A hydrogel material for antibacterial and tissue repair, and its preparation method and application

Abstract

The present invention discloses a hydrogel material for antibacterial and tissue repair and its preparation method and application, which belongs to the field of biomedical material technology. The preparation method of the hydrogel material includes: deionized water dissolving phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, heating in a water bath under a light-proof environment, obtaining a photoinitiator solution, adding phenylboronic acid grafted methacryloyl gelatin, heating and fully dissolving, obtaining a bio-ink precursor A; polyvinyl alcohol is added to the photoinitiator solution, heated and stirred and fully dissolved, obtaining a bio-ink precursor B, adding magnesium manganese doped vacancy bismuth oxide nanomaterial and mixing evenly, obtaining a bio-ink precursor C, mixing with the bio-ink precursor A, and the obtained mixed bio-ink is formed by injection extrusion and light curing. The hydrogel material of the present invention can increase ROS production and enzyme-like activity, realize antibacterial sequential treatment, and has important application potential in promoting the repair of infected tissues.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a hydrogel material for antibacterial and tissue repair, and a preparation method and application thereof. Background Art

[0002] Bacterial infection remains a serious complication of open wounds, such as fractures and diabetic wounds. Long-term, high-dose antibiotic therapy is currently a common clinical treatment, but it is also associated with the prevalence of drug-resistant bacteria, poor treatment efficiency, and high recurrence rates. Sonodynamically powered inorganic nanomaterials for antimicrobial efficacy have been a hot topic in biomedical research. However, strategies based on sonodynamic and enzyme-like activity to generate reactive oxygen species (ROS) can induce bacterial oxidative stress, indiscriminately disrupt bacterial membrane permeability, and poorly treat drug-resistant bacteria. In materials with wide band gaps, the energy required for electrons to transition from the valence band to the conduction band is high, resulting in low carrier concentrations (electrons and holes) at room temperature or lower, thus affecting their conductivity and sonodynamic activity. Defect engineering can significantly shorten the band gap by introducing oxygen vacancies to create defect states within Bi₂O₃, thereby addressing the high energy required for valence state transitions. Excessively narrow band gaps can lead to rapid electron-hole pair recombination, hindering efficient ROS generation.

[0003] Ultrasound has been widely used as an excitation source in the field of defect-rich biomaterials due to its advantages such as high safety, high body tolerance, strong penetration, and on-demand regulation. Increasing the piezoelectric potential through ultrasound can promote the separation of electron-hole pairs, enhance catalytic activity, and improve antibacterial efficacy. However, at the site of infection, anti-inflammatory and tissue repair exist in a spatiotemporal continuum. Therefore, a bidirectional regulatory strategy is particularly important to maintain normal ROS levels after generating ROS for antibacterial purposes to promote tissue repair.

[0004] Oxygen vacancies have electron affinity and act as electron traps due to local electron abundance, thus having strong light absorption ability in the near-infrared (NIR) range. It can also convert the absorbed light energy into heat energy through the localized surface plasmon resonance (LSPR) effect, realizing the photothermal effect. The photothermal effect can promote the piezoelectric effect on ·OH and ·O2 to a certain extent. - The effects produced synergistically enhance the antibacterial efficacy.

[0005] Manganese has multiple oxidation states. This variability facilitates electron transfer during catalysis, promoting the decomposition of ROS. It possesses catalase (CAT) and superoxide dismutase (SOD)-like enzyme activities, making it widely used in enzyme-mimicking processes related to redox reactions. Magnesium is a highly abundant element in the human body and a key cofactor for adenosine triphosphate (ATP) and numerous enzymes. Studies have shown that magnesium can alleviate oxidative stress, slow telomere shortening, promote cell proliferation, and improve cellular aging. It can also regulate immune homeostasis and promote the polarization of macrophages toward the anti-inflammatory M2 phenotype.

[0006] At present, there is no product that integrates clinical needs and material characteristics to control the antibacterial treatment effect while promoting tissue repair and achieving the regulation of the pathological microenvironment, which is particularly suitable for the repair of infected tissues. Summary of the Invention

[0007] The main purpose of the present invention is to provide a hydrogel material for antibacterial and tissue repair, which contains magnesium-manganese-doped vacancy bismuth oxide nanomaterials (MMBO x ), MMBO x With the programmable ROS capability, a large amount of ROS can be generated on demand. The hydrogel material has antibacterial activity and promotes tissue repair.

[0008] Another object of the present invention is to provide a method for preparing the hydrogel material for antibacterial and tissue repair, which adopts a one-step hydrothermal method and is simple and efficient.

