LU504863B1

LU504863B1 - Triple-network intelligent response hydrogel and preparation method and use thereof

Info

Publication number: LU504863B1
Application number: LU504863A
Authority: LU
Inventors: Peiyuan Li
Original assignee: Univ Guangxi Chinese Medicine
Priority date: 2022-10-20
Filing date: 2023-08-03
Publication date: 2024-02-08
Also published as: CN115990133A

Abstract

The present invention discloses a triple-network intelligent response hydrogel and a preparation method and use thereof, and belongs to the technical field of hydrogel biomaterials. A mass concentration of a gelatin-curcumin inclusion compound contained in the hydrogel is 6 mg/mL to 40 mg/mL. According to the invention, a mechanical property of the hydrogel is adjusted by controlling an addition amount of the gelatin-curcumin inclusion compound, and a tensile property of the hydrogel is improved, so that the hydrogel can be firmly attached to a skin surface to adapt to violent and frequent movements of wrist, and is used in a drug for wrist wound healing, and epithelium and hair follicle regeneration.

Description

TRIPLE-NETWORK INTELLIGENT RESPONSE HYDROGEL

AND PREPARATION METHOD AND USE THEREOF

TECHNICAL FIELD

The present invention relates to the technical field of hydrogel biomaterials, and particularly to a triple-network intelligent response hydrogel and a preparation method and use thereof.

BACKGROUND

At present, traditional hydrogels or injectable hydrogels widely used have the problem of burst release of drug at an initial stage, which may cause excessive drug concentration in local sites to produce certain toxic and side effects. In this regard, a sustained-release carrier is studied in the prior art to slow down the release rate of curcumin, such as a curcumin micelle hydrogel with pH response and a preparation method thereof (publication number CN 109820815 B) in the prior art, which is used for oral intestinal sustained release, and does not overcome the problem of attachment mechanics of in-vitro wounds. For the hydrogel attached in vitro, the prior art further discloses a polyacrylamide hydrogel dressing and a preparation method and application thereof (publication number CN 111437437 B), which can improve the mechanical property of hydrogel and has a certain release effect, but a large amount of acrylamide is used as a raw material, and the inhalation of acrylamide dust through respiratory tract or direct contact with an acrylamide solution through skin will lead to poisoning, thus being not conducive to safe preparation and use. Therefore, the effective release in lesion locations, intelligent regulation, simple preparation and safe use of a drug with a mechanical property are improved to comprehensively solve the technical problems.

SUMMARY

One object of the present invention is to solve at least the above defects and technical problems and to provide at least the advantages that will be described hereinafter.

According to the present invention, a mechanical property of a hydrogel may be adjusted by controlling an addition amount of a gelatin-curcumin inclusion compound, and a tensile property of the hydrogel is improved, so that the hydrogel can be firmly attached to a skin surface to adapt to violent and frequent movements of wrist.

The present invention provides a triple-network intelligent response hydrogel, wherein a mass concentration of a gelatin-curcumin inclusion compound contained in the hydrogel is 6 mg/mL to 40 mg/mL.

The present invention further provides a preparation method of the triple-network intelligent response hydrogel above, wherein the method comprises: dissolving chitosan in an acetic acid solution, and then adding the gelatin-curcumin inclusion compound for dissolution to obtain a first solution;

dissolving agarose in pure water, and adding a sodium hydroxide solution for mixing to obtain a second solution; and adding the first solution into the second solution for uniformly mixing, and then carrying out ultrasonic vibration on the mixture for defoaming to obtain the triple-network intelligent response hydrogel; wherein, a mass ratio of the chitosan to the gelatin-curcumin inclusion compound and the agarose is 1: 1.2 to 8: 0.34.

Preferably, a ratio of the sodium hydroxide to the agarose is 0.12 mol: 30 gto 35 g.

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

Preferably, the gelatin-curcumin inclusion compound is prepared by the following steps of: step 1: adding gelatin in pure water for dissolution, then adding a

NaOH solution to adjust a pH value to be 12, adding curcumin, and sealing and uniformly mixing the mixture in the dark; wherein a mass ratio of the gelatin to the curcumin is 2 g: 10 mg to 20 mg; step 2: adding HCL into the mixed solution in the step 1 to adjust a pH value of the solution to be 6, uniformly mixing the solution, and then carrying out ultrasonic vibration on the solution for defoaming; step 3: centrifuging the liquid subjected to the ultrasonic vibration in the step 2, and removing the curcumin not compounded at a bottom layer; and step 4: freeze-drying the upper layer solution obtained in the step 3 to form a foamed solid, thus obtaining the gelatin-curcumin inclusion compound.

Preferably, the mixed liquid of the first solution and the second solution is further added with a gold nanorod coated with mesoporous silica, and a mass ratio of the gold nanorod coated with the mesoporous silica to the chitosan is 240 ug: 0.1 g.

