A tobacco mosaic virus nanocarrier-based kidney injury diagnosis and treatment integrated nanocomposite, a preparation method thereof, a pharmaceutical composition and application thereof

Abstract

The application discloses a tobacco mosaic virus nanocarrier-based kidney injury diagnosis and treatment integrated nanocomposite, a preparation method thereof, a pharmaceutical composition and application, belongs to the technical field of biological drugs, and aims to develop a kidney injury diagnosis and treatment integrated nanocomposite which can realize long-time circulation in vivo, efficiently enriches at a damaged kidney part, and simultaneously has high-contrast imaging capability and sustained drug release function, and application of the kidney injury diagnosis and treatment integrated nanocomposite in preparation of the following kidney disease related drugs; the drugs include drugs for kidney disease diagnosis, drugs for kidney targeted delivery or drugs for kidney disease diagnosis and treatment integration; the kidney injury diagnosis and treatment integrated nanocomposite comprises a tobacco mosaic virus, a fluorescent dye and a therapeutically active ingredient.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a nanocomposite for the diagnosis and treatment of kidney injury based on tobacco mosaic virus nanocarrier, its preparation method, pharmaceutical composition, and application. Background Technology

[0002] Acute kidney injury (AKI) is a critical clinical condition characterized by a rapid decline in renal function over a short period, typically accompanied by a significant decrease in glomerular filtration rate, accumulation of metabolic waste products, electrolyte disturbances, and enhanced oxidative stress and inflammatory responses. This disease has a high incidence and mortality rate among intensive care patients and significantly increases the risk of developing chronic kidney disease, making it a significant medical challenge that urgently needs to be addressed. However, current methods for sensitive and reliable early diagnosis of AKI, as well as effective targeted therapies, severely limit the effectiveness of clinical interventions.

[0003] In diagnosis, current technologies primarily rely on biochemical indicators such as serum creatinine (CREA) and blood urea nitrogen (BUN) to assess kidney function. However, these indicators typically only show significant increases after severe kidney damage has occurred, exhibiting a clear lag and hindering early disease warning. Furthermore, these indicators are overall functional indicators and cannot provide spatial distribution information of the damaged site, making real-time, dynamic monitoring of disease progression difficult. In recent years, fluorescence imaging technology, due to its non-invasive, real-time, and high-sensitivity advantages, has been considered an important direction for early diagnosis of AKI. However, existing nano-fluorescent probe systems (such as small-molecule fluorescent dyes and quantum dots) still have many limitations. First, small molecule probes (such as free Cy5 dyes) have short circulating half-lives in vivo and are easily metabolized and cleared, making it difficult to provide a continuous and effective imaging window. Second, traditional nanoprobe designs often follow the principle of "small size first" (usually 1-10 nm), which, while beneficial for renal clearance, results in a significantly insufficient retention time in damaged renal tissue, making it impossible to achieve effective enrichment. This leads to low signal-to-noise ratio and poor contrast in imaging, making it difficult to meet the needs of accurate diagnosis. In addition, some inorganic nanomaterials (such as quantum dots) also have potential biotoxicity and long-term safety issues, further limiting their application prospects in clinical practice.

[0004] In terms of treatment, the commonly used antioxidant intervention strategy is represented by N-acetylcysteine ​​(NAC). Although it can alleviate kidney damage to some extent by scavenging reactive oxygen species, it still suffers from problems such as rapid metabolism, low bioavailability, and poor targeting, making it difficult to maintain an effective drug concentration at the site of kidney damage, thus resulting in limited overall treatment efficacy. In addition, traditional drug delivery methods often rely on systemic distribution and lack a precise delivery mechanism targeting the lesion site, which further reduces treatment efficiency.

[0005] From the perspective of nanomaterial applications, existing research reports have shown that natural plant virus nanoparticles (such as Tobacco Mosaic Virus, TMV) are used as carriers in drug delivery systems.

[0006] Relevant non-patent literature retrieved: Journal name: 《Chinese Journal of POLYMER SCIENCE》, literature name: 《Single-wavelength Excited Ratiometric Fluorescence pH Probe to Image Intracellular Trafficking of Tobacco Mosaic Virus》, 2020, 6, 587 - 592. This literature discloses the following content: As a typical plant virus with biocompatibility and high transfection efficiency, tobacco mosaic virus (TMV) has shown broad application potential in the drug or gene delivery field...... Here, we report a single-wavelength excited ratiometric fluorescent pH probe. This probe is constructed by simultaneously coupling pH-sensitive fluorescein isothiocyanate (FITC) and pH-insensitive rhodamine B isothiocyanate (RBIRC) onto the inner surface of TMV. The fluorescence intensity ratio of FITC to RBITC excited at 488 nm responds specifically towards pH value over other interferential agents. By taking use of this single-wavelength excited ratiometric pH probe and confocal laser scanning microscopy, it is shown that the endocytosed TMV is located in a pH decreasing microenvironment and eventually enters lysosomes.This work may provide important guidance on construction of TMV-based nanocarriers. The Chinese translation is: The journal *Chinese Journal of Polymer Science*, titled "Single-wavelength excitation ratiometric fluorescent pH probe for imaging intracellular transport of tobacco mosaic virus," published in 2020, Vol. 6, pp. 587-592, discloses the broad application potential of TMV in drug and gene delivery. It reports a single-wavelength excitation ratiometric fluorescent pH probe constructed by simultaneously coupling pH-sensitive fluorescein isothiocyanate (FITC) and pH-insensitive rhodamine isothiocyanate B (RBITC) to the inner surface of TMV. Under 488 nm excitation, the fluorescence intensity ratio of FITC to RBITC specifically responds to pH values, unaffected by other interfering substances. Combining this single-wavelength excitation ratiometric pH probe with laser confocal microscopy, it was confirmed that the endocytosed TMV exists in a microenvironment with continuously decreasing pH and eventually enters the lysosome. This work can provide important guidance for constructing TMV-based nanocarriers.

