PL243058B1 - Hydrogel skin substitute based on agarose and chitosan and method of its production - Google Patents

Abstract

The subject of the application is a hydrogel skin substitute based on chitosan and agarose, characterized in that chitosan is dissolved in a 0.5% (v/v) aqueous solution of acetic acid and agarose in a 0.1% (w/v) aqueous solution of sodium hydroxide, the weight proportions of the solid components are 2 - 4% (w/v) of chitosan in relation to acetic acid and 4% (w/v) of agarose in relation to sodium base, respectively, and the components are mixed in a 1:1 ratio (v :v) giving a final concentration in the final product of 1 - 2% (w/v) chitosan and 2% (w/v) agarose. In addition, the biomaterial alternatively contains bioactive substances such as vitamins, antibiotics, hydrocortisone, curcumin. The application also includes a method for the production of a skin substrate, which consists in combining a 1:1 (v:v) solution of chitosan in acetic acid with a suspension of agarose in sodium base, heating the mixture at a temperature of 70 - 90°C, and then drying it in air in room temperature. An alternative method of producing the bioactive skin substitute according to the invention consists in combining a 1:1 (v:v) solution of chitosan in acetic acid with a suspension of agarose in sodium base, heating the mixture at a temperature of 70 - 90°C, and cooling it to a temperature of 45 - 65°C. °C, adding bioactive compounds such as vitamins, antibiotics, hydrocortisone, and then air drying at room temperature. The biomaterial developed according to the invention can be used in regenerative medicine as a bioactive skin substitute for covering superficial wounds or as a functional equivalent of the skin inhabited by the patient's cells in vitro.

Description

Description of the invention

The subject of the invention is a hydrogel bioactive skin substitute being a preparation based on chitosan and agarose for use in skin regenerative medicine and a method for its production.

Skin substitutes are a heterogeneous group of materials used to cover the wound surface to provide temporary or permanent physiological wound closure, including protection against mechanical trauma, bacterial colonization, and to provide a moist wound environment. The new generation of bioactive wound dressings, which are usually made using synthetic or natural polymers, should primarily provide oxygen, prevent the wound from drying out and promote healing. Bioactive skin substitutes should have some key features, such as high fluid absorption capacity, biocompatibility, biodegradability, non-toxicity and the ability to stimulate the skin regeneration process (Dai et al., Expert Rev Anti Infect Ther, 2011,9:857-79; Dhivya et al., BioMedicine, 2015, 5(4):22).

Agarose is a biocompatible polysaccharide polymer derived from seaweed (Bhat et al., J Biosci Bioeng, 2012, 114(6):663-70; Hu et al., Materials, 2016, 9(10):816). Agarose is widely used in the field of biomedical engineering for the production of biomaterials supporting wound healing, cartilage repair and regeneration of nervous tissue (Bhat et al., J R Soc Interface, 2010, 8(57):540-54). However, agarose has the disadvantage of not being able to promote the adhesion of eukaryotic cells. The ability of the material to stimulate cell adhesion to its surface is a key feature when using a biomaterial as a skin substitute populated with patient cells (Cao et al., Biomacromol, 2009, 10(10):2954-59). Therefore, the combination of agarose with other biopolymers, e.g. chitosan, is necessary to obtain a biomaterial surface that promotes cell adhesion (Felfel et al., Carbohydr Polym, 2019, 204:59-67). Chitosan is a natural cationic polymer derived from chitin. Chitosan is soluble in an acidic aqueous environment and is often used to prepare hydrogels, films, fibers or sponges - thus the majority of biomaterials used in biomedical engineering, for which biocompatibility is a key feature. The most important features of chitosan include: biocompatibility, lack of toxicity, biodegradability, the ability to accelerate the wound healing process, the ability to retain fluids and antibacterial properties (Rinaudo, Prog Polym Sci, 2006, 31(7):603-32). In addition, chitosan is able to form hydrogen bonds with other polymers due to the presence of polar groups such as -OH and -NH2 (Chaudhary et al., ACS App Mater Interface, 2015, 7(44):24957-62). For this reason, chitosan has also been successfully used in combination with various natural and synthetic polymers such as alginate, collagen, hyaluronic acid, agarose, cellulose, starch, gelatin, polycaprolactone, polylactic acid and polyvinyl alcohol (Lewandowska et al., Inter J. Biol Macromol, 2014, 65:534-41; Zheng et al., Macromol Biosci, 2014, 14(5):655-66; Lewandowska et al., J Molec Liq, 2015, 212:879-84).

