Anti-oxidation gel for wound healing as well as preparation method and application of anti-oxidation gel

Abstract

The invention relates to antioxidant gel for wound healing as well as a preparation method and application of the antioxidant gel. The antioxidant gel is prepared from the following components in parts by mass: 0.01 to 20 parts of hyaluronic acid, 0.01 to 20 parts of cysteine and 1 to 50 parts of polyethylene glycol carboxyl activated ester. The cysteine modified hyaluronic acid and the polyethylene glycol carboxyl activated ester are cross-linked to form a hydrogel network, the tissue adhesion capacity of the hyaluronic acid gel is remarkably improved, the antioxidant capacity and the anti-inflammatory capacity are further improved, and the hydrogel material is simple in component, good in biocompatibility, stable in cross-linked network and biosafe.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical materials, and in particular to an antioxidant gel for wound healing and a preparation method and application thereof. Background Art

[0002] Oxidative stress (OS) refers to the imbalance between oxidant production and antioxidant defense in the body, which leads to neutrophil inflammatory infiltration, increased protease secretion, and the production of a large number of oxidative intermediates, causing damage to biological systems. Oxidative stress is a negative effect produced by free radicals in the body and is considered an important factor leading to aging and disease.

[0003] Rapid wound healing, especially the promotion of healing of refractory wounds, is very important, which can significantly reduce complications and improve the quality of life of patients. Among the causes of refractory wounds, excessive oxidative stress plays an important role. Excessive oxidative stress will delay the healing process, adversely affect the growth, proliferation and repair function of cells, thereby delaying the healing process of refractory wounds. At the same time, oxidative stress will damage the cell structure, lead to lipid peroxidation of the cell membrane, damage the cell membrane structure, make the cells vulnerable to the external environment, and thus affect the repair of the wound. Oxidative stress will also aggravate the inflammatory response, affect the regulation and repair function of inflammatory cells, and further hinder the healing of refractory wounds. Therefore, effectively inhibiting oxidative stress can improve the efficiency of wound healing. At present, hyaluronic acid gel is a good wound dressing and has been widely used in clinical practice. However, the traditional hyaluronic acid dressing has poor tissue adhesion ability and insufficient antioxidant capacity, which limits its effectiveness.

[0004] Therefore, how to achieve the tissue adhesion ability of hyaluronic acid and further enhance the antioxidant effect has become a problem that needs to be solved urgently. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides an antioxidant gel for wound healing, a preparation method and application thereof. The present invention forms a hydrogel network by cross-linking cysteine-modified hyaluronic acid with polyethylene glycol carboxyl activated ester, which significantly improves the tissue adhesion ability of the hyaluronic acid gel, and further improves the antioxidant and anti-inflammatory abilities. The above hydrogel material has simple components and good biocompatibility, and the cross-linked network is stable and biosafe.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides an antioxidant gel for wound healing, wherein the antioxidant gel comprises, by mass, 0.01-20 parts of hyaluronic acid, 0.01-20 parts of cysteine, and 1-50 parts of polyethylene glycol carboxyl activated ester.

[0008] The 0.01-20 parts can be, for example, 0.01 parts, 1 parts, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, 17 parts, 18 parts, 19 parts or 20 parts, etc.

[0009] The 1-50 parts can be, for example, 1 part, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, 17 parts, 18 parts, 19 parts, 20 parts, 21 parts, 22 parts, 23 parts, 24 parts, 25 parts, 26 parts, 27 parts, 28 parts, 29 parts, 30 parts, 31 parts, 32 parts, 33 parts, 34 parts, 35 parts, 36 parts, 37 parts, 38 parts, 39 parts, 40 parts, 41 parts, 42 parts, 43 parts, 44 parts, 45 parts, 46 parts, 47 parts, 48 ​​parts, 49 parts or 50 parts, etc.

[0010] In the present invention, hyaluronic acid is modified by cysteine ​​to prepare hyaluronic acid modified by cysteine. At the same time, polyethylene glycol carboxyl activated ester and modified hyaluronic acid are micro-crosslinked by thioesterification reaction of carboxyl activated ester and thiol group. The retention capacity of hyaluronic acid is improved. On the one hand, the unreacted thiol of cysteine ​​is chemically bonded to the tissue surface to enhance the adhesion of the hydrogel, and the uncrosslinked polyethylene glycol carboxyl activated ester site and the tissue surface protein are cross-linked, thereby fixing the antioxidant gel on the tissue, further improving the tissue adhesion of the hydrogel. On the other hand, the antioxidant effect of the thiol in cysteine ​​is utilized to slow down the oxidative stress of the wound surface, thereby accelerating the healing of the refractory wound surface. Compared with traditional oxidative stress control schemes, such as nanozymes, drugs, etc., this scheme is simple and has better biocompatibility. The crosslinking of polyethylene glycol carboxyl activated ester and hyaluronic acid is utilized to form a gel to improve the overall stability.

