Dendritic mesoporous silica loaded with shikonin, preparation method and antibacterial effect

Abstract

This invention provides a dendritic mesoporous silica and its preparation method that can inhibit key pathogenic processes such as growth, acid production, sugar production, and biofilm formation of cariogenic bacteria and achieve inhibitory effects on cariogenic bacteria in complex microbial communities. This invention does not aim to simply kill all bacteria, but rather utilizes the acidic microenvironment of caries to trigger drug release. By inhibiting key pathogenic processes such as growth, acid production, sugar production, and biofilm formation of cariogenic bacteria, it reduces the overall cariogenic toxicity of the biofilm. This "functional inhibition" strategy is theoretically more conducive to maintaining the stability of the microbial community structure while reducing the pathogenicity of the biofilm.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a dendritic mesoporous silica with shikonin loaded on its surface, its preparation method, and its uses. Background Technology

[0002] Dental caries is a major public health problem affecting oral health worldwide, and its root cause lies in the dynamic imbalance of the dental plaque microecology. Under physiological conditions, the oral cavity is a sophisticated ecosystem composed of hundreds of microorganisms. Symbiotic bacteria and opportunistic pathogens maintain a dynamic balance through complex interactions, jointly forming the first biological barrier against pathogen invasion. This fragile ecological balance is easily disrupted by modern dietary patterns. Frequent intake of fermentable carbohydrates provides excessive metabolic substrates for cariogenic microorganisms such as Streptococcus mutans and Lactobacillus, leading to abnormally active glycolysis pathways and the production and accumulation of large amounts of organic acids. This process not only directly lowers the local pH value at the plaque-enamel interface, triggering the initial chemical process of enamel demineralization, but also causes microbial ecological plaque imbalance. Plaque imbalance is not only about increased bacterial count, but also about the comprehensive activation of cariogenic virulence phenotypes, such as biofilm formation, acid production and acid tolerance, polysaccharide synthesis, and enhanced expression of related genes. A persistently low pH microenvironment selectively promotes the competitive advantage of acid-tolerant and acid-producing bacteria while inhibiting the growth of beneficial bacteria sensitive to acid, ultimately leading to an irreversible transformation of the plaque community structure from a healthy symbiotic model to a cariogenic pathological one. This imbalanced state of plaque microecology, characterized by the dominance of specific cariogenic bacteria, significantly enhanced acid production and tolerance, denser biofilm structure, and abnormal metabolic activity, is the initiating factor and continuous driving force behind the development of dental caries.

[0003] Faced with this etiological challenge, existing clinical intervention strategies have significant theoretical and practical limitations. Traditional mechanical removal methods are effective to some extent, but their effectiveness heavily depends on patient compliance and operator technique, and cannot achieve 24-hour dynamic control of plaque. While chemical antimicrobial agents such as chlorhexidine can broadly inhibit microbial growth, long-term use may lead to oral flora imbalance, taste disturbances, and increase the risk of microbial resistance. Their non-selective killing mode can also damage the self-recovery potential of the oral microecology. Most current caries prevention agents lack ecological selectivity, and modern caries prevention emphasizes regulation rather than eradication. An ideal intervention strategy should precisely inhibit the virulence expression of cariogenic bacteria, controlling their numbers and metabolic activity within a harmless range, while protecting or promoting the growth of beneficial bacteria, guiding the entire biofilm ecosystem back to a non-pathogenic state. In the complex and variable physiological environment of the oral cavity, including saliva rinsing, pH fluctuations, and temperature changes, how to target and deliver active ingredients to the tightly structured cariogenic biofilm and achieve controlled release and sustained action within a specific microenvironment is a major scientific challenge facing existing technologies.

[0004] Despite its significant advantages in antibacterial activity, shikonin's application in the complex oral environment still faces certain limitations. For example, the molecule has poor water solubility and limited stability in the oral environment, and it lacks the ability to directly regulate tooth mineralization. Therefore, shikonin alone is insufficient to address the microecological issues in the caries process, necessitating the use of appropriate delivery systems to improve its stability and targeting efficiency, particularly nanotechnology-driven intelligent drug delivery systems. Summary of the Invention

[0005] This invention provides a dendritic mesoporous silica that can inhibit key pathogenic processes such as growth, acid production, sugar production, and biofilm formation of cariogenic bacteria, and can achieve inhibitory effects on cariogenic bacteria in complex bacterial communities, and provides an intelligent drug delivery system.

[0006] The present invention provides a dendritic mesoporous silica with shikonin loaded on its surface. Shikonin is loaded on the surface of the raw dendritic mesoporous silica, and the surface of the shikonin-loaded mesoporous silica is coated with a polydopamine coating and a polyethylene glycol modification layer.

[0007] The raw material dendritic mesoporous silica in this invention does not refer to dendritic mesoporous silica with shikonin loaded on its surface, but rather to dendritic mesoporous silica that has not yet been loaded with shikonin.

[0008] Furthermore, the specific surface area of ​​the aforementioned dendritic mesoporous silica loaded with shikonin is 38-44 m². 2 / g, pore volume is 0.1-0.15cm³ 3 / g, with a diameter of 53.10±8.07nm.

[0009] Furthermore, the preparation method of the above-mentioned raw material dendritic mesoporous silica includes the following steps: (1) Preparation of alkaline catalyst solution: Weigh triethanolamine into a container and place it in a water bath to form a homogeneous alkaline catalyst solution; (2) Constructing a surfactant film system: Hexadecyltrimethylammonium bromide and sodium salicylate were added sequentially to the solution formed in (1), and stirred at 70-90℃ for 0.5-1.5h to form a triethanolamine / hexadecyltrimethylammonium bromide / sodium salicylate composite micelle system with hexadecyltrimethylammonium bromide as template and sodium salicylate as pore expander; (3) Silicon source hydrolysis condensation and crystal nucleus growth: Tetraethyl orthosilicate is added to the composite micelles formed in (2) to form a dendritic mesoporous structure with central radial channel characteristics; (4) Separate the product and purify it to obtain dendritic mesoporous silica for later use.

[0010] Furthermore, the mass ratio of the above-mentioned triethanolamine, hexadecyltrimethylammonium bromide, sodium salicylate, and tetraethyl orthosilicate is 1: 5-6: 2-3: 53-55.

[0011] The present invention also provides a method for preparing the above-mentioned dendritic mesoporous silica with shikonin loaded on its surface, comprising the following steps: (1) Shikonin loaded onto dendritic mesoporous silica: The dendritic mesoporous silica prepared above was dispersed in another alcohol solution, and shikonin was also dissolved in another alcohol solution. Under the conditions of room temperature and protection from light, the shikonin solution was added dropwise to the dendritic silica and stirred for 20-30 h. After the reaction was completed, a mixture was obtained. The mixture was centrifuged, and after being dispersed in a solution, it was washed and purified to obtain the final precipitate, which was stored in the dark for later use. (2) Polydopamine surface modification: The precipitate purified in (1) was dispersed in a buffer solution, and dopamine hydrochloride was also dissolved in the same other buffer solution. Under the conditions of room temperature and protection from light, dopamine hydrochloride was added dropwise to the dispersion of the precipitate purified in (1), and stirred for 20-30 hours. After the reaction was completed, a mixture was obtained. The mixture was centrifuged, washed and purified to obtain the final precipitate, which was stored in the dark for later use. (3) Polyethylene glycol surface modification: The precipitate purified in (2) was dispersed in a buffer solution, and the methoxy polyethylene glycolamine was also dissolved in another buffer solution. Under the conditions of room temperature and protection from light, the methoxy polyethylene glycolamine was added dropwise to the dispersion of the precipitate purified in (2), and stirred for 20-30 hours. After the reaction was completed, a mixture was obtained, washed and purified, and used for later use.

