Nano preparation for periodontitis and periimplant periarthritis as well as preparation method and application of nano preparation

Abstract

The invention provides a nano preparation for treating periodontitis and periodontitis as well as a preparation method and application of the nano preparation. The nano preparation can simultaneously play multiple roles of photo-thermal antibiosis, anti-inflammation and osteogenesis promotion. The hydrogel nano preparation is prepared from 0.15 to 0.3 weight percent of punicalagin (PUN), 0.25 to 0.35 weight percent of mesoporous polydopamine (MPDA), 1 to 3 weight percent of hyaluronic acid-phenylboronic acid-polyvinyl alcohol injectable hydrogel matrix and the balance of water, has the characteristics of photo-thermal antibacterial property, anti-inflammatory property and osteogenesis promoting property, and has good biocompatibility; the problems of incomplete removal of bacterial plaque and drug resistance caused by combined use of existing mechanical debridement and antibiotics are solved, the efficiency and effect of treating periodontitis and peri-implantitis are remarkably improved, and the traditional Chinese medicine composition has important clinical application value and wide market prospects. The preparation method is simple and convenient, raw materials are easy to obtain and store, the treatment effect is remarkable, and industrial large-scale production, popularization and application are facilitated.

Description

Technical Field

[0001] This invention relates to the field of oral medical materials technology, specifically to a nano-formulation for periodontitis and peri-implantitis, its preparation method, and its application. Background Technology

[0002] Periodontitis is a chronic inflammatory disease driven by multiple pathogenic factors, affecting both the soft and hard tissues of the teeth. It is one of the leading causes of natural tooth loss in adults and is highly recurrent, considered one of the most prevalent chronic diseases globally. Dental plaque biofilm is a major initiating factor in periodontitis; the virulence factors it releases can activate host cells to produce inflammatory mediators and proteolytic enzymes, leading to irreversible damage to the periodontal ligament and alveolar bone. Therefore, achieving effective plaque removal and inflammation control is central to the treatment of periodontitis.

[0003] With the popularization of dental implant technology, the incidence of peri-implantitis has been rising year by year, becoming a key factor affecting the long-term success rate of implants. The etiology of peri-implantitis is similar to that of periodontitis, both closely related to plaque biofilm. However, due to the threaded structure and rough surface of implants, bacteria adhere more easily, making the biofilm more difficult to remove completely, leading to more rapid disease progression and more destructive inflammatory bone resorption. Therefore, its treatment also faces problems such as high infection persistence, difficult debridement, and high recurrence rate.

[0004] Currently, both periodontitis and peri-implantitis are primarily treated clinically with mechanical debridement combined with antibiotics. However, the complex anatomical structures of tooth roots, such as bifurcation and depressions, as well as the micropores and grooves on the implant surface, make it difficult for traditional debridement methods to thoroughly remove plaque and inflamed cementum from deep or hidden areas. Furthermore, the overuse and repeated use of antibiotics can easily induce bacterial resistance, reducing treatment effectiveness and affecting local microenvironment homeostasis. Therefore, existing treatment methods still have significant shortcomings in completely controlling infection and preventing tissue destruction.

[0005] In recent years, phototherapy, as a treatment method relying on natural or artificial light sources, has attracted attention in the field of oral infection, mainly including photothermal therapy (PTT) and photodynamic therapy (PDT). Among them, photothermal therapy is considered to have potential clinical application value due to its advantages such as not relying on antibiotics, controllable treatment, low toxicity, and no drug resistance. Photothermal agents can convert light energy into heat energy, achieving bactericidal effects by disrupting the redox balance, enzyme activity, and DNA structure of bacteria. However, traditional PTT requires temperatures to be raised above 60 ℃ to achieve significant antibacterial effects, which often leads to thermal damage to periodontal and peri-implant tissues, limiting its clinical usability. Based on this, mild photothermal therapy (mild PTT) has become a more promising strategy in recent years. This method effectively weakens bacterial cell membrane stability and increases their sensitivity to heat stimulation by controlling the local temperature in a lower range (approximately 42-50 ℃), while avoiding thermal damage to surrounding tissues. At the same time, mild photothermal stimulation can induce the expression of heat shock proteins, activate local immune regulation, and promote tissue repair, which is particularly important for the delicate tissues around periodontal and implants. Therefore, mild photothermal therapy is considered a safer and more transformative optical strategy for treating periodontitis and peri-implantitis.

[0006] Besides controlling infection, the regulation of the immune microenvironment also plays a crucial role in disease treatment. Studies have shown that in both periodontitis and peri-implantitis lesions, the proportion of M1 pro-inflammatory macrophages is significantly increased, producing large amounts of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α, activating effector cells such as neutrophils and osteoclasts, leading to persistent alveolar bone destruction. Conversely, the number of M2 macrophages is insufficient, making it difficult to effectively promote inflammation resolution and tissue repair. Therefore, regulating the transformation of macrophages from M1 to M2 types to achieve anti-inflammatory, repair-promoting, and bone resorption-inhibiting effects is considered one of the key strategies for treating these two types of diseases.

