- Email: [email protected]
Encapsulation of crosslinked subtilisin microcrystals in hydrogel beads for controlled release applications
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 2 ( 2 0 0 7 ) 17–23
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/ejps
Encapsulation of crosslinked subtilisin microcrystals in hydrogel beads for controlled release applications Chandroth Kalyad Simi, Tholath Emilia Abraham ∗ Chemical Sciences and Technology Division, Regional Research Laboratory (CSIR), Thiruvananthapuram, India
a r t i c l e
i n f o
a b s t r a c t
Article history:
Enzymes are less stable in harsh conditions and hence to overcome this nature, several
Received 12 March 2007
methodologies are being developed. It was found that crosslinked enzyme crystals are the
Received in revised form 3 May 2007
most promising strategy for the stabilization of the enzymes [Emilia Abraham, T., Jegan
Accepted 4 May 2007
Roy, J., Bindhu, L.V., Jayakumar, K.K., 2004. Crosslinked enzyme crystals of glucoamylase
Published on line 22 May 2007
as a potent catalyst for biotransformations. Carbohydr. Res. 339, 1099–1104; Navia, M., St. Clair, N., 1997. Crosslinked enzyme crystals. Biosens. Bioelectron. 12, 7]. A cost effective
Keywords:
methodology of crystallization of protease (Bacillus subtilis) with ammonium sulphate (65%,
Protease
w/v) and then crosslinking the crystals with glutaraldehyde (4%, v/v) in isopropanol for
CLEC
20 min gave a stable and active enzyme. SEM studies showed that the protease is in small
Crosslinked subtilisin crystals
cubic shaped crystals of 1–2 m size. Crosslinked enzyme crystal (CLEC) of protease has
Controlled protein release
good stability in polar and nonpolar organic solvents, such as hexane, toluene, benzene
Alginate–guar gum composite beads
and carbon tetrachloride and it had high thermal stability up to 60 ◦ C and hence can be
Hydrogel
used as a catalyst for the biotransformation of compounds which are not soluble in aqueous medium. The CLECs were entrapped in the alginate:guar gum (3:1) composite beads which were resistant to low pH conditions in the stomach and hence was found to be useful for the oral drug delivery. This method can be used to deliver the protein and peptide drugs which require high concentrations at the delivery stage, and which normally degrades in the stomach before reaching the jejunum. Application of these pH-sensitive beads for the controlled release of subtilisin in in vitro was studied and found to be a viable strategy. © 2007 Elsevier B.V. All rights reserved.
1.
Introduction
Most of the drugs in the recent research and developments are peptides or peptide-like synthetic organic compounds, as a result of its high selectivity and its ability for effective action (Morishita and Peppas, 2006). Drug delivery by oral route is the most widely used method although it is not feasible for proteins and peptide drugs. These peptide drugs are quickly broken down into smaller units in the gut, mostly by proteolytic enzymes in the stomach. Much has been learned about
∗
Corresponding author. Tel.: +91 471 2515253; fax: +91 471 2491712. E-mail address: [email protected] (T. Emilia Abraham). 0928-0987/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2007.05.003
the oral delivery of proteins and its absorption in gastrointestinal tract. Various strategies have been used to overcome the difficulties and to develop safe and effective oral delivery systems for proteins (Mahato, 2003). Enzymes are not only used as digestive drugs but also for biotransformation. The development of robust biocatalysts with increased stability and catalytic activity in organic media is a major challenge in industrial biocatalysis (Govardhan, 1999; Fagain, 2003). Several methodologies are developed to overcome this difficulty (Emilia Abraham et al., 2004). Intermolecular crosslinking of
18
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 2 ( 2 0 0 7 ) 17–23
enzyme is one of the most exciting developments in the area of biocatalysis (Jegan Roy and Emilia Abraham, 2004, 2006; Margolin, 1996) and drug delivery. Crosslinked protein crystals are novel type of biocatalysts produced by stepwise crystallization followed by crosslinking by bifunctional reagents to form strong covalent bond between free amino acid groups in the protein molecule to preserve the crystalline structure (Haring and Schreier, 1999). Some of the advantages of CLEC are (1) increased stability, high activity and selectivity in organic media, (2) high mechanical stability, (3) easy recovery and recycling and (4) stable against proteolysis and self-digestion. Crosslinked protein and peptide crystals can be advantageously used in industrial, clinical and research settings. Proteases are one of the most important groups of industrial enzymes accounting for at least a quarter of the total global enzyme market (Adil and Mohammed, 1998). Biosynthetic application of protease includes esterification of sugars and oligosaccharides for the synthesis of novel peptides and proteins (Chen et al., 1998; Perez-Victoria and Morales, 2006; Potier et al., 2000; Pedersen et al., 2003). The active site of the serine proteases contains three amino acids: serine, histidine and aspartate called the catalytic triad and are capable of self-digestion. Protein crystalline drugs are carrier-free, pure, stable and storable at room temperature and are in the most concentrated form. CLECs are ideally suited for diagnostic and therapeutic applications by further encapsulating in polymer matrices to enhance the stability and bioactivity. Hydrogels of non-toxic biopolymers are attractive delivery system for proteins and other drugs because these materials combine a good biocompatibility with possibilities of manipulating the release characteristic of the protein (Chen et al., 1995; Hennink et al., 1997; Kim et al., 1992; Park et al., 1993) or drugs. Encapsulation of soluble enzymes in polymer matrices has been done in the past (Anjani et al., 2007). But the enzymes leak out of the matrix due to the larger pore size of the matrices, such as alginate, carrageenan and agar. The crosslinked enzyme crystal has a size of a few microns, larger than the normal pore size of the hydrogels and does not leak out of the matrix under normal conditions. Here, the encapsulation of the crosslinked protein/enzyme crystal in a natural hydrogel is done for the first time and is a novel concept for the oral delivery of peptides, proteins and enzymes and also for use in biotransformation. In this paper, the optimized conditions for the crystallization and crosslinking of the protease, subtilisin, to get robust enzyme crystals are given. The utility of the CLECs as biocatalyst was evaluated by studying its thermal and solvent stability. These crosslinked crystals were immobilized in a non-toxic, hydrophilic, biodegradable natural biopolymer matrix to form beads, which are pH sensitive. The orally delivered encapsulated enzyme crystal for its controlled release behavior was studied at in vitro conditions.
