Liquid metal-based anti-inflammatory drug as well as preparation method and application thereof

Abstract

The invention discloses a liquid metal-based anti-inflammatory drug as well as a preparation method and application thereof, and relates to the technical field of biomedicine. The preparation method of the liquid metal nanoparticles comprises the steps that liquid metal is placed in a solvent, a dispersing agent is added, ultrasonic dispersion and gradient centrifugation are conducted, and the liquid metal nanoparticle suspension preparation is obtained. The concentration of the liquid metal in the liquid metal nanoparticle suspension preparation is 5-30 mg / mL, and the size of the liquid metal nanoparticles is 50-500 nm; the liquid metal is gallium, indium, tin or alloy thereof; the dispersing agent is ligand molecules for realizing separation of the liquid metal nanoparticles by forming an end-capping layer on newly-generated liquid drops in at least one mode of static electricity, cross-linking and covalent replacement. The liquid metal nanoparticles can be directly used for treating infectious bone defect diseases, and other auxiliary agents can also be added to prepare anti-inflammatory drugs; meanwhile, the antibacterial and osteogenesis characteristics are achieved, and the dual requirements for treating infectious bone defects, inhibiting infection and promoting tissue regeneration are met.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a liquid metal-based anti-inflammatory drug and a preparation method and application thereof. Background Art

[0002] Infected bone defect is a bone injury or bone defect caused by microorganisms. Its treatment needs to solve two key problems: one is how to control infection, and the other is how to promote tissue regeneration. Antimicrobial treatment can effectively eliminate harmful bacteria and reduce the risk of infection, thereby preventing further tissue damage. Tissue regeneration aims to repair damaged tissue until its original structure and function are restored; especially the support function of bone tissue is restored.

[0003] For the treatment of infected bone defect diseases in the oral cavity, the most common clinical method is local mechanical debridement, supplemented by antibacterial therapy, which usually involves the use of antibiotics, antifungal or antiviral drugs. Through the combination of mechanical removal and drug therapy, bacterial growth and reproduction can be directly inhibited, thereby alleviating the symptoms of infection. However, this treatment method is often accompanied by a long course of treatment, a high recurrence rate, narrow-spectrum antibacterial and potential risks such as drug resistance. More importantly, current clinical technology is still unable to promote periodontal tissue regeneration. Nanomedicines, with their unique antibacterial mechanism, have become the preferred choice to break the limitations of traditional antibiotics, but they are still insufficient in promoting tissue regeneration. Therefore, the development of multifunctional nanomedicines that have both osteogenesis and antibacterial activity has clinical and scientific significance for the repair of infected bone defects.

[0004] In recent years, many “all-in-one” nanomedicines, i.e., by combining multiple components to synergize all the required properties, have been frequently reported to synergistically treat infected bone defects; for example, multidrug delivery systems, modified hydrogels, and engineered membranes. However, the structural complexity of these drugs increases the risk of drug antagonism, material instability, and technical sensitivity. In addition, the active ingredients commonly incorporated (e.g., zinc and magnesium, etc.) are quite unstable in physiological environments and release toxic ions, raising concerns about biocompatibility. Therefore, there is a strong demand for the design of “one-to-many” nanomedicines with simple ingredients, integrated functions, and high biosafety. Summary of the invention

[0005] In view of the deficiencies of the above prior art, the present invention provides a liquid metal-based anti-inflammatory drug and its preparation method and application. The liquid metal-based anti-inflammatory drug provided by the present invention realizes the integration of antibacterial and osteogenic properties at the nanoscale, aims to repair infected bone defects, and meets the dual needs of inhibiting infection and promoting tissue regeneration when treating infected bone defects. The preparation method of the anti-inflammatory drug is simple and has a wide range of applications. It is specifically achieved through the following technologies.

[0006] The first aspect of the present invention provides a method for preparing a liquid metal nanoparticle suspension, comprising the following steps: placing liquid metal in a solvent, adding a dispersant, ultrasonically dispersing, and gradient centrifuging to obtain a liquid metal nanoparticle suspension;

[0007] The concentration of liquid metal in the liquid metal nanoparticle suspension preparation is 5-30 mg / mL, and the size of the liquid metal nanoparticles is 50-500 nm;

[0008] The liquid metal is at least one of gallium, indium, and tin, or an alloy of at least two of gallium, indium, and tin; the dispersant is a ligand molecule that forms a capping layer on newly generated liquid metal nanoparticles through at least one of electrostatics, crosslinking, and covalent substitution to achieve separation of liquid metal nanoparticles.

