Enzyme polymer composite composition and application thereof

Abstract

The invention provides an enzyme polymer composite composition and application, and relates to the technical field of medical materials. The enzyme polymer composite composition consists of a hydrogen peroxide scavenging enzyme / SOD (superoxide dismutase) compound and dispersion liquid, the hydrogen peroxide scavenging enzyme / SOD compound is obtained by carrying out polymerization reaction, dialysis and drying on raw material components containing hydrogen peroxide scavenging enzyme, SOD, chitosan containing carbon-carbon double bonds and derivatives thereof, and a water-soluble or acid-soluble polymerizable monomer; the dispersion liquid is selected from water or hydrogel. The enzyme polymer composite composition can be used as a material for wound recovery and healing.

Description

Technical Field

[0001] This invention belongs to the field of medical materials technology and relates to an enzyme polymer composite composition and its application. Background Technology

[0002] In the early stages of wound formation, immune cells, including macrophages, engulf pathogens and clear necrotic tissue. However, this process also generates a large number of superoxide anion free radicals, which not only attack invading bacteria but also severely damage healthy cells and tissues around the wound, causing symptoms such as redness, swelling, and pain, thus delaying the wound healing process. Superoxide dismutase (SOD) can effectively catalyze the conversion of superoxide anion free radicals into hydrogen peroxide. Chinese patent CN116173283A discloses a chitosan-polyvinyl alcohol (PVA) SOD enzyme hydrogel dressing. The hydrogel layer is composed of chitosan-PVA hydrogel, SOD enzyme solution, gelatin, and agar, and has the advantages of good biocompatibility, good anti-allergy properties, and rapid wound healing.

[0003] The applicant believes that the above technology still needs further improvement and optimization. Summary of the Invention

[0004] Superoxide anion free radicals can be converted into hydrogen peroxide and oxygen under the action of SOD, but high concentrations of hydrogen peroxide are still detrimental to wound healing. Hydrogen peroxide scavenging enzymes, represented by catalase (CAT), can effectively remove hydrogen peroxide, which is beneficial to wound healing. Based on this, the present invention provides an enzyme polymer composite composition and its application.

[0005] The technical solution of the present invention is as follows:

[0006] An enzyme polymer complex composition comprising a hydrogen peroxide scavenging enzyme / SOD complex and a dispersion; The hydrogen peroxide scavenging enzyme / SOD complex is obtained by polymerization, dialysis and drying of a raw material component containing hydrogen peroxide scavenging enzyme, SOD, chitosan containing carbon-carbon double bonds and its derivatives, and water-soluble or acid-soluble polymerizable monomers. The dispersion contains water or a hydrogel; The weight ratio of the hydrogen peroxide scavenging enzyme / SOD complex to the dispersion is 1-20:100.

[0007] Preferably, the chitosan containing carbon-carbon double bonds and its derivatives are at least one of maleamide compounds, acrylamide compounds, acrylate compounds and maleate ester compounds of chitosan and its derivatives.

[0008] Preferably, the water-soluble or acid-soluble polymerizable monomer is composed of monofunctional polymerizable monomers and polyfunctional polymerizable monomers; The monofunctional polymerizable monomer comprises one or a combination of two or more of the following: polymerizable monomers containing amino or quaternary ammonium salts, acrylamide monomers, hydroxyalkyl acrylates, (meth)acrylic acid and its salts, vinyl benzoic acid and its salts, and polyethylene glycol monoacrylates.

[0009] More preferably, the multifunctional polymerizable monomer is selected from one or a combination of two or more of acrylamide monomers and polyethylene glycol diacrylate.

[0010] More preferably, the weight ratio of the monofunctional polymerizable monomer to the polyfunctional polymerizable monomer is 10-100:1.

[0011] Preferably, the weight ratio of the hydrogen peroxide scavenging enzyme, the SOD, the carbon-carbon double bond-containing chitosan and its derivatives, and the polymerizable monomer is 10-0.5:1:0.1-5:10-120.

[0012] Preferably, the hydrogen peroxide scavenging enzyme is selected from at least one of catalase, peroxidase, peroxidase, sulfur-oxidizing protein peroxidase, acetaldehyde dehydrogenase, superoxide dismutase, glutathione peroxidase, lipid peroxidase, plasma enzyme, and collagenase.