Preferably, the gold nanorod coated with the mesoporous silica is prepared by the following method of: mixing a first CTAB solution with a HAuCl solution, then adding a

NaBH4 solution, uniformly mixing the mixture, and standing to form a nanogold seed; wherein, 36.5mg/mL first CTAB: 0.01 M HAuCl: 0.675 mg/mL NaBH4 = 20 ML: 1 mL: 1 mL; then adding 231 mL of 15.15 mg/mL second CTAB solution, 12.5 mL of 0.01 mol/L HAuCL4, 4.1 mL of 0.01 M AgNOs;, 750 uL of concentrated HCL and 400 uL of 0.1 mol/L. ascorbic acid in sequence, and uniformly mixing the mixture to prepare a growth solution of the gold nanorod; adding 100 pL of nanogold seed into the growth solution, uniformly mixing the mixture, and standing at 28°C for 5 hours to obtain the fully grown gold nanorod; centrifugally washing the prepared gold nanorod with Milli-Q water, and then redispersing the gold nanorod into 20 mL of water; adjusting a pH value of the gold nanorod solution to be 9 to 11 with a NaOH solution under stirring; and then adding 3 mL of 10%

TEOS/methanol solution to react under a mild condition for 24 hours to 5 obtain the gold nanorod coated with the mesoporous silica.

The present invention further provides a use of the triple-network intelligent response hydrogel above, wherein the hydrogel is used in a drug for wound healing promotion, and epithelium and hair follicle regeneration.

Preferably, the hydrogel is used in a drug for wrist wound healing, and epithelium and hair follicle regeneration, and used in preparation of a drug resisting drug-resistant bacteria.

The present invention achieves at least the following beneficial effects: 1. The curcumin is a safe and natural drug with a variety of pharmacological effects, but due to low bioavailability, low targeting ability to lesion locations, poor stability and water solubility in vivo, and other disadvantages of the curcumin, it is difficult to exert a therapeutic effect of the drug while improving an adhesion mechanical property, so that practical application of the curcumin is greatly affected. Therefore, according to the present invention, a gelatin-curcumin nanocomposite is added to an agarose-chitosan single-network system to form a double-network system, so that the mechanical property and viscoelasticity of the hydrogel are improved, and the bioavailability and therapeutic effect of insoluble traditional Chinese medicine components are improved, and meanwhile, the mechanical property of the hydrogel may be adjusted by controlling the addition amount of the gelatin-curcumin inclusion compound.

2. According to the present invention, the gold nanorod coated with the mesoporous silica is also introduced, which may also cooperatively regulate a drug release rate through pH and thermal response while ensuring the adhesion mechanical property, so as to overcome an influence of the improved mechanical property on drug release, thus improving the antibacterial ability and mechanical property of the hydrogel.

3. According to the present invention, the mechanical property of the triple-network intelligent response hydrogel is improved, which affects the swelling rate, and although the drug release rate can be reduced, the effect of improving the antibacterial ability and mechanical property of the hydrogel is achieved by cooperatively and intelligently regulating the release rate through pH and thermal response, and moreover, non-toxic,

easily available and cheap raw materials are adopted to improve the safety of preparation and use, thus having great popularization and application values.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows solubility comparison between curcumin in water and curcumin in Gel@Cur in water.

FIG. 2 shows property investigation of AC-Gel@Cur (6 mg/mL, 12 mg/mL and 40 mg/mL) hydrogels with different concentrations, wherein: (a) shows bending photos of the AC-Gel@Cur (6 mg/mL, 12 mg/mL and 40 mg/mL) hydrogels with different concentrations; (b) shows swelling rates of Ac-Gel@Cur hydrogels with different Gel@Cur contents (6 mg/mL, 12 mg/mL, 24 mg/mL and 40 mg/mL), (c) shows antibacterial rates of the Ac-Gel@Cur hydrogels with different Gel@Cur contents against Staphylococcus aureus, (d) shows compressive stress-strain curves of the Ac-Gel@Cur hydrogels with different Gel@Cur contents; (e) shows photos of the AC-Gel@Cur (12 mg/mL) hydrogel used for a human wrist movement wound and a photo of an AC-Gel@CUR (12 mg/mL) frozen gel attached to a centrifuge tube.

FIG. 3 shows photos of gel-sol transition of an Agarose hydrogel, an

Agarose-CS hydrogel and an AC-Gel@Cur and AC-Gel@Cur-Au hydrogel in a heating-cooling process.

FIG. 4 shows compressive stress-strain curves of various hydrogels.

FIG. 5 shows photos of the bent AC-Gel@Cur-Au hydrogel and photos of the hydrogel adhered to a finger.

FIG. 6 shows swelling rates of various hydrogels.

