A biomimetic iron-doped ceria nanoprobe targeting atherosclerotic plaques and a preparation method and application thereof

Abstract

The application belongs to the technical field of nano probes, and relates to a kind of bionic iron-doped cerium oxide nano probes targeting atherosclerotic plaque and its preparation method and application.The application discloses a kind of bionic iron-doped cerium oxide nano probes targeting atherosclerotic plaque, which takes iron-doped cerium oxide nanoparticles as core, and outer layer is coated with the mixed film formed by macrophage membrane and liposome, the mixed film includes the membrane protein targeting atherosclerotic plaque;The liposome contains active oxygen responsive sulfur ketal bond.The probe is targeted and enriched in atherosclerotic plaque, and in the local active oxygen microenvironment of plaque, the sulfur ketal bond responds and breaks, the mixed film structure disintegrates, and releases iron-doped cerium oxide nanoparticles.

Description

Technical Field

[0001] This invention belongs to the field of nanoprobe technology, and relates to a biomimetic iron-doped cerium oxide nanoprobe targeting atherosclerotic plaques, its preparation method and application. Background Technology

[0002] Cerium oxide (CeO2) based nanomaterials are characterized by their unique CeO2 content. 3+ / Ce 4+ Reversible redox cycles and their associated oxygen vacancy structures have shown great potential in the field of biomimetic antioxidant catalysis. They can simultaneously mimic the dual functions of superoxide dismutase (SOD) and catalase (CAT), achieving a cascade scavenging of reactive oxygen species (ROS) such as superoxide anions and hydrogen peroxide. Therefore, they have attracted widespread attention in the biotherapy of oxidative stress-related diseases (such as atherosclerosis). However, existing cerium oxide nanomaterials still face several bottlenecks in practical applications. First, simply prepared cerium oxide materials often have insufficient oxygen vacancy concentrations, resulting in limited antioxidant enzyme activity and poor therapeutic effects, often requiring invasive drug delivery. Second, existing materials suffer from poor size control or are prone to aggregation, making them easily captured by the reticuloendothelial system after intravenous injection, resulting in poor target enrichment. Furthermore, most studies rely on the passive targeting effect of the materials, lacking the ability to actively identify and accumulate in inflammatory lesions (such as atherosclerotic plaques), severely limiting their diagnostic and therapeutic efficacy.

[0003] To improve diagnostic and treatment outcomes, existing technologies attempt to endow materials with multimodal imaging and therapeutic capabilities through elemental doping or the construction of composite systems. For example, some studies have used gadolinium (Gd) doping to impart cerium oxide with magnetic resonance imaging (MRI) capabilities, or constructed iron composites to achieve T2-weighted imaging. However, Gd... 3+ Potential leaching can cause serious biosafety issues; and T2 effect-based imaging is prone to signal artifacts, affecting the accurate delineation of lesions. Other studies have constructed metal polyphenol networks to encapsulate cerium oxide to achieve dual-modal CT / MRI imaging, but their delivery methods and designs often fail to meet the particle size, stability, and targeting requirements for intravenous injection. Although existing technologies have attempted to improve the biocompatibility and cycle time of nanomaterials through biomimetic strategies (such as cell membrane encapsulation), a systematic solution that effectively integrates highly active antioxidant therapy, highly safe dual-modal imaging, and precise active targeting capabilities is still lacking.

[0004] In conclusion, developing a cerium oxide-based nanomedicine platform that combines highly efficient antioxidant enzyme activity, high-safety dual-modal imaging performance, and active inflammatory lesion targeting capability is of great significance for achieving precise diagnosis and treatment of diseases such as atherosclerosis. Summary of the Invention

[0005] The purpose of this invention is to address the aforementioned problems in existing technologies by constructing a biomimetic iron-doped cerium oxide nanoprobe through the synergistic design of biomimetic modification and iron doping. This nanoprobe combines active targeting, immune escape, enhanced antioxidant therapy, and dual-modal MRI / CT imaging functions, significantly improving the targeting, imaging performance, treatment efficiency, and biosafety of existing cerium oxide nanomaterials. It has outstanding application prospects and clinical translation potential in the field of precision nanodiagnosis and treatment of atherosclerosis.

[0006] One objective of this invention is achieved through the following technical solution: A biomimetic iron-doped cerium oxide nanoprobe targeting atherosclerotic plaques, wherein the biomimetic iron-doped cerium oxide nanoprobe MM / TK@Fe-CeO2 has iron-doped cerium oxide nanoparticles Fe-CeO2 as the core and is coated with a hybrid membrane composed of macrophage membrane and liposomes, the hybrid membrane including membrane proteins that target atherosclerotic plaques. The liposomes contain reactive oxygen species-responsive thioacetal bonds; The Fe:Ce molar ratio in the iron-doped cerium oxide nanoparticles is (3~9):100.

[0007] In this invention, the hybrid membrane composed of macrophage membrane and liposomes mimics the lipid bilayer structure of natural exosomes, fully preserving the inflammatory chemokine receptors on the macrophage membrane surface, thus endowing the nanoprobes with the ability to actively target atherosclerotic plaques and possess immune escape capabilities. Simultaneously, the liposomes contain reactive oxygen species-responsive thioketal bonds, exhibiting responsive release properties within the lesion microenvironment.

[0008] Preferably, after the biomimetic iron-doped cerium oxide nanoprobe MM / TK@Fe-CeO2 targeting atherosclerotic plaques is enriched in the atherosclerotic plaques, the thioketal bonds break in response to the local reactive oxygen microenvironment of the plaques, the mixed membrane structure disintegrates, and the iron-doped cerium oxide nanoparticles are released.