The invention solves the problem of obtaining a hydrogel, thin, flexible skin substitute that will provide temporary physiological closure of the wound, protection against pathogens and accelerated regeneration or reconstruction of the skin in the case of prior colonization of the material with the patient's cells.

So far, a hydrogel skin substitute based on chitosan and agarose, in the form of a thin, flexible film, has not been developed. In the available scientific literature in the field of regenerative medicine, there are only reports describing the method of production and the supposed use of materials based on chitosan and agarose, in the form of three-dimensional scaffolds, hydrogels for local injections or hydrogel drug carriers. The aim of the invention is to obtain a bioactive skin substitute based on chitosan and agarose in the form of a thin, hydrogel film, which - due to the valuable biological properties of its polysaccharides - will ensure physiological wound closure and accelerate the regeneration of damaged tissue. From the French patent description No. FR2843972 (A1) there is known a method of obtaining a skin substitute based on a chitosan matrix populated with fibroblasts and/or keratinocytes for use as a bioactive cell dressing for the treatment of chronic and acute wounds and for use in in vitro tests in toxicology. The method of obtaining a skin substitute consists in preparing a solution of chitosan in maleic acid or in another acid with or without the addition of polyvinyl alcohol, glycerol, porogenic agent (sodium chloride, glucose, sucrose), and then drying or freeze-drying and stabilizing the matrix in an alkaline solution, rinsing in aqueous isopropanol, in PBS and/or in a culture medium, followed by in vitro cell culture on a chitosan matrix.

From the Czech patent description No. CZ2007683 (A3) there is known a method for obtaining the chitosan-schizophylan complex (mushroom e-1,3-D-glucan) or its salts, alone or in combination with one or more other natural polysaccharides, for use as preparation for accelerating wound healing and preventing the bandage from sticking to the wound. The preparation method is characterized in that the chitosan and schizophyllan and the antiseptic are dispersed in sterile water and then subjected to air drying or freezing and lyophilization.

An injectable hydrogel based on chitosan and agarose for skin regeneration applications is also known. The hydrogel production method consists in dissolving deacetylated chitosan (at a concentration of 0.75% to 2.5%) in 1% acetic acid, adding agarose (3%) in the form of powder and mixing the resulting suspension at 50°C. The resulting gel is used for local injection into the wound site and has the ability to set at the human body temperature - 37°C (Miguel et al., Carbohydr Polym, 2014, 111:366-73).

There is a known method of obtaining a potential hydrogel dressing material based on chitosan and agar, which consists in dissolving chitosan in a 1.5% acetic acid solution, adding deionized water, agar powder and optionally TMPGDE as a cross-linking agent. The suspension prepared in this way is subjected to a temperature of 120°C in an autoclave (Sahiner et al., Curr Appl Polym Sci, 2017, 1(1):52-62).

A method for producing a three-dimensional scaffold based on chitosan, agarose and gelatin for tissue engineering applications, including cartilage tissue regeneration, is known. The biomaterial production method involves dissolving chitosan in a 1% aqueous solution of acetic acid, adding gelatin, mixing the resulting solution with an aqueous solution of agarose at 50°C, and then adding glutaraldehyde (cross-linking agent). The resulting solution is poured into syringes and incubated for 16 hours in a cooling bath at -12°C. After incubation, the gels are thawed in deionized water at room temperature, washed with deionized water and air dried (Bhat et al., J R Soc Interface, 2010, 8(57):540-54; Bhat et al., J Biosci Bioeng, 2012 ,

114(6):663-70).

