Preparation method and application of CuPB@HA nanodrug

Abstract

This invention provides a method for preparing and applying a hyaluronic acid-coated copper-doped Prussian blue nanozyme CupB@HA. The preparation method includes the following steps: first, Prussian blue nanoparticles are synthesized, then copper is doped to obtain copper-doped Prussian blue nanoparticles (CuPB), and the surface of these nanoparticles is modified with hyaluronic acid to obtain the CupB@HA nanozyme. The CupB@HA nanozyme can target and accumulate in macrophages with high CD44 expression in atherosclerotic lesions, thereby improving uptake efficiency at the lesion site. This nanozyme also has lipid metabolism regulation functions, reducing the expression of the lipid uptake receptor CD36 and upregulating the expression of cholesterol efflux-related proteins ABCA1 and ABCG1, thereby reducing foam cell formation and promoting macrophage phenotype transformation favorable to inflammation resolution. Simultaneously, the CupB@HA nanozyme also possesses reactive oxygen species scavenging capabilities, effectively alleviating oxidative stress in the plaque microenvironment, thereby further inhibiting inflammatory responses and improving plaque stability. Animal experiments show that the CupB@HA nanozyme can significantly reduce atherosclerotic plaque burden and improve plaque stability, demonstrating promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and nanomaterials technology, specifically relating to a hyaluronic acid-coated copper-doped Prussian blue nanozyme CuPB@HA, its preparation method, and its applications. Particularly, it relates to its application in the preparation of drugs for the prevention and / or treatment of atherosclerosis, aiming to address problems such as macrophage oxidative stress, inflammatory response, and cholesterol metabolism imbalance in atherosclerotic lesions. Background Technology Atherosclerosis is a cardiovascular disease driven by lipid metabolism disorders, chronic inflammation, and oxidative stress. Its development is closely related to abnormal macrophage activation and foam cell formation. Current treatments have limited intervention in key pathological processes such as local plaque oxidative stress, inflammatory response, and cholesterol metabolism imbalance, and lack targeted intervention.

[0002] In recent years, nanomedicines have shown promising potential in the treatment of atherosclerosis. Hyaluronic acid can bind to the CD44 receptor, enabling targeted delivery to lesion macrophages; copper-doped Prussian blue nanozymes possess excellent reactive oxygen species scavenging capabilities and can improve lipid metabolism imbalances. Therefore, developing a nanomedicine with lesion-targeting, antioxidant, and lipid-regulating effects is of great significance for the prevention and treatment of atherosclerosis. Summary of the Invention To address the shortcomings of existing technologies, this invention provides a method for preparing CuPB@HA nanomedicines and their applications.

[0003] Technical solution: A method for preparing CuPB@HA nanomedicine includes the following steps: Step 1, Synthesis of copper-doped Prussian blue nanozyme CuPB: Polyvinylpyrrolidone was added to distilled water and dissolved completely under magnetic stirring. Potassium ferricyanide was then added and stirred until fully dissolved. Concentrated hydrochloric acid was added to adjust the acidity of the reaction system, and stirring continued. Copper chloride was then added, and the reaction was continued under stirring. The resulting mixture was then placed under heating conditions. After the reaction was complete, the product was collected by centrifugation and purified by washing with deionized water and ethanol, respectively, to obtain copper-doped Prussian blue nanozyme CuPB.

[0004] Step 2, Preparation of hyaluronic acid-coated copper-doped Prussian blue nanozyme CuPB@HA: Hyaluronic acid was dissolved in water, and then an aqueous solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide was added dropwise to the hyaluronic acid solution for activation under stirring. Subsequently, the activated hyaluronic acid solution was added dropwise to the Cubab dispersion obtained in step 1, and the reaction was continued under light-protected and stirring conditions. After the reaction was completed, the mixture was collected by centrifugation, and the resulting precipitate was washed with deionized water, purified, and then freeze-dried to obtain hyaluronic acid-coated copper-doped Prussian blue nanozyme CupB@HA.

[0005] Preferably, in step 1, the polyvinylpyrrolidone is PVP K30 with a molecular weight of approximately 50,000; preferably, it is added to 40 mL of distilled water, and the amount of material added is 4 g.

[0006] Preferably, in step 1, the amount of potassium ferricyanide added is 132 mg, and the stirring time after addition is 1 h.

[0007] Preferably, in step 1, the amount of concentrated hydrochloric acid added is 33 μL, and stirring is continued for 15 min after addition.

