CN112057618A - Fe (III) -ART nano particle, preparation method and application thereof - Google Patents

Abstract

The invention discloses Fe (III) -ART nano particles, a preparation method and application thereof, wherein the preparation method comprises the following steps: (1) dispersing artemisinin in ethanol, adding NaOH, stirring at 45-55 ℃ for 15 minutes to 1 hour, adding water with the same volume as that of the ethanol, and adjusting the pH value to be 5-7 by using acetic acid; (2) dripping the solution obtained in the step (1) into FeCl₃Continuously stirring for 0.5-1.5 h in water solution, centrifuging, washing the solid product with water, and freeze-drying to obtain powder, namely artemisininNaOH and FeCl₃In a molar ratio of 1:2: 1. The nano-drug can be used for treating GSH (the content (10 mM) of GSH in cancer cells is about 2-4 times of that of normal cells) in iron-artemisinin nano-particles³⁺Reduction to Fe²⁺，Fe²⁺Reacting with artemisinin to generate free radicals, and the consumption of GSH can enhance the oxidative stress effect in cells.

Description

A kind of Fe(III)-ART nanoparticle, its preparation method and application

technical field

The invention belongs to the field of nano-medicine, and particularly relates to a Fe(III)-ART nanoparticle, a preparation method and application thereof.

Background technique

Reactive oxygen species (ROS, including superoxide anion free radicals, hydroxyl free radicals and hydroperoxide free radicals, etc.) are the main molecules produced by the body during oxidative stress. In recent years, studies have found that ROS can promote tumor cell apoptosis. It has anti-cancer effects in several aspects, including tumor cell necrosis, induction of autophagic death, and so on. For example, photodynamic therapy using specific wavelengths of light to activate photosensitizers to generate ROS to kill cancer cells and sonodynamic therapy using specific frequency and intensity of ultrasound to activate sonosensitizers to generate ROS to kill cancer cells has become a popular alternative to surgery, radiotherapy, and chemotherapy. After a new cancer treatment technology.

On this basis, the researchers proposed the concept of chemical kinetics, using endogenous responsive stimuli to generate ROS to treat cancer. Based on the Fenton reaction, they utilize iron ions to catalyze the generation of ROS from H ₂ O ₂ in vivo, which can achieve anti-tumor effects (Angew. Chem. Int. Ed. Engl., Vol. 128, p. 2141). However, the amount of _H2O2 expressed in _tumors was too low to effectively initiate the Fenton reaction to generate enough ROS to kill cancer cells. In addition, the Fenton reaction is demanding on the pH of the environment (pH ~ 3), and the reaction efficiency is low in vivo, and its cancer treatment effect is not ideal. How to overcome the low reaction efficiency of Fenton reaction in vivo becomes the key to the application of chemodynamic therapy to tumor treatment.

Some studies have found that artemisinin compounds may generate free radicals by reacting with iron, thereby achieving anti-tumor effects (Cancer Letters, Vol. 179, pp. 151-156). In addition, because artemisinin is insoluble in water, its solubility in common pharmaceutical solvents such as polyoxyethylene castor oil is not large, resulting in low bioavailability, which seriously affects its efficacy.

SUMMARY OF THE INVENTION

In order to overcome the low reaction efficiency of the Fenton reaction in vivo, and to generate sufficient free radicals to kill cancer cells to achieve effective cancer treatment, the technical problem to be solved by the present invention is to provide a Fe(III)-ART nanometer Particles, methods for their preparation and applications. Fe(III)-ART nanoparticles (iron-artemisinin nanoparticles coordinating and self-assembled by Fe ³⁺ and artemisinin) are independent of H ₂ O ₂ and pH, and can effectively improve the intratumoral tumorigenesis by regulating the dosage. Free radical yield, enabling highly efficient treatment of cancer with chemokinetic therapy.

