A multi-target metabolic intervention diagnosis and treatment integrated system based on tetrahedral DNA nanostructure and application thereof

Abstract

The application discloses a kind of multi-target metabolic intervention diagnosis and treatment integrated system based on tetrahedral DNA nanostructure and application thereof, provide a kind of set "targeting-treatment-diagnosis" in one DNA tetrahedron nano device (TDN-siRNAs-Apt-P). The delivery obstacle of siRNA is overcome by tetrahedral DNA framework, precise targeting is realized by using aptamer, real-time monitoring of tumor markers is realized by using probe, and three kinds of metabolic inhibition siRNA are simultaneously targeted and delivered, which cooperatively block three "supply lines" of glucose, amino acid and lipid metabolism, thereby solving the problem of drug resistance caused by metabolic plasticity of cancer cells and the defect of lack of visual monitoring of traditional carriers.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a multi-target metabolic intervention and diagnosis system based on tetrahedral DNA nanostructures and its application. Background Technology

[0002] Cervical cancer (CC) is a common gynecological malignancy, and its progression is closely related to metabolic remodeling. Unlike normal cells, cancer cells exhibit unique metabolic characteristics. They not only preferentially perform glycolysis (dependent on GLUT1 for glucose transport) when oxygen is plentiful, but also highly rely on glutamine (transported via ASCT2) for precursors and significantly upregulate de novo lipid synthesis pathways (regulated by SREBP1) to meet the demands of rapid proliferation.

[0003] Metabolic plasticity allows cervical cancer cells to evade targeted therapy by dynamically adjusting their dependence on glucose, glutamine, and lipids. Overcoming this metabolic adaptation requires multi-stage intervention, but precisely combining multiple therapeutic agents with diagnostic feedback remains a significant challenge.

[0004] Current metabolic therapies mostly focus on single targets (such as simple glycolysis inhibitors), but cancer cells have extremely high metabolic plasticity. For example, blocking glycolysis alone can lead to compensatory enhancement of glutamine or fatty acid oxidation in cancer cells, failing to completely cut off the tumor's energy supply.

[0005] Small interfering RNA (siRNA) technology holds great potential in silencing disease-related genes by precisely targeting and interfering with metabolism. However, naked siRNA faces significant obstacles in clinical applications, including its susceptibility to enzymatic degradation in the physiological environment, extremely poor cellular uptake, and low endosome escape efficiency. Suitable delivery vectors are needed to protect and deliver it. However, most traditional nanodelivery vectors lack specific targeting capabilities, are prone to off-target toxicity, and cannot achieve real-time visibility of the drug delivery process or prognostic monitoring. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0007] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0008] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide an integrated system for diagnosis and treatment of multi-target metabolic intervention based on tetrahedral DNA nanostructures.

[0009] To address the aforementioned technical problems, this invention provides the following technical solution: a multi-target metabolic intervention and diagnostic integrated system based on tetrahedral DNA nanostructures, characterized in that: the system comprises: A tetrahedral DNA nanostructure backbone, a targeting aptamer attached to the backbone, a small interfering RNA loaded on the backbone, and a strand displacement probe hybridized to the backbone; The targeting aptamer is the AS1411 aptamer, which is used to specifically target tumor cells; The small interfering RNAs include those that target and silence the ASCT2, GLUT1, and SREBP1 genes. The chain displacement probe can be triggered by miR-21, which is highly expressed in tumor cells, and generate a detectable signal.

[0010] As a preferred embodiment of the multi-target metabolic intervention and integrated diagnosis and treatment system of the present invention, the small interfering RNA includes siASCT2 which targets and silences the ASCT2 gene, siGLUT1 which targets and silences the GLUT1 gene, and siSREBP1 which targets and silences the SREBP1 gene.

[0011] As a preferred embodiment of the multi-target metabolic intervention and integrated diagnosis and treatment system of the present invention, wherein: the small interfering RNA is connected to the vertices of the tetrahedral DNA nanostructure backbone through a DNA / RNA hybrid strand.

[0012] As a preferred embodiment of the multi-target metabolic intervention and integrated diagnosis and treatment system of the present invention, the chain displacement probe contains a fluorescent reporter group and a quencher group. When it does not bind to miR-21, the fluorescence is quenched. When it binds to miR-21, a chain displacement reaction occurs, causing the fluorescent reporter group and the quencher group to separate and generate a fluorescent signal.

[0013] As a preferred embodiment of the multi-target metabolic intervention and integrated diagnosis and treatment system of the present invention, the tetrahedral DNA nanostructure backbone is formed by the self-assembly of four single-stranded DNA molecules through complementary base pairing.