[0008] Preferably, in step 1, the amount of copper chloride added is 6.86 mg, and stirring is continued for 1 h after addition.

[0009] Preferably, in step 1, the heating reaction temperature is 80 °C and the reaction time is 24 h.

[0010] Preferably, in step 1, after the reaction is completed, the sample is washed three times by centrifugation with deionized water and ethanol, respectively, under centrifugation conditions of 18000 rpm for 15 min.

[0011] Preferably, in step 2, the amount of hyaluronic acid added is 47 mg, dissolved in 5 mL of water.

[0012] Preferably, in step 2, the amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 15.4 mg, the amount of N-hydroxysuccinimide is 9.6 mg, and both are dissolved in 10 mL of water.

[0013] Preferably, in step 2, the aqueous solution containing EDC and NHS is added dropwise to the hyaluronic acid solution under vigorous magnetic stirring, and the activation reaction time is 3 h.

[0014] Preferably, in step 2, when the activated hyaluronic acid solution is added dropwise to the CuPB dispersion, the volume of the CuPB dispersion is 8 mL.

[0015] Preferably, in step 2, the activated hyaluronic acid and CupB react continuously for 3 days under conditions of light protection and vigorous stirring.

[0016] Preferably, in step 2, after the reaction is completed, the centrifugation conditions are 15,000 rpm for about 30 min; the resulting precipitate is dispersed in 40 mL of deionized water and washed three times, and then freeze-dried for storage.

[0017] CupB@HA nanomedicine 45 was obtained according to the above preparation method.

[0018] The aforementioned CuPB@HA nanozyme can be used to prepare products for the prevention or improvement of atherosclerosis. This application is a non-diagnostic or non-therapeutic method.

[0019] Preferably, the CuPB@HA nanozyme can target and accumulate in macrophages in atherosclerotic lesions, thereby improving cellular uptake efficiency.

[0020] Preferably, the CuPB@HA nanozyme can downregulate the lipid uptake receptor CD36 and promote the expression of cholesterol efflux-related transport proteins ABCA1 and ABCG1, thereby inhibiting foam cell formation.

[0021] Preferably, the CuPB@HA nanozyme can upregulate NRF2 and SOD1 expression, reduce iNOS, IL-1β, TNF-α and IL-6 levels, and increase ARG1 and IL-10 expression, thereby reshaping the lesion microenvironment.

[0022] Preferably, the CuPB@HA nanozyme can reduce the area of ​​atherosclerotic plaques, lipid deposition and necrotic core area, and increase collagen content, thereby improving plaque stability.

[0023] By adopting the above solution, the present invention has the following beneficial effects: The CuPB@HA nanozyme provided by this invention enhances the enrichment and uptake efficiency of hyaluronic acid-mediated CD44 targeting in macrophages of atherosclerotic lesions, thereby strengthening the local intervention effect. Simultaneously, copper-doped Prussian blue endows the nanozyme with excellent multi-enzyme-like antioxidant activity, effectively scavenging excess reactive oxygen species at the lesion site and alleviating oxidative stress. Furthermore, this nanozyme can inhibit CD36-mediated lipid uptake, promote ABCA1 and ABCG1-mediated cholesterol efflux, restore macrophage lipid homeostasis, and reduce inflammatory responses. Animal experiments show that the CuPB@HA nanozyme of this invention can significantly reduce atherosclerotic plaque burden, decrease lipid deposition and necrotic core area, increase collagen deposition, and exhibits good in vivo biocompatibility, providing a novel nanomedicine strategy for precise intervention in atherosclerosis. Attached Figure Description

[0024] Figure 1Characterization of the basic properties of CuPB@HA in Example 1 of this invention.

[0025] Figure 2 This study investigates the multi-enzyme-like antioxidant properties of CupB@HA in Example 1 of the present invention.

[0026] Figure 3 This serves as a verification of in vivo targeted imaging of CupB@HA and enrichment of CD44-overexpressing macrophages in Example 2 of the present invention.

[0027] Figure 4 Example 2 of this invention demonstrates the in vivo efficacy of CupB@HA in improving atherosclerosis.

[0028] Figure 5 The CupB@HA magnetic resonance imaging used in Example 2 of this invention is used for the assessment of carotid atherosclerotic plaques.

