A palladium cerium-based nanoparticle, and a preparation method and application thereof

Abstract

The application discloses a palladium cerium-based nanoparticle and a preparation method and application thereof, and relates to the technical field of biological medicines.The palladium cerium-based nanoparticle disclosed by the application comprises a palladium cerium-based nanoparticle core and a neutrophil membrane coated on the surface of the nanoparticle core, and has good active oxygen scavenging, inflammation targeting and / or blood-brain barrier crossing functions; the palladium cerium-based nanoparticle can be used for relieving or treating perioperative neurocognitive disorders, and provides a new strategy and means for precise intervention of the disease.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a palladium-cerium-based nanoparticle, its preparation method, and its application. Background Technology

[0002] Perioperative neurocognitive impairment (PND) is a common central nervous system complication in elderly patients after anesthesia and surgery, leading to postoperative cognitive decline, prolonged hospital stay, and poor long-term prognosis, thus becoming a significant clinical problem in perioperative medicine. Current research indicates that the occurrence of PND is closely related to postoperative peripheral inflammation amplification, blood-brain barrier damage, neuroinflammatory activation, and oxidative stress imbalance. In particular, the entry of inflammatory signals into the brain and excessive accumulation of reactive oxygen species can further exacerbate abnormal microglial activation, synaptic damage, and neuronal dysfunction. Although anti-inflammatory drugs, antioxidants, and neuroprotective strategies are currently used to intervene in PND, problems remain, including poor targeting of brain tissue, limited blood-brain barrier penetration, insufficient lesion enrichment, and systemic side effects. Furthermore, there is a lack of precise intervention methods that combine targeted delivery of inflammation with efficient reactive oxygen species scavenging capabilities. Therefore, there is an urgent need to develop novel targeted therapeutic strategies.

[0003] In view of this, the present invention is proposed. Summary of the Invention

[0004] The present invention aims to provide a palladium-cerium-based nanoparticle, its preparation method and application, wherein the palladium-cerium-based nanoparticle has good reactive oxygen species scavenging, inflammation targeting and / or blood-brain barrier crossing functions; the palladium-cerium-based nanoparticle can be used to alleviate or treat perioperative neurocognitive impairment, providing a new strategy and means for precise intervention of this disease.

[0005] This invention is implemented as follows:

[0006] Existing technologies for the treatment of perioperative neurocognitive disorders have many shortcomings, as follows:

[0007] 1) Insufficient targeting of brain tissue: Traditional anti-inflammatory drugs or antioxidants are difficult to effectively accumulate in the brain injury area after systemic administration, resulting in low drug concentration at the lesion site and easy to cause systemic adverse reactions;

[0008] 2) Low blood-brain barrier penetration efficiency: Most therapeutic molecules and nanoparticles have difficulty effectively crossing the blood-brain barrier, resulting in limited central delivery efficiency;

[0009] 3) Insufficient intervention by a single mechanism: Treatment strategies that focus solely on anti-inflammation or simply on clearing reactive oxygen species are unlikely to simultaneously improve multiple pathological changes, such as blood-brain barrier damage, amplified neuroinflammation, abnormal microglial activation, and synaptic damage.

[0010] 4) Lack of precise delivery methods: Current technologies lack biomimetic nanotherapy systems that combine inflammation targeting, immune escape, and efficient reactive oxygen species removal capabilities, and in particular, there is a lack of effective strategies for precise intervention of PND in elderly individuals.

[0011] Based on this, the present invention provides palladium-cerium-based nanoparticles to overcome or solve one or all of the above-mentioned defects.

[0012] On one hand, the present invention provides a palladium-cerium-based nanoparticle, wherein the palladium-cerium-based nanoparticle includes a nanoparticle core and a neutrophil membrane coated on the surface of the nanoparticle core; wherein the nanoparticle core (Pd / hCeO2 nanoparticle) includes a CeO2 nanoparticle with a hollow structure and palladium metal particles deposited on the surface of the CeO2 nanoparticle.

[0013] Cerium-based nanomaterials depend on Ce³⁺ / Ce 4 ⁺ While possessing a reversible cyclic and oxygen vacancy structure, exhibiting reactive oxygen species (ROS) scavenging capabilities, traditional cerium-based nanomaterials still suffer from limited catalytic activity and insufficient lesion delivery efficiency. This invention addresses this by introducing palladium (Pd) metal particles onto the surface of hollow CeO2 nanoparticles to construct a heterostructure, which is then coated with neutrophil membranes. This enhances the material's catalytic activity and ROS scavenging capacity, while also endowing the palladium-cerium-based nanoparticles with inflammatory targeting and / or blood-brain barrier specificity, making them suitable for intervention and treatment of PND-related oxidative stress microenvironments.

[0014] Experimental results from embodiments of the present invention show that the palladium-cerium-based nanoparticles provided by the present invention exhibit a variety of beneficial effects beyond the expectations of those skilled in the art; such as better reactive oxygen species scavenging ability, restoration of atresia zona 1 (ZO-1) and occludin in the hippocampal microvessels and hippocampal tissue of PND mouse models, effective reduction of abnormal activation of microglia in the hippocampus of PND model mice, inhibition of glycolytic metabolic reprogramming, and reduction of apoptosis damage in the hippocampus of PND model mice; targeting brain tissue and effectively improving the learning and recognition memory functions of model mice, etc.

[0015] Optionally, in some embodiments of the present invention, the above-mentioned palladium-cerium-based nanoparticles have a particle size of 80-250 nm as detected by DLS; optionally, they have a particle size of 100-220 nm; and optionally, they have a particle size of 190-200 nm.

