MicroRNA response type nano diagnosis and treatment platform as well as preparation method and application thereof

Abstract

The invention discloses a microRNA (Ribonucleic Acid) response type nano diagnosis and treatment platform as well as a preparation method and application thereof. The preparation method comprises the following steps: 1, dissolving DNAzyme (Dz) containing a self-blocking structure in a Tris-HCl buffer solution, heating and annealing, and slowly cooling to form a stable secondary structure; 2, Fe < 2 + > ions and a DNAzyme solution are mixed under the acidic condition, and a metal-nucleic acid framework (DzM) is formed through self-assembly; the outer layer of DzM is coated with a metal-phenol network formed by Fe < 3 + >, Mn < 2 + > and tannic acid (TA), and the DNAzyme-metal-tannic acid nano-composite (DzMT) with a stable structure is obtained. The nano platform integrates microRNA recognition, fluorescence imaging and self-enhancement treatment functions, has high selectivity, high sensitivity and good biological safety, and provides a new strategy for precise diagnosis and treatment of tumors.

Description

Technical Field

[0001] This invention belongs to the field of nanobiomedicine and tumor molecular diagnostics and therapy technology, specifically relating to a microRNA-responsive nanotherapeutic platform, its preparation method, and its applications. This nanoplatform achieves precise intracellular imaging and synergistic chemodynamic therapy (CDT) through a multi-layered regulatory mechanism triggered by microRNA. Background Technology

[0002] The occurrence and development of tumors is a complex, multifactorial process. Current clinical treatments, including surgery, radiotherapy, chemotherapy, and immunotherapy, while improving patient survival to some extent, still have significant side effects, drug resistance, and recurrence risks. In recent years, chemodynamic therapy (CDT), as an emerging non-invasive tumor treatment strategy, has gradually become an important direction for precision oncology due to its unique mechanism of generating hydroxyl radicals (•OH) through Fenton or Fenton-like reactions based on endogenous tumor-derived hydrogen peroxide (H2O2) to induce apoptosis in cancer cells. Traditional Fenton reactions typically rely on Fe... 2+ As a catalyst, it decomposes H2O2 into •OH, which has strong oxidizing activity, under acidic conditions. However, CDT faces several limitations in practical applications: (1) the concentration of H2O2 in tumor cells is limited, making it difficult to continuously drive an efficient Fenton reaction; (2) Fe 3+ Reduced to Fe 2+ The rate of release is extremely low, resulting in limited reaction cycle efficiency; (3) There is a lack of effective tumor targeting and real-time imaging methods, making it difficult to monitor the treatment process; (4) Non-specific reactions may cause oxidative damage in normal tissues, resulting in side effects. In order to improve the efficiency of the Fenton reaction and tumor selectivity, researchers have proposed a variety of strategies, such as introducing exogenous H2O2 donors (such as CuO2, CaO2 nanoparticles) or H2O2 generating enzymes (such as glucose oxidase, lactate oxidase), but these methods often have problems such as difficulty in controlling the release rate, poor stability and high systemic toxicity. In addition, the traditional CDT system lacks a "smart response" mechanism and cannot autonomously regulate itself according to specific biomarkers in the tumor, which limits its precision treatment capabilities.

[0003] MicroRNAs (miRNAs) are a class of non-coding small RNA molecules, approximately 20–24 nucleotides in length, that regulate post-transcriptional gene expression by binding to target mRNAs, playing a crucial role in cell proliferation, differentiation, and apoptosis. Numerous studies have shown that miRNAs are significantly aberrantly expressed in various tumors. miR-21, a typical oncogenic miRNA, is highly expressed in breast, lung, liver, and cervical cancers, but at extremely low levels in normal cells. Therefore, miR-21 can serve as a tumor-specific molecular marker for precise tumor identification and response regulation. DNAzymes (deoxyribonucleases) are catalytically active single-stranded DNA molecules capable of specifically recognizing and cleaving RNA substrates. Compared to proteases, DNAzymes offer advantages such as structural stability, ease of synthesis, and high programmability, and have been widely used in biosensoring and gene regulation. However, the activity of DNAzymes depends on specific metal ions (such as Mg²⁺). 2+ Mn 2+ As a cofactor, its catalytic efficiency within cells is limited; simultaneously, DNAzymes are susceptible to degradation by cellular nucleases and lack targeted delivery capabilities, restricting their in vivo applications. To address these issues, a design strategy combining metal-nucleic acid frameworks (MNAF) and metal-phenolic networks (MPN) has emerged in recent years. By utilizing the coordination interactions between metal ions and the phosphate backbone and phenolic hydroxyl groups of nucleic acids, structurally stable and controllably released nanoplatforms can be constructed for drug delivery and tumor therapy. Tannic acid (TA) is a natural polyphenol molecule with excellent metal chelating ability and reducing properties, capable of binding Fe... 3+ Reduced to Fe 2+ Accelerate Fe 2+ / Fe 3+ This cycle significantly increases the Fenton reaction rate.

