poly(acrylic acid) composite membrane

Polymer Gels and Networks 1 (1993) 247-255

Electrically Controlled Protein Permeation through a Poly(vinyl alcohol)/Poly(acrylic acid) Composite Membrane Takeshi Yamauchi, Etsuo Kokufuta* Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305, Japan

& Yoshihito Osada Faculty of Polymer Science, Hokkaido University, Sapporo, Hokkaido 060, Japan (Received 9 July 1993; accepted 16 July 1993)

A B S T R A CT This paper reports on the preparation and permeability of an electrically activated polyelectrolyte gel membrane which allows the diffusion of a protein as a high-molecular-weight ionic solute. The membrane was prepared through iterative freezing-thawing of an aqueous solution containing poly(vinyl alcohol) (PVA) and poly(acrylic acid) (PAA). The permeability as a function of electric field was studied in a 150 m M phosphate buffer ( p H 6.8), using trypsin with a molecular weight of 23 268 Da as the protein solute. The permeation rate of trypsin increased with an increase in the applied electric field; this was more remarkable when trypsin was transported toward an electrode opposite in sign to the protein charges. This means that pore channels, the sizes of which were large enough to permit the diffusion of trypsin, were formed within the membrane as a result of isometric contraction under the applied electric field. Using the P V A / P A A gel membrane, the initiation-termination control of protein permeation could be accomplished by switching a direct current source on and off. There was little loss in the enzymatic activity of the trypsin molecules during the permeation process under the electric fields'. INTRODUCTION The regulation of the diffusional release of solutes from polymeric matrices by external s t i m u l a t i o n h a s recently drawn much attention in * To whom all correspondence should be addressed. 247

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Takeshi Yamauchi, Etsuo Kokufuta, Yoshihito Osada

connection with the development of drug delivery devices. 1 Toward this end, a number of studies have been focused on stimuli-sensitive polymers in the form of gels and membranes, the structural or chemical properties of which vary in response to changes in environmental conditions. However, many previously reported model systems have been limited to the release control of low-molecular-weight solutes such as methyl orange, 2 glutamate, 3 indomethacin, 4, 5 and vitamin B I 2 . 4" 5 A few studies have dealt with the controlled release of high-molecularweight solutes: the glucose-stimulated release of glycosylated insulin from a membrane-separated aqueous phase containing its complex with Concanavalin A (Con A); 6 the D-mannose-responsive release of exo-1, 4-~-D-glucosidase from a Con A-loaded agarose gel in the form of beads; 7 and the electrically controlled release of insulin from an interpolymer complex of poly(ethyloxazolin) with poly(methacrylic acid) or poly(acrylic acid) (PAA). ~ Osada has reported on several polyelectrolyte gels that undergo changes in shape due to the swelling or shrinking of their cross-linked chain networks in response to an electric field applied to the gel environments. 9 When an electric field was applied to such polyelectrolyte gels in such a way that their dimensions were kept constant without allowing for any change in shape, isometrically contractile stress was generated in the gel and this expanded the pore channels through which solutes and solvents could permeate. 9 For example, a composite gel membrane consisting of P A A and poly(vinyl alcohol) (PVA) was useful in the electrical regulation of the permeation of water through its pore channels, 1° the sizes of which could be made to expand by turning on a direct current (DC) or caused to contract by turning off the current. These results have prompted us to further examine the permeability of a P V A / P A A gel membrane with respect to a highmolecular-weight solute under different conditions of applied electric fields. Through such examinations, we have attempted to determine the potential of the gel for regulating the diffusional release of polymeric drugs such as proteins. Trypsin with an absolute molecular weight of 23 268 Da and an apparent Stokes diameter of 12 nm was chosen for use as the high-molecular-weight solute in view of the information available on its structural and enzymological properties. '~ E X P E R I M E N T A L SECTION Materials P V A (Mw = 17 000; Kurare Chemical Co., Japan) and P A A (Mw = 25 000; Wako Chemical Co., Japan) were commercial products. Trypsin (from bovine pancreas, Sigma type XIII) was the same as the sample

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used in the previous study. 12 Ntx-Benzoyl-DL-arginine-p-nitroanilide ( B A N A ) used as the substrate for the assay of the enzymatic activity of trypsin was purchased from Sigma Chemical Co.

Membrane preparation The P V A / P A A composite m e m b r a n e was prepared through the iterative freezing-thawing of an aqueous solution containing 10% of each polymer. The polymer solution was poured into the space (c. 0.02 cm) between two glass plates and cooled from room temperature to - 3 0 ° C in a refrigerator. The frozen polymer solution was then thawed at r o o m temperature for 5 h. Such freezing and thawing procedures were repeated 15 times at suitable time intervals. The gel m e m b r a n e obtained was washed thoroughly with distilled water and subjected to permeation experiments. The m e m b r a n e thickness in a swollen state was 0.02 + 0.005 cm, as estimated using a m e m b r a n e thickness gauge. The rupture of the m e m b r a n e took place at a tensile stress of 2.08 + 0.51 kg when a ribbon-like sample (3 cm length; 1 cm wide; and 0.02 cm thickness) was subjected to the tensile test, which was carried out at room temperature and at an extension speed of 0.3 cm/s. This tensile stress was about 6.6 times larger than that for a low-density

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250

Takeshi Yamauchi, Etsuo Kokufuta, Yoshihito Osada

poly(ethylene) film measured under the same conditions as used for the membrane sample.