FIG. 7 shows "intelligent" response drug release of the

AC-Gel@Cur-Au hydrogel, wherein: (a) and (b) show Cur cumulative release curves of the AC-Gel@Cur-Au hydrogel under different conditions; (c) shows swelling rates of the AC-Gel@Cur-Au hydrogel at different pH values; (d) shows a Cur cumulative release curve of the

AC-Gel@Cur-Au hydrogel at a pH value of 5 and under NIR laser irradiation, and (e) shows an ultraviolet absorption spectrum of a release medium PBS; and (f) shows an UV-Vis absorption spectrum of

AuNRs@S102 and AuNRs@SiO2 released by the AC-Gel@Cur-Au hydrogel under different conditions.

FIG. 8 shows in-vitro antibacterial effects of various hydrogels, wherein: (a) shows Agar plate photos of Staphylococcus aureus and (b) shows Agar plate photos of Escherichia coli, and (c) shows corresponding antibacterial rates of Staphylococcus aureus and (f) shows corresponding antibacterial rates of Escherichia coli; (d) shows survival rates of

Staphylococcus aureus treated with AC-Gel@cur-Au under different 405+808 nm irradiation intervals (0 minute to 10 minutes), and (e) shows survival rates of Staphylococcus aureus treated with AC-Gel@cur-Au under different irradiation conditions (405 nm, 808 nm and 405+808 nm); and (g) shows survival rates of Escherichia coli treated with

AC-Gel@cur-Au under different 405+808 nm irradiation intervals (0 minute to 10 minutes), and (h) shows survival rates of Escherichia coli treated with AC-Gel@cur-Au under different irradiation conditions (405 nm, 808 nm and 405+808 nm).

FIG. 9 shows an effect of the hydrogel on wound healing in an infected KM mouse evaluated in vivo, wherein: (a) shows experimental procedures of bacterial infection of a wound; (b) shows photos of a wound infected by Staphylococcus aureus in a KM mouse subjected to different treatments on the 0% 39 6% and 9% days; (c) shows corresponding wound healing rates; (d) shows weight curves of the mouse in different treatment groups; (e) shows total numbers of

Staphylococcus aureus bacteria of the wound on LB agar plates on the 9 day; and (f) shows collagen contents in the groups evaluated by Masson's trichromatic staining.

FIG. 10 shows histological analysis on a wound tissue of a mouse infected with Staphylococcus aureus on the 9 day by HE and Masson's trichrome staining,

DETAILED DESCRIPTION

The present invention is further described in detail hereinafter with reference to the embodiments, so that those skilled in the art can implement according to the text of the specification.

It should be noted that experimental methods involved in the following embodiments are all conventional methods unless otherwise specified. All the reagents and materials can be obtained commercially unless otherwise specified.

Embodiment 1

A triple-network intelligent response hydrogel of the present invention was prepared as follows. (1) 2 g of gelatin was added into 100 mL of pure water and mixed uniformly, the mixture was lightly poured into a dry 250 mL conical flask and stirred to be dissolved in a water bath at 37°C, a NaOH solution was added into the conical flask to adjust a pH value of the solution to be 12 and added with 10 mg of curcumin, a bottle mouth of the conical flask was sealed with plastic wrap, and the mixture was stirred in the dark for 30 minutes. (2) HCL was added into the conical flask in the step (1) to adjust a pH value of the solution to be 6 and continuously stirred for 10 minutes, and the conical flask was put into a numerical control ultrasonic cleaner for ultrasonic vibration for 5 minutes. (3) The liquid subjected to the ultrasonic vibration in the conical flask in the step (2) was centrifuged, and the curcumin not compounded at a bottom layer was removed. (4) The upper layer solution obtained in the step (3) was freeze-dried to form a foamed solid, thus obtaining a gelatin-curcumin inclusion compound, which was recorded as Gel@Cur. (5) 0.1 g of chitosan was added into 5 mL of 1% acetic acid solution,

heated and stirred to dissolve, and added with 0.24 g of the gelatin-curcumin inclusion compound obtained in the step (4), and the mixture was heated and stirred to dissolve. (6) 0.034 g of agarose was added into 3 mL of pure water solution, heated and stirred to dissolve, and added with 120 uL of sodium hydroxide solution (1 mol/L). (7) 1 mL of the solution obtained in the step (5) was added into the mixed solution obtained in the step (6), stirred for 5 minutes, and subjected to ultrasonic vibration in an ultrasonic cleaner for 2 minutes to obtain the triple-network intelligent response hydrogel in which a mass concentration of the gelatin-curcumin inclusion compound was 12 mg/mL, which was recorded as AC-Gel@Cur.

Embodiment 2

The preparation method of the triple-network intelligent response hydrogel of the present invention was the same as that of Embodiment 1, with the only difference in that: in step (5), 0.12 g of the gelatin-curcumin inclusion compound obtained in the step (4) was added into the mixed solution obtained in the step (5).

