A polymer, a preparation method thereof, a hydrogel and a wound dressing

Abstract

The present invention provides a polymer and a preparation method thereof, a hydrogel and a wound dressing. The polymer is formed by a Schiff base reaction between a block copolymer and a glycosaminoglycan aldehyde derivative. Among them, the block copolymer includes an end-aminated polyethylene glycol segment and a polyamino acid chain segment, and the polyamino acid chain segment includes a poly-L-methionine segment and a poly-L-ornithine segment. The hydrogel formed by the above polymer of the present invention has an immunomodulatory effect and can reduce the high reactive oxygen species level in the wound microenvironment. At the same time, the two components of the block copolymer and the glycosaminoglycan aldehyde derivative in the hydrogel can enhance the phagocytic function of macrophages and promote the transformation of macrophages into the M2 phenotype to regulate the wound microenvironment and achieve the purpose of promoting the healing of chronic wounds.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical dressings, and particularly relates to a polymer and a preparation method thereof, a hydrogel and a wound dressing. Background Art

[0002] Chronic wounds refer to wounds that still do not heal completely or show no tendency to heal after 4 weeks or more of standardized and complete treatment, mainly occurring in patients with diabetes, long-term bedridden conditions, trauma, etc. According to incomplete statistics, about 100 million person-times of patients need wound treatment in China every year, and chronic wound patients account for about 30%. Chronic wounds are mainly characterized by a local persistent inflammatory response. As the main effector cells of the inflammatory response, the function and state of macrophages determine the duration of inflammation. Among them, the role of the efferocytosis function and phenotypic conversion disorder of macrophages in the persistent state of chronic wounds has been increasingly emphasized. Some studies have shown that the phagocytic function of macrophages can remove cell debris that may cause inflammation and immune responses, providing a clean repair environment for the wound. Macrophages secrete different cytokines through phenotypic conversion to achieve multiple key steps of wound healing. Generally, pro-inflammatory cytokines such as TNF-α and IL-1 secreted by macrophages during the inflammatory phase can induce an inflammatory response in the early stage of the wound, attract immune cells to the wound site, clear pathogens and necrotic tissues, and create conditions for wound healing. Macrophages in the proliferative phase secrete some anti-inflammatory cytokines, such as interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1), etc. These cytokines can inhibit the inflammatory response, reduce inflammatory symptoms, and promote wound healing. However, it has been reported that the long-term inflammatory environment of chronic wounds leads to macrophage dysfunction and phenotypic dysregulation, resulting in macrophages being unable to perform their original functions to complete wound treatment.

[0003] As is well known, reactive oxygen species (ROS) are key regulators of macrophage-mediated inflammatory responses and cell proliferation during wound healing. In particular, high levels of ROS can inhibit the phagocytic ability of macrophages and weaken their ability to transform the pro-inflammatory M1 phenotype into the anti-inflammatory M2 phenotype, resulting in macrophages being unable to perform their original functions to complete wound treatment.

[0004] Currently, in the treatment of chronic wounds, although methods of regulating macrophages with cytokines, drugs, and stem cells have certain curative effects, due to the influence of dosage, source, and individual differences, they generally cannot appropriately regulate the efferocytosis function and phenotypic conversion of macrophages.

[0005] Therefore, it is particularly important to provide a material that can reduce the high ROS level at the wound site and regulate the efferocytosis function and phenotypic conversion of macrophages to clear the wound inflammatory response and complete the repair of chronic wounds. Summary of the Invention

[0006] In view of this, the object of the present invention is to provide a polymer, a preparation method thereof, a hydrogel and a wound dressing. The hydrogel can reduce the level of reactive oxygen species (ROS) in the wound surface and provide a suitable microenvironment for wound repair.

[0007] To achieve this purpose, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides a polymer formed by reacting a block copolymer with a glycosaminoglycan aldehyde derivative;

[0009] The block copolymer includes an end-aminated polyethylene glycol segment and a polyamino acid chain segment, and the polyamino acid chain segment includes a poly-L-methionine segment and a poly-L-ornithine segment.

[0010] Preferably, the glycosaminoglycan aldehyde derivative is selected from one or more aldehyde derivatives of heparin, heparin derivatives, sulfated dextran, sulfated dextran derivatives, sulfated hyaluronic acid, sulfated hyaluronic acid derivatives, hyaluronic acid, hyaluronic acid derivatives, chondroitin sulfate or chondroitin sulfate derivatives.

[0011] Preferably, the degree of oxidation of the glycosaminoglycan aldehyde derivative is 5% - 90%.

[0012] Preferably, the molar ratio of the amino group in the block copolymer to the aldehyde group in the glycosaminoglycan aldehyde derivative is 2:1 - 1:2.

[0013] Preferably, the molar number of the poly-L-methionine segment is 50% - 99% of the polyamino acid chain segment.

[0014] In the second aspect, the present invention provides a preparation method of the above polymer, including the following steps:

[0015] S1: React end-aminated polyethylene glycol with L-methionine-N-carboxylic acid anhydride to obtain an intermediate;

[0016] S2: React the intermediate with L-ornithine-N-carboxylic acid anhydride with protected amino group, and then deprotect to obtain a block copolymer;

[0017] S3: React the block copolymer with a glycosaminoglycan aldehyde derivative to obtain a polymer.

