Equol-loaded tetrahedral framework nucleic acid complex and application thereof in preparation of medicine for relieving fatty liver disease

Abstract

The application discloses a tetrahedral framework nucleic acid complex loaded with equol and application of the complex in preparation of a medicine for relieving fatty liver disease. The complex is obtained by stable combination of tetrahedral framework nucleic acid and equol through hydrogen bond and hydrophobic interaction; the equol is loaded on the tetrahedral framework nucleic acid structure, and can be slowly released, so that the bioavailability of the equol is improved, and the problems of poor water solubility and low stability of the equol are effectively overcome. Meanwhile, the complex can inhibit lipid accumulation in HepG2 cells, relieve lipid deposition of liver cells, and reduce accumulation of TG and T-CHO in cells, and can be used for relieving fatty liver, and preparing more and more efficient fatty liver treatment medicines, and has wide application potential in intervention of lipid metabolism disorder and related metabolic diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a tetrahedral framework nucleic acid complex loaded with estrol and its application in the preparation of drugs to alleviate fatty liver disease. Background Technology

[0002] Hepatic steatosis has become one of the most common chronic liver metabolic disorders worldwide, and is an early manifestation of non-alcoholic fatty liver disease (NAFLD) and a common pathological feature of many chronic metabolic diseases. In recent years, with the Westernization of dietary structures and the increase in high-fat, high-sugar, and high-energy-density diets, the prevalence of NAFLD has continued to rise, particularly among obese, overweight, and metabolically abnormal individuals. Currently, NAFLD intervention mainly relies on lifestyle management, control of metabolic risk factors, and some drug therapies, but these still face challenges such as limited applicability, significant individual variability in efficacy, and a lack of widely applicable intervention programs. Meanwhile, NAFLD is closely related to obesity, insulin resistance, type 2 diabetes, and dyslipidemia, and its complex pathogenesis further increases the difficulty of disease prevention and treatment. Therefore, developing intervention strategies to reverse steatosis is of great significance for prevention and treatment.

[0003] Equol (Eq), a highly active compound produced from soy isoflavones through gut microbiota metabolism, shows potential efficacy in regulating lipid metabolism, alleviating oxidative stress, and maintaining metabolic homeostasis. However, inherent limitations such as poor water solubility, chemical stability, and limited cellular uptake efficiency severely restrict its application in cellular function regulation and intervention studies. Current research has employed traditional delivery systems such as liposomes and polymer nanoparticles to improve the bioavailability of equol, but these methods still suffer from low drug loading, uncontrollable release behavior, complex preparation processes, and potential biocompatibility risks, making it difficult to meet the demands for efficient, stable, and safe delivery.

[0004] Tetrahedral framework nucleic acids (tFNAs) are a class of nanomaterials formed by the self-assembly of DNA. Compared to other nanomaterials, tFNAs have advantages such as uniform structure, controllable size, low cytotoxicity, good biocompatibility, and biodegradability. More importantly, tFNAs can not only function directly as functional nanomaterials, but also serve as drug delivery carriers, efficiently loading small molecule drugs and other active substances through non-covalent interactions, while protecting the loaded substances from degradation and promoting cellular uptake and delivery across biological barriers. However, there are currently no research reports on using tetrahedral framework nucleic acids to load equaphenone or other hepatitis treatment drugs to alleviate lipid deposition in hepatocytes. Therefore, this invention application is filed. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the problems of poor water solubility, low stability and lack of efficacy of existing equol, as well as the shortcomings of drugs for relieving fatty liver disease. The present invention provides a tetrahedral framework nucleic acid complex loaded with equol and its application in the preparation of drugs for relieving fatty liver disease.

[0006] The first objective of this invention is to provide a tetrahedral framework nucleic acid complex loaded with equadol.

[0007] A second objective of this invention is to provide a method for preparing a tetrahedral framework nucleic acid complex loaded with estrol.

[0008] A third objective of this invention is to provide the application of tetrahedral framework nucleic acid complexes loaded with estrol.

