Lipase release through semi-interpenetrating polymer network hydrogels based on chitosan, acrylamide, and citraconic acid

Abstract

In this study, a series of semi-interpenetrating polymer network (IPN) hydrogels were prepared as a support material for lipase immobilization. Hydrogels were synthesized via free radical polymerization in different compositions of chitosan (Cs), acrylamide (AAm), and citraconic acid (CA). The swelling values of the hydrogels were found to be 240–400%. Depending on the swelling results, Cs–P(AAm-co-CA)-2 hydrogel was chosen for lipase immobilization. Three different types of immobilization technique were carried out. Lipase release behaviors were investigated, and immobilization yields of three immobilization methods were compared, and the maximum immobilization yield value was determined for entrapment method.

Keywords::

• ethylene glycol dimethacrylate
• immobilization yields
• IPN hydrogel
• lipase immobilization
• swelling

Introduction

Hydrogels are hydrophilic three-dimensional networks held together by cross-linked chemical or physical bonds (Elvira et al. 2002). They have been extensively studied and used for a large number of applications in medicine, such as controlled drug release matrices (Hoffman 2002), enzyme and yeast cell immobilizations (Aykut et al. 1988), and blood-contact applications (Pulat and Asıl 2009). Interpenetrating polymer networks (IPNs) are mixtures of two cross-linked polymers. IPNs are preferred in a number of biotechnological and biomedical applications because of their certain unique biophysical properties such as ease of fabrication into various geometrical forms, a soft and rubbery texture, minimal mechanical irritation to the surrounding tissues, and unusual stability against biofluids (Bajpai et al. 2001). IPN structures are also used for controlling the overall hydrogel hydrophilicity and drug release kinetics ( Pulat and Asıl 2009, Bae and Kim 2001). A wide range of the so-called semi-IPNs have been prepared through the dissolution of a preformed linear polymer in a hydrophilic monomer and cross-linking agent mixture, which is subsequently polymerized ( Pulat et al. 2011). In this way, a synthetic hydrogel network is formed around a primary polymer chain, which modifies the behavior of the hydrogel (Kayaman et al. 1999). The use of semi-IPNs in pH-sensitive or temperature- sensitive drug delivery systems has been well documented (Kim et al. 2003).

Because of its easy polymerization and biocompatible properties, acrylamide (AAm) is widely used to prepare hydrogels designed for drug release. In an ionic polymeric network containing carboxylic acid or amide groups, ionization takes place as the pH of the external swelling medium changes. Numerous studies about preparing anionic or cationic hydrogels have been found in the literature (Pulat and Eksi 2006). Besides many studies about poly(acrylamide) (PAAm) hydrogels, some investigations have focused on copolymeric hydrogels containing different vinyl monomers, such as hydroxyethyl methacrylate, N-isopropylacrylamide, crotonic acid, itaconic acid, and acrylonitrile (Karadag et al. 1996). Citraconic acid (CA) is known as a suitable monomer for radical copolymerization with vinyl monomers (Switała and Zeliazkow 2002). Because of its two carboxylate groups, CA-containing hydrogels are favorable for swelling at high pH values. However, at low pH values, carboxylate groups cannot be easily ionized and swelling values of the structure were less than the expected values. Therefore, various copolymeric conformations can be used to arrange the swelling response of the hydrogels.

Chitosan (Cs) is a natural polymer obtained through the alkaline deacetylation of chitin; it exhibits excellent biological properties such as biodegradation in the human body and immunological, antibacterial, and wound-healing activity. Cs has also been found to be a good candidate as a support material for gene delivery, cell culture, and tissue engineering (Macleod et al. 1999). Moreover, Cs has antacid and anti-ulcer activity, and can prevent or weaken drug-induced irritations in the stomach. These interesting properties make Cs an ideal candidate for use in controlled drug release formulations. However, this naturally abundant material also exhibits limitations in its reactivity and processability. One strategy to overcome these shortcomings is to incorporate Cs into IPN hydrogels (El-Sherbiny et al. 2005).

Enzymes are large biological molecules responsible for the thousands of chemical inter-conversions that sustain life. They are highly selective catalysts, greatly accelerating both the rate and the specificity of metabolic reactions, from the digestion of food to the synthesis of DNA. Enzymes are used in the chemical industries and in other industrial applications, when extremely specific catalysts are required, such as biofuel, food, and paper industries. Their commercial availability at high purity levels makes them very attractive for mass production of enzyme sensors (Arslan and Arslan 2011, Cete and Bal 2013). The main limitations of enzymes are pH, ionic strength, chemical inhibitors, and temperature that affect their activity. Most enzymes lose their activity when exposed to temperatures above 60°C. Thus, enzymes are often immobilized onto solid supports to increase their operational stability and recoverability (Pulat and Akalin 2013, Pulat and Akalin Görgülü et al. 2013, Ates and Icli 2012). Immobilization methods can be divided into two general classes: chemical and physical methods. Enzymes have been immobilized at the surface of the transducer by adsorption, covalent attachment, and entrapment in a gel or an electrochemically generated polymer.