[0012] Furthermore, in the above (1), the mass ratio of dendritic mesoporous silica to shikonin is 4-6:1, and the volume ratio of the alcohol solution dissolving dendritic mesoporous silica to the alcohol solution dissolving shikonin is the same as the mass ratio of dendritic mesoporous silica to shikonin.

[0013] Furthermore, in the above (2), the mass ratio of the precipitate purified by (1) to dopamine hydrochloride is 2-3:1, and the volume ratio of the buffer solution for dissolving the precipitate purified by (1) to the buffer solution for dissolving dopamine hydrochloride is 4-6:1.

[0014] Furthermore, in the above (3), the mass ratio of the precipitate purified by (2) to the methoxy polyethylene glycolamine is 1:2-3, and the volume ratio of the buffer solution for dissolving the precipitate purified by (2) to the buffer solution for dissolving the methoxy polyethylene glycolamine is 1-3:1.

[0015] The present invention also provides the use of dendritic mesoporous silica loaded with shikonin for inhibiting cariogenic bacteria, including Streptococcus mutans, Streptococcus distantly, and Streptococcus sanguinis.

[0016] Furthermore, the aforementioned dendritic mesoporous silica loaded with shikonin exhibits inhibitory effects against Streptococcus mutans, Streptococcus pyogenes, and Streptococcus sanguinis in mixed bacterial communities.

[0017] Healthy oral plaque is not simply a buildup of bacteria, but a complex micro-ecosystem composed of various microorganisms. Different bacterial species maintain a relatively stable dynamic balance through nutrient competition, metabolic interactions, and spatial occupancy. When this balance is disrupted, the relative abundance of acid-producing and acid-tolerant bacteria, such as *S. mutans*, increases abnormally, and the plaque micro-ecosystem gradually shifts towards a cariogenic state. This is considered an important biological basis for the development of dental caries. This invention shifts from simply pursuing broad-spectrum bactericidal action to the targeted regulation of the plaque micro-ecosystem. Its core is not to completely eliminate oral flora, but to inhibit the dominant expansion of cariogenic bacteria, maintaining plaque in a relatively non-pathogenic homeostasis. This regulatory model helps preserve the basic diversity of the oral microbiota and reduces excessive disturbance to the normal micro-ecosystem while lowering the risk of caries.

[0018] The MSPP provided by this invention still exhibits a considerable total drug solubility under organic solvent-free conditions, the particle surface tends to be electrically neutral, and it has better long-term dispersion stability and biocompatibility. Furthermore, it is a drug delivery system with targeted enamel.

[0019] This invention does not aim to simply kill all bacteria, but rather to use the acidic microenvironment of caries to trigger drug release. By inhibiting key pathogenic processes such as the growth, acid production, sugar production, and biofilm formation of cariogenic bacteria, it reduces the overall cariogenic toxicity of the biofilm. This "functional inhibition" strategy is theoretically more conducive to maintaining the stability of the bacterial community structure while reducing the pathogenicity of the biofilm.

[0020] In a mixed bacterial community simulating dental caries, the MSPP of this invention exhibits significant antibacterial activity, effectively inhibiting the metabolic activity of cariogenic streptococci such as *S. mutans*, *S. sanguinis*, and *S. sobrinus*, and reducing their acid-producing capacity. Acid production metabolism and extracellular polysaccharide synthesis are considered the two most critical virulence phenotypes of cariogenic bacteria, which respectively determine the formation of the acidic environment of plaque and the structural stability of biofilm. MSPP can simultaneously inhibit the acid-producing capacity and extracellular polysaccharide synthesis capacity of cariogenic bacteria, thereby weakening the cariogenic potential of biofilms at both the metabolic and structural levels. Attached Figure Description

[0021] Figure 1 for Figure 1 Immediate (A), 24h (B), and 48h (C) liquid appearance morphology of Shikonin (1% DMSO)①, Shikonin (DDW)②, and MSPP (DDW)③; Figure 2The images show the morphological characteristics of MSPP under transmission and scanning electron microscopy. (A) Transmission electron microscopy image of MSPP (scale bar = 100 nm); (B) Scanning electron microscopy images of MSPP at different magnifications (5000×, scale bar = 200 nm; 10000×, scale bar = 100 nm). Figure 3 Fourier transform infrared spectra of MSPP, DMSNs, and Shikonin; Figure 4 Figure 1 shows the results of CCK-8 experiments on HOK cells and MC3T3-E1 cells using MSPP at pH 7.0 and pH 5.5. Figure 5 Figure 1 shows the results of MSPP staining of live and dead cells in HOK and MC3T3-E1 cells; Figure 6 The graph shows the determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of MSPP against cariogenic bacteria. (A) Bacterial growth after treatment with different concentrations of MSPP at pH 7.0 and pH 5.5 for 24 h, plate plating results, with dashed boxes indicating the corresponding culture dishes for MBC determination; (B) Changes in absorbance (OD630) measured by an ELISA reader at a wavelength of 630 nm. Figure 7 The diagram shows the acid production kinetics of cariogenic bacteria under MSPP intervention: (A) acid production kinetic curve; (B) pH change in the culture medium ΔpH. Different superscript letters indicate significant differences between groups (P<0.05). Figure 8 The effect of intervention on sugar production and adhesion ability of cariogenic bacteria is shown in the figure. (A) Production of insoluble extracellular polysaccharides (IEPS); (B) Bacterial adhesion rate. Different superscript letters indicate significant differences between groups (P<0.05). Figure 9 Scanning electron microscope images of biofilms after MSPP intervention; Figure 10 The images show the fluorescence staining analysis of live and dead bacteria in S. mutans biofilms treated with MSPP. (A) CLSM three-dimensional structural reconstruction and Z-axis layered image (interlayer spacing = 1 μm); (B) Packing map of live and dead bacteria ratio based on ImageJ analysis (scale bar = 200 μm). Green fluorescence indicates live bacteria, and red fluorescence indicates dead bacteria. Figure 11 The image shows the fluorescence staining analysis of live and dead bacteria in S. sanguinis biofilms under MSPP treatment; (A) CLSM 3D structural reconstruction and Z-axis layered image (layer spacing = 1 μm); (B) Stacking map of live and dead bacteria ratio based on ImageJ analysis (scale bar = 200 μm). Green fluorescence represents live bacteria, and red fluorescence represents dead bacteria; Figure 12 Fluorescence staining analysis of live and dead bacteria in S. sobrinus biofilms treated with MSPP; (A) CLSM three-dimensional structural reconstruction and Z-axis layered image (interlayer spacing = 1 μm); (B) Packing map of live and dead bacteria ratio based on ImageJ analysis (scale bar = 200 μm). Green fluorescence indicates live bacteria, and red fluorescence indicates dead bacteria; Figure 13 A fluorescence staining analysis of live and dead bacteria in a mixed bacterial biofilm under MSPP treatment; (A) CLSM 3D structural reconstruction and Z-axis layered image (layer spacing = 1 μm); (B) Stacked map of live and dead bacteria ratio based on ImageJ analysis (scale bar = 200 μm). Green fluorescence represents live bacteria, and red fluorescence represents dead bacteria. Detailed Implementation

[0022] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to examples. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained in the art without creative effort should fall within the scope of protection of the present invention.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0024] Example 1: Synthesis and water solubility determination of dendritic mesoporous silica with surface-loaded shikonin 1.1 Synthesis of raw material dendritic mesoporous silica (DMSNS) (1) Preparation of alkaline catalyst solution: Accurately weigh 0.068 g of triethanolamine (TEA) into a 250 mL three-necked round-bottom flask, add 25 mL of deionized water, and place in an 80 °C constant temperature water bath. Preheat and stir evenly at a stirring rate of 150 rpm for 1 h to form a uniform alkaline catalytic environment.

