A mitochondrial-targeting copper nanocluster based on natural polyphenols and its preparation and application

Abstract

This invention provides a mitochondrial-targeting copper nanocluster, comprising copper nanoclusters and polyphenol ligand molecules attached to the surface of the copper nanoclusters, with a size between 1-20 nm. The polyphenol ligand molecules are selected from any one or a combination of two or more of gallic acid, epigallocatechin gallate, epicatechin, chlorogenic acid, or tannic acid. This invention's mitochondrial-targeting copper nanocluster uses a polyphenol structure as the mitochondrial-targeting functional group and the copper nanoclusters as an antioxidant, effectively targeting and scavenging mitochondrial overload reactive oxygen species, inhibiting oxidative stress and apoptosis in renal tubular epithelial cells, and exhibiting good therapeutic effects for acute kidney injury.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine, and in particular to a copper nanocluster based on natural polyphenols, which has mitochondrial targeting and is extremely small in size, its preparation method, and its application in the treatment of acute kidney injury. Background Technology

[0002] Acute kidney injury (AKI) is a serious clinical condition that threatens patients' lives, with high morbidity and mortality. The pathological mechanisms of AKI are complex, involving multiple cellular and molecular pathways. Currently, clinical treatment for AKI mainly relies on symptomatic supportive measures, such as maintaining electrolyte balance and renal replacement therapy. However, these methods can only alleviate symptoms and cannot fundamentally prevent or reverse the disease. Existing research indicates that mitochondrial dysfunction and excessive production of reactive oxygen species (ROS) play a crucial role in the occurrence and development of AKI. As the center of cellular energy metabolism, mitochondrial dysfunction leads to insufficient cellular energy supply, further exacerbating cellular damage. Simultaneously, excessive ROS production causes oxidative stress, resulting in damage to cellular structure and function.

[0003] The role of antioxidants in the treatment of acute kidney injury (AKI) has been extensively studied. Existing antioxidants are mainly classified into small-molecule antioxidants (such as N-acetylcysteine, NAC), artificial nanozymes (such as cerium nanoparticles), and natural antioxidant enzymes (such as superoxide dismutase (SOD) and catalase (CAT)). Among these, small-molecule antioxidants, due to their lack of specific targeting ability to damaged renal tubular epithelial cells or mitochondria, are difficult to efficiently concentrate in the kidneys or mitochondria, thus limiting the actual therapeutic effect of NAC. Natural antioxidant enzymes, due to their excellent ROS scavenging efficiency and biocompatibility, have been widely studied for the treatment of various inflammatory diseases. However, these antioxidant enzymes are difficult to maintain stability in the physiological environment and have low efficiency during endocytosis, making it difficult to reach the sites of ROS generation.

[0004] Therefore, it is necessary to develop a nanomedicine that can target mitochondria and has good antioxidant properties and high stability, so that it can stably reach the site of ROS generation and clear ROS during endocytosis. Summary of the Invention

[0005] The purpose of this invention is to provide a copper nanocluster based on natural polyphenols, which has mitochondrial targeting capability and is extremely small in size, and a method for preparing the same. The copper nanocluster, modified with polyphenolic compounds, has good mitochondrial targeting ability and can efficiently remove ROS, thus exhibiting excellent therapeutic effects on acute kidney injury.

[0006] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0007] In a first aspect, the present invention provides a method for preparing mitochondrial-targeted copper nanoclusters, comprising:

[0008] 1) Under stirring conditions, slowly add divalent copper aqueous solution to polyphenol aqueous solution, controlling the molar ratio of polyphenol to divalent copper to be 1-2:2-3, to obtain a mixed solution;

[0009] 2) Slowly add alkali solution to the mixed solution obtained in 1), with the molar ratio of the added alkali solution to the polyphenols described in 1) being no less than 1:50. Then continue to react under stirring conditions for 1-4 hours. After the reaction is completed, cool to room temperature, adjust the pH of the solution to weakly alkaline, and remove unreacted polyphenols and copper ions to obtain mitochondrial-targeted copper nanoclusters.

[0010] In the present invention, to further improve the yield of the target product, the polyphenol aqueous solution and the divalent copper aqueous solution described in 1) can be controlled at appropriate concentrations. In a preferred embodiment, the concentration of the polyphenol aqueous solution is controlled at 50-100 mM, and the concentration of the divalent copper aqueous solution is controlled at 100-150 mM; in a more preferred embodiment, the concentration of the polyphenol aqueous solution is controlled at 100 mM, and the concentration of the divalent copper aqueous solution is controlled at 150 mM.

