A ros-responsive metal nucleic acid coordination nanodelivery system and a preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a ROS-responsive metal nucleic acid coordination nano drug delivery system, which comprises a nano inner core, the nano inner core is obtained by self-assembly of polyvalent metal ions and functional nucleic acid molecules, and the outer layer of the nano inner core is modified with a ROS-responsive double-carboxyl thio-ketone ligand and a hydrophilic polymer stabilizer; the polyvalent metal ions can inhibit the activation of an NF-kappa B inflammatory pathway, the functional nucleic acid molecules can specifically silence an enteritis pathogenic target gene GAL3ST1, and the ROS-responsive double-carboxyl thio-ketone ligand contains a thio-ketone sensitive skeleton. The nano drug delivery system of the application is specifically disintegrated under a high ROS microenvironment at an acute radiation enteritis inflammation site, releases the polyvalent metal ions and the functional nucleic acid molecules, realizes triple synergistic treatment of 'ROS removal, gene silencing and anti-inflammatory pathway inhibition', and the preparation method of the application is simple, has good repeatability, has excellent biocompatibility, and can accurately target treatment of acute radiation enteritis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a ROS-responsive metal-nucleic acid coordination nanoparticle drug delivery system, its preparation method, and its application. Background Technology

[0002] During radiotherapy for pelvic and abdominal malignant tumors, intestinal tissue is highly susceptible to ionizing radiation damage, leading to acute radiation enteritis, one of the most common complications in on-site radiotherapy. Acute radiation enteritis mainly manifests as intestinal mucosal congestion and edema, erosion and ulceration, inflammatory cell infiltration, and intestinal barrier damage, severely affecting patients' radiotherapy compliance and quality of life. Currently, there is no radical treatment. Existing clinical treatment regimens are mostly symptomatic, including anti-inflammatory drugs, mucosal protectants, and antioxidants, which have significant technical limitations: 1) These drugs lack intestinal inflammation targeting, resulting in large distribution in normal tissues after systemic administration, low treatment efficiency, and a high risk of toxic side effects; 2) The intestinal inflammatory microenvironment contains high levels of ROS-mediated oxidative stress damage and a continuously activated NF-κB inflammatory pathway, making it impossible for conventional drugs to simultaneously achieve antioxidant, pathway inhibition, and targeted gene intervention; 3) siRNA-based gene drugs have poor stability, are easily degraded by nucleases in the body, are difficult to deliver efficiently to intestinal inflammatory sites, exhibit significant off-target effects, and cannot achieve precise silencing of pathogenic genes.

[0003] GAL3ST1 is a key pathogenic gene regulating intestinal inflammation and mucosal repair impairment in acute radiation enteritis. Specific silencing of GAL3ST1 can effectively reverse intestinal pathological damage. However, there is currently a lack of efficient, stable, and responsive GAL3ST1 siRNA delivery vectors that address the inflammatory microenvironment, hindering the controlled release and synergistic treatment of gene drugs. Furthermore, single anti-inflammatory, antioxidant, or gene therapy modalities offer limited efficacy and are insufficient to halt the multidimensional pathological progression of acute radiation enteritis. Therefore, developing a nanomedicine delivery system that responds to the intestinal inflammatory microenvironment and integrates ROS clearance, inflammatory pathway inhibition, and targeted gene silencing is a crucial technological direction for overcoming the clinical treatment bottlenecks of acute radiation enteritis. Summary of the Invention

[0004] The purpose of this invention is to provide a ROS-responsive metal-nucleic acid coordination nanoparticle drug delivery system, aiming to achieve a triple synergistic treatment of acute radiation enteritis through oxidative stress clearance, inflammatory pathway inhibition, and target gene silencing.

[0005] To achieve the above objectives, the present invention provides a ROS-responsive metal-nucleic acid coordination nanoparticle drug delivery system, comprising a nanocore, wherein the nanocore is obtained by self-assembly of a multivalent metal ion and a functional nucleic acid molecule, the outer layer of the nanocore chelates a ROS-responsive dicarboxylated thioacetate ligand, and the outer layer of the nanocore is modified with a hydrophilic polymeric stabilizer; the multivalent metal ion is selected from divalent or trivalent transition metal ions, and the multivalent metal ion is used to specifically inhibit the overactivation of the NF-κB inflammatory signaling pathway; the functional nucleic acid molecule is selected from any one or more combinations of DNA, mRNA, siRNA, shRNA, miRNA, antisense oligonucleotide ASO, and CRISPR guide gRNA, and the functional nucleic acid molecule is used to target and silence the pathogenic gene of radiation enteritis; the ROS-responsive dicarboxylated thioacetate ligand contains a thioacetate-sensitive backbone.

