Microenvironment-responsive ROS antibacterial photodynamic therapy for periodontitis and its preparation method

Abstract

This invention belongs to the field of antibacterial photodynamic therapy (PDPT), providing a microenvironment-responsive ROS-modulated antibacterial photodynamic therapy for periodontitis and its preparation method. It includes: preparing tin sulfide oxide using sodium dodecylbenzenesulfonate, tin chloride pentahydrate, and L-cysteine ​​as raw materials; preparing nanozymes using zinc nitrate, copper nitrate, urea, and thioacetamide as raw materials; and preparing antibacterial photodynamic therapy using indocyanine green, bacterial targeting peptides, nanozymes, tin sulfide oxide, and dihydromyricetin as raw materials. This invention constructs a microenvironment-responsive antibacterial photodynamic therapy targeting the infection and treatment characteristics of periodontitis. It proposes a strategy combining microenvironment-responsive programmed regulation of ROS in bacterial infection, precise and long-lasting treatment, and immune regulation to construct antibacterial photodynamic therapy, thereby improving and expanding the efficacy and application scenarios of antibacterial photodynamic therapy for periodontitis.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial photodynamic technology, and particularly relates to antibacterial photodynamic therapy for periodontitis using microenvironment-responsive regulation of ROS and its preparation method. Background Technology

[0002] Periodontitis can lead to loose teeth and even tooth loss, and it is also a risk factor for systemic diseases such as cardiovascular disease, diabetes, and hypertension. Pathogenic bacteria within the periodontal pockets of periodontitis can spread through the bloodstream and other routes, causing infections in distant organs. Epidemiological surveys show that the periodontal health rate among adults in my country is less than 10%. Therefore, timely and effective treatment of periodontitis and long-term control of pathogenic bacteria within the periodontal pockets are crucial.

[0003] Antibacterial photodynamic therapy has been applied in clinical practice. However, the antibacterial photodynamic therapy currently used in the treatment of periodontitis has many shortcomings: (1) The hypoxic microenvironment in the periodontal pocket and the self-aggregation characteristics of photosensitizers limit the efficiency of ROS production by antibacterial photodynamic therapy; (2) The short lifespan of ROS (nanosecond to microsecond) and the limited diffusion distance result in a limited effective time for antibacterial photodynamic therapy to exert its antibacterial effect; (3) Antibacterial photodynamic therapy cannot responsively generate ROS (exerting antibacterial effect) and clear ROS (relieving tissue oxidative stress) according to the characteristics of the infection microenvironment; (4) The ROS generated by antibacterial photodynamic therapy has insufficient bacterial targeting, affecting the antibacterial efficiency; (5) Antibacterial photodynamic therapy focuses on antibacterial activity, and its ability to regulate immune cells and osteoclasts in the periodontal inflammatory microenvironment is limited. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides an antibacterial photodynamic therapy for periodontitis using microenvironment-responsive ROS and its preparation method, aiming to solve the problems mentioned in the background art.

[0005] In a first aspect, the present invention provides a method for preparing antibacterial photodynamic therapy for periodontitis using microenvironment-responsive ROS, comprising the following steps:

[0006] Step 1: Sodium dodecylbenzenesulfonate, tin chloride pentahydrate, and L-cysteine ​​are mixed in a mixed solution of ethylene glycol and water. After stirring at room temperature in the dark, a hydrothermal reaction is carried out. After the reaction is completed and cooled to room temperature, the precipitate is collected by centrifugation and washed. After freeze-drying, tin sulfide oxide is obtained.

[0007] Step 2: Dissolve zinc nitrate, copper nitrate and urea in deionized water, heat and stir, then raise and maintain the temperature. After the reaction is complete, centrifuge, wash and dry the reaction product, then heat to 350-450℃ to obtain the precursor; then dissolve the precursor and thioacetamide in ethanol at a mass ratio of 1:4, stir, centrifuge, wash and dry the reaction product to obtain nanozyme.

[0008] Step 3: Indocyanine green and bacterial targeting peptide UBI29-41 were dissolved in ultrapure water at a mass ratio of 10:1 and stirred in the dark. The mixture was then centrifuged and freeze-dried to obtain the conjugate product of photosensitizer indocyanine green and bacterial targeting peptide UBI29-41. Nanozymes and tin sulfide oxide were added to the ultrapure water at a mass ratio of 2:1 and stirred overnight. After stirring, the mixture was centrifuged and freeze-dried to obtain tin sulfide oxide loaded with nanozymes. Tin sulfide oxide loaded with nanozymes, dihydromyricetin, and the conjugate product were stirred overnight in the dark at a mass ratio of 2:1:1. After the reaction was completed, the reaction product was centrifuged, washed, and freeze-dried to obtain antibacterial photodynamic therapy.

[0009] Further, in step one, 22.4 mmol sodium dodecylbenzenesulfonate, 1.0 mmol tin chloride pentahydrate, and 8.0 mmol L-cysteine ​​are mixed in a mixed solution of ethylene glycol and water.

[0010] Furthermore, in step one, the hydrothermal reaction temperature is 150-170℃, and the time is 8-12h.

[0011] Further, in step two, 10.1 mmol zinc nitrate, 12.4 mmol copper nitrate and 83.2 mmol urea are dissolved in deionized water.

[0012] Further, in step two, the mixture is heated and stirred, and then the temperature is raised and maintained. Specifically, the mixture is stirred at 65-85°C for 0.5 hours, and then the reaction temperature is raised to 95°C and maintained for 3 hours.

