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Two Dimensional Auto-Organized Nanostructure Formation of Hyaluronate on Bovine Serum Albumin Monolayer and Its Surface Tension Tsuyoshi Nonogaki, Shouhong Xu, Shin-ichi Kugimiya, Shizuko Sato, Isamu Miyata, and Masakatsu Yonese* Faculty of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori Mizuho-ku Nagoya 467, Japan Received April 12, 1999. In Final Form: January 31, 2000 The layer-by-layer interaction between bovine serum albumin BSA and sodium hyaluronate NaHA was studied by a quartz crystal microbalance QCM method. The surface structures of the BSA layer and the NaHA layer and their surface tensions were investigated using atomic force microscopy AFM. BSA showed Langmuir type adsorption on poly(γ-methyl-L-glutamate) PMLG thin film and were found to be a monolayer in the saturated adsorption state. The adsorption of NaHA on the BSA monolayer was also the Langmuir type. From their linear reciprocal plots, adsorption constants K and saturated adsorption masses Γ∞ were determined. The BSA molecules on the PMLG film surface could be imaged using AFM in the monolayer state. Furthermore, the surface structure of NaHA adsorbed on the BSA monolayer was found to form hexagonal-like networks. From the adhesion force Fad between the AFM tip and the surfaces of the BSA layers or the NaHA layers, their surface tensions γs were estimated. The surface tension of the PMLG films increased with increasing the adsorption of BSA but the surface tension of the saturated adsorption BSA layer was found to decrease with increasing the adsorption of NaHA.
Introduction A protein and an acid polysaccharide interact electrostatically and form soluble and/or insoluble complexes depending on the pH and the ionic strength of solutions. The kinds of the charged groups of acid polysaccharides and the combination affect their characteristics, such as counterion bindings1 and complex formation with a protein.2 The interaction between a protein and a polyelectrolyte and its macrostructure in solutions have been studied widely using a static light scattering, SLS, a dynamic light scattering, DLS, and an electrophoretic light scattering, ELS.3,4 Recently, layer-by-layer interactions between polyelectrolytes have attracted special interests pursuing to elucidate the interfacial functions and have been studied using the L-B method,5,6 a surface plasmon resonance method7,8 and a quartz crystal microbalance, QCM, method.9,10 QCM is a useful quantitative technique to provide nanogram mass changes. In this paper, the formation of bovine serum albumin BSA monolayer on poly(R-amino acid):poly(γ-methyl-L-
glutamate) PMLG thin film11 and the layer-by-layer interaction between BSA and sodium hyaluronate NaHA were studied by the QCM method. Furthermore, the surface structures of the BSA layer and the NaHA layer were investigated using atomic force microscopy, AFM. AFM has been employed to examine the topography of sample surfaces with atomic or molecular resolution. Tapping-mode AFM has the advantages of imaging soft biological molecules and live cells directly without using any complicated chemical method to immobilize samples onto the substrate.12-15 AFM has been also a useful tool in studying surface interactions, and a great deal of work has been carried out on theoretical and experimental issues.16,17 AFM is able to obtain force (>1 pN)-distance (lateral, >1 Å; vertical, >0.1 Å) curves from surfaces with high resolution.18 The BSA molecules adsorbed on the PMLG film and the surface structures of NaHA adsorbed on the BSA monolayer were imaged using AFM. Furthermore, from the adhesion force Fad between the AFM tip and the surfaces of the BSA layers or the NaHA layers, their surface tensions γs were estimated. Experimental Section
(1) Yonese, M.; Tsuge, H.; Kishimoto, H. J. Phys. Chem. 1987, 91, 1971. (2) Yonese, M.; Yano, M.; Kishimoto, H. Bull Chem. Soc. Jpn. 1991, 64, 1814. (3) Yonese, M.; Xu, S. H.; Kugimiya, S.; Sato S.; Miyata, I. Prog. Colloid Polym. Sci. 1997, 106, 252. (4) Cheng, J. Y.; van de Wetering, P.; Talsma, H.; Crommelin, D. J.; Hennink, W. E. Pharm. Res. 1996, 13, 1038. (5) Ge, S.; Kojio, K.; Takahara, A.; Kajiyama, T. J. Biomater. Sci. Polym. Ed. 1998, 9 (2), 131. (6) Esker, A. R.; Mengel, C.; Wegner, G. Science, 1998, 280, 892. (7) Yamamoto, S.; Tsujii, Y.; Yamada, K.; Fukuda, T.; Miyamoto, T. Langmuir 1996, 12, 3671. (8) Green, R. J.; Davies, J.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. Biomaterials 1997, 18, 8, 405. (9) Niikura, K.; Nagata, K.; Okahata, Y. Chem. Lett. 1996, 863 (10) Ariga, K.; Lvov, Y.; Kunitake, T. J. Am. Chem. Soc. 1997, 119, 2224. (11) Hara, M.; Higuchi, M.; Minoura, N.; Ohuchi, S.; Cho, C. S.; Akaike, T.; Higuchi, A. Nippon Kagaku Kaishi 1996, 5, 483.
