A method for realizing tumor cell targeted therapy by microRNA and APE1 enzyme double-triggered nanoparticle self-assembly and application thereof

Abstract

The application discloses a kind of microRNA and APE1 enzyme double trigger's nanoparticle self-assembly realizes the method and application of tumor cell targeted therapy, belong to biomedical technology field.The application designs hairpin probe H1 and H2, and H1 and H2 are loaded on photo-thermal carrier material.H1 contains AP site, can be cut by APE1;H2 is labeled with fluorescent donor group and fluorescent quenching group.In the tumor cell of the simultaneous presence of APE1 and miRNA-21, hybridization chain reaction (HCR) is started, forms long-chain polymer, produces photo-thermal effect under near-infrared light source, to ablate tumor.Meanwhile, the double detection of APE1 enzyme and miRNA-21 can also be realized.The method can realize the diagnosis and treatment integration of lung cancer, liver cancer, breast cancer and other APE1 / miRNA-21 high expression tumors.

Description

Technical Field

[0001] This invention relates to a method and application of tumor cell targeted therapy achieved by the self-assembly of nanoparticles triggered by both microRNA and APE1 enzyme, belonging to the biomedical field. Background Technology

[0002] Photothermal therapy (PTT), with its minimally invasive nature and excellent spatiotemporal controllability, has become a promising new strategy for cancer treatment and has attracted widespread attention from the academic community. Compared with traditional treatment modalities such as surgery, chemotherapy, and radiotherapy, PTT has significant advantages such as ease of operation, minimal trauma, high safety, and short treatment cycle. Its core mechanism lies in the efficient conversion of the light energy of the excitation laser into local heat energy through non-radiative transitions using photothermal agents (PTAs), thereby achieving specific killing of tumor cells.

[0003] The basic process of photothermal therapy involves delivering a photothermal agent to tumor tissue, then radiating the agent with light of a specific wavelength to raise the local temperature of the tumor cells, thereby killing them. Currently, there are many types of photothermal agents used in photothermal therapy, mainly including photosensitive polymers, metal nanoparticles, carbon nanomaterials, and metal sulfides. Among them, gold nanoparticles (AuNPs) have advantages such as simple preparation process, good biocompatibility, easy surface modification, and controllable photothermal conversion efficiency. By modifying the surface of spherical gold nanoparticles with specific nucleic acid functional units (DNA), the base complementary pairing interaction can mediate the controllable and orderly self-assembly of the gold nanoparticles, thereby endowing the system with diverse photothermal response characteristics and achieving selective regulation of the photothermal conversion effect.

[0004] However, existing photothermal therapy technologies based on gold nanoparticles still face many problems that urgently need to be solved: (1) The contradiction between excitation wavelength and tissue penetration depth. Most AuNPs photothermal reagents have excitation wavelengths concentrated in the visible light region (500~600 nm), but the light in this band has weak penetration ability into human skin and deep tissues, which seriously limits the full utilization of photothermal conversion efficiency and affects the treatment effect of deep tumors.

[0005] (2) The dilemma of “enrichment-penetration” caused by size effect. Although small-sized AuNPs (<20 nm) can easily penetrate tissues and accumulate in tumor sites, their absorption peaks are still located in the visible light region, resulting in poor tissue penetration and easy expulsion by tumor cells; the absorption peaks of large-sized AuNPs (100~200 nm) can be red-shifted to the near-infrared region (about 800 nm), significantly improving tissue penetration, but the enrichment efficiency in tumor tissues is significantly reduced.

[0006] (3) Insufficient targeting and systemic toxicity. Currently, the accumulation of AuNPs in tumor sites mainly relies on passive targeting through the EPR effect (high permeability and retention effect). This method cannot completely avoid the distribution of AuNPs in normal tissues, which can easily cause damage to normal cells and trigger systemic toxicity. Although simple intratumoral injection can increase the local concentration in the tumor in the short term, it cannot be used for metastatic or deep tissue tumors and carries the risk of tumor spread.

[0007] For example, Chinese invention patent CN113637669B discloses a photothermal-gene combined therapy system based on DNA self-assembly technology. This system utilizes miR-21 overexpressed in tumor cells as a trigger chain to induce a cascade hybridization reaction in three hairpin structures (H1, H2, H3) modified on the surface of gold nanoparticles, leading to AuNP aggregation and generating a photothermal effect only under 660 nm laser irradiation. Simultaneously, siRNA targeting VEGF is generated in situ during the assembly process, achieving photothermal-gene combined therapy for cervical cancer. This research provides a new approach for combined tumor therapy. However, this technical solution still uses 660 nm near-infrared light irradiation, which has limited penetration into deep tissues, making it difficult to apply to the treatment of deep tumors or metastases. Furthermore, this system relies entirely on miR-21 as a triggering factor. Although miR-21 is overexpressed in various tumors, it also has a certain expression level in some normal tissues or inflammatory states, which may lead to non-specific activation, causing photothermal damage to normal tissues or off-target gene silencing effects.

[0008] It is evident that existing photothermal-gene combined therapy systems based on DNA self-assembly still have significant shortcomings in terms of tissue penetration depth. Therefore, developing a novel photothermal-gene combined therapy platform with longer excitation wavelengths (such as near-infrared II), more precise targeting, controllable aggregation behavior, simple preparation, and anti-metastasis capabilities has important clinical significance and application value.

[0009] APE1 participates in DNA repair and redox regulation. DNA oxidative damage caused by various endogenous and exogenous factors is mainly repaired through the BER pathway, and APE1 is a core functional enzyme in this pathway that cleaves the AP site. Studies have shown that APE1 is highly expressed in various malignant tumor cells, with expression levels significantly higher than in normal tissue cells. This difference in expression provides an ideal triggering target for tumor-specific therapy.

