A rapamycin-loaded targeted hollow mesoporous prussian blue nanoparticle, a preparation method thereof and application thereof in treatment of ischemia-reperfusion-induced acute kidney injury

Abstract

The present application provides a kind of rapamycin-loaded targeted hollow mesoporous prussian blue nanoparticles and its preparation method and application in the treatment of ischemia-reperfusion-induced acute kidney injury.The nanoparticles, including hollow mesoporous prussian blue nanoparticles, rapamycin is loaded in the hollow mesoporous prussian blue nanoparticles, and the surface is modified with amino hyaluronic acid.Amino hyaluronic acid modification endows the nanoparticles with kidney targeting property;Hollow mesoporous prussian blue nanoparticles and rapamycin play a dual role in promoting mitochondrial autophagy and reducing oxidative stress.Therefore, the nanoparticles of the present application can effectively restore renal function, reduce histopathological changes, inhibit oxidative stress and inflammatory response, and ultimately protect kidney cells from apoptotic damage, which has great application potential in clinic.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nanoparticle preparation and medicine, and particularly relates to a rapamycin-loaded targeted hollow mesoporous Prussian blue nanoparticle, a preparation method thereof and application of the nanoparticle in treatment of ischemia / reperfusion-induced acute kidney injury. BACKGROUND

[0002] Acute kidney injury (AKI) is a sudden onset of renal dysfunction or failure, a clinical syndrome of rapid decline in renal function within a short period of time caused by various causes. The diagnostic criteria include a three-fold increase in serum creatinine or a urine output of less than 0.3 mL / kg / h within 24 hours. AKI can lead to serious complications such as electrolyte imbalance, fluid overload and accumulation of metabolic waste, and symptoms may include a rapid decline in glomerular filtration rate within a short period of time, rapid increase in blood creatinine and urea nitrogen, decreased urine output, fatigue, edema, mental confusion, nausea, etc. In severe cases, AKI can develop into end-stage renal disease, requiring treatment such as dialysis or kidney transplantation. At the same time, AKI patients have a higher risk of developing chronic kidney disease and cardiovascular disease. In addition, AKI places a huge economic burden on individuals and society due to increased costs of hospitalization, dialysis and long-term care. A series of supportive strategies are commonly used in clinical practice to treat AKI, including minimizing or avoiding the use of nephrotoxic drugs, providing nutritional support, increasing circulating blood volume and renal replacement therapy, etc. There is currently no approved drug therapy specifically targeting AKI.

[0003] Ischemia / reperfusion (I / R), sepsis, nephrotoxic drugs and other pathogenic factors can induce AKI, among which I / R is the main clinical cause of AKI. AKI caused by I / R is a complex pathophysiological condition characterized by temporary interruption of blood flow (ischemia) followed by restoration of blood supply to the kidney (reperfusion). This phenomenon can occur in various clinical settings such as kidney transplantation, trauma, cardiac surgery and shock. Oxidative stress begins with ischemia, which is the reduction of blood flow in the kidney leading to insufficient supply of oxygen and nutrients related to cell metabolism. This ischemia promotes cells, especially sensitive renal tubular epithelial cells, to enter an anaerobic metabolism state, resulting in accumulation of metabolic byproducts and insufficient adenosine triphosphate (ATP) production. Subsequent reperfusion aims to restore blood flow, but paradoxically triggers further harmful cascades. The sudden influx of oxygen becomes a substrate for xanthine oxidase and nicotinamide adenine dinucleotide phosphate oxidase, leading to the production of excess reactive oxygen species (ROS) such as superoxide ions (·O2 -), hydrogen peroxide (H2O2), and hydroxyl radicals (·OH). Under physiological conditions, ROS are neutralized by antioxidant defense systems, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). However, during reperfusion, the antioxidant system is overwhelmed, leading to the occurrence of oxidative stress. The interaction between oxidative stress and I / R-induced AKI is bidirectional and multifaceted. On one hand, oxidative stress promotes inflammation, apoptosis, and endothelial dysfunction, thereby exacerbating kidney injury. On the other hand, kidney injury itself can further enhance oxidative stress through the release of damage-associated molecular patterns, activation of intracellular signaling pathways, and induction of mitochondrial dysfunction, thereby forming a vicious cycle. Understanding this relationship is helpful for developing therapeutic strategies aimed at improving the kidney's antioxidant capacity or reducing ROS generation, thereby protecting the kidney during and after I / R. Various catalysts, such as Au, CeO2, RuO, and CuO, can scavenge ROS and have shown efficacy in treating AKI. However, the mitigating effect of catalysis is short-lived because the catalysts are metabolized over time, which can lead to a rapid recovery of ROS to their original levels. Therefore, while these catalysts can temporarily reduce ROS levels, they may not fundamentally address the problem of persistent overproduction of ROS. Thus, simultaneously eliminating excess ROS and the source of ROS can play a dual role in the treatment of AKI.

[0004] Mitochondria are commonly referred to as the "powerhouse" of the cell because they play a crucial role in generating ATP through oxidative phosphorylation. In addition to energy production, mitochondria are involved in various cellular processes, including calcium ion balance, ROS generation, and apoptosis. As a high-energy-consuming organ of the human body, the kidney has a relatively high content of mitochondria and oxygen consumption (approximately 7% of the total oxygen consumption of the human body). In I / R-induced AKI, excessive ROS leads to an imbalance between ROS generation and antioxidant defense mechanisms in mitochondria, inducing mitochondrial damage. More harmful is that the disruption of the electron transport chain in damaged mitochondria not only hinders the normal operation of the oxidative phosphorylation process but also promotes the continuous generation of highly destructive ·O2 - , further exacerbating oxidative stress and ultimately leading to apoptosis. As AKI progresses, the vicious cycle of inflammation, oxidative stress, mitochondrial damage, and apoptosis further exacerbates kidney injury.

[0005] Currently, the treatment of AKI mainly relies on supportive therapy, which can enhance the blood perfusion of the damaged kidney through volume expansion, or serve as a functional replacement therapy for the kidney through hemodialysis and filtration. However, the clinical efficacy of the supportive therapy can only delay the pathological progression of AKI, and cannot reverse the already occurred kidney tissue damage and promote the repair of the kidney tissue. Therefore, it is of great significance and clinical value to develop a strategy that can quickly reverse the kidney tissue damage, effectively restore the kidney function, and significantly improve the survival rate of patients. For I / R-induced AKI, which is a complex acute and critical illness, although there is currently a lack of safe and effective therapeutic drugs, some intervention drugs are often used in clinic to relieve oxidative damage and inflammatory response. However, these drugs are difficult to quickly correct the oxidative stress and mitochondrial damage closely related to cell apoptosis from the source, that is, it is difficult to achieve comprehensive regulation of multiple pathogenesis, and thus it is difficult to effectively reverse the kidney cell damage, repair the structure of the damaged kidney tissue, and finally achieve the complete recovery of the kidney function. In addition, the drugs lack specific targeting to the damaged kidney, and thus it is difficult to accumulate in the lesion site. The toxicity of the drugs and the accumulation in non-target organs also limit the drug dosage, and thus seriously affect the treatment results. At present, with the deepening understanding of the pathophysiology of ischemia-reperfusion AKI, a variety of intelligent-microenvironment responsive nanodrug delivery systems have been developed, such as polymer nanoparticles, DNA nanorobots, inorganic nanoparticles (gold, cerium dioxide, ruthenium oxide, cuprous oxide with ROS scavenging function) and the like. Although the currently developed nanodrug delivery system can improve the efficacy of the drugs to some extent, it still cannot achieve the repair and regeneration of the kidney tissue and the complete recovery of the kidney function, which may be related to the fact that it still targets the single pathogenesis of I / R-induced AKI for anti-inflammatory and antioxidant treatment, and cannot quickly improve the oxidative stress and mitochondrial damage closely related to cell damage. Therefore, for I / R-induced AKI, which is a rapidly occurring and complex pathogenesis, it is urgent to provide a therapeutic drug and method that can quickly reverse the kidney tissue damage, restore the kidney function, and significantly improve the survival rate of patients. SUMMARY

[0006] The present application aims to overcome the above-mentioned defects and deficiencies in the prior art, and provides a rapamycin-loaded targeted hollow mesoporous Prussian blue nanoparticle.

[0007] A second object of the present application is to provide a preparation method of the above-mentioned rapamycin-loaded targeted hollow mesoporous Prussian blue nanoparticle.

[0008] A third object of the present application is to provide an application of the above-mentioned rapamycin-loaded targeted hollow mesoporous Prussian blue nanoparticle in the preparation of a drug for treating acute kidney injury caused by ischemia-reperfusion.

[0009] A fourth object of the present application is to provide a drug for treating acute kidney injury caused by ischemia-reperfusion, comprising the rapamycin-loaded targeted hollow mesoporous Prussian blue nanoparticle described above.

[0010] The above objects of the present application are achieved by the following technical solutions:

[0011] The present application provides a rapamycin-loaded targeted hollow mesoporous Prussian blue nanoparticle, which comprises a hollow mesoporous Prussian blue nanoparticle, wherein rapamycin is loaded in the hollow mesoporous Prussian blue nanoparticle, and the surface of the hollow mesoporous Prussian blue nanoparticle is modified with amino hyaluronic acid.

[0012] The kidney is an organ with a high content of mitochondria and a high demand for oxygen in the body, so it is prone to a series of injury events under ischemia-reperfusion conditions, including oxidative stress, mitochondrial damage, adenosine triphosphate depletion, and inflammatory response, which ultimately collectively cause acute kidney injury (AKI). Currently, in addition to supportive treatment, there is no approved therapy for AKI in clinical practice. Although many studies focus on removing excess reactive oxygen species (ROS) at the lesion site to reduce AKI, little attention is paid to inhibiting the generation of ROS from the source. Mitophagy is a selective autophagy pathway that degrades dysfunctional or excessive mitochondria through lysosomal phagocytosis. This mechanism maintains cellular homeostasis by ensuring the removal of defective mitochondria, thereby reducing oxidative stress and preventing the accumulation of damaged organelles. Defects in mitochondrial autophagy function are associated with a range of conditions, including AKI, which provides a new target for the treatment of AKI.