[0025] (2) Construction of surfactant template system: While maintaining the alkaline catalyst solution in a water bath with stirring, 380 mg of hexadecyl trimethyl ammonium bromide (CTAB) and 168 mg of sodium salicylate (NaSal) were added sequentially to the solution. This mixing process was continued at 80°C for 1 hour with stirring to ensure complete dissolution of CTAB and NaSal, and to allow for self-assembly to form a stable CTAB / NaSal / TEA composite micelle system with CTAB as a template and NaSal as a pore-expanding agent.

[0026] (3) Silicon source hydrolysis condensation and DMSNs nucleation growth: Using a constant-pressure dropping funnel, 4 mL of tetraethyl orthosilicate (TEOS) was slowly and uniformly added dropwise to the CTAB / NaSal / TEA composite micelle aqueous solution prepared in step two. The dropping rate was controlled at approximately 0.5 mL / min, and the entire process lasted for approximately 8 min to avoid excessively high local concentrations of TEOS, which could lead to homogeneous nucleation and the formation of amorphous silica impurities. After the addition was complete, the system temperature was maintained at 80 °C, and the reaction was continued for 2 h with stirring at 300 rpm. During this process, the silicate species generated by the hydrolysis of TEOS under alkaline conditions and the positively charged CTA⁺-Sal⁻ composite micelles undergo synergistic self-assembly through electrostatic interactions. The silica network condenses and solidifies around the micelle template, ultimately forming a dendritic mesoporous structure with central radial channels.

[0027] (4) Separation and purification of the product: After the reaction was complete, the milky white reaction solution was quickly transferred to 50 mL centrifuge tubes and placed in a high-speed refrigerated centrifuge pre-cooled to 4°C. Centrifuged at 11,000 rpm for 10 min at 4°C, the supernatant was carefully discarded, and the bottom white precipitate was collected. The white precipitate was redispersed with 40 mL of deionized water and centrifuged at 11,000 rpm for 10 min at 4°C. This process was repeated until the pH of the supernatant was measured to be 7.0 to completely remove residual ions and catalyst. Subsequently, the precipitate was dispersed and washed with 40 mL of anhydrous ethanol and centrifuged (4°C, 11,000 rpm, 10 min), repeated three times to displace the water between the particles, improve the subsequent drying efficiency, and obtain a loose product.

[0028] (5) Post-treatment and template removal: The alcohol-washed wet product was transferred to a lyophilization bottle and initially dried in a vacuum freeze dryer at -50°C and <10 Pa for 24 h to obtain a white crude DMSNs containing a CTAB template. The lyophilized sample was placed in an alumina ceramic crucible and further dried in a temperature-controlled muffle furnace. Under static air, the temperature was increased to 550°C at a rate of 2°C / min and calcined at this temperature for 4 h. This process aimed to completely decompose and remove the CTAB template agent in the channels, while further condensing the silicon framework to enhance its structural stability. After calcination, the product was allowed to cool naturally to room temperature, finally obtaining a powdered DMSNs final product with open mesoporous channels, which was then stored in a desiccator for later use.

[0029] 1.2 Synthesis of Dendritic Mesoporous Silica (MSPP) with Shikonin Loaded on its Surface (1) Shikonin loaded onto DMSN 50 mg of DMSNs were dispersed in 50 mL of anhydrous ethanol and sonicated for 30 min until completely dispersed. 10 mg of shikonin was dissolved in 10 mL of anhydrous ethanol and sonicated until clear. The shikonin anhydrous ethanol solution (10 mL, 1 mg / mL) was placed in a syringe and added dropwise at a constant rate of 0.8 mL / min to the continuously stirred (300 rpm) DMSNs ethanol dispersion using a syringe pump. The entire addition process was carried out at room temperature in the dark. After the shikonin addition was complete, the mixture was kept on a thermostatic magnetic stirrer and stirred at 300 rpm for 24 h at room temperature in the dark.

[0030] (2) Purification of crude DMSNs-Shikonin After the reaction was complete, the mixture was centrifuged at 12,000 rpm for 15 min to collect the loaded nanoparticles. To remove unencapsulated shikonin, the precipitate was washed with 5 mL of anhydrous ethanol: after adding ethanol, the precipitate was first gently vortexed for 10 s to initially disperse it, and then immediately placed in an ice-water bath and sonicated at 100 W for 1 min to ensure complete dispersion. The entire washing process was completed within 5 min, followed immediately by centrifugation at 12,000 rpm for 15 min and discarding the supernatant. This washing process was repeated three times. Finally, the precipitate was washed twice with PBS buffer in the same manner to replace the dispersion medium. The final precipitate was stored at 4°C in the dark for later use.

[0031] (3) Polydopamine surface-modified structure (DMSNs-Shikonin@PDA) The purified DMSNs-Shikonin precipitate was dispersed in 50 mL of Tris-HCl buffer (10 mM, pH 8.5) and sonicated in an ice-water bath for 20 min until completely dispersed. 20 mg of dopamine hydrochloride was weighed and dissolved in 10 mL of the same buffer, then sonicated until clear. The dopamine hydrochloride solution (10 mL, 2 mg / mL) was placed in a syringe and added dropwise at a constant rate of 1.0 mL / min to the continuously stirred (300 rpm) DMSNs-Shikonin dispersion using a syringe pump. The entire addition process was carried out at room temperature in the dark. After the addition was complete, the reaction system was continued to be stirred at 300 rpm for 24 h at room temperature in the dark.

[0032] After the reaction was complete, the mixture was centrifuged at 12,000 rpm for 15 min, and the supernatant was discarded. To remove unreacted monomers and byproducts, the precipitate was washed with deionized water: after adding deionized water, the precipitate was vortexed and resuspended, and then centrifuged under the same conditions. This washing process was repeated three times. The final precipitate was stored at 4°C in the dark for later use.

[0033] (4) Polyethylene glycol surface modification (DMSNs-Shikonin@PDA-PEG) The purified DMSNs-Shikonin@PDA precipitate was dispersed in 50 mL of Tris-HCl buffer (10 mM, pH 8.5) and sonicated in an ice-water bath for 20 min until completely dispersed. 125 mg of methoxypoly(ethylene glycol) amine (mPEG-NH2, Mw = 2000 Da) was weighed and dissolved in 25 mL of the same buffer solution, and sonicated until clear. The mPEG-NH2 solution (25 mL, 6 mg / mL) was placed in a syringe and added dropwise at a constant rate of 0.5 mL / min to the continuously stirred (300 rpm) DMSNs-Shikonin@PDA dispersion using a syringe pump. The entire addition process was carried out at room temperature in the dark. After the addition was complete, the reaction system was continued to be stirred at 300 rpm for 24 h at room temperature in the dark.

