BG112942A - Polyzwitterion hydrogels for dressings for chronic and necrotic wound treatment - Google Patents

Abstract

The invention relates to polyzwitterion hydrogels for dressings for treatment of chronic and necrotic wounds, including heavily exuding wounds. The developed dressings from polyzwitterion hydrogel or two-layer ones with a top layer of chitosan and an inner layer from polyzwitterion hydrogel simultaneously (a) have ultra low bioadhesion and antibiofilm-forming properties (b) show the so-called anti-poly electric effect - they swell more in saline than when in water, including wound exudate, (c) show antibacterial properties, and (d) have proteolytic activity due to an enzyme with proteolytic action immobilized therein to decompose the necrotic tissue and find application in pharmacy and medicine.

Description

POLYCVITERIONAL HYDROGELS FOR BANDAGES FOR THE TREATMENT OF CHRONIC AND NECROTIC WOUNDS

Field of technology

The invention relates to polyviterione hydrogels for dressings for the treatment of chronic and necrotic wounds and finds application in pharmacy and medicine.

BACKGROUND OF THE INVENTION

Skin tissue loss from burns, injuries, diabetes and more. leads to the formation of wounds. Its recovery is the result of complex processes, involving cells, extracellular matrix and soluble mediators (growth factors, etc.). At the same time, moisture, heat and the nutrient medium, together with the reduced functioning of the immune system in the wound, favor the formation of necrotic tissue, which is an ideal environment for bacterial growth.

There are various wound dressings designed to protect the wound from bacterial infections and other adverse effects, or to help regenerate injured tissue. Traditional gauze bandages protect the wound to some extent from bacteria, but this protection is lost when wet. When the wound dries, they stick to it, which causes pain and discomfort when changing dressings and disrupts the natural healing process. Wound dressings have been developed, consisting of an upper layer that prevents the penetration of bacteria and a lower layer that absorbs the exudate. However, dressings that absorb the exudate from the wound tend to grow bacteria inside them, which subsequently form a biofilm. This necessitates the development of wound dressings, which have a local antimicrobial effect, most often achieved by the release of germicidal compounds. Such dressings are, for example, those containing silver. However, their effectiveness has been questioned, as topically applied silver may delay wound healing due to its toxic effect on regenerating keratinocytes [Punjataewakupt A., Napavichayanun S., Aramwit R., The downside of antimicrobial agents for wound healing. European Journal of Clinical Microbiology & Infectious Diseases, 2019, 38 (1), 39-54; Szmyd R., Goralczyk AG, Skalniak L, Cierniak A., Lipert B., Filon FL, CroseraM., Borowczyk J., Laczna E., Drukala J., Klein A., Jura J. Effect of silver nanoparticles on human primary keratinocytes. Biol Chern 2013, 394 (1): 113-123]. However, when bacteria form a biofilm, the use of antibiotic dressings is required, and it is known that in order to kill bacteria in a biofilm, antibiotic concentrations thousands of times higher are required than for free-moving bacteria. At the same time, the problem of antibiotic resistance to a large number of bacterial strains is well known, which further reduces the chances of fighting a bacterial infection. These disadvantages are overcome by the present invention, because the polyviterione hydrogels, due to their hydrophilicity, absorb the exudate of the wound, and due to their low bioadhesiveness, do not create conditions for (do not allow) the formation of a biofilm, ie. the development of bacteria. This does not require the inclusion of germicidal compounds, thus completely avoiding the side effects of their application. Moreover, it does not reach the stage of including antibiotic treatment due to the fact that polyviterione hydrogels do not allow the formation of bacterial biofilms, ie. have antibiofilm-forming activity. In addition, the polyviterion hydrogel maintains the necessary moisture for normal tissue healing, does not stick to it, which would allow easy and patient-friendly dressing change.

Normal wound healing occurs when it is well cleared of necrotic tissue. Therefore, wound preparations containing proteolytic enzymes that break down only necrotic tissue without affecting healthy cells have been developed. Such are the products in the form of wires and ointments created by a team of Prof. Nedkov and currently available on the market [I. Topalov, G. Fichev, R. Nedkov „Treatment of purulent, necrotic and atonic wounds with necroiytin Comptes rendus de l'Academie bulgare des Sciences, 54 127-130 (2000)]. These products can be easily washed off, especially when applied to copiously exuding wounds, or unwanted mechanically removed, which requires special attention on the part of the patient when applying them and creates additional inconvenience while reducing their healing effect. Unlike lubricants, the crosslinked polyviterion hydrogel is a non-flowing and elastic material that can be the basis for a mechanically stable dressing. Such a dressing does not leak, does not wash off, does not break easily when moving and does not require special care on the part of the patient when it needs to be applied.

Polyviterions (PCs) are polymers that are characterized by ultra-low protein adsorption. PCs contain monomer units with covalently linked positively and negatively loaded groups. Depending on the type of functional groups carrying the charge, they are polycarboxybetaines (PCBs), polyphosphobetaines (PFBs) and polysulphobetaines (PSBs). PCBs have the lowest protein adsorption measured so far for a biomaterial - lower than 0.3 ng / cm ² , thus exceeding the gold standard so far - polyethylene glycol (PEG) [Z. Zhang, N. Vaisocherova, G. Cheng, W. Yang, H. Xue, S. Jiang „Nonfouling Behavior of Polycarboxybetaine-Grafted Surfaces: Structural and Environmental Effects Biomacromolecules 9, 2686-2692 (2008)]. In addition to the extremely low bioadhesiveness, PCYs show the so-called anti-polyelectrolyte effect, ie. swell more strongly in saline solutions than in pure water. These properties determine their biocompatibility and make them extremely suitable for the development of wound dressings. Electrofiber PSB has been shown to be resistant to bacterial adhesion in gram-negative P. aeruginosa and gram-positive S. epidermidis tests and is a potential material for antimicrobial dressings [R.

Lalani, L. Liu „Electrospun Zwitterionic Poly (Sulfobetaine Methacrylate) for Nonadherent, Superabsorbent, and Antimicrobial Wound Dressing Applications Biomacromolecules 13, 1853-1863 (2012)].