[0009] The fourth object of this invention is to provide a drug.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a tetrahedral framework nucleic acid complex loaded with equol, which is obtained by combining tetrahedral framework nucleic acid with equol; the tetrahedral framework nucleic acid is obtained by self-assembly of four single-stranded DNAs with nucleotide sequences as shown in SEQ ID NO. 1~4; the molar ratio of the tetrahedral framework nucleic acid to equol is (1-5):(100-700).

[0011] This invention constructs a tetrahedral framework nucleic acid complex (tFNAs-Eq) loaded with equol. This complex efficiently loads equol through non-covalent bonds (hydrogen bonds and hydrophobic interactions), significantly improving the water solubility, stability, and cellular uptake of equol, and prolonging its duration of action through nuclease-responsive sustained-release behavior. In vitro cell experiments confirmed that the tFNAs-Eq complex significantly enhances the alleviating effect of sodium oleate (NaOL)-induced lipid deposition in HepG2 cells, with better efficacy than free equol. It can be used to alleviate fatty liver and to prepare more and more effective drugs for the treatment of fatty liver. This invention provides a novel therapeutic agent for nutritional intervention in NAFLD and has promising application prospects.

[0012] Preferably, the molar ratio of the tetrahedral framework nucleic acid to equadol is (1-3):(150-650).

[0013] More preferably, the molar ratio of the tetrahedral framework nucleic acid to equadol is 1:(160-640).

[0014] Preferably, the tetrahedral framework nucleic acid is prepared by adding four single-stranded DNAs to TM buffer, maintaining the temperature at 90-98°C for 5-15 min, and then rapidly cooling the temperature to 0-5°C and maintaining the temperature at 0-5°C for 20-30 min.

[0015] This invention provides a method for preparing the above-mentioned complex, which involves dissolving tetrahedral framework nucleic acid and equadol, incubating them, and then purifying them by centrifugation.

[0016] Preferably, the incubation conditions are 0~5℃ for 12~24 h.

[0017] More preferably, the reaction temperature is 4°C and the reaction time is 24 h. Preferably, the dissolving solvent is one or more of PBS and DMSO.

[0018] Preferably, the purification is performed using a 10-50 kDa ultrafiltration membrane.

[0019] This invention provides the application of the above-mentioned complex in the preparation of drugs for alleviating fatty liver disease.

[0020] Preferably, the fatty liver disease is non-alcoholic fatty liver disease or alcoholic fatty liver disease.

[0021] The present invention also provides a drug comprising the above-mentioned complex.

[0022] Preferably, the drug can alleviate lipid deposition in hepatocytes.

[0023] Preferably, the drug further contains pharmaceutically acceptable excipients or formulations.

[0024] Preferably, the dosage form of the drug is tablets, powders, or injections.

[0025] The present invention has the following beneficial effects: This invention provides a tetrahedral framework nucleic acid complex loaded with equol. Equol is loaded onto the tetrahedral framework nucleic acid via groove binding. Using the tetrahedral framework nucleic acid as a delivery carrier improves the stability, bioactivity, and bioavailability of equol. Simultaneously, this complex exhibits a certain sustained-release property, which helps prolong the duration of action of equol. Furthermore, the excellent cellular uptake capacity of the tetrahedral framework nucleic acid promotes the entry of equol into cells and the exertion of its biological functions. The complex provided by this invention can inhibit lipid accumulation in HepG2 cells, alleviate lipid deposition in hepatocytes, and reduce the accumulation of intracellular TG and T-CHO, thus enabling its use in alleviating fatty liver and developing more effective and efficient drugs for the treatment of fatty liver. Therefore, the tetrahedral framework nucleic acid complex loaded with equol provided by this invention has good application value in the treatment of NAFLD and shows promising application prospects as a novel therapeutic agent. Attached Figure Description