[0004] Based on this, the present invention combines microRNA-responsive DNAzymes with multivalent metal ions (Fe). 2+ Fe 3+ Mn 2+A DNAzyme-metal-tannic acid nanocomposite (DzMT) with multiple gating properties was constructed by synergistic assembly of DNAzyme and tannic acid (TA), enabling a multi-level, programmable tumor-selective therapeutic strategy. This system dissociates in the acidic tumor microenvironment, releasing the DNAzyme and metal components. Overexpressed miR-21 in the tumor specifically binds to the DNAzyme, triggering its catalytic core activation and generating a fluorescent signal for miRNA visualization. Simultaneously, the activated DNAzyme recognizes and cleaves catalase (CAT) mRNA, inhibiting CAT expression and preventing H2O2 decomposition, thereby increasing intracellular hydrogen peroxide concentration and providing sufficient substrate for the subsequent Fenton reaction. The released Fe... 2+ Further catalyzing the decomposition of H2O2 to generate hydroxyl radicals (•OH), inducing cellular oxidative stress and apoptosis; while tannic acid can reduce Fe 3+ Reduced to Fe 2+ Fe 2+ / Fe 3+ A self-circulating system is used to achieve a continuously enhanced Fenton response. Compared with traditional chemokinetic therapy (CDT) systems, this method has the following significant advantages: (1) It achieves precise response and highly selective treatment within tumor cells by triggering DNAzyme activation through miRNA, significantly reducing off-target effects; (2) It enhances the driving force of the Fenton response by increasing intracellular H2O2 levels through the CAT gene silencing strategy; (3) It enhances the driving force of the Fenton response by increasing the intracellular H2O2 level through Fe 2+ / Fe 3+ The cycling and TA reduction process constructs a self-enhancing reaction system to achieve long-term •OH generation; (4) the activated DNAzyme generates a fluorescent signal to realize dynamic visualization imaging of miRNA, which facilitates real-time monitoring of the treatment process. In summary, the invented microRNA-responsive nanotherapeutic platform provides an integrated, controllable, and highly selective new strategy for tumor molecular diagnosis and treatment, which is expected to break through the bottlenecks of low efficiency and poor specificity of traditional CDT, and provide a new technical path for precise tumor imaging and targeted chemokinetics. Summary of the Invention

[0005] The purpose of this invention is to provide a microRNA-responsive nanotherapeutic platform, its preparation method, and its applications. This platform utilizes a microRNA-specific activation mechanism, a synergistic catalytic effect of metal ions, and a stable encapsulation by a phenol-hydroxyl coordination network to achieve multiple gating controls within tumor cells. It can simultaneously perform microRNA-specific recognition, fluorescence imaging, and Fenton reaction-driven chemokinetic therapy (CDT).

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for preparing a microRNA-responsive nanotherapeutic platform.

[0008] A method for preparing a microRNA-responsive nanotherapeutic platform includes the following steps:

[0009] Step 1: Dissolve the DNAzyme (Dz) containing the self-blocking structure in Tris-HCl buffer, heat and anneal the resulting solution, and then slowly cool it to form a DNAzyme solution with a stable secondary structure;

[0010] Step 2: Add Fe 2+ Ions are mixed with the DNAzyme solution obtained in step 1 under acidic conditions to self-assemble into metal-nucleic acid framework (DzM) nanoparticles; the outer layer of the metal-nucleic acid framework (DzM) is coated with Fe 3+ Mn 2+ A metal-phenol network was formed with tannic acid (TA) to obtain a DNAzyme-metal-tannic acid nanocomposite (DzMT) with a stable structure.

[0011] Preferably, in step 1, the self-blocking DNAzyme contains the following three functional regions:

[0012] (1) RNA cutting core region (8-17 DNAzyme) is used to achieve sequence-specific cleavage reaction;

[0013] (2) microRNA recognition region, used to bind to miR-21, which is highly expressed in tumors, to release the self-blocking structure of the catalytic core;

[0014] (3) Target mRNA recognition region, used to specifically recognize and cleave catalase (CAT) mRNA.

[0015] Preferably, in step 2, DNAzyme and FeCl2·4H2O are mixed and self-assembled under acidic conditions to form a metal-nucleic acid framework (DzM); then the pH is adjusted to 7.0 and the mixture is shaken and mixed, and FeCl3·6H2O, MnCl2·4H2O and tannic acid are added in sequence to form a metal-phenol network coating layer, thereby obtaining a stable DNAzyme-metal-tannic acid nanocomposite (DzMT).

[0016] More preferably, in step 2, 200 μM DNAzyme and 20 mM FeCl2·4H2O are mixed under acidic conditions to self-assemble into a metal-nucleic acid framework (DzM); then the pH is adjusted to 7.0 and the mixture is shaken to mix, and FeCl3·6H2O (10 mg / mL), MnCl2·4H2O (10 mg / mL) and tannic acid (40 mg / mL) are added sequentially to form a metal-phenol network coating layer, thereby obtaining a stable DNAzyme-metal-tannic acid nanocomposite (DzMT).

[0017] Preferably, the DNAzyme sequence is: 5′- GCT AAA GGT CAA CAT CAG TCT GAT AAGCTA CAG GGA GGC CCC TGT CCG AGC CGG TCG AAC CTT TAG CA-3′.

[0018] Preferably, the sequence of miR-21 is: 5′- UAG CUU AUC AGA CUG AUG UUG A-3′.

[0019] Preferably, the sequence of the catalase (CAT) mRNA is: 5′-UGC UAA AGG AGC AGG GGC CU-3′.

[0020] Secondly, the present invention provides a microRNA-responsive nanodiagnostic and therapeutic platform prepared by the method described above.

[0021] Preferably, the DNAzyme-metal-tannic acid nanocomposite (DzMT) has an average particle size of 140 nm, exhibits good dispersibility and structural stability at pH 7.4, and can rapidly decompose and release active components at pH 5.5.

[0022] Thirdly, the present invention provides an application of the aforementioned microRNA-responsive nanotherapeutic platform in the preparation of pharmaceutical compositions.

[0023] The pharmaceutical composition is used for fluorescence imaging of microRNA overexpression in tumors, catalase gene silencing, and Fenton reaction-driven chemokinetic therapy, achieving integrated imaging and treatment.

