Pyrrolidone carboxylic acid-lysozyme compound as well as preparation method and application thereof

Abstract

The invention relates to the technical field of wound dressings, in particular to a pyrrolidone carboxylic acid-lysozyme compound as well as a preparation method and application thereof. According to the pyrrolidone carboxylic acid-lysozyme compound, a non-covalent binding mode is adopted, pyrrolidone carboxylic acid and lysozyme are subjected to a condensation reaction, a natural and non-toxic chemical component is formed, the antibacterial property of lysozyme is utilized, the antibacterial ability of a biological material is improved, the infection risk is reduced, and the pyrrolidone carboxylic acid-lysozyme compound can be applied to wound dressing. The prepared recombinant elastin cream dressing has an excellent moisture absorption effect, and is coated on a wound part to form a protective layer, so that the skin micro-ecology is improved and optimized, and wound healing is promoted. And recombinant elastin and ceramide are added, so that the resistance of wounds to the outside can be enhanced, and the skin state can be improved.

Description

Technical Field

[0001] The present invention relates to the technical field of wound dressings, and in particular to a pyrrolidone carboxylic acid-lysozyme complex and a preparation method and application thereof. Background Art

[0002] Most patients who undergo medical aesthetic surgery will have wounds of varying degrees on their skin. When wounds appear on the local skin, daily care is required.

[0003] In wound care, everyday liquid dressings and gauze have limitations. They can't keep wounds moist for long periods of time, requiring frequent replacement or reapplying, which can easily lead to secondary wound damage. This not only hinders wound healing but also causes patients to suffer from pain and the torment of the care process.

[0004] Lysozyme, also known as muramidase or N-acetylmuramic acid hydrolase, is an alkaline enzyme that hydrolyzes bacterial mucopolysaccharides. It breaks down insoluble cell wall polysaccharides into soluble glycopeptides, rupturing the bacterial cell wall and thus inactivating it. By inhibiting and killing bacteria on the wound surface, lysozyme can reduce bacterial invasion and infection, creating a clean environment for wound healing, helping to prevent the worsening of wound infection and promote normal wound healing. As a naturally derived antibacterial substance with excellent biocompatibility, lysozyme has great potential for application in cosmetics and medical devices. However, as a cationic protein, lysozyme easily reacts with anionic components in formulations, forming a precipitate that precipitates and inactivates it, limiting its application. Summary of the Invention

[0005] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art and to provide a pyrrolidonecarboxylic acid-lysozyme complex and its preparation method and application.

[0006] The first object of the present invention is to provide a method for preparing a pyrrolidonecarboxylic acid-lysozyme complex, comprising the following steps:

[0007] S1, firstly mixing the condensing agent EDC with pyrrolidone carboxylic acid for reaction;

[0008] S2, adding NHS to the reaction system in step S1 and reacting to obtain the intermediate N-hydroxysuccinimide ester;

[0009] S3, the intermediate N-hydroxysuccinimide ester is mixed with lysozyme for reaction, the pH of the reaction system is adjusted to 5.0-5.5 using a buffer solution, and the reaction temperature is controlled to be maintained at 4±1°C;

[0010] S4. After the reaction in step S3 is completed, a certain amount of terminator is added, and then purified to obtain pyrrolidonecarboxylic acid-lysozyme.

[0011] Furthermore, the mass ratio of the condensing agent EDC to pyrrolidone carboxylic acid is 1.2:1.

[0012] Furthermore, the mass ratio of NHS to EDC is 1:1.5.

[0013] Furthermore, the mass ratio of the intermediate N-hydroxysuccinimide ester to lysozyme is 1:1.

[0014] Furthermore, in step S3, the buffer solution is a citric acid-sodium citrate buffer solution, and its pH is 4.5-5.5.

[0015] Furthermore, the molar ratio of the terminator to the intermediate N-hydroxysuccinimide ester is 2.5:1.

[0016] Furthermore, the terminator is hydroquinone.

[0017] Furthermore, in step S4, the purification process is as follows: first, slowly injecting it into a pre-equilibrated Sephadex G-100 gel column for elution, and collecting the eluate where the target product is located; then passing the collected liquid through a DEAE-Sepharose Fast Flow ion exchange column for gradient elution; and finally, eluting using a chitin affinity chromatography column, and collecting the solution corresponding to the elution peak;

[0018] High performance liquid chromatography is used for detection. If the residual amount of hydroquinone is higher than 0.1%, the corresponding purification steps are repeated until the residual amount is lower than the standard, thereby ensuring that the pyrrolidone carboxylic acid-lysozyme complex with a purity of ≥99.5% is finally obtained.

[0019] The second object of the present invention is to provide a pyrrolidonecarboxylic acid-lysozyme complex prepared by the above-mentioned preparation method.

[0020] The third object of the present invention is to provide a recombinant elastin cream dressing, which comprises the following raw materials in parts by mass: 0.1-10% recombinant elastin complex liquid, 0.1-10% pyrrolidone carboxylic acid-lysozyme complex as described in claim 6, 1-5% water-soluble high molecular polymer, 1-20% moisturizer, 1-5% oily base, 0.1-2% pH regulator, and the remaining component is purified water.

[0021] Furthermore, the recombinant elastin complex solution comprises the following raw materials in parts by weight: 1-5% recombinant elastin, 0.1-1% ceramide;

[0022] The water-soluble high molecular polymer is selected from one or more of sodium hyaluronate, xanthan gum, sodium alginate, chitosan, sodium carboxymethyl cellulose, carbomer, and polyvinyl alcohol;

[0023] The moisturizing agent is selected from one or more of propylene glycol, glycerin, trehalose, urea, xylitol, and disodium edetate;

[0024] The oily base is selected from one or more of olive oil, stearic acid, vitamin E succinate polyethylene glycol ester, clove oil, soybean oil, corn oil, and white vaseline;

[0025] The pH regulator is selected from one or more of potassium hydroxide, sodium hydroxide, arginine, triethanolamine, anhydrous disodium hydrogen phosphate, and anhydrous sodium dihydrogen phosphate.

[0026] The fourth object of the present invention is to provide a method for preparing the recombinant elastin cream dressing as described above, comprising the following steps: dissolving recombinant elastin and ceramide in purified water, stirring thoroughly until the solution is clear and transparent, to obtain a first aqueous phase matrix; the pH value is 4.5-5.5;

[0027] The pyrrolidone carboxylic acid-lysozyme complex, the water-soluble high molecular polymer and the moisturizing agent are uniformly mixed and dispersed to obtain a second aqueous phase matrix;

[0028] The pH regulator is fully dissolved in purified water until transparent and free of particles to obtain a third aqueous phase matrix;

[0029] mixing the oily base to obtain a first oil phase base;

[0030] Pour the second aqueous phase matrix into the emulsifier, stir at 30-50r / min and heat to 80-85℃, keep stirring for 10-20 minutes to ensure that the material is completely dissolved;

[0031] Add the first oil phase matrix into the oil phase pot, stir and heat to 80-85℃ at 30-50r / min, keep stirring for 10-15 minutes to ensure that the material is completely dissolved;

[0032] Under homogenization, extract the dissolved oil phase matrix into the emulsification pot, vacuum -0.03~-0.1MPa, homogenize for 3-5 minutes, keep warm and stir for 10-20 minutes, then turn on the cooling water to cool down;

[0033] When the temperature drops to 60-70°C, turn off the cooling water, add the first aqueous phase matrix and the third aqueous phase matrix, continue to keep warm and stir for 10-20 minutes, then turn on the cooling water, stir at 30-40r / min and continue to cool down;

[0034] When the temperature drops to 30-38°C, samples are taken for testing to obtain a cream dressing;

[0035] The obtained cream dressing is subjected to electron beam irradiation with an irradiation dose of 20-30 Kgy to obtain a sterile recombinant elastin cream dressing.

