Tetrahedral framework nucleic acid-metformin compound as well as preparation method and application thereof

Abstract

The invention provides a tetrahedral framework nucleic acid-metformin compound as well as a preparation method and application thereof, and belongs to the field of medicines. A novel nano-drug system is constructed for the first time, metformin is efficiently delivered to macrophages based on a delivery system of tetrahedral framework nucleic acid, the NLRP3-mediated pyroptosis and AMPK / NF-kappa B signal axis are effectively adjusted, the cellular uptake and treatment effect of metformin in treatment of diabetic periodontitis are remarkably enhanced, and the metformin nano-drug has a good application prospect in treatment of diabetic periodontitis. And the method has a good industrial application prospect.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to a tetrahedral framework nucleic acid-metformin complex and a preparation method and use thereof. Background Art

[0002] Diabetic periodontitis is a serious complication of diabetes, characterized by enhanced inflammatory response, accelerated tissue destruction, and increased susceptibility to periodontal disintegration. Metabolic disturbances caused by hyperglycemia interact with chronic low-grade inflammation, leading to periodontal tissue damage and posing a challenge to treatment. Conventional treatments have limited effects on diabetic patients, and innovative approaches are needed to address the inflammatory and metabolic aspects of the disease. Exploring molecular pathways such as NF-κB, AMPK, and pyroptosis has become a promising therapeutic direction.

[0003] The NF-κB signaling pathway drives the inflammatory response by upregulating proinflammatory factors such as IL-6 and TNF-α. Under hyperglycemia, AGEs accumulation continuously activates NF-κB, forming a chronic inflammatory cycle and driving macrophages to develop into a proinflammatory M1 phenotype. As a metabolic regulator, AMPK reduces oxidative stress, restores cellular energy balance, promotes tissue repair, and promotes M2 macrophage polarization to alleviate periodontal inflammation by counteracting NF-κB signals. Pyroptosis is induced by oxidative stress and AGEs accumulation, releasing IL-1β and IL-18, further exacerbating inflammation. NF-κB activation, AMPK inhibition, and pyroptosis together constitute the pathological network of diabetic periodontitis. Regulating these pathways is expected to provide an effective treatment strategy.

[0004] Metformin (Met) is an anti-diabetic drug and AMPK activator with anti-inflammatory and antioxidant properties. By activating AMPK, metformin can inhibit NF-κB, thereby reducing the production of pro-inflammatory factors, inhibiting cell pyroptosis-related signals, and reducing inflammation and tissue damage. However, under the condition of hyperglycemia, the cellular uptake of metformin is limited, and its bioavailability and intracellular delivery efficiency are often insufficient. Summary of the invention

[0005] The purpose of the present invention is to provide a tetrahedral framework nucleic acid-metformin complex and a preparation method and use thereof.

[0006] The invention provides a tetrahedral framework nucleic acid-metformin complex, which is composed of a mixture of tetrahedral framework nucleic acid and metformin, wherein the molar ratio of the tetrahedral framework nucleic acid to metformin is 1:50-900.

[0007] Furthermore, the molar ratio of the tetrahedral framework nucleic acid to metformin is 1:100-800, preferably 1:600.

[0008] Furthermore, the tetrahedral framework nucleic acid is self-assembled from four DNA single strands, and the sequences of the four single strands are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 respectively.

[0009] The present invention also provides a method for preparing the tetrahedral framework nucleic acid-metformin complex, which comprises the following steps: mixing the tetrahedral framework nucleic acid and metformin, reacting them, and filtering them to obtain the complex.

[0010] Furthermore, the reaction temperature is 30-45° C. and the reaction time is 2-4 hours.

[0011] Furthermore, the reaction temperature is 37° C. and the reaction time is 3 hours.

[0012] The present invention also provides use of the tetrahedral framework nucleic acid-metformin complex in preparing a drug for preventing and / or treating periodontitis.

[0013] Furthermore, the periodontitis is diabetic periodontitis.

[0014] Furthermore, the diabetes is type II diabetes.

[0015] Furthermore, the drug is a drug that inhibits NLRP3-mediated pyroptosis and NF-κB signaling pathway.

[0016] The tetrahedral framework nucleic acid (tFNA)-based delivery system of the present invention efficiently delivers metformin (Met) to macrophages, effectively regulates key inflammatory pathways, including NLRP3-mediated pyroptosis and AMPK / NF-κB signaling axis, significantly enhances the cellular uptake and therapeutic effect of Met in the treatment of diabetic periodontitis, and has good prospects for industrial application.

