pH-responsive antibacterial-biomimetic mineralization material and preparation method and application thereof

Abstract

The application belongs to the technical field of nanomaterials, and particularly relates to a pH-responsive antibacterial-biomimetic mineralization material and a preparation method and application thereof. + The biomimetic mineralization material is cooperatively assembled by DNA-stabilized silver nanoclusters and amorphous calcium phosphate; the iM + The DNA-stabilized silver nanoclusters are silver nanoclusters synthesized and stabilized by DNA as a template; the iM + The DNA-stabilized silver nanoclusters are silver nanoclusters synthesized and stabilized by DNA as a template; the iM + The DNA is an oligonucleotide containing an i-Motif forming domain, and a nucleotide sequence thereof is shown as SEQ ID NO. 1. The antibacterial-biomimetic mineralization material designed by the application is expected to achieve specific removal of cariogenic plaque without destroying normal oral flora, promote enamel remineralization, and thus achieve the purpose of efficient prevention and treatment of dental caries.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials technology, specifically relating to a pH-responsive antibacterial-biomimetic mineralization material, its preparation method, and its application. Background Technology

[0002] Dental caries is a common oral disease with a high incidence rate in clinical practice. The development of dental caries is a dynamic process involving alternating enamel demineralization and remineralization, with early clinical manifestations including enamel caries. Cariogenic biofilms are the root cause of dental caries. Cariogenic bacteria colonize the tooth surface and gradually mature into cariogenic biofilms, metabolizing and producing lactic acid, which significantly lowers the pH within the biofilm, leading to enamel demineralization. Tooth enamel possesses a highly mineralized and orderly hierarchical crystalline structure, making it the hardest tissue in the human body, and it undertakes a series of important oral physiological functions such as chewing, speech, and facial aesthetics. However, its lack of organic matter also makes it almost impossible for enamel tissue to repair itself once damaged. Without intervention, enamel continues to demineralize, dental caries progresses rapidly, and ultimately leads to serious consequences such as tooth defects, pulpitis, and tooth loss. Therefore, how to effectively prevent and treat early enamel caries is a key issue in controlling the progression of dental caries and reducing the burden on public health.

[0003] Currently, fluoride remains a commonly used clinical treatment, but it suffers from limitations such as insufficient antibacterial properties, limited remineralization induction ability, and potential fluorosis. Therefore, recent research has focused on using biomimetic mineralization strategies to reconstruct the structure and properties of tooth enamel. Based on the understanding of the enamel biomineralization process, current biomimetic mineralization strategies primarily mimic the function of enamel matrix proteins to mediate the formation of amorphous mineral precursors. These precursors then undergo in-situ phase transformation crystallization, further transforming into highly ordered apatite crystals to achieve enamel repair. However, unlike the sterile, static, and mild in vitro mineralization environment, the oral cavity is a dynamic environment with a rich microbiome. Therefore, the aforementioned biomimetic mineralization strategies often fail to achieve ideal results in practical applications. Existing antibacterial-remineralization bifunctional materials often rely on complex broad-spectrum antibacterial components. While these can effectively inhibit bacteria, they may disrupt the balance of normal oral flora, leading to the risk of oral microecological dysbiosis. Meanwhile, the dense structure of cariogenic biofilms severely hinders remineralizing materials from reaching the demineralization sites and exerting their remineralization effect. Furthermore, biomimetic mineralizing materials, such as ACP, face the risk of being dissolved by acid in localized acidic microenvironments. In summary, existing antibacterial-remineralizing materials struggle to effectively penetrate biofilms, exert specific antibacterial effects against cariogenic bacteria, and achieve effective remineralization in the presence of cariogenic biofilms. Summary of the Invention

[0004] To address the challenges of existing antibacterial-remineralization materials failing to effectively penetrate biofilms, exert specific antibacterial effects against cariogenic bacteria, and achieve effective remineralization in the presence of cariogenic biofilms, the development of functional materials capable of specifically eliminating cariogenic bacteria without affecting normal flora is of great significance. This invention provides a pH-responsive antibacterial-biomimetic mineralization material, its preparation method, and its applications. To achieve the above objectives, this invention adopts the following technical solution.

[0005] This invention provides a pH-responsive antibacterial-biomimetic mineralizing material, wherein the antibacterial-biomimetic mineralizing material is composed of iM + DNA-stable silver nanoclusters and amorphous calcium phosphate are synergistically assembled.

[0006] The iM + DNA-stabilized silver nanoclusters are based on iM + Silver nanoclusters were synthesized and stabilized using DNA as a template via a sodium borohydride chemical reduction method; the iM + The DNA is an oligonucleotide containing an i-Motif forming domain, and the nucleotide sequence of the oligonucleotide is shown in SEQ ID NO.1: 5'-GGGCCCCGGGCCCTAACCCTAACCCTAACCCTAACCCGGGGCCC-3', wherein the i-Motif forming domains (the core region forming the i-Motif) are located at positions 17-19 (CCC), 23-25 ​​(CCC), 29-31 (CCC), and 35-37 (CCC) from 5'. These four cytosine contiguous regions (CCC) - The tract undergoes hemiprotonation at acidic pH, forming antiparallel C·C bonds in the following manner. + Hydrogen bond linkage between base pairs:

[0007] C in positions 17 through 19 - tract and C at positions 35-37 - tract pairing.

[0008] The C in the 17th position forms three C·C pairs, the C in the 35th position forms three C·C pairs, the C in the 18th position forms three C·C pairs, and the C in the 19th position forms three C·C pairs. + Base pairs.

[0009] C in positions 23 to 25 - tract and C at positions 29-31 - tract pairing.

[0010] The C in position 23 forms three C·C pairs, the C in position 24 forms three C·C pairs, and the C in position 25 forms three C·C pairs. + Base pairs.

[0011] The above pairings make four Cs - The tracts are arranged antiparallel and intercalated to form a stable four-stranded i-Motif structure. The non-C bases connecting the pairing regions (TAA at positions 14-16, 20-22, 26-28, and 32-34) form a loop. Other C-tracts at the ends of the sequence (positions 4-7, 11-13, and 42-44) do not participate in core pairing but can serve as flanking protrusions and do not affect the formation of the core i-Motif.

[0012] After cariogenic bacteria form plaque biofilms, they produce acid in the local environment, significantly lowering the pH and accelerating enamel demineralization and the progression of caries. Designing a biomimetic mineralizing material that can efficiently penetrate biofilms and respond to low pH environments holds promise for specifically inhibiting cariogenic bacteria while promoting enamel remineralization. Based on this, this invention utilizes iM... + The synergistic assembly of DNA-stabilized silver nanoclusters and amorphous calcium phosphate (ACP) simultaneously solves three major technical challenges: permeability of cariogenic biofilms, responsive antibacterial activity, and biomimetic remineralization. The specific mechanism is as follows: iM + DNA possesses a pH-responsive i-Motif structure, which can serve as a template to mediate the formation of small silver nanoclusters with a size of approximately 2 nm; in the acidic microenvironment of cariogenic biofilms, iMotif... + The i-Motif structure of DNA undergoes a conformational change, reducing its binding affinity to silver nanoclusters (AgNCs), thereby responsively releasing the silver nanoclusters to achieve specific killing of cariogenic biofilms; simultaneously, iM + DNA, as a template, can also act as a mineral precursor stabilizer, inducing the formation of amorphous calcium phosphate (ACP); this iM + DNA-induced ACP can penetrate deep into cariogenic biofilms and induce demineralized enamel through non-classical crystallization pathways to achieve biomimetic remineralization. This effectively overcomes the technical defects of existing antibacterial-remineralization materials, such as difficulty in penetrating cariogenic biofilms, inability to achieve specific antibacterial effects on cariogenic biofilms, and difficulty in achieving effective remineralization in the presence of cariogenic biofilms.

[0013] The present invention also provides a method for preparing the aforementioned antibacterial-biomimetic mineralized material, comprising the following steps: With the iM + Using DNA as a template, iM +DNA was mixed with an aqueous solution of silver nitrate and incubated at a low temperature (3℃~5℃). Then, sodium borohydride (reducing agent) was added to carry out a chemical reduction reaction, yielding iM. + DNA-stable silver nanoclusters, denoted as iM + DNA-AgNC.

[0014] To the iM + DNA-AgNC was added to a supersaturated calcium phosphate solution, and iM was used. + DNA phosphate backbone for Ca 2+ Electrostatic enrichment and the heterogeneous nucleation-inducing effect of AgNCs lead to Ca 2+ With phosphate in iM + In situ deposition of DNA-AgNC on the surface forms amorphous calcium phosphate (ACP), thereby obtaining iM + DNA-AgNC / ACP composite material, namely the antibacterial-biomimetic mineralized material.