[0009] Another object of the present invention is to provide a use of the hydrogel material in preparing products for antibacterial and tissue repair promotion.

[0010] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0011] A first aspect of the present invention provides a method for preparing a hydrogel material for antibacterial and tissue repair, comprising the following steps:

[0012] (a) dissolving lithium phenyl (2,4,6-trimethylbenzoyl) phosphate in deionized water and heating in a water bath in a dark environment to obtain a photoinitiator solution;

[0013] (b) adding phenylboronic acid grafted methacryloyl gelatin (GelMA-PBA) to the photoinitiator solution, heating, and fully dissolving to obtain bio-ink precursor A;

[0014] (c) adding polyvinyl alcohol to the photoinitiator solution, heating and stirring to fully dissolve the solution, thereby obtaining a bio-ink precursor B;

[0015] (d) Adding MMBO to the bio-ink precursor Bx , mixed evenly to obtain bio-ink precursor C;

[0016] (e) The bio-ink precursor A and the bio-ink precursor C are mixed evenly to obtain a mixed bio-ink; the mixed bio-ink is formed by injection extrusion and then photocuring.

[0017] Preferably, in step (a), the water bath heating temperature is 37-45° C., the water bath heating time is 30-60 min, and the mixture is shaken every 5-15 min during the heating process.

[0018] Preferably, in step (b), the GelMA-PBA is prepared by the following method:

[0019] (b1) Adding methacryloylated gelatin (GelMA) to deionized water, heating and stirring in a dark environment to obtain a transparent GelMA solution;

[0020] (b2) Phenylboronic acid (PBA) is dissolved in N,N-dimethylformamide (DMF) to obtain precursor solution A; 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM) is dissolved in water to obtain precursor solution B; precursor solution A is slowly added dropwise to precursor solution B to obtain a mixed solution, which is activated at room temperature to obtain a PBA solution;

[0021] (b3) adding the PBA solution dropwise to the GelMA solution, adjusting the pH value of the solution, and reacting at room temperature to obtain a reaction solution;

[0022] (b4) The reaction solution is centrifuged and the supernatant is collected, dialyzed, the pH value of the solution is adjusted, and freeze-dried to obtain the product.

[0023] More preferably, in step (b1), the volume ratio of GelMA to deionized water is 1:50;

[0024] And / or in step (b2), the precursor solution A is a solution formed by dissolving 2-3 mmol PBA in 9 mL DMF, the precursor solution B is a solution formed by dissolving 2-3 mmol DMTMM in 6 mL water, and the activation time at room temperature is 30-45 min;

[0025] And / or in step (b3), the pH of the solution is adjusted to 4.5-6, and the reaction time at room temperature is 20-25 h;

[0026] And / or in step (b4), centrifuging at 3000-7000 rpm for 10-30 min, dialyzing against 0.1-0.3 mol / L sodium chloride solution, and adjusting the pH of the solution to 7-7.4.

[0027] Preferably, in step (b), the heating temperature is 37-45°C, the heating time is 30-60 min, and the mixture is shaken every 5-15 min during the heating process; the bio-ink precursor A is filtered through a 0.22 μm sterile filter;

[0028] and / or in step (c), the heating temperature is 60-90°C, the heating time is 30-60 min, and the stirring speed is 100-1000 rpm;

[0029] And / or in step (d), the bio-ink precursor B is added to MMBO x Before filtration, the MMBO was filtered using a 0.22 μm sterile filter head. x Carry out UV sterilization in advance;

[0030] And / or in step (e), the mixed bio-ink comprises the following raw material components in a weight-to-volume ratio (w / v): 8%-15% GelMA-PBA, 1%-10% polyvinyl alcohol (PVA), 0.15%-0.3% lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), 0.01%-1% MMBO x , w / v means mg / mL.

[0031] Preferably, in step (d), the MMBO x Obtained by a preparation method comprising the following steps:

[0032] (1) Dissolve NaBiO3 and NaOH in deionized water, add soluble magnesium salt and soluble manganese salt, and stir at room temperature to form a suspension;

[0033] (2) The suspension is transferred to a reactor, heated for reaction, and the obtained reaction product is separated, washed, and dried to obtain magnesium-manganese-doped vacancy bismuth oxide;

[0034] (3) The magnesium-manganese-doped vacancy bismuth oxide is dispersed in a mixed solution of deionized water and ethanol, ultrasonically crushed with ice water, centrifuged to obtain the supernatant, centrifuged again, and the precipitate is washed and dried to obtain the product.