[0029] Figure 6 This is an evaluation of the in vivo metabolic clearance and biosafety of CupB@HA in Example 3 of the present invention. Detailed Implementation

[0030] To further illustrate the technical means and effects of the present invention, the technical solution of the present invention will be further explained below with reference to the accompanying drawings and specific embodiments. However, the present invention is not limited to the scope of the embodiments.

[0031] To further illustrate the technical means and effects of the present invention, the technical solution of the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. However, the present invention is not limited to the scope of the following embodiments.

[0032] Example 1: Synthesis, basic property characterization and antioxidant performance evaluation of CuPB@HA nanozyme Step 1: Synthesis and Characterization of CuPB@HA Nanozymes The preparation method of CuPB@HA nanozyme provided in this embodiment includes the following steps: (1.1) Preparation of CupB nanozyme: 4 g of polyvinylpyrrolidone was added to 40 mL of deionized water and magnetically stirred until completely dissolved. Then, 132 mg of potassium ferricyanide K3[Fe(CN)6] was added and stirring was continued for 1 h. 33 μL of concentrated hydrochloric acid was added to the mixture and stirred for 15 min. Then, 6.86 mg of copper chloride was added and stirring was continued for 1 h. The resulting mixture was placed at 80 ℃ for 24 h. After the reaction was completed, the mixture was centrifuged at 18,000 rpm for 15 min and washed three times with deionized water and anhydrous ethanol, respectively, to obtain copper-doped Prussian blue nanozyme CupB.

[0033] (1.2) Preparation of CupB@HA nanozyme: 47 mg of hyaluronic acid was dissolved in 5 mL of deionized water; 15.4 mg of EDC and 9.6 mg of NHS were dissolved in 10 mL of deionized water, and the hyaluronic acid solution was added dropwise, and the reaction was stirred for 3 h. Subsequently, the activated hyaluronic acid solution was added dropwise to 8 mL of CuPB aqueous dispersion, and the mixture was stirred for 3 days under light-protected conditions. After the reaction was completed, the mixture was centrifuged at 15,000 rpm for about 30 min. The resulting precipitate was washed three times with deionized water and then freeze-dried to obtain CupB@HA nanozyme. Basic properties of CuPB@HA nanozymes were characterized: Transmission electron microscopy characterization results as follows Figure 1 As shown in A, CuPB@HA exhibits a regular cubic structure with relatively uniform particle morphology.

[0034] High-angle annular dark-field scanning transmission electron microscopy and elemental surface scanning results are as follows: Figure 1 As shown in Figure B, Fe, Cu, C, K, N and O elements are distributed in the nanoparticles, with Fe and Cu elements being more uniformly distributed, indicating that copper elements were successfully introduced into the Prussian blue structure.

[0035] Dynamic light scattering detection results are as follows Figure 1 As shown in C, PB, CuPB, and CuPB@HA all have relatively uniform particle size distributions; compared with CuPB, the hydrated particle size of CuPB@HA is increased, indicating that hyaluronic acid was successfully modified on the CuPB surface.

[0036] X-ray photoelectron spectroscopy results are as follows: Figure 1 As shown in D, Cu is present in the material. + Cu² + and Fe² + Fe³ + With different valence states, the variable valence metal ions provide a basis for electron transfer and reactive oxygen species scavenging.

[0037] Step 2: Detection of multi-free radical scavenging ability and biomimetic enzyme activity (2.1) ABTS + • Scavenging ability test: Dissolve 30 mg ABTS in 7 mL of deionized water, and dissolve 7 mg potassium persulfate in 10 mL of deionized water; mix 500 μL of each solution and react in the dark for 12 h, then dilute to 30 mL to obtain ABTS. + • Working solution. PB, CuPB, and CuPB@HA were respectively mixed with ABTS. + • The working solutions were mixed, and the concentration of each material was 100 μg / mL. After reacting for 10 min, the absorbance at 734 nm was measured.

[0038] like Figure 2 As shown in A, PB, CupB, and CupB@HA can all reduce ABTS. + The characteristic absorption of · is shown in the figure, with the CupB@HA group exhibiting the lowest absorbance, indicating that it has strong ABTS. + • Clearance ability.

[0039] (2.2) DPPH· scavenging ability test: DPPH was dissolved in anhydrous ethanol to prepare a working solution of 0.03 mg / mL. PB, CuPB and CuPB@HA were added to a final concentration of 100 μg / mL respectively, and the absorbance at 519 nm was measured after reacting in the dark for 10 min.

