Preparation and application of gold cluster compound for targeted therapy of acute kidney injury disease

Abstract

The invention discloses preparation and application of a gold cluster compound for targeted therapy of acute kidney injury diseases, and relates to the field of nephropathy treatment medicines. The molecular composition (Au) is x (S) y, x is equal to 3-144, y is equal to 1-140, and S is a sulfydryl-containing renal injury target protein binding peptide or a sulfydryl-modified renal injury target protein binding peptide. The method comprises the following steps: mixing a gold salt solution with a sulfydryl-containing ligand to form a mixed solution; under the conditions of certain temperature and pH, the mixed solution system is subjected to a reduction reaction, high-valence Au ions in the gold salt are reduced into Au atoms or monovalent Au ions, and Au forms a gold cluster compound through physical or chemical interaction; the cluster compound disclosed by the invention has remarkable treatment activity in a cis-platinum induced acute kidney injury model, and the acute kidney injury is treated in a targeted manner by inhibiting over transcription and expression of pro-inflammatory cytokines and removing reactive oxygen species (ROS).

Description

Technical Field

[0001] This invention relates to the field of treatment of acute kidney injury (AKI), specifically to a method for preparing a cluster compound containing thiol (Au) and the application of such cluster compounds in kidney diseases such as acute kidney injury. Background Technology

[0002] Renal injuries are damage to kidney tissue caused by various internal and external factors, and their incidence is increasing year by year. Because the kidneys are richly vascularized, damage can easily lead to bleeding and extravasation of urine into the interstitial spaces, causing shock and infection, and triggering critical conditions. Acute kidney injury (AKI) is a common clinical syndrome, formerly known as acute renal failure. It refers to a clinical syndrome caused by a rapid decline in kidney function due to various etiologies. Its main manifestations include oliguria or anuria, swelling, and loss of appetite. In later stages, it easily progresses to chronic kidney disease (CKD) and can even lead to the failure of other organs, further increasing the harm to human health. AKI has a high morbidity and mortality rate, with more than 13 million new cases and 1.7 million deaths worldwide each year, causing a huge clinical and socioeconomic burden. Currently, there is no specific treatment for AKI; the main clinical approach is prevention and supportive strategies, focusing on correcting the underlying cause. Although the pathogenesis of AKI is not fully understood, oxidative stress and inflammation are considered major factors in the occurrence and progression of AKI, and anti-inflammatory and antioxidant therapies have been shown to have good protective effects on the kidneys in AKI patients. However, the reported small molecule drugs have limited therapeutic efficacy due to low bioavailability, poor renal accumulation efficiency, short renal retention time, and high toxicity at high doses. Furthermore, most of these drugs lack the ability to target AKI lesions and pose potential toxic side effects. Therefore, there are currently no specific drugs available for targeted therapy of AKI.

[0003] Several animal models can partially represent the pathogenesis of AKI. Intraperitoneal injection of cisplatin is widely used in AKI research. Cisplatin is a broad-spectrum anticancer drug, but while possessing high anticancer activity, it also produces toxic side effects, particularly nephrotoxicity and damage. Animal models of intraperitoneal cisplatin injection show elevated levels of reactive oxygen species (ROS) in mouse kidney tissue. Pathological examination of the mouse kidneys reveals epithelial edema, unclear structure, and inflammatory cell infiltration in the interstitium. These pathogenic characteristics are highly similar to the pathogenesis of human AKI, making it an ideal model for AKI research. Studies have shown that nanotechnology can improve drug solubility and bioavailability, especially by modifying the kidneys to increase targeting, bringing great hope for AKI treatment. Gold clusters are smaller than the glomerular filtration barrier (typically less than 2 nm), allowing them to freely pass through the glomerulus and directly reach the diseased kidney cells, enabling passive targeting of AKI lesions. Due to their unique structure, gold clusters exhibit antioxidant nanozyme activity, effectively scavenging excessive reactive oxygen species (ROS) both in vivo and in vitro. Furthermore, gold clusters bound to peptides can be actively targeted in vivo through the design of surface-targeting peptides, thereby synergizing with passive targeting caused by the size effect and improving the efficiency of AKI renal targeted therapy. However, there are currently no reports on the synthesis methods of gold-linked peptide complexes that bind to renal injury target proteins and their application in the targeted therapy of renal injury diseases. Summary of the Invention

[0004] This invention provides a gold (Au) cluster compound, the active core of which is an Au cluster. This compound exhibits good therapeutic activity in a cisplatin-induced AKI model and also possesses anti-inflammatory and antioxidant activities, thereby enabling rapid and precise treatment of kidney injury.