[0035] More preferably, in step (1), the soluble magnesium salt is MgCl2, and the soluble manganese salt is MnCl2·4H2O; and / or the mass ratio of NaBiO3, soluble magnesium salt and soluble manganese salt is 1-2:0.01-0.025:0.03-0.045; and / or the stirring time at room temperature is 1-3 h;

[0036] And / or in step (2), the heating reaction temperature is 160-190°C, the heating reaction time is 3-6 h, and the drying temperature is 50-70°C;

[0037] And / or in step (3), the volume ratio of deionized water to ethanol in the mixed solution is 1:1, the mass volume ratio of the magnesium-manganese-doped vacancy bismuth oxide to the mixed solution is 0.4-0.7 g / 30 mL, the ice water ultrasonic crushing temperature is 0-10°C, the ultrasonic time is 2 h; the first centrifugal speed is 2000-4000 rpm, the time is 10-20 min; the second centrifugal speed is 8000-9000 rpm, and the time is 15-30 min.

[0038] Preferably, in step (e), the mixed bio-ink is injected and extruded and then photocured, the process comprising: adding the mixed bio-ink into a syringe, extruding, and placing it under a blue light source with a wavelength of 405 nm and a power of 3 W for photocuring for 30 to 120 s.

[0039] In a second aspect, the present invention provides a hydrogel material for antibacterial and tissue repair, which is obtained by any of the aforementioned methods for preparing a hydrogel material for antibacterial and tissue repair.

[0040] The third aspect of the present invention provides use of the hydrogel material in preparing products for antibacterial and tissue repair promotion.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] The present invention adopts a one-step hydrothermal method to synthesize MMBO x , the preparation method is efficient and simple, MMBO x It is nano-doped with Mn, Mg ions and bismuth oxide containing oxygen vacancies, which has ROS programmable ability. It can promote the production of ROS by improving the sonodynamic / enzyme activity through piezoelectric effect and oxygen vacancy incorporation, and upregulate ROS capacity. x The mixed bio-ink compounded with GelMA-PBA was injection molded, and the resulting product was placed under a blue light source for photocuring to obtain a hydrogel material, which has the ability to regulate ROS and buffer the ROS production rate under the dual effects of ultrasound and near-infrared light (NIR), which can reduce tissue damage. The preparation of products for antibacterial and tissue repair promotion has important clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Conventional transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM) and lattice images of the samples in the examples.

[0044] Figure 2 is the X-ray diffraction (XRD) pattern of the sample in the embodiment.

[0045] Figure 3The X-ray photoelectron spectroscopy (XPS) spectrum of the samples in the examples is shown in FIG.

[0046] Figure 4 The electron paramagnetic resonance (EPR) spectrum of the samples in the examples is shown in FIG.

[0047] Figure 5 These are the results of measuring ROS production under ultrasonic action on the materials in the examples.

[0048] Figure 6 These are the results of photothermal and ROS generation measurements of the materials in the examples under infrared action.

[0049] Figure 7 These are the enzyme-like activity results of the materials in the examples.

[0050] Figure 8 For the hydrogel material in the embodiment, anti-Staphylococcus aureus ( S. aureus )ability.

[0051] Figure 9 For the hydrogel material in the embodiment, the anti-Escherichia coli ( E. coli )ability.

[0052] Figure 10 In vivo anti- S. aureus ability.

[0053] Figure 11 This is the ability of the hydrogel material in the examples to promote the repair of skin defects in infected rats. DETAILED DESCRIPTION

[0054] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0055] The GelMA-PBA used in the following examples was prepared by the following method: 0.5 g of methacrylated gelatin was added to 25 mL of deionized water and heated with stirring at 40°C in the dark to obtain a transparent GelMA solution. Precursor solution A was obtained by dissolving 2 mmol of DMTMM in 6 mL of water, and 2 mmol of PBA was dissolved in 9 mL of DMF to obtain precursor solution B. Precursor solution A was slowly added dropwise to precursor solution B to obtain PBA solution, and the PBA solution was activated at room temperature for 30 minutes. The activated PBA solution was added dropwise to the GelMA solution, and the pH of the solution was adjusted to 4.5 with concentrated hydrochloric acid. The reaction was allowed to proceed at room temperature for 24 hours. The resulting solution was centrifuged at 4000 rpm for 10 minutes. The supernatant was removed and dialyzed against 0.1 mol / L sodium chloride solution for 3 days to adjust the pH to 7.4. The solution was then lyophilized to obtain GelMA-PBA.

