Periodontal microneedle loaded with near-infrared responsive composite bilirubin nanoparticles and preparation method thereof

Abstract

The present invention provides a periodontal microneedle loaded with near-infrared responsive composite bilirubin nanoparticles and a preparation method thereof, which belongs to the technical field of composite materials. In the present invention, bilirubin-gelatin complex nanoparticles have good antioxidant activity and promote macrophage repolarization to M2 by removing ROS; folic acid can specifically bind to FA receptors on the surface of M1 macrophages, giving nanoparticles good M1 macrophage targeting properties and improving the bioavailability of the material; organic-metal coordination supramolecular network coating has both anthocyanin and iron ion properties, showing anti-inflammatory and antioxidant therapeutic effects; at the same time, it has both photothermal properties, can convert near-infrared light energy into thermal energy, and induce M1 macrophage apoptosis. Therefore, the near-infrared responsive composite bilirubin nanoparticles provided by the present invention can reshape the periodontitis immune homeostasis by the dual functions of stimulating M1 macrophage apoptosis through photothermal effect and removing ROS to induce M1 macrophage M2 polarization.

Description

Technical Field

[0001] The present invention relates to the technical field of composite materials, and in particular to a periodontal microneedle carrying near-infrared responsive composite bilirubin nanoparticles and a preparation method thereof. Background Art

[0002] Periodontitis is a chronic inflammatory disease prevalent worldwide. It can continuously destroy the supporting tissues of the periodontium. It is the main cause of loose and falling teeth and a potential factor in causing systemic inflammation. Currently, periodontitis has become the sixth most common disease in humans. According to statistics, approximately 796 million adults worldwide suffer from periodontal health problems, which has brought a great economic burden to patients and the medical system. Plaque microorganisms are the initiating factors of periodontitis. Currently, the main treatment for periodontitis is to control the development of periodontitis by removing bacterial plaque. However, because the main cause of periodontal destruction - the host's dysregulated immune inflammatory response - has not been improved, the tissue regeneration capacity is impaired, and the regeneration effect of periodontal tissue is not ideal.

[0003] With the deepening of relevant research in recent years, it has become increasingly clear that the damage to periodontal tissues caused by host immune homeostasis imbalance in the pathological environment of periodontitis is obvious. The underlying mechanisms involve mitochondrial and endoplasmic reticulum dysfunction leading to the accumulation of reactive oxygen species (ROS), and an overactivated immune response that triggers the release of proinflammatory cytokines. This ultimately affects periodontal tissue repair, reduces the efficiency and quality of new bone formation, and activates bone resorption.

[0004] Macrophages play a key role in regulating the dynamic balance of host immunity. Normally, inactive macrophages (macrophages) from the circulatory system and tissues are ) recruit and activate a large number of macrophages to differentiate into different phenotypes - pro-inflammatory macrophages (M1) and anti-inflammatory macrophages (M2), which are respectively involved in the process of identifying and eliminating pathogens and tissue regeneration and repair. However, in the periodontal tissues at the site of inflammation, due to the dysfunction of mitochondria and endoplasmic reticulum, ROS accumulates in large quantities, resulting in the disruption of the dynamic balance of M1 / M2 macrophages. Macrophages are unable to repolarize from a pro-inflammatory phenotype to an anti-inflammatory phenotype, resulting in the secretion of a large number of pro-inflammatory cytokines, promoting osteoclast differentiation, and ultimately leading to alveolar bone resorption, hindering periodontal regeneration and repair. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a periodontal microneedle loaded with near-infrared responsive composite bilirubin nanoparticles and a preparation method thereof. The near-infrared responsive composite bilirubin nanoparticles provided by the present invention can reshape the immune homeostasis of periodontitis and have a good therapeutic effect on periodontitis.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a near-infrared responsive composite bilirubin nanoparticle, comprising a bilirubin-gelatin composite nanoparticle, wherein the surface of the bilirubin-gelatin composite nanoparticle is sequentially coated with a macrophage cell membrane and an organic-metal coordination supramolecular network coating; the outer surface of the near-infrared responsive composite bilirubin nanoparticle is modified with phospholipid polyethylene glycol folic acid;

[0008] The bilirubin and gelatin in the bilirubin-gelatin complex nanoparticles are covalently bound via an amide bond;

[0009] The organic ligand of the organic-metal coordination supramolecular network coating is anthocyanin, and the coordinated metal ion is a trivalent iron ion.

[0010] Preferably, the particle size of the bilirubin-gelatin complex nanoparticles is 100 to 300 nm;

[0011] The particle size of the near-infrared responsive composite bilirubin nanoparticles is 100-500 nm.

[0012] The present invention provides a method for preparing the above-mentioned near-infrared responsive composite bilirubin nanoparticles, comprising the following steps:

[0013] Bilirubin, gelatin, a carboxyl activator and an organic solvent are mixed and subjected to an amidation reaction to obtain a bilirubin-gelatin complex;

[0014] mixing the aqueous dispersion of the bilirubin-gelatin complex with an organic solvent for self-assembly to obtain bilirubin-gelatin complex nanoparticles;

[0015] co-extruding the bilirubin-gelatin complex nanoparticles with macrophage cell membranes to obtain cell membrane-coated bilirubin-gelatin complex nanoparticles;

[0016] Mixing the cell membrane-coated bilirubin-gelatin complex nanoparticles aqueous dispersion, anthocyanin, a soluble trivalent iron source, and water, and adjusting the pH value of the resulting mixture to 7.5-8 to obtain bilirubin-gelatin complex nanoparticles coated with an organic-metal coordination supramolecular network coating;

[0017] The aqueous dispersion of the bilirubin-gelatin complex nanoparticles coated with the organic-metal coordination supramolecular network coating is mixed with phospholipid polyethylene glycol folic acid and incubated to obtain near-infrared responsive complex bilirubin nanoparticles.

