Process for the preparation of copper-azosalicylate nanoplatelets for cyclooxygenase-2 inhibition

Abstract

The application discloses a preparation method of copper-azo salicylic acid nanosheet for cyclooxygenase-2 inhibition, which comprises the following steps: (1) dissolving copper nitrate trihydrate and azo salicylic acid in an N,N-dimethylformamide solution of ethanol, performing ultrasonic dissolution, and then performing hydrothermal reaction, cooling to room temperature after reaction, and centrifuging the reaction solution to collect the solid; (2) dispersing the solid in water again, obtaining uniform two-dimensional nanosheet after ultrasonic centrifugation, and washing and vacuum drying the nanosheet to obtain the product. The nanosheet is prepared by coordinating the multidentate ligand formed by 5-aminosalicylic acid through an azo bond and Cu 2+ The nanosheet avoids the toxic side effects brought by directly taking COX-2 inhibitors, and can efficiently deliver 5-aminosalicylic acid to a tumor site through enhanced permeability and retention effect, so as to be beneficial to inhibiting the expression of COX-2 / PGE2, and the released Cu 2+ Also can catalyze Fenton-like reaction to produce hydroxyl radicals to kill tumor cells.

Description

Technical Field

[0001] This invention relates to a method for preparing copper-azosalicylic acid nanosheets for cyclooxygenase-2 inhibition. Background Technology

[0002] Cyclooxygenase-2 (COX-2) and its downstream product prostaglandin E2 (PGE2), as a pair of pro-inflammatory lipid mediators, are overexpressed at various stages of cancer. Overexpression of COX-2 / PGE2 proteins can promote tumor progression, inhibit dendritic cell migration and cytotoxic T lymphocyte infiltration into tumor tissues, leading to tumor immune tolerance (Cell 2015, 162(6), 1257-1270; Nano Lett. 2020, 20(9), 6272-6280.). In addition, Santiago Zelenay's group, by screening a compound library containing 1280 approved drugs, found that all types of chemotherapeutic drugs can enhance COX-2 transcription while inhibiting cancer cell proliferation, which is one of the reasons for chemotherapeutic drug resistance. COX-2 / PGE2 expression on dead cancer cells is a major obstacle to cytotoxic therapy-driven tumor immunotherapy (Nat. Commun. 2022, 13(1), 2063). This discovery reveals that inhibiting the COX-2 / PGE2 pathway can serve as a target for remodeling the tumor immune microenvironment for anti-tumor immunotherapy.

[0003] However, high doses of COX-2 / PGE2 inhibitors can cause gastrointestinal bleeding and cardiotoxicity. For example, 5-aminosalicylic acid (5-ASA), a traditional nonsteroidal anti-inflammatory drug (NSAID) and a COX-2 inhibitor, can cause gastrointestinal bleeding when taken orally in high doses. Therefore, further research is needed on how to rationally use COX-2 inhibitors without increasing toxic side effects or keeping them within tolerable limits. Summary of the Invention

[0004] Objective of this invention: This invention aims to provide a method for preparing copper-azosalicylic acid nanosheets for cyclooxygenase-2 inhibition. The copper-azosalicylic acid nanosheets prepared by this method can, under the hypoxic conditions of the tumor microenvironment, have their azo bonds degraded by hypoxic azoreductase, releasing Cu. 2+ Together with 5-aminosalicylic acid (5-ASA), it inhibits cyclooxygenase-2 expression and induces tumor cell death.

[0005] Technical solution: The preparation method of copper-azosalicylic acid nanosheets for cyclooxygenase-2 inhibition according to the present invention includes the following steps:

[0006] (1) Dissolve copper nitrate trihydrate and azo salicylic acid in ethanol in N,N-dimethylformamide (DMF) solution, sonicate to dissolve, and then carry out hydrothermal reaction. After the reaction, cool to room temperature and collect the solid by centrifugation.

[0007] (2) The solid is redispersed in water, and uniform two-dimensional nanosheets are obtained by repeated ultrasonic energy vibration. The two-dimensional nanosheets are then washed and vacuum dried.

