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Alternating Bioactivity of Polymeric Layer-by-Layer Assemblies: Anti- vs Procoagulation of Human Blood on Chitosan and Dextran Sulfate Layers Takeshi Serizawa, Miyuki Yamaguchi, Takahisa Matsuyama, and Mitsuru Akashi* Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065, Japan Received February 21, 2000; Revised Manuscript Received April 14, 2000
Introduction. The fabrication of ultrathin polymer films on material surfaces is important for various scientific and biomedical fields in order to modify or improve the intact characteristics of these surfaces, which can be exposed to various environments, including biological systems. The effective coating of biomedical materials with polymers thus results in a potentially drastic change in biological affinity. In most cases, the material characteristics seem to be governed by the chemical composition of the surface. Layerby-layer assembly, which can be achieved by the alternate immersion of certain substrate into oppositely charged watersoluble polymers, is a promised methodology that uses electrostatic interaction (as polyion complexes) to fabricate polyelectrolyte multilayers on substrates.1 Other interactions such as hydrogen bonds,2 charge transfer,3 and stereocomplex4 formation between significant polymers have also been utilized. On the basis of this concept, we have fabricated ultrathin polymer films using the repetitive adsorption/drying of a substrate process.5 These assembly-coated substrates have considerable potential in biomedical applications. The layer-by-layer assembly process creates a monolayer of adsorbed polymers at each assembly step. It is important whether the outermost monolayer shows any specific properties similar to that of the bulk film against a given biochemical environment. If possible, alternating biological activities of the layers in the assembly should be constructed. Hubbell et al.6 prepared polyelectrolyte multilayers on biological surfaces to obtain bioinert surfaces, in which cell interactions with the underlying surface were suppressed by their coating. However, no alternating biological activity of the multilayers was demonstrated. To our knowledge, there is no research on the activity of these assemblies against biological functions. In the present paper, the alternating anti- vs procoagulant activity of ultrathin polymer films prepared by the layerby-layer assembly technique against human blood was studied. The introduction of anti- and procoagulant activities onto material surfaces is a current topic in the biomedical field. Dextran sulfate (Dex)7 and chitosan8 were selected as polymers with anti- and procoagulant activities, respectively. The layer-by-layer assembly of these polymers was quantitatively analyzed by a quartz crystal microbalance (QCM), which had been utilized extensively for investigating the process of assembly.4,5 The films were then prepared onto a
cell disk, and then the anti- vs procoagulant activity was studied. We also focused on the salt concentration in the polymer aqueous solutions for the assembly, which leads to thicker films. An advantage of the present study is performing a final control of multiplayer biocompatibility. Experimental Section. Materials. Dex (Mw 500 000), chitosan (Mw 1 200 000), and formic acid were purchased from Wako Pure Chemical Industries, Ltd. and utilized without further purification. The chitosan contained 10-25% N-acetylglucosamine units (as chitin) according to the data sheet. The XPS analysis (see below) of the nitrogen peaks of the chitosan-Dex assembly showed that it contained 20% chitin units from the peak ratio between the amide nitrogen (400.2 eV) and the amine one (402.2 eV). Sodium chloride (NaCl) (guaranteed reagent) was purchased from Nacalai Tesque and utilized 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 QCM was performed essentially as described in our previous studies.4,5 An AT-cut quartz crystal with a parent frequency of 9 MHz was obtained from USI. A crystal (9 mm in diameter) was coated on both sides with silver 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 rubber gel in order to prevent degradation during immersion in the aqueous solutions. The amount of polymers adsorbed, ∆m, could be calculated by measuring the frequency decrease in the QCM, ∆F, using Sauerbrey’s equation9 as follows -∆F )
