Preparation method and application of uracil modified gold nano conjugate
By preparing uracil-modified gold nanoconjugates with a particle size ≤2 nm, the problems of insufficient biofilm penetration and pathogen targeting in local treatment of periodontitis were solved, achieving highly efficient antibacterial and tissue repair effects.
Patent Information
- Application Number
- CN202610086770.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-22
- Publication Date
- 2026-02-17
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
Existing topical treatments for periodontitis have weak ability to penetrate biological membrane barriers, poor targeting of pathogens, and lack of virulence regulation, making it difficult to effectively fight bacteria while reducing tissue damage and blocking pathological processes.
Uracil-modified gold nanoparticles with a particle size ≤2 nm were prepared. Surface modification and pH responsiveness were achieved by binding uracil derivatives through Au-S bonds. The nanoparticles were purified by ultrafiltration centrifugation, dialysis, and polyacrylamide gel electrophoresis for use in the preparation of periodontitis treatment products.
It achieves deep biofilm penetration, selective enrichment of pathogens, significantly inhibits proton kinetic potential and virulence factor secretion, reduces inflammatory factor expression, promotes tissue repair, and has good biocompatibility.
Smart Images
Figure CN121534079A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biological medicine, and particularly relates to a preparation method of a uracil-modified gold nanocomposite and application thereof. BACKGROUND
[0002] Periodontitis is a common oral disease characterized by chronic inflammation and destruction of periodontal support tissue, which can not only lead to alveolar bone resorption and tooth loosening and shedding, but also is related to cardiovascular diseases, diabetes and other systemic diseases. At present, the clinical treatment mainly uses mechanical scaling, periodontal pocket irrigation and local or systemic application of antibacterial drugs to control periodontal pathogen load and block tissue destruction. In order to improve the effect of local treatment, various sustained-release local drug delivery systems have been developed in the prior art, such as fiber strips, films, gels and microspheres loaded with chlorhexidine, tetracycline or metronidazole antibacterial drugs by using high molecular materials such as chitosan and polylactic acid-glycolic acid copolymer, which can achieve drug release by being placed in the periodontal pocket, so as to maintain the local drug concentration and reduce the systemic side effects.
[0003] However, the existing local drug delivery system still has the following outstanding defects: first, periodontal pathogenic bacteria exist in the form of biofilm, and after the existing preparation releases the drug, the drug molecules are difficult to effectively penetrate the dense bacterial outer polysaccharide matrix, resulting in insufficient effective concentration in the pathogenic bacteria and limited antibacterial effect; second, the existing strategy relies on broad-spectrum bactericidal mechanism and lacks selective recognition and targeting ability for the main pathogenic bacteria of periodontitis, which can easily disturb the balance of bacterial flora, damage normal tissues and induce drug resistance; third, the existing system mostly only focuses on inhibiting bacterial growth and lacks effective regulation of the secretion of key virulence factors such as gingival protease and the corresponding metabolic pathways, so it cannot block the vicious cycle of "virulence secretion-tissue destruction-inflammation continuation".
[0004] In summary, the current local treatment products for periodontitis generally have the problems of weak biofilm penetration ability, poor pathogenic bacteria targeting and lack of virulence regulation, and it is difficult to effectively inhibit bacteria while reducing tissue damage and blocking the pathological process. Therefore, it is urgent to develop a new local treatment strategy that can effectively break through the biofilm barrier, selectively target pathogenic bacteria and inhibit their virulence secretion. SUMMARY
[0005] The purpose of the embodiments of the present application is to provide a preparation method of a uracil-modified gold nanocomposite and application thereof, which aims to solve the problems proposed in the background.
[0006] The embodiments of the present application are implemented in the following way: a preparation method of a uracil-modified gold nanocomposite, comprising the following steps: Step 1: controlled reduction of gold precursor; In the presence of a reducing agent, a uracil ligand solution was slowly added to a gold precursor solution to carry out a controlled reduction reaction, forming gold nanoconjugates with an average particle size ≤2 nm. Step 2: Surface modification and control; A thiolized uracil derivative was added to the dispersion of the gold nanoconjugated material obtained in step 1, and the pH of the reaction system was adjusted to alkaline so that the thiolized uracil derivative was bound to the surface of the gold nanoconjugated material through Au-S bonds, thereby obtaining uracil-modified gold nanoconjugated material.