[0015] Among them, to the iM + DNA-AgNC was added to a supersaturated calcium phosphate solution, and iM was used. + The negative charge of the phosphate groups on the DNA backbone affects Ca2+. 2+ The electrostatic enrichment effect of AgNCs, and the silver ions (Ag) on ​​the AgNCs surface + / Ag 0 As a heterogeneous nucleation site, it induces Ca in supersaturated calcium-phosphorus solutions. 2+ With PO4 3- / HPO4 2- In iM + Heterogeneous nucleation occurs on the surface of DNA-AgNC, forming amorphous calcium phosphate (ACP); the ACP passes through Ca... 2+ With iM + Ionic bonding of the DNA phosphate backbone, and Ca 2+ Coordination adsorption with AgNCs surface, in-situ deposition coating on iM + DNA-AgNC surface, to obtain iM + DNA-AgNC is a core-shell composite material with ACP as the shell, denoted as iM. + DNA-AgNC / ACP, namely the antibacterial-biomimetic mineralized material.

[0016] Preferably, the molar ratio of calcium ions to phosphate ions in the supersaturated calcium-phosphorus solution is 1.66 to 1.68, and the pH of the supersaturated calcium-phosphorus solution is 6.97 to 7.03.

[0017] Preferably, the reaction temperature for forming amorphous calcium phosphate is 36℃~38℃, and the reaction time is 25min~35min.

[0018] The present invention also provides the application of the antibacterial-biomimetic mineralizing material in the preparation of oral medical materials for preventing tooth decay, antibacterial purposes, or promoting tooth remineralization.

[0019] Preferably, the oral medical material includes the antibacterial-biomimetic mineralized material.

[0020] Preferably, the dosage form of the oral medical material is selected from toothpaste, mouthwash, dental varnish, fissure sealant, or dental adhesive.

[0021] Preferably, the oral medical material is used for the prevention and treatment of early enamel caries.

[0022] The present invention also provides an oral medical material for preventing tooth decay, antibacterial purposes, or promoting tooth remineralization, comprising the antibacterial-biomimetic mineralizing material.

[0023] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a pH-responsive antibacterial biomimetic mineralizing material. The biomimetic mineralizing material provided by this invention is composed of iM... + DNA-stabilized silver nanoclusters and amorphous calcium phosphate synergistically constitute; iM + DNA-stabilized silver nanoclusters are based on iM + Silver nanoclusters were synthesized and stabilized using DNA as a template via a sodium borohydride chemical reduction method; iM + The DNA is an oligonucleotide containing an i-Motif forming domain. This invention, by introducing an i-Motif forming domain into the DNA sequence, enables a response to low pH environments; the DNA can also serve as a template to stably form Ag nanoclusters with a particle size of approximately 2 nm, exhibiting good dispersibility and stability, thus achieving an antibacterial effect; simultaneously, the DNA can stabilize the formation of amorphous calcium phosphate induced by supersaturated calcium phosphate, possessing both enamel affinity and mineralization induction capabilities. Compared to traditional macromolecular polyanionic compounds, low molecular weight oligonucleotides, due to their small size and high permeability, are more suitable for entering the biofilm of cariogenic bacteria to exert their effects. This invention utilizes a special oligonucleotide sequence containing an i-Motif domain, leveraging its template function to synthesize iM... + DNA-AgNC, and further stabilizes the formation of supersaturated calcium and phosphorus iM + DNA-AgNC / ACP combines biofilm permeability, pH-responsive antibacterial properties, and enamel-inspired biomineralization, achieving a functional integration of caries prevention materials. This invention utilizes iM... + The synergistic assembly of DNA-stabilized silver nanoclusters and amorphous calcium phosphate simultaneously solves three major challenges: biomembrane permeability, responsive antibacterial activity, and biomimetic remineralization. +DNA possesses a pH-responsive i-Motif structure and can also serve as a template to mediate the formation of small-sized silver nanoclusters (~2 nm); iM + DNA undergoes conformational changes in the acidic microenvironment of cariogenic biofilms, leading to iM + The decreased binding affinity between DNA and AgNC leads to the responsive release of silver nanoclusters, achieving specific killing of cariogenic biofilms; simultaneously, this DNA template can act as a mineral precursor stabilizer, inducing the formation of amorphous calcium phosphate; iM + DNA-induced ACP can penetrate deep into the biofilm and induce biomimetic remineralization of demineralized enamel through a non-classical crystallization pathway. This solves the problems in existing technologies where existing antibacterial-remineralization materials cannot effectively penetrate into the biofilm, exert the specific antibacterial effect of cariogenic biofilms, and achieve effective remineralization in the presence of cariogenic biofilms.

[0024] 2. The novel material designed by this invention is expected to achieve the specific elimination of cariogenic bacteria and promote enamel remineralization without disrupting the normal oral flora, thereby achieving the goal of highly effective prevention and treatment of dental caries. Attached Figure Description

[0025] Figure 1 Characterization of a pH-responsive antibacterial-biomimetic mineralizing material designed for this invention, wherein: A is iM + A schematic diagram of a DNA sequence and its conformational change under acidic conditions to form an i-Motif structure; When B is at pH 7.4 and 5.0, iM + Circular dichroism (CD) spectrum of DNA. C represents iM under neutral and acidic conditions. + Non-denaturing gel electrophoresis image of DNA; D is iM + The fluorescence spectra of thioflavin T (ThT) in DNA at pH 7.4 and 5.0, respectively; E for iM + A schematic diagram of DAgACP preparation; F is iM + High-resolution transmission electron microscopy (HRTEM) images, EDS elemental maps (calcium, phosphorus, oxygen, nitrogen, and silver), and selected area electron diffraction (SAED) characterization of DAgACP; G is obtained from the statistical analysis of transmission electron microscopy images. + Particle size distribution of DAgACP; H is iM + EDS spectrum of DAgACP; I, J, and K are iM + DNA, iM +DAg, iM + DACP, iM + The UV-Vis spectrum, XPS full spectrum, and XPS magnified spectra of Ag 3d and Ca 2p of DAgACP; L~N represent the iM values ​​that highlight the main vibrational peaks, in sequence. + DNA, iM + DAg, iM + DACP, iM + Fourier transform infrared (FTIR) spectrum of DAgACP.

[0026] Figure 2 To target this antibacterial-biomimetic mineralization material (iM⁺DAgACP nanoassemblies or iM + Explanation of the mechanism of pH-responsive antibacterial properties of DNA-AgNC / ACP composite material: under acidic conditions, iM + After DNA undergoes a conformational change, its binding affinity to AgNC decreases, leading to the responsive release of AgNC and thus exhibiting pH-responsive antibacterial properties; among which: A represents the penetration of the iM⁺DAgACP nanoassemblies into Streptococcus mutans. Streptococcus mutans A schematic diagram illustrating the process of responsively releasing silver nanoclusters (AgNCs) from within a biofilm under acidic conditions; B and C are three-dimensional reconstruction results obtained by tomographic scanning along the depth direction of the biofilm after 60 min of treatment with calcein blue-labeled iM⁺DAgACP and HMWN-DAgACP stably formed from high molecular weight natural (HMWN) DNA, respectively, to compare the permeation and distribution of the materials in the variable chain biofilm. D represents the ThT fluorescence emission spectra of iM⁺DAgACP under pH 7.4 and pH 5.0 conditions, used to characterize the structural changes in pH-responsiveness of this nanoassembly; E represents the quantitative results of Ag⁺ release from iM⁺DAgACP nanoassemblies determined by inductively coupled plasma mass spectrometry (ICP-MS) in HEPES buffer systems at pH 7.4 and pH 5.0 (n=3). F represents a representative image of the DNA-silver nanocluster complex structural model obtained from molecular dynamics (MD) simulations, including: a linear DNA conformation model under neutral conditions and an i-Motif DNA conformation model under acidic conditions; where, under neutral pH conditions, DNA (represented in red) exhibits an extended, flexible linear conformation and provides multiple binding sites for AgNCs (represented in white spheres); under acidic conditions, DNA folds to form a compact i-Motif quadruplex structure, which is stabilized by protonated cytosine-cytosine (CC⁺) base pairing; Na⁺ is represented by blue spheres; the system is solvated using the TIP3P water model (represented by transparent boxes). G~K are the curves showing the changes in structural parameters over time during a 100 ns molecular dynamics simulation of the DNA-AgNC complex under neutral and acidic conditions. The structural parameters include: G is the root mean square deviation (RMSD) of DNA heavy atoms; H is the radius of gyration (Rg); I is the solvent-accessible surface area; J is the number of intramolecular hydrogen bonds; and K is the binding energy of the DNA-AgNC complex. L represents the comparison of the average absolute binding energy in the DNA-AgNC model under neutral and acidic conditions; M represents the molecular dynamics simulation images of the linear DNA-AgNC and i-Motif DNA-AgNC complexes at 0, 25 ns, 50 ns, 75 ns, and 100 ns, respectively, used to characterize the dynamic evolution of the complex conformation over time; the DNA strand is represented in red, AgNCs in white, and Na⁺ in blue, and the simulation box is displayed with a transparent surface for comparison.

