A siRNA-loaded metal cobalt-based single-atom nanoszyme particle, a preparation method and application thereof

Abstract

The present application is suitable for the technical field of tumor treatment drugs, and provides a metal cobalt-based single-atom nanozyme particle loaded with siRNA and a preparation method and application thereof. The particle is formed by electrostatic combination of siRNA for knocking down a c-Myc gene and a polyethylene glycol modified cobalt-based single-atom nanozyme, wherein the cobalt-based single-atom nanozyme is prepared by calcining Co-doped ZIF-8, cobalt elements are dispersed in a nitrogen-doped carbon carrier in the form of single atoms, and cobalt-nitrogen coordination active sites are formed. The preparation process includes three steps of synthesis of the cobalt-based single-atom nanozyme, polyethylene glycol modification and siRNA loading, and is simple in process, convenient in operation, does not require complex and expensive equipment, and is easy for industrialized production. It is verified by experiments that the particle has reliable safety and significant effectiveness in the treatment of cholangiocarcinoma, can effectively inhibit the growth of cholangiocarcinoma solid tumors, is suitable for the treatment of malignant tumors, and has a broad application prospect.

Description

Technical Field

[0001] This invention belongs to the field of tumor therapeutic drug technology, and particularly relates to a cobalt-based single-atom nanozyme particle loaded with siRNA, its preparation method and application. Background Technology

[0002] Cholangiocarcinoma (CCA) is a malignant tumor originating from the epithelium of the bile ducts. It is highly malignant, has an insidious onset, and a strong ability to invade and metastasize in the early stages, leading to most patients being diagnosed at an advanced stage and missing the opportunity for radical surgery. Although surgical resection remains a potentially curative treatment, its applicability is limited and the recurrence rate is high. For advanced patients who are not candidates for surgery, systemic chemotherapy (commonly used drugs include gemcitabine and cisplatin) is the standard therapy, but it has inherent drawbacks such as poor targeting, significant toxic side effects, and a tendency to develop drug resistance. Radiotherapy is limited in its application due to the low sensitivity of cholangiocarcinoma and the potential for radiation damage. Overall, the efficacy of traditional treatments has reached a bottleneck, and the prognosis for cholangiocarcinoma patients is extremely poor, with a 5-year survival rate of less than 10%. There is an urgent need to develop new, highly effective, and low-toxicity treatment strategies.

[0003] In recent years, the development of nanomedicine has brought new ideas to cancer treatment. Among them, single-atom nanozymes, as a cutting-edge nanomaterial, possess highly efficient catalytic activity similar to natural enzymes due to their atomically dispersed metal active centers, and exhibit good biocompatibility. Under specific stimulation of the tumor microenvironment, these materials can catalyze the generation of reactive oxygen species (ROS), inducing tumor cell death, and are expected to enhance therapeutic effects by regulating the immune microenvironment. However, the application of single-atom nanozymes in the treatment of cholangiocarcinoma still faces a series of key challenges: First, the catalytic performance of existing single-atom nanozymes may be unstable in complex in vivo environments, and the reactive oxygen species they catalyze are singular and have limited intensity, so their killing efficacy against cholangiocarcinoma cells needs further improvement; second, most studies focus on the catalytic mechanism of the material itself, lacking active functionalization design targeting the characteristics of cholangiocarcinoma (such as its unique tumor microenvironment and molecular pathological features), resulting in insufficient targeting and therapeutic specificity; third, the existing systems have relatively simple functions, making it difficult to overcome the multidrug resistance and immune escape mechanisms of tumors, which can limit the therapeutic effect. To address these issues, this invention proposes a cobalt-based single-atom nanozyme particle loaded with siRNA, its preparation method, and its application. Summary of the Invention

[0004] The purpose of this invention is to provide a cobalt-based single-atom nanozyme particle loaded with siRNA, its preparation method, and its application, aiming to solve the problems of limited efficacy and serious adverse reactions of existing treatments for cholangiocarcinoma.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] A cobalt-based single-atom nanozyme particle loaded with siRNA includes siRNA for knocking down the cMyc gene and a polyethylene glycol-modified cobalt-based single-atom nanozyme; the cobalt element in the cobalt-based single-atom nanozyme is obtained by calcining Co-doped ZIF-8, and the cobalt element is dispersed in the form of single atoms in the nitrogen-doped carbon support derived from ZIF-8, forming cobalt-nitrogen coordination active sites.

[0007] Furthermore, the cobalt-based single-atom nanozyme has a dodecahedral structure with a diameter of 50-200 nm; preferably 100 nm.

[0008] Furthermore, the siRNA sequence includes:

[0009] Chain of Justice (F): CUGAGACAGAUCAGCAACATT (as shown in SEQ ID NO.1 of the sequence list);

[0010] Antisense chain (R): UGUUGCUGAUCUGUCUCAGTT (as shown in SEQ ID NO.2 of the sequence listing).

