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Adsorption of Bovine Serum Albumin on Template-Polymerized Chitosan/Poly(methacrylic acid) Complexes C. L. de Vasconcelos, P. M. Bezerril, T. N. C. Dantas, M. R. Pereira, and J. L. C. Fonseca* Departamento de Quı´mica, UniVersidade Federal do Rio Grande do Norte, Campus UniVersita´ rio, Lagoa NoVa, Natal, RN 59078-970, Brazil ReceiVed February 23, 2007. In Final Form: April 16, 2007 Particulate systems composed of polyelectrolyte complexes (PEC) based on chitosan and poly(methacrylic acid) were obtained via template polymerization. The resultant particles were characterized as having regions with different charge densities: chitosan predominating in the core and poly(methacrylic acid) at the surface, the particles being negatively charged, as a consequence. Albumin was adsorbed on these particles (after cross-linking with glutardialdehyde), and pH was controlled to obtain two conditions: (i) adsorption of positively charged albumin and (ii) adsorption of albumin at its isoelectric point. Adsorption isotherms and ζ-potential measurements showed that albumin adsorption was controlled by hydrogen bonding/van der Waals interactions and that brush-like structures may enhance the adsorption of albumin on these particles. It was also found that shearing can induce desorption of albumin from the PEC surface, depending on the continuous phase albumin concentration.
1. Introduction The technique of template polymerization mimics bioprocesses such as self-replication of DNA and biosynthesis of proteins.1,2 In this type of reaction, the propagation of the growing polymer occurs along a preformed linear macromolecule that was previously added to the reaction system. The polymeric template shapes the resultant macromolecule via cooperative interactions, such as van der Waals forces, hydrogen bonding, and electrostatic attractions between oppositely charged groups of monomer molecules and templates.3 The template polymerization technique has been used in the development of mucoadhesive polymeric carriers of biomolecules, and chitosan has frequently been used as a template in this process.4,5 Its suitability comes from the fact that this natural linear copolymer, consisting of β-(1 f 4)-2-amino-2-deoxy-Dglucopyranose and β-(1 f 4)-2-acetamido-2-deoxy-D-glucopyranose units, is biocompatible, also being a polyelectrolyte with polycationic character.6 Ahn et al.7,8 and Choi et al.9 have prepared mucoadhesive polymers by template polymerization of acrylic acid, AA, in the presence of different templates, such as poly(ethylene glycol), silk sericin, and chitosan. These researchers found that chitosan-based template particles exhibited a strong adhesive force and limited aqueous solubility, FTIR spectroscopy and DMTA indicating that the polymer complex was formed by hydrogen bonding.7 When using chitosan-derived materials in medical applications, the issue of surface interaction with cells and proteins is of fundamental importance. Zhou et al. have used chitosan with PEO grafts to reduce specific protein adsorption, with potential * To whom correspondence should be addressed. Tel.: +55 84 3215 3828, ext. 215. Fax: +55 84 3211 9224. E-mail:
[email protected]. (1) Polowinski, S. Prog. Polym. Sci. 2002, 27, 537. (2) Tan, Y. Y. Prog. Polym. Sci. 1994, 19, 561. (3) Serizawa, T.; Hamada, K.; Akashi, M. Nature 2004, 429, 52. (4) Ahn, J. S.; Choi, H. K.; Chun, M. K.; Ryu, J. M.; Jung, J. H.; Kim, Y. U.; Cho, C. S. Biomaterials 2002, 23, 1411. (5) Chun, M. K.; Cho, C. S.; Choi, H. K. J. Controlled Release 2002, 81, 327. (6) Muroga, Y.; Yoshida, T.; Kawaguchi, S. Biophys. Chem. 1999, 81, 45. (7) Ahn, J. S.; Choi, H. K.; Cho, C. S. Biomaterials 2001, 22, 923. (8) Ahn, J. S.; Choi, H. K.; Lee, K. H.; Nahm, J. H.; Cho, C. S. J. Appl. Polym. Sci. 2001, 80, 274. (9) Choi, H. K.; Kim, O. J.; Chung, C. K.; Cho, C. S. J. Appl. Polym. Sci. 1999, 73, 2749.