[0009] In the above-mentioned preparation method provided by the present invention, the purpose of the dispersant is to ensure the uniform dispersion of liquid metal nanoparticles and maintain dispersion stability during the ultrasonic nano-nano process. The dispersant is a ligand molecule, and its working principle is to form a capping layer on the newly generated droplets by means of static electricity, cross-linking, covalent substitution, etc., thereby helping the separation of liquid metal nanoparticles and maintaining stability in the suspension system. The addition of a dispersant also significantly improves the functionality of liquid metal nanoparticles. For example, liquid metal surface-modified with blue light-responsive graphene quantum dots not only obtains antibacterial properties enhanced by chairside blue light, but also the osteogenic differentiation effect of graphene quantum dots further ensures the effectiveness of liquid metal nanoparticles in regulating bone homeostasis. The modification of polydopamine causes gallium oxidation to be concentrated on the outer surface of the nanoparticles, which can serve as a gallium ion reservoir to enhance the release of active metal ions. Tannic acid, as a natural polyphenol antioxidant, can disrupt the internal metabolism of bacteria and exert a bactericidal effect. Therefore, its surface modification will significantly improve the antibacterial properties of liquid metal nanoparticles.

[0010] Compared with liquid metal nanoparticles that have not been modified with a dispersant, the liquid metal nanoparticles of the present invention have higher stability, smaller size and narrower size distribution, and better antibacterial and osteogenic activity.

[0011] Preferably, the liquid metal is a gallium-indium alloy, and the mass ratio of gallium to indium is in the range of (1-9):1.

[0012] Furthermore, the mass ratio of the dispersant to the liquid metal is (0.3-4):100.

[0013] Furthermore, the dispersant is any one or more of graphene quantum dots (GQDs), polydopamine, sodium alginate, and tannic acid.

[0014] Furthermore, during ultrasonic dispersion, the ultrasonic frequency is 20-25 KHz, the ultrasonic power is 180-290 W, the ultrasonic time is 0.5-6 h, and the temperature is 0-15°C.

[0015] Furthermore, the solvent is any one or more of methanol, ethanol, ethylene glycol and water.

[0016] Furthermore, the specific method of the gradient centrifugation is: centrifuging the ultrasonically dispersed system at 500-1000 rpm for 5-10 min, 1000-3000 rpm for 5-10 min, 3000-5000 rpm for 5-10 min, 5000-9000 rpm for 5-10 min, and 9000-15000 rpm for 5-10 min.

[0017] The second aspect of the present invention provides a liquid metal nanoparticle suspension preparation, which is prepared by the above-mentioned preparation method.

[0018] The third aspect of the present invention provides a liquid metal-based anti-inflammatory drug, including the above-mentioned liquid metal nanoparticle suspension preparation.

[0019] Furthermore, the liquid metal-based anti-inflammatory drug is prepared by preparing the liquid metal nanoparticle suspension preparation with physiological saline or phosphate buffer.

[0020] Furthermore, the administration form of the liquid metal-based anti-inflammatory drug includes injection, spray, aerosol, ointment, cream, film or paste.

[0021] Preferably, it is an injection, spray, film-coating or aerosol;

[0022] More preferably, it is an injection;

[0023] Optimally, the concentration of the liquid metal nanoparticles in the injection is 0.5-3 mg / mL.

[0024] The liquid metal nanoparticle suspension preparation or liquid metal-based anti-inflammatory drug provided by the present invention has the characteristics of integrating the dual functions of antibacterial and osteogenesis.

[0025] The fourth aspect of the present invention provides a liquid metal nanoparticle suspension preparation or the application of a liquid metal-based anti-inflammatory drug.

[0026] Furthermore, liquid metal nanoparticle suspensions or liquid metal-based anti-inflammatory drugs can be used to prepare drugs for treating infectious bone defect diseases.

[0027] The infectious bone defect disease includes but is not limited to bone tissue damage caused by inflammation and infection in various parts of the human or animal body such as the oral cavity and limbs.

[0028] Preferably, the infectious bone defect disease includes but is not limited to periodontitis, necrotizing ulcerative periodontitis, peri-implantitis, osteomyelitis, suppurative arthritis and the like.

[0029] The liquid metal nanoparticle suspension provided by the present invention can not only be used alone and directly to treat diseases or symptoms such as oral infectious bone defects, osteomyelitis, suppurative arthritis, etc., but can also be selected from other auxiliary materials or auxiliary reagents permitted in clinical medicine to be further formulated into corresponding drug dosage forms for scientific research or treatment, and can also be used in combination with other therapeutic drugs.