[0007] Current research on TMV nanocarriers mainly focuses on fields such as oncology, with limited applications in kidney diseases. In particular, the "enhanced permeability and retention-like effect" of increased vascular permeability and enhanced local retention in renal tissue during acute kidney injury has not been fully utilized to achieve passive targeted enrichment of nanomaterials. Furthermore, the "theranostics" design, which combines viral nanocarriers for both imaging and treatment, is still in the exploratory stage, and related systematic research is relatively lacking.

[0008] Therefore, developing a complex of nano-therapeutic platforms that can achieve long-term circulation in vivo, efficiently accumulate in damaged kidney sites, and simultaneously possess high-contrast imaging capabilities and continuous drug release functions is of great significance for overcoming the bottlenecks of existing AKI diagnosis and treatment technologies, and also constitutes a key technical problem that urgently needs to be solved in this field. Summary of the Invention

[0009] The purpose of this invention is to provide: A nanocomposite for the diagnosis and treatment of kidney injury based on tobacco mosaic virus nanocarrier, and related technologies, to address technical problems such as the short in vivo circulating half-life of small molecule probes, their easy rapid metabolism and clearance, and their difficulty in providing a continuous and effective imaging window, or a combination thereof.

[0010] Terminology Explanation: Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms, the definition provided in this chapter shall prevail.

[0011] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0012] The definitions of standard chemical terms can be found in the references “Fluorescent Dyes and Their Biomedical Applications”, Chemical Industry Press, March 2022, and “Small Animal In Vivo Optical Imaging Technology and Applications”, Shanghai Jiaotong University Press, November 2023.

[0013] Unless otherwise stated, conventional methods within the scope of the art, such as ultraviolet-visible absorption spectroscopy, fluorescence emission spectroscopy, dynamic light scattering, transmission electron microscopy, etc., shall be used for detection.

[0014] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0015] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0016] The term "therapeutic integration" as used in this article refers to an integrated technological model that combines disease diagnosis and treatment functions within a single system, formulation, or device, enabling precise identification, real-time monitoring, and targeted therapy of lesions. This model achieves precise localization and diagnosis through specific identification of lesion microenvironment signals, synchronously or sequentially releases therapeutically active substances for intervention, and allows for dynamic monitoring and feedback evaluation of the treatment process and efficacy. This improves diagnostic accuracy, enhances treatment effectiveness, reduces adverse reactions, and optimizes the treatment process, making it a common technological approach in the fields of biomedicine, nanoparticles, targeted delivery, and precision medicine. Nanomedicines, multifunctional carriers, and intelligent responsive formulations, which combine imaging / detection and therapeutic functions, are all conventional therapeutic integration methods in this field.

[0017] The term "cyanine dyes" used in this article refers to a class of synthetic organic cationic fluorescent dyes belonging to the polymethine family. Their core structural feature is that two nitrogen-containing heterocycles (such as indole, benzothiazole, quinoline, etc.) are conjugated by an odd number of carbons through polymethine (-CH=CH-). n The bridging chains connect the positive charges, which resonate and delocalize between the two quaternary nitrogen atoms, forming a large π-conjugated chromogenic system. It is mainly used for fluorescent labeling of biomolecules (nucleic acids, proteins, antibodies), near-infrared imaging, therapeutic probes, photodynamic / photothermal therapy, and nanocarrier tracing. It is a conventional fluorescent dye in the fields of biomedicine and analytical detection, with typical examples being Cy3, Cy5, Cy7, and indocyanine green (ICG).

[0018] The term "rhodamine-type dyes" as used in this article refers to a class of basic xanthracene-based fluorescent dyes with an oxanthracene core. Their basic structure consists of an oxanthracene parent ring and a 9-position benzene ring (or a substituted benzene ring) conjugated together. The molecules typically contain amino substituents, allowing for modulation of fluorescence signals through protonation, deprotonation, or structural modification. They possess a rigid conjugated planar structure, high fluorescence quantum yield, good photostability, and emission wavelengths mostly in the visible light region. These dyes exhibit excellent fluorescence properties and good biocompatibility, and are widely used in cell staining, ion detection, biomolecular labeling, in vivo imaging, and fluorescent probe construction. They are commonly used organic fluorescent dyes in analytical chemistry and biomedicine. Typical examples include rhodamine B, rhodamine 6G, rhodamine 123, tetramethylrhodamine (TAMRA), and its derivatives.

[0019] The term FITC-type dyes used in this article refer to a class of acidic sulfonate fluorescent dyes with fluorescein as the parent backbone. Their basic structure consists of a benzopyranone parent ring and an aromatic ring conjugated together. The molecules typically contain reactive groups such as isothiocyanates and carboxyl groups, allowing for modulation of the fluorescence signal through substituent modification, pH changes, or alterations in environmental polarity. These dyes exhibit high fluorescence quantum yield, stable excitation and emission performance, and emission wavelengths predominantly in the green and visible light regions. They are highly efficient at labeling, possess excellent biocompatibility, and are widely used in immunofluorescence detection, flow cytometry, biomolecule labeling, in vivo tracking, and fluorescent probe construction. They are commonly used small-molecule fluorescent dyes in biomedicine and analytical detection. Typical examples include fluorescein isothiocyanate (FITC), fluorescein carboxylate (FAM), tetrachlorofluorescein (TET), hexachlorofluorescein (HEX), and their derivatives.

