RU2637634C1 - Antibacterial protein sponge for chemotherapy of infected wounds and method of its production - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: antibacterial protein sponge for chemotherapy of infected wounds contains protein basis, including serum albumin or serum total protein or plasma total protein and medicinal agent or agents possessing antibiotic activity against pathogens causing purulent inflammations. It also provides a method of obtaining antibacterial protein sponge, including freezing solution containing serum albumin or serum total protein or plasma total protein, keeping in frozen state, defrosting, washing resulting protein sponge from not included in its composition components, mechanical spin of free liquid from washed protein sponge swelling of squeezed protein sponge in the solution of one or more agents with antibiotic activity, freezing swelled protein sponge and its drying by lyophilization.

EFFECT: new effective medical material - antibacterial protein sponge, designed for chemotherapy of infected wounds.

3 cl, 5 dwg, 1 tbl, 3 ex

Description

The invention relates to interdisciplinary technical solutions combining the chemistry of macromolecular compounds, medicine, biotechnology and the development of functional biomaterials based on natural biopolymers - proteins. More specifically, the invention relates to macroporous protein sponges having antibacterial activity and methods for their preparation. In particular, the claimed anti-bacterial sponge includes either a major plasma protein — serum albumin, or total serum protein, or total plasma protein [Peters T. All About Albumin: Biochemistry, Genetics, and Medical Application. // London: Academic Press; 1995. 432 p.], As well as one or more agents with antibiotic activity, manifesting their action against pathogenic microorganisms that cause purulent inflammation. The most effectively claimed material can be used for biomedical purposes, for example, as a means for the treatment of purulent-necrotic wounds.

In particular, various polymeric spongy materials are known, including sponges based on substances of protein nature, which are used in the treatment of wounds and burns [M.I. Shtilman. Introduction to polymer technology for biomedical applications. M.: From RKhTU, 2000. 247 p.]. These sponges have a common specific property - pronounced sorption ability with respect to fluid exudate secreted by a wound or burn [http://www.terra-medica.spb.ru/st1_2003/balin.htm]. Known examples of protein materials of this type are hemostatic collagen sponges [http://dic.academic.ru/dic.nsf/meditem/486]. As a rule, the absorption capacity of such coatings for wounds and burns is due to a system of capillaries that penetrate a polymer matrix that is well wetted by biological fluid. In this case, the soluble substances initially included in the composition of such sponges, for example hemostatic agents, are released into the liquid phase.

A number of serum albumin-based biomedical materials are known that are prepared in various ways, most often by gelation of solutions of this protein, for example, caused by heat [Gosal W.S., Ross-Murphy S.V. Globular protein gelation // Curr. Opinion Coll. Interface Sci. 2000. V. 5. No. 3-4. P. 188-194; Clark A.H., Tuffnell C.D. Small-angle X-ray-scattering studies of thermally-induced globular protein gels // Int. J. Peptide Protein Res. 1980. V. 16. No. 4. P. 339-351], exposure to high pressure [Galazka V.B., Dickinson E., Ledward D.A. Influence of high pressure processing on protein solutions and emulsions // Curr. Opinion Coll. Interface Sci. 2000. V. 5. No. 3-4. P. 182-187], protein denaturation with surfactants or emulsification of the aqueous phase in hydrophobic liquids [Autushenko S. S., Nikolaev B. P., Shlyakov A.M. Structuring of proteins in water-oil emulsions according to 1 H-NMR data // Coll. g. 1993.V. 55. No. 1. C.3-5], as well as covalent crosslinking of protein macromolecules by chemical cross-agents or irradiation [Hopwood D. A comparison of the crosslinking abilities of glutaraldehyde, formaldehyde and alpha-hydroxyadipaldehyde with bovine serum albumin and casein. // Histochemistry 1969. V. 17. No. 2. P. 151-161; Vermonden T., Censi R., Hennik W.E. Hydrogels for protein delivery // Chem. Revs. 2012. V. 112. No. 5. P. 2853-2888; Bychkova A.V., Rosenfeld M.A., Leonova V.B., Sorokina O.N., Lomakin S.M., Kovarsky A.L. Free radical crosslinking of serum albumin molecules on the surface of magnetite nanoparticles in an aqueous dispersion // Coll. g. 2013.V. 75. No. 1. S. 9-16]. It is proposed to use such protein materials as carriers for the controlled release of drugs, as well as biospecific sorbents, sorption matrices of extracorporeal devices for blood detoxification, etc. [Lundblad R.L. Biotechnology of Plasma Proteins. // CRC Press, Boca Raton e.a., 2013. P. 83-182].

The structure of this type of protein materials is characterized by gel microporosity, i.e. if any soluble agents exhibiting a medicinal effect are included in the volume of the gel matrix, then when such material is placed in excess fluid (in particular, blood, exudate, wound or burn medium), the release rate of such medicinal agents (for example, painkillers, antibiotics and etc.) to the external liquid will be low, determined by their slow diffusion in the gel mass and further from it. When a similar gel containing soluble substances in its volume is dried and only then placed in excess liquid, the intensity of drug release is even more reduced, because additional time is required for the slow swelling of the dry polymer. The indicated properties of such gel systems containing medicinal agents and called their “depot forms” are a disadvantage of microporous protein gels in the case of the use of such materials in biomedical systems.

