Mitochondrial targeting copper nanocluster based on natural polyphenol as well as preparation and application of mitochondrial targeting copper nanocluster

Abstract

The invention provides a mitochondrial targeting copper nanocluster which structurally comprises a copper nanocluster and polyphenol ligand molecules attached to the surface of the copper nanocluster, and the size of the copper nanocluster is 1-20 nm. The polyphenol ligand molecule is selected from any one or a composition of more than two of gallic acid, epigallocatechin gallate, epicatechin, chlorogenic acid or tannic acid. According to the mitochondrial targeting copper nanocluster, a polyphenol structure serves as a mitochondrial targeting functional group, the nanocopper cluster serves as an antioxidant drug, mitochondrial overload active oxygen can be efficiently cleared in a targeted mode, oxidative stress and apoptosis of renal tubular epithelial cells are inhibited, and the mitochondrial targeting copper nanocluster has a good treatment effect on acute kidney injury diseases.

Description

Technical Field

[0001] The present invention relates to the field of nanomedicine, and in particular to a copper nanocluster based on natural polyphenols, having mitochondrial targeting and ultra-small size, as well as a preparation method thereof and application in the treatment of acute kidney injury. Background Art

[0002] Acute Kidney Injury (AKI) is a clinical condition that seriously threatens the life and health of patients, with high morbidity and mortality. The pathological mechanism of AKI is complex, involving multiple cellular and molecular pathways. At present, the clinical treatment of AKI mainly relies on symptomatic supportive measures, such as maintaining water and electrolyte balance and renal replacement therapy, but these methods can only relieve symptoms and cannot fundamentally prevent or reverse the disease. Existing studies have shown that mitochondrial dysfunction and excessive production of reactive oxygen species (ROS) play a key role in the occurrence and development of AKI. As the center of cellular energy metabolism, mitochondria dysfunction can lead to insufficient cellular energy supply, further aggravating cell damage. At the same time, excessive ROS generation can cause oxidative stress, leading to damage to cell structure and function.

[0003] The role of antioxidants in the treatment of AKI has been widely studied. Existing antioxidants are mainly divided 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 them, small molecule antioxidants lack the ability to specifically target damaged renal tubular epithelial cells or mitochondria, making it difficult to efficiently concentrate in the kidney or mitochondrial sites, which limits the actual therapeutic effect of NAC. Natural antioxidant enzymes have been widely studied for the treatment of various inflammatory diseases due to their excellent ROS scavenging efficiency and biocompatibility. However, these antioxidant enzymes are difficult to maintain stability in a physiological environment, and their efficiency is low during cellular endocytosis, making it difficult to reach the relevant sites of ROS generation.

[0004] In view of this, it is necessary to develop a nanodrug that can target mitochondria and has good antioxidant properties and high stability, which can stably reach the site of ROS generation and eliminate ROS during cellular endocytosis. Summary of the invention

[0005] The purpose of the present invention is to provide a copper nanocluster based on natural polyphenols, with mitochondrial targeting and ultra-small size, and a preparation method thereof. The copper nanocluster is modified by polyphenol compounds, has good mitochondrial targeting ability, can efficiently remove ROS, and has excellent therapeutic effect on acute kidney injury.

[0006] The above-mentioned object of the present invention is achieved by the following technical solutions:

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

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

[0009] 2) slowly adding alkali solution to the mixed solution obtained in 1), wherein the molar ratio of the added alkali solution to the polyphenols described in 1) is not less than 1:50, and then continuing to react for 1-4 hours under stirring conditions. After the reaction is completed, cooling to room temperature, adjusting the pH value of the solution to weak alkalinity, and removing unreacted polyphenols and copper ions, thereby obtaining mitochondrial-targeted copper nanoclusters.

[0010] In the scheme of the present invention, in order 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 scheme, 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 scheme, 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 the present invention, 1) the polyphenol aqueous solution can be prepared by dissolving any one or a mixture of two or more of natural polyphenols such as gallic acid, epigallocatechin gallate, epicatechin, chlorogenic acid or tannic acid in water;

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

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

[0014] In the scheme described in the present invention, the temperature, stirring speed and the speed of adding divalent copper mentioned in 1) will have an important influence on the size of the generated copper nanoclusters. In order to obtain ultra-small copper nanoclusters as much as possible, in the preferred scheme, the temperature when obtaining the mixed solution mentioned in 1) is controlled at 60-90°C, the stirring speed mentioned in 1) is controlled at 500-900rpm, and the slow addition mentioned in 1) is dropwise addition; in the more preferred scheme, the temperature when obtaining the mixed solution mentioned in 1) is controlled at 80°C, the stirring speed mentioned in 1) is controlled at 700rpm, and the slow addition mentioned in 1) is dropwise addition at a speed of 1 drop / second.

