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Interaction of Organic Molecules with Weak Amphoteric Charged Membrane Surfaces: Effect of Interfacial Charge Structure Hidetoshi Matsumoto,† Yoshiyuki Koyama,‡ and Akihiko Tanioka*,† Department of Organic and Polymeric Materials, and International Research Center of Macromolecular Science, Tokyo Institute of Technology, 2-12-1 Ookayama, Meguro-ku, Tokyo 152-8552, Japan, and Department of Home Economics, Otsuma Women’s University, 12 Sanban-cho, Chiyoda-ku, Tokyo 102-8357, Japan Received October 15, 2001. In Final Form: January 22, 2002 Weak amphoteric charged membranes were prepared by graft copolymerization of poly(ethylene glycol) (PEG) derivatives with pendant ionizable groups onto porous polyethylene (PE) membranes. Two types of weak amphoteric charged membranes and two types of weak single charged membranes were prepared and then characterized by zeta (ζ) potential and dynamic contact angle measurements. To identify the characteristics of interactions between organic molecules and membrane surfaces, we used four kinds of model organic molecules: pyrene (highly hydrophobic), 1-pyrenesulfonic acid (having an anionic group), 1-pyrenemethylamine (having a cationic group), and 1-aminopyrene (having a polar group). The time dependence of the adsorbed amount on the membrane was investigated with fluorescence spectroscopy. The results demonstrated that ionic PEG chains on the membrane surfaces reduced the adsorption of hydrophobic adsorbents and interacted with polar groups by hydrogen bonding. On the other hand, for ionic adsorbents, electrostatic interaction played an important role on the surfaces of oppositely charged PEG chain grafted membranes. The electrostatic interactions were correlated with the interfacial charge properties obtained from the ζ potential measurements.
1. Introduction Weak amphoteric charged membranes are expected to be the next-generation charged membranes for the following features: controllability of the charge property by changing the pH of the outer solution and potentiality as an antifouling material that prevents adsorption of organic molecules and biological macromolecules on the surface.1-12 These properties can be applied to medical devices, drug delivery systems, and separation of ionic drugs and proteins.5-12 We have proposed the architecture of the novel amphoteric charged membranes by surface modification using poly(ethylene glycol) (PEG) derivatives with pendant ionizable groups.12 The ionic PEG derivatives were synthesized by addition of mercaptans to the double bond pendants of copoly(allyl glycidyl ether/ethylene oxide) * To whom correspondence should be addressed. Telephone: +813-5734-2426.Fax: +81-3-5734-3659.E-mail:
[email protected]. † Tokyo Institute of Technology. ‡ Otsuma Women’s University. (1) Elmidaoui, A.; Boutevin, B.; Belcadi, S.; Gavach, C. J. Polym. Sci. B: Polym. Phys. 1991, 29, 705. (2) Saito, K.; Tanioka, A. Polymer 1996, 37, 2299. (3) Saito, K.; Ishizuka, S.; Higa, M.; Tanioka, A. Polymer 1996, 37, 2493. (4) Ramı´rez, P.; Alcaraz, A.; Mafe´, S. J. Electroanal. Chem. 1997, 436, 119. (5) Jimbo, T.; Higa M.; Minoura, N.; Tanioka, A. Macromolecules 1998, 31, 1277. (6) Jimbo, T.; Tanioka A.; Minoura, N. Langmuir 1998, 14, 7112. (7) Jimbo, T.; Tanioka A.; Minoura, N. Colloids Surf. A 1999, 159, 459. (8) Jimbo, T.; Tanioka A.; Minoura, N. Langmuir 1999, 15, 1829. (9) Jimbo, T.; Ramı´rez, P.; Tanioka, A.; Mafe´, S.; Minoura, N. J. Colloid Interface Sci. 2000, 225, 447. (10) Uematsu, I.; Jimbo, T.; Tanioka, A. J. Colloid Interface Sci. 2002, 245, 319. (11) Matsumoto, H.; Koyama, Y.; Tanioka, A. J. Colloid Interface Sci. 2001, 239, 467. (12) Matsumoto, H.; Koyama, Y.; Tanioka, A. Langmuir 2001, 17, 3375.