[0056] MMBO used in the following examples x The product was prepared by the following method: 1.58 g of NaBiO₃ and 1.12 g of NaOH were dispersed in 30 mL of deionized water. 0.02 g of anhydrous magnesium chloride and 0.04 g of MnCl₂·4H₂O were added to the mixture and stirred at room temperature for 2 h. The solution was then transferred to a stainless steel hydrothermal reactor lined with polytetrafluoroethylene, sealed, and reacted at 180°C for 5 h. After the reaction, the reactor was removed and allowed to cool to room temperature. The lid was opened, the product was removed, filtered, washed several times with deionized water and anhydrous ethanol, and dried at 60°C under atmospheric pressure to obtain the pre-product. 0.5 g of the pre-product was dispersed in 30 mL of a mixture of deionized water and ethanol (1:1 volume ratio), ultrasonically dispersed in ice water for 2 h, and then centrifuged at 3000 rpm for 10 min. The supernatant was collected and centrifuged at 8000 rpm for 30 min, and the resulting precipitate was dried at 60°C under atmospheric pressure to obtain the final product, MMBO. x .

[0057] Unless otherwise specified, other raw materials or processing techniques are conventional commercially available raw materials or conventional operations in this field.

[0058] Example 1

[0059] The hydrogel material was prepared in this embodiment, and the specific steps are as follows:

[0060] 1. Material ratio: Bio-ink precursor A was prepared by 10% (w / v) phenylboronic acid grafted methacryloyl gelatin and 0.25% (w / v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate; bio-ink precursor B was prepared by 10% (w / v) polyvinyl alcohol and 0.25% (w / v) phenyl (2,4,6-trimethylbenzoyl) lithium phosphate.

[0061] 2. Dissolve phenyl (2,4,6-trimethylbenzoyl) lithium phosphate with deionized water to prepare a 0.25% (w / v) solution. Place it in a light-proof glass container, shake it by hand to mix it, and heat it in a 37°C water bath. Shake it by hand every 10 minutes. After 30 minutes, observe it to ensure that it is fully dissolved. This is recorded as the photoinitiator solution.

[0062] 3. Weigh 10% (w / v) of phenylboronic acid-grafted methacryloyl gelatin and add it to the above photoinitiator solution. Shake to mix thoroughly and heat in a 37°C water bath. Shake to mix thoroughly every 10 minutes. After 60 minutes, observe and confirm that the solution is fully dissolved. Filter it through a 0.22 μm sterile filter and record it as bio-ink precursor A.

[0063] 4. Weigh 10% (w / v) polyvinyl alcohol and add it to the above photoinitiator solution. Heat and stir at 80°C and 500 rpm. After 60 min, observe whether it is fully dissolved. Filter it through a 0.22 μm sterile filter head and record it as bio-ink precursor B.

[0064] 5. Use UV sterilization to sterilize MMBO x Sterilize and aseptically weigh 0.5% (w / v) MMBO x Add it to the bio-ink precursor B and mix it evenly by ultrasonication, which is recorded as bio-ink precursor C.

[0065] 6. Bio-ink precursor A and bio-ink precursor C were mixed evenly in a volume ratio of 3:1, and recorded as mixed bio-ink.

[0066] 7. The mixed bio-ink was added to a syringe, injected and extruded, and then placed under a blue light source with a wavelength of 405 nm and a power of 3 W for photocuring for 30 to 120 s to obtain a hydrogel material, which was recorded as MGA.

[0067] Comparative Example 1

[0068] Compared with Example 1, most of the steps are the same, except that MMBO is omitted. x The obtained hydrogel material was recorded as GA.

[0069] Figure 1 TEM, HRTEM and lattice images of the obtained samples are shown. x (Left) and MMBO x (Right) There is no obvious difference in morphology, and the particle size is about 50nm. TEM HRTEM also observed that due to the generation of oxygen vacancies, the interplanar spacing attributed to (200) in BO x The MMBO x The interplanar spacing of BO is slightly reduced (0.283nm) due to the influence of doping. x and MMBO x It is also observed that disordered lattice defects are generated due to the influence of oxygen vacancies and doping. This lattice distortion can increase polarity and destroy the central symmetric structure, thus showing strong piezoelectricity. The corresponding mapping diagram shows that Bi and O in BO x and MMBO x Evenly distributed in MMBO, Mn and Mg elements x Evenly distributed.

[0070] Figure 2 is the XRD pattern of the obtained sample. x and MMBO xThe diffraction peaks match those of bismuth oxide with space group Fm3m (JCPDS#47-1057) ( Figure 2 A). with BO x In comparison, MMBO x The crystallinity of the diffraction peak decreases slightly, and the diffraction peak shifts to a higher angle ( Figure 2 B), which may be due to the further introduction of oxygen defects by the doping of manganese and magnesium and the smaller ionic radius of manganese and magnesium than that of bismuth.