[0014] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for preparing a multi-target metabolic intervention integrated diagnostic and therapeutic system, characterized by comprising the following steps: Four equimolar amounts of single-stranded DNA were dissolved in PBS buffer containing 0.05 mM MgCl2·6H2O at pH 7.4. The mixture was heated and then cooled to allow the single-stranded DNA to self-assemble into a tetrahedral DNA nanostructure TDN-Apt carrying the AS1411 aptamer. Small interfering RNA was mixed with pre-annealed strand displacement probes and TDN-Apt, and then the mixture was incubated at 37°C for 20 minutes to obtain a nanotherapeutic system loaded with small interfering RNA and probes.

[0015] As a preferred embodiment of the preparation method described in this invention, the heating and cooling process involves heating at 95°C for 10 minutes, followed by cooling to 25°C at a constant rate of 0.1°C / s.

[0016] In a preferred embodiment of the preparation method described in this invention, the molar ratio of the small interfering RNA to the pre-annealed strand displacement probe to the tetrahedral DNA nanostructure is 1:1.

[0017] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of a multi-target metabolic intervention diagnostic and therapeutic integrated system in the preparation of drugs for the treatment of cervical cancer.

[0018] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of a multi-target metabolic intervention integrated diagnostic and therapeutic system in the preparation of diagnostic reagents for real-time monitoring of miR-21 levels in tumor cells.

[0019] Beneficial effects of this invention: (1) High structural stability and biocompatibility: The rigid framework of TDN effectively protects the easily degradable siRNA. The system can still maintain excellent structural integrity and functional specificity after being placed in PBS for 7 days.

[0020] (2) Strong targeted uptake and specific imaging capabilities: The introduction of AS1411 increased the relative fluorescence intensity of tumor cells (HeLa) uptake of nanomedicine by 1.5 times; the probe can be specifically activated by the cervical cancer marker miR-21, and the fluorescence half-life exceeds 12 hours in the in vivo or situ tumor model, realizing real-time tumor monitoring with high signal-to-noise ratio.

[0021] (3) Significant effects of metabolic blockade and apoptosis promotion: Multiple gene silencing downregulated the expression levels of ASCT2, GLUT1, and SREBP1 by more than 50%, resulting in a residual glucose concentration in the culture medium that was 2.9 times higher than that in the control group, an intracellular lactate decrease of about 50%, and a 50% reduction in triglycerides. The resulting energy crisis directly led to the complete collapse of the PI3K-AKT signaling axis, a significant decrease in mitochondrial membrane potential, a 4-fold increase in the late-stage apoptosis rate of tumor cells, and a significant decrease in migration rate to 30.39%.

[0022] (4) High tumor inhibition rate in vivo and no systemic toxicity: In the orthotopic mouse model of cervical cancer, the TDN-siRNAs-Apt-P treatment group almost completely inhibited tumor growth, with an average tumor weight inhibition rate of up to 80% (from 1.75g ​​to 0.35g). At the same time, the mice maintained stable weight throughout the treatment, and no abnormalities were found in liver function and blood routine tests, with no obvious organ damage. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 The synthesis and characterization of functional tetrahedra for the TDN-siRNAs-Apt-P nanotherapeutic system included: (A) flowchart of functional tetrahedron preparation; (B) 8% native PAGE verification of functional tetrahedron assembly; (C) DLS and TEM detection of functional tetrahedron particle size; (D) Zeta potential detection of hydrated particle size of functional tetrahedron; and (E) storage stability of functional tetrahedron and fluorescence effect of probe after miR-21 addition.

[0024] Figure 2 To achieve live-cell miR-21 diagnosis of functional tetrahedrons, the following were determined: (A) the diagnostic mechanism of functional tetrahedron miR-21; (B) flow cytometry detection of the fluorescence intensity of functional tetrahedrons in H8 and HeLa cells; (C) native PAGE verification of the miR-21 chain substitution specificity of functional tetrahedrons; and (D) the luminescence of functional tetrahedrons under treatment with gradient concentrations of miR-21 inhibitors / mimics.

[0025] Figure 3 The effects of functional tetrahedrons on HeLa cells were investigated, including (A) scratch assay to detect cell migration ability after 48 hours; (B) colony formation assay to detect cell proliferation ability; (C) ROS (reactive oxygen species) assay; (D) flow cytometry assay to detect mitochondrial membrane potential; and (E) flow cytometry assay to detect apoptosis.