[0013] Furthermore, in step two, after centrifugation, washing, and drying of the reaction product, specifically: the reaction product is centrifuged, washed, and dried at 50-70℃ for 3-5 hours;

[0014] The reaction product was stirred, centrifuged, washed, and dried to obtain nanozymes. Specifically, the reaction product was stirred at 85-95℃ for 0.5-1.5h, centrifuged, washed, and dried at 35-45℃ for 5-7h to obtain nanozymes.

[0015] Further, in step three, indocyanine green and bacterial targeting peptide UBI29-41 are dissolved in ultrapure water at a mass ratio of 10:1 and stirred in the dark for 12-24 hours.

[0016] Secondly, the present invention provides an antibacterial photodynamic therapy for periodontitis that uses microenvironment-responsive regulation of ROS, prepared by a method for preparing an antibacterial photodynamic therapy for periodontitis using microenvironment-responsive regulation of ROS.

[0017] Thirdly, this invention provides the application of antibacterial photodynamic therapy of ROS in treating periodontitis in the preparation of products for treating periodontitis, including formulations, medical materials or medical devices.

[0018] Furthermore, the product uses microenvironment-responsive programmed regulation of ROS to target bacteria and continuously generate ROS, thus treating periodontitis.

[0019] This invention offers the following advantages: it constructs a microenvironment-responsive antibacterial photodynamic therapy targeting the infection and treatment characteristics of periodontitis. It proposes a strategy combining microenvironment-responsive programmed regulation (generating and clearing ROS at specific times and spaces), precise (bacterial targeting), long-lasting (continuous ROS generation) treatment, and immune regulation to construct antibacterial photodynamic therapy, thereby improving and expanding the efficacy and application scenarios of antibacterial photodynamic therapy for periodontitis. Specifically, the nanozyme exhibits pH responsiveness, efficiently generating ROS for antibacterial activity at the infection site (pH=5.0) and clearing excess ROS in a normal environment (pH=7.4), avoiding tissue damage; the bacterial-targeting peptide precisely binds to pathogenic bacteria; and the photocatalytic oxygen production from tin sulfide oxidation improves the hypoxic environment, continuously generating ROS and prolonging the antibacterial effect. Attached Figure Description

[0020] Exemplary embodiments of the present invention can be more fully understood by referring to the following figures:

[0021] Figure 1 These are the characterization results of tin sulfide (OS) oxide from Example 4 of the present invention, wherein:

[0022] Figure 1 In the image, A is a scanning electron microscope image of tin sulfide oxide;

[0023] Figure 1 B in the image is a transmission electron microscope (TEM) image of tin sulfide oxide.

[0024] Figure 1 C in the image is an atomic force microscope image of tin sulfide oxide;

[0025] Figure 1 D in the diagram is the X-ray diffraction pattern of tin sulfide oxide;

[0026] Figure 1 E in the figure represents the photocatalytic oxygen production of tin sulfide oxide.

[0027] Figure 2 These are the characterization results of the nanozyme (ZC) in Example 4 of this invention, wherein:

[0028] Figure 2 In the image, A is a scanning electron microscope image of the nanozyme;

[0029] Figure 2 B in the diagram is the X-ray diffraction pattern of the nanozyme.

[0030] Figure 2 C in the image represents the Fourier transform infrared spectrum of the nanozyme.

[0031] Figure 2In the diagram, DH is the X-ray photoelectron spectrum of the nanozyme (ZC), D is the full X-ray photoelectron spectrum of the nanozyme (ZC), E is the X-ray photoelectron spectrum of zinc, F is the X-ray photoelectron spectrum of copper, G is the X-ray photoelectron spectrum of sulfur, and H is the X-ray photoelectron spectrum of oxygen.

[0032] Figure 3 The results show the enzyme-like activity of the nanozyme (ZC) in Example 4 of this invention, wherein:

[0033] Figure 3 In this context, A represents the catalase-like activity of nanozymes at different pH values, characterized by hydrogen peroxide.

[0034] Figure 3 In this context, B represents the catalase-like activity of the nanozyme at different pH values ​​using ammonium molybdate.

[0035] Figure 3 C in the figure represents the peroxidase-like activity of nanozymes at different pH values ​​through OPD characterization.

[0036] Figure 3 D in the figure is used to characterize the peroxidase-like activity of nanozymes at different pH values ​​via TMB.

[0037] Figure 4 The characterization results of the antibacterial photodynamic therapy (IU-DZ@OS) in Example 4 of this invention are as follows:

[0038] Figure 4 In the diagram, A is the X-ray diffraction pattern of tin sulfide oxide (ZC@OS) loaded with nanozymes;

[0039] Figure 4 B in the diagram represents the reactive oxygen species generation of antibacterial photodynamic therapy (IU-DZ@OS).

[0040] Figure 4 C and D in the figure represent Fourier transform infrared spectroscopy analysis of antibacterial photodynamics.

[0041] Figure 5 This is the in vitro biocompatibility experiment result of the antibacterial photodynamic therapy (IU-DZ@OS) of Example 5 of the present invention, namely, the live and dead staining images of osteoblast precursor cells and macrophages.

[0042] Figure 6 This is the in vitro antioxidant experiment result (intracellular reactive oxygen species level detection) of the antibacterial photodynamic (IU-DZ@OS) of Example 5 of the present invention.

[0043] Figure 7 This is the result of the in vitro macrophage polarization detection experiment of antibacterial photodynamic therapy (IU-DZ@OS) in Example 5 of the present invention.

[0044] Figure 8 These are the bacterial targeting test results of the antibacterial photodynamic therapy (IU-DZ@OS) and the non-bacterial targeted photodynamic therapy system (I-DZ@OS) of Example 5 of the present invention.

