Bionic nano platform as well as preparation method and application thereof

Abstract

The invention specifically discloses a bionic nano platform as well as a preparation method and application thereof, and relates to the technical field of biological medicines. According to the bionic nano platform disclosed by the invention, efficient conversion from ultrasonic energy to chemical energy (CO release) is realized by constructing a Schottky heterojunction structure of Ni and LDH. In addition, by means of tumor cell membrane coating, homologous recognition characteristics are endowed to the nano-material, so that the nano-material can actively target brain glioma and penetrate through BBB, and precise delivery and deep tumor enrichment are achieved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a biomimetic nanoplatform, its preparation method, and its application. Background Technology

[0002] Glioblastoma (GBM) is one of the most aggressive and malignant primary tumors of the central nervous system, characterized by high heterogeneity, strong invasiveness, and high drug resistance. Current standard clinical treatments primarily include surgical resection, radiotherapy, and chemotherapy regimens primarily based on temozolomide (TMZ). However, due to indistinct tumor boundaries, the blood-brain barrier (BBB) ​​hindering drug delivery, and tumor cell resistance to radiotherapy and chemotherapy, the overall median survival for patients remains less than 15 months, with a recurrence rate approaching 90%. Therefore, achieving effective BBB crossing, precise drug delivery, and simultaneous regulation of tumor metabolism and the immune microenvironment has become a critical scientific challenge in the treatment of GBM.

[0003] In recent years, nanotechnology has provided new insights into the precision diagnosis and treatment of tumors. Various inorganic nanomaterials, such as metal oxides, sulfides, and layered double hydroxides (LDHs), have been widely used in drug delivery, photothermal therapy (PTT), photodynamic therapy (PDT), and sonodynamic therapy (SDT). However, existing nanotherapy systems still face significant limitations in clinical translation: (1) Limited penetration and non-specific distribution: Traditional nanomedicines have difficulty effectively penetrating the BBB, have low enrichment efficiency in the body, and are easily cleared by the reticuloendothelial system, resulting in insufficient therapeutic dose and increased toxic side effects.

[0004] (2) Low energy conversion efficiency: Most acoustic or photosensitive nanomaterials rely on oxygen-involved energy conversion processes, which exhibit severe efficacy attenuation in the hypoxic tumor microenvironment. Oxygen-dependent mechanisms lead to insufficient generation of reactive oxygen species (ROS), limiting the therapeutic effect on deep solid tumors (especially brain tumors).

[0005] (3) Lack of precise and controllable treatment triggering mechanism: Traditional chemical or optical stimulation methods are difficult to achieve precise spatial and temporal control, often causing damage to non-target tissues. In contrast, ultrasound is considered a more clinically promising external energy source due to its good tissue penetration and focusability, but its energy coupling efficiency with nanocatalytic materials still needs to be improved.

[0006] (4) Insufficient synergy between metabolic regulation and immune activation: Most current nanotherapy systems only target the direct killing of tumor cells, neglecting the close link between abnormal tumor metabolism and immunosuppression. Abnormal mitochondrial oxidative phosphorylation (OXPHOS) in tumor cells not only maintains their high energy demands but also induces immune escape through metabolic byproducts (such as lactate and adenosine), keeping the tumor microenvironment (TME) in a state of continuous immunosuppression. How to simultaneously disrupt tumor metabolism and remodel the TME during treatment is the core issue for which there is currently no effective solution. Summary of the Invention

[0007] (a) Technical problems to be solved Therefore, one of the main objectives of this invention is to provide a Ni / LDH nanoparticle. By constructing a Schottky heterojunction structure of Ni and LDH, a highly efficient conversion of ultrasonic energy into chemical energy (CO release) is achieved. Furthermore, by encapsulating it on a tumor cell membrane, it is endowed with "homology recognition" properties, enabling it to actively target gliomas and cross the brain border (BBB), achieving precise delivery and deep tumor accumulation.

[0008] (II) Technical Solution To achieve the above objectives, the present invention provides Ni / LDH nanoparticles, including carbonyl-supported Ni nanosheets and FeNi-LDH nanosheets.

[0009] In one embodiment, the mass ratio of the carbonyl-supported Ni nanosheets to the FeNi-LDH nanosheets is 0.75:0.25 to 0.25:0.75.

[0010] In one embodiment, the mass ratio of the carbonyl-supported Ni nanosheets to the FeNi-LDH nanosheets is 0.33:0.67.

[0011] In another aspect, the present invention provides a method for preparing the above-mentioned Ni / LDH nanoparticles, comprising: S1: The nickel complex is reacted with the metal carbonyl complex to obtain the carbonyl-supported Ni nanosheets; S2: The FeNi-LDH nanosheets are obtained by reacting a solution of iron nitrate hydrate and nickel nitrate hydrate with an alkaline solution; S3: The Ni / LDH nanoparticles are obtained by electrostatic self-assembly of carbonyl-loaded Ni nanosheets and FeNi-LDH nanosheets.

