Injectable multifunctional glycopeptide hydrogel, preparation method and application thereof

Abstract

The invention relates to an injectable multifunctional glycopeptide hydrogel, a preparation method and an application thereof, and relates to the technical field of biomedical materials and related medical device research and development. Prepared antimicrobial peptide hyaluronic acid, catechol peptide hyaluronic acid, angiogenic peptide hyaluronic acid and amino hyaluronic acid are uniformly mixed in a molar ratio of (0.1-1): (0.1-1): (0.1-1): (0.3-5), reacted at room temperature for 5-30 minutes, and allowed to stand to obtain a multifunctional glycopeptide hydrogel. The multifunctional hydrogel is prepared by matching a wound repair process, and different functions play a synergistic role, thereby better promoting wound healing and regeneration. The prepared hydrogel has a simple preparation process, is degradable, has good biocompatibility, and has good commercial prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials and related medical device research and development, and in particular to an injectable multifunctional glycopeptide hydrogel, a preparation method and application thereof. Background Art

[0002] Skin is the tissue on the surface of the body that wraps around the muscles. It is the largest organ in the human body and is responsible for protecting the body, perspiring, sensing heat and cold, and pressure. Skin damage is prone to infection, especially chronic wound infection, such as diabetic wounds, burns, pressure sores, acne, etc.; infection will further hinder wound healing and is a major clinical problem. The abuse of antibiotics has led to bacterial resistance, which has brought great difficulties to the treatment of infections. With the rapid development of materials science, biology, and medicine, many new types of medical antibacterial dressings have emerged. They are endowed with certain antibacterial activity through physical or chemical methods, providing a sterile environment for the wound, which is conducive to wound repair.

[0003] As a new type of wound dressing, hydrogel has a three-dimensional network structure, good moisture retention, and good biocompatibility. It can be completely degraded, does not require reoperation, and will not cause secondary damage to the wound. Adding antimicrobial agents to hydrogels can give them antibacterial properties, such as nanosilver, antibiotics, and quaternary ammonium salts, but their high cytotoxicity, easy to produce drug resistance, and enrichment in the body limit their widespread application.

[0004] Hyaluronic acid is an important component of human tissues and organs. It has good biocompatibility, degradability, and non-immunogenicity. It is widely used in clinical practice. It can also regulate the polarization of pro-inflammatory M1 macrophages into anti-inflammatory M2 cells, providing a better microenvironment for tissue repair. Antimicrobial peptides have good antibacterial activity. Catechol peptides are reducing and can remove free radicals from wound sites and reduce inflammation. Angiogenic peptides can promote angiogenesis at the site of trauma and provide nutrients and oxygen for cell proliferation. However, small polypeptide molecules are prone to rapid degradation and cannot function for a long time. At present, many studies are preparing hydrogels with different properties by adjusting the physical and chemical properties of hydrogels. Some studies are giving hydrogels a single property. Few studies prepare multifunctional hydrogels by matching the wound repair process to better promote wound healing and regeneration.

[0005] Therefore, without the use of antibiotics, cytokines or therapeutic cells, it is urgent to develop a multifunctional synergistic hydrogel with good biocompatibility, good antibacterial activity, free radical scavenging, inflammation reduction, angiogenesis promotion, and wound healing. How to provide an injectable multifunctional glycopeptide hydrogel and its preparation method is a problem that those skilled in the art need to solve urgently. Summary of the invention

[0006] The technical problem to be solved by the present invention is an injectable multifunctional glycopeptide hydrogel, a preparation method and its application. The purpose is to prepare a multifunctional hydrogel by matching the wound repair process, so that different functions play a synergistic role, thereby better promoting wound healing and regeneration. The prepared hydrogel has a simple preparation process, is degradable, has good biocompatibility, and has good commercial prospects.

[0007] In order to solve the above technical problems, the first object of the present invention is to provide a method for preparing an injectable multifunctional glycopeptide hydrogel, wherein the prepared antimicrobial peptide hyaluronic acid, catechol peptide hyaluronic acid, angiogenic peptide hyaluronic acid and amino hyaluronic acid are uniformly mixed in a molar ratio of (0.1-1): (0.1-1): (0.1-1): (0.3-5), reacted at room temperature for 5-30 minutes, and allowed to stand to obtain a multifunctional glycopeptide hydrogel, wherein the antimicrobial peptide hyaluronic acid has a structural formula as shown in Formula I, the catechol peptide hyaluronic acid has a structural formula as shown in Formula II, the angiogenic peptide hyaluronic acid has a structural formula as shown in Formula III, and the amino hyaluronic acid has a structural formula as shown in Formula IV;

