Fiber membrane composite hydrogel with antibacterial and antioxidant dual effects as well as preparation and application of fiber membrane composite hydrogel

Abstract

The invention relates to fiber membrane composite hydrogel with antibacterial and antioxidant dual effects and preparation and application of the fiber membrane composite hydrogel. Invasion of external pathogens can be blocked through a hydrophobic nanofiber membrane, and a moist environment of a wound surface is maintained by utilizing the hydrogel; meanwhile, the fiber membrane loaded with the gold nanorods and the hydrogel loaded with the luteolin can provide photo-thermal antibacterial and antioxidant functions respectively, the synergistic effect of promoting chronic wound healing is achieved, and a novel treatment scheme can be provided for chronic wounds.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a fiber membrane composite hydrogel with dual antibacterial and antioxidant effects, and the preparation and application of the hydrogel. Background Art

[0002] Chronic wounds such as those caused by diabetes are susceptible to bacterial infection and overexpression of reactive oxygen species (ROS), which leads to a slow healing process and a serious threat to the health of patients. Traditional dressings can only physically isolate the wound surface and cannot effectively inhibit bacterial infection and inflammatory response. Therefore, the treatment of chronic wounds requires more intelligent new dressings that can simultaneously meet the dual functional requirements of antibacterial and antioxidant. Photothermal materials can convert light energy into thermal energy and kill bacteria through local high temperature, and have become an emerging means of photothermal therapy. Natural flavonoids have good ROS scavenging ability and can exert anti-inflammatory and antioxidant functions. At the same time, they have attracted widespread attention in the medical community due to their good biocompatibility. At the same time, hydrogels with ROS-responsive properties are expected to achieve on-demand drug release. In recent years, the incidence of diabetes has increased year by year. The development of a safe and efficient antibacterial and antioxidant multifunctional dressing has become an urgent need that needs to be addressed in the field of medical materials. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a fiber membrane composite hydrogel with dual antibacterial and antioxidant effects, as well as its preparation and application.

[0004] The present invention provides a method for preparing a fiber membrane composite hydrogel, comprising:

[0005] (1) mixing a phenylboronic acid grafted polymer solution and a phenolic hydroxyl group-containing drug solution to obtain a precursor;

[0006] (2) Placing a polymer nanofiber membrane loaded with gold nanorods on the surface of the precursor and allowing it to stand to obtain a fiber membrane composite hydrogel.

[0007] In the step (1), the phenylboronic acid grafted polymer is one or more of phenylboronic acid grafted hyaluronic acid and phenylboronic acid grafted sodium alginate.

[0008] The preparation of the phenylboronic acid grafted polymer includes: dissolving the polymer in a morpholineethanesulfonic acid buffer solution, continuously stirring until completely dissolved, then adding a carboxyl activator 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride, stirring to activate the carboxyl group, then adding 3-aminophenylboronic acid to perform a coupling reaction; dialyzing, freeze-drying, and obtaining the phenylboronic acid grafted polymer; wherein the polymer includes one or more of hyaluronic acid and sodium alginate.

[0009] The solvent of the phenylboronic acid grafted polymer solution in step (1) is one of deionized water and PBS buffer.

[0010] The phenolic hydroxyl-containing drug in step (1) is a flavonoid drug; wherein the flavonoid drug is one or more of luteolin, quercetin, and morusin;

[0011] The solvent of the phenolic hydroxyl group-containing drug solution is one of deionized water and PBS buffer.

[0012] The concentration of the phenylboronic acid grafted polymer solution in step (1) is 0.5-2.5% (w / v); the concentration of the phenolic hydroxyl group-containing drug in the precursor is 0-2 mg / mL, and further, the concentration is 0.5-2 mg / mL.

[0013] In the step (1), the volume ratio of the phenylboronic acid grafted polymer solution to the phenolic hydroxyl group-containing drug solution is 2:1 to 1:2.

[0014] The preparation of the gold nanorod-loaded polymer nanofiber membrane in step (2) comprises mixing a polymer, an organic solvent, and gold nanorods to obtain a spinning solution, and electrospinning to obtain the gold nanorod-loaded polymer nanofiber membrane.

[0015] The electrospinning process parameters include: the spinning solution is placed in a syringe, the injection speed is 0.5-2 mL / h, the spinning voltage is 8-16 kV, the receiving distance is 10-20 cm, and the electrospun nanofiber membrane is vacuum dried.