[0026] Figure 1The diagram shows the preparation and characterization results of the complex (A in the figure is a schematic diagram of the synthesis process of the tFNAs-Eq complex; B is the PAGE characterization result of tFNAs; C is the particle size result of tFNAs and tFNAs-Eq complex measured by DLS; D is the Zeta potential result of tFNAs and tFNAs-Eq complex measured by DLS; E is the TEM image of tFNAs, scale bar: 100nm, 20nm; F is the stability of tFNAs and tFNAs-Eq complex at 37℃ and 10% FBS and at 4℃; G is the Hoechst 33342 (λ) of tFNAs). ex = 350 nm) fluorescence and fluorescence density of the tFNAs-Eq complex*** P <0.001 vsHochest 33342, ### P <0.001 vs tFNAs (n = 3); H represents the encapsulation efficiency of tFNAs-Eq at different ratios (1:160-1:640); I represents the binding mode of tFNAs loaded with equadolol verified by molecular docking; J represents the in vitro release rate of the equadolol-tFNAs-Eq complex in 10% FBS (pH 7.4, 37℃); K represents the in vitro release rate of the equadolol-tFNAs-Eq complex in PBS (pH 7.4, 37℃); L represents the release rate of the tFNAs-Eq complex in BSA, DNase I, and Mg 2+ In vitro release rate).

[0027] Figure 2 Figure A shows the CLSM imaging results of HepG2 cells taking up CY5-labeled tFNAs and the tFNAs-Eq complex after 4 hours of co-incubation. Red represents CY5-labeled tFNAs or the tFNAs-Eq complex, green represents the cytoskeleton, and blue represents the cell nucleus. The scale bar is 20 μm. Figure B shows the fluorescence intensity of single-chain S1, tFNAs, and the tFNAs-Eq complex in HepG2 cells as shown by flow cytometry. Data are expressed as mean ± standard deviation. P <0.05、** P <0.01、*** P <0.001 and **** P <0.0001 indicates a statistically significant difference between the marked groups.

[0028] Figure 3 The effect of equol and tFNAs-Eq complex on alleviating lipid deposition in HepG2 cells (A and B in the figure show the effect of different concentrations of equol and tFNAs on HepG2 cell viability, *compared with the control group:*) P<0.05,** P <0.01, *** P <0.001, **** P <0.0001, ns indicates no statistically significant difference; C and D show the results of Oil Red O staining of HepG2 cells and the degree of Oil Red O staining measured by absorbance; E and F show the effects of estrol, tFNAs, and tFNAs-Eq complex on the T-CHO and TG content of HepG2 cell adipocytes, #compared with the control group:# P <0.05, ### P <0.001, *Comparison with the model group:* P <0.05, ** P <0.01, *** P <0.001). Detailed Implementation

[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0030] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0031] The equol (Eq) used in the examples was a commercially available product with a purity of ≥98%, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0032] Example 1 Synthesis of a tetrahedral framework nucleic acid complex loaded with estrol (tFNAs-Eq) 1. Synthesis of tetrahedral framework nucleic acids (tFNAs) The four single-stranded DNA molecules of the tetrahedral framework nucleic acid used in this embodiment are described in the prior art: Javier V, Akul P, Richard AC, et al. The role of size in biostability of DNAtetrahedra.[J]. Chemical communications (Cambridge, England), 2023, 59(34):DOI:10.1039 / D3CC01123B. Tetrahedral framework nucleic acids (tFNAs) are synthesized by self-assembly of four sequence-specific single-stranded DNA molecules (ssDNA) through PCR amplification (maintaining 95℃ for 10 min, rapidly cooling to 4℃ and maintaining for 20-30 min). The specific preparation method is as follows: Four single-stranded DNAs, S1, S2, S3, and S4, with their specific sequences shown in Table 1, were dissolved in TM buffer at equimolar ratios to achieve a final concentration of 250 nM for each single-stranded DNA. After thorough mixing, the mixture was rapidly heated to 95°C and maintained for 10 min, followed by rapid cooling to 4°C and maintained for 20-30 min, thus achieving self-assembly synthesis of tetrahedral framework nucleic acids (tFNAs).

[0033] Table 1. Specific sequences of four single-stranded DNAs

[0034] 2. Synthesis of estrol-loaded tetrahedral framework nucleic acid complexes (tFNAs-Eq) Equol (Eq) was added to the tFNAs prepared above and incubated. The concentration of equol was 40 μM, the concentration of tFNAs was 250 nM, and the molar ratio of tFNAs to Eq was 1:160. The mixed solution was incubated at 4 °C for 24 h, and then ultrafiltered at 10000 rpm (30 kDa molecular weight membrane) for 10 min to remove residual ssDNA and free equol, yielding the tFNAs-Eq complex.

