Use of tetrahedral framework nucleic acid-curcumin complex in the preparation of drugs for the prevention and / or treatment of diabetic osteoporosis

Abstract

This invention provides the use of a tetrahedral framework nucleic acid-curcumin complex in the preparation of drugs for the prevention and / or treatment of diabetic osteoporosis, belonging to the pharmaceutical field. The tetrahedral framework nucleic acid-curcumin complex is prepared from tetrahedral framework nucleic acid and curcumin as raw materials, wherein the molar ratio of tetrahedral framework nucleic acid to curcumin is 1:(50-800). The tetrahedral framework nucleic acid-curcumin complex of this invention improves the water solubility, stability in physiological fluids, and in vivo utilization efficiency of the natural product curcumin, overcoming the shortcomings of curcumin such as poor water solubility, low bioavailability, and instability in physiological media. In the obtained complex, curcumin and tetrahedral framework nucleic acid exert a synergistic effect, targeting NRF2, inhibiting ferroptosis of bone marrow mesenchymal stem cells in the diabetic bone microenvironment, exerting hypoglycemic and osteopromoting effects, and can be used for the prevention and / or treatment of diabetic osteoporosis, showing good application prospects.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to the use of tetrahedral framework nucleic acid-curcumin complex in the preparation of drugs for the prevention and / or treatment of diabetic osteoporosis. Background Technology

[0002] Diabetic osteoporosis (DOP) is a major cause of bone loss and microstructural damage in diabetic patients, leading to increased susceptibility to fractures. It is currently considered a systemic metabolic bone disease. A 2021 report in *Nat Rev Endocrinol* showed that diabetic patients have a 32% increased risk of fractures compared to non-diabetic individuals, and a 1.23-fold increased mortality rate compared to those with type 2 diabetes mellitus (T2DM). This results in significant morbidity and mortality rates and imposes a substantial medical burden. Over the past decade, research on the long-term effects of diabetes on bone pathology has increased. A 2022 report in *Lancet Diabetes Endocrinol* reported that one of the key reasons for fragility fractures due to changes in the bone microenvironment is ferroptosis in bone mesenchymal stem cells (BMSCs). More specifically, increased ferroptosis inhibits the expression of osteogenic transcription factors osterix (OSX; also known as SP7) and Runt-associated transcription factor 2 (RUNX2), disrupting the balance between osteogenic commitment and differentiation in BMSCs, thereby negatively impacting bone homeostasis. Therefore, targeting ferroptosis has become a new strategy for treating diabetic osteoporosis.

[0003] Ferroptosis, a recently discovered form of programmed cell death, differs from traditional apoptosis, autophagy, and pyroptosis, and is characterized by iron-dependent accumulation and lipid peroxidation. Glutathione peroxidase 4 (GPX4), an antioxidant enzyme negatively regulated by endoplasmic reticulum (ER) stress, scavenges excess lipid peroxides and is a key upstream regulator of ferroptosis. However, in the diabetic microenvironment, the content and activity of intracellular GPX4 protein are significantly reduced, leading to increased reactive oxygen species (ROS) and iron (Fe2+) levels. 2+Excessive accumulation of ROS and ectopic expression of lipid hydroperoxides and ferroptosis-related proteins are observed. Recent studies have shown that direct application of the ferroptosis inhibitor ferrostatin-1 (Fer-1) in a DOP mouse model can reduce bone loss, indicating that ferroptosis is closely related to the pathogenesis of DOP and is a potential therapeutic target. Nuclear factor E2-related factor 2 (NRF2) can inhibit ferroptosis by upregulating GPX4 expression. Therefore, designing and developing a potent NRF2-based GPX4 agonist to effectively eliminate ROS and regulate lipid peroxidation, inhibit ferroptosis, and increase bone formation is of great significance for alleviating diabetic osteoporosis.

[0004] Curcumin, a polyphenol derived from the medicinal plant turmeric, has been reported to activate NRF2. Furthermore, curcumin has been extensively studied in vitro for its anti-inflammatory, antioxidant, lipid-lowering, and osteoclast-inhibiting pharmacological effects. Unfortunately, curcumin has a short circulation time, and repeated administration increases toxicity, while its absorption rate and bioavailability are low. With the rapid development of nanotechnology, nanomaterials, possessing good biocompatibility, stability, and multifunctional design, show broad application prospects in the biomedical field. However, the application of ferroptosis nano-inhibitors in the treatment of diabetic osteoporosis remains a gap.

[0005] Tetrahedral framework nucleic acids (tFNAs) are self-assembled from four single-stranded DNA (ssDNA) molecules through temperature regulation, enabling them to freely cross cell membranes. They possess excellent biocompatibility, safety, editability, and stability, making them a DNA nanomaterial with broad application potential. Patent application number 202211183683.X discloses a drug delivery system using tetrahedral framework nucleic acids to load curcumin. This system enhances the water solubility, stability, and bioavailability of curcumin, and can be used for the prevention or treatment of radiation-induced oral mucositis. However, whether this tetrahedral framework nucleic acid-loaded curcumin drug delivery system can be effectively used to treat diabetic osteoporosis remains unclear and requires further investigation. Summary of the Invention

[0006] The purpose of this invention is to provide the use of a tetrahedral framework nucleic acid-curcumin complex in the preparation of a medicament for the prevention and / or treatment of diabetic osteoporosis.