[0013] Rapamycin (Rapa) is a potent mammalian target of rapamycin inhibitor and also an effective immunomodulator, originally used to treat transplant rejection and certain autoimmune diseases. In addition, some studies have found other effects of Rapa, including neuroprotection, relief of osteoarthritis, maintenance of muscle vitality, etc. As mentioned above, oxidative stress can cause damage to a large number of mitochondria in the kidney, thereby forming a source of excess ROS, and Rapa can regulate cellular homeostasis and cell viability by activating the autophagy mechanism. According to the different organelles that are selectively degraded, autophagy can be divided into mitochondrial autophagy, endoplasmic reticulum autophagy, and ribosome autophagy, etc. Therefore, it is speculated that Rapa can selectively remove damaged mitochondria in AKI, fundamentally inhibit the excessive production of ROS, thereby reducing the damage caused by oxidative stress, and ultimately play a role in the treatment of AKI. However, the physicochemical properties of Rapa (poor water solubility, short half-life in vivo, lack of specific targeting to the lesion site, etc.) limit its application in vivo.

[0014] Prussian blue (PB) is an antidote approved by the U.S. Food and Drug Administration for the radioactive elements thallium and cesium, with good biological safety. Hollow mesoporous prussian blue (HMPB) nanoparticles have unique physicochemical properties, including hollow mesoporous structure, high specific surface area, easy surface modification, and responsiveness to external stimuli, especially in an acidic microenvironment, thus attracting attention as a potential drug delivery platform. In addition, the multiple enzyme-like properties (peroxidase POD, catalase CAT, and superoxide dismutase SOD) of HMPB endow it with the ability to scavenge ROS, which has great potential in the treatment of oxidative stress-related diseases, including neurodegenerative diseases, inflammatory bowel disease, acute pancreatitis, etc. Using HMPB as a carrier not only can effectively load Rapa, but also can scavenge excess ROS produced by ischemia / reperfusion (I / R)-induced AKI. However, the unique anatomical structure of the kidney hinders the effective aggregation of nanoparticles at the injury site.

[0015] Hyaluronic acid (HA) is a naturally occurring polysaccharide commonly used to modify nanoparticles, achieving active targeted drug delivery through specific interaction between HA and CD44 receptors. In the pathological state of AKI, CD44 receptors are highly expressed on damaged renal tubular epithelial cells. After HA binds to CD44, nanoparticles can be taken up by damaged cells through endocytosis. Therefore, using HA modification can achieve more accurate and effective treatment of AKI.

[0016] The rapamycin-loaded targeted hollow mesoporous prussian blue nanoparticles provided by the present application can effectively scavenge the original ROS and the source of ROS (a large number of damaged mitochondria) through the dual action of the carrier hollow mesoporous prussian blue and the drug rapamycin, thereby preventing further outbreaks of ROS. Aminated hyaluronic acid is modified on the surface of the rapamycin-loaded hollow mesoporous prussian blue nanoparticles through chelation of amino groups and iron ions. The rapamycin-loaded and hyaluronic acid-modified hollow mesoporous prussian blue nanoparticles can clear a large number of damaged mitochondria by activating the mitochondrial autophagy signaling pathway (PINK-1 / Parkin-P62-LC3), cut off the conduction of the endogenous apoptosis pathway mediated by the Bax-Cyt-c-Cleaved Casp-3 signal axis, and reduce the expression of the renal tubular marker kidney injury molecule-1 (KIM-1). Therefore, the rapamycin-loaded targeted hollow mesoporous prussian blue nanoparticles are expected to become a drug and therapy for treating ischemia / reperfusion-related diseases.

[0017] Further, the water dynamic diameter of the rapamycin-loaded targeted hollow mesoporous Prussian blue nanoparticle is 50-200 nm, and the surface charge is -10 to -40 mV.

[0018] Preferably, the water dynamic diameter of the rapamycin-loaded targeted hollow mesoporous Prussian blue nanoparticle is 119.5 nm, and the surface charge is -30.3 mV.

[0019] Further, the drug loading amount of the rapamycin-loaded hollow mesoporous Prussian blue nanoparticle is 5-50%.

[0020] Preferably, the drug loading amount of the rapamycin-loaded hollow mesoporous Prussian blue nanoparticle is 40%.

[0021] The present application also provides a preparation method of the above-mentioned rapamycin-loaded targeted hollow mesoporous Prussian blue nanoparticle, which comprises adding an amino hyaluronic acid solution to a dispersion solution of the rapamycin-loaded hollow mesoporous Prussian blue nanoparticle, and stirring to obtain the rapamycin-loaded targeted hollow mesoporous Prussian blue nanoparticle.

[0022] Further, the preparation method of the hollow mesoporous Prussian blue nanoparticle comprises reacting bismuth nitrate, potassium ferricyanide and polyvinylpyrrolidone in an acidic solution.

[0023] Further, the preparation method of the rapamycin-loaded hollow mesoporous Prussian blue nanoparticle comprises mixing the hollow mesoporous Prussian blue nanoparticle and rapamycin in ethanol at different mass ratios, and stirring to obtain the rapamycin-loaded hollow mesoporous Prussian blue nanoparticle.

[0024] Further, the mass ratio of the hollow mesoporous Prussian blue nanoparticle to rapamycin is 1:1-10.

[0025] Further, the preparation method of the amino hyaluronic acid comprises reacting hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in a mixed solution, and then adding ethylenediamine to obtain the amino hyaluronic acid.

[0026] Further, the mixed solution is DMSO / water with a volume ratio of 3:1.

[0027] In the present application, HMPB is synthesized by hydrothermal method, then Rapa-loaded HMPB nanoparticles (HMPB-Rapa) are prepared by pore adsorption, and finally the amino-modified HA is modified on the surface of HMPB-Rapa by chelation of amino group and iron ions to obtain the final preparation (HA-HMPB-Rapa). In the I / R-induced AKI rat model, after single administration through tail vein at 6 hours after ischemia-reperfusion, HA-HMPB-Rapa can effectively restore renal function, reduce histopathological changes, inhibit oxidative stress and inflammatory response, and ultimately protect renal cells from apoptotic damage.

[0028] Therefore, the present application also provides the use of the above-mentioned any rapamycin-loaded targeted hollow mesoporous Prussian blue nanoparticles in the preparation of a medicament for treating acute kidney injury caused by ischemia-reperfusion.

[0029] The present application also provides a medicament for treating acute kidney injury caused by ischemia-reperfusion, which comprises rapamycin-loaded targeted hollow mesoporous Prussian blue nanoparticles.

[0030] Compared with the prior art, the present application has the following beneficial effects:

[0031] The present application provides a rapamycin-loaded targeted hollow mesoporous Prussian blue nanoparticle, which comprises a hollow mesoporous Prussian blue nanoparticle, and the hollow mesoporous Prussian blue nanoparticle is loaded with rapamycin and modified with amino-modified hyaluronic acid on the surface. The amino-modified hyaluronic acid is used to modify the hollow mesoporous Prussian blue nanoparticle, which endows the nanoparticle with kidney targeting property, so that HA-HMPB-Rapa can actively target the CD44 receptor overexpressed on the surface of damaged renal tubular epithelial cells; the unique hollow mesoporous structure of HMPB can effectively load Rapa, and the drug loading capacity of HA-HMPB-Rapa is as high as 40%; HMPB can be degraded in the acidic pathological microenvironment of AKI, ensuring the rapid release of the drug, and can also effectively eliminate the existing excess ROS; Rapa can eliminate a large number of damaged mitochondria in the damaged kidney, i.e. the main source of excess ROS, by promoting mitochondrial autophagy, thereby preventing further outbreak of ROS. Therefore, HA-HMPB-Rapa has a double effect in reducing oxidative stress.

[0032] Compared with the prior art, it is difficult to quickly correct the phenomena of oxidative stress and mitochondrial damage closely related to apoptosis from the source, that is, it is difficult to achieve comprehensive regulation of multiple pathogenesis, and thus it is difficult to effectively reverse kidney cell damage, repair the structure of damaged kidney tissue, and ultimately achieve complete recovery of kidney function. The nanoparticles of the present application have satisfactory therapeutic effects in I / R-induced AKI rat models, mainly manifested by improved survival rate of rats, recovered kidney function, reduced kidney tissue lesions, and inhibited oxidative stress, apoptosis, and inflammatory response. In addition, a single low-dose tail vein administration of HA-HMPB-Rapa (Rapa: 1 mg / kg) at 6 h after I / R can effectively alleviate AKI and restore kidney function, reduce histopathological changes, inhibit oxidative stress and inflammatory response, and ultimately protect kidney cells from apoptotic damage. This experimental design is consistent with the clinical situation and reflects the window period between the onset of pathology and treatment. Low dose also indicates a reduction in cost and kidney toxicity in future clinical applications. In summary, HA-HMPB-Rapa has the advantages of simple preparation process, good safety, high drug loading, good targeting, rapid drug release, long storage time, good hemolytic property, acid-responsive drug release behavior, and multiple enzyme activities. From the "multi-target, multi-pathway, multi-link" regulation of various pathological mechanisms such as oxidative stress, mitochondrial damage, and inflammatory response, it can promote cell viability, mitochondrial membrane potential (MMP), and ATP levels to quickly reverse kidney tissue damage, restore kidney function, significantly improve patient survival rate, break the vicious cycle of AKI pathology, effectively alleviate I / R-induced AKI, and has the potential for further clinical transformation. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The flow chart for the preparation of nanoparticles and its mechanism of action.