[0008]

[0009] The beneficial effects of the present invention are as follows: the present invention forms an injectable hydrogel with antimicrobial peptide hyaluronic acid, catechol peptide hyaluronic acid and vascular peptide hyaluronic acid through dynamic covalent bonds and hydrogen bonds with amino hyaluronic acid. Antimicrobial peptides with good antimicrobial activity, catechol peptides and angiogenesis-promoting peptides that scavenge oxygen free radicals, and hyaluronic acid that regulates macrophage polarization are used as raw materials to prepare a multifunctional glycopeptide hydrogel; the hydrogel acts on bacteria, destroys the integrity of the cell membrane, causes the contents to leak, and thus kills the bacteria; acts with active oxygen to scavenge free radicals, reduces further damage to cells and tissues; and can act on VEGF receptors to promote angiogenesis. In addition, hyaluronic acid can induce M1 macrophages to polarize into M2 macrophages, secrete various anti-inflammatory cytokines, and is beneficial to wound healing. Therefore, the hydrogel of the present invention has good biocompatibility, can be completely degraded, and finally discharged from the body. In addition, the prepared hydrogel has a microporous structure, can promote cell proliferation, and is beneficial to wound healing. Therefore, the hydrogel of the present invention can match the wound healing process, kill bacteria, remove free radicals, promote angiogenesis, and regulate local immune cells, and has great potential for difficult-to-heal wounds.

[0010] Based on the above technical solution, the present invention can also be improved as follows.

[0011] Furthermore, the prepared antimicrobial peptide hyaluronic acid, catechol peptide hyaluronic acid, angiogenic peptide hyaluronic acid and amino hyaluronic acid are uniformly mixed in a molar ratio of (0.5-1): (0.5-1): (0.5-1): (0.3-3), reacted at room temperature for 5-30 minutes, and allowed to stand to obtain a multifunctional glycopeptide hydrogel with a molar concentration of 0.01-10wt%.

[0012] The beneficial effect of the technical solution further adopted by the present invention is that the activity of the multifunctional glycopeptide hydrogel is optimized by adjusting the ratio of various raw materials.

[0013] Furthermore, the preparation method of the antimicrobial peptide hyaluronic acid is as follows: dissolving oxidized hyaluronic acid in a buffer solution (e.g., 0.1MMES) at pH=5.0±0.3, then adding EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC·HCL) and NHS (N-hydroxysuccinimide) according to a molar ratio of oxidized hyaluronic acid to EDC and NHS of 1:(1-5):(1:5) to activate the carboxyl group of the oxidized hyaluronic acid, and then adding the antimicrobial peptide according to a molar ratio of oxidized hyaluronic acid to antimicrobial peptide of 1:0.01-1, reacting at room temperature for 24-48 hours, and obtaining the antimicrobial peptide hyaluronic acid; the amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO:1.

[0014] Furthermore, the preparation method of the catechol peptide hyaluronic acid is as follows: oxidized hyaluronic acid and 3-maleimidopropionic acid are reacted at a molar ratio of 1:1.2-3 at room temperature for 24-72 hours, dialyzed for 72-120 hours, and freeze-dried for 24-120 hours to obtain an intermediate product, the intermediate product is dissolved in a pH = 5.0 ± 0.3 buffer (for example, 0.1M MES), and then catechol peptide is added at a molar ratio of the intermediate product to catechol peptide of 1:0.05-1, and reacted at room temperature for 24-48 hours to obtain catechol peptide hyaluronic acid; the amino acid sequence of the catechol peptide is shown in SEQ ID NO:2.

[0015] Furthermore, the preparation method of the angiogenic peptide hyaluronic acid is to dissolve the intermediate product in a pH = 5.0 ± 0.3 buffer solution (e.g., 0.1M MES), add angiogenic peptide according to a molar ratio of the intermediate product to the angiogenic peptide of 1:0.05-1, and react at room temperature for 24-48 hours to obtain angiogenic peptide hyaluronic acid; the amino acid sequence of the angiogenic peptide is shown in SEQ ID NO:3.

[0016] Furthermore, the preparation method of the amino hyaluronic acid is to react hyaluronic acid with adipic acid dihydrazide at room temperature for 24-72 hours, the molar ratio of hyaluronic acid to adipic acid dihydrazide is 1:1-30, dialyze for 72-120 hours, and freeze-dry for 24-120 hours to obtain the amino hyaluronic acid.