[0011] Preferably, the polyethylene glycol carboxyl activated ester may be any one of Formula I, Formula II, Formula III and Formula IV:

[0012]

[0013] Wherein, in each formula, Z is a natural number between 20 and 300, and can be 29 to 228, 29, 34, 38, 45, 57, 136, 181 or 228;

[0014] The group R is a formula A or a formula B, wherein M represents a nitrogen atom, an oxygen atom, a sulfur atom, or a phosphorus atom, preferably an oxygen atom, a nitrogen atom, or a sulfur atom, connected to the end of the polyethylene glycol, and X or Y is an integer between 1 and 5, such as 0, 2, or 3; the groups R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 and R 16 Each of them is independently a hydrogen atom, a hydroxyl group, an alkyl group having 1 to 10 carbon atoms, a phenyl group or a sulfonic acid group.

[0015] Preferably, the polyethylene glycol carboxyl activated ester may be any one of the following 1)-8):

[0016] 1) As shown in formula I, wherein R is formula A, in formula A, X is 2 to 3, M is an oxygen atom, Z is 136, and R1, R2, R3 and R4 are all hydrogen atoms;

[0017] 2) As shown in formula I, wherein R is formula B, in formula B, Y is 1 to 2, M is an oxygen atom, Z is 228, and R1, R2, R3 and R4 are all hydrogen atoms;

[0018] 3) As shown in formula II, wherein R is formula A, in formula A, X is 2 to 3, M is an oxygen atom, Z is 57, R1, R2, R3 and R4 are all hydrogen atoms, and R5, R6, R7 and R8 are all hydroxyl groups;

[0019] 4) As shown in formula II, wherein R is formula B, in formula B, Y is 1 to 2, M is an oxygen atom, Z is 57, R1, R2, R3 and R4 are all hydrogen atoms, and R5, R6, R7 and R8 are all hydroxyl groups;

[0020] 5) As shown in formula III, wherein R is formula A, in which X is 2 to 3, M is a nitrogen atom, Z is 38, R1, R2, R3 and R4 are all hydrogen atoms, R5, R6, R7 and R8 are all methyl groups, R9, R 10 , R 11 and R 12 is phenyl;

[0021] 6) As shown in formula III, wherein R is formula B, in which Y is 1 to 2, M is a sulfur atom, Z is 29, R1, R2, R3 and R4 are all hydrogen atoms, R5, R6, R7 and R8 are all sulfonic acid groups, R9, R 10 , R 11 and R 12 is phenyl;

[0022] 7) As shown in Formula IV, wherein R is Formula A, in which X is 3 to 5, M is a nitrogen atom, Z is 29, R1, R2, R3, R4, R5, R6, R7 and R8 are all sulfonic acid groups, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 and R 16 is a hydrogen atom;

[0023] 8) As shown in Formula IV, wherein R is Formula B, in Formula B, Y is 2 to 4, M is a nitrogen atom, Z is 29, R1, R2, R3, R4, R5, R6, R7 and R8 are all sulfonic acid groups, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 and R 16 A hydrogen atom.

[0024] Preferably, the molecular weight of the polyethylene glycol backbone is 2000-10000. For example, it can be 2000, 4000, 6000, 8000 or 10000.

[0025] Preferably, the molecular weight of the hyaluronic acid is (20-150)×10 4 , the (20-150) can be, for example, 20-40, 40-80, 80-150, etc.

[0026] Preferably, the mass ratio of cysteine ​​to hyaluronic acid is 1:(0.01-3). The (0.01-3) may be, for example, 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5 or 3.

[0027] In the present invention, within the above range, the feed ratio of cysteine ​​is too low, the hyaluronic acid grafting rate is low, and the function improvement is not obvious; cysteine ​​completely modifies the hyaluronic acid carboxyl group, maximizes the hyaluronic acid grafting rate, and further improves the tissue adhesion and antioxidant capacity of the hydrogel.

[0028] In a second aspect, the present invention provides a method for preparing the antioxidant gel for wound healing as described in the first aspect, the preparation method comprising:

[0029] Add hyaluronic acid to the solution and stir for the first time until it is completely dissolved. After dissolution, add a catalyst and continue stirring for the second time to activate the carboxyl group of the hyaluronic acid. Then add cysteine ​​to react. After the reaction is completed, the reaction solution is dialyzed and freeze-dried to obtain cysteine-modified hyaluronic acid freeze-dried powder. The modified hyaluronic acid freeze-dried powder and polyethylene glycol carboxyl activated ester are dissolved separately and mixed evenly to obtain an antioxidant gel.

[0030] Preferably, the solvent of the hyaluronic acid solution comprises sterile water and / or buffer.

[0031] Preferably, the buffer comprises any one or a combination of at least two of phosphate buffer, phosphate-buffered saline, Tris-hydrochloric acid buffer, glycine-hydrochloric acid buffer, disodium hydrogen phosphate-sodium citrate buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, citric acid-sodium citrate buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, boric acid-borax buffer, borax-sodium hydroxide buffer or sodium carbonate-sodium bicarbonate buffer.

[0032] Preferably, the pH of the hyaluronic acid solution is 4-7, for example, it can be 4, 4.5, 5, 5.5, 6, 6.5 or 7.

[0033] Preferably, the catalyst comprises a combination of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and a protecting agent.

