PDA-Ga < 3 + > nanoparticles as well as preparation method and application thereof

Abstract

The invention provides PDA-Ga < 3 + > nanoparticles and a preparation method and application thereof, and is characterized in that the preparation method comprises the following steps: S1, dissolving dopamine hydrochloride and gallium chloride in deionized water, and stirring to obtain a pre-doped solution; s2, adding a buffer solution into the pre-doped solution, controlling the pH value of a reaction system to be alkaline, carrying out a stirring reaction to form a black suspension, and continuing the stirring reaction; and S3, centrifuging and washing the reaction system in the step S2 to obtain the PDA-Ga < 3 + > nanoparticles. The PDA-Ga < 3 + > nanoparticles provided by the invention have multiple characteristics of pH responsiveness, antibacterial property, anti-inflammatory property and bidirectional regulation and control of osteogenesis and osteoclast differentiation, and are expected to realize collaborative treatment of periodontitis, alleviation of inflammatory response of periodontium, reduction of alveolar bone resorption and induction of bone tissue regeneration, so that the success rate of periodontitis treatment is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials, and particularly to a PDA-Ga 3+ nanoparticle and its preparation method and application. Background Art

[0002] Periodontitis is a chronic inflammatory disease caused by local microbiota and host immune responses, which can lead to damage of periodontal tissues and even tooth loss. Its overall prevalence rate is 45-50%, and 11.2% of the world's population suffers from severe periodontitis. Plaque biofilm is the initiating factor of periodontitis, while the imbalance of pro- / anti-inflammatory cytokines is an important factor for the exacerbation of periodontitis. Pro-inflammatory cytokines, such as IL-1β, IL-6 and TNF-α, activate inflammation-related transcription factors or activate related signaling pathways, accelerating the occurrence process of periodontitis. Anti-inflammatory cytokines IL-10, TGF-β and IL-11 can down-regulate the expression levels of pro-inflammatory factors, protect periodontal tissues and inhibit the occurrence of periodontitis. In the treatment of periodontitis, regulating the secretion of pro- / anti-inflammatory cytokines is crucial for restoring immune balance. In addition, an important feature of periodontitis is alveolar bone resorption, and the formation of osteoclasts will exacerbate alveolar bone resorption. The mutual regulation between osteoclasts and osteoblasts is the basis for achieving the balance of bone formation and bone resorption in the bone remodeling process. Therefore, inhibiting the formation of osteoclasts and promoting osteogenic differentiation are of great significance for alveolar bone regeneration and bone homeostasis of periodontal tissues.

[0003] The conventional non-surgical treatment methods for periodontitis are scaling and root planing combined with antibiotic treatment. However, due to the complex terrain in the periodontal pocket, the limitation of instrument models and bacterial drug resistance, etc., the clinical efficacy of this method is limited. In clinical drug treatment, novel and effective treatment regimens are still lacking. Currently, the combination of advanced nanoparticles and new treatment strategies is the most active research field in the treatment of periodontitis. Nanotherapy achieves stable cell targeting, oral retention and intelligent release by changing the chemical composition or physical properties of nanoparticles. At the same time, the high surface area / volume ratio of nanoparticles enables high drug loading capacity, ensuring significant treatment effects. In the strategy of antibacterial treatment for periodontitis alone, metal nanoparticles and nanomaterials loaded with photosensitizers / antibiotics have been developed to exert bactericidal effects. In the strategy of immune regulation treatment for periodontitis alone, nanomaterials that can reshape macrophage polarization, regulate the secretion of pro-inflammatory / anti-inflammatory cytokines and restore the balance of Th17 / Treg cells have been studied to regulate the host immune system and reduce the inflammatory response of periodontal tissues.

[0004] With the continuous exploration of the pathogenesis of periodontitis, the relationship between dental plaque, host immune response and periodontal tissue damage is being closely elucidated from multiple perspectives. Separate antibacterial or immunomodulatory strategies are not suitable for completely controlling the progression of periodontitis. Therefore, there is an urgent need to explore multifunctional nanomaterials with antibacterial, anti-inflammatory, osteogenic differentiation-promoting and osteoclast differentiation-inhibiting properties to improve the treatment effect of periodontitis with a synergistic treatment strategy. Summary of the Invention

[0005] Aiming at the defects of the prior art, the purpose of the present invention is to provide a PDA-Ga 3+ nanoparticle and its preparation method and application.

[0006] The technical solution of the present invention is as follows: In the first aspect of the present invention, a preparation method of PDA-Ga 3+ nanoparticles is provided, and the preparation method includes the following steps: S1. Dissolve dopamine hydrochloride and gallium chloride in deionized water, and stir to obtain a pre-doped solution; S2. Add a buffer solution to the pre-doped solution, control the pH of the reaction system to be alkaline, stir and react to form a black suspension, and continue to stir and react; S3. Centrifuge and wash the reaction system in step S2 to obtain PDA-Ga 3+ nanoparticles.

[0007] Further, in step S1, the mass ratio of dopamine hydrochloride to gallium chloride is 45:0.6-20.

[0008] Further, in step S1, the stirring time is 0.5-1 h.

[0009] Further, in step S2, the buffer solution is a Tris solution.

[0010] Further, the concentration of the Tris solution is 2.25-75 mg / mL.

[0011] Further, in step S2, first stir for 0.5-1 h to form a black suspension, and continue to stir for 1.5-24 h.

[0012] Further, in step S3, the centrifugation parameters are to centrifuge at 12000-15000 rpm for 20-30 min first, and then perform differential centrifugation at 2000-4000 rpm for 20-30 min.

[0013] In the second aspect of the present invention, a PDA-Ga 3+ nanoparticle is provided, and the nanoparticle is prepared according to the preparation method of the above-mentioned PDA-Ga 3+ nanoparticle.

[0014] In the third aspect of the present invention, there is provided the use of the above-mentioned PDA-Ga³⁺ nanoparticles in the preparation of products for treating periodontitis and in the preparation of products for inhibiting alveolar bone resorption.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The PDA-Ga 3+ nanoparticles provided by the present invention have multiple properties such as pH responsiveness, antibacterial property, anti-inflammatory property, and bidirectional regulation of osteogenic and osteoclastic differentiation, overcoming the drawback of the single effect of the treatment strategy for periodontitis, and are expected to achieve the synergistic treatment of periodontitis, reduce the inflammatory response of periodontal tissues, reduce alveolar bone resorption, induce bone tissue regeneration, and thus improve the success rate of periodontitis treatment.