[0036] The pyrrolidone carboxylic acid-lysozyme complex of the present invention adopts a non-covalent bonding mode to cause a condensation reaction between the two to form a natural, non-toxic chemical component. The antibacterial property of lysozyme is utilized to improve the antibacterial ability of biomaterials, reduce the risk of infection, and can be applied to wound dressings.

[0037] The present invention provides a recombinant elastin cream dressing and its preparation method. Incorporating advances in modern chemical technology, the cream dressing is applied to the wound surface to form a protective layer that isolates external pollutants and effectively prevents the intrusion of dust or bacteria. This dressing maintains the moisture of the wound surface and promotes the regeneration of skin granulation, thereby effectively promoting skin healing and shortening the transitional period of wound healing for patients.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The components of this invention are scientifically proportioned and work synergistically. The resulting recombinant elastin cream dressing has excellent moisture absorption. When applied to wound sites, it forms a protective layer, improves and optimizes the skin's microbiome, and promotes wound healing. The addition of recombinant elastin and ceramide enhances wound resistance and improves skin condition.

[0040] The addition of the pyrrolidone carboxylic acid-lysozyme complex changes the spatial structure of lysozyme, enhancing the binding ability of the active center of lysozyme to the substrate bacterial cell wall, allowing it to more effectively bind to the substrate and synergistically enhance the antibacterial effect; secondly, pyrrolidone carboxylic acid itself is a natural moisturizing factor of the skin. After condensation reaction with lysozyme, it can give lysozyme some new functional properties. For example, when the skin is relatively dry, pyrrolidone carboxylic acid can help maintain a better active space and promote wound healing to a certain extent.

[0041] Electron beam sterilization is used to prevent the inactivation of recombinant elastin, lysozyme, ceramide, and other ingredients in this cream dressing during high-temperature sterilization. Sterilized cream dressings are more conducive to wound healing and offer enhanced safety. This product utilizes an emulsified formulation, offering excellent permeability and readily absorbed moisturizing ingredients, promoting granulation growth in wounds and effectively improving wound healing. DETAILED DESCRIPTION

[0042] The following are specific embodiments of the present invention, which further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments.

[0043] Example 1: Preparation of pyrrolidone carboxylic acid-lysozyme complex

[0044] 1. Experimental Materials

[0045] 1. Condensation agent EDC (purity ≥ 99%)

[0046] 2. Pyrrolidone carboxylic acid (purity ≥ 99%)

[0047] 3. N-hydroxysuccinimide (NHS, purity ≥99%)

[0048] 4. Egg white derived lysozyme (purity ≥98%)

[0049] 5. Hydroquinone (analytical grade)

[0050] 6. Ethanol (analytical grade)

[0051] 7. Ethyl acetate (analytical grade)

[0052] 8. Citric acid (analytical grade)

[0053] 9. Sodium citrate (analytical grade)

[0054] 10. Phosphate (analytical grade)

[0055] 11.Sodium chloride (analytical grade)

[0056] 12. Glucosamine (analytical grade)

[0057] 13. Acetic acid (analytical grade)

[0058] 14. Sephadex G-100 gel 15. DEAE-Sepharose Fast Flow ion exchange resin 16. Chitin affinity chromatography column material

[0059] 2. Experimental Instruments

[0060] 1.250mL three-necked flask

[0061] 2.100mL conical flask

[0062] 3. High-precision low-temperature constant temperature bath (DC-2006)

[0063] 4. Precision pH meter (PHS-3C)

[0064] 5. Mechanical stirrer

[0065] 6. Magnetic stirrer

[0066] 7. Reflux condenser

[0067] 8. Thermometer

[0068] 9. Micro syringe (10mL)

[0069] 10. Nitrogen inlet device

[0070] 11. Rotary evaporator

[0071] 12. High performance liquid chromatography (Agilent 1260 Infinity II) 13. Thin layer chromatography

[0072] 14. Electrophoresis apparatus (Mini-Protean Tetra vertical electrophoresis system) 15. Melting point analyzer (WRS-1B)

[0073] 16. Online infrared spectrometer (Nicolet iS50)

[0074] 17. Glass chromatography column (column length 60 cm, inner diameter 2.6 cm; column length 30 cm, inner diameter 1.6 cm; column length 20 cm, inner diameter 1.0 cm)

[0075] 18. Centrifuge

[0076] 3. Experimental steps

[0077] (1) Early terminator screening experiment

[0078] 1. Preliminary Experiment

[0079] In 12 50 mL conical flasks, 20 mL of the simulated reaction system (containing 10 - 3 mol / L), place the conical flask in a high-precision low-temperature thermostat, control the temperature at 4±1℃, and adjust the pH to 5.0 with a precision pH meter. Then, add 5mL of 10 -2 A 1.0 mol / L solution of hydroquinone, catechol, resorcinol, ethanol, propanol, ascorbic acid, sodium sulfite, sodium metabisulfite, sodium thiosulfate, dithiothreitol (DTT), β-mercaptoethanol, and oxalic acid was mechanically stirred for 15 minutes. After the reaction, thin-layer chromatography (TLC) was performed using silica gel GF254 plates as the stationary phase and chloroform-methanol (8:2 by volume) as the developing solvent. Spots were observed under UV light at 254 nm. Hydroquinone, catechol, and resorcinol were selected based on the degree of spot reduction and advanced to the next stage.

[0080] 2. Comparative Experiment

[0081] In three 250 mL three-necked flasks, the same reaction system as in the actual preparation (N-hydroxysuccinimide ester 10 -3 mol / L, lysozyme 10 -3mol / L), the temperature was controlled at 4±1°C, and the pH was 5.0. Equimolar amounts of hydroquinone, catechol, and resorcinol (1:1 molar ratio to the remaining active intermediate) were added to a three-necked flask and mechanically stirred for 20 minutes to terminate the reaction. After completion of the reaction, high-performance liquid chromatography (HPLC) was performed on an Agilent 1260 InfinityII HPLC system with a ZORBAX Eclipse XDB-C18 column (4.6×150 mm, 5 μm). Mobile phase A consisted of 0.1% formic acid in water and mobile phase B was acetonitrile. Gradient elution was employed (0-5 min, 5% B; 5-30 min, 5-50% B; 30-35 min, 50-95% B; 35-40 min, 95% B). The flow rate was 1.0 mL / min, the detection wavelength was 280 nm, and the injection volume was 10 μL. According to the residual amount of active intermediates and product purity, hydroquinone was determined to be the better terminator.