[0016] Furthermore, the spinning solution is prepared by mixing the polymer and the organic solvent, stirring for 4-12 hours, then adding the gold nanorods, ultrasonically dispersing for 10-30 minutes, and stirring for 1-3 hours.

[0017] The polymer is a high molecular polymer, wherein the high molecular polymer is one or more of polylactic acid, polyglycolic acid, and polylactic acid-glycolic acid copolymer; the organic solvent is one or more of hexafluoroisopropanol, chloroform, dichloromethane, and NN dimethylformamide.

[0018] The concentration of the polymer in the spinning solution is 10-20% (w / v); the gold nanorods account for 0.1-0.5 wt% of the mass of the polymer.

[0019] The step (2) is to stand at room temperature for 10-60 minutes.

[0020] The invention provides a fiber membrane composite hydrogel prepared by the method. The fiber membrane composite hydrogel consists of an upper layer of polymer nanofiber membrane loaded with gold nanorods and a lower layer of drug-loaded hydrogel, wherein the lower layer of drug-loaded hydrogel is a skin-attaching layer.

[0021] The fiber membrane composite hydrogel has the dual effects of antibiosis and antioxidation.

[0022] The application provides application of the fiber membrane composite hydrogel in preparation of a chronic wound repair dressing.

[0023] Note: w / v in the application is g / mL.

[0024] The fiber membrane composite hydrogel has the dual effects of antibiosis and antioxidation. The composite hydrogel is composed of an upper electrospun nanofiber membrane and a lower drug-loaded hydrogel, wherein the nanofiber membrane is loaded with gold nanorods, and the hydrogel is loaded with luteolin with antioxidation activity. The final fiber membrane composite hydrogel can realize the synergistic effect of antibiosis and antioxidation, accelerate the chronic wound healing process, and provide a new solution for the treatment of chronic wounds such as diabetic wounds.

[0025] Beneficial effects

[0026] (1) The fiber membrane composite hydrogel can effectively block the penetration of external moisture, reduce the direct contact of the wound with external pathogens, and reduce the risk of infection, based on the hydrophobic characteristics of the nanofiber membrane. Meanwhile, the hydrophobic fiber membrane can reduce the volatilization of water in the hydrogel, which is conducive to maintaining a moist healing environment and accelerating wound healing.

[0027] (2) The fiber membrane composite hydrogel can be stably heated to 58.5 DEG C within 5 minutes after NIR irradiation, and has significant photothermal properties. In addition, after undergoing 5 NIR irradiation-natural cooling cycles, the fiber membrane composite hydrogel does not show photothermal performance attenuation.

[0028] (3) The fiber membrane composite hydrogel can significantly kill Staphylococcus aureus through auxiliary NIR irradiation, and the bacterial survival rate is only 0.12%, which exhibits excellent antibacterial performance.

[0029] (4) The fiber membrane composite hydrogel can release drugs to remove ROS, and the DPPH removal rate is 84.86%, which can effectively remove free radicals and has excellent antioxidant function. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 SEM diagram between fiber membrane composite hydrogel layers;

[0031] Figure 2 Water contact angle of both sides of the fiber membrane composite hydrogel;

[0032] Figure 3Photothermal properties of fiber membrane composite hydrogel: (a) temperature change curve of single irradiation, (b) temperature change curve of cyclic irradiation;

[0033] Figure 4 Antibacterial effect of fiber membrane composite hydrogel: (a) antibacterial image, (b) bacterial survival rate;

[0034] Figure 5 DPPH scavenging effect of fiber membrane composite hydrogel: (a) color change diagram, (b) DPPH scavenging rate. DETAILED DESCRIPTION

[0035] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0036] Example 1

[0037] Preparation of fiber membrane composite hydrogel:

[0038] (1) Preparation of spinning solution:

[0039] Polylactic acid (Mw ~ 80000, Shanghai MacLean Biochemical Technology Co., Ltd.) was dissolved in hexafluoroisopropanol solvent and stirred for 6 hours to form a homogeneous solution; then gold nanorods were added, ultrasonically dispersed for 30 minutes, and stirred for 2 hours until uniformly dispersed to obtain a spinning solution; wherein the concentration of polylactic acid in the spinning solution was 15% (w / v), and the gold nanorods accounted for 0.3wt% of the mass of the polylactic acid.

[0040] (2) Preparation of electrospun fiber membrane:

[0041] The spinning solution prepared in step (1) was placed in a syringe, and a nanofiber membrane was prepared by electrospinning technology with an injection speed of 1 mL / h, a spinning voltage of 12 kV, a receiving distance of 15 cm, an ambient temperature of 25 ± 1 ° C, and an ambient humidity of 50% ± 5%; the prepared nanofiber membrane was then vacuum dried at 37 ° C for 3 h and stored for later use.

