A multifunctional hydrogel and its applications

Abstract

The present invention discloses a multifunctional hydrogel and its application, belonging to the field of biomedical technology. The multifunctional hydrogel is crosslinked by ketothiol with hydrazide groups at both ends and oxidized dextran to form a crosslinked network containing dynamic covalent bonds. The ketothiol with hydrazide groups at both ends is dissolved in deionized water to obtain a ketothiol solution, the oxidized dextran is dissolved in deionized water to obtain an oxidized dextran solution, and the ketothiol solution and the oxidized dextran solution are mixed and allowed to stand to obtain the multifunctional hydrogel. The multifunctional hydrogel of the present invention has dual pH / ROS responsiveness, can respond to the inflammatory microenvironment and low pH of infectious wounds, scavenge reactive oxygen species and release therapeutic drugs, regulate macrophage polarization and reduce bacterial infection, thus playing a key role in the inflammatory, proliferative and remodeling stages of wound healing to accelerate wound healing. Adding liposomes encapsulating curcumin to the multifunctional hydrogel can further improve the antibacterial effect.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a multifunctional hydrogel and its applications. Background Art

[0002] The skin is an important tissue organ of the human body and plays an important role in protecting the stability of the internal environment of the human body. A series of problems brought about by skin injuries affect human health. During the wound healing process, wound infections caused by microorganisms such as bacteria and fungi pose a great hazard, and the abuse of antibiotics has made wound infections become more and more normal. Therefore, it is of great significance to prepare a wound dressing with high antibacterial ability, effective prevention of wound infection, and promotion of wound healing.

[0003] The wound healing process is a complex physiological process, and restoring the barrier function at the wound site involves multiple factors. Traditional wound dressings, such as gauze, sponges, and bandages, have been widely used in clinical practice, but they usually cannot fully meet the various requirements of the complex wound healing process, such as providing a moist environment, good adaptability to the shape of the wound, etc. Therefore, multifunctional wound dressings are crucial for providing a stable healing environment. With the development of the medical and health field and the in-depth study of wound pathology and healing mechanisms by people, a large number of dressings have been researched and developed for the cure of different wounds, and a variety of commercial products have been formed. Currently, existing wound dressings can be divided into two categories: traditional dressings and new dressings. Among them, the new dressings can be roughly divided into film types, foam types, fiber types, hydrocolloid types, alginate types, and hydrogel types according to their own material characteristics. Among them, hydrogel dressings have become one of the most competitive dressing varieties due to their good liquid absorption and moisture retention, biocompatibility, self-adhesion, structural flexibility, and effective absorption and release characteristics of therapeutic agents. However, simple hydrogel dressings can only play the role of physical isolation and creating a moist environment, and cannot meet the increasing requirements for the performance of wound dressings in clinical practice. Developing multifunctional hydrogel wound dressings to meet various needs of wound repair has become the mainstream trend in the design of hydrogel dressings. In addition, targeted wound repair materials should be used according to different skin wound conditions to better promote wound healing. Therefore, it is still an important scientific issue to effectively promote wound healing by preparing hydrogel dressings with good mechanical properties, excellent biocompatibility, and inherent antibacterial and antioxidant properties through a simple and rapid method. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of the present invention is to provide a multifunctional hydrogel and its application. The present invention crosslinks oxidized dextran with ketothiol hydrazide at both ends to obtain a hydrogel with dual pH / ROS responsiveness, which can respond to the inflammatory microenvironment and low pH of infectious wounds, release therapeutic drugs to neutralize reactive oxygen species, regulate macrophage polarization and reduce bacterial infection, thereby playing a key role in the inflammatory, proliferative and remodeling stages of wound healing to accelerate wound healing.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] In the first aspect of the present invention, a multifunctional hydrogel is provided. The multifunctional hydrogel is crosslinked by ketothiol hydrazide at both ends and oxidized dextran to form a crosslinked network containing dynamic covalent bonds, which is the multifunctional hydrogel.

[0007] The oxidized dextran is prepared by the following reaction:

[0008] Dissolve dextran in water, add NaIO 4 Perform an oxidation reaction to obtain oxidized dextran.

[0009] The content of ketothiol hydrazide at both ends in the multifunctional hydrogel is 5.0 - 7.5 mg / mL, and the content of oxidized dextran is 75 - 90 mg / mL.

[0010] Preferably, the ketothiol hydrazide at both ends is prepared by the following method:

[0011] (1) Add butyl 3-mercaptopropionate and anhydrous acetone to dioxane hydrochloride, and react under a protective atmosphere to obtain dibutyl thione dipropionate;

[0012] (2) React dibutyl thione dipropionate with hydrazine hydrate to obtain ketothiol hydrazide at both ends.

[0013] Preferably, the oxidation degree of the oxidized dextran is 40 - 55%.

[0014] Preferably, the mass ratio of dextran to NaIO 4 is 5:6.56; the time of the oxidation reaction is 2 - 10 h.

[0015] Preferably, the multifunctional hydrogel is prepared by the following method:

[0016] Dissolve ketothiol hydrazide at both ends in deionized water to obtain a ketothiol solution, dissolve oxidized dextran in deionized water to obtain an oxidized dextran solution, mix the ketothiol solution and the oxidized dextran solution, and let it stand to obtain the multifunctional hydrogel.

[0017] Preferably, the concentration of the ketothiol solution is 10 - 15 mg / mL; the concentration of the oxidized dextran solution is 150 - 180 mg / mL; the volume ratio of the ketothiol solution to the oxidized dextran solution is 1:1.

[0018] In the second aspect of the present invention, there is provided the use of the multifunctional hydrogel in at least one of the following 1) - 5):

[0019] 1) Encapsulating and sustainedly releasing drugs;

[0020] 2) Preparing drugs or dressings for scavenging ROS;

[0021] 3) Preparing antibacterial drugs or dressings;

[0022] 4) Dual response to acidity and ROS;

[0023] 5) Preparing degradable drugs or dressings.

