Gold-copper nanocluster with efficient multi-source active oxygen generation performance and preparation method and application of gold-copper nanocluster

Abstract

The invention provides a gold-copper nanocluster with efficient multi-source active oxygen generation performance and a preparation method and application of the gold-copper nanocluster, and belongs to the field of biomedical materials. The gold-copper nanocluster is synthesized through mediation of Tf, and Tf is full transferrin, iron-saturated transferrin or siderophore. The gold-copper nanocluster with Tf as the ligand is synthesized by adopting a simple one-pot method, and the problems that in the prior art, a multi-source active oxygen generation system is complex to construct and a preparation method is tedious are solved; the gold-copper nanocluster synthesized by taking Tf as a ligand can be used as a simple multi-source active oxygen generation system, superoxide anions can be generated through photosensitized oxidation activity sensitization oxygen, and hydroxyl radicals can be generated through peroxidase-like activity catalysis hydrogen peroxide, so that efficient generation of multi-source active oxygen is realized; and the effect of enhancing tumor treatment is achieved.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and in particular relates to a gold-copper nanocluster with high-efficiency multi-source active oxygen generation performance, a preparation method and application thereof. Background Art

[0002] Reactive oxygen species (ROS) are a class of substances derived from O2 that are more active than O2 itself, mainly including superoxide anions (O2 ·- ), hydroxyl radical (·OH) and singlet oxygen ( 1 O2) is an important signal and regulatory molecule for the redox balance of the human body. When the ROS concentration exceeds the body's tolerance threshold, it will cause oxidative damage to life-related molecules. Therefore, increasing the level of ROS in diseased tissues and enhancing the oxidative damage effect on diseased tissues is one of the effective means of treating diseases.

[0003] Photodynamic therapy (PDT) is a ROS-mediated treatment method, the principle of which is that photosensitizers sensitize oxygen to ROS under the excitation of a light source of a specific wavelength. Studies have shown that on the basis of PDT, the overexpressed H2O2 (hydrogen peroxide) in the diseased tissue reacts to generate OH, which can further increase the level of ROS, and then use multi-source ROS to enhance the therapeutic effect of the disease. For example, Xu et al. from Harbin Engineering University (Xu, Jiating et al., All-in-One Theranostic Nanomedicine with Ultrabright Second Near-Infrared Emission for Tumor-Modulated Bioimaging and Chemodynamic / Photodynamic Therapy. ACS Nano, 2020.14(8): p.9613-9625.) used mesoporous silica as a shell to coat the pre-synthesized lanthanum-doped nanoparticle (LDNPs) core, and then converted it into copper / manganese silicate nanospheres (CMSNs) by a hydrothermal method, and finally modified it with PEG to obtain a PEG / LDNPs@CMSNs nanoformulation. Mn release from CMSNs in PEG / LDNPs@CMSNs 2+ and Cu + A Fenton-like reaction with H2O2 generates ·OH, and LDNPs are generated by PDT 1O2, multi-source ROS synergistically enhance the inhibitory effect on tumors. In addition, Wang et al. from Shandong University (Wang, Man et al., Au2Pt-PEG-Ce6 nanoformulation with dual nanozyme activities for synergistic chemodynamic therapy / phototherapy. Biomaterials, 2020.252:120093.) combined SH-PEG-NH2 with the pre-synthesized Au2Pt nanozyme, and then covalently linked the photosensitizer Ce6 through NHS / EDC reaction, and finally constructed an Au2Pt-PEG-Ce6 nanoformulation. The Au2Pt in Au2Pt-PEG-Ce6 catalyzes the H2O2 reaction to generate ·OH through peroxidase-like (POD) activity, and Ce6 generates through PDT 1 O2 also improves the tumor efficacy through the synergistic effect of multi-source ROS. However, the preparation steps of the above-mentioned nanoformulations are cumbersome and complicated, and there are many components. It is urgent to build a simple multi-source reactive oxygen generation system.

[0004] Gold nanoclusters (AuNCs) are new molecular aggregates synthesized from several to hundreds of gold (Au) atoms under the protection of ligands such as proteins, peptides or reducing small molecules. They have good biocompatibility, simple system and easy preparation. In recent years, they have been widely used in biomedical fields such as bioimaging, biodetection and disease diagnosis and treatment. At present, it has been reported that AuNCs have good photosensitivity and oxidation activity and can be used as a new type of nano photosensitizer for PDT. For example, AuNCs with dihydrolipoic acid as ligand (Han, Rongcheng et al., Super-efficient in Vivo Two-Photon Photodynamic Therapy with a Gold Nanocluster as a Type I Photosensitizer. ACS Nano, 2020.14(8): p.9532-9544.) can sensitize oxygen to O2 after being excited by light. ·-, thereby killing tumor cells. In addition, studies have shown that AuNCs with histidine, glutathione or 3-mercaptopropionic acid as ligands (Li, Yueyuan et al., An enzymatic activity regulation-based clusterzyme sensor array for high-throughput identification of heavy metal ions. Journal of Hazardous Materials, 2023. 454: 131501.) all have POD-like activity, which is expected to provide the possibility for the generation of multi-source reactive oxygen species. However, the catalytic activity of the POD-like system based on AuNCs is low, which limits its application as a multi-source reactive oxygen generation system. In order to improve the POD-like activity of AuNCs, Chen et al. from Shanghai Jiao Tong University (Chen, Xiaoliang et al., Single-Stranded DNA-Encoded Gold Nanoparticle Clusters as Programmable Enzyme Equivalents. Journal of the American Chemical Society, 2022. 144(14): p. 6311-6320.) designed and synthesized a series of single-stranded DNA scaffolds with periodically repeating polyadenine (polyA) ring handle units through the rolling circle amplification (RCA) method. By regulating the number and spatial spacing of polyA ring handle units, the POD-like activity of the synthesized AuNCs can be effectively improved. Han et al. from Tianjin University (Han, Lu et al., Improvement of mimetic peroxidase activity of gold nanoclusters on the luminol chemiluminescence reaction by surface modification with ethanediamine. Luminescence, 2018. 33 (4): p. 751-758.) mixed ethylenediamine with a pH of 4.5 with pre-synthesized AuNCs, and prepared AuNCs with positive surface charge under the action of EDC, whose POD-like activity was significantly improved. However, the above-mentioned RCA and charge regulation methods are relatively complicated, and the cost of DNA chains is relatively high. Summary of the invention