[0008] The product, copper-azosalicylic acid nanosheets, has a length of 80–140 nm, a width of 30–80 nm, and a thickness of 2–8 nm. It is uniform in size and has good dispersibility.

[0009] In step (1), the azo salicylic acid is prepared by the following method: 5-nitrososalicylic acid and sodium hydroxide are added to a mixed solvent of water and methanol, zinc powder is added, the mixture is stirred and refluxed overnight, filtered while hot, washed, and the methanol is removed by rotary evaporation. The resulting filtrate is adjusted to pH 2-3 by adding dilute hydrochloric acid under stirring. The solid precipitate is filtered, and the filtered solid product is washed and vacuum dried to obtain azo salicylic acid.

[0010] The mass ratio of 5-nitrososalicylic acid to sodium hydroxide is 1:1.

[0011] The mass ratio of 5-nitrososalicylic acid to zinc powder is 6:5.

[0012] Among them, filtration is carried out while the temperature is still hot when it drops to no less than 60°C.

[0013] In step (1), the molar ratio of copper nitrate trihydrate to azo salicylic acid is 2:3.

[0014] In step (1), the ultrasound time is not less than 30 minutes.

[0015] In step (1), the temperature of the hydrothermal reaction is not lower than 100℃ and the reaction time is 12-14h.

[0016] In step (2), the ultrasonic power is 450W, the ultrasonic process is to turn on for 1 second and turn off for 2 seconds, and repeat the above operation in sequence; the ultrasonic time is 2 hours.

[0017] In step (2), the vacuum drying temperature is 25°C, the vacuum degree is 0.1 MPa, and the vacuum drying time is 24 hours.

[0018] The copper-azo salicylic acid nanosheets of this invention can degrade azo bonds in the hypoxic environment of tumor sites, releasing Cu. 2+And 5-ASA. 5-Aminosalicylic acid (5-ASA) is linked by azo bonds to form hypoxia-responsive azosalicylic acid, and the synthesized azosalicylic acid is a polydentate coordination, which coordinates with copper to form Cu-azosalicylic acid. Based on a specific molar ratio, hydrothermal reaction temperature, and reaction time, two-dimensional nanosheets (Cu-NSs) are generated. The nanosheet structure of Cu-NSs itself enhances its bioavailability. After reaching the tumor site, Cu-NSs are rapidly degraded under the action of hypoxia azo reductase, releasing 5-ASA and Cu. 2+ The released 5-ASA can inhibit the expression of COX-2 and its downstream product PGE2 overexpressed in tumor sites, while Cu 2+ It can catalyze Fenton-like reactions to induce tumor cell death and enhance anti-cancer efficiency; in addition, Cu-NSs nanosheets have a large specific surface area, which is beneficial to the uptake by tumor cells and can also increase the loading of 5-ASA.

[0019] The tumor-killing mechanism of copper-azosalicylic acid nanosheets based on the catalytic Fenton reaction:

[0020] Cu Ⅱ -NSs+GSH→Cu I -NSs+GSSG

[0021] Cu I -NSs+H2O2→Cu Ⅱ -NSs+·OH+OH - .

[0022] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: The method of this invention can prepare hypoxia-responsive copper-azosalicylic acid nanosheets with uniform size and good dispersibility. The nanosheet shape enhances its bioavailability and permeability in vivo (easier for tumor cells to take up), resulting in a large accumulation of Cu-NSs at the tumor site, thereby achieving the purpose of releasing high concentrations of 5-ASA at the tumor site. The nanosheets have a length of 80-140 nm, a width of 30-80 nm, and a thickness of 2-8 nm. The uniform size and good dispersibility of the nanosheets prevent aggregation and precipitation in solutions (PBS or physiological saline), thus avoiding the problem of inactivation. The nanosheets prepared by coordinating Cu with a polydentate ligand formed by azo bonds of 5-aminosalicylic acid avoid the toxic side effects of directly taking COX-2 inhibitors and can efficiently deliver 5-aminosalicylic acid to the tumor site, which is beneficial for downregulating COX-2 / PGE2 expression. The copper-azosalicylic acid nanosheets of this invention are rapidly degraded and release Cu under the action of tumor hypoxia azo reductase. 2+ And 5-aminosalicylic acid, releasing Cu 2+ It can also catalyze Fenton-like reactions to generate hydroxyl radicals that kill tumor cells. Attached Figure Description

[0023] Figure 1 This is a transmission electron microscope image of copper-azosalicylic acid nanosheets from Example 1.