In this embodiment, the triple-network intelligent response hydrogel in which a mass concentration of the gelatin-curcumin inclusion compound was 6 mg/mL was obtained.

Embodiment 3

The preparation method of the triple-network intelligent response hydrogel of the present invention was the same as that of Embodiment 1, with the only difference in that: in step (5), 0.48g of the gelatin-curcumin inclusion compound obtained in the step (4) was added into the mixed solution obtained in the step (5).

In this embodiment, the triple-network intelligent response hydrogel in which a mass concentration of the gelatin-curcumin inclusion compound was 24 mg/mL was obtained.

Embodiment 4

The preparation method of the triple-network intelligent response hydrogel of the present invention was the same as that of Embodiment 1, with the only difference in that: in step (5), 0.8g of the gelatin-curcumin inclusion compound obtained in the step (4) was added into the mixed solution obtained in the step (5).

In this embodiment, the triple-network intelligent response hydrogel in which a mass concentration of the gelatin-curcumin inclusion compound was 40 mg/mL was obtained.

Embodiment 5

A preparation method of a blank double-network hydrogel was the same as that of Embodiment 1, with the only difference in that: in step (5), the gelatin-curcumin inclusion compound obtained in the step (4) was not added into the mixed solution obtained in the step (5).

In this embodiment, a blank double-network hydrogel matrix was obtained, which was recorded as Agarose-CS. A mass concentration of the gelatin-curcumin inclusion compound was 0%.

Embodiment 6

A preparation method of a blank single-network hydrogel was the same as that of Embodiment 1, with the only difference in that: in step (7), the mixed solution in the step (5) was added to the step (6). In this embodiment, a blank single-network hydrogel matrix was obtained, which was recorded as Agarose.

Embodiment 7

The preparation method of the triple-network intelligent response hydrogel of the present invention was the same as that of Embodiment 1, with the only difference in that: in step (7), the gold nanorod coated with the mesoporous silica was also added. Specifically, in the step (7), 1 mL of the solution obtained in the step (5) was added into the mixed solution obtained in the step (6), and 240 ug of the gold nanorod coated with the mesoporous silica was also added, stirred for 5 minutes, and subjected to ultrasonic vibration in an ultrasonic cleaner for 2 minutes to obtain a hydrogel, which was recorded as AC-Gel@Cur-Au. The gold nanorod coated with the mesoporous silica was prepared by the following method. 20 mL of CTAB solution (36.5 mg/mL) was mixed with 1 mL of 0.01 M HAuCl, and then 1 mL of NaBH; solution (0.675 mg/mL)

subjected to an ice bath was added into the above solution, stirred for 2 minutes and then allowed to stand at 25°C for 2 hours to form a nanogold seed. 231 mL of CTAB solution (15.15 mg/mL), 12.5 mL of 0.01 mol/L

HAuCL4, 4.1 mL of 0.01 M AgNOs, 750 ul of concentrated HCL and 400 pL of 0.1 mol/L ascorbic acid were added into a reaction vessel in sequence, and stirred for 30 minutes to prepare a growth solution of the gold nanorod. 100 pL. of the above nanogold seed was added into the growth solution, stirred for 2 minutes, and then allowed to stand at 28°C for 5 hours to obtain the fully grown gold nanorod. The prepared gold nanorod was centrifugally washed with Milli-Q water (40 mL per tube, rotating speed of 9,500 r/min, 25 minutes), and then redispersed in 20 mL of water. The gold nanorod solution was added with 0.1 mol/L. NaOH solution under stirring to adjust a pH value to be about 10.0. Then, 3 mL of 10% TEOS/methanol solution was added at a certain speed to react at 37°C for 24 hours to obtain the gold nanorod coated with the mesoporous silica, which was recorded as AUNR@ SiO».

Embodiment 8

The preparation method of the hydrogel was the same as that of

Embodiment 5, with the only difference in that: in step (7), the gold nanorod coated with the mesoporous silica was also added. Specifically, in the step (7), 1 mL of the solution obtained in the step (5) was added into the mixed solution obtained in the step (6), and 240 ug of the gold nanorod coated with the mesoporous silica was also added, stirred for 5 minutes, and subjected to ultrasonic vibration in an ultrasonic cleaner for 2 minutes to obtain a hydrogel, which was recorded as AC-Au. The used gold nanorod coated with the mesoporous silica was prepared by the same preparation method of the gold nanorod coated with the mesoporous silica in Embodiment 7.