Based on the above object, the present invention adopts the following technical solutions:

A preparation method of Fe(III)-ART nanoparticles, the process is as follows:

(1) Disperse artemisinin in ethanol, add NaOH, stir at 45 ℃ ~ 55 ℃ for 15 minutes ~ 1 hour, then add the same volume of water as ethanol, and adjust pH=5~7 with acetic acid;

(2) Drop the solution obtained in step (1) into the FeCl ₃ aqueous solution, continue stirring for 0.5 h to 1.5 h, centrifuge, wash the solid product with water, and freeze-dry it into powder, that is, artemisinin, NaOH and FeCl ₃ The molar ratio is 1:2:1.

Further, 50 mL of ethanol is required per 1 mmol of artemisinin, and the volume _of FeCl aqueous solution is equal to the sum of the volumes of water and ethanol in step (1).

Fe(III)-ART nanoparticles prepared by the above preparation method.

The application of the above Fe(III)-ART nanoparticles in the preparation of antitumor drugs.

Preferably, the antitumor drug is a drug for the treatment of lung adenocarcinoma.

After Fe(III)-ART nanoparticles enter the tumor site, they will decompose and release Fe ³⁺ and artemisinin in the lysosome of cancer cells. Fe ³⁺ can be reduced to Fe ²⁺ by intracellular GSH, and consumption of GSH enhances the Oxidative stress, while Fe ²⁺ catalyzes the decomposition of artemisinin to generate free radicals, killing cancer cells. The cancer cells are A549 cells.

Compared with the prior art, the nanomedicine for chemodynamic therapy provided by the present invention does not need to introduce additional nanocarriers, and can be enriched at the tumor site through passive targeting effect (EPR), and the nanomedicine generates free radicals in the body. independent of pH and H ₂ O ₂ .

The invention adopts the coordination self-assembly method of carboxyl group and Fe ³⁺ to prepare iron-artemisinin nanoparticles. Through the modification of Fe ³⁺ , the water solubility of artemisinin is greatly improved, and its circulation time in the body is also effectively prolonged. . The nanoparticles can be enriched at the tumor site through the EPR effect, improve the concentration of the therapeutic drug at the lesion site, and avoid the toxic and side effects caused by the use of drug carriers. The nanomedicine is responsive to the tumor intracellular environment, and GSH in cancer cells (about 2-4 times the content (~10 mM) in normal cells) can reduce Fe ³⁺ in iron-artemisinin nanoparticles to Fe ²⁺ , Fe ²⁺ reacts with artemisinin to generate free radicals, and the consumption of GSH can enhance the oxidative stress in cells. The nanomedicine has high efficiency and good selectivity for cancer treatment, and can be excreted through renal metabolism, avoiding the toxicity caused by the long-term retention of nanoparticles in the body.

Description of drawings

Fig. 1 is a scanning electron microscope (SEM) picture of Fe(III)-ART nanoparticles prepared in the embodiment of the present invention;

Fig. 2 is the Fe(III)-ART nanoparticle X-ray energy dispersive spectrum (EDS) diagram prepared in the embodiment of the present invention;

Fig. 3 shows that Fe(III)-ART nanoparticles prepared in the embodiment of the present invention are dispersed in glutathione (GSH) solutions of different concentrations for a period of time, the supernatant is taken out by centrifugation, and potassium ferricyanide (K ₃ [ Picture of the color change of the solution after Fe(CN) ₆ ],) or potassium ferrocyanide (K ₄ Fe(CN) ₆ );

Figure 4 is the ultraviolet-visible (UV-Vis) absorption spectrum of the remaining methylene blue after the Fe(III)-ART nanoparticles provided in the embodiment of the present invention catalyzed degradation of methylene blue in solutions containing different concentrations of glutathione (GSH) picture;

Figure 5 is an electron paramagnetic resonance (ESR) diagram of Fe(III)-ART nanoparticles provided in an embodiment of the present invention in a solution with or without glutathione (GSH);

Figure 6 is a picture of the cytotoxicity test results of Fe(III)-ART nanoparticles and pure artemisinin drugs provided in the embodiment of the present invention, in Figure 6, ns represents no statistical significance;