[0016] 2. The preparation method of the PDA-Ga 3+ nanoparticles provided by the present invention is simple and has a short preparation period, which is beneficial to the large-scale preparation of PDA-Ga 3+ nanoparticles, suitable for industrial scale-up production, and has good commercial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] By reading the following detailed description of the non-limiting embodiments with reference to the accompanying drawings, other features, objects, and advantages of the present invention will become more apparent: Figure 1 It is a flowchart of the preparation method of the PDA-Ga 3+ nanoparticles provided by the embodiment of the present invention; Figure 2 It is a guiding diagram of the experimental idea for verifying the potential of PDA-Ga 3+ nanoparticles to treat periodontitis provided by the experimental example of the present invention; Figure 3 It is a SEM image of the PDA-Ga 3+ nanoparticles and a schematic diagram of the Mapping elemental analysis results provided by the embodiment of the present invention ( Figure 3 in a: SEM image of the PDA-Ga 3+ nanoparticles; Figure 3 in b: schematic diagram of the Mapping elemental analysis results of the PDA-Ga 3+ nanoparticles); Figure 4 It is an XPS wide-spectrum scan image of the PDA-Ga 3+ nanoparticles and an XPS high-resolution scan spectrum of Ga 2p provided by the embodiment of the present invention ( Figure 4 in a: XPS wide-spectrum scan image of the PDA-Ga 3+ nanoparticles; Figure 4where b is: the high-resolution XPS scanning spectrum of Ga 2p); Figure 5 PDA-Ga provided by the embodiment of the present invention 3+ pH-responsive ion release curve of nanoparticles; Figure 6 PDA-Ga provided by the experimental example of the present invention 3+ Schematic diagram of the results of the scanning electron microscope for the in vitro anti-P. gingivalis biofilm experiment of PDA-Ga nanoparticles; Figure 7 PDA-Ga provided by the experimental example of the present invention 3+ Schematic diagram of the results of the CLSM staining for the in vitro anti-P. gingivalis biofilm experiment of PDA-Ga nanoparticles; Figure 8 PDA-Ga with different concentrations provided by the experimental example of the present invention 3+ Schematic diagram of the results of the qualitative and quantitative detection of DPPH radical scavenging by PDA-Ga nanoparticles (“*” compared with the NC group, “&” compared with the PC group, “$” compared with the PG20 group, “#” compared with the PG40 group; * / & / $ / # P < 0.05, ** / && / $$ / ## P < 0.01, *** / &&& / $$$ / ### P < 0.001); Figure 9 PDA-Ga with different concentrations provided by the experimental example of the present invention 3+ Schematic diagram of the results of the expression levels of inflammation-related genes after treatment with PDA-Ga nanoparticles and RAW264.7 cells (“*” compared with the PG0 group, “&” compared with the PG20 group, “$” compared with the PG40 group; * / & / $ P < 0.05, ** / && / $$ P < 0.01, *** / &&& / $$$ P < 0.001); Figure 10 PDA-Ga provided by the experimental example of the present invention 3+ Schematic diagram of the results of the in vitro biocompatibility detection of PDA-Ga nanoparticles ( Figure 10 where a is: CCK8 results of BMSCs cells after treatment with PDA-Ga 3+ nanoparticles; Figure 10 where b is: CCK8 results of BMDM cells after treatment with PDA-Ga 3+ nanoparticles; “*” compared with the PG0 group, “&” compared with the PG20 group, “$” compared with the PG40 group; * / & / $ P < 0.05, ** / && / $$ P < 0.01, *** / &&& / $$$ P < 0.001); Figure 11PDA-Ga provided for the experimental examples of the present invention 3+ Schematic diagram of the detection results of the effects of nanoparticles on osteoblast-related genes in cells ("*" compared with the PG0 group, "&" compared with the PG20 group, "$" compared with the PG40 group; * / & / $ P < 0.05, ** / && / $$ P < 0.01, *** / &&& / $$$ P < 0.001); Figure 12 PDA-Ga provided for the experimental examples of the present invention 3+ Schematic diagram of the results of the effects of nanoparticles on alkaline phosphatase activity and extracellular matrix mineralization in cells ("*" compared with the PG0 group, "&" compared with the PG20 group, "$" compared with the PG40 group; * / & / $ P < 0.05, ** / && / $$ P < 0.01, *** / &&& / $$$ P < 0.001); Figure 13 PDA-Ga provided for the experimental examples of the present invention 3+ Schematic diagram of the detection results of the effects of nanoparticles on osteoclast-related genes in cells ("*" compared with the NC group, "&" compared with the PC group, "$" compared with the PG20 group, "#" compared with the PG40 group; * / & / $ / # P < 0.05, ** / && / $$ / ## P < 0.01, *** / &&& / $$$ / ### P < 0.001); Figure 14 PDA-Ga provided for the experimental examples of the present invention 3+ Schematic diagram of the detection results of the effects of nanoparticles on osteoclast differentiation; Figure 15 PDA-Ga provided for the experimental examples of the present invention 3+ Schematic diagram of the detection results of the anti-inflammatory effect of nanoparticles in vivo ("*" compared with the NC group, "&" compared with the PC group; * / & P < 0.05, ** / && P < 0.01, *** / &&& P < 0.001); Figure 16 PDA-Ga provided for the experimental examples of the present invention 3+ Schematic diagram of the results of three-dimensional reconstruction and quantitative analysis of Micro CT of inhibiting alveolar bone resorption in periodontitis mice by nanoparticles ("*" compared with the NC group, "&" compared with the PC group; * / & P < 0.05, ** / && P < 0.01, *** / &&& P < 0.001); Figure 17 PDA-Ga provided for the experimental examples of the present invention after 3+Schematic diagram of the results of the changes in the supporting tissues around the maxillary second molar of mice after treatment with nanoparticles (the distance between the black horizontal lines represents the distance of the cementoenamel junction); Figure 18 For the experimental example of the present invention, after PDA-Ga 3+ Schematic diagram of the results of the formation of osteoclasts in the alveolar bone around the maxillary second molar of mice after treatment with nanoparticles; Figure 19 For the experimental example of the present invention, after PDA-Ga 3+ Schematic diagram of the results of the changes in the expression of osteogenesis-related proteins in the alveolar bone around the maxillary second molar of mice after treatment with nanoparticles; Figure 20 For the experimental example of the present invention, after PDA-Ga 3+ Schematic diagram of the results of the pathological changes in the important organs of mice after treatment with nanoparticles. Detailed implementation manners

[0018] The present invention will be described in detail below with reference to specific embodiments.

