CN114010584A

CN114010584A - Antibacterial wound repair gel and preparation method thereof

Info

Publication number: CN114010584A
Application number: CN202111322063.5A
Authority: CN
Inventors: 庄心蕊; 李漫; 李琳; 陈乃猛; 张燕; 刘建建
Original assignee: Bloomage Biotech Co Ltd
Current assignee: Bloomage Biotech Co Ltd
Priority date: 2021-11-09
Filing date: 2021-11-09
Publication date: 2022-02-08
Anticipated expiration: 2041-11-09
Also published as: CN114010584B

Abstract

The invention provides an antibacterial wound repair gel which comprises a pseudoprotein-hyaluronic acid gel and a bioactive component dispersed in the pseudoprotein-hyaluronic acid gel, wherein the pseudoprotein-hyaluronic acid gel is formed by polymerizing first hyaluronic acid or a salt thereof and a pseudoprotein biomaterial. The bacteriostatic wound repair gel has film forming property, avoids the invasion of microorganisms and plays a role of barrier; can provide a wound surface moist environment and create a microenvironment beneficial to tissue growth; the release of the effective factors is continuous, the cell activity is stimulated, the cell healing is promoted, the duration is long, and the dressing change frequency is reduced. Can be widely applied to the repair of various skin wounds caused by operation wounds, burns, scalds and daily physical activities.

Description

Antibacterial wound repair gel and preparation method thereof

Technical Field

The invention belongs to the technical field of gel, and particularly relates to bacteriostatic wound repair gel and a preparation method thereof.

Background

The skin is the largest organ of the human body, is the first barrier between the body and the nature, and has the main function of protection, and the invasion of pathogenic microorganisms in the environment and the loss of body fluid of the body are prevented. The skin is comprised of three major parts: epidermal, dermal and subcutaneous tissue. Wherein the epidermis layer is the outermost layer of the skin, and the epidermis layer is an important guarantee for the skin to play a protective function. The division, proliferation and migration capability of epithelial cells are important guarantees for wound healing.

The normal skin is subjected to external factors such as surgery, external forces, chemicals and intrinsic factors such as local blood supply disorder, etc. to cause trauma on the skin surface, which is accompanied by the destruction of the skin integrity and the loss of certain normal tissues. At the same time, the normal function of the skin is affected and impaired to some extent.

The wound healing process mainly comprises a series of pathophysiological processes of local tissue regeneration, repair and reconstruction and repair after tissue loss caused by the action of a wound-causing factor. The process of wound healing is based on a series of biological activities of inflammatory cells, neutrophils, repair cells and the like, and simultaneously, cell matrixes are involved. When skin soft tissue is seriously damaged and can not be completely repaired by self, fibrous tissue replaces and repairs to form scars, so that the integrity and the attractiveness of the skin are affected, and therefore, products beneficial to wound repair are often used in the wound repair process.

In the early 90 s, Turner reports that moist environment treatment significantly accelerated the rate of wound area reduction in patients, with a large amount of granulation tissue formation and rapid epithelial regeneration; knighton also found that the use of occlusive dressings to keep the wound bed moist and create a hypoxic environment, favoring capillary growth and regeneration; wheeland reports that the wound does not scab under the humid environment, is beneficial to keeping the activity of tissue cells and the migration of epithelial cells on a smooth surface, and accelerates the healing speed

At present, a wound repair product mainly represents medical wound gel, and the use method is to paint the gel on a wound to-be-repaired area, but the wound repair product has two defects: 1) the characteristics of high molecular weight and hydrophilicity of some biomacromolecules and the barrier property of skin enable the activity of the gel to be low, and the speed of the skin absorbing gel effective factors is reduced, so that the wound repair efficiency is reduced; 2) the gel is non-film-forming, so that the invasion of external microorganisms is not blocked, and the repair efficiency is reduced; 3) as a wound dressing, the gel has certain antibacterial property to avoid wound infection, and the traditional gel loses antibacterial property in the later period after the antibacterial agent is exuded in the earlier period, so that the antibacterial effect cannot be achieved in the later period, the healing speed of the wound is slowed down, even the wound infection is caused, and the repair of the wound is not facilitated.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a gel for promoting bacteriostatic wound repair and a preparation method thereof.