2F02 AxFqµq
× ∆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 dyne cm-2). This equation was reliable when the measurements were performed in air as described in this study. Since the mass of the solvents was never detected as a frequency shift, and the effects of the viscosity of the absorbent on the frequency can be ignored. Before the assembly measurements, the QCM electrodes were treated 3 times with a piranha solution (H2SO4:H2O2
10.1021/bm000006g CCC: $19.00 © 2000 American Chemical Society Published on Web 06/17/2000
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) 3:1) for 1 min each time, followed by rinsing with pure water and drying with N2 gas in order to clean its surface. The cleaned QCM electrodes were then immersed in an aqueous chitosan solution (1 mg mL-1) containing 25%HCOOH for 15 min at ambient temperature, rinsed thoroughly with pure water, and then dried under N2 gas. The frequency decrease was then measured. The QCM electrodes were immersed again into an aqueous Dex solution (1 mg mL-1), and the same procedure was repeated. This alternate cycling was repeated for the entire film assembly. NaCl was added at a given concentration in the polymer solutions. The mean film thickness of the resulting assemblies was estimated from the frequency shifts, assuming the film density10 to be 1.2 g cm-3 and the film surface to be flat. In the present study, the assembly was started from a chitosan step. Another assembly should be investigated using an initial Dex step, because the first step of the assembly is caused by nonspecific physisorption onto a silver surface. Coagulation Assay. The anti- vs procoagulant activity assay was performed basically according to our previous study.11 A cell disk of 13.5 mm diameter (normally utilized as a cell-culture plastic disk prepared by poly(ethylene terephthalate)) (Sumilon, Japan), which has been already sterilized, 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 immersed in whole blood (1 mL), which was supplied by T.M., using 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. Others. The static contact angle was measured by water dropping of 3 µL at ambient temperature. The angle data were collected at 50 and 70 s after the dropping. X-ray photoelectron spectra (XPS) were obtained with a Shimadzu ESCA 1000 apparatus employing Mg KR radiation. The peaks were referenced to carbon at 285.0 eV to account for the sample charging. The spectra were obtained at 50-time scans. The summed peaks at 400.2 (amide) and 402.2 eV (amine), and of 169.0 eV were assigned to N1s and S2p3/2, respectively. Results and Discussion. Chitosan-Dex Assembly. In the present study, chitosan was selected as the cationic polymer because it has already been utilized in layer-bylayer assembly with certain anionic polymers.12 Dex was selected because the sulfuric acid residue on the polymer is known to be effective for the assembly, and also because of its anticoagulant activity,7 although this polymer has not been previously applied to this assembly process.1 To make the films thicker, we added NaCl to the polymer solution in accordance with previous reports.13 Figure 1 shows the frequency shifts against the assembly step of the layer-bylayer assemblies in chitosan and Dex solutions in the absence and presence of 1 M NaCl at ambient temperature. The frequencies shifted with the increasing assembly step, thus indicating stepwise polymer deposition. The amount (corresponding to -∆F) of polymer assembled obviously increased by the addition of NaCl into the solutions. The
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Figure 1. Frequency shifts of the QCM plotted against the assembly step for the chitosan-Dex assembly: (a) prepared in the absence of NaCl; (b) prepared in the presence of 1 M NaCl. The open and closed symbols represent the chitosan and Dex steps, respectively.
