pubs.acs.org/Langmuir © 2009 American Chemical Society
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Effect of Surface Morphology and Charge on the Amount and Conformation of Fibrinogen Adsorbed onto Alginate/Chitosan Microcapsules Hong G. Xie,†,‡ Jia N. Zheng,†,‡ Xiao X. Li,†,‡ Xiu D. Liu,§ Jing Zhu,† Feng Wang, Wei Y. Xie,† and Xiao J. Ma*,† †
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Laboratory of Biomedical Material Engineering, Biotechnology Division, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, People’s Republic of China, ‡Graduate School of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China, §College of Environment and Chemical Engineering, Dalian University, Dalian Economic Technological Development Zone, Dalian 116622, People’s Republic of China, and Laboratory of Optical Fabrication and Coating, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, People’s Republic of China Received October 12, 2009. Revised Manuscript Received November 2, 2009
We report the influence of surface morphology and charge of alginate/chitosan (ACA) microcapsules on both the amount of adsorbed protein and its secondary structural changes during adsorption. Variations in surface morphology and charge were controlled by varying alginate molecular weight and chitosan concentration. Plasma fibrinogen (Fgn) was chosen to model this adsorption to foreign surfaces. The surface of ACA microcapsules exhibited a granular structure after incubating calcium alginate beads with chitosan solution to form membranes. The surface roughness of ACA microcapsule membranes decreased with decreasing alginate molecular weight and chitosan concentration. Zeta potential measurements showed that there was a net negative charge on the surface of ACA microcapsules which decreased with decreasing alginate molecular weight and chitosan concentration. The increase in both surface roughness and zeta potential resulted in an increase in the amount of Fgn adsorbed. Moreover, the higher the zeta potential was, the stronger the protein-surface interaction between fibrinogen and ACA microcapsules was. More protein molecules adsorbed spread and had a greater conformational change on rougher surfaces for more surfaces being available for protein to attach.
1. Introduction Protein adsorption from a biological environment onto implanted biomaterials is recognized as the first event when integrating an implanted device into living tissue. The adsorbed proteins then determine the subsequent cellular interactions, which can significantly impact the performance of biomaterials in a biological environment. Recent studies have suggested that the preservation of the native secondary structure of protein adsorbed is important for biological applications.1-4 For example, proteins adsorbed on films of low-density polyethylene, isotactic polypropylene,5 and poly(2-hydroxyethylmethacrylate)6 in their native conformational state would not induce platelet adhesion. In order to manipulate protein adsorption and design biocompatible materials, the mechanisms underlying protein-surface interactions, especially how surface properties of materials induce conformational changes of adsorbed proteins, need to be well understood. In fact, protein adsorption is a complex process, and it is influenced by various factors such as the circumstances that the materials are facing, surface properties of materials, and so on. *To whom correspondence should be addressed: E-mail:
[email protected]. Telephone: 86-411-84379139. Fax: 86-411-84379096. (1) Steiner, G.; Tunc, S.; Maitz, M.; Salzer, R. Anal. Chem. 2007, 79(4), 1311– 1316. (2) Sivaraman, B.; Fears, K. P.; Latour, R. A. Langmuir 2009, 25(5), 3050–3056. (3) Clarke, M. L.; Wang, J.; Chen, Z. J. Phys. Chem. B 2005, 109(46), 22027– 22035. (4) Schwinte, P.; Ball, V.; Szalontai, B.; Haikel, Y.; Vogel, J. C.; Schaaf, P. Biomacromolecules 2002, 3(6), 1135–1143. (5) Hylton, D. M.; Shalaby, S. W.; Latour, R. A. J. Biomed. Mater. Res., Part A 2005, 73(3), 349–358. (6) Tanaka, M.; Motomura, T.; Kawada, M.; Anzai, T.; Kasori, Y.; Shiroya, T.; Shimura, K.; Onishi, M.; Mochizuki, A. Biomaterials 2000, 21(14), 1471–1481.
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Environmental parameters such as ionic strength and pH have great effects on the surface charge and free energy of materials, which in turn affect the amount of protein adsorbed.7-14 Recently, the effect of surface morphology on protein adsorption was also studied experimentally and theoretically.15-20 Some studies15,16 showed that the amount of adsorbed protein was independent of surface roughness. It is widely believed that a greater surface roughness increases the effective surface area if the indentation is larger than the protein.18 Thus, more surfaces are available for protein attachment through nonspecific interactions. However, previous studies have focused mainly on surfaces with a regular (7) Lvov, Y.; Decher, G.; Sukhorukov, G. Macromolecules 1993, 26(20), 5396– 5399. (8) Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Langmuir 1997, 13(13), 3427–3433. (9) Ladam, G.; Schaaf, P.; Decher, G.; Voegel, J. C.; Cuisinier, F. J. G. Biomol. Eng. 2002, 19, 273–280. (10) Gergely, C.; Bahi, S.; Szalontai, B.; Flores, H.; Schaaf, P.; Voegel, J. C.; Cuisinier, F. J. G. Langmuir 2004, 20(13), 5575–5582. (11) Noh, H.; Vogler, E. A. Biomaterials 2006, 27(34), 5780–5793. (12) Xu, L. C.; Siedlecki, C. A. Biomaterials 2007, 28(22), 3273–3283. (13) Hoven, V. P.; Tangpasuthadol, V.; Angkitpaiboon, Y.; Vallapa, N.; Kiatkamjornwong, S. Carbohydr. Polym. 2007, 68(1), 44–53. (14) Pasche, S.; V€or€os, J.; Griesser, H. J.; Spencer, N. D.; Textor, M. J. Phys. Chem. B 2005, 109(37), 17545–17552. (15) Han, M.; Sethuraman, A.; Kane, R. S.; Belfort, G. Langmuir 2003, 19(23), 9868–9872. (16) Cai, K.; Bossert, J.; Jandt, K. D. Colloids Surf., B 2006, 49(2), 136–144. (17) Rechendorff, K.; Hovgaard, M. B.; Foss, M.; Zhdanov, V. P.; Besenbacher, F. Langmuir 2006, 22(26), 10885–10888. (18) Blanco, E. M.; Horton, M. A.; Mesquida, P. Langmuir 2008, 24(6), 2284– 2287. (19) Toscano, A.; Santore, M. M. Langmuir 2006, 22(6), 2588–2597. (20) Hovgaard, M. B.; Rechendorff, K.; Chevallier, J.; Foss, M.; Besenbacher, F. J. Phys. Chem. B 2008, 112(28), 8241–8249. (21) Vertegel, A. A.; Siegel, R. W.; Dordick, J. S. Langmuir 2004, 20(16), 6800– 6807.