[0009] Preferably, the average particle size of the iron-doped cerium oxide nanoparticles (Fe-CeO2) is 1~30 nm.

[0010] Preferably, the average particle size of the biomimetic iron-doped cerium oxide nanoprobe MM / TK@Fe-CeO2 is 60~180nm.

[0011] Preferably, in the biomimetic iron-doped cerium oxide nanoprobe targeting atherosclerotic plaques, the molar ratio of trivalent cerium to tetravalent cerium in the cerium oxide lattice is (0.8~1.2):1.

[0012] Preferably, the membrane protein targeting atherosclerotic plaques is derived from the macrophage membrane.

[0013] Preferably, the method for preparing the hybrid membrane includes the following steps: Macrophage membranes were mixed with iron-doped cerium oxide nanoparticles coated with liposomes. After co-incubation, sonication, and physical extrusion, the macrophage membranes and liposome membranes were fused to form a hybrid membrane coating the outer layer of iron-doped cerium oxide nanoparticles.

[0014] Further preferably, the method for preparing the liposome-coated iron-doped cerium oxide nanoparticles includes the following steps: Lipid materials are combined with iron-doped cerium oxide nanoparticles via a thin-film hydration method to form liposome-coated iron-doped cerium oxide nanoparticles; wherein the lipid materials include phospholipids, cholesterol, and reactive oxygen species-responsive amphiphilic polymers.

[0015] The second objective of this invention is achieved through the following technical solution: A method for preparing a biomimetic iron-doped cerium oxide nanoprobe targeting atherosclerotic plaques includes the following steps: (1) Add citric acid monohydrate, cerium salt and iron salt in a molar ratio of 1:1:(0.03~0.12) to water, add ammonia water dropwise at 30~70℃ and react for 12~48h, then transfer to a reaction vessel and hydrothermally react at 75~90℃ for 12~48h; after dialysis purification, concentration and dispersion, Fe-CeO2 nanoparticle dispersion is obtained; (2) Dissolve lecithin, cholesterol and ROS-responsive amphiphilic polymer DSPE-TK-PEG2000 in an organic solvent, evaporate to form a film, add Fe-CeO2 nanoparticle dispersion from step (1), hydrate and sonicate to obtain TK@Fe-CeO2; (3) The macrophage membrane was co-incubated with TK@Fe-CeO2 in step (2), and then subjected to ultrasonication and physical extrusion to obtain biomimetic iron-doped cerium oxide nanoprobes MM / TK@Fe-CeO2 with an average particle size of 60~180nm.

[0016] Preferably, the biomimetic iron-doped cerium oxide nanoprobe that targets atherosclerotic plaques targets atherosclerotic plaques.

[0017] Preferably, in step (1), the molar ratio of citric acid monohydrate, cerium salt and iron salt is 1:1:0.09.

[0018] Preferably, in step (1), the molar ratio of Fe:Ce in the iron salt and cerium salt is (3~9):100.

[0019] Further optimization is made in step (1), where the molar ratio of Fe:Ce in the iron salt and cerium salt is 9:100.

[0020] Preferably, in step (1), the concentration of ammonia is 1~10mM.

[0021] Preferably, in step (1), the volume ratio of ammonia to water is (0.8~1.3):1.

[0022] Preferably, in step (1), the cerium salt includes at least one of cerium chloride, cerium nitrate, cerium acetate, and cerium sulfate.

[0023] Preferably, in step (1), the iron salt includes at least one of ferric chloride, ferric nitrate, ferric sulfate, ferrous chloride, ferrous sulfate, and ferric citrate.

[0024] Preferably, in step (2), the mass ratio of lecithin DPPC, cholesterol and DSPE-TK-PEG2000 is (2~10):(1~5):1.

[0025] Preferably, in step (2), the organic solvent is a mixed solution of dichloromethane and methanol with a volume ratio of (5~10):1.

[0026] More preferably, the organic solvent is a mixture of dichloromethane and methanol in a volume ratio of 9:1.

[0027] Preferably, in step (2), the rotary evaporation temperature is 30~50℃ and the rotation speed is 90~120r / min.

[0028] Preferably, in step (2), the hydration ultrasonic power is 80~150W and the time is 1~30min.

[0029] Preferably, in step (2), the average particle size of the TK@Fe-CeO2 is 90~200nm.

[0030] Preferably, in step (3), the macrophage membrane is obtained by freezing and thawing macrophages, hypotonic treatment, disruption and fractionation centrifugation.

[0031] Preferably, in step (3), the method for preparing the macrophage membrane includes: washing macrophages with PBS and freezing them at -80°C; after thawing, centrifuging at 800-2000 rpm to collect the precipitate; adding Tris solution containing 0.1-2 mM sucrose; and letting it stand overnight at 4°C; after disruption by a cell disruptor, centrifuging at 800-2000 rpm and 8000-30000 rpm in sequence to collect the macrophage membrane in the supernatant.

[0032] Preferably, in step (3), the mass ratio of the macrophage membrane to the liposomes in the TK@Fe-CeO2 is 1:(6~12).

[0033] Further preferably, the mass ratio of the macrophage membrane to the lipids in the TK@Fe-CeO2 is 1:10.

[0034] Preferably, in step (3), the co-incubation temperature is 4°C and the time is 6~24h.

[0035] Preferably, in step (3), the power of the ultrasound is 80~150W and the time is 1~30min.

[0036] Preferably, in step (3), the physical extrusion is performed using a physical extrusion device, including a liposome extruder.

[0037] The third objective of this invention is achieved through the following technical solution: An MRI / CT dual-modal contrast agent comprising 0.1-100 wt% of the aforementioned biomimetic iron-doped cerium oxide nanoprobes targeting atherosclerotic plaques.