[0030] For example, the liquid metal nanoparticle suspension preparation provided by the present invention can be mixed with physiological saline or phosphate buffer to prepare an injection.

[0031] Taking the treatment of oral infectious bone defects (periodontitis) as an example, the injection can be prepared according to the above method and delivered to the diseased area through precise microinjection technology; then it can be irradiated with chairside blue light to activate its antibacterial and osteogenic effects, thereby effectively repairing the inflammation caused by oral bacterial infection and related bone tissue defects.

[0032] In actual research, the therapeutic effect of liquid metal nanoparticle suspensions or liquid metal-based anti-inflammatory drugs can be evaluated through disease indicators such as gingival bleeding index (GBI), quantification of alveolar bone atrophy, and changes in subgingival flora.

[0033] Optionally, when the liquid metal nanoparticle suspension provided by the present invention is mixed with phosphate buffer to prepare an injection, the concentration of the functionally integrated material is 0.5-3 mg / mL; the wavelength of the chairside blue light is 450 nm, and the power density is 0.4-1 W / cm 2 , the irradiation time is 1-10 min.

[0034] In addition to the above-mentioned application methods, according to the differences in drug dosage forms in terms of administration methods, sites of action, etc., the liquid metal nanoparticle suspension preparation or the corresponding liquid metal-based anti-inflammatory drug provided by the present invention can also be used in other ways to act on the corresponding treatment area of ​​the human or animal body.

[0035] Compared with the prior art, the present invention is beneficial in that:

[0036] 1. The present invention provides a liquid metal nanoparticle suspension preparation that can simultaneously achieve antibacterial and osteogenic properties, and a corresponding liquid metal-based anti-inflammatory drug, which meets the dual needs of treating infected bone defects to inhibit infection and promote tissue regeneration.

[0037] 2. The preparation method of the liquid metal nanoparticle suspension preparation and the corresponding medicine provided by the present invention is simple and convenient, and can be used not only in the oral cavity, but also in the treatment of infectious bone defect diseases in most other parts of the human or animal body, and has a wide range of applications.

[0038] Taking periodontitis as an example, this disease meets the needs of both infection control and bone defect reconstruction in oral treatment, providing a simple and efficient solution strategy for the biological characteristics of pathogenic bacteria in inflammatory bone defects and the physicochemical properties of bone defects; it effectively reduces oral inflammation and promotes tissue repair, emphasizing its potential benefits as a nanomedicine in the special application scenario of the oral cavity.

[0039] 3. The liquid metal nanoparticle suspension preparation and corresponding drugs provided by the present invention have simple preparation process, high biosafety, integrated therapeutic and repair functions, and chairside blue light enhanced response activity, and have good practical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Transmission electron microscope images of liquid metal nanoparticles prepared in Example 1, Comparative Example 1 and Comparative Example 2.

[0041] Figure 2 Element distribution diagram of liquid metal nanoparticles in Example 1 (scale bar = 25 nm).

[0042] Figure 3 The particle size distribution of the liquid metal nanoparticles prepared in Example 1 and Comparative Example 1.

[0043] Figure 4 In vitro cell compatibility of the liquid metal nanoparticles prepared in Example 1 and Comparative Example 1 with rat primary bone marrow mesenchymal stem cells (BMSC) and L929 mouse fibroblasts (scale bar = 100 μm).

[0044] Figure 5 The in vitro antibacterial activity of the liquid metal nanoparticles prepared in Example 1 and Comparative Example 1 against Streptococcus mutans (S. mutans) and Porphyromonas gingivalis (P. gingivalis).

[0045] Figure 6 Alkaline phosphatase staining (ALP) and Alizarin red staining (ARS) of the liquid metal nanoparticles prepared in Example 1 and Comparative Example 1 after co-culture with rat primary bone marrow mesenchymal stem cells, and tartrate-resistant acid phosphatase staining (TRAP) of the liquid metal nanoparticles co-cultured with rat primary bone marrow macrophages (BMMs) for osteoclast differentiation (scale bar = 200 μm).

[0046] Figure 7In vivo therapeutic effect evaluation of the liquid metal nanoparticles prepared in Example 1 and Comparative Example 1 in treating periodontitis rats (scale bar = 1 mm). DETAILED DESCRIPTION

[0047] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] In some embodiments of the present invention, the preparation method of the liquid metal nanoparticle suspension is to place the liquid metal in a solvent, add a dispersant, perform ultrasonic dispersion, and perform gradient centrifugation to obtain the liquid metal nanoparticle suspension.