[0012] In one embodiment, the molar ratio of nickel to carbonyl in S1 is 1:1.8.

[0013] In one embodiment, the nickel complex in S1 is Ni(acac)2, and the metal carbonyl complex is W(CO)6.

[0014] In one embodiment, the molar ratio of Ni(acac)2 to W(CO)6 is 1:0.3.

[0015] In one embodiment, Ni(acac)2 and W(CO)6 are dissolved in an organic solvent to react.

[0016] In one embodiment, the organic solvent is oleylamine (OAm).

[0017] In one embodiment, the reaction conditions are: reacting at 60°C for 20 min, then at 5°C for 1 min. -1 The heating rate was slowly increased to 180℃, and the reaction was carried out at 180℃ for 1 hour.

[0018] In one embodiment, the molar ratio of nickel to iron in S1 is 1:1.

[0019] In one embodiment, the iron nitrate hydrate is Fe(NO3)3·9H2O, and the nickel nitrate hydrate is Ni(NO3)2·6H2O.

[0020] In one embodiment, the molar ratio of Fe(NO3)3·9H2O and Ni(NO3)2·6H2O is 1:1.

[0021] In one embodiment, the solution of iron nitrate hydrate and nickel nitrate hydrate is mixed with an alkaline solution and aged for 1.5 hours, and then reacted in a reactor at 80°C for 24 hours.

[0022] In another aspect, the present invention also provides Ni / LDH nanoparticles obtained by the above preparation method.

[0023] In another aspect, the present invention also provides a biomimetic nanoplatform (Ni / LDH@M), comprising the above-mentioned Ni / LDH nanoparticles and cancer cell membranes.

[0024] In one embodiment, the mass ratio of the Ni / LDH nanoparticles to the cancer cell membrane is 1:2 to 2:1.

[0025] In one embodiment, the mass ratio of the Ni / LDH nanoparticles to the cancer cell membrane is 1:1.

[0026] In one embodiment, the cancer cell membrane is a glioma cell membrane.

[0027] In one embodiment, the cancer cell membrane is a GL261 cell membrane.

[0028] In another aspect, the present invention provides a pharmaceutical composition comprising: (1) Therapeutic effective amount of the above-mentioned Ni / LDH nanoparticles and / or biomimetic nanoplatform; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

[0029] In another aspect, the present invention provides a pharmaceutical preparation comprising the above-described pharmaceutical composition.

[0030] In another aspect, the present invention also provides a pharmaceutical product comprising the above-described pharmaceutical preparation.

[0031] In one embodiment, the pharmaceutical product is a vial or box.

[0032] In another aspect, the present invention also provides the use of the above-mentioned Ni / LDH nanoparticles, biomimetic nanoplatforms, pharmaceutical compositions, pharmaceutical formulations and / or pharmaceutical products in the preparation of drugs for the prevention and / or treatment of glioblastoma.

[0033] (III) Beneficial Effects This invention provides a biomimetic nanoplatform, its preparation method, and its applications. Compared with existing technologies, it has the following advantages: 1. A highly efficient energy conversion Ni / LDH heterojunction structure was established to achieve precise CO release driven by acoustic energy: By modulating the Schottky barrier structure between Ni nanosheets and LDH, the electron-hole separation efficiency was significantly improved. Under ultrasonic excitation, Ni acts as an electron trapping center, forming a directional potential gradient that promotes electron migration to the Ni surface and reduces carbonyl groups to generate CO. Simultaneously, LDH conduction band electrons can activate oxygen generation. 1 O2 further promotes the carbonyl oxidation reaction. This system can efficiently produce CO under both oxygen-rich and oxygen-deficient environments. In particular, under tumor hypoxic conditions, the CO release rate is nearly twice that under normal oxygen conditions, demonstrating excellent catalytic adaptability.