[0024] Those skilled in the art will understand that the "pharmaceutical composition" described in this invention encompasses all suitable forms for delivering the microRNA-responsive nanotherapeutic platform as an active ingredient. Depending on the application, the pharmaceutical composition can be configured in various types, including but not limited to:

[0025] (1) Therapeutic preparations: preparations for systemic or local administration, such as injectable solutions, lyophilized powder injections, or gels and sprays for local administration, which are intended to directly treat tumors through chemokinetics.

[0026] (2) Diagnostic agents: Agents used for in vitro or in vivo diagnostics. For example, they can be used to prepare in vitro diagnostic kits for fluorescence imaging and detection of microRNA in biological samples (such as tissue sections and cell cultures) from subjects; they can also be used as in vivo contrast agents, provided that clinical permission permits.

[0027] (3) Therapeutic preparations: These are compositions that simultaneously contain diagnostic and therapeutic functions, enabling tumor localization, imaging, and treatment within the same preparation, which aligns with the advanced medical concept of therapeutic integration.

[0028] These pharmaceutical compositions typically contain a pharmaceutically acceptable carrier, the choice of which is appropriate for the type of formulation (e.g., injection, lyophilized, topical, etc.) and the intended use (e.g., treatment or diagnosis). Those skilled in the art can determine suitable carriers and formulation methods based on conventional technical knowledge without inventive effort.

[0029] Preferably, the application is based on the following mechanism: the nanotherapeutic platform selectively accumulates in tumor tissue by enhancing penetration and retention effects, and dissociates in the acidic tumor microenvironment; the released DNAzyme is activated by miR-21 overexpressed in tumor cells, activating its catalytic activity.

[0030] More preferably, the DNAzyme-metal-tannic acid nanocomposite (DzMT) is delivered to the test subject via cellular uptake, selectively enriched in tumor tissue using the enhanced permeation and retention effect (EPR), and dissociated in the acidic tumor microenvironment, releasing DNAzyme and responding to microRNA activation, thereby achieving tumor-specific imaging and synergistic therapy.

[0031] The DNAzyme-metal-tannic acid nanocomposite (DzMT) exhibits significant fluorescence imaging and therapeutic effects in tumor cells, effectively inhibiting tumor growth and showing no obvious toxicity to normal tissues.

[0032] Preferably, the Fenton reaction is achieved through the following pathway: Fe 2+ + H2O2 → Fe 3+ + •OH + OH − Tannic acid (TA) can convert the generated Fe 3+ Reduced to Fe 2+ Fe 2+ / Fe 3+The self-circulating system continuously generates hydroxyl radicals (•OH), thereby inducing oxidative stress damage and apoptosis in tumor cells.

[0033] Preferably, the DNAzyme is activated after binding with miR-21. On the one hand, it activates its catalytic activity, leading to the recovery of fluorescence signals and enabling spatial visualization imaging of microRNAs in tumor cells. On the other hand, the activated DNAzyme can specifically cleave CAT mRNA, inhibit the expression of catalase in cells, and promote the enrichment of H2O2 in cells, thereby significantly enhancing the efficiency of the Fenton reaction.

[0034] Beneficial effects: Compared with the prior art, the present invention has the following advantages and innovations: (1) It achieves specific activation of DNAzyme through microRNA response, significantly improving the selective recognition ability in tumor cells; (2) DNAzyme cleaves CAT mRNA to inhibit H2O2 decomposition, enhancing Fenton reaction kinetics at the molecular level; (3) Tannic acid synergistically enhances Fe 2+ / Fe 3+ (3) By cycling and constructing a self-enhancing Fenton reaction system, continuous •OH generation is achieved; (4) The microRNA recognition, fluorescence imaging, gene silencing and CDT therapy functions are integrated to achieve multi-level synergistic diagnosis and treatment; (5) The DzMT structure has high stability and controllable release, making it suitable for complex tumor microenvironments. The microRNA-responsive nanodiagnosis and treatment platform proposed in this invention provides a novel, efficient, safe and programmable strategy for precise molecular imaging and synergistic tumor treatment, and has broad clinical application prospects. Attached Figure Description

[0035] Figure 1 Workflow diagram of microRNA-responsive nanodiagnostic platform.

[0036] Figure 2 (A) Schematic diagram of DNAzyme structure; (B) The activation process of DNAzyme is broken down into two energy conversion steps (1–2); (C) Activation efficiency of DNAzyme.

[0037] Figure 3 Schematic diagram of the construction and characterization of the microRNA-responsive nanotherapeutic platform; where: (A) Fe 2+ A schematic diagram of the metal-nucleic acid coordination structure formed with Dz; (B) Fe 2+ (C) Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images of Dz coordinated with Dz, scale bar: 200 nm; (D) TEM elemental distribution map of DzM, scale bar: 200 nm; (F) Tannic acid (TA) and Fe in DzMT. 2+ / Fe3+ Schematic diagram of the metal-phenol coordination network; (E) TEM and SEM images of DzMT, scale bar: 200 nm; (F) TEM elemental distribution map of DzMT, scale bar: 100 nm.

[0038] Figure 4 Analysis of miR-21 response and fluorescence signal of DzMT under acidic conditions; including: (A) Schematic diagram of pH-responsive DzMT release process; (B) Fluorescence emission spectrum of DzMT in response to miR-21 at pH 5.5; (C) Fluorescence change of DzMT in response to substrate at pH 5.5; (D) Kinetic curve of Fenton reaction in DzMT under different pH conditions; (E) Mutant miR-21 sequence; (F) Fluorescence signal heatmap of Cy5 induced by mutant miR-21; (G) Fluorescence emission spectrum at different concentrations of miR-21; (H) Correlation curve between Cy5 fluorescence intensity and miR-21 concentration; (I) Linear fitting relationship between Cy5 fluorescence intensity and logarithmic value of miR-21 concentration, data are mean ± standard deviation (n=3).