[0049] The concentration of liquid metal in the liquid metal nanoparticle suspension preparation is 5-30 mg / mL, and the size of the liquid metal nanoparticles is 50-500 nm;

[0050] The liquid metal is at least one of gallium, indium, and tin, or an alloy of at least two of gallium, indium, and tin; the dispersant is a ligand molecule that forms a capping layer on newly generated liquid metal nanoparticles through at least one of electrostatics, crosslinking, and covalent substitution to achieve separation of liquid metal nanoparticles.

[0051] Specifically, the dispersant can be any one or more of graphene quantum dots, polydopamine, sodium alginate, and tannic acid. The above dispersants can use common specifications and models currently available on the market and medically permitted. In a specific implementation case of the present invention, the ultrasonic frequency during ultrasonic dispersion is 20-25 KHz, the ultrasonic power is 180-290 W, the ultrasonic time is 0.5-6 h, and the temperature is 0-15°C.

[0052] In a specific implementation case of the present invention, the solvent is any one or more of methanol, ethanol, ethylene glycol, and water.

[0053] In a specific implementation case of the present invention, the centrifugal speed of the gradient centrifugation is 3000-15000 rpm, and the time is 5-15 min.

[0054] In other embodiments of the present invention, the liquid metal nanoparticle suspension provided by the present invention can not only be used alone and directly to treat oral infectious bone defects, osteomyelitis, suppurative arthritis and other diseases or symptoms, but can also be selected from other auxiliary materials or auxiliary reagents permitted in clinical medicine, and further formulated into corresponding drug dosage forms (i.e., liquid metal-based anti-inflammatory drugs) for scientific research or treatment, and can also be used in combination with other therapeutic drugs.

[0055] Specifically, when other auxiliary materials or auxiliary reagents are used to prepare liquid metal-based anti-inflammatory drugs, the auxiliary reagents can be physiological saline or phosphate buffer.

[0056] Specifically, the formulated pharmaceutical dosage form may be an injection, spray, aerosol, ointment, cream, film-coat or paste.

[0057] Phosphate buffered saline may be prepared according to any existing medically acceptable formulation.

[0058] Optionally, the composition of the phosphate buffer is: 1.44 g / L disodium hydrogen phosphate, 0.24 g / L sodium dihydrogen phosphate, 8 g / L sodium chloride, and pH value = about 7.4.

[0059] In other embodiments of the present invention, liquid metal nanoparticle suspensions or corresponding liquid metal-based anti-inflammatory drugs can be used to treat various infectious bone defect diseases.

[0060] Example 1

[0061] The liquid metal nanoparticle suspension preparation prepared in this embodiment is prepared as follows:

[0062] (1) Placing liquid metal gallium (Ga) in ethylene glycol;

[0063] (2) Graphene quantum dots (particle size <100 nm) were then added at a ratio of 0.3% of the mass of the liquid metal, and ultrasonic dispersion treatment and gradient centrifugation were performed in sequence. Finally, a uniformly dispersed liquid metal nanoparticle suspension [GaNP(GQD)] was successfully collected.

[0064] The ultrasonic treatment conditions were as follows: ultrasonic treatment for 4 h at 0 °C, an ultrasonic frequency of 25 kHz, and an ultrasonic power of 290 W.

[0065] The specific method of gradient centrifugation is: the system subjected to ultrasonic dispersion treatment is centrifuged at 1000 rpm for 5 min, 3000 rpm for 5 min, 5000 rpm for 5 min, 9000 rpm for 5 min, and 15000 rpm for 5 min.

[0066] Example 2

[0067] The liquid metal nanoparticle suspension prepared in this embodiment is prepared by the following method:

[0068] (1) placing a liquid metal gallium-indium alloy (containing 75.5% gallium and 24.5% indium) in ethanol;

[0069] (2) Dopamine hydrochloride was then added at a ratio of 4% of the mass of the liquid metal. After ultrasonic dispersion, 0.1 mL of 60 mg / mL tris(hydroxymethyl)aminomethane buffer was added to the suspension to trigger the self-polymerization of dopamine into polydopamine. The polymerization process lasted for 1 hour at 20°C. Gradient centrifugation was performed, and finally a uniformly dispersed liquid metal nanoparticle suspension [GaInNP(PDA)] was successfully collected.

[0070] The ultrasonic treatment conditions were as follows: ultrasonic treatment for 2 h at 0 °C, an ultrasonic frequency of 25 kHz, and an ultrasonic power of 180 W.

