Preparation of a curcumin / nitric oxide / photothermal synergistic antibacterial nanodrug composite dynamic cross-linked hydrogel

Abstract

The present invention provides a preparation method for a nano-drug composite dynamic cross-linked hydrogel with curcumin / nitric oxide / photothermal synergistic antibacterial effect. The present invention uses copper salt to chelate curcumin to form first nanoparticles, and then uses polydopamine to coat the first nanoparticles to form nano-drug P Cur Sodium nitroprusside and sodium phytate were used as raw materials and nano-coprecipitation method was used to prepare second nanoparticles; polydopamine was then used to coat the second nanoparticles to prepare a core-shell structure nano drug P NO ; The nano drug P cur and P NO The nanocomposite hydrogel is mixed evenly with carboxymethyl chitosan and oxidized hyaluronic acid to form a nanocomposite dynamically cross-linked hydrogel. In addition to its significant antibacterial properties, the nanocomposite hydrogel of the present invention also has significant advantages such as high antibacterial efficacy, good biosafety, convenient preparation, and low cost. The present invention also combines drug, photothermal, and gas therapy in a single device, facilitating synergistic and precise bacterial elimination.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of antibacterial hydrogel, and particularly relates to a preparation of a curcumin / nitric oxide / photothermal synergistic antibacterial nanomedicine composite dynamic crosslinking hydrogel. BACKGROUND

[0002] The information disclosed in this Background section is for the purpose of increasing the understanding of the background of the application without admitting that such information forms prior art.

[0003] Human health is a key link to achieve sustainable development. Bacterial infection is one of the major diseases faced by mankind, and is the second largest global death cause next to ischemic heart disease, which poses a challenge to global public health. However, traditional treatment methods, such as inorganic antibacterial agents, can cause serious systemic toxicity, and antibiotics can cause drug resistance. At the same time, most of these materials only have a single function, and the antibacterial effect is not ideal. Therefore, with the increasing number of drug-resistant bacteria, it is crucial to develop new antibacterial therapies.

[0004] Curcumin is one of the most widely used natural synthetic drugs in the world, which is a photosensitive pigment molecule with anti-aging, food colorant, free radical scavenging, lipid-lowering, anti-inflammatory, antioxidant, antibacterial, anticancer and other effects. Due to the poor water solubility and structural instability of curcumin, the bioavailability is poor, and direct application is difficult to fully exert its ideal medicinal value.

[0005] Nitric oxide (NO) is a short-lived, naturally occurring reactive oxidative gas with strong antibacterial properties through oxidative stress and inactivation of lipid, protein and DNA targets. NO has good antifungal, antiviral and antiparasitic properties through similar cellular mechanisms, and has no cytotoxicity to mammalian cells at specific antibacterial dose levels. More importantly, there is no evidence to support the tendency of NO to induce bacterial resistance.

[0006] In recent years, photothermal therapy has been used to effectively eradicate bacteria and biofilms by generating heat therapy due to its advantages of broad-spectrum and high-efficiency antibacterial, good controllability, non-invasive, prevention of multi-drug resistant bacteria, etc. Many inorganic materials (such as gold nanoparticles, graphene, carbon nanotubes and transition metal disulfides) and organic materials (such as small molecule preparations of indocyanine green (ICG), IR-825 and polymeric materials such as polypyrrole, polyaniline, dopamine (PDA)) have been reported as PTT biomaterials. As a naturally occurring melanin analogue, PDA has the advantages of simple synthesis method, antioxidant performance, adhesion performance and biocompatibility, and is most widely used in PTT.

[0007] Hydrogel has a three-dimensional network structure, which is comparable to the natural extracellular matrix in physical and functional aspects, making it suitable for nutrient delivery, cell communication and cell regeneration. Hydrogel also has good moisture retention, good biocompatibility, and designable physical structure, and is widely used in wound dressings. More importantly, hydrogel is also the best drug carrier, and by introducing structures that respond to different conditions (pH, ROS, glucose, etc.), intelligent drug release can be achieved. In addition, hydrogel can be made into an injection form, which has attracted the interest of many researchers because of its many advantages, including the ability to fill uneven spaces in wounds, adhere to wounds, and encapsulate bioactive chemicals and cells in situ, all of which are essential for skin tissue regeneration.