[0040] like Figure 2 As shown in Figure B, CupB@HA exhibits the most significant reduction in DPPH· characteristic absorption, indicating its excellent DPPH· scavenging ability. (2.3) Detection of superoxide anion free radical scavenging: 5,5-dimethyl-1-pyrroline-N-oxide was used as a free radical scavenger, and superoxide anion free radicals ·O2 were generated using a riboflavin / ultraviolet irradiation system. - Electron paramagnetic resonance (EPR) detection was performed after adding 100 μg / mL of PB, CupB, and CupB@HA, respectively.

[0041] like Figure 2 As shown in C, the Control group exhibits a significant O2 content. - The characteristic signals of PB and CuPB gradually weakened after processing, with the CuPB@HA group showing the lowest signal, indicating that CuPB@HA can effectively remove O2. - .

[0042] (2.4) Hydroxyl radical scavenging detection: Fe²⁺ was used. + The Fenton reaction system composed of H2O2 generates hydroxyl radicals ·OH. Using 5,5-dimethyl-1-pyrrolline-N-oxide as a scavenger, 100 μg / mL of PB, CupP, or CupP@HA is added before electron paramagnetic resonance detection.

[0043] like Figure 2 As shown in D, the ·OH characteristic signal of the CuPB@HA group is significantly lower than that of the Control, PB and CuPB groups, indicating that CuPB@HA has a strong hydroxyl radical scavenging ability.

[0044] Furthermore, the superoxide dismutase-like activity of the material was detected using the riboflavin / UV irradiation combined with the nitroblue tetrazolium method, and the catalase-like activity catalyzing the decomposition of H2O2 was evaluated using the dissolved oxygen assay. The results showed that CuPB@HA possesses both SOD-like and CAT-like enzyme activities, enabling synergistic scavenging of various reactive oxygen species.

[0045] Example 2: In vivo targeted therapy of CupB@HA for atherosclerosis: an experimental study Step 1: Establishment of the atherosclerosis model and drug administration Select male ApoE - / - A atherosclerosis model was established in mice by feeding them a high-fat diet for 12 weeks; age-matched C57BL / 6 mice served as healthy controls. The model mice were randomly divided into Saline group, PB group, CupB group, and CupB@HA group.

[0046] PB, CupB, and CupB@HA were all administered via tail vein injection at a dose of 0.5 mg / kg, based on Fe content, twice weekly for a total of 5 treatments.

[0047] Step 2: In vivo targeted imaging and plaque enrichment verification of CuPB@HA (2.1) In vivo and in vitro fluorescence imaging: Free Cy5.5, Cy5.5-labeled CupB and Cy5.5-labeled CupB@HA were injected into the model mice, and in vivo fluorescence imaging of the small animals was performed 12 h and 24 h after injection.

[0048] like Figure 3 As shown in Figure A, the CupB@HA group exhibited more obvious fluorescence signals in the heart and abdominal regions, and some signal retention could still be observed at 24 h.

[0049] After imaging, the heart, liver, spleen, lungs, and kidneys were sampled for in vitro fluorescence detection. For example... Figure 3 As shown in Figure B, the material is mainly distributed in the liver, while the CupB@HA group has a more obvious fluorescence signal in the heart region, indicating that it can be enriched in the diseased cardiovascular region.

[0050] (2.2) Plaque colocalization detection: Aortic root tissue from mice injected with Cy5.5-labeled CupB@HA was subjected to immunofluorescence staining. Green fluorescence represents CD68 or CD44, red fluorescence represents Cy5.5-CuPB@HA, and yellow fluorescence represents colocalization of the two.

[0051] like Figure 3As shown in C in the figure, CupB@HA exhibits significant co-localization with both CD68-positive macrophages and CD44-positive regions, indicating that CupB@HA can accumulate within atherosclerotic plaques and preferentially distribute in macrophage regions with high CD44 expression. The scale bar in the figure is 50 μm.

[0052] Step 3: Evaluation of the efficacy of CuPB@HA in improving atherosclerosis After treatment, complete aortic and aortic root tissues were taken and subjected to Oil Red O staining, hematoxylin-eosin staining, Masson staining and CD36 immunohistochemical staining, respectively.

[0053] like Figure 4 As shown in Figure A, significant Oil Red O positive lipid deposition was observed in the entire aorta and aortic root of the Saline group. After treatment with PB and CupP, lipid deposition was reduced, with the CupP@HA group showing the smallest aortic plaque area and aortic root lipid area.

