Tri-network intelligent responsive hydrogel and its preparation method and uses

Abstract

The present invention discloses a three-network intelligent response hydrogel, its preparation method, and uses, belonging to the technical field of hydrogel biomaterials. The hydrogel of the present invention contains a gelatin-curcumin complex at a mass concentration of 6-40 mg / mL. By controlling the amount of gelatin-curcumin complex added, the present invention adjusts the mechanical properties of the hydrogel, improving its tensile properties. The hydrogel can firmly adhere to the skin surface and adapt to intense and frequent wrist movements. It is used as a pharmaceutical for wrist wound healing and epithelial and hair follicle regeneration.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrogel biomaterials, and in particular to a three-network intelligent response hydrogel and a preparation method and application thereof. Background Art

[0002] Currently, conventional hydrogels or injectable hydrogels, which are commonly used, suffer from the problem of an initial burst of drug release, resulting in excessively high local drug concentrations and potentially toxic side effects. To address this, prior art has developed sustained-release carriers to slow the release rate of curcumin, such as a pH-responsive curcumin micellar hydrogel and its preparation method (Announcement No. CN 109820815 B). However, while its oral intestinal sustained-release effect does not overcome the mechanical adhesion issues of wounds in vitro, prior art also discloses a polyacrylamide hydrogel dressing and its preparation method and application (Announcement No. CN 111437437 B). While this hydrogel can improve the mechanical properties of the hydrogel and exhibit a certain release effect, it uses a large amount of acrylamide as a raw material. Inhalation of acrylamide dust through the respiratory tract or direct skin contact with its aqueous solution can cause poisoning, making it unsafe to prepare and use. Therefore, achieving effective drug release at the lesion site with improved mechanical properties, intelligent control, simple preparation, and safe use requires a comprehensive solution. Summary of the Invention

[0003] An object of the present invention is to solve at least the above-mentioned disadvantages and technical problems and to provide at least the advantages to be described hereinafter.

[0004] The present invention can adjust the mechanical properties of the hydrogel by controlling the addition amount of the gelatin-curcumin complex, thereby improving the tensile properties of the hydrogel, enabling the hydrogel to firmly adhere to the skin surface and adapt to intense and frequent wrist movements.

[0005] The present invention provides a three-network intelligent response hydrogel, which contains a gelatin-curcumin complex with a mass concentration of 6-40 mg / mL.

[0006] The present invention also provides a method for preparing the above-mentioned three-network intelligent response hydrogel, which is as follows:

[0007] Dissolving chitosan and acetic acid solution, and then adding gelatin-curcumin complex to dissolve to obtain a first solution;

[0008] Dissolving agarose and pure water, and adding sodium hydroxide solution to mix, to obtain a second solution;

[0009] The first solution is added to the second solution and mixed evenly, and then subjected to ultrasonic vibration to defoam, thereby obtaining a three-network intelligent responsive hydrogel;

[0010] Wherein, the mass ratio of the chitosan: gelatin-curcumin complex: agarose is 1:1.2-8:0.34.

[0011] Preferably, the ratio of sodium hydroxide to agarose is 0.12 mol:30-35 g.

[0012] Preferably, the ratio of chitosan to 1% acetic acid solution is 0.1 g:5 mL; the ratio of agarose to pure water is 0.034 g:3 mL; the ratio of agarose to 1 mol / L sodium hydroxide solution is 0.034 g:120 μL.

[0013] Preferably, the gelatin-curcumin complex is prepared by the following steps:

[0014] Step 1: Dissolve gelatin in pure water, then add NaOH aqueous solution to adjust the pH to 12, add curcumin, seal and protect from light, and mix well; wherein the mass ratio of gelatin to curcumin is 2g:10-20mg.

[0015] Step 2: Add HCL to the mixed solution of step 1, adjust the solution pH to 6, mix well, and then defoam by ultrasonic vibration;

[0016] Step 3: centrifuge the liquid after ultrasonic vibration in step 2 to remove the uncomplexed curcumin at the bottom layer;

[0017] Step 4: freeze-drying the upper layer solution obtained in step 3 to form a foamy solid to obtain a gelatin-curcumin complex.

[0018] Preferably, mesoporous silica-coated gold nanorods are further added to the mixture of the first solution and the second solution, and the mass ratio of the mesoporous silica-coated gold nanorods to chitosan is 240 ug:0.1 g.