applications in biomedical and diagnostic fields.10 Immobilization of enzymes via adsorption on chitosan-based materials can be used in biotechnology and biodevices.11 When using chitosan in wound healing, Hamilton et al. have found that surface properties influence tissue and fibroblast responses, which are very important for the application of these materials.12 As a consequence, efforts should be driven toward studying protein adsorption on these template polymers, mainly because this sort of study has been carried out, for the most part, with inorganic surfaces.13-16 Protein adsorption is a complex process involving van der Waals, hydrogen bonding, and electrostatic interactions and is dependent on the chemical and physical characteristics of the surface. To monitor protein adsorption and conformational changes in different colloids, techniques such as quartz crystal microbalance (QCM) and electrochemical impedance system,17 optical waveguide light spectroscopy,18 ellipsometry and ATRFTIR spectroscopy,19,20 radiolabeling,21 atomic force microscopy (AFM),22 time-of-flight secondary ion mass spectroscopy (TOFSIMS),23 and total internal reflection fluorescence microscopy (TIRFM)24 have been used. Besides, other important techniques, such as ζ-potential analysis and UV-vis spectroscopy, have (10) Zhou, Y.; Liedberg, B.; Gorochovceva, N. A.; Makuska, R.; Dedinaite, A.; Claesson, P. M. J. Colloid Interface Sci. 2007, 305, 62. (11) Lu, H. Y.; Hu, N. F. J. Phys. Chem. B 2006, 110, 23710. (12) Hamilton, V.; Yuan, Y.; Rigney, D. A.; Puckett, A. D.; Ong, J. L.; Yang, Y.; Elder, S. H.; Bumgardner, J. D. J. Mater. Sci.: Mater. Med. 2006, 17, 1373. (13) Rezwan, K.; Studart, A. R.; Voros, J.; Gauckler, L. J. J. Phys. Chem. B 2005, 109, 14469. (14) Kandori, K.; Miyagawa, K.; Ishikawa, T. J. Colloid Interface Sci. 2004, 273, 406. (15) Peng, Z. G.; Hidajat, K.; Uddin, M. S. Colloids Surf., B 2004, 35, 169. (16) Kandori, K.; Mukai, M.; Yasukawa, A.; Ishikawa, T. Langmuir 2000, 16, 2301. (17) Zhang, Y. Y.; Fung, Y. S.; Sun, H.; Zhu, D. R.; Yao, S. Z. Sens. Actuators, B 2005, 108, 933. (18) Hook, F.; Voros, J.; Rodahl, M.; Kurrat, R.; Boni, P.; Ramsden, J. J.; Textor, M.; Spencer, N. D.; Tengvall, P.; Gold, J.; Kasemo, B. Colloids Surf., B 2002, 24, 155. (19) McClellan, S. J.; Franses, E. I. Colloids Surf., A 2005, 260, 265. (20) McClellan, S. J.; Franses, E. I. Langmuir 2005, 21, 10148. (21) Moulton, S. E.; Barisci, J. N.; Bath, A.; Stella, R.; Wallace, G. G. J. Colloid Interface Sci. 2003, 261, 312. (22) Ying, P. Q.; Yu, Y.; Jin, G.; Tao, Z. L. Colloids Surf., B 2003, 32, 1. (23) Henry, M.; Dupont-Gillain, C.; Bertrand, P. Langmuir 2003, 19, 6271. (24) Vaidya, S. S.; Ofoli, R. Y. Langmuir 2005, 21, 5852.
10.1021/la700537t CCC: $37.00 © 2007 American Chemical Society Published on Web 06/05/2007
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Figure 1. Chemical structures of (a) chitosan and (b) poly(methacrylic acid).
also been used for gathering direct information on adsorption modes and changes of surface charge at colloid and protein interfaces.25 Bovine serum albumin (BSA) has been widely used as a model protein in adsorption studies on several surfaces. This protein consists of 583 amino acid residues and three domains in a single polypeptide chain.26 The secondary structure of BSA is composed of nine helical loops connected with 17 disulfide bridges (the three protein domains each contain one small and two large helical loops), leading to a secondary structure of approximately 67% helical content with the remainder consisting of ∼23% extended conformations (β-strands) and ∼10% β-turns.27 The aim of this work is to elucidate the nature of the adsorption process in terms of hydrogen bonding and hydrophobic and electrostatic interactions between BSA and chitosan/poly(methacrylic acid) (CS/PMAA) complex surfaces (obtained via template polymerization) at different pH values. This combination of adsorbate and adsorbent was chosen because, to our knowledge, adsorption of BSA onto polylelectrolyte particles obtained via template polymerization has not been reported in the literature yet. 2. Experimental Procedures 2.1. Materials. Chitosan (Polymar Ltd.) was used in this work with a deacetylation degree of ca. 90% and a molecular weight Mv ≈ 2.9 × 105 g mol-1 (determined using the Mark-Howink-Sakurada equation from viscometric data28). Methacrylic acid (Aldrich), potassium peroxydisulfate (P.A., Vetec), glutardialdehyde (P.A., 25%, Vetec), and potassium chloride (P.A., Vetec) were used as received. Bovine serum albumin was purchased from Sigma-Aldrich and used as received. The chemical structures of chitosan and methacrylic acid are shown in Figure 1. 2.2. Template Polymerization. Chitosan, CS, was dissolved in a 2 wt % acetic acid solution under magnetic stirring at room (25) Rezwan, K.; Meier, L. P.; Rezwan, M.; Voros, J.; Textor, M.; Gauckler, L. J. Langmuir 2004, 20, 10055. (26) Murayama, K.; Tomida, M. Biochemistry 2004, 43, 11526. (27) Wang, S. C.; Lee, C. T. J. Phys. Chem. B 2006, 110, 16117. (28) Fernandes, A. L. P.; Morais, W. A.; Santos, A. I. B.; de Araujo, A. M. L.; dos Santos, D. E. S.; dos Santos, D. S.; Pavinatto, F. J.; Oliveira, O. N.; Dantas, T. N. C.; Pereira, M. R.; Fonseca, J. L. C. Colloid Polym. Sci. 2005, 284, 1.
