RU2769243C1 - Method for producing heterogeneous enzyme preparation based on ficin and low-molecular chitosan - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, in particular to a method for producing a heterogeneous enzyme preparation based on ficin and low molecular weight 50-190 kDa chitosan.

EFFECT: invention allows to reduce the consumption of anti-inflammatory, wound-healing agents due to the prolonged action and high stability of the resulting drug.

1 cl, 6 dwg, 1 ex

Description

The invention relates to the chemical and pharmaceutical industry and can be used in the manufacture of medicines with a wound healing effect. The drug is stable during storage, has antimicrobial and adsorbing properties.

In modern medicine, the problem of treating purulent wounds of various etiologies remains relevant. Enzyme-based drugs used in the treatment of wounds are represented by an insignificant group of drugs, since the use of free forms of enzymes in solutions does not give a high effect due to the fact that the protein is unstable under such conditions, is quickly inactivated and does not provide long-term contact with wound surface. The most common compositions are gels, ointments and dressings, which, when applied locally to a wound, simultaneously influence the pathogenic microflora and stimulate healing processes.

Ficin (EC 3.4.22.3) belongs to the group of cysteine proteases, has a wide substrate specificity, preferentially hydrolyzes tyrosine and phenylalanine bonds in proteins. Shows high activity in the pH range 6.5-9.5. Its isoelectric point is 9.0. The enzyme is used in medicine, pharmaceuticals, accelerates the healing of wounds, trophic ulcers and bedsores, helping to cleanse them of necrotic masses, accelerates the cleansing of the wound from pathogenic microflora, creates optimal conditions for reparative processes [L. Feijoo-Siota, T.G. Villa, Native and biotechnologically engineered plant proteases with industrial applications, Food Bioprocess Technol. 4 (2011) 1066-1088]. Ficin has anti-adhesive activity against oral plaque bacteria [Patent RU 2393845 C2, IPC A61K 8/66, A61Q 11/00, publ. 10.07.2010, Bull. No. 19]. It is used in medicines for the treatment and/or prevention of eye diseases: age-related macular degeneration, choroidal revascularization, Hippel-Lindau disease, revascularization of the iris [Patent RU 2472523 C2, IPC A61K 38/48, A61P 27/02, publ. . 20.01.2013, Bull. No. 2]. Preparations containing ficin can be used to reduce the concentration or eliminate the allergen Fel dl from the environment: the enzyme decomposes allergenic epitopes [Patent RU 2706206 C1, IPC A61K 38/48, publ. 11/14/2019, Bull. No. 32].

Direct introduction of free enzymes into the wound leads to their rapid inactivation, which reduces the therapeutic effect; therefore, there are numerous methods for immobilizing various enzymes, including ficin. The use of physical immobilization to obtain a complex of a copolymer of vinyl alcohol, vinyl glutarant and vinyl acetate and protease contributes to the prolongation of its lytic action due to the depot effect by slowly releasing the enzyme into the wound area [Patent RU 2014086, IPC A61L 15/12, publ. 06/15/1994].

Chitosan is a biocompatible and biodegradable polymer that exhibits antibacterial activity, which allows it to be used in various fields of medicine, including for rapid wound healing [Chitin and Chitosan. Obtaining, properties and application. Ed. Academician of the Russian Academy of Agricultural Sciences K.G. Scriabin. The science. 2002. 365 pages].