[0038] Preferably, the preparation method of the MRI / CT dual-modal contrast agent includes: adding a biomimetic iron-doped cerium oxide nanoprobe targeting atherosclerotic plaques to a 0.9% sodium chloride solution.

[0039] Preferably, the MRI / CT dual-modal contrast agent is used to visualize atherosclerotic plaques under the influence of an external magnetic field (for MRI) and / or X-rays (for CT).

[0040] The fourth objective of this invention is achieved through the following technical solution: An anti-atherosclerotic drug comprising 0.1 to 100 wt% of the aforementioned biomimetic iron-doped cerium oxide nanoprobes targeting atherosclerotic plaques.

[0041] Preferably, the application of the anti-atherosclerotic drug includes: in the microenvironment of atherosclerotic plaques with elevated reactive oxygen species (ROS) levels, the drug targets the inflammatory site of the plaque via macrophage membrane proteins (MM); simultaneously, under ROS triggering, thioacetyl (TK) bonds break, achieving responsive drug release; under physiological and inflammatory pathological pH conditions, the drug exhibits superoxide dismutase (SOD)-like and catalase-like activities, thereby achieving precise regulation and treatment of oxidative stress.

[0042] Compared with the prior art, the present invention has the following beneficial effects: 1. The biomimetic iron-doped cerium oxide nanoprobe MM / TK@Fe-CeO2 of the present invention has a small particle size. This size range effectively avoids large-scale capture by the reticuloendothelial system such as the liver after intravenous injection, significantly improving the stability and retention time of the nanoprobe in blood circulation. It also endows the material with good passive targeting penetration ability, effectively penetrating the vascular endothelial barrier and accumulating in inflammatory lesions such as atherosclerotic plaques, providing a favorable basis for subsequent active targeting recognition.

[0043] 2. This invention constructs a macrophage membrane-liposome fusion biomimetic carrier, enabling iron-doped cerium oxide nanoprobes to acquire active target-seeking, immune escape, and exosome-mimicking functions. This overcomes the problems of traditional cerium oxide nanomaterials, such as reliance on passive accumulation, insufficient targeting, and difficulty in effectively enriching in plaque areas. The biomimetic probe of this invention utilizes specific receptors on the surface of macrophage membranes, which can actively chemotactically attract and enrich in inflammatory lesions, achieving a leap from "passive accumulation" to "active target-seeking," significantly improving lesion targeting efficiency and diagnostic accuracy.

[0044] 3. This invention precisely controls the Ce content of cerium oxide through iron doping. 3+ / Ce 4+ Valence state and oxygen vacancy distribution significantly enhance the antioxidant enzyme activity of the material, endow it with MRI / CT dual-modal imaging capabilities, and improve the biosafety of the material in vivo by replacing toxic gadolinium with biosafe iron, thus achieving an essential fusion of diagnostic and therapeutic functions.

[0045] 4. This invention employs a biomimetic modification strategy of macrophage membranes, which retains the recognition function of natural membrane proteins, endows the nanoprobes with excellent immune escape ability, significantly prolongs blood circulation time, and improves the stability and biodistribution characteristics of the material in vivo.

[0046] 5. This invention integrates enhanced antioxidant therapy and MRI / CT dual-modal imaging into the same biomimetic nanoprobe, which is actively and precisely delivered to atherosclerotic plaques, achieving integrated synergy of diagnosis-targeting-treatment, and providing a systematic nano-solution for the precision diagnosis and treatment of atherosclerosis. Attached Figure Description

[0047] Figure 1 The images are TEM images of the biomimetic iron-doped cerium oxide nanoprobe MM / TK@Fe-CeO2 at different magnifications in Example 3 of this invention.

[0048] Figure 2 The images show the STEM-HAADFF diagram and mapping diagram of the biomimetic iron-doped cerium oxide nanoprobe MM / TK@Fe-CeO2 in Example 3 of this invention.

[0049] Figure 3XPS images of the biomimetic iron-doped cerium oxide nanoprobes MM / TK@Fe-CeO2 in Examples 1-4 of this invention.

[0050] Figure 4 The images are DLS diagrams of iron-doped cerium oxide nanoparticles from Examples 1-4 of this invention.

[0051] Figure 5 These are physical images of the iron-doped cerium oxide nanoparticle dispersions in Examples 1-4 of this invention.

[0052] Figure 6 The image shows the in vitro CT imaging results and the calculation diagram of concentration and CT signal intensity in Example 1 of the present invention.

[0053] Figure 7 This is an MRI imaging image from Example 1 of the present invention.

[0054] Figure 8 The in vitro MRI imaging image and the corresponding intensity visualization pseudocolor image are shown in Example 1 of the present invention.

[0055] Figure 9 This is a graph showing the evaluation and comparison results of the antioxidant capacity of ABTs in Application Example 2 of the present invention.

[0056] Figure 10 This is a TEM image of hydrogen peroxide cracking and release in Application Example 3 of the present invention.

[0057] Figure 11 This refers to the cell survival rate in Example 3 of the present invention.

[0058] Figure 12 This is a comparison chart of fluorescence intensity in application example 3 of the present invention.

[0059] Figure 13 This is a staining image of reactive oxygen species in Application Example 3 of the present invention.

[0060] Figure 14 This is a diagram showing the expression of CD86 and CD206 in macrophages in Example 4 of the present invention.

[0061] Figure 15 This is an Oil Red O staining image of macrophages in Application Example 4 of the present invention.

[0062] Figure 16 This is a live fluorescence imaging image from application example 5 of the present invention.

