Off Behavior for Protein Transport

Sep 3, 2008 - Key Laboratory for Green Technology of Ministry of Education, ... or open channels for protein transport under different NaCl concentrat...
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J. Phys. Chem. B 2008, 112, 11923–11928

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Smart Zwitterionic Membranes with On/Off Behavior for Protein Transport Yanlei Su,* Lili Zheng, Chao Li, and Zhongyi Jiang Key Laboratory for Green Technology of Ministry of Education, School of Chemical Engineering and Technology, Tianjin UniVersity, Tianjin 300072, China ReceiVed: February 19, 2008

Poly(acrylonitrile) (PAN)-based zwitterionic membranes, composed of PAN and poly(N,N-dimethyl-Nmethacryloxyethyl-N-(3-sulfopropyl) copolymer, are electrolyte-sensitive smart membranes. The hydrophilicity was increased and protein adsorption was remarkably decreased for the membranes in response to environmental stimuli. FTIR spectroscopic analysis directly provided molecular-level observation of the enhanced dissociation and hydration of zwitterionic sulfobetaine dipoles at higher electrolyte concentrations. The smart PAN-based zwitterionic membranes can close or open channels for protein transport under different NaCl concentrations. The electrolyte-sensitive switch of on/off behavior for protein transport is reversible. Introduction Protein transport through channels is an interesting and fundamental science problem. Efforts have been devoted to enable on/off function for protein transport through channels in the synthetic membranes. Yu et al. have reported that Au nanotubule membranes can show good selectivity for separation of proteins on the basis of molecule size by controlling the inside diameter of the nanotubules, where protein adsorption was eliminated by chemisorbing a poly(ethylene glycol) thiol to the Au nanotubules.1,2 Chun et al. have studied protein transport in nanoporous polycarbonate track-etched (PCTE) membranes modified with self-assembled thiols on electroless gold deposition. The fluxes of bovine serum albumin (BSA) and bovine hemoglobin (BHb) through the membranes showed maximum values at the isoelectric points (pI) of the proteins. At pH values above and below the pI, electrostatic interactions between the proteins, their counterions, and the pore surfaces led to a decrease in flux.3,4 It is easy to prepare ultrafiltration membranes by the phase inversion method. However, few studies of controlled protein transport through channels in ultrafiltration membranes have been carried out. The on/off behavior with ultrafiltration membranes is highly desired in order to investigate the intrinsic permeability and develop smart membranes with controllable transport. In recent years, the rapid developments in the field of smart materials have attracted more and more attention. These materials exhibit a distinct change of properties in response to external stimuli, such as pH, temperature, light, and chemical additions. Csetneki et al. have fabricated smart nanocomposite polymer membranes with on/off switching control. The channels were designed to contain an ordered array of core-shell type magnetic polystyrene latex particles. The temperature-sensitive collapse and swelling transition of the poly(N-isopropylacrylamide) (PNIPA) shell affected the BSA permeation pattern from the on to off state.5 However, there are few reports in the literature about the electrolyte-sensitive smart materials to achieve on/off behavior for protein transport. Zwitterionic groups, such as phosphorylcholine, sulfobetaine, and carboxybetaine, contain an equal number of cationic and * Corresponding author. Fax: 86-22-27890882. E-mail: suyanlei@ tju.edu.cn.

