Application of albumin functionalized tantalum sulfide nanodot in preparation of medicine for treating acute kidney injury

Abstract

The invention relates to application of albumin functionalized tantalum sulfide nanodots in preparation of drugs for treating acute kidney injury, and belongs to the technical field of new application of nano-drugs. The albumin functionalized tantalum sulfide nanodot used in the invention is formed by simply and efficiently assembling an ideal carrier albumin which is safe, non-toxic and free of immunogenicity and a layered transition metal tantalum sulfide which is high in biocompatibility; the protein can be specifically accumulated in the kidney by passing through a glomerular filtration barrier by virtue of the ultra-small particle size of the protein, is actively internalized to proximal tubule epithelial cells by virtue of the specific binding of albumin and a giant protein receptor, and is sequentially positioned to mitochondria by virtue of the high affinity of albumin to the mitochondria. The nanodot can efficiently remove active oxygen, protect mitochondria and limit cell apoptosis, immune activation and aseptic inflammation through active valence change of tantalum ions in a pathological injury microenvironment of acute kidney injury, so that near-end tubule epithelial cell injury is relieved, and kidney functions are effectively recovered.

Description

Technical Field

[0001] The present invention relates to the technical field of new uses of nano-drugs, and particularly relates to the application of albumin-functionalized tantalum sulfide nanodots in the preparation of drugs for treating acute kidney injury. Background Art

[0002] Acute kidney injury (AKI) is manifested as a sudden disorder or loss of kidney function, accompanied by the over-accumulation of metabolic wastes such as serum creatinine (CRE) and blood urea nitrogen (BUN). AKI is a multi-factorial clinical syndrome, often secondary to potential nephrotoxic factors such as drug exposure, sepsis, or ischemia-reperfusion injury in the body. The annual number of patients is over 13 million. The efficacy of existing supportive therapies and kidney transplantation is limited, and it is difficult to prevent the disease from progressing to intractable irreversible fibrosis, bringing a heavy economic burden. The global death burden related to AKI has far exceeded that of high-risk severe diseases such as breast cancer. The development of new and potent kidney protection drugs that can precisely target AKI is crucial and highly concerned.

[0003] Heterogeneous AKI has a common therapeutic target cell: damaged proximal tubular epithelial cells (PTECs). PTECs have a high tendency to be damaged due to their unique location distribution and high-intensity reabsorption function. During AKI, they are exposed to concentrated metabolic toxins and continuously stimulated by risk factors, causing mitochondrial electron transport chain disorders and generating excessive reactive oxygen species (ROS). Toxic ROS attack the endogenous antioxidant defense and then turn back on the mitochondria, causing oxidative stress and releasing damage products: the electron transport carrier cytochrome c (Cyt c) and damage-associated molecular patterns such as mitochondrial DNA (mtDNA). Among them, Cyt c activates endogenous apoptosis, and the increase in the apoptosis rate of PTECs directly weakens the kidney function of AKI patients; mtDNA initiates a pattern recognition cascade reaction, triggering the activation of the cGAS / STING innate immune axis and inflammatory storm, which exacerbates kidney injury. The imbalanced redox ecology and intense inflammatory response reinforce each other. The continuous high-stress state and the uncurbed vicious cycle are reflected in the sudden loss of kidney function in AKI patients, and then progress to a poor prognosis.

[0004] Based on the above pathophysiological mechanism of AKI, eliminating the excessive ROS, the key factor in the AKI disease process, in target cells PTECs should be an effective means to break the pathological cycle and achieve the treatment of AKI. However, existing therapies or nanotechnologies face various dilemmas in drug administration and development: ① Small molecule antioxidants have a narrow therapeutic window and low bioavailability; ② Artificial nanomaterials with insufficient biocompatibility are prone to eliciting immunogenic reactions and accelerating their own clearance, making it difficult to exert and maintain drug efficacy; ③ Raw material-improved nanodrugs with good biocompatibility lack the precise delivery ability for the complex and delicate anatomical structure of the kidney; ④ Multilayered nanoplatforms with cumbersome assembly are difficult to ensure that all functional response events before reaching the lesion are completed in a linked manner, and the inevitable increase in their particle size also makes them subject to the physiological block of the glomerular filtration barrier (GFB). Therefore, optimizing the design or finding a new nanoplatform with a minimal number of raw materials, which can organically integrate different functional modules such as biocompatible components, targeting binding signals, and antioxidant groups, is a potential strategy to break through the treatment limitations. Summary of the Invention

[0005] The present invention aims to solve the technical problems in the prior art, such as insufficient biocompatibility, lack of targeting ability, and / or complex assembly and too large particle size of nanoplatforms applied in the treatment of AKI, and provides an application of albumin-functionalized tantalum sulfide nanodots in the preparation of drugs for treating acute kidney injury. The albumin-functionalized tantalum sulfide nanodots used in the present invention are simply and efficiently assembled from albumin, an ideal carrier that is safe, non-toxic, and non-immunogenic, and layered transition metal tantalum sulfide with high biocompatibility; they can cross the GFB by virtue of their ultra-small particle size, actively internalize into PTECs by using the affinity between albumin and the megalin receptor, and sequentially reach the mitochondria depending on the tropism of albumin for mitochondria. In terms of the mechanism of action, the tantalum ions serving as the antioxidant module in the nanodots efficiently scavenge ROS and protect mitochondria through active valence changes, limit apoptosis, immune activation, and sterile inflammation, and ultimately achieve injury mitigation and effective treatment during AKI.