Figure 7 shows Fe(III)-ART nanoparticles and pure artemisinin drugs provided in the embodiment of the present invention after co-culturing with A549 cells for a period of time, and then staining the cells with a hydroxyl radical fluorescent probe (DCFH-DA) Post-fluorescence imaging pictures;

Fig. 8 is the tumor size change curve of tumor model mice after treatment with Fe(III)-ART nanoparticles and saline or artemisinin suspension provided in the embodiment of the present invention;

Fig. 9 is the average mass of the tumor taken out from the tumor model mouse after treatment with Fe(III)-ART nanoparticles and saline or artemisinin suspension provided in the embodiment of the present invention;

Figure 10 is the H&E staining diagram of the main organs of the tumor model mouse after treatment with Fe(III)-ART nanoparticles and saline or artemisinin suspension provided in the embodiment of the present invention;

In Figure 6, Figure 8, Figure 9, when p < 0.05, there is statistical significance * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Detailed ways

The technical solutions of the present invention will be described in further detail below with reference to specific embodiments and accompanying drawings, but the protection scope of the present invention is not limited thereto.

Example 1

A preparation method of Fe(III)-ART nanoparticles, the process is as follows:

Disperse 1 mmol artemisinin in 50 mL of ethanol, add 0.08 g NaOH, stir at 50 °C for 30 min, then continue to add 50 mL of water, and adjust the pH of the solution to about 5 with CH ₃ COOH, add the above solution dropwise to 100 mL of FeCl ₃ aqueous solution with a concentration of 0.01 mmol/mL, and continued stirring for 1 h; centrifuged, and the solid product was centrifuged and washed three times with deionized water and freeze-dried (-30 °C, freeze dryer, and dried to powder. , the specific time is 24 h), and the product is obtained.

The scanning electron microscope image of the product of Example 1 is shown in Figure 1, and the X-ray energy spectrum analysis spectrum is shown in Figure 2. It can be seen from Figure 1 that the product is a small particle with a size of about 80 nm. The X-ray energy spectrum of the product (Fig. 2) shows that the product mainly contains Fe, O, and C elements.

Example 2

Detection of redox reactions between the obtained iron-artemisinin nanoparticles and glutathione.

Disperse 1 mg of the sample prepared by the method described in Example 1 in 2 mL of glutathione solution with concentrations of 0, 2, 4, 6, 8, and 10 mM, respectively. After 30 min of reaction, the product was centrifuged. Remove the supernatant and add 1 mL of potassium ferricyanide solution (1 mmol/mL) for detecting Fe ³⁺ or 1 mL of potassium ferricyanide solution (1 mmol/mL) for detecting Fe ²⁺ to the supernatant, respectively. , Fe ³⁺ will turn the colorless potassium ferrocyanide solution blue. Fe ²⁺ will turn the yellow potassium ferricyanide solution blue, the results are shown in Figure 3.

According to the color change diagram of the supernatant of this Example 2 (Fig. 3), as the concentration of glutathione increased, the color of the supernatant added with potassium ferricyanide changed from yellow to blue to dark blue, correspondingly The color of the supernatant added with potassium ferrocyanide changed from colorless to blue to light blue, indicating that glutathione would release Fe ³⁺ from iron-artemisinin nanoparticles first, and then Fe ³⁺ is reduced to Fe ²⁺ .

Example 3

The ability of the obtained iron-artemisinin nanoparticles to generate free radicals in glutathione solution was examined.

Disperse 1 mg of the sample prepared by the method described in Example 1 in 2 mL of glutathione aqueous solution with concentrations of 0, 2, 4, 6, 8, and 10 mM (0 mM is ultrapure water, without GSH). ), and 0.02 mL of methylene blue solution (1 mg/mL) was added to the solution. After 3 h of reaction, the solution was centrifuged, and UV-Vis was used to detect the absorption peak intensity of the supernatant at 664 nm. weaker, the higher the yield of hydroxyl radicals in solution, the results are shown in Figure 4.