[0071] Figure 3 is the XPS spectrum of the obtained sample, where the XPS total spectrum ( Figure 3 A) and the fine maps of Mn, Bi, Mg, and O ( Figure 3 BF). In the XPS total spectrum ( Figure 3 A) Medium, BO x and MMBO x Signal peaks of Bi and O elements were observed. x The signal peaks of Mn and Mg elements were further observed. Figure 3 B) shows that Mn element exists mainly in divalent and tetravalent forms, indicating that MMBO x It has strong nanozyme activity and potential for MRI imaging. High-resolution Bi 4f spectroscopy ( Figure 3 C) shows that doping causes the binding energy of trivalent bismuth to shift to higher angles. High-resolution Mg 1s spectrum ( Figure 3 D) indicates that Mg exists in a divalent form. The high-resolution O 1s spectrum analysis shows that the BO x ( Figure 3 E) compared to (49.53%), doping ( Figure 3 F) leads to an increase in the oxygen vacancy content (63.1%).

[0072] Figure 4 is the EPR spectrum of the obtained sample. x An oxygen vacancy signal of unpaired electrons is shown at g=2.002, indicating the generation of oxygen vacancies. The peak intensity is further enhanced after doping, which is consistent with the XPS results.

[0073] Figure 5 The ROS production of the obtained sample under ultrasound was determined by ESR. Figure 5 A) and O2 - ( Figure 5 B) the generation of BO x and MMBO x The results showed that after 5 min of ultrasound (US) irradiation, BO x and MMBO xThe signal intensity increased significantly, and MMBO x The signal absorption peak is the strongest. Further detection of methylene blue (MB) (·OH indicator, Figure 5 C) and 1,3-diphenylisobenzofuran (DPBF) (·O2 - indicator, Figure 5 D) The UV absorption peak intensity of the indicator shows that MMBO x With the lowest absorbance, it means MMBO x It has the best ultrasonic catalytic performance.

[0074] Figure 6 The photothermal and ROS generation of the obtained sample under infrared action is measured. In a dry environment, after irradiation for 200 seconds, BO x and MMBO x The temperature rose rapidly from 22.5℃ to 104.7℃ and 109.0℃ respectively ( Figure 6 A); In a humid environment, BO x and MMBO x The temperature increased from 25.2℃ to 47.8℃ and 57.0℃ respectively ( Figure 6 B); Five near-infrared laser on / off cycles to test MMBO x Photothermal reversibility ( Figure 6 C), MMBO x The absorbance of DPBF + US + NIR (420 nm) is significantly lower than that of MMBO x +US, indicating that near-infrared irradiation effectively accelerates ultrasound-induced O2 - Spawn Rate ( Figure 6 D); MMBO x The absorbance of MB (664 nm) in the +US+NIR system is significantly lower than that of MMBO. x +US system, indicating that near-infrared radiation-induced hyperthermia effectively accelerates ultrasound-induced ·OH generation ( Figure 6 E); MMBO x The absorbance of +US in a water bath (55°C) was significantly lower than that at room temperature (25°C) over time ( Figure 6 F) Verify MMBO x The photothermally enhanced ROS generation under +US irradiation was caused by the increase in temperature.

[0075] Figure 7 The enzyme-like activity of the material is due to the formation of oxygen vacancies and the inherent multivalence of manganese. x and MMBO xIt may exhibit mimetic enzyme activity. Using 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate, the OXD-like activity was studied at pH 5.5. Figure 7 As shown in A, MMBO x The UV-visible absorption peak is higher than that of BO x , indicating that manganese doping enhances OXD-like activity. After US stimulation, the sound piezoelectric catalysis and piezoelectric effect drive BO x and MMBO x Electrons migrate to the surface and participate in redox reactions under the action of the inherent electric field. x +US's O2 - The generation rate is higher than that of BOx+US. x pH responsiveness of OXD-like activity was investigated by setting different pH values ​​(4.5, 5.5, 6.5, 7.4) ( Figure 7 B). As the pH value gradually decreases, the UV absorption peak at 652nm becomes stronger and stronger. These results show that MMBO x Can intelligently trigger OXD-like activity to produce O2 in an acidic environment - , and was synergistically enhanced under piezoelectric effect and US stimulation. In addition, TMB was used as an indicator to evaluate POD-like activity in the presence of H2O2. Figure 7 As shown in C and 7D, similar to OXD-like activity, MMBO x The enzyme activity is stronger under acidic conditions and is significantly enhanced under US drive. Since both OXD-like activity and sonic electrocatalysis require oxygen as a substrate for reaction, H2O2 was used as a substrate and a dissolved oxygen meter was used to detect CAT-like activity under different pH conditions. Figure 7 As shown in E, MMBO x These results indicate that MMBO-based x The valence state conversion of Mn ions ( ) can generate oxygen through CAT-like activity, which is then converted into O2 by OXD-like activity and sonication electrocatalysis. - , forming a cascade catalysis to promote ROS production. In addition, acoustic piezoelectric catalysis can synergistically enhance the activities of OXD-like, POD-like, and CAT-like to produce a large amount of ROS.