[0020] The term "near-infrared fluorescent dye" as used in this article refers to a class of functional fluorescent molecules or fluorescent carriers with a conjugated aromatic system as their core framework. Their basic structure consists of a fluorophore, a recognition unit, and a linker group conjugated together. The molecules typically contain hydrophilic groups or targeting modification groups, enabling specific regulation of fluorescence signals through recognition of the lesion microenvironment (such as pH, reactive oxygen species, or specific enzymes) or targeted binding. They possess a large π-conjugated planar structure, resulting in deep tissue penetration, low background fluorescence interference, and minimal photodamage to biological samples. Their absorption and / or emission wavelengths are mostly in the near-infrared region (700-900 nm). These dyes exhibit sensitive fluorescence response and controllable targeting, and are widely used in in vivo deep tissue imaging, precise lesion localization, real-time disease monitoring, integrated diagnosis and treatment, and biomolecular detection. They are commonly used fluorescence detection tools in precision medicine, bioanalysis, and biopharmaceutical fields.

[0021] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides: a nanocomposite for the diagnosis and treatment of kidney injury based on tobacco mosaic virus nanocarrier, comprising tobacco mosaic virus, fluorescent dye and therapeutic active ingredient.

[0022] The integrated nanocomposite for the diagnosis and treatment of kidney injury of the present invention has at least one of the following properties: (1) Fluorescence emission wavelength range 550-800nm; (2) In vivo circulation time ≥ 6-24h; (3) Kidney retention time ≥12-48h; (4) The signal enhancement factor in the lesion area is ≥2 times; (5) Cell viability ≥ 85%.

[0023] Preferably, the fluorescence emission wavelength range is selected from 550nm, 560nm, 570nm, 580nm, 590nm, 600nm, 610nm, 620nm, 630nm, 640nm, 650nm, 660nm, 670nm, 680nm, 690nm, 700nm, 710nm, 720nm, 730nm, 740nm, 750nm, 760nm, 770nm, 780nm, 790nm, 800nm, or a range derived therefrom.

[0024] Preferably, the in vivo circulation time is selected from 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, or a range derived therefrom.

[0025] Preferably, the renal retention time is selected from 12h, 14h, 16h, 18h, 20h, 22h, 24h, 26h, 28h, 30h, 32h, 34h, 36h, 38h, 40h, 42h, 44h, 46h, 48h, or a range derived therefrom.

[0026] Preferably, the signal enhancement factor of the lesion area is selected from 2 times, 3 times, 4 times, 5 times, or a range derived therefrom.

[0027] Preferably, the cell viability is selected from 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a range derived therefrom.

[0028] In some embodiments, the fluorescent dye includes at least one of cyanine dyes, rhodamine dyes, FITC dyes, and near-infrared fluorescent dyes.

[0029] Preferably, the cyanine dyes include at least one of Cy3, Cy5, Cy5.5, Cy7 and their derivatives.

[0030] In some embodiments, the therapeutically active ingredient includes at least one of an antioxidant, an anti-inflammatory drug, an anti-apoptotic drug, and a growth factor that promotes renal tubular repair.

[0031] Preferably, the antioxidant drug includes at least one of a thiol-containing antioxidant, a vitamin, and an antioxidant enzyme; more preferably, the thiol-containing antioxidant includes at least one of aramidin (SS-31), N-acetylcysteine ​​(NAC), glutathione (GSH), and cysteine.

[0032] Preferably, the anti-inflammatory drug includes dexamethasone.

[0033] Preferably, the anti-apoptotic drug includes Z-VAD-FMK.

[0034] Preferably, the growth factors include HGF and / or EGF.

[0035] In some embodiments, the molar ratio of the tobacco mosaic virus to the fluorescent dye is 5-50:1, for example 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1. 1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, or ranges derived therefrom.

[0036] Preferably, the molar ratio of the tobacco mosaic virus to the fluorescent dye is 10-20:1.

[0037] In some embodiments, the molar ratio of the tobacco mosaic virus to the therapeutic active ingredient is 1:0.05-10, for example 1:0.05, 1:0.10, 1:0.15, 1:0.20, 1:0.25, 1:0.30, 1:0.35, 1:0.40, 1:0.45, 1:0.50, 1:0.55, 1:0.60, 1:0.65, 1:0.70, 1:0.75, 1:0.80, 1:0.85, 1:0.90, 1:0.95, 1:1.00, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, or a range derived therefrom.

[0038] Preferably, the molar ratio of the tobacco mosaic virus to the therapeutic active ingredient is 1:0.5-3.

[0039] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: The first priority option is the molar ratio of tobacco mosaic virus to the therapeutic active ingredient. This technical approach further enhances the above-mentioned effects by achieving passive enrichment and long-term retention of nanoparticles in damaged kidneys, thereby significantly improving imaging signal intensity and local drug concentration.

[0040] The second preferred approach: the molar ratio of tobacco mosaic virus to the therapeutic active ingredient. This approach further enhances the above-mentioned effects by achieving passive enrichment and long-term retention of nanoparticles in damaged kidneys, thereby significantly improving imaging signal intensity and local drug concentration.

[0041] Secondly, the present invention provides a method for preparing the integrated nanocomposite for the diagnosis and treatment of kidney injury, comprising the following steps: (1) Disperse tobacco mosaic virus in buffer solution to obtain TMV suspension; (2) Add the fluorescent dye to the TMV suspension for fluorescent labeling reaction, purify and collect to obtain TMV-dye; (3) Resuspend the TMV-dyed, add the therapeutic active ingredient load, purify and collect to obtain the product.

[0042] In some implementations, the pH of the buffer solution in step (1) is 6.5-7.5; for example, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or a range derived therefrom.

[0043] Preferably, the buffer solution includes phosphate-buffered saline (PBS) or KP buffer.

[0044] In some embodiments, the concentration of the TMV suspension in step (1) is 5-20 mg / mL, for example 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, or a range derived therefrom.

