Natural Electroactive Hydrogel from Soy Protein Isolation

A natural electroactive protein hydrogel was prepared from soy protein isolate ... behavior under strong acidic (pH = 2 − 3) or basic (pH = 11 − 1...
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Biomacromolecules 2010, 11, 3638–3643

Natural Electroactive Hydrogel from Soy Protein Isolation Kun Tian, Zhengzhong Shao, and Xin Chen* The Key Laboratory of Molecular Engineering of Polymers of MOE, Department of Macromolecular Science, Laboratory of Advanced Materials, Fudan University, Shanghai, 200433, People’s Republic of China Received September 14, 2010; Revised Manuscript Received November 1, 2010

A natural electroactive protein hydrogel was prepared from soy protein isolate (SPI) solution by cross-linking with epichlorohydrin. Under electrical stimulus, such SPI hydrogel quickly bends toward one electrode, showing a good electroactivity. Because of its amphoteric nature, the SPI hydrogel bends either toward the anode (pH < 6) or cathode (pH > 6), depending on the pH of the electrolyte solution. Other factors, such as electric field strength, ionic strength and gel thickness also influence the electromechanical behavior of the SPI hydrogels. Moreover, this SPI hydrogel exhibits a good electroactive behavior under strong acidic (pH ) 2 - 3) or basic (pH ) 11 - 12) solutions, which is a significant improvement over two other kinds of natural electroactive hydrogels, i.e., chitosan/carboxymethylcellulose and chitosan/carboxymethylchitosan hydrogel, which we reported previously. The wide pH range and good electroactivity of this natural protein hydrogel suggests its great potential for microsensor and actuator applications, especially in the biomedical field, and also to increase the scope of natural polymer-based electroactive hydrogels.

Introduction Smart hydrogels have potential applications in many fields, such as sensors, actuators, tissue engineering, drug delivery, microfluidic control, artificial organs, and wound dressing. These hydrogels normally have a three-dimensional cross-linked macromolecular network structure and can deform quickly and reversibly under environmental stimulus, such as pH, temperature, light, electric/magnetic field, and so forth.1-4 In recent years, there has been increasing interest in studying electroactive hydrogels because their electrical response can be controlled relatively easily in a number of useful application geometries, which transform the electrical energy into mechanical work by swelling, shrinking, or bending in an electric field. However, most of the electroactive hydrogels developed recently are from synthetic polymers, such as poly(acrylic acid)/poly(vinyl alcohol) copolymer,5 poly(vinyl alcohol)/poly(sodium maleate-co-sodium acrylate),6 poly(acrylic acid)/poly(vinyl sulfonic acid) copolymer,7 and sulfonated polystyrene.8 Due to the poor biocompatibility and latent toxic effect of synthetic polymers, the application of these hydrogels in biological and pharmaceutical fields is quite limited. Therefore, more and more natural polymers have been used to prepare synthetic/natural polymer blend materials for better biocompatibility and less latent toxicity, such as alginate/poly(methacrylic acid),9 hyaluronic acid/poly(vinyl alcohol),10 and chitosan/polyaniline hydrogels.11 Moreover, most of the electroactive hydrogels reported are made from either polycationic or polyanionic material so they only show the electrical response in a specific pH range. In our previous work on natural amphoteric electroactive hydrogels, we have successfully prepared two such materials from polysaccharides, namely, chitosan/carboxymethylcellulose (CS/CMC) hydrogel12 and chitosan/carboxymethylchitosan (CS/CMCS) hydrogel.13 They all exhibit a good electromechanical response over a wide pH range and show the maximum equilibrium bending angle at pH ) 6. However, these reported hydrogels did not work well under strong acidic or basic conditions. To overcome these limi* To whom correspondence should be addressed. Tel.: +86-21-65642866. Fax: +86-21-5163-0300. E-mail: [email protected].

