pH- and temperature-sensitive permeation through polyelectrolyte complex films composed of chitosan and polyalkyleneoxide–maleic acid copolymer

Journal of Membrane Science 241 (2004) 347–354

pH- and temperature-sensitive permeation through polyelectrolyte complex films composed of chitosan and polyalkyleneoxide–maleic acid copolymer Toru Yoshizawa, Yoshitsune Shin-ya∗ , Kyung-Jin Hong, Toshio Kajiuchi Department of International Development Engineering, Tokyo Institute of Technology, 2-12-1 Ookayama, Ishikawadai 4th Building, Meguro-ku, Tokyo 152-8550, Japan Received 30 January 2004; accepted 2 June 2004

Abstract The pH- and temperature-effects on drug permeation from polyelectrolyte complex (PEC) films composed of a cationic polymer, chitosan (CS) and an anionic polymer, polyalkyleneoxide–maleic acid copolymer (PAOMA), have been investigated. In this study, we prepared and investigated PEC films for utility as on–off switches in drug transport. PEC films were prepared by a casting/solvent evaporation method. PEC films swelled at low pH and low temperature. This swelling behavior was reversible with the films responding rapidly to a change in environmental pH and temperature. Drug permeation rates were tested with pH 3.8 and 6.2 at 25 and 50 ◦ C. Salicylic acid, phenol and glucose were selected as model drugs to examine the effect of molecular weight. A decrease in pH from 6.2 to 3.8 and an increase in temperature from 25 to 50 ◦ C resulted in a corresponding increase in the rate of drug permeation. Of the three model drugs tested, glucose, with the highest molecular weight showed the lowest effective diffusion coefficient. © 2004 Elsevier B.V. All rights reserved. Keywords: Polyelectrolyte complex film; Chitosan; Polyalkyleneoxide–maleic acid copolymer; Stimuli-sensitive permeation; Swelling

1. Introduction Stimuli-responsive biodegradable and biocompatible films and hydrogels have recently shown enormous potential in controlled release drug delivery applications [1–6]. Temperature-sensitive hydrogels are one of the most commonly studied classes of stimuli-sensitive systems [7,8]. Poly(N-isopropylacrylamide) (PNIPAAm) is a typical polymer among the temperature-sensitive polymers investigated to date [9–13]. PNIPAAm has a lower critical solution temperature (LCST) in the range of 25–32 ◦ C. However, the clinical applications of PNIPAAm hydrogels are limited due to the carcinogenic or teratogenic toxicity of the monomer and crosslinkers used in PNIPAAm synthesis [1]. In addition, PNIPAAm and its derivatives are not biodegradable [1].

∗ Corresponding author. Tel.: +81-3-5734-3246; fax: +81-3-5734-3315. E-mail address: [email protected] (Y. Shin-ya).

0376-7388/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.memsci.2004.06.006

Chitosan (CS), a naturally occurring biodegradable and biocompatible cationic polysaccharide has found wide utility in applications ranging from tissue engineering to drug delivery [14–19]. Polyalkyleneoxide–maleic acid copolymer (PAOMA) is an anionic polymer with excellent biocompatibility and unique amphiphilic properties. Especially, a series of PAOMA has also various LCST with changing the alkyleneoxide (AO) chain composition. Using these unique polymeric materials, enzyme modification [20,21] and protein separation [22] have been conducted. Electrostatic interactions between the cationic CS and the anionic PAOMA polyelectrolytes result in the formation of a polyelectrolyte complex (PEC). The swelling, drug permeation and release properties of PEC can be controlled by pH and temperature changes. The high biocompatibility of PEC films provides potential for therapeutic applications as a stimuli-responsive drug delivery carrier. In this study, we prepared PEC films composed of CS and PAOMA by a casting/solvent evaporation method to investigate the films’ pH- and temperature-responsive behavior on permeation of three model drugs; salicylic acid, phenol

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T. Yoshizawa et al. / Journal of Membrane Science 241 (2004) 347–354

O

H

H

O H

O

HO

HO NH2

CH2OH

H

O H

O H

HO

CH2OH

H

CH2OH

H

H

NH2

H

HO

H

H

NH2

OH

n Fig. 1. Chitosan structure.

and glucose. Whilst previous studies have developed materials that are either pH sensitive or temperature-sensitive, this material holds significant potential for greater control over drug delivery because of the ability to control solute permeation through both pH and temperature changes [23].

