Multifunctional MXMoS-DNA hydrogel for infection-responsive photo-thermal / immunoregulation / osteogenesis and preparation method of multifunctional MXMoS-DNA hydrogel

Abstract

The invention discloses multifunctional MXMoS2-DNA hydrogel for infection responsive photo-thermal / immunoregulation / osteogenesis and a preparation method of the multifunctional MXMoS2-DNA hydrogel. The preparation method comprises the following steps: (1) preparing a Ti < 3 > C < 2 > T < x > MXene dispersion liquid; (2) preparing a MoS2 quantum dot solution; (3) doping the MoS2 quantum dots with Ti < 3 > C < 2 > T < x > MXene; (4) preparing DNA hydrogel; and (5) preparation of the MXMoS2-DNA composite hydrogel. The hydrogel prepared by the invention can improve the photothermal conversion efficiency and stability of MXene, is suitable for a physiological environment, realizes accurate drug release triggered by pathogenic enzyme (such as DNase), regulates an immune microenvironment, promotes polarization of M2 type macrophages, activates a Wnt pathway, promotes osteogenic differentiation and mineralization, and has a good application prospect. The comprehensive treatment requirements of infectious jaw defect repair can be met.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical gel materials, and specifically relates to an infection-responsive photothermal / immunomodulatory / osteogenic multifunctional MXMoS2-DNA hydrogel and a preparation method thereof. Background Art

[0002] Mandibular osteomyelitis, a severe maxillofacial infection often caused by Staphylococcus aureus, is difficult to treat due to its complex anatomy; poor blood supply, which hinders antibiotic penetration; and pathogenic biofilms that impede drug penetration. Traditional treatments, such as long-term antibiotic use or surgical debridement, often have limited efficacy and can lead to residual infection, chronic inflammation, and poor bone healing. Furthermore, the growing prevalence of antibiotic resistance necessitates the development of antibiotic-free, localized treatments.

[0003] In recent years, photothermal therapy (PTT) has attracted attention due to its ability to kill bacteria through near-infrared (NIR)-induced thermal effects without the need for antibiotics. Among them, the two-dimensional material MXene (Ti3C2Tx) has excellent photothermal properties and electrical conductivity and has been used to construct photothermal antibacterial materials. However, the single component of the two-dimensional material MXene has the following main defects: (1) poor stability, easy oxidation, easy stacking, and narrow absorption band; (2) photothermal conversion efficiency depends on LSPR and is easily affected by the physiological environment; (3) it only has bactericidal function and lacks immune regulation and osteogenesis; (4) lacks infection-responsive release mechanism: most systems release non-selectively, which may cause thermal damage to non-infected areas; (5) it cannot recognize pathogen-specific enzymes such as bacterial DNase. To improve these deficiencies, some studies have attempted to composite the two-dimensional material MXene with other materials, such as MoS2 (a narrow-bandgap semiconductor with broad spectral absorption and non-radiative relaxation capabilities), to enhance its photothermal efficiency. Furthermore, DNA hydrogels have been used as novel smart materials for controlled drug release, but research into multifunctional synergistic treatments, such as infection responsiveness, immune regulation, and osteogenesis, remains limited. Furthermore, traditional anti-infective bone repair materials struggle to balance antibacterial, anti-inflammatory, and osteogenesis functions. While osteogenic induction factors such as BMP-2 have repair capabilities, they are prone to failure in infected environments. Therefore, constructing an MXMoS2-DNA hydrogel system with photothermal sterilization, immune regulation, osteogenesis promotion, and infection-triggered response capabilities to achieve multifunctional synergistic treatment and accurately and efficiently repair infected jaw defects is a technical challenge that needs to be addressed. Summary of the Invention