[0017] The present invention has achieved the following beneficial effects:

[0018] (1) A novel nanodrug system, tFNA-Met complex (TMC), was constructed for the first time and demonstrated efficient Met loading (about 70%) and rapid cellular internalization, reaching high concentrations in macrophages within 3 hours;

[0019] (2) In in vitro and in vivo experiments, TMC nano-drug delivery system was used to observe macrophages cultured in a high-glucose environment. TMC significantly inhibited the expression of pro-inflammatory cytokines (IL-1β, IL-18, IL-6, TNF-α) and osteoclast differentiation markers (MMP9).

[0020] (3) In in vitro and in vivo experiments, TMC inhibited NLRP3-mediated pyroptosis by downregulating macrophage NLRP3, Caspase-1, Caspase-11, and GSDMD, thereby reducing the production of IL-1β and IL-18. In addition, TMC activated AMPK and inhibited downstream NF-κB signaling, thereby alleviating the inflammatory response in type II diabetic periodontitis;

[0021] (4) In vivo experiments, TMC treatment demonstrated protective effects on periodontal tissues in type II diabetic mice by intraperitoneal injection of TMC, such as reduced inflammatory cell infiltration, collagen degradation, and osteoclast formation, and alleviated alveolar bone loss.

[0022] (5) TMC showed no obvious toxic effects in both in vitro and in vivo experiments.

[0023] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0024] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the synthesis of tFNA-Met complex (TMC).

[0026] Figure 2 (A) is the fluorescence intensity of Hochest 33342 fluorescence competition experiment; (B) is the absorbance of pure tFNA, pure Met, and TMC at different wavelengths.

[0027] Figure 3 (A) Transmission electron microscope (TEM) image of TMC, (B) Atomic force microscope (AFM) image of TMC, and (C) tFNA and potential detection of TMC.

[0028] Figure 4 The proliferation activity of macrophages after TMC treatment obtained by synthesizing different concentrations of Met and the same concentration of tFNA (such as 1:100, Met:tFNA=25μM:250nM in the solution) was detected by CCK-8.

[0029] Figure 5Confocal microscopy was used to detect the uptake of TMC in macrophages: (A) Cy5-tFNA; (B) Cy5-TMC.

[0030] Figure 6 Flow cytometry was used to detect the effect of TMC uptake in macrophages.

[0031] Figure 7 Fluorescence confocal microscopy shows the expression of inflammatory factors IL-1β, IL-18, TNF-α, and IL-6 in macrophages after TMC treatment in vitro (1: blank group; 2: control group; 3: tFNA treatment group only; 4: Met treatment group only; 5: TMC treatment group).

[0032] Figure 8 The fluorescence semi-quantitative expression of inflammatory factors (I) IL-1β, (II) IL-18, (III) TNF-α, and (IV) IL-6 in macrophages after TMC treatment in vitro (1: blank group; 2: control group; 3: tFNA treatment group only; 4: Met treatment group only; 5: TMC treatment group).

[0033] Fig. 9 WB is the expression of inflammatory factors IL-1β, IL-18, TNF-α, and IL-6 in macrophages after TMC treatment in vitro.

[0034] Fig.10 The expression of inflammatory factors (I) IL-1β, (II) IL-18, (III) TNF-α, and (IV) IL-6 in macrophages after TMC treatment in vitro by PCR (1: blank group; 2: control group; 3: tFNA treatment group only; 4: Met treatment group only; 5: TMC treatment group).

[0035] Fig.11 Activation of multinucleated giant cells after TMC treatment in vitro (1: blank group; 2: control group; 3: tFNA treatment group only; 4: Met treatment group only; 5: TMC treatment group).

[0036] Fig.12 WB results: (A) protein expression of MMP9 in multinuclear giant cells after TMC treatment; (B) mRNA expression of MMP9 in multinuclear giant cells after TMC treatment. (1: blank group; 2: control group; 3: tFNA treatment group only; 4: Met treatment group only; 5: TMC treatment group).

[0037] Fig.13The results of qPCR show the protein expression of MMP9 in multinuclear giant cells after TMC treatment. (1: blank group; 2: control group; 3: tFNA treatment group only; 4: Met treatment group only; 5: TMC treatment group).

[0038] Fig.14 WB was used to detect the expression of pyroptotic body-related ligands (NLRP3, Caspase-11, Caspase-1, cleaved Caspase-1, GSDMD and GSDMD-N). (1: blank group; 2: control group; 3: tFNA treatment group only; 4: Met treatment group only; 5: TMC treatment group).

[0039] Fig.15 The figure is a semi-quantitative analysis of the expression of pyroptotic body-related ligands after TMC treatment ((I) NLRP3; (II) Caspase-11; (III) Caspase-1; (IV) cleaved Caspase-1; (V) GSDMD and (VI) GSDMD-N). (1: blank group; 2: control group; 3: tFNA treatment group only; 4: Met treatment group only; 5: TMC treatment group).