[0019] Preferably, the mesoporous silica-coated gold nanorods are prepared by the following method:

[0020] Mix the first CTAB aqueous solution with the HAuCl solution, then add the NaBH4 solution, mix thoroughly, and allow to stand to form gold nanoparticles. The ratio of the first CTAB (36.5 mg / mL): HAuCl (0.01 M): NaBH4 (0.675 mg / mL) = 20 mL: 1 mL: 1 mL.

[0021] Next, 231 mL of a 15.15 mg / mL second CTAB aqueous solution, 12.5 mL of 0.01 mol / L HAuCl₄, 4.1 mL of 0.01 M AgNO₃, 750 μL of concentrated HCl, and 400 μL of 0.1 mol / L ascorbic acid were added sequentially and mixed thoroughly to prepare a gold nanorod growth solution. 100 μL of the gold nanoseeds were added to the growth solution, mixed thoroughly, and allowed to stand at 28°C for 5 hours to obtain fully grown gold nanorods. The prepared gold nanorods were washed by centrifugation with Milli-Q water and then redispersed in 20 mL of water. The gold nanorod aqueous solution was adjusted to a pH of 9-11 with NaOH solution while stirring. 3 mL of a 10% TEOS / methanol solution was then added, and the mixture was reacted under mild conditions for 24 hours to obtain mesoporous silica-coated gold nanorods.

[0022] The present invention also provides the use of the above-mentioned three-network intelligent response hydrogel, which is used as a medicine to promote wound healing, epithelial and hair follicle regeneration.

[0023] Preferably, the hydrogel is used for wrist wound healing, epithelial and hair follicle regeneration, and the preparation of drugs for resisting drug-resistant bacteria.

[0024] The present invention achieves at least the following beneficial effects:

[0025] 1. Curcumin is a safe natural medicine with multiple pharmacological effects. However, due to its low bioavailability, low targeting ability to the lesion site, poor stability and water solubility in the body, it is difficult to exert the therapeutic effect of the drug while improving the adhesion mechanical properties, which greatly affects its practical application. Therefore, the present invention adds gelatin-curcumin nanocomposite to the single-network system of agarose-chitosan to form a double-network system, increase the mechanical properties and viscoelasticity of the hydrogel, improve the bioavailability and therapeutic effect of the poorly soluble traditional Chinese medicine ingredients, and simultaneously adjust the mechanical properties of the hydrogel by controlling the amount of gelatin-curcumin complex added.

[0026] 2. The present invention also introduces mesoporous silica-coated gold nanorods, which can not only ensure the adhesion mechanical properties, but also synergistically regulate the drug release rate through pH and thermal response, overcome the effect of improved mechanical properties on drug release, and thus improve the antibacterial ability and mechanical properties of the hydrogel.

[0027] 3. The three-network intelligent response hydrogel of the present invention improves mechanical properties and affects the swelling rate. Although it will reduce the drug release rate, the release rate is increased through the coordinated intelligent regulation of pH and thermal response, which has the effect of improving the antibacterial ability and mechanical properties of the hydrogel. In addition, it uses non-toxic, easily available and inexpensive raw materials to improve the safety of preparation and use, and has great promotion and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Comparison of the solubility of curcumin in water and curcumin in Gel@Cur in water.

[0029] Figure 2 Performance evaluation of AC-Gel@Cur hydrogels at different concentrations (6, 12, and 40 mg / mL). (a) Photographs of the bending of AC-Gel@Cur hydrogels at different concentrations (6, 12, and 40 mg / mL); (b) swelling ratio, (c) antibacterial efficiency against Staphylococcus aureus, and (d) compressive stress-strain curves of AC-Gel@Cur hydrogels with different Gel@Cur contents (6, 12, 24, and 40 mg / mL); (e) Photograph of AC-Gel@Cur (12 mg / mL) hydrogel applied to a human wrist sports wound and AC-Gel@CUR (12 mg / mL) cryogel attached to a centrifuge tube.

[0030] Figure 3 Photos of the gel-sol transition of Agarose hydrogel, Agarose-CS hydrogel, AC-Gel@Cur and AC-Gel@Cur-Au hydrogel during the heating-cooling process.

[0031] Figure 4 Compression stress-strain curves of various hydrogels.

[0032] Figure 5 Photos of AC-Gel@Cur-Au hydrogel bending and adhesion on the finger.

[0033] Figure 6 is the swelling ratio of various hydrogels.