[0019] The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several changes and improvements can still be made, and these all belong to the protection scope of the present invention.

[0020] Embodiment This embodiment provides a PDA-Ga 3+ nanoparticle (PDA-Ga 3+ NPs) and its preparation method. As Figure 1 shown, the preparation method includes the following steps: S1. The mass ratio of dopamine hydrochloride to gallium chloride is 45:0.6 - 20. In this embodiment, the mass ratio of dopamine hydrochloride to gallium chloride is preferably 45:20. By the pre-doping method, 45 mg of dopamine hydrochloride and 20 mg of gallium chloride are dissolved in 130 mL of deionized water and stirred for 1 h to completely dissolve, obtaining a pre-doped solution; S2. Add 20 mL of Tris buffer solution (the concentration of the Tris solution is 2.25 - 75 mg / mL, and the concentration of the Tris buffer solution is preferably 75 mg / mL in this embodiment) to the pre-doped solution in step S1. It can be observed that the color of the solution immediately changes to light brown, indicating that dopamine is oxidized to form intermediates such as o-quinone; after stirring and reacting for 0.5 h, the color of the solution gradually turns black, indicating that more dopamine molecules are oxidized and polymerized to form longer polydopamine molecular chains and larger aggregates; continue stirring for 1.5 h to obtain polydopamine with a mature structure.

[0021] S3. Centrifuge the reaction system in step S2. The centrifugation parameters are centrifuging at 15,000 rpm for 20 min to precipitate the nanoparticles, and differential centrifuging at 4,000 rpm for 20 min to separate the target nanoparticles. Then wash them 3 times with deionized water and redisperse them in 1 mL of deionized water to obtain PDA-Ga 3+ nanoparticles (PDA is obtained by the self-polymerization of dopamine hydrochloride (DA) in an alkaline solution).

[0022] For the PDA-Ga 3+ nanoparticles provided in this example, the following characterizations are carried out: 1) Use scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) to analyze the morphology, size, and elemental composition of PDA-Ga 3+ nanoparticles: Take 1 mL of the nanoparticle aqueous solution and coat it on tin foil. Dry it in an oven, sputter with gold, and use SEM to scan the PDA-Ga 3+ nanoparticles on the tin foil, and take pictures to record the nanoparticle morphology. Then locate the area to be analyzed in the SEM image and start the EDS probe for area scanning (Element Mapping) to analyze the elemental distribution. Use X-ray photoelectron spectroscopy to analyze the distribution of functional groups grown on PDA-Ga 3+ nanoparticles: Take 1 mL of the PDA-Ga 3+ nanoparticle aqueous solution and coat it on tin foil. Dry it in an oven, sputter with gold, and start the XPS system for full-spectrum scanning and high-resolution narrow scanning of Ga. The excitation source of XPS is Al Kα, 1486.6 eV, the voltage is 12.5 kV, and the resistance is 16 mA. The results are as Figure 3 shown.

[0023] Figure 3 As shown in a, it can be seen that the nanoparticles are in a uniform spherical morphology, with a particle size of about 86.012 ± 15.274 nm; the elemental distribution map ( Figure 3 b) shows the co-localization of C, N, O, and Ga, confirming that PDA-Ga 3+ NPs are nanostructures with metal ion binding. The EDS results (Table 1) show that the elemental contents of nitrogen (N), gallium (Ga), carbon (C), and oxygen (O) in PDA-Ga NPs are 6.10 ± 0.51%, 13.07 ± 0.12%, 67.65 ± 0.40%, and 13.17 ± 0.15% respectively, indicating that Ga ions are successfully incorporated into PDA. The XPS full-spectrum ( 3+ of PDA-Ga Figure 4 a) and the Ga 2p spectrum ( Figure 4 b) experimental results show that Ga exists in the form of Ga in PDA-Ga 3+ nanoparticles, and Ga 3+ in the form of3+ is the most stable form of gallium oxide, and this form of Ga 3+ endows the nanoparticles with excellent chemical stability, thus providing a structural basis for maintaining the long-term stability of their pharmacological activities. This property is of great significance for ensuring the efficacy consistency of nano-drug formulations.

[0024] 2) Prepare 1 mL of PDA-Ga 3+ nanoparticle solutions with different pH values (7.4, 6.5, 5.5). The solvent is PBS solution. The concentration of PDA-Ga 3+ nanoparticles is 1 mg / mL. Place them in a dialysis bag with a cut-off molecular weight of 14 kDa. Tie both ends of the dialysis bag tightly and immerse it in 100 mL of PBS solution (the pH value of the PBS solution is the same as that of the above PDA-Ga 3+ nanoparticle solution). Shake it on a constant temperature shaker at 37 °C at a speed of 180 rpm / min for 0, 2, 4, 8, 12, and 24 h, and then take out 2 mL. To ensure that the total release volume remains unchanged, add 2 mL of PBS solution after each sampling. Use inductively coupled plasma mass spectrometry (ICP-MS) to detect the concentration of gallium ions in the solutions collected at different time points.

[0025] The results are as Figure 5 shown. The acidic microenvironment can significantly increase the release rate and release amount of Ga ions in PDA-Ga 3+ nanoparticles, and PDA-Ga 3+ is relatively stable in a neutral environment. Only 0.957 ± 0.063 μg of Ga ions are released per milligram of PDA-Ga 3+ nanoparticles after 24 h of dialysis. It shows that the release of Ga ions in PDA-Ga 3+ nanoparticles has pH responsiveness and can trigger the efficient release of Ga 3+ in the acidic microenvironment (pH 4.5 - 5.5) of dental plaque, significantly enhancing the local antibacterial effect; while it remains stable in the neutral physiological environment. This intelligent release property can not only reduce the effective antibacterial concentration but also selectively act on the lesion area, thus significantly reducing the potential toxicity to normal periodontal tissues and ensuring good biosafety.

[0026] Experimental Examples Characterize the biological properties of the PDA-Ga 3+ nanoparticles provided in the examples through the following experiments to verify that the PDA-Ga 3+ nanoparticles have great potential for treating periodontitis. The verification idea is as Figure 2 shown.

