Multifunctional hydrogel and application thereof

Abstract

The invention discloses multifunctional hydrogel and application thereof, and belongs to the technical field of biological medicine. According to the multifunctional hydrogel, ketal mercaptan with two hydrazide ends and oxidized dextran are cross-linked to form a cross-linked network containing dynamic covalent bonds. The preparation method comprises the following steps: dissolving ketal mercaptan with two hydrazide ends in deionized water to obtain a ketal mercaptan solution, dissolving oxidized dextran in deionized water to obtain an oxidized dextran solution, mixing the ketal mercaptan solution with the oxidized dextran solution, and standing to obtain the multifunctional hydrogel. The multifunctional hydrogel has pH / ROS dual responsiveness, can respond to inflammation microenvironment and low pH of infectious wounds, remove active oxygen, release therapeutic drugs, regulate macrophage polarization and relieve bacterial infection, thereby playing a key role in inflammation, proliferation and remodeling stages of wound healing to accelerate wound healing. The curcumin-encapsulated liposome is added into the multifunctional hydrogel, so that the antibacterial effect can be further improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a multifunctional hydrogel and application thereof. Background Art

[0002] Skin is an important tissue organ of the human body and plays an important role in protecting the stability of the human body's internal environment. A series of problems caused by skin damage affect human health. In the process of wound healing, wound infections caused by bacteria, fungi and other microorganisms are seriously harmful, and the abuse of antibiotics has made wound infections more and more normalized. Therefore, it is of great significance to prepare wound dressings with high antibacterial ability, effectively prevent wound infection and promote wound healing.

[0003] The wound healing process is a complex physiological process, and restoring the barrier function of the wound involves many factors. Traditional wound dressings, such as gauze, sponges and bandages, have been widely used in clinical practice, but they usually cannot fully meet the multi-faceted requirements of the complex wound healing process, such as providing a moist environment and good adaptability to the wound shape. Therefore, multifunctional wound dressings are essential for providing a stable healing environment. With the development of the medical and health field and people's in-depth research on wound pathology and healing mechanisms, a large number of dressings have been studied and developed to target the healing of different wounds, and a variety of commercial products have been formed. At present, the 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, foam, fiber, hydrocolloid, alginate and hydrogel according to their own material properties. 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 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 clinical requirements for wound dressing performance. The development of multifunctional hydrogel wound dressings to meet the various needs of wound repair has become the mainstream trend in hydrogel dressing design. In addition, targeted wound repair materials should be used for different skin wound conditions to better promote wound healing. Therefore, it is still an important scientific issue to prepare hydrogel dressings with good mechanical properties, excellent biocompatibility, and inherent antibacterial and antioxidant properties through a simple and rapid method to effectively promote wound healing. 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 uses oxidized dextran and hydrazide-terminated thioketal to cross-link to obtain a hydrogel with dual pH / ROS response, which can respond to the inflammatory microenvironment and low pH of infected wounds, release therapeutic drugs to neutralize reactive oxygen, regulate macrophage polarization and reduce bacterial infection, thereby playing a key role in the inflammation, proliferation and remodeling stages of wound healing to accelerate wound healing.

[0005] To achieve the above object, the present invention adopts the following technical solutions: In a first aspect of the present invention, a multifunctional hydrogel is provided, wherein the multifunctional hydrogel is cross-linked with oxidized dextran by hydrazide-modified ketalthiols at both ends to form a cross-linked network containing dynamic covalent bonds, that is, the 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.

[0006] Preferably, the thioketal with 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) Dibutyl dipropionate thioketone reacts with hydrazine hydrate to obtain ketal thiohydride with hydrazide at both ends.

[0007] Preferably, the oxidation degree of the oxidized dextran is 40-55%.

[0008] Preferably, the mass ratio of dextran to NaIO4 is 5:6.56; and the oxidation reaction time is 2 to 10 hours.

[0009] Preferably, the multifunctional hydrogel is prepared by the following method: The thioketal with hydrazide at both ends is dissolved in deionized water to obtain a thioketal solution, the oxidized dextran is dissolved in deionized water to obtain an oxidized dextran solution, the thioketal solution and the oxidized dextran solution are mixed, and the multifunctional hydrogel is obtained after standing.

[0010] Preferably, the concentration of the thioketal solution is 10-15 mg / mL; the concentration of the oxidized dextran solution is 150-180 mg / mL; and the volume ratio of the thioketal solution to the oxidized dextran solution is 1:1.

[0011] The second aspect of the present invention provides the use of the multifunctional hydrogel in at least one of the following 1) to 5): 1) Encapsulation and sustained release of drugs; 2) Preparation of drugs or dressings for scavenging ROS; 3) Preparation of antimicrobial drugs or dressings; 4) Dual response to acidity and ROS; 5) Preparation of biodegradable drugs or dressings.

[0012] A third aspect of the present invention provides a medicine for treating infected wounds, wherein the medicine uses a multifunctional hydrogel as a dressing, and the dressing contains liposomes encapsulating curcumin.

