CN112057618A

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

Info

Publication number: CN112057618A
Application number: CN202010999993.3A
Authority: CN
Inventors: 陈健; 高永举; 王晓博; 杨保成
Original assignee: Huanghe Science and Technology College
Current assignee: Huanghe Science and Technology College
Priority date: 2020-09-22
Filing date: 2020-09-22
Publication date: 2020-12-11
Anticipated expiration: 2040-09-22
Also published as: CN112057618B

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

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

Technical Field

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

Background

Reactive Oxygen Species (ROS) is a main molecule generated during oxidative stress of the body, and in recent years, research shows that ROS can play an anticancer role in several aspects of promoting tumor cell apoptosis, causing tumor cell necrosis, inducing cell autophagic death, and the like. For example, photodynamic therapy using light of a specific wavelength to activate a photosensitizer, which generates ROS to kill cancer cells, and sonodynamic therapy using ultrasonic waves of a specific frequency and intensity to activate a sonosensitizer, which generates ROS to kill cancer cells, have become a new cancer treatment technique following surgery, radiotherapy, and chemotherapy.

On this basis, researchers have proposed the concept of chemokinetics, which use endogenous responses to stimulate the production of ROS to treat cancer. They are based on Fenton reaction and utilize iron ions to catalyze in vivo H₂O₂ROS production, an antitumor effect can be achieved (angelw. chem. int. ed. engl., volume 128, page 2141). However, expression of H within tumors₂O₂Too low an amount is not sufficient to effectively initiate the Fenton reaction to generate sufficient ROS to kill cancer cells. In addition, the Fenton reaction has strict requirements on the pH of the environment (pH-3), the reaction efficiency in organisms is low, and the cancer treatment effect is not ideal. How to overcome the problem of low response efficiency of Fenton reaction in organisms becomes a key point for applying chemo-kinetic therapy to tumor treatment.

It has been found that artemisinin compounds can generate free radicals by reacting with iron, thereby achieving anti-tumor effect (Cancer Letters, volume 179, page 151-156). In addition, because the artemisinin is insoluble in water, the solubility of the artemisinin in common pharmaceutical solvents such as polyoxyethylene castor oil and the like is not high, the bioavailability of the artemisinin is low, and the exertion of the drug effect of the artemisinin is seriously influenced.

Disclosure of Invention

In order to overcome the problem of low reaction efficiency of Fenton reaction in organisms and generate enough free radicals to kill cancer cells so as to realize effective treatment of cancer, the invention aims to provide Fe (III) -ART nanoparticles, a preparation method and application thereof. Fe (III) -ART nanoparticles (Fe)³⁺Iron-artemisinin nanoparticles self-assembled by coordination with artemisinin) are independent of H₂O₂And pH, the free radical yield in the tumor is effectively improved by regulating and controlling the administration dosage, and the high-efficiency cancer treatment by the chemo-kinetic therapy is realized.

Based on the purpose, the invention adopts the following technical scheme:

a preparation method of Fe (III) -ART nano particles 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 into powder to obtain artemisinin, NaOH and FeCl₃In a molar ratio of 1:2: 1.

Further, 50 mL of ethanol, FeCl, is required per 1 mmol of artemisinin₃The volume of the aqueous solution is equal to the sum of the volumes of water and ethanol in the step (1).

Fe (III) -ART nano-particles prepared by the preparation method.

The Fe (III) -ART nano-particle is applied to the preparation of antitumor drugs.

Preferably, the anti-tumor drug is a drug for treating lung adenocarcinoma.

After Fe (III) -ART nano-particles enter a tumor site, Fe is released in lysosomes of cancer cells³⁺And artemisinin, Fe³⁺Can be reduced to Fe by intracellular GSH²⁺Depletion of GSH to enhance oxidative stress, with Fe²⁺Catalyzing artemisinin to decompose and generate free radicals to kill cancer cells. The cancer cells are A549 cells.

