CN112472731B

CN112472731B - Cucumber exosome-like vesicle containing cucurbitacin B and capable of serving as anti-cancer drug

Info

Publication number: CN112472731B
Application number: CN202011418370.9A
Authority: CN
Inventors: 陈敬华; 陈婷婷; 夏垚坤; 曾鲁鹏; 马丙香; 王华英; 施婉华
Original assignee: Fujian Medical University
Current assignee: Fujian Medical University
Priority date: 2020-12-06
Filing date: 2020-12-06
Publication date: 2022-07-19
Anticipated expiration: 2040-12-06
Also published as: CN112472731A

Abstract

The invention discloses a cucumber exosome-like vesicle containing cucurbitacin B and capable of being used as an anti-cancer drug. Cucurbitacin B can be used to inhibit proliferation of a variety of cancer cells, but its extraction process is complicated and time consuming. The cucurbitacin B obtained by the method for separating the cucumber exosome-like vesicles has the advantages of simplicity in operation, rapidness and the like, and the lipid bilayer of the exosome-like vesicles is used as a natural protective film of cucurbitacin B, so that the stability of cucurbitacin B can be maintained for a long time. Moreover, the existence of the lipid bilayer also improves the uptake rate of cucurbitacin B by cells to a certain extent through the principle of similar intermiscibility. When the cucurbitacin B-like exosome is acted on adenocarcinoma human alveolar basal epithelial cells (A549 cells), the maximum cell activity inhibition rate of the cucumber exosome-like vesicles is 2 times that of cucurbitacin B standard products, the dosage is lower, the toxicity to normal cells and the drug resistance of cancer cells can be reduced, and the cucurbitacin B-like exosome has good anticancer potential and industrialization prospect and has important significance in clinical tumor treatment application.

Description

Cucumber exosome-like vesicle containing cucurbitacin B and capable of being used as anti-cancer drug

Technical Field

The invention relates to the technical field of separation and extraction of plant exosome-like vesicles and research of anti-cancer drugs, in particular to extraction of cucumber exosome-like vesicles containing active drug ingredients and feasibility research of the cucumber exosome-like vesicles as anti-cancer drugs.

Background

1. Exosomes are nanoscale vesicle structures actively secreted by cells, and in recent years, the exosomes are popular for research as drug carriers and can participate in various biological processes such as intercellular communication. Moreover, the lipid bilayer outside the exosome can effectively protect the internal active ingredients and ensure that the active ingredients are not decomposed before reaching the target site. In addition, the presence of lipid bilayers provides modification sites for a variety of functional molecules.

2. However, the isolation of animal exosomes by harvesting still presents some problems in practical applications. For example, obtaining exosomes by cell culture has the disadvantages of being long in time, limited in number, high in cost and the like. Furthermore, tumor cell-derived exosomes, not only may themselves promote tumor spread, but also may elicit a severe immune response in the body. This greatly limits their application in the field of drug carrier research.

3. The plant exosome-like vesicle has a lipid bilayer structure like an animal exosome, can protect the internal active ingredients, and can be used for treating various diseases by containing natural pharmacological active ingredients. Meanwhile, the separation has the advantages of high speed, high yield, low cost and the like. In addition, some studies have shown that exosome-like vesicles secreted by edible plants do not elicit immune responses in humans. Based on the advantages, the research and application of the plant exosome-like vesicle in the drug carrier have great potential.

4. The cucumber is a daily vegetable, has a long medicinal history, has medicinal values recorded in book records of Qianjin marrow prescription, Yi Lin Ji Yao and the like, contains various pharmacologically active components such as cucurbitacin, vitamins and the like, and has pharmacological effects of resisting cancer, resisting inflammation and the like. At present, the separation of cucurbitacin B and other active ingredients from cucumber has been studied. However, cucurbitacin B has poor stability and is prone to structural change due to environmental factors such as temperature and humidity, and thus pharmacological activity of cucurbitacin B is affected. Moreover, cucurbitacin B has low water solubility, which greatly limits its clinical application. The cucumber exosome-like vesicle has a lipid bilayer structure similar to exosome, is a natural protective film and can reduce the influence of the external environment on the internal components of the cucumber exosome-like vesicle. In addition, the existence of lipid bilayer also increases water solubility and biocompatibility of cucurbitacin B to some extent.