[0011] A method for preparing cobalt-based single-atom nanozyme particles loaded with siRNA according to the above-described method includes the following steps:

[0012] Step 1: A methanol solution of 2-methylimidazole was mixed with a methanol solution of Zn(NO3)2·6H2O and Co(NO3)2·6H2O under stirring and then transferred to a reaction vessel for further reaction. After the reaction was completed, the precipitate was collected by centrifugation. The precipitate was washed and dried to obtain Co-doped ZIF-8. The Co-doped ZIF-8 was calcined under an inert gas atmosphere to obtain a cobalt-based single-atom nanozyme.

[0013] Step 2: Weigh cobalt-based single-atom nanozymes and polyethylene glycol according to the preset mass ratio; under ice bath conditions, ultrasonically disperse the cobalt-based single-atom nanozymes in PBS buffer to obtain a cobalt-based single-atom nanozyme dispersion; add polyethylene glycol solution to the cobalt-based single-atom nanozyme dispersion and react on a shaker. After the reaction is completed, centrifuge to collect the precipitate and wash it with PBS buffer to obtain PEG-Co-SA.

[0014] Step 3: Mix the siRNA solution with the PEG-Co-SA solution to allow the siRNA molecules to fully bind with the PEG-Co-SA through electrostatic interaction. Centrifuge to remove the free siRNA, and the resulting precipitate is the cobalt-based single-atom nanozyme particle loaded with siRNA.

[0015] Furthermore, in step 1, the calcination temperature is 900℃, the heating rate is 5℃ / min, and the calcination time is 3h; the inert gas is nitrogen.

[0016] Furthermore, in step 2, the mass ratio of cobalt-based single-atom nanozyme to polyethylene glycol is (0.9-1.1):1, preferably 1:1;

[0017] The conditions for ultrasonic dispersion of cobalt-based single-atom nanozymes are: ultrasonic disruptor power of 50-300W and ultrasonic disruption time of 30-60min; preferably, ultrasonic disruptor power of 100W and ultrasonic disruption time of 60min.

[0018] Furthermore, in step 2, the reaction conditions for the cobalt-based single-atom nanoenzyme dispersion and the polyethylene glycol solution are: reacting at 220 rpm on a shaker at 20-25℃ for 12 hours, with the preferred reaction temperature being 25℃.

[0019] Furthermore, in step 3, the volume ratio of siRNA solution to PEG-Co-SA solution is (0.5-1.0):1, preferably 0.6:1;

[0020] The reaction time after mixing siRNA solution and PEG-Co-SA solution is 15-30 min, preferably 20 min; the reaction temperature is 20-25℃, preferably 25℃; and the pH of the reaction system is 7.0-7.4, preferably 7.4.

[0021] Application of a cobalt-based single-atom nanozyme particle loaded with siRNA as described above in the preparation of antitumor drugs.

[0022] Furthermore, the drug was administered via combined ultrasound irradiation (frequency 1 MHz, intensity 1-2 W·cm). -2 To achieve synergistic anti-tumor therapy; tumors include cholangiocarcinoma.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] The cobalt-based single-atom nanozyme particles loaded with siRNA provided by this invention (siRNA@Co-SA) achieve several superior effects: They exert gene knockdown function through the loaded siRNA, targeting and knocking down c-Myc gene expression, thus interfering with nutrient metabolism (especially amino acid metabolism) in tumor cells; simultaneously, the carrier PEG-Co-SA itself possesses multiple enzymatic activities, generating reactive oxygen species (ROS) at the tumor site; the synergistic effect of these two factors promotes oxidative stress in tumor cells, ultimately inducing tumor cell senescence and apoptosis. The preparation process of siRNA@Co-SA is simple and convenient, requiring no complex and expensive equipment, making it easy to achieve industrial production; it possesses both highly efficient targeting of cholangiocarcinoma cells and fully leverages the unique advantages of single-atom nanozymes; and experimental verification shows that siRNA@Co-SA has reliable safety and significant efficacy in the treatment of cholangiocarcinoma, effectively inhibiting the growth of solid tumors in cholangiocarcinoma, making it suitable for the treatment of malignant tumors and possessing broad application prospects. Attached Figure Description

[0025] Figure 1 Characterization images of cobalt-based single-atom nanozymes (Co-SA). A: Transmission electron microscopy (TEM) image of Co-SA; B: Scanning electron microscopy (SEM) image of Co-SA; C: High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) image of Co-SA, showing atomically dispersed cobalt atoms, marked with red circles; D: Energy dispersive spectroscopy-elemental mapping image and elemental distribution of Co-SA; E: High-resolution X-ray photoelectron spectrum of Co in Co-SA; F: High-resolution X-ray photoelectron spectrum of N in Co-SA; G: High-resolution X-ray photoelectron spectrum of C in Co-SA; H: X-ray diffraction (XRD) pattern of Co-SA; I: Raman spectroscopy results of Co-SA.