[0071] The specific method of gradient centrifugation is: centrifuge the system after ultrasonic dispersion treatment at 500 rpm for 5 minutes, 1000 rpm for 5 minutes, 3000 rpm for 5 minutes, 5000 rpm for 5 minutes, and 9000 rpm for 5 minutes.

[0072] Example 3

[0073] The liquid metal nanoparticle suspension prepared in this embodiment is prepared by the following method:

[0074] (1) placing liquid metal gallium-indium alloy (containing 75.5% gallium and 24.5% indium) in deionized water;

[0075] (2) Then, sodium alginate was added at a ratio of 0.3% of the mass of the liquid metal, and after ultrasonic dispersion treatment and gradient centrifugation, a uniformly dispersed liquid metal nanoparticle suspension [GaInNP(Alg)] was successfully collected.

[0076] The ultrasonic treatment conditions were as follows: ultrasonic treatment for 6 h at 15 °C, an ultrasonic frequency of 20 KHz, and an ultrasonic power of 200 W.

[0077] The specific method of gradient centrifugation is: centrifuge the system after ultrasonic dispersion treatment at 500 rpm for 10 min, 1000 rpm for 10 min, 3000 rpm for 10 min, 5000 rpm for 10 min, and 9000 rpm for 10 min.

[0078] Example 4

[0079] The liquid metal nanoparticle suspension prepared in this embodiment is prepared by the following method:

[0080] (1) Placing liquid metal gallium in deionized water;

[0081] (2) Tannic acid was then added at a ratio of 0.3% of the mass of the liquid metal, and after ultrasonic dispersion treatment and gradient centrifugation, a uniformly dispersed liquid metal nanoparticle suspension [GaNP(TA)] was successfully collected.

[0082] The ultrasonic treatment conditions were as follows: ultrasonic treatment for 4 h at 10 °C, an ultrasonic frequency of 25 KHz, and an ultrasonic power of 250 W.

[0083] The specific method of gradient centrifugation is: the system subjected to ultrasonic dispersion treatment is centrifuged at 800 rpm for 5 min, 1200 rpm for 5 min, 2800 rpm for 5 min, 4800 rpm for 5 min, and 9000 rpm for 5 min.

[0084] Comparative Example 1

[0085] The liquid metal nanoparticle suspension prepared in this comparative example is prepared by the following method:

[0086] (1) Placing liquid metal gallium in ethylene glycol;

[0087] (2) Ultrasonic dispersion treatment and gradient centrifugation were performed in sequence, and finally a uniformly dispersed liquid metal nanoparticle suspension GaNP was successfully collected.

[0088] The ultrasonic treatment conditions were as follows: ultrasonic treatment for 4 h at 0 °C, an ultrasonic frequency of 25 kHz, and an ultrasonic power of 290 W.

[0089] The specific method of gradient centrifugation is: the system subjected to ultrasonic dispersion treatment is centrifuged at 1000 rpm for 5 min, 3000 rpm for 5 min, 5000 rpm for 5 min, 9000 rpm for 5 min, and 15000 rpm for 5 min.

[0090] Comparative Example 2

[0091] The liquid metal nanoparticle suspension prepared in this comparative example is prepared by the following method:

[0092] (1) Placing liquid metal gallium in ethanol;

[0093] (2) Then, polyethylene glycol (PEG 5000) was added at a ratio of 9% of the mass of the liquid metal, and ultrasonic dispersion treatment and gradient centrifugation were performed in sequence. Finally, a uniformly dispersed liquid metal nanoparticle suspension [GaNP(PEG)] was successfully collected.

[0094] The ultrasonic treatment conditions were as follows: ultrasonic treatment for 4 h at 0 °C, an ultrasonic frequency of 25 kHz, and an ultrasonic power of 290 W.

[0095] The specific method of gradient centrifugation is: the system subjected to ultrasonic dispersion treatment is centrifuged at 1000 rpm for 5 min, 3000 rpm for 5 min, 5000 rpm for 5 min, 9000 rpm for 5 min, and 15000 rpm for 5 min.

[0096] Test example: Characterization test of liquid metal nanoparticles

[0097] The transmission electron microscopy images of the liquid metal nanoparticles of Example 1, Comparative Example 1 and Comparative Example 2 are as follows: Figure 1 As shown. It can be seen that compared with the bare GaNP (Comparative Example 1) and GaNP (PEG) (Comparative Example 2) containing only a 0.5-3 nm thin oxide layer, the GaNP (GQD) nanoparticles with a typical core-shell structure were successfully prepared by the method of Example 1, and the physical adsorption between graphene quantum dots and gallium-based nanoparticles contributed to the formation of this core-shell structure. The lattice spacing of the shell determined by high-resolution transmission electron microscopy images is 0.23 nm, corresponding to the (100) plane of graphene.