[0063] Figure 17 This is an MRI image of a mouse in Application Example 5 of the present invention. Detailed Implementation

[0064] The technical solution of the present invention will be further described and illustrated below through specific embodiments. It should be understood that the specific embodiments described herein are only for the purpose of helping to understand the present invention and are not intended to limit the present invention.

[0065] Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commonly used in the art, and the methods used in the embodiments are all conventional methods in the art.

[0066] In this article, the raw materials include: ROS-responsive amphiphilic polymer, purchased from Weihua Biotechnology, WH0018000405 DSPE-TK-PEG2000; RAW264.7 cells were purchased from Wuhan Pronosei Life Science Technology Co., Ltd.

[0067] In this paper, the biomimetic iron-doped cerium oxide nanoprobe targeting atherosclerotic plaques has iron-doped cerium oxide nanoparticles as the core and is coated with a hybrid membrane composed of macrophage membrane and liposomes. This hybrid membrane includes membrane proteins that target atherosclerotic plaques. Its preparation method includes the following steps: (1) Add citric acid monohydrate, cerium salt and iron salt in a molar ratio of 1:1:0.09 to water, add 20 mL of 4 mM ammonia water dropwise at 50 °C and react for 24 h, then transfer to a reaction vessel and hydrothermally react at 80 °C for 24 h; after dialysis purification, concentration and dispersion, Fe-CeO2 nanoparticle dispersion is obtained; (2) Dissolve lecithin, cholesterol and ROS-responsive amphiphilic polymer DSPE-TK-PEG2000 in 5 mL of a mixed solution of dichloromethane and methanol in a volume ratio of 9:1 with a mass ratio of 15:5:3. After rotary evaporation to form a film, add 5 mL of Fe-CeO2 nanoparticle dispersion from step (1), hydrate and sonicate to obtain TK@Fe-CeO2; (3) The macrophage membrane and the TK@Fe-CeO2 obtained in step (2) are mixed at a mass ratio of 1:10, where the mass ratio is the mass ratio of the macrophage membrane to the lipid component in TK@Fe-CeO2. The mixture is co-incubated at 4°C and then subjected to ultrasonication and physical extrusion to obtain biomimetic iron-doped cerium oxide nanoprobes MM / TK@Fe-CeO2 with an average particle size of 60~180nm.

[0068] Example 1

[0069] The preparation method of the biomimetic iron-doped cerium oxide nanoprobe in this embodiment includes the following steps: (1) Weigh citric acid monohydrate (2.10 g, 0.01 mol) and dissolve it in 20 mL of deionized water. After stirring and dissolving, add CeCl3·7H2O (3.73 g, 0.01 mol) and FeCl3·6H2O (81.09 mg, 0.0003 mol) in sequence and stir well. Heat to 50℃ and slowly add 20 mL of 4 mM ammonia solution. After reacting for 24 h, transfer to a 50 mL polytetrafluoroethylene-lined reactor and hydrothermally react at 80℃ for 24 h. After the reaction is completed, cool to room temperature and put into a dialysis bag with a molecular weight cutoff of 10000. Dialyze in deionized water for 72 hours, changing the water three times during the process. Then concentrate by rotary evaporation and disperse in 10 mL of PBS buffer (pH 7.14) to obtain an iron-doped cerium oxide nanoparticle dispersion, denoted as 3% Fe-CeO2 (denoted as CF).

[0070] (2) Dissolve 15 mg of lecithin DPPC (19.23 μmol), 5 mg of cholesterol (12.93 μmol) and 3 mg of DSPE-TK-PEG2000 (1.07 μmol) in 5 mL of a mixed solution of dichloromethane and methanol (volume ratio 9:1), evaporate the solution at 40 °C to form a film, add 5 mL of iron-doped cerium oxide nanoparticle dispersion from (1), complete the lipid coating by ultrasonication in a water bath, and then hydrate until clear to obtain TK@Fe-CeO2 liposomes (denoted as TK).

[0071] (3) Raw264.7 macrophages were cultured to a height of 1.0 × 10⁻⁶. 7 Cells / mL, take 5 mL of this cell and wash with PBS and freeze at -80℃; after thawing, centrifuge at 1000 rpm for 4 min to collect the pellet, add Tris solution containing 0.25 mM sucrose, and let stand overnight at 4℃; after disruption by a 100 W cell disruptor, centrifuge at 1000 rpm and 15000 rpm respectively, collect the macrophage cell membrane in the supernatant, and store at 4℃.

[0072] (4) 0.5 mg of macrophage membrane from (3) and 5 mg (based on liposome composition) of TK@Fe-CeO2 liposomes were co-incubated at 4℃ for 4 h. After being sonicated at 100 W for 10 min, the mixture was homogenized by physical extrusion using an Avanti liposome extruder to obtain biomimetic iron-doped cerium oxide nanoprobe MM / TK@Fe-CeO2 (denoted as TM).

[0073] Example 2

[0074] The preparation method of the biomimetic iron-doped cerium oxide nanoprobe in this embodiment includes the following steps: (1) Weigh citric acid monohydrate (2.10 g, 0.01 mol) and dissolve it in 20 mL of deionized water. After stirring and dissolving, add CeCl3·7H2O (3.73 g, 0.01 mol) and FeCl3·6H2O (162.18 mg, 0.0006 mol) in sequence and stir well. Heat to 50℃ and slowly add 20 mL of 4 mM ammonia solution. After reacting for 24 h, transfer to a 50 mL polytetrafluoroethylene-lined reactor and hydrothermally react at 80℃ for 24 h. After the reaction is completed, cool to room temperature and put into a dialysis bag with a molecular weight cutoff of 10000. Dialyze in deionized water for 72 hours, changing the water three times during the process. Then concentrate by rotary evaporation and disperse in PBS buffer (pH 7.14) to obtain an iron-doped cerium oxide nanoparticle dispersion, denoted as 6%Fe-CeO2 (denoted as CF).