[0040] Fig.16 WB was used to detect the expression of AMPK / NF-κB pathway in macrophages after TMC treatment. (1: blank group; 2: control group; 3: tFNA treatment group only; 4: Met treatment group only; 5: TMC treatment group).

[0041] Fig.17 Immunofluorescence was used to detect the expression of AMPK / NF-κB pathway in macrophages after TMC treatment. (1: blank group; 2: control group; 3: tFNA treatment group only; 4: Met treatment group only; 5: TMC treatment group).

[0042] Fig.18 WB semi-quantitative analysis of AMPK / NF-κB pathway protein expression in macrophages after TMC treatment (A: AMPKα; B: P-AMPK; C: P65; D: P-P65). (1: blank group; 2: control group; 3: tFNA treatment group only; 4: Met treatment group only; 5: TMC treatment group).

[0043] Fig.19 In vivo imaging of TMC drug release and distribution in animals: (I) Distribution and metabolism of TMC in the maxilla after different injection methods (top: intraperitoneal injection; bottom: intravenous injection); (II) Distribution and metabolism of TMC in major organs (heart, liver, and kidney) after different injection methods; (III) Statistical graph of Cy5 fluorescence changes in the maxilla after two different injection methods (1: intravenous injection; 2: intraperitoneal injection).

[0044] Fig. 20 (A) Histological observation of inflammation degree images (HE sections) and (B) inflammation score results. (1: control group; 2: T2D-ligation group; 3: T2D-ligation + tFNA group; 4: T2D-ligation + Met group, and 5: T2D-ligation + TMC group).

[0045] Fig.21 Immunofluorescence was used to observe histological pyroptosis and the expression of inflammatory factors. (1: control group; 2: T2D-ligation group; 3: T2D-ligation + tFNA group; 4: T2D-ligation + Met group, and 5: T2D-ligation + TMC group).

[0046] Fig. 22 Fluorescence semi-quantitative histological pyroptosis and inflammatory factor expression. (1: control group; 2: T2D-ligation group; 3: T2D-ligation + tFNA group; 4: T2D-ligation + Met group, and 5: T2D-ligation + TMC group).

[0047] Fig.23 To observe the degree of pyroptosis and inflammation by histology (IHC). (1: control group; 2: T2D-ligation group; 3: T2D-ligation + tFNA group; 4: T2D-ligation + Met group, and 5: T2D-ligation + TMC group).

[0048] Fig.24 Semi-quantitative analysis of IHC inflammatory factor expression. (1: control group; 2: T2D-ligation group; 3: T2D-ligation + tFNA group; 4: T2D-ligation + Met group, and 5: T2D-ligation + TMC group).

[0049] Fig.25 For imaging observation of the jaw level (Micro-CT): (I) 3D reconstruction of the jaw; (II) XY-axis plane of the maxillary ligature area; (III) XZ-axis plane of the maxillary ligature area. (1: control group; 2: T2D-ligature group; 3: T2D-ligature + tFNA group; 4: T2D-ligature + Met group, and 5: T2D-ligature + TMC group).

[0050] Fig.26 Bone volume fraction (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) were analyzed for imaging (1: control group; 2: T2D-ligation group; 3: T2D-ligation + tFNA group; 4: T2D-ligation + Met group, and 5: T2D-ligation + TMC group).

[0051] Fig. 27The degree of osteoclast chemotaxis (Trap) was analyzed histologically. (1: control group; 2: T2D-ligation group; 3: T2D-ligation + tFNA group; 4: T2D-ligation + Met group, and 5: T2D-ligation + TMC group).

[0052] Fig.28 Periodontal collagen fibers (Masson) were analyzed for histology. (1: control group; 2: T2D-ligation group; 3: T2D-ligation + tFNA group; 4: T2D-ligation + Met group, and 5: T2D-ligation + TMC group).

[0053] Fig.29 This is a schematic diagram of the present invention. DETAILED DESCRIPTION

[0054] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0055] Statistical analysis of the present invention: All experimental data were analyzed using GraphPad Prism 9.5.1 software (San Diego, CA, USA). The statistical method for comparison between groups involved one-way analysis of variance (ANOVA) and post hoc analysis (Sidak test). The experimental results are expressed as mean ± standard deviation (n ≥ 3), and P < 0.05 is considered statistically significant.

[0056] Example 1. Preparation of the tetrahedral framework nucleic acid-metformin complex (TMC) of the present invention

[0057] 1. tFNA synthesis: Four specific single-stranded DNAs (ssDNA) S1, S2, S3, and S4 (see Table 1) were added in equimolar amounts to a TM buffer composed of Tris-HCl and MgCl2, and then heated (95°C, 10 minutes) and cooled (4°C, 20 minutes) to obtain self-assembled DNA tetrahedrons (tFNA).