[0005] The object of the present invention is to provide a gold-copper nanocluster with high-efficiency multi-source reactive oxygen generation performance, and a preparation method and use thereof. Compared with the above-mentioned other methods for constructing a system with multi-source reactive oxygen generation performance, the copper (Cu) ion doping method adopted by the gold-copper nanocluster has the advantages of being simple, easy to operate and low cost. In addition, through copper ion doping, the POD-like activity of the gold-copper nanocluster is significantly enhanced, and it also has good photosensitizing oxidation activity, which meets its needs as a high-efficiency multi-source reactive oxygen generation system and achieves a synergistic effect on tumor treatment.

[0006] The invention provides a gold-copper nanocluster synthesized in one step with Tf as a ligand, wherein the gold-copper nanocluster is a copper-doped gold nanocluster; the gold-copper nanocluster uses gold atoms and copper atoms as metal cores and Tf as a ligand shell; the Tf is full transferrin, iron-saturated transferrin or ferritin, with an English name of Holo-Transferrin, CAS number: 11096-37-0, and MDL number: MFCD00131337.

[0007] The present invention provides a method for synthesizing gold-copper nanoclusters using Tf as a ligand. The method is to add copper ions into the process of synthesizing gold nanoclusters using Tf as a ligand, and synthesize the gold-copper nanoclusters in one step. The specific steps are:

[0008] (1) placing Tf in a clean reaction container, adding ultrapure water, and placing it in a heat-collecting constant-temperature magnetic stirrer at a temperature of less than or equal to 37° C. and heating and stirring the mixture to fully dissolve the mixture and prepare a suspension;

[0009] (2) First, slowly add tetrachloroauric acid (HAuC14) solution, and then add copper sulfate pentahydrate (CuSO4·5H2O) solution;

[0010] (3) then adding ascorbic acid solution, and then adding NaOH solution to adjust the pH value to 10-13, and maintaining constant temperature heating and stirring for 6-8 hours to obtain a reaction solution, until the reaction solution is observed to exhibit red fluorescence under ultraviolet light;

[0011] (4) Centrifuge the reaction solution at a temperature less than or equal to 4° C., and then ultrafilter the reaction solution to obtain the product.

[0012] The amount of Tf is greater than or equal to 20 mg, and the amount of ultrapure water is greater than or equal to 3 mL. The present invention has no strict restrictions on the amount of ultrapure water. It is only necessary to fully dissolve Tf, but it cannot be added too much, because adding too much will cause the concentration of tetrachloroauric acid and copper sulfate pentahydrate added thereto to be low, resulting in slow subsequent reactions. When 20 mg of Tf is used, it is recommended to use 3 mL of ultrapure water. When the amount of Tf is increased, the amount of ultrapure water can be increased in proportion, or the proportion of ultrapure water can be adjusted within a range of 30%. For example, 6 mL of ultrapure water can be used when 40 mg of Tf is used, and it can also be selected to use within the range of 4.2 to 7.8 mL of ultrapure water.

[0013] Tf is a protein, and its optimum temperature is 25°C to 37°C. Therefore, the preferred temperature of the heat collecting constant temperature magnetic stirrer is 37°C.

[0014] The mass ratio of Tf to ascorbic acid is 1:0.00017-0.00018, and the concentration of the ascorbic acid solution is 3.5 mg / mL.

[0015] Preferably, copper sulfate pentahydrate is added to the solution 5 minutes before the ascorbic acid solution is added.

[0016] Preferably, NaOH solution is added to adjust the pH value to 10-13 10 minutes after ascorbic acid is added to the solution.

[0017] The ratio of Tf to HAuC14 is 0.005-0.006 mmol HAuC14 per 20 mg Tf, and preferably the concentration of the HAuC14 solution is 100 mmol / L.

[0018] The molar mass ratio of the HAuC14 and CuSO4 is 1:(0.05-0.3), and the concentration of the CuSO4·5H2O solution is preferably 100 mmol / L.

[0019] The concentration of the NaOH solution is 0.5 mol / L; the pH value is within the range of 10 to 13, and the stirring time is within 6 to 8 hours, and the reaction solution can be prepared;

[0020] The stirring is preferably carried out at a constant speed of 800 to 1200 rpm, preferably at a speed of 800 rpm, and at a temperature of 25°C to 37°C, preferably at a constant temperature of 37°C.

[0021] Preferably the ultrafiltration tube is a 10KD ultrafiltration tube, which can filter out the product.

[0022] Centrifugation is preferably performed at 4000 rpm and 4°C for 20 minutes.

[0023] When the molar mass ratio of HAuC14 to CuSO4 is 1:0, the synthesized clusters are copper-free gold nanoclusters, which are referred to as Tf@AuNCs.

[0024] The clusters synthesized by the present invention using HAuC14 and CuSO4 in a ratio of 1: (0.05-0.3) are all copper-doped gold nanoclusters, i.e., gold-copper nanoclusters; the clusters synthesized in a ratio of 1:0.2 are gold-copper nanoclusters with the best properties, which are referred to as Tf@ACNCs.

[0025] The present invention also provides the use of the aforementioned gold-copper nanoclusters in tumor treatment, wherein the gold-copper nanoclusters are an efficient multi-source reactive oxygen generation system used in tumor treatment; the multi-source reactive oxygen generation system means that ROS can be generated through multiple reaction pathways to increase ROS production.

[0026] Compared with the existing technology, the present invention has the following beneficial effects:

[0027] The method for preparing gold-copper nanoclusters mediated by Tf is simple, has mild reaction conditions, strong operability, and low cost. The prepared gold-copper nanoclusters have good biocompatibility, low toxicity, good stability, good tumor targeting, stable fluorescence performance, good POD-like activity for catalyzing H2O2 to generate ·OH, and sensitizing oxygen to generate O2 ·- The photosensitized oxidation activity of the drug increased the amount of ROS generated, enhanced the tumor efficacy, and provided new ideas and methods for tumor treatment.

