An amphiphilic functionalized d-form-alanine, a preparation method and application thereof, and a self-targeting carbon monoxide nanogenerator and a preparation method and application thereof

Abstract

The application provides an amphiphilic functionalized D-form-alanine and a preparation method and application thereof, and a self-targeting carbon monoxide nanogenerator and a preparation method and application thereof, and belongs to the technical field of antibacterial materials. The application provides an amphiphilic functionalized D-form-alanine. The self-targeting carbon monoxide nanogenerator prepared by using the amphiphilic functionalized D-form-alanine changes the permeability of the outer membrane of the cell wall of gram-negative bacteria, is metabolically utilized by bacteria in a specific manner, generates CO under the action of light to kill bacteria, removes biofilms that are not easy to touch and are hidden in deep periodontal pockets, simultaneously relieves inflammation by inducing macrophages to polarize from an M1 phenotype to an M2 phenotype, further promotes alveolar bone regeneration, and avoids damage to normal tissues.

Description

Technical Field

[0001] This invention relates to the field of antibacterial materials technology, and in particular to an amphiphilic functionalized D-configuration alanine and its preparation method and application, as well as a self-targeted carbon monoxide nanogenerator and its preparation method and application. Background Technology

[0002] Periodontitis is a chronic, multifactorial inflammatory disease caused by an imbalance in the oral microbiota. It can lead to tooth loss and systemic inflammation, resulting in a significant economic burden and a major impact on patients' overall health and quality of life. The key to preventing and treating periodontitis lies in removing plaque, reducing inflammation, and promoting the regeneration of damaged periodontal tissues.

[0003] Due to the complex anatomy of teeth, microorganisms are hidden deep within periodontal pockets and difficult to access. Traditional mechanical treatments are often insufficient to completely remove pathogenic microorganisms from these pockets; therefore, mechanical removal is frequently combined with antibiotic therapy. However, in the early stages of periodontitis, bacteria aggregate to form plaque biofilms, increasing their resistance to antibiotics by 1000 times. Antibiotics not only struggle to penetrate the lesions but also easily induce bacterial resistance, leading to an imbalance in the oral microbiome and potentially triggering systemic side effects.

[0004] The local delivery of gaseous signaling molecules using nanotechnology has become a new direction in the treatment of bacterial-associated infections. Commonly used gaseous molecules include nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S), which play crucial roles in regulating human physiological and pathological processes. Among them, CO exhibits excellent activity in promoting tissue regeneration, immunomodulation, and anti-inflammatory and antibacterial effects. Two types of CO-releasing molecules have been reported in related technologies: one is metal carbonyl compounds, which can efficiently release CO and have good broad-spectrum antibacterial activity, but they are highly toxic and their antibacterial mechanism is unclear; the second is 3-hydroxyflavone derivatives, which have better biocompatibility and can efficiently and rapidly release CO under light and self-report CO release. However, nanoparticles composed of these compounds can only selectively bind to Gram-positive bacteria, which greatly limits their efficacy in periodontitis, where Gram-negative bacteria are the main pathogens. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide an amphiphilic functionalized D-configuration-alanine, its preparation method and application, and a self-targeting carbon monoxide nanogenerator, its preparation method and application. The amphiphilic functionalized D-configuration-alanine of this invention can specifically eliminate Gram-positive and Gram-negative bacteria.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides an amphiphilic functionalized D-configuration alanine (HBT) having the structure shown in Formula I:

[0008]

[0009] This invention also provides a method for preparing the amphiphilic functionalized D-configuration alanine described in the above technical solution, comprising the following steps:

[0010] 3-hydroxy-2-naphthoic acid was mixed with lithium methyl and subjected to an addition hydrolysis reaction to give compound 2;

[0011] Under alkaline conditions, compound 2, 4-methylbenzoic acid, and hydrogen peroxide were mixed and subjected to a cyclization reaction to obtain compound 3;

[0012] The compound 3 was mixed with BOC-D-alanine and coupled to obtain compound 4.

[0013] Compound 4 was mixed with trifluoroacetic acid and subjected to a BOC deprotection reaction to obtain the amphiphilic functionalized D-configuration alanine.

[0014] The structures of compounds 2, 3, and 4 are shown below:

[0015]

[0016] The present invention also provides the application of the amphiphilic functionalized D-configuration-alanine described in the above technical solution in the preparation of a self-targeted carbon monoxide nanogenerator.

[0017] The present invention also provides a self-targeted carbon monoxide nanogenerator, which is prepared by self-assembly of the amphiphilic functionalized D-configuration alanine and the amphiphilic surfactant described in the above technical solution.

[0018] Preferably, the amphiphilic surfactant comprises hexadecyltrimethylammonium bromide (CTAB).

[0019] Preferably, the mass ratio of the amphiphilic functionalized D-configuration alanine to the amphiphilic surfactant is 8-10:0-2.

[0020] Preferably, the mass ratio of the amphiphilic functionalized D-configuration alanine to the amphiphilic surfactant is 10:0, 9.5:0.5, 8:2, or 9:1.

[0021] This invention also provides a method for preparing the self-targeted carbon monoxide nanogenerator described in the above technical solution, comprising the following steps:

[0022] The amphiphilic functionalized D-configuration alanine, amphiphilic surfactant, and organic solvent are mixed to obtain a mixture.

[0023] The mixture is injected into water for self-assembly to obtain the self-targeted carbon monoxide nanogenerator.

[0024] Preferably, the self-assembly temperature is 40–60°C.

[0025] The present invention also provides the application of the self-targeting carbon monoxide nanogenerator described in the above technical solution in the preparation of drugs for treating periodontitis.

