Nano indicator for targeted induction of mitochondrial apoptosis and real-time monitoring of Cyt c release as well as preparation method and application of nano indicator

Abstract

The invention discloses a nano indicator for targeted induction of mitochondrial apoptosis and real-time monitoring of Cyt c release as well as a preparation method and application of the nano indicator. The nano indicator comprises a two-dimensional material modified with hyaluronic acid, and a chemotherapeutic drug and a carbon-based quantum dot-Cyt c aptamer compound which are loaded on the two-dimensional material modified with hyaluronic acid, the carbon-based quantum dot-Cyt c aptamer compound comprises a carbon-based quantum dot and a Cyt c aptamer modified on the carbon-based quantum dot. The hyaluronic acid in the nano indicator enables the nano indicator to target AML cells, and the chemotherapeutic drug can induce mitochondrial apoptosis so as to trigger the release of Cyt c. Meanwhile, the two-dimensional material and the carbon-based quantum dot-Cyt c aptamer compound are paired to quench the fluorescence, and when Cyt c is recognized, the carbon-based quantum dot and the two-dimensional material are separated to restart the fluorescence. Therefore, the nano indicator provided by the invention can monitor the release of Cyt c in the same cell in real time while inducing mitochondrial apoptosis by targeted delivery of chemotherapeutic drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a nanometer indicator for inducing mitochondrial apoptosis in a targeted manner and monitoring Cyt c release in real time, and a preparation method and application thereof. Background Art

[0002] Acute myeloid leukemia (AML) is a hematological malignancy caused by the uncontrolled proliferation of myeloid blasts, with an overall poor prognosis. To date, chemotherapy remains the most effective clinical treatment strategy; however, the inherent heterogeneity of AML cells leads to differences in response to leukemia drugs, resulting in significant differences in treatment outcomes between individuals. Currently, the monitoring of chemotherapy efficacy in AML cells mainly involves batch evaluation of cell population marker levels after cell drug treatment to obtain average information. However, this method may mask the proportion of drug-resistant cells in the cell population, leading to misjudgment of the effect of chemotherapy drugs and missing the best treatment opportunity. Therefore, monitoring efficacy-related markers while dynamically releasing drugs is crucial for early evaluation of the therapeutic effect of chemotherapy drugs and timely adjustment of medication strategies. In recent years, bionanomaterials have shown unique advantages in drug delivery, pharmacokinetics and biosensing due to their unique size and surface reactivity. Among them, two-dimensional (2D) nanomaterials have attracted widespread attention due to their rich functional groups and large specific surface area. As an emerging two-dimensional nanomaterial, MXenes have attracted increasing attention in the field of nanodiagnosis and therapy due to their excellent photoquenching properties and amphiphilicity. However, research combining its pharmacological advantages with optical properties for drug delivery and simultaneous efficacy monitoring remains limited.

[0003] Therefore, the existing technology still needs to be improved and developed. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a nano-indicator for targeted induction of mitochondrial apoptosis and real-time monitoring of Cyt c release, as well as a preparation method and application thereof, in order to solve the problem that relevant markers cannot be monitored in real time and in situ when chemotherapy drugs induce mitochondrial apoptosis to treat AML.

[0005] The technical solution of the present invention is as follows:

[0006] In a first aspect, a nano-indicator for inducing mitochondrial apoptosis and monitoring Cyt c release in real time is provided, wherein the nano-indicator comprises: a two-dimensional material modified with hyaluronic acid, and a chemotherapeutic drug and a carbon-based quantum dot-Cyt c aptamer complex loaded on the two-dimensional material modified with hyaluronic acid;

[0007] The carbon-based quantum dot-Cyt c aptamer complex comprises: carbon-based quantum dots and Cyt c aptamers modified on the carbon-based quantum dots.

[0008] According to a preferred technical solution, the hyaluronic acid is modified on the two-dimensional material through an esterification reaction.

[0009] In a preferred technical solution, the chemotherapy drug is loaded on the two-dimensional material modified with hyaluronic acid through π-π stacking and / or hydrogen bonding.

[0010] According to a preferred technical solution, the carbon-based quantum dot-Cyt c aptamer complex is loaded on the two-dimensional material modified with hyaluronic acid through hydrogen bonding and / or van der Waals forces.

[0011] According to a preferred technical solution, the Cyt c aptamer is an amino-modified Cyt c aptamer, and the Cyt c aptamer is modified on the carbon-based quantum dots through an amide condensation reaction.

[0012] In a preferred technical solution, the two-dimensional material is MXene.

[0013] In a preferred technical solution, the chemotherapy drug is dihydroartemisinin.

[0014] In a preferred technical solution, the carbon-based quantum dots are graphene quantum dots.

[0015] In a preferred technical solution, the Cyt c aptamer is 5'-NH 2 -CCGTGTCTGGGGCCGACCGGCGCATTGGGTACGTTGTTGC-3'.

[0016] In a second aspect, a method for preparing the nano-indicator as described in the first aspect is provided, comprising the steps of:

[0017] providing a dispersion of the two-dimensional material;

[0018] Adding hyaluronic acid to the dispersion of the two-dimensional material to react and obtain a two-dimensional material modified with hyaluronic acid;

[0019] Adding chemotherapy drugs to the dispersion of the two-dimensional material modified with hyaluronic acid, mixing and stirring, to obtain a two-dimensional material loaded with chemotherapy drugs and modified with hyaluronic acid;

[0020] The carbon-based quantum dot-Cyt c aptamer complex is added to the dispersion liquid of the two-dimensional material loaded with chemotherapy drugs and modified with hyaluronic acid to react and obtain the nano indicator.

[0021] According to a preferred technical solution, the mass ratio of hyaluronic acid to the two-dimensional material is (1-10):1; the mass ratio of the two-dimensional material modified with hyaluronic acid to the chemotherapeutic drug is (0.25-1.25):1; the ratio of the two-dimensional material loaded with chemotherapeutic drugs and modified with hyaluronic acid to the carbon-based quantum dot-Cyt c aptamer complex is (50-200) mg:1 μmol.

[0022] A preferred technical solution is a method for preparing the carbon-based quantum dot-Cyt c aptamer complex, comprising the steps of:

[0023] Adding an amide condensation reagent to the carbon-based quantum dot solution, stirring, and then adding a Cyt c aptamer to react to obtain the carbon-based quantum dot-Cyt c aptamer complex;

[0024] Wherein, the ratio of the carbon-based quantum dots to the Cyt c aptamer is (0.5-10) g:1 mmol.