coiled polymers seemed to adsorb more in the presence of NaCl, possibly because of a relaxation of the electrostatic repulsion in the polymers dissolved in an aqueous phase, thus resulting in thicker films at each assembly step. The frequency shifts after 10-step assemblies were -163 and -9946 Hz, which translated to mean film thicknesses of 3.7 and 227 nm, respectively. The error of the frequency shifts after 10-step assemblies were within 30 and 2%, respectively. The former large error could be attributed to a small frequency shift, which was too small for the QCM to analyze accurately. The former thickness at each step corresponded to 0.37 nm, which seemed to be less than the cross-sectional area of each polymer. This suggests that the incomplete surface coverage of the polymers was occurred at each filmforming step, indicating the difficulty of the alternating bioactivity (see the following section). The positive frequency shift at the first step in Figure 1a indicates the desorption of some impurity on the QCM. The frequency in Figure 1b decreased exponentially with the increasing assembly step. It is difficult to explain why we observed this process. The apparent surface area for polymer assembly on the QCM might be increased with increasing the assembly step. Although further studies on this type of assembly are necessary, we utilized the assembly for the bioactive assay. Consequently, we found that ultrathin chitosan-Dex films could be prepared via a layer-by-layer assembly onto a substrate, and that the thickness of the assembly could be altered by changing the NaCl concentration of the solution. Furthermore, the assembly could be fabricated from the first step of the physisorption of chitosan onto the substrate surface, thus indicating that polymer physisorption as a first step is sufficient for a layer-by-layer assembly using stepwise polyion complex formation. Accordingly, the assembly can be formed on a cell disk as discussed below. In our previous papers, we have also shown that polymer physisorption as a first step was sufficient for stepwise assemblies.4,5,12b,13 Blood Anti- vs Procoagulant Activity on a ChitosanDex Assembly. In our research group, the anti- or procoagulant activities of various polymeric films to which certain polymers were grafted have been studied by simple immersion in human blood for an adequate period of time.11 Here, we studied the anti- and procoagulant activity of human blood
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Figure 3. Anti- vs procoagulation activity against human blood of the following assemblies: (a) [(chitosan-Dex)2-chitosan]-Dexchitosan; (b) (chitosan-Dex)3-chitosan-Dex, in which [(chitosanDex)2-chitosan] and (chitosan-Dex)3 were prepared in the presence of 1 M NaCl and in the absence of NaCl, respectively. CT represents chitosan.
Figure 2. Anti- vs procoagulation activity against human blood of the following assemblies: (a) cell disk; (b) (chitosan-Dex)2-chitosan and (c) (chitosan-Dex)3 prepared in the absence of NaCl; (d) (chitosan-Dex)2-chitosan and (e) (chitosan-Dex)3 prepared in the presence of 1 M NaCl. CT represents chitosan.
on chitosan vs Dex surfaces in the chitosan-Dex assemblies. To construct a suitable assay system, we utilized a cell disk as a substrate, and assembled on it in a manner similar to that described above. The static contact angle measurement is a significant tool for confirming the alternate change in chemical compositions in layer-by-layer assemblies.4a,b,14 The angles of the surfaces of the (chitosan-Dex)2-chitosan and (chitosan-Dex)3 assemblies prepared in the absence of NaCl were 32.8 ( 0.8 and 19.3 ( 0.6°, respectively, indicating the alternate assembly of the different polymers (the angle of the cell disk was 46.1 ( 0.8°). In fact, the angles of the cast films of chitosan and Dex were 26.1 ( 0.6 and less than 10°, respectively. Note that the angles of the assemblies prepared in the presence of 1 M NaCl were less than 10°,