Experimental Example 1

Solubility test of curcumin and curcumin inclusion compound mL of distilled water was added into two dry 100 mL beakers 10 respectively, the same amount of curcumin and gelatin curcumin inclusion compound obtained in the step (4) of Embodiment 1 were added into the beakers respectively and stirred with a glass rod, and dissolution conditions in the two beakers were observed. If the curcumin and the gelatin curcumin inclusion compound were both dissolved, the same amount of curcumin and curcumin inclusion compound were continuously added into the beakers until one of them was completely dissolved and the other was precipitated. Dissolution conditions of the curcumin and the gelatin curcumin inclusion compound were observed by direct observation, and solubilities of the curcumin and the gelatin curcumin inclusion compound were calculated by an ultraviolet spectrophotometer. A curcumin-gelatin nanocomposite was prepared by alkali dissolution and acid neutralization with gelatin as a carrier. Because the curcumin was difficult to dissolve in water, a curcumin dispersion was in a suspended state, and insoluble drug particles were clearly seen at a bottom portion after standing. A Gel@Cur-based solution was yellowish orange. A solubility of the curcumin in Gel@Cur shown in FIG. 1 could reach 104 ug/mL, and a solubility of the curcumin in water was 2.1 ug/mL. The solubility of the curcumin was increased to 50 times of an original drug after the compound was formed, which could significantly improve the water solubility of the curcumin and was conductive to the exertion of biological activity potential. Experimental results are shown in FIG. 1 below.

Experimental Example 2

Mechanical properties, swelling properties and antibacterial effects of different dosages of gelatin-curcumin inclusion compounds were tested, and performance parameters of the single/double/triple-network hydrogels prepared in Embodiments 1 to 8 were detected.

I. Screening of Gel@Cur concentration

Mechanical properties, swelling properties and antibacterial effects of hydrogels with different Gel@Cur concentrations in Embodiments 1 to 4 were tested. Results are shown in FIG. 2. Gel@Cur hydrogels with four different concentrations were prepared (the Gel@Cur concentrations were 6 mg/mL, 12 mg/mL, 24 mg/mL and 40 mg/mL respectively). The mechanical properties of the hydrogels could be adjusted through a gelatin-curcumin concentration, and the greater the concentration was, the more the number of hydrogen bonding entanglement points between chains was, and the greater the compressive property was. The addition of

Gel@Cur enhanced the strength of the hydrogels, so that the hydrogels had slight elasticity and flexibility. However, a denser network structure could also increase the resistance of water molecules to enter a gel structure, thus reducing the swelling rate. Generally speaking, the physical and chemical properties of the hydrogels could be finely adjusted by changing the amount of Gel@Cur. In addition, a sample with the Gel@Cur concentration of 12 mg/mL had the highest antibacterial activity. Although the mechanical strength of the hydrogel could be improved by continuously increasing Gel@Cur, the antibacterial effect and swelling rate of the hydrogel were reduced, which was not conducive to an application as a biomedical material in tissue engineering. The compressive property and elongation at break of the sample of 12 mg/mL were sufficient to maintain the integrity of the hydrogel during activities of a patient suffering from skin infection, and the sample hydrogel of 12 mg/mL had good tensile property, thus being able to be firmly attached to a skin surface to adapt to violent and frequent movements of wrist.

Moreover, the gel showed a certain adhesion ability. Therefore, based on the analysis of mechanical property, antibacterial effect and swelling property, the gelatin-curcumin concentration of 12mg/mL was selected as the follow-up experiment. 1. Characteristics: the intelligent gels of the gelatin-curcumin inclusion compound prepared in Embodiments 1 to 6 were colorless to yellow transparent gels, with uniform and delicate texture and suitable adhesion. Results were shown in FIG. 3. 2. pH: a few samples of Embodiments 1 to 8 were taken, in which pH values were measured to range from 6 and 8 with precision pH test paper. 3. Mechanical property: the hydrogels prepared in Embodiments 5 to 8 were adhered to skin to test adhesion effects of the hydrogels as shown in FIG. 4. In Embodiments 1 to 4 and Embodiments 5 to 8, the compressive properties, elongations at break and adhesive strengths of the single, double and triple-network hydrogel dressings were tested by an universal testing machine respectively. Results of Embodiments 1 to 4 are shown in the compression stress-strain curves in FIG. 2(d), and results of

Embodiments 5 to 8 are shown in FIG. 4. When a hot agar solution was cooled to room temperature, sol-gel transition occurred, and agarose self-assembled to form a first network structure under the induction of hydrogen-bond interaction. Meanwhile, chitosan chains were embedded in a slightly acidic agar network. In Embodiment 5, after the weakly acidic chitosan/agarose gel was treated with NaOH, the chitosan chains in an agarose matrix were physically crosslinked to form a double-network structure under the induction of pH, so that the mechanical property of the hydrogel was significantly improved. In Embodiments 1 to 4, Gel@Cur was added to form a triple-network structure on the basis of agarose-chitosan double network, so that the mechanical property was further improved, and the compressibility and elongation at break were sufficient to maintain the integrity of the hydrogel during the activities of the patient suffering from skin infection. Therefore, on this basis,