According to the UV-Vis absorption spectrum of the supernatant in Example 3 (Figure 4), after the same reaction time, the absorption peak intensity of the methylene blue solution weakened with the increase of the glutathione concentration, indicating that the iron-artemisinin nanoparticle The particles can generate free radicals under the action of glutathione, and the yield of free radicals is related to the concentration of glutathione.

Example 4

Signal detection of the obtained iron-artemisinin nanoparticles generating free radicals in glutathione solution.

Disperse 1 mg sample or 0.8 mg artemisinin and 0.58 g FeCl ₃ mixture (referred to as Fe ³⁺ & ART) prepared by the method described in Example 1 in 2 mL of 0 mM and 10 mM glutathione aqueous solutions, respectively. , and added 0.2 mmol of the radical scavenger 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), which was directly detected after the addition, and then the free radicals in the solution were detected by electron paramagnetic resonance spectrometer. signal, the results are shown in Figure 5.

According to the ESR signal of the reactant obtained in Example 4 (Fig. 5), in the presence of glutathione, iron-artemisinin nanoparticles have a free radical signal, and in the absence of glutathione, iron-green Artemisinin nanoparticles have no free radical signal, indicating that iron-artemisinin nanoparticles generate free radicals only under the action of glutathione.

Example 5

A549 cells were selected to study the anti-tumor effect of iron-artemisinin nano-drugs by CCK-8 experiment.

Using A549 cells as target cells, the antitumor activity of the sample prepared according to Example 1 and artemisinin was evaluated by the commonly used CCK-8 method. A549 cells were grown in a 96-well plate (Corning Glass Works) in monolayer form, with a cell density of 100,000 cells/well. When the cell density reached 50%, the corresponding samples were added for co-culture. The experiment was divided into 3 groups with different concentrations. According to the sample solution prepared in Example 1, the artemisinin solution and the control group without any material, (the control group is phosphate buffered solution PBS), the sample volume was 10 μL, and the sample concentrations were 100, 500, 1000, and 1500, respectively. , 2000 μg/mL (the corresponding artemisinin concentrations in this series of concentration samples are 80, 400, 800, 1200, 1600 μg/mL), and the artemisinin concentrations are 80, 400, 800, 1200, 1600 μg/mL ( Both are saline solutions, Figure 6 shows the final concentration of the sample in the cell culture well, which itself contains 90 μL of cell culture medium). Cells and particles were cultured for 24 hours and then replaced with fresh medium (DEME high-glucose medium with 10% fetal bovine serum and 1% penicillin-streptomycin), and 10 μL CCK-8 solution was added for 4 hours. The optical intensity of the derived CCK-8 solution was measured by ELISA, which indirectly reflected the concentration of living cells. The same concentration of artemisinin and iron-artemisinin nanoparticles were compared. The results are shown in Figure 6. . The picture (Fig. 6) shows that in the samples with different concentrations, compared with the same group, the growth inhibition rate of the samples prepared according to Example 1 on A549 cells is higher than that of artemisinin on A549 cells. 50, 100, 150, and 200 μg/mL, with significant differences, especially indicating that iron-artemisinin nanoparticles can effectively improve the anticancer effect of artemisinin.

Example 6

The samples prepared according to Example 1 and artemisinin were examined for the generation of free radicals in cells by fluorescence imaging technology. A549 cells were grown in a monolayer in a 24-well plate with a cell density of 700,000 cells/well. When the cell density reached 50%, the corresponding samples were added for co-culture. The experiment was divided into 3 groups, which were the sample solution prepared according to Example 1 (iron-artemisinin nanoparticle physiological saline solution, 10 μL, 2000 μg/mL), artemisinin solution (artemisinin physiological saline solution) , 10 μL, 1600 μg/mL) and the control group without any material (the control group was normal saline). After culturing the cells and particles for 4 hours, a free radical fluorescent probe (DCFH-DA) (10 μL, 10 mM) was added to the medium, and the cells were incubated for 15 min. The cells were then washed twice with PBS buffer, and the fluorescence of the cells (λex = 480 nm, λem = 525 nm) was observed on a laser scanning confocal microscope (Zeiss LSM 710). The results are shown in Figure 7.