[0027] Figure 3 The antibacterial-biomimetic mineralization material (iM⁺DAgACP nanoassemblies or iM) designed for this invention + Characterization of the antibacterial properties of DNA-AgNC / ACP composite material; among which: A and B are statistical graphs showing the bacterial growth rate of Streptococcus mutans when co-incubated with iM⁺DAgACP or iM⁺DACP under different pH conditions, where the growth rate is expressed as a percentage relative to the PBS control group (n=3). C~E are representative images of bacterial suspensions plated after treatment with PBS, iM⁺DACP, chlorhexidine (CHX) or iM⁺DAgACP, and quantitative results of Streptococcus mutans viability obtained by plate plating colony counting method (n=3). F is a live / dead staining fluorescence image of the biofilm formed after Streptococcus mutans suspension was co-incubated with PBS, iM⁺DACP, CHX or iM⁺DAgACP for 48 h. G represents the quantitative result of the biofilm formation inhibition rate (n=3); H represents the CLSM imaging results of the pre-formed biofilm after 24 h of treatment with various treatment groups such as PBS, iM⁺DACP, CHX, or iM⁺DAgACP; I represents the quantitative results of the degree of biofilm disruption / removal (n=3); In the figure, asterisks indicate statistically significant differences: * p <0.05,** p <0.01, *** p <0.001, **** p <0.0001.

[0028] Figure 4 The antibacterial-biomimetic mineralization material (iM⁺DAgACP nanoassemblies or iM) designed for this invention + Characterization of the biomimetic mineralization properties of DNA-AgNC / ACP composite material; among which: A is a schematic diagram of the remineralization experiment under sterile conditions; B is a scanning electron microscope (SEM) image of the surface and corresponding cross-sectional morphology of tooth enamel after one to three cycles of iM⁺DAgACP-mediated remineralization; C represents the statistical results of the thickness of the remineralized enamel layer formed by different remineralization cycles; D represents the energy dispersive spectroscopy (EDS) results of the remineralized glaze, used to characterize its elemental composition; E and F represent the X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD) characterization results of natural tooth enamel, acid-etched tooth enamel, and iM⁺DAgACP remineralized tooth enamel under different treatment conditions, respectively. G and H are schematic diagrams and SEM images of the enamel surface and its corresponding cross-sectional morphology after remineralization in the presence of Streptococcus mutans biofilm, respectively; the processing groups corresponding to the images include PBS, chlorhexidine (CHX), iM⁺DACP and iM⁺DAgACP; Figure 5 To verify the anti-caries effect of the material designed in this invention; wherein: A is a schematic diagram of the process for establishing a dental caries model, administering medication, and evaluating the effects. B shows representative growth patterns of oral swabs from rats in the blank group (BS), CHX group, and iM⁺DAgACP group on MSB agar plates; C and D represent the counts and statistical analysis of Streptococcus mutans colony-forming units (CFU) on days 14, 30, and 42 (n=5 for each group). E is a stereomicroscopic image of the rat maxillary molar after purpurate ammonium staining, showing enamel lesions (E), mild dentin lesions (Ds), moderate dentin lesions (Dm), and severe dentin lesions (Dx). F~H represent the caries scores for smooth and fissure surfaces based on the modified Keyes scoring system (n=12 slices per group). Image I is a sagittal tomographic image and three-dimensional reconstruction of the maxillary molar of a rat using micro-CT. The red arrows indicate carious lesions, and the blue arrows represent enamel tissue with a Hausfield unit >4200.

[0029] Data are expressed as mean ± standard deviation; ns: no significant difference; * p <0.05,** p <0.01, *** p <0.001. Detailed Implementation

[0030] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0031] The experimental materials used in the examples are as follows: Calcium nitrate tetrahydrate (Ca(NO3)2·4H2O; ACS reagent, ≥99%), dipotassium hydrogen phosphate (K2HPO4; ACS reagent, ≥98%), sodium fluoride (NaF; ACS reagent, ≥99%), silver nitrate (AgNO3; ACS reagent, ≥99%), and 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) were all purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Calcium chloride dihydrate (CaCl2·2H2O; ACS reagent, ≥99%), potassium chloride (KCl; ACS reagent, ≥99%), sodium borohydride (NaBH4, ≥98%), sodium acetate (NaAc), glacial acetic acid (HAc), tris(hydroxymethyl)aminomethane (Tris), hydrochloric acid (HCl), sodium chloride (NaCl), and ammonium purpurate (C8H8N6O6) were all purchased from Aladdin (Shanghai, China). Sodium hydroxide (NaOH; ACS reagent, 97%) and thymol (C 10 H 14 O (analytical grade, 98%) was purchased from Macklin Reagents, Inc. (Shanghai, China). Phosphoric acid (H3PO4, analytical grade, ≥85%) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Salmon sperm DNA (high molecular weight (HMW) linear DNA) was purchased from Coolaber Biotech Ltd. (Beijing, China).

[0032] The experiment used two artificially synthesized oligonucleotide chains: It can form i-motif forming sequences (iM) + DNA, 5'-GGGCCCCGGGCCCTAACCCTAACCCTAACCCTAACCCGGGGCCC-3', nucleotide sequence as shown in SEQ ID NO.1) and a C-free non-iMotif forming sequence (iM) without cytosine. -DNA, 5'-TTTAATTTTAATTTTAATTTTAATTTTAATTTTAATTTTAATTT-3' (nucleotide sequence as shown in SEQ ID NO.2). All oligonucleotides were synthesized by Genewiz Biotechnology Co., Ltd. (Suzhou, China) with a purity ≥95% (high performance liquid chromatography purification) to avoid interference from impurities in secondary structure formation. A 100 μM stock solution was prepared using nuclease-free water.

[0033] Dulbecco's modified Eagle's medium (DMEM), phosphate-buffered saline (PBS), penicillin-streptomycin solution (10000 U / ml), and fetal bovine serum (FBS; 99%) were purchased from Gibco BRL, Thermo Fisher Scientific (Grand Island, NY, USA). Cell counting kit 8 (CCK8) was purchased from Elabscience (Wuhan, China). Brain heart infusion (BHI) and BHI agar were purchased from Hopebio (Qingdao, China). The live / dead bacterial staining kit was purchased from Invitrogen, Thermo Fisher Scientific (Carlsbad, California, USA). Crystal violet staining solution was purchased from BeyotiMe (Shanghai, China). Sucrose (analytical grade), sodium penicillin, and streptomycin sulfate were purchased from Solarbio (Beijing, China). Cariogenic feed 2000 was purchased from Shuyu Biotech (Shanghai, China). Mitis salivarius agar (MSB), bacitracin solution, and potassium tellurite solution were purchased from Tuopu Biol Engineering Co. (Shandong, China). Nuclease-free ultrapure water (resistivity ≥18.2 MΩ·cm) was used throughout the experiment to prevent DNA degradation, and all solutions were filtered through a 0.22 μm microporous membrane before use.

[0034] Example I. Experimental Methods: 1. iM under acidic conditions + DNA structure detection iM +DNA oligonucleotides were purified by high-performance liquid chromatography to a purity ≥95%. Two buffer solutions were prepared to assess pH-dependent i-motif formation, and the ionic strength was adjusted with NaCl to simulate the physiological environment.

[0035] Neutral control buffer (pH 7.0): Dissolve 20 mM Tris and 100 mM NaCl in nuclease-free water, and adjust the pH to 7.0 with HCl (calibrated with a precision pH meter, accuracy ±0.01) as a negative control.

[0036] Acidic detection buffer (pH 5.0): Dissolve 20 mM NaAc and 100 mM NaCl in nuclease-free water, and adjust the pH to 5.0 with glacial acetic acid to provide the optimal environment for i-motif folding.

[0037] 1.1 Circular dichroism (CD) detection iM was prepared separately using either acidic detection buffer or neutral control buffer. + DNA was diluted to a final concentration of 5 μM, with a total sample volume of 200 μL. Circular dichroism chromatograms of synthesized DNA at different pH values ​​(7.4 and 5.0) were collected using a J1500 circular dichroism chromatogram (JASCO, Japan).

[0038] 1.2. Non-denaturing polyacrylamide gel electrophoresis (native PAGE) DNA (iMn) was reacted with either acidic detection buffer or neutral control buffer. + DNA and iM - The DNA was diluted to a final concentration of 10 μM. 6× non-denaturing loading buffer was added at a 1:5 (v / v) ratio. A 12% polyacrylamide gel was prepared using gel polymerization buffer. 15 μL of DNA solution was loaded into each well, with a separate well containing a 500 bp DNA marker as a molecular weight reference. Electrophoresis was performed at a constant voltage of 100 V for 90 min, until the bromophenol blue indicator migrated to approximately 2 / 3 of the gel length. After electrophoresis, the gel was stained with SYBR Gold nucleic acid dye and observed using a 302 nm UV transilluminator.

[0039] 1.3 Detection of Thioflavin T (ThT) fluorescence signal Add the sample to each well of a 96-well plate, with a total volume of 200 μL per well. The system ratio is: 1 μM iM +DNA oligonucleotides, 6 μM ThT, 100 mM buffers at different pH levels (make up the volume, neutral control buffer (pH 7.0) and acidic assay buffer (pH 5.0)). Place the 96-well plate in a microplate reader, and collect fluorescence spectra using a Hitachi F4600 fluorescence spectrophotometer. Excitation wavelength was 425 nm, and emission spectra from 455 nm to 650 nm were recorded. All assays were performed at 25°C.

