An ultrasonic controllable anti-pancreatic cancer c-Pd@Pt enyne nanosensor, a preparation method and application thereof

Abstract

An ultrasound-controlled c-Pd@Pt ene nanozyme for treating pancreatic cancer, its preparation method, and its application; belonging to the field of pharmaceuticals for treating pancreatic cancer. This invention addresses the challenges of obtaining two-dimensional sheet-like structures and constructing bimetallic core-shell structures using Pd-based nanomaterials in existing technologies. In the application of this nanozyme material in the preparation of drugs for treating pancreatic cancer, ultrathin c-Pd@Pt core-shell bimetallic ene with uniform size was successfully prepared using ultrathin Pd nanosheets as seed crystals through precise epitaxial growth technology. The Pt shell layer achieved good epitaxial growth on the Pd seed crystal surface, forming a structurally stable core-shell structure. Due to lattice mismatch between the core and shell, the Pt shell layer introduced approximately 0.7% compressive strain. This invention's material can simultaneously overcome the two major challenges of the hypoxic tumor microenvironment and the insufficient efficacy of single-therapy approaches, achieving multi-mechanism synergistic tumor treatment under ultrasound excitation.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals for treating pancreatic cancer, specifically to an ultrasound-controlled activated anti-pancreatic cancer c-Pd@Pt bimetallic olefin nanoenzyme material, its preparation method, and its application in the preparation of drugs for treating pancreatic cancer. Background Technology

[0002] While noble metal nanomaterials and their nanozyme systems have been extensively studied in catalysis, biosensing, and tumor therapy, they still have shortcomings in areas such as controllable fabrication of two-dimensional ultrathin structures, stability of bimetallic interfaces, reaction efficiency in hypoxic tumor microenvironments, and controllable activation of deep tumors. These shortcomings are mainly reflected in the following aspects: 1. Two-dimensional Pd ultrathin metalenes are difficult to obtain stably. Pd nanomaterials readily form three-dimensional particles, such as spheres or polyhedra, during conventional synthesis. Driven by strong interactions between metal atoms and surface energy, they tend to grow and aggregate along the thickness direction, resulting in limited specific surface area and insufficient exposure of active sites. Although strategies such as ligand or gas molecule modulation have been reported, challenges remain in achieving size uniformity, thickness controllability, dispersion stability, and reproducibility, making it difficult to obtain ultrathin two-dimensional Pd metalloalkenes that can serve as reliable templates.

[0003] 2. It is difficult to simultaneously maintain the core-shell integrity and phase purity of bimetallic structures such as Pd@Pt. During the preparation of bimetals, Pt precursors are prone to homogeneous nucleation to generate free Pt nanoparticles, or interdiffusion under high temperature conditions leading to alloying / mixing, which weakens the clarity and controllability of the core-shell structure; discontinuous shell or insufficient interfacial bonding can also affect the stability and long-term catalytic performance of the material in complex media.

[0004] 3. Single-metal nanozymes have limited function and efficiency, and insufficient adaptability to the microenvironment. Although single-component nanozymes (such as Pd or Pt) have certain enzyme-like activities, they often face problems such as limited catalytic efficiency, low site utilization, and insufficient stability in complex systems. Furthermore, they are difficult to simultaneously meet the multifunctional requirements of oxygen production, oxygen activation, and reactive oxygen species (ROS) generation.

[0005] 4. Under hypoxic tumor conditions, OXD-like reactions are substrate-limited and lack an effective self-oxygenation cascade design. OXD-like activity is significantly oxygen-dependent, and the reaction rate is prone to decrease and ROS production is insufficient in the hypoxic environment of tumors. Although tumor tissues are often accompanied by high H2O2 levels, cascade nanozyme systems that effectively couple catalase-like oxygen production with OXD-like oxygen-based ROS generation are still insufficient.