[0034] Figure 7 "Smart" responsive drug release from AC-Gel@Cur-Au hydrogel. (a) and (b) Cumulative release curves of Cur from AC-Gel@Cur-Au hydrogel under different conditions; (c) Swelling ratio of AC-Gel@Cur-Au hydrogel at different pH values; (d) Cumulative release curve of Cur from AC-Gel@Cur-Au hydrogel at pH 5 and under NIR laser irradiation, and (e) UV absorption spectrum of the release medium, PBS; (f) UV-Vis absorption spectra of AuNRs@SiO2 and AuNRs@SiO2 released from AC-Gel@Cur-Au hydrogel under different conditions.

[0035] Figure 8The in vitro antibacterial effects of various hydrogels. (a) Photographs of Staphylococcus aureus and (b) Escherichia coli agar plates and the corresponding antibacterial rates of (c) Staphylococcus aureus and (f) Escherichia coli. Survival rates of Staphylococcus aureus treated with AC-Gel@cur-Au were observed under (d) different 405+808 nm irradiation intervals (0–10 min) and (e) different illumination conditions (405 nm, 808 nm, and 405+808 nm). Survival rates of Escherichia coli treated with AC-Gel@cur-Au were observed under (g) different 405+808 nm irradiation intervals (0–10 min) and (h) different illumination conditions (405 nm, 808 nm, and 405+808 nm).

[0036] Figure 9 In vivo evaluation of the hydrogel's effect on wound healing in KM mice. (a) Experimental procedure for bacterially infected wounds; (b) Photographs of S. aureus-infected wounds in KM mice treated with different treatments on days 0, 3, 6, and 9; (c) Corresponding wound healing rates; (d) Body weight curves of mice in different treatment groups; (e) Total S. aureus counts on LB agar plates on day 9 after wounding. (f) Assessment of collagen content in each group by Masson's trichrome staining.

[0037] Figure 10 The wound tissues of mice infected with Staphylococcus aureus on the 9th day were histologically analyzed using HE and Masson's trichrome staining. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below with reference to the embodiments so that those skilled in the art can implement the invention with reference to the description.

[0039] It should be noted that the experimental methods involved in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0040] Example 1

[0041] The preparation of the three-network intelligent response hydrogel of the present invention is as follows:

[0042] (1) Add 2 g of gelatin to 100 mL of pure water and mix well. Pour the mixture lightly into a dry 250 mL conical flask and stir to dissolve in a 37°C water bath. Add NaOH aqueous solution to the conical flask to adjust the pH to 12, add 10 mg of curcumin, seal the conical flask with plastic wrap, and stir in the dark for 30 min.

[0043] (2) Add HCL to the conical flask in step (1) to adjust the solution pH to 6, continue stirring for 10 minutes, and place the conical flask in a CNC ultrasonic cleaner for ultrasonic vibration for 5 minutes;

[0044] (3) Centrifuge the liquid in the conical flask after ultrasonic vibration in step (2) to remove the uncomplexed curcumin at the bottom layer

[0045] (4) freeze-drying the upper layer solution obtained in step (3) to form a foamy solid, thereby obtaining a gelatin-curcumin complex, which is designated as Gel@Cur;

[0046] (5) Weigh 0.1 g of chitosan and add it to 5 mL of 1% acetic acid solution, heat and stir to dissolve, add 0.24 g of the gelatin-curcumin complex obtained in step (4), heat and stir to dissolve;

[0047] (6) Weigh 0.034 g of agarose and add it to 3 mL of pure water solution. Heat and stir to dissolve. Add 120 μL of sodium hydroxide solution (1 mol / L).

[0048] (7) 1 mL of the solution obtained in step (5) was added to the mixed solution obtained in step (6), stirred for 5 min, and ultrasonically vibrated in an ultrasonic cleaner for 2 min to obtain a three-network intelligent responsive hydrogel with a gelatin-curcumin complex mass concentration of 12 mg / mL, which was recorded as AC-Gel@Cur.

[0049] Example 2

[0050] The preparation method of the three-network intelligent response hydrogel of the present invention is the same as that of Example 1, except that in step (5), 0.12 g of the gelatin-curcumin complex obtained in step (4) is added to step (5) to obtain a mixed solution.

[0051] The three-network intelligent responsive hydrogel obtained in this example has a gelatin-curcumin complex mass concentration of 6 mg / mL.