[0028] That is, the present invention adopts a simple one-pot method to synthesize gold-copper nanoclusters with Tf as a ligand, which overcomes the problems of complex multi-source active oxygen generation system and cumbersome preparation method in the prior art; compared with copper-free doped gold nanoclusters synthesized with Tf as a ligand, the gold-copper nanoclusters synthesized with Tf as a ligand in the present invention can be used as a simple multi-source active oxygen generation system, which can generate superoxide anions by sensitizing oxygen through photosensitive oxidation activity and generate hydroxyl radicals by catalyzing hydrogen peroxide through peroxidase-like activity, thereby solving the defect of poor catalytic activity of peroxidase-like system based on gold nanoclusters, realizing efficient generation of multi-source active oxygen, achieving the effect of enhancing tumor treatment, and having good application prospects.

[0029] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the optimization of the gold-copper nanoclusters synthesized by Tf-mediated synthesis in the present invention.

[0031] Figure 2 This is the particle size distribution diagram of Tf@ACNCs of the present invention.

[0032] Figure 3 Transmission electron microscope image and element distribution map of Tf@ACNCs of the present invention.

[0033] Figure 4 3D fluorescence spectrum and fluorescence emission spectrum of Tf@ACNCs of the present invention.

[0034] Figure 5 The TMB oxidation results of Tf@ACNCs of the present invention: A is the spectrum of TMB oxidation by H2O2 catalyzed by Tf@ACNCs; B is the Lineweaver-Burk fitting curve of TMB oxidation by H2O2 catalyzed by Tf@ACNCs; C is the spectrum of TMB oxidation by Tf@ACNCs under 488nm visible light excitation; D is the spectrum of TMB oxidation by Tf@ACNCs jointly catalyzed by H2O2 and 488nm visible light excitation.

[0035] Figure 6 The EPR detection results of Tf@ACNCs: ABC is the EPR spectrum of Tf@ACNCs under 488nm visible light excitation, A is O2 ·- ; B is ·OH; C is 1 O2; D is the EPR spectrum of H2O2 catalyzed by Tf@ACNCs.

[0036] Figure 7 The results of Tf@ACNCs generating reactive oxygen species in melanoma cells A375 under 488 nm illumination and without illumination: A is HPF staining; B is DHE staining; C is SOSG staining.

[0037] Figure 8 The live / dead cell results of A375 cells treated with Tf@ACNCs under 488 nm illumination and without illumination.

[0038] Fig. 9 The results of Tf@ACNCs inducing A375 apoptosis under 488 nm illumination and without illumination: A is the Annexin V-FITC / PI flow cytometry clustering diagram; B is the Annexin V-FITC / PI flow cytometry apoptotic cell statistical analysis diagram. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments and experimental examples. It should be understood that the specific embodiments and experimental examples described herein are only used to explain the present invention and are not intended to limit the present invention. The raw materials and equipment used in the specific implementation of the present invention are all known products and are obtained by purchasing commercially available products.

[0040] In the present invention, Tf is holo-transferrin, iron-saturated transferrin or ferritin, English name: Holo-Transferrin, CAS number: 11096-37-0, MDL number: MFCD00131337;

[0041] Example 1: Preparation of gold-copper nanoclusters synthesized by Tf-mediated synthesis according to the present invention. The specific process steps are as follows:

[0042] (1) Soak the glass bottle in aqua regia, clean it, and dry it for later use.

[0043] (2) Take 3 mL of ultrapure water and add it to a clean glass bottle, weigh 20 mg of Tf and dissolve it in the ultrapure water, stir to make it fully dissolved, and then place it in a 37°C heat-collecting constant temperature magnetic stirrer for heating and stirring, first slowly add 50 μL of 100 mmol / L HAuC14 solution, and then add 100 mmol / L CuSO4·5H2O solution, the molar mass ratio of HAuC14 and CuSO4·5H2O is 1:0.05, 5 minutes after the addition of CuSO4·5H2O solution, add 1 μL of 3.5 mg / mL ascorbic acid solution, 10 minutes after the addition of ascorbic acid solution, add 0.5 mol / L NaOH solution to adjust the pH value to 10, stir at a constant speed of 800 rpm and 37°C until the solution in the glass bottle is observed to exhibit red fluorescence under ultraviolet light, and the stirring time is 6.5 h.

[0044] (3) After the reaction, the sample was transferred to a 10KD ultrafiltration tube and centrifuged at 4000 rpm for 20 minutes at 4°C to obtain the final product, Tf-mediated synthesis of gold-copper nanoclusters (centrifuged precipitate).

[0045] Example 2

[0046] The preparation method of the Tf-mediated synthesis of gold-copper nanoclusters in this example is basically the same as that in Example 1, except that the molar mass ratio of HAuC14 and CuSO4·5H2O is in the range of 1:0.1.

[0047] Example 3

[0048] The preparation method of the Tf-mediated synthesis of gold-copper nanoclusters in this example is basically the same as that in Example 1, except that the molar mass ratio of HAuC14 and CuSO4·5H2O is in the range of 1:0.2.

[0049] Example 4

[0050] The preparation method of the Tf-mediated synthesis of gold-copper nanoclusters in this example is basically the same as that in Example 1, except that the molar mass ratio of HAuC14 and CuSO4·5H2O is in the range of 1:0.3.

[0051] Example 5. Preparation of Tf-mediated synthesis of gold nanoclusters

[0052] The preparation method described in Example 1 was adopted, the molar mass ratio of HAuC14 and CuSO4·5H2O was in the range of 1:0, the pH value was 10, and the synthesis time was within 6.5 hours to prepare Tf-mediated synthesized gold nanoclusters, namely Tf@AuNCs.