[0006] 5. Pancreatic cancer is limited by the barrier and deep localization of the tumor, and there is a lack of externally controllable activation methods. Pancreatic cancer suffers from poor blood supply and fibrotic stroma forming a dense barrier, limiting the deep penetration and retention of nanosystems. Meanwhile, tumors are often located deep within the body, requiring external signals with tissue penetration capabilities to achieve temporal and tertiary control of nanoenzyme reactions, in order to balance efficacy and safety.

[0007] In summary, existing technologies still have shortcomings in the controllable preparation of ultrathin Pd metalloenes, the stable and controllable construction of Pd@Pt core-shell structures and phases, the cascade catalysis of hypoxic tumors with self-oxygenation, and the external controllable activation of deep pancreatic cancer lesions. It is necessary to propose new material design and preparation schemes to address these shortcomings. Summary of the Invention

[0008] The purpose of this invention is to address the problems in the prior art of obtaining two-dimensional sheet-like structures and constructing bimetallic core-shell structures for Pd-based nanomaterials, and to provide an ultrasound-controlled activated c-Pd@Pt bimetallic ene nanoenzyme material for treating pancreatic cancer, its preparation method, and its application in the preparation of drugs for treating pancreatic cancer.

[0009] To address the aforementioned technical problems, the present invention adopts the following technical solution: The purpose of this invention is to provide a method for preparing an ultrasound-controlled c-Pd@Pt nanoenzyme for treating pancreatic cancer, comprising the following steps: Step 1: Add palladium acetylacetonate, hexadecyltrimethylammonium bromide, and PVP to a mixed solvent consisting of DMF and deionized water, mix thoroughly, introduce CO gas, seal, and react in an oil bath. Centrifuge, discard the supernatant, wash the precipitate, and disperse it in deionized water to obtain a Pd metal olefin dispersion. Step 2: Take the Pd metal olefin dispersion obtained in Step 1, add formaldehyde, PVP and deionized water, then add platinum acetylacetone, mix well, heat to react, cool naturally after the reaction is complete, centrifuge, wash the precipitate with anhydrous ethanol and then with water, dry, and obtain the nanozyme.

[0010] Further specifying, in step 1, 50 mg of palladium acetylacetonate, 185 mg of cetyltrimethylammonium bromide and 160 mg of PVP are added to 12 mL of mixed solvent, and the precipitate is dispersed in 7.5 mL of deionized water after precipitation treatment.

[0011] Further specifying, in step 1, the volume ratio of DMF to deionized water in the mixed solvent is 10:2.

[0012] Further specifying, in step 1, the oil bath temperature is 40°C.

[0013] To further specify, in step 1, the oil bath reaction time is 9 hours.

[0014] To further specify, in step 1, centrifugation is performed at 13,000 rpm.

[0015] To further specify, in step 1, the centrifugation process is performed for 8 minutes.

[0016] Further specifying, in step 2, take 2.5 mL of the Pd metal olefin dispersion obtained in step 1, add 0.5 mL of formaldehyde, 100 mg of PVP and 10 mL of deionized water, and then add 5 mg of platinum acetylacetone.

[0017] Further specifying, in step 2, the reaction is carried out under heating conditions of 150℃-180℃.

[0018] Further specifying, in step 2, the heating reaction must last for at least 4 hours.

[0019] Further specifying, in step 2, centrifugation is performed at 13,000 rpm.

[0020] To further specify, in step 2, the centrifugation process is performed for 8 minutes.

[0021] To further specify, in step 2, the precipitate is purified by washing once with anhydrous ethanol and then washing two to three times with water.

[0022] Another objective of this invention is to provide an ultrasound-controlled anti-pancreatic cancer c-Pd@Pt nanoenzyme prepared by any of the above preparation methods.

[0023] Furthermore, the use of the ultrasound-controlled anti-pancreatic cancer c-Pd@Pt nanoenzyme of the present invention is also provided, for use in a drug for treating pancreatic cancer.