[0018] Preferably, the mass ratio of bilirubin to gelatin is 1:10-15;

[0019] The amidation reaction temperature is room temperature and the time is 8 to 10 hours.

[0020] Preferably, the mass ratio of the bilirubin-gelatin complex nanoparticles to the macrophage cell membrane is 1 to 2:1.

[0021] Preferably, the mass ratio of the anthocyanin, the soluble trivalent iron source and the cell membrane-coated bilirubin-gelatin complex nanoparticles is 5:1:4-5.

[0022] Preferably, the mass ratio of the bilirubin-gelatin complex nanoparticles coated with the organic-metal coordination supramolecular network coating to the phospholipid polyethylene glycol folic acid is 4 to 10:1;

[0023] The incubation temperature is 35-37° C., and the incubation time is 0.5-2 h.

[0024] The present invention provides the use of the above-mentioned near-infrared responsive composite bilirubin nanoparticles in the preparation of a drug for treating periodontitis.

[0025] The present invention provides a near-infrared responsive composite bilirubin nanoparticle periodontitis drug-loaded microneedle, comprising a drug-loaded microneedle tip body and a microneedle base. The raw materials for preparing the microneedle tip body include methacrylated hydrogel, a photoinitiator and the above-mentioned near-infrared responsive composite bilirubin nanoparticles.

[0026] Preferably, the mass ratio of the methacrylated hydrogel, the photoinitiator and the near-infrared responsive composite bilirubin nanoparticles is 200:5:8-10.

[0027] The present invention provides near-infrared-responsive composite bilirubin nanoparticles, comprising bilirubin-gelatin complex nanoparticles, the surfaces of which are sequentially coated with a macrophage cell membrane and an organic-metal coordination supramolecular network coating; the outer surface of the near-infrared-responsive composite bilirubin nanoparticles is modified with phospholipid polyethylene glycol folic acid; the bilirubin and gelatin in the bilirubin-gelatin complex are covalently bound via an amide bond; the organic ligand of the organic-metal coordination supramolecular network coating is anthocyanin, and the coordinated metal ion is a trivalent iron ion. In the present invention, the bilirubin-gelatin complex nanoparticles have good antioxidant activity and promote macrophage repolarization to M2 by scavenging ROS. The present invention coats the surface of bilirubin-gelatin complex nanoparticles with an organic-metal coordination supramolecular network coating. The organic-metal coordination supramolecular network coating is obtained by coordinating anthocyanin (Cyanidin-3-O-β-glucoside, C3G) and trivalent iron ions. The two form an organic-metal coordination supramolecular network coating through coordination bonds, which combines the characteristics of anthocyanin and iron ions, showing anti-inflammatory and antioxidant therapeutic effects; at the same time, it has photothermal properties and can convert near-infrared (NIR) light energy into thermal energy to induce apoptosis of M1 macrophages. Therefore, the near-infrared responsive composite bilirubin nanoparticles provided by the present invention can reshape the immune homeostasis of periodontitis through the dual functions of stimulating M1 macrophage apoptosis through photothermal effect and scavenging ROS to induce M2 polarization of M1 macrophages. Phospholipid polyethylene glycol folic acid (FA) can pass through the pores of the organic-metal coordination supramolecular network coating and insert into the phospholipid bilayer of the macrophage cell membrane, thereby modifying the outer surface of the composite nanoparticles. Since folic acid can specifically bind to the FA receptor on the surface of M1 macrophages, it gives the nanoparticles good M1 macrophage targeting properties, thereby improving the bioavailability of the material.

[0028] The present invention provides a method for preparing the above-mentioned near-infrared responsive composite bilirubin nanoparticles. The method is simple to operate and easy to realize industrial batch production.

[0029] The present invention provides a near-infrared responsive composite bilirubin nanoparticle periodontitis drug-loaded microneedle, comprising a drug-loaded microneedle tip body and a microneedle base; the raw materials for preparing the microneedle tip body include methacrylated hydrogel, a photoinitiator and the above-mentioned near-infrared responsive composite bilirubin nanoparticles. Due to the complex and changeable anatomical structure of the oral periodontium and the influence of multiple factors such as chewing, saliva secretion, and bacterial infection, it is difficult to achieve long-term and stable drug delivery. The near-infrared responsive composite bilirubin nanoparticle periodontitis drug-loaded microneedle provided by the present invention has good mechanical properties, can penetrate deep periodontal tissues to achieve efficient delivery, and can reshape the periodontitis immune homeostasis by regulating and stimulating the apoptosis of M1 macrophages and scavenging ROS to induce M1 macrophage repolarization, ultimately promoting the regeneration and repair of periodontal tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The preparation process of near-infrared responsive composite bilirubin nanoparticles and periodontitis drug-loaded microneedles;