36 wt.% aqueous acetic acid were mixed and stirred for 3 days. The molar ratio of carboxyl group in PAOMA to amino group in CS was set at 1. The mixture was cast on a polystyrene petridish (135 mm × 95 mm) and dried in vacuo at 50 ◦ C until the film reached a constant weight. The dried films were cut into disks (φ, 10 mm) and stored in a refrigerated desiccator until use.

2. Experimental 2.3. Swelling tests 2.1. Materials CS (degree of acetylation, 0.085; average molecular weight, 106 ) was provided from Kyowa Tecnos Co. Ltd., Japan. PAOMA (AEM-0530, AKM-0530) were supplied from Nippon Oil & Fats Co., Japan. The structures of the polymers used are shown in Figs. 1 and 2. Table 1 shows the PAOMA copolymer composition, molecular weight and cloud point. Cloud point was majored as follows; PAOMA aqueous solution (2 g dm−3 , 7 cm3 ) was prepared and settled at temperature-constant bath. Temperature of the bath was increased at a rate of 1–3 ◦ C/10 min. The LCST was defined as the temperature at the beginning to become clouded. Salicylic acid, phenol and glucose were purchased from Wako Pure Chemical Industries Ltd., Japan. All other reagents were of analytical grade.

Dried films were carefully weighed and immersed in buffer media (50 cm3 ) with pH values ranging from 2.2 to 7.5 at 37 ◦ C, or with temperature ranging from 0 to 60 ◦ C at pH 3.8. At predetermined time intervals (12 h), swollen films were taken out, and the excess water was blotted with filter paper at the surface. This was then weighed on a sensitive balance (AEU-210, Shimadzu). For cyclic stimuli-responsive swelling tests, the PEC films were exposed to the different environments repeatedly. The pH-responsive swelling of PEC films was investigated between pH 6.2 and 3.8. The temperature-responsive swelling was investigated between 50 ◦ C (above the LCST of AEM-0530) and 25 ◦ C (below the LCST of AEM-0530). The following equation was used to determine the swelling degree (DS).

2.2. Preparation of PEC films

DS =

PAOMA solution (30 wt.%) was purified by dialysis (MWCO:3500, SPECTRUM) against distilled water for 4 days. PAOMA solution (90 g dm−3 , 18.5 cm3 ) dissolved in distilled water and CS solution (2 wt.%, 50 g) dissolved in

H C

H2 C

H C

H C n

CH2

O C

O EO i EO= CH2

CH2 O

PO

(1)

Here, W0 and Wet are the film weights of dry and swollen films. All the data were averages of three independent experiments. 2.4. Drug permeation tests The permeability of model drugs through PEC films prepared from CS and AEM-0530 was measured at pH 3.8

C O

O

O

H

H

j

Wet − W0 W0

CH3

PO= CH2 CH O CH3

Fig. 2. PAOMA structure.

Table 1 PAOMA copolymer composition

AEM-0530 AKM-0530

EO ratioa

MW

Cloud point (◦ C)

0.4 1.0

20000 20000

35 Over 70

a EO ratio means molar fraction of ethylene oxide in alkylene oxide chain defined as the following equation; i/(i + j) where i and j are the random copolymerization degree of PAOMA copolymer shown in Fig. 2.

T. Yoshizawa et al. / Journal of Membrane Science 241 (2004) 347–354

and 6.2 and at temperatures 25 and 50 ◦ C by a modified diffusion-vessels method [24]. The apparatus used in this study was shown in Fig. 3. First, the bottle cap of a smaller vial was punched and the PEC film was sandwiched between the cap and packing seal. Then, the model drug solution was placed into a smaller vial (donor). The vial was then capped with the punched cap and placed into a larger vial (receptor) containing the buffer solution and shaken by an orbit shaker. Shaking speed was set at 150 rpm to eliminate a boundary layer effect. Filter paper between PEC film and packing seal was used to support the PEC film. The initial donor concentration was 2.0 g dm−3 and the effective surface area for flux was 0.785 cm2 . UV–vis spectroscopy (U-3210, Hitachi, Japan) was used to measure solute concentration of salicylic acid (234 nm) and phenol (270 nm). Glucose was measured using glucose-testwako (Wako, Japan). The temperature in cyclic thermo-responsive permeability measurements though the PEC film fluctuated between 25 and 50 ◦ C. The lower and higher temperatures were below and above the LCST of AEM-0530. 2.5. Determination of permeation and diffusion coefficients