It was found that a copolymer film of PCBs and polybutyl methacrylate (PBMA) accelerates wound healing, and its effect is comparable to that of preparations containing basic fibroblast growth factor, polyvinylpyrrolidone-iodine complex, deproteinized hemodialysate vitamin E [veal blood or Fujishita, S. Inaba, S Tada, M. Gemmei-lde, H. Kitano, Y. Saruwatari Effect of Zwitterionic Polymers on Wound Healing Biol. Pharm. Bull. 31, 2309—2315 (2008)]. An in vivo study of a PCI-modified wound model with a poly (tetrafluoroethylene) membrane showed that the wound healed in 14 days, with the histology of the restored skin close to that of normal skin [J.-F. Jhong, A. Venault, S.-S. Howe, S.-H. Chen, T.-S. Wei, J. Zheng, J. Huang, Y. Chang „Surface Zwitterionization of Expanded Poly (tetrafluoroethylene) Membranes via Atmospheric Plasma-Induced Polymerization for Enhanced Skin Wound Healing ACS Appl. Mater. Interfaces 5, 6732-6742 (2013)]. Modification of the poly (tetrafluoroethylene) film with PSB increases the hydrophilic properties of the surface and its ability to retain moisture. The hydration capacity of the film was determined to be 10.3 mg / cm ² Although significant, due to the limitation of moisture being sorbed only by the surface layer of the dressing, these materials are not suitable for abundantly exuding wounds. With the present invention this disadvantage is overcome by the possibility of water, respectively the wound exudate to be absorbed in the whole volume of the hydrogel. In comparison, their hydration capacity is 4-5 times higher for a bandage comparable in thickness. In addition, the hydrogel is a 3D object and the amount of water absorbed, respectively exudate, can be controlled by the film thickness, the type of PCJ, the crosslinking density. For example, 1 cm ³ of dry PCBs absorbs and retains more than 1500 mg of water and 5 times more saline. This large absorption capacity of aqueous solutions defines PCJ hydrogels as suitable dressings for the treatment of chronic wounds. By absorbing the exudate of the wound, the hydrogel reduces the amount of metalloproteases in the wound, the high content of which causes the wound to become chronic. Thus, by reducing the amount of metalloproteases without inhibiting their activity, the dressing creates favorable conditions for tissue repair processes.

Chitosan is a natural polysaccharide that has antifungal and antimicrobial properties, as well as oxygen permeability - characteristics that make it a suitable and researched material for the treatment of various types of wounds [Chitosan: A potential biopolymer for wound management. Banoa I., Arshad M., Yasin T., Ghauri M.A., Younus M. International Journal of Biological Macromolecules 102 (2017) 380-383].

Therapeutic protein treatment is an attractive approach to treating various diseases. However, proteins are often unstable under physiological conditions, which reduces their bioavailability and activity and requires the use of higher doses.

Polymer grafting to proteins is often used to increase protein stability [Peptide and Protein Delivery, Chris Van der Walle, 2011 Elsevier Inc. Chapter 11. Stefano Salmaso, Paolo Caliceti „Peptide and Protein Bioconjugation: A Useful Tool to Improve the Biological Performance of Biotech Drugs]. Conjugation of proteins with PCBs improves their stability commensurate with the effect of PEG, as the conjugates retain and even increase their activity [A. J. Keefe, S. Jiang Poly (zwitterionic) protein conjugates offer increased stability without sacrificing binding affinity or bioactivity Nature Chemistry, 4, 59-63 (2012)]. PFBs have also been used to conjugate proteins, for example to interferon [A. Lewis, Y. Tang, S. Brocchini, J. Choi, A. Godwin ,, Poly (2-methacryloyloxyethyl phosphorylcholine) for Protein Conjugation Bioconjugate Chern. 19, 2144-2155 (2008)]. Conjugation of proteins with sulfobetaines has not been reported to date.

A silicone dry emulsion with immobilized serine protease from the subtilisin group has been developed for enzymatic wound cleansing, which has been shown to be more effective than known enzyme ointments [Richard Bott; James Crissman, Csilla Kollar, Mae Saldajeno, Grant Ganshaw, Xavier Thomas, Thomas H. Lane, Paal Klykken, Jeffrey M. Davidson, Lillian B. Nanney, A silicone-based controlled-release device for accelerated proteolytic debridement of wounds. Wound Rep Reg (2007) 15 227-235]. It is included in a matrix of poly (vinyl alcohol) and the resulting composite material is placed on an adhesive carrier. Under the action of the exudate in the wound, poly (vinyl alcohol) dissolves and within 8 hours releases into the wound a silicone emulsion of serine protease. In this way, the enzymatic cleansing of model wounds on pig skin is ensured. However, these dressings do not provide absorption of wound exudate, and the authors do not comment on their action in abundantly exuding wounds, where due to the large amount of fluid, the silicone emulsion with protease would dissolve much faster and similar to lubricating wires, this would until the active ingredient serine protease is washed away.

EP 0194647 B1 discloses a biocompatible, occlusive or semi-occlusive surgical adhesive tape with low moisture permeability and containing enzyme in powder form and silver sulfadiazine, resistant to sterilization and storage at temperatures below 40 ° C. Compared to the polyviterione hydrogels of the present invention, it is again unable to absorb wound exudate. Excessively dry wounds (eg from burns) require special water treatment of the wound so that the band can begin to release the protease.

There are no known in the literature for the treatment of chronic wound dressings from polyviterion hydrogel or two-layer ones with a top layer of chitosan and inner from polyviterion hydrogel, which (a) have ultra low bioadhesion and antibiofilm-forming properties, (b) exhibit t the so-called anti-polyelectrolyte effect, i.e. to absorb larger amounts of saline solutions, incl. wound exudate than water, (c) exhibit antibacterial properties, and (d) have proteolytic activity due to an enzyme immobilized therein with proteolytic action to degrade necrotic tissue.

Description of the invention

It is an object of the present invention to provide polyviterione hydrogels for dressings for the treatment of chronic and necrotic wounds, including abundantly exuding wounds, which have the necessary properties, namely: the ability to swell more in saline solutions and body fluids than in pure water. , i.e. to absorb abundantly exudate from the wound; extremely low bioadhesiveness, ie not to allow the formation of a bacterial biofilm, which can be enhanced by the antibacterial properties of the second layer of chitosan hydrogel, while having proteolytic activity due to the enzymes with proteolytic action included in the composition of polyviterione hydrogel, aimed at degrading necrotic tissue. chronic wounds. In addition, the hydrogel provides diffusion of gases, and its mechanical properties (modulus of elasticity) are very close to those of the skin, which creates additional comfort for patients and contributes to the normal healing of the wound.

Single-layer polyviterion hydrogels and two-layer polyviterion / chitosan hydrogels have been developed for dressings for the treatment of chronic and / or abundantly exuding wounds, which are a solution to the set task.

Hydrogels from crosslinked polyviterions with immobilized protease offer a complex of functionalities and are suitable for the development of dressings for the treatment of chronic and / or abundantly exuding wounds. The included enzyme with proteolytic action, immobilized in the pores of the hydrogel or on its surface, degrades the necrotic tissue and facilitates the processes of tissue repair without being washed away by wound fluids. The polyviter network absorbs the exudate, and it can absorb more than 300 times more exudate than the dry weight of the dry membrane. The swollen network maintains the necessary moisture to repair the damaged tissue, which in combination with the low bioadhesiveness of the hydrogel prevents the formation of a biofilm of bacteria and protects the wound from bacterial infection, and the addition of a second layer of chitosan enhances the antibacterial activity of the dressing. This eliminates the need for antibiotic or other germicidal treatment. The low bioadhesion of the polyviterione hydrogel prevents the dressing from sticking to the tissue, which together with its mechanical strength and elastic properties close to those of the skin, contribute to the patient's comfort during wound treatment and when removing / changing the dressing.