[0031] Figure 2 Fluorescence colocalization image of BRNP and mBRNP;

[0032] Figure 3 BRNP, mBRNP@C3G-Fe 3+ Transmission electron microscope images of

[0033] Figure 4 BRNP, mBRNP and mBRNP@C3G-Fe 3+ Particle size distribution diagram;

[0034] Figure 5 mBRNP@C3G-Fe 3+ FA-mBRNP@C3G-Fe 3+ Particle size diagram;

[0035] Figure 6 mBRNP@C3G-Fe 3+ FA-mBRNP@C3G-Fe 3+ Potential diagram of

[0036] Figure 7 Different cells react with FA-mBRNP@C3G-Fe 3+ The swallowing result;

[0037] Figure 8 The temperature variation curve over time in photothermal imaging;

[0038] Figure 9 is the light-to-heat conversion efficiency curve;

[0039] Figure 10 This is the result of photothermal-induced apoptosis of M1 macrophages;

[0040] Figure 11 Annexin V-FITC cell apoptosis detection results;

[0041] Figure 12 Results of intracellular reactive oxygen species testing after LPS stimulation induced M1 polarization of macrophages;

[0042] Figure 13 This is the expression result of M1 marker IL-1β;

[0043] Figure 14 The expression results of M2 marker IL-10;

[0044] Figure 15 The microscopic morphology of MN;

[0045] Figure 16 The results of the tissue penetration test of MN;

[0046] Figure 17 is the mechanical strength test result of MN;

[0047] Figure 18 FA-mBRNP@C3G-Fe in MN 3+ The co-localization observation results;

[0048] Figure 19 FA-mBRNP@C3G-Fe in MN 3+ The sustained release results;

[0049] Figure 20 The alveolar bone resorption and treatment effects of each group. DETAILED DESCRIPTION

[0050] The present invention provides a near-infrared responsive composite bilirubin nanoparticle, comprising a bilirubin-gelatin composite nanoparticle, wherein the surface of the bilirubin-gelatin composite nanoparticle is sequentially coated with a macrophage cell membrane and an organic-metal coordination supramolecular network coating; the outer surface of the near-infrared responsive composite bilirubin nanoparticle is modified with phospholipid polyethylene glycol folic acid;

[0051] The bilirubin and gelatin in the bilirubin-gelatin complex are covalently bound via an amide bond;

[0052] The organic ligand of the organic-metal coordination supramolecular network coating is anthocyanin, and the coordinated metal ion is a trivalent iron ion.

[0053] In the present invention, the particle size of the bilirubin-gelatin complex nanoparticles is 100-300 nm, preferably 200-300 nm; the particle size of the near-infrared responsive complex bilirubin nanoparticles is 100-500 nm, preferably 200-400 nm.

[0054] In the present invention, the bilirubin and gelatin in the bilirubin-gelatin complex are covalently bound via an amide bond, and the mass ratio of the bilirubin to gelatin is preferably 1:10.

[0055] In the present invention, the preparation method of near-infrared responsive composite bilirubin nanoparticles comprises the following steps:

[0056] Bilirubin, gelatin, a carboxyl activator and an organic solvent are mixed and subjected to an amidation reaction to obtain a bilirubin-gelatin complex;

[0057] mixing an aqueous dispersion of the bilirubin-gelatin complex with an organic solvent for self-assembly to obtain bilirubin-gelatin complex nanoparticles;

[0058] co-extruding the bilirubin-gelatin complex nanoparticles with macrophage cell membranes to obtain cell membrane-coated bilirubin-gelatin complex nanoparticles;

[0059] Mixing the cell membrane-coated bilirubin-gelatin complex nanoparticles aqueous dispersion, anthocyanin, a soluble trivalent iron source, and water, and adjusting the pH value of the resulting mixture to 7.5-8 to obtain bilirubin-gelatin complex nanoparticles coated with an organic-metal coordination supramolecular network coating;

[0060] The aqueous dispersion of the bilirubin-gelatin complex nanoparticles coated with the organic-metal coordination supramolecular network coating is mixed with phospholipid polyethylene glycol folic acid and incubated to obtain near-infrared responsive complex bilirubin nanoparticles.

[0061] The present invention mixes bilirubin, gelatin, a carboxyl activator, and an organic solvent, and performs an amidation reaction to obtain a bilirubin-gelatin complex. In the present invention, the carboxyl activator is preferably N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). In the present invention, the mass ratio of bilirubin to gelatin is 1:10-15, preferably 1:12-14; in the present invention, the molar ratio of bilirubin to N-hydroxysuccinimide is 1:1-2, preferably 1:1, and the molar ratio of bilirubin to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 2:7-14, preferably 2:10-12.

[0062] In the present invention, the organic solvent is preferably dimethyl sulfoxide.

[0063] In the present invention, the mixing method is preferably: first, the bilirubin, the carboxyl activator and the organic solvent are stirred and mixed, and then the gelatin is added. In the present invention, the stirring and mixing time is preferably 30 minutes.

[0064] In the present invention, the amidation reaction is preferably carried out under anaerobic conditions. In the present invention, the amidation reaction is carried out at room temperature for 8 to 10 hours, preferably 9 hours.