[0076] Excessive ROS in inflamed tissues is an important factor that hinders tissue repair. x and MMBO x CAT-like enzyme activity, the results showed that ( Figure 7 F), MMBO x The most oxygen is produced, which is attributed to the increase of manganese ion doping and oxygen vacancies. MMBO was verified by 5 cycles of reaction. x Stable CAT activity ( Figure 7 G). MMBO was calculated by Michaelis-Menten equation x Km and Vmax ( Figure 7 H), were 11.29 mM and 3.36 mg·L -1 min -1 BO was detected using SOD kit. x and MMBO x The results showed that MMBO x Shows better than BO x Higher O2 - Clearance ability ( Figure 7 I) and there is cyclic stability ( Figure 7 J). MMBO was further calculated x The SOD-like activity unit is 112.5U / mg ( Figure 7 K). BO was detected by using DPPH as the reaction substrate. x and MMBO x The results showed that MMBO has the ability to scavenge total free radicals. x The lowest absorption peak intensity means the best total free radical scavenging ability ( Figure 7 L). These results indicate that the valence state of manganese ions is converted ( ) and the multiple reaction sites provided by oxygen vacancies can remove H2O2 and O2 - etc. ROS, alleviating ROS disorder in the process of tissue repair.

[0077] Figure 8 The obtained hydrogel is anti-Staphylococcus aureus ( S. aureus ) ability. Low concentration of H2O2 was used to simulate the presence of low concentration of ROS in bacterial infection environment. Under ultrasound, the MGA+H2O2 group S. aureus The survival rate was significantly reduced, only 24.5% of the control group ( Figure 8 A, 8C). Under the combined intervention of US and NIR, the MGA+H2O2 group had the strongest antibacterial activity, and its activity was only 3.3% of that of the control group, proving that MMBO xIts excellent POD enzyme activity and photoacoustic dynamics are used to achieve excellent antibacterial effects. Persistent infections usually have defensive biofilms that show strong resistance to antibacterial effects, hindering the efficacy of antibacterial interventions and ultimately leading to the emergence of refractory bacterial infections. Crystal violet staining and live / dead staining were used to evaluate the destructive effect of MGA on biofilms under US, NIR and combined interventions. Under different interventions, the staining intensity of the biofilm showed a trend of gradually decreasing from the GA group, MGA group to the MGA+H2O2 group, and from no external field intervention, NIR, US to combined intervention ( Figure 8 B). The absorbance results at 590nm showed that the corresponding absorbance of the bacterial biofilm in the MGA+H2O2 group combined with US and NIR intervention was only 20%, which was the most destructive to the biofilm ( Figure 8 D). Live / dead staining results showed that the green fluorescence intensity of the MGA+H2O2 group under the intervention of US and NIR was significantly reduced, and the bacterial biofilm was severely damaged and fragmented ( Figure 8 E). SEM evaluated the post-treatment S. aureus Membrane integrity and morphology ( Figure 8 F). The bacteria in the MGA group were intact and round. Some bacteria in the MGA+H2O2 group, US, and NIR-treated MGA and MGA+H2O2 groups showed structural damage, with proteins leaking out of the membrane. Under combined NIR and US treatment, most bacteria in the MGA and MGA+H2O2 groups showed surface cracks, with internal substances leaking out of the membrane.

