Application of polyethylene glycol modified black phosphorus nanosheets in prevention and treatment of acute liver injury

Abstract

The application relates to the field of nanobiomedical technology, and particularly relates to application of polyethylene glycol modified black phosphorus nanosheets in prevention and treatment of acute liver injury. The polyethylene glycol modified black phosphorus nanosheets not only have a significant active oxygen scavenging capacity, but also have calcium ion chelation and buffering capacity, can simultaneously regulate intracellular oxidation-reduction homeostasis and calcium homeostasis, inhibit intracellular calcium overload, especially mitochondrial calcium overload, maintain mitochondrial function, and further reduce liver cell damage, apoptosis and inflammatory response, and play a liver protection role. The application finds that the black phosphorus nanosheets are used in calcium ion chelation and inhibition of cell calcium overload, and breaks through the limitation that the black phosphorus nanosheets are mainly used as active oxygen scavenging materials in the prior art. The material can also adsorb copper ions and realize radioactivity copper labeling without chelating agent, and can be used for liver enrichment tracing and integrated diagnosis and treatment application. The material has a significant protective effect on paracetamol-induced acute liver injury.

Description

Technical Field

[0001] This invention relates to the field of nanobiomedicine technology, specifically to the application of polyethylene glycol-modified black phosphorus nanosheets in regulating redox-calcium homeostasis imbalance and preventing and / or treating acute liver injury, and also to its application in liver photoacoustic imaging and positron emission tomography. Background Technology

[0002] Acute liver injury is a severe clinicopathological process characterized by rapid onset and progression, which can further develop into acute liver failure, seriously threatening patients' lives and health. Acetaminophen overdose is one of the important causes of acute liver injury. Excessive acetaminophen is metabolized in the body to produce the highly reactive intermediate N-acetyl-p-benzoquinone imine, which can lead to glutathione depletion, excessive generation of reactive oxygen species, mitochondrial dysfunction, and amplified inflammatory responses, ultimately causing hepatocyte necrosis and apoptosis. Therefore, developing safe and effective materials or formulations for the prevention and treatment of acute liver injury is of great significance.

[0003] Further research indicates that the development of acute liver injury is not driven solely by oxidative stress, but is closely related to intracellular calcium homeostasis disturbances. Oxidative stress can induce endoplasmic reticulum-derived calcium... 2+ Abnormal release causes cytoplasmic calcium 2+ Increase; excess Ca 2+ Further entry into the mitochondria triggers mitochondrial calcium overload, decreased membrane potential, impaired energy metabolism, and the release of pro-apoptotic factors. Simultaneously, impaired mitochondrial function further amplifies the generation of reactive oxygen species (ROS), leading to the formation of ROS and calcium. 2+ A positive feedback injury loop exists. Therefore, redox imbalance and calcium homeostasis imbalance synergistically participate in and jointly promote the progression of acute liver injury.

[0004] Currently, N-acetylcysteine ​​is a commonly used antidote for acetaminophen poisoning in clinical practice. However, it mainly works by supplementing glutathione precursors, which has limitations such as a limited therapeutic window and a relatively singular intervention target, making it difficult to simultaneously address the multiple pathological processes of oxidative stress, calcium overload, mitochondrial damage, and the amplification of the inflammatory cascade. Therefore, it is necessary to develop a drug that can simultaneously regulate ROS imbalance and calcium... 2+ Anomalous multi-mechanism synergistic intervention strategies have significant clinical implications. Summary of the Invention

[0005] The purpose of this invention is to provide a use for polyethylene glycol-modified black phosphorus nanosheets (BPP) to overcome the problems of existing technologies that have a single target for intervention in acute liver injury and insufficient attention to abnormal calcium homeostasis.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides the use of polyethylene glycol-modified black phosphorus nanosheets in the preparation of a medicament for the prevention and treatment of acute liver injury.

[0007] The polyethylene glycol-modified black phosphorus nanosheets exert a liver-protective effect by simultaneously scavenging reactive oxygen species and binding excess calcium ions, thereby restoring intracellular redox homeostasis and calcium homeostasis.

[0008] The calcium ions are bound to the surface of the polyethylene glycol-modified black phosphorus nanosheets or the active sites of the polyethylene glycol-modified black phosphorus nanosheets through coordination, adsorption, chelation or buffering.

[0009] The polyethylene glycol-modified black phosphorus nanosheets can reduce cytoplasmic calcium ion levels and mitochondrial calcium ion levels, thereby inhibiting intracellular calcium overload and mitochondrial calcium overload.

