Rapamycin-loaded targeted hollow mesoporous Prussian blue nanoparticles, preparation method thereof and application of nanoparticles in treatment of ischemia-reperfusion-induced acute kidney injury

Abstract

The invention provides targeted hollow mesoporous Prussian blue nanoparticles loaded with rapamycin as well as a preparation method and application thereof in treating acute kidney injury induced by ischemia reperfusion. The nano particles comprise hollow mesoporous Prussian blue nano particles, rapamycin is loaded in the hollow mesoporous Prussian blue nano particles, and aminated hyaluronic acid is modified on the surfaces of the hollow mesoporous Prussian blue nano particles. The nanoparticles are endowed with kidney targeting property through aminated hyaluronic acid modification; the hollow mesoporous Prussian blue nano particles and the rapamycin play a dual role in promoting mitochondrial autophagy and relieving oxidative stress reaction. Therefore, the nanoparticles can effectively recover renal functions, relieve pathological changes of tissues and inhibit oxidative stress and inflammatory response in treatment of acute kidney injury induced by ischemia-reperfusion, and finally protect kidney cells from apoptosis injury, and have great application potential clinically.

Description

Technical Field

[0001] The invention relates to the field of nanoparticle preparation and medical treatment, and in particular to a targeted hollow mesoporous Prussian blue nanoparticle loaded with rapamycin and a preparation method thereof, and application thereof in treating acute kidney injury induced by ischemia-reperfusion. Background Art

[0002] Acute kidney injury (AKI) refers to the sudden onset of renal dysfunction or failure, a clinical syndrome caused by a rapid decline in renal function in a short period of time due to a variety of causes. 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. Symptoms may include a sharp drop in glomerular filtration rate in a short period of time, a rapid increase in serum creatinine and urea nitrogen, decreased urine output, fatigue, edema, 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 survivors are at higher risk of chronic kidney disease and cardiovascular disease. In addition, AKI imposes a huge economic burden on individuals and society due to the increased costs of hospitalization, dialysis, and long-term care. A series of supportive strategies are usually used clinically to treat AKI, including minimizing or avoiding the use of nephrotoxic drugs, providing nutritional support, increasing circulating blood volume, and renal replacement therapy. There is currently no approved drug therapy specifically for AKI.

[0003] Ischemia / reperfusion (I / R), sepsis, nephrotoxic drugs, and other pathogenic factors can induce AKI, of which I / R is the main clinical cause of AKI. AKI caused by I / R is a complex pathophysiological condition characterized by a temporary interruption of blood flow (ischemia) followed by the restoration of blood supply to the kidney (reperfusion). This phenomenon can occur in a variety of clinical settings such as kidney transplantation, trauma, cardiac surgery, and shock. Oxidative stress begins with ischemia, where reduced blood flow in the kidneys leads to an insufficient supply of oxygen and nutrients related to cellular metabolism. This ischemia prompts cells, especially sensitive renal tubular epithelial cells, to enter an anaerobic metabolic state, resulting in the accumulation of metabolic byproducts and insufficient production of adenosinetriphosphate (ATP). The subsequent reperfusion is intended to restore blood flow, but paradoxically triggers a further harmful cascade. The sudden influx of oxygen becomes a substrate for xanthine oxidase and nicotinamide adenine dinucleotide phosphate oxidase, resulting in the production of excessive 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 such as 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 the one hand, oxidative stress promotes inflammation, apoptosis, and endothelial dysfunction, thereby aggravating renal injury. On the other hand, renal injury itself can further enhance oxidative stress by releasing damage-associated molecular patterns, activating intracellular signaling pathways, and inducing mitochondrial dysfunction, thus forming a vicious cycle. Understanding this relationship will help develop therapeutic strategies aimed at improving the antioxidant capacity of the kidney 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 may cause ROS to quickly return to their original levels. Therefore, although these catalysts can temporarily reduce ROS levels, they may not necessarily fundamentally solve the problem of continued excessive ROS production. Therefore, eliminating both excess ROS and the source of ROS may play a two-pronged role in the treatment of AKI.

[0004] Mitochondria are often referred to as the "powerhouses" of cells because they play a key role in producing ATP through oxidative phosphorylation. In addition to producing energy, mitochondria are also involved in a variety of cellular processes, including calcium ion balance, ROS generation, and apoptosis. As a high-energy-consuming organ in the human body, the mitochondrial content and oxygen demand of the kidney (accounting for about 7% of the total oxygen consumption of the human body) are relatively higher than other organs. In I / R-induced AKI, excessive ROS can lead to an imbalance between ROS generation and antioxidant defense mechanisms in mitochondria, inducing mitochondrial damage. Even 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 extremely destructive ·O2 - The continued generation of inflammatory cytokines further exacerbates oxidative stress and ultimately leads to cell apoptosis. As AKI develops, the vicious cycle of inflammatory response, oxidative stress, mitochondrial damage, and cell apoptosis further exacerbates renal injury.

[0005] At present, the treatment of AKI mainly relies on supportive therapy, which is to increase the blood perfusion of damaged kidneys through volume expansion, or to use hemodialysis and filtration as a functional replacement therapy for the kidneys. However, the efficacy of clinical supportive therapy can only delay the pathological progression of AKI, and cannot reverse the renal tissue damage that has occurred and promote the repair of renal tissue. Therefore, it is of great research significance and clinical value to develop strategies that can quickly reverse renal tissue damage, effectively restore renal function, and significantly improve the survival rate of patients. For I / R-induced AKI, an acute and critical disease with a complex pathogenesis, although there is currently a lack of safe and effective therapeutic drugs, some interventional drugs are often used clinically to alleviate oxidative damage and inflammatory responses. However, these drugs are difficult to quickly correct the oxidative stress and mitochondrial damage that are closely related to cell apoptosis from the source, that is, it is difficult to achieve comprehensive regulation of multiple pathogenesis, and thus cannot effectively reverse renal cell damage and repair the structure of damaged renal tissue, and ultimately it is difficult to achieve complete recovery of renal function; in addition, the drug itself lacks specific targeting to the damaged kidney, making it difficult to enrich in the lesion site; the toxicity of the drug and the adverse reactions caused by the accumulation in non-target organs also limit its dosage, which seriously affects its treatment results. At present, with the deepening of the understanding of the pathophysiology of ischemia-reperfusion AKI, a variety of intelligent-microenvironment-responsive nanodrug delivery systems have been developed one after another, such as polymer nanoparticles, DNA nanorobots, inorganic nanoparticles (gold, cerium dioxide, ruthenium oxide, cuprous oxide with ROS scavenging function), etc. Although the currently developed nano-drug delivery system can improve the efficacy of drugs to a certain extent, it still cannot achieve the repair and regeneration of renal tissue and the complete recovery of renal function. This may be related to the fact that it is still an anti-inflammatory and antioxidant treatment for the single pathogenesis of I / R-induced AKI, and cannot quickly improve the oxidative stress and mitochondrial damage closely related to cell damage from the root. Therefore, for ischemia-reperfusion-induced AKI, which has a rapid onset and complex pathological mechanism, it is urgent to provide a therapeutic drug and method that can quickly reverse renal tissue damage, restore renal function, and significantly improve patient survival. Summary of the invention

[0006] The purpose of the present invention is to overcome the above defects and shortcomings in the prior art and provide a targeted hollow mesoporous Prussian blue nanoparticle loaded with rapamycin.