[0024] In the third aspect of the present invention, there is provided a drug for treating infectious wounds, wherein the drug uses the multifunctional hydrogel as a dressing, and the dressing contains liposomes encapsulating curcumin.

[0025] Preferably, the liposomes encapsulating curcumin are prepared by the following method:

[0026] (1) Dissolve egg yolk lecithin and cholesterol in ethanol to obtain a lipid solution, and dissolve curcumin in ethanol to obtain a curcumin solution; mix the lipid solution and the curcumin solution evenly to obtain a mixture;

[0027] (2) Drop the mixture into the preheated phosphate buffer solution, stir and react, evaporate and remove ethanol by rotary evaporation, and filter through a 0.22 μm filter membrane to obtain liposomes encapsulating curcumin.

[0028] Preferably, the drug is prepared by the following method:

[0029] Dissolve the ketothiol with acyl hydrazide groups at both ends and the liposomes encapsulating curcumin in deionized water to obtain a drug-loading solution, dissolve oxidized dextran in deionized water to obtain an oxidized dextran solution, mix the drug-loading solution and the oxidized dextran solution, and let it stand to obtain a drug for treating infectious wounds.

[0030] Advantages of the present invention:

[0031] (1) In the present invention, crosslinking is carried out by oxidized dextran and ketothiol with acyl hydrazide groups at both ends to obtain a hydrogel with dual pH / ROS response. This hydrogel can not only encapsulate drugs but also sustainably release drugs, and this hydrogel also has the functions of reducing ROS and antibacterial action, and is a multifunctional hydrogel. It can be used by various methods such as injection and smearing.

[0032] (2) The present invention prepares a pH / ROS dual-responsive hydrogel dressing through a simple and rapid method, which can respond to the inflammatory microenvironment and low pH of infectious wounds, release therapeutic drugs to neutralize reactive oxygen species, regulate macrophage polarization and reduce bacterial infection, thereby playing a key role in the inflammatory, proliferative and remodeling stages of wound healing to accelerate wound healing.

[0033] (3) The preparation method of the present invention is simple, and the prepared hydrogel has good biocompatibility, stable structure and performance. The drug prepared with this hydrogel has a hemolysis rate of less than 0.5% on red blood cells, has a minimal impact on the viability of L929 cells and RAW 264.7 cells, can effectively scavenge various types of reactive oxygen species, effectively kill bacteria, continuously inhibit inflammation, accelerate blood vessel formation, and promote wound healing. Brief Description of the Drawings

[0034] Figure 1 : ODex, TKNN and TKNN x / ODex y Characterization diagrams of the hydrogel; where (a) is the comparison diagram of the nuclear magnetic resonance hydrogen spectrum of ODex and dextran (hereinafter referred to as Dex); (b) is the infrared spectrum of ODex and Dex; (c) is the nuclear magnetic resonance hydrogen spectrum of TKNN; (d) is the infrared spectrum of TKNN; (e) is the infrared spectrum of ODex, TKNN and freeze-dried hydrogel; (f) is the precursor solution and TKNN 1 / ODex 15 Picture of the hydrogel; (g) is the freeze-dried TKNN at a scale of 200 μm 1 / ODex 15 SEM diagram of the hydrogel; (h) is the freeze-dried TKNN at a scale of 50 μm 1 / ODex 15 SEM diagram of the hydrogel;

[0035] Figure 2 : Prepared liposomes and TKNN x (Cur-lip) / ODex y Characterization analysis diagrams of the hydrogel; where (a) is the particle size distribution diagram of unloaded liposomes (hereinafter referred to as liposome); (b) is the particle size distribution diagram of curcumin-loaded liposomes (hereinafter referred to as Cur-liposome); (c) is the cryo-TEM diagram of unloaded liposomes (i.e., liposome) at a scale of 100 μm; (d) is the cryo-TEM diagram of curcumin-loaded liposomes (i.e., Cur-liposome) at a scale of 100 μm; (e) is the ultraviolet-visible absorption curves of curcumin (hereinafter referred to as Cur), liposome and Cur-liposome; (f) is the freeze-dried TKNN at a scale of 100 μm1 (Cur-lip) / ODex 15 SEM images of the hydrogel; (g) Freeze-dried TKNN at a scale of 100 μm 1.5 (Cur-lip) / ODex 15 SEM images of the hydrogel; (h) Freeze-dried TKNN at a scale of 100 μm 1.5 (Cur-lip) / ODex 18 SEM images of the hydrogel;

[0036] Figure 3 : TKNN x (Cur-lip) / ODex y Functional display diagrams of the hydrogel; among them, (a) shows the injectability of the hydrogel; (b) shows the self-healing performance and healing principle of the hydrogel; (c) shows the adhesion of the hydrogel to different mouse organs; (d) shows the adhesion mechanism of the hydrogel;

[0037] Figure 4 : TKNN x / ODex y Biocompatibility of the hydrogel; among them, (a) shows different compositions of TKNN x / ODex y Hemolysis rate of the hydrogel; (b) shows the cell survival rate of L929 cells co-cultured with different TKNN x / ODex y after 24 h, 36 h, and 48 h; (c) shows the live / dead cell staining fluorescence images of different compositions of TKNN x / ODex y after co-incubation with L929 cells for 24 h, 36 h, and 48 h at a scale of 200 μm;

[0038] Figure 5 : TKNN x / ODex y ROS scavenging and antibacterial tests of the hydrogel; among them, (a) shows the cell survival rate of RAW 264.7 cells co-incubated with TKNN x / ODex y after 24 h; (b) shows the intracellular ROS content of cells after intervention with TKNN x / ODex y hydrogel; (c) shows TKNN at a scale of 200μm x / ODex yFluorescence images of cells stained with DCFH-DA after hydrogel intervention; (d) shows the inhibition rates of gels with different compositions against Escherichia coli; (e) shows the antibacterial rates of gels with different compositions against Staphylococcus aureus; (f) shows the survival of Escherichia coli and Staphylococcus aureus after treatment with different gels.