[0058] 2. Complex reaction of metformin (Met) and tFNA: Met solution and tFNA solution were mixed, and the final concentration of Met was controlled to be 600 μM and the final concentration of tFNA was 1 μM, and the mixture was shaken at 37°C for 3 hours. The remaining ssDNA was then removed by ultrafiltration (30 kDa molecular weight filter, Millipore, USA) to obtain tetrahedral framework nucleic acid-metformin complex (tFNA-Met, abbreviated as TMC).

[0059] Table 1. Sequences of single-stranded DNA

[0060]

[0061] The following is a control sample preparation.

[0062] Comparative Example 1: Preparation of Cy5-labeled tetrahedral framework nucleic acid and tetrahedral framework nucleic acid-metformin complex

[0063] 1. Synthesis of Cy5-labeled tFNA: Refer to the method of Example 1, except that four specific single-stranded DNAs (ssDNA) S1-Cy5, S2, S3, and S4 (see Table 1) are selected to obtain Cy5-labeled tFNA.

[0064] 2. Synthesis of Cy5-labeled tetrahedral framework nucleic acid-metformin complex: Referring to the method of Example 1, the Cy5-labeled tetrahedral framework nucleic acid-metformin complex was prepared using the Cy5-labeled tFNA obtained in step 1.

[0065] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0066] Experimental Example 1: Characterization of tFNA and TMC

[0067] 1. Experimental methods

[0068] Microplate reader (Varioskan TM The fluorescence intensity of Hoechst33342 was measured by ultramicrospectrometer ( N50, IMPLEN, Germany) was used to draw the absorption curves of tFNA, free Met and TMC. The size and Zeta potential of tFNA and TMC were measured by dynamic light scattering (DLS, Nano ZS, Malvern, UK). The encapsulation efficiency of TMC was determined by ultramicrospectroscopy using the following formula:

[0069] Met encapsulation efficiency = [(load concentration of tFNA and Met - residual Met concentration) / initial Met concentration] × 100%

[0070] The successful synthesis of tFNA was verified by 8% PAGE. The size and shape of TMC were measured by transmission electron microscopy (TEM) and atomic force microscopy (AFM) (Tecnai G2 F20 S-TWIN, FEI, USA; SPM-Nanoa, SHIMADZU, Japan).

[0071] 2. Experimental results

[0072] Hochest 33342 was mixed with deionized water, free tFNA, free Met and TMC (tFNA:Met = 1:600) respectively. Hochest 33342 fluorescence competition experiment ( Figure 2A) shows that with the addition of Met, Hochest 33342 (λ ex =346nm) decreases in fluorescence intensity ( Figure 2 B), indicating that Met replaced Hochest 33342 bound to tFNA. The spectrum showed that the characteristic absorption peak of TMC was close to tFNA (260nm) and Met (233nm), indicating that Met was successfully loaded on tFNA ( Figure 2 B).

[0073] In addition, dynamic light scattering (DLS) analysis showed that the particle size of tFNA was approximately 10.34 nm, while that of TMC was approximately 16.84 nm ( Figure 3 A, B). The Zeta potential of tFNA is about -7.38 mV, while the Zeta potential of TMC is about -3.57 mV ( Figure 3 C). Transmission electron microscopy (TEM) and atomic force microscopy (AFM) images show that the diameter of TMC is between 15 and 20 nm and has a tetrahedral structure ( Figure 3 A, B).

[0074] The above results indicate that the present invention successfully combines Met with tFNA to synthesize TMC.

[0075] Experimental Example 2: In vitro cell testing of TMC

[0076] 1. Experimental methods

[0077] (1) Cell culture: RAW264.7 cells were cultured in complete medium containing high-glucose Dulbecco's modified Eagle's medium, fetal bovine serum (10%, HyClone, USA), and penicillin-streptomycin solution (1%, HyClone, USA). The concentration of LPS (Sigma-Aldrich, Missouri, USA) in the medium was 1 μg mL -1 , ATP (proteintech, Wuhan) at a concentration of 5 mM was used to induce inflammation and activate NLRP3 inflammasome. To confirm the anti-inflammatory effect of the drugs, cells were pretreated with tFNA, Met, and TMC for 2 h. Except for the blank group, LPS was added for 6 h, and then ATP was treated for 0.5 h. To study the effect of TMC on osteoclast differentiation in vitro, RAW264.7 cells were cultured in six-well plates containing 10% fetal bovine serum (1 × 10 cells per well). ^4 -5×10 ^4Cells were added to each well, and 2 mL of liquid was added to each well. The groups were divided into: 1: blank group; 2: control group; 3: tFNA group (250 nM); 4: Met group (150 μM); TMC group (tFNA: Met = 1: 600), a total of five groups, and each experiment was repeated three times. Except for the blank group, 100 ng / mL of RANKL (Hua'an Biotechnology) was used to induce osteoclast differentiation for 6 days, and the time points for changing the liquid were 0, 3, and 5 days, respectively. Osteoclasts could be observed on the 6th day for further experiments.