[0039] Figure 5 (A) Schematic diagram of the mechanism of intracellular miR-21 imaging; (B) Fluorescence imaging of DzMT co-cultured with MCF-7 cells at different incubation times; (C) Fluorescence imaging of DzMT co-cultured with MCF-10A cells at different incubation times; (D) Quantitative analysis of the average fluorescence intensity of a single MCF-7 cell; (E) Quantitative analysis of the average fluorescence intensity of a single MCF-10A cell; (F) Fluorescence imaging of DzMT in MCF-7, A549, MDA-MB-231, HeLa and MCF-10A cells; (G) Fluorescence imaging of miR-21 in MCF-7 cells after treatment with anti-miR-21, control and miR-21 mimics; (H) Quantitative analysis of the average fluorescence intensity of a single cell in different cell lines; (I) Quantitative analysis of the average fluorescence intensity of a single cell in MCF-7 cells after treatment with anti-miR-21, control and miR-21 mimics.

[0040] Figure 6(A) Schematic diagram of the mechanism of DzMT-induced apoptosis; (B) Confocal laser scanning microscopy (CLSM) images of reactive oxygen species (ROS) generated in MCF-7 cells after 24 h of incubation with different treatment groups; (C) CLSM images of lipid peroxidation (LPO) generation in MCF-7 cells treated with DzMT and nDzMT for 24 h; (D) Mitochondrial membrane potential of MCF-7 cells measured by JC-1, scale bar: 25 μm; (E) Relative CAT mRNA levels in MCF-7 and MCF-10A cells after treatment with different concentrations of DzMT by RT-qPCR; (F) Western blot analysis of catalase protein expression; (G) Cell viability of MCF-7 and MCF-10A cells after treatment with different concentrations of DzMT; (H) Live and dead cell imaging of MCF-7 cells stained with Calcein-AM / PI; (I) Flow cytometry apoptosis analysis of MCF-7 cells after different treatment groups stained with Annexin V-FITC / PI. Detailed Implementation

[0041] The present invention will be further explained below with reference to the accompanying drawings and embodiments.

[0042] Reagents and materials:

[0043] All HPLC-purified oligonucleotides were synthesized by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). SYBR Gold was purchased from Life Technologies (Carlsbad, California, USA). Tris-HCl (pH 8.0) and magnesium chloride (MgCl2) were purchased from Sigma-Aldrich (St. Louis, USA). Ferrous chloride tetrahydrate (FeCl2·4H2O), ferric chloride hexahydrate (FeCl3·6H2O), and manganese chloride tetrahydrate (MnCl2·4H2O) were purchased from Maclean Chemical Reagent Co., Ltd. (Shanghai, China). Ribonuclease inhibitors, tannic acid (TA), and diethyl pyrocarbonate (DEPC) for water treatment were purchased from Shanghai Sangon Biotech Co., Ltd. Hoechst 33342, Calcein-AM / PI cell viability-toxicity assay kit, Annexin V-FITC apoptosis assay kit, LysoTracker Green, CCK-8, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), and JC-1 were all purchased from Beyotime Biotechnology (Shanghai, China). Lipofectamine TM 3000 transfection reagent and Opti-MEM ®Low-serum culture medium was purchased from Thermo Fisher Scientific (Massachusetts, USA). Human breast cancer cell lines (MCF-7 and MDA-MB-231), human normal breast cell line (MCF-10A), human cervical cancer cell line (HeLa), and human lung cancer cell line (A549) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). This invention has been approved by the Ethics Committee of Nanjing Drum Tower Hospital. Ultrapure water was obtained from a Millipore filtration system (Milford, Massachusetts, USA).

[0044] Detection by fluorescence spectroscopy

[0045] To investigate the effect of microRNA-21 (miR-21) on DNAzyme activation and its substrate cleavage ability, and to evaluate the self-assembly and response performance of DzMT nanocomposites under different pH conditions, fluorescence spectroscopy was used for detection. The fluorescence spectra of Cy5 were recorded at room temperature using a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan) equipped with a xenon lamp as the excitation source. The excitation wavelength was set to 635 nm, and the excitation and emission slit widths were both 7.0 nm. The recording wavelength range was 650–750 nm. Quantitative analysis of fluorescence intensity was performed at 668 nm.

[0046] Cell Culture and Imaging Experiments

[0047] MCF-7, MDA-MB-231, MCF-10A, HeLa, and A549 cells were seeded in 20 mm glass-bottomed culture dishes and cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37 °C in an incubator containing 5% carbon dioxide. Cell counts were performed using a Countstar IC 1000 automated cell counter (Innovent Biologics, Delaware, USA). Total RNA was extracted using the SteadyPure Rapid RNA Extraction Kit (Aikorite Biotechnology Co., Ltd., Hunan, China). Total RNA concentration was determined using a NanoDrop 2000 fluorescence spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA).

[0048] For cell imaging, adherent cells were washed twice with 1×PBS buffer, and then the DzMT nanocomposite was diluted in 1 mL of Opti-MEM medium (i.e., serum-depleted transfection medium). The treated cells were then incubated at 37 °C for 6 h in a 5% CO2 incubator. After incubation, cells were washed twice with 1×PBS buffer, and then culture dishes were filled with fresh DMEM medium containing 10% fetal bovine serum (FBS). Cell images were acquired using a confocal laser scanning microscope (CLSM) with a 40× objective lens. Cy5 fluorescence was excited by a 635 nm laser. Quantitative analysis of 200 × 200 pixel regions was performed using ImageJ software.