100 g of tyrosine for 60 min at pH 7) was purchased from Amano Japan. Glutaraldehyde (Sigma, St. Louis, MO, USA), ammonium sulphate, Guar gum (MW, 220 KDa) and sodium alginate (MW, 200 KDa) (Sisco Research Laboratory, Mumbai, India) were of enzyme quality. Casein and Folin phenol reagent (SD fine chemicals, Mumbai, India) was used. All the other chemicals used are of analytical grade. The experimental results are expressed as an average of five independent measurements.
2.2.
Enzyme assay
The protease assay was done at 40 ◦ C in glycine–NaOH buffer (50 mM, pH 10.5) using 1% casein as substrate. One millilitre of approximately diluted enzyme was incubated with 1 ml of 1% (w/v) casein solution for 10 min at 40 ◦ C. The reaction was stopped by the addition of 5% trichloroacetic acid and was centrifuged for 10 min at 8000 g. One millilite of the supernatant was taken and the products were measured by adding 0.4 M sodium carbonate followed by the addition of Folin phenol reagent. The optical density of the samples was taken at 660 nm in a spectrophotometer (Shimadzu, Tokyo, Japan) against appropriate blank.
2.3.
Protein estimation
Lowry’s method was followed for the protein estimation (Lowry et al., 1951). In Lowry’s method, protein is first treated with alkaline copper sulphate in the presence of tartarate. This incubation is then followed by addition of the Folin phenol reagent. Colour of the reaction occurs when the tetradentate copper complexes transfer electrons to the phosphomolybdic acid/phosphotungstic acid complexes. BSA was used as the standard.
2.4.
Crystallisation of protease
Protease was extracted in 0.1 M phosphate buffer of pH 7.0. Ammonium sulphate (50, 60, 65, 70, 75, 80%, w/v) was added stepwise at regular intervals with magnetic stirring to get the pure fraction. Enzyme activity of crystals was measured at all the ammonium sulphate concentrations. Crystallization was done with 65% ammonium sulphate at 4 ± 1 ◦ C for 24 h. The crystals formed were separated by centrifugation at 8000 g for 10 min.
2.5.
Crosslinking of crystals
2.
Materials and methods
Protease crystals were crosslinked with glutaraldehyde solution of different concentration (1–5%, v/v) in isopropanol for 20 min at 25 ◦ C. After crosslinking, it was washed thrice with 0.1 M phosphate buffer of pH 7. Enzyme activity was measured as described earlier. The above experiment was repeated at different duration keeping both glutaraldehyde concentration and temperature constant.
2.1.
Materials
2.6.
Protease from Bacillus subtilis (150,000 units/g; 1 unit is the amount of enzyme which produces amino acid equivalent to
Crystal morphology
Crystal morphology was observed under scanning electron microscope (JEOL, model JSM 5600 LV, Tokyo, Japan). Samples
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 2 ( 2 0 0 7 ) 17–23
were sputtered with gold and scanned at an accelerating voltage of 10 kV.
2.7.
Thermal stability
Thermal stability of CLEC protease at different temperature (40, 50, 60, 70 ◦ C) compared to that of native protease at different time intervals.
2.8.
Organic solvent stability
Organic solvent stability of CLEC protease was done by incubating 1 mg of CLEC in various organic solvents for 24 h. The organic solvents were chosen according to the dielectric constant and log P values. After 24 h, the activity of CLEC protease was done as described earlier. Activity was compared with the original activity and the percentage of activity retention was calculated.
2.9. Preparation of CLEC-entrapped alginate–guar gum beads Guar gum (1%, w/v) and alginate (3%, w/v) was separately dissolved in deionised water and equal quantities were mixed. An appropriate amount of CLEC is added to this mixture and kept for some time to remove air bubbles. Beads were made by dropping the solution using syringe in to 0.5 M calcium chloride solution. The resultant ∼2 mm beads were cured for 1 h and washed with distilled water and stored in 0.05 M calcium chloride solution.
2.9.1.
Determination of entrapment efficiency
An appropriate amount of sample was dissolved in phosphate buffer of pH 7.4 (0.1 M) and then centrifuged. The supernatant was treated with oxalate to precipitate calcium ions, which interferes with the protein estimation. The protein estimation was done by Lowry’s method.
2.9.2.
3.
Results and discussion
3.1.