[0006] To solve the above technical problems, the technical solutions of the present invention are specifically as follows:

[0007] An application of albumin-functionalized tantalum sulfide nanodots in the preparation of drugs for treating acute kidney injury.

[0008] In the above technical solution, preferably, the albumin-functionalized tantalum sulfide nanodots are spherical in shape and have a particle size between 4 and 6 nm.

[0009] In the above technical solution, preferably, in the albumin-functionalized tantalum sulfide nanodots, the tantalum sulfide nanodots are the core and albumin is the outer coating carrier.

[0010] In the above technical solution, preferably, the albumin-functionalized tantalum sulfide nanodots are obtained by stirring and self-assembling tantalum sulfide nanodots and an albumin aqueous solution.

[0011] In the above technical solution, more preferably, the stirring and self-assembling time is 2 to 3 h, the gas condition is argon, and the temperature condition is room temperature.

[0012] In the above technical solution, more preferably, the tantalum sulfide nanodots are prepared by top-down grinding of tantalum sulfide powder and liquid-phase ultrasonic exfoliation, and the albumin aqueous solution is 10 mg / mL human serum albumin or bovine serum albumin.

[0013] In the above technical solution, more preferably, the grinding time of the tantalum sulfide powder is 2 to 3 h, the liquid-phase ultrasonic exfoliation time is 1 to 2 w, and the liquid-phase solvent is N-methylpyrrolidone.

[0014] In the above technical solution, more preferably, the volume ratio of the tantalum sulfide nanodots to the albumin aqueous solution is 1:10.

[0015] The beneficial effects of the present invention are as follows:

[0016] The patent application with publication number CN119258232 A discloses that albumin-functionalized tantalum sulfide nanosheets can be used for the treatment of acute liver injury. However, the present invention for the first time discovers that albumin-functionalized tantalum sulfide nanodots can be used for the treatment of acute kidney injury, which is a new use different from the uses disclosed in the prior art. What the present invention uses are albumin-functionalized tantalum sulfide nanodots, rather than the nanosheet structure. With its ultra-small particle size, it specifically accumulates in the kidneys by crossing the GFB, actively internalizes into PTECs by the specific binding of albumin to megalin, and sequentially localizes to mitochondria depending on the high affinity and tropism of albumin for mitochondria. These nanodots efficiently scavenge ROS and protect mitochondria in the pathological injury microenvironment of AKI through the active valence change of tantalum ions, limit apoptosis, immune activation, and sterile inflammation, thereby achieving the alleviation of PTECs injury and effectively restoring kidney function. More specifically:

[0017] The albumin-functionalized tantalum sulfide nanodots used in the present invention are obtained by a simple, convenient, and efficient self-assembly method. The raw materials used are inexpensive and easily available, the synthesis method is simple, and they all have high biocompatibility.

[0018] The albumin-functionalized tantalum sulfide nanodots used in the present invention are prepared by stirring and self-assembling using tantalum sulfide and albumin as raw materials. They are spherical-like, have a small size ( Figure 1 ) and are enriched with negative charges on the surface ( Figure 2 ), and have good water solubility and dispersibility.

[0019] The albumin-functionalized tantalum sulfide nanodots used in the present invention have strong broad-spectrum free radical scavenging activity ( Figure 3 ).

[0020] The albumin-functionalized tantalum sulfide nanodots used in the present invention can specifically accumulate in the kidneys of mice ( Figure 4 ).

[0021] The albumin-functionalized tantalum sulfide nanodots used in the present invention can be actively recognized and internalized by human renal tubular epithelial cells (HK-2), and this specific drug uptake is inhibited by megalin receptor inhibitors ( Figure 5 ).

[0022] The albumin-functionalized tantalum sulfide nanodots used in the present invention can sequentially localize to the mitochondria of HK-2 cells ( Figure 6 ).

[0023] The albumin-functionalized tantalum sulfide nanodots used in the present invention can effectively restore the renal function of AKI mice and alleviate the pathological changes of renal tissues ( Figure 7 ).

[0024] The albumin-functionalized tantalum sulfide nanodots used in the present invention can effectively reduce the ROS level in the renal tissues of AKI mice and reverse oxidative stress ( Figure 8 ).

[0025] The albumin-functionalized tantalum sulfide nanodots used in the present invention can effectively reduce the apoptosis rate of renal tissues in AKI mice and inhibit apoptosis ( Figure 9 ).