[0034] After the reaction, the mixture was centrifuged at 12,000 rpm for 15 min, and the supernatant was discarded. To remove unreacted monomers and byproducts, the precipitate was washed with deionized water: after adding deionized water, the precipitate was vortexed and resuspended, and then centrifuged under the same conditions. This washing process was repeated three times. To facilitate long-term storage and maintain the structural integrity of the nanoparticles, the final washed precipitate was dispersed in phosphate buffered saline (PBS) containing 5% (w / v) mannitol to prepare a homogeneous dispersion with a concentration of 10 mg / mL. The dispersion was aliquoted into sterile lyophilization bottles and pre-frozen at -80°C for 4 h. Subsequently, it was transferred to a freeze dryer and freeze-dried at -50°C and 0.05 mBar for 48 h to obtain loose and porous DMSNs-Shikonin@PDA-PEG solid powder. The lyophilized powder was sealed in a light-proof container and stored in a dry and light-proof environment at -20°C for long-term storage. Before use, take an appropriate amount of lyophilized powder, resuspend it in the corresponding buffer solution, and briefly sonicate it to restore it to a uniform dispersion.

[0035] 1.3 Water solubility determination In the physical characterization of the MSPP nanoparticle drug delivery system, the dissolution behavior of the active ingredient shikonin and the effect of the nanocarrier on its solubility were first systematically evaluated. Free shikonin exhibits extremely low water solubility in deionized water (…). Figure 1 During the observation period ( Figure 1 None of them showed enhanced solubility, suggesting that the drug is difficult to apply directly to aqueous media before being loaded into a nanosystem.

[0036] After being encapsulated by a nanocarrier, the solubility characteristics of the active ingredient changed significantly. The prepared drug-loaded nano-dispersion rapidly formed a homogeneous and transparent solution system in deionized water, without visible precipitation or phase separation. Figure 1 Time-dependent observations show () Figure 1 MSPP remained stable during a 48-hour standing period, without drug recrystallization, particle aggregation, or increased solution turbidity, demonstrating that nanocarriers can significantly improve the water dispersibility of drugs and impart good time stability. Furthermore, compared to free shikonin dissolved in 1% DMSO (… Figure 1 MSPP still exhibits a considerable total drug solubility even without organic solvents, and has better long-term dispersion stability and biocompatibility.

[0037] Example 2 Characterization of MSPP 2.1 Characterization Methods 2.1.1 Transmission electron microscopy analysis and scanning electron microscopy analysis Approximately 0.5 mg of MSPP lyophilized powder was dispersed in 1 mL of anhydrous ethanol and subjected to ultrasonic treatment in an ice-water bath (100 W, 1 min) to prepare a highly diluted and homogeneous suspension. 5 μL of this suspension was pipetted and slowly added dropwise to the center of a copper mesh carbon film, and allowed to air dry at room temperature for 10 min. The dried sample-carrying copper mesh was then placed into the TEM sample holder. The morphology, particle size, dispersibility, and core-shell structure of the nanoparticles were observed at different magnifications (×20 kV, ×50 kV, ×100 kV) under an accelerating voltage of 120 kV.

[0038] The silicon wafer was ultrasonically cleaned with anhydrous ethanol and then air-dried. A small amount of MSPP powder was evenly sprinkled onto the sample holder with conductive adhesive, and any unadhered particles were gently blown away with a syringe. To enhance conductivity, a platinum film of approximately 5 nm thickness was deposited on the sample surface using an ion sputtering instrument.

[0039] 2.1.2 Fourier Transform Infrared Spectroscopy Analysis Approximately 1 mg of MSPP lyophilized powder and 100 mg of dry KBr powder were thoroughly mixed and ground in an agate mortar until homogeneous and extremely fine. An appropriate amount of the mixture was placed into a tableting mold and pressed into a transparent or translucent sheet under a hydraulic press at 10 MPa for 1 minute. The KBr tablet was placed in the sample chamber, and transmission spectra were acquired using a pure KBr tablet as the background at a wavenumber range of 4000-400 cm⁻¹, a resolution of 4 cm⁻¹, and 32 scans.

[0040] 2.1.3 Dynamic light scattering and Zeta potential analysis Accurately weigh 1 mg of MSPP lyophilized powder, disperse it in 10 mL of PBS buffer to prepare a homogeneous suspension of 0.1 mg / mL, and briefly vortex.

[0041] Hydrated particle size and polydispersity index (PDI) measurement: Inject approximately 1 mL of sample into a plastic cuvette and place it in the instrument. Set the temperature to 25 °C and the equilibration time to 2 min. Select the "Size Measurement" mode and run at least 3 measurements, taking the average value to obtain the hydrodynamic diameter distribution and PDI value of the nanoparticles in the solution, in order to evaluate their dispersion stability.

[0042] Zeta potential measurement: The sample is injected into a folded capillary electrophoresis pool, ensuring no air bubbles are present. The same temperature is set, the "Zeta potential" mode is selected, and the "Smoluchowski" model is used. At least five measurements are run, and the average value is taken to obtain particle surface charge data, verifying the change in surface charge after PEGylation.

[0043] 2.1.4 Specific Surface Area and Pore Structure Analysis Approximately 50 mg of MSPP lyophilized powder was accurately weighed and placed into a clean, dry sample tube. The sample tube was then placed in a degassing station and degassed under vacuum at 120°C for 6 kJ to completely remove adsorbed moisture and gas from the sample surface. The degassed sample tube was then transferred to the analysis station. N2 adsorption-desorption isotherms were measured at liquid nitrogen temperature. The relative pressure (P / P0) ranged from 0.01 to 0.99. The BET specific surface area (SBET) of the sample was calculated using the Brunauer-Emmett-Teller (BET) model. The pore size distribution was calculated from the adsorption branch data using the Barrett-Joyner-Halenda (BJH) model.

[0044] 2.2 Characterization Results 2.2.1 Transmission electron microscopy and scanning electron microscopy analysis of MSPP Transmission electron microscopy (TEM) was used to characterize the structural morphology of the nanosystem. TEM images ( Figure 2 The data clearly shows that DMSNs exhibit a typical dendritic mesoporous structure with open channels and a high specific surface area. After surface modification with PDA-PEG, the outer edge of the DMSNs is coated with a uniform and continuous amorphous layer. This coating layer completely covers the surface of the DMSNs without obvious cracking or aggregation, indicating that the PDA-PEG composite layer was successfully constructed and has a uniform morphology. The coated nanoparticles still maintain good monodispersity without significant morphological damage or structural collapse, confirming that the surface modification process did not affect the integrity of the mesoporous framework of the DMSNs.

[0045] 2.1.2 Fourier Transform Infrared Spectroscopy Analysis The chemical structures of Shikonin, DMSNs and MSPP were characterized by FTIR. Figure 3 ) Spectral coverage 4000-1500cm -1 Senses area and 1500-400 cm -1 Fingerprint area 196.

[0046] Sensual area, Shikonin at 3450cm -1 The characteristic absorption of phenolic hydroxyl groups is observed at this location; DMSNs exhibit a broad but weak band in this region due to surface silanol groups and adsorbed water. In MSPP, this absorption peak shows a significant increase in intensity, broadening in shape, and a red shift of approximately 15 cm⁻¹, indicating that the hydroxyl group participates in a multiple hydrogen bond network: involving interfacial interactions between Shikonin phenolic hydroxyl groups, PDA catechol / amino groups, and DMSNs surface silanol groups. 2950 cm⁻¹ -1 With 2850cm -1 The increased intensity of the CH stretching vibration peak in MSPP is attributed to the methylene structure of the PEG-modified layer, confirming successful PEG grafting.

