Transport Properties of EVAl-Starch-Î± Amylase Membranes

Biomacromolecules 2005, 6, 1389-1396

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Transport Properties of EVAl-Starch-r Amylase Membranes M. L. Coluccio, N. Barbani, A. Bianchini, D. Silvestri, and R. Mauri* Department of Chemical Engineering, DICCISM, University of Pisa, Via Diotisalvi 2, 56126 Pisa, Italy Received October 26, 2004; Revised Manuscript Received February 3, 2005

We investigated the influence of various physicochemical parameters on the morphology and time-porosity formation of membranes composed of ethylene-vinyl alcohol, starch, and R-amylase. In particular, we determined that (1) it is possible to obtain a membrane with desired porosity by phase inversion in an appropriate water-ethanol mixture and (2) the enzymatic bioerosion is controlled by the amount of R-amylase present in the blend. Although no experiments involving drugs were carried out, the delivery properties of the film were determined by measuring the Darcy permeability, the effective diffusivity, and the mean reaction rate of the membranes, relating them to the modality of membrane preparation, the amount of enzyme present within the membrane, and the incubation time of the samples in a buffer solution. Simple theoretical models of the delivery properties of the membranes were developed, leading to predictions that were in good agreement with the experimental results. Introduction In the biomedical industry, various devices have been developed to provide zero-order drug release. Such devices must necessarily be designed to overcome Fick’s law since, as the drug diffuses out, its concentration decreases with time. To this end, one approach has been to split the mass transfer into two steps, such that the drug must pass, first, through a holding volume and then out to the delivery site. In this way, the flux of the drug out can be kept nearly constant by providing a relatively large surface area for the first step and a small area for the second step.1,2,3 Another approach is to design devices where diffusion is coupled with a second mechanism of mass transfer, so that the combination of the two rate processes can provide a relatively flat release profile. Examples of these devices include swellable polymers,4 desorpting hydrogels5 and other devices where the mass transfer of drugs is controlled through multiple resistances in series.6-8 In this work, we wanted to develop an enzymatically controlled system, where the decrease of the driving force for drug diffusion is compensated by an increase in the Darcy permeability (and the porosity as well) of the matrix, due to enzymatic erosion. This idea of using enzymatically erodible systems for controlled drug release is not new in the biomedical and environmental fields.9,10 In particular, polysaccaridic biodegradable matrixes, such as starch and starchbased blends, have been used as substrate, as starch is inexpensive and available in large quantities and, above all, its degradation occurs naturally in the human body.11 In fact, starch is naturally hydrolyzed by several amylolytic enzymes, such as R-amylase, producing oligosaccharides, dextrins, and maltose.12 Using this property, an enzymatically controlled release systems was recently developed by adding R-amylase * To whom correspondence should be addressed. E-mail: r.mauri@ ing.unipi.it. Tel: ++39-050-511248. Fax: ++39-050-511266.

to cross-linked amylose tablets.13 The properties of this delivery system were successively investigated,14 studying the influence on enzymatic hydrolysis and on drug release of several physicochemical parameters, such as pH, ionic strength, enzyme concentration, and compression force. From previous studies, it was known that the kinetics of the starch hydrolysis by R-amylase depends on three factors: the type of R-amylase, the properties and structure of the starch substrate, and the mode of interaction between enzyme and polymeric chain. The type of R-amylase determines the type and distribution of the hydrolysis products. This is important because some of these products, such as maltose, inhibit the successive hydrolytic action of R-amylase.15 The structure of the starch substrate, such as its crystallinity, is probably the most important factor affecting the kinetics of starch hydrolysis, since R-amylase can more easily degrade amorphous regions than crystalline regions.16 Surface area is also important, as a high surface favors the migration of the enzyme to the substrate and therefore tends to increase the rate of hydrolysis. Finally, the kinetics of starch hydrolysis is influenced by the mode of interaction between enzyme and polymer, since, for example, the hydrolysis of an insoluble starch substrate is obviously very different than that of a soluble substrate.17 In addition, the hydrolysis of starch is drastically reduced as the reaction progresses, due to the inhibition of the enzymatic degradation by reaction byproducts.16,17 In our work, we exploited the properties of R-amylasecontrolled starch-based matrixes to develop membranes to be used for controlled drug release and for hemodialysis. To do that, however, the matrix cannot be compose of starch alone, as its mechanical resistance is not sufficient. This is why we used bio-artificial polymeric materials, which are blends of biological macromolecules and hydrophilic synthetic polymers, as these materials combine the good biocompatibility of the biological component with the good