[0026] Figure 2 The following are the enzyme activity results for Co-SA. A: Colorimetric results of TMB under different pH conditions with and without H2O2; B: POD-like activity spectra under different pH conditions with and without H2O2; C: Absorption spectra of TMB after treatment with different Co-SA concentrations and different ultrasonic times; D: Absorption spectra of DPBF after treatment with different Co-SA concentrations and different ultrasonic times; E: Absorption spectra of ABDA after treatment with different Co-SA concentrations and different ultrasonic times; F: ESR results of ·OH generated at different ultrasonic times and different Co-SA concentrations; G: ·O2 generated at different ultrasonic times and different Co-SA concentrations. - ESR results; H: Genes generated at different ultrasound times and different Co-SA concentrations 1 ESR results for O2.

[0027] Figure 3 Characterization of the bioactivity of cobalt-based single-atom nanozyme particles loaded with siRNA (siRNA@Co-SA) on cholangiocarcinoma. A: Effects of different concentrations of Co-SA and siRNA@Co-SA on cell viability in the normal intrahepatic bile duct epithelial HIBEC cell line (n=3); B: Effects of different concentrations of Co-SA and siRNA@Co-SA on cell viability in the cholangiocarcinoma cell line HUCCC-9810 (n=3); C: Effects of Co-SA, siRNA@Co-SA, and siRNA@Co-SA+US (ultrasound irradiation combined with siRNA@Co-SA treatment) on cell viability in the cholangiocarcinoma cell line HUCCC-9810 (n=3); D: Observation of different treatment groups (Control, siRNA, US (ultrasound irradiation treatment)) by PI staining. Effects of Co-SA, siRNA@Co-SA, and siRNA@Co-SA+US (ultrasound irradiation combined with siRNA@Co-SA treatment) on the nuclei of HUCCC-9810 cells (n=3). In the figure, red represents dead cells and green represents live cells; E: Confocal microscopy observation of the effects of different treatment groups on intracellular ROS generation in HUCCC-9810 cells. In the figure, green represents intracellular ROS; F: Quantitative map of intracellular ROS generation in HUCCC-9810 cells by different treatment groups (n=3); G: Confocal microscopy observation of the effects of different treatment groups on mitochondrial membrane potential (ΔΨm) of HUCCC-9810 cells by JC-1 staining. In the figure, red represents polymer fluorescence and green represents monomer fluorescence.

[0028] Figure 4 This diagram illustrates the effect of siRNA@Co-SA on inhibiting the growth of cholangiocarcinoma. A: Schematic diagram of mouse subcutaneous cholangiocarcinoma model establishment; B: Tumor images of different treatment regimens (categorized by treatment group: Control, siRNA, Ultrasound, Co-SA, siRNA@Co-SA, siRNA@Co-SA+US, ultrasound irradiation combined with siRNA@Co-SA treatment)); C: Effect of different treatment regimens on tumor weight; D: Effect of different treatment regimens on tumor volume; E: Changes in tumor volume in mice under different treatment regimens; F: H&E staining of tumor tissues under different treatment regimens (scale bar: 50 μm); G: TUNEL staining of tumor tissues under different treatment regimens (scale bar: 50 μm); H: Ki67 immunohistochemical staining results of tumor sites under different treatment regimens (scale bar: 50 μm). Detailed Implementation

[0029] To provide a clearer understanding of the technical features, objectives, and beneficial effects of this invention, the technical solution of this invention is described in detail below, but this should not be construed as limiting the scope of implementation of this invention. Unless otherwise specified, the methods used in this invention are conventional methods in this technical field. In this invention, materials, reagents, or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0030] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0031] Example 1: Preparation of siRNA-loaded cobalt-based single-atom nanozyme particles (siRNA@Co-SA) and characterization of cobalt-based single-atom nanozymes (Co-SA).

[0032] 1.1 Preparation of siRNA@Co-SA;

[0033] Step 1: Synthesis of Co-SA;

[0034] First, 0.8733 g of 2-methylimidazole was dissolved in 20 mL of methanol under stirring to obtain solution A. Then, 0.8032 g of Zn(NO3)2·6H2O and 0.064 g of Co(NO3)2·6H2O were dissolved in 10 mL of methanol under stirring at 1000 rpm to obtain solution B. Next, solution A was rapidly added to solution B under vigorous stirring at 5000 rpm, and stirring was continued for 30 min. Afterward, the mixture was transferred to a 50 mL high-pressure reactor and stored in a 120 °C oven for 4 h. After the reaction was complete, the resulting mixture was centrifuged at 4750 rpm for 15 min to collect the precipitate. The precipitate was washed three times with methanol and dimethylformamide (DMF) and then dried under vacuum at 80 °C for 12 h to obtain Co-doped ZIF-8 (Co / ZIF-8). Finally, the dried Co / ZIF-8 powder was placed in a tube furnace and heated to 900°C at a heating rate of 5°C / min under nitrogen atmosphere, and calcined for 3 hours to obtain a black solid powder, which is Co-SA.