[0078] (2) Cell viability of RAW264.7 cells: RAW264.7 cells were cultured in 96-well plates (1×10 ^4 Then, the cells were pretreated with different ratios of tFNA-Met (tFNA:Met = 1:100, 1:200, 1:400, 1:600, 1:800, tFNA concentration was 250 nM) for 2 hours, and then LPS (1 μg mL -1 ) for 6 hours, and ATP (5 mM) was added for 0.5 hours. Subsequently, the cells were cultured in 10% CCK-8 solution (KeyGEN Biotech) at 37°C for 1 hour. Cell viability was measured by OD value at 450 nm.

[0079] (3) Cellular uptake of Cy5-labeled tFNA and TMC: To study the cellular internalization of tFNA and TMC, RAW264.7 cells were cultured in six-well plates (1×10 ^5 Cells) were treated with Cy5-labeled tFNA and TMC for 3 hours. Then fluorescein isothiocyanate (FITC) was used for staining, and the cytoskeleton and nucleus were counterstained with DAPI. Finally, the distribution of Cy5-labeled tFNA and TMC in cells was observed using a confocal microscope (FV3000; Olympus, Tokyo, Japan). The proportion of cells with Cy5 fluorescence to total cells was determined using a flow cytometer (CytoFLEX, Beckman Coulter Inc., Brea, USA).

[0080] (4) Western Blot analysis: After the cells were treated as described above, they were washed three times with PBS and proteins were extracted using a whole cell lysis reagent (KeyGen Biotech, Nanjing, China). The purified samples were centrifuged at 12,000 rpm for 10 min and their concentrations were determined. The samples were mixed with loading buffer (Beyotime, Shanghai, China), heated for 15 min (100°C), and finally stored at -80°C. The target proteins were separated by SDS-PAGE gel, transferred to polyvinylidene fluoride (PVDF) membranes, blocked with blocking buffer for 15 min, and then incubated with various primary antibodies, including anti-IL-1β, anti-TNF-α, anti-IL-6, etc., at 4°C overnight. After washing, they were incubated with secondary antibodies for 1 h, and finally the protein bands were visualized by a chemiluminescence detection system (iBright 1500, Invitrogen, USA).

[0081] (5) Real-time polymerase chain reaction (RT-PCR): Total RNA was lysed with Trizol (Thermo Fisher Scientific, MA, USA), purified, and converted into cDNA using an RNA purification kit (Thermo Fisher Scientific, MA, USA). Green I master mix was used to detect target mRNA in RT-PCR system (ABIQuantStudio 7, Thermo Fisher, USA), and β-actin was used as a control for all target mRNAs.

[0082] (6) Immunofluorescence staining: In order to more intuitively observe the expression and distribution of the target protein in the cells, immunofluorescence experiments were performed. After the previous treatment, the cells were fixed with 4% paraformaldehyde for 0.5 h and then treated with 0.5% Triton X-100 at 37°C for 10 min to perforate the cell membrane. Then, they were blocked with 5% goat serum for 20 min and incubated with the primary antibody overnight. The next day, they were incubated with the secondary antibody for 1 h, and then the cytoskeleton and cell nucleus were stained with phalloidin and DAPI, respectively. Finally, the cells were observed using a laser confocal microscope (FV3000; Olympus, Tokyo, Japan).

[0083] 2. Experimental results

[0084] (1) Biocompatibility

[0085] To determine the optimal concentrations of tFNA and Met, the present invention calculated the encapsulation efficiency of various compositions and verified their biocompatibility by CCK-8 experiment ( Figure 4). The concentration of tFNA in the solution was set to 250 nM, and the ratio range of tFNA:Met was set to 1:100, 1:200, 1:400, 1:600, and 1:800. CCK-8 results showed that cell viability increased with the increase of Met concentration from 25 μM to 200 μM, and no significant difference in cell viability was observed between 100, 150, and 200 μM concentrations. Based on the above results, a tFNA:Met concentration ratio of 1:600 ​​was selected for subsequent experiments.

[0086] (2) tFNA’s excellent delivery performance

[0087] Using confocal microscopy and flow cytometry, the present invention studied the uptake of Cy5-labeled tFNA and TMC in RAW264.7 cells. Confocal microscopy results ( Figure 5 ) showed that Cy5-labeled tFNA and TMC were widely distributed in cells, and the content of TMC was higher. Flow cytometry yielded similar results ( Figure 6 ), indicating that the addition of Met did not affect the excellent cellular uptake and delivery performance of tFNA.