Materials. Bovine serum albumin, BSA, was purified according to R. F. Chen by delipidizing a BSA Fraction V (Seikagaku Kougyou); i.e., (1) the BSA solution (10 w/v%) was prepared and an activated carbon (5 g, Darco Ci. Ltd., G-60) was added to 100 (12) You, H. X.; Yu, L. Biophys. J. 1997, 73, 3299. (13) Drake, B.; Prater, C. B.; Weisenhorn, A. L.; Gould, S. A. C.; Albrecht, T. R.; Quate, C. F.; Cannell, D. S.; Hansma, H. G.; Hansma, P. K. Science 1989, 243, 1586. (14) Radmacher, M.; Tillmann, R. W.; Fritz, M.; Gaub, H. E. Science 1992, 257, 1900. (15) Henderson, E.; Haydon, P. G.; Sakaguchi, D. S. Science 1992, 1944. (16) Boland, T.; Ratner, B. D. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 5297. (17) Dammer, U.; Popescu, O.; Wagner, P.; Anselmetti, D.; Guntherodt, H. J.; Misevic, G. N. Science 1995, 267, 1173. (18) Cappella, B.; Baschieri, P.; Frediani, C.; Miccoli, P.; Ascoli, C. IEEE Eng. Med. Biol. Mag. 1997, 58.
10.1021/la990444c CCC: $19.00 © 2000 American Chemical Society Published on Web 04/07/2000
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Figure 1. Time course of resonance frequency of PMLG tip in water. PMLG tip: quartz crystal microbalance (QCM) tip covered by PMLG layer.
a
Figure 2. Time courses of resonance frequency changes of QCM with BSA adsorption to the PMLG cast film: 9, 0.1 × 10-6 kg dm-3 BSA solution; b, 1.0 × 10-6 kg dm-3 BSA solution; 2, 10 × 10-6 kg dm-3 BSA solution. cm3 of the BSA solution. (2) After the solution was adjusted to pH ) 3, the solution was stirred in an ice bath for 2 h and centrifuged under 24000g for 30 m. (3) The supernatant was filtered and adjusted at pH ) 5 by adding NaOH (0.2 mol dm-3). (4) Then, the solution was passed through a mixed column (cationexchange resin:anion-exchange resin ) 1:1) and then freezedried. Cation and anion-exchange resins were of Amberlight IR120B and IRA-400 (Organo Co.). Weight-average molecular weight Mw was determined to be 70 000 using the static light scattering (Otsuka Electronics Co., DLS-700Ar). Sodium hyaluronate NaHA (Seikagaku Kougyou, MW ) 850 000) and poly(γ-methyl-L-glutamate) PMLG (SIGMA Co., MW ) 307 000) were of a commercial origin and were used without any purification. Casting solution on the quartz crystal disk tip was prepared by adding 10 mg of PMLG to 10 mL of 1,2-dichloroethane (Wako Co., the special grade). β-Sheet PMLGs do not dissolve in 1,2dichloroethane.11 So the PMLG solution was used, after filtrating it through the filter (pore size 0.5 µm Millipore Co.), as the casting solution. Other reagents were all special grade (Katayama Co.) and water was distilled and deionized. Methods. Preparation of PMLG Cast Film on Quartz Crystal Disk Tip. PMLG cast film was prepared by spreading of the casting solution (2 µL) on the quartz crystal microbalance QCM (USI System Co.) disk tip on which Au is covered by evaporation. The tip is denoted by PMLG tip. After drying the tip under conditions of room temperature and atmospheric pressure, the adsorbed amounts of PMLG on the tip were determined from the deviations of the resonance frequency of QCM as described below. Measurement of Adsorption of BSA on PMLG Cast Film by QCM. The adsorbed amounts of BSA on the PMLG cast film
b