[0027] 1) PDA-Ga at different concentrations 3+ Effect of nanoparticles on the biofilm formation of Porphyromonas gingivalis Inoculate 1 mL of Porphyromonas gingivalis suspension with a bacterial density of 1×10 7 CFU / mL onto the cell culture slides in a 24-well plate, and simultaneously add PDA-Ga 3+ nanoparticles at different concentrations (20 / 40 / 80 μg / mL), and co-culture for 48 hours under anaerobic conditions at 37°C. Discard the bacterial suspension in the wells and wash 3 times with PBS. Then add 250 μL of 2.5% glutaraldehyde to each well and incubate overnight at 4°C. After washing 3 more times with PBS, the cell culture slides are sequentially dehydrated through gradient dehydration with 30%, 50%, 70%, 85%, 95% and 100% ethanol, and subjected to carbon dioxide critical point drying. After ion sputtering and gold plating treatment of the Porphyromonas gingivalis biofilm on the glass slides, SEM is used for scanning and photography records are taken. According to the manufacturer's instructions, the LIVE / DEAD BacLight Bacterial Viability Detection Kit (L-7012, Invitrogen, USA) is used to stain the live and dead bacteria in the biofilm. According to the manufacturer's instructions, mix 1.5 μL of SYTO 9 and 1.5 μL of PI dye with 1 mL of deionized water. Then, add 200 μL of the dye mixture to the surface of each cell culture slide and incubate in the dark at room temperature for 20 minutes. Subsequently, remove the staining solution. After staining, the biofilm is observed using a confocal laser scanning microscope (CLSM). SYTO 9 emits green fluorescence at an excitation wavelength of 488 nm, and PI emits red fluorescence at an excitation wavelength of 543 nm.

[0028] SEM results show that the addition of PDA-Ga 3+ nanoparticles can effectively inhibit the biofilm formation of Porphyromonas gingivalis, and its inhibitory effect is concentration-dependent. The PG80 group has the most significant effect, completely inhibiting the microbial communication between Porphyromonas gingivalis cells, and these cells appear as independent bacterial individuals ( Figure 6 ). The CLSM staining images obtained by live / dead staining show that as the concentration of PDA-Ga 3+ nanoparticles increases, the bacteria are eliminated, and the formation of Porphyromonas gingivalis biofilm is gradually hindered ( Figure 7 ).

[0029] PG0: Do not perform any treatment on the cells; PG20: The addition amount of PDA-Ga 3+ nanoparticles is 20 μg / mL; PG40: The addition amount of PDA-Ga 3+ nanoparticles is 40 μg / mL; PG80: PDA-Ga 3+ The addition amount of nanoparticles was 80 μg / mL; 2) PDA-Ga at different concentrations 3+ Qualitative and quantitative detection of DPPH radical scavenging by NPs Weigh DPPH and prepare a 0.04 mg / mL DPPH solution (Yesan, Shanghai) with absolute ethanol. Take 0.5 mL of PDA-Ga 3+ nanoparticle solutions at different concentrations (20 / 40 / 80 μg / mL), add 0.5 mL of DPPH solution to each respectively, mix well, take optical photos after standing at room temperature for 30 min, then centrifuge at 5000 r / min for 10 min, and measure the absorbance of the supernatant at 528 nm. The scavenging rate of the sample for DPPH radicals was calculated using the following formula: Scavenging rate = (A1 - A2) / (A0 - A2) × 100%.

[0030] A0 refers to the absorbance of 0.5 mL of absolute ethanol + 0.5 mL of DPPH solution; A1 refers to the absorbance of 0.5 mL of PDA-Ga 3+ nanoparticle solution + 0.5 mL of DPPH solution; A2 refers to the absorbance of 0.5 mL of PDA-Ga 3+ nanoparticle solution + 0.5 mL of absolute ethanol.

[0031] The results are as Figure 8 shown: After adding PDA-Ga 3+ NPs to the DPPH solution, DPPH was reduced and the solution color changed from purple to yellow; the scavenging rates of reactive nitrogen species (RNS) radicals of 20 μg / mL, 40 μg / mL, and 80 μg / mL PDA-Ga 3+ NPs were 83.05 ± 1.11%, 91.15 ± 0.48%, and 86.90 ± 1.21% respectively, indicating that the three concentrations of PDA-Ga 3+ nanoparticles all had good antioxidant properties.

[0032] 3) Expression levels of inflammation-related genes in RAW264.7 cells treated with PDA-Ga 3+ NPs (qPCR detection) Seed RAW 264.7 cells at 5 × 10 5Cells were seeded in 6-well plates at a density of cell / mL and stimulated with 100 ng / mL of Pg-LPS (Porphyromonas gingivalis lipopolysaccharide), and 20 μg / mL, 40 μg / mL, and 80 μg / mL of PDA-Ga were added simultaneously. 3+ After 6 h of treatment with the nanoparticles, total RNA was extracted using RNAiso Plus, and the relevant cDNA was synthesized using the PrimeScript™ RT reagent Kit. qPCR analysis was performed using SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) and the Roche LightCycler 480 detection system to evaluate the effects of different concentrations of PDA-Ga 3+ nanoparticles on the expression of inflammation-related genes, specifically including the expression of IL-1β, TNF-α, IL-6, and IL-10. The primer sequences of the inflammation-related genes used in qPCR are shown in Table 2.

[0033] The qPCR detection results are as Figure 9 shown: Compared with the NC group without any treatment, the pro-inflammatory related genes TNF-α, IL-1β, and IL-6 in the PC group with only LPS added were upregulated by 4.21 ± 0.52, 718.57 ± 93.84, and 41.57 ± 6.30 times, respectively, and the anti-inflammatory related gene IL-10 was downregulated by 2.85 ± 0.17 times; while the three concentrations of PDA-Ga 3+ NPs groups could all downregulate the expression of TNF-α, IL-1β, and IL-6 after LPS stimulation and upregulate the expression level of IL-10; among them, the expression level of IL-10 in the PG40 group even exceeded that of the NC group and was 1.42 ± 0.03 times that of the NC group. This indicates that PDA-Ga 3+ NPs can effectively inhibit the inflammatory response of macrophages caused by Porphyromonas gingivalis (Pg-LPS) and alleviate the progression of periodontitis.