[0034] Preferably, the protective agent comprises N-hydroxysuccinimide and / or N-hydroxysulfosuccinimide.

[0035] Preferably, the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added in an amount of 0.005-30 parts by mass, for example, 0.005 parts, 0.05 parts, 0.5 parts, 1 parts, 5 parts, 10 parts, 15 parts, 20 parts, 25 parts or 30 parts.

[0036] Preferably, the protective agent is added in an amount of 0.006-34 parts by mass, for example, 0.006 parts, 0.05 parts, 0.5 parts, 1 parts, 5 parts, 10 parts, 15 parts, 20 parts, 25 parts, 30 parts or 34 parts.

[0037] Preferably, the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to the protective agent in the catalyst is (1-3):(2-5). The (1-3) may be, for example, 1, 2 or 3. The (2-5) may be, for example, 2, 3, 4 or 5.

[0038] Preferably, the first speed is 200-1000 rpm, and the time is 40 min-12 h, and the 40 min-12 h can be, for example, 40 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h or 12 h, etc. The 200-1000 rpm can be, for example, 200 rpm, 400 rpm, 600 rpm, 800 rpm or 1000 rpm.

[0039] Preferably, the second stirring speed is 200-1000 rpm, and the time is 20-40 min, and the 20-40 min can be, for example, 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, 32 min, 34 min, 36 min, 38 min or 40 min, etc. The 200-1000 rpm can be, for example, 200 rpm, 400 rpm, 600 rpm, 800 rpm or 1000 rpm.

[0040] Preferably, the gas environment of the reaction includes any one of air, nitrogen, argon or helium, or a combination of at least two of them.

[0041] Preferably, the reaction time is 4-12 h, for example, 4 h, 6 h, 8 h, 10 h or 12 h.

[0042] Preferably, the dialysis solution comprises any one of ultrapure water, deionized water, purified water or dialysis buffer or a combination of at least two thereof.

[0043] Preferably, the dialysis buffer comprises any one or a combination of at least two of phosphate buffer, phosphate-buffered saline, Tris-hydrochloric acid buffer, HEPES zwitterionic buffer or amino acid buffer.

[0044] Preferably, the pH of the dialyzed solution is 4-7, for example, 4, 4.5, 5, 5.5, 6, 6.5 or 7.

[0045] Preferably, the molecular weight cutoff of the dialysis bag for dialysis is 1000-25000, for example, it can be 1000, 2000, 3500, 5000, 7000, 8000, 10000, 15000, or 25000.

[0046] Preferably, the dialysis time is 2-5 days, for example, 2 days, 3 days, 4 days or 5 days.

[0047] Preferably, the freeze-drying method comprises packaging the purified reaction solution and subjecting it to freezing treatment at low temperature, and after freezing, using a freeze dryer to sublimate and dry the solvent in the reactant to obtain a dry product.

[0048] The solvent for dissolving the hyaluronic acid freeze-dried powder includes sterile water and / or buffer 1.

[0049] Preferably, the buffer 1 comprises any one or a combination of at least two of phosphate buffer, phosphate-buffered saline, Tris-hydrochloric acid buffer, glycine-hydrochloric acid buffer, disodium hydrogen phosphate-sodium citrate buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, citric acid-sodium citrate buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, boric acid-borax buffer, borax-sodium hydroxide buffer or sodium carbonate-sodium bicarbonate buffer.

[0050] Preferably, the solvent for dissolving the polyethylene glycol carboxyl activated ester comprises sterile water and / or buffer 2.

[0051] Preferably, the buffer 2 comprises any one or a combination of at least two of phosphate buffer, phosphate-buffered saline, Tris-hydrochloric acid buffer, glycine-hydrochloric acid buffer, disodium hydrogen phosphate-sodium citrate buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, citric acid-sodium citrate buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, boric acid-borax buffer, borax-sodium hydroxide buffer or sodium carbonate-sodium bicarbonate buffer.

[0052] Preferably, the mass concentration of the polyethylene glycol carboxyl activated ester is 1-50%, for example, it can be 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%.

[0053] Preferably, the mass concentration of the modified hyaluronic acid lyophilized powder is 0.01-20%, for example, it can be 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%, etc.

[0054] Preferably, the volume ratio of the polyethylene glycol carboxyl activated ester solution to the modified hyaluronic acid solution in the mixture is 1:(0.1-5), and the (0.1-5) can be, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4 or 5, etc.

[0055] In a third aspect, the present invention provides a use of the antioxidant gel for wound healing described in the first aspect in alleviating oxidative stress for non-therapeutic purposes or in preparing a product for alleviating oxidative stress.

[0056] The products may include drugs for wound healing, etc.

[0057] Compared with the prior art, the present invention has at least the following beneficial effects:

[0058] 1. The present invention uses cysteine ​​to modify hyaluronic acid. The cysteine-modified hyaluronic acid is cross-linked with polyethylene glycol carboxyl activated ester to form a hydrogel network. The unreacted thiol groups of cysteine ​​are chemically bonded to the tissue surface. At the same time, the uncross-linked polyethylene glycol carboxyl activated ester sites are cross-linked with the tissue surface proteins, thereby fixing the antioxidant gel on the tissue and improving the tissue adhesion of the hydrogel.