temperature for 24 h and filtered through a Millipore Millex 41 µm membrane. Chitosan was precipitated using NaOH solution 1.25 M, washed and dried in a vacuum at room-temperature prior to use. 2.2.1. Polyelectrolyte Complexes. Polyelectrolyte complexes, PEC, of chitosan/poly(methacrylic acid), CS/PMAA, were obtained from template polymerization of methacrylic acid, MAA, using CS as a template. CS was solubilized in an MAA solution (0.06 mol L-1) at a given MAA/aminoglucoside molar ratio, rCOOH/NH2, under magnetic stirring, at room temperature, for 18 h. The polymerization was carried out at 70 °C, under magnetic stirring, after the addition of K2S2O8 (final concentration, 0.002 mol L-1) until an opalescent unstable dispersion was obtained. The resulting CS/PMAA dispersion was filtered in a polyester screen to remove particle aggregates, and the final dispersion underwent centrifugation (Himac CR21G, Himac Inc.) at 15 000 rpm and 25 °C for 40 min to separate the particles, which were dried in a vacuum at room temperature. 2.2.2. Particle Covalent Cross-Linking. Particles used in adsorption experiments (Section 2.4.) were covalently cross-linked using glutardialdehyde. This was done by adding 5 g of wet PEC particles to 10 mL of a 2.5% aqueous solution of glutardialdehyde, the resultant dispersion being left under magnetic stirring for 6 h. Finally, the material was washed, centrifuged, dried at room temperature in a vacuum for 48 h, ground, and sieved. A brownish powder, insoluble at alkaline and acidic pH, was obtained using this procedure. 2.3. Particle Characterization. 2.3.1. Scanning Electron Microscopy (SEM). Scanning electron microscopy (SEM) images of the coagulated particles were obtained using a Phillips XL30 electron microscope (filament, tungsten; voltage, 25.0 kV). Prior to the analysis, all samples were metalized with gold. 2.3.2. Fourier Transform Infrared Spectroscopy (FTIR). FTIR spectra were carried out in a Thermo Nicolet Nexus 470 spectrometer using disks made of CS/PMAA particles pressed with KBr (obtained as described in Section 2.2.). The operational parameters were number of scans, 32 and resolution, 4 cm-1. 2.3.3. X-ray Diffractometry. X-ray diffractometry (Shimadzu LabX XRD-6000 X-ray diffractometer) of PEC particles was obtained using a Cu KR target at 30 kV and 30 mA. The diffraction angle was varied within the range of 5° < 2θ < 50° at a scan rate of 2° min-1. 2.3.4. Electrophoretic Mobility. Electrophoretic mobility measurements were carried out at room temperature for PEC particles from the redispersion of these particles in 10-3 mol L-1 potassium
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chloride, KCl, aqueous solution, to maintain a constant ionic strength. The effect of pH on PEC particle electrophoretic behavior was evaluated in the range of pH 2.35-10.64. A Micronal pH meter, model B474, was used for pH determination of the PEC dispersions at room temperature. Five buffer systems [0.1 M potassium hydrogen phthalate/0.1 M HCl buffer (pH 2.35, 3.61), 0.1 M potassium hydrogen phthalate/0.1 M NaOH buffer (pH 4.17, 4.85, 5.77), 0.1 M potassium dihydrogen phosphate/0.1 M NaOH buffer (pH 6.36, 7.01), 0.025 M borax/0.1 M HCl buffer (pH 8.12), and 0.025 M borax/0.1 M NaOH buffer (pH 9.33, 10.64)] were used in these experiments. For these experiments, a PEC particle concentration of 0.5 g L-1 was used. Experiments of electrophoretic mobility, µE, were carried out using a Zeta-Meter System 3.0+. The ζ-potential of the PEC particles was calculated from µE by employing the Smoluchowski relationship29 ζ)
µ Eη 0r
(1)
where 0 is the permittivity of a vacuum, r is the relative dielectric permittivity of the medium, and η is the viscosity of the dispersing phase. 2.3.5. Particle Size. The average diameter size of chitosan/poly(methacrylic acid) cross-linked particles, dhp, was determined using a laser granulometer Cilas 920L at room temperature, using water as the continuous phase and an ultrasonication time of 60 s. 2.3.6. Particle Density. The cross-linked particle density was determined by classic water picnometry (using Archimedes principle30). A given mass of particles, mp, was added to a picnometer of volume VT (this volume was determined using bidistilled water as a standard liquid). Water was added to the picnometer to complete its volume, and the mass of added water, mw, was determined. The particle density was related to VT using the relationship VT ) Vp + Vw )