[0026] The albumin-functionalized tantalum sulfide nanodots used in the present invention can effectively inhibit the activation of the cGAS pathway and inflammatory cell infiltration in the renal tissues of AKI mice ( Figure 10 ).

[0027] The albumin-functionalized tantalum sulfide nanodots used in the present invention have good biosafety, and no obvious morphological lesions were observed in the major organs (heart, liver, spleen, lung, and kidney) of mice after single high-dose administration in the short term or multiple therapeutic-dose administrations in the long term ( Figure 11 ). BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The present invention will be further described in detail below with reference to the drawings and specific embodiments.

[0029] Figure 1 Transmission electron microscope image (A) and particle size statistical chart (B) of the albumin-functionalized tantalum sulfide nanodots prepared in Example 1.

[0030] Figure 2Zeta potential map of the albumin-functionalized tantalum sulfide nanodots prepared in Example 1.

[0031] Figure 3 UV absorption spectra of in vitro free radical scavenging of the albumin-functionalized tantalum sulfide nanodots prepared in Example 1, including superoxide anion (A), hydrogen peroxide (B), hydroxyl radical (C), and peroxynitrite (D).

[0032] Figure 4 Representative fluorescence images of major organs (kidney, liver, lung, heart, and spleen) of control or AKI group mice after intravenous injection of the albumin-functionalized tantalum sulfide nanodots prepared in Example 1 loaded with fluorescent labels 9 h after injection.

[0033] Figure 5 Representative fluorescence images of the albumin-functionalized tantalum sulfide nanodots prepared in Example 1 loaded with fluorescent labels being taken up by HK-2 cells.

[0034] Figure 6 Colocalization fluorescence images of the albumin-functionalized tantalum sulfide nanodots prepared in Example 1 loaded with fluorescent labels with various cell organelles (endoplasmic reticulum, nucleus, mitochondria, lysosome, and Golgi apparatus) in HK-2 cells.

[0035] Figure 7 Renal function index graphs (A: CRE level; B: BUN level) and renal tissue morphological staining graphs (C) of normal control mice, AKI mice, and AKI mice injected with the albumin-functionalized tantalum sulfide nanodots prepared in Example 1.

[0036] Figure 8 ROS fluorescence staining graphs of renal tissues of normal control mice, AKI mice, and AKI mice injected with the albumin-functionalized tantalum sulfide nanodots prepared in Example 1.

[0037] Figure 9 TUNEL staining graphs of renal tissues of normal control mice, AKI mice, and AKI mice injected with the albumin-functionalized tantalum sulfide nanodots prepared in Example 1.

[0038] Figure 10 Immunohistochemical staining graphs of cGAS (A) and F4 / 80 (B) of renal tissues of normal control mice, AKI mice, and AKI mice injected with the albumin-functionalized tantalum sulfide nanodots prepared in Example 1.

[0039] Figure 11 Tissue morphological staining graphs of major organs (heart, liver, spleen, lung, and kidney) of normal mice after single short-term high-dose (A) or long-term multiple treatment-dose (B) administration of the albumin-functionalized tantalum sulfide nanodots prepared in Example 1. Detailed implementation manners

[0040] The inventive concept of the present invention is as follows: In order to solve the technical problems of insufficient biocompatibility, lack of targeting ability, and / or complex assembly and too large particle size of the nanoformulations applied to the treatment of AKI in the prior art, the present invention provides albumin-functionalized tantalum sulfide nanodots for the treatment of AKI. The albumin-functionalized tantalum sulfide nanodots used in the present invention are simply and efficiently stirred and self-assembled from albumin, an ideal carrier that is safe, non-toxic, and non-immunogenic, and layered transition metal tantalum sulfide with high biocompatibility. The nanodots are spherical-like, have ultra-small particle sizes, abundant surface negative charges, and broad-spectrum free radical scavenging activities. The albumin-functionalized tantalum sulfide nanodots used in the present invention penetrate the GFB by virtue of their ultra-small particle sizes and specifically accumulate in the kidneys, actively internalize into PTECs by using the specific binding of albumin to megalin, and sequentially localize to mitochondria depending on the high affinity of albumin for mitochondria. The nanodots efficiently scavenge ROS and protect mitochondria through the active valence change of tantalum ions in the pathological injury microenvironment of AKI, limit apoptosis, immune activation, and sterile inflammation, and thus relieve PTECs injury and effectively restore kidney function.

[0041] The present invention provides an application of albumin-functionalized tantalum sulfide nanodots in the preparation of drugs for treating acute kidney injury.

[0042] Furthermore, it is the application of albumin-functionalized tantalum sulfide nanodots in achieving the treatment of AKI by integrating biocompatible components, PTECs mitochondrial targeting binding signals, and antioxidant groups.