[0011] In the scheme described in this invention, 1) the polyphenol aqueous solution can be prepared by dissolving any one or a mixture of two or more of the natural polyphenols such as gallic acid, epigallocatechin gallate, epicatechin, chlorogenic acid or tannic acid in water.

[0012] In the present invention, 1) the divalent copper aqueous solution can be any one or a mixture of two or more of copper nitrate aqueous solution, copper sulfate aqueous solution or copper chloride aqueous solution.

[0013] In a preferred embodiment of the present invention, in step 1), the molar ratio of polyphenols to divalent copper in the resulting mixed solution is controlled to be 1:2-3.

[0014] In the scheme described in this invention, the temperature, stirring speed, and the rate at which divalent copper is added all have a significant impact on the size of the generated copper nanoclusters. In order to obtain copper nanoclusters of the smallest possible size, in the preferred scheme, the temperature when obtaining the mixed solution in the first step is controlled at 60-90℃, the stirring speed in the second step is controlled at 500-900 rpm, and the slow addition in the third step is dropwise addition; in a more preferred scheme, the temperature when obtaining the mixed solution in the first step is controlled at 80℃, the stirring speed in the second step is controlled at 700 rpm, and the slow addition in the third step is dropwise addition at a rate of 1 drop / second.

[0015] In the scheme described in this invention, the addition of the alkaline solution in step 2) is to promote the deprotonation and complexation of polyphenols with metal ions. The added alkaline solution can be any existing alkaline solution, preferably NaOH solution or KOH solution; more preferably NaOH solution; and most preferably a 1M NaOH solution.

[0016] In the present invention, to avoid excessive oxidation of the polyphenols, it is necessary to control the rate at which the alkali solution is added to the mixed solution obtained in step 1) and the stirring speed in step 2). Preferably, the alkali solution in step 2) is added dropwise, and the stirring speed is controlled at 500-900 rpm, more preferably at 700 rpm.

[0017] In the scheme described in this invention, adjusting the pH of the solution to a weakly alkaline state as described in step 2) is to promote the deprotonation of the polyphenols modified on the surface of the prepared copper nanoclusters, increase their negative potential, and thus enhance the stability of the ultrasmall nanoclusters in various physiological solvents. In a preferred embodiment of this invention, adjusting the pH of the solution to a weakly alkaline state as described in step 2) means adjusting it to a final pH of 8; more preferably, this is achieved by adding an alkaline solution to the solution cooled to room temperature after the reaction; and even more preferably, the alkaline solution is a 1M NaOH aqueous solution.

[0018] In the present invention, the removal of unreacted polyphenols and copper ions described in 2) can be achieved by various existing means. In a preferred embodiment, unreacted polyphenols and copper ions are removed by filtration and dialysis. In a more preferred embodiment, the filtration is performed using a filter membrane with a pore size of 0.22 micrometers, the dialysis has a molecular weight cutoff of 8000-14000 Daltons, and the dialysis time is 1-3 days.

[0019] In a preferred embodiment of the present invention, the solution obtained by removing unreacted polyphenols and copper ions in step 2) is freeze-dried to obtain mitochondrial-targeted copper nanocluster powder.

[0020] In a second aspect, the present invention provides mitochondrial-targeting copper nanoclusters prepared by the method of the first aspect of the present invention, the structure of which includes copper nanoclusters and polyphenol ligand molecules attached to the surface of the copper nanoclusters, wherein the polyphenol ligand molecules are selected from any one or a combination of two or more of gallic acid, epigallocatechin gallate, epicatechin, chlorogenic acid or tannic acid.

[0021] The mitochondrial-targeting copper nanoclusters described in this invention have a size between 1 and 20 nm. The modification with natural polyphenol ligand molecules gives them excellent mitochondrial targeting ability and makes them stable in various solvents.

[0022] Thirdly, the present invention also provides the application of the mitochondrial-targeted copper nanoclusters described in the second aspect of the present invention in the in vivo treatment of acute kidney injury (AKI).

[0023] In the applications described in this invention, the mitochondrial-targeted copper nanoclusters can be prepared into various formulations such as lyophilized powder or injection solution.

