Process Biochemistry 42 (2007) 884–888 www.elsevier.com/locate/procbio
Short communication
Immobilization of trypsin on polysaccharide film from Anacardium occidentale L. and its application as cutaneous dressing Flaviane Maria Floreˆncio Monteiro a,b, Germana Michelle de Medeiros e Silva a, Juciene Bezerra Rodrigues da Silva a, Camila Souza Porto a, Luiz Bezerra de Carvalho Jr.a,c, Jose´ Luiz de Lima Filho a,c, Ana Maria dos Anjos Carneiro-Lea˜o a,d, Maria das Grac¸as Carneiro-da-Cunha a,c, Ana Lu´cia Figueiredo Porto a,d,* a b
Laborato´rio de Imunopatologia Keizo Asami (LIKA), UFPE, Brazil Programa de Po´s-Graduac¸a˜o em Cieˆncia Veterina´ria, UFRPE, Brazil c Departamento de Bioquı´mica, UFPE, Brazil d Departamento de Morfologia e Fisiologia Animal, UFRPE, Brazil
Received 11 August 2006; received in revised form 9 January 2007; accepted 10 January 2007
Abstract Polysaccharide obtained from Anacardium occidentale L. gum was used for trypsin entrapment using cellulose (gaze) as a support and this preparation was applied as cutaneous wound healing. Trypsin release in vitro and the influence of pH and temperature on activity, stability and storage time of entrapped enzyme were evaluated. The preparation showed that it was still capable to release enzyme even after 48 h. Entrapped enzyme presented an optimal pH and temperature of 8.6 and 55 8C, respectively. Also, it was stable at high temperature (45 8C for 60 min) and wide range of pH, retaining 80% of its initial activity when stored for 28 days at 25 8C. Histopathological analysis of mice skin wound healing under the entrapped trypsin preparation treatment showed an acceleration of fibroblast proliferation, neovascularization of granulation tissue and stimulating effect on the epithelium formation compared to the skin wound under the treatment using preparations without trypsin. These results demonstrate that the trypsin–polysaccharide–cellulose preparation could be used in cutaneous dressing applications for wound healing. # 2007 Elsevier Ltd. All rights reserved. Keywords: Immobilization; Anacardium occidentale L.; Polymeric films; Trypsin; Wound healing; Cutaneous dressing
1. Introduction Polysaccharide films have been widely used in the pharmaceutical field. For instance, films prepared using chitin or chitosan have been developed as wound dressings, oral mucoadhesive and water-resisting adhesive by virtue of their release characteristics and adhesion [1]. Also, they have been applied in the development of biosensors, biological membranes, immunological experiments and suture threads as they are biodegradable and biocompatible [2]. A polysaccharide with molecular mass of 1.6 105 Da presenting a main chain formed by units of D-Galp joined by glycoside links b-(1 ! 3) substituted in O-6 has been obtained
* Corresponding author at: Departamento de Morfologia e Fisiologia Animal, UFRPE, Brazil. Tel.: +55 81 21268484; fax: +55 81 21268485. E-mail address:
[email protected] (A.L.F. Porto). 1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2007.01.006
from Anacardium occidentale (cashew tree) gum, abundant in Northeast region of Brazil [3]. This polysaccharide has been reported as a potential constituent agent of films and thickening [3,4] in addition to its confirmed anti tumours, anti parasitic and cicatrizing effect [5–7]. The easy access to this inexpensive, nontoxic, hydrophilic, biocompatible and biodegradable polysaccharide, presenting interesting biological activity and good rheological properties are factors that make viable its potential use as immobilization carrier for drug delivery systems. Filmsentrapped proteases could be promising drugs for wound therapy. For instance, matrix metalloproteinases play an important role in the degradation process of extracellular matrix and this is an important feature for the development, morphogenesis, tissue repair and remodelling [8]. Recently, in our laboratory trypsin was covalently immobilized onto a membrane of a cellulosic exopolysaccharide produced by Zoogloea sp. in sugarcane. The preparations showed to be thermal stable and reusable [9]. Previously, lipase
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was covalently immobilized onto a chitosan produced by a native Mucoralean strain, Syncephalastrum racemosum, isolated from herbivorous dung [10]. Thus the aim of this work was to entrap trypsin on the polysaccharide from A. occidentale, to investigate the effect of pH, temperature on the preparation, its stability and to evaluate it as a drug delivery system in cutaneous dressings for wound healing.
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determination of the water soluble released products. One unit of activity (U) was defined as the amount of enzyme that produces an increase in optical density of 1.0 in 1 h at 440 nm. The specific activity was obtained by the ratio between the enzymatic activity and protein concentration (mg/mL). Data from experiments in triplicate were expressed as average standard deviation. The concentration of protein was determined using bovine serum albumin (0– 100 mg/mL) as the standard according to the method of Bradford [13].