The developed polyviterione hydrogels are single-layer or double-layer, with components and structure, schematically presented in Figure 1, Figure 2, Figure 3, Figure 4, Figure 5 and Figure 6.

The subject of the invention are the following hydrogels:

Single layer hydrogel composed of a polyviterite network of poly (sulfobetaine methacrylate) (PSBMA) and crosslinking segments of polyethylene glycol diacrylate (PEGDA) with a molecular weight of 575 g / mol, the content of PEGDA is in the range of 0.2 wt.% To 8%. . The water content varies from 0% to 200%.

A single layer hydrogel composed of a polyviterite network of poly (carboxybetaine methacrylate) (PCBMA) with crosslinking segments of polyethylene glycol diacrylate (PEGDA) with a molecular weight of 575 g / mol, with a PEGDA content ranging from 7.5% to 17% by weight. . The water content varies from 0% to 910%.

Single layer hydrogel composed of a polyviterite network of (PSBMA) and crosslinking segments of polyethylene glycol diacrylate (PEGDA) with a molecular weight of 575 g / mol, the content of PEGDA ranging from 0.2 wt.% To 8.2 wt.%. Subtilisin protease is included in the pores of the network in amounts from 10 mg / g to 87 mg / g, based on the weight of the dry network. The water content varies from 0% to 200%.

A single layer hydrogel composed of a polyviterite network of poly (carboxybetaine methacrylate) (PCBMA) with crosslinking segments of PEGDA with a molecular weight of 575 g / mol, with a PEGDA content ranging from 7.5% to 17.6% by weight. Subtilisin protease is included in the pores of the network in amounts from 10 mg / g to 106 mg / g, relative to the weight of the dry network. The water content varies from 0% to 900%.

Single layer hydrogel composed of a polyviterite network of poly (sulfobetaine methacrylate) (PSBMA) and crosslinking segments of polyethylene glycol diacrylate (PEGDA) with a molecular weight of 575 g / mol, the content of PEGDA is in the range of 0.2 wt.% To 8%. . On the surface of the hydrogel is immobilized protease - Subtilisin with grafted chains of PSBMA with a molecular weight of 8000 g / mol to 12,000 g / mol. Immobilization is performed by physical connection, ie. interaction between zwitterionic residues of the PSBMA enzyme graft and those of the hydrogel. The amount of immobilized enzyme is from 0.4 mg / cm ² to 0.8 mg / cm ² . The water content varies from 0% to 150%.

A polyviterionic two-layer hydrogel composed of an upper layer that is not in contact with the wound and a lower layer designed to be in contact with the injured tissue. The top layer is of un crosslinked or crosslinked chitosan with added poly (ethylene oxide) in amounts of from 0% to 20% by weight. The lower layer is a polyviterion hydrogel of PSBMA and crosslinking segments of PEGDA with a molecular weight of 575 g / mol and a content in the range from 0.2 wt.% To 8.2 wt.% Or of PCBMA with crosslinking segments of PEGDA with a molecular weight of 575 g / mol and content from 7.5% by weight to 17.6% by weight. Subtilisin protease is included in the pores of the polyviterione hydrogel in amounts of 10 mg / g to 87 mg / g or 10 mg / g to 106 mg / g, based on the dry weight of the PSBMA or PCBMA, respectively. The two layers are in weight ratios upper: lower layer from 5:95 to 30:70. The water content varies from 0% to 190% by weight and from 0% to 850% by weight, respectively, and

A polyviterionic two-layer hydrogel composed of an upper layer that is not in contact with the wound and a lower layer designed to be in contact with the injured tissue. The top layer is of un crosslinked or crosslinked chitosan with added poly (ethylene oxide) in amounts of from 0% to 20% by weight. The lower layer is a polyviterion hydrogel of PSBMA and crosslinking segments of PEGDA with a molecular weight of 575 g / mol and a content of 0.2 wt% to 8.2 wt%. On the surface of the lower layer is immobilized protease Subtilisin with grafted chains of PSBMA with a molecular weight of 8000 g / mol to 12,000 g / mol. Immobilization is performed by physical interaction between the zwitterionic residues of the PSBMA enzyme graft and the same from the hydrogel. The amount of immobilized enzyme is from 0.4 mg / cm ² to 0.8 mg / cm ² . The two layers are in weight ratios upper: lower layer from 5:95 to 30:70. The water content varies from 0% to 150%.

Poly (sulfobetaine methacrylate) and poly (cabroxybetaine methacrylate) networks were synthesized for the development of polyviterione hydrogels for dressings for the treatment of chronic and abundantly exuding wounds, using the corresponding monomers and crosslinking agent polyethylene glycol dialects. 575 g / mol. The preparation of the PSBMA network was performed in an aqueous medium at 60 ° C, initiator K ₂ 5 ₂ O ₈ (0.1 mol.%) And PEGDA in an amount of 0.1 mol% to 4.0 mol% relative to the SBMA monomer.

The PCBMA network was prepared in a mixed solvent of ethylene glycol / ethanol / water (3: 1: 1 by volume) at 60 ° C, sodium metabisulfite initiator (0.664 mol%) and ammonium persulfate (1.445 mol%). The amount of PEGDA varies from 3.0 mol% to 7.0 mol%.

A two-layer hydrogel composed of a first layer of non-crosslinked or crosslinked chitosan with added poly (ethylene oxide) in amounts of 0% to 20% by weight was obtained. Crosslinking of chitosan is performed with crosslinking agents known in the literature [Reddy, N., Reddy, R., Jiang, Q. Crosslinking biopolymers for biomedical applications. Trends in Biotechnology. 2015, 33 (6), 362-369] - epichlorohydrin, ethylene glycol diglycidyl ether, glutaraldehyde, in amounts of from 1% to 10% by weight. The second layer is from a network of PSBMA or PCBMA, prepared as described above.

PSBMA with a molecular weight in the range of 8000 g / mol h-12000 g / mol and final reactive groups - cyclic carbonate (PSBMA-1) and succinimide active ester (PSBMA-2) were obtained by polymerization by transfer of an SBMA atom in the presence of initiators of 2-oxo-1,3-dioxolan-4-yl- (methyl-2-bromo-2-methylpropanoate) or N-succinimidyl- (methyl-2-bromo-2-methylpropanoate), CuBr catalyst and ligand, respectively 2,2'-dipyridyl in trifluoroethanol at 60 ° C for 14 h.

It was obtained modified with linear PSBMA subtilisin. The modification of subtilisin with the synthetic polyviterions described above was performed in glycerol solution at 25 ° C to give two subtilisin-PSBMA-1 and subtilisin PSBMA-2 conjugates, with an average of one PSBMA chain grafted.