[0034] NC: No treatment was performed on the cells; PC: Lipopolysaccharide (LPS, 100 ng / mL) was added and co-cultured with the cells without adding PDA-Ga 3+ nanoparticles; PG20: Lipopolysaccharide (LPS, 100 ng / mL) was added, and 20 μg / mL of PDA-Ga 3+ nanoparticles were added simultaneously; PG40: Lipopolysaccharide (LPS, 100 ng / mL) was added, and 40 μg / mL of PDA-Ga 3+ nanoparticles were added simultaneously; PG80: Lipopolysaccharide (LPS, 100 ng / mL) was added, and 80 μg / mL PDA-Ga 3+ nanoparticles were added simultaneously; 4) Experiment on the ability of PDA-Ga 3+ nanoparticles to regulate osteoblast / osteoclast differentiation 4.1) Experiment on the ability of DA-Ga 3+ nanoparticles to regulate osteoblast differentiation Before exploring the ability of PDA-Ga 3+ nanoparticles to regulate osteoblast differentiation, its cytotoxic effect on BMSCs cells was first explored: 0.1 mL of BMSCs cell suspension was inoculated into a 96-well plate at a density of 1×10 5 cells / mL, and after overnight incubation, different concentrations of PDA-Ga 3+ nanoparticles (20 / 40 / 80 μg / mL) were added and the cells were cultured for another 3 days. Then, the CCK-8 cell counting kit was used according to the manufacturer's instructions to evaluate the cell proliferation ability. Subsequently, qPCR was used to analyze the osteogenic gene expression levels of BMSCs in each group: 2 mL of BMSCs cell suspension was inoculated into a 6-well plate at a density of 1×10 5 cells / mL, and after overnight incubation, osteogenic induction medium containing different concentrations of PDA-Ga 3+ nanoparticles (20 / 40 / 80 μg / mL) was added and the cells were cultured for 3 days. After 3 days, the total RNA of the cells in each group was extracted using RNAiso Plus, and the relevant cDNA was synthesized using the PrimeScript™ RT reagent Kit. SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) and the Roche LightCycler480 detection system were used for qPCR experiments to analyze the differences in the expression levels of osteogenesis-related genes Runx2, ALP, and OCN in cells of different groups. Next, ALP staining and quantitative analysis were used to detect the alkaline phosphatase expression of BMSCs: 0.5 mL of BMSCs cell suspension was inoculated into a 24-well plate at a density of 1×10 5 cells / mL, and after overnight incubation, osteogenic induction medium containing different concentrations of PDA-Ga 3+ nanoparticles (20 / 40 / 80 μg / mL) was added and the cells were cultured for 3 days. After 3 days, the osteogenic differentiation performance of BMSCs treated with different concentrations of nanoparticles was evaluated using an alkaline phosphatase detection kit and a BCIP / NBT alkaline phosphatase color development kit. Finally, ARS staining and quantitative analysis were used to detect the extracellular matrix mineralization ability of BMSCs in each group: 0.5 mL of BMSCs cell suspension was inoculated into a 24-well plate at a density of 1×10 5Inoculate at a density of cells / mL into a 24-well plate. After overnight incubation, add osteogenic induction medium containing different concentrations of PDA-Ga 3+ nanoparticles (20 / 40 / 80 μg / mL) and continue culturing for 14 days. After 14 days, aspirate the old culture medium from each group, wash 3 times with PBS; fix with 250 μL of 4% paraformaldehyde fixative at room temperature for 20 minutes, and wash 3 times with PBS (5 minutes each time); add 250 μL of alizarin red solution to each well and incubate at room temperature for 30 minutes; rinse thoroughly with a large amount of running water to remove excess dye, and use a scanner to obtain the staining images of each group; for quantitative analysis, add 250 μL of 10% cetylpyridinium chloride solution to each well to dissolve the alizarin red dye bound to calcium nodules, then transfer the supernatant to a new 96-well plate and measure the OD value at a wavelength of 570 nm.

[0035] The CCK-8 detection results are as Figure 10 shown in A: After BMSCs cells were co-cultured with 20 μg / mL, 40 μg / mL, and 80 μg / mL PDA-Ga 3+ NPs for 3 days, their proliferation ability was up-regulated (PG40>PG20>PG80>PG0), and there were statistical differences between the PG20 and PG40 groups and the PG0 group. However, as the concentration increased to PG80, the cell number decreased. Overall, the results still indicate that PDA-Ga3+ NPs have good biocompatibility and no obvious toxicity to BMSCs cells.

[0036] After 3 days of osteogenic induction, the expression levels of osteogenesis-related genes Runx2, ALP, and OCN in the PG20 group, PG40 group, and PG80 group were higher than those in the PG0 group. Except for the Runx2 gene expression levels in the PG20 group and PG80 group, which showed no obvious statistical differences from those in the PG0 group, there were obvious statistical significances in the expression of osteogenic genes in the other groups compared with the PG0 group ( Figure 11 ). The ALP staining results are as Figure 12 shown. As the concentration of PDA-Ga 3+ nanoparticles increased, the number of ALP-positive cells increased, and the ALP activity detection results were consistent with this (PG80>PG40>PG20>PG0). After 14 days of osteogenic induction, the ARS staining and quantitative results are as Figure 12 shown: The cell matrix mineralization ability of the PG20 group, PG40 group, and PG80 group was stronger than that of the PG0 group, and there were statistical differences (PG40>PG80>PG20>PG0).

[0037] PG0: Do not perform any treatment on the cells; PG20: The addition amount of PDA-Ga3+ nanoparticles is 20 μg / mL; PG40: The addition amount of PDA-Ga3+ nanoparticles is 40 μg / mL; PG80: The addition amount of PDA-Ga3+ nanoparticles is 80 μg / mL; The above results show that PDA-Ga 3+ nanoparticles have good biocompatibility and do not reduce the cell viability of BMSCs; 20 μg / mL, 40 μg / mL, and 80 μg / mL PDA-Ga 3+ nanoparticles can all promote the osteogenic differentiation of BMSCs.