[0049] This invention provides a microRNA-responsive nanotherapeutic platform, its preparation method, and its applications. This microRNA-responsive nanotherapeutic platform is used for the specific detection of microRNA in living cells, catalase gene silencing, and Fenton reaction-driven chemokinetic therapy, achieving integrated imaging and treatment. Compared with existing technologies, this invention has the following advantages and innovations: (1) Specific activation of DNAzyme is achieved through microRNA response, significantly enhancing the selective recognition ability within tumor cells; (2) DNAzyme cleaves CAT mRNA to inhibit H2O2 decomposition, enhancing Fenton reaction kinetics at the molecular level; (3) Tannic acid synergistically enhances Fe... 2+ / Fe 3+ (3) By cycling and constructing a self-enhancing Fenton reaction system, continuous •OH generation is achieved; (4) The microRNA recognition, fluorescence imaging, gene silencing and CDT therapy functions are integrated to achieve multi-level synergistic diagnosis and treatment; (5) The DzMT structure has high stability and controllable release, making it suitable for complex tumor microenvironments. The microRNA-responsive nanodiagnosis and treatment platform proposed in this invention provides a novel, efficient, safe and programmable strategy for precise molecular imaging and synergistic tumor treatment, and has broad clinical application prospects.

[0050] Example 1:

[0051] The microRNA-responsive nanotherapeutic platform was prepared in this embodiment, and the steps are as follows:

[0052] Step 1: Dissolve the DNAzyme (Dz) containing the self-blocking structure in Tris-HCl buffer, heat and anneal the resulting solution, and then slowly cool it to form a DNAzyme solution with a stable secondary structure;

[0053] The self-blocking DNAzyme contains the following three functional regions:

[0054] (1) RNA cutting core region (8-17 DNAzyme) is used to achieve sequence-specific cleavage reaction;

[0055] (2) microRNA recognition region, used to bind to miR-21, which is highly expressed in tumors, to release the self-blocking structure of the catalytic core;

[0056] (3) Target mRNA recognition region, used to specifically recognize and cleave catalase (CAT) mRNA.

[0057] Step 2: 200 μM DNAzyme and 20 mM FeCl2·4H2O were self-assembled under acidic conditions to form a metal-nucleic acid framework (DzM); then the pH was adjusted to 7.0 and the mixture was shaken and coated with FeCl3·6H2O (10 mg / mL), MnCl2·4H2O (10 mg / mL) and tannic acid (40 mg / mL) to obtain a stable DNAzyme-metal-tannic acid nanocomposite (DzMT).

[0058] The DNAzyme sequence is: 5′- GCT AAA GGT CAA CAT CAG TCT GAT AAG CTA CAGGGA GGC CCC TGT CCG AGC CGG TCG AAC CTT TAG CA-3′.

[0059] The miR-21 sequence is: 5′- UAG CUU AUC AGA CUG AUG UUG A-3′.

[0060] The sequence of CAT mRNA is: 5′-UGC UAA AGG AGC AGG GGC CU-3′.

[0061] Step 3: The DNAzyme-metal-tannic acid nanocomposite (DzMT) is delivered to the test subject through cellular uptake. It selectively accumulates in tumor tissue using the enhanced permeation and retention effect (EPR) and dissociates in the acidic tumor microenvironment, releasing DNAzyme and responding to microRNA activation, thereby achieving tumor-specific imaging and synergistic therapy.

[0062] Example 2: Multilevel regulation and synergistic imaging therapy mechanism of microRNA-responsive nanodiagnostic and therapeutic platform.

[0063] The multi-level regulation and synergistic imaging therapeutic mechanism of the microRNA-responsive nanotherapeutic platform, such as... Figure 1As shown. This invention uses breast cancer as a model for validation studies. Breast cancer is the most common malignant tumor in women, and its highly expressed microRNA-21 (miR-21) is selected as an endogenous trigger signal for tumor-specific recognition and response regulation. The miRNA-responsive 8-17 DNAzyme (Dz) is labeled with the fluorescent group Cy5 at the 5′ end and the quencher group BHQ2 at the 3′ end, and consists of three functional domains: (i) the catalytic core region of the 8-17 DNAzyme with RNA cleavage activity ( Figure 1 (ii) Catalase (CAT) mRNA recognition region (green); Figure 1 (iii) miR-21 binding domain (purple); Figure 1 (Blue). In the absence of miR-21, the catalytic core is blocked by the miR-21 recognition region, and the DNAzyme is inactive, unable to cleave the target mRNA or produce a fluorescent signal. Dz and Fe 2+ Ion coordination forms a metal-nucleic acid framework (DzM), which exhibits high metal loading capacity but limited structural stability. To improve its stability, the outer layer of the DzM is further coated with Fe. 3+ Mn 2+ A metal-phenol network constructed with tannic acid (TA) forms a stable DNAzyme-metal-tannic acid nanocomposite (DzMT) with acidic dissociation characteristics. After cellular uptake, DzMT selectively accumulates in tumor tissue through enhanced permeability and retention (EPR) effects. In the acidic tumor microenvironment, DzMT dissociates, releasing DNAzyme, metal ions, and tannic acid. The released DNAzyme is specifically recognized and bound by miR-21 overexpressed in the tumor, releasing its blocking structure, activating the catalytic core, separating Cy5 and BHQ2, and restoring fluorescence, achieving highly sensitive imaging of miR-21. Simultaneously, the activated DNAzyme in Mn... 2+ As a cofactor, it specifically cleaves CAT mRNA, inhibits catalase expression, and prevents the breakdown of intracellular H2O2, thereby promoting H2O2 accumulation. The accumulated H2O2 then accumulates in Fe... 2+ Under ion catalysis, the Fenton reaction occurs, generating hydroxyl radicals (•OH), which trigger cell membrane lipid peroxidation, mitochondrial damage, and apoptosis.