[0010] miR-21 is an oncogenic miRNA that is widely overexpressed in various malignant tumors. It promotes tumor cell proliferation, invasion, and drug resistance by targeting tumor suppressor genes such as PTEN and PDCD4. Based on the difference in miR-21 expression between tumor cells and normal cells, using miR-21 as a smart response trigger has become an important strategy for building a tumor-specific diagnosis and treatment system. Summary of the Invention

[0011] The present invention aims to construct a smart self-assembly system of gold nanoparticles triggered by tumor-specific depurinyl / depyrimidine endonuclease 1 (APE1 enzyme) and microRNA, for the purpose of achieving selective and precise photothermal therapy of tumors.

[0012] APE1 enzyme exists in small amounts in the cytoplasm. In the early stages of treatment, the APE1 enzyme in the cytoplasm can cleave the AP site, thereby synergistically initiating a hybridization chain reaction (HCR) with miRNA-21. The expression of miRNA-21 differs significantly between tumor cells and normal cells, exhibiting low or no expression in normal cells, while being highly expressed in tumor cells. At the same time, HCR can achieve a highly efficient response to trace amounts of biomarkers through a chain amplification effect.

[0013] To address the problems of the existing technology, this invention provides a composition for integrated tumor diagnosis and treatment based on hybridization chain reaction and photothermal agents. The composition comprises two hairpin probes, H1 and H2, loaded onto gold nanoparticles. H1 contains an AP site, which can be recognized and cleaved by the APE1 enzyme, exposing a single-stranded region. This single-stranded region of H1 can achieve base complementarity with miRNA-21. The single-stranded region of H2 can bind complementaryly to the unwound single-stranded region of H1. Furthermore, the newly generated single-stranded fragment after the hybridization and ring opening of H2 and H1 can also complementarily pair with the single-stranded region of H1. After determining the probe base sequence, the complementary double-stranded regions of the hairpin structure H2 are labeled with a fluorescent group Cy3 (fluorescent donor, excitation wavelength 466 nm) and a quencher group BHQ2 (fluorescent acceptor), respectively, while H1 is not fluorescently labeled. When H2 maintains its hairpin conformation and the complementary double strands are not opened, the fluorescence energy emitted by Cy3 is efficiently absorbed by the neighboring BHQ2, resulting in no significant fluorescence signal output from the system. When APE1 enzyme and miRNA-21 are added to the system, HCR can be initiated. APE1 enzyme, acting as an initiator, first cleaves H1 to form a single-stranded region. Then, miRNA-21 binds to the single-stranded region of H1 and opens its hairpin structure, exposing a new single-stranded region. This region can further complement the single-stranded region of H2, thereby opening the hairpin structure of H2 and exposing another new single strand. This newly generated single strand can complement the initial initiation site sequence, thus initiating the next round of hybridization and ring-opening.

[0014] This invention provides an integrated tumor diagnosis and treatment composition based on hybridization chain reaction, the composition comprising: a first hairpin probe and a second hairpin probe; The first hairpin probe H1 and the second hairpin probe H2 are respectively modified on the photothermal therapy material; In one embodiment of the present invention, the nucleotide sequence of the first hairpin probe H1 contains a depurinyl / depyrimidine site that can be recognized and cleaved by the APE1 enzyme, and the single-stranded region exposed after H1 is cleaved by the APE1 enzyme is completely complementary to the sequence of miRNA-21.

[0015] In one embodiment of the present invention, the hairpin structure double-stranded region of the second hairpin probe H2 is marked with a fluorescent donor group and a fluorescent quencher group, respectively.

[0016] The composition uses the tumor cell-specific APE1 enzyme and highly expressed miRNA-21 as endogenous dual trigger molecules. It precisely responds to APE1 enzyme and miRNA-21 signals in the tumor cell microenvironment to initiate hematopoietic regeneration (HCR). Specifically, the APE1 enzyme cleaves the AP site on H1, exposing the region that miRNA-21 can recognize. Subsequently, the highly expressed miRNA-21 in tumor cells acts as an initiator of HCR, driving the orderly and controllable self-assembly of dispersed gold nanoparticles within the cell, forming appropriately sized and structurally stable nanoaggregates. Photothermal therapy (PTT) induces tumor cell apoptosis mainly through the mitochondrial-mediated endogenous apoptosis pathway. Under near-infrared light irradiation, the photothermal agent converts light energy into local heat energy, rapidly increasing the intracellular temperature of tumor cells, triggering significant thermal stress and intracellular oxidative stress. This leads to a decrease in mitochondrial membrane potential, opening of the membrane permeability transition pore, and the release of pro-apoptotic factors such as cytochrome C into the cytoplasm. The released cytochrome C assembles with Apaf-1 and Caspase-9 to form apoptotic bodies, which sequentially activate downstream Caspase-9 and executive caspase-3 / 7, ultimately cleaving intracellular substrate proteins and degrading DNA, inducing typical apoptotic morphological changes and programmed cell death in tumor cells. Simultaneously, high fever can further amplify apoptotic signals by activating the p53 signaling pathway, upregulating pro-apoptotic proteins such as Bax, and downregulating anti-apoptotic proteins such as Bcl-2, thus achieving highly efficient clearance of tumor cells.

[0017] In one embodiment of the present invention, the fluorescent donor group is selected from Cy3, FAM, HEX, and ROX, and the fluorescent quencher group is selected from BHQ1, BHQ2, BHQ3, and Dabcyl.

[0018] Preferably, the fluorescent donor group is Cy3 and the fluorescent quencher group is BHQ2.

[0019] In one embodiment of the present invention, the photothermal therapy material is selected from at least one of indocyanine green (ICG), IR780, gold nanoparticles, gold nanorods, polydopamine, Prussian blue nanoparticles, and graphene quantum dots; preferably, gold nanoparticles.

[0020] In one embodiment of the present invention, the nucleotide sequence of H1 is shown in SEQ ID NO.2.

[0021] In one embodiment of the present invention, the nucleotide sequence of H2 is shown in SEQ ID NO.3.

[0022] The present invention also provides a system for targeted therapy of tumor cells, the system comprising the composition and a light irradiation device.

[0023] In one embodiment of the present invention, the light source emitted by the illumination device is a near-infrared light source.