[0006] Furthermore, the multivalent metal ions are selected from Zn. 2+ Fe 2+ Fe 3+ Cu 2+ Mn 2+ Co 2+ Mg 2+ Ga 3+ Ca 2+ One or more of them.

[0007] Furthermore, the functional nucleic acid molecule is a small interfering RNA siGAL3ST1 that targets the GAL3ST1 gene.

[0008] Furthermore, the ROS-responsive dicarboxylated thioacetate ligand is 2,2'-[propane–2,2-dimethylbis(thio)]diacetic acid.

[0009] Furthermore, the hydrophilic polymeric stabilizer is selected from one or more of polyvinylpyrrolidone, polyethylene glycol, hyaluronic acid, and dextran.

[0010] Furthermore, the particle size of the nano-drug delivery system is 100–300 nm.

[0011] This invention also provides a method for preparing a ROS-responsive metal-nucleic acid coordination nanoparticle drug delivery system, comprising the following steps: S1. Nanocore self-assembly: Mixing a multivalent metal ion solution with a functional nucleic acid molecule solution induces coordination self-assembly of the bases of the multivalent metal ions and the functional nucleic acid molecules to obtain the start-up mixed system; S2, Annealing and Curing and Isothermal Coordination: After water bath annealing of the mixed system, it was incubated at an isothermal temperature to obtain a mixed system I containing a structurally stable nanocore; S3. Surface modification of dual ligands: Under light-proof and mild stirring conditions, ROS-responsive dicarboxylic thioacetate ligand solution and hydrophilic polymer stabilizer solution were added dropwise to mixture system I to obtain mixture system II containing target nanoparticles.

[0012] Furthermore, it also includes the following steps: S4. Purification: Mixture system II is subjected to ultracentrifugation or ultrafiltration to obtain a precipitate. The precipitate is resuspended in enzyme-free water to obtain a purified ROS-responsive metallo-nucleic acid coordination nanoparticle drug delivery system.

[0013] Furthermore, in step S1, the solvents for both the polyvalent metal ion solution and the functional nucleic acid molecule solution are DEPC-free enzyme-free water, the concentration of the polyvalent metal ion solution is 6–15 mM, the concentration of the functional nucleic acid molecule solution is 1–10 mg / mL, the mixing method is vortex oscillation, and the mixing time is 6–20 min.

[0014] Furthermore, in step S2, the water bath annealing temperature is 45–85°C, the heating time is 3–6 min, the constant temperature incubation temperature is 37–40°C, and the constant temperature incubation time is 6–10 h.

[0015] Furthermore, in step S3, the stirring speed is 600–700 rpm, the stirring time is 12–15 h, and the reaction temperature is room temperature.

[0016] Further, in step S4, mixture system II is ultracentrifuged at 60000×g~70000×g and 4~5℃ for 15~25 min to obtain precipitate, and the precipitate is resuspended and stored at 4℃ in the dark.

[0017] The ROS-responsive metal-nucleic acid coordination nanoparticle drug delivery system described in this invention can be used in the preparation of drugs for treating radiation-induced intestinal injury, including acute radiation enteritis induced by abdominal / pelvic radiotherapy, chronic radiation-induced intestinal mucosal damage, and radiation-induced intestinal ulcers.

[0018] This invention utilizes the multi-coordination properties of multivalent metal ions. On one hand, it strongly coordinates with the bases of functional nucleic acid molecules, tightly encapsulating the functional nucleic acid drug and resisting its degradation by nucleases in vivo. On the other hand, it forms metal-carboxyl coordination with the carboxyl groups at both ends of a ROS-responsive dicarboxylated thioacetate ligand, constructing an outer ROS-responsive functional shell. A hydrophilic polymer stabilizer is added to enhance the dispersibility and biocompatibility of the nanoparticles. In the low-ROS environment of normal intestinal tissue, the nanoparticle structure of this invention is highly stable with no drug leakage. In the high-ROS lesion area of ​​acute radiation enteritis, the thioacetate backbone in the ROS-responsive dicarboxylated thioacetate ligand on the outer layer of the nanoparticle is specifically oxidized and broken by ROS, disrupting the nanoparticle coordination network and triggering in-situ disintegration of the nanoparticles. This precisely releases the functional nucleic acid molecule and multivalent metal ions, demonstrating strong targeting. Thus, the released functional nucleic acid molecule (siGAL3ST1) specifically silences the GAL3ST1 pathogenic gene, blocking the pathological process of enteritis; the released multivalent metal ions (Zn... 2+ It effectively inhibits the excessive activation of the NF-κB inflammatory pathway, suppresses the intestinal inflammatory cascade from the source, and achieves a triple synergistic therapeutic effect.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Precise ROS response and site-specific drug release: Utilizing the high ROS characteristics of radiation enteritis lesions, the drug is released specifically at the inflammatory site through the ROS sensitivity of the TK thioacetate backbone, avoiding drug leakage from normal tissue and reducing toxic side effects.