[0025] In a third aspect, a use of the nano-indicator as described in the first aspect in the preparation of a drug for treating acute myeloid leukemia is provided.

[0026] Beneficial effects: The present invention develops an integrated nano-indicator, wherein the nano-indicator is modified with hyaluronic acid on a two-dimensional material and loaded with chemotherapeutic drugs. The hyaluronic acid enables it to target AML cells, and the chemotherapeutic drugs can induce mitochondrial apoptosis, thereby triggering the release of cytochrome c (Cyt c). Furthermore, the present invention constructs an "on-type" Cyt c nano-indicator by pairing the two-dimensional material with carbon-based quantum dots modified with Cyt c aptamers to quench the fluorescence. When the nano-indicator recognizes Cyt c, the carbon-based quantum dots and the two-dimensional material are separated, and then fluorescence is emitted. Therefore, the nano-indicator of the present invention can monitor the release of Cyt c in the same cell in real time while targeted delivery of chemotherapeutic drugs to induce mitochondrial apoptosis. This bifunctional nano-indicator with excellent targeting and high biocompatibility realizes the simultaneous evaluation of chemotherapy-induced mitochondrial apoptosis and drug effects in AML cells, and has good potential in the fields of new drug development and personalized diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the preparation process and detection process of the nano-indicator of the present invention; wherein A is a schematic diagram of the preparation process of DA-MXene in Example 1 of the present invention; B is a schematic diagram of the targeted chemotherapy of AML cells by DA-MXene and the synchronous in situ monitoring of Cyt c release induced by mitochondrial apoptosis.

[0028] Figure 2is the characterization of DA-MXene in Example 1 of the present invention; wherein A is the TEM image of MXene; B is the AFM image of MXene; C is the corresponding thickness profile of MXene; D is the hydrated particle size distribution of MXene before and after ultrasonic treatment; E is the drug loading efficiency test result of DHA on MXene, and the error bar represents the standard deviation (n=3); F is the molecular weight distribution of MXene, GQD, Apt GQD and UV-vis absorption spectra of DA-MXene; G is the zeta potential of MXene, DHA@MXene and DA-MXene; H and I are the full and high-resolution spectra of X-ray electron energy spectra of MXene and DA-MXene; J is the X-ray diffraction pattern of MXene and DA-MXene; K is the image of MXene, DHA@MXene-HA and DA-MXene after incubation in water, PBS, RPMI 1640 medium and PBS buffer containing 20% ​​FBS for 24 hours; L is the in vitro release curve of DHA from DA-MXene at 37°C under acidic (pH 5.5, 6.3) and neutral (pH 7.4) conditions.

[0029] Figure 3 is the quantitative detection and analysis result of Cyt c based on photoluminescence (PL) spectroscopy test in Example 1 of the present invention; wherein A is Apt GQD With concentrations of 0-600mg mL -1 A is the PL spectra of MXene-HA after co-incubation; B is the PL spectra of MXene-HA with different concentrations and Apt GQD Fluorescence intensity and quenching efficiency after co-incubation; C is the fluorescence intensity and quenching efficiency of DA-MXene with different concentrations (0-10U mL -1 ) PL spectrum after co-incubation with Cyt c; D is the relative fluorescence intensity of DA-MXene and Cyt c (Fr represents the fluorescence intensity after co-incubation of DA-MXene and Cyt c, and Fq represents the fluorescence intensity of DA-MXene without adding target Cyt c); E is the linear relationship fitting between the relative fluorescence intensity and the Cyt c concentration, and the data are expressed as the mean ± standard deviation (n = 3).

[0030] Figure 4 DA-MXene nanoindicator in Example 1 of the present invention is the experimental result of targeted cell uptake in HL-60 cells; wherein A is the quantitative analysis of flow cytometry and incubation of HL-60, THP-1 and K562 cells with CD44 antibody (blue line) and IgG (yellow line); B is the fluorescence image of HL-60 cells after incubation with HA, HA+DA-MXene and DA-MXene for 24 hours, wherein the cell nucleus is stained with Hoechst (blue), and Cyt c is stained with Apt QGDIndicates (red), scale bar: 20 μm; C is the Apt of HL-60 cells after HA, HA+DA-MXene and DA-MXene treatment for 24 hours GQD Fluorescence intensity (FI) analysis, data are expressed as mean ± standard deviation (n = 3), ***P < 0.001.

[0031] Figure 5 The CCK-8 detection results and flow cytometry analysis results of MXene-HA, DHA, DHA@MXene and DA-MXene in Example 1 of the present invention are shown; wherein, A is the CCK-8 detection result of HL-60 cells after incubation with MXene, DHA, DHA@MXene and DA-MXene for 24 hours, and the data are expressed as mean ± standard deviation (n=3); B is the CCK-8 detection result of K562 and THP-1 cells after incubation with DA-MXene; C is the flow cytometry analysis result of HL-60 cells after incubation with PBS, K562, THP-2 and HL-60 with DA-MXene for 24 hours; D is the live / dead cell staining imaging of the HL-60 cell blank group and the HL-60 cells after incubation with DHA, DHA@MXene and DA-MXene for 24 hours, scale bar: 100 μm.

[0032] Figure 6 It is the fluorescence imaging of ΔΨm, statistical analysis of fluorescence, statistical analysis of ROS level and WB detection of apoptosis-related key proteins in the blank group of HL-60 cells and HL-60 cells co-incubated with DHA, DHA@MXene, and DA-MXene in Example 1 of the present invention; wherein, A is the fluorescence imaging of ΔΨm; B is the intracellular fluorescence imaging of the ROS level probe of the blank group of HL-60 cells and HL-60 cells treated with DHA (4.0 μM), DHA@MXene, and DA-MXene (4.0 μM DHA equivalent) for 24 hours: 100 μm; C is the statistical analysis of ROS level; Figure D is the statistical analysis of ΔΨm fluorescence; E is the Western blotting of apoptosis-related proteins Bcl-2, Bax and internal reference GAPDH in the blank group of HL-60 cells and HL-60 cells incubated with DHA, DHA@MXene, and DA-MXene (4.0 μM DHA equivalent) for 24 hours. Blot analysis, data are expressed as mean ± SD (n = 3), ***P < 0.001, **P < 0.01, *P < 0.05; F is Western blot gray value analysis.