[0045] In some embodiments, the concentration of the fluorescent dye in step (2) is 0.1-5 mM, for example 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.1 mM, 2.2 mM, 2.3 mM. M, 2.4mM, 2.5mM, 2.6mM, 2.7mM, 2.8mM, 2.9mM, 3.0mM, 3.1mM, 3.2mM, 3.3mM, 3.4mM, 3.5mM, 3.6mM, 3.7mM, 3.8mM, 3.9mM, 4.0mM, 4.1mM, 4.2mM, 4.3mM, 4.4mM, 4.5mM, 4.6mM, 4.7mM, 4.8mM, 4.9mM, 5.0mM, or ranges derived therefrom.

[0046] In some embodiments, the fluorescent labeling reaction in step (2) is carried out in the dark at 15-37°C for 0.5-6 hours; for example, the reaction temperature can be selected from 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, or a range derived therefrom; the reaction time can be selected from 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, or a range derived therefrom.

[0047] In some embodiments, the concentration of the therapeutic active ingredient in step (3) is 0.5-20 mg / mL, for example 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 5.5 mg / mL, 6 mg / mL, 6.5 mg / mL, 7 mg / mL, 7.5 mg / mL, 8 mg / mL, 8.5 mg / mL, 9 mg / mL, 9.5 mg / mL, 1 0 mg / mL, 10.5 mg / mL, 11 mg / mL, 11.5 mg / mL, 12 mg / mL, 12.5 mg / mL, 13 mg / mL, 13.5 mg / mL, 14 mg / mL, 14.5 mg / mL, 15 mg / mL, 15.5 mg / mL, 16 mg / mL, 16.5 mg / mL, 17 mg / mL, 17.5 mg / mL, 18 mg / mL, 18.5 mg / mL, 19 mg / mL, 19.5 mg / mL, 20 mg / mL, or ranges derived therefrom.

[0048] In some implementations, the loading method in step (3) includes at least one of electrostatic adsorption, covalent coupling, and physical loading. Preferably, the loading time is 1-6 hours, for example, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, or a range derived therefrom.

[0049] In some implementations, the pH of the buffer solution used for resuspension in step (3) is 6.5-7.5; for example, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or a range derived therefrom.

[0050] Preferably, the buffer solution includes phosphate-buffered saline (PBS) or KP buffer.

[0051] The purification and collection methods involved in this invention include at least one of dialysis, ultrafiltration centrifugation, and gel filtration. Preferably, the molecular weight cutoff for dialysis is 10-100 kDa; and the rotation speed for ultrafiltration centrifugation is 2000-10000 rpm.

[0052] Thirdly, the present invention provides a pharmaceutical composition comprising the therapeutic nanocomposite for kidney injury or the therapeutic nanocomposite for kidney injury prepared by the preparation method and pharmaceutically acceptable excipients.

[0053] The dosage form of the drug includes powder, granules, capsules, tablets, pills, suspensions, or solutions.

[0054] Fourthly, the present invention provides the application of the integrated nanocomposite for the diagnosis and treatment of kidney injury, the integrated nanocomposite for the diagnosis and treatment of kidney injury prepared by the preparation method, or the pharmaceutical composition in the preparation of the following drugs related to kidney diseases.

[0055] The drugs include drugs for the diagnosis of kidney diseases, drugs for targeted delivery to the kidneys, or drugs for integrated diagnosis and treatment of kidney diseases.

[0056] In some implementations, the kidney disease includes acute kidney injury, kidney fibrosis, or kidney cancer.

[0057] The beneficial effects of this invention are as follows: This invention is the first to use tobacco mosaic virus for the diagnosis of acute kidney injury or the construction of an integrated diagnostic and therapeutic system. It makes full use of the regularity of its structure, high specific surface area and easy functionalization modification to achieve multi-component synergistic loading. Fluorescent dyes and antioxidant drugs are synergistically integrated on the same nanoplatform to achieve the integrated function of "diagnosis + treatment" (Theranostics). It can perform simultaneous treatment while realizing real-time imaging, significantly improving the overall intervention efficiency.

[0058] Compared to existing technologies, the tobacco mosaic virus (TMV)-based integrated nanocomposite for the diagnosis and treatment of kidney injury provided by this invention exhibits significant advantages in imaging performance, biosafety, and therapeutic efficacy, as detailed below: (a) Excellent imaging performance 1. High signal strength and long duration The integrated nanocomposite for the diagnosis and treatment of kidney injury of the present invention exhibits a significantly prolonged circulation time and imaging window in vivo. Compared with traditional small molecule fluorescent probes, which are not easily cleared rapidly in vivo and whose signals decay rapidly within hours after injection in animal models, the integrated nanocomposite for the diagnosis and treatment of kidney injury of the present invention can still detect obvious fluorescent signals in the kidney area within 24 hours after injection, demonstrating excellent in vivo stability and sustained imaging capability.

[0059] 2. Strong kidney targeting and high imaging contrast In an acute kidney injury model, the therapeutic nanocomposite of this invention can achieve significant enrichment at the site of kidney injury, with its signal intensity in the diseased kidney being approximately 2-3 times higher than that in normal tissue, and the proportion of fluorescence signal in the kidney region to the overall distribution is significantly increased (up to approximately 60%). These results demonstrate that this invention can achieve highly specific and high-contrast imaging of injured kidneys, significantly superior to traditional nanoprobes.

[0060] 3. Strong imaging stability and adaptability Because this invention employs a scalable fluorescent labeling strategy, it is applicable to a variety of fluorescent dye systems (including visible and near-infrared bands), and can flexibly adjust the imaging wavelength according to different application scenarios, reduce biological background interference, and improve imaging sensitivity.

[0061] (ii) Good biosafety 1. Low cytotoxicity In vitro cell experiments showed that the integrated nanocomposite for the diagnosis and treatment of kidney injury of the present invention exhibited good biocompatibility over a wide concentration range. After co-incubation with renal tubular epithelial cells at high concentrations (e.g., 30 mg / mL) for 48 hours, the cell viability remained above 90%, and no significant toxic effects were observed.