[0016] Optionally, in some embodiments of the present invention, the surface zeta potentials of the palladium-cerium-based nanoparticles (CeO2 nanoparticles), the palladium-cerium-based nanoparticle core, and the palladium-cerium-based nanoparticles are 26.05 mV, -12.69 mV, and -20.89 mV, respectively.

[0017] The Ce³⁺ ions and Ce in the core of the nanoparticles 4 The molar ratio of ⁺ ions (Ce³⁺ / Ce) 4 The value is 0.40-0.45.

[0018] Optionally, in some embodiments of the present invention, the particle size of the nanoparticles is determined by dynamic light scattering (DLS), and the results are expressed as mean ± standard deviation. This method can be referenced in ISO 22412 "Particle size analysis—Dynamic light scattering (DLS)" and its current version. The zeta potential of the nanoparticle surface is determined by electrophoretic light scattering (ELS) or laser Doppler electrophoresis, and the results are expressed in mV. This method can be referenced in ISO 13099 "Colloidal systems—Methods for zeta-potential determination" and its current version. Ce³⁺ / Ce 4 The molar ratio was determined using X-ray photoelectron spectroscopy (XPS).

[0019] Optionally, in some embodiments of the present invention, the neutrophil membrane described above is derived from the cell membrane of inflammation-induced neutrophils.

[0020] Neutrophils are among the first immune cells recruited to lesion sites during inflammatory responses, possessing innate inflammatory tropism, transendothelial migration, and lesion recognition capabilities. This invention utilizes an inflammation-induced homologous neutrophil membrane to coat Pd / hCeO2 nanoparticles. Compared to ordinary neutrophil membranes, this inflammation-induced homologous neutrophil membrane exhibits higher levels of inflammation-targeting and endothelial adhesion-related membrane proteins, including β2-integrin (β2-integrin / CD18), CD11a (integrin αL subunit), CD11b (integrin αM subunit), and / or CD44; the expression levels of these membrane proteins can be characterized by Western blot analysis. Therefore, while retaining the reactive oxygen species scavenging ability of the nanoparticles, this invention further endows the nanoparticles with the ability to target inflammation, escape immune responses, and cross the blood-brain barrier, thereby improving their delivery efficiency in perioperative brain injury areas.

[0021] The methods for preparing inflammation-induced neutrophils and neutrophil membranes involved in the embodiments of the present invention can refer to the methods and steps described in Example 1 of the present invention; in other embodiments, they can also be prepared by referring to other conventional methods in the art (WANG X, JIAO M, TIAN F, et al. A Biomimetic Nanoplatformwith Improved Inflammatory Targeting Behavior for ROS Scavenging-Based Treatment of Ulcerative Colitis [J]. Adv Healthc Mater, 2023, 12(29):e2301450.); this is obvious to those skilled in the art;

[0022] Optionally, in some embodiments of the present invention, the neutrophil membrane surface retains one or more inflammation-targeting-related adhesion molecules, including one or more of β2-integrin, CD11a, CD11b and CD44.

[0023] Optionally, in some embodiments of the present invention, the levels of inflammation-targeting-related membrane proteins on the neutrophil membrane are higher than those on the non-inflammatory-induced neutrophil membrane.

[0024] Optionally, in some embodiments of the present invention, the palladium-cerium-based nanoparticles described above have one or more of the following activities: superoxide anion (•O2⁻) scavenging activity, hydroxyl radical (•OH) scavenging activity, and hydrogen peroxide decomposition activity.

[0025] The detection methods for superoxide anion (•O2⁻) scavenging activity, hydroxyl radical (•OH) scavenging activity, and hydrogen peroxide decomposition activity involved in the embodiments of the present invention are as follows: •O2⁻ and •OH scavenging activities can be detected using electron spin resonance (ESR) combined with spin trapping, preferably using DMPO as the spin trapping agent. •OH can be generated from the Fe²⁺ / H₂O₂ Fenton reaction system, and the •OH scavenging ability is evaluated by detecting changes in the characteristic signal intensity of DMPO-•OH; •O2⁻ can be generated from the KO₂ source system, and the •O2⁻ scavenging ability is evaluated by detecting changes in the characteristic signal intensity of DMPO-OOH. Hydrogen peroxide decomposition activity can be detected using UV-Vis method or a hydrogen peroxide detection kit, and the H₂O₂ decomposition ability of the sample is evaluated by measuring the residual H₂O₂ or its characteristic absorbance change after the reaction.

[0026] On the other hand, the present invention provides the use of palladium-cerium-based nanoparticles as described in any of the preceding claims in the preparation of medicaments for treating perioperative neurocognitive disorders.

[0027] Optionally, in some embodiments of the present invention, when the above-mentioned drug is applied to the individual in need of treatment, it may produce one or more of the following effects: reducing surgery-induced blood-brain barrier damage, reducing neuroinflammation, inhibiting microglia glycolytic metabolic reprogramming, reducing synaptic damage, and reducing abnormal microglia activation.

[0028] Optionally, in some embodiments of the present invention, the aforementioned perioperative neurocognitive impairment includes one or more of postoperative learning and memory impairment, postoperative cognitive recovery delay, and postoperative neurocognitive impairment.

[0029] Optionally, in some embodiments of the present invention, the individual to be treated is an elderly patient; such as a patient aged 65 years or older.

[0030] On the other hand, the present invention provides a medicament for alleviating or treating perioperative neurocognitive disorders, the medicament comprising palladium-cerium-based nanoparticles as described in any of the preceding claims and a pharmaceutically acceptable carrier.