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Table 1. Melting Point and Enthalpy of Fusion of EVAl, EVAl/ Starch, E25, E50, and E100 Membranes

EVA1 30/70 starch/EVA1 E25 E50 E100

Tm (°C)

∆H (J/g)

156 162 163 164 164

86 41 52 51 53

material properties of the synthetic polymer.18,19 In our case, the membranes are blends of EVAl (i.e., a copolymer of ethylene and vinyl alcohol) and starch, the latter acting also as the biological substrate of the eroding enzyme, that is R-amylase. Similar systems have been studied in recent articles,20,21,22 showing that the biological activity of the enzyme is unaltered when it is embedded within a EVAlstarch matrix. However, it was stressed that the potential use of these films for application as drug delivery systems is severely limited by high film dissolution after contact with aqueous media.In this work, after describing in the next section the properties of the materials and the methods that were used in our experiments, in section 3 we present the results, describing the influence of several physicochemical parameters such as R-amylase concentration and phase inversion characteristics on the physical properties of the resulting membranes, such as its Darcy permeability and the effective reaction rate, describing the strength of the enzymatic hydrolysis. Materials and Methods Reagents. Poly(ethylene-co-vinyl alcohol) with 40% ethylene content, denoted as clarene R20 and supplied by Solvay, together with a soluble starch supplied by Carlo Erba, Italy, were used as received. Dimethyl sulfoxide (DMSO, Riedel-de-Haen), 2-propanol (Carlo Erba, Italy), and R-amylase from Bacillus subtilis (Sigma, St Louis, MO) (MW ) 96000) were also used without further purification. Membrane Preparation. The membranes were produced by phase inversion from a mixture of EVAl and a starch polymer solution containing R-amylase. Concretely, first we dissolved 2.5 g of EVAl into 10 mL of DMSO and added 1 g of starch; then, from this homogeneous mixture, we prepared three different solutions by adding 25, 50, and 100 mg of enzyme. The resulting solution was spread uniformly on a glass plate using a knife machine “Separem Type SP L1”, forming a 50 µm-thick film. Subsequently, the film was, first, quickly immersed in a 50% 2-propanol/50% water solution bath for 1 h at room temperature, and then the resulting membrane was immersed in a 2-propanol bath for 15 h, again at room temperature. Finally, the membranes were kept in an oven at 30 °C until the evaporation of the solvent was complete. In the following, the membranes are named TQ, E25, E50, and E100 when, respectively, no enzyme, 25, 50, and 100 mg of enzyme are added. Thermal Properties and Membrane Microstructure. The thermal behavior of the membranes was studied with a Perkin-Elmer DSC7 differential scanning calorimeter. Scans were carried out at 10 °C/min, using samples of a few

milligrams, in the presence of excess water (wet samples), that were hermetically sealed into aluminum pans. Membranes were fractured in liquid nitrogen, sputtered with gold, and then observed at 10 kV, by a scanning electron microscopy (SEM), using a JEOL JSM 5600 microscope. Release Tests. Release tests were carried out to evaluate the amounts of starch, maltose, and RA that were released from the membranes in the inversion bath in order to evaluate the stability of the matrix. To do that, first, the 2-propanol that was present in the release solution was evaporated in a ventilated oven at 40 °C for 48 h. Then, we formed a mixture of 2 mL of the resulting solution and 1.5 mg of enzyme, let it react for 1 h at 37 °C, and then measured the maltose concentration using the DNS method. The latter consists of adding DNS acid which, by reacting with the hydrolysis starch products, forms nitroamminosalicilic acid, which in turn can be detected measuring the absorbance of the solution at a wavelengthh of 546 nm. At this point, the same measurements were repeated using a system without enzyme, so that at the end the amount of starch that was digested by RA could be determined. Finally, the amount of released RA was evaluated by measuring the absorbance of the solution at a wavelength of at 280 nm, thereby determining the enzyme concentration. Absorbance measurements were performed using Shimadzu UV-2100 dual beam spectrophotometer. Darcy Permeability and Effective Diffusivity Measurement. A simple device was built to measure the Darcy permeability of the matrix. It consists of a conduit, where the membrane was clamped transversally; on one side, water was kept at a given pressure, whereas at the other side, it was continuously removed and weighted. Considering that in a homogeneous material the pressure drop and the induced fluid flow are related linearly through Darcy’s law, the mean velocity of the fluid, V, flowing through the membrane can be written as V)