[0045] Figure 9 The results of the in vitro antibacterial colony coating experiment of the antibacterial photodynamic therapy (IU-DZ@OS) in Example 5 of this invention are as follows:

[0046] Figure 9 In this context, A represents the in vitro antibacterial colony plating experiment.

[0047] Figure 9 B in the graph represents the bacterial survival rate.

[0048] Figure 10 This is the result of the in vitro antibacterial live / dead staining experiment of antibacterial photodynamic (IU-DZ@OS) in Example 5 of the present invention.

[0049] Figure 11 This is a scanning electron microscope image of the antibacterial photodynamic therapy (IU-DZ@OS) of Example 5 of the present invention after in vitro antibacterial treatment.

[0050] Figure 12 The results of the in vivo periodontitis treatment efficacy evaluation experiment of antibacterial photodynamic therapy (IU-DZ@OS) in Example 6 of this invention are as follows:

[0051] Figure 12 In this context, A represents a MicroCT image.

[0052] Figure 12 B in the diagram represents the distance between the alveolar ridge crest and the cementoenamel junction.

[0053] Figure 12 C in the graph represents the bone volume fraction.

[0054] Figure 12 D in the diagram represents the trabecular separation.

[0055] Figure 12 E in the image represents the trabecular bone thickness.

[0056] Figure 12 F in the graph represents a heatmap of the expression levels of genes related to bone formation and bone resorption. Detailed Implementation

[0057] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0058] 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 invention pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0059] This invention provides a method for preparing antibacterial photodynamic therapy for periodontitis using microenvironment-responsive ROS, comprising the following steps:

[0060] Step 1: Sodium dodecylbenzenesulfonate, tin chloride pentahydrate, and L-cysteine ​​are mixed in a mixed solution of ethylene glycol and water. After stirring at room temperature in the dark, a hydrothermal reaction is carried out. After the reaction is completed and cooled to room temperature, the precipitate is collected by centrifugation and washed. After freeze-drying, tin sulfide oxide is obtained.

[0061] Step 2: Dissolve zinc nitrate, copper nitrate and urea in deionized water, heat and stir, then raise and maintain the temperature. After the reaction is complete, centrifuge, wash and dry the reaction product, then heat to 350-450℃ to obtain the precursor; then dissolve the precursor and thioacetamide in ethanol at a mass ratio of 1:4, stir, centrifuge, wash and dry the reaction product to obtain nanozyme.

[0062] Step 3: Indocyanine green and bacterial targeting peptide UBI29-41 were dissolved in ultrapure water at a mass ratio of 10:1 and stirred in the dark. The mixture was then centrifuged and freeze-dried to obtain the conjugate product of photosensitizer indocyanine green and bacterial targeting peptide UBI29-41. Nanozymes and tin sulfide oxide were added to the ultrapure water at a mass ratio of 2:1 and stirred overnight. After stirring, the mixture was centrifuged and freeze-dried to obtain tin sulfide oxide loaded with nanozymes. Tin sulfide oxide loaded with nanozymes, dihydromyricetin, and the conjugate product were stirred overnight in the dark at a mass ratio of 2:1:1. After the reaction was completed, the reaction product was centrifuged, washed, and freeze-dried to obtain antibacterial photodynamic therapy.

[0063] In some embodiments, in step one, 22.4 mmol sodium dodecylbenzenesulfonate, 1.0 mmol tin chloride pentahydrate, and 8.0 mmol L-cysteine ​​are mixed in a mixed solution of ethylene glycol and water.

[0064] In some embodiments, in step one, the hydrothermal reaction temperature is 150-170°C and the time is 8-12 hours.

[0065] In some embodiments, in step two, 10.1 mmol zinc nitrate, 12.4 mmol copper nitrate and 83.2 mmol urea are dissolved in deionized water.

[0066] In some embodiments, in step two, the mixture is heated and stirred, and then the temperature is raised and maintained. Specifically, the mixture is stirred at 65-85°C for 0.5 h, and then the reaction temperature is raised to 95°C and maintained for 3 h.

[0067] In some embodiments, in step two, after centrifugation, washing and drying of the reaction product, the reaction product is specifically centrifuged, washed and dried at 50-70°C for 3-5 hours.

[0068] The reaction product was stirred, centrifuged, washed, and dried to obtain nanozymes. Specifically, the reaction product was stirred at 85-95℃ for 0.5-1.5h, centrifuged, washed, and dried at 35-45℃ for 5-7h to obtain nanozymes.

[0069] In some embodiments, in step three, indocyanine green and bacterial targeting peptide UBI29-41 are dissolved in ultrapure water at a mass ratio of 10:1 and stirred in the dark for 12-24 hours.

[0070] In some embodiments, the present invention provides an antibacterial photodynamic therapy for periodontitis that is regulated by microenvironment-responsive ROS, prepared by a method for preparing an antibacterial photodynamic therapy for periodontitis that is regulated by microenvironment-responsive ROS.

[0071] In some embodiments, the present invention provides the application of antibacterial photodynamic therapy of ROS in the treatment of periodontitis in the preparation of products for treating periodontitis, including formulations, medical materials or medical devices.

[0072] In some embodiments, the product treats periodontitis by microenvironmentally responsively and programmatically regulating ROS, targeting bacteria, and continuously generating ROS.

[0073] Example 1:

[0074] Synthesizing tin sulfide (OS), specific preparation method:

[0075] 2.4 mmol sodium dodecylbenzenesulfonate, 1.0 mmol tin chloride pentahydrate, and 8.0 mmol L-cysteine ​​were mixed in a mixture of ethylene glycol and water. A hydrothermal reaction was carried out at room temperature, protected from light, with stirring, at a temperature of 150-170°C for 8-12 hours. After the reaction was completed and cooled to room temperature, the precipitate was collected by centrifugation, washed, and freeze-dried to obtain tin sulfide oxide (OS).