[0018] Preferably, the molar ratio of the end-aminated polyethylene glycol to L-methionine-N-carboxylic acid anhydride is 1:1 - 1:600.

[0019] Preferably, the molar ratio of the intermediate to L-ornithine-N-carboxylic acid anhydride with protected amino group is 1:1 - 1:600.

[0020] Preferably, the temperature of the reaction in step S1 is room temperature, and the time is 2 to 4 days.

[0021] Preferably, the temperature of the reaction in step S2 is room temperature, and the time is 2 to 4 days.

[0022] Preferably, the temperature of the reaction in step S3 is 2 to 6 °C, and the time is 2 to 4 days.

[0023] In the present invention, the room temperature refers to a temperature of "10 to 30 °C", preferably "15 to 25 °C".

[0024] In a third aspect, the present invention provides a hydrogel formed from the above polymer.

[0025] In a fourth aspect, the present invention provides a wound dressing comprising the above hydrogel.

[0026] Preferably, the wound dressing further comprises auxiliary materials.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] The present invention provides a polymer formed by a Schiff base reaction between a block copolymer and a glycosaminoglycan aldehyde derivative. Among them, the introduction of the glycosaminoglycan aldehyde derivative can regulate the transformation of macrophages into the M2 phenotype. In the present invention, the block copolymer comprises an amino-terminated polyethylene glycol segment and a polyamino acid chain segment. Among them, the amino-terminated polyethylene glycol segment can improve the water solubility of the polyamino acid; the polyamino acid chain segment comprises a poly-L-methionine segment and a poly-L-ornithine segment. Among them, the presence of the poly-L-methionine segment can eliminate excessive ROS, and the presence of the poly-L-ornithine segment can enhance the continuous efferocytosis behavior of macrophages.

[0029] The hydrogel formed from the above polymer in the present invention has an immunomodulatory effect and can reduce the high ROS level in the wound microenvironment. At the same time, the two components of the block copolymer and the glycosaminoglycan aldehyde derivative in the hydrogel can synergistically regulate the function of macrophages through different pathways, promote the transformation of macrophages into the M2 phenotype, regulate the wound microenvironment, and achieve the purpose of promoting the healing of chronic wounds. Animal experiments have verified the effectiveness of chronic wound treatment. Therefore, the hydrogel provided by the present invention helps to reconstruct vascular function, deposit collagen, and epithelialize, and solves the problem of difficult healing of clinical chronic wounds. Description of the Drawings

[0030] Figure 1 1H NMR spectrum of mPEG 2k -b-(Met 25 -b-Orn5) obtained in Example 1;

[0031] Figure 2 1H NMR spectrum of aldehyde - modified hyaluronic acid;

[0032] Figure 3 CD spectra of mPMO / OHA hydrogel, mPML / OHA hydrogel, and mPMO / ODEX hydrogel;

[0033] Figure 4 Live / Dead staining results of mPMO / OHA hydrogel, mPML / OHA hydrogel, and mPMO / ODEX hydrogel;

[0034] Figure 5 CCK - 8 results of mPMO / OHA hydrogel, mPML / OHA hydrogel, and mPMO / ODEX hydrogel;

[0035] Figure 6 CCK - 8 results of mPMO / OHA hydrogel, mPML / OHA hydrogel, and mPMO / ODEX hydrogel under 0.2 mM H2O2 condition;

[0036] Figure 7 Flow cytometry detection of macrophage cell phenotypes after co - culturing mPMO / OHA hydrogel, mPML / OHA hydrogel, and mPMO / ODEX hydrogel with RAW264.7 cells;

[0037] Figure 8 Macrophage efferocytosis ability after co - culturing mPMO / OHA hydrogel, mPML / OHA hydrogel, and mPMO / ODEX hydrogel with RAW264.7 cells. Detailed implementation manners

[0038] Next, in combination with the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0039] The present invention provides a polymer formed by reacting a block copolymer with a glycosaminoglycan aldehyde derivative.

[0040] In the present invention, a Schiff base reaction occurs between the amino group in the block copolymer and the aldehyde group in the aldehyde group-modified derivative of glycosaminoglycan, thereby connecting the block copolymer and the aldehyde group-modified derivative of glycosaminoglycan. Therefore, the aldehyde group content in the aldehyde group-modified derivative of glycosaminoglycan is crucial, and the aldehyde group content is positively correlated with the degree of oxidation. In the present invention, the degree of oxidation in the aldehyde group-modified derivative of glycosaminoglycan is 5% - 90%, preferably about 20%. If the aldehyde group content is too low, the modulus of the hydrogel prepared subsequently will be too low. On the contrary, if it is too high, the biological function of glycosaminoglycan will be affected.

[0041] In some embodiments of the present invention, the molar ratio of the amino group in the block copolymer to the aldehyde group in the aldehyde group-modified derivative of glycosaminoglycan is 2:1 - 1:2, specifically, it can be 2:1, 1:1, 1:2, etc.

