阅读说明：本技术 一种改善肿瘤微环境的钌基抗肿瘤纳米药物的制备方法 (Preparation method of ruthenium-based anti-tumor nano-drug for improving tumor microenvironment ) 是由 高大威 李春慧 丛聪 张旭武 刘志伟 李晓玲 于 2021-01-29 设计创作，主要内容包括：本发明涉及一种改善肿瘤微环境的钌基抗肿瘤纳米药物的制备方法,属于纳米药物技术领域,称取大豆卵磷脂、胆固醇和阿托伐醌,加无水乙醇溶解制得含阿托伐醌的脂质体脂相混合液,然后将其旋蒸使脂相成膜；将钌纳米颗粒用水重悬并向其中加入磷酸缓冲液,混匀后加入吐温80得到含钌的水相混合液；将水相混合液加至脂相膜中,混匀后超声。本发明可以阻断线粒体的呼吸链,缓解肿瘤组织的缺氧情况；释放的纳米酶能够利用增加的氧气消耗肿瘤部位的葡萄糖,减少ATP的产生,在对肿瘤部位进行饥饿治疗同时产生过氧化氢,同时利用肿瘤微环境的过氧化氢进行类芬顿反应产羟基自由基,从而实现良好的抗肿瘤效果；本发明制备方法简便易行,可以规模化生产。(The invention relates to a preparation method of a ruthenium-based anti-tumor nano-drug for improving a tumor microenvironment, which belongs to the technical field of nano-drugs, and comprises the steps of weighing soybean lecithin, cholesterol and atovaquone, adding absolute ethyl alcohol to dissolve the soybean lecithin, the cholesterol and the atovaquone to prepare a liposome lipid phase mixed solution containing the atovaquone, and then carrying out rotary evaporation on the liposome lipid phase mixed solution to form a membrane; re-suspending the ruthenium nanoparticles with water, adding a phosphate buffer solution into the re-suspended ruthenium nanoparticles, uniformly mixing, and adding tween 80 to obtain a ruthenium-containing water-phase mixed solution; adding the water phase mixed solution into the lipid phase membrane, mixing uniformly and performing ultrasonic treatment. The invention can block the respiratory chain of mitochondria and relieve the hypoxia condition of tumor tissues; the released nano enzyme can consume glucose at the tumor part by using increased oxygen, reduce the generation of ATP, generate hydrogen peroxide while performing starvation treatment on the tumor part, and generate hydroxyl free radicals by using the hydrogen peroxide in a tumor microenvironment to perform Fenton-like reaction, so that a good anti-tumor effect is realized; the preparation method is simple and easy to implement, and can be used for large-scale production.)

1. A preparation method of ruthenium-based anti-tumor nano-drug for improving tumor microenvironment is characterized in that: the ruthenium-based anti-tumor nano-drug is a clear and transparent ruthenium/atovaquone liposome, and the preparation method comprises the following steps:

(1) respectively weighing a certain amount of soybean lecithin, cholesterol and atovaquone, and dissolving with anhydrous ethanol to obtain liposome lipid phase mixed solution containing atovaquone;

(2) placing the liposome lipid phase mixed solution containing atovaquone prepared in the step (1) into a rotary evaporator for rotary evaporation to enable the lipid phase to form a film;

(3) after the ruthenium nanoparticles are resuspended by using a certain amount of water, a certain proportion of phosphate buffer is added into the ruthenium nanoparticles, a certain amount of tween 80 is added after the ruthenium nanoparticles are uniformly mixed, and the ruthenium-containing water-phase mixed solution is obtained after the ruthenium nanoparticles are uniformly mixed;

(4) adding the ruthenium-containing water-phase mixed solution obtained in the step (3) into the membrane prepared in the step (2);

(5) and (4) uniformly mixing the liquid obtained in the step (4), and performing ultrasonic treatment to obtain a clear and transparent ruthenium/atovaquone liposome.

2. The preparation method of the ruthenium-based anti-tumor nano-drug for improving the tumor microenvironment according to claim 1, which is characterized in that: the ratio of the soybean lecithin, the cholesterol, the atovaquone and the absolute ethyl alcohol in the lipid phase mixed solution in the step (1) is 40-70 mg: 4-7 mg: 0.85-1.5 mg: 8-12 mL.