anionic species on the same monomer residue, which form a dipole moment for every zwitterionic group. Zwitterionic groups prefer to have an antiparallel orientation with respect to each other, so that the electrostatic energy and net dipole moments are minimized.6 The homopolymer of sulfobetaine is insoluble in deionized water, which is attributed to the attractive dipole electrostatic interactions. These interactions can be shielded by the addition of electrolytes resulting in solubilization of the polymer in water.7,8 Because of their biocompatibilities, Jiang et al. also have fabricated superlow fouling surfaces with sulfobetaine polymes on glass and gold surfaces.9,10 We have fabricated poly(acrylonitrile) (PAN)-based zwitterionic membranes with sulfobetaine copolymers. The controlled adsorption and desorption of BSA on the zwitterionic membranes were achieved through adjusting NaCl concentrations in solutions.11 In the present work, protein transport through the channels in PAN-based zwitterionic membranes was carefully examined, which displayed the smart on/off feature under different electrolyte concentrations. The on/off behavior for protein transport is related to the electrostatic interactions between proteins and sulfobetaine groups. It was also found that the electrolyte-sensitive switch of on/off behavior for protein transport is reversible. The characteristic property of controlled protein transport endows the smart PAN-based zwitterionic membranes with potential applications in protein separation and purification, controlled release, and other biotechnologies. Experimental Section Materials. Acrylonitrile (AN) was purchased from Damao Chemical Co. (Tianjin, China) and distilled before use. 2-(Dimethylamino)ethyl methacrylate (DMAEMA) was purchased from Xinyu Chemical Co. (Wuxi, China). 1,3-Propane sultone was purchased from Sigma and used without any pretreatment. Azobis(isobutyronitrile) (AIBN), dimethyl sulfoxide (DMSO), and polyvinyl alcohol (PVA) were purchased from Kewei Chemical Reagent Co. (Tianjin, China). Hen egg white lysozyme and bovine serum albumin (BSA) were purchased from Institute of Hematology, Chinese Academy of Medical Science (Tianjin, China). Water used in all experiments was deionized water at pH 5.8. Synthesis of PAN-Based Zwitterionic Copolymers. Poly(acrylonitrile and 2-(dimethylamino)ethyl methacrylate) (PAN-

10.1021/jp804422t CCC: $40.75  2008 American Chemical Society Published on Web 09/03/2008

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Figure 1. Chemical structure of PAN-based zwitterionic copolymer.

DMAEMA) copolymer was first synthesized by free radical polymerization. AN (34.0 g), DMAEMA (8.5 g), dispersant PVA (0.1 g), and 150 mL of deionized water were introduced into a three-necked round-bottom flask placed in a thermostatted water bath at temperature of 60 °C. The reaction mixture was protected by continuously bubbling nitrogen and initiated with AIBN (0.2 g). After the desired reaction time of 8 h, PANDMAEMA copolymer was precipitated, washed, and dried in vacuum subsequently. The fraction of DMAEMA groups in PAN-DMAEMA copolymer was 8.4 mol%. PAN-DMAEMA copolymer was then reacted with 1,3propane sultone for preparation of the zwitterionic copolymer. The synthesized PAN-DMAEMA copolymer (13.0 g) and 1,3propane sultone (2.16 g) were dissolved in 150 mL of DMSO and continuously stirred for 16 h at temperature of 60 °C. After betainization, PAN and poly(N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl) (PDMMAS) copolymer, PAN-based zwitterionic sulfobetaine copolymer, was precipitated in excess deionized water. The precipitation was collected, washed with deionic water, and dried in vacuum. The weight percentages of C, N, and S elements in PAN-DMMSA copolymer were 61.14, 19.41, and 3.72 wt% from an elemental analyzer (Vario EL, Element Co, Germany), respectively. Therefore, the composition of PAN-based zwitterionic copolymer is 91.6 mol% AN and 8.4 mol% DMMAS. The chemical structure of PAN-based zwitterionic copolymer was given in Figure 1. Preparation and Characterization of PAN-Based Zwitterionic Membranes. PAN-based zwitterionic polymer was dissolved in DMSO at a concentration of 15 wt% to prepare casting solution. The solution was stirred at 60 °C for about 4 h to ensure the homogeneous mixing and then left for 4 h to allow complete release of bubbles. After being cooled to room temperature, the solution was spread on glass plates with a casting knife and then immediately immersed in a coagulation bath of deionized water. After being peeled off from the glass plates, PAN-based zwitterionic membranes were rinsed with deionized water to remove residual DMSO and kept in water before use. The cross-section morphology of PAN-based zwitterionic membrane was observed by scanning electron microscopy (SEM) (Philips XL30E). The membrane frozen in liquid nitrogen was broken and sputtered with gold before SEM analysis. Fourier transform infrared (FTIR) spectroscopic analysis was used to study the molecular mechanism for the dissociation and hydration of sulfobetaine groups in the membranes. The dry and wet PAN-based zwitterionic membranes were measured directly on an FTIR spectrometer (Nicolet 560, Nicolet Co.) in transmission mode, respectively. Each spectrum was collected by cumulating eight scans at a resolution of 2 cm-1. In order to observe the influence of electrolyte on the dissociation and hydration of sulfobetaine dipoles, PAN-based zwitterionic membranes were immersed in aqueous solutions with different NaCl concentrations before FTIR spectrum measurement.