[0004] In order to address the deficiencies in the prior art, the present invention aims to provide an infection-responsive photothermal / immunomodulatory / osteogenic multifunctional MXMoS2-DNA hydrogel and a preparation method thereof.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: A method for preparing an infection-responsive photothermal / immunomodulatory / osteogenic multifunctional MXMoS2-DNA hydrogel comprises the following steps: Step S1, Ti3C2T X Preparation of MXene dispersion: Weigh Ti3AlC2 powder and slowly add it into the pre-made HF solution. Stir magnetically at room temperature for 24 hours to complete the selective etching of Al element and generate Ti3C2T X MXene nanosheets, the Ti3C2T X MXene nanosheets were prepared and the supernatant was separated by centrifugation until the pH value of the washing liquid was close to neutral (pH value was 6-7) to obtain Ti3C2T X MXene precipitation, the Ti3C2T X The MXene precipitate was resuspended in deionized water to obtain a Ti3C2T3O4 solution with a concentration of 12 mg / mL. X MXene dispersion, set aside; Step S2, preparation of MoS2 quantum dot solution: adding MoCl5 or MoO3 to deionized water, adjusting the pH with NaOH solution, ultrasonically treating to form a clear solution, sequentially adding bovine serum albumin solution and Na2S solution to the clear solution, and then adjusting the pH with HCl solution to obtain a MoS2 quantum dot solution for later use; Step S3, MoS2 quantum dots doped with Ti3C2T X MXene: The MoS2 quantum dot solution obtained in step S2 is slowly added to the Ti3C2T X The MXene dispersion was ultrasonically stirred to uniformly adsorb / bind the MoS2 quantum dots to the MXene surface to form a mixed solution, which was then transferred to a tetrafluoroethylene autoclave for high-temperature reaction. After the high-temperature reaction was completed, the mixture was naturally cooled, centrifuged, and washed three times to obtain a MoS2 quantum dot-doped MXene dispersion. Ti3C2T X MXene composite nanomaterials, namely MXMoS2 composite nanomaterials, are reserved; Step S4, preparation of DNA hydrogel: dissolve the short-chain DNA in PBS buffer, slowly add a cross-linking agent, and allow to stand to form a transparent homogeneous DNA hydrogel for later use; Step S5, preparation of MXMoS2-DNA composite hydrogel: the MXMoS2 composite nanomaterial obtained in step S3 was dispersed in PBS buffer at a concentration of 12 mg / mL to obtain MXMoS2 dispersion, the MXMoS2 dispersion is slowly added to the MXMoS2 dispersion obtained in step S4 The DNA hydrogel is stirred and mixed evenly, a cross-linking agent is added to induce cross-linking and gel formation, and the gel is allowed to stand at a constant temperature to form an MXMoS2-DNA hydrogel.

[0006] Furthermore, in step S1, the mass volume ratio of Ti3AlC2 powder to HF solution is 1 g:10 mL; the centrifugal speed is 3500 rpm, and the centrifugal time is 10 min.

[0007] Furthermore, the specific method for preparing the MoS2 quantum dot solution in step S2 is as follows: first, MoCl5 or MoO3 is added to deionized water, and then the pH is adjusted to 11 with NaOH solution, and ultrasonic treatment is performed to form a clear solution; then 1 mL of the clear solution is added to 39 mL of 1 mg / mL bovine serum albumin solution, and the mixture is stirred at room temperature to mix evenly, 0.2 mL of 0.5 M Na2S solution is added thereto, and the mixture is vigorously stirred for 5 minutes to obtain a mixed solution, and then 1 M HCl solution is added to the mixed solution to adjust the pH to 6-7, and a clear light yellow MoS2 quantum dot solution is formed during the reaction; finally, the obtained clear light yellow MoS2 quantum dot solution is subjected to ultrafiltration or dialysis to remove free ions, and concentrated to obtain MoS2 quantum dot solution.

[0008] Furthermore, in step S3, the MoS2 quantum dot solution and Ti3C2T X The volume ratio of the MXene dispersion is 1:(3-5); the ultrasonic time is 30 minutes; the high-temperature reaction temperature in a tetrafluoroethylene autoclave is 180°C, and the reaction time is 6-12 hours.

[0009] Furthermore, the specific process of preparing the DNA hydrogel in step S4 is as follows: First, the short-chain DNA is dissolved in PBS buffer and its concentration is adjusted to 10 mg / mL DNA solution; then, slowly add PEG-dithio crosslinker is used to form a transparent homogeneous DNA hydrogel at 4°C or room temperature. Finally, DNA hydrogel products of different forms are prepared by freeze-drying or microinjection.

[0010] Furthermore, the short-chain DNA is purchased from the market at a concentration of 5-20 mg / mL; the molar ratio of the DNA solution to the PEG-dithio crosslinker is 1:1; and the standing time is 30-60 min.

[0011] Furthermore, in step S5, the mass ratio of the MXMoS2 composite nanomaterial, PBS buffer and DNA hydrogel is 1:51:10.

[0012] Furthermore, a multifunctional infection-responsive photothermal / immunomodulatory / osteogenic The MXMoS2-DNA hydrogel is prepared by the above-mentioned preparation method of the infection-responsive photothermal / immunomodulatory / osteogenic multifunctional MXMoS2-DNA hydrogel.

[0013] Compared with the prior art, the present invention has the following positive and beneficial effects: (1) The present invention enhances the NIR absorption bandwidth and non-radiative relaxation ability of MXene by in-situ doping with MoS2, which not only improves the photothermal conversion efficiency of the two-dimensional material MXene (Ti3C2Tx), but also significantly improves its oxidation stability and layered structure retention ability, achieving key performances such as rapid heating, concentration dependence, and low thermal fatigue.