[0031] On the other hand, the present invention provides a method for relieving or treating perioperative neurocognitive impairment, comprising: administering a therapeutically effective amount of the drug as described above to the individual requiring treatment.

[0032] On the other hand, the present invention provides a method for preparing palladium-cerium-based nanoparticles as described in any of the preceding claims, comprising: depositing palladium metal particles on the surface of CeO2 nanoparticles to obtain a palladium-cerium-based nanoparticle core, and then coating the palladium-cerium-based nanoparticle core with a neutrophil cell membrane to obtain palladium-cerium-based nanoparticles.

[0033] Palladium-cerium-based nanoparticle cores can also be prepared using existing techniques (A Nanocapsule System Combats Aging by Inhibiting Age-Related Angiogenesis Deficiency and Glucolipid Metabolism Disorders; Bo Li et al.).

[0034] Optionally, in some embodiments of the present invention, the above-mentioned neutrophil membrane is coated onto the surface of the core of the above-mentioned palladium-cerium-based nanoparticles by means of extrusion, ultrasonication, mechanical fusion or a combination thereof. Attached Figure Description

[0035] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 The image shows a transmission electron microscope (TEM) image of the hCeO2 nanospheres in Example 1 of this invention. As can be seen from the image, the hCeO2 nanospheres have a uniform spherical structure and obvious hollow morphological features.

[0037] Figure 2 The image shows a TEM image of the Pd / hCeO2 nanoparticles in Example 1 of this invention. As can be seen from the image, Pd was successfully deposited on the surface of the hCeO2 nanospheres to form Pd / hCeO2 heterostructure nanoparticles with a diameter of approximately 100 nm.

[0038] Figure 3 The X-ray diffraction (XRD) patterns of Pd / hCeO2 nanoparticles and hCeO2 nanospheres in Example 1 of this invention are shown. The results show that in addition to retaining the characteristic diffraction peaks of CeO2, the Pd / hCeO2 nanoparticles also exhibit characteristic peaks of Pd around 40°, 46° and 68°, indicating that Pd was successfully loaded onto the hCeO2 surface and formed a Pd / hCeO2 heterostructure.

[0039] Figure 4 The image shows the X-ray photoelectron spectroscopy (XPS) spectra of Pd / hCeO2 nanoparticles and hCeO2 nanospheres in Example 1 of this invention. The results show the Ce³⁺ / Ce content of the Pd / hCeO2 nanoparticles. 4 The ⁺ molar ratio is approximately 0.43, which is higher than the 0.17 of hCeO2 nanospheres, indicating that the introduction of Pd can increase the Ce³⁺ ratio and oxygen vacancy level on the material surface, thereby enhancing its interfacial electronic modulation effect.

[0040] Figure 5 The image shows the dynamic light scattering (DLS) patterns of hCeO2 nanospheres, Pd / hCeO2 nanoparticles, and PCN (Pd / hCeO2 coated with neutrophil membrane and highly expressing adhesion receptors) in Example 1 of this invention. The results show that the particle sizes of hCeO2 nanospheres, Pd / hCeO2, and PCN are 142.7, 175.7, and 194.9 nm, respectively.

[0041] Figure 6 The image shows a TEM image of PCN in Example 2 of this invention. As can be seen from the image, there is a clear membrane structure around the Pd / hCeO2 nanoparticles, indicating that the adhesion receptor-expressing homologous neutrophil membrane was successfully coated on the surface of the nanoparticles.

[0042] Figure 7 The image shows the Zeta potentials of hCeO2, Pd / hCeO2, and PCN in Example 2 of this invention. The results show that the Zeta potentials of hCeO2, Pd / hCeO2 nanoparticles, and PCN are 26.05, -12.69, and -20.89 mV, respectively, indicating that PCN was successfully constructed.

[0043] Figure 8 The removal of •O from hCeO2, Pd / hCeO2 and PCN obtained in Examples 1 and 2 2− Electron spin resonance (ESR) plots of Pd / hCeO2 and PCN significantly reduced the electron spin resonance of •OH. 2− The characteristic peak intensity of •OH is higher than that of hCeO2 nanospheres.

[0044] Figure 9 This is a Western blot image of the adhesion receptor highly expressing homologous neutrophil membrane-related proteins in Example 2 of the present invention. The results show that the extracted neutrophil membrane retains inflammation-targeting-related membrane proteins.

[0045] Figure 10 Image (a) is an immunofluorescence assay of tight junction protein expression in the hippocampus of mice in different treatment groups in Example 3 of this invention. Figure 10 (b) shows the Western blot analysis of tight junction protein expression in the hippocampus of mice in different treatment groups. The results showed that the expression of ZO-1 and Occludin proteins in the hippocampus of mice in the PND group was significantly downregulated, and the expression was restored after treatment with Pd / hCeO2 and PCN, with the PCN group showing a more significant recovery.

[0046] Figure 11 The images show the abnormal activation of microglia in different treatment groups in Example 4 of this invention. The results show that PCN has the strongest ability to inhibit the pro-inflammatory activation of microglia induced by anesthesia and surgery, which is significantly better than Pd / hCeO2.

[0047] Figure 12 The image shows the expression of glycolysis-related molecules in different treatment groups in Example 4 of this invention. The results show that PCN has the strongest ability to inhibit the reprogramming of glucose metabolism in microglia, which is significantly better than Pd / hCeO2.