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surface morphology, for example, silica beads of various sizes,21 nanopyramids,22 or regular patterns,23 leaving the understanding of how random surface roughness affects protein less explored. Furthermore, how surface morphology and surface roughness affect the conformation state of the adsorbed proteins is poorly understood. It has been demonstrated that a polyanion/polycation complex film is a classic membrane with random surface morphology.24 The surface roughness of the film could be controlled easily by changing the complex composition. The membrane of alginate/ chitosan (ACA) microcapsules is just a polyelectrolyte complex membrane composed of alginate [copolymer of (1f4) linked R-Lguluronic acid (G block) and β-D-mannuronic acid (M block)] and chitosan [polymeric amine composed of randomly distributed β-(1-4)-linked D-glucosamine and N-acetyl-D-glucosamine].25,26 ACA microcapsules have been widely investigated for applications such as cell transplantation, drug delivery systems, and biosensors for the nonimmunogenic response properties of both alginate and chitosan.27-30 Studies have been carried out to characterize their surface chemical properties,26 which suggested that ACA membranes made of chitosan and alginates with different composition and molecular weight had similar wettability but different surface charge, while the membrane surface morphology of ACA microcapsules and its effect on protein adsorption are still unclear. Because plasma fibrinogen (Fgn) is the most relevant protein in the body that adsorbs to foreign materials, it was selected for this study. Fgn takes part in blood coagulation and facilitates adhesion as well as aggregation of platelets, which are very important properties in the processes of both homeostasis and thrombosis.31 Human plasma Fgn is a 340 kDa diametric protein with dimensions of about 5.0 5.0 47.0 nm3.32,33 In this study, we investigated the influence of alginate molecular weight and chitosan concentration on the surface morphology and surface charge of alginate/chitosan microcapsules. Then, we characterized the amount of adsorbed protein and its secondary structural changes during adsorption on ACA microcapsules to analyze the mechanisms underlying protein-surface interactions. The membranes were characterized via scanning electron microscope (SEM) measurements, white-light profilometric measurements, and zeta potential measurements. The secondary structures of Fgn adsorbed were evaluated by using diffuse reflectance Fourier transform infrared (FTIR) spectrometry. (22) M€uller, B.; Riedel, M.; Michel, R.; De Paul, S. M.; Hofer, R.; Heger, D.; Gr€utzmacher, D. J. Vac. Sci. Technol., B 2001, 19(5), 1715–1720. (23) Galli, C.; Coen, M. C.; Hauert, R.; Katanaev, V. L.; Gr€oning, P.; Schlapbach, L. Colloids Surf., B 2002, 26(3), 255–267. (24) Quinn, A.; Tjipto, E.; Yu, A.; Gengenbach, T. R.; Caruso, F. Langmuir 2007, 23(9), 4944–4949. (25) Liu, X. D.; Xue, W. M.; Liu, Q.; Yu, W. T.; Fu, Y. L.; Xiong, X.; Ma, X. J.; Yuan, Q. Carbohydr. Polym. 2004, 56(4), 459–464. (26) Xie, H. G.; Li, X. X.; Lv, G. J.; Xie, W. Y.; Zhu, J.; Luxbacher, T.; Ma, R.; Ma, X. J. J. Biomed. Mater. Res., Part A, published online April 7, http://dx.doi.org/ 10.1002/jbm.a.32437. (27) Sun, L.; Zhang, Hn.; Wang, W.; Ma, X. J. Chin. J. Clin. Rehabil. 2005, 9 (30), 87–89. (28) Ramadas, M.; Paul, W.; Dileep, K. J.; Anitha, Y.; Sharma, C. P. J. Microencapsulation 2000, 17(4), 405–411. (29) Qi, W. T.; Ma, J.; Yu, W. T.; Xie, Y. B.; Wang, W.; Ma, X. J. Enzyme Microb. Technol. 2006, 38(5), 697–704. (30) Lawrie, G.; Keen, I.; Drew, B.; Chandler-Temple, A.; Rintoul, L.; Fredericks, P.; Groendahl, L. Biomacromolecules 2007, 8(8), 2533–2541. (31) Cacciafesta, P.; Humphris, A. D. L.; Jandt, K.; Miles, M. J. Langmuir 2000, 16(17), 8167–8175. (32) Sigal, G. B.; Mrksich, M.; Whitesides, G. M. J. Am. Chem. Soc. 1998, 120 (14), 3464–3473. (33) Hall, C. E.; Slayter, H. S. J. Biophys. Biochem. Cytol. 1959, 5(1), 11–16.
