CN107875124B - Method for extracting and purifying cell vesicles wrapping medicine from cell suspension - Google Patents

Method for extracting and purifying cell vesicles wrapping medicine from cell suspension Download PDF

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CN107875124B
CN107875124B CN201610866539.4A CN201610866539A CN107875124B CN 107875124 B CN107875124 B CN 107875124B CN 201610866539 A CN201610866539 A CN 201610866539A CN 107875124 B CN107875124 B CN 107875124B
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CN107875124A (en
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张一�
李凯
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Hubei Soundny Bio Tech Co ltd
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Abstract

The invention provides a method for extracting and purifying cell vesicles wrapping a drug from a cell suspension, which comprises the following steps: 1) filtering the cell suspension, and collecting filtrate; 2) centrifuging the filtrate collected in the step 1), and collecting precipitates to obtain cell vesicles coated with the drug; wherein the filtration is to filter the cell suspension through a filter membrane or a filter element with the pore diameter of 5-10 μm; the centrifugation is high-speed centrifugation with the centrifugal force of 100-100000 g; the cell vesicle for coating the drug is a microparticle which is released by apoptotic cells and contains a tumor therapeutic drug, and the cell suspension is a mixed solution containing the cells, cell fragments and the cell vesicle. The method has the advantages of relatively simple process, small loss to instruments, no pollution, high yield of the cell vesicles and contribution to realizing industrial production, and the cell vesicles coated with the medicine obtained by the method have higher medicine content and better treatment effect.

