A bimetallic copper-palladium nanoscale enzyme and a preparation method and application thereof

Abstract

The present application relates to a kind of bimetallic copper palladium nanoscale enzyme and its preparation method and application, the bimetallic copper palladium nanoscale enzyme surface is decorated with cysteamine.The bimetallic copper palladium nanoscale enzyme described in the present application can be enriched in tumor site by passive targeting, can be used as radiotherapy sensitizer to inhibit the radiotherapy resistance of tumor, and improve the treatment effect of radiotherapy.

Description

Technical Field

[0001] This invention relates to the field of radiotherapy drug technology, specifically to a bimetallic copper-palladium nanozyme, its preparation method, and its application. Background Technology

[0002] In clinical practice, radiotherapy is commonly used to treat breast cancers. Due to the unique microenvironment of tumors—hypoxia, weak acidity, high expression of H2O2, and the influence of glutathione—tumor cells are less sensitive to radiation, leading to less than ideal killing effects and even recurrence. High-dose radiotherapy can also cause radiation damage to normal tissues and organs surrounding the tumor target area, resulting in radiotherapy side effects. Radiosensitizers work by increasing the radiosensitivity of the tumor target area without damaging surrounding normal tissues.

[0003] In recent years, the research and application of nanozymes have received widespread attention. Compared with natural enzymes, nanozymes have advantages such as stable properties, high catalytic activity, and low cost, and are therefore widely used in energy, chemical engineering, and biomedical engineering. The tumor microenvironment contains higher levels of H2O2 and glutathione than normal tissues. Utilizing this characteristic, many studies have focused on constructing nanozymes to catalyze the production of O2 from H2O2, thereby alleviating the hypoxic microenvironment within tumors. Nanozymes with peroxidase activity can directly convert H2O2 into linear reactive oxygen species, inducing tumor cell apoptosis; some nanozymes can oxidize glutathione, inhibiting the repair of radiation damage to tumor cells caused by glutathione. Therefore, nanozymes show great potential for application in enhancing the radiosensitization of tumors.

[0004] In addition, nano-sensitizers need to avoid causing side effects in the body, and at the same time, they need to ensure that the nano-sensitizers are enriched at the tumor site to better enhance the radiotherapy effect. Summary of the Invention

[0005] The purpose of this invention is to provide a copper-palladium nanozyme coated with cysteamine and its preparation method. This nanosystem can effectively target tumor sites and release copper-palladium nanozymes therein, which can alleviate tumor hypoxia, enhance the tumor-killing effect of radiation, and at the same time consume glutathione in tumor cells, inhibiting their self-repair, thereby improving the radiosensitivity of tumors.

[0006] The solution adopted by the present invention to solve the above-mentioned technical problems is as follows:

[0007] A nanozyme, wherein the nanozyme is a bimetallic copper-palladium nanozyme with cysteine ​​surface modified.

[0008] Preferably, the cysteine ​​loading is 4 wt% to 11 wt% of the total weight of the nanozyme, more preferably 4.5 to 10.3 wt%, for example 4.5 wt%, 5.0 wt%, 6.0 wt%, 7.0 wt%, 8.0 wt%, 9.0 wt%, 10.0 wt%, and 10.3 wt%.

[0009] Preferably, the weight ratio of copper to palladium in the nanozyme is 1:6 to 6:1, more preferably 1:5.6 to 4.5:1, for example 1:5.6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 4.5:1.

[0010] This invention also provides a method for preparing nanozymes, comprising the following steps:

[0011] (1) Prepare palladium compound solution and copper compound solution respectively;

[0012] (2) Mix the palladium compound solution and the copper compound solution, add a reducing agent to the mixed solution, and carry out a reduction reaction;

[0013] (3) After the reduction reaction is completed, the reaction solution is centrifuged to collect the precipitate, washed and dried to obtain bare bimetallic copper palladium;

[0014] (4) The obtained bare bimetallic copper palladium was modified with cysteine ​​in solution;

[0015] (5) After the reaction is complete, centrifuge to collect the precipitate, wash and dry it to obtain cysteamine-modified bimetallic copper-palladium nanozyme.

[0016] Preferably, the palladium compound is selected from one or more of Na2PdCl4, K2PdCl4, (NH4)2PdCl4, and Pd(acac)2.

[0017] Preferably, the copper compound is selected from one or more of CuCl2 and Cu(acac)2.

[0018] Preferably, the reducing agent is selected from one or more of KBH4, NaBH4, and ascorbic acid.