[0008] However, the industry has not yet found a medical dressing with good multiple synergistic antibacterial effect. SUMMARY

[0009] To solve the above problems, the present application provides a preparation of a curcumin / nitric oxide / photothermal synergistic antibacterial nano-drug composite dynamic crosslinking hydrogel. The present application first designs and prepares two kinds of nano-drugs. Natural Chinese herbal medicine curcumin is used as an antibacterial agent, and copper ions are used to chelate curcumin to form nanoparticles, which are then coated with a polydopamine shell to form nanospheres P Cur . Sodium nitroprusside is used as a NO donor, and nano-precipitation is used to prepare nanoparticles, which are then coated with silica and then polydopamine to prepare nanomedicine P NO . On this basis, carboxymethyl chitosan and oxidized hyaluronic acid are used as raw materials to prepare an injectable hydrogel by Schiff base crosslinking and to compound the nanomedicine P cur and P NO (Gel@Cur+NO). The present application has the following advantages: (1) The nanoparticles of curcumin are prepared by a metal coordination method, and the polydopamine with photothermal effect is coated to prepare the nanomedicine P cur . This solves the problems of poor water solubility and poor stability of curcumin; (2) The nanomedicine P NO is prepared by a nano-precipitation method, and a two-step coating of silica and polydopamine is used to prepare a nanomedicine with a core-shell structure. P NO plays a dual role as a NO donor and a photothermal agent. (3) The hydrogel system Gel@Cur+NO combines Chinese herbal antibacterial, NO gas therapy and photothermal antibacterial multiple antibacterial in an organic way.

[0010] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0011] The first aspect of the application provides a preparation method of a curcumin / nitric oxide / photothermal synergistic antibacterial nano-drug composite dynamic crosslinking hydrogel, comprising the following steps:

[0012] The copper salt is chelated with curcumin to form first nanoparticles, and then the first nanoparticles are coated with polydopamine to form a nano-drug P Cur ;

[0013] The second nanoparticles are prepared by using nanoscale coprecipitation with sodium nitroprusside and sodium phytate as raw materials; and then the second nanoparticles are coated with polydopamine to prepare the nano-drug P NO ;

[0014] The nano-drugs P cur and P NO are mixed with carboxymethyl chitosan and oxidized hyaluronic acid to form a nano composite dynamic crosslinking hydrogel.

[0015] In some embodiments, the mass ratio of the copper salt to curcumin is 1:2-2.5. Curcumin has a rare beta-diketone structure, which is easy to interact with metal ions to form a complex. After curcumin is combined with metal ions, the structural stability is greatly improved, the anti-damage ability in the body is enhanced, and the bioavailability is improved to a certain extent. At the same time, the curcumin metal complex has the same anti-inflammatory, antibacterial, antioxidant, antitumor and other medicinal properties as curcumin. Research has found that the antibacterial performance of curcumin copper complex is significantly improved compared with curcumin alone.

[0016] In some embodiments, the mass ratio of curcumin to dopamine hydrochloride is 1:1-1.1.

[0017] In some embodiments, the mass ratio of sodium phytate to sodium nitroprusside is 5:1-1.5.

[0018] In some embodiments, the mass ratio of sodium nitroprusside to dopamine hydrochloride is 1:2-2.2.

[0019] In some embodiments, the mass ratio of the nano-drugs Pcur and P NO is 1:1-1.5.

[0020] In some embodiments, the mass ratio of carboxymethyl chitosan to oxidized hyaluronic acid is 1:2-6.

[0021] The second aspect of the application provides a curcumin / nitric oxide / photothermal synergistic antibacterial nano-drug composite dynamic crosslinking hydrogel prepared by the above method.

[0022] In a third aspect of the present application, the curcumin / nitric oxide / photothermal synergistic antibacterial nanomedicine composite dynamic crosslinking hydrogel is applied to the preparation of an antibacterial dressing or an antibacterial drug.

[0023] In a fourth aspect of the present application, the curcumin / nitric oxide / photothermal synergistic antibacterial nanomedicine composite dynamic crosslinking hydrogel is applied to the drug / NO / chemical kinetics / photothermal synergistic antibacterial.

[0024] Advantages of the present application

[0025] (1) The present application designs and develops a new copper ion coordination curcumin into spherical nanoparticles by the chelation reaction of curcumin (Cur) and copper ions and coats a dopamine shell on the basis. The water solubility and bioavailability of curcumin after complexation are greatly improved, which is used for effective synergistic, precise drug-photothermal antibacterial. NO The silica nanoparticles co-doped with sodium nitroprusside and phytic acid and the PDA modification are used to generate NO and have good photothermal effect under near-infrared (NIR) laser irradiation. Therefore, bacteria can be effectively killed by gas therapy in cooperation with PTT. In addition, the biocompatible OHA / CMC hydrogel has a unique injectable self-healing property and can be used as a promising drug and multifunctional nanoparticle delivery system. The nanocomposite hydrogel prepared by combining the hydrogel with the nanoparticles can well release the nanoparticles. Under 808 nm near-infrared laser irradiation, the hydrogel loaded nanoparticles exhibit ideal antibacterial performance on Staphylococcus aureus and Escherichia coli through the synergistic effect of PTT and NO generation, and the antibacterial experiments in vitro and in vivo are verified. In addition to the significant antibacterial performance, the nanocomposite hydrogel has obvious advantages such as high antibacterial effect, good biological safety, convenient preparation and low cost, and is an ideal bactericidal material for treating in vivo and in vitro wound infections. The hydrogel loaded nanoparticles of the present application combine drugs, photothermal and gas therapy in one device, which helps to synergistically and accurately eliminate bacteria.