Description

Method for extracting and purifying cell vesicles wrapping medicine from cell suspension
Technical Field
The invention relates to a method for extracting and purifying cell vesicles coated with a drug from a cell suspension.
Background
Tumors are a persistent disease that seriously threatens human life. In recent years, radiotherapy and chemotherapy are mainly used as treatment methods. However, the two conventional tumor treatment methods can kill normal tissue cells while eliminating tumor cells, damage the immune system of a patient, cause the immune function to be deficient or reduced, greatly reduce the life quality of the patient, bring intolerable toxic and side effects to the patient and cause failure of radiotherapy and chemotherapy. Aiming at the toxic and side effects of radiotherapy and chemotherapy, people think that the method of wrapping the chemotherapeutic drug by the carrier enables the chemotherapeutic drug to be selectively released at the tumor part and even selectively enter the tumor cells, thereby achieving the effect of selectively killing the tumor cells. However, the exogenous carrier also has certain toxic and side effects on organisms, and the particle size of the clinically used carrier particles also enables the carrier particles to easily penetrate cell membranes of normal cells, so that the toxic and side effects of the carrier and the chemotherapeutic drugs wrapped by the carrier on the organisms are increased.
In order to overcome the defects of the radiotherapy and chemotherapy tumor treatment method and the method for treating tumors by wrapping chemotherapeutic drugs by adopting exogenous carriers, Chinese patent CN102302784A discloses a tumor chemotherapeutic drug preparation and a preparation method thereof. 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. CN102302784A also discloses a preparation method of the tumor chemotherapeutic drug preparation, wherein microparticles containing cell vesicles and encapsulated chemotherapeutic drugs are extracted and purified from tumor cell culture fluid treated with tumor chemotherapeutic drugs by using a gradient centrifugation method (see experimental step 2(3) of example 1 of CN102302784A specification). Specifically, the tumor cell culture solution was gradually centrifuged, and the centrifuged supernatant was recentrifuged at 500rpm, 1000rpm, and 5000rpm for 10 minutes, followed by centrifugation at 14000g for 1 minute to remove cells and debris, and centrifugation at 14000g for 1 hour to obtain the microparticles.
Cell debris can be selectively removed by the gradient centrifugation method, but part of the vesicles are lost, so that the number of the finally collected cell vesicles is reduced. And the centrifugal volume is large during high-speed centrifugation, so that the efficiency of collecting vesicles through gradient centrifugation is reduced, the difficulty of industrial production is high, and large-scale clinical medication cannot be met.
Disclosure of Invention
Aiming at the problem of low vesicle collection efficiency by gradient centrifugation, the invention aims to provide a method for preparing cell vesicles coated with a drug from a cell suspension.
In order to achieve the above object, the present invention provides a method for extracting and purifying cell vesicles encapsulating a therapeutic agent for tumor from a cell suspension, comprising the steps of:
1) filtering the cell suspension, and collecting filtrate;
2) centrifuging the filtrate collected in the step 1), and collecting precipitates to obtain cell vesicles coated with the drug;
wherein the filtration in the step 1) is to filter the cell suspension through a filter membrane or a filter element with the pore size of 5-10 μm;
the centrifugation in the step 2) is high-speed centrifugation with the centrifugal force of 100-100000 g;
the preparation method of the cell suspension comprises the following steps:
apoptosis of cells is caused by chemical, physical or biological methods, the apoptotic cells are contacted with a tumor therapeutic agent, so that the tumor therapeutic agent is encapsulated into the apoptotic cells, the drug-encapsulated cell vesicles are microparticles containing the tumor therapeutic agent and released by the apoptotic cells, and the cell suspension is a mixed solution containing the cells, cell debris and cell vesicles.
The cell suspension of the invention is a cell culture solution containing cell vesicles, wherein the cells include but are not limited to various tumor cells, red blood cells and the like.
The method for inducing apoptosis of cells is preferably a method of irradiating cells with ultraviolet rays, or a method of contacting cells directly with a chemotherapeutic agent, or a method of contacting cells with radiation.
The invention uses a microfiltration method to further extract and purify the cell suspension, and obtains the cell vesicles which are suitable for the used encapsulated drugs.
Preferably, the microfiltration is membrane filtration or cartridge filtration. The particle size of the cell vesicle is between 100 and 1000nm, so that filter membranes or filter elements with different pore sizes of 1-10 μm are adopted to filter the cell suspension to remove cell debris and purify the cell suspension. The inventors of the present invention tried to filter cell suspension with filter membrane or filter element having pore size of 1 μm, 2.5 μm and less than 1 μm respectively, and found that cell vesicles can easily pass through micropores, and in addition, large cell debris is present, and as the filtration volume increases, the large cell debris overlaps with each other, blocking the micropores, resulting in a smaller final filtration volume, and the purity of vesicles collected by high speed centrifugation is low. The inventor filters the cell suspension by using a filter membrane or a filter element with the pore diameter of 5-10 μm, and finds that both cell vesicles and cell debris can pass through micropores, but as the filtering volume is increased, large cell debris is overlapped at the micropores to form a smaller micropore structure, so that the large cell debris is retained, and the cell vesicles can easily pass through, so that the filtering volume is increased, and finally, the number of the cell vesicles collected by high-speed centrifugation is larger.
Preferably, the microporous filtration of the invention is to use a filter membrane or a filter element with a pore size of 5-10 μm to filter the cell suspension.
Preferably, the material of the filter membrane or the filter element is selected from polypropylene, polyvinylidene fluoride, polytetrafluoroethylene and nylon, and the cell vesicles coated with the drugs extracted by the filter membrane or the filter element made of the material have no obvious difference in purity and quantity.
Preferably, the filter membrane according to the invention is selected from one of polypropylene J100047050, polyvinylidene fluoride SVWG04700, polytetrafluoroethylene LSWG04700, JCWP04700 and nylon NCG 047100.
Preferably, the filter element of the present invention is polypropylene KA3J100P1 from PALL corporation.
Preferably, the step of collecting the cell vesicles coated with the drug by high-speed centrifugation is to collect the cell vesicles coated with the drug by centrifuging the filtrate collected after filtration with a centrifugal force of 5000-.
Preferably, the step of collecting the cell vesicles coated with the drug by high-speed centrifugation is to collect the cell vesicles coated with the drug by centrifuging the filtrate collected after filtration for 30-90 minutes at a centrifugal force of 100-100000 g.
Preferably, the step of collecting the cell vesicles coated with the drug by high-speed centrifugation is to collect the cell vesicles coated with the drug by centrifuging the filtrate collected after filtration for 40-80 minutes at a centrifugal force of 100-100000 g.
According to the invention, a nano-particle analyzer, a high performance liquid chromatograph and a flow cytometer are adopted to represent the cell vesicles coated with the medicine extracted and purified by the method, and the result shows that more cell vesicles coated with the medicine can be obviously obtained by the method, and the medicine content of each vesicle is higher.
In-vivo animal experiments and in-vitro apoptosis experiments show that the cell vesicles coated with the medicine obtained by the method have better treatment effect.
The method for extracting and purifying the cell vesicles coated with the tumor chemotherapeutic drug from the cell suspension, disclosed by the invention, has the advantages that the large cell fragments in the cell suspension can be better removed by microfiltration, the loss of the vesicles is reduced, and most of the vesicles can be collected by high-speed centrifugation. The method has the advantages of relatively simple process, less loss to instruments, no pollution, less vesicle loss and high collection efficiency, needs to replace filter membranes or filter elements regularly, is favorable for realizing industrial production, and the cell vesicles coated with the medicine obtained by the method have higher medicine content and better treatment effect.
Drawings
FIG. 1 is a scanning electron micrograph of vesicles from a group of experimental cells;
FIG. 