[0075] (2~4) Following the steps (2~4) of Example 1, the biomimetic iron-doped cerium oxide nanoprobe MM / TK@Fe-CeO2 (denoted as TM) was obtained.

[0076] Example 3

[0077] The preparation method of the biomimetic iron-doped cerium oxide nanoprobe in this embodiment includes the following steps: (1) Weigh citric acid monohydrate (2.10 g, 0.01 mol) and dissolve it in 20 mL of deionized water. After stirring and dissolving, add CeCl3·7H2O (3.73 g, 0.01 mol) and FeCl3·6H2O (243.27 mg, 0.0009 mol) in sequence and stir well. Heat to 50℃ and slowly add 20 mL of 4 mM ammonia solution. After reacting for 24 h, transfer to a 50 mL polytetrafluoroethylene-lined reactor and hydrothermally react at 80℃ for 24 h. After the reaction is completed, cool to room temperature and put into a dialysis bag with a molecular weight cutoff of 10000. Dialyze in deionized water for 72 hours, changing the water three times during the process. Then concentrate by rotary evaporation and disperse in PBS buffer (pH 7.14) to obtain an iron-doped cerium oxide nanoparticle dispersion, denoted as 9%Fe-CeO2 (denoted as CF).

[0078] (2~4) Following the steps (2~4) of Example 1, the biomimetic iron-doped cerium oxide nanoprobe MM / TK@Fe-CeO2 (denoted as TM) was obtained.

[0079] Example 4

[0080] The preparation method of the biomimetic iron-doped cerium oxide nanoprobe in this embodiment includes the following steps: (1) Weigh citric acid monohydrate (2.1 g, 0.01 mol) and dissolve it in 20 mL of deionized water. After stirring and dissolving, add CeCl3·7H2O (3.73 g, 0.01 mol) and FeCl3·6H2O (324.36 mg, 0.0012 mol) in sequence and stir well. Heat to 50℃ and slowly add 20 mL of 4 mM ammonia solution. After reacting for 24 h, transfer to a 50 mL polytetrafluoroethylene-lined reactor and hydrothermally react at 80℃ for 24 h. After the reaction is completed, cool to room temperature and put into a dialysis bag with a molecular weight cutoff of 10000. Dialyze in deionized water for 72 hours, changing the water three times during the process. Then concentrate by rotary evaporation and disperse in PBS buffer (pH 7.14) to obtain an iron-doped cerium oxide nanoparticle dispersion, denoted as 12%Fe-CeO2 (denoted as CF).

[0081] (2~4) Following the steps (2~4) of Example 1, the biomimetic iron-doped cerium oxide nanoprobe MM / TK@Fe-CeO2 (denoted as TM) was obtained.

[0082] Comparative Example 1 The preparation method of the cerium oxide nanoprobe in this comparative example includes the following steps: (1) Weigh 2.1 g (0.01 mol) of citric acid monohydrate and dissolve it in 20 mL of deionized water. After stirring and dissolving, add CeCl3·7H2O (3.73 g (0.01 mol) and stir well. Heat to 50 °C and slowly add 20 mL of 4 mM ammonia solution. After reacting for 24 h, transfer to a 50 mL polytetrafluoroethylene-lined reactor and hydrothermally react at 80 °C for 24 h. After the reaction is completed, cool to room temperature and place in a dialysis bag with a molecular weight cutoff of 10,000. Dialyze in deionized water for 72 hours, changing the water three times during the process. Then concentrate by rotary evaporation and disperse in PBS buffer (pH 7.14) to obtain a pure cerium oxide nanoparticle dispersion, denoted as CeO2.

[0083] (2~4) Follow the steps (2~4) of Example 1 to obtain the cerium oxide nanoprobe MM / TK@CeO2.

[0084] Application Example 1 The biomimetic iron-doped cerium oxide nanoprobes MM / TK@Fe-CeO2 in Examples 1-3 and the cerium oxide nanoprobes MM / TK@CeO2 in Comparative Example 1 were subjected to MRI and CT imaging tests, and the results are shown in Table 1.

[0085] (1) CT Imaging: CF and TM samples from Example 3 were used, and iohexol, a commonly used CT contrast agent, was used as a control. A Bruker small animal CT imaging instrument was used, with imaging conditions set to 80 kVp tube voltage and 400-500 μA tube current. The above samples and iohexol controls were subjected to CT scans under the same imaging conditions to obtain their respective CT images. The CT values ​​(Henness units) of each sample and iohexol control in the CT images were measured. The CT-mass concentration relationship curves of the samples and iohexol were plotted with mass concentration as the abscissa and CT value as the ordinate, and linear fitting was performed to obtain the quantitative conversion relationship between CT value and mass concentration. See Figure 6 .

[0086] (2) MRI imaging: Using iron concentration as the sole uniform variable, TM samples with different iron concentrations from Examples 1, 2, and 3 were selected for imaging tests (see...). Figure 7 A MAGNETOM 3.0 T magnetic resonance imaging (MRI) system was used, with the imaging sequence parameters set as follows: repetition time (TR) of 15 ms and echo time (TE) of 2.1 ms. Under the same imaging parameters, MRI scans were performed on TM samples with different iron concentrations, and corresponding MRI images were acquired. Based on the acquired MRI images, see... Figure 8 The effects of different iron concentrations on the intensity or relaxation characteristics of magnetic resonance signals were analyzed.