[0026] (2) The preparation method of the present application is simple, practical and easy to popularize. BRIEF DESCRIPTION OF DRAWINGS

[0027] The drawings accompanying the specification of the present application serve to provide further understanding of the present application, and the exemplary embodiments of the present application and the description thereof serve to explain the present application and do not constitute an improper limitation on the present application.

[0028] Figure 1 P cur , P NO and a preparation schematic diagram of the hydrogel.

[0029] Figure 2 P cur(a) SEM image of the nanoparticles (scale bar = 3.0 μm); (b) TEM image of the nanoparticles (scale bar = 100 nm); (c) P cur Dopamine shell of the nanoparticles after ethanol dissolution (scale bar = 100 nm); (d) Size distribution; (e) FTIR spectrum; (f) UV-Vis absorption spectrum; (g) Fluorescence spectrum; (h) Cur standard curve; (i) Cur maximum absorption peak and P cur concentration relationship.

[0030] Figure 3 Cur and P cur Optical images of the nanoparticles dissolved in water and DMSO, respectively.

[0031] Figure 4 (a) SiO2@PA+SNP TEM image (scale bar = 200 nm); P NO (b) TEM image of the nanoparticles (scale bar = 200 nm); (b) SEM image of the nanoparticles (scale bar = 5.0 μm); (d) and (e) Size distribution of SiO2@SNP+PA and P NO ; (f) Monodisperse hollow structure of P NO ; (g) FTIR spectrum; (h) Optical images of SiO2@SNP+PA and P NO in water.

[0032] Figure 5 (a) 1H NMR spectra of OHA and HA in deuterium water; (b) FTIR spectrum of the hydrogel; (c) Gelation time of the blank hydrogel.

[0033] Figure 6 (a) Gelation time of the nanocomposite hydrogel; (b) Injectability of the hydrogel with methylene blue; (c) Injectability of the nanocomposite hydrogel.

[0034] Figure 7 (a) Photographs of the self-healing performance of the hydrogel with methylene blue and the nanocomposite hydrogel; (b) and (d) are SEM images of No. 3, 8 blank hydrogels; (c) and (e) are SEM images of No. 4, 9 nanocomposite hydrogels; (f) Near-infrared radiation (10 mg / ml) as a function of power and (g) the corresponding images recorded by the thermal imager; (h) Near-infrared radiation (0.9 W / cm 2 ) as a function of concentration and (i) the corresponding images recorded by the thermal imager; (j) Irradiation cycle experiment of the nanocomposite hydrogel.

[0035] Figure 8(a) Oscillation-frequency sweep mode of No. 3, 4, 8, 9 hydrogels; (b) Time sweep mode of No. 3, 8 hydrogels; (c) Time sweep mode of No. 8, 9 hydrogels; (d) Oscillation-amplitude sweep mode of No. 3 hydrogel; (e) Oscillation-amplitude sweep mode of No. 8 hydrogel; (f) Oscillation-amplitude sweep mode of No. 4 hydrogel; (g) Oscillation-amplitude sweep mode of No. 9 hydrogel.

[0036] Figure 9 (a) Oscillation-time sweep mode of No. 3 hydrogel; (b) Oscillation-time sweep mode of No. 8 hydrogel; (c) Oscillation-time sweep mode of No. 4 hydrogel; (d) Oscillation-time sweep mode of No. 9 hydrogel; (e) Photos of E. coli and (g) S. aureus forming colonies; (f) Corresponding bacterial survival rates of E. coli and (h) S. aureus under different treatments. DETAILED DESCRIPTION

[0037] It should be noted that the following detailed description is exemplary in nature and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0038] The application will be described in further detail below with reference to specific embodiments. It should be noted that the specific embodiments described are illustrative in nature and are not intended to limit the application.

[0039] Example 1 Preparation of nanodrugs

[0040] 1. Preparation of P cur

[0041] Copper sulfate pentahydrate (10 mg) was dissolved in deionized water (20 ml), and after complete dissolution, anhydrous ethanol (20 ml) was added and ultrasonically dissolved; curcumin (20 mg) was dissolved in dimethyl sulfoxide (2.5 ml), and then added dropwise to the mixed solution of copper sulfate, and after stirring at room temperature for 5 h, dopamine hydrochloride (20 mg) was added, and after 1 h, ammonia water (100 μL) was added, and the polymerization was carried out in the dark for 12 h. After the reaction was completed, centrifugation was performed, and deionized water was used for washing 3 times, and finally freeze-drying was performed to obtain a black powder solid P cur (0.0367 g).