[0048] Figure 13 The results of TUNEL staining in the dentate gyrus of the hippocampus of mice in different treatment groups showed that the number of TUNEL-positive cells in the dentate gyrus of the hippocampus of mice in the PND group was significantly increased. After treatment with Pd / hCeO2 and PCN, the number of TUNEL-positive cells decreased, with the improvement being more significant in the PCN group.

[0049] Figure 14 The results showed that the PCN could actively recruit surgically induced inflammatory brain regions and remain there for at least 24 hours.

[0050] Figure 15 Figure 1 shows the results of the water maze test in mice under different treatment groups; among them, Figure 15 (a) shows the escape latency results of mice in different treatment groups during the water maze training period. Figure 15 (b) shows the results of the time mice spent in the target quadrant of the water maze in different treatment groups. The results showed that the learning and memory abilities of mice in the PND group were significantly reduced; after PCN treatment, the escape latency of mice was shortened, the time spent in the target quadrant was increased, and the learning and memory abilities were significantly improved.

[0051] Figure 16 The results of the novel object recognition experiment in mice under different treatment groups show that PCN can improve the novel object recognition ability and cognitive function of PND model animals, and the effect is significantly better than Pd / hCeO2. Detailed Implementation

[0052] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. It should be understood that the following embodiments are only for illustrating the present invention and are not intended to limit the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all conventional products that can be purchased commercially.

[0053] All publications, patent applications, patents and other references mentioned in this article are incorporated herein by reference in their entirety.

[0054] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention pertains. The singular terms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise.

[0055] As used in this article, unless otherwise specified, the word “or” is used in an inclusive sense of “and / or” rather than in an exclusive sense of “either / or”.

[0056] As used herein, the term "comprising" means that other elements may be present in addition to the defined elements. The use of "comprising" indicates inclusion, not limitation. In other words, the term "comprising" means "primarily includes, but not necessarily only includes." Furthermore, variations of the word "comprising" (e.g., "including," "containing") have the same meaning accordingly. On the one hand, the techniques described herein relate to the compositions, systems, methods, and their respective components, which are essential to the invention but may also include unspecified necessary or non-essential elements ("comprising").

[0057] The term "therapeutic effective dose" as used herein can refer to an amount sufficient to achieve the desired therapeutic effect or to act on adverse symptoms but generally insufficient to cause adverse side effects. For any given patient, the specific therapeutic effective dose level will depend on a variety of factors, including the disease being treated and its severity; the specific composition used; the patient's age, weight, general health, sex, and diet; the time of administration; the route of administration; the excretion rate of the specific compound used; the duration of treatment; drugs used in combination with or concurrently with the specific compound used; and similar factors well known in the medical field. For example, those skilled in the art know that a compound can be started at a dose less than that required to achieve the desired therapeutic effect and the dose can be gradually increased until the desired effect is achieved. If necessary, the effective daily dose can be divided into multiple doses for administration. Therefore, a single-dose composition may contain such a dose or submultiples of such a dose to constitute a daily dose. A physician may adjust the dose individually if there are any contraindications. Dosage can be varied and can be administered as follows: once or multiple times daily, for one or several days, once weekly, twice weekly, etc. Guidelines for appropriate dosages for specific classes of medicines can be found in the literature.

[0058] As used in this article, “pharmaceutically acceptable” can mean any dosage form of compound, preparation, or composition suitable for contact with human subjects or their tissues and, as needed, for animal use, without excessive toxicity or irritation, and with reduced side effects or complications when used as a consumable or administered, and with a reasonable benefit / risk ratio.

[0059] Generally, the properties of a pharmaceutically acceptable carrier will depend on the specific route of administration. For example, parenteral preparations typically contain injectable fluids, including pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, glucose solutions, glycerol, etc., as solvents. For solid compositions (e.g., in powder, pill, tablet, or capsule form), conventional non-toxic solid carriers may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition to be administered may also contain small amounts of non-toxic excipients, such as wetting agents or emulsifiers, preservatives, and pH buffers, such as sodium acetate or sorbitol monolaurate.

[0060] The terms “individual,” “subject,” “host,” “animal,” and “patient” used in this article are used interchangeably to refer to any subject or any mammal for diagnosis, treatment, prevention, or therapy as required; for example, humans (e.g., adults, adolescents, young children, older adults, children, infants, and fetuses), companion animals (e.g., pets, horses), livestock, or other animals.

[0061] The term "treatment" as used herein can refer to both therapeutic procedures and preventative or preventative measures aimed at preventing, reducing, mitigating, inhibiting, or eliminating adverse physiological changes, symptoms, diseases, or conditions (such as cancer). The features and performance of the invention are further described in detail below with reference to embodiments.

[0062] Example 1

[0063] Preparation and characterization of Pd / hCeO2 nanoparticles

[0064] In this embodiment, hollow CeO2 nanospheres are first prepared, and then Pd is loaded on their surface to construct Pd / hCeO2 (nanoparticle core) heterostructure nanoparticles.

[0065] 1. Preparation of hollow CeO2 nanospheres

[0066] The preparation of hCeO2 was based on previous research reports with appropriate optimizations. The specific steps are as follows:

[0067] 1) Weigh 1 g of Ce(NO3)3·6H2O and 0.4 g of PVP, add them to 30 mL of ethylene glycol, and dissolve them completely;

[0068] 2) Add 2 mL of 1 M HCl to the above mixture and mix thoroughly to obtain a homogeneous reaction solution;

[0069] 3) Transfer the resulting mixed solution to a polytetrafluoroethylene-lined reactor and react at 160 °C for 3 h;

[0070] 4) After the reaction is complete, cool to room temperature and collect the precipitate by centrifugation;

[0071] 5) Wash the precipitate three times with acetone and deionized water in sequence, freeze-dry it and set it aside for later use to obtain hCeO2 nanospheres.