[0013] Preferably, the hydrogel is a thermosensitive hydrogel, and preferably, the phase transition temperature of the hydrogel is 10-41℃.

[0014] More preferably, the polymer material of the hydrogel is selected from one or a combination of two or more of PEG-PPG, PEG-PPG-PEG, PCL-PEG-PCL, PCL-PEG, PEG-PLGA, PCL-PEG-PLGA, PLGA-PEG-PLGA, PNIPAMAm-PEG, PNIPAMAm-PEG-PPG, PNIPAMAm, PNIPAMAm-PVA, PDLLA-PEG-PDLLA, and PNIPAMAm-PAA.

[0015] Preferably, the dispersion further contains hyaluronic acid with a molecular weight not exceeding 100 kDa; The hyaluronic acid in the dispersion has a weight percentage of 0.1-1%.

[0016] The enzyme polymer composite composition described in any of the above embodiments is used as a material for wound treatment.

[0017] The beneficial effects of this invention are: (1) The enzyme polymer composite composition of the present invention contains SOD and hydrogen peroxide scavenging enzyme. SOD can convert superoxide anion free radicals into hydrogen peroxide and oxygen. Hydrogen peroxide scavenging enzyme then converts and removes hydrogen peroxide. This not only removes superoxide anion free radicals, but also avoids the adverse effects of hydrogen peroxide on wound recovery and healing. Moreover, the use of CAT can increase the oxygen content of the micro-oxygen environment that is conducive to wound recovery and healing, thereby improving the wound healing speed and effect.

[0018] (2) The enzyme polymer composite composition of the present invention also contains chitosan and its derivatives. Chitosan and its derivatives have good antibacterial properties and are beneficial to wound recovery, which can further improve the effect of the enzyme polymer composite composition on wound recovery and healing. Moreover, by cross-linking, SOD, hydrogen peroxide scavenging enzyme and chitosan and its derivatives are aggregated and combined together, resulting in a better effect on wound healing.

[0019] (3) The present invention further adds low molecular weight hyaluronic acid (molecular weight ≤100kDa) to the enzyme polymer composite composition, which can further enhance the inhibitory effect on superoxide anion free radicals and is more conducive to wound recovery. Detailed Implementation

[0020] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0021] To improve the effect on wound recovery and healing, this invention proposes an enzyme polymer complex composition, which consists of a hydrogen peroxide scavenging enzyme / SOD complex and a dispersion. The above-mentioned hydrogen peroxide scavenging enzyme / SOD complex is obtained by polymerization, dialysis and drying of a raw material component containing hydrogen peroxide scavenging enzyme, SOD, chitosan containing carbon-carbon double bonds and its derivatives, and water-soluble or acid-soluble polymerizable monomers. The above dispersion contains water or a hydrogel; The weight ratio of the hydrogen peroxide scavenging enzyme / SOD complex to the dispersion is 1-20:100.

[0022] In this invention, the hydrogen peroxide scavenging enzyme / SOD complex has the following characteristics: (1) For the presence of a large number of superoxide anion free radicals at the wound site, SOD can convert superoxide anion free radicals into hydrogen peroxide and oxygen, and hydrogen peroxide scavenging enzyme can continue to convert and remove hydrogen peroxide, avoiding the adverse effects of hydrogen peroxide accumulation on wound recovery and healing; (2) Chitosan and its derivatives have good antibacterial properties, biocompatibility and wound healing effects, which can further improve the effect of the complex on wound recovery and healing. Chitosan carries a positive charge and can promote the binding of hydrogen peroxide scavenging enzyme and SOD through electrostatic adsorption, thereby improving the binding effect of the complex; it was also unexpectedly found that the chemical cross-linking of chitosan and SOD can improve the removal efficiency of superoxide anion free radicals; (3) The complex is bound together by polymerization, and the binding is tight and highly stable, which can better exert the synergistic effect of hydrogen peroxide scavenging enzyme, SOD and chitosan and its derivatives.

[0023] For example, the weight ratio of the hydrogen peroxide scavenging enzyme / SOD complex to the dispersion can be any value from 1:100, 2:100, 3:100, 4:100, 5:100, 6:100, 7:100, 8:100, 9:100, 10:100, 12:100, 15:100, 18:100, 20:100, etc., without any particular restrictions.