[0039] Figure 6 : TKNN x (Cur-lip) / ODex y Swelling, degradation, and response properties of the hydrogel; among them, (a) shows the swelling rate curves of hydrogels with different compositions over time; (b) shows the degradation experimental results of hydrogels with different compositions; (c) shows the pH response and oxidation response display of the hydrogel; (d) shows the curcumin release of hydrogels with different compositions in different concentrations of H 2 O 2 solution; (e) shows the curcumin release of hydrogels with different compositions in different pH buffer solutions.

[0040] Figure 7 : TKNN x (Cur-lip) / ODex y In vitro antibacterial test of the hydrogel; among them, (a) shows the inhibition rates of gels with different compositions against Escherichia coli; (b) shows the antibacterial rates of gels with different compositions against Staphylococcus aureus; (c) shows the survival of Escherichia coli and Staphylococcus aureus after treatment with different gels.

[0041] Figure 8 : TKNN x (Cur-lip) / ODex y Study on the wound healing of mice infected with Staphylococcus aureus by the hydrogel; among them, (a) shows the wound healing pictures of mice after intervention with different hydrogels; (b) shows the wound healing rate pictures after intervention with different hydrogels. Detailed implementation mode

[0042] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0043] As introduced in the background art section, existing wound dressings can be divided into two major categories: traditional dressings and new dressings. Among them, new dressings can be roughly divided into film type, foam type, fiber type, hydrocolloid type, alginate type, and hydrogel type according to their own material characteristics. Among them, hydrogel dressings have become one of the most competitive dressing varieties due to their good liquid absorption and moisturizing properties, biocompatibility, self-adhesion, structural flexibility, and effective absorption and release characteristics of therapeutic agents. However, simple hydrogel dressings can only play the role of physical isolation and creating a moist environment, and cannot meet the increasing requirements for the performance of wound dressings in clinical practice.

[0044] Based on this, the purpose of the present invention is to provide a multifunctional hydrogel and its application. In the present invention, oxidized dextran and ketothiol with hydrazide groups at both ends are crosslinked, so that the amino group at the end of ketothiol reacts with the aldehyde group formed by ring opening on oxidized dextran to form a dynamic covalent bond, thereby grafting ketothiol onto oxidized dextran to form a crosslinked network and obtaining a hydrogel. In addition to controlling the concentrations of oxidized dextran and ketothiol with hydrazide groups at both ends, the degree of oxidation of oxidized dextran is also one of the key factors for gel formation. If the degree of oxidation is too low, gel formation cannot occur; if the degree of oxidation is too high, dextran cannot play a role in the hydrogel. Through research by the inventors, it is found that the degree of oxidation of oxidized dextran needs to be controlled at 40 - 55%. The ketothiol in the hydrogel can reduce ROS, thereby playing a role in scavenging ROS. In addition, the hydrogel also has antibacterial effects. The present invention also provides a drug for treating infectious wounds. Curcumin is encapsulated in liposomes and then loaded into the hydrogel. The hydrogel can be applied to the wound in various ways such as injection or smearing. In the inflammatory environment and acidic environment of the wound, the ketothiol structure and dynamic covalent bond of the hydrogel are gradually destroyed, and the hydrogel changes from a gel state to a sol state, realizing the release of the active molecule curcumin. The curcumin in the hydrogel is gradually released to form a sustained release. The released drug neutralizes reactive oxygen species, regulates macrophage polarization, and reduces bacterial infection, thereby playing a key role in the inflammatory, proliferative, and remodeling stages of wound healing to accelerate wound healing.

[0045] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with specific embodiments.

[0046] Note: In the present invention, the degree of oxidation of oxidized dextran = the molar amount of aldehyde groups in oxidized dextran / the molar amount of hydroxyl groups in dextran × 100%.

[0047] The test materials used in the examples of the present invention are all conventional test materials in the art and can be obtained through commercial channels.

[0048] Example 1: Preparation of multifunctional hydrogel

[0049] (1) Dissolve 5 g of dextran (MW = 40000 Da) in 62.5 mL of water. After complete dissolution, add 6.56 g of NaIO 4 , and stir in the dark for 6 h. Then add 2 mL of ethylene glycol and stir for 1 h to terminate the reaction. Dialyze in pure water for 4 days, and then freeze-dry to obtain a white product, which is oxidized dextran (ODex), and its oxidation degree is about 50%.

[0050] (2) Add butyl 3-mercaptopropionate (8.00 g) and anhydrous acetone (6.00 g) to a flask, use dioxane hydrochloride as a solvent, and react at room temperature for 15 h under N 2 protection. After removing the solvent under reduced pressure, dilute the residue with ether, transfer it to a separatory funnel, and extract it successively with saturated NaHCO 3 solution and water, and retain the organic phase. Dry it with anhydrous NaSO 4 for 24 h, and then rotary evaporate to obtain dibutyl thione dipropionate as a light amber oil.

[0051] Dissolve dibutyl thione dipropionate (5.00 g) in methanol (100 mL), add hydrazine hydrate (6.30 g), stir at room temperature for 18 h, rotary evaporate to remove the solvent, dilute with water and transfer to a separatory funnel, extract twice with anhydrous ether, retain the aqueous phase, and rotary evaporate to remove water to obtain a white solid product, that is, ketothiol hydrazide (TKNN) with hydrazide groups at both ends.

[0052] (3) Weigh TKNN precisely and dissolve it in deionized water to obtain a TKNN precursor solution with a concentration of 10 mg / mL (x = concentration of TKNN / 10, when the concentration of TKNN is 10 mg / mL, x = 1.0); weigh ODex precisely and dissolve it in deionized water to obtain an ODex precursor solution with a concentration of 150 mg / mL (y = concentration of ODex / 10, when the concentration of ODex is 150 mg / mL, y = 15); mix the above two precursor solutions in a volume ratio of 1:1, and let it stand for a few seconds to obtain a multifunctional hydrogel denoted as TKNN 1 / ODex 15 hydrogel.