[0040] 2. Preparation and characterization of nanocomposites 2.1 Preparation of different nanocomposite materials 2.1.1, iM + DAgACP composite material (iM) + DNA-AgNC / ACP composite material) iM + DAgACP nanocomposite materials (iM) + The DNA-AgNC / ACP composite material was prepared according to the following method: (1) Preparation of solution A: Dissolve 165.2 mg of calcium nitrate tetrahydrate (Ca(NO3)2·4H2O) in 100 mL of nuclease-free ultrapure water and stir for 5 min; then add iM + Add DNA and gently stir for 5 minutes; then add silver nitrate (AgNO3) and continue to gently stir for 5 minutes, followed by incubation at 4°C for 30 minutes; add sodium borohydride (NaBH4), mix thoroughly, and let stand at room temperature (RT) for 30 minutes to obtain solution A.

[0041] (2) Preparation of solution B: Dissolve 73.2 mg of dipotassium hydrogen phosphate (K2HPO4) in 100 mL of nuclease-free ultrapure water to obtain solution B.

[0042] Add solution B dropwise to an equal volume of solution A, and stir at low speed to obtain iM. + DAgACP nanocomposite materials (iM) + DNA-AgNC / ACP composite material).

[0043] 2.1.2, iM + DAg composite materials iM + The preparation method of DAg and iM + The DAgACP composite material is similar, but without the addition of calcium nitrate and dipotassium hydrogen phosphate. The simplified steps are: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] + DNA was dissolved in nuclease-free ultrapure water, AgNO3 was added, and the mixture was gently stirred for 5 min. After incubation at 4°C for 30 min, NaBH4 was added, and the mixture was thoroughly mixed. The reaction was continued at room temperature for another 30 min to form silver nanoclusters (M). + DNA-stable silver nanoclusters, denoted as iM+ DNA-AgNC, abbreviated as iM + DAg), to obtain iM + DAg composite material.

[0044] 2.1.3, iM + DACP composite materials iM + Preparation method of DACP composite material and iM + The DAgACP composite material is similar, but without the addition of silver nitrate and subsequent reduction steps. + DNA-stable amorphous calcium phosphate, denoted as iM + DNA-ACP, abbreviated as iM + DACP.

[0045] The nanocomposite materials obtained above can be freeze-dried into powder using a freeze dryer (SP Industries, Inc., Warminster, PA, USA) for subsequent characterization.

[0046] 2.2 High-resolution transmission electron microscopy characterization The iM was studied using a high-resolution transmission electron microscope (HRTEM, JEM200, NEC Corporation, Tokyo, Japan). + The morphology, selected area electron diffraction (SAED) pattern, and energy-dispersive X-ray spectroscopy (EDS) elemental distribution of the DAgACP composite material were characterized.

[0047] 2.3 Zeta potential and dynamic light scattering analysis iM was determined using a Zetasizer Nano ZS potentiometric particle size analyzer (Malvin Instruments Ltd., Worcestershire, UK). + Zeta potential and hydrated particle size of DAgACP composite materials. Hydrated particle size was measured using dynamic light scattering (DLS) with a 4mW helium-neon laser (wavelength 633nm) and a detection angle of 173°. Zeta potential testing conditions: temperature 25℃±0.1℃, equilibration time 2min, 3 tests per sample, 10-15 scans per test; calculations were performed using the Smoluchowski model with an applied voltage of 50V.

[0048] 2.4 Spectral Characterization Ultraviolet-Visible Spectroscopy: Measured using a Shimadzu UV2600 spectrometer. Fourier Transform Infrared Spectroscopy (FTIR): Measured using an Agilent Cary 630 infrared spectrometer (USA), scanning range 4000 cm⁻¹ to 400 cm⁻¹. X-ray Diffraction (XRD): Measured using a Rigaku SmartLabSE X-ray diffractometer (Japan), CuKα radiation (λ=0.154056 nm), operating voltage 40 kV, current 30 mA, scanning range 5° to 70°, scanning rate 2° / min. X-ray Photoelectron Spectroscopy (XPS): Measured using a Thermo Fisher KAlpha photoelectron spectrometer, excitation source Al Kα (hv=1486.6 eV).

[0049] 2.5 Transmission Electron Microscopy (TEM) Take 2 μL iM + DAgACP was dropped onto a carbon-supported copper mesh (400 mesh, China Microscope Instruments, Beijing, China) and dried for 5 min. Transmission electron microscopy images were acquired at 110 kV using a JEM1230 transmission electron microscope (JEOL, Tokyo, Japan).

[0050] 2.6 ThT fluorescence detection 1 μM iM under different pH conditions (7.4 and 5.0) + The DAgACP composite material was mixed with 6 μM ThT and added to a 96-well plate. The 96-well plate was then placed in a microplate reader, and the detection parameters were the same as in Section 1.3.

[0051] 2.7 Inductively Coupled Plasma Mass Spectrometry (ICP-MS) To explore iM + The silver ion release behavior of DAgACP composite material at different pH values ​​(7.4 and 5.0) was investigated by adding 2 mL of the prepared iM... +DAgACP solution was loaded into dialysis membranes (purchased from Solarbio Science, Beijing, China) and immersed in 50 mL of 20 mM HEPES buffer (pH 7.4 and 5.0, respectively). At preset time points (1 h, 3 h, 5 h, 8 h, 12 h, and 24 h), 1.5 mL of buffer was sampled and replenished with an equal volume of fresh 20 mM HEPES buffer at the same pH, maintaining a total volume of 50 mL. The silver ion release concentration at each time point was determined using a PerkinElmer NEXION 1000G inductively coupled plasma mass spectrometer.

[0052] 2.8 Molecular Dynamics Simulation To compare conformational differences under different protonation states, two initial DNA models were directly constructed: aprotonated (neutral / basic) conformation and protonated (acid-related) conformation. The latter was not generated by displaying low pH or dynamic protonation in molecular dynamics (MD) simulations, but was predefined as the starting structure and then simulated using the same MD workflow. The i-Motif initial structure was constructed based on crystal structures from the Protein Data Bank (PDB) (PDB ID: 1ELN). Using 1ELN as a template, the target DNA sequence was structurally predicted and optimized using the Protenix server online platform (https: / / protenix-server.com / ), with homology modeling used for fine-tuning of DNA loop regions. Spherical silver nanoclusters (AgNCs) were constructed using the nanomaterials modeling module of the CHARMM GUI online platform (https: / / charmm-gui.org). The force field parameters for the silver nanoclusters were derived from the INTERFACE ForceField (IFF), which is specifically optimized for interactions between metal nanoparticles and biomolecules. The parameters were obtained from the INTERFACE MD database (https: / / bionanostructures.com / interface-md / ). A DNA-AgNCs composite system was constructed using the PACKMOL program.

[0053] The simulation chamber was set as a cube; DNA molecules were described using the AMBER BSC1 force field, processed by the Ambertools toolkit, and exported via AcPyPE. All molecular dynamics simulations were performed using the GROMACS 2022.5 software package, and the system was solvated using the TIP3P water model. System energy was determined in two steps: 5000 steps using the steepest descent method to eliminate high-energy conformations and unreasonable atomic contacts; and 2500 steps using the conjugate gradient method to further optimize the system energy to a local minimum. The convergence criterion was that the maximum force component was below 1000 kJ·mol⁻¹.-1 ·nm -2 Long-range electrostatic interactions were calculated using the Particle Mesh Ewald (PME) method; van der Waals interactions were described using the Lennard-Jones potential with a cutoff distance of 10 Å.

[0054] The simulation was conducted at an isothermal temperature of 300 K, with temperature control using a velocity-scaled thermostat and a coupling time constant of 0.1 ps; pressure was controlled using a Parrinello-Rahman pressure regulator. During the 300 ps NVT equilibrium phase, positional confinement (force constant 1000 kJ·mol⁻¹) was applied to the DNA and AgNCs. -1 ·nm -2 The system was then subjected to 500ps NPT equilibration; formal production simulation was run for 100ns under the NPT system. Trajectory analysis was performed using GROMACS built-in tools, including root-mean-square deviation (RMSD), radius of gyration (Rg), and hydrogen bond analysis, and energy calculations were performed using the gmx energy tool.