[0042] 2. Preparation of P NO

[0043] ​​Phytate sodium (100 mg) was dissolved in deionized water (1.2 ml), and sodium nitroprusside (SNP, 20 mg) was dissolved in deionized water (0.6 ml). The two solutions were mixed and then added dropwise into anhydrous ethanol (35 ml). After the precipitation was completed, tetraethyl orthosilicate (1.5 ml) and ammonia water (2 ml) were added. The reaction was carried out at 40°C for 6 h in the dark. After the reaction was completed, centrifugation was performed, and the white solid was washed with anhydrous ethanol three times. The white solid was dispersed in anhydrous ethanol (100 ml) and ultrasonicated for 30 min. Dopamine hydrochloride (40 mg) was added, and ultrasonication was continued for 20 min. Ethanolamine (0.48 g) was added. The reaction was carried out in the dark for 14 h. After the reaction was completed, centrifugation was performed, and the gray-brown powder was washed with anhydrous ethanol three times. Finally, the P NO (0.332 g).

[0044] Example 2 Preparation of hydrogel

[0045] 1. Preparation of oxidized hyaluronic acid: Hyaluronic acid (1 g) was dissolved in deionized water (100 ml). Then, sodium periodate (0.5 g) was added, and the reaction was carried out in the dark for 5 h. Ethylene glycol (2 ml) was added, and the reaction was continued for 2 h. The resulting solution was dialyzed against deionized water in a dialysis bag with a molecular weight cutoff of 3500 for 24 h. After dialysis, rotary evaporation was performed, and finally, freeze-drying was carried out to obtain white flocculent oxidized hyaluronic acid (OHA, 0.9819 g).

[0046] 2. Preparation of blank hydrogel: Carboxymethyl chitosan was mixed with PBS (7.4) at a concentration of 20 mg / ml. Then, OHA (40 mg / ml, 60 mg / ml, 80 mg / ml, 100 mg / ml, and 120 mg / ml) was added in equal proportions to obtain the blank hydrogel.

[0047] 3. Preparation of nanocomposite hydrogel Gel@Cur+NO: The two nanomedicines P cur and P NO were added in a 1:1 ratio to the carboxymethyl chitosan solution, ultrasonicated, and then OHA was added. After mixing, the nanocomposite hydrogel was obtained.

[0048] The numbers of the hydrogels are shown in Tables 1 and 2.

[0049] Example 1 Basic test characterization

[0050] Spectral analysis: Quantitative analysis was performed using ultraviolet-visible spectroscopy (UV2800S, China), and the absorption spectrum was recorded in the wavelength range of 300-900 nm. Fluorescence spectroscopy was analyzed using a fluorescence spectrometer (F97 Pro, China).

[0051] Infrared analysis: Fourier transform infrared (FTIR, Nicolet NEXUS 670, China) was used to analyze the composition of raw materials and nanoparticles. The sample was mixed with potassium bromide to make a tablet, and then measured in the range of 4000-400 cm -1 with a resolution of 4 cm -1 .

[0052] Particle size and morphology analysis: Dynamic light scattering (DLS, Zetasizer Nano ZS90, UK) was used to measure the size of nanoparticles and the dispersion index. Scanning electron microscopy (SEM, TESCAN, Czech Republic) was used to observe the morphology of the freeze-dried hydrogel. Before characterization, a layer of gold was sputtered on the hydrogel to improve electrical conductivity. Transmission electron microscopy (TEM, JEM 2100, Japan) and scanning electron microscopy were used to observe the micro-morphology of P cur and P NO .

[0053] Nuclear magnetic resonance analysis: A 400 MHz nuclear magnetic resonance (NMR) spectrometer (HNMR, JNM-ECZL400S, Japan) was used to obtain the 1 H nuclear magnetic resonance spectrum of HA and OHA with D2O as the solvent. 1

[0054] Rheological property characterization

[0055] The mechanical properties and self-healing ability of the blank hydrogel and nanocomposite hydrogel were characterized by a rheometer. The mechanical properties and self-healing ability of the gel were evaluated by time sweep mode, oscillation-frequency sweep mode, oscillation-amplitude sweep mode and oscillation-time sweep mode, respectively.

[0056] Photothermal property characterization

[0057] A near-infrared (NIR) laser emitter (Xi'an Laize Electronics Technology Co., Ltd.) with an emission wavelength of 808 nm was used as the light source. The FLIR thermal imaging device was used to record the temperature change under near-infrared irradiation to study the photothermal properties of the nanocomposite hydrogel. The nanocomposite hydrogel containing different concentrations of drugs (P cur and P NO )(0, 5 mg / ml and 15 mg / ml) was irradiated with a power density of 0.9 W / cm 2 for 10 minutes to investigate the concentration dependence of the photothermal effect. The nanocomposite hydrogel (P cur and P NO )(10 mg / ml) was irradiated with different power densities (0.5, 1.0, 1.5, 2.5 W / cm 2 ​The hydrogels were then irradiated with near-infrared radiation for 10 minutes to investigate the effect of laser power on their photothermal efficiency. The laser irradiation cooling process was repeated five times to evaluate the photothermal stability of the hydrogels.