For example, a group of protein materials based on serum albumin chemically crosslinked with pectin and containing copper ions as an antibacterial agent is known [R. Harris, W. Nasi. Gels for use in wound management // WO 2008015475]. This type of protein material having antibacterial activity is obtained from an aqueous solution of a mixture of copper salt containing a mixture of serum albumin and pectin by chemical crosslinking of these biopolymers with water-soluble carbodiimide. At certain ratios of these ingredients, hydrogels are formed, which are proposed to be used to treat various, including infected, wounds. This technical solution has several disadvantages. Thus, the obtained gel protein material has a microporous structure, which provides only a slow diffusion-limited release of an antibacterial agent, in this case, copper ions, or their transition to a soluble state during biodegradation of the protein base of the material under the action of the patient's proteinases. Therefore, to create high therapeutic concentrations of the antibacterial agent necessary in the case of severely infected wounds, it is required to use large volumes of gel material, which often technically complicates the surgical procedures (surgery, dressings, etc.), and is also expensive from an economic point of view. For gelation through chemical crosslinking of the biopolymers that make up the material, water-soluble carbodiimide (specifically, 1-ethyl-3- (dimethylaminopropyl) -carbodimide) is used, the unreacted part of which remains in the gel volume and is not removed by any washing. Therefore, when placing the formed gel in the wound, there is a danger of toxic effects of such carbodiimide on the patient's body due to undesirable chemical modification of the body's own proteins and peptides. Since this technical solution provides for obtaining the final protein material only in a wet form, the storage of such a product is low, and the cost of transporting the material increases sharply, because in its composition you have to transport just 80-90% of the water.

In turn, in cases where it is necessary to quickly provide a high therapeutic dose of a medicinal agent, large-porous materials are best suited, in which the main transport function is provided not by slow diffusion, but by convection of fluid through a system of interconnected pores of capillary size.

In particular, polymeric, including protein, materials obtained by the so-called cryostructuring methods, such as cryotropic gelation, lyophilization, cryoextraction, possess such a specific microstructure [Lozinsky V.I. Cryogels based on natural and synthetic polymers: production, properties and applications // Uspekhi Khimii. 2002.V. 71. No. 6. S.559-585; Lozinsky V.I., Okay O. Basic principles of cryotropic gelation // Adv. Polym. Sci. 2014. V. 263. R. 49-101]. A characteristic feature of all types of cryogels and cryostructures is their macroporosity, formed by polycrystals of a frozen solvent, after either melting, or sublimation, or cryoextraction of which communicating pores of capillary size remain in the sample mass [Lozinsky V.I. A new family of macroporous and supermacroporous materials for biotechnological purposes - polymer cryogels. // Bulletin of the Russian Academy of Sciences, Ser. Chem. 2008. No5. S.996-1013; Okay O., Lozinsky. V.I. Synthesis, structure-property relationships of cryogels. // Adv. Polym. Sci. 2014. V. 263. R. 103-157].

For example, in medical practice, a hemostatic native plasma sponge is used, obtained by lyophilization of blood plasma, the main protein component of which, as you know, is serum albumin [Lundblad R.L. Biotechnology of Plasma Proteins. // CRC Press, Boca Raton e.a., 2013. P. 83-182]. Such a sponge has a pronounced hemostatic effect, but is very fragile and also quickly dissolves in the biological fluid of the wound [http://znaiu.ru/art/4002390600.php], which is a functional disadvantage of this and similar materials.

Known macroporous albumin lyophilized composition having a sponge texture and having activity to induce an immune response to flavivirus. [D.K. Wellom, D.E. Voisvillo, P. Dedgeorge, P. Charametaro. Lyophilized composition for inducing an immune response to flavivirus, composition and method for its preparation // Pat. RF No 2541784 (2007); B.I. No. 5 (2015)]. This protein sponge is obtained from a composition comprising certain concentrations of live attenuated flavivirus (a biologically active ingredient of the composition), a stabilizer (as such, serum albumin is used in this known invention), components of a buffer solution, lactose and amorphous mannitol. The corresponding liquid composition is frozen in a multi-stage mode and freeze-dried, while drying also stepwise changing the parameters (vacuum and temperature) of the process. The result is a macroporous protein product, which is an albumin sponge containing a medicinal agent (in this case, attenuated viral particles), which has the target biological activity - the ability to induce the body's immune response to infection with flaviviruses. The main advantages of such a protein sponge are transport stability and good storage of a biologically significant component.

This technical solution, as the closest to the claimed invention in relation to the type of protein base of the sponge, i.e. serum albumin and the content of a medicinal agent in it, as well as a production method involving the lyophilization of the original liquid composition, is taken as a prototype.

However, such a biologically active protein sponge and its production method have several disadvantages:

1. One of the main disadvantages is the fast solubility of the albumin sponge in physiological environments (this does not allow to prolong the manifestation of biological activity).

2. The complex composition of the frozen composition with special requirements for the type of one of the components — the use of exclusively amorphous mannitol significantly increases the cost of the final product.