Optimization of protease crystallization
19
Protease enzyme, subtilisin, which was purified by stepwise ammonium sulphate precipitation, was crystallized at pH 7. Effect of concentration of ammonium sulphate on the crystallization yield was studied. The results were shown in Fig. 1. The maximum activity and yield of good crystals were obtained with 65% ammonium sulphate. Crystallized enzymes, which are more concentrated and are purer shows high activity. Crystallization of macromolecules requires the creation of supersaturated state. At 65% ammonium sulphate concentration, protease precipitated out which is shown by the higher enzyme activity. Enzyme crystals formed were crosslinked with 1–5% glutaraldehyde (v/v) in isopropanol. Effect of glutaraldehyde concentration on the crosslinking of crystals was shown in Fig. 2. Chemical crosslinking does not disturb the crystal lattice structure but provides additional stability. Enzyme in a crosslinked crystal is stabilized by links throughout its three-dimensional structure. Maximum activity yield was obtained when the crystals were crosslinked with 4% glutaraldehyde for 20 min. Activity decreases with increase in duration of crosslinking mainly due to the denaturation of the enzyme by the prolonged exposure to glutaraldehyde. CLEC protease has less activity compared to crystal because of the denaturation occured in the presence of glutaraldehyde. Crosslinked enzyme crystal was associated with some quantity of water. But in the case of enzyme, crystal binding of water is absent, so dilution is less. Therefore, activity of same quantity of CLEC has lower activity compared to enzyme crystal. This is also a factor for decreasing the activity of CLEC compared to enzyme crystal. CLEC shows good activity at 4% (v/v) glutaraldehyde concentration due to the dense crosslinking of the enzyme and thereby the enzyme concentration per unit volume is high. At high concentration of glutaraldehyde, activity decreases because of denaturation and excessive crosslinking, which may lead to
Swelling characteristics
Vacuum dried samples were kept in 5 ml of buffer of pH 1.2 (HCl–KCl buffer 0.1 M) and a solution of pH 7.4 (phosphate buffer 0.1 M). Every 30 min intervals, the samples were taken out, blotted with paper to absorb excess water adhering on the surface and weighed.
Swelling ratio (Qs ) = (Ws − Wd )/Wd . Ws : weight of swollen sample Wd : weight of dry sample
2.9.3.
Protein release studies
Five grams of the hydrogel beads were kept in 10 ml 0.1 M phosphate buffer of pH 7.4 and 0.1 M HCl–KCl buffer of pH 1.2. The medium was stirred at a uniform rate of 1300 rpm using a suitable paddle. At regular intervals certain amount of medium was taken and the protein released was determined by a UV–vis spectrophotometer at 660 nm.
Fig. 1 – Effect of ammonium sulphate (w/v) concentration on the crystallization of enzyme.
20
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 2 ( 2 0 0 7 ) 17–23
3.2.
Crystal morphology
Scanning electron micrograph studies showed very small cubic crystals of the enzyme having the size of 1–2 m. The crosslinked enzyme was also seen as clumps of crystals (Fig. 3).
3.3.
Fig. 2 – Effect of glutaraldehyde concentration on crosslinking.
aggregation, protein precipitation and distortion of the crystal lattice. The CLEC formed had good structural rigidity and hence there was no noticeable self-degradation of the protease in terms of activity and was stable for many days (Navia and St. Clair, 1997). The enzyme crystals, which are normally very fragile become more sturdy and robust after crosslinking. Combination of crystalline lattice contacts and the covalent crosslinking results increase in enzyme stability toward thermal deactivation and organic solvent denaturation.
Thermal stability of CLEC protease
CLEC protease was thermostable up to 60 ◦ C. Above 60 ◦ C, the enzyme activity started decreasing when the incubation time was increased (Fig. 4). Figs. 5 and 6 show the thermal stability of native protease and CLEC protease at different temperature and time intervals. Results showed that thermal stability of CLEC protease was higher than the native enzyme. The enzyme crystal maintains its native conformation at high temperature mainly because in CLEC, the enzyme molecules are symmetrically arranged and crosslinked to stabilize their native conformations (Lanne et al., 1987). The increased thermal stability may be due to (1) the ordered arrangement of the molecules by inter and intramolecular crosslinks within and between the crystals, and gives the rigidity of the three-dimensional arrangement of the molecules in the CLEC and (2) due to additional ionic and hydrophobic contacts between the enzyme molecule. When an enzyme is transferred from a solution to crystalline form, an increase in the number of both polar and hydrophobic interactions among the enzyme molecules may significantly enhance the stability against heat by preventing unfolding, aggregation or dissociation (Islam et al., 1990; Mozhaev, 1993). The increased thermal stability of CLEC protease is a useful property needed for organic reactions to be conducted at an elevated temperature.
Fig. 3 – SEM of protease crystal (a and b) and CLEC protease (c and d).
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 2 ( 2 0 0 7 ) 17–23
Fig. 4 – Thermal stability of CLEC protease at different temperature.
3.4.
21
Fig. 6 – Thermal stability of CLEC protease.
Solvent stability of CLEC protease
Stability of CLEC protease in different solvents was studied and is shown in Table 1. The enzyme activity in organic solvents is intimately related to the water content, size and morphology of the catalyst particle and to the enzyme microenvironment. High biocatalytic activity was found in solvents having log P value between 2 and 4. CLEC proteases have high activity in nonpolar solvents like hexane, toluene, benzene and carbon tetra chloride, due to their low dielectric constant and higher log P values. Acetone, a nonpolar solvent is an exceptional case. The decrease in activity of CLEC in polar solvents compared to the nonpolar solvents is due to the stripping of water from the surface of the protein by competing strongly for hydrogen bonds between the protein molecules (Marcela et al., 2002; Thomas et al., 2002; Reinhammer and Oda, 1979).
Fig. 5 – Thermal stability of native protease.
Fig. 7 – Swelling characteristics of encapsulated CLEC protease.