[0026] Figure 4To induce cellular metabolic reprogramming in patients with functional tetrahedrons, the following steps were taken: (A) qPCR verification of functional tetrahedron gene silencing levels; (B) Western blotting to detect changes in related proteins; (C) changes in glucose (Glu) levels in the culture medium and cells; (D) changes in lactate (LD) levels in the culture medium; (E) changes in triglyceride (TG) levels in cells; (F) changes in α-ketoglutarate (α-KG) levels in cells; (G) a bar chart showing differentially expressed KEGG pathway genes between the TDN-siRNAs-Apt-P and Control groups; and (H) a bar chart showing differentially expressed GO pathway genes between the TDN-siRNAs-Apt-P and Control groups. (Blue highlights indicate pathways / functions related to metabolism / apoptosis.) Figure 5 This study aims to investigate the antitumor treatment and monitoring of cervical cancer in situ using functional tetrahedrons. The results include: (A) a flowchart of in vivo antitumor treatment and monitoring; (B) in vivo detection of functional tetrahedrons based on miR-21; (C) mouse body weight monitoring during treatment; (D) mouse tumor volume monitoring during treatment; (E) mouse tumor anatomy after treatment; (F) H&E staining of mouse tumor tissue after treatment; (G) Ki67 immunofluorescence detection of mouse tumor tissue after treatment; and (H) TUNEL immunofluorescence detection of mouse tumor tissue after treatment.

[0027] Figure 6 For the targeted uptake and cell imaging of functional tetrahedrons, (A) confocal laser scanning microscopy images of H8 and HeLa cells after application of TDN-P or TDN-Apt-P, respectively; (B) quantitative analysis of Cy3 fluorescence intensity in different treatment groups. Detailed Implementation

[0028] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0029] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0030] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0031] The raw materials used in this invention were: chloroform, isopropanol, and magnesium chloride hexahydrate (MgCl2·6H2O), purchased from Beijing Chemical Plant (Beijing, China). Tris was purchased from Beijing Bio-Tech Co., Ltd. (Beijing, China). TRIzol reagent was purchased from Invitrogen (USA). SafeBlue dye was purchased from TransGen Biotech Co., Ltd. (Wuhan, China). All oligonucleotides used in this study were custom-synthesized and purified by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China), and their specific sequences are shown in Table 1. The sequences of S1, S2, S3, T4, and S4 are shown in SEQ ID No. 1~5.

[0032] Table 1

[0033] The instruments used in these embodiments of the invention were as follows: DNA concentration was measured using a Nanodrop OneC spectrophotometer (ThermoFisher Scientific, Inc., Wilmington, DE, USA). UV-Vis and fluorescence spectra were acquired using a Spark™ multi-well microplate reader (Tecan, Männedorf, Switzerland). Gel images were acquired using an iBright FL1000 imaging system (Thermo Fisher Scientific, Inc., Wilmington, DE, USA). Cell imaging was performed using a laser confocal scanning microscope (CLSM, Leica TCS SP2, Leica Microsystems, Mannheim, Germany).

[0034] Detection method in this embodiment of the invention: (1) Cell culture and cell viability detection Human cervical cell lines (including non-tumorigenic H8 cells and tumorigenic HeLa cells) were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were cultured in a humidified incubator at 37°C with 5% CO2. To determine the optimal treatment concentration, HeLa cells were cultured at a rate of 2 × 10⁶ cells / year. 3The crystals were seeded at a density of [number] per well in 96-well plates. After incubation with the drug for 24 hours, each well was washed with PBS. Then, 200 μL of MTT solution (final concentration 0.5 mg / mL, prepared in culture medium) was added to each well, and the plates were incubated at 37°C for another 4 hours. After discarding the MTT solution, 100 μL of DMSO was added to each well to fully dissolve the formazan crystals. Finally, the optical density (OD value) of each well was measured using a Spark™ multi-well microplate reader.

[0035] (2) qRT-PCR analysis HeLa and H8 cells were mixed at a ratio of 1×10 6 Seeded at a density of [number] cells / well in 6-well plates. Total intracellular RNA was extracted using TRIzol reagent. cDNA samples were synthesized via reverse transcription using First-Strand cDNA Synthesis Super Mix for qPCR (TransGen Biotech Ltd.). Quantitative PCR analysis was then performed using NovoStart™ SYBR Green PCR SuperMix Plus (Novoprotein Scientific Inc.). The specific PCR primer sequences used in this experiment are detailed in Table 2.

[0036] Table 2

[0037] (3) Characterization and stability analysis The successful construction of TDN-Apt, TDN-Apt-P, and TDN-siRNAs-Apt-P was verified by 8% non-denaturing polyacrylamide gel electrophoresis (PAGE) at 80 V for 120 min. The morphology and particle size of the nanostructures were observed using transmission electron microscopy (TEM). Hydration particle size and Zeta potential at each assembly stage were measured using dynamic light scattering (DLS). For stability assessment, the siRNA mixture and TDN-siRNAs-Apt-P were placed in 1×PBS and incubated at 37 °C for different durations. Structural integrity was then assessed by 10% non-denaturing PAGE. Furthermore, the intracellular sensing function of this platform was verified using probes labeled with Cy3 and BHQ2.