Specifically, the present invention relates to the following aspects:

1. the bacteriostatic wound repair gel is characterized by comprising a pseudoprotein-hyaluronic acid gel and a bioactive component dispersed in the pseudoprotein-hyaluronic acid gel, wherein the pseudoprotein-hyaluronic acid gel is formed by polymerizing first hyaluronic acid or a salt thereof and a pseudoprotein biomaterial.

2. The bacteriostatic wound repair gel according to item 1, wherein the mass ratio of hyaluronic acid or a salt thereof to the pseudoprotein biomaterial in the pseudoprotein-hyaluronic acid gel is 1: 1-3, preferably, in the bacteriostatic wound repair gel, the mass ratio of the pseudoprotein-hyaluronic acid gel is 0.5-3%, the mass ratio of the bioactive component is 0.03-0.1%, further preferably, the molecular weight of the first hyaluronic acid or the salt thereof is 100-300kDa, preferably 200-260 kDa.

3. The bacteriostatic wound repair gel according to item 1, wherein the pseudoprotein biomaterial comprises a polymer of amino acids, preferably a polyester of amino acids, and further preferably an arginine-based polyester urethane.

4. The bacteriostatic wound repair gel according to item 1, wherein the bioactive component comprises second hyaluronic acid or a salt thereof, and one or more of bletilla striata, ectoin and panthenol, preferably comprises second hyaluronic acid or a salt thereof, and bletilla striata, ectoin and panthenol.

5. A method for preparing bacteriostatic wound repair gel, which comprises the following steps:

dissolving a first hyaluronic acid or a salt thereof and a pseudoprotein biomaterial in a mixed solution of dimethyl sulfoxide and water to form a solution A,

the solution A is polymerized at high temperature to obtain gel B,

fully swelling the gel in purified water to obtain gel C,

and adding a bacterium filtering aqueous solution of a bioactive component into the gel C to obtain the bacteriostatic wound repair gel.

6. The method according to item 5, wherein the step of polymerizing the solution A at a high temperature comprises: reacting for 4-6 hours at 60-90 ℃.

7. The method according to item 5, wherein the mass ratio of the first hyaluronic acid or salt thereof to the pseudoprotein biomaterial in solution A is 1: 1-3, preferably the mass ratio of the gel B to the bioactive component is 0.03-0.1%, further preferably the molecular weight of the first hyaluronic acid or the salt thereof is 100-300kDa, preferably 200-260 kDa.

8. The method according to item 5, wherein the pseudoprotein biomaterial comprises a polymer of amino acids, preferably a polyester of amino acids, further preferably an arginine-based polyester urethane.

9. The method according to item 5, wherein the bioactive component comprises one or more of second hyaluronic acid or a salt thereof, and bletilla striata, ectoin and panthenol, and preferably comprises second hyaluronic acid or a salt thereof, and bletilla striata, ectoin and panthenol.

10. The method according to item 5, wherein the method is used for preparing the bacteriostatic wound repair gel according to any one of items 1 to 4.

The bacteriostatic wound repair gel has film forming property, avoids the invasion of microorganisms and plays a role of barrier; can provide a wound surface moist environment and create a microenvironment beneficial to tissue growth; the release of the effective factors is continuous, the cell activity is stimulated, the cell healing is promoted, the duration is long, and the dressing change frequency is reduced. Can be widely applied to the repair of various skin wounds caused by operation wounds, burns, scalds and daily physical activities.

Detailed Description

The present invention is further illustrated by the following examples, which are intended to be purely exemplary of the invention and are not intended to be limiting.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in experimental or practical applications, the materials and methods are described below. In case of conflict, the present specification, including definitions, will control, and the materials, methods, and examples are illustrative only and not intended to be limiting. The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.

Aiming at the problems of the existing wound repair products, the invention provides an antibacterial wound repair gel which comprises a pseudoprotein-hyaluronic acid gel and a bioactive component dispersed in the pseudoprotein-hyaluronic acid gel, wherein the pseudoprotein-hyaluronic acid gel is formed by polymerizing first hyaluronic acid or a salt thereof and a pseudoprotein biological material.