[0010] The polyethylene glycol-modified black phosphorus nanosheets can reduce intracellular and mitochondrial reactive oxygen species levels, maintain mitochondrial membrane potential, and inhibit mitochondrial-mediated apoptosis.

[0011] Preferably, the drug is an injectable preparation.

[0012] Preferably, the drug is administered via intravenous injection.

[0013] Preferably, the polyethylene glycol-modified black phosphorus nanosheets are the only effective ingredient in the drug.

[0014] Preferably, the effective amount of the polyethylene glycol-modified black phosphorus nanosheets in the drug is 2.5~2000 μg / mL.

[0015] Preferably, the acute liver injury is acetaminophen-induced acute liver injury.

[0016] Preferably, the polyethylene glycol-modified black phosphorus nanosheets are prepared by modifying black phosphorus nanosheets with amino-terminated polyethylene glycol. The amino-terminated polyethylene glycol is NH2-PEG-NH2, and the molecular weight of NH2-PEG-NH2 is 500 Da-30 kDa; The lateral dimensions of the black phosphorus nanosheets are 50–400 nm, preferably 100–200 nm; The thickness of the polyethylene glycol-modified black phosphorus nanosheets is 3–10 nm, preferably 3–5 nm.

[0017] In a second aspect, the present invention provides a medicament for the prevention and treatment of acute liver injury, comprising polyethylene glycol-modified black phosphorus nanosheets and a pharmaceutically acceptable carrier.

[0018] A third aspect of the present invention provides 64Application of Cu-labeled polyethylene glycol-modified black phosphorus nanosheets in the preparation of formulations for liver imaging tracking.

[0019] A fourth aspect of the present invention provides a formulation for integrated liver enrichment tracing and diagnosis, wherein it comprises radioactive... 64 Cu-labeled polyethylene glycol-modified black phosphorus nanosheets and pharmaceutically acceptable carriers.

[0020] Compared with the prior art, the present invention has at least the following beneficial effects: (1) This invention is the first to propose the application of black phosphorus nanosheets in calcium ion chelation and buffering. Existing technologies mainly focus on the ROS scavenging performance of black phosphorus nanosheets, while this invention is the first to discover and systematically verify its application in calcium ion chelation and buffering. 2+ The potential regulatory effects expand the functional boundaries and application scope of black phosphorus nanosheets; (2) The BPP described in this invention can simultaneously scavenge reactive oxygen species and bind excess Ca. 2+ It can synergistically restore intracellular redox homeostasis and calcium homeostasis, overcoming the limitations of existing antioxidant strategies that only target ROS and cannot simultaneously address calcium. 2+ Exceptional limitations; (3) The BPP described in this invention can significantly reduce cytoplasmic calcium. 2+ Levels and mitochondrial Ca 2+ Levels of activity inhibit intracellular calcium overload and mitochondrial calcium overload, thereby interrupting ROS-Ca2+ metabolism. 2+ Positive feedback damage loop; (4) The BPP described in this invention can maintain mitochondrial membrane potential, alleviate mitochondrial dysfunction, reduce cytochrome c release, regulate the expression of key apoptosis proteins B-cell lymphoma / leukemia-2 (Bcl-2) and Bcl-2-related X protein (Bax) and caspase-3 activation, and inhibit mitochondrial-mediated apoptosis. (5) The BPP described in this invention can upregulate nuclear transcription-related factor 2 (Nrf2) related antioxidant defense, improve indicators such as superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH), and reduce the level of the lipid peroxidation marker malondialdehyde (MDA). At the same time, it can inhibit the expression of inflammatory factors and the infiltration of inflammatory cells, and exert a multi-mechanism synergistic liver protection effect. (6) The BPP described in this invention can be rapidly enriched in the liver in vivo and has a long retention time, which is beneficial for achieving passive targeted therapy for acute liver injury. (7) The BPP described in this invention also has good photoacoustic imaging capabilities and can be adsorbed 64Cu enables chelate-free positron emission tomography (PET), thereby constructing an integrated diagnostic and therapeutic platform; (8) The BPP described in this invention has good biocompatibility and biodegradability in vivo and in vitro, and has broad application prospects. Attached Figure Description

[0021] Figure 1 This is a transmission electron microscope (TEM) image of polyethylene glycol-modified black phosphorus nanosheets (BPP) in Example 1 of the present invention.