[0087] Application Example 2 The biomimetic iron-doped cerium oxide nanoprobes MM / TK@Fe-CeO2 from Examples 1-3 and the biomimetic iron-doped cerium oxide nanoprobes MM / TK@Fe-CeO2 from Comparative Example 1 were subjected to in vitro ABTs antioxidant enzyme activity tests, and the results are shown in Table 1.

[0088] The specific steps are as follows: Preheat the ABTs working solution and adjust its absorbance at 734 nm to 0.70 ± 0.02 using a microplate reader. Mix the MM / TK@Fe-CeO2 from Examples 1-3 and the MM / TK@CeO2 from Comparative Example 1 with the prepared ABTs working solution at different concentrations (based on Ce elemental mass concentration, gradient 12.5~200 μg / mL) in a 96-well plate and react at room temperature in the dark for 30 minutes. After the reaction, measure the absorbance at 734 nm using a microplate reader and calculate the ABTs free radical scavenging rate. See [link to relevant documentation]. Figure 9 .

[0089] Application Example 3 The biomimetic iron-doped cerium oxide nanoprobes MM / TK@Fe-CeO2 in Examples 1-3 and the cerium oxide nanoprobes MM / TK@CeO2 in Comparative Example 1 were subjected to ROS-responsive degradation performance tests and intracellular reactive oxygen species scavenging ability tests.

[0090] (1) The specific steps are as follows: Under the condition of 125 μM hydrogen peroxide (H2O2), take the TM samples from Examples 1-3 with a Ce concentration of 200 μg / mL, mix them with hydrogen peroxide solution, and incubate at 37°C for 30 minutes. After the reaction, take the supernatant and drop it onto a copper grid to prepare a transmission electron microscope (TEM) air-dried sample. Observe and photograph the sample using a biological transmission electron microscope (TEM). See below. Figure 10 .

[0091] (2) The specific steps are as follows: RAW264.7 macrophages in the logarithmic growth phase were seeded in culture plates. A blank control group and an experimental group were set up. The experimental group was supplemented with biomimetic iron-doped cerium oxide nanoprobes MM / TK@Fe-CeO2 prepared in Examples 1-3 and cerium oxide nanoprobes MM / TK@CeO2 prepared in Comparative Example 1, at concentrations of 100 μg / mL and 200 μg / mL, respectively. All groups were incubated at 37℃ in a 5% CO2 incubator for the same time. Cell viability was detected using the CCK-8 assay. The results are shown below. Figure 11 .

[0092] (3) The specific steps are as follows: RAW264.7 macrophages in the logarithmic growth phase were seeded in culture plates and divided into three groups: Treatment group: Complete culture medium containing lipopolysaccharide (LPS) was added to construct an oxidative stress cell model; TM sample prepared in Example 3 was added to make the cerium concentration in the culture medium 50 μg / mL; Model group: Complete culture medium containing lipopolysaccharide (LPS) was added to construct an oxidative stress cell model, and an equal volume of phosphate-buffered saline (PBS) was added to the oxidative stress cell model. Control group: No lipopolysaccharide (LPS) added, normal culture conditions maintained; phosphate-buffered saline (PBS) added in the same volume as the treatment group.

[0093] The three groups of cells were incubated at 37°C and 5% CO2, respectively. After incubation, the culture medium was discarded, the cells were washed with PBS buffer, and then incubated with the DCFH-DA fluorescent probe to detect the intracellular total reactive oxygen species (ROS) level.

[0094] Finally, following the operating procedures of the fluorescent probe kit, the changes in fluorescence intensity of cells in each group were detected using a fluorescence microplate reader or flow cytometry to quantitatively assess the residual reactive oxygen species level at the cellular level. Figure 12 The fluorescence characteristics of the three groups of cells are shown in the figure. Figure 13 .

[0095] Application Example 4 The biomimetic iron-doped cerium oxide nanoprobe MM / TK@Fe-CeO2 from Example 3 was used to test its cell bioactivity.

[0096] (1) Western Blot protein expression analysis The specific steps are as follows: RAW264.7 macrophages were seeded and induced with lipopolysaccharide (LPS) at a final concentration of 1 μg / mL. Simultaneously, three different stages of nanomaterials (CF, TK, and TM, prepared in Example 3, all with a cerium concentration of 50 μg / mL) were added and co-incubated at 37°C in a 5% CO2 incubator for 24 h. Cells from each group were collected, and total protein was extracted using lysis buffer. Equal volumes of protein samples were separated by SDS-PAGE electrophoresis and then transferred to PVDF or NC membranes. After blocking with blocking buffer, the cells were sequentially incubated with specific primary antibodies (M1 macrophage marker CD86 and M2 macrophage marker CD206) and horseradish peroxidase (HRP)-labeled secondary antibodies. Finally, enhanced chemiluminescence (ECL) assay was used for color development, and the protein expression levels of cell polarization markers in each group were detected and quantitatively analyzed using an imaging system. The results are shown in [Figure 1]. Figure 14 .

[0097] (2) Oil Red O (ORO) staining The specific steps are as follows: RAW264.7 macrophages were seeded and induced with oxidized low-density lipoprotein (ox-LDL) solution at a final concentration of 50 μg / mL, while simultaneously adding 50 μg / mL of TM from Example 3. The cells were incubated at 37°C in a 5% CO2 incubator for 48 h to construct a foam cell model. After incubation, the original culture medium was discarded, and the cells were gently washed with phosphate-buffered saline (PBS). The cells were fixed and then stained with Oil Red O staining kit. After staining, the distribution of red lipid droplets in the cytoplasm was observed under an optical microscope. Figure 15 The percentage of Oil Red O staining positive area was quantitatively analyzed using ImageJ software to evaluate the inhibitory effect of nanomaterials on macrophage foaming.