[0013] Preferably, 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.

[0014] Preferably, 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.

[0015] Beneficial effects of the present invention: (1) The present invention cross-links dextran by oxidation and thioketal with hydrazide at both ends to obtain a hydrogel with dual pH / ROS response. The hydrogel can not only encapsulate drugs but also release drugs slowly. The hydrogel also has the effects of reducing ROS and antibacterial. It is a multifunctional hydrogel. It can be used in various ways such as injection and application.

[0016] (2) The present invention prepares a pH / ROS dual-responsive hydrogel dressing through a simple and rapid method. The hydrogel dressing can respond to the inflammatory microenvironment and low pH of infected wounds, release therapeutic drugs to neutralize reactive oxygen species, regulate macrophage polarization and alleviate bacterial infection, thereby playing a key role in the inflammation, proliferation and remodeling stages of wound healing to accelerate wound healing.

[0017] (3) The preparation method of the present invention is simple, and the prepared hydrogel has good biocompatibility, stable structure and performance. The drug prepared using the hydrogel has a hemolysis rate of less than 0.5% on red blood cells, has little effect on the vitality of L929 cells and RAW 264.7 cells, can effectively remove various types of reactive oxygen species, effectively kill bacteria, continuously inhibit inflammation, accelerate the formation of blood vessels, and promote wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : ODex, TKNN and TKNN x / ODex y Characterization diagram of hydrogel; (a) is the comparison diagram of H NMR spectra of ODex and dextran (hereinafter referred to as Dex); (b) is the infrared spectra of ODex and Dex; (c) is the H NMR 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 TKNN1 / ODex 15 Pictures of hydrogels; (g) freeze-dried TKNN1 / ODex at a scale of 200 μm 15 SEM image of hydrogel; (h) freeze-dried TKNN1 / ODex at 50 μm scale 15 SEM images of hydrogels; Figure 2 : Prepared liposomes and TKNN x (Cur-lip) / ODex y Characterization analysis diagram of hydrogel; (a) is the particle size distribution diagram of unloaded liposomes (hereinafter referred to as liposomes); (b) is the particle size distribution diagram of liposomes loaded with curcumin (hereinafter referred to as Cur-liposomes); (c) is the cryo-TEM image of unloaded liposomes (i.e., liposomes) at a scale of 100 μm; (d) is the cryo-TEM image of liposomes loaded with curcumin (i.e., Cur-liposomes) at a scale of 100 μm; (e) is the UV-visible absorption curve of curcumin (hereinafter referred to as Cur), liposomes and Cur-liposomes; (f) freeze-dried TKNN1 (Cur-lip) / ODex at a scale of 100 μm 15 SEM image of hydrogel; (g) freeze-dried TKNN at 100 μm scale 1.5 (Cur-lip) / ODex 15 SEM image of hydrogel; (h) freeze-dried TKNN at 100 μm scale 1.5 (Cur-lip) / ODex 18 SEM images of hydrogels; Figure 3 :TKNNx (Cur-lip) / ODex y Functional demonstration diagram of hydrogel; (a) is the injectable performance of hydrogel; (b) is the self-healing performance and healing principle of hydrogel; (c) is the adhesion of hydrogel to different mouse organs; (d) is the adhesion mechanism of hydrogel; Figure 4 :TKNN x / ODex y Biocompatibility of hydrogels; (a) is TKNN with different compositions x / ODex y Hemolysis rate of hydrogel; (b) L929 cells and different TKNN x / ODex y Cell survival rate after 24 h, 36 h, and 48 h of hydrogel co-culture; (c) is the TKNN with different compositions at a scale of 200 μm x / ODex y Cell live / dead / staining fluorescence images after hydrogel and L929 cells were co-incubated for 24 h, 36 h, and 48 h; Figure 5 :TKNN x / ODex y Hydrogel ROS removal and antibacterial test; (a) RAW 264.7 cells and TKNN x / ODex y Cell survival rate after 24 h of hydrogel co-incubation; (b) is TKNN x / ODex y The content of intracellular ROS after hydrogel intervention; (c) is TKNN at a scale of 200 μm x / ODex y Fluorescence image of cells after DCFH-DA staining after hydrogel intervention; (d) is the inhibition rate of Escherichia coli by gels of different compositions; (e) is the inhibition rate of Staphylococcus aureus by gels of different compositions; (f) is the survival graph of Escherichia coli and Staphylococcus aureus after treatment with different gels; Figure 6 :TKNN x (Cur-lip) / ODex y Swelling, degradation and response properties of hydrogels; (a) is the curve of swelling rate of hydrogels with different compositions changing with time; (b) is the graph of degradation test results of hydrogels with different compositions; (c) is the graph showing pH response and oxidation response of hydrogels; (d) is the curcumin release of hydrogels with different compositions in H2O2 solutions with different concentrations; (e) is the curcumin release of hydrogels with different compositions in buffer solutions with different pH values; Figure 7 :TKNN x (Cur-lip) / ODex y In vitro antibacterial test of hydrogels; (a) is the inhibition rate of gels with different compositions on Escherichia coli; (b) is the inhibition rate of gels with different compositions on Staphylococcus aureus; (c) is the survival chart of Escherichia coli and Staphylococcus aureus after treatment with different gels; Figure 8 :TKNN x (Cur-lip) / ODex y Study on the effect of hydrogel on wound healing of mice infected with Staphylococcus aureus; (a) is a graph of wound healing in mice after intervention with different hydrogels; (b) is a graph of wound healing rate after intervention with different hydrogels. DETAILED DESCRIPTION