[0007] In summary, the clinical treatment of periodontitis and peri-implantitis still faces key technical bottlenecks, including the difficulty in completely removing plaque, increasing antibiotic resistance, insufficient inflammatory regulation, and irreversible bone resorption. Developing a hydrogel nanoparticle formulation based on a gentle photothermal strategy, possessing multiple functions including antibacterial, anti-inflammatory, and ossification, for the treatment of periodontitis and peri-implantitis and promoting the repair of periodontal and peri-implant tissues, is a crucial technical problem urgently needing to be solved in this field. Summary of the Invention

[0008] To overcome the shortcomings of existing technologies, this invention aims to provide a nano-formulation for periodontitis and peri-implantitis, its preparation method, and its application.

[0009] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a nano-formulation for periodontitis and peri-implantitis, comprising 0.15-0.3 wt% punicin (PUN), 0.25-0.35 wt% mesoporous polydopamine (MPDA), 1-3 wt% hyaluronic acid-phenylboronic acid-polyvinyl alcohol injection hydrogel matrix, and water; Furthermore, the formulation is an injectable preparation; A second aspect of the present invention provides a method for preparing any of the above-mentioned nano-formulations for periodontitis and peri-implantitis, comprising the following steps: Synthesis of S1 mesoporous polydopamine: A mixed solution of anhydrous ethanol and deionized water was prepared at a volume ratio of 1~2:1. Pluronic F-127 and dopamine hydrochloride were added, and the mixture was magnetically stirred at room temperature and 400~600 rpm to obtain a clear solution. Then, 1,3,5-trimethylbenzene and concentrated ammonia were slowly added dropwise to the solution to form a nanoemulsion system. The reaction was carried out at room temperature, in the dark, and with a stirring rate of 400~600 rpm for 3~5 h. The precipitate was then collected by centrifugation at 10,000~14,000 rpm for 20~50 min. The precipitate was washed alternately with an ethanol-acetone mixture and deionized water, and then freeze-dried to obtain black mesoporous polydopamine. Synthesis of S2 pungent glycoside-mesoporous polydopamine: Pungent glycoside and mesoporous polydopamine prepared in the above steps were added to anhydrous ethanol and stirred in the dark at room temperature for 10-14 h. The precipitate was collected, washed alternately with anhydrous ethanol and deionized water, and freeze-dried to obtain black pungent glycoside-mesoporous polydopamine. Preparation of S3 hyaluronic acid-phenylboronic acid-polyvinyl alcohol injection hydrogel: Hyaluronic acid was slowly added to deionized water and, after it was fully dissolved, phenylboronic acid and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride were added. The pH was adjusted to 6-7 using 1-2 mol / L hydrochloric acid. The mixture was stirred at 400-600 rpm for 70-76 h at room temperature. The solution was poured into a 6-8 kDa dialysis bag and dialyzed in deionized water for 90-100 h. The dialysis liquid was collected and freeze-dried to obtain hyaluronic acid-phenylboronic acid powder. The hyaluronic acid-phenylboronic acid and polyvinyl alcohol obtained above were then dissolved in PBS solution with a pH of 7-8 to obtain PBS solution of hyaluronic acid-phenylboronic acid and PBS solution of polyvinyl alcohol, respectively. The PBS solution of hyaluronic acid-phenylboronic acid and the PBS solution of polyvinyl alcohol were then mixed to obtain hyaluronic acid-phenylboronic acid-polyvinyl alcohol injection hydrogel. Preparation of S4 nano-formulation: weigh the black pungent glycoside-mesoporosis polydopamine obtained in S2 and disperse it in the hyaluronic acid-phenylboronic acid-polyvinyl alcohol injection hydrogel prepared in S3, mix evenly, and the nano-formulation is obtained. Furthermore, in S1, the mass ratio of Pluronic F-127 to dopamine hydrochloride is 2~1:1; Furthermore, in S1, the amounts of 1,3,5-trimethylbenzene and concentrated ammonia added are 0.015 to 0.025 of the solution volume, respectively. Furthermore, in S1, the volume ratio of ethanol to acetone in the ethanol-acetone mixture is (1~2):1; Furthermore, in S2, the mass ratio of the added pungent glycoside to mesoporous polydopamine is 1:(1~2). Furthermore, in S3, the mass ratio of the added hyaluronic acid, phenylboronic acid, and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride is 1:(1~2):1; even further, in S3, the mass ratio of the added hyaluronic acid, phenylboronic acid, and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride is 1:1:1; Furthermore, in S3, the mass fraction of hyaluronic acid-phenylboronic acid in the PBS solution is 1-2%; preferably, the mass fraction of hyaluronic acid-phenylboronic acid is 1.5%. Furthermore, in S3, the polyvinyl alcohol in the PBS solution has a mass fraction of 1-2%; even further, in S3, the polyvinyl alcohol in the PBS solution has a mass fraction of 1.5%. Furthermore, in S3, the volume ratio of the hyaluronic acid-phenylboronic acid PBS solution to the polyvinyl alcohol PBS solution is 1~4:1; Furthermore, in S4, the concentration of black pungent glycoside-mesoporphin polydopamine in the nano-formulation is 5~5.5 mg / mL; A third aspect of the present invention provides the use of any of the above-mentioned nano-formulations for periodontitis and peri-implantitis in the preparation of medicaments or medical devices for treating periodontitis and / or peri-implantitis. The beneficial effects of this invention are: Mesoporous polydopamine (MPDA) is a class of biomimetic nanomaterials formed by the spontaneous oxidative polymerization of dopamine under alkaline conditions. Its highly ordered mesoporous structure and abundant surface-active groups such as phenyl, amino, and hydroxyl groups enable it to efficiently load various types of drugs through multiple non-covalent interactions such as π-π stacking and hydrogen bonding, exhibiting excellent biocompatibility, biodegradability, and drug loading stability. Furthermore, MPDA possesses significant antioxidant properties; its reversible catechol / quinone structure effectively scavenge excess reactive oxygen species (ROS), thereby alleviating oxidative stress and helping to maintain mitochondrial homeostasis. On the other hand, due to its melanin-like molecular structure, MPDA exhibits strong near-infrared absorption and high photothermal conversion efficiency, enabling rapid and controllable heat generation under light irradiation. This heat-ablation antibacterial process achieves antibacterial action by disrupting bacterial cell membrane structure and inducing the leakage of cell contents. This process does not rely on antibiotics, avoiding the drug resistance problems associated with existing topical antibiotic applications. Therefore, MPDA is considered an ideal basic material for constructing photothermal antibacterial nanoparticle carriers.