A hydrogel based on chitosan and agarose is also known for use as a drug carrier. The hydrogel production method consists in dissolving agarose at 60°C in a chitosan solution prepared in an aqueous solution of acetic acid. Then, 5-Fluorouracil (a drug with anticancer activity) is added to the prepared solution of chitosan and agarose at 60°C, the whole thing is poured into teflon molds and cooled at 4°C (Zamora-Mora et al., Carbohydr Polym, 2014, 111 :348-55).

The subject of the invention is a bioactive skin substitute based on chitosan and agarose, characterized in that chitosan is dissolved in a 0.5% (v/v) aqueous solution of acetic acid and agarose in a 0.1% (w/v) aqueous solution of sodium hydroxide , the weight proportions of the solid components are 2-4% (w/v) of chitosan in relation to acetic acid and 4% (w/v) of agarose in relation to sodium base, respectively, and the components are mixed in a 1:1 ratio ( v:v) giving a final concentration of 1-2% (w/v) chitosan and 2% (w/v) agarose in the final product.

Preferably, chitosan is used in an amount of 3% (w/v) based on acetic acid.

Preferably, the invention contains, in addition to chitosan and agarose, in the amounts described above, bioactive substances such as vitamins, antibiotics, hydrocortisone, curcumin, where as vitamins it contains vitamin C and vitamin E, and as antibiotics it contains vancomycin, gentamicin, daptomycin, tigecycline.

The method of producing the leather substitute according to the invention consists in adding 1:1 (v:v) to 2-4% (w/v) chitosan solution prepared in 0.5% (v/v) aqueous acetic acid solution. 4% (w/v) agarose suspension prepared in 0.1% (w/v) aqueous solution of sodium hydroxide, the whole is mixed on a magnetic stirrer with a heating plate at a temperature of 70-90°C, preferably 75°C, for 10- 25 minutes or until the agarose is completely dissolved, then the resulting mixture is spread in the form of a thin 0.5-2 mm layer on the surface of the mold and air-dried at room temperature.

Preferably, the obtained mixture is brought to a temperature of 45-65°C and bioactive compounds such as vitamins, antibiotics, hydrocortisone, curcumin are added, preferably when vitamin C and vitamin E are used as vitamins, and vancomycin, gentamicin, daptomycin, tigecycline as antibiotics .

Preferably, vitamin C (ascorbic acid) is used in the amount of 0.05-2 mg per 1 ml of the chitosan/agarose mixture or/and vitamin E in the amount of 0.05-2 mg per 1 ml of the chitosan/agarose or/and hydrocortisone mixture in the amount of 1-20 μg per 1 ml of the chitosan/agarose and/or curcumin mixture in the amount

0.5-50 mg per 1 ml of chitosan/agarose or/and gentamicin in the amount of 1-20 μg per 1 ml of chitosan/agarose and/or vancomycin in the amount of 1-20 μg per 1 ml of chitosan/agarose and/or daptomycin in the amount of 0.5-10 μg per 1 ml of the chitosan/agarose mixture or/and tigecycline in the amount

0.5-10 μg per 1 ml of chitosan/agarose mixture.

The biomaterial obtained according to the invention can be used as:

- a functional equivalent of the skin - a biocompatible and biodegradable biomaterial settled in vitro with the patient's own cells, which will constitute a barrier against pathogens, will ensure a moist environment at the wound site and free gas exchange,

- bioactive skin substitute - biocompatible and biodegradable biomaterial providing a moist environment at the wound site, allowing easy transport of oxygen and nutrients, which will isolate the wound from the external environment, preventing infection.