[0062] 2. No obvious toxic side effects in the body. In vivo experimental results showed that after treatment with the integrated nanocomposite for the diagnosis and treatment of kidney injury of the present invention, no obvious histopathological damage was observed in the major organs (including heart, liver, spleen, lung and kidney) of the experimental animals; at the same time, serum biochemical indicators (such as ALT, AST, BUN and CREA) were not significantly different from those of the control group, indicating that the integrated nanocomposite for the diagnosis and treatment of kidney injury has good in vivo safety and biological tolerability.

[0063] (iii) Significantly enhanced therapeutic effect 1. Improve the targeted delivery efficiency of antioxidant active substances This invention utilizes TMV nanocarriers to achieve effective enrichment and sustained release of therapeutic active ingredients in the kidney injury area, significantly increasing the local concentration of the drug at the lesion site and thus enhancing its efficacy.

[0064] 2. Significantly improves renal function indicators In an animal model of acute kidney injury, the integrated nanocomposite for kidney injury diagnosis and treatment of this invention can significantly improve kidney function indicators. For example, compared with the free antioxidant treatment group, the serum creatinine (CREA) and blood urea nitrogen (BUN) levels in the integrated nanocomposite group were significantly reduced and approached normal levels, indicating that it has a better effect in alleviating kidney function damage.

[0065] 3. Enhance the sustainability of treatment effects Because the integrated nanocomposite for the diagnosis and treatment of kidney injury of this invention has a longer retention time in the kidney, it can achieve continuous drug release, which significantly prolongs the duration of treatment and improves the overall efficacy compared with traditional drug delivery methods.

[0066] (iv) Integrated diagnosis and treatment and real-time monitoring of treatment efficacy This invention presents a nanocomposite for the diagnosis and treatment of kidney injury, possessing both imaging and therapeutic functions, enabling dynamic monitoring of disease progression. In animal models, the untreated group showed persistently strong fluorescent signals in the kidney region, while after effective treatment, the signal in the kidney region significantly decreased and approached normal levels, indicating that this nanocomposite for the diagnosis and treatment of kidney injury can non-invasively and in real-time reflect the process of kidney function recovery.

[0067] (v) Comprehensive technological advantages In summary, this invention achieves the following effects by constructing a TMV-based nanodiagnostic and therapeutic platform: 1. Strong imaging signal and long duration; 2. High-specificity enrichment in areas of kidney injury; 3. Excellent biosafety; 4. Significantly enhanced antioxidant therapeutic effect; 5. Integration of imaging and treatment, and visualization monitoring of therapeutic effects; This effectively overcomes the problems of short imaging time, poor targeting, low drug utilization, and lack of real-time monitoring capabilities in existing technologies. Attached Figure Description

[0068] Figure 1 The image shows the UV-Vis absorption spectrum of TMV-Cy3-SS-31.

[0069] Figure 2 The fluorescence emission spectrum of TMV-Cy3-SS-31 is shown.

[0070] Figure 3 This is a DLS diagram of TMV-Cy3-SS-31.

[0071] Figure 4 This is a TEM image of TMV-Cy3-SS-31.

[0072] Figure 5 This is a statistical graph showing the cytotoxicity of TMV-Cy3-SS-31.

[0073] Figure 6 This image shows the in vivo fluorescence imaging and temporal distribution of TMV-Cy3-SS-31 in a cisplatin-induced AKI mouse model. In the image, the left side represents normal mice, and the right side represents AKI mice.

[0074] Figure 7 The figure shows the distribution and fluorescence intensity comparison of TMV-Cy3-SS-31 in organs of normal and AKI mice. In the figure, A is the distribution map of TMV-Cy3-SS-31 in organs of normal and AKI mice, and B is the fluorescence intensity statistical map.

[0075] Figure 8HE staining images of the major organs (heart, liver, spleen, lung, and kidney) of TMV-Cy3-SS-31 in normal and AKI model mice.

[0076] Figure 9 The figure shows the effect of TMV-Cy3-SS-31 on improving renal function indicators (CREA, BUN) in AKI mice and its therapeutic effect. In the figure, A is a statistical chart of serum creatinine (CREA) level in mice and B is a statistical chart of blood urea nitrogen (BUN) level in mice.

[0077] Figure 10 TMV-Cy3-SS-31 in vivo fluorescence imaging was used to monitor AKI treatment outcomes in real time. Detailed Implementation

[0078] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0079] All numerical values ​​or expressions relating to component amounts, process conditions, etc., used in this invention shall be understood to be modified by the word "about" in all cases. When referring to a quantity or range of values, the quantity or range is an approximation within experimental variability (or within statistical experimental error). In this invention, the term "about" shall have the meaning of being within 10%, preferably within 5%, of a specified value or range. Unless otherwise specified, percentages are mass percentages.

[0080] In this invention, "room temperature" refers to ambient temperature, ranging from about 15°C to about 37°C. In some embodiments, "room temperature" refers to a temperature ranging from about 20°C to about 30°C; in other embodiments, "room temperature" refers to a temperature ranging from about 25°C to about 30°C; and in still other embodiments, "room temperature" refers to 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, etc.

[0081] The term "pharmaceutically acceptable" means that a carrier, delivery substance, excipient, diluent, and / or the salt formed therefrom is generally chemically or irrationally compatible with other components constituting a drug dosage form and physiologically compatible with receptors.

[0082] The term "pharmaceuticalally acceptable excipient" refers to carriers that do not cause significant irritation to the body and do not impair the biological activity and properties of the active compound. This includes, but is not limited to, any diluents, disintegrants, binders, glidants, and wetting agents commonly used in the art for human or animal use.

