CN111617053A

CN111617053A - Method for preparing vesicle by inducing apoptosis with high osmotic pressure

Info

Publication number: CN111617053A
Application number: CN202010555537.XA
Authority: CN
Inventors: 尹江峰; 杨帆; 张一�
Original assignee: Hubei Soundny Bio Tech Co ltd
Current assignee: Hubei Soundny Bio Tech Co ltd
Priority date: 2020-06-17
Filing date: 2020-06-17
Publication date: 2020-09-04

Abstract

The invention provides a method for preparing vesicles by inducing apoptosis with hypertonic solution and a method for preparing drug-loaded vesicles by wrapping chemotherapeutic drugs with the vesicles. The osmotic pressure of the hypertonic solution is 500-650mOSM/kg, preferably formulated from trehalose. The number and the particle size uniformity of the vesicles obtained by the invention are superior to those of the vesicles generated by ultraviolet irradiation. The drug-loaded vesicle prepared from the vesicle generated by the induction of the high-permeability solution has satisfactory killing effect on in-vitro tumor cells.

Description

Method for preparing vesicle by inducing apoptosis with high osmotic pressure

Technical Field

The invention belongs to the technical field of biology, and relates to a method for preparing vesicles by inducing apoptosis with high osmotic pressure.

Background

Cytoskeleton is an important structure by which eukaryotic cells maintain their basic morphology, and cells, after being stimulated exogenously or endogenously, cause rearrangement of the cytoskeleton, resulting in uneven local stress on the cell membrane. The abnormally stressed cytoplasm expands outwards, and then is randomly coated with part of cell contents and released to the outside of the cell in the form of vesicles. This particular subcellular structure, with a diameter of about 0.1-1 μm, is called a "cell vesicle".

Chinese patent CN102302784B and its derived patents disclose a method for incubating tumor chemotherapy drug preparation at room temperature by using cell vesicles obtained by irradiating tumor cells with ultraviolet rays and chemotherapy drugs. The medicine-carrying vesicle prepared by the method has the characteristics of low toxicity, strong targeting property and the like, and can effectively activate the immune response of an organism. The preparation is prepared by taking a cell vesicle derived from an apoptotic tumor cell as a carrier and coating a tumor chemotherapeutic drug with the cell vesicle. The preparation can directly reach tumor part, improve the drug effect of chemotherapeutic drugs, and overcome the toxic and side effects on organism due to exogenous vector administration. The preparation method of the tumor chemotherapy pharmaceutical preparation can induce apoptosis by ultraviolet irradiation and collect cell vesicles, and then incubate the cell vesicles with chemotherapy drugs as active ingredients to enable the chemotherapy drugs to be wrapped by the cell vesicles to form the pharmaceutical preparation.

However, the ultraviolet ray penetration is not strong, and when the cell density is high, the cells are piled up, and the cells piled up inside cannot be irradiated with the ultraviolet ray, but only the cells on the surface layer can be irradiated to generate vesicles. This poses a great challenge to batch stability of vesicle preparation. While irradiation with low density cells, although uniform, limits the capacity of vesicles. In the field of vesicle preparations of tumor chemotherapeutic drugs, development of other novel preparation methods is urgently needed to improve efficiency.

Disclosure of Invention

The invention provides a method for inducing apoptosis to generate vesicles by preparing a hypertonic solution, and further a drug-loaded vesicle prepared by wrapping tumor chemotherapy drugs with the vesicles is used as a tumor chemotherapy drug preparation. Osmotic pressure is easily adjusted through the concentration of an osmotic pressure regulator, cells are evenly suspended in a hypertonic solution in a stirring mode for induction, and the generated vesicles are good in uniformity and convenient for stable mass preparation. The osmotic pressure regulator used in the invention is a pharmaceutical adjuvant recorded in pharmacopoeia, and has good safety.

According to one aspect of the invention, a method of inducing vesicle production by tumor cells with a hypertonic solution is investigated. Suspending cells in a hypertonic solution prepared from sodium chloride, trehalose, sucrose, maltose and D-sorbitol to prepare cell vesicles; through detecting the number and the particle size of the vesicles generated by induction, the cell vesicles generated by the induction of hypertonic solution are found to have uniform particle size and higher yield compared with the vesicles generated by the induction of ultraviolet irradiation. Among them, the hypertonic solution is preferably prepared by dissolving trehalose in purified water.