[0065] In the present invention, after the amidation reaction, the obtained amidation reaction solution is dialyzed and dried to obtain a bilirubin-gelatin complex solid. In the present invention, the dialysis includes a first dialysis and a second dialysis performed sequentially, wherein the dialysate used in the first dialysis is a 10 mM sodium hydroxide solution, and the first dialysis duration is 4 to 8 hours, preferably 5 to 6 hours; the dialysate used in the second dialysis is water, and the second dialysis duration is preferably 24 to 36 hours.

[0066] In the present invention, the drying method is preferably freeze-drying; the present invention has no special requirements for the freeze-drying method, and the freeze-drying method well known to those skilled in the art can be used.

[0067] The present invention mixes an aqueous dispersion of a bilirubin-gelatin complex with an organic solvent and self-assembles to produce bilirubin-gelatin complex nanoparticles. In the present invention, the concentration of the bilirubin-gelatin complex in the aqueous dispersion is 1 to 5 mg / mL, preferably 2 to 4 mg / mL. In the present invention, the organic solvent is preferably chloroform. In the present invention, the mixing method is preferably stirring, and the stirring time is preferably 15 minutes.

[0068] The present invention preferably performs the self-assembly under ultrasonic conditions. In the present invention, the ultrasonic power is 100-150W, the frequency is 40-60kHz, preferably 50kHz, and the duration is 20-30min, preferably 25min.

[0069] After the self-service loading, the present invention preferably performs solid-liquid separation on the resulting solution. In the present invention, the solid-liquid separation is preferably performed by centrifugation. In the present invention, the centrifugation speed is 12,000 to 14,000 rpm, and the time is 8 to 12 minutes, preferably 10 minutes.

[0070] In the present invention, the bilirubin-gelatin complex nanoparticles are co-extruded with macrophage cell membranes to obtain cell membrane-coated bilirubin-gelatin complex nanoparticles. In the present invention, the macrophage cell membrane is preferably the cell membrane of the mouse macrophage cell line Raw 264.7. As a specific embodiment of the present invention, the macrophages are extracted according to the instructions provided by the Beyotime Biotechnology Membrane Protein Extraction Kit.

[0071] In the present invention, the mass ratio of the bilirubin-gelatin complex nanoparticles to the macrophage cell membrane is preferably 1 to 2: 1. In the present invention, a micro extruder is preferably used for the co-extrusion.

[0072] In the present invention, after the co-extrusion, the co-extruded product is preferably centrifuged, the centrifugal force of the centrifugation is 12000g, and the time is 10 to 15 minutes, preferably 12 to 14 minutes.

[0073] The present invention mixes the cell membrane-coated bilirubin-gelatin complex nanoparticles aqueous dispersion, anthocyanin, a soluble trivalent iron source, and water, and adjusts the pH of the resulting mixture to 7.5 to 8 to obtain bilirubin-gelatin complex nanoparticles coated with an organic-metallic coordination supramolecular network coating. In the present invention, the concentration of the cell membrane-coated bilirubin-gelatin complex nanoparticles aqueous dispersion is preferably 50 μg / mL.

[0074] In the present invention, the soluble ferric iron source is preferably ferric chloride, more preferably ferric chloride hexahydrate. In the present invention, the mass ratio of the anthocyanin, the soluble ferric iron source and the cell membrane-coated bilirubin-gelatin complex nanoparticles is preferably 5:1:4-5.

[0075] The present invention preferably adjusts the pH value of the obtained mixed solution to 7.5-8. In the present invention, the pH adjuster used to adjust the pH value is preferably dilute hydrochloric acid and sodium hydroxide solution.

[0076] In the present invention, after adjusting the pH value of the obtained mixed solution to 7.5-8, the anthocyanin is subjected to a coordination reaction with trivalent iron ions. The temperature of the coordination reaction is preferably room temperature, and the time is 3-5 minutes, preferably 4 minutes.

[0077] In the present invention, after the coordination reaction, the coordination reaction solution is preferably centrifuged, and the centrifugal force of the centrifugation is 12000g, and the time is 10 to 15 minutes, preferably 12 to 14 minutes.

[0078] In the present invention, an aqueous dispersion of bilirubin-gelatin complex nanoparticles coated with an organic-metal coordination supramolecular network coating is mixed with phospholipid polyethylene glycol folic acid and incubated to obtain near-infrared responsive composite bilirubin nanoparticles. In the present invention, the concentration of the aqueous dispersion of bilirubin-gelatin complex nanoparticles coated with an organic-metal coordination supramolecular network coating is preferably 200 μg / mL.

[0079] In the present invention, the mass ratio of the bilirubin-gelatin complex nanoparticles coated with the organic-metal coordination supramolecular network coating to the phospholipid polyethylene glycol folic acid is 4:1; in the present invention, the incubation temperature is 35-37°C, and the time is 0.5-2 hours, preferably 1-1.5 hours. In the present invention, after the incubation, the resulting liquid is preferably centrifuged at a centrifugal force of 12,000 g for 10-15 minutes, preferably 12-14 minutes.

[0080] The present invention provides the use of the above-mentioned near-infrared responsive composite bilirubin nanoparticles in the preparation of a drug for treating periodontitis.