[0047] Fingerprint area, Shikonin at 1650-1550cm -1 The characteristic C=C and N–H absorptions are retained in MSPP, but with broadened peak shapes, indicating that the active material is loaded within the pores and the bulk structure is intact. MSPP shows better absorption than DMSNs at 1250 cm⁻¹. -1 and 1100cm -1 The newly added absorption peaks correspond to the COC ether bond of PEG and the CN stretching vibration of the aromatic amine of PDA, respectively, providing direct evidence for PDA encapsulation and PEG modification. These results collectively confirm the successful construction of drug-loaded nanoparticles MSPP, that is, the active substance is effectively loaded onto the DMSNs carrier, and further functionalized through PDA encapsulation and PEG surface modification.

[0048] 2.1.3 Dynamic light scattering and Zeta potential analysis Based on dynamic light scattering and Zeta potential analysis, the physicochemical properties of nanoparticles (DMSNs, DMSNs-Shikonin, DMSNs-Shikonin@PDA, and DMSNs-Shikonin@PDA-PEG) at four stages of MSPP construction were systematically characterized. Particle size analysis showed that the hydrodynamic diameter of the nanoparticles gradually increased from 118.77±4.46 nm for DMSNs to 153.10±8.07 nm for MSPP, indicating that the Shikonin loading, PDA coating, and PEG modification processes were all successful without causing significant particle aggregation. Zeta potential analysis showed that the surface potential gradually increased from -14.35±0.96 mV for DMSNs to -7.38±1.11 mV for MSPP (results shown in Table 1), indicating that PEG modification effectively neutralized the surface charge, making the particle surface more electrically neutral. This property helps reduce non-specific interactions with negatively charged components in biological systems, improving the colloidal stability and blood compatibility of the nanoparticles.

[0049] Table 1 Physicochemical properties of MSPP nanoparticles constructed in four stages polymer Size (nm) Zeta potential (mV) Specific surface area (m² / g) Pore ​​volume (cm3 / g) Aperture (nm) DMSNs 118.77±4.46 -14.35±0.96 268.21 0.59 2.67 DMSNs-Shikonin 130.21±3.37 -8.56±2.19 117.56 0.37 2.08 DMSNs-Shikonin@PDA 140.32±4.49 -10.44±3.29 41.28 0.12 - DMSNs-Shikonin@PDA-PEG 153.10±8.07 -7.38±1.11 42.63 0.10 - 2.1.4 Specific Surface Area and Pore Structure Analysis The specific surface area and pore structure of samples at each stage of the construction process of DMSNs and MSPP were systematically characterized using nitrogen adsorption-desorption technology. As shown in Table 1 above, DMSNs exhibited a high specific surface area (268.21 m² / g), a large pore volume (0.59 cm³ / g), and a uniform mesopore size (2.67 nm). This structural feature provided sufficient physical space and diffusion channels for efficient loading of Shikonin. After loading Shikonin (DMSNs-Shikonin), the specific surface area decreased to 117.56 m² / g, the pore volume decreased to 0.37 cm³ / g, and the pore size decreased to 2.08 nm, indicating that Shikonin molecules had successfully entered and occupied part of the pore space. After PDA coating (DMSNs-Shikonin@PDA), the specific surface area further decreased to 41.28 m² / g, the pore volume decreased to 0.12 cm³ / g, and the pore size data was no longer significant, confirming that the PDA layer formed a dense coating on the particle surface, effectively sealing the mesoporous channels. In the PEG-modified stage (DMSNs-Shikonin@PDA-PEG), the specific surface area (42.63 m² / g) and pore volume (0.10 cm³ / g) remained essentially unchanged, indicating that PEG modification mainly acted on the outer surface of the particles and did not further affect the internal pore structure.

[0050] 3. Evaluation of in vitro cell activity and toxicity of the MSPP system 3.1 Experimental Methods 3.1.1 Cell Culture and Passaging Take cryopreserved HOK and MC3T3-E1 cell cryovials, thaw rapidly in a 37°C water bath, centrifuge, and culture; wash and passage; take cells in logarithmic growth phase, digest and centrifuge, and resuspend in the appropriate complete culture medium. Count cells using a Countstar IC1000 automated cell counter to ensure a viable cell rate >95%. Dilute the cell suspension to the working concentration for later use.

[0051] 3.1.2 CCK-8 assay for cell proliferation activity Follow the instructions in the kit's instructions.

[0052] 3.1.3 Live / Dead Cell Double Staining Assay Cell treatment: HOK and MC3T3-E1 cells were seeded into 24-well plates and cultured for 24 hours until adherence. The original culture medium was then discarded. 500 μL of complete culture medium containing different concentrations of MSPP (20.0, 10.0, 5.0, 2.5, 1.25, 0.625, 0.3125 mg / mL) was added to each well, with three replicates for each concentration. A cell control group was also included, and the cells were cultured for another 24 hours.

[0053] Staining and observation: After incubation, the culture medium was discarded, and the cells were washed once with PBS. 300 μL of staining working solution consisting of Calcein-AM (2 μM) and propidium iodide (PI, 4.5 μM) was added to each well, and the cells were incubated at 37°C in the dark for 20 min. Immediately after incubation, the cells were observed using an inverted fluorescence microscope: green fluorescent live cells were observed using the FITC channel (excitation / emission: 494 / 517 nm), and red fluorescent dead cells were observed using the TRITC channel (excitation / emission: 535 / 617 nm). Three non-overlapping fields of view from each well were randomly selected for photographing and recording.

[0054] 3.2 Experimental Results In vitro cell viability and toxicity were evaluated using the CCK-8 assay and live / dead cell fluorescence staining. HOK and MC3T3-E1 cells were treated with MSPP at concentrations ranging from 20 to 0.3125 mg / mL under pH 5.5 and pH 7.0 conditions. The CCK-8 assay results showed ( Figure 4 At concentrations ≤ 5 mg / mL, the viable cell ratio of both cell types was above 80% under both pH conditions; at concentrations ≤ 2.5 mg / mL, the viable cell ratio exceeded 90%. Further results from live and dead cell fluorescence staining indicated (…). Figure 5 At a concentration of 20,000 mg / mL, the vast majority of cells exhibited green fluorescence (live cells), with only a few red fluorescent positive cells (dead cells) observed in the 20 mg / mL and 10 mg / mL treatment groups. This indicates that within the experimental concentration range, MSPP did not show significant cytotoxicity to HOK and MC3T3-E1 cells, and exhibited good biocompatibility at concentrations ≤5 mg / mL.

[0055] 4. The impact of MSPP on the plaque microecology of major cariogenic bacteria The standard strains used in this study include: Streptococcus mutans (Streptococcus mutans) Streptococcus mutans, S. mutans UA159 (ATCC 700610), distant streptococci ( Streptococcus sobrinus, S. sobrinus )6715 ((ATCC6715) and Streptococcus sanguinis ( Streptococcus sanguinis, S. sanguinis, )10556 (ATCC 10556) were all purchased from Guangdong Provincial Microbial Culture Collection Center.

[0056] 4.1 Experimental Methods 4.1.1 MSPP Planktonic Bacterial Inhibition Experiment 4.1.1.1 Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of MSPP against three major cariogenic bacteria MSPP was prepared using PBS at pH 5.5 and pH 7.0 via a two-fold dilution method in BHI-sucrose liquid medium (BHI-sucrose, BHIS) containing 1% sucrose. The concentration gradient of MSPP was set from 20 to 0.0625 mg / mL. The following control groups were established: 0.12% CHX as a positive control, BHIS bacterial suspension alone as a negative control, and BHIS medium without bacterial suspension as a blank control.