[0035] Example 2 Characterization and performance determination of tFNAs-Eq complex 1. Characterization of tFNAs-Eq The successful synthesis of the tFNAs-Eq complex was verified by polyacrylamide gel electrophoresis (PAGE). Subsequently, dynamic light scattering (DLS) was used to measure the particle size and zeta potential of the tFNAs and tFNAs-Eq complex, and transmission electron microscopy (TEM) was used to observe their size and shape.

[0036] The synthesis steps of tFNAs-Eq are as follows: Figure 1 As shown in Figure A, tFNAs are self-assembled from four equimolar amounts of ssDNA, and then the tFNAs form a complex by being incubated with equadol at 4°C for 24 h.

[0037] PAGE measurement results are as follows: Figure 1 As shown in Figure B, tFNAs were successfully synthesized. DLS measurement results are as follows... Figure 1 As shown in C and D, the particle size of tFNAs is 10.1 nm, while the particle size of the tFNAs-Eq complex is 21.04 nm. The Zeta potential of equol is -8.71 mV, that of tFNAss is -9.86 mV, and that of tFNAs-Eq is -32.97 mV. TEM images are shown below. Figure 1As shown in Figure E, the tFNAs have a size of 10-20 nm and a tetrahedral structure. In summary, the results indicate that the tFNAs-Eq complex was successfully synthesized and is relatively stable compared to tFNAs.

[0038] 2. tFNAs-Eq Encapsulation Rate First, the absorption peak of equol was measured using a UV spectrophotometer. Then, the absorbance of equol solutions at different concentrations (gradient concentrations: 0, 50, 100, 200, 300, 400, 500 μM) was measured to obtain a standard curve for equol. tFNAs (250 nM) were incubated with different concentrations of equol to obtain tFNAs-Eq. The tFNAs-Eq solutions were then centrifuged at 10,000 rpm for 10 min in 30 kD ultrafiltration tubes to separate loaded and free equol. Measurements were performed at 280 nm using a microplate reader. The concentration of free equol in the tFNAs-Eq solution was calculated based on the standard curve. Finally, the loading efficiency of tFNAs-Eq was determined based on the total equol concentration and the unloaded concentration.

[0039] Load factor (%) = (Total Equol) Free Equol / Total Equol × 100.

[0040] The results are as follows Figure 1 As shown in Figure H, by measuring and analyzing the encapsulation efficiency of equol to tFNAs at different ratios, the results showed that with the increase of the equol ratio, the loading rate of equol on tFNAs first increased, then decreased, and then increased slightly, indicating that the binding sites gradually became saturated. The maximum encapsulation efficiency was reached at 80 μM, which was 59.58 ± 0.51%.

[0041] 3. In vitro release rate and stability of tFNAs-Eq ①In vitro release rate of tFNAs-Eq: 1 mL of equol (100 μM) and tFNAs-Eq solution (tFNAs:Eq = 1:400, tFNAs = 250 nM) were added to a dialysis membrane as the inner solution (30 kDa). This solution was then immersed in a centrifuge tube containing 30 mL of PBS. The membrane was incubated at 37°C and 150 rpm on a shaker. At regular intervals, 200 μL of the outer solution was taken and the absorbance was measured at 280 nm. The concentration of equol in the outer solution was determined based on the equol standard curve. Then, 200 μL of PBS was added back to continue the slow release process.

[0042] Q=[V0C i +V t ∑C （i-1） ] / m×100% Where: V0 is the total volume of the release medium (mL) - 30mL; C iV represents the concentration of Eq (μg / mL) at time point i; t The sample volume (mL) is 0.2 mL; m is the mass of equadol (μg).