[0010] Punicalagin (as shown in Formula I) is a natural ellagic tannin polyphenolic compound derived from pomegranate. It exhibits low toxicity, good biocompatibility, and significant antioxidant and anti-inflammatory activities. Its highly hydroxylated molecular structure endows it with a potent ability to scavenge free radicals, thereby effectively alleviating oxidative stress. Studies have shown that punicalagin can inhibit the polarization of macrophages towards the pro-inflammatory M1 phenotype and promote their conversion to the anti-inflammatory and pro-repair M2 phenotype, thereby downregulating the expression of various inflammatory factors and improving the local inflammatory microenvironment. Furthermore, punicalagin can inhibit the formation and differentiation of osteoclasts by regulating key inflammatory and bone metabolism-related signaling pathways such as NF-κB and MAPK, thus being considered a potential therapeutic candidate molecule for chronic bone resorption-related diseases. It is worth emphasizing that punicalagin possesses both anti-inflammatory and osteogenic effects, promoting alveolar bone regeneration while inhibiting excessive inflammatory responses, aligning with the dual therapeutic goals of "effective anti-inflammation + promotion of alveolar bone regeneration" urgently needed in the treatment of periodontitis and peri-implantitis. (I).

[0011] Hyaluronic acid (HA) is a naturally occurring non-immune linear glycosaminoglycan found in the extracellular matrix, playing a crucial role in maintaining tissue homeostasis, regulating cell behavior, mediating cell signaling, and promoting wound healing. The HA-PBA-PVA hydrogel system, constructed based on HA, exhibits rapid gelation under mild conditions due to the presence of reversible borate ester bonds, and displays chemical and biomechanical properties similar to living tissue, which is beneficial for remodeling the microenvironment surrounding cells. The introduction of borate ester bonds also endows this hydrogel with the ability to effectively scavenge hydrogen peroxide (H2O2) without the need for additional catalysts and without producing cytotoxic byproducts.

[0012] It is worth noting that HA-PBA-PVA hydrogel exhibits excellent responsiveness to both pH and ROS, and the pathological microenvironment of periodontitis and peri-implantitis is characterized by elevated acidity and oxidative stress levels. This allows the hydrogel to achieve more precise drug release at the lesion site. Therefore, based on its excellent biocompatibility, injectability, sustained-release properties, hydrophilicity, and antioxidant properties, HA-PBA-PVA hydrogel is considered an ideal sustained-release carrier for local drug delivery in periodontitis and peri-implantitis.

[0013] However, current clinical treatment still faces significant challenges: on the one hand, the uncontrollable drug release in traditional drug delivery methods may expose healthy tissue to excessively high drug concentrations, potentially causing damage, while the effective drug concentration in the lesion area is difficult to maintain; on the other hand, simple mechanical debridement cannot completely reach the deep periodontal pockets and complex structures such as implant grooves, resulting in incomplete removal of plaque biofilm, leading to insufficient drug delivery and limited efficacy. Therefore, there is an urgent need to develop a novel drug delivery system that can accurately and continuously release drugs and adapt to the characteristics of the lesion microenvironment to improve local treatment efficacy and reduce the risk of side effects.

[0014] This invention utilizes Pluronic F-127 as a triblock surfactant to self-assemble into micelles in an ethanol / water system, stabilizing a trimethylbenzene nanoemulsion. Pluronic F-127 acts as a soft template and pore structure guide, facilitating the polymerization of dopamine at its confined interface. Subsequent washing with alternating ethanol-acetone and deionized water removes Pluronic F-127 and trimethylbenzene, yielding mesoporous polydopamine. An injectable hydrogel nanoparticle system is then constructed based on mesoporous polydopamine, punicalin, and hyaluronic acid-phenylboronic acid-polyvinyl alcohol, achieving a dual sustained-release structure of MPDA and a hydrogel network. The MPDA nanoparticles loaded with punicalin are uniformly embedded in the hydrogel polymer network. Drug release requires diffusion through the mesoporous channels of MPDA before penetrating the outer hydrogel network, forming a "double-layered diffusion barrier," thus significantly delaying the release of punicalin and greatly extending its sustained-release time.