Figure 3. (a) Adsorption behavior of BSA to the PMLG cast film: CBSA, concentration of BSA; ΓBSA, adsorption mass of BSA. (b) Reciprocal plot of CBSA and ΓBSA. were measured by immersing the PMLG tip into various concentrations of BSA solutions (CBSA/10-6 kg dm-3 ) 0.1, 0.3, 0.5, 1.0, 3.0, 5.0, 10), and the time courses of the adsorption of BSA were obtained from the decrease of the resonance frequency of QCM. BSA solutions were gently stirred in order to avoid the effect of diffusion as much as possible in the condition without losing the stability of the QCM frequency at pH ) 6.8 and 25 °C. Measurement of Adsorption of NaHA on Saturated Adsorption Film of BSA Using QCM. The adsorbed amounts of NaHA on the saturated adsorption film of BSA were measured by immersing the saturated adsorption film of BSA on the PMLG tip into various concentrations of NaHA solutions (CHA/10-6 kg dm-3 ) 0.1, 0.3, 0.5, 1.0, 3.0, 5.0, 10), and the time courses of the adsorption of NaHA were determined from the decrease of the resonance frequency of QCM. NaHA solutions were gently stirred as in the cases of the adsorption of BSA.
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Table 1. Adsorption Characteristics of BSA on PMLG Cast Film and NaHA on BSA Monolayer adsorption const (K)/106 dm3 kg-1 satd adsorption mass (Γ∞)/10-6 kg m-2 acid polysaccharide/BSA repeating units/BSA electrolytic dissociation groups/BSA
BSA
NaHA
1.2 2.7
3.5 2.9 1/10 232 232
Analysis of Adsorbed Amounts of PMLG, BSA, and NaHA Using QCM. Adsorbed amounts of PMLG, BSA and NaHA were obtained using QCM, whose resonance frequency is 9 MHz. The diameter of its quartz crystal disk tip is 4 mm, and Au electrodes are deposited on both sides. The QCM was connected to an oscillator circuit to drive the quartz at its resonance frequency. The frequency changes were measured using a frequency counter attached to a personal computer system. According to Sauerbrey’s eq 1,19,20 a frequency decrease of 1 Hz corresponded to a mass increase of 1 ng on the QCM electrodes.
∆F )
2F02∆m
xµqFqA
b
(1)
Here ∆F is the frequency change (Hz), F0 the fundamental frequency of the QCM (9 × 106 Hz), µq the shear modulus of quartz (2.95 × 1011 dyn cm-2), Fq the density of quartz (2.65 g cm- 3), and A the electrode area (0.13 cm2). Observation of Surface Structures Using AFM. Surface structures of PMLG cast film and the adsorption layer of BSA and NaHA were observed using AFM (NanoscopeIII, Digital Instruments Co.). After the samples of PMLG cast film and the adsorption layer of BSA and NaHA on the QCM tip under conditions of room temperature and atmospheric pressure were dried, the surface structures were observed by a tapping mode and a phase-difference mode. An AFM cantilever was made of Si monocrystal (the radius of curvature of probe: 10 nm) and was used without any surface treatment. Estimation of Surface Tension Using Force Mode of AFM. Adhesion force Fad between the AFM tip and sample surface can be given directly from force-distance curves using force mode AFM. The value of Fad equals the pull-off force, i.e., the product of jump-off-contact cantilever deflection. Fad can be related to the surface tension of AFM tip (γt) and sample surface (γs) by the Derjaguin-Muller-Toporov (DMT) theory taking LennardJones interactions into account.18,21,22 When the tip-sample system is sketched as a sphere on a flat surface, Fad is given by eq 2. R is tip radius. The values of γs were estimated from the
Fad ) -4πRxγtγs
a
(2)
experimental results of Fad using γt to be 1044.75 mN m-1 which is the surface tension of Si3N4 probe in hydrogen gas. Then, the values of γs are relative ones.