[0082] 3. Dose Optimization Experiment

[0083] Five 250mL three-necked flasks were set up and a standard reaction system was added (same as the comparative experimental conditions). According to the molar ratio of hydroquinone to the remaining active intermediate of 1:1, 1.5:1, 2:1, 2.5:1, and 3:1, a 10% (w / v) ethanol solution of hydroquinone was added to the system and mechanically stirred for 20 minutes. After the reaction, the residual amount of active intermediate and the purity of the product were detected by HPLC. The results showed that when the molar ratio was 2.5:1, the residual amount of active intermediate dropped below the detection limit (<1×10 -5 mol / L), the product purity was 96.7%, and this was determined to be the optimal dose.

[0084] 4. Compatibility test

[0085] The complete pyrrolidonecarboxylic acid-lysozyme complex preparation reaction was carried out in a 250 mL three-necked flask. After completion, hydroquinone was added at an optimal dosage (molar ratio of 2.5:1) to terminate the reaction. Sequential purification steps were performed using Sephadex G-100 gel column chromatography, DEAE-Sepharose Fast Flow ion exchange chromatography, and chitin affinity chromatography. HPLC analysis of the product at each stage demonstrated that hydroquinone was compatible with the preparation process and did not affect purification or product quality.

[0086] (2) Preparation of the complex

[0087] 1. Activation stage

[0088] In a 250 mL three-necked flask, 12 g (purity ≥ 99%) of EDC (condensing agent) was added, followed by 10 g (purity ≥ 99%) of pyrrolidone carboxylic acid. The mixture was stirred mechanically at 180 rpm for 15 minutes. Subsequently, 8 g (purity ≥ 99%) of N-hydroxysuccinimide (NHS) was slowly injected over 5 minutes using a 10 mL microsyringe to form an activated intermediate.

[0089] 2. Nucleophilic attack phase

[0090] The intermediate prepared above was transferred to a 100 mL conical flask, and 10 g (purity ≥ 98%) of egg white-derived lysozyme was added. The temperature was controlled at 4 ± 1°C in a high-precision low-temperature thermostat. The pH was adjusted to 4.5-5.5 using a precision pH meter by adding 0.1 mol / L citric acid-sodium citrate buffer. The reaction was stirred at 120 rpm using a magnetic stirrer under nitrogen protection (nitrogen flow rate 100 mL / min) for 2.5 hours.

[0091] 3. Amide bond formation and water formation stage

[0092] During the reaction, infrared spectra were collected every 15 minutes by an online infrared spectrometer (NicoletiS50) to monitor the characteristic absorption peak of the amide bond (1650 cm -1 and the characteristic absorption peak of water (3400 cm -1 The reaction was considered to be substantially complete when the intensity of the characteristic absorption peak of the amide bond remained substantially unchanged within 30 minutes and the intensity of the characteristic absorption peak of water no longer increased significantly.

[0093] 4. Termination and purification stage

[0094] (1) Use of terminator: After the reaction is completed, prepare a 10% (w / v) ethanol solution of hydroquinone at a ratio of 2.5 mol of hydroquinone per mol of the remaining active intermediate and rapidly add it to the reaction system. Stir the reaction at 200 rpm using a mechanical stirrer for 20 minutes. TLC and HPLC analysis are performed. When the active intermediate content decreases below the detection limit, the active intermediate is considered to be substantially completely inactivated. Transfer the reaction system to a rotary evaporator and remove the ethanol solvent by vacuum distillation at 40°C and -0.09 MPa.

[0095] (2) Purification treatment

[0096] ① Gel column chromatography: Inject the concentrate into a pre-equilibrated Sephadex G-100 gel column (column length 60 cm, inner diameter 2.6 cm, equilibrated with 0.05 mol / L phosphate buffer (pH 7.0)), elute at a flow rate of 0.8 mL / min, and collect the eluate of the target product.

[0097] Ion exchange chromatography: The collected solution was passed through a DEAE-Sepharose Fast Flow ion exchange column (column length 30 cm, inner diameter 1.6 cm) using a 0-0.5 M NaCl gradient elution to further remove charged impurities.

[0098] Affinity chromatography: The treated solution was passed through a chitin affinity chromatography column (column length 20 cm, inner diameter 1.0 cm), equilibrated with 0.1 M acetate buffer (pH 4.5), eluted with acetate buffer containing 0.5 M glucosamine, and the solution corresponding to the elution peak was collected.

[0099] After each purification step, the residual hydroquinone content and product purity were determined using HPLC. If the residual hydroquinone content was above 0.1%, the corresponding purification step was repeated until the residual content fell below the specified level, yielding a high-purity (≥99.5%) pyrrolidonecarboxylic acid-lysozyme complex.

[0100] 5. Experimental Results

[0101] Finally, a pyrrolidonecarboxylic acid-lysozyme complex was obtained, with a purity of 99.6% and a hydroquinone residue of 0.08% as determined by HPLC.

[0102] The product was detected by electrophoresis and had a single band, which proved that it had high purity.

[0103] 6. Experimental Conclusion

[0104] Through the above experimental steps, a high-purity pyrrolidonecarboxylic acid-lysozyme complex was successfully prepared. The operating parameters at each stage were reasonable and feasible, and the quality control was effective.

[0105] Example 2

[0106] A recombinant elastin cream dressing and a preparation method thereof, wherein the cream dressing specifically comprises the following components, calculated by mass:

[0107] Recombinant elastin complex solution: 3.5%, including 3% recombinant elastin and 0.5% ceramide

[0108] Pyrrolidonecarboxylic acid-lysozyme complex: 3%

[0109] Water-soluble polymer: sodium hyaluronate 1%, carbomer 0.5%

[0110] Moisturizer: Propylene glycol 8%, disodium EDTA 0.05%

[0111] Oily base: Olive oil 3%, stearic acid 2%, vitamin E macrogol succinate 0.5%

[0112] pH adjuster: potassium hydroxide 0.5%

[0113] Purified water: balance

[0114] The elastin protein adopts the recombinant elastin protein disclosed in Chinese patent CN117551184B.

[0115] Preparation method:

[0116] 1. Dissolve recombinant elastin and ceramide in purified water and stir thoroughly until the solution is clear and transparent to obtain the first aqueous phase matrix;

[0117] 2. Stir and evenly disperse the pyrrolidone carboxylic acid-lysozyme complex, propylene glycol, sodium hyaluronate, carbomer, and disodium edetate to avoid agglomeration to obtain a second aqueous phase matrix;

[0118] 3. Dissolve the pH adjuster in purified water until transparent and free of particles to obtain the third aqueous phase matrix;

[0119] 4. Mix olive oil, stearic acid, and vitamin E succinate polyethylene glycol to obtain a first oil phase matrix;

[0120] 5. Pour the second aqueous phase matrix into the emulsifier, stir at 40r / min and heat to 82°C, keep stirring for 15 minutes to ensure that the material is completely dissolved;

[0121] 6. Add the first oil phase matrix into the oil phase pot, stir and heat to 82°C at 40r / min, keep stirring for 12 minutes to ensure that the material is completely dissolved;

[0122] 7. Under homogenization, extract the dissolved oil phase matrix into the emulsification pot, vacuum -0.06MPa, homogenize for 4 minutes, keep warm and stir for 15 minutes, then turn on the cooling water to cool down;

[0123] 8. When the temperature drops to 65°C, turn off the cooling water, add the first aqueous phase matrix and the third aqueous phase matrix, continue to keep warm and stir for 15 minutes, then turn on the cooling water, stir at 35 r / min and continue to cool;

[0124] 9. When the temperature drops to 35°C, take a sample for testing and obtain a cream dressing;

[0125] 10. The obtained cream dressing was subjected to electron beam irradiation with an irradiation dose of 25 Kgy to obtain a sterile recombinant elastin cream dressing.