[0052] Example 3

[0053] The preparation method of the three-network intelligent response hydrogel of the present invention is the same as that of Example 1, except that in step (5), 0.48 g of the gelatin-curcumin complex obtained in step (4) is added to step (5) to obtain a mixed solution.

[0054] The three-network intelligent responsive hydrogel obtained in this example has a gelatin-curcumin complex mass concentration of 24 mg / mL.

[0055] Example 4

[0056] The preparation method of the three-network intelligent response hydrogel of the present invention is the same as that of Example 1, except that in step (5), 0.8 g of the gelatin-curcumin complex obtained in step (4) is added to step (5) to obtain a mixed solution.

[0057] The three-network intelligent responsive hydrogel obtained in this example has a gelatin-curcumin complex mass concentration of 40 mg / mL.

[0058] Example 5

[0059] The preparation method of the blank double network hydrogel is the same as that of Example 1, except that the gelatin-curcumin complex obtained in step (4) is not added to the mixed solution obtained in step (5).

[0060] The blank double-network hydrogel matrix obtained in this example is designated as Agarose-CS, wherein the gelatin-curcumin complex has a mass concentration of 0%.

[0061] Example 6

[0062] The preparation method of the blank single-network hydrogel is the same as that of Example 1, except that the mixed solution of step (5) is not added to step (6) in step (7). In this example, a blank single-network hydrogel matrix is obtained, which is recorded as Agarose.

[0063] Example 7

[0064] The preparation method of the three-network intelligent response hydrogel of the present invention is the same as that of Example 1, except that: in step (7), mesoporous silica-coated gold nanorods are further added. Specifically, in step (7), 1 mL of the solution obtained in step (5) is added to the mixed solution obtained in step (6), and 240 μg of mesoporous silica-coated gold nanorods are added. The mixture is stirred for 5 minutes and ultrasonically vibrated in an ultrasonic cleaner for 2 minutes to obtain a hydrogel, which is recorded as AC-Gel@Cur-Au. The mesoporous silica-coated gold nanorods are prepared by the following method:

[0065] Mix 20 mL of CTAB aqueous solution (36.5 mg / mL) with 1 mL of 0.01 M HAuCl, then add 1 mL of ice-cooled NaBH4 solution (0.675 mg / mL) to the above solution, stir for 2 minutes, and then let it stand at 25°C for 2 hours to form gold nanoparticles.

[0066] A 15.15 mg / mL CTAB aqueous solution, 12.5 mL 0.01 mol / L HAuCl₄, 4.1 mL 0.01M AgNO₃, 750 μL concentrated HCl, and 400 μL 0.1 mol / L ascorbic acid were stirred for 30 minutes to prepare a gold nanorod growth solution. 100 μL of the aforementioned gold nanoparticles was added to the growth solution, stirred for 2 minutes, and then allowed to stand at 28°C for 5 hours to obtain fully grown gold nanorods. The prepared gold nanorods were washed by centrifugation with Milli-Q water (40 mL per tube, 9500 rpm for 25 minutes) and then redispersed in 20 mL of water. While stirring, the gold nanorod aqueous solution was adjusted to a pH of approximately 10.0 using 0.1 mol / L NaOH solution. After adding 3 mL of 10% TEOS / methanol solution at a certain speed, the mixture was reacted at 37°C for 24 hours to obtain mesoporous silica-coated gold nanorods, which were designated as AuNR@SiO2.

[0067] Example 8

[0068] The preparation method of the hydrogel is the same as that of Example 5, except that mesoporous silica-coated gold nanorods are further added in step (7). Specifically, in step (7), 1 mL of the solution obtained in step (5) is added to the mixed solution obtained in step (6), and 240 μg of mesoporous silica-coated gold nanorods are also added. The mixture is stirred for 5 minutes and ultrasonically vibrated in an ultrasonic cleaner for 2 minutes to obtain a hydrogel, which is designated as AC-Au. The mesoporous silica-coated gold nanorods used are prepared by the same method for preparing mesoporous silica-coated gold nanorods as in Example 7.