3. The multi-stage freeze-lyophilization regime of the initial composition greatly complicates the process as a whole and reduces its manufacturability.

4. In addition, the composition of this spongy protein material does not provide for the use of any agents with antibacterial activity, which increases the risk of microbial contamination of both the material itself and the surgical field, where it is introduced during surgery into the body.

In this regard, the objectives of the invention are both the creation of a new effective material for medical use - an antibacterial protein sponge intended for chemotherapy of infected wounds, and the development of a more technologically advanced method for producing this material compared to analogs and a prototype.

These problems are solved by the fact that the inventive antibacterial protein sponge for chemotherapy of infected wounds contains one or more medicinal agents, the protein base of the sponge or serum albumin, or total blood serum protein, or total blood plasma protein, and the drug agent or agents have antibiotic activity against pathogenic microorganisms causative agents of purulent inflammation. The inventive antibacterial protein sponge is formed by freezing a solution containing either serum albumin, or a total blood serum protein, or a total blood plasma protein, keeping it frozen, then thawing, washing the formed protein sponge from components not included in it, followed by mechanical extraction of free liquid from the washed protein sponge, swelling of the washed and squeezed protein sponge in a solution of one or more agents with antibiotic activity, then freezing a swollen protein sponge and drying it by lyophilization. While freezing a solution containing 3-6 wt. % or serum albumin, or total serum protein, or total plasma protein, 6-18 wt. % urea and 0.1-0.2 wt. % cysteine is carried out at -10 ... -30 ° C for 6-36 h, and the swelling of the washed and squeezed protein sponge in a solution of one or more agents with antibiotic activity and selected from the group consisting of vancomycin, lincomycin, clarithromycin, torbamycin, gentamicin or mixtures thereof, at a concentration of such agents equal to or higher than 2 wt. %, carried out at 5-35 ° C for 3-24 hours

The antibacterial protein sponge obtained by this method can be made of any geometric shape: in the form of blocks, plates, disks, granules, irregularly shaped particles (obtained by grinding the block), tubes, etc., and one or more substances with antibiotic activity included in its composition , are not limited to the above options, and their nomenclature can be easily expanded depending on the type of infection, the specific type of such an antibacterial sponge is intended to suppress.

The specific parameters of the proposed technical solution are determined by the following factors:

1) The composition of the claimed antibacterial protein sponge and solutions used in its preparation.

The sponge protein base consists of either serum albumin, or total serum protein, or total blood plasma protein, which are non-toxic absolutely biocompatible components that do not need to be removed from the wound, can be left where they are subsequently gradually absorbed by proteolytic enzymes the patient’s body itself.

The medicinal substances that make up such a sponge, which have antibiotic activity against pathogenic microorganisms that cause purulent inflammations, belong to different groups of antibiotics that are allowed by domestic and international Pharmacopoeias for use in medical practice.

The concentration of albumin or the sum of plasma and serum proteins in the initial solution was found experimentally and is in the range of 3-6 wt. % With less than 3 wt. % of the resulting protein sponge has low strength, poorly retains its shape and integrity during manipulations in physiological environments. If the concentration of protein in the initial solution exceeds 6 wt. %, the resulting protein sponge, on the contrary, is too hard and brittle in a dry form, and poorly absorbs liquid in the aquatic environment due to insufficient porosity.

In addition to protein components, urea and cysteine additives in the amount of 6-18 wt. % and 0.1-0.2 wt. % respectively. These substances are necessary for cryotropic gelation of proteins with the formation of intermolecular cystine bridges, which ensures the integrity of the sponge in aqueous media and its rather slow biodegradation when located in the wound space. The indicated concentration ranges of these substances were found experimentally: when their content in the initial solution is less than the lower limits, the protein sponge does not maintain its integrity after thawing the frozen system, and if the upper limits are exceeded, a viscous turbid colloidal solution is obtained.

Medicinal substances included in the inventive antibacterial protein sponge having antibiotic activity against pathogenic microorganisms causing purulent inflammation are introduced into the protein base after its formation, washing and squeezing from free fluid, namely at the stage of sponge swelling in a solution of antibiotics or a mixture of antibiotics. Their concentration in such a solution was also found experimentally and is determined, firstly, by the solubility of a particular substance or mixture of substances and, secondly, by the amount of antibiotic or mixture of antibiotics that must be introduced into the final product. By varying the concentration of such a solution, it is easy to regulate the drug content in the resulting antibacterial protein sponge. According to the proposed technical solution, the concentration of the antibiotic in the swelling solution is 2 wt. % and, when necessary, higher. At a lower concentration than the lower value, the amount of antibiotic in the sponge is insufficient to achieve a positive therapeutic effect. The upper limit of concentration in a solution for swelling a spongy protein base depends on the solubility of a particular antibiotic or a mixture thereof.

2) Modes for implementing the proposed technical solution

The process of obtaining the inventive antibacterial protein sponge includes the following stages:

- preparation of the initial solution;

- its freezing, keeping in a frozen state for a certain time, followed by thawing;

- washing the formed protein sponge from components not included in its composition;

- mechanical extraction of free fluid from the washed sponge;

- swelling of pressed material in a solution of one or more substances with antibiotic activity;

- freezing a swollen sponge and drying it by lyophilization.

