Preparation method and application of carbon dot-stabilized copper peroxide

Abstract

The application discloses a preparation method and application of carbon dot-stabilized copper peroxide, and belongs to the field of nanometer material preparation. The application takes citric acid and urea as raw materials, and takes deionized water as a solvent. The citric acid and the urea are ultrasonically dispersed in the deionized water, and then the solution is placed in a hydrothermal synthesis kettle to react for a certain time under high-temperature conditions. After the reaction is completed, a reaction liquid is taken, a volume of acetone is added, a precipitate is centrifugally separated, and the precipitate is redissolved with deionized water, so that a carbon dot (CD) aqueous solution is obtained. An appropriate amount of the CD aqueous solution is taken, an appropriate amount of copper chloride is added, and stirring is performed for a certain time. The pH is adjusted to alkaline by using sodium hydroxide, hydrogen peroxide is added in an ice bath, and stirring is performed for a certain time. After purification, carbon dot-stabilized copper peroxide (CuO2@CD) is obtained.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of preparation and application of nanomaterials, and particularly relates to a preparation method of carbon dot-stabilized copper peroxide (CuO2@CD) and application thereof in anti-tumor treatment. BACKGROUND

[0002] Cancer seriously threatens human life and health. Traditional cancer treatment methods include surgery, chemotherapy and radiotherapy. Due to poor specificity, these treatment methods often cause damage to normal tissues, resulting in serious side effects. Photothermal therapy (PTT) can achieve precise treatment of deep tumors due to the strong penetration ability of near-infrared laser, and can achieve ablation of deep tumors without damaging normal cells, having the advantage of small side effects. Chemodynamic therapy (CDT) can kill tumor cells by converting endogenous hydrogen peroxide in tumor cells into strong cytotoxic •OH, and can specifically kill deep tumors. However, the tumor region is in an oxygen-deficient state, and the content of endogenous hydrogen peroxide is limited, which limits the chemodynamic therapy. In addition, the high level of glutathione in the tumor region further weakens the effect of •OH. Therefore, there is an urgent need for a kind of nanomaterial that can improve the oxygen deficiency of tumor, the limited hydrogen peroxide and the high level of glutathione to enhance the application of photothermal therapy (PTT) and chemodynamic therapy (CDT) in the field of anti-tumor.

[0003] Some scholars have prepared copper peroxide nanoparticles by using the complexation of copper ions and hydrogen peroxide assisted by hydroxyl ions. The copper peroxide nanoparticles have the characteristics of acid response, and can release hydrogen peroxide and copper ions with Fenton-like catalytic activity under weak acid conditions. The Fenton-like reaction that accompanies can effectively generate hydroxyl radicals, thereby efficiently killing cells and showing good tumor chemodynamic efficacy (J. Am. Chem. Soc. 2019, 141, 25, 9937-9945).

[0004] Carbon dots (CDs) are a new type of carbon nanomaterial, which has the advantages of photothermal property, biocompatibility, permeability, low toxicity, etc. In recent years, researchers have used the photothermal properties of carbon dots for antibacterial therapy (CN113549448A, CN117208893A), or combined with photodynamic and photothermal therapy (CN110339357A, CN110151995A) to become an effective and small side effect phototherapeutic photosensitizer for new photothermal therapy.

[0005] However, the existing copper peroxide has poor water dispersibility. In order to improve the poor dispersibility of copper peroxide and the limited photothermal performance, the present application loads copper peroxide on carbon dots, which is a feasible strategy. SUMMARY

[0006] The present application aims to overcome the deficiencies of the prior art, and provides a preparation method and application of carbon dot-stabilized copper peroxide. Carbon dots are hydrothermally synthesized from citric acid and urea, and then treated with acetone to in-situ synthesize copper peroxide under alkaline conditions to obtain carbon dot-stabilized copper peroxide (CuO2@CD). The particle size of CuO2@CD is uniform, and it has excellent photo-thermal performance, oxygen production performance in an acid environment, peroxide enzyme (POD-like) activity, and glutathione oxidase (GSHox-like) activity. It has considerable application potential in the photothermal treatment and chemical kinetic treatment of cancer.