[0034] Figure 2 Characterization of HMPB, HMPB-Rapa, and HA-HMPB-Rapa. Among them, Figure 2 A is the particle size distribution graph; B is the potential graph; C is the potential distribution graph of HA-HMPB-Rapa; D is the transmission electron microscope picture of HMPB-Rapa; E is the transmission electron microscope picture of HA-HMPB-Rapa.

[0035] Figure 3 UV absorption spectra of Rapa, HMPB, HMPB-Rapa, and HA-HMPB-Rapa.

[0036] Figure 4The effect of the mass ratio of Rapa to HMPB on drug loading and the pH-responsive drug release curve of HA-HMPB-Rapa.

[0037] Figure 5 The storage stability of the nanoparticles at 4℃.

[0038] Figure 6 The relative hemolysis rate of HA-HMPB-Rapa at different concentrations.

[0039] Figure 7 The detection of the ability of HA-HMPB-Rapa to scavenge reactive oxygen species. Among them, Figure 7 A is the scavenging rate of DPPH by HA-HMPB-Rapa at different concentrations; B is the scavenging rate of ·OH by HA-HMPB-Rapa at different concentrations; C is the scavenging rate of ·O2- by HA-HMPB-Rapa at different concentrations; D is the picture of oxygen production catalyzed by HA-HMPB-Rapa; E is the amount of oxygen produced by HA-HMPB-Rapa at different concentrations within 10 min; F is the POD-like activity of HA-HMPB-Rapa at 100 μg / mL.

[0040] Figure 8 The cell survival rate of HK-2 cells incubated with different doses of HA-HMPB and HA-HMPB-Rapa for 12 or 24 h.

[0041] Figure 9 The fluorescence images and semi-quantitative results of the cellular uptake of HMPB-RhB and HA-HMPB-RhB by H2O2-stimulated HK-2 cells after different incubation times.

[0042] Figure 10 The identification of the protective effect of HA-HMPB-Rapa on H2O2-stimulated HK-2 cells. Among them, Figure 10 A is the cell survival rate of H2O2-stimulated HK-2 cells 24 h after treatment; B and C are the fluorescence images of AM / PI staining of HK-2 cells in different groups and the semi-quantitative results of PI-positive cells, respectively.

[0043] Figure 11 The intracellular ATP content of H2O2-stimulated HK-2 cells 24 h after treatment.

[0044] Figure 12 The evaluation of the antioxidant capacity of the cells. Among them, Figure 12 A and B are the fluorescence images and semi-quantitative results of intracellular ROS of H2O2-stimulated HK-2 cells 24 h after treatment, respectively; C is the flow cytometry result.

[0045] Figure 13 Fluorescent images of the changes of mitochondrial membrane potential in H2O2-stimulated HK-2 cells at 24 h after treatment.

[0046] Figure 14 Pictures of the color changes of the kidneys during ischemia and reperfusion.

[0047] Figure 15 Pictures of the color changes of the kidneys during ischemia and reperfusion. Figure 15 The left panel in FIG. 7 shows the fluorescent images of the major organs (heart, liver, spleen, lung and kidney) of AKI rats at different time points (1 or 6 h) after intravenous injection of HMPB-RhB and HA-HMPB-RhB. The right panel in FIG. 7 shows the semi-quantitative results of the fluorescent intensity of the kidneys of AKI rats at different time points (1 or 6 h) after intravenous injection of HMPB-RhB and HA-HMPB-RhB.

[0048] Figure 16 The left panel in FIG. 7 shows the fluorescent images of the major organs (heart, liver, spleen, lung and kidney) of AKI rats at different time points (1 or 6 h) after intravenous injection of HMPB-RhB and HA-HMPB-RhB. The right panel in FIG. 7 shows the semi-quantitative results of the fluorescent intensity of the kidneys of AKI rats at different time points (1 or 6 h) after intravenous injection of HMPB-RhB and HA-HMPB-RhB.

[0049] Figure 17 The left panel in FIG. 7 shows the fluorescent images of the major organs (heart, liver, spleen, lung and kidney) of AKI rats at different time points (1 or 6 h) after intravenous injection of HMPB-RhB and HA-HMPB-RhB. The right panel in FIG. 7 shows the semi-quantitative results of the fluorescent intensity of the kidneys of AKI rats at different time points (1 or 6 h) after intravenous injection of HMPB-RhB and HA-HMPB-RhB.

[0050] Figure 18 The left panel in FIG. 7 shows the fluorescent images of the major organs (heart, liver, spleen, lung and kidney) of AKI rats at different time points (1 or 6 h) after intravenous injection of HMPB-RhB and HA-HMPB-RhB. The right panel in FIG. 7 shows the semi-quantitative results of the fluorescent intensity of the kidneys of AKI rats at different time points (1 or 6 h) after intravenous injection of HMPB-RhB and HA-HMPB-RhB.

[0051] Figure 19 The left panel in FIG. 7 shows the fluorescent images of the major organs (heart, liver, spleen, lung and kidney) of AKI rats at different time points (1 or 6 h) after intravenous injection of HMPB-RhB and HA-HMPB-RhB. The right panel in FIG. 7 shows the semi-quantitative results of the fluorescent intensity of the kidneys of AKI rats at different time points (1 or 6 h) after intravenous injection of HMPB-RhB and HA-HMPB-RhB.

[0052] Figure 20 The left panel in FIG. 7 shows the fluorescent images of the major organs (heart, liver, spleen, lung and kidney) of AKI rats at different time points (1 or 6 h) after intravenous injection of HMPB-RhB and HA-HMPB-RhB. The right panel in FIG. 7 shows the semi-quantitative results of the fluorescent intensity of the kidneys of AKI rats at different time points (1 or 6 h) after intravenous injection of HMPB-RhB and HA-HMPB-RhB.

[0053] Figure 21 The left panel in FIG. 7 shows the fluorescent images of the major organs (heart, liver, spleen, lung and kidney) of AKI rats at different time points (1 or 6 h) after intravenous injection of HMPB-RhB and HA-HMPB-RhB. The right panel in FIG. 7 shows the semi-quantitative results of the fluorescent intensity of the kidneys of AKI rats at different time points (1 or 6 h) after intravenous injection of HMPB-RhB and HA-HMPB-RhB.

[0054] Figure 22 The left panel in FIG. 7 shows the fluorescent images of the major organs (heart, liver, spleen, lung and kidney) of AKI rats at different time points (1 or 6 h) after intravenous injection of HMPB-RhB and HA-HMPB-RhB. The right panel in FIG. 7 shows the semi-quantitative results of the fluorescent intensity of the kidneys of AKI rats at different time points (1 or 6 h) after intravenous injection of HMPB-RhB and HA-HMPB-RhB.

[0055] Figure 23 The left panel in FIG. 7 shows the fluorescent images of the major organs (heart, liver, spleen, lung and kidney) of AKI rats at different time points (1 or 6 h) after intravenous injection of HMPB-RhB and HA-HMPB-RhB. The right panel in FIG. 7 shows the semi-quantitative results of the fluorescent intensity of the kidneys of AKI rats at different time points (1 or 6 h) after intravenous injection of HMPB-RhB and HA-HMPB-RhB.

[0056] Figure 24 The left panel in FIG. 7 shows the fluorescent images of the major organs (heart, liver, spleen, lung and kidney) of AKI rats at different time points (1 or 6 h) after intravenous injection of HMPB-RhB and HA-HMPB-RhB. The right panel in FIG. 7 shows the semi-quantitative results of the fluorescent intensity of the kidneys of AKI rats at different time points (1 or 6 h) after intravenous injection of HMPB-RhB and HA-HMPB-RhB.

[0057] Figure 25The levels of (A) TNF-a, (B) IL-6 and (C) IL-1 β in the serum of rats in different treatment groups.

[0058] Figure 26 The expression levels and semi-quantitative results of (A) PINK-1, (B) Parkin, (C) LC3 I / II, (D) P62, (E) Bcl-2, (F) Bax, (G) Cyt-c, (H) Cleaved-Casp-3 and (I) KIM-1; (J) schematic diagram of the treatment mechanism of nanoparticles.

[0059] Figure 27 The (A) hematology test index heat map and (B) H&E staining images of the main organs of rats in different treatment groups.

[0060] Figure 28 The body weight changes of rats within 7 days. DETAILED DESCRIPTION

[0061] The present application will be further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0062] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0063] Example 1 Preparation and characterization of HA-HMPB-Rapa

[0064] 1. Preparation method

[0065] (1) Preparation of aminated hyaluronic acid

[0066] 250 mg of hyaluronic acid (HA) (21 kDa) was reacted with 759 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 910 mg of N-hydroxysuccinimide in a mixed solution of 6 mL (DMSO / water = 3:1, v / v) for 0.5 h, then 1.3 g of ethylenediamine was mixed with the activated hyaluronic acid solution and stirred at room temperature overnight. Finally, dialysis and freeze-drying were performed to obtain aminated hyaluronic acid.

[0067] (2) Preparation of hollow mesoporous Prussian blue (HMPB) nanoparticles

[0068] Bismuth nitrate (243 mg), potassium ferricyanide (396 mg) and polyvinylpyrrolidone (5 g) were dissolved in 40 mL of hydrochloric acid (1 M) solution. After reacting at 80°C for 24 h, the unreacted raw materials were removed by high-speed centrifugation and washing the solution with water and ethanol several times. Finally, HMPB was obtained by freeze-drying.

[0069] (3) Preparation of HMPB-Rapa

[0070] Rapa and HMPB were mixed in ethanol with different mass ratios. After stirring overnight, HMPB-Rapa was obtained. Free Rapa was removed by centrifugation and ethanol washing.

[0071] (4) Preparation of HA-HMPB-Rapa

[0072] 1 mL of aminated hyaluronic acid solution (2 mg / mL) was added dropwise to the HMPB-Rapa dispersion (1 mL, 1 mg / mL), and then stirred overnight. The reaction was centrifuged and washed with water three times to remove excess HA.