[0057] Three single bacterial suspensions (1×10⁷ CFU / mL) and their proportionally mixed suspensions were inoculated with the drug solution at a 1:1 volume ratio into 96-well plates. After shaking with a micro-shaker for 1 min, the plates were incubated at 37°C and 5% CO₂ for 24 h in the dark. The absorbance (OD630) of each well was measured using a microplate reader at 630 nm. The inhibition rate was calculated as [1 - (A630 experimental group / A630 negative control group)] × 100%. The MIC50 was determined as the drug concentration that inhibited growth by 50%.

[0058] For each well with a drug concentration higher than the MIC, 20 μL was spread onto BHI solid medium and cultured for another 24 hours under the same conditions. The lowest drug concentration with fewer than 3-5 colonies was defined as the MBC.

[0059] 4.1.1.2 Effect of MSPP on Acid Production Levels of Three Major Cariogenic Bacteria MSPP solution was prepared in BHIS liquid medium using a two-fold dilution method, with the concentration gradient set from 20 to 0.0625 mg / mL. 0.12% CHX was used as the positive control group, BHIS bacterial suspension alone as the negative control group, and BHIS medium without bacterial suspension as the blank control group.

[0060] Three single-strain bacterial suspensions (1×10⁷ CFU / mL) and their equal-ratio mixed suspensions with the drug solution (V:V=1:1) were inoculated into test tubes and cultured at 37℃ and 5% CO₂ in a constant-temperature shaker at 160 rpm for 72 h. During the culture, samples were taken at 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24, 48, and 72 h. Equal volumes of culture were centrifuged at 4℃ and 8000 rpm for 30 min, and the supernatant was collected. The cell pellet was flash-frozen in liquid nitrogen and stored at -80℃ for subsequent RNA isolation.

[0061] The pH value of the supernatant was measured using a precision digital pH meter, and acid production curves of three major cariogenic bacteria were plotted. To compare the pH-lowering abilities of different groups, the pH change difference over 72 hours was calculated (ΔpH = initial pH - final pH). 4.1.1.3 Effects of MSPP on the synthesis of water-insoluble extracellular polysaccharide (IEPS) by three major cariogenic bacteria Plotting the standard curve for the determination of dextran using the sulfuric acid-anthrone method: Measurement of IEPS: Three single-species bacterial suspensions (1×10⁷ CFU / mL) and their proportionally mixed suspensions were prepared with the corresponding minimum inhibitory concentrations (MICs) selected in the antibacterial experiment, and BHI-containing liquid culture media were prepared at concentrations of 2MIC, 1MIC, 1 / 2MIC, and 1 / 4MIC. The bacterial suspensions and drug-containing culture media were mixed in test tubes at a volume ratio of V:V=1:1 and incubated at 37℃, 5% CO₂, and 160 rpm for 48 h.

[0062] The obtained culture was centrifuged at 12000 rpm and 4℃ for 30 min, and the supernatant was collected. The precipitate was washed with sterile distilled water and centrifuged twice. The washed precipitate was resuspended in 4 mL of 0.4 mol / L NaOH, washed, and centrifuged three times. The supernatants were combined and further centrifuged, filtered, and sterilized.

[0063] A suitable amount of supernatant was used to determine the content of water-insoluble extracellular polysaccharides in bacteria using the sulfuric acid-anthrone method: 200 μL of supernatant was mixed with 600 μL of anthrone reagent and reacted at 95 °C for 10 min; after cooling, the OD value at 625 nm was measured, and the content of water-insoluble extracellular polysaccharides was calculated using a standard curve.

[0064] 4.1.1.4 Effect of MSPP on the adhesion ability of cariogenic bacteria MSPP solution was prepared in BHIS liquid medium using a two-fold dilution method, with the concentration gradient set from 20 to 0.0625 mg / mL. 0.12% CHX was used as the positive control group, BHIS bacterial suspension alone as the negative control group, and BHIS medium without bacterial suspension as the blank control group.

[0065] Three single bacterial suspensions (1×10⁻⁶) 7 The bacterial suspension (CFU / mL) and its equal-ratio mixed bacterial suspension were added to sterile glass tubes at a volume ratio of 1:1 with MSPP solutions of different concentrations. The tubes were then incubated at 37°C and 5% CO2 for 2 hours to allow the bacteria to adhere to the glass wall surface.

[0066] After incubation, the supernatant was carefully discarded, and the inner wall of the test tube was gently washed three times with sterile PBS buffer to remove unadhered bacteria. Then, a certain volume of sterile PBS was added to the test tube, and the bacteria adhering to the glass wall surface were detached and suspended by thorough shaking. The resulting bacterial suspension was serially diluted and spread onto BHI solid medium plates, and incubated at 37°C and 5% CO2 for 24 hours. The number of colonies formed (CFU) was counted, and the adhesion rate of bacteria in each group was calculated.

[0067] The adhesion rate is calculated using the following formula: Adhesion rate (%) = CFU of inoculated bacteria / CFU of adherent bacteria × 100.

[0068] 4.1.2 MSPP Biomembrane Inhibition Experiment 4.1.2.1 Scanning electron microscopy detection of MSPP biofilm formation The three main cariogenic bacteria in the logarithmic growth phase were centrifuged at 3000 rpm for 10 min, the supernatant was discarded, and the suspensions were resuspended in BHIS medium. The bacterial concentration was adjusted to OD630 = 0.2. The three bacterial suspensions were then mixed in equal volumes to prepare a mixed bacterial solution.

[0069] Place a sterile coverslip in each well of a 6-well plate, add 2 mL of mixed bacterial solution, and incubate statically at 37 °C and 5% CO2 for 24 h to construct a mature biofilm.

[0070] After biofilm formation, 2 mL of the appropriate concentration of drug solution was added to each well, and incubation continued for 24 h under the same conditions. After the intervention was completed, the cells were gently washed twice with pre-warmed sterile PBS. Drug preparation and grouping were the same as in Experiment 1.2.2.4.

[0071] After drug intervention, carefully aspirate the culture medium and drug, and gently wash three times with 0.1M phosphate buffer (pH 7.0). Add 2.5% glutaraldehyde solution and fix overnight at 4°C. After fixation, wash three times with PBS for 10 min each time.

[0072] The samples were subjected to gradient dehydration using 30%, 50%, 70%, 80%, 90%, 95%, and 100% ethanol solutions, with each stage lasting 15 minutes. After critical point drying, coverslips were attached to the sample stage and coated with platinum.

[0073] The three-dimensional structure of the biofilm and the morphology of the bacteria were observed using a scanning electron microscope at different magnifications (×2000, ×5000), and representative field-of-view images were collected.

[0074] 4.1.2.2 MSPP staining for removing live and dead bacteria from biofilms that have already formed. Three major cariogenic bacteria in logarithmic growth phase were centrifuged at 3000 rpm for 10 min, the supernatant was discarded, and the suspensions were resuspended in BHIS medium, adjusted to OD630 = 0.2, and the three bacterial suspensions were mixed in equal volumes. Sterile cell smears were placed in each well of a 6-well plate, 2 mL of the mixed bacterial suspension was added, and the plates were incubated statically for 24 h under the same conditions.

[0075] The experimental groups included: drug intervention groups (1.250, 0.625, 0.3125 mg / mL), a positive control group (0.12% CHX), and a negative control group (BHIS medium). After biofilm formation, 2 mL of the corresponding concentration of drug solution was added to each well, and incubation was continued for 24 h under the same conditions. After completion, the cells were gently washed twice with pre-warmed sterile PBS.

