Quaternary ammonium salt-modified recombinant collagen gel dressing and method for preparing the same

Abstract

The application discloses a kind of quaternary ammonium salt modified recombinant collagen gel dressings and preparation method thereof.The method is grafted to quaternary ammonium salt containing epoxy group on the molecular chain of recombinant collagen under alkaline condition by ring-opening reaction, quaternary ammonium salt modified recombinant collagen is obtained, then with tannic acid as crosslinking agent, quaternary ammonium salt modified recombinant collagen gel dressing is obtained.The quaternary ammonium salt recombinant collagen gel dressing of the application has excellent biocompatibility, suitable mechanical properties and adhesion properties and antibacterial properties, as dressing use can effectively promote the tissue regeneration and functional recovery of wound surface.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and particularly relates to a quaternary ammonium salt-modified recombinant collagen gel dressing and a preparation method thereof. Background Art

[0002] A "wound" refers to a state in which the normal skin structure is disrupted due to physical, chemical, thermal injury, or physiological pathology. Wounds that fail to heal normally, or that heal slowly or stagnantly within 3-6 weeks are considered chronic wounds. For chronic wounds, prolonged healing times increase the likelihood of pathogens invading the wound, leading to a deteriorating wound environment and hindering all stages of wound healing.

[0003] Dressings are designed to provide a suitable environment to promote wound healing and prevent bacterial infection. Traditional dressings have been gradually replaced by new medical dressings, such as hydrocolloids, scaffolds, and hydrogels, due to their defects such as dryness, poor adhesion, lack of antibacterial properties, and susceptibility to secondary wound damage. Hydrogels have high water absorption and help promote wound healing through their water exchange activities. They can also provide a relatively cool environment, relieve wound pain, and form an optimal environment between the wound and the dressing. They have been successfully used to treat wounds such as skin defects, bacterial infections, burns, and diabetic foot. However, hydrogel dressings usually only have a single biological function, which limits their multiple promotion effects on wound healing. In view of the problem that the use of antibiotic-loaded hydrogels will produce bacterial resistance, it is more advantageous to use cationic polymers with antibacterial properties and broad-spectrum bactericidal properties as antibacterial materials.

[0004] Chinese patent CN201910015358.4 discloses a recombinant human collagen gel dressing for wounds, its preparation method, and its use method. The dressing includes a regenerative agent and a cross-linking agent, and a hydrogel state is formed on the wound surface through a cross-linking reaction between the two. Chinese patent CN202211656646.6 discloses a recombinant collagen gel dressing and its preparation method. The recombinant collagen cross-linked particles obtained by cross-linking and crushing with a cross-linking agent are mixed with an aqueous solution of a thickener and a phosphate to obtain a recombinant collagen gel dressing. However, none of the above gel dressings can inhibit pathogenic microorganisms invading the wound. Summary of the Invention

[0005] The invention provides a quaternary ammonium salt modified recombinant collagen gel dressing and a preparation method thereof.

[0006] The technical solutions of the present invention are as follows:

[0007] The preparation method of the quaternary ammonium salt modified recombinant collagen gel dressing comprises the following specific steps:

[0008] (1) Dissolve the recombinant collagen in water at a mass ratio of 2wt%-30wt% of the mass of water, and adjust the pH value to 10-11.5 with alkali, heat the recombinant collagen solution to 40°C-65°C, then slowly add a quaternary ammonium salt solution dropwise, and stir to react for 2-24 hours, wherein the mass ratio of quaternary ammonium salt to recombinant collagen is 1:1-1:2. After the reaction is completed, cool to room temperature, adjust the pH value of the solution to 2-6 with acid, concentrate by rotary evaporation, and slowly pour it into anhydrous ethanol to precipitate solids, filter to obtain a white filter cake, and vacuum dry to obtain quaternized recombinant collagen;

[0009] (2) dissolving quaternized recombinant collagen and tannic acid in water, and performing a cross-linking reaction to obtain a quaternary ammonium salt-modified recombinant collagen gel, wherein the mass of the quaternized recombinant collagen is 20wt%-40wt% of the mass of water, and the mass of the tannic acid is 1wt%-10wt% of the mass of water.