[0053] The beneficial effects of the present invention are demonstrated by specific test examples below:

[0054] Experimental Example 1: Optimization of Tf-mediated synthesis of gold-copper nanoclusters of the present invention

[0055] 1. Test methods

[0056] The CCK-8 test was used to verify the effect of the nanoclusters synthesized in Examples 1 to 4 and Example 5 on inhibiting the proliferation of A375 under 488 nm illumination and without illumination. The specific test method is as follows:

[0057] (1) Plating: Adjust the concentration of the digested A375 cell suspension to 5.0×10 4 / well, inoculated into 96-well plates and incubated for 24 hours.

[0058] (2) Adding drugs: Add 0 / 5 / 10 / 20 / 40 μmol / L of the nanoclusters synthesized in Examples 1 to 5 to the well plate, respectively. Place the well plate in an incubator and culture for 12 hours. Set up a group without drugs + no light (blank group), a group without drugs + light, a group with different concentrations of drugs + no light, and a group with different concentrations of drugs + light. There are 3 sub-wells in each group. The drug refers to the nanoclusters prepared in Examples 1 to 5. The blank group (i.e., Control in the figure, the same for subsequent experiments) is ultrapure water. The concentrations expressed in a similar manner as "0 / 5 / 10 / 20 / 40 μmol / L" in the present invention are understood to be 0 μmol / L, 5 μmol / L, 10 μmol / L, 20 μmol / L, and 40 μmol / L as multiple parallel concentrations.

[0059] (3) Illumination: The experimental group that needs illumination is illuminated with LED light with an excitation wavelength of 488 nm. The power is 1 W and the illumination is for 15 minutes. After the illumination is completed, incubate for 4 hours.

[0060] (4) CCK-8 color development: Wash the cells once with PBS, and then react with the cells with CCK-8 working solution for 1 hour.

[0061] (5) Detection: Take out the 96-well plate and use an ELISA reader to detect the absorbance at 450 nm. Incubation time for each group: When the absorbance value of the blank group is between 1 and 2, the incubation is completed and each group is tested.

[0062] (6) Screening: Cell survival rate (%) = [(absorbance of drug-treated group - absorbance of blank group) / (absorbance of control group - absorbance of blank group)] × 100%. The lower the cell survival rate, the stronger the inhibitory effect on cell proliferation. The gold-copper nanoclusters with the strongest inhibitory effect are the most preferred gold-copper nanoclusters, which are named Tf@ACNCs. The data are expressed as mean ± standard deviation (Mean ± SD), and two-way ANOVA was performed using GraphPad Prism software. When the p value was less than 0.05, it was statistically significant. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0063] 2. Test results

[0064] The screening results of the gold-copper nanoclusters synthesized by Tf-mediated synthesis of the present invention are as follows: Figure 1 As shown. Figure 1 As shown, clusters doped with copper in different proportions can inhibit the proliferation of A375 without illumination, and the inhibitory effect is further enhanced after illumination, indicating that it plays a role in enhancing tumor treatment. Among them, the gold-copper nanocluster with a molar mass ratio of HAuC14 to CuSO4·5H2O of 1:0.2 has the best effect of inhibiting A375 without illumination, and the cell survival rate is still the lowest after illumination, indicating that it has the strongest effect of inhibiting the proliferation of A375, and is the most preferred gold-copper nanocluster, which is named Tf@ACNCs, that is, the product prepared in Example 3 is Tf@ACNCs.

[0065] Experimental Example 2: Characterization of the physical and chemical properties of the gold-copper nanoclusters synthesized by Tf-mediated synthesis of the present invention

[0066] 1. Test methods

[0067] (1) Test 1: The Tf@ACNCs prepared in Example 3 were diluted 1000 times with ultrapure water and ultrasonically shaken at room temperature for 30 min. The mixture was then added to a cuvette and the particle size distribution of the Tf@ACNCs was measured using a dynamic light scattering instrument.

[0068] (2) Test 2: The Tf@ACNCs prepared in Example 3 were diluted 1000 times with ultrapure water at room temperature, and 10 μL was dropped on a clean nickel mesh. After ventilation and drying at room temperature, the mixture was observed and imaged using a transmission electron microscope, and then elemental analysis was performed and an element distribution map was collected.

[0069] (3) Test 3: 1.5 mL of Tf@ACNCs prepared in Example 3 was added to a rectangular square transparent quartz colorimetric blood. The emission wavelength of the sample was first measured, and then its appropriate excitation wavelength was measured according to the emission wavelength to obtain a 3D fluorescence spectrum.

[0070] (4) Test 4: 1.5 mL of Tf@AuNCs prepared in Example 5 was added to a rectangular square transparent quartz colorimetric solution. The emission wavelength of the sample was first measured, and then its appropriate excitation wavelength was measured according to the emission wavelength to obtain a 3D fluorescence spectrum.

[0071] (5) Test 5: 1.5 mL of Tf@ACNCs prepared in Example 3 was added to a rectangular square transparent quartz colorimetric solution, and the fluorescence emission spectrum of the sample was detected.

[0072] (6) Test 6: 1.5 mL of Tf@AuNCs prepared in Example 5 was added to a rectangular square transparent quartz colorimetric solution, and the fluorescence emission spectrum of the sample was detected.

[0073] 2. Test results

[0074] Depend on Figure 2 Particle size distribution and Figure 3 A transmission electron microscopy image shows that the particle size of Tf@ACNCs prepared by the present invention is about 11.8nm. Figure 3 From the element distribution diagram B, we can see that gold and copper are evenly distributed in the nanocluster particles, and the distribution of gold and copper overlaps, indicating that copper is adsorbed on the surface of gold and is closely combined with gold. It can be seen that the gold-copper nanocluster is composed of gold atoms and copper atoms as the metal core and Tf as the ligand shell. Figure 4 A is the 3D fluorescence spectrophotometer detection result of Tf@ACNCs and Tf@AuNCs synthesized by Tf in the present invention. Figure 4 A shows that both Tf@ACNCs and Tf@ACNCs have strong absorption in the range of 400-550nm, and both have obvious absorption peaks at 488nm. The emission wavelength range of Tf@ACNCs and Tf@ACNCs is both between 600 and 800nm, and the strongest emission peaks are both around 690nm, which indicates that the doping of copper ions has no obvious effect on the fluorescence of gold nanoclusters.