[0017] Furthermore, the preparation method of the oxidized hyaluronic acid is as follows: according to the molar ratio of hyaluronic acid to sodium periodate of 1:0.05-1.2, hyaluronic acid and sodium periodate are dissolved in distilled water, reacted in the dark for 1-24 hours at room temperature, and then excess ethylene glycol is added to terminate the reaction, the molar ratio of sodium periodate to ethylene glycol is 1:2-10, reacted at room temperature for 0.5-2 hours, then dialyzed for 48-120 hours, and freeze-dried for 24-120 hours to obtain oxidized hyaluronic acid.

[0018] The beneficial effect of the technical solution further adopted by the present invention is that: under room temperature conditions, glycopeptide hydrogel is obtained through esterification reaction, amidation reaction, Michael addition reaction and Schiff base reaction, the reaction conditions are mild, the time is short and the efficiency is high, and the polypeptide and polysaccharide are cross-linked through dynamic chemical bonds and hydrogen bonds to form a hydrogel, and no additional cross-linking agent is required. The hydrogel has low cost, good biocompatibility and good clinical application prospects.

[0019] Furthermore, the molecular weight of the hyaluronic acid is 1000-5000k Da.

[0020] The second purpose is to provide an injectable multifunctional glycopeptide hydrogel, which is prepared as described above.

[0021] The beneficial effects of the technical solution adopted by the present invention are as follows: the multifunctional glycopeptide hydrogel of the present invention has the following advantages: (1) In terms of material source, natural biological materials are used, which have good biocompatibility, and the degradation products are amino acids and glycosaminoglycans, which are substances existing in the body and have no potential safety issues; (2) In terms of preparation process, the glycopeptide material is obtained by addition reaction, and then an injectable hydrogel is formed by dynamic covalent bonds and hydrogen bonds; (3) In terms of scaffold function, the prepared hydrogel has a porous structure, which is conducive to cell proliferation and migration; and contains a large amount of water, which can provide a moist environment for the wound site; it has antibacterial, anti-inflammatory and angiogenesis-promoting effects, which is conducive to wound healing and tissue regeneration.

[0022] The third purpose is to provide an application of an injectable multifunctional glycopeptide hydrogel, wherein the multifunctional glycopeptide hydrogel is applied to treat infected wounds and / or promote tissue repair.

[0023] The beneficial effect of the technical solution adopted by the present invention is that the glycopeptide hydrogel can exert good biological activity at the site of wound infection and provide a sterile environment for wound healing.

[0024] Furthermore, the dialysis condition is to dialyze in distilled water for 72 to 120 hours at room temperature using a dialysis bag with a molecular weight cutoff of 3500Da.

[0025] Furthermore, the temperature conditions involved in the present invention refer to 20-30°C. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The synthetic route of the HA-AMP polymer of the present invention;

[0027] Figure 2 The synthetic route of the HA-CAP polymer of the present invention;

[0028] Figure 3 The synthetic route of the HA-SLanc polymer of the present invention;

[0029] Figure 4 is the circular dichroism spectrum of the HA-AMP polymer of the present invention;

[0030] Figure 5 is the circular dichroism spectrum of the HA-CAP polymer of the present invention;

[0031] Figure 6 is the circular dichroism spectrum of the HA-SLanc polymer of the present invention;

[0032] Figure 7 The synthetic route of the HA-P hydrogel of the present invention;

[0033] Figure 8 is a picture of the HA-P hydrogel of the present invention;

[0034] Fig. 9 is a SEM image of the HA-P hydrogel of the present invention;

[0035] Fig.10 is the antibacterial activity of the HA-P hydrogel of the present invention;

[0036] Fig.11 The cell compatibility of the HA-P hydrogel of the present invention;

[0037] Fig.12 The tubularity of the HA-P hydrogel of the present invention;

[0038] Fig.13 The HA-P hydrogel of the present invention promotes the healing of infected wounds;

[0039] Fig.14 These are H&E and Masson's staining pictures of the wound site of the present invention. DETAILED DESCRIPTION

[0040] Example 1

[0041] This embodiment relates to a method for preparing an injectable multifunctional glycopeptide hydrogel, comprising the following steps (eg Figures 1 to 3 and 7):

[0042] S1: reacting hyaluronic acid with sodium periodate at a molar ratio of 5:1 for 3 h in a dark environment, adding excess ethylene glycol to terminate the reaction, the molar ratio of sodium periodate to ethylene glycol is 1:2, reacting for 0.5 h, dialyzing for 72 h, and freeze-drying for 72 h to obtain oxidized hyaluronic acid (HA-CHO), reacting HA-CHO with an antimicrobial peptide (amino acid sequence such as SEQ ID NO: 1) at a molar ratio of 10:1, and reacting at room temperature for 24 h to obtain an antimicrobial peptide hyaluronic acid (HA-AMP);