[0055] 3. Evaluation of antibacterial properties 3.1 Resistance to airborne bacteria To evaluate iM + The inhibitory effect of DAgACP composite material on the growth of planktonic Streptococcus mutans was investigated through the following antibacterial experiments: Streptococcus mutans ( Streptococcus mutans , S. mutans (ATCC 700610, American Collection of Type Cultures, Manassas, USA) The culture was carried out in Brain Heart Infusion (BHI) medium under microaerophilic conditions (85% N2, 10% H2, 5% CO2) at 37°C. The concentration of the culture in the logarithmic growth phase was adjusted to 1 × 10⁻⁶. 6 Colony-forming units per milliliter (CFU / mL). This bacterial suspension was then compared with different groups of iM... + DAgACP composites (pH values ​​of 7.4, 6.5, 6, 5.5, 5, and 4) were mixed at predetermined volume ratios (50%, 75%, 87.5%, 93.75%, and 96.875%, v / v) and incubated at 37°C for 12 hours. The optical density (OD) of each mixture at 600 nm was measured using a spectrophotometer. 600 ). (with OD) 600The value is used to evaluate the growth rate of planktonic bacteria; the lower the value, the stronger the antibacterial effect of the composite material.

[0056] To evaluate iM + To investigate the pH-responsive antibacterial activity of DAgACP against Streptococcus mutans, parallel experiments were conducted, and iM + DACP composite material served as a control. Different target pH values ​​(7.4, 6.5, 6.0, 5.5, 5.0, and 4.0) were used to prepare iM solutions. + DAgACP composite materials and iM + DACP composite material was mixed with Streptococcus mutans bacterial suspension at a fixed volume ratio of 50% and incubated at 37°C under microaerophilic conditions. OD values ​​for each group were measured using a spectrophotometer at preset time points (0h, 0.5h, 1h, 3h, 5h, 10h, 18h, and 24h). 600 .

[0057] To quantitatively evaluate the antibacterial effects of different treatment groups on *Streptococcus planktonicus*, a serial dilution plating method was used for colony counting, and corresponding control groups were set up for comparison. A total of four experimental groups were set up: (1) iM + DAgACP group; (2) iM + DACP group; (3) 0.2% chlorhexidine (CHX, positive control group); (4) phosphate-buffered saline (PBS, negative control group). The materials of each group were mixed with the bacterial suspension at a volume ratio of 1:1 and incubated for 6 h. After incubation and dilution, 50 μL of bacterial suspension was spread on BHI agar plates and cultured for another 48 h. The number of visible colonies on each plate was counted, and the bacterial concentration was calculated in terms of colony forming units per milliliter (CFU / mL). Representative plates were photographed to record the colony morphology and density.

[0058] 3.2. Resistance to Streptococcus mutans biofilm formation 3.2.1, iM + Biomembrane permeability of DAgACP To explore iM + The biomembrane permeability of DAgACP composite materials was investigated, and the effect of DNA molecular weight on this performance was evaluated. Two types of DNA were used in the experiments: iM + DNA (low molecular weight) and commercial salmon sperm DNA (high molecular weight, as a control).

[0059] The method for constructing a Streptococcus mutans biofilm is as follows: 1×10 6CFU / mL bacterial suspension was inoculated into 24-well plates containing sterile coverslips, and BHI medium was added to each well. The plates were statically incubated at 37°C under microaerophilic conditions for 48 hours until the biofilm matured. After biofilm formation, the coverslips were gently washed three times with sterile PBS to remove any unattached airborne bacteria.

[0060] Fluorescent labeling of the composite material: Calcein Blue (54375-47-2; Meilun Pharmaceutical Co., Ltd., USA) was incorporated into iM during the synthesis process. + DAgACP composite material. The pretreated biofilm was then mixed with various calcium chlorophyll blue-labeled iM... + The biofilm was co-incubated with DAgACP suspension at 37°C under microaerophilic conditions for 60 min. After incubation, the biofilm was washed three times with sterile PBS to remove unbound composite material. The biofilm was stained with SYTO™ 9 and propidium iodide (PI) using a live / dead bacterial staining kit (LIVE / DEAD™ BacLight™ Bacterial Viability Kit, Thermo Fisher Scientific, USA), and imaged using a confocal laser scanning microscope (CLSM, Leica Microsystems, Wetzlar, Germany). The calcein blue labeled iM was analyzed. + The penetration depth and distribution of DAgACP within biofilms were compared, and the penetration capabilities of the two systems were examined.

[0061] 3.2.2 Inhibition of biofilm formation To evaluate iM + The inhibitory effect of DAgACP composite material on the formation of Streptococcus mutans biofilm was investigated, with three control groups: iM + DACP (material control, excluding the influence of amorphous calcium phosphate alone), 0.2% CHX (antibacterial positive control), and PBS (negative control with no inhibitory effect). Prepare a Streptococcus mutans suspension as described above, adjusting the final concentration to 1×10⁻⁶. 6 CFU / mL, compared with each test substance (PBS, 0.2% CHX, iM + DACP, iM + DAgACP was mixed at a 1:1 volume ratio. The mixture was inoculated into 24-well plates and statically cultured at 37°C under microaerophilic conditions for 48 hours to allow biofilm formation. Staining was performed using a live / dead bacterial staining kit, and observation was performed using CLSM imaging.

[0062] 3.2.3 Biofilm removal Establish a biofilm clearance model: Using a biofilm clearance model with a concentration of 1×10⁻⁶ during the logarithmic growth phase... 6A CFU / mL Streptococcus mutans suspension was inoculated into 24-well plates with sterile coverslips, and BHI medium was added to each well. The plates were then statically incubated at 37°C under microaerophilic conditions for 24 hours to allow mature biofilm formation. Equal volumes of different test substances (PBS, 0.2% CHX, iM) were added to each well. + DACP, iM + (DAgACP), continue incubation for 24 hours. Stain using a live / dead bacterial staining kit and observe using CLSM imaging.

[0063] 4. Enamel Remineralization Experiment 4.1 Acid etching treatment of tooth enamel samples With the approval of the Ethics Committee of the Affiliated Stomatological Hospital of the School of Stomatology, Air Force Medical University (Approval No.: KQ-YJ-2023-144) and after obtaining written informed consent from the patients, freshly extracted, caries-free permanent teeth were collected. The teeth were stored in a 0.2% (w / w) thymol solution at 4°C to prevent microbial contamination. Enamel blocks were cut using a water-cooled, low-speed diamond cutter to avoid thermal damage. The enamel samples were then polished sequentially with 600, 800, 1200, 2000, and 2500 grit silicon carbide sandpaper to obtain a smooth surface, followed by ultrasonic cleaning with deionized water for 30 minutes to remove polishing residue.

[0064] Acid etching: Enamelled sections were treated with 37% (w / w) phosphoric acid (H3PO4) for 30 seconds to simulate demineralization of early caries samples. After acid etching, the sections were sonicated in deionized water for 15 minutes to thoroughly remove residual acid and contaminants, and then placed in deionized water for later use.

[0065] 4.2 In vitro remineralization of tooth enamel To evaluate the remineralization effect, 100 μL of freshly prepared iM solution was used. + DAgACP composite material, sodium fluoride (NaF, 1500 ppm, remineralization positive control), or deionized water (negative control) were added dropwise to the acid-etched enamel area. The sample was immersed in 10 mL of preheated modified simulated oral fluid MSOF (pH 7.00±0.03; composition: 1.5 mM CaCl2·2H2O, 0.9 mM K2HPO4, 15 ppm NaF, 130 mM KCl, 1 mM NaN3, 20 mM HEPES buffer) and incubated at 37°C for 24 h. The MSOF was replaced every 12 h to maintain a stable ion concentration. After incubation, the sample was thoroughly rinsed with deionized water, air-dried, and characterized. The cyclic remineralization experiment repeated the above steps for three cycles to simulate the long-term remineralization effect under oral conditions.

[0066] 4.3 Characterization Methods 4.3.1 Scanning Electron Microscopy and Energy-Dispersive X-ray Spectroscopy After platinum sputtering, the morphology of the enamel surface (top view) and the structure of the remineralized layer (cross-section) were observed using a scanning electron microscope (SEM, S-4800, Hitachi, Tokyo, Japan) at an accelerating voltage of 5 kV. The elemental composition and distribution of the remineralized layer were analyzed using EDX on the SEM.

[0067] 4.3.2 X-ray photoelectron spectroscopy The chemical valence state and elemental composition of the remineralized glaze surface were analyzed at room temperature using XPS (Thermo Fisher K-Alpha) with Al Kα (hν=1486.6eV) as the excitation source.

[0068] 4.3.3 X-ray diffraction The crystal structure of the remineralized glaze was characterized using XRD (Rigaku SmartLab SE, Japan) with CuKα radiation (λ = 1.5418 Å), operating voltage 30 kV, and current 20 mA. The crystal structure was identified by scanning at a rate of 2° / min within the range of 2θ = 5° to 70°.