The pseudoprotein biomaterial is a novel, degradable, biologically active material synthesized based on poly (ester, amide, urea or polyurethane) polymers and amino acids (such as arginine, lysine, proline, serine, etc.). The biological material not only has the characteristics of protein, but also has the characteristics of non-protein, and the main chemical structure of the material is mainly formed by peptide chains and non-peptide chains, so that the biological material has unique biological characteristics, such as inhibiting inflammatory reaction, promoting cell growth and healing wounds.

The pseudoprotein biomaterial comprises a polymer of amino acids, such as amino acid based polyesters, polyamides, ureas or polyurethanes, preferably amino acid based polyesters. The amino acid can be selected from one of arginine, lysine, serine, proline, leucine and glycine. Further preferred is an arginine-based polyester urethane (Arg-PEUU).

Hyaluronic Acid (HA) is a viscoelastic biomaterial with different molecular weights, and it HAs no antigenicity, no sensitization, no immunoreaction, no pyrogen, no colloid permeability, and stable physicochemical properties. Exogenous hyaluronic acid can be combined with fibrin to form an intercellular matrix framework, and the extracellular matrix framework regulates the proliferation of fibroblasts and the formation of granulocytes, so that soft tissues are repaired, and meanwhile, HA is also an important component of the extracellular matrix and HAs the characteristics of resisting inflammation and promoting wound healing. Research shows that the high molecular weight HA can inhibit inflammation and promote cell migration, and the low molecular weight HA can promote cell proliferation and is beneficial to wound healing.

The hyaluronate can be sodium salt, potassium salt, zinc salt, magnesium salt, calcium salt, gold salt and the like of hyaluronic acid.

The first hyaluronic acid or salt thereof is a hyaluronic acid or salt thereof having a relatively high molecular weight and is used to polymerize with the pseudoprotein biomaterial to form a gel. In a specific embodiment, the first hyaluronic acid or salt thereof has a molecular weight of 100-300kDa, such as 100kDa, 110kDa, 120kDa, 130kDa, 140kDa, 150kDa, 160kDa, 170kDa, 180kDa, 190kDa, 200kDa, 210kDa, 220kDa, 230kDa, 240kDa, 250kDa, 260kDa, 270kDa, 280kDa, 290kDa, 300kDa, preferably 200-260 kDa.

The mass ratio of hyaluronic acid or salt thereof to the pseudoprotein biomaterial in the pseudoprotein-hyaluronic acid gel is 1:1 to 3, for example, 1:1, 1:2, 1: 3.

In a specific embodiment, in the bacteriostatic wound repair gel, the pseudoprotein-hyaluronic acid gel accounts for 0.5% to 3% by mass, for example, may be 0.5%, 1%, 2%, 3%, and the bioactive component accounts for 0.03% to 0.1% by mass, for example, may be 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%.

Wherein the bioactive component comprises second hyaluronic acid or its salt, and one or more of rhizoma Bletillae, ectoin, and panthenol, including one, two, three, or four thereof.

Ectoin (Ectoin) is derived from high halophilic bacteria (Halomonas Elongata), is an amino acid derivative, belongs to an extreme enzyme component, and can protect halophilic bacteria from being damaged under extreme conditions of high salt, high temperature and high ultraviolet radiation. Ectoin is also a strongly hydrophilic substance, and these small amino acid derivatives bind to water molecules around it and produce the so-called "Ectoin hydrorecombination". These complexes then once again surround cells, enzymes, proteins and other biomolecules, forming a protective, nourishing and stable hydrated shell around them. Studies have shown that ectoin has a highly potent restorative protective effect, and also calms and soothes irritated and damaged skin, and that the regeneration process of skin tissue is significantly increased due to its excellent anti-inflammatory properties.

Bletilla striata is also called as bletilla striata, and tubers of the bletilla striata have a plurality of effects of disinfection, hemostasis, wound infection prevention and the like, and have good bactericidal and anticancer effects.