This process flow chart provides the production of the inventive material, while it is preferable to carry out these stages in sterile conditions.

The modes of freezing the initial solutions and keeping them in the frozen state were determined experimentally. The inventive method for producing an antibacterial protein sponge provides for the implementation of these processes at -10 ... -30 ° C for 6-36 hours. The upper limit of the claimed temperature range is due to the fact that at higher subzero temperatures due to supercooling effects the compositions of the claimed composition often do not freeze , and the use of temperatures below -30 ° C is impractical, because in this case, the efficiency of cryotropic gelation in these systems sharply decreases, which is manifested in a decrease in the yield of cross-linked protein cryogel and in the deterioration of its spongy texture.

The washing of the obtained protein sponge from protein components not included in its composition, as well as from the addition of urea and cysteine, is carried out by known methods, preferably using sterile water. The free liquid is squeezed out of the washed sponge under loads not leading to crushing and destruction of the soft spongy protein material.

The inventive conditions for the swelling of the washed and squeezed from the free liquid of the protein sponge in a solution of one or a mixture of several antibiotics are due to the needs of a particular process. If it is desirable to carry out the process quickly, but not achieve equilibrium of swelling and maximum saturation of the sponge with medicine, swelling can be carried out with moderate heating (i.e. at 30-35 ° C) for 3-5 hours. If the maximum possible saturation of the sponge with antibiotic is necessary , then a longer swelling period (up to 24 hours) and its holding at a low (10 ... 5 ° C) positive temperature are preferable.

The freezing of a protein sponge swollen in an antibiotic solution and its freeze drying is carried out in known modes; the main condition of this stage of the proposed method is the strict prevention of thawing of the samples and the preservation of their sterility when the vacuum is released after drying.

The inventive antibacterial protein sponge intended for chemotherapy of infected wounds; the method of obtaining such material, as well as the claimed combination of features, were not previously known, that is, the proposed technical solution meets the criterion of "novelty."

Typical examples of the implementation of the claimed technical solution are described below, which illustrate but do not limit its scope, the remaining examples are summarized in the table, and the figures below contain the following information:

FIG. 1. Results of determining the in vitro activity (disco-diffusion method) of antibacterial protein sponges obtained in Example 1 (a-c) from bovine serum albumin (BSA) and loaded with vancomycin (B), gentamicin (G) and clarithromycin (K); test culture - Staphylococcus aureus MRSA.

FIG. 2. In-vivo biotesting of antibacterial protein sponges obtained in example 1 (a-b) from bovine serum albumin (BSA) and loaded with gentamicin and vancomycin:

a) Fixation of the laboratory animal on the stage and removal of the hairline.

b) Preparation of the site for the introduction of the Teflon ring.

c) The appearance of the surgical field after implantation of a teflon ring.

d) Wound infection by a pathogenic culture of a methicellin-resistant strain of Staphylococcus aureus MRSA.

e) Isolation of the wound from the external environment using a cotton-gauze swab.

f) Appearance of the wound after 5 days and sampling for microbiological analysis.

g) Insertion of an BSA sponge loaded with gentamicin into the infected wound.

h) Insertion of an BSA sponge loaded with vancomycin into the infected wound.

i) The appearance of the surgical field with a BSA sponge loaded with gentamicin, 10 days after the start of treatment.

j) Sampling for microbiological analysis from the surgical field with a BSA sponge loaded with gentamicin 10 days after the start of treatment.

k) The appearance of the surgical field with a BSA sponge loaded with vancomycin, 10 days after the start of treatment.

m) Sampling for microbiological analysis from the surgical field with a BSA sponge loaded with vancomycin 10 days after the start of treatment.

FIG. 3. The results of determining the in vitro activity (disco-diffusion method) of antibacterial protein sponges obtained in Example 2 (b-d) from total sheep serum protein (CCO) and loaded with vancomycin (B), gentamicin (G) and clarithromycin (K ); test culture - Escherichia coli sp.

FIG. 4. Results of determining the in vitro activity (disco-diffusion method) of antibacterial protein sponges obtained in Example 3 (c-d) from total human blood plasma protein (HSP) and loaded with vancomycin (B), gentamicin (G) and clarithromycin (K ); test culture - Pseusomonas aeruginosa Escherichia coli sp.