3. The preparation method of the ruthenium-based anti-tumor nano-drug for improving the tumor microenvironment according to claim 1, which is characterized in that: and (3) in the step (2), the rotary evaporation temperature of the rotary evaporator is 40-50 ℃, and the rotary evaporation time is 0.5-4 h.

4. The preparation method of the ruthenium-based anti-tumor nano-drug for improving the tumor microenvironment according to claim 1, which is characterized in that: the volume ratio of the phosphate buffer solution, the water and the Tween 80 of the aqueous phase mixed solution containing ruthenium in the step (3) is 400-500: 9500-9600: 5-15.

5. The preparation method of the ruthenium-based anti-tumor nano-drug for improving the tumor microenvironment according to claim 1, which is characterized in that: the ruthenium nanoparticles in the step (3) are obtained by reducing ruthenium trichloride with sodium borohydride, and the size of the ruthenium nanoparticles is 5-20 nm.

6. The preparation method of the ruthenium-based anti-tumor nano-drug for improving the tumor microenvironment according to claim 1, which is characterized in that: the ultrasonic power in the step (5) is 200-800W, and the ultrasonic time is 5-20 min.

7. The preparation method of the ruthenium-based anti-tumor nano-drug for improving the tumor microenvironment according to claim 1, which is characterized in that: the average particle size of the ruthenium-based anti-tumor nano-drug is 150-250 nm.

Technical Field

The invention relates to a preparation method of a ruthenium-based anti-tumor nano-drug for improving a tumor microenvironment, belonging to the technical field of nano-drugs.

Background

About 380 million new malignant tumors are developed in China every year, and about 30 ten thousand people die. Although the research on cancer has progressed, cancer is still one of the diseases with higher incidence. The common treatment modes at present are surgical treatment, chemotherapy, radiotherapy, targeted treatment, immunotherapy, gene therapy, phototherapy and the like. Although all the treatment methods can achieve certain effects, all the treatment methods have limitations or have certain toxic and side effects.

The nano enzyme has enzyme activity and nano material property as a promising natural enzyme substitute. Ruthenium (Ru) is a member of the platinum group metals, but its cost is low; the ruthenium-based nanoenzyme is a newly emerging nanoenzyme, glycolysis in tumor cells is active, glucose can be utilized to generate more energy and metabolites in a short time, and the ruthenium-based nanoenzyme can catalyze glucose and O₂Reaction, consuming the energy source of the tumor cells. However, the reaction process needs oxygen to be involved, however, the research finds that the tumor tissue and the periphery are an extremely anoxic environment, and the insufficient oxygen content greatly limits the effect of the nano enzyme treatment.

Therefore, the method can be used for exploring the nano-drug with the anti-tumor effect, which can cut off the energy supply of the tumor based on blocking the glycolysis reaction in the tumor cell and can improve the tumor hypoxia environment, can realize good anti-tumor effect and has important social significance.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a preparation method of ruthenium-based anti-tumor nano-drug for improving tumor microenvironment.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a preparation method of a ruthenium-based anti-tumor nano-drug for improving a tumor microenvironment is provided, wherein the ruthenium-based anti-tumor nano-drug is a clear and transparent ruthenium/atovaquone liposome, and the preparation method comprises the following steps:

(1) respectively weighing a certain amount of soybean lecithin, cholesterol and atovaquone, and dissolving with anhydrous ethanol to obtain liposome lipid phase mixed solution containing atovaquone;

(2) placing the liposome lipid phase mixed solution containing atovaquone prepared in the step (1) into a rotary evaporator for rotary evaporation to enable the lipid phase to form a film;

(3) after the ruthenium nanoparticles are resuspended by using a certain amount of water, a certain proportion of phosphate buffer is added into the ruthenium nanoparticles, a certain amount of tween 80 is added after the ruthenium nanoparticles are uniformly mixed, and the ruthenium-containing water-phase mixed solution is obtained after the ruthenium nanoparticles are uniformly mixed;

(4) adding the ruthenium-containing water-phase mixed solution obtained in the step (3) into the membrane prepared in the step (2);

(5) and (4) uniformly mixing the liquid obtained in the step (4), and performing ultrasonic treatment to obtain a clear and transparent ruthenium/atovaquone liposome.