Panthenol is also known as panthenol, provitamin B5. Colorless to yellowish, transparent and viscous liquids with a slight odor. Panthenol is a vitamin B nutritional supplement which is widely applied, and is widely applied in the fields of medicines, foods, feeds, cosmetics and the like, and skin medicaments, skin care and hair cosmetics are widely applied.

In a particular embodiment, the bioactive ingredient comprises a second hyaluronic acid or a salt thereof.

In a particular embodiment, the bioactive ingredient includes a second hyaluronic acid or salt thereof and ectoin.

In a particular embodiment, the bioactive ingredient comprises a second hyaluronic acid or a salt thereof, bletilla striata, ectoin.

In a preferred embodiment, the bioactive ingredient comprises a second hyaluronic acid or a salt thereof, bletilla striata, ectoin, panthenol.

In a preferred embodiment, the bioactive ingredient consists of a second hyaluronic acid or a salt thereof, bletilla striata, ectoin, panthenol.

The second hyaluronic acid or a salt thereof may be the same as or different from the first hyaluronic acid or a salt thereof. In a particular embodiment, the second hydrolyzed hyaluronic acid or salt thereof has a molecular weight of 5-20kDa, e.g. may be 5kDa, 6kDa, 7kDa, 8kDa, 9kDa, 10kDa, 11kDa, 12kDa, 13kDa, 14kDa, 15kDa, 16kDa, 17kDa, 18kDa, 19kDa, 20kDa, preferably 8-15 kDa.

The invention also provides an antibacterial wound repair gel, which consists of the pseudoprotein-hyaluronic acid gel, and bioactive components and water dispersed in the pseudoprotein-hyaluronic acid gel. The description of the pseudoprotein-hyaluronic acid gel and the bioactive ingredient is as described above.

In a specific embodiment, the bacteriostatic wound repair gel consists of a pseudoprotein-hyaluronic acid gel, and dispersed therein a second hyaluronic acid or a salt thereof, bletilla striata, ectoin, panthenol, and water. The description of the pseudoprotein-hyaluronic acid gel and the bioactive ingredient is as described above.

The invention also provides a method for preparing the bacteriostatic wound repair gel, which is characterized by comprising the following steps of:

the solution A is polymerized at high temperature to obtain gel B,

fully swelling the gel in purified water to obtain gel C,

Wherein the pseudoprotein biomaterial comprises a polymer of amino acids, such as amino acid based polyesters, polyamides, urea or polyurethanes, preferably amino acid based polyesters. The amino acid can be selected from one of arginine, lysine, serine, proline, leucine and glycine. Further preferred is an arginine-based polyester urethane (Arg-PEUU).

The first hyaluronic acid or salt thereof has a relatively high molecular weight, and in a specific embodiment, the molecular weight of the first hyaluronic acid or salt thereof is 100-300kDa, such as 100kDa, 110kDa, 120kDa, 130kDa, 140kDa, 150kDa, 160kDa, 170kDa, 180kDa, 190kDa, 200kDa, 210kDa, 220kDa, 230kDa, 240kDa, 250kDa, 260kDa, 270kDa, 280kDa, 290kDa, 300kDa, preferably 200-260 kDa.

In one embodiment, the step of polymerizing the solution a at high temperature is: reacting for 4-6 hours at 60-90 ℃.

In solution a, the mass ratio of the first hyaluronic acid or salt thereof to the pseudoprotein biomaterial is 1: 1-3.

The purpose of fully swelling the gel B is that the three-dimensional molecular network of the gel can be fully extended, so that the gel has certain viscoelasticity, and in addition, the active substances can enter three-dimensional pore channels to slowly release to play a role. Specifically, the gel B is soaked in purified water, the temperature is controlled to be 25-30 ℃, the pH value is adjusted to be 5-7, and the purified water is replaced every 2-3 hours to obtain the fully swollen gel C.

In a specific embodiment, the mass ratio of the gel B to the bioactive component is 50: 3.

the bioactive components comprise second hyaluronic acid or its salt, and one or more of rhizoma Bletillae, ectoin, and panthenol, i.e. one, two, three or four of them.

Wherein the molecular weight of the second hydrolyzed hyaluronic acid or salt thereof is 5-20kDa, preferably 8-15 kDa.