[0024] In one embodiment of the present invention, the wavelength of the near-infrared light source is 700~1000 nm. After the gold nanoparticles are irradiated by the near-infrared light source, they realize the conversion of light energy into heat energy, thereby increasing the local temperature of the tumor and killing tumor cells by utilizing the photothermal effect.

[0025] The wavelength of the near-infrared light source is preferably 750~850 nm, more preferably 800~808 nm.

[0026] The present invention also provides a dual detection method for detecting APE1 enzyme and miRNA-21 expression levels using the aforementioned composition, comprising the following steps: (a) Anneal the first hairpin probe H1 and the second hairpin probe H2 respectively; (b) The annealed H1 and H2 from (a) were mixed with the sample to be tested in a reaction buffer and incubated. The change in fluorescence intensity of the reaction system was then detected.

[0027] When both APE1 enzyme and miRNA-21 are present in the sample to be tested, they jointly trigger a hybridization chain reaction, causing H1 and H2 to hybridize and assemble into a long-chain polymer, which spatially separates the fluorescent donor and the quencher group, and the fluorescence signal is restored. When at least one of APE1 enzyme or miRNA-21 is missing in the sample to be tested, the hybridization chain reaction cannot be started and no obvious fluorescence signal is restored.

[0028] In one embodiment of the present invention, H1 and H2 are diluted with a buffer solution and then annealed.

[0029] In one embodiment of the present invention, H1 and H2 are diluted to 20 μM using HEPES buffer. In one embodiment of the present invention, the annealing is performed by heating to 95 °C and holding for 10 min, followed by natural cooling to room temperature, so that H1 and H2 can fully form a stable hairpin structure.

[0030] In one embodiment of the present invention, in (b), the final concentrations of H1 and H2 in the reaction system are each independently 10 nM to 1 μM, preferably 100 nM to 300 nM.

[0031] In one embodiment of the present invention, the incubation temperature is 25~42 ℃, preferably 37 ℃; the incubation time is 30~180 min, preferably 90~120 min.

[0032] In one embodiment of the present invention, the sample to be tested is a cell lysate, tissue extract, serum, plasma, or an in vitro reaction mixture.

[0033] In one embodiment of the present invention, the method further includes using a real-time quantitative PCR instrument or a fluorescence spectrophotometer to dynamically monitor the fluorescence intensity, and quantitatively detecting the concentration of miRNA-21 based on the linear relationship between the fluorescence intensity and the concentration of the target miRNA-21.

[0034] The method combines direct detection and indirect deduction to achieve precise characterization and semi-quantitative analysis of the expression abundance of APE1 enzyme and miRNA-21. Specifically, this dual detection method works as follows: First, by utilizing changes in the Cy3 fluorescence signal of a specific molecular probe, the expression differences of APE1 enzyme and miRNA-21 within cells are directly captured. Changes in fluorescence intensity visually determine the relative expression levels of APE1 enzyme and miRNA-21, enabling direct detection and quantitative analysis of the target miRNA. Second, by precisely quantifying the apoptosis and death ratio of tumor cells, the expression levels of endogenous APE1 enzyme and miRNA-21 are inversely deduced and assessed. Combined with differences in the physiological responses of cells during treatment, the expression abundance of APE1 enzyme and miRNA-21 is indirectly characterized, completing a semi-quantitative determination of their expression levels. This dual detection method enables dual verification through "direct detection + indirect deduction." It can rapidly reflect the real-time expression status of APE1 enzyme and miRNA-21 through changes in fluorescence signals, and indirectly verify the expression differences of APE1 enzyme and miRNA-21 through quantitative analysis of cell apoptosis and death ratios. This allows for a comprehensive and accurate assessment of the expression levels of the two tumor markers, APE1 enzyme and miRNA-21, providing a reliable basis for optimizing subsequent treatment plans and evaluating efficacy.

[0035] The present invention also provides a kit for dual detection of APE1 enzyme and miRNA-21, comprising the aforementioned tumor diagnostic and therapeutic composition.

[0036] In one embodiment of the present invention, the kit further comprises a reaction buffer.

[0037] The present invention also provides for the use of the composition, or the system, or the kit in (a) or (b) below: (a) Products for detecting or assessing the level of cancer treatment; (b) Products for the treatment or adjuvant treatment of tumors.

[0038] In one embodiment of the present invention, the tumor is a tumor with upregulated expression levels of APE1 and / or miRNA-21.

[0039] In one embodiment of the present invention, the tumor includes one or more of the following: lung cancer, liver cancer, colorectal cancer, stomach cancer, breast cancer, pancreatic cancer, ovarian cancer, esophageal cancer, prostate cancer, bladder cancer, cervical cancer, glioma, and osteosarcoma.

[0040] [Beneficial Effects] 1. Highly targeted, precise and safe treatment: Targeting the APE1 enzyme in the cytoplasm and highly expressed miRNAs in tumor cells. 21 is an endogenous dual trigger factor that initiates HCR self-assembly only in the tumor microenvironment; in normal cells, it is triggered by miRNAs. Low APE1 levels make it difficult to trigger a response, enabling selective photothermal killing of tumor cells while significantly reducing toxic side effects on normal tissues and improving treatment safety. Furthermore, as tumor cell apoptosis progresses, the APE1 enzyme in the cell nucleus undergoes significant subcellular localization changes, migrating in large quantities from the nucleus to the cytoplasm. Accumulated APE1 in the cytoplasm can further trigger and enhance the HCR response, amplifying biological effects through this regulatory pathway and thus significantly improving overall therapeutic efficacy.

[0041] 2. Significantly improved photothermal therapy efficacy: Small-sized gold nanoparticles in APE1 enzyme and miRNA Under 21 induction, they are ordered to assemble into aggregates, which greatly improves the near-infrared light absorption and photothermal conversion efficiency. This effectively solves the problems of poor tissue penetration and limited photothermal effect of single small-diameter AuNPs, and can efficiently convert light energy into heat energy to precisely kill tumor cells.

[0042] 3. Dual anti-tumor effects of apoptosis and immune activation: PTT induces tumor cell apoptosis through the mitochondrial-mediated endogenous apoptosis pathway and can activate the p53 signaling pathway to amplify the apoptosis effect; at the same time, it induces immunogenic cell death, releases related signaling molecules to activate the body's anti-tumor immunity, and achieves a synergistic effect of local treatment and systemic immune response.