[0035] Step 2: Preparation of PEG-Co-SA (PEG-modified cobalt-based single-atom nanozyme);

[0036] Co-SA and polyethylene glycol (PEG) undergo a cross-linking reaction via electrostatic adsorption. The specific procedure is as follows: Raw materials are weighed according to a Co-SA:PEG ratio of 1:1. Under conditions of a Φ6 operating lever, 100W power, and ultrasonication for 60 minutes (4 seconds on, 4 seconds off), 1 mg of Co-SA is pre-dispersed in 1 mL of PBS buffer using an ultrasonic homogenizer in an ice bath environment to prepare a Co-SA dispersion with a concentration of 1 mg / mL. Then, 1 mL of PEG solution (1 mg / mL, obtained by adding PEG to PBS buffer) is added to the Co-SA dispersion, and the mixture is reacted on a shaker at 220 rpm for 12 hours at 25°C. After the reaction, the precipitate is collected by centrifugation at 10000 rpm for 20 minutes and washed three times with PBS buffer (pH 7.4) to obtain PEG-Co-SA.

[0037] Step 3: Synthesis of siRNA@Co-SA;

[0038] Small interfering RNA (siRNA) knocked down the expression of the c-Myc gene (Gene ID: 4609; Standard Transcript Accession Number: NM_002467.6; Corresponding Protein Accession Number: NP_002458.2).

[0039] A 10 μM siRNA solution was prepared by dissolving 2 OD (Optical Density) siRNA (Germ Genetics Co., Ltd., catalog number A10001) in 500 μL of sterile, enzyme-free water (DEPC water) and storing it at -20°C. A 1 mg / mL PEG-Co-SA solution was prepared by dispersing PEG-Co-SA in sterile, enzyme-free water (DEPC). 20 μL of the PEG-Co-SA solution was added to 12 μL of the 10 μM siRNA solution, and the mixture was gently mixed in a 25°C water bath for 20 min to allow the siRNA molecules to fully bind to the PEG-Co-SA via electrostatic interaction. The mixture was then centrifuged at 10,000 rpm for 20 min, and the supernatant was discarded to remove unbound free siRNA. The resulting precipitate was the target product siRNA@Co-SA, which was stored at -20°C for later use.

[0040] 1.2 Morphological characteristics of Co-SA;

[0041] The morphology and size of the nanostructure were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results showed that the nanozyme exhibited a dodecahedral structure of MOF (Metal-Oxide-Factory). Figure 1Images A and B in the image fully demonstrate its good dispersibility and excellent morphological characteristics. The average hydrodynamic diameter of Co-SA is approximately 103.5 ± 2.3 nm. In aberration-corrected high-angle annular dark-field scanning electron microscopy (HAADF-STEM) images, cobalt atoms can be observed to exist as isolated single atoms on the support (marked with red circles), rather than clusters or nanoparticles. Figure 1 The presence of carbon (C) further illustrates the dispersion of single atoms. The corresponding energy-spectral mapping (ESD) image further shows that carbon, nitrogen, and cobalt are uniformly distributed in Co-SA, and the combined elemental image further clarifies the relevant structure. Figure 1 (Middle D). High-resolution X-ray photoelectron spectroscopy (XPS) results suggest that Co 2p 3 / 2 The peak was corrected to 780.07 eV, accompanied by a satellite peak of approximately 786 eV, indicating that Co... 2+ The ion exists in the Co–N–C single-atom structure in a nitrogen-coordinated form; Co 2p 1 / 2 Peak corrected to 795.56 eV, Co 2p 3 / 2 With Co 2p 1 / 2 The binding energy spacing between them is approximately 15.56 eV, indicating that only one pair of spin-orbit doublets appears; no characteristic peak of metallic Co (approximately 778.1 eV) was detected, further confirming that cobalt is atomically dispersed; at the same time, the spectrum does not contain a CoO shoulder peak above 780.9 eV or a Co3O4 satellite peak near 786 eV, indicating that this binding energy value corresponds to a Co-N4 structure ( Figure 1 The electron binding energy position of nitrogen is 398.06 eV, corresponding to pyridinic nitrogen (N), which accounts for 45.64%, providing sufficient sites for the anchoring of cobalt single atoms. Pyrrolic nitrogen (N) exists at an electron binding energy position of 400.30 eV, and its proportion is lower than that of pyridinic nitrogen. It can help provide additional electron density and stabilize Co sites, further verifying the high catalytic activity and structural stability of Co-SA. Figure 1 (F). In addition, Sp 2 The characteristic peak of C=C corresponding to hybrid carbon was corrected to 284 eV, accounting for 76.73%, and there were no metal carbide peaks near 283 eV; the proportions of CN / CO, C=O, and OC=O peaks were all low. Figure 1 The X-ray diffraction (XRD) pattern of Co-SA showed no characteristic diffraction peaks belonging to metallic cobalt or its oxides, only two broad peaks at 26° and 44°, corresponding to the crystal planes of carbon. No crystal diffraction peaks of Co or CoO were detected, confirming that Co exists in a single-atom form. Figure 1 (H). The D-peak represents defects in the C atom lattice, and the G-peak represents sp atoms of the C atom. 2Hybrid in-plane stretching vibrations are clearly visible in the Co-SA Raman results, with D and G bands appearing at ~1362 cm⁻¹. -1 and ~1590cm -1 At this location, and with the intensity ratio of the D-peak to the G-peak (I(D) / I(G)) being approximately between 0.9 and 1.0, it is indicated that the introduction of stably atomically dispersed Co sites adds additional defects to the graphitic carbon matrix, which is beneficial for the formation of high-density Co-N4 active sites; simultaneously at 2700 cm⁻¹... -1 The peak shape at this location is broad and weakly enclosed, indicating a moderate degree of graphitization, still dominated by multilayered disordered carbon, consistent with the characteristics of a single-atom carrier. Figure 1 Middle I).