[0007] In a further technical solution, in step 1, the molar ratio of uracil ligand in the uracil ligand solution to the gold precursor in the gold precursor solution is 2:1; the controlled reduction reaction is carried out under stirring at a reaction temperature of 26±0.5℃, and the uracil ligand solution is added by a syringe pump at a rate of 0.50 mL / s.
[0008] In a further technical solution, in step 2, the pH of the reaction system is adjusted to 11±0.1 to carry out the surface modification reaction.
[0009] Further technical solutions also include step 3: purification treatment; The product obtained in step 2 was purified to remove free ligands and unreacted precursors.
[0010] A further technical solution is that the purification process includes one or more of ultrafiltration centrifugation, dialysis, and polyacrylamide gel electrophoresis separation.
[0011] A further technical solution involves drying the obtained uracil-modified gold nanoparticles using a low-temperature freeze-drying process; the dried uracil-modified gold nanoparticles are then redispersed in a buffer solution or carrier system before use.
[0012] Another objective of this invention is to provide an application of a uracil-modified gold nanoconjugate, wherein the uracil-modified gold nanoconjugate prepared by the above method is used in the preparation of products for the treatment or prevention of periodontitis.
[0013] A further technical solution is that the product is used to inhibit the formation of periodontal pathogenic bacteria biofilm, interfere with bacterial proton dynamic potential (PMF), and inhibit the secretion of bacterial virulence factors.
[0014] In a further technical solution, the periodontal pathogen is *Porphyromonas gingivalis*.
[0015] The present invention provides a method for preparing uracil-modified gold nanoparticles and their applications, the beneficial effects of which are as follows: (1) Ultra-small and uniform size: particle size ≤2 nm, narrow distribution (PDI about 0.18), can penetrate the nanopores of biomembrane to achieve deep penetration.
[0016] (2) Stable and controllable surface function: Uracil is stably modified through Au-S bond, and has pH responsiveness, and can be protonated in a slightly acidic environment.
[0017] (3) The preparation process has good reproducibility: key parameters (ratio, pH, feeding rate) are precisely controllable and the batch-to-batch consistency is high (RSD 1–2%).
[0018] (4) Selective pathogen enrichment: It can be actively taken up by periodontal pathogens and its enrichment efficiency in bacteria is more than 200 times higher than that of normal cells, with low off-target damage.
[0019] (5) Dual interference with energy metabolism: Simultaneously dissipates bacterial transmembrane pH gradient and membrane potential, inhibits proton dynamic potential (PMF) by more than 90%, and blocks virulence secretion.
[0020] (5) Highly effective antibacterial and tissue repair: It significantly removes biofilm, inhibits bone resorption, reduces inflammatory factors, and promotes periodontal collagen fiber reconstruction.