[0014] (2) The present invention utilizes DNA enzyme (DNase) secreted by Staphylococcus aureus as a triggering factor to achieve selective degradation of DNA hydrogel in the lesion area, control the release of MXMoS2, and exert therapeutic effects only in the infection environment, with higher localization and biosafety.

[0015] (3) The hydrogel prepared by the present invention is an injectable hydrogel with good rheological properties and biocompatibility. It can be precisely injected into the infected site by minimally invasive means and is particularly suitable for diseases such as mandibular osteomyelitis. In addition, while achieving photothermal sterilization, it can also regulate the polarization of macrophages from M1 to M2, reduce local inflammation, and effectively promote the osteogenic differentiation and mineralization of MC3T3-E1 by activating the bone formation signaling pathway with Wnt3a as the core. It has obvious advantages of "bone immune regulation" and "antibacterial-osteogenic synergy".

[0016] (4) The present invention adopts a strategy of combining photothermal + intelligent release + immune regulation, which does not require the use of traditional antibiotics and can effectively reduce the risk of drug resistance and antibiotic side effects, and is more in line with the development trend of antibiotic-free infection treatment in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the preparation process of MXMoS2 composite nanomaterial of the present invention; Figure 2 Schematic diagram of the preparation process of MXMoS2-DNA hydrogel of the present invention; Figure 3 This is a photothermal response test diagram of the MXMoS2 composite nanomaterial of the present invention; wherein, Figure 3 a is the MXMoS2 composite nanomaterial of the present invention and MXene, MoS2 at a wavelength of Infrared thermal image of infrared light irradiation at 808nm and power of 2.0W / cm2 for more than 120s; Figure 3b is a temperature rise curve of the MXMoS2 composite nanomaterial of the present invention at different concentrations under infrared light irradiation; Figure 3 c is a photothermal cycle performance diagram of the MXMoS2 composite nanomaterial of the present invention in five on / off infrared light cycles; Figure 4 is a structural representation diagram of the MXMoS2 composite nanomaterial of the present invention; wherein, FIG. 4a is the SEM image of MXene material; Figure 4 b is the SEM image of MoS2 material; Figure 4 c is the SEM image of MXMoS2 composite nanomaterials; Figure 4 d is the Fourier transform infrared spectrum of MXMoS2 composite nanomaterials; Figure 4 e is the X-ray diffraction (XRD) pattern of MXMoS2 composite nanomaterials; Figure 5 Figure 2 is a diagram of Staphylococcus aureus (S. aureus) co-cultured in different hydrogels with or without infrared light irradiation; Figure 5 a is an image of Staphylococcus aureus (S. aureus) colonies co-cultured on different hydrogels with or without infrared light irradiation; Figure 5 b is Figure 5 a Statistical analysis of the viability of cultured Staphylococcus aureus (S. aureus); Figure 6 a) RAW264.7 cells treated with different hydrogels after 24 hours of inflammation qRT-PCR results of Nos2, b) Il6, c) Arg1, and d) Il10 gene expression levels; Figure 7 a is the qRT-PCR results of Alp, Runx2, and Sp7 levels in osteogenic differentiated MC3T3-E1 cells treated with different conditions for 7 days; Figure 7 b is the SEM image of ALP staining of MC3t3-E1 cells treated with different conditions for 7 days; Figure 7 c is a quantitative analysis of ALP staining in MC3t3-E1 cells treated with different conditions for 7 days; Figure 8 a is the GO analysis graph related to MXMoS2 and osteoblast differentiation and Wnt signaling pathway; Figure 8 b is a box diagram of some genes in the Wnt signaling pathway that were significantly upregulated in the MXMoS2 group; Figure 9 a is a schematic diagram of the treatment process of mice with suppurative osteomyelitis; Figure 9 b is a picture of bacterial colonies in the wounds of mice with suppurative osteomyelitis before and after infrared light irradiation treatment; Figure 9 c is Figure 9 Statistical graph of bacterial colony counts in b; Figure 9 d is a 3D reconstruction of the maxillary bone by micro-CT after 4 weeks of treatment with different hydrogels, and representative images of the mandible of mice stained with HE and Masson staining; Figure 9 e is the BMD map of newly formed bone calculated based on micro-CT; Figure 10 HE staining images of the heart, liver, spleen, lung and kidney of mice in the control group, DNA hydrogel group and MXMoS2-DNA hydrogel group. DETAILED DESCRIPTION

[0018] The technical solutions of the present invention are further specifically described below through examples and drawings. These examples are provided for the purpose of illustrating the present invention and are not intended to limit the present invention. All other examples obtained by persons of ordinary skill in the art based on the examples in this application without creative work are intended to fall within the scope of protection of this application.