[0024] In some embodiments, chitosan and its derivatives containing carbon-carbon double bonds are at least one of maleamide compounds, acrylamide compounds, acrylate compounds, and maleate ester compounds of chitosan and its derivatives. Chitosan and its derivatives have hydroxyl and primary amino groups in their structure. Both hydroxyl and primary amino groups are highly reactive and can react with acryloyl chloride, maleic anhydride, acrylic anhydride, methacrylic anhydride, etc., thereby grafting polymerizable carbon-carbon double bonds onto chitosan and its derivatives. Methods for preparing chitosan and its derivatives containing carbon-carbon double bonds can be found in Chinese patents CN119081216A, CN119120444A, and CN119219848A.

[0025] In some embodiments, the water-soluble or acid-soluble polymerizable monomers are composed of monofunctional polymerizable monomers and polyfunctional polymerizable monomers. In this invention, functionality refers to the number of carbon-carbon double bonds capable of polymerization. A monofunctional polymerizable monomer has only one polymerizable carbon-carbon double bond in its structure and acts as a chain extender, improving the polymerization effect. A polyfunctional polymerizable monomer has two or more polymerizable carbon-carbon double bonds in its structure and acts as a crosslinking agent, stably binding SOD, hydrogen peroxide scavenging enzyme, and chitosan and its derivatives.

[0026] In some embodiments, the monofunctional polymerizable monomer comprises one or a combination of two or more of the following: polymerizable monomers containing amino or quaternary ammonium salts, acrylamide monomers, hydroxyalkyl acrylates, (meth)acrylic acid and its salts, vinyl benzoic acid and its salts, and polyethylene glycol monoacrylates. The polymerizable monomers containing amino or quaternary ammonium salts carry a positive charge, which can promote the binding of SOD and hydrogen peroxide scavenging enzyme before the polymerization reaction, thus facilitating the acquisition of a tightly bound hydrogen peroxide scavenging enzyme / SOD complex. For example, polymerizable monomers containing amino or quaternary ammonium salts can be dimethylaminoethyl (meth)acrylate, dimethylaminoethyl acrylate, dimethylaminobutyl (meth)acrylate, dimethylaminopropyl acrylate, aminoethyl acrylate, aminoethyl methacrylate, aminopropyl acrylate, aminopropyl methacrylate, N-methylaminoethyl acrylate, N-methylaminoethyl methacrylate, N-hydroxyethylaminoethyl acrylate, N-hydroxyethylaminoethyl methacrylate, N-ethylaminoethyl acrylate, acryloyloxyethyltrimethylammonium chloride, methacryloyloxyethyltrimethylammonium chloride, methacryloyloxypropyltrimethylammonium chloride, methacryloyloxybutyltrimethylammonium chloride, etc.; acrylamide monomers can be acrylamide, N,N-dimethylacrylamide, 2 - Methacrylamide, N-methacrylamide, N-ethylacrylamide, N,N-diethylacrylamide, N-hydroxyethylacrylamide, N-methyl-2-methylacrylamide, etc.; (meth)acrylate hydroxyalkyl esters can be hydroxyethyl methacrylate, hydroxyethyl acrylate, hydroxypropyl methacrylate, hydroxypropyl acrylate, etc.; (meth)acrylic acid and its salts can be acrylic acid, sodium acrylate, ammonium acrylate, sodium methacrylate, etc.; vinyl benzoic acid and its salts can be vinyl benzoic acid, sodium vinyl benzoate, potassium vinyl benzoate, etc.; polyethylene glycol monoacrylate can be polyethylene glycol (200) monoacrylate, polyethylene glycol (200) monomethacrylate, polyethylene glycol (400) monoacrylate, triethylene glycol monoacrylate, tetraethylene glycol monomethyl ether acrylate, etc.

[0027] In some embodiments, the multifunctional polymerizable monomer is selected from one or more combinations of acrylamide monomers and polyethylene glycol diacrylates. For example, the acrylamide monomer may be selected from N,N'-methylenediacrylamide, N,N'-ethylenediacrylamide, N,N'-propylenediacrylamide, etc. More preferably, the acrylamide monomer may be N,N'-methylenediacrylamide. For example, the polyethylene glycol polyacrylate may be difunctional, such as polyethylene glycol (200) diacrylate, polyethylene glycol (400) diacrylate, polyethylene glycol (200) dimethacrylate, etc., or trifunctional or more, such as tetra-arm polyethylene glycol tetraacrylate, hexa-arm polyethylene glycol hexaacrylate, etc.

