TECHNICAL FIELD

The subject-matter of the invention is a method for obtaining a chitosan-based bioactive hemostatic agent suitable for use as a biomaterial that inhibits bleeding of various origins and intensities, as well as an element of hemostatic dressings and systems for controlled delivery and release of bioactive substances, including hydrophobic ones. Further subject-matter of the invention is a chitosan-based bioactive hemostatic agent suitable for use as a hemostatic biomaterial, an element of hemostatic dressings and systems for controlled delivery and release of bioactive substances, including hydrophobic ones.

BACKGROUND OF THE INVENTION

Chitosan is a biopolymer obtained by a chemical or enzymatic deacetylation reaction of chitin, which results in obtaining a polymer with a degree of deacetylation of at least 60%. Its chain is formed by two types of monomer units: N-acetylaminoglucose and glucosamine They are linked by beta-glycosidic bonds. Chitosan in its chemical structure contains many hydroxyl and amino groups, due to which it can undergo a number of modifications. This biopolymer, as a result of physical or chemical cross-linking reactions, forms a hydrogel which has the capability to absorb large amounts of water.

Additionally, native chitosan has hemostatic properties resulting from the presence of free amino groups that interact with erythrocytes and platelets.

Therefore, chitosan finds application as an element of hemostatic agents, which are biomaterials used to stop the hemorrhages by sorbing aqueous fraction of blood and consequently concentrating components responsible for the induction of the blood coagulation cascade.

The best known methods for obtaining chitosan-based hemostatic agents include:

1. Dissolving the chitosan in an aqueous medium of pH less than 6.3 using acetic acid followed by lyophilization;

2. Physically cross-linking the chitosan by polyphosphate compounds followed by lyophilization; 3. Chemically cross-linking the chitosan by an organic compound containing at least two carboxyl or aldehyde groups in its structure, e.g., glutaraldehyde, followed by lyophilization;

4. Forming a composite of chitosan with a polymer or an inorganic compound after prior dissolving the chitosan in an aqueous acid solution;

5. Coating the gauze with a native chitosan in powder form.

Chitosan and its derivatives, due to their antibacterial properties and biocompatibility, are often used in medicine and pharmacy. It is known that the chitosan derivatives for medical and pharmaceutical use must be of high purity.

The known acylation methods require the use of toxic substances, which are very difficult, or even impossible, to remove completely from the product. Therefore, new solutions are being sought, allowing for a simple and short synthesis of a biocompatible acylated chitosan with controlled properties.

Among the known methods for obtaining a chitosan-based hemostatic agent, the most similar to the embodiment according to the present invention is the method presented in US patent No. 8106030 B2, which discloses a method for obtaining a hemostatic agent by dissolving chitosan in an aqueous acid solution and forming a salt soluble in an aqueous medium and adding a second component that does not undergo gelification, e.g. cellulose, alginate, silica or clay.

Chinese patent CN 101053669 B presents a method for obtaining a hemostatic agent, wherein water- soluble chitosan derivatives, e.g., carboxymethyl chitosan, are dissolved in water, and then subjected to lyophilization for 24-60 hours.

US patent No. 8715719 B2 describes a method for obtaining a hemostatic agent, wherein a solution of chitosan having an average molar mass of less than 600,000 g/mol is prepared in a solvent other than water, a cross-linking agent in the form of an organic acid, e.g. lactic acid, is added, and then the lyophilization is performed.

As indicated by the specialist literature and patent reports, there is currently no known method for obtaining a chitosan-based hemostatic agent with enhanced bioactivity by a physical cross-linking reaction using poly(aspartic acid), carried out without the use of toxic substances, and with lysozyme incorporated. AIM OF THE INVENTION

The aim of the invention is to develop a method for producing a chitosan-based bioactive hemostatic agent by a physical cross-linking reaction of chitosan using biocompatible modifiers, and then lysozyme incorporation, and lyophilization of the resulting agent, and to obtain a bioactive hemostatic agent which is a product of the physical cross-linking reaction of chitosan and the lysozyme incorporation into the structure of the cross-linked chitosan. This aim is achieved by the method of the invention.

SUMMARY OF THE INVENTION

The gist of the embodiment according to the invention is that, in the method for obtaining a chitosan- based bioactive hemostatic agent, the chitosan is subjected to the physical cross-linking reaction using poly(aspartic acid) as a cross-linking agent, in an aqueous medium, and then lysozyme is added to the reaction product and the resulting composite is subjected to lyophilization.