[0024] Figure 2 Selected area electron diffraction pattern of copper-azosalicylic acid nanosheets in Example 1;

[0025] Figure 3 Elemental mapping (Cu, C, N, O) of the energy dispersive X-ray spectrum of copper-azo salicylic acid nanosheets in Example 1;

[0026] Figure 4 The energy dispersive X-ray spectrum of copper-azosalicylic acid nanosheets in Example 1;

[0027] Figure 5 X-ray diffraction pattern of copper-azosalicylic acid nanosheets in Example 1;

[0028] Figure 6 The UV absorption spectra of copper-azo salicylic acid nanosheets degrading and releasing 5-aminosalicylic acid under different time periods (15, 30, 60, 120 min) in an in vitro simulated hypoxic environment are shown.

[0029] Figure 7 Comparison of cell viability of mouse breast cancer 4T1 cells under normoxic and hypoxic conditions with different concentrations of Cu, azosalicylic acid and copper-azosalicylic acid nanosheets.

[0030] Figure 8 To quantitatively analyze the inhibitory effects of Cu, azosalicylic acid, and copper-azosalicylic acid nanosheets on cyclooxygenase-2 (COX-2) protein in 4T1 cells;

[0031] Figure 9 To quantitatively detect prostaglandin E2 (PGE2) secreted by 4T1 cells treated with Cu, azosalicylic acid and copper-azosalicylic acid nanosheets;

[0032] Figure 10 The stability of 100 μg / mL copper-azosalicylic acid nanosheets dissolved in PBS solution (pH=7.4) in Example 1 was tested over 14 days.

[0033] Figure 11 TEM image of the product of Comparative Example 1;

[0034] Figure 12 TEM image of the product of Comparative Example 2;

[0035] Figure 13 This is a TEM image of the product of Comparative Example 3. Detailed Implementation

[0036] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0037] Example 1

[0038] The present invention provides a method for preparing copper-azosalicylic acid nanosheets for cyclooxygenase-2 inhibition, comprising the following steps:

[0039] (1) Preparation of azosalicylic acid: 7.2 g sodium hydroxide and 7.2 g 5-nitrosalicylic acid were dissolved in a mixed solvent of 20 mL deionized water and 35 mL methanol. Then 6 g reduced zinc powder was added to the solution, and the mixture was stirred and refluxed overnight. The solution was cooled to 60 °C and filtered while hot. The solution was then washed three times with a small amount of methanol. The filtrate and washings were further concentrated by a rotary evaporator to remove methanol. The pH of the resulting filtrate was adjusted to 2-3 by adding dilute hydrochloric acid under stirring until no more precipitate was produced. The solid precipitate was filtered, and the yellow product (the solid product after filtration) was collected through a Buchner funnel. The product was washed with deionized water and vacuum dried for 24 h (the vacuum drying temperature was 25 °C and the vacuum degree was 0.1 MPa) to obtain the azosalicylic acid multidentate ligand prodrug (azosalicylic acid linked to 5-aminosalicylic acid through an azo bond to form a multidentate ligand prodrug).

[0040] (2) Preparation of copper-azosalicylic acid nanosheets: 48.2 mg of copper nitrate trihydrate and 91 mg of azosalicylic acid were weighed according to a molar ratio of 2:3 and dissolved in 25 mL of N,N-dimethylformamide solution in ethanol. The solution was sonicated for 30 min and then transferred to a reaction vessel and heated to 100 °C for 12 h. The solution was then cooled to room temperature and the solid was collected by centrifugation. The solid was then redispersed in water and sonicated for 2 h to obtain uniform two-dimensional nanosheets. The sonication power was 450 W, with the cycle time being 1 s and 2 s respectively. The nanosheets were centrifuged (the centrifugation speed was 13000 rpm and the centrifugation time was 15 min). The copper-azosalicylic acid nanosheet product was washed with water and ethanol respectively. After washing, the product was vacuum dried for 24 h (the vacuum drying temperature was 25 °C and the vacuum degree was 0.1 MPa).