[0059] 2. The antioxidant effect of the thiol group of the present invention reduces the oxidative stress of the wound surface, thereby accelerating the healing of refractory wounds. Compared with traditional oxidative stress control schemes, such as nanozymes, drugs, etc., this scheme is simple and has better biocompatibility. The cross-linking of polyethylene glycol carboxyl activated ester and hyaluronic acid is used to form a gel, which improves the overall stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is a graph showing the in vitro anti-inflammatory ability of the antioxidant hydrogel.

[0061] Figure 2 This is a graph showing the in vitro antioxidant capacity of the antioxidant hydrogel.

[0062] Figure 3 This is a diagram showing the results of antioxidant hydrogel promoting wound healing in vivo. DETAILED DESCRIPTION

[0063] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0064] Sources of reagents and consumables used in the following examples:

[0065] Dialysis bags were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0066] Example 1

[0067] This embodiment provides an antioxidant gel for wound healing

[0068] 5 parts of hyaluronic acid (molecular weight 800,000-1.5 million, McLean, H909938-25g) were added to sterile water with a pH of 5, stirred until completely dissolved, the speed was 400rpm, and the catalyst was added after dissolution and continued to stir for 30min, the speed was 400rpm, the catalyst EDC and NHS were added in 8 parts and 12 parts, and the molar ratio of EDC and NHS was 2:3. Then 4 parts of cysteine ​​were added, and the reaction was carried out for 8h in a nitrogen environment. After the reaction, the above reaction solution was loaded into a dialysis bag with a molecular weight cutoff of 10000, dialyzed with ultrapure water at a pH of 5, and lyophilized after dialysis for 3 days to obtain hyaluronic acid freeze-dried powder modified with cysteine, and the modified hyaluronic acid freeze-dried powder and two-arm polyethylene glycol succinimide succinate 6000 (Formula 1) were dissolved in phosphate buffer solutions with a pH of 9 and a pH of 7, respectively, with a mass concentration of 5% and 10%, respectively, and the two solutions were mixed in equal volumes to obtain an antioxidant gel.

[0069]

[0070] Example 2

[0071] This embodiment provides an antioxidant gel for wound healing

[0072] 15 parts of hyaluronic acid (molecular weight 800,000-1.5 million, McLean, H909938-25g) were added to sterile water with a pH of 4, stirred until completely dissolved at a speed of 500 rpm, and after dissolution, the catalyst was added and stirred for 40 minutes at a speed of 500 rpm. The catalysts EDC and NHS were added in 20 parts and 25 parts respectively, and the molar ratio of EDC to sulfo-NHS was 2:2.5. Then 6 parts of cysteine ​​were added and reacted for 12 hours in a nitrogen environment. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 10,000, dialyzed with ultrapure water at a pH of 4, and lyophilized after 4 days of dialysis to obtain cysteine-modified hyaluronic acid lyophilized powder. The modified hyaluronic acid lyophilized powder and two-arm polyethylene glycol succinimidyl valerate 6000 (Formula 2) were dissolved in phosphate buffer solutions at pH 9 and pH 4, respectively, with mass concentrations of 10% and 25%, respectively. The two solutions were mixed in equal volumes to obtain an antioxidant gel.

[0073]

[0074] Example 3

[0075] This embodiment provides an antioxidant gel for wound healing

[0076] 3 parts of hyaluronic acid (molecular weight 400,000-800,000, McLean, H909938-25g) were added to sterile water with a pH of 4.5, stirred until completely dissolved at a speed of 600 rpm, and after dissolution, the catalyst was added and stirred for 20 minutes at a speed of 600 rpm. The catalysts EDC and NHS were added in 6 and 8 parts respectively, and the molar ratio of EDC to NHS was 3:4. Then 2 parts of cysteine ​​were added and reacted in a nitrogen environment for 10 hours. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 10,000, dialyzed with ultrapure water at a pH of 4.5, and lyophilized after 5 days of dialysis to obtain cysteine-modified hyaluronic acid lyophilized powder. The modified hyaluronic acid lyophilized powder and two-arm polyethylene glycol succinimidyl succinate 6000 (Formula 1) were dissolved in phosphate buffer solutions at pH 9 and pH 7, respectively, with mass concentrations of 8% and 15%, respectively. The two solutions were mixed in equal volumes to obtain an antioxidant gel.

[0077] Example 4

[0078] This embodiment provides an antioxidant gel for wound healing, which differs from Embodiment 1 in that 10 parts of hyaluronic acid and 0.2 parts of cysteine ​​are used, that is, the mass ratio of hyaluronic acid to cysteine ​​is 5:0.1, and the rest is consistent with Embodiment 1.

[0079] Example 5

[0080] This embodiment provides an antioxidant gel for wound healing. The difference between this embodiment and embodiment 1 is that 10 parts of hyaluronic acid and 2 parts of cysteine ​​are used, that is, the mass ratio of hyaluronic acid to cysteine ​​is 5:1, and the rest is consistent with embodiment 1.