[0072] Figure 1 The image shows a TEM image of the hCeO2 nanospheres in this embodiment. As can be seen from the image, the hCeO2 nanospheres have a uniform spherical structure and obvious hollow morphological features.

[0073] 2. Preparation of Pd / hCeO2 nanoparticles

[0074] Based on the above-mentioned hCeO2 nanospheres, Pd / hCeO2 heterostructure nanoparticles were further constructed. The specific steps are as follows:

[0075] 1) Take 40 mg of hCeO2 nanospheres, add 20 mg of Na2PdCl4, 20 mg of PVP, 20 mg of ascorbic acid (AA) and 65 mg of KCl, and then add 8 mL of deionized water;

[0076] 2) Disperse the resulting mixture using ultrasound to ensure a homogeneous reaction system;

[0077] 3) The reaction system was then heated at 80 °C for 3 h;

[0078] 4) After the reaction is complete, centrifuge to collect the product and wash it with deionized water to remove free components;

[0079] 5) Finally, Pd / hCeO2 nanoparticles were obtained.

[0080] Figure 2 The image shows a TEM image of the Pd / hCeO2 nanoparticles in this embodiment. As can be seen from the image, Pd was successfully deposited on the surface of the hCeO2 nanospheres to form Pd / hCeO2 heterostructure nanoparticles with a diameter of approximately 100 nm.

[0081] Figure 3 The XRD results for the Pd / hCeO2 nanoparticles and hCeO2 nanospheres in this embodiment show that, in addition to retaining the characteristic diffraction peaks of CeO2, the Pd / hCeO2 nanoparticles also exhibit characteristic peaks of Pd around 40°, 46°, and 68°. This indicates that the Pd micro-nanostructure was successfully loaded onto the hCeO2 surface in a particulate form, forming a Pd / hCeO2 heterostructure. Pd was successfully loaded onto the hCeO2 surface, forming a Pd / hCeO2 heterostructure.

[0082] Figure 4The above are XPS images of Pd / hCeO2 nanoparticles and hCeO2 nanospheres in this embodiment. The results show the Ce³⁺ / Ce content of the Pd / hCeO2 nanoparticles. 4 The ⁺ molar ratio is approximately 0.43, which is higher than the 0.17 of hollow CeO2 nanospheres, indicating that the introduction of Pd can increase the Ce³⁺ ratio and oxygen vacancy level on the material surface, thereby enhancing its interfacial electronic modulation effect.

[0083] 3. Basic characterization of Pd / hCeO2 nanoparticles

[0084] The particle size, surface potential, and reactive oxygen species scavenging ability of the hCeO2 nanospheres and Pd / hCeO2 nanoparticles obtained in this embodiment were detected.

[0085] Figure 5 The following are DLS images of hCeO2 nanospheres and Pd / hCeO2 nanoparticles in this embodiment. The results show that the particle sizes of hCeO2 nanospheres and Pd / hCeO2 nanoparticles are 142.7 nm and 175.7 nm, respectively.

[0086] Figure 8 The ESR diagrams of hCeO2 nanospheres and Pd / hCeO2 nanoparticles obtained in this embodiment for removing •O2⁻ and •OH are shown. The results show that Pd / hCeO2 nanoparticles significantly reduced the characteristic peak intensities of •O2⁻ and •OH, and their effect was better than that of hCeO2 nanospheres.

[0087] In summary, this embodiment successfully prepared Pd / hCeO2 heterostructured nanoparticles. The obtained Pd / hCeO2 nanoparticles possess uniform morphology, stable structure, and good reactive oxygen species scavenging ability, and can be used for the subsequent construction of biomimetic nano-formulations PCN.

[0088] Example 2

[0089] Based on the Pd / hCeO2 nanoparticles obtained in Example 1, this embodiment extracts the adhesion receptor-highly expressed homologous neutrophil membrane and constructs Pd / hCeO2 biomimetic nanoparticles (PCN) coated with homologous neutrophil membranes.

[0090] The first step is to obtain homologous neutrophils that highly express adhesion receptors:

[0091] 1) Two-month-old C57BL / 6 mice were selected and intraperitoneally injected with LPS to induce neutrophil activation in vivo;

[0092] 2) Six hours after LPS treatment, the mice were anesthetized and euthanized by cervical dislocation. The bilateral tibias and femurs were removed, and the attached soft tissues and skin were peeled off.

[0093] 3) Soak the bone tissue in 1×HBSS-0.38% sodium citrate buffer solution for later use;

[0094] 4) Cut off the epiphyses at both ends of the tibia and femur, repeatedly flush the bone marrow cavity with buffer solution using a syringe, and blow and agitate to obtain a bone marrow cell suspension until the bone fragment turns white;

[0095] 5) After filtering the obtained cell suspension through a 70 μm nylon filter, centrifuge at 230 ×g for 6 min and collect the cell pellet;

[0096] 6) After discarding the supernatant, resuspend the cells in 2 mL of buffer, add buffer to 40 mL, centrifuge at 230 ×g for 6 min, discard the supernatant and resuspend to obtain bone marrow cell suspension.