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

[0016] In the scheme of the present invention, in order to avoid excessive oxidation of the polyphenols, it is necessary to control the speed of adding the alkali solution to the mixed solution obtained in 1) and the stirring speed in 2). In a preferred scheme, the alkali solution in 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 the present invention, the pH value of the solution is adjusted to a weak alkaline state in 2) to promote the deprotonation of the prepared copper nanocluster surface-modified polyphenols, increase its negative potential, and thus enhance the stability of the ultra-small nanoclusters in various physiological solvents. In a preferred scheme of the present invention, the pH value of the solution is adjusted to a weak alkaline state in 2) to a final pH value of 8; further preferably, the pH value is adjusted to a final pH value of 8 by adding an alkali solution to the solution cooled to room temperature after the reaction is completed; in a more preferred scheme, the alkali solution is a 1M NaOH aqueous solution.

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

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

[0020] In a second aspect, the present invention provides mitochondrial-targeted copper nanoclusters prepared by the method of the first aspect of the present invention, wherein the structure 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 of the present invention have a size between 1 and 20 nm, and the modification of natural polyphenol ligand molecules enables them to have excellent mitochondrial targeting ability and to be dispersed and stably in a variety of solvents.

[0022] In a third aspect, the present invention further provides use 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 application of the present invention, the mitochondria-targeted copper nanoclusters can be prepared into a variety of preparations such as freeze-dried powder or injection.

[0024] The lyophilized powder or injection can be enriched in the kidneys by intravenous administration, and after being taken up by renal tubular endothelial cells, the mitochondria are located, and mitochondrial ROS are accurately removed. Meanwhile, the copper nanoclusters of the present invention have good biocompatibility and safety.

[0025] The present invention has developed a new type of mitochondrial targeting group - polyphenol, and used 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 cellular internalization, these nanoclusters can effectively target mitochondria and remove mitochondrial ROS. Compared with small molecule antioxidants and antioxidant enzymes, the ultra-small antioxidant nanoclusters provided by the present invention have better antioxidant properties, higher stability, and excellent biodistribution and bioavailability. Through the present invention, the limitations of existing antioxidants and nanomedicines are overcome, and a treatment method with efficient mitochondrial targeting and antioxidant ability is provided, which has important clinical application value.

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

[0027] The raw materials used in the present invention include copper salts and natural polyphenols that are widely present in nature. These ingredients are affordable and easy to obtain, which significantly reduces the production cost. In addition, the introduction of natural polyphenols not only enhances the stability of ultra-small nanoclusters, but also due to their negative electrical properties, there is no risk of excessive electrostatic effects interfering with mitochondrial membrane homeostasis compared to commonly used cationic targeting agents. The copper nanoclusters in the present invention are synthesized using copper, an essential element in the body, which not only improves its biocompatibility, but also due to its ultra-small size, it can be metabolized by the kidneys, avoiding the risk of heavy metal accumulation, and providing a safe and bio-friendly new therapeutic material for the treatment of AKI. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic diagram of the synthesis of polyphenol copper nanoclusters prepared by the present invention.

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

[0030] Figure 3It is a dynamic light scattering hydrated particle size distribution diagram of the polyphenol copper nanoclusters prepared by the present invention in different solvents.

[0031] Figure 4 The invention discloses a Fourier transform infrared spectrum of the polyphenol copper nanoclusters prepared by the invention and the raw materials thereof.

[0032] Figure 5 It is the X-ray photoelectron spectrum of the polyphenol copper nanoclusters prepared by the present invention.

[0033] Figure 6 It is a high-resolution Cu 2p X-ray photoelectron spectrum of the polyphenol copper nanoclusters prepared by the present invention.

[0034] Figure 7 The invention discloses an X-ray excited Auger electron spectrum of the polyphenol copper nanoclusters prepared by the invention.

[0035] Figure 8 The results show that the polyphenol copper nanoclusters prepared in Example 1 have the ability to scavenge the main active oxygen species, a: superoxide anion scavenging rate, b: hydrogen peroxide scavenging rate, c: hydroxyl radical scavenging rate.