The invention also researches the influence of different osmotic pressures on the generation of vesicles by inducing apoptosis. When the osmotic pressure of the hypertonic solution is 500-650mOSM/kg, the apoptosis rate of cells and the yield of vesicles are highest, and preferably, the osmotic pressure of the hypertonic solution for inducing apoptosis is 550 mOSM/kg.

The invention also researches the influence of the induction time on the vesicle yield, the yield of the cell vesicles induced by the hypertonic solution increases along with the increase of the induction time, and after 24 hours, the yield increases gradually, and the induction time is preferably 24 hours.

The invention researches the comparison between the functions of the cell vesicles generated by the induction of hypertonic solution and the vesicles generated by ultraviolet irradiation, and the result shows that the chemotaxis of the vesicles induced by the two modes on the neutrophils has no obvious difference.

In another aspect of the present invention, a method for preparing a drug-loaded vesicle by incubating a cell vesicle induced by a hypertonic solution with a chemotherapeutic drug is provided, wherein the drug encapsulated by the drug-loaded vesicle is a tumor chemotherapeutic drug selected from methotrexate, adriamycin, cisplatin, paclitaxel, 5-fluorouracil, gemcitabine or 10-hydroxycamptothecin; preferably, the drug-loaded vesicle-encapsulated drug is methotrexate.

The invention researches the yield and the drug-loading rate of the drug-loaded vesicle prepared by the cell vesicle generated by the induction of the hypertonic solution, and compared with the drug-loaded vesicle prepared by the induction of ultraviolet irradiation and the induction of chemotherapeutic drugs, the yield and the drug-loading rate of the drug-loaded vesicle are obviously improved. The invention also verifies the contrast of the uptake effect of the tumor cells on the vesicles prepared by the three modes and the in vitro uptake effect on the A549 human lung adenocarcinoma cell line,

Compared with the killing effects of OVCAR-3 human ovarian cancer cell line, HepG2 human liver cancer cell line, MCF-7 human breast cancer cell line and HCT-8 human colon cancer cell line tumors, the drug-loaded vesicles prepared by hypertonic solution induction are obviously superior to chemotherapeutic drug induction methods and are equivalent to ultraviolet irradiation methods.

The invention has the beneficial effects that: provides a method for efficiently preparing cell vesicles, which is simple to operate, and the prepared vesicles are safe and have uniform particle sizes. The vesicle can be used for wrapping tumor chemotherapy drugs to prepare drug-loaded vesicles. The drug-loaded vesicle can be well taken by tumors, has obvious tumor killing effect, and can be used as a chemotherapeutic drug preparation. The vesicle can also be used for wrapping other medicines to prepare a medicine carrying vesicle, and has wide application prospect in the field of medicine preparations.

Drawings

FIG. 1 is a diagram showing the number of vesicles induced by five hypertonic solutions.

FIG. 2 shows the detection of vesicle size distribution induced by hypertonic solution and irradiated by UV light.

FIG. 3 Effect of osmotic pressure on tumor cell apoptosis rate.

FIG. 4 effect of trehalose osmolality on the induction of vesicle production.

Figure 5 effect of hypertonic solution induction time on vesicle production.

FIG. 6 chemotaxis of neutrophils by trehalose hypertonic solution induced vesicles.

Figure 7 comparison of vesicle numbers of drug-loaded vesicles prepared by inducing apoptosis in different ways.

FIG. 8 is a comparison of drug loading of drug-loaded vesicles prepared by inducing apoptosis in different ways.

FIG. 9 comparison of uptake by tumor cells of drug-loaded vesicles prepared by different ways of inducing apoptosis.

FIG. 10 comparison of in vitro killing of tumor cells by drug-loaded vesicles prepared by inducing apoptosis in different ways.

Detailed Description

The following examples are merely illustrative of embodiments of the present invention and do not limit the scope of the present invention in any way.