[0042] (3) Synthesis of phenylboronic acid grafted hyaluronic acid:

[0043] Hyaluronic acid (molecular weight 800,000-1.5 million, Shanghai MacLean Biochemical Technology Co., Ltd.) was dissolved in morpholineethanesulfonic acid buffer (pH 5.5) and stirred for 3 hours until completely dissolved. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (Shanghai Myrel Biochemical Technology Co., Ltd.) was then added and stirred for 30 minutes. Then, 3-aminophenylboronic acid (Shanghai Shaoyuan Reagent Co., Ltd.) was added and reacted for 24 hours. The resulting solution was dialyzed against deionized water for 5 days using a dialysis bag with a molecular weight cutoff of 8-14 kDa and finally freeze-dried for 2 days to obtain phenylboronic acid-grafted hyaluronic acid.

[0044] (4) Preparation of precursor:

[0045] The phenylboronic acid-grafted hyaluronic acid synthesized in step (3) was dissolved in deionized water and stirred to obtain a solution 1 with a concentration of 2.5% (w / v);

[0046] Luteolin (purity ≥98%, Shanghai Aladdin Biochemical Technology Co., Ltd.) was dissolved in PBS buffer solution (pH=10) and stirred until the solution was homogeneous to obtain Solution 2.

[0047] Solution 1 and solution 2 were mixed at a volume ratio of 1:1 to obtain a precursor, wherein the concentration of luteolin in the precursor was 1 mg / mL.

[0048] (5) Preparation of fiber membrane composite hydrogel:

[0049] The electrospun nanofiber membrane prepared in step (2) is placed on the surface of the precursor prepared in step (4), and allowed to stand at room temperature for 20 minutes to fully crosslink and composite, thereby obtaining a fiber membrane composite hydrogel.

[0050] The interlayer morphology of the fiber membrane composite hydrogel prepared in Example 1 was characterized by scanning electron microscopy. Figure 1 As shown in the figure, the upper nanofiber membrane and the lower hydrogel interface are well bonded, and no obvious delamination or gaps are observed.

[0051] The hydrophilicity and hydrophobicity of both sides of the fiber membrane composite hydrogel prepared in Example 1 were characterized by a contact angle meter. Figure 2 As shown, the droplets on the hydrogel side surface showed a rapid spreading trend and were highly hydrophilic, while the contact angle of the nanofiber membrane side surface was 129.16°, showing hydrophobicity.

[0052] The photothermal properties of the fiber membrane composite hydrogel prepared in Example 1 were characterized by near infrared light NIR irradiation method. The specific operation steps are as follows: (1) using NIR (808nm, 2W / cm 2) irradiate for 5 minutes, and use an infrared thermal imager to record the temperature of the sample every 1 minute; (2) use the same NIR power, perform 5 cycles of irradiation, each cycle of NIR irradiation for 5 minutes, turn off for 5 minutes, and use an infrared thermal imager to record the temperature change. Figure 3 As shown in (a), the surface temperature of the fiber membrane composite hydrogel gradually increases with the extension of irradiation time, and the temperature can rise to 58.5℃ in 5 minutes, showing excellent photothermal performance. Figure 3 As shown in (b), after 5 cycles of NIR irradiation-natural cooling, the maximum temperature of the fiber membrane composite hydrogel remained stable at 52-57 °C, showing good photothermal stability.

[0053] The photothermal antibacterial effect of the fiber membrane composite hydrogel prepared in Example 1 was evaluated by in vitro antibacterial experiments. Sterile PBS buffer was used as the control group to compare the survival rate of Staphylococcus aureus in the fiber membrane composite hydrogel with or without NIR irradiation. Figure 4 As shown in (a), when the fiber membrane composite hydrogel is not irradiated with NIR, there are still a large number of bacterial colonies in the culture dish, but after NIR irradiation, the number of bacteria is significantly reduced, as shown in Figure 4 As shown in (b), after NIR irradiation, the bacterial survival rate of the fiber membrane composite hydrogel group was 0.12%, showing excellent antibacterial effect.

[0054] Comparative Example 1

[0055] Preparation method of hydrogel: The phenylboronic acid-grafted hyaluronic acid of Example 1 was dissolved in deionized water and stirred evenly to obtain solution 1 with a concentration of 2.5% (w / v). No luteolin solution was added. The solution was allowed to stand at room temperature for 20 minutes, and only the phenylboronic acid-grafted hyaluronic acid was self-crosslinked to form a gel to obtain a hydrogel.