Recently, many materials and medical products based on natural and synthetic polymers have been developed. A composition is known that accelerates the healing of wounds of various etiologies without inflammatory complications, containing chitosan and/or the salt form of chitosan or its derivatives with gold nanoparticles and amino acids [Patent RU 2666599 C1, IPC A61L 15/18, A61L 15/22, A61L 15/28 , A61K 31/722, A61P 17/02, publ. 09/11/2018, Bull. No. 26]. Chitosan is part of the wound dressing. It is an elastic, water-insoluble film that does not contain toxic substances, has good adsorbing capacity, antibacterial and wound healing properties [Patent RU 2582220 C1, IPC A61L 15/20, A61L 15/28, A61L 15/44, A61F 13/00, publ. 20.04.2016, Bull. No. 11]. On the basis of chitosan, a method has been developed for producing a biodegradable film for its use in pharmaceuticals, medicine, veterinary medicine, food or cosmetic industries [Patent RU 2656502 C1, IPC C08J 5/18, C08L 5/08, C08L 3/02, A61L 15/22, publ. . 06/05/2018, Bull. No. 16; Patent RU 2613112 C2, IPC A61L 15/44, A61L 15/28, A61L 15/20, A61P 17/02, publ. 03/15/2017, Bull. No. 8]. Chitosan is part of the gel for external use, which results in the acceleration of wound healing processes and a decrease in the severity of scars [Patent RU 261 1400 C2, IPC A61K 31/722, A61K 31/095, A61K 31/17, A61K 9/06, A61P 17/ 02, publ. 21.02.2017, Bull. No. 6]. Chitosan is added to cosmetic gels for moisturizing and softening the skin of the face and body [Patent RU 2085187 C1, IPC A61K 7/48, publ. 07/27/1997].

Low molecular weight forms of chitosan are promising polymers for the development and creation of a new class of dosage forms with wound healing and antibacterial properties. It has been shown that the antibacterial properties of chitosan strongly depend on its molecular weight parameters [Kulikov S.N., Tyurin Yu.A., Fassakhov R.S., Varlamov V.P. / Antibacterial and antimycotic activity of chitosan: mechanisms of action and the role of structure // Journal of Microbiology, Epidemiology and Immunobiology, 2009, No. 5, p. 91-98]. Chitosan is a copolymer of glucosamine and acetylglucosamine, is a heterogeneous group of substances that differ in molecular weight, degree of deacetylation, location of residual acetylated units along the polymer chain, and due to the wide variety of these parameters of polymer samples, chitosan is characterized by significant variations in the manifestation of antibacterial properties [Kumar A.B.V., Varadaraj M.C., Tharanathan R.N. Low molecular weight chitosan - preparation with the aid of pepsin, characterization, and its bactericidal activity. biochim. Biophys. acta. 2004, 1670(2): 137-146].

For aesthetic medicine and cosmetology, a chitosan-containing hydrogel for cosmetic skin care with low molecular weight chitosan in the composition, used to moisturize the skin, reduce wrinkles, and implement a lifting effect, has been created [Patent RU 2667130 C1, IPC A61K 8/04, A61K 8/73, A61K 8 /36, A61Q 19/08, publ. 09/14/2018, Bull. No. 26]. For use in medicine, a preparation has been developed that is a gel of the following composition (in g): low molecular weight chitosan - 0.05-2.00; methylcellulose-100 - 1.0-3.0; glycerin - 10.0-20.0; nipagin - 0.1-0.3; water - up to 100.0, where low molecular weight chitosan has a molecular weight from 5 to 72 kDa with a degree of deacetylation from 20 to 85-89% at concentrations from 0.05 to 2.0%. This composition provides a high wound healing effect [Patent RU 2440122 C1, IPC A61K 31/722, A61K 31/717, A61K 47/10, A61R 31/04, publ. 01/20/2012, Bull. No. 2]. For the pharmaceutical industry, an antibacterial composition has been created, including a water-soluble low molecular weight chitosan used in a wide range of acidity levels [Patent RU 2494746 C1, A61K 31/722, A61K 47/48, A61P 31/04, publ. 10.10.2013, Bull. No. 28]. A preparation in powder form for regeneration of soft tissues with low molecular weight chitosan with a molecular weight of 5-72 kDa has been developed [Patent RU 2485959 C1, IPC A61K 31/722, A61K 33/30, A61K 33/34, A61K 9/14, A61P 17/02 , A61P 31/04, B82B 1/00, B82Y 5/0, publ. 06/27/2013, Bull. No. 18].