[0041] Figure 1 This is a transmission electron microscope image of the copper-azosalicylic acid nanosheets prepared in Example 1. Figure 1 The middle frame image shows a sample of reddish-brown copper-azosalicylic acid nanosheets; the length of the two-dimensional nanosheets is 80–140 nm and the width is 30–80 nm.

[0042] Stability test: A PBS solution (pH = 7.4) containing 100 μg / mL copper-azosalicylic acid nanosheets was prepared. After 14 days, the nanosheets remained monodisperse in the solution, showing good particle size and polymer dispersibility index, with no aggregation observed. Figure 10 As shown.

[0043] The copper-azosalicylic acid nanosheets prepared in Example 1 had a 5-aminosalicylic acid (5-ASA) loading of 92.6%, Cu 2+ The content of copper-azo salicylic acid nanosheets is 7.4%.

[0044] Comparative Example 1

[0045] The preparation method of Comparative Example 1 is basically the same as that of Example 1, the only difference being that copper nitrate trihydrate and azosalicylic acid are mixed in a molar ratio of 1:1. Figure 11 As shown, the obtained product consists of a mixture of nanosheets and rods, with significant aggregation. The loading of 5-aminosalicylic acid (5-ASA) in the product is 78%.

[0046] Comparative Example 2

[0047] The preparation method of Comparative Example 2 is basically the same as that of Example 1, the only difference being that the hydrothermal temperature is 160℃. Figure 12 As shown, the obtained product is spherical, with large particle size and severe aggregation. The loading of 5-aminosalicylic acid (5-ASA) in the product is 82.3%.

[0048] Comparative Example 3

[0049] The preparation method of Comparative Example 3 is basically the same as that of Example 1, the only difference being that the hydrothermal reaction time is 18 hours. Figure 13 As shown, the obtained product has an irregular shape and poor monodispersity. The loading of 5-aminosalicylic acid (5-ASA) in the product is 79.4%.

[0050] The products obtained in Comparative Examples 1-3 exhibited morphologies of rod-like and nanosheet-like structures, spherical and irregular shapes, respectively, and showed poor monodispersity. The loading of 5-aminosalicylic acid (5-ASA) in copper-azosalicylic acid was 78%, 82.3%, and 79.4%, respectively. In summary, the nanoformulations prepared in Comparative Examples 1-3, due to their poor dispersibility, are not suitable for further use in tumor treatment. Furthermore, the azosalicylic acid content in Comparative Examples 1-3 was lower than the 92.6% in Example 1. Clearly, at the same concentration, the copper-azosalicylic acid nanosheets in Example 1 showed a more significant inhibitory effect on COX-2 in 4T1 cells.

[0051] Comparative Example 4

[0052] The preparation method of Comparative Example 4 was basically the same as that of Example 1, the only difference being that the ultrasonic time for obtaining uniform nanoparticles by ultrasonication was 0.5 h. The resulting product had an irregular shape and poor monodispersity. The loading of 5-aminosalicylic acid (5-ASA) in the product was 80.1%.

[0053] Comparative Example 5

[0054] The preparation method of Comparative Example 5 was basically the same as that of Example 1, the only difference being that the ultrasonic power was 350W, on for 1 second and off for 2 seconds. The resulting product had an irregular shape and poor monodispersity. The loading of 5-aminosalicylic acid (5-ASA) in the product was 82.9%.

[0055] Figure 2 Selected area electron diffraction pattern of copper-azo salicylic acid nanosheets under transmission electron microscopy, such as... Figure 2 As shown, copper-azosalicylic acid nanosheets are amorphous materials.