[0042] In the present invention, the block copolymer includes an end-amino group modified polyethylene glycol segment and a polyamino acid chain segment. Among them, the molecular weight of polyethylene glycol in the end-amino group modified polyethylene glycol segment is between 200 - 40000 kDa, preferably 2000 kDa.

[0043] The polyamino acid chain segment includes a poly-L-methionine segment and a poly-L-ornithine segment. The molar number of the poly-L-methionine segment accounts for 50% - 99% in the polyamino acid chain segment, preferably 5 / 6. The reason for defining the proportion of the above L-methionine in the present invention is that if the proportion of methionine is too high, the gelation temperature of the hydrogel will be too low, which is not conducive to storage. If the proportion of methionine is too low, the gelation temperature will be too high, which is not conducive to practical applications.

[0044] Among them, in the present invention, by using the end-amino group modified polyethylene glycol segment, the amino group can attack the carbonyl carbon atom, thereby initiating a ring-opening reaction to achieve the combination of polyethylene glycol and L-methionine. Subsequently, through the ring-opening reaction, L-ornithine is connected to L-methionine, thereby obtaining the block copolymer.

[0045] In the present invention, the aldehyde group-modified derivative of glycosaminoglycan is selected from one or more aldehyde group-modified derivatives of heparin, heparin derivatives, sulfated dextran, sulfated dextran derivatives, sulfated hyaluronic acid, sulfated hyaluronic acid derivatives, hyaluronic acid, hyaluronic acid derivatives, chondroitin sulfate or chondroitin sulfate derivatives, preferably an aldehyde group-modified derivative of hyaluronic acid, that is, aldehyde group-modified hyaluronic acid. In some embodiments of the present invention, when the aldehyde group-modified derivative of glycosaminoglycan is selected from aldehyde group-modified hyaluronic acid, the polymer can be represented as a polymer shown in Formula I:

[0046] Formula I;

[0047] Among them, x is any integer from 5 to 900, y is any integer from 1 to 600, and z is any integer from 1 to 600;

[0048] “ ” represents a linking site for linking glycosaminoglycan or polyamino acid segments, and “b” represents block copolymerization.

[0049] The present invention also provides a method for preparing the above polymer, comprising the following steps:

[0050] S1: Reacting the amino-terminated polyethylene glycol with L-methionine-N-carboxylic acid anhydride to obtain an intermediate;

[0051] S2: Reacting the intermediate with L-ornithine-N-carboxylic acid anhydride with protected amino group, preferably benzyloxycarbonyl-L-ornithine-N-carboxylic acid anhydride, and then deprotecting to obtain a block copolymer;

[0052] S3: Reacting the block copolymer with a glycosaminoglycan aldehyde derivative to obtain a polymer.

[0053] According to the present invention, first, the amino-terminated polyethylene glycol is reacted with L-methionine-N-carboxylic acid anhydride to obtain an intermediate. In the present invention, generally, the amino-terminated polyethylene glycol is synthesized first. The molecular weight of the polyethylene glycol is generally between 200 and 40,000 kDa, preferably 2,000 kDa. The present invention has no particular limitation on the method for synthesizing the amino-terminated polyethylene glycol, and it can be carried out according to the means well-known to those skilled in the art. The L-methionine-N-carboxylic acid anhydride can be prepared according to the method well-known to those skilled in the art, or can be directly purchased as a commercially available product. In some embodiments of the present invention, the molar ratio of the amino-terminated polyethylene glycol to L-methionine-N-carboxylic acid anhydride is 1:1 to 1:600, preferably 1:10 to 1:50. The temperature of the reaction is room temperature, and the time is 2 to 4 days, preferably 3 days.

[0054] Then, according to the present invention, the intermediate is reacted with L-ornithine-N-carboxylic acid anhydride with protected amino group, preferably benzyloxycarbonyl-L-ornithine-N-carboxylic acid anhydride, and then deprotected to obtain a block copolymer. The L-ornithine-N-carboxylic acid anhydride with protected amino group can be prepared according to the method well-known to those skilled in the art, or can be directly purchased as a commercially available product.

[0055] In some embodiments of the present invention, preferably, the reaction is carried out according to the molar ratio of the intermediate to L-ornithine-N-carboxylic acid anhydride with protected amino group of 1:1 to 1:600, preferably 1:5 to 1:200, to obtain a block copolymer. The reaction is preferably carried out at room temperature, and the reaction time is 2 to 4 days, preferably 3 days.

[0056] In some preferred embodiments of the present invention, after generally dehydrating the amino-terminated polyethylene glycol, it is then reacted with L-methionine-N-carboxylic acid anhydride. The method of dehydrating can be any method well-known to those skilled in the art without special limitations. The present invention preferably dehydrates the amino-terminated polyethylene glycol and toluene by azeotropic distillation and then removes toluene; the temperature of the azeotropic distillation is preferably 110~125 °C; the time of the azeotropic distillation is preferably 2~3 h; the method of removing toluene is preferably vacuum pumping; this reaction is preferably carried out in an organic solvent; the organic solvent can be any organic solvent well-known to those skilled in the art without special limitations. The present invention preferably uses dry anhydrous N,N-dimethylformamide (Dimethylformamide, DMF); the temperature of this reaction is room temperature, preferably 10~40 °C, more preferably 15~25 °C; the time of this reaction is preferably 2~4 days, more preferably 3 days.