The cell, reagent and raw material sources used in the method are as follows:

the K562 cells, A549 human lung adenocarcinoma cell line, OVCAR-3 human ovarian cancer cell line, HepG2 human liver cancer cell line, MCF-7 human breast cancer cell line, and HCT-8 human colon cancer cell line cells used in the examples were purchased from the China center for type culture Collection. The chemotherapeutic agents used in the examples, Methotrexate (MTX), doxorubicin hydrochloride (DOX), Cisplatin (CDDP), 10-hydroxycamptothecin (10-HCPT) were purchased from Dalian Meiren Biotechnology Ltd. 1640 medium was purchased from Biological Industries. Annexin V-FITC apoptosis detection kit was purchased from Sigma-Aldrich. The rest of the reagents are all commercial products.

Example 1

The inventor firstly researches the function of different osmotic pressure regulators for inducing apoptosis to generate vesicles, selects 5 osmotic pressure regulators, respectively adds purified water into sodium chloride, trehalose, sucrose, maltose and D-sorbitol to prepare a hypertonic solution with the osmotic pressure of 450mOSM/kg, detects the osmotic pressure by adopting a freezing point osmometer OM806 produced by German Roze (LOSER), regulates the pH to 7.0 by using 3M NaOH solution, filters and sterilizes the solution for later use, and respectively takes 2 × 10⁸The individual K562 cells were suspended in 50ml of the above hypertonic solution, and a sterilized magnetic stirrer was added, set at a stirring speed of 100rpm, and stirred overnight in a refrigerator at 4 ℃.

Take 2 × 10⁸The K562 cells were suspended in RPMI1640 medium, UV-irradiated for 1h, and then incubated overnight in a carbon dioxide incubator at 37 ℃. After overnight incubation, vesicles were collected by gradient centrifugation and analyzed for number and size using a marvens NS300 particle tracking analyzer.

The experimental results show that the number of vesicles induced by the hypertonic solution prepared from sodium chloride and trehalose is higher than that induced by ultraviolet (figure 1). The trehalose-induced vesicles have narrow and single peak types on the concentration-particle size detection report, and the vesicle particle sizes are more uniform (as shown in fig. 2).

Example 2

The osmotic pressure of the trehalose hypertonic solution to induce apoptosis-producing vesicles was further optimized according to the study of example 1.

Preparing hypertonic trehalose solution with osmotic pressure of 400,450,500,550,600,650,700mOSM/kg by the same method as in example 1, adjusting pH to 7.0 with 3M NaOH solution, filtering, sterilizing, and collecting 2 × 10⁸The individual K562 cells were suspended in 50ml of the above hypertonic solution, and a sterilized magnetic stirrer was added, set at a stirring speed of 100rpm, and stirred overnight in a refrigerator at 4 ℃.

Then carrying out apoptosis detection and vesicle yield detection. The apoptosis detection method comprises the steps of taking the treated K562 cells to be suspended in Annexin V binding buffer, adding apoptosis detection reagents Annexin V-FITC and PI, incubating for 30min, and then using a BD FACSCAnto II flow cytometer to carry out apoptosis detection, wherein Annexin V positive grouping is apoptotic cells. And collecting vesicles from the treated K562 cells by gradient centrifugation, and analyzing the number and the particle size of the vesicles by using a Malvern NS300 particle tracking analyzer.

The results show that the apoptosis rate gradually increases with the increase of osmotic pressure, and the apoptosis rate does not increase after the osmotic pressure exceeds 600mOSM/kg (shown in figure 3). The number of vesicles induced by 550mOSM/kg trehalose solution was the greatest (FIG. 4).

Example 3

The time to trehalose-induced apoptosis was further optimized based on the study of example 2.

Preparing trehalose hypertonic solution with osmotic pressure of 550mOSM/kg, adjusting pH to 7.0 with 3M NaOH solution, filtering, sterilizing, and collecting 2 × 10⁸Suspending the K562 cells in 50ml of the hypertonic solution, adding a sterilized magnetic stirrer, setting the stirring speed at 100rpm, stirring for 4,8,16,24,36 and 48 hours in a refrigerator at 4 ℃, collecting the vesicles through gradient centrifugation, and analyzing the number and the particle size of the vesicles by using a Malvern NS300 particle tracking analyzer. The result shows that the vesicle yield increases along with the increase of the induction time, the vesicle yield increases slowly after the time exceeds 24h, and the convenience of both the yield and the actual production can be consideredThe induction time was set at 24h (see FIG. 5).