mp m w + Fp Fw
(2)
where Vw and Vp are, respectively, the volumes occupied by water and particles and Fp and Fw are the densities of the particles and water, respectively. Rearranging eq 2, it follows that
(
Fp ) mp VT -
)
mw Fw
-1
(3)
2.4. Adsorption Experiments. Cross-linked particles (Section 2.2.2.) were used in these experiments. Starting from particles with rCOOH/NH2 ) 5.6 at pH ) 3.04, the influence of the following parameters on BSA adsorption was studied: increase in rCOOH/NH2 to 11.1, pH remaining constant (pH ) 3.04) and increase in pH to 5.09, rCOOH/NH2 remaining constant (rCOOH/NH2 ) 5.6). The buffer system used in these experiments was made of 0.2 M sodium hydrogen phosphate/0.1 M citric acid. The adsorption process was studied in terms of adsorption isotherm and surface charge monitoring, as described in the next sections. 2.4.1. Adsorption Isotherms. Adsorption of bovine serum albumin on CS/PMAA particles was carried out at 22 ( 2 °C by mixing VA ) 5 mL of albumin solution at a given concentration C0 to a mass (mp ) 500 mg) of cross-linked particles (Section 2.2.2.) in tubes. The mixture was maintained for 48 h at 22 ( 2 °C to reach equilibrium (being manually shaken from time to time) and fed into centrifuge cuvettes. Subsequently, the cuvettes were twice centrifuged for 10 min at 4000 rpm and 22 ( 2 °C (Hermle centrifuge, model Z200A, Hermle Labortechnik). After centrifugation, the solid particles were collected for surface charge analysis, as described in Section 2.4.2., and the protein concentration in the supernatant, CE, was determined using a UV spectrophotometer (Genesys 10-UV/vis, (29) de Vasconcelos, C. L.; de Moura, K. T.; Morais, W. A.; Dantas, T. N. C.; Pereira, M. R.; Fonseca, J. L. C. Colloid Polym. Sci. 2005, 283, 413. (30) Saliba, C. C.; Orefice, R. L.; Rubens, J.; Carneiro, G.; Duarte, A. K.; Schneider, W. T.; Fernandes, M. R. F. Polym. Test 2005, 24, 819.
Thermo Electron Corporation) from the intensity of a band at 278 nm and previously built calibration curves for BSA solutions. The values of absorbance were determined using as a blank the supernatant resultant from the interaction of 5 mL of the pure buffer solution with 500 mg of the PEC particles. The surface density, Γ, of the protein was calculated as Γ)
(C0 - CE)VAFpdhp 6mp
(4)
where Fp is the density of the particle and dhp is the average diameter of the particle. When analyzing the adsorption dependence with time, a similar procedure as described previously was carried out, with the following differences: (i) The albumin solution volume was 100 mL (PEC particle mass being proportionally increased to 1 g). (ii) The mixture was submitted to mechanical stirring (2100 rpm) during all adsorption experiments, to discard from the kinetic analysis phenomena such as albumin percolation through sedimented PEC particles, as well as to ensure that particles would be homogeneously distributed through the continuous phase when collecting the sample at a given time t. As previously pointed out, at a given time t, a volume of 5 mL was collected from the dispersion and submitted to centrifugation. At the same time, a sample with the same volume was collected from another dispersion prepared exactly in the same way but without albumin (pure buffer replaced the albumin solution). This dispersion was also centrifuged, and the supernatant was used as a blank for the determination of the supernatant absorbance in the albumin adsorption experiment. The determination of Γ was carried out using eq 4. 2.4.2. Surface Charge Monitoring. The particles obtained as a result of a given centrifugation carried out as described in the previous section were redispersed in the same buffer used for the preparation of the original dispersion. Electrophoretic mobility was immediately measured as described in Section 2.3.4., to estimate the effect of adsorption of albumin on the particle ζ-potential, in other words, on the particle surface charge.