The technical scheme of the invention is further improved as follows: the ratio of the soybean lecithin, the cholesterol, the atovaquone and the absolute ethyl alcohol in the lipid phase mixed solution in the step (1) is 40-70 mg: 4-7 mg: 0.85-1.5 mg: 8-12 mL.

The technical scheme of the invention is further improved as follows: and (3) in the step (2), the rotary evaporation temperature of the rotary evaporator is 40-50 ℃, and the rotary evaporation time is 0.5-4 h.

The technical scheme of the invention is further improved as follows: the volume ratio of the phosphate buffer solution, the water and the Tween 80 of the aqueous phase mixed solution containing ruthenium in the step (3) is 400-500: 9500-9600: 5-15.

The technical scheme of the invention is further improved as follows: the ruthenium nanoparticles in the step (3) are obtained by reducing ruthenium trichloride with sodium borohydride, and the size of the ruthenium nanoparticles is 5-20 nm.

The technical scheme of the invention is further improved as follows: the ultrasonic power in the step (5) is 200-800W, and the ultrasonic time is 5-20 min.

The technical scheme of the invention is further improved as follows: the average particle size of the ruthenium-based anti-tumor nano-drug is 150-250 nm.

Due to the adoption of the technical scheme, the invention has the technical progress that:

the invention can block the respiratory chain of mitochondria, thereby relieving the hypoxia condition of tumor tissues; the released nano enzyme can consume glucose at the tumor part by using increased oxygen, reduces the generation of ATP, generates hydrogen peroxide while performing starvation treatment on the tumor part, and generates hydroxyl radicals by performing Fenton-like reaction by using the hydrogen peroxide in the tumor microenvironment, thereby realizing good anti-tumor effect.

The invention has good biocompatibility, can realize the slow release of the drug through the liposome, and the released atovaquone can also influence the activity of the dihydrolactate dehydrogenase to prevent the synthesis of pyrimidine and further cause a lethal effect on tumor cells.

The Ru NPs in the ruthenium/atovaquone liposome are obtained by a simple one-step reduction method and are wrapped in the water phase of the liposome, and the preparation method is simple, convenient and feasible and can be produced in a large scale.

Drawings

FIG. 1 is a TEM image of ruthenium-based anti-tumor nano-drug obtained in example 1 of the present invention;

FIG. 2 is a particle size distribution diagram of ruthenium-based anti-tumor nano-drug obtained in example 1 of the present invention;

FIG. 3 is a zeta potential diagram of ruthenium-based anti-tumor nano-drug obtained in example 1 of the present invention;

FIG. 4 is a TEM image of the release of ruthenium nanoparticles and atovaquone under acidic conditions of the ruthenium-based anti-tumor nano-drug obtained in example 2 of the present invention;

FIG. 5 is a graph showing the cell viability of Hela cells after co-incubation with culture medium and ruthenium-based anti-tumor nano-drug obtained in example 3 of the present invention;

FIG. 6 shows the body weight change of mice after ruthenium-based anti-tumor nano-drug treatment obtained in example 2 of the present invention;

FIG. 7 shows the change of tumor weight after the ruthenium-based anti-tumor nano-drug obtained in example 3 of the present invention is treated.

Detailed Description

The technical solutions of the present invention will be described in detail below with reference to specific embodiments and drawings to facilitate understanding for those skilled in the art, but the present invention is not limited in any way. The specific conditions are not indicated in the implementation case, and the implementation is carried out according to the conventional conditions or the conditions suggested by the manufacturer; the reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1

Weighing 40 mg of soybean lecithin, 4 mg of cholesterol and 0.85 g of atovaquone, adding 8mL of absolute ethyl alcohol to dissolve to obtain a liposome lipid phase mixed solution containing atovaquone, then placing the liposome lipid phase mixed solution on a rotary evaporator (Shanghai Shen Sheng science and technology Co., Ltd.), and carrying out rotary evaporation at 50 ℃ for 0.5 h, so that a lipid phase forms a film at the bottom of a bottle; uniformly mixing 400 mu L of phosphate buffer solution with pH of 6.5 and 9.6 mL of ultrapure water resuspended ruthenium nanoparticles, adding 5 mu L of Tween 80, uniformly mixing, adding the mixture into a lipid phase membrane, rotationally mixing for 30 min, and ultrasonically treating the obtained solution for 5 min under the condition of 800W by using a cell disruptor to obtain the ruthenium nanoparticle and atovaquone loaded nanoliposome Ru @ ATO @ Lip.