[0007] The second object of the invention is to provide a method for preparing the above-mentioned targeted hollow mesoporous Prussian blue nanoparticles loaded with rapamycin.

[0008] The third object of the present invention is to provide a use of the above-mentioned targeted hollow mesoporous Prussian blue nanoparticles loaded with rapamycin in the preparation of a drug for treating acute kidney injury caused by ischemia-reperfusion.

[0009] The fourth object of the present invention is to provide a drug for treating acute kidney injury caused by ischemia-reperfusion, comprising the above-mentioned targeted hollow mesoporous Prussian blue nanoparticles loaded with rapamycin.

[0010] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0011] The invention provides a targeted hollow mesoporous Prussian blue nanoparticle loaded with rapamycin. The particle comprises a hollow mesoporous Prussian blue nanoparticle, 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 rich in mitochondria and with a high oxygen demand. Therefore, it is prone to a series of damage events under ischemia-reperfusion conditions, including oxidative stress, mitochondrial damage, adenosine triphosphate depletion, and inflammatory response, which ultimately lead to acute kidney injury (AKI). Currently, there are no approved therapies for AKI in clinical practice except supportive treatment. Although many studies have focused on clearing excess reactive oxygen species (ROS) at the lesion site to alleviate AKI, little attention has been paid to inhibiting the generation of ROS from the source. Mitochondria are a selective autophagy pathway that degrades dysfunctional or excess mitochondria through lysosomal phagocytosis. This mechanism maintains cellular homeostasis by ensuring the removal of defective mitochondria, thereby alleviating oxidative stress and preventing the accumulation of damaged organelles. Defects in mitochondrial autophagy 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 rapamycin target inhibitor and 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, and maintenance of muscle vitality. As mentioned above, oxidative stress can damage a large number of mitochondria in the kidneys, thus forming a source of excessive ROS. Rapa can regulate cell homeostasis and cell viability by activating the autophagy mechanism. Depending on the selectively degraded organelles, autophagy can be divided into mitochondrial autophagy, endoplasmic reticulum autophagy, and ribosomal autophagy. Therefore, it is speculated that Rapa can selectively remove damaged mitochondria in AKI, fundamentally inhibit the excessive production of ROS, thereby alleviating 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 lesions, etc.) limit its application in vivo.

[0014] Prussian blue (PB) is a US Food and Drug Administration-approved detoxifier for radioactive elements thallium and cesium with good biosafety. 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 acidic microenvironments, and have attracted much attention as a potential drug delivery platform. In addition, the multi-type enzyme properties of HMPB (peroxidase POD, catalase CAT, and superoxide dismutase SOD) give it the ability to scavenge ROS, which is very promising in the treatment of oxidative stress-related diseases (including neurodegenerative diseases, inflammatory bowel disease, acute pancreatitis, etc.). Using HMPB as a carrier can not only effectively load Rapa, but also clear the 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 site of injury.

[0015] Hyaluronic acid (HA) is a naturally occurring polysaccharide that is often used to modify nanoparticles to achieve active targeted drug delivery through the 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, the use of HA modification can achieve more precise and effective AKI treatment.

[0016] The targeted hollow mesoporous Prussian blue nanoparticles of the load rapamycin provided by the present invention, through the dual effects of the carrier hollow mesoporous Prussian blue and the drug rapamycin, can effectively remove the original ROS and the source of ROS (a large number of damaged mitochondria), thereby preventing the further outbreak of ROS.Aminated hyaluronic acid is modified on the surface of the hollow mesoporous Prussian blue nanoparticles of the load rapamycin by the chelation of amino and iron ions.And the hollow mesoporous Prussian blue nanoparticles of the load rapamycin and the modified hyaluronic acid can remove 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 (Kidney injury molecule-1, KIM-1).So, the targeted hollow mesoporous Prussian blue nanoparticles of the load rapamycin are expected to become a medicine and therapy for the treatment of ischemia-reperfusion related diseases.

[0017] Furthermore, the hydrodynamic diameter of the targeted hollow mesoporous Prussian blue nanoparticles loaded with rapamycin is 50 to 200 nm, and the surface charge is -10 to -40 mV.

[0018] Preferably, the targeted hollow mesoporous Prussian blue nanoparticles loaded with rapamycin have a hydrodynamic diameter of 119.5 nm and a surface charge of -30.3 mV.

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

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

[0021] The present invention also provides a method for preparing the above-mentioned targeted hollow mesoporous Prussian blue nanoparticles loaded with rapamycin, which comprises adding an amino hyaluronic acid solution to a dispersion of the hollow mesoporous Prussian blue nanoparticles loaded with rapamycin, stirring, and obtaining the targeted hollow mesoporous Prussian blue nanoparticles loaded with rapamycin.

[0022] Furthermore, the preparation method of the hollow mesoporous Prussian blue nanoparticles is to react bismuth nitrate, potassium ferrocyanide and polyvinyl pyrrolidone in an acidic solution to obtain the hollow mesoporous Prussian blue nanoparticles.

[0023] Furthermore, the preparation method of the hollow mesoporous Prussian blue nanoparticles loaded with rapamycin is to mix the hollow mesoporous Prussian blue nanoparticles and rapamycin in ethanol at different mass ratios and stir to obtain the obtained product.

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

[0025] Furthermore, the preparation method of the amino hyaluronic acid is to react hyaluronic acid with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in a mixed solution, and then add ethylenediamine and mix to obtain the amino hyaluronic acid.

[0026] Furthermore, the mixed solution has a volume ratio of DMSO / water = 3:1.

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

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

[0029] The present invention also provides a medicine for treating acute kidney injury caused by ischemia-reperfusion, wherein the medicine comprises targeted hollow mesoporous Prussian blue nanoparticles loaded with rapamycin.

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

[0031] The present invention provides a targeted hollow mesoporous Prussian blue nanoparticle loaded with rapamycin, including hollow mesoporous Prussian blue nanoparticles, which are loaded with rapamycin and surface-modified with amino hyaluronic acid. The hollow mesoporous Prussian blue nanoparticles are modified with amino hyaluronic acid, which gives the nanoparticles kidney targeting, so that HA-HMPB-Rapa can actively target the CD44 receptors 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 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 drugs, and can also effectively remove existing excess ROS; Rapa can remove a large number of damaged mitochondria in damaged kidneys, i.e., the main source of excessive ROS, by promoting mitochondrial autophagy, thereby preventing further outbreaks of ROS. Therefore, HA-HMPB-Rapa has a two-pronged effect in alleviating oxidative stress.

[0032] Compared with the prior art, it is difficult to quickly correct the oxidative stress and mitochondrial damage phenomenon closely related to apoptosis from the source, that is, it is difficult to achieve the comprehensive regulation of multiple pathogenesis, and then it is impossible to effectively reverse renal cell damage and repair the structure of damaged renal tissue, and finally it is difficult to achieve the problem of complete recovery of renal function. The nanoparticles of the present invention have a satisfactory therapeutic effect in the I / R-induced AKI rat model, and the dual effects of carrier and drug produce a satisfactory therapeutic effect, which is mainly manifested in the improvement of rat survival rate, recovery of renal function, reduction of renal tissue lesions, and inhibition of oxidative stress, apoptosis, and inflammatory response. In addition, 6h after I / R, a single low-dose tail vein administration of HA-HMPB-Rapa (Rapa: 1mg / kg) can effectively alleviate AKI and restore renal function, reduce tissue pathological changes, inhibit oxidative stress and inflammatory response, and finally protect renal cells from apoptotic damage. This experimental design is consistent with the clinical situation and reflects the window period between pathological onset and treatment. Low doses also indicate a reduction in cost and nephrotoxicity 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 hemolysis, acid-responsive drug release behavior and multiple types of enzyme activity. It regulates various pathological mechanisms such as oxidative stress, mitochondrial damage, and inflammatory response from the perspective of "multi-target, multi-pathway, and multi-link", and can promote the recovery of cell viability, mitochondrial membrane potential (MMP) and ATP levels, so as to quickly reverse renal tissue damage, restore renal function, significantly improve patient survival rate, break the vicious cycle of AKI pathology, and effectively alleviate I / R-induced AKI. It has the potential for further clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The preparation flow chart of nanoparticles and their mechanism of action.