[0088] (3) Inhibitory effect of TMC on LPS-induced macrophage inflammation and osteoclast differentiation

[0089] Periodontitis is an inflammatory disease associated with a variety of inflammatory cytokines, including the IL-1 family (IL-1β, IL-18), the IL-6 family, and TNF-α. Reducing the levels of these cytokines is an effective method for treating periodontitis. The present invention constructed an in vitro inflammatory microenvironment model of T2D periodontitis by stimulating RAW264.7 macrophages cultured in a high-sugar environment with LPS+ATP. qPCR analysis ( Fig.10 ) showed that LPS+ATP treatment significantly upregulated the mRNA levels of IL-1β, IL-18, TNF-α, and IL-6. tFNA and Met treatment effectively downregulated the expression of these inflammatory markers, and the anti-inflammatory effect of TMC was better than that of other groups. Protein level analysis, including immunofluorescence staining ( Figure 7 , 8 ) and Western Blot( Fig. 9 ), confirmed the above results, indicating that TMC exhibited stronger anti-inflammatory properties compared with free tFNA and Met, indicating that tFNA successfully delivered Met and synergistically enhanced the inhibition of the inflammatory microenvironment.

[0090] In order to study the inhibitory effect of TMC on alveolar bone destruction, TRAP staining was performed to evaluate the differentiation of osteoclasts ( Fig.11). After RANKL treatment, the number of osteoclasts increased significantly; free tFNA slightly reduced osteoclast differentiation, while free Met had the least inhibitory effect. In contrast, TMC showed a significantly stronger inhibitory effect on osteoclast differentiation. Further analysis showed that ( Fig.12 , 13 ), tFNA and Met both effectively reduced the expression of MMP9, which plays an important role in extracellular matrix degradation and bone resorption in periodontitis, and TMC showed more significant effects at the mRNA and protein levels.

[0091] (4) TMC’s inhibitory effect on macrophage pyroptosis

[0092] Pyroptosis is a lytic proinflammatory cell death form associated with NLRP3. To study the effect of TMC on pyroptosis in vitro, the present invention activated the NLRP3 inflammasome in RAW264.7 cells by LPS+ATP. Immunoblotting ( Fig.14 ) and immunofluorescence staining results ( Fig.15 ) showed that the intracellular levels of NLRP3, Caspase-1, cleaved Caspase-1, GSDMD, and GSDMD-N increased after LPS+ATP induction. Compared with free tFNA and Met, TMC significantly inhibited the expression of these markers, indicating that TMC inhibited the activation of NLRP3 inflammasome through the classical pathway, thereby significantly reducing the expression of GSDMD-N and inhibiting pyroptosis. Caspase-11 is an important mediator of the non-classical activation pathway of NLRP3 inflammasome. Immunoblotting and immunofluorescence staining showed that TMC inhibited the expression of Caspase-11, indicating that TMC also inhibits pyroptosis through the non-classical pathway.

[0093] (5) TMC inhibits inflammation by targeting NF-κB

[0094] NF-κB activation plays a key role in chronic inflammation, which is characterized by macrophage M1 polarization and upregulated expression of proinflammatory cytokines (TNF-α, IL-6). AMPK is an enzyme closely related to metabolism, which can inhibit the expression of NF-κB by phosphorylation at the Thr172 site of the AMPKα subunit. Studies have shown that free tFNA can produce anti-inflammatory effects by regulating IκBα / NF-κB expression, while free Met can regulate macrophage inflammation by regulating NF-κB signaling through AMPK; however, the effect of free tFNA on AMPK is still unclear. Therefore, TMC may produce a powerful anti-inflammatory effect similar to Met by regulating AMPK in macrophages. In order to verify this hypothesis, the present invention used immunofluorescence staining ( Fig.17 ) and Western Blot( Fig.16) The expression of total AMPKα, p-AMPKα (activated AMPKα), p65, and p-p65 (activated p65) were analyzed ( Fig.18 ). Both tFNA and Met promoted the expression and activation of AMPKα, while inhibiting the expression and phosphorylation of p65. However, TMC significantly enhanced the activation of AMPKα and the inhibitory effect of NF-κB. These results suggest that TMC inhibits LPS+ATP-induced inflammation by regulating AMPK to inhibit the expression of NF-κB.

[0095] The above results indicate that the TMC formed by the combination of tFNA and Met has good biocompatibility and excellent cell uptake and delivery properties. TMC can inhibit LPS-induced macrophage inflammation and osteoclast differentiation, and effectively downregulate the mRNA levels of IL-1β, IL-18, TNF-α and IL-6. In addition, TMC can also inhibit macrophage pyroptosis and inhibit inflammation by targeting NF-κB.