[0043] Furthermore, in the albumin-functionalized tantalum sulfide nanodots, the tantalum sulfide nanodots are the core and albumin is the outer coating carrier. The albumin-functionalized tantalum sulfide nanodots are prepared by stirring and self-assembling using tantalum sulfide and albumin as raw materials, are spherical-like, and have a particle size between 4 and 6 nm. The specific preparation process is as follows:

[0044] Step (1) Synthesis:

[0045] Weigh 0.2 g of tantalum sulfide powder and grind it in an agate mortar for 2 - 3 h. Measure 20 mL of N-methylpyrrolidone, mix the two, and perform ultrasonic exfoliation for 1 - 2 w. Centrifuge at 8000 rpm for 10 min and take the supernatant, and stir and self-assemble it with the albumin aqueous solution at room temperature under an argon gas condition for 2 - 3 h;

[0046] The volume ratio of the tantalum sulfide nanodots to the albumin aqueous solution is 1:10; the albumin aqueous solution is 10 mg / mL human serum albumin or bovine serum albumin.

[0047] Step (2) Purification:

[0048] Centrifuge the sample obtained in step (1) at a high speed. The centrifugation conditions are 13,000 rpm for 10 min. Retain the precipitate, resuspend it repeatedly with ultrapure water and centrifuge and wash it 3 times to remove unreacted impurities. The centrifugation conditions are the same as before, i.e., 13,000 rpm for 10 min.

[0049] Step (3) Storage:

[0050] Argonize the sample purified in step (2) and store it in the dark at 4 °C to obtain the albumin-functionalized tantalum sulfide nanodot solution.

[0051] The technical solutions of the present invention are clearly and completely described below through examples. However, it should be understood that the following examples do not limit the protection scope of the present invention.

[0052] Example 1

[0053] (1) Synthesis of albumin-functionalized tantalum sulfide nanodots:

[0054] Weigh 0.2 g of tantalum sulfide powder and grind it in an agate mortar for 2 h. Measure 20 mL of N-methylpyrrolidone. After mixing the two, ultrasonically exfoliate for 1 week, centrifuge at 8,000 rpm for 10 min, and take the supernatant. Stir and self-assemble it with a 10 mg / mL bovine serum albumin aqueous solution in a round-bottom flask at a volume ratio of tantalum sulfide nanodots to bovine serum albumin aqueous solution of 1:10. The temperature is room temperature, the gas condition is argon, and the duration is 3 h;

[0055] (2) Purification:

[0056] Centrifuge the sample obtained in step (1) at 13,000 rpm for 10 min. Retain the precipitate, resuspend it repeatedly with ultrapure water and centrifuge and wash it 3 times to remove unreacted impurities. The centrifugation conditions are the same as above, i.e., centrifuge at 13,000 rpm for 10 min.

[0057] (3) Storage:

[0058] Argonize the sample purified in step (2) and store it in the dark at 4 °C to obtain albumin-functionalized tantalum sulfide nanodots (labeled: fTANS).

[0059] Example 2

[0060] (1) Synthesis of albumin-functionalized tantalum sulfide nanodots:

[0061] Weigh 0.2 g of tantalum sulfide powder and grind it in an agate mortar for 2 h. Measure 20 mL of N-methylpyrrolidone. After mixing the two, ultrasonically exfoliate for 2 weeks, centrifuge at 8,000 rpm for 10 min, and take the supernatant. Stir and self-assemble it with a 10 mg / mL bovine serum albumin aqueous solution in a round-bottom flask at a volume ratio of tantalum sulfide nanodots to bovine serum albumin aqueous solution of 1:10. The temperature is room temperature, the gas condition is argon, and the duration is 3 h;

[0062] (2) The purification and storage steps are the same as those in Example 1, and the obtained product is called albumin-functionalized tantalum sulfide nanodots 2.

[0063] Example 3

[0064] (1) Synthesis of albumin-functionalized tantalum sulfide nanodots:

[0065] Weigh 0.2 g of tantalum sulfide powder and grind it in an agate mortar for 3 h. Measure 20 mL of N-methylpyrrolidone, mix the two, and ultrasonically exfoliate for 2 w. Centrifuge at 8000 rpm for 10 min and take the supernatant. Self-assemble it by stirring in a round-bottom flask with a 10 mg / mL bovine serum albumin aqueous solution at a volume ratio of tantalum sulfide nanodots to bovine serum albumin aqueous solution of 1:10. The temperature is at room temperature, the gas condition is argon, and the duration is 2 h;

[0066] (2) The purification and storage steps are the same as those in Example 1, and the obtained product is called albumin-functionalized tantalum sulfide nanodots 3.

[0067] Example 4

[0068] (1) Synthesis of albumin-functionalized tantalum sulfide nanodots:

[0069] Weigh 0.2 g of tantalum sulfide powder and grind it in an agate mortar for 2 h. Measure 20 mL of N-methylpyrrolidone, mix the two, and ultrasonically exfoliate for 1 w. Centrifuge at 8000 rpm for 10 min and take the supernatant. Self-assemble it by stirring in a round-bottom flask with a 10 mg / mL human serum albumin aqueous solution at a volume ratio of tantalum sulfide nanodots to human serum albumin aqueous solution of 1:10. The temperature is at room temperature, the gas condition is argon, and the duration is 3 h;

[0070] (2) The purification and storage steps are the same as those in Example 1, and the obtained product is called albumin-functionalized tantalum sulfide nanodots 4.