349

where J is the overall flux; P is the overall permeation coefficient; CR is the solute concentration in the receptor vial at time t and CD is the initial solute concentration of the donor vial. The local mass fluxes, in filter paper and film are given by Jp = Pp (Cp,2 − CR )

(3)

Jf = Pf (Cf,1 − Cf,2 )

(4)

The solute partition coefficient, kf , is calculated from the following equation. kf =

Cf,1 Cf,2 = CD Cp,2

(5)

Substitution of this expression into Eq. (4) gives Jf = kf Pf (CD − Cp,2 )

(6)

On physical grounds, at steady state the flux in all regions will be the same. Hence, J = JP = Jf

(7)

Integration of these equations gives finally The concentration profiles of drug around an ideal flat film and filter paper for physical permeation are illustrated in Fig. 3b. On the assumption inherent to the permeate process, the effect of laminar film is negligible. The overall equation of the permeation is J = P(CD − CR )

(2)

J=

CD − CR (1/Pp ) + (1/(kf Pf ))

(8)

Combination of this equation with Eq. (2) gives 1 1 1 = + Pp k f Pf P

(9)

Permeation of drugs is assumed to obey Fickian’s law. As such, permeability coefficients are determined based on the following equation [13]:

 CD − CR 1 1 ln = −A + Pt (10) CD VD VR where VD and VR is the volume of donor and receptor vial, respectively (VA = 80 cm3 , VB = 33 cm3 ), A is the effective area of permeation (A = 0.785 cm2 ). Permeation coefficient of filter paper, Pp , is determined in the same way that it is described at Section 2.4 by using filter paper instead of PEC films and calculated by

 CD − CR 1 1 ln = −kp A Pp t + (11) CD VD VR where the solute partition coefficient of filter paper, kp , was negligible. For further elucidation, the effective diffusion coefficient was calculated using the following equation: D = Pf l

Fig. 3. (a) Experimental apparatus. (b) Concentration profiles around ideal flat film and filter paper.

(12)

where D is the effective diffusion coefficient and l is the film thickness in the swollen state at constant pH and temperature. Each experiment was repeated three times and the results expressed as the mean of the three independent results.

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tosan against pH. The chitosan got insoluble in water above pH 6.5, and therefore, the insolubilized chitosan molecule had inflexible structure so that the DS does not change over pH 6.5.

3. Results and discussion 3.1. PEC films The resultant PEC films were transparent and had a thickness of ca. 60 ␮m. The surface morphology was quite smooth and uniform, and the cross-sectional morphology was equally dense and homogeneous. 3.2. Effect of pH on swelling For characterization of PEC film response to changes in pH, samples were equilibrated in an aqueous swelling medium between pH 2.2 and 7.5. The weight of all films increased rapidly and equilibrated within 6 h. The swollen PEC films were transparent over the whole pH range. The effect of external pH on the swelling behavior of PEC films is shown in Fig. 4. The results indicate that PEC films swell significantly at low pH; as the pH value increases, the DS decreases sharply. This deswelling behavior can be attributed to the electrostatic interaction between the protonation of amino groups in CS and carboxyl groups in the PAOMA. The pKa of CS is 6.5 and the pKa of PAOMA is 4.8. At a low pH, most of the amino groups in CS are in the form of NH3 + and most of the carboxyl groups in PAOMA are in the form of COOH. Therefore, the repulsive force between positive charges of CS molecules resulted in longer intermolecular distances. As the pH of the medium increases, the carboxylic acid groups become ionized, and the electrostatic attraction between NH3 + and COO− gradually becomes stronger with a corresponding decrease in DS. In the pH range of 4.8–6.5, PEC films shrink because electrostatic interaction between NH3 + and COO− is very strong. After pH 6.5, most of amino groups in CS become NH2 and carboxylic groups in PAOMA are still ionized. Therefore, DS would be expected to increase at high pH owing to the repulsive force between COO− groups in PAOMA. However, DS reduction could not be confirmed in the present study. This contradiction can be explained by the solubility of the chi-