[0005] This invention relates to the preparation of a cluster compound formed by multiple gold atoms and a thiol-containing ligand, and its application in the preparation of drugs targeting kidney injury, specifically in the treatment of kidney diseases. The invention involves mixing a gold salt solution with a thiol-containing ligand at a specific temperature, followed by the addition of a reducing agent. The high-valence gold is reduced to gold atoms and low-valence gold ions, forming a cluster compound through physical and / or chemical interactions. This invention can be used to treat kidney diseases such as acute kidney injury, chronic kidney injury, and renal failure. The mechanism of action of this cluster compound is related to the regulation of cytokine expression.

[0006] A gold cluster compound for targeted therapy of kidney injury, characterized in that the molecular composition of the cluster compound is (Au)x(S)y, x = 3 to 144, y = 1 to 140, wherein S is a kidney injury target protein binding peptide containing thiol or a kidney injury target protein binding peptide modified with added thiol, and Au reacts with the thiol group in the polypeptide to form a complex.

[0007] The method for preparing the gold cluster is characterized by comprising the following steps: mixing a gold salt solution with a thiol-containing ligand to form a mixed solution; adding a reducing agent under certain temperature and pH conditions to cause a reduction reaction in the mixed solution system; and forming the (Au)x(S)y cluster by reacting with the thiol group of the ligand.

[0008] The gold salt is a trivalent gold compound, such as chloroauric acid, wherein the trivalent gold is reduced to gold atoms and / or monovalent gold.

[0009] The sulfhydryl-containing ligand is a sulfhydryl-containing kidney injury target protein binding peptide or a sulfhydryl-modified kidney injury target protein binding peptide, which can be a natural protein or a synthetic protein, preferably the kidney injury target protein binding peptide TSP (purity 95.0%).

[0010] The reducing agent is sodium borohydride (NaBH4), sodium hydroxide (NaOH), vitamin C, carbon monoxide (CO), or trisodium citrate dihydrate, etc., and the preferred agent is selected based on the actual synthesis.

[0011] In the mixed solution, the concentration of the thiol-containing ligand is 0.1 μM-10 M, and the concentration of the gold salt is 0.01 M-10 M. The preferred molar ratio of ligand to gold salt is 1:0.5 to 1:5, which can be optimized based on actual synthesis.

[0012] The mixture undergoes a reduction reaction at a temperature of 20–80°C and a pH condition that is weakly acidic, neutral, or alkaline, which can be adjusted and optimized as needed.

[0013] The mixture undergoes a reduction reaction and is stirred at 20–80°C in the dark for 1–10 hours. The solution color changes from colorless to light yellow, and then changes color again. AuS clusters of different molecular weights are obtained by purification methods such as ultrafiltration and dialysis. The hydrated particle size of the AuS clusters is generally less than 5 nm.

[0014] The gold cluster compound is used to prepare drugs for treating acute kidney injury. The kidney diseases mentioned include, but are not limited to, acute kidney injury, chronic kidney injury, and renal failure, among other kidney diseases.

[0015] The mechanism of action of the gold cluster compound includes, but is not limited to, inhibiting pro-inflammatory factors and reducing reactive oxygen species (ROS).

[0016] The cluster compounds of this invention exhibit significant therapeutic activity in a cisplatin-induced acute kidney injury model. They effectively scavenge ROS levels induced by H2O2, cisplatin, or bacterial lipopolysaccharide (LPS), protecting cells from subsequent apoptosis. Furthermore, they effectively inhibit the expression of inflammatory factors such as IL-6 and IL-1β induced by LPS or cisplatin. The ligand-gold cluster compounds described in this invention offer numerous advantages for the treatment of kidney diseases, exhibiting excellent biocompatibility and biosafety. Long-term use does not result in significant toxic side effects, and they can accumulate more extensively in the kidneys, thus improving treatment efficiency. Attached Figure Description

[0017] Figure 1a The UV-Vis absorption spectra of TSP and TSPAuNCs in Example 1 of this invention are shown below.

[0018] Figure 1b The fluorescence spectrum of TSPAuNCs in Example 1 of this invention is shown below. EX represents the excitation spectrum and EM represents the emission spectrum.