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Xie et al. Table 1. Molecular Weight, Chemical Composition, and Sequence Parameters of the Used Alginatesa alginates
FG
FGG
FMG
FMM
MW (kDa)
sample 1 0.34 0.17 0.17 0.50 270 sample 2 0.34 0.17 0.16 0.50 430 sample 3 0.35 0.19 0.17 0.48 490 sample 4 0.36 0.19 0.17 0.48 590 a FG, FGG, FMG, and FMM are the fraction of G blocks, GG blocks, MG blocks, and MM blocks, respectively.
2. Materials and Methods 2.1. Materials. Chitosan with molecular weight of 50 kDa and deacetylation degree of 96-98% was modified from raw material (Yuhuan Chemical Plant, Zhejiang, China) in our laboratory. Sodium alginates were purchased from Qingdao Crystal Salt Bioscience and Technology Corporation (Qingdao, China). All sodium alginate samples were purified using a protocol originally published by Kl€ ock et al.34 The compositions of the alginates were characterized by 1H NMR based on data published by Grasdalen et al.35,36 Table 1 shows data obtained for the alginate samples. Bovine plasma fibrinogen was purchased from Sigma Aldrich Co., and KBr (spectral purity) was purchased from Tianjin Guangfu Fine Chemical Research Institute. All other chemicals used were of analytical grade reagent or the best available quality, and double distilled water was used throughout the experiments. 2.2. Preparation of ACA Microcapsules. ACA microcapsules were prepared according to the method developed in our lab.25 Simply, all alginate solutions (1.5% w/v) were prepared by dissolving sodium alginate in 0.9% (w/v) NaCl solution. Subsequently, the solutions were filtered successively over 1.2, 0.8, 0.45, and 0.22 μm microfilters. The solution was then extruded through a 0.5 mm needle into a 100 mmol/L CaCl2 solution, using an electrostatic droplet generator, to form calcium alginate gel beads. After being gelled for 30 min, the beads were incubated with 0.5% (w/v) chitosan solution dissolved in 0.1 mol/L sodium acetate-acetic buffer solution at the ratio of 1:5 (beads/solution) to form membranes. After being rinsed with 0.9% NaCl solution, 0.15% alginate solution was added to counteract excessive positive charges on the membrane, followed by rinsing with 0.9% NaCl solution. Then, the microcapsules were incubated with chitosan solution and alginate solution again to form dense membranes. The diameter of ACA microcapsules prepared by this method was about 370 μm. The coefficient of variance (CV) in diameter of ACA microcapsules was less than 5%. 2.3. Preparation of ACA Membranes. ACA membranes were prepared for surface morphology and roughness studies. Alginate solution was cast onto a dry glass slide. The glass slides with sodium alginate solution were then immersed in a 100 mmol/ L CaCl2 solution to form calcium alginate gels. After being gelled for 30 min, the slides with gel were incubated in 0.5% chitosan solution to form a membrane on the gel surface. The slides with a membrane were subsequently rinsed with 0.9% NaCl solution. A 0.15% alginate solution was added to counteract excessive charges on the membrane. The membranes were then washed with a large amount of 0.9% NaCl solution. At last, the membranes were incubated with chitosan solution and alginate solution again to form dense membranes. 2.4. Surface Morphology Measurements. The surface morphology of ACA microcapsules was analyzed first using a scanning electron microscope (SEM, JSM-5600 LV). Quantitative analysis of surface roughness was then performed with ACA membranes by using a noncontact, three-dimensional optical (34) Kl€ock, G.; Frank, H.; Houben, R.; Zekorn, T.; Horcher, A.; Siebers, U.; Wohrle, M.; Federlin, K.; Zimmermann, U. Appl. Microbiol. Biotechnol. 1994, 40 (5), 638–643. (35) Grasdalen, H.; Larsen, B.; Smidsroed, O. Carbohydr. Res. 1979, 68(1), 23–31. (36) Grasdalen, H. Carbohydr. Res. 1983, 118, 255–260.
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interferometer (New-View 5020, ZYGO). The roughness factor was subsequently determined using the commercial scanning probe image processor software (SPIP, Image Metrology ApS, version 4.8, Lyngby, Denmark). Before starting the measurement, free water on the surface of membranes was removed with absorbent paper from one side of the gel/membrane. Average values were obtained from multiple roughness values (at least five) on different areas of each sample. 2.5. Zeta Potential Measurements. Zeta potential is an important and useful indicator of surface charge. The zeta potentials of ACA microcapsules were determined using a SurPASS Electrokinetic analyzer (Anton Paar GmbH, Austria) equipped with a cylindrical cell. The streaming potential was measured by using Ag/AgCl electrodes. For each measurement, approximately 0.5 mL of wet microcapsules was transferred into the glass cylinder of the measuring cell. Before starting the measurement, the microcapsules were rinsed with double distilled water to remove NaCl. A background electrolyte of 1 mmol/L KCl solution was used. The zeta potential, ζ, was obtained from the streaming potential measurements based on the Smoluchowski equation37,38 as follows: ζ¼
dUstr ηKB dp ε0 εr
ð1Þ
where Ustr is the streaming potential, p is the pressure drop across the streaming channel, ε0 is the vacuum permittivity (8.854 10-12 J-1 3 C2 3 m-1), εr is the dielectric constant of the aqueous solution (78.3), η is the electrolyte solution viscosity (0.8902 mPa 3 s), and κB is the electrical conductivity of the bulk solution. Since the surface conductivity of the microcapsules cannot be determined directly, the zeta potential obtained from the streaming potential measurement using eq 1 is considered an apparent value. 2.6. Protein Adsorption Measurements. Protein adsorption measurements were carried out based on a method described previously.26,39 Simply, the adsorption experiments were performed in 67 mmol/L phosphate buffer (KH2PO4/Na2HPO4, pH 7.4) in order to keep a constant pH during the adsorption process. ACA microcapsules (about 0.1 mL) were incubated with 0.3 mL of buffer solution containing 1.0 mg/mL protein at 37 °C for 24 h to achieve adsorption equilibrium. To rule out the possibility of protein sedimentation, a blank experiment with protein only was carried out. Protein concentration of the centrifuged supernatant was measured by the Bradford method. The protein adsorbed on ACA microcapsules was calculated using the following equation: q¼