[0064] Due to Fe 3+ To Fe 2+ The reduction rate is low (approximately 0.002-0.01 M). -1 ·s -1 ), TA, as a natural polyphenol reducing agent, can reduce Fe 3+ Efficient reduction to Fe 2+Establish Fe 2+ / Fe 3+ The cyclic system enables self-reinforcement of the Fenton reaction. This autocatalytic process continuously generates •OH, creating a positive feedback amplification effect for chemokinetic therapy (CDT), thereby significantly improving the treatment efficiency and selectivity of tumors. In contrast, in normal cells, due to the extremely low expression level of miR-21, the DNAzyme cannot be unlocked, resulting in neither the generation of fluorescent signals nor the cleavage of CAT mRNA or the triggering of the Fenton reaction, thus avoiding non-specific cell damage and ensuring high specificity and safety of the treatment.

[0065] In summary, the microRNA-responsive nanotherapeutic platform provided by this invention achieves a synergistic integration of miRNA-specific imaging and self-enhanced chemokinetic therapy through a multi-level coupling mechanism of "miRNA triggering – gene silencing – metal cycling – Fenton amplification".

[0066] Example 3: Synthesis and Characterization of DzMT

[0067] The DNAzyme-metal-tannic acid nanocomposite (DzMT) of this invention is constructed through coordination self-assembly between metal ions and DNAzymes. Firstly, Fe... 2+ Ions coordinate with phosphate groups, nitrogen atoms, and oxygen atoms in DNAzyme molecules, self-assembling to form a metal-nucleic acid framework (DzM). Figure 2 (A). Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) observations showed that DzM exhibited a uniform spherical morphology with an average particle size of 114.8 nm. Figure 2 (B). High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and energy-dispersive elemental mapping (EDS) results show that C, N, O, P, and Fe elements are uniformly distributed in DzM. Figure 2 The coordination structure between the metal and nucleic acid was confirmed by the study of C. To further improve its structural stability and physiological adaptability, Fe was coated onto the surface of DzM. 3+ Mn 2+ A metal-phenol network shell (MT) formed by co-assembling with tannic acid (TA) was used to prepare a structurally stable composite nanomaterial, DzMT, with acid dissociation properties. Figure 2 (D). TEM and SEM results showed that the particle size of DzMT increased to approximately 147.5 nm. Figure 2 The presence of Fe (E) indicates successful outer coating. Elemental mapping analysis further confirmed the coexistence and uniform distribution of elements such as Fe, Mn, C, and O in DzMT, confirming the formation of the composite structure. Figure 2 F).

[0068] Example 4: Reaction Thermodynamics Study

[0069] Dz ( Figure 3 In section A), structural stability and conformational switchability must be maintained to prevent nonspecific signal leakage and ensure efficient activation after miRNA triggering. For example... Figure 3 As shown in Figure B, the process of miR-21 activating Dz can be divided into three main steps. The free energy change (ΔG) of the reaction was theoretically calculated using NUPACK software (http: / / www.nupack.org / ). Figure 3 (C). And the value of ΔG for the overall reaction is obtained according to Hess's law (ΔG = ΔG1 + ΔG2 + ΔG3).

[0070] The results show that the total free energy change ΔG = -33.67 kcal / mol, indicating that the reaction is thermodynamically highly spontaneous. When the miR-21 concentration is 20 nM, the theoretical conversion efficiency reaches 99.99%. Figure 3 The results (D) indicate that miR-21 can efficiently trigger DNAzyme activation and subsequent target cleavage.

[0071] Example 5: Fluorescence Spectroscopy and Kinetic Analysis

[0072] To evaluate the responsiveness and multifunctionality of DzMT under acidic conditions, this invention employs an FLS1000 fluorescence spectrometer for systematic analysis. Figure 4 (A). To verify whether Dz released from DzMT under acidic conditions can still produce a functional response to miR-21 ( Figure 4 In study B), the 5' end of a DNAzyme was labeled with the fluorescent group Cy5, and the 3' end with the quencher group BHQ2, to monitor its miR-21-triggered fluorescence recovery. Under acidic conditions at pH 5.5, the fluorescence signal of Cy5 significantly increased after the addition of miR-21 following the decomposition of DzMT and the release of Dz. Figure 4 (Middle B, purple curve), while without miR-21, only a weak background signal was detected ( Figure 4 (B, blue curve); under neutral or alkaline conditions (pH 7.4), no significant fluorescence recovery was observed regardless of the presence of miR-21. Figure 4 The green and red curves (Figure B) indicate that DzMT possesses excellent acid-responsive release characteristics and specific responsiveness. Further experiments verified the catalytic cleavage function of Dz after activation (…). Figure 4 In a method involving the substrate probe (C), Cy5 fluorescent dye was embedded inside, and BHQ2 quencher was attached to the 5' end to monitor fluorescence recovery after cleavage. The addition of miR-21 under acidic conditions significantly enhanced the Cy5 signal, indicating that Dz activity was restored and could cleave CAT mRNA.

[0073] Furthermore, the DzMT-mediated Fenton reaction kinetics were monitored in PBS buffers at pH 5.5 and pH 7.4. Figure 4 (D). Under pH 7.4 conditions, the initial rate (V) m The calculated value is 1.71 × 10⁻⁶. -7 M·s -1 ( Figure 4 (Middle D, gray curve), while under pH 5.5 conditions, V m Significantly increased to 5.22×10 -6 M·s -1 ( Figure 4 The pink curve (D) indicates that an acidic environment can significantly enhance the Fenton reaction efficiency of DzMT and increase the •OH generation efficiency.