Microbial control of the bioactive ingredient is required prior to its addition to gel C. Specifically, the bioactive ingredient may be dissolved in purified water and filtered through a 0.45 μm filter.

The method can be used for preparing the bacteriostatic wound repair gel.

The bacteriostatic wound repair gel disclosed by the invention takes a pseudo-protein biomaterial, particularly arginine-based polyester urethane as the pseudo-protein biomaterial, and the pseudo-protein biomaterial and high-molecular sodium hyaluronate generate a polymerization reaction under a specific condition to form hydrogel with a three-dimensional pore structure, and the hydrogel is coated on the surface of a wound, so that a layer of film can be formed on the outer surface layer, the wound part is protected, the interference of microorganisms on the external part is blocked, and a clean repair environment is provided for the wound. In addition, the unique guanidyl side group of the arginine-based polyester urethane has strong cationic property in polymerization reaction, and can weaken inflammatory reaction and promote cell growth and wound healing. The high molecular hyaluronic acid is combined with the pseudoprotein biomaterial, and has good biocompatibility. The added other bioactive components have synergistic effect, and are more favorable for improving the activity of cell repair factors and accelerating the repair of surface wounds.

Examples

The experimental methods used in the following examples are all conventional methods, unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1

(1) Weighing 0.5g of first hyaluronic acid (sodium hyaluronate, molecular weight: 260kDa) and 0.5g of arginine-based polyester urethane (Arg-PEUU) in 150ml of mixed solution of DMSO and water (wherein the volume ratio of the DMSO to the water is 2: 3), and uniformly stirring until uniform solution A is formed;

(2) weighing bioactive components 0.06g, containing: dissolving 0.02g of second hyaluronic acid (sodium hyaluronate, molecular weight: 9kDa), 0.02g of ectoin, 0.01g of bletilla striata and 0.01g of panthenol in 50g of purified water, and filtering with 0.45 μm filter membrane to obtain solution B;

(3) placing the uniform solution A in a constant-temperature water bath at 80 ℃ for reacting for 5 hours to generate a polymerization reaction, thereby obtaining gel B;

(4) soaking the gel B into purified water, controlling the temperature to be 25-30 ℃, adjusting the pH value to be 5-7 by 0.25mol/L sodium hydroxide, and replacing the purified water every 2-3 hours to obtain gel C;

(5) and mixing the gel C with the prepared solution B, and uniformly stirring at room temperature to obtain the repair gel. Wherein the mass ratio of the pseudoprotein-hyaluronic acid gel in the repair gel is 0.67%.

Examples 2 to 4

Examples 2 to 4 are different from example 1 in that the amount of the first hyaluronic acid added and the amount of the arginine-based polyester urethane added are different, and the amounts of the other materials added and the reaction conditions are the same as in example 1. Specifically, as shown in Table 1, the amount of the first hyaluronic acid (sodium hyaluronate, molecular weight: 260kDa) added in example 2 was 0.33g, and the amount of the arginine-based polyester urethane was 0.67 g; the amount of the first hyaluronic acid (sodium hyaluronate, molecular weight: 260kDa) added in example 3 was 0.25g, the amount of the arginine-based polyester urethane was 0.75g, and the amount of the first hyaluronic acid (sodium hyaluronate, molecular weight: 260kDa) added in example 4 was 0.1g, and the amount of the arginine-based polyester urethane was 0.9 g. In each example, the mass ratio of the pseudoprotein-hyaluronic acid gel in the repair gel was 0.67%.

Examples 5 to 7

Examples 5 to 7 are different from example 1 in the molecular weight of the first hyaluronic acid, and the addition amount of other substances and reaction conditions are the same as in example 1. Specifically, as shown in table 1, the molecular weight of the first hyaluronic acid (sodium hyaluronate) in example 5 is 300 kDa; the molecular weight of the first hyaluronic acid (sodium hyaluronate) in example 6 was 100 kDa; the molecular weight of the first hyaluronic acid (sodium hyaluronate) in example 7 was 20 kDa.

In each example, the mass ratio of the pseudoprotein-hyaluronic acid gel in the repair gel was 0.67%.