[0038] (4) Cellular uptake and intracellular localization In the cell uptake experiment, H8 and HeLa cells were mixed at a ratio of 5 × 10⁻⁶. 4Cells were seeded at a density of 1 cells / well in 12-well plates. After 24 hours of culture, cells were treated for 4 hours with 125 nM Cy3-labeled TDN-P (composed of S1, S2, S3, T4, and Probe, without the A41411 functional sequence) or TDN-Apt-P (composed of S1, S2, S3, S4, and Probe, containing the A41411 functional sequence). After washing three times with PBS, cell samples were collected and resuspended for flow cytometry analysis to quantify cell internalization. In intracellular localization experiments, HeLa cells were seeded at a density of 5 × 10⁶ cells / well. 5 Cells were seeded at a density of 1 cell / well in confocal culture dishes and co-incubated with Cy3-labeled nanostructures for 4 hours. Subsequently, the nuclei and cytoskeleton were stained with DAPI. Fluorescence signals were acquired using laser confocal scanning microscopy (CLSM) with excitation wavelengths set to DAPI 405 nm and Cy3 561 nm. Quantitative analysis of the CLSM images was performed using ImageJ software.

[0039] (5) Validation of miR-21 detection based on non-denaturing PAGE This study evaluated the feasibility of detecting miR-21 using a single-stranded DNA sequence (A) representing the S4 tail region (containing the linker sequence and the AS1411 sequence). A probe prepared as a hybridization complex (Probe / A or F / A, final concentration 1 μM) was reacted with 1 μM miR-21 at 37°C for 20 minutes. The resulting reaction products were separated by 20% non-denaturing polyacrylamide gel electrophoresis (Native PAGE). After Cy3 fluorescence imaging, the gel was stained with SafeBlue for 20 minutes to observe the total DNA bands.

[0040] (6) Construction of HeLa cell models with gradient miR-21 concentrations In homogeneous solutions, the fluorescence intensity of the probe showed a strong linear correlation with miR-21 concentration. To verify that this concentration-dependent response remained valid in the intracellular environment, HeLa cells were transfected with different concentrations of miR-21 mimics or inhibitors. Twenty-four hours after transfection, the probe was added to the culture medium and incubated for another four hours, after which its fluorescence intensity was measured.

[0041] (7) Cell scratch and colony formation assay To evaluate the inhibitory effect of TDN-siRNAs-Apt-P on tumor cell metastasis, HeLa cells were seeded in confocal culture dishes and cultured to 60-70% confluence. A vertical scratch was made along the center of the culture dish using a sterile pipette tip, and the initial bright-field image was recorded under a microscope. Subsequently, cells were treated with different reagents for 48 hours, and the results were observed and recorded again to quantitatively analyze the scratch closure rate. In the colony formation assay, HeLa cells were seeded at a density of 200 cells / well in 12-well plates. Cells were cultured for 7-14 days, with the culture medium changed and the drug replenished every 3-4 days. After the formation of visible colonies, the cells were washed with PBS, fixed with 4% paraformaldehyde for 15 minutes, and then stained with crystal violet for 15 minutes. Finally, images were acquired to record the clonal morphology and count the cells.

[0042] (8) Detection of intracellular reactive oxygen species (ROS) and mitochondrial membrane potential Intracellular ROS levels induced by different treatments were measured using a reactive oxygen species (ROS) detection kit (Beyotime Biotech, S0033S, China). HeLa cells were co-incubated with DCFH-DA (diluted 1:1000) for 20 minutes, followed by fluorescence imaging using a laser confocal scanning microscope (CLSM). Mitochondrial membrane potential (MMP) was assessed using a Rhodamine 123 detection kit (Beyotime Biotech, C2008S, China). After treatment with specific drugs, cells were stained with Rhodamine 123 for 15 minutes, washed three times with PBS, and finally analyzed by flow cytometry (Becton Dickinson, USA).

[0043] (9) Apoptosis detection After drug treatment, cells were collected and centrifuged at 1000 rpm. The resulting cell pellet was washed with PBS and then resuspended in 195 μL of Annexin V-FITC binding buffer. Next, 5 μL of Annexin V-FITC and 10 μL of propidium iodide (PI) were added sequentially. The mixture was incubated at room temperature in the dark for 10–20 minutes, and finally, the apoptosis rate was quantitatively analyzed using flow cytometry.

[0044] (10) Western blot analysis Protein concentrations were determined using a BCA protein assay kit (Boster, Wuhan, China). Total protein (35 µg per sample) was separated by SDS-PAGE and transferred to a PVDF membrane (Bio-Rad Laboratories Inc, USA). Primary antibodies against GLUT1, ASCT2, SREBP1, AKT, NF-κB, GSK3β, and their phosphorylated variants (p-AKT, p-GSK3β, p-NF-κB) were incubated at the manufacturer's recommended dilution ratios. β-actin was used as an internal control. Finally, the immunoblot bands were developed and visualized using an ECL advanced chemiluminescence assay kit (Invitrogen, USA).