[0020] 2. Triple synergistic treatment mechanism: TK clears local ROS and polyvalent metal ions (Zn). 2+ It inhibits the NF-κB inflammatory pathway, silences pathogenic target genes through functional nucleic acid molecules (siGAL3ST1), and significantly improves the treatment effect of enteritis through a synergistic effect of oxidative stress regulation, inflammatory pathway inhibition, and gene-targeted intervention.

[0021] 3. Highly efficient nucleic acid protection: Multivalent metal ions with multifunctional coordination can stably encapsulate functional nucleic acid molecules, effectively resisting degradation by nucleases in vivo, and significantly improving the in vivo stability and duration of action of nucleic acid drugs. 4. Excellent biocompatibility: This invention uses biosafe zinc ions, medical-grade polyvinylpyrrolidone (PVP), and small molecule ligand TK, resulting in low biotoxicity.

[0022] 5. Simple and controllable process: The nano-drug delivery system in this invention has a simple preparation method, mild conditions, good reproducibility, and is easy to scale up for production. Attached Figure Description

[0023] Figure 1This is a schematic diagram of the assembly of Zn-siGAL3ST1@TK nanoparticles in this invention; Figure 2 The particle size distribution and transmission electron microscopy (TEM) morphology of the Zn-siGAL3ST1@TK nanoparticles in this invention are shown in the images (a: particle size distribution; b: TEM morphology). Figure 3 The following are physicochemical and functional characterization diagrams of the Zn-siGAL3ST1@TK nanoparticles in this invention: (a: bar chart of DPPH free radical scavenging ability of each group of samples under different X-ray irradiation doses; b: transmission electron microscopy image of Zn-siGAL3ST1@TK nanoparticles after X-ray irradiation; c: cumulative release curve of zinc ions from the nanocarrier under different X-ray irradiation doses). Figure 4 The following graphs illustrate the functional verification of the nano-drug delivery system in this invention at the in vitro cellular level in protecting against radiation-induced intestinal injury: (a: Bar graph showing cell viability of NCM460 and IEC-6 intestinal cells at different concentrations of carrier; b: Statistical graph of intestinal cell survival rate under different X-ray irradiation doses; c: Quantitative graph showing internalization and uptake of Zn-siGAL3ST1@TK nanocarrier by cells at different incubation times; d: Bar graph showing relative expression of GAL3ST1 gene in each group of cells; e: Bar graph showing quantitative detection of sulfatide content in each group of cells; f: Graph showing NF-κB pathway-related proteins in each group; g: Bar graph showing relative quantitative expression of phosphorylated IκBα protein; h: Bar graph showing relative quantitative expression of phosphorylated p65 protein; i: Relative expression of inflammatory factor IL-6 mRNA in each group of cells; j: Relative expression of inflammatory factor IL-1β in each group of cells.) relative mRNA expression level; k, l: ROS (reactive oxygen species) fluorescence staining map and fluorescence intensity quantitative bar chart of each group of cells; m: γH2AX (DNA damage marker) fluorescence staining map of each group of cells (scale bar 20 μm); n: γH2AX fluorescence intensity quantitative statistical bar chart). Figure 5Figure 1 shows the in vivo efficacy evaluation results of the nano-drug delivery system in this invention for alleviating acute radiation enteritis (a: schematic diagram of the experimental process of in vivo drug administration, X-ray irradiation and sample collection in C57BL / 6 mice; b: statistical curve of weight change in mice in each group over a 6-day period; c: statistical curve of weight loss rate in mice in each group; d: box plot of DAI score for intestinal disease activity index in mice in each group; e: box plot of pathological damage score for intestinal tissue in mice in each group; f: bar plot of relative expression level of GAL3ST1 gene in mouse intestinal tissue; g: bar plot of sulfatide content in mouse intestinal tissue; h: H&E pathological staining of mouse colon tissue; i: bar plot of IL-6 inflammatory factor concentration in mouse intestinal tissue; j: bar plot of IL-1β inflammatory factor concentration in mouse intestinal tissue; k: bar plot of ROS fluorescence staining in colon tissue; l: bar plot of γH2AX fluorescence staining for DNA damage marker in colon tissue; m: ROS immunofluorescence staining in colon tissue; n: γH2AX DNA damage immunofluorescence staining in colon tissue). Figure 6 The following are in vivo biosafety evaluation diagrams of the nano-drug delivery system in this invention: (a: Schematic diagram of animal experiment process for mouse enema administration and sample collection; b: 6-day weight change curves of mice in the control group and the treatment group; c: Box plot of colon length statistics of mice in the two groups; d: H&E pathological sections of major organs of mice (heart, liver, spleen, lung, and kidney); e: Bar chart of serum liver function index ALT; f: Bar chart of serum liver function index AST; g: Bar chart of kidney function index BUN; h: Bar chart of kidney function index CREA; i: Bar chart of blood routine red blood cell count; j: Bar chart of blood routine platelet count; k: Bar chart of blood routine white blood cell count; l: Bar chart of blood routine thrombus cell percentage). Detailed Implementation