5. Anticancer active ingredients often need to be combined with appropriate drug delivery vehicles in order to exert their anticancer effects better. This often requires a complex loading procedure. For example, covalent attachment of a drug to a carrier unit by chemical synthesis and formation of a drug-carrier complex by self-assembly usually require stringent experimental conditions and skilled operators. In recent years, the development of DNA self-assembly technology makes DNA nanostructures become popular drug carrier research targets, and researchers only need to incubate drugs and DNA to obtain drug-carrier complexes. However, DNA is expensive, complex in design, poor in vivo stability and easy to be digested, which limits the application of DNA self-assembly technology in drug carriers to a certain extent. A simpler assembly method is to mix the synthesized lipid structure with the active drug, prepared by an additional extrusion process. The cucumber exosome-like vesicle can be directly and efficiently used for inhibiting the proliferation of cancer cells without any assembly condition. This is because it contains both lipid bilayer structure which can be used as a drug carrier and cucurbitacin B as an anticancer active ingredient. The lipid bilayer is used as a natural drug carrier, and can improve the uptake rate of cucurbitacin B by cancer cells, thereby achieving better anticancer effect.

Disclosure of Invention

The invention aims to provide a cucumber exosome-like vesicle containing cucurbitacin B and capable of being used as an anti-cancer drug, and a preparation method and application thereof.

(1) The invention relates to an exosome-like vesicle obtained by separating cucumber, which contains cucurbitacin B anticancer active ingredients and lipid bilayers capable of protecting the internal active ingredients, and can effectively maintain the stability of the internal active ingredients;

(2) the cucumber exosome-like vesicle obtained by the invention can obviously inhibit the activity of A549 cells, the highest cell activity inhibition rate is twice of that of cucurbitacin B standard substance, the dosage is less, and the problems of normal cytotoxicity, cancer cell drug resistance and the like caused by high-dose anticancer drugs can be reduced;

(3) the cucumber exosome-like vesicle obtained by the invention can inhibit the proliferation of various cancer cells.

Solution technical scheme

In order to realize the purpose of the invention, the invention provides the following technical scheme:

step a, preparation of cucumber exosome-like vesicles:

(1) juicing cucumbers by using a juicer and filtering by using warp cloth to obtain filtrate;

(2) centrifuging the filtrate and obtaining a supernatant by removing the precipitate;

(3) and after ultracentrifugation is carried out on the supernatant, a proper amount of PBS is used for heavy suspension and precipitation, and then cucumber exosome-like vesicles are obtained.

The juice squeezing in the step (1) needs to be carried out by mixing precooled PBS or PBS ice blocks with cucumbers.

The centrifugal separation in the step (2) is carried out for 10 min at 5000 g and 35 min at 10000 g.

The ultracentrifugation in the step (3) is performed at 30000 g for 30 min.

B, an analysis pretreatment method of active ingredients in the cucumber exosome-like vesicles comprises the following steps:

(1) centrifuging the cucumber exosome-like vesicle obtained in the step a, and removing the supernatant PBS to obtain cucumber exosome-like vesicle sediment;

(2) resuspending cucumber exosome-like vesicle sediment by using a proper amount of methanol to obtain cucumber exosome-like vesicle suspension, and performing ultrasonic treatment;

(3) centrifuging the suspension subjected to ultrasonic treatment, precipitating cucumber exosome-like vesicles for later use, and collecting supernatant;

(4) repeating the steps (2) and (3) twice, and combining the supernatant collected for 3 times.

The ratio of the total amount of methanol to the total protein of the cucumber exosome-like vesicle in the steps (2) and (4) is 1 mL to 6 mg (the total protein of the cucumber exosome-like vesicle is measured by using a BCA protein quantification kit).

And (3) the ultrasonic parameters in the step (2) are 500W and 15 min.

C, analyzing important parameters of active ingredients in the cucumber exosome-like vesicles by using high performance liquid chromatography:

(1) mobile phase conditions were acetonitrile-water (45: 55);

(2) the column temperature is 30 ℃;

(3) the detection wavelength was 228 nm.