[0045] (11) Analysis of cell metabolite content Forty-eight hours after treatment, intracellular and extracellular glucose, lactate, triglycerides, and α-ketoglutarate were quantitatively analyzed using commercially available assay kits. The glucose (o-toluidine method), lactate, and triglyceride assay kits were purchased from Beyotime Biotech and Nanjing Jiancheng Bioengineering Institute; the α-ketoglutarate assay kit was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0046] (12) Tumor xenotransplantation and in vivo imaging Five-week-old female BALB / c nude mice were selected, and each mouse was injected with 1×10 7 A subcutaneous HeLa cell xenograft tumor model was constructed using [number] cells. Tumor volume was monitored using calipers and calculated according to the formula V=1 / 2ab. 2 Calculations were performed (where a and b represent the length and width of the tumor, respectively). In in vivo imaging experiments, 100 μL of TDN-siRNAs-Apt-P was injected via the tail vein, and fluorescence signals were recorded at different time points (0, 1, 2, 4, 6, 12, and 24 hours) using the Davinch Invivo Imaging HR system.

[0047] (13) Histological examination and statistical analysis Tumor tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 5 μm thick sections for hematoxylin-eosin (H&E) staining. Statistical analysis was performed using Origin 2021 software, and results are expressed as mean ± standard deviation.

[0048] Example 1 This embodiment describes the synthesis, characterization, and stability analysis of a multi-target metabolic intervention and therapeutic system based on tetrahedral DNA nanostructures (TDN-siRNAs-Apt-P nanotherapeutic system), specifically including the following steps: (1) Synthesis of TDN-siRNAs-Apt-P nanotherapeutic system: such as Figure 1 As shown in Figure A, four equimolar amounts of single-stranded DNA (S1, S2, S3, S4) were dissolved in PBS buffer containing 0.05 mM MgCl2·6H2O (pH 7.4).

[0049] The mixture was heated at 95°C for 10 minutes and then cooled to 25°C at a constant rate of 0.1°C / s to induce the self-assembly of the tetrahedral DNA backbone (TDN-Apt).

[0050] Subsequently, the targeting siRNAs (siASCT2, siGLUT1, and siSREBP1) and the pre-annealed fluorescent probes were mixed with TDN-Apt at a 1:1 molar ratio.

[0051] Finally, the mixture was incubated at 37°C for 20 minutes to obtain the fully functionalized TDN-siRNAs-Apt-P nanostructure.

[0052] (2) Structural and particle size characterization of the TDN-siRNAs-Apt-P nanotherapeutic system: like Figure 1 In sample B, 8% non-denaturing polyacrylamide gel electrophoresis (PAGE) showed that the molecular weight increased stepwise with the assembly of the TDN-siRNAs-Apt-P nanotherapeutic system, confirming successful assembly.

[0053] like Figure 1 C Figure 1 Dynamic light scattering (DLS) and nanopotential meter measurements in D showed that with the loading of probes and siRNAs, the particle size of the nanotetrahedrons increased to 28.7 ± 5.2 nm, and the zeta potential decreased to -26.1 ± 2.0 mV.

[0054] The nanostructures observed by transmission electron microscopy (TEM) were uniformly dispersed and had a consistent particle size, which was consistent with the results of DLS measurements.

[0055] (3) Stability characterization of the TDN-siRNAs-Apt-P nanotherapeutic system: The siRNAs mix and TDN-siRNAs-Apt-P were placed in 1×PBS and incubated at 37°C for different durations.

[0056] like Figure 1C. The structural integrity was assessed using 10% non-denaturing PAGE. The sensing function of the platform was verified using probes labeled with Cy3 and BHQ2, and statistical analysis was performed using grayscale analysis. The results are as follows: Figure 1 As shown in Figure D, after one week, TDN-siRNAs-Apt-P still retained about 80% of its structural integrity and functionality, indicating that it has good physiological stability.

[0057] Example 2 This embodiment is a live-cell targeting and specificity diagnostic experiment on the TDN-siRNAs-Apt-P nanotherapeutic system prepared in Example 1, specifically including the following steps: (1) Targeting of the TDN-siRNAs-Apt-P nanotherapeutic system: like Figure 2 As shown in Figure A, the designed TDN-siRNAs-Apt-P nanotherapeutic system can specifically release fluorescence under the action of miR-21.

[0058] The effects of flow cytometry on cervical cancer cells (HeLa) and normal cervical cells (H8) after co-incubation with the TDN-siRNAs-Apt-P nanotherapy system for 4 hours were compared. Figure 2 B shows that the system loaded with the probe specifically exhibited high-intensity fluorescence release in HeLa cells, while no obvious fluorescence signal was observed in H8 cells.

[0059] (2) Specificity of the TDN-siRNAs-Apt-P nanotherapeutic system: Its specificity was assessed using 20% ​​non-denaturing PAGE, and the results are as follows: Figure 2 As shown in Figure C, Cy3 fluorescence results show that the F chain in the Probe structure exhibits miR-21 responsive fluorescence recovery due to the fluorescence resonance transfer effect.