[0024] The lyophilized powder or injection solution can be concentrated in the kidneys via intravenous administration, and after uptake by renal tubular endothelial cells, it targets the mitochondria for precise removal of mitochondrial ROS. Simultaneously, the copper nanoclusters of this invention exhibit good biocompatibility and safety.

[0025] This invention develops a novel mitochondrial-targeting group—polyphenol—and uses polyphenol modification to prepare ultra-small antioxidant nanoclusters. These nanoclusters can be effectively enriched in the kidneys and taken up by renal tubular epithelial cells. After internalization, these nanoclusters can effectively target mitochondria and scavenge mitochondrial ROS. Compared with small molecule antioxidants and antioxidant enzymes, the ultra-small antioxidant nanoclusters provided by this invention have better antioxidant properties, higher stability, and excellent biodistribution and bioavailability. This invention overcomes the limitations of existing antioxidants and nanomedicines, providing a therapeutic approach with highly efficient mitochondrial targeting and antioxidant capacity, and has significant clinical application value.

[0026] By means of the above solution, the present invention has the following advantages:

[0027] The raw materials used in this invention include copper salts and natural polyphenols, which are widely found in nature. These components are affordable and readily available, significantly reducing production costs. Furthermore, the introduction of natural polyphenols not only enhances the stability of the ultrasmall nanoclusters but also, due to their negatively charged properties, avoids the risk of excessive electrostatic interaction and disruption of mitochondrial membrane homeostasis compared to commonly used cationic targeting agents. The copper nanoclusters in this invention are synthesized using copper, an essential element in living organisms. This not only improves their biocompatibility but also, due to their ultrasmall size, allows them to be metabolized by the kidneys, avoiding the risk of heavy metal accumulation. This provides a safe and bio-friendly novel therapeutic material for the treatment of AKI (Acute Kidney Disease). Attached Figure Description

[0028] Figure 1 This is a schematic diagram illustrating the synthesis of the polyphenol copper nanoclusters prepared in this invention.

[0029] Figure 2A , Figure 2B This is a high-resolution transmission electron microscope (HRTEM) image of the polyphenol copper nanoclusters prepared in this invention.

[0030] Figure 3This is a dynamic light scattering hydration particle size distribution diagram of the polyphenol copper nanoclusters prepared in this invention in different solvents.

[0031] Figure 4 The Fourier transform infrared spectra of the polyphenol copper nanoclusters and their raw materials prepared in this invention are shown.

[0032] Figure 5 This is the X-ray photoelectron spectrum of the polyphenol copper nanoclusters prepared in this invention.

[0033] Figure 6 This is the high-resolution Cu 2p X-ray photoelectron spectroscopy of the polyphenol copper nanoclusters prepared in this invention.

[0034] Figure 7 This is the X-ray excited Auger electron spectroscopy of the polyphenol copper nanoclusters prepared in this invention.

[0035] Figure 8 This demonstrates the scavenging ability of the polyphenol copper nanoclusters prepared in Example 1 against major reactive oxygen species: a: scavenging rate of superoxide anions, b: scavenging rate of hydrogen peroxide, and c: scavenging rate of hydroxyl radicals.

[0036] Figure 9 The images are in vitro fluorescence images of the polyphenol copper nanoclusters prepared in Example 1, injected into the tail vein of mice. a: In vitro fluorescence images of the main tissues of healthy or AKI mice after tail vein injection of ICG-labeled GA-CuNCs at a specified time point. b: The fluorescence intensity quantification histogram corresponding to a).

[0037] Figure 10 This is a confocal imaging image of cells co-localized with mitochondria and polyphenol copper nanoclusters prepared in Example 1.

[0038] Figure 11 This study demonstrated the levels of CRE and BUN in the serum of AKI mice after tail vein injection of the polyphenol copper nanoclusters prepared in Example 1.

[0039] Figure 12 This study demonstrates the effect of tail vein injection of polyphenol copper nanoclusters prepared in Example 1 on the morphology of kidney mitochondria in mice undergoing ischemia-reperfusion. Detailed Implementation

[0040] The specific embodiments of the present invention will be described in further detail below with reference to the examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0041] This invention provides a method for preparing mitochondrial-targeting copper nanoclusters, specifically comprising the following steps:

[0042] Under stirring, a 100-150 mM divalent copper aqueous solution was slowly added to a 50-100 mM natural polyphenol aqueous solution. An appropriate amount of alkaline solution was added to promote the deprotonation and complexation of metal ions by the polyphenols. Stirring was continued for 1-4 hours, followed by pH adjustment, filtration, dialysis, and lyophilization to obtain ultra-small copper nanoclusters based on natural polyphenols and exhibiting mitochondrial targeting. The volume ratio of the natural polyphenol aqueous solution to the divalent copper aqueous solution was 1:1.