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

[0035] Figure 3 Ultraviolet 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 is the storage stability of the nanoparticles at 4°C.

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

[0039] Figure 7 This is a test of the ability of HA-HMPB-Rapa to remove reactive oxygen species. Figure 7 A is the scavenging rate of DPPH by a series of HA-HMPB-Rapa concentrations; B is the scavenging rate of ·OH by a series of HA-HMPB-Rapa concentrations; C is the scavenging rate of ·O2- by a series of HA-HMPB-Rapa concentrations; D is a picture of oxygen production from H2O2 catalyzed by HA-HMPB-Rapa; E is the amount of oxygen produced by H2O2 catalyzed by HA-HMPB-Rapa at different concentrations within 10 min; F is the POD-like activity of 100 μg / mL HA-HMPB-Rapa.

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

[0041] Fig. 9 Fluorescence images of cellular uptake of HMPB-RhB and HA-HMPB-RhB by HK-2 cells stimulated with H2O2 after different incubation times, as well as semi-quantitative results of fluorescence intensity.

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

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

[0044] Fig.12 To evaluate the antioxidant capacity of cells. Fig.12 A and B are the ROS fluorescence images and semi-quantitative results of HK-2 cells stimulated by H2O2 24 hours after treatment; C is the flow cytometry result.

[0045] Fig.13 Fluorescence images of changes in mitochondrial membrane potential in HK-2 cells stimulated with H2O2 24 hours after treatment.

[0046] Fig.14 These are pictures of the color changes in the kidneys during ischemia and reperfusion.

[0047] Fig.15 This is a study on the distribution of nanoparticles in the body. Fig.15 A in the figure is the fluorescence image of the main 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; B is the semi-quantitative analysis result of the fluorescence intensity in the kidney of AKI rats at different time points (1 or 6 h) after intravenous injection of HMPB-RhB and HA-HMPB-RhB.

[0048] Fig.16 The results are semi-quantitative analysis of the fluorescence intensity of the main 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.

[0049] Fig.17 Figure 2 shows the animal experiment flow chart and the renal coefficients of AKI rats after treatment with different preparations.

[0050] Fig.18 Figure 3 shows the changes in the survival rate of rats in each group 7 days after treatment.

[0051] Fig.19 Figure 3 shows the changes in body weight of rats in each group 7 days after treatment.

[0052] Fig. 20 (A) BUN, (B) CRE and (C) LDH levels in the serum of rats in each group after treatment.

[0053] Fig.21 H&E staining images of rat kidney sections in different treatment groups.

[0054] Fig. 22 Fluorescence images of ROS in the kidneys of rats in different treatment groups and semi-quantitative results of the relative fluorescence intensity of DHE.

[0055] Fig.23 The levels of (A) SOD, (B) GSH and (C) MDA in the kidneys of rats in different treatment groups.

[0056] Fig.24 Fluorescence images of TUNEL staining of renal tissue sections and the corresponding semi-quantitative results.

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

[0058] Fig.26 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 nanoparticle therapeutic mechanism.

[0059] Fig. 27 (A) Heat map of hematological test indicators and (B) H&E staining images of major organs of rats in different treatment groups.

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

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

[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 amino 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 6 mL of a mixed solution (DMSO / water = 3:1, v / v) for 0.5 h, and 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 amino hyaluronic acid.

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

[0068] Bismuth nitrate (243 mg), potassium ferrocyanide (396 mg) and polyvinyl pyrrolidone (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 the solution was washed with water and ethanol several times. Finally, HMPB was obtained by freeze drying.

[0069] (3) Preparation of HMPB-Rapa

[0070] Rapamycin (Rapa) and HMPB were mixed in ethanol at different mass ratios and stirred overnight to obtain HMPB-Rapa. Free Rapa was removed by centrifugation and ethanol washing.

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

[0072] 1 mL of the amino 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 three times with water to remove excess HA.

[0073] 2. Characterization methods

[0074] (1) After free Rapa was separated by centrifugation, it was 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 / total mass of Rapa) × 100%.

[0076] ② Drug loading (%) = mass of loaded Rapa / mass of HA-HMPB-Rapa × 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 ultraviolet absorption spectra of the nanoparticles and Rapa were measured using an ultraviolet spectrophotometer (UV-2600i, Shimadzu, 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) with different pH values ​​(7.4, 6.5, 5.5) at 37°C. Samples were collected at designated time points (0.25h, 0.5h, 1h, 2h, 6h, 12h and 24h, respectively). The samples were then analyzed by high performance liquid chromatography at a wavelength of 268nm to calculate the cumulative release of Rapa.

[0079] (3) Stability testing of HA-HMPB-Rapa: To determine the long-term storage stability of HA-HMPB-Rapa, the samples were stored at 4°C, and the hydrodynamic size and polydispersity index of the nanoparticles were monitored for 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 (Triton X-100) and PBS (0.01M, pH 7.4) were used as positive and negative controls, respectively. After the incubation, the samples were centrifuged at 4000 rpm for 5 min, the supernatant was collected, and the released hemoglobin was detected at a wavelength of 560 nm using an enzyme reader (Biotek SYNERGY H1 Instruments, Inc.). The relative hemolysis rate was calculated according to the formula. Relative hemolysis rate (%) = (A 样品 -A PBS ) / (A TritonX-100 -A PBS )×100%.

[0081] 3. Results

[0082] (1) HA-HMPB-Rapa preparation process Figure 1 As shown, the present invention mixes polyvinyl pyrrolidone, potassium ferrocyanide and bismuth nitrate in an acidic solution and synthesizes HMPB by a one-pot hydrothermal method. During the formation of HMPB, bismuth ions react with ferrocyanide to form degradable Bi 3+ -Fe(CN)6 3- , which promoted the formation of hollow mesoporous structures in nanoparticles, and further prepared Rapa-loaded HMPB (HMPB-Rapa), which was further modified with HA (named HA-HMPB-Rapa) to achieve effective treatment of I / R-induced AKI.

[0083] (2) Dynamic light scattering analysis results are as follows Figure 2 As shown, Figure 2 A and B show that the average size of HMPB is 97.4 nm, the size distribution is uniform (polydispersity index (PDI): 0.126), and the surface charge is -24.8 mV. In the present invention, HMPB is loaded with Rapa through pore adsorption and hydrophobic interaction between the hydrophobic cavity and the drug. After loading Rapa, the hydrodynamic diameter of HMPB-Rapa increased slightly (about 106.2 nm), but its surface charge did not change significantly. Through the chelation of iron and amino groups, the amino-modified HA is modified on the surface of HMPB-Rapa, so that the nanoparticles have active targeting ability to 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 with HA, the hydrodynamic diameter of HA-HMPB-Rapa increased to about 119.5 nm and the surface charge decreased to -30.3 mV. 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) UV absorption spectrum results are as follows Figure 3 As shown in Figure 3, the broad absorption of HA-HMPB-Rapa in the range of 600 nm to 800 nm is consistent with the characteristic absorption of HMPB, which is attributed to the electron transfer in Fe-CN-Fe. Drug loading and HA modification did not change the UV-visible absorption spectrum of HMPB, indicating that HA-HMPB-Rapa still retains the catalytic properties of HMPB.