[0019] Figure 1c This is a hydration particle size diagram of TSPAuNCs in Example 1 of the present invention;

[0020] Figure 1d This is the MALDI diagram of TSPAuNCs in Embodiment 1 of the present invention;

[0021] Figure 2a This is a graph showing the effect of Example 1 of the present invention on the viability of HEK293T cells;

[0022] Figure 2b This is a graph showing the effect of Example 1 of the present invention on the viability of HEK293T cells stimulated with cisplatin;

[0023] Figure 3a This is a biological distribution diagram of Example 1 of the present invention in mice;

[0024] Figure 4a This is a diagram showing the effect of Example 1 of the present invention on the reactive oxygen species level in kidney tissue sections in a mouse model of cisplatin-induced acute kidney injury;

[0025] Figure 4b The diagram shows the effect of adding 100 μM TSPAuNCs in Example 1 of this invention on inflammatory factors in a mouse model of cisplatin-induced acute kidney injury.

[0026] Figure 4c Representative histopathological sections and pathological scores of mouse kidneys stained with hematoxylin and eosin in the AKI model. Detailed Implementation

[0027] Typical embodiments embodying the features and advantages of the present invention will be described in detail in the following description. It should be understood that the present invention can have various variations in different embodiments without departing from the scope of the present invention, and the descriptions and illustrations herein are for illustrative purposes only and not intended to limit the present invention.

[0028] This invention provides a method for preparing a gold-ligand cluster compound and its application in acute kidney injury. Essentially, it involves the reaction of gold with thiol groups in small peptides to form gold-sulfur bonds. The method includes providing a gold salt solution, mixing the gold salt solution with a ligand solution, and subjecting the mixture to a reduction reaction under specific temperature, pH, and reducing agent conditions. This reduces the high-valence gold in the gold salt to gold atoms or low-valence gold ions, with the thiol groups provided by the ligand.

[0029] Preferably, a weak base can be added to the mixture to provide a weakly alkaline environment, thereby giving the reducing agent reducing properties and thus reducing gold ions.

[0030] The gold salt solution used in this invention is a trivalent gold salt solution, such as chloroauric acid solution. During the preparation process, trivalent gold is reduced to gold atoms or monovalent gold ions.

[0031] The ligands selected in this invention are proteins containing thiol groups, which can be natural proteins or artificially synthesized proteins, preferably the kidney injury target protein binding peptide TSP (purity 95.0%, Hubei Qiangyao Biotechnology Co., Ltd.).

[0032] The method for preparing TSPAuNCs clusters according to an embodiment of the present invention includes:

[0033] The gold salt solution was mixed with the glutathione solution containing thiol groups and stirred for 5-10 minutes to ensure full contact between the gold salt and the ligand, forming a mixture. A reducing agent was added to the mixture to induce a reduction reaction, the pH was adjusted, and the reaction was carried out in the dark to finally obtain a gold-peptide cluster.

[0034] Furthermore, the above-mentioned clusters can be purified by using a high-speed centrifuge to remove large particles, followed by purification using an ultrafiltration tube to remove unreacted alkali and unreacted gold salts, and the pH can be adjusted to concentrate for later use.

[0035] The molar ratio of gold salt to thiol ligand is 1:0.5-1:5, preferably 1:1. The reducing agent used can be sodium borohydride (NaBH4), sodium hydroxide (NaOH), vitamin C, etc., preferably trisodium citrate. The concentration of gold salt can be 0.01 μM to 10 M, for example, a 25 mM chloroauric acid solution, mixed with a thiol peptide with a concentration of 0.1 μM to 10 M, for example, 1 μM, at an optimal molar ratio (e.g., 1:1).

[0036] The preparation method of the present invention produces clusters with a hydrated particle size of 1-5 nm. Each Au-peptide cluster has a precise molecular composition (Au)x(S)y, x = 3-144, y = 1-140, and exhibits photofluorescence characteristics.

[0037] The cluster compound prepared by the method of the present invention has scavenging reactive oxygen species and anti-inflammatory activity, and can treat kidney diseases caused by oxidative stress and inflammation.

[0038] The method for preparing TSPAuNCs clusters of the present invention uses the thiol-containing kidney injury target protein binding peptide TSP as a ligand and utilizes the affinity of gold for thiol groups to synthesize Au-peptide conjugates. The one-step reduction synthesis method is simple, and the obtained clusters have high stability, making them particularly suitable for treating excessive inflammatory responses.

[0039] In this invention, a thiol-containing kidney injury target protein-binding peptide is selected as a ligand to directly add to Au salt, forming a thiol-Au conjugate. Furthermore, when preparing this type of cluster compound, its biocompatibility, water solubility, chemical stability, size, and surface properties can be independently designed and modified to meet the requirements of specific biomedical applications.

[0040] The gold cluster compound of the present invention has many advantages for the treatment of kidney injury: it has broad-spectrum anti-inflammatory activity; it has good ROS scavenging ability; it has low toxicity and side effects; it can accumulate in kidney tissue, greatly enhancing the treatment efficiency, etc.