[0043] The results are as follows Figure 1 As shown in Figure J, sustained-release performance is a key characteristic of nanodelivery systems. In PBS buffer (pH 7.4, 37°C) containing 10% FBS, free equol exhibited a burst release characteristic, subsequently stabilizing after 24 h and reaching a maximum cumulative release of 99.37 ± 0.59% at 48 h. In contrast, the release curve of tFNAs-Eq was slower, with a maximum cumulative release of 97.86 ± 0.73% at 48 h. Notably, the release level of tFNAs-Eq at 24 h was roughly equivalent to that of equol at 12 h, indicating that the loading of tFNAs significantly delayed the release of equol. Both groups showed relatively high release rates, which may be due to the proteins, enzymes, and Mg in the FBS. 2+ Weakening or disrupting the non-covalent interaction between tFNAs and equol promotes the release of equol.

[0044] ②tFNAs-Eq stability: Equal amounts of tFNAs and tFNAs-Eq were incubated in 10% fetal bovine serum (FBS) at 37°C for 0, 2, 4, 6, 8, 12, and 24 h. Alternatively, they were stored at 4°C for 1, 2, 3, 4, 5, and 6 days, and the stability of tFNAs and tFNAs-Eq was observed by PAGE.

[0045] The measurement results are as follows Figure 1 As shown in Figure F, tFNAs at 37°C in the presence of 10% FBS began to show band blurring and degradation after 12 h. In contrast, tFNAs-Eq remained stable until 24 h, at which point signs of degradation appeared. When stored in TM buffer at 4°C, tFNAs began to degrade after 5 days, while tFNAs-Eq only began to degrade after 6 days. This indicates that the stability of tFNAs in the tFNAs-Eq complex is significantly enhanced.

[0046] 4. The binding mechanism between tFNAs and equadol To further determine the binding site of equadol, a Hoechst 33342 fluorescence competition assay was performed. Hoechst 33342 can bind to tFNAs, and then tFNAs and tFNAs-Eq were stained with Hoechst 33342.

[0047] The fluorescent dye Hoechst 33342 was added to tFNAs (250 nM) and tFNAs-Eq solutions (250 nm ~ 100 μM), respectively. The mixtures were thoroughly mixed and incubated at room temperature in the dark for 30 min. Then, the ELISA reader was used to read the λ... ex = The binding mechanism between estrol and tFNAs was analyzed by detecting the fluorescence absorbance of the sample at a wavelength of 350 nm.

[0048] The measurement results are as follows Figure 1 As shown in G, the fluorescence intensity (λ) of Hoechst 33342 is displayed. ex = 350 nm) decreased with the addition of eequine, indicating that Hoechst 33342, which binds to tFNAs, was replaced by eequine. This means that the site where eequine interacts with tFNAs is the same double-stranded DNA double helix groove region as Hoechst 33342.

[0049] 5. Molecular simulation analysis of the tFNAs-Eq binding mechanism First, sequence information was retrieved from the Uniprot database (https: / / www.uniprot.org / ). The sequences were submitted to the ITASSER online server (https: / / zhanggroup.org / I-TASSER / ) for initial structure prediction. Energy optimization and local conformational adjustments were performed using the Rosetta Relax module to obtain the DNA structure for molecular docking. The three-dimensional structure of equol was obtained from the PubChem database. Molecular docking was performed on the nucleic acid binding site between equol and tFNAs using Autodock Vina. Ligand-receptor interaction analysis was performed using the PLIP online server (https: / / plip-tool.biotec.tudresden.de / plip-web / plip / index). The three-dimensional conformation of the ligand-receptor complex was visualized using PyMOL software.

[0050] The results are as follows Figure 1 As shown in Figure I, equol can insert into the DNA double helix structure, forming a stable binding conformation (binding energy: -7.16 kcal / mol). Equol primarily forms hydrophobic interactions with bases A10, G11, T26, A9, A27, G28, and T8, while forming hydrogen bonds with bases T12 and G29. Based on the docking conformation, it is theoretically estimated that a single tFNA molecule can bind approximately 30-36 equol molecules (i.e., approximately 1.5 × 10⁻⁶ molecules per mole of tFNAs). -9 (mol equadol). The experimentally measured loading was 4.8 × 10⁻⁶. 5The mol Eq / mol tFNAs ratio is significantly higher than the theoretical estimate, indicating that the system has a high loading capacity. The results suggest that the binding of equadol to tFNAs is not limited to a single groove binding mode, but may also involve interactions within the minor / major groove and the angular regions at the junctions of the six DNA double-stranded fragments.