[0010] Preferably, in step (1), the recombinant collagen is produced by fermentation of Pichia pastoris with a deposit number of CGMCC No. 5021, which has been fully disclosed in Chinese Patent No. 201110327865.5.

[0011] Preferably, in step (1), the mass of the recombinant collagen is 5wt%-20wt% of the mass of water.

[0012] Preferably, in step (1), the base is sodium hydroxide solution or sodium carbonate solution, more preferably 0.5 mol / L sodium hydroxide solution.

[0013] Preferably, in step (1), the quaternary ammonium salt is selected from antibacterial quaternary ammonium salts containing epoxy groups such as N-2,3-epoxypropyltrimethylammonium chloride, N-2,3-epoxypropyldimethylbutylammonium chloride, N-2,3-epoxypropyldimethyloctylammonium chloride or N-2,3-epoxypropyldimethyldodecylammonium chloride. In a specific embodiment of the present invention, N-2,3-epoxypropyltrimethylammonium chloride is taken as an example.

[0014] Preferably, in step (1), the mass ratio of the quaternary ammonium salt to the recombinant collagen is 1:1.2-1:1.8.

[0015] Preferably, in step (1), the heating method is water bath or oil bath heating, the heating temperature is 45° C.-55° C., and the stirring time is 3-12 h.

[0016] Preferably, in step (1), the acid is selected from hydrochloric acid solution, acetic acid solution, etc., more preferably 4 mol / L dilute hydrochloric acid.

[0017] Preferably, in step (1), the pH value of the solution is adjusted to 3-5 with acid.

[0018] Preferably, in step (2), the cross-linking reaction time is 4-12 hours.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] (1) The recombinant collagen used in the present invention has good tissue compatibility and a three-dimensional cross-linked network structure that can absorb water and swell while maintaining a relatively fixed shape, which can cool wounds and relieve pain. At the same time, collagen has good adhesion to the wound surface, not only stopping bleeding but also promoting tissue regeneration and functional recovery.

[0021] (2) The present invention reacts the epoxy-containing quaternary ammonium salt with the amino group of the recombinant collagen through ring opening, thereby fixing the antibacterial group on the recombinant collagen molecular chain. While maintaining the antibacterial properties, it also improves its chemical stability, making it less likely to penetrate into the skin.

[0022] (3) The crosslinking agent tannic acid used in the present invention is a natural water-soluble polyphenol with high biological safety. The polyphenolic hydroxyl groups of tannic acid react with the amino groups of collagen to produce a Schiff base reaction, thereby obtaining crosslinked recombinant collagen. Furthermore, the tannic acid that has not undergone the crosslinking reaction also has broad-spectrum antibacterial activity. The synergistic effect of the antibacterial effect of tannic acid and the bactericidal effect of quaternary ammonium salts exerts a better antibacterial activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The results of the anti-Escherichia coli test in Example 3 and the blank control group are shown;

[0024] Figure 2 The results of Example 3 and the blank control group against Staphylococcus aureus are shown;

[0025] Figure 3 This is the HE staining result of the wound damage in diabetic rats in Example 3. DETAILED DESCRIPTION

[0026] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0027] Example 1

[0028] Dissolve 4g of recombinant collagen in 200ml of water, adjust the pH to 10.3 with 0.5mol / L sodium hydroxide solution, and heat the mixture to 40°C in a water bath. Dissolve 3.3g of N-2,3-epoxypropyltrimethylammonium chloride in 10ml of water and slowly add it dropwise to the recombinant collagen solution using a dropper. Stir magnetically for 24 hours. After cooling the reaction solution to room temperature, adjust the pH to 6 with 4mol / L hydrochloric acid. After rotary evaporation and concentration, slowly pour the solution into anhydrous ethanol to precipitate a white flocculent solid. Filter the mixture to obtain a white filter cake, which is then vacuum-dried to obtain the quaternized recombinant collagen. Separately, dissolve 4g of quaternized recombinant collagen and 0.2g of tannic acid in 20ml of purified water and cross-link at room temperature for 24 hours to obtain a quaternary ammonium salt-modified recombinant collagen gel dressing.