[0015] Building upon this, the phenylboronic acid-diol bonds in the hydrogel framework provide a dual pH / ROS response: a locally slightly acidic environment accelerates the hydrolysis of phenylboronic acid bonds, while excessive ROS further promotes the oxidative breakage of the phenylboronic acid structure, both leading to the selective deconstruction of the hydrogel network. When tissues are in a state of inflammation or high oxidative stress, the outer hydrogel barrier undergoes controlled loosening, allowing for more effective triggering of the mesoporous diffusion process of the inner MPDA layer. Thus, the system maintains long-lasting sustained release under normal conditions, while achieving stimulus-responsive enhanced release in the lesion microenvironment. This three-tiered regulatory mechanism ensures higher effective drug exposure in the lesion area while reducing the release rate in normal tissue, further minimizing the risk of burst release and improving therapeutic efficiency.

[0016] Therefore, the hydrogel nanoformulation of this invention not only possesses good biocompatibility, anti-inflammatory and osteogenic properties, but also exhibits excellent photothermal antibacterial efficiency due to the photothermal characteristics of MPDA. Its preparation method is simple, the raw materials are stable and easy to store, and it has good potential for large-scale production and application. Attached Figure Description

[0018] Figure 1 Microstructure of mesoporous polydopamine and punicin-mesopamine nanoparticles Figure 2 The specific surface area of ​​mesoporous polydopamine and pungent glycoside-mesopamine nanoparticles Figure 3 Microstructure of hyaluronic acid-phenylboronic acid-polyvinyl alcohol hydrogel and injectable hydrogel nanoparticles (punicin-mesoporosis polydopamine@hyaluronic acid-phenylboronic acid-polyvinyl alcohol hydrogel) Figure 4 The state of hyaluronic acid-phenylboronic acid-polyvinyl alcohol hydrogel and injectable hydrogel nanoparticles before and after curing. Figure 5 Drug release behavior of pungent glycoside in different systems Figure 6 Evaluation of the photothermal properties of injectable hydrogel nanoformulations Figure 7 Photothermal antibacterial effect of injectable hydrogel nanoparticles Figure 8 Anti-inflammatory effects of injectable hydrogel nanoparticles Figure 9 Osteogenesis of Injectable Hydrogel Nanoparticles Figure 10 The efficacy of injectable hydrogel nanoparticles in treating periodontitis in rats Figure 11The efficacy of injectable hydrogel nanoparticles in treating peri-implantitis in rats Detailed Implementation

[0019] The following examples are provided to better understand the preparation method of this hydrogel nanoformulation and the advantages and effects of this invention. They are not intended to limit the scope of protection for the preparation and application of this invention by being limited to the specific steps. Any product with the same or similar characteristics as this invention, obtained through inspiration or combination thereof, is within the scope of protection of this invention.

[0020] (I) Implementation Examples Example 1: Preparation of mesoporous polydopamine Weigh 2 g of Pluronic F-127 and 1 g of dopamine hydrochloride and add them to a mixture of 100 mL of anhydrous ethanol and 100 mL of deionized water. Stir the mixture at 500 rpm using a magnetic stirrer at room temperature until Pluronic F-127 and dopamine hydrochloride dissolve and the liquid becomes completely clear. While the solution is still stirred, slowly add 4 mL of 1,3,5-trimethylbenzene dropwise through a constant-pressure dropping funnel, stirring continuously for 30 minutes to form a homogeneous nanoemulsion system. Then, add 10 mL of 27 v / v% concentrated ammonia dropwise through a constant-pressure dropping funnel, and continue the reaction at room temperature, in the dark, and at 500 rpm for 4 hours. Subsequently, centrifuge the system at 12,000 rpm for 25 minutes to collect the precipitate, and wash the precipitate alternately with a mixture of 40 mL of ethanol and 20 mL of acetone and deionized water. After washing, the precipitate was collected and freeze-dried to obtain black mesoporous polydopamine nanoparticles.

[0021] Example 2: Preparation of pungent glycoside-mesoporous polydopamine Weigh 1 g of punicalin and 2 g of mesoporous polydopamine, add them to 20 mL of anhydrous ethanol, and stir with a magnetic stirrer at 500 rpm at room temperature in the dark for 12 h. After the reaction is complete, collect the solution into a centrifuge tube, centrifuge at 12,000 rpm for 10 minutes, collect the precipitate, freeze-dry it, and obtain punicalin-mesoporous polydopamine powder.

[0022] Example 3: Preparation of hyaluronic acid-phenylboronic acid-polyvinyl alcohol hydrogel Weigh 100 mg of hyaluronic acid and slowly add it to 20 mL of deionized water. After it is fully dissolved, add 47 mg of phenylboronic acid and 80 mg of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride. Adjust the pH to 6.5 with 1M hydrochloric acid and stir at 500 rpm for 72 h at room temperature. Pour the resulting solution into a 6-8 kDa dialysis bag and dialyze it in 1 L of deionized water for 96 h. Change the deionized water 3 times a day. Collect the dialyzed liquid and freeze-dry it to obtain hyaluronic acid-phenylboronic acid powder. Weigh out 45 mg of hyaluronic acid-phenylboronic acid and 15 mg of polyvinyl alcohol and dissolve them in 3 mL and 1 mL of PBS (pH 7.4) solution, respectively, to prepare hyaluronic acid-phenylboronic acid solution and polyvinyl alcohol solution with a mass fraction of 1.5 wt%. Then, mix 3 mL of solution containing 45 mg of hyaluronic acid-phenylboronic acid with 1 mL of solution containing 15 mg of polyvinyl alcohol to obtain 4 mL of hyaluronic acid-phenylboronic acid-polyvinyl alcohol hydrogel.