[0007] To achieve the above-mentioned application purposes, the technical scheme adopted by the present application is as follows:

[0008] A preparation method of carbon dot-stabilized copper peroxide, specifically comprising the following steps:

[0009] (1) Ultrasonically disperse citric acid and urea in deionized water, and add them into a hydrothermal synthesis kettle to obtain solution I through hydrothermal reaction; add several times the volume of acetone into solution I, and centrifugally separate the precipitate to obtain CD.

[0010] In the formula, the molar ratio of citric acid to urea is 1-2:0.5-1. The temperature of the hydrothermal synthesis reaction is 100-200 °C, and the time is 3-12 h. The volume of acetone added into solution I is 2-5 times that of solution I.

[0011] Here, the citric acid can also be folic acid, amino acid, or other water-soluble weak organic acids, and the urea can also be thiourea, ethylenediamine, or other binary amines.

[0012] (2) Disperse CD obtained in step (1) in deionized water to obtain a CD dispersion liquid, add an appropriate amount of copper salt solution to stir, and adjust the pH to alkaline with sodium hydroxide to obtain solution II.

[0013] In the formula, the mass ratio of copper salt to CD is 0.01-0.1:1, and the pH of solution II is adjusted to 8-10 with sodium hydroxide. Here, the copper salt can be copper chloride, copper nitrate, copper sulfate, or the like.

[0014] Preferably, the volume ratio of the CD dispersion liquid to the copper salt solution is 1:1.

[0015] (3) Add hydrogen peroxide solution to solution II in an ice bath and stir for a certain period of time to obtain solution III.

[0016] In the formula, the hydrogen peroxide must be added dropwise under ice bath, and the final concentration of hydrogen peroxide is 200-500 mM. The stirring time is 0.5-3 h.

[0017] (5) Purify solution III by dialysis, and centrifugally separate the precipitate to obtain carbon dot-stabilized copper peroxide.

[0018] The dialysis time is 12-24 h.

[0019] The application also provides the application of the CuO2@CD in anti-tumor treatment. After acetone treatment, the photothermal performance of the CD is significantly enhanced, so that the CuO2@CD has excellent photothermal effect. In addition, the copper peroxide synthesized in situ in the CD can be decomposed into hydrogen peroxide and copper ions in the weak acid environment of the tumor, the hydrogen peroxide can not only continue to decompose to generate oxygen under acidic conditions and help to improve the tumor hypoxic environment, but also can be catalyzed by the peroxidase-like activity of the copper ions to form •OH, so as to achieve specific killing of tumor cells. Finally, the copper ions can oxidize glutathione and reduce its content, so as to achieve enhanced chemical kinetic therapy effect. In combination with the above performances, the CuO2@CD has considerable application potential in PTT and CDT anti-tumor treatment.

[0020] The photothermal performance of the CuO2@CD provided by the application is evaluated by detecting the temperature rise of the CuO2@CD under 808 nm laser irradiation by using a thermal imager. Within 5 min, 500 μg / mL CuO2@CD is raised from 15°C to 60.0°C under 808 nm laser irradiation. While the CuO2@CD not treated by acetone and the nano-CuO2 alone are raised to 48.3°C and 38.3°C respectively under the same conditions, which indicates that the photothermal performance of the CuO2@CD is greatly enhanced by loading carbon dots and acetone treatment, and the CuO2@CD has good PTT potential.

[0021] The oxygen production capacity of the CuO2@CD in an acid environment provided by the application is evaluated by detecting the oxygen content of the CuO2@CD aqueous solution at pH 6.5 by using a portable dissolved oxygen meter. 500 μg / mL CuO2@CD can raise the oxygen content in the system from 2 mg / L to 11 mg / L within 20 min. It indicates that the CuO2@CD can produce a large amount of oxygen under weak acidic conditions, which is conducive to the improvement of the tumor hypoxic environment by the CuO2@CD.