Figure 2. The cross-sectional morphology of the PAN-based zwitterionic membrane.

Static contact angles of the membrane surfaces were measured using the captive air bubble technique. Wet PAN-based zwitterionic membranes were glued in plastic slides, which were then inverted and floated in aqueous solutions with different NaCl concentrations. Air bubbles were placed in contact with the membrane surfaces. Static contact angles were measured using a contact angle goniometer (JC2000C Contact Angle Meter, Powereach Co., Shanghai, China). At least six water contact angles at different locations on one surface were averaged to get a reliable value. Protein Adsorption Experiments. Circular pieces cut from PAN-based zwitterionic membrane with an appropriate area of 18.15 cm2 were put into vials containing 10 mL volume 0.3 mg/mL lysozyme or 1.0 mg/mL BSA aqueous solutions and incubated at room temperature of 20 ( 1 °C, respectively. The pH value was maintained at 5.8 (deionized water’s pH), while the ionic strength was adjusted with NaCl. After incubation of 5 h, protein concentrations in the solutions were analyzed with a UV spectrophotometer (Hitach UV-2800, Japan), and then the amount of adsorbed BSA or lysozyme on the PAN-based zwitterionic membrane was calculated. Protein Transport Experiments. Protein transport experiments were carried out in a two-compartment glass diffusion cell system at room temperature of 20 ( 1 °C. A circular piece of PAN-based zwitterionic membrane was cut and mounted tightly between the two chambers. At the beginning of experiment, the donor chamber was filled with 0.3 mg/mL lysozyme or 1.0 mg/mL BSA solutions at different NaCl concentrations with a water pH of 5.8. The receptor chamber was filled with similar NaCl solutions free of protein. Magnetic stirring bars were put in each chamber and kept rotating in the process of protein transport experiments. At a given interval time, protein concentrations in the receptor chamber were analyzed with a UV-vis spectrophotometer. Results and Discussion Dissocation and Hydration of Sulfobetaine Groups. PANbased zwitterionic membranes were prepared by a phase inversion method. PAN is the membrane matrix, and zwitterionic sulfobetaine groups are arranged on the membrane surfaces and channel walls.11 Figure 2 shows the cross-sectional morphology of the PAN-based zwitterionic membrane obtained by SEM. A typical structure of an asymmetric ultrafiltration membrane with a dense skin layer, a support layer with a sponge-like structure, and macrovoids appear in the middle of the membranes.

Smart Zwitterionic Membranes

Figure 3. The symmetric stretch vibration of sulfonate groups in PANbased zwitterionic membranes after immersion into aqueous solutions at different NaCl concentrations. The peak can be resolved into two components corresponding to dissociated and undissociated sulfonate groups.