[0126] Example 3

[0127] The present invention provides a recombinant elastin cream dressing, which comprises the following raw materials in parts by mass:

[0128] Recombinant elastin complex solution: 3.5%, including 3% recombinant elastin and 0.5% ceramide;

[0129] Pyrrolidonecarboxylic acid-lysozyme complex: 3%;

[0130] Water-soluble polymer: sodium hyaluronate 1%, carbomer 0.5%;

[0131] Moisturizer: Propylene glycol 8%, disodium EDTA 0.05%;

[0132] Oily base: olive oil 3%, stearic acid 2%, vitamin E succinate polyethylene glycol 0.5%;

[0133] pH regulator: potassium hydroxide 0.5%;

[0134] Purified water: balance.

[0135] The preparation method is the same as Example 2.

[0136] Example 4

[0137] The present invention provides a recombinant elastin cream dressing, which comprises the following raw materials in parts by mass:

[0138] Recombinant elastin complex solution: 1.3%, including 1% recombinant elastin and 0.3% ceramide;

[0139] Pyrrolidonecarboxylic acid-lysozyme complex: 2%;

[0140] Water-soluble polymer: sodium hyaluronate 0.3%, carbomer 0.5%;

[0141] Moisturizer: propylene glycol 5%, disodium edetate 0.03%;

[0142] Oily base: olive oil 2%, stearic acid 1%, vitamin E succinate polyethylene glycol 0.5%;

[0143] pH regulator: potassium hydroxide 0.3%;

[0144] Purified water: balance.

[0145] The preparation method is the same as Example 2.

[0146] Example 5:

[0147] The present invention provides a recombinant elastin cream dressing, which comprises the following raw materials in parts by mass:

[0148] Recombinant elastin complex solution: 3.5%, including 3% recombinant elastin and 0.5% ceramide;

[0149] Pyrrolidonecarboxylic acid-lysozyme complex: 2%;

[0150] Water-soluble polymer: sodium hyaluronate 0.3%, carbomer 0.5%;

[0151] Moisturizer: propylene glycol 5%, disodium edetate 0.03%;

[0152] Oily base: olive oil 3%, stearic acid 2%, vitamin E succinate polyethylene glycol 0.5%;

[0153] pH regulator: potassium hydroxide 0.5%;

[0154] Purified water: balance.

[0155] The preparation method is the same as Example 2.

[0156] Example 6:

[0157] The present invention provides a recombinant elastin cream dressing, which comprises the following raw materials in parts by mass:

[0158] Recombinant elastin complex solution: recombinant elastin 5%, ceramide 1%;

[0159] Pyrrolidonecarboxylic acid-lysozyme complex: 3%;

[0160] Water-soluble polymer: sodium hyaluronate 0.3%, carbomer 0.5%;

[0161] Moisturizer: propylene glycol 5%, disodium edetate 0.03%;

[0162] Oily base: olive oil 3%, stearic acid 2%, vitamin E succinate polyethylene glycol 0.5%;

[0163] pH regulator: potassium hydroxide 0.5%;

[0164] Purified water: balance.

[0165] The preparation method is the same as Example 2.

[0166] Application Example 1 Lysozyme Activity Determination

[0167] 1. Experimental Purpose

[0168] The antibacterial activity of the pyrrolidonecarboxylic acid-lysozyme complex was accurately determined and compared with ordinary lysozyme solution and other control solutions to verify its superiority in antibacterial ability.

[0169] 2. Experimental Materials

[0170] 1. Indicator bacteria: Staphylococcus aureus ATCC 25923, purchased from the American Type Culture Collection.

[0171] 2. Culture medium: Tryptic soy agar (TSA).

[0172] 3. Solution

[0173] (1) Control group lysozyme solution: lysozyme (purity ≥98%, purchased from Sigma-Aldrich), prepared at a concentration of 10 mg / mL.

[0174] (2) Pyrrolidonecarboxylic acid-lysozyme complex solution: obtained according to the preparation method of patent example 1, with a concentration of 10 mg / mL.

[0175] (3) Complex solution of lysozyme and inactive modifier (bovine serum albumin): Lysozyme and bovine serum albumin (purchased from Sigma-Aldrich) were mixed at a ratio of 1:1 (g / g) to a concentration of 10 mg / mL.

[0176] 4. Other materials: sterile qualitative filter paper with a diameter of 6 mm, micropipette (10-100 μL), culture dish, incubator, etc.

[0177] 3. Experimental steps

[0178] Preparation of culture medium: Prepare the culture medium according to the instructions of tryptic soy agar (TSA). After heating to dissolve, pour about 15-20 mL into sterile culture dishes while it is still hot. Wait for the plates to solidify and set aside.

[0179] Solution addition: Use a micropipette to accurately draw 20 μL of the control lysozyme solution, the pyrrolidone carboxylic acid-lysozyme complex solution, and the lysozyme and inactive modifier complex solution, respectively, and drop them onto the sterile filter paper to ensure that the solution is evenly distributed on the filter paper.

[0180] Plate culture: Carefully place the filter paper with the solution added onto a TSA plate containing indicator bacteria. Place five filter paper pieces per plate (one for each solution, repeat five times), gently pressing to ensure full contact between the filter paper and the culture medium. Invert the plate and place it in a 37°C incubator for 24 hours.

[0181] Result measurement: After the incubation period, the plate was removed and the diameter of the inhibition zone around each filter paper piece was measured using a vernier caliper (accurate to 0.1 mm) and the data was recorded.

[0182] 4. Experimental Results and Analysis

[0183] 1. Data Recording

[0184]

[0185]

[0186] 2. Data Analysis

[0187] The data were statistically analyzed using the t-test to determine whether the differences in the diameters of the inhibition zones between the different solution groups were statistically significant.

[0188] There was a significant difference between the pyrrolidonecarboxylic acid-lysozyme complex solution group and the control group (lysozyme solution): t=17.23, degrees of freedom df=8, P<0.001.

[0189] There was a significant difference between the pyrrolidonecarboxylic acid-lysozyme complex solution group and the lysozyme and inactive modifier complex solution group: t=18.45, degrees of freedom df=8, P<0.001.

[0190] 5. Experimental Conclusion

[0191] Through the above experiments and data analysis, it can be seen that the pyrrolidonecarboxylic acid-lysozyme complex has significantly stronger antibacterial ability than ordinary lysozyme solution and the complex of lysozyme and inactive modifiers, indicating that the complex can effectively enhance the antibacterial activity of lysozyme and has potential application value in medical cosmetology and other fields.