AuNR@ S102 was introduced into the AC-Gel@Cur hydrogel to form an injectable AC-Gel@Cur-AU hydrogel with drug release by NIR/PH intelligent control, and the obtained hydrogel was brownish yellow, and inherited similar gel-sol transition through a heating-cooling process. The

AC-Gel@Cur hydrogel still had a certain viscoelasticity, and the addition of AuNR@ S10: did not significantly affect the physical and chemical properties of AC-gel @ cur (FIG. 5). 4. Measurement of swelling rate: the stability of the hydrogel was determined by measuring swelling ratios (SR) of different samples. The freeze-dried hydrogel sample formed was soaked in a PBS solution (2 mL, pH=7.4) at 37°C for 24 hours, then the hydrogel sample was taken out, and surface water was removed with filter paper. Finally, the hydrogel was weighed, and the swelling rate was calculated by the following formula: swelling rate=(Wt-W0)/W0x100%, wherein WO and Wt respectively represented an initial freeze-dried weight of the hydrogel and a wet gel with swelling balance. The measurement of the hydrogels of

Embodiment 1 and Embodiments 5 to 8 was shown in FIG. 6, and with the increase of viscoelasticity, the swelling rate was gradually reduced.

Experimental Example 3

In-vitro "intelligent" curcumin release experiment of

AC-Gel@Cur-Au hydrogel (1) pH and thermal release: the hydrogel was placed in 100 mL of release medium (PBS phosphate buffer, pH 5.0 and 7.4), and rotated under 100 r/min at 25°C, 37°C and 50°C respectively. The hydrogel was contacted with the release medium, centrifuged, and then added with fresh release medium for further Cur release analysis, and samples were collected at different time points. A supernatant was taken, an absorbance was measured at 425 nm by an ultraviolet spectrophotometer, and a drug cumulative release rate was calculated. The hydrogel of the present invention showed obvious pH and thermal response drug release behaviors (FIG. 7a and FIG. 7b). Under the mechanism, there might be different swelling rates at different pH values (FIG. 7c). (2) Near-infrared light triggered drug release: the hydrogel was immersed in 50 mL of PBS solution, then irradiated with 808 nm NIR laser at an irradiation intensity of 0.5 W/cm? for 10 minutes, and then irradiated for 10 minutes after an interval of 30 minutes, which was repeated for three times. Meanwhile, 1 mL of soaking solution was taken before and after each NIR light irradiation for detection. In order to investigate the influence of NIR laser, we immersed the hydrogel in the same volume of PBS solution without using NIR laser treatment and sampled every 10 minutes for detection. In this experiment, the ultraviolet spectrophotometer was used to quantitatively analyze the drug release. As shown in FIG. 7de, a near-infrared "on-off" phenomenon was obvious after irradiation with 808 nm NIR laser for three times. The near-infrared response Cur release of AC-Gel@Cur-Au was related to the near-infrared triggered gel-sol transition. Due to multiple noncovalent interactions between Cur and nanofibers, Cur was tightly wrapped in the

AC-Gel@Cur-Au hydrogel, and difficult to diffuse out. The gel-sol transition occurring under irradiation led to physical crosslinking fracture, thus accelerating the diffusion of drug from a loose network. Importantly, due to the interaction between AuNR@ SiIO2R and the hydrogel network,

AuNR@ S102 was prevented from leaking out of the hydrogel (FIG. 7f).

The product of the present invention showed obvious near-infrared light triggered response drug release behavior.

Results show that the diffusion of Cur from inside to outside can be accelerated under near-infrared light irradiation and low pH. On one hand, a local drug concentration and an action time may be maintained conveniently; and on the other hand, drug release on demand can be realized. These results show that a synergistic effect of AC-Gel @Cur-Au,

pH reaction and near-infrared reaction may control a release amount of

Cur in the hydrogel, so that the hydrogel may be effectively used for clinical treatment of skin wound.

Experimental Example 4

In-vitro chemotherapy/photodynamic/photothermal triple antibacterial experiment of hydrogel

According to the operation of "Technical Standard For Disinfection” in 2002 version of the Ministry of Health, an in-vitro antibacterial experiment was carried out on the hydrogels prepared in Embodiment 1 and Embodiments 5 to 8 by an experimental method of: preparing a bacterial suspension with a concentration of 107 CFU/mL to108 CFU/mL. 0 .1 mL of bacterial suspension was added into a specified amount of sample and mixed uniformly, and after counting for 30 minutes, the mixture was irradiated with 405+808 nm lasers for 10 minutes and diluted. 40 pL of the solution was put in a sterile agar medium to culture at 37°C for 24 hours, and bacteria were counted. Experimental results are shown in FIG. 8, and when a bactericidal rate is great than or equal to 99.9%, the sample has a bactericidal effect.