[0022] Figure 2 The graph shows the test results of BPP's ability to scavenge ABTS free radicals in Embodiment 1 of the present invention.

[0023] Figure 3 In Example 1 of this invention, Ca was adsorbed. 2+ TEM image and elemental distribution of BPP (CaBPP).

[0024] Figure 4 BPP and radionuclides in Example 1 of this invention 64 The graph shows the test results for the binding ability of Cu.

[0025] Figure 5 In Embodiment 1 of the present invention 64 Positron emission tomography (PET) image of Cu-BPP in healthy mice.

[0026] Figure 6 This is a graph showing the therapeutic effect of BPP on a mouse model of acute liver injury, as assessed by serum alanine aminotransferase (ALT) concentration in Example 1 of this invention. Detailed Implementation

[0027] This invention provides an application of polyethylene glycol-modified black phosphorus nanosheets in the prevention and treatment of acute liver injury. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0028] Black phosphorus nanosheets, as a novel two-dimensional nanomaterial, possess a large specific surface area, abundant surface active sites, good reactivity, and the characteristic of gradual degradation under oxidative environments, attracting widespread attention in the biomedical field. Current research mainly focuses on the reactive oxygen species scavenging, antioxidant, and tumor therapy functions of black phosphorus nanosheets, generally regarding them as an antioxidant nanomaterial. However, regarding whether black phosphorus nanosheets possess calcium ion binding capacity, whether they can be used to regulate intracellular calcium homeostasis, and especially whether they can inhibit intracellular and mitochondrial calcium overload, there is a lack of systematic research and clear application reports in the current technology.

[0029] Therefore, if a method could be developed that not only utilizes the reactive oxygen species scavenging function of black phosphorus nanosheets but also further explores their role in Ca2+, it would be beneficial. 2+ Nanomaterials with potential applications in regulation hold promise for constructing a novel intervention system for acute liver injury that combines antioxidant properties, calcium homeostasis restoration, mitochondrial protection, and inflammation suppression. In particular, intravenously injected nanomaterials tend to accumulate in the liver, a characteristic that facilitates passive targeted therapy of the liver; further endowing these materials with imaging and tracing capabilities could enable integrated liver diagnosis and treatment.

[0030] Therefore, it is necessary to develop a method that can simultaneously scavenge ROS and chelate or buffer Ca. 2+ The discovery of black phosphorus nanosheets that restore redox-calcium stability has significant scientific and application value.

[0031] Specifically, this invention provides an application of polyethylene glycol-modified black phosphorus nanosheets in the preparation of a drug for the prevention and treatment of acute liver injury.

[0032] Studies have found that polyethylene glycol-modified black phosphorus nanosheets, in addition to their reactive oxygen species scavenging function, also possess calcium ion chelation and buffering capabilities. They can be used to simultaneously restore intracellular redox homeostasis and calcium homeostasis, thereby achieving multi-mechanism synergistic protection against acute liver injury.

[0033] In this embodiment, the polyethylene glycol-modified black phosphorus nanosheets simultaneously scavenge reactive oxygen species and bind excess calcium ions (Ca). 2+ It restores intracellular redox homeostasis and calcium homeostasis, thereby exerting a hepatoprotective effect.

[0034] The calcium ions can bind to the surface of the polyethylene glycol-modified black phosphorus nanosheets or the active sites of the polyethylene glycol-modified black phosphorus nanosheets through coordination, adsorption, chelation or buffering.

[0035] In this embodiment, the polyethylene glycol-modified black phosphorus nanosheets can reduce cytoplasmic calcium. 2+ Levels and mitochondrial Ca 2 + Levels of calcium levels inhibit intracellular calcium overload and mitochondrial calcium overload.

[0036] In this embodiment, the polyethylene glycol-modified black phosphorus nanosheets can reduce intracellular and mitochondrial reactive oxygen species levels, maintain mitochondrial membrane potential, and inhibit mitochondrial-mediated apoptosis.

[0037] In this embodiment, the polyethylene glycol-modified black phosphorus nanosheets can upregulate Nrf2 and / or increase one or more antioxidant indicators among SOD, CAT, and GSH, while reducing MDA levels.

[0038] In this embodiment, the polyethylene glycol-modified black phosphorus nanosheets can reduce one or more inflammation-related markers among tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and myeloperoxidase (MPO), and promote macrophage polarization to an anti-inflammatory phenotype.