ðCi - Cf ÞV A
ð2Þ
where Ci and Cf are the initial protein concentration and the protein concentration in the supernatant after adsorption studies, respectively, V is the total volume of the solution (0.3 mL), and A is the total sphere surface area of ACA microcapsules added into the solution (about 15 cm2). Each experiment was performed in triplicate. 2.7. Fgn Conformational Changes Measurements. After protein adsorption, the protein solutions over the surface of ACA microcapsules were infinitely diluted with pure buffer solution to remove the bulk protein solution and wash away any loosely adherent protein. Following this infinite dilution step, ACA microcapsules were lyophilized at 20 Pa and -50 °C for 24 h. (37) Lyons, J. S.; Furlong, D. N.; Homola, A.; Healy, T. W. Aust. J. Chem. 1981, 34(6), 1167–1175. (38) Werner, C.; K€orber, H.; Zimmermann, R.; Dukhin, S.; Jacobasch, H. J. J. Colloid Interface Sci. 1998, 208(1), 329–346. (39) Xie, H. G.; Li, X. X.; Lin, J. Z.; Xie, W. Y.; Ma, X. J. CIESC J. 2009, 60(4), 929–935.
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The conformational changes in Fgn induced by adsorption were analyzed by diffuse reflectance Fourier transform infrared (FTIR) spectroscopy with a VECTOR 22 spectrometer (Bruker). Infrared spectra were measured with 1.0 mg samples in 99.0 mg of KBr. An adsorption spectrum was recorded in the range of 400-4000 cm-1. Background measurements were based on 64 scans, and sample measurements were based on 32 scans. The data obtained by FTIR were processed using the options of OPUS version 4.0. The collected spectra were prepared for analysis by subtracting the background spectrum taken for each kind of ACA microcapsules without the adsorbed protein from the spectrum of the corresponding kind of ACA microcapsules with adsorbed protein. The resultant difference represented the contribution to the overall spectrum from the adsorbed protein layer alone. The criterion used for subtraction was a straight baseline in the region of 2500-1800 cm-1.40,41 The subtraction factor was set as 1.0 in all cases. The resultant protein spectra were smoothed by application of the Savitsky-Golay algorithm employing nine smoothing points to remove the possible noise.42 In order to enhance the resolution, the adsorbed protein spectra were deconvolved using Fourier self-deconvolution by application of a Lorentzian line shape and a noise reduction factor of 0.5.43 The deconvolution factor was determined individually for each spectrum by increasing the value until the point of oscillation. Secondary derivative spectra were obtained with the Savitsky-Golay derivative function for a five data point window. The possible overlapping bands in the spectra were separated by a fitting procedure which assumes the adsorption bands for the different structural components to be Gaussian shaped.44 The number of components and their peak positions, determined by second derivation, were used as starting parameters. The frequency range of the fit was chosen to be the local minimum surrounding the amide I band (∼17201580 cm-1). The secondary structure content was calculated from the areas of the individual assigned bands and their fraction of the total area in the amide I region. 2.8. Statistical Analysis. Data were expressed as mean ( standard error of the mean. Statistical comparisons were performed using a one-way analysis of variance (ANOVA). Pvalues smaller than 0.05 were considered to indicate statistical significance.
3. Results and Discussion 3.1. Surface Morphology Analysis. 3.1.1. Surface Morphology of Calcium Alginate Gels. The surface roughness of calcium alginate gels made of four alginate samples was analyzed using a white-light profilometer, and the quantification is shown in Table 2. At magnification in the white-light profilometer, all the surfaces of calcium alginate gels appeared to be uniformly structured with almost similar irregularities (Figure 1). The surface roughness of gels made of four alginates was 67, 58, 51, and 58 nm, respectively, and showed no statistically difference. The morphology of calcium alginate gel has been characterized via SEM in several reports.45-47 However, few reports have been found to study the surface roughness of calcium alginate gels in the wet state. In this study, the surface roughness of wet calcium alginate gels was measured to serve as a control group to compare (40) Cheng, S. S.; Chittur, K. K.; Sukenik, C. N.; Culp, L. A.; Lewandowska, K. J. Colloid Interface Sci. 1994, 162(1), 135–143. (41) Fu, K.; Griebenow, K.; Hsieh, L.; Klibanov, A. M.; Langer, R. J. Controlled Release 1999, 58(3), 357–366. (42) Savitsky, A.; Golay, J. E. Anal. Chem. 1964, 36(8), 1628–1639. (43) Holm, N. K.; Jespersen, S. K.; Thomassen, L. V.; Wolff, T. Y.; Sehgal, P.; Thomsen, L. A.; Christiansen, G.; Andersen, C. B.; Knudsen, A. D.; Otzen, D. E. Biochim. Biophys. Acta 2007, 1774(9), 1128–1138. (44) Dong, A.; Huang, P.; Caughey, W. S. Biochemistry 1992, 31(1), 182–189. (45) Kim, W. T.; Chung, H.; Shin, I. S.; Yam, K. L.; Chung, D. Carbohydr. Polym. 2008, 71(4), 566–573. (46) Zvitov, R.; Nussinovitch, A. Food Hydrocolloids 2001, 15(1), 33–42. (47) Lei, J.; Kim, J. H.; Jeon, Y. S. Macromol. Res. 2008, 16(1), 45–50.