The samples (AC-Gel@Cur, AC-Gel@Cur-Au and AC-Au) irradiated with the 405+808 nm lasers showed a stronger antibacterial effect on Staphylococcus aureus and Escherichia coli. With the increase of irradiation time of the 405+808 nm lasers, an antibacterial rate of

AC-Gel@Cur to Escherichia coli and Staphylococcus aureus was increased gradually. A bacterial colony removal rate of irradiation with the 405 nm laser and the 808 nm laser was lower than that of irradiation with 808+405 nm dual light sources. It was indicated that an antibacterial effect of the 405+808 nm lasers used at the same time was better than that of the 405 nm laser and the 808 nm laser used separately. A high temperature and ROS produced by the AC-Gel@Cur-Au hydrogel for

Escherichia coli and Staphylococcus aureus under short-term near-infrared radiation were the reasons why the hydrogel had antibacterial activity. Low antibacterial activity under non-irradiation might be attributed to the low concentration of curcumin released in the dark for 40 minutes and the antibacterial effect of chitosan itself, and the

AC-Gel@Cur-Au hydrogel could kill bacteria through the synergistic treatment of PTT, PDT and chemotherapy.

Experimental Example 5

Experiment on skin wound healing of mouse

The hydrogels of Embodiments 7 and 8 were used to test a wound of a mouse by a test scheme as follows.

Male KM mice (weighing 22 g to 24 g) were randomly divided into three groups (n=8), comprising a control group, and AC-Au and

AC-Gel@Cur-Au hydrogel irradiation group. Before surgery, pentobarbital sodium (ImL/kg) was injected intraperitoneally for anesthesia. After shaving the back of the mouse, a wound was formed on the back. The wound was infected with a Staphylococcus aureus suspension (100 pL, 1.0x10$ CFU/mL) to establish a Staphylococcus aureus-infected mouse model. 24 hours after infection, the hydrogel was injected into the infected wound. In the irradiation group, the wound was irradiated with the 808+405 nm lasers for 10 minutes. Wound images were recorded on different days respectively, and wound healing (%) was calculated according to the following formula: wound healing (%)=[(Co-C)/Co]* 100%, wherein Co was an initial wound area (0* day) and C; was a wound healing time.

In-vivo antibacterial analysis: under a sterile condition, an infected wound tissue was taken and put into a sterile centrifuge tube filled with 2 mL of normal saline. After homogenization, the diluted bacterial suspension was smeared on a LB agar plate, and a number of remaining bacteria was measured. A specimen was cut on the 9% day and fixed with 4% formaldehyde solution. Then, the specimen was embedded with paraffin and sliced into a 5 um thick slide. The sample was subjected to hematoxylin-eosin (H&E) and Masson's trichrome staining, and then observed by a fluorescence microscope.

A Staphylococcus aureus-infected full-thickness skin wound model was used to evaluate the antibacterial effect and wound healing property of AC-Gel@Cur-Au under laser irradiation in vivo (FIG. 9). After laser irradiation, typical images of the wound were recorded by a digital camera on the 0* day, the 3 day, the 6 day and the 9 day. As shown in the figure, a wound shrinkage percentage was calculated according to comparison between a healed wound area on the 0* day and an original wound area. On the 9% day after surgery, a wound area of the

AC-Gel@Cur-Au irradiation group was significantly smaller than those of the other two groups (P<0.05). It was indicated that a wound healing speed of the AC-Gel@Cur-Au hydrogel combined with light irradiation was the fastest. A CFU number of residual Staphylococcus aureus of the

AC-Gel@Cur-Au group was significantly lower than those of the other two groups (P<0.05).

The wound was subjected to H&E staining on the 9% day (FIG. 10).

On the 9" day, new blood vessels could be seen in the AC-Gel@Cur-Au irradiation group. À large number of inflammatory cells could still be seen in the control group and the AC-Au irradiation group. The Masson's trichrome staining (FIG. 10) was used to evaluate collagen fibers and blood vessels formed, and a collagen deposition level on the 9% day of the

AC-Gel@Cur-Au irradiation group was significantly higher than those of the control group and the AC-Au irradiation group (P<0.01). It was indicated that the AC-Gel@Cur-Au hydrogel could effectively promote collagen deposition during wound healing. All mice had normal weight and normal survival. During the whole experiment, the weights of all mice with different treatments were increased normally, which indicated that side effects of all treatments could be ignored.

The above results verify that the combination of wound dressing

AC-Gel@Cur-Au triggered by the 808+405 nm with PTT and PDT not only has an ability to resist broad-spectrum bacteria, but also can promote wound healing, epithelization and hair follicle regeneration. It is indicated that the wound dressing shows great hope in treating a postoperative severe infectious abscess wound and has great potential in a clinical challenge of resisting drug-resistant bacteria.