2.4. Trypsin release from TRYPSIN–POLICAJU–CELLULOSE preparation
2. Materials and methods 2.1. Materials Polysaccharide from A. occidentale L. tree gum (collected in South coast of Pernambuco, Brazil) was obtained according to Menestrina et al. [4] and termed POLICAJU. The cellulose (gaze) used as matrix for the POLICAJU was obtained from Cremer1. The swine pancreas trypsin (E.C. 3.4.21.4) and azocasein substrate were obtained from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA). All other chemicals were of analytical grade.
2.2. Film preparation and enzyme entrapment The POLICAJU film was prepared as described by Carneiro-da-Cunha et al. [11] at 10% (w/v) and solubilized in either 4% (v/v) acetic acid or 150 mM NaCl under constant agitation for 16 h at 25 8C pH adjusted to 5.0 with a 1 M NaOH solution. Trypsin (10 mg) was added to the solubilized POLICAJU (10 mL) prepared in acetic acid and then mixed for 1h. Afterwards, 200 mL of each solution were placed upon a surface of 1.0 cm2 of the cellulosic matrix (gaze) and films were obtained at 50 5 8C, containing 200 mg of enzyme per cm2 and termed TRYPSIN–POLICAJU–CELLULOSE preparations (Fig. 1). Preparations without trypsin were also prepared in acetic acid or NaCl and named CELLULOSE preparations (controls).
2.3. Determination of enzymatic activity and protein It was carried out according to the method of Leighton et al. [12]. Briefly, free enzyme (150 mL) was incubated in 1.0% (w/v) azocasein (250 mL) prepared in Tris–HCl buffer with 0.1 M of CaCl2, pH 7.2; whereas the TRYPSIN–POLICAJU–CELLULOSE preparations (1 cm2 containing 200 mg) in azocasein (1000 mL), under light protection for 1 h at 25 2 8C. Afterwards, samples (400 mL) of each assay were used for spectrophotometric
The preparation was incubated in 1000 mL of buffer solution of 0.2 M citrate–phosphate, pH 5.0 for 72 h. Every 24 h samples of 150 mL were collected from the supernatant for the determination of enzymatic activity and protein concentration.
2.5. Effect of pH and temperature on the activity of free enzyme and TRYPSIN–POLICAJU–CELLULOSE preparation The pH effect was evaluated by incubating samples of free trypsin and TRYPSIN–POLICAJU–CELLULOSE preparation with 1% (w/v) azocasein prepared in 0.2 M buffer, pH 5.0–5.4 (citrate–phosphate); pH 6.2–6.8 (sodium– phosphate); pH 7.2–9.0 (Tris–HCl) and pH 9.4–10.2 (glycine–NaOH), containing 0.1 M CaCl2. At time intervals samples were removed and enzyme activity determined as described in Section 2.3. The temperature effect was investigated by incubating the free trypsin and TRYPSIN–POLICAJU–CELLULOSE preparation with 1% (w/v) azocasein prepared in 0.2 M Tris–HCl buffer with 0.1 M of CaCl2, pH 7.2, at temperatures ranging from 25 to 65 8C and measuring the enzyme activities as above described.
2.6. Effect of pH and temperature on the stability of TRYPSIN– POLICAJU–CELLULOSE preparation TRYPSIN–POLICAJU–CELLULOSE preparation samples were incubated at pH varying from 5.0 to 10.2 using different buffer solutions according to their pKa values for 60 min at 25 8C. Afterwards, the preparations were collected and incubated for 1 h in azocasein (1% w/v) solution with 0.2 M Tris–HCl buffer, pH 7.2 at 25 8C. Then the products released were estimated at 440 nm as described in Section 2.3. For the thermal stability determination the preparations were incubated at 25, 35, 45, 55 and 65 8C for 60 min. Afterwards they were withdrawn and their activity determined at 25 8C as previously described (Section 2.3).
2.7. Stability of TRYPSIN–POLICAJU–CELLULOSE in storage The storage stability of the TRYPSIN–POLICAJU–CELLULOSE preparation was determined by keeping the preparations at 25 3 8C and determining their activities at the 2nd, 7th, 14th, 21st and 28th days. In each determination the immobilized trypsin was incubated in 1000 mL of azocasein (1.0% (w/v) in 0.2 M Tris–HCl buffer with 0.1 M of CaCl2, pH 7.2 for 1 h at 25 8C) and then the enzymatic activity established as described above.
2.8. Histopathological evaluation of TRYPSIN–POLICAJU– CELLULOSE preparation as wound healing in a cutaneous excision model
Fig. 1. The TRYPSIN–POLICAJU–CELLULOSE preparation.