Results PMLG Film on the Quartz Crystal Disk Tip. PMLG of R-helix type can dissolve in 1,2-dichloroethane.11 PMLG solution (2 µL, 10 mg of PMLG in 10 mL of 1,2dichloroethane) after filtrating was used as the casting solution and cast on the quartz crystal disk tip covered by Au (area: 1.3 × 10-1 cm2) without the leak from the edges. As the β-sheet type of PMLG was removed by filtration, only the R-helix type is considered to cover the Au electrode surface. After the cast solution was dried under conditions of room temperature and atmospheric (19) Okahata, Y.; Kimura, K.; Ariga, K. J. Am. Chem. Soc. 1989, 111, 9190. (20) Masuda, H.; Baba, N. Surface 1990, 28, 222. (21) Derjaguin, B. V.; Muller, V. M.; Toporov, Y. P. J. Colloid Interface Sci. 1974, 53, 314. (22) Blackman, G. S.; Mate, C. M.; Philpott, M. R. Phys. Rev. Lett. 1990, 65, 2270.
Figure 4. (a) Adsorption behavior of NaHA to BSA monolayer: CHA, concentration of NaHA; ΓHA, adsorption mass of NaHA. (b) Reciprocal plot of CHA and ΓHA.
pressure, the average mass of PMLG on both sides of the tip was determined to be 1.6 µg (5.2 × 10-12 mol) from the decreases of the resonance frequency of QCM. The tip is denoted by PMLG tip. The PMLG tip was immersed in water and the time course of the resonance frequency F was measured at 25 °C. As shown in Figure 1, the values of F were almost constant. Before and after immersion of PMLG tip in water, the resonance frequencies of QCM were constant beyond 10 min. Then, after immersion of the PMLG tip in water for 2 h, the adsorptions of BSA were measured. Adsorption of BSA on the PMLG Tip. Adsorption mass of BSA on the PMLG surface was measured by immersing the PMLG tip into BSA solutions. As shown in Figure 2, increasing the adsorption of BSA on the PMLG surface, the frequencies of the tip decreased and attained constant values in the range 10-30 min depending on the concentrations of BSA, CBSA. Equilibrium adsorption masses Γ were obtained from the decreases of the frequencies ∆F at equilibrium states, and the results were shown in Figure 3a as a function of CBSA. The adsorption was found to be Langmuir type. Langmuir adsorption equation is expressed by eq 3 and the reciprocal plot is expressed by eq 4, where Γ is adsorption mass, K
Γ)
Γ∞KC 1 + KC
1 1 1 ) + Γ Γ∞KC Γ∞
(3) (4)
represents adsorption constants, Γ∞ is the saturation adsorption mass, and C is the concentration of solution.
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Figure 5. AFM image of Au surface on QCM tip.
Figure 6. AFM image of surface of PMLG cast film (2 µm × 2 µm).