[0029] Example 2

[0030] Dissolve 10g of recombinant collagen in 200ml of water, adjust the pH to 10.5 with 0.5mol / L sodium hydroxide solution, and heat the mixture to 55°C in a water bath. Dissolve 10g of N-2,3-epoxypropyltrimethylammonium chloride in 10ml of water and slowly add it dropwise to the recombinant collagen solution using a dropper. Stir magnetically for 12 hours. After cooling the reaction solution to room temperature, adjust the pH to 2 with 4mol / L hydrochloric acid. After rotary evaporation and concentration, slowly pour the solution into anhydrous ethanol to precipitate a white flocculent solid. Filter the mixture to obtain a white filter cake, which is then vacuum-dried to obtain the quaternized recombinant collagen. Separately, dissolve 5g of quaternized recombinant collagen and 2g of tannic acid in 20ml of purified water and cross-link at room temperature for 4 hours to obtain a quaternary ammonium salt-modified recombinant collagen gel dressing.

[0031] Example 3

[0032] Dissolve 30g of recombinant collagen in 200ml of water, adjust the pH to 10 with 0.5mol / L sodium hydroxide solution, and heat the mixture to 50°C in a water bath. Dissolve 20g of N-2,3-epoxypropyltrimethylammonium chloride in 10ml of water and slowly add it dropwise to the recombinant collagen solution using a dropper. Stir magnetically for 5h. After cooling the reaction solution to room temperature, adjust the pH to 3 with 4mol / L hydrochloric acid. After rotary evaporation and concentration, slowly pour the solution into anhydrous ethanol to precipitate a white flocculent solid. Filter the mixture to obtain a white filter cake, which is then vacuum-dried to obtain the quaternized recombinant collagen. Separately, dissolve 6g of quaternized recombinant collagen and 1g of tannic acid in 20ml of purified water and cross-link at room temperature for 8h to obtain a quaternary ammonium salt-modified recombinant collagen gel dressing.

[0033] Example 4

[0034] Dissolve 40g of recombinant collagen in 200ml of water, adjust the pH to 10.8 with 0.5mol / L sodium hydroxide solution, and heat the mixture to 60°C in a water bath. Dissolve 20g of N-2,3-epoxypropyltrimethylammonium chloride in 10ml of water and slowly add it dropwise to the recombinant collagen solution using a dropper. Stir magnetically for 8h. After cooling the reaction solution to room temperature, adjust the pH to 3 with 4mol / L hydrochloric acid. After rotary evaporation and concentration, slowly pour the solution into anhydrous ethanol to precipitate a white flocculent solid. Filter the mixture to obtain a white filter cake, which is then vacuum-dried to obtain the quaternized recombinant collagen. Separately, dissolve 7g of quaternized recombinant collagen and 0.4g of tannic acid in 20ml of purified water and cross-link at room temperature for 12h to obtain a quaternary ammonium salt-modified recombinant collagen gel dressing.