[0051] 6. Analysis of the tFNAs-Eq release mechanism To evaluate albumin (BSA) activity, DNase I activity, and Mg 2+ The effect of tFNAs-Eq complex release behavior on physiological conditions was investigated through in vitro release experiments. Specifically, the tFNAs-Eq complex was dispersed in BSA solution (2.5 mg BSA / mL PBS) to simulate the effect of complex entry into serum and the influence of the protein corona on the release rate. Different concentrations of DNase I (0.1, 0.5, 1 U / mL) and 12.5 mM MgSO4 were used. 2+ To simulate typical nuclease activity levels in human serum, we used 12.5 mM Mg²⁺. + Phosphate-buffered saline (PBS, pH 7.4) was used to simulate the ionic environment of plasma. The PBS system served as a control to assess which factors primarily affected in vitro release. Subsequently, samples were incubated at 37°C with gentle shaking at 150 rpm to simulate physiological conditions. Samples were collected at predetermined time points, and the nuclease reaction was immediately terminated by adding 5 mM EDTA.

[0052] The release of equadol was evaluated under different component conditions. The results are as follows: Figure 1 K and L indicate bovine serum albumin (BSA), DNase I, and Mg. 2+ All enzymes promoted equol release to varying degrees, with DNase I showing the strongest effect (release rate of 60.10 ± 1.41%). Furthermore, the release rate increased with increasing DNase I concentration. The tested DNase I concentration range (0.5-3 U / mL) is close to the reported average concentration of nucleases in human blood (3.6 U / mL). Previous studies have shown that enzymatic degradation is a major driver of nucleic acid structure destruction, with DNase I playing a particularly important role, suggesting that DNase I may be a major influencing factor in equol release. DNase I is a divalent metal ion-dependent endonuclease, Mg... 2+It can enhance its enzymatic activity. With increasing DNase I concentration, the cleavage of the nucleic acid phosphodiester backbone is enhanced, leading to a significant increase in equadol release. In contrast, BSA has a relatively small effect on the release behavior, which may be because BSA forms a protein crown on the tFNAs-Eq surface, producing a partial shielding effect, thereby reducing the degradation of the complex by DNase I.

[0053] In summary, this invention successfully prepared a nanomaterial—tFNAs-Eq composite, which has good loading capacity, good sustained-release behavior and excellent structural stability.

[0054] Example 3 Evaluation of the uptake capacity of tFNAs-Eq complex in HepG2 cells 1. Assay of cellular uptake capacity of tFNAs and tFNAs-Eq complex CY5 was ligated to the 5' end of the specific single-stranded DNA (ssDNA) S1 of tetrahedral framework nucleic acids (tFNAs) to obtain CY5-labeled S1 (as shown in Table 1, CY5-S1). Using CY5-S1, CY5-labeled tFNAs and CY5-labeled tFNAs-Eq complexes (molar ratio of tFNAs to equadol was 1:240) were prepared respectively for the following experiments.

[0055] After HepG2 liver cancer cells adhered and grew in 6-well plates, the cells were co-incubated with CY5-S1, CY5-tFNAs, and CY5-tFNAs-Eq for 4 h. After 4 h of incubation, the cells were washed with PBS, fixed with 4% paraformaldehyde, and then treated with 4',6-diaminobenzene... 2 Phenylindole (DAPI) and microfilament green fluorescent probe (Actin-Tracker Green-488) staining were performed. To assess the uptake of S1, tFNAs, and tFNAs-Eq cells, results were obtained using confocal microscopy and flow cytometry.

[0056] The results are as follows Figure 2 As shown in Figure A, images from laser confocal microscopy reveal weak fluorescence in the CY5-S1 group, while strong fluorescence was detected in the tFNAs and tFNAs-Eq groups, with tFNAs and tFNAs-Eq being widely distributed in the cytoplasm. This indicates that single-stranded tFNAs have difficulty entering cells, while tFNAs and tFNAs-Eq have a strong ability to enter cells.