[0057] Then, L-ornithine-N-carboxylic acid anhydride with protected amino is added for reaction. It is preferred to purge with nitrogen 2~3 times before this reaction. The temperature of this reaction is room temperature, preferably 10~40 °C, more preferably 15~25 °C; the time of this reaction is preferably 2~4 days, more preferably 3 days. After the reaction is completed, it is preferably precipitated with ice ether, filtered by a Buchner funnel, and vacuum dried to obtain a light yellow polymer, and weighed.

[0058] Then, the obtained product is deprotected, and the means of deprotection can be carried out according to the means well-known to those skilled in the art. After deprotection, the present invention preferably dissolves the product in trifluoroacetic acid (Trifluoroacetic acid, TFA) and reacts with hydrobromic acid / acetic acid solution for 1 h, then precipitates with ice ether again, filters by a Buchner funnel, dissolves the solid in an appropriate amount of DMF, loads it into a dialysis bag, dialyzes in deionized water and then lyophilizes to obtain the block copolymer shown in formula C. The dialysis bag is preferably a 3500 Da dialysis bag; the dialysis time is preferably 2~4 days, more preferably 3 days.

[0059] In the present invention, the glycosaminoglycan aldehyde derivative is preferably prepared according to the following method. The glycosaminoglycan is fully dissolved in water, preferably Milli-Q water, and sodium periodate is slowly added dropwise to the glycosaminoglycan solution in the dark. The reaction is carried out at room temperature for 1~5 h, preferably 2~4 h. Ethylene glycol is added to react with the remaining sodium periodate, and stirring is continued for 1~5 h to terminate the reaction. It is placed in a dialysis bag, dialyzed in deionized water and then lyophilized to obtain the glycosaminoglycan aldehyde derivative. Among them, the dialysis bag is preferably a 3500 Da dialysis bag; the dialysis time is preferably 1~4 days, more preferably 2 days.

[0060] As described in the relevant content above, in the present invention, the degree of aldehyde group substitution in the glycosaminoglycan aldehyde derivative is crucial. Therefore, the present invention determines the degree of aldehyde group substitution in the glycosaminoglycan aldehyde derivative by the hydroxylamine hydrochloride method to ensure that the degree of aldehyde group substitution is within the target range.

[0061] According to the present invention, after obtaining the block copolymer and the glycosaminoglycan aldehyde derivative, it is preferred to react the block copolymer with the glycosaminoglycan aldehyde derivative to obtain a polymer. Among them, the block copolymer and the glycosaminoglycan aldehyde derivative are preferably reacted according to the molar ratio of the amino group in the block copolymer to the aldehyde group in the glycosaminoglycan aldehyde derivative of 1:(1-2). The reaction is preferably carried out under solvent conditions. The temperature of the reaction is 2-6°C, preferably 4°C; the time is 2-4 days, preferably 3 days.

[0062] In some embodiments of the present invention, when the glycosaminoglycan aldehyde derivative is aldehyde group-substituted hyaluronic acid, the preparation method of the polymer is as follows:

[0063] S1: React the compound shown by formula A with L-methionine-N-carboxy anhydride to obtain the compound shown by formula B;

[0064] S2: React the compound shown by formula B with L-ornithine-N-carboxy anhydride with protected amino group, and then deprotect to obtain the block copolymer shown by formula C;

[0065] S3: React the block copolymer shown by formula C with aldehyde group-substituted hyaluronic acid to obtain the polymer shown by formula I;

[0066] Formula A; Formula B; Formula C;

[0067] Among them, b represents block copolymerization.

[0068] The specific parameters in the above method are as described in the relevant content above, and will not be elaborated here.

[0069] In some embodiments of the present invention, in the block copolymer shown by formula C obtained, x can take any integer from 5 to 900, y takes any integer from 1 to 600, and z takes any integer from 1 to 100

[0070] Specifically, in some embodiments of the present invention, in the block copolymer shown by formula C obtained, x = 45, y = 25, z = 5;

[0071] Or x = 45, y = 40, z = 1;

[0072] Or x = 45, y = 15, z = 25;

[0073] Or x = 180, y = 100, z = 25;

[0074] Or x = 180, y = 160, z = 4;

[0075] Or x = 180, y = 60, z = 100.

[0076] The present invention also provides a hydrogel formed from the above-mentioned polymer (such as the polymer shown in Formula I).

[0077] In some embodiments of the present invention, the hydrogel is prepared by the following method:

[0078] Prepare a solution of the polymer in a phosphate buffered saline solution, add the corresponding mass of the glycosaminoglycan aldehyde derivative according to the molar ratio of aldehyde group to amino group, and stir at 2 - 6 °C for 2 - 4 days.

[0079] It should be noted that the concentration of the polymer solution can be adjusted according to the actual situation. If the polymer is to form a gel at low temperature, a polymer solution with a high degree of polymerization of methionine and a high concentration can be preferably used. If the polymer is to form a gel at high temperature, a polymer solution with a low degree of polymerization of methionine and a low concentration can be preferably used.