Crosslinked enzyme crystals were encapsulated in a composite alginate:guar gum (3:1) hydrogel beads and then studied its controlled protein release efficiency which will be useful parameter for its usage as microreactor in biotransformation and in therapeutic oral delivery of the enzymes. Protein analysis of CLEC encapsulated beads, which is ∼2 mm in diameter showed a loading of 65.49% protein. The enzyme activity yield is 69.67% in wet beads. Swelling characteristics of samples in two different pHs (1.2 and 7.4) were shown in Fig. 7. Sample has a very rapid swelling at pH 7.4 which is the pH encountered in the duodenum area where the drug has to be released. Sample at pH 1.2 shows slow swelling, the normal pH of the stomach where the drug releases should not take place. The protein released at pH 1.2 was found to be slow. At pH 7.4, rapid release of protein was taking place as expected from a rapidly swelling hydrogel, and hence the entrapped protein will also be released rapidly. Results of the in vitro studies are shown in Fig. 8. The protein release from the hydrogel at pH
22
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 2 ( 2 0 0 7 ) 17–23
Table 1 – Stability of CLEC protease after incubation with organic solvent (100%) for 24 h (retention of activity) Solvent Hexane Ethyl acetate Acetone Methanol Acetonitrile Toluene DMF DMSO Benzene Carbon tetrachloride
Dielectric constant 1.9 6.0 20.7 32.7 37.5 2 38.3 47.2 2.28 2.24
log P
Polarity
4.0 0.73 −0.24 −0.74 −0.34 2.73 −1.038 −1.378 2.0 3.0
Nonpolar Dipolar aprotic Dipolar aprotic Polar aprotic Dipolar aprotic Nonpolar Dipolar aprotic Dipolar aprotic Nonpolar Nonpolar
100% Solvent 97.11 30.76 69.90 27.36 19.93 66.82 21.03 7.67 30.77 45.81
Residual activity (U/mg) 0.1489 0.0987 0.09248 0.09163 0.08748 0.1093 0.07648 0.01798 0.09008 0.0856
Activity of crosslinked protease enzyme used was 0.318 U/mg.
protease. High biocatalytic activity was found in solvents having log P value between 2 and 4, hence could be ideally used as a catalytic microreactor in biotransformation. CLEC protease was further encapsulated in alginate:guar gum hydrogel and is used successfully for the controlled release of enzymes in appropriate environment. CLEC hydrogel beads released low amounts of protein at pH 1.2 and a much higher level of protein release was observed at pH 7.4, which is the situation encountered in the human gastrointestinal tract. Alginate is the pH-sensitive component of the bead and releases the drug at the pH prevalent in the jejunum. These non-toxic hydrogel beads were found to be ideal for the oral delivery of peptide and bioactive protein drugs, which is required at high concentrations at the delivery stage.
Acknowledgement Fig. 8 – Release studies of CLEC at pH 7.4 and pH 1.2.
1.2 was found to be lesser than that at pH 7.4 and this may be mainly due to the pH-sensitive behaviour of alginate component than the guar gum. The addition of guar gum, which is a neutral polysaccharide, has facilitated the controlled release of protein at pH 7.4. Enzyme assay was done by using standard procedure. Activity of enzyme at different pH was studied. Crystallisation and crosslinking experiments were done at pH 7. There is no remarkable change in the activity of enzyme at pH 7 and 7.4. The main aim is to study the release property of enzyme at pH 7.4, which is the pH of the duodenum area. At pH 1.2 release of enzyme is poor compared to 7.4. So we think that there is no need to study the activity of enzyme at pH 1.2.
4.
Conclusion
Crosslinked protease crystal with high stability than native enzyme was prepared and its crystallization and crosslinking parameters were optimized. A 65% ammonium sulphate concentration was the optimum for the crystallization. CLEC protease has a high activity when crosslinked with 4% glutaraldehyde in isopropanol. It was stable in polar and nonpolar organic solvents and at a high temperature than native
We are very grateful to the Council of Scientific and Industrial Research (CSIR, India) for the financial support.
references
Adil, A., Mohammed, S., 1998. Alkaline proteases: a review. Bioresour. Technol. 64, 175–183. Anjani, K., Kailasapathy, K., Phillips, M., 2007. Microencapsulation of enzymes for potential application in acceleration of cheese ripening. Int. Dairy J. 17, 79–86. Chen, T., Jo, S., Park, K., 1995. Polysaccharide hydrogels for protein drug delivery. Carbohydr. Polym. 28, 69–76. Chen, Y.-X., Zhang, X.-Z., Zheng, K., Chen, S.-M., Wang, Q.-C., Wu, X.-X., 1998. Protease-catalyzed synthesis of precursor dipeptides of RGD with reverse micelles. Enzyme Microb. Technol. 23, 243–248. Emilia Abraham, T., Jegan Roy, J., Bindhu, L.V., Jayakumar, K.K., 2004. Crosslinked enzyme crystals of glucoamylase as a potent catalyst for biotransformations. Carbohydr. Res. 339, 1099–1104. Fagain, C.O., 2003. Enzyme stabilization—recent experimental progress. Enzyme Microb. Technol. 33, 137–149. Govardhan, C.P., 1999. Crosslinking of enzymes for improved stability and performance. Curr. Opin. Biotechnol. 10, 331–335. Haring, D., Schreier, P., 1999. Curr. Opin. Chem. Biotechnol. 13, 35. Hennink, W.E., Franssen, O., van Dijk-Wolthuis, W.N.E., Talsma, H., 1997. Dextran hydrogels for the controlled release of protein. J. Controlled Release 48, 107–114.