[0024] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes ultrathin Pd nanosheets as seed crystals and employs precise epitaxial growth techniques to successfully prepare c-Pd@Pt core-shell bimetallic olefins with uniform size (average lateral dimension approximately 31.91 nm) and ultrathin thickness (average thickness only approximately 2.22 nm). The Pt shell layer achieves excellent epitaxial growth on the Pd seed crystal surface, forming a structurally stable core-shell structure. Due to the lattice mismatch between the core and shell, the Pt shell layer introduces approximately 0.7% compressive strain. This unique compressive strain effect, combined with the electronic (ligand) effect between Pd and Pt, constitutes the key structural basis for enhancing its intrinsic catalytic activity.

[0025] In terms of catalase-like activity, the rate and total amount of O2 generated by the decomposition of H2O2 by c-Pd@Pt were significantly higher than those of pure Pd nanosheets, demonstrating that it can more efficiently improve the hypoxic microenvironment of tumors and provide sufficient oxygen substrate for subsequent reactions.

[0026] In terms of OXD-like activity, c-Pd@Pt exhibits significantly stronger catalytic activity for TMB oxidation than pure Pd, with a specific activity as high as 1.92 U mg. -1 It is pure Pd nanosheets (0.44 U mg). -1 The effect of the bimetallic core-shell structure and compressive strain on catalytic performance is approximately 4.4 times greater than that of the catalytic core-shell structure, confirming the significant enhancing effect of the bimetallic core-shell structure and compressive strain on catalytic performance.

[0027] Enzyme kinetic analysis showed that c-Pd@Pt had a larger maximum reaction rate (Vt). max and better substrate affinity (smaller K+) m (Value), catalytic efficiency is comprehensively improved.

[0028] c-Pd@Pt can achieve highly efficient CAT-OXD cascade catalysis under hypoxic conditions. That is, it uses the O2 generated by its own CAT-like activity as the substrate for its OXD-like activity, and constructs a self-oxygen-supplying and self-consuming catalytic circuit, which fundamentally overcomes the bottleneck of limited activity of traditional OXD-like enzymes in the hypoxic microenvironment of tumors.

[0029] This invention reveals that c-Pd@Pt core-shell bimetallic olefins also possess excellent acoustic sensitizing properties. Under ultrasonic excitation, the c-Pd@Pt+US group generates superoxide anion radicals (•O2). - The strongest signal indicates that its sonodynamic therapy effect is significant. This sonodynamic effect can not only generate reactive oxygen species to kill tumor cells, but also synergize with the cascade enzyme catalytic reaction of the material itself, forming a dual-modal synergistic enhancement mechanism of sonodynamic therapy (SDT) and enzyme catalytic therapy.

[0030] In vitro cell experiments showed that, under ultrasound assistance, c-Pd@Pt exhibited strong concentration-dependent killing effects on both PANC-1 and MIA PaCa-2 pancreatic cancer cell lines, and could induce large-scale apoptosis / necrosis, demonstrating the broad-spectrum effectiveness of this strategy.

[0031] In vivo animal experiments showed that the c-Pd@Pt+US synergistic treatment group exhibited the most significant inhibitory effect on tumor growth in mice, with the smallest tumor volume, and showed a statistically significant difference compared to other control groups (P<0.01). This strongly confirms that the "compressive strain core-shell bimetallic ene-mediated sonodynamic-enzyme catalytic synergistic treatment strategy" constructed in this invention possesses excellent anti-tumor performance at the in vivo level.

[0032] This invention, through ingenious material design and structural control, creates a novel nanomaterial integrating compressive strain effect, bimetallic synergy, highly efficient cascade enzyme catalysis, and acoustic sensitization. This material can simultaneously overcome the two major challenges of the hypoxic tumor microenvironment and the insufficient efficacy of single therapies, achieving multi-mechanism synergistic tumor treatment under ultrasound stimulation. This provides new ideas and material basis for developing highly efficient, low-side-effect nanozyme-based tumor therapy platforms.