Permeation experiments The permeability of the membrane for trypsin was investigated at room temperature using the permeation cell shown in Fig. 1. The cell consisted of two connected compartments; one side (source phase; S-cell) with a 150 mM phosphate buffer (pH 6.8, 70 ml) containing 2 mg/ml trypsin and the other side (receiving phase; R-cell) containing the same buffer (70 ml) without the trypsin. The membrane, having an effective area of 0.785 cm 2, was fixed tightly between the two sides. Circular platinum mesh electrodes with a diameter of 1 cm were placed in both the source and receiving phases separated by the membrane, very near to but not in contact with it. The electrodes were connected to a DC source (HA-501 potentiostat, Hokuto Debkou Co., Japan). The isoelectric point of trypsin has been determined to be 10.8 (see Ref.ll); therefore, it is assumed that the protein carried the positive charges in the buffer solution (pH 6.8) used. The transport of the positively charged trypsin molecules was thus effected in two ways (see Fig. 1): (1) 'electrically enhanced (EE)' transport, in which the solute migrated toward the oppositely charged electrode as a result of both the potential gradient between the two electrodes and the concentration gradient between the R and S cells; and (2) 'electrically impeded (El)' transport, in which the solute moved toward the receiving side as a result of the concentration gradient alone, in competition with the potential gradient.

Measurements of concentration and activity for trypsin The protein concentration in the R-cell was determined at suitable time intervals (10 min) by measuring the absorbance of the sample solution at 280 nm using a Hitachi U-3200 spectrophotometer. To investigate the denaturing of the enzyme during permeation under electric fields, the total enzymatic activity of the receiving solution was assayed after the finish of the permeation experiments. The activity measurement was carried out using B A N A as the substrate in the manner described previously? 2 RESULTS AND DISCUSSION

Permeation characteristics Preliminary experiments revealed that the membrane exhibited permeability only under an applied electric field. It was thus found that

Protein permeation through a PVA/ PAA composite membrane 3O

251

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Fig. 2. Time dependence of trypsin concentration in the R cell during the EE and EI transport processes at different applied electric fields. Changes in the protein concentration in the S cell before and after the transport experiments were within 1 % in the largest case (EE transport at 8 V).

the application of an electric field brought a b o u t the formation of pore channels within the m e m b r a n e , due to isometric contraction, through which the trypsin molecules could p e r m e a t e from the S cell to the R cell. The time d e p e n d e n c e of the trypsin concentrations in the R-cell under different electric fields is shown in Fig. 2, from which the p e r m e a t i o n rates (P) were estimated and summarised in Table 1. In the estimation of P values, we used the thickness (0.02 cm) of the m e m b r a n e in a swollen state. Actually, it cannot be said that there was no change in the m e m b r a n e thickness before and after the application of an electric field, because the present gel m e m b r a n e shrank when a D C current was applied under conditions in which the m e m b r a n e was allowed to change in shape. F r o m our previous study, j3 however, we believe that any error due to this uncertainty regarding the m e m b r a n e thickness in the estimation of the P values is less than 8% and therefore irrelevant to the following discussion. It can be seen from Fig. 1 and Table 1 that the E E transport process elicited a larger p e r m e a t i o n rate than the EI transport process. Since the diffusion constant (D, 1.1 x 10 6 cm2/s) of trypsin is known, ~ the rate of trypsin p e r m e a t i o n through the m e m b r a n e due solely to the thermal diffusion mechanism can be calculated using the relation P =

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252

TABLE 1 P e r m e a b i l i t y of P A A / P V A C o m p o s i t e Gel M e m b r a n e and E n z y m a t i c Activities o f Trypsin T r a n s p o r t e d t h r o u g h the M e m b r a n e

Voltage (V)

0 2 4 6 8

Permeation constant x 105 (cm/s)

Relative activity (rs/r,) x 100 ( % )