[0035] Example 5

[0036] Dissolve 50g of recombinant collagen in 200ml of water, adjust the pH to 11.1 with 0.5mol / L sodium hydroxide solution, and heat the mixture to 65°C in a water bath. Dissolve 27.8g of N-2,3-epoxypropyltrimethylammonium chloride in 10ml of water and slowly add it dropwise to the recombinant collagen solution using a dropper. Stir magnetically for 8h. After cooling the reaction solution to room temperature, adjust the pH to 3 with 4mol / L hydrochloric acid. After rotary evaporation and concentration, slowly pour the solution into anhydrous ethanol to precipitate a white flocculent solid. Filter the mixture to obtain a white filter cake, which is then vacuum-dried to obtain the quaternized recombinant collagen. Separately, dissolve 8g of quaternized recombinant collagen and 1.5g of tannic acid in 20ml of purified water and cross-link at room temperature for 2h to obtain a quaternary ammonium salt-modified recombinant collagen gel dressing.

[0037] Example 6

[0038] Dissolve 60g of recombinant collagen in 200ml of water, adjust the pH to 11.5 with 0.5mol / L sodium hydroxide solution, and heat the mixture to 40°C in a water bath. Dissolve 30g of N-2,3-epoxypropyltrimethylammonium chloride in 10ml of water and slowly add it dropwise to the recombinant collagen solution using a dropper. Stir magnetically for 2h. After cooling the reaction solution to room temperature, adjust the pH to 5 with 4mol / L hydrochloric acid. After rotary evaporation and concentration, slowly pour the solution into anhydrous ethanol to precipitate a white flocculent solid. Filter the mixture to obtain a white filter cake, which is then vacuum-dried to obtain the quaternized recombinant collagen. Separately, dissolve 4g of quaternized recombinant collagen and 0.5g of tannic acid in 20ml of purified water and cross-link at room temperature for 10h to obtain a quaternary ammonium salt-modified recombinant collagen gel dressing.

[0039] Comparative Example 1

[0040] A quaternary ammonium salt-modified recombinant collagen gel dressing was prepared according to the method of Example 3, except that the amount of recombinant collagen added was 1 g.

[0041] Comparative Example 2

[0042] A quaternary ammonium salt-modified recombinant collagen gel dressing was prepared according to the method of Example 3, except that heating was not performed during the modification with N-2,3-epoxypropyltrimethylammonium chloride.

[0043] Comparative Example 3

[0044] A quaternary ammonium salt-modified recombinant collagen gel dressing was prepared according to the method of Example 3, except that the amount of N-2,3-epoxypropyltrimethylammonium chloride added was 1 g.

[0045] Comparative Example 4

[0046] The quaternary ammonium salt-modified recombinant collagen gel dressing was prepared according to the method of Example 3, except that the magnetic stirring time was 1 h.

[0047] Comparative Example 5

[0048] A quaternary ammonium salt-modified recombinant collagen gel dressing was prepared according to the method of Example 3, except that the pH of the alkaline solution was adjusted to 8.

[0049] Comparative Example 6

[0050] A quaternary ammonium salt-modified recombinant collagen gel dressing was prepared according to the method of Example 3, except that the solution was not adjusted to acidic.

[0051] Comparative Example 7

[0052] A quaternary ammonium salt-modified recombinant collagen gel dressing was prepared according to the method of Example 3, except that the amount of quaternary ammonium salt-modified recombinant collagen added was 0.5 g.

[0053] Comparative Example 8

[0054] The quaternary ammonium salt modified recombinant collagen gel dressing was prepared according to the method of Example 3, except that the amount of tannic acid added was 0.05 g.

[0055] Comparative Example 9

[0056] A quaternary ammonium salt-modified recombinant collagen gel dressing was prepared according to the method of Example 3, except that the cross-linking time was 1 h.

[0057] Comparative Example 10

[0058] Dissolve 6 g of recombinant collagen and 1 g of tannic acid in 20 ml of purified water, respectively, and crosslink at room temperature for 8 h to obtain recombinant collagen gel. Weigh 15 g of recombinant collagen gel and place it in 100 ml of purified water, and adjust the pH to 10 with 0.005 mol / L sodium hydroxide solution. Place it in a water bath and heat to 50°C. Slowly add N-2,3-epoxypropyltrimethylammonium chloride solution (20 g of N-2,3-epoxypropyltrimethylammonium chloride) to the recombinant collagen solution with a dropper, and magnetically stir for 5 h. After the reaction solution cools to room temperature, adjust the pH of the solution to 3 with 4 mol / L hydrochloric acid. Because the gel structure is destroyed and is not in a liquid state, it is not possible to prepare quaternary ammonium salt-modified recombinant collagen gel dressing.