The reciprocal plot was linear as shown in Figure 3b. The values of K and Γ∞ are summarized in Table 1. The shape of a BSA molecule is reported to be an ellipsoid having a cross section of 4.1 × 14.1 nm2 by X-ray structure analysis.23 Assuming BSA molecules adsorb on the surface of PMLG cast film side-on and in a monolayer state, the (23) Peters, T. J. Adv. Protein Chem. 1985, 37, 161.
saturated adsorption mass should be 2.56 × 10-6 kg m-2. As the result of the saturation adsorption mass (2.7 × 10-6 kg m-2) was almost equal to the calculated one, the immobilized BSA molecules were confirmed to adsorb on the PMLG surface in the monolayer state. Adsorption of NaHA on the BSA Monolayer. The adsorbed amounts of NaHA on the saturated adsorption film of BSA were measured by immersing the tip covered
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Figure 7. AFM image of surface of BSA monolayer (2 µm × 2 µm) An arrow shows a BSA molecule.
Figure 8. AFM image of surface of NaHA layer (2 µm × 2 µm).
by the saturated BSA monolayer (BSA tip) into NaHA solutions (pH ) 6.8). The time courses of the adsorption of NaHA were determined from the decrease of the resonance frequency of the tip and the equilibrium adsorptions were attained in the region of 30-120 min as well as the adsorption of BSA to the PMLG surface. The equilibrium adsorption masses of NaHA on the BSA tip are shown in Figure 4(a) as a function of the concentrations of NaHA CNaHA. The adsorption of NaHA
was found to be the Langmuir adsorption isotherm. The adsorption constants K and the saturation adsorption mass Γ∞ obtained from the reciprocal plot (Figure 4b) were summarized in Table 1 with the results of BSA adsorbed on the PMLG film. The values of the molecular numbers and the repeating units of NaHA adsorbed on a BSA molecule in the saturated monolayer state are also shown in Table 1. The result shows one NaHA molecule adsorbs on 10 BSA molecules.
Nanostructure of Hyaluronate on BSA Monolayer
Figure 9. Force-distance curves of PMLG cast film (0), BSA monolayer (O), and NaHA layer (4).
Analysis of Membrane Surface Structure Using AFM. AFM Image of the PMLG Cast Film and the BSA Monolayer. Surface structures of PMLG cast film on the Au-evaporated layer of the QCLM tip were observed using AFM (NanoscopeIII, Digital Instruments Co.) by a tapping mode and a phase-difference mode. Furthermore, the surface structures in the saturated adsorption of the BSA monolayer on the PMLG film and the NaHA monolayer on the BSA layer were observed in the same manner. Figure 5 shows the surface structure of Au layer as the basis. Au particles in the range of 40-100 nm were found to arrange closely with about 10 nm differences in the height. After the samples of PMLG cast film and the saturated adsorption monolayer of BSA on the PMLG film were dried, their AFM images were observed and are shown in Figures 6 and 7 on the same scale as in Figure 5. Smooth unevenness was found in macrostructure of PMLG film and is considered to result from the cast method. The AFM images of the saturated adsorption monolayer of BSA on the PMLG cast film are shown in Figure 7, and an individual BSA molecule was observed as an ellipsoid shown by an arrow. The surface coverage of BSA molecules was relatively low. The mean diameter of the BSA molecule was 38.3 nm as a corresponding sphere, and the height was 6.4 nm. These values were larger than those of X-ray structure analysis, 14.1 × 4.1 nm. The convolution effects of the tip24 are considered to be one factor for the difference. Another factor could result from the association of BSA such as dimer, trimer, et al. BSA molecules are considered to associate during the drying process of the sample. AFM Image of the Saturated Adsorption Layer of NaHA. Surface structure of the saturated NaHA adsorption layer on the saturated BSA adsorption monolayer was observed using AFM after drying the samples in the same manner as in the case of the BSA. As shown in Figure 8, a network structure was found to spread over the BSA monolayer that was almost a hexagon in shape. The average mesh size was 153.4 nm. The average dimension of the strand was 29.4 nm in width and 3.5 nm in height. The strand was wide and should be formed by the cluster of HA molecules.25,26,27 BSA molecules could be seen as particles under the NaHA network and they were found to spread uniformly. (24) Hansma, H. G. Biophys. J. 1995, 68, 3. (25) Mikelsaar, R. H.; Scott, J. E. J. Glycoconj. 1994, 11, 65. (26) Scott, J. E.; Cummings, C.; Brass, A.; Chen, Y. Biochem. J. 1991, 274, 699. (27) Hadler, N. M.; Dourmashikin, R. R.; Nermut, M. V.; Williams, L. D. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 307.