[0043] S2: Hyaluronic acid and sodium periodate were reacted at a molar ratio of 5:1, and the reaction was carried out under light-proof conditions for 3 hours. Excess ethylene glycol was added to terminate the reaction, dialyzed for 72 hours, and freeze-dried for 72 hours to obtain oxidized hyaluronic acid. HA-CHO was reacted with 3-maleimidopropionic acid to obtain intermediate I, which was then reacted with catechol peptide at a molar ratio of 10:1 and reacted at room temperature for 24 hours to obtain catechol peptide hyaluronic acid (HA-CAP);

[0044] S3: reacting hyaluronic acid with sodium periodate at a molar ratio of 5:1 for 3 h in a dark environment, adding excess ethylene glycol to terminate the reaction, dialyzing for 72 h, and freeze-drying for 72 h to obtain oxidized hyaluronic acid, reacting HA-CHO with 3-maleimidopropionic acid to obtain intermediate II, and then reacting with angiogenic peptide at a molar ratio of 10:1, and reacting at room temperature for 24 h to obtain angiogenic peptide hyaluronic acid (HA-SLanc);

[0045] S4: Hyaluronic acid and adipic acid dihydrazide were reacted at a molar ratio of 1:5 for 24 h, then dialyzed for 72 h, and freeze-dried for 72 h to obtain amino hyaluronic acid (HA-NH 2 ).

[0046] S5: Combine HA-AMP, HA-CAP, HA-SLanc and HA-NH 2 The mixture was mixed in a molar ratio of 0.5:0.5:0.5:2, reacted at room temperature for 5 minutes, and allowed to stand to obtain a 1 wt% glycopeptide hydrogel.

[0047] Example 2

[0048] This embodiment relates to a method for preparing an injectable multifunctional glycopeptide hydrogel, comprising the following steps (eg Figures 1 to 3 and 7):

[0049] S1, reacting hyaluronic acid with sodium periodate at a molar ratio of 5:2, reacting for 4 hours under light-proof conditions, adding excess ethylene glycol to terminate the reaction, dialyzing for 72 hours, and freeze-drying for 72 hours to obtain oxidized hyaluronic acid (HA-CHO), reacting HA-CHO with antimicrobial peptide at a molar ratio of 10:2, and reacting at room temperature for 24 hours to obtain antimicrobial peptide hyaluronic acid (HA-AMP);

[0050] S2, reacting hyaluronic acid with sodium periodate at a molar ratio of 5:2, reacting for 4 hours under light-proof conditions, adding excess ethylene glycol to terminate the reaction, dialyzing for 72 hours, and freeze-drying for 72 hours to obtain oxidized hyaluronic acid, reacting HA-CHO with 3-maleimidopropionic acid to obtain intermediate I, and then reacting with catechol peptide at a molar ratio of 10:2, reacting at room temperature for 24 hours to obtain catechol peptide hyaluronic acid (HA-CAP);

[0051] S3, reacting hyaluronic acid with sodium periodate at a molar ratio of 5:2, reacting for 4 hours in a dark environment, adding excess ethylene glycol to terminate the reaction, dialyzing for 72 hours, and freeze-drying for 72 hours to obtain oxidized hyaluronic acid, reacting HA-CHO with 3-maleimidopropionic acid to obtain intermediate II, and then reacting with angiogenic peptide at a molar ratio of 10:2, reacting at room temperature for 24 hours to obtain angiogenic peptide hyaluronic acid (HA-SLanc);

[0052] S4, hyaluronic acid and adipic acid dihydrazide were reacted at a molar ratio of 1:6 for 24 h, then dialyzed for 72 h, and freeze-dried for 72 h to obtain amino hyaluronic acid (HA-NH 2 ).

[0053] S5, HA-AMP, HA-CAP, HA-SLanc and HA-NH 2 The mixture was mixed in a molar ratio of 0.6:0.6:0.6:2, reacted at room temperature for 6 min, and allowed to stand to obtain a 1 wt% glycopeptide hydrogel, such as Figure 8 shown.