Lipases, which catalyze the hydrolysis of esters such as glyceride, are industrially useful enzyme finding wide use in oil processing, production of surfactants, and preparation of enantioselective pharmaceutics. For their industrial applications, immobilized lipases have been specifically studied due to their enhanced stability, easy separation, and reusability. Various immobilization methods and supports have been developed in order to improve their activity (Pulat and Akalin 2013, Ates et al. 2013).

The aim of this study is to develop a suitable support material for lipase immobilization based on semi-IPN hydrogels prepared using Cs, AAm, and CA. We planned to investigate the effects of the monomer composition on the swelling behaviors and morphological structures of the hydrogels. Therefore, convenient hydrogels were chosen for lipase immobilization studies. Three different types of immobilization method were used: adsorption, entrapment and covalent bonding. Another target of the study was to determine the lipase immobilization yields depending on loading and releasing percentages.

Methods

Materials

Cs, AAm, CA, acetic acid, EGDMA, and carbodiimide (CDI) were obtained from Aldrich, Seelze, Germany. Lipase (from Candida rugosa), (NH₄)₂S₂O₈/Na₂S₂O₅, and phosphate buffer (PB) tablets at different pH values were purchased from Merck. Britton-Robinson buffer (BRB) solutions were prepared as given in the literature (Britton and Robinson 1931).

Preparation of the hydrogels

An aqueous solution mixture (2.5 mL) of AAm and CA at a certain molar ratio (Table I) was placed into a glass tube. A 5.0 mL solution (1.0%) of Cs-acetic acid, the cross-linking agent ethylene glycol dimethacrylate (EGDMA; 0.1 mL), and the initiator (NH₄)₂S₂O₈/Na₂S₂O₅ (redox pair; 0.05 g/0.05 g) were added to the monomer mixture solution. The reaction proceeded for 24 h at room temperature. The fresh hydrogel rods that were obtained were cut into pieces (0.5 cm long). The gel discs were washed several times with distilled water and dried first in air and then in a vacuum oven at 37°C, and they were stored for further use (Pulat and Asıl 2009, Blanco et al. 2003). The dimensions of the dried poly(acryl amide-co-citraconic acid) [P(AAm-co-CA)] hydrogels were measured with a micrometer. The average thickness was 0.35 ± 0.05 cm, with the radius differing between 0.40 and 0.50 cm according to the content of the gel matrix. The average amount of Cs per gel disc was calculated to be 5 mg.

Table I. Amounts of AAm and CA in the monomer mixture solutions used to form the hydrogels.

Hydrogel	AAm (mol/L)	CA (mol/L)	Gel formation (%)
Cs–P(AAm-co-CA)-1	5	1	92
Cs–P(AAm-co-CA)-2	4	2	86
Cs–P(AAm-co-CA)-3	3	3	74

The gel formation percentages of the samples were gravimetrically determined as follows (Pulat and Cetin 2008, Chen et al. 2005):

Dried hydrogel were weighed and then placed in distilled water for 48 h to extract the unreacted monomers. The hydrogels were then taken out from the extraction medium and dried in a vacuum oven at 40°C to a constant weight. The gel formations percentages were determined using the following formula:

where M is the weight of the dried hydrogel after extraction and M₀ is the weight of the dried hydrogel before extraction. All measurements were performed in triplicate.

Swelling and degradation

Swelling tests of hydrogel discs were gravimetrically carried out in three steps. In the first step, dried discs were left to swell in a BRB solution (pH = 7.4) at 37°C. Swollen gels, removed from the swelling medium at regular intervals, were dried superficially with a filter paper, weighed, and placed into the same bath. The measurements were performed until a constant weight was reached for each sample. The swelling percentage (S% values) was calculated with the following equation (Pulat and Asıl 2009, Hsiue et al. 2001):

where M_w is the wet weight of the sample and M_d is the dry weight of the sample before swelling. The incubation times for all gels were approximately 24 h. In the second step, the dried hydrogel discs were swollen in BRB (pH = 7.4) solutions at different temperatures ranging from 20 to 60°C, so that we could investigate the effect of temperature on swelling behaviors. At the end of 24-h incubation, the swollen discs were removed from the swelling medium, dried superficially with a filter paper, and weighed. S% values were calculated with Eq. (2).