thus indicating that the surfaces significantly swell water. The assembly of both polymers on the substrate was also confirmed by the XPS analysis. The analysis showed the presence of nitrogen and sulfur, which were not detected in the substrate. For example, the S/N peak ratio of a 20-step chitosan-Dex assembly prepared in the absence of NaCl was 1.3. The ratio from the total frequency shifts for each polymer estimated from the QCM results in Figure 1a was calculated to be 0.68. This ratio should then be compared to the quantitative QCM results. However, the observed mass in the QCM analysis included the mass of the counterions not joined to the polyion complex as well as possibly 1020 wt % swelling water.15 In addition, the XPS data might include experimental errors because the film thickness was less than 10 nm (the XPS could analyze the surface of approximately 10 nm in depth). Consequently, we assumed that assemblies similar to these on the QCM surface could be observed on the cell disk substrate. Figure 2 shows photographs of the anti- vs procoagulant activity against whole blood of the (chitosan-Dex)2chitosan and (chitosan-Dex)3 assemblies (chitosan and Dex surfaces, respectively), which were prepared in the absence and presence of 1 M NaCl, for the given incubation times at 37 °C. Blood coagulation on the assemblies differed with increasing incubation time. No coagulation was observed on any of the assemblies, including the cell disk, after 5 min. We observed slight coagulation on the cell disk after 15 min and strong coagulation after 30 min, as shown in Figure 2a. On the chitosan and Dex surfaces prepared in the absence of NaCl, moderate coagulation was observed even after 15 min, and strong coagulation was observed after 30 min as shown in parts b and c of Figure 2. These activities were different from those observed in Figure 2a, thus indicating indirect evidence for polymer assembly on the cell disk. Although the chitosan surface may show strong coagulant activity, the Dex surface should not. The surface of the assemblies prepared in the absence of NaCl did not seem to retain any of the intact bioactivity of each layer, although the chitosan surface might show its nature. This may be caused by the incomplete surface coverage of the polymers described above. On the other hand, the assemblies prepared in the presence of 1 M NaCl showed clear, alternating antiand procoagulant activity. Whole blood was gradually coagulated on the chitosan surface with increasing incubation time, as shown in Figure 2d. This process was similar to that observed in parts b and c of Figure 2. However, we could not observe any coagulation on the Dex surface, as
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shown in Figure 2e. Note that it is impossible to perform the anticoagulant activity by the Dex dissolved in the blood from the assembly, because the amount is too small to show any anticoagulant activity.7a It is difficult to explain why we observed alternate bioactivity only on the assembly prepared in the presence of salt. It is possible that the charged and thicker assembly of each layer could induce these results. In our previous paper,13 the electrostatic adsorption of anionic polystyrene nanospheres onto the surface of a cationic poly(allylamine hydrochloride) (PAH) layer prepared by the layer-by-layer assembly of PAH and poly(sodium styrenesulfonate) was analyzed with respect to the NaCl concentration in the polymer solutions for assembly. Since the adsorption process was strongly enhanced by the addition of salt, we concluded that there were excess charges (not joined in complex) on the film surface which promoted the electrostatic adsorption of the nanospheres. In addition, it is well-known that the anticoagulant activity of Dex is derived from its charged sulfate group. As a consequence, the presence of an effective charge due to a thicker layer in the Dex surface, which was prepared in the presence of salt, resulted in anticoagulant activity against human blood. As shown in the QCM study, the mean film thickness was altered by the addition of salt. There is a possibility that the anti- vs procoagulant activity was affected by the total thickness of the assembly. In fact, the thickness of the assembly prepared in the presence of 1 M NaCl was much larger than that prepared in its absence. To analyze the thickness effect, the coagulation activity of the assemblies [(chitosan-Dex)2-chitosan]-Dex-chitosan and (chitosanDex)3-chitosan-Dex, in which [(chitosan-Dex)2-chitosan] and (chitosan-Dex)3 were prepared in the presence of 1 M NaCl and subsequently in the absence of NaCl, respectively, was analyzed. As shown