[0069] Test Example 1

[0070] Solubility test of curcumin and curcumin inclusion complex

[0071] Add 10 mL of distilled water to two dry 100 mL beakers, add equal amounts of curcumin and the gelatin-curcumin complex obtained in step (4) of Example 1 to each beaker, stir with a glass rod, and observe the dissolution in the two beakers. If both dissolve, continue to add equal amounts of curcumin and curcumin inclusion complex to the beaker until one of the two dissolves completely and the other precipitates. Observe the dissolution of both by direct observation, and calculate the solubility of both using a UV spectrophotometer. Using the alkali dissolution and acid neutralization method, gelatin was used as a carrier to prepare a curcumin-gelatin nanocomposite. Since curcumin is poorly soluble in water, the curcumin aqueous dispersion is in a suspended state, and the insoluble drug particles at the bottom are clearly visible after standing. The Gel@Cur solution appears yellow-orange. Figure 1 The solubility of curcumin in Gel@Cur can reach 104ug / mL, while the solubility of curcumin in water is 2.1ug / mL. After forming the complex, the solubility of curcumin increases to 50 times that of the original drug, which can significantly improve the water solubility of curcumin and help realize its biological activity potential. The experimental results are as follows Figure 1 .

[0072] Test Example 2

[0073] The mechanical properties, swelling properties and antibacterial effects of the hydrogels were tested by adjusting the amount of gelatin-curcumin complex. The performance parameters of the single / double / triple network hydrogels prepared in Examples 1 to 8 were tested.

[0074] 1. Screening of Gel@Cur Concentration

[0075] The mechanical properties, swelling properties and antibacterial efficacy of the hydrogels with different Gel@Cur concentrations in Examples 1-4 were tested. Figure 2 As shown. Four different concentrations of Gel@Cur hydrogels were prepared (the concentrations of Gel@Cur were 6, 12, 24, and 40 mg / ml, respectively). The mechanical properties of the hydrogel can be adjusted by the concentration of gelatin curcumin. The higher the concentration, the more hydrogen bond entanglement points between the chains increase, and the greater the compression performance. The addition of Gel@Cur enhances the strength of the hydrogel, and they have slight elasticity and flexibility. However, the denser the network structure, the greater the resistance to water molecules entering the gel structure, and the lower its swelling rate. In general, the physical and chemical properties of the hydrogel can be finely adjusted by changing the amount of Gel@Cur. In addition, the sample with a Gel@Cur concentration of 12 mg / ml had the highest antibacterial activity. Although further increasing the Gel@Cur content can improve the mechanical strength of the hydrogel, its antibacterial effect and swelling rate decreased, which is not conducive to its application as a biomedical material in tissue engineering. The compression properties and elongation at break of the 12 mg / ml sample are sufficient to maintain the integrity of the hydrogel during patient activities after skin infection. The 12 mg / ml sample hydrogel has good tensile properties and can firmly adhere to the skin surface, adapting to the intense and frequent wrist movements. The gelatin also exhibits a certain degree of adhesion. Therefore, based on the comprehensive analysis of its mechanical properties, antibacterial effect, and swelling properties, a gelatin curcumin concentration of 12 mg / mL was selected for subsequent experiments.

[0076] 1. Properties: The gelatin-curcumin complex smart gel prepared in Examples 1 to 6 is a colorless to yellow, transparent gel with a uniform and fine texture and suitable adhesion. Figure 3 shown.

[0077] 2. pH: Take a small amount of each sample from Examples 1 to 8 and measure the pH with precision pH test paper. The pH is between 6 and 8.

[0078] 3. Mechanical properties: The hydrogels prepared in Examples 5-8 were adhered to the skin to test their adhesion effects. Figure 4 The compression performance, elongation at break and adhesion strength of single, double and triple network hydrogel dressings were tested by universal testing machine respectively in Examples 1-4 and 5-8. Figure 2 (d) Compressive stress-strain curves. The results of Examples 5-8 are as follows: Figure 4 As shown, when the hot agar solution cools to room temperature, a sol-gel transition occurs. Agarose self-assembles into a primary network structure under the guidance of hydrogen bonding, while simultaneously embedding chitosan chains within the slightly acidic agar network. In Example 5, after the weakly acidic chitosan / agarose hydrogel was treated with NaOH, the chitosan chains within the agarose matrix physically cross-linked under pH-induced conditions to form a dual network structure, significantly improving the hydrogel's mechanical properties. In Examples 1-4, Gel@Cur was added to the agarose-chitosan double network to form a triple network structure, further improving its mechanical properties. The compression performance and elongation at break were sufficient to maintain the integrity of the hydrogel during the activity of patients after skin infection, meeting the requirements. On this basis, AuNR@SiO2 was introduced into the AC-Gel@Cur hydrogel to form an injectable AC-Gel@Cur-AU hydrogel with NIR / PH intelligent controlled drug release. The resulting hydrogel was brown-yellow and inherited a similar gel-sol transformation through the heating-cooling process. The AC-Gel@Cur hydrogel still had a certain degree of viscoelasticity. The addition of AuNR@SiO2 did not significantly affect the physicochemical properties of AC-Gel@Cur ( Figure 5 ).