[0019] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0020] As introduced in the background technology section, existing wound dressings can be divided into two categories, traditional dressings and new dressings. Among them, new dressings can be roughly divided into film, foam, fiber, hydrocolloid, alginate and hydrogel according to their own material properties. 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.

[0021] Based on this, the purpose of the present invention is to provide a multifunctional hydrogel and its application. The present invention cross-links oxidized dextran and thioketal with hydrazide at both ends, so that the amino group at the end of thioketal reacts with the aldehyde group formed by ring opening on the oxidized dextran to form a dynamic covalent bond, so that thioketal is grafted onto the oxidized dextran to form a cross-linked network to obtain a hydrogel. In addition to controlling the concentration of oxidized dextran and thioketal with hydrazide at both ends, the oxidation degree control of oxidized dextran is also one of the key factors for gelation. If the oxidation degree is too low, gelation cannot be achieved. If the oxidation degree is too high, dextran cannot play a role in the hydrogel. The inventor found that the oxidation degree of oxidized dextran needs to be controlled at 40~55%. The thioketal in the hydrogel can reduce ROS, thereby playing a role in removing ROS. In addition, the hydrogel also has an antibacterial effect. The present invention also provides a drug for treating infected wounds, wherein curcumin is encapsulated in liposomes and then loaded in hydrogels. The hydrogels can be applied to wounds in various ways such as injection or smearing. In the inflammatory environment and acidic environment of the wound, the ketalthiolate structure and dynamic covalent bonds of the hydrogel are gradually destroyed, and the hydrogel is transformed from a gel state to a sol state, thereby achieving 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, regulates macrophage polarization and reduces bacterial infection, thereby playing a key role in the inflammation, proliferation and remodeling stages of wound healing to accelerate wound healing.

[0022] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0023] Note: The oxidation degree of the oxidized dextran in the present invention = the molar amount of aldehyde groups in the oxidized dextran / the molar amount of hydroxyl groups in the dextran × 100%.

[0024] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0025] Example 1: Preparation of multifunctional hydrogel (1) Dissolve 5 g of dextran (MW = 40000Da) in 62.5 mL of water. After complete dissolution, add 6.56 g of NaIO4 and stir for 6 h in the dark. Then add 2 mL of ethylene glycol and stir for 1 h to terminate the reaction. After dialysis in pure water for 4 days, freeze-dry to obtain a white product, which is oxidized dextran (ODex), with an oxidation degree of about 50%.

[0026] (2) Add butyl 3-mercaptopropionate (8.00 g) and anhydrous acetone (6.00 g) to a flask with dioxane hydrochloride as solvent and react at room temperature for 15 h under N2 protection. After removing the solvent under reduced pressure, dilute the residue with ether and transfer it to a separatory funnel. Extract it with saturated NaHCO3 solution and water in sequence, and retain the organic phase. Dry it with anhydrous NaSO4 for 24 h and then rotary evaporate it to obtain dibutyl propionate as a light amber oil.

[0027] Dissolve dibutylthiodipropionate (5.00 g) in methanol (100 mL), add hydrazine hydrate (6.30 g), stir at room temperature for 18 h, remove the solvent by rotary evaporation, dilute with water and transfer to a separatory funnel, extract twice with anhydrous ether, retain the aqueous phase, and remove water by rotary evaporation to obtain a white solid product, that is, thioketal (TKNN) with hydrazide at both ends.

[0028] (3) Accurately weigh TKNN and dissolve it in deionized water to obtain a TKNN precursor solution with a concentration of 10 mg / mL (x = TKNN concentration / 10, when TKNN concentration is 10 mg / mL, x = 1.0); accurately weigh ODex and dissolve it in deionized water to obtain an ODex precursor solution with a concentration of 150 mg / mL (y = ODex concentration / 10, when ODex concentration is 150 mg / mL, y = 15); mix the above two precursor solutions in a volume ratio of 1:1, and after standing for a few seconds, a multifunctional hydrogel is obtained, which is recorded as TKNN1 / ODex. 15 Hydrogel.

[0029] Example 2 The difference from Example 1 is that in step (3), the concentration of TKNN precursor solution is 15 mg / mL (x= 1.5); the concentration of ODex is 180 mg / mL (y=18). The multifunctional hydrogel finally prepared is denoted as TKNN 1.5 / ODex 18 Hydrogel.