[0098] Application Example 5 The biomimetic iron-doped cerium oxide nanoprobe MM / TK@Fe-CeO2 from Example 3 was used to perform in vivo performance tests in mice.

[0099] (1) In vivo fluorescence imaging The specific steps are as follows: ApoE were selected from those fed a high-fat diet. - / - Atherosclerotic mice were used as an animal model. MM / TK@Fe-CeO2 nanomaterials labeled with the near-infrared fluorescent dye DiD were injected into mice via tail vein (200 μL). In vivo fluorescence imaging was performed at 0, 1, 2, 4, and 6 h post-administration to record the distribution of fluorescence throughout the body. Figure 16 The study focused on fluorescence signals in the aortic region and used accompanying software to quantitatively analyze the average fluorescence intensity of the region of interest (ROI) to evaluate the targeted enrichment ability of nanomaterials in atherosclerotic plaques.

[0100] (2) In vivo MRI imaging The specific steps are as follows: Select ApoE - / - A mouse model of atherosclerosis was established by tail vein injection of 200 μL of 2 mg / mL MM / TK@Fe-CeO2 nanomaterial PBS dispersion. Mice were scanned using a small animal magnetic resonance imaging system before administration and at 0, 1, 2, 3, 4, and 6 h after administration, acquiring T1-weighted images. The signal enhancement in the aortic root and aortic arch regions was particularly observed. Figure 17 MRI imaging was performed using a MAGNETOM 3.0 T magnetic resonance imaging system, with a TR of 577 ms and a TE of 6.1 ms.

[0101] Table 1. Performance data of nanoprobes in the examples and comparative examples.

[0102] according to Figures 1-5 It can be seen that this invention successfully prepared a biomimetic iron-doped cerium oxide nanoprobe (MM / TK@Fe-CeO2) with a distinct "core-shell" structure. TEM ( Figure 1 ) and STEM-HAADF and Mapping ( Figure 2 Visual evidence confirms that Ce, Fe, and O are uniformly distributed, with Fe uniformly doped within the CeO2 core lattice, and the outer layer successfully encapsulates the hybrid membrane of TK liposomes and macrophages. XPS analysis ( Figure 3 Further investigation revealed that as the Fe doping concentration increased, the surface Ce...3+ The significantly increased doping ratio (reaching 53.03% at a preferred 9% doping level) induces a large number of oxygen vacancies, providing the probe with excellent antioxidant catalytic activity. (DLS data) Figure 4 The results show that its hydrated particle size is concentrated in the range of 100~200 nm, possessing a good basis for in vivo circulation and targeted physiological function. Macroscopic image ( Figure 5 The study verified that the probe exhibited a highly uniform and stable colloidal state within the doping range of 3-9%, while significant precipitation occurred at 12% doping. Therefore, 9% iron doping was determined to be the optimal ratio balancing catalytic activity and colloidal stability. Figure 6 It is known that the biomimetic iron-doped cerium oxide nanoprobe of this invention has good in vitro CT imaging ability, and its CT value increases linearly with concentration, with imaging contrast superior to that of iohexol commonly used in clinical practice. According to Figure 7 It can be seen that among Examples 1-3 and Comparative Example 1 as a control, Example 3 has a significant T1 imaging effect and is the optimal imaging choice. According to Figure 9 It can be seen that, at different concentrations, the ABTs free radical scavenging efficiency of the biomimetic iron-doped cerium oxide nanoprobe of Example 3 of the present invention is significantly enhanced. According to... Figure 10 It is known that the biomimetic iron-doped cerium oxide nanoprobe cleaves in a hydrogen peroxide environment, demonstrating that when it reaches atherosclerotic plaques, it can cleave in the lesion microenvironment, releasing iron-doped cerium oxide nanoparticles to exert MRI / CT dual-modal imaging capabilities and provide antioxidant, anti-inflammatory, and anti-foaming treatment. According to Figure 11 It can be seen that, at a range of concentrations, the cell biosafety of Example 3 is significantly better than that of other examples and comparative examples. Figure 12 The intracellular fluorescence of macrophages treated with the probe was extremely weak, indicating that it could efficiently remove excess ROS induced by LPS. Figure 13 This further demonstrates that the LPS-induced RAW264.7 macrophage model™ (treatment group) significantly reduced reactive oxygen species and restored them to normal levels (control group). According to... Figure 14 Protein expression analysis showed that the biomimetic iron-doped cerium oxide nanoprobe of this invention effectively inhibited the expression of the M1 macrophage marker (CD86) and promoted the expression of the M2 macrophage marker (CD206), successfully inducing macrophages to shift from a pro-inflammatory to an anti-inflammatory state. Figure 15 It is evident that the biomimetic iron-doped cerium oxide nanoprobe of this invention has a significant intervention and therapeutic effect on the foaming process of macrophages. According to... Figure 8 , 16 As shown in 17, the biomimetic iron-doped cerium oxide nanoprobes were demonstrated in vitro and in ApoE. - / -The MRI imaging results in mice with atherosclerosis showed that the Fe-CeO2 core provided excellent T1-weighted imaging signals, especially in the aortic plaque area of ​​mice, which achieved clear imaging, demonstrating that the probe has good plaque homing ability and MRI / CT dual-modal diagnostic performance.

[0103] In summary, this invention, through the synergistic design of biomimetic modification and iron doping, constructs a biomimetic iron-doped cerium oxide nanoprobe that combines active targeting, immune escape, enhanced antioxidant therapy, and dual-modal MRI / CT imaging functions. This significantly improves the targeting, imaging performance, therapeutic efficiency, and biosafety of existing cerium oxide nanomaterials, and has outstanding application prospects and clinical translation potential in the field of precision nanodiagnosis and treatment of atherosclerosis.