[0075] The optimal excitation wavelength was detected to be 488 nm, and subsequent experiments were performed at this excitation wavelength.

[0076] Figure 4 B is the fluorescence emission spectra of Tf@ACNCs and Tf@AuNCs synthesized by Tf in the present invention. Figure 4 B shows that the fluorescence emission wavelength range of Tf@ACNCs and Tf@ACNCs is consistent, both have the strongest emission peak at about 660nm, and there is no significant difference in the fluorescence intensity between the two, which once again shows that copper ion doping does not affect the fluorescence emission of gold nanoclusters.

[0077] Experimental Example 3: TMB (3,3',5,5'-tetramethylbenzidine) oxidation test of gold-copper nanoclusters synthesized by Tf-mediated synthesis of the present invention

[0078] 1. Test methods

[0079] In order to verify the performance of Tf@ACNCs prepared in Example 3 in catalyzing the oxidation of TMB by H2O2 and the oxidation of TMB under 488nm visible light excitation, a TMB oxidation test was carried out. The specific test method is as follows:

[0080] (1) Reagent preparation: prepare 0.5 mol / L Na2HPO4 / NaH2PO4 buffer at pH 5, 0.05 mol / L HAc / NaAc buffer at pH 4, 40 mmol / L TMB, 20 / 40 / 60 / 80 / 160 / 240 / 320 mmol / L H2O2. The solvent is ultrapure water.

[0081] (2) Test 1: In a 96-well plate, 48 μL of Na2HPO4 / NaH2PO4 buffer, 5 μL of ultrapure water, 20 μmol / L Tf@ACNCs and 2 μL of TMB (3,3',5,5'-tetramethylbenzidine) were added to each well in sequence, and then 25 μL of H2O2 of different concentrations were added, and mixed thoroughly to avoid bubbles. Ultrapure water was used as a blank control. There were 3 wells in each group. After the addition of the sample, the absorbance at 652 nm was measured using an ELISA reader at 0 / 1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 minutes. In the present invention, the time recorded in a similar manner as "0 / 1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 minutes" is understood to be 0 minutes, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes and multiple parallel time points.

[0082] (3) Test 2: In a 96-well plate, 50 μL of HAc / NaAc buffer, 45 / 35 / 25 / 15 μL of ultrapure water, 0 / 10 / 20 / 30 μmol / L Tf@ACNCs and 5 μL of TMB were added to each well in sequence, and the mixture was thoroughly mixed to avoid bubbles. Ultrapure water was used as a blank control, and 488 nm 3W LED light was used for 20 minutes. There were 3 pairs of wells in each group. After the illumination was completed, the absorption spectrum in the range of 500 to 800 nm was measured using an ELISA reader. In the present invention, the volumes recorded in a similar manner as "45 / 35 / 25 / 15 μL" are understood to be multiple parallel volumes of 45 μL, 35 μL, 25 μL, and 15 μL.

[0083] (4) Test 3: In a 96-well plate, 76 μL of HAc / NaAc buffer, 20 μmol / L Tf@ACNCs and 4 μL TMB were added to each well in turn, mixed thoroughly to avoid bubbles, and illuminated with 360 nm 1W LED light for 3 minutes. Then, 10 μL of 60 mmol / L H2O2 was added. The wells were divided into the non-illumination + H2O2 group, the illumination + non-H2O2 group, and the non-illumination + non-H2O2 group as a control. There were 3 duplicate wells in each group. After 10 minutes, the absorption spectrum in the range of 500-800 nm was measured using an ELISA reader. The absorption spectrum in the range of 500-800 nm was measured using the same method using Tf@AuNCs instead of Tf@ACNCs.

[0084] (5) Calculation: Draw the Lineweaver-Burk fitting curve of test 1 according to the Beer-Lambert law and the Michaelis-Menten equation to determine the maximum reaction rate. The specific formula is: 1 / v0 = K M / V max [S]+1 / V max , v0 is the initial reaction rate, V max is the maximum reaction rate, [S] is the initial concentration of substrate, K M is the Michaelis-Menten constant.

[0085] 2. Test results

[0086] The performance of the gold-copper nanoclusters synthesized by Tf-mediated oxidation of TMB is as follows: Figure 5 shown. Figure 5 A is the curve of absorbance variation over time of Tf@ACNCs catalyzing H2O2 with different concentrations to oxidize TMB. The results show that Tf@ACNCs can catalyze H2O2 to oxidize TMB. Figure 5 B is in Figure 5 The Lineweaver-Burk fitting curve of Tf@ACNCs catalyzing H2O2 was drawn based on A, and Vmax =4.64×10 -8 Ms -1 , indicating that it can be used as an efficient POD mimic. Figure 5 A and Figure 5 B The results show that Tf@ACNCs have POD-like activity. Figure 5 C is the absorption spectrum of TMB oxidation under 488nm visible light excitation when Tf@ACNCs concentration is 10 / 20 / 30μmol / L. The results show that Tf@ACNCs can oxidize TMB under 488nm visible light excitation, and the oxidation performance gradually increases with the increase of concentration, indicating that Tf@ACNCs has good photosensitized oxidation activity. Figure 5 D is the absorption spectrum of Tf@ACNCs catalyzing H2O2 and 488nm visible light to oxidize TMB. The results show that Tf@ACNCs has the strongest performance in oxidizing TMB under the combined conditions of catalyzing H2O2 and 488nm visible light excitation. This indicates that after copper doping, Tf@ACNCs has better POD-like activity in catalyzing H2O2, and its oxidation performance can be further enhanced by combining with photosensitized oxidation, generating reactive oxygen species efficiently and from multiple sources, playing a synergistic role.

[0087] Experimental Example 4: EPR (Electron Paramagnetic Resonance) Detection Test of Gold-Copper Nanoclusters Synthesized by Tf Mediated by the Present Invention

[0088] 1. Test methods

[0089] The EPR detection test was used to explore the types of active oxygen generated by the Tf@ACNCs prepared in Example 3 after being photoexcited. The specific test method is as follows:

[0090] (1)O2 ·- Detection: A certain volume of Tf@ACNCs was freeze-dried and dissolved with an equal volume of DMSO. 20 μmol / L DMSO-dissolved Tf@ACNCs was added to 80 μL DMSO. 1 μL DMPO (5,5-dimethyl-1-pyrroline-N-oxide) was added and then irradiated with 3W 360nm LED light for 10 minutes. Then, an appropriate amount of sample was drawn with a capillary tube and O2 was detected using an EPR (electron paramagnetic resonance) instrument. ·- The same method was used to detect O2 using Tf@AuNCs instead of Tf@ACNCs. ·- .