[0098] The particle size distribution of the liquid metal nanoparticles in Example 1 and Comparative Example 1 is as follows: Figure 2 As shown, Figure 2 It shows that both GaNP and GaNP(GQD) exhibit a unimodal size distribution. The particle size of liquid metal nanoparticles is significantly reduced after surface coating with graphene quantum dots. The average size of Ga NPs is 106.79 nm, while the average size of GaNC is 71.24 nm, and the particle size distribution is more uniform than that of GaNP.

[0099] The element distribution of the liquid metal nanoparticles of Example 1 is as follows: Figure 3 As shown. The results show that the distribution density of oxygen (O) and carbon elements presents a hollow structure, and the distribution of gallium elements presents a high density in the core and a low density on the surface. These results indicate that GaNP (GQD) nanoparticles are composed of slightly oxidized liquid metal gallium nanospheres wrapped by graphene quantum dots.

[0100] Application example: Testing of the biocompatibility, anti-infection, and tissue regeneration effects of liquid metal nanoparticle suspensions

[0101] 1. Biocompatibility verification

[0102] Cytotoxicity was detected using the Alamar Blue cell viability assay kit. The experimental method is as follows:

[0103] (1) L929 mouse fibroblasts and rat primary bone marrow mesenchymal stem cells were cultured at a rate of approximately 5 × 10 cells / well. 3 The cells were seeded at a density of 100 cells / mL in 96-well culture plates and cultured overnight.

[0104] (2) After refreshing the culture medium with different concentrations of nanoparticles, the culture plates were illuminated with blue light (1 W / cm 2 ) irradiate for 5 minutes.

[0105] (3) The fluorescence intensity at an excitation wavelength of 530 nm and an emission wavelength of 590 nm was measured at the designated time points (7 days). The experiment was repeated three times, and the relative cell viability was calculated as the percentage of surviving cells compared with the control cells in complete medium without the addition of nanoparticles.

[0106] Figure 4 In vitro cell compatibility of the liquid metal nanoparticles prepared in Example 1 and Comparative Example 1 with rat primary bone marrow mesenchymal stem cells and L929 mouse fibroblasts (scale bar = 100 μm). Under blue light irradiation, after 7 days of co-culture, the cell viability remained above 80%. This shows that the liquid metal nanoparticles prepared in Example 1 and Comparative Example 1 have good biocompatibility.

[0107] 2. In vitro verification of antibacterial activity

[0108] The plate colony counting method was used to evaluate the antibacterial activity of liquid metal nanoparticles. The experimental method is as follows:

[0109] (1) Place 1 mL of Streptococcus mutans (approximately 10 6 CFU / mL) and Porphyromonas gingivalis (about 10 9 CFU / mL) suspensions were independently mixed with 1 mL of liquid culture medium (control group), equal concentrations of GaNPs and GaNP(GQDs) to make the final concentration of liquid metal nanoparticles in the mixture reach 0.75 mg / mL, and blue light was irradiated for 5 min or not irradiated as a control.

[0110] (2) Spread the suspension on an agar plate. The antibacterial rate of the nanomaterial is determined by counting the colony forming units (CFU) on the plate. Antibacterial rate = [(CFU ctrl – CFU exp ) / CFU ctrl ] × 100%. In the formula, CFU ctrl and CFU exp These are the CFU of the control group and the experimental group, respectively.

[0111] Figure 5In vitro antibacterial activity of liquid metal nanoparticles prepared in Example 1 and Comparative Example 1 against Streptococcus mutans and Porphyromonas gingivalis. GaNP and GaNP (GQD) both showed antibacterial efficacy enhanced by chairside blue light response against typical oral pathogens. The antibacterial rates of GaNP against Streptococcus mutans and Porphyromonas gingivalis under blue light irradiation were 41.45 ± 5.38% and 86.27 ± 6.71%, respectively. The antibacterial activity of GaNP (GQD) against Streptococcus mutans and Porphyromonas gingivalis under blue light irradiation was 75.00 ± 1.77% and 97.15 ± 3.02%, respectively.

[0112] 3. In vitro verification of the regulatory effect on osteoblasts and osteoclasts

[0113] The experimental approach to evaluate the osteoblast-modulating ability of liquid metal nanoparticles was as follows:

[0114] (1) The culture medium of rat primary bone marrow mesenchymal stem cells was replaced with osteogenic differentiation medium supplemented with 0.1 μM dexamethasone, 10 mM sodium β-glycerophosphate, and 50 μg / mL L-ascorbic acid.