[0034] 2. Achieving non-invasive, ultrasound-controlled synergistic therapy of tumor metabolic inhibition and immune activation: In an orthotopic glioma mouse model, the Ni / LDH@M+US group exhibited the most significant anti-tumor effect, with tumor volume reduced by 96% and median survival extended to 52 days. H&E and TUNEL staining showed a significant increase in tumor tissue necrosis and apoptosis. Immunohistochemistry and flow cytometry further revealed that this treatment significantly increased the proportion of tumor-infiltrating CD8⁺ T cells and decreased the proportion of regulatory T cells (Tregs) and M2 macrophages, indicating that Ni / LDH@M can induce immunogenic cell death and remodel the immune microenvironment. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 Here are schematic diagrams of the preparation and characterization of Ni / LDH@M: (A) EDS elemental distribution map of Ni / LDH; (B) High-resolution transmission electron microscopy (HRTEM) image of Ni / LDH, showing clear lattice fringes; (C) HRTEM image of Ni / LDH@M; (D) Co-localization image of FITC-labeled Ni / LDH (green) and DiD-labeled GL261 cell membrane (red) under confocal laser scanning microscopy (CLSM); Figure 2 The following are catalytic performance analysis diagrams for Ni / LDH@M: (A) Valence band XPS spectra of LDH and Ni / LDH@M; (B) Mott–Schottky curves of LDHNSs and Ni / LDH NPs; (C) Tauc curves obtained based on UV–vis diffuse reflectance spectroscopy (DRS), used to estimate the optical band gap; (D) Schematic diagram of band structure evolution after Ni introduction and assembly; (E) Electrochemical impedance spectroscopy (EIS) Nyquist curve; (F) Current-time response under ultrasonic triggering; (G~I) Ultraviolet photoelectron spectroscopy (UPS) results used to determine the work function; (J) Schematic diagram of Schottky junction formation and charge transfer pathway under ultrasonic excitation; (K) CO and... 1 O2 generation; (L) Steady-state photoluminescence spectroscopy characterizing carrier recombination behavior; (E) Evaluation based on DPBF decolorization detection results at 410 nm. 1 O2 yield, and comparison with TiO2; (K, L legend: I, Ni; II, Ni) 0.75 / LDH 0.25 @M;III,Ni 0.67 / LDH 0.33 @M;IV, Ni 0.50 / LDH 0.50 @M;V,Ni 0.33 / LDH 0.67 @M;VI,Ni 0.25 / LDH 0.75 @M; VII, LDH); Figure 3 This is a schematic diagram of the catalytic mechanism of Ni / LDH@M; Figure 4The following are in vitro therapeutic evaluation images of Ni / LDH@M: (A) Intracellular CO levels after different treatments analyzed by flow cytometry; (B) Cytochrome c oxidase activity of GL261 cells after different treatments (PBS, Ni / LDH@M, Ni+US, Ni / LDH@M+US), expressed as a percentage relative to the untreated control; (C) Oxygen consumption rate (OCR) of cells after Ni / LDH@M+US treatment (n=8) measured using a Seahorse analyzer; (D) Confocal images showing the release of cytochrome c after different treatments; (E) Confocal images showing the expression of activated caspase-3 in GL261 cells after different treatments. Figure 5 Here are the blood-brain barrier permeability analysis diagrams of Ni / LDH@M: (A) Schematic diagram of the proposed mechanism of Ni / LDH@M crossing the blood-brain barrier (BBB); (B) In vivo fluorescence imaging of mice after intravenous injection of Cy5.5 Ni / LDH@M at different time points; Figure 6 The in vivo antitumor effect of Ni / LDH@M is shown in the following figures: (A) In vivo bioluminescence imaging of GL261-Luc tumor-bearing mice at different time points; (B) MRI images of the brains of representative mice in each treatment group. Figure 7 Here are the immune response effects of Ni / LDH@M against glioma: (A) Schematic diagram of the experimental procedure for flow cytometry (FCM) and immunofluorescence (IF) analysis of brain tissue and cervical lymph nodes in C57BL / 6 mice bearing GBM in situ; (B-D) IF images of the GBM region, showing Cyt c Release, CRT exposure, HMGB1, Ki-67, and cleavage caspase-3; scale bar 50 µm; (E–I) Quantitative analysis of immune cell populations in mouse brain tissue after four treatments: E) M1 glioma-associated microglia / macrophages (M1-like GAMs); F) M2 glioma-associated microglia / macrophages (M2-like GAMs); G) mature dendritic cells (mature DCs); H) CD8⁺ T cells; I) regulatory T cells (Tregs); data are expressed as mean ± standard deviation (mean ± SD, n = 3 mice / group); statistical analysis: all experiments were repeated three times, and data are expressed as mean ± SD; (E–I) one-way ANOVA was used. P<0.05, P<0.01, P<0.001, P<0.0001; Figure 8Here is a biosafety analysis chart of Ni / LDH@M: (A) Different doses (4~30 mg·kg) -1 (a) Evaluation of systemic toxicity of Ni / LDH@M in mice; (b) Absorbance of supernatant after treatment with different concentrations of Ni / LDH@M, deionized water (positive control), and PBS (negative control); (c) From left to right: H2O, Ni / LDH@M (12.5, 25, 50, 100, 200, 300, and 400 μg·mL⁻¹) -1 (D) Evaluation of systemic toxicity-related biochemical and hematological parameters; in PBS (control) or Ni / LDH@M (20 mg·kg⁻¹) -1 On day 14 after treatment, liver and kidney function indicators and complete blood cell count (CBC) of mice were measured; data are expressed as mean ± SD (n=3). Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Terms and Definitions As used in this article, the term "blood-brain barrier" is composed of tightly connected brain microvascular endothelial cells and is a physiological barrier that prevents most therapeutic drugs from entering brain tissue.