[0073] 2. Characterization methods

[0074] (1) Free Rapa was separated by centrifugation and measured by high performance liquid chromatography at the characteristic absorption peak (268 nm) of Rapa. The encapsulation efficiency and drug loading of Rapa in HA-HMPB-Rapa were calculated according to the following formulas ① and ②.

[0075] ① Encapsulation efficiency (%) = (1 - mass of free Rapa / mass of total Rapa) x 100%.

[0076] ② Drug loading (%) = mass of loaded Rapa / mass of HA-HMPB-Rapa x 100%.

[0077] HA-HMPB, HMPB-RhB and HA-HMPB-RhB were prepared by a similar method. HMPB and Rhodamine B (RhB) were dispersed in water and stirred overnight. The hydrodynamic size and zeta potential of the nanoparticles were measured using a Malvern Zetasizer Nano instrument (ZEN3600, UK). The morphology of HMPB-Rapa and HA-HMPB-Rapa was characterized using a transmission electron microscope (Hitachi-HT7700, Japan). The UV absorption spectra of the nanoparticles and Rapa were measured using a UV spectrophotometer (UV-2600i, Japan).

[0078] (2) pH-responsive drug release behavior of HA-HMPB-Rapa. The HA-HMPB-Rapa solution was placed in a dialysis bag and dialyzed in phosphate-buffered saline (PBS) at different pH values (7.4, 6.5, 5.5) at 37°C. Samples were collected at designated time points (0.25 h, 0.5 h, 1 h, 2 h, 6 h, 12 h, and 24 h, respectively). Then the samples were subjected to high-performance liquid chromatography analysis at a wavelength of 268 nm, and the cumulative release amount of Rapa was calculated.

[0079] (3) Stability detection of HA-HMPB-Rapa. To determine the long-term storage stability of HA-HMPB-Rapa, the sample was stored at 4°C, and the hydrodynamic size and polydispersity index of the nanoparticles were monitored within one week using a Malvern Zetasizer Nano instrument (ZEN3600, UK).

[0080] (4) Blood compatibility of HA-HMPB-Rapa. Different concentrations of HA-HMPB-Rapa (from 25 µg / mL to 1000 µg / mL) were incubated with an equal volume of 4% red blood cell suspension at 37°C for 2 h. 1% Triton X-100 and PBS (0.01M, pH 7.4) were used as positive and negative controls, respectively. After incubation, the samples were centrifuged at 4000 rpm for 5 min, and the supernatant was collected. The released hemoglobin was detected using a microplate reader (Biotek SYNERGY H1 Instruments, Inc.) at a wavelength of 560 nm. The relative hemolysis rate was calculated according to the formula. Relative hemolysis rate (%) = (A 样品 -A PBS ) / (A TritonX-100 -A PBS ) x 100%.

[0081] 3、Results

[0082] (1) The preparation process of HA-HMPB-Rapa is shown in Figure 1 . In the present application, polyvinylpyrrolidone, potassium ferricyanide, and bismuth nitrate are mixed in an acidic solution, and HMPB is synthesized by one-pot hydrothermal method. In the process of HMPB formation, bismuth ions and ferricyanide form degradable Bi 3+ -Fe (CN) 6 3- , which promotes the formation of a hollow mesoporous structure in the nanoparticles, further preparing HMPB loaded with Rapa (HMPB-Rapa), and further modifying it with HA (named HA-HMPB-Rapa) to achieve effective treatment of I / R-induced AKI.

[0083] (2) Results of dynamic light scattering analysis are as follows Figure 2 As shown, Figure 2 A and B indicate that HMPB has an average size of 97.4 nm, a uniform size distribution (polydispersity index (PDI): 0.126), and a surface charge of -24.8 mV. In this invention, HMPB loads Rapa through pore adsorption and hydrophobic interactions between the drug and the hydrophobic cavity. After Rapa loading, the hydrodynamic diameter of HMPB-Rapa increases slightly (approximately 106.2 nm), but its surface charge does not change significantly. Through iron-amino chelation, aminolated HA is modified on the surface of HMPB-Rapa, thereby enabling the nanoparticles to actively target CD44 (a receptor highly expressed on damaged renal tubular epithelial cells), such as... Figure 2 As shown in A, B, and C, after loading Rapa and modifying it with HA, the hydrodynamic diameter of HA-HMPB-Rapa increased to approximately 119.5 nm, while the surface charge decreased to -30.3 mV. Transmission electron microscopy results are shown below. Figure 2 As shown in D and E, both HMPB-Rapa and HA-HMPB-Rapa are spherical, and a clear layer of hyaluronic acid can be observed around HA-HMPB-Rapa.

[0084] (3) The results of the ultraviolet absorption spectrum are as follows Figure 3 As shown, the broad absorption in the 600 nm to 800 nm range of HA-HMPB-Rapa is consistent with the characteristic absorption of HMPB, which is attributed to electron transfer in Fe-CN-Fe. Drug loading and HA modification do not change the UV-Vis absorption spectrum of HMPB, indicating that HA-HMPB-Rapa still retains the catalytic performance of HMPB.

[0085] (4) Results of Rapa drug loading and pH-responsive drug release are as follows Figure 4 As shown, high drug loading is crucial for the efficacy of nanomedicines. Studies on the effect of the Rapa / HMPB mass ratio on the drug loading of HA-HMPB-Rapa revealed that the drug loading increases with increasing Rapa / HMPB mass ratio. Figure 4 As shown in Figure A, a plateau was eventually reached (drug loading: 40%); simultaneously, as... Figure 4 As shown in B, the cumulative release of Rapa within 24 hours under acidic conditions was significantly higher than that under physiological conditions, and the release increased as the pH decreased (pH 7.4: 15.4%, pH 6.5: 28.0%, pH 5.5: 43.0%). This pH-responsive drug release behavior allows the drug to be rapidly released from damaged renal cells, which are pathologically weakly acidic.

[0086] (5) Storage stability results are as followsFigure 5 As shown, the hydrodynamic diameter and PDI of HA-HMPB-Rapa remained stable over a period of up to one week, indicating its suitability for long-term storage.

[0087] (6) Hemolysis test results are shown in Figure 6 As shown, the hemolysis rate of HA-HMPB-Rapa was still below the threshold of 5% at a concentration of up to 1000 μg / mL, verifying the excellent hemocompatibility of HA-HMPB-Rapa.

[0088] Example 2 Ability of HA-HMPB-Rapa to scavenge reactive oxygen species

[0089] 1. Method

[0090] (1) 1,1-Diphenyl-2-picrylhydrazyl (DPPH) scavenging experiment. DPPH is a stable free radical compound that exhibits a purple color when in free radical form, which fades upon reduction by antioxidants. This property can be used to quantify the ability of a substance to scavenge free radicals, helping to assess the potential of the substance as an antioxidant. After mixing different concentrations of HA-HMPB-Rapa with an equal volume of DPPH solution, the reaction was carried out in the dark for 0.5 h. Then the absorbance of DPPH free radicals at 517 nm was measured using a microplate reader.

[0091] (2) ·OH scavenging experiment. ·OH is one of the most reactive and destructive free radicals in biological systems. Its high reactivity and ability to initiate chain reactions make ·OH particularly harmful to the integrity and function of cell membranes, and thus associated with various pathological conditions, including neurodegenerative diseases and age-related diseases. The deoxyribose method was used to evaluate the ·OH scavenging ability of HA-HMPB-Rapa. Different concentrations of HA-HMPB-Rapa (from 25 µg / mL to 1000 µg / mL) were mixed with PBS, deoxyribose (50 mM), Na2EDTA (1 mM), FeCl3 (3.2 mM), and H2O2 (50 mM) in a volume ratio of 2:8:1:1:1:1. Then, ascorbic acid (1.8 mM, 50 μL) was used to initiate the reaction, and incubated at 50°C for 20 min. Finally, a mixture of 10% trichloroacetic acid and 5% thiobarbituric acid (5:3, v / v) was added to the reaction system, and then incubated at 100°C for another 15 min. After cooling to room temperature, the absorbance of the reaction solution at 532 nm was read using a microplate reader.

[0092] (3) Superoxide anion (O2") scavenging experiment. O2" is a major ROS produced in cellular respiration and other metabolic processes. SOD is an important antioxidant enzyme that catalyzes the conversion of O2" to oxygen and H2O2. This enzymatic reaction is a major defense mechanism against oxidative stress by neutralizing excess O2", thus preventing deleterious effects on cellular components. In this example, O2" was generated by a xanthine and xanthine oxidase reaction system, which can react with water-soluble tetrazolium salt-1 to form a yellow precipitate. The addition of HA-HMPB-Rapa to the reaction system resulted in a significant color change, so the O2" scavenging ability of HA-HMPB-Rapa was determined using a SOD kit (SO101S, Biyun Tian) according to the instructions. The SOD-like activity of HA-HMPB-Rapa is derived from the reducing ability and low redox potential of its nanocore HMPB.

[0093] (4) CAT-like activity assay. CAT is a key antioxidant enzyme present in cells (especially peroxisomes) that catalyzes the decomposition of H2O2 into water and O2, thus protecting organisms from oxidative stress caused by H2O2. HA-HMPB-Rapa was mixed with H2O2 to study the CAT-like activity of HA-HMPB-Rapa. Dissolved oxygen analyzer (Strater 400D, Ohaus) was used to detect the generated O2. In addition, the mixing of HA-HMPB-Rapa (100 μg / mL, 1 mL) with H2O2 (30%, 0.5 mL) can directly observe the generation of gas bubbles.