[0081] Example 6

[0082] This embodiment provides an antioxidant gel for wound healing, which is different from Example 1 in that the molar ratio of EDC to NHS in the catalyst is 0.5:1, and the rest is consistent with Example 1.

[0083] Example 7

[0084] This embodiment provides an antioxidant gel for wound healing, which is different from Example 1 in that the molar ratio of EDC to NHS in the catalyst is 0.1:1, and the rest is consistent with Example 1.

[0085] Example 8

[0086] This embodiment provides an antioxidant gel for wound healing, which differs from Embodiment 1 in that the pH of the hyaluronic acid solution is 8, and the rest is consistent with Embodiment 1.

[0087] Example 9

[0088] This embodiment provides an antioxidant gel for wound healing. The difference between this embodiment and embodiment 1 is that the pH of the hyaluronic acid solution is 10, and the rest is consistent with embodiment 1.

[0089] Comparative Example 1

[0090] This comparative example provides an antioxidant gel for wound healing, which differs from Example 1 in that hyaluronic acid is not modified with cysteine, and hyaluronic acid is used to replace the hyaluronic acid modified with cysteine ​​in Example 1 and mixed with two-arm polyethylene glycol succinimidyl succinate 6000 (Formula 1), and the rest is the same as Example 1.

[0091] Comparative Example 2

[0092] This comparative example provides an antioxidant gel for wound healing, which differs from Example 1 in that hyaluronic acid is not added, and an equal amount of cysteine ​​is used to replace the hyaluronic acid modified with cysteine ​​in Example 1 and mixed with two-arm polyethylene glycol succinimidyl succinate 6000 (Formula 1), and the rest is the same as Example 1.

[0093] Comparative Example 3

[0094] This comparative example provides an antioxidant gel for wound healing, which differs from Example 1 in that two-arm polyethylene glycol succinimidyl succinate 6000 is not added, and the rest is the same as Example 1.

[0095] Comparative Example 4

[0096] This comparative example provides an antioxidant gel for wound healing, which differs from Example 1 in that 20 parts of polyethylene glycol carboxyl activated ester and 0.001 parts of hyaluronic acid are used, and the rest is consistent with Example 1.

[0097] Comparative Example 5

[0098] This comparative example provides an antioxidant gel for wound healing, which differs from Example 1 in that 25 parts of polyethylene glycol carboxyl activated ester and 0.005 parts of hyaluronic acid are used, and the rest is consistent with Example 1.

[0099] Test Example 1

[0100] This test case is used to test the antioxidant capacity

[0101] (1) ABTS+ free radical scavenging rate test

[0102] Add 25 mL of 7.4 mM ABTS +The (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) free radical solution was mixed with 25 mL of 2.45 mM potassium persulfate solution and incubated at 25 °C in the dark for 12 h to prepare ABTS. + Free radical stock solution. Take 50 μL of ABTS + free radical stock solution and add it to 3 mL of water to dilute it to obtain ABTS + Free radical solution. 50 mg of the antioxidant gel of Examples 1-11 and Comparative Examples 1-3 were added to 3 mL of diluted ABTS + After incubation in the free radical solution at 25°C in the dark for 30 min, the sample absorbance was measured at 734 nm using an enzyme reader.

[0103] ABTS + Free radical scavenging rate (%) = [(A0-A1) / A0] × 100, where A0 is the absorbance of the control group and A1 is the absorbance of the sample group. The experiment was repeated three times for each concentration. The specific test results are shown in Table 1.

[0104] (2) DPPH free radical scavenging rate detection

[0105] The DPPH (2,2-diphenyl-1-trinitrohydrazine) free radical was dissolved in methanol to prepare a DPPH (2,2-diphenyl-1-trinitrohydrazine) free radical solution with a concentration of 0.1 mg / mL. 50 mg of the antioxidant gel of Example 1-11 and Comparative Example 1-3 were added to 3 mL of the DPPH free radical solution and incubated at 25° C. in the dark for 2 h. The absorbance of the sample solution at 517 nm was detected using an ELISA instrument.

[0106] DPPH free radical scavenging rate (%) = [(A0-A1) / A0] × 100, where A0 is the absorbance of the control and A1 is the absorbance of the sample group. The experiment was repeated three times at each concentration. The specific test results are shown in Table 1.

[0107] Table 1

[0108]

[0109]

[0110] The test results show that:

[0111] (1) By comparing Example 1 with Examples 4-5, it can be seen that the mass ratio of cysteine ​​to hyaluronic acid is outside the range specified by the invention, the hyaluronic acid grafting rate is low, the antioxidant capacity is reduced, and the free radical scavenging rate of the hydrogel is further reduced.

[0112] (2) By comparing Example 1 with Examples 6-7, it can be seen that when the mass ratio of EDC to NHS in the catalyst is outside the range specified by the invention, the activation ability of the hyaluronic acid carboxyl group is reduced, the grafting rate is reduced, and the free radical scavenging rate of the hydrogel is reduced.

[0113] (3) By comparing Example 1 with Examples 8-9, it can be seen that when the pH range of the hyaluronic acid solution is outside the range specified by the invention, the thiol groups are more likely to be converted into disulfide bonds, the antioxidant capacity is reduced, and the free radical scavenging rate of the hydrogel is reduced.