The morphology of the ruthenium and atovaquone loaded nanoliposome Ru @ ATO @ Lip is characterized by using a transmission electron microscope, as shown in figure 1, ruthenium nanoparticles are loaded in the liposome, and the particle size of the nanoliposome Ru @ ATO @ Lip is about 140 nm approximately. The ruthenium and atovaquone loaded nanoliposome Ru @ ATO @ Lip is characterized by a laser particle size analyzer, as shown in figure 2, the average particle size of the ruthenium and atovaquone loaded nanoliposome Ru @ ATO @ Lip is about 150 nm, and the result is consistent with that of a transmission electron microscope. As shown in FIG. 3, the zeta potential of the ruthenium nanoparticles was 18 mV, while the zeta potential of Ru @ ATO @ Lip was-7.5 mV. From the change in potential, it was confirmed that the ruthenium nanoparticles were encapsulated in the liposome.

Example 2

Weighing 52 mg of soybean lecithin, 5.6 mg of cholesterol and 1.0 mg of atovaquone, adding 10mL of absolute ethyl alcohol to dissolve to obtain a liposome lipid phase mixed solution containing atovaquone, then placing the liposome lipid phase mixed solution on a rotary evaporator (Shanghai Shen Sheng science and technology Co., Ltd.), and carrying out rotary evaporation at 45 ℃ for 2.5 h to form a lipid phase film at the bottom of a bottle; and uniformly mixing 450 mu L of phosphate buffer solution with the pH value of 6.5 and 9.55 mL of ruthenium nano particles resuspended in ultrapure water, adding 10 mu L of Tween 80, uniformly mixing, adding into a lipid phase, rotationally mixing for 35 min, and treating the obtained solution for 10 min under the condition of 500W by using a cell disruptor to obtain the ruthenium and atovaquone loaded nano liposome Ru @ ATO @ Lip.

The primary morphology characterization of the nanometer liposome Ru @ ATO @ Lip loaded with ruthenium and atovaquone is carried out by using a transmission electron microscope, so that the ruthenium nanometer particle is loaded in the liposome, and the particle size of the nanometer liposome Ru @ ATO @ Lip is about 140 nm approximately. The ruthenium and atovaquone loaded nano liposome Ru @ ATO @ Lip is characterized by a laser particle size analyzer, and the average particle size of the ruthenium and atovaquone loaded nano liposome Ru @ ATO @ Lip is about 150 nm and is consistent with the transmission electron microscope result. The laser particle size analyzer is used for characterizing the nanometer liposome Ru @ ATO @ Lip loaded with ruthenium and atovaquone, the zeta potential of the ruthenium nanometer particles is 18 mV, and the zeta potential of the Ru @ ATO @ Lip is-7.5 mV. The change of the potential can be used to show that the ruthenium nano-particles are coated in the liposome.

In order to prove that the ruthenium particles can generate Fenton-like reaction under acidic conditions, so that the liposome structure is damaged to achieve the purpose of drug release, a transmission electron microscope is adopted to perform morphological characterization on the ruthenium and atovaquone loaded nanoliposome Ru @ ATO @ Lip processed under acidic conditions, and as shown in figure 4, the damage of the nanoliposome Ru @ ATO @ Lip structure and the release of internal drugs can be seen under acidic conditions.