in Figure 3, blood coagulation was observed on both chitosan and Dex surfaces. Therefore, we concluded that the outermost surface was the key factor in the anticoagulant activity observed rather than the total film thickness. On the other hand, the anti- vs procoagulant activity was also analyzed on cast chitosan and Dex films (0.1 mg/cell disk, corresponding to a mean film thickness of approximately 1 µm). In the former case, moderate blood coagulation was observed after 30 min. The coagulant activity seemed to be slightly weaker than the chitosan surface of the assembly. It is important to note that chitosan is not dissolved in an aqueous phase at physiological pH, and thus a detailed analysis of blood coagulation will require careful technique. In the latter case, strong blood coagulation was observed after 30 min, thus indicating that Dex was dissolved in the blood. Conclusion. The coagulation of human blood on a layerby-layer assembly constructed of chitosan and Dex was analyzed in terms of the outermost surface layer of the assembly and in the presence of NaCl in the polymer solution. Alternating bioactivity was observed, for the first
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time, on the assembly prepared in the presence of NaCl. The introduction of anti- vs procoagulant activity on a polymer surface is important for the modification of the surface characteristics in biomedical research. We showed that the assembly was effective for this purpose. Studies on the critical salt concentrations in the polymer solution against their effect on coagulation activity still remain to be performed. To discuss the mechanism responsible for this effect, platelet adhesion or the activation of coagulant factors should be studied in detail. This layer-by-layer assembly protocol will be applied to other in vitro or in vivo systems, such as alternating cell adhesion activity, in the near future. Acknowledgment. The authors would like to acknowledge Dr. A. Kishida (National Cardiovascular Center, Japan) for helpful discussions and Med. Dr. Y. Maeda (Kagoshima University, Japan) for his technical support. This work was financially supported in part by Grant-in-Aid for Scientific Researches in the Priority Area of “Molecular Synchronization for Design of New Materials System” (No. 404/ 11167270) from the Ministry of Education, Science, Sports, and Culture, Japan. References and Notes (1) (a) Decher, G.; Hong, J.-D. Makromol. Chem., Macromol. Symp. 1991, 46, 321. (b) Decher, G.; Hong, J.-D. Ber. Bunsen-Ges. Phys. Chem. 1991, 95, 1430. (c) Decher, G. Compr. Supramol. Chem. 1996, 9, 507. (d) Decher, G. Science 1997, 277, 1232. (2) Stockton, W. B.; Rubner, M. F. Macromolecules 1997, 30, 2717. (3) (a) Shimazaki, Y.; Mitsuishi, M.; Ito, S.; Yamamoto, M. Langmuir 1997, 13, 1385. (b) Shimazaki, Y.; Mitsuishi, M.; Ito, S.; Yamamoto, M. Langmuir 1998, 14, 2768. (c) Shimazaki, Y.; Mitsuishi, M.; Ito, S.; Yamamoto, M. Macromolecules 1999, 32, 8220. (4) (a) Serizawa, T.; Hamada, K.; Kitayama, T.; Fujimoto, N.; Hatada, K.; Akashi, M. J. Am. Chem. Soc. 2000, 9, 1891. (b) Serizawa, T.; Hamada, K.; Kitayama, T.; Katsukawa, K.; Hatada, K.; Akashi, M. Langmuir, in press. (5) (a) Serizawa, T.; Yamamoto, K.; Akashi, M. Langmuir 1999, 15, 4682. (b) Serizawa, T.; Hashiguchi, S.; Akashi, M. Langmuir 1999, 15, 5363. (c) Serizawa, T.; Akashi, M. J. Polym. Sci., Part A: Polym. Chem. 1999, 37, 1903. (6) Elbert, D. L.; Herbert, C. B.; Hubbell, J. A. Langmuir 1999, 15, 5355. (7) (a) Akashi, M.; Sakamoto, N.; Suzuki, K.; Kishida, A. Bioconjugate Chem. 1996, 7, 393. (b) Sakamoto, N.; Shioya, T.; Serizawa, T.; Akashi, M. Bioconjugate Chem. 1999, 10, 538. (8) Muzzarelli, R. A. A. Chitin; Pergamon Press: Oxford, England, 1997. (9) Sauerbrey, G. Z. Phys. 1959, 155, 206. (10) Lvov, Y.; Ichinose, I.; Ariga, K.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117. (11) Sakamoto, N.; Shioya, T.; Kishida, A.; Akashi, M. Macromol. Symp. 1997, 120, 159. (12) (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., in press. (13) (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., in press. (14) Yoo, D.; Shiratori, S. S.; Rubner, M. F. Macromolecules 1998, 31, 4309. (15) Farhat, T.; Yassin, G.; Dubas, S. T.; Schlenoff, J. B. Langmuir 1999, 15, 6621.
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