According to the invention, the method for obtaining the chitosan-based bioactive hemostatic agent comprising converting a polysuccinimide by alkaline hydrolysis to poly(aspartic acid), which is purified before further use in the process, and dissolving the chitosan in an aqueous acid solution, precipitating the chitosan with an alkali, purifying it and subjecting it to the physical cross-linking reaction in an aqueous medium with poly(aspartic acid), and then cooling the reaction suspension to a temperature not higher than 5°C and adding an aqueous lysozyme solution, freezing, and subjecting to lyophilization, is based upon that the polysuccinimide (PSI) is hydrolyzed by introducing an aqueous alkali solution addition to the aqueous suspension of the polysuccinimide, with constant stirring and adding the alkali solution portionwise until the pH of the mixture reaches 11.

Next, the cations in the resulting poly(aspartic acid) salt are exchanged for protons using an ion- exchange resin.

However, independently of the process of converting the polysuccinimide to poly(aspartic acid), chitosan having an average molar mass of 2,000 to 800,000 g/mol and a degree of deacetylation of 60- 100% is dissolved in an aqueous acid solution at a concentration of 1 to 5 wt%, after which the chitosan is precipitated with an aqueous alkali solution added until a neutral pH of the mixture is obtained. Next, the resulting salt of the acid used is removed from the mixture by membrane dialysis using MWCO dialysis membranes.

The precipitated, purified chitosan in the form of an aqueous suspension is placed in a reaction vessel, an aqueous solution of the obtained poly(aspartic acid) is introduced as a cross-linking agent, while maintaining continuous stirring, in the amount of 0.1 to 6 g of poly(aspartic acid) per 1 g of chitosan, and after the chitosan has been cross-linked, the temperature of the mixture is lowered to no more than 5°C and an aqueous lysozyme solution is added while stirring, then the mixture containing the cross- linked chitosan with the lysozyme incorporated into its structure is subjected to freezing and lyophilization at a temperature of -40°C to -60°C under a pressure of less than 1 mbar.

Preferably, the polysuccinimide obtained by either aspartic acid or maleic anhydride and urea polycondensation is used as a source of poly(aspartic acid).

Preferably, an aqueous solution of sodium or potassium hydroxide at a concentration of at least 1 wt%, and most preferably 5 wt%, is used as an agent hydrolyzing the polysuccinimide.

Preferably, the process for obtaining poly(aspartic acid) employs an aqueous suspension which contains 10 wt% of the polysuccinimide.

Preferably, the alkaline hydrolysis process of the polysuccinimide is carried out with continuous stirring using a magnetic or mechanical stirrer at a speed of 300 rpm.

Preferably, the alkali solution is added portionwise to the polysuccinimide suspension while stirring until the pH of the mixture reaches 11.

Preferably, the cations derived from the alkali used are removed from the poly(aspartic acid) salt solution using a strong sulfonic acid ion-exchange resin.

Preferably, for obtaining chitosan in an amorphous form, an aqueous chitosan solution at a concentration of 1 to 10 wt% is used.

Preferably, the chitosan is dissolved in a 2-5 wt% solution of hydrochloric acid.

Preferably, a chitosan having an average molar mass of 100,000 to 400,000 g/mol is used.

Preferably, a chitosan having a degree of deacetylation of more than 75% is used.

Preferably, a chitosan of fungal origin is used.

Preferably, the chitosan is precipitated with a 2 wt% aqueous sodium or potassium hydroxide solution.

Preferably, the chitosan suspension is purified from the salts formed using dialysis membranes with MWCO (Molecular Weight Cut-Off) of 10,000-12,000 Da.

Preferably, the poly(aspartic acid) is added portionwise to the chitosan using continuous stirring at speed of 300-400 rpm. Preferably, the physical cross-linking of the chitosan is carried out by maintaining a ratio of 0,5-2,25 g of poly(aspartic acid) to 1 g of chitosan.

Preferably, the suspension containing the cross-linked chitosan is cooled to 3°C.

Preferably, the lysozyme is added to the suspension with the cross-linked chitosan at 3°C with constant stirring.

Preferably, 12.5 to 25 mg of lysozyme per 1 g of chitosan is used.

Preferably, chicken egg white lysozyme is used.

Preferably, freezing of the composite of cross-linked chitosan with incorporated lysozyme is carried out at a temperature below 0°C, preferably at -20°C.