[0019] Example 1 A method for preparing an infection-responsive photothermal / immunomodulatory / osteogenic multifunctional MXMoS2-DNA hydrogel comprises the following steps: Step S1, Ti3C2T X Preparation of MXene dispersion: Weigh Ti3AlC2 powder and slowly add it into a pre-made 40% HF solution. Stir magnetically at room temperature for 24 hours to complete the selective etching of Al element and generate Ti3C2T X MXene nanosheets, Ti3C2T washed with deionized water X MXene nanosheets were separated by centrifugation at a speed of 3500 rpm for 10 min until the pH value of the washing solution was close to neutral. (pH value is 6-7), Ti3C2T X MXene precipitation, the Ti3C2T X The MXene precipitate was resuspended in deionized water to obtain a Ti3C2T3O4 solution with a concentration of 12 mg / mL. X MXene dispersion, set aside; wherein, the mass volume ratio of Ti3AlC2 powder to HF solution is 1g:10mL; Step S2, preparation of MoS2 quantum dot solution: First, add MoCl5 or MoO3 into deionized water, adjust the pH to 11 with NaOH solution, and perform ultrasonic treatment to form a clear solution; then take 1 mL of the clear solution and add it to 39 mL of 1 mg / mL bovine serum albumin solution, stir and mix at room temperature, and add 0.2 mL of 1 mg / mL bovine serum albumin solution. 0.5M Na2S solution was stirred vigorously for 5 minutes to obtain a mixed solution, and then 1M HCl solution was added to the mixed solution to adjust its pH to 6-7. During the reaction, a clear light yellow MoS2 quantum dot solution was formed; finally, the obtained clear light yellow MoS2 quantum dot solution was ultrafiltered or dialyzed to remove free ions and concentrated to obtain a MoS2 quantum dot solution for later use; Step S3, MoS2 quantum dots doped with Ti3C2T X MXene: The MoS2 quantum dot solution obtained in step S2 is slowly added to the Ti3C2T X In the MXene dispersion, MoS2 quantum dot solution and Ti3C2T X The volume ratio of MXene dispersion was 1:3, and ultrasonic dispersion was performed for 30 min to make MoS2 quantum dots uniformly adsorbed / bound to the MXene surface to form a mixed solution. The mixed solution was transferred to a tetrafluoroethylene autoclave for high-temperature reaction at 180 ° C for 6 h. After the high-temperature reaction was completed, it was naturally cooled, centrifuged, and washed 3 times to obtain MoS2 quantum dot-doped Ti3C2T X MXene composite nanomaterials, namely MXMoS2 composite nanomaterials, are reserved; Step S4, preparation of DNA hydrogel: Dissolve the short-chain DNA in PBS buffer and adjust the DNA concentration to 10 mg / mL of DNA solution; then, slowly add PEG-dithio crosslinker to the DNA solution at a molar ratio of 1:1, and let it stand at 4°C or room temperature for 30 minutes to form a transparent homogeneous DNA hydrogel; finally, prepare DNA hydrogel products of different forms by lyophilization or microinjection for later use; wherein, the short-chain DNA is purchased commercially at a concentration of 5-20 mg / mL; Step S5, preparation of MXMoS2-DNA composite hydrogel: the MXMoS2 composite nanomaterial obtained in step S3 was dispersed in PBS buffer at a concentration of 12 mg / mL to obtain MXMoS2 dispersion, the MXMoS2 dispersion is slowly added to the MXMoS2 dispersion obtained in step S4 The DNA hydrogel was stirred and mixed evenly, a cross-linking agent was added to induce cross-linking and gelation, and the gel was allowed to stand at constant temperature to form an infection-responsive photothermal / immunomodulatory / osteogenic multifunctional MXMoS2-DNA hydrogel; wherein the mass ratio of MXMoS2 composite nanomaterial, PBS buffer and DNA hydrogel was 1:51:10.