(copoly(AGE/EO)).12-14 These polyelectrolytes have both the advantages from the polyether backbone structure, such as high hydrophilicity, biocompatibility, and nonspecific adsorption resistance,15-21 and functionality from the pendant charge groups. We have prepared ionic PEG chain grafted type weak amphoteric charged membranes by the radiation-induced graft copolymerization of polyelectrolytes onto porous polyethylene (PE) membranes, and characterized the pH dependence of their interfacial charge properties by zeta (ζ) potential measurements.12 The interfacial charge structure of weak amphoteric charged membranes, which contain both weak acidic groups and weak basic groups, is so complex that the interfacial properties have not been elucidated in detail. Particularly, there have been few physicochemical studies on the correlation between interfacial properties of weak amphoteric charged membranes and their interactions with organic molecules. In this study, we prepared two types of ionic PEG chain grafted weak amphoteric charged (13) Koyama, Y.; Umehara, M.; Mizuno, A.; Itaba M.; Yasukouchi, T.; Natsume, K.; Suginaka, A. Bioconjugate Chem. 1996, 7, 298. (14) Koyama, Y.; Ito, T.; Matsumoto, H.; Tanioka, A.; Okuda, T.; Aoyagi, H.; Niidome, T. Submitted for publication in J. Biomater. Sci. Polym. Ed. (15) Poly(Ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications; Harris, J. M., Ed.; Plenum Press: New York, 1992. (16) Poly(Ethylene Glycol): Chemical and Biological Applications; Harris, J. M., Zalipsky, S., Eds.; ACS Symposium Series 680; American Chemical Society: Washington, DC, 1997. (17) Jeon, S. I.; Lee, J. H.; Andrade J. D.; De Gennes, P. G. J. Colloid Interface Sci. 1991, 142, 149. (18) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714. (19) Bergstro¨m, K.; Holmberg, K.; Safranji, A.; Hoffman, A., S.; Edgell, M. J.; Kozlowski, A.; Hovanes, B. A.; Harris, J. M. J. Biomed. Mater. Res. 1992, 26, 779. (20) O ¨ sterberg, E.; Bergstro¨m, K.; Holmberg, K.; Riggs, J. A.; Van Alstine, J. M.; Schuman, T. P.; Burns, N. L.; Harris, J. M. Colloid Surf. A 1993, 77, 159. (21) O ¨ sterberg, E.; Bergstro¨m, K.; Holmberg, K.; Schuman, T. P.; Riggs, J. A.; Burns, N. L.; Emoto, K.; Van Alstine, J. M.; Harris, J. M. J. Biomed. Mater. Res. 1995, 29, 741.
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Figure 2. Chemical structures of organic molecules. Figure 1. Schematic representation of ideal morphological model for grafted polymer chains on the pore surface of (a) ASC-type amphoteric charged membrane, (b) MGC-type amphoteric charged membrane, (c) acidic-type single charged membrane, and (d) basic-type single charged membrane, in the ionized state.
membranesthe amphoteric ion pair side chain (ASC) type and the mixed grafted chain (MGC) typesand two types of the weak single charged membranes acidic type and basic type (Figure 1). Their grafted polymer chains on the surface are expected to exclude hydrophobic interaction and to have a function based on electrostatic interaction. To identify the characteristics of interactions between organic molecules and the ionic PEG chain grafted membrane surfaces (electrostatic interaction, hydrophobic interaction, or hydrogen bonding), we carefully selected four kinds of model organic molecules: pyrene (highly hydrophobic), 1-pyrenesulfonic acid (having an anionic group), 1-pyrenemethylamine (having a cationic group), and 1-aminopyrene (having a polar group). We chose pyrene and its derivatives for two reasons. First, the pyrene ring is highly hydrophobic. Second, their strong fluorescence allows accurate determination of the degree of adsorption at the membrane/solution interface, even at very low concentrations.22 The time dependence of the adsorbed amount of these organic molecules onto the membranes was investigated with fluorescence spectroscopy. We present here the first findings of the interactions between the organic molecules and amphoteric charged membrane surfaces under the condition without other electrolytes. The aims of this paper are (1) to identify the characteristics of interactions between organic molecules and the ionic PEG chain grafted membranes and (2) to examine the correlation between the interfacial charge properties of ionic PEG chain grafted membranes and their interactions with charged molecules. 2. Experimental Section 2.1. Materials. Pyrene (extra-pure grade) and 1-pyrenesulfonic acid, sodium salt, were purchased from Wako Pure Chemical, Japan, and Molecular Probes, USA, respectively. 1-Aminopyrene (97% purity) and 1-pyrenemethylamine hydro-