[0039] Preferably, the drug is in the form of an injection, and more preferably, it is administered via intravenous injection. Studies have found that intravenous injection facilitates the rapid entry of the polyethylene glycol-modified black phosphorus nanosheets into the systemic circulation and their accumulation in the liver, thereby helping to improve its efficacy in preventing and treating acute liver injury. Preferably, the polyethylene glycol-modified black phosphorus nanosheets are the sole active ingredient in the drug, and the effective amount of the polyethylene glycol-modified black phosphorus nanosheets in the drug is 2.5–2000 μg / mL, preferably 5–500 μg / mL.

[0040] The polyethylene glycol-modified black phosphorus nanosheets in this embodiment will be described in detail below.

[0041] In this embodiment, the polyethylene glycol-modified black phosphorus nanosheets are prepared by surface modification of black phosphorus nanosheets with amino-terminated polyethylene glycol. This surface modification improves the dispersion stability of black phosphorus nanosheets in aqueous systems, reduces the tendency for material aggregation, enhances its biocompatibility, and helps delay non-specific degradation during storage and application, thereby improving the stability and reproducibility of the material during in vitro and in vivo use. The polyethylene glycol-modified black phosphorus nanosheets can also gradually degrade into phosphorus-containing oxidation products and / or phosphates in oxidizing environments, thus exhibiting good degradability and biosafety.

[0042] The amino-terminated polyethylene glycol is NH2-PEG-NH2. Studies have found that amino-terminated polyethylene glycol can form stable adsorption or interactions with the surface of black phosphorus nanosheets through its terminal amino groups. This improves the hydrophilicity and dispersion stability of the material surface while maintaining adequate exposure of active sites on the black phosphorus nanosheets, which is beneficial for its subsequent reactive oxygen species scavenging and calcium ion chelation or buffering functions.

[0043] Preferably, the molecular weight of the NH2-PEG-NH2 is 500 Da-30 kDa, such as 500 Da, 1 kDa, 2 kDa, 5 kDa, 10 kDa, 20 kDa, 30 kDa, etc. Other specific values ​​within the above range are also acceptable and will not be elaborated here. Further preferably, it is 1 kDa to 10 kDa; even further preferably, it is 2 kDa to 5 kDa; and most preferably, it is 2 kDa.

[0044] When the molecular weight of amino-terminated polyethylene glycol is too low, its coverage and steric hindrance protection on the surface of black phosphorus nanosheets are relatively limited, resulting in little improvement in the material's dispersibility and stability in the aqueous phase. Aggregation is also likely, thus affecting its bioavailability and efficacy. While a higher molecular weight can further improve dispersion and stability, the thicker polymer coating may shield the active sites on the black phosphorus nanosheet surface, hindering their contact with reactive oxygen species and calcium ions, thereby affecting their reactive oxygen species scavenging ability and calcium ion chelation efficiency. Controlling the molecular weight of NH2-PEG-NH2 within the aforementioned preferred range, especially within the range of 2 kDa to 5 kDa, can better balance material dispersibility, structural stability, exposure of surface active sites, and biological function, thus improving its therapeutic effect in the prevention and treatment of acute liver injury.

[0045] Preferably, the lateral dimensions of the black phosphorus nanosheets are 50–400 nm, such as 50 nm, 60 nm, 80 nm, 100 nm, 150 nm, 200 nm, 300 nm, 400 nm, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here. More preferably, the lateral dimensions are 80–250 nm; even more preferably, the lateral dimensions are 100–200 nm.

[0046] The lateral size of black phosphorus nanosheets affects the material's dispersion stability, cellular uptake efficiency, in vivo distribution, and biological activity. If the size is too small, although the material has a large specific surface area, its structural stability is relatively reduced, making it prone to rapid degradation and hindering the maintenance of sustained efficacy. Furthermore, excessively small particles may be cleared more quickly during in vivo circulation. If the size is too large, the material's cellular uptake efficiency may decrease, and its dispersibility may worsen, affecting its ability to reach the site of injury and exert its therapeutic effect. Controlling the lateral size of black phosphorus nanosheets within the aforementioned preferred range, especially within the 100–200 nm range, is more conducive to balancing material stability, cellular uptake efficiency, liver accumulation capacity, and therapeutic efficacy, thereby improving its overall performance in the prevention and treatment of acute liver injury.

[0047] Preferably, the thickness of the polyethylene glycol-modified black phosphorus nanosheets is 3–10 nm, such as 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here. More preferably, it is 3–6 nm; even more preferably, it is 3.5–5 nm.