[0020] Example 2 A method for preparing an infection-responsive photothermal / immunomodulatory / osteogenic multifunctional MXMoS2-DNA hydrogel comprises the following steps: Step S1, Ti3C2T X Preparation of MXene dispersion: Weigh Ti3AlC2 powder and slowly add it into a pre-made 40% HF solution. Stir magnetically at room temperature for 24 hours to complete the selective etching of Al element and generate Ti3C2T X MXene nanosheets, Ti3C2T washed with deionized water X MXene nanosheets were separated by centrifugation at a speed of 3500 rpm for 10 min until the pH value of the washing solution was close to neutral. (pH value is 6-7), Ti3C2T X MXene precipitation, the Ti3C2T X The MXene precipitate was resuspended in deionized water to obtain a Ti3C2T3O4 solution with a concentration of 12 mg / mL. X MXene dispersion, set aside; wherein, the mass volume ratio of Ti3AlC2 powder to HF solution is 1g:10mL; Step S2, preparation of MoS2 quantum dot solution: First, add MoCl5 or MoO3 into deionized water, adjust the pH to 11 with NaOH solution, and perform ultrasonic treatment to form a clear solution; then take 1 mL of the clear solution and add it to 39 mL of 1 mg / mL bovine serum albumin solution, stir and mix at room temperature, and add 0.2 mL of 1 mg / mL bovine serum albumin solution. 0.5M Na2S solution was stirred vigorously for 5 minutes to obtain a mixed solution, and then 1M HCl solution was added to the mixed solution to adjust its pH to 6-7. During the reaction, a clear light yellow MoS2 quantum dot solution was formed; finally, the obtained clear light yellow MoS2 quantum dot solution was ultrafiltered or dialyzed to remove free ions and concentrated to obtain a MoS2 quantum dot solution for later use; Step S3, MoS2 quantum dots doped with Ti3C2T X MXene: The MoS2 quantum dot solution obtained in step S2 is slowly added to the Ti3C2T X In the MXene dispersion, MoS2 quantum dot solution and Ti3C2T X The volume ratio of MXene dispersion was 1:4, and ultrasonic dispersion was performed for 30 min to make MoS2 quantum dots uniformly adsorbed / bound to the MXene surface to form a mixed solution. The mixed solution was transferred to a tetrafluoroethylene autoclave for high-temperature reaction at 180 ° C for 9 h. After the high-temperature reaction was completed, it was naturally cooled, centrifuged, and washed 3 times to obtain MoS2 quantum dot-doped Ti3C2TX MXene composite nanomaterials, namely MXMoS2 composite nanomaterials, are reserved; Step S4, preparation of DNA hydrogel: dissolve the short-chain DNA in PBS buffer and adjust the DNA concentration to 10 mg / mL DNA solution; then, slowly add PEG-dithio crosslinker to the DNA solution, with the molar ratio of DNA solution to PEG-dithio crosslinker being 1:1, and let it stand at 4°C or room temperature for 45 minutes to form a transparent homogeneous DNA hydrogel; finally, prepare DNA hydrogel products of different forms by freeze-drying or microinjection for later use; wherein, the short-chain DNA is purchased from the market and its concentration is 5-20 mg / mL; Step S5, preparation of MXMoS2-DNA composite hydrogel: the MXMoS2 composite nanomaterial obtained in step S3 was dispersed in PBS buffer at a concentration of 12 mg / mL to obtain MXMoS2 dispersion, the MXMoS2 dispersion is slowly added to the MXMoS2 dispersion obtained in step S4 The DNA hydrogel was stirred and mixed evenly, a cross-linking agent was added to induce cross-linking and gelation, and the gel was allowed to stand at constant temperature to form an infection-responsive photothermal / immunomodulatory / osteogenic multifunctional MXMoS2-DNA hydrogel; wherein the mass ratio of MXMoS2 composite nanomaterial, PBS buffer and DNA hydrogel was 1:51:10.