[0078] Figure 9 The obtained hydrogel is anti-Escherichia coli ( E. coli ) ability. First, the bacterial coating method was used to evaluate the effects of different hydrogel materials on Escherichia coli ( E. coli ) in vitro antibacterial activity, low concentration of H2O2 was used to simulate the presence of low concentration of ROS in the bacterial infection environment. Under ultrasound, the MGA+H2O2 group E. coli The survival rate was significantly reduced, only 20.4% of the control group ( Figure 9 A, 9B). Under the combined intervention of US and NIR, the MGA+H2O2 group had the strongest antibacterial ability. E. coli The activity of the biofilm was only 1.3% of that in the control group. Crystal violet staining was used to evaluate the destructive effect of MGA on biofilms under US, NIR, and combined intervention. Under different interventions, the staining intensity of the biofilm showed a trend of gradual decrease from the GA group, MGA group to the MGA+H2O2 group, and from no external field intervention, NIR, US to combined intervention ( Figure 9 C). The absorbance results at 590nm showed that the corresponding absorbance of the bacterial biofilm in the MGA+H2O2 group combined with US and NIR intervention was only 20%, which was the most destructive to the biofilm ( Figure 9 D). SEM evaluated the post-treatment E. coliMembrane integrity and morphology ( Figure 9 E). The bacteria in the MGA group were intact and round. Some bacteria in the MGA+H2O2 group, US, and NIR-treated MGA and MGA+H2O2 groups showed structural damage, with proteins leaking out of the membrane. Under combined NIR and US treatment, most bacteria in the MGA and MGA+H2O2 groups showed surface cracks, with internal substances leaking out of the membrane.

[0079] Figure 10 Hydrogel in vivo S. aureus Ability. Inoculation in the back of rats S. aureus A full-thickness infected skin defect model was constructed. After implantation of the composite hydrogel, different external field interventions were applied to investigate the therapeutic efficacy of the hydrogel material based on the ROS regulation strategy in promoting infection-related tissue repair. Three days after US and NIR treatment, interstitial fluid from the wound tissue was collected and bacterial plating was performed to evaluate in vivo antimicrobial efficacy. The MGA+NIR+US group showed minimal bacterial growth, only 10% of the control group.

[0080] Figure 11 The ability of hydrogel to promote the repair of skin defects in infected rats. On day 0, we established full-thickness skin defects and placed 10 6 CFU S. aureus Bacterial suspension was dripped onto the wound surface. After 12 hours of infection induction, different groups of composite hydrogel materials were implanted into the full-thickness skin defect of rats. The wound healing process was recorded every two days to better evaluate the comprehensive effect of the hydrogel material in vivo in terms of antibacterial and repair promotion. Typical wound repair pictures are shown below. Figure 11 As shown in A and 11B, the wound area of ​​all groups showed a downward trend with the extension of time. On the 1st day, the hydrogel was evenly coated on the wound surface. In addition, the Control and MGA groups did not show obvious repair effects, which may be attributed to the failure to effectively control their wound infections. In the following 3-9 days, the wounds of rats in each group showed an accelerated healing process, and the experimental groups were better than the control group. Specifically, on the 9th day, the wound area gradually decreased from the MGA group, MGA+NIR, MGA+US to MGA+NIR+US. This trend indicates that MGA has effective antibacterial and tissue repair capabilities, and is activated under the action of NIR and US. In addition, in order to further determine the skin repair effect, the rats were euthanized on the 9th day after surgery, and the wound tissue was collected for histological analysis. Hematoxylin and eosin (H&E) staining showed that the defect healing in the Control and MGA groups was poor, with obvious neutrophil infiltration. The MGA+NIR and MGA+US groups showed a certain degree of healing. The MGA+US+NIR group basically completed epithelialization, with the least neutrophil infiltration. Masson staining results showed that the MGA+US+NIR group showed the most collagen deposition ( Figure 11 C).

[0081] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A method for preparing a hydrogel material for antibacterial and tissue repair, characterized in that: The following steps are involved: (a) dissolving lithium phenyl (2,4,6-trimethylbenzoyl) phosphate in deionized water and heating in a water bath in a dark environment to obtain a photoinitiator solution; (b) adding phenylboronic acid-grafted methacryloyl gelatin to the photoinitiator solution, heating, and fully dissolving to obtain bio-ink precursor A; (c) adding polyvinyl alcohol to the photoinitiator solution, heating and stirring to fully dissolve the solution, thereby obtaining a bio-ink precursor B; (d) Adding magnesium manganese doped vacancy bismuth oxide nanomaterials, abbreviated as MMBO, to the bio-ink precursor B x , mixed evenly to obtain bio-ink precursor C; (e) The bio-ink precursor A and the bio-ink precursor C are mixed evenly to obtain a mixed bio-ink; the mixed bio-ink is formed by injection extrusion and then photocuring.

2. The method for preparing the hydrogel material according to claim 1, wherein: In step (a), the water bath heating temperature is 37-45°C, the water bath heating time is 30-60 min, and the mixture is shaken every 5-15 min during the heating process.