[0076] Example 2:

[0077] Synthetic nanozymes (ZC), specific preparation method:

[0078] First, 10.1 mmol of zinc nitrate, 12.4 mmol of copper nitrate, and 83.2 mmol of urea were dissolved in deionized water, and the mixture was stirred at 75 °C for 0.5 h. The reaction temperature was then increased to 95 °C and maintained for 3 h. The product was washed by centrifugation and then dried at 60 °C for 3 h. The dried product was heated to 400 °C to obtain the precursor. The precursor and thioacetamide were then dissolved in ethanol at a mass ratio of 1:4. The mixture was stirred at 90 °C for 1 h, washed by centrifugation, and then dried at 40 °C for 6 h to obtain the nanozyme (ZC).

[0079] Example 3:

[0080] Synthetic microenvironment-responsive ROS-modulated antibacterial photodynamic therapy for periodontitis (IU-DZ@OS), specific preparation method:

[0081] First, the photosensitizer indocyanine green (ICG) and the bacterial targeting peptide UBI29-41 were dissolved in ultrapure water at a mass ratio of 10:1 and stirred in the dark for 24 hours. The resulting product (IU) was obtained by centrifugation and freeze-drying. Then, nanozymes (ZC) and tin sulfide (OS) were added to the ultrapure water at a mass ratio of 2:1 and stirred overnight (10-12 hours). The resulting product was then centrifuged and freeze-dried to obtain nanozyme-loaded tin sulfide (ZC@OS). Finally, nanozyme-loaded tin sulfide (ZC@OS), dihydromyricetin (DH), and IU were stirred overnight (10-12 hours) in the dark at a mass ratio of 2:1:1. The precipitate was then washed by centrifugation and freeze-dried to obtain antibacterial photodynamic therapy (IU-DZ@OS) with microenvironment-responsive ROS for periodontitis treatment.

[0082] Example 4: Characterization Experiment

[0083] 1. Characterization experiments of tin sulfide oxide (OS):

[0084] The morphology of tin sulfide oxide was observed by scanning electron microscopy and transmission electron microscopy; the thickness and surface roughness of tin sulfide oxide were characterized by atomic force microscopy; the crystal phase structure of tin sulfide oxide was analyzed by X-ray diffraction; and the catalytic oxygen production capacity of tin sulfide oxide was detected by oxygen generation probe.

[0085] The characterization results of tin sulfide (OS) are as follows: Figure 1 As shown, the results indicate that tin sulfide oxide, as revealed by scanning electron microscopy and transmission electron microscopy, exhibits a nanosheet structure with a large specific surface area. Figure 1 A and B in the image); the atomic force microscopy image of tin sulfide oxide shows that the edges of tin sulfide oxide are clear and the thickness is relatively uniform. Figure 1The X-ray diffraction pattern of tin sulfide oxide shows that it contains the characteristic crystal phase structure of tin sulfide, proving the successful preparation of tin sulfide oxide. Figure 1 (D in the image); the oxygen probe intensity map of tin sulfide oxide shows that tin sulfide oxide can catalyze the production of oxygen under photocatalysis (D). Figure 1 (E in the text).

[0086] 2. Characterization experiments of nanozymes (ZC):

[0087] (1) The morphology and structure of nanozymes were observed by scanning electron microscopy; the crystal phase structure of nanozymes was analyzed by X-ray diffraction; the characteristic absorption peaks of nanozymes were detected by Fourier transform infrared analysis; and the elemental and valence state composition of nanozymes was detected by X-ray photoelectron spectroscopy.

[0088] The characterization results of nanozymes (ZC) are as follows: Figure 2 As shown, the results indicate that the scanning electron microscope images of the nanozyme show that the nanozyme has a nanoscale structure. Figure 2 A); X-ray diffraction pattern of nanozymes (in the image). Figure 2 (B) shows the characteristic crystalline phase structure of the nanozyme containing zinc oxide and copper sulfide, verifying the successful preparation of the nanozyme; Fourier transform infrared spectrum of the nanozyme ( Figure 2 C) Verification of the successful preparation of nanozymes; X-ray photoelectron spectroscopy of nanozymes ( Figure 2 The DH (dihydrotesting) data show that the nanozyme is composed of zinc, copper, sulfur, and oxygen.

[0089] (2) The catalase-like activity of the nanozyme was characterized by hydrogen peroxide and ammonium molybdate; the peroxidase-like activity of the nanozyme was characterized by o-phenylenediamine (OPD) and 3,3',5,5'-tetramethylbenzidine (TMB).

[0090] The catalase-like activity results of nanozymes (ZC) are as follows: Figure 3 As shown, the results indicate that the catalase-like activity of nanozymes at different pH values ​​(pH=5.0 or pH=7.4) was characterized by hydrogen peroxide oxidation. The nanozymes exhibited stronger catalase-like activity at pH=7.4. Figure 3 (A in the text); The catalase-like activity of the nanozyme at different pH (pH=5.0 or pH=7.4) was characterized by the ammonium molybdate method. The catalase-like activity of the nanozyme was stronger at pH=7.4. Figure 3 (B in the text); The peroxidase-like activity of nanozymes at different pH (pH=5.0 or pH=7.4) was characterized by the o-phenylenediamine method. The peroxidase-like activity of nanozymes at pH=5.0 was stronger. Figure 3The peroxidase-like activity of the nanozyme at different pH values ​​(pH=5.0 or pH=7.4) was characterized by the 3,3',5,5'-tetramethylbenzidine method. The nanozyme exhibited stronger peroxidase-like activity at pH=5.0. Figure 3 (D in the text). This indicates that nanozymes (ZC) have the function of generating and scavenging ROS in a pH-responsive manner in the infectious microenvironment.