[0042] Example 2: Enzyme activity analysis experiment of Co-SA.

[0043] To evaluate the ability of Co-SA as a Fenton catalyst to convert H2O2 to ·OH, a colorimetric method based on the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by H2O2 was used for detection. This example verified the effect of the reaction medium pH on the catalytic activity of Co-SA. Under conditions of TMB alone, TMB and H2O2 in the presence of TMB (TMB+H2O2), no oxidized TMB (blue) was observed. When the pH was acidic (pH=5, pH=6), a significant blue color was observed in the reaction system, indicating the large-scale production of oxidized TMB, and the intensity of the blue color increased as the pH decreased. Figure 2 (A). This suggests that Co-SA can efficiently decompose H2O2 to generate oxidized substances under acidic conditions, and its activity increases with decreasing solution pH. However, no significant color change or wavelength change was observed under neutral conditions (pH=7). Figure 2 (Figure B). This indicates that Co-SA exhibits higher catalytic activity in acidic environments in vivo (e.g., tumor site pH 5.5-6.0, H2O2 50-100 μM), while normal tissue (pH 7.4, H2O2 < 1 μM) hardly triggers the catalytic reaction, thus achieving lesion-specific catalytic activity. Different Co-SA concentrations (Co-SA 20, 50, 100 μg / mL) and different ultrasound irradiation (frequency 1 MHz, intensity 1-2 W·cm) were used to further enhance the catalytic activity. -2 The TMB results, obtained at 1, 3, and 5 min time intervals, indicate that the generation of ·OH from Co-SA in the presence of H2O2 also exhibits significant concentration-dependent and ultrasound-time-dependent effects. Figure 2 (C)

[0044] The cleavage experiment of 1,3-diphenylisobenzofuran (DPBF) was used to evaluate the ·O2. - The generation of O2. -Co-SA undergoes a cycloaddition reaction with DPBF to generate an unstable internal peroxide intermediate, which subsequently undergoes ring-opening cleavage to oxidized products such as 1,2-dibenzoylbenzene (DB). This reaction disrupts the conjugated structure of DPBF, resulting in significant changes to its UV-Vis absorption spectrum. An "ultrasound-catalytic cascade amplification" system was constructed for Co-SA under ultrasonic conditions. Under acidic conditions, Co-SA... 2+ / Co 3+ Valence cycle (Co) 2+ +H₂O₂→Co 3+ +·OH+OH - Co 3+ +H₂O₂→Co 2+ +·O2 - +2H + This process significantly accelerates the reaction, endowing Co-SA with pH-dependent Fenton-like reactivity. Furthermore, ultrasonic irradiation raises the local hotspot temperature, inducing "hot electron" emission at the Co-N4 site, lowering the D-band center of Co, optimizing the H2O2 adsorption energy, and reducing the O–O bond cleavage barrier, thus enhancing the multi-enzyme activity of Co-SA. Simultaneously, the local high electric field generated by ultrasound causes transient charge separation at the Co-N4 site, forming an "acousto-electrocatalytic" micro-battery that drives the continuous single-electron redox cycle of H2O2, achieving the reaction of ·OH and ·O2. - The synchronous outbreak. For example... Figure 2 As shown in Figure D, Co-SA under ultrasonic irradiation (frequency 1 MHz, intensity 1-2 W·cm) -2 The induced generation of a large amount of O2 - Furthermore, the amount produced exhibits significant ultrasonic time- and concentration-dependent characteristics. Similarly, ABDA (9,10-anthratridimyl-bis(methylene)dicarboxylic acid), as a singlet oxygen indicator, is oxidized upon contact with singlet oxygen to form corresponding internal peroxides. Its conjugated system is disrupted, leading to a significant decrease in UV absorption, particularly the gradual reduction or even disappearance of its characteristic absorption peaks at 380 nm and 400-420 nm. By monitoring this change in absorption signal, singlet oxygen can be detected. After detecting singlet oxygen generation using ABDA, it was found that Co-SA produces ·O2. - The ability to enhance with increasing material concentration and with increasing ultrasonic time exhibits significant concentration- and ultrasonic time-dependent properties. Figure 2 (E).