[0033] Figure 7The results of using DA-MXene for targeted chemotherapy and in situ monitoring of Cyt c in Example 1 of the present invention; wherein, A is a schematic diagram of using DA-MXene for mitochondrial apoptosis and simultaneous monitoring of Cyt c; B is mitochondrial staining and Cyt c detection of HL-60 cells treated with DA-MXene (4.0 μM DHA equivalent) after 0, 1, 5, and 10 hours, scale bar: 10 μm; C is mitochondrial apoptosis and Apt c detection of HL-60 cells after incubation with DA-MXene (4.0 μM DHA equivalent) at different treatment times. GQD Statistical analysis of fluorescence intensity (FI). DETAILED DESCRIPTION

[0034] The present invention provides a nanometer indicator for inducing mitochondrial apoptosis and monitoring Cyt c release in real time, and a preparation method and application thereof. In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below.

[0035] The embodiment of the present invention provides a nano-indicator for inducing mitochondrial apoptosis in a targeted manner and monitoring Cyt c release in real time, wherein the nano-indicator comprises: a two-dimensional material modified with hyaluronic acid, and a chemotherapeutic drug and a carbon-based quantum dot-Cyt c aptamer complex loaded on the two-dimensional material modified with hyaluronic acid;

[0036] The carbon-based quantum dot-Cyt c aptamer complex comprises: carbon-based quantum dots and Cyt c aptamers modified on the carbon-based quantum dots.

[0037] Specifically, in AML chemotherapy, mitochondrial apoptosis is the main pathway of apoptosis, and the activation of cytochrome c (Cyt c) is one of its important markers. Therefore, the present invention has developed a nano-indicator based on two-dimensional materials, which can target the delivery of chemotherapy drugs, thereby inducing mitochondrial apoptosis and detecting the release of Cyt c in the cytoplasm at the same time. In order to detect Cyt c, the present invention uses two-dimensional materials as energy acceptors and carbon-based quantum dots as fluorescence donors to construct a fluorescence resonance energy transfer (FRET) indicator. Based on carbon-based quantum dots to ensure the biocompatibility of nano-indicators, it is beneficial to dynamic monitoring in active cells. In addition, the two-dimensional material is modified with hyaluronic acid (HA), which can target the CD44 receptor overexpressed on the surface of AML cells. This bifunctional nano-indicator makes full use of the surface loading and optical quenching effect of two-dimensional materials, and has outstanding biocompatibility. It can monitor drug action and efficacy while AML cells remain active, and shows good potential in the future opening of new drugs and drug screening.

[0038] In one embodiment, the hyaluronic acid is modified on the two-dimensional material through an esterification reaction.

[0039] In one embodiment, the chemotherapeutic drug is loaded on the two-dimensional material modified with hyaluronic acid through π-π stacking and / or hydrogen bonding.

[0040] In one embodiment, the carbon-based quantum dot-Cyt c aptamer complex is loaded on the two-dimensional material modified with hyaluronic acid through hydrogen bonding and / or van der Waals forces.

[0041] In one embodiment, the Cyt c aptamer is an amino-modified Cyt c aptamer, and the Cyt c aptamer is modified on the carbon-based quantum dots through an amide condensation reaction.

[0042] In one embodiment, the two-dimensional material is MXene.

[0043] In one embodiment, the chemotherapy drug is dihydroartemisinin, but is not limited thereto, and other chemotherapy drugs that can induce mitochondrial apoptosis may also be used.

[0044] In one embodiment, the carbon-based quantum dots are graphene quantum dots, but are not limited thereto, and other carbon-based quantum dots with good biocompatibility may also be used.

[0045] In one embodiment, the Cyt c aptamer is 5'-NH 2 -CCGTGTCTGGGGCCGACCGGCGCATTGGGTACGTTGTTGC-3', but not limited thereto, other Cytc aptamers with the function of specifically recognizing and binding to Cyt c may also be used.

[0046] The present invention provides a method for preparing the nano-indicator as described above, comprising the steps of:

[0047] providing a dispersion of the two-dimensional material;

[0048] Adding hyaluronic acid to the dispersion of the two-dimensional material to react and obtain a two-dimensional material modified with hyaluronic acid;

[0049] Adding chemotherapy drugs to the dispersion of the two-dimensional material modified with hyaluronic acid, mixing and stirring, and obtaining a two-dimensional material loaded with chemotherapy drugs and modified with hyaluronic acid;

[0050] The carbon-based quantum dot-Cyt c aptamer complex is added to the dispersion liquid of the two-dimensional material loaded with chemotherapy drugs and modified with hyaluronic acid to react and obtain the nano indicator.

[0051] In one embodiment, the mass ratio of the two-dimensional material to the hyaluronic acid is (1-10):1.

[0052] In one embodiment, the mass ratio of the two-dimensional material modified with hyaluronic acid to the chemotherapeutic drug is (0.25-1.25):1.

[0053] In one embodiment, the ratio of the two-dimensional material loaded with chemotherapy drugs and modified with hyaluronic acid to the carbon-based quantum dot-Cyt c aptamer complex is (50-200) mg:1 μmol.

[0054] In one embodiment, the method for preparing the carbon-based quantum dot-Cyt c aptamer complex comprises the steps of:

[0055] Adding an amide condensation reagent to the carbon-based quantum dot solution, stirring, and then adding a Cyt c aptamer to react to obtain the carbon-based quantum dot-Cyt c aptamer complex;

[0056] Wherein, the ratio of the carbon-based quantum dots to the Cyt c aptamer is (0.5-10) g:1 mmol.

[0057] In a more specific embodiment, the amide condensation reagent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

[0058] An embodiment of the present invention provides a use of the nano-indicator as described above in the preparation of a drug for treating acute myeloid leukemia.

[0059] The present invention will be further described below by means of specific examples.