[0036] Fig. 9 These are in vitro fluorescence imaging images of polyphenol copper nanoclusters prepared in Example 1 injected into the tail vein of mice, a: in vitro fluorescence imaging of major tissues at designated time points after injection of ICG-labeled GA-CuNCs into the tail vein of healthy or AKI mice, b: corresponding fluorescence intensity quantification histogram in a).

[0037] Fig.10 This is a cell confocal imaging image of the co-localization of polyphenol copper nanoclusters and mitochondria prepared in Example 1.

[0038] Fig.11 It reflects the levels of CRE and BUN in the serum of mice with AKI induced by ischemia-reperfusion after the polyphenol copper nanoclusters prepared in Example 1 were injected into the tail vein.

[0039] Fig.12 The results show the effect of tail vein injection of the polyphenol copper nanoclusters prepared in Example 1 on the morphology of renal mitochondria in ischemia-reperfusion mice. DETAILED DESCRIPTION

[0040] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0041] The present invention provides a method for preparing mitochondria-targeted copper nanoclusters, which specifically comprises the following steps:

[0042] Slowly add a 100-150 mM divalent copper aqueous solution to a 50-100 mM natural polyphenol aqueous solution under stirring, and add an appropriate amount of alkaline solution to promote the deprotonation of polyphenols to complex metal ions. Continue stirring for 1-4 hours, then perform pH adjustment, filtration, dialysis and freeze-drying in sequence to obtain natural polyphenol-based, mitochondrially targeted, and ultra-small copper nanoclusters, wherein the volume ratio of the natural polyphenol aqueous solution to the divalent copper aqueous solution is 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 1 M, which is slowly added dropwise to the stirring mixed solution to avoid excessive oxidation of the polyphenol compounds; in some specific embodiments, the alkaline solution is preferably NaOH, and the volume is preferably 2 mL.

[0045] The present invention further found that, without adding additional stabilizers, the reaction stirring speed and hydrothermal temperature control are key parameters to prevent 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 found that adjusting the pH of the prepared nanoclusters to a weak alkaline state can promote the deprotonation of polyphenols modified on the surface of the clusters, increase their negative potential, and thus enhance the stability of the ultra-small nanoclusters in various physiological solvents. In some specific embodiments, a 1M NaOH aqueous solution is selected for pH adjustment, and the final pH is preferably 8.0.

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

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

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

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

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

[0052] The second object of the present invention is to claim protection for an ultra-small copper nanocluster based on natural polyphenols prepared by the above-mentioned preparation method, comprising a copper nanocluster and a polyphenol ligand molecule attached to the surface of the copper nanocluster. The size of the copper nanocluster is between 1-20 nm, and the polyphenol ligand molecule is modified to have excellent mitochondrial targeting ability.

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

[0054] The third object of the present invention is to provide the use of the ultrasmall copper nanoclusters described in the present invention in the in vivo treatment of AKI.

[0055] Furthermore, in the application of the present invention, the ultrasmall copper nanoclusters can be prepared into various preparations such as freeze-dried powder or injection.

[0056] The injection can be enriched in the kidneys through intravenous administration, locate mitochondria after being taken up by renal tubular endothelial cells, and accurately remove mitochondrial ROS. At the same time, the nanoclusters have good biocompatibility and safety.

[0057] Based on the description of the above specific implementation methods, the present invention also provides the following preferred embodiments and is described in detail with reference to the accompanying drawings as follows.

[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 Figure 1 As shown, the preparation steps are as follows:

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

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

[0062] (3) Slowly add the copper solution to the gallic acid solution at a rate of 1 drop / second while stirring at 700 rpm. After mixing evenly, add 2 mL of 1 M NaOH alkaline solution to promote partial deprotonation of polyphenols. Continue the reaction at 80°C for 2 h and then return to room temperature. Use 1 M NaOH aqueous solution to adjust the pH of the reaction solution to 8.0;

[0063] (4) After the reaction is completed, filtering through a filter membrane with a pore size of 0.22 μm to obtain a gallic acid-modified copper nanocluster suspension;

[0064] (5) The copper nanocluster suspension was placed in 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 taken out and freeze-dried for 5 days to obtain gallic acid-modified ultrasmall copper nanoclusters GA-CuNC ( Figure 1 “GA-Cu” is used as the representative).