[0079] 4. Swelling ratio determination: The stability of the hydrogel was determined by measuring the swelling ratio (SR) of different samples. The freeze-dried hydrogel sample was soaked in PBS solution (2 mL, pH = 7.4) at 37 ° C for 24 h, then the hydrogel sample was taken out and the surface water was removed with filter paper. Finally, the hydrogel was weighed and the swelling ratio was calculated using the following formula:

[0080] Swelling ratio = (Wt-W0) / W0×100%, where W0 and Wt represent the initial hydrogel freeze-dried weight and the wet gel with swelling equilibrium, respectively. Figure 6 As shown in Figure 3, as the viscoelasticity increases, the swelling rate gradually decreases.

[0081] Test Example 3

[0082] AC-Gel@Cur-Au hydrogel in vitro "intelligent" release of curcumin

[0083] (1) pH and thermal release: The hydrogel was placed in 100 mL of release medium (PBS phosphate buffer, pH 5.0 and 7.4), placed at 25°C, 37°C and 50°C, respectively, and rotated at 100 r / min. The hydrogel was contacted with the release medium, centrifuged, and then added with fresh release medium for further Cur release analysis. Samples were collected at different time points. The supernatant was taken and the absorbance was measured at 425 nm using a UV spectrophotometer to calculate the cumulative drug release rate. The hydrogel of the present invention exhibited obvious pH and thermal responsive drug release behavior ( Figure 7 a, b). The mechanism may be that different pH values have different swelling rates ( Figure 7 c).

[0084] (2) Near-infrared light-triggered drug release: The hydrogel was immersed in 50 mL of PBS solution and then exposed to 808 nm NIR laser at 0.5 W / cm -2 The hydrogel was irradiated with a light intensity of 10 min, and then irradiated for 10 min after an interval of 30 min, and repeated three times. At the same time, 1 mL of immersion liquid was taken for detection before and after NIR light irradiation. In order to investigate the influence of NIR laser, without using NIR laser treatment, we immersed the hydrogel in an equal volume of PBS solution and took samples every 10 min for detection. In this experiment, an ultraviolet spectrophotometer was used to quantitatively analyze the release of the drug. Figure 7 As shown in de, an obvious near-infrared "on-off" phenomenon occurs after three irradiations with 808 nm NIR laser. The near-infrared responsive Cur release of AC-Gel@Cur-Au is related to its near-infrared triggered gel-sol transformation. Due to the multiple non-covalent interactions between Cur and nanofibers, Cur is tightly wrapped in the AC-Gel@Cur-Au hydrogel and is difficult to diffuse out. The gel-sol transition occurs under irradiation, resulting in the breaking of physical cross-links, thereby accelerating the diffusion of drugs from the loose network. Importantly, due to the interaction between AuNR@SiO2R and the hydrogel network, the leakage of AuNR@SiO2 from the hydrogel is prevented ( Figure 7 f). The product of the present invention exhibits obvious near-infrared light-triggered drug release behavior.

[0085] The results showed that near-infrared light irradiation and low pH conditions can accelerate the diffusion of Cur from the inside out. This facilitates maintaining local drug concentration and duration of action while also enabling on-demand drug release. These results suggest that the synergistic effects of AC-Gel@Cur-Au, pH response, and near-infrared reaction can control the release of Cur from the hydrogel, making it an effective clinical treatment for skin wounds.

[0086] Test Example 4

[0087] In vitro chemotherapy / photodynamic / photothermal triple antibacterial experiment of hydrogel

[0088] According to the 2002 edition of the Ministry of Health's "Technical Specifications for Disinfection", the hydrogels prepared in Example 1 and Examples 5 to 8 were subjected to in vitro antibacterial experiments. The experimental method was as follows: a concentration of 10 7 ~10 8 CFU / mL bacterial suspension. Add 0.1mL of bacterial suspension to the specified amount of sample, mix well and time for 30 minutes, irradiate with 405+808nm laser light for 10 minutes, dilute, take 40μL and place it in sterile agar medium, culture at 37℃ for 24 hours, and count the bacteria. The experimental results are as follows Figure 8 As shown in the figure, the sample has a bactericidal effect only when the sterilization rate is ≥99.9%.