[0091] (2) ·OH detection: 20 μmol / L Tf@ACNCs were added to 80 μL ultrapure water, and 1 μL DMPO was added. The mixture was illuminated with 3W 360nm LED light for 10 minutes. Then, an appropriate amount of sample was drawn with a capillary tube and ·OH was detected using an EPR instrument. The same method was used to detect ·OH using Tf@AuNCs instead of Tf@ACNCs.

[0092] (3) 1 O2 detection: Add 20μmol / L Tf@ACNCs to 80μL ultrapure water, add 1μLTEMP (2,2,6,6-tetramethylpiperidine) and illuminate with 3W 360nm LED light for 10 minutes, then draw an appropriate amount of sample with a capillary and detect with an EPR instrument 1 O2. The same method was used to detect Tf@AuNCs instead of Tf@ACNCs. 1 O2.

[0093] The EPR detection test was used to investigate whether the Tf@ACNCs prepared in Example 3 catalyzed H2O2 to generate ·OH. The specific test method is as follows:

[0094] Take 50μL Na2HPO4 / NaH2PO4 buffer, add 20μL ultrapure water and 20μL 50mmol / LH2O2, then add 10μmol / L Tf@ACNCs, add 1μL DMPO, react for 2 minutes, then use a capillary to draw an appropriate amount of sample, and use an EPR instrument to detect ·OH. Use Tf@AuNCs instead of Tf@ACNCs to detect ·OH using the same method.

[0095] 2. Test results

[0096] Figure 6 This is the EPR detection test result of the gold-copper nanoclusters synthesized by Tf mediation of the present invention.

[0097] like Figure 6 As shown in A, Tf@ACNCs and Tf@AuNCs appeared O2 after being excited by 360nm light. ·- The characteristic peaks of O2 ·- .

[0098] like Figure 6 As shown in B, neither Tf@ACNCs nor Tf@AuNCs showed the characteristic peak of ·OH after being excited by 360nm light, indicating that ·OH was not generated.

[0099] like Figure 6 As shown in C, Tf@ACNCs and Tf@AuNCs did not show any 1 The characteristic peak of O2 indicates that no1 O2.

[0100] The above results show that Tf@ACNCs and Tf@AuNCs are both Type I photosensitizers, and copper doping does not affect the photosensitized oxidation mechanism and performance of gold nanoclusters.

[0101] like Figure 6 As shown in D, after Tf@ACNCs catalyzed H2O2, a characteristic peak of ·OH appeared, indicating that ·OH was generated, and it had POD-like activity, and its performance was significantly stronger than that of Tf@AuNCs, indicating that copper doping enhanced its POD-like activity. The above results show that Tf@ACNCs has multi-source active oxygen generation performance, which can generate O2 through the photosensitive oxidation mechanism. ·- , and can catalyze the generation of ·OH through POD-like activity.

[0102] Experimental Example 5: Effect of the gold-copper nanoclusters synthesized by Tf-mediated synthesis on the generation of reactive oxygen species in melanoma cells A375 under 488 nm illumination and without illumination

[0103] 1. Test methods

[0104] The reactive oxygen species specific probe was used to explore the types and effects of reactive oxygen species generated by Tf@ACNCs prepared in Example 3 in melanoma cells A375 under 488 nm illumination and without illumination. The specific experimental method is as follows:

[0105] (1) Plating: Adjust the concentration of the digested A375 cell suspension to 1.0×10 5 / well, seeded into 24-well plates and incubated for 24 hours.

[0106] (2) Adding drugs: Add 20 μmol / L Tf@ACNCs to the plate. Place the plate in an incubator and incubate for 12 hours. Set up a group without drug and without light, a group without drug and light, a group with drug and without light, and a group with drug and light. Use Tf@AuNCs instead of Tf@ACNCs to set up the same groups.

[0107] (3) Illumination: Use LED light with an excitation wavelength of 488 nm to illuminate the experimental group that needs illumination. The power is 1.5 W, and the illumination time is 15 minutes. Incubate for 4 hours.

[0108] (4) Staining: The culture medium was discarded, and the cells were washed once with PBS. Then, DHE (dihydroethidium, a superoxide anion fluorescent probe) / Hoechst 33342 (dibenzhydrylamine, a cell nucleus stain), HPF (hydroxyphenylfluorescein, a hydroxyl free radical fluorescent probe) / Hoechst 33342, and SOSG (singlet oxygen green fluorescent probe) / Hoechst 33342 working solutions (DHE was 5 μmol / L, Hoechst 33342 was 10 μmol / L, HPF was 10 μmol / L, and SOSG was 5 μmol / L) were added to the cells for reaction in the dark for 30 min.

[0109] (5) Image acquisition: The cells were washed once with PBS and images were acquired using an inverted fluorescence microscope. Figure 7 The Merge in represents the superposition of two images, and the subsequent experiments are the same.

[0110] 2. Test results

[0111] The results of the Tf-mediated synthesis of gold-copper nanoclusters in the present invention generating reactive oxygen species in melanoma cells A375 under 488 nm light irradiation and no light irradiation are as follows: Figure 7 As shown. Figure 7 As shown in A, compared with the other groups, cells in the Tf@ACNCs+light-irradiated group and the Tf@AuNCs+light-irradiated group showed red fluorescence, indicating that O2 ·- .like Figure 7 As shown in Figure B, compared with other groups, cells in the Tf@ACNCs+non-illumination group and the Tf@ACNCs+illumination group showed green fluorescence, indicating that they catalyzed the overexpressed H2O2 in tumor cells to generate ·OH, while no cells in the Tf@AuNCs+non-illumination group and the Tf@AuNCs+illumination group showed green fluorescence, which may be due to insufficient ·OH generated by Tf@AuNCs in cells, indicating that its performance in catalyzing H2O2 to produce ·OH is low. Figure 7 As shown in C, all groups showed no green fluorescence, indicating that no 1 The above results show that Tf@ACNCs can produce ·OH in A375 cells without illumination and can also produce O2 after illumination. ·- , indicating that it has the ability to generate multiple sources of reactive oxygen species in cells.