[0021] (6) Excellent biocompatibility: high cell survival rate, no obvious toxicity observed in animals, suitable for local application. Attached Figure Description
[0022] Figure 1 This is a schematic diagram of uracil surface modification (where a represents a decrease in pH and b represents an increase in pH). Figure 2 A synthetic route for uracil-modified gold nanoconjugates is provided in this embodiment of the invention. Figure 3 Transmission electron microscopy (TEM) images of U-AuNCs (where a is a TEM image and b is a frequency map of the size distribution of U-AuNCs determined by TEM). Figure 4 The image shows the UV-Vis absorption spectrum of U-AuNCs. Figure 5 The Fourier transform infrared (FT-IR) spectrum of U-AuNCs is shown below. Figure 6 The results show the changes in Zeta potential of U-AuNCs under different pH conditions; Figure 7 The distribution of U-AuNCs in biofilms is shown (where a is a confocal image of U-AuNCs permeating the biofilm matrix, and b is a colocalization analysis of U-AuNCs and the biofilm matrix). Figure 8The distribution of U-AuNCs in pathogenic bacteria and host cells is compared (where a is a biological transmission electron microscopy image of bacteria treated with U-AuNCs, b is a confocal image of oral epithelial cells treated with U-AuNCs, and c is an ICP-MS statistical graph of the content of U-AuNCs-treated bacteria and oral epithelial cells). Figure 9 The results are for host cell viability testing (where a is the cell viability test result of fibroblasts at 1 / 3 day, and b is the cell viability test result of oral epithelial cells at 1 / 3 day). Figure 10 The results of the bacterial proton dynamic potential (PMF) inhibition effect detection are shown (where a is a statistical graph of the change in pH in bacteria after U-AuNCs treatment, and b is the bacterial PMF inhibition rate after U-AuNCs treatment). Figure 11 The results of the detection of changes in bacterial virulence factor secretion are shown in Figure 1 (where a is a statistical graph of the inhibition rate of bacterial virulence factor Rgp after U-AuNCs treatment, and b is a statistical graph of the inhibition rate of bacterial virulence factor Kgp after U-AuNCs treatment). Figure 12 The results are the quantitative analysis of bacterial viability and metabolic activity within the biofilm (where a is the quantitative analysis of bacterial viability within the biofilm, b is the quantitative analysis of biofilm disruption, and c is the analysis of biofilm metabolic activity). Figure 13 The time response results for PMF inhibition are shown (where a is the time response result of bacterial pH after U-AuNCs treatment, b is the time response result of bacterial membrane potential after U-AuNCs treatment, and c is the PMF inhibition rate of bacteria after U-AuNCs treatment). Figure 14 The results of the biofilm gingival protease activity assay are shown (where a is a statistical graph of the inhibition rate of the biofilm bacterial virulence factor Rgp, and b is a statistical graph of the inhibition rate of the biofilm bacterial virulence factor Kgp). Figure 15 The results are for detecting virulence secretion-related functions and the protective effect of the host epithelial barrier (where a is the immunofluorescence staining of the host epithelial barrier, b is the quantitative analysis of the immunofluorescence staining of the epithelial barrier protein ZO-1, and c is the quantitative analysis of the immunofluorescence staining of the epithelial barrier protein Occludin). Figure 16 The results of the Micro-CT bone loss analysis are as follows (where a is a schematic diagram of Micro-CT, b is a quantitative analysis of mesial buccal bone resorption, c is a quantitative analysis of mesial lingual bone resorption, d is a quantitative analysis of distal buccal bone resorption, e is a quantitative analysis of distal lingual bone resorption, f is Tb.Th analysis, and g is BV / TV analysis). Figure 17Histological staining results of periodontal tissues (where a is HE staining analysis and b is Masson staining analysis); Figure 18 The results are the immunofluorescence detection results of the expression of inflammation-related factors (where a is the immunofluorescence of the expression of inflammation-related factors, b is the quantitative analysis of IL-6, and c is the quantitative analysis of TNF-α). Figure 19 The results are histopathological examinations of major organs. Detailed Implementation
[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.
[0024] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.
[0025] like Figure 1 a, Figure 1 b and Figure 2 The image shows a method for preparing uracil-modified gold nanoconjugated materials according to an embodiment of the present invention, comprising the following steps: Step 1: Controlled reduction of gold precursor (forming ≤2 nm ultra-small gold nanoconjugates); The uracil derivative was dissolved in an alkaline aqueous solution (e.g., 0.2 mol / L NaOH), and the solution was vortexed and sonicated to obtain a clear and transparent ligand solution for later use. Chloroauric acid was dissolved in deionized water as a gold precursor solution, and the mixture was stirred under constant temperature conditions, preferably at 26 ± 0.5 °C, at a stirring rate of approximately 600 rpm.