[0033] For a deeper understanding of the features and technical content of this invention, please refer to the accompanying detailed description and drawings. It should be noted that the drawings are provided for illustrative purposes only and are not intended to limit the scope of the invention. Attached Figure Description

[0034] Figure 1 This is a low-magnification transmission electron microscope (TEM) image of c-Pd@Pt bimetallic olefin. Figure 2 This is a high-magnification TEM image; Figure 3 This is a statistical analysis chart of particle size and thickness; Figure 4 XRD patterns comparing c-Pd@Pt nanosheets and pure Pd nanosheets; Figure 5 XANES spectrum of synchrotron radiation; Figure 6 This is a synchrotron radiation EXAFS spectrum; Figure 7 This is a comparison chart of the enzyme catalytic performance and sonodynamic performance of the c-Pd@Pt core-shell bimetallic olefin prepared in the embodiments of the present invention and the control sample (Pd metallene). Figure 7 In the diagram, a is the dissolved oxygen generation curve of c-Pd@Pt bimetallic olefin prepared in this invention and pure Pd metal olefin in the presence of hydrogen peroxide; b is a comparison diagram of CAT-like oxygen production activity; c is a spectrum of TMB oxidation catalyzed by oxidase-like enzyme (OXD-like); d is a radar diagram of enzyme kinetic parameters; e is a verification diagram of CAT-OXD cascade reaction under hypoxic conditions; and f is an ESR spectrum under ultrasonic (US) excitation. Figure 8 This is a graph illustrating the in vitro cell-killing activity and in vivo anti-pancreatic cancer effect of the compressive strain c-Pd@Pt core-shell bimetallic olefin prepared according to embodiments of the present invention under ultrasound (US) assistance. Figure 8In the figure, a represents the CCK-8 cell viability test results of PANC-1 pancreatic cancer cells under different treatment conditions, b represents the CCK-8 cell viability test results of MIA PaCa-2 pancreatic cancer cells, c represents the flow cytometry analysis of PANC-1 and MIA PaCa-2 cells after Annexin V-FITC / PI double staining, d represents the matrix of ex vivo tumor solid images of mice in each treatment group after the end of the treatment cycle, and e represents the statistical analysis of tumor volume in mice in each treatment group. Detailed Implementation

[0035] The present invention will be described in detail below with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but should not be considered as limiting 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. These all fall within the protection scope of the present invention.

[0036] Example 1 In this embodiment, the ultrasound-controlled activated anti-pancreatic cancer c-Pd@Pt bimetallic olefin nanoenzyme material was prepared according to the following steps: Step 1: Weigh 250 mg of palladium acetylacetonate (Pd(acac)), 185 mg of cetyltrimethylammonium bromide (CTAB), and 160 mg of polyvinylpyrrolidone (PVP). Add them to a mixed solvent consisting of 10 mL of DMF and 2 mL of deionized water to obtain a reaction solution. Then, transfer the obtained reaction solution to a pressure-resistant bottle and introduce saturated CO gas into the system. Seal the bottle and place it in an oil bath (reaction temperature 40℃) for 9 h. After the reaction is completed, centrifuge the product at 13000 rpm for 8 min. Discard the supernatant, dry the precipitate, and disperse it in 7.5 mL of deionized water to obtain a Pd metal olefin dispersion.

[0037] Step 2: Take 2.5 mL of the Pd metal ene dispersion prepared in Step 1, add 0.5 mL of formaldehyde, 100 mg of PVP and 10 mL of deionized water, then add 5 mg of platinum acetylacetonate Pt(acac)2, mix well, transfer to a 25 mL high-pressure reactor, and react at 160 ℃ for 4 h. After the reaction is completed, cool naturally and centrifuge the product at 13000 rpm for 8 min. The obtained precipitate is purified by washing once with anhydrous ethanol and 2-3 times with water to obtain the c-Pd@Pt bimetallic ene nanozyme material.