The equilibrium degree of swelling of the synthesized PSBMA polyviterione networks in water and aqueous sodium chloride solution with a concentration of 0.1 M to 5.0 M was determined by gravimetric analysis. For the polyviterione hydrogels formed in this way, their modulus of elasticity at a corresponding equilibrium degree of swelling in water is determined by the Hertz contact theory. Vickers microhardness of dry polyviter nets was determined.

The polyvinyl hydrogels based on PSBMA and PKBMA and crosslinked with PEGDA with a molecular weight of 575 g / mol and a content of 0.2 wt% to 8.2 wt% or from 7.5 wt% to 17.6 wt%, respectively, include the enzyme Subtilisin in enzyme solution concentration of 0.813 rng / ml and at a temperature of 4 ° C. The activity of subtilisin immobilized in the polyviterione hydrogels on casein substrate was then determined.

The enzymatic activity of the two conjugates subtilisin-PSBMA-1 and subtilisin-PSBMA-2 was determined using the same substrate.

The cytotoxicity of the PSBMA networks was studied and the cytological changes in cells incubated with polyviterione hydrogels were studied. These hydrogels were found to be non-cytotoxic, and the cell viability in the presence of PSBMA hydrogels was even greater than that of the cells in the control sample, where there were no polyviterione hydrogels. PEGDA was also found to have no effect on cell viability.

The equilibrium rate of swelling of the synthesized PSBMA polyviterione networks in water and aqueous sodium chloride solution with a concentration of 0.1 M to 5.0 M varies from 200% to 2300%, depending on both the amount of crosslinking segments and the concentration of saline. The measured modulus of elasticity of PSBMA polyviterione hydrogels is between 1 kPa and 3 kPa, i. in the region of the modulus of elasticity measured in vivo for human skin [S. Pailler-Mattei, S. Bec, N. Zahouani In vivo measurements of the elastic mechanical properties of human skin by indentation tests Medical Engineering & Physics 30 (2008) 599-606]. The temperature dependence of the ability of PSBMA polyviterion networks to absorb water (in the temperature range 20-90 ° C) was studied. The swelling of the same networks in Kokubo's solution at 37 ° C was studied in order to simulate the conditions in the human body. The rheological properties of PSBMA hydrogels in their equilibrium degree of swelling in water were studied, and these measurements proved the mechanical stability and strength of the hydrogels thus obtained.

The equilibrium degree of swelling of the synthesized PKBMA polyviterione networks in water and in sodium chloride solutions with a concentration from 0.1 M to 5.0 M was determined gravimetrically. The degree of swelling varies from 200% to 1000%, depending on both the amount of crosslinking segments and the salt concentration in the solution. The kinetics of swelling in water of PKBMA polyviterion networks was studied by gravimetric analysis. For the PKBMA polycviterion hydrogels formed in this way, their modulus of elasticity at a corresponding equilibrium degree of swelling was determined by means of the Hertz contact theory, as it is again in the range from 1 to 5 kPa, i. again fully consistent with the modulus of elasticity measured in vivo for human skin. The pH dependence of the ability of PKBMA polyviterion networks to swell in the range pH = 2 to 10 was studied. The rheological properties of PKBMA hydrogels in their equilibrium degree of swelling in water were studied, and these measurements proved the mechanical stability and strength of the hydrogels thus obtained.

When comparing the equilibrium swelling of the synthesized polyviterite networks in water, it was found that the swelling capacity of PCBMA is several times higher than that of PSBMA, while this is not the case in sodium chloride solution. The reason for the difference in the behavior of the two types of networks in saline solutions is the strong tendency of PSBMA to form zwitterionic clusters, which play the role of a physical network and which decompose in saline solutions and not in pure water. As a result, PSBMA has a more pronounced tendency to swell in saline solutions than PCBMA. It is also known that the modulus of elasticity of networks is proportional to the number of physical and / or chemical bonds in them. There is a relationship between the equilibrium swelling ratio and the modulus of elasticity of the synthesized polyvitertone networks.

A relationship has been established between the number of physical and / or chemical bonds in polyviter nets and microhardness. The more connections in the networks, the greater the microhardness. The microhardness of PSBMA decreased with increasing PEGDA concentration to 8.2% by weight, while the microhardness of PCBMA increased with increasing PEGDA concentration from 7.5% by weight to 17.6% by weight.

The observed dependences in the polyviterite networks show that the concentration of PEGDA affects the properties of the networks, as in the PSBMA network the modulus of elasticity and microhardness decreases due to disintegration of the physical nodes of the network and increase of its hydrophilicity, and in the network the PCBMA effect - Increasing the density of chemical crosslinking leads to an increase in its elasticity and microhardness. In this sense, the concentration of PEGDA is used to control the properties of polysviterion networks and fine-tuning the properties of polysviterion hydrogels as immobilization matrices.

To impart proteolytic properties to polyviterione hydrogels for dressings for the treatment of chronic and profusely exuding wounds, the proteolytic enzyme subtilisin is included in the polyviterion hydrogels obtained according to the above description. The method by which the enzyme is immobilized in the polyviterione hydrogel is physical incorporation, due to its easy feasibility and the fact that it does not change the inherent properties of the enzyme, nor does it use chemical transformations. Thus, a polyviterion hydrogel for dressings for the treatment of chronic and / or necrotic wounds is formed, in which the subtilisin is located in the pores of the hydrogels of PSBMA or PKBMA. The concentration of PEGDA used for the crosslinking of PSBMA or PCBMA does not significantly affect the incorporation of subtilisin into the hydrogels if sufficient time is provided, such as for immobilization in PSBMA the hydrogels are sufficient for 48 hours to achieve equilibrium efficient enzymatic incorporation. 45-50%. 66 hours are required to achieve equilibrium enzymatic incorporation into PCBMA hydrogels, achieving an efficiency of 55-60%. The increased time and higher loading efficiency of the enzyme in PKBMA hydrogel is related to its ability to swell more, as described above.

Immobilization of subtilisin in the polysvitertviterion hydrogel helps the enzyme to remain active at all times while the dressing is applied to the wound. The enzyme immobilized in PKBMA retains its activity after its incorporation into the hydrogel, which gradually decreases from 100% to 50% after 6 cycles of application. Due to the lower equilibrium degree of swelling, the enzyme involved in PSBMA exhibits 60% of the activity of the non-immobilized enzyme, which decreases to 30% of the 6th cycle.

The modification of subtilisin with polysulphobetaine chains with terminal cyclic carbonate (PSBMA-1) or with succinimidester groups (PSBMA-2) is carried out by interaction of amino groups of the enzyme and the terminal reaction groups of polysulphobetaine with the formation of a bond or urethane. On average, one PSBMA chain per subtilisin molecule was grafted. After modification, the subtilisin-PSBMA-1 and subtilisin-PSBMA-2 conjugates retain 56% and 67% of the unmodified subtilisin activity, respectively, measured on a casein substrate. Immobilization of the conjugates is performed by adding a solution of the conjugate in saline to a network of PSBMA. The modified subtilisin is immobilized on the surface of the PSBMA hydrogel by zwitterionic clusters formed between the network and the PSBMA enzyme graft.