[0019] Preferably, the solvents used in the palladium compound solution and copper compound solution are organic solvents, and more preferably ethanol, acetone, and chloroform.

[0020] Preferably, the molar ratio of palladium to copper in the mixed solution in step (2) is 1:5 to 5:1, for example, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1.

[0021] Preferably, the molar amount of the reducing agent added in step (2) is 15 to 25 times the sum of the molar amounts of palladium and copper, for example, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times, or 25 times.

[0022] Preferably, the solution used in step (4) is an organic solvent, and more preferably ethanol, acetone, or chloroform.

[0023] Preferably, in step (4), the mass ratio of the bare bimetallic copper palladium to the added cysteine ​​is 1:(5-20), for example, 1:5, 1:8, 1:9, 1:10, 1:11, 1:12, 1:15, or 1:20. The modification method involves dispersing and ensuring sufficient contact between the two substances in a solvent. Dispersion methods include, but are not limited to, ultrasonication and stirring. The preferred reaction time is 2-10 hours, for example, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, or 10 hours. The cysteine ​​loading in the obtained nanozyme is 4 wt% to 11 wt% of the total weight of the nanozyme, for example, 4.0 wt%, 4.5 wt%, 5.0 wt%, 6.0 wt%, 7.0 wt%, 8.0 wt%, 9.0 wt%, 10.0 wt%, 10.3 wt%, or 11.0 wt%. Within a certain range, prolonged reaction time and increased cysteine ​​dosage will lead to increased cysteine ​​loading. When the reaction time exceeds 6 hours and / or the mass ratio of cysteine ​​to bare bimetallic copper-palladium is greater than 6, further increasing the reaction time or the ratio of the two will not significantly increase the cysteine ​​loading.

[0024] The present invention also provides the use of the nanozymes or nanozymes obtained by the above-described preparation methods in the preparation of radiosensitizers.

[0025] The bimetallic copper-palladium nanozyme obtained in this invention can be passively targeted and enriched at the tumor site, thereby inhibiting tumor radiotherapy resistance and improving the therapeutic effect of radiotherapy. Attached Figure Description

[0026] Figure 1 This is a transmission electron microscope (TEM) image of the bare bimetallic copper-palladium obtained in Example 1;

[0027] Figure 2 This is a transmission electron microscope image of the bimetallic copper-palladium nanozyme radiosensitizer obtained in Example 1;

[0028] Figure 3 This is a particle size distribution diagram of the bimetallic copper-palladium nanozyme radiosensitizer obtained in Example 1;

[0029] Figure 4 The absorption spectrum of the bimetallic copper-palladium nanozyme radiosensitizer obtained in Example 1 is shown below.

[0030] Figure 5XPS spectra of the bimetallic copper-palladium nanozyme radiosensitizer obtained in Example 1;

[0031] Figure 6 The GSH oxidation capacity of the bimetallic copper-palladium nanozyme radiotherapy sensitizer obtained in Example 1 was tested.

[0032] Figure 7 The results show the cellular-level sensitization effect of the bimetallic copper-palladium nanozyme radiotherapy sensitizer obtained in Example 1. Detailed Implementation

[0033] To better understand the present invention, the following embodiments are further illustrations of the present invention, but the content of the present invention is not limited to the following embodiments.

[0034] Example 1

[0035] (1) Dissolve 0.1g Na2PdCl4 in 10ml of ethanol and name the resulting solution A;

[0036] (2) Dissolve 0.09g of Cu(acac)2 in 50ml of ethanol and name the resulting solution B;

[0037] (3) Add B to A and stir until the solution is homogeneous;

[0038] (4) Dissolve 0.43g of NaBH4 powder in 20ml of ethanol and name the resulting solution C;

[0039] (5) Add C to the mixture of A and B, and then heat the mixture to 40°C and react for 2 hours;

[0040] (6) Let the precipitation stand overnight, discard the supernatant, and wash the precipitate with ethanol 2-3 times to obtain a black product;

[0041] (7) The obtained black product was vacuum dried overnight to obtain a black powder, namely bare bimetallic copper palladium;

[0042] (8) Take 0.02g of black powder and disperse it in 100ml of ethanol, take 0.2g of cysteine ​​and disperse it in 10ml of ethanol, add the latter to the former, and sonicate for 6h.

[0043] (9) The reaction solution was centrifuged to collect the precipitate, which was then washed with ethanol 2-3 times. The product was then vacuum dried to obtain the bimetallic copper-palladium nanozyme radiosensitizer. The cysteine ​​loading of the product was measured to be 11 wt%.