[0081] The present invention provides a near-infrared responsive composite bilirubin nanoparticle periodontitis drug-loaded microneedle, comprising a drug-loaded microneedle tip and a microneedle base; the preparation raw materials of the microneedle tip include methacrylated hydrogel (GelMA), a photoinitiator and the above-mentioned near-infrared responsive composite bilirubin nanoparticles.

[0082] In the present invention, the photoinitiator is preferably phenyl (2,4,6-trimethylbenzoyl) lithium phosphate. In the present invention, the mass ratio of the methacrylated hydrogel, the photoinitiator and the near-infrared responsive composite bilirubin nanoparticles is preferably 200:5:8-10.

[0083] In the present invention, the raw material for preparing the microneedle base preferably includes gelatin. In the present invention, the gelatin is preferably provided in the form of an aqueous solution, and the concentration of the gelatin aqueous solution is preferably 10 wt%.

[0084] In the present invention, the method for preparing the near-infrared responsive composite bilirubin nanoparticle periodontitis drug-loaded microneedles preferably comprises the following steps:

[0085] mixing methacrylated hydrogel, a photoinitiator, and near-infrared responsive composite bilirubin nanoparticles to obtain a mixture;

[0086] The mixture is added to a drug-loaded microneedle negative mold, gelatin is spread on the negative mold, and ultraviolet light curing is performed to obtain near-infrared responsive composite bilirubin nanoparticle periodontitis drug-loaded microneedles.

[0087] In the present invention, the wavelength of the UV curing is 360-480 nm, and the intensity is 50 W / cm 2 ; The UV curing time is 3 to 5 minutes, preferably 4 minutes.

[0088] In the present invention, after the UV curing, the obtained cured product is preferably placed at room temperature in a dark environment for 24 hours.

[0089] The preparation process of the near-infrared responsive composite bilirubin nanoparticles and periodontitis drug-loaded microneedles of the present invention is as follows: Figure 1 shown.

[0090] The periodontal microneedles loaded with near-infrared responsive composite bilirubin nanoparticles and the preparation method thereof provided by the present invention are described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0091] Example 1

[0092] (1) Preparation of bilirubin-gelatin complex (BR-GA)

[0093] 20 μmol of BR, 20 μmol of N-hydroxysuccinimide, and 70 μmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were added to 4 mL of dimethyl sulfoxide and stirred at room temperature for 30 minutes. 100 mg of GA was then added to the solution and incubated in an anaerobic incubator overnight. 6 mL of 10 mM sodium hydroxide solution was added and the solution was dialyzed against 10 mM sodium hydroxide for 4 hours, followed by further dialysis against distilled water for 24 hours. Finally, BR-GA was lyophilized to obtain.

[0094] (2) Preparation of bilirubin-gelatin complex nanoparticles (BRNP)

[0095] Dissolve 5 mg of BR-GA in 1 mL of distilled water and slowly add 0.25 mL of chloroform. Stir the resulting solution at room temperature for 15 minutes, then sonicate (40 kHz, 100 W) for 30 minutes. Collect the supernatant and centrifuge (12,000 rpm, 10 minutes) to obtain the precipitate, which is BRNP.

[0096] (3) Preparation of cell membrane-coated bilirubin-gelatin complex nanoparticles (mBRNP)

[0097] Cell membranes of the mouse macrophage cell line Raw264.7 were extracted using the Biotech Membrane Protein Extraction Kit instructions. The cell membranes and BRNPs were co-extruded using a microextruder. Gradient centrifugation at 3000 g for 30 minutes and then at 12000 g for 30 minutes was performed to precipitate the mBRNPs.

[0098] (4) Bilirubin-gelatin complex nanoparticles coated with organic-metal coordination supramolecular network coating (mBRNP@C3G-Fe 3+ )

[0099] The mixed solution was prepared in proportion to make the concentrations of 0.04 mg / mL C3G, 0.008 mg / mL ferric chloride hexahydrate, and 50 μg mBRNP, and the pH value was adjusted to 8.0. The supernatant was collected and centrifuged (12000 rpm, 10 min) to obtain mBRNP@C3G-Fe 3+ .

[0100] (5) Preparation of near-infrared responsive composite bilirubin nanoparticles (FA-mBRNP@C3G-Fe 3+ )

[0101] Take 1mL 200μg mBRNP@C3G-Fe 3+The mixture was incubated with 50 μg of phospholipid polyethylene glycol folic acid at 37 °C for 1 h, and then centrifuged (10000 g, 10 min) to collect the precipitate to obtain FA-mBRNP@C3G-Fe 3+ .

[0102] Structural characterization

[0103] Fluorescence co-localization images of BRNP and mBRNP are shown in Figure 2. Figure 2 As shown in the figure, it can be seen that the cell membrane is successfully coated with nanoparticles, and the macrophage membrane (red fluorescence) and BRNP (green fluorescence) can be well co-localized.

[0104] BRNP, mBRNP@C3G-Fe 3+ Transmission electron microscopy images of Figure 3 As shown in Figure 2, BRNP is a regular round shape with a particle size of about 200 nm. 3+ A composite coating is formed on the surface, showing an obvious core-shell structure, with a slightly increased material particle size and clearer surface boundaries.