[0022] The peroxidase-like activity of the CuO2@CD provided by the application is evaluated by the detection results of the hydroxyl radical detection reagent methylene blue (MB). GSH is added to the acetic acid buffer containing MB, and the final concentration of GSH is controlled to be 100 μM. Then CuO2@CD is added to the mixed solution and the concentration is controlled to be 100 μg / mL. After 10 min of reaction, the absorption of the solution at a wavelength of 650 nm is detected. The CuO2@CD can eliminate the absorption of MB at 650 nm in a short time, which indicates that a large amount of •OH is generated, and the CuO2@CD has application potential in chemical kinetic therapy.

[0023] The CuO2@CD provided by the application is measured by detecting the ultraviolet characteristic absorption change of glutathione detection reagent DTNB in a 200 μM glutathione system at different reaction times. 100 μg / mL of CuO2@CD can remove GSH in the system within 120 min, which embodies excellent glutathione peroxidase-like activity, which helps to alleviate the excessive expression of glutathione in the tumor microenvironment and improve the effect of chemical kinetics treatment.

[0024] The beneficial effects of the application are:

[0025] The application synthesizes citric acid urea carbon dots (CD) by a hydrothermal synthesis method, and the photo-thermal performance of the CD is greatly improved by acetone treatment. Compared with nano copper peroxide, the copper peroxide loaded with carbon dots is not easy to aggregate in aqueous solution and has good dispersity. The photo-thermal performance of CuO2@CD is significantly improved, and good photo-thermal performance can be obtained at a lower concentration, thereby reducing the toxicity of high-concentration copper peroxide to the body. The in-situ synthesis of copper peroxide in an ice bath makes CuO2@CD have uniform particle size. When the mass ratio of copper chloride to CD is 1:10 and the stirring time is 2 h, CuO2@CD has the best enzyme-like activity. CuO2@CD can be decomposed into copper ions and hydrogen peroxide in a weak acid environment of a tumor, and the hydrogen peroxide can continue to decompose into oxygen to improve the tumor hypoxic environment, and can also be catalyzed by copper ions to produce highly toxic ·OH to kill tumor cells. Finally, the copper ions can oxidize GSH into GSSG, weaken the antioxidant stress ability of tumor cells, and achieve the enhancement of the effect of chemical kinetics treatment. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The TEM image of CD.

[0027] Figure 2 The TEM image of CuO2@CD.

[0028] Figure 3 The XPS full spectrum of CuO2@CD.

[0029] Figure 4 The O 1s orbital XPS spectrum of CuO2@CD.

[0030] Figure 5 The Cu 2p orbital XPS spectrum of CuO2@CD.

[0031] Figure 6 The photo-thermal heating curve of CuO2@CD, CuO2@CD*.

[0032] Figure 7 The acid environment oxygen production curve of CuO2@CD, CuO2@CD#.

[0033] Figure 8 POD-like activity chart of CuO2@CD, CuO2@CD#.

[0034] Figure 9 GSHox-like activity chart of CuO2@CD. DETAILED DESCRIPTION

[0035] In order to better understand the present application, the content of the present application is further illustrated below in combination with examples, but the content of the present application is not limited only to the following examples. Example 1

[0036] Preparation of CuO2@CD: 1 g of citric acid and 0.7 g of urea were taken in a beaker, 30 mL of deionized water was added, and it was ultrasonically dissolved. The solution was transferred to a hydrothermal synthesis kettle and reacted at 150°C for 5 h. After the reaction was completed, 3 times the volume of acetone was added to the reaction solution, and the precipitate was collected by centrifugation. The precipitate was redissolved with deionized water to obtain CD. 5 mL of CD (1 mg / mL) was stirred with 5 mL of CuCl2 (100 μg / mL) for 2 h, and 1M NaOH solution was added to adjust the pH of the mixture to 10. The solution was placed in an ice bath and continuously added with hydrogen peroxide under stirring until the final concentration was 500 mM. After stirring for 30 min, the solution was dialyzed for 12 h and the precipitate was separated by centrifugation to obtain CuO2@CD.