k ∆P µL

(1)

where k is the Darcy permeability of the membrane, µ is the viscosity of the solution, and L is the membrane thickness. The Darcy permeability, which is measured in Darcy (1 Darcy ) 1 µm2), is a quantitative measure of the ability of the fluid to be convected through the membrane and depends only on the microstructure of the matrix. In fact, according to Blake-Kozeny’s semiempirical correlation,23 we have k)

δ23 150(1 - )2

(2)

where is the porosity and δ indicates, approximately, the pore size. Therefore, from the Darcy permeability k and the porosity , both of which can be determined experimentally, we can determine the microscopic pore size δ. In a second set of measurements, we determined the effective diffusivity D of a solute, i.e., maltose, dissolved in a carrier fluid, i.e., water and diffusing through the membrane. This can be done easily considering that the mass flux of the solute, Jc, induced by the difference of solute

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Properties of EVAl-Starch-R Amylase Membranes

concentration, ∆c ) cs, between the two sides of the membrane, follows Fick’s law D Jc ) cs L

(3)

In the absence of a flow field across the membrane, D will be roughly equal to the product of the molecular diffusivity, Dm, and an O(1) constant, depending only on the microstructure of the porous material. As a double check of our measurements, we also measured the mass flux, Jc, of a solute diffusing and being convected through the membrane, i.e., being driven both by the difference of solute concentration, ∆c ) cs, and by the pressure drop, ∆P, that are imposed between the two sides of the membrane, i.e. dc dz

Jc ) cV - D

(4)

Here z is the distance from the membrane surface (i.e., the longitudinal coordinate), c ) c(z) is the local solute concentration, D is the effective diffusivity, and V is the uniform mean velocity of the carrier fluid, which in turn is driven by the pressure drop, according to eq 1. It should be stressed that here the effective diffusivity D is not the same as that appearing in eq 3, as it depends strongly on the mean fluid velocity V. In fact when convection dominates diffusion at the microscale, i.e., when the Peclet number is very large, NPe ) Vδ/Dm . 1, the effective diffusivity depends linearly on the mean fluid velocity, i.e. D ) R-1DmNPe ) R - 1Vδ, when NPe ) Vδ/Dm . 1

(5)

where R > 1 is a sort of tortuosity coefficient23 that depends only on the microstructure of the porous material.24,25,26,27 Therefore, we see that here D can be several order of magnitude larger than the molecular diffusivity Dm. At steady state, the problem {dJc}/{dz} ) 0 can be solved with boundary conditions c(0) ) cs and c(L) ) 0, finding NSh )

Jc VL L N and N ) ) , with NSh ) -N Dc /L D Rδ 1-e s

Here NSh denotes the Sherwood number, representing the ratio between the solute mass flux and its convection-driven diffusive component at the macroscale, whereas N indicates the ratio between macro- and microscale. Note that N can also be interpreted as a macroscale Peclet number, representing the ratio between convective and diffusive mass fluxes at the macroscale. Since in our case N . 1, we obtain lim NSh ) N, that is lim Jc )

N.1

N.1

kcs ∆P µL

(6)

Using these results, we can determine again the Darcy permeability k by measuring the solute mass flux induced by different values of ∆P. Enzymatic Erosion and Enzymatic ActiVity. Enzymatic degradation was investigated by immersing the membranes in a stock solution containing a phosphate buffer (pH 6.9) at 37 °C. These conditions are ideal for the activity of