[0024] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. Specific examples are given below to illustrate the present invention in detail. It should also be understood that the following examples are only for specific illustration of the present invention and should not be construed as limiting the scope of protection of the present invention. Non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention are all within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are only examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0025] Example 1 This embodiment provides a method for preparing a ROS-responsive metal-nucleic acid coordination nanoparticle drug delivery system, the main process of which is as follows: Figure 1 As shown, the entire process employs aseptic and RNase-free techniques to avoid nucleic acid degradation and impurity contamination, ensuring the stability of nanoparticle assembly and the efficacy of the drug. The specific steps are as follows: (1) Preparation of sterile raw material solution DEPC-treated sterile ultrapure water was used as the solvent to prepare solutions for each component. All solutions were filtered through a 0.22 μm sterile filter membrane to remove impurities and bacteria, including nucleases and particulate matter. Specific preparation parameters were as follows: 6 mM sterile zinc sulfate aqueous solution, 74.90 μM TK molecule (2,2'-[propane-2,2-dimethylbis(thio)diacetic acid) Tris-HCl sterile buffer (pH=7.4), 0.0659 mmol / L GAL3ST1 siRNA sterile aqueous solution, and 0.02 mg / mL PVP molecule Tris-HCl sterile buffer. All solutions were stored at low temperature and protected from light for later use. Among them, DEPC-treated sterile ultrapure water was purchased from Summers, zinc sulfate heptahydrate, 2,2'-[propane-2,2-dimethylbis(thio)]diacetic acid and polyvinylpyrrolidone were purchased from Maclean, the specific small interfering RNA (siGAL3ST1) targeting the GAL3ST1 gene and the negative control siRNA were purchased from Jiangsu Saisofe Biotechnology Co., Ltd., and Tris-HCL was purchased from White Shark Biotechnology Co., Ltd.

[0026] (2) Nanonucleus self-assembly Take a 1.5 mL RNase-free EP tube, add 50 μL of 6 mM zinc sulfate aqueous solution and 130 μL of DEPC sterile water in sequence, mix well, then add 20 μL of 1 mg / mL GAL3ST1 siRNA sterile aqueous solution, vortex vigorously for 6 min to initiate the coordination interaction between zinc ions and siRNA bases, and obtain the initiation mixture.

[0027] (3) Annealing, curing and isothermal coordination The starting mixture was heated in a 65°C water bath for 5 min to complete nucleic acid annealing, and then naturally cooled to 37°C for 6 h of constant temperature incubation to promote full coordination of zinc ions with siRNA and form a structurally stable nanocore (Zn-siGAL3ST1), thus obtaining mixture system I with stable nanocore.

[0028] (4) Surface modification of dual ligands Mixture system I was placed in a magnetic stirrer and slowly added dropwise at room temperature, 600 rpm and in the dark, with 1 mL of TK molecule Tris-HCl sterile buffer (prepared in step (1)) and 1 mL of PVP molecule Tris-HCl sterile buffer (prepared in step (1)) added dropwise. After the addition was completed, the mixture was stirred for 12 h. The carboxyl groups at both ends of TK (2,2'-[propane-2,2-dimethylbis(thio)diacetic acid)) were metal coordinated with the zinc ions exposed on the surface of the nanocore, thus completing the modification of the ROS-responsive shell of the nanoparticles and obtaining mixture system II containing a large number of free zinc ions, free TK, free siRNA and target nanoparticles (Zn-siGAL3ST1@TK).