[0028] In some embodiments, the weight ratio of monofunctional polymerizable monomers to polyfunctional polymerizable monomers is 10-100:1. For example, the weight ratio can be any value from 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, 100:1, etc., without any particular limitation.

[0029] In this invention, the polymerization method described above is not particularly limited. For example, polymerization can be carried out at a polymerization temperature of 5-35°C and in a buffer solution. Examples of buffer solutions include PBS buffer solution with pH 7.0, 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution with pH 7.0, Tris-HCl buffer solution with pH 7.0, Tris-HCl buffer solution with pH 7.5, sodium carbonate buffer solution with pH 9.3, borax-boric acid buffer solution with pH 8.6, and borate buffer solution with pH 9.18. A polymerization temperature of 5-35°C ensures the reactivity of hydrogen peroxide scavenging enzyme, SOD, and the polymerization product hydrogen peroxide scavenging enzyme / SOD complex, preventing inactivation. The polymerization reaction of this invention can be a free radical polymerization reaction. There are no particular limitations on the free radical initiator used; preferably, it can be obtained by combining ammonium persulfate (APS) and tetramethylethylenediamine (TEMED). APS is the initiator, and TEMED catalyzes the generation of free radicals from APS at room temperature, accelerating the polymerization reaction of carbon-carbon double bonds. The polymerization reaction can be achieved at room temperature to obtain a hydrogen peroxide scavenging enzyme / SOD complex. In this invention, the polymerization reaction time at room temperature using the APS and TEMED initiator can be 2 hours, 2.5 hours, 3 hours, etc., and can be adjusted according to the amount of initiator and the APS / TEMED ratio. The molecular weight cutoff of the dialysis bag used for dialysis can be 2000-10000, such as 3000, 3500, 4000, 4500, etc. The dialysis medium can be a neutral buffer solution, and the dialysis time can be 12-48 hours. Drying can be achieved using a freeze-drying method.

[0030] In some embodiments, the weight ratio of hydrogen peroxide scavenging enzyme, SOD, chitosan containing carbon-carbon double bonds and its derivatives, and polymerizable monomers is 10-0.5:1:0.1-5:10-120. For example, the weight ratio can be 0.5:1:0.1:10, 1:1:0.2:10, 1:1:0.5:30, 3:1:0.5:50, 3:1:0.5:20, 3:1:0.5:70, 3:1:0.5:100, 3:1:0.5:120, 5:1:0.5:50, 5:1:2:100, 5 Any value from :1:5:100, 5:1:2:120, 5:1:5:120, 10:1:0.2:20, 10:1:0.2:50, 10:1:0.2:70, 10:1:0.2:80, 10:1:0.2:100, 10:1:0.1:120, 10:1:0.5:120, 10:1:1:20, 10:1:1:50, 10:1:1:80, 10:1:1:120, 10:1:3:120, 10:1:5:120, etc., without any special restrictions.

[0031] In some embodiments, the hydrogen peroxide scavenging enzyme is selected from at least one of catalase, peroxidase, peroxidase, thiooxidase protein peroxidase, acetaldehyde dehydrogenase, superoxide dismutase, glutathione peroxidase, lipid peroxidase, plasma enzyme, and collagenase. Preferably, the hydrogen peroxide scavenging enzyme is selected from catalase (CAT). CAT can convert hydrogen peroxide into water and oxygen, thus removing hydrogen peroxide and increasing the oxygen content of the micro-oxygen environment on the wound surface, thereby improving wound recovery and healing.