FTIR spectroscopic analysis can provide important information relating to molecular structure and process on a picosecond time scale. For the dry membranes, CN vibration has a characteristic absorbance at 2244 cm-1, the adsorption peak at 1732 was attributed to CdO groups, and the adsorption peaks at 1209 and 1037 cm-1 were ascribed to the asymmetric and symmetric stretch vibrations of sulfonate groups12 (Figure S2 in the Supporting Information). For the wet membranes, there was no change on the CN vibration that remains at 2244 cm-1. However, there was remarkable change in the stretch vibrations of sulfonate groups due to wetness. The asymmetric stretch vibration of sulfonate groups was split into several peaks at 1217, 1200 (main peak), 1190, 1181, and 1171 cm-1 peak, respectively. Two peaks at 1037 and 1042 cm-1 appeared simultaneously in the range of the symmetric stretch vibration of sulfonate groups (Figure 3, in 0 mol/L NaCl solution). More attention was focused on the symmetric stretch vibration of sulfonate groups at different NaCl concentrations. It can be seen in Figure 3 that the intensity of peak at 1042 cm-1 was further enhanced and the peak at 1037 cm-1 was almost disappeared when the membranes were immersed into 0.50 mol/L NaCl solutions. It was reasonable to assume that two species of sulfonate groups exist in the wet membranes. The peak at 1037 cm-1 was assigned to the undissociated sulfonate groups where the sulfobetaine dipoles are oriented in an antiparallel fashion as that in the dry membranes, and the band at 1042 cm-1 was associated with dissociated and hydrated sulfonate groups surrounded with a counterion atmosphere, where the sulfobetaine dipole assemblies are destroyed because of penetration of water molecules inside the zwitterions and the formation of hydrogen bonds.7,8,13,14 To obtain quantitative information, the spectral pattern of the symmetric stretch vibration of sulfonate groups was resolved into two components: one for the undissociated sulfonate dipoles, the other for dissociated sulfonate groups (one representative fitted curve was given in Figure 3 in 0.50 mol/L NaCl solution). The total peak areas of undissociated and dissociated sulfonate groups were normalized to 1.15,16 The normalized integrated peak area of each component corresponded to the fraction of sulfonate groups in undissociated and dissociated states. For the wet PAN-based zwitterionic membranes, the

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Figure 4. Effect of NaCl concentrations on water contact angles of PAN-based zwitterionic membranes.

fraction of sulfonate groups in the dissociated and hydrated state was about 53.5% in the absence of electrolyte. When the membranes were soaked in NaCl solutions, the peak intensity of the dissociated sulfonate groups was further increased, which means that the fraction of dissociated and hydrated sulfobetaine groups was further increased. The fraction of sulfonate groups in the dissociated and hydrated state was about 79.3% when the membranes were soaked in 0.50 mol/L NaCl solution. The added electrolyte screens the positive and negative charges of dipoles, so that the dipole moments of the sulfobetaine groups are minimized, the attractively electrostatic interactions between sulfobetaine dipoles are weakened, and the sulfobetaine groups are easily dissociated.13,14 To our knowledge, this is the first report regarding quantitative information on the dissociation of the dipole-dipole self-assemblies of zwitterions. Enhanced Hydrophilicity after Environmental Stimuli. There are attractive dipole electrostatic interactions among zwitterionic sulfobetaine groups. The interactions can be effectively shielded by the addition of electrolytes.7,8,13,14 Water contact angles were measured to evaluate the hydrophilicity after environmental stimuli. It is convenient to measure the water contact angles of wet membranes at different electrolyte concentrations using the captive air bubble technique. The contact angles of the air-water-solid triphase lines of PANbased zwitterionic membranes at different NaCl concentrations are shown in Figure 4. As expected, the wettability of PANbased zwitterionic membranes was tunable in response to environmental stimuli.14,17 The water contact angle of PANbased zwitterionic membranes was 43 ( 3° in the absence of electrolyte. There was a detectable decrease of contact angle when PAN-based zwitterionic membranes were immersed in aqueous NaCl solutions. The water contact angle of PAN-based zwitterionic membranes was decreased to 28 ( 2° after immersion into 0.50 mol/L NaCl solution. The smaller water contact angle means a higher hydrophilicity for the zwitterionic membranes. The enhanced hydrophilicity of PAN-based zwitterionic membranes after immersion into aqueous NaCl solutions is related to the dissociation and hydration of zwitterionic sulfobetaine groups. More sulfobetaine groups are dissociated and hydrated in the condition of higher electrolyte concentrations.13,14 Since the dissociated sulfobetaine groups are surrounded with a hydration shell (a counterion atmosphere),13 PAN-based zwitterionic membranes have higher hydrophilicity after environment stimuli.

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Figure 5. Effect of NaCl concentrations on the amount of adsorbed lysozyme and BSA on PAN-based zwitterionic membranes.