[0115] (2) The cells were co-cultured with GaNPs and GaNP(GQDs) at a concentration of 0.75 mg / mL, respectively, and irradiated with blue light for 5 min or without irradiation as a control.

[0116] (3) The osteogenic induction medium was replaced every 2 days. After 7 and 14 days of co-culture, the cells under different treatment conditions were stained with an alkaline phosphatase staining kit to visualize early markers of osteogenic differentiation, and images were taken under a stereomicroscope.

[0117] (4) After 21 days of osteogenic induction, the mineralization of primary rat bone marrow mesenchymal stem cells was evaluated by Alizarin Red S staining to visualize late markers of osteogenic differentiation. The cells were fixed with 4% paraformaldehyde and stained with 1% Alizarin Red S solution for 15 min. After three rinses with distilled water, the stained calcium nodules were observed under a stereomicroscope.

[0118] The experimental approach to evaluate the osteoclast modulating ability of liquid metal nanoparticles is as follows:

[0119] (1) Rat primary bone marrow macrophages were used to determine the biological effects of GaNPs and GaNPs (GQDs) on osteoclast differentiation. To induce rat primary bone marrow macrophages to differentiate into osteoclasts, RANKL (50 ng / mL) and m-CSF (30 ng / mL) were supplemented in the culture medium. After co-culture with liquid metal nanoparticles at a concentration of 0.75 mg / mL, rat primary bone marrow macrophages were irradiated with blue light for 5 min or without illumination as a control.

[0120] (2) After 7 days of co-culture, osteoclast formation was monitored by tartrate-resistant acid phosphatase staining, and images of stained cells were obtained under a stereomicroscope.

[0121] Figure 6 The in vitro evaluation results of the osteogenesis and osteoclast regulation of the liquid metal nanoparticles prepared in Example 1 and Comparative Example 1 (scale bar = 200 μm). Compared with the control group, the liquid metal nanoparticle treatment group showed significant effects on the expression of early markers of osteogenesis and the staining of late markers. In particular, the osteogenic staining results of the graphene quantum dot-modified GaNP (GQD) were more intense than those of the unmodified GaNP, indicating that GaNP (GQD) has a more outstanding ability to promote osteogenic differentiation. The control group showed many huge multinucleated cells, i.e., positively stained osteoclasts, under osteoclast induction, while the liquid metal nanoparticle treatment group showed a significant decrease in the number of osteoclasts resistant to tartrate acid phosphatase staining, and the positive staining was significantly weakened, showing excellent inhibition of osteoclast activity. The results show that the surface modification of graphene quantum dots enables liquid metal nanoparticles to exhibit better bidirectional regulation of bone homeostasis.

[0122] 4. Animal experiment verification of the therapeutic effect on periodontitis

[0123] The specific experimental steps to evaluate the in vivo therapeutic effect of liquid metal nanoparticles on periodontitis are as follows:

[0124] (1) Eighteen rats were randomly divided into three groups, namely, healthy group, saline group, GaNP group, and GaNP(GQD) group, with 6 rats in each group.

[0125] (2) The rats were placed under general anesthesia with 4-0 non-absorbable sutures placed under the gums on both sides of the maxillary second molars. Every other day, 50 μL of 10 9 CFU / mL suspension of Porphyromonas gingivalis was injected into the gingival sulcus around the molars. Silk ligature was maintained for 7 days and checked daily to induce periodontitis.

[0126] (3) After the ligature was removed, 50 μL of sterile saline was injected into the periodontal pocket as a control group for periodontitis treatment, referred to as the saline group. In the GaNP and GaNP(GQD) groups, 50 μL of 0.75 mg / mL GaNP and GaNP(GQD) solutions were injected into the periodontal pocket, respectively, and the injected area was then irradiated with blue light for 5 min.

[0127] (4) After 14 days of administration, the extent of periodontal inflammation, alveolar bone resorption, and in vivo bactericidal effect were evaluated.

[0128] Figure 7Results of in vivo therapeutic effect evaluation of the liquid metal nanoparticles prepared in Example 1 and Comparative Example 1 in treating periodontitis rats (scale bar = 1 mm).