[0054] like Figure 4 As shown in B, the Saline group had a larger plaque area at the aortic root, accompanied by significant wall thickening and necrotic core formation; after CupB@HA treatment, the plaque and necrotic core areas were significantly reduced.

[0055] like Figure 4 As shown in C, the collagen fiber content in the plaques of the CupB@HA group increased, suggesting that it can improve plaque tissue structure and enhance plaque stability.

[0056] like Figure 4 As shown in Figure D, CD36 expression was significantly positive in the plaques of the Saline group. CD36 expression decreased after CupB@HA treatment, indicating that CupB@HA can inhibit the uptake of oxidized low-density lipoprotein by macrophages, thereby reducing foam cell formation and lipid accumulation.

[0057] Step 4: Magnetic resonance imaging to evaluate carotid artery plaques After the fifth treatment, carotid artery MRI was performed on mice in each group without the use of additional MRI contrast agents.

[0058] A 9.4 T magnetic resonance imaging system and a fast spin echo sequence were used for scanning. The scanning parameters were: repetition time 1154 ms, echo time 24.9 ms, field of view 17 mm × 36 mm, slice thickness 0.6 mm, and a total of 10 slices were acquired.

[0059] like Figure 5As shown, the carotid artery wall in the Saline group was significantly thickened and formed plaques; the wall thickness and plaque volume in the PB and CupP groups were reduced; the degree of wall thickening and plaque volume in the CupP@HA group were further reduced, indicating that magnetic resonance imaging can be used for non-invasive evaluation of the therapeutic effect of CupP@HA on carotid atherosclerotic plaques.

[0060] Example 3: In vivo distribution and clearance evaluation of Cupb and Cupb@HA Normal C57BL / 6 mice were selected and injected once via tail vein with CupP or CupP@HA. Samples were collected before administration and at 7, 14 and 28 days after administration, with 3 mice at each time point.

[0061] Heart, liver, spleen, lung, and kidney tissues were collected separately. After weighing, the tissues were digested with acid, and the Fe and Cu content in each tissue was measured. The results are expressed as the elemental content per gram of wet tissue.

[0062] like Figure 6 As shown in Figure A, the content of Fe in the heart, liver, spleen, lungs, and kidneys showed a time-dependent change after drug administration. Among them, the liver, spleen, and lungs were the organs with the highest Fe content. The Fe content in the lung tissue decreased in the later stages, while the Fe content in the heart and kidneys changed relatively little.

[0063] like Figure 6 As shown in B, after administration, Cu was mainly distributed in the liver and lung tissues, reaching a high level around 14 days and decreasing significantly at 28 days; the Cu content in the heart, spleen and kidney tissues did not show a sustained increase.

[0064] The above results indicate that after Cubat and Cubat@HA enter the body, they are mainly distributed in organs such as the liver, spleen, and lungs, and undergo redistribution and partial clearance over time. No neutral element accumulation was observed in the heart and kidneys.

[0065] The above description of the embodiments is provided to facilitate understanding and use of the present invention by those skilled in the art. Those skilled in the art can make corresponding adjustments, substitutions, or improvements to the above embodiments without departing from the basic principles of the present invention. Therefore, the present invention is not limited to the above embodiments, and equivalent improvements and modifications made according to the principles of the present invention should fall within the protection scope of the present invention.

Claims

1. A method for preparing CuPB@HA nanomedicine, characterized in that, Includes the following steps: Step 1, Synthesis of copper-doped Prussian blue nanozyme CupB: Polyvinylpyrrolidone was dissolved in water, then potassium ferricyanide was added and stirred until fully dissolved; then concentrated hydrochloric acid was added to adjust the acidity of the reaction system, and then copper chloride was added to continue the reaction; then the resulting mixture was heated to react, and after the reaction was completed, it was centrifuged and washed to obtain copper-doped Prussian blue nanozyme CupB. Step 2, Preparation of hyaluronic acid-coated copper-doped Prussian blue nanozyme CupB@HA: Hyaluronic acid was dissolved in water, and then an aqueous solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide was added dropwise to the hyaluronic acid solution for activation; subsequently, the activated hyaluronic acid solution was added dropwise to the CupB dispersion obtained in Step 1, and the reaction was carried out under light-protected and stirred conditions; after the reaction was completed, the solution was centrifuged, washed, purified, and lyophilized to obtain hyaluronic acid-coated copper-doped Prussian blue nanozyme CupB@HA.