[0038] Comparative Example: A method for preparing an ultrathin Pd metallene material, comprising the following steps: Weigh 250 mg of palladium acetylacetonate (Pd(acac)), 185 mg of cetyltrimethylammonium bromide (CTAB), and 160 mg of polyvinylpyrrolidone (PVP), and add them to a mixed solvent consisting of 10 mL of DMF and 2 mL of deionized water to obtain a reaction solution. Then, transfer the obtained reaction solution to a pressure-resistant bottle and introduce saturated CO gas into the system. Seal the bottle and place it in an oil bath (reaction temperature 40℃) for 9 h. After the reaction is complete, centrifuge the product at 13000 rpm for 8 min. Discard the supernatant and centrifuge the precipitate at 13000 rpm for 8 min. Purify the product by washing once with anhydrous ethanol and 2–3 times with water to obtain Pd metal olefins.

[0039] The morphology of the material was characterized using transmission electron microscopy (TEM). A low-magnification TEM image of the c-Pd@Pt bimetallic olefin is shown below. Figure 1 As shown in the figure, the c-Pd@Pt bimetallic olefin nanoenzyme material exhibits an ultrathin sheet-like morphology with good dispersion and uniform size. The crystal structure of the material was observed using transmission electron microscopy (TEM). Figure 2 In the high-magnification TEM image, the layered structure at the edge of the nanosheet and the lattice stripes on the surface are clearly identifiable. These features confirm that the Pt shell has achieved good epitaxial growth on the surface of the Pd seed crystal. Image processing and analysis were performed using Imag J software to obtain statistical analysis diagrams of the particle size and thickness of c-Pd@Pt nanosheets. Figure 3 Data shows that the c-Pd@Pt nanosheets prepared by the method in Example 1 have an average lateral dimension of about 31.91 nm and an average thickness of only 2.22 nm. This ultrathin structure greatly increases the exposure of surface active atoms. The phase composition of the material was determined using X-ray diffraction (XRD). Figure 4 The XRD patterns of c-Pd@Pt nanosheets and pure Pd nanosheets are shown. The spectral results show that c-Pd@Pt maintains the lattice structure characteristics of Pd very well, confirming that Pd nanosheets were used as seed crystals for epitaxial growth during the synthesis process, and that the Pt shell was successfully covered on its surface, forming a stable core-shell structure. The chemical states of the elements were analyzed using synchrotron X-ray absorption near-edge structure (XANES). The synchrotron XANES spectra are shown below. Figure 5 As shown, the results indicate that the intensity of the white line peak of c-Pd@Pt is close to that of Pt foil, suggesting that Pt in the sample is close to the metallic state. The local coordination environment was detected using synchrotron extended X-ray absorption fine structure (EXAFS) spectroscopy, as shown in the synchrotron EXAFS spectrum. Figure 6As shown, the results indicate that the Pt-Pt bond length in c-Pd@Pt is slightly shorter than that in standard Pt foil, which is due to core-shell lattice mismatch and introduces approximately 0.7% compressive strain into the Pt shell. The compressive strain effect and the ligand effect of Pd and Pt are the key structural basis for its superior catalytic performance compared to Pd monometallic olefins. Figure 7 This is a comparison of the enzyme catalytic performance and sonodynamic performance of the c-Pd@Pt core-shell bimetallic olefin prepared in the embodiments of the present invention and the control sample (Pd metal olefin). The oxygen generation during the reaction was monitored in real time using a dissolved oxygen analyzer. The dissolved oxygen generation curves of the c-Pd@Pt bimetallic ene prepared in this invention and pure Pd metal ene in the presence of hydrogen peroxide are shown below. Figure 7 As shown in a, its catalase-like activity was characterized. Under the same conditions, the oxygen production rate and oxygen production of c-Pd@Pt were significantly higher than those of single metal Pd, demonstrating that it can more efficiently improve the hypoxic microenvironment of tumors and generate sufficient substrates for OXD-like reactions. The absorbance change after the substrate reaction was measured using a UV-Vis spectrophotometer. The spectrum of TMB oxidation catalyzed by OXD-like enzymes is shown below. Figure 7 As shown in b, the characteristic absorption peak intensity of c-Pd@Pt is much higher than that of Pd, indicating that the bimetallic olefin structure has a stronger substrate oxidation ability and can generate sufficient •O2. - reactive oxygen species; The comparison of OXD-like specific activities between c-Pd@Pt bimetallic ene and pure Pd metal ene is shown in the figure below. Figure 7 As shown in figure c. The data in the figure, calculated using Prism software based on the kinetic data from the UV-Vis absorption spectra, shows that the specific activity of c-Pd@Pt is as high as 1.92 U mg. -1 The single-metal Pd is only 0.44 U mg. -1 The former is about 4.4 times that of the latter, which strongly confirms the significant enhancing effect of bimetallic core-shell structure and lattice strain effect on catalytic performance; The enzyme kinetic parameters radar plot is obtained by fitting steady-state kinetic tests with UV-Vis absorption spectroscopy data, as shown below. Figure 7 As shown in d. Calculations using Origin software show that c-Pd@Pt has a larger V compared to Pd. max (Maximum reaction rate) and better K m (Miele constant) shows that its affinity for the substrate and catalytic rate are both optimized; The absorbance changes of the reaction system were monitored in real time using a UV-Vis spectrophotometer, and the results were displayed using Origin software. The verification diagram of the CAT-OXD cascade reaction under hypoxic conditions is shown below. Figure 7As shown in e, the absorbance of the c-Pd@Pt group increased rapidly after the addition of H2O2, proving that it successfully constructed a "self-oxygen supply-self consumption" cascade catalytic circuit, effectively overcoming the dependence of OXD-like activity on the hypoxic tumor microenvironment of pancreatic cancer; The reactive oxygen species in the system were detected using electron spin resonance spectroscopy (ESR) combined with spin trapping technology. The ESR spectrum under ultrasonic (US) excitation is shown below. Figure 7 As shown in f. Compared to the simple ultrasound or Pd+US group, the c-Pd@Pt+US group detected the strongest superoxide anion (•O2). - The signal confirms that the material has excellent acoustic sensor properties and can achieve synergistic enhancement of acoustic dynamics and enzyme catalysis.