[0007] This invention provides the use of a tetrahedral framework nucleic acid-curcumin complex in the preparation of a medicament for the prevention and / or treatment of diabetic osteoporosis; the tetrahedral framework nucleic acid-curcumin complex is prepared from tetrahedral framework nucleic acid and curcumin as raw materials, wherein the molar ratio of tetrahedral framework nucleic acid to curcumin is 1:(50-800).

[0008] Furthermore, the drug is a drug that inhibits ferroptosis of bone marrow mesenchymal stem cells in the diabetic bone microenvironment.

[0009] Furthermore, the drug is a drug that targets and binds to NRF2.

[0010] Furthermore, the drug is a drug that lowers blood sugar and / or promotes bone formation.

[0011] Furthermore, the molar ratio of the tetrahedral framework nucleic acid to curcumin is 1:100.

[0012] Furthermore, the tetrahedral framework nucleic acid is formed by four DNA single strands through complementary base pairing, and the sequences of the four DNA single strands are shown in SEQ ID NO. 1 to 4.

[0013] Furthermore, the preparation method of the tetrahedral framework nucleic acid includes the following steps: dissolving four DNA single strands in equimolar amounts in TM buffer, maintaining at 85-105℃ for 5-15 min, and then maintaining at 2-8℃ for 10-30 min;

[0014] Preferably, the method for preparing the tetrahedral framework nucleic acid includes the following steps: dissolving four DNA single strands in equimolar amounts in TM buffer, maintaining the solution at 95°C for 10 min, and then maintaining the solution at 4°C for 20 min.

[0015] Furthermore, the TM buffer contains 10 mM Tris-HCl, 50 mM MgCl2, and pH 8.0; the concentration of the four DNA single strands is 1 μM.

[0016] Furthermore, the preparation method of the tetrahedral framework nucleic acid-curcumin complex includes the following steps:

[0017] Curcumin and tetrahedral framework nucleic acids are incubated at a specific molar ratio to obtain the product.

[0018] Preferably, the incubation temperature is 20–40°C, and the incubation time is 1–8 hours.

[0019] Furthermore, the drug is an injectable formulation.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] This invention prepares a tetrahedral framework nucleic acid-curcumin complex, which improves the water solubility, stability in physiological fluids, and in vivo utilization efficiency of the natural product curcumin, overcoming the shortcomings of curcumin such as poor water solubility, low bioavailability, and instability in physiological media. In the obtained complex, curcumin and tetrahedral framework nucleic acid exert a synergistic effect, targeting NRF2 to inhibit ferroptosis of bone marrow mesenchymal stem cells in the diabetic bone microenvironment, thereby exerting hypoglycemic and osteopromoting effects. It can be used for the prevention and / or treatment of diabetic osteoporosis and has good application prospects.

[0022] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0023] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0024] Figure 1 The synthesis characterization results of the tetrahedral framework nucleic acid-curcumin complex (tFNA-Cur) are as follows: A is a schematic diagram of the tFNA-Cur synthesis; B is a dissolution image of tFNA, Cur, and tFNA-Cur; C is a PAGE image verifying the successful synthesis of tFNA; D is a PAGE image verifying the successful synthesis of tFNA-Cur; E is the absorption spectrum of tFNA, Cur, and tFNA-Cur; F is the particle size and Zeta potential of tFNA, Cur, and tFNA-Cur; G is the AFM detection result of tFNA-Cur; H is the encapsulation efficiency of tFNA-Cur; I is the drug release curve of tFNA-Cur; J is the 6-h cell penetration ability of Cur and tFNA-Cur; K is the 12-h cell penetration ability of Cur and tFNA-Cur; L is the serum stability gel electrophoresis result of tFNA and tFNA-Cur; M is the serum stability statistical result of tFNA and tFNA-Cur.

[0025] Figure 2The effect of tetrahedral framework nucleic acid-curcumin complex (tFNA-Cur) on osteogenic capacity of bone marrow mesenchymal stem cells (BMSCs) in the diabetic microenvironment was investigated: A shows the effect of different concentrations of tFNA on BMSC cell viability as detected by CCK-8 assay; B shows the effect of different concentrations of Cur on BMSC cell viability as detected by CCK-8 assay; C shows the effect of different concentrations of tFNA-Cur on BMSC cell viability as detected by CCK-8 assay; D shows the effect of different concentrations of advanced aging substances (AGEs) on BMSC cell viability as detected by CCK-8 assay; E shows the alkaline phosphatase (ALP) staining results; F shows the alizarin red staining results; G shows the quantitative statistical results of alkaline phosphatase (ALP); H shows the alizarin red staining results. The results are as follows: I represents the quantitative statistical results of osteogenic gene ALP detected by RT-qPCR; J represents the expression results of osteogenic gene Runx2 detected by RT-qPCR; K represents the expression results of osteogenic gene Osx detected by RT-qPCR; L represents the expression results of osteogenic gene Opn detected by RT-qPCR; M represents the expression results of osteogenic proteins ALP, RUNX2, OSX, and OPN detected by Western blotting; N represents the statistical results of osteogenic protein ALP, RUNX2, OSX, and OPN expression detected by Western blotting; O represents the expression results of osteogenic protein ALP detected by immunofluorescence; P represents the expression results of osteogenic protein RUNX2 detected by immunofluorescence; and Q represents the expression results of osteogenic protein OSX detected by immunofluorescence.