The method of producing a hydrogel skin substitute developed according to the invention, using the neutralization of an acid chitosan solution with an alkaline agarose suspension, is a method not yet used in the process of creating biomaterials, due to the well-known mechanism of gelation and precipitation of an acid solution of chitosan in an alkaline environment. All the methods of production of agarose-chitosan materials described so far assume mixing an aqueous agarose solution with an acid solution of chitosan or adding agarose powder to an acid solution of chitosan. The method of production of the material according to the invention involves the addition of a suspension of agarose in sodium base to an acid solution of chitosan. The production method is unique because it takes into account such concentrations of individual components (chitosan and agarose) and solvents (acetic acid and sodium hydroxide) that enable the neutralization of the acid chitosan solution while maintaining a uniform mixture without signs of chitosan precipitation/gelling. Another novelty is the possibility of adding (immobilizing by trapping) bioactive compounds at the production stage: antibiotics, hormones, vitamins or plant extracts.

The advantage of the method of producing a bioactive skin substitute developed according to the invention is obtaining a biocompatible biomaterial in the form of a thin, flexible, absorbent, hydrogel film, which is biodegradable and non-toxic to eukaryotic cells. In addition, the surface of the material promotes the adhesion and proliferation of skin fibroblasts, keratinocytes and mesenchymal stem cells. The biomaterial also promotes the formation of the extracellular matrix, including collagen fibers, which contributes to the acceleration of the process of rebuilding the injured tissue. The bioactive and biodegradable skin substitute according to the invention will provide a physiological closure of the wound, protecting against the development of infection, and thanks to the ability to retain exudate, it will maintain a moist wound environment, creating optimal conditions for the regeneration process.

The bioactive skin substitute being the subject of the invention, due to its biological properties, will provide appropriate conditions to support skin regeneration. The developed biomaterial can be used in regenerative medicine as a bioactive skin substitute for covering superficial wounds or as a functional equivalent of skin populated with patient cells in vitro.

The subject of the invention is illustrated by the following examples:

Example I

2 ml of 0.5% (v/v) aqueous acetic acid solution was added to 0.06 g of chitosan and mixed until uniform. 2 ml of 0.1% (w/v) sodium hydroxide was added to 0.08 g of agarose and mixed to obtain a uniform mass. Both prepared solutions were combined and stirred for 15 minutes on a magnetic stirrer at a temperature of 75°C, then spread in the form of a thin (1.5 mm) layer on the surface of a flat polystyrene mold with an area of 20 cm ² and air dried at room temperature.

The obtained biomaterial has the structure of an absorbent hydrogel film, is non-toxic, biodegradable and its surface promotes the adhesion and proliferation of skin fibroblasts, keratinocytes and mesenchymal stem cells.

Example II

1 ml of 0.5% (v/v) aqueous acetic acid solution was added to 0.02 g of chitosan and mixed until uniform. 1 ml of 0.1% (w/v) sodium hydroxide was added to 0.04 g of agarose and mixed to obtain a uniform mass. Both prepared solutions were combined and stirred for 10 minutes on a magnetic stirrer at 70°C. The mixture was cooled to 45°C, 25 μl of a solution of vitamin C (3-o-ethyl-L-ascorbic acid) at a concentration of 24 mg/ml (final concentration in the material 300 μg/ml) and 5 μl of a hydrocortisone solution at a concentration of 4 mg/ml (final concentration in the material 10 μg/ml), then spread in a thin (1.5 mm) layer on the surface of a flat polystyrene mold with an area of 10 cm ² and air dried at room temperature.

The obtained biomaterial has the structure of an absorbent hydrogel film, releases bioactive compounds, is non-toxic, biodegradable and its surface promotes the adhesion and proliferation of skin fibroblasts, keratinocytes and mesenchymal stem cells.

Example III

1 ml of 0.5% (v/v) aqueous acetic acid solution was added to 0.03 g of chitosan and mixed until uniform. 1 ml of 0.1% (w/v) sodium hydroxide was added to 0.04 g of agarose and mixed to obtain a uniform mass. Both prepared solutions were combined and stirred for 10 minutes on a magnetic stirrer at 75°C. The mixture was cooled to 50°C, 3 mg of vitamin E (final concentration in the material 1.5 mg/ml) and 5 µl of an antibiotic solution (vancomycin, gentamicin, daptomycin or tigecycline) at a concentration of 4 mg/ml (final concentration in the material) were added 10 μg/ml), and then spread as a thin (1.5 mm) layer on the surface of a flat polystyrene mold with an area of 10 cm ² and air dried at room temperature.