2. The preparation method according to claim 1, characterized in that, In step 1, the polyvinylpyrrolidone is PVPK30 with a molecular weight of approximately 50,000; and / or, The amount of polyvinylpyrrolidone added is 4 g, and the volume of the solvent water is 40 mL; and / or, The polyvinylpyrrolidone was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

3. The preparation method according to claim 1, characterized in that, In step 1, the amount of potassium ferricyanide added is 132 mg; and / or, The potassium ferricyanide is stirred for 1 hour after addition to ensure complete dissolution; and / or, The potassium ferricyanide was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

4. The preparation method according to claim 1, characterized in that, In step 1, the amount of concentrated hydrochloric acid added is 33 μL; and / or, The stirring time after adding hydrochloric acid is 15 min; and / or, The hydrochloric acid was purchased from Chengdu Kelong Chemical Co., Ltd.

5. The preparation method according to claim 1, characterized in that, In step 1, the amount of copper chloride fed is 6.86 mg; and / or, The stirring time after adding copper chloride is 1 hour; and / or, The copper chloride was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

6. The preparation method according to claim 1, characterized in that, In step 1, the heating reaction temperature is 80°C, and the reaction time is 24 h; and / or, After the reaction is completed, the mixture is centrifuged and washed with deionized water and ethanol, respectively; and / or, The centrifugal washing conditions are 18,000 rpm for 15 min; and / or, The washing process is repeated three times.

7. The preparation method according to claim 1, characterized in that, In step 2, the amount of hyaluronic acid added is 47 mg, dissolved in 5 mL of water; and / or, The hyaluronic acid is HA; and / or, The hyaluronic acid was purchased from Shanghai McLean Biochemical Technology Co., Ltd.

8. The preparation method according to claim 1, characterized in that, In step 2, the amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide added is 15.4 mg, and the amount of N-hydroxysuccinimide added is 9.6 mg, both dissolved in 10 mL of water; and / or, The 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is EDC, and the N-hydroxysuccinimide is NHS; and / or, The EDC and NHS were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

9. The preparation method according to claim 1, characterized in that, In step 2, the aqueous solution containing EDC and NHS is added dropwise to the hyaluronic acid solution under vigorous magnetic stirring; and / or, The activation reaction time is 3 h; and / or, When the activated hyaluronic acid solution is added dropwise to the CuPB dispersion, the volume of the CuPB dispersion is 8 mL.

10. The preparation method according to claim 1, characterized in that, In step 2, the activated hyaluronic acid reacts with CupB under conditions of light protection and vigorous stirring for 3 days; and / or, After the reaction is completed, the centrifugation conditions are 15000 rpm for approximately 30 min; and / or, The resulting precipitate was dispersed in 40 mL of deionized water; and / or, Wash three times with deionized water; and / or, After purification, the sample was freeze-dried.

11. The hyaluronic acid-coated copper-doped Prussian blue nanozyme CuPB@HA prepared by the preparation method according to any one of claims 1-10.

12. The hyaluronic acid-coated copper-doped Prussian blue nanozyme CuPB@HA according to claim 11, characterized in that, The nanozyme comprises a copper-doped Prussian blue core and a hyaluronic acid layer coating its surface.

13. The hyaluronic acid-coated copper-doped Prussian blue nanozyme CuPB@HA according to claim 11, characterized in that, The hyaluronic acid coating can mediate the nanozyme's targeted recognition of CD44-overexpressing cells and enhance the cell's uptake capacity.

14. The hyaluronic acid-coated copper-doped Prussian blue nanozyme CuPB@HA according to claim 11, characterized in that, The nanozyme has the ability to scavenge reactive oxygen species and can regulate the expression of cholesterol uptake and efflux-related proteins, thereby reducing foam cell formation.

15. The use of the hyaluronic acid-coated copper-doped Prussian blue nanozyme CuPB@HA according to claim 11 in the preparation of drugs for the prevention and / or treatment of atherosclerosis.

16. The application according to claim 15, characterized in that, The CupB@HA reduces atherosclerotic plaque burden and improves plaque stability by scavenging reactive oxygen species, inhibiting inflammatory responses, downregulating the expression of cholesterol uptake-related proteins, and upregulating the expression of cholesterol efflux-related proteins.

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