[0060] Example 1

[0061] 1. Materials and Methods

[0062] 1.1 Materials and reagents

[0063] Human leukemia cell line (HL-60 cells), human immortalized myeloid leukemia cell line (K652 cells) and human monocytic cell line (THP-1 cells) were purchased from the cell bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Roswell Park Memorial Institute-1640 (RPMI-1640) medium and penicillin / streptomycin were purchased from Gibco (New York, USA). Cell counting kit-8 (CCK-8) and phosphate buffered saline (PBS) were purchased from Biyontime Biotech (Shanghai, China). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 98%) and N-hydroxysuccinimide (NHS, 98%) were purchased from Macklin Reagent Inc (Shanghai, China). Ti, which contains abundant carboxyl groups and emits red fluorescence at a wavelength of 641 nm, was used to generate the 3D-conjugated TiO2 antibody. 3 AlC 2 and graphene quantum dots (GQDs) were synthesized and purified by Jiangsu Pioneer Nanomaterials Technology Co., Ltd. Cytochrome c (Cyt c, 95%) was purchased from Sigma-Aldrich (Shanghai, China). Hydrogen fluoride (HF) and tetrahydrofuran (THF) were purchased from Macklin (Shanghai, China). Hyaluronic acid (HA: molecular weight = 240 kDa) was purchased from Bloomage Biotechnology (China). Mito-Tracker Green was purchased from Yesheng Technology (Shanghai, China). Hoechst 33258 and LIVE / DEAD cell imaging kit were purchased from Invitrogen (Carlsbad, CA, USA). Human Bcl-2 polyclonal antibody, Bax polyclonal antibody, and Caspase 3 / p17 / p19 monoclonal antibody were purchased from Proteintech (Wuhan, China). All chemical reagents were obtained from commercial suppliers without further purification. Distilled deionized water was used in all experiments.

[0064] 1.2 Preparation of DA-MXene nanoindicators

[0065] Ti 3 C 2 The preparation method of MXene nanosheets is as follows: First, 1 gram of Ti 3 AlC 2 The powder was slowly added to 20 ml of hydrogen fluoride (HF) solution, and the mixture was stirred at 40 °C for 24 hours. Then, the mixture was repeatedly washed with distilled deionized water and centrifuged several times until the pH value of the supernatant reached 6. The final product was dissolved in deionized water for later use. The suspension was purged with nitrogen and then sonicated in a bath ultrasonic cleaner for 1 hour. Next, the solution was centrifuged at 3500 rpm for 8 minutes to separate the monolayer Ti3 C 2 After removing the precipitate, the uniformly dispersed Ti in the supernatant was obtained. 3 C 2 MXene nanosheets (abbreviated as Ti 3 C 2 MXene or MXene).

[0066] Ti 3 C 2 The MXene dispersion was ultrasonically treated in an ice bath for 1 hour to obtain a uniform MXene dispersion. The carboxyl groups of the HA molecules were pre-activated using EDC / NHS. First, 200 mg of HA was dispersed in 20 mL of deionized water, and then EDC (38 mg) and NHS (46 mg) were added and stirred continuously overnight. The product was purified using an ultrafiltration tube (Amicon Ultracel-10k) to obtain activated HA. 5 mg of the purified HA product was added to the MXene dispersion (0.25 mg mL -1 , 4 mL) for 30 min and kept stirring overnight. Centrifuge at 10,000 rpm for 10 min using a 10-K ultrafiltration tube to obtain the MXene-HA product. The final product was stored at 4 degrees Celsius for subsequent use.

[0067] DHA (1.0 mg mL -1 , 8 mL) was dissolved in dimethyl sulfoxide (DMSO) and added to the MXene-HA dispersion (1.0 mg mL -1 , 1.25mL). Then, the mixture was stirred at room temperature for 10 hours. Ultrafiltration centrifugation was used to remove excess DHA in the mixture. The sample was loaded into an Amicon Ultra-0.5 centrifugal filter unit (Amicon Ultracel-10k; EMD Millipore, Billerica, Massachusetts, USA) and centrifuged at 10000rpm for 30 minutes to collect DHA@MXene-HA. In addition, DHA@MXene was also prepared in this embodiment, and its preparation steps and proportions were the same as those of DHA@MXene-HA, except that MXene was replaced by MXene-HA, that is, DHA@MXene did not contain HA.

[0068] In order to determine the optimal loading capacity of MXene for DHA, this example configured DHA and MXene with different mass ratios (i.e., 0.25:1, 0.5:1, 0.75:1, 1:1, and 1.25:1) according to the previously described method, and ultrafiltration was used to collect free DHA at each loading ratio. Then, high performance liquid chromatography (HPLC) was used to analyze the drug loading capacity (DL) of DHA. DHA was dissolved in anhydrous ethanol and separated using a C18 reverse phase chromatographic column, and the detection wavelength was set to 238nm. By using DHA standard solutions of known concentrations (16, 32, 64, 128, 256, and 400 μg mL -1 ) A standard curve was established. The drug loading capacity (DL) of DHA on MXene nanosheets was calculated by the following formula: DL = mass of DHA / mass of MXene × 100%.

[0069] The DHA mass loading rate was calculated based on the DHA concentration and solution volume determined by HPLC. DHA =DHA weight / (DHA weight +MXene weight )×100%, DHA weight It is the total mass of DHA minus the mass of free DHA after ultrafiltration.

[0070] Next, the Cyt c DNA aptamer (5'-NH 2 -CCG TGT CTG GGG CCG ACC GGC GCA TTGGGT ACG TTG TTG C-3', 100 μM, 1 mL) was conjugated to graphene quantum dots (GQDs) via EDC / NHS chemical reaction. Briefly, 2.4 mg EDC and 3.6 mg NHS were added to 1 mL of graphene quantum dot solution (1 mg mL -1 ) and the mixture was stirred for 15 minutes. Then, the aptamer (Apt) was added to the graphene quantum dot solution at a final concentration of 1 mM. The mixture was then shaken overnight at room temperature. The product was purified by ultrafiltration (Amicon Ultra-0.5, 30KD, Millipore, Billerica, Massachusetts, USA). The mixture was transferred to an ultrafiltration tube and centrifuged at 5000 rpm for 30 minutes. This process was repeated three times to obtain the purified carbon-based quantum dot-Cyt c aptamer complex (Apt GQD ).

[0071] Then Apt GQD Added into DHA@MXene-HA dispersion to synthesize DA-MXene (DHA / Apt GQD@MXene-HA) nanoindicator. In order to evaluate the effect of MXene nanosheets on Apt GQD The quenching efficiency of the molecule and optimization of Apt GQD The ratio of Apt to MXene is GQD (final concentration was 5 μM) and different concentrations (final concentration was 0-600 μg mL -1 ) was mixed in ultrapure water at room temperature in the dark for 30 minutes. Then, the fluorescence intensity was measured by fluorescence spectrometer, with an excitation wavelength of 560 nm and an emission wavelength of 580 nm to 800 nm. According to the formula Qe (%) = (F 0 -Fq) / F 0 Calculated quenching efficiency (Qe), where F 0 is the fluorescence signal of MXene, and Fq represents Apt GQD Fluorescence intensity after quenching by MXene. According to the Qe analysis results, 5μM Apt GQD With 600 μg mL -1 MXene was used for the preparation of DA-MXene.