[0043] 4. Predicting therapeutic effects by relative fluorescence intensity: The relative fluorescence intensity in cells and living tissues can be used to directly reflect the activation level of the hybridization chain reaction mediated by APE1 enzyme and miRNA-21 and the degree of intracellular self-assembly of gold nanoparticles. Higher fluorescence intensity indicates better nanoparticle aggregation effect and stronger near-infrared photothermal conversion ability at the tumor site. Based on this, the cell apoptosis efficiency and in vivo tumor inhibition effect can be effectively predicted.

[0044] 5. Controllable reaction and stable system: The hybridization chain reaction can be continuously repeated, and the self-assembly process of nanoparticles is ordered and spatiotemporally controllable; AuNPs are prepared by a cryogenic method. DNA enhances DNA modification efficiency and stability, ensuring the uniformity and reliability of probes and nanosystems.

[0045] 6. Mature experimental methods with wide applicability: The preparation of gold nanoparticles, probe design, HCR reaction and assembly conditions are all standardized, and the operation is simple and highly reproducible; it can not only be used for tumor-targeted photothermal therapy, but also provide a stable research platform for the study of APE1 enzyme and miRNA function, the analysis of tumor development and apoptosis molecular mechanisms.

[0046] 7. Providing new ideas for novel intelligent nano-formulations: Constructing a stimulus-responsive self-assembly system provides a novel design strategy and technical support for developing a new generation of precise, efficient, and low-toxicity tumor photothermal therapy agents (AuNPs@HCR), which has good potential for scientific research and clinical translation. Attached Figure Description

[0047] Figure 1 These are TEM images of AuNPs and AuNPs@HCR of the present invention; Figure 2 To demonstrate the feasibility of solution phase analysis for this invention: fluorescence curve of H1 + H2 + miRNA-21; Figure 3 Agarose gel analysis for HCR; Figure 4 This is the particle size distribution curve of the AuNPs of this invention; Figure 5 The zeta potential diagrams for AuNPs and AuNPs@HCR are shown. Figure 6The UV absorption spectrum of AuNPs@HCR and the absorbance change curve after AuNPs@HCR undergoes HCR self-assembly; Figure 7 Cell viability was determined by CCK-8 assay under both light and dark conditions for cells treated with different concentrations of AuNPs@HCR. Figure 8 Images of confocal fluorescence uptake by cells after incubation with AuNPs@HCR for 0, 2, 4, and 6 h; Figure 9 Images of co-localized fluorescence after AuNPs@HCR incubation; Figure 10 Double staining for apoptosis in AuNPs@HCR drugs under different light conditions; Figure 11 To monitor changes in intracellular Cy3 fluorescence in live cells after treatment with different doses of AuNPs@HCR drug using fluorescence microscopy; Figure 12 For in vivo anti-tumor therapy; Figure 13 H&E staining analysis of tumor tissue. Detailed Implementation

[0048] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0049] The raw materials used in the following examples: Reagents: PBS buffer was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). N,N-Dimethylformamide (DMF) was purchased from Aladdin Reagents Ltd. (Shanghai, China). Lysosomal staining (Lyso Tracker Red), Cell Viability Assay Kit-8 (CCK-8), Hoechst 33342, and Calcein / PI Apoptosis Double Staining Kit were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). HEPES buffer and DNA probes were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The water used in the experiment was ultrapure water (18.2 MΩ). Glass-bottomed culture dishes were purchased from Cellvis (Hangzhou, China). Opti-MEM was purchased from Gibco (USA). Cell culture medium (DMEM), fetal bovine serum (FBS), trypsin-EDTA, streptomycin, penicillin, and phosphate-buffered saline (PBS) were purchased from Vicente Biotechnology Co., Ltd. (Nanjing, China). APE1 endonuclease was purchased from Geneplus Ltd. (Beijing, China). Mice were purchased from Vital River Laboratory Animal Co., Ltd. (Beijing).

[0050] Example 1: Synthesis of AuNPs and AuNPs@HCR and their TEM images 1. Synthesis of AuNPs: Prepare aqua regia (concentrated hydrochloric acid and concentrated nitric acid mixed at a volume ratio of 3:1) in a fume hood. Immerse the 200 mL double-necked flask, magnetic stir bar, glass stopper, and condenser used for AuNPs synthesis in the aqua regia for 15 min. After immersion, rinse the flask three times with plenty of deionized water and air dry it for later use to avoid impurities in the instrument affecting the purity of AuNPs synthesis.

[0051] Preparation of the reaction system: Add 98 mL of ultrapure water and 2 mL of chloroauric acid (50 mM, HAuCl4) aqueous solution to a pretreated double-necked flask to obtain a final concentration of 1 mM HAuCl4 aqueous solution. Place a magnetic stir bar in the flask, connect a condenser, and seal the other neck of the flask with a glass stopper. The chloroauric acid aqueous solution is prepared as follows: Weigh 2.059 g of chloroauric acid tetrahydrate (HAuCl4). Dissolve 4H2O in a small amount of ultrapure water in a beaker and stir until completely clear; transfer to a 100 mL volumetric flask and dilute to the mark with pure water; store in the dark at 4 ℃.

[0052] Heating and Reaction Initiation: Place the double-necked flask in a silicone oil bath and heat with stirring until the solution is under reflux. Once the reflux is stable, remove the glass stopper and quickly add 12 mL of 34 mM sodium citrate solution. Immediately replace the glass stopper and continue stirring under reflux at 200 rpm. After adding the sodium citrate solution, the solution color will gradually change from light yellow to dark red within 1 minute, indicating that AuNPs have been formed. Continue reflux for 30 minutes to ensure the reaction is complete. Then, turn off the heating device and continue stirring until the solution cools to room temperature, at which point stirring should be stopped.