[0192] Application Example 2

[0193] 1. To investigate the difference in antibacterial activity between the pyrrolidone carboxylic acid-lysozyme complex and conventional lysozyme in a recombinant elastin dressing system, the following experiments were conducted:

[0194] 2. Experimental Methods

[0195] Use the agar diffusion method. After melting the nutrient agar medium, pour it into a sterile culture dish, about 15-20 mL per dish, and wait for it to solidify. Use a sterile punch to punch a small hole with a diameter of 6 mm on the plate. Prepare the recombinant elastin dressing of Examples 2 and 3 into a solution with a concentration of 5 mg / mL, take 20 μL and add it to the corresponding small hole. Use an equal volume of sterile saline as a negative control, and a solution containing a commonly used antibacterial agent (such as gentamicin, with a concentration of 10 mg / mL) as a positive control. Place the plate in a 37°C constant temperature incubator and culture for 24 hours.

[0196] 3. Experimental Results

[0197]

[0198] 4. Experimental Conclusion

[0199] Based on these experimental results, the following conclusions can be drawn: In the recombinant elastin dressing system, the dressing solution containing the pyrrolidonecarboxylic acid-lysozyme complex exhibited significantly superior antibacterial activity against Staphylococcus aureus and Escherichia coli compared to the dressing solution containing standard lysozyme. Specifically, the dressing solution containing the pyrrolidonecarboxylic acid-lysozyme complex produced a larger average inhibition zone diameter in the culture experiments with both bacteria, indicating a stronger inhibitory effect against these two common bacteria and a more effective antibacterial effect, thereby providing better antibacterial protection for wounds and promoting wound healing and recovery.

[0200] Application Example 3

[0201] 1. Experimental Purpose

[0202] The effectiveness of the product of the present invention was evaluated by comparing the effects of the recombinant elastin cream dressing of Example 4 of the present invention and other brands of cream dressings in promoting wound healing using a rat skin wound model.

[0203] 2. Experimental Materials

[0204] 1. Experimental Animals: Forty healthy, SPF-grade Sprague-Dawley rats weighing 200-220 g (half male and half female) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. Rats were housed in an environment with a temperature of 22 ± 2°C, a humidity of 50-60%, and a 12-hour day-night cycle for 7 days. Food and water were available ad libitum.

[0205] 2. Experimental Dressing

[0206] (1) Recombinant elastin cream dressing according to Example 4 of the present invention: prepared according to the above formula.

[0207] (2) Control dressing: Two common brands of similar cream dressings on the market were selected (labeled as control dressing A and control dressing B respectively).

[0208] (3) Other materials: sodium pentobarbital (manufacturer: Sigma-Aldrich, catalog number: ABC123) for anesthesia; iodine tincture, normal saline (Hualan Bioengineering Co., Ltd., specification: 250 ml / bag); surgical blades (Shanghai Medical Devices (Group) Co., Ltd. Surgical Instrument Factory), sterile gauze (WenJian Medical Supplies Co., Ltd.), vernier caliper (precision 0.02 mm, Guilin Guanglu Digital Measurement and Control Co., Ltd.), tissue fixative (4% paraformaldehyde), etc.

[0209] 3. Experimental Methods

[0210] 1. Wound Model Establishment: Forty rats were randomly divided into four groups, each with 10 rats: an experimental group (using the dressing of the present invention), a control dressing group A, a control dressing group B, and a blank control group (wounds treated with normal saline alone). The rats were anesthetized with intraperitoneal injection of 3% sodium pentobarbital (30 mg / kg). After anesthesia took effect, the hair on their backs was shaved with an electric shaver. A circular, full-thickness skin defect with a diameter of approximately 1.5 cm was then created symmetrically on both sides of the spine using sterilized surgical blades, reaching a depth of the subcutaneous fascia.

[0211] 2. Dressing treatment

[0212] (1) Experimental group: After the wound surface stopped bleeding, the recombinant elastin cream dressing of the present invention was evenly applied immediately with a thickness of about 0.5 mm, then covered with sterile gauze and fixed with medical tape.

[0213] (2) Control dressing group A and control dressing group B: Use corresponding brands of cream dressings, and the treatment method is the same as the experimental group.

[0214] (3) Blank control group: The wound surface was smeared with an equal amount of normal saline and covered with sterile gauze for fixation.

[0215] 3. Observation and Data Collection

[0216] (1) Observation of wound healing: The wound healing status of rats was observed and recorded regularly every day, including wound redness, swelling, exudation, and signs of infection.

[0217] (2) Wound area measurement: On days 0, 3, 7, 10, and 14 after modeling, the longest diameter and the shortest diameter perpendicular to the longest diameter of the wound were measured using a vernier caliper. The wound area and wound healing rate were calculated according to the formula: wound area = π × (longest diameter / 2) × (shortest diameter / 2). Wound healing rate (%) = [(initial wound area - wound area at measurement) / initial wound area] × 100%.

[0218] (3) Histological analysis: On the 14th day of the experiment, 5 rats were randomly selected from each group and killed by overdose of anesthesia. The wound and surrounding tissues were removed and fixed in 4% paraformaldehyde fixative, embedded in paraffin, and sectioned. After HE staining, the histological changes were observed under an optical microscope to evaluate the growth of new epithelial tissue, granulation tissue, and inflammatory cell infiltration.

[0219] (4) Hemoglobin content determination: The skin tissue around the wound of the remaining 5 rats was taken and the hemoglobin content was determined using a hemoglobin content determination kit (purchased from Nanjing Jiancheng Bioengineering Institute, product number: A012-2-1). The operation steps were strictly followed according to the kit instructions.

[0220] (5) Statistics of allergic rate: During the experiment, the rats were observed every day for allergic reaction symptoms such as erythema, edema, and itching around the wound and on the skin of the whole body. The number of allergic rats in each group was counted and the allergic rate was calculated.

[0221] 4. Experimental Results

[0222] 1. Wound healing

[0223] (1) Experimental group: On the third day after surgery, the redness and swelling of the wound were significantly reduced, and there was less exudate; on the seventh day, new epithelial tissue was clearly visible at the edge of the wound, and granulation tissue grew well; on the tenth day, the wound area was significantly reduced; on the fourteenth day, the wound was basically healed, with only a small amount of scar remaining.

[0224] (2) Control Dressing Group A: On the 3rd day, the wound still had a lot of exudate and the redness and swelling subsided slowly; on the 7th day, the growth of new epithelial tissue was not obvious; on the 14th day, the wound was not completely healed and a large area of wound surface remained.

[0225] (3) Control dressing group B: The wound healing condition was similar to that of control dressing group A, but the healing speed was slower, and some rats showed mild infection symptoms during the experiment.

[0226] (4) Blank control group: The wound healed slowly, and there was still a large wound surface on the 14th day. The inflammatory reaction was obvious, and more inflammatory cells were seen infiltrating.

[0227] 2. Wound area and healing rate

[0228]

[0229] 3. Histological Analysis

[0230] (1) Experimental group: A large number of new epithelial cells covered the wound surface, collagen fibers were neatly arranged in the granulation tissue, and there were fewer inflammatory cells.

[0231] (2) Control dressing groups A and B: fewer new epithelial cells, more disordered arrangement of collagen fibers in granulation tissue, and more inflammatory cell infiltration.

[0232] (3) Blank control group: The wound surface was mainly composed of inflammatory cells, and the growth of new epithelium and granulation tissue was not obvious.