[0057] In addition, flow cytometry yielded similar results, such as... Figure 2As shown in Figure B, after 4 h of incubation, 14.5% CY5 fluorescence was detectable in the S1 group cells, while the fluorescence of the tFNAs and tFNAs-Eq groups was even stronger, reaching 83.6% and 84.3%, respectively. These results demonstrate the suitability of tFNAs as a carrier and their excellent cellular uptake capacity.

[0058] Example 4: Effect of tFNAs-Eq complex on alleviating lipid deposition in HepG2 cells 1. Cell Culture and Processing HepG2 cells were cultured in DMEM complete medium (37℃, 5% CO2) in an adherent state. The optimal NaOL intervention concentration was determined based on CCK-8, Oil Red O staining, and triglyceride (TG) content. Cells were then co-cultured with 0.48 mM NaOL for 24 h to establish a NaOL-induced HepG2 cell steatosis model (non-alcoholic fatty liver disease (NAFLD)). Subsequent experiments included a control group, a model group, an estrol group, a tFNAs group, and a tFNAs-Eq group, with cell intervention treatment lasting 24 h.

[0059] 2. Measurement of cell viability With 1×10 4 HepG2 cells were seeded into 96-well plates at a density of cells / well and incubated at 37°C with 5% CO2 for 24 h. Cells were then divided into groups with equol concentrations of 0, 20, 40, 60, 80, and 100 μM and tFNA concentrations of 0, 62.5, 125, 250, and 500 nM. The cells were then cultured in complete medium containing the components of the aforementioned groups. After 24 h, CCK-8 solution was added, and the cells were incubated at 37°C for 0.5 h. Absorbance was measured at 450 nm using a multi-wavelength microplate reader, and relative cell viability was calculated.

[0060] CCK-8 assays were performed to assess its biocompatibility, and the results were as follows: Figure 3 As shown in Figure A, cell viability increased at equadol concentrations of 60 μM and below, and the encapsulation efficiency of equadol was also improved. Figure 1 Therefore, 60 μM was chosen as the working concentration of equadol for in vitro studies. Meanwhile, tFNA concentration screening experiments showed that 250 nM was the optimal working concentration for tFNAs. Figure 3 B), and thus a ratio of 1:240 (tFNAs:Eq) was chosen for subsequent experiments.

[0061] 3. Oil Red O staining, TG content and T-CHO content determination Press 1×10 5HepG2 cells were seeded into 6-well plates at a density of cells / well and cultured for 24 h. After 24 h of culture, the cells were treated with 0.48 mM NaOL, followed by treatment with each experimental group for another 24 h. Cells were then washed twice with PBS and fixed with 4% paraformaldehyde for 30 min. After washing twice with distilled water, the cells were immersed in 60% isopropanol for 30 s, stained with Oil Red O solution in the dark for 20 min, washed with distilled water, counterstained with Mayer's hematoxylin for 1 min, and then blued with PBS for 1 min. Intracellular lipid droplets were observed using an inverted microscope. After observation, 1 mL of 60% isopropanol was added to each well, and the cells were incubated at room temperature for 30 min. The absorbance of each well was measured at 510 nm to quantify lipid content. Additionally, the cell pellet was collected, lysed with 2% Triton X-100 in an ice bath for 30 min, and intracellular TG and total cholesterol (T-CHO) levels were detected using a reagent kit.

[0062] Oil Red O staining results as follows Figure 3 As shown in Figure C, compared with the control group, the model group showed significant accumulation of red lipid droplets, indicating that the fatty degeneration model had been successfully constructed. Compared with the model group, tFNAs did not significantly reduce lipid droplet accumulation. However, the accumulation of red lipid droplets in cells was significantly reduced and the color became lighter in the equol and tFNAs-Eq groups, with the tFNAs-Eq group showing a more significant reduction in lipid droplets than the equol group. The results indicate that both equol and tFNAs-Eq can reduce the level of lipid accumulation in HepG2 cells, with tFNAs-Eq showing a better effect.