[0053] Example 2

[0054] The difference from Example 1 is that in step (3), the concentration of the TKNN precursor solution is 15 mg / mL (x = 1.5); the concentration of ODex is 180 mg / mL (y = 18). Finally, a multifunctional hydrogel denoted as TKNN 1.5 / ODex 18 hydrogel is prepared.

[0055] Example 3: Preparation of a drug for treating infectious wounds

[0056] (1) Weigh accurately 50 mg of egg yolk lecithin and 12.5 mg of cholesterol, and dissolve them in an appropriate amount of ethanol. Dissolve curcumin in ethanol to prepare a 1 mg / mL curcumin solution. Measure 2 mL of the curcumin solution and add it to the above solution and mix well to obtain a mixed solution. Dropwise add the mixed solution (1 - 2 drops per second) into a phosphate buffer solution (pH 7.4) at 60 °C, stir for 1 h, then remove ethanol by rotary evaporation, and filter through a 0.22 μm filter membrane to obtain a liposome (Cur-liposome) solution encapsulating curcumin.

[0057] (2) Weigh accurately TKNN and dissolve it in the above Cur-liposome solution to obtain a TKNN precursor solution with a concentration of 10 mg / mL (x = 1.0); weigh accurately ODex and dissolve it in deionized water to obtain an ODex precursor solution with a concentration of 150 mg / mL (y = 15); mix the above two precursor solutions in a volume ratio of 1:1, and let it stand for a few seconds to obtain a drug for treating infectious wounds, denoted as TKNN 1 (Cur-lip) / ODex 15 Hydrogel.

[0058] Example 4

[0059] The difference from Example 3 is that in step (2), a TKNN precursor solution with a concentration of 15 mg / mL (x = 1.5) is obtained; an ODex precursor solution with a concentration of 180 mg / mL (y = 18) is obtained. Finally, a drug for treating infectious wounds is prepared, denoted as TKNN 1.5 (Cur-lip) / ODex 18 Hydrogel.

[0060] Example 5

[0061] The difference from Example 3 is that in step (2), a TKNN precursor solution with a concentration of 15 mg / mL (x = 1.5) is obtained; an ODex precursor solution with a concentration of 150 mg / mL (y = 15) is obtained. Finally, a drug for treating infectious wounds is prepared, denoted as TKNN 1.5 (Cur-lip) / ODex 15 Hydrogel.

[0062] Example 6: TKNN prepared in Example 1 1 / ODex 15 Characterization of the hydrogel

[0063] It is determined by nuclear magnetic resonance hydrogen spectrum and infrared spectrum that dextran is successfully oxidized and ring-opened after the reaction. The experimental results are as Figure 1 , from Figure 1 of (a) 1It can be seen from the 1H NMR spectrum that after the oxidation reaction, the peak at 4.9 ppm disappeared, indicating that the adjacent hydroxyl groups of dextran were oxidized. In addition, from Figure 1 As can be seen from the FT-IR spectrum of (b), compared with Dex, a stretching vibration peak of C=O appears at 1716 cm -1 in ODex, further proving that dextran was successfully oxidized and ring-opened to form aldehyde. The oxidation degree of ODex was measured to be 51.3% by the hydroxylamine hydrochloride titration method.

[0064] The molecular structure of TKNN was determined by 1H NMR and IR spectra. The experimental results are as shown in Figure 1 (c) and Figure 1 (d). By comparison, TKNN was successfully synthesized.

[0065] Figure 1 Figure (e) shows the FT-IR spectra of ODex, TKNN, and freeze-dried (-50 °C, 12 h) TKNN 1 / ODex 15 hydrogels. A new absorption peak appears at 1658 cm -1 in the hydrogel sample. This absorption peak belongs to the characteristic absorption of the hydrazone bond (-N=C-) formed between -NH-NH 2 on the TKNN molecular chain and -CHO of ODex, further indicating that the hydrogel network was successfully constructed.

[0066] Figure 1 Figure (f) shows the pictures of the precursor solution and TKNN 1 / ODex 15 hydrogels. The gel can be rapidly formed after mixing equal volumes of the TKNN precursor solution and the ODex precursor solution.

[0067] Figure 1 Figures (g) and Figure 1 Figure (h) show the SEM images of freeze-dried TKNN 1 / ODex 15 hydrogels. The interior of the gel presents a loose and porous structure. This structure is beneficial for the exchange of substances between the hydrogel and the wound surface and for absorbing the exudate from the wound surface to maintain a clean healing environment for the wound.

[0068] Example 7: Characterization of the TKNN x (Cur-lip) / ODex y hydrogels prepared in Examples 3-5

[0069] Figure 2 Figure (a) shows the test results of the particle size of the drug-free liposomes, with an average diameter of 124.567 ± 3.244 nm. Figure 2(b) shows the particle size test results of Cur-liposome. After encapsulating curcumin, the average diameter increased to 185.4 ± 2.933 nm. The encapsulation efficiency of curcumin liposome was 87.373% ± 6.015%.

[0070] Figure 2 (c) and Figure 2 (d) are the TEM images of liposome and Cur-liposome respectively. It can be seen that the prepared liposomes are spherical or spherical-like structures.

[0071] Figure 2 (e) is the UV-visible absorption spectra of Cur (curcumin), liposome and Cur-liposome. The absorption peak of curcumin appears at 427 nm in the spectrum of Cur-liposome, indicating that curcumin was successfully encapsulated into the liposome.

[0072] Figure 2 (f)- Figure 2 (g) shows the SEM images of the TKNN x (Cur-lip) / ODex y hydrogel (lyophilized, -50 °C, 12 h). The interior of the gel presents a loose and porous structure. As the content of ODex and TKNN in the hydrogel increases, the internal structure of the hydrogel becomes more compact. This is because as the number of aldehyde groups in the system increases, the degree of crosslinking within the hydrogel increases, resulting in a denser internal framework structure of the hydrogel.