[0076] The fluorescent staining solution was prepared according to the kit instructions: 3 μL each of SYTO 9 and PI staining solution were added to 3 mL of sterile PBS and mixed thoroughly in the dark. 500 μL of staining solution was added to each slide, and the slide was incubated at 37 °C in the dark for 15 min. After washing with pre-warmed sterile PBS, the slide was placed on a glass slide.

[0077] Observation was performed using an inverted fluorescence microscope (SYTO 9 excitation / emission wavelength 480 / 500 nm; PI excitation / emission wavelength 490 / 635 nm), with 10 fields of view randomly selected for each sample.

[0078] 4.2 Experimental Results 4.2.1 MSPP Planktonic Bacterial Inhibition Experiment 4.2.1.1 MIC and MBC The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of MSPP against S. mutans, S. sanguinis, S. sobrinus and their equal-proportion mixed strains were determined using the micro-broth dilution method combined with plate plating validation at pH 7.0 and pH 5.5.

[0079] Under neutral conditions at pH 7.0, the plate coating results showed ( Figure 6 MSPP showed only a slight inhibitory effect on the growth of *S. mutans* at a concentration of 10 mg / mL; it had some inhibitory effect on *S. sobrinus* and mixed strains at 5 mg / mL, but the inhibitory trend on *S. sanguinis* was not obvious. These results indicate that the direct antibacterial activity of MSPP in a neutral environment is relatively weak.

[0080] Under acidic conditions of pH 5.5, the results of plate coating and absorbance of the microplate reader were obtained. Figure 6All studies showed that with increasing MSPP concentration, the growth of each tested strain was inhibited to varying degrees, exhibiting a concentration-dependent trend. Based on this, the MICs of MSPP against *S. mutans*, *S. sanguinis*, *S. sobrinus*, and their mixtures were determined to be 0.625, 0.625, 1.25, and 0.625 mg / mL, respectively, at pH 5.5, with corresponding MBCs of 5, 10, 2.5, and 10 mg / mL. These results indicate that the antibacterial activity of MSPP varies under different pH conditions, exhibiting stronger inhibitory activity under acidic conditions.

[0081] 4.2.1.2 Effect of MSPP on Acid Production Levels of Three Major Cariogenic Bacteria

[0082] The statistical analysis results of the terminal (72h) ΔpH value are shown in the figure. Figure 7 The control group showed the largest ΔpH range due to a significant decrease in culture medium pH caused by bacterial metabolism; the ΔpH value of the MSPP-treated group gradually decreased with increasing drug concentration. Under 2MIC concentration intervention, the ΔpH values ​​of the three strains and the mixed bacterial community were significantly lower than those of the control group (P<0.05): S. mutans decreased from 3.26±0.01 to 1.73±0.02, S. sanguinis from 1.93±0.01 to 0.45±0.03, S. sobrinus from 2.75±0.02 to 0.40±0.07, and the mixed bacterial community from 2.65±0.01 to 0.54±0.06.

[0083] The above results indicate that MSPP can inhibit the glycolysis and acid production process of cariogenic bacteria, and has an inhibitory effect on both single and mixed bacterial systems at different concentrations.

[0084] Effects of MSPP on sugar production capacity and adhesion ability of cariogenic bacteria (4.2.1.3 and 4.2.1.4) The IEPS production of three cariogenic bacteria and their mixtures after treatment with different concentrations of MSPP was determined using the sulfuric acid-anthrone method to analyze the effect of MSPP on extracellular polysaccharide production. The results showed that ( Figure 8 As the MSPP concentration increased, the overall IEPS production of each strain showed a decreasing trend, suggesting that MSPP has a concentration-dependent inhibitory effect on extracellular polysaccharide production.

[0085] Statistical analysis showed that under higher concentrations of MSPP treatment, the IEPS yield of all strains was significantly lower than that of the negative control group (P<0.05). At a concentration of 2×MIC, the IEPS yield of *S. mutans* decreased from 0.526±0.015 mg / mL to 0.246±0.039 mg / mL, approximately 46.7% of the control group; the IEPS yields of *S. sanguinis* and *S. sobrinus* decreased from 0.218±0.026 mg / mL and 0.218±0.026 mg / mL to 0.050±0.020 mg / mL and 0.049±0.020 mg / mL, respectively, representing 22.8% and 22.7% of the control group. The mixed microbial community (MIX) showed the most significant decrease in IEPS yield under MSPP intervention, decreasing from 0.364±0.020 mg / mL to 0.050±0.020 mg / mL, only 13.7% of the control group. Even at a sub-inhibitory concentration of 1 / 8 MIC, a slight decrease in IEPS production was observed, suggesting that MSPP can interfere with bacterial extracellular polysaccharide production at low concentrations.

[0086] Adhesion assays showed that at higher concentrations (MIC and 2 × MIC), the adhesion rates of each strain were lower than those of the negative control group. Specifically, the adhesion rate of *S. mutans* decreased from 100% of the control group to 20% (2 × MIC); *S. sanguinis* and *S. sobrinus* decreased from 100% of the control group to 15% and 14%, respectively; the mixed microbiota (MIX) showed the most significant decrease in adhesion ability under high concentrations, decreasing from 100% of the control group to 12%. Even at a sub-inhibitory concentration of 1 / 8 MIC, a slight decrease in adhesion rate was observed, suggesting that the intervention agent can interfere with the adhesion of cariogenic bacteria even at low concentrations.

[0087] 4.2.2 MSPP Biomembrane Inhibition Experiment 4.2.2.1 Observation of the three-dimensional morphology and structure of biomembranes Scanning electron microscopy was used to observe the surface morphology changes of biofilms from three cariogenic streptococci and their mixed flora. Figure 9 In the control group, all bacterial species exhibited typical chain-like arrangement characteristics, with tightly aggregated bacterial cells, dense biofilm structure, and a large amount of extracellular matrix-like material visible between the bacterial cells, connecting the bacteria to form a continuous biofilm structure.

[0088] With increasing MSPP concentration, the biofilm structure of all bacterial species underwent significant changes. For *S. mutans* biofilm, treatment with 0.3125 mg / mL MSPP shortened bacterial chain length, increased intercellular spacing, and weakened intercellular connections. When the concentration increased to 1.25 mg / mL, the biofilm structure became significantly fragmented, the bacterial chain structure essentially broke down, the bacteria became dispersed, and the extracellular matrix significantly decreased. *S. sanguinis* biofilm showed high sensitivity to MSPP, exhibiting significant structural loosening and reduced bacterial aggregation even at 0.3125 mg / mL. With further increases in concentration, the biofilm continuity was disrupted, and bacteria existed primarily as single cells or small clusters. The structural changes in *S. sobrinus* biofilm followed a similar trend to the two bacteria mentioned above; with increasing MSPP concentration, the biofilm matrix gradually decreased, and the bacterial arrangement gradually shifted from tightly aggregated to dispersed.

[0089] In mixed bacterial biofilms, MSPP treatment also led to significant structural damage. The untreated biofilm exhibited a dense, aggregated state, while under the influence of 0.3125 mg / mL MSPP, the biofilm structure showed significant loosening. With increasing concentration, the bacterial aggregation gradually disintegrated, the bacterial distribution became more dispersed, and the original biofilm structure was essentially destroyed. These results indicate that MSPP can significantly alter the surface structure of cariogenic bacterial biofilms, leading to reduced bacterial aggregation, loosening of the biofilm structure, and gradual disintegration.