[0094] (5) POD-like activity assay. POD is an enzyme present in plant and animal tissues that catalyzes the oxidation of various substrates with H2O2 as a co-substrate. To evaluate the POD-like activity of HA-HMPB-Rapa, the chromogenic substrate 2,2'-azinobis-3-ethylbenzthiazoline-6-sulphonate (ABTS) was chosen. Under the catalysis of HA-HMPB-Rapa, H2O2 will oxidize ABTS to form a non-toxic substrate and a specific ultraviolet absorption peak will appear at a wavelength of 415 nm. Acetic acid-sodium acetate buffer (pH 3.6), different concentrations of HA-HMPB-Rapa (from 25 µg / mL to 1000 µg / mL), H2O2 and ABTS were mixed in a volume ratio of 200:10:10:15. After 30 min of reaction in the dark at room temperature, the absorbance value at 415 nm was measured. In addition, the UV-visible absorption spectrum of the reaction solution was recorded at different reaction time intervals (2, 4, 8, 15, 20, 25 and 30 min, respectively) using a UV spectrophotometer (UV-2600i, Shimadzu, Japan).

[0095] 2. Results

[0096] (1) The results of the DPPH scavenging experiment are shown in Figure 7 A, when the concentration was 25 µg / mL, HA-HMPB-Rapa had more than 20% DPPH scavenging effect. Subsequently, as the concentration of HA-HMPB-Rapa increased, its ability to scavenge DPPH gradually increased, reaching 58.7% at a concentration of 400 µg / mL, which indicated that it had excellent DPPH scavenging ability.

[0097] (2) The results of the ·OH scavenging experiment are shown in Figure 7 B, the ·OH scavenging ability was positively correlated with the concentration of HA-HMPB-Rapa, with a maximum scavenging rate of 63.4%, which further demonstrated the antioxidant ability of HA-HMPB-Rapa.

[0098] (3) The results of the ·O2- scavenging experiment are shown in Figure 7 C, when the concentration of HA-HMPB-Rapa was 400 µg / mL, ·O2- was almost completely scavenged.

[0099] (4) The results of the CAT-like activity assay are shown in Figure 7 D, there were a large number of bubbles in the cuvettes containing the mixed solution, while there were almost no bubbles in the cuvettes containing only nanoparticles and H2O2. As shown in Figure 7E shows that the amount of oxygen is positively correlated with the concentration of HA-HMPB-Rapa and the co-incubation time. The control group containing only H2O2 produced very little oxygen (13.2 ± 1.4 mg / L) within 10 min. In contrast, after the addition of HA-HMPB-Rapa at a concentration of 1000 μg / mL, 44.4 ± 1.5 mg / L of oxygen was produced within the same time, thanks to the CAT-like activity of HA-HMPB-Rapa.

[0100] (5) The results of the POD-like activity determination are shown in Figure 7 F shows that the absorbance increases with the extension of the co-incubation time when the concentration of HA-HMPB-Rapa is 100 μg / mL, which indicates that HA-HMPB-Rapa has a POD-like activity.

[0101] In summary, these results show that HA-HMPB-Rapa has multiple enzyme-like activities and a strong ability to scavenge ROS, indicating that HA-HMPB-Rapa is a potential antioxidant for the treatment of I / R-induced AKI.

[0102] Example 3 Cell toxicity and cellular uptake

[0103] 1. Method

[0104] (1) Cell toxicity. One of the main concerns in the application of nanomedicines for the treatment of AKI is that their potential nephrotoxicity can cause further damage to the injured kidney. To assess the potential nephrotoxicity of HA-HMPB-Rapa, HK-2 cells were cultured in MEM-α medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (100 IU / mL) at 37°C and 5% CO2. The cells were seeded into 96-well plates (1 × 10 4 cells per well) and incubated for 24 h. After 24 h, the culture medium was aspirated and washed with PBS, and different concentrations of HA-HMPB and HA-HMPB-Rapa (concentrations of 6, 12, 25, 50, 75, 100, 200, and 300 μg / mL) were added, and incubated for 12 and 24 h, and the cell viability was determined by the thiazolyl blue method.

[0105] (2) Cellular uptake. Injured HK-2 cells overexpress the CD44 receptor, which provides a pathway for the active targeting of the kidney by HA-modified nanoparticles. The present application used RhB-loaded nanoparticles (HMPB-RhB and HA-HMPB-RhB) to investigate the uptake behavior of H2O2-stimulated HK-2 cells. The cells were seeded in 6-well plates at a density of 1 × 10 5Cells were seeded into 12-well plates at a density of 1 x 105 cells per well and cultured for 24 h. HMPB-RhB or HA-HMPB-RhB was incubated with H2O2 (800 μM) stimulated cells for 6 or 12 h. Then, cells were washed with PBS and stained with 4', 6-diamidino-2-phenylindole (DAPI). Cells were observed under a fluorescence microscope (CYT7UMW, Agilent, USA) and 6 fields were randomly selected for photography.

[0106] 2. Results

[0107] (1) The results of the cytotoxicity experiment are shown in Table 1. The cytotoxicity of HA-HMPB and HA-HMPB-Rapa at 12 h and 24 h can be ignored within a certain concentration range (from 6 μg / mL to 300 μg / mL), and the cell survival rate is more than 85%. Figure 8

[0108] (2) The results of the cell uptake experiment are shown in Table 2. Within the same incubation time, H2O2 stimulated HK-2 cells uptook more HA-HMPB-RhB than HMPB-RhB (6 hours: about 2.1 times, 12 hours: 3.3 times), which verified the active targeting effect of HA. Figure 9

[0109] Example 4 Evaluation of the protective ability of HA-HMPB-Rapa on H2O2 stimulated cells

[0110] 1. Methods

[0111] (1) Protective effect of HA-HMPB-Rapa on H2O2 stimulated HK-2 cells. In I / R induced AKI, tubular injury is significantly earlier than glomerular injury. Since the renal tubule is the main damage site during and after I / R induced AKI, the damage and recovery of the renal tubule has a significant impact on the pathological process of AKI. This experiment aims to study the effect of different preparations on H2O2 stimulated HK-2 cells. Cells were seeded into 96-well plates (1 x 10 4 ​​HK-2 cells were stimulated with H2O2 for 24 h. After washing with PBS, the cells were incubated with PBS, Rapa (120 pg / mL), HA-HMPB (180 pg / mL), HMPB-Rapa (Rapa: 120 pg / mL) and HA-HMPB-Rapa (Rapa: 120 pg / mL) for another 24 h. Cell viability was determined by the thiazolyl blue method. Normal cells were used as negative control. To determine the anti-apoptotic effect of HA-HMPB-Rapa, Calcein-AM / PI staining was performed according to the manufacturer's instruction using Calcein-AM / PI kit (C2015M, Biyun Tian). The cells were observed under an inverted fluorescence microscope (CYT7UMW, Agilent, U.S.A) and six random fields were photographed. The percentage of PI-positive cells was quantified using ImageJ software.

[0112] (2) Intracellular ATP level evaluation. ATP is the main energy currency in biological systems, providing necessary energy for various cellular processes. Therefore, the change in ATP level will have a profound impact on cell function and viability. In the I / R-induced AKI microenvironment, the disruption of respiratory chain and the disturbance of energy metabolism in renal tubular epithelial cells eventually lead to cell death. To investigate the effect of nanoparticles on energy metabolism, the intracellular ATP level was determined using ATP assay kit (S0027, Biyun Tian). After incubation of the formulations with H2O2-stimulated cells for 24 h, the intracellular ATP level was determined according to the manufacturer's instruction.

[0113] (3) Assessment of antioxidant capacity at the cellular level. The relationship between mitochondria, oxidative stress, and I / R-induced AKI is crucial. Mitochondria are indispensable in maintaining cellular energy metabolism and redox homeostasis. In I / R-induced AKI, mitochondria play a dual role as both initiators and recipients of oxidative stress. Ischemia induces mitochondrial dysfunction, reduces ATP synthesis, and promotes ROS generation, thereby initiating the oxidative stress pathway. After reperfusion, the restoration of oxygen supply exacerbates ROS production, further impairing mitochondrial function and aggravating oxidative stress-mediated kidney injury. This complex interaction highlights the importance of therapeutic interventions targeting mitochondrial dysfunction and oxidative stress for improving I / R-induced AKI. The above studies have demonstrated that HA-HMPB-Rapa can scavenge multiple ROS and increase intracellular ATP levels in damaged cells. Based on these findings, the ability of HA-HMPB-Rapa to maintain intracellular redox homeostasis in H2O2-stimulated HK-2 cells was investigated. Intracellular ROS levels were assessed using the ROS indicator 2′,7′-dichlorofluorescein diacetate (DCFH-DA). This substance readily enters cells and, under the influence of ROS, is converted into a fluorescent compound called DCF. Cells stimulated with H₂O₂ were co-incubated with the formulation for 24 h, followed by co-incubation at 37°C with DCFH-DA (10 μM) in the dark for 0.5 h. After staining with DAPI (10 μg / mL), cells were observed and photographed under an inverted fluorescence microscope (CYT7UMW, Agilent, USA). Semi-quantitative analysis of the fluorescence images was performed using ImageJ software.

[0114] (4) Mitochondrial membrane potential (MMP) assay. The ability of nanoparticles to restore mitochondrial membrane potential (MMP) was verified using a kit for the mitochondrial membrane potential probe 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine (JC-1) (C2003S, Beyotime). ROS stimulation led to MMP depolarization, and JC-1 monomers with green fluorescence were observed in damaged mitochondria. In contrast, JC-1 aggregated and emitted red fluorescence in normal mitochondria with high MMP. The assay was performed at 1 × 10⁻⁶ per well. 5 Cells were seeded at a density of 100 cells / day in confocal culture dishes and cultured for 24 hours. Then, the cells were co-incubated with the above-mentioned preparation and H2O2-stimulated cells for another 24 hours. Finally, the cells were stained with the JC-1 probe in the dark at 37°C. Cells were observed under a confocal laser scanning microscope, and six fields of view were randomly selected for photographs.