[0026] Figure 3 Results of tetrahedral framework nucleic acid-curcumin complex (tFNA-Cur) activating the NRF2 / GPX4 pathway to inhibit ferroptosis: A and E show the fluorescence detection and statistical results of reactive oxygen species (ROS); B and F show the fluorescence detection and statistical results of mitochondrial membrane potential (MMP); C and G show the fluorescence detection and statistical results of intracellular ferrous ion (FerroOrange); D shows the mitochondrial morphological changes detected by TEM; H shows the expression results of gene Gpx4 detected by RT-qPCR; I shows the expression results of gene Acsl4 detected by RT-qPCR; J shows the expression results of gene Nrf2 detected by RT-qPCR; K and L show the expression and statistical results of proteins GPX4, ACSL4, NRF2, and KEAP1 detected by WB; M shows the expression results of protein GPX4 detected by immunofluorescence; N shows the expression results of protein ACSL4 detected by immunofluorescence; O shows the expression results of protein NRF2 detected by immunofluorescence; P shows the molecular docking simulation binding site of curcumin (Cur) and NRF2.

[0027] Figure 4The therapeutic effects of tetrahedral framework nucleic acid-curcumin complex (tFNA-Cur) on diabetic osteoporosis are shown in the following diagrams: A is a schematic diagram of tFNA-Cur treatment for DOP; B is the HE section of pancreatic tissue; C and E are the results of micro-CT reconstruction and quantitative analysis of the femur; D and F are the results of micro-CT reconstruction and quantitative analysis of the tibia; G is the HE staining results of the femur and tibia; H is the Masson staining results of the femur and tibia.

[0028] Figure 5 Results of the tetrahedral framework nucleic acid-curcumin complex (tFNA-Cur) activating the NRF2 / GPX4 pathway in vivo to inhibit ferroptosis and promote osteogenic formation of BMSCs: A and D are the expression of tibial tissue protein ALP detected by immunofluorescence; B and E are the expression of tibial tissue protein GPX4 detected by immunofluorescence; C and F are the expression of tibial tissue protein NRF2 detected by immunofluorescence. Detailed Implementation

[0029] The raw materials and equipment used in the specific embodiments of the present invention are all known products, obtained by purchasing commercially available products.

[0030] Curcumin (Cur for short) is a commercially available product with a purity of >98%.

[0031] Example 1: Preparation of tetrahedral framework nucleic acid-curcumin complex: tFNA-Cur

[0032] The synthetic route for preparing the tetrahedral framework nucleic acid-curcumin complex is shown below. Figure 1 As shown in Figure A.

[0033] 1. Preparation of tetrahedral framework nucleic acids (tFNA)

[0034] The four DNA single strands (S1, S2, S3, S4) shown in Table 1 were added to TM buffer (10 mM Tris-HCl, 50 mM MgCl2, pH 8.0) in an equimolar ratio, with a final concentration of 1 μM for the four DNA single strands. The reaction solution was heated to 95 °C and maintained for 10 min, then rapidly cooled to 4 °C and maintained for 20 min to obtain tetrahedral framework nucleic acid, named tFNA.

[0035] Table 1. Sequences of the four DNA single strands

[0036]

[0037] 2. Preparation of tetrahedral framework nucleic acid-curcumin (tFNA-Cur) complex

[0038] Curcumin was dissolved in cell culture grade DMSO to obtain a 10 mM curcumin solution. 2 μl of the 10 mM curcumin solution was added to 200 μl of tFNA solution (1000 nM) (molar ratio of tetrahedral framework nucleic acid to curcumin was 1:100). The mixture was incubated with shaking at room temperature for 3 hours, and then centrifuged using an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kDa to remove residual free curcumin, yielding the tetrahedral framework nucleic acid-curcumin complex (tFNA-Cur).

[0039] 3. Characterization of tetrahedral framework nucleic acids and tetrahedral framework nucleic acid-curcumin complex

[0040] The successful synthesis of tFNA and tFNA-Cur was verified by polyacrylamide gel electrophoresis (PAGE); the absorption spectra of tFNA, Cur, and tFNA-Cur were analyzed using an ultramicro spectrophotometer (UV5 Nano, Mettler Toledo, Switzerland); the particle size and zeta potential of tFNA, Cur, and tFNA-Cur were detected using a Malvern Zetasizer nanoparticle size analyzer (Malvern, UK); and the three-dimensional nanostructure of tFNA-Cur was observed by transmission electron microscopy (TEM) and atomic particle microscopy.

[0041] Following the above preparation method, tetrahedral framework nucleic acid (TBA) and curcumin were prepared at molar ratios of 1:50, 1:100, 1:200, 1:400, and 1:800 to form TBA-curcumin complexes (TBA concentrations were 1000 nM, 200 μl; curcumin concentrations were 10 mM, 1 μl, 2 μl, 4 μl, 8 μl, and 16 μl). The encapsulation efficiency of the complexes prepared at each curcumin concentration was then measured.