[0114] (4) By comparing Example 1 with Comparative Examples 1-3, it can be seen that cysteine, hyaluronic acid and polyethylene glycol carboxyl activated ester all play an important role in improving the antioxidant capacity of the hydrogel.

[0115] (5) By comparing Example 1 with Comparative Examples 4-5, it can be seen that when the mass ratio of polyethylene glycol carboxyl activated ester to modified hyaluronic acid is outside the range specified by the invention, the thiol content of the hydrogel decreases, the antioxidant capacity decreases, and the free radical scavenging rate of the hydrogel is further reduced.

[0116] Test Example 2

[0117] This test case is used to detect anti-inflammatory ability

[0118] RAW264.7 cells were used to evaluate the anti-inflammatory ability of the hydrogel. RAW264.7 cells were seeded in 48-well plates (3 × 10 4 Cells / well) were incubated at 37°C and 5% CO2 overnight. The next day, 1 μL of 1 mg / mL LPS solution was added to induce inflammation. 50 mg of the antioxidant gel of Example 1 and Comparative Example 1 and 1 μL of 1 mg / mL LPS solution were added to the cell culture medium at the same time and incubated at 37°C and 5% CO2. After 24 hours, the culture medium was removed, immunofluorescence staining (CD206, CD86) was performed, and cell nuclei were stained with DAPI. The staining results were observed under an inverted fluorescence microscope. The specific results are shown in Figure 2. Figure 1 As shown, CD206 can stain anti-inflammatory M2 macrophages (red fluorescence), and CD86 can stain pro-inflammatory M1 macrophages (green fluorescence).

[0119] The results show that compared with Comparative Example 1, the expression level of red fluorescent CD206 in Example 1 is higher, the expression of green fluorescent CD86 is not obvious, and the macrophages are more likely to exist in the M2 polarization state. The hydrogel within the scope of the present invention has good anti-inflammatory ability.

[0120] Test Example 3

[0121] This test example detects the ability to rescue oxidatively damaged cells

[0122] NIH-3T3 cells were used to evaluate the ability of hydrogels to rescue oxidatively damaged cells. NIH-3T3 mouse fibroblasts were seeded in 48-well plates (2×10 4 cells / well), and incubated overnight at 37°C and 5% CO2. The next day, 100 μM tert-butyl peroxide (tBOOH) was added to induce oxidative stress. Before induction, 50 mg of the antioxidant gel of Examples 1-11 and Comparative Examples 1-3 were mixed with tBOOH, pretreated for 20 min, added to the cell culture medium, and incubated at 37°C and 5% CO2. After 5 h, the cell metabolic activity was detected by CCK8 and live / dead cell staining kit.

[0123] Cell viability (%) = (A1 / A0) × 100, where A0 is the absorbance of the control group and A1 is the absorbance of the sample group. The experiment was repeated three times for each group.

[0124] The cell metabolic activity is shown in Table 2, and the staining results are shown in Figure Figure 2 shown.

[0125] Table 2

[0126]

[0127]

[0128] The test results show that:

[0129] (1) By comparing Example 1 with Examples 4-5, it can be seen that the mass ratio of cysteine ​​to hyaluronic acid is outside the range specified by the invention, the hyaluronic acid grafting rate is low, the antioxidant capacity is reduced, and the oxide removal rate of the hydrogel is reduced.

[0130] (2) By comparing Example 1 with Examples 6-7, it can be seen that when the mass ratio of EDC to NHS in the catalyst is outside the range specified by the invention, the activation ability of the hyaluronic acid carboxyl group is reduced, the grafting rate is reduced, and the free radical scavenging rate of the hydrogel is reduced.

[0131] (3) By comparing Example 1 with Examples 8-9, it can be seen that when the pH range of the hyaluronic acid solution is outside the range specified by the invention, the thiol groups are more likely to be converted into disulfide bonds, the antioxidant capacity is reduced, and the free radical scavenging rate of the hydrogel is reduced.

[0132] (4) By comparing Example 1 with Comparative Examples 1-3, it can be seen that cysteine, hyaluronic acid and polyethylene glycol carboxyl activated ester all play an important role in improving the antioxidant capacity of the hydrogel.

[0133] (5) By comparing Example 1 with Comparative Examples 4-5, it can be seen that when the mass ratio of polyethylene glycol carboxyl activated ester to modified hyaluronic acid is outside the range specified by the invention, the thiol content of the hydrogel decreases, the antioxidant capacity decreases, and the free radical scavenging rate of the hydrogel is further reduced.

[0134] Test Example 4

[0135] This test case performs cytotoxicity testing

[0136] The CCK8 method was used to detect the cytotoxicity of the hydrogel. Before the test, the hydrogel was sterilized under ultraviolet light for 30 minutes and then placed in fresh culture medium until it was completely degraded to obtain a degradation solution (100 mg / mL), which was diluted to different concentrations (20 mg / mL, 2 mg / mL). Mouse fibroblasts (NIH-3T3) were cultured at 3×10 3 The cells / well were inoculated in a 96-well plate and cultured at 37°C and 5% CO2. After the cells adhered, the cell culture medium was replaced with degradation solutions of different concentrations. After 24 hours, 10% CCK8 detection solution was added to each well. After the reaction changed color, the absorbance of the sample solution was detected at 450nm using an enzyme reader.