[0073] Example 8: The TKNN prepared in Example 3 1 (Cur-lip) / ODex 15 Characterization of injectability, self-healing and adhesion properties of the hydrogel

[0074] According to the method in step (3) of Example 3, a trace amount of rhodamine B dye was added to the ODex precursor solution. After vortexing evenly with the TKNN(Cur-lip) solution to form a gel, it was then uniformly loaded into a syringe and injected into water to observe whether continuous injection was possible, so as to evaluate the injectability of the hydrogel. The results are as Figure 3 (a) shows that the hydrogel has good injectability. The newly prepared hydrogel was stained with methylene blue and rhodamine B dyes respectively. The two stained gels were evenly separated with a knife, and then the two cross-sections were brought into contact to observe whether the hydrogel could be picked up with tweezers without breaking to evaluate the self-healing performance of the hydrogel. The results are as Figure 3As shown in (b), the cut surface of the hydrogel can heal after 1 hour of contact, indicating that the hydrogel has good self-healing properties. This is mainly due to the dynamic exchange of acylhydrazone bonds and the intermolecular hydrogen bond interactions. As a natural polysaccharide, dextran has excellent viscosity. In addition, the presence of liposomes also increases the adhesion ability of the hydrogel. Figure 3 (c) shows that the hydrogel has good adhesion ability to various biological materials such as mouse organs. Figure 3 (d) shows the adhesion mechanism. There are a large number of hydroxyl groups on the dextran molecule, which have strong hydrogen bond interactions with the hydroxyl groups and amino groups on the tissue surface, equivalent to forming physical crosslinks between the hydrogel and the tissue surface.

[0075] Test Example 1: TKNN x / ODex y Biocompatibility test of hydrogel

[0076] (1) The blood compatibility of the hydrogel can be evaluated by in vitro hemolysis experiment. The test is divided into four groups: PBS as the negative control group, distilled water as the positive control group, TKNN 1 / ODex 15 hydrogel prepared in Example 1 and TKNN 1.5 / ODex 18 hydrogel prepared in Example 2 as the experimental groups; the hydrogels in the experimental groups are freeze-dried at -50 °C for 12 h, and each group of experiments is tested in parallel 3 times.

[0077] Weigh 50 mg of the materials in the experimental groups and place them in 5 mL of PBS buffer solution, incubate at 37 °C for 24 h to obtain the hydrogel leaching solution. Draw fresh mouse blood from the anticoagulant tube, discard the supernatant after centrifuging at 3000 rpm for 15 min, then resuspend the blood cells with sterile normal saline, and repeat the resuspension and centrifugation operations multiple times until the supernatant becomes clear and then stop. Subsequently, dilute the blood cells with normal saline to a 5% blood cell suspension for standby. Finally, mix the blood cell suspension and the hydrogel leaching solution in equal volumes, incubate at 37 °C for 2 h, centrifuge and take 200 µL of the supernatant, and measure the OD value at 540 nm with an enzyme-labeled instrument.

[0078] Hemolysis rate (%) = ( A s - A p ) / ( A t - A p ) × 100%;

[0079] where A s is the OD value measured for the gel leaching solution of the experimental group; Ap OD value measured for the negative control group; A t OD value measured for the positive control group. The results are as Figure 4 shown in (a). The insets in the figure are the positive control group (distilled water), negative control group (PBS), TKNN 1 / ODex 15 hydrogel, TKNN 1.5 / ODex 18 hydrogel group and photos after incubation with the erythrocyte suspension. It was observed that the positive control group showed obvious redness, indicating that hemolysis occurred to erythrocytes under the influence of internal and external osmotic pressures. The upper layers of the negative control and hydrogel groups were light yellow, and there were erythrocyte precipitates in the lower layers. All hydrogel groups showed slight hemolysis, which were 0.12% and 0.07% respectively, both lower than 5%, meeting the biosafety standards of national materials. Based on the above results, it is shown that the hydrogels all exhibit good blood compatibility.

[0080] (2) Evaluate cell viability by the MTT method. First, select L929 cells (mouse fibroblasts) in the logarithmic growth phase for digestion, counting and dilution. Add the diluted cell suspension to a 96-well plate and culture adherently for 24 h. Place the freeze-dried gels (TKNN 1 / ODex 15 hydrogel prepared in Example 1 and TKNN 1.5 / ODex 18 hydrogel prepared in Example 2) in MEM complete medium and soak in an incubator for 24 h to obtain the hydrogel leaching solution. Discard the old medium, wash 3 times with sterile PBS buffer and then replace it with the hydrogel leaching solution. Use the wells with MEM complete medium as the control group. Place the plate in the incubator and culture for 24 h, 36 h, 48 h. When the predetermined culture time is reached, suck out the hydrogel leaching solution, wash three times with PBS buffer, then add 100 µL of MEM complete medium to each well and add 20 µL of MTT solution under light-shielded conditions. Incubate in the incubator for 4 h and then discard the supernatant. Subsequently, add 100 µL of DMSO solution to each well, shake the plate for 10 min under light-shielded conditions and then use an enzyme-linked immunosorbent assay (ELISA) reader to detect the OD value at 490 nm. Each group of experiments is operated in parallel three times.

[0081] Cytotoxicity (%) = ( A s - A 0 ) / ( A c - A 0 ) × 100%;

[0082] Among them, As is the absorbance value after culturing with the hydrogel leachate; A c is the absorbance value after culturing with MEM medium; A 0 is the absorbance value of PBS. The results are as shown in Figure 4 (b). After co-incubating the hydrogel leachate prepared from the hydrogels of Example 1 and Example 2 with L929 cells for 48 h, it was found that the relative cell survival rate was still above 90%, indicating that the hydrogel meets the international material usage standards during use.