[0060] Furthermore, SafeBlue chemiluminescence results demonstrated that only miR-21 could achieve the substitution of the Q chain in the Probe structure, while miR-122 (also a biomarker highly expressed in cervical cancer) could not.

[0061] (3) Concentration dependence of TDN-siRNAs-Apt-P nanotherapeutic system: When the intracellular miR-21 concentration is altered (by transfection with inhibitors or mimics), such as Figure 2 As shown in Figure D, the intracellular Cy3 fluorescence intensity exhibits a highly concentration-dependent change, demonstrating its excellent intracellular target diagnostic capability.

[0062] Example 3 This embodiment is an in vitro cell function inhibition experiment of the TDN-siRNAs-Apt-P nanotherapeutic system prepared in Example 1. Cell experiments were conducted using different drug components at a concentration of 125 nM, and specifically included the following steps: (1) Effects of TDN-siRNAs-Apt-P nanotherapeutic system on cell migration: like Figure 3 As shown in Figure A, the scratch assay showed that after 48 hours of treatment, the migration rate of HeLa cells in the TDN-siRNAs-Apt-P nanotherapy system treatment group decreased to 30.39% (compared to 66.66% in the control group).

[0063] (2) Effects of the TDN-siRNAs-Apt-P nanotherapeutic system on cell proliferation: like Figure 3 As shown in Figure B, the colony formation experiment showed that after 14 days of treatment, the colony formation ability of HeLa cells treated with the TDN-siRNAs-Apt-P nanotherapeutic system was significantly inhibited.

[0064] (3) Effects of TDN-siRNAs-Apt-P nanotherapeutic system on cellular ROS: like Figure 3 As shown in Figure C, the reactive oxygen species (ROS) detection experiment showed that after 48 hours of treatment, the ROS level of HeLa cells treated with the TDN-siRNAs-Apt-P nanotherapy system was significantly increased.

[0065] (4) Effects of the TDN-siRNAs-Apt-P nanotherapeutic system on mitochondrial membrane potential: like Figure 3 As shown in Figure D, the mitochondrial membrane potential detection experiment showed that after 48 hours of treatment, the mitochondrial membrane potential of HeLa cells treated with the TDN-siRNAs-Apt-P nanotherapeutic system decreased by 60%.

[0066] (5) Effects of the TDN-siRNAs-Apt-P nanotherapeutic system on apoptosis: like Figure 3 As shown in E, the cell live / dead staining experiment showed that after 48 hours of treatment, the apoptosis rate of HeLa cells in the TDN-siRNAs-Apt-P nanotherapy system treatment group increased to 53.75% (compared to 16.77% in the control group), and the late apoptosis rate increased to 51.5% (compared to 11.9% in the control group).

[0067] Example 4 This embodiment is an in vitro tumor cell metabolic reprogramming experiment of the TDN-siRNAs-Apt-P nanotherapeutic system prepared in Example 1. Cells were treated with different drug components at a concentration of 125 nM, and after 48 hours, cells and culture medium were collected for the experiment. The specific steps include: (1) Effects of TDN-siRNAs-Apt-P nanotherapeutic system on cellular gene and protein expression: like Figure 4 As shown in A and 4B, qPCR and Western Blot analysis confirmed that the expression levels of ASCT2, GLUT1, and SREBP1 were downregulated by more than 50%.

[0068] (2) Effects of the TDN-siRNAs-Apt-P nanotherapeutic system on cell metabolism: like Figure 4 As shown in C, compared with the control group, the intracellular glucose content in the TDN-siRNAs-Apt-P nanodiagnostic system treatment group was significantly reduced, and the amount of glucose remaining in the culture medium was significantly increased. like Figure 4 As shown in Figure D, the metabolite detection showed a sharp decrease in lactic acid (LD) in the culture medium; like Figure 4 As shown in E and 4F, intracellular triglycerides (TG) and ketoglutarate (α-KG) were significantly reduced.

[0069] (2) Effects of the TDN-siRNAs-Apt-P nanotherapeutic system on cellular pathways and functions: The effects of this nanosystem were assessed at the whole-genome level through transcriptome sequencing analysis, such as... Figure 4 As shown in G, the KEGG enrichment analysis of differentially expressed genes (DEGs) revealed that multiple sub-metabolic pathways of sugars, lipids, and amino acids, such as “Nitrogen metabolism,” “Lipid and atherosclerosis,” and “Glycine, serine, and threonine metabolism,” all showed extremely high rich factors, confirming that multiple gene silencing triggered a wide range of metabolic chain reactions.

[0070] At the same time, such as Figure 4 As shown in H, GO functional enrichment analysis revealed the destructive effect of this energy crisis on cell structure. A large number of DEGs were concentrated in items such as "organelle organization" and "intracellular anatomical structure", indicating that energy deprivation led to organelle dysfunction and loss of structural integrity.