[0083] The term "pharmaceutical composition" means a composition comprising the renal injury diagnostic and therapeutic nanocomposite described in this disclosure and at least one pharmaceutically acceptable ingredient selected from the following, depending on the method of administration and dosage form: carrier, diluent, adjuvant, excipient, preservative, filler, disintegrant, wetting agent, emulsifier, suspending agent, sweetener, flavoring agent, fragrance, antibacterial agent, antifungal agent, lubricant, dispersant, thermosensitive material, temperature regulator, adhesive, stabilizer, suspending agent, etc.

[0084] The "pharmaceutical composition" described in this invention can be prepared by any method known in pharmaceutical science. Generally, these preparation methods involve associating the renal injury diagnostic and therapeutic nanocomposite (hereinafter referred to as the active ingredient) with a carrier and / or one or more other auxiliary ingredients, and then, if desired and / or expected, shaping and / or packaging the product into desired single-dose or multi-dose units.

[0085] The "pharmaceutical composition" of the present invention can be prepared according to known methods, such as those described in the general rules for preparation in the Chinese Pharmacopoeia 2025, the Japanese Pharmacopoeia 16th edition, the United States Pharmacopoeia, and the European Pharmacopoeia 9th edition. The specific preparation method depends on the dosage form.

[0086] The active ingredient and pharmaceutically acceptable excipients in the "pharmaceutical composition" described in this invention will vary depending on the identity, body type, and / or condition of the treated subject and further on the route of administration of the active ingredient. The pharmaceutical composition may contain between 0.1% and 100% (w / w) of the active ingredient.

[0087] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0088] The extraction and purification method of TMV described in this embodiment refers to "1.1 TMV extraction" in Example 1 of Patent Document CN121243378A (publication date: January 2, 2026).

[0089] Specifically, take tobacco leaves from the wild when they show typical mosaic virus lesions but have not yet entered the senescence stage, and quickly place them in a suitable environment. Store frozen at 80°C to prevent viral degradation.

[0090] Cryopreserved leaflets infected with TMV were removed and ground into a fine powder using a cryogenic grinder to ensure complete disruption of cell structure. Then, 0.1M KP buffer (containing 0.2% (v / v) β-mercaptoethanol), pre-cooled to 4°C, was added at a leaflet weight to buffer volume ratio of 1:3, and the mixture was thoroughly homogenized. The homogenate was first filtered through two layers of gauze to remove large particles, then centrifuged at 11,000×g for 20 minutes, and the supernatant was collected. This supernatant was then passed through two layers of gauze again to ensure clarity. Next, an equal volume of a 1:1 mixture of chloroform and n-butanol was added to the supernatant, and the mixture was stirred in an ice bath for 30 minutes to thoroughly mix and promote the separation of proteins and impurities, avoiding foaming. The mixture was then centrifuged at 4,500×g for 10 minutes to separate the organic and aqueous phases. The TMV-containing upper aqueous phase was collected and kept on ice for later use. Subsequently, 0.2M sodium chloride, 8% (w / v) polyethylene glycol 8000 (PEG 8000), and 1% (v / v) Triton X-100 were added sequentially to the aqueous phase. The mixture was slowly stirred in an ice bath for 30 minutes to promote virus aggregation and impurity removal. The mixture was placed in a 4°C refrigerator and allowed to stand for at least 1 hour, followed by centrifugation at 22,000×g for 15 minutes to obtain a virus precipitate. The virus precipitate was resuspended in 0.1M KP buffer and gently shaken overnight at 4°C to fully dissolve and stabilize the virus particles. Discontinuous sucrose gradients of 20% (w / v), 40% (w / v), and 80% (w / v) were prepared. The resuspended TMV protein solution was loaded onto the gradient solution and centrifuged at 10,000×g for 4.5 hours. After centrifugation, clear blue TMV bands were visible. The blue TMV characteristic bands in the gradient were collected and centrifuged multiple times at 3000 rpm for 15 minutes each time to gradually concentrate to the target volume or concentration. Finally, the TMV concentration was accurately determined using a NanoDrop spectrophotometer, providing a precise basis for sample quantification in subsequent experiments and applications. The TMV concentration used in all subsequent experiments was based on the NanoDrop measurement results.

[0091] Example 1 Preparation and characterization of TMV-Cy3-SS-31 nanoprobes preparation: TMV was suspended in 0.01M potassium phosphate buffer (pH 7.0) to obtain a TMV solution with a concentration of 15 mg / mL.

[0092] A 1 mM Cy3-NHS solution was prepared by dissolving Cy3-NHS ester in anhydrous DMSO.

[0093] Take 1 mL of TMV solution (15 mg / mL) and add an appropriate amount of Cy3-NHS solution at a TMV capsid protein subunit to Cy3 molar ratio of 10:1. Incubate at room temperature in the dark for 2 h. Centrifuge the reaction solution through a 100 kDa ultrafiltration tube (4℃, 4000 g, 20 min) and wash three times with KP buffer to remove free Cy3, obtaining purified TMV-Cy3. Resuspend the prepared TMV-Cy3 in KP buffer, add an appropriate amount of SS-31 (0.5 mg / mL) at a TMV capsid protein subunit to SS-31 molar ratio of 1:1, and incubate at room temperature in the dark for 3 h. Purify the final reaction solution through a 100 kDa ultrafiltration tube (4℃, 4000 g, 20 min) and wash three times with KP buffer to remove free SS-31, obtaining the final product TMV-Cy3-SS-31 composite nanomaterial.

[0094] Characterization: 1. Ultraviolet-Visible Absorption Spectroscopy The detection results show that TMV-Cy3-SS-31 exhibits a characteristic absorption peak of Cy3 at 550 nm, such as... Figure 1 As shown.