[0074] To verify the system's specificity, this invention designed three mutant miR-21 sequences (M1, M2, M3) Figure 4 (E). For example Figure 4 As shown in Figure F, the mutant or blank control group produced only very weak fluorescence signals, while the signal intensities induced by miR-21 were 3.08 times, 5.41 times, and 6.82 times that of the M1, M2, and M3 groups, respectively (t-test, p < 0.001), demonstrating that the system has high specificity for miR-21 recognition.

[0075] Under optimal conditions, this invention evaluated the quantitative detection performance of the nanoplatform by monitoring the changes in Cy5 fluorescence signal caused by different concentrations of miR-21. Figure 4 G, H, and miR-21 induced an increase in Cy5 fluorescence intensity in a dose-dependent manner, and there was a significant linear correlation between Cy5 fluorescence intensity (F) and the logarithm of miR-21 concentration (C), within a concentration range of 5 × 10⁻⁶. −14 Up to 1 × 10 −9 M ( Figure 4 In equation I, the equation is F = 17887.04 log 10 C + 271286 (R 2 The detection limit (LOD) is 24.2 fM (0.994), indicating that the platform has ultra-high sensitivity and excellent quantitative detection performance.

[0076] Example 6: In situ real-time imaging of miR-21 in living cells

[0077] DzMT was co-cultured with the breast cancer cell line MCF-7 (highly expressing miR-21) and the normal breast epithelial cell line MCF-10A (lowly expressing miR-21), and the images were analyzed using confocal laser scanning microscopy (CLSM). Figure 5(A). The results showed that the Cy5 fluorescence signal in MCF-7 cells significantly increased with incubation time ( Figure 5 In B and D cells, only weak background fluorescence was observed in MCF-10A cells ( Figure 5 The results (C and E) indicate that DzMT can be effectively taken up by cancer cells and achieve miR-21-specific activation in tumor cells.

[0078] To further verify the miRNA response selectivity of DzMT, this invention applied it to cell lines with different miR-21 expression levels ( Figure 5 The results showed that cancer cell lines MCF-7, HeLa, MDA-MB-231, and A549 all exhibited significantly enhanced Cy5 fluorescence signals, while the normal cell line MCF-10A showed only low-level fluorescence (F). Figure 5 (H). This result indicates that DzMT can distinguish cells with different miR-21 expression levels, achieving highly selective imaging of cancer cells.

[0079] Considering the dynamic changes in miRNA expression levels during tumorigenesis and progression, this invention further evaluated the sensitivity of DzMT to changes in intracellular miR-21 expression. The miR-21 level in MCF-7 cells was regulated by transfection with a miR-21 mimic (mimic-miR-21) or a miR-21 inhibitor (anti-miR-21). Results showed that miR-21 expression was upregulated by approximately 1.88-fold in the mimic-miR-21 group, and Cy5 fluorescence signal was significantly enhanced. Figure 5 In the anti-miR-21 group, miR-21 expression decreased by approximately 2.45-fold, and the fluorescence signal was significantly weakened. Figure 5 Middle I).

[0080] In summary, the results show that the microRNA-responsive nanotherapeutic platform of the present invention can achieve highly sensitive, dynamic, and real-time imaging of miR-21 in living cells, and has the ability to accurately reflect changes in intracellular miRNA expression levels, providing a visualization tool for early molecular diagnosis of cancer.

[0081] Example 7: Mechanism of DzMT-induced cell death in in vitro experiments

[0082] This invention further investigates the cytotoxic effects and mechanisms of a microRNA-responsive nanotherapeutic platform in the breast cancer MCF-7 cell line. Figure 6 (A). In the experiment, cells were randomly divided into five groups and treated with PBS, free Dz, DzM, mutant Dz (nDzMT), and DzMT, respectively.

[0083] First, intracellular reactive oxygen species (ROS) levels were detected using a 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) kit. Confocal laser scanning microscopy (CLSM) images showed that cells treated with DzMT exhibited the strongest green fluorescence signal. Figure 6 The presence of B indicates that it can significantly induce the accumulation of intracellular ROS. Increased ROS (especially hydroxyl radicals •OH) can trigger lipid peroxidation (LPO) and induce ferroptosis. To further verify this phenomenon, this invention uses BODIPY... TM Intracellular LPO levels were detected using the 581 / 591 C11 fluorescent probe. The emission spectrum of the oxidized BODIPY probe shifted from red (~590 nm) to green (~510 nm). Results showed that cells treated with DzMT exhibited a stronger green fluorescence signal, significantly enhanced compared to the nDzMT control group. Figure 6 (C) confirmed that DzMT can significantly promote intracellular oxidative stress and lipid peroxidation accumulation.

[0084] Furthermore, the continuous accumulation of ROS disrupts mitochondrial membrane structure, leading to decreased membrane potential and disordered energy metabolism. Changes in mitochondrial membrane potential were analyzed using the JC-1 kit. Results showed that JC-1 aggregates (red fluorescence) were significantly reduced in the DzMT-treated group, while monomers (green fluorescence) were significantly enhanced, and the red / green fluorescence ratio decreased in a dose-dependent manner. Figure 6 The results (D) indicate that DzMT can induce significant mitochondrial damage.

[0085] This invention used real-time quantitative PCR technology to determine the CAT mRNA levels in MCF-7 cells and MCF-10A cells treated with different concentrations of DzMT. Figure 6 As shown in Figure E, compared with MCF-10A cells, DzMT significantly inhibited CAT mRNA expression in MCF-7 cells. Western blot analysis further confirmed that DzMT downregulated catalase protein expression (…). Figure 6 (F). This highlights the tumor-specific gene silencing capability of this nanoplatform.