The fluorescence imaging picture (Fig. 7) showed that the fluorescence intensity of cells co-cultured with iron-artemisinin nanoparticles was the strongest, indicating that iron-artemisinin nanoparticles can generate a large number of free radicals in cells and kill cancer cells.

Example 7

The antitumor effect of iron-artemisinin nanomedicines was evaluated in tumor model mice. 0.1 mL of cell suspension (containing 5×10 ⁶ human lung adenocarcinoma cancer cells (A549)) was injected into the root of the forelimb of BLAB/c mice (female mice, 5 weeks old, 10-20 g), and then cultured for 15 days. The tumor model mice were randomly divided into three groups. The tail vein was injected with 0.1 mL of normal saline, artemisinin suspension (artemisinin in saline solution, 0.1 mL, 1.6 mg/mL) and iron-artemisinin nanoparticle solution (iron-artemisinin nanoparticle physiological salt), respectively. Aqueous solution, 0.1 mL, 2 mg/mL) into the tumor transplant model mice, administered once every three days, and the long and short diameters of the tumors were measured, the tumor size was calculated, the survival rate of nude mice was monitored, and the body weight of nude mice was weighed , draw the survival curve and tumor growth curve, and evaluate the tumor therapeutic effect and toxic and side effects of the nanoparticles. After the treatment, the main organs and tumors were excised, and the health status of each organ of the mice treated by this method was evaluated by histopathological analysis (H&E). The results are shown in Figures 8 to 10.

The change curve of tumor size in mice (Fig. 8) and the average mass of tumors taken out after treatment (Fig. 9) showed that the tumors of mice treated with iron-artemisinin nanomedicine became the smallest, indicating that iron-artemisinin nanomedicine has better anticancer efficacy. The results of H&E staining (Fig. 10) showed that the morphology of the main organs of the mice treated with the iron-artemisinin nanomedicine did not change significantly, which indicated that the iron-artemisinin nanomedicine itself had no obvious toxic and side effects. The above examples are only used to illustrate the preferred embodiments of the present invention, but the present invention is not limited to the above-mentioned embodiments. Any modifications made within the spirit and principles of the present invention are within the knowledge scope of those of ordinary skill in the field. , equivalent substitutions and improvements, etc., shall be regarded as the protection scope of this application.

Claims (7)

1. a preparation method of Fe(III)-ART nanoparticle, is characterized in that, process is as follows: (1) Disperse artemisinin in ethanol, add NaOH, stir at 45 ℃ ~ 55 ℃ for 15 minutes ~ 1 hour, then add the same volume of water as ethanol, and adjust pH=5~7 with acetic acid; (2) Drop the solution obtained in step (1) into the FeCl3 aqueous solution, continue stirring for 0.5 h to 1.5 h, centrifuge, wash the solid product with water, and freeze-dry it into powder, that is, the moles of artemisinin, NaOH and FeCl3 The ratio is 1:2:1. 2. The preparation method of Fe(III)-ART nanoparticles according to claim 1, characterized in that, every 1 mmol of artemisinin needs 50 mL of ethanol, and the volume of FeCl aqueous solution is equal to the volume of water and ethanol in step (1). Sum. 3. Fe(III)-ART nanoparticles obtained by the preparation method of claim 1 or 2. 4. The application of Fe(III)-ART nanoparticles according to claim 3 in the preparation of antitumor drugs. 5. The application according to claim 4, wherein the antitumor drug is a drug for the treatment of lung adenocarcinoma. 6. application according to claim 4, is characterized in that, after Fe (III)-ART nano-particle enters tumor site, can decompose and release Fe ³⁺ and artemisinin in cancer cell lysosome, Fe ³⁺ It can be reduced to Fe ²⁺ by intracellular GSH, and the consumption of GSH enhances oxidative stress. At the same time, Fe ²⁺ catalyzes the decomposition of artemisinin to generate free radicals, killing cancer cells. 7. The use according to claim 6, wherein the cancer cells are A549 cells.

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