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Islam, S.A., Weaver, D.L., 1990. Molecular interactions in protein crystals: solvent accessible surface and stability. Proteins: Struct. Funct. Genet. 8, 1–5. Jegan Roy, J., Emilia Abraham, T., 2006. Preparation and characterization of cross-linked enzyme crystals of laccase. J. Mol. Catal. B: Enzym. 38, 31–36. Jegan Roy, J., Emilia Abraham, T., 2004. Strategies in making cross-linked enzyme crystals. Chem. Rev. 104, 3705–3722. Kim, S.W., Bae, Y.H., Okano, T., 1992. Hydrogels: swelling, drug loading and release. Pharm. Res. 9, 283–290. Lanne, C., Boeren, L., Vos, K., Veeger, C., 1987. Rules for optimization of biocatalysis in organic solvents. Biotechnol. Bioeng. 30, 81–87. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with Folin–phenol reagent. J. Biol. Chem. 193, 265–275. Mahato, R.I., 2003. Emerging trends in oral delivery of peptide and proteins. Crit. Rev. Ther. Drug Carrier Syst. 20, 153–214. Marcela, A., Eduardo, H., Michael, A.P., Rafael, V., 2002. Cross-linked crystals of chloroperoxidase. Biochem. Biophys. Res. Commun. 295, 828–831. Margolin, A.L., 1996. Novel crystalline catalysts. Trends Biotechnol. 14, 223–230. Morishita, M., Peppas, N.A., 2006. Is the oral route possible for peptide and protein drug delivery? Drug Discov. Today 11, 905–910.
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Mozhaev, V.V., 1993. Mechanism-based strategies for protein thermostabilization. Trends Biotechnol. 11, 88–95. Navia, M., St. Clair, N., 1997. Crosslinked enzyme crystals. Biosens. Bioelectron. 12, 7. Pedersen, N.R., Wimmer, R., Matthiesen, R., Pedersen, L.H., Gessesse, A., 2003. Synthesis of sucrose laurate using a new alkaline protease. Tetrahedron: Asymmetry 14, 667–673. Perez-Victoria, I., Morales, J.C., 2006. Complementary regioselective esterification of non-reducing oligosaccharides catalyzed by different hydrolases. Tetrahedron 62, 878–886. Park, K., Shalaby, W.S.W., Park, H., 1993. Biodegradable Hydrogels for Drug Delivery. Technomic Publishing, Basel, Switzerland. Potier, P., Bouchu, A., Descotes, G., Queneau, Y., 2000. Proteinase N-catalysed transesterifications in DMSO–water and DMF–water: preparation of sucrose monomethacrylate. Tetrahedron Lett. 41, 3597–3600. Reinhammer, B., Oda, Y., 1979. Spectroscopic and catalytic properties of Rhus vernicifera laccase depleted in type 2 copper. J. Inorg. Biochem. 11, 115–127. Thomas, B., Claude, J., Pierre, B., Eliane, C., Nathalie, J., Catherine, M., Christian, M., 2002. Crystal structure of a four-copper laccase complexed with an arylamine: insights into substrate recognition and correlation with kinetics. Biochemistry 41, 7325–7333.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/ejps
Encapsulation of crosslinked subtilisin microcrystals in hydrogel beads for controlled release applications Chandroth Kalyad Simi, Tholath Emilia Abraham ∗ Chemical Sciences and Technology Division, Regional Research Laboratory (CSIR), Thiruvananthapuram, India
a r t i c l e
i n f o
a b s t r a c t
Article history:
Enzymes are less stable in harsh conditions and hence to overcome this nature, several
Received 12 March 2007
methodologies are being developed. It was found that crosslinked enzyme crystals are the
Received in revised form 3 May 2007
most promising strategy for the stabilization of the enzymes [Emilia Abraham, T., Jegan
Accepted 4 May 2007
Roy, J., Bindhu, L.V., Jayakumar, K.K., 2004. Crosslinked enzyme crystals of glucoamylase
Published on line 22 May 2007
as a potent catalyst for biotransformations. Carbohydr. Res. 339, 1099–1104; Navia, M., St. Clair, N., 1997. Crosslinked enzyme crystals. Biosens. Bioelectron. 12, 7]. A cost effective
Keywords:
methodology of crystallization of protease (Bacillus subtilis) with ammonium sulphate (65%,
Protease
w/v) and then crosslinking the crystals with glutaraldehyde (4%, v/v) in isopropanol for
CLEC
20 min gave a stable and active enzyme. SEM studies showed that the protease is in small
Crosslinked subtilisin crystals
cubic shaped crystals of 1–2 m size. Crosslinked enzyme crystal (CLEC) of protease has
Controlled protein release
good stability in polar and nonpolar organic solvents, such as hexane, toluene, benzene
Alginate–guar gum composite beads
and carbon tetrachloride and it had high thermal stability up to 60 ◦ C and hence can be
Hydrogel
used as a catalyst for the biotransformation of compounds which are not soluble in aqueous medium. The CLECs were entrapped in the alginate:guar gum (3:1) composite beads which were resistant to low pH conditions in the stomach and hence was found to be useful for the oral drug delivery. This method can be used to deliver the protein and peptide drugs which require high concentrations at the delivery stage, and which normally degrades in the stomach before reaching the jejunum. Application of these pH-sensitive beads for the controlled release of subtilisin in in vitro was studied and found to be a viable strategy. © 2007 Elsevier B.V. All rights reserved.
1.