3.3. Effect of temperature on swelling The swelling behavior of PEC films prepared from CS and PAOMA (AEM-0530, AKM-0530) was investigated as a function of temperature. The transparent CS/AEM-0530 films turned slightly white around 42 ◦ C (>LCST), while the color of CS/AKM-0530 was not changed at all temperatures. In consistent with this observation, the DS values were found to be independent of temperature as shown in Fig. 5. In the case of AKM-0530, this is due to the lack of LCST in the temperature range examined. In contrast, DS of PEC films prepared with AEM-0530 were sensitive to temperatures between 30 and 60 ◦ C. The relatively broad transition was attributed to the fact that the PAOMA could have a broad molecular weight distribution and an unhomogeneous EO/PO ratio [25]. The AEM-0530 aqueous solution has LCST at ca. 35 ◦ C, as indicated by a reversible phase transition from soluble to insoluble states. The temperature-responsive soluble–insoluble transition of AEM-0530 can be explained by the dissociation of ordered water molecules surrounding hydrophobic propylene oxide groups over 35 ◦ C. Due to this phenomenon, the PEC films composed of CS and AEM-0530 show a volume phase transition around the LCST of AEM-0530. In contrast, AKM-0530 does not have a LCST below 100 ◦ C because the ethylene oxide groups are strongly hydrophilic. As a result, the hydrated water is highly ordered around these groups at the temperatures examined. 3.4. Cyclic stimuli-responsive swelling In addition to the equilibrium swelling experiments, stimuli-responsive swelling tests were carried out 12

20 AEM-0530 AKM-0530

18 16

10 8 DS

DS

14 12

6

10 8

4

AEM-0530 AKM-0530

6 4

2 0

2 1

2

3

4

5

6

7

8

20

40

60

80

o

Temperature [ C]

pH Fig. 4. pH-dependent swelling of PEC films prepared from different types of PAOMA-copolymer. Each value represents the mean ± S.D. (n = 3).

Fig. 5. Temperature-dependent swelling of PEC films prepared from different types of PAOMA copolymer. Each value represents the mean ± S.D. (n = 3).

T. Yoshizawa et al. / Journal of Membrane Science 241 (2004) 347–354

8 7

9 (a)

(b)

8

6

Table 2 Permeability and effective diffusion coefficients of model drugs through CS/AEM-0530 film (n = 3) pH

Temperature (◦ C)

Model drug

Pf × 10−5 (cm/s)

3.8 3.8 6.2 6.2 3.8 3.8 3.8 3.8

25 50 25 50 25 50 25 50

Salicylic Salicylic Salicylic Salicylic Phenol Phenol Glucose Glucose

1.12 3.21 0.44 1.92 1.43 4.49 0.39 0.68

DS

DS

5 4

7

3 6

2 1

5

123456789

1234567

Repeated run number

Repeated run number

Fig. 6. (a) pH ((䊉) pH 6.2 or (䊊) pH 3.8)-responsive and (b) temperature ((䊉) 50 ◦ C or (䊊) 25 ◦ C)-responsive swelling of PEC film prepared from CS and AEM-0530 at 37 ◦ C. Each value represents the mean ± S.D. (n = 3).

consecutively under defined conditions of pH (6.2 and 3.8) and temperature (25 and 50 ◦ C) to confirm the response and reversibility of the swelling process. The results of the stimuli-responsive swelling test are shown in Fig. 6. Depending on pH condition, the swelling was changed reversibly. As a result, the film could be expected to control drug permeability in response to environmental pH. When the temperature-responsive swelling test was carried out at pH 3.8, the swelling was also changed reversibly in regard to temperature. We note, however, that the difference in DS between 25 and 50 ◦ C was not so significant. The pH-responsive swelling observed here is consistent with alternative hydrogel models [26]. Furthermore, the thermo-responsive swelling behavior is similar to that observed in hydrogels composed of PNIPAAm [27]. 3.5. Effect of pH on permeation and diffusion coefficients

0.06 0.05 0.04 0.03 0.02 0.01 0.00 0

2

4

6

8

10 12 14

Time [h] Fig. 7. Salicylic acid concentration in permeate through the PEC films prepared from AEM-0530 at different pH at 25 ◦ C. Each value represents the mean ± S.D. (n = 3).