[0066] like Figure 1 As shown, in the prepared copper nanocluster GA-CuNC, the center is a 0-valent copper atom, and the periphery is a stable shell layer of 1-valent and / or 2-valent copper complexed with gallic acid ligands.

[0067] The high-resolution transmission electron microscope image of the polyphenol copper nanocluster GA-CuNC prepared in Example 1 is as follows: Figure 2A , Figure 2B As shown in the figure, the microscopic morphology of a single GA-CuNC is spherical, and it can be seen that the size of the nanocluster is less than 10nm, which is an ultra-small copper nanocluster.

[0068] Figure 3 The dynamic light scattering hydrated particle size distribution diagram of the polyphenol copper nanoclusters GA-CuNC prepared in Example 1 dispersed in different physiological solvents (including water, PBS buffer and cell culture medium DMEM), with a particle size of about 10 nm. And these copper nanocluster particles all show similar hydrated sizes in the above-mentioned several different solvents, indicating that they have excellent particle stability in various biologically relevant media.

[0069] Figure 4 The polyphenol copper nanoclusters GA-CuNC ( Figure 4 Fourier transform infrared (FTIR) spectra of CuNCs and GA (represented by the red line in the figure). Spectral analysis showed that the -1 The absorption band of 1683cm indicates the presence of phenolic hydroxyl groups. The red shift of the Ar─OH peak may be due to the coordination bond between copper and phenolic hydroxyl groups. -1 and 1384cm -1The peaks at 1590 cm-1 correspond to the stretching vibrations of C=O and C─OH in GA. -1 、1540cm -1 , and 1500cm -1 The peak at is the characteristic absorption peak of benzene ring.

[0070] Figure 5 X-ray photoelectron spectrum (XPS) of the polyphenol copper nanoclusters GA-CuNC prepared in Example 1 (the orange line GA-CuNCs represents the GA-CuNC in Example 1, and the green line GA represents gallic acid). The XPS scanning spectrum confirms the coexistence of C1s (283.86 eV), O 1s (530.32 eV) and Cu 2p (932.64 eV). Figure 6 This is the high-resolution Cu 2p spectrum of the polyphenol copper nanocluster GA-CuNC prepared in Example 1. The main peaks of Cu 2p are located at 932.73 and 952.38 eV, and the spin-orbit splitting is 19.65 eV, which is attributed to Cu 2p3 / 2 and Cu 2p1 / 2. The 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 ) main peaks correspond to Cu 0 (917.50 eV), Cu + (915.50eV) and Cu 2+ The three peaks at (916.70 eV) were fitted, confirming the existence of three different valence states in Cu-TA NM. Therefore, copper ions have successfully formed a stable chemical crystal structure after the reduction and capping stabilization of GA. These results also show that GA-CuNC has three different valence states (Cu 0 , Cu + and Cu 2+ ), and the presence of these redox pairs also endows GA-CuNC with excellent redox ability towards ROS.

[0071] Figure 8 The polyphenol copper nanoclusters GA-CuNC prepared in Example 1 react with typical active oxygen species (O2· - The results showed that the scavenging efficiency of GA-CuNC on the above three ROS was positively correlated with their concentrations.

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

[0073] GA-CuNCs were stained with Cy5.5 and colocalized using a commercial Mito-Tracker. Fig.10 As shown in the figure, it can be seen that after 3 hours of co-incubation, the fluorescence signal of Cy5.5 labeled GA-CuNC has a high degree of overlap with mitochondria. This result shows that GA-CuNC ( Fig.10 The GA-Cu (represented by “GA-Cu” in the literature) can better target mitochondria, thereby precisely regulating mitochondrial oxidative stress.

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

[0075] In this example, copper nanoclusters modified with tannic acid are prepared, which have mitochondrial targeting and antioxidant functions. The specific preparation steps are as follows:

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

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

[0078] (3) Slowly add the copper solution to the gallic acid solution at a rate of 1 drop / second while stirring at 700 rpm. After mixing evenly, add 2 mL of 1 M NaOH alkaline solution to promote partial deprotonation of polyphenols. Continue the reaction at 80°C for 2 h and then return to room temperature. Use 1 M NaOH aqueous solution to adjust the pH of the reaction solution to 8.0;

[0079] (4) After the reaction is completed, filtering through a filter membrane with a pore size of 0.22 μm to obtain a tannic acid-modified copper nanocluster suspension;

[0080] (5) The tannic acid-modified copper nanocluster 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 min, then taken out and freeze-dried for 5 days to finally obtain tannic acid-modified ultrasmall copper nanoclusters TA-CuNC.