[0048] The thickness of polyethylene glycol-modified black phosphorus nanosheets is closely related to its number of layers, surface reactivity, and degradation behavior. If the thickness is too small, the material may exhibit high reactivity but insufficient structural stability, making it prone to rapid degradation during preparation, storage, or application. If the thickness is too large, its specific surface area and surface active sites are relatively reduced, potentially affecting its efficiency in scavenging reactive oxygen species and its ability to bind calcium ions. Controlling the thickness within the aforementioned preferred range, especially within the range of 3.5–5 nm, allows the material to possess high surface activity, good structural stability, and a suitable degradation rate, thereby improving its antioxidant and calcium homeostasis regulation effects.

[0049] Preferably, the mass ratio of the black phosphorus nanosheets to the amino-terminated polyethylene glycol is 1:5 to 10:1, such as 1:5, 2:1, 3:1, 5:1, 8:1, 10:1, etc. Other specific ratios within the above range are also acceptable and will not be elaborated here. Further preferred is 1:2 to 5:1; even further preferred is 1:1 to 3:1.

[0050] The ratio of black phosphorus nanosheets to amino-terminated polyethylene glycol (PEG) significantly affects the surface modification degree, dispersion stability, and active site exposure of the final material. When the amount of PEG is too low, the surface modification of the black phosphorus nanosheets is insufficient, resulting in poor dispersibility and storage stability in aqueous systems. Conversely, when the amount of PEG is too high, excessive surface coating may impair the interaction between the black phosphorus nanosheets and reactive oxygen species (ROS) and calcium ions, thereby weakening its biological function. Controlling the mass ratio of black phosphorus nanosheets to PEG within the aforementioned optimal range allows for the formation of a suitable PEG coating layer on the material surface. This improves stability and biocompatibility while better preserving the intrinsic ROS scavenging and calcium ion chelating abilities of the black phosphorus nanosheets, thus contributing to enhanced efficacy in the prevention and treatment of acute liver injury.

[0051] Comparative studies on amino-terminated polyethylene glycol of different molecular weights, black phosphorus nanosheets of different sizes and thicknesses, and different feed ratios revealed that the above-mentioned preferred range can achieve a better balance between dispersibility, stability, exposure of surface active sites, reactive oxygen species scavenging ability, calcium ion chelation ability, and the effect of preventing and treating acute liver injury.

[0052] The polyethylene glycol-modified black phosphorus nanosheets described in this application can simultaneously have one or more of the following beneficial effects: improved aqueous dispersibility and storage stability, improved cellular uptake efficiency and liver enrichment capacity, improved reactive oxygen species scavenging efficiency, improved calcium ion chelation or buffering capacity, reduced intracellular calcium overload and mitochondrial calcium overload, improved mitochondrial function protection, and improved prevention and treatment of acute liver injury.

[0053] In this embodiment, the preparation method of the polyethylene glycol modified black phosphorus nanosheets includes the following steps: (1) Preparation of black phosphorus nanosheets; (2) The black phosphorus nanosheets are mixed with amino-terminated polyethylene glycol, and after ultrasonic and stirring treatment, they are centrifuged and washed to obtain polyethylene glycol modified black phosphorus nanosheets.

[0054] Step (1) may include: dispersing black phosphorus powder in N-methyl-2-pyrrolidone, followed by ultrasonic exfoliation and centrifugation to obtain black phosphorus nanosheets. The black phosphorus nanosheets are prepared from bulk black phosphorus by liquid phase exfoliation and have a two-dimensional sheet structure.

[0055] For example, 20 mg of black phosphorus powder was added to 50 mL of N-methyl-2-pyrrolidone and sonicated with a probe for 24 h under ice bath conditions; unpeeled particles were first removed by centrifugation at 4000 rpm for 30 min, and then black phosphorus nanosheets were collected by centrifugation at 10000 rpm and 4℃ for 10 min.

[0056] Step (2) can specifically include: adding 10 mg NH2-PEG-NH2 (molecular weight of 2 kDa) to 10 mL of black phosphorus nanosheet aqueous dispersion with a concentration of 0.2 mg / mL, ultrasonicating in a bath at 4℃ for 30 min, stirring at room temperature for 2 h, centrifuging at 10000 rpm and 4℃ for 10 min and washing to obtain polyethylene glycol modified black phosphorus nanosheets.

[0057] This invention provides a drug for the prevention and treatment of acute liver injury, comprising polyethylene glycol-modified black phosphorus nanosheets and a pharmaceutically acceptable carrier.