[0072] 1. Physicochemical Characterization of 3DA-MXene

[0073] The particle size and Zeta potential of the prepared nanoparticles were determined by dynamic light scattering (DLS) using a MalvernZetasizer ZSP instrument. Atomic force microscopy (AFM) images of MXene nanosheets were obtained using a Bruker DimensionIcon microscope (Bruker Dimension Icon, USA). Transmission electron microscopy (TEM) imaging of MXene nanosheets was observed using a Talos L120C transmission electron microscope (Thermo Scientific, Eugene, Oregon, USA). Ultraviolet-visible spectra (UV-Vis) were measured by a UV-Vis spectrophotometer (TU1901, Puxi General Instrument Co., Ltd., Beijing, China). The hyaluronic acid (HA) modification of MXene was confirmed by X-ray photoelectron spectroscopy (XPS, K-α, Thermo Fisher Scientific, USA) and X-ray diffraction (XRD, Rigaku Ultima IV, Japan).

[0074] 1.4 Quantitative analysis of DHA release

[0075] The DHA release was quantitatively analyzed by equilibrium dialysis. GQD @MXenes(0.40mg mL -1, 4mL) was added to a 100kDa dialysate and dialyzed with sodium borate buffer of pH 7.4, pH 6.3, and pH 5.5. The three groups of samples were stirred in the dark at 37°C, and 200μL samples were taken for HPLC analysis at 1, 4, 8, 12, 24, and 36 hours. The DHA concentration in the sample was compared with the standard curve to calculate the released DHA concentration. The drug release rate was calculated according to the following formula: Release rate (%) = cumulative drug release mass / total mass of drug load × 100%.

[0076] 1.5 Quantitative detection and analysis of Cyt c

[0077] 200 μl of DA-MXene nanoindicator (1.0 mg mL -1 ) were mixed with different concentrations of Cyt c dilutions (final concentrations: 0, 0.05, 0.10, 0.20, 0.50, 1.00, 3.00, 5.00, and 10 U mL -1 ) were mixed. The mixture was then incubated at 37°C in the dark for 2 h, and then the fluorescence intensity of the sample was measured. After the reaction was completed, the photoluminescence spectrum of each sample was measured by excitation at 560 nm and scanning emission at 580 nm to 800 nm. The detection limit (LOD) of the DA-MXene nanoindicator was calculated by the following formula: LOD = 3σ / k (where σ is the overall standard deviation of the blank sample and k is the slope of the calibration curve). Each measurement was repeated three times independently.

[0078] 1.6 Analysis of CD44 expression in different cells

[0079] In order to investigate the ability of nanoparticles to selectively target AML cells, this example used flow cytometry to evaluate the expression of CD44 on different leukemia cell lines. The cell lines included HL-60 (acute myeloid leukemia), THP-1 (acute monocytic leukemia), and K562 (chronic myeloid leukemia). The three cell lines were cultured at 1×10 6 The cells were resuspended in PBS at a concentration of 10 cells / mL. The cells were incubated with FITC-conjugated CD44 antibody at 4°C in the dark for 30 minutes. After incubation, the cells were washed twice with PBS to remove excess antibody. The stained cells were then analyzed using flow cytometry to quantify the percentage of CD44-positive cells. The gating strategy was determined based on the isotype control staining, and the data were analyzed using FlowJo software to compare the expression levels of CD44 in HL-60, THP-1, and K562 cells.

[0080] 1.7 Targeted cytotoxicity of DA-MXene

[0081] HL-60, THP-1 and K562 cells were seeded into 96-well microplates (2 × 10 cells per well). 5 cells) and cultured in 1640 medium with 10% FBS in 5% CO 2 Incubate overnight at 37 °C in an incubator. The cytotoxicity of MXenes, DHA, DHA@MXene, and DA-MXene was studied using the CCK-8 assay. HL-60 cells were incubated with different concentrations of MXene, DHA, DHA@MXene, and DA-MXene for 24 hours. The CCK-8 assay requires the addition of 10 μL of CCK-8 reagent to each well, followed by incubation in the dark for 4 hours. The optical density (OD) was then measured at 450 nm using a microplate reader (BioTek Synergy H1). Cell viability was calculated using the following formula: Cell Viability (%) = Control group OD value - Blank group OD value / Sample OD value - Blank group OD value × 100%. The same method was used to detect the cell viability of K562 and THP-1 cells treated with different concentrations of DA-MXene.

[0082] DA-MXene-induced cell apoptosis was evaluated using flow cytometry. HL-60, K562, and THP-1 cells were cultured with 100 μg mL-1 of DA-MXene for 24 hours. After treatment, the cells were collected, resuspended in PBS, and stained using the Annexin V-FITC / PI Apoptosis Detection Kit (Cat. #C1062S, Beyotime). The stained cells were analyzed by flow cytometry using a BD AccuriC6 Plus flow cytometer. Apoptosis was quantified by measuring the percentage of Annexin V-positive cells to determine the apoptosis rate. The effect of DA-MXene on cell activity was tested using the LIVE / DEAD Cell Imaging Kit (488 / 528). HL-60 cells were cultured at 2×10 3 The cells were seeded in a 96-well plate at a density of 10 cells / well. MXene, DHA or DHA@MXene were added to the cells and co-cultured for 24 hours. Using the LIVE / DEAD cell imaging kit, live cells were distinguished by the green fluorescence of calcein AM, while dying and dead cells were distinguished by the red fluorescence of propidium iodide.

[0083] 1.8 Detection of mitochondrial membrane potential

[0084] The pre-cultured HL-60 cells were incubated in culture flasks for 24 h. After three washes with PBS, the culture medium was replaced with 100 μg mL -1Serum-free RPMI 1640 solution of DA-MXene nanoparticles was added and incubated for another 4 hours. After washing with PBS, JC-1 working solution was added and the cells were incubated for 20 minutes. After that, they were washed three times with PBS and the fluorescence changes caused by changes in mitochondrial membrane potential were captured using laser scanning confocal microscopy.

[0085] 1.9 Detection of intracellular ROS

[0086] In order to evaluate mitochondrial apoptosis and cell damage in HL-60 cells treated with DHA, DHA@MXene, and DA-MXene, mitochondrial activity and intracellular ROS generation were detected in this example. HL-60 cells were seeded in 96-well plates (1.0×10 4 cells) and cultured at 37°C, 5% CO 2 The cells were then incubated overnight under the conditions of DHA, DHA@MXene, or DA-MXene for 12 hours, with a DHA concentration of 4 μM in each treatment group. Mitochondrial activity was assessed by incubating the cells for 30 minutes using a fluorescent mitochondrial activity assay kit. At the same time, the intracellular ROS level was assessed using a fluorescent ROS assay kit, and the fluorescence intensity was quantified after 30 minutes of incubation.