± ± ± ± ± ± ± ±

0.02 0.62 0.03 0.63 0.22 0.99 0.04 0.11

5.18 ± 1.45 11.9 ± 2.49 2.51 ± 0.16 9.80 ± 3.22 6.15 ± 0.97 18.0 ± 3.97 1.66 ± 0.15 4.23 ± 0.67

3.6. Effect of temperature on permeation and diffusion coefficients Fig. 8 shows the typical permeation behavior of salicylic acid through PEC films at different temperatures and the

-3

pH 3.8 pH 6.2

acid acid acid acid

D × 10−7 (cm2 /s)

Fig. 7 shows the typical permeation behavior of salicylic acid through PEC films at different pH media at 25 ◦ C. From the slope of the linear line in Fig. 7, permeation coefficient, Pf , was calculated using Eqs. (9) and (10). For further elucidation of salicylic acid permeation through PEC film, the effective diffusion coefficient, D, was calculated using Eq. (12). The values of Pf and D for CS/AEM-0530 film are summarized in Table 2. There is a direct correlation between decreasing Pf and D and increasing pH. The dependence of Pf and D on pH is explained by free volume in the film. In polymer hydrogels, the effective free volume is derived from the free volume of water, and the transport of solutes is presumed to permeate through the free-water region in the swollen film [28]. As shown Fig. 4, PEC films swell at low pH. Consequently, as the total volume of pores or channels in a unit volume of film increase, the amount of solute transport increases. In contrast, as the volume of pores decrease at high pH, Pf and D also decreased. CS/AKM films also showed pH-dependent Pf and D trends (data is not shown).

Permeated drug conc. [g dm ]

-3

Permeated drug conc. [g dm ]

The permeation of drugs through PEC films composed of CS and PAOMA was studied in buffer solutions at pH 3.8 and 6.2.

351

0.12 o

25 C o 50 C

0.10 0.08 0.06 0.04 0.02 0.00 0

2

4

6

8

10 12 14

Time [h] Fig. 8. The concentration of salicylic acid in permeate through the PEC films prepared from AEM-0530 at different temperature in pH 3.8 media. Each value represents the mean ± S.D. (n = 3).

T. Yoshizawa et al. / Journal of Membrane Science 241 (2004) 347–354

3.7. Cyclic stimuli-responsive permeation Fig. 10 illustrates the effect of repeated change in buffer temperatures between 50 and 25 ◦ C at pH 3.8 on salicylic acid permeation through the PEC films prepared from CS and AEM-0530. External temperature conditions were changed every 3 h. Permeability changes in

Drug Conc.

Flux

1 10-6

0.2

8 10-7 6 10-7

0.1 -7

4 10 0.05

2

0.15

Flux [g/dm s]

-3

values of Pf and D for CS/AEM-0530 films are summarized in Table 2. It was found that Pf and D increased with increasing temperature. This positive permeation behavior is similar to that observed by Hendri et al. [29]. Though this dependence of Pf and D on temperature was discussed by the activation effect of temperature [30], we believe this phenomenon can be attributed to the hydrophobicity of the AEM-0530. As illustrated in Fig. 9a, at lower temperature, PAOMA could be swollen in the network structure of CS/PAOMA films due to the increasing hydrophilicity of PAOMA under LCST. To the contrary, PAOMA aggregates because of the increasing hydrophobicity above LCST with a shrinkage of the network strugcture of CS/PAOMA films (Fig. 9b). Hence, the lower rate of drug permeation at 25 ◦ C (under LCST) could be attributed to the obstruction effect of both PAOMA and its bound water in the CS/AEM-0530 film as if a valve is closed (Fig. 9a). The rate of drug permeation at 50 ◦ C (above LCST) was higher because the AEM-0530 molecule shrunk in the film, resulting in increasing void space that drug solution could permeate (Fig. 9b). Curiously, CS/AKM-0530 film also showed temperaturedependent Pf and D trends (data is not shown). The reason for this temperature-dependency is unclear but may be related to the differences in phase transition temperature between the PAOMA aqueous solution and the CS/PAOMA polyelectrolyte complex. Further studies on the phase transition temperature and microstructure of PEC films will be conducted to clarify the temperature effect on solute permeation.