[0069] 4.4 In vitro remineralization of tooth enamel in the presence of Streptococcus mutans biofilm To evaluate iM + The remineralization capacity of DAgACP in enamel in the presence of mature Streptococcus mutans biofilm. All samples were subjected to... 60 Sterilization was performed using Co gamma rays. Enamellable samples were first immersed in artificial saliva for 30 minutes to promote acquired membrane formation. Streptococcus mutans was then sterilized at a concentration of 1×10⁻⁶. 6 CFU / mL inoculum was seeded onto the enamel surface and cultured in BHI medium supplemented with 5% (m / v) sucrose at 37°C for 24 h to construct a mature biofilm. The enamel slides covering the biofilm were treated with the following reagents: PBS, 0.2% CHX, sodium fluoride (NaF), and iM... + DACP, iM + DAgACP nanoassemblies. 100 μL of each treatment solution was locally applied to the biofilm surface and allowed to stand for 2 hours. The samples were then transferred to 10 mL of fresh artificial saliva and maintained at 37°C for 24 hours. To simulate a dynamic oral environment, the remineralization solution was changed every 6 hours. After the experiment, the samples were fixed with 2.5% (v / v) glutaraldehyde at 4°C for 24 hours, dehydrated stepwise with a gradient of ethanol (30%–100%, v / v), dried, and platinum-sprayed. Scanning electron microscopy was used to observe changes in enamel morphology and the structural characteristics of the adhered biofilm.

[0070] 5. Animal experiments 5.1 Animal preparation and ethical approval Male SD rats (8 weeks old, weighing 200g-300g) were randomly divided into 4 groups (n=3 per group) corresponding to 4 treatment conditions: deionized water (negative control group), sodium fluoride (NaF, 1500ppm, remineralization positive control group), and iM + DACP composite material (material control group), iM + DAgACP composite material (experimental group). All animal experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Fourth Military Medical University (approval number: IACUC20250096) and strictly followed the guidelines for the breeding and use of laboratory animals.

[0071] 5.2 Rat dental caries model and evaluation of treatment effects To evaluate iM + To investigate the in vivo preventive and therapeutic effects of DAgACP composite material on dental caries in rats and to verify its systemic and local biocompatibility, the following experiment was conducted: Eighteen 3-week-old male specific pathogen-free (SPF) Sprague Dawley (SD) rats were selected. All animal experiments were approved by the Laboratory Animal Care and Use Committee (IACUC) of the Fourth Military Medical University (approval number: IACUC20250096) and followed international laboratory animal welfare standards.

[0072] To reduce oral background flora and facilitate Streptococcus mutans colonization, rats were given water supplemented with a non-enteric pathogenic antibiotic [sodium penicillin + streptomycin sulfate, 1.0 g / kg body weight / day] for 3 consecutive days. After drug withdrawal, rats were given 0.3 mL of water containing Streptococcus mutans (1×10⁻⁶) daily. 6 Infection was performed by gavage with fresh brain heart infusion (BHI) culture medium (CFU / mL) for 10 consecutive days. Infection was performed after a 2-hour fast to enhance bacterial adhesion. Successful colonization of *Streptococcus mutans* was confirmed on day 10 by oral swabs: the swab was streaked onto a bacitracin (MSB) agar plate and incubated at 37°C for 48 hours; the appearance of black colonies (characteristic colonies of *Streptococcus mutans* on MSB plates) was considered a successful infection.

[0073] Rats that were successfully infected were randomly divided into 3 groups (n=6 in each group): PBS group: negative control group (no treatment effect); 0.2% CHX group: positive control group (known antibacterial / anti-caries effect); iM +DAgACP group: Experimental group. The drug was applied topically once daily using a sterile dental microbrush for 4 consecutive weeks: 150 μL of the test agent was applied to the buccal and occlusal surfaces of all maxillary and mandibular molars, maintaining contact for 2 min. After treatment, rats were fasted and deprived of water for 30 min to prevent immediate ingestion of the material. During infection and treatment, all rats were fed a cariogenic Keyes2000 diet and had free access to 5% sucrose water to maintain a cariogenic environment.

[0074] Four weeks after treatment, the Streptococcus mutans load was assessed by oral swab culture. SD rats were euthanized by intraperitoneal anesthesia with sodium pentobarbital (100 mg / kg body weight). Maxillary molars were scanned using a microcomputed tomography (MicroCT) system (Bruker SkyScan 1176, Belgium) with parameters of 80 kV voltage, 100 μA current, 10 μm pixel size, and the entire crown of the molar being scanned. Three-dimensional reconstruction of the crown and hemicrown was performed using CTVOL software (ver. 2.3.2.0, Bruker MicroCT, USA). The volume of caries (demineralized area) was quantified using CTAn software (Bruker MicroCT, USA), and a fixed grayscale threshold was used to distinguish between caries and healthy enamel / dentin.

[0075] The maxilla was stained with 0.1% ammonium purpurate solution (pH 8.3) at room temperature for 10 min, and rinsed with distilled water to visualize caries. The molars were cut mesiodistally using a water-cooled low-speed diamond cutter, and cross-sectional photographs were taken using a stereomicroscope (Nikon SM218, Japan). The severity of caries was assessed blinded by two independent pathologists using the Keyes scoring system: E: enamel caries only; Ds: dentin caries, involving 1 / 4–3 / 4 of the dentin; Dm: dentin caries, involving >3 / 4 of the dentin.

[0076] 6. Statistical Analysis All data are expressed as mean ± standard deviation (mean ± SD). Statistical analysis was performed using GraphPad Prism 8.0 software (La Jolla, CA, USA). Normality was verified using the Shapiro-Wilk test before parametric tests, and homogeneity of variance was verified using the corrected Levene test. Differences among multiple groups were analyzed using one-way or two-way ANOVA, followed by the Bonferroni multiple comparison test; nonparametric data were analyzed using the Kruskal-Wallis H test, followed by the Dunn multiple comparison test. The statistical significance level was set at α = 0.05. p Value label: * p<0.05,** p <0.01, *** p <0.001.

[0077] II. Results: 1. Design and characterization of pH-responsive DNA nanoassemblies This invention designs an oligonucleotide (iM) capable of forming an i-Motif structure. + DNA), design process such as Figure 1 As shown in A in the figure. Multiple characterization methods were used to verify iM. + pH-responsive i-Motif structures are formed in DNA.

[0078] Circular dichroism (CD) results ( Figure 1 B) shows that at pH 7.4, iM + DNA exhibits a weak positive peak at approximately 275 nm, indicating a random coil conformation; however, under pH 5.0 conditions, iM + The DNA exhibited typical i-Motif CD characteristic signals, with a strong positive peak at 288 nm and a distinct negative peak at 260 nm, confirming intramolecular C-C⁺ base pairing. - The DNA was a control oligonucleotide that did not contain cytosine, could not be protonated, and could not form an i-motif (sequence: 5′TTTAATTTTAATTTTAATTTTAATTTTAATTTTAATTTTT3′, nucleotide sequence as shown in SEQ ID NO.2). Non-denaturing polyacrylamide gel electrophoresis (PAGE) results showed ( Figure 1 C in (iM) + DNA migrates faster at pH 5.0 than at pH 7.4, while the migration rate of iM DNA remains essentially unchanged under both pH conditions. + DNA migration is accelerated under acidic conditions, possibly due to folding to form i-Motif structures with smaller hydrodynamic radii. Thioflavin T (ThT) fluorescence assay (… Figure 1 The D-values ​​in the study show that fluorescence is significantly enhanced at pH 5.0, further confirming the formation of the i-Motif structure.

[0079] iM + The preparation process of DAgACP nanoassemblies is as follows: Figure 1E is shown in the figure. High-resolution transmission electron microscopy (HRTEM) and energy dispersive X-ray spectroscopy (EDS) surface scan results (…). Figure 1 The F) in the image shows that calcium (Ca), phosphorus (P), oxygen (O), nitrogen (N), and silver (Ag) are uniformly distributed; the selected area electron diffraction (SAED) shows a diffuse ring, indicating that iM + DAgACP has an amorphous structure. Particle size distribution ( Figure 1 The G spectrum shows that the average particle size is approximately 65.76 nm. Energy dispersive spectroscopy (EDS) Figure 1 The presence of silver was confirmed by H in the sample; quantitative analysis of silver content showed that only iM + DAgACP can detect silver (mass fraction 0.92 wt%). UV-Vis absorption spectrum ( Figure 1 The I) in the image shows that iM + DAg and iM + The DAgACP sample exhibits a characteristic absorption peak of silver nanoclusters at approximately 430 nm.

[0080] Using X-ray photoelectron spectroscopy (XPS) and peak fitting ( Figure 1 Analysis of the chemical valence states of elements (J and K) in silver: Ag appears in the 3d orbitals at 368.2 eV and 374.2 eV. 0 Characteristic peaks; characteristic peaks appear in the calcium 2p orbitals at approximately 347 eV and 351 eV, confirming the presence of Ca in ACP. 2+ The existence of. Figure 1 In the image, L~N represent locally magnified Fourier transform infrared (FTIR) spectra, showing a significant peak shift in DNA functional groups, including approximately 1645 cm⁻¹. -1 The C=O and C=N stretching vibration peaks are located at approximately 1220 cm⁻¹. -1 The peak of the asymmetric stretching vibration of PO2⁻ is approximately 1056 cm⁻. -1 The peak of the COP symmetrical stretching vibration is observed. The aforementioned shift indicates that iM... + DAgACP contains DNAAg and DNACa. 2+ Interacting. Simultaneously, iM + DACP and iM + All DAgACP groups were at 500cm -1 ~600cm -1The presence of ACP characteristic peaks in the region confirms the successful synthesis of ACP. In summary, the above results collectively confirm iM + The DAgACP nanoassembly was successfully constructed.