tations and enlarge the application fields of electroactive hydrogels, we considered a pure natural amphoteric polyelectrolyte, protein, which contains a wide range of acidic and basic amino acid residues. We propose that this would be an ideal material to prepare natural polymer-based electroactive hydrogels, as the design and structure to function and application of peptide- and protein-based materials is really the hot spot nowadays.14 Soy protein isolate (SPI), the most important component of soybeans, contains more than 90% protein content with two major components: glycinin (11S, approximately 52% of the total protein content) and β-conglycinin (7S, approximately 35% of the total protein content).15 Glycinin is made of six subunits, each consisting of a basic polypeptide (β-polypeptide) with a molecular mass of about 20 kDa and an acidic polypeptide (Rpolypeptide) with a molecular mass of about 32 kDa, which are connected by a single disulfide bond forming the Rβ subunit.16 β-Conglycinin has a trimeric structure and is composed of three kinds of subunits: R (∼67 kDa), R′ (∼71 kDa), and β (∼50 kDa).17 Owing to its sustainability, abundance, low cost, and functionality, soy protein has attracted great research interest for the development of environmentally-friendly protein materials with potentially good properties, such as regeneration, biocompatibility, biodegradability, and so on.18 To date, numerous soy protein-based materials have been studied that can be divided into several main groups of plastics,19,20 gels,21-23 films,24,25 and additives or coatings.26,27 However, most of these soy protein-based materials are modified chemically or blended with other synthetic/natural polymers to overcome their fundamental limitations, such as water sensitivity, poor processability, and low mechanical strength.19,28,29 As far as we are aware, there have not been any studies on electroactive protein hydrogels made of pristine soy protein. The motivation for the selection of SPI as an electroactive material lies on the fact that there is a large amount of polar amino acid residues in soy protein peptide chains,30 which can generate charges in different pH buffer solutions. The present study shows that SPI hydrogel exhibits a good electroactivity over a wide range of pH, especially under strong acidic or basic environmental conditions,

10.1021/bm101094g  2010 American Chemical Society Published on Web 11/18/2010

Electroactive Hydrogel from Soy Protein Isolation

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which suggests the great potential for its applications as a natural polymer-based electroactive hydrogel.

Experimental Section Preparation of SPI Films. SPI powder (Shanghai Shenyuan Food Co., Ltd., protein content >90%) was dissolved in 6 mol/L guanidine hydrochloride (Sinopharm Chemical Reagent Co., Ltd.) aqueous solution and then stirred at room temperature for 3 h, while adding 25 mmol/L dithiothreitol (Shanghai Boyun Biotechnology Co., Ltd., imported package from Sigma) to break the disulfide bonds. After dialysis against NaOH aqueous solution (pH ) 11.5, diluted from 2 mol/L NaOH aqueous solution) for two days and then deionized water for another day at room temperature, the solution was centrifuged at a speed of 9000 r/min for 10 min to obtain a clear supernatant. The concentration of SPI solution was about 2% (w/w), analyzed by the gravity method. The details of the solution process has been described elsewhere31 and results in a very stable protein solution with almost no degradation of the protein chains relative to more conventional alkaline processes, for example, which is important for ongoing work on improved structural mechanical properties of soy proteins. To prepare SPI films, the solution was first concentrated to 6% (w/w) by using reverse dialysis against 10% (w/w) polyethylene glycol solution. Then, 5 mL of this concentrated SPI solution was transferred to a 5 × 5 cm2 polystyrene weighing boat and allowed to dry at room temperature. Finally, the dried films were put into 70% (v/v) ethanol aqueous solution with 1% (v/v) epichlorohydrin to cross-link the soy protein polypeptide chains at 60 °C for 2 h. The resulting cross-linked SPI films were washed repeatedly with deionized water to remove unreacted epichlorohydrin. To study the effect of the cross-linking agent on the properties of SPI films, five epichlorohydrin concentrations were chosen: 0.2, 0.5, 1.0, 1.5, and 2.0%. Swelling of SPI Films. The SPI films were dried in a vacuum oven for two days to constant weight and then immersed in Britton-Robinson buffer solutions with different pH values (from 2 to 12), but constant ionic strength (0.1 M), or with different ionic strengths (from 0 to 0.3 M), but constant pH value (pH ) 4.0 or 10.0), respectively.32 After the excess solution on the surface had been removed with filter paper, the weight of swollen samples was measured immediately. The swelling ratio was determined as follows:

swelling ratio ) (Ws - Wd)/Wd

(1)