Because dressings are applied directly to the wound and have direct contact with the cells of both healthy and injured tissues, the established lack of cytotoxicity of polyviterione hydrogels is essential for the effectiveness of the dressings. The viability of murine embryonic fibroblasts (L929 cell line) incubated with PSBMA was found to be equal to and higher than that of the control. The lack of cytotoxicity has also been demonstrated by human fibroblast tests, with cell survival of about 80% when incubated with PSBMA and 80-90% when incubated with PCBMA.

Microscopic examination revealed that inoculated cells from murine fibroblasts cell line L929 had low bioadhesion to the hydrobel of PSBMA, which causes their poor adhesion and easy leaching from the surface of the hydrogel. However, they do not change shape and remain round, unlike the same cells seeded at the bottom of wells in a 48-well plate, which adhere and change shape to the typical spindle-shaped fibroblasts. This is an important property that is important for easier removal of the dressing when it needs to be replaced or when it is no longer needed to treat the wound.

As a result of the analyzes, it was found that the developed polyviterione hydrogels with immobilized subtilisin have characteristics that make them suitable materials for wound dressings, which are characterized by the ability to absorb wound exudate, while enzymatic degradation of necrotic tissue and protection of the wound from infections, ie. dressings with significantly accelerated and highly effective healing effect.

Description of the attached figures

Figures 1 to 6 show schematically the structure and type of components that make up the polyviterione hydrogels.

Figure 1. Single layer hydrogel of crosslinked PCBMA

Where:

Position 1: PCBMA chain

Position 2: PEGDA cross-linking segment

Figure 2. Single-layer crosslinked PSBMA hydrogel containing Subtilisin in the hydrogel pores

Where:

Position 2: PEGDA cross-linking segment

Position 3: PSBMA chain

Position 4: Subtilisin

Figure 3. Single-layer crosslinked PCBMA hydrogel containing Subtilisin in the hydrogel pores

Where:

- Position 1: PCBMA chain

Position 2: PEGDA cross-linking segment

Position 4: Subtilisin

Figure 4. Single layer hydrogel of crosslinked PSBMA with surface immobilized Subtilisin with grafted PSBMA

Where:

- Position 2: PEGDA cross-linking segment

Position 3: PSBMA chain

- Item 5: Immobilized to the surface Subtilisin with grafted PSBMA chain

Figure 5. Two-layer hydrogel of chitosan layer and polyviterion layer

Where:

Position 6: Chitosan layer

Item 7: Polyviterion hydrogel containing Subtilisin in hydrogel pores

Figure 6. Two-layer hydrogel of chitosan layer and layer of polyviterione hydrogel of PSBMA with surface immobilized Subtilisin with grafted PSBMA

Where:

- Position 2: PEGDA cross-linking segment

Position 3: PSBMA chain

- Item 5: Immobilized to the surface Subtilisin with grafted PSBMA chain

Position 6: Chitosan layer

Figure 7. Light microscopy of murine embryonic fibroblasts (cell line L929), incubated at the bottom of a 48-well plate for 72 hours (Microscope Olympus SK40, CAM 2800-XP), left image magnification 20 times (20x) and right image magnification 40 times (40x).

Figure 8. Light microscopy of murine embryonic fibroblasts (cell line L929) incubated with PSBMA hydrogel crosslinked with 0.2 wt.% PEGDA (A) and PSBMA hydrogel crosslinked with 1 wt.% PEGDA (Olympus) (Bus-28) XP, 20x)

Embodiments of the invention

The presented examples do not exhaust the possible exemplary embodiments.

Example 1

Polyviterion hydrogel from crosslinked PSBMA

Poly (sulfobetaine methacrylate) (PSBMA), crosslinked with polyethylene glycol diacrylate (PEGDA) with a molecular weight of 575 g / mol and a content of crosslinking segments of PEGDA 2 wt.%, With a water content of 110% at equilibrium swelling.

Example 2

Polyviterion hydrogel from crosslinked PKBMA

Poly (carboxybetaine methacrylate) (PCBMA), crosslinked with polyethylene glycol diacrylate (PEGDA) with a molecular weight of 575 g / mol and a content of crosslinking segments of PEGDA 15% by weight with a water content of 530% with equilibrium swelling.

Example 3

Polyviterion hydrogel of crosslinked PSBMA containing subtilisin

Poly (sulfobetaine methacrylate) (PSBMA), crosslinked with polyethylene glycol diacrylate (PEGDA) with a molecular weight of 575 g / mol and content of crosslinking segments of PEGDA 2 wt.%, Water 110% and immobilized protease content Subtilisin 66 mg / g the weight of the dry net.

Example 4

Polyviterion hydrogel from crosslinked PCBMA containing subtilisin

Poly (carboxybetaine methacrylate) (PCBMA), crosslinked with polyethylene glycol diacrylate (PEGDA) with a molecular weight of 575 g / mol and a content of crosslinking segments of PEGDA 15 wt.%, Water 530% and immobilized protease content Subtilisin 104 mg / g the weight of the dry net.

Example 5

Polysviterionic crosslinked PSBMA hydrogel containing modified subtilisin on the surface of the hydrogel

Poly (sulfobetaine methacrylate) (PSBMA), crosslinked with polyethylene glycol diacrylate (PEGDA) with a molecular weight of 575 g / mol and a content of crosslinking segments of PEGDA 3 wt.%, Water 120% and a content of immobilized on the surface of the hydrogel 0.8 mg Subtil cm ² , which was modified with grafted PSBMA chains with a molecular weight of 10,500 g / mol.

Example 6

Polyviterionic bilayer hydrogel of crosslinked PSBMA and chitosan containing subtilisin

The bilayer hydrogel is composed of an upper layer that is not in contact with the wound and a lower layer designed to be in contact with the injured tissue. The upper layer is of non-crosslinked or crosslinked chitosan and poly (ethylene oxide) (PEO) in a weight ratio of chitosan: PEO = 9: 1. The lower layer is a polyviterione hydrogel of PSBMA crosslinked with PEGDA with a molecular weight of 575 g / mol and a content of crosslinking segments of 2 wt.%. The lower layer contains the included protease Subtilisin 66 mg / g relative to the dry weight of PSBMA. Total water content 105%. The two layers have an upper: lower layer weight ratio of 15:85.

Example 7

Polyviterionic bilayer hydrogel of crosslinked PCBMA and chitosan containing subtilisin

The bilayer hydrogel is composed of an upper layer that is not in contact with the wound and a lower layer designed to be in contact with the injured tissue. The top layer is of un crosslinked or crosslinked chitosan and poly (ethylene oxide) (PEO) in technical terms chitosan: PEO = 9: 1. The lower layer is a polyvinyl hydrogel of PKBMA crosslinked with PEGDA with a molecular weight of 575 g / mol and a crosslinking segment content of 15% by weight. The lower layer contains the included protease Subtilisin 104 mg / g relative to the weight of the dry network of PCBMA. Total water content 500%. The two layers have an upper weight ratio: PCJ layer of 15:85.