[0032] In some embodiments, the hydrogel is a thermosensitive hydrogel, preferably with a phase transition temperature of 10-41°C. When using a thermosensitive hydrogel, during storage, the temperature is below the phase transition temperature of the hydrogel, and the enzyme polymer composite composition is in a liquid state with good fluidity, allowing for application by spraying or other methods. When the enzyme polymer composite composition is applied to the wound surface, because the wound temperature is higher than the phase transition temperature of the hydrogel, the hydrogel changes from a liquid state to a gel state and tightly covers the wound surface. For example, the phase transition temperature of the hydrogel can be any value from 10°C, 12°C, 14°C, 15°C, 18°C, 20°C, 22°C, 25°C, 27°C, 28°C, 30°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, etc., without particular limitation. Further, the phase transition temperature of the hydrogel can be 20-38°C. In some embodiments, the polymer material of the hydrogel is selected from one or a combination of two or more of PEG-PPG, PEG-PPG-PEG, PCL-PEG-PCL, PCL-PEG, PEG-PLGA, PCL-PEG-PLGA, PLGA-PEG-PLGA, PNIPAMAm-PEG, PNIPAMAm-PEG-PPG, PNIPAMAm, PNIPAMAm-PVA, PDLLA-PEG-PDLLA, and PNIPAMAm-PAA, and the concentration of the polymer material in the hydrogel can be 1-30 wt%. The above-mentioned hydrogels exhibit good biocompatibility and a fast phase transition response rate within a suitable phase transition temperature range.

[0033] In some embodiments, the dispersion further contains hyaluronic acid with a molecular weight not exceeding 100 kDa; The weight percentage of hyaluronic acid in the dispersion is 0.1-1%. This invention also found that adding low molecular weight hyaluronic acid to the enzyme polymer composite composition can further enhance the inhibitory effect of the enzyme polymer composite composition on superoxide anion free radicals, which is more beneficial to wound healing. The possible reason is that, under the action of SOD and hydrogen peroxide scavenging enzyme, chitosan and hyaluronic acid synergistically exert an inhibitory effect on superoxide anion free radicals. For example, the weight percentage of hyaluronic acid in the dispersion can be 0.1%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, etc. Further, the weight percentage of hyaluronic acid in the dispersion can be 0.1-0.5%.

[0034] On the other hand, the present invention proposes the application of the enzyme polymer composite composition described in any of the above embodiments as a material for wound treatment. Addressing issues such as bacterial infection and inflammation in wounds, the enzyme polymer composite composition of the present invention synergistically exerts the effects of SOD, hydrogen peroxide scavenging enzyme, and chitosan (and also includes hyaluronic acid), thus promoting wound recovery and healing. Furthermore, the use of a hydrogel enhances the practicality of the enzyme polymer composite composition.

[0035] The technical solution of the present invention will be further described and illustrated below with reference to various embodiments. Unless otherwise specified, the parts mentioned in the following embodiments are parts by weight.

[0036] Preparation Examples 1-4: Preparation of Hydrogen Peroxide Scavenging Enzyme / SOD Complex Preparation Example 1 10 mg CAT, 1 mg SOD, 1 mg acrylated chitosan (chitosan number-average molecular weight 10,000), and 20 mg acrylamide were dissolved in 10 ml of PBS buffer (20 mM, pH 7.0) and stirred until homogeneous. Then, 1 ml of DMSO solution containing 2 mg N,N'-methylenediacrylamide was added and stirred until homogeneous. The reaction system was purged with nitrogen for 30 min. Immediately after adding 0.5 ml of APS aqueous solution (15 mg / ml), 0.5 ml of TEMED aqueous solution (30 μL / ml) was added, and the polymerization reaction was carried out at room temperature for 3 h. The reaction product was dialyzed for 18 h in PBS buffer solution at pH 7.0 using a dialysis bag with a molecular weight cutoff of 4000, with the water changed every 2 h. After dialyzing, the product was freeze-dried under vacuum to obtain the CAT / SOD complex, denoted as P-1.

[0037] Preparation Example 2 The difference between this preparation example and Preparation Example 1 is that in Preparation Example 1, CAT was changed from 10 mg to 5 mg, acrylated chitosan was changed from 1 mg to 3 mg, and 20 mg acrylamide was changed to a combination of 80 mg acrylamide and 20 mg hydroxyethyl acrylate. The remaining steps remained unchanged. A CAT / SOD complex was obtained, denoted as P-2.

[0038] Preparation Example 3 The difference between this preparation example and Preparation Example 1 is that in Preparation Example 1, CAT was adjusted from 10 mg to 1 mg, and acrylated chitosan was adjusted from 1 mg to 0.2 mg. The remaining steps remained unchanged. A CAT / SOD complex was obtained, denoted as P-3.