[0026] This invention provides an amphiphilic functionalized D-configuration alanine, and compared with the prior art, the beneficial effects of this invention are as follows:

[0027] Molecular orthogonal metabolic labeling technology utilizes the differences in metabolism between bacteria and mammalian cells to selectively fluorescently label bacteria. Among these, the D-alanine metabolic labeling strategy leverages the characteristic of alanine as a key component of bacterial peptidoglycan, specifically incorporating and labeling bacterial peptidoglycan with high selectivity, high biocompatibility, and wide applicability. However, because Gram-negative bacteria have an outer membrane surrounding their peptidoglycan layer, D-alanine has difficulty inserting into this layer. Hexadecyltrimethylammonium chloride, by utilizing the electrostatic attraction between its positively charged ammonium group and the negatively charged membrane component, can permeate the outer membrane of Gram-negative bacteria, thereby improving the labeling efficiency of D-alanine for Gram-negative bacteria. Therefore, the HBT provided in this invention, a 3-hydroxyflavone derivative, serves as the core of a CO nanogenerator. Addressing the challenge of its inability to target and kill Gram-negative bacteria, this invention modifies the permeability of the outer membrane of Gram-negative bacterial cell walls using metabolic labeling technology to construct a non-metallic CO nanogenerator with spectral antibacterial activity. This invention couples a 3-hydroxyflavonoid derivative with D-alanine to produce an amphiphilic functionalized D-configuration-alanine (HBT) with red fluorescence, which selectively targets bacteria and can specifically eliminate Gram-positive and Gram-negative bacteria.

[0028] This invention also provides a self-targeted carbon monoxide nanogenerator that, by altering the permeability of the outer membrane of Gram-negative bacterial cell walls and combining it with molecular orthogonal metabolic labeling technology, can be specifically metabolized and utilized by Gram-positive and Gram-negative bacteria. Under light irradiation, it generates CO to kill bacteria and remove biofilms hidden deep in periodontal pockets that are difficult to access. At the same time, the 3-hydroxyflavone derivative HBT and its released CO can alleviate inflammation by inducing macrophages to polarize from the M1 phenotype to the M2 phenotype, thereby promoting alveolar bone regeneration and avoiding damage to normal tissues. This achieves comprehensive treatment of periodontitis and is a novel treatment system that integrates antibacterial, anti-inflammatory, and bone-promoting effects, applicable to the treatment of periodontitis.

[0029] Data from the embodiments show that the self-targeting carbon monoxide nanogenerator provided by the present invention can generate a large amount of CO under white light irradiation, which can efficiently and safely remove biofilm in periodontal pockets, reduce inflammation and inhibit alveolar bone destruction. Attached Figure Description

[0030] Figure 1 Schematic diagram of a self-targeting carbon monoxide nanogenerator used for the treatment of periodontitis; Figure 2 For HBT 1 H NMR spectrum; Figure 3 (a) shows the diameter distribution of self-targeted carbon monoxide nanogenerators composed of different ratios of CTAB and HBT; (b) shows the transmission electron microscope images of self-targeted carbon monoxide nanogenerators composed of different ratios of CTAB and HBT (the scale bars in the figures are all 50 nm); (c) to (d) show the red fluorescence intensity of Staphylococcus aureus and Porphyromonas gingivalis at 618 nm and the growth curve of bacteria at 600 nm after co-culturing with self-targeted carbon monoxide nanogenerators composed of different ratios of CTAB and HBT, respectively; (e) shows the zeta potential before and after irradiation with CTAB / nHBT (CTAB is 10 wt%); (f) to (g) show the fluorescence spectrum and ultraviolet-visible absorption spectrum of HBT and CTAB / nHBT (CTAB is 10 wt%), respectively. Figure 4 (a) shows a schematic diagram of HBT releasing CO under hypoxic conditions and visible light irradiation; (b) shows the change of the UV-Vis spectrum of HBT with irradiation time under visible light irradiation; (c) shows the change of CO release from HBT with irradiation time under visible light irradiation; and (d) shows a confocal image of macrophages treated with PBS, HBT, CTAB / nHBT, and CTAB / nHBT loaded with FITC protein. Figure 5 (a) is a CLSM micrograph of the Porphyromonas gingivalis biofilm after experimental treatment; (b) is a semi-quantitative analysis of red and green fluorescence in CLSM imaging; (c) is the residual biofilm; (d) is the thickness of the Porphyromonas gingivalis biofilm in CLSM imaging; (e) is the biofilm biomass in the CV staining test; and (f) is the number of CFUs extracted per unit area from the Porphyromonas gingivalis biofilm. Figure 6 (a) to (c) show the levels of TNF-α, IL-6, and IL-10 in the supernatant of RAW 264.7 cells treated with different methods as determined by ELISA Kit; (d) shows the percentage of m2-like macrophages in the co-culture system; and (e) to (i) show representative flow cytometry data of m1-related markers (CD286) and m2-related markers (CD206) after different treatments. Figure 7 (a) to (c) are periodontal clinical indicators of rats after different treatments (negative control group, positive control group, CTAB / nHBT light-illuminated group, CTAB / nHBT non-light-illuminated group), (d) is the colony count of gingival crevicular fluid of rats after different treatments, (e) is the micro-CT image of the left maxillary second molar of rats after different treatments, and (f) to (i) are the corresponding bone-related indicators; Figure 8 (a) shows Masson staining images of rat periodontal tissues after different treatments, (b) shows HE staining images of rat periodontal tissues in each group, and (c) to (f) show the levels of TNF-α, IL-6, IL-10 and IL-4 in periodontal tissues after different treatments as determined by ELISA-Kit. Figure 9 (a) shows the change in body weight of rats in different groups after administration over time; (b) to (f) show the blood analysis of rats in the normal group after administration; (g) shows the cytotoxicity of HBT and CTAB / nHBT under dark and light conditions evaluated by CCK-8; and (h) shows the hemolysis experiment of different concentrations of CTAB / nHBT and representative photos. Figure 10 This diagram illustrates the mechanism by which 3-hydroxyflavone derivatives release CO under light exposure in different oxygen environments. Detailed Implementation