[0023] Example 4: Preparation of hyaluronic acid-phenylboronic acid-polyvinyl alcohol hydrogel loaded with mesoporous polydopamine 16 mg of mesoporous polydopamine powder was weighed and dispersed in 4 mL of hyaluronic acid-phenylboronic acid-polyvinyl alcohol hydrogel to obtain 4 mL of hyaluronic acid-phenylboronic acid-polyvinyl alcohol hydrogel loaded with mesoporous polydopamine.

[0024] Example 5: Preparation of injectable hydrogel nanoformulations 20.7 mg of punicin-mesoporosis polydopamine powder was weighed and dispersed in 4 mL of hyaluronic acid-phenylboronic acid-polyvinyl alcohol hydrogel to obtain 4 mL of injectable hydrogel nanoformulation.

[0025] (II) Experimental Examples Experimental methods: 1. Microstructure of mesoporous polydopamine and punicin-mesopamine nanoparticles The surface morphology and internal structure of the prepared mesoporous polydopamine (MPDA) and its complex loaded with punicin were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

[0026] 2. Specific surface area of ​​mesoporous polydopamine and pungent glycoside-mesopamine nanoparticles The specific surface area and pore structure of mesoporous polydopamine and punicalin-loaded mesoporous polydopamine nanoparticles were determined using the nitrogen adsorption-desorption (BET) method. Before testing, the samples were degassed under vacuum conditions: MPDA was degassed at 150 °C for 6 h, and punicalin-loaded mesoporous polydopamine nanoparticles were degassed at 80 °C for 10 h to prevent drug decomposition. Adsorption-desorption isotherms were measured at liquid nitrogen temperature (77 K), the specific surface area was calculated according to the BET equation, and the pore size distribution was analyzed using the BJH method.

[0027] 3. Microstructure of hyaluronic acid-phenylboronic acid-polyvinyl alcohol hydrogel and injectable hydrogel nanoparticles (punicin-mesoporosis polydopamine@hyaluronic acid-phenylboronic acid-polyvinyl alcohol hydrogel) Hyaluronic acid-phenylboronic acid-polyvinyl alcohol hydrogel and injectable hydrogel nanoparticles were prepared into hydrogels, which were then rapidly cooled with liquid nitrogen and fractured to obtain cross sections. After freeze-drying using a vacuum freeze dryer, the microstructure was observed using a scanning electron microscope.

[0028] 4. Measurement of the curing state of hyaluronic acid-phenylboronic acid-polyvinyl alcohol hydrogel and injectable hydrogel nanoformulation. Place 1 ml of hydrogel in a small glass vial, tilt it, and take a picture. After the hyaluronic acid-phenylboronic acid-polyvinyl alcohol hydrogel and injectable hydrogel nanoparticles have solidified, observe their state, cut them at an angle, invert them, and take a picture. Load the uncured pure hydrogel and hydrogel nanoparticles into a 1 ml syringe, and push them to observe whether the hydrogel is injectable.

[0029] 5. Drug release behavior of pungent glycoside in different systems To evaluate the release regulation and sustained-release effects of different carriers on punicalin, punicalin-mesoporous polydopamine, punicalin@hyaluronic acid-phenylboronic acid-polyvinyl alcohol hydrogel, and punicalin-mesoporous polydopamine@hyaluronic acid-phenylboronic acid-polyvinyl alcohol hydrogel were placed in dialysis bags and transferred to PBS (pH 7.4) at 37 ℃ with shaking for in vitro release experiments. Samples were taken at 0, 2, 4, 8, 12, 24, 36, 48, 60, and 72 h, and 1 mL of the released solution was taken and replenished with an equal volume of fresh PBS. The concentration of punicalin in the samples at each time point was determined by high-performance liquid chromatography (HPLC), and the relative cumulative release (%) was calculated based on the standard curve. Release curves were plotted to compare the sustained-release performance of different systems.

[0030] 6. Evaluation of the photothermal properties of injectable hydrogel nanoformulations An injectable hydrogel nanoparticle formulation with a mesoporous polydopamine concentration of 4 mg / mL was placed in a quartz cuvette and subjected to a power density of 1.5 W / cm². 2The photothermal response performance of injectable hydrogel nanoformulations was evaluated by recording the temperature rise curve under 808 nm near-infrared laser irradiation using an infrared thermal imager. Simultaneously, the laser was turned off after the sample reached its maximum temperature, allowing it to cool naturally back to its initial temperature. This process was repeated three times to perform a laser on-off cycle experiment.

[0031] 7. Photothermal antibacterial effect of injectable hydrogel nanoparticles 1×10 6 CFU / ml suspensions of *Porphyromonas gingivalis* and *Fusobacterium nucleatum* were inoculated into 48-well plates, and injectable hydrogel nanoparticles of mesoporous polydopamine at a concentration of 4 mg / mL were added. The light-treated group received a power density of 1.5 W / cm². 2 The bacteria were irradiated with an 808 nm near-infrared laser for 15 min and then cultured together with the dark group under anaerobic conditions at 37°C for 12 h. 20 μL of bacterial suspension from each well was plated and cultured under the aforementioned conditions for 72 h. The culture results were compared, and photographs were taken.