[0115] 2. Results

[0116] (1) The protective effect of HA-HMPB-Rapa on H2O2-stimulated HK-2 cells is as follows: Figure 10 As shown in Figure A, after 24 hours of co-incubation with H2O2 (800 μM), only 8.4% of the cells survived. Free Rapa had no significant protective effect against H2O2-stimulated HK-2 cells, possibly due to low cellular uptake of the free drug. Conversely, HA-HMPB and HMPB-Rapa protected cells from oxidative stress-induced cell death. HA-HMPB-Rapa almost restored cell viability to normal levels (approximately 94.3%). Figure 10 As shown in B and C, after PI staining, a large number of apoptotic cells (red fluorescence) appeared in the model group, while almost no apoptotic cells were detected in the HA-HMPB-Rapa group (approximately 5.7%). These results indicate that HA-HMPB-Rapa can effectively inhibit H2O2-stimulated apoptosis in HK-2 cells and promote the recovery of cell viability.

[0117] (2) Results of intracellular ATP level assessment as follows Figure 11 As shown, the ATP level in the model group cells stimulated by H2O2 decreased significantly, to only 1 / 8 of that in normal HK-2 cells. Conversely, HMPB-Rapa significantly restored the ATP level in damaged cells from 4.4 nM / mg prot to 21.5 nM / mg prot. After treatment with HA-HMPB-Rapa, the intracellular ATP level almost returned to normal (29.5 nM / mg prot).

[0118] (3) Results of antioxidant capacity assessment at the cellular level, as follows Figure 12 As shown in Figure A, compared with the H2O2-stimulated model group, the fluorescence intensity of the HA-HMPB, HMPB-Rapa, and HA-HMPB-Rapa groups was significantly reduced, especially the HA-HMPB-Rapa group. Figure 12 The semi-quantitative analysis shown in B confirms this; as... Figure 12 As shown in Figure C, flow cytometry results also validated the ability of HA-HMPB-Rapa to scavenge intracellular ROS. The powerful antioxidant capacity of HA-HMPB-Rapa stems from the multi-enzyme activity of HMPB and the autophagy-promoting effect of Rapa in clearing damaged mitochondria. This two-pronged approach effectively removes existing excess ROS while eliminating its source (numerous damaged mitochondria), thus preventing the onset of oxidative stress.

[0119] (4) Results of mitochondrial membrane potential measurement are as follows Figure 13As shown, a strong green fluorescence signal was observed in the model group, indicating MMP depolarization under H2O2 stimulation. In contrast, MMP levels returned to normal after HA-HMPB-Rapa treatment, demonstrating superior efficacy compared to other prescriptions.

[0120] Example 5: Establishment of an I / R-induced AKI rat model

[0121] Sprague-Dawley (SD) rats (6-7 weeks old, 130-150g, male) were provided by Guangzhou Mindray Bio-Technology Co., Ltd. All animal experiments complied with the "Guidelines for the Protection and Utilization of Laboratory Animals" and the experimental protocols approved by the Animal Protection and Utilization Committee of Sun Yat-sen University.

[0122] The method for establishing an I / R-induced AKI rat model is as follows: After fasting overnight, SD rats were anesthetized and placed on a heating pad (37±2℃) to maintain body temperature. Abdominal villi were removed, and both kidneys were exposed through a midline abdominal incision. After blunt dissection of the renal artery and renal vein, the renal artery was clamped with a non-traumatic vascular clamp for 40 min to induce ischemia, and then the vascular clamp was removed to induce reperfusion.

[0123] like Figure 14 As shown, ischemia manifests as a rapid change in kidney color to yellowish-brown, and if the color returns to normal (light reddish-brown) within a short time (5-6 minutes) after the clamp is removed, then reperfusion is considered successful.

[0124] Example 6: Kidney targeting study of HA-HMPB-Rapa

[0125] Six hours after establishing an I / R-induced AKI rat model, the in vivo biodistribution of nanoparticles was investigated by tail vein injection of HMPB-RhB or HA-HMPB-RhB. Rats were euthanized at predetermined time points (1 h and 6 h after intravenous injection), and major organs were collected. In vitro organ imaging was performed using a small animal optical imaging system (PerkinElmer IVIS Lumina Series III, USA), and the fluorescence intensity of different organs was quantified.

[0126] The distribution results of nanoparticles in biological bodies are as follows: Figure 15 As shown, HA-modified nanoparticles rapidly accumulated in the kidneys 1 hour after intravenous injection, and the fluorescence signal in the kidneys remained at a high level over time. The fluorescence intensity of the HA-HMPB-RhB group was significantly higher than that of the HMPB-RhB group, further confirming the active targeting effect of HA-modified nanoparticles on damaged kidneys. Meanwhile, as... Figure 16As shown, the accumulation of HMPB-RhB and HA-HMPB-RhB in the liver and spleen was limited. Previous studies have shown that particles with a size between 10 nm and 250 nm are easily captured by the mononuclear phagocyte system, resulting in accumulation in the liver and spleen. For HMPB-RhB, it is possible that the PVP passivated the surface of HMPB, reducing the possibility of protein corona formation during blood circulation and avoiding being captured by macrophages. However, further studies are needed to elucidate the underlying mechanisms. For HA-HMPB-RhB, the HA on its surface not only provides targeting, but also improves biocompatibility to some extent. Previous studies have shown that negatively charged nanoparticles can hinder the formation of protein corona, reducing the interaction with macrophages, thereby further avoiding the capture of nanoparticles by the mononuclear phagocyte system. The therapeutic window of AKI refers to the most effective time limit for intervention to reduce kidney damage and improve prognosis. The therapeutic window of AKI is usually narrow, emphasizing timely detection and intervention to prevent irreversible kidney damage. Therefore, after the diagnosis of AKI, it is of great clinical value to quickly and accurately deliver drugs to the damaged kidney to prevent the development of AKI, thereby preventing end-stage renal disease.

[0127] Example 7 In vivo pharmacodynamic evaluation of HA-HMPB-Rapa in I / R-induced AKI rats

[0128] 1. Method

[0129] SD rats were randomly divided into seven groups: (i) normal group (Control); (ii) sham operation group (Sham); (iii) AKI+PBS (Model); (iv) AKI+Rapa group (Rapa: 1 mg / kg); (v) AKI+HA-HMPB group (HA-HMPB: 1.5 mg / kg); (vi) AKI+HMPB-Rapa group (Rapa: 1 mg / kg); (vii) AKI+HA-HMPB-Rapa group (Rapa: 1 mg / kg). Blood samples were collected by retro-orbital venous plexus puncture before modeling (0 h) and at specific time points (12 and 24 h, respectively). After 24 h, rats were weighed and sacrificed. Kidneys were collected, washed with PBS and weighed. Kidney index was calculated according to the formula. In the case of AKI, an increase in kidney coefficient usually indicates pathological enlargement of the kidney relative to body weight. The increase in kidney weight can be attributed to various factors, including kidney congestion, inflammation, cell proliferation, or compensatory response to kidney injury or dysfunction. Kidney coefficient is often used as an indicator to evaluate the efficacy of experimental treatment or intervention on AKI-related kidney injury. Fresh right kidneys were collected and stored at -80°C for subsequent Western blot and tissue homogenate detection. Part of the left kidney was embedded and frozen, then cut into 5 μm sections for dihydroethidium (DHE) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Another part was fixed with 4% paraformaldehyde together with other major organs (heart, liver, spleen, lung). Kidney coefficient (%) = kidney weight (mg) / body weight (g) x 100%.

[0130] (1) Survival rate monitoring. I / R-induced AKI rats were randomly divided into five groups (n=12) and treated with different formulations (including PBS, Rapa, HA-HMPB, HMPB-Rapa and HA-HMPB-Rapa), respectively. The body weight changes and survival of SD rats within 7 days were recorded. Normal SD rats were used as controls.

[0131] (2) Renal function assessment. Blood urea nitrogen (BUN) and creatinine (CRE) are key indicators for assessing AKI, and their changes reflect the changes in glomerular filtration rate. Lactate dehydrogenase (LDH) normally exists in cells and is released into the blood due to cell damage and necrosis. AKI can trigger an inflammatory response in the kidney, including the secretion of pro-inflammatory cytokines and the recruitment of immune cells, leading to further cell damage and LDH release. The levels of BUN, CRE and LDH in serum were detected using BUN detection kit (BC1535, Solabio), CRE detection kit (C011-2-1, Nanjing Jiancheng) and LDH detection kit (A020-2-2, Nanjing Jiancheng) according to the instructions.

[0132] (3) Histological analysis. The kidneys fixed with 4% paraformaldehyde were dehydrated and embedded with paraffin, and then cut into 5 μm thick sections. After hematoxylin-eosin (H&E) staining, the sections were imaged under a microscope (CYT7UMW, Agilent, U.S.A), and six random fields were selected to observe the histological structure of the kidney.

[0133] (4) In vivo antioxidant performance evaluation. The relationship between Superoxide Dismutase (SOD), Glutathione (GSH) and Malondialdehyde (MDA) and I / R-induced AKI strongly illustrates the complex dynamics of oxidative stress and antioxidant defense during the process of kidney injury. After I / R, the production of ·O2- significantly increases due to electron leakage from the mitochondrial electron transport chain. SOD can catalyze the conversion of ·O2- to O2, reducing the damage of ·O2- to the kidney. Therefore, SOD plays a key role in reducing ROS-mediated oxidative stress damage during AKI. The activity and expression level of SOD are often used as indicators of the state of oxidative stress and antioxidant defense in AKI. GSH is a tripeptide and the main antioxidant in cells, playing an important role in reducing oxidative stress by directly scavenging ROS and maintaining the reduced form of other antioxidants, including vitamins C and E. MDA is the end product of lipid peroxidation reactions, in which ROS such as ·OH attack polyunsaturated fatty acids within the cell membrane. MDA is often used as a biomarker to assess the degree of oxidative stress and lipid peroxidation. In I / R-induced AKI, the increase in MDA levels not only reflects the increase in ROS production, but also reflects the damage to the cell membrane, leading to cell death and tissue damage. The levels of SOD (BC165, Solarbio), GSH (BC1175, Solarbio) and MDA (BC0025, Solarbio) in kidney tissue homogenate were determined using the corresponding detection kits.