[0039] Preparation Example 4 The difference between this preparation example and Preparation Example 1 is that in Preparation Example 1, 20 mg of acrylamide was changed to a combination of 30 mg acrylamide and 30 mg dimethylaminoethyl methacrylate, and N,N'-methylenediacrylamide was changed from 2 mg to 4 mg. The remaining steps remained unchanged. A CAT / SOD complex was obtained, denoted as P-4.

[0040] Comparative Preparation Example 1 The difference between this comparative preparation example and Preparation Example 1 is that acrylated chitosan was not added in Preparation Example 1. All other steps remained unchanged. A CAT / SOD complex was obtained, denoted as C-1.

[0041] Comparative Preparation Example 2 The difference between this comparative preparation example and Preparation Example 1 is that in Preparation Example 1, acrylated chitosan was replaced with carboxymethyl chitosan (degree of deacetylation 98%, degree of substitution 80%, number average molecular weight 10,000). The remaining steps remained unchanged. A CAT / SOD complex was obtained, denoted as C-2.

[0042] Comparative preparation example 3 The difference between this comparative preparation example and Preparation Example 1 is that CAT was not added in Preparation Example 1. All other steps remained unchanged. The SOD complex was obtained and designated C-3.

[0043] Example 1 The enzyme polymer composite composition was prepared by combining the CAT / SOD complex P-1 from Preparation Example 1 and Pluronic F127 hydrogel (15 wt% concentration, phase transition temperature 37°C) at a weight ratio of 1:100. The CAT / SOD complex P-1 was added to Pluronic F127 at a temperature not exceeding 20°C and stirred until uniformly dispersed to obtain the enzyme polymer composite composition.

[0044] Example 2 The difference between this embodiment and Embodiment 1 is that in Embodiment 1, the weight ratio of the CAT / SOD complex P-1 and the Pluronic F127 hydrogel was adjusted from 1:100 to 5:100. The remaining steps remain unchanged.

[0045] Example 3 The difference between this embodiment and Embodiment 1 is that in Embodiment 1, the weight ratio of the CAT / SOD complex P-1 and the Pluronic F127 hydrogel was adjusted from 1:100 to 10:100. The remaining steps remain unchanged.

[0046] Example 4 The difference between this embodiment and Embodiment 1 is that in Embodiment 1, the weight ratio of the CAT / SOD complex P-1 and the Pluronic F127 hydrogel was adjusted from 1:100 to 20:100. The remaining steps remain unchanged.

[0047] Comparative Example 1 The difference between this comparative example and Example 1 is that in Example 1, the CAT / SOD complex P-1 was replaced with an equal weight of the CAT / SOD complex C-1 from Comparative Preparation Example 1. All other steps remained unchanged.

[0048] Comparative Example 2 The difference between this comparative example and Example 1 is that in Example 1, the CAT / SOD complex P-1 was replaced with an equal weight of the CAT / SOD complex C-2 from Comparative Preparation Example 2. All other steps remained unchanged.

[0049] Comparative Example 3 The difference between this comparative example and Example 1 is that in Example 1, the CAT / SOD complex P-1 was replaced with an equal weight of the SOD complex C-3 from Comparative Preparation Example 3. All other steps remained unchanged.

[0050] Comparative Example 4 10 mg CAT, 1 mg SOD and 2 mg cetyltrimethylammonium bromide were added to 260 mg of Pluronic F127 hydrogel from Example 1 at a temperature not exceeding 20°C and stirred until homogeneous to obtain an enzyme polymer composite composition.

[0051] Comparative Example 5 At a temperature not exceeding 20°C, 10 mg CAT, 1 mg SOD, 1 mg carboxymethyl chitosan from Comparative Preparation Example 2, and 2 mg cetyltrimethylammonium bromide were added to 280 mg of Pluronic F127 hydrogel from Example 1 and stirred until homogeneous to obtain an enzyme polymer composite composition.

[0052] Example 5 The enzyme polymer complex composition was prepared by combining the CAT / SOD complex P-2 from Preparation Example 2 and Pluronic F127 hydrogel (15 wt% concentration, phase transition temperature 37 °C) in a weight ratio of 5:100. It was prepared according to the method of Example 1.