[0105] BRNP, mBRNP, and mBRNP@C3G-Fe 3+ The particle size distribution diagram is as follows Figure 4 The particle size distribution results show that the prepared BRNP particle size is mainly concentrated around 200 nm, and the particle size of the prepared BRNP is mainly concentrated around 200 nm when coated with macrophage membrane (mBRNP) and organic-metallic supramolecular network coating (mBRNP@C3G-Fe 3+ ), the particle size of the material increased slightly, proving that the material biomodification was successful.

[0106] mBRNP@C3G-Fe 3+ FA-mBRNP@C3G-Fe 3+ The particle size diagram is as follows Figure 5 As shown, the potential diagram is Figure 6 Particle size analysis showed that folic acid modification did not affect the nanoparticle size, with an average particle size of approximately 230 nm. Potential analysis showed that the nanoparticle potential was approximately -27 mV, close to the cell membrane potential, confirming that folic acid modification did not disrupt the cell membrane structure on the nanoparticle surface.

[0107] Performance Testing

[0108] (1) To demonstrate that M1 macrophages specifically take up FA-mBRNP@C3G-Fe 3+ ability to fibroblasts and Macrophages were used as the control group, and M1 macrophages were used as the experimental group to detect the ability of different cells to absorb nanoparticles. First, 1 μg / mL lipopolysaccharide (LPS) was used to pre-induce macrophage M1 polarization for 24 hours as the experimental group. 10 μM FA-mBRNP@C3G-Fe was labeled with red fluorescent Dil. 3+ Nanomaterials were added to fibroblasts, After co-culture in 37℃ incubator for 8 h, the unphagocytosed FA-mBRNP@C3G-Fe 3+ Finally, the cells were fixed with 4% paraformaldehyde solution for 10 min, the cell nuclei were stained with DAPI, and the cells were observed under a fluorescence microscope. Figure 7 As shown. With fibroblasts and Compared with M1 macrophages, M1 macrophages showed a strong effect on FA-mBRNP@C3G-Fe 3+ The phagocytic level of FA-mBRNP@C3G-Fe 3+ It has good targeting of M1 macrophages.

[0109] (2) mBRNP@C3G-Fe with a concentration of 80 μM 3+ Place in EP tube, then at 1.5Wcm -2 The NIR irradiation was carried out for 10 minutes, and the irradiation was stopped and the temperature was naturally cooled to 37°C. Four cycles were repeated and the infrared thermal imager was used to collect the thermal image and record the temperature change. The temperature change curve over time is shown in FIG. Figure 8 As shown, the photothermal conversion efficiency is Figure 9 shown.

[0110] Depend on Figure 8 It can be seen that under 808nm NIR irradiation, mBRNP@C3G-Fe 3+ The considerable heat generated caused the temperature to rise rapidly to about 80 °C within 10 min, and a relatively stable and repeatable heating-cooling process was maintained during the four cycles, indicating that mBRNP@C3G-Fe 3+ It has stable light-to-heat conversion capability. Figure 9 It can be seen that the heat transfer time constant calculated by a single cycle is 448.63. 3+ It has excellent photothermal conversion performance and significant photothermal stability and is an effective photothermal agent.

[0111] (3) The present invention evaluated FA-mBRNP@C3G-Fe 3+ The photothermal-induced apoptosis of M1 macrophages was investigated by setting up a blank control group (Control) without drug addition and an experimental group (FA-mBRNP@C3G-Fe 3+). The samples were treated with or without NIR light under the same conditions (NIR parameters: 808 nm 1.5 W cm -2 ). CCK-8 was then used to evaluate the activity of M1 macrophages in each group.

[0112] The results of photothermal-induced apoptosis of M1 macrophages are as follows Figure 10 As shown in Figure 2, it can be seen that the control group had no significant effect on the activity of M1 macrophages regardless of whether NIR light was given or not; while the addition of FA-mBRNP@C3G-Fe 3+ After that, there was no significant effect on cell activity without NIR light, indicating that the prepared nanoparticles had no obvious toxic side effects on cells and had good biocompatibility. 3+ After NIR light stimulation, the cell activity of the group decreased by about 40%, indicating that M1 macrophages took up FA-mBRNP@C3G-Fe 3+ After being stimulated by NIR light, the cell activity is reduced.

[0113] Annexin V-FITC cell apoptosis detection kit was used to stain the cells. Figure 11 As shown, FA-mBRNP@C3G-Fe 3+ After NIR light irradiation, a large number of cells in the experimental group highly expressed the apoptosis marker Annexin V with intact cell membranes, confirming that the prepared FA-mBRNP@C3G-Fe 3+ The nanomaterials were able to perform photothermal conversion and successfully induce apoptosis of M1 macrophages.

[0114] (4) Mouse macrophage cell line RAW264.7 was stimulated with 1 μg / mL LPS to induce M1 polarization, and BRNP, mBRNP, and mBRNP@C3G-Fe were added. 3+ The cells were cultured in a cell incubator for 24 hours. The Biyuntian Cell Reactive Oxygen Species Detection Kit was used for detection. According to the instructions, 10 μM DCFH-DA reactive oxygen species fluorescent probe was used to stain the cells. The results were then observed using a fluorescence microscope. Figure 12 The results showed that after LPS stimulation induced macrophage M1 polarization, intracellular reactive oxygen species increased significantly; while the addition of BRNP, mBRNP, mBRNP@C3G-Fe 3+ Afterwards, they could reduce the intracellular reactive oxygen species to a certain extent, among which mBRNP@C3G-Fe 3+ The reactive oxygen species in the treated group cells were able to return to a near-normal level, indicating that the near-infrared responsive composite bilirubin nanoparticles prepared by the present invention have excellent antioxidant capacity.