[0134] The kidney cryosections were incubated with DHE solution (10 mM) at 37 °C in the dark for 30 min, then washed with PBS three times to remove unbound dye. After staining with DAPI (10 pg / mL), the slides were mounted with an anti-fluorescence quenching mounting medium. The red fluorescence within the tissue was observed under an inverted fluorescence microscope (CYT7UMW, Agilent, U.S.A).

[0135] (5) In vivo anti-apoptosis effect evaluation. Apoptosis of renal tubular epithelial cells is the main form of cell death in I / R-induced AKI. Although apoptosis is a natural process to eliminate damaged cells, a large amount of uncontrolled apoptosis during AKI leads to loss of nephron and further to renal dysfunction. Renal cryosections were fixed in 4% paraformaldehyde for 45 min and then treated with Triton X-100 (0.5%) for 5 min at room temperature. Afterwards, the sections were incubated with TUNEL working solution at 37°C for 1 h in the dark according to the instructions. The sections were stained with DAPI (10 pg / mL) and observed under a fluorescence microscope (CYT7UMW, Agilent, U.S.A). Six fields were randomly selected for photography, and the TUNEL-positive cells representing apoptosis in the fields were quantified using ImageJ software.

[0136] (6) In vivo anti-inflammatory effect evaluation. Inflammation exacerbates kidney injury in the later stage of AKI. Inflammatory cells, especially neutrophils and monocytes, are recruited to the damaged kidney tissue, promoting the release of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), thereby increasing vascular permeability, leading to vascular leakage and edema, and further damaging the kidney. Enzyme-linked immunosorbent assay kit was used to detect serum TNF-α, IL-6 and IL-1β according to the instructions.

[0137] (7) Western blot analysis. As highly active organelles of metabolism, mitochondria are responsible for ATP production through oxidative phosphorylation. The kidney possesses a large number of mitochondria, second only to the heart. The distribution of mitochondria in the kidney is not uniform, but is related to the specific functions of each part of the kidney. For example, the mitochondria in the proximal tubules, which are responsible for most of the reabsorption and secretion processes, are dense, reflecting their high metabolic activity and energy demand. This is the reason why the proximal tubule is one of the main lesion sites of AKI induced by I / R. Mitochondrial dysfunction is a key event in I / R-induced AKI. ATP production decreases, ROS production increases, and MMP depolarization are the main causes of cell damage and apoptosis. Mitophagy is a selective autophagy process that is a major quality control mechanism that specifically targets and degrades damaged mitochondria. This highly regulated pathway ensures that only damaged or excess mitochondria are targeted for degradation, which is crucial for maintaining cellular homeostasis, especially in cells with high energy demands such as neurons and kidney cells. To study the molecular mechanisms of the formulation in the treatment of AKI induced by I / R in vivo, the expression of related key proteins in the model of AKI induced by I / R was evaluated, including PINK-1, Parkin, LC3 and P62 proteins in the mitochondrial autophagy signaling pathway, and Bcl-2, Bax, Cyt-c, Cleaved Casp-3 proteins in the apoptosis signaling pathway.

[0138] The protein in the kidney tissue homogenate was quantified by BCA kit. The protein solution was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then electrotransferred to a polyvinylidene fluoride membrane. After blocking with 5% skim milk solution for 2 h, the membrane was incubated with anti-PINK-1 (1:500), Parkin (1:1000), P62 (1:5000), LC3 (1:1000), Bcl-2 (1:2000), Bax (1:6000), Cyt-c (1:1000), Casp-3 (1:500) and KIM-1 (1:1000) antibodies at 4°C overnight. Anti-β-actin antibody (1:5000) and anti-GAPDH antibody (1:20000) were used as internal controls. After co-incubation of the polyvinylidene fluoride membrane with horseradish peroxidase-conjugated secondary antibody, chemiluminescence solution was added uniformly on the membrane, and images were collected using a multifunctional gel imaging system (ChemiDoc™ Imaging System, Bio-Rad, USA). The intensity of the protein bands was semi-quantitatively analyzed using ImageJ software. All quantitative data from Western blot analysis were from three independent experiments.

[0139] (8) In vivo biocompatibility evaluation. Kidney is one of the most important organs to maintain homeostasis in vivo, which is often exposed to high concentrations of metabolic waste. Therefore, it is essential to ensure the biocompatibility of the preparation for the treatment of AKI. SD rats were randomly divided into three groups: (i) PBS group, (ii) HA-HMPB group (1.5 mg / kg) and HA-HMPB-Rapa group (Rapa: 1 mg / kg). The rats were injected with the above preparations via the tail vein every other day. After 7 days, the rats were sacrificed, and whole blood and serum samples were collected for biochemical detection. At the same time, the main organs of the rats were collected for H&E staining to evaluate the histological changes.

[0140] 2. Results

[0141] (1) The animal experiment process and the results of rat kidney coefficient detection are shown in Figure 17 , the kidney coefficient of rats treated with HA-HMPB-Rapa was significantly lower than that of the model group and the free Rapa group, close to the normal level. This indicates that HA-HMPB-Rapa can effectively alleviate I / R-induced AKI and reduce kidney swelling.

[0142] (2) The results of rat survival rate monitoring are shown in Figure 18 and Figure 19 , I / R-induced AKI resulted in a 50% mortality rate in the model group within 7 days, and the body weight of the rats in the model group decreased significantly within two days after modeling. The tail vein injection of HA-HMPB-Rapa (Rapa: 1 mg / kg) after 6 h can significantly improve the survival rate within 7 days (91.7%). In addition, both HA-HMPB and HMPB-Rapa showed therapeutic effects, with survival rates of 58.3% and 66.7%, respectively.

[0143] (3) The results of rat kidney function evaluation are shown in Figure 20 A and 20B, after 40 min of ischemia and 12 h of reperfusion, the BUN and CRE levels in the serum of the model group increased significantly, which were 6.3 times and 6.1 times of the control group, respectively. In addition, the CRE content showed an upward trend with the extension of time, indicating the deterioration of AKI. In the Sham group, both indicators showed no significant fluctuations, indicating that BUN and CRE levels were not affected by other operations in addition to I / R-induced AKI. Single administration of HA-HMPB or HMPB-Rapa alleviated AKI to some extent. Compared with the model group, the BUN levels of these two groups decreased by 40.2% and 55.3%, respectively, and the CRE levels decreased by 39.2% and 46.3%, respectively, after 24 h. In addition, HA-HMPB-Rapa showed the best therapeutic effect. Compared with the model group, the BUN and CRE levels decreased by 70.2% and 79.3%, respectively, close to the normal level. Figure 20As shown in FIG. C, 24 h after modeling, the LDH level of the model group was 1.4 times that of the control group. Single administration of HA-HMPB-Rapa effectively reduced the LDH level of the AKI rats to the normal range, which indicated that HA-HMPB-Rapa could maintain the integrity of the cell membrane of the kidney cells and avoid the leakage of intracellular LDH.

[0144] (4) The results of histopathological analysis are shown in FIG. Figure 21 As shown in FIG. C, 24 h after modeling, the LDH level of the model group was 1.4 times that of the control group. Single administration of HA-HMPB-Rapa effectively reduced the LDH level of the AKI rats to the normal range, which indicated that HA-HMPB-Rapa could maintain the integrity of the cell membrane of the kidney cells and avoid the leakage of intracellular LDH.

[0145] (5) The results of in vivo antioxidant performance evaluation are shown in FIG. Figure 22 As shown in FIG. C, 24 h after modeling, the LDH level of the model group was 1.4 times that of the control group. Single administration of HA-HMPB-Rapa effectively reduced the LDH level of the AKI rats to the normal range, which indicated that HA-HMPB-Rapa could maintain the integrity of the cell membrane of the kidney cells and avoid the leakage of intracellular LDH. Figure 23 As shown in FIG. A, after HA-HMPB-Rapa treatment, the SOD level in the damaged kidney was restored to 75.6% of the control group, which was 2.1 times that of the model group (HA-HMPB-Rapa group: 231.6 U / mg prot v.s. Model group: 112.8 U / mg prot). As shown in FIG. B, I / R-induced AKI produced a large amount of ROS, which significantly reduced the GSH content of the model group (401.1 μg / g tissue), which was more than 50% lower than the normal value (1054 μg / g tissue). HMPB-Rapa was more effective than free Rapa in restoring the GSH content of the damaged kidney, which indicated that Rapa could achieve more kidney accumulation under the protection of HMPB to promote mitochondrial autophagy to remove damaged mitochondria. In addition, after HA-HMPB-Rapa treatment, the GSH content was restored to near normal levels, which was not statistically different from the control group and the sham treatment group. Figure 23 B).Figure 23 As shown in Fig. 7C, the MDA content of the model group was 2.3 times that of the control group, indicating that I / R-induced oxidative stress caused a significant increase in the level of lipid peroxidation in kidney cells. In contrast, after a single dose of HA-HMPB-Rapa, the MDA content was reduced by 47.6% compared to the model group, close to the normal value.

[0146] (6) The results of the anti-apoptosis effect evaluation in vivo are shown in Fig. 8. Figure 24 As shown in Fig. 8, a large number of TUNEL-positive cells (green fluorescence, with an apoptosis rate of up to 72.4%) were observed in the model group. In contrast, HA-HMPB and HMPB-Rapa could reduce the apoptosis rate to 25.4% and 19.6%, respectively. The apoptosis rate of the HA-HMPB-Rapa group was the lowest (3.7%), close to that of the control group, indicating that it could prevent kidney failure by protecting kidney cells from a large amount of apoptosis.