[0032] 8. In vitro anti-inflammatory effects of injectable hydrogel nanoparticles RAW264.7 cells were seeded, and an in vitro inflammation model was established using 100 ng / mL lipopolysaccharide (LPS). Hyaluronic acid-phenylboronic acid-polyvinyl alcohol hydrogel, hyaluronic acid-phenylboronic acid-polyvinyl alcohol hydrogel loaded with mesoporous polydopamine, and hyaluronic acid-phenylboronic acid-polyvinyl alcohol hydrogel loaded with punicin-mesopamine were then added to the hydrogel. The control group received no LPS stimulation and no drug treatment; the LPS group received 100 ng / mL LPS stimulation but no drug treatment; the hydrogel group received 100 ng / mL LPS stimulation followed by 200 ppm hyaluronic acid-phenylboronic acid-polyvinyl alcohol hydrogel treatment; the mesoporous polydopamine-hydrogel group received 100 ng / mL LPS stimulation followed by 200 ppm mesoporous polydopamine-loaded hyaluronic acid-phenylboronic acid-polyvinyl alcohol hydrogel treatment; and the hydrogel nanoparticle group received 100 ng / mL LPS stimulation followed by 200 ppm punicin-mesoporous polydopamine-loaded hyaluronic acid-phenylboronic acid-polyvinyl alcohol hydrogel treatment. RNA was extracted on days 3 and 5 of culture and reverse transcribed. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, and interleukin levels were calculated by real-time quantitative PCR. Il-6 ), tumor necrosis factor-α ( ) Tnf-α ), interleukin-10 ( Il-10 ) and transforming growth factor-β ( Tgf-β The relative expression levels of genes.

[0033] 9. Injectable hydrogel nanoparticles promote osteogenic effects in vitro. RAW 264.7 cells were seeded, and the corresponding hydrogel was added 12 h later. The culture medium from the wells was aspirated daily, centrifuged at 1200 rpm for 10 min at 4°C, and the supernatant was collected and filtered. The filtered RAW 264.7 cell supernatant was indirectly co-cultured with MC3T3-E1 cells. On days 14 and 21 after co-culture, 2% Alizarin Red staining solution was added, and the mineralization of MC3T3-E1 cells was observed and recorded under an optical microscope after 5 min.

[0034] 10. The efficacy of injectable hydrogel nanoparticles in the in vivo treatment of periodontitis A rat model of periodontitis was established in the left maxillary second molar. After mechanically removing plaque, tartar, and food debris around the affected tooth, sterile saline (periodontitis group), minocycline ointment (minocycline group), and an injectable hydrogel nanoparticle formulation were injected into the gingival sulcus of the affected tooth, once a week for a total of four times. The injectable hydrogel group was immediately irradiated with an 808 nm near-infrared laser for 15 min after injection. On day 28 post-treatment, the animals were euthanized, and maxillary alveolar bone and jawbone specimens were obtained. High-resolution micro-CT scanning was used to perform three-dimensional imaging of each bone sample to evaluate the efficacy of the periodontitis treatment.

[0035] 11. The efficacy of injectable hydrogel nanoparticles in the in vivo treatment of peri-implantitis A rat model of peri-implantitis was established. Sterile saline (peri-implantitis group) and injectable hydrogel nanoparticles were injected around the implants every two days for a total of five times. The injectable hydrogel nanoparticle group received 808 nm near-infrared laser irradiation for 15 minutes immediately after injection. On day 21 post-treatment, the animals were euthanized, and maxillary alveolar bone and jawbone specimens with implants were obtained. High-resolution micro-CT scanning was used to perform three-dimensional imaging of the bone samples containing implants to evaluate the effectiveness of the treatment for peri-implantitis.

[0036] Experimental results: 1. Microstructure of mesoporous polydopamine and punicin-mesopamine nanoparticles like Figure 1 As shown, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of mesoporous polydopamine reveal that the nanoparticles are approximately 100 nm in size, with abundant mesoporous structures on their surface. SEM and TEM images of pungent glycoside-mesoporous polydopamine show that the nanoparticles are approximately 100 nm in size. SEM images reveal that, compared to mesoporous polydopamine, the pore size of pungent glycoside-mesoporous polydopamine is reduced, indicating that pungent glycoside was successfully incorporated into the porous structure of the mesoporous polydopamine.

[0037] 2. Specific surface area of ​​mesoporous polydopamine and pundicin-mesopamine nanoparticles like Figure 2 As shown, BET surface area analysis results indicate that the specific surface area of ​​the mesoporous polydopamine is 53.73 m² / g, and the pore volume is 0.21 cm³ / g. In contrast, the specific surface area and pore volume of punicalin-mesoporous polydopamine decreased to 40.09 m² / g and 0.17 cm³ / g, respectively. This decrease is attributed to the successful entry and occupation of punicalin molecules into the mesoporous pores of the mesoporous polydopamine, proving that punicalin was successfully loaded into the channels of the mesoporous polydopamine.

[0038] 3. Microstructure of hyaluronic acid-phenylboronic acid-polyvinyl alcohol hydrogel and injectable hydrogel nanoformulation (punicin-mesoporosis polydopamine@hyaluronic acid-phenylboronic acid-polyvinyl alcohol hydrogel) like Figure 3 As shown, both the hyaluronic acid-phenylboronic acid-polyvinyl alcohol hydrogel and the injectable hydrogel nanoparticles exhibit a uniformly connected three-dimensional porous network, which is conducive to water diffusion and nutrient exchange. Among them, the injectable hydrogel nanoparticles have a more complete and stable pore network, which provides spatial support for the stable loading and uniform distribution of punicin-mesoporous polydopamine nanoparticles, and helps to achieve continuous release and avoid sudden release.