[0039] As used herein, the term "pharmaceutical composition" refers to a composition comprising Ni / LDH@M formulated together with one or more pharmaceutically acceptable carriers.

[0040] The formulation of a pharmaceutical composition can be tailored to the application. In particular, pharmaceutical compositions can be formulated using methods known in the art to provide rapid, continuous, or delayed release of the active ingredient upon administration to mammals. For example, the formulation can be selected from any of the following: plasters, granules, lotions, liniments, powders, syrups, liquids and solutions, aerosols, sprays, extracts, elixirs, ointments, fluid extracts, emulsions, suspensions, decoctions, infusions, tablets, suppositories, injections, alcoholic preparations, capsules, creams, lozenges, tinctures, pastes, pills, and soft or hard gelatin capsules.

[0041] As used herein, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or animals without excessive adverse effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0042] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to a carrier used for the administration of therapeutic agents, encompassing a variety of excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity upon administration. Suitable carriers are well known to those skilled in the art, and a thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0043] Pharmaceutically acceptable carriers in a composition include any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption-delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, flow aids, pH adjusters, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, etc., compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition may contain other active compounds that provide complementary, additional, or enhanced therapeutic functions. Solid carriers or excipients, such as lactose, starch, or talc, or liquid carriers, such as water, fatty oils, or liquid paraffin, are possible. Other examples of carriers include culture media, such as DMEM or RPMI; and cryogenic storage media containing components that scavenge free radicals, provide pH buffering, osmotic / osmotic support, energy substrates, and ion concentrations to balance intracellular states at low temperatures; and mixtures of organic solvents with water.

[0044] The active substance in the product disclosed in this invention accounts for 0.001-99.9 wt% of the total weight of the composition, with the remainder being pharmaceutically acceptable carriers and other additives.

[0045] The pharmaceutical compositions of the present invention can be administered using any known method. One of a variety of methods known to those skilled in the art can be used to administer the substance, compound, or agent to a subject using the terms "give" or "apply".

[0046] For example, compounds or agents can be administered intranasally (e.g., by inhalation), intrathecally (into the spinal canal or subarachnoid space), intraarterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ocularly, sublingually, orally (by ingestion), intracerebrally, and transdermally (by absorption, e.g., through a skin catheter). Compounds or agents can also be suitably introduced via rechargeable or biodegradable polymeric devices or other devices (e.g., patches and pumps or formulations) that provide prolonged, slowed, or controlled release of the compound or agent. Administration can also be performed, for example, once, multiple times, and / or over one or more prolonged periods.

[0047] As used herein, the term “therapeutic effective dose” refers to a dose sufficient to treat a disease with a reasonable benefit / risk ratio suitable for medical treatment, and the effective dose level includes subject type and severity, age, sex, drug activity, drug sensitivity, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant drugs, and other factors known in the medical field.

[0048] As used herein, the term “treatment” for a symptom or patient refers to steps taken to achieve a beneficial or desired outcome, including clinical outcomes. Beneficial or desired clinical outcomes include, but are not limited to, eliminating, substantially inhibiting, slowing, or reversing the progression of a disease, symptom, or condition; substantially improving or alleviating the clinical or aesthetic symptoms of a symptom; substantially preventing the clinical or aesthetic symptoms of a disease, symptom, or condition; and avoiding harmful or unpleasant symptoms. Treatment also refers to achieving one or more of the following: (a) reducing the severity of the symptom; (b) limiting the development of characteristic symptoms of the symptom being treated; (c) limiting the exacerbation of characteristic symptoms of the symptom being treated; (d) limiting the recurrence of the symptom in patients who previously had the symptom; and / or (e) limiting the recurrence of symptoms in patients who previously did not have symptoms of the symptom.

[0049] The term "prevention" refers to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptoms.

[0050] A subject’s “effective response” to a drug or treatment, or a subject’s “responsiveness” and similar terms, refers to a clinically or therapeutically beneficial effect conferred on a subject who is at risk of or has a disease or condition such as cancer. In one respect, such benefit includes one or more of the following: prolongation of survival (including overall survival and progression-free survival); objective response (including CR or PR); or improvement of signs or symptoms of cancer.

[0051] Subjects who “do not respond effectively” to treatment are defined as patients who do not have any of the following: prolonged survival (including overall survival and progression-free survival); objective response (including CR or PR); or improvement in signs or symptoms of cancer.

[0052] As used herein, the term “reduction or inhibition” refers to the ability to cause an overall reduction of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more. Reduction or inhibition can refer to the symptoms of the treated condition (e.g., cancer), the presence or size of metastases, or the size of the primary tumor.