[0147] (7) The results of the anti-inflammatory effect evaluation in vivo are shown in Fig. 9. Figure 25 As shown in Fig. 9, a single dose of HMPB-Rapa had a certain anti-inflammatory effect, with TNF-a, IL-1b, and IL-6 levels reduced by 61.4%, 20.6%, and 25.3%, respectively, compared to the model group. In addition, HA-HMPB-Rapa had the best anti-inflammatory effect, with the levels of the three inflammatory factors significantly lower than those of the model group.

[0148] (8) The results of Western blot analysis are shown in Fig. 10. Figure 26 As shown in Fig. 10A, after treatment with HMPB-Rapa or HA-HMPB-Rapa, the expression of PINK-1 in the kidney was 1.5 times and 1.8 times that of the model group, respectively, indicating that both formulations could upregulate the expression of PINK-1, thereby promoting the recognition of damaged mitochondria and initiating the subsequent mitochondrial autophagy process. Figure 26 As shown in Fig. 10B, upregulation of Parkin expression was also observed in the HMPB-Rapa and HA-HMPB-Rapa groups, indicating that when PINK1 accumulates on the outer membrane of damaged mitochondria, Parkin is recruited to it, thereby preparing for the subsequent mitochondrial autophagy. Figure 26 As shown in Figs. 10C and 10D, treatment with HA-HMPB-Rapa resulted in an increase in the LC3 I / LC3 II ratio and a decrease in the expression level of P62, which are two established biomarkers of autophagy activation. Previous studies have shown that an increase in ROS levels hinders the degradation of P62, thereby hindering the autophagy process. Therefore, the multiple enzyme activities of the nanocore endow HA-HMPB with the ability to scavenge ROS, thereby enabling it to promote the orderly progress of autophagy. The results of the study indicate that HA-HMPB-Rapa can effectively regulate the expression of key proteins in the mitochondrial autophagy signaling pathway, ensuring the timely and effective removal of damaged mitochondria.

[0149] Damaged mitochondria produce excess ROS, which exceeds the antioxidant capacity of renal cells, leading to the occurrence of oxidative stress. The excessive production of ROS and the depolarization of MMPs lead to the transcriptional or post-transcriptional regulation of apoptosis-related proteins of the Bcl-2 family, resulting in renal cell apoptosis. Therefore, the effect of HA-HMPB-Rapa on the expression of downstream anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax after inducing autophagy of damaged mitochondria was studied. As shown in Figure 26 E and F, a large number of damaged mitochondria in the model group led to overexpression of Bax and a decrease in the expression level of Bcl-2, indicating that the apoptosis pathway was initiated and activated in damaged renal cells, and the expression of the two key proteins in the signaling pathway was reversed by the preparation treatment. Compared with the model group, HA-HMPB-Rapa can significantly reduce the expression of Bax (decreased by 64.8%) and up-regulate the expression of Bcl-2 (increased by 3.1 times). At the same time, HMPB-Rapa also inhibited the occurrence of apoptosis, and compared with the model group, the level of Bax decreased by 35.4%, and the level of Bcl-2 increased by 2.6 times. Studies have shown that the increase in the Bax / Bcl-2 ratio further damages the integrity of the mitochondrial membrane. Once activated, Bax undergoes a conformational change and migrates from the cytoplasm to the outer membrane of the mitochondria, oligomerizes to form a pore structure, leading to the release of Cyt-c into the cytoplasm. In contrast, the anti-apoptotic protein Bcl-2 can inhibit this process by binding to activated Bax, or by sequestering "BH3-only" pro-apoptotic proteins and preventing them from activating Bax. After entering the cytoplasm, Cyt-c will bind to Apoptotic protease activating factor-1 (Apaf-1), causing Apaf-1 to undergo a conformational change and form oligomers. Then, the Cyt-c / Apaf-1 complex recruits caspase-9 precursors to form a large multimeric complex called apoptosome. Subsequently, the caspase-9 enzyme is catalytically activated, leading to the cleavage and activation of the apoptosis factor Casp-3. As shown in Figure 26 G, the expression of Cyt-c in the model group increased significantly, which was 1.7 times that of the control group. In contrast, after a single dose of HA-HMPB, HMPB-Rapa or HA-HMPB-Rapa, the content of Cyt-c reversed, which decreased by 25.2%, 25.2% and 45.8% compared with the model group, respectively. These findings are consistent with the semi-quantitative results of Bax and Bcl-2 described above, indicating that the decrease in the Bax / Bcl-2 ratio inhibits the release of Cyt-c. Subsequently, the expression of Cleaved Casp-3, the "executor" of the apoptosis pathway, in each group was studied. As shown in Figure 26H shows that AKI caused a 1.6-fold increase in Cleaved Casp-3 levels, indicating that the apoptosis signaling pathway was highly activated in the model group. In addition, the expression of Cleaved Casp-3 was inhibited after treatment with HMPB-Rapa or HA-HMPB-Rapa, with the best effect being observed for HA-HMPB-Rapa. The above results show that HA-HMPB-Rapa can clear damaged mitochondria by promoting mitophagy, thereby inhibiting the release of pro-apoptotic factors and preventing apoptosis of kidney cells. As shown in Figure 26 I shows that KIM-1 is rapidly and specifically upregulated upon kidney injury, making KIM-1 a reliable indicator of the early stages of kidney injury. In addition to its diagnostic role, KIM-1 can also be used as an indicator for assessing prognosis. An increase in KIM-1 levels indicates that the injury is worsening and is associated with the progression of chronic kidney disease. I / R-induced AKI caused a significant increase in the expression of KIM-1 in the model group, which was 1.8 times that of the control group, indicating that the kidney injury was severe. Compared with the model group, HA-HMPB-Rapa treatment reduced the expression of KIM-1 by 63.9%, which means that HA-HMPB-Rapa can effectively protect the kidney from injury and inhibit the progression of AKI to chronic kidney disease.

[0150] In summary, as shown in Figure 26 J, HA-HMPB-Rapa can act on the PINK-1 / Parkin signaling pathway, promote mitophagy, and promptly remove a large number of damaged mitochondria caused by AKI, thereby inhibiting a series of catastrophic cell damage induced by damaged mitochondria from the source. Prompt removal of damaged mitochondria can restore the expression of anti-apoptotic protein Bcl-2 and inhibit the expression of pro-apoptotic protein Bax. In addition, this process also inhibits the release of Cyt-c after mitochondrial membrane rupture and the subsequent activation of Cleaved Casp-3, thereby inhibiting the initiation of the apoptosis pathway. Through these interrelated effects, enhancing mitophagy can serve as a protective mechanism to maintain cellular homeostasis and prevent the activation of cell death pathways, thereby reducing the extent of AKI injury after I / R.

[0151] (9) In vivo biocompatibility evaluation results As shown in Figure 27 hematology analysis showed that neither HA-HMPB nor HA-HMPB-Rapa caused significant changes in blood parameters compared with the control group, indicating that their systemic toxicity was minimal. As shown in Figure 27 A, neither of the two preparations had a significant effect on liver and kidney function, as indicated by the levels of glutamic oxalacetic transaminase, glutamic pyruvic transaminase, BUN, and CRE. In addition, as shown in Figure 27 B, H&E results of major organs (heart, liver, spleen, lung, and kidney) showed that both preparations had minimal effect on organ histological morphology. In addition, as shown in Figure 28As shown, intravenous injection of these two preparations also did not cause significant changes in the body weight of rats. The above results show that HA-HMPB-Rapa has good biocompatibility, laying a foundation for its clinical application.

Claims

1. A rapamycin-loaded targeted hollow mesoporous Prussian blue nanoparticle, characterized in that, The hollow mesoporous Prussian blue nanoparticles are loaded with rapamycin, and the surface of the hollow mesoporous Prussian blue nanoparticles is modified with amino hyaluronic acid. The preparation method of the rapamycin-loaded targeted hollow mesoporous Prussian blue nanoparticles comprises adding an amino hyaluronic acid solution to a dispersion of the rapamycin-loaded hollow mesoporous Prussian blue nanoparticles, and stirring to obtain the rapamycin-loaded targeted hollow mesoporous Prussian blue nanoparticles. The preparation method of the hollow mesoporous Prussian blue nanoparticles comprises reacting bismuth nitrate, potassium ferricyanide and polyvinylpyrrolidone in an acidic solution. The preparation method of the rapamycin-loaded hollow mesoporous Prussian blue nanoparticles comprises mixing the hollow mesoporous Prussian blue nanoparticles and rapamycin in ethanol, and stirring to obtain the rapamycin-loaded hollow mesoporous Prussian blue nanoparticles; and the mass ratio of the hollow mesoporous Prussian blue nanoparticles to rapamycin is 1:1-10. The preparation method of the amino hyaluronic acid comprises reacting hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in a mixed solution, and then adding ethylenediamine to obtain the amino hyaluronic acid.

2. The rapamycin-loaded, targeted hollow mesoporous prussian blue nanoparticle of claim 1, wherein, The hydrodynamic diameter of the rapamycin-loaded targeted hollow mesoporous Prussian blue nanoparticles is 50-200 nm, and the surface charge is -10 to -40 mV.

3. The rapamycin-loaded, targeted hollow mesoporous prussian blue nanoparticle of claim 1, wherein, The drug loading amount of the rapamycin-loaded hollow mesoporous Prussian blue nanoparticles is 5-50%.

4. Use of the rapamycin-loaded targeted hollow mesoporous Prussian blue nanoparticles in the preparation of a medicament for treating acute kidney injury caused by ischemia-reperfusion.

5. A medicament for treating acute kidney injury caused by ischemia-reperfusion, characterized by, The rapamycin-loaded targeted hollow mesoporous Prussian blue nanoparticles.

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