[0056] Figure 3 The elemental mapping diagram of EDS in copper-azosalicylic acid nanosheets is shown below. Figure 3 As shown, the prepared nanosheets contain four elements: Cu, C, N, and O, which are consistent with the elements coordinated with azosalicylic acid and Cu.

[0057] Figure 4 The energy spectrum of EDS in copper-azo salicylic acid nanosheets, and Figure 3 The results were consistent, indicating that the nanosheets contain four elements: Cu, C, N, and O.

[0058] Figure 5 The X-ray diffraction pattern of copper-azosalicylic acid nanosheets is consistent with the SAED results, indicating that the nanosheets generally exhibit moderate crystallinity.

[0059] In vitro degradation of copper-azosalicylic acid nanosheets under simulated hypoxic conditions:

[0060] A PBS solution (pH = 7.4) containing 100 μg / mL copper-azosalicylic acid nanosheets was prepared and stirred under N2 protection. Then, rat liver microsomes (80 μg / mL) and NADPH (50 μM) were added to the solution as co-stimulatory reductases. The mixture was stirred for another 2 h. Samples of the solution were taken at 0 min, 15 min, 30 min, 60 min and 120 min, and the degradation and release of 5-aminosalicylic acid from the copper-azosalicylic acid nanosheets were measured by UV spectrophotometer.

[0061] like Figure 6 As shown, with the extension of incubation time, the absorbance intensity at 300 nm after degradation continuously increases, which is due to the release of 5-aminosalicylic acid during degradation. This corresponds to the absorbance of pure 5-aminosalicylic acid, which fully demonstrates that copper-azosalicylic acid nanosheets can be degraded to release 5-aminosalicylic acid under in vitro hypoxia reductase environment.

[0062] To investigate the cytotoxic effects of copper-azosalicylic acid nanosheets on murine breast cancer 4T1 cells, 4T1 cells were seeded at 5000 cells per well and incubated for 24 h. Subsequently, 4T1 cells were incubated with Cu, azosalicylic acid, and copper-azosalicylic acid nanosheets (Cu-NSs) under normoxic and hypoxic conditions for 24 h, respectively. The drug concentrations, calculated based on nanoparticle concentration, were 10, 20, 40, 80, and 160 μg / mL. The normoxic conditions were (74% N2, 5% CO2, 21% O2), and the hypoxic conditions were (94% N2, 5% CO2, 1% O2). Then, 10 μL of CCK-8 was added to each well, and incubation continued for 2.5 h. Cytotoxicity was calculated using a microplate reader at 450 nm.

[0063] like Figure 7 As shown, Cu and azosalicylic acid exhibited negligible toxicity to tumor cells; however, Cu-NSs showed a concentration-dependent toxicity relationship, and, under hypoxic conditions, Cu-NSs demonstrated greater cytotoxicity, due to the release of Cu. 2+ This is caused by the highly toxic hydroxyl radicals generated by the Fenton-like mediated reaction.

[0064] To evaluate the inhibitory effect of copper-azosalicylic acid nanosheets on COX-2 in 4T1 cells under hypoxic conditions, 4T1 cells were seeded at 200,000 cells per well and incubated for 24 h. Subsequently, 4T1 cells were incubated for 24 h under hypoxic conditions with Cu (14.8 μg / mL), azosalicylic acid (185.2 μg / mL), and copper-azosalicylic acid nanosheets (200 μg / mL), respectively. Cells were then collected by centrifugation and washed three times with cold PBS. COX-2 protein was extracted using RIPA lysis buffer. COX-2 protein levels in each group were detected by Western blotting.

[0065] like Figure 8 As shown, under hypoxic conditions (94% N2, 5% CO2, 1% O2), Cu-NSs significantly inhibited COX-2 protein compared with the control group, Cu group and azosalicylic acid group.

[0066] To measure the inhibitory effect of copper-azosalicylic acid nanosheets on PGE2 in 4T1 cells under hypoxic conditions, 4T1 cells were seeded at 100,000 cells per well and incubated for 24 h. Subsequently, 4T1 cells were incubated for 24 h under hypoxic conditions with Cu (14.8 μg / mL), azosalicylic acid (185.2 μg / mL), and copper-azosalicylic acid nanosheets (200 μg / mL), respectively. The supernatant from each group was obtained by centrifugation, and the PGE2 content in the supernatant was measured using a PGE2 ELISA kit according to the instruction manual.