[0112] Experimental Example 6: Effect of the gold-copper nanoclusters synthesized by Tf-mediated synthesis of the present invention on the inhibition of A375 proliferation under 488 nm illumination and without illumination

[0113] 1. Test methods

[0114] Calcein-AM (calcein acetyl methyl ester, live cell indicator) / PI (propidium iodide, dead cell indicator) staining was used to detect the live / dead cell results of A375 treated with Tf@ACNCs prepared in Example 3 under no illumination and 488 nm illumination conditions. The specific test method is as follows:

[0115] (1) Plating: Adjust the concentration of the digested A375 cell suspension to 1.0×10 5 / well, seeded into 24-well plates and incubated for 24 hours.

[0116] (2) Adding drugs: Add 20 μmol / L Tf@ACNCs to the plate. Place the plate in an incubator and incubate for 12 hours. Set up a group without drug and without light, a group without drug and light, a group with drug and without light, and a group with drug and light. Use Tf@AuNCs instead of Tf@ACNCs to set up the same groups.

[0117] (3) Illumination: The experimental group that needs illumination was illuminated with LED light with an excitation wavelength of 488 nm. The power was 1.5 W and the illumination lasted for 15 minutes. Incubate for 4 hours.

[0118] (4) Calcein-AM / PI staining: discard the culture medium, wash the cells once with PBS, and then react with the cells with Calcein-AM / PI staining working solution (Calcein-AM is 2 μmol / L, PI is 8 μmol / L) in the dark for 30 minutes.

[0119] (5) Image acquisition: The cells were washed once with PBS and images were acquired using an inverted fluorescence microscope.

[0120] Annexin V-FITC (fluorescein isothiocyanate labeled annexin V) / PI flow cytometry was used to detect the effect of Tf@ACNCs prepared in Example 3 on inducing A375 apoptosis under no illumination and 488 nm illumination conditions. The specific test method is as follows:

[0121] (1) Plating: Adjust the concentration of digested A375 cell suspension to 3.0×10 5 / well, seeded into 6-well plates and incubated for 24 hours.

[0122] (2) Adding drugs: Add 20 μmol / L Tf@ACNCs to the well plate. Place the well plate in an incubator and incubate for 12 hours. Set up a group without drug and without light, a group without drug and light, a group with drug and without light, and a group with drug and light. Each group has 3 duplicate wells. Use Tf@AuNCs instead of Tf@ACNCs to set up the same groups.

[0123] (3) Illumination: Use LED light with an excitation wavelength of 488 nm to illuminate the experimental group that needs illumination. The power is 2 W, and the illumination time is 15 minutes. Incubate for 4 hours.

[0124] (4) Annexin V-FITC / PI staining: Collect the culture medium, wash the cells once with PBS and collect them, centrifuge them together with the digested cells, resuspend the cells with 100 μL of staining solution, add 5 μL of Annexin V-FITC and 10 μL of PI staining solution. Mix well and react for 15 minutes, then add 400 μL of staining solution.

[0125] (5) Detection: Detection of Annexin V-FITC by flow cytometry — / PI — (Normal cells, Q4), AnnexinV-FITC + / PI — (early apoptotic cells, Q3), Annexin V-FITC + / PI + (late apoptotic cells, Q2), Annexin V-FITC — / PI + (necrotic cells, Q1) ratio.

[0126] (6) Calculation: The higher the apoptosis rate, the stronger the inhibitory effect on proliferation. Cell apoptosis rate (%) = early apoptotic cell rate (%) + late apoptotic cell rate (%). Data are expressed as mean ± standard deviation (Mean ± SD) and analyzed by one-way ANOVA using GraphPad Prism software. When the p value is less than 0.05, it is considered statistically significant. *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001.

[0127] 2. Test results

[0128] The results of the live / dead cells of A375 cells treated with the gold-copper nanoclusters synthesized by Tf-mediated treatment of the present invention under no illumination and 488 nm illumination are as follows: Figure 8 As shown. Figure 8As shown in the figure, compared with the Tf@ACNCs+non-illumination group and the Tf@AuNCs+non-illumination group, the cells produced red fluorescence, indicating that Tf@ACNCs can kill A375 without illumination, which may be due to catalyzing the overexpressed H2O2 in tumor cells to produce reactive oxygen. Compared with other groups, the Tf@ACNCs+illumination group had the least green fluorescent cells and the most red fluorescent cells, indicating that Tf@ACNCs had a better effect in killing A375 without illumination, and further killed A375 after illumination, which may be through the synergy of photosensitive oxidation and H2O2 catalysis, generating reactive oxygen from multiple sources, and enhancing the inhibitory effect on tumors.

[0129] The results of the gold-copper nanoclusters synthesized by Tf-mediated induction of A375 apoptosis under no illumination and 488 nm illumination are as follows: Fig. 9 As shown. Fig. 9 As shown in A, compared with the Tf@ACNCs+non-illumination group and the Tf@AuNCs+non-illumination group, the number of apoptotic cells increased, indicating that Tf@ACNCs induced A375 apoptosis, which may be caused by catalyzing the overexpressed H2O2 in tumor cells to produce reactive oxygen species. Compared with other groups, the Tf@ACNCs+illumination group had the strongest effect in inducing A375 apoptosis, indicating that Tf@ACNCs further induced A375 apoptosis under the condition of illumination, which may be through the production of reactive oxygen species through photosensitive oxidation, which synergized with the reactive oxygen species produced by the catalytic effect of H2O2, enhancing the effect of inducing A375 apoptosis. Fig. 9 The statistical analysis of B also further demonstrated this trend.