[0039] 4. The state of injectable hyaluronic acid-phenylboronic acid-polyvinyl alcohol hydrogel and injectable hydrogel nano-formulation before and after curing. like Figure 4 As shown, both the hydrogel nanoformulation of the present invention and the simple hyaluronic acid-phenylboronic acid-polyvinyl alcohol hydrogel are in a liquid state before curing. The hydrogel nanoformulation appears as a black substance containing pungent glycoside-mesoporous polydopamine. After curing, both are in a stable solid state, and the hydrogel nanoformulation remains injectable.

[0040] 5. Drug release behavior like Figure 5 As shown, the in vitro release behavior of punicalin in different carrier systems exhibited significant rate differences. The results indicated that punicalin was released fastest in mesoporous polydopamine (MPDA) alone; the release rate decreased in the hyaluronic acid-phenylboronic acid-polyvinyl alcohol (HA-PBA-PVA) system; and the release was slowest in the composite hydrogel nanoformulation (punicalin-MPDA@HA-PBA-PVA). In the composite system, MPDA particles are embedded in a polymer network, and the drug must first diffuse through the MPDA pores before permeating through the outer hydrogel network, forming a "double diffusion barrier," which significantly delays the release process. This multi-layered control mechanism of the composite hydrogel nanoformulation ensures continuous and stable drug release, helps maintain effective drug concentrations, and reduces the risk of burst release.

[0041] 6. Evaluation of the photothermal properties of injectable hydrogel nanoformulations like Figure 6 As shown, the injectable hydrogel nanoformulation with a mesoporous polydopamine concentration of 4 mg / ml can reach a temperature of 55.4℃ after irradiation with an 808 nm near-infrared laser for 15 min, exhibiting photothermal response properties. Furthermore, in three laser on-off cycles, the temperature rise of the injectable hydrogel nanoformulation remained stable, with no temperature decrease observed. Figure 6 (a) and (b) represent the photothermal heating curve of the hydrogel nanoparticle formulation and the heating-cooling curve of the three-on-off test, respectively.

[0042] 7. Photothermal antibacterial effect of injectable hydrogel nanoparticles like Figure 7 As shown, under dark conditions, the injectable hydrogel nanoparticle formulation with a mesoporous polydopamine concentration of 4 mg / ml exhibited almost no antibacterial activity against *Porphyromonas gingivalis* and *Fusobacterium nucleatum*. Under 808 nm near-infrared laser irradiation, the bacterial colonies in the injectable hydrogel nanoparticle formulation group were significantly lower than those in the control group, indicating that the injectable hydrogel has excellent photothermal antibacterial activity. Figure 7 (a) and (b) represent the antibacterial plate coating diagrams of hydrogel nanoparticles against Porphyromonas gingivalis and Fusobacterium nucleatum, respectively.

[0043] 8. Anti-inflammatory effects of injectable hydrogel nanoparticles like Figure 8 As shown, real-time quantitative PCR results indicate that after treatment with injectable hydrogel nanoparticles, pro-inflammatory genes in RAW264.7 cells were reduced. Il-6 and Tnf-α Expression was significantly downregulated, while anti-inflammatory genes... Il-10 and Tgf-β Significant upregulation of expression can effectively suppress inflammation. Figure 8 (a)-(f) represent IL-6 and TNF-α, respectively. α CD86, IL-10, TGF- β The relative expression levels of MRC1 / CD206 mRNA.

[0044] 9. Osteogenesis of Injectable Hydrogel Nanoparticles like Figure 9 As shown, after 14 and 21 days of treatment with the injectable hydrogel nanoparticles, the amount of calcium nodule deposition was significantly higher than that in the control group and the LPS group, indicating that the injectable hydrogel nanoparticles can promote osteoblast differentiation under inflammatory conditions and have an osteogenic effect.

[0045] 10. The efficacy of injectable hydrogel nanoparticles in treating periodontitis like Figure 10 As shown, the results of Micro-CT three-dimensional reconstruction indicate that the distance from the cementoenamel junction to the alveolar crest in rats treated with injectable hydrogel nanoparticles was significantly reduced in the periodontitis group, demonstrating excellent inhibitory effects on bone loss and promotion of bone regeneration in vivo.

[0046] 11. The efficacy of injectable hydrogel nanoparticles in treating peri-implantitis Figure 11 These are Micro-CT reconstruction images of rat jawbones with implants, in vitro. The peri-implantitis group represents untreated samples, while the hydrogel nanoparticle group represents samples taken 21 days after treatment with the hydrogel nanoparticle. High-brightness cylindrical images (high-density images) represent pure titanium dental implants, medium-brightness images (medium-density images) represent peri-implant mineralized jawbone, and low-gray to black areas mainly represent low-attenuation components (such as soft tissues like granulation tissue and fibrous capsules, background / air, and voids). CT results show that the hydrogel nanoparticle group has a larger area of ​​medium-density mineralized bone near the implant, while the peri-implantitis group has a larger area of ​​low-grayness granulation tissue near the implant. This indicates that the hydrogel nanoparticle group has more peri-implant mineralized bone and less inflammatory granulation / fibrous capsule soft tissue. This demonstrates that injectable hydrogel nanoparticles have significant advantages in controlling peri-implantitis-related bone destruction: treatment with hydrogel nanoparticles increases peri-implant bone density, reduces the extent of bone defects, decreases low-density inflammatory granulation / fibrous capsule tissue, and results in a more continuous overall bone wall structure.