[0097] The second step is the isolation of neutrophils:

[0098] 1) Slowly add 72%, 64%, and 52% Percoll solution sequentially to the centrifuge tube;

[0099] 2) Gently spread the above bone marrow cell suspension on top of the 52% Percoll layer;

[0100] 3) Centrifuge at 4 ℃ and 1545 ×g for 30 min;

[0101] 4) Collect cellular components located at the 64% and 72% Percoll interface;

[0102] 5) Wash the obtained cell solution with 1×HBSS-0.38% sodium citrate buffer, centrifuge at 1545×g for 5 min, and repeat twice;

[0103] 6) Then add three volumes of red blood cell lysis buffer, mix gently and incubate at room temperature for about 3 minutes;

[0104] 7) Add 3 mL of 1×PBS to terminate the lysis, centrifuge at 1000 rpm for 3 min, and wash once with 1×PBS to obtain a suspension of mouse bone marrow-derived neutrophils.

[0105] Next was the extraction of adhesion receptor-highly expressed homologous neutrophil membranes:

[0106] The isolated neutrophils were subjected to hypotonic lysis, followed by differential centrifugation and membrane purification to separate cell membrane components, yielding the inflammatory-induced homologous neutrophil membrane, i.e., the homologous neutrophil membrane with high expression of adhesion receptors.

[0107] Figure 9This is a Western blot image showing the high expression of adhesion receptor-related proteins of homologous neutrophil membranes in this embodiment. The results show that the extracted neutrophil membranes retained inflammation-targeting membrane proteins. Western blot was used to further verify the expression of β-integrin, CD11a, CD11b, and CD44 in neutrophil membranes (uninduced by inflammation), inflammatory-induced neutrophil membranes, and PCN. The results indicate that PCNs retain characteristic membrane proteins derived from neutrophil membranes, and their expression levels are significantly higher than those in uninduced neutrophil membranes.

[0108] Finally, the preparation of PCN:

[0109] 1) The Pd / hCeO2 nanoparticles prepared in Example 1 were mixed with the above-mentioned adhesion receptor-overexpressing homologous neutrophil membrane at a mass ratio of 1:5;

[0110] 2) Ultrasonic treatment for 3 min under 60 W ice water bath conditions to fully fuse the membrane material with Pd / hCeO2 nanoparticles;

[0111] 3) The mixed solution was then filtered sequentially through 800 nm, 400 nm and 200 nm polycarbonate membranes to remove agglomerated particles and improve coating uniformity;

[0112] 4) Finally, Pd / hCeO2 biomimetic nanoparticles (PCN) coated with homologous neutrophil membranes that highly express adhesion receptors were obtained.

[0113] Figure 6 The image shows a TEM image of the PCN in this embodiment. As can be seen from the image, there is a clear membrane structure around the Pd / hCeO2 nanoparticles, indicating that the adhesion receptor-expressing homologous neutrophil membrane was successfully coated on the surface of the nanoparticles.

[0114] 5. Basic Characterization and Functional Testing of PCN

[0115] The PCN obtained in this embodiment was tested for particle size, surface potential, and reactive oxygen species scavenging ability.

[0116] Figure 5 The following are DLS images of hCeO2 nanospheres, Pd / hCeO2 nanoparticles, and PCN in this embodiment. The results show that the particle sizes of hCeO2 nanospheres, Pd / hCeO2 nanoparticles, and PCN are 142.7 nm, 175.7 nm, and 194.9 nm, respectively.

[0117] Figure 7The image shows the Zeta potential diagrams of hCeO2 nanospheres, Pd / hCeO2 nanoparticles, and PCN in this embodiment. The results show that the surface potentials of hCeO2 nanospheres, Pd / hCeO2 nanoparticles, and PCN change sequentially, indicating that PCN was successfully constructed.

[0118] Figure 8 The images show the ESR diagrams of hCeO2 nanospheres, Pd / hCeO2 nanoparticles, and PCN obtained in Examples 1 and 2 for scavenging •O2⁻ and •OH. The results show that both Pd / hCeO2 nanoparticles and PCN significantly reduced the characteristic peak intensities of •O2⁻ and •OH, and their effects were better than those of hCeO2 nanospheres.

[0119] In summary, this embodiment successfully prepared Pd / hCeO2 biomimetic nanoparticles (PCNs) coated with homologous neutrophil membranes that highly express adhesion receptors. The obtained PCNs have a stable membrane-coated structure, suitable particle size and surface potential, and highly express characteristic membrane proteins of neutrophil membranes. They also possess good reactive oxygen species scavenging ability and can be used for subsequent in vivo and in vitro functional verification related to perioperative neurocognitive impairment.

[0120] Example 3

[0121] To verify the ameliorative effects of Pd / hCeO2 and PCN on blood-brain barrier damage in PND model mice as described in Examples 1 and 2 of this invention, this example uses immunofluorescence staining and Western blot experiments to detect the expression of tight junction proteins ZO-1 and Occludin in hippocampal microvessels. The specific experimental methods are as follows:

[0122] 1) An 18-month-old male C57BL / 6 mouse model of pulmonary dysplasia (PND) was established. Mice were anesthetized with 4–5% isoflurane inhalation in an anesthesia induction chamber. After entering a stable state of anesthesia, the anesthesia was maintained with 2.1% isoflurane, and intramedullary fixation of tibial fractures was performed under 30% oxygen conditions. Control group mice received the same anesthesia and fixation treatment but did not undergo fracture surgery.

[0123] 2) The experimental animals were randomly divided into a control group, a PND group, and a PND+Pd / hCeO2 group. Figure 10 PC) and PND+PCN group ( Figure 10 (PCN). At 2 h and 24 h postoperatively, Pd / hCeO2 or PCN was administered via the tail vein, while the control group and PND group received an equal volume of PBS.