Comparative example 1

The repair gel of comparative example 1 differs from example 1 only in that it does not contain a bioactive ingredient. Specifically, the preparation steps are as follows:

(2) placing the uniform solution A in a constant-temperature water bath at 80 ℃ for reacting for 5 hours to generate a polymerization reaction, thereby obtaining gel B;

(3) soaking the gel B into purified water, controlling the temperature to be 25-30 ℃, adjusting the pH value to be 5-7 by 0.25mol/L sodium hydroxide, and replacing the purified water every 2-3 hours to obtain repair gel;

wherein the mass ratio of the pseudoprotein-hyaluronic acid gel in the repair gel is 0.67%.

Comparative example 2

(1) 1g of first hyaluronic acid (sodium hyaluronate, molecular weight: 260kDa) was weighed and sufficiently swelled in 150ml of purified water to obtain gel A.

(2) Weighing bioactive components 0.06g, containing: 0.02g of second hyaluronic acid (sodium hyaluronate, molecular weight: 9kDa), 0.02g of ectoin, 0.01g of bletilla striata, and 0.01g of panthenol were dissolved in 50g of purified water, and filtered through a 0.45-micrometer filter membrane for use, thereby obtaining a solution B.

(3) And mixing the gel A with the prepared solution B, and uniformly stirring at room temperature to obtain the repair gel.

Wherein the mass ratio of the hyaluronic acid gel in the repair gel is 0.67%.

The pseudoprotein itself does not form a gel.

The conditions of the above examples and comparative examples are shown in table 1.

TABLE 1 reaction conditions for the different examples and comparative examples

Test examples

Test example 1 biological test

According to the regulations of the national standard GB/T16886.1-2011 medical instrument biological evaluation and the requirements of the appendix A "biological evaluation experiment", refer to the section 5 of "GB/T16886.5-2017 medical instrument biological evaluation": ex vivo cytotoxicity assay, "GB/T16886.10-2017 medical device biology evaluation" part 10: and (3) stimulation and sensitization tests, and biological evaluation test items are carried out on the product. The items include: cytotoxicity, skin sensitization, and intradermal stimulation

1.1 test items

1) Cytotoxicity

The products of the examples and comparative examples were taken as 0.2g sample: 1mL of leaching medium, wherein the leaching medium is MEM medium containing serum, and the test solution is prepared at (37 +/-1) DEG C and (24 +/-2) h and is taken according to the leaching solution method specified in GB/T16886.5-2017.

2) Skin sensitization

Taking the products of the examples and comparative examples, the weight ratio of 0.2g sample: 1mL of leaching medium, namely 0.9% of sodium chloride injection and cottonseed oil respectively, (121 +/-2) DEG C, (1 +/-0.1) h, preparing test solution, and taking the test solution to perform the maximum dose test method specified in GB/T16886.10-2017.

3) Intradermal stimulation

Taking the products of the examples and the comparative examples, adding 0.9% of sodium chloride injection and cottonseed oil according to the proportion of 0.2g/mL respectively, leaching at the temperature of 37 +/-1 ℃ for 72 +/-2 h to prepare leaching liquor, standing, taking supernate as test liquid, and taking the test liquid to test according to the method specified in GB/T16886.10-2017.

1.2 assay analysis

1) Cytotoxicity

The test is carried out by adopting a leaching liquor test-MTT method, and the test result shows that 100 percent of leaching liquor of the medical wound dressing has no cytotoxicity.

2) Skin sensitization

The skin sensitization test was conducted by a closed application test method, and the results showed that the skin of the test animals of examples and comparative examples did not show erythema and edema at the excitation site, and the rating was less than 1 according to Magnusson and Kligman rating standards, and the test results showed that the products of examples and comparative examples did not cause skin sensitization.

3) Intradermal stimulation

The intradermal stimulation test is carried out by adopting an experimental method of injecting the product into a mouse, after the intradermal stimulation test is carried out for (72 +/-2) h, the result analysis is carried out on a test sample, and the evaluation is carried out according to an intradermal reaction scoring system, so that the result shows that the injection part of the mouse of the experimental group of examples and comparative examples does not have erythema and edema, the experimental result shows that the products of the examples and comparative examples do not cause the intradermal stimulation reaction of the mouse, and the products have no intradermal stimulation reaction.