[0058] This invention provides a 64 Application of Cu-labeled polyethylene glycol-modified black phosphorus nanosheets in the preparation of formulations for liver imaging tracking.

[0059] The imaging tracer may include photoacoustic imaging and / or positron emission tomography.

[0060] The polyethylene glycol-modified black phosphorus nanosheets can be combined with Cu 2+ In combination, the polyethylene glycol-modified black phosphorus nanosheets can also react with radioactive... 64Cu forms a non-chelating agent labeled product.

[0061] This invention provides a formulation for integrated liver enrichment tracing and diagnosis, wherein it contains radioactivity. 64 Cu-labeled polyethylene glycol-modified black phosphorus nanosheets and pharmaceutically acceptable carriers.

[0062] Compared with existing technologies, the polyethylene glycol-modified black phosphorus nanosheets (BPP) of this embodiment have the following technical advantages: (1) The BPP has both reactive oxygen species scavenging ability and calcium ion chelation and buffering ability; (2) The BPP can reduce cytoplasmic calcium. 2+ Levels and mitochondrial Ca 2+ Levels, inhibiting intracellular Ca 2+ Overload and mitochondrial calcium overload; (3) The BPP can reduce intracellular ROS and mitochondrial ROS levels, maintain mitochondrial membrane potential, and inhibit mitochondrial-mediated apoptosis. (4) The BPP can be used to prepare drugs for the prevention and / or treatment of acute liver injury, especially suitable for acetaminophen-induced acute liver injury; (5) The BPP can also adsorb Ca 2+ Formation of calcium ion-loaded products, adsorption of Cu 2+ Formation of copper ion-loaded products; the BPP can be further made chelate-free. 64 Cu marking; (6) The BPP and its 64 Cu-labeled products can be used for liver photoacoustic imaging and / or positron emission tomography, enabling integrated liver enrichment tracking and diagnosis.

[0063] The present invention will be further described below through specific embodiments.

[0064] Example 1 (1) Preparation of black phosphorus nanosheets Weigh 20 mg of black phosphorus powder and add it to 50 mL of N-methyl-2-pyrrolidone. Sonicate the mixture with a probe for 24 h under ice bath conditions. Centrifuge the resulting dispersion at 4000 rpm for 30 min to remove unpeeled particles. Collect the supernatant and then centrifuge at 10000 rpm and 4℃ for 10 min to collect the precipitate. Wash the precipitate five times with deionized water and then redisperse it in deionized water to obtain an aqueous dispersion of black phosphorus nanosheets.

[0065] (2) Preparation of BPP Take 10 mL of a 0.2 mg / mL aqueous dispersion of black phosphorus nanosheets, add 10 mg of NH2-PEG-NH2 (molecular weight 2 kDa), and sonicate in a bath at 4℃ for 30 min, followed by stirring at room temperature for 2 h. After the reaction is complete, collect the product by centrifugation at 10000 rpm and 4℃ for 10 min, wash several times with deionized water, and redisperse in deionized water to obtain polyethylene glycol-modified black phosphorus nanosheets, denoted as BPP.

[0066] (3) Characterization and performance testing 1) Structural characterization of BPP The morphology of BPP was observed using transmission electron microscopy. Figure 1 The results showed that it maintained a two-dimensional layered structure; its thickness was measured to be approximately 3.79–3.89 nm using atomic force microscopy; dynamic light scattering results showed that its hydrated particle size was approximately 134.09 nm; Zeta potential and Fourier transform infrared spectroscopy indicated that the PEG modification was successful. BPP exhibits broad-spectrum ultraviolet-visible-near-infrared (UV-vis-NIR) absorption characteristics.

[0067] 2) Oxidative degradation performance of BPP BPP was incubated in deionized water or H2O2 solutions of different concentrations, and the absorbance at 808 nm was measured at different time points. The results showed that BPP can be gradually degraded in an oxidizing environment, and the higher the H2O2 concentration, the faster the degradation. Fourier transform infrared spectroscopy (FT-IR) analysis of the degradation products revealed characteristic peaks related to PO and P=O, indicating that the degradation products include phosphorus oxides and / or phosphates.

[0068] 3) Free radical scavenging performance of BPP The following are used: 2,2'-adiazon-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazine (DPPH), and superoxide anion (O2· - The antioxidant properties of BPP were evaluated by testing and electron spin resonance (ESR) assay. Figure 2 The results showed that BPP can efficiently scavenge ABTS radicals, DPPH radicals, and O2· - It also significantly weakens hydroxyl radicals (·OH) and O2· - The ESR characteristic signal indicates that it has excellent broad-spectrum ROS scavenging ability.