[0059] Comparative Example 11

[0060] Prepare quaternary ammonium salt-modified recombinant collagen gel dressing according to the method of Example 3, except that the crosslinking agent tannic acid is replaced by tea polyphenol.

[0061] Comparative Example 12

[0062] Prepare recombinant collagen gel dressing according to the method of Example 3, except that the collagen is not modified with quaternary ammonium salt.

[0063] Performance Test 1

[0064] The antibacterial activity of quaternary ammonium salt-modified recombinant collagen gel dressing against Escherichia coli (gram-positive bacteria) and Staphylococcus aureus (gram-negative bacteria) was studied using plate counting method. All glassware and experimental instruments were sterilized by high temperature and high pressure for 30 min before testing. The gel dressing was cut into 20 mm*20 mm squares and sterilized under a UV lamp for 2 h. Prepare a bacterial solution with a concentration of 10 7 CFU / mL, and mix 1 mL of the bacterial solution with 9 mL of sterile physiological saline to dilute it. Immerse the sterilized gel dressing in the diluted bacterial solution and incubate it at 37°C for 12 h. After taking it out, count the colonies growing on the plate, and calculate the antibacterial rate according to the formula: CFU (control) is the number of colonies on the blank control plate, and CFU (sample)=The number of colonies on the plate containing the sample. Three parallel experiments were performed for each sample and the average value was taken. The specific experimental results are shown in Table 1. Compared with Comparative Examples 1-6, the recombinant collagens of Comparative Examples 1-6 failed to be effectively quaternized, and were mainly based on tannic acid antibacterial, so the antibacterial rate was significantly reduced. Comparative Examples 7-8 were not able to form gels because the addition of quaternized collagen was too little, the addition of cross-linking agent was too little, and the cross-linking reaction time was short, so no sampling was performed for antibacterial testing. Comparative Example 10 was first cross-linked and then modified with quaternary ammonium salt. Because the experimental conditions of quaternary ammonium salt modification destroyed the structure of the gel, no effective sampling was performed for antibacterial testing, so no antibacterial testing was performed. Compared with the Examples, Comparative Example 11 had a decreased antibacterial rate because the cross-linking agent did not have antibacterial properties. Compared with the Examples, Comparative Example 12 had a decreased antibacterial rate because the collagen was not modified with quaternary ammonium salt. The experimental results of the embodiment and the comparative example show that, compared with tannic acid, quaternary ammonium salt plays the main antibacterial role, followed by tannic acid, and the two work together to exert a more excellent antibacterial effect. The antibacterial graphs of Example 3 and the blank control group are shown as follows: Figure 1 (anti-E. coli) and Figure 2 (anti-Staphylococcus aureus) as shown.

[0065] Table 1 Antibacterial rate of Examples and Comparative Examples (%)

[0066]

[0067]