[0053] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0054] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0055] Example 1: Preparation of Ni / LDH@M: 1. Synthesis of Ni nanosheets (Ni): 1 mmol of Ni(acac)₂ and 0.3 mmol of W(CO)₆ were dissolved in 7 mL of oleylamine (OAm) in a three-necked flask under argon protection. The solution was heated to 60°C with stirring and held for 20 min to ensure complete dissolution, followed by heating at 5°C for 1 min. -1 The heating rate was increased to 180°C, and the reaction was carried out at 180°C for 1 h to promote the nucleation and lateral growth of ultrathin nanosheets. After the reaction was completed and naturally cooled to room temperature, the reaction solution was added to a mixed solvent of ethanol / n-hexane with a volume ratio of 8:1, centrifuged at 8000 rpm for 10 min, and washed three times with the same mixed solvent to remove organic residues. The final product was dried under vacuum to obtain Ni nanosheets with an average lateral size of approximately 70 nm.

[0056] 2. Synthesis of FeNi-LDH nanosheets (LDH): Fe(NO3)3·9H2O (0.5 mmol) and Ni(NO3)2·6H2O (0.5 mmol) were dissolved in 50 mL of deoxygenated ultrapure water. 0.4 mL of concentrated nitric acid was added to ensure complete dissolution of the metal ions, yielding solution A. NaOH (0.05 mol) was dissolved in 50 mL of deoxygenated water to obtain solution B. Under nitrogen-purged three-necked flask conditions at room temperature, solutions A and B were simultaneously added dropwise to the deoxygenated ultrapure water reaction system with vigorous stirring. After the addition was complete, the mixture was aged for 1.5 h to ensure uniform co-precipitation. The mixture was then transferred to a 100 mL polytetrafluoroethylene-lined reactor and reacted at 80°C for 24 h. After the reaction was complete, the precipitate was collected by centrifugation (6000 rpm, 5 min), washed four times with nitrogen-saturated water, and dried under vacuum to obtain LDH. All aqueous systems were purged with N2 for 30 minutes before use to deoxygenate them, thereby minimizing the generation of oxidation defects during the synthesis process.

[0057] 3: Preparation of Ni / LDH nanoparticles (Ni / LDH): Under constant magnetic stirring, 1 mL of 15 mg / mL Ni / LDH nanoparticles was prepared. -1 Ni nanosheets were added dropwise to a concentration of 15 mg / mL in ethanol / water (volume ratio 1:1) to a final volume of 2 mL. -1 The Ni:LDH composite was prepared in an LDH aqueous suspension and aged for 24 h in the dark to promote interfacial adhesion and complex formation. It was then centrifuged at 10000 rpm for 10 min, washed three times with ultrapure water, and redispersed for later use. To investigate the effect of component ratio on performance, four Ni:LDH composites with different mass ratios were prepared and labeled as Ni... 0.75 LDH 0.25 @M、Ni 0.67 LDH 0.33 @M、Ni 0.50 LDH 0.50 @M、Ni 0.33 LDH 0.67 @M and Ni 0.25 LDH 0.75 @M. Optimized Ni ratio 0.33 LDH 0.67 @M exhibits the highest interface electron transfer efficiency.

[0058] In all samples, Ni 0.33 / LDH 0.67 @M exhibited the strongest DPBF consumption and the highest FL-CO-1 fluorescence signal, confirming its optimal performance. 1 O2 and CO generation capacity.

[0059] 4. Preparation of the biomimetic nanoplatform (Ni / LDH@M): GL261 cells were first collected and washed three times with pre-chilled PBS. The cells were then resuspended in hypotonic lysis buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.2 mM PMSF) and sonicated on ice. The lysis buffer was centrifuged at 8000 rpm for 10 min to remove nuclei and unlysed cells, followed by ultracentrifugation at 100000 g for 1 h to collect the cell membrane fraction. The separated cell membrane was washed and resuspended in PBS to a final concentration of 1 mg / mL. -1 Subsequently, the Ni / LDH dispersion (1 mg m -1 The mixture was combined with the cell membrane suspension in equal volume ratio and extruded repeatedly 11 times using a mini extruder (Avanti Polar Lipids) equipped with a 200 nm polycarbonate filter membrane to obtain Ni / LDH@M with a cell membrane coating. The obtained Ni / LDH@M was purified by centrifugation, resuspended, and stored at 4°C for later use.

[0060] Transmission electron microscopy (TEM) revealed that Ni / LDH@M has a nearly circular shape. Energy dispersive spectroscopy (EDS) analysis showed that Ni, Fe, and O are all distributed in Ni / LDH@M, proving that Ni / LDH@M was successfully prepared.

[0061] High-resolution transmission electron microscopy (HRTEM) images show interplanar spacings of approximately 0.26 nm and 0.18 nm, corresponding to the (009) plane of LDH and the (200) plane of face-centered cubic Ni, respectively, confirming close contact between the two components.

[0062] Energy-dispersive X-ray spectroscopy (EDS) elemental mapping showed that Ni, Fe, and O were uniformly distributed, consistent with the uniform anchoring of Ni nanosheets on the LDH surface.