[0083] (3) The biocompatibility of the hydrogel was further evaluated by a live / dead cell staining experiment. First, L929 cells in the logarithmic growth phase were seeded into a 6-well plate. After they were completely adherent, the old medium was replaced with the hydrogel leachate prepared from the hydrogels of Example 1 and Example 2, and the control group was still cultured with complete medium. Subsequently, it was placed in an incubator for 24 h, 36 h, and 48 h. When the preset culture time was reached, it was washed 3 times with sterile PBS buffer, then the cell live / dead staining working solution was added and incubated in the dark for 30 min, and then the cells were washed with PBS buffer. An inverted fluorescence microscope was used to observe and take images. The experimental results are as shown in Figure 4 (c). It was observed that there were a large number of live cells showing green fluorescence and only a few dead cells showing red fluorescence in each group. The results of the cell live / dead staining were consistent with the results of the MTT experiment, further verifying that the hydrogel has excellent cell compatibility and does not affect cell viability during cell culture.

[0084] Test Example 2: TKNN x / ODex y ROS Scavenging and Antibacterial Tests of Hydrogel

[0085] (1) When there is an excessive amount of ROS in cells, it will cause the wound surface to remain in the inflammatory stage, and the wound is difficult to fully heal. Therefore, detecting the ROS content in cells can also reflect the inflammation level. First, the cytotoxicity of the hydrogels prepared in Example 1 and Example 2 against Raw 264.7 cells (mouse mononuclear macrophage leukemia cells) was detected. Raw 264.7 cells need to be cultured in serum-free DMEM high-glucose medium (purchased from Beijing Solarbio Science & Technology Co., Ltd.). The wells supplemented with complete medium were used as the control group, and the leachates of TKNN 1 / ODex 15 and TKNN 1.5 / ODex 18 hydrogels were used as the experimental groups. The experimental steps were carried out according to the L929 cell cytotoxicity test in (1) of Test Example 1. The experimental results are as shown in Figure 5As shown in (a), after co-incubating the hydrogel leachates with different compositions with Raw 264.7 cells for 48 h, the relative cell viability remained above 90%, indicating that the cell viability of Raw 264.7 cells was not affected during cell culture.

[0086] (2)An inflammatory environment was simulated by inducing Raw 264.7 cells with lipopolysaccharide (LPS), and the anti-inflammatory performance of the hydrogel was evaluated by studying the scavenging effect of the hydrogel on intracellular ROS. First, 1 mg of LPS was accurately weighed and added to 1 mL of sterile PBS to prepare a stock solution of 1 mg / mL. The solution was filtered through a 0.22 μm filter membrane to ensure sterility, and finally the stock solution was diluted to a 1 μg / mL LPS solution with serum-free high-glucose DMEM medium. Raw 264.7 cells in the logarithmic growth phase were selected and seeded into the well plate so that each well contained 10 5 cells, and they were cultured for 12 h to allow them to adhere completely; subsequently, the original old medium was replaced with 100 μL of serum-free high-glucose DMEM medium for starvation treatment for 12 h; then 1 μg / mL LPS solution (200 μL) was added for induction (i.e., the LPS group). The experimental group was the hydrogel leachate prepared by adding the hydrogels of Example 1 and Example 2, and the experimental group with serum-free high-glucose DMEM medium without LPS was used as the control group. All groups were incubated at 37 °C for 24 h; after the culture was completed, the supernatant was discarded, and the cells were washed 3 times with sterile PBS buffer, and then 5 μM of ROS fluorescent probe (DCFH-DA) solution was added and incubated at 37 °C in the dark for 30 min; finally, the cells were washed 2 times with sterile PBS buffer, and the fluorescence intensity in the cells was detected using a multifunctional microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0087] ROS scavenging rate (%) = ( A t - A 0 ) / A c × 100%;

[0088] where A t is the fluorescence intensity containing LPS and the hydrogel leachate; A 0 is the fluorescence intensity of only serum-free high-glucose DMEM medium; A c is the fluorescence intensity containing only LPS. The experimental results are as shown in Figure 5 (b). The induction of LPS caused a sudden increase in the intracellular ROS level, and its content reached about 3 times that of the normal group; after intervention with the hydrogel leachate, the expression of ROS content decreased significantly.

[0089] (3) In addition, to more intuitively observe the changes in intracellular ROS, the Raw 264.7 cell line was inoculated into a 6-well plate and cultured in the same manner as the above operation. After treatment with DCFH-DA, it was observed and photographed using an inverted fluorescence microscope. Figure 5 In (c), when only LPS treatment was performed (i.e., the LPS group), a large amount of ROS was generated in the cells at this time, and the cells showed strong green fluorescence expression; when intervened with the hydrogel leachate, an obvious fluorescence quenching phenomenon occurred. This is because a large number of hydroxyl groups in the hydrogel skeleton can scavenge a certain amount of ROS, having a certain antioxidant property, and can effectively solve the problem of excessive intracellular ROS.

[0090] (4) Staphylococcus aureus ( S.aureus , a Gram-positive bacterium) and Escherichia coli ( E.coli , a Gram-negative bacterium) were selected as model bacteria for the antibacterial experiment of the hydrogel. 50 mg of the hydrogels prepared in Example 1 and Example 2 (lyophilized, -50 °C, 12 h) were placed in the bacterial solution and co-cultured at 37 °C and 180 rpm for 24 h, and then the bacterial solution was diluted for plating; the bacterial solution without hydrogel intervention was used as the control group. The bacterial plates were placed in an incubator at 37 °C for 12 h, and the colony growth on the LB plates of each group was observed. Figure 5 (d) and Figure 5 (e) show that the hydrogels prepared in Example 2 have bactericidal rates of up to 75% and 93% against E.coli and S.aureus , indicating good antibacterial ability. Figure 5 (f) is a photo of the antibacterial experiment, showing that the number of bacterial colonies attached to the agar surface in the experimental group treated with the hydrogel is less, further verifying the good antibacterial activity of the prepared hydrogel.