[0044] Transmission electron microscopy was used to characterize the bare bimetallic copper-palladium obtained during the preparation process and the final product, the bimetallic copper-palladium nanozyme radiosensitizer. The results are as follows: Figure 1 and 2 As shown, both the cysteine-modified and unmodified bimetallic copper-palladium structures are nanostructures.

[0045] The particle size of the bimetallic copper-palladium nanozyme obtained in this embodiment was analyzed, and the results are as follows: Figure 3 As shown, the median particle size is 6 nm.

[0046] Figure 4 The absorption spectrum of the bimetallic copper-palladium nanozyme obtained in this embodiment shows that the bimetallic copper-palladium nanozyme does not have obvious characteristic peaks.

[0047] Figure 5 The XPS spectrum of the bimetallic copper-palladium nanozyme obtained in this embodiment shows that the obtained bimetallic copper-palladium nanozyme contains copper and palladium elements.

[0048] The bimetallic copper-palladium nanozyme obtained in this embodiment was used to test its GSH oxidation capacity. The experimental procedure involved adding the bimetallic copper-palladium nanozyme to a mixed solution containing 0.1 mmol / L GSH and 25 mmol / L H₂O₂. The generated hydroxyl radicals were then tested with methylene blue. The hydroxyl radicals consumed the methylene blue, and the results are as follows: Figure 6 As shown, compared to the control group which only added PBS, the bimetallic copper-palladium nanozyme can generate hydroxyl radicals and consume methylene blue in a simulated tumor microenvironment with GSH and H2O2 concentrations.

[0049] The bimetallic copper-palladium nanozyme obtained in this embodiment was used to test the cellular sensitization effect. The experimental procedure involved seeding 4T1 cells in the logarithmic growth phase at a density of 5000 cells / well in 96-well plates. After cell attachment, different treatments were administered, including a control group, a bimetallic copper-palladium nanozyme group, a radiotherapy-only group, and a bimetallic copper-palladium nanozyme plus radiotherapy group. CCK8 reagent was then added, and after color development, the absorbance at 450 nm was measured using an ELISA reader. The results are as follows: Figure 7 As shown, compared with the bimetallic copper-palladium nanozyme group or the radiotherapy group alone, the bimetallic copper-palladium nanozyme plus radiotherapy group can effectively kill tumor cells, and there is a therapeutic synergistic effect between nanozyme and radiotherapy.

[0050] Example 2

[0051] (1) Dissolve 0.1g Pd(acac)2 in 50ml ethanol and name the resulting solution A;

[0052] (2) Dissolve 0.1g Cu(acac)2 in 50ml of ethanol and name the resulting solution B;

[0053] (3) Add B to A and stir until the solution is homogeneous;

[0054] (4) Dissolve 3g of ascorbic acid in 50ml of ethanol and name the resulting solution C;

[0055] (5) Add C to the mixture of A and B, and then heat the mixture to 30°C and react for 2 hours;

[0056] (6) Let the precipitation stand overnight, discard the supernatant, and wash the precipitate with ethanol 2-3 times to obtain a black product;

[0057] (7) The obtained black product was vacuum dried overnight to obtain a black powder, namely bare bimetallic copper palladium;

[0058] (8) Take 0.02g of black powder and disperse it in 100ml of ethanol, take 0.2g of cysteine ​​and disperse it in 10ml of ethanol, add the latter to the former, and sonicate for 6h.

[0059] (9) Centrifuge the reaction solution to collect the precipitate, and wash the precipitate with ethanol 2-3 times. Dry the product under vacuum to obtain the bimetallic copper palladium nanozyme radiosensitizer.

[0060] Example 3

[0061] (1) Dissolve 0.02g Na2PdCl4 in 10ml ethanol and name the resulting solution A;

[0062] (2) Dissolve 0.1g of Cu(acac)2 in 50ml of ethanol and name the resulting solution B;

[0063] (3) Add B to A and stir until the solution is homogeneous;

[0064] (4) Dissolve 0.40g NaBH4 powder in 20ml ethanol and name the resulting solution C;

[0065] (5) Add C to the mixture of A and B, and then heat the mixture to 40°C and react for 2 hours;

[0066] (6) Let the precipitation stand overnight, discard the supernatant, and wash the precipitate with ethanol 2-3 times to obtain a black product;

[0067] (7) The obtained black product was vacuum dried overnight to obtain a black powder, namely bare bimetallic copper palladium;

[0068] (8) Take 0.02g of black powder and disperse it in 100ml of ethanol, take 0.2g of cysteine ​​and disperse it in 10ml of ethanol, add the latter to the former, and sonicate for 6h.