Example 8

Polyviterionic bilayer hydrogel of crosslinked PSBMA and chitosan containing modified subtilisin on the surface of the lower zwitterionic layer

The bilayer hydrogel is composed of an upper layer that is not in contact with the wound and a lower layer designed to be in contact with the injured tissue. The top layer is of un crosslinked or crosslinked chitosan and poly (ethylene oxide) (PEO) in technical terms chitosan: PEO = 9: 1. The lower layer is a polyvinyl hydrogel of PSBMA crosslinked with PEGDA with a molecular weight of 575 g / mol and a content of crosslinking segments of 3 wt.%. On the surface of the lower layer, which does not border the chitosan layer, is immobilized protease Subtilisin with grafted chains of PSBMA with a molecular weight of 10,500 g / mol. The content of immobilized subtilisin is 0.8 mg / cm ² . Total water content 108%. The two layers have an upper: lower layer weight ratio of 15:85.

Example 9

Preparation of hydrogel from PSBMA

[2- (Methacryloyloxy) ethyl] -dimethyl (3-sulfopropyl) ammonium hydroxide (from the sulfobetaine group, SBMA) was dissolved in distilled water to give a 1M solution, to which was added PEGDA (1 mol% relative to the monomer). SBMA). The crosslinking polymerization was initiated with potassium persulfate (K ₂ 5 ₂ 0 ₈ , 0.1 mol.%) And carried out at 60 ° C for 6 hours. The resulting mesh was immersed in a large volume of distilled water for 2 weeks and the water was changed daily to remove unreacted monomer and PEGDA. The wash water is checked by UV spectroscopy to ensure that the network is purified. The dry net is placed in water until equilibrium swelling. Thus, a polyviterione hydrogel network with the composition disclosed in Example 1 was obtained.

Example 10

Preparation of hydrogel from PKBMA

The carboxybetaine methacrylate (KBMA) monomer was dissolved in a mixed solvent of ethylene glycol / ethanol / water (3: 1: 1 by volume) to give a 1M solution. PEGDA 6 mol% relative to the SBMA monomer was added. The polymerization was initiated by a mixed initiator consisting of 0.664 mol% sodium metabisulphite (Na ₂ S ₂ 0s) and 1,445 mol% ammonium persulphate, (MNq 3), and carried out at 60 ° C for 15 hours. volume of distilled water for 2 weeks and the water is changed daily to remove residual unreacted monomer and crosslinking agent.The wash water is checked with a UV spectrometer to ensure that the mesh is purified.The dry mesh is placed in water until In this way, a polyviterione hydrogel with the composition disclosed in Example 2 was obtained.

Example 11

Synthesis of subtilisin-PSBMA conjugate

Subtilisin and PSBMA-1 or PSBMA-2 in a weight ratio of 1: 2 were dissolved in glycerol and stirred for 2 days at room temperature. The solution is then dialyzed against saline to remove unreacted polymer.

Example 12

Determination of equilibrium degree of swelling

The equilibrium swelling (EDS) of polyviterion nets is determined gravimetrically. For this purpose, disks with a defined size of dry polymer mesh are weighed (Wo) and then immersed in distilled water at a temperature of 20 ° C. The weight of the swollen disks is measured for several days until an equilibrium constant value is reached (We ). To determine the equilibrium degree of swelling according to equation (1), the values for Wo and We for at least three disks of each sample are used and averaged.

EDS = [We-Wo) / Wo] xl00 (1)

Example 13

Immobilization of subtilisin in the hydrogel

Disc samples of a certain size from both PSBMA or PCBMA networks were incubated in the enzyme solution at a concentration of 0.813 mg / ml at 4 ° C. The absorbance of the enzyme solution in which the loading is performed is measured at the 24th, 48th and 66th hour after immersion of the polymer samples. The enzyme loading efficiency (SES) for each zwitterionic hydrogel is calculated by the formula:

SES = [(m ₀ -m _t ) / m ₀ ] * 100 (2) where it is the amount of enzyme in the stock solution and m _t is the amount of enzyme in the solution after time t from immersion of the network in the solution.

V- Example 14

Absorption of collagenase by PCBMA hydrogels

A dry polyviterite PCBMA mesh (30 mg) was immersed in a 600 μί solution of collagenase (1 mg / mL) in phosphate buffer pH 7.4 at 37 ° C for 72 h. Then, 30 μί of the incubated solution was mixed with 270 μί of Comassie Brilliant Blue G (Bradford® kit). The mixture was left at room temperature for 5 minutes and then the absorbance of the solution was measured at 595 nm. Three independent measurements were performed for each sample and the results were averaged. The amount of collagenase in the solution before and after incubation with PCBMA hydrogel was obtained using a calibration curve obtained with bovine serum albumin with equation Y = 0.637x - 0.078 (R = 0.9987). The percentage of absorbed collagenase (EA) from the PCBMA network is then calculated using the equation:

EA = (m ₀ - m _t ) / m ₀ ) * 100 where m ₀ is the concentration of collagenase in the stock solution (1 mg / mL), am _t is the residual enzyme concentration measured after 72 h of incubation with the hydrogel. Depending on the degree of crosslinking, PKBMA hydrogels absorb between 30 and 40% of the collagenase in the stock solution. These values demonstrate the potential of PCBMA hydrogels to physically remove collagenase from chronic wounds, one of the enzymes that is too high in chronic wounds.

Similar results were obtained after the same experiment with PSBMA hydrogels, which showed the ability to reabsorb 30 to 40% of the collagenase in the stock solution. These values demonstrate the potential of PSBMA hydrogels to physically remove collagenase from chronic wounds.

Example 15

In vitro determination of the ability of PCBMA hydrogels to inhibit collagenase activity

Fluorescence analysis determined the inhibitory activity of PCBMA hydrogels against collagenase using the EnzCheck® gelatinase / collagenase kit. Dry webs of PCBMA (30 mg) were incubated in 600 μΙ 32 U / ml collagenase solution at 37 ° C. After 24 h, 100 μL of the collagenase solution was mixed with 80 μL of phosphate buffer. Collagenase activity assay was initiated by the addition of 20 μL of gelatin substrate (250 pg / mL) and subsequent measurement of the fluorescence of the resulting solution at 528 nm. all measurements are performed three times. Results are expressed as the percentage of residual collagenase activity incubated with PCBMA hydrogel compared to collagenase control solution that was not incubated with PSBMA hydrogel. The results show that collagenase retains 70% of its activity after incubation with PCBMA hydrogels. This result indicates that the polyviterione hydrogel does not inhibit collagenase activity, which is beneficial for the healing of chronic wounds in which collagenase is an integral part of the remodeling process.