[0082] Example 3. Preparation of Epigallocatechin Gallate-modified Copper Nanoclusters

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

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

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

[0086] (3) Slowly add the copper solution to the gallic acid solution at a rate of 1 drop / second while stirring at 700 rpm. After mixing evenly, add 2 mL of 1 M NaOH alkaline solution to promote partial deprotonation of polyphenols. Continue the reaction at 80°C for 2 h and then return to room temperature. Use 1 M NaOH aqueous solution to adjust the pH of the reaction solution to 8.0;

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

[0088] (5) placing the epigallocatechin gallate-modified copper nanocluster suspension into a dialysis bag with a molecular weight cutoff of 3500 Daltons and dialyzing for 3 days;

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

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

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

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

[0093] To label GA-CuNC with ICG, GA-CuNC and DSPE-PEG2000-ICG were stirred at room temperature overnight. The solution was then dialyzed with deionized water for 12 h using a dialysis bag with a molecular weight cutoff of 3500 Da to obtain the labeled drug, which was recorded 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. Among them, AKI mice were injected with cisplatin for modeling, and GA-CuNC@ICG (10 mg / kg) was intravenously injected 30 min after modeling. Subsequently, the mice were euthanized at selected time points after administration, and major organs were obtained. In vivo fluorescence imaging was performed using an IVIS Lumina imaging system (Xenogen Corporation, Alameda, California, USA). The fluorescence signal of each organ was quantified using ImageJ software. ICG: λem=808nm, λex=900nm.

[0094] like Fig. 9 As shown in the right side of parts a and b, the imaging results show that GA-CuNC@ICG is mainly distributed in the liver (Li) and kidney (Ki) of healthy mice. The fluorescence signal in the kidney reaches a peak at about 0.5 hours after injection. Fig. 9 As shown in the left side of parts a and b, consistent with the imaging results of healthy mice, GA-CuNC@ICG is also mainly enriched in the liver (Li) and kidney (Ki) of cisplatin-induced AKI mice. The results show that intravenously administered copper nanocluster particles can quickly concentrate on damaged kidney tissue.

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

[0096] Twenty 7-week-old male C57 mice were randomly divided into 4 groups after 1 week of adaptive feeding, with 5 mice in each group, namely, sham group (Sham), renal ischemia-reperfusion model group (Saline), N-acetylcysteine ​​group (NAC) and polyphenol-modified copper nanoclusters group (GA-Cu). The sham group (Sham) was intraperitoneally injected with normal saline every day; the renal ischemia-reperfusion model group (Saline) was subjected to unilateral dorsal incision, blunt separation of the renal pedicle, and clamping of the renal pedicle with a micro-arterial hemostatic clamp. After 45 minutes of ischemia, the hemostatic clamp was removed, the kidney was reset, the bilateral dorsal wounds were sutured, and renal perfusion was restored for 24 hours. Two hours after the establishment of the renal ischemia-reperfusion model in the NAC group, 100 mg / kg of N-acetylcysteine ​​was injected into the tail vein; two hours after the establishment of the renal ischemia-reperfusion model in the GA-Cu group, 10 mg / kg of GA-CuNC was injected into the tail vein.

[0097] Sample collection

[0098] 24 hours after the renal ischemia-reperfusion model was established in the above-mentioned Saline group, NAC group and GA-Cu group, the eyeballs of all mice were removed and blood was collected. The serum was collected by centrifugation and then frozen in a -80°C refrigerator; the kidneys of all mice were removed, fixed with 4% paraformaldehyde and stored at room temperature.

[0099] Assessment of renal function

[0100] The blood urea nitrogen (BUN) and serum creatinine (CRE) concentrations of the collected serum samples were tested to evaluate renal function indicators. The concentrations of BUN and CRE were determined by BUN detection kit (Nanjing Jiancheng Bioengineering Institute) and CRE detection kit (Nanjing Jiancheng Bioengineering Institute), respectively.

[0101] like Fig.11As shown, the renal function indicators (BUN and CRE) of mice in the Saline group were increased; in contrast, the acute renal injury of mice in the GA-Cu group was alleviated after administration of the polyphenol copper nanoclusters GA-CuNC prepared in Example 1; it is worth mentioning that compared with the NAC group, the serum BUN and CRE levels in the 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 solution containing 2.5% glutaraldehyde and 4% formaldehyde, and the samples were processed in the transmission electron microscopy room of Zhejiang University School of Medicine. The microscopic structure of mitochondria in renal tubular epithelial cells was photographed using a transmission electron microscope (TEM) (Shimadzu Corporation, Tokyo, Japan).