Remarkable Decrease of Protein Adsorption after EnvironmentalStimuli.Thereareelectrostaticinteractions(charge-dipole and dipole-dipole) between the charged amino acid residues in proteins with dipole moments of sulfobetaine groups at lower ionic strength, which possess adequate force to induce the adherence of proteins on PAN-based zwitterionic membranes.11 Figure 5 presents the influence of NaCl concentrations on the amount of absorbed lysozyme and BSA on PAN-based zwitterionic membranes. Because of the porous structure, high water content, and the specifically favorable interactions, the amount of adsorbed lysozyme and BSA on PAN-based zwitterionic membranes was relatively high at 0.001 mol/L NaCl in aqueous solution. The amount of adsorbed lysozyme and BSA was dramatically decreased with an increase of NaCl concentration. When NaCl concentration in solution reached to 0.10 mol/L, the amount of adsorbed lysozyme and BSA was nearly decreased to zero, showing an excellent antifouling property of zwitterionic sulfobetaine materials.9,10 The remarkable reduction of protein adsorption was attributed to the electrostatic interactions between the sulfobetaine groups and proteins, which are completely screened by the addition of electrolyte.11 The results of protein adsorption resistance are in good agreement with the enhanced hydrophilicity of PAN-based zwitterionic membranes after environmental stimuli. On/Off Behavior for Protein Transport. Protein diffusion experiments can give important information about the intrinsic permeability of PAN-based zwitterionic membranes. The diffusion experiments utilize a concentration gradient to drive the proteins transport across the membranes. Figure 6 shows the transport process of lysozymes through the channels in PANbased zwitterionic membranes at different NaCl concentrations. When NaCl concentrations in the solutions are below 0.02 mol/ L, only a small quantity of lysozyme can penetrate the membrane. Lysozyme concentrations in the receptor chamber did not increase gradually with the elapsed diffusion time. It was deduced that the channels in the membranes are closed in the later diffusion stages, so that lysozyme cannot penetrate the membranes again. However, lysozyme molecules can easily pass through the channels when NaCl concentrations are above 0.03 mol/L. The channels in the membranes are always open, and lysozyme concentrations in the receptor chamber increased continuously with elapsed diffusion time. Figure 6 also shows BSA penetration through the channels in PAN-based zwitterionic membranes at different NaCl concentrations. The channels in PAN-based zwitterionic membranes

Figure 6. Diffusion experiments with 0.3 mg/mL lysozyme and 1.0 mg/mL BSA aqueous solutions at different NaCl concentrations at pH 5.8. The concentrations of lysozyme and BSA in the receptor chamber were plotted as a function of the elapsed diffusion time.

are close for BSA transport when NaCl concentrations are below 0.04 mol/L. The channels are open for BSA transport when NaCl concentrations are above 0.05 mol/L. It is certain that there are channels across PAN-based zwitterionic membranes and the pore sizes are larger than the sizes of lysozyme and BSA molecules. The controlled on/off behavior of the channels is related to the adjustment of permeability by the addition of electrolyte. Protein adsorption can occur on both membrane surfaces and channel walls in PAN-based membranes at lower NaCl concentrations. Protein molecules adhere to the pore walls so that the channels are narrowed; therefore, proteins cannot pass through the channels in PAN-based zwitterionic membranes at lower NaCl concentrations. The addition of NaCl in solution weakens the electrostatic interactions between proteins and sulfobetaine dipoles and enhances hydrophilicity of the zwitterionic membranes;7,8,13,14 therefore, protein adsorption is suppressed and the channels are always open, which ensures free passage of protein molecules through the channels in PANbased zwitterionic membranes. In Figure 6, it can be seen that a small quantity of protein molecules is transported through the channels in the initial diffusion stages at lower NaCl concentrations, which means that the process of channel blocking is not very fast. Huisman et al. suggested that protein-membrane and protein-protein interac-