Compared with the prior art, the nano-drug for the chemokinetic treatment provided by the invention does not need to additionally introduce a nano-carrier, can be enriched at a tumor part by a passive targeting Effect (EPR), and does not depend on pH and H when generating free radicals in vivo₂O₂。

The invention adopts carboxyl and Fe³⁺Preparing iron-artemisinin nano particles by coordination self-assembly method through Fe³⁺The modification of (2) greatly improves the water solubility of the artemisinin, and effectively prolongs the circulation time in the body. The nanoparticles can be enriched at tumor sites through EPR effect, improve the concentration of therapeutic drugs at the focus sites, and avoid the toxic and side effects caused by the use of drug carriers. The nano-drug has responsiveness to the environment in tumor cells, and GSH (the content (10 mM) of GSH in cancer cells is about 2-4 times of that of normal cells) in the cancer cells can react Fe in the 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. The nano-drug has high efficiency and good selectivity for cancer treatment, and can be discharged out of the body through kidney metabolism, thereby avoiding the toxicity caused by long-term retention of the nano-particles in the body.

Drawings

FIG. 1 is a Scanning Electron Microscope (SEM) picture of Fe (III) -ART nanoparticles prepared by the embodiment of the invention;

FIG. 2 is an X-ray energy spectrum (EDS) chart of Fe (III) -ART nanoparticles prepared by the example of the present invention;

FIG. 3 shows Fe (III) -ART nanoparticles prepared in the example of the present invention dispersed in Glutathione (GSH) solutions of different concentrations for a certain period of time, centrifuged to remove the supernatant and added with potassium ferricyanide (K)₃[Fe(CN)₆]B) or potassium ferrocyanide (K)₄Fe(CN)₆) Pictures of the color change of the post-solution;

FIG. 4 is a graph showing the ultraviolet-visible (UV-Vis) absorption spectrum of the residual methylene blue after the Fe (III) -ART nanoparticles provided by the embodiment of the invention are used for catalyzing and degrading the methylene blue in the solutions containing different concentrations of Glutathione (GSH);

FIG. 5 is a graph of electron paramagnetic resonance (ESR) spectra of Fe (III) -ART nanoparticles in a solution with or without Glutathione (GSH) provided in an embodiment of the present invention;

FIG. 6 is a graph showing the result of cytotoxicity test of Fe (III) -ART nanoparticles and pure artemisinin drug provided by the embodiment of the present invention, wherein ns in FIG. 6 represents no statistical significance;

FIG. 7 is a fluorescent image of the Fe (III) -ART nanoparticles and pure artemisinin drugs co-cultured with A549 cells for a period of time, followed by staining the cells with hydroxyl radical fluorescence probe (DCFH-DA) according to the embodiment of the present invention;

FIG. 8 is a graph showing the change of tumor size of tumor model mice treated with Fe (III) -ART nanoparticles and physiological saline or artemisinin suspension according to the embodiment of the present invention;

FIG. 9 shows the mean mass of tumors taken from tumor model mice after treatment with Fe (III) -ART nanoparticles and saline or artemisinin suspensions according to an embodiment of the present invention;

FIG. 10 is a graph of H & E staining of major organs of tumor model mice after treatment with Fe (III) -ART nanoparticles and saline or artemisinin suspensions according to the embodiment of the present invention; the scale of each graph in figure 10 is 100 μm,

in FIG. 6, FIG. 8, FIG. 9, when p is<0.05, statistical significanceP < 0.05 , ** P < 0.01, *** P< 0.001, **** P < 0.0001。

Detailed Description

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

Example 1

dispersing 1 mmol of artemisinin in 50 mL of ethanol and adding 0.08 g of NaOH, stirring at 50 ℃ for 30 min, then adding 50 mL of water and adding CH₃Adjusting the pH of the solution to about 5 with COOH, and gradually adding the solutionAdding FeCl of which the concentration is 0.01 mmol/mL into 100 mL in a dropwise manner₃Stirring the mixture in the aqueous solution for 1 hour; centrifuging, washing the solid product with deionized water for 3 times, and freeze-drying at (-30 deg.C, for 24 hr) to obtain the final product.

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

Example 2

And detecting the oxidation-reduction reaction between the obtained iron-artemisinin nano particles and glutathione.

1 mg of the sample prepared as described in example 1 was dispersed in 2mL of glutathione solutions having concentrations of 0, 2, 4, 6, 8, and 10 mM, respectively, and after 30 min of reaction, the product was centrifuged to take out the supernatant, and 1 mL of Fe to be detected was added to the supernatant, respectively³⁺Potassium ferrocyanide solution (1 mmol/mL) or 1 mL for detecting Fe²⁺Potassium ferricyanide solution (1 mmol/mL), Fe³⁺The colorless potassium ferrocyanide solution turns blue. Fe²⁺The yellow potassium ferricyanide solution turned blue, and the results are shown in detail in figure 3.