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Table 2. Surface Roughness of ACA Membranes Made of Varying Alginate and Chitosan (Measured by Using a Three-Dimensional Optical Interferometer) surface roughness (nm) calcium alginate gels
alginate/ chitosan membrane
roughness factor of alginate/chitosan membrane
120 ( 26 197 ( 28 293 ( 42 375 ( 39
5.5 7.6 9.9 12.0
270 67 ( 18 alginate 430 58 ( 14 molecular weight (kDa) 490 51 ( 15 590 73 ( 21 chitosan concentration (%)
0.25 0.50 1.00
76 ( 29 197 ( 28 242 ( 34
3.3 7.1 8.4
with surface roughness of ACA microcapsules. As we know, calcium alginate hydrogels are the cross-linked networks containing a large fraction of water. There are open and porous gel networks in the surface layer, and besides the polymer concentration is higher at the surface than in the inner section.48 So, the gel surface appeared to be uniformly structured with almost similar irregularities at magnification. As a result, the surface roughness of gels made of four alginates showed no statistical difference. 3.1.2. Effect of Molecular Weight of Alginate on Surface Roughness of ACA Microcapsules. The surface morphologies of ACA microcapsules made of alginates with different molecular weight were first characterized via SEM. As shown in Figure 2, the surface of ACA microcapsules exhibited an obvious granular structure. The size of the granular structure on the surface decreased with decreasing alginate molecular weight. As a result, ACA microcapsules made of alginate with a molecular weight of 270 kDa have the smoothest surface and ACA microcapsules made of alginate with a molecular weight of 590 kDa have the roughest surface. The SEM results were confirmed by a further quantitative analysis using a white-light profilometer. As shown in Table 2, the surface roughness of ACA membranes decreased significantly (p < 0.05) from 375 to 120 nm when the molecular weight of alginate decreased from 590 to 270 kDa. A membrane with greater surface roughness could provide a more effective surface for protein adsorption. The roughness factor results showed that the surface area increased 117% when the surface roughness of ACA membranes increased from 120 to 375 nm. The influence of surface morphology on protein adsorption began to receive keen interest.49 ACA microcapsule surfaces with random surface morphology were chosen for this study. We characterized the surface roughness of calcium alginate gels and ACA membranes made of different alginates first. Compared with the surface of a calcium alginate gel, it can be concluded that the surface of an ACA membrane was roughened to a varied extent corresponding to the molecular weight of the alginate used, when calcium alginate gel was incubated with chitosan solution to form a membrane. It has been demonstrated30 that, in the process of calcium alginate gels being incubated with chitosan solution to form membranes, negatively charged carboxylic groups of alginate were bonded by positively charged amino groups of chitosan. As a result, electrostatic repulsion force caused by negatively charged carboxylic groups among the gel strands decreased, and gel networks in the membrane rearranged and assembled (48) Gaseroed, O.; Smidsroed, O.; Skjak-Braek, G. Biomaterials 1998, 19(20), 1815–1825. (49) Song, W.; Chen, H. Chin. Sci. Bull. 2007, 52(23), 3169–3173.
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into granular structures (Figure 3). The intermolecular force of alginate with lower molecular weight in solution is less50 than that of alginate with higher molecular weight. Thus, when the former alginate was formed into calcium alginate gels, the number of cross-linking points between two alginate molecules was less.51 That is the reason for the more open and porous gel networks48 and higher solute diffusivity52,53 in the gels made of alginate with lower molecular weight. So, when calcium alginate gels were incubated with chitosan solution to form membranes, a gel skeleton made of alginate with lower molecular weight would assemble into a smaller sized granular structure. This is the reason that the surface of an ACA membrane made of alginate with lower molecular weight was smoother than that of a membrane made of alginate with higher molecular weight. 3.1.3. Effect of Chitosan Concentration on Surface Roughness of ACA Microcapsules. The effect of chitosan concentration on the surface morphology of ACA microcapsules was also investigated in the study. The surface morphologies of ACA microcapsules made of alginate with a molecular weight of 430 kDa and chitosan at different concentrations were characterized and analyzed. As shown in Figure 4, all the surfaces of ACA membranes made of chitosan at three concentrations exhibited a granular structure with varied size. The quantification results showed that the surface roughness of ACA membranes made of chitosan at concentrations of 0.25%, 0.5%, and 1.0% was 76, 197, and 242 nm, respectively. The roughness factor results showed the surface area increased 155% when surface roughness increased from 76 to 242 nm. The formation of an ACA membrane is essentially the result of electrostatic interaction between negatively charged carboxylic groups of alginate and positively charged amino groups of chitosan. However, not all the ionic groups on the polyelectrolyte participate in electrostatic interaction due to steric effects.54 Thus, the structure of the ACA microcapsule membrane can be represented as alternative sequences of ionic interchain bonds and looplike regions incorporating the uncoupled units of both chains. When calcium alginate gels were transferred into chitosan solution at higher concentration, more positively charged chitosan molecules were available for negatively charged carboxylic groups in calcium alginate gel networks to bind at the beginning for a kinetic effect. The steric effect was weakened to some extent in this case. So, electrostatic repulsion force caused by negatively charged carboxylic groups between the gel strands decreased to a greater extent. The gel skeleton in gels assembled into larger sized granular structures, and a rougher surface was produced. In fact, the effect of chitosan concentration on the properties of ACA membranes has been reported by several studies.25,55,56 All the studies showed that more chitosan molecules could bind to calcium alginate gels in the case of higher chitosan concentration, resulting in more thick and tight ACA membranes. The results in this study showed that the ACA membrane made of chitosan at higher concentration was rougher than that of lower chitosan concentration. 3.2. Zeta Potential Analyses. Charged interfaces are often characterized by their zeta potentials which are the potentials at (50) Chikahisa, Y. J. Phys. Soc. Jpn. 1964, 19(1), 92–100. (51) Martinsen, A.; Storroe, I.; Skjak-Braek, G. Biotechnol. Bioeng. 1992, 39(2), 186–194. (52) Kikuchi, A.; Kawabuchi, M.; Sugihara, M.; Sakurai, Y.; Okano, T. J. Controlled Release 1997, 47(1), 21–29. (53) Amsden, B.; Turner, N. Biotechnol. Bioeng. 1999, 65(5), 605–610. (54) Bromberg, L. E. J. Membr. Sci. 1991, 62(2), 117–236. (55) Bartkowiak, A.; Hunkeler, D. Chem. Mater. 1999, 11(9), 2486–2492. (56) Lyu, S. Y.; Kwon, Y. J.; Joo, H. J.; Park, W. B. Arch. Pharm. Res. 2004, 27 (1), 118–126.