3. Results and Discussion 3.1. FTIR Spectroscopy. FTIR spectra for PMAA, PEC particles, and CS are shown in Figure 2. The FTIR spectrum of CS itself showed characteristic peaks of amide groups: amide I and amide II bands at 1647 and 1598 cm-1, respectively. The intense band at around 3420 cm-1 should be assigned to the stretching vibration of O-H and/or N-H, as well as to intermolecular hydrogen bonding within the polysaccharide.31 The absorption bands at 1154 (antisymmetric stretching of the C-O-C bridge), 1078, and 1031 cm-1 (skeletal vibrations involving the C-O stretching) are characteristic of chitosan’s saccharide structure.32 PMAA, in acidic medium, is characterized by a hypercoiled compact structure, stabilized by hydrophobic interactions of R-methyl groups and the formation of intramolecular hydrogen bonding,33 characterized in this work by a wider band at 3453 cm-1 in the spectrum of PMAA. One can see in the FTIR spectrum of PMAA the presence of bands at 1715-1175 cm-1, assigned to C-O and C-H stretching vibrations.31 The absorption band at 1716 cm-1 in the FTIR spectrum of PMAA has been assigned to the CdO stretching vibration in the carboxylic groups. One can see in the spectrum of the PEC particles of CS/PMAA a shift from 1716 to 1701 cm-1, attributed to the carboxyl absorption band from PMAA; the amide bands in the CS spectrum disappear (31) Sun, T.; Xu, P. X.; Liu, Q.; Xue, J. A.; Xie, W. M. Eur. Polym. J. 2003, 39, 189. (32) Peniche, C.; Elvira, C.; Roman, J. S. Polymer 1998, 39, 6549. (33) Kudryashova, E. V.; Gladilin, A. K.; Izumrudov, V. A.; van Hoek, A.; Visser, A. J. W. G.; Levashov, A. V. Biochim. Biophys. Acta 2001, 1550, 129.
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Figure 2. FTIR spectra of CS, PMAA, and PEC (rCOOH/NH2 ) 5.6).
(the amide II band could be overlapped by the unprotonated NH2 bending band, which would disappear, as the amino groups were protonated), and a new distinct band appears at 1637 cm-1, which can be assigned to the absorption band of NH3+ from CS. Furthermore, the absorption bands at 1541 and 1388 cm-1, present in the spectrum of CS/PMAA particles, resulted from asymmetrical and symmetrical [OdC-O]- stretching vibrations.34,35 Chitosan is insoluble in pure water, complete solubilization taking effect when MAA is added to the solution. It indicates that, like acetic acid in ordinary chitosan solutions, MAA protonates NH2 groups from chitosan. As polymerization begins, the propagation of the PMAA chains results in an increase in charge density: as it becomes critical, insoluble polyelectrolyte complex particles between chitosan and PMAA are formed through electrostatic interactions. Of course, hydrophobic interactions and hydrogen bonding cannot be disregarded. FTIR results confirm polyelectrolyte complexation between the dissociated carboxylic groups of PMAA and the protonated amino groups of CS. 3.2. XRD Measurements. Figure 3 depicts X-ray diffractograms for CS, PEC particles of different rCOOH/NH2, and PMAA. The X-ray diffraction profile of chitosan consists of two reflection falls at 2θ ) 11.6 and 20.1°, which are in agreement with data already reported in the literature.36,37 Chitosan has two distinct crystal forms I and II. The crystal form I is orthorhombic, having a unit cell with a ) 7.76 Å, b ) 10.91 Å, and c ) 10.30 Å. The reflection fall at 2θ ) 11.6° has been assigned to crystal form I, which corresponds to the diffraction angle on the (100) plane. The strongest reflection at 2θ ) 20.1° has been assigned to crystal form II, which also corresponds to the diffraction angle on the (100) plane. The crystal form II also is orthorhombic (34) Peniche, C.; Arguelles-Monal, W.; Davidenko, N.; Sastre, R.; Gallardo, A.; San, Roman, J. Biomaterials 1999, 20, 1869. (35) Hu, Y.; Jiang, X. Q.; Ding, Y.; Ge, H. X.; Yuan, Y. Y.; Yang, C. Z. Biomaterials 2002, 23, 3193. (36) Wu, Y.; Zheng, Y. L.; Yang, W. L.; Wang, C. C.; Hu, J. H.; Fu, S. K. Carbohydr. Polym. 2005, 59, 165. (37) Zong, Z.; Kimura, Y.; Takahashi, M.; Yamane, H. Polymer 2000, 41, 899.
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Figure 3. X-ray diffractograms of chitosan (a), PEC [rCOOH/NH2 ) 5.6 (b) and rCOOH/NH2 ) 11.1 (c)], and PMAA (d).