[0041] The application of the gold-binding peptide clusters to target proteins of kidney injury is for the treatment of kidney diseases. These kidney diseases include, but are not limited to, acute kidney injury, chronic kidney injury, and kidney failure.

[0042] The TSPAuNCs cluster compound, its preparation method, and its applications are further described below with reference to specific embodiments. In these embodiments, the gold salt used is trivalent chloroauric acid; the functional protein is a kidney injury target protein-binding peptide. The UV-Vis absorption and fluorescence properties of the synthesized TSPAuNCs were characterized using UV and fluorescence spectroscopy. The in vitro protective effect and mechanism of TSPAuNCs were studied using HEK293T kidney cells, and the in vivo effects of the TSPAuNCs cluster compound were evaluated using an acute kidney injury mouse model, demonstrating the feasibility of using this cluster compound for kidney diseases induced by oxidative stress and inflammation.

[0043] Example 1: Preparation and Characterization of TSPAuNCs Clusters

[0044] 1) At a certain temperature (preferably 55℃ in this example), 0.005g of kidney injury target protein binding peptide TSP powder is dissolved in 25.7mL of ultrapure water, and a stir bar is added and stirred on a magnetic stirrer.

[0045] 2) Add 102 μL of chloroauric acid solution (25 mM) dropwise to the above solution and stir for 2 minutes;

[0046] 3) Immediately add 500 μL of NaOH (0.5 M) solution dropwise to adjust the pH of the solution to alkaline, and continue stirring for 6 hours;

[0047] The stir bar was removed using a magnetic rod, yielding gold clusters emitting orange fluorescence. Free metal ions and unreacted ligands were removed using an ultrafiltration tube with a molecular weight cutoff of 3000 Da. The synthesized clusters appeared brownish-yellow under visible light and red under ultraviolet light.

[0048] like Figure 1a As shown, TSP exhibits a strong absorption peak at 293 nm, while the cluster compound shows no absorption peak around 290 nm, indicating that TSP has undergone a reaction. Figure 1b As shown, the fluorescence spectrum of this cluster compound indicates that the optimal excitation wavelength is 348 nm and the optimal emission wavelength is 698 nm. Figure 1c The hydrated particle size of Example 1 of the present invention is 2.7 nm. Figure 1d The MALDI diagram of this invention shows that the cluster compound has approximately 26 gold atoms.

[0049] The tests in the following embodiments all used the TSPAuNCs clusters obtained in Example 1.

[0050] Example 2: Detection of cytotoxicity of TSPAuNCs on normal HEK293T cells and cisplatin-stimulated HEK293T cells at the cellular level.

[0051] Normal HEK293T cells and cisplatin-stimulated HEK293T cells were divided into 5 × 10⁶ cells per well. 3 Cells were seeded at a density of 100 μM into 96-well plates and incubated at 37°C for 24 hours. Then, the cells were treated with 5, 20, 50, and 100 μM gold clusters for 24 hours each. Next, 10 μL of CCK-8 reagent diluted with 100 μL of culture medium was added to each well, and the cells were incubated for 60 minutes; the absorbance at 450 nm was measured using a microplate reader.

[0052] Figure 2a Figure 1 shows the effect of this invention on the viability of normal HEK293T cells. Figure 2bThe diagram illustrates the effect of this invention on the viability of HEK293T cells stimulated with cisplatin. The schematic results show that even at a concentration of 100 μM, this gold cluster compound does not cause any significant cytotoxicity to normal HEK293T cells or HEK293T cells stimulated with cisplatin.

[0053] Example 3: Biodistribution of TSPAuNCs in mice

[0054] To clarify the kidney targeting of Example 1 of the present invention, normal healthy mice were used as controls, and an acute kidney injury model induced by cisplatin (Beijing Honghu United Chemical Products Co., Ltd.) was used. This model was established by intraperitoneal injection of Cisplatin solution.

[0055] The model group was stimulated with intraperitoneal injection of Cisplatin (10 mg / kg), followed by intraperitoneal injection of TSPAuNCs (10 mg / kg) 2 hours later. The control group received only TSPAuNCs without Cisplatin stimulation. Mice were weighed daily during the modeling period to monitor weight changes. Mice were sacrificed 72 hours after Cisplatin stimulation, and samples from major organs (heart, liver, spleen, lungs, and kidneys) were taken and weighed.