[0030] Example 3: Preparation of a drug for treating infected wounds (1) Accurately weigh 50 mg of egg yolk lecithin and 12.5 mg of cholesterol, and add an appropriate amount of ethanol to dissolve. Dissolve curcumin in ethanol to prepare a 1 mg / mL curcumin solution, measure 2 mL of the curcumin solution, add it to the above solution and mix well to obtain a mixed solution. Add the mixed solution dropwise (1-2 drops / second) to a 60°C phosphate buffer solution (pH 7.4), stir for 1 hour, then remove the ethanol by rotary evaporation, and filter through a 0.22 μm filter membrane to obtain a liposome solution encapsulating curcumin (Cur-liposome).

[0031] (2) Accurately weigh 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); accurately weigh 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 after standing for a few seconds, the drug for treating infected wounds is obtained, which is recorded as TKNN1(Cur-lip) / ODex 15 Hydrogel.

[0032] Example 4 The difference from Example 3 is that in step (2), a TKNN precursor solution with a concentration of 15 mg / mL is obtained (x=1.5); and an ODex precursor solution with a concentration of 180 mg / mL is obtained (y=18). Finally, a drug for treating infected wounds is prepared, which is denoted as TKNN. 1.5 (Cur-lip) / ODex 18 Hydrogel.

[0033] Example 5 The difference from Example 3 is that in step (2), a TKNN precursor solution with a concentration of 15 mg / mL is obtained (x=1.5); and an ODex precursor solution with a concentration of 150 mg / mL is obtained (y=15). Finally, a drug for treating infected wounds is prepared, which is denoted as TKNN. 1.5 (Cur-lip) / ODex 15 Hydrogel.

[0034] Example 6: TKNN1 / ODex prepared in Example 1 15 Characterization of hydrogels The nuclear magnetic resonance spectroscopy and infrared spectroscopy confirmed that the dextran was successfully oxidized and ring-opened after the reaction. Figure 1 ,from Figure 1 (a) 1 It can be seen from the H NMR spectrum that after the oxidation reaction, the peak at 4.9 ppm disappears, proving that the ortho-hydroxyl group of dextran is oxidized. Figure 1 From the FT-IR spectrum of (b), we can see that compared with Dex, ODex has a -1 The stretching vibration peak of C=O appeared at , which further proved that the dextran was successfully oxidized to aldehyde. The oxidation degree of ODex was 51.3% measured by hydroxylamine hydrochloride titration.

[0035] The molecular structure of TKNN was determined by NMR hydrogen spectrum and infrared spectrum. The experimental results are as follows Figure 1 (c) and Figure 1 As shown in (d), after comparison, TKNN was successfully synthesized.

[0036] Figure 1 (e) ODex, TKNN and freeze-dried (-50 ℃, 12 h) TKNN1 / ODex 15 FT-IR spectrum of hydrogel. The hydrogel sample has a wavelength of 1658 cm -1 A new absorption peak appears at . This absorption peak is attributed to the characteristic absorption of the acylhydrazone bond (-N=C-) formed between -NH-NH2 on the TKNN molecular chain and -CHO of ODex, further indicating that the hydrogel network was successfully constructed.

[0037] Figure 1 (f) Precursor solution and TKNN1 / ODex 15 Image of hydrogel. TKNN precursor solution and ODex precursor solution can quickly form a gel after mixing equal volumes.

[0038] Figure 1 (g) and Figure 1 (h) Lyophilized TKNN1 / ODex 15 The SEM image of the hydrogel shows a loose and porous structure inside the gel. This structure is conducive to the exchange of substances between the hydrogel and the wound surface and facilitates the absorption of exudate from the wound surface to maintain a clean healing environment for the wound.

[0039] Example 7: TKNN prepared in Examples 3 to 5 x (Cur-lip) / ODex y Characterization of hydrogels Figure 2 (a) is the particle size test result of unloaded liposomes, with an average diameter of 124.567±3.244 nm. Figure 2 (b) is the particle size test result 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%.

[0040] Figure 2 (c) and Figure 2 (d) TEM images of liposome and Cur-liposome, respectively. It can be seen that the prepared liposomes are spherical or quasi-spherical structures.

[0041] Figure 2 (e) is the UV-visible absorption spectra of Cur (curcumin), liposome and Cur-liposome. The absorption peak of curcumin appears at 427nm in the Cur-liposome spectrum, indicating that curcumin is successfully encapsulated into the liposome.

[0042] Figure 2 (f) - Figure 2 (g) TKNN prepared in Examples 3 to 5 x (Cur-lip) / ODex y The SEM images of the hydrogel (freeze-dried, -50 °C, 12 h) show that the gel has a loose and porous structure. As the ODex content and TKNN content in the hydrogel increase, the internal structure of the hydrogel becomes more compact. This is because as the aldehyde groups in the system increase, the degree of cross-linking in the hydrogel increases, resulting in a denser skeleton structure inside the hydrogel.