2 efficacy test

2.1 test subjects: 6-8 week old mice

2.2 Experimental quantities: 100, including 10 each of the test groups of examples 1-7 and comparative examples 1-2, and 10 of the blank control groups.

2.3 wound surface model preparation:

2.3.1 depilation

3 days before the wound surface is manufactured, the mouse is anesthetized by using a mouse respiratory anesthesia machine, and after the mouse is completely anesthetized, four limbs of the mouse are fixed on a mouse board in a bending manner. Depilatory is carried out by means of a depilatory cream.

2.3.2 wound preparation

The mouse is anesthetized by a mouse animal respiratory anesthesia machine and then placed on an operation table experiment pad in a lying state, the back skin of the mouse is ensured to be flat as much as possible, the back skin of the mouse is wiped for 3-5 times by a 75% sterilized alcohol cotton sheet, the back of the mouse is scratched by gentle external force, the superficial scratch of the back of the mouse is caused, and a single wound surface of 4 x 4cm is formed.

2.4 dressing contact

Dividing the mice into 10 groups, 9 groups of test groups and 1 group of control groups; the 9 test groups were coated with the gels of examples 1 to 7, comparative example 1 and comparative example 2, respectively, and the control group was not coated with any gel as a blank.

Control group: after the wound surface is manufactured, the medical plaster is used as a contrast surface for protection;

test groups: after the wound surface is manufactured, the wound surface is covered by the wound surface repairing gel, and the wound surface is protected by the application, so that the influence of other uncontrollable variables except a single variable, namely the wound surface dressing, on the wound surface is controlled as much as possible.

The times of dressing change: change twice a day, once in the morning and evening

2.5 Observation

When changing the medicine, the mouse state, the wound infection condition, the bleeding condition, the wound healing condition and the wound infection condition are observed and recorded. The results are shown in Table 2.

Table 2 efficacy test recording table

Note: 1. the mouse status is divided into five grades, from top to bottom: excellent, good, better, general, poor. The state of the mouse is judged according to the activity degree of the mouse, and the higher the activity degree is, the higher the rating is.

2. The wound healing condition is evaluated according to the score of 1-10, the better the healing condition is, the higher the score is. The judgment of the wound healing condition is judged according to the wound narrowing range, the smaller the wound is, the better the healing degree is, and the higher the score is.

3. Whether the wound is infected or not is judged according to whether the mouse wound has swelling, red swelling and pus. The severity is divided into three levels, in order from heavy to light: severe infection (swelling, pus), mild infection (red swelling), no infection.

Test example 2 study of bacteriostatic properties of bacteriostatic wound repair dressing

The test method comprises the following steps: flask shaking method

Test materials: staphylococcus aureus

Test temperature: 25 deg.C

Test rotating speed: 200rpm/min

The specific method comprises the following steps: 2g of each of the gels of the above examples and comparative examples was taken and placed in a sterile Erlenmeyer flask as a sample group; meanwhile, preparing a blank control group without a sample, adding 70ml of 0.03mol/l PBS and 5ml of staphylococcus aureus bacteria liquid into 10 groups respectively, performing shake culture for 1h at 25 ℃ and 200rpm/min, sampling for 0h and 1h respectively, and calculating the change of the number of bacteria. The test of each sample group and the control group was repeated 3 times, and the average inhibition rate was calculated. The results are shown in Table 3.

Calculating the bacteriostatic rate: the formula X is (A-B)/A100%, and X is the bacteriostasis rate; a is the average colony number of the sample before oscillation; b is the average colony number of the sample after oscillation. And (4) judging the standard: the colony number before and after oscillation of the group without the added sample is within 10 percent, the difference between the bacteriostasis rate of the test sample and the bacteriostasis rate of the blank reference control group is more than 26 percent, and the product can be judged to have the bacteriostasis function.