[0091] 3. Characterization experiment of antibacterial photodynamic therapy (IU-DZ@OS) of ROS in the treatment of periodontitis by microenvironment response regulation:

[0092] The successful preparation of tin sulfide oxide (ZC@OS) loaded with nanozymes was analyzed by X-ray diffraction; the reactive oxygen species generation capacity of the antibacterial photodynamic therapy was detected by DPBF probe; the morphology and structure of the antibacterial photodynamic therapy were observed by scanning electron microscopy; the successful preparation of the antibacterial photodynamic therapy was verified by Fourier transform infrared spectroscopy analysis of the characteristic absorption peaks of the antibacterial photodynamic therapy and its components (ZC, POS, DH, and ICG); and the successful preparation of the antibacterial photodynamic therapy was verified by detecting the ZETA potentials of the antibacterial photodynamic therapy and its components (UBI27-41, UI, ZC, ZC@OS, and DH).

[0093] The characterization results of the antibacterial photodynamic therapy (IU-DZ@OS) are as follows: Figure 4 As shown, the results indicate that X-ray diffraction analysis ( Figure 4 A) shows that the characteristic crystalline phase structures of nanozymes and tin sulfide oxide also exist in tin sulfide oxide (ZC@OS) loaded with nanozymes, proving the successful preparation of tin sulfide oxide loaded with nanozymes; the DPBF reactive oxygen species detection probe shows that antibacterial photodynamic therapy can generate reactive oxygen species (ROS). Figure 4 (B in the text); Fourier transform infrared spectroscopy analysis showed that the characteristic peak structures of OS, ZC, DH and ICG also existed in the antibacterial photodynamic therapy, proving the successful synthesis and preparation of the antibacterial photodynamic therapy. Figure 4 (C and D in the text).

[0094] Example 5: In vitro experiment of antibacterial photodynamic therapy (IU-DZ@OS) with microenvironment response regulation of ROS for the treatment of periodontitis

[0095] 1. In vitro biocompatibility experiment of antibacterial photodynamic therapy (IU-DZ@OS) with microenvironment response regulation for the treatment of periodontitis:

[0096] Osteoblast progenitor cells (MC3T3-E1) and macrophages (RAW264.7) were evaluated using a live-dead staining method. Cells in the logarithmic growth phase were stained with 2 × 10⁻⁶ cells. 4Cells were seeded in 24-well plates and cultured overnight in complete α-MEM and DMEM media until adherence, respectively. Cells were then grouped according to experimental criteria. Control group: normally cultured cells; Control group + light: cells cultured in media irradiated with 808nm laser; IU-DZ@OS group: cells co-cultured in complete media containing antibacterial photodynamic therapy (APPT); IU-DZ@OS group + light: cells co-cultured in complete media containing APT and irradiated with 808nm laser. After co-culturing APT with osteoblast precursor cells and macrophages for 1 and 3 days, respectively, cells were stained using a cell viability / death staining kit and observed under an inverted fluorescence microscope to obtain images.

[0097] The in vitro biocompatibility test results of antibacterial photodynamic therapy (IU-DZ@OS) are as follows: Figure 5 As shown in the results, the live / dead staining images of osteoblast precursor cells and macrophages indicate that the cells in each group are in good condition and have normal morphology. This suggests that the antibacterial photodynamic therapy (IU-DZ@OS) has good in vitro biocompatibility.

[0098] 2. In vitro antioxidant activity experiment of antibacterial photodynamic therapy (IU-DZ@OS) regulated by microenvironment response to treat periodontitis:

[0099] Macrophages (RAW264.7) were assessed using the cellular reactive oxygen species detection probe 2,7-dichlorofluorescein diacetate (DCFH-DA). Cells in the logarithmic growth phase were divided into groups of 2 × 10⁻⁶ cells. 4 Cells were seeded in 24-well plates and cultured overnight in DMEM complete medium until adherence. Cells were then treated according to experimental groups: Control group: normally cultured cells; Lipopolysaccharide group: cells cultured in lipopolysaccharide-containing complete medium for 24 h to simulate oxidative stress and inflammation; IU-DZ@OS group: cells cultured in lipopolysaccharide-containing complete medium for 24 h followed by co-culture in DMEM complete medium containing antibacterial photodynamic therapy (PDPT); IU-DZ@OS group + light: cells cultured in lipopolysaccharide-containing complete medium for 24 h followed by co-culture in DMEM complete medium containing PDPT and then irradiated with 808 nm light. After treatment, cells were stained with 2,7-dichlorofluorescein diacetate (DCFH-DA) according to the kit instructions, and then observed and images were acquired under an inverted fluorescence microscope.

[0100] The in vitro antioxidant activity results of antibacterial photodynamic therapy (IU-DZ@OS) are as follows: Figure 6As shown, the results indicated that compared with the normal control group, the 2,7-dichlorofluorescein diacetate (DCFH-DA) staining fluorescence intensity in the lipopolysaccharide group was significantly enhanced, indicating a higher level of reactive oxygen species (ROS) within the cells. After antibacterial photodynamic therapy (IU-DZ@OS), the 2,7-dichlorofluorescein diacetate (DCFH-DA) staining fluorescence intensity was significantly reduced. This suggests that antibacterial photodynamic therapy (IU-DZ@OS) has a good antioxidant effect and can effectively reduce the level of ROS in cells under inflammatory conditions.