The aim of the invention is to develop a method for obtaining a heterogeneous enzyme preparation based on ficin and low molecular weight chitosan 50-190 kDa, which has both anti-inflammatory and regenerative effects in combination with an antibacterial effect. Previously, we carried out the immobilization of ficin on medium molecular weight 200 kDa and high molecular weight 350 kDa chitosans, which themselves did not show significant antibacterial activity [Diana R. Baidamshina, Victoria A. Koroleva, Elena Yu. Trizna, Svetlana M. Pankova, Mariya N. Agafonova, Milana N. Chirkova, Olga S. Vasileva, Nafis Akhmetov, Valeriya V. Shubina, Andrey G. Porfiryev, Elena V. Semenova, Oskar A. Sachenkov, Mikhail I. Bogachev, Valeriy G. Artyukhov, Tatyana V. Baltina, Marina G. Holyavka, Airat R. Kayumov. Anti-biofilm and wound-healing activity of chitosan-immobilized Ficin // International Journal of Biological Macromolecules, 2020, V. 164, P. 4205-4217]. At the same time, there are data in the literature that indicate an increase in the antibacterial properties of chitosan with a decrease in its molecular weight, which is associated with better solubility of low molecular weight forms of the polymer, so we chose low molecular weight 50-190 kDa chitosan as a matrix for ficin immobilization.

Known methods for producing heterogeneous enzyme preparations based on proteases, in particular bromelain [Patent RU 2691611, IPC C12N 11/04, C12N 11/10, publ. 06/14/2019 Bull. No. 17] and papain [Patent RU 2712690, IPC C12N 11/04, C12N 11/10, publ. 01/30/2020 Bull. No. 4] and gels of food chitosan and chitosan succinate. In contrast to them, we propose to use ficin as an active substance, which has a slightly different (preferably hydrolyzes tyrosine and phenylalanine bonds in proteins, at the same time, the process of cleavage along arginine and lysine bonds is not observed at all), but at the same time a rather wide substrate specificity and hydrolyzes casein at a faster rate than papain, bromelain or trypsin [Workshop on biotechnology: immobilized biological objects in the laboratory work system / M.G. Kholyavka, M.A. Nakvasina, V.G. Artyukhov. - Voronezh State University. - Voronezh: VSU Publishing House, 2017. 161 p.]. In addition, ficin effectively hydrolyzes the structural components of the biofilm matrix of Staphylococcus aureus and Staphylococcus epidermidis already at a concentration of 10 μg/ml, almost completely destroying the biofilm at a concentration of 1 mg/ml. After treatment with ficin, the biofilm structure becomes porous and with reduced viscosity. Treatment with ficin enhances the antimicrobial effect of ciprofloxacin, gentamicin and benzalkonium chloride against cells inside biofilms of Staphylococcus aureus and Staphylococcus epidermidis [Baidamshina D.R., Trizna E.Y., Holyavka M.G., Bogachev M.I., Artyukhov V.G., Akhatova F.S., Rozhina E.V., Fakh.R.mov.Ka Targeting microbial bioflms using Ficin, a nonspecifc plant protease roof // Scientific Reports, 2017, 7:46068, DOI: 10.1038/srep46068].

The prototype was a method for obtaining a heterogeneous enzyme preparation based on ficin, which has wound healing and regenerating properties [Patent RU 2677858 C2, IPC A61K 31/74, A61K 38/46, A61P 17/02, C12N 11/08, publ. 01/10/2019, Bull. No. 1], which includes the immobilization of ficin in a buffer solution on a matrix of ion-exchange fibers VION KN-1 in a ratio of 20 ml of ficin buffer solution at a concentration of 1 mg/ml per 1 g of fibers, while 0.05 M Tris-HCl buffer is used as a buffer solution with pH 7.0; then carry out incubation for 24 hours at room temperature with occasional stirring; and then the formed precipitate is washed with 0.05 M Tris-HCl buffer pH 7.0 until no ficin is present in the washings.

The disadvantage of the prototype is the use of synthetic fibers that can stick to the surface of the wound.