[0124] 3) Mice were sacrificed at the specified time points after surgery, and hippocampal tissue was quickly collected and frozen sections were prepared. Immunofluorescence staining was used to detect the expression of tight junction proteins ZO-1 and Occludin, and co-stained with the vascular endothelial cell marker CD31 to observe changes in tight junction structures in hippocampal microvessels.

[0125] 4) At the same time, total protein was extracted from the hippocampus tissue of mice in each group, and the expression levels of ZO-1 and Occludin proteins were detected by Western blot to further verify the damage to the blood-brain barrier structure and its improvement.

[0126] Figure 10 (a) shows the immunofluorescence detection of tight junction protein expression in the hippocampus microvessels of mice in different treatment groups. Figure 10 (b) shows the Western blot results of tight junction protein expression in the hippocampus of mice in different treatment groups. The results showed that the expression of ZO-1 and Occludin proteins in the hippocampal microvessels and hippocampal tissue of mice in the PND group was significantly downregulated; after treatment with Pd / hCeO2 and PCN, the expression of the above proteins was restored, with the PCN group showing a more significant recovery.

[0127] Example 4

[0128] This example illustrates the regulatory effects of Pd / hCeO2 nanoparticles prepared in Example 1 and PCN prepared in Example 2 on abnormal activation of microglia and glycolytic metabolic reprogramming in PND model mice.

[0129] 1) The animal model, grouping, and administration methods described in Example 3 were used. Mice were sacrificed at specified time points after surgery, and brain tissue was isolated and frozen sections were prepared. Immunofluorescence staining was used to detect the expression of the microglial marker Iba1 and pro-inflammatory phenotype-related markers to evaluate the activation status of microglia in different treatment groups.

[0130] 3) At the same time, total protein was extracted from the hippocampus tissue of mice in each group, and the expression levels of glycolysis-related molecules HK2, PFKM, PKM2 and LDHA were detected by Western blot to evaluate the reprogramming of glycolysis metabolism in microglia in different treatment groups.

[0131] Figure 11 The images show the abnormal activation of microglia in different treatment groups. The results show that the pro-inflammatory activation of microglia in the hippocampus of mice in the PND group was significantly enhanced. After treatment with Pd / hCeO2 nanoparticles and PCN, the abnormal activation of microglia was reduced, with the PCN group showing more significant improvement.

[0132] Figure 12The images show the expression of glycolysis-related molecules in different treatment groups. The results show that the expression of glycolysis-related molecules in the hippocampus of mice in the PND group was significantly increased. After treatment with Pd / hCeO2 nanoparticles and PCN, the expression of the above molecules decreased, with the PCN group showing a more significant inhibitory effect.

[0133] In summary, this embodiment demonstrates that PCN can effectively alleviate abnormal activation of microglia in the hippocampus of PND model mice and inhibit glycolytic metabolic reprogramming, thereby exerting a good regulatory effect on neuroinflammation.

[0134] Example 5

[0135] This example illustrates the effect of Pd / hCeO2 nanoparticles prepared in Example 1 and PCN prepared in Example 2 on improving apoptosis damage in the hippocampus of aged PND model mice.

[0136] The animal model, grouping, and administration methods described in Example 3 were used. Mice were sacrificed at specified time points post-surgery, and brain tissue was rapidly separated. Hippocampal tissue was collected and frozen sections were prepared. TUNEL staining was used to detect hippocampal cell apoptosis in different treatment groups, and TUNEL-positive cells were statistically analyzed to evaluate the protective effects of Pd / hCeO2 nanoparticles and PCN against surgery-induced brain tissue apoptosis.

[0137] Figure 13 The results show the TUNEL staining results in the hippocampus of mice in different treatment groups. The results showed that the number of TUNEL-positive cells in the hippocampus of mice in the PND group was significantly increased, suggesting that anesthesia and surgery can induce apoptosis and damage in the hippocampus. After treatment with Pd / hCeO2 nanoparticles and PCN, the number of TUNEL-positive cells decreased, with the PCN group showing more significant improvement.

[0138] In summary, this embodiment demonstrates that PCN can effectively reduce hippocampal cell apoptosis damage in PND model mice, suggesting that it has a good protective effect against surgery-induced brain tissue damage.

[0139] Example 6

[0140] This example illustrates the distribution characteristics, brain enrichment, and cognitive-improving effects of the PCN prepared in Example 2 in aged PND model mice.

[0141] The animal model, grouping, and administration methods described in Example 3 were used. To observe the enrichment of PCN in the brain, Cy5.5-labeled PCN was injected via the tail vein, and in vivo fluorescence imaging was performed on mice in each group at 12 h and 24 h post-administration. Mice were anesthetized and placed in a small animal in vivo imaging system during imaging. The fluorescence signal intensity of brain regions was recorded, and differences in brain fluorescence enrichment among different groups were compared to evaluate the brain-targeted delivery capability of PCN under postoperative inflammatory conditions.

[0142] Simultaneously, behavioral tests were performed on mice in each group at set postoperative time points. The water maze test was used to evaluate the spatial learning and memory abilities of mice in different treatment groups; the new object recognition test was used to evaluate the recognition and memory abilities of mice in different treatment groups, and the changes in cognitive function of each group were compared.

[0143] Figure 14 The images show in vivo fluorescence imaging of the brains of mice from different groups. The results showed that PCN exhibited stronger fluorescence enrichment in the surgically induced inflamed brain regions and remained there for at least 24 hours, suggesting that PCN has a good ability to target and enrich in inflamed brain regions.