FIG. 5. In-vivo biotesting of antibacterial protein sponges obtained in Example 3 (c-d) from total human blood plasma protein (PCP) and loaded with gentamicin and vancomycin:

a) Fixation of a laboratory animal on a stage and removal of a section of hair on the hind limb.

b) Appearance of a longitudinal incision fixed by a retractor after dissection of the muscle fascia.

c) Wound infection by a pathogenic culture of a methicellin-resistant strain of Staphylococcus aureus MRSA.

g) The appearance of the wound 3 days after infection.

d) The introduction into the infected wound of the PKC-sponge loaded with gentamicin.

f) Insertion of a vancomycin-loaded sponge into the infected wound.

g) Appearance of a sutured wound with an implanted PCC sponge loaded with gentamicin.

h) Appearance of a sutured wound with an implanted PCC sponge loaded with vancomycin.

i) Appearance of the scar (without signs of inflammation) in the area of surgical intervention 12 days after implantation in the wound of the RCC sponge loaded with gentamicin.

j) The appearance of the scar (without signs of inflammation) in the area of surgical intervention 12 days after implantation in the wound of the RCC sponge loaded with vancomycin.

k) Scap decapitation for microbiological control of a model of a purulent wound treated with an implanted PKC-sponge loaded with gentamicin.

m) Scap decapitation for microbiological control of a model of a purulent wound treated with an implanted RCC sponge loaded with vancomycin.

m) Sampling for microbiological analysis from a decapitated scar after treatment of a model of a purulent wound using an implanted PKC-sponge loaded with gentamicin.

o) Sampling for microbiological analysis from a decapitated scar after treatment of a purulent wound model using an implanted RCC sponge loaded with vancomycin.

Example 1. An antibacterial protein sponge formed according to the protocol No. 1 (a-b) of the table of examples.

A) Obtaining a spongy protein base

An aqueous solution containing 4 wt. % bovine serum albumin (BSA), 12 wt. % urea and 0.1 wt. % cysteine is poured with a layer 0.5 cm high into flat-bottomed glass bottles with an inner diameter of 1 cm, which are placed in the chamber of an F-32 ultra-cryostat (Julabo, Germany), where the contents of the bottles are frozen at -20 ° C for 18 hours, and then thawed room temperature. The spongy albuminous disks formed in this way are washed from soluble components with sterile water.

B) Saturation of a protein sponge with a drug substance that has antibiotic activity against pathogenic microorganisms that cause purulent inflammation

Each water-swollen disk is placed on a glass filter and the unbound liquid is squeezed out under a vacuum of a water-jet pump for 1 min. Then the disks are placed in either an aqueous solution (4 wt.%) Of vancomycin (Elli Lilly, USA), or gentamicin (Borisov factory of medicinal products, Belarus), or clarithromycin (Abbott, France), where they are incubated for 6 h at 10 ° C with periodic by stirring. Spongy disks swollen in an antibiotic solution are transferred to a Petri dish, frozen by known methods, and freeze-dried using an ALPHA 1-2 LD plus freeze-dryer (Martin Christ, Germany). The resulting dry preparations are stored in an airtight container at -20 ° C.

C) In vitro bioassay of an antibacterial protein sponge

To assess the antimicrobial activity of samples of albumin sponges obtained according to Project 1a-c, a test culture of a methicellin-resistant microorganism belonging to the species: Staphylococcus aureus was used. A microbial suspension was prepared on physiological saline in a concentration corresponding to a turbidity standard of 0.5 Mack Farland, and then applied to the surface of Muller-Hinton agar in Petri dishes. Evenly distributed and dried identical to the method for determining the antibiotic resistance of microorganisms by the disk-diffuse method. The studied samples of albumin sponges (diameter 10 mm, thickness 8 mm) loaded with antibiotics were applied to the surface of the agar. The cups were incubated in a thermostat at a temperature of 35 ° C for 18-24 hours. The results were taken into account by measuring (in mm) the growth inhibition zone of the test culture of the microorganism around the test sample. It was found (Fig. 1) that around the albumin sponge with vancomycin, cell growth retardation was St. aureus MRSA was 27 mm, sponges with gentamicin - 24 mm; sponges with clarithromycin - 0 mm (this was due to the resistance of the culture to this antibiotic).

D) In vivo bioassay of the antibacterial protein sponge

Experimental studies were performed on non-linear male rats of the Wistar breed, weighing 250 ± 10 g, in which a purulent wound was simulated to ascertain and objectively evaluate the wound process.

Under intramuscular calypsol anesthesia, the animal was fixed on a stage, hair was removed on a 3 × 3 cm site (Figs. 2a and 2b), the skin and subcutaneous fatty tissue 2 cm in diameter (314 mm ² ) were excised, and a teflon ring was sutured to the site of the skin defect. with a side 1 cm of the same diameter (Fig. 2B). After that, the fascia and the underlying muscle tissue, inside the cap, were crushed by a kocher. About 1 ml of a solution with pathogenic flora of methicellin-resistant Staphylococcus aureus (Staphylococcus aureus MRSA) in the amount of 10 ⁷ cells (Fig. 2d) was injected into the cavity of the cap with a syringe, after which the ring was isolated from the external environment (Fig. 2e). On the 5th day after the simulation of a purulent wound, all animals were lethargic, inhibited, with reduced aggression on external stimuli, appetite was reduced. Locally observed perifocal edema, hyperemia of the underlying skin along the edge of the wound. In the wound area pronounced purulent discharge, fibrinous plaque with areas of necrosis (Fig. 2E). Microbiological examination of biopsy samples from the wound site revealed that the degree of dissemination of Staphylococcus aureus MRSA per gram of tissue was 10 ⁹ cells and corresponded to a pronounced purulent process. After that, in animals after treatment of the wound surface with physiological saline, local treatment of purulent wounds was started with albumin sponges loaded with gentamicin (Fig. 2g) and vancomycin (Fig. 2h).