[0040] The following experiments were used to verify the effectiveness of the invention: Experiment 1: Effects of c-Pd@Pt nanomaterials and their combination with ultrasound on the viability of pancreatic cancer cells. (1) Experimental materials: PANC-1 cells and MIA PaCa-2 cells were both obtained from the ATCC cell bank; CCK-8 kit was purchased from Dojindo Laboratories, Japan.

[0041] (2) Cell seeding: Take PANC-1 or MIA PaCa-2 cells in the logarithmic growth phase, adjust the cell suspension concentration, and seed them in 96-well plates at a density of 6000 cells / well. Add 100 μl of complete culture medium to each well and incubate in a constant temperature incubator at 37℃ and 5% CO2 for 24 hours.

[0042] (3) Drug treatment and ultrasound intervention: The old culture medium was removed and replaced with 100 μl of freshly prepared culture medium containing different concentrations of Pd or c-Pd@Pt material. For the ultrasound treatment group, ultrasound irradiation (power density 1.0 W / cm²) was administered immediately after drug administration. After treatment, the cells were cultured in an incubator for 24 hours.

[0043] (4) Detection and Analysis: After culture, 10 μl of CCK-8 solution was added to each well. The 96-well plate was incubated at 37°C in the dark for 90 minutes. The absorbance of each well was measured at 450 nm using a microplate reader. Cell-free medium containing 10% CCK-8 was used as a blank control, and cell viability was calculated. Cell viability is shown in the figure below. Figure 8 As shown in ab.