[0029] (5) Purification Mixture system II was transferred to a 100 kDa ultrafiltration centrifuge tube and subjected to low-temperature centrifugation and ultrafiltration to effectively retain intact nanoparticles and thoroughly remove uncoordinated free zinc ions, free TK, free siRNA and trace impurities from the system. The retained product was collected and fully resuspended in sterile RNase-free ultrapure water to obtain a purified ROS-responsive zinc-based nucleic acid coordination nanoparticle drug delivery system, which was stored at 4°C in a light-protected environment for later use.

[0030] Physicochemical characterization of nanoparticles (Zn-siGAL3ST1@TK): The particle size, morphology, and dispersibility of the nanoparticles (Zn-siGAL3ST1@TK) prepared in this example were detected, and the results are as follows: Figure 2 As shown. The results indicate that the nanoparticles prepared in this embodiment have a regular morphology and exhibit a uniform spherical structure. Figure 2 b) The particle size is concentrated in the range of 150–200 nm, and the particle size distribution is uniform; the polymer dispersion index (PDI) is <0.2. Figure 2 a) It exhibits no obvious particle aggregation, excellent water dispersibility, and good structural stability under normal temperature and physiological conditions, meeting the requirements for in vivo drug delivery.

[0031] Example 2: ROS Response Performance Evaluation of Nanoparticle Drug Delivery System To verify the microenvironment response characteristics of the nano-drug delivery system prepared in Example 1, the free radical scavenging ability of each nano-formulation under different irradiation doses was detected by a DPPH free radical scavenging experiment. The results are as follows: Figure 3 As shown in figure a. After irradiating the Zn-siGAL3ST1@TK nanoparticles with 10 Gy, their microstructure was observed again using TEM. The irradiation response fracture of the TK ligands is shown in figure a. Figure 3As shown in b. The in vitro zinc ion release behavior of Zn-siGAL3ST1@TK nanoparticles was investigated using dialysis. Specifically, unirradiated, 8 Gy, 10 Gy, and 12 Gy X-ray irradiated Zn-siGAL3ST1@TK nanoparticles were sealed in dialysis bags with a molecular weight cutoff of 3000 Da and placed in a release medium. Samples were taken at set time points, and the zinc ion concentration in the release medium was detected using inductively coupled plasma mass spectrometry (Perkin Elmer NexION 300D ICP-MS, USA). The results are shown in Figure b. Figure 3 As shown in c.

[0032] The above experimental results show that in the high ROS environment following high-dose radiotherapy, the thioacetyl-sensitive backbone of TK molecules on the surface of Zn-siGAL3ST1@TK nanoparticles can specifically respond to high concentrations of ROS, exhibiting the strongest DPPH scavenging ability. Simultaneously, TEM observation revealed that the outer layer of Zn-siGAL3ST1@TK nanoparticles detached and dispersed after radiotherapy. Furthermore, in the high ROS inflammatory environment following high-dose radiotherapy, the thioacetyl-sensitive backbone of TK molecules on the nanoparticle surface can specifically respond to high concentrations of ROS and rapidly undergo oxidative cleavage, disrupting the internal coordination network of the nanoparticles and inducing controlled disintegration of the nanoparticles, achieving site-specific and rapid release of zinc ions and GAL3ST1 siRNA. These experimental results confirm that the nano-drug delivery system prepared in Example 1 possesses excellent ROS-specific recognition and lesion-controlled drug release capabilities, effectively avoiding drug leakage and toxic side effects from normal tissues, and ensuring the effectiveness of targeted therapy.

[0033] Example 3: Evaluation of the in vitro anti-inflammatory and gene silencing effects of the nano-drug delivery system This embodiment uses an ionizing radiation-induced intestinal epithelial cell damage model to verify the toxicity of nanomaterials in vitro. The results are as follows: Figure 4 As shown in a; simultaneously, the inhibitory effect of nanomaterials on the radiation-induced enteritis cell model was verified by CCK-8 cell viability assay, and the results are as follows. Figure 4 As shown in b; subsequently, cell model experiments verified the cellular uptake of the nanomaterials, and the results are as follows. Figure 4As shown in c. Then, the in vitro antioxidant, anti-inflammatory and gene silencing effects of the nano-drug delivery system were verified. Specifically, six parallel control groups were set up: blank control group, radiotherapy alone group, Zn-siGAL3ST1 group, Zn-siGAL3ST1@TK group, radiotherapy + Zn-siGAL3ST1 group, and radiotherapy + Zn-siGAL3ST1@TK group. Among them, the blank control group used PBS buffer (pH= 7.2~7.4); Zn-siGAL3ST1 was the mixture system I prepared in step (3) of Example 1, and the purified Zn-siGAL3ST1 resuspension obtained in step (5) of Example 1. In the Zn-siGAL3ST1 group, Zn-siGAL3ST1@TK group, radiotherapy + Zn-siGAL3ST1 group, and radiotherapy + Zn-siGAL3ST1@TK group, the concentration of the nano-formulation was 150 μg / mL.