[0101] 3. In vitro macrophage polarization assay of antibacterial photodynamic therapy (IU-DZ@OS) regulated by microenvironment response in the treatment of periodontitis:

[0102] Macrophages (RAW264.7) were evaluated using fluorescent probes of 4',6-diamidino-2-phenylindole (DAPI), mannose receptor (CD206), and nitric oxide synthase (iNOS). Cells in the logarithmic growth phase were analyzed at a concentration of 2 × 10⁻⁶ cells / cells. 4 Cells were seeded in 24-well plates and cultured overnight in DMEM complete medium until adherence. Cells were then treated according to experimental groups: Control group: normally cultured cells; Lipopolysaccharide group: cells cultured in lipopolysaccharide-containing complete medium for 24 h to simulate oxidative stress and inflammation; IU-DZ@OS group: cells cultured in lipopolysaccharide-containing complete medium for 24 h followed by co-culture in DMEM complete medium containing antibacterial photodynamic therapy (PDPT); IU-DZ@OS group + light: cells cultured in lipopolysaccharide-containing complete medium for 24 h followed by co-culture in DMEM complete medium containing PDPT and then irradiated with 808 nm light. After treatment, cells were stained using 4',6-diamidinyl-2-phenylindole (DAPI), mannose receptor (CD206), and nitric oxide synthase (iNOS) fluorescent probes according to the kit instructions, and then observed and imaged under a laser confocal fluorescence microscope.

[0103] The results of the in vitro macrophage polarization assay for antibacterial photodynamic therapy (IU-DZ@OS) are as follows: Figure 7 As shown, the results indicated that compared with the normal control group, the nitric oxide synthase (iNOS) fluorescent probe in the lipopolysaccharide group was significantly enhanced, indicating that macrophages were polarized towards pro-inflammatory M1 macrophages and were in an inflammatory state. After antibacterial photodynamic therapy (IU-DZ@OS), the iNOS fluorescent probe was significantly weakened, while the mannose receptor (CD206) fluorescent probe was significantly enhanced, indicating that macrophage polarization towards pro-inflammatory M1 macrophages was weakened, while polarization towards anti-inflammatory M2 macrophages was enhanced. This suggests that antibacterial photodynamic therapy (IU-DZ@OS) can effectively promote macrophage polarization towards anti-inflammatory M2 macrophages and reduce the level of inflammation.

[0104] 4. Microenvironment-responsive regulation of ROS in the treatment of periodontitis: Antibacterial photodynamic therapy (IU-DZ@OS) bacterial targeting assay:

[0105] (1) Synthesizing a non-bacterial targeted photodynamic therapy system (I-DZ@OS) without the bacterial targeting peptide UBI29-41. The specific preparation method is as follows: First, nanozyme (ZC) and tin sulfide oxide (OS) are added to an ultrapure aqueous solution at a mass ratio of 2:1. After stirring overnight, tin sulfide oxide loaded with nanozyme (ZC@OS) is obtained by centrifugation and freeze-drying. ZC@OS, dihydromyricetin (DH) and photosensitizer indocyanine green (ICG) are stirred overnight in a light-protected environment at a mass ratio of 2:1:1. Then, the precipitate is washed by centrifugation and freeze-dried to obtain a non-bacterial targeted photodynamic therapy system (I-DZ@OS) without the bacterial targeting peptide UBI29-41.

[0106] (2) IU-DZ@OS and I-DZ@OS were labeled with red fluorescent dyes, while methicillin-resistant Staphylococcus aureus, Escherichia coli, Porphyromonas gingivalis and Fusobacterium nucleatum were labeled with green fluorescent dyes. IU-DZ@OS and I-DZ@OS were co-cultured with the four bacteria, and then observed and images were obtained under a fluorescence microscope.

[0107] The bacterial targeting assay results for the antibacterial photodynamic therapy (IU-DZ@OS) and the non-bacterial targeted photodynamic therapy system (I-DZ@OS) are as follows: Figure 8 As shown, the results indicate that compared to the non-bacterial targeted photodynamic therapy system (I-DZ@OS) which does not contain the bacterial targeting peptide UBI29-41, the antibacterial photodynamic therapy (IU-DZ@OS) exhibits stronger binding ability with the four bacteria, specifically a larger overlap area of ​​red and green fluorescence (yellow fluorescence). This suggests that the antibacterial photodynamic therapy (IU-DZ@OS) possesses better bacterial targeting and binding capabilities.

[0108] 5. In vitro antibacterial colony-smear experiment of antibacterial photodynamic therapy (IU-DZ@OS) with microenvironment response regulation for ROS treatment of periodontitis:

[0109] Bacterial suspensions of methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, Porphyromonas gingivalis, and Fusobacterium nucleatum in the logarithmic growth phase were treated with antibacterial agents according to groups. Control group: normal culture; Control group + light: bacteria irradiated with 808nm laser; IU-DZ@OS group: bacteria co-cultured with antibacterial photodynamic extract; IU-DZ@OS group + light: bacteria co-cultured with antibacterial photodynamic extract followed by 808nm laser irradiation. The treated bacterial suspensions were spread onto bacterial culture plates and incubated overnight. Colony forming units (CFU) were then observed in each group. Bacterial survival rate (100%) = CFU of experimental group / CFU of control group × 100%.