[0031] This invention provides an amphiphilic functionalized D-configuration alanine (HBT) having the structure shown in Formula I:

[0032]

[0033] Figure 10 The diagram illustrates the mechanism of CO release from 3-hydroxyflavone derivatives under light irradiation in different oxygen environments. It shows that under light irradiation, 3-hydroxyflavone derivatives can be oxidized by oxygen or singlet oxygen in an oxygen-rich environment, releasing CO. In an oxygen-deficient environment, the structure of 3-hydroxyflavone derivatives changes under light irradiation and further decomposes, releasing CO.

[0034] This invention also provides a method for preparing the amphiphilic functionalized D-configuration alanine described in the above technical solution, comprising the following steps:

[0035] 3-hydroxy-2-naphthoic acid was mixed with lithium methyl and subjected to an addition hydrolysis reaction to give compound 2;

[0036] Under alkaline conditions, compound 2, 4-methylbenzoic acid, and hydrogen peroxide were mixed and subjected to a cyclization reaction to obtain compound 3;

[0037] The compound 3 was mixed with BOC-D-alanine and coupled to obtain compound 4.

[0038] Compound 4 was mixed with trifluoroacetic acid and subjected to a BOC deprotection reaction to obtain the amphiphilic functionalized D-configuration alanine.

[0039] The structures of compounds 2, 3, and 4 are shown below:

[0040]

[0041] Unless otherwise specified, all raw materials used in this invention are commercially available products in the field.

[0042] In this invention, 3-hydroxy-2-naphthoic acid (compound 1) is mixed with methyllithium and subjected to an addition hydrolysis reaction to obtain compound 2 (1-(3-hydroxynaphtho-2-yl)acetone).

[0043] In this invention, the preferred mass ratio of compound 1 to methyllithium is 2:0.7015.

[0044] In this invention, the temperature of the addition hydrolysis reaction is preferably 0°C, and the time is preferably 3 hours; the addition hydrolysis reaction is preferably carried out under a protective atmosphere, which is preferably N2.

[0045] In a specific embodiment of the present invention, hydroxy-2-naphthoic acid is preferably dissolved in ultra-dry tetrahydrofuran (THF) at 0°C under N2 environment and stirred for 30 min. Methyllithium (tetrahydrofuran solvent) is added and stirred at 0°C for 3 h. The reaction is then quenched with 0.5 M hydrochloric acid. The resulting addition hydrolysis product is subjected to vacuum distillation to remove THF. The residue is diluted with distilled water and then extracted three times with dichloromethane. The resulting organic layer is collected, dried with anhydrous sodium sulfate, filtered, and the solution is removed by vacuum distillation to obtain crude compound 2. Then, it is purified by gradient silica gel column chromatography to obtain a yellow powder, namely compound 2. The eluent used in the silica gel column chromatography is a mixture of petroleum ether and ethyl acetate, wherein the volume ratio of petroleum ether to ethyl acetate in the mixture is 10:1.

[0046] After obtaining compound 2, the present invention carries out a cyclization reaction by mixing compound 2, 4-methylbenzoic acid and hydrogen peroxide under alkaline conditions to obtain compound 3 (4-(3-hydroxy-4-oxo-4H-benzo[g]chromium-2-yl)benzoic acid).

[0047] In this invention, the alkaline condition is preferably an inorganic alkaline solution, the inorganic alkaline solution is preferably a sodium hydroxide solution, and the concentration of the sodium hydroxide solution is preferably 5 mol / L.

[0048] In this invention, the hydrogen peroxide is preferably used in the form of a hydrogen peroxide solution, wherein the volume percentage of hydrogen peroxide in the hydrogen peroxide solution is preferably 30%.

[0049] In this invention, the preferred ratio of the amount of compound 2,4-methylbenzoic acid and hydrogen peroxide solution is 1g:0.8068g:5.00mL.

[0050] In this invention, the preferred temperature for the cyclization reaction is 0°C, and the preferred time is 8–12 h.

[0051] In a specific embodiment of the present invention, preferably, compound 2 is dissolved in ethanol, sodium hydroxide solution is added, and the mixture is stirred for 30 min. Then, 4-methylbenzoic acid is added, and the mixture is stirred overnight. The resulting mixture is cooled to 0°C in an ice bath, and hydrogen peroxide solution is added dropwise. The mixture is stirred overnight, and the resulting mixture is acidified with 0.5 mol / L hydrochloric acid to pH 6.5, producing a bright yellow precipitate. Finally, the bright yellow precipitate is filtered and washed with cold ethanol to obtain compound 3.

[0052] After obtaining compound 3, the present invention performs a coupling reaction between compound 3 and BOC-D-alanine to obtain compound 4 (tert-butyl(R)-2-((tert-butyloxycarbonyl)amino)-3-(4-(3-hydroxy-4-oxo-4H-benzo[g]chrom-2-yl)benzamide)propionic acid).

[0053] In this invention, the preferred mass ratio of compound 3 to BOC-D-alanine is 395.2:400.

[0054] In this invention, the coupling reaction is preferably carried out at 0°C and for 8 to 12 hours; the coupling reaction is preferably carried out under a protective atmosphere, preferably N2.

[0055] In this invention, the coupling reaction is preferably carried out in a mixed solution of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU)-1-hydroxy-7-azabenzotriazole (HoAt)-N,N-diisopropylethylamine (DIEA), and the solvent of the mixed solution of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate-1-hydroxy-7-azabenzotriazole-N,N-diisopropylethylamine is preferably ultra-dry dichloromethane.