According to the invention, the chitosan-based bioactive hemostatic agent containing lysozyme is characterized in that it is a product of the implementation of the method of the invention, which comprises converting the polysuccinimide, obtained either from aspartic acid or maleic anhydride and urea, by alkaline hydrolysis, to poly(aspartic acid), dissolving the chitosan in an aqueous acid solution, precipitating the chitosan with an alkali, purifying the chitosan and subjecting it to physical cross-linking reaction in an aqueous medium with the obtained poly(aspartic acid), and then cooling the reaction suspension and adding an aqueous lysozyme solution, freezing and subjecting to lyophilization the suspension containing cross-linked chitosan with lysozyme incorporated into its structure, wherein the polysuccinimide is hydrolyzed by introducing an aqueous alkali solution addition to the aqueous polysuccinimide suspension, with constant stirring and adding the alkali solution portionwise, until the pH of the mixture reaches 11, then the cations are removed from the obtained poly(aspartic acid) salt by ion exchange on an ion-exchange resin, and the chitosan with an average molar mass of 2,000 to 800,000 g/mol and a degree of deacetylation of 60-100% is dissolved in a 1-5 wt% acid solution, then the chitosan is precipitated with an alkali solution added until a neutral pH of the mixture is reached, and then the salt formed of the acid used is removed from the mixture by membrane dialysis, then the precipitated chitosan in the form of an aqueous suspension is placed in a reaction vessel, an aqueous solution of the obtained poly(aspartic acid) is introduced while maintaining constant stirring and the mass ratio of chitosan : poly(aspartic acid) of 1:0.1 to 1:6, and after cross-linking the chitosan, the mixture is cooled to not more than 5°C and an aqueous lysozyme solution is added with constant stirring, maintaining the mass ratio of chitosan to lysozyme of 1000:12.5-25, wherein the mixture containing the cross-linked chitosan with lysozyme incorporated into its structure is subjected to freezing and then lyophilization at a temperature of -40° to -60°C under a pressure of less than 1 mbar.

The bioactive hemostatic agent, based on physically cross-linked chitosan with lysozyme incorporated, is characterized by a high sorption capacity of aqueous solutions, exhibits antibacterial activity and is easily biodegradable and non-cytotoxic as well.

The embodiment of the invention is illustrated in the following examples, not limiting the scope of its protection.

EXAMPLES

Example 1

To 5 g of polysuccinimide, obtained by polycondensation of aspartic acid, 50 mL of water was added, the solution was stirred with a mechanical stirrer, and then, while stirring, portions of 5 wt% NaOH solution were added dropwise, until the pH of the solution reached 11. The hydrolysis reaction was carried out for 1 hour. Next, the entire reaction mixture was transferred to an ion-exchange column to remove the sodium ions from the reaction mixture and convert the sodium salt of poly(aspartic acid) to poly(aspartic acid). The resulting solution was diluted so that the total amount of aqueous poly(aspartic acid) solution was 50 ml. The obtained aqueous poly(aspartic acid) solution showed pH = 2.6, sodium ion concentration was less than 15 mg/L.

In the meantime, to prepare chitosan in the amorphous form 10 g of chitosan with an average molar mass of 300,000 g/mol, obtained from shrimp, was added to 2000 mL of distilled water (conductivity 10 mS/cm) and while stirring, small portions of 5 wt% HCI solution were added dropwise, until pH 5 was reached. The mixture was heated to 50°C until the chitosan dissolved completely. Next, 5 wt% NaOH solution was added to the solution while stirring until pH 7 was reached. Chitosan was obtained in the amorphous form, which was then purified with dialysis membrane with MWCO in the range of 10,000-12,000 Da. The final concentration of chitosan suspension in water was 10 g/L.

The hemostatic agent was prepared using a mechanical stirrer (400 rpm). 4 mL of aqueous poly(aspartic acid) solution was added dropwise over 10 minutes, while stirring, to 40 mL of chitosan suspension at a concentration of 10 g/L. The product formed was left to stand for 20 minutes and then decanted. The aqueous suspension of such cross-linked chitosan was cooled to 3°C and then 5 mg of lysozyme in the form of an aqueous solution at a concentration of 1 mg/ml was added while stirring. Composite material formed was frozen and then subjected to lyophilization at a temperature -40°C to -60°C under a pressure of less than 1 mbar to obtain the finished hemostatic product.