[0053] Purification: The synthesized AuNPs mixture was transferred into an ultrafiltration tube and centrifuged at 4000 rpm for 8 min. The supernatant was discarded, and the precipitate was resuspended in ultrapure water. The washing was repeated 4 times to complete the purification of AuNPs and obtain AuNPs with a particle size of 13 nm.

[0054] 2. Synthesis of AuNPs@HCR: Thiol activation: Mix 0.8 μL of TCEP solution (dissolve 1.0 mg of TCEP in 8 μL of 60 nM acetate buffer) with 10 μL of H1 solution (or H2 solution, 100 μM) and incubate at 25 °C for 1 h to complete the thiol activation of H1 and H2.

[0055] Table 1 Sequence

[0056] Note: -SH and HS- represent DNA-modified thiol groups, which are linked to the gold surface through Au-S covalent coordination bonds; / idSp / represents the AP site, which can be cleaved by APE1.

[0057] Transmission electron microscopy (TEM) characterization: by Figure 1 It can be clearly seen that the prepared AuNPs, AuNPs-H1, and AuNPs-H2 have uniform particle size distribution and good dispersibility. The TEM images show that the AuNPs and AuNPs@HCR particles have a particle size of about 13 nm.

[0058] 3. Measurement of hydrated particle size and potential (ζ) Disperse appropriate amounts of AuNPs and AuNPs@HCR in 1 mL of water, respectively. After ultrasonic dispersion, immediately take 1 mL of sample and place it in a particle size measuring dish. Test the hydrated particle size of the particles at 25 °C using a nanoparticle size analyzer.

[0059] AuNPs and AuNPs@HCR were dispersed in 1 mL of water, and immediately placed in a potential measuring dish after ultrasonic dispersion. The potential ζ of the particles was measured using a Zeta potential analyzer.

[0060] The hydrodynamic size distribution of AuNPs is as follows: Figure 4 As shown. Figure 5 As shown, the zeta potentials of AuNPs and AuNPs@HCR are -2.6 ± 0.5 mV and -6.7 ± 1.1 mV, respectively, indicating that the surface of AuNPs remains negatively charged after encapsulating DNA.

[0061] Example 2: Fluorescence Spectroscopy Analysis The occurrence of hybridization chain reaction (HCR) is determined by changes in fluorescence signal. The specific method is as follows: 1. In vitro HCR reaction AuNPs-H1 (10 μL, 100 μM) and AuNPs-H2 (10 μL, 100 μM) prepared in Example 1 were placed in HEPES buffer (40 μL, 20 mM HEPES, 100 mM NaCl, 5 mM MgCl2, pH 7.4) to prepare a final concentration of 10 μM stock solution. The solution was heated to 95°C and maintained for 5 minutes, and then cooled to room temperature.

[0062] In a 2000 μL reaction system, add the following: annealed AuNPs-H1 (final concentration 10 nM), annealed AuNPs-H2 hairpin (final concentration 10 nM), APE1 enzyme (2 U / mL), and miRNA-21 (set final concentration gradient: 0, 1, 3, 5, 10 nM). Make up the reaction buffer to 2000 μL, incubate for 2 h, and then place in a shaker at room temperature and shake continuously at 200 rpm for 90 min.

[0063] 2. Results Analysis After incubation, 500 μL of the reaction solution was transferred to a quartz dish. Fluorescence spectra were detected using a fluorescence spectrometer (excitation wavelength 550 nm, excitation and emission slit widths both 10.0 nm, scan rate 800 nm / min). The 5′ end of H2 was labeled with Cy3, and the 3′ end with BHQ2. When H2 maintained its hairpin conformation and the complementary double strands were not opened, the fluorescence energy emitted by Cy3 was efficiently absorbed by the neighboring BHQ2, resulting in no significant fluorescence signal output. The HCR reaction was initiated upon the addition of APE1 enzyme and miRNA-21. APE1 cleaved H1 to form a single-stranded region. Subsequently, miRNA-21 bound to the single-stranded region of H1 and opened its hairpin structure, exposing a new single-stranded region. This region could further complement the single-stranded region of H2, thereby opening the hairpin structure of H2. At this point, Cy3 and BHQ2 spatially separated, and fluorescence was restored. The fluorescence intensity was positively correlated with the degree of HCR reaction.

[0064] A curve was plotted with miRNA-21 concentration on the x-axis and relative fluorescence intensity on the y-axis to evaluate the triggering efficiency of miRNA-21 for HCR.

[0065] Table 2 Concentration ratio of each substance in the final detection system

[0066] like Figure 2 As shown, with miRNA As the concentration of 21 increases, the fluorescence signal gradually intensifies, and the response intensity is similar to that of miRNA. The concentration of miRNA-21 showed a good linear relationship. This indicates that the reaction system has good detection sensitivity and response specificity for miRNA-21, and can accurately distinguish targets at different concentrations.

[0067] Example 3: HCR gel electrophoresis analysis Agarose gels were prepared using 1× TBE buffer (3% by mass).

[0068] H1 (10 μL, 10 μM) and H2 (10 μL, 10 μM) were placed in HEPES buffer (20 mM HEPES, 100 mM NaCl, 5 mM MgCl2, pH 7.4), heated to 95 °C and maintained for 5 minutes, and then cooled to room temperature.

[0069] In a 200 μL reaction system, add the following: annealed H1 (final concentration 1 μM), annealed H2 hairpin (final concentration 1 μM), APE1 enzyme (2 U / mL), and miRNA-21 (final concentration 0.2 μM), respectively. Add HEPES reaction buffer to a final volume of 200 μL, incubate for 2 h, and then place in a shaker at room temperature and shake continuously at 200 rpm for 90 min.

[0070] The reaction mixture was added to 2 μL of loading buffer (6 ×), and agarose gel electrophoresis was performed at 110 V for 60 min. Images were acquired using a BioRad imaging system.

[0071] The reaction mixture was subjected to agarose gel electrophoresis, and the results are as follows: Figure 3 As shown.