[0085] (4) Rapa drug loading and pH-responsive drug release results Figure 4 As shown in Figure 2, high drug loading is crucial for the efficacy of nanomedicines. When studying the effect of the mass ratio of Rapa / HMPB on the drug loading of HA-HMPB-Rapa, it was found that the drug loading increased with the increase of the mass ratio of Rapa / HMPB, as shown in Figure 2. Figure 4 As shown in A, it finally reaches a plateau (drug loading: 40%); Figure 4 As shown in Figure B, the cumulative release of Rapa within 24 hours under acidic conditions is much higher than that under physiological conditions, and the release increases with decreasing pH (pH 7.4: 15.4%, pH 6.5: 28.0%, pH 5.5: 43.0%). This pH-responsive drug release behavior enables the drug to be rapidly released in pathologically weakly acidic damaged renal cells.

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

[0087] (6) Hemolysis test results Figure 6 As shown, the hemolysis rate of HA-HMPB-Rapa was still below the threshold of 5% when the concentration was as high as 1000 μg / mL, verifying the excellent blood compatibility of HA-HMPB-Rapa.

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

[0089] 1. Methods

[0090] (1) 1,1-Diphenyl-2-picrylhydrazyl (DPPH) scavenging experiment. DPPH is a stable free radical compound that appears purple when in the form of a free radical and fades under the reducing action of an antioxidant. This property can be used to quantify the ability of a substance to scavenge free radicals, which helps to evaluate the potential of a substance as an antioxidant. HA-HMPB-Rapa of different concentrations was mixed with an equal volume of DPPH solution and reacted in the dark for 0.5 h. The absorbance of the DPPH free radical at 517 nm was then measured using an ELISA reader.

[0091] (2) ·OH scavenging assay. ·OH is one of the most reactive and destructive free radicals in biological systems. The high reactivity and ability to initiate chain reactions make ·OH particularly detrimental to the integrity and function of cell membranes and is therefore associated with various pathological conditions, including neurodegenerative diseases and diseases associated with aging. The ·OH scavenging ability of HA-HMPB-Rapa was evaluated using the deoxyribose method. 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, the reaction was initiated with ascorbic acid (1.8 mM, 50 μL) and incubated at 50°C for 20 min. Finally, a mixed solution of 10% trichloroacetic acid and 5% thiobarbituric acid (5:3, v / v) was added to the reaction system, and then incubated for another 15 min at 100° C. After cooling to room temperature, the absorbance of the reaction solution at 532 nm was read using an ELISA reader.

[0092] (3) ·O2- scavenging experiment. ·O2- is a major ROS produced during cellular respiration and other metabolic processes. SOD is an important antioxidant enzyme that catalyzes the conversion of ·O2- into oxygen and H2O2. This enzymatic reaction neutralizes excess ·O2-, thereby preventing harmful effects on cellular components, and is the main defense mechanism against oxidative stress. In this embodiment, ·O2- is generated by xanthine and xanthine oxidase reaction system, which can react with water-soluble tetrazolium salt-1 to form a yellow precipitate. Adding HA-HMPB-Rapa to the reaction system will result in a significant color change, so the scavenging ability of HA-HMPB-Rapa for ·O2- was measured using a SOD kit (SO101S, Bio-Tech) according to the instructions. The SOD-like activity of HA-HMPB-Rapa originates from the reducing ability and low redox potential of its nano-core HMPB.

[0093] (4) CAT-like activity assay. CAT is a key antioxidant enzyme present in cells (especially peroxisomes), which can catalyze the decomposition of H2O2 into water and O2, thereby protecting the organism from oxidative stress caused by H2O2. HA-HMPB-Rapa was mixed with H2O2 to study the CAT-like activity of HA-HMPB-Rapa. The generated O2 was detected using a dissolved oxygen analyzer (Strater 400D, Ohaus). In addition, HA-HMPB-Rapa (100 μg / mL, 1 mL) was mixed with H2O2 (30%, 0.5 mL), and the generation of bubbles could be directly observed.

[0094] (5) POD-like activity assay. POD is an enzyme present in plant and animal tissues that catalyzes the oxidation of various substrates using H2O2 as a co-substrate. In order to evaluate the POD-like activity of HA-HMPB-Rapa, the chromogenic substrate 2,2'-azinobis-3-ethylbenzthiazoline-6-sulphonate (ABTS) was selected. Under the catalytic action of HA-HMPB-Rapa, H2O2 oxidizes ABTS to form a non-toxic substrate with a specific UV absorption peak at 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 reacting for 30 min in a dark environment at room temperature, the absorbance value at a wavelength of 415 nm was measured. In addition, the UV-visible absorption spectra of the reaction solution at different reaction time intervals (2, 4, 8, 15, 20, 25 and 30 min, respectively) were recorded using an UV spectrophotometer (UV-2600i, Shimadzu Corporation, Japan).

[0095] 2. Results

[0096] (1) DPPH scavenging test results are as follows Figure 7 As shown in Figure A, when the concentration was 25 μg / mL, the scavenging effect of HA-HMPB-Rapa on DPPH exceeded 20%. 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, indicating that it has an excellent ability to scavenge DPPH.

[0097] (2) OH removal test results are as follows Figure 7 As shown in Figure B, the ·OH scavenging ability was positively correlated with the concentration of HA-HMPB-Rapa, and the maximum scavenging rate was 63.4%, which further demonstrated the antioxidant capacity of HA-HMPB-Rapa.

[0098] (3) O2- removal test results are as follows Figure 7 As shown in Figure C, when the concentration of HA-HMPB-Rapa was 400 μg / mL, ·O2- was almost completely cleared.

[0099] (4) CAT-like activity assay results Figure 7 As shown in D, a large number of bubbles are generated in the cuvette containing the mixed solution, while there are almost no bubbles in the cuvette containing only nanoparticles and H2O2. Figure 7As shown in E, the amount of oxygen was 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 adding HA-HMPB-Rapa at a concentration of 1000 μg / mL, 44.4±1.5 mg / L of oxygen was produced in the same time, which was attributed to the CAT-like activity of HA-HMPB-Rapa.

[0100] (5) The results of POD-like activity determination are as follows Figure 7 As shown in F, when the concentration of HA-HMPB-Rapa was 100 μg / mL, the absorbance increased with the extension of the co-incubation time, indicating that HA-HMPB-Rapa had POD-like activity.

[0101] Taken together, these results indicate that HA-HMPB-Rapa possesses multiple types of enzyme activities and a strong ability to scavenge ROS, suggesting that HA-HMPB-Rapa is a potential antioxidant for the treatment of I / R-induced AKI.