[0044] The results of CCK-8 cell viability assays for PANC-1 pancreatic cancer cells under different treatment conditions are as follows: Figure 8As shown in figure a; the results showed that the c-Pd@Pt+US group exhibited significant cytotoxicity in a concentration-dependent manner, proving that ultrasound can effectively stimulate nanozyme activity to kill cancer cells. The results of the CCK-8 cell viability assay for MIA PaCa-2 pancreatic cancer cells are as follows: Figure 8 As shown in b; consistent with the results of PANC-1, the c-Pd@Pt+US group showed the lowest cell viability at all concentrations, confirming that this treatment strategy has a broad-spectrum killing effect on different pancreatic cancer cell lines.

[0045] Experiment 2: Detection of apoptosis induced by c-Pd@Pt nanomaterials in pancreatic cancer cells (1) Experimental reagents and instruments: Annexin V-FITC / PI apoptosis detection kit, purchased from Beyotime; flow cytometer (Epics Altra II), purchased from Beckman Coulter.

[0046] (2) Cell seeding and treatment: Take PANC-1 or MIA PaCa-2 cells and seed them at a rate of 4 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 μg / mL in 6-well plates and cultured overnight at 37°C and 5% CO2. Subsequently, cells were treated with Pd or c-Pd@Pt at a final concentration of 40 μg / mL; the sonication group received ultrasound irradiation (1.0 W / cm²) after drug administration. 2 Each group of cells was cultured for another 24 hours.

[0047] (3) Cell collection and staining: After treatment, the culture medium was aspirated, the cells were washed with PBS, digested with trypsin, and the cells were collected. After counting, 1×10⁶ cells were collected. 5 The cells were resuspended in 250 μl Annexin V binding buffer. Then, 5 μl Annexin V-FITC and 10 μl propidium iodide (PI) were added sequentially, mixed well, and incubated at room temperature in the dark for 20 minutes.

[0048] (4) Results Analysis: Immediately after incubation, the cells were analyzed by flow cytometry to detect and determine the apoptosis rate of each group. The apoptosis rates of each group are shown below. Figure 8 As shown in c.

[0049] Flow cytometry analysis of PANC-1 and MIA PaCa-2 cells after Annexin V-FITC / PI double staining is shown in the figure. Figure 8As shown in c; compared with the control group and the single therapy group, the proportion of Q2 quadrant cells in the c-Pd@Pt+US group was significantly increased (71.5% for PANC-1 and 83.1% for MIA PaCa-2), confirming that the material can induce large-scale death of cancer cells under ultrasound. Experiment 3: Mouse pancreatic cancer cells and animal experiments (1) Cell type: PANC-1 human pancreatic cancer cells, derived from the ATCC cell bank.

[0050] (2) Mouse type: 6-week-old female BALB / c Nude nude mice, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0051] (3) Thirty-six 6-week-old female BALB / c Nude nude mice were used to establish an orthotopic pancreatic cancer model after one week of acclimatization. The specific procedures were as follows: The mice were placed on a sterile operating table, and the surgical area was disinfected with povidone-iodine. Sodium pentobarbital was injected intraperitoneally at a dose of 10 μl / g body weight for anesthesia. After anesthesia took effect, a 1 cm longitudinal incision was made in the left upper abdomen of the mouse, and the pancreas was exposed using the spleen as an anatomical landmark. PANC-1 cell suspension was slowly injected into the pancreas using a microsyringe, with an inoculation volume of 5 × 10⁶ cells. 5 One cell per animal. After injection, the layers are sutured together, and the procedure is strictly performed in accordance with animal ethics guidelines.