[0043] Example 8: TKNN1 (Cur-lip) / ODex prepared in Example 3 15 Characterization of hydrogel injectability, self-healing and adhesion properties According to the method of step (3) of Example 3, a trace amount of rhodamine B dye was added to the ODex precursor solution, vortexed with the TKNN (Cur-lip) solution to form a gel, and then evenly loaded into a syringe and injected in water to observe whether it can be injected continuously, so as to evaluate the injectability of the hydrogel. Figure 3 As shown in (a), the hydrogel has good injectability. The newly prepared hydrogel was dyed with methylene blue and rhodamine B dyes respectively, and the two dyed gels were evenly separated with a knife. The two cross sections were then contacted to observe whether the hydrogel can be picked up with tweezers without breaking to evaluate the self-healing performance of the hydrogel. The results are shown in Figure 3 As shown in (b), the hydrogel can heal after one 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 hydrogen bond interaction between molecules. Dextran, as a natural polysaccharide, has excellent viscosity. In addition, the presence of liposomes also increases the adhesion ability of the hydrogel. Figure 3 (c) Demonstrates that the hydrogel has good adhesion ability to various biomaterials such as mouse organs. Figure 3 (d) is the adhesion mechanism. There are a large number of hydroxyl groups on the dextran molecules, which have strong hydrogen bond interactions with the hydroxyl and amino groups on the tissue surface, which is equivalent to forming a physical cross-link between the hydrogel and the tissue surface.

[0044] Test Example 1: TKNN x / ODex y Biocompatibility test of hydrogel (1) The blood compatibility of the hydrogel can be evaluated by an in vitro hemolysis experiment. The experiment was divided into four groups: PBS as a negative control group, distilled water as a positive control group, and TKNN1 / ODex prepared in Example 1 as a positive control group. 15 Hydrogel and TKNN prepared in Example 2 1.5 / ODex 18The hydrogels in the experimental group were freeze-dried at -50 °C for 12 h, and each group was tested three times in parallel.

[0045] Weigh 50 mg of the experimental group's material and place it in 5 mL of PBS buffer, incubate at 37 °C for 24 h to obtain the hydrogel extract. Draw fresh blood from the mouse from the anticoagulant tube, centrifuge at 3000 rpm for 15 min, discard the supernatant, resuspend the blood cells with sterile saline, repeat the resuspension and centrifugation operation several times until the supernatant becomes clear and stop. The blood cells are then diluted with saline to a 5% blood cell suspension for later use. Finally, the blood cell suspension and the hydrogel extract were mixed in equal volumes, incubated at 37 °C for 2 h, centrifuged to obtain 200 µL of the supernatant, and the OD value was measured at 540 nm by an enzyme marker.

[0046] Hemolysis rate (%) = A s - A p ) / ( A t - A p )×100%; in A s The OD value was measured for the gel extract of the experimental group; A p OD value measured for the negative control group; A t is the OD value measured in the positive control group. Figure 4 As shown in (a), the illustrations in the figure are the positive control group (distilled water), the negative control group (PBS), TKNN1 / ODex 15 Hydrogel, TKNN 1.5 / ODex 18 Photos of the hydrogel group after incubation with red blood cell suspension. It was observed that the positive control group showed obvious red color, that is, the red blood cells were affected by the internal and external osmotic pressure and hemolysis occurred, while the upper layer of the negative control and hydrogel groups was light yellow, and there was red blood cell precipitation in the lower layer. All hydrogel groups showed slight hemolysis, which was 0.12% and 0.07% respectively, both lower than 5%, which met the national material biosafety standards. The above results show that the hydrogels all show good blood compatibility.

[0047] (2) Cell viability was assessed using the MTT method. First, L929 cells (mouse fibroblasts) in the logarithmic growth phase were selected for digestion, counting, and dilution. The diluted cell suspension was added to a 96-well plate and cultured for 24 h. The freeze-dried gel (TKNN1 / ODex prepared in Example 1) was 15 Hydrogel and TKNN prepared in Example 21.5 / ODex 18 The hydrogel was placed in MEM complete medium and placed in an incubator for 24 h to obtain a hydrogel extract. The old medium was discarded, washed three times with sterile PBS buffer and replaced with the hydrogel extract. The wells with MEM complete medium were used as the control group. The plate was placed in an incubator for 24 h, 36 h, and 48 h. When the predetermined culture time was reached, the hydrogel extract was removed and washed three times with PBS buffer. Then 100 µL of MEM complete medium was added to each well and 20 µL of MTT solution was added under light-proof conditions. After incubation in the incubator for 4 h, the supernatant was discarded. Then 100 µL of DMSO solution was added to each well. After the plate was vibrated for 10 min in a light-proof environment, the OD value was detected at 490 nm using an enzyme reader. Each group of experiments was performed in parallel three times.

[0048] Cytotoxicity (%) = ( A s - A 0 ) / ( A c - A 0 )×100%; in, A s is the absorbance value after incubation with hydrogel extract; A c is the absorbance value after culture in MEM medium; A 0 is the absorbance value of PBS. Figure 4 As shown in (b), after the hydrogel extract prepared by the hydrogels of Example 1 and Example 2 was incubated with L929 cells for 48 hours, it was found that the relative cell survival rate was still above 90%, indicating that the hydrogel met the international material use standards during use.