[0080] The present invention also provides a wound dressing, comprising the above-mentioned hydrogel and optional excipients, and the excipients can be antibacterial agents, humectants, hemostatic agents, breathable materials, adhesives, drug carriers or components promoting cell growth (such as bioactive components such as fibroblast growth factor (FGF) and collagen).

[0081] To further illustrate the present invention, the following examples are provided for detailed description. The experimental raw materials used in the following examples of the present invention are all commercially available products.

[0082] Example 1 Synthesis of poly(ethylene glycol)-block-poly(L-methionine-block-L-ornithine) (methoxypoly(ethylene glycol)-block-poly(L-methionine-block-L-Ornithine), abbreviated as: mPEG 2k -b-P(Met 25 -b-Orn5))

[0083] This example provides an mPEG 2k -b-P(Met 25 -b-Orn5), and its synthetic route is as follows:

[0084] ;

[0085] Wherein, x = 45, y = 25, z = 5;

[0086] The specific steps are as follows:

[0087] (1) Synthesize amino-terminal mPEG 2k : Dissolve 40 g of mPEG 2k in 200 mL of dichloromethane completely. Add 9.6 g of p-toluenesulfonyl chloride (PTSC) and 2.8 g of potassium hydroxide in sequence. Stir for 7 days and then pour into a separatory funnel. Wash with ice-saturated sodium chloride solution for 6 times to make the lower organic phase product as clear as possible. After liquid separation, dry the lower product with anhydrous magnesium sulfate (MgSO4) at room temperature overnight. The next day, filter off the magnesium sulfate with a sintered glass funnel, concentrate by rotary evaporation, then precipitate the liquid with ice-cold diethyl ether. After stirring for 15 min, filter with a Buchner funnel and dry in vacuo at room temperature for 12 h to obtain the polymer. Dissolve the polymer obtained from the reaction in ammonia water with a mass-to-volume ratio of 10 times that of the polymer, add an equal mass of ammonium chloride, and react at room temperature for 7 days. Extract the organic phase with dichloromethane, then wash the organic phase with ice-saturated sodium chloride solution twice, and dry with anhydrous MgSO4 at room temperature overnight. The next day, filter off the magnesium sulfate with a sintered glass funnel, then precipitate again with ice-cold diethyl ether, filter dry with a Buchner funnel, and dry in vacuo at room temperature to obtain the final product mPEG 2k -NH2 (wherein, mPEG 2k means the molecular weight of mPEG is 2000 kDa);

[0088] (2) Synthesize two kinds of amino acid NCAs: Prepare an anhydrous and dry three-necked round-bottom flask, purge with nitrogen, add 300 mL of tetrahydrofuran (THF). Weigh 15 g of L-methionine (L-Met) / L-ornithine (L-Orn(z)-OH) and 20 g of bis(trichloromethyl) carbonate (BTC) and add them to the flask. Heat in an oil bath at 48 °C. The liquid becomes transparent after 1 h. Increase the nitrogen flow rate to blow away THF as much as possible. When there are 20 - 30 mL of THF remaining, precipitate with ice-cold n-hexane. Keep stirring rapidly during the precipitation process. Filter with a Buchner funnel to obtain the solid product. Then dissolve the solid with an appropriate amount of ice-cold ethyl acetate, wash three times with anhydrous saturated sodium chloride solution, add to a conical flask, add an appropriate amount of anhydrous magnesium sulfate and dry at -20 °C overnight. The next day, filter with a G4 sintered glass funnel, connect the filtrate to a cold trap and pump away the solvent with a vacuum pump, and then perform recrystallization purification (methionine does not need recrystallization) to obtain L-methionine-N-carboxyanhydride (L-Met NCA) and benzyloxycarbonyl-L-ornithine-N-carboxyanhydride (Z-L-Orn NCA) respectively;

[0089] (3) Synthesis of mPEG2k-b-P(Met25-b-Orn5): Add 1 g of mPEG-NH2 into 200 mL of dry toluene, and azeotropically remove water at 125 °C for 2 h. Then pump dry the toluene with a vacuum pump. Subsequently, add 40 mL of dry anhydrous N,N-dimethylformamide (DMF) and 2.17 g of L-Met NCA, and react at room temperature for 3 days. Replace the nitrogen three times by evacuation and filling, then add 0.73 g of Z-L-Orn NCA and continue to react at room temperature for 3 days. Precipitate with ice-cold diethyl ether, filter with a Buchner funnel, and dry in vacuum to obtain a light yellow polymer, and weigh it. After that, deprotect the protecting groups. Dissolve the product in trifluoroacetic acid (TFA) with a mass-to-volume ratio of 10 times, add hydrobromic acid / acetic acid solution with a mass-to-volume ratio of three times, and react for 1 h. Then precipitate with ice-cold diethyl ether again, filter with a Buchner funnel. Dissolve the solid in an appropriate amount of DMF, load it into a 3500 Da dialysis bag, dialyze in deionized water for 3 days, and then lyophilize to obtain mPEG 2k -b-(Met 25 -b-Orn5) (abbreviation: mPMO).