During dynamic observation of animals, on the 4th day from the start of treatment, it was revealed that the animals retained contour infiltration around the wounds, moderate hyperemia, slight edema, slight purulent discharge, there were areas of necrosis along the wound. But all these symptoms were less pronounced compared with the period before applying the sponge. On the 4th, 10th and 15th day from the start of treatment, wound planimetry was performed. At the same time, biopsy material was taken for microbiological examination. The average time for cleansing wounds from purulent and necrotic masses using trypsin solution in the first phase of traumatic inflammation (control group) was 14 ± 0.5 days, and the time for complete healing was 17 ± 0.3 days. In the group of animals treated with albumin sponges with gentamicin, the wounds were cleaned at 6 ± 0.3 days, and healing took place at 13 ± 0.4 days. By 11 ± 0.6 days in the group of animals, where albumin sponges with vancomycin were used for treatment, the wounds were completely cleansed of purulent-necrotic masses. On 5 ± 0.4 days, in the animals of this group, against the background of wound cleansing, the appearance of fine-grained granulation tissue took place. Animals during these periods were more active, in response to aggression, most rats vigorously resisted, food consumption increased. By 4.5 ± 0.5 days of observation, hyperemia around the wounds was not observed, the skin was easily collected, i.e. a decrease in diameter was noted. On the 10th day, marginal epithelization began to be noted, while a significant decrease in the size of the sponges was detected by almost 70% with both gentamicin (Fig. 2i and 2k) and vancomycin (Fig. 2l and 2m), i.e. their gradual resorption. In the study of microbiological material, the growth of pathological microflora was not detected. Thus, the effectiveness of the use of antibiotic-loaded albumin sponges for chemotherapy of infected wounds has been demonstrated.

Example 2. An antibacterial protein sponge formed according to protocol No. 2 (b-d) of the table of examples.

A) Obtaining a spongy protein base

Freshly obtained sheep blood serum with a total protein content of 7.6 wt. % diluted with sterile water so that the protein concentration was 4.5 wt. % Urea and cysteine are added to the resulting solution in an amount corresponding to their concentration in a mixture of 14 and 0.18 wt. % respectively. Such a liquid system is dispensed in portions of 0.4 ml into 10 mm wells of a plastic tablet, which are placed in an RL 48RRCSW freezer (Samsung, South Korea), where the contents of the wells are frozen at -18 ° C for 24 hours and then thawed at room temperature . The spongy protein disks thus formed are washed from soluble components with sterile water.

B) Saturation of a protein sponge with a drug substance that has antibiotic activity against pathogenic microorganisms that cause purulent inflammation

The washed protein disks are squeezed from unbound water on filter paper and placed either in an aqueous solution (3.5 wt.%) Of vancomycin (Teva, Israel), or gentamicin (Borisov factory of medications, Belarus), or clarithromycin (Abbott, France), where they are incubated 24 hours at 5 ° C with occasional stirring. Spongy disks swollen in an antibiotic solution are transferred to a Petri dish, frozen by known methods, and freeze-dried using an ALPHA 1-2 LD plus freeze-dryer (Martin Christ, Germany). The resulting dry preparations are stored in an airtight container at -20 ° C.

C) In vitro biotesting of an antibacterial protein sponge. To evaluate the antimicrobial activity of protein sponge samples obtained according to Project 2b-d, a test culture of a microorganism belonging to the species Escherichia coli sp. Further manipulations are carried out similarly to the methods described in paragraph "B" of example 1. It was found (Fig. 3) that around the protein sponge with gentamicin cell growth retardation of Escherichia coli sp. amounted to 36 mm, and sponges with vancomycin and clarithromycin showed a minimal effect, this is due to the high resistance of the intestinal microflora to these antibiotics. Thus, the effectiveness of protein sponges prepared from total serum protein and loaded with an antibiotic active against intestinal microorganisms is shown when using such depot forms as antibacterial drugs.

Example 3. An antibacterial protein sponge formed according to the protocol No. 3c-d of the table of examples.

A) Obtaining a spongy protein base

Quick-frozen human blood plasma with a total protein content of 7.8 wt. % thawed at 30 ° C and diluted with sterile water so that the total protein concentration was 4 wt. % Urea and cysteine are added to the resulting solution in an amount corresponding to their concentration in a mixture of 13 and 0.13 wt. % respectively. Such a liquid system is dosed in 0.15 ml portions into conical plastic bottles, which are sealed and placed in the chamber of a Proline RP 1840 ultra-cryostat (Lauda, Germany), where the contents of the bottles are frozen at -17.5 ° С for 12 h, and then thawed at 4 ° С. Thus formed protein sponges, which are in the form of a cone, are washed from soluble components with sterile water.

B) Saturation of a protein sponge with a drug substance that has antibiotic activity against pathogenic microorganisms that cause purulent inflammation

The washed protein sponges are squeezed from unbound water on filter paper and placed either in an aqueous solution of vancomycin (Teva, Israel) (5 wt.%), Or gentamicin (Borisov Plant of Medications, Belarus) (4 wt.%), Or clarithromycin (Abbott , France) (6 wt.%), Where they are incubated for 18 hours at 18 ° C with periodic stirring. Protein sponges swollen in an antibiotic solution are labeled on a Petri dish, frozen by known methods and freeze-dried using a Freezone 1L freeze-drying machine (Labconco, USA). The resulting dry preparations are stored in an airtight container at -20 ° C.