R-amylase which, by degrading endogenous starch, increases the matrix porosity. Different incubation times were used, to study its effects on the enzymatic activity and the matrix morphology, which, in turn, determine the transport properties of the membrane. The enzymatic activity was evaluated by measuring the amount of maltose that was produced, thereby determining the amount of substrate that was digested by the enzyme. To do that, a part (0.5×0.5 cm2) of the E25, E50, and E100 membranes were immersed in 30 mL of a 1% substrate solution (i.e., Zulkowsky starch, a very soluble starch obtained from potatoes). For comparison, the same amounts of free enzyme (i.e., with no membrane) were added to 30 mL of the same solution. After incubation at 37 °C at different times, the enzymatic activity was measured by extracting 1 mL of each solution and measuring the absorbance of the nitroamminosalicilic acid that was formed during the maltose-DNS reaction at a 546 nm wavelength. The process of enzymatic degradation takes place in two successive steps: first the starch penetrates the membrane and reaches the pore walls and then hydrolysis takes place as starch reacts with the enzyme, which is either free or entrapped within the matrix. Accordingly, since the characteristic time of the process, τs, is the sum of the penetration time, τp, and the reaction time τr, and considering that we can safely assume that the rate of hydrolysis, Rs (i.e., the amount of starch that reacts per unit time and per unit membrane volume), is proportional to the starch bulk concentration cs, we obtain Rs ) rscs, where rs )

1 1 ) τs τp + τr

(7)

In addition, we can assume that the reaction rate rr ) τr-1 depends on the enzyme concentration, ce, via a Langmuir isotherm28 ce rr ) kr Keq + ce

(8)

where kr and Keq are the specific rate constant and the equilibrium constant between free and adsorbed enzyme, respectively.16 Clearly, the kinetics of enzymatic degradation is controlled by the slowest process between penetration and reaction. In our case, since τr , τp, we obtain Rs ) rscs ≈

cs τp

(9)

Now, initially, the membrane is dry and therefore water (with starch dissolved into it) enters the membrane by capillary convection, that is driven by a pressure drop induced by the curvature of the meniscus interface within the pores, i.e., ∆P ) 2σ/δ, where σ is the surface tension and we have assumed that the pore side walls are wettable by water (i.e., the contact angle is 0°). Now, consider that the mean fluid velocity (in the longitudinal direction z) is Vσ )

(∆P)δ2 8µRz

(10)

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Figure 3. Enzymatic kinetics of entrapped amylase in E100 membranes and enzymatic kinetics of free amylase.

Figure 1. Mean water speed as a function of the pressure drop through E25, E50, and E100 membranes in phosphate buffers at 37 °C. The straight lines correspond to Darcy permeabilities equal to k ) 1.5 × 10-4 µm2, k ) 1.2 × 10-4 µm2, and k ) 1.0 × 10-4 µm2, respectively.

Figure 4. Hydrolysis starch rate by entrapped amylase in E100 membranes as a function of the incubation time in phosphate buffer solutions (PBS) at 37 °C.

Figure 2. Mean water speed as a function of the pressure drop through E100 membranes in phosphate buffers at 37 °C for incubation times of 4, 8, 16, and 24 h. The straight lines correspond to Darcy permeabilities k ) 0.7 × 10-4 µm2, k ) 1.1 × 10-4 µm2, k ) 1.5 × 10-4 µm2, and k ) 1.6 × 10-4 µm2, respectively.

where z is the penetration depth, that is the distance covered by the meniscus along the longitudinal direction, and R is a tortuosity coefficient, which is roughly equal to the O(1) coefficient R defined in eq 5. Therefore, since the mean fluid velocity equals the mean speed with which the meniscus moves through the medium, i.e., {d(Rz)}/{dt} ) Vσ, we obtain dz σδ ) dt 4µR2z and by integration we can calculate the penetration depth after a time t and from it the mean velocity z)

x

σδt w Vσ ) 2R2µ

x

σδ 8R2µt

(11)