[0072] Example 4: Ultraviolet Absorption Spectroscopy First, H1 was cleaved with APE1 enzyme (2 U / mL). Then, AuNPs-H1, AuNPs-H2, and miRNA-21 reactants were precisely prepared in centrifuge tubes and placed in a shaker at room temperature at 200 rpm for 90 min to promote a hybridization chain reaction of the three substances in the solution phase, thereby triggering the self-assembly of nanoparticles. The specific steps are as follows: AuNPs-H1 (10 μL, 100 μM) and AuNPs-H2 (10 μL, 100 μM) prepared in Example 1 were placed in HEPES buffer (20 mM HEPES, 100 mM NaCl, 5 mM MgCl2, pH 7.4) to prepare a final concentration of 10 nM stock solution. The solution was heated to 95 °C and maintained for 5 minutes, and then cooled to room temperature.

[0073] In a 200 μL reaction system, add the following: annealed AuNPs-H1 (final concentration 1 nM), annealed AuNPs-H2 hairpin (final concentration 1 nM), APE1 enzyme (2 U / mL), and miRNA-21 (final concentration 0.2 nM), and then add HEPES reaction buffer to a final volume of 200 μL.

[0074] Before the reaction (0 h) and after the reaction (1 h), 60 µL of the reaction solution was placed in a cuvette and the absorption spectrum of the solution was measured in the wavelength range of 400–800 nm.

[0075] like Figure 6 As shown, before the HCR reaction, AuNPs@HCR exhibited a strong characteristic absorption peak at only 520 nm, corresponding to the surface plasmon resonance absorption of dispersed small-sized gold nanoparticles. However, after HCR assembly was triggered by APE1 enzyme and miRNA-21, the UV-Vis absorption spectrum of the system changed significantly. The absorption peak at 520 nm was significantly reduced, and a new absorption peak appeared at around 800 nm, showing a significant redshift of the absorption peak.

[0076] The above structure indicates that, under the action of hybridization chain reaction mediated by APE1 enzyme and miRNA-21, dispersed AuNPs@HCR undergo orderly self-assembly and form nanoaggregates, resulting in a significant redshift of its surface plasmon resonance absorption peak. This successfully achieves optical absorption regulation from the visible light region to the near-infrared light region, laying a good optical foundation for subsequent near-infrared light-mediated tumor photothermal therapy.

[0077] Example 5: AuNPs@HCR Cytotoxicity Assay HeLa cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin at 37 °C in a 5% CO2 incubator.

[0078] HeLa cells were seeded in 96-well plates (1 × 10⁶ cells per well). 4 (Number of cells). After culturing for 24 h, the cells were washed with PBS. Fresh cell culture medium containing different mass concentrations (0, 5, 10, 20, 30, 50, 70, 100 μg / mL) of AuNPs@HCR (a mixture of AuNPs-H1 and AuNPs-H2 at equal concentrations) was added. After incubation for 4 hours, the cells were washed to remove any uninternalized nanomaterials.

[0079] The cells were divided into two groups: one group was not exposed to light (0 min) and the other group was exposed to an 808 nm laser (1 W / cm²). 2 Irradiate for 5 min. After culturing for another 24 h, add 100 μL of fresh culture medium containing 10% CCK-8 and incubate for 2 h. Measure the absorbance at 450 nm using a microplate reader.

[0080] Formula for calculating cell growth viability: Cell Viability (%) = [(As-Ab) / (Ac-Ab)] × 100%, where, As represents the OD value of the treatment group, Ac represents the OD value of the control group, and Ab represents the OD value of the blank well (n = 3).

[0081] To verify the antitumor therapeutic effect of AuNPs@HCR, the cytotoxicity of AuNPs@HCR was first verified at the cellular level, and the killing effect of AuNPs@HCR on tumor cells was analyzed using CCK-8 assay. Figure 7 As shown, when the light is applied for 5 minutes (808nm, 1 W / cm²), 2 As the dose of AuNPs@HCR increased, cell viability continued to decline. However, in the 0 min light-illuminated group, cells incubated with AuNPs@HCR showed no cytotoxicity, confirming that AuNPs@HCR self-assembles under the induction of APE1 enzyme and miRNA-21 in cells. The AuNPs@HCR that self-assembles and aggregates in light-illuminated cells undergoes PTT to effectively kill cancer cells. Furthermore, the cytotoxicity of AuNPs@HCR increases significantly with increasing concentration, demonstrating that the AuNPs@HCR aggregates after light irradiation has a concentration-dependent tumor cell killing effect.

[0082] When the concentration of AuNPs@HCR reached 100 μg / mL, the cell survival rate in the light-illuminated group decreased to about 8.27%, further verifying the system's efficient tumor cell killing ability under near-infrared light irradiation. Meanwhile, the high cell viability in the unilluminated group also indicates that the material itself has good biocompatibility and will not produce non-specific toxic side effects on normal cells.

[0083] Example 6: Cellular Uptake Analysis Confocal fluorescence microscopy (CLSM): HeLa cells were seeded in a 35 mm glass-bottomed confocal dish (1×10⁻⁶). 5 Cells were incubated overnight in Opti-MEM medium (cells / plate) until 80%–90% confluence was achieved. Free DNA (150 nM of H1 and Cy3-labeled H2) or AuNPs@HCR (50 µg / mL, equal volumes of AuNPs-H1 25 µg / mL and AuNPs-H2 25 µg / mL, where H2 was labeled with Cy3 only) were added separately and incubated for 0, 2, 4, and 6 h. Cells were then washed with pre-warmed PBS to remove uninternalized DNA and NPs.

[0084] Cell nuclei were stained with Hoechst 33342 (5 μg / mL) for 5 min.

[0085] The fluorescence signals were analyzed using laser confocal scanning microscopy. The Cy3 collection channel was located at 560 nm–610 nm (λex = 552 nm) and the Hoechst 33342 collection channel was located at 425 nm–475 nm (λex = 405 nm).

[0086] The ability of AuNPs@HCR to co-deliver DNA probes to tumor cells was investigated using confocal imaging. Results are as follows: Figure 8 As shown, CLSM images of HeLa cells incubated with Cy3-labeled AuNPs@HCR for 4 h showed strong fluorescence, while only negligible fluorescence was observed in HeLa cells incubated with DNA for 4 h.