[0047] The above description is merely a preferred embodiment of the present invention, but the present invention should not be limited to the content disclosed in this embodiment. Therefore, any equivalent or modified versions made without departing from the spirit of the present invention fall within the scope of protection of the present invention.

Claims

1. A nano-formulation for periodontitis and peri-implantitis, characterized in that, It is composed of 0.15~0.3 wt% punicin (PUN), 0.25~0.35 wt% mesoporous polydopamine (MPDA), 1~3 wt% hyaluronic acid-phenylboronic acid-polyvinyl alcohol injection hydrogel matrix and water.

2. The nano-formulation for periodontitis and peri-implantitis according to claim 1, characterized in that, The preparation is an injectable formulation.

3. A method for preparing a nano-formulation for periodontitis and peri-implantitis as described in any one of claims 1 to 2, characterized in that, Includes the following steps: Synthesis of S1 mesoporous polydopamine: A mixed solution of anhydrous ethanol and deionized water was prepared at a volume ratio of (1~2):

1. Pluronic F-127 and dopamine hydrochloride were added, and the mixture was magnetically stirred at room temperature and 400~600 rpm to obtain a clear solution. Then, 1,3,5-trimethylbenzene and concentrated ammonia were slowly added dropwise to the solution to form a nanoemulsion system. The reaction was carried out at room temperature, in the dark, and with a stirring rate of 400~600 rpm for 3~5 h. The precipitate was then collected by centrifugation at 10,000~14,000 rpm for 20~50 min. The precipitate was washed alternately with an ethanol-acetone mixture and deionized water, and then freeze-dried to obtain black mesoporous polydopamine. Synthesis of S2 pungent glycoside-mesoporous polydopamine: Pungent glycoside and mesoporous polydopamine prepared in the above steps were added to anhydrous ethanol and stirred in the dark at room temperature for 10-14 h. The precipitate was collected, washed alternately with anhydrous ethanol and deionized water, and freeze-dried to obtain black pungent glycoside-mesoporous polydopamine. Preparation of S3 hyaluronic acid-phenylboronic acid-polyvinyl alcohol injection hydrogel: Hyaluronic acid was slowly added to deionized water and, after it was fully dissolved, phenylboronic acid and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride were added. The pH was adjusted to 6-7 using 1-2 mol / L hydrochloric acid. The mixture was stirred at 400-600 rpm for 70-76 h at room temperature. The solution was poured into a 6-8 kDa dialysis bag and dialyzed in deionized water for 90-100 h. The dialysis liquid was collected and freeze-dried to obtain hyaluronic acid-phenylboronic acid powder. The hyaluronic acid-phenylboronic acid and polyvinyl alcohol obtained above were then dissolved in PBS solution with a pH of 7-8 to obtain PBS solution of hyaluronic acid-phenylboronic acid and PBS solution of polyvinyl alcohol, respectively. The PBS solution of hyaluronic acid-phenylboronic acid and the PBS solution of polyvinyl alcohol were then mixed to obtain hyaluronic acid-phenylboronic acid-polyvinyl alcohol injection hydrogel. Preparation of S4 nano-formulation: Weigh the black pungent glycoside-mesoporosis polydopamine obtained in S2 and disperse it in the hyaluronic acid-phenylboronic acid-polyvinyl alcohol injection hydrogel prepared in S3, mix evenly, and the nano-formulation is obtained.

4. The method for preparing the nano-formulation for periodontitis and peri-implantitis according to claim 3, characterized in that, In S1, the mass ratio of Pluronic F-127 to dopamine hydrochloride is (2~1):

1.

5. The method for preparing the nano-formulation for periodontitis and peri-implantitis according to claim 3, characterized in that, In S1, the amounts of 1,3,5-trimethylbenzene and concentrated ammonia added are 0.015 to 0.025 of the solution volume, respectively.

6. The method for preparing the nano-formulation for periodontitis and peri-implantitis according to claim 3, characterized in that, In S1, the volume ratio of ethanol to acetone in the ethanol-acetone mixture is (1~2):

1.

7. The method for preparing the nano-formulation for periodontitis and peri-implantitis according to claim 3, characterized in that, In S2, the mass ratio of added pungent glycoside to mesoporous polydopamine is 1:(1~2).

8. The method for preparing the nano-formulation for periodontitis and peri-implantitis according to claim 3, characterized in that, In S3, the mass ratio of the added hyaluronic acid, phenylboronic acid and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride is 1:(1~2):1; In S3, the hyaluronic acid-phenylboronic acid PBS solution contains 1-2% hyaluronic acid-phenylboronic acid by mass. In S3, the polyvinyl alcohol in the PBS solution contains 1-2% polyvinyl alcohol by mass. In S3, the volume ratio of the hyaluronic acid-phenylboronic acid PBS solution to the polyvinyl alcohol PBS solution is (1~4):

1.

9. The method for preparing the nano-formulation for periodontitis and peri-implantitis according to claim 3, characterized in that, In S4, the concentration of black pungent glycoside-mesoporphin polydopamine is 5~5.5 mg / mL.

10. The use of the nano-formulation for periodontitis and peri-implantitis as described in any one of claims 1 to 2 in the preparation of drugs or medical devices for treating periodontitis and / or peri-implantitis.

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