[0087] 1.10 Immunoblotting analysis of apoptosis proteins

[0088] HL-60 cells were collected after washing with pre-cooled PBS, and lysis buffer containing PMSF was added. After shaking thoroughly, the cells were incubated on ice for 30 minutes to ensure complete cell lysis. Then, the samples were centrifuged at 4°C and 12,000 rpm for 30 minutes to collect total protein. After the samples were incubated with the BCA protein detection kit working solution at 37°C for 30 minutes, the absorbance at 562 nm was measured to determine the protein concentration. After protein quantification, the samples were mixed with 5× loading buffer at a ratio of 1:4 and boiled in boiling water for 5 minutes to denature the proteins. After pre-electrophoresis, the standard (Marker) and the sample to be analyzed were added in sequence. After the addition of samples, the proteins were separated by 15% SDS-PAGE gel electrophoresis and wet transferred to polyvinylidene fluoride membrane (PVDF). The membrane was washed with TBST solution for 5 minutes, repeated twice, and then placed in 5% BSA blocking solution and blocked on a shaker for 2 hours or overnight at 4°C. Next, the primary antibody was diluted 1:500 and incubated on a shaker at room temperature (50 rpm) for 4 hours or at 4°C overnight, followed by washing with TBST. The secondary antibody was diluted 1:10,000, incubated on a shaker at room temperature for 1 hour and washed again. Finally, the ECL chemiluminescent reagent was coated on the membrane surface, and after waiting for 1-2 minutes, the KwikQuant digital imager was used for imaging.

[0089] 1.11 In situ detection of intracellular Cyt c by DA-MXene

[0090] HL-60 cells were cultured at 1×10 4 The cells were seeded in 6-well plates at a density of 100 cells / mL and incubated in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO. 2 To study the release of Cyt c induced by DA-MXene, 100 μg mL -1 DA-MXene was used to treat cells for different time points (0, 1, 5, and 10 hours). Mitochondrial apoptosis was assessed using the mitochondrial probe MitoTracker Green, and Apt GQD Cyt c release was monitored. After incubation at different time points, cells were collected and live cell imaging was performed using confocal laser scanning microscopy (CLSM). The excitation / emission wavelengths of MitoTracker Green were 490 / 523 nm, and Apt GQD The excitation / emission wavelengths are 594 / 640 nm.

[0091] 2. Results and Discussion

[0092] 2.1 Principle of DA-MXenes nanoindicator for mitochondrial apoptosis and simultaneous detection of Cyt c

[0093] Based on the advantages of 2D MXene nanosheets in surface modification and optical absorption, this embodiment designed a MXene-based composite nanoindicator for DHA targeted delivery and mitochondrial damage-induced Cyt c detection. Figure 1 As shown, MXene nanosheets were first modified with hyaluronic acid (MXene-HA for short) to target the CD44 receptor overexpressed on the surface of AML cells and improve its stability in a physiological environment. Subsequently, DHA was loaded onto MXene-HA through π-π stacking and hydrogen bonding to obtain DHA@MXene-HA.

[0094] In this example, MXene is used as a receptor and organic fluorescent GQD is used as a donor to design a highly biocompatible FRET nanoprobe. GQD is modified on the Cyt c aptamer (Apt) to obtain a carbon-based quantum dot-Cyt c aptamer complex (Apt GQD ), then Apt GQD It is loaded onto MXene-HA through hydrogen bonding and van der Waals forces to form DHA / Apt GQD@MXene nanocomposite particles (DA-MXene nanoindicators for short). In the initial state, the fluorescence of GQD is quenched by MXene through the FRET effect, resulting in "SIGNAL OFF". When DA-MXenes are engulfed by AML cells, DHA is released from the MXene surface, inducing mitochondrial dysfunction. Along with mitochondrial apoptosis, Cyt c is released into the cytoplasm and interacts with Apt GQD Combined, Apt GQD Stay away from MXene, thus triggering the turning on of the fluorescence signal.

[0095] 2.2 Preparation and characterization of DA-MXene nanoindicators

[0096] Two-dimensional MXene Ti was synthesized by hydrofluoric acid etching and aqueous exfoliation. 3 C 2 Nanosheets. The synthesized nanosheets exhibit a clear two-dimensional thin layer morphology ( Figure 2 A), with a thickness ranging from 2 to 3 nanometers ( Figure 2 B and C), the hydrated particle size distribution of the nanosheets before and after ultrasonic treatment is shown in Figure 2 D in the figure. Subsequently, the MXene nanosheets were modified with hyaluronic acid (HA) molecules (MXene-HA) via EDC / NHS coupling chemistry. Next, DHA was loaded onto MXene-HA (DHA@MXene-HA) via π-π stacking and hydrogen bonding interactions. High-performance liquid chromatography (HPLC) analysis showed that the optimal loading amount of DHA on MXene was 90.35% (mass ratio), with a linear range of 0.001 to 0.046 mM ( Figure 2 Middle E).

[0097] GQDs were labeled with a DNA aptamer (Apt) that recognizes cytochrome c (Cyt c) through EDC / NHS chemical reaction, thereby obtaining Apt GQD Then, Apt GQD The hydrogen bonds and van der Waals interactions between MXene molecules were loaded onto DHA@MXene-HA to form DHA / Apt GQD @MXene-HA nanocomposite (abbreviated as DA-MXene nanoindicator). Due to the optical absorption ability of MXene, MXene quenches the fluorescence of GQD through Fourier transform spectroscopy resonance energy transfer (FRET), resulting in a "SIGNAL OFF" state. Ultraviolet-visible spectroscopy (UV-vis) shows that MXene has a broad absorption band in the range of 200 to 800nm, which is consistent with the Apt GQD The fluorescence emission (emission peak at 641nm) overlaps well ( Figure 2 (F). Compared with the unmodified MXene, the zeta potential of DA-MXene shifted to -41.2 mV, which may provide better dispersibility for the nanocomposite ( Figure 2 G in the figure). The total X-ray photoelectron spectra of MXene and DA-MXene are as follows: Figure 2 The peaks of the O1s and C1s spectra of the DA-MXene sample shift to lower binding energy values, which are 529.9 eV and 283.3 eV, respectively. Figure 2 This may be due to the coordination effect between HA and MXene.