[0068] Performance Test 2

[0069] A wound repair model for type 1 diabetic rats was constructed, specifically including: (1) type 1 diabetic rat model: the rats were fasted for 12 hours and then intraperitoneally injected with streptozotocin (STZ). The blood glucose level was measured 1 week after the injection. When the tail vein blood glucose level reached 16.7 mmol and typical diabetic symptoms (weight loss, polyuria, polydipsia, and polyphagia) appeared, the diabetic model was considered to be successfully established; (2) wound repair model: chloral hydrate powder was dissolved in physiological saline to prepare a 10% chloral hydrate solution. The solution was prepared to a thickness and intraperitoneally injected into the rats. The injection volume was 1 ml / 300 g. After the rats were anesthetized, all the hair on the back of the rats was shaved off with a shaving machine. The backs were then disinfected with iodine tincture. A circular full-thickness skin defect wound with a diameter of 18 mm was made on the back. The sample of Example 3 was sterilized and placed on the wound on the back of the rats. The wound was then covered with a 3M transparent dressing. The HE staining results of the wound tissue of the diabetic rats 14 days after surgery in Example 3 are shown as follows. Figure 3As shown in the figure, the skin is damaged over a large area, necrotic tissue and inflammatory exudate can be seen in the epidermis (black arrows), the epidermis is lost over a large area at the damaged area, and a large new epidermis is seen at the edge, which is thicker than the normal epidermis (red arrows). The dermis and fat layer disappear, and the hair follicles and sebaceous glands disappear, and are replaced by a large amount of granulation tissue. The granulation tissue has a compact structure and mainly contains a small amount of inflammatory cells (yellow arrows), a large number of new capillaries (green arrows), and a large amount of new fibrous tissue (black arrows). This shows that the quaternary ammonium salt modified recombinant collagen gel dressing prepared by the present invention can promote tissue regeneration and functional recovery of the wound from below.

Claims

1. A method for preparing a quaternary ammonium salt modified recombinant collagen gel dressing, characterized in that: The specific steps are as follows: (1) The recombinant collagen is dissolved in water at a mass of 2wt%-30wt% of the mass of water, and the pH value is adjusted to 10-11.5 with alkali. The recombinant collagen solution is heated to 40℃-65℃, and then the quaternary ammonium salt solution is slowly added dropwise, and the reaction is stirred for 2-24h, wherein the mass ratio of the quaternary ammonium salt to the recombinant collagen is 1:1-1:

2. After the reaction is completed, the solution is cooled to room temperature, and the pH value is adjusted to 2-6 with acid. After rotary evaporation and concentration, the solution is slowly poured into anhydrous ethanol to precipitate solids, and filtered to obtain a white filter cake, which is vacuum dried to obtain quaternized recombinant collagen. The recombinant collagen is prepared from Pichia pastoris with a preservation number of CGMCC No. 5021. produced by fermentation of Benzyl alcohol, wherein the quaternary ammonium salt is selected from N-2,3-epoxypropyltrimethylammonium chloride, N-2,3-epoxypropyldimethylbutylammonium chloride, N-2,3-epoxypropyldimethyloctylammonium chloride or N-2,3-epoxypropyldimethyldodecylammonium chloride; (2) The quaternized recombinant collagen and tannic acid are dissolved in water and cross-linked to obtain a quaternary ammonium salt-modified recombinant collagen gel, wherein the mass of the quaternized recombinant collagen is 20wt%-40wt% of the mass of water, and the mass of the tannic acid is 1wt%-10wt% of the mass of water.

2. The preparation method according to claim 1, characterized in that In step (1), the mass of the recombinant collagen is 5wt%-20wt% of the mass of water.

3. The preparation method according to claim 1, characterized in that In step (1), the base is sodium hydroxide solution or sodium carbonate solution.

4. The preparation method according to claim 1, characterized in that In step (1), the mass ratio of the quaternary ammonium salt to the recombinant collagen is 1:1.2-1:1.

8.

5. The preparation method according to claim 1, characterized in that In step (1), the heating method is water bath or oil bath heating, the heating temperature is 45°C-55°C, and the stirring time is 3-12h.

6. The preparation method according to claim 1, characterized in that In step (1), the acid is selected from hydrochloric acid solution or acetic acid solution.

7. The preparation method according to claim 1, characterized in that In step (1), the pH value of the solution is adjusted to 3-5 with acid; in step (2), the cross-linking reaction time is 4-12 hours.

8. The quaternary ammonium salt-modified recombinant collagen gel dressing prepared according to the preparation method according to any one of claims 1 to 7.

Images