[0089] Samples irradiated with 405+808 nm laser light (AC-Gel@Cur, AC-Gel@Cur-Au, and AC-Au) exhibited enhanced antibacterial activity against both Staphylococcus aureus and Escherichia coli. The inhibition rate of AC-Gel@Cur against E. coli and S. aureus gradually increased with increasing 405+808 nm irradiation time. The bacterial colony removal rate of 405 nm and 808 nm laser irradiation was lower than that of 808+405 nm dual-light irradiation. This suggests that the antibacterial effect of the combined 405+808 nm laser light is superior to that of the 405 nm and 808 nm laser light alone. The high temperature and ROS generated by the AC-Gel@Cur-Au hydrogel under short-term near-infrared irradiation contribute to the hydrogel's antibacterial activity against E. coli and S. aureus. The smaller antibacterial activity under non-illumination may be attributed to the smaller concentration of curcumin released in the dark for 40 min and the antibacterial effect of chitosan itself. AC-Gel@Cur-Au hydrogel can kill bacteria through the synergistic treatment of PTT, PDT and chemotherapy.

[0090] Test Example 5

[0091] Mouse skin wound healing experiment

[0092] The hydrogels of Examples 7 and 8 were used to test wounds in mice. The test protocol is as follows:

[0093] Male KM mice (weighing 22-24 g) were randomly divided into three groups (n=8): a control group and an AC-Au and AC-Gel@Cur-Au hydrogel illumination group. Before surgery, mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (1 mL / kg). After shaving the back, a wound was created on the back. Staphylococcus aureus suspension (100 μL, 1.0 × 10 8 CFU / mL) to establish a mouse model of Staphylococcus aureus infection. Twenty-four hours after infection, the hydrogel was injected into the infected wound. The light irradiation group irradiated the wound with 808+405 nm laser light for 10 minutes. Wound images were recorded on different days, and wound healing percentage was calculated using the following formula:

[0094] Wound healing (%) = [(C0-C t ) / C0]×100%, where C0 is the initial wound area (day 0), C t Wound healing time.

[0095] In vivo antimicrobial analysis: Under sterile conditions, infected wound tissue was collected and placed in a sterile centrifuge tube containing 2 mL of normal saline. After homogenization, the diluted bacterial suspension was spread on LB agar plates, and the remaining bacterial count was determined. On day 9, specimens were removed and fixed in 4% formaldehyde. They were then embedded in paraffin and sectioned into 5-μm-thick slides. After staining with hematoxylin and eosin (H&E) and Masson's trichrome, the samples were observed using a fluorescence microscope.

[0096] A full-thickness skin wound model infected with Staphylococcus aureus was used to evaluate the antibacterial effect and wound healing performance of AC-Gel@Cur-Au under laser irradiation in vivo. Figure 9 ). After laser irradiation, typical images of the wound were recorded with a digital camera on days 0, 3, 6, and 9. As shown in the figure, the percentage of wound contraction was calculated based on the comparison of the healed wound area on day 0 with the original wound area. On the 9th day after surgery, the wound area in the AC-Gel@Cur-Au light-irradiated group was significantly smaller than that in the other two groups (P<0.05). This shows that the wound healing speed of AC-Gel@Cur-Au hydrogel combined with light irradiation is the fastest. The number of residual Staphylococcus aureus CFU in the AC-Gel@Cur-Au group was significantly lower than that in the other two groups (p<0.05).

[0097] H&E staining was performed on the wound surface on day 9 ( Figure 10 On day 9, new blood vessels were observed in the AC-Gel@Cur-Au illumination group. A large number of inflammatory cells were still observed in the control group and the AC-Au illumination group. Masson's trichrome staining ( Figure 10) evaluated the collagen fibers and blood vessels formed. The AC-Gel@Cur-Au light-exposed group showed significantly higher collagen deposition levels on day 9 compared to the control and AC-Au light-exposed groups (P<0.01), indicating that the AC-Gel@Cur-Au hydrogel effectively promotes collagen deposition during wound healing. All mice maintained normal body weight and survival. Throughout the experiment, all mice treated with different treatments experienced normal weight gain, indicating negligible side effects from all treatments.