[0071] Example 5

[0072] (1) Synthesis of albumin-functionalized tantalum sulfide nanodots:

[0073] Weigh 0.2 g of tantalum sulfide powder and grind it in an agate mortar for 2 h. Measure 20 mL of N-methylpyrrolidone, mix the two, and ultrasonically exfoliate for 2 w. Centrifuge at 8000 rpm for 10 min and take the supernatant. Self-assemble it by stirring in a round-bottom flask with a 10 mg / mL human serum albumin aqueous solution at a volume ratio of tantalum sulfide nanodots to human serum albumin aqueous solution of 1:10. The temperature is at room temperature, the gas condition is argon, and the duration is 3 h;

[0074] (2) The purification and storage steps are the same as those in Example 1, and the obtained product is called albumin-functionalized tantalum sulfide nanodots 5.

[0075] Example 6

[0076] (1) Synthesis of albumin-functionalized tantalum sulfide nanodots:

[0077] Weigh 0.2 g of tantalum sulfide powder and grind it in an agate mortar for 3 h. Measure 20 mL of N-methylpyrrolidone. After mixing the two, perform ultrasonic exfoliation for 2 w. Centrifuge at 8000 rpm for 10 min and take the supernatant. Stir and self-assemble it with a 10 mg / mL aqueous solution of human serum albumin in a round-bottom flask at a volume ratio of tantalum sulfide nanodots to bovine serum albumin aqueous solution of 1:10. The temperature is room temperature, the gas condition is argon, and the duration is 2 h;

[0078] (2) The purification and storage steps are the same as those in Example 1, and the obtained product is called albumin-functionalized tantalum sulfide nanodots 6.

[0079] The grinding time of tantalum sulfide powder, the liquid-phase ultrasonic exfoliation time, and the stirring self-assembly duration in the above examples can also be any values within the aforementioned defined ranges, and no further examples will be given here.

[0080] The albumin-functionalized tantalum sulfide nanodots synthesized in Example 1 were characterized in terms of morphology, physicochemical properties, AKI efficacy evaluation, and biosafety verification below. The albumin-functionalized tantalum sulfide nanodots synthesized in Example 1 in the specification drawings are all represented by fTANS.

[0081] Example 7

[0082] Taking the albumin-functionalized tantalum sulfide nanodots fTANS synthesized in Example 1 as an example, its morphology, particle size, surface potential, and in vitro free radical scavenging performance were characterized. The specific steps are as follows:

[0083] (1) Morphology characterization: Take the transmission electron microscope image of fTANS with a TECNAI G2 high-resolution transmission electron microscope. The results are as Figure 1 shown. fTANS is spherical-like in water (A), has an ultra-small particle size and good dispersibility, and the average particle size is 4.06 nm (B).

[0084] (2) Surface potential characterization: Detect the surface potential of fTANS with a Zeta potential analyzer. The results are as Figure 2 shown. fTANS has abundant negative charges, and its surface potential is -11.0 mV.

[0085] (3) Determination of in vitro free radical scavenging performance:

[0086] The scavenging ability of fTANS against superoxide anions was determined by the nitroblue tetrazolium chloride (NBT)-ultraviolet method. The reaction system consisted of 0.1 M methionine, 0.1 M riboflavin, 0.01 M NBT, 0.1 M PBS (pH 7.4), different concentrations of fTANS (45, 90, 180, 360 μg / mL), and ultrapure water. The mixed solution was added to a cuvette and irradiated under an ultraviolet lamp for 5 min, and the absorbance of the reaction product was measured in the wavelength range of 450 - 850 nm.

[0087] The scavenging ability of fTANS against hydrogen peroxide was determined by the ultraviolet method. The reaction system consisted of different concentrations of H2O2 solutions (3.125, 6.25, 12.5, 25, 50 mM) and a fixed concentration of fTANS (25 μg / mL). After reacting in the dark for 12 h, the absorbance of the reaction product was measured in the wavelength range of 250 - 300 nm.

[0088] The scavenging ability of fTANS against hydroxyl radicals was determined by the 3,3’,5,5’-tetramethylbenzidine (TMB)-ultraviolet method. The reaction system consisted of 0.05 mM FeSO4·7H2O, 0.1 mM TMB, 0.01 M PBS (pH 7.4), and different concentrations of fTANS (2, 4, 8, 16 μg / mL). The mixed solution was added to a cuvette and reacted at room temperature in the dark for 6 min, and the absorbance of the reaction product was measured in the wavelength range of 500 - 800 nm.

[0089] The scavenging ability of fTANS against peroxynitrite was determined by the ultraviolet method. The reaction system consisted of different concentrations of ONOO - solutions (0.1, 0.2, 0.5, 1, 2 μM) and a fixed concentration of fTANS (25 μg / mL). After reacting in the dark for 12 h, the absorbance of the reaction product was measured at 275 nm.