[0096] Experimental Example 3: In vivo animal testing of TMC

[0097] 1. Experimental methods

[0098] (1) Establishment and treatment of animal model: Animal experiments followed the guidelines of the Animal Ethics Committee of West China School of Stomatology, Sichuan University. Six-week-old male C57BL / 6J mice of similar weight were randomly divided into 5 groups (n=6): 1: control group; 2: T2D-ligation group; 3: T2D-ligation + tFNA group; 4: T2D-ligation + Met group, and 5: T2D-ligation + TMC group. After 7 days of adaptation, mice except the control group were fed a high-fat diet (HFD). One month later, fasting mice were injected with 1% streptozotocin (STZ, Aladdin, Shanghai, China) for 7 consecutive days. Fasting blood glucose concentration exceeded 16.7 mmol L -1 The model was successfully established. A 5-0 silk thread was used to ligate between the first and second molars of the left maxillary teeth for 9 days. In the drug injection treatment, the tFNA group, the Met group, and the TMC group were given corresponding drugs by intraperitoneal injection once every two days, with concentrations of tFNA 250nM, metformin 250mg kg -1 The control group and T2D-ligation group were injected with an equal volume of normal saline.

[0099] (2) Trap staining: The differentiation and activation of osteoclasts are closely related to inflammatory factors in periodontal tissues. The present invention explored the activity level of osteoclasts by Trap staining. The staining steps were performed according to the instructions of the Trap staining kit (Servicebio, Wuhan, China), and positive cells were observed at 20× magnification using Aperio Imaging Mirror software.

[0100] (3) Micro-CT analysis: The left maxillary bone was examined using a SCANCO Medical Micro-CT 50 (70 kV, 200 μA, 300 ms, 10 μm), and the region of interest (ROI) was located between the left maxillary first and second molars.

[0101] (4) In vivo biodistribution: To study the distribution and metabolism of the drug in vivo, Cy5-labeled TMC was injected via the tail vein. Mice were killed at different time points after injection (1 h, 3 h, 6 h, 12 h, 24 h), and the main organs (left maxillary bone, intestine, heart, liver, spleen, lung and kidney) were collected and the distribution and metabolism of the drug were observed using a fluorescence imaging system (IVIS Lumina III Series, PerkinElmer, USA).

[0102] (5) Histological analysis: The samples were immersed in 10% EDTA decalcification solution for 1 month, and the solution was changed twice a week. After decalcification, the samples were dehydrated, paraffin-embedded, and sectioned (3 μm thickness), followed by H&E, Masson, and Trap staining. In addition, the samples were subjected to immunofluorescence (IF) and immunohistochemistry (IHC) staining using the anti-IL-1β, anti-TNF-α, anti-IL-6, and anti-NLRP3 antibodies described above.

[0103] 2. Experimental results

[0104] (1) Inhibitory effect of TMC on periodontal tissue inflammation in ligature-induced T2D periodontitis

[0105] result( Fig.19 ) showed that after injection via both routes, TMC was distributed throughout the body within 24 hours and was detected in the maxilla for up to 6 hours, indicating that TMC can provide long-term protection for periodontal tissues. Semi-quantitative fluorescence analysis showed that the concentration of TMC in the maxilla was higher after intraperitoneal injection. Clinically, intraperitoneal injection is usually easier for patients to accept than intravenous injection, making it a possible route to effectively inhibit T2D periodontitis. Hematoxylin and eosin (H&E) staining images ( Fig. 20 ) showed that TMC inhibited the infiltration of inflammatory cells and bleeding through periodontal ligation.

[0106] In order to evaluate the anti-inflammatory effect of TMC in vivo, the present invention performed immunofluorescence staining. Immunofluorescence images ( Fig.21 ) and semi-quantitative fluorescence ( Fig. 22) analysis showed that the levels of IL-1β, IL-6 and TNF-α in periodontal tissues increased significantly after ligation, while tFNA, Met and TMC treatments inhibited these inflammatory factors, among which TMC had the most significant effect. In addition, the present invention evaluated the expression of NLRP3, a pyroptosis marker that was upregulated after ligation. tFNA, Met and TMC treatments all effectively reversed this increase, and TMC had the strongest inhibitory effect. Immunohistochemistry (IHC) staining images ( Fig.23 ) and semi-quantitative ( Fig.24 ) analysis further confirmed the immunofluorescence results, showing that the expression patterns of IL-1β, IL-6, TNF-α, and NLRP3 were consistent in both methods, thus validating these findings. Fig.24 The results showed that at the same dosage, TMC's effect in inhibiting IL-1β and NLRP3 was better than the sum of the effects of tFNA and Met alone, playing a synergistic role and achieving unexpected technical effects. In conclusion, TMC significantly inhibited the inflammation of T2D periodontal soft tissue after ligation, inhibited the expression of the key inflammatory factor IL-1β through the pyroptosis pathway, and thus fought against periodontal inflammation.