This first stage lasts until the penetration depth equals half membrane thickness, that is when t ht, there is no more convection, and therefore, the starch penetrates the membrane by



Figure 6. Thermograms of EVAl, EVAl/starch, E25, E50, and E100 membranes.

diffusion. Therefore, considering that τp ) R2L2/Dm, where R is a tortuosity constant, from eq 9 we obtain Rs ) rscs, where rs )

Dm 1 ) 2 2 τp R L

(14)

Clearly, as the enzymatic reaction proceeds, the tortuosity constant R will decrease and therefore we expect that at larger time t > ht the rate of hydrolysis will increase in time, albeit slowly. Results and Discussion Membrane Stability. The enzyme release for the E25, E50 and E100 membrane in the first inversion bath was of 41.6, 42.5, and 43.37%, respectively. On the other hand, no enzyme release was observed in the second inversion bath. Both the first and the second bath did not present any trace of starch and maltose. These results show that the alcoholic environment of the phase inversion bath does inactivate the enzyme. Darcy Permeability Measurements. At first, we checked that, as expected, the TQ, E25, E50 and E100 membranes result impermeable to water because of their dense skin and compact structure. After treatment in phosphate buffer at 37 °C, we saw that the volumetric flux of water flowing through the E25, E50 and E100 membranes (the TQ membrane remained impermeable, as expected) increased as the amount of entrapped enzyme and the incubation time increased (see Figures 1 and 2). The tests were performed in a range of pressures drops (∆P ) 60÷180 mmHg) between the diastolic and systolic pressure. Using these results and applying eqs 1 and 2, we determined the Darcy permeability as a function of enzyme concentration and incubation time. In particular, we see that the Darcy permeability increases as the matrix becomes more porous, until after 16 h of incubation time it starts to level off, reaching an asymptote after 24 h.

Stability of Entrapped r-Amylase. In Figure 3, the amount of maltose produced by E100 membranes is reported as a function of the incubation time in phosphate buffer over a period of 1 h. We see that the maltose profile increases slowly with the incubation time, as the matrix porosity increases and starch can gradually reach the entrapped R-amylase. At the end, after about 2 h, the maltose produced reaches an asymptotic value, that remains constant in time. On the other hand, when we measure the amount of maltose produced in a solution containing free enzyme, the asymptotic value is reached much sooner, as one would expect. The stability of the entrapped enzyme was also evaluated after incubation of E100 in phosphate buffer for different time intervals. In the model of eqs 7-14, we have considered that the activity of the membrane is the result of two successive phenomena: first the starch penetrates the membrane and reaches the pore walls, and then hydrolysis takes place as starch reacts with the enzymes. The effect of these two events implies an initial decrease of the membrane activity, until all of the membrane pores are wet, and then membrane activity increases with porosity. These predictions are in qualitative agreement with the experimental results described in Figure 4. Unfortunately, a quantitative comparison between theoretical predictions and experimental results is not possible, as many of the parameters of eq 13 are not known. However, by applying eq 13 based on both reasonable estimates (i.e., ) 0.3, R ) 10) and experimental measurements (i.e., σ ) 70 dyn/cm, L ) 0.05 cm, δ ) 0.5 µm obtained from eq 2), we predict that rs ) O(10-3 s) at t ) 4 h, in agreement with the results of Figure 4. Repeatability Tests. Repeatability tests were carried out by dipping samples of the membranes in a series of baths containing 1% starch solutions. The samples were kept in the first bath for 10 min and, after an intermediate washing, were transferred to the second bath. Repeating this operation for 6 times, we evaluated the ability of the enzyme to keep its activity by UV analysis.

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Figure 7. SEM images of 30/70 starch/EVAl membranes: (A and B) surface and section of the membrane obtained by phase inversion into a 2-propanol bath; (C and D) surface and section of the membrane obtained by phase inversion in a water bath; (E and F) surface and section of the membrane obtained by phase inversion in a 2-propanol/water (50/50 vol.) bath.