[0063] To verify the fusion of cell membranes with Ni / LDH nanoparticles, Ni / LDH nanoparticles were labeled with fluorescein isothiocyanate (FITC), and tumor cell membranes (M) were added at different mass ratios. The resulting mixtures were extruded through a 0.2 µm polycarbonate membrane to promote membrane-nanoparticle fusion, and their fluorescence emission spectra were recorded on a fluorescence spectrophotometer (excitation wavelength 480 nm, emission wavelength range 600–720 nm). Furthermore, tumor cell membranes were stained with DiD (DiD-M) and mixed with FITC-labeled Ni / LDH (FITC-Ni / LDH). The co-localization of DiD and FITC signals was observed using a confocal laser scanning microscope (CLSM) to further confirm the cell membrane coating on the Ni / LDH surface. Figure 1 ).

[0064] Example 2: Catalytic performance of Ni / LDH@M: Valence band X-ray photoelectron spectroscopy (XPS) results show that the valence band top of LDH is located at 1.41 eV, while the valence band top of Ni / LDH@M is located at 1.31 eV.

[0065] The flat band potentials obtained by Mott–Schottky testing are -0.68V for LDH and -0.97V for Ni / LDH@M.

[0066] The results were then validated for consistency through bandgap testing. UV-vis diffuse reflectance spectroscopy and Tauc fitting analysis showed that the bandgap of LDH and Ni / LDH@M were 1.87 eV and 1.53 eV, respectively. The bandgap narrowed after Ni assembly lowered the excitation threshold and facilitated carrier generation. The conduction band potential of Ni / LDH@M remained negative compared to O2 / 1 The redox potential of O2 (−0.16 eV vs. NHE) is thermodynamically favorable for electron transfer from the conduction band to O2, thereby generating... 1 O2.

[0067] The work functions of each component were determined by ultraviolet photoelectron spectroscopy (UPS): the work functions of LDH, Ni, and Ni / LDH@M heterostructure were 4.02 eV, 4.43 eV, and 4.12 eV, respectively. Since the work function of Ni is higher than that of LDH, electrons will flow from LDH to Ni until their Fermi levels reach equilibrium. This process induces upward band bending on the LDH side and forms a Schottky barrier at the interface, while simultaneously generating a built-in electric field that effectively suppresses electron backflow, thereby maintaining directional charge separation under ultrasonication.

[0068] Electrochemical impedance spectroscopy (EIS) results showed that the Nyquist plot semicircle of Ni / LDH@M was significantly smaller than that of the original LDH, indicating a significant reduction in its interfacial charge transfer resistance. Furthermore, ultrasonically triggered transient photocurrent measurements revealed that Ni / LDH@M exhibited a stronger and more persistent response during multiple on / off cycles, suggesting enhanced carrier migration and suppressed recombination. Mechanistically, electrons enriched at the Ni interface catalyze the reduction of carbonyl groups on the surface to CO, while the conduction band electrons of LDH simultaneously activate molecular oxygen to generate CO under ultrasonic excitation. 1 O2. Overall, the constructed Ni / LDH@M heterojunction achieved bandgap narrowing, efficient charge separation and transport, and significantly improved ultrasound-induced CO and O2 concentrations. 1 O2 dual-channel yield ( Figure 2 , Figure 3 ).

[0069] Example 3: In vitro therapeutic evaluation of Ni / LDH@M: To evaluate its antitumor effect in glioma cells, the treatment methods for each group were as follows: Control group: Cells were added only with an equal volume of PBS and were not sonicated to exclude background interference, serving as a control group.

[0070] US Group: Cells were added to PBS solution and sonicated (1.0 MHz, 50% duty cycle, 1.0 W·cm⁻¹). - The cells were treated with ultrasound for 3 min (2, 3 min) to evaluate the effects of ultrasound alone on cells.

[0071] Ni + US group: Add Ni nanosheets (50 μg·mL⁻¹) to cell culture medium -1 Subsequently, the samples were subjected to ultrasonic irradiation under the same conditions to evaluate the catalytic or physical effects of Ni alone under acoustic activation.

[0072] LDH + US group: Add LDH nanosheets (50 μg·mL) to cell culture medium -1 Subsequently, the LDH was subjected to ultrasound irradiation under the same conditions to verify its biological effects and substrate contribution under acoustic treatment.

[0073] Ni / LDH@M group: Cells were treated with Ni / LDH@M (50 μg·mL) -1 However, no sonication was performed to assess the biocompatibility of the nanocomposite itself and its cytotoxicity under unexcited conditions.

[0074] Ni / LDH@M + US group: Cells were treated with Ni / LDH@M (50 μg·mL) -1 Afterwards, ultrasonic treatment was performed (1.0 MHz, 50% duty cycle, 1.0 W·cm). -2 (3 min). This group was used to evaluate the synergistic therapeutic effect of Ni / LDH@M under ultrasound stimulation, including the combined antitumor effect of CO2 generation-induced mitochondrial function inhibition and reactive oxygen species (ROS) upregulation.