Figure 4. SEM image of a set of PEC particles (rCOOH/NH2 ) 5.6).
having a unit cell of a ) 4.4 Å, b ) 10.0 Å, and c ) 10.3 Å. Wan et al.38 reported that chitosan crystallinity results from hydrogen bonding within chitosan chains, which is connected to the chitosan deacetylation degree: as the deacetylation degree increases, macromolecular chains become more flexible (better packing), with a higher number of NH2 groups (higher number of hydrogen bonds), increasing crystallinity. The X-ray diffraction profile of PEC showed that the complexation reaction modified the crystallinity of chitosan. One can see that for rCOOH/NH2 ) 5.6 and 11.1, the diffractograms are identical to the one for pure PMAA. The strongest broad reflection falls at 2θ ≈ 16 and 32°, as well as the lack of sharp peaks in (38) Wan, Y.; Creber, K. A. M.; Peppley, B.; Bui, V. T. Polymer 2003, 44, 1057.
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Figure 5. Diameter distributions for the cross-linked PEC particles. Circles: rCOOH/NH2 ) 5.6. Squares: rCOOH/NH2 ) 11.1. Figure 7. Isothermal adsorption curves for BSA adsorbed on CS/ PMAA complexes. Circles: rCOOH/NH2 ) 5.6 and pH ) 3.04. Squares: rCOOH/NH2 ) 5.6 and pH ) 5.09. Triangles: rCOOH/NH2 ) 11.1 and pH ) 3.04.
the diffractograms of the complexes, and pure PMAA has been found elsewhere for amorphous PMAA microgels.39 3.3. ζ-Potential of Noncross-Linked Particles. Figure 6 shows the ζ-potential as a function of pH for different values of rCOOH/NH2. One can see that the particles presented negative surface charge at the used pH range. This result is consistent with a complexation mechanism based on electrostatic interactions between the
oppositely charged polyelectrolytes, in which positive chitosan chains are covered by layers of negatively charged chains of poly(methacrylic acid), PMAA.40 One can also observe that the ζ-potential became more negative as the pH was increased. This is the result of both neutralization of PMAA carboxyl groups and deprotonation of NH2 groups from chitosan. One can also observe that, at constant pH, the ζ-potential seemed to be independent of rCOOH/NH2 (within the associated experimental errors). Finally, it was found that particles were not stable (they were resolubilized) out of the pH range used in Figure 6 (from 4.3 to 5.6). If one has in mind that pKa ) 6.5 and 4.75 for CS and PMAA, respectively,4 in stronger acidic conditions, such as pH < 3.5, most of the carboxylic groups of PMAA are not ionized (although chitosan would be fully protonated), resulting in a low formation of complex. On the other hand, in conditions pH > 6.0, although part of the carboxyl groups is neutralized, amino groups from CS are not protonated, disfavoring the formation of polyelectrolyte complex. 3.4. BSA Adsorption on Cross-Linked Particles. Crosslinking with glutardialdehyde turned out the particles resistant to pH at which they were previously soluble. This is the reason as to why adsorption experiments were carried out with these particles. These particles had rather broad bimodal size distribution, with an average diameter of 45 µm (rCOOH/NH2 ) 5.6) and 29 µm (rCOOH/NH2 ) 11.1) and were both irregularly shaped. As a consequence, it was assumed that differences in adsorption behavior mainly could be related to surface chemistry. Figure 4 shows a characteristic SEM image obtained for the cross-
(39) Zhang, Y.; Fang, Y.; Wang, S.; Lin, S. Y. J. Colloid Interface Sci. 2004, 272, 321.
(40) de Vasconcelos, C. L.; Bezerril, P. M.; dos Santos, D. E. S.; Dantas, T. N. C.; Pereira, M. R.; Fonseca, J. L. C. Biomacromolecules 2006, 7, 1245.
Figure 6. ζ-Potential of PEC as a function of pH. Circles: rCOOH/NH2 ) 5.6. Squares: rCOOH/NH2 ) 8.3. Diamonds: rCOOH/NH2 ) 11.1.
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Figure 8. Scheme representing proposed brush-like structure occurring on PEC particle’s surface.