[0107] (2) Protective effect of TMC on periodontal tissue destruction in ligature-induced diabetic periodontitis

[0108] Alveolar bone resorption is a major problem in periodontitis, and 3D reconstruction of the maxilla allows for direct assessment of alveolar bone loss. Images showed significant alveolar bone loss between the first and second molars after ligation, confirming the success of the model construction. Compared with the model group, both the free tFNA and free Met groups showed a reduction in alveolar bone loss, suggesting a certain protective effect against alveolar bone destruction. However, TMC was significantly more effective with minimal bone loss. Micro-CT images from the XY and XZ planes further confirmed the optimal bone protective effect of TMC ( Fig.25 ). To quantify bone changes, bone volume parameters including bone volume fraction (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) were analyzed. Higher BV / TV, Tb.Th, and Tb.N values ​​indicate greater bone volume, while higher Tb.Sp reflects lower bone density. Fig.26 As shown in Figure 3, the BV / TV, Tb.Th, and Tb.N values ​​of the affected maxillary molar area were higher in the TMC group than in the other groups, while Tb.Sp was significantly lower, indicating that the alveolar bone loss was minimal after TMC treatment. Histological analysis evaluated the number of osteoclasts by TRAP staining ( Fig. 27). Free tFNA and free Met reduced osteoclast activity and number to some extent, but TMC treatment significantly reduced the presence of osteoclasts and almost completely eliminated them. The periodontal ligament (PDL) fibrous connective tissue located in the matrix between the tooth root and the alveolar bone undergoes degradation and collagen loss during inflammation. The present invention uses Masson staining to examine the histological changes of PDL ( Fig.28 ). Inflammation caused the PDL structure of the control group to be sparse, and the collagen content was significantly reduced, while TMC treatment maintained the integrity of the PDL, presenting a denser structure, with an increased number of collagen fibers that were more uniform than the control group. Overall, TMC showed a more significant protective effect on periodontal tissues compared with free tFNA and Met, indicating that TMC has significant application potential in the treatment of T2D periodontitis.

[0109] The above results show that TMC can inhibit periodontal inflammation in ligature-induced T2D periodontitis and protect damaged periodontal tissues. Moreover, at the same dosage, TMC's inhibitory effects on IL-1β and NLRP3 are better than the sum of the effects of tFNA and Met alone, playing a synergistic role and achieving unexpected technical effects.

[0110] In summary, the present invention provides a tetrahedral framework nucleic acid-metformin complex and its preparation method and use. The present invention constructs a new type of nanodrug system for the first time. The delivery system based on the tetrahedral framework nucleic acid efficiently delivers metformin to macrophages, effectively regulates NLRP3-mediated pyroptosis and AMPK / NF-κB signaling axis, and significantly enhances the cell uptake and therapeutic effect of metformin in the treatment of diabetic periodontitis, which has good prospects for industrial application.

Claims

1. A tetrahedral framework nucleic acid-metformin complex, characterized in that: The invention is composed of a mixture of tetrahedral framework nucleic acid and metformin, wherein the molar ratio of the tetrahedral framework nucleic acid to the metformin is 1:50-900.

2. The tetrahedral framework nucleic acid-metformin complex according to claim 1, characterized in that: The molar ratio of the tetrahedral framework nucleic acid to metformin is 1:100-800, preferably 1:

600.

3. The tetrahedral framework nucleic acid-metformin complex according to claim 1, characterized in that: The tetrahedral framework nucleic acid is self-assembled from four DNA single strands, and the sequences of the four single strands are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 respectively.

4. A method for preparing the tetrahedral framework nucleic acid-metformin complex according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: mixing tetrahedral framework nucleic acid and metformin, reacting the mixture, and filtering the mixture to obtain the product.

5. The method according to claim 4, characterized in that The reaction temperature is 30-45° C. and the reaction time is 2-4 hours.

6. The method according to claim 5, characterized in that The reaction temperature was 37°C and the reaction time was 3 hours.

7. Use of the tetrahedral framework nucleic acid-metformin complex according to any one of claims 1 to 3 in the preparation of a medicament for preventing and / or treating periodontitis.

8. The use according to claim 7, characterized in that The periodontitis is diabetic periodontitis.

9. The use according to claim 8, characterized in that The diabetes is type II diabetes.

10. The use according to claim 7, characterized in that The drug is a drug that inhibits NLRP3-mediated pyroptosis and NF-κB signaling pathway.

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