[0090] 4.2.2.2 Biofilm activity and ratio of live to dead bacteria The effect of MSPP on the survival status of bacteria within a biofilm was observed using live / dead bacterial fluorescence staining combined with CLSM, and semi-quantitative analysis of the fluorescence signal was performed using ImageJ. CLSM three-dimensional reconstruction images showed (…). Figure 10 (A) As the MSPP concentration increased from 0.312 mg / mL to 1.25 mg / mL, the green fluorescence in the *S. mutans* biofilm gradually decreased, while the red fluorescence significantly increased, indicating a significant decrease in bacterial survival within the biofilm. ImageJ fluorescence area and intensity analysis results ( Figure 10 B) Further, it was shown that MSPP treatment decreased the proportion of live bacteria and increased the proportion of dead bacteria in a concentration-dependent manner, with red fluorescence signal dominating in the 1.25 mg / mL treatment group.

[0091] exist S. sanguinis and S. sobrinus In biomembrane systems ( Figure 11 and 12MSPP treatment also showed a similar trend. With increasing drug concentration, the overall structure of the biofilm gradually loosened, spatial continuity decreased, and green fluorescence signal decreased while red fluorescence increased. ImageJ semi-quantitative analysis showed that the viable bacterial ratio of both bacteria continuously decreased with increasing MSPP concentration, reaching near the detection limit in the 1.25 mg / mL treatment group, indicating that MSPP has a significant inhibitory effect on bacteria in the formed biofilm.

[0092] In a mixed microbial biofilm composed of S. mutans, S. sanguinis and S. sobrinus ( Figure 13 MSPP treatment resulted in more significant biofilm structural damage, manifested as reduced biofilm thickness and disintegration of its spatial structure. Fluorescence signal analysis showed that with increasing MSPP concentration, green fluorescence gradually decreased while red fluorescence significantly increased. ImageJ semi-quantitative results ( Figure 13 B) Further, it was shown that the proportion of dead bacteria in the mixed bacterial biofilm was relatively high under the same concentration conditions, suggesting that MSPP has a strong interfering effect on multi-species biofilm systems.

[0093] The CLSM observation results were largely consistent with the inhibitory trend of biofilm metabolic activity detected by MTT, but differed somewhat from the MBRC values ​​measured by crystal violet staining. These results suggest that bacterial metabolic activity within the biofilm was significantly reduced under low concentrations of MSPP, while the extracellular polysaccharide backbone was partially preserved; therefore, some biofilm structure could still be observed in crystal violet staining.

Claims

1. A dendritic mesoporous silica with shikonin loaded on its surface, characterized in that: Shikonin is loaded onto the surface of the raw material dendritic mesoporous silica, and the surface of the mesoporous silica loaded with shikonin is coated with a polydopamine coating and a polyethylene glycol modification layer.

2. The dendritic mesoporous silica with shikonin loaded on its surface as described in claim 1, characterized in that: The specific surface area of the surface loaded dendritic mesoporous silica with shikonin is 38-44 m 2 / g, the pore volume is 0.1-0.15 cm 3 / g, and the diameter is 53.10±8.07 nm.

3. The dendritic mesoporous silica with shikonin loaded on its surface as described in claim 1, characterized in that: The preparation method of the raw material dendritic mesoporous silica includes the following steps: (1) Preparation of alkaline catalyst solution: Weigh triethanolamine into a container and place it in a water bath to form a homogeneous alkaline catalyst solution; (2) Constructing a surfactant film system: Hexadecyltrimethylammonium bromide and sodium salicylate were added sequentially to the solution formed in (1), and stirred at 70-90℃ for 0.5-1.5h to form a triethanolamine / hexadecyltrimethylammonium bromide / sodium salicylate composite micelle system with hexadecyltrimethylammonium bromide as template and sodium salicylate as pore expander; (3) Silicon source hydrolysis condensation and crystal nucleus growth: Tetraethyl orthosilicate is added to the composite micelles formed in (2) to form a dendritic mesoporous structure with central radial channel characteristics; (4) Separate the product and purify it to obtain dendritic mesoporous silica as raw material for later use.

4. The dendritic mesoporous silica with shikonin loaded on its surface as described in claim 3, characterized in that: The mass ratio of triethanolamine, hexadecyltrimethylammonium bromide, sodium salicylate, and tetraethyl orthosilicate is 1: 5-6: 2-3: 53-55.

5. A method for preparing dendritic mesoporous silica with surface-loaded shikonin as described in any one of claims 1-4, characterized in that: Includes the following steps: (1) Shikonin loaded onto dendritic mesoporous silica: The raw material dendritic mesoporous silica prepared according to claim 3 or 4 is dispersed in an alcohol solution, and shikonin is also dissolved in another alcohol solution of the same kind. Under the conditions of room temperature and protection from light, the shikonin solution is added dropwise to the dendritic mesoporous silica and stirred for 20-30 h. After the reaction is completed, a mixture is obtained. The mixture is centrifuged, and after being dispersed in a solution, it is washed and purified to obtain the final precipitate, which is stored in the dark for later use. (2) Polydopamine surface modification: The precipitate purified in (1) was dispersed in a buffer solution, and dopamine hydrochloride was also dissolved in the same other buffer solution. Under the conditions of room temperature and protection from light, dopamine hydrochloride was added dropwise to the dispersion of the precipitate purified in (1), and stirred for 20-30 hours. After the reaction was completed, a mixture was obtained. The mixture was centrifuged, washed and purified to obtain the final precipitate, which was stored in the dark for later use. (3) Polyethylene glycol surface modification: The precipitate purified in (2) was dispersed in a buffer solution, and the methoxy polyethylene glycolamine was also dissolved in another buffer solution. Under the conditions of room temperature and protection from light, the methoxy polyethylene glycolamine was added dropwise to the dispersion of the precipitate purified in (2), and stirred for 20-30 hours. After the reaction was completed, a mixture was obtained, washed and purified, and used for later use.

6. The method as described in claim 5, characterized in that: In (1), the mass ratio of dendritic mesoporous silica to shikonin is 4-6:1, and the volume ratio of the alcohol solution dissolving the dendritic mesoporous silica to the alcohol solution dissolving the shikonin is the same as the mass ratio of dendritic mesoporous silica to shikonin.

7. The method as described in claim 5, characterized in that: In (2), the mass ratio of the precipitate purified by (1) to dopamine hydrochloride is 2-3:1, and the volume ratio of the buffer solution for dissolving the precipitate purified by (1) to the buffer solution for dissolving dopamine hydrochloride is 4-6:

1.

8. The method as described in claim 5, characterized in that: The mass ratio of the purified precipitate (2) to the methoxy polyethylene glycolamine in (3) is 1:2-3, and the volume ratio of the buffer solution for dissolving the purified precipitate (2) to the buffer solution for dissolving the methoxy polyethylene glycolamine is 1-3:

1.

9. The use of the dendritic mesoporous silica with surface-loaded shikonin as described in any one of claims 1-4, or the dendritic mesoporous silica with surface-loaded shikonin prepared by any one of methods 5-8, for inhibiting cariogenic bacteria, characterized in that: The cariogenic bacteria include Streptococcus mutans, Streptococcus distantly related to the virus, and Streptococcus sanguinis.

10. The use as described in claim 9, characterized in that: The dendritic mesoporous silica exhibits inhibitory effects on Streptococcus mutans, Streptococcus pyogenes, and Streptococcus sanguinis in mixed bacterial communities.

Images