D, using cucurbitacin B and cucumber exosome-like vesicles for killing various cancer cells:

(1) uniformly adding the cancer cell suspension into a 96-well plate, and sucking and removing the culture solution after the cells adhere to the wall;

(2) administration: adding the cucurbitacin B standard substance and the cucumber exosome-like vesicles into different holes of the 96-hole plate respectively, reacting for 24 hours, and then absorbing and discarding;

(3) adding MTT solution into the 96-well plate, reacting for 4 h, and then sucking away;

(4) dimethyl sulfoxide (DMSO) was added to the above 96-well plate, and after shaking, absorbance was measured with a microplate reader to calculate the cell survival rate.

In the step (1), the concentration of the cell suspension is 10 ten thousand cells/mL, and the volume of the cell suspension in each hole is 100 mu L.

In the step (2), the administration concentration of the cucurbitacin B standard substance is 0, 12.5, 25, 50, 100, 200, 400, 800, 1600 and 3200 nM. The concentrations of cucumber exosome-like vesicles administered were 0, 0.078, 0.16, 0.31, 0.62, 1.25, 2.5, 5, 10, 20 nM (here the concentrations are of cucumber exosome-like vesiculosin B).

In the step (3), the concentration of MTT was 0.5 mg/mL, and the volume added per well was 100 uL.

In the step (4), the volume of DMSO is 150 mu L. The detection wavelength was 570 nm.

And e, detecting the generation condition of ROS in A549 cells after incubation of the cucurbitacin B standard product and the cucumber exosome-like vesicle by using an ROS (reactive oxygen species) detection kit:

(1) uniformly adding the A549 cell suspension into a 24-pore plate, and sucking and removing the culture solution after the cells adhere to the wall;

(2) adding a fluorescent probe (DCFH-DA) into the 24-well plate, incubating for 1 h, and then sucking away;

(3) adding serum-free RPMI-1640 cell culture medium, Rosu (positive control), cucurbitacin B and cucumber exosome-like vesicles into different wells of the 24-well plate respectively, incubating for 15 min, and then removing;

(4) a proper amount of serum-free RPMI-1640 cell culture medium was added to the 24-well plate, and the fluorescence intensity between cells of different groups was observed.

In the step (1), the concentration of the A549 cell suspension is 5 ten thousand cells/mL, and the volume of the cell suspension per hole is 800 mu L.

In the step (2), the concentration of the fluorescent probe DCFH-DA is 10 μ M. After being discarded, the cells were washed 3 times with serum-free RPMI-1640 cell culture medium.

In the step (3), the dilution ratio of the Rosu is 1:200, the concentration of cucurbitacin B is 500 nM, and the concentration of cucumber exosome-like vesicles is 20 nM (here, the concentration is the concentration of cucumber exosome-like vesiculocucurbitacin B).

The fluorescence excitation wavelength in the step (4) is 488 nm.

Advantageous effects

Compared with the prior art, the invention has the advantages that:

(1) the preparation method for separating the exosome-like vesicles from the cucumbers is simple and convenient to operate, and compared with animal exosomes, the preparation method has the advantages of higher yield, higher speed and lower cost;

(2) the cucumber exosome-like vesicle prepared by the invention contains cucurbitacin B anticancer active ingredients, and the natural lipid bilayer structure contained in the cucumber exosome-like vesicle can maintain the stability of cucurbitacin B for a long time;

(3) the cucumber exosome-like vesicle simultaneously contains a carrier and an anticancer active ingredient, can be directly used for inhibiting the proliferation of cancer cells as a natural anticancer drug, and when the cucumber exosome-like vesicle acts on A549 cells, the maximum cell activity inhibition rate (95.1%) of the cucumber exosome-like vesicle is 2 times that of a cucurbitacin B standard product (45.7%).

(4) High doses of anticancer drugs tend to cause death of normal cells and increase resistance of cancer cells. The A549 cytotoxicity test result shows that when the same cell activity inhibition rate is achieved, the concentration of cucurbitacin B in the cucumber exosome-like vesicle is far lower than that of a cucurbitacin B standard product, so that the cucumber exosome-like vesicle can effectively solve the problems of normal cytotoxicity, cancer cell drug resistance and the like caused by a high-dose cucurbitacin B standard product.