TABLE 3 bacteriostatic ratio (%) of the gels of examples and comparative examples against Staphylococcus aureus

And (4) analyzing results: based on the judgment standard whether the product is bacteriostatic or not, the bacteriostatic repair gels of 7 examples and 2 comparative examples have bacteriostatic effects on staphylococcus aureus within 1 hour. Of examples 1-3, example 2 was the most effective in inhibiting bacterial growth, where the ratio of the first hyaluronic acid to the arginine-based polyester urethane was 1: 2. the bacteriostatic rate of the bacteriostatic repairing gel of the example 4 is not much different from that of the examples 1 and 3, and the weight ratio of the first sodium hyaluronate to the arginine-based polyester urethane in the bacteriostatic repairing gel is 1: 9, which indicates that the first sodium hyaluronate and arginine-based polyester urethane can be controlled in the ratio of 1: 1-3, the weight proportion of the two is more or less, and the bacteriostatic effect is not obviously improved.

The results of the bacteriostatic rate of the bacteriostatic repair gels in the embodiments 5 and 6 are similar to those of the bacteriostatic rate of the bacteriostatic repair gel in the embodiment 1, which shows that the first sodium hyaluronate and the arginine-based polyester urethane in the range of 100-300KDa have good bacteriostatic ability in polymerization reaction.

Example 7 had a poor bacteriostatic ability relative to the bacteriostatic repair gels of the other examples, which may be that the first sodium hyaluronate had too low a molecular weight to polymerize with the arginine-based polyester urethane, thereby reducing bacteriostatic action.

The bacteriostatic rate of comparative example 1 is slightly lower than that of example 1, which is probably because the bioactive ingredient is absent in comparative example 1, so that the bacteriostatic ability of the bacteriostatic repairing gel is reduced, and the bacteriostatic rate is reduced compared with that of the example.

Comparative example 2 the calculated result according to the judgment standard of bacteriostasis was 26%, and the bacteriostasis effect was not ideal. The polymerization of the first sodium hyaluronate and the arginine-based polyester urethane is proved to be capable of effectively improving the bacteriostasis effect of the bacteriostasis restoration gel and meeting the bacteriostasis requirement of the bacteriostasis restoration gel. The lower bacteriostatic rate of comparative example 2 may be due primarily to the action of the bioactive ingredient, indicating that the bioactive ingredient acts synergistically in the bacteriostatic repair gel.

Claims

2. The bacteriostatic wound repair gel according to claim 1, wherein the mass ratio of hyaluronic acid or a salt thereof to the pseudoprotein biomaterial in the pseudoprotein-hyaluronic acid gel is 1: 1-3, preferably, in the bacteriostatic wound repair gel, the mass ratio of the pseudoprotein-hyaluronic acid gel is 0.5-3%, the mass ratio of the bioactive component is 0.03-0.1%, further preferably, the molecular weight of the first hyaluronic acid or the salt thereof is 100-300kDa, preferably 200-260 kDa.

3. Bacteriostatic wound repair gel according to claim 1, characterized in that the pseudoprotein biomaterial comprises a polymer of amino acids, preferably a polyester of amino acids, further preferably an arginine-based polyester urethane.

4. The bacteriostatic wound repair gel according to claim 1, wherein the bioactive component comprises one or more of second hyaluronic acid or its salt, bletilla striata, ectoin and panthenol.

the solution A is polymerized at high temperature to obtain gel B,

fully swelling the gel in purified water to obtain gel C,

6. The method of claim 5, wherein the step of polymerizing solution A at an elevated temperature comprises: reacting for 4-6 hours at 60-90 ℃.

7. The method of claim 5, wherein the mass ratio of the first hyaluronic acid or salt thereof to the pseudoprotein biomaterial in solution A is 1: 1-3, preferably the mass ratio of the gel B to the bioactive component is 0.03-0.1%, further preferably the molecular weight of the first hyaluronic acid or the salt thereof is 100-300kDa, preferably 200-260 kDa.

8. The method according to claim 5, wherein the pseudoprotein biomaterial comprises a polymer of amino acids, preferably a polyester of amino acids, further preferably an arginine-based polyester urethane.

9. The method according to claim 5, wherein the bioactive component comprises one or more of second hyaluronic acid or its salt, bletilla striata, ectoin, and panthenol.

10. The method according to claim 5, wherein the method is used for preparing the bacteriostatic wound repair gel according to any one of claims 1-4.