where Ws and Wd are the weights of the samples in swollen and dry states, respectively. Mechanical Properties of SPI Hydrogels. The SPI films were first put into pH buffer solutions for 12 h to reach swelling equilibrium to form the hydrogel. Then the hydrogel sample (5 × 30 mm) was held unstrained on the clamps within the custom-built water chamber, which was integrated with the Instron 5565 mechanical testing instrument. We used this mechanical testing instrument (at 20 ( 0.5 °C; gauge length: 15 mm; cross-head speed: 20 mm/min) to test the mechanical properties of SPI hydrogels. The physical dimensions of hydrogel samples were measured using vernier caliper. At least three replicates from individual hydrogels were used for the mechanical testing. Wide Angle X-ray Diffraction (WAXD) Measurement of SPI Films. Wide-angle X-ray diffraction measurements were performed with an X’Pert PRO diffractometer, using Ni-filtered Cu KR radiation (λ ) 1.5406 Å) with 2θ range between 5 and 60° at 40 kV and 40 mA. Measurement of Bending of SPI Hydrogels in an Electric Field. A schematic diagram of the equipment used for studying the electrical response of SPI hydrogels is shown in Figure 1, as in our previous work.12,13 All hydrogel strips (10 mm long and 2 mm wide) were first swollen to equilibrium in an electrolyte solution (BrittonRobinson pH buffer solution) to be used later in the bending experiments. The thickness of the hydrogel in a swollen state was about 0.135 mm (if not specifically mentioned). Two parallel carbon

Figure 1. Schematic diagram for measuring the bending behavior of SPI hydrogels.

electrodes, 50 mm apart (if not specifically mentioned), were immersed in the electrolyte solutions, and the hydrogel strip under investigation was mounted centrally between them. Upon application of a dc electric field, the degree of bending, θ, was measured by reading the angle of deviation from the vertical position. We define the value of the bending angle as being positive when the hydrogel bends toward the anode and negative when it bends toward the cathode. The bending behavior was recorded with a digital video camera (Sony, Japan).

Results and Discussion Cross-linking of SPI Films. Water sensitivity and poor mechanical properties (brittleness) are important reasons that prevent soy protein materials from being used in structural applications. To prepare a useful SPI hydrogel, we used epichlorohydrin as a cross-linking agent in the preparation of SPI film to enhance its water stability and mechanical strength. Epichlorohydrin is a common cross-linking agent for biomacromolecules, such as protein33,34 and polysaccharide,35,36 as it can react with a number of amino and hydroxyl groups in the macromolecular chains. We found the SPI film dissolved in water if we did not use epichlorohydrin or its content was very low ( 6, especially in pH > 11 buffer solutions (Figure 4a), because of the repulsion between ionized carboxyl groups on the protein chains. A higher pH gives a higher swelling ratio owing to the increased number of -COO- ions in the hydrogel. The appearance of the high swelling ratio in strong basic solution (pH > 11) is due to the large numbers of -COO- ions from 20% acidic amino acid in soy protein.28 With the same tendency of swelling ratio, the equilibrium bending angle of the SPI hydrogels increases gradually by decreasing or increasing the pH value from the isoelectric point of SPI (Figure 4b). They show a large equilibrium bending angle (>60°) when pH ) 2-4 and pH ) 10-12, and they can even bend near to 90° in pH ) 2 and pH ) 12 buffer solutions at 20 V. In addition, the SPI hydrogel can also bend toward the cathode in a neutral environment (pH ) 7) with an equilibrium bending angle of 21° at 20 V. All these characteristics of SPI hydrogel make it an excellent natural electroactive hydrogel with a wide pH range of bending behavior, which is greater than the natural electroactive hydrogels(CS/CMCandCS/CMCShydrogel)wereportedpreviously.12,13 It is worth mentioning that CS/CMC and CS/CMCS hydrogels show the best electrical response at pH ) 5-6, so the SPI hydrogel complements these materials very well. Effect of Electrical Field Strength on the Bending Behavior of SPI Hydrogels. The key force for the electroactive hydrogels to bend in an electrolyte solution comes from the electric field acting on the charges on the hydrogels. The ions in the solution move faster as the electric field strength increases, thereby generating an ionic gradient more quickly. This then results in increases in both bending rate and angle.12 Figure 5 shows the influence of applied voltage (the distance between two electrodes kept constant) on the bending rate of the SPI hydrogels. We use exponential decay functions to fit the kinetic