[0042] Release and stability of tFNA-Cur: PBS (pH 7.4, 0.01 mol / L) was used as the medium. The dialysis bag (30 kDa; Solarbio, Beijing, China) was divided into an external liquid (30 ml) and an internal liquid (3 ml). Equimolar concentrations of Cur (200 μl, 20 μM) and tFNA-Cur (200 μl, where the molar ratio of tetrahedral framework nucleic acid to curcumin was 1:100 and the curcumin concentration was 20 μM) were dissolved in the internal fluid at 37 °C with continuous stirring (150 rpm). The OD values ​​of curcumin released from the external liquid were measured at different time points using an ultramicro spectrophotometer. tFNA and tFNA-Cur were incubated with 2% or 10% serum for different times (0h, 2h, 4h, 6h, 8h, 10h, 12h), and the stability results were analyzed by agarose gel electrophoresis. Finally, images of tFNA and tFNA-Cur were captured using a UV exposure device (Bio-Rad, Hercules, USA).

[0043] BMSCs uptake of Cur and tFNA-Cur: BMSCs were seeded in confocal culture dishes (10,000 cells) and treated with Cur and tFNA-Cur for 6 and 12 hours. Images of Cur and tFNA-Cur cell uptake were captured using a confocal microscope (Olympus, Tokyo, Japan).

[0044] Depend on Figure 1 A indicates that tFNA-Cur is synthesized in a two-step process, which is simple; according to... Figure 1 As shown in the visual image B, curcumin has poor solubility in water, while tFNA-Cur has good solubility in water, which can significantly improve the water solubility of Cur and solve the problem of poor water solubility of Cur limiting its clinical application.

[0045] Figure 1 C, 1D, and 1E indicate that tFNA and tFNA-Cur were successfully synthesized.

[0046] Figure 1 F and 1G indicate that the prepared tFNA-Cur particles have a size of 40nm, which falls between 1-100nm, meeting the definition of nanomedicines. After entering the body through blood circulation, it can eliminate the limitations of special biological barriers on drug action, such as the blood-brain barrier, blood-eye barrier, and cell biomembrane barrier, and be absorbed by tissues and cells through endocytosis, improving bioavailability. In addition, tFNA-Cur is negatively charged, resulting in fewer biological side effects.

[0047] Figure 1H and 1I indicate that when the molar ratios of tetrahedral framework nucleic acid (TBA) and curcumin are 1:50, 1:100, and 1:200 (tetrahedral framework nucleic acid concentration of 1000 nM, 200 μl; curcumin concentration of 10 mM, 1 μl, 2 μl, and 4 μl), the tFNA-Cur drug loading efficiencies are high, at 91.8259%, 84.2881%, and 82.226%, respectively. Figure 3 Following the results of C, a tFNA-Cur complex prepared with a tetrahedral framework nucleic acid and curcumin molar ratio of 1:100 was subsequently selected for further research. The tFNA-Cur complex prepared in this invention is slowly released in the physiological environment, exerting a sustained biological effect.

[0048] Figure 1 J and 1K indicate that cells have a strong ability to enter tFNA-Cur and that its bioavailability is better than that of free Cur, thus enabling Cur to exert its biological effects more efficiently.

[0049] Depend on Figure 1 L and 1M indicate that tFNA-Cur improves the stability of Cur in physiological fluids.

[0050] The following specific experimental examples demonstrate the beneficial effects of the present invention.

[0051] Example 1: Tetrahedral framework nucleic acid-curcumin complex (tFNA-Cur) promotes osteogenic capacity of BMSCs in the diabetic microenvironment.

[0052] 1. Experimental Methods

[0053] Effects of different concentrations of tFNA, Cur, and tFNA-Cur, as well as AGEs, on BMSCs activity: BMSCs were seeded at a density of 20,000 cells / well in 96-well plates and cultured at 37°C and 5% CO2 for 24 hours. Different concentrations of tFNA, Cur, and tFNA-Cur were then added and the cells were cultured for another 12 hours, or different concentrations of AGEs were added and the cells were cultured for another 24 hours. Cell viability was then assessed using CCK8. tFNA and tFNA-Cur were prepared according to the method described in Example 1, with a molar ratio of tetrahedral framework nucleic acid to curcumin of 1:100 (tetrahedral framework nucleic acid concentration: 1000 nM, 200 μl; curcumin concentration: 10 mM, 2 μl).

[0054] Advanced glycation end products (AGEs) are formed by the non-enzymatic glycation reaction between ketoses such as glucose, fructose, and glucose-6-phosphate and protein amino groups. Once formed, they are irreversible. Diseases such as diabetes, inflammation, and aging accelerate the production and accumulation of AGEs in the body; the accumulation of AGEs, in turn, exacerbates diabetes, inflammation, and aging, thus creating a vicious cycle that ultimately leads to various chronic complications of diabetes. This study uses AGEs to treat bone mesenchymal stem cells (BMSCs) in vitro to simulate the bone microenvironment of diabetes.

[0055] Alkaline phosphatase (ALP) and Alizarin Red staining: BMSCs were seeded at a density of 200,000 cells / well in 6-well plates and cultured at 37°C and 5% CO2 for 24 hours. BMSCs were then pretreated with tFNA-Cur (tetrahedral framework nucleic acid and curcumin molar ratio of 1:100) prepared in Example 1 for 12 hours, followed by replacement with osteogenic induction medium and treatment with 150 μg / mL AGEs for 7 days. BMSCs were then fixed with 4% paraformaldehyde (4°C, 20 min) and stained with ALP staining solution (C3250S, Beyotime, China) at 37°C for 10 minutes. After 14 days of the same treatment, cells were incubated with Alizarin Red staining solution at room temperature for 5 minutes. ALP activity and mineralized nodules were observed and photographed under an optical microscope.