[0034] pass Figure 4 As can be seen from the in vitro cell experiments, the nano-drug delivery system of this invention is safe and non-toxic compared to the control groups, and can improve cell viability after radiotherapy, with high cell uptake 2 hours after radiotherapy. Furthermore, RT-qPCR (RT-qPCR kit purchased from Cisco) and ELISA (ELISA kit purchased from Edison Biotechnology Co., Ltd.) results confirmed that Zn-siGAL3ST1@TK can efficiently silence GAL3ST1 gene expression, significantly reduce intracellular thioglycoside levels, and the intervention effect is significantly better than that of the free drug group. Western blot results showed that ionizing radiation can significantly activate the NF-κB signaling pathway, promoting NF-κB p65 and IκBα phosphorylation; free Zn-siGAL3ST1 can inhibit pathway activation, while Zn-siGAL3ST1@TK can significantly block NF-κB pathway activation, downregulate the expression levels of phosphorylated p65 and phosphorylated IκBα proteins, such as... Figure 4 As shown in fh. qPCR results further confirmed that Zn-siGAL3ST1@TK significantly inhibited the abnormal upregulation of radiation-induced pro-inflammatory factors IL-6 and IL-1β, and its anti-inflammatory effect was significantly better than that of the free drug group, such as... Figure 4 As shown in Figure ij. Immunofluorescence results of ROS and γ-H2AX showed that ionizing radiation can induce a large accumulation of reactive oxygen species and DNA double-strand breaks; Zn-siGAL3ST1@TK can effectively scavenge ROS, reduce the fluorescence signal intensity of γ-H2AX, and significantly alleviate radiation-induced oxidative stress and DNA damage, while the protective effect of the free drug Zn-siGAL3ST1 is weaker, such as... Figure 4 As shown in kn.

[0035] In summary, the Zn-siGAL3ST1@TK nanoparticles can efficiently clear excessive ROS accumulation in radiation-damaged intestinal epithelial cells, significantly alleviating cellular oxidative stress damage. Simultaneously, they can effectively block the abnormal activation of the intracellular NF-κB inflammatory signaling pathway, significantly downregulating the expression of pro-inflammatory factors such as IL-6 and TNF-α, thus inhibiting the intestinal inflammatory cascade from its source. At the gene intervention level, this nanodelivery system can efficiently deliver siGAL3ST1 into target cells, precisely silencing the pathogenic target gene GAL3ST1 of acute radiation enteritis, achieving a gene silencing efficiency of up to 85%, significantly superior to other groups. This fully demonstrates that the nanodelivery system of this invention, relying on a triple synergistic mechanism of "ROS clearance, inflammatory pathway inhibition, and target gene silencing," possesses excellent in vitro anti-inflammatory therapeutic effects.

[0036] Example 4: Evaluation of the efficacy of in vivo treatment for acute radiation enteritis In this embodiment, a mouse model of acute radiation enteritis caused by localized ionizing radiation in the peritoneal cavity was constructed. Sixty C57BL / 6 mice (6-8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were randomly divided into six groups of 10 mice each: blank control group, radiotherapy alone group, Zn-siGAL3ST1 group, Zn-siGAL3ST1@TK group, radiotherapy + Zn-siGAL3ST1 group, and radiotherapy + Zn-siGAL3ST1@TK group. Among them, Zn-siGAL3ST1 is the mixture system I prepared in step (3) of Example 1, and the purified Zn-siGAL3ST1 resuspension obtained in step (5) of Example 1. Each group of mice was administered the corresponding formulation via colonic enema. Taking the Zn-siGAL3ST1@TK group as an example, Zn-siGAL3ST1@TK nanoparticle formulation (200 μL, 10 mg / kg) was administered via colonic enema for preventive intervention, thus establishing an in vivo model of radiation enteritis induced by a single 12 Gy abdominal X-ray irradiation. Figure 5 a).