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Figure 10. Changes of surface excess free energy of PMLG cast film with BSA adsorption: b, γPMLG (surface excess free energy of PMLG cast film); O, ΓBSA(adsorption mass of BSA). CBSA: concentration of BSA.
Figure 11. Changes of surface excess free energy of BSA monolayer with NaHA adsorption: b, γBSA (surface excess free energy of BSA monolayer); O, ΓHA (adsorption mass of NaHA). CHA: concentration of NaHA.
Surface Tension of Membranes. Adhesion forces Fad between the AFM tip and the surfaces of the BSA layer or the NaHA layer were obtained from the force-distance curves using the force mode of AFM. As typical results, the force-distance curves of the BSA and the NaHA layers in the saturated adsorption states are shown in Figure 9. The value of Fad equals the pull-off force, i.e., the product of the jump-off-contact cantilever deflection and the shear modulus of cantilever. From the values of Fad, the surface tensions of the BSA and the NaHA layer γs were obtained by eq 2, assuming the tip-sample system is a sphere on a flat surface. As shown in Figure 10, the surface tension of the PMLG film was found to increase with increasing adsorption mass of BSA. On the other hand, as shown in Figure 11, the surface tension of the saturated adsorption BSA monolayer was found to decrease with increasing adsorption of NaHA. Discussion Adsorption Mechanism of BSA on the PMLG Layer. The PMLG sample is almost R-helix type but contains slightly the β-sheet type. By filtration of the PMLG solution dissolved in 1,2-dichloroethane, the β-sheet type was removed from the casting solution. Then, the R-helix type thin film was formed on the Au surface of the QCM tip by hydrophobic interaction. On the PMLG surface, BSA molecules were adsorbed according to the Langmuir type adsorption. BSA molecule is reported to compose of about 70% R-helix and slight β-sheet struc-
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Figure 12. Schematic representation of formation of networks as NaHA adsorption.
tures.28 Then, in the interaction between BSA molecules and the PMLG cast film, the helix-helix interaction11 makes a primary roll, and the adsorption monolayer of BSA molecules is considered to be formed on the PMLG surface. Formation of Network Structure as Adsorption of NaHA on the BSA Monolayer. NaHA molecules adsorbed on the BSA monolayer form the network structure as shown in Figure 8. The NaHA solutions were almost pH ) 6.8 at which their carboxylic groups were in the completely dissociated state. BSA molecules at pH ) 6.8 were slightly negative in the charge amounts as isoelectric point was pH ) 5.2.3 Using the results of pH (28) Reed, R. G.; Feldhoff, R. C.; Clute, O. L.; Peters, T. J. Biochemistry 1975, 14, 4578.
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titration of NaHA and BSA solutions, the charge amounts of NaHA and BSA per 1 mol were -2.93 × 10-9 and -3.86 × 10-9 mol at pH ) 6.8. NaHA molecules interact with the outer surfaces of BSA molecules. Although the charge amounts of the outer surfaces could not be determined from our experimental results, the charge amounts should be negative. Then, NaHA molecules do not interact electrostatically with the BSA monolayer primarily, but interact hydrophobically and/or sterically. NaHA molecules are reported to form a double helical structure due to the hydrophobic interaction, and the diameter is observed to be 2 nm.27 The dimension of the strands of the NaHA network was 30 nm in width and 3.5 nm in height on average. Then, the strands are considered composed of the aggregation of 15 NaHA double helixes in width and mono or double layers of the helixes in height. The model of the NaHA network structure on the BSA monolayer is shown in Figure 12. The surface tension of the saturated adsorption BSA monolayer decreased with increasing adsorption of NaHA as shown in Figure 11. With increasing adsorption of NaHA, the surface charge amounts increase negatively. Then, the decreases of γs might result from the increase of the surface charge amounts. The precise details should be studied further. LA990444C