[0026] The uracil ligand solution was added slowly to the gold precursor solution at a molar ratio of ligand to gold precursor of approximately 2:1 using a precision syringe pump at a rate of approximately 0.50 mL / s, achieving controlled reduction of gold ions in the presence of a reducing agent. By comprehensively adjusting the addition rate, ligand ratio, and mild reduction conditions, excessive particle growth could be suppressed, keeping the average particle size of the resulting gold nanoconjugated nanoparticles within an ultra-small range of no more than 2 nm. TEM revealed uniform, near-spherical particles, and UV-Vis spectra did not show characteristic absorption peaks (approximately 510–530 nm) associated with large-diameter gold nanoparticles.
[0027] Step 2: Surface modification and particle size / electrical regulation of uracil (formation of a stable functional layer); After the gold nanoconjugated material is formed, a thiolized uracil derivative is added to the dispersion of the gold nanoconjugated material. By adjusting the pH of the reaction system to a suitable alkaline range (such as pH 10-12, preferably 11±0.1), the uracil ligand is deprotonated, and the thiol group forms a stable Au–S bond with the gold surface, thereby achieving surface functionalization coverage.
[0028] By adjusting the molar ratio of uracil ligand to gold, the final particle size and surface charge can be precisely controlled. For example, with a ligand:gold molar ratio of 2:1, the resulting material has an average particle size of approximately 1.5 ± 0.3 nm and a PDI of approximately 0.18. The material exhibits overall negative charge under neutral to weakly alkaline conditions (pH 6.5–8.0) (confirmed by Zeta potential testing), which is beneficial for water dispersion stability and biocompatibility. In the slightly acidic environment associated with periodontitis (pH 5.5–6.5), the uracil functional sites can undergo localized and reversible protonation regulation, thereby endowing the material with pH-responsive properties.
[0029] Step 3: Purification and stabilization treatment (removing impurities and ensuring material homogeneity); The functionalized reaction system requires multi-step purification to remove free ligands, small molecule impurities, and unreacted precursors. The purification steps may include: Step 3.1: Ultrafiltration centrifugation (preferred); Using ultrafiltration centrifuge tubes with a molecular weight cutoff of 10 kDa, and centrifuging at a rate of approximately 3000 rpm for 30 minutes for 2-3 cycles, unbound uracil ligands and small molecule byproducts can be effectively removed. Step 3.2: Dialysis (optional); The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 1 kDa, and deionized water was used as the dialysis medium (replaced every 2 hours) for 12-24 hours. This step can effectively remove unreacted small molecule uracil derivatives, inorganic salts and low molecular weight byproducts, ensuring that most of the product consists of conjugated nanomaterials. Step 3.3: PAGE (polyacrylamide gel electrophoresis) purification (optional, used to improve homogeneity); functionalized gold nanoparticles and free ligands are separated in the gel by PAGE (12% in situ gel), and the target band can be recovered as needed, improving the purity and modification consistency of the material.
[0030] Step 3.4: Combination verification (Au–S bond conjugation confirmation); The purified product was detected by Fourier transform infrared spectroscopy (FT-IR): unreacted uracil ligands were detected at ~2550 cm⁻¹. -1The peaks at the position of the free thiol (–SH) stretching vibration are obvious. In the purified uracil-modified gold nanoparticle conjugates (U-AuNCs), the peaks completely disappear or only a weak signal remains, indicating that the thiol groups have all participated in Au–S bond binding. At the same time, the ligand skeleton vibration peaks (such as C=O, N–H) can still be observed, proving that the functional groups are completely preserved.
[0031] This comprehensive purification and validation system ensured that the uracil ligands were almost completely attached to the surface of the gold nanoparticle conjugate, resulting in U-AuNCs with uniform structure, extremely low impurity content, and good stability.
[0032] Step 4: Stabilization and drying for preservation; Low-temperature freeze-drying (e.g., -50 ℃, 0.1 mbar) can be used for drying to avoid ligand desorption caused by high temperature. The dried uracil-modified gold nanoconjugate can be redispersed in a buffer solution or carrier system before use and adjusted to a physiological or periodontal suitable weak acid to neutral range to maintain a high specific surface area and active interface.