[0037] Dynamic monitoring of mouse body weight showed that mice in the ionizing radiation (IR) group experienced progressive weight loss with a significantly increased rate of weight loss. Intervention with free Zn-siGAL3ST1 only mildly alleviated radiation-induced weight loss, while the weight loss in the radiotherapy + Zn-siGAL3ST1@TK group was significantly less severe than in the radiotherapy-only group. This indicates that Zn-siGAL3ST1@TK can significantly reverse radiation-induced weight loss, effectively maintaining normal body weight in mice, and its intervention effect is significantly superior to the free drug group. Figure 5bc). Disease Activity Index (DAI) and intestinal HE pathological damage score results indicated that clinical symptoms such as diarrhea and bloody stools were significantly aggravated in the IR group mice, and the degree of intestinal tissue damage was increased, with both DAI and pathological scores significantly upregulated; after Zn-siGAL3ST1@TK intervention, the above scores were significantly reduced, which can effectively improve the clinical and pathological damage manifestations related to radiation enteritis. Figure 5 de, h). Molecular level detection results showed that, compared with the blank control group, the expression of GAL3ST1 mRNA and the content of thioglycosides in the intestinal tissue of IR group mice were significantly increased; free Zn-siGAL3ST1 could only mildly inhibit GAL3ST1 expression and thioglycoside synthesis, while Zn-siGAL3ST1@TK could efficiently silence the GAL3ST1 gene and significantly downregulate the level of intestinal thioglycosides, showing a superior in vivo gene silencing and target molecule inhibition effect. Figure 5 fg). The results of pro-inflammatory factor detection showed that IR stimulation significantly induced the release of large amounts of pro-inflammatory factors such as IL-6 and IL-1β from mouse intestinal tissue; Zn-siGAL3ST1@TK significantly inhibited radiation-mediated upregulation of pro-inflammatory factors, and its in vivo anti-inflammatory effect was significantly better than that of the free drug Zn-siGAL3ST1 (fg). Figure 5 Immunofluorescence results of ROS and γ-H2AX confirmed that ROS accumulated in large quantities in the intestinal tissue of the IR group, and the γ-H2AX fluorescence signal was significantly enhanced, indicating that the intestine was subjected to severe oxidative stress and extensive DNA breakage damage; Zn-siGAL3ST1@TK can clear excess ROS in the intestine, release siGAL3ST1 in response, inhibit the activation of the GAL3ST1 thioglycoside axis from upstream, and thus block the downstream inflammatory cascade reaction and reduce radiation damage. Figure 5 kn).

[0038] Compared to the control groups, the nanomedicine (Zn-siGAL3ST1@TK) of this invention can precisely target and accumulate in the inflammatory sites of radiation-induced intestinal damage in mice, effectively improving typical pathological damage such as intestinal mucosal congestion, edema, erosion, and ulceration; significantly reducing inflammatory cell infiltration in intestinal tissue, continuously inhibiting abnormal activation of the NF-κB inflammatory pathway, efficiently silencing the expression of the pathogenic GAL3ST1 gene in intestinal tissue, and effectively repairing the intestinal mucosal barrier function. The rate of weight recovery and intestinal tissue pathological scores in mice were significantly better than those in the control groups, demonstrating a significant improvement in disease prevention.

[0039] In addition, 12 C57BL / 6 mice (6–8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were randomly divided into two groups of 6 mice each: a blank control group and a Zn-siGAL3ST1 group, for biosafety testing of the nanoformulation. Mice in the blank control group were administered PBS buffer (200 μL) via colonic enema, while mice in the Zn-siGAL3ST1 group were administered Zn-siGAL3ST1@TK nanoformulation (200 μL, 10 mg / kg) via colonic enema. Figure 6 As shown in a. Biosafety test results show ( Figure 6 During the treatment period, mice showed no significant adverse reactions, and no significant pathological damage was observed in major organs such as the heart, liver, and kidneys. Figure 6 bd), during the treatment period, the liver and kidney function and blood routine indicators of mice were normal ( Figure 6 (el), confirming that the nano-drug delivery system of the present invention has excellent biocompatibility in vivo, no obvious systemic toxicity, and good prospects for safe in vivo application, and can be safely used for targeted treatment of acute radiation enteritis.