[0069] 4) BPP's calcium ion binding capacity Add CaCl2 solution to the BPP aqueous dispersion, stir at room temperature, and centrifuge to collect the product, obtaining Ca 2+ The combined BPP is denoted as CaBPP. Figure 3TEM and elemental mapping showed that Ca could be uniformly distributed on the surface of the nanosheets; energy-dispersive X-ray spectroscopy (EDS) detected a significant Ca signal; Raman and X-ray photoelectron spectroscopy (XPS) results further proved that BPP could interact with Ca. 2+ The binding occurs. This result indicates that, in addition to its antioxidant properties, BPP also possesses a clear calcium ion binding capacity.

[0070] 5) Protective effect of BPP against oxidative stress-induced hepatocyte damage Normal mouse hepatocytes AML12 were pretreated with different concentrations of BPP and then stimulated with H2O2. CCK-8 cell proliferation assays and live / dead staining results showed that BPP significantly improved cell survival rate and reduced cell death rate under oxidative stress, indicating that it has a significant protective effect on hepatocytes.

[0071] 6) BPP's effect on intracellular ROS and Ca 2+ Dual regulatory role of steady state AML12 cells were pretreated with BPP and then stimulated with H2O2 or ionomycin, respectively. Cytoplasmic ROS and mitochondrial ROS were detected using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and the mitochondrial superoxide fluorescent probe (MitoSOX Red), respectively. Cytoplasmic calcium ion fluorescent probe (Fluo-4 AM) was used to detect cytoplasmic calcium ions. 2+ The concentration of mitochondrial calcium was detected using a combination of a mitochondrial calcium ion fluorescent probe (Rhod-2 AM) and a mitochondrial green fluorescent probe (MitoTracker Green). 2+ The results showed that BPP not only significantly reduced intracellular and mitochondrial ROS levels, but also significantly reduced cytoplasmic calcium levels. 2+ and mitochondrial Ca 2+ The results demonstrate that BPP can simultaneously inhibit intracellular calcium overload and mitochondrial calcium overload.

[0072] 7) BPP restores mitochondrial function and interrupts ROS-Ca. 2+ Positive feedback damage loop Mitochondrial membrane potential was detected using a mitochondrial membrane potential probe (JC-1). Results showed that H2O2 treatment caused a decrease in mitochondrial membrane potential, while BPP pretreatment significantly maintained it. (The text also mentions ROS and Ca, but this seems unrelated to the main topic and is likely a separate, incomplete sentence.) 2+ The test results show that BPP simultaneously removes ROS and binds excess Ca. 2+ Interruption of ROS-Ca 2+ A positive feedback amplification loop is used to restore mitochondrial function.

[0073] 8) Copper ion binding of BPP and64 Cu without chelating agent labeling Adding CuCl2 solution to BPP yields CuBPP. TEM, EDS, and XPS results show that Cu successfully binds to BPP. Further... 64 CuCl2 was mixed and incubated with BPP. Figure 4 TLC and centrifugation results show that BPP can achieve efficient chelation-free separation. 64 Cu labeling, i.e., obtaining 64 Cu-BPP.

[0074] 9) In vivo PET imaging and biodistribution of BPP Will 64 Cu-BPP was intravenously injected into mice for PET imaging. Figure 5 The results showed that BPP could rapidly accumulate in the liver and spleen, and maintain high uptake and long retention time in the liver, before being gradually degraded and cleared via the hepatobiliary pathway.

[0075] 10) In vivo photoacoustic imaging of BPP In vivo photoacoustic imaging studies were conducted on BPP. The results showed that BPP exhibited good concentration-dependent photoacoustic signals, and significant signal enhancement was observed in the liver region after intravenous injection, further demonstrating its ability to accumulate in the liver and its imaging tracking capabilities.

[0076] 11) Therapeutic effect of BPP in acute liver injury model An acute liver injury model in mice was established using acetaminophen. Figure 6 The results showed that after BPP treatment, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) decreased significantly, the area of ​​liver tissue necrosis was significantly reduced, and the number of positive cells for apoptosis detection by terminal deoxynucleotidyl transferase deoxyuridine triphosphate (TUNEL) was significantly reduced. The overall therapeutic effect was better than that of the N-acetylcysteine ​​control group.