[0045] Furthermore, using electron spin resonance (ESR) spectroscopy and with the aid of a commonly used trapping agent (5,5-dimethyl-1-pyrrolline-N-oxide, DMPO), it was demonstrated that Co-SA could withstand ultrasonic irradiation (frequency 1 MHz, intensity 1-2 W·cm).-2 The conversion of H2O2 into a large amount of ·OH (the appearance of DMPO-OH four-line spectrum in the ESR spectrum with a signal intensity ratio of 1:2:2:1) demonstrates excellent Fenton characteristics. Figure 2 (F). Similarly, ESR technology has also verified O2. - The formation of DMPO-·O2 (the appearance of DMPO-·O2 in the ESR spectrum) - The six-line spectrum showed a signal intensity ratio of 1:1:2:2:1:1, and the stable nitroxide radical TEMPO was generated through the reaction of TEMP (2,2,6,6-tetramethyl-4-piperidinol) with singlet oxygen, exhibiting a 1:1:1 triplet split peak. Experimental results indicate that Co-SA, under ultrasonic irradiation (frequency 1 MHz, intensity 1-2 W·cm), exhibits stable nitroxide radical TEMPO. -2 Highly efficient O2 was detected under these conditions. - and 1 O2 generation ( Figure 2 (G and H). This confirms that Co-SA converts H2O2 to ·O2 under ultrasonic conditions. - and 1 Its highly efficient catalytic activity in the O2 process, exhibiting significant concentration-dependent and ultrasound-time-dependent characteristics, highlights its broad application prospects in the biomedical field.

[0046] Example 3: Effect of siRNA@Co-SA on the bioactivity of cholangiocarcinoma (CCA).

[0047] To verify the in vitro anticancer activity of siRNA@Co-SA, the cytotoxicity of Co-SA and siRNA@Co-SA was assessed using the Cell Count Kit-8 (CCK-8) assay. Figure 3 As shown in Figure A, Co-SA and siRNA@Co-SA are almost non-toxic to normal cells (HIBEC cells of intrahepatic bile duct epithelium). Even after treatment with high concentrations of 100 μg / mL Co-SA and siRNA@Co-SA for 24 h, the proliferation of normal cells was not significantly inhibited, demonstrating their safety to normal cells. Under the same culture conditions, with increasing concentrations of Co-SA and siRNA@Co-SA, the proliferation of HCCC-9810 cells (cholangiocarcinoma cells) was inhibited to varying degrees, exhibiting concentration-dependent cytotoxicity to this tumor cell line. Figure 3(B) This may be because Co-SA itself possesses various enzyme activities, generating reactive oxygen species (ROS) in the tumor microenvironment through Fenton-like reactions and oxidative dismutation-like reactions, inducing apoptosis. This suggests that the ROS-generating capacity of Co-SA may be directly proportional to its concentration. Notably, under the same concentration conditions, the CCA cell mortality rate was higher in the siRNA@Co-SA treatment group, indicating that siRNA@Co-SA exhibits stronger toxicity to CCA cells than Co-SA. This may be because the loaded siRNA knocks down the expression of the c-Myc gene. The protein encoded by the c-Myc gene, as an important transcription factor in vivo, directly transcribes and activates key and rate-limiting enzymes in the serine synthesis pathway (SSP), maintaining the de novo serine-glycine synthesis pathway. Once the expression of the c-Myc gene is knocked down, the above regulatory axis is disrupted, leading to a comprehensive downregulation of the expression of key SSP enzymes, thereby inhibiting one-carbon metabolism, disrupting redox homeostasis, and nucleic acid synthesis. This demonstrates the synergistic effect of enzymatic and targeted c-Myc knockdown therapy, ultimately limiting tumor cell proliferation.