[0095] 2. Fluorescence emission spectrum The detection results show that TMV-Cy3-SS-31 has a strong emission peak in the near-infrared region, and its fluorescence intensity is higher than that of free Cy3. Figure 2 As shown.

[0096] 3. Dynamic Light Scattering (DLS) The detection results show that the hydrated particle size of TMV-Cy3-SS-31 is approximately 197.6 nm, with a uniform distribution (PDI < 0.2). Figure 3 As shown.

[0097] 4. Transmission electron microscope (TEM) The detection results showed that TMV-Cy3-SS-31 maintained a typical rod-like morphology, approximately 300 nm in length, and was well dispersed with no obvious aggregation. Figure 4 As shown.

[0098] 5. In vitro biosafety assessment After treating HK-2 cells with different concentrations of TMV-Cy3-SS-31 for 24 and 48 hours, the results are as follows: Figure 5 As shown in the figure. The results showed that after treatment with TMV-Cy3-SS-31, the cell survival rate remained at a high level (close to 100%), and no obvious cytotoxic effects were observed, indicating that TMV-Cy3-SS-31 has good safety and no obvious toxicity.

[0099] Example 2 In vivo imaging diagnosis of TMV-Cy3-SS-31 in AKI mouse model AKI model establishment: Eight-week-old male Balb / c nude mice were selected and AKI model was induced by a single intraperitoneal injection of cisplatin (20 mg / kg body weight). The control group was injected with an equal volume of physiological saline.

[0100] In vivo imaging: 24 hours after modeling, TMV-Cy3-SS-31 (7.5 mg / kg) was injected via the tail vein. Continuous imaging was performed at 1 min, 5 min, 10 min, 30 min, 1 h, and 2 h after injection using a small animal in vivo imaging system (excitation 540-560 nm, emission 580-600 nm).

[0101] In vivo fluorescence imaging and temporal distribution of TMV-Cy3-SS-31 in a cisplatin-induced AKI mouse model are shown in Figure 6. Comparison of TMV-Cy3-SS-31 distribution and fluorescence intensity in organs of normal and AKI mice is also shown in Figure 6. Figure 7 As shown.

[0102] The results showed that the kidney region of mice in the AKI group continuously exhibited strong fluorescence signals for 2 hours after injection, while the fluorescence signals of mice in the normal control group were mainly concentrated in the liver. Two hours after injection, mice were sacrificed and their organs were extracted for imaging. The fluorescence intensity of the kidneys in the AKI group was more than three times that of the normal group. This indicates that TMV-Cy3-SS-31 can effectively distinguish between AKI and normal kidneys, achieving high-contrast diagnosis.

[0103] HE staining images of TMV-Cy3-SS-31 in major organs (heart, liver, spleen, lung, and kidney) of normal and AKI model mice. Results are as follows: Figure 8 As shown.

[0104] The results showed that HE staining revealed the histopathological effects of TMV-Cy3-SS-31 on the heart, liver, spleen, lungs, and kidneys of mice. Compared with the normal control group, no obvious pathological damage was observed in any organ in the TMV-Cy3-SS-31 treatment group, indicating that it has good biocompatibility. The kidneys of mice in the AKI model group showed typical pathological changes of acute kidney injury, confirming the successful establishment of the model.

[0105] Example 3 Evaluation of the therapeutic effect of TMV-Cy3-SS-31 on AKI Grouping and Treatment: Cisplatin (20 mg / kg body weight)-induced AKI mice were randomly divided into three groups: AKI model group (saline, 100 mg / kg / day, intraperitoneal injection), free SS-31 treatment group (SS-31, 100 mg / kg / day, intraperitoneal injection), and TMV-Cy3-SS-31 treatment group (dose equivalent to SS-31, 100 mg / kg / day, intraperitoneal injection). A control group (saline, 100 mg / kg / day, intraperitoneal injection) was also included. Treatment was administered for 3 consecutive days.

[0106] Kidney function tests: 72 hours after the last administration, blood was collected from mice to test serum creatinine (CREA) and blood urea nitrogen (BUN).

[0107] The effects of TMV-Cy3-SS-31 on improving renal function indicators (serum creatinine, blood urea nitrogen BUN) in AKI mice and its therapeutic efficacy are as follows: Figure 9 As shown. The results showed that, compared with the normal control group (Control), the levels of CREA and BUN in the model group (AKI) mice were significantly reduced (P < 0.01 or 0.0001), indicating that cisplatin-induced AKI modeling was successful.

[0108] After drug intervention, compared with the AKI group, TMV-Cy3-SS-31 treatment significantly reduced CREA and BUN levels (P < 0.01 or 0.001), while SS-31 treatment showed no statistically significant difference in CREA and BUN levels (P > 0.05). This indicates that TMV-Cy3-SS-31 has excellent therapeutic effects on AKI.

[0109] Meanwhile, compared with the control group, there was no statistically significant difference in CREA and BUN levels after TMV-Cy3-SS-31 treatment (P>0.05), indicating that AKI mice had reached a healthy level after TMV-Cy3-SS-31 treatment, confirming that TMV-Cy3-SS-31 can effectively repair kidney damage.

[0110] Example 4 Monitoring the efficacy of AKI treatment using TMV-Cy3-SS-31 Methods: AKI mice were treated according to the protocol in Example 3 (divided into free SS-31 group and TMV-Cy3-SS-31 group). After the treatment ended (day 3), all mice were injected with TMV-Cy3-SS-31 (7.5 mg / kg) via the tail vein and in vivo fluorescence imaging was performed (method as in Example 2).

[0111] TMV-Cy3-SS-31 in vivo fluorescence imaging to assess AKI treatment outcomes, such as Figure 10 As shown.