Drawings

Fig. 1 is a graph showing the characterization results of cucumber exosome-like vesicles isolated in example 1. In the figure: (A) detecting the particle size distribution of the cucumber exosome-like vesicle by dynamic light scattering; (B) the appearance of the cucumber exosome-like vesicle is characterized by a transmission electron microscope; (C) zeta potential of cucumber exosome-like vesicles.

Fig. 2 is a hplc analysis chart of cucurbitacin B and cucurbitacin B standards in cucumber exosome-like vesicles of example 2.

FIG. 2-1 is a linear analysis chart of the cucurbitacin B standard in example 2.

Fig. 3 is a graph representing ROS production by a549 cells by the cucurbitacin B standard and cucumber exosome-like vesicles in example 4. Bright field pictures (A) and fluorescence field pictures (B) of A549 cells which are not treated by any medicine under a fluorescence microscope; bright field (C) and fluorescence field (D) pictures of the ROSs (ROS positive control) -treated a549 cells; bright field (E) and fluorescence field (F) pictures of A549 cells treated by the cucurbitacin B standard substance; bright field (G) and fluorescence field (H) pictures of a549 cells treated with cucumber exosome-like vesicles.

Detailed Description

In order to explain the technical contents, characterization and performance analysis methods of the present invention in detail, the embodiments of the present invention will be explained in detail below with reference to the accompanying drawings.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the reagents used are commercially available.

Example 1:

example 1 of the present invention provides a method for separating exosome-like vesicles from cucumber, which comprises the following preparation steps:

(1) juicing the cucumbers by a juicer and filtering by using warp cloth to obtain filtrate;

(3) and (3) carrying out ultracentrifugation on the supernatant, and then carrying out heavy suspension precipitation by using a proper amount of PBS to obtain cucumber exosome-like vesicles.

The juicing in the step (1) needs to be carried out by mixing precooled PBS or PBS ice blocks with cucumbers.

The ultracentrifugation in the step (3) is performed at 30000 g for 30 min.

The characterization result of the obtained cucumber exosome-like vesicles is shown in figure 1, wherein (A) the particle size distribution of the cucumber exosome-like vesicles is detected by dynamic light scattering; (B) the appearance of the cucumber exosome-like vesicle is characterized by a transmission electron microscope; (C) zeta potential of cucumber exosome-like vesicles.

Example 2:

the process of analyzing the active ingredients in the cucumber exosome-like vesicles prepared in example 1 by high performance liquid chromatography was as follows:

(1) centrifuging the cucumber exosome-like vesicles prepared in the example 1, and removing the supernatant PBS to obtain cucumber exosome-like vesicle precipitates;

(2) using a proper amount of methanol to resuspend cucumber exosome-like vesicle precipitates to obtain a cucumber exosome-like vesicle suspension, and carrying out ultrasonic treatment;

In the steps (2) and (4), the ratio of the total amount of the methanol to the total protein of the cucumber exosome-like vesicle is 1 mL to 6 mg (the total protein of the cucumber exosome-like vesicle is measured by using a BCA protein quantification kit).

In the step (2), the ultrasonic parameters are 500W and 15 min.

Filtering the supernatant obtained in the step (4) by using a 0.22 mu m filter membrane for later use.

The ultrasonic extracts of the cucurbitacin B standard substance and the cucumber exosome-like vesicle are analyzed and detected at 228 nm under the conditions that the mobile phase is acetonitrile-water (45: 55), the flow rate is 1 mL/min, and the column temperature is 30 ℃.

The HPLC analysis chart of cucurbitacin B and cucurbitacin B standard substance in cucumber exosome-like vesicle is shown in FIG. 2; the linear analysis of the cucurbitacin B standard is shown in FIG. 2-1.