[0115] (5) PCR was used to detect the effect of the near-infrared responsive composite bilirubin nanoparticles prepared by the present invention on macrophage M2 polarization. After the mouse macrophage cell line RAW264.7 was stimulated with 1 μg / mL LPS to simulate the inflammatory state and induce M1 polarization, BRNP, mBRNP, and mBRNP@C3G-Fe were added respectively. 3+ and FA-mBRNP@C3G-Fe 3+ After further culturing for 24 h, cells from each group were collected for PCR detection.

[0116] The expression results of M1 marker IL-1β were as follows Figure 13 The expression results of M2 marker IL-10 are shown in Figure 14 As shown in Figure 3, the expression of M1 marker IL-1β increased by about 26 times in the LPS group, while the expression of IL-1β was significantly reduced after the addition of nanoparticles. 3+ After treatment, the expression of M2 marker IL-10 increased significantly, indicating that FA-mBRNP@C3G-Fe 3+ It can promote the repolarization of M1 macrophages to M2.

[0117] Example 2

[0118] Preparation of drug-loaded microneedles (MN) for periodontitis

[0119] Add photoinitiator, FA-mBRNP@C3G-Fe 3+ , GelMA, photoinitiator and FA-mBRNP@C3G-Fe 3+ The mass ratio of 200:5:8 was fully mixed and poured into the MN female mold to form the MN body. A 10wt% GA aqueous solution was evenly spread on the top to form the MN base and irradiated under ultraviolet light (wavelength: 360-480nm, intensity: 50W / cm 2 After irradiation for 5 min at room temperature in the dark for 24 h, the mold was removed to obtain the drug-loaded MN.

[0120] The microscopic morphology of the prepared MN was observed by stereo microscope, and the results were as follows: Figure 15 As shown in Figure 2, MN is conical in shape, uniformly filled, and regularly arranged.

[0121] Performance Testing

[0122] (1) The mechanical properties of microneedles are the key factor in whether they can penetrate periodontal tissue. Pig jaws were used to simulate the in vivo application scenario of MN and test the tissue penetration of MN. The results are as follows Figure 16 The results showed that MN could smoothly penetrate the pig gingival tissue (shown in the red box).

[0123] The mechanical strength of MN was further tested by compression test. The tip of MN was placed vertically on a stainless steel plate and pressed against another stainless steel plate at a constant speed of 0.5 mm / min. The relationship between the applied pressure and the deformation of MN was recorded. The results were as follows: Figure 17 As shown. It can be seen that the addition of FA-mBRNP@C3G-Fe 3+ The mechanical strength of the microneedles was slightly decreased and had sufficient mechanical strength to penetrate freshly extracted porcine gingival tissue.

[0124] (2) FA-mBRNP@C3G-Fe in MN 3+ Colocalization observation was performed using a fluorescence microscope, and the results were as follows: Figure 18 As shown in the fluorescence image, FA-mBRNP@C3G-Fe 3+ Evenly distribute on the needle tip.

[0125] FA-mBRNP@C3G-Fe 3+ The microneedles were placed in 1 mL of PBS and the in vitro sustained release performance was evaluated at a constant temperature of 37 ° C. The supernatant of the samples was taken at different time points (1, 3, 5, 7, 9, 11, 13, 15, and 17 days), and the absorbance was measured at a wavelength of 450 nm using a UV-visible spectrophotometer to calculate the FA-mBRNP@C3G-Fe 3+ The release amount of FA-mBRNP@C3G-Fe 3+ The sustained release results are as follows Figure 19 According to the sustained-release curve, the nanoparticles released approximately 70% of the drug within one week and then continued to release slowly for two weeks, demonstrating good sustained-release properties.

[0126] (3) The mice were randomly divided into 4 groups: control group, periodontitis group, microneedle treatment group, and microneedle photothermal treatment group. The mouse periodontitis model was established according to the standard. After the model was successfully established, microneedles were inserted into the palatal gingiva of the mice in the microneedle treatment group and the microneedle photothermal treatment group. One day later, the microneedle photothermal treatment group was irradiated with NIR for 5 minutes. After 7 days of treatment, the mice were euthanized, and the mandibular alveolar bone specimens were collected. The specimens were photographed with a stereo microscope and scanned with micro-CT. Finally, the specimens were decalcified and paraffin sections were stained with H&E to evaluate the alveolar bone resorption and treatment effect of each group.

[0127] Alveolar bone resorption and treatment effects in each group Figure 20As shown in the figure, using the control group as a reference, the periodontitis group experienced the greatest alveolar bone resorption, while the microneedle treatment group experienced partial improvement. The microneedle treatment group experienced the least alveolar bone resorption, which was closest to the control group. This demonstrates that the near-infrared-responsive composite bilirubin nanoparticle microneedles prepared by the present invention are effective in treating periodontitis in mice, and the therapeutic effect can be further enhanced when combined with photothermal therapy.