[0112] The results showed that the kidneys of mice in the AKI group had strong fluorescent signals. However, the fluorescence signal intensity in the kidney region of mice that underwent effective treatment (especially the TMV-Cy3-SS-31 treatment group) was significantly reduced, indicating that TMV-Cy3-SS-31 can not only diagnose AKI, but also assess the therapeutic effect on AKI mice, and the signal intensity was negatively correlated with the degree of renal function recovery.

[0113] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. The application of a nanocomposite for the diagnosis and treatment of kidney injury and its pharmaceutical composition in the preparation of drugs related to the following kidney diseases, characterized in that, The drugs include drugs for the diagnosis of kidney diseases, drugs for targeted delivery to the kidneys, or drugs for integrated diagnosis and treatment of kidney diseases. The integrated nanocomposite for the diagnosis and treatment of kidney injury comprises tobacco mosaic virus, fluorescent dye, and therapeutic active ingredient, and possesses at least one of the following properties: (1) Fluorescence emission wavelength range 550-800nm; (2) In vivo circulation time ≥ 6-24h; (3) Kidney retention time ≥12-48h; (4) The signal enhancement factor in the lesion area is ≥2 times; (5) Cell viability ≥ 85%.

2. The application according to claim 1, characterized in that, The fluorescent dye includes at least one of cyanine dyes, rhodamine dyes, FITC dyes, and near-infrared fluorescent dyes; the therapeutic active ingredient includes at least one of antioxidant drugs, anti-inflammatory drugs, anti-apoptotic drugs, and growth factors that promote renal tubular repair.

3. The application according to claim 2, characterized in that, The fluorescent dye is a cyanin dye, and the therapeutic active ingredient is an antioxidant drug.

4. The application according to claim 3, characterized in that, The cyanine dyes include at least one of Cy3, Cy5, Cy5.5, Cy7 and their derivatives; the antioxidant is a thiol-containing antioxidant, which includes at least one of aramidin, N-acetylcysteine, glutathione and cysteine.

5. The application according to claim 1, characterized in that, The pharmaceutical composition is made from the therapeutic nanocomposite for kidney injury and pharmaceutically acceptable excipients.

6. The application according to claim 1, characterized in that, The molar ratio of the tobacco mosaic virus to the fluorescent dye is 5-50:

1.

7. The application according to claim 1, characterized in that, The molar ratio of the tobacco mosaic virus to the therapeutic active ingredient is 1:0.05-10.

8. A nanocomposite for the diagnosis and treatment of kidney injury based on tobacco mosaic virus nanocarrier, characterized in that, It includes tobacco mosaic virus, fluorescent dyes, and therapeutically active ingredients, and possesses at least one of the following properties: (1) Fluorescence emission wavelength range 550-800nm; (2) In vivo circulation time ≥ 6-24h; (3) Kidney retention time ≥12-48h; (4) The signal enhancement factor in the lesion area is ≥2 times; (5) Cell viability ≥ 85%.

9. The integrated nanocomposite for the diagnosis and treatment of kidney injury according to claim 8, characterized in that, The molar ratio of the tobacco mosaic virus to the fluorescent dye is 5-50:

1.

10. The integrated nanocomposite for the diagnosis and treatment of kidney injury according to claim 8, characterized in that, The molar ratio of the tobacco mosaic virus to the fluorescent dye is 10-20:

1.

11. The integrated nanocomposite for the diagnosis and treatment of kidney injury according to any one of claims 8-10, characterized in that, The fluorescent dye includes at least one of cyanine dyes, rhodamine dyes, FITC dyes, and near-infrared fluorescent dyes; the therapeutic active ingredient includes at least one of antioxidant drugs, anti-inflammatory drugs, anti-apoptotic drugs, and growth factors that promote renal tubular repair.

12. The integrated nanocomposite for the diagnosis and treatment of kidney injury according to claim 11, characterized in that, The fluorescent dye is a cyanine dye, and the therapeutic active ingredient is an antioxidant drug, which includes at least one of thiol-containing antioxidants, vitamins, and antioxidant enzymes.

13. The integrated nanocomposite for the diagnosis and treatment of kidney injury according to claim 12, characterized in that, The cyanine dyes include at least one of Cy3, Cy5, Cy5.5, Cy7 and their derivatives; the thiol-containing antioxidants include at least one of aramidin, N-acetylcysteine, glutathione and cysteine.

14. The method for preparing the integrated nanocomposite for diagnosis and treatment of kidney injury according to any one of claims 8-13, characterized in that, Includes the following steps: (1) Disperse tobacco mosaic virus in buffer solution to obtain TMV suspension; (2) Add the fluorescent dye to the TMV suspension for fluorescent labeling reaction, purify and collect to obtain TMV-dye; (3) Resuspend the TMV-dyed, add the therapeutic active ingredient load, purify and collect to obtain the product.

15. The preparation method according to claim 14, characterized in that, The pH of the buffer solution in step (1) is 6.5-7.

5.

16. The preparation method according to claim 14, characterized in that, The buffer solution mentioned in step (1) includes phosphate buffer or KP buffer.

17. The preparation method according to claim 14, characterized in that, The concentration of the TMV suspension in step (1) is 5-20 mg / mL.

18. The preparation method according to claim 14, characterized in that, The concentration of the fluorescent dye in step (2) is 0.1-5 mM.

19. The preparation method according to claim 14, characterized in that, The fluorescent labeling reaction described in step (2) is carried out in the dark at 15-37℃ for 0.5-6 hours.

20. The preparation method according to claim 14, characterized in that, The concentration of the therapeutic active ingredient in step (3) is 0.5-20 mg / mL.

21. The preparation method according to claim 14, characterized in that, The load time mentioned in step (2) is 1-6 hours.

22. A pharmaceutical composition, characterized in that, It is made from the integrated nanocomposite for diagnosis and treatment of kidney injury as described in any one of claims 8-13 or the preparation method described in any one of claims 14-21, and pharmaceutically acceptable excipients.

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