[0038] 4.2) Experiment on the ability of PDA-Ga 3+ nanoparticles to regulate osteoclast differentiation Before exploring the ability of PDA-Ga 3+ nanoparticles to regulate osteoclast differentiation, first explore their toxic effects on BMDM: Adjust the BMDM cell suspension to a density of 5 × 10 5 cell / mL, inoculate 0.1 mL of this cell suspension into a 96-well plate, place it in a cell culture incubator at 37°C and containing 5% CO2 for overnight culture. Subsequently, discard the old culture medium, wash it once with PBS, and then add different concentrations of PDA-Ga 3+ nanoparticles (20 / 40 / 80 μg / mL) and continue to culture for 3 days. And use the CCK-8 cell counting kit to analyze the cell proliferation according to the instructions provided by the manufacturer. Subsequently, qPCR was used to analyze the osteoclast gene expression levels of BMDM in each group (the primer sequences are shown in Table 3): Inoculate 2 mL of BMDM cell suspension at a density of 5 × 10 5 cell / mL into a 6-well plate. Different groups were induced to differentiate into osteoclasts by 25 ng / mL M-CSF and 50 ng / mL RANKL, and at the same time, 20 / 40 / 80 μg / mL PDA-Ga 3+The nanoparticles were applied for 2 days. After 2 days, the cells were washed three times with PBS, and 1 mL of RNAiso Plus was added to each well to extract total RNA. Relevant cDNA was synthesized using the PrimeScript™ RT reagent Kit. qPCR was performed using SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) and the Roche LightCycler480 detection system to detect the differential expression levels of genes closely related to osteoclast differentiation, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K (CtsK), cellular oncogene fos (C-fos), and nuclear factor of activated T cells (NFATc1) in cells of different groups. Finally, TRAP staining was used to detect the formation of mature osteoclasts in each group: BMDMs were seeded in 96-well plates at a density of 1×10 5 cells / well. Cells in different groups were induced with RANKL (50 ng / mL) and M-CSF (25 ng / mL) for 5 - 8 days. When multinucleated giant cells were visible under the microscope, TRAP staining was performed according to the instructions of the TRAP staining kit, and observations were made under an optical microscope. Cells with three or more nuclei were considered mature osteoclasts.

[0039] The CCK-8 assay results are shown in Figure 10 Figure B: After co-culturing BMDMs with 20 μg / mL, 40 μg / mL, and 80 μg / mL PDA-Ga 3+ NPs for 3 days, their proliferation ability was upregulated compared with the PG0 group (PG40 > PG20 > PG80 > PG0), and there were statistically significant differences. The number of cells in the PG80 group decreased compared with the PG20 and PG40 groups. However, the overall results still indicate that PDA-Ga 3+ NPs have good biocompatibility and no obvious toxicity to BMDMs.

[0040] The qPCR results ( Figure 13 ) showed that 20 μg / mL, 40 μg / mL, and 80 μg / mL PDA-Ga 3+ NPs downregulated the expression levels of osteoclast-related genes NFATc1, c-fos, TRAP, and Ctsk, and there were statistically significant differences; among them, PDA-Ga 3+ NPs regulated the expression of TRAP and Ctsk genes in a concentration-dependent manner. The results of TRAP staining (Figure 14 )Display: Compared with BMDM without RANKL stimulation (NC group), BMDM in the PC group can gradually fuse and form mature osteoclasts under the induction of 25 ng / mL M-CSF and 50 ng / mL RANKL, while 20 μg / mL, 40 μg / mL, and 80 μg / mL PDA-Ga 3+ After being endocytosed by cells, NPs can inhibit the cell fusion and osteoclast differentiation trend induced by RANKL.

[0041] NC: Only add 25 ng / mL M-CSF to maintain the growth of BMDM cells; PC: Add 25 ng / mL M-CSF and 50 ng / mL RANKL to stimulate and induce osteoclast differentiation in cells, without adding PDA-Ga 3+ nanoparticles; PG20: Add 25 ng / mL M-CSF and 50 ng / mL RANKL to stimulate and induce osteoclast differentiation in cells, and simultaneously add 20 μg / mL PDA-Ga3+ nanoparticles to cells; PG40: Add 25 ng / mL M-CSF and 50 ng / mL RANKL to stimulate and induce osteoclast differentiation in cells, and simultaneously add 40 μg / mL PDA-Ga 3+ nanoparticles; PG80: Add 25 ng / mL M-CSF and 50 ng / mL RANKL to stimulate and induce osteoclast differentiation in cells, and simultaneously add 80 μg / mL PDA-Ga 3+ nanoparticles; The above results show that PDA-Ga 3+ nanoparticles have good biocompatibility and do not reduce the cell viability of BMDM; 20 μg / mL, 40 μg / mL, and 80 μg / mL PDA-Ga 3+ nanoparticles can all inhibit the cell fusion and osteoclast differentiation trend induced by RANKL.

[0042] 6) Detection of the anti-inflammatory effect of PDA-Ga 3+ NPs in vivo After 12 male C57BL / 6 mice aged 6 - 8 weeks were adaptively fed for 1 week, they were randomly divided into three groups (N = 4 / group); NC group: control group, mice without ligation; PC group: mice with ligature-induced periodontitis untreated; PG group: mice with ligature-induced periodontitis treated with PDA-Ga 3+ NPs.

[0043] Anesthetize the mice by intraperitoneal injection of avertin (dose: 0.2 mL of anesthetic per 10 g body weight). Then, tie silk ligatures (nylon 4-0) around the necks of the second maxillary molars on both sides of the mice in the PC group and the PG group to induce periodontitis for 2 weeks. Subsequently, inject 0.2 mg / spot PDA-Ga 3+ NPs for treatment into the mesial, middle, and distal buccal / palatal sides of the second maxillary molars on both sides of the mice in the PG group every other day; the PC group was injected with an equal volume of ddH2O as a control. One week after treatment, euthanize all the mice. First, collect the gingival tissues around the second maxillary molars on both sides of each group of mice into 1.5 mL EP tubes, cut them into pieces with scissors, add 1 mL of RNAiso Plus to each tube to extract their total RNA, and use the PrimeScript™ RT reagent Kit to synthesize the relevant cDNA. Use SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) and the Roche LightCycler480 detection system to perform qPCR to detect the differences in the expression levels of inflammation-related genes IL-1β and IL-6 (primer sequences are shown in Table 2) in the gingival tissues of different groups. Then, take out the maxilla and fix it with 4% paraformaldehyde. Use Micro-CT (SkyScan 1176, Bruker micro-CT, Belgium) to observe the changes in the morphology, quantity, and quality of the alveolar bone. Reconstruct and obtain three-dimensional digital images and sectional images in the proximal-distal and proximal-lingual directions of different parts (CTvox software and DataViewer software). Select the bone area around the second maxillary molar as the region of interest (ROI), and quantitatively measure the bone volume / tissue volume (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) through CTAn software; measure the distance from the alveolar crest (ABC) between the first and second molars to the cementoenamel junction (CEJ) of the second molar of the mice through Image-pro-plus 6.0 software. Finally, after the specimens completed with Micro CT scanning are decalcified with 10% EDTA, embed them in paraffin and make sections for histological evaluation. Perform HE staining, Masson trichrome staining, TRAP staining, and immunohistochemical staining (Col1α and OPN) according to the instructions of the manufacturer (Servicebio, China).