Introduction
Most of the drugs in the recent research and developments are peptides or peptide-like synthetic organic compounds, as a result of its high selectivity and its ability for effective action (Morishita and Peppas, 2006). Drug delivery by oral route is the most widely used method although it is not feasible for proteins and peptide drugs. These peptide drugs are quickly broken down into smaller units in the gut, mostly by proteolytic enzymes in the stomach. Much has been learned about
∗
Corresponding author. Tel.: +91 471 2515253; fax: +91 471 2491712. E-mail address: [email protected] (T. Emilia Abraham). 0928-0987/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2007.05.003
the oral delivery of proteins and its absorption in gastrointestinal tract. Various strategies have been used to overcome the difficulties and to develop safe and effective oral delivery systems for proteins (Mahato, 2003). Enzymes are not only used as digestive drugs but also for biotransformation. The development of robust biocatalysts with increased stability and catalytic activity in organic media is a major challenge in industrial biocatalysis (Govardhan, 1999; Fagain, 2003). Several methodologies are developed to overcome this difficulty (Emilia Abraham et al., 2004). Intermolecular crosslinking of
18
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 2 ( 2 0 0 7 ) 17–23
enzyme is one of the most exciting developments in the area of biocatalysis (Jegan Roy and Emilia Abraham, 2004, 2006; Margolin, 1996) and drug delivery. Crosslinked protein crystals are novel type of biocatalysts produced by stepwise crystallization followed by crosslinking by bifunctional reagents to form strong covalent bond between free amino acid groups in the protein molecule to preserve the crystalline structure (Haring and Schreier, 1999). Some of the advantages of CLEC are (1) increased stability, high activity and selectivity in organic media, (2) high mechanical stability, (3) easy recovery and recycling and (4) stable against proteolysis and self-digestion. Crosslinked protein and peptide crystals can be advantageously used in industrial, clinical and research settings. Proteases are one of the most important groups of industrial enzymes accounting for at least a quarter of the total global enzyme market (Adil and Mohammed, 1998). Biosynthetic application of protease includes esterification of sugars and oligosaccharides for the synthesis of novel peptides and proteins (Chen et al., 1998; Perez-Victoria and Morales, 2006; Potier et al., 2000; Pedersen et al., 2003). The active site of the serine proteases contains three amino acids: serine, histidine and aspartate called the catalytic triad and are capable of self-digestion. Protein crystalline drugs are carrier-free, pure, stable and storable at room temperature and are in the most concentrated form. CLECs are ideally suited for diagnostic and therapeutic applications by further encapsulating in polymer matrices to enhance the stability and bioactivity. Hydrogels of non-toxic biopolymers are attractive delivery system for proteins and other drugs because these materials combine a good biocompatibility with possibilities of manipulating the release characteristic of the protein (Chen et al., 1995; Hennink et al., 1997; Kim et al., 1992; Park et al., 1993) or drugs. Encapsulation of soluble enzymes in polymer matrices has been done in the past (Anjani et al., 2007). But the enzymes leak out of the matrix due to the larger pore size of the matrices, such as alginate, carrageenan and agar. The crosslinked enzyme crystal has a size of a few microns, larger than the normal pore size of the hydrogels and does not leak out of the matrix under normal conditions. Here, the encapsulation of the crosslinked protein/enzyme crystal in a natural hydrogel is done for the first time and is a novel concept for the oral delivery of peptides, proteins and enzymes and also for use in biotransformation. In this paper, the optimized conditions for the crystallization and crosslinking of the protease, subtilisin, to get robust enzyme crystals are given. The utility of the CLECs as biocatalyst was evaluated by studying its thermal and solvent stability. These crosslinked crystals were immobilized in a non-toxic, hydrophilic, biodegradable natural biopolymer matrix to form beads, which are pH sensitive. The orally delivered encapsulated enzyme crystal for its controlled release behavior was studied at in vitro conditions.
100 g of tyrosine for 60 min at pH 7) was purchased from Amano Japan. Glutaraldehyde (Sigma, St. Louis, MO, USA), ammonium sulphate, Guar gum (MW, 220 KDa) and sodium alginate (MW, 200 KDa) (Sisco Research Laboratory, Mumbai, India) were of enzyme quality. Casein and Folin phenol reagent (SD fine chemicals, Mumbai, India) was used. All the other chemicals used are of analytical grade. The experimental results are expressed as an average of five independent measurements.
2.2.
Enzyme assay
The protease assay was done at 40 ◦ C in glycine–NaOH buffer (50 mM, pH 10.5) using 1% casein as substrate. One millilitre of approximately diluted enzyme was incubated with 1 ml of 1% (w/v) casein solution for 10 min at 40 ◦ C. The reaction was stopped by the addition of 5% trichloroacetic acid and was centrifuged for 10 min at 8000 g. One millilite of the supernatant was taken and the products were measured by adding 0.4 M sodium carbonate followed by the addition of Folin phenol reagent. The optical density of the samples was taken at 660 nm in a spectrophotometer (Shimadzu, Tokyo, Japan) against appropriate blank.
2.3.
Protein estimation
Lowry’s method was followed for the protein estimation (Lowry et al., 1951). In Lowry’s method, protein is first treated with alkaline copper sulphate in the presence of tartarate. This incubation is then followed by addition of the Folin phenol reagent. Colour of the reaction occurs when the tetradentate copper complexes transfer electrons to the phosphomolybdic acid/phosphotungstic acid complexes. BSA was used as the standard.
2.4.
Crystallisation of protease
Protease was extracted in 0.1 M phosphate buffer of pH 7.0. Ammonium sulphate (50, 60, 65, 70, 75, 80%, w/v) was added stepwise at regular intervals with magnetic stirring to get the pure fraction. Enzyme activity of crystals was measured at all the ammonium sulphate concentrations. Crystallization was done with 65% ammonium sulphate at 4 ± 1 ◦ C for 24 h. The crystals formed were separated by centrifugation at 8000 g for 10 min.
2.5.
Crosslinking of crystals
2.
Materials and methods
Protease crystals were crosslinked with glutaraldehyde solution of different concentration (1–5%, v/v) in isopropanol for 20 min at 25 ◦ C. After crosslinking, it was washed thrice with 0.1 M phosphate buffer of pH 7. Enzyme activity was measured as described earlier. The above experiment was repeated at different duration keeping both glutaraldehyde concentration and temperature constant.
2.1.
Materials
2.6.