[0021] Example 3 A method for preparing an infection-responsive photothermal / immunomodulatory / osteogenic multifunctional MXMoS2-DNA hydrogel comprises the following steps: Step S1, Ti3C2T X Preparation of MXene dispersion: Weigh Ti3AlC2 powder and slowly add it into a pre-made 40% HF solution. Stir magnetically at room temperature for 24 hours to complete the selective etching of Al element and generate Ti3C2T X MXene nanosheets, Ti3C2T washed with deionized water X MXene nanosheets were separated by centrifugation at a speed of 3500 rpm for 10 min until the pH value of the washing solution was close to neutral. (pH value is 6-7), Ti3C2T X MXene precipitation, the Ti3C2T X The MXene precipitate was resuspended in deionized water to obtain a Ti3C2T3O4 solution with a concentration of 12 mg / mL. XMXene dispersion, set aside; wherein, the mass volume ratio of Ti3AlC2 powder to HF solution is 1g:10mL; Step S2, preparation of MoS2 quantum dot solution: First, add MoCl5 or MoO3 into deionized water, adjust the pH to 11 with NaOH solution, and perform ultrasonic treatment to form a clear solution; then take 1 mL of the clear solution and add it to 39 mL of 1 mg / mL bovine serum albumin solution, stir and mix at room temperature, and add 0.2 mL of 1 mg / mL bovine serum albumin solution. 0.5M Na2S solution was stirred vigorously for 5 minutes to obtain a mixed solution, and then 1M HCl solution was added to the mixed solution to adjust its pH to 6-7. During the reaction, a clear light yellow MoS2 quantum dot solution was formed; finally, the obtained clear light yellow MoS2 quantum dot solution was ultrafiltered or dialyzed to remove free ions and concentrated to obtain a MoS2 quantum dot solution for later use; Step S3, MoS2 quantum dots doped with Ti3C2T X MXene: The MoS2 quantum dot solution obtained in step S2 is slowly added to the Ti3C2T X In the MXene dispersion, MoS2 quantum dot solution and Ti3C2T X The volume ratio of MXene dispersion was 1:5, and ultrasonic dispersion was performed for 30 min to make MoS2 quantum dots uniformly adsorbed / bound to the MXene surface to form a mixed solution. The mixed solution was transferred to a tetrafluoroethylene autoclave for high-temperature reaction at 180 ° C for 12 h. After the high-temperature reaction was completed, it was naturally cooled, centrifuged, and washed three times to obtain MoS2 quantum dot-doped Ti3C2T X MXene composite nanomaterials, namely MXMoS2 composite nanomaterials, are reserved; Step S4, preparation of DNA hydrogel: dissolve the short-chain DNA in PBS buffer and adjust the DNA concentration to 10 mg / mL DNA solution; then, slowly add PEG-dithio crosslinker to the DNA solution, with the molar ratio of DNA solution to PEG-dithio crosslinker being 1:1, and let it stand at 4°C or room temperature for 60 minutes to form a transparent homogeneous DNA hydrogel; finally, prepare DNA hydrogel products of different forms by freeze-drying or microinjection for later use; wherein, the short-chain DNA is purchased from the market and its concentration is 5-20 mg / mL; Step S5, preparation of MXMoS2-DNA composite hydrogel: the MXMoS2 composite nanomaterial obtained in step S3 was dispersed in PBS buffer at a concentration of 12 mg / mL to obtain MXMoS2 dispersion, the MXMoS2 dispersion is slowly added to the MXMoS2 dispersion obtained in step S4 The DNA hydrogel was stirred and mixed evenly, a cross-linking agent was added to induce cross-linking and gelation, and the gel was allowed to stand at constant temperature to form an infection-responsive photothermal / immunomodulatory / osteogenic multifunctional MXMoS2-DNA hydrogel; wherein the mass ratio of MXMoS2 composite nanomaterial, PBS buffer and DNA hydrogel was 1:51:10.

[0022] Test example (1) Photothermal response test The MXMoS2 composite nanomaterial prepared by the present invention, MXene, and MoS2 were irradiated with a near-infrared laser of wavelength 808 nm for more than 120 s and heated rapidly (much higher than the single MXene group). Figure 3 As shown in a, the heating rate of MXMoS2 composite nanomaterials is positively correlated with their concentration, indicating that they have good photothermal response sensitivity. Figure 3 As shown in b, the MXMoS2 composite nanomaterials still maintain thermal effects after multiple rounds of heating cycles, as shown in Figure 3 As shown in Figure c, it shows that the MXMoS2 composite nanomaterial prepared by the present invention exhibits low thermal fatigue and good stability.

[0023] (2) Structural characterization The present invention confirms that MoS2 is successfully doped in MXene surface. Figure 4 a. Figure 4 b. Figure 4 c shows that the material morphology is regular, maintaining the MXene layered structure while improving the conductivity and surface absorption capacity; Figure 4 e It can be seen that the XRD pattern of MXene@MoS2 composite material shows a prominent peak at 2θ≈6°, corresponding to Ti3C2T X The (002) plane of MXene indicates that it has a characteristic layered structure. In addition, the obvious peaks at 2θ≈32.7° and 58.9° confirm the presence of MoS2, which correspond to the (100) and (110) planes, respectively. These peaks are consistent with the hexagonal structure of 2H-MoS2, indicating that MoS2 is successfully in situ hydrogenated on the MXene surface without destroying the overall crystallinity of any component. Figure 4As can be seen in Figure d, the MXene@MoS2 composite exhibits characteristic absorption peaks attributed to both components. For the MXene material, the peaks at 1630 cm⁻¹ and ~1050 cm⁻¹ are attributed to Ti-O bending vibrations and C-F stretching vibrations, respectively, indicating surface terminations (–O, –OH, –F). The MoS2 component exhibits Mo-S stretching vibrations around 470 cm⁻¹. Furthermore, broad peaks around 3400 cm⁻¹ and 2900 cm⁻¹ correspond to OH and C-H stretching vibrations, respectively, indicating the presence of residual BSA on the MoS2. These spectra confirm the successful hybridization and coexistence of MXene and MoS2 surface functionalities.