[0056] In a specific embodiment of the present invention, preferably, the BOC-D-alanine is mixed with a mixed solution of compound 3 and HATU-HoAt-DIEA at 0°C under N2 protection to carry out the coupling reaction, stirred overnight, and distilled water is added at 0°C to produce a bright yellow precipitate. The bright yellow precipitate is then filtered and washed with ice-cold distilled water to obtain compound 4.

[0057] After obtaining compound 4, the present invention mixes compound 4 with trifluoroacetic acid to carry out boc deprotection reaction to obtain the amphiphilic functionalized D-configuration-alanine.

[0058] In this invention, the preferred ratio of compound 4 to trifluoroacetic acid (TFA) is 400 mg: 5 mL.

[0059] In this invention, the boc deprotection reaction is preferably carried out in dichloromethane (DCM), and the ratio of compound 4 to dichloromethane is preferably 400 mg: 5 mL.

[0060] In a specific embodiment of the present invention, it is preferable to dissolve compound 4 in dichloromethane, add trifluoroacetic acid (TFA), stir for 1 h, then remove dichloromethane by vacuum distillation, add the resulting residual solution to ice-cold ether to produce a yellow precipitate, and collect the final yellow-brown product by centrifugation to obtain the HBT.

[0061] The present invention also provides the application of the amphiphilic functionalized D-configuration-alanine described in the above technical solution in the preparation of a self-targeted carbon monoxide nanogenerator.

[0062] The present invention also provides a self-targeted carbon monoxide nanogenerator, which is prepared by self-assembly of the amphiphilic functionalized D-configuration alanine and the amphiphilic surfactant described in the above technical solution.

[0063] In this invention, the amphiphilic surfactant includes hexadecyltrimethylammonium bromide (CTAB).

[0064] In this invention, the mass ratio of the amphiphilic functionalized D-configuration alanine to the amphiphilic surfactant is 8-10:0-2, specifically 10:0, 9.5:0.5, 8:2 or 9:1. The use of a low dose of amphiphilic surfactant is for safety. On the basis of ensuring safety, a suitable ratio is found that can play the role of a permeabilizing agent for the outer membrane of Gram-negative bacteria.

[0065] This invention also provides a method for preparing the self-targeted carbon monoxide nanogenerator described in the above technical solution, comprising the following steps:

[0066] The amphiphilic functionalized D-configuration alanine, amphiphilic surfactant, and organic solvent are mixed to obtain a mixture.

[0067] The mixture is injected into water for self-assembly to obtain the self-targeted carbon monoxide nanogenerator.

[0068] In this invention, the self-assembly temperature is preferably 40 to 60°C, specifically 40, 50 or 60°C.

[0069] In this invention, the organic solvent is preferably tetrahydrofuran. This invention does not have a special limitation on the amount of the organic solvent used, as long as it can ensure the dissolution of the amphiphilic functionalized D-configuration-alanine and the amphiphilic surfactant.

[0070] In a specific embodiment of the present invention, HBT and an amphiphilic surfactant are preferably dissolved in tetrahydrofuran, the resulting mixture is injected into deionized water, stirred to carry out the self-assembly, and then dialyzed to remove the tetrahydrofuran to obtain the self-targeted carbon monoxide nanogenerator.

[0071] The present invention also provides the application of the self-targeting carbon monoxide nanogenerator described in the above technical solution in the preparation of drugs for treating periodontitis.

[0072] The present invention does not impose any special limitation on the specific method of application, and any method known to those skilled in the art can be used.

[0073] In this invention, when the self-targeting carbon monoxide nanogenerator is applied to the treatment of periodontitis, it is preferably used in conjunction with light irradiation, preferably white light irradiation, and the intensity of the white light irradiation is preferably 28 mW / cm². -2 The preferred time is 5 minutes.

[0074] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0075] Unless otherwise specified in the embodiments and accompanying drawings of this invention, CTAB / nHBT refers to a self-targeted carbon monoxide nanogenerator formed by a mass ratio of HBT to CTAB of 9:1.

[0076] The principle for preparing amphiphilic functionalized D-configuration -alanine in this embodiment of the invention is shown in the following formula:

[0077]

[0078] Example 1

[0079] A method for preparing amphiphilic functionalized D-configuration alanine (HBT) includes the following steps:

[0080] Hydroxy-2-naphthoic acid (compound 1, 2 g) was dissolved in ultra-dry tetrahydrofuran (45 mL) at 0 °C under N2 environment and stirred for 30 min. Methyllithium (701.5 mg, tetrahydrofuran solvent, 33.88 mL) was added, and the mixture was stirred at 0 °C for 3 h. The reaction was quenched dropwise with 0.5 mol / L hydrochloric acid (25 mL). THF was removed by vacuum distillation. The residue was diluted with distilled water (25 mL), extracted with dichloromethane (25 mL × 3), and the resulting organic layer was collected, dried over anhydrous sodium sulfate, and filtered off. The solution was then vacuum distilled to remove dichloromethane, yielding 1-(3-hydroxynaphthyl-2-yl)acetone (crude compound 2). The solution was purified by gradient silica gel column chromatography (eluent: petroleum ether: ethyl acetate (v / v)) to give 1.73 g of yellow powder (compound 2, 93%).