[0052] (4) Three weeks after modeling, the tumor-forming mice were randomly divided into 6 groups of 6 mice each, namely: control group, control + ultrasound (control + US), Pd, Pd + ultrasound (Pd + US), c-Pd@Pt (c-Pd@Pt), and c-Pd@Pt + ultrasound (c-Pd@Pt + US). According to the experimental groups, mice were administered saline, Pd, or c-Pd@Pt via tail vein injection. The ultrasound group received ultrasound irradiation treatment (power density 1.0 W / cm²) after drug administration. 2 The above treatment lasted for 4 weeks.

[0053] (5) After treatment, mice in each group were sacrificed under anesthesia. The abdominal cavity was opened, the in situ pancreatic tumor tissue was separated, the tumor volume was calculated, and the treatment effect was evaluated. The treatment effect was as follows: Figure 8 As shown in de.

[0054] The matrix of ex vivo tumor images of mice in each treatment group after the end of the treatment cycle is shown in the figure. Figure 8 As shown in d, the tumor volume in the c-Pd@Pt+US group is the smallest, while the tumor volume in the control group and the single therapy group is larger, which strongly confirms that the material has excellent tumor inhibition ability at the in vivo level.

[0055] Statistical analysis of tumor volume in mice of each treatment group is shown in the figure below. Figure 8 As shown in e; the results showed that tumor growth in the c-Pd@Pt+US group was significantly inhibited (P<0.01), and its tumor inhibition rate was much higher than that of the chemotherapy or ultrasound alone group, further verifying the high efficiency of sonodynamic and cascade enzyme catalysis synergistic therapy.

[0056] The specific embodiments of the present invention have been described in detail above. It should be noted that the present invention is not limited to the specific embodiments described above. Various modifications or alterations can be made by those skilled in the art without departing from the scope of protection defined by the claims, and all such modifications or alterations fall within the technical solutions of the present invention.

Claims

1. A method for preparing an ultrasound-controlled c-Pd@Pt olefin nanozyme for pancreatic cancer, characterized in that, Includes the following steps: Step 1: Add palladium acetylacetonate, hexadecyltrimethylammonium bromide, and PVP to a mixed solvent consisting of DMF and deionized water, mix thoroughly, introduce CO gas, seal, and react in an oil bath. Centrifuge, discard the supernatant, wash the precipitate, and disperse it in deionized water to obtain a Pd metal olefin dispersion. Step 2: Take the Pd metal olefin dispersion obtained in Step 1, add formaldehyde, PVP and deionized water, then add platinum acetylacetone, mix well, heat and react at 150℃-180℃, cool naturally after the reaction is completed, centrifuge, wash the precipitate with anhydrous ethanol and then with water, dry, and obtain the nanozyme. In this nanozyme, Pt is continuously epitaxially grown on the surface of the Pd metalloene nanosheets to form a complete Pt shell. Due to the lattice mismatch between the core and shell, a compressive strain of 0.7% is introduced into the Pt shell. Furthermore, the nanozyme generates superoxide anion radicals (•O2) under ultrasonic excitation. - .

2. The method of claim 1, wherein, In step 1, 50 mg of palladium acetylacetonate, 185 mg of cetyltrimethylammonium bromide and 160 mg of PVP were added to 12 mL of mixed solvent, and the precipitate was dispersed in 7.5 mL of deionized water after precipitation.

3. The method of claim 1, wherein, In the mixed solvent in step 1, the volume ratio of DMF to deionized water is 10:

2.

4. The method of claim 1, wherein, The oil bath temperature is 40℃.

5. The method of claim 1, wherein, Centrifuge at 13,000 rpm.

6. The method of claim 2, wherein, In step 2, take 2.5 mL of the Pd metal olefin dispersion obtained in step 1, and add 0.5 mL of formaldehyde, 100 mg of PVP and 10 mL of deionized water.

7. The method according to claim 6, characterized in that, In step 2, the dosage of acetylacetone platinum is 5 mg.

8. A nanozyme prepared by the method according to any one of claims 1-7.

9. The use of a nanozyme prepared by the method of any one of claims 1-7 in the preparation of a drug for treating pancreatic cancer.

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