C) In vitro bioassay of an antibacterial protein sponge

To assess the antimicrobial activity of protein sponge samples obtained according to Project 3 (c-d), a Pseusomonas aeruginosa Pseudomonas aeruginosa test culture is used. Further manipulations are carried out similarly to the methods described in paragraph “B” of Example 1. It was found (Fig. 4) that growth retardation of Ps cells around a protein sponge with gentamicin. aeruginosa was 44 mm, sponges with clarithromycin - 23 mm. The preparation of a protein sponge loaded with vancomycin did not affect the test culture, which is due to the high resistance of Pseudomonas aeruginosa to this antibiotic. Thus, the effectiveness of protein sponges prepared from total plasma protein and loaded with antibiotics active against putrefactive microorganisms is shown when using such depot forms as antibacterial drugs.

D) In vivo bioassay of the antibacterial protein sponge

Experimental studies were performed on non-linear male rats of the Wistar breed, weighing 250 ± 10 g, in which a purulent wound was simulated to ascertain and objectively evaluate the wound process.

Under intramuscular calypsol anesthesia, the animal was fixed on a stage and the hairline on the hind limb (thigh) was removed (Fig. 5a). Then, with a scalpel, a longitudinal skin incision of 1 cm was performed, the muscle fascia was dissected, and the muscles were accessed. The open wound was fixed with a retractor. (Fig. 5b). About 1 ml of a solution of pathogenic microflora of meticelin-resistant Staphylococcus aureus St. Was injected into the wound cavity with a syringe. aureus MRSA in the amount of 10 ⁷ cells (Fig. 5B) and the infected limb was ligated. After 72 hours, the dressing was removed and a sample was taken for microbiological examination, which showed the degree of contamination with the pathogenic microflora St. aureus MRSA at 10 ⁹ cells per gram of tissue, which corresponded to a pronounced purulent process (Fig. 5g). The wound cavity was washed with 20 ml of physiological saline, after which protein sponges were implanted with gentamicin (Fig. 5e) and vancomycin (Fig. 5e) into the wound cavity. Next, the wound was sutured in layers with surgical suture material polyglycolide-co-lactide, braided purple, USP 1 (4metric) (Figs. 5g and 5h).

During dynamic observation of animals from the next day from the start of treatment, it was found that the animals were active, from 2-3 days almost all the animals vigorously resisted in response to aggression, food consumption increased. By 5 ± 0.5 days of observation, hyperemia around the wounds was not observed; after a week, signs of inflammation decreased. After 7 days, the sutures were removed, the surface of the skin a day after removing the sutures became smooth. After 12 days, all animals fully recovered, a picture of complete healing was observed, the scars in the area of surgical intervention were without signs of inflammation (Fig. 5i and 5k). Further, all animals were withdrawn from the experiment, decapitation was performed under ether anesthesia (Figs. 5l and 5m) for microbiological control of the treated model of a purulent wound (Figs. 5n and 5o). After 14 days during microbiological examination of the material, the growth of pathological microflora was not detected, and the sponge itself underwent biodegradation by almost 95-100%.

Observations of the clinic of experimental purulent wounds showed that the protein sponges used in the experiments, loaded with gentamicin and vancomycin, as well as other antibiotics or their mixtures (table of examples), have greater clinical efficacy. Thus, the applicability of such antibacterial protein sponges loaded with antibiotics for chemotherapy of infected wounds has been demonstrated.

The claimed technical solution has the following advantages over analogues and prototype:

1. Compared with analogs of antibacterial depots based on finely porous protein gels, the inventive broad-porous antibacterial sponges loaded with antibiotics can quickly achieve high drug concentrations in an infected wound, which creates conditions for the intensive destruction of purulent microflora in it and promotes effective wound cleansing. This is ensured by the specific structure of the inventive sponges with a large number of interconnecting macropores of capillary size, which allows one to achieve high therapeutic concentrations of the antibacterial agent necessary in the case of severely infected wounds, and it does not require the use of large (as in the case of analogues) volumes of material, and this is essential simplifies ongoing surgical procedures (surgery, dressings, etc.), and also reduces treatment costs.

2. In contrast to the known analogues, where water-soluble carbodiimide is used for gelation through chemical crosslinking of the biopolymers included in the material, the unreacted part of which remains in the gel volume and is not removed from it, that is, there is a risk of toxic effects of such carbodiimide on the patient’s body due to undesirable chemical modification of their own proteins and peptides of the body, in the present method, only non-toxic, actually biogenic, ingredients (protein, urea, cysteine) are used, the soluble part of which is removed by washing the sponge after its formation. Thus, the risk of introducing toxic substances with the material of the sponge is excluded, and its subsequent loading with antibiotics permitted for biomedical use is determined only by the permissible concentrations of these substances.