[0048] To further elucidate the synergistic effect of ultrasound irradiation combined with siRNA@Co-SA, a CCK-8 assay was performed. The results are as follows: Figure 3 The results showed that the cytotoxic effect of Co-SA or siRNA@Co-SA alone on CCA was concentration-dependent, with 10 mg / mL of Co-SA and siRNA@Co-SA causing approximately 50% cell death. However, ultrasound irradiation (frequency 1 MHz, intensity 1-2 W·cm⁻¹) did not significantly reduce the cytotoxicity. -2 In the siRNA@Co-SA treatment group (siRNA@Co-SA+US), lower doses of siRNA@Co-SA resulted in the same cytotoxicity. This indicates that siRNA@Co-SA exerts stronger antitumor activity under ultrasound irradiation. Furthermore, the additional effect of ultrasound irradiation gradually weakened with increasing material concentration, which may be related to the gradually increasing cytotoxicity of siRNA@Co-SA itself. These experimental results further demonstrate the synergistic antitumor effect of combined ultrasound irradiation and siRNA@Co-SA.

[0049] Fluorescence imaging further elucidated the effect of siRNA@Co-SA on cell viability. In this experiment, cells co-stained with calcein-AM / PI showed significant differences: the control group mainly showed bright green fluorescence, indicating viable cells; while cells treated with Co-SA and siRNA@Co-SA alone showed moderate cytotoxicity, with some dead cells marked with red fluorescence visible; however, ultrasound irradiation combined with siRNA@Co-SA treatment caused the death of a large number of cancer cells, highlighting the synergistic effect of enzymatic and targeted knockdown of c-Myc therapy under ultrasound irradiation. Figure 3 (D).

[0050] To explore the potential mechanisms of cell death, this study investigated the generation of intracellular reactive oxygen species (ROS) after treatment. Fluorescence imaging after DCFH-DA treatment showed that, compared with the siRNA@Co-SA group and Co-SA alone, the group irradiated with ultrasound combined with siRNA@Co-SA showed a significant increase in intracellular ROS in HCCC-9810 cells, indicating that ultrasound synergistically enhanced the ROS production capacity of the nanomaterials themselves. Figure 3 (E and F in the middle).

[0051] In the mitochondrial membrane potential (ΔΨm) detection experiment, the JC-1 fluorescent probe was used to detect changes in mitochondrial membrane potential in CCA cells of different treatment groups: Compared with the control group, after Co-SA treatment, the red polymer fluorescence weakened and the green monomer fluorescence strengthened, indicating progressive depolarization of mitochondrial membrane potential; in the siRNA@Co-SA group, the green monomer fluorescence was further enhanced; and in the siRNA@Co-SA group excited by ultrasound irradiation, the strongest green fluorescence and the weakest red fluorescence were observed, indicating that the depolarization of mitochondrial membrane potential was further aggravated, indicating that ultrasound irradiation combined with siRNA@Co-SA induces mitochondrial apoptosis by damaging ΔΨm. Figure 3 (G).

[0052] Example 4: The effect of siRNA@Co-SA on inhibiting the growth of bile duct cancer.

[0053] To evaluate the efficacy of tumor suppression, this study used a HuCC-T1 cell subcutaneous tumor model to investigate the tumor-suppressive effects of different treatment regimens, including PBS buffer (Control), siRNA, ultrasound irradiation, Co-SA, siRNA@Co-SA, and ultrasound irradiation combined with siRNA@Co-SA (siRNA@Co-SA+US). The mice used in this experimental model were BALB / c-nu athymic male nude mice (Hangzhou Medical College Animal Center, 6-8 weeks old, SPF, weighing 20-25g). Throughout the experiment, the health status of the mice and tumor growth were monitored. If tumor ulceration, infection, or necrosis occurred, the experiment was immediately terminated and the animals were euthanized. PBS buffer, siRNA, Co-SA, and siRNA@Co-SA were administered via tail vein injection on days 1, 3, and 5, respectively. The ultrasound irradiation combined with siRNA@Co-SA group and the ultrasound irradiation alone group underwent ultrasound irradiation (frequency 1MHz, intensity 1-2W·cm) on days 2, 4, and 6, respectively. -2 Tumor volume changes were recorded in detail twice daily after the start of treatment in each group. Figure 4 (A)

[0054] Tumor volume and weight measurements indicated that siRNA or ultrasound irradiation alone had little inhibitory effect on tumor growth throughout the treatment period. Co-SA alone showed some inhibitory effect on tumor size, and siRNA@Co-SA exhibited a further enhanced tumor-inhibiting effect compared to Co-SA. In contrast, the ultrasound irradiation combined with siRNA@Co-SA group showed the best tumor-inhibiting effect, with significant reductions in both tumor volume and weight. Figure 4 (medium BD), and the intragroup variation in tumor volume within each group was small ( Figure 4 (E), demonstrating the stability of the treatment regimen's effectiveness.