[0081] Compound 2 (1 g) was dissolved in ethanol (10 mL), and sodium hydroxide (5.37 mL, 5 mol / L) was added. The mixture was stirred for 30 min. 4-Methylbenzoic acid (806.8 mg) was added, and the mixture was stirred overnight. The mixture was cooled to 0 °C in an ice bath, and hydrogen peroxide solution (5 mL, 30%, v / v) was added dropwise. The mixture was stirred overnight, and the mixture was acidified with 0.5 mol / L hydrochloric acid to pH 6.5, producing a bright yellow precipitate. Finally, the bright yellow precipitate was filtered and washed with cold ethanol to give 1.37 g of 4-(3-hydroxy-4-oxo-4H-benzo[g]chrom-2-yl)benzoic acid (compound 3, 77%).

[0082] BOC-D-alanine (395.2 mg) was reacted with a mixed solution of compound 3 (460 mg), HATU (577.6 mg), HoAt (206.9 mg), and DIEA (356.7 mg) (solvent: ultra-dry dichloromethane, 20 mL) at 0 °C under N2 protection. The mixture was stirred overnight, and distilled water was added at 0 °C, producing a bright yellow precipitate. The precipitate was then filtered and washed with ice-cold distilled water (50 mL) to give tert-butyl(R)-2-((tert-butyloxycarbonyl)amino)-3-(4-(3-hydroxy-4-oxo-4H-benzo[g]chrom-2-yl)benzamide)propionic acid (compound 4, 669 mg, 84%).

[0083] Compound 4 (400 mg) was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (5 mL) was added. The mixture was stirred for 1 h, and the dichloromethane was removed by vacuum distillation. The remaining solution was added to ice-cold diethyl ether, which produced a yellow precipitate. The final yellow-brown product was collected by centrifugation to obtain the target compound HBT (327 mg, 90%).

[0084] Preparation of self-targeted carbon monoxide nanogenerator: HBT and CTAB (HBT to CTAB mass ratio 9:1) were dissolved in tetrahydrofuran, and the resulting mixture was injected into deionized water. The mixture was stirred at 50°C to form CTAB / nHBT, where CTAB was 10 wt%. Then, tetrahydrofuran was removed by dialysis.

[0085] Figure 1 The diagram below shows the self-targeted carbon monoxide nanogenerator used in the treatment of periodontitis in Example 1. (a) is a flowchart of the self-assembly of HBT and CTAB to form a nano-carbon monoxide generator, and (b) is a schematic diagram of the self-targeted nano-carbon monoxide generator eradicating Porphyromonas gingivalis biofilm, regulating periodontal immune response and alveolar bone regeneration. In (b), a) it shows that CTAB / nHBT is non-fluorescent and non-toxic; b) it shows that CTAB / nHBT can penetrate biofilms and selectively target bacteria; c) it shows that CTAB / nHBT can regulate macrophage polarization, relieve inflammation, and further promote alveolar bone regeneration; d) it shows that photostimulation can enable CTAB / nHBT to generate CO and eradicate biofilms.

[0086] Figure 2 For HBT 1 The HNMR spectrum indicates that HBT was successfully prepared.

[0087] Self-targeted carbon monoxide nanogenerators formed by different ratios of CTAB and HBT: HBT and CTAB (mass ratios of HBT to CTAB were 10:0, 9.5:0.5, 9:1, and 8:2, respectively) were dissolved in tetrahydrofuran. The resulting mixture was injected into deionized water and stirred at 50°C to allow for self-assembly. Then, the tetrahydrofuran was removed by dialysis to obtain self-targeted carbon monoxide nanogenerators formed by different ratios of CTAB and HBT, denoted as nHBT, CTAB / nHBT (5wt%), and CTAB / nHBT (20wt%). nHBT refers to a mass ratio of HBT to CTAB of 10:0, CTAB / nHBT (5wt%) refers to a mass ratio of HBT to CTAB of 9.5:5, and CTAB / nHBT (20wt%) refers to a mass ratio of HBT to CTAB of 8:2.

[0088] Self-targeting carbon monoxide nanogenerators formed by different ratios of CTAB and HBT were subjected to dynamic light scattering (DLS) measurements using a Malvern Instruments Nano-ZS microscope to determine their diameter and zeta potential of CTAB / nHBT (10 wt%) in the dark and under 5 min illumination conditions. Cryo-TEM images were obtained using a Talos L120C G2 microscope. A fluorescence spectrophotometer was used to detect wavelengths of 500–700 nm (excitation wavelength 418 nm), and a UV spectrophotometer was used to detect wavelengths of 310–540 nm. Gram-positive Staphylococcus aureus and Gram-negative Porphyromonas gingivalis were co-cultured with different nano-carbon monoxide generators, and the red fluorescence intensity at 618 nm and the growth curves of the bacteria at 600 nm were measured within 12 h.

[0089] Figure 3 (a) shows the diameter distribution of self-targeted carbon monoxide nanogenerators composed of different ratios of CTAB and HBT (CTAB contents of 0 wt%, 5 wt%, 10 wt%, and 20 wt%). (b) shows transmission electron microscopy images of the self-targeted carbon monoxide nanogenerators composed of different ratios of CTAB and HBT (scale bars are 50 nm in all figures). (c) to (d) show the red fluorescence intensity of Staphylococcus aureus and Porphyromonas gingivalis at 618 nm and the growth curve of bacteria at 600 nm after co-culturing with the self-targeted carbon monoxide nanogenerators composed of different ratios of CTAB and HBT, respectively. (e) shows the zeta potential before and after irradiation with CTAB / nHBT (10 wt% CTAB). (f) to (g) show the fluorescence spectrum and UV-Vis absorption spectrum of HBT and CTAB / nHBT (10 wt% CTAB), respectively. It can be concluded that the self-targeted carbon monoxide nanogenerators composed of different ratios of CTAB and HBT have small and relatively uniform particle sizes, exhibiting a uniform spherical shape. The presence or absence of CTAB in the nano-carbon monoxide generator did not significantly affect the penetration effect on Gram-positive bacteria, while only nano-carbon monoxide generators carrying at least 10 μM CTAB could penetrate the outer membrane of Gram-negative bacteria and perform labeling. The surface charge of CTAB / nHBT remained positive both before and after visible light irradiation. Furthermore, the fluorescence emission intensity of CTAB / nHBT decreased by 87%, and the maximum emission wavelength blue-shifted to 581 nm. The UV absorption peak of CTAB / nHBT blue-shifted to 412 nm, and the absorption intensity decreased by 24%. This shift is attributed to the combined effects of aggregation-induced quenching (ACQ) and excited-state intramolecular proton transfer (ESIPT), which are crucial for enhancing photostability and reducing phototoxicity.