[0102] Example 3 Cytotoxicity and cellular uptake

[0103] 1. Methods

[0104] (1) Cytotoxicity. A major concern about the use of nanomedicines to treat AKI is that their potential nephrotoxicity can cause further damage to damaged kidneys. To evaluate 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 cells / well). 4 After 24 h, the culture medium was discarded and washed with PBS, and different concentrations of HA-HMPB and HA-HMPB-Rapa (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. Damaged HK-2 cells overexpress CD44 receptors, which provides a way for HA-modified nanoparticles to actively target the kidneys. The present invention uses RhB-loaded nanoparticles (HMPB-RhB and HA-HMPB-RhB) to explore the uptake behavior of HK-2 cells stimulated by H2O2. 5The cells were seeded into 12-well plates at a density of 10 cells and cultured for 24 h. HMPB-RhB or HA-HMPB-RhB was co-incubated with H2O2 (800 μM) stimulated cells for 6 or 12 h. Then, the cells were washed with PBS and stained with 4', 6-diamidino-2-phenylindole (4', 6-diamidino-2-phenylindole, DAPI). The cells were observed under a fluorescence microscope (CYT7UMW, Agilent, USA) and 6 fields of view were randomly selected for photography.

[0106] 2. Results

[0107] (1) Cytotoxicity test results Figure 8 As shown, within a certain concentration range (from 6 μg / mL to 300 μg / mL), the cytotoxicity of HA-HMPB and HA-HMPB-Rapa at 12 h and 24 h was negligible, and the cell survival rates were both over 85%.

[0108] (2) Cell uptake experimental results are as follows Fig. 9 As shown, within the same incubation time, H2O2-stimulated HK-2 cells took up more HA-HMPB-RhB than HMPB-RhB (6 h: about 2.1 times, 12 h: 3.3 times), which verified the active targeting effect of HA.

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

[0110] 1. Methods

[0111] (1) Protective effect of HA-HMPB-Rapa on HK-2 cells stimulated by H2O2. In I / R-induced AKI, tubular injury occurs significantly earlier than glomerular injury. Since the renal tubules are the main site of injury during and after I / R-induced AKI, tubular injury and recovery have a significant impact on the pathological process of AKI. This experiment was designed to study the effects of different preparations on HK-2 cells stimulated by H2O2. Cells were seeded into 96-well plates (1×10 4cells) and cultured for 24 h. After aspirating the culture medium and washing with PBS, PBS, Rapa (120 μg / mL), HA-HMPB (180 μg / mL), HMPB-Rapa (Rapa: 120 μg / mL) and HA-HMPB-Rapa (Rapa: 120 μg / mL) were co-incubated with H2O2-stimulated HK-2 cells for 24 h. Cell viability was determined by the thiazolyl blue method. Normal cells were used as negative controls. To determine the anti-apoptotic effect of HA-HMPB-Rapa, cells were stained with Calcein-AM / PI using the Calcein-AM / PI kit (C2015M, Beyotime) according to the manufacturer's instructions. Cells were observed under an inverted fluorescence microscope (CYT7UMW, Agilent, USA) and six fields of view were randomly selected for photography, and the percentage of PI-positive cells was quantified using ImageJ software.

[0112] (2) Assessment of intracellular ATP levels. ATP is the main energy currency in biological systems and provides the necessary energy for various cellular processes. Therefore, changes in ATP levels can have a profound impact on cell function and vitality. In the I / R-induced AKI microenvironment, the destruction of the respiratory chain and the disorder of energy metabolism in renal tubular epithelial cells ultimately lead to cell death. The intracellular ATP level was measured using an ATP detection kit (S0027, Bio-Tech) to study the effect of nanoparticles on energy metabolism. After the preparation was incubated with H2O2-stimulated cells for 24 hours, the intracellular ATP level was measured according to the method provided by the manufacturer.

[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 the maintenance of cellular energy metabolism and redox homeostasis. Mitochondria play a dual role in I / R-induced AKI, both as initiators and recipients of oxidative stress. Ischemia induces mitochondrial dysfunction, reduces ATP synthesis, and promotes ROS generation, thereby initiating oxidative stress pathways. After reperfusion, the restoration of oxygen supply exacerbates ROS production, further impairs mitochondrial function, and aggravates oxidative stress-mediated renal injury. This complex interaction highlights the importance of therapeutic intervention targeting mitochondrial dysfunction and oxidative stress to improve I / R-induced AKI. The above studies have demonstrated that HA-HMPB-Rapa can scavenge multiple ROS and increase ATP levels in damaged cells. In view of 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), which easily enters cells and becomes a fluorescent compound called DCF under the action of ROS. The preparations were incubated with H2O2-stimulated cells for 24 h, and then incubated with DCFH-DA (10 μM) at 37°C in the dark for 0.5 h. After staining with DAPI (10 μg / mL), the cells were observed and photographed under an inverted fluorescence microscope (CYT7UMW, Agilent, USA). The fluorescence images were semi-quantitatively analyzed using ImageJ software.

[0114] (4) Mitochondrial membrane potential measurement. The mitochondrial membrane potential probe 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine (JC-1) kit (C2003S, Biotech) was used to verify the ability of nanoparticles to restore the mitochondrial membrane potential (MMP) of cells. ROS stimulation leads to MMP depolarization, and JC-1 monomers with green fluorescence can be observed in damaged mitochondria. In contrast, JC-1 aggregates in normal mitochondria with high MMP and emits red fluorescence. 1×10 5 The cells were inoculated into a confocal culture dish at a density of 10 cells. After culturing for 24 hours, the above preparations were co-incubated with H2O2-stimulated cells for 24 hours, and then stained with JC-1 probe under 37°C dark conditions. The cells were observed under a confocal laser scanning microscope and six fields of view were randomly selected for photography.

[0115] 2. Results

[0116] (1) The protective effect of HA-HMPB-Rapa on HK-2 cells stimulated by H2O2 Fig.10 As shown in A, after incubation with H2O2 (800 μM) for 24 h, only 8.4% of the cells survived. Free Rapa had no obvious protective effect on HK-2 cells stimulated by H2O2, which may be due to the low uptake of free drugs by cells. In contrast, HA-HMPB and HMPB-Rapa can protect cells from oxidative stress-induced cell death. HA-HMPB-Rapa almost restored cell viability to normal (about 94.3%). Fig.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 (about 5.7%) were detected in the HA-HMPB-Rapa group. The above results show that HA-HMPB-Rapa can effectively inhibit the apoptosis of HK-2 cells stimulated by H2O2 and promote the recovery of cell viability.

[0117] (2) The results of intracellular ATP level assessment are as follows Fig.11 As shown in the figure, the ATP level in the model group cells stimulated by H2O2 decreased significantly, only 1 / 8 of that in normal HK-2 cells. In contrast, HMPB-Rapa can significantly restore the ATP level of damaged cells from 4.4nM / mg prot to 21.5nM / mg prot. After treatment with HA-HMPB-Rapa, the intracellular ATP level almost returned to normal levels (29.5nM / mg prot).

[0118] (3) The results of antioxidant capacity evaluation at the cellular level are as follows Fig.12 As shown in A, compared with the model group stimulated by H2O2, the fluorescence intensity of the HA-HMPB, HMPB-Rapa, and HA-HMPB-Rapa groups was significantly reduced, especially in the HA-HMPB-Rapa group. Fig.12 This was confirmed by the semiquantitative analysis shown in B; Fig.12 As shown in Figure C, the results of flow cytometry also verified the ability of HA-HMPB-Rapa to remove intracellular ROS. The powerful antioxidant capacity of HA-HMPB-Rapa comes from the multi-type enzyme activity of HMPB and the removal of damaged mitochondria by Rapa's autophagy-promoting effect. This two-pronged approach can effectively remove existing excess ROS and eliminate its source (a large number of damaged mitochondria), thereby avoiding the outbreak of oxidative stress.