The technical result consists in developing a method for producing a heterogeneous drug based on ficin and low molecular weight chitosan 50-190 kDa, which makes it possible to reduce the consumption of an anti-inflammatory, wound healing agent due to the prolonged action and high stability of the resulting drug with high antibacterial activity due to ficin, which is included in the composition of ficin, exceeding, respectively, 2 and 2.5 times the activity of ficin immobilized on medium molecular weight 200 kDa and high molecular weight 350 kDa chitosan; remaining after two days of incubation is not less than 46% of the initial catalytic capacity.

The active substance of the agent we propose is ficin, which has anti-inflammatory and wound healing properties, as a carrier for the immobilization of the enzyme, we chose low-molecular chitosan with a molecular weight of 50-190 kDa, which, as a carrier for immobilization, can not only increase the stability of ficin with respect to various denaturing factors , but it also has a regenerating and higher antibacterial activity compared to medium and high molecular weight chitosan, which is associated with better solubility of low molecular weight forms of the polymer. In addition, low molecular weight chitosan protects the treated skin area from drying out, retaining moisture, and makes it easy to remove the drug along with pus and exudate.

The technical result is achieved by the fact that in the method for obtaining a heterogeneous enzyme preparation based on ficin and low molecular weight chitosan, including the immobilization of the enzyme on the carrier matrix in a buffer solution in a ratio of 20 ml of the enzyme buffer solution at a concentration of 1 mg/ml per 1 g of the carrier, incubation for 24 hours at room temperature with periodic stirring and washing, according to the invention, ficin immobilization is carried out on a matrix of low molecular weight chitosan with a molecular weight of 50-190 kDa; as a buffer solution for immobilization, 0.05 M borate buffer with 0.1 M KCl in the composition with pH 8.0 is used; the formed precipitate is washed with 0.05 M borate buffer solution with 0.1 M KCl in the composition with pH 8.0 until the absence of ficin in the washing water.

Fig. Fig. 1. Diagram of protein content (mg/ml) in ficin preparations immobilized on a matrix of low molecular weight chitosan: 1 - 0.05 M phosphate buffer, 2 - 0.05 M borate buffer with 0.1 M KCl in the composition, 3 - 0.05 M tris-glycine buffer, 4 - 0.05 M glycine buffer.

Fig. 2. Diagram of the total activity (u/ml) of ficin preparations immobilized on a matrix of low molecular weight chitosan: 1 - 0.05 M phosphate buffer, 2 - 0.05 M borate buffer with 0.1 M KCl in the composition, 3 - 0.05 M tris-glycine buffer, 4 - 0.05 M glycine buffer.

Fig. Fig. 3. Diagram of the specific activity (u/mg of protein) of ficin preparations immobilized on a matrix of low molecular weight chitosan: 1 - 0.05 M phosphate buffer, 2 - 0.05 M borate buffer with 0.1 M KCl in the composition, 3 - 0.05 M tris-glycine buffer, 4 - 0.05 M glycine buffer.

Fig. Fig. 4. Diagram of dependence of catalytic activity (%) of free (1) and immobilized (2) ficin on incubation time.

Fig. Fig. 5. Percentage of retention of the amount of ficin in the sorbent phase during incubation of preparations: 1 - in 50 mM Tris-HCl buffer, pH 7.5; 2 - in saline.

Fig. 6. Amount of biomass of Escherichia coli (K-12 MKD910) under different culture conditions.

An example of the implementation of the method.

Ficin from Sigma-Aldrich was chosen as the object of study, azocasein from Sigma-Aldrich served as a substrate for hydrolysis, and low-molecular-weight chitosan with a molecular weight of 50–190 kDa from Sigma-Aldrich served as a carrier for immobilization. To obtain heterogeneous biocatalysts based on ficin immobilized on a matrix of low-molecular-weight chitosan, we used the following buffer solutions as an immobilization medium: 0.05 M glycine, 0.05 M tris-glycine, 0.05 M phosphate, pH 5.8 to 10.0.