[0144] Figure 15 Figures showing the results of the water maze test in mice under different treatment groups. Figure 15 (a) shows the escape latency results of mice in different treatment groups during the water maze training period. Figure 15 (b) shows the results of the time mice spent in the target quadrant of the water maze in different treatment groups. The results showed that the learning and memory abilities of mice in the PND group were significantly reduced; after PCN treatment, the learning and memory abilities of mice were significantly improved.

[0145] Figure 16 The results of the novel object recognition experiment in mice under different treatment groups are shown in the figure. The results show that the novel object recognition ability of mice in the PND group is significantly reduced; after PCN treatment, the recognition and memory ability of mice is significantly improved.

[0146] In summary, this embodiment demonstrates that PCN can achieve good enrichment in the brains of aged PND model mice and improve the learning and recognition memory functions of the model mice, thus exhibiting good central delivery ability and cognitive protection.

[0147] The Pd / hCeO2 biomimetic nanoparticles coated with homologous neutrophil membranes and highly expressing adhesion receptors provided in this invention are innovative in the following aspects:

[0148] 1. Construction of a biomimetic delivery vector for neutrophil membranes with high expression of adhesion receptors:

[0149] By utilizing adhesion receptors that highly express inflammation-adhesion-related membrane proteins retained in the membranes of homologous neutrophils, nanoformulations possess the ability to target inflammation, escape immune signals, and cross the blood-brain barrier, thereby achieving precise delivery to surgically induced inflammatory brain regions and improving the enrichment efficiency at lesion sites.

[0150] 2. Functional design of Pd / hCeO2 heterostructure nanoparticles:

[0151] By introducing Pd onto the surface of hollow CeO2 nanospheres, Pd / hCeO2 heterostructure nanoparticles are constructed, which enhances the Ce³⁺ ratio and oxygen vacancy level on the material surface, improves the interfacial electronic regulation effect and the ability to scavenge various reactive oxygen species, and thus more effectively copes with the PND-related oxidative stress microenvironment.

[0152] 3. Multi-mechanism synergistic intervention targeting key pathological stages of PND:

[0153] The PCN provided by this invention can not only remove excess reactive oxygen species and reduce oxidative stress damage, but also protect the integrity of the blood-brain barrier structure, inhibit abnormal activation of microglia and glycolytic metabolic reprogramming, reduce hippocampal cell apoptosis damage, and improve the learning and memory abilities of model animals, thereby achieving multi-stage synergistic intervention for perioperative neurocognitive impairment.

[0154] This invention overcomes the shortcomings of existing intervention strategies, such as insufficient targeting of brain tissue, low blood-brain barrier penetration efficiency, and difficulty in covering complex pathological processes with a single mechanism of action, and provides a new technical solution for the precise prevention and treatment of PND in the elderly.

[0155] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A palladium-cerium-based nanoparticle, characterized in that, The palladium-cerium-based nanoparticles include a palladium-cerium-based nanoparticle core and a neutrophil membrane coated on the surface of the nanoparticle core; wherein, the palladium-cerium-based nanoparticle core includes CeO2 nanoparticles with a hollow structure and palladium metal particles deposited on the surface of the CeO2 nanoparticles.

2. The palladium-cerium-based nanoparticles according to claim 1, characterized in that, The palladium-cerium-based nanoparticles have a particle size of 80-250 nm as detected by DLS; optionally, they have a particle size of 100-220 nm, and optionally, they have a particle size of 190-200 nm.

3. The palladium-cerium-based nanoparticles according to claim 1, characterized in that, The surface zeta potentials of the CeO2 nanoparticles, the palladium-cerium-based nanoparticle core, and the palladium-cerium-based nanoparticles are 26.05 mV, -12.69 mV, and -20.89 mV, respectively.

4. The palladium-cerium-based nanoparticles according to claim 1, characterized in that, The Ce³⁺ ions and Ce in the core of the nanoparticles 4 The molar ratio of ⁺ ions (Ce³⁺ / Ce) 4 The value is 0.40-0.

45.

5. The palladium-cerium-based nanoparticles according to any one of claims 1-4, characterized in that, The neutrophil membrane is derived from the cell membrane of inflammation-induced neutrophils; Optionally, the neutrophil membrane has one or more of the following inflammation-targeting membrane proteins: β2-integrin, CD11a, CD11b, and CD44; Optionally, the levels of inflammation-targeting membrane proteins on the neutrophil membrane are higher than those on the non-inflammatory-induced neutrophil membrane.

6. The palladium-cerium-based nanoparticles according to claim 5, characterized in that, The palladium-cerium-based nanoparticles possess one or more of the following activities: superoxide anion scavenging activity, hydroxyl radical scavenging activity, and hydrogen peroxide decomposition activity.

7. The use of the palladium-cerium-based nanoparticles according to any one of claims 1-6 in the preparation of a medicament for treating perioperative neurocognitive disorders.

8. The application according to claim 7, characterized in that, When the drug is administered to the individual to be treated, it can produce one or more of the following effects: reducing surgery-induced blood-brain barrier damage, reducing neuroinflammation, reducing synaptic damage, and reducing abnormal microglial activation.

9. A medicament for relieving or treating perioperative neurocognitive disorders, characterized in that, The drug comprises palladium-cerium-based nanoparticles as described in any one of claims 1-5 and a pharmaceutically acceptable carrier.

10. A method for preparing palladium-cerium-based nanoparticles as described in any one of claims 1-6, characterized in that, It includes: depositing palladium metal particles on the surface of CeO2 nanoparticles to obtain a palladium-cerium-based nanoparticle core, and then coating the palladium-cerium-based nanoparticle core with a neutrophil cell membrane to obtain palladium-cerium-based nanoparticles.

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