linked PEC particles used in this work, and Figure 5 depicts their broad bimodal size distributions. 3.4.1. Adsorption Isotherms. Figure 7 shows BSA surface density, Γ, as a function of BSA equilibrium concentration in the supernatant, CE, for the adsorption of BSA on CS/PMAA complexes with rCOOH/NH2 and pH values used in this work. For rCOOH/NH2 ) 5.6 and pH ) 3.04, one can see that there is a maximum with Γmax ≈ 0.28 g m-2 at CE ≈ 1 g dm-3. A similar behavior has been predicted in some models relating to the adsorption of supercritical fluids on oxides, using pseudo-latticebased approaches involving the energy of interaction adsorbateadsorbate, , and adsorbate-surface, s.41 Since at this pH chitosan is highly protonated and PMAA is not ionized, in electrostatic terms, there is repulsion between the positively charged particles and the positively charged BSA. Adsorption will occur mainly via hydrogen bonding and/or van der Waals interactions. On the other hand, the decrease in Γ may be analyzed as a consequence that, in the continuous phase, there are negatively charged polyvalent species (e.g., phosphate and citrate, from the buffer), which increase the possibility of adsorbent-adsorbent interactions (via the formation of a bridge), as the concentration of protein is increased. Another possibility is the occurrence of depletion flocculation,42 as a result of the increase in protein concentration. Increasing the pH to 5.09 did not have a marked effect on the isotherm, as one may confirm by analyzing Figure 7. The maximum in adsorption still is at 1 g dm-3; Γmax, however, is around 0.55 g m-2, indicating that adsorption was much more effective as the pH was increased. The isoelectric point of BSA is at pH 4.7-5.0,25 which means that, regarding our experiments, the decrease in net surface charge of BSA resulted in a higher adsorption. Peng et al.43 found that the maximum adsorption of
BSA on positively charged magnetic particles occurred at the isoelectric point of BSA. Roach et al.44,45 found that the adsorption of albumin onto the hydrophobic (CH3-terminated) surface was higher than that onto the hydrophilic (OH-terminated) surface,
(41) Donohue, M. D.; Aranovich, G. L. AdV. Colloid Interface Sci. 1998, 77, 137. (42) de Vasconcelos, C. L.; Pereira, M. R.; Fonseca, J. L. C. J. Dispers. Sci. Technol. 2005, 26, 59.
(43) Peng, Z. G.; Hidajat, K.; Uddin, M. S. J. Colloid Interface Sci. 2004, 271, 277. (44) Roach, P.; Farrar, D.; Perry, C. C. J. Am. Chem. Soc. 2005, 127, 8168. (45) Roach, P.; Farrar, D.; Perry, C. C. J. Am. Chem. Soc. 2006, 128, 3939.
Figure 9. Albumin adsorption density, Γ, as a function of time, t, for PEC particles with rCOOH/NH2 ) 5.6 at pH ) 5.09. Circles: C0 ) 0.37 g dm-3. Squares: C0 ) 2.5 g dm-3.
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Figure 11. Scheme representing a possible interaction via hydrogen bonding between a carbonyl group from albumin and a carboxyl group from a PEC particle. As a result of this interaction, the equilibrium is shifted in the direction of protonation of carboxylate groups from PEC particles.
Figure 10. ζ-Potential of PEC particles as a function of CE. Circles: rCOOH/NH2 ) 5.6 and pH ) 3.04. Squares: rCOOH/NH2 ) 5.6 and pH ) 5.09. Triangles: rCOOH/NH2 ) 11.1 and pH ) 3.04.
due to a higher binding affinity toward the hydrophobic surface. Their work, however, was carried out at pH ) 7.4 (albumin was negatively charged, contrary to the conditions of the present work), and there were now data on the surface charge on the solid surface (via formation of a double layer). In other words, there is the possibility that the higher adsorption in a less hydrophilic surface was, in fact, a higher adsorption of a negatively charged particle in a less negative surface. Despite that, using the same arguments pointed out by Roach et al., the increase in albumin adsorption could be due to the formation of a less hydrophilic particle surface since NH3+ would be deprotonated by increasing pH. Conversely, new COOH groups from the particle would be neutralized, resulting in a more hydrophilic surface: in this case, however, the interaction with positively charged regions of albumin (although the net charge of BSA is near zero) with the structure generated by the neutralization of the carboxyl groups could increase adsorption. Regarding data related to the adsorption of BSA at pH ) 3.04 and rCOOH/NH2 ) 11.1, one also can see in Figure 7 that the increase in rCOOH/NH2 modified the isotherm profile: there is a more pronounced rise in adsorption at lower concentrations and the occurrence of a plateau between CE ) 0.1 and 0.6 g dm-3. On the other hand, there is also a maximum at CE ) 1 g dm-3, the value of Γmax being 0.35 g m-2, which is higher than the value found for rCOOH/NH2 ) 5.6, followed by a continuous increase in Γ. The increase in rCOOH/NH2 results in the increase of the possibility of a brush-like structure46 made of PMAA chains (a possible structure is pictorially represented in Figure 8): these structures would increase albumin adsorption via hydrogen bonding/van der Waals interactions. The adsorption of albumin (46) Uhlmann, P.; Houbenov, N.; Brenner, N.; Grundke, K.; Burkert, S.; Stamm, M. Langmuir 2007, 23, 57.