[0119] (4) Mitochondrial membrane potential measurement results Fig.13As shown, a strong green fluorescence signal can be found in the model group, indicating that MMP depolarization under H2O2 stimulation. In contrast, MMP returned to normal levels after HA-HMPB-Rapa treatment, and its efficacy was better than other prescriptions.

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

[0121] Sprague-Dawley (SD) rats (6–7 weeks old, 130–150 g, male) were provided by Guangzhou Mindray Biotechnology Co., Ltd. All animal experiments were in accordance with the Guide for the Care and Use of Laboratory Animals and experimental protocols approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University.

[0122] The I / R-induced AKI rat model was established as follows: After an overnight fast, SD rats were anesthetized and placed on a heating pad (37±2°C) to maintain body temperature. The abdominal villi were removed, and the 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 minutes to induce ischemia, and then the vascular clamp was removed to induce reperfusion.

[0123] like Fig.14 As shown, the manifestation of ischemia is that the color of the kidney quickly changes to khaki, and if it returns to normal blood color (light reddish brown) within a short time (5-6 minutes) after the clamp is removed, reperfusion is considered successful.

[0124] Example 6 Study on kidney targeting of HA-HMPB-Rapa

[0125] After 6 h of establishing the I / R-induced AKI rat model, the in vivo biodistribution of the nanoparticles was studied by tail vein injection of HMPB-RhB or HA-HMPB-RhB. At the predetermined time points (1 h and 6 h after intravenous injection), the rats were euthanized and the main organs were collected. The small animal optical imaging system (PerkinElmer IVIS Lumina Series III, USA) was used for in vitro organ imaging, and the fluorescence intensity of different organs was quantified.

[0126] The distribution of nanoparticles in vivo is shown in Fig.15 As shown in the figure, HA-modified nanoparticles can accumulate rapidly in the kidneys 1 hour after intravenous injection, and the fluorescence signal in the kidneys remains at a high level over time. The fluorescence intensity of the HA-HMPB-RhB group is significantly higher than that of the HMPB-RhB group, further confirming the active targeting effect of HA-modified nanoparticles on damaged kidneys. Fig.16As shown, limited accumulation of HMPB-RhB and HA-HMPB-RhB in the liver and spleen was observed. Previous studies have shown that particles with a size between 10 nm and 250 nm are easily captured by the mononuclear phagocyte system and accumulate in the liver and spleen. For HMPB-RhB, it may be that PVP passivates the surface of HMPB, thereby reducing its possibility of forming a protein corona during blood circulation and avoiding large-scale capture by macrophages. However, further studies are needed to elucidate its underlying mechanism. For HA-HMPB-RhB, the HA on its surface can not only provide targeting, but also improve biocompatibility to a certain extent. At the same time, previous studies have shown that negatively charged nanoparticles may hinder the formation of protein corona and reduce 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 renal damage and improve prognosis. The therapeutic window of AKI is usually narrow, emphasizing timely detection and intervention to prevent irreversible renal damage. Therefore, after AKI is diagnosed, rapid and precise delivery of drugs to the damaged kidneys to prevent the development of AKI has important clinical value for end-stage renal disease.

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

[0128] 1. Methods

[0129] SD rats were randomly divided into seven groups: (i) normal group (Control); (ii) sham 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 retroorbital venous plexus puncture before modeling (0h) and at specific time points (12 and 24h, respectively). After 24h, the rats were weighed and killed. The kidneys were collected, washed with PBS and weighed. The renal index was calculated according to the formula. In the case of AKI, an increase in the renal index generally indicates a pathological enlargement of the kidney relative to body weight. The increase in kidney weight can be attributed to a variety of factors, including renal congestion, inflammation, cell proliferation, or compensatory responses to renal injury or dysfunction. The renal coefficient is often used as an indicator to evaluate the efficacy of experimental treatments or interventions on AKI-related renal injury. Fresh right kidneys were collected and stored at -80°C for subsequent protein immunoblotting and tissue homogenate detection. Part of the left kidney was embedded and frozen, then cut into 5 μm slices for dihydroethidium (DHE) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The other part was fixed with 4% paraformaldehyde together with other major organs (heart, liver, spleen, and lungs). Renal coefficient (%) = kidney weight (mg) / body weight (g) × 100%.

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

[0131] (2) Assessment of renal function. Blood urea nitrogen (BUN) and creatinine (CRE) are key indicators for assessing AKI, and their changes reflect changes in glomerular filtration rate. Lactate dehydrogenase (LDH), which is normally present in cells, is released into the blood due to cell damage and necrosis. AKI can trigger an inflammatory response in the kidney, including the secretion of proinflammatory 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 measured using a BUN detection kit (BC1535, Solebao), a CRE detection kit (C011-2-1, Nanjing Jiancheng), and a LDH detection kit (A020-2-2, Nanjing Jiancheng), respectively, according to the instructions.

[0132] (3) Histological analysis. The kidneys fixed with 4% paraformaldehyde were dehydrated and embedded in paraffin, and then cut into 5 μm thick sections. The sections were stained with hematoxylin-eosin (H&E) and imaged under a microscope (CYT7UMW, Agilent, USA). Six fields of view were randomly selected to observe the histological structure of the kidneys.

[0133] (4) Evaluation of in vivo antioxidant properties. 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 renal injury. After I / R, the production of ·O2- increased significantly 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 alleviating ROS-mediated oxidative stress injury during AKI. The activity and expression level of SOD are often used as indicators of oxidative stress and antioxidant defense status in AKI. GSH is a tripeptide and the main antioxidant in cells. It plays an important role in reducing oxidative stress by directly scavenging ROS and maintaining the reduced forms of other antioxidants (including vitamins C and E). MDA is the end product of lipid peroxidation, in which ROS such as ·OH attack polyunsaturated fatty acids in cell membranes. MDA is commonly used as a biomarker to assess the extent of oxidative stress and lipid peroxidation. In I / R-induced AKI, the increase in MDA levels reflects not only the increase in ROS generation but also the destruction of cell membranes, leading to cell death and tissue damage. The levels of SOD (BC165, Solebold), GSH (BC1175, Solebold), and MDA (BC0025, Solebold) in renal tissue homogenates were determined using the corresponding detection kits.

[0134] Kidney cryosections were incubated with DHE solution (10 μM) at 37°C in the dark for 30 min, and then washed three times with PBS to remove unbound dye. After DAPI (10 μg / mL) staining, the sections were sealed with anti-fluorescence quenching sealing agent. The red fluorescence in the tissue was observed under an inverted fluorescence microscope (CYT7UMW, Agilent, USA).

[0135] (5) Evaluation of in vivo anti-apoptotic effect. 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 can lead to the loss of nephrons and renal dysfunction. Kidney cryosections were fixed in 4% paraformaldehyde for 45 min and then treated with TritonX-100 (0.5%) for 5 min at room temperature. The sections were then incubated with TUNEL working solution at 37°C in the dark for 1 h according to the instructions. The sections were stained with DAPI (10 μg / mL) and observed under a fluorescence microscope (CYT7UMW, Agilent, USA). Six fields of view were randomly selected for photography, and the TUNEL-positive cells representing apoptosis in the field of view were quantified using ImageJ software.