[0137] Cell viability (%) = (A1 / A0) × 100, where A0 is the absorbance of the control group and A1 is the absorbance of the sample group. The experiment was repeated three times for each group.

[0138] The specific results are shown in Table 3.

[0139] Table 3

[0140]

[0141] According to ISO standards, if the cell viability exceeds 70%, the biomaterial is non-toxic. The cell viability results of Examples 1-9 and Comparative Examples 1-5 show that hyaluronic acid, cysteine ​​and bi-arm polyethylene glycol succinimidyl succinate 6000 are all materials with good cell compatibility and are biosafe.

[0142] Test Example 5

[0143] This test case is used to detect the healing of in vivo refractory wounds

[0144] The experimental dogs after tumor resection were used as test cases. The wounds on the hind legs and buttocks of the animals were too large to heal, which seriously affected the animals' normal life. The antioxidant gel of Example 1 was applied to the wound surface of the animals once a day, and the growth of the wound surface was observed and recorded. The specific results are shown in the figure. Figure 3 shown.

[0145] The results showed that after one month of treatment with the antioxidant gel of Example 1, the wound area began to shrink; after two months of treatment, new tissue appeared around the wound, hair regenerated, and the wound further shrank; after three months of treatment, most of the wound healed and the hair regenerated in the healed part of the wound; after four months of treatment, the wound was completely healed. The antioxidant hydrogel within the scope of the present invention can effectively promote the healing of refractory wounds.

[0146] In summary, the present invention forms a hydrogel network by cross-linking cysteine-modified hyaluronic acid with polyethylene glycol carboxyl activated ester, which significantly improves the tissue adhesion ability of hyaluronic acid gel and further improves the antioxidant and anti-inflammatory abilities. The above-mentioned combined hydrogel material has simple components and better biocompatibility, and the cross-linked network is stable and biosafe.

[0147] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. An antioxidant gel for wound healing, characterized in that: The antioxidant gel comprises, by weight, 0.01-20 parts of hyaluronic acid, 0.01-20 parts of cysteine ​​and 1-50 parts of polyethylene glycol carboxyl activated ester.

2. The antioxidant gel according to claim 1, characterized in that The polyethylene glycol carboxyl activated ester can be any one of Formula I, Formula II, Formula III and Formula IV: Wherein, in each formula, Z is a natural number between 20 and 300, and can be 29 to 228, 29, 34, 38, 45, 57, 136, 181 or 228; The group R is a formula A or a formula B, wherein M represents a nitrogen atom, an oxygen atom, a sulfur atom, or a phosphorus atom, preferably an oxygen atom, a nitrogen atom, or a sulfur atom, connected to the end of the polyethylene glycol, and X or Y is an integer between 1 and 5, such as 0, 2, or 3; the groups R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 and R 16 Each of them is independently a hydrogen atom, a hydroxyl group, an alkyl group having 1 to 10 carbon atoms, a phenyl group or a sulfonic acid group. Preferably, the polyethylene glycol carboxyl activated ester may be any one of the following 1)-8): 1) As shown in formula I, wherein R is formula A, in formula A, X is 2 to 3, M is an oxygen atom, Z is 136, and R1, R2, R3 and R4 are all hydrogen atoms; 2) As shown in formula I, wherein R is formula B, in formula B, Y is 1 to 2, M is an oxygen atom, Z is 228, and R1, R2, R3 and R4 are all hydrogen atoms; 3) As shown in formula II, wherein R is formula A, in formula A, X is 2 to 3, M is an oxygen atom, Z is 57, R1, R2, R3 and R4 are all hydrogen atoms, and R5, R6, R7 and R8 are all hydroxyl groups; 4) As shown in formula II, wherein R is formula B, in formula B, Y is 1 to 2, M is an oxygen atom, Z is 57, R1, R2, R3 and R4 are all hydrogen atoms, and R5, R6, R7 and R8 are all hydroxyl groups; 5) As shown in formula III, wherein R is formula A, in which X is 2 to 3, M is a nitrogen atom, Z is 38, R1, R2, R3 and R4 are all hydrogen atoms, R5, R6, R7 and R8 are all methyl groups, R9, R 10 , R 11 and R 12 is phenyl; 6) As shown in formula III, wherein R is formula B, in which Y is 1 to 2, M is a sulfur atom, Z is 29, R1, R2, R3 and R4 are all hydrogen atoms, R5, R6, R7 and R8 are all sulfonic acid groups, R9, R 10 , R 11 and R 12 is phenyl; 7) As shown in Formula IV, wherein R is Formula A, in which X is 3 to 5, M is a nitrogen atom, Z is 29, R1, R2, R3, R4, R5, R6, R7 and R8 are all sulfonic acid groups, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 and R 16 is a hydrogen atom; 8) As shown in Formula IV, wherein R is Formula B, in Formula B, Y is 2 to 4, M is a nitrogen atom, Z is 29, R1, R2, R3, R4, R5, R6, R7 and R8 are all sulfonic acid groups, R9, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 and R 16 is a hydrogen atom; Preferably, the molecular weight of the polyethylene glycol backbone is 2000-10000; Preferably, the molecular weight of the hyaluronic acid is (20-150)×10 4 .