[0098] These results demonstrate that the 808+405nm laser-triggered wound dressing AC-Gel@Cur-Au combined with PTT and PDT not only protects against a broad spectrum of bacteria but also promotes wound healing, epithelialization, and hair follicle regeneration. This suggests that the wound dressing shows great promise in treating severe postoperative infected abscess wounds and has significant potential in combating the clinical challenge of drug-resistant bacteria.

[0099] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. Triple-network intelligent responsive hydrogel, characterized by: The hydrogel contains a gelatin-curcumin complex at a mass concentration of 6-40 mg / mL, wherein the gelatin-curcumin complex is prepared by the following steps: Step 1: Dissolve gelatin in pure water, then add NaOH aqueous solution to adjust the pH to 12, then add curcumin, seal and protect from light, and mix well; wherein the mass ratio of gelatin to curcumin is 2g:10-20mg; Step 2: Add HCL to the mixed solution of step 1, adjust the solution pH to 6, mix well, and then defoam by ultrasonic vibration; Step 3: centrifuge the liquid after ultrasonic vibration in step 2 to remove the uncomplexed curcumin at the bottom layer; Step 4: freeze-drying the upper layer solution obtained in step 3 to form a foamy solid, thereby obtaining a gelatin-curcumin complex; Wherein, the hydrogel is prepared by the following method: Dissolving chitosan and acetic acid solution, and then adding gelatin-curcumin complex to dissolve to obtain a first solution; Dissolving agarose and pure water, and adding sodium hydroxide solution to mix, to obtain a second solution; The first solution is added to the second solution and mixed evenly, and then subjected to ultrasonic vibration to defoam, thereby obtaining a three-network intelligent responsive hydrogel; Among them, the mass ratio of chitosan: gelatin curcumin complex: agarose is 1:1.2-8:0.34, and mesoporous silica-coated gold nanorods are also added to the liquid mixed with the first solution and the second solution. The mass ratio of the mesoporous silica-coated gold nanorods to chitosan is 240ug:0.1g.

2. The three-network intelligent response hydrogel according to claim 1, characterized in that: The ratio of sodium hydroxide to agarose is 0.12 mol:30-35 g.

3. The three-network intelligent response hydrogel according to claim 1, characterized in that: The ratio of the chitosan to the 1% acetic acid solution is 0.1 g:5 mL; the ratio of the agarose to pure water is 0.034 g:3 mL; and the ratio of the agarose to the 1 mol / L sodium hydroxide solution is 0.034 g:120 μL.

4. The three-network intelligent response hydrogel according to claim 1, characterized in that The mesoporous silica-wrapped gold nanorods are prepared by the following method: Mix the first CTAB aqueous solution with the HAuCl solution, then add the NaBH4 solution, mix thoroughly, and allow to stand to form gold nanoparticles. The ratio of the first CTAB (36.5 mg / mL): HAuCl (0.01 M): NaBH4 (0.675 mg / mL) = 20 mL: 1 mL: 1 mL. Then, 231 mL of a 15.15 mg / mL second CTAB aqueous solution, 12.5 mL of 0.01 mol / L HAuCL4, 4.1 mL of 0.01 M AgNO3, 750 μL of concentrated HCL, and 400 μL of 0.1 mol / L ascorbic acid were added in sequence and mixed evenly to prepare a gold nanorod growth solution; 100 μL of the gold nanoseeds were added to the growth solution, mixed evenly, and allowed to stand at 28°C for 5 hours to obtain fully grown gold nanorods; the prepared gold nanorods were centrifuged and washed with Milli-Q water and then redispersed in 20 mL of water; the gold nanorod aqueous solution was adjusted to pH 9-11 with NaOH solution under stirring; and 3 mL of a 10% TEOS / methanol solution was added, and the mixture was reacted under mild conditions for 24 hours to obtain mesoporous silica-coated gold nanorods.

5. Use of the triple-network intelligent response hydrogel according to any one of claims 1, 2, 3 or 4 in preparing a drug for promoting wound healing.

6. Use of the triple-network intelligent response hydrogel according to any one of claims 1, 2, 3 or 4 in preparing a wrist wound healing drug.

7. Use of the triple-network intelligent response hydrogel according to any one of claims 1, 2, 3 or 4 in preparing drugs for resisting Staphylococcus aureus.

Images