Figure 6. Effect of ionic strength on the swelling ratio of SPI films (a) and the equilibrium bending angle of the SPI hydrogels at 15 V (b) in pH ) 4.0 buffer solutions.

curves in order to determine kinetic parameters here, as in our previous work.12,43 The kinetic curves in Figure 5 can be fitted quite well with a first-order exponential decay function (eq 2), and the time constant for the bending decreased from 10.5 to 6.6 s with increasing voltage (a lower time constant means a faster bending rate)

bending angle ) A0(1 - e-t/τ)

(2)

where A0 is the equilibrium bending angle, t is the time, and τ is the time constant. In addition, the equilibrium bending angle increased from 23 to 63° (absolute value) as the voltage increased, thus confirming that a higher applied electrical field strength gives a faster bending rate and a greater equilibrium bending angle. Compared with the bending behavior of CS/ CMC and CS/CMCS hydrogels,12,13 the bending rate of SPI hydrogel is faster, for example, the time constant of CS/CMC hydrogel is 25.6 s at 20 V, but SPI hydrogel here is only 6.6 s, as shown above. This indicates the SPI hydrogel has greater electrosensitivity. In addition, we measured the bending behavior of SPI hydrogel under the same electrical field strength with different electrode distance by adjusting the electric voltage. The result shows that the SPI hydrogels have the same bending behavior under the same electrical field strength (Figure S2). Effect of Ionic Strength on the Bending Behavior of SPI Hydrogels. We investigated the influence of the ionic strength on the swelling ratio and equilibrium bending angle of the SPI hydrogels, as shown in Figure 6. With increases in ionic strength (I), the swelling ratio of the SPI hydrogel first increases from I ) 0 to 0.1 M and then goes down slightly until it reaches an equilibrium state (Figure 6a). The appearance of a maximum

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Figure 7. Bending behavior of SPI hydrogels with various thickness in swollen state in at 15 V (pH ) 4; ionic strength ) 0.1 M): (a) 0.092, (b) 0.135, (c) 0.225, (d) 0.425 mm.

swelling ratio at I ) 0.1 M can be explained as follows. At first, the addition of salt weakens the electrostatic attractions between oppositely charged ionic groups (for instance, -NH3+ and -COO-) on the polypeptide chains (which is similar to the salting-in effect), so that the hydrogel becomes less compact and the swelling ratio increases. However, with the further increase in ionic strength, the charges on polypeptide chains is somewhat shielded by the increased number of salt ions (which is similar to the salting-out effect), so that the repulsion between polypeptide chains in the hydrogel is weakened, resulting in the decrease in the swelling ratio. The effect of ionic strength on the equilibrium bending angle of the SPI hydrogel has the same trend, but is more pronounced than that on swelling ratio. As ionic strength increases, the equilibrium bending angle first increases, reaching the maximum value when I ) 0.1 M (from approximately 10-50°) and then decreases slightly to reach an equilibrium bending angle with further increases in ionic strength (Figure 6b). It electrolyzes heavily if more salt is added into the buffer solution when I ) 0.3 M. The bending angle shows a maximum value when I ) 0.1 M, corresponding to the effective free charges on the hydrogel being a maximum according to the swelling ratio data discussed above. Effect of Thickness on the Bending Behavior of SPI Hydrogels. Figure 7 shows the influence of thickness on the bending behavior of thin SPI hydrogel strips. A thin hydrogel (0.092 mm, curve a) had a high bending rate (27 s to reach the bending equilibrium) and a large equilibrium bending angle (more than 70°). However, if the thickness is increased, both the bending rate and the equilibrium bending angle decrease. When the thickness of the hydrogel was about 4-fold of curve a in Figure 7 (0.425 mm, curve d), the bending angle was only 13° after 45 s and seemed still not to have reached equilibrium. The effect of thickness on the bending behavior of SPI hydrogel is quite similar to the CS/CMC and CS/CMCS hydrogels12,13 we reported previously. Changing thickness of the films also gives complex changes in other interdependent physical properties such as swelling, and hence elastic modulus, so the main body of the work reports results specifically on one film thickness of 0.135 mm. Reversible Bending Behavior of SPI Hydrogels. Figure 8 shows an example of the reversible bending behavior of the SPI hydrogels by alternately applying an electric voltage. The SPI hydrogels bend and return to their initial position quickly as the electric field is turned on and off several times. The shape of each cycle is very similar, which suggests that SPI hydrogels

Tian et al.