The FT-IR (Fourier-Transform Infrared Spectroscopy) spectrum of the obtained hemostatic agent is shown in the accompanying drawing in Fig. 1 (Example 1). The spectrum shows the characteristic bands for chitosan originating from free hydroxyl (3351 cm ¹ ) and amine (1585 cm ¹ ; 1149 cm ¹ ) groups, bands typical of aliphatic moieties (2917 cm ¹ ; 2879 cm ¹ ) of glycosidic bonds between chitosan mer units (1063 cm ¹ ) and glucopyranose rings (895 cm ¹ ). Additionally, bands originating from carboxyl groups derived from poly(aspartic acid) (1706 cm ¹ ) are present.

Example 2

To 5 g of polysuccinimide, obtained by polycondensation of urea and maleic anhydride in a microwave radiation field, 50 mL of water was added. The solution was stirred with a mechanical stirrer, and then, while stirring, portions of 2 wt% KOH solution were added dropwise, until the pH of the solution reached 11. The hydrolysis reaction was carried out for 1 hour and 30 minutes. Next, the mixture was transferred to an ion-exchange column to remove the potassium ions from the reaction mixture and convert the potassium salt of poly(aspartic acid) to poly(aspartic acid). The resulting solution was diluted so that the total amount of aqueous poly(aspartic acid) solution was 50 mL. The obtained aqueous poly(aspartic acid) solution showed pH = 2.6, potassium ion concentration was less than 15 mg/L.

In the meantime, to prepare chitosan in the amorphous form, 10 g of chitosan with an average molar mass of 200,000 g/mol, obtained from mushrooms ( Agaricus ), was added to 2000 mL of distilled water (conductivity 10 mS/cm) and while stirring, small portions of 5 wt% acetic acid solution were added dropwise, until pH 5 was reached. The mixture was heated to 60°C until the chitosan dissolved completely. Next, 2 wt% KOH solution was added to the solution while stirring until pH 7 was reached. Chitosan was obtained in the amorphous form, which was then purified with dialysis membrane with MWCO in the range of 10,000-12,000 Da. The final concentration of chitosan suspension in water was 10 g/L.

The hemostatic agent was prepared using a mechanical stirrer (300 rpm). 4 mL of aqueous poly(aspartic acid) solution was added dropwise over 10 minutes, while stirring, to 40 mL of chitosan suspension at a concentration of 10 g/L. The product formed was left to stand for 20 minutes and then decanted. The aqueous suspension of such cross-linked chitosan was cooled to 5°C and then 10 mg of lysozyme in the form of an aqueous solution at a concentration of 2 mg/ml was added while stirring. Composite material formed was frozen and then subjected to lyophilization at a temperature of -40°C to -60°C under a pressure of less than 1 mbar to obtain the finished hemostatic product.

The FT-IR spectrum of the obtained hemostatic agent is shown in the accompanying drawing in Fig. 2 (Example 2). The spectrum shows the characteristic bands for chitosan originating from free hydroxyl (3350 cm ¹ ) and amine (1525 cm ¹ ; 1156 cm ¹ ) groups, bands typical of aliphatic moieties (2957 cm ¹ ; 2880 cm ¹ ) of glycosidic bonds between chitosan mer units (1061 cm ¹ ) and glucopyranose rings (901 cm ¹ ). Additionally, bands originating from carboxyl groups derived from poly(aspartic acid) (1705 cm ¹ ) are present.

ADVANTAGEOUS EFFECTS OF THE INVENTION

The invention makes it possible to obtain a chitosan-based bioactive hemostatic agent containing lysozyme, characterized by more advantageous biological properties, increased biodegradability, sorptive and antibacterial capabilities in comparison with native chitosan-based hemostatic agents.

In the process carried out by the method of the invention, the physical cross-linking of chitosan is carried out with the use of a cross-linking agent which is non-toxic, but unfortunately unstable over time, i.e. poly(aspartic acid), which requires performing the entire process of the hemostatic agent production in such a way that it is possible to obtain chitosan in an amorphous form and poly(aspartic acid) practically simultaneously, in two process steps independent of each other. Thus, it is possible to obtain a chitosan hydrogel, which has a chemically unmodified structure, has free amino groups responsible for the beneficial biological properties of chitosan and does not negatively affect living eukaryotic cells.

Cross-linked chitosan produced by the method of the invention contains the lysozyme enzyme, which is stabilized in the three-dimensional structure of the cross-linked chitosan by hydrogen bonds. Modification of chitosan by lysozyme introduction allows increasing the biodegradation rate of chitosan material.

The method of the invention makes it possible to modify the degree of chitosan hydrogel cross-linking by changing the amount of cross-linking agent and the final product biodegradation rate by changing the amount of lysozyme.

Moreover, an advantageous feature of the method of the invention is the lack of harmful by-products.