[0110] The in vitro antibacterial colony-coated plate experiment results of antibacterial photodynamic therapy (IU-DZ@OS) are as follows: Figure 9 As shown in the results, compared with the control group, antibacterial photodynamic therapy (IU-DZ@OS) had better antibacterial effects against methicillin-resistant Staphylococcus aureus, Escherichia coli, Porphyromonas gingivalis, and Fusobacterium nucleatum.

[0111] 6. In vitro antibacterial live / dead staining experiment of ROS in the treatment of periodontitis by regulating the microenvironment response:

[0112] Bacterial suspensions of *Porphyromonas gingivalis* and *Fusobacterium nucleatum* in the logarithmic growth phase were subjected to antibacterial treatment according to groups. Control group: normal culture; Control group + light: bacteria irradiated with 808nm laser; IU-DZ@OS group: bacteria co-cultured with antibacterial photodynamic extract; IU-DZ@OS group + light: bacteria co-cultured with antibacterial photodynamic extract followed by 808nm laser irradiation. The treated bacterial suspensions were stained with green fluorescent nucleic acid staining solution and propidium iodide, and the survival of bacteria in each group was observed under an inverted fluorescence microscope.

[0113] Results of in vitro antibacterial live / dead staining assay using photodynamic therapy (IU-DZ@OS) are as follows: Figure 10 As shown in the results, compared with the control group, antibacterial photodynamic therapy (IU-DZ@OS) can significantly exert its antibacterial effect against Porphyromonas gingivalis and Fusobacterium nucleatum by disrupting the integrity of the bacterial membrane.

[0114] 7. Scanning electron microscopy experiments of in vitro antibacterial effects of IU-DZ@OS (Illuminated by microenvironment response-regulated ROS in the treatment of periodontitis) after antibacterial action:

[0115] Methicillin-resistant Staphylococcus aureus (MRSA) and Porphyromonas gingivalis in the logarithmic growth phase were subjected to antibacterial treatment according to the following groups: Control group: normal culture; Control group + light: bacteria irradiated with 808 nm laser; IU-DZ@OS group: bacteria co-cultured with antibacterial photodynamic extract; IU-DZ@OS group + light: bacteria co-cultured with antibacterial photodynamic extract and then irradiated with 808 nm laser. The treated bacterial cultures were fixed with electron microscopy fixative, then air-dried, sputter-coated with gold, and the morphology and structure of the bacteria were observed under a scanning electron microscope.

[0116] Scanning electron microscope image of antibacterial photodynamic therapy (IU-DZ@OS) after in vitro antibacterial treatment is shown below. Figure 11 As shown in the results, compared with the control group, the bacterial morphology changed significantly after treatment with antibacterial photodynamic therapy (IU-DZ@OS), with the bacterial membrane structure shrinking and losing its normal morphology. This indicates that antibacterial photodynamic therapy (IU-DZ@OS) has a good antibacterial effect.

[0117] Example 6: In vivo experiment of antibacterial photodynamic therapy (IU-DZ@OS) with microenvironment response regulation of ROS for the treatment of periodontitis

[0118] 1. In vivo periodontology efficacy evaluation experiment of antibacterial photodynamic therapy (IU-DZ@OS) regulated by microenvironment response for periodontitis:

[0119] First, an in vivo animal model of periodontitis was established using 8-week-old female SD rats. After one week of acclimatization, the SD rats were randomly assigned to groups for periodontitis model establishment. The rats were anesthetized during the experiment. Then, 4-0 non-absorbable sutures were placed subgingivally on both sides of the maxillary second molars. The rats were fed a high-sugar solution (10% sucrose), and *Porphyromonas gingivalis* was applied around the sutures to establish the rat periodontitis model. After successful model establishment, the sutures were removed. The SD rats were randomly assigned to groups and given different treatments. The normal control group and the periodontitis group were fed normally; the periodontitis group + phototherapy: rats were fed normally and irradiated with an 808nm laser; the IU-DZ@OS group: rats were fed normally and treated with antibacterial photodynamic therapy; the IU-DZ@OS group + phototherapy: rats were fed normally and treated with antibacterial photodynamic therapy and irradiated with an 808nm laser. After four weeks of treatment, all rats were euthanized with an overdose of anesthetic. Maxillary alveolar bone was collected and preserved in 4% paraformaldehyde solution for subsequent analysis. Alveolar bone samples were scanned using MicroCT. The acquired image data were imported into Materialise Mimics 21.0 software for 3D reconstruction and bone parameter analysis. Subsequently, Bone-analysis 2.0 software was used to calculate and analyze bone-related parameters such as the distance between the alveolar ridge crest and cementoenamel junction, bone volume fraction, trabecular separation, and trabecular thickness in the bone defect areas defined in the reconstructed model.

[0120] The results of the in vivo periodontitis treatment efficacy evaluation experiment of antibacterial photodynamic therapy (IU-DZ@OS) are as follows: Figure 12 As shown, the results indicated that compared with the normal group, the periodontitis group rats showed significant alveolar bone resorption, and the distance between the alveolar ridge crest and cementum enamel junction in the periodontitis group was statistically significantly different from that in the normal group. This suggests that the periodontitis model was successfully established. Figure 12 (A and B in the text). After antibacterial photodynamic therapy for periodontitis, which utilizes programmed regulation of ROS in response to the microenvironment for precise and long-lasting treatment, the damaged alveolar bone showed significant recovery. Figure 12 (A) The relevant bone parameters were significantly restored and improved. Figure 12 (BF in the middle). This indicates that antibacterial photodynamic therapy (IU-DZ@OS) has a good in vivo periodontitis treatment effect and can promote the recovery and regeneration of damaged alveolar bone.