[0033] In embodiments of the present invention, such as Figure 3 a and Figure 3 As shown in b, the prepared U-AuNCs are ultra-small gold nanomaterials with a particle size of no more than 2 nm, and their average particle size is controlled at about 1.2–1.5 nm with a narrow size distribution; no plasmon resonance peak of about 520 nm appears in the ultraviolet-visible spectrum to rule out the possibility of forming large-sized gold nanoparticles. Figure 4 Fourier transform infrared spectroscopy (FT-IR, Thermo Scientific Nicolet iS20) showed that the characteristic peak of the free thiol (–SH) stretching vibration at approximately 2550 cm⁻¹ completely disappeared, indicating that the U₂SH ligand has been stably bound to the surface of the gold nanoparticle conjugated material via Au–S bonds, and there is essentially no free ligand residue. Figure 5 In PBS at pH 8, the Zeta potential of the material was –(38±1.5) mV. When the pH of the medium was adjusted to 6, the Zeta potential changed to –(25±1.2) mV. This change indicates that the functional sites on the material surface are responsive to the ambient pH, providing experimental evidence for its differential surface electrical properties under different pH conditions. Figure 6 ).
[0034] The particle size, zeta potential and functional indicators (such as PMF inhibition rate) of different batches of U-AuNCs are shown in Table 1 below. It can be seen that different batches of U-AuNCs have good batch-to-batch consistency, and the relative standard deviation can be controlled at about 1%-2%.
[0035] Table 1 Table 1. Parameter indicators of different batches of U-AuNCs
[0036] It should be noted that in this invention, the particle size of the gold nanoconjugated material is specifically controlled to an ultra-small scale of no more than 2 nm, and the surface is functionalized with uracil. The purpose is to enable the material to be preferentially taken up by periodontal pathogens through the nanoscale pores of the periodontal biofilm EPS via pyrimidine-related transport pathways, thereby obtaining the deep penetration ability and active pathogen uptake characteristics that traditional 10-100 nm gold nanoparticles do not possess, providing the basic conditions for subsequent selective enrichment and energy metabolism intervention.
[0037] The working process of the prepared U-AuNCs can be summarized as three consecutive stages: biofilm penetration, selective enrichment of pathogens, dual-mode PMF interference, and virulence blocking. This is used to solve the problems of insufficient biofilm penetration, poor antibacterial targeting, and imperfect virulence blocking mechanism in the existing technology.
[0038] During the biofilm penetration phase, U-AuNCs diffuse within the periodontal pocket after local administration; due to their particle size being no larger than 2 nm, they can freely pass through the approximately 10-20 nm pore structure of the periodontal pathogenic bacteria biofilm EPS. Confocal microscopy imaging shows ( Figure 7 a and Figure 7 (b) The material can penetrate to a depth of approximately 80% of the biofilm thickness, thereby significantly improving accessibility to deep-seated residual pathogens.
[0039] During the pathogen selective enrichment stage, uracil modification enables U-AuNCs to be recognized as a pyrimidine / nitrogen source by periodontal pathogens such as *P. gingivalis*, and actively taken up by their related transport systems, achieving highly efficient enrichment within the pathogens. Experimental results show that the enrichment efficiency of U-AuNCs within bacteria can be at least 200 times higher than that within cells. Figure 8 a, Figure 8 b and Figure 8 c), cell viability remains above 80% ( Figure 9 a and Figure 9 (b) thereby significantly reducing off-target damage to normal periodontal tissues while ensuring antibacterial and detoxifying effects.