[0056] Real-time quantitative RT-PCR analysis: BMSCs were seeded at a density of 200,000 cells / well in 6-well plates and cultured at 37°C and 5% CO2 for 24 hours. BMSCs were then pretreated with tFNA-Cur (tetrahedral framework nucleic acid and curcumin molar ratio of 1:100) prepared in Example 1 for 12 hours, followed by treatment with osteogenic induction medium and AGEs at 150 μg / mL for 7 days. Transcriptional levels of Alp, Runx2, Osx, and Opn were assessed using reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, MA, USA). cDNA synthesis was performed using Premix Ex Taq II (Perfect Real Time Kit; Takara, Dalian, China). The cDNA was synthesized using the kit from Q7. Green I master mixture (ABIQuantStudio 7, Thermo Fisher, USA) was used to measure target genes. 2 -ΔΔCT The results were analyzed using relative quantitative methods, with β-Actin used as a control gene. All experiments were repeated three times.

[0057] Western blot analysis (WB): BMSCs were treated the same as for RT-PCR. BMSCs were lysed using a cell protein extraction reagent (KEYGENBiotech, Nanjing, China), then mixed with loading buffer at a 4:1 (v / v) ratio and boiled for 5 minutes. Proteins were separated using 10% SDS-PAGE and transferred to PVDF membranes. The membranes were then blocked with blocking buffer (ThermoFisher Scientific, MA, USA) for 10 minutes and incubated overnight at 4°C with anti-ALP (1:500, Huaan Biotechnology, Zhejiang, China), anti-RUNX2 (1:500, Huaan Biotechnology, Zhejiang, China), anti-OSX (1:500, Huaan Biotechnology, Zhejiang, China), and anti-OPN (1:1000, Abcam, Cambridge, UK). The next day, after washing with TBST, the bands were incubated with rabbit secondary antibody (1:3000, BEYOTIME, Shanghai, China) for 1 hour. The results were visualized and quantitatively analyzed using an ECL chemiluminescence detection system (Bio-Rad, Hercules, CA, USA).

[0058] Immunofluorescence (IF) staining: BMSCs were seeded at a density of 50,000 cells / well in confocal dishes and cultured at 37°C and 5% CO2 for 24 hours. BMSCs were then pretreated with tFNA-Cur (tetrahedral framework nucleic acid and curcumin molar ratio of 1:100) prepared in Example 1 for 12 hours, followed by treatment with osteogenic induction medium and AGEs at 150 μg / mL for 4 days. BMSCs were fixed with 4% paraformaldehyde solution (4°C, 25 min), perforated onto the cell membrane with 0.5% Triton X-100 (RT, 20 min), and blocked with 5% goat serum (37°C, 20 min). They were then incubated overnight at 4°C with osteogenic-associated antibodies (ALP, RUNX2, and OSX). The next day, BMSCs were incubated with secondary rabbit antibody (1:200, Invitrogen, Carlsbad, USA) at 37°C for 1 hour. The cytoskeleton was stained with phenocrystin (FITC) (37℃, 20 min), and the cell nuclei were stained with DAPI (37℃, 10 min). Images were taken using a confocal microscope (Olympus, Tokyo, Japan).

[0059] 2. Experimental Results

[0060] Figure 2A-2D show the effects of different concentrations of each drug on cell viability. The results indicate that tFNA and Cur inhibit BMSCs at a concentration concentration. Optimal cell viability was observed when Cur was 20 μM and tFNA was 200 nM. The complex tFNA-Cur, prepared with 20 μM Cur and 200 nM tFNA, was used for further experiments. AGEs showed a concentration-dependent effect on BMSCs; low concentrations of AGEs promoted BMSC proliferation, while high concentrations inhibited it. Good BMSC activity was observed at an AGEs concentration of 150 μg / ml, and this concentration was selected for subsequent experiments. Figure 2 In C, when the tFNA:Cur ratio is 1:100, the optimal concentration ratio for preparing tFNA-Cur is 200 nM for tFNA and 20 μM for Cur, which also results in the best cell activity.

[0061] Figure 2 E and 2G represent the results of alkaline phosphatase (ALP) staining. Figure 2 F and 2H represent the results of alizarin red staining, from... Figure 2 The results of E-2H showed that BMSCs treated with 150 μg / ml AGEs in a simulated diabetic microenvironment had reduced osteogenic capacity and calcium nodule formation. After pretreatment with tFNA, Cur, and tFNA-Cur, the osteogenic capacity and calcium nodule formation of BMSCs were reversed. Among them, the effect of the tFNA-Cur treatment group was significantly better than that of the tFNA and Cur groups alone.

[0062] Figure 2 I-2L shows the results of RT-qPCR detection of osteogenic genes Alp, Runx2, Osx, and Opn. The results indicate that osteogenic gene expression in BMSCs is significantly suppressed in the diabetic microenvironment. Pretreatment with tFNA, Cur, and tFNA-Cur reverses osteogenic gene expression. Among them, the tFNA-Cur treatment group showed the best effect on promoting osteogenic gene expression, followed by the Cur group, and the tFNA group also showed some reversal.

[0063] Figure 2 M and Figure 2 N represents the results of Western blotting (WB) detection of osteogenic proteins ALP, RUNX2, OSX, and OPN. These results indicate that osteogenic protein expression decreases in the diabetic microenvironment. Pretreatment with tFNA, Cur, and tFNA-Cur reversed the expression of ALP, RUNX2, OSX, and OPN. Among these, the tFNA-Cur treatment group showed significantly better results than the tFNA and Cur groups alone.