The results of the repeatability tests are shown in Figure 5, representing the amount of maltose produced (and therefore the amount of starch consumed) in each test. We see that maltose production remains fairly constant in time, confirming the repeatability of our experimental results. Thermal Analysis (DSC). In Figure 6, we reported the thermograms of E25, E50, and E100 membranes (i.e., membranes composed of an EVAl, starch, and R-A blend), comparing them with those of pure EVAl, which are shown in Table 1. We see that the presence of the biological components determines a decrease of the enthalpy of fusion, indicating a corresponding reduction of cristallinity of the matrix, and a shift of the melting point toward higher values, indicating an interaction between the components. From these results, we can conclude that in our bio-artificial polymeric materials the synthetic material and the synthetic polymer have been fused together successfully.

Morphology of Membranes. The membrane microstructure was observed by SEM (see Figure 7), showing the surface of the membranes obtained by phase inversion in the presence of isopropyl alcohol, with porosity ) 0.3. The porosity was calculated applying eq 2, where δ and k were measured from micrographs and permeability tests, respectively. On the other hand, the surface of the membranes obtained by phase inversion in aqueous baths does not show any porosity. These results prove our early conjecture that the presence of isopropyl alcohol in the inversion bath causes a slower phase inversion process, thereby obtaining a microporous membrane, whereas when using an aqueous inversion bath, the process is very rapid and the pores do not have the time to form. In addition, morphological analysis after phosphate buffer treatment is in agreement with the Darcy permeability results,



Figure 8. SEM images of E100 membranes as a function of incubation time in phosphate buffer solution: (A) surfaces and section after 0 h of incubation; (B) surfaces and section after 4 h of incubation; (C) surfaces and section after 8 h of incubation; (D) surfaces and section after 16 h of incubation; (E) surfaces and section after 24 h of incubation.

showing an increase of porosity that is consistent with the results of Figure 8.

depended on the enzyme concentration and the incubation time in phosphate buffer.

Conclusions

The next phase will be to proceed to membranes containing drug and to test them as controlled enzymatic drug release systems.

The objective of this study was to produce and characterize bio-artificial polymeric systems with an enzymatic controlled porosity that are usable in biomedical applications as controlled drug release systems. Concretely, our system consisted of a blend of EVAl (i.e., a copolymer of ethylene and vinyl alcohol), starch, and R-amylase, which is a starcheroding enzyme. During the membrane preparation, first we devoted a particular attention to deactivate the enzyme, and then we performed a controlled reactivation by incubation in a phosphate buffer at 37 °C. An inversion bath containing isopropyl alcohol allows a reversible inactivation of R-amylase; however, as soon as the membranes were put into an aqueous solution, by SEM analysis and Darcy permeability experiments, we observed an increasing in porosity, which we linked to the enzymatic degradation of endogenous starch. In fact, the increase in porosity and Darcy permeability

Acknowledgment. This work was supported by the Italian Ministry for University Education and Research (MIUR). References and Notes (1) Hsieh, D. S. T.; Rhine, W. D.; Langer, R. S. Zero-order controlled release polymer matrixes for micro- and macromolecules. J. Pharm. Sci. 1983, 72, 17. (2) Brooke, D.; Washkuhn, R. J. Zero-order drup delivery system: theory and preliminary testing. J. Pharm. Sci. 1977, 66, 159. (3) Lipper, R. A.; Higuchi, W. I. Analysis of theoretical behavior of a proposed zero-order drug delivery system. J. Pharm. Sci. 1977, 66, 163. (4) Petropoulos, J. H.; Papadokostaki, K. G.; Amarantos, S. G. A general model for the release of active agents incorporated in swellable polymeric matrixes. J. Polymer. Sci. B: Polym. Phys. 1992, 30, 717.