[0056] Comparative Example 2

[0057] The preparation method of the drug-loaded hydrogel is as follows: the phenylboronic acid-grafted hyaluronic acid of Example 1 is dissolved in deionized water and stirred evenly to obtain solution 1 with a concentration of 2.5% (w / v); luteolin (purity ≥98%, Shanghai Aladdin Biochemical Technology Co., Ltd.) is dissolved in PBS buffer solution (pH = 10) and stirred until the solution is uniform to obtain solution 2.

[0058] Solution 1 and Solution 2 were mixed at a volume ratio of 1:1 to obtain a precursor, which was allowed to stand at room temperature for 20 minutes to obtain a drug-loaded hydrogel. The concentration of luteolin in the precursor was 0.5 mg / mL.

[0059] Comparative Example 3

[0060] The drug-loaded hydrogel was prepared according to the method of Comparative Example 2, except that the concentration of luteolin in the precursor was 2 mg / mL.

[0061] The antioxidant activity of the lower drug-loaded hydrogel prepared in Example 1 and the hydrogels prepared in Comparative Examples 1, 2, and 3 was evaluated by DPPH radical scavenging experiments. As shown in Figure 5 (a), as the amount of luteolin added gradually increased, the color of the solution gradually changed from deep purple to bright yellow, as shown in Figure 5 (b), compared with the drug-loaded hydrogel prepared in Example 1 (84.86%), the DPPH scavenging rates of the hydrogels prepared in Comparative Examples 1, 2, and 3 were 12.83%, 46.48%, and 86.79%, respectively. The hydrogels prepared in Comparative Examples 1 and 2 had poor antioxidant performance and could not effectively scavenge free radicals, while the drug-loaded hydrogel prepared in Comparative Example 3 had a doubled antioxidant drug concentration compared with the drug-loaded hydrogel prepared in Example 1, but the antioxidant performance was not much different.

Claims

1. A method for preparing a fiber membrane composite hydrogel, comprising: (1) mixing a phenylboronic acid grafted polymer solution and a phenolic hydroxyl group-containing drug solution to obtain a precursor; (2) Placing a polymer nanofiber membrane loaded with gold nanorods on the surface of the precursor and allowing it to stand to obtain a fiber membrane composite hydrogel.

2. The preparation method according to claim 1, characterized in that In the step (1), the phenylboronic acid grafted polymer is one or more of phenylboronic acid grafted hyaluronic acid and phenylboronic acid grafted sodium alginate; The solvent of the phenylboronic acid grafted polymer solution in step (1) is one of deionized water and PBS buffer.

3. The preparation method according to claim 1, characterized in that: The phenolic hydroxyl-containing drug in step (1) is a flavonoid drug; wherein the flavonoid drug is one or more of luteolin, quercetin, and morusin; The solvent of the phenolic hydroxyl group-containing drug solution is one of deionized water and PBS buffer.

4. The preparation method according to claim 1, characterized in that The concentration of the phenylboronic acid grafted polymer solution in step (1) is 0.5-2.5% (w / v); the concentration of the phenolic hydroxyl group-containing drug in the precursor is 0-2 mg / mL; In the step (1), the volume ratio of the phenylboronic acid grafted polymer solution to the phenolic hydroxyl group-containing drug solution is 2:1 to 1:

2.

5. The preparation method according to claim 1, characterized in that: The preparation of the gold nanorod-loaded polymer nanofiber membrane in step (2) comprises mixing a polymer, an organic solvent, and gold nanorods to obtain a spinning solution, and electrospinning to obtain the gold nanorod-loaded polymer nanofiber membrane.

6. The preparation method according to claim 5, characterized in that: The polymer is one or more of polylactic acid, polyglycolic acid, and polylactic acid-glycolic acid copolymer; the organic solvent is one or more of hexafluoroisopropanol, chloroform, dichloromethane, and NN dimethylformamide.

7. The preparation method according to claim 5, characterized in that: The concentration of the polymer in the spinning solution is 10-20% (w / v); the gold nanorods account for 0.1-0.5 wt% of the mass of the polymer.

8. The preparation method according to claim 1, characterized in that: The step (2) is to allow the mixture to stand at room temperature for 10-60 minutes.

9. A fiber membrane composite hydrogel prepared by the method according to claim 1.

10. Use of the fiber membrane composite hydrogel according to claim 9 in a chronic wound repair dressing.

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