chloride (95% purity) were purchased from Aldrich Chemical, USA. All reagents were used without further purification. The chemical structures are shown in Figure 2. Copoly(allyl glycidyl ether/ethylene oxide) (copoly(AGE/EO),13 AGE/EO ) 13.6/86.4 in molar ratio, M h n ) 3260, M h w/M h n ) 1.05) was provided by NOF, Japan. L-Cysteine hydrochloride monohydrate, 2-aminoethanethiol hydrochloride, 3-mercaptopropionic acid, methanol, chloroform, diethyl ether, acetone, and potassium chloride were purchased from Wako Pure Chemical, Japan. These reagents were of extra-pure grade. High-density polyethylene (PE) porous membranes (Hipore N720, 25 µm thick and 50 nm average pore size) were obtained from Asahi Kasei, Japan. 2.2. Synthesis of PEG Derivatives with Ionizable Groups. We synthesized the PEG derivatives with pendant ionizable groups by adding weak electrolytes having a highly reactive mercapto group to the double bond side chains of copoly(AGE/ EO).12-14 Here, the PEG derivatives having amino groups (PEGA), carboxyl groups (PEG-C), and the amphoteric ion pair, cysteine residues (PEG-Cys), were synthesized using 2-aminoethanethiol, 3-mercaptopropionic acid, and L-cysteine, respectively, as previously reported.12 The structures of ionic PEG derivatives used in this study are shown in Figure 3. 2.3. Membrane Preparation. Two types of amphoteric charged membranes, the ASC type and the MGC type, and two types of single charged membranes, the acidic type and the basic type, were prepared by radiation-induced graft copolymerization as previously described.12 PEG-Cys, the mixture of PEG-A and PEG-C (mixture ratio of -NH2 in PEG-A to -COOH in PEG-C was 1:1 in moles), PEG-C, and PEG-A were used as the grafting polymer for the ASC-type and MGC-type amphoteric charged membranes and the acidic-type and basic-type single charged membranes, respectively. The PE porous membranes were used as the substrate on which the PEG derivatives were graft polymerized. The circular PE membrane samples (area ) 17.35 cm2) were extracted in methanol for 12 h before use. After soaking in methanol and then in deionized water, these membranes were immersed in 16 wt % polymer aqueous solution on a glass plate. This glass plate was covered with another one, and then the plates were sealed with cellophane tape. The plates were irradiated with 1.17 and 1.33 eV γ-rays using a 60Co generalpurpose irradiation device (Model RE-1012, Toshiba, Japan) at (22) Oya, T.; Enoki, T.; Grosberg, A. Y.; Masamune, S.; Sakiyama, T.; Takeoka, Y.; Tanaka, K.; Wang, G.; Yilmaz, Y.; Feld, M. S.; Dasari, R.; Tanaka, T., Science 1999, 286, 1543.
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Matsumoto et al. Table 1. Physicochemical Properties of Unmodified and Grafted Membranes
membrane ASC type MGC type acidic type basic type unmodified PE
water contact ζ DGa permeabilityb anglec potentiald 2 (w/w %) (mL/m ‚h‚cmH2O) (deg) (mV) 4.5 4.5 2.7 5.7
36 47 52 34 72e
86 85 85 79 102
-5.4 -0.2 -7.9 +2.9 -4.8e
a DG ) (W - W )/W , where W ) the weight of the dried 2 1 1 1 membrane before graft copolymerization and W2 ) the weight of the dried membrane after graft copolymerization. b Measured in deionized water at pH 5.6. All water permeability values were reproducible within (2 mL/m2‚h‚cmH2O. c Dynamic sessile drop method, Measured with distilled water at pH 5.6. All contact angle values were reproducible within (3°. d Measured in 1mM KCl at pH 5.6. All ζ potential values were reproducible within (1.2 mV. e Measured after immersion in MeOH.