[0090] The in vitro free radical scavenging results are as Figure 3 shown. The albumin-functionalized tantalum sulfide nanodots fTANS prepared in the present invention have broad-spectrum free radical scavenging performance and can efficiently scavenge superoxide anions (A), hydrogen peroxide (B), hydroxyl radicals (C), and peroxynitrite (D).

[0091] Example 8

[0092] Taking the albumin-functionalized tantalum sulfide nanodots fTANS synthesized in Example 1 as an example, the distribution of fTANS in the main organs of mice in the control group or the AKI group was explored. The specific steps are as follows:

[0093] Synthesis of fluorescently labeled nano-drug (FITC-fTANS): Mix and stir FITC solution (1 mg powder dissolved in 1 mL DMSO) and fTANS at a volume ratio of 1:5 in the dark at room temperature for 5 h, and then transfer to a 3.5 kD dialysis bag for dialysis. Replace the ultrapure water outside the bag every 8 h during dialysis. After 1 day, centrifuge at 13000 rpm for 10 min, and the obtained supernatant is FITC-fTANS.

[0094] Animal feeding: Kunming mice (male, 4 - 6 weeks old, 18 - 22 g) are raised in standard-grade animal rooms, maintaining a room temperature of 24°C ± 2°C and a 12 h day-night light cycle. After 1 week of regular and quantitative adaptive feeding, the experiment is carried out.

[0095] Animal model establishment, drug administration and drug distribution detection: Mice are randomly divided into Control and AKI groups and are deprived of water for 15 h. After 2 h of intramuscular injection of 50% glycerol (8 mL / kg) to induce the AKI model, both groups of mice are given an equal amount of FITC-fTANS by tail vein injection, and the main organs of the mice (kidney, liver, lung, heart and spleen) are collected at 9 h after drug administration. After rinsing the surface of the organs with normal saline, wipe them clean with filter paper and take bright-field and fluorescence images under the microscope.

[0096] The results are as Figure 4 shown. The albumin-functionalized tantalum sulfide nanodots fTANS prepared by the present invention can specifically accumulate in the kidney tissues of mice.

[0097] Example 9

[0098] Taking the albumin-functionalized tantalum sulfide nanodots fTANS synthesized in Example 1 as an example, explore its targeting ability to PTECs. The specific steps are as follows:

[0099] The steps for synthesizing the fluorescently labeled nano-drug (FITC-fTANS) are the same as those in Example 8.

[0100] Culture of HK-2 cells: Incubate with DMEM / F12 cell culture medium containing 10% fetal bovine serum and place in a 5% CO2, 37°C constant temperature incubator for culture.

[0101] Verification of megalin receptor targeting of fTANS: At 3×10 4Inoculate HK-2 cells at a density of [number] / well in a 24-well cell culture plate. After incubating the cells in a constant temperature CO2 incubator for 24 h, pretreat them with PBS, gentamicin (GEN), ethylenediaminetetraacetic acid (EDTA), or bovine serum albumin (BSA) for 30 min respectively. Subsequently, gently rinse three times with PBS, aspirate and discard the megalin receptor endocytosis inhibitor in the three groups, and then add FITC-fTANS to co-incubate with each group of cells at 37 °C for 30 min. Finally, rinse the bottom of the wells three times with Hank's buffered salt solution, and observe the uptake of the nanodrug by HK-2 cells and capture fluorescence images under an inverted fluorescence microscope.

[0102] The results are as Figure 5 shown that FITC-fTANS can be effectively taken up by HK-2 cells, and this uptake rate significantly decreases after pretreatment with the megalin receptor endocytosis inhibitor, indicating that the internalization and absorption of fTANS by PTECs through megalin is reliable.

[0103] Example 10

[0104] Taking the albumin-functionalized tantalum sulfide nanodots fTANS synthesized in Example 1 as an example, explore its distribution in various organelles of HK-2 cells. The specific steps are as follows:

[0105] The steps for synthesizing the fluorescently labeled nanodrug (FITC-fTANS) are the same as those in Example 8.

[0106] The steps for culturing HK-2 cells are the same as those in Example 9.

[0107] Organelle distribution experiment of fTANS: Inoculate HK-2 cells at a density of 1×10 4 cells / well in a 24-well cell culture plate, and pre-stain the cells with FITC-fTANS for 1 h. After rinsing three times with cell culture medium, specifically label each organelle with a fluorescent probe or dye: mitochondria (Mito-Tracker Red probe, dilution ratio 1:5000, co-incubate at 37 °C for 20 min), endoplasmic reticulum (ER-Tracker Red probe, dilution ratio 1:2000, co-incubate at 37 °C for 30 min), Golgi apparatus (Golgi-Tracker Red probe, dilution ratio 1:100, co-incubate at 4 °C for 30 min), lysosome (Lyso-Tracker Red probe, dilution ratio 1:15000, co-incubate at 37 °C for 50 min), and nucleus (Hoechst 33342 dye, dilution ratio 1:100, co-incubate at room temperature for 30 min). Repeat the rinsing step three times, observe and collect the co-localization images of the nanodrug and subcellular structures using an inverted fluorescence microscope, and scan and calculate the correlation coefficient with Image J.