[0024] (3) In vitro antibacterial and infection response tests In vitro antibacterial ability test: Figure 5 a and Figure 5 b It can be seen that in vitro bacterial activity detection of Staphylococcus aureus (S. aureus) was performed. Under near-infrared light (NIR) irradiation, the bacterial survival rate of the MXMoS2 group decreased significantly (>90% sterilization rate), which was significantly better than that of Ti3C2T3 without MoS2. X MXene control group.

[0025] Infection response performance: In the presence of Staphylococcus aureus (S. aureus) secreted Under DNA enzyme (DNase) conditions, the DNA hydrogel degrades rapidly, while under non-infection conditions, the hydrogel maintains a stable structure, achieving the effect of "infection trigger-selective release".

[0026] (4) Immunomodulation and inflammation relief effect test Macrophage polarization experiment: Figure 6 a- Figure 6 d It can be seen that when RAW264.7 cells were treated with the infection-responsive photothermal / immunomodulatory / osteogenic multifunctional MXMoS2-DNA hydrogel prepared by the present invention, qPCR and immunofluorescence showed that the expression of M1 markers (Nos2, Il6) decreased, and the expression of M2 markers increased. (Il10, Arg1) were significantly increased, indicating that the infection-responsive photothermal / immunomodulatory / osteogenic multifunctional MXMoS2-DNA hydrogel has effective anti-inflammatory and immune remodeling functions.

[0027] (5) In vitro osteogenic induction ability test Osteogenesis-related gene expression: Figure 7 a It can be seen that in MC3T3-E1 osteoblast precursor cells, the infection-responsive photothermal / immunomodulatory / osteogenic multifunctional MXMoS2-DNA hydrogel treatment group significantly upregulated bone formation genes such as Runx2, Sp7 and ALP; Figure 7 b and Figure 7 c As can be seen, ALP staining and mineralized nodule staining showed enhanced bone formation activity.

[0028] Signaling pathway analysis: Figure 8 a and Figure 8 b As can be seen, tissue transcriptome sequencing showed that the Wnt signaling pathway was activated; among them, Wnt3a was the key upregulated gene, verifying that the infection-responsive photothermal / immunomodulatory / osteogenic multifunctional MXMoS2-DNA hydrogel has the ability to regulate the bone immune microenvironment.

[0029] (6) In vivo efficacy test in a mouse mandibular osteomyelitis model Therapeutic effect: In the mouse mandibular infection model induced by Staphylococcus aureus (S. aureus), Figure 9 a- Figure 9 c It can be seen that after the infection-responsive photothermal / immunomodulatory / osteogenic multifunctional MXMoS2-DNA hydrogel prepared by the present invention was injected and irradiated with near-infrared light, the bacterial load in the infected tissue was significantly reduced; Figure 9 d. Figure 9 e It can be seen that CT and tissue staining showed that the new bone formation in the bone defect area was significantly better than that in the control group.

[0030] Biosafety: Figure 10 It can be seen that the infection-responsive photothermal / immunomodulatory / osteogenic multifunctional MXMoS2-DNA hydrogel prepared by the present invention showed no obvious toxicity in major organs in histological analysis; body weight was stable and liver and kidney function indicators were normal, indicating that the infection-responsive photothermal / immunomodulatory / osteogenic multifunctional MXMoS2-DNA hydrogel prepared by the present invention has good biocompatibility.