[0090] FITC, HBT and CTAB (mass ratio 5:9:1) were dissolved in tetrahydrofuran, injected into deionized water, and stirred moderately at 50°C to form FITC-CTAB / nHBT. Tetrahydrofuran was then removed by dialysis.

[0091] HBT was dissolved in DMSO to a concentration of 100 μM. Changes in the UV-Vis spectrum over time after HBT stimulation were detected. Using an HBT standard curve, these changes in the UV-Vis spectrum were quantified as CO2 release. Mouse RAW264.7 cells were injected with 1×10⁻⁶ cells... 5 Cells were seeded at a density of 1 / 2 well in 24-well plates. Once the cells reached 70%–80% confluence, 100 μL of PBS, HBT, CTAB / nHBT, and FITC-CTAB / nHBT were added respectively, and incubated for 12 h. Cells were washed three times with PBS and fixed for 10 min with commercially available paraformaldehyde fixative (4% by volume). After removing the fixative and washing three times with PBS, cells were stained with 5 mg / L DAPI solution for 5 min. After removing the solution, cells were washed twice with PBS. Cell uptake was observed using a confocal microscope. HBT showed red fluorescence, FITC showed green fluorescence, and DAPI stained macrophage nuclei showed blue fluorescence.

[0092] Figure 4 (a) shows a schematic diagram of CO release from HBT under anaerobic conditions and visible light irradiation; (b) shows the change in the UV-Vis spectrum of HBT with irradiation time under visible light irradiation; (c) shows the change in CO release from HBT with irradiation time under visible light irradiation; and (d) shows confocal imaging of macrophages treated with PBS, HBT, CTAB / nHBT, and CTAB / nHBT loaded with FITC protein. The conclusion is that HBT undergoes visible light-induced rearrangement under anaerobic conditions, leading to CO release. In an anaerobic environment, the absorbance of HBT decreases and CO release increases with increasing visible light irradiation time. Both HBT and FITC-CTAB / nHBT can enter macrophages and emit strong fluorescence, while only weak red fluorescence is observed in macrophages co-incubated with CTAB / nHBT, indicating that CTAB / nHBT cannot be specifically utilized by mammalian cells, and mammalian cells cannot metabolize d-alanine derivatives.

[0093] Gram-negative Porphyromonas gingivalis preserved at -80℃ was inoculated onto TSA plates containing 5 wt% sheep blood and incubated upside down at 37℃ for 5 days until black colonies appeared. The experimental strains that had been incubated overnight were picked and inoculated into BHI liquid medium and incubated at 37℃ for 24 hours.

[0094] Adjust the suspension concentration to 1×10 8CFU / mL, take 1 mL of bacterial suspension into two confocal microplates, incubate for 1 h, change the medium and continue culturing for 36 h, discard the culture medium, add 1 mL of culture medium containing PBS and 1 mL of culture medium containing CTAB / nHBT (50 μM), and continue culturing for 12 h. After culturing, wash the biofilm twice with PBS, add 1.5 μL of green fluorescent dye (SYTO 9), stain for 15 min, wash twice with PBS, and take confocal images.

[0095] Adjust the suspension concentration to 1×10 8 CFU / mL, 1 mL of bacterial suspension was taken and placed in three confocal microplates respectively. The plates were incubated for 1 h, the medium was changed, and the plates were cultured for another 36 h. The culture medium was discarded, and 1 mL each of PBS-containing, CTAB / nHBT-containing (50 μM), and CTAB / nHBT-containing (100 μM) culture medium were added. The plates were cultured for another 12 h. After the culture, the biofilm was washed twice with PBS, and 1.5 μL of green fluorescent dye (SYTO 9) was added and stained for 15 min. The plates were washed twice with PBS, and confocal microscopy images were taken. After 10 min of illumination, confocal microscopy images were taken again. The biofilm residue in each treatment group was then measured.

[0096] Adjust the suspension concentration to 1×10 8 CFU / mL was used to culture biofilms in 48-well plates for 36 h. After washing with PBS, culture medium containing PBS, CTAB / nHBT medium (50 μM), and CTAB / nHBT medium (100 μM) were added, and the biofilms were cultured for another 12 h under light / no light conditions. The biofilms were washed with PBS. The biofilms were stained with 0.1 wt% crystal violet solution for 15 min. After washing three times, the dye was extracted with 30 wt% acetic acid for 10 min. Finally, the extract was transferred to a 96-well plate, and the OD value was read at 660 nm using a microplate reader (n=3).

[0097] Adjust the suspension concentration to 1×10 8 CFU / mL was used to culture biofilms in 48-well plates for 36 h. After washing with PBS, culture medium containing PBS, CTAB / nHBT medium (50 μM), and CTAB / nHBT medium (100 μM) were added respectively, and culture was continued for 12 h under light / no light conditions. The biofilms were washed with PBS and serially diluted to be planted on 5 wt% sheep blood TSA plates for colony counting.