[0108] The results are as follows Figure 6 As shown, FITC-fTANS showed a high correlation with mitochondria, with a Pearson correlation coefficient of 0.6701, indicating that fTANS has a high affinity for mitochondria.

[0109] Example 11

[0110] Taking the albumin-functionalized tantalum sulfide nanodots fTANS synthesized in Example 1 as an example, its AKI treatment effect was evaluated, including the detection of kidney function indicators and the observation of kidney tissue morphology. The specific steps are as follows:

[0111] The steps of animal feeding and AKI model establishment were the same as those in Example 8.

[0112] Animal grouping, administration, and detection of kidney function indicators: Control + PBS group: Normal mice were intravenously injected with an equal intervention dose of sterile PBS through the tail vein; AKI + PBS group: AKI mice were intravenously injected with an equal intervention dose of sterile PBS through the tail vein; AKI + fTANS group: AKI mice were intravenously injected with 2 mg / kg fTANS through the tail vein. Each group contained 6 mice, which were randomly captured, labeled, and then grouped. 24 hours after model establishment, the mice in each group were anesthetized and blood was collected from the eyeballs. During this period, the mouse heart could be gently pressed to promote blood pumping. After the collected whole blood of the mice was allowed to stand for 3 hours, it was centrifuged at 5000 rpm for 7 minutes to retain the upper clear supernatant. The pure serum obtained by repeating the centrifugation 3 times was used as the test sample. Creatinine (CRE) and blood urea nitrogen (BUN) detection kits were used to evaluate the kidney function of the mice.

[0113] Observation of kidney tissue morphology (H&E staining): After euthanizing the mice, the bilateral kidneys of the mice were removed, rinsed with PBS, wiped clean with filter paper, immersed in 4% paraformaldehyde for fixation, and then dehydrated with ultrapure water - gradient concentration ethanol, cleared with xylene, infiltrated with wax, and embedded in sequence to obtain paraffin tissue blocks. Paraffin tissue sections were made using a microtome. The kidney tissue sections of each group of mice were dewaxed and rehydrated, washed 3 times with PBS, and then hematoxylin stain was added dropwise to stain the tissue for 10 minutes. After washing the sections with double-distilled water, they were differentiated with 1% hydrochloric acid ethanol and washed again. Subsequently, a weakly alkaline blue-promoting solution was used to make the cell nuclei blue again. After washing again, eosin stain was added dropwise to immerse the tissue for 3 minutes, and the excess dye was washed away with double-distilled water. The tissue sections were dehydrated by immersing them in gradient ethanol - xylene, and after the sections were air-dried, they were sealed with neutral gum. The histopathological changes of the tissue were observed and photographed under an optical biological microscope.

[0114] The results are as follows Figure 7 As shown, fTANS can effectively restore the kidney function of AKI mice (A - B) and relieve the pathological changes of the kidney tissue (C).

[0115] Example 12

[0116] Taking the albumin-functionalized tantalum sulfide nanodots fTANS synthesized in Example 1 as an example, evaluate its inhibitory effect on oxidative stress during AKI, including the detection of ROS levels. The specific steps are as follows:

[0117] The steps of animal feeding and establishing the AKI model are the same as those in Example 8.

[0118] The steps of animal grouping and drug administration are the same as those in Example 11.

[0119] ROS staining of kidney tissue: Use the dihydroethidium (DHE) fluorescent probe. First, perform pre-treatment of frozen sections on tissue samples: After freezing the tissue in liquid nitrogen for 5 min, embed it with a cryo-embedding agent to obtain a frozen tissue block; Equilibrate the tissue block in a thermostat at -20 °C for 1 h, and cut 10-μm-thick frozen sections with a cryostat. DHE staining: Let the frozen sections stand for 20 min to return to room temperature, stain the nuclei with DAPI for 3 min; Wash with TBST (Tris-buffered saline solution containing 0.5% Tween-20) for 5 min, and repeat 3 times; Drop 100 μL of DHE working solution (DHE stock solution:TBST = 1:400 dilution) on each group, incubate in the dark at room temperature for 1 h; Wash 5 times, mount with an anti-fluorescence quenching mounting medium, and observe the ROS level in the tissue and capture images under an upright fluorescence microscope.

[0120] The results are as Figure 8 shown, fTANS can effectively reduce the ROS level in the kidney tissue of AKI mice and reverse oxidative stress.

[0121] Example 13

[0122] Taking the albumin-functionalized tantalum sulfide nanodots fTANS synthesized in Example 1 as an example, evaluate its inhibitory effect on apoptosis during AKI, including TUNEL staining. The specific steps are as follows:

[0123] The steps of animal feeding and establishing the AKI model are the same as those in Example 8.