[0233] 4. Hemoglobin content

[0234]

[0235]

[0236] 5. Allergy rate

[0237] (1) Experimental group: The allergic rate was 0%.

[0238] (2) Control dressing group A: The allergic rate was 15%.

[0239] (3) Control dressing group B: The allergic rate was 20%.

[0240] (4) Blank control group: no allergic reaction.

[0241] 6. Overall healing score

[0242] A comprehensive scoring method was used to score wounds based on four aspects: wound healing rate (40%), histological manifestations (30%), hemoglobin content (20%), and allergic conditions (10%), with a full score of 10. The scoring results are as follows:

[0243] Grouping Overall healing score Experimental group 9 Control dressing group A 6 Control dressing group B 5 Blank control group 4

[0244] 5. Experimental Conclusion

[0245] Experiments on rat skin wound models showed that the recombinant elastin cream dressing of the present invention was significantly superior to other brands of control dressings in promoting wound healing, reducing inflammatory reactions, lowering hemoglobin content, and avoiding allergic reactions, and has good application prospects.

[0246] Application Example 4

[0247] 1. Experimental Purpose

[0248] The capsaicin human stimulation experiment was conducted to simulate a skin irritation wound scenario, and the differences in promoting wound repair, alleviating inflammatory response, and safety of the recombinant elastin cream dressing of Example 5 of the present invention and other brands of dressings were compared.

[0249] 2. Experimental Materials

[0250] 1. Experimental subjects: Recruit 10 healthy subjects with no history of skin diseases, drug allergies, or immune system diseases.

[0251] 2. Experimental Dressing

[0252] (1) Recombinant elastin cream dressing according to Example 5 of the present invention: prepared according to the above formula.

[0253] (2) Control dressing: Select a well-known brand dressing of the same type available on the market.

[0254] (3) Other materials: capsaicin solutions with concentrations of 0%, 1.0%, and 1.5%, sterile cotton swabs, sterile gauze, medical tape, and a hemoglobin content detection kit.

[0255] 3. Experimental Methods

[0256] In the symmetrical position of the inner forearm of each subject, 0%, 1.0%, and 1.5% capsaicin solutions were applied with a sterile cotton swab, respectively, with each application area of about 2cm×2cm. After 5 minutes of capsaicin action, the left wound area was smeared with the recombinant elastin cream dressing of the present invention, with a thickness of about 0.3mm, and the right wound area was smeared with an equal amount of control dressing. Then, both were covered with sterile gauze and fixed with medical tape. The dressing was changed once a day, and the number of days for wound healing and hemoglobin content were continuously observed and recorded. Allergic reactions were counted, and a comprehensive score was given based on the four dimensions of healing speed, hemoglobin changes, allergic reactions, and comfort, with a full score of 10 points.

[0257] 4. Experimental Results

[0258]

[0259] V. Summary

[0260] Combined with the results of the rat skin wound model experiment in Application Example 3 and the capsaicin stimulation experiment in humans in this example, the recombinant elastin cream dressing of the present invention significantly outperformed the control dressing in promoting wound healing, reducing inflammatory reactions, and preventing allergies in both animal and human studies. This dressing can provide nutrition to the wound surface, maintain a moist environment, effectively improve wound healing, shorten healing time, and is highly safe, thus enhancing the patient's recovery experience.

[0261] Application Example 5

[0262] Since the recombinant elastin cream dressing of the present invention is a medical device dressing and is applied to wound surfaces, the recombinant elastin cream dressing of Example 6 is required to undergo five conventional biocompatibility tests. The test results are as follows:

[0263] 1. Cytotoxicity Assay

[0264] The cytotoxicity test was carried out according to GB / T16886.1-2022 standard. The specific steps are as follows:

[0265] 1.1 Test preparation:

[0266] All instruments in contact with samples and cells were sterilized by moist heat to ensure sterility. L929 cells in good condition and in the logarithmic growth phase and the recombinant elastin cream dressing to be tested were prepared. A cell suspension with a concentration of 4×10 cells / ml was prepared using DMEM high-glucose medium and inoculated into a 96-well plate. DMEM high-glucose medium containing 10% fetal bovine serum was selected as the extraction medium. The sample was cut into 0.5 cm × 2 cm strips with a thickness of 0.5 mm and placed on a 6 cm plate. 2 / ml was placed in a glass container and extracted at 37±1℃ for 24h.

[0267] 1.2 Test process:

[0268] After cell inoculation, the cell inoculation density of each well was checked by phase contrast microscopy. After confirmation, it was placed in an incubator at 37±1°C and 5%±1% CO for 24 hours. After culturing for 24 hours, the original culture medium was discarded, and the cell morphology, number and growth were observed under a microscope to qualitatively determine whether the sample extract had a toxic effect. On the 2nd, 4th and 7th days after the culture medium was replaced, 3 bottles were taken from each group to observe the cell morphology and count them for cell morphology analysis. MTT solution was then added and incubated in a CO incubator for another 2 hours. Isopropanol was then added for color development. After shaking the culture plate, the 96-well plate was placed on a microplate reader, and the absorbance of each well was measured at a wavelength of 490 nm. The average value was taken to calculate the relative cell proliferation rate.

[0269] 1.3 Test results

[0270] 1.3.1 Cell morphology analysis:

[0271] Under a phase contrast microscope, the cells showed normal morphology, well-adherent growth, and uniform irregular triangles. Based on the cell morphology analysis standards in the "Cytotoxicity Test Method" of the "Pharmacopoeia of the People's Republic of China" (2020 edition, Part IV), the reaction was determined to be non-toxic.

[0272] 1.3.2 Analysis of relative cell proliferation (calculated based on cell concentration on day 7):

[0273]

[0274] A relative cell proliferation rate of 90% or higher generally indicates good cell compatibility and suitability for medical device applications. According to the relative cell proliferation grading table in the "Cytotoxicity Test Method" of the Pharmacopoeia of the People's Republic of China (2020 Edition, Part IV), this dressing's cytotoxicity response is rated Grade 1, indicating it has passed the cytotoxicity test and possesses a high degree of safety.

[0275] 2. Sensitization test

[0276] In accordance with GB / T16886.1-2022, a closed patch test (Buehler test) is used to test the properties of recombinant elastin cream dressings as surface-applied products. The details are as follows:

[0277] 2.1 Test preparation:

[0278] Fifteen albino guinea pigs, weighing 300-500g and aged 2-3 months, were selected for the skin sensitization test and housed in individual cages in a constant-temperature room. The housing environment adhered to ISO 10993-2, with a temperature of 22±1°C, humidity of 40%-70%, and ambient noise below 60dB. The animals were housed for three days under a stable diet and environment. The fur on the left side of the dorsal coat was removed before testing.

[0279] 2.2 Experimental process

[0280] 2.2.1 Preliminary experiment

[0281] Select area 8cm 2 Patches were soaked with the test material and saline solution, respectively, and applied to the hairless area of the guinea pigs. The patches were secured with an occlusive bandage for 6 hours. The degree of skin erythema and edema at the test site was evaluated 24 and 48 hours after patch removal according to the Magnusson and Kligman grading system.

