. On the other hand, the Dex surface prepared in the presence of less than 0.2 M NaCl seemed to lead the pass, due to the fewer amounts of free sulfate groups on the surface (see the following section). Therefore, direct observation of adsorbed platelets as well as other mechanistic analyses will be undertaken soon. In summary, we found that the antivs procoagulant activity on Dex-chitosan assemblies was dependent on the NaCl concentration in the polymer solution, as well as the step number of the layer-by-layer assembly. Dye Adsorption onto a Dex-Chitosan Assembly. In previous sections, it was demonstrated that the alternating blood antivs procoagulant activity of the layer-by-layer assembly between Dex and chitosan was realized when the polymers were assembled in the presence of suitable concentrations of salt. In the present section, the relative charge amount on the film surface was directly analyzed by the adsorption of cationic and anionic dyes onto the films with a Dex or chitosan outermost surface, to follow the above results. Figure 6 shows the dependence of the absorbance at 600 nm against the NaCl concentration during the assembly, when methylene blue was adsorbed onto the (chitosan-Dex)3 (six steps) and (chitosan-Dex)2-chitosan (five steps) assemblies prepared at various concentrations of NaCl. Methylene blue was clearly adsorbed onto the films with an anionic Dex surface. Almost no adsorption was observed onto the chitosan surface. This observation indicates that methylene blue was electrostatically adsorbed to Dex on the film, following the presence of anionic charges on the (chitosan-Dex)3 assembly. In addition, the relative adsorption amount of methylene blue onto the Dex surface was


increased with increased NaCl concentration. As shown in the previous section, the anticoagulant activity was observed above the NaCl concentration of 0.5 M. The excess charge that was analyzed by the adsorption of methylene blue seems to induce the anticoagulant activity of Dex on the film surface. The relative adsorption amount onto the (chitosanDex)2 (four steps) assembly prepared in the presence of 1 M NaCl, which did not show the anticoagulant activity, was smaller than that onto the (chitosan-Dex)3 (six steps). This also suggests that this film does not have enough sulfate groups on the film surface for the expression of the anticoagulant activity. The dye adsorption test used was initially developed for the layer-by-layer assembly containing a weak polyacid such as poly(acrylic acid) and polyamine.10 It is necessary for the present study to consider whether methylene blue stained only the surface or diffused into the film. When the absorbance in Figure 6 was potted against the total polymer amount, the outermost Dex amount, and the total Dex amount, almost linear relationships were observed in all cases. Accordingly, we could not determine whether methylene blue stained only the surface or not. However, the fivestep assembly with a chitosan surface prepared in the presence of 1 M NaCl was not stained, although the internal structure was similar to that of the six-step assembly with a Dex surface. This suggests that only the surface was stained under the present conditions. On the other hand, the adsorption was also analyzed by using anionic dyes such as methyl orange, trypan blue, brilliant blue, and bromophenol blue, which might electrostatically interact with chitosan. However, the adsorption was not observed onto both films even at the concentration of 10 mM. This observation suggests that a small amount of cationic charge of chitosan remained on the film surface. This cationic charge possibly leads to the adsorption of plasma proteins on the film surface, followed by the platelet aggregation with fibrin polymer formation, as previously mentioned. Chitosan-Heparin Assembly and the Bioactivity. Heparin is a well-known sulfated polysaccharide which possesses strong anticoagulant activity4a and can be used in layer-by-layer assembly with cationic chitosan. We prepared a chitosan-heparin assembly and analyzed the antivs procoagulant activity in a manner similar to the method described above. A NaCl concentration of 1 M in the polymer solution was selected because alternating activity was observed in the Dex-chitosan system. The fabrication process of the layer-by-layer assembly of chitosan and heparin was also analyzed by QCM, and a stepwise frequency shift was observed (original data not shown). The total thickness of the film for a six-step assembly was estimated to be 3.9 nm, which is 15 times smaller than that prepared with Dex under the same conditions. The thickness was similar to the Dex film thickness prepared in the absence of NaCl. This may also be due to the difference in molecular weight between Dex and heparin. The molecular weight of heparin is 25 times smaller than that of Dex. This smaller molecular weight will result a smaller coiled state; hence the apparent thickness of each heparin layer becomes smaller

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Figure 7. The antivs procoagulation activity of chitosan-heparin assemblies prepared in the presence 1 M NaCl with the step number against whole human blood.