[0043] Furthermore, the concentration of the natural polyphenol aqueous solution is preferably 100 mM, and the concentration of the divalent copper aqueous solution is preferably 150 mM.

[0044] Furthermore, the alkaline solution is NaOH or KOH with a concentration of 1M, and is added slowly dropwise to the stirred mixed solution to avoid excessive oxidation of the polyphenolic compounds; in some specific embodiments, the alkaline solution is preferably NaOH with a volume of 2 mL.

[0045] This invention further reveals that, without the addition of additional stabilizers, the reaction stirring speed and hydrothermal temperature control are key parameters for preventing the growth of nanoclusters. In some preferred embodiments, the stirring speed is 500-900 rpm and the temperature is 60-90°C; the addition rate of the divalent copper aqueous solution is 1 drop / s. In some specific embodiments, the stirring speed is preferably 700 rpm and the temperature is preferably 80°C.

[0046] This study further discovered that adjusting the pH of the prepared nanoclusters to a slightly alkaline state can promote the deprotonation of the polyphenols modified on the cluster surface, increase their negative potential, and thus enhance the stability of the ultrasmall nanoclusters in various physiological solvents. In some specific embodiments, a 1M NaOH aqueous solution was selected for pH adjustment, and the final pH was preferably 8.0.

[0047] Furthermore, the filtration uses a filter membrane with a pore size of 0.22 micrometers.

[0048] Furthermore, the molecular weight cutoff for dialysis is 8000-14000 Daltons, and the dialysis time is 1-3 days. The purpose of the dialysis procedure is to remove unreacted polyphenols and copper ions, as well as some oligomers that have not grown into nanoclusters.

[0049] Furthermore, the freeze-drying conditions are as follows: the dialysate is frozen in liquid nitrogen for 10-20 minutes, then immediately removed and placed in a freeze dryer for 3-7 days under conditions of cold trap temperature less than -50°C and vacuum degree less than 10 Pa, finally obtaining mitochondrial-targeted ultra-small nanocluster powder based on natural polyphenols.

[0050] Furthermore, the natural polyphenols may be selected from any one or a combination of two or more of gallic acid, epigallocatechin gallate, epicatechin, chlorogenic acid, or tannic acid.

[0051] Furthermore, the divalent copper solution can be any one or a mixture of two or more of the following: copper nitrate aqueous solution, copper sulfate aqueous solution, or copper chloride aqueous solution.

[0052] The second objective of this invention is to claim protection for an ultrasmall copper nanocluster based on natural polyphenols prepared by the above-described preparation method, comprising copper nanoclusters and polyphenol ligand molecules attached to the surface of the copper nanoclusters, wherein the size of the copper nanoclusters is between 1-20 nm, and the polyphenol ligand molecules are modified to give it excellent mitochondrial targeting ability.

[0053] The polyphenol ligand molecules attached to the surface of copper nanoclusters can be selected from any one or a combination of two or more of gallic acid, epigallocatechin gallate, epicatechin, chlorogenic acid, or tannic acid.

[0054] A third objective of this invention is to provide the application of the ultrasmall copper nanoclusters described herein in the in vivo treatment of AKI.

[0055] Furthermore, in the applications described in this invention, the ultrasmall copper nanoclusters can be prepared into various formulations such as lyophilized powder or injection solution.

[0056] The aforementioned injectable formulation can be administered intravenously, where it accumulates in the kidneys. After being taken up by renal tubular endothelial cells, it targets and precisely removes mitochondrial ROS. Simultaneously, the nanoclusters exhibit good biocompatibility and safety.

[0057] Based on the description of the specific embodiments above, the present invention also provides the following preferred embodiments, which are described in detail below with reference to the accompanying drawings.