Ficin was immobilized on a matrix of low molecular weight chitosan with a molecular weight of 50–190 kDa in a buffer solution in a ratio of 20 ml of an enzyme buffer solution at a concentration of 1 mg/ml per 1 g of carrier. Incubation was carried out for 24 hours at room temperature with occasional stirring; washing was carried out with a buffer solution used as an immobilization medium until no ficin was present in the washings.

The protein content in the preparations was determined by the Lowry method [Lowry O.N., Rosebrough N.J., Faar A.L., Randall R.J. Protein measurement with folin-phenol reagent// J. Biol. Chem. - 1951.-V. 193.-p. 265-275]. The level of proteolytic activity of ficin was measured on the substrate azocasein [Garcia-Carreno, F. L. The digestive proteases of langostilla (Pleuroncodes planipes, Decapoda): their partial characterization and the effect of feed on their composition // Comparative Biochemistry and Physiology Part B: Comparative Biochemistry - 1992. - V. 103. - P. 575-578]. To 50 mg of the sample, 200 μl of Tris-HCl buffer pH 7.5 and 800 μl of azocasein at a concentration of 0.5% in 50 mM Tris-HCl buffer pH 7.5 were added and incubated for 2 hours at 37°C. Next, 800 μl of trichloroacetic acid (TCA) at a concentration of 5% was added, incubated for 10 minutes at -4°C, then centrifuged for 3 minutes at 11,700 g to remove non-hydrolyzed azocasein. To 1200 μl of the supernatant, 240 μl of 3% NaOH was added to neutralize the acid, after which the optical density of the test sample was measured at 410 nm in a 1 cm cuvette. The control sample contained 800 µl of azocasein, 800 µl of TCA, 50 mg of sample, and 200 µl of Tris-HCl buffer. A unit of catalytic activity was the amount of ficin that hydrolyzes 1 μM of azocasein in 1 min under the experimental conditions. The specific proteolytic activity of ficin was calculated by the formula:

A=D*1000/120/200/C,

where A is proteolytic activity, µM/min per 1 mg of protein,

D is the optical density of the sample at 410 nm,

C - protein concentration in the sample, mg / ml, measured by the Lowry method,

120 - incubation time in minutes,

200 - sample volume, in µl,

1000 - recalculation in microns.

The study of the stability of free and immobilized ficin was carried out according to the following scheme: incubation of samples at 37°C with measurement of enzymatic activity after 1, 2, 4, 8, 24, 48, 72, 96, 120, 144, 168 hours. To measure the percentage of ficin desorption from the matrix of low molecular weight chitosan, 200 μl of 50 mM Tris-HCl buffer with pH 7.5 or saline was added to 50 mg of the sample, incubation was carried out at 37°C for 1, 2, 3, 4, 5, 24, 48 hours followed by measurement of the protein content in the solution by the Lowry method.

The antibacterial activity of the compounds was determined against a representative of the opportunistic flora: collection strain Escherichia coli (K-12 MKD910). The bacteria were grown on LB medium [Bowdish D.M.E., Davidson D.J., Hancock R.E.W. A revaluation of the role of host defense peptides in mammalian immunity // Curr. Protein Pept. sci. 2005. V.6. P. 35-51] (%): tryptone - 1.0; yeast extract - 0.5; NaCl - 0.5; pH 8.5. Agar medium LBA includes an additional 2% agar. Sterile glucose was added to the medium at the rate of 1 ml of the medium: 25 μl of glucose. A culture was added to a dish with enzymes (free, 1 mg/ml; immobilized, 75 mg/ml) and glucose. Then they were incubated in a thermostat at 37°C for a day. Culture growth was monitored by determining the change in optical density (OD600) of the culture. The increase in biomass was determined nephelometrically on a spectrophotocolorimeter at a wavelength of 600 nm, diluting the culture liquid, if necessary, so that the optical density was 0.1. The absorbance in a 1 cm cuvette equal to unity was taken as a unit of biomass. The amount of biomass was expressed in units of optical density.