[0104] like Fig.12 As shown, the mitochondrial morphology in the renal tubular endothelial cells of mice in the Sham group is highly ordered, rod-shaped, and the cristae of the mitochondria are clearly visible. In the kidneys of mice in the Saline group, a large number of abnormal mitochondrial changes can be observed, including swelling, reduced or disappeared cristae, and even mitochondrial fragmentation, indicating that mitochondrial function in renal tubular endothelial cells is fatally damaged under the background of the disease. In contrast, in the kidneys of mice in the GA-Cu group, the mitochondrial morphology of renal tubular epithelial cells is close to normal, indicating that the administration of polyphenol copper nanoclusters GA-CuNC prepared in Example 1 effectively inhibits the swelling and fission of mitochondrial tubular epithelial cells. In addition, in the kidneys of mice in the NAC group, the abnormal mitochondrial morphology was not improved, and it can be seen that the therapeutic effect of N-acetylcysteine ​​injected in this group on acute renal injury is not as good as that of GA-CuNC injected in the GA-Cu group.

[0105] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the technical principles of the present invention, and these improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing mitochondrial-targeted copper nanoclusters, comprising: 1) slowly adding a divalent copper aqueous solution to a polyphenol aqueous solution under stirring conditions, controlling the molar ratio of polyphenol to divalent copper to be 1-2:2-3, preferably 1:2-3, to obtain a mixed solution; the polyphenol aqueous solution is prepared by dissolving any one or a mixture of two or more of natural polyphenols such as gallic acid, epigallocatechin gallate, epicatechin, chlorogenic acid or tannic acid in water; 2) slowly adding alkali solution to the mixed solution obtained in 1), wherein the molar ratio of the added alkali solution to the polyphenols described in 1) is not less than 1:50, and then continuing to react for 1-4 hours under stirring conditions. After the reaction is completed, cooling to room temperature, adjusting the pH value of the solution to weak alkalinity, and removing unreacted polyphenols and copper ions, thereby obtaining mitochondrial-targeted copper nanoclusters.

2. The method according to 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; in a preferred embodiment, 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.

3. The method according to claim 1, characterized in that: 1) The divalent copper aqueous solution is any one of a copper nitrate aqueous solution, a copper sulfate aqueous solution or a copper chloride aqueous solution or a mixture of two or more thereof.

4. The method according to claim 1, characterized in that: 1) the temperature of the mixed solution is controlled at 60-90°C, 1) the stirring speed is controlled at 500-900rpm, and 1) the slow addition is dropwise addition; in the preferred embodiment, 1) the temperature of the mixed solution is controlled at 80°C, 1) the stirring speed is controlled at 700rpm, and 1) the slow addition is dropwise addition at a speed of 1 drop / second.

5. The method according to claim 1, characterized in that: 2) The alkali solution is any one of NaOH solution or KOH solution or a mixture of two or more thereof; preferably NaOH solution; most preferably NaOH solution with a concentration of 1 M.

6. The method according to claim 1, characterized in that: 2) The alkali solution is added dropwise, and the stirring speed is controlled at 500-900 rpm, more preferably at 700 rpm.

7. The method according to claim 1, characterized in that: 2) The step of adjusting the pH value of the solution to weak alkalinity is to adjust the pH value to a final value of 8; preferably, the pH value is adjusted to a final value of 8 by adding an alkali solution to the solution cooled to room temperature after the reaction is completed; in a more preferred embodiment, the alkali solution is a 1M NaOH aqueous solution.

8. The method according to claim 1, characterized in that: The method also includes freeze-drying the solution obtained by removing unreacted polyphenols and copper ions in 2) to obtain mitochondrial-targeted copper nanocluster powder.

9. A mitochondrial-targeted copper nanocluster, characterized in that: The method is prepared by any one of claims 1 to 8, and its structure includes copper nanoclusters and polyphenol ligand molecules attached to the surface of the copper nanoclusters, with a size between 1 and 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.

10. Use of the mitochondrial targeted copper nanoclusters according to claim 9 in the preparation of an in vivo drug for treating acute kidney injury (AKI); in a preferred application, the mitochondrial targeted copper nanoclusters are prepared as a lyophilized powder or an injection.

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