[0058] Example 1. Preparation of gallic acid-modified copper nanoclusters

[0059] This embodiment provides a gallic acid-based copper nanocluster with mitochondrial targeting and antioxidant functions. See [link to previous document]. Figure 1 As shown, the preparation steps are as follows:

[0060] (1) Dissolve gallic acid in 50 mL of deionized water and sonicate for 30 minutes to obtain an aqueous solution with a concentration of 100 mM;

[0061] (2) Dissolve copper chloride in 50 mL of deionized water to obtain a copper solution with a concentration of 150 mM;

[0062] (3) Add the copper solution slowly to the gallic acid solution at a rate of 1 drop / second, while stirring at 700 rpm. After mixing thoroughly, add 2 mL of 1M NaOH alkaline solution to promote partial deprotonation of the polyphenols. Continue the reaction at 80℃ for 2 hours, then return to room temperature. Adjust the pH of the reaction solution to 8.0 using 1M NaOH aqueous solution.

[0063] (4) After the reaction was completed, the mixture was filtered through a filter membrane with a pore size of 0.22 micrometers to obtain a suspension of gallic acid-modified copper nanoclusters;

[0064] (5) The copper nanocluster suspension was placed into a dialysis bag with a molecular weight cutoff of 3500 Daltons and dialyzed for 3 days.

[0065] (6) The dialysate was frozen in liquid nitrogen for 15 minutes, then removed and freeze-dried for 5 days to finally obtain gallic acid-modified ultrasmall copper nanoclusters GA-CuNC. Figure 1 (The Chinese version uses "GA-Cu" to represent it).

[0066] like Figure 1 As shown, the prepared copper nanoclusters GA-CuNC have a central zero-valent copper atom surrounded by a stable shell composed of monovalent and / or divalent copper complexed with gallic acid ligands.

[0067] The high-resolution transmission electron microscope image of the polyphenol copper nanoclusters GA-CuNC prepared in Example 1 is shown below. Figure 2A , Figure 2B As shown in the figure, the microstructure of a single GA-CuNC is spherical, and the size of the nanoclusters is less than 10 nm, which belongs to ultra-small copper nanoclusters.

[0068] Figure 3 The image shows the dynamic light scattering hydration particle size distribution of the polyphenol copper nanoclusters GA-CuNC prepared in Example 1 dispersed in different physiological solvents (including water, PBS buffer, and DMEM cell culture medium), with a particle size of approximately 10 nm. Furthermore, these copper nanoclusters exhibited similar hydration sizes in the aforementioned different solvents, indicating excellent particle stability in various biologically relevant media.

[0069] Figure 4 The polyphenol copper nanoclusters GA-CuNC prepared in Example 1 ( Figure 4 The Fourier transform infrared (FTIR) spectrum of the sample (represented by the red line and GA-CuNCs) is shown. Spectral analysis reveals the image at approximately 3500 cm⁻¹. -1 The absorption band indicates the presence of phenolic hydroxyl groups. The red shift of the Ar-OH peak may be due to coordination bonding between copper and the phenolic hydroxyl groups. Furthermore, at 1683 cm⁻¹... -1 and 1384cm -1The peaks at 1590 cm⁻¹ correspond to the stretching vibrations of C=O and C─OH in GA, respectively. -1 1540cm -1 and 1500cm -1 The peak at that location is a characteristic absorption peak of the benzene ring.

[0070] Figure 5 The image shows the X-ray photoelectron spectroscopy (XPS) of the polyphenol copper nanoclusters GA-CuNC prepared in Example 1 (the orange line GA-CuNCs represents GA-CuNC from Example 1, and the green line GA represents gallic acid). The XPS scanning spectrum confirmed the coexistence of C1s (283.86 eV), O 1s (530.32 eV), and Cu 2p (932.64 eV). Figure 6 The high-resolution Cu 2p spectrum of the polyphenol copper nanoclusters GA-CuNC prepared in Example 1 is shown. The main peaks of Cu 2p are located at 932.73 and 952.38 eV, with spin orbital splitting at 19.65 eV, attributed to Cu 2p³ / 2 and Cu 2p¹ / ². Other peaks at 940.80, 943.92, and 962.32 eV are attributed to Cu 2+ Satellite peaks. X-ray Auger spectrum of Cu-LMM ( Figure 7 The main peaks correspond to Cu 0 (917.50 eV) and Cu 0, respectively. + (915.50eV) and Cu 2+ The fitting of three peaks (916.70 eV) confirmed the existence of three different valence states in Cu-TA NM. Therefore, copper ions successfully formed a stable chemical crystal structure after reduction and capping stabilization with GA. These results also indicate that GA-CuNC possesses three different valence states (Cu... 0 Cu + and Cu 2+ Furthermore, the presence of these redox pairs endows GA-CuNC with excellent redox capabilities for ROS.