[0064] Figure 2O, 2P, and 2Q were detected by immunofluorescence for the expression of osteoblast proteins ALP, RUNX2, and OSX, respectively. The results were consistent with those detected by Western blotting, indicating that tFNA-Cur can significantly reverse the suppressed osteoblast protein expression in the diabetic microenvironment, and its effect is better than that of tFNA and Cur alone.

[0065] Figure 2 The results showed that tFNA-Cur pretreatment reversed the impaired osteogenic capacity of BMSCs in the diabetic microenvironment, increased the expression of osteogenic-related genes and proteins ALP, RUNX2, OSX and OPN, and increased calcium nodule formation, with significantly better effects than tFNA alone and free Cur.

[0066] Experiment 2: Tetrahedral framework nucleic acid-curcumin complex (tFNA-Cur) activates the NRF2 / GPX4 pathway and inhibits ferroptosis.

[0067] 1. Experimental Methods

[0068] Detection of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) levels: BMSCs were first pretreated with tFNA-Cur (the molar ratio of tetrahedral framework nucleic acid to curcumin was 1:100, with 20 μM Cur and 200 nM tFNA) for 12 h, followed by treatment with 150 μg / mL AGEs for 24 h. Subsequently, cells were incubated with Hoechst 33342 (1X, C1028, Beyotime, China) for 10 min and with DCFH-DA (10 μM, S0033S, Beyotime, China) for 20 min to detect ROS levels. Additionally, they were incubated with Rhodamine 123 (1X, C2008S, Beyotime, China) for 20 min to assess MMP levels. After washing with PBS, images of ROS and MMP were obtained using confocal microscopy.

[0069] Fe 2+ Detection (FerroOrange): Using Fe 2+ Probes (1 μM, MkBio, Mx4559) were used to assess intracellular Fe. 2+ After the same treatment as described above, the cells were incubated with Hoechst 33342 for 10 minutes and FerroOrange for 20 minutes. Subsequently, fluorescence images of the BMSCs were captured using confocal microscopy.

[0070] Transmission electron microscopy (TEM) was used to observe mitochondrial morphological changes: After the above treatment, BMSCs were fixed with 3% glutaraldehyde solution at 4°C for 16 hours. Subsequently, the BMSCs were dehydrated with acetone, embedded in Epon 812, sectioned (60-90 nm), and stained with uranyl acetate and lead citrate. Finally, the mitochondrial morphology of each treatment group was examined and captured using a JEM-1400FLASH TEM.

[0071] Detection of GPX4, ACSL4, NRF2, and KEAP1 gene and protein levels: BMSCs were first pretreated with tFNA-Cur (the molar ratio of tetrahedral framework nucleic acid to curcumin was 1:100, Cur was 20 μM, and tFNA was 200 nM) for 12 hours, and then treated with 150 μg / mL AGEs for 24 hours. The detection method was the same as in Experiment 1.

[0072] Cur and NRF2 molecular docking: The predicted NRF2 structure was generated using Alphafold. The protonation state of the small molecule was set to pH 7.4, and Cur was expanded into a 3D structure using Open Babel. A series of preparations were performed on the receptor protein and ligand using the AutoDock tool (ADT3). The docking box was generated using the AutoGrid program, followed by molecular docking using Autodock Vina (1.2.0). The optimal binding conformation was selected to analyze interactions. Finally, a protein-ligand interaction diagram was generated using PyMOL. The NRF2 protein is represented as a dark blue cartoon model, the ligands as cyan stick models, and their binding sites as magenta stick structures. Nonpolar hydrogen atoms were omitted. Hydrogen bonds, ionic interactions, and hydrophobic interactions are depicted as yellow, magenta, and green dashed lines, respectively.

[0073] 2. Experimental Results

[0074] Figure 3 A and 3E represent the results of reactive oxygen species (ROS) detection. Figure 3 B and 3F represent mitochondrial membrane potential (MMP). Figure 3 C and 3G represent the intracellular ferrous ion (FerroOrange) detection results. These results indicate that 150 μg / ml AGEs treatment of BMSCs, simulating a diabetic microenvironment, produces excessive ROS, alters mitochondrial membrane permeability, and increases intracellular Fe... 2+ Excessive accumulation. Pretreatment with tFNA, Cur, and tFNA-Cur can remove excessively accumulated ROS and Fe. 2+ The tFNA-Cur group showed better results than tFNA alone and free Cur.

[0075] Figure 3D represents the morphological changes in mitochondria detected by TEM. These results indicate that in the diabetic microenvironment, mitochondria shrink, become denser, and have fewer cristae. Pretreatment with tFNA, Cur, and tFNA-Cur reversed these mitochondrial changes, with the tFNA-Cur group showing the best results.

[0076] Figure 3 H-3J shows the expression results of genes Gpx4, Acsl4, and Nrf2, respectively. The experimental results indicate that the expression of ferroptosis-related genes Gpx4, Acsl4, and Nrf2 is significantly inhibited in the diabetic microenvironment. After pretreatment with tFNA, Cur, and tFNA-Cur, the gene expression levels were reversed. Among them, the tFNA-Cur group was more effective than tFNA alone and free Cur.