[0054] Example 3

[0055] This embodiment relates to a method for preparing an injectable multifunctional glycopeptide hydrogel, comprising the following steps (eg Figures 1 to 3 and 7):

[0056] S1, reacting hyaluronic acid with sodium periodate at a molar ratio of 5:3, reacting for 5 hours under light-proof conditions, adding excess ethylene glycol to terminate the reaction, dialyzing for 72 hours, and freeze-drying for 72 hours to obtain oxidized hyaluronic acid (HA-CHO), reacting HA-CHO with antimicrobial peptide at a molar ratio of 10:3, and reacting at room temperature for 24 hours to obtain antimicrobial peptide hyaluronic acid (HA-AMP);

[0057] S2, reacting hyaluronic acid with sodium periodate at a molar ratio of 5:3, reacting for 5 hours in a dark environment, adding excess ethylene glycol to terminate the reaction, dialyzing for 72 hours, and freeze-drying for 72 hours to obtain oxidized hyaluronic acid, reacting HA-CHO with 3-maleimidopropionic acid to obtain intermediate I, and then reacting with catechol peptide at a molar ratio of 10:3, reacting at room temperature for 24 hours to obtain catechol peptide hyaluronic acid (HA-CAP);

[0058] S3, reacting hyaluronic acid with sodium periodate at a molar ratio of 5:3, reacting for 5 hours under light-proof conditions, adding excess ethylene glycol to terminate the reaction, dialyzing for 72 hours, and freeze-drying for 72 hours to obtain oxidized hyaluronic acid, reacting HA-CHO with 3-maleimidopropionic acid to obtain intermediate II, and then reacting with angiogenic peptide at a molar ratio of 10:3, reacting at room temperature for 24 hours to obtain angiogenic peptide hyaluronic acid (HA-SLanc);

[0059] S4, hyaluronic acid and adipic acid dihydrazide were reacted at a molar ratio of 1:7 for 24 h, then dialyzed for 72 h, and freeze-dried for 72 h to obtain amino hyaluronic acid (HA-NH 2 ).

[0060] S5, HA-AMP, HA-CAP, HA-SLanc and HA-NH 2 The mixture was mixed in a molar ratio of 0.7:0.7:0.7:3, reacted at room temperature for 7 minutes, and allowed to stand to obtain a 1 wt% glycopeptide hydrogel.

[0061] Example 4

[0062] This embodiment relates to a method for preparing an injectable multifunctional glycopeptide hydrogel, comprising the following steps (eg Figures 1 to 3 and 7):

[0063] S1, reacting hyaluronic acid with sodium periodate at a molar ratio of 5:4, reacting for 6 hours in a dark environment, adding excess ethylene glycol to terminate the reaction, dialyzing for 72 hours, and freeze-drying for 72 hours to obtain oxidized hyaluronic acid (HA-CHO), reacting HA-CHO with an antimicrobial peptide at a molar ratio of 10:4, and reacting at room temperature for 24 hours to obtain an antimicrobial peptide hyaluronic acid (HA-AMP);

[0064] S2, reacting hyaluronic acid with sodium periodate at a molar ratio of 5:4, reacting for 6 hours in a dark environment, adding excess ethylene glycol to terminate the reaction, dialyzing for 72 hours, and freeze-drying for 72 hours to obtain oxidized hyaluronic acid, reacting HA-CHO with 3-maleimidopropionic acid to obtain intermediate I, and then reacting with catechol peptide at a molar ratio of 10:4, reacting at room temperature for 24 hours to obtain catechol peptide hyaluronic acid (HA-CAP);

[0065] S3, reacting hyaluronic acid with sodium periodate at a molar ratio of 5:4, reacting for 6 hours under light-proof conditions, adding excess ethylene glycol to terminate the reaction, dialyzing for 72 hours, and freeze-drying for 72 hours to obtain oxidized hyaluronic acid, reacting HA-CHO with 3-maleimidopropionic acid to obtain intermediate II, and then reacting with angiogenic peptide at a molar ratio of 10:4, reacting at room temperature for 24 hours to obtain angiogenic peptide hyaluronic acid (HA-SLanc);

[0066] S4, hyaluronic acid and adipic acid dihydrazide were reacted at a molar ratio of 1:8 for 24 h, then dialyzed for 72 h, and freeze-dried for 72 h to obtain amino hyaluronic acid (HA-NH 2 ).

[0067] S5, HA-AMP, HA-CAP, HA-SLanc and HA-NH 2 The mixture was mixed in a molar ratio of 0.8:0.8:0.8:3, reacted at room temperature for 7 minutes, and allowed to stand to obtain a 1 wt% glycopeptide hydrogel.