[0081] 2. iM + Biomembrane penetration ability and pH response behavior of DAgACP nanoassemblies After successfully synthesizing iM + Following DAgACP, this invention systematically evaluated its biofilm penetration ability and pH response behavior, such as... Figure 2 As shown in A in the figure. To evaluate iM + The penetration effect of DAgACP on mature biofilms was investigated using calcein blue fluorescent labeling. Streptococcus mutans biofilms were subjected to calcein blue-labeled iM... + Co-incubation with DAgACP followed by live / dead bacterial staining. Images obtained using confocal laser scanning microscopy (CLSM). Figure 2 B in Figure 2 C) shows: After 1 hour of processing, iM + DAgACP can be uniformly distributed throughout the entire Streptococcus mutans biofilm, showing fluorescent signals in both the surface and deep layers; while complexes assembled from high-molecular-weight natural (HMWN) DNA are almost unable to penetrate, mainly confined to the biofilm surface. These results confirm that iM + DAgACP exhibits stronger biomembrane penetration capability, an advantage that can be attributed to iM + DNA at the nanoscale. Compared to natural nucleic acids, its smaller size significantly reduces steric hindrance, making it easier to penetrate deep into biological membranes.

[0082] CLSM cross-sectional images reveal a significant phenomenon: compared to the HMWNDAgACP-treated group, low molecular weight iM + The biofilm in the DAgACP-treated group exhibited stronger red fluorescence, indicating that the former led to a higher proportion of bacterial death. These results suggest that iM + DNA can respond to the acidic microenvironment within biological membranes; once activated, it promotes the release of Ag⁺, thereby enhancing iM. + The bactericidal effect of DAgACP. In contrast, the AgNCs in HMWNDAgACP, which is not pH-responsive, mostly remain in a stable DNA-bound state, exhibiting weaker antibacterial activity and therefore weaker red fluorescence. To further verify this hypothesis, ThT fluorescence spectroscopy was used (…). Figure 2D) and inductively coupled plasma-mass spectrometry (ICPMS) Figure 2 (E) The structural transformation and pH-responsive silver release performance were examined. ThT spectroscopy showed that the fluorescence intensity at pH 5.0 was significantly higher than at pH 7.4, confirming the presence of pH-dependent i-Motif structure formation. ICP-MS results indicated that the cumulative silver release at pH 5.0 was 48.80% ± 0.05%, approximately 2.6 times that at pH 7.4 (18.59% ± 0.07%). These results demonstrate that i-Motif... + The DAgACP nanoassemblies exhibit acid-triggered, on-demand release behavior.

[0083] To elucidate the structural basis of pH-responsive silver release, this invention employs molecular dynamics (MD) simulations. Since directly modeling variable protonation states at different pH levels is computationally extremely costly, a representative conformation strategy is adopted: linear DNA and i-Motif DNA structures are constructed separately to simulate the DNA-AgNC complex under neutral and acidic conditions, allowing for direct comparison of the interactions between different DNA structures and AgNCs. Figure 2 (F in the original text). During a 100 ns simulation, the changes in structural parameters over time can quantitatively reflect the conformational transitions of DNA at different pH levels. For example... Figure 2 As shown in G, the root mean square deviation (RMSD) of DNA heavy atoms increases rapidly within the first 20 ns, then gradually stabilizes. The equilibrium RMSD of linear DNA is 5–6 Å, higher than that of i-Motif DNA (3–4 Å), indicating its more flexible conformation; while the lower RMSD of i-Motif DNA reflects its greater rigidity and structural stability under acidic conditions. Consistent with the RMSD trend, the radius of gyration (Rg) of linear DNA is 6.0–7.0 Å, while the Rg of i-Motif DNA is stable at 4.3–4.9 Å. Figure 2 The smaller Rg in i-Motif DNA quantitatively reflects its dense, highly folded structure, a direct result of CC⁺ base pairing. Solvent-accessible surface area (SASA) analysis (… Figure 2I) in the above analysis supports the conclusion that linear DNA has a larger SASA (375–420 Ų, average ≈400 Ų), indicating that the bases and phosphate backbone are largely exposed to the solvent; while i-Motif DNA has a significantly reduced SASA (315–330 Ų, average ≈325 Ų), indicating that it has a dense folded conformation with a large number of hydrophilic groups embedded within. The quantitative result of a SASA reduction of approximately 75 Ų strongly supports the shielding effect caused by folding: this effect reduces the accessibility of AgNCs to the binding sites of solvent molecules. Hydrogen bond analysis ( Figure 2 The results (J) show that the number of intramolecular hydrogen bonds in i-Motif DNA (150–200, average ≈175) is significantly higher than that in linear DNA (25–65, average ≈45). This dense hydrogen bond network makes the i-Motif structure more stable and compact; while linear DNA has fewer hydrogen bonds and is relatively flexible overall. The formation of new hydrogen bonds after acidification highlights the crucial role of cytosine protonation and subsequent C⁺ pairing in driving DNA structural rearrangement.

[0084] Binding energy analysis ( Figure 2 K~ Figure 2 The L in the figure indicates that the DNAAgNC complexes under both conditions reached near-stability after approximately 20 ns. Compared to the i-Motif DNAAgNC complex, the linear DNAAgNC complex exhibited a stronger binding energy. The higher absolute binding energy of the linear complex suggests that its open conformation and larger surface area facilitate multiple coordination and electrostatic interactions with silver atoms; conversely, the dense i-Motif structure restricts the entry of AgNCs, leading to weakened binding affinity. In summary, these results indicate that pH-induced DNA folding can modulate AgNC binding strength, thereby controlling the controlled release of silver from the nanoassembly.

[0085] Finally, a dynamic snapshot during the 100ns simulation process ( Figure 2 The molecular dynamics results (M) visually demonstrate the above behavior: In the i-Motif system, AgNCs mainly remain on the periphery of the DNA structure, constrained by steric hindrance and electrostatic interactions, forming a stable but limited binding conformation; while in the linear DNA system, AgNCs gradually approach and embed into the major and minor grooves of the DNA, forming multiple coordination modes and a wider binding interface. These molecular dynamics results collectively reveal the iM +The molecular mechanism of pH-responsive interaction between DNA and AgNCs: Under neutral or weakly alkaline conditions, the DNA strand exhibits an extended linear conformation with a flexible backbone. A large number of bases and phosphate groups are exposed to the solvent and AgNCs, providing multiple potential binding sites and a large contact area for AgNCs. However, under acidic pH conditions, cytosine protonation induces DNA folding into a dense i-Motif quadruplex, forming a close-packed structure, significantly reducing the accessibility of AgNCs. These structural understandings corroborate experimental results, confirming the i-Motif interaction. + DAgACP nanoassemblies exhibit pH-triggered, structure-dependent response characteristics.

[0086] 3. iM + pH-responsive antibacterial activity of DAgACP To evaluate its pH-responsive antibacterial properties, planktonic Streptococcus mutans was mixed with iM + DAgACP or iM + DACP was co-cultured within the pH range of 7.4–4.0, and bacterial growth was monitored by measuring OD600. Figure 3 A and Figure 3 (B in the text). Compared to neutral and weakly acidic conditions (pH 7.4 and 6.5), iM + DAgACP nanoassemblies exhibited significantly enhanced antibacterial activity under acidic conditions (pH < 6.5). In contrast, iM + DACP showed similar antibacterial efficacy to the PBS control group under all tested pH conditions. Plate count experiments further validated these results. Figure 3 C~ Figure 3 E in: Under neutral conditions, iM + The growth of Streptococcus mutans in the DAgACP group was significantly lower than that in the PBS and iM groups. + The DACP group showed a decrease, but the difference was not statistically significant (p=0.0815). This indicates that iM + DAgACP does not exhibit strong antibacterial activity in a neutral environment and demonstrates good safety in scenarios where sterilization is not required. However, under acidic conditions, it shows better performance compared to PBS and iM... + Compared to the DACP group, iM + DAgACP treatment can reduce viable bacterial count by more than 90%. It is worth noting that iM + DAgACP differs fundamentally from the traditional bactericide chlorhexidine (CHX) in its mechanism of action: CHX treatment results in OD... 600 It remains largely stable, consistent with its mechanism of causing intracellular material coagulation without causing cell lysis; while iM + DAgACP group OD 600The significant reduction suggests that it may have strong lytic activity, which can directly cause physical rupture of Streptococcus mutans cells.