[0090] Analyze mPEG 2k -b-(Met 25 -b-Orn5) by nuclear magnetic resonance, and obtain its 1H NMR spectrum as shown in Figure 1 the figure, which proves the successful synthesis of mPMO.

[0091] Example 2 Synthesis of OHA

[0092] Dissolve 1 g of hyaluronic acid in 100 mL of Milli-Q water. Dissolve 1.01 g, 1.35 g, 1.52 g, 1.69 g, 1.92 g, 2.14 g, and 2.37 g of sodium periodate in 30 mL of Milli-Q water respectively. Slowly add the sodium periodate solution dropwise to the hyaluronic acid solution in the dark, react at room temperature for 2 h, add 2 mL of ethylene glycol, continue to stir for 1 h to terminate the reaction, put it into a 3500 Da dialysis bag, dialyze in deionized water for 2 days, and then lyophilize to obtain seven kinds of hyaluronic acids with different oxidation degrees.

[0093] Analyze the aldehyde-functionalized hyaluronic acid by nuclear magnetic resonance, and obtain its 1H NMR spectrum as shown in Figure 2 the figure. It can be seen that the stretching vibration of the aldehyde group appears at 1735 cm -1 position, which proves the successful synthesis of OHA.

[0094] The oxidation degree of OHA and the aldehyde group content were determined by the hydroxylamine hydrochloride method. The aldehyde groups in OHA reacted with hydroxylamine hydrochloride to generate a compound oxime while releasing one molecule of hydrochloric acid. The oxidation degree and aldehyde group content of OHA could be indirectly measured by determining the pH value through acid-base titration.

[0095] Among them, the oxidation degree (%) of oxidized hyaluronic acid:

[0096] ;

[0097] V: the volume of NaOH consumed by oxidized hyaluronic acid (mL), V0: the volume of NaOH consumed by unoxidized hyaluronic acid (mL);

[0098] M: the molar concentration of NaOH (mol / L), W: the mass of oxidized hyaluronic acid (g);

[0099] M W : the molar mass of the structural unit of hyaluronic acid (g / mol).

[0100] After testing, when adding 1.01 g, 1.35 g, 1.52 g, 1.69 g, 1.92 g, 2.14 g, and 2.37 g of sodium periodate, the oxidation degrees of the obtained oxidized hyaluronic acids were 19.0%, 28.4%, 45.5%, 47.4%, 64.5%, 73.3%, 80.8% respectively, and the aldehyde group contents were 38.0%, 56.8%, 91.0%, 94.8%, 129.0%, 146.6%, 161.6% respectively.

[0101] Since polysaccharides with an oxidation degree of about 20% can provide the maximum number of aldehyde group units under the condition of retaining their biological functions. Therefore, when preparing the mPMO / OHA hydrogel subsequently, OHA with an oxidation degree of 19% was used, that is, the aldehyde group content was 38%.

[0102] Example 3 Synthesis of mPMO / OHA hydrogel

[0103] A solution with a polymer (i.e., mPMO) concentration of 8 wt% was prepared in a phosphate buffered saline solution, and the corresponding mass of OHA was added according to the molar ratio of aldehyde group to amino group of 1:1, and stirred at 4 °C for 3 days.

[0104] Comparative Example 1 Synthesis of mPML / OHA hydrogel

[0105] (1) Synthesis of lysine NCA: Prepare an anhydrous and dry three-necked round-bottom flask, purge with nitrogen, add 300 mL of tetrahydrofuran (THF), weigh 15 g of L-lysine (L-Lys(z)-OH) and 20 g of bis(trichloromethyl) carbonate (BTC) and add them to the flask. Heat in an oil bath at 48 °C. After 1 h, the liquid becomes transparent. Increase the nitrogen flow rate to blow away as much THF as possible. When there are 20 - 30 mL of THF remaining, precipitate with ice-cyclohexane. Continuously stir rapidly during the precipitation process. Filter using a Buchner funnel to obtain a solid product. Then dissolve the solid in an appropriate amount of ice-ethyl acetate, wash three times with anhydrous saturated sodium chloride solution, transfer to a conical flask, add an appropriate amount of anhydrous magnesium sulfate and dry overnight at -20 °C. The next day, filter using a G4 sintered glass funnel, connect the filtrate to a cold trap and pump away the solvent with a vacuum pump, and then perform recrystallization purification to obtain carbobenzoxy-L-lysine-N-carboxyanhydride (Z-L-Lys NCA);

[0106] (2) Synthesis of mPEG 2k -b-P(Met 25 -b-Lys5): Add 1 g of mPEG-NH2 to 200 mL of dry toluene, azeotropically remove water at 125 °C for 2 h, pump dry the toluene with a vacuum pump. Sequentially add 40 mL of dry anhydrous N,N-dimethylformamide (DMF) and 2.17 g of L-Met NCA, react at room temperature for 3 days, evacuate and replace nitrogen three times, then add 0.77 g of Z-L-Lys NCA and continue to react at room temperature for 3 days. Precipitate with ice-ether, filter using a Buchner funnel, and dry in vacuo to obtain a light yellow polymer and weigh it. Then deprotect the protecting groups. Dissolve the product in 10 times the mass-volume of trifluoroacetic acid (TFA), add three times the mass-volume of hydrobromic acid / acetic acid solution and react for 1 h. Then precipitate again with ice-ether, filter using a Buchner funnel. Dissolve the solid in an appropriate amount of DMF, load it into a 3500 Da dialysis bag, dialyze in deionized water for 3 days and then lyophilize to obtain mPEG 2k -b-(Met 25 -b-Lys5) (abbreviation: mPML).