[0040] During the dual-mode PMF interference and virulence blocking phase, in the slightly acidic environment of periodontitis (pH approximately 5.5-6.5), uracil N3 sites undergo reversible protonation, interfering with the distribution of protons across the bacterial membrane and disrupting the transmembrane pH gradient. The surface charge and electron distribution of the ultrasmall gold nanoconjugates decreased significantly; at the same time, changes in the surface charge and electron distribution of the ultrasmall gold nanoconjugates could cause changes in the bacterial membrane potential. Dissipation. The combined effect of these two factors allows the overall suppression rate of the proton dynamic potential (PMF) to reach or exceed 90%. Figure 10 a and Figure 10 b). Under conditions of significant PMF inhibition, the function of the PMF-dependent type IX secretion system (T9SS) is impaired, and the secretion of key virulence factors such as gingival protease can be reduced by at least 80%, consistent with the aforementioned results of significant PMF inhibition. Figure 11 a and Figure 11 (b) The overall activity of the pathogenic bacteria was reduced to about 15% of that in the control group, thereby effectively weakening its continuous damage to periodontal tissues.
[0041] This invention achieves deep penetration of periodontal pathogens, selective enrichment of pathogens, and dual intervention of energy metabolism and virulence output, which helps to break the vicious cycle of "biofilm barrier - drug off-target - continuous virulence secretion - tissue damage" in periodontitis.
[0042] Several specific embodiments are provided below to verify the effectiveness of this method.
[0043] Example 1: Evaluation of the efficacy of U-AuNCs in clearing and blocking the virulence of Porphyromonas gingivalis biofilm; The aim is to quantitatively evaluate the comprehensive performance of U-AuNCs in overcoming biofilm barriers, interfering with bacterial proton dynamics, and inhibiting the secretion of virulence factors by simulating the periodontitis infection microenvironment in vitro.
[0044] The specific experimental design is as follows: Biofilm model: Porphyromonas gingivalis (ATCC 33277) was cultured in brain and heart extract medium supplemented with heme (5 µg / mL) and vitamin K (1 µg / mL) for 72 hours at 37 °C under an anaerobic atmosphere (85% N2, 10% H2, 5% CO2) to form a mature biofilm in 24-well plates or confocal culture dishes.
[0045] Intervention treatment: Four groups were established (n=3): Blank control group: Add an equal volume of sterile PBS; Ligand control group: Fresh culture medium containing 100 µg / mL U was added; Positive control group: Add an equal volume of 0.2% (w / v) chlorhexidine solution; Experimental group: Fresh culture medium containing 100 µg / mL U-AuNCs was added; Co-culture and detection: After co-culturing each group with the biofilm for 24 hours, the following analyses were performed: Biofilm permeability: Z-stack scans (0.5 µm thickness) of Cy3-labeled U-AuNCs-treated biofilms were performed using a confocal laser scanning microscope (CLSM, Zeiss LSM 880).
[0046] Bacterial survival rate: Staining was performed using the Live / Dead™ BacLight™ Bacterial Viability Kit (Invitrogen), and the total amount of biofilm was quantified using a microplate reader (crystal violet staining method).
[0047] Proton dynamic system inhibition: The PMF inhibition rate was calculated by monitoring the fluorescence intensity changes of the membrane potential-sensitive fluorescent probe DISC3(5) (1 µM) using a fluorescence spectrophotometer.
[0048] Virulence factor activity: Collect the culture supernatant, add the gingival protease-specific fluorescent substrate (Boc-Phe-Ser-Arg-MCA), react at 37 ℃ in the dark for 30 minutes, and then detect its fluorescence values at 405 nm excitation and 460 nm emission.
[0049] result: Deep penetration: CLSM 3D reconstruction images show that U-AuNCs signals can penetrate deep into the biofilm. Figure 7 ).
[0050] Highly effective sterilization: Quantitative analysis of live / dead bacteria staining showed that the bacterial survival rate within the biofilm of the experimental group decreased to (4.44±1.73)%, and its sterilization efficiency was 1.35 times that of the positive control group (chlorhexidine, (29.63±5.63)%) (p < 0.01). Crystal violet staining and MTT assay results showed that the experimental group significantly inhibited biofilm formation and reduced the metabolic activity of bacteria within the biofilm, with a sterilization effect superior to the chlorhexidine control group (…). Figure 12 a, Figure 12 b and Figure 12 c).