[0071] Figure 8 The polyphenol copper nanoclusters GA-CuNC prepared in Example 1 are effective against typical reactive oxygen species (O2·). - The scavenging capacity of GA-CuNC for the above three ROS (H2O2 and ·OH, represented by parts a, b, and c in the figure, respectively) was tested. The results showed that the scavenging efficiency of GA-CuNC for these three ROS was positively correlated with their concentration.

[0072] Verification of mitochondrial targeting of the polyphenol copper nanoclusters GA-CuNC prepared in Example 1:

[0073] GA-CuNC was stained with Cy5.5 and co-localized using the commercial Mito-Tracker. The results are as follows: Figure 10 As shown in the figure, after 3 hours of co-incubation, the fluorescence signal of Cy5.5-labeled GA-CuNC showed a high degree of overlap with that of mitochondria. This result indicates that GA-CuNC ( Figure 10 The substance (represented by "GA-Cu") can effectively target mitochondria, thereby precisely regulating mitochondrial oxidative stress.

[0074] Example 2. Preparation of tannic acid-modified copper nanoclusters

[0075] This embodiment prepares copper nanoclusters based on tannic acid modification, which have mitochondrial targeting and antioxidant functions. The specific preparation steps are as follows:

[0076] (1) Dissolve tannic acid in 50 mL of deionized water and sonicate for 30 minutes to obtain an aqueous solution with a concentration of 100 mM;

[0077] (2) Dissolve copper chloride in 50 mL of deionized water to obtain a copper solution with a concentration of 150 mM;

[0078] (3) Add the copper solution slowly to the gallic acid solution at a rate of 1 drop / second, while stirring at 700 rpm. After mixing thoroughly, add 2 mL of 1M NaOH alkaline solution to promote partial deprotonation of the polyphenols. Continue the reaction at 80℃ for 2 hours, then return to room temperature. Adjust the pH of the reaction solution to 8.0 using 1M NaOH aqueous solution.

[0079] (4) After the reaction was completed, the mixture was filtered through a filter membrane with a pore size of 0.22 micrometers to obtain a suspension of tannic acid-modified copper nanoclusters.

[0080] (5) The tannic acid-modified copper nanoclusters suspension was placed in a dialysis bag with a molecular weight cutoff of 3500 Daltons and dialyzed for 3 days.

[0081] (6) The dialysate was frozen in liquid nitrogen for 15 minutes and then taken out and freeze-dried for 5 days to finally obtain tannic acid modified ultra-small copper nanoclusters TA-CuNC.

[0082] Example 3. Preparation of copper nanoclusters modified with epigallocatechin gallate

[0083] This embodiment prepares copper nanoclusters modified with epigallocatechin gallate, which have mitochondrial targeting and antioxidant functions. The specific preparation steps are as follows:

[0084] (1) Dissolve epigallocatechin gallate in 50 mL of deionized water and sonicate for 30 minutes to obtain an aqueous solution with a concentration of 100 mM.

[0085] (2) Dissolve copper chloride in 50 mL of deionized water to obtain a copper solution with a concentration of 150 mM;

[0086] (3) Add the copper solution slowly to the gallic acid solution at a rate of 1 drop / second, while stirring at 700 rpm. After mixing thoroughly, add 2 mL of 1M NaOH alkaline solution to promote partial deprotonation of the polyphenols. Continue the reaction at 80℃ for 2 hours, then return to room temperature. Adjust the pH of the reaction solution to 8.0 using 1M NaOH aqueous solution.

[0087] (4) After the reaction was completed, the mixture was filtered through a filter membrane with a pore size of 0.22 micrometers to obtain a suspension of copper nanoclusters modified with epigallocatechin gallate;

[0088] (5) The copper nanoclusters modified with epigallocatechin gallate were placed in a dialysis bag with a molecular weight cutoff of 3500 Daltons and dialyzed for 3 days.

[0089] (6) The dialysate was frozen in liquid nitrogen for 15 minutes and then taken out and freeze-dried for 5 days to finally obtain the ultra-small copper nanoclusters EGCG-CuNC modified with epigallocatechin gallate.