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Figure 1. Three-dimensional surface morphology of calcium alginate gels made of alginates with different molecular weights (measured by using a three-dimensional optical interferometer, images 0.73 0.53 mm2): (A) 270 kDa, (B) 430 kDa, (C) 490 kDa, and (D) 590 kDa.
Figure 2. SEM images (a-d) and three-dimensional surface morphology (A-D, 0.73 0.53 mm2) of ACA membranes made of alginates with different molecular weights: (a, A) 270 kDa, (b, B) 430 kDa, (c, C) 490 kDa, and (d, D) 590 kDa.
Figure 3. Schematic diagram of surface roughening process: (A) surface of calcium alginate gel, and (B) surface of alginate/chitosan microcapsule. Dotted lines show the gel skeleton.
the hydrodynamic slipping plane adjacent to the phase boundary. ACA microcapsules made of alginate with molecular weight of 270, 430, 490, and 590 kDa exhibited zeta potentials (ζ) of -6.5 ( 0.3, -5.3 ( 0.2, -3.4 ( 0.3, and -2.1 ( 0.1 mV, respectively, at pH 7.4. ACA microcapsules made of chitosan at concentrations of 0.25%, 0.5%, and 1.0% showed zeta potentials of -6.7 ( 0.4, -5.7 ( 0.3, and -4.6 ( 0.3 mV, respectively, at pH 7.4. The negative zeta potentials indicated that ACA microcapsules had a net negative charge at neutral pH. The varying zeta potential of ACA microcapsules with the molecular weight of alginate and concentration of chitosan indicated a varying degree Langmuir 2010, 26(8), 5587–5594
of net negatively charged groups on the surface of ACA microcapsules. It has been found that electrostatic interaction between protein and the substrate plays an important role in protein adsorption.57,58 Positively charged groups of chitosan on the surface of ACA microcapsules were found to increase the amount of adsorbed Fgn at neutral pH.26 ACA microcapsule membranes were (57) Patil, S.; Sandberg, A.; Heckert, E.; Self, W.; Seal, S. Biomaterials 2007, 28 (31), 4600–4607. (58) Burns, N. L.; Holmberg, K.; Brink, C. J. Colloid Interface Sci. 1996, 178(1), 116–122.
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Figure 4. SEM images (a-c) and three-dimensional surface morphology (A-C, 0.73 0.53 mm2) of ACA membranes made of chitosans with different concentration: (a, A) 0.25%, (b, B) 0.50%, and (c, C) 1.00%.
fabricated by the electrostatic interaction between negatively charged carboxylic groups of alginate and positively charged amino groups of chitosan, followed by a neutralization of excess positively charged chitosans with alginate solution. So, there will be a large amount of negatively charged carboxylic groups on the microcapsule membrane surface. As a result, the zeta potentials of ACA microcapsules were negative at neutral pH. Meanwhile, chitosan fragments are also exposed on the surface of ACA membranes.26 Amino groups of chitosan could be protonated and carry positive charge at neutral pH, although chitosan has a pKa value of 6.3-6.8.59 ACA microcapsules made of alginate with high molecular weight have a less open and porous gel network,48 leading to less efficient binding between chitosan and calcium alginate beads.51,53 Therefore, there were more amino groups being uncovered. This is the reason that the zeta potential of ACA microcapsules increased with increasing molecular weight of alginate used. With more amino groups being not bonded, it would have more effect on protein adsorption. 3.3. Fgn Adsorption onto ACA Microcapsules. Protein adsorption onto ACA microcapsules made of alginates with different molecular weights and chitosan at different concentrations was studied at pH 7.4. The results (Figure 5A) showed that the amount of adsorbed Fgn increased with an increase in alginate molecular weight, from 1.87 ( 0.08 μg/cm2 on ACA microcapsules made of alginate with a molecular weight of 270 kDa to 2.85 ( 0.08 μg/cm2 on ACA microcapsules made of alginate with a molecular weight of 590 kDa. In the case of ACA microcapsules made of chitosan at different concentrations, the amount of adsorbed Fgn increased from 1.75 ( 0.13 μg/cm2 on ACA microcapsules made of chitosan at a concentration of 0.25% to 2.24 ( 0.05 μg/cm2 on ACA microcapsules made of chitosan at a concentration of 1.0% (Figure 5B). The differences were statistically significant. Protein adsorption on some materials has been widely studied. It has been found that factors such as surface morphology, surface free energy, and surface charge played important roles in protein adsorption. A greater surface roughness increases the effective surface area. Thus, more surfaces are available for protein attachment. Therefore, the amount of adsorbed Fgn on ACA microcapsules increased with the increase in surface roughness. The results were consistent with the observations made by other (59) Tomihata, K.; Ikada, Y. Biomaterials 1997, 18(7), 567–575.