3. The method for preparing the hydrogel material according to claim 1, wherein: In step (b), the phenylboronic acid grafted methacryloyl gelatin is prepared by the following method: (b1) GelMA was added to deionized water and heated with stirring in a dark environment to obtain a transparent GelMA solution; (b2) Dissolving phenylboronic acid (PBA) in DMF to obtain precursor solution A; dissolving DMTMM in water to obtain precursor solution B; slowly adding precursor solution A dropwise to precursor solution B to obtain a mixed solution, and activating the solution at room temperature to obtain a PBA solution; (b3) adding the PBA solution dropwise to the GelMA solution, adjusting the pH value of the solution, and reacting at room temperature to obtain a reaction solution; (b4) The reaction solution is centrifuged and the supernatant is collected, dialyzed, the pH value of the solution is adjusted, and freeze-dried to obtain the product.

4. The method for preparing the hydrogel material according to claim 3, wherein: In step (b1), the volume ratio of GelMA to deionized water was 1:50; And / or in step (b2), the precursor solution A is a solution formed by dissolving 2-3 mmol PBA in 9 mL DMF, the precursor solution B is a solution formed by dissolving 2-3 mmol DMTMM in 6 mL water, and the activation time at room temperature is 30-45 min; And / or in step (b3), the pH of the solution is adjusted to 4.5-6, and the reaction time at room temperature is 20-25 h; And / or in step (b4), centrifuging at 3000-7000 rpm for 10-30 min, dialyzing against 0.1-0.3 mol / L sodium chloride solution, and adjusting the pH of the solution to 7-7.

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5. The method for preparing the hydrogel material according to claim 1, wherein: In step (b), the heating temperature is 37-45° C., the heating time is 30-60 min, and the mixture is shaken every 5-15 min during the heating process; the bio-ink precursor A is filtered through a 0.22 μm sterile filter; and / or in step (c), the heating temperature is 60-90°C, the heating time is 30-60 min, and the stirring speed is 100-1000 rpm; And / or in step (d), the bio-ink precursor B is added to MMBO x Before filtration, the MMBO was filtered using a 0.22 μm sterile filter head. x Carry out UV sterilization in advance; And / or in step (e), the mixed bio-ink comprises the following raw material components in a mass volume ratio: 8% to 15% of phenylboronic acid grafted methacryloyl gelatin, 1% to 10% of polyvinyl alcohol, 0.15% to 0.3% of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, 0.01% to 1% of MMBO x , mass-to-volume ratio is mg / mL.

6. The method for preparing the hydrogel material according to claim 1, wherein: In step (d), the MMBO x Obtained by a preparation method comprising the following steps: (1) Dissolve NaBiO3 and NaOH in deionized water, add soluble magnesium salt and soluble manganese salt, and stir at room temperature to form a suspension; (2) The suspension is transferred to a reactor, heated for reaction, and the obtained reaction product is separated, washed, and dried to obtain magnesium-manganese-doped vacancy bismuth oxide; (3) The magnesium-manganese-doped vacancy bismuth oxide is dispersed in a mixed solution of deionized water and ethanol, ultrasonically crushed with ice water, centrifuged to obtain the supernatant, centrifuged again, and the precipitate is washed and dried to obtain the product.

7. The method for preparing the hydrogel material according to claim 6, characterized in that: In step (1), the soluble magnesium salt is MgCl2, and the soluble manganese salt is MnCl2·4H2O; and / or the mass ratio of NaBiO3, soluble magnesium salt and soluble manganese salt is 1-2:0.01-0.025:0.03-0.045; and / or the stirring time at room temperature is 1-3 h; And / or in step (2), the heating reaction temperature is 160-190°C, the heating reaction time is 3-6 h, and the drying temperature is 50-70°C; And / or in step (3), the volume ratio of deionized water to ethanol in the mixed solution is 1:1, the mass volume ratio of the magnesium-manganese-doped vacancy bismuth oxide to the mixed solution is 0.4-0.7 g / 30 mL, the ice water ultrasonic crushing temperature is 0-10 ° C, the ultrasonic time is 2 h; the first centrifugal speed is 2000-4000 rpm, the time is 10-20 min; the second centrifugal speed is 8000-9000 rpm, and the time is 15-30 min.

8. The method for preparing the hydrogel material according to claim 1, wherein: In step (e), the mixed bio-ink is injected and extruded and then photocured, the process comprising: adding the mixed bio-ink into a syringe, extruding, and photocuring under a blue light source with a wavelength of 405 nm and a power of 3 W for 30 to 120 seconds.

9. A hydrogel material for antibacterial and tissue repair, characterized in that: The hydrogel material is obtained by the preparation method of any one of claims 1 to 8 for antibacterial and tissue repair.

10. Use of the hydrogel material according to claim 9 in preparing products for antibacterial and tissue repair promotion.

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