[0053] Example 6 The enzyme polymer complex composition consisted of the CAT / SOD complex P-3 from Preparation Example 3 and Pluronic F127 hydrogel (15 wt% concentration, phase transition temperature 37 °C) in a weight ratio of 5:100. It was prepared according to the method of Example 1.

[0054] Example 7 The enzyme polymer complex composition consisted of the CAT / SOD complex P-4 from Preparation Example 4 and Pluronic F127 hydrogel (15 wt% concentration, phase transition temperature 37 °C) in a weight ratio of 5:100. It was prepared according to the method of Example 1.

[0055] Example 8 The difference between this embodiment and Embodiment 7 is that in Embodiment 7, the weight ratio of the CAT / SOD complex P-4 and the Pluronic F127 hydrogel was adjusted from 5:100 to 12:100. The remaining steps remain unchanged.

[0056] Example 9 The difference between this embodiment and Embodiment 7 is that in Embodiment 7, the weight ratio of the CAT / SOD complex P-4 and the Pluronic F127 hydrogel was adjusted from 5:100 to 20:100. The remaining steps remain unchanged.

[0057] Example 10 The enzyme polymer complex composition consisted of the CAT / SOD complex P-4 from Preparation Example 4 and Pluronic F127 hydrogel (15 wt% concentration, phase transition temperature 37 °C) in a weight ratio of 5:100. It was prepared according to the method of Example 1.

[0058] Pluronic F127 hydrogel also contains 0.1 wt% hyaluronic acid, with a molecular weight of 10 kDa.

[0059] Example 11 The enzyme polymer complex composition consisted of the CAT / SOD complex P-4 from Preparation Example 4 and Pluronic F127 hydrogel (15 wt% concentration, phase transition temperature 37 °C) in a weight ratio of 5:100. It was prepared according to the method of Example 1.

[0060] Pluronic F127 hydrogel also contains 0.8 wt% hyaluronic acid, with a molecular weight of 10 kDa.

[0061] Example 12 The enzyme polymer complex composition consisted of the CAT / SOD complex P-4 from Preparation Example 4 and Pluronic F127 hydrogel (15 wt% concentration, phase transition temperature 37 °C) in a weight ratio of 5:100. It was prepared according to the method of Example 1.

[0062] Pluronic F127 hydrogel also contains 0.5 wt% hyaluronic acid, with a molecular weight of 100 kDa.

[0063] Comparative Example 6 The difference between this comparative example and Example 10 is that in Example 10, the CAT / SOD complex P-4 of Preparation Example 4 was replaced with an equal weight of the CAT / SOD complex C-1 of Comparative Preparation Example 1. The remaining steps remained unchanged.

[0064] Comparative Example 7 The difference between this comparative example and Example 10 is that in Example 10, the CAT / SOD complex P-4 of Preparation Example 4 was replaced with an equal weight of the CAT / SOD complex C-2 of Comparative Preparation Example 2. All other steps remained unchanged.

[0065] Comparative Example 8 The difference between this comparative example and Example 10 is that in Example 10, hyaluronic acid was replaced with a medium-to-high molecular weight hyaluronic acid with a molecular weight of 500 kDa. The remaining steps remain unchanged.

[0066] Superoxide anion free radicals (O2) -Scavenging ability test: Add 4.5 ml of 50 mmol / L Tris-HCl buffer solution (pH 8.2) and 4.2 ml of ultrapure water to a test tube, mix well, and incubate in a 25°C water bath for 20 min. Immediately after incubation, add 0.5 ml of preheated 3 mmol / L pyrogallol at 25°C, shake quickly, and measure the absorbance at 325 nm every 30 s. Plot a regression equation for absorbance versus time, with the slope representing the pyrogallol auto-oxidation rate V. A 10 mmol / L HCl solution was used as a blank control.

[0067] Determination of the auto-oxidation rate of pyrogallol after adding the test complex sample: Add 0.5 ml of the test complex sample, reduce the amount of water accordingly, and keep the other steps the same. Measure the absorbance. Repeat 3 times for each sample.

[0068] Formula for calculating the inhibition rate of superoxide anion free radicals by the test complex sample: Inhibition rate = (V1 - V2) / V1 × 100% Where V1 is the auto-oxidation rate of pyrogallol, and V2 is the auto-oxidation rate of pyrogallol after the sample to be tested is added.

[0069] One hour after adding ultrapure water and the sample to be tested to the above solutions, the oxygen content in the solutions was measured.