[0124] The steps of animal grouping and drug administration are the same as those in Example 11.

[0125] The steps of paraffin embedding and sectioning of kidney tissue are the same as those in Example 11.

[0126] TUNEL staining: Drop 20 μg / mL proteinase K without DNase, incubate at 37 °C for 20 min. Wash with HBSS 3 times, 5 min each time. Drop 50 μL of TUNEL detection solution (prepared according to the volume ratio of terminal deoxynucleotidyl transferase:fluorescent labeling solution = 1:9), incubate at 37 °C in the dark for 60 min. Repeat washing 3 times, mount with an anti-fluorescence quenching mounting medium, and observe the positive rate of TUNEL in cells under an upright fluorescence microscope.

[0127] The results are asFigure 9 As shown, fTANS can effectively reduce the apoptosis rate of kidney tissues in AKI mice and inhibit apoptosis.

[0128] Example 14

[0129] Taking the albumin-functionalized tantalum sulfide nanodots fTANS synthesized in Example 1 as an example, its inhibitory effect on inflammation activation and outbreak during AKI was evaluated, including cGAS and F4 / 80 immunohistochemistry. The specific steps are as follows:

[0130] The steps of animal feeding and establishing the AKI model were the same as those in Example 8.

[0131] The steps of animal grouping and drug administration were the same as those in Example 11.

[0132] The steps of paraffin embedding and sectioning of kidney tissues were the same as those in Example 11.

[0133] Immunohistochemistry: The kidney tissue sections of each group of mice were successively dewaxed and rehydrated, antigen repaired, endogenous enzyme blocked, blocked, incubated with cGAS or F4 / 80 primary antibody, and incubated with HRP-labeled secondary antibody. Subsequently, DAB (HRP enzyme reaction substrate) chromogenic solution was incubated for 3 min, and the reaction was terminated by soaking in tap water. The nucleus was stained with hematoxylin for 30 s, differentiated with 1% hydrochloric acid ethanol for 1 s, and blued by soaking in tap water for 3 min. Finally, dehydration, xylene transparency, and neutral resin sealing were performed, and images were observed and taken under a biological microscope.

[0134] The results are as Figure 10 shown, fTANS effectively inhibits the activation of the cGAS pathway (A) and inflammatory cell infiltration (B) in the kidney tissues of AKI mice.

[0135] Example 15

[0136] Taking the albumin-functionalized tantalum sulfide nanodots fTANS synthesized in Example 1 as an example, its short-term and long-term biosafety in vivo was explored. The specific steps are as follows:

[0137] The animal feeding steps were the same as those in Example 8.

[0138] Biosafety assessment: After short-term (single high-dose administration of fTANS, 32 mg / kg) and long-term (multiple therapeutic-dose administrations of fTANS, 2 mg / kg, once a week for one month) administrations, the main organs (heart, liver, spleen, lung, and kidney) of mice were collected for H&E staining to observe tissue morphological changes.

[0139] The H&E staining steps were the same as those in Example 11.

[0140] The results are as Figure 11As shown, fTANS has good biosafety, and no obvious morphological lesions were observed in the major organs (heart, liver, spleen, lungs, and kidneys) of mice after a single high-dose administration in the short term (A) or multiple therapeutic-dose administrations in the long term (B).

[0141] In the experimental methods of Examples 7-15 above, experimental reagents not clearly given, such as working solutions, fixing solutions, primary antibodies, secondary antibodies, etc., are all commonly used reagents in the art.

[0142] Obviously, the above examples are merely illustrative for clear explanation and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. Application of albumin-functionalized tantalum sulfide nanodots in the preparation of drugs for treating acute kidney injury.

2. The application according to claim 1, characterized in that, The albumin-functionalized tantalum sulfide nanodots are spherical in shape and have a particle size between 4 - 6 nm.

3. The application according to claim 1, wherein In the albumin-functionalized tantalum sulfide nanodots, the tantalum sulfide nanodots are the core and albumin is the outer coating carrier.

4. The application according to any one of claims 1-3, characterized in that The albumin-functionalized tantalum sulfide nanodots are obtained by stirring and self-assembling tantalum sulfide nanodots and an albumin aqueous solution.

5. The application according to claim 4, wherein The stirring and self-assembling time is 2 - 3 h, the gas condition is argon, and the temperature condition is room temperature.

6. The application according to claim 4, wherein The tantalum sulfide nanodots are prepared by top-down grinding of tantalum sulfide powder and liquid-phase ultrasonic exfoliation. The albumin aqueous solution is a 10 mg / mL human serum albumin or bovine serum albumin.

7. The application according to claim 6, wherein The grinding time of the tantalum sulfide powder is 2 - 3 h, the liquid-phase ultrasonic exfoliation time is 1 - 2 w, and the liquid-phase solvent is N-methylpyrrolidone.

8. The application according to claim 6, characterized in that, The volume ratio of the tantalum sulfide nanodots to the albumin aqueous solution is 1:10.

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