[0136] (6) Evaluation of in vivo anti-inflammatory effects. In the late stage of AKI, inflammatory responses can exacerbate kidney damage. Inflammatory cells, especially neutrophils and monocytes, are recruited into damaged kidney tissues, promoting the massive release of proinflammatory 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 causing further damage to the kidneys. Serum TNF-α, IL-6, and IL-1β were detected using an enzyme-linked immunosorbent assay kit according to the instructions.

[0137] (7) Western blot analysis. As highly metabolically active organelles, mitochondria are responsible for producing ATP through oxidative phosphorylation. The kidney has 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 proximal tubules, which are responsible for most of the reabsorption and secretion processes, are densely populated with mitochondria, reflecting their vigorous metabolic activity and high energy requirements. This is why the proximal tubule is one of the main lesions in I / R-induced AKI. Mitochondrial dysfunction is a key event in I / R-induced AKI. Reduced ATP production, increased ROS production, and MMP depolarization are the main causes of cell damage and apoptosis. Mitochondrial autophagy is a selective autophagic 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 critical for maintaining cellular homeostasis, especially in cells with high energy demands such as neurons and kidney cells. To investigate the molecular mechanism by which the preparation exerts its therapeutic effect in vivo, the expression of relevant key proteins in the I / R-induced AKI model was evaluated, including PINK-1, Parkin, LC3, and P62 proteins in the mitochondrial autophagy signaling pathway, and Bcl-2, Bax, Cyt-c, and Cleaved Casp-3 proteins in the apoptosis signaling pathway.

[0138] The protein in the kidney tissue homogenate was quantified using a BCA kit. The protein solution was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electroblotted onto a polyvinylidene difluoride membrane. After blocking with 5% skim milk powder solution for 2 h, the membrane was incubated with antibodies against 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) at 4°C overnight. Anti-β-actin antibody (1:5000) and anti-GAPDH antibody (1:20000) were used as internal controls. After the polyvinylidene fluoride membrane was incubated with the secondary antibody coupled to horseradish peroxidase, the chemiluminescent solution was evenly added to the membrane and the multifunctional gel imaging system (ChemiDoc TM Images were collected using the ELISA Imaging System (Bio-Rad, USA). The intensity of protein bands was semi-quantitatively analyzed using ImageJ software. All quantitative data for immunoblotting analysis were from three independent experiments.

[0139] (8) In vivo biocompatibility evaluation. The kidney is one of the most important organs for maintaining homeostasis and is often exposed to high concentrations of metabolic waste. Therefore, ensuring the biocompatibility of the preparation is crucial 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 through the tail vein every other day. After 7 days, the rats were killed and whole blood and serum samples were collected for biochemical testing. At the same time, the main organs of the rats were collected for H&E staining to evaluate histological changes.

[0140] 2. Results

[0141] (1) Animal experiment process and rat kidney coefficient test results Fig.17 As shown in the figure, the renal 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 renal swelling.

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

[0143] (3) The results of rat renal function evaluation are as follows Fig. 20 As shown in A and 20B, after 40 minutes of ischemia and 12 hours of reperfusion, the levels of BUN and CRE in the serum of the model group increased significantly, which were 6.3 times and 6.1 times that of the control group, respectively. In addition, the CRE content showed an upward trend with the extension of time, indicating the worsening of AKI. In the Sham group, there was no obvious fluctuation in these two indicators, indicating that BUN and CRE levels were not affected by other operations except I / R-induced AKI. A single administration of HA-HMPB or HMPB-Rapa alleviated AKI to a certain 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 hours. In addition, HA-HMPB-Rapa had the best therapeutic effect. Compared with the model group, the BUN and CRE levels decreased by 70.2% and 79.3%, respectively, which were close to normal levels. As shown Fig.21As shown in C, 24 hours after modeling, the LDH level in the model group was 1.4 times that in the control group. A single administration of HA-HMPB-Rapa can effectively reduce the LDH level of AKI rats to the normal range, indicating that HA-HMPB-Rapa can maintain the integrity of the cell membrane of kidney cells and prevent the leakage of intracellular LDH.

[0144] (4) Histopathological analysis results Fig.21 As shown, obvious pathological changes were observed in the kidney sections in the model group, including tubular epithelial cell shedding, tubular dilatation, cavitation, and the formation of intratubular protein casts and cell casts. In addition, abnormal phenomena such as glomerular atrophy and cracking were observed in the model group. In contrast, after treatment with HA-HMPB and HMPB-Rapa, the kidneys recovered to a certain extent, as shown by reduced vacuolar degeneration and glomerular cracking. In addition, only mild tubular damage (very little vacuolar degeneration) was found in the kidneys treated with HA-HMPB-Rapa, indicating that HA-HMPB-Rapa can effectively protect the kidneys from histopathological changes caused by I / R.

[0145] (5) In vivo antioxidant performance evaluation results Fig. 22 As shown, significantly higher ROS levels were observed in the model group than in the other groups (ROS combined with DHE dye to show a large amount of red fluorescence). HA-HMPB showed antioxidant activity, and the DHE fluorescence intensity in the HA-HMPB group decreased by 67.8% compared with the model group, which was attributed to the accumulation of antioxidant nanocore HMPB in damaged kidneys. More importantly, a single dose of HA-HMPB-Rapa significantly cleared ROS in damaged kidneys, with only slight red fluorescence in the field of view, and the fluorescence intensity was only 15.5% of that in the model group. Fig.23 As shown in A, after HA-HMPB-Rapa treatment, the SOD level in the damaged kidneys recovered 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 vs. model group: 112.8 U / mg prot). Fig.23 As shown in B, the large amount of ROS produced by I / R-induced AKI caused a significant decrease in the GSH content in the model group (401.1 μg / g tissue), which was more than 50% lower than the normal value (1054 μg / g tissue). HMPB-Rapa is more effective than free Rapa in restoring the GSH content in damaged kidneys, indicating that under the protection of HMPB, Rapa is able to achieve more kidney accumulation and promote mitochondrial autophagy to clear damaged mitochondria. In addition, after HA-HMPB-Rapa treatment, the GSH content returned to near normal levels, with no statistical difference from the control group and the sham treatment group. Fig.23As shown in Figure C, the MDA content in the model group was 2.3 times that in the control group, indicating that I / R-induced oxidative stress led to a significant upregulation of lipid peroxidation levels in renal cells. In contrast, after a single administration of HA-HMPB-Rapa, the MDA content decreased by 47.6% compared with the model group, close to the normal value.

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

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

[0148] (8) Western blot analysis results Fig.26 As shown in A, after treatment with HMPB-Rapa or HA-HMPB-Rapa, the expression of PINK-1 in the kidney was 1.5 and 1.8 times higher than that in the model group, respectively, indicating that both agents can upregulate the expression of PINK-1, thereby promoting the recognition of damaged mitochondria and initiating the subsequent mitophagy process. Fig.26 As shown in B, upregulation of Parkin expression was also observed in the HMPB-Rapa and HA-HMPB-Rapa groups. These results suggest that when PINK1 accumulates on the outer membrane of damaged mitochondria, Parkin is recruited there, thus preparing for subsequent mitophagy. Fig.26 As shown in C and D, treatment with HA-HMPB-Rapa resulted in an increase in the LC3 I / LC3 II ratio and a downregulation of P62 expression levels, two established biomarkers of autophagy activation. Previous studies have shown that increased levels of ROS can hinder the degradation of P62, thereby hindering the autophagic process. Therefore, the multi-species enzyme activity of the nanocore endows HA-HMPB with the ability to scavenge ROS, thereby enabling it to promote the orderly progress of autophagy. The results of this study indicate that HA-HMPB-Rapa can effectively regulate the expression of key proteins in the mitophagy signaling pathway, ensuring the timely and effective removal of damaged mitochondria.