[0107] 3) Synthesis of mPML / OHA hydrogel: Prepare a solution with an mPML polymer concentration of 8% in phosphate buffered saline, add the corresponding mass of OHA according to a molar ratio of aldehyde group to amino group of 1:1, and stir at 4 °C for 3 days.

[0108] Comparative Example 2 Synthesis of mPMO / ODEX hydrogel

[0109] (1) Synthesis of oxidized dextran (ODEX):

[0110] 1 g of dextran was fully dissolved in 100 mL of secondary distilled water (Milli-Q). 131 mg, 261 mg, 524 mg, and 786 mg of sodium periodate were respectively dissolved in 30 mL of secondary distilled water (Milli-Q). Sodium periodate was slowly added dropwise to the hyaluronic acid solution in the dark. The reaction was carried out at room temperature for 2 h. Then, 2 mL of ethylene glycol was added, and stirring was continued for 1 h to terminate the reaction. The mixture was placed in a 3500 Da dialysis bag and dialyzed in deionized water for 2 days and then freeze-dried to obtain 4 kinds of dextrans with different oxidation degrees.

[0111] The oxidation degree and aldehyde group content of ODEX were determined by the hydroxylamine hydrochloride method. The aldehyde groups in ODEX reacted with hydroxylamine hydrochloride to generate a compound oxime while releasing one molecule of hydrochloric acid. The oxidation degree and aldehyde group content of OHA could be indirectly measured by determining the pH value through acid-base titration.

[0112] Among them, the oxidation degree (%) of oxidized dextran:

[0113] ;

[0114] V: the volume (mL) of NaOH consumed by oxidized dextran, V0: the volume (mL) of NaOH consumed by unoxidized dextran;

[0115] M: the molar concentration (mol / L) of NaOH, W: the mass (g) of oxidized dextran;

[0116] M W : the molar mass (g / mol) of the structural unit of dextran.

[0117] After testing, when 131 mg, 261 mg, 524 mg, and 786 mg of sodium periodate were added, the oxidation degrees of the obtained oxidized dextrans were 18.4%, 32.4%, 56.7%, and 75.3% respectively, and the aldehyde group contents were 36.8%, 64.8%, 113.4%, and 150.6% respectively.

[0118] 2) Synthesis of mPMO / ODEX hydrogel: A solution with an mPMO polymer concentration of 8% was prepared in a phosphate buffered saline solution. The corresponding mass of ODEX was added according to a molar ratio of aldehyde groups to amino groups of 1:1, and stirred at 4 °C for 3 days.

[0119] Circular dichroism tests were carried out on the mPMO / OHA hydrogel (OHA oxidation degree was 19%), mPML / OHA hydrogel, and mPMO / ODEX (ODEX oxidation degree was 18.4%) hydrogels obtained in Example 3 and Comparative Examples 1 - 2 to study the secondary structure in the hydrogels. The results are as Figure 3As shown, it can be seen that all three hydrogels have a positive peak at 195 nm and two negative peaks at 210 nm and 225 nm, which are the characteristic peak patterns of α-helix. By fitting the proportion of the secondary structure, it is found that there is no obvious difference in the proportion of the secondary structure of the three hydrogels, indicating that the methionine segment dominates the formation of the secondary structure, and the polysaccharide and lysine segments do not have an obvious effect on the secondary structure of the hydrogel. Moreover, as the temperature increases from 10 °C to 70 °C, the intensities of the three absorption peaks all decrease accordingly, indicating that with the increase of temperature, the change of the polyamino acid chain segment will promote the solution-gel phase transition of the material, and the secondary structure remains stable all the time.

[0120] In this invention, HaCat cells are used. After they adhere to the wall, they are co-cultured with mPML / OHA hydrogel, mPMO / OHA hydrogel, and mPMO / ODEX hydrogel materials for 24 h. The cytotoxicity of the materials is detected by the Calcein-AM / PI live / dead cell staining method and the CCK-8 method. The results are as Figure 4 shown. In each field of view, the number of apoptotic cells in each group is similar, and the cell viability of different hydrogel material groups is higher than that of the blank group (Control group, that is, single HaCat cells without adding hydrogel), indicating that the hydrogel does not produce obvious toxicity to cells. The results are as Figure 5 shown.