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Figure 5. Fibrinogen adsorption onto microcapsules made of different alginates (A) and chitosan (B) at pH 7.4. The amount of adsorbed protein on membranes made of different alginates was graphed as mean ( standard error (n = 3).
groups.17,22 Also, it can be seen that the amount of adsorbed Fgn increased with the increase of zeta potential. This is because Fgn has an isoelectric point of 5.1, and is negatively charged at pH 7.4.60 In fact, it has been observed several times that electrostatic repulsion between a surface and a protein does not prevent adsorption.9,10,61 The results were in good accordance with our previous study26 for electrostatic interaction being one of the driving forces for protein adsorption on ACA microcapsules. The results in this study suggest that there are stronger protein-surface interactions between Fgn and ACA microcap-
(60) Bajpai, A. K. J. Mater. Sci. 2008, 19(1), 343–357. (61) Norde, W.; Lyklema, J. J. Biomater. Sci., Polym. Ed. 1991, 2(3), 183–202.
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Figure 6. Schematic to show adsorption orientation of fibrinogen on a smoother surface (A) and rougher surface (B).
sules made of alginate with higher molecular weight or chitosan at higher concentration. A previous study has suggested that Fgn, a rodlike shaped molecule, can adsorb in two possible orientations: side-on, with its long axis parallel to the particle radius, or end-on, with its long axis perpendicular to the radius.62 According to Sivaraman et al.,2 the amount of a close-packed layer of end-on adsorbed Fgn is about 2.26 μg/cm2 (25 nm2 per adsorbed molecule of Fgn), whereas the amount of a side-on adsorption configuration is 0.24 μg/cm2 (235 nm2 per adsorbed molecule of Fgn). Considering the fact that surface roughness increases geometrical area, we can conclude from the Fgn adsorption results that the surfaces of ACA microcapsules were saturated with Fgn, and these protein molecules were arranged on the surface in a mixture of side-on and end-on configurations, as the values for the amount of adsorbed Fgn, with two exceptions, lay between the theoretical values for side-on and end-on protein adsorption. The results of surface roughness showed that the surface area increased 117% when surface roughness increased from 120 to 375 nm. Correspondingly, the amount of Fgn adsorbed increased 52%. Because the roughness features were more than 2 times larger than Fgn, the Fgn molecules would experience a largely smooth surface.18 Therefore, we can conclude that there were more protein molecules that arranged on the rougher surface in the side-on configuration (Figure 6). Fgn adsorption on ACA microcapsules made of different concentrations of chitosan had the same trend. Fgn arranged on the surface in side-on configuration may have greater conformational changes. 3.4. Conformational Assessment. Fourier transform infrared spectroscopy (FTIR) has been used for analysis of adsorbed protein conformation. Protein infrared spectra contain peaks arising mainly from amide bond vibrations. The amide I band centered at ∼1700-1600 cm-1 is largely due to CdO stretching vibrations. This band is sensitive to changes in secondary structure and has therefore been widely used for protein conformational studies.1,62-64 Amide bonds within different secondary structures, R-helix, β-sheet, β-turn, and unordered, give rise to (62) Roach, P.; Farrar, D.; Perry, C. C. J. Am. Chem. Soc. 2005, 127(22), 8168– 8173. (63) Roach, P.; Farrar, D.; Perry, C. C. J. Am. Chem. Soc. 2006, 128(12), 3939– 3945. (64) Wei, T.; Kaetwtathip, S.; Shing, K. J. Phys. Chem. C 2009, 113(6), 2053– 2062.
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Figure 7. Separation of the amide I band of fibrinogen into its structural components. The dark solid line shows the original FTIR spectrum. The gray solid line shows the deconvolved spectrum. The dark gray solid line shows the second-deritive spectrum. In all cases, the sum of the separated band components (wide dashed curve) fits the deconvolved band contour very well. (A) Free fibrinogen, (B) fibrinogen adsorbed onto ACA microcapsules made of alginate with molecular weight of 270 kDa, and (C) fibrinogen adsorbed onto ACA microcapsules made of alginate with molecular weight of 590 kDa.
specific vibrational bands. These component bands are largely overlapping, and all contribute to the characteristic broad amide I band observed in infrared spectra.1,62-64 From previous studies,65 component bands with maxima at 1688-1683 cm-1 and at 1625-1616 cm-1 have been assigned to intermolecular β-sheet structures, at 1670-1665 cm-1 to β-turn structures, at 16551650 cm-1 to R-helices, at 1640-1633 cm-1 to intramolecular β-sheet structures, and at 1610-1600 cm-1 to random chains (Figure 7). Curve fitting analysis of component bands showed that with an increase in surface roughness of ACA microcapsules the content of R-helix structure of Fgn adsorbed decreased (Figure 7), shown by a decrease in the percent area of the component band centered at ∼1653 cm-1. An increasing contribution from bands at lower (65) Schwinte, P.; Voegel, J. C.; Picart, C.; Haikel, Y.; Schaaf, P.; Szalontai, B. J. Phys. Chem. B 2001, 105(47), 11906–11916.