[0068] Example 5

[0069] This embodiment relates to a method for preparing an injectable multifunctional glycopeptide hydrogel, comprising the following steps (eg Figures 1 to 3 and 7):

[0070] S1, reacting hyaluronic acid with sodium periodate at a molar ratio of 5:6, reacting for 7 hours in a dark environment, adding excess ethylene glycol to terminate the reaction, dialyzing for 72 hours, and freeze-drying for 72 hours to obtain oxidized hyaluronic acid (HA-CHO), reacting HA-CHO with an antimicrobial peptide at a molar ratio of 1:1, and reacting at room temperature for 24 hours to obtain an antimicrobial peptide hyaluronic acid (HA-AMP);

[0071] S2, reacting hyaluronic acid with sodium periodate at a molar ratio of 5:6, reacting for 7 hours in a dark environment, adding excess ethylene glycol to terminate the reaction, dialyzing for 72 hours, and freeze-drying for 72 hours to obtain oxidized hyaluronic acid, reacting HA-CHO with 3-maleimidopropionic acid to obtain intermediate I, and then reacting with catechol peptide at a molar ratio of 1:1, reacting at room temperature for 24 hours to obtain catechol peptide hyaluronic acid (HA-CAP);

[0072] S3, reacting hyaluronic acid with sodium periodate at a molar ratio of 5:6, reacting for 7 hours in a dark environment, adding excess ethylene glycol to terminate the reaction, dialyzing for 72 hours, and freeze-drying for 72 hours to obtain oxidized hyaluronic acid, reacting HA-CHO with 3-maleimidopropionic acid to obtain intermediate II, and then reacting with angiogenic peptide at a molar ratio of 1:1, reacting at room temperature for 24 hours to obtain angiogenic peptide hyaluronic acid (HA-SLanc);

[0073] S4, hyaluronic acid and adipic acid dihydrazide were reacted at a molar ratio of 1:10 for 24 h, then dialyzed for 72 h, and freeze-dried for 72 h to obtain amino hyaluronic acid (HA-NH 2 ).

[0074] S5, HA-AMP, HA-CAP, HA-SLanc and HA-NH 2 The mixture was mixed in a molar ratio of 1:1:1:5, reacted at room temperature for 10 min, and allowed to stand to obtain a 10 wt% glycopeptide hydrogel.

[0075] Example 6: HA-AMP, HA-CAP, and HA-Slanc prepared in Examples 1-5 1 HNMR analysis

[0076] This example uses deuterated water as solvent and is tested on a nuclear magnetic resonance spectrometer Bruker 400. The results are as follows: the chemical shift of 3.0-4.5 ppm is the characteristic absorption peak of hyaluronic acid; the chemical shift of 6.2 ppm is the characteristic absorption peak of antimicrobial peptide. The above analysis proves the correctness of the chemical structure of antimicrobial peptide hyaluronic acid.

[0077] This example uses deuterated water as solvent and is tested on a nuclear magnetic resonance spectrometer Bruker 400. The results are as follows: the chemical shift of 3.0-4.5 ppm is the characteristic absorption peak of hyaluronic acid; the chemical shift of 6.5 ppm is the characteristic absorption peak of catechol peptide. The above analysis proves the correctness of the chemical structure of catechol peptide hyaluronic acid.

[0078] This example uses deuterated water as a solvent and is tested on a nuclear magnetic resonance spectrometer Bruker 400. The results are as follows: the chemical shift of 3.0-4.5 ppm is the characteristic absorption peak of hyaluronic acid; the chemical shift of 5.9 ppm is the characteristic absorption peak of angiogenic peptide. The above analysis proves the correctness of the chemical structure of angiogenic peptide hyaluronic acid.

[0079] This example uses deuterated water as solvent and is tested on a nuclear magnetic resonance spectrometer Bruker 400. The results are as follows: the chemical shift of 3.0-4.5 ppm is the characteristic absorption peak of hyaluronic acid; the chemical shift of 1.5-2.5 ppm is the characteristic absorption peak of adipic acid dihydrazide. The above analysis proves the correctness of the chemical structure of amino hyaluronic acid.

[0080] Example 7: Circular dichroism analysis

[0081] Circular dichroism spectrometry was used to analyze the secondary structure of peptides and glycopeptides, such as Figure 4-6 As shown, the secondary structure of the polypeptide did not change significantly before and after the reaction, indicating that this method is feasible and will not affect the structure of the polypeptide, thereby maintaining the biological activity of the polypeptide.

[0082] The hydrogel obtained in Example 1, Figure 8 The hydrogel sample was freeze-dried to obtain a freeze-dried sample, and the microstructure was observed by SEM, as shown in Fig. 9 As shown, the hydrogel has a highly cross-linked three-dimensional network structure with pore sizes ranging from a few microns to several hundred microns.