[0090] Example 4. Biodistribution and efficacy tests of GA-CuNC

[0091] The biodistribution of gallic acid-modified copper nanoclusters GA-CuNC obtained in Example 1 of this invention in mice and the in vivo experimental process of treating acute kidney injury in mice include the following steps:

[0092] (1) Biodistribution of GA-CuNC in vivo

[0093] To label GA-CuNC with ICG, GA-CuNC was stirred with DSPE-PEG2000-ICG overnight at room temperature. The solution was then dialyzed against deionized water for 12 hours using a dialysis bag with a molecular weight cutoff of 3500 Da to obtain the labeled drug, denoted as "GA-CuNC@ICG". Finally, GA-CuNC@ICG was lyophilized and stored for future use. In the biodistribution study, 30 C57 mice were randomly divided into healthy and AKI mice. AKI mice were injected with cisplatin to induce the model, and GA-CuNC@ICG (10 mg / kg) was intravenously injected 30 minutes after modeling. Subsequently, the mice were euthanized at selected time points after administration, and major organs were harvested. In vivo fluorescence imaging was performed using the IVIS Lumina imaging system (Xenogen Corporation, Alameda, California, USA). The fluorescence signal of each organ was quantified using ImageJ software. ICG: λem = 808 nm, λex = 900 nm.

[0094] like Figure 9 As shown on the right side of sections a and b, imaging results indicate that GA-CuNC@ICG is mainly distributed in the liver (Li) and kidney (Ki) of healthy mice. The fluorescence signal in the kidney peaks approximately 0.5 hours after injection. Figure 9 As shown on the left side of sections a and b, consistent with imaging results from healthy mice, GA-CuNC@ICG was also primarily enriched in the liver (Li) and kidney (Ki) of cisplatin-induced AKI mice. The results indicate that intravenously administered copper nanoclusters can rapidly concentrate on damaged kidney tissue.

[0095] (2) Animal grouping and experimental protocol for in vivo experiments on the treatment of acute kidney injury

[0096] Twenty 7-week-old male C57 mice were randomly divided into four groups of five mice each after a one-week acclimatization period: sham operation group (Sham), renal ischemia-reperfusion model group (Saline), N-acetylcysteine ​​group (NAC), and polyphenol-modified copper nanoclusters group (GA-Cu). The sham operation group (Sham) received daily intraperitoneal injections of saline. The renal ischemia-reperfusion model group (Saline) underwent a unilateral dorsal incision, blunt dissection of the renal pedicle, and clamping of the pedicle with a micro-arterial hemostatic clamp. After 45 minutes of ischemia, the clamp was removed, the kidney was repositioned, and both dorsal wounds were sutured. Renal perfusion was restored for 24 hours. Two hours after establishing the renal ischemia-reperfusion model, the NAC group received a tail vein injection of 100 mg / kg N-acetylcysteine; two hours after establishing the renal ischemia-reperfusion model, the GA-Cu group received a tail vein injection of 10 mg / kg GA-CuNC.

[0097] Sample collection

[0098] Twenty-four hours after establishing the renal ischemia-reperfusion model in the Saline, NAC, and GA-Cu groups, blood was collected from all mice by enucleation, and the serum was collected by centrifugation and then frozen at -80°C. The kidneys of all mice were removed, fixed with 4% paraformaldehyde, and stored at room temperature.

[0099] Kidney function assessment

[0100] Renal function indicators were assessed by measuring blood urea nitrogen (BUN) and serum creatinine (CRE) concentrations in collected serum samples. BUN and CRE concentrations were measured using a BUN assay kit (Nanjing Jiancheng Biotechnology Institute) and a CRE assay kit (Nanjing Jiancheng Biotechnology Institute), respectively.

[0101] like Figure 11As shown, the renal function indicators (BUN and CRE) of mice in the Saline group were elevated; in contrast, the GA-Cu group, after administration of the polyphenol copper nanoclusters GA-CuNC prepared in Example 1, alleviated acute kidney injury in mice; it is worth mentioning that, compared with the NAC group, the serum BUN and CRE levels in mice in the GA-Cu group were significantly reduced.

[0102] mitochondrial morphology of renal tubular epithelial cells

[0103] Kidney tissues collected from mice in each group were immersed in a transmission electron microscopy (TEM) solution containing 2.5% glutaraldehyde and 4% formaldehyde. The samples were processed in the TEM laboratory at Zhejiang University School of Medicine. The microscopic structure of mitochondria in renal tubular epithelial cells was imaged using a TEM microscope (Shimadzu Corporation, Tokyo, Japan).