[0091] Test Example 3: TKNN x (Cur-lip) / ODex y Degradability and sustained release of the hydrogel

[0092] (1) The TKNN 1 (Cur-lip) / ODex 15 hydrogels, TKNN 1.5 (Cur-lip) / ODex 18 hydrogels, and TKNN 1.5 (Cur-lip) / ODex 15 hydrogels prepared in Examples 3 to 5 were lyophilized (-50 °C, 12 h), then weighed and recorded as W 0, and then placed in a PBS (pH 7.4) buffer solution at 37 °C. Samples were taken at the set time points (1 min, 3 min, 5 min, 10 min, 30 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, and 5 h), and the excess solution on the surface was blotted with absorbent paper. Weigh again and record at this time as W s . Each experiment was performed in parallel three times. TKNN x (Cur-lip) / ODex y The swelling ratio (SR) of the hydrogel was calculated by the following formula:

[0093] SR (%) = W s / W 0 × 100%;

[0094] The experimental results are as shown in Figure 6 (a). All the prepared hydrogels could absorb a solution twice as heavy as their own weight, showing excellent swelling performance. The hydrogels rapidly absorbed water and swelled within the first hour and basically reached equilibrium after 2 hours. Moreover, the higher the content of TKNN or ODex, the lower the swelling ratio at equilibrium. Since the degree of crosslinking is higher and the internal structure becomes denser, the resistance to the passage of deionized water increases, resulting in a decrease in the swelling ratio. Although the swelling ratio decreased among the groups, they still maintained strong water absorption performance. They could still effectively absorb exudate on the wound bed and maintain a clean environment required for healing, thus playing a role in promoting wound healing.

[0095] (2) The TKNN x (Cur-lip) / ODex y hydrogels prepared in Examples 3 - 5 were freeze-dried and weighed, and recorded as W 0 . They were placed in 10 mL of PBS (pH 7.4) buffer solution and then placed in a thermostatic shaker (100 rpm, 37 °C). Samples were taken at the preset time points (5 min, 20 min, 1 h, 3 h, 4 h, 5 h, 6 h, and 7 h), freeze-dried, and weighed, and recorded as W s . The degradation rate (%) of the samples was calculated by the following formula:

[0096] Degradation rate (%) = W s / W 0 × 100%;

[0097] The experimental results are as shown in Figure 6As shown in (b), the degradation rate of the hydrogel is affected by the TKNN content. The lower the TKNN content, the weaker the crosslinking degree inside the gel, and the faster the degradation rate.

[0098] (3) Macroscopic experiments were carried out on TKNN x (Cur-lip) / ODex y The pH and ROS response properties of the hydrogel in vitro were tested. The pH response of the hydrogel was detected by dropping weak acidic and weak alkaline solutions to adjust the pH value of the system; by dropping H 2 O 2 The ROS response property of the hydrogel was tested by simulating the reactive oxygen environment at the wound site. The vial inversion method was used to observe whether the gel-sol transition occurred. As Figure 6 shown in (c), a certain amount of HCl solution (0.01 mol / L) was added to the prepared hydrogel and left standing at room temperature. The gel gradually transformed into a sol. This is because the dynamic hydrazone bond hydrolyzes in an acidic environment, and the gel skeleton collapses, resulting in the gel-to-sol transition; when the hydrochloric acid was neutralized with NaOH solution and placed at room temperature, the sample changed from a solution to a gel state again, that is, it was observed that it did not flow after being inverted for 30 s. This is because new bonds are formed in a weak alkaline environment for dynamic crosslinking, and the gel skeleton is restored. In addition, the ketothiol structure in TKNN has ROS responsiveness. After adding H 2 O 2 solution (200 mM), the ketothiol structure was destroyed and lost its crosslinking effect, and the gel gradually transformed into a sol.

[0099] (4) The TKNN x (Cur-lip) / ODex y hydrogels prepared in Examples 3 to 5 were respectively immersed in H 2 O 2 solutions with different concentrations (200 mM, 50 mM) or PBS solutions with different pH values (7.4, 5.0), then placed in a thermostatic shaker (100 rpm, 37 °C), and samples were taken at specific time points (10 min, 30 min, 1 h, 3 h, 5 h, 8 h, 12 h, 24 h, and 30 h). The release amount of Cur was determined by ultraviolet spectrophotometry. Figure 6 (d) shows that in 200 mM H 2 O 2 solution, the hydrogel has a greater cumulative drug release amount, and the lower the crosslinking degree, the higher the cumulative drug release amount. The total release amount of curcumin within 30 hours can reach up to 33.67%. Figure 6(e) The in vitro curcumin release behavior of the hydrogel in buffer solutions with different pH values (pH = 5.0, 7.4) was shown. Within 2 hours, there was an obvious drug burst release phenomenon in the drug-loaded (curcumin) hydrogels at different pH values. As time extended, the curcumin release tended to be stable. At the same time point, the cumulative curcumin release amount of the hydrogel was more in the environment with a lower pH value. The total curcumin release amount of the hydrogel was 33.91% in the environment with pH = 5.0 after 30 hours. The above results indicated that the hydrogel had excellent pH / ROS-responsive release ability, which was because the internal skeleton of the hydrogel formed by the cross-linking of hydrazone bonds and ketothiol structures was more easily destroyed in an acidic and ROS-enriched environment. After the gel skeleton was destroyed, the liposomes loaded with curcumin were released from inside the gel.

[0100] Test Example 4: TKNN x (Cur-lip) / ODex y In vitro antibacterial test of the hydrogel

[0101] 50 mg of TKNN prepared in Example 2 1.5 / ODex 18 and TKNN prepared in Example 4 1.5 (Cur-lip) / ODex 18 The hydrogels were freeze-dried and then placed in a bacterial solution of Escherichia coli or Staphylococcus aureus (concentration: 10 5 CFU / mL), and co-cultured at 37 °C and 180 rpm for 24 h. Then the bacterial solution was diluted for plating. The bacterial solution without hydrogel intervention was used as the control group. The bacterial plates were placed in an incubator at 37 °C for 12 h, and the colony growth on the LB plates of each group was observed. Figure 7 (a) showed that, compared with the control group, the hydrogel group showed good antibacterial ability against Escherichia coli, and the antibacterial ability of the TKNN 1.5 (Cur-lip) / ODex 18 hydrogel was higher, up to 90%. In addition, Figure 7 (b) showed that the hydrogels prepared in Example 2 and Example 4 also showed good antibacterial rates against Staphylococcus aureus, both higher than 90%. However, there was no significant difference in the antibacterial rates between the TKNN 1.5 / ODex 18 hydrogel and the TKNN 1.5 (Cur-lip) / ODex 18 hydrogel, and the TKNN 1.5 / ODex 18 hydrogel could also well inhibit Staphylococcus aureus. Figure 7 (c) was the photo of the antibacterial experiment, showing that after treatment with TKNN 1.5 / ODex 18 and TKNN1.5 (Cur-lip) / ODex 18 In the experimental group treated with the hydrogel, fewer bacterial colonies adhered to the surface of the agar, further verifying the good antibacterial activity of the prepared hydrogel.