According to the color change chart of the supernatant of 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, and the color of the supernatant added with potassium ferrocyanide correspondingly changed from colorless to blue to light blue, indicating that glutathione can cause Fe³⁺Firstly, the Fe is released from the iron-artemisinin nano particles, and then the Fe is released³⁺Reduction to Fe²⁺。

Example 3

The obtained iron-artemisinin nano-particles are tested for the capability of generating free radicals in glutathione solution.

1 mg of the sample prepared as described in example 1 was dispersed in 2mL of an aqueous glutathione solution (0 mM is ultrapure water without GSH) at concentrations of 0, 2, 4, 6, 8, 10 mM, respectively, and 0.02 mL of a methylene blue solution (1 mg/mL) was added to the solution, and after 3 hours of reaction, the solution was centrifuged, and the intensity of the absorption peak of the supernatant at 664 nm was measured using UV-Vis, the weaker the absorption peak, the higher the yield of hydroxyl radicals in the solution, and the results are shown in FIG. 4.

According to the UV-visible absorption spectrum (FIG. 4) of the supernatant of this example 3, the intensity of the absorption peak of methylene blue solution decreased with the increase of glutathione concentration after the same reaction time, indicating that iron-artemisinin nanoparticle can generate free radical under the action of glutathione, and the yield of free radical is related to the concentration of glutathione.

Example 4

And (3) detecting signals of free radicals generated by the obtained iron-artemisinin nano particles in a glutathione solution.

1 mg of sample prepared as described in example 1 or 0.8 mg of artemisinin with 0.58 g of FeCl₃Mixture (noted as Fe)³⁺ART) was dispersed in 2mL of an aqueous solution of glutathione at concentrations of 0 mM and 10 mM, respectively, and 0.2 mmol of a radical trapping agent 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) was added thereto, and direct detection was performed after the addition, and then signals of radicals in the solution were detected using an electron paramagnetic resonance spectrometer, and the results are shown in fig. 5.

According to the ESR signal of the reactant obtained in this example 4 (fig. 5), the iron-artemisinin nanoparticle has a free radical signal in the presence of glutathione, and the iron-artemisinin nanoparticle has no free radical signal in the absence of glutathione, indicating that the iron-artemisinin nanoparticle generates free radicals under the action of glutathione.

Example 5

A549 cells are selected, and the anti-tumor effect of the iron-artemisinin nano-medicament is researched through a CCK-8 experiment.

The samples prepared according to example 1 and artemisinin were evaluated for anti-tumor activity using the commonly used CCK-8 method with A549 cells as target cells. A549 cells were grown as monolayers in 96-well plates (Corning Glass Works) at a cell density of 100000 cells/well, when the cell density reached 50%, the corresponding samples were added and incubated together, and the experiment was divided into 3 groups, namely, the sample solution prepared according to example 1, artemisinin solution and the control group without any material (the control group is phosphate buffered saline PBS), in different concentrations, the sample volume was 10. mu.L, the sample concentration was 100, 500, 1000, 1500, 2000. mu.g/mL (the corresponding artemisinin concentration in the samples of this series of concentrations was 80, 400, 800, 1200, 1600. mu.g/mL), the artemisinin concentration was 80, 400, 800, 1200, 1600. mu.g/mL (all physiological saline solution, FIG. 6 shows the final concentration of the sample in the cell culture well, and 90. mu.L of cell culture medium was also present in the culture well itself). After 24 hours of culture, the cells and particles were replaced with fresh medium (DEME high-sugar medium, 10% fetal bovine serum and 1% penicillin-streptomycin were added), 10. mu.L of CCK-8 solution was added and the culture was continued for 4 hours, and finally the optical intensity of the derived CCK-8 solution was measured by an enzyme-linked immunosorbent assay (ELISA), indirectly reflecting the concentration of viable cells, and the results of comparison between artemisinin and iron-artemisinin nanoparticles of the same concentration were shown in FIG. 6. The graph (fig. 6) shows that, in samples with different concentrations, compared with the samples in the same group, the growth inhibition rate of the sample prepared according to example 1 on the a549 cells is higher than that of artemisinin on the a549 cells, and the concentrations are 50, 100, 150 and 200 μ g/mL, which have significant differences, especially, the iron-artemisinin nanoparticles can effectively improve the anti-cancer effect of artemisinin.