Smart Zwitterionic Membranes tions influence the performance during ultrafiltration of protein solutions over polymer membranes.18 In a similar way in the initial diffusion stages at lower electrolyte concentrations, attractive electrostatic interactions between proteins and sulfobetaine dipoles result in protein adsorption (protein-membrane interactions). The protein adsorption causes conformational changes in the proteins,19 and the denatured proteins lead to protein aggregation20,21 and finally block the channels in PANbased zwitterionic membranes (protein-protein interactions). These may be the detailed events for the close of channels in PAN-based zwitterionic membranes at lower NaCl concentrations. Another phenomenon was also noted in the protein diffusion experiments. The protein fluxes (the slopes of protein concentrations in the receptor chamber versus time plots) were increased with an increase of NaCl concentration (Figure 6, lysozyme transport in 0.03, 0.05, 0.10, and 0.50 mol/L NaCl solutions, BSA transport in 0.05, 0.10, and 0.50 mol/L NaCl solutions). It was difficult to exactly explain the mechanism of enhanced fluxes. The probable reasons included that the interactions between proteins and membranes are further decreased in the nanoscale channels, the diffusion coefficients of proteins are increased with an increase of electrolyte concentration,22 and pore sizes in PAN-based zwitterionic membranes are enlarged at higher NaCl concentration. An on/off phenomenon of protein transport clearly demonstrated that electrolytes play an important role in controlling the permeability of smart PAN-based zwitterionic membranes. Switch of On/Off Behavior. In order to evaluate the reversible switch of on/off behavior of PAN-based zwitterionic membranes, protein transport experiments using the same membrane were carried out continuously at lower and higher NaCl concentrations, respectively. In the initial diffusion experiment, the channels in PAN-based zwitterionic membranes are close due to blocking by the adsorbed protein at a lower NaCl concentration. The glass diffusion cell system was then emptied and rinsed three times with deionized water. The cells were refilled, and the diffusion experiment was carried out again at a higher NaCl concentration. The channels were open for lysozyme and BSA transport since protein adsorption was suppressed.11 The results of controlled protein transport experiments at lower and higher NaCl concentrations, respectively, are shown in Figure 7. It was found that the electrolyte-sensitive switch of on/off behavior for protein transport is fast and reversible. The reversible switch of on/off behavior for protein transport through smart PAN-based zwitteronic membranes was achieved by a simple method through adjusting electrolyte concentration. The switching is attributed to the pronounced effect of electrolyte on protein adsorption. Protein adsorption on PAN-based membranes was controlled by the addition of electrolyte, which affected the protein permeation pattern from off to on state. The electrolyte-sensitive switch of on/off behavior for protein transport makes smart PAN-based zwitteronic membranes that have potential applications in protein separation and purification, controlled release, and other biotechnologies.

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Figure 7. The reversible switch of on/off behavior for lysozyme (0.3 mg/mL lysozyme solution) and BSA (1.0 mg/mL BSA solution) transport experiments at different NaCl concentrations at pH 5.8. The diffusion experiments were first carried out in 0.01 mol/L NaCl solution, the glass diffusion cell system was then emptied and washed with deionized water, the cells were refilled, and the diffusion experiment was carried out again in 0.10 mol/L NaCl solution.

lower NaCl concentrations. The electrostatic interactions are screened at higher NaCl concentrations, so that the membranes can resist protein adsorption, the channels are open, and proteins can easily transport through the channels in PAN-based zwitterionic membranes. Acknowledgment. This research was supported by Tianjin Natural Science Foundation (No. 07JCYBJC00900), the Program of Introducing Talents of Discipline to Universities (No. B06006), and Doctoral Fund of Ministry of Education of China for New Teachers (No. 20070056041). Supporting Information Available: The detained synthesis process and chemical structure for PAN-based zwitterionic copolymers; FTIR spectra of dry and wet PAN-based zwitterionic copolymer. This material is available free of charge via the Internet at http://pubs.acs.org.

Conclusions In conclusion, smart PAN-based zwitterionic ultrafiltration membranes displayed an on/off feature for protein transport. The on/off behavior can be controlled through adjusting electrolyte concentrations. There are attractive electrostatic interactions between proteins and sulfobetaine dipoles, which induce the adherence of proteins to the membranes and the blockage of channels in PAN-based zwitterionic membranes at

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