[0071] In summary, transcriptomic evidence elucidates the mechanism of action of this nanosystem at the whole-genome level: through precise metabolic checkpoint blockade, it induces irreversible adaptive metabolic reprogramming in tumor cells, ultimately leading to inactivation of cell signaling and structural disintegration.

[0072] Example 5 To evaluate the in vivo therapeutic efficacy of the TDN-siRNAs-Apt-P nanotherapeutic system, a subcutaneous xenograft orthotopic model of HeLa cells was established, following the procedures outlined below. Figure 5 The procedure shown in A is used to diagnose and treat the model when the tumor volume reaches 100 mm. 3 At that time, 100 μL of the drug was injected via the tail vein on days 0, 4, 8, and 12.

[0073] (1) In vivo diagnostic efficacy of the TDN-siRNAs-Apt-P nanodiagnostic system: This diagnostic and treatment system is administered via tail vein injection for in vivo monitoring and treatment. For example... Figure 5 As shown in Figure B, in vivo bioluminescence imaging revealed that the tumor region released strong, specific fluorescence approximately 4 hours after injection, with a half-life exceeding 12 hours.

[0074] (2) In vivo therapeutic effects of the TDN-siRNAs-Apt-P nanotherapy system: like Figure 5 As shown in C, 5D, and 5E, after two weeks of continuous drug administration, the tumor volume in the control group exceeded 800 mm. 3 In contrast, tumor growth was almost completely suppressed in the TDN-siRNAs-Apt-P treatment group (volume <100 mm). 3 ).

[0075] like Figure 5 As shown in F, histological H&E staining revealed that, compared with the control group, the tumors of mice treated with TDN-Apt alone showed no obvious histological damage, while siRNAs and DNA tetrahedrons carrying siRNAs both showed obvious nuclear shrinkage and reduced cell area.

[0076] at the same time, Figure 5 G and 5H immunofluorescence results showed that, compared with the control, siRNAs and DNA tetrahedrons carrying siRNAs significantly reduced the expression of Ki-67 in tumor tissues and induced apoptosis.

[0077] Figure 6A comparative study using confocal laser scanning microscopy images directly compared the uptake of two samples (TDN-P and TDN-Apt-P) in normal cervical cells (H8) and cervical cancer cells. The results showed that in H8 cells, TDN-P showed no obvious signal, while TDN-Apt-P displayed a red signal. In HeLa cells, both TDN-P and TDN-Apt-P showed detectable red signals, indicating successful uptake of the molecules. Figure 6 B. Quantitative analysis of fluorescence intensity in different treatment groups showed that TDN-Apt-P exhibited the highest absolute fluorescence intensity and the highest relative fluorescence intensity in the HeLa cell line, significantly higher than other groups. In contrast, the fluorescence intensity of both samples was low in the H8 cell line, especially TDN-P, which was not effectively detected in H8 cells. This figure demonstrates that the introduction of the AS1411 aptamer significantly enhanced the uptake efficiency of nanomedicines in tumor cells, fully illustrating the excellent tumor-targeting specificity of the TDN-siRNAs-Apt-P nanotherapeutic system.

[0078] The multifunctional diagnostic and therapeutic system (TDN-siRNAs-Apt-P) provided by this invention consists of a tetrahedral DNA nanostructure (TDN), a DNA aptamer (AS1411), three small interfering RNAs (siASCT2, siGLUT1, and siSREBP1) targeting metabolic genes, and a strand displacement probe responsive to miR-21. The TDN vector backbone is formed by the self-assembly of four single-stranded DNA strands (S1, S2, S3, and S4) based on the base complementarity pairing principle, resulting in a three-dimensional tetrahedral structure (TDN) with excellent cell membrane permeability and structural rigidity. A DNA aptamer, AS1411, is extended from one edge of the TDN to specifically target tumor cells and increase the vector's entry efficiency. The vertices of the TDN are designed to connect hybrid DNA / RNA strands, loaded with siASCT2, siGLUT1, and siSREBP1, respectively. After entering the cell, these hybrid strands can be specifically recognized and degraded by RNase H nuclease, thereby intelligently releasing siRNA and silencing the corresponding metabolic genes. The linker portion of TDN contains a chain displacement probe. When this system enters cervical cancer cells and encounters the highly expressed marker miR-21, it triggers a chain displacement reaction, releasing a fluorescent signal, enabling real-time monitoring and diagnosis of the target.

[0079] This invention precisely and simultaneously integrates an AS1411 targeting module, three synergistic siRNA therapeutic modules, and a miR-21-responsive diagnostic module on a single rigid DNA tetrahedral backbone, achieving a high degree of structural and functional synergy. Unlike traditional single-target inhibition, this system simultaneously silences GLUT1 (glucose metabolism), ASCT2 (amino acid metabolism), and SREBP1 (lipid metabolism), triggering a wide-ranging metabolic chain reaction that leads to complete energy depletion in tumor cells. Furthermore, this system does not rely on external stimulation; instead, it utilizes a miR-21 trigger chain displacement probe, which is endogenously highly expressed in tumor cells, for specific diagnosis and leverages endogenous nucleases to release therapeutic drugs, achieving a seamless integration of dynamic biosensing and targeted therapy.