[0049] (3) Live / dead cell staining experiment was used to further evaluate the biocompatibility of the hydrogel. First, L929 cells in the logarithmic growth phase were inoculated into a 6-well plate. After they were completely attached to the wall, the old culture medium was replaced with the hydrogel extract prepared from the hydrogels of Example 1 and Example 2. The control group was still cultured with complete culture medium. Then, the cells were placed in an incubator and cultured for 24 h, 36 h, and 48 h. When the preset culture time was reached, they were washed three times with sterile PBS buffer, and then the cell live / dead staining working solution was added and incubated in the dark for 30 min. The cells were then washed with PBS buffer and observed and imaged using an inverted fluorescence microscope. The experimental results are shown in Figure 2. Figure 4(c) As shown. It was observed that there were a large number of live cells showing green fluorescence in each group, and only a few dead cells showing red fluorescence. The live / dead staining results of cells were consistent with the results of the MTT experiment, which once again verified that the hydrogel had excellent cell compatibility and would not affect cell viability during cell culture.

[0050] Test Example 2: TKNN x / ODex y Hydrogel ROS scavenging and antibacterial assays (1) When there is an excessive amount of ROS in the cells, the wound surface will remain in the inflammatory stage, and the wound will be difficult to fully heal. Therefore, detecting the ROS content in the cells can also reflect the level of inflammation. First, the cytotoxicity of the hydrogels prepared in Example 1 and Example 2 to 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 Solebow Technology Co., Ltd.). The wells with complete culture medium were used as the control group, and TKNN1 / ODex 15 and TKNN 1.5 / ODex 18 The hydrogel extract was used as the experimental group, and the experimental steps were carried out according to the L929 cell toxicity test in Experimental Example 1 (1). The experimental results are shown in Figure 5 As shown in (a), after incubating Raw 264.7 cells with hydrogel extracts of different compositions for 48 h, the relative survival rate of the cells was still above 90%, indicating that the cell viability of Raw 264.7 cells was not affected during the cell culture process.

[0051] (2) Raw 264.7 cells were induced by lipopolysaccharide (LPS) to simulate the inflammatory environment, and the anti-inflammatory properties of the hydrogel were 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 1 mg / mL stock solution. The solution was filtered through a 0.22 μm filter to ensure sterility, and finally the stock solution was diluted to a 1 μg / mL LPS solution using serum-free DMEM high-glucose medium. Raw 264.7 cells in the logarithmic growth phase were selected and inoculated into the well plate so that each well contained 10 5cells, cultured for 12 h to allow them to completely adhere to the wall; then, the original old culture medium was replaced with 100 μL serum-free DMEM high-glucose culture medium for starvation treatment for 12 h; then 1 μg / mL LPS solution (200 μL) was added for induction (i.e., LPS group), the experimental group was the hydrogel extract prepared by adding the hydrogels of Example 1 and Example 2, and the experimental group with serum-free DMEM high-glucose culture medium without LPS was used as the control group, and all groups were incubated normally at 37°C for 24 h; after the culture was completed, the supernatant was discarded, and the cells were washed three times with sterile PBS buffer, and then 5 μM ROS fluorescent probe (DCFH-DA) solution was added, and incubated at 37°C in the dark for 30 min; finally, the cells were washed twice 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.

[0052] ROS clearance rate (%) = ( A t - A 0 ) / A c ×100%; in, A t is the fluorescence intensity of the leachate containing LPS and hydrogel; A 0 is the fluorescence intensity of serum-free DMEM high-glucose medium only; A c is the fluorescence intensity of LPS only. Figure 5 As shown in (b), LPS induction caused a sharp increase in the level of ROS in the cells, and its content reached about 3 times that of the normal group; after intervention with gel extract, the expression of ROS content was significantly reduced.

[0053] (3) In addition, in order 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 above. After being treated with DCFH-DA, the cells were observed using an inverted fluorescence microscope and photographed. Figure 5 In (c), when only LPS was treated (i.e., LPS group), a large amount of ROS was produced in the cells, and the cells showed strong green fluorescence expression; when the hydrogel extract was used for intervention, obvious fluorescence quenching occurred. This is because the large amount of hydroxyl groups in the hydrogel skeleton can remove certain ROS, have certain antioxidant properties, and can effectively solve the problem of excessive ROS in cells.

[0054] (4) Select Staphylococcus aureus ( S. aureus , Gram-positive species) and Escherichia coli ( E. coli, Gram-negative bacteria) as the model bacteria for the hydrogel antibacterial experiment. 50 mg of the hydrogel prepared in Example 1 and Example 2 (lyophilized, -50 ℃, 12 h) was placed in the bacterial solution and cultured at 37 ℃ 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 plate was placed in a 37 ℃ incubator for 12 h, and the colony growth on the LB plate of each group was observed. Figure 5 (d) and Figure 5 (e) shows that the hydrogel prepared in Example 2 E. coli and S. aureus The bactericidal rates can reach 75% and 93%, indicating good antibacterial ability. Figure 5 (f) is a photo of the antibacterial experiment, showing that in the experimental group treated with hydrogel, the number of bacterial colonies attached to the agar surface is even smaller, further verifying the good antibacterial activity of the prepared hydrogel.