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Xie et al. Table 3. Changes in Secondary Structure of Fgn Adsorbed on ACA Microcapsulesa ACA microcapsules
R-helix
intermolecular β-sheet
intramolecular β-sheet
alginate molecular weight (kDa)
270 430 490 590
12.6 ( 1.2 10.1 ( 0.4 4.4 ( 0.9 4.2 ( 0.6
20.6 ( 0.9 18.9 ( 1.1 34.4 ( 1.7 37.9 ( 3.7
23.3 ( 2.2 46.2 ( 2.7 27.2 ( 1.4 26.0 ( 1.8
chitosan concentration (%)
0.25 0.50 1.00
17.7 ( 2.8 11.8 ( 2.7 10.0 ( 2.2
26.4 ( 0.9 28.2 ( 1.4 33.0 ( 2.5
20.0 ( 1.6 13.4 ( 1.9 7.0 ( 0.6
32.8 ( 2.5
16.3 ( 1.4
6.1 ( 0.7
fibrinogen (native) a Structure percentage values are given.
or higher wavenumber indicated a concurrent increase in both the intermolecular and intramolecular β-sheet structure components. When protein adsorption on surfaces happened, the intermolecular β-sheet structures would originate from surface-protein interactions. The percentage of intramolecular β-sheet content decreased with increasing surface roughness. Nevertheless, the percentage of intramolecular β-sheet content of Fgn adsorbed on ACA microcapsules was higher than that of free Fgn (Table 3). These findings suggested that Fgn was somewhat denatured on interaction with the surface of ACA microcapsules, gaining a large degree of their intermolecular and intramolecular β-sheet structures at the expense of the helical secondary structure. This change in conformation may arise from a stronger interaction with a rougher surface, a feature that has been previously reported for both BGG and fribrinogen along with other proteins.17,22 Protein-surface interactions can induce conformational changes in the adsorbing protein molecules. It has been reported previously that Fgn undergo greater conformational disordering when adsorbing onto different surfaces. The effect of a regular surface morphology on protein adsorption has already been studied experimentally and theoretically,17,66 leaving the understanding of how random surface roughness affects protein adsorption less explored. Here we described protein-surface interactions by both the amount of adsorbed Fgn and its secondary structure changes during adsorption onto ACA microcapsules with random surface roughness. The structure of Fgn consists of an elongated, symmetrical dimer, and its distinguishable regions include two negatively charged outer domains (D domains) connected to a central globular domain (E domain) through a pair of three nonidentical R-helix coils.19,67,68 It can adsorb in side-on or end-on configurations62 and spread on surfaces.69 The current study suggested that side-on orientation was favored on rougher surfaces, indicating that upon adsorption the protein molecules would extensively spread on the surfaces with D domains, E domain, and R-helix coiled-coil regions interacting with surfaces. Besides, protein-surface interactions between Fgn and ACA microcapsules with rougher surfaces were stronger. Therefore, protein structural perturbation would be expected to be greater on rougher surfaces, as was observed with the loss of the helical component and with the increase of intermolecular β-sheet content to a greater extent. With adsorption onto smoother surfaces, actual surface area may be limited for all the protein molecules spreading. Proteins (66) Lee, N. K.; Vilgis, T. A. Phys. Rev. E 2003, 67(5), 050901. (67) Evans-Nguyen, K. M.; Tolles, L. R.; Gorkun, O. V.; Lord, S. T.; Schoenfisch, M. H. Biochemistry 2005, 44(47), 15561–15568. (68) Desroches, M. J.; Omanovic, S. Phys. Chem. Chem. Phys. 2008, 10(18), 2502–2512. (69) Santore, M. M.; Wertz, C. F. Langmuir 2005, 21(22), 10172–10178.
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adsorbed were restricted in the end-on configuration. Thus, there was a decrease in the degree of loss of the helical component in Fgn adsorbed. The results agreed well with the previous conclusion2,20 that an increase in protein concentration caused an increase in the amount of protein adsorbed but a decrease in the degree of conformational change. From the results presented, it is clear that orientation and packing density of adsorbed proteins are also dependent upon surface roughness.
4. Conclusions Specifically, the influences of alginate molecular weight and chitosan concentration on surface morphology and charge of ACA microcapsules, and the effects of surface roughness and charge on protein adsorption have been studied. On the basis of the results from our present studies, we concluded that the surface morphology and charge of ACA microcapsules were influenced greatly by the two factors. ACA microcapsules made of alginate with lower molecular weight or chitosan at lower concentration have smoother surfaces and lower zeta potentials. Greater surface roughness and zeta potential played more significant roles in influencing protein adsorption. There were stronger proteinsurface interactions between Fgn and ACA microcapsules with higher zeta potentials. A membrane with greater surface roughness could provide more effective surfaces for protein adsorption. More protein molecules could adsorb on it, and more of them would spread and show a greater conformational change. Recent studies have shown that the conformation of the adsorbed protein layer, not just the amount of adsorbed protein, is an important determinant of cellular response to biomaterial surfaces. The present study showed that surface morphology and charge of ACA microcapsules had great effects on both the amount and conformation of adsorbed protein. Future studies are planned to correlate cellular response to ACA microcapsules with different surface roughness in order to develop a more comprehensive understanding of how surface roughness, surface charge, and protein adsorption influence the biocompatibility of implanted biomaterials and, most importantly, to gain further insight into how surfaces can be engineered to control protein adsorption behavior and thereby direct biological response. Acknowledgment. The authors are grateful to Dr. Thomas Luxbacher at Anton Paar GmbH for his critical reading of the manuscript. This study was supported by the National Key SciTech Special Project of China (Grant Number 20082 10002019), the National Basic Research Program of China (Grant 2007CB714305), the National Natural Science Foundation of China (Grant 20736006), and the Hi-Tech Research and Development (863) Program of China (Grant 2006AA02A140).
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