[0077] Figure 3 K and 3L represent the expression results of proteins GPX4, ACSL4, NRF2, and KEAP1 detected by Western blotting. These results indicate that the expression of ferroptosis-related proteins GPX4, ACSL4, and NRF2 is significantly inhibited in the diabetic microenvironment. The protein expression levels were reversed after pretreatment with tFNA, Cur, and tFNA-Cur. Among them, the tFNA-Cur group was more effective than tFNA alone and free Cur.

[0078] Figure 3 M, 3N, and 3O represent the expression results of immunofluorescence detection of proteins GPX4, ACSL4, and NRF2, which are consistent with the Western blot results.

[0079] Figure 3 P represents the molecular docking simulation site for the binding of curcumin (Cur) and NRF2. This result indicates that Cur and NRF2 directly form multiple sets of interactions, such as the hydrogen bond formed between Asp408 of NRF2 and Cur. Under the influence of these interactions, the binding energy of the small protein complex is -7.1 kcal / mol, exhibiting excellent overall performance. Cur activates NRF2 to exert its biological effects.

[0080] Figure 3 The results showed that treatment of BMSCs with 150 μg / mL AGEs in a simulated diabetic microenvironment increased ferroptosis in BMSCs, mainly manifested as increased lipid peroxidation (increased ROS expression) and Fe2+. 2+Elevated levels of tFNA-Cur inhibited GPX4 expression and altered mitochondrial morphology (mitochondrial atrophy, increased membrane permeability, and loss of cristae). Molecular docking simulations revealed that Cur in tFNA-Cur formed a hydrogen bond with the Asp408 of NRF2, with a binding energy of -7.1 kcal / mol. Since NRF2 is a target protein of the ferroptosis inhibition pathway, this demonstrates that the synthesized tFNA-Cur can target and inhibit ferroptosis. Experimental results also confirmed that tFNA-Cur pretreatment activated NRF2 expression and upregulated GPX4, thereby inhibiting ferroptosis. More excitingly, tFNA-Cur significantly inhibited ferroptosis more effectively than free Cur.

[0081] Experimental Example 3: Treatment of Diabetic Osteoporosis with Tetrahedral Framework Nucleic Acid-Curin Complex (tFNA-Cur)

[0082] 1. Experimental Methods

[0083] Animal Experiments: All animal experiments were approved by the Animal Ethics Committee of West China Hospital of Stomatology, Sichuan University. Four-week-old male C57BL / 6J mice were purchased from Jicui Pharmaceutical Co., Ltd. (Nanjing, China) and housed under pathogen-free conditions at 55% ± 5% humidity and 24 ± 2℃. First, the mice were randomly divided into five groups: control group, DOP, DOP+tFNA, DOP+curcumin, and DOP+tFNA-Cur (n = 6). Throughout the experiment, the control group mice were fed a normal diet (10% kcal from fat), while the model and treatment groups were fed a high-fat diet (HFD) (60% kcal from fat). After four weeks, except for the control group, all groups were injected intraperitoneally with streptozotocin (STZ) (35 mg / kg) for 7 days to induce diabetes. The control group mice received citrate-buffered saline injections. Subsequently, mice with high blood glucose levels (11.1 mmol / L) accompanied by polyphagia, polydipsia, and polyuria were considered diabetic mice and used in subsequent experiments. The treatment group received intraperitoneal injections three times a week for 8 weeks of tFNA (1 μM, 200 μL), curcumin (40 μM, 200 μL), or tFNA-Cur (tFNA: 1 μM, Cur: 40 μM, 200 μL; when preparing tFNA-Cur, the tFNA concentration was 1 μM and the Cur concentration was 40 μM). In addition, the control group and the DOP group received injections of physiological saline (0.9%, 200 μL). Body weight and fasting blood glucose levels were monitored every two weeks. Finally, after euthanasia, long bones (femur and tibia), blood, pancreas, and tissues such as heart, liver, spleen, lungs, and kidneys were collected from mice anesthetized with pentobarbital for subsequent experiments.

[0084] Micro-CT analysis: The femur and tibia were scanned using a SCANCO medical micro-computed tomography scanner 50 (70kV, 200μa, 300ms, 10μm). Regions of interest (ROIs) for the femur and tibia were determined 2mm below the epiphysis.

[0085] Bone tissue and serum AGEs examination: Blood samples were incubated at 4°C for 30 min, and then centrifuged at 3000 rpm for 10 min to obtain serum. The levels of AGEs in serum and bone samples were determined using a mouse AGEs ELISA kit (YKW-20124, Shanghai, China).

[0086] Histological analysis (H&E, Masson, and IF): Specimens from different treatment groups were fixed in 4% paraformaldehyde for 72 hours. One month after decalcification, the bones were dehydrated, embedded in paraffin, and sectioned (3 μm thick). The sections were then stained with H&E, Masson, and IF to observe tissue morphology, structural components, and protein expression.

[0087] 2. Experimental Results

[0088] Figure 4 B represents the HE section results of pancreatic tissue. Figure 4 B showed that the islets of Langerhans in the diabetic osteoporosis model group were destroyed and inflammatory cells infiltrated; the tFNA, Cur and tFNA-Cur treatment groups showed reversal, among which tFNA-Cur treatment had the best effect.