The obtained biomaterial has the structure of an absorbent hydrogel film, releases bioactive compounds, is non-toxic, biodegradable and its surface promotes the adhesion and proliferation of skin fibroblasts, keratinocytes and mesenchymal stem cells.

Example IV

1 ml of 0.5% (v/v) aqueous acetic acid solution was added to 0.03 g of chitosan and mixed until uniform. 1 ml of 0.1% (w/v) sodium hydroxide was added to 0.04 g of agarose and mixed to obtain a uniform mass. Both prepared solutions were combined and stirred for 10 minutes on a magnetic stirrer at 80°C. The mixture was cooled to 55°C, 30 mg of curcumin was added (final concentration in the material 15 mg/ml), then spread in the form of a thin (1.5 mm) layer on the surface of a flat polystyrene mold with an area of 10 cm ² and air dried. in room temperature.

The obtained biomaterial has the structure of an absorbent hydrogel film, releases bioactive compounds, is non-toxic, biodegradable and its surface promotes the adhesion and proliferation of skin fibroblasts, keratinocytes and mesenchymal stem cells.

Claims (6)

1. Hydrogel skin substitute based on chitosan and agarose, characterized in that it is chitosan dissolved in 0.5% (v/v) aqueous acetic acid solution and agarose dissolved in 0.1% (w/v) aqueous sodium hydroxide solution , and the weight proportions of the solid components are 2-4%, preferably 3% (w/v) chitosan in relation to acetic acid and 4% (w/v) agarose in relation to sodium base, where the components are mixed together in a ratio 1:1 (v:v) while maintaining a concentration of 1-2% (w/v) chitosan and 2% (w/v) agarose in the obtained product. 2. The hydrogel substitute according to claim The composition of claim 1, characterized in that it contains bioactive substances such as vitamins, antibiotics, hydrocortisone, curcumin, where as vitamins it contains vitamin C and vitamin E, and as antibiotics it contains vancomycin, gentamicin, daptomycin, tigecycline. 3. A method of producing a hydrogel skin substitute based on chitosan and agarose, characterized in that 2-4% (w/v) chitosan solution prepared in 0.5% (v/v) aqueous acetic acid solution is added in a ratio of 1: 1 (v:v) 4% (w/v) agarose suspension prepared in 0.1% (w/v) aqueous sodium hydroxide solution, the whole is stirred at 70-90°C, preferably 75°C, for 10- 25 minutes or until the agarose is completely dissolved, then the resulting mixture is spread thin and dried. 4. The method of claim 3, characterized in that chitosan is used in an amount of 3% (w/v) with respect to acetic acid. 5. The method of claim The process according to claim 3, characterized in that the resulting mixture is brought to a temperature of 45-65°C and bioactive compounds such as vitamins, antibiotics, hydrocortisone, curcumin are added, the vitamins being preferably vitamin C and vitamin E, and the antibiotics vancomycin , gentamicin, daptomycin, tigecycline. 6. The method of claim 4, characterized in that the bioactive compounds are used in the following amounts: vitamin C (ascorbic acid) in the amount of 0.05-2 mg per 1 ml of the chitosan/agarose mixture or/and vitamin E in the amount of 0.05-2 mg per 1 ml of the chitosan/agarose and/or hydrocortisone mixture in the amount of 1-20 μg per 1 ml of the chitosan/agarose and/or curcumin mixture in the amount of 0.5-50 mg per 1 ml of the chitosan/agarose and/or gentamicin mixture in the amount of 1-20 μg per 1 ml of the chitosan/agarose mixture or/and vancomycin in the amount of 1-20 μg per 1 ml of the chitosan/agarose mixture and/or daptomycin in the amount of 0.5-10 μg per 1 ml of the chitosan/agarose mixture and/or tigecycline in the amount 0.5-10 μg per 1 ml of chitosan/agarose mixture.