[0031] Finally, it should be noted that although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and spirit of the present invention, and that all equivalent changes and improvements made within the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing an infection-responsive photothermal / immunomodulatory / osteogenic multifunctional MXMoS2-DNA hydrogel, characterized in that: The steps include: Step S1, Ti3C2T X Preparation of MXene dispersion: Ti3AlC2 powder was weighed and slowly added to the pre-made HF solution, and magnetically stirred at room temperature for 24 h to generate Ti3C2T X MXene nanosheets, the Ti3C2T X MXene nanosheets were separated by centrifugation until the pH value of the washing solution was close to neutral to obtain Ti3C2T X MXene precipitation, the Ti3C2T X The MXene precipitate was resuspended in deionized water to obtain a Ti3C2T3O4 solution with a concentration of 12 mg / mL. X MXene dispersion, set aside; Step S2, preparation of MoS2 quantum dot solution: adding MoCl5 or MoO3 to deionized water, adjusting the pH with NaOH solution, ultrasonically treating to form a clear solution, sequentially adding bovine serum albumin solution and Na2S solution to the clear solution, and then adjusting the pH with HCl solution to obtain a MoS2 quantum dot solution for later use; Step S3, MoS2 quantum dots doped with Ti3C2T X MXene: The MoS2 quantum dot solution obtained in step S2 is slowly added to the Ti3C2T X The MXene dispersion was ultrasonically stirred to form a mixed solution, which was then transferred to a tetrafluoroethylene autoclave for high-temperature reaction. After the reaction was completed, the mixture was naturally cooled, centrifuged, and washed three times to obtain MoS2 quantum dots doped with Ti3C2T X MXene composite nanomaterials, namely MXMoS2 composite nanomaterials, are reserved; Step S4, preparation of DNA hydrogel: dissolve the short-chain DNA in PBS buffer, slowly add a cross-linking agent, and allow to stand to form a transparent homogeneous DNA hydrogel for later use; Step S5, preparation of MXMoS2-DNA composite hydrogel: the MXMoS2 composite nanomaterial obtained in step S3 was dispersed in PBS buffer at a concentration of 12 mg / mL to obtain MXMoS2 dispersion, the MXMoS2 dispersion is slowly added to the MXMoS2 dispersion obtained in step S4 The DNA hydrogel is stirred and mixed evenly, a cross-linking agent is added to induce cross-linking and gel formation, and the gel is allowed to stand at a constant temperature to form an MXMoS2-DNA hydrogel.

2. The method for preparing an infection-responsive photothermal / immunomodulatory / osteogenic multifunctional MXMoS2-DNA hydrogel according to claim 1, characterized in that: In step S1, the mass volume ratio of Ti3AlC2 powder to HF solution is 1 g:10 mL; the centrifugal speed is 3500 rpm, and the centrifugal time is 10 min.

3. The method for preparing an infection-responsive photothermal / immunomodulatory / osteogenic multifunctional MXMoS2-DNA hydrogel according to claim 1, characterized in that: The specific method for preparing the MoS2 quantum dot solution in step S2 is as follows: first, MoCl5 or MoO3 is added to deionized water, the pH is adjusted to 11 with NaOH solution, and ultrasonic treatment is performed to form a clear solution; then 1 mL of the clear solution is added to 39 mL of 1 mg / mL bovine serum albumin solution, stirred at room temperature to mix evenly, 0.2 mL of 0.5 M Na2S solution is added thereto, and vigorously stirred for 5 minutes to obtain a mixed solution, and then 1 M HCl solution is added to the mixed solution to adjust its pH to 6-7. A clear light yellow MoS2 quantum dot solution is formed during the reaction; finally, the obtained clear light yellow MoS2 quantum dot solution is subjected to ultrafiltration or dialysis to remove free ions and concentrated to obtain a MoS2 quantum dot solution.

4. The method for preparing an infection-responsive photothermal / immunomodulatory / osteogenic multifunctional MXMoS2-DNA hydrogel according to claim 1, characterized in that: In step S3, the MoS2 quantum dot solution and the Ti3C2T X The volume ratio of the MXene dispersion is 1:(3-5); the ultrasonic time is 30 minutes; the high-temperature reaction temperature in a tetrafluoroethylene autoclave is 180°C, and the reaction time is 6-12 hours.

5. The method for preparing an infection-responsive photothermal / immunomodulatory / osteogenic multifunctional MXMoS2-DNA hydrogel according to claim 1, characterized in that: The specific process of preparing the DNA hydrogel in step S4 is as follows: first, the short-chain DNA is dissolved in PBS buffer and its concentration is adjusted to 10 mg / mL of DNA solution; then, PEG-dithio crosslinker is slowly added to the DNA solution, and the solution is allowed to stand at 4°C or room temperature to form a transparent homogeneous DNA hydrogel; finally, the DNA hydrogel products of different forms are prepared by freeze-drying or microinjection.

6. The method for preparing an infection-responsive photothermal / immunomodulatory / osteogenic multifunctional MXMoS2-DNA hydrogel according to claim 5, characterized in that: The short-chain DNA was purchased commercially with a concentration of 5-20 mg / mL; the molar ratio of the DNA solution to the PEG-dithio crosslinker was 1:1; and the standing time was 30-60 min.

7. The method for preparing an infection-responsive photothermal / immunomodulatory / osteogenic multifunctional MXMoS2-DNA hydrogel according to claim 1, characterized in that: In step S5, the mass ratio of the MXMoS2 composite nanomaterial, the PBS buffer and the DNA hydrogel is 1:51:

10.

8. An infection-responsive photothermal / immunomodulatory / osteogenic multifunctional MXMoS2-DNA hydrogel, characterized by: The multifunctional MXMoS2-DNA hydrogel with infection-responsive photothermal / immunomodulatory / osteogenic properties is prepared by the preparation method according to any one of claims 1 to 7.

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