[0128] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A near-infrared responsive composite bilirubin nanoparticle, comprising a bilirubin-gelatin complex nanoparticle, wherein the surface of the bilirubin-gelatin complex nanoparticle is sequentially coated with a macrophage cell membrane and an organic-metal coordination supramolecular network coating; the outer surface of the near-infrared responsive composite bilirubin nanoparticle is modified with phospholipid polyethylene glycol folic acid; The bilirubin and gelatin in the bilirubin-gelatin complex nanoparticles are covalently bound via an amide bond; The organic ligand of the organic-metal coordination supramolecular network coating is anthocyanin, and the coordinated metal ion is trivalent iron ion; The phospholipid polyethylene glycol folic acid passes through the pores of the organic-metal coordination supramolecular network coating and is inserted into the phospholipid bilayer of the macrophage cell membrane; The preparation method of the near-infrared responsive composite bilirubin nanoparticles comprises the following steps: Bilirubin, gelatin, a carboxyl activator and an organic solvent are mixed and subjected to an amidation reaction to obtain a bilirubin-gelatin complex; mixing the aqueous dispersion of the bilirubin-gelatin complex with an organic solvent for self-assembly to obtain bilirubin-gelatin complex nanoparticles; co-extruding the bilirubin-gelatin complex nanoparticles with macrophage cell membranes to obtain cell membrane-coated bilirubin-gelatin complex nanoparticles; Mixing the cell membrane-coated bilirubin-gelatin complex nanoparticles aqueous dispersion, anthocyanin, a soluble trivalent iron source, and water, and adjusting the pH value of the resulting mixture to 7.5-8 to obtain bilirubin-gelatin complex nanoparticles coated with an organic-metal coordination supramolecular network coating; The aqueous dispersion of the bilirubin-gelatin complex nanoparticles coated with the organic-metal coordination supramolecular network coating is mixed with phospholipid polyethylene glycol folic acid and incubated to obtain near-infrared responsive complex bilirubin nanoparticles.

2. The near-infrared responsive composite bilirubin nanoparticles according to claim 1, characterized in that The particle size of the bilirubin-gelatin complex nanoparticles is 100 to 300 nm; The particle size of the near-infrared responsive composite bilirubin nanoparticles is 100-500 nm.

3. The method for preparing the near-infrared responsive composite bilirubin nanoparticles according to claim 1 or 2, comprising the following steps: Bilirubin, gelatin, a carboxyl activator and an organic solvent are mixed and subjected to an amidation reaction to obtain a bilirubin-gelatin complex; mixing the aqueous dispersion of the bilirubin-gelatin complex with an organic solvent for self-assembly to obtain bilirubin-gelatin complex nanoparticles; co-extruding the bilirubin-gelatin complex nanoparticles with macrophage cell membranes to obtain cell membrane-coated bilirubin-gelatin complex nanoparticles; Mixing the cell membrane-coated bilirubin-gelatin complex nanoparticles aqueous dispersion, anthocyanin, a soluble trivalent iron source, and water, and adjusting the pH value of the resulting mixture to 7.5-8 to obtain bilirubin-gelatin complex nanoparticles coated with an organic-metal coordination supramolecular network coating; The aqueous dispersion of the bilirubin-gelatin complex nanoparticles coated with the organic-metal coordination supramolecular network coating is mixed with phospholipid polyethylene glycol folic acid and incubated to obtain near-infrared responsive complex bilirubin nanoparticles.

4. The preparation method according to claim 3, characterized in that The mass ratio of bilirubin to gelatin is 1:10-15; The amidation reaction temperature is room temperature and the time is 8 to 10 hours.

5. The preparation method according to claim 3, characterized in that The mass ratio of the bilirubin-gelatin complex nanoparticles to the macrophage cell membrane is 1-2:

1.

6. The preparation method according to claim 3, characterized in that The mass ratio of the anthocyanin, the soluble trivalent iron source and the cell membrane-coated bilirubin-gelatin complex nanoparticles is 5:1:4-5.

7. The preparation method according to claim 3, characterized in that The mass ratio of the bilirubin-gelatin complex nanoparticles coated with the organic-metal coordination supramolecular network coating to the phospholipid polyethylene glycol folic acid is 4 to 10:1; The incubation temperature is 35-37° C., and the incubation time is 0.5-2 h.

8. Use of the near-infrared responsive composite bilirubin nanoparticles according to claim 1 or 2 or the near-infrared responsive composite bilirubin nanoparticles prepared by the preparation method according to any one of claims 3 to 7 in the preparation of drugs for treating periodontitis.

9. A near-infrared responsive composite bilirubin nanoparticle periodontitis drug-loaded microneedle, comprising a drug-loaded microneedle tip and a microneedle base, characterized in that: The raw materials for preparing the microneedle tip body include methacrylated hydrogel, a photoinitiator, and the near-infrared responsive composite bilirubin nanoparticles according to claim 1 or 2 or the near-infrared responsive composite bilirubin nanoparticles prepared by the preparation method according to any one of claims 3 to 7.

10. The near-infrared responsive composite bilirubin nanoparticle periodontitis drug-loaded microneedles according to claim 9, characterized in that: The mass ratio of the methacrylated hydrogel, the photoinitiator and the near-infrared responsive composite bilirubin nanoparticles is 200:5:8-10.

Images