In the last step, the dried hydrogel discs were swollen in different BRB solutions at various pH values between 2 and 12 so that we could investigate the effect of pH on the swelling behaviors. The temperature and swelling time were kept constant (37°C and 24 h, respectively). The swollen discs were removed from the swelling medium, dried superficially with filter paper, and weighed. S% values were calculated with Eq. (2). Behind the swelling tests, standard degradation tests were also performed in accordance with the literature (Tan et al. 2009). Reproducible results for all swelling studies were obtained with triplicate measurements.

Scanning electron microscopy

The hydrogel disc samples, swollen to equilibrium in water at room temperature, were removed and placed in a deep freezer at − 20°C for 24 h and then transferred into a Christ-Alfa 2–4 freeze dryer (Martin Christ GmbH, Germany) at − 85°C for 1 week. Besides lipase-entrapped discs, the dried and swollen discs were coated with 200 A˚ of Au. Surface micrographs of the samples were obtained with a JEOL Mark JSM 840A scanning electron microscope (SEM, Hitachi, Japan).

Immobilization of lipase on Cs–P(AAm-co-CA)-2 hydrogel

Depending on morphological structure and swelling test results, Cs–P(AAm-co-CA)-2 hydrogel was chosen for lipase immobilization. Three different types of immobilization technique were carried out: physical adsorption, covalent bonding and entrapment. Hydrogel discs were swollen in 25 mL of phosphate buffer solution (PBS) at pH 6.9 for 24 h. The samples were taken out and left in 20.0 mL of 1.0% lipase solution (1.0 g lipase/100 mL PBS) at 25°C. After 2 h, adsorption bath was hold at + 4°C for 24 h. Then, the adsorption procedure was ended and hydrogel samples were lead out (Ye et al. 2007). The amount of unbound enzyme in immobilization solution was determined by spectrophotometric measurements at 206 nm.

Lipase immobilization by covalent bonding was carried out as follows: First, the functional groups of Cs were activated with CDI coupling agent (Chiou and Wu 2004, Hung et al. 2003). Dried disc was left to swell in 25.0 mL of PBS (pH 6.9) at 24 h, and then it was put into 20.0 mL of 1% (w/v) CDI solution (pH 6.9) at 25°C. The system was shaken for 4 h and then left overnight at room temperature. Cs–P(AAm-co-CA)-2 disc was removed from activation medium and washed several times with PBS. Activated disc was put into 20.0 mL of 1% (w/v) lipase solution, and immobilization process was performed at 25°C, 2 h. After this period, the mixture was left for 24 h at 4°C. Finally, immobilized disc was taken out from the medium, dried in air conditions, and then stored at 4°C for further use later (Xiao-Jun et al. 2007). The amount of unbound enzyme in immobilization solution was determined by spectrophotometric measurements at 206 nm.

The entrapment of model enzyme in any cross-linked polymer networks can be accomplished with copolymerization/cross-linking in the presence of the enzyme. In this work, particular amounts of lipase (72 mg/disc) were added to Cs–P(AAm-co-CA)-2 hydrogels during copolymerization/cross-linking reactions ( Luo et al. 2009, Obara et al. 2005). Enzyme-entrapped hydrogel discs were dried, and they were stored for further use.

Release of lipase from the hydrogels

In this part of the study, lipase release behaviors were investigated and immobilization yields were compared for different immobilization methods. Lipase immobilized hydrogel discs were placed into a vessel containing 100 mL of PBS (pH = 7.4). At different times, aliquots of 100 μL were drawn from the medium to follow the lipase release; a maximum of 30 aliquots were taken, so the vessel volume could be considered constant. The release always maintained sink conditions (Bajpai et al. 2001, Blanco et al. 1996, Pulat et al. 2008). Lipase release was determined spectrophotometrically with a Unicam UV-2100 spectrophotometer at a wavelength of 206 nm for 48 h. Reproducible results were obtained with triplicate measurements. The cumulative release (%) of lipase was calculated with the following equation:

where W_t is the weight of the released lipase in the releasing medium at any time and W_total is the initial total weight of lipase taken by the gel system (Bajpai and Sonkusley 2002).