[0121] Hydrogen peroxide (H2O2) is an important reactive oxygen species (ROS). It plays a dual role in the life activities of cells. On the one hand, under normal physiological conditions, ROS (including hydrogen peroxide) are produced in cells and play an important signal transduction role; on the other hand, when ROS are produced excessively or removed insufficiently, it will cause oxidative stress to cells, leading to cell damage and disease occurrence. In this invention, the CCK-8 method is used to detect the protective ability of mPML / OHA hydrogel, mPMO / OHA hydrogel, and mPMO / ODEX hydrogel on HaCat cells exposed to 200 μM H2O2. The results are as Figure 6 shown (where the Control group is single HaCat cells without adding hydrogel, and the H2O2 group is HaCat cells co-cultured with hydrogel in a medium containing 0.2 mM H2O2). It can be seen that there is no obvious difference in cell viability among different hydrogel groups, but they are all higher than the Control group. This is because L-methionine can play a protective role, indicating that the hydrogels provided by this invention have excellent antioxidant effects, ensuring that the hydrogels can be used for subsequent experiments on living organisms. The results are as Figure 6 shown.

[0122] Lipopolysaccharide (LPS) is a bacterial cell wall component that can stimulate immune cells, including macrophages. In chronic wounds, LPS can trigger a persistent inflammatory response by activating macrophages, thereby affecting the wound healing process. In this invention, RAW264.7 cells were co-cultured with mPML / OHA hydrogel, mPMO / OHA hydrogel, and mPMO / ODEX hydrogel in an LPS and H2O2 environment for 1 day, then digested into single-cell suspensions for flow cytometry detection. The gating process was as follows: after removing adhesions, viable cells were selected, and CD80 or CD206 positive cells were selected among the F4 / 80 strongly positive cells, which were the M1 or M2 macrophage gates. The results are as Figure 7 shown (where the Control group, that is, single HaCat cells, without adding hydrogel and LPS and H2O2; the LPS + H2O2 group, that is, HaCat cells were co-cultured with LPS and H2O2 without adding hydrogel). It can be seen that the mPMO / OHA hydrogel can effectively increase the proportion of M2 macrophages.

[0123] In this invention, bone marrow-derived neutrophils were apoptotic-treated and co-cultured with RAW264.7 cells exposed to H2O2 and LPS and mPML / OHA hydrogel, mPMO / OHA hydrogel, and mPMO / ODEX hydrogel to verify the regulation of different hydrogels on macrophage efferocytosis. The results are as Figure 8 shown (where the Control group, that is, single HaCat cells, without adding hydrogel and LPS and H2O2, and the LPS + H2O2 group, that is, HaCat cells were co-cultured with LPS and H2O2 without adding hydrogel). The mPM / OHA hydrogel can significantly improve the phagocytic ability of macrophages to apoptotic cells.

[0124] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the broadest scope consistent with the principles and novel features disclosed herein.

Claims

1. A polymer, characterized in that, The polymer is formed by reacting a block copolymer with a glycosaminoglycan aldehyde derivative; The block copolymer includes a terminal amino group - modified polyethylene glycol segment and a polyamino acid chain segment, and the polyamino acid chain segment includes a poly - L - methionine segment and a poly - L - ornithine segment; The glycosaminoglycan aldehyde derivative is selected from one or more aldehyde derivatives of heparin, heparin derivatives, sulfated dextran, sulfated dextran derivatives, sulfated hyaluronic acid, sulfated hyaluronic acid derivatives, hyaluronic acid, hyaluronic acid derivatives, chondroitin sulfate or chondroitin sulfate derivatives.

2. The polymer according to claim 1, wherein The degree of oxidation of the glycosaminoglycan aldehyde derivative is 5% - 90%.

3. The polymer according to claim 1, characterized in that, The molar ratio of the amino group in the block copolymer to the aldehyde group in the glycosaminoglycan aldehyde derivative is 2:1 - 1:

2.

4. The polymer according to claim 1, wherein The molar number of the poly - L - methionine segment is 50% - 99% of the polyamino acid chain segment.

5. A method for preparing a polymer according to any one of claims 1 to 4, characterized in that, It includes the following steps: S1: React the terminal amino group - modified polyethylene glycol with L - methionine - N - carboxy - anhydride to obtain an intermediate; S2: React the intermediate with L - ornithine - N - carboxy - anhydride with protected amino group, and then deprotect to obtain the block copolymer; S3: React the block copolymer with the glycosaminoglycan aldehyde derivative to obtain the polymer.

6. The preparation method according to claim 5, characterized in that, The molar ratio of the terminal amino group - modified polyethylene glycol to L - methionine - N - carboxy - anhydride is 1:1 - 1:600; The molar ratio of the intermediate to L - ornithine - N - carboxy - anhydride with protected amino group is 1:1 - 1:600; The temperature of the reaction in step S1 is room temperature, and the time is 2 - 4 days; The temperature of the reaction in step S2 is room temperature, and the time is 2 - 4 days; The temperature of the reaction in step S3 is 2 - 6 °C, and the time is 2 - 4 days.

7. A hydrogel, characterized in that, It is formed by the polymer described in any one of claims 1 - 4 or the polymer prepared by the preparation method described in any one of claims 5 and 6.

8. A wound dressing, characterized in that, It includes the hydrogel described in claim 7.

9. The wound dressing according to claim 8, wherein, The wound dressing further includes excipients.

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