[0121] Example 7:

[0122] (1) Synthesize tin sulfide oxide. The specific preparation steps are the same as in Example 1, except that the reaction temperature is 150℃ and the reaction time is 8h.

[0123] (2) Synthesize nanozymes. The specific preparation steps are the same as in Example 2, except that: the above mixture is stirred at 65°C for 0.5 h; the product is washed by centrifugation and then dried at 50°C for 4 h; the dried product is heated to 350°C to obtain the precursor; the above mixed solution is stirred at 85°C for 0.5 h, the product is washed by centrifugation and then dried at 35°C for 5 h.

[0124] (3) Synthesize antibacterial photodynamic therapy for periodontitis by regulating ROS in response to microenvironment. The specific preparation steps are the same as in Example 3, except that: firstly, the photosensitizer indocyanine green and the bacterial targeting peptide UBI29-41 are dissolved in ultrapure water at a mass ratio of 10:1 and stirred in the dark for 12 hours.

[0125] Example 8:

[0126] (1) Synthesize tin sulfide oxide. The specific preparation steps are the same as in Example 1, except that the reaction temperature is 170℃ and the reaction time is 12h.

[0127] (2) Synthesize nanozymes. The specific preparation steps are the same as in Example 2, except that: the above mixture is stirred at 85°C for 0.5 h; the product is washed by centrifugation and then dried at 70°C for 5 h; the dried product is heated to 450°C to obtain the precursor; the above mixed solution is stirred at 95°C for 1.5 h, the product is washed by centrifugation and then dried at 45°C for 7 h.

[0128] (3) Synthesize antibacterial photodynamic therapy for periodontitis by regulating ROS in response to microenvironment. The specific preparation steps are the same as in Example 3, except that: firstly, the photosensitizer indocyanine green and the bacterial targeting peptide UBI29-41 are dissolved in ultrapure water at a mass ratio of 10:1 and stirred in the dark for 20 hours.

[0129] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing an antibacterial photodynamic system for treating periodontitis using ROS responsive to microenvironment, characterized in that, Includes the following steps: Step 1: Sodium dodecylbenzenesulfonate, tin chloride pentahydrate, and L-cysteine ​​are mixed in a mixed solution of ethylene glycol and water. After stirring at room temperature in the dark, a hydrothermal reaction is carried out. After the reaction is completed and cooled to room temperature, the precipitate is collected by centrifugation and washed. After freeze-drying, tin sulfide oxide is obtained. Step 2: Dissolve zinc nitrate, copper nitrate and urea in deionized water, heat and stir, then raise and maintain the temperature. After the reaction is complete, centrifuge, wash and dry the reaction product, then heat to 350-450℃ to obtain the precursor; then dissolve the precursor and thioacetamide in ethanol at a mass ratio of 1:4, stir, centrifuge, wash and dry the reaction product to obtain nanozyme. Step 3: Combine indocyanine green with the bacterial targeting peptide UBI 29-41 Dissolved in ultrapure water at a mass ratio of 10:1 and stirred in the dark, the photosensitizer indocyanine green and the bacterial targeting peptide UBI were obtained by centrifugation and freeze-drying. 29-41 The conjugation product was obtained by adding nanozymes and tin sulfide oxide in an ultrapure aqueous solution at a mass ratio of 2:1, stirring overnight, centrifuging and freeze-drying to obtain tin sulfide oxide loaded with nanozymes; tin sulfide oxide loaded with nanozymes, dihydromyricetin and the conjugation product were stirred overnight in the dark at a mass ratio of 2:1:

1. After the reaction was completed, the reaction product was centrifuged, washed and freeze-dried to obtain an antibacterial photodynamic system.

2. The preparation method according to claim 1, characterized in that: In step one, 2.4 mmol sodium dodecylbenzenesulfonate, 1.0 mmol tin chloride pentahydrate, and 8.0 mmol L-cysteine ​​are mixed in a mixed solution of ethylene glycol and water.

3. The preparation method according to claim 1, characterized in that: In step one, the hydrothermal reaction temperature is 150-170℃ and the time is 8-12 hours.

4. The preparation method according to claim 1, characterized in that: In step two, 10.1 mmol zinc nitrate, 12.4 mmol copper nitrate and 83.2 mmol urea are dissolved in deionized water.

5. The preparation method according to claim 1, characterized in that: In step two, the mixture is heated and stirred, and then the temperature is raised and maintained. Specifically, the mixture is stirred at 65-85℃ for 0.5 hours, and then the reaction temperature is raised to 95℃ and maintained for 3 hours.

6. The preparation method according to claim 1, characterized in that: In step two, after centrifugation, washing and drying of the reaction product, the reaction product is specifically centrifuged, washed and dried at 50-70℃ for 3-5 hours. The reaction product was stirred, centrifuged, washed, and dried to obtain nanozymes. Specifically, the reaction product was stirred at 85-95℃ for 0.5-1.5h, centrifuged, washed, and dried at 35-45℃ for 5-7h to obtain nanozymes.

7. The preparation method according to claim 1, characterized in that: In step three, indocyanine green is combined with the bacterial targeting peptide UBI. 29-41 Dissolve in ultrapure water at a mass ratio of 10:1 and stir in the dark for 12-24 hours.

8. A microenvironment-responsive ROS antibacterial photodynamic system for treating periodontitis, characterized by: It was prepared by the method described in any one of claims 1-7 for the preparation of an antibacterial photodynamic system for treating periodontitis by regulating ROS in response to microenvironment.

9. The application of the microenvironment-responsive ROS antibacterial photodynamic system for treating periodontitis as described in claim 8 in the preparation of products for treating periodontitis, characterized in that: Products include formulations, medical materials, or medical devices.

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