[0087] Based on its therapeutic effect against planktonic Streptococcus mutans, the efficacy of iM was further investigated. + The inhibitory and scavenging abilities of DAgACP on biofilms. For example... Figure 3 As shown in F, with iM + After co-incubation with DAgACP for 48 hours, the biofilm accumulation was significantly reduced, and the inhibition rate on Streptococcus mutans biofilm formation was approximately 72%. Figure 3 (G in the text). Furthermore, iM + When DAgACP is applied to a pre-formed mature biofilm and cultured for another 24 hours, significant disruption of the biofilm structure can be observed. Figure 3 (H in the PBS group). Using the PBS group as a reference, iM + DAgACP has a biofilm clearance rate of approximately 62% ( Figure 3 (I in the text). In summary, iM + DAgACP nanoassemblies possess pH-responsive antibacterial properties, which can respond to the acidic microenvironment within cariogenic biofilms, kill Streptococcus mutans, and disrupt the biofilm structure, thereby effectively preventing caries-related demineralization processes.

[0088] 4. Based on iM + In vitro enamel remineralization with DAgACP To evaluate iM + To assess the remineralization capacity of DAgACP, this invention establishes two in vitro models to simulate different levels of oral hygiene and microbial complexity, corresponding to different clinical scenarios: (1) a sterile remineralization environment (bacteria-free), simulating the ideal oral state after professional teeth cleaning; and (2) a mature biofilm-mediated model, simulating the oral microenvironment where interdental spaces are difficult to clean and plaque easily accumulates. By gradually increasing the biocomplexity of the models, the aim is to comprehensively evaluate the effects of the material under different physiological conditions.

[0089] First, evaluate iM in a sterile environment. + The remineralization ability of DAgACP ( Figure 4 (A) This model simulates the ideal oral cavity state after teeth cleaning. Natural tooth enamel has a smooth and dense surface with regularly arranged hydroxyapatite crystals; however, after acid etching, the enamel exhibits severe demineralization, destruction of the enamel prism structure, and surface porosity. Based on the mineralization precursor role of ACP and the excellent affinity of DNA for hydroxyapatite, this invention designs iM... + DAgACP can adhere to acid-etched enamel surfaces and induce epitaxial regeneration of crystals. Consistent with the hypothesis, this nanoassembly can effectively penetrate and bind to enamel prisms, subsequently transforming into oriented crystals consistent with the orientation of natural tooth enamel. SEM results ( Figure 4 Confirmed by B) in iM+ After DAgACP treatment, a dense and regularly arranged mineralized layer was formed, with a seamless interface between the newly formed layer and the natural layer, indicating successful epitaxial regeneration of enamel crystals. Furthermore, the thickness of the regenerated layer increased with the number of treatment cycles: 1.9 ± 0.1 μm for one cycle, 3.3 μm ± 0.4 μm for two cycles, and 5.6 μm ± 0.5 μm for three cycles. Figure 4 B and Figure 4 (C in the text). In contrast, enamel treated with modified simulated oral fluid (MSOF) or fluoride only showed sparse flaky or needle-like deposits, with limited structural repair effects.

[0090] The chemical composition and crystallinity of the regenerated layer were further verified. EDS and XPS analysis were performed. Figure 4 D and Figure 4 The E-ray (XRD) shows that the main elements are C, O, Ca, and P, consistent with the composition of natural tooth enamel. Notably, no Ag signal was detected, indicating that AgNCs were not incorporated into the newly formed crystals, ensuring the purity of the mineralized phase. (XRD pattern) Figure 4 The F in the sample exhibits distinct hydroxyapatite characteristic diffraction peaks (002, 210, 211, 202, 213, 004), matching the standard card in PDF 090432, confirming that the regenerated layer has a hydroxyapatite-like structure. Meanwhile, iM... + The absence of obvious characteristic peaks in the DAg group indicates that ACP is indispensable for crystal growth.

[0091] More importantly, to evaluate its effectiveness in difficult-to-clean areas where plaque easily accumulates, a mature biofilm-mediated model was used ( Figure 4 G in the control group (PBS, iM). + SEM images of DACP and NaF show ( Figure 4 In the case of H), acid etching of the enamel surface covers a thick, mature biofilm of Streptococcus mutans. While CHX treatment can destroy the biofilm, it does not provide mineralization or repair. In contrast, iM... + DAgACP can effectively penetrate mature biofilms, exerting a strong antibacterial effect and significantly degrading biofilms; simultaneously, the nanoassemblies can reach the underlying acid-etched enamel surface to induce remineralization. In summary, the above results indicate that iM + DAgACP nanoassemblies can effectively promote the formation of dense hydroxyapatite layers under both neutral (sterile) and acidic (bacterial infection) conditions, laying a solid foundation for subsequent in vivo applications.

[0092] 5. Based on iM + In vivo enamel remineralization of DAgACP To verify iM +The therapeutic potential of DAgACP under physiological conditions; this invention establishes a rat caries model ( Figure 5 (A) The colonization of *Streptococcus mutans* was monitored using *Mitis salivarius* agar (MSB) plates to confirm the successful establishment of the model. Four days after antibiotic treatment, no bacterial growth was observed on the plates; however, ten days after inoculation and colonization with bacterial suspension, numerous colonies appeared, confirming the successful establishment of the *Streptococcus mutans* infection model. Figure 5 (B~D in the text). After 4 weeks of continuous treatment, compared with the PBS group, iM + Both the DAgACP and CHX groups showed a significant reduction in colony-forming units (CFU), indicating that the nanocomposite material can effectively inhibit bacterial proliferation in vivo and has a good antibacterial effect.

[0093] Furthermore, effective caries prevention depends not only on antibacterial activity but also on remineralization capacity. Therefore, this invention uses ammonium purpurate staining and Keyes scoring to assess the extent of caries damage. Stereomicroscopic observation ( Figure 5 The E) results showed that both the PBS and CHX groups exhibited significant dentin caries (Ds / Dm / Dx); while the iM + The DAgACP group primarily presented with mild enamel caries (E), without deep structural damage. iM + The DAgACP group showed a significant decrease in total Keyes scores on both smooth and fissure surfaces, and a significant reduction in the incidence of severe caries. Figure 5 F~ Figure 5 (H in the text). The above results confirm that iM + DAgACP nanoassemblies exhibit excellent anti-caries effects.

[0094] Micro-CT analysis provides direct structural evidence for the above conclusions. Figure 5 (I) Two-dimensional sagittal sections and three-dimensional reconstructed images showed that the PBS group and CHX group exhibited significant demineralization and enamel defects; while iM + The enamel structure of the molars treated with DAgACP remained intact, with minimal caries. Imaging results were consistent with microbiological and histological evaluations, confirming that the nanocomposite material effectively inhibits bacterial colonization while maintaining enamel mineralization density.

[0095] In summary, the in vivo experimental results indicate that iM + DAgACP can effectively inhibit Streptococcus mutans colonization, reduce the occurrence of dental caries, and protect the integrity of tooth enamel, showing potential as a novel anti-caries nanomedicine for clinical translation.

[0096] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0097] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. A pH-responsive antibacterial-biomimetic mineralizing material, characterized in that, The antibacterial-biomimetic mineralizing material is made of iM + DNA-stable silver nanoclusters and amorphous calcium phosphate are synergistically assembled; The iM + DNA-stabilized silver nanoclusters are based on iM + Silver nanoclusters were synthesized and stabilized using DNA as a template via a sodium borohydride chemical reduction method; the iM + The DNA is an oligonucleotide containing an i-Motif forming domain, and its nucleotide sequence is shown in SEQ ID NO.

1.

2. The method for preparing the antibacterial-biomimetic mineralized material according to claim 1, characterized in that, Includes the following steps: With the iM + Using DNA as a template, iM + DNA was mixed with an aqueous solution of silver nitrate and incubated at 3°C–5°C. Sodium borohydride was then added for chemical reduction to obtain iM. + DNA-stable silver nanoclusters, denoted as iM + DNA-AgNC; To the iM + DNA-AgNC was added to a supersaturated calcium phosphate solution, and iM was used. + DNA phosphate backbone for Ca 2+ Electrostatic enrichment and the heterogeneous nucleation-inducing effect of AgNCs lead to Ca 2+ With phosphate in iM + In situ deposition of amorphous calcium phosphate on the surface of DNA-AgNC, thus obtaining iM + DNA-AgNC / ACP composite material, namely the antibacterial-biomimetic mineralized material.

3. The preparation method according to claim 2, characterized in that, The molar ratio of calcium ions to phosphate ions in the supersaturated calcium-phosphorus solution is 1.66 to 1.68, and the pH of the supersaturated calcium-phosphorus solution is 6.97 to 7.

03.

4. The preparation method according to claim 2, characterized in that, The reaction temperature for the formation of amorphous calcium phosphate is 36℃~38℃, and the reaction time is 25min~35min.

5. The use of the antibacterial-biomimetic mineralizing material according to claim 1 in the preparation of oral medical materials for preventing tooth decay, antibacterial purposes, or promoting tooth remineralization.

6. The application according to claim 5, characterized in that, The oral medical materials include toothpaste, mouthwash, dental varnish, fissure sealant, or dental adhesive.

7. The application according to claim 6, characterized in that, The oral medical materials are used for the prevention and treatment of early enamel caries.

8. An oral medical material for preventing tooth decay, antibacterial purposes, or promoting tooth remineralization, characterized in that, It includes the antibacterial-biomimetic mineralization material as described in claim 1.

Images