[0058] Experimental Example 4 Characterization of in vitro antibacterial properties

[0059] First, the in vitro antibacterial activity of the hydrogel (Gel@Cur+NO) was determined by the plating method. The bacterial suspension was diluted to 10 8 CFU / mL, and bacterial dispersion was obtained. 150 μL bacterial suspension was added to different concentrations of No. 1 hydrogel (P cur and P NO The antibacterial activity of hydrogel (Gel@Cur+NO) was investigated in 150 μL of 0, 2.5, and 7.5 mg / ml of the drug. cur and P NO The bacterial dispersion was mixed with 0, 2.5, and 7.5 mg / ml of the mixture and the concentration was 0.9 W / cm 2 Irradiation / no irradiation with 808nm near-infrared laser for 10min.

[0060] After different treatments, the mixed solutions were incubated for 3 h at 37°C. After the above treatments, the obtained bacterial suspension (100 μL) was spread on Luria-Bertani (LB) agar plates and incubated at 37°C for 12–16 h. The number of colonies was counted and the bacterial survival rate was determined.

[0061] The test results of Experimental Examples 1 to 4 are as follows:

[0062] 1P cur Synthesis and characterization of

[0063] The β-diketone structural element in curcumin can isomerize into ketoenol form and chelate Cu in a mixed solution of anhydrous ethanol and aqueous solution at a ratio of 2:1. 2+ ions. P was prepared by oxidative polymerization of dopamine (DA) under weak alkaline conditions. cur PDA-assisted surface coating formed by oxidative polymerization of dopamine in weakly alkaline solution is a simple, safe, effective, and economical surface functionalization method with diverse properties, including high chemical reactivity, strong photothermal performance, and biocompatibility.

[0064] The synthesized P was observed by SEM and TEM. cur The morphology and particle size, such as Figure 2 As shown in a. SEM image shows P cur It is a monodisperse sphere with a smooth surface. TEM images show that P cur It is a regular monodisperse spherical structure.cur The morphology of the dopamine shell, e.g. Figure 2 As shown in Figure c, a uniform PDA coating thickness appeared on the surface of the curcumin copper complex, which also proved that dopamine was successfully oxidized and polymerized on the surface of the curcumin copper complex to form a regular and uniform spherical core-shell structure. Figure 2 Dynamic light scattering (DLS) analysis showed that P cur The particle size is about 105.0 nm, with a small polydispersity index of 0.210, which indicates that the nanoparticles are small in size and have a relatively uniform particle size distribution. Figure 2 e stands for P cur FT-IR spectra of nanoparticles before and after coating. The PDA-coated nanoparticles have a wavelength of 3300-3500 cm -1 The characteristic peak at corresponds to the vibration of the amine group in dopamine, indicating that PDA was successfully coated on the surface of the nanoparticles. cur The optical properties of P cur and UV absorption and fluorescence spectra of curcumin. Figure 2 As shown in f, at about 435nm, P cur The absorption spectrum of curcumin is similar to that of free Cur. Determining the curcumin standard curve is helpful to analyze the relationship between curcumin concentration and absorbance. The results of the curcumin standard curve are as follows: Figure 2 The results showed that curcumin had a high activity in the concentration range of 0.313-10.000 μg mL -1 ) has a good linear relationship with its corresponding 435nm UV absorbance value, and the linear equation is y=0.1671x+0.004875(R 2 =1.000). The results show that the selected curcumin has good quality and stable UV absorption, and can be used for experimental research on the photostability of curcumin. At the same time, the maximum absorption peak and P cur The relationship between concentration, such as Figure 2 As shown in Figure 1, the results also show that P cur In the determination concentration range (0.62~4.45μgmL -1 ) has a good linear relationship with its corresponding ultraviolet absorbance value at about 431nm. In order to test the refractive index of Cur particles of different sizes dispersed in water through the Tyndall effect, the present invention uses Cur and P cur Dissolved in water and DMSO (such as Figure 3 Cur is soluble in DMSO but insoluble in water, while P cur Soluble in water and DMSO, which indicates that Cur and Cu 2+ The binding of P changes the polarity of Cur. curbioavailability than Cur because better water solubility means better bioavailability. In conclusion, the present application successfully prepared P cur .