Although the implementations of the present invention have been disclosed above, the implementations are not limited to the applications listed in the specification and the embodiments, and can be fully applied to various fields suitable for the present invention, and additional modifications can be easily implemented by those skilled in the art.

Therefore, the present invention is not limited to the specific details without departing from the general concept defined by the claims and the equivalent scope.

Claims

1. À triple-network intelligent response hydrogel, wherein a mass concentration of a gelatin-curcumin inclusion compound contained in the hydrogel is 6 mg/mL to 40 mg/mL.

2. A preparation method of a triple-network intelligent response hydrogel, wherein the triple-network intelligent response hydrogel according to claim 1 is prepared by the following method of: dissolving chitosan in an acetic acid solution, and then adding the gelatin-curcumin inclusion compound for dissolution to obtain a first solution; dissolving agarose in pure water, and adding a sodium hydroxide solution for mixing to obtain a second solution; and adding the first solution into the second solution for uniformly mixing, and then carrying out ultrasonic vibration on the mixture for defoaming to obtain the triple-network intelligent response hydrogel; wherein, a mass ratio of the chitosan to the gelatin-curcumin inclusion compound and the agarose is 1: 1.2 to 8: 0.34.

3. The preparation method of the triple-network intelligent response hydrogel according to claim 2, wherein a ratio of the sodium hydroxide to the agarose is 0.12 mol: 30 gto 35 g.

4. The preparation method of the triple-network intelligent response hydrogel according to claim 3, wherein a ratio of the chitosan to 1%

acetic acid solution is 0.1 g: 5 mL; a ratio of the agarose to the pure water is 0.034 g: 3 mL; and a ratio of the agarose to 1 mol/L sodium hydroxide solution is 0.034 g: 120 pL.

5. The preparation method of the triple-network intelligent response hydrogel according to claim 2 or 3 or 4, wherein the gelatin-curcumin inclusion compound is prepared by the following steps of: step 1: adding gelatin in pure water for dissolution, then adding a NaOH solution to adjust a pH value to be 12, adding curcumin, and sealing and uniformly mixing the mixture in the dark; wherein a mass ratio of the gelatin to the curcumin is 2 g: 10 mg to 20 mg; step 2: adding HCL into the mixed solution in the step 1 to adjust a pH value of the solution to be 6, uniformly mixing the solution, and then carrying out ultrasonic vibration on the solution for defoaming; step 3: centrifuging the liquid subjected to the ultrasonic vibration in the step 2, and removing the curcumin not compounded at a bottom layer; and step 4: freeze-drying the upper layer solution obtained in the step 3 to form a foamed solid, thus obtaining the gelatin-curcumin inclusion compound.

6. The preparation method of the triple-network intelligent response hydrogel according to claim 5, wherein the mixed liquid of the first solution and the second solution is further added with a gold nanorod coated with mesoporous silica, and a mass ratio of the gold nanorod coated with the mesoporous silica to the chitosan is 240 ug: 0.1 g.

7. The preparation method of the triple-network intelligent response hydrogel according to claim 6, wherein the gold nanorod coated with the mesoporous silica is prepared by the following method of: mixing a first CTAB solution with a HAuCl solution, then adding a NaBH4 solution, uniformly mixing the mixture, and standing to form a nanogold seed; wherein, 36.5 mg/mL first CTAB: 0.01 M HAuCl: 0.675 mg/mL NaBH4 = 20 mL: 1 mL: 1 mL; then adding 231 mL of 15.15 mg/mL second CTAB solution, 12.5 mL of 0.01 mol/L HAuCL4, 4.1 mL of 0.01 M AgNOs;, 750 uL of concentrated HCL and 400 uL of 0.1 mol/L. ascorbic acid in sequence, and uniformly mixing the mixture to prepare a growth solution of the gold nanorod; adding 100 pL of nanogold seed into the growth solution, uniformly mixing the mixture, and standing at 28°C for 5 hours to obtain the fully grown gold nanorod; centrifugally washing the prepared gold nanorod with Milli-Q water, and then redispersing the gold nanorod into mL of water; adjusting a pH value of the gold nanorod solution to be 9 to 11 with a NaOH solution under stirring; and then adding 3 mL of 10% 20 TEOS/methanol solution to react under a mild condition for 24 hours to obtain the gold nanorod coated with the mesoporous silica.

8. A use of a triple-network intelligent response hydrogel, wherein the hydrogel according to any one of claim 1 or 2 or 3 or 4 or 6 or 7 is used in a drug for wound healing promotion, and epithelium and hair follicle regeneration.

9. The use of the triple-network intelligent response hydrogel according to claim 8, wherein the hydrogel is used in a drug for wrist wound healing, and epithelium and hair follicle regeneration.

10. The use of the triple-network intelligent response hydrogel according to claim 9, wherein the hydrogel is used in preparation of a drug resisting drug-resistant bacteria.