Protease from Bacillus subtilis (150,000 units/g; 1 unit is the amount of enzyme which produces amino acid equivalent to
Crystal morphology
Crystal morphology was observed under scanning electron microscope (JEOL, model JSM 5600 LV, Tokyo, Japan). Samples
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 2 ( 2 0 0 7 ) 17–23
were sputtered with gold and scanned at an accelerating voltage of 10 kV.
2.7.
Thermal stability
Thermal stability of CLEC protease at different temperature (40, 50, 60, 70 ◦ C) compared to that of native protease at different time intervals.
2.8.
Organic solvent stability
Organic solvent stability of CLEC protease was done by incubating 1 mg of CLEC in various organic solvents for 24 h. The organic solvents were chosen according to the dielectric constant and log P values. After 24 h, the activity of CLEC protease was done as described earlier. Activity was compared with the original activity and the percentage of activity retention was calculated.
2.9. Preparation of CLEC-entrapped alginate–guar gum beads Guar gum (1%, w/v) and alginate (3%, w/v) was separately dissolved in deionised water and equal quantities were mixed. An appropriate amount of CLEC is added to this mixture and kept for some time to remove air bubbles. Beads were made by dropping the solution using syringe in to 0.5 M calcium chloride solution. The resultant ∼2 mm beads were cured for 1 h and washed with distilled water and stored in 0.05 M calcium chloride solution.
2.9.1.
Determination of entrapment efficiency
An appropriate amount of sample was dissolved in phosphate buffer of pH 7.4 (0.1 M) and then centrifuged. The supernatant was treated with oxalate to precipitate calcium ions, which interferes with the protein estimation. The protein estimation was done by Lowry’s method.
2.9.2.
3.
Results and discussion
3.1.
Optimization of protease crystallization
19
Protease enzyme, subtilisin, which was purified by stepwise ammonium sulphate precipitation, was crystallized at pH 7. Effect of concentration of ammonium sulphate on the crystallization yield was studied. The results were shown in Fig. 1. The maximum activity and yield of good crystals were obtained with 65% ammonium sulphate. Crystallized enzymes, which are more concentrated and are purer shows high activity. Crystallization of macromolecules requires the creation of supersaturated state. At 65% ammonium sulphate concentration, protease precipitated out which is shown by the higher enzyme activity. Enzyme crystals formed were crosslinked with 1–5% glutaraldehyde (v/v) in isopropanol. Effect of glutaraldehyde concentration on the crosslinking of crystals was shown in Fig. 2. Chemical crosslinking does not disturb the crystal lattice structure but provides additional stability. Enzyme in a crosslinked crystal is stabilized by links throughout its three-dimensional structure. Maximum activity yield was obtained when the crystals were crosslinked with 4% glutaraldehyde for 20 min. Activity decreases with increase in duration of crosslinking mainly due to the denaturation of the enzyme by the prolonged exposure to glutaraldehyde. CLEC protease has less activity compared to crystal because of the denaturation occured in the presence of glutaraldehyde. Crosslinked enzyme crystal was associated with some quantity of water. But in the case of enzyme, crystal binding of water is absent, so dilution is less. Therefore, activity of same quantity of CLEC has lower activity compared to enzyme crystal. This is also a factor for decreasing the activity of CLEC compared to enzyme crystal. CLEC shows good activity at 4% (v/v) glutaraldehyde concentration due to the dense crosslinking of the enzyme and thereby the enzyme concentration per unit volume is high. At high concentration of glutaraldehyde, activity decreases because of denaturation and excessive crosslinking, which may lead to
Swelling characteristics
Vacuum dried samples were kept in 5 ml of buffer of pH 1.2 (HCl–KCl buffer 0.1 M) and a solution of pH 7.4 (phosphate buffer 0.1 M). Every 30 min intervals, the samples were taken out, blotted with paper to absorb excess water adhering on the surface and weighed.
Swelling ratio (Qs ) = (Ws − Wd )/Wd . Ws : weight of swollen sample Wd : weight of dry sample
2.9.3.
Protein release studies
Five grams of the hydrogel beads were kept in 10 ml 0.1 M phosphate buffer of pH 7.4 and 0.1 M HCl–KCl buffer of pH 1.2. The medium was stirred at a uniform rate of 1300 rpm using a suitable paddle. At regular intervals certain amount of medium was taken and the protein released was determined by a UV–vis spectrophotometer at 660 nm.
Fig. 1 – Effect of ammonium sulphate (w/v) concentration on the crystallization of enzyme.
20
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3.2.
Crystal morphology
Scanning electron micrograph studies showed very small cubic crystals of the enzyme having the size of 1–2 m. The crosslinked enzyme was also seen as clumps of crystals (Fig. 3).
3.3.
Fig. 2 – Effect of glutaraldehyde concentration on crosslinking.
aggregation, protein precipitation and distortion of the crystal lattice. The CLEC formed had good structural rigidity and hence there was no noticeable self-degradation of the protease in terms of activity and was stable for many days (Navia and St. Clair, 1997). The enzyme crystals, which are normally very fragile become more sturdy and robust after crosslinking. Combination of crystalline lattice contacts and the covalent crosslinking results increase in enzyme stability toward thermal deactivation and organic solvent denaturation.