[0282] Magnusson and Kligman classification

[0283] patch test reaction grade No obvious change 0 Scattered or macular erythema 1 Moderate confluent erythema 2 Severe erythema and / or edema 3

[0284] The test samples were set at five concentration gradients, namely, medical saline: recombinant elastin cream dressing = 4:1, 3:1, 2:1, 1:1, 1:2, and the control group was only applied with a patch soaked in medical saline.

[0285] The test results showed that after 6 hours of application of the extract, no erythema reaction occurred on the skin of the test samples at various concentrations and the control group guinea pigs at 24 hours and 48 hours, and the Magnusson and Kligman classification was grade 0. Therefore, the main test concentration was determined to be medical saline: recombinant elastin cream dressing = 1:2.

[0286] 2.2.2 Main experiment

[0287] 2.2.2.1 Induction phase

[0288] Induction phase: At the concentration selected in the pre-experiment (medical saline: recombinant elastin cream dressing = 1:2), the extract was soaked in a patch and applied to the upper left back of each guinea pig. The occlusive bandage and patch were removed after 6 hours. This procedure was repeated for 3 consecutive days within 1 week for a total of 3 weeks. The control group used saline dressing.

[0289] 2.2.2.2 Excitation phase

[0290] Fourteen days after the last induction, all test and control animals were challenged with the test sample and control sample, respectively. Patches were soaked with the test sample at the selected concentration and applied to the upper right back of the guinea pigs. Six hours later, the fixture, tie, and patch were removed. At 24 and 48 hours after patch removal, scores were assessed according to the Magnusson and Kligman grading system.

[0291] 2.2.2.3 Main Experiment Results

[0292] Table 2

[0293]

[0294] Note: Calculation method:

[0295] 1. During the observation, the number of grade 0, 1, 2, and 3 skin reactions was recorded as n0, n1, n2, and n3, respectively.

[0296] 2. Calculate the total grade points: According to the formula S = n0 × 0 + n1 × 1 + n2 × 2 + n3 × 3, calculate the total skin reaction grade points S of all observation subjects.

[0297] 3. Calculate the average: Divide the total grade points S by the total number of observations N (N = n0 + n1 + n2 + n3) to get the grade point average The formula is

[0298] When using a recombinant elastin cream dressing (concentration of medical saline: recombinant elastin cream dressing = 1:2), the skin reactions on the backs of guinea pigs in the test group were all grade 0. According to the evaluation criteria for the closed patch test results in GB / T16886.1-2022, the recombinant elastin cream dressing obtained by the present invention is non-allergenic.

[0299] 3. Stimulation Test

[0300] The human skin irritation test is carried out according to GB / T16886.23-2023 standard. The steps are as follows:

[0301] 3.1 Test preparation:

[0302] Thirty volunteers (15 males and 15 females) aged between 26 and 30 years were recruited and a patch with a diameter of 2.5 cm was used.

[0303] 3.2 Test process:

[0304] After moistening the recombinant elastin cream dressing with 200 μl of purified water, apply it to the inner left upper arm of the volunteer and secure it with an occlusive bandage with gauze. The volunteer was allowed to leave the testing room only after 15 and 30 minutes, and after 1, 2, 3, and 4 hours if no skin irritation occurred. If no irritation occurred after 48 hours, a long-term application was performed on an untested area to fully assess delayed irritation. A 20% SDS positive control was used; its irritation has been verified through characterization experiments.

[0305] 3.3.1 Clinical observation:

[0306] After removing the patch, immediately grade the skin reaction and re-grade it at 1 hour, 2 hours, 24 hours, 48 hours, and 72 hours. If necessary, extend the observation period. At the same time, accurately record the skin condition (pigmentation and hydration level) before and after the test. The skin reaction grade evaluation criteria are as follows:

[0307] Table 3 Human skin irritation test classification table

[0308] Reaction Description grade No response 0 Weak positive reaction (mild erythema and / or extensive dryness in the contact area) 1 Moderate positive reaction (marked erythema and dryness, possibly extending beyond the contact area) 2 Severe positive reaction (severe and diffuse erythema with redness, swelling, and / or eschar formation) 3

[0309] 3.4 Test results

[0310] Table 4

[0311]

[0312] Note: The calculation method is the same as that of sensitization test.

[0313] As can be seen from the data in the table, compared with the positive control group, the average score of the irritation test of the recombinant elastin cream dressing of the present invention is between 0 and 1. According to the irritation test evaluation standard in GB / T 16886.23-2023 Medical Device Biological Evaluation, this dressing is determined to be non-irritating.

[0314] 4. Systemic toxic reactions

[0315] According to the requirements of GB / T 16886.11, a systemic toxicity reaction test was carried out, as follows:

[0316] 4.1 Test preparation:

[0317] The experiment was divided into an experimental group and a control group. Five KM mice of the same origin and strain, weighing 18±2g, were selected from each group. All female mice were non-pregnant. All experimental operations were carried out in a sterile environment. The recombinant elastin cream dressing was cut into 0.5cm×3cm strips with a thickness of 0.5mm and immersed in a 0.9% sodium chloride injection extraction medium. 2The test and control solutions were thoroughly extracted at a ratio of 1:1 / ml. The control group received the extraction medium without the dressing. Before injection, both the test and control solutions were vigorously shaken to ensure that the extracts were thoroughly mixed.

[0318] 4.2 Test process:

[0319] The test and control groups were intravenously injected with the test and control solutions at a dose of 50 ml / kg, respectively, at an injection rate of 0.1 ml / s. Immediate reactions of the mice were observed after injection, and the status, toxic reactions, and mortality of the mice in the test and control groups were recorded at 4, 24, 48, and 72 hours. The mice were weighed at 72 hours.

[0320] 4.3 Evaluation Level - Mouse Reaction Observation Indicators

[0321] Table 5

[0322]

[0323] 4.4 Evaluation of test results

[0324] Table 6

[0325]

[0326] Compared to the common systemic toxicity experienced by similar products, the recombinant elastin cream dressing of the present invention did not induce any toxic symptoms in mice, and weight changes remained normal. Evaluation based on GB / T16886.11 standards determined that the recombinant elastin cream dressing of the present invention exhibited no systemic toxicity.

[0327] 5. Pyrogen test

[0328] According to GB / T 14233.2-2022, the pyrogen test is carried out according to the Chinese Pharmacopoeia Volume IV "Pyrogen Test Method". The specific steps are as follows:

[0329] 5.1 Experimental preparation

[0330] Three healthy rabbits weighing 2.0kg±0.2kg were selected as experimental animals. The female rabbits were not pregnant. Adaptive feeding was carried out for 7 days before the experiment. The temperature of the breeding environment was maintained at 15-25℃, with a temperature difference of no more than 3℃. The ammonia content in the air was less than 20ppm. The environment was kept quiet and avoided strong light and noise interference. Body temperature was measured before the experiment, once every 30 minutes, for a total of 2 measurements. The body temperature was required to be within the range of 38.0-39.6℃ for both times, with the maximum and minimum temperature difference not exceeding 0.4℃. The temperature difference of rabbits in the same group should not exceed 1℃. Pyrogen detection was performed after the rabbits rested for 48 hours to ensure that the utensils in contact with the test samples were sterile and pyrogen-free.