[0063] To more accurately and objectively evaluate intracellular lipid accumulation levels, isopropanol was used to extract lipids from cells and absorbance was measured. The results are as follows: Figure 3 As shown in Figure D, compared with the control group (0.58±0.01), the absorbance of the model group (0.76±0.01) increased significantly ( P <0.001), consistent with the results observed by Oil Red O staining. Both equol and tFNAs-Eq treatments reduced the absorbance of intracellular lipids, with tFNAs-Eq showing the lowest absorbance (0.38±0.04). These results further demonstrate that equol and tFNAs-Eq can inhibit lipid accumulation in HepG2 cells and alleviate lipid deposition in hepatocytes; compared to equol, tFNAs-Eq showed a better effect in alleviating lipid deposition in hepatocytes.

[0064] Meanwhile, the results of the measured total cholesterol (T-CHO) and TG levels are as follows: Figure 3As shown in E and F, compared with the control group, the TG and T-CHO contents in the model group cells were significantly increased. However, compared with the model group, the TG and T-CHO contents in HepG2 cells treated with equadol and tFNAs-Eq were decreased, with the tFNAs-Eq treatment group showing a highly significant decrease in both TG and T-CHO contents. P The value <0.01 indicates that both equol and tFNAs-Eq can reduce the accumulation of intracellular TG and T-CHO, with tFNAs-Eq showing better improvement than free equol. Both have the effect of alleviating lipid deposition in hepatocytes and can be used to alleviate fatty liver, and to develop more and more effective drugs for the treatment of fatty liver.

[0065] In summary, this invention provides a tetrahedral framework nucleic acid complex loaded with equol. Equol is loaded onto the tetrahedral framework nucleic acid via groove binding. Using the tetrahedral framework nucleic acid as a delivery carrier improves the stability, bioactivity, and bioavailability of equol. Simultaneously, this complex exhibits a certain sustained-release property, which helps prolong the duration of action of equol. Furthermore, the excellent cellular uptake capacity of the tetrahedral framework nucleic acid promotes the entry of equol into cells and the exertion of its biological functions. The complex provided by this invention can inhibit lipid accumulation in HepG2 cells, alleviate lipid deposition in hepatocytes, and reduce the accumulation of intracellular TG and T-CHO, thus enabling its use in alleviating fatty liver and facilitating the preparation of more and more effective drugs for the treatment of fatty liver. Therefore, the tetrahedral framework nucleic acid complex loaded with equol described in this invention has potential application value in the treatment of NAFLD and can serve as a novel therapeutic agent with promising application prospects.

[0066] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A tetrahedral framework nucleic acid complex loaded with estrol, characterized in that, It is obtained by combining tetrahedral framework nucleic acid with equadol; the tetrahedral framework nucleic acid is obtained by self-assembly of four single-stranded DNAs with nucleotide sequences as shown in SEQ ID NO. 1~4; the molar ratio of the tetrahedral framework nucleic acid to equadol is (1-5):(100-700).

2. The complex according to claim 1, characterized in that, The molar ratio of the tetrahedral framework nucleic acid to equadol is (1-3):(150-650).

3. The complex according to claim 1, characterized in that, The preparation method of the tetrahedral framework nucleic acid is as follows: add four single-stranded DNAs to TM buffer, maintain at 90~98℃ for 5~15 min, and rapidly cool to 0~5℃ for 20~30 min to obtain the nucleic acid.

4. A method for preparing the complex according to any one of claims 1 to 3, characterized in that, The tetrahedral framework nucleic acid and equadol were dissolved, incubated, and purified by centrifugation.

5. The preparation method according to claim 4, characterized in that, The incubation conditions are 0~5℃, 12~24 h.

6. The preparation method according to claim 4, characterized in that, The purification process uses a 10-50 kDa ultrafiltration membrane for ultrafiltration purification.

7. The use of the complex according to any one of claims 1 to 3 in the preparation of a drug for relieving fatty liver disease.

8. The application according to claim 7, characterized in that, The fatty liver disease mentioned is non-alcoholic fatty liver disease.

9. A drug, characterized in that, Contains the complex according to any one of claims 1 to 3.

10. The drug according to claim 9, characterized in that, The drug also contains pharmaceutically acceptable excipients or preparations.

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