[0065] 2P NO synthesis and characterization

[0066] Biocompatible P NO was prepared by a method of nanocomprecipitation followed by polymerization which can be dispersed in ethanol. Figure 4 TEM images showed that the morphology and size of the prepared SiO2@PA+SNP nanoparticles were uniform, as shown in a of Figure 4 Dynamic light scattering (DLS) analysis showed that the particle size of SiO2@PA+SNP was about 279.2 nm with a small polydispersity index of 0.016, as shown in d of Figure 4 After coating dopamine, the particle size of P NO increased to 322.0 nm, and TEM images 4b also showed that SiO2@PA+SNP successfully coated a layer of dopamine shell. NO The morphology and particle size of the synthesized P NO were observed by SEM, and SEM images showed that P -1 was monodisperse spheres. As shown in f of Figure 4 TEM images showed that the present application obtained SiO2@PA+SNP monodisperse hollow structure by washing SiO2@PA+SNP with water. As shown in g of Figure 4 FT-IR spectra of SiO2@PA+SNP showed strong and wide absorption bands at 1113 cm NO which were anti-symmetric stretching vibrations of Si-O-Si, all proving the successful compounding of the silica shell. In order to test the refractive index of SiO2@PA+SNP and P NO dispersed in water by the Tyndall effect, the present application dissolved SiO2@PA+SNP and P NO in water (h of Figure 4 ). SiO2@PA+SNP and P NO were both easily soluble in water, which indicated that SiO2@PA+SNP and P 1 both had good bioavailability.

[0067] 3Hydrogel synthesis and characterization

[0068] Carboxymethyl chitosan (CMC) was dissolved in PBS (pH=7.4) at a concentration of 2% (w / v). Oxidized hyaluronic acid was dissolved in PBS (pH=7.4) at a concentration of 2%-6% w / v. Oxidized hyaluronic acid solutions with concentrations of 2%, 3%, 4%, 5% and 6% (w / v) were prepared respectively. The 2% (w / v) CMC solution and the OHA solutions of different concentrations were vortexed for 10 seconds to obtain a uniform mixture. Five injectable hydrogels of different concentrations were obtained. Proton nuclear magnetic resonance ( 1 HNMR) spectrum confirmed the successful synthesis of OHA. 1 HNMR spectrum, new spectra were found at 5.18, 5.08 and 4.97 ppm ( Figure 5 In a), this is attributed to the formation of aldehyde groups in OHA. In addition, the signal at around 2.01 ppm corresponds to the acetyl group (-COCH3) connected to the amino group in the HA backbone. Figure 5 The FT-IR spectrum of the injectable hydrogel is shown in (b). 1600 cm -1 The characteristic absorption peak at is attributed to the imine bond (RC=NR) formed by the Schiff base reaction between the aldehyde group in OHA and the amino group in CMC.

[0069] 3.1 Gelation time of injectable hydrogel

[0070] like Figure 5 As shown in Figure c, after placing the CMC / OHA mixed solution in a glass bottle tube, place it on a vortex mixer and vortex it at high speed for 10 seconds before starting the timer. Observe the glass bottle and turn it upside down every 10 seconds to observe the gel state until there is no liquid flow in the tube and maintain it for 1 minute, indicating that the solution has formed a hydrogel. The time required for this process is recorded as the gel time of the injectable hydrogel. In addition, it can be clearly observed that the gel time shortens with the increase of the molar ratio of -CHO / -NH2. The gel time of the nanoparticles loaded is faster than the ordinary gel time ( Figure 6 (a) This may be due to the -NH2 in dopamine, which shortens the gelation time.

[0071] Table 1 Hydrogel gelation time

[0072]

[0073] Table 2 Hydrogel formation time after loading nanoparticles

[0074]

[0075] 3.2 Injectability of injectable hydrogels

[0076] like Figure 6As shown in Figures b and c, the precursor solutions of the injectable hydrogel (CMC+OHA+methylene blue and CMC+OHA+NPs) were placed in a syringe and then slowly squeezed to observe whether the precursor solution could pass through the needle without clogging, thereby testing whether the injectable hydrogel had injectable properties. The precursor solutions of CMC+OHA and CMC+OHA+NPs could pass through the syringe needle smoothly without clogging, which is due to the shear thinning effect of the hydrogel (viscosity decreases with increasing shear stress). This experiment confirmed that the hydrogel has good injectable properties.

[0077] 3.3 Healability of injectable hydrogels

[0078] like Figure 7 As shown in a, a macroscopic self-healing experiment is used to verify the self-healing properties of the injectable hydrogel. In the macroscopic self-healing experiment, CMC+OHA+methylene blue and CMC+OHA+NPs are taken as examples. A hydrogel dyed with bromophenol blue and a hydrogel loaded with nanoparticles are cut into two gel blocks of equal volume, and then the two hydrogel blocks of different colors are alternately combined into a mixed overall hydrogel. After the combined overall hydrogel is placed in a humid environment at room temperature for 2 hours, the movement of the bromophenol blue dye is observed, and the healed hydrogel is picked up with tweezers to observe the healing ability of the hydrogel. Figure 7 As can be clearly observed in Figures I-IV of Figure a, after the two different colored gel blocks were combined to form a single hydrogel, the boundary between the two gel blocks became blurred over time, indicating molecular motion between adjacent gel blocks. Furthermore, after two injectable hydrogel blocks of different colors were combined for 2 hours, a complete hydrogel was formed, which was picked up with tweezers without any gel blocks falling off (IV). These phenomena confirm the excellent self-healing properties of the nanocomposite hydrogel.