[0044] The specific steps of HE staining are as follows: ① Bake the slices: Bake the slices on a baking machine for 30 min; ② Deparaffinize: Pass through xylene three times, 15 min each time; ③Rehydration: Pass through anhydrous ethanol twice, 10 minutes for each pass, then pass through 90% and 80% ethanol, 5 minutes for each pass; rinse with water for 3 minutes; ④Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 5 minutes, then rinse with running water for 5 minutes; ⑤Differentiation: Immerse the sections in the differentiation solution for 3 - 5 seconds, then rinse with running water for 5 minutes; ⑥Blueing: Treat with 85% and 95% gradient alcohol for 5 minutes each; ⑦Eosin staining: Immerse the sections in eosin staining solution for 3 minutes; ⑧Pass through anhydrous ethanol twice, 5 minutes for each pass; ⑨Clearing: Clear with xylene for 5 minutes; ⑩Mounting: Drop neutral resin on the sections and mount. Observe and take pictures under the microscope.

[0045] The specific steps of Masson trichrome staining are as follows: ①Section preparation: Dewax and rehydrate the paraffin sections according to the above steps; ②Immerse the sections in Masson A solution at room temperature overnight (about 15 hours).

[0046] ③Incubate the sections in Masson A solution in an oven at 65°C for 30 minutes, and wash with tap water for 30 seconds until the yellow color on the tissue fades. At the same time, preheat Masson D solution and Masson F solution in an oven at 65°C.

[0047] ④Mix Masson B solution and Masson C solution in equal volume (prepare and use immediately, do not prepare and store in advance), immerse the sections in the mixed solution for 1 minute, and wash slightly with running water.

[0048] ⑤Differentiate the sections with 1% hydrochloric acid alcohol (concentrated hydrochloric acid: anhydrous ethanol = 1:100) for about 1 minute until the cell nuclei are gray - black and the background is almost colorless or light gray.

[0049] ⑥Wash slightly with tap water, slightly drain the excess water on the sections, immerse the sections in Masson D solution for 6 minutes. At this time, the tissue shows bright red. If the red color is too light, the staining time can be appropriately extended. Then rinse the sections with running tap water for about 20 seconds until the water flowing from the sections is colorless.

[0050] ⑦Slightly drain the water from the sections (do not let the sections dry), immerse the sections in Masson E solution for about 1 minute. This step is for differentiation. Differentiate until the collagen fibers are light red and the fibers are red. The time of Masson E solution can be adjusted according to the staining depth as needed, generally 1 - 2 minutes.

[0051] ⑧ After the sections are slightly drained of Masson E solution, without washing with water, directly immerse them in Masson F solution for staining for 30 s.

[0052] ⑨ Rinse and differentiate the sections in three consecutive cylinders of 1% acetic acid aqueous solution for about 7 s in each cylinder. The purpose is to differentiate the excess aniline blue. Examine under the microscope during the rinse in the third cylinder of 1% acetic acid aqueous solution to avoid over-differentiation of the blue color.

[0053] ⑩ Dehydrate the sections in three consecutive cylinders of absolute ethanol for about 3 s, 5 s, and 5 s respectively. Clear in xylene for 5 min, and mount with neutral balsam. Observe and photograph under the microscope.

[0054] The steps of TRAP staining are as follows: ① Preparation of TRAP staining working solution: Preparation of substrate reaction solution B: Before use, pipette reagent B2 into reagent B1 and dissolve and mix well to prepare solution A; pipette reagent B4 into reagent B3 and dissolve and mix well to prepare solution B. Mix solution A and solution B to obtain the final solution.

[0055] Preparation of substrate reaction solution C: Before use, pipette reagent C2 into reagent C1 and dissolve and mix well.

[0056] Preparation of substrate reaction solution E: Before use, pipette reagent E2 into reagent E1 and dissolve and mix well.

[0057] Preparation of substrate incubation solution: Add 30 mL of distilled water to a staining cylinder and incubate in a water bath at 37 °C for 10 minutes. Sequentially add substrate reaction solutions C, D, and E to the pre-warmed distilled water and mix well.

[0058] ② Dewax the sections: Pass through xylene for 3 times, 15 min each time; ③ Rehydrate the sections: Immerse in absolute ethanol for 5 min, then pass through 90% and 70% ethanol for 2 min each, and immerse in distilled water for 2 min; ④ Incubation: Before the sections are completely dry, place them in the substrate incubation solution and incubate in a 37 °C incubator in the dark for 60 min; ⑤ Counterstain: After incubation, take out the sections and rinse with running water for 1 min. Counterstain before the sections are completely dry. Then counterstain with hematoxylin for 1 - 2 min. Dehydrate the sections in three consecutive cylinders of absolute ethanol for about 3 s, 5 s, and 5 s respectively. Clear in xylene for 5 min, and mount with neutral balsam.

[0059] ⑥ Microscopic examination: Observe and photograph under an optical microscope.

[0060] The specific steps of the immunohistochemistry experiment are as follows: ① Deparaffinize the paraffin sections to water: sequentially place the sections in xylene I for 15 min, xylene II for 15 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, 85% alcohol for 5 min, 75% alcohol for 5 min, and wash with distilled water.

[0061] ② Dropwise add an endogenous peroxidase blocker (3% H2O2), incubate at room temperature for 10 min to inactivate endogenous enzymes, wash with distilled water 3 times, 2 min each time; ③ Antigen retrieval: Place the tissue sections in a retrieval box filled with EDTA antigen retrieval buffer and perform antigen retrieval in a microwave oven. Medium heat for 8 min, stop for 8 min, turn to medium-low heat for 7 min, and after natural cooling, place the slides in PBS and wash 3 times, 5 min each time.

[0062] ④ Draw a circle: After slightly drying the sections, use a histochemical pen to draw a circle around the tissue.

[0063] ⑤ Serum blocking: Drop 5% BSA blocking solution within the circle, incubate at room temperature for 10 min, discard the excess liquid, and do not wash with distilled water.

[0064] ⑥ Add primary antibody: Drop the primary antibody (rabbit IgG) prepared in a certain proportion with PBS on the sections, and place the sections flat in a wet box and incubate overnight at 4°C.