[0056] Organs (heart, liver, spleen, lung, and kidney) from mice were collected, and the Au content in the tissues was detected using inductively coupled plasma mass spectrometry (ICP-MS). The results showed that ( Figure 3a Compared with the Control+Cisplatin group, the same dose of TSPAuNCs significantly increased kidney accumulation in AKI mice, indicating that TSPAuNCs can more effectively target the kidneys.

[0057] Example 4: TSPAuNCs inhibit Cisplatin-induced acute kidney injury in mice at the animal level.

[0058] To clarify the in vivo relevance of the protective activity of Example 1 of the present invention, a Cisplatin-induced acute kidney injury mouse model was also used, with normal mice as normal controls. Mice were sacrificed 72 hours after Cisplatin stimulation, and kidney tissue was obtained.

[0059] We first assessed the levels of reactive oxygen species (ROS) in the kidneys after Cisplatin stimulation. Figure 4aAs shown, compared with the normal group, the ROS level in the kidney tissue of mice stimulated by Cisplatin was significantly increased, indicating that the mice had renal function impairment. Treatment with TSPAuNCs significantly reduced ROS levels, and statistical results showed that TSPAuNCs had a significant therapeutic effect. In addition, kidney tissue homogenate was obtained by grinding kidney tissue, and enzyme-linked immunosorbent assay (ELISA) analysis revealed that injection of 100 μT TSPAuNCs significantly reduced the increase in Cisplatin-induced TNF-α and IL-6 in the kidneys of mice. Figure 4b This indicates that TSPAuNCs can inhibit the release of Cisplatin-induced pro-inflammatory factors. Histopathological scoring in ear sections stained with hematoxylin and eosin (H&E) confirmed the protective effect of the cluster compound against acute kidney injury. Figure 4c The study observed that the Cisplatin-induced model group showed significant interstitial inflammatory cell infiltration in kidney tissue sections, along with eosinophilic degeneration and shedding of renal tubular epithelial cells and luminal dilation, resulting in severe tubular damage. In contrast, the treatment group showed no significant damage in its kidney tissue sections. TSPAuNCs can effectively reduce inflammatory cell infiltration, thereby protecting the kidneys and demonstrating a good therapeutic effect on acute kidney injury. These data suggest that TSPAuNCs are a potential candidate drug for the treatment of acute kidney injury.

Claims

1. A gold cluster compound, characterized in that, Molecular composition (Au)x(S)y, x = 3-144, y = 1-140, wherein S is a thiol-containing renal injury target protein binding peptide or a thiol-modified renal injury target protein binding peptide.

2. The gold cluster complex according to claim 1, characterized in that The hydrated particle size of the cluster is between 1-5 nm.

3. A method of preparing a gold cluster compound according to any one of claims 1-2, characterized in that, The method comprises the following steps: The gold salt solution is mixed with the thiol-containing ligand to form a mixed solution, and a reducing agent is added under certain temperature and pH conditions to cause a reduction reaction in the mixed solution system, and Au is combined with the thiol of the ligand to form the (Au)x(S)y cluster.

4. The method of claim 3, wherein, The gold salt is a trivalent gold compound, such as chloroauric acid, wherein the trivalent gold is reduced to gold atoms or / and monovalent gold.

5. The method of claim 3, wherein, The thiol-containing ligand is a thiol-containing renal injury target protein binding peptide or a thiol-modified renal injury target protein binding peptide, which is a natural protein or an artificially synthesized protein, and is preferably a renal injury target protein binding peptide TSP (purity 95.0%).

6. The method of claim 3, wherein, The reaction temperature is 20-80°C, and the pH condition is weakly acidic, neutral or alkaline.

7. The method of claim 3, wherein, The reducing agent is selected from sodium borohydride (NaBH4), sodium hydroxide (NaOH), vitamin C, carbon monoxide (CO) or trisodium citrate dihydrate.

8. The method of claim 3, wherein, In the mixed solution, the concentration of the thiol-containing ligand molecules is 0.1 μM-10M, and the concentration of the Au compound is 0.01M-10M, and preferably the molar ratio of the thiol-containing ligand molecules to the gold salt is 1:0.5-1:

5.

9. The method of claim 3, wherein, The mixed solution is subjected to a reduction reaction, and is stirred at 20-80°C for 1-10 hours. After the color of the solution changes from colorless to light yellow, the color changes again. Different molecular weight TSP AuNCs clusters are obtained by purification methods such as ultrafiltration and dialysis.

10. The gold cluster of any one of claims 1-2 is used for preparing a drug for treating acute kidney injury, wherein the kidney diseases include but are not limited to acute kidney injury, chronic kidney injury, renal failure and the like.

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