[0070] The results are shown in Table 1 below.

[0071] Table 1

[0072] The data results above show that the CAT / SOD enzyme polymer composite composition of the present invention has a good removal effect on superoxide anion free radicals and can create a micro-oxygen environment, thereby improving the recovery and healing effect on wounds. Further addition of low molecular weight hyaluronic acid to the enzyme polymer composite composition can further enhance its ability to scavenge superoxide anion free radicals.

[0073] As described above, the basic principles, main features, and advantages of the present invention have been shown and described. Those skilled in the art should understand that the present invention is not limited to the above embodiments, which are merely preferred embodiments and should not be construed as limiting the scope of the invention. All equivalent changes and modifications made in accordance with the scope of the patent and the description should still fall within the scope of the present invention. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. An enzyme polymer complex composition, characterized in that, Composed of a hydrogen peroxide scavenging enzyme / SOD complex and a dispersion; The hydrogen peroxide scavenging enzyme / SOD complex is obtained by polymerization, dialysis and drying of a raw material component containing hydrogen peroxide scavenging enzyme, SOD, chitosan containing carbon-carbon double bonds and its derivatives, and water-soluble or acid-soluble polymerizable monomers. The dispersion contains water or a hydrogel; The weight ratio of the hydrogen peroxide scavenging enzyme / SOD complex to the dispersion is 1-20:

100.

2. The enzyme polymer composite composition according to claim 1, characterized in that, The carbon-carbon double bond-containing chitosan and its derivatives are at least one of maleamide compounds, acrylamide compounds, acrylate compounds, and maleate ester compounds of chitosan and its derivatives.

3. The enzyme polymer composite composition according to claim 1, characterized in that, The water-soluble or acid-soluble polymerizable monomers are composed of monofunctional polymerizable monomers and polyfunctional polymerizable monomers; The monofunctional polymerizable monomer comprises one or a combination of two or more of the following: polymerizable monomers containing amino or quaternary ammonium salts, acrylamide monomers, hydroxyalkyl acrylates, (meth)acrylic acid and its salts, vinyl benzoic acid and its salts, and polyethylene glycol monoacrylates.

4. The enzyme polymer composite composition according to claim 3, characterized in that, The multifunctional polymerizable monomer is selected from one or a combination of two or more of the following: acrylamide monomers and polyethylene glycol diacrylate.

5. The enzyme polymer composite composition according to claim 3, characterized in that, The weight ratio of the monofunctional polymerizable monomer to the polyfunctional polymerizable monomer is 10-100:

1.

6. The enzyme polymer composite composition according to claim 1, characterized in that, The weight ratio of the hydrogen peroxide scavenging enzyme, the SOD, the carbon-carbon double bond-containing chitosan and its derivatives, and the polymerizable monomer is 10-0.5:1:0.1-5:10-120.

7. The enzyme polymer complex composition according to any one of claims 1-6, characterized in that, The hydrogen peroxide scavenging enzyme is selected from at least one of catalase, peroxidase, peroxidase, sulfur-oxidizing protein peroxidase, acetaldehyde dehydrogenase, superoxide dismutase, glutathione peroxidase, lipid peroxidase, plasma enzyme, and collagenase.

8. The enzyme polymer composite composition according to claim 1, characterized in that, The hydrogel is a thermosensitive hydrogel; The phase transition temperature of the hydrogel is 10-41℃.

9. The enzyme polymer composite composition according to claim 8, characterized in that, The polymer material of the hydrogel is selected from one or a combination of two or more of PEG-PPG, PEG-PPG-PEG, PCL-PEG-PCL, PCL-PEG, PEG-PLGA, PCL-PEG-PLGA, PLGA-PEG-PLGA, PNIPAMAm-PEG, PNIPAMAm-PEG-PPG, PNIPAMAm, PNIPAMAm-PVA, PDLLA-PEG-PDLLA, and PNIPAMAm-PAA.

10. The enzyme polymer complex composition according to claim 1, characterized in that, The dispersion also contains hyaluronic acid with a molecular weight not exceeding 100 kDa; The hyaluronic acid in the dispersion has a weight percentage of 0.1-1%.

11. The application of the enzyme polymer complex composition according to any one of claims 1-10, characterized in that, As a material for wound treatment.

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