Thermal stability of CLEC protease
CLEC protease was thermostable up to 60 ◦ C. Above 60 ◦ C, the enzyme activity started decreasing when the incubation time was increased (Fig. 4). Figs. 5 and 6 show the thermal stability of native protease and CLEC protease at different temperature and time intervals. Results showed that thermal stability of CLEC protease was higher than the native enzyme. The enzyme crystal maintains its native conformation at high temperature mainly because in CLEC, the enzyme molecules are symmetrically arranged and crosslinked to stabilize their native conformations (Lanne et al., 1987). The increased thermal stability may be due to (1) the ordered arrangement of the molecules by inter and intramolecular crosslinks within and between the crystals, and gives the rigidity of the three-dimensional arrangement of the molecules in the CLEC and (2) due to additional ionic and hydrophobic contacts between the enzyme molecule. When an enzyme is transferred from a solution to crystalline form, an increase in the number of both polar and hydrophobic interactions among the enzyme molecules may significantly enhance the stability against heat by preventing unfolding, aggregation or dissociation (Islam et al., 1990; Mozhaev, 1993). The increased thermal stability of CLEC protease is a useful property needed for organic reactions to be conducted at an elevated temperature.
Fig. 3 – SEM of protease crystal (a and b) and CLEC protease (c and d).
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Fig. 4 – Thermal stability of CLEC protease at different temperature.
3.4.
21
Fig. 6 – Thermal stability of CLEC protease.
Solvent stability of CLEC protease
Stability of CLEC protease in different solvents was studied and is shown in Table 1. The enzyme activity in organic solvents is intimately related to the water content, size and morphology of the catalyst particle and to the enzyme microenvironment. High biocatalytic activity was found in solvents having log P value between 2 and 4. CLEC proteases have high activity in nonpolar solvents like hexane, toluene, benzene and carbon tetra chloride, due to their low dielectric constant and higher log P values. Acetone, a nonpolar solvent is an exceptional case. The decrease in activity of CLEC in polar solvents compared to the nonpolar solvents is due to the stripping of water from the surface of the protein by competing strongly for hydrogen bonds between the protein molecules (Marcela et al., 2002; Thomas et al., 2002; Reinhammer and Oda, 1979).
Fig. 5 – Thermal stability of native protease.
Fig. 7 – Swelling characteristics of encapsulated CLEC protease.
Crosslinked enzyme crystals were encapsulated in a composite alginate:guar gum (3:1) hydrogel beads and then studied its controlled protein release efficiency which will be useful parameter for its usage as microreactor in biotransformation and in therapeutic oral delivery of the enzymes. Protein analysis of CLEC encapsulated beads, which is ∼2 mm in diameter showed a loading of 65.49% protein. The enzyme activity yield is 69.67% in wet beads. Swelling characteristics of samples in two different pHs (1.2 and 7.4) were shown in Fig. 7. Sample has a very rapid swelling at pH 7.4 which is the pH encountered in the duodenum area where the drug has to be released. Sample at pH 1.2 shows slow swelling, the normal pH of the stomach where the drug releases should not take place. The protein released at pH 1.2 was found to be slow. At pH 7.4, rapid release of protein was taking place as expected from a rapidly swelling hydrogel, and hence the entrapped protein will also be released rapidly. Results of the in vitro studies are shown in Fig. 8. The protein release from the hydrogel at pH
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Table 1 – Stability of CLEC protease after incubation with organic solvent (100%) for 24 h (retention of activity) Solvent Hexane Ethyl acetate Acetone Methanol Acetonitrile Toluene DMF DMSO Benzene Carbon tetrachloride
Dielectric constant 1.9 6.0 20.7 32.7 37.5 2 38.3 47.2 2.28 2.24
log P
Polarity
4.0 0.73 −0.24 −0.74 −0.34 2.73 −1.038 −1.378 2.0 3.0
Nonpolar Dipolar aprotic Dipolar aprotic Polar aprotic Dipolar aprotic Nonpolar Dipolar aprotic Dipolar aprotic Nonpolar Nonpolar
100% Solvent 97.11 30.76 69.90 27.36 19.93 66.82 21.03 7.67 30.77 45.81
Residual activity (U/mg) 0.1489 0.0987 0.09248 0.09163 0.08748 0.1093 0.07648 0.01798 0.09008 0.0856
Activity of crosslinked protease enzyme used was 0.318 U/mg.
protease. High biocatalytic activity was found in solvents having log P value between 2 and 4, hence could be ideally used as a catalytic microreactor in biotransformation. CLEC protease was further encapsulated in alginate:guar gum hydrogel and is used successfully for the controlled release of enzymes in appropriate environment. CLEC hydrogel beads released low amounts of protein at pH 1.2 and a much higher level of protein release was observed at pH 7.4, which is the situation encountered in the human gastrointestinal tract. Alginate is the pH-sensitive component of the bead and releases the drug at the pH prevalent in the jejunum. These non-toxic hydrogel beads were found to be ideal for the oral delivery of peptide and bioactive protein drugs, which is required at high concentrations at the delivery stage.
Acknowledgement Fig. 8 – Release studies of CLEC at pH 7.4 and pH 1.2.
1.2 was found to be lesser than that at pH 7.4 and this may be mainly due to the pH-sensitive behaviour of alginate component than the guar gum. The addition of guar gum, which is a neutral polysaccharide, has facilitated the controlled release of protein at pH 7.4. Enzyme assay was done by using standard procedure. Activity of enzyme at different pH was studied. Crystallisation and crosslinking experiments were done at pH 7. There is no remarkable change in the activity of enzyme at pH 7 and 7.4. The main aim is to study the release property of enzyme at pH 7.4, which is the pH of the duodenum area. At pH 1.2 release of enzyme is poor compared to 7.4. So we think that there is no need to study the activity of enzyme at pH 1.2.
4.
Conclusion
Crosslinked protease crystal with high stability than native enzyme was prepared and its crystallization and crosslinking parameters were optimized. A 65% ammonium sulphate concentration was the optimum for the crystallization. CLEC protease has a high activity when crosslinked with 4% glutaraldehyde in isopropanol. It was stable in polar and nonpolar organic solvents and at a high temperature than native
We are very grateful to the Council of Scientific and Industrial Research (CSIR, India) for the financial support.
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