[0083] Example 8: Antibacterial activity and biocompatibility test of the hydrogel prepared in Example 1

[0084] 1. Antibacterial activity test of hydrogel

[0085] The hydrogel prepared in Example 1 was mixed with bacteria (10 6 CFU / mL, MRSA) for 2 h, and then the blank control group, chitosan group (purchased from Qilikang Skin Pharmaceutical) and experimental group were evenly spread on LB solid culture medium and observed after 24 h of culture. Fig.10 As shown, there were no obvious colonies in the experimental group, while there were obvious colonies in the other groups, indicating that the hydrogel can significantly inhibit the growth of bacteria and has good antibacterial activity.

[0086] 2. Evaluation of Hydrogel Cytotoxicity in Vitro

[0087] The present invention uses CCK-8 to detect the cytotoxicity of hydrogel to fibroblasts. The blank control group, chitosan group and hydrogel prepared in Example 1 were co-cultured with cells, incubated for a certain period of time, and then treated with CCK-8 for 4 hours, and the absorbance was detected at 405 nm. Fig.11 As shown, the cell survival rate was above 95%, indicating that the hydrogel had no obvious cytotoxicity.

[0088] The experimental process specifically refers to "GB / T 16886.5-2003, Biological Evaluation of Medical Devices Part 5: In vitro Cytotoxicity Test". From the experimental results, it can be seen that the cell survival rate is greater than 95% and the cytotoxicity classification is level 0. Therefore, the material has no cytotoxicity.

[0089] 3. Evaluation of Hemolysis of Hydrogel

[0090] According to the requirements of GB / T 16886.4-2003 / ISO 10993-4:2002, Biological evaluation of medical devices Part 4: Selection of experiments for interaction with blood, the hemolysis rate of the hydrogel was calculated according to the hemolysis rate calculation method. As shown in Table 1, the experimental results show that the hemolysis rate of the hydrogel is <5%, which meets the requirements for the use of medical devices.

[0091] Table 1 Hemolysis rate of hydrogel

[0092]

[0093] 4. Hydrogel Tube Formation Experiment

[0094] The effect of hydrogel on promoting tube formation of umbilical vein endothelial cells was evaluated through tube formation experiments. Fig.12 As shown, the hydrogel has a good angiogenic effect on umbilical vein endothelial cells and is beneficial to the formation of blood vessels.

[0095] 5. Research on the repair of skin wounds caused by bacterial infection in vivo

[0096] SD rats were generally anesthetized, and after skin preparation of the surgical area on the back, the rats were fixed on the operating table. An open full-thickness skin defect with a diameter of 1 cm was made on the back, and the wound was infected with MRSA bacteria to prepare a bacterial infection wound model.

[0097] The SD rats with bacterial infection wound model were randomly divided into three groups: a blank control group, a chitosan group, and a hyaluronic acid peptide experimental group, with 6 rats in each group; the blank control group was smeared with normal saline, the chitosan group was treated with chitosan purchased from Qilikang Skin Pharmaceutical, and the hyaluronic acid peptide experimental group was treated with the hyaluronic acid peptide prepared in Example 1. The dressings were changed every other day, and the wound healing was observed in real time. Fig.13 As shown in the figure, after 12 days of treatment, the wounds in the experimental group were basically completely healed, which was significantly better than that in the control group. This shows that the hydrogel can significantly promote wound healing. And pathological analysis was performed, such as Fig.14 As shown in the figure, after treatment, a large number of collagen fibers have been formed in the experimental group, inflammatory cells have been significantly reduced, there is a relatively complete epidermis, and the dermis tissue repair is relatively complete and close to normal tissue. This shows that the multifunctional hydrogel can quickly kill bacteria, remove free radicals, and promote angiogenesis, thereby accelerating the repair and regeneration of wounds.

[0098] In summary, the multifunctional glycopeptide hydrogel has antibacterial properties, removes ROS, reduces inflammation, promotes angiogenesis, and accelerates wound repair. Compared with chitosan hydrogel, it has better biological activity and is a hydrogel suitable for the treatment of chronic infected wounds.