[0149] Damaged mitochondria produce excessive ROS, which exceeds the antioxidant capacity of renal cells, leading to the occurrence of oxidative stress. Excessive production of ROS and depolarization of MMPs lead to renal cell apoptosis by regulating apoptosis-related proteins of the Bcl-2 family through transcription or post-transcription. 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. Fig.26 As shown in E and F, the large number of damaged mitochondria in the model group led to overexpression of Bax and decreased expression levels of Bcl-2, indicating that the apoptotic pathway was initiated and activated in damaged renal cells, and the agent treatment was able to reverse the expression of these two key proteins in the signaling pathway. Compared with the model group, HA-HMPB-Rapa significantly reduced Bax expression (reduced by 64.8%) and upregulated Bcl-2 expression (increased by 3.1 times). At the same time, HMPB-Rapa also inhibited the occurrence of apoptosis, with Bax levels reduced by 35.4% and Bcl-2 levels increased by 2.6 times compared with the model group. Studies have shown that an increase in the Bax / Bcl-2 ratio further damages the integrity of the mitochondrial membrane. Once activated, Bax undergoes conformational changes and migrates from the cytoplasm to the outer membrane of the mitochondria, oligomerizing to form pore-like structures, resulting in 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 binds to Apoptoticprotease activating factor-1 (Apaf-1), causing Apaf-1 to undergo conformational changes and form oligomers. The Cyt-c / Apaf-1 complex then recruits caspase-9 precursors to form a large multimeric complex called the apoptosome. Subsequently, the caspase-9 enzyme is catalytically activated, leading to the cleavage and activation of the apoptotic factor Casp-3. Fig.26 As shown in 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 administration of HA-HMPB, HMPB-Rapa or HA-HMPB-Rapa, the changes in the Cyt-c content were reversed, decreasing by 25.2%, 25.2% and 45.8% respectively compared with the model group. These findings are consistent with the semi-quantitative results of Bax and Bcl-2 mentioned above, indicating that the decrease in the Bax / Bcl-2 ratio inhibited the release of Cyt-c. Subsequently, the expression of Cleaved Casp-3, the "executor" of the apoptotic pathway, was studied in each group. As Fig.26As shown in Figure 3, AKI caused the level of Cleaved Casp-3 to increase by 1.6 times, indicating that the apoptosis signaling pathway was highly activated in the model group. In addition, after treatment with HMPB-Rapa or HA-HMPB-Rapa, the expression of Cleaved Casp-3 was inhibited, among which HA-HMPB-Rapa had the best effect. The above results indicate that HA-HMPB-Rapa can promote mitochondrial autophagy to clear damaged mitochondria, thereby inhibiting the release of pro-apoptotic factors, thereby preventing the apoptosis of renal cells. Fig.26 As shown in Figure 1, KIM-1 is specifically and rapidly upregulated when the kidney is injured, making KIM-1 a reliable indicator in the early stages of kidney injury. In addition to its diagnostic role, KIM-1 can also be used as an indicator to assess prognosis. Increased levels of KIM-1 indicate increased injury and are associated with the progression of chronic kidney disease. I / R-induced AKI resulted in a significant increase in the expression of KIM-1 in the model group, which was 1.8 times that of the control group, indicating severe kidney damage. 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 development of AKI to chronic kidney disease.

[0150] In summary, if Fig.26 As shown in Figure 1, HA-HMPB-Rapa can act on the PINK-1 / Parkin signaling pathway, promote mitochondrial autophagy, 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. Timely removal of damaged mitochondria can restore the expression of the anti-apoptotic protein Bcl-2 and inhibit the expression of the 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 apoptotic pathway. Through these interrelated effects, enhancing mitochondrial autophagy can serve as a protective mechanism to maintain cell homeostasis and prevent the activation of cell death pathways, thereby reducing the degree of damage in AKI after I / R.

[0151] (9) In vivo biocompatibility evaluation results such as Fig. 27 As shown, hematological 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. Fig. 27 As shown in A, the two preparations had no significant effect on liver and kidney function in terms of AST, ALT, BUN, and CRE levels. Fig. 27 As shown in B, the H&E results of major organs (heart, liver, spleen, lung, and kidney) showed that the two preparations had minimal effect on the histological morphology of the organs. Fig.28As shown, intravenous injection of these two preparations did not cause significant changes in rat body weight. The above results indicate that HA-HMPB-Rapa has good biocompatibility, laying a foundation for its clinical application.

Claims

1. A targeted hollow mesoporous Prussian blue nanoparticle loaded with rapamycin, characterized in that: The invention comprises hollow mesoporous Prussian blue nanoparticles, wherein rapamycin is loaded in the hollow mesoporous Prussian blue nanoparticles, and the surface of the hollow mesoporous Prussian blue nanoparticles is modified with amino hyaluronic acid.

2. The targeted hollow mesoporous Prussian blue nanoparticles loaded with rapamycin according to claim 1, characterized in that: The hydrodynamic diameter of the targeted hollow mesoporous Prussian blue nanoparticles loaded with rapamycin is 50 to 200 nm, and the surface charge is -10 to -40 mV.

3. The targeted hollow mesoporous Prussian blue nanoparticles loaded with rapamycin according to claim 1, characterized in that: The drug loading amount of the hollow mesoporous Prussian blue nanoparticles loaded with rapamycin is 5-50%.

4. The method for preparing the targeted hollow mesoporous Prussian blue nanoparticles loaded with rapamycin according to any one of claims 1 to 3, characterized in that: The amino hyaluronic acid solution is added to the dispersion of hollow mesoporous Prussian blue nanoparticles loaded with rapamycin, and stirred to obtain targeted hollow mesoporous Prussian blue nanoparticles loaded with rapamycin.

5. The preparation method according to claim 4, characterized in that: The preparation method of the hollow mesoporous Prussian blue nanoparticles is to react bismuth nitrate, potassium ferrocyanide and polyvinyl pyrrolidone in an acidic solution to obtain the hollow mesoporous Prussian blue nanoparticles.

6. The preparation method according to claim 4, characterized in that: The preparation method of the hollow mesoporous Prussian blue nanoparticles loaded with rapamycin is as follows: the hollow mesoporous Prussian blue nanoparticles and rapamycin are mixed in ethanol and stirred to obtain the obtained product.

7. The preparation method according to claim 6, characterized in that: The mass ratio of the hollow mesoporous Prussian blue nanoparticles to rapamycin is 1:1-10.

8. The preparation method according to claim 4, characterized in that: The preparation method of the amino hyaluronic acid is to react hyaluronic acid with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in a mixed solution, and then add ethylenediamine and mix to obtain the amino hyaluronic acid.

9. Use of the targeted hollow mesoporous Prussian blue nanoparticles loaded with rapamycin according to any one of claims 1 to 3 in the preparation of a drug for treating acute kidney injury caused by ischemia-reperfusion.

10. A drug for treating acute kidney injury caused by ischemia-reperfusion, characterized in that: The targeted hollow mesoporous Prussian blue nanoparticles containing rapamycin according to any one of claims 1 to 3.

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