[0102] Test Example 5: TKNN x (Cur-lip) / ODex y The hydrogel on mice S.aureus Wound healing test for infection

[0103] The model animals used in the experiment were all healthy 6-week-old female Kunming (KM) mice (purchased from Jinan Pengyue Laboratory Animal Breeding Co., Ltd.). Before the experiment started, all mice were adaptively fed for about 1 week and could eat and drink freely. The mice were anesthetized by intraperitoneal injection of sodium pentobarbital (0.9 mg / g), and a full-thickness wound with a diameter of 0.8 cm was established on the back of the mice and evenly smeared with Staphylococcus aureus bacterial solution. When yellow exudate flowed out from the wound, the infection was successful.

[0104] The mice were randomly divided into 6 groups, with 12 mice in each group. They were respectively treated with a commercial dressing (Tegaderm) (3M hydrocolloid dressing, Minnesota Mining and Manufacturing Co., Ltd. Taiwan, specification 90022T 10*10 cm) group, the TKNN 1.5 / ODex 18 hydrogel group prepared in Example 2, the TKNN 1.5 (Cur-lip) / ODex 18 hydrogel group prepared in Example 4. The wounds were treated with the hydrogels of each group, and the wound area was recorded. The untreated mice were used as the control group. The wound sites of the mice in each group were photographed on days 0, 3, 7, 11, and 14, and the changes in the wound size were observed. The ImageJ software was used to analyze the wound healing situation.

[0105] Wound healing rate (%) = ( S 0 - S t ) / S 0 × 100%;

[0106] where S 0 is the wound area size on day 0, S t is the wound area size observed within the predetermined time. The experimental results are as Figure 8 shown. Compared with other groups, TKNN 1.5 (Cur-lip) / ODex 18The wound healing rate of the hydrogel group was faster and almost completely healed on the 14th day. Quantitative analysis of the wound healing rate showed that the wound healing in the hydrogel group was better than that in the control group, indicating that the TKNN prepared by the present invention x (Cur-lip) / ODex y The hydrogel can effectively promote the healing of bacterially infected wounds.

[0107] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A multifunctional hydrogel, characterized in that: The multifunctional hydrogel is cross-linked by thioketal with hydrazide at both ends and oxidized dextran to form a cross-linked network containing dynamic covalent bonds, that is, a multifunctional hydrogel; The oxidized dextran is prepared by the following reaction: Dissolve dextran in water, add NaIO4 to carry out oxidation reaction, and obtain oxidized dextran; The content of thioketal with hydrazide at both ends in the multifunctional hydrogel is 5.0-7.5 mg / mL, and the content of oxidized dextran is 75-90 mg / mL; The thioketal hydrazide at both ends is prepared by the following method: (1) adding butyl 3-mercaptopropionate and anhydrous acetone to dioxane hydrochloride, and reacting under a protective atmosphere to obtain dibutylthiodipropionate; (2) Dibutylthiodipropionate reacts with hydrazine hydrate to obtain ketalthiohydride with hydrazide at both ends; The multifunctional hydrogel is prepared by the following method: Dissolving thioketal with hydrazide at both ends in deionized water to obtain a thioketal solution, dissolving oxidized dextran in deionized water to obtain an oxidized dextran solution, mixing the thioketal solution and the oxidized dextran solution, and allowing to stand to obtain a multifunctional hydrogel; the volume ratio of the thioketal solution to the oxidized dextran solution is 1:1; The oxidation degree of the oxidized dextran is 40-55%.

2. The multifunctional hydrogel according to claim 1, characterized in that The mass ratio of the dextran to NaIO4 is 5:6.56; the oxidation reaction time is 2 to 10 hours.

3. The multifunctional hydrogel according to claim 1, characterized in that The concentration of the thioketal solution is 10-15 mg / mL; the concentration of the oxidized dextran solution is 150-180 mg / mL.

4. Use of the multifunctional hydrogel according to any one of claims 1 to 3 in at least one of the following 1) to 4): 1) Encapsulation and sustained release of drugs; 2) Preparation of drugs or dressings for scavenging ROS; 3) Preparation of antimicrobial drugs or dressings; 4) Preparation of biodegradable drugs or dressings.

5. A drug for treating infected wounds, characterized in that: The drug is a dressing of the multifunctional hydrogel according to any one of claims 1 to 3, wherein the dressing contains liposomes encapsulating curcumin.

6. The drug according to claim 5, characterized in that The liposome encapsulating curcumin is prepared by the following method: (1) adding egg yolk lecithin and cholesterol into ethanol to dissolve to obtain a lipid solution, and adding curcumin into ethanol to obtain a curcumin solution; and uniformly mixing the lipid solution and the curcumin solution to obtain a mixed solution; (2) The mixed solution was dropped into a preheated phosphate buffer solution, stirred for reaction, and the ethanol was removed by rotary evaporation. The mixture was filtered through a 0.22 μm filter membrane to obtain liposomes encapsulating curcumin.

7. The drug according to claim 5, characterized in that The drug is prepared by the following method: The thioketal with hydrazide at both ends and the liposome encapsulating curcumin are dissolved in deionized water to obtain a drug-loaded solution, the oxidized dextran is dissolved in deionized water to obtain an oxidized dextran solution, the drug-loaded solution and the oxidized dextran solution are mixed, and the mixture is allowed to stand to obtain a drug for treating infected wounds.

Images