than that of Dex. Subsequently, the thickness of each chitosan layer will also be smaller because there is a smaller amount of interactive polymer heparin present on the film surface. Figure 7 shows the antivs procoagulation of human blood on the outermost surface of chitosan or heparin for 5- to 10-step assemblies prepared in the presence of 1 M NaCl after a 30 min incubation at 37 °C. Anticoagulant activity was observed on all the films. In the case of a Dex-chitosan assembly of the similar thickness (prepared in the absence of NaCl), coagulation was observed without any alternating anticoagulant activity. It is difficult to account for this obvious difference between these two systems. The heparin used with a smaller molecular weight might interpenetrate the outermost chitosan layer and exist at the surface, resulting in the anticoagulation. The intact bioactivity (strong or weak) of the polymers and/or the molecular weight seemed to affect the antivs procoagulation. In other words, the alternating bioactivity of the layer-by-layer assembly would be governed by the above parameters. In fact, the adsorption of cationic methylene blue on the assembly even with a heparin surface was not detected by UV-vis spectroscopy. This means that the small amount of heparin on the surface showed the remarkable anticoagulant activity. Note that the antivs procoagulation activity was observed on the heparin and chitosan surfaces after 12 h of incubation, respectively. Consequently, we found that the chitosan-heparin assembly showed the strong anticoagulant activity. Conclusion Ultrathin Dex-chitosan films were prepared by the layerby-layer assembly technique using polyion complex formation in the absence or presence of salt in the polymer solution.

The fabrication process was quantitatively analyzed by the frequency shifts of a QCM as the substrate. The antivs procoagulant activity of the films was dependent on the salt concentration and the number of steps of the assembly. There was a critical concentration (apparent film thickness) for the alternating bioactivity, and the alternating activity was observed after a suitable underlying assembly of the polymers. The adsorption of a cationic dye revealed that there was an essential charge density of sulfates remaining on the film surface for the anticoagulant activity. This assembly method was also applied to a chitosan-heparin system, and the bioactivity was obviously different from the above system. The surface of the chitosan-heparin assembly showed strong anticoagulant activity. A suitable selection of polymers and/or conditions for the assembly is required to generate the antivs procoagulant activity on the material surface. The present system will be useful for coating various biomedical materials which contact blood. Other important characteristics of layer-by-layer assemblies, including their biodegradability and cell adhesion properties, as well as other interesting polymer combinations, will be studied soon. Acknowledgment. The authors acknowledge Dr. A. Kishida (National Cardiovascular Center, Japan) for grateful discussions and to Med. Dr. Y. Maeda (Kagoshima University, Japan) for his technical support. This work was financially supported in part by Grant-in-Aid for Scientific Research in the Priority Area of “Molecular Synchronization for Design of New Materials System” (No. 404/11167270, 13022258) and by Grant-in-Aid for Scientific Researches (No. 476/12750802, 851/14780643) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.


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Biomacromolecules, Vol. 3, No. 4, 2002 731 (7) (a) Lvov, Y.; Onda, M.; Ariga, K.; Kunitake, T. J. Biomater. Sci., Polym. Ed. 1998, 9, 345. (b) Serizawa, T.; Goto, H.; Kishida, A.; Endo, T.; Akashi, M. J. Polym. Sci., Part A: Polym. Chem. 1999, 36, 801. (c) Techaboonyakiat, W.; Serizawa, T.; Endo, T.; Akashi, M. Polym. J. 2000, 32, 481. (8) Lvov, Y.; Ichinose, I.; Ariga, K.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117. (9) Sakamoto, N.; Shioya, T.; Kishida, A.; Akashi, M. Macromol. Symp. 1997, 120, 159. (10) (a) Shiratori, S. S.; Rubner, M. F. Macromolecules 2000, 33, 4213. (b) Chung, A. J.; Rubner, M. F. Langmuir 2002, 18, 1176. (11) (a) Serizawa, T.; Takeshita, H.; Akashi, M. Chem. Lett. 1998, 487. (b) Serizawa, T.; Takeshita, H.; Akashi, M. Langmuir 1998, 14, 4088. (c) Serizawa, T.; Kamimura, S.; Akashi, M. Colloids Surf. 2000, 164, 237.

BM0200027

Alternating Bioactivity of Polymeric Layer-by-Layer Assemblies

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