[0069] (9) Centrifuge the reaction solution to collect the precipitate, wash the precipitate with ethanol 2-3 times, and vacuum dry the product to obtain the bimetallic copper palladium nanozyme radiosensitizer.

[0070] Example 4

[0071] (1) Dissolve 0.1g Na2PdCl4 in 10ml of ethanol and name the resulting solution A;

[0072] (2) Dissolve 0.02g of Cu(acac)2 in 50ml of ethanol and name the resulting solution B;

[0073] (3) Add B to A and stir until the solution is homogeneous;

[0074] (4) Dissolve 0.40g NaBH4 powder in 20ml ethanol and name the resulting solution C;

[0075] (5) Add C to the mixture of A and B, and then heat the mixture to 40°C and react for 2 hours;

[0076] (6) Let the precipitation stand overnight, discard the supernatant, and wash the precipitate with ethanol 2-3 times to obtain a black product;

[0077] (7) The obtained black product was vacuum dried overnight to obtain a black powder, namely bare bimetallic copper palladium;

[0078] (8) Take 0.02g of black powder and disperse it in 100ml of ethanol, take 0.2g of cysteine ​​and disperse it in 10ml of ethanol, add the latter to the former, and sonicate for 6h.

[0079] (9) Centrifuge the reaction solution to collect the precipitate, wash the precipitate with ethanol 2-3 times, and vacuum dry the product to obtain the bimetallic copper palladium nanozyme radiosensitizer.

[0080] Table 1 shows the ICP-OES analysis results of the bimetallic copper-palladium nanozyme prepared in Example 3. As can be seen from Table 1, the copper content in the sample is 53.0 wt% and the palladium content is 11.6 wt%.

[0081] Table 1. ICP-OES analysis results of the bimetallic copper-palladium nanozyme prepared in Example 3.

[0082]

[0083] Table 2 shows the inductively coupled plasma optical emission spectrometry (ICP-OES) analysis results of the bimetallic copper-palladium nanozyme prepared in Example 4. As can be seen from Table 2, the copper content in the sample is 9.25 wt%, and the palladium content is 51.8 wt%.

[0084] Table 2. ICP-OES analysis results of the bimetallic copper-palladium nanozyme prepared in Example 4.

[0085]

[0086] The above description is merely a preferred embodiment of the present invention, and should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A method for preparing a bimetallic copper-palladium nanozyme with a surface modified with cysteine, characterized in that, The steps include the following: (1) Prepare palladium compound solution and copper compound solution respectively; the palladium compound is selected from any one or more of Na2PdCl4, K2PdCl4, (NH4)2PdCl4, and Pd(acac)2; the copper compound is selected from any one or more of CuCl2 and Cu(acac)2; (2) Mix the palladium compound solution and the copper compound solution, add a reducing agent to the mixed solution, and carry out a reduction reaction. The molar ratio of palladium to copper in the mixed solution is 1:5 to 5:1; the amount of reducing agent added is 15 to 25 times the sum of the molar amounts of palladium and copper. (3) After the reduction reaction is completed, the reaction solution is centrifuged to collect the precipitate, washed and dried to obtain bare bimetallic copper palladium; (4) The obtained bare bimetallic copper palladium was modified with cysteine ​​in solution; wherein the mass ratio of bare bimetallic copper palladium to added cysteine ​​was 1:5~20; the reaction time was 2~10 hours; and the cysteine ​​loading in the obtained nanozyme was 4 wt%~11 wt% of the total weight of the nanozyme. (5) After the reaction was completed, the precipitate was centrifuged, washed and dried to obtain cysteamine-modified bimetallic copper-palladium nanozyme. The reducing agent is selected from any one or more of KBH4, NaBH4, and ascorbic acid.

2. The preparation method according to claim 1, characterized in that, The solvents used in the palladium compound solution and copper compound solution are organic solvents.

3. A bimetallic copper-palladium nanozyme with a surface modified with cysteine, characterized in that, The nanozyme was prepared using the preparation method described in any one of claims 1-2.

4. The nanozyme according to claim 3, characterized in that, The weight ratio of copper to palladium in the nanozyme is 1:6 to 6:

1.

5. The application of the nanozyme obtained by the preparation method according to any one of claims 1 to 2 or the nanozyme according to any one of claims 3 to 4 in the preparation of radiotherapy sensitizers.

Images