[0079] 3.4 SEM characterization of injectable hydrogels

[0080] The freeze-dried injectable hydrogel was treated with liquid nitrogen and then fractured to expose the cross section of the hydrogel. The cross section of the hydrogel was sprayed with gold and then the morphology of the hydrogel was observed by SEM. Figure 7 b) Blank hydrogel No. 3, ( Figure 7 d) No. 8 nanocomposite hydrogel and ( Figure 7 c) No. 4 blank hydrogel, ( Figure 7 e) The morphology of the injectable hydrogel of nanocomposite hydrogel No. 9. It was found that hydrogel No. 3, nanocomposite hydrogel No. 8, hydrogel No. 4 and nanocomposite hydrogel No. 9 all have regular three-dimensional network structures.

[0081] 3.5 Photothermal properties of injectable hydrogels loaded with nanoparticles

[0082] Near-infrared laser-induced photothermal therapy has the ability to inhibit microorganisms, increase local microcirculation blood flow, and reduce inflammation. Considering the photothermal properties of PDA, the temperature changes under near-infrared irradiation were recorded using a FLIR thermal imaging device to study the photothermal properties of hydrogel loaded with nanoparticles at different concentrations. 1 ml of hydrogel was placed in a 2 mL centrifuge tube, and the temperature of the hydrogel was recorded every 30 s using an infrared thermal imager under the irradiation of 808 nm laser. First, the temperature rise of the hydrogel loaded with different concentrations of nanoparticles (0, 5 (P cur and P NO Each 7.5 mg) 15 mg / mL) OHA + CMC + P cur + P NO The temperature rise of the hydrogel under the irradiation of 808 nm near-infrared laser with a power density of 0.9 W / cm 2 for 10 min. The infrared camera (FLIR ONE) recorded the temperature change (ΔT) of each group every 2 min. As shown in Figure 7 , the temperature rise of the hydrogel (10 mg / mL) at concentration No. 4 under the irradiation of 808 nm near-infrared laser with different power densities (0.5, 1.0, 1.5, 2.5 W / cm 2 ) for 10 min was measured. The infrared camera (FLIR ONE) recorded the temperature change (ΔT) of each group every 2 min. As shown in Figure 7 , especially at a concentration of 15 mg / mL, the temperature change was 12.7°C. This finding indicates that the hydrogel loaded with nanoparticles can effectively convert 808 nm laser energy into local heat. The temperature of the hydrogel loaded with nanoparticles further proves that the photothermal effect is positively correlated with laser power, showing power-dependent photothermal properties. The temperature change of the hydrogel loaded with nanoparticles was measured by on / off cycle illumination to study its photothermal stability. As shown in Figure 8 , the hydrogel loaded with nanoparticles showed good photothermal stability after 5 cycles, and the final temperature remained basically unchanged. In summary, the hydrogel loaded with nanoparticles has typical photothermal effect and good photothermal stability, and can be used for photothermal therapy.

[0083] 3.6 Rheological properties of hydrogel

[0084] No. 3, No. 4 blank hydrogel and No. 8, No. 9 nanocomposite hydrogel (P cur + P NO 15 mg / ml) were prepared into hydrogel discs with a diameter of 40 mm and a height of 1000 um, and then the hydrogel discs were placed at the center of the sample stage bottom plate of the TA rheometer (2000 EX) to detect the relevant data. As shown in Figure 8As shown in Figure a, the storage modulus and loss modulus of blank hydrogels No. 3 and No. 4 and nanocomposite hydrogels No. 8 and No. 9 were tested with a fixed strain of 1.0% and an angular frequency sweep range of 0.1 rad / s to 100 rad / s. Throughout the measurement process, G″ for all injectable hydrogels was less than G′, indicating that the hydrogels were in a gel state. Figure 8 The time sweep test of the blank hydrogels No. 3 and No. 8 and the nanocomposite hydrogels No. 4 and No. 9 (Fig. c) was performed with the temperature set at 37°C, the frequency set at 1Hz, and the strain set at 1%, thereby proving the stability of the blank hydrogels No. 3 and No. 8 and the nanocomposite hydrogels No. 4 and No. 9. In different time periods, G′ was greater than G″, indicating that the blank hydrogels No. 3 and No. 4 and the nanocomposite hydrogels No. 8 and No. 9 were always in the gel state. By setting the fixed frequency to 1Hz and the strain sweep range to γ ​​= 0.01% - 500%, the blank hydrogels No. 3 and No. 4 ( Figure 8 d and e) and No. 8 and No. 9 nanocomposite hydrogels ( Figure 9 The strain at the gel-sol transition point in (f and h) is shown. The storage modulus and loss modulus curves are related to a critical strain of approximately 200%. As the pressure increases, the G′ value suddenly drops below G″, indicating that the hydrogel network has been destroyed. In order to analyze the effects of the blank hydrogels No. 3 and No. 4 ( Figure 9 a and c) and No. 8 and No. 9 nanocomposite hydrogels ( Figure 9 Self-healing properties were tested in Figures b and d) by cycling the samples at 1% strain (100-second intervals) and 700% strain (100-second intervals) at a fixed frequency of 1 Hz. The strain results for the hydrogels show that the three-dimensional network structures of the blank hydrogels Nos. 3 and 4 and the nanocomposite hydrogels Nos. 8 and 9 can autonomously repair themselves after damage, demonstrating excellent self-healing capabilities.