3. In contrast to the well-known analogue, providing for the receipt of the final protein material only in the wet form, the claimed technical solution provides the preparation of a dry form of an antibacterial protein sponge, which dramatically improves the storage performance of the final product and reduces the cost of its transportation.

4. In contrast to medicinal components of rapidly dissolving albumin sponges based on simply lyophilized-dried albumin as in the prototype, the claimed technical solution allows to obtain protein sponges not only from isolated serum albumin, but also from more affordable blood serum and whole blood plasma, as well as provides slow absorption of the protein sponge in vivo, i.e. has a prolonged effect.

5. In the claimed technical solution does not use any special forms of precursors such as exclusively amorphous mannitol, as in the prototype method, which simplifies the process of obtaining the target product.

6. The presence in the inventive protein sponges of high concentrations of substances with antibacterial activity, in particular antibiotics, provides reliable protection of drugs against microbial infection.

7. The advantages of the claimed antibacterial protein sponges also include: a) simplicity and ease of use for doctors and patients; b) the effectiveness of the application, safety and non-invasiveness of changing dressings; c) the ability of such drugs to destroy the bacterial biofilm both on implants and on biological tissues; d) the ability to provide a high concentration of antibiotic in areas with low perfusion of blood in tissues; e) due to the high concentration of aminoglycosides and cyclic glycopeptides (for example, gentamicin, vancomycin or clarithromycin), do not allow resistant bacterial strains to develop in wounds; e) the local hemostatic effect observed in the experiments; e) since the claimed protein sponges are biodegradable materials, their use does not require further manipulations to remove the sponges from the scar of the wound that has already been treated.

8. The inventive antibacterial protein sponges exhibit a durant effect due to the slow release of active components (antibiotics) as the protein base biodegrades. Possessing sorption, necrolytic, antibacterial, stimulating properties, the preparations contribute to a multicomponent directed action on the processes of reparative regeneration of purulent wounds, which makes it possible to recommend such antibacterial protein sponges for clinical use.

Technical result

The new biocompatible, non-toxic, absorbable in the wound over time and effectively cleans it of pathogenic microorganisms antibacterial protein sponge, as well as a convenient and economical way to obtain such material.

The most effectively claimed technical solution and the antibacterial protein sponges obtained according to it can be used in the following areas

practical medicine: a) in purulent surgery (treatment of abscesses and phlegmon); b) traumatology-orthopedics (prevention and treatment of post-traumatic and hematogenous osteomyelitis - to fill the cavities after trepanation and resection sequestronecrectomy of the affected bones, infectious damage to the soft tissues, paraprosthetic infections of large and small joints); c) in disaster medicine and combustology; d) in general surgery (mastectomy, in the treatment of diabetic feet, trophic ulcers, with hepatectomy and pancreotomy for the prevention and treatment of postoperative infectious complications; when performing operations to remove part of the gastrointestinal tract, hepatectomy, pancreotomy and appendectomy); e) in cardiac surgery (prevention and treatment of sternal osteomyelitis and mediastinitis, as well as various infectious complications); e) in otolaryngology and dentistry.

* BSA - bovine serum albumin; CCA - pig serum albumin; HSA - human serum albumin; SCS - total serum protein of cattle; SKO - total serum protein of sheep; SKH - total human serum protein; PCT - total protein of the blood plasma of the calf; PKL - total horse protein; PCC is a common plasma protein of human blood.

** Data on the determination of in vitro activity (disco-diffusion method) of antibacterial protein sponges (test culture / diameter of the region of growth inhibition of cells).

*** The healing time of the model of external purulent wound (MNGR) or the model of cavity purulent wound (MPHR) infected with the Staphylococcus aureus Staphylococcus aureus strain is indicated. aureus MRSA.

**** No experiments have been conducted.

Claims (3)

1. An antibacterial protein sponge for chemotherapy of infected wounds containing one or more medicinal agents, characterized in that the protein base of the sponge includes either serum albumin, or total blood serum protein, or total blood plasma protein, and the drug agent or agents have antibiotic activity in relation to pathogenic microorganisms causative agents of purulent inflammation. 2. A method of obtaining an antibacterial protein sponge according to claim 1, including freezing a solution containing either serum albumin, or a total blood serum protein, or a total blood plasma protein, holding in a frozen state, thawing, washing the formed protein sponge from those not included in it the composition of the components, mechanical extraction of free liquid from the washed protein sponge, swelling of the pressed protein sponge in a solution of one or more agents with antibiotic activity, freezing of the swollen protein howling sponge and its drying by lyophilization. 3. The method according to p. 2, characterized in that the freezing of a solution containing 3-6 wt. % or serum albumin, or total serum protein, or total plasma protein, 6-18 wt. % urea and 0.1-0.2 wt. % cysteine is carried out at -10 ... -30 ° C, followed by keeping it frozen for 6-36 hours, and the swollen washed and squeezed protein sponge in a solution of one or more agents with antibiotic activity and selected from the group comprising vancomycin, lincomycin, clarithromycin, torbamycin, gentamicin or a mixture thereof, at a concentration of such agents equal to or higher than 2 wt. %, carried out at 5-35 ° C for 3-24 hours

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