[0055] H&E staining of tumor cells further confirmed the therapeutic potential of siRNA@Co-SA, especially under the combined effect of ultrasound irradiation. The H&E staining results of the ultrasound irradiation combined with siRNA@Co-SA group showed extensive nuclear fragmentation and karyolysis in tumor cells, significantly more pronounced than in other groups. Figure 4 (F). TUNEL results showed that ultrasound irradiation combined with siRNA@Co-SA treatment significantly increased tumor cell apoptosis, further confirming its significant tumor-inhibiting effect. Figure 4Immunohistochemical analysis of Ki67 in tumor tissues also showed that after ultrasound irradiation combined with siRNA@Co-SA treatment, the expression of Ki67 in tumor cells was significantly reduced compared with other groups, suggesting the excellent anti-tumor proliferation properties of siRNA@Co-SA synergistic therapy with ultrasound irradiation. Figure 4 (H). The above results fully demonstrate the potential and safety of ultrasound irradiation combined with siRNA@Co-SA as a clinical intervention for cancer treatment.

[0056] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the present invention.

Claims

1. A cobalt-based single-atom nanozyme particle loaded with siRNA, characterized in that, It includes siRNA for knocking down the cMyc gene and polyethylene glycol-modified cobalt-based single-atom nanozymes; the cobalt element in the cobalt-based single-atom nanozymes is obtained by calcining Co-doped ZIF-8, and the cobalt element is dispersed in the form of single atoms in the nitrogen-doped carbon support derived from ZIF-8, forming cobalt-nitrogen coordination active sites.

2. The cobalt-based single-atom nanozyme particles loaded with siRNA according to claim 1, characterized in that, The cobalt-based single-atom nanozyme has a dodecahedral structure with a diameter of 50-200 nm.

3. The cobalt-based single-atom nanozyme particles loaded with siRNA according to claim 1, characterized in that, The sequence of the siRNA includes: Justice Chain: CUGAGACAGAUCAGCAACATT; Antonyms: UGUUGCUGAUCUGUCUCAGTT.

4. A method for preparing cobalt-based single-atom nanozyme particles loaded with siRNA according to any one of claims 1-3, characterized in that, Includes the following steps: Step 1: A methanol solution of 2-methylimidazole was mixed with a methanol solution of Zn(NO3)2·6H2O and Co(NO3)2·6H2O under stirring and then transferred to a reaction vessel for further reaction. After the reaction was completed, the precipitate was collected by centrifugation. The precipitate was washed and dried to obtain Co-doped ZIF-8. The Co-doped ZIF-8 was calcined under an inert gas atmosphere to obtain a cobalt-based single-atom nanozyme. Step 2: Weigh cobalt-based single-atom nanozymes and polyethylene glycol according to the preset mass ratio; under ice bath conditions, ultrasonically disperse the cobalt-based single-atom nanozymes in PBS buffer to obtain a cobalt-based single-atom nanozyme dispersion; add polyethylene glycol solution to the cobalt-based single-atom nanozyme dispersion and react on a shaker. After the reaction is completed, centrifuge to collect the precipitate and wash it with PBS buffer to obtain PEG-Co-SA. Step 3: Mix the siRNA solution with the PEG-Co-SA solution to allow the siRNA molecules to fully bind with the PEG-Co-SA through electrostatic interaction. Centrifuge to remove the free siRNA, and the resulting precipitate is the cobalt-based single-atom nanozyme particle loaded with siRNA.

5. The preparation method according to claim 4, characterized in that, In step 1, the calcination temperature is 900℃, the heating rate is 5℃ / min, and the calcination time is 3h; the inert gas is nitrogen.

6. The preparation method according to claim 4, characterized in that, In step 2, the mass ratio of cobalt-based single-atom nanozyme to polyethylene glycol is (0.9-1.1):1; The conditions for ultrasonic dispersion of cobalt-based single-atom nanozymes are: ultrasonic disruptor power of 50-300W and ultrasonic disruption time of 30-60min.

7. The preparation method according to claim 4, characterized in that, In step 2, the reaction conditions for the cobalt-based single-atom nanoenzyme dispersion and the polyethylene glycol solution are: reacting at 220 rpm on a shaker at 20-25℃ for 12 hours.

8. The preparation method according to claim 4, characterized in that, In step 3, the volume ratio of siRNA solution to PEG-Co-SA solution is (0.5-1.0):1; The reaction time for the mixture of siRNA solution and PEG-Co-SA solution was 15-30 min, the reaction temperature was 20-25℃, and the pH of the reaction system was 7.0-7.

4.

9. The use of a cobalt-based single-atom nanozyme particle loaded with siRNA according to any one of claims 1-3 in the preparation of antitumor drugs.

10. The application according to claim 9, characterized in that, The drug achieves synergistic anti-tumor therapy through combined ultrasound irradiation. The ultrasound irradiation conditions are: frequency 1MHz, intensity 1-2W·cm. -2 The tumors mentioned include cholangiocarcinoma.

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