Figure 3. Chemical structures of PEG derivatives with ionizable groups. dose rates of 2.47 kGy/h and total doses of 50 kGy. The grafted membranes were washed and then rinsed for 12 h with deionized water to sufficiently remove the nongrafted polymers. The degree of polymerization (DG) and physicochemical properties of the unmodified and grafted membranes are listed in Table 1. 2.4. Measurements. 2.4.1. Hydraulic Permeability Measurements. The hydraulic permeability of the membranes was calculated from the flow rate of deionized water, which was eluted for 20 min under an applied constant hydrostatic pressure difference of 75.0 cmH2O across the membrane. The area of the membrane exposed to the flow was 4.9 cm2. All measurements were done five times at 25 ( 0.1 °C and were reproducible to within (2 mL/m2‚h‚cmH2O. 2.4.2. ζ Potential Measurements. The experimental setup is the same as that previously mentioned.5-7,11,12 The membrane was placed between two 64-cm3 compartments. Hydrostatic pressure across the membrane was applied by changing the solution levels of both reservoirs. The area of the membrane exposed to the flow was 1.8 cm2. The electrodes were made of Ag/AgCl. By varying the applied pressure (∆P) ranging from 15 to 120 cmH2O, the streaming potential (∆E), which had been generated by a flow of ions due to ∆P, was measured with a digital multimeter (HP3458A, Hewlett-Packard, USA), and the data were recorded using a microcomputer. The ζ potential was obtained from the slope of a ∆E-∆P plot using the following Helmholtz-Smoluchowski equation:5-7,11,12,23
Figure 4. Schematic diagram of adsorption experiment.
where η is the solution viscosity and and λ are the permittivity and electrical conductivity of the solution, respectively. The KCl concentration in the outer solution was 1 mmol/L at pH 5.6 throughout the measurements. All measurements were carried out five times at 25 ( 0.1 °C and were reproducible to within (1.2 mV. 2.4.3. Dynamic Contact Angle (DCA) Measurements. The dynamic contact angle measurements were carried out on the freeze-dried membranes using an automatic dynamic contact angle meter (DCA-VZ, Kyowa Interface Science, Japan) employing the sessile drop technique with distilled water at pH 5.6. This instrument is equipped with automatic image acquisition and computation software for the time dependence of the contact angle. Dynamic contact angles were measured by reading automatically the angle between the tangent at the contact point and the horizontal line of the solid at 33-ms intervals and recording the data using a microcomputer from 100 to 1600 ms
after a drop. All measurements were carried out at least three times at 25 ( 1 °C and were reproducible within 3°. 2.4.4. Determination of Amount of Adsorbed Molecules. Before the adsorption experiments, we developed calibration curves for the model adsorbents. The fluorescence agent (equivalent weight of 200 mL of 2.5 µmol/L solution) was first dissolved in 1 mL of acetone (for pyrene) or methanol (for other pyrene derivatives). The solution was spread on the wall of a pear-shaped flask, and then the solvent was evaporated for removal under reduced pressure. After 200 mL of deionized water was added to the flask, the solution was ultrasonicated for 15 min and heated in a thermostated bath at 60 °C for 30 min to give 2.5 µmol/L aqueous solution. We measured the fluorescence of these adsorbent solutions and confirmed the absence of excimer emission peak of pyrene derivatives near 470 nm.24 The calibration curve was made at five measurement points from 2.5 × 10-8 to 2.5 × 10-7 mol/L: for pyrene, with 336 nm fluorescence excitation and 377 nm emission wavelength; for 1-pyrenesulfonic acid, with 347 nm fluorescence excitation and 379 nm emission wavelength; for 1-pyrenemethylamine, with 343 nm fluorescence excitation and 377 nm emission wavelength; and for 1-aminopyrene, with 355 nm fluorescence excitation and 442 nm emission wavelength. Fluorescence spectra were measured with a spectrofluorometer (FP-750, JASCO, Japan), using a 1-cm2 quartz cell (F10-UV-10, GL-Science, Japan). The emission and excitation slit widths equaled 5 and 5 nm, respectively. The schematic diagram of the adsorption experiments is shown in Figure 4. A freeze-dried test membrane (1 × 1 cm2) was immersed into 3 mL of 2.5 × 10-7 mol/L model adsorbent solution in the quartz cell. At the same time, fluorescence measurements were started and continued for 10-25 h. The concentration of the pyrene and pyrene derivatives in solution was determined by the calibration curves. The adsorbed organic molecules on the membrane were estimated from the decreased amount of organic molecules in solution. The adsorbed amount on the quartz cell was also deduced as a blank. Pyrene and pyrene derivative solutions were protected from light except for the fluorescence
(23) Hunter, R. J. Zeta Potential in Colloid Science: Principles and Applications; Academic Press: London, 1981.