Results

According to the literature, the general mechanism shown in Figure 1 could be suggested for semi-IPN formation from Cs P(AAm-co-CA) polymers (Mahdavinia et al. 2004, Solomons 1996). This reaction mechanism has been promoted by other researchers (Pulat and Asıl 2009, Ravikumar 2000). The persulfate initiator is reduced to the (SO₄)− anion radical. This radical abstracts hydrogen from the monomer to form vinyl radicals. Thus, the radically initiated copolymerization reaction between AAm and CA is performed (Pulat and Cetin 2008, Lichen et al. 2007). It can be thought that P(AAm-co-CA) is the host polymer in this IPN system. Intermolecular forces between the polymer molecules in semi-IPN hydrogels are also shown in Figure 1. Gel formation percentages of the hydrogels calculated using Eq. (1) are given in Table I. In accordance with standard degradation tests (Tan et al. 2009), these hydrogels were found to be stable in neutral media and acidic media of pH 4.0 at 37°C for 30 days.

Figure 1. General mechanism for semi-IPN formation through Cs and P(AAm-co-CA).

Figure 2 represents the variation of S% values with time at pH 7.4 and 37°C. S% increased with time initially and then remained constantly close to 24 h. S% values were determined to be 400% for the most swollen hydrogel Cs–P(AAm-co-CA)-1, and 240% for least swollen hydrogel Cs–P(AAm-co-CA)-3.

Figure 2. The variation in S% values with time at 37°C and pH 7.4.

Figure 3 represents the variation of S% values of hydrogels with temperature at pH 7.4 for 24 h. As seen from this figure, all the hydrogels swell much more at high temperatures than at low temperatures.

Figure 3. The variation in S% values with temperature at pH 7.4 for 24 h.

The variations in S% values with pH at 37°C for 24 h were also given in Figure 4. As explained in experimental part, Cs–P(AAm-co-CA)-2 hydrogel was chosen for lipase immobilization and three different types of immobilization technique were carried out: physical adsorption, covalent bonding, and entrapment. Lipase release was measured spectrophotometrically, and cumulative release (%) was calculated using Eq. (3). The release profiles of lipase immobilized using three different methods are represented in Figure 6.

Figure 4. The variation of S% values with pH at 37°C for 24 h.

Figure 6. The cumulative release of immobilized lipase from Cs–P(AAm-co-CA)-2 hydrogel.

Discussion

Gel formation

It is well known that EGDMA is the selective cross-linker for vinyl polymers and couldn't be used for cross-linking of Cs (Beppu et al. 2007). While EGDMA provide the cross-linking of vinyl polymer chains P(AAm-co-CA), Cs molecules are placed as guest polymer in the hydrogel network. Gel formation percentages of the hydrogels calculated using Eq. (1) are given in Table I. In general, high gel formation values were mostly obtained so this procedure is convenient for preparing the semi-IPN type hydrogels with Cs and vinyl polymers. The highest and the lowest gel formation percentages were obtained for Cs–P(AAm-co-CA)-1 and Cs–P(AAm- co-CA)-3 hydrogels, respectively. Increase in CA resulted in the decrease in gel formation percentage. This result might be caused from the opposite–ionic interactions in between PAAm and PCA. As is known PAAm is a positively charged polymer in contrast with PCA. This complicated copolymeric structure might interfere the diffusion of Cs molecules into the hydrogel. Gel formation between Cs and copolymeric chains could be simplified by the decrease in the number of CA molecules; thus, gel formation of Cs–P(AAm-co-CA)-1 hydrogels would be favorable than that of Cs–P(AAm-co-CA)-3.

Swelling behaviors of the hydrogels

Swelling behaviors of the hydrogels could be interpreted, which is shown in Figures 2–4. The variation in S% values with time can be explained with the component of copolymer (Figure 2). In general, the increase in CA negatively affects the swelling values. As is known, PCA is an anionic polymer having two -COOH units and PAAm is called as a cationic polymer. These two opposite ionic charges could negatively affect the expansion of polymer chains and swelling capacity of bulk structure decreases. Different swelling values between 80% and 400% have been reported in the literature for similar hydrogels, depending on the composition or monomer ratio, polymerization route, type of cross-linker, density of cross-linker, and so forth. The ‘‘ionic charge content’’ concept was emphasized in those studies (El-Sherbiny et al. 2005). Instead of the swelling value of pure Cs hydrogels (nearly 100%), Cs hydrogels containing ionic components were reported to present much more swelling behavior (Liu et al. 2007). The results are in agreement with those given in the literature.

It is known that the swelling of PCA and PAAm hydrogels is positively dependent on the temperature (Figure 3). As the temperature increases, the thermal mobility of the polymer chains also increases, H-bonds are broken, and the hydrogels can easily swell (Xiao et al. 2005, Tasdelen et al. 2004).