[0067] Since PGE2 is a downstream product of COX-2, COX-2 protein is significantly inhibited under hypoxic conditions (94% N2, 5% CO2, 1% O2). Figure 9 As shown, compared with the control group, Cu group and azosalicylic acid group, Cu-NSs had a significant inhibitory effect on PGE2 secretion, which is consistent with the inhibitory effect on COX-2 protein.

[0068] The Cu-NSs nanosheets of this invention can be used to downregulate COX-2 / PGE2 expression and kill tumors. The morphology of the nanosheets can enhance their permeability and bioavailability in vivo. They can be highly enriched at the tumor site by utilizing the enhanced tumor permeability and retention effect. The coordination of azosalicylic acid with Cu without other excipients allows Cu-NSs to accumulate at the tumor site, ensuring its efficient release of 5-ASA. 5-ASA can downregulate COX-2 / PGE2 expression, avoiding the side effects such as gastrointestinal bleeding caused by direct oral administration of high doses of COX-2 inhibitors.

Claims

1. A method for preparing copper-azosalicylic acid nanosheets for cyclooxygenase-2 inhibition, characterized in that, Includes the following steps: (1) Copper nitrate trihydrate and azosalicylic acid were dissolved in N,N-dimethylformamide solution in ethanol, and after ultrasonic dissolution, a hydrothermal reaction was carried out. After the reaction, the mixture was cooled to room temperature, and the solid was collected by centrifugation. The molar ratio of copper nitrate trihydrate and azosalicylic acid was 2:

3. The temperature of the hydrothermal reaction was 100℃ and the reaction time was 12h. The azosalicylic acid was prepared by the following method: 5-nitrosalicylic acid and sodium hydroxide were added to a mixed solvent of water and methanol, and zinc powder was added to it. The mixture was stirred and refluxed overnight. The mixture was filtered while hot, washed, and the methanol was removed by rotary evaporation. The resulting filtrate was adjusted to pH 2-3 by adding dilute hydrochloric acid under stirring. The solid precipitate was filtered, and the solid product was washed and vacuum dried to obtain azosalicylic acid. (2) The solid is redispersed in water, and uniform two-dimensional nanosheets are obtained by repeated ultrasonic energy vibration. The two-dimensional nanosheets are then washed and vacuum dried. The ultrasonic power is 450W, and the ultrasonic process is to turn on for 1s and turn off for 2s, and repeat the above operation in sequence. The ultrasonic time is 2h. The two-dimensional nanosheets have a length of 80–140 nm, a width of 30–80 nm, and a thickness of 2–8 nm; the loading of 5-aminosalicylic acid in the two-dimensional nanosheets is 92.6%, and Cu... 2+ The content of copper-azo salicylic acid nanosheets is 7.4%.

2. The method for preparing copper-azosalicylic acid nanosheets for cyclooxygenase-2 inhibition according to claim 1, characterized in that: The mass ratio of 5-nitrososalicylic acid to sodium hydroxide is 1:

1.

3. The method for preparing copper-azosalicylic acid nanosheets for cyclooxygenase-2 inhibition according to claim 1, characterized in that: The mass ratio of 5-nitrososalicylic acid to zinc powder is 6:

5.

4. The method for preparing copper-azosalicylic acid nanosheets for cyclooxygenase-2 inhibition according to claim 1, characterized in that: Filter while hot when the temperature drops to no less than 60°C.

5. The method for preparing copper-azosalicylic acid nanosheets for cyclooxygenase-2 inhibition according to claim 1, characterized in that: In step (1), the ultrasound time is not less than 30 minutes.

6. The method for preparing copper-azosalicylic acid nanosheets for cyclooxygenase-2 inhibition according to claim 1, characterized in that: In step (2), the vacuum drying temperature is not lower than 25°C, the vacuum degree is 0.1 MPa, and the vacuum drying time is not lower than 24 hours.

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