Example 4

This example investigates the in vitro chemotactic effect of tumor cell vesicles induced by a hypertonic trehalose solution on neutrophils.

Preparing trehalose hypertonic solution with osmotic pressure of 550mOSM/kg, adjusting pH to 7.0 with 3M NaOH solution, filtering, sterilizing, and collecting 2 × 10⁸Suspending the K562 cells in 50ml of the above hypertonic solution, adding sterilized magnetic stirrer, setting stirring speed at 100rpm, stirring at 4 deg.C for 24 hr in refrigerator, and taking 2 × 10⁸The K562 cells were suspended in RPMI1640 medium, irradiated with UV light for 1h, and then incubated overnight in a carbon dioxide incubator at 37 ℃. After overnight incubation, vesicles were collected by gradient centrifugation and chemotaxis of neutrophils by the vesicles was examined. The measuring method comprises the following steps: the upper chamber of the Transwell chamber is added with human peripheral blood separated neutrophils and serum-free culture medium, the lower chamber is respectively added with trehalose hypertonic solution and ultraviolet induced vesicles, incubation is carried out for 1h, and the lower chamber neutrophils are collected and counted.

The result shows that the trehalose hypertonic solution and the ultraviolet-induced vesicle have obvious chemotactic effect on the neutrophils, and the chemotactic capabilities of the trehalose hypertonic solution and the ultraviolet-induced vesicle are not obviously different. The PBS control group had no chemotactic effect on neutrophils (see figure 6).

Example 5

In the embodiment, the drug-loaded vesicles are prepared by inducing apoptosis with trehalose hypertonic solution, and the number of the vesicles and the drug-loaded amount of the vesicles are detected.

1) Preparing trehalose hypertonic solution with osmotic pressure of 550mOSM/kg, adjusting pH to 7.0 with 3M NaOH solution, filtering, sterilizing, and collecting 2 × 10⁸Each K562 cell was suspended in 50ml of the above hypertonic solution, and the following chemotherapeutic drugs, Methotrexate (MTX)2mg/ml, doxorubicin hydrochloride (DOX) 200. mu.g/ml, Cisplatin (CDDP) 200. mu.g/ml, and 10-hydroxycamptothecin (10-HCPT) 500. mu.g/ml, were added, respectively. A sterilized magnetic stirrer was added thereto, and the mixture was stirred at a stirring speed of 100rpm in a refrigerator at 4 ℃ for 24 hours.

Meanwhile, the drug-loaded vesicle is prepared by directly inducing tumor cell apoptosis with chemotherapeutic drugs, the trehalose hypertonic solution is replaced by 0.9% sodium chloride injection, the concentration of the chemotherapeutic drugs is the same, and the conditions and the time for stirring and incubating are the same as those of the experiment 1). Another group of K562 cells was taken, UV-irradiation induced apoptosis was performed by the method described in example 2 of CN102302784B, and after vesicles were extracted, the drug-loaded vesicles were prepared by adding 2mg/ml of Methotrexate (MTX), 200. mu.g/ml of doxorabicin hydrochloride (DOX), 200. mu.g/ml of Cisplatin (CDDP), and 500. mu.g/ml of 10-hydroxycamptothecin (10-HCPT).

The number of vesicles in each group was detected by Malvern NS300 particle tracking analyzer, and the results showed that the number of drug-loaded vesicles prepared by induction with trehalose hypertonic solution was significantly higher than those in the ultraviolet light irradiation group and the chemotherapy drug direct induction group (shown in FIG. 7). The unit vesicles (1 × 10) were detected by using Thermo Ultimate 3000 HPLC¹⁰In the method, referring to the pharmacopoeia 2015 edition of the people's republic of China (chromatographic column is C18250 × 4.6.6 mm), the HPLC detection conditions of 10-hydroxycamptothecin are that the mobile phase methanol is water which is 50:50(v/v), and the chromatographic column is Thermo Acclaim^TM120, column temperature 30 ℃, flow rate: 1ml/min, detection wavelength: 266 nm. The results are shown in FIG. 8.