[0087] Example 7: Colocalization Analysis Colocalization of AuNPs@HCR was investigated at the subcellular level using CLSM.

[0088] HeLa cells were seeded in 35 mm glass-bottom confocal dishes and incubated overnight to achieve 80-90% confluence. HeLa cells were then incubated in Opti-MEM medium for 4 hours with an equal mixture of AuNPs@HCR (50 µg / mL, AuNPs-H1 25 µg / mL, and AuNPs-H2 25 µg / mL, where H2 was labeled only with Cy3). Cells were then washed with pre-warmed PBS to remove uninternalized NPs. Lysosomes were stained red with Lyso Tracker Red (100 nM) for 20 min. Cell nuclei were stained with Hoechst 33342 (5 μg / mL, 5 min). The Lyso Tracker Red channel collects 650-700 nm (λex = 638 nm), the Cy 3 channel collects 560 nm~610 nm (λex = 552 nm), and the Hoechst 33342 channel collects 425 nm~475 nm (λex = 405 nm).

[0089] To further investigate the ability of AuNPs@HCR to escape from endosomes after internalization by tumor cells, subcellular localization analysis was performed. Results are as follows: Figure 9 As shown, colocalization studies indicate that the Cy3 fluorescence signal of the DNA probe and the lysozyme fluorescence signal overlapped poorly after 4 h of co-incubation, with a Pearson correlation coefficient of 0.47, suggesting that a sufficient number of AuNPs@HCRs escaped from the lysosomes into the cytoplasm.

[0090] Example 8: Apoptosis Double Staining HeLa cells were used at a density of 8 × 10⁸ cells per well. 4Cells were seeded into confocal culture dishes and cultured at 37 °C, 5% CO2, and 21% O2 (normal cell culture) for 12 hours. Subsequently, AuNPs@HCR (0, 20, 50, and 100 µg / mL) were added, and HeLa cells were incubated in Opti-MEM medium for 4 hours. All cells were washed three times with PBS. The cells were then incubated under near-infrared light (808 nm, 1 W / cm²). 2） Cells were irradiated for 5 min, with three replicates per group (n = 3). Cell viability was assessed using the Calcein / PI Cell Viability and Cytotoxicity Assay Kit with dual-fluorescence staining.

[0091] Preparation of Calcein AM / PI detection working solution: Calcein AM (1000×, 1µL), PI (1000×, 1µL), detection buffer (1mL). Incubate at 37 ℃ in the dark for 30 min. After staining, rinse three times with PBS, add OPT medium, and perform confocal microscopy imaging.

[0092] like Figure 10 As shown, the apoptosis rate was only 2.3 ± 1.1% in the cell-free DNA group, 20.3 ± 4.1% in the AuNPs@HCR (20 µg / mL) group, 54.3 ± 2.2% in the AuNPs@HCR (50 µg / mL) group, and 92.3 ± 5.1% in the AuNPs@HCR (100 µg / mL) group. Flow cytometry apoptosis detection results showed that the apoptosis level under different treatment conditions exhibited a significant concentration-dependent difference.

[0093] Example 9: Fluorescence microscopy monitoring of intracellular Cy3 fluorescence changes in live cells after treatment with different doses of AuNPs@HCR drug HeLa cells were seeded into 35 mm confocal culture dishes and incubated overnight until 80%–90% confluence was achieved. Then, free DNA, AuNPs@HCR (20 µg / mL), and AuNPs@HCR (50 µg / mL) were added, and the cells were incubated in Opti-MEM medium for 4 h. Cells were then washed with pre-warmed PBS to remove uninternalized DNA and NPs. All groups were treated with near-infrared light (808 nm, 1 W / cm²). 2） Irradiate for 5 minutes.

[0094] The fluorescence signal of Cy3 was analyzed using confocal laser scanning microscopy. The Cy3 channel ranged from 560 nm to 610 nm (λex = 552 nm), while the Hoechst 33342 channel ranged from 425 nm to 475 nm (λex = 405 nm). Figure 11As shown, no fluorescence signal was observed in the cell-free DNA group, while a weak fluorescence signal was observed in the AuNPs@HCR (20 µg / mL) group, and a strong fluorescence signal was observed in the AuNPs@HCR (50 µg / mL) group. Quantitative fluorescence analysis using ImageJ revealed that, compared to the cell-free DNA group, the fluorescence intensity of the AuNPs@HCR (20 µg / mL) and AuNPs@HCR (50 µg / mL) groups increased by 1.5-fold and 3.2-fold, respectively.

[0095] The above results indicate that with gradually increasing drug concentration, the HCR reaction triggered by APE1 enzyme and miRNA-21 is continuously enhanced, resulting in the release of a large number of quenched fluorescent groups on the probe and a significant increase in intracellular relative fluorescence intensity. Simultaneously, the enhanced fluorescence signal further reflects the improved nanoparticle assembly efficiency and photothermal therapeutic effect, thereby leading to a simultaneous and significant increase in the apoptosis rate of tumor cells. These results directly confirm that changes in intracellular fluorescence can effectively indicate the HCR response process and the degree of therapeutic response, and that with increasing drug dosage, the photothermal killing effect mediated by nanoparticle assembly is continuously enhanced, ultimately leading to a significant increase in the apoptosis rate.

[0096] Example 10: In vivo antitumor therapy The experimental animals used in this embodiment were 7-week-old female BALB / c Nude mice (weighing 18 g to 22 g). The experimental animals, sterile bedding, and sterile feed were all provided by Beijing Vital River Co., Ltd. All mice were housed in an SPF-grade animal facility, with environmental parameters strictly controlled at a temperature of 20 ℃ ± 1 ℃, relative humidity of 60% ± 5%, and a 12 h / 12 ​​h light / dark cycle. The experimental protocol strictly followed the "Guidelines for Ethical Review of Laboratory Animal Welfare" formulated by the Chinese Association for Laboratory Animal Science and was reviewed and approved by the Laboratory Animal Welfare and Ethics Committee of Cangzhou Medical College (SYDW-20260130).