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(5) Singh, M.; Lumpkin, J. A.; Rosenblatt, J. Mathematical modeling of drug release from hydrogel matrixes via a diffusion coupled with desorption mechanism. J. Controlled Release 1994, 32, 17. (6) Langer, R. S. Polymeric delivery systems for controlled drug release. Chem. Eng. Commun. 1980, 6, 1. (7) Langer, R. S.; Peppas, N. A. Biomaterials 1981, 2, 201. (8) Varelas, C. G.; Dixon, D. G.; Steiner, C. A. Zero-order release from biphasic polymer hydrogels. J. Controlled Release 1995, 34, 185. (9) Heller, J.; Baker, R. W. Theory and practice of controlled drug delivery from bioerodible polymers. In Controlled Release of BioactiVe Materials; Baker, R. W., Ed.; Academic Press: New York, 1980; pp 1-17. (10) Heller, J. Use of enzymes and bioerodible polymers in self-regulated and triggered drug delivery systems. In Pulse and Self-Regualated Drug DeliVery; Kost, J., Ed.; CRC Press: Boca Raton, 1990; pp 93108. (11) Kost, J.; Shefer, S. Chemically modified polysaccharides for enzymatically controlled oral drug delivery. Biomaterials 1990, 11, 695. (12) Biliaderis, C. G. The structure and interactions of starch with food constituents. Can. J. Physiol. Pharmacol. 1991, 69, 60. (13) Dumoulin, Y.; Cartilier, L. H.; Mateescu, M. A. Cross-linked amylose tablets containing R-amylase: an enzymatically controlled drug release system. J. Controlled Release 1999, 60, 161. (14) Rahmouni, M.; Chouinard, F.; Nekka, F.; Lenaerts, V.; Leroux, J. C. Enzymatic degradation of cross-linked high amylose starch tablets and its effect on in vitro release of sodium diclofenac. Eur. J. Pharm Biopharm. 2001, 51, 191. (15) MacGregor, E. A. R-amylase structure and activity. J. Protein Chem. 1988, 7, 399. (16) Leloup, V. M.; Colonna, P.; Ring, S. G. R-amylase adsorption on starch crystallite. Biotechnol. Bioeng. 1991, 38, 127. (17) Leloup, V. M.; Colonna, P.; Ring, S. G. Physicochemical aspects of resistant starch. J. Cereal Sci. 1992, 16, 253.

Coluccio et al. (18) Giusti, P.; Lazzeri, L.; Lelli, L. Bioartificial polymeric materials: a new method to design biomaterials by using both biological and synthetic polymers. Trends Polym. Sci. 1993, 1 (9), 261. (19) Lazzeri, L. Progress in bioartificial polymeric materials. Trends Polym. Sci. 1996, 4 (8), 249. (20) Cristallini, C.; Barbani, N. Physical and biological stability of dehydro-thermally cross-linked R-amylase-poly(vinyl alcohol) blends. Polym. Int. 1998, 44, 491. (21) Cristallini, C.; Lazzeri, L.; Cascone, M. G.; Polacco, G.; Lupinacci, D.; Barbani, N. Enzyme-based bioartificial polymeric materials: the R-amylase-poly(vinyl alcohol) system. Polym. Int. 1997, 44 (4), 510. (22) Giusti, P.; Lazzeri, L.; Barbani, N.; Narducci, P.; Bonaretti, A.; Palla, M.; Lelli, L. Enzyme-based bioartificial polymeric materials: the R-amylase-poly(vinyl alcohol) system. J. Mater. Sci. Mater. Med. 1993, 4 (6), 538. (23) Bird, R. B.; Stewart, W. E.; Lightfoot, E. N. Transport Phenomena; Wiley: New York, 1960; p 199. (24) Koch, D. L.; Brady, J. F. The symmetry properties of the effective diffusivity tensor in anisotropic porous media. Phys. Fluids 1987, 30, 642. (25) Shapiro, M.; Brenner, H. Dispersion of a chemically reactive solute in a spatially periodic model of a porous medium. Chem. Eng. Sci. 1988, 43, 551. (26) Mauri, R. Dispersion, convection and reaction in porous media. Phys. Fluids A 1991, 3, 743. (27) Mauri, R. Heat and mass transport in random velocity fields with application to dispersion in porous media. J. Eng. Math 1995, 29, 77. (28) Sattler, W.; Esterbaur, H.; Blatter, O.; Steiner, W. The effect of enzyme concentration on the rate of the hydrolysis of cellulose. Biotechnol. Bioeng. 1989, 33, 1221.

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Transport Properties of EVAl-Starch-Î± Amylase Membranes

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