[0075] Flow cytometry and confocal fluorescence imaging confirmed that ultrasound irradiation triggered significant CO2 production in Ni / LDH@M treated glioma cells. Enzyme-linked immunosorbent assay (ELISA) results showed that cytochrome c oxidase activity was significantly inhibited in the Ni / LDH@M+US group. Cellular energy metabolism was immediately impaired upon ETC blockade. Correspondingly, the oxygen consumption rate of cells in the Ni / LDH@M+US group was significantly reduced, indicating a marked inhibition of oxidative respiration. Released Cyt c It then interacts with apoptotic protease activators, triggering a caspase-3-dependent apoptotic cascade. Figure 4 ).

[0076] Example 4: Blood-brain barrier permeability of Ni / LDH@M: Mice were intravenously injected with Cy5.5-labeled Ni / LDH@M (200 μL; Cy5.5 dose was 1 mg·kg⁻¹). -1 At preset time points, the fluorescence distribution of Cy5.5 in mice was monitored using an in vivo imaging system (IVIS, PerkinElmer, USA).

[0077] Following intravenous injection, whole-body fluorescence imaging showed that Cy5.5-labeled Ni / LDH@M rapidly accumulated in the brain region, reaching a peak at 3 hours post-administration and maintaining a detectable signal for up to 72 hours. Figure 5 ).

[0078] Example 5: In vivo antitumor effect of Ni / LDH@M: An orthotopic intracranial glioblastoma model was established using 6-week-old C57BL / 6 mice. Both male and female mice were used in the experiment; sex was not considered as a biological variable. GL261-Luc cells (5 × 10⁻⁶) were used. 4 (Each mouse) was stereotactically injected into the right striatum of mice with the assistance of a stereotactic instrument (equipped with a mouse restraint device, RWD Life Science, Shenzhen, China). The specific injection coordinates were: 3.0 mm anterior to the anterior fontanelle, 2.5 mm lateral to the midline, and a depth of 3.0 mm. Tumor progression was monitored using bioluminescence imaging (IVIS Spectrum, PerkinElmer, USA) and magnetic resonance imaging (MRI, uMR 9.4T, United Imaging Healthcare, China).

[0079] On day 10 post-inoculation, mice were randomly divided into five groups and administered PBS, Ni / LDH@M (5 mg / kg) to the other two groups, respectively. -1 ), bevacizumab (BEV, 5 mg·kg) -1 ), Temozolomide (TMZ, 5 mg·kg) -1) or Ni / LDH@M+US (1.0MHz, 50% duty cycle, 1.0W·cm) -2 Treatment. All formulations were administered via tail vein injection every 3 days for a total of 4 doses. For the Ni / LDH@M+US group, the tumor area was subjected to 6 minutes of ultrasound irradiation 24 hours after each injection.

[0080] As expected, the tumor suppression effects of the BEV and TMZ groups were limited, with no significant difference compared to the PBS group; in contrast, Ni / LDH@M+US treatment induced a significant anti-tumor response. Figure 6 ).

[0081] Example 6: Immune response of Ni / LDH@M against glioma: In an orthotopic GBM mouse model, we investigated how CO released from Ni / LDH@M regulates ICD and its subsequent immune effects. Figure 7 Tumor-bearing mice were randomly divided into five groups: PBS, Ni / LDH@M, BEV, TMZ, and Ni / LDH@M+US. The treatment methods for each group were the same as in Example 5. After treatment, tumor tissue was collected for immunofluorescence and flow cytometry analysis.

[0082] Immunofluorescence staining results showed that Ni / LDH@M+US treatment induced severe mitochondrial damage in glioma cells, characterized by Cyt c The massive release of mitochondria into the cytoplasm, coupled with a significant decrease in intracellular ATP levels, suggests that the mitochondrial electron transport chain was effectively blocked, leading to energy depletion. Simultaneously, CRT exposure on the tumor cell membrane surface increased significantly, while HMGB1 nuclear translocation was inhibited, indicating successful induction of ICD. Furthermore, the strong positive signal of cleavage caspase-3 and the significant decrease in the proliferation marker Ki67 jointly demonstrate that Ni / LDH@M+US triggered mitochondrial-dependent apoptosis rather than necrosis. These intracellular events collectively laid the foundation for antigen presentation and subsequent immune activation.

[0083] Further immunofluorescence co-localization analysis showed that Ni / LDH@M+US significantly reduced Foxp within the tumor lesion. 3+ Regulatory T cells (Tregs) and CD206 + The proportion of M2 macrophages was significantly increased, while CD11c was also significantly increased. + CD86 + Mature dendritic cells (DCs) and M1 macrophages. This shift indicates that the tumor immune microenvironment has been remodeled from an immunosuppressive state to a pro-inflammatory state.