[0098] The X-ray diffraction (XRD) patterns of MXene and DA-MXene show that DA-MXene retains the crystal characteristics of MXene, indicating that the modification of HA mainly occurs on the surface of MXene and has no significant effect on its internal crystal structure ( Figure 2 In order to study the stability of DA-MXene in physiological environment, the dispersion stability of MXene, DHA@MXene-HA and DA-MXene in water, PBS, RPMI 1640 medium and PBS buffer containing 20% ​​FBS was tested in this example. Figure 2 Middle K). After 24 h of incubation, MXene exhibited obvious agglomeration, while DA-MXene maintained excellent dispersion.

[0099] Glycolytic metabolism, hypoxia, and insufficient blood perfusion of AML cells lead to the formation of an acidic tumor microenvironment. In an acidic environment, the protonation effect weakens the hydrogen bonding force on the surface of MXene nanosheets and triggers changes in the distribution of π-π electron clouds, leading to the release of DHA. In addition, DHA containing enol and alcohol groups has better solubility in acidic environments. DA-MXene released about 69.24% of DHA at pH 5.5, showing a higher release rate than that at pH 7.4 and pH 6.3 ( Figure 2 Middle L).

[0100] 2.3DA-MXene nanoindicator for quantitative detection of cytochrome C

[0101] exist Figure 3 In this example, the sensing ability of DA-MXene nanoindicator for cytochrome C detection was quantitatively analyzed by fluorescence photoluminescence spectroscopy. Quenching efficiency is an important basis in FRET detection. In order to optimize the quenching efficiency (Qe), this example used Apt GQD With different concentrations of MXene (0-600 μg mL -1 )adaptation( Figure 3 (A).

[0102] The results show that Apt GQD The fluorescence intensity (FI) of MXene gradually decreases with the increase of MXene concentration, and the maximum quenching efficiency (Qe) reaches 91.74% ( Figure 3 This ensures that the subsequent detection has low background noise. This example uses PL spectroscopy to quantitatively analyze the DA-MXene nanoindicator to 0-10 U mL -1 Detection of Cyt c in the concentration range ( Figure 3 C and D). In the range of 0 to 0.5 U mL -1 In the concentration range of Apt GQD There is a good linear relationship between the recovered fluorescence intensity (FI) and the Cyt c concentration (R 2 =0.951)( Figure 3 (E). The limit of detection (LOD) of Cyt c assay was calculated to be 0.049 U mL -1 , meeting the concentration requirement for intracellular Cyt c monitoring. LOD was calculated by the formula 3σ / slope, where σ represents the standard deviation of the blank signal.

[0103] 2.4 Targeted uptake of DA-MXene nanoindicators by AML cells

[0104] The CD44 receptor is a glycoprotein highly expressed on the surface of acute myeloid leukemia (AML) cells, and hyaluronic acid is composed of glucuronic acid and N-acetylglucosamine. The CD44 molecule contains a binding site rich in glycosaminoglycans (GAGs), which strongly binds to the repeating disaccharide units of hyaluronic acid (HA). Here, this embodiment designs HA-modified DA-MXene nanoindicators to specifically identify HL-60 acute myeloid leukemia (AML) cells.

[0105] This example further studies its ability to selectively target acute myeloid leukemia (AML) cells. First, the expression of native CD44 in acute leukemia cell lines HL-60 and THP-1 and chronic leukemia cell line K562 was examined. Flow cytometry analysis showed that the percentages of CD44+ cells in HL-60, THP-1 and K562 cells were 94.82%, 58.12% and 0.29%, respectively ( Figure 4 Figure (A) confirmed the specific overexpression of CD44 on the surface of HL-60 acute myeloid leukemia (AML) cells. Subsequently, HL-60 cells were treated with HA, HA+DA-MXene, and DA-MXene for 24 hours, and the Cyt c signal was detected by confocal fluorescence imaging. Significant Cyt c expression was detected in the DA-MXene nanoindicator treatment group, which was expressed as Apt GQD Signal( Figure 4 The quantitative fluorescence intensity (FI) of the DA-MXene treatment group was 1975.5±386.9 au, which was 1.8 times that of the HA+DA-MXene treatment group ( Figure 4 Middle C).

[0106] 2.5DA-MXene nanoindicator induces mitochondrial apoptosis in HL-60 cells

[0107] DHA can induce a decrease in mitochondrial membrane potential and an increase in membrane permeability, thereby triggering the release of Cyt c and inducing apoptosis through the mitochondrial apoptosis pathway.

[0108] First, this example used CCK-8 detection and flow cytometry to evaluate the overall cell viability of HL-60 cells after DA-MXene treatment. Figure 5 Figure A shows that the viability of HL-60 cells gradually decreased with the increase of DA-MXene concentration, with 100 μg mL -1 DA-MXene can reduce cell viability to 20.31%. However, the viability of K562 and THP-1 cells was only reduced to 48.89% and 36.63%, respectively, indicating that DA-MXene has better targeted cytotoxicity against HL-60 cells ( Figure 5 In flow cytometry analysis, 100 μg mL-1 of DA-MXene induced about 90.3% of cell apoptosis ( Figure 5 Middle C). Figure 5 Center D shows the Calcein / AM staining results of HL-60 cells treated with or without DA-MXene, where DA-MXene caused 87.67% cell death.

[0109] Mitochondrial membrane potential (ΔΨm) refers to the potential difference between the two sides of the mitochondrial membrane. The decrease in ΔΨm is accompanied by an increase in membrane permeability. In this example, JC-1 (mitochondrial membrane potential probe) was used to detect the ΔΨm of HL-60 cells treated with control group, DHA, DHA@MXene and DA-MXene. HL-60 cells treated with DAMXene showed the lowest mitochondrial membrane potential, with a red / green fluorescence intensity ratio of 23.67% ( Figure 6 In addition, this example analyzed mitochondrial activity and intracellular ROS release to confirm the level of mitochondrial apoptosis in HL-60 cells treated with DHA, DHA@MXene, and DA-MXene, and to determine whether these treatments successfully induced mitochondrial apoptosis and cell damage. DA-MXene triggered the highest level of ROS release, reaching 6.1 times that of the control group ( Figure 6 (B and D).