[0055] Test Example 3: TKNN x (Cur-lip) / ODex y Degradability and sustained release of hydrogels (1) TKNN1 (Cur-lip) / ODex prepared in Examples 3 to 5 15 Hydrogel, TKNN 1.5 (Cur-lip) / ODex 18 Hydrogel, TKNN 1.5 (Cur-lip) / ODex 15 The hydrogel was freeze-dried (-50 °C, 12 h) and then weighed and recorded as W 0 Then, the samples were placed in a PBS (pH 7.4) buffer solution at 37 °C. The samples were taken out at 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). The excess solution on the surface was wiped off with absorbent paper and the samples were weighed again. The time at this time was recorded as W s Each experiment was performed in parallel three times. x (Cur-lip) / ODex y The hydrogel swelling ratio (SR) was calculated by the following formula: SR(%)= W s / W 0 ×100%; The experimental results are as follows Figure 6As shown in (a), the prepared hydrogels can absorb solutions that are twice heavier than their own weight, showing excellent swelling properties. 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 rate of the gel at equilibrium. As the degree of cross-linking increases and the internal structure becomes denser, the resistance to the passage of deionized water increases, resulting in a decrease in the swelling rate. Although the swelling rate between the groups has decreased, it can still maintain a strong water absorption performance. It can still effectively absorb exudate on the wound bed and maintain the clean environment required for healing, thereby promoting wound healing.

[0056] (2) The TKNN prepared in Examples 3 to 5 x (Cur-lip) / ODex y The hydrogel was freeze-dried and weighed, and recorded as W 0 . Place in 10 mL of PBS (pH 7.4) buffer and then place in a constant temperature shaker (100 rpm, 37 °C). Take out the samples for freeze drying and weigh at the preset time points (5 min, 20 min, 1 h, 3 h, 4 h, 5 h, 6 h and 7 h) and record them as W s The degradation rate (%) of the sample was calculated by the following formula: Degradation rate (%) = W s / W 0 ×100%; The experimental results are as follows Figure 6 As shown in (b), the degradation rate of the hydrogel is affected by the TKNN content. The lower the TKNN content, the weaker the internal cross-linking of the gel and the faster the degradation rate.

[0057] (3) TKNN through macro experiments x (Cur-lip) / ODex y The pH and ROS response performance of the hydrogel in vitro were tested. The pH value of the system was adjusted by dripping weak acidic solution and weak alkaline solution to test the pH responsiveness of the hydrogel; the ROS response performance of the hydrogel was tested by dripping H2O2 to simulate the active oxygen environment at the wound site. The vial inversion method was used to observe whether the gel-sol transition occurred. Figure 6As shown in (c), a certain amount of HCl solution (0.01 mol / L) was added to the prepared hydrogel, and the gel gradually transformed into a sol when it was left standing at room temperature. This is because the dynamic acylhydrazone bond was hydrolyzed in an acidic environment, and the gel skeleton collapsed, resulting in a transition from gel to sol; when the hydrochloric acid was neutralized with NaOH solution and placed at room temperature, the sample again transformed from a solution to a gel state, that is, it was observed that it did not flow when inverted for 30 seconds. This is because the bonds were re-formed in a weakly alkaline environment, dynamic cross-linking was performed, and the gel skeleton was restored. In addition, the ketalthiolate structure in TKNN is ROS-responsive. After adding H2O2 solution (200 mM), the ketalthiolate structure was destroyed and lost the cross-linking effect, and the gel gradually transformed into a sol.

[0058] (4) The TKNN prepared in Examples 3 to 5 x (Cur-lip) / ODex y The hydrogels were immersed in H2O2 solutions of different concentrations (200 mM, 50 mM) or PBS solutions of different pH values ​​(7.4, 5.0), and then placed in a constant temperature 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 of Cur was determined by UV spectrophotometry. Figure 6 (d) shows that in 200 mM H2O2 solution, the hydrogel has a larger cumulative drug release, and the lower the cross-linking degree, the higher the cumulative drug release, and the total curcumin release can reach up to 33.67% within 30 hours. Figure 6 (e) shows the in vitro curcumin release behavior of the hydrogel in buffer solutions with different pH values ​​(pH=5.0, 7.4). Within 2 hours, the drug-loaded (curcumin) hydrogels at different pH values ​​all showed obvious drug burst release. As time went on, the curcumin release tended to be stable. At the same time point, the lower the pH value, the more the cumulative curcumin release of the hydrogel. The total curcumin release of the hydrogel at pH=5.0 for 30 hours was 33.91%. The above results show that the hydrogel has excellent pH / ROS responsive release ability, which is because the internal skeleton of the hydrogel formed by cross-linking of acylhydrazone bonds and ketalthiols is more easily destroyed in an acidic environment rich in ROS. After the gel skeleton is destroyed, the curcumin-loaded liposomes are released from the inside of the gel.