Figure 4 shows the variation in S% values with pH at 37°C for 24 h. In general, swelling values are not affected by pH values. Our previous studies showed that PAAm-Cs IPN type hydrogels swell much more than the hydrogels prepared in this study (Pulat et al. 2011). While PAAm-Cs hydrogels swell near 1800%, the maximum swelling value of Cs–P(AAm-co-CA) hydrogel is near 450%. When the amount of CA increases, the swelling value decreases. This behavior could be explained by opposite ionic charges of AAm and CA molecules. The dissociation constant of CA is pKa₁ = 6.17, whereas the dissociation of acid groups is nearly completed. But ionization effect is not observed because of the opposite charge of amide groups. So the copolymeric hydrogels synthesized in this study are not sensitive to the variations in pH.

SEM analysis

SEM micrographs of dry, swollen, and lipase-entrapped hydrogels are presented in Figure 5. The morphological differences between dry and wet states of the hydrogels can be clearly observed. The dry hydrogel showed a nonporous surface with a smooth structure. The porosity values of hydrogels were directly determined from SEM micrographs and are presented in Table II (Pulat and Şenvar 1995). As seen from this table, the average pore density of Cs–P(AAm-co-CA)-1 hydrogel is very low, but its large pores result in the highest swelling value. Cs–P(AAm-co-CA)-2 hydrogel presented a regular homogeneous porous morphology, and this hydrogel shows the maximum average pore density value. The least swollen hydrogel, Cs–P(AAm-co-CA)-3, displays less porosity relative to others. There are some considerably large craters on the surface, but the actual number of pores is very less.

Figure 5. SEM micrographs of surfaces of (a) dry Cs–P(AAm-co-CA)-2, (b) swollen Cs–P(AAm-co-CA)-1, (c) swollen Cs–P(AAm-co-CA)-2, (d) swollen Cs–P(AAm-co-CA)-3, and (e) lipase-entrapped dry Cs–P(AAm-co-CA)-2 hydrogels.

Table II. Pore characteristics of the hydrogels.

Hydrogel	Average pore density (no. of pores/cm^2)	Average pore radius (μm)
Cs–P(AAm-co-CA)-1	6.4 × 10^2	215 ± 4.2
Cs–P(AAm-co-CA)-2	2.5 × 10^4	32.2 ± 1.4
Cs–P(AAm-co-CA)-3	1.8 × 10^2	38.5 ± 2.5

Besides the differences in the porosity values, chemical structures also affect the swelling behaviors of the hydrogels. The increasing amount of CA molecules in the copolymer structure results in opposite ionic effect on the positive ions of AAm molecules. Ionizable segments of AAm and CA could not sufficiently impact upon the swelling of hydrogel. So, Cs–P(AAm-co-CA)-3, the most CA included hydrogel, is the less swollen hydrogel.

The rough structures of lipase-entrapped Cs–P(AAm- co-CA)-2 hydrogel are also given in the same figure.

Release of Lipase from the Hydrogels and Comparison of the Immobilization Methods

As seen from Figure 6, the amount of lipase releasing from Cs–P(AAm-co-CA)-2 hydrogel slightly rises up and is complied at near 30 h. Maximum cumulative release and release rate values are observed for adsorption method. As is known, adsorption is relatively easy method than other immobilization techniques. At the end of 30 h, 30% of immobilized lipase is prevented. The release rates of covalent bonding and entrapment methods seem to be similar, but the cumulative release values are different. At the end of 30 h, 40% and 60% of immobilized lipase are prevented for covalently bonding and entrapment methods, respectively. Immobilization date could be commented by taking into consideration the amount of lipase to be loaded and released. Both of these values are given in Table III. As seen, the maximum loading and minimum release values were obtained for entrapment method. Depending on loading and release results, the method of entrapping lipase into Cs–P(AAm-co-CA)-2 hydrogel is found to present the maximum immobilization yield.

Table III. Loading and release percentages of lipase of three immobilization methods.

Immobilization methods	Loading (%)	Cumulative release (%)
Adsorption	39	70
Covalent bonding	56	60
Entrapment	100	40

Conclusion

Semi-IPN hydrogels in different compositions were prepared as a support material for lipase immobilization. P(AAm-co-CA) copolymers were synthesized using free radical polymerization and used as host polymer while Cs is placed into the structure as guest polymer. The variations in swelling percentages of the hydrogels with time, temperature, and pH were measured, and swelling values were found to be in between 240 and 400%. Depending on swelling results, Cs–P(AAm-co-CA)-2 hydrogel was chosen for lipase immobilization. The following three different types of immobilization technique were carried out: physical adsorption, covalent bonding, and entrapment. Depending on release results, it can be concluded that the entrapment method of lipase into Cs–P(AAm-co-CA)-2 hydrogel presents the maximum immobilization yield.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the article.