E1 transport

EE transport

E1 transport

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0 0.15 0.55 2.03 5.30

0 1.36 3.92 4.69 10-90

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D/g, where ~ is the membrane thickness (0.02 cm) in a swollen state. We obtained a P value of 5.5 x 10 5 cm/s under conditions in which the trypsin was assumed to be transported from the S to R cells through the membrane without any effect from the applied electric field. However, this value is about half the P value (10.90 x 10 5 cm/s) observed in the E E permeation experiment at 8 V. The difference between the calculated and observed results is still significant even when the error based on the membrane thickness was taken into consideration. In E E transport, trypsin was carried toward the oppositely charged electrode (see Fig. 1); therefore, an electrophoretic effect due to the potential gradient between both the electrodes could facilitate the permeation of trypsin with positive charges. The electrically facilitated trypsin permeation through the present membrane would provide a useful tool in constructing a drug delivery device. In addition, this could also play a part in the field of enzyme immobilisation since the study and development of polymeric supports that function to promote the diffusion of polymeric substrates such as polysaccharides and proteins are now required (for example, see Refs 14 and 15). In the E1 permeation at 8 V, on the other hand, the observed P value (5.30 x 10 -5 cm/s) was close to the calculated rate of trypsin permeation. This could indicate that the application to the present membrane of electric fields >- 8 V results in the formation of pore channels with sizes large enough to permit trypsin permeation. At voltages < 6, then, the P values obtained in both the E E and E1 transport experiments were smaller than the calculated P value. Such reduced permeation rates can be related to the steric hindrance effect of small pore channels; that is, the membrane hinders trypsin permeation

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when the size of the pore channels is smaller than the molecular size of the protein. This is the case even in the EE process, in which the permeation was facilitated by the electrophoretic effect. As the main conclusion to be drawn from Table 1, it is apparent that the application of electric fields -> 8 V results in the formation of pore channels within the m e m b r a n e due to electrochemically generated contractile stress--pore channels with sizes large enough to permit trypsin permeation. U n d e r such conditions, the EE process has been found to elicit a permeation rate which is larger than that in the thermal diffusion process based on a concentration gradient. However, the P values for the EI permeation were smaller than those for the EE permeation. This seems to be an interference effect of the potential difference, which was opposite in direction to the concentration gradient (see Fig. 1); the potential difference developed in this way may thus inhibit the diffusion of trypsin molecules even when the pore channels have expanded to allow their permeation.

Enzymatic activity It was known that the application of an electric field was needed to activate the m e m b r a n e system for protein permeation. At present, however, it is not clear to what extent the biochemical activity of proteins is maintained under applied electric fields. For example, the effect of an electric field on insulin activity has not been studied in an electrically activated polymer system from which insulin release was successfully c o n t r o l l e d / Thus, changes in the enzymatic activity of trypsin during the permeation under an applied electric field will become an important problem with respect to the application of the m e m b r a n e in a drug delivery device. The activity of trypsin after the completion of the permeation experiments was examined and compared with that of the native enzyme at the same protein concentration (Table 1). The relative activity was defined as (rs/r.) × 100, where rs and rn are the BANA-hydrolysing rates of the sample and native enzymes determined under the same assay conditions, respectively. As can be seen from Table 1, there is no serious difference in the enzymatic activity before and after the permeation of trypsin through the membrane. This result means that denaturing of the enzyme did not occur in either the EE or EI transport process.

Electric on/off control of trypsin permeation The features of the present m e m b r a n e have been applied in the on/off control of trypsin permeation through the application and removal of

Takeshi Yamauchi, Etsuo Kokufuta, Yoshihito Osada

254

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an electric field (8 V) (see Fig. 3). The control experiments were carried out for both the E E and EI transport processes. The transport of trypsin to the R cell was initiated when a DC source was switched on, then immediately terminated by switching off the current. Such initiation-termination control could be repeated reversibly at least three times during a single run when the experiment with freshly prepared trypsin solution was repeated in a series of seven runs. The permeation rate under the 'on' state was usually larger for E E transport than for EI transport because of the electrophoretic effect, but remained unaltered by repetition of the 'on' and 'off' states in both the E E and EI transport processes. As a result, it was found that the present membrane expands and contracts its pore size reversibly in response to an electrical stimulus. In addition, such expansion and contraction was comparatively rapid enough to allow the control of the initiation and termination of trypsin permeation by switching the current on and off.

CONCLUSION A composite polyelectrolyte gel membrane consisting of PVA and P A A is capable of electrically regulating the permeation of trypsin as the high-molecular-weight ionic solute without serious loss in enzymatic activity. Facilitated permeation was observed when the protein was transported toward the electrode opposite in sign to the charge of the protein. These beneficial features of the present membrane system are due to the electrically and reversibly controlled expansion-contraction

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of the pore channels which are formed by electrochemically generated contractile stress over the gel membrane. The concept introduced here, however, should be applicable in general, and various types of polyelectrolytes could be used to construct membranes that function to regulate the permeation of polymeric solutes.

ACKNOWLEDGMENTS The authors wish to thank R. Craig for his critical reading of this manuscript and Drs K. Yamada and S. Tatekawa of Nihon University for m e a s u r e m e n t of tensile stress. This research was supported in part at University of Tsukuba by a grant to E. K. through a grant from the New Energy and Industrial Technology Development Organization (NEDO).

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