Figure 8. Reversible bending behavior of the SPI hydrogels at 15 V (pH ) 10; ionic strength ) 0.1 M).

Figure 9. Schematic figure of SPI hydrogel loops when curling (a) and loosing (b) under a circle electric field at 15 V (pH ) 4.0, ionic strength ) 0.1 M; the diameter of the anode is 7 mm; the outside diameter and the thickness of the cathode are 50 and 3 mm, respectively). Table 2. Distance from the SPI Hydrogel Loop to the Central Electrode with the Change of Time under the Circular Electric Fielda electric field

0s

4s

8s

12 s

turn on turn off

11 mm 1.5 mm

7 mm 3 mm

4 mm 6 mm

1.5 mm 8 mm

a

pH, 4.0; ionic strength, 0.1 M; electric voltage, 15 V.

with a longer life have the potential in applications such as microsensors, actuators, artificial muscles, and so on. Trial of SPI Electroactive Hydrogel Loops. To increase the possible applications of the electroactive protein hydrogels, we looked at the electrical response of a SPI hydrogel loop under a circular electric field, because it is ideally suited to the strong bending mode deformation in our electroactive gel films rather than the stronger tensile deformation of other electroactive materials.44 If we put the SPI hydrogel loop in an acidic environment and set the central electrode as anode, we found the SPI loop curled up toward the center very quickly when the electric field was turned on (Figure 9a); correspondingly, it loosened from the center to the circumference if the voltage was removed (Figure 9b). We have measured the distance from the loop to central electrode (d) with the time, and the result is presented in Table 2. The SPI hydrogel loops moved very fast in this trial experiment. It only took about 12 s to circle from 11 to 1.5 mm in the curling process and from 1.5 to 8 mm in the loosing process. Though this trial experiment is rather simple at the current stage, it implies that our SPI electroactive hydrogels could be used in diverse applications, and we will explore the practical use of such a protein electroactive hydrogel in our future work.

Electroactive Hydrogel from Soy Protein Isolation

Conclusions We conclude that an effective natural electroactive protein hydrogel can be prepared from SPI solution by cross-linking with epichlorohydrin. The amphoteric nature of the protein means the SPI hydrogel can bend either toward the anode (pH < 6) or cathode (pH > 6), depending on the pH of the electrolyte solution. The equilibrium bending angles of SPI hydrogels are influenced by pH, electric field strength, ionic strength, and gel thickness. SPI hydrogel exhibits a linear electrical response with both pH and electric field over a wide range of 2 e pH e 12, where the effective limits are defined by the equilibrium bending angle reaching about 90° in buffer solutions with an optimized ionic strength of 0.1 M and the highest applied electric field of 400 V/m used in our experiments. This broad operating window increases the scope of potential applications of natural electroactive hydrogels over previously reported materials such as CS/ CMC and CS/CMCS. Acknowledgment. This work is supported by the National Natural Science Foundation of China (No. 20674011), the Program for New Century Excellent Talents in University of MOE of China (NCET-06-0354), and the Program for Changjiang Scholars and Innovative Research Team in University of MOE of China (IRT-06-12). Special thanks to Prof. David Porter from the University of Oxford for valuable discussions and help with preparation of the manuscript. We also thank Dr. Jingrong Yao from Fudan University and Miss Juan Guan who got her Master’s degree at Fudan University and is now a Ph.D. candidate in the University of Oxford for helpful advice and discussions. Supporting Information Available. The effect of epichlorohydrin content on the swelling ratio of SPI films, the realtime photographs of SPI hydrogel bending, and the bending behavior of the SPI hydrogel under the same electric field strength by adjusting electric voltage and the distance between two electrodes are reported. This material is available free of charge via the Internet at http://pubs.acs.org.

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