[0051] Energy disintegration: PMF detection results showed that the experimental group inhibited bacterial proton kinetics immediately after drug administration and remained stable within 30 minutes, with an inhibition rate as high as (92.3±3.1)%, indicating that its dual-mode interference mechanism can significantly inhibit bacterial energy metabolism. Figure 13 a, Figure 13 b and Figure 13 c).
[0052] Source reduction: The activity of gingival protease in the supernatant of the experimental group decreased by (91.7±4.2)% compared with the negative control group. Figure 14 a and Figure 14(b) The host epithelial barrier structure and function are protected, confirming that PMF interference can effectively block the function of the T9SS secretory system at the source. Figure 15 a, Figure 15 b and Figure 15 c, where Figure 15 b and Figure 15 The bar chart color of c and Figure 15 (The horizontal row name of 'a' corresponds to the color).
[0053] Example 2: Evaluation of the efficacy and biosafety of U-AuNCs in a rat model of periodontitis; The aim was to evaluate the therapeutic efficacy of U-AuNCs gel formulation for experimental periodontitis in vivo, explore its anti-inflammatory effects through histological and molecular indicators, and systematically examine its biosafety.
[0054] The specific experimental design is as follows: Animal model construction: SPF-grade male SD rats (n=25, weighing 200-220g) were selected. The bilateral maxillary first molars were ligated using the orthodontic wire ligation method, and 1×10⁻⁶ oz. was injected into the ligation area for 3 consecutive days. 9 A CFU suspension of Porphyromonas gingivalis (ATCC 33277) was used for 4 weeks to establish a robust periodontitis model.
[0055] Grouping and drug administration: Rats that successfully developed the model were randomly divided into the following four groups (n=5): Healthy control group: No ligation or other treatment was performed; Model control group: Blank gel matrix was administered using the same method; U treatment group: U (100 μg / mL) was injected locally into the periodontal pocket, 5 μL per side, once a week, for a total of 2 times; Positive control group: 0.2% (w / v) chlorhexidine gel was administered using the same method; U-AuNCs treatment group: U-AuNCs (100 μg / mL) were injected locally into the periodontal pocket, 5 μL per side, once a week, for a total of 2 times.
[0056] Evaluation methods and time points: The following analyses were performed 2 weeks after the last dose: Imaging assessment: Maxillary bone blocks were harvested and scanned at 10 μm resolution using a high-resolution mini-CT scanner (Skyscan 1272, Bruker) to quantify the bone loss volume from the alveolar ridge crest to the cementoenamel junction (CEJ).
[0057] Histological and immunofluorescence evaluation: After decalcification and paraffin embedding, jawbone samples were sectioned and subjected to the following evaluations: Hematoxylin-eosin (H&E) staining: to assess tissue morphology and the degree of inflammatory cell infiltration.
[0058] Masson Trichrome staining: to observe the arrangement and content of collagen fibers in the periodontal ligament and to assess tissue repair.
[0059] Immunofluorescence staining: Using anti-TNF-α and anti-IL-6 antibodies, the expression levels of key pro-inflammatory factors in periodontal tissues were observed and semi-quantitatively analyzed by confocal microscopy.
[0060] Systematic safety assessment: Histopathological section analysis of major organs such as the heart, liver, spleen, lungs, and kidneys was performed.
[0061] The results are as follows: Reduced bone loss: Micro-CT analysis showed that, compared with the model group, the U-AuNCs treatment group effectively inhibited alveolar bone resorption, significantly reduced bone loss volume, and the alveolar bone morphology was closer to that of the healthy control group, indicating that the preparation can effectively reverse the bone destruction caused by experimental periodontitis. Figure 16 a, Figure 16 b、 Figure 16 c. Figure 16 d、 Figure 16 e Figure 16 f and Figure 16 g, where Figure 16 b to Figure 16 The bar chart color of g and Figure 17 (The column names in column 'a' correspond to specific colors).