In the experimental surgical procedure mice were intraperitoneally anaesthetized with 2% xilazine chloride and ketamine chloridate (10 and 115 mg/kg, respectively) [14]. The antisepsis of dorsal thoracic region was made with iodopovidone and sterile saline solution 0.15 M NaCl. An aseptic dermal wound (1.33 0.2 cm2) was made by skin incision and divulsion of epidermal layer. After the surgery the animals were randomly divided into three groups (n = 15) according to the treatment: CELLULOSE in acetic acid preparation; CELLULOSE in NaCl preparation and TRYPSIN–POLICAJU–CELLULOSE preparation. Immediately after the surgery the curative was applied once only on the wounds. Fragments of skin were collected from five anaesthetized animals on the 2nd and 12th days. Soon after the mice were intraperitoneal euthanasiaded
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with sodium pentobarbital super dosage (200 mg/kg). Excised wound sites were formalin fixed, routinely processed and embedded into wax. Thick sections were stained using haematoxylin and eosin (HE) and Masson’s Trichrome stains and examined by light microscopy [15]. All animal procedures were in accordance with the Cole´gio Brasileiro de Experimentac¸a˜o em Animal (COBEA) and the Animal Ethical Committee of the Universidade Federal de Pernambuco approved the experimental protocol.
3. Results and discussion 3.1. Trypsin release from TRYPSIN–POLICAJU– CELLULOSE preparation The trypsin release from the TRYPSIN–POLICAJU– CELLULOSE preparation was followed during 72 h in the supernatant. It was observed an increase from 7.17 to 15.89 U/ mg (121.7%) in the first 24 h. After that, there was a decrease and then remained constant until the end of experiment (Fig. 2). Even so, 12 U/mg of specific activity represents an increase of about 70% in relation to the zero time (initial). The preparation allowed the release of enzyme to the external medium and the initial increase is probably due to the cumulative release of those molecules allocated near the preparation surface. Studies carried out by Markvicheva et al. [16] with bovine trypsin immobilized by entrapment in PVCL–Ca alginate and PVCL– chitosan–chitosan sulphate polymers both magnetized showed a partial enzyme release from the beads in the supernatant and the loss of relative activity was of 80% and 70%, respectively. 3.2. Effect of pH on activity of the free enzyme and TRYPSIN–POLICAJU–CELLULOSE preparation and on the stability of the entrapped enzyme The effect of pH on the enzymatic activity is illustrated in Fig. 3A. Both immobilized trypsin and free enzyme presented similar behaviour at tested pH values range (5.0–10.2) and same optimum pH value (8.6–9.0). Similar activity profiles were reported for free trypsin and covalently immobilized on aminopropyl-Celite and succinamidopropyl-Celite [17] and on chitosan–silica gel (CTS–SiO2) with different activators [18]. In addition, it was observed that both the immobilized trypsin
Fig. 2. Time course of enzyme release from the TRYPSIN–POLICAJU– CELLULOSE preparation. The preparation was incubated in buffer solution 0.2 M citrate–phosphate, pH 5.0 at 25 8C and at the indicated time samples were withdrawn and the enzyme activity and protein content established. Data presented as average standard deviation (triplicates).
Fig. 3. Effect of pH (A) and temperature (B) on the activity of free enzyme (*) and TRYPSIN–POLICAJU–CELLULOSE preparation (&). Buffer solutions: pH 5.0 and 5.4 (0.2 M citrate–phosphate), pH 6.2 and 6.8 (0.2 M sodium– phosphate, pH 7.2, 7.8, 8.6 and 9.0 (0.2M Tris–HCl) and pH 9.4 and 10.2 (0.2 M glycine–NaOH). Inserts show the effect of pH and temperature on the stability of TRYPSIN–POLICAJU–CELLULOSE preparation. Samples were incubated at the indicated pH and temperature for 60 min and their activities determined. Each data point of all figures represents the average of three experiments and the error bars show the standard deviation.