[0037] The TEM of the CD prepared in this example is shown in Figure 1 , and the particle size is within 5 nm. The TEM of CuO2@CD is shown in Figure 2 , and the particle size is about 30 nm. According to the XPS spectrum analysis of Figure 3 , 4 , 5, CuO2@CD mainly contains four elements of C, N, O and Cu. The characteristic peak of O-O bond appears in the XPS spectrum of O 1s orbit, indicating the presence of peroxyl group, and the doublet in the XPS spectrum of Cu 2p orbit also proves the existence of Cu 2+ , indicating the successful preparation of CuO2@CD.

[0038] Performance detection method of CuO2@CD:

[0039] Measurement of the photothermal performance of CuO2@CD:

[0040] Take 200 μL CuO2@CD (500 μg / mL) in a transparent centrifuge tube, use an 808 nm laser (P = 1.5W) to irradiate the liquid for 5 min, record the maximum temperature of the solution every 30 s with a thermal imager, and finally plot the time as the horizontal coordinate and the temperature as the vertical coordinate to obtain the photothermal heating curve of CuO2@CD. CuO2@CD has excellent photothermal performance and can be heated to 60 °C within 5 min under 808 nm laser (P = 1.5) irradiation. See Figure 6 .

[0041] Detection of oxygen production performance of CuO2@CD in acid environment:

[0042] Prepare 10 mL of CuO2@CD at 500 μg / mL with pH 6.5 acetic acid buffer solution, and detect the oxygen content in the solution under constant stirring with a portable dissolved oxygen meter. Plot the time as the horizontal coordinate and the oxygen content as the vertical coordinate to obtain the oxygen production curve of CuO2@CD in acid environment (Figure 6). Figure 7 .

[0043] Detection of CuO2@CD peroxidase-like (POD-like) activity:

[0044] Prepare CuO2@CD at 500 μg / mL with pH 6.5 acetic acid buffer solution, add glutathione (100 μM), mix well for 5 min, then add methylene blue (MB) as a hydroxyl radical detection reagent, mix thoroughly, and detect the ultraviolet full wavelength absorption of the mixed solution after 20 min, while setting up a MB + GSH control group. Finally, plot the wavelength as the horizontal coordinate and the absorbance as the vertical coordinate to obtain the peroxidase-like (POD-like) activity spectrum of CuO2@CD (Figure 7). Figure 8 .

[0045] Detection of CuO2@CD glutathione oxidase-like (GSHox-like) activity:

[0046] Prepare CuO2@CD aqueous solution, take an appropriate amount of CuO2@CD aqueous solution and add to PBS buffer solution (pH 7.2) containing 200 μM GSH to make the final concentration of CuO2@CD 100 μg / mL, the reaction time is from 0 min to 120 min, then add the thiol detection reagent DTNB, mix well for 1 min, and then detect the characteristic absorption at wavelength 412 nm. Finally, plot the wavelength as the horizontal coordinate and the absorbance as the vertical coordinate to obtain the glutathione oxidase-like (GSHox-like) activity spectrum of CuO2@CD (Figure 8). Figure 9 .