[0080] In summary, this invention provides a programmable tetrahedral DNA nanostructure (TDN) platform (TDN-siRNAs-Apt-P) for precise integrated treatment and diagnosis of cervical cancer.

[0081] By leveraging the geometric rigidity of the DNA tetrahedron to protect the load from nuclease degradation and enhance intracellular delivery, this nanodevice simultaneously coordinates the silencing of three key metabolic hubs: GLUT1, ASCT2, and SREBP1. This structure is further functionalized via the AS1411 aptamer for active tumor targeting and enables real-time intracellular molecular imaging through a miR-21-triggered strand displacement probe.

[0082] In in vitro experiments, simultaneous disruption of glucose, glutamine, and lipid supply lines leads to a severe energy crisis, triggers profound metabolic network remodeling, inhibits the nutrient-sensitive PI3K-AKT survival signaling pathway, and induces intense apoptosis.

[0083] In vivo evaluation in xenograft models showed excellent tumor aggregation, sustained diagnostic imaging, and a significant reduction in tumor weight of approximately 80%, with no significant systemic toxicity detected.

[0084] By structurally combining multidimensional metabolic blockade with dynamic biomarker monitoring, this TDN platform successfully overcomes the metabolic adaptation of tumors, providing an efficient and effective blueprint for next-generation therapeutic and diagnostic nanomedicines.

[0085] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A multi-target metabolic intervention and diagnostic system based on tetrahedral DNA nanostructures, characterized in that: include, A tetrahedral DNA nanostructure backbone, a targeting aptamer attached to the backbone, a small interfering RNA loaded on the backbone, and a strand displacement probe hybridized to the backbone; The targeting aptamer is the AS1411 aptamer, which is used to specifically target tumor cells; The small interfering RNAs include those that target and silence the ASCT2, GLUT1, and SREBP1 genes. The chain displacement probe can be triggered by miR-21, which is highly expressed in tumor cells, and generate a detectable signal.

2. The integrated diagnostic and therapeutic system for multi-target metabolic intervention as described in claim 1, characterized in that: The small interfering RNAs include siASCT2, which targets and silences the ASCT2 gene; siGLUT1, which targets and silences the GLUT1 gene; and siSREBP1, which targets and silences the SREBP1 gene.

3. The integrated diagnostic and therapeutic system for multi-target metabolic intervention as described in claim 1, characterized in that: The small interfering RNA is connected to the apex of the tetrahedral DNA nanostructure backbone via a DNA / RNA hybrid strand.

4. The integrated diagnostic and therapeutic system for multi-target metabolic intervention as described in claim 1, characterized in that: The chain displacement probe contains a fluorescent reporter group and a quencher group. When it does not bind to miR-21, its fluorescence is quenched. When it binds to miR-21, a chain displacement reaction occurs, causing the fluorescent reporter group to separate from the quencher group and generate a fluorescent signal.

5. The integrated diagnostic and therapeutic system for multi-target metabolic intervention as described in claim 1, characterized in that: The tetrahedral DNA nanostructure backbone is formed by the self-assembly of four single-stranded DNA molecules through complementary base pairing.

6. The preparation method of the multi-target metabolic intervention integrated diagnostic and therapeutic system according to any one of claims 1 to 5, characterized in that, Includes the following steps: Four equimolar amounts of single-stranded DNA were dissolved in PBS buffer containing 0.05 mM MgCl2·6H2O at pH 7.

4. The mixture was heated and then cooled to allow the single-stranded DNA to self-assemble into a tetrahedral DNA nanostructure TDN-Apt carrying the AS1411 aptamer. Small interfering RNA was mixed with pre-annealed strand displacement probes and TDN-Apt, and then the mixture was incubated at 37°C for 20 minutes to obtain a nanotherapeutic system loaded with small interfering RNA and probes.

7. The preparation method according to claim 6, characterized in that: The heating and cooling process involves heating at 95°C for 10 minutes, followed by cooling to 25°C at a constant rate of 0.1°C / s.

8. The preparation method according to claim 6, characterized in that: The molar ratio of the small interfering RNA to the pre-annealed strand displacement probe to the tetrahedral DNA nanostructure is 1:

1.

9. The application of the multi-target metabolic intervention diagnostic and therapeutic integrated system as described in any one of claims 1 to 5 in the preparation of a drug for treating cervical cancer.

10. The application of the multi-target metabolic intervention diagnostic and therapeutic system as described in any one of claims 1 to 5 in the preparation of diagnostic reagents for real-time monitoring of miR-21 levels in tumor cells.

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