(24) Galla, H.-J.; Sackmann, E. Biochim. Biophys. Acta 1974, 339, 103.
∆E ζ ) ∆P ηλ
(1)
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Figure 5. Fluorescence spectra of aqueous solutions at pH 5.6 and their calibration curves of (a) pyrene (λex ) 336 nm, λem ) 377 nm), (b) 1-pyrenesulfonic acid (λex ) 347 nm, λem ) 379 nm), (c) 1-pyrenemethylamine (λex ) 343 nm, λem ) 377 nm), and (d) 1-aminopyrene (λex ) 355 nm, λem ) 442 nm), emission and excitation slit widths ) 5 and 5 nm, respectively. measurements, because their solutions are photolytic. All experiments were carried out in the pH 5.6 solution at 25 ( 1 °C. The experiments were carried out three times for each membrane, and the mean value ((standard deviation) of each membrane is indicated.
3. Results and Discussion 3.1. Characterization of Interfacial Physicochemical and Charge Properties. The degree of graft polymerization (DG), water permeability, contact angle, and ζ potential of the prepared membranes are listed in Table 1. Here, the PE membrane is so hydrophobic that the water permeability and ζ potential measurements could be carried out after immersion in MeOH. The ASCtype, MGC-type, and basic-type membranes had similar DG values of about 5%, while the acidic-type membrane had a 2.7% DG. The difference in DG between the acidic type and the other types of membranes was caused by the
difference in the grafting efficiency during graft copolymerization. All of the grafted membranes have a smaller water permeability than an unmodified PE membrane. Ionic PEG chain grafting also facilitated the wettability and reduced contact angles. These results indicated that the pore surface of the PE porous membranes were covered with the ionic PEG chains by graft copolymerization. The interfacial charge properties of the membranes were evaluated from the ζ potentials in 1 mmol/L KCl at pH 5.6. The ζ potential of an unmodified PE membrane was -4.8 mV. Acidic-type (PEG-C grafted) and basic-type (PEG-A grafted) membranes were more negative, -7.9 mV, and more positive, +2.9 mV, than the PE membrane, respectively. The ASC- and MGC-type membranes had a -5.4 mV (similar to the PE membrane) and -0.2 mV ζ potential (more positive than the PE membrane), respec-
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Figure 6. Adsorbed amount of (a) pyrene (λex ) 336 nm, λem ) 377 nm), (b) 1-pyrenesulfonic acid (λex ) 347 nm, λem ) 379 nm), (c) 1-pyrenemethylamine (λex ) 343 nm, λem ) 377 nm), and (d) 1-aminopyrene (λex ) 355 nm, λem ) 442 nm) on unmodified and grafted membranes as a function of time at pH 5.6.
tively. For the ASC-type (PEG-Cys grafted) membrane, PEG-Cys chains on the pore surface are neutral at pH 5.6; however, the contribution of the negative charge on the pore surface of the original PE membrane is not negligible. On the other hand, for the MGC-type (PEGA/PEG-C grafted) membrane, this originated in the conformational difference between PEG-A and PEG-C chains: PEG-A chains would be more extended than PEG-C chains on the pore surface at pH 5.6. More detailed discussions were described in our previous paper.12 3.2. Adsorption Experiments. 3.2.1. Fluorescence Spectra and Calibration Curves of Organic Molecules. Fluorescence spectra and calibration curves of four kinds of organic molecules are shown in Figure 5. All calibration curves had good linearity (correlation factor R2 nearly equals 0.999). The concentration of the organic molecules in the solutions can be determined from the maximum fluorescence intensity at 377 nm for pyrene, 379 nm for 1-pyrenesulfonic acid, 377 nm for 1-pyrenemethylamine, and 442 nm for 1-aminopyrene on the basis of each calibration curve. 3.2.2. Adsorption Behavior of Organic Molecules. Figure 6 shows the adsorbed amount of organic molecules on
unmodified and grafted membranes as a function of time. In this section, we try to estimate the characteristics of the interfacial interaction based on the properties from the pyrene ring and the functional group of the pyrene derivatives. (a) Pyrene: Pyrene has high hydrophobicity. Adsorption amounts of pyrene on the membranes were remarkably reduced by ionic PEG chain grafting in short time periods (