[0084] To determine whether local immune remodeling is accompanied by systemic immune activation, flow cytometry analysis was performed on immune cells in cervical lymph nodes. As expected, the Ni / LDH@M+US treatment group showed a high concentration of M1 macrophages, mature dendritic cells (DCs), and CD8+ cells. + The proportion of cytotoxic T lymphocytes (CTLs) was highest, while M2 macrophages and Tregs were significantly reduced. In contrast, BEV and TMZ showed only mild immunomodulatory effects, thus highlighting the key role of CO released by Ni / LDH@M in amplifying the antitumor immune response.

[0085] Example 7: Biosafety of Ni / LDH@M: 1. Systemic toxicity: 6-8 week old SPF-grade C57BL / 6 mice were randomly divided into several groups (e.g., control group and different dosage groups, n=5 in each group) after acclimatization for 7 days. The control group received an equal volume of PBS via tail vein injection, while the experimental groups received the designed dosage (e.g., 4-70 mg / kg). -1 The test nanoformulation was injected via tail vein, and a single-dose acute toxicity experiment was conducted. During the administration period, the general condition of mice (activity, diet, coat, respiration, behavior, etc.), weight changes, and the occurrence of death or significant toxic reactions were observed and recorded daily. Twenty-four hours after the last administration, blood was collected under anesthesia via the retro-orbital or cardiac route and placed in tubes containing anticoagulants or procoagulants. Plasma or serum was separated by centrifugation at 4°C and 500×g for 10 min. Hematological parameters such as white blood cell count, red blood cell count, and platelet count were measured using an automated hematology analyzer. Serum biochemical indicators related to liver and kidney function, such as ALT, AST, ALP, BUN, and CREA, were measured using commercially available biochemical kits to evaluate systemic toxicity and organ dysfunction.

[0086] After four consecutive administrations, blood samples were collected from tumor-bearing mice under anesthesia via the heart. Plasma was separated by centrifugation at 4°C and 500×g for 10 min. Hematological parameters such as white blood cells, red blood cells, and platelets were detected using an automated hematology analyzer. The levels of alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA) in plasma were measured using commercially available diagnostic kits. These results demonstrate that Ni / LDH@M exhibits good biocompatibility.

[0087] 2. Hemolysis test: Blood compatibility of the material was assessed using fresh mouse whole blood. 300 μL of whole blood was mixed with 1 mL of Ni / LDH@M dispersions of different concentrations (12.5~200 μg·mL⁻¹). -1The samples were incubated at 37°C for 4 hours. Deionized water and PBS were used as positive and negative controls, respectively. After incubation, the samples were centrifuged at 2000 rpm for 10 minutes, and the absorbance of the supernatant at 570 nm was measured. The hemolysis rate of all samples was less than 5%, meeting the international biocompatibility standards. Figure 8 ).

[0088] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0089] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Ni / LDH nanoparticle, characterized in that, This includes carbonyl-supported Ni nanosheets and FeNi-LDH nanosheets.

2. The Ni / LDH nanoparticles according to claim 1, characterized in that, The mass ratio of the carbonyl-supported Ni nanosheets to FeNi-LDH nanosheets is 0.75:0.25~0.25:0.

75.

3. A method for preparing Ni / LDH nanoparticles as described in claim 1 or 2, characterized in that, include: S1: The nickel complex is reacted with the metal carbonyl complex to obtain the carbonyl-supported Ni nanosheets; S2: The FeNi-LDH nanosheets are obtained by reacting a solution of iron nitrate hydrate and nickel nitrate hydrate with an alkaline solution; S3: The Ni / LDH nanoparticles are obtained by electrostatic self-assembly of carbonyl-loaded Ni nanosheets and FeNi-LDH nanosheets.

4. A Ni / LDH nanoparticle, characterized in that, It is obtained by the preparation method described in claim 3.

5. A biomimetic nanoplatform, characterized in that, Includes the Ni / LDH nanoparticles and cancer cell membranes as described in claim 1, 2 or 4.

6. The biomimetic nanoplatform according to claim 5, characterized in that, The mass ratio of the Ni / LDH nanoparticles to the cancer cell membrane is 1:2 to 2:

1.

7. A pharmaceutical composition, characterized in that, include: (1) A therapeutically effective amount of the Ni / LDH nanoparticles of claim 1, 2 or 4 and / or the biomimetic nanoplatform of claim 5 or 6; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

8. A pharmaceutical preparation, characterized in that, Includes the pharmaceutical composition of claim 7.

9. A pharmaceutical product, characterized in that, Includes the pharmaceutical preparation described in claim 8.

10. The use of the Ni / LDH nanoparticles of claim 1, 2, or 4, the biomimetic nanoplatform of claim 5 or 6, the pharmaceutical composition of claim 7, the pharmaceutical formulation of claim 8, and / or the pharmaceutical product of claim 9 in the preparation of a drug for the prevention and / or treatment of glioblastoma.

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