[0110] Along with mitochondrial apoptosis, cytochrome c (Cyt c) is released into the cytoplasm and induces apoptosis by activating the proteases of the caspase family. In this example, Western blotting (WB) was used to detect the key apoptosis-related proteins Bax and Bcl-2, with GAPDH as the internal reference. The results are shown in Figure 2. Figure 6 As shown in E and F. Figure 6 As shown in Figures E and F, DA-MXene induced a stronger mitochondrial-cytochrome c-mediated apoptosis effect, with the Bax / Bcl-2 expression ratio of 3.64, which was significantly higher than that of the control group.

[0111] 2.6 Targeted chemotherapy and simultaneous in situ monitoring of Cyt c in living AML cells

[0112] In AML cells, the release of cytochrome c (Cyt c) triggered by mitochondrial apoptosis can accurately and rapidly reflect the level of cell apoptosis, making Cyt c an important target for evaluating the efficacy of chemotherapy. Therefore, this embodiment designs a multifunctional nanoindicator based on the FRET effect for targeted chemotherapy and simultaneous in situ monitoring of mitochondrial apoptosis ( Figure 7 (A).

[0113] First, the secretion of Cyt c in vitro after DA-MXene-induced mitochondrial apoptosis was studied. HL-60 cells were incubated with 100 μg mL-1 of DA-MXene for 0, 1, 5, and 10 hours, respectively. Significant loss of mitochondrial structure and disruption of membrane morphology were observed at 5 hours, indicating that DA-MXene caused effective damage to the cells (P<0.001, compared with the negative control group). Next, the ability of DA-MXene to monitor Cyt c in vitro was evaluated. HL-60 cells incubated with DA-MXene exhibited time-dependent changes in fluorescence intensity, with significant red fluorescence recovery detected after 1 hour (491.08±123.81 au) and 1824.99±226.27 au ( Figure 7 (B and C). The nanoindicator detected the Cyt c red signal almost in the synchronous time window of mitochondrial structural destruction, and there was no need to introduce other detection reagents again to increase unnecessary interference.

[0114] 3. Conclusion

[0115] The present invention develops a multifunctional bio-nano indicator for in situ monitoring of mitochondrial apoptosis and the release of Cyt c in active acute myeloid leukemia (AML) cells. The high complexity and heterogeneity of AML cell populations result in significant differences in the responses of different cells to treatment. Current strategies for monitoring tumor efficacy rely mainly on the analysis of large sample average signals. Although this method can provide an overall trend of drug action, it may mask the differences in drug response between different cells, making it difficult to identify unresponsive resistant cells or over-responsive sensitive cells. This may lead to misjudgment of the therapeutic effect and miss the best time to adjust the treatment regimen. Therefore, monitoring the chemotherapy effect of AML at the single cell level is crucial for accurately evaluating the treatment outcome. This multifunctional bio-nano indicator overcomes the limitations of current large sample tumor efficacy monitoring methods.

[0116] Due to the good biocompatibility of HA-modified MXene, the bio-nano indicator can track the dynamic cell behavior of single living cells from drug treatment to marker release in real time. This method provides a new technical approach for studying the dynamic behavior of heterogeneous cells and may improve the effect of personalized treatment of AML. This innovative method has great potential in future drug development and personalized cancer diagnosis. This study still needs more clinical validation in the future.

[0117] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A nanometer indicator for inducing mitochondrial apoptosis and monitoring Cyt c release in real time, characterized in that: The nano indicator comprises: a two-dimensional material modified with hyaluronic acid, and a chemotherapeutic drug and a carbon-based quantum dot-Cyt c aptamer complex loaded on the two-dimensional material modified with hyaluronic acid; The carbon-based quantum dot-Cyt c aptamer complex comprises: carbon-based quantum dots and Cyt c aptamers modified on the carbon-based quantum dots.

2. The nano indicator according to claim 1, characterized in that: The hyaluronic acid is modified on the two-dimensional material through an esterification reaction.

3. The nano indicator according to claim 1, characterized in that: The chemotherapy drug is loaded on the two-dimensional material modified with hyaluronic acid through π-π stacking and / or hydrogen bonding.

4. The nano indicator according to claim 1, characterized in that: The carbon-based quantum dot-Cyt c aptamer complex is loaded on the two-dimensional material modified with hyaluronic acid through hydrogen bonds and / or van der Waals forces.

5. The nano indicator according to claim 1, characterized in that: The Cyt c aptamer is an amino-modified Cyt c aptamer, and the Cyt c aptamer is modified on the carbon-based quantum dots through an amide condensation reaction.

6. The nano indicator according to claim 1, characterized in that: The two-dimensional material is MXene; And / or, the chemotherapy drug is dihydroartemisinin; And / or, the carbon-based quantum dots are graphene quantum dots; And / or, the Cyt c aptamer is 5'-NH2-CCGTGTCTGGGGCCGACCGGCGCAT TGGGTACGTTGTTGC-3'.

7. A method for preparing a nano indicator according to any one of claims 1 to 6, characterized in that: Includes steps: providing a dispersion of the two-dimensional material; Adding hyaluronic acid to the dispersion of the two-dimensional material to react and obtain a two-dimensional material modified with hyaluronic acid; Adding chemotherapy drugs to the dispersion of the two-dimensional material modified with hyaluronic acid, mixing and stirring, to obtain a two-dimensional material loaded with chemotherapy drugs and modified with hyaluronic acid; The carbon-based quantum dot-Cyt c aptamer complex is added to the dispersion liquid of the two-dimensional material loaded with chemotherapy drugs and modified with hyaluronic acid to react and obtain the nano indicator.

8. The preparation method according to claim 7, characterized in that: The mass ratio of the hyaluronic acid to the two-dimensional material is (1-10):1; the mass ratio of the two-dimensional material modified with hyaluronic acid to the chemotherapy drug is (0.25-1.25):1; the ratio of the two-dimensional material loaded with chemotherapy drugs and modified with hyaluronic acid to the carbon-based quantum dot-Cyt c aptamer complex is (50-200) mg:1 μmol.

9. The method for preparing the nano indicator according to claim 8, characterized in that: The method for preparing the carbon-based quantum dot-Cyt c aptamer complex comprises the steps of: Adding an amide condensation reagent to the carbon-based quantum dot solution, stirring, and then adding a Cyt c aptamer to react to obtain the carbon-based quantum dot-Cyt c aptamer complex; Wherein, the ratio of the carbon-based quantum dots to the Cyt c aptamer is (0.5-10) g:1 mmol.

10. Use of the nano-indicator according to any one of claims 1 to 6 in the preparation of a drug for treating acute myeloid leukemia.

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