[0098] Figure 5(a) is a CLSM micrograph of the *Porphyromonas gingivalis* biofilm after experimental treatment; (b) is a semi-quantitative analysis of red-green fluorescence in CLSM imaging; (c) is the residual biofilm; (d) is the thickness of the *Porphyromonas gingivalis* biofilm in CLSM imaging; (e) is the biofilm biomass in the CV staining test; and (f) is the number of CFUs extracted per unit area from the *Porphyromonas gingivalis* biofilm. The conclusions are: positive surface charge facilitates the targeting and accumulation of CTAB / nHBT in the biofilm; CTAB / nHBT can be metabolized by the *Porphyromonas gingivalis* biofilm. After 10 min of visible light irradiation, the integrity of the biofilm was disrupted, the biofilm thickness decreased significantly, and the number of biofilm colonies decreased significantly. Crystal violet staining also showed that after light irradiation, the biofilm biomass of *Porphyromonas gingivalis* in the CTAB / nHBT group was significantly reduced.

[0099] RAW 264.7 cells were cultured in 24-well plates (2 × 10⁶ cells per well). 5 The cells were cultured in a culture medium (10 ng / mL) for 24 h. Lipopolysaccharide (LPS) was then added to the medium. -1 RAW264.7 cells were induced to transform into the M1 subtype by inducing them at 37℃ for 24 h. Groups were then treated with PBS-containing medium and HBT-containing medium (DMSO, 50 μM), under either light or no light exposure. An ELISA kit was used to detect that HBT light exposure inhibited the pro-inflammatory cytokines TNF-α and IL-6, and induced the secretion of the anti-inflammatory cytokine IL-10.

[0100] RAW 264.7 cells were cultured in 24-well plates (2 × 10⁶ cells per well). 5 Incubate in a culture medium containing 10 ng / mL cells for 24 hours. Add LPS (10 ng / mL). -1 M1-related markers (CD286) and M2-related markers (CD206) were added, and the cells were cultured for another 24 hours. The medium was changed, and PBS-containing medium and HBT-containing medium (DMSO for dissolution, 50 μM) were added respectively. The cells were cultured under light and no light. Flow cytometry analysis showed that CTAB / nHBT could regulate macrophage polarization and increase the proportion of M2-like macrophages.

[0101] Figure 6Images (a)–(c) show the levels of TNF-α, IL-6, and IL-10 in the supernatant of RAW264.7 cell culture medium treated with different methods, as determined using an ELISA Kit. Image (d) shows the percentage of m2-like macrophages in the co-culture system. Images (e)–(i) show representative flow cytometry data of m1-related markers (CD286) and m2-related markers (CD206) after different treatments. The conclusion is that HBT can exert its physiological function by inhibiting the expression of pro-inflammatory factors TNG-α and IL-6, promoting the expression of the anti-inflammatory factor IL-10, and facilitating the transformation of macrophages from the M1 phenotype to the M2 phenotype, thus effectively alleviating inflammation.

[0102] Twenty-four six-week-old SD rats were randomly divided into four groups of six after one week of acclimatization. Three groups were used to establish a periodontitis model. Under isoflurane anesthesia, the rats' mouths were opened, and 0.22 mm orthodontic ligatures were tied to the bilateral maxillary second molars. The remaining group received no treatment and served as the negative control. The ligatures were checked and weighed periodically after model establishment. The ligatures were removed after three weeks. The negative control group was injected with PBS, while the other three groups were injected with PBS, CTAB / nHBT, and CTAB / nHBT, respectively, serving as the positive control, CTAB / nHBT light-exposed group, and CTAB / nHBT non-light-exposed group. Each rat received 100 μL of PBS twice weekly for three weeks. The CTAB / nHBT light-exposed group was exposed to light for 5 minutes 6 hours after administration.

[0103] After treatment, periodontal clinical indicators such as periodontal probing depth, attachment loss, and gingival bleeding index were recorded in rats. Gingival crevice fluid from both maxillary second molars of rats was collected using a No. 25 absorbent paper tip for 30 seconds and placed in 100 μL PBS. 10 μL of this fluid was used for plate colony counting. Blood samples from normal group rats were collected for analysis of red blood cells (RBC), hemoglobin (HGB), red blood cell specific volume (HCT), and platelets (PLT).

[0104] Rats were euthanized, and the maxilla was removed and fixed in paraformaldehyde fixative (4% by volume, commercially available) for 24 hours. After fixation, alveolar bone resorption was analyzed using micro-CT. Three-dimensional digital and tomographic images of the alveolar bone were reconstructed using CT Vox and Data Viewer software, and the distance between the alveolar ridge (ABC) and the enamel-pulp junction (CEJ) was measured. Bone mineral density (BMD), relative bone volume (BV / TV), and trabecular bone thickness (Tb.Th) around the ligated molars were calculated using CTAn software.

[0105] Rats were euthanized, and periodontal tissues from the treatment sites were fixed in paraformaldehyde fixative (4% by volume, commercially available) and embedded in paraffin. Hematoxylin and eosin (H&E) staining and Masson's trichrome staining were used to further assess the inflammatory status of the periodontal tissues. The stained sections were observed at 100x and 400x magnification using an optical microscope (Olympus, Tokyo, Japan) to detect the number of inflammatory cells and assess collagen degradation. The levels of TNF-α, IL-6, IL-10, and IL-4 in periodontal tissues after different treatments were measured using an ELISA kit.

[0106] Figure 7 Images (a)–(c) show periodontal clinical indices in rats after different treatments (negative control group, positive control group, CTAB / nHBT light-treated group, and CTAB / nHBT non-light-treated group); image (d) shows the colony count in the gingival crevicular fluid of rats after different treatments; image (e) shows the micro-CT image of the left maxillary second molar of rats after different treatments; and images (f)–(i) show the corresponding bone-related indices. The conclusion is that after 3 weeks of CTAB / nHBT injection treatment, the relevant periodontal clinical indices in rats with periodontitis improved, with more significant improvement in the light-treated group. CTAB / nHBT treatment significantly improved alveolar bone parameters (BV / TV, Tb.Th, BMD, and ABC-CEJ distance) at the lesion site in rats, indicating that CTAB / nHBT can promote osteogenesis.

[0107] Figure 8 (a) shows Masson staining images of periodontal tissues from rats after different treatments; (b) shows HE staining images of periodontal tissues from each group; (c)–(f) show the levels of TNF-α, IL-6, IL-10, and IL-4 in periodontal tissues after different treatments, measured using an ELISA kit. The conclusions are as follows: The NS group experienced significant collagen loss, while CTAB / nHBT treatment enhanced collagen deposition; inflammatory cells were clearly present in the NS group, and CTAB / nHBT treatment improved these abnormalities, especially after visible light irradiation, which significantly reduced inflammatory cells. The expression levels of IL-6 and TNF-α decreased in the CTAB / nHBT group, while the expression levels of IL-4 and IL-10 increased. This indicates that CTAB / nHBT has a good anti-inflammatory effect.

[0108] RAW264.7 cells were cultured in 96-well plates at a density of 5000 cells per well. Once the cells reached 80% confluence, they were divided into five groups. Two groups were incubated with CTAB / nHBT medium (2 μM, 4 μM, 8 μM, 16 μM, 32 μM, 64 μM, 128 μM, 256 μM, and 512 μM), one under light and the other without. Two other groups were incubated with HBT medium (2 μM, 4 μM, 8 μM, 16 μM, 32 μM, 64 μM, 128 μM, 256 μM, and 512 μM), one under light and the other without. A third group was incubated with medium (n=3). All five groups were co-incubated for 24 h. Cells were washed with PBS, and then 20 μL of CK-8 solution was added to 200 μL of α-MEM in each plate. After incubation at 37°C for 2 hours, OD 450 was measured using a microplate reader.

[0109] Appropriate blood volumes were collected from SD rats and added to PBS at a 1:2 volume ratio. The mixture was thoroughly mixed and centrifuged multiple times until the supernatant was colorless and transparent, leaving red blood cells. Red blood cells were then added to PBS at a 1:9 volume ratio to obtain a 10 vol% red blood cell (RBC) suspension. The obtained RBC suspension was mixed with equal volumes of PBS and deionized water as negative and positive controls, respectively. Additionally, the RBC suspension was mixed with equal volumes of 16 μM, 32 μM, 64 μM, 128 μM, 256 μM, and 512 μM CTAB / nHBT solutions and incubated for 3 h. The resulting supernatant was collected by centrifugation to measure its optical density at 541 nm. The hemolysis rate of red blood cells in each group was calculated using the following formula: Hemolysis rate = (OD sample - OD negative) / (OD positive - OD negative).

[0110] Figure 9 (a) shows the change in body weight of rats in different groups after drug administration over time; (b)–(f) show the blood analysis of rats in the normal control group after drug administration; (g) shows the cytotoxicity of HBT and CTAB / nHBT under dark and light conditions evaluated by CCK-8; and (h) shows the hemolysis experiment and representative photographs of different concentrations of CTAB / nHBT. The conclusion is that CTAB / nHBT has no toxic side effects in vivo or in vitro and has good safety.

[0111] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An amphiphilic functionalized D-configuration alanine having the structure shown in Formula I:

2. The method for preparing amphiphilic functionalized D-configuration -alanine according to claim 1, characterized in that, Includes the following steps: 3-hydroxy-2-naphthoic acid was mixed with lithium methyl and subjected to an addition hydrolysis reaction to give compound 2; Under alkaline conditions, compound 2, 4-methylbenzoic acid, and hydrogen peroxide were mixed and subjected to a cyclization reaction to obtain compound 3; The compound 3 was mixed with BOC-D-alanine and coupled to obtain compound 4. Compound 4 was mixed with trifluoroacetic acid and subjected to a BOC deprotection reaction to obtain the amphiphilic functionalized D-configuration alanine. The structures of compounds 2, 3, and 4 are shown below:

3. The application of the amphiphilic functionalized D-configuration-alanine of claim 1 in the preparation of a self-targeted carbon monoxide nanogenerator.

4. A self-targeting carbon monoxide nanogenerator, characterized in that, It is prepared by self-assembly of the amphiphilic functionalized D-configuration alanine as described in claim 1 and an amphiphilic surfactant.

5. The self-targeting carbon monoxide nanogenerator according to claim 4, characterized in that, The amphiphilic surfactant includes hexadecyltrimethylammonium bromide.

6. The self-targeting carbon monoxide nanogenerator according to claim 4 or 5, characterized in that, The mass ratio of the amphiphilic functionalized D-configuration alanine to the amphiphilic surfactant is 8–10:0–2.

7. The self-targeting carbon monoxide nanogenerator according to claim 6, characterized in that, The mass ratio of the amphiphilic functionalized D-configuration alanine to the amphiphilic surfactant is 10:0, 9.5:0.5, 8:2, or 9:

1.

8. The method for preparing the self-targeted carbon monoxide nanogenerator according to any one of claims 4 to 7, characterized in that, Includes the following steps: The amphiphilic functionalized D-configuration alanine, amphiphilic surfactant, and organic solvent are mixed to obtain a mixture. The mixture is injected into water for self-assembly to obtain the self-targeted carbon monoxide nanogenerator.

9. The preparation method according to claim 8, characterized in that, The self-assembly temperature is 40–60°C.

10. The use of the self-targeting carbon monoxide nanogenerator according to any one of claims 4 to 7 in the preparation of drugs for treating periodontitis.

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