Example 6

This example studies the in vitro anti-tumor effect of drug-loaded vesicles.

Separately, trehalose hypertonic solution-induced, chemotherapeutic (MTX) direct-induced and ultraviolet-induced Methotrexate (MTX) drug-loaded vesicles were prepared according to the method of example 5, and stained using PKH 26. the vesicles were resuspended in Diluent C solution, 2. mu.l of PKH26 ethanol solution was added, incubation was performed at 37 ℃ for 30min, then an equal volume of fetal bovine serum was added to stop staining, 14000g was centrifuged for 30min to remove free dye, and PKH 26-labeled vesicles were resuspended using physiological saline.A 549 human lung adenocarcinoma cell line, OVCAR-3 human ovarian cancer cell line, HepG2 human liver cancer cell line, MCF-7 human breast cancer cell line and HCT-8 human colon cancer cell line were selected, and 24, 1 × 10 per well, 24 per well were selected⁵And (4) cells. 5% CO at 37 ℃₂Incubate overnight in incubator, add equal amount of the three PKH26 labeled vesicles per well, incubate for 3h, digest the cells in the well and transfer to flow tube using BDFACSCANTO II flowThe vesicle uptake detection is carried out by a cytometer. PKH26 was detected using the PE channel and the results were expressed as the percentage of PE positive cells (see figure 9 for results).

Three groups of A549 human lung adenocarcinoma cell lines, OVCAR-3 human ovarian cancer cell lines, HepG2 human liver cancer cell lines, MCF-7 human breast cancer cell lines and HCT-8 human colon cancer cell lines are respectively selected and planted into 48-pore plates, and each pore is 1.5 × 10⁴And (4) cells. 5% CO at 37 ℃₂After incubation in an incubator overnight, MTX drug-loaded vesicles prepared by three methods with the same amount are respectively added into the same cell line of each pore, and the mixture is incubated at 37 ℃ and 5% CO₂Apoptosis was detected after 48h incubation in the incubator, as described in reference example 2. The in vitro tumor killing results are shown in fig. 10. The results show that the drug-loaded vesicles prepared by the three modes have no obvious difference in-vitro anti-tumor function.

In conclusion, the drug-loaded vesicle prepared by incubating the vesicle prepared by inducing apoptosis with the hypertonic solution and the chemotherapeutic drug can be taken by tumor cells, has good in-vitro anti-tumor effect, and can be used as a tumor chemotherapeutic drug preparation.

Claims

1. A method for preparing medicine-carrying vesicles by inducing apoptosis with hypertonic solution comprises uniformly suspending cells in hypertonic solution by stirring, continuously stirring at 4 deg.C to induce apoptosis to generate vesicles, and collecting vesicles by gradient centrifugation; and incubating the vesicle with a tumor chemotherapy drug to obtain a drug-loaded vesicle.

2. The method of claim 1, wherein the hypertonic solution is prepared by dissolving sodium chloride, trehalose, sucrose, maltose or D-sorbitol in purified water.

3. The method of claim 2, wherein the hypertonic solution is formulated with trehalose.

4. The method as claimed in claim 1, wherein the osmotic pressure of the hypertonic solution is 500-650 mOSM/kg.

5. The method of claim 4, wherein the hypertonic solution has an osmotic pressure of 550 mOSM/kg.

6. A process according to any one of claims 1 to 5, wherein the stirring time at 4 ℃ is 24 h.

7. The method of claim 1, wherein the drug-loaded vesicle encapsulated drug is selected from the group consisting of methotrexate, doxorubicin, cisplatin, paclitaxel, 5-fluorouracil, gemcitabine, and 10-hydroxycamptothecin.

8. The method of claim 7, wherein the drug-loaded vesicle encapsulated drug is methotrexate.

9. The use of the drug-loaded vesicles prepared according to the method of claim 1 as a pharmaceutical formulation for tumor chemotherapy.

10. A drug-loaded vesicle, characterized in that it is prepared according to the method of claim 1.