[0130] The above test results show that compared with undoped copper gold nanoclusters (Tf@AuNCs), the gold-copper nanoclusters synthesized by Tf-mediated synthesis of the present invention enhance the effect of catalyzing H2O2 to produce reactive oxygen species and thus inhibit tumors under non-illumination conditions, and can also synergize with the photosensitive oxidation under illumination conditions to produce reactive oxygen species to inhibit tumors, thereby enhancing the tumor efficacy.

[0131] From the test results of the above test examples, it can be seen that the POD-like activity of the gold-copper nanoclusters synthesized by Tf-mediated synthesis in catalyzing H2O2 to produce ·OH is significantly improved, so its effect of inhibiting tumor proliferation is also significantly improved. Compared with the gold nanoclusters not doped with copper, the gold-copper nanoclusters synthesized by Tf-mediated synthesis of the present invention can efficiently generate reactive oxygen from multiple sources through H2O2 catalysis and photosensitive oxidation, thereby enhancing the inhibitory effect on tumors.

[0132] In summary, the present invention adopts a simple one-pot method to synthesize gold-copper nanoclusters with Tf as a ligand, which overcomes the problems of complex multi-source reactive oxygen generation systems and cumbersome preparation methods in the prior art; compared with copper-free doped gold nanoclusters synthesized with Tf as a ligand, the gold-copper nanoclusters synthesized with Tf as a ligand in the present invention can be used as a simple multi-source reactive oxygen generation system, which can generate superoxide anions by sensitizing oxygen through photosensitive oxidation activity and generate hydroxyl radicals by catalyzing hydrogen peroxide through peroxidase-like activity, thereby solving the defect of poor catalytic activity of peroxidase-like systems based on gold nanoclusters, realizing efficient generation of multi-source reactive oxygen, achieving the effect of enhancing tumor treatment, and having good application prospects.

[0133] Although the invention has been described herein with reference to illustrative embodiments thereof, it should be understood that numerous other modifications and implementations can be devised by those skilled in the art that will fall within the scope and spirit of the principles disclosed herein.

Claims

1. A gold-copper nanocluster with high-efficiency multi-source active oxygen generation performance, characterized in that: The gold-copper nanocluster is synthesized using Tf as a ligand; the Tf is full transferrin, iron-saturated transferrin or ferroferrin, and the element distribution map obtained by transmission electron microscopy shows that the distribution areas of gold and copper in the nanocluster particles are overlapping and uniform.

2. The gold-copper nanoclusters with high-efficiency multi-source active oxygen generation performance according to claim 1, characterized in that: The molar mass ratio of the tetrachloroauric acid to copper sulfate pentahydrate is 1:(0.05-0.3).

3. The gold-copper nanoclusters with high-efficiency multi-source active oxygen generation performance according to claim 1, characterized in that: The gold-copper nanocluster has gold atoms and copper atoms as metal cores and Tf as ligand shells.

4. A method for preparing gold-copper nanoclusters with high-efficiency multi-source active oxygen generation performance, characterized in that: The gold-copper nanoclusters are prepared from tetrachloroauric acid, copper sulfate pentahydrate, Tf, ascorbic acid and sodium hydroxide as raw materials; The molar mass ratio of the tetrachloroauric acid to copper sulfate pentahydrate is 1:(0.05-0.3).

5. The method for preparing gold-copper nanoclusters with high-efficiency multi-source active oxygen generation performance according to claim 4, characterized in that: The molar mass ratio of the tetrachloroauric acid to copper sulfate pentahydrate is 1:0.

2.

6. The method for preparing gold-copper nanoclusters with high-efficiency multi-source active oxygen generation performance according to claim 4, characterized in that: The preparation method of the gold-copper nanoclusters comprises the following steps: (1) dissolving Tf in ultrapure water to prepare a suspension; (2) adding tetrachloroauric acid solution, copper sulfate pentahydrate solution, ascorbic acid solution and sodium hydroxide solution to the suspension in sequence, mixing and reacting to obtain a reaction solution; (3) Centrifuging and ultrafiltering the reaction solution to obtain gold-copper nanoclusters.

7. The method for preparing gold-copper nanoclusters with high-efficiency multi-source active oxygen generation performance according to claim 6, characterized in that: The amount of Tf used in step (1) is greater than or equal to 20 mg, and the amount of ultrapure water used is greater than or equal to 3 mL; a heat-collecting constant temperature magnetic stirrer with a temperature less than or equal to 37° C. is used for constant temperature heating and stirring; The ratio of Tf to HAuC14 in step (2) is 0.005-0.006 mmol HAuC14 per 20 mg Tf, the molar mass ratio of HAuC14 to CuSO4 is 1:(0.05-0.3), and the mass ratio of Tf to ascorbic acid is 1:0.00017-0.00018; Using the sodium hydroxide solution to adjust the pH value to 10-13; Stirring during the reaction, the stirring time is 6 to 8 hours, and the temperature is 25°C to 37°C; In step (3), centrifugation is performed at a temperature less than or equal to 4°C.

8. The method for preparing gold-copper nanoclusters with high-efficiency multi-source active oxygen generation performance according to claim 7, characterized in that: The concentration of the HAuC14 solution is 100 mmol / L, and the concentration of the CuSO4·5H2O solution is 100 mmol / L; The concentration of the ascorbic acid solution is 3.5 mg / mL; the ascorbic acid solution is added 5 minutes after the copper sulfate pentahydrate solution is added; Ten minutes after the addition of ascorbic acid, NaOH solution was added to adjust the pH; The concentration of the NaOH solution is 0.5 mol / L; Stir at a constant speed of 800-1200 rpm and a constant temperature of 37°C; Ultrafiltration uses 10KD ultrafiltration tube; Centrifuge at 4000 rpm and 4°C for 20 minutes.

9. Use of the gold-copper nanoclusters with high-efficiency multi-source active oxygen generation performance as claimed in any one of claims 1 to 3 in tumor treatment drugs.

10. Use of the gold-copper nanoclusters obtained by the method for preparing the gold-copper nanoclusters with high-efficiency multi-source active oxygen generation performance according to any one of claims 4 to 8 in tumor treatment drugs.

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