[0040] The above are merely embodiments of the present invention. The invention is not limited to the fields covered by these embodiments. Commonly known structures and characteristics are not described in detail here. Those skilled in the art are aware of all common technical knowledge in the field prior to the application date or priority date, are aware of all existing technologies in that field, and have the ability to apply conventional experimental methods prior to that date. Those skilled in the art can improve and implement this solution based on the guidance provided in this application and their own capabilities. Some typical known structures or methods should not be obstacles for those skilled in the art to implement this application. It should be noted that those skilled in the art can make several modifications and improvements without departing from the structure of the present invention. These should also be considered within the scope of protection of the present invention, and will not affect the effectiveness or practicality of the invention. The scope of protection claimed in this application should be determined by the content of its claims. The specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A ROS-responsive metal-nucleic acid coordination nanoparticle drug delivery system, characterized in that: The device includes a nanocore, which is self-assembled from multivalent metal ions and functional nucleic acid molecules. The outer layer of the nanocore chelates a ROS-responsive dicarboxythioacetate ligand and is modified with a hydrophilic polymeric stabilizer. The multivalent metal ions are selected from divalent or trivalent transition metal ions and are used to specifically inhibit the overactivation of the NF-κB inflammatory signaling pathway. The functional nucleic acid molecules are selected from any one or more combinations of DNA, mRNA, siRNA, shRNA, miRNA, antisense oligonucleotide ASO, and CRISPR guide gRNA and are used to target and silence genes causing radiation enteritis. The ROS-responsive dicarboxythioacetate ligand contains a thioacetate-sensitive backbone.

2. The ROS-responsive metal-nucleic acid coordination nanoparticle drug delivery system according to claim 1, characterized in that: The multivalent metal ions are selected from Zn. 2+ Fe 2+ Fe 3+ Cu 2+ Mn 2+ Co 2+ Mg 2+ Ga 3+ Ca 2+ One or more of them.

3. The ROS-responsive metal-nucleic acid coordination nanoparticle drug delivery system according to claim 1, characterized in that: The functional nucleic acid molecule is a small interfering RNA siGAL3ST1 that targets the GAL3ST1 gene.

4. The ROS-responsive metal-nucleic acid coordination nanoparticle drug delivery system according to claim 1, characterized in that: The ROS-responsive dicarboxylated thioacetate ligand is 2,2'-[propane–2,2-dimethylbis(thio)]diacetic acid.

5. The ROS-responsive metal-nucleic acid coordination nanoparticle drug delivery system according to claim 1, characterized in that: The hydrophilic polymeric stabilizer is selected from one or more of polyvinylpyrrolidone, polyethylene glycol, hyaluronic acid, and dextran.

6. The ROS-responsive metal-nucleic acid coordination nanoparticle drug delivery system according to claim 1, characterized in that: The particle size of the nano-drug delivery system is 100–300 nm.

7. The preparation method of the ROS-responsive metal-nucleic acid coordination nanoparticle drug delivery system according to any one of claims 1-6, characterized in that: Includes the following steps: S1. Nanocore self-assembly: Mixing a multivalent metal ion solution with a functional nucleic acid molecule solution induces coordination self-assembly of the bases of the multivalent metal ions and the functional nucleic acid molecules to obtain the start-up mixed system; S2, Annealing and Curing and Isothermal Coordination: After water bath annealing of the mixed system, it was incubated at an isothermal temperature to obtain a mixed system I containing a structurally stable nanocore; S3. Surface modification of dual ligands: Under light-proof and mild stirring conditions, ROS-responsive dicarboxylic thioacetate ligand solution and hydrophilic polymer stabilizer solution were added dropwise to mixture system I to obtain mixture system II containing target nanoparticles.

8. The preparation method of the ROS-responsive metal-nucleic acid coordination nanoparticle drug delivery system according to claim 7, characterized in that: It also includes the following steps: S4. Purification: Mixture system II is subjected to ultracentrifugation or ultrafiltration to obtain a precipitate. The precipitate is resuspended in enzyme-free water to obtain a purified ROS-responsive metallo-nucleic acid coordination nanoparticle drug delivery system.

9. The preparation method of the ROS-responsive metal-nucleic acid coordination nanoparticle drug delivery system according to claim 8, characterized in that: In step S1, the solvent for both the polyvalent metal ion solution and the functional nucleic acid molecule solution is DEPC-free water. The concentration of the polyvalent metal ion solution is 6–15 mM, and the concentration of the functional nucleic acid molecule solution is 1–10 mg / mL. The mixing method is vortexing, and the mixing time is 6–20 min. And / or, in step S2, the water bath annealing temperature is 45-85℃, the heating time is 3-6 min, the constant temperature incubation temperature is 37-40℃, and the constant temperature incubation time is 6-10 h; And / or, in step S3, the stirring speed is 600-700 rpm, the stirring time is 12-15 h, and the reaction temperature is room temperature; And / or, in step S4, mixture system II is ultracentrifuged at 60000×g~70000×g and 4~5℃ for 15~25min to obtain precipitate, and the precipitate is resuspended and stored at 4℃ in the dark.

10. The application of the ROS-responsive metal-nucleic acid coordination nanodelivery system as described in any one of claims 1-6 in the preparation of drugs for the treatment of radioactive intestinal injury.

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