Statistical processing of the obtained results was carried out at a significance level of 5% using Student's t-test.

During the experiment to determine the protein content, it was found that the largest amount of the enzyme (in mg per g of carrier) in the preparations of ficin immobilized on a matrix of low molecular weight chitosan is observed when the process is carried out in a phosphate buffer with pH 7.8 (Fig. 1).

High overall activity (in units per ml of solution) was shown by ficin preparations prepared using borate buffer pH 8.0 with 0.1 M KCl in the composition (Fig. 2).

The maximum specific activity (in units per mg of protein) of a heterogeneous biocatalyst based on ficin adsorbed on a chitosan matrix was recorded using a borate buffer with 0.1 M KCl in the composition with pH 8.0–8.5 (Fig. 3).

Thus, in terms of the ratio of such parameters as protein content, total and specific activity of the biocatalyst, the most promising buffer for ficin immobilization on low molecular weight chitosan is a borate buffer with 0.1 M KCl in the composition with pH 8.0.

One of the main requirements for immobilized enzymes is their resistance to various denaturing agents (temperature, pH, action of microorganisms, etc.) and the time of their action on the catalyst. Analysis of the catalytic activity of the native enzyme and heterogeneous enzyme preparation showed that soluble ficin is inactivated by 30% after 4 hours of incubation and retains no more than 50% of the initial activity after two days of incubation. Ficin adsorbed on the matrix of low molecular weight 50-190 kDa chitosan is highly active even after two days of incubation, retaining at least 46% of the initial catalytic capacity (Fig. 4).

Desorption of ficin from the sorbent phase in 50 mM Tris-HCl buffer with pH 7.5 after 48 hours of incubation did not exceed 60%. When the drug was placed in physiological saline, 40% desorption was detected after 3 hours of incubation; after incubation for 48 hours, desorption was 80% (Fig. 5).

Thus, immobilization of ficin on low molecular weight 50-190 kDa chitosan prevents its rapid inactivation and increases the half-life of the enzyme.

By the amount of biomass of Escherichia coli (K-12 MKD910) under various cultivation conditions, we determined the antibacterial activity of low molecular weight chitosan 50-190 kDa and ficin immobilized on low molecular weight chitosan 50-190 kDa, in comparison with the antibacterial activity of other types of chitosan and ficin immobilized on them. (Fig. 6). It has been established that the antibacterial activity of low molecular weight 50-190 kDa chitosan is about 2 times higher than that of medium molecular weight 200 kDa and high molecular weight 350 kDa chitosan. The antibacterial activity of ficin immobilized on low molecular weight 50–190 kDa chitosan is 2 and 2.5 times higher, respectively, than that of ficin immobilized on medium molecular weight 200 kDa and high molecular weight 350 kDa chitosan.

We have developed a method for obtaining a wound healing and regenerating heterogeneous drug for medical and pharmaceutical purposes based on ficin and low molecular weight 50-190 kDa chitosan, which has a pronounced antimicrobial and adsorbing effect on bacterial cultures, is stable during storage and use, which characterizes it as an effective means of combating with purulent complications of wounds caused by microorganisms.

Claims (1)

A method for producing a heterogeneous enzyme preparation based on ficin and low molecular weight chitosan, including immobilizing ficin on a carrier matrix in a buffer solution in a ratio of 20 ml of an enzyme buffer solution at a concentration of 1 mg/ml per 1 g of carrier, incubation for 24 hours at room temperature with occasional stirring and washing, characterized in that the immobilization of ficin is carried out on a matrix of low molecular weight chitosan with a molecular weight of 50-190 kDa; as a buffer solution for immobilization, 0.05 M borate buffer with 0.1 M KCl in the composition with pH 8.0 is used; the formed precipitate is washed with 0.05 M borate buffer solution with 0.1 M KCl in the composition with pH 8.0 until the absence of ficin in the washing water.

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