[0059] Test Example 4: TKNN x (Cur-lip) / ODex y In vitro antibacterial test of hydrogel 50 mg of TKNN prepared in Example 2 1.5 / ODex 18 and TKNN prepared in Example 41.5 (Cur-lip) / ODex 18 The hydrogel was freeze-dried and then placed in a bacterial solution of Escherichia coli or Staphylococcus aureus (concentration of 10 5 CFU / mL) and co-cultured at 37°C and 180 rpm for 24 h, then the bacterial solution was diluted and plated. The bacterial solution without hydrogel intervention was used as the control group. The bacterial plate was placed in a 37°C incubator for 12 h, and the growth of colonies on the LB plate of each group was observed. Figure 7 (a) shows that compared with the control group, the hydrogel group showed good antibacterial ability against Escherichia coli, and TKNN 1.5 (Cur-lip) / ODex 18 The antibacterial ability of hydrogel is higher, up to 90%. In addition, Figure 7 (b) It can be seen that the hydrogels prepared in Example 2 and Example 4 also showed good antibacterial rates against Staphylococcus aureus, both higher than 90%. 1.5 / ODex 18 Hydrogel and TKNN 1.5 (Cur-lip) / ODex 18 There was no significant difference in the antibacterial rate of hydrogels. 1.5 / ODex 18 The hydrogel can also effectively inhibit Staphylococcus aureus. Figure 7 (c) is a photo of the antibacterial experiment, showing that the 1.5 / ODex 18 and TKNN 1.5 (Cur-lip) / ODex 18 In the experimental group treated with hydrogel, the number of bacterial colonies attached to the agar surface was even smaller, further verifying the good antibacterial activity of the prepared hydrogel.

[0060] Test Example 5: TKNN x (Cur-lip) / ODex y Hydrogel for mice S. aureus Infected wound healing assay The model animals used in the experiment were healthy 6-week-old female Kunming (KM) mice (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.). Before the experiment, all mice were adaptively raised for about 1 week and were free to eat and drink. 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 mouse and Staphylococcus aureus liquid was evenly applied. The infection was successful when yellow exudate flowed out of the wound.

[0061] The mice were randomly divided into 6 groups, with 12 mice in each group. The commercial dressing (Tegaderm) (3M hydrophilic colloid dressing, Minnesota Mining Manufacturing Co., Ltd., Taiwan, specification 90022T 10*10 cm) group, TKNN prepared in Example 2 group, and the TKNN prepared in Example 2 group were used respectively. 1.5 / ODex 18 Hydrogel group, TKNN prepared in Example 4 1.5 (Cur-lip) / ODex 18 In the hydrogel group, the wound was treated with each group of hydrogels, and the wound area was recorded. The untreated mice served as the control group. On days 0, 3, 7, 11, and 14, the wound sites of the mice in each group were photographed and the changes in wound size were observed. The wound healing was analyzed using ImageJ software.

[0062] Wound healing rate (%) = ( S 0 - S t ) / S 0 ×100%; in S 0 is the wound area size on day 0, S t is the size of the wound area observed within a predetermined time. Figure 8 As shown, compared with other groups, TKNN 1.5 (Cur-lip) / ODex 18 The 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 of the hydrogel group was better than that of the control group, indicating that the TKNN prepared by the present invention x (Cur-lip) / ODex y Hydrogels can effectively promote the healing of bacterially infected wounds.

[0063] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. 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.

2. The multifunctional hydrogel according to claim 1, characterized in that 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) Dibutyl dipropionate thioketone reacts with hydrazine hydrate to obtain ketal thiohydride with hydrazide at both ends.

3. The multifunctional hydrogel according to claim 1, characterized in that The oxidation degree of the oxidized dextran is 40-55%.

4. 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.

5. The multifunctional hydrogel according to claim 1, characterized in that: The multifunctional hydrogel is prepared by the following method: The thioketal with hydrazide at both ends is dissolved in deionized water to obtain a thioketal solution, the oxidized dextran is dissolved in deionized water to obtain an oxidized dextran solution, the thioketal solution and the oxidized dextran solution are mixed, and the multifunctional hydrogel is obtained after standing.

6. The multifunctional hydrogel according to claim 5, 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; and the volume ratio of the thioketal solution to the oxidized dextran solution is 1:

1.

7. Use of the multifunctional hydrogel according to any one of claims 1 to 6 in at least one of the following 1) to 5): 1) Encapsulation and sustained release of drugs; 2) Preparation of drugs or dressings for scavenging ROS; 3) Preparation of antimicrobial drugs or dressings; 4) Dual response to acidity and ROS; 5) Preparation of biodegradable drugs or dressings.

8. 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 6, wherein the dressing contains liposomes encapsulating curcumin.

9. The drug according to claim 8, 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.

10. The drug according to claim 8, 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.

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