[0104] All aspects, embodiments, and features of this invention should be considered illustrative in all respects and not limiting of the invention; the scope of the invention is defined only by the claims. Other embodiments, modifications, and uses will become apparent to those skilled in the art without departing from the spirit and scope of the invention as claimed.

[0105] In the preparation method of this invention, the order of the steps is not limited to the listed order. For those skilled in the art, variations in the order of the steps without creative effort are also within the scope of protection of this invention. Furthermore, two or more steps or actions can be performed simultaneously.

[0106] Finally, it should be noted that the specific embodiments described herein are merely illustrative examples of the invention and are not intended to limit the implementation of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them; it is neither necessary nor possible to exemplify all embodiments here. However, these obvious variations or modifications derived from the essential spirit of the invention still fall within the scope of protection of the invention, and interpreting them as any additional limitation would contradict the spirit of the invention.

Claims

1. A biomimetic iron-doped cerium oxide nanoprobe targeting atherosclerotic plaques, characterized in that, The biomimetic iron-doped cerium oxide nanoprobe MM / TK@Fe-CeO2 has iron-doped cerium oxide nanoparticles Fe-CeO2 as its core, and is coated with a hybrid membrane composed of macrophage membrane and liposomes. This hybrid membrane includes membrane proteins that target atherosclerotic plaques. The liposomes contain reactive oxygen species-responsive thioacetal bonds; The Fe:Ce molar ratio in the iron-doped cerium oxide nanoparticles is (3~9):

100.

2. The biomimetic iron-doped cerium oxide nanoprobe targeting atherosclerotic plaques according to claim 1, characterized in that, The average particle size of the iron-doped cerium oxide nanoparticles Fe-CeO2 is 1~30 nm; The average particle size of the biomimetic iron-doped cerium oxide nanoprobe MM / TK@Fe-CeO2 is 60~180nm.

3. The biomimetic iron-doped cerium oxide nanoprobe targeting atherosclerotic plaques according to claim 1, characterized in that, In the biomimetic iron-doped cerium oxide nanoprobe targeting atherosclerotic plaques, the molar ratio of trivalent cerium to tetravalent cerium in the cerium oxide lattice is (0.8~1.2):

1.

4. The biomimetic iron-doped cerium oxide nanoprobe targeting atherosclerotic plaques according to claim 1, characterized in that, The method for preparing the hybrid membrane includes the following steps: mixing macrophage membranes with iron-doped cerium oxide nanoparticles coated with liposomes, and then co-incubating, sonicating, and physically extruding the mixture to fuse the macrophage membranes with the liposome membranes, forming a hybrid membrane coated with the outer layer of iron-doped cerium oxide nanoparticles.

5. A method for preparing a biomimetic iron-doped cerium oxide nanoprobe targeting atherosclerotic plaques as described in claim 1, characterized in that, The preparation method includes the following steps: (1) Add citric acid monohydrate, cerium salt and iron salt in a molar ratio of 1:1:(0.03~0.12) to water, add ammonia water dropwise at 30~70℃ and react for 12~48h, then transfer to a reaction vessel and hydrothermally react at 75~90℃ for 12~48h; after dialysis purification, concentration and dispersion, Fe-CeO2 nanoparticle dispersion is obtained; (2) Dissolve lecithin, cholesterol and ROS-responsive amphiphilic polymer DSPE-TK-PEG2000 in an organic solvent, evaporate to form a film, add Fe-CeO2 nanoparticle dispersion from step (1), hydrate and sonicate to obtain TK@Fe-CeO2; (3) The macrophage membrane was co-incubated with TK@Fe-CeO2 in step (2), and then subjected to ultrasonication and physical extrusion to obtain biomimetic iron-doped cerium oxide nanoprobes MM / TK@Fe-CeO2 with an average particle size of 60~180nm.

6. The method for preparing biomimetic iron-doped cerium oxide nanoprobes targeting atherosclerotic plaques according to claim 5, characterized in that, In step (1), the cerium salt includes at least one of cerium chloride, cerium nitrate, cerium acetate, and cerium sulfate; the iron salt includes at least one of ferric chloride, ferric nitrate, ferric sulfate, ferrous chloride, ferrous sulfate, and ferric citrate.

7. The method for preparing biomimetic iron-doped cerium oxide nanoprobes targeting atherosclerotic plaques according to claim 5, characterized in that, In step (2), the mass ratio of lecithin DPPC, cholesterol and DSPE-TK-PEG2000 is (2~10):(1~5):1; the organic solvent is a mixed solution of dichloromethane and methanol with a volume ratio of (5~10):

1.

8. The method for preparing biomimetic iron-doped cerium oxide nanoprobes targeting atherosclerotic plaques according to claim 5, characterized in that, In step (3), the mass ratio of the macrophage membrane to the liposomes in the TK@Fe-CeO2 is 1:(6~12).

9. An MRI / CT dual-modal contrast agent, characterized in that, It includes 0.1 to 100 wt% of biomimetic iron-doped cerium oxide nanoprobes targeting atherosclerotic plaques as described in any one of claims 1 to 4; The MRI / CT dual-modal contrast agent, under the action of an external magnetic field and / or X-rays, can visualize atherosclerotic plaques.

10. An anti-atherosclerotic drug, characterized in that, It includes 0.1 to 100 wt% of biomimetic iron-doped cerium oxide nanoprobes targeting atherosclerotic plaques as described in any one of claims 1 to 4.

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