[0099] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for preparing an injectable multifunctional glycopeptide hydrogel, characterized in that: The prepared antimicrobial peptide hyaluronic acid, catechol peptide hyaluronic acid, angiogenic peptide hyaluronic acid and amino hyaluronic acid are uniformly mixed in a molar ratio of (0.1-1): (0.1-1): (0.1-1): (0.3-5), reacted at room temperature for 5-30 minutes, and allowed to stand to obtain a multifunctional glycopeptide hydrogel, wherein the antimicrobial peptide hyaluronic acid has a structural formula as shown in Formula I, the catechol peptide hyaluronic acid has a structural formula as shown in Formula II, the angiogenic peptide hyaluronic acid has a structural formula as shown in Formula III, and the amino hyaluronic acid has a structural formula as shown in Formula IV; 2. The method for preparing an injectable multifunctional glycopeptide hydrogel according to claim 1, characterized in that: The prepared antimicrobial peptide hyaluronic acid, catechol peptide hyaluronic acid, angiogenic peptide hyaluronic acid and amino hyaluronic acid are uniformly mixed in a molar ratio of (0.5-1): (0.5-1): (0.5-1): (0.3-3), reacted at room temperature for 5-30 minutes, and allowed to stand to obtain a multifunctional glycopeptide hydrogel with a molar concentration of 0.01-10wt%.

3. The method for preparing an injectable multifunctional glycopeptide hydrogel according to claim 1 or 2, characterized in that: The preparation method of the antimicrobial peptide hyaluronic acid comprises dissolving oxidized hyaluronic acid in a buffer solution having a pH of 5.0±0.3, then adding EDC and NHS at a molar ratio of oxidized hyaluronic acid to EDC and NHS of 1:(1-5):(1:5) to activate the carboxyl group of the oxidized hyaluronic acid, and then adding the antimicrobial peptide at a molar ratio of oxidized hyaluronic acid to antimicrobial peptide of 1:0.01-1, reacting at room temperature for 24-48 hours, and obtaining the antimicrobial peptide hyaluronic acid; the amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO:

1.

4. The method for preparing an injectable multifunctional glycopeptide hydrogel according to claim 3, characterized in that: The preparation method of the catechol peptide hyaluronic acid comprises reacting oxidized hyaluronic acid and 3-maleimidopropionic acid at a molar ratio of 1:1.2-3 at room temperature for 24-72 hours, dialyzing for 72-120 hours, and freeze-drying for 24-120 hours to obtain an intermediate product, dissolving the intermediate product in a pH=5.0±0.3 buffer, then adding catechol peptide at a molar ratio of the intermediate product to the catechol peptide of 1:0.05-1, and reacting at room temperature for 24-48 hours to obtain the catechol peptide hyaluronic acid; the amino acid sequence of the catechol peptide is shown in SEQ ID NO:

2.

5. The method for preparing an injectable multifunctional glycopeptide hydrogel according to claim 4, characterized in that: The preparation method of the angiogenic peptide hyaluronic acid is to dissolve the intermediate product in a pH = 5.0 ± 0.3 buffer, add the angiogenic peptide according to the molar ratio of the intermediate product to the angiogenic peptide of 1:0.05-1, react at room temperature for 24-48 hours, and obtain the angiogenic peptide hyaluronic acid; the amino acid sequence of the angiogenic peptide is shown in SEQ ID NO:

3.

6. The method for preparing an injectable multifunctional glycopeptide hydrogel according to claim 1, characterized in that: The preparation method of the amino hyaluronic acid comprises reacting hyaluronic acid with adipic acid dihydrazide at room temperature for 24-72 hours, wherein the molar ratio of hyaluronic acid to adipic acid dihydrazide is 1:1-30, dialyzing for 72-120 hours, and freeze-drying for 24-120 hours to obtain the amino hyaluronic acid.

7. The method for preparing an injectable multifunctional glycopeptide hydrogel according to claim 4, characterized in that: The preparation method of the oxidized hyaluronic acid is as follows: the molar ratio of hyaluronic acid to sodium periodate is 1:0.05-1.2, hyaluronic acid and sodium periodate are dissolved in distilled water, reacted in the dark for 1-24 hours at room temperature, and then excessive ethylene glycol is added to terminate the reaction, the molar ratio of sodium periodate to ethylene glycol is 1:2-10, reacted at room temperature for 0.5-2 hours, then dialyzed for 48-120 hours, and freeze-dried for 24-120 hours to obtain the oxidized hyaluronic acid.

8. The method for preparing an injectable multifunctional glycopeptide hydrogel according to claim 6 or 7, characterized in that: The molecular weight of the hyaluronic acid is 1000-5000k Da.

9. An injectable multifunctional glycopeptide hydrogel, characterized in that: A multifunctional glycopeptide hydrogel prepared by the method for preparing an injectable multifunctional glycopeptide hydrogel as described in any one of claims 1 to 8.

10. The use of an injectable multifunctional glycopeptide hydrogel according to claim 9, characterized in that: The multifunctional glycopeptide hydrogel is used in preparing materials for treating infected wounds and / or promoting tissue repair.

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