[0097] 1. AuNPs@HCR Antitumor Level Detection To construct a xenograft tumor model, HeLa cells (1 × 10⁻⁶) in the logarithmic growth phase were used. 7 Each tumor cell was resuspended in 50 μL of PBS and thoroughly mixed with an equal volume (50 μL) of matrix gel. The mixture was then injected subcutaneously into the right axilla of BALB / c nude mice using a syringe. Tumor volume was monitored daily, and when the tumor volume reached 100 mm², the tumor was counted. 3In this study, tumor-bearing mice were randomly divided into four groups (n = 4 / group), and each group received an intratumoral injection of PBS (control), cell-free DNA (25 nmol / kg), AuNPs@HCR (5 mg / kg), or AuNPs@HCR (10 mg / kg), respectively, to treat the tumor. All groups received a single intratumoral injection. 1.5 h post-injection, all groups were treated with an 808 nm wavelength laser (1 W / cm²). 2 Irradiate for 20 minutes. Measure the tumor's long diameter (L) and short diameter (W) every 2 days, and calculate the volume V = L × W. 2 / 2, and the tumor volume of mice was monitored and recorded over 14 days.

[0098] like Figure 12 The experimental results showed that, because the cell-free DNA (25 nmol / kg) group did not contain AuNPs, the cell-free DNA could not inhibit tumor growth, and the tumor volume reached 1398 mm² at the treatment endpoint. 3 The AuNPs@HCR (5 mg / kg) and AuNPs@HCR (10 mg / kg) groups significantly inhibited tumor growth, and the final tumor volume was negatively correlated with the in vivo dosage. Specifically, the AuNPs@HCR (10 mg / kg) group achieved a tumor volume of only 291 mm² at the treatment endpoint. 3 It was much lower than the control group (1547 mm). 3 This provides direct evidence that the nanosystem also exhibits concentration-dependent antitumor effects in living organisms.

[0099] The body weight of mice was monitored daily throughout the treatment process. No significant abnormal fluctuations in body weight were observed in any group of mice, further demonstrating that the treatment regimen had low systemic toxicity and good in vivo biosafety in mice.

[0100] 2. H&E staining analysis of tumor tissue After 14 days of treatment, mice were euthanized, and tumor tissue was collected and fixed in 4% paraformaldehyde solution. The tumor tissue was further embedded in paraffin, sectioned, and fixed onto glass slides. The antitumor effect of AuNPs@HCR was evaluated using hematoxylin and eosin (H&E) staining.

[0101] like Figure 13 As shown, the AuNPs@HCR (5 mg / kg) and AuNPs@HCR (10 mg / kg) groups induced a certain degree of nuclear death and apoptosis, while the PBS group and the cell-free DNA (25 nmol / kg) group did not induce apoptosis, indicating that AuNPs@HCR can induce apoptosis after laser irradiation.

[0102] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A tumor diagnostic and therapeutic composition based on hybridization chain reaction, characterized in that, The composition comprises: a first hairpin probe and a second hairpin probe; The nucleotide sequence of H1 is shown in SEQ ID NO.2; The nucleotide sequence of H2 is shown in SEQ ID NO.3; The first hairpin probe H1 and the second hairpin probe H2 are respectively modified on the photothermal therapy material; The nucleotide sequence of the first hairpin probe H1 contains a depurinyl / depyrimidine site that can be recognized and cleaved by the APE1 enzyme, and the single-stranded region exposed after H1 is cleaved by the APE1 enzyme is completely complementary to the sequence of miRNA-21. The hairpin structure of the second hairpin probe H2 is marked with fluorescent donor groups and fluorescent quencher groups, respectively.

2. The integrated tumor diagnosis and treatment composition according to claim 1, characterized in that, The fluorescent donor group is selected from Cy3, FAM, HEX, and ROX, and the fluorescent quencher group is selected from BHQ1, BHQ2, BHQ3, and Dabcyl. Preferably, the fluorescent donor group is Cy3 and the fluorescent quencher group is BHQ2.

3. The integrated tumor diagnosis and treatment composition according to claim 1, characterized in that, The photothermal therapy material is selected from at least one of indocyanine green, IR780, gold nanoparticles, gold nanorods, polydopamine, Prussian blue nanoparticles, and graphene quantum dots; preferably, it is gold nanoparticles.

4. A system for targeted therapy of tumor cells, characterized in that, The tumor cell targeted therapy system includes the composition of any one of claims 1 to 3 and an illumination device whose emission source is a near-infrared light source.

5. The system according to claim 4, characterized in that, The wavelength of the near-infrared light source is 700~1000 nm, preferably 750~850 nm, and more preferably 800~808 nm.

6. A dual detection method for detecting the expression levels of APE1 enzyme and miRNA-21 using the integrated tumor diagnostic and therapeutic composition according to any one of claims 1 to 3, characterized in that, Includes the following steps: (a) Anneal the first hairpin probe H1 and the second hairpin probe H2 respectively; (b) The annealed H1 and H2 from (a) were mixed with the sample to be tested in a reaction buffer and incubated. The change in fluorescence intensity of the reaction system was then detected.

7. The method according to claim 6, characterized in that, The sample to be tested is a cell lysate, tissue extract, serum, plasma, or an in vitro reaction mixture.

8. A kit for dual detection of APE1 enzyme and miRNA-21, characterized in that, The tumor diagnosis and treatment composition comprising any one of claims 1 to 3.

9. The use of the tumor diagnostic and therapeutic composition according to any one of claims 1 to 3, or the tumor cell targeted therapy system according to any one of claims 4 or 5, or the kit according to claim 8, in the following (a) or (b): (a) Products for detecting or assessing the level of cancer treatment; (b) Products for the treatment or adjuvant treatment of tumors.

10. The application according to claim 9, characterized in that, The tumor is a tumor in which the expression levels of APE1 and / or miRNA-21 are upregulated; Preferably, the tumor includes one or more of the following: lung cancer, liver cancer, colorectal cancer, stomach cancer, breast cancer, pancreatic cancer, ovarian cancer, esophageal cancer, prostate cancer, bladder cancer, cervical cancer, glioma, and osteosarcoma.

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