o Temperature [ C] Permeated drug conc. [g dm ]

352

2 10-7

0 60 50 40 30 20 10

0

0

5

10 15 Time [h]

20

25

Fig. 10. Salicylic acid permeation through PEC film in response to stepwise temperature change between 50 and 25 ◦ C.

flux usually occurred and the salicylic acid permeation was controlled by the changing temperature. This results in a decrease of drug concentration in permeates below the LCST. This is consistent with the results of the permeation behavior at different temperatures (Section 3.5). Permeability flux also decreased below the LCST. The decreasing tendency of difference in flux between 25 and 50 ◦ C seems to be related to the film swelling property as shown Fig. 6. Cyclic temperature-responsive permeation with this same behavior was also seen at pH 6.2 (data was not shown). 3.8. Effect of types of drug on permeation and diffusion coefficients Three different types of model drugs, phenol (MW = 94.11), salicylic acid (MW = 138.12) and glucose (MW = 180.16) were used for permeation tests to investigate the effect of drug molecular weight. Diffusion coefficients, D, of the model drugs are summarized in Table 2. At similar temperature levels, phenol had the largest D and glucose the smallest indicating that lower MW drugs permeate faster. This relationship between drug molecular weight and effective diffusion coefficient is similar to that of CS/gelatin films [28].

Fig. 9. Mechanism of chemical valve phenomena induced by changing temperature in thermo-sensitive PEC films: (a) swollen state of the PAOMA-copolymer at temperature < LCST and (b) shrunken state of the PAOMA-copolymer at temperature > LCST.

4. Conclusion PEC films were prepared from CS and PAOMA. Equilibrium pH-swelling studies showed that the films swelled

T. Yoshizawa et al. / Journal of Membrane Science 241 (2004) 347–354

at low pH and shrunk at high pH regardless of the type of PAOMA. In contrast, equilibrium temperature-swelling studies of PEC films prepared from CS and AEM-0530 showed that the film swelled at 25 ◦ C and shrunk at 50 ◦ C. Cyclic stimuli-responsive swelling tests with alternating pH between 6.2 and 3.8 highlighted the reversibility and response of the swelling process. However, the difference in responsive swelling between 25 and 50 ◦ C was not so significant. PEC films prepared from CS and AEM-0530 were shown to change drug permeability in response to changes in environmental pH and temperature condition. Permeation studies showed that an increase in temperature from 25 to 50 ◦ C yielded an increase in the rate of drug permeation because of shrinking PAOMA in the film. The effective diffusion coefficient of drug permeation was found to be correlated to molecular weight.

Acknowledgements We wish to thank Nippon Oil & Fats Co. and Kyowa Tecnos Co. Ltd. for providing PAOMA and chitosan, respectively. We are grateful to Prof. S. Hirose at International Student Center, Tokyo Institute of Technology, for his helpful discussion. Gratitude is expressed to Dr. A.K. Salem for a critical reading of the manuscript.

Nomenclature A Ci Ci,j DS D J Ji ki l P Pi T Vi W

effective contact area (m2 ) concentration of i (mol m−3 ) concentration of i in phase j (mol m−3 ) swelling degree (–) effective diffusion coefficient (m2 h−1 ) overall flux (mol m−2 h−1 ) local flux of component i (mol m−2 h−1 ) solute partition coefficient of component i (–) film thickness (m) overall permeation coefficient (m h−1 ) local permeation coefficient of component i (m h−1 ) time (h) volume of i (m3 ) weight of PEC film (g)

Subscripts 0 initial 1 interface 1 2 interface 2 et equilibrium at time t D donor R receptor f film p filter paper

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