Similar results were obtained after conducting the same experiment with PSBMA hydrogels, with collagenase retaining almost 100% of its activity after incubation with PSBMA hydrogels. These values demonstrate the potential of PSBMA hydrogels to physically remove collagenase from chronic wounds without altering its enzymatic activity, which is required in the wound healing process.

Example 16

Ex vivo determination of the ability of PCBMA hydrogels to inhibit collagenase activity in wound exudates

Early exudate is extracted from a UrgoClean dressing of a patient with a chronic leg wound. For this purpose, 1 g of a sample of the dressing is immersed in 5 mL of distilled water for 15 minutes. The dressing was then removed and the mixture was stirred for 2 minutes and centrifuged for 5 minutes at 10,000 rpm. The precipitate is removed and the liquid is centrifuged again under the same conditions. The extracted exudate was stored at 4 ° C until used for the determination itself.

The effectiveness of collagenase inhibition in real wound exudate by PCBMA hydrogels was tested with wound exudate diluted 8 times. Dry disc-shaped PCBMA nets (30 mg) were incubated in 600 μL of exudate at 37 ° C for 24 h. Then 100 μι of the incubated solution was mixed with 80 μL of phosphate buffer pH 6.5. A sample of wound exudate, without added PCBMA hydrogel, was used as a control. Residual collagenase activity was determined after adding 20 μι of gelatin substrate (250 pg / mL) and monitoring the fluorescence following the reaction at 493/528 nm. All measurements are performed three times.

The results are in complete agreement with the results of the in vitro experiment described in Example 14, showing that wound exudate collagenase retains 70% of its activity after incubation with PCBMA hydrogels, which is beneficial for the healing of chronic wounds in which collagenase is irreversible. part of the remodeling process.

The results of the ex vivo determination of the ability of PSBMA hydrogels to inhibit collagenase activity in wound exudates again coincide with the results obtained from in vitro tests, i. collagenase from wound exudate retains almost 100% of its activity after incubation with PSBMA hydrogel.

Example 17

Evaluation of the cytotoxicity of hydrogels by PCBMA

Human fibroblasts are used to assess the cytotoxicity of PCBMA hydrogels. Cell cultures were maintained at 37 ° C under 5% CO ₂ atmosphere in DMEM (Dulbecco's Modified Eagle Medium) medium. The cultural environment changes every day. Cells were harvested using trypsin-EDTA solution, seeded at a density of 2 x 10 ⁴ cells / well in a sterile 24-well polystyrene plate. 24 hours later, PCBMA hydrogels (30 mg) and a silicone sample (positive control) were incubated with the cells and left for an additional 24 hours at 37 ° C under 5% CO ₂ atmosphere. The cells were then washed twice with phosphate buffer and mixed with 500 μΙ 10% (v / v) AlamarBlue reagent in DMEM. The resulting solutions were left at 37 ° C for 4 hours. Finally, their absorption at 570 nm was measured. The relative viability of BJ5ta cells (%) was determined after incubation with PCBMA hydrogel, compared to cell viability when incubated with silicone and DMEM controls. All results are reported as averages of three independent measurements.

The results show that between 80 and 90% of fibroblast cells survive after incubation with PCBMA hydrogels, i. the latter are not cytotoxic.

Analogous tests with PSBMA hydrogels show that the survival of human fibroblasts after incubation with PSBMA hydrogels is between 70 and 90% depending on the degree of crosslinking of the PSBMA hydrogel. Thus, again, PSBMAs are classified as non-cytotoxic materials.

Example 18

Determination of cytotoxicity of PSBMA hydrogels with mouse fibroblasts

The effect of PSBMA hydrogels on cell viability and proliferation was also examined by MTT test (thiazolyl blue tetrazolium bromide). For this purpose, mouse embryonic fibroblasts (BALB / with 3T3 and L929 constant cell lines) were used as model systems. Cells were grown as monolayer cultures in DMEM medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin and 100 pg / mL streptomycin at 37 ° C in a humidified CO ₂ incubator, respectively. Cells were isolated using a mixture of 0.05% trypsin and 0.02% EDTA.

The MTT test was performed by first swelling the dry PSBMA nets in sterile distilled water for 48 hours and then placing them in DMEM medium for several hours. They are then cut into small homogeneous pieces (discs) and transferred to a 48-well tissue culture plate, where they are sterilized by ultraviolet radiation for 1 hour. A cell suspension with a density of 5 x 10 cells per mL was added to each well. Cell-seeded hydrogels were maintained at 37 ° C below 5% CO ₂ for 24 and 48 hours. The cells were then carefully removed by washing several times with phosphate buffer (pH = 7.2). Finally, each hydrogel was incubated with a solution of thiazolyl blue tetrazolium bromide in DMEM (5 mg in 10 mL DMEM) for 3 hours at 37 ° C under 5% carbon dioxide and 95% air (CO ₂ incubator), followed by extraction with a mixture of absolute ethanol.

The results show that PSBMA are non-cytotoxic as the survival of mouse fibroblasts is at least 100% and reaches 120, which is a sign of cell proliferation on PSBMA hydrogels.

Example 19

Evaluation of cytopathological changes in murine fibroblast cells incubated with PSBMA hydrogels

Two PSBMA hydrogels containing PEGDA crosslinking segments 0.2 wt% and 1.0 wt% were incubated in cell medium on sterile cover plates in 6-well plates. Cells in wells not containing PSBMA hydrogels served as controls. After 24h and 48h incubation, the cover bags were removed and the ability of the hydrogels to induce cytopathological changes was assessed using the double staining method with acridine orange (AO) and propidium iodide (PI) and the Papenheim (May - Grunwald-Giemsa) method. according to the standard procedure. The cells were examined with an Olympus CK40 microscope, CAM 2800-XP.

Light microscopy characterized: (i) murine embryonic fibroblasts (L929 cell line) incubated at the bottom of a 48-well plate (Figure 7) and (I) L929 cells incubated on PSBMA hydrogels with varying degrees of crosslinking (Figure 8). .

Microscopic images clearly show the ability of the cells to adhere to the surface of the wells and form monolayers. The cells grown at the bottom of the 48-well plates have the elongated spindle shape that is characteristic of fibroblasts and form dense monolayers due to their good adhesion to the plate surface (Figure 7). In contrast, the cells grown on the surfaces of the two PSBMA hydrogels (Figure 8) do not spread on their surface and have a round shape, forming clusters rather than monolayers. The tendency of cells to proliferate in clusters means that hydrogels do not provoke cell death. On the other hand, no monolayers of cells are formed, which shows less cellular adhesion to hydrogels than to the surface of the plate. The cell monolayer attached to the surface of the 48-well plate is difficult to remove - 4-5 minutes of prolonged treatment with a solution containing EDTA and trypsin, while the cells attached to the surface of the hydrogels are easily washed.