Example 3:

the MTT method is used for verifying the cytotoxicity effect of the cucurbitacin B standard product and cucumber exosome-like vesicles on cancer cells, and the specific operation method is as follows:

(1) uniformly adding the cancer cell suspension into a 96-well plate, and sucking and removing a culture solution after the cells are attached to the wall;

(2) administration: adding the cucurbitacin B standard product and the cucumber exosome-like vesicle prepared in the example 1 into different wells of the 96-well plate respectively, reacting for 24 h, and then sucking and discarding;

(3) adding MTT solution into the 96-well plate, reacting for 4 h, and then sucking and discarding;

(4) dimethyl sulfoxide (DMSO) was added to the above 96-well plate, shaken, and then absorbance was measured by a microplate reader to calculate the cell survival rate.

As shown in table 1, firstly, compared to the cucurbitacin B standard, the cucumber exosome-like vesicle prepared in example 1 has better killing effect on a549 cells, the highest cell activity inhibition rate (95.1%) of the cucumber exosome-like vesicle is 2 times that of the cucurbitacin standard (45.7%), and the dosage of the cucumber exosome-like vesicle is lower; secondly, the cucumber exosome-like vesicle prepared in example 1 has a good killing effect on various cancer cells.

TABLE 1 cucurbitacin B standard and cucumber exosome-like vesicles as anti-cancer drugs for tumor cells

Result of killing

Example 4:

and verifying the condition that the cucurbitacin B standard product and the cucumber exosome-like vesicle enable the A549 cell to generate the ROS by using a ROS detection kit. The method comprises the following specific steps:

(1) uniformly adding the A549 cell suspension into a 24-well plate, and absorbing and removing a culture solution after the cells are attached to the wall;

(2) adding a fluorescent probe (DCFH-DA) into the 24-well plate, incubating for 1 h, and then absorbing and discarding;

(3) serum-free RPMI-1640 cell culture media, Rosu (positive control), cucurbitacin B and the cucumber exosome-like vesicles prepared in example 1 are respectively added into different wells of the 24-well plate, incubated for 15 min and then discarded;

(4) a suitable amount of serum-free RPMI-1640 cell culture medium was added to the above 24-well plate, and the fluorescence intensity between cells of different groups was observed.

The concentration of the A549 cell suspension in the step (1) is 5 ten thousand cells/mL, and the volume of the cell suspension in each hole is 800 mu L.

The concentration of the fluorescent probe DCFH-DA in the step (2) is 10 MuM. After the aspiration, the cells were washed 3 times with serum-free RPMI-1640 cell culture medium.

In the step (3), the dilution ratio of the Rosu in the step (3) is 1:200, the concentration of cucurbitacin B is 500 nM, and the concentration of cucumber exosome-like vesicles is 20 nM (here, the concentration is the concentration of cucurbitacin B in cucumber exosome-like vesicles).

The fluorescence excitation wavelength in the step (4) is 488 nm.

As shown in fig. 3, cucumber exosome-like vesicles and cucurbitacin B standard were able to make a549 cells produce ROS. Bright field (a) and fluorescence field (B) pictures of a549 cells without any drug treatment under a fluorescence microscope; bright field (C) and fluorescence field (D) pictures of the ROSs (ROS positive control) -treated a549 cells; bright field (E) and fluorescence field (F) pictures of A549 cells treated by the cucurbitacin B standard substance; bright field (G) and fluorescence field (H) pictures of a549 cells treated with cucumber exosome-like vesicles.

Claims

1. The application of cucumber exosome-like vesicles containing cucurbitacin B in preparation of killing drugs for various cancer cells is as follows: a549 cells; HGC27 cells; HepG2 cells; a HeLa cell; MDA-MB-231 cells; 4T1 cells;

the preparation method of the cucumber exosome-like vesicle containing cucurbitacin B comprises the following steps:

(1) juicing cucumbers by using a juicer, filtering by using warp cloth to obtain filtrate, and mixing precooled PBS (phosphate buffer solution) or PBS ice blocks with the cucumbers in the juicing process;

(2) centrifuging the filtrate and obtaining supernatant by removing precipitate, wherein the centrifugation is carried out for 10-35 min between 5000 and 10000 g;

(3) and centrifuging the supernatant at 30000 g for 30 min, and then carrying out heavy suspension precipitation by using a proper amount of PBS to obtain cucumber exosome-like vesicles.