on latex particles with brush-structured surfaces already has been reported in the literature.47 A kinetic analysis can be carried out at different regions of a given adsorption isotherm to obtain a more detailed description of the adsorption process. From Figure 7, we chose to perform this analysis with the albumin adsorption isotherm at pH ) 5.09 on PEC particles with rCOOH/NH2 ) 5.6 (due to the strong albumin adsorption). Since this isotherm presents a very pronounced maximum, we chose to perform the kinetic analysis at two regions: before the adsorption maximum (at CE ≈ 0.1 g dm-3) and after the adsorption maximum (at CE ≈ 2 g dm-3). The initial albumin solution concentrations for these two situations were, respectively, C0 ) 0.37 g dm-3 and C0 ) 2.5 g dm-3. Figure 9 shows the dependence of Γ in relation to time at the mentioned concentrations. It can be seen that in the region before the adsorption maximum, Γ continuously increases and tends to a value around 0.3 g m-2, which is the same observed in the equilibrium experiments. Conversely, for the region after the adsorption maximum, there is no adsorption at all: it does not agree with the equilibrium experiments, which yielded Γ ≈ 0.2 g m-2 at the same value of C0. What, at a first glance, seemed to be an inconsistency, though, is more evidence of the depletion effect. As pointed out in the Experimental Procedures, equilibrium experiments were carried out in the absence of high shear, while kinetic experiments were performed with strong mechanical stirring: if depletion was important, stirring could provide kinetic energy to break weak albumin-PEC particle surface interactions through shearing. As a result, there would be no albumin adsorption in this region. Anastassopoulos et al. have also found that shearing can induce desorption in systems based on polymer brushes.48 To be sure of the role of electrostatic interactions on the process of albumin adsorption, an analysis on the surface charge of the particles during adsorption must be performed. Such an analysis is described in the next section. (47) Wittemann, A.; Ballauff, M. Anal. Chem. 2004, 76, 2813. (48) Anastassopoulos, D. L.; Spiliopoulos, N.; Vradis, A. A.; Toprakcioglu, C.; Baker, S. M.; Menelle, A. Macromolecules 2006, 39, 8901.
7694 Langmuir, Vol. 23, No. 14, 2007
3.4.2. Surface Charge Analysis. Figure 10 shows the ζ-potential as a function of CE for BSA adsorption at pH and rCOOH/NH2 values used in this work. Regarding the medium with pH ) 3.04 and rCOOH/ NH2 ) 5.6, one can see that the particles presented a positive charge at this pH, which is expected since chitosan is fully protonated and ionization in PMAA is minimal (as pointed out in Section 3.3.). As BSA is adsorbed on the particle surface, the surface charge tends to more positive values, which is consistent with the positive charge of BSA at this pH. The effect of increasing pH to 5.09, rCOOH/NH2 being maintained at 5.6, also can be seen in Figure 10: the particle surface now is negative, which is consistent with the ionization of the COOH external groups. The ζ-potential becomes less negative as albumin is adsorbed, so that two hypotheses can be drawn: (i) BSA is positively charged at this pH; this does not seem to be the case since BSA is very close to its isoelectric point and (ii) BSA interacts (via hydrogen bonding) with nonionized carboxyl groups from PMAA, resulting in a decrease in carboxylate concentration. Figure 11 shows a possibility of interaction between carbonyl groups from albumin and carboxyl groups from PEC particles: as a result of this interaction, the PEC particle surface would be less negative. Finally, a third glance at Figure 10 shows the result of increasing rCOOH/NH2 to 11.1 (pH ) 3.04) on the ζ-potential. It can be seen that, prior to the adsorption of BSA (CE ) 0), the particles had ζ around their isoelectric point, in opposition to the situation in which rCOOH/NH2 ) 5.6, in which the particles were positively charged. The reason for the decrease in charge is the increase in carboxyl groups from PMAA: the decrease in charge would enhance nonelecrostatic adsorption of albumin, apart from the
de Vasconcelos et al.
occurrence of brush structures, as was already described in the latter section.
4. Conclusion Particles made of cross-linked CS/PMAA were obtained via template polymerization, these particles being resistant to solubilization even at a pH near 3. The ζ-potential measurements of the particles before cross-linking showed that the particles had their external layers rich in PMAA. BSA was adsorbed on cross-linked CS/PMAA particles both when BSA was positively charged and when near its isoelectric point. Adsorption occurred despite the fact that CS/PMAA particles were negatively or positively charged, showing the importance of nonelectrostatic interactions in the process of BSA adsorption onto these particles. an increase in the PMAA content in the particles, which favors the occurrence of brush structures on them, also favored BSA adsorption. On the other hand, there were regions in the adsorption isotherm where the increase in the continuous phase albumin concentration favored the occurrence of depletion from the PEC surface, shearing contributing to enhance this effect. Acknowledgment. The authors thank Brazil’s Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Ministe´rio da Cieˆncia e Tecnologia (MCT), Fundac¸ a˜o Coordenac¸ a˜o de Aperfeic¸ oamento de Pessoal de Nı´vel Superior (CAPES), FINEP, CT-PETRO, and Pro´-Reitoria de Pesquisa da Universidade Federal do Rio Grande do Norte (PROPESQ-UFRN) for financial support during the course of this work. LA700537T