[0129] The gingival bleeding index of each group was measured using a periodontal probe. The scoring criteria were as follows: "0" means no bleeding on probing, and "1" means bleeding on probing. The gingival bleeding index score of the healthy group was significantly lower than that of the saline group (P<0.0001), while the gingival bleeding index score of the liquid metal nanoparticle-treated group was significantly lower than that of the saline group (P<0.0001), and there was no significant difference between the healthy group and the group treated with liquid metal nanoparticles.

[0130] Micro-computed tomography (Micro-CT) analysis was performed to evaluate the extent of alveolar bone resorption. The vertical distance between the alveolar bone ridge (ABC) and the cemento-dentinal junction (CEJ) was used as a treatment indicator. Compared with the healthy group, the CEJ-ABC distance in the saline group was the longest, indicating significant alveolar bone loss. In contrast, the prepared liquid metal nanoparticle treatment shortened the CEJ-ABC distance to a great extent. Among them, the CEJ-ABC in the GaNP(GQD) group was not significantly different from that in the healthy group.

[0131] The in vivo bactericidal effect of each test group was quantitatively evaluated using a standard plate count method. Compared with the high bacterial infection observed in the saline group, colony forming unit counts showed that the prepared liquid metal nanoparticle treatment significantly reduced the bacterial load. Among them, the GaNP (GQD) treatment group had the lowest colony count.

[0132] In summary, the reduction of alveolar bone loss and bacterial infection together indicate that the liquid metal nanoparticle preparation prepared in Example 1 has a good in vivo therapeutic effect on periodontitis. Compared with the experimental results of Comparative Example 1, the antibacterial and osteogenic functions of the preparation are optimized by modification of the dispersant graphene quantum dots, thereby improving its therapeutic effect on periodontitis.

[0133] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A method for preparing a liquid metal nanoparticle suspension, characterized in that: The following steps are involved: Placing liquid metal in a solvent, adding a dispersant, performing ultrasonic dispersion, and gradient centrifugation to obtain a liquid metal nanoparticle suspension preparation; The concentration of liquid metal in the liquid metal nanoparticle suspension preparation is 5-30 mg / mL, and the size of the liquid metal nanoparticles is 50-500 nm; The liquid metal is at least one of gallium, indium, and tin, or an alloy of at least two of gallium, indium, and tin; the dispersant is a ligand molecule that forms a capping layer on newly generated liquid metal nanoparticles through at least one of electrostatics, crosslinking, and covalent substitution to achieve separation of liquid metal nanoparticles.

2. The method for preparing the liquid metal nanoparticle suspension according to claim 1, characterized in that: The mass ratio of the dispersant to the liquid metal is (0.3-4):

100.

3. The method for preparing the liquid metal nanoparticle suspension according to claim 1, characterized in that: The dispersant is any one or more of graphene quantum dots, polydopamine, sodium alginate and tannic acid.

4. The method for preparing the liquid metal nanoparticle suspension according to claim 1, characterized in that: The ultrasonic frequency during ultrasonic dispersion is 20-25 KHz, the ultrasonic power is 180-290 W, the ultrasonic time is 0.5-6 h, and the temperature is 0-15°C.

5. The method for preparing the liquid metal nanoparticle suspension according to claim 1, characterized in that: The solvent is any one or more of methanol, ethanol, ethylene glycol and water.

6. The method for preparing the liquid metal nanoparticle suspension according to claim 1, characterized in that: The specific method of the gradient centrifugation is: centrifuging the ultrasonically dispersed system at 500-1000 rpm for 5-10 min, 1000-3000 rpm for 5-10 min, 3000-5000 rpm for 5-10 min, 5000-9000 rpm for 5-10 min, and 9000-15000 rpm for 5-10 min.

7. A liquid metal nanoparticle suspension prepared by the preparation method according to any one of claims 1 to 5.

8. A liquid metal-based anti-inflammatory drug, characterized in that: It comprises the liquid metal nanoparticle suspension preparation as claimed in claim 6; preferably, the liquid metal-based anti-inflammatory drug is prepared by preparing the liquid metal nanoparticle suspension preparation with physiological saline or phosphate buffer.

9. The liquid metal-based anti-inflammatory drug according to claim 8, characterized in that: The administration form of the liquid metal-based anti-inflammatory drug includes injection, spray, aerosol, ointment, cream, film or paste; Preferably, it is an injection, spray, film-coating or aerosol; More preferably, it is an injection; Optimally, the concentration of the liquid metal nanoparticles in the injection is 0.5-3 mg / mL.

10. An application of the liquid metal nanoparticle suspension preparation according to claim 6, or the liquid metal-based anti-inflammatory drug according to claim 8 or 9. Characterized in that: Used for preparing medicines for treating infectious bone defect diseases.

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