The antitumor effect of the ruthenium and atovaquone loaded nanoliposome Ru @ ATO @ Lip on a cervical cancer U14 tumor-bearing mouse is researched, the drug is administered for 14 days in a tail vein mode, the daily dosage is 200 mu L, the weight change of the mouse is recorded during the drug administration period, the result is shown in figure 6, and the weight change curve of the mouse shows that the mouse injected with the ruthenium and atovaquone loaded nanoliposome Ru @ ATO @ Lip has no obvious difference with the weight of a normal saline solution group, so that the nanoliposome Ru @ ATO @ Lip can not damage the body of the mouse, and the biological safety is high.

Example 3

Weighing 74 mg of soybean lecithin, 7.0 mg of cholesterol and 1.5 mg of atovaquone, adding 12 mL of absolute ethyl alcohol to dissolve to obtain a liposome lipid phase mixed solution containing atovaquone, then placing the liposome lipid phase mixed solution on a rotary evaporator (Shanghai Shen Sheng science and technology Co., Ltd.), and carrying out rotary evaporation at 40 ℃ for 4 hours, wherein the lipid phase forms a film at the bottom of a bottle; and uniformly mixing 500 mu L of phosphate buffer solution with pH6.5 and 9.5 mL of ultrapure water-resuspended ruthenium nanoparticles, adding 15 mu L of Tween 80, uniformly mixing, adding into a lipid phase, rotationally mixing for 30 min, and treating the obtained solution for 20 min under the condition of 200W by using a cell disruptor to obtain the ruthenium and atovaquone-loaded nanoliposome Ru @ ATO @ Lip.

The primary morphology characterization of the nano-liposome drug loaded with ruthenium and atovaquone is carried out by using a transmission electron microscope, and as can be seen from a transmission electron microscope result picture, the ruthenium nano-particle is loaded on the liposome, and the particle size of the nano-liposome Ru @ ATO @ Lip is about 140 nm approximately. The ruthenium and atovaquone loaded nano liposome Ru @ ATO @ Lip is characterized by a laser particle size analyzer, and the average particle size of the ruthenium and atovaquone loaded nano liposome Ru @ ATO @ Lip drug is about 150 nm and is consistent with the transmission electron microscope result. The laser particle size analyzer is used for characterizing the ruthenium and atovaquone loaded nano liposome Ru @ ATO @ Lip, the zeta potential of the ruthenium nano particles is 18 mV, and the zeta potential of the Ru @ ATO @ Lip is-7.5 mV., so that the ruthenium nano particles are encapsulated in the liposome according to the potential change condition.

In order to prove that the ruthenium particles can generate Fenton-like reaction under acidic conditions to damage the liposome structure and further achieve the purpose of drug release, a transmission electron microscope is adopted to perform morphology characterization on the ruthenium and atovaquone loaded nano liposome Ru @ ATO @ Lip processed under acidic conditions, and the damage of the nano liposome structure and the release of the internal drug can be seen from an electron microscope result picture.

HeLa cells are incubated with a culture medium and different groups of drugs (nano liposome Ru @ ATO @ Lip loaded with ruthenium and atovaquone, nano liposome Ru @ Lip loaded with ruthenium, nano liposome ATO @ Lip loaded with atovaquone, ruthenium and atovaquone) respectively to characterize the survival capability of the cells, as shown in figure 5, it can be seen that the single ruthenium and single atovaquone groups have a certain killing effect on the cells, but after being encapsulated in the liposome, the biological safety is improved, which indicates that the toxic and side effects of the drugs on normal cells can be reduced by the encapsulation of the liposome; after the liposome entraps ruthenium and atovaquone and hybridizes with a cell membrane, the cell survival rate is obviously reduced, which shows that the ruthenium and the atovaquone have the capability of mutually promoting and killing cancer cells.

In order to characterize the in vivo tumor treatment effect of the drug, the prepared nano drug, normal saline, Ru @ Lip and ATO @ Lip are respectively injected into a cervical cancer U14 tumor-bearing mouse, the administration is carried out for 14 days, and the daily administration amount is 200 mu L, as shown in figure 7, the tumor weight of the mouse injected with the nano liposome Ru @ ATO @ Lip loaded with ruthenium and atovaquone is obviously smaller than that of the normal saline group phase, which indicates that the nano liposome Ru @ ATO @ Lip loaded with ruthenium and atovaquone has good tumor inhibition effect.