and free enzyme presented a second peak at the pH 10.2, probably, due to contamination of the employed trypsin by traces of another protease. The trypsin used in this work was contaminated with traces of chymotrypsin according to information from the producer Sigma (St. Louis, MO, USA). Chong et al. [19] reported a similar situation for proteases from fish intestine with two peak activity regions between pH 7.5–9.0 and at a higher pH of 11.5–12.5 indicating the existence of two groups of alkaline proteases functioning in the digestive tract. The trypsin used in this work probably has also traces of chymotrypsin as contaminat. The pH stability experiments (insert of Fig. 3A). The loss of activity varied from 10% to 30% of that estimated at the initial time. This stability is compatible with the tested environment medium. For instance, at pH 5.0, usually found at the wound tissue, after 60 min the enzyme retained 88% of the initial activity. 3.3. Effect of temperature on activity of the free enzyme and TRYPSIN–POLICAJU–CELLULOSE preparation and on the stability of the entrapped enzyme The effect of temperature on enzymatic activity was also similar for free enzyme and immobilized trypsin (Fig. 3B) with
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the same optimum (100%) at 55 8C for both. Nevertheless, the immobilized trypsin preserved a higher activity (92%) at 65 8C than the free enzyme (69%). Similar behaviour was reported by Xi et al. [18] in the comparison of thermal stability of free and covalently immobilized trypsin on chitosan–silica gel activated by diazotization. In the thermal stability evaluation of the immobilized trypsin activity there was no marked difference inside the tested temperature range (insert of Fig. 3B). Furthermore, at 65 8C the enzyme showed 94% of the initial activity. From this it is also possible to deduce that immobilized trypsin is especially resistant to high temperatures. Fig. 4. Stability of immobilized TRYPSIN–POLICAJU–CELLULOSE preparation over the 28 days of storage at room temperature (25 3 8C). Each data point represents the average of three experiments and the error bars show the standard deviation.
3.4. Stability of the TRYPSIN–POLICAJU–CELLULOSE preparation The stability of immobilized trypsin was evaluated over the 28 days of storage at room temperature (25 3 8C) in a view to
Fig. 5. Microscopic aspects of the evolution of cutaneous repair process after 2 days (A–C) and 12 days (D–F) under treatment with dressings containing 0.15 M NaCl, 4% acetic acid and immobilized trypsin. Masson’s Trichrome stain. Original enlargement 100. (A) and (D) topically treated with preparation containing CELLULOSE in NaCl; (B) and (E) with preparation containing CELLULOSE in acetic acid; (C) and (F) with preparation containing TRYPSIN–POLICAJU– CELLULOSE in acetic acid.
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a possible application as dermal dressing. On the storage day and on the 2nd, 7th, 14th, 21st and 28th days were collected samples for determination of enzymatic activity (Fig. 4). A decline (17%) of activity was found in the first seven days and after that remained stable with activity retention of 86%. The stability of a trypsin covalently immobilized directly and through bovine serum albumin on a membrane of a cellulosic exopolysaccharide produced by Zoogloea sp. was evaluated over 54 days by Cavalcante et al. [9]. These authors found retention of 89.17% and 99.12%, respectively. Once more the results found in this work suggest that the immobilized trypsin in POLICAJU presents characteristics to be used in the dermal cicatrization process. 3.5. Histopathological evaluation of the TRYPSIN– POLICAJU–CELLULOSE preparation Immobilized trypsin was applied in the healing cutaneous surgically caused in mice. Before the euthanasia on the 2nd and 12th days, were collected fragments from tissue biopsy, processed and histopathologically analyzed. In Fig. 5 are displayed microscopic aspects of the evolution of cutaneous repair process after 2 days (A–C) and 12 days (D–F) of treatment with dressings containing CELLULOSE preparations in 0.15 M NaCl (A and D); CELLULOSE preparations in 4% acetic acid (B and E); TRYPSIN–POLICAJU–CELLULOSE preparations (C and F). In the evaluation on 2nd day it was found the presence of typical elements of inflammatory phase. Nevertheless, the TRYPSIN–POLICAJU–CELLULOSE preparations groups presented bigger tissue fragility with characteristic of a process less developed. After 12 days of treatment the animals from TRYPSIN–POLICAJU–CELLULOSE preparations group presented a tissue pattern more developed, due to big quantity of collagen meanwhile formed and by the organization pattern of these fibres establishing by evidence a remodelling process more advanced than in the CELLULOSE preparations in 4% acetic acid and CELLULOSE preparations in 0.15 M NaCl groups. Markvicheva et al. [16], worked with entrapped trypsin in hydrogel film and they found a markedly accelerated healing of excision skin wounds in a mice model. The results found in this work also gave evidence of stimulating effect of TRYPSIN–POLICAJU– CELLULOSE preparations in a cicatricial process. In conclusion, the findings in this work suggest that the polysaccharide from A. occidentale L. tree gum could be considered as: a suitable, inexpensive and abundant, biodegradable and biocompatible matrix for biomolecules immobilization like trypsin and an efficient drug delivery system. Furthermore, the entrapped trypsin in this polysaccharide and cellulose (gaze) showed to be a promising wound healing dressing to be used in clinical therapy. Acknowledgements Flaviane Maria Floreˆncio Monteiro and Camila Souza Porto are very grateful to scholarships from CAPES for development
of PhD and CNPq for Scientific Initiation Programmes, respectively. The authors acknowledge the ALFA-VALNATURA Programme (AML/B7-311/97/0666/II0440-FA).
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