[0085] 4 Antibacterial experiment

[0086] Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) were used as typical bacteria to evaluate the antibacterial properties of the hydrogel. The experimental equipment and samples were sterilized with a high-pressure steam sterilizer before the experiment. The plate counting method was used to detect the in vitro antibacterial effect of the nanocomposite hydrogel on the two bacteria under 808nm near-infrared irradiation. After incubating the two bacterial solutions with the nanocomposite hydrogel sample for 3 hours (37°C), the diluted bacterial solution was smeared on a solid culture dish. The colonies on the culture dish were photographed and counted, and the antibacterial rate was calculated. ​As shown in e-h, the nanocomposite hydrogel + near-infrared showed strong antibacterial activity against E. coli (gram-negative bacteria) and S. aureus (gram-positive bacteria). The number of bacterial colonies in the PBS group decreased compared to the blank hydrogel group. This may be due to the antibacterial effect of chitosan in the hydrogel. Most notably, a satisfactory antibacterial effect was observed in the nanocomposite hydrogel + NIR group, with a nearly 99% reduction in bacterial colonies, which can be attributed to the synergistic eradication of bacteria by photothermal and NO treatment.

[0087] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a curcumin / nitric oxide / photothermal synergistic antibacterial nanomedicine composite dynamic cross-linked hydrogel, characterized in that: include: Copper salt is used to chelate curcumin to form first nanoparticles, and then polydopamine is used to coat the first nanoparticles to form nanodrug P Cur ; Sodium nitroprusside was used as a NO donor and co-precipitated with sodium phytate to prepare nanoparticles. Silica was then polymerized on the surface of the nanoparticles, and then dopamine was polymerized to prepare a core-shell structured nanodrug P. NO ; The nano drug P cur and P NO It is mixed evenly with carboxymethyl chitosan and oxidized hyaluronic acid to form a nanocomposite dynamic cross-linked hydrogel.

2. The method for preparing the curcumin / nitric oxide / photothermal synergistic antibacterial nanomedicine composite dynamic cross-linked hydrogel according to claim 1, wherein: The mass ratio of the copper salt to curcumin is 1:2-2.

5.

3. The method for preparing the curcumin / nitric oxide / photothermal synergistic antibacterial nanomedicine composite dynamic cross-linked hydrogel according to claim 1, wherein: The mass ratio of the curcumin to dopamine hydrochloride is 1:1-1.

1.

4. The method for preparing the curcumin / nitric oxide / photothermal synergistic antibacterial nanomedicine composite dynamic cross-linked hydrogel according to claim 1, wherein: The mass ratio of the sodium phytate to the sodium nitroprusside is 5:1-1.

5.

5. The method for preparing the curcumin / nitric oxide / photothermal synergistic antibacterial nanomedicine composite dynamic cross-linked hydrogel according to claim 1, wherein: The mass ratio of the sodium nitroprusside to dopamine hydrochloride is 1:2-2.

2.

6. The method for preparing the curcumin / nitric oxide / photothermal synergistic antibacterial nanomedicine composite dynamic cross-linked hydrogel according to claim 1, wherein: The nano drug P cur and P NO The mass ratio is 1:1-1.

5.

7. The method for preparing the curcumin / nitric oxide / photothermal synergistic antibacterial nanomedicine composite dynamic cross-linked hydrogel according to claim 1, characterized in that: The mass ratio of the carboxymethyl chitosan to the oxidized hyaluronic acid is 1:2-6.

8. A curcumin / nitric oxide / photothermal synergistic antibacterial nanomedicine composite dynamic cross-linked hydrogel prepared by the method according to any one of claims 1 to 7.

9. Use of the curcumin / nitric oxide / photothermal synergistic antibacterial nanomedicine composite dynamic cross-linked hydrogel according to claim 8 in the preparation of antibacterial materials or antibacterial drugs.

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