3. The antioxidant gel according to claim 1 or 2, characterized in that: The mass ratio of cysteine ​​to hyaluronic acid is 1:(0.01-3).

4. A method for preparing an antioxidant gel for wound healing as claimed in any one of claims 1 to 3, characterized in that: The preparation method comprises: Add hyaluronic acid to the solution and stir for the first time until it is completely dissolved. After dissolution, add a catalyst and continue stirring for the second time to activate the carboxyl group of the hyaluronic acid. Then add cysteine ​​to react. After the reaction is completed, the reaction solution is dialyzed and freeze-dried to obtain cysteine-modified hyaluronic acid freeze-dried powder. The modified hyaluronic acid freeze-dried powder and polyethylene glycol carboxyl activated ester are dissolved separately and mixed evenly to obtain an antioxidant gel.

5. The preparation method according to claim 4, characterized in that: The solvent of the hyaluronic acid solution includes sterile water and / or buffer; Preferably, the buffer comprises any one or a combination of at least two of phosphate buffer, phosphate-buffered saline, Tris-hydrochloric acid buffer, glycine-hydrochloric acid buffer, disodium hydrogen phosphate-sodium citrate buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, citric acid-sodium citrate buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, boric acid-borax buffer, borax-sodium hydroxide buffer or sodium carbonate-sodium bicarbonate buffer; Preferably, the pH of the hyaluronic acid solution is 4-7; Preferably, the catalyst comprises a combination of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and a protecting agent; Preferably, the protective agent comprises N-hydroxysuccinimide and / or N-hydroxysulfosuccinimide; Preferably, the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added in an amount of 0.005-30 parts by mass fraction; Preferably, the protective agent is added in an amount of 0.006-34 parts by mass; Preferably, the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to the protective agent in the catalyst is (1-3):(2-5); Preferably, the first stirring speed is 200-1000 rpm, and the time is 40 min-12 h; Preferably, the second stirring speed is 200-1000 rpm, and the time is 20-40 min; Preferably, the gas environment of the reaction includes any one of air, nitrogen, argon or helium, or a combination of at least two thereof; Preferably, the reaction time is 4-12 hours.

6. The preparation method according to claim 4 or 5, characterized in that: The dialysis solution includes any one of ultrapure water, deionized water, purified water or dialysis buffer or a combination of at least two thereof; Preferably, the dialysis buffer comprises any one or a combination of at least two of phosphate buffer, phosphate-buffered saline, Tris-hydrochloric acid buffer, HEPES zwitterion buffer or amino acid buffer; Preferably, the pH of the dialyzed solution is 4-7; Preferably, the molecular weight cut-off of the dialysis bag for dialysis is 1000-25000; Preferably, the dialysis time is 2-5 days.

7. The preparation method according to any one of claims 4 to 6, characterized in that: The freeze-drying method comprises packaging the purified reaction solution and placing it under low temperature for freezing treatment, and after freezing, using a freeze dryer to sublimate and dry the solvent in the reactant to obtain a dry product.

8. The preparation method according to any one of claims 4 to 7, characterized in that: The solvent for dissolving the hyaluronic acid freeze-dried powder includes sterile water and / or buffer 1; Preferably, the buffer 1 comprises any one or a combination of at least two of phosphate buffer, phosphate-buffered saline, Tris-hydrochloric acid buffer, glycine-hydrochloric acid buffer, disodium hydrogen phosphate-sodium citrate buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, citric acid-sodium citrate buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, boric acid-borax buffer, borax-sodium hydroxide buffer or sodium carbonate-sodium bicarbonate buffer; Preferably, the solvent for dissolving the polyethylene glycol carboxyl activated ester comprises sterile water and / or buffer 2; Preferably, the buffer 2 comprises any one or a combination of at least two of phosphate buffer, phosphate-buffered saline, Tris-hydrochloric acid buffer, glycine-hydrochloric acid buffer, disodium hydrogen phosphate-sodium citrate buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, citric acid-sodium citrate buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, boric acid-borax buffer, borax-sodium hydroxide buffer or sodium carbonate-sodium bicarbonate buffer.

9. The preparation method according to any one of claims 4 to 8, characterized in that: The mass concentration of the polyethylene glycol carboxyl activated ester is 1-50%; Preferably, the mass concentration of the modified hyaluronic acid freeze-dried powder is 0.01-20%; Preferably, the volume ratio of the polyethylene glycol carboxyl activated ester solution to the modified hyaluronic acid solution in the mixed solution is 1:(0.1-5).

10. Use of the antioxidant gel for wound healing according to any one of claims 1 to 3 in inhibiting oxidative stress for non-therapeutic purposes or in preparing products for inhibiting oxidative stress.

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