[0077] 12) Anti-apoptotic and antioxidant mechanisms of BPP Western blot results showed that BPP reduced cytochrome c release, downregulated Bax and cleaved caspase-3, and upregulated Bcl-2. Immunofluorescence and biochemical assays indicated that BPP restored Nrf2 levels, increased SOD, CAT, and GSH levels, and decreased MDA levels.

[0078] 13) Anti-inflammatory and immunomodulatory effects of BPP Immunofluorescence and ELISA results showed that BPP could reduce abnormal infiltration of macrophages and neutrophils, decrease the levels of TNF-α, IL-1β, IL-6 and MPO, and promote the transformation of macrophages to an anti-inflammatory phenotype, thereby improving the liver inflammatory microenvironment.

[0079] 14) In vivo safety of BPP In healthy mice, after intravenous injection of BPP, pathological findings in major organs and blood biochemical parameters were measured at different time points. The results showed no obvious toxic damage to any major organs, and no significant abnormalities were observed in serum biochemical and routine blood parameters, indicating that BPP has good in vivo safety.

[0080] Conclusion: This invention provides an application of black phosphorus nanosheets (BPP). This material not only exhibits excellent reactive oxygen species (ROS) scavenging properties but also demonstrates, for the first time, calcium ion chelation and buffering capabilities, significantly inhibiting intracellular calcium overload, particularly in mitochondria. This simultaneously restores redox homeostasis and calcium homeostasis, protecting mitochondrial function and alleviating acute liver injury. This invention overcomes the limitations of existing research, which primarily focuses on the ROS scavenging effects of black phosphorus nanosheets, providing a new technical pathway and theoretical basis for the application of black phosphorus nanosheets in calcium homeostasis regulation and the prevention and treatment of drug-induced acute liver injury.

[0081] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. The application of polyethylene glycol-modified black phosphorus nanosheets in the preparation of drugs for the prevention and treatment of acute liver injury.

2. The application according to claim 1, characterized in that, The polyethylene glycol-modified black phosphorus nanosheets exert a liver-protective effect by simultaneously scavenging reactive oxygen species and binding excess calcium ions, thereby restoring intracellular redox homeostasis and calcium homeostasis.

3. The application according to claim 2, characterized in that, The calcium ions bind to the surface of the polyethylene glycol-modified black phosphorus nanosheets or the active sites of the polyethylene glycol-modified black phosphorus nanosheets through coordination, adsorption, chelation, or buffering.

4. The application according to claim 1, characterized in that, The polyethylene glycol-modified black phosphorus nanosheets can reduce cytoplasmic calcium ion levels and mitochondrial calcium ion levels, inhibiting intracellular calcium overload and mitochondrial calcium overload; furthermore, the polyethylene glycol-modified black phosphorus nanosheets can reduce intracellular reactive oxygen species (ROS) levels and mitochondrial ROS levels, maintain mitochondrial membrane potential, and inhibit mitochondrial-mediated apoptosis.

5. The application according to claim 1, characterized in that, The drug is an injectable form; The drug is administered via intravenous injection; The polyethylene glycol-modified black phosphorus nanosheets are the only effective ingredient in the drug. The effective amount of the polyethylene glycol-modified black phosphorus nanosheets in the drug is 2.5~2000 μg / mL.

6. The application according to claim 1, characterized in that, The acute liver injury mentioned is acetaminophen-induced acute liver injury.

7. The application according to claim 1, characterized in that, The polyethylene glycol-modified black phosphorus nanosheets were prepared by modifying black phosphorus nanosheets with amino-terminated polyethylene glycol. The amino-terminated polyethylene glycol is NH2-PEG-NH2, and the molecular weight of NH2-PEG-NH2 is 500 Da-30 kDa; The lateral dimensions of the black phosphorus nanosheets are 50–400 nm. The thickness of the polyethylene glycol-modified black phosphorus nanosheets is 3–10 nm.

8. A drug for the prevention and treatment of acute liver injury, characterized in that, It contains polyethylene glycol-modified black phosphorus nanosheets and a pharmaceutically acceptable carrier.

9. A kind 64 Application of Cu-labeled polyethylene glycol-modified black phosphorus nanosheets in the preparation of formulations for liver imaging tracking.

10. A formulation for integrated liver enrichment tracing and diagnosis, characterized in that, Contains radioactivity 64 Cu-labeled polyethylene glycol-modified black phosphorus nanosheets and pharmaceutically acceptable carriers.

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