Example 20

Determination of the enzymatic activity of the protease loaded in PSBMA hydrogels

The determination is performed in 0.06 M Tris buffer with pH 7.4. A casein solution obtained by dissolving 3 g of casein in 100 ml of buffer is used as a substrate. _{0.5M HCl 4} was used as precipitant to stop the enzymatic reaction and precipitate the large casein peptides.

Three tubes were used for each measurement. In tube 1, the reaction is carried out between the enzyme (in the form of a liqueur, i.e. in pure state or loaded into PSBMA hydrogel, Example 1) and the substrate sodium caseinate. In a tube 2 and a tube 3 are placed 3 ml of precipitate to fix the beginning and end of the enzymatic reaction. In tube 1, mix 2.5 ml of casein solution, 1.5 ml of Tris buffer and 1 ml of enzyme liqueur containing 45.6 μs of enzyme. This is the 'control in which the activity of the enzyme in solution is measured. All tubes were immersed in a water bath at 37.5 ± 0.2 ° C. To record the start of the enzymatic reaction, after the first minute of incubation, a 2 ml sample was withdrawn from tube 1 and poured into tube 2. After At 31 minutes incubation, a second 2 ml sample was removed from tube 1 and poured into tube 3. After centrifugation at 2000 rpm for 10 minutes, the supernatants of tubes 2 and 3 were measured at 275 nm. The enzyme activity is calculated using the following equation:

A = 3,156. 27Ε ₂₇₅ where A is the activity of the enzyme in CTA units per ml, ΔΕ275 θ the difference between the absorbance reported after 31 minutes and after the first minute from the beginning of the enzymatic reaction.

The measurement of the activity of the enzyme loaded in the PSBMA hydrogels is determined in the same way, with the difference that in tube 1 instead of pure enzyme a hydrogel loaded with an equivalent amount of enzyme is placed.

The results show that the loading of subtilis in PSBMA hydrogels leads to 60% activity of the loaded enzyme compared to the enzyme in solution. The load in PKBMA keeps the enzyme activity at 100%. The difference is due to the different behavior of the two types of polyviterione hydrogels in water. PSBMA, due to its strong tendency to form a physical network, does not allow casein macromolecules to enter it so easily, and hence the enzymatic action is not as effective. PCBMA does not form additional dipole clusters (physical network) and casein easily enters the gel where part of the enzymatic reaction takes place. Thus, the loading of subtilisin in PKBMA hydrogel does not affect their enzymatic activity.

Claims (7)

Patent claims Polycviterion dressing hydrogel for the treatment of chronic and necrotic wounds, characterized in that it is a single layer hydrogel composed of a polyviterionic network of poly (sulfobetaine methacrylate) (PSBMA) and crosslinking segments of polyethylene glycol molecular diacrylate g / mol, the PEGDA content being in the range of 0.2 wt% to 8.2 wt%, and the amount of water ranging from 0 wt% to 200 wt%. 2. Polyviterion hydrogel for dressings for the treatment of chronic and necrotic wounds, characterized in that it is a single-layer hydrogel composed of a polyviterion network of poly (carboxybetaine methacrylate) (PCBMA) and crosslinking segments of polyethylene glycol diacrylate (5) g / mol, the PEGDA content ranging from 7.5% to 17.6% by weight and the amount of water ranging from 0% to 910% by weight. 3. Polyviterion hydrogel for dressings for the treatment of chronic and necrotic wounds, characterized in that it is a single layer hydrogel composed of a polyviterion network of poly (sulfobetaine methacrylate) (PSBMA) and crosslinking segments of polyethylene glycol molecular diacrylate g / mol, the content of PEGDA in the range from 0.2 wt.% to 8.2 wt.%, and in the pores of the network is included protease Subtilisin in amounts from 10 mg / g to 87 mg / g, relative to the weight of the dry network , and water from 0% by weight to 200% by weight. 4. Polyviterion hydrogel for dressings for the treatment of chronic and necrotic wounds, characterized in that it is a single-layer hydrogel composed of a polyviterion network of poly (carboxybetaine methacrylate) (PCBMA) with crosslinking segments of polyethylene glycol molecular diacrylate5 g / mol, the content of PEGDA is in the range from 7.5 wt.% to 17.6 wt.%, and in the pores of the network is included protease Subtilisin in amounts from 10 mg / g to 106 mg / g, relative to the weight of the dry network , and water from 0% by weight to 900% by weight. 5. Polyviterion hydrogel for dressings for the treatment of chronic and necrotic wounds, characterized in that it is a single-layer hydrogel composed of a polyviterion network of poly (sulfobetaine methacrylate) (PSBMA) and crosslinking segments of polyethylene glycol molecular diacrylate g / mol, the content of PEGDA in the range from 0.2 wt.% to 8.2 wt.%, on the surface of the hydrogel is immobilized protease Subtilisin with grafted chains of PSBMA with a molecular weight of 8000 g / mol to 12,000 g / mol, in an amount of Subtilisin from 0.4 mg / cm ² flo 0.8 mg / cm ² and water from 0 wt.% to 150 wt.%. Polyviterion hydrogel for dressings for the treatment of chronic and necrotic wounds according to claims 3 and 4, characterized in that it is covered with an additional layer, wherein the hydrogel is a two-layer hydrogel composed of an upper layer and a lower layer, wherein the upper layer layer is of non-crosslinked or crosslinked chitosan with added poly (ethylene oxide) in amounts of 0 wt.% to 20 wt.%, and the bottom layer is polyviterione hydrogel of PSBMA and crosslinking segments of PEGDA with a molecular weight of 575 g / mol and content in the range from 0.2 wt.% to 8.2 wt.% or from PKBMA with crosslinking segments of PEGDA with a molecular weight of 575 g / mol and a content in the range from 7.5 wt.% to 17.6 wt.%, in the pores of the polyviterion hydrogel is included protease Subtilisin in quantities from 10 mg / g to 87 mg / g or from 10 mg / g to 106 mg / g, based on the dry weight of PSBMA or PCBMA respectively, at upper: lower layer weight ratios from 5:95 to 30:70 , and respectively total water content from 0 wt.% to 190 wt.% and from 0 wt wt.% to 850 wt.%. The polyviterionic dressing hydrogel for the treatment of chronic and necrotic wounds according to claim 5, characterized in that it is coated with an additional layer, wherein the hydrogel is a two-layer hydrogel composed of an upper layer and a lower layer, wherein the upper layer is of non-crosslinked or crosslinked chitosan with added poly (ethylene oxide) in amounts of 0 wt.% to 20 wt.%, and the lower layer is a polyviterione hydrogel of PSBMA and crosslinking segments of PEGDA with a molecular weight of 575 g / mol and a content of 0.2 wt. % to 8.2 wt.%, on the surface of the hydrogel is immobilized protease Subtilisin with grafted chains of PSBMA with a molecular weight of 8000 g / mol to 12,000 g / mol, with an amount of Subtilisin from 0.4 mg / cm ² to 0.8 mg / cm ² , at upper: lower layer weight ratios of 5:95 to 30:70, and a total water content of 0% to 150% by weight.