[0047] It can be seen that the CuO2@CD prepared in the embodiment has excellent oxygen production capacity in acid environment, peroxidase-like activity and glutathione oxidase-like activity. Example 2

[0048] Preparation of CuO2@CD*: The difference between the present embodiment and Example 1 is that no acetone is added after the hydrothermal reaction of citric acid and urea, and the reaction solution is directly centrifuged and the precipitate is collected. The precipitate is redissolved with deionized water to obtain CD*. 5 mL of CD* (1 mg / mL) is stirred with 5 mL of CuCl2 (50 μg / mL) for 2 h, and CuO2@CD* is prepared according to the method of Example 1. The CuO2@CD* prepared in the present embodiment has poorer photothermal performance than CuO2@CD because it is not treated with acetone, as shown in Figure 6 . It is indicated that the treatment of CD with acetone helps to enhance the photothermal performance. Example 3

[0049] Preparation of CuO2@CD#: The difference between the present embodiment and Example 1 is that the concentration of CuCl2 is 10 μg / mL, i.e. the mass ratio of CuCl2 to CD is 0.05 mg:5 mg. The oxygen production capacity in acid environment and peroxidase-like activity of CuO2@CD# prepared in the present embodiment are shown in Figure 7 , 8 . Because the proportion of copper ions decreases in the synthesis, the CuO2 in CuO2@CD# decreases, resulting in the decrease of oxygen production capacity and enzyme activity. Therefore, the appropriate ratio of raw materials is very important for the performance of CuO2@CD. Comparative Example 1

[0050] Preparation of nano-CuO2: 0.5 g of PVP is dissolved in 5 mL of 0.01 M CuCl2·2H2O aqueous solution, and then 5 mL of 0.02 M NaOH solution is added to adjust the pH. Then 100 μL of 30% H2O2 is slowly added dropwise under ice bath condition. After stirring for 30 min, the precipitate is washed by centrifugation to obtain nano-CuO2. The photothermal performance of nano-CuO2 prepared in the present embodiment is shown in Figure 6 .

[0051] The above is only a basic description of the inventive concept, and several simple deductions or substitutions can be made without departing from the inventive concept, which should be regarded as falling within the protection scope of the present application.

Claims

1. A method for preparing carbon point-stabilized copper peroxide, characterized in that, Includes the following steps: (1) Dissolve a water-soluble organic weak acid and a diamine in deionized water and perform a hydrothermal synthesis reaction to obtain solution I; add excess acetone to solution I, centrifuge to collect the precipitate, and obtain CD; the volume of acetone added is 2 to 5 times the volume of solution I; (2) Disperse the CD obtained in step (1) in deionized water to obtain CD dispersion, add copper salt solution and stir to dissolve, adjust the pH value to 8~10 to obtain solution II; the mass ratio of copper salt to CD is 0.1-0.01:1; (3) Add hydrogen peroxide solution to solution II under ice bath conditions and stir for 0.5-3 h to obtain solution III; (4) Purify solution III to obtain carbon point stable copper peroxide.

2. The method for preparing carbon point stabilized copper peroxide according to claim 1, characterized in that, The molar ratio of the water-soluble organic weak acid to the diamine is 1~2:0.5~1.

3. The method for preparing carbon point stabilized copper peroxide according to claim 1, characterized in that, The hydrothermal synthesis reaction is carried out at a temperature of 100~200 ºC for 3~12 h.

4. The method for preparing carbon point stabilized copper peroxide according to claim 1, characterized in that, The final concentration of hydrogen peroxide is 200-500 mmol / L.

5. The method for preparing carbon point stabilized copper peroxide according to claim 1, characterized in that, The purification process in step (4) includes dialysis, centrifugation, and ultrafiltration.

6. The method for preparing carbon point stabilized copper peroxide according to claim 1, characterized in that, The water-soluble organic weak acid includes one or more of citric acid, folic acid, and amino acids; the diamine includes one or more of urea, ethylenediamine, and thiourea; and the copper salt includes one or more of copper chloride, copper nitrate, and copper sulfate.

7. A carbon point-stabilized copper peroxide prepared by the method according to any one of claims 1-6.

8. The use of carbon point stabilized copper peroxide as described in claim 7 in the preparation of pharmaceutical formulations for photothermal therapy and / or chemikinetic therapy.

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