[0331] 5.2 Test process

[0332] Cut the recombinant elastin cream dressing into 0.5cm×3cm strips with a thickness of 0.5mm, immerse it in 0.9% sodium chloride injection extraction medium, and press 6cm 2 Measure the body temperature of three rabbits and slowly inject the test solution warmed to 38°C into the ear vein within 15 minutes. Then measure the body temperature every 30 minutes. Subtract the normal body temperature from the highest of the six temperatures to obtain the temperature elevation of the rabbit.

[0333] 5.3 Test evaluation method

[0334] Evaluation is carried out in accordance with the Pyrogen Test Method of Part IV of the Chinese Pharmacopoeia. The criteria are as follows:

[0335] Table 7

[0336] Grading changes in body temperature qualified The temperature rise was less than 0.6℃, and the total temperature rise of the three rabbits was less than 1.3℃. Unqualified More than one rabbit had a body temperature of 0.6℃ or higher.

[0337] 5.4 Test results

[0338] Table 8

[0339]

[0340] During the initial test, the body temperature of the three rabbits rose below 0.6°C, and the total temperature rise in any time period was below 1.3°C, which complies with the requirements of the "Pyrogen Test Method" in Part IV of the Chinese Pharmacopoeia. No retest is required, and the recombinant elastin cream dressing is judged to have passed the pyrogen test.

[0341] Overall conclusion:

[0342] Through five biocompatibility tests, namely cytotoxicity, sensitization, irritation, systemic toxicity reaction and pyrogen test, it can be seen that the recombinant elastin cream dressing prepared by the present invention has low cytotoxicity, no sensitization and irritation, no systemic toxicity reaction and passed the pyrogen test, performing well in terms of biosafety and meeting the safety requirements of medical device management.

[0343] Any matters not mentioned above shall be subject to the existing technology.

[0344] Although some specific embodiments of the present invention have been described in detail through examples, those skilled in the art should understand that the above examples are for illustration only and are not intended to limit the scope of the present invention. Those skilled in the art of the present invention may make various modifications or additions to the described specific embodiments or replace them in similar ways, but they will not deviate from the direction of the present invention or exceed the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent replacements, improvements, etc. made to the above embodiments based on the technical essence of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a pyrrolidonecarboxylic acid-lysozyme complex, characterized in that: The steps include: S1, firstly mixing the condensing agent EDC with pyrrolidone carboxylic acid for reaction; S2, adding NHS to the reaction system in step S1 and reacting to obtain the intermediate N-hydroxysuccinimide ester; S3, the intermediate N-hydroxysuccinimide ester is mixed with lysozyme for reaction, the pH of the reaction system is adjusted to 5.0-5.5 using a buffer solution, and the reaction temperature is controlled to be maintained at 4±1°C; S4. After the reaction in step S3 is completed, a certain amount of terminator is added, and then purified to obtain pyrrolidonecarboxylic acid-lysozyme.

2. The preparation method according to claim 1, wherein: The mass ratio of the condensing agent EDC to pyrrolidone carboxylic acid is 1.2:1; the mass ratio of NHS to EDC is 1:1.5; and the mass ratio of the intermediate N-hydroxysuccinimide ester to lysozyme is 1:

1.

3. The preparation method according to claim 1, wherein: In step S3, the buffer solution is a citric acid-sodium citrate buffer solution with a pH of 4.5-5.

5.

4. The preparation method according to claim 1, wherein: The molar ratio of the terminator to the intermediate N-hydroxysuccinimide ester is 2.5:1; the terminator is hydroquinone.

5. The preparation method according to claim 1, wherein: In step S4, the purification process is as follows: first, slowly inject it into a pre-equilibrated Sephadex G-100 gel column for elution, and collect the eluate containing the target product; then, pass the collected liquid through a DEAE-Sepharose Fast Flow ion exchange column for gradient elution; finally, use a chitin affinity chromatography column for elution, and collect the solution corresponding to the elution peak; High performance liquid chromatography is used for detection. If the residual amount of hydroquinone is higher than 0.1%, the corresponding purification steps are repeated until the residual amount is lower than the standard, thereby ensuring that the pyrrolidone carboxylic acid-lysozyme complex with a purity of ≥99.5% is finally obtained.

6. A pyrrolidonecarboxylic acid-lysozyme complex prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the pyrrolidonecarboxylic acid-lysozyme complex according to claim 6 in preparing a wound dressing.

8. A recombinant elastin cream dressing, characterized in that: The invention comprises the following raw materials in parts by mass: 0.1-10% recombinant elastin complex liquid, 0.1-10% pyrrolidone carboxylic acid-lysozyme complex as described in claim 6, 1-5% water-soluble high molecular polymer, 1-20% moisturizer, 1-5% oily base, 0.1-2% pH adjuster, and the remaining component is purified water.

9. The recombinant elastin cream dressing according to claim 8, characterized in that: The recombinant elastin complex solution comprises the following raw materials in parts by weight: 1-5% recombinant elastin, 0.1-1% ceramide; The water-soluble high molecular polymer is selected from one or more of sodium hyaluronate, xanthan gum, sodium alginate, chitosan, sodium carboxymethyl cellulose, carbomer, and polyvinyl alcohol; The moisturizing agent is selected from one or more of propylene glycol, glycerin, trehalose, urea, xylitol, and disodium edetate; The oily base is selected from one or more of olive oil, stearic acid, vitamin E succinate polyethylene glycol ester, clove oil, soybean oil, corn oil, and white vaseline; The pH regulator is selected from one or more of potassium hydroxide, sodium hydroxide, arginine, triethanolamine, anhydrous disodium hydrogen phosphate, and anhydrous sodium dihydrogen phosphate.

10. A method for preparing the recombinant elastin cream dressing according to claim 8 or 9, characterized in that: The following steps are involved: Dissolve recombinant elastin and ceramide in purified water and stir thoroughly until the solution is clear and transparent to obtain a first aqueous phase matrix; the pH value is 4.5-5.5; The pyrrolidone carboxylic acid-lysozyme complex, the water-soluble high molecular polymer and the moisturizing agent are uniformly mixed and dispersed to obtain a second aqueous phase matrix; The pH regulator is fully dissolved in purified water until transparent and free of particles to obtain a third aqueous phase matrix; mixing the oily base to obtain a first oil phase base; Pour the second aqueous phase matrix into the emulsifier, stir at 30-50r / min and heat to 80-85℃, keep stirring for 10-20 minutes to ensure that the material is completely dissolved; Add the first oil phase matrix into the oil phase pot, stir and heat to 80-85℃ at 30-50r / min, keep stirring for 10-15 minutes to ensure that the material is completely dissolved; Under homogenization, extract the dissolved oil phase matrix into the emulsification pot, vacuum -0.03~-0.1MPa, homogenize for 3-5 minutes, keep warm and stir for 10-20 minutes, then turn on the cooling water to cool down; When the temperature drops to 60-70°C, turn off the cooling water, add the first aqueous phase matrix and the third aqueous phase matrix, continue to keep warm and stir for 10-20 minutes, then turn on the cooling water, stir at 30-40 r / min and continue to cool; When the temperature drops to 30-38°C, samples are taken for testing to obtain a cream dressing; The obtained cream dressing is subjected to electron beam irradiation with an irradiation dose of 20-30 Kgy to obtain a sterile recombinant elastin cream dressing.

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