[0104] like Figure 12 As shown, the mitochondria in the renal tubular endothelial cells of mice in the Sham group exhibited highly ordered morphology, with rod-shaped structures and clearly visible cristae. In contrast, numerous abnormal mitochondrial changes were observed in the kidneys of mice in the Saline group, including swelling, reduced or absent cristae, and even mitochondrial fragmentation, indicating fatal damage to mitochondrial function in renal tubular endothelial cells under disease conditions. In comparison, the mitochondrial morphology in the kidneys of mice in the GA-Cu group was nearly normal, demonstrating that the application of the polyphenol copper nanoclusters GA-CuNC prepared in Example 1 effectively inhibited mitochondrial swelling and division in renal tubular epithelial cells. Furthermore, the abnormal mitochondrial morphology in the kidneys of mice in the NAC group was not improved, indicating that the therapeutic effect of N-acetylcysteine ​​injected in this group on acute kidney injury was less than that of GA-CuNC injected in the GA-Cu group.

[0105] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing mitochondrial-targeted copper nanoclusters, comprising: 1) Under stirring conditions, slowly add divalent copper aqueous solution to polyphenol aqueous solution, and control the molar ratio of polyphenol to divalent copper to be 1-2:2-3 to obtain a mixed solution; the polyphenol aqueous solution is prepared by dissolving any one or more of gallic acid, epigallocatechin gallate, epicatechin, chlorogenic acid or tannic acid in water. 2) Add alkali solution slowly dropwise to the mixed solution obtained in 1), with the molar ratio of the added alkali solution to the polyphenols described in 1) not less than 1:

50. Then continue to react under stirring at 500-900 rpm for 1-4 h. After the reaction is completed, cool to room temperature, adjust the pH of the solution to 8, and remove unreacted polyphenols and copper ions to obtain mitochondrial-targeted copper nanoclusters.

2. The method as described in claim 1, characterized in that: 1) Control the molar ratio of the polyphenols and divalent copper to be 1:2-3.

3. The method as described in claim 1, characterized in that: 1) The concentration of the polyphenol aqueous solution is controlled at 50-100 mM, and the concentration of the divalent copper aqueous solution is controlled at 100-150 mM.

4. The method as described in claim 1, characterized in that: 1) The concentration of the polyphenol aqueous solution is controlled at 100 mM, and the concentration of the divalent copper aqueous solution is controlled at 150 mM.

5. The method as described in claim 1, characterized in that: 1) The divalent copper aqueous solution is any one or a mixture of two or more of copper nitrate aqueous solution, copper sulfate aqueous solution or copper chloride aqueous solution.

6. The method as described in claim 1, characterized in that: 1) The temperature of the mixed solution is controlled at 60-90℃, 1) The stirring speed is controlled at 500-900rpm, 1) The slow addition is dropwise addition.

7. The method as described in claim 1, characterized in that: 1) The temperature of the mixed solution is controlled at 80℃, 1) The stirring speed is controlled at 700rpm, and 1) The slow addition is at a rate of 1 drop / second.

8. The method as described in claim 1, characterized in that: 2) The alkaline solution is any one or a mixture of two or more of NaOH solution or KOH solution.

9. The method as described in claim 1, characterized in that: 2) The alkaline solution mentioned is a 1 M NaOH solution.

10. The method as described in claim 1, characterized in that: 2) The stirring speed is controlled at 700 rpm.

11. The method as described in claim 1, characterized in that: The adjustment of the solution pH to 8 mentioned in 2) is achieved by adding an alkaline solution to the solution after the reaction has been completed and cooled to room temperature.

12. The method as described in claim 11, characterized in that: The alkaline solution is a 1 M NaOH aqueous solution.

13. The method as described in claim 1, characterized in that: It also includes freeze-drying the solution obtained after removing unreacted polyphenols and copper ions in step 2) to obtain mitochondrial-targeted copper nanocluster powder.

14. A mitochondrial-targeting copper nanocluster, characterized in that: Prepared by the method according to any one of claims 1-8, the structure includes copper nanoclusters and polyphenol ligand molecules attached to the surface of the copper nanoclusters, with a size between 1-20 nm; the polyphenol ligand molecules are selected from any one or a combination of two or more of gallic acid, epigallocatechin gallate, epicatechin, chlorogenic acid or tannic acid.

15. The use of the mitochondrial-targeting copper nanoclusters of claim 14 in the preparation of an in vivo drug for treating acute kidney injury.

16. The application as described in claim 15, characterized in that: The mitochondrial-targeted copper nanoclusters were prepared as lyophilized powder or injection solution.

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