[0065] ⑦ Add secondary antibody: Place the slides in PBS and wash 3 times, 5 min each time. After slightly drying the sections, drop the secondary antibody Anti-Rabbit IgG (HRP-Polymer) prepared in a certain proportion corresponding to the primary antibody within the circle to cover the tissue, and incubate at room temperature for 60 min. Rinse with PBS 3 times, 2 min each time.

[0066] ⑧ DAB color development: Use a DAB color development kit, develop color at room temperature, control the reaction time under the microscope, generally 5 - 30 s.

[0067] ⑨ Counterstain and mount: Place the slides in hematoxylin stain for 30 s - 3 min, wash with distilled water for 3 min; 85% and 95% ethanol for 5 min each, dehydrate through two passages of absolute ethanol, 5 min each passage; clear with xylene for 5 min, and mount with neutral gum.

[0068] ⑩ Microscopic examination: Observe and take pictures under an optical microscope.

[0069] PDA-Ga 3+ The anti-inflammatory results of PDA-Ga Figure 15 nanoparticles are as 3+After nanoparticle treatment, the expression levels of pro-inflammatory genes IL-6 and IL-1β in the PG group decreased. Among them, there were significant statistical differences in the IL-6 expression between the PC group and the NC group, and between the PG group and the PC group. From the Micro-CT image ( Figure 16 ), it can be seen that compared with the NC group, the alveolar bone resorption around the maxillary second molar in the PC group was obvious, while the PG group could significantly inhibit alveolar bone resorption. The specific quantitative measurement and analysis values were as follows: the distances from the cementoenamel junction (CEJ) to the alveolar crest (ABC) of the maxillary second molar in the NC, PC, and PG groups were 0.257 ± 0.037 mm, 0.559 ± 0.091 mm, and 0.0354 ± 0.059 mm, respectively; the bone volume fractions (BV / TV) of the NC, PC, and PG groups were 56.197 ± 4.331%, 26.793 ± 3.070%, and 43.233 ± 5.755%, respectively; the trabecular bone thicknesses (Tb.Th) of the NC, PC, and PG groups were 0.136 ± 0.013 mm, 0.096 ± 0.009 mm, and 0.118 ± 0.008 mm, respectively; the trabecular bone numbers (Tb.N) of the NC, PC, and PG groups were 4.172 ± 0.637 / mm, 2.805 ± 0.259 / mm, and 3.665 ± 0.507 / mm, respectively; the trabecular bone separations (Tb.Sp) of the NC, PC, and PG groups were 0.096 ± 0.017 mm, 0.142 ± 0.027 mm, and 0.107 ± 0.016 mm, respectively.

[0070] HE staining and MASSON staining ( Figure 17 ) showed that the alveolar bone around the maxillary second molar in the NC group was plump, and there was no obvious hyperplasia of the gingival papilla; the alveolar bone resorption around the target tooth in the PC group was obvious, with osteoporosis, the gingival epithelium hyperplasia presented papillary, and there were a large number of inflammatory cell infiltrations in the hyperplastic fibrous connective tissue; the pathological manifestations in the PG group were significantly alleviated compared with the PC group, but there were still mild gingival hyperplasia and bone resorption compared with the NC group.

[0071] The results of TRAP staining ( Figure 18 ) showed that compared with the NC group, the number of osteoclasts in the periodontal bone resorption area of the maxillary second molar in the PC group increased significantly, while the number of osteoclasts in the PG group decreased significantly, indicating that PDA-Ga 3+ NPs could reduce alveolar bone resorption.

[0072] The results of immunohistochemistry showed ( Figure 19 ) that the expressions of osteogenesis-related proteins Col1α and OPN in the bone resorption area of the PC group decreased significantly, while the expressions of Col1α and OPN proteins in the PG group were the highest, indicating that PDA-Ga 3+ NPs could promote bone tissue regeneration.

[0073] HE staining was performed on the important organs of each group of mice ( Figure 20 ), and no obvious pathological changes were found, further proving the in vivo safety of PDA-Ga 3+ NPs.

[0074] The above experimental results indicate that PDA-Ga 3+ nanoparticles have anti-inflammatory properties, can reduce alveolar bone resorption, and have the ability to induce bone tissue regeneration. Therefore, they can improve the success rate of periodontitis treatment. Moreover, the PDA-Ga 3+ nanoparticles provided by the present invention also possess good biological activity.

[0075] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. Without conflict, the embodiments of the present application and the features in the embodiments can be arbitrarily combined with each other.

Claims

1. A preparation method of PDA-Ga 3+ nanoparticles, characterized in that The preparation method includes the following steps: S1. Dissolve dopamine hydrochloride and gallium chloride in deionized water, and stir to obtain a pre-doped solution; S2. Add a buffer solution to the pre-doped solution, control the pH of the reaction system to be alkaline, stir to form a black suspension, and then continue stirring for reaction; S3. Centrifuge and wash the reaction system in step S2 to obtain PDA-Ga 3+ nanoparticles.

2. The preparation method of PDA-Ga nanoparticles according to claim 1, characterized in that 3+ In step S1, the mass ratio of dopamine hydrochloride to gallium chloride is 45:0.6 - 20. ​ 3. The preparation method of the PDA-Ga 3+ nanoparticles, characterized in that In step S1, the stirring time is 0.5 - 1 h.

4. The preparation method of PDA-Ga nanoparticles according to claim 1, characterized in that, 3+ In step S2, the buffer solution is Tris solution. ​ 5. The preparation method of the PDA-Ga 3+ nanoparticles, characterized in that The concentration of the Tris solution is 2.25 - 75 mg / mL.

6. The preparation method of the PDA-Ga 3+ nanoparticles, characterized in that In step S2, first stir for 0.5 - 1 h to form a black suspension, and then continue stirring for 1.5 - 24 h.

7. The preparation method of the PDA-Ga 3+ nanoparticles, characterized in that In step S3, the parameters of centrifugation are to centrifuge at 12000 - 15000 rpm for 20 - 30 min first, and then centrifuge at 2000 - 4000 rpm for differential centrifugation for 20 - 30 min.

8. A PDA-Ga 3+ nanoparticle, characterized in that The nanoparticles are prepared according to the preparation method of the nanoparticles described in any one of claims 1-7 of PDA-Ga 3+ ​ 9. Use of the PDA-Ga³ + nanoparticles in the preparation of a product for treating periodontitis.

10. Use of the PDA-Ga³ + nanoparticles in the preparation of a product for inhibiting alveolar bone resorption.

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