[0089] Figure 4 C and 4E represent the results of femoral micro-CT reconstruction and quantitative analysis. These results indicate that the modeling group mice developed severe osteoporosis, trabecular bone resorption, and fracture. The tFNA, Cur, and tFNA-Cur treatment groups were able to reverse bone destruction, with tFNA-Cur showing the best treatment effect.

[0090] Figure 4 D and 4F represent the results of micro-CT reconstruction and quantitative analysis of the tibia. The trend of the tibial experimental results is consistent with that of the femur. The tFNA-Cur treatment group can significantly reduce osteoporosis caused by diabetes and increase bone formation.

[0091] Figure 4 G represents the HE staining results of the femur and tibia. Figure 4 H represents the Masson staining results of the femur and tibia. The experimental results show that the modeling group mice experienced trabecular bone resorption and fracture, and increased medullary cavity fat. The tFNA, Cur, and tFNA-Cur treatment groups were able to reverse bone destruction and reduce medullary cavity fat, with the tFNA-Cur group showing the best effect.

[0092] Figure 5A and 5D show the results of immunofluorescence detection of ALP expression in tibial tissue. The results indicate that osteoblast protein expression was significantly reduced in the modeling group mice, and a large amount of bone marrow fat accumulated. After treatment with tFNA, Cur, and tFNA-Cur, osteoblast protein expression was increased in vivo, with the tFNA-Cur group showing the best effect.

[0093] Figure 5 B and 5E show the results of immunofluorescence detection of GPX4 protein expression in tibial tissue. These results indicate that GPX4 protein expression was significantly reduced in the modeling group mice. Treatment with tFNA, Cur, and tFNA-Cur reversed ferroptosis in the diabetic microenvironment, with the tFNA-Cur group showing the best effect.

[0094] Figure 5 C and 5F show the results of immunofluorescence detection of NRF2 protein expression in tibial tissue. The results indicate that Cur and tFNA-Cur can significantly activate NRF2 protein expression, with tFNA-Cur being more effective than free Cur.

[0095] Figure 4 and Figure 5 The results confirmed that the tFNA-Cur of this invention has achieved excellent effects in the treatment of diabetic osteoporosis, and verified that in vitro tFNA-Cur inhibits ferroptosis by targeting and activating the NRF2 / GPX4 pathway, promotes the osteogenic capacity of BMSCs, and increases bone formation, thereby treating diabetic osteoporosis. The therapeutic effect of tFNA-Cur is significantly better than that of simple tFNA and free Cur.

[0096] In summary, this invention prepared a tetrahedral framework nucleic acid-curcumin complex. This complex improves the water solubility, stability in physiological fluids, and in vivo utilization efficiency of the natural product curcumin, overcoming the shortcomings of curcumin such as poor water solubility, low bioavailability, and instability in physiological media. In the obtained complex, curcumin and tetrahedral framework nucleic acid exert a synergistic effect, targeting NRF2 to inhibit ferroptosis of bone marrow mesenchymal stem cells in the diabetic bone microenvironment, thereby exerting hypoglycemic and osteopromoting effects. It can be used for the prevention and / or treatment of diabetic osteoporosis and has good application prospects.

Claims

1. The use of a tetrahedral framework nucleic acid-curcumin complex in the preparation of a medicament for the prevention and / or treatment of diabetic osteoporosis; wherein the tetrahedral framework nucleic acid-curcumin complex is prepared from tetrahedral framework nucleic acid and curcumin as raw materials, wherein, The molar ratio of tetrahedral framework nucleic acid to curcumin is 1:(50-800); The tetrahedral framework nucleic acid is formed by four DNA single strands through complementary base pairing, and the sequences of the four DNA single strands are shown in SEQ ID NO.1 to 4.

2. The use according to claim 1, characterized in that: The drug is a drug that inhibits ferroptosis of bone marrow mesenchymal stem cells in the diabetic bone microenvironment.

3. The use according to claim 2, characterized in that: The drug is a drug that targets and binds to NRF2.

4. The use according to claim 1, characterized in that: The drug is a hypoglycemic agent and / or a bone-promoting agent.

5. The use according to any one of claims 1 to 4, characterized in that: The molar ratio of the tetrahedral framework nucleic acid to curcumin is 1:

200.

6. The use according to any one of claims 1 to 4, characterized in that: The preparation method of the tetrahedral framework nucleic acid includes the following steps: dissolving four DNA single strands in equimolar amounts in TM buffer, maintaining at 85-105℃ for 5-15 min, and then maintaining at 2-8℃ for 10-30 min.

7. The use according to claim 6, characterized in that: The preparation method of the tetrahedral framework nucleic acid includes the following steps: dissolving four DNA single strands in equimolar amounts in TM buffer, maintaining at 95°C for 10 min, and then maintaining at 4°C for 20 min.

8. The use according to claim 7, characterized in that: The TM buffer solution contains 10 mM Tris-HCl, 50 mM MgCl2, and pH 8.0; the four DNA single strands have a concentration of 1 μM.

9. The use according to any one of claims 1 to 4, characterized in that: The preparation method of the tetrahedral framework nucleic acid-curcumin complex includes the following steps: Curcumin is incubated with tetrahedral framework nucleic acids at a specific molar ratio to obtain the final product.

10. The use according to claim 9, characterized in that: The incubation temperature is 20–40°C, and the incubation time is 1–8 hours.

11. The use according to any one of claims 1 to 4, characterized in that: The drug is an injectable preparation.

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