Example 6

The samples prepared according to example 1 and artemisinin were examined for the intracellular generation of free radicals using fluorescence imaging techniques. A549 cells were grown in a monolayer in 24-well plates at a cell density of 700000 cells/well, and when the cell density reached 50%, the corresponding samples were added and co-cultured. The experiment was divided into 3 groups, namely a sample solution (iron-artemisinin nanoparticle physiological saline solution, 10. mu.L, 2000. mu.g/mL), an artemisinin solution (artemisinin physiological saline solution, 10. mu.L, 1600. mu.g/mL) and a control group (control group is physiological saline) without any material prepared according to example 1. After 4 hours of cell and particle culture, free radical fluorescent probe (DCFH-DA) (10. mu.L, 10 mM) was added to the medium and the cells were cultured for an additional 15 min. The cells were then washed twice with PBS buffer and observed for fluorescence on a laser scanning confocal microscope (Zeiss LSM 710) (λ ex = 480 nm, λ em = 525 nm) with the results shown in figure 7.

The fluorescence imaging picture (fig. 7) shows that the fluorescence intensity of the cells cultured with the iron-artemisinin nanoparticles is strongest, which indicates that the iron-artemisinin nanoparticles can generate a large amount of free radicals in the cells to kill cancer cells.

Example 7

The anti-tumor effect of the iron-artemisinin nano-drug is evaluated on a tumor model mouse. 0.1 mL of cell suspension (containing 5X 10 cells)⁶Human lung adenocarcinoma cells (A549)) were injected into the roots of forelimbs of BLAB/c mice (female mice, 5 weeks old, 10-20 g), followed by culture for 15 days. Tumor model mice were randomly divided into three groups. Injecting 0.1 mL of physiological saline, artemisinin suspension (artemisinin physiological saline solution, 0.1 mL, 1.6 mg/mL) and iron-artemisinin nanoparticle solution (iron-artemisinin nanoparticle physiological saline solution, 0.1 mL, 2 mg/mL) into a tumor transplantation model mouse through tail vein respectively, administering for 1 time every three days, measuring the long diameter and short diameter of the tumor, calculating the size of the tumor, monitoring the survival rate of the nude mouse and weighing the weight of the nude mouse, drawing a survival curve and a tumor growth curve, and evaluating the tumor treatment effect and the toxic and side effect of the nanoparticles. After the treatment, the major organs and tumors were harvested and analyzed by histopathological analysis (H)&E) The health of each organ of the mice treated by this method was evaluated, and the results are shown in FIGS. 8 to 10.

The mouse tumor size change curve (figure 8) and the average mass of the tumors taken out after treatment (figure 9) show that the tumors of the mice treated by the iron-artemisinin nano-drug group become minimum, which indicates that the iron-artemisinin nano-drug has better anti-cancer efficacy. The H & E staining results (fig. 10) show that the major organ morphology of mice treated with the iron-artemisinin nanomedicine was not significantly altered, indicating that the iron-artemisinin nanomedicine itself had no significant toxic side effects. The above embodiments are only for illustrating the preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention within the knowledge of those skilled in the art should be considered as the protection scope of the present application.

Claims

1. A preparation method of Fe (III) -ART nano particles is characterized by comprising the following steps:

(2) and (2) dropping the solution obtained in the step (1) into a FeCl3 aqueous solution, continuously stirring for 0.5-1.5 h, centrifuging, washing the solid product with water, and freeze-drying into powder to obtain the artemisinin, NaOH and FeCl3 with the molar ratio of 1:2: 1.

2. The method for preparing Fe (III) -ART nanoparticles as claimed in claim 1, wherein 50 mL of ethanol is required for each 1 mmol of artemisinin, and the volume of FeCl3 aqueous solution is equal to the sum of the volumes of water and ethanol in step (1).

3. Fe (III) -ART nanoparticles prepared by the preparation method of claim 1 or 2.

4. The use of Fe (III) -ART nanoparticles as claimed in claim 3 for preparing antitumor drugs.

5. The use according to claim 4, wherein the antineoplastic drug is a drug for the treatment of lung adenocarcinoma.

6. The use according to claim 4, wherein Fe (III) -ART nanoparticles, after entering the tumor site, are responsible for the release of Fe in cancer cell lysosomes³⁺And artemisinin, Fe³⁺Can be reduced to Fe by intracellular GSH²⁺Depletion of GSH to enhance oxidative stress, with Fe²⁺Catalyzing artemisinin to decompose and generate free radicals to kill cancer cells.

7. The use of claim 6, wherein the cancer cells are A549 cells.