[0086] This invention uses the CCK-8 assay to assess cell viability. The viability of normal breast MCF-10A cells is not affected by DzMT. Figure 6 The presence of G indicates that DzMT has excellent selectivity for cancer cells. To evaluate the cell-killing effect of DzMT, cell viability was analyzed using a Calcein-AM / PI double staining method. The results showed that red fluorescence was significantly enhanced and green fluorescence was significantly weakened in the DzMT-treated group. Figure 6The presence of H+ in the cells indicated a significant decrease in cell viability. Further analysis of apoptosis was performed using Annexin V-FITC / PI double staining combined with flow cytometry. The results showed that the proportion of early (Q2) and late (Q4) apoptotic / necrotic cells was significantly increased in the DzMT-treated group, with the total apoptosis rate (Q2+Q4) reaching 55.44%. Figure 6 The concentration of I in the middle ions was significantly higher than that in the control group.

[0087] The results in summary demonstrate that the microRNA-responsive nanotherapeutic platform prepared in this invention induces cancer cell death through multiple regulatory mechanisms: first, DNAzyme-mediated silencing of the CAT gene, increasing intracellular H2O2 levels; second, metal ions (Fe... 2+ / Fe 3+ / Mn 2+ (1) The Fenton reaction is driven to generate •OH and form a self-reinforcing cycle; (2) Oxidative stress and mitochondrial dysfunction jointly lead to the synergistic occurrence of apoptosis and ferroptosis. This mechanism provides strong experimental evidence for precise chemokinetic therapy (CDT) based on miRNA response.

[0088] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a microRNA-responsive nanotherapeutic platform, characterized in that: The method comprises the following steps: Step 1: dissolving the DNAzyme containing a self-blocking structure in a Tris-HCl buffer, annealing the obtained solution, and slowly cooling to form a DNAzyme solution with a stable secondary structure; Step 2: Fe 2+ ions were mixed with the DNAzyme solution obtained in Step 1 under acidic conditions to self-assemble into metal-nucleic acid framework nanoparticles; the outer layer of the metal-nucleic acid framework was coated with a metal-phenol network formed by Fe 3+ , Mn 2+ and tannic acid to obtain a DNAzyme-metal-tannic acid nanocomposite with a stable structure.

2. The method of claim 1, wherein: In step 1, the DNAzyme containing a self-blocking structure comprises the following three functional regions: (1) an RNA cleavage core region for realizing a sequence-specific cleavage reaction; (2) a microRNA recognition region for binding to miR-21 highly expressed in tumors to remove the self-blocking structure of the catalytic core; (3) a target mRNA recognition region for specifically recognizing and cleaving catalase mRNA.

3. The method of claim 1, wherein: In step 2, the DNAzyme is mixed with FeCl2·4H2O under acidic conditions to form a metal-nucleic acid framework; then the pH is adjusted to 7.0 and the mixture is shaken, and FeCl3·6H2O, MnCl2·4H2O and tannic acid are sequentially added to form a metal-phenol network coating layer, thereby obtaining a stable DNAzyme-metal-tannic acid nanocomposite.

4. The method of claim 1 or 2, wherein: The sequence of the DNAzyme is: 5'-GCT AAA GGT CAA CAT CAG TCT GAT AAG CTA CAG GGA GGC CCC TGT CCG AGC CGG TCG AAC CTT TAG CA-3'; The sequence of miR-21 is: 5'-UAG CUU AUC AGA CUG AUG UUG A-3'; The sequence of catalase mRNA is: 5'-UGC UAA AGG AGC AGG GGC CU-3'.

5. A microRNA-responsive nanodiagnosis and treatment platform prepared by the method of claim 1. 6.The microRNA-responsive nanodiagnostic and nanotherapeutic platform according to claim 5, wherein: The average particle size of the DNAzyme-metal-tannic acid nanocomposite is 140 nm, and it has good dispersibility and structural stability at pH 7.4, but can be quickly decomposed and release active components at pH 5.

5.

7. The use of the microRNA-responsive nanodiagnostic and nanotherapeutic platform of claim 5 or 6 in the preparation of a pharmaceutical composition, characterized in that: The pharmaceutical composition is used for fluorescence imaging of overexpressed microRNAs in tumors, catalase gene silencing, and Fenton reaction-driven chemical kinetics therapy, realizing integration of imaging and treatment.

8. Use according to claim 7, characterized in that: The application is based on the following mechanism: the nanodiagnosis and treatment platform selectively enriches in tumor tissues by enhancing the permeability and retention effect, and dissociates in the acidic tumor microenvironment; the released DNAzyme is activated by the overexpressed miR-21 in tumor cells.

9. Use according to claim 7, characterized in that: The Fenton reaction is realized by the following pathways: Fe 2+ + H2O2 → Fe 3+ + •OH + OH − wherein the tannic acid reduces the generated Fe 3+ to Fe 2+ , forming Fe 2+ / Fe 3+ autocycle system, continuously generating hydroxyl radicals, thereby inducing tumor cell oxidative stress damage and apoptosis.

10. Use according to claim 7, characterized in that: After binding with miR-21, the DNAzyme is activated, on the one hand, to activate its catalytic activity, resulting in recovery of the fluorescence signal and realizing spatial visualization imaging of microRNAs in tumor cells; on the other hand, the activated DNAzyme can specifically cleave CAT mRNA, inhibit the expression of catalase in cells, promote the enrichment of H2O2 in cells, and thus enhance the efficiency of the Fenton reaction.

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