[0062] Inflammation Relief and Enhanced Repair: H&E staining showed that the area of inflammatory cell infiltration in the periodontal tissues of the U-AuNCs treatment group was reduced by approximately 78% compared to the model control group, and the tissue structure was closer to that of the healthy control group. Masson staining results showed that, compared with the model control group, the periodontal ligament region of the U-AuNCs treatment group exhibited richer and more orderly arranged blue collagen fibers, indicating that it can effectively promote the repair and reconstruction of periodontal supporting tissues. Figure 17 a and Figure 18 b).
[0063] Downregulation of pro-inflammatory factors: Semi-quantitative immunofluorescence analysis confirmed that the fluorescence signal intensity of TNF-α and IL-6 in periodontal tissues of the U-AuNCs treatment group was downregulated by (72.5±8.3)% and (68.9±7.1)%, respectively, revealing its significant anti-inflammatory mechanism at the molecular level. Figure 18 a, Figure 18 b and Figure 18 c, where Figure 18 b and Figure 18 The bar chart color of c and Figure 19 (The horizontal row name of 'a' corresponds to the color).
[0064] Biocompatibility: No drug-related pathological changes were found in histopathological sections of major organs, confirming the good in vivo biocompatibility of U-AuNCs. ).
[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A method for preparing uracil-modified gold nanoconjugated materials, characterized in that, Includes the following steps: Step 1: Controlled reduction of gold precursor; In the presence of a reducing agent, a uracil ligand solution was slowly added to a gold precursor solution to carry out a controlled reduction reaction, forming gold nanoconjugates with an average particle size ≤2 nm. Step 2: Surface modification and control; A thiolized uracil derivative was added to the dispersion of the gold nanoconjugated material obtained in step 1, and the pH of the reaction system was adjusted to alkaline so that the thiolized uracil derivative was bound to the surface of the gold nanoconjugated material through Au-S bonds, thereby obtaining uracil-modified gold nanoconjugated material.
2. The method for preparing uracil-modified gold nanoconjugated materials according to claim 1, characterized in that, In step 1, the molar ratio of uracil ligand in the uracil ligand solution to gold precursor in the gold precursor solution is 2:1; the controlled reduction reaction is carried out under stirring at a reaction temperature of 26±0.5℃, and the uracil ligand solution is added by a syringe pump at a rate of 0.50 mL / s.
3. The method for preparing uracil-modified gold nanoconjugated materials according to claim 2, characterized in that, In step 2, the pH of the reaction system is adjusted to 11±0.1 to carry out the surface modification reaction.
4. The method for preparing uracil-modified gold nanoconjugated materials according to claim 1, characterized in that, It also includes step 3: purification treatment; The product obtained in step 2 was purified to remove free ligands and unreacted precursors.
5. The method for preparing uracil-modified gold nanoconjugated materials according to claim 4, characterized in that, The purification process includes one or more of ultrafiltration centrifugation, dialysis, and polyacrylamide gel electrophoresis separation.
6. The method for preparing uracil-modified gold nanoconjugated materials according to claim 1, characterized in that, The obtained uracil-modified gold nanoconjugates were dried using a low-temperature freeze-drying process; the dried uracil-modified gold nanoconjugates were redispersed in a buffer solution or carrier system before use.
7. An application of a uracil-modified gold nanoconjugated compound, wherein the uracil-modified gold nanoconjugated compound prepared by the method for preparing the uracil-modified gold nanoconjugated compound according to any one of claims 1-6, is characterized in that, The uracil-modified gold nanoconjugate is used in the preparation of products for the treatment or prevention of periodontitis.
8. The application of the uracil-modified gold nanoconjugated material according to claim 7, characterized in that, The product is used to inhibit the formation of periodontal pathogenic bacteria biofilm, interfere with bacterial proton kinetic potential, and inhibit the secretion of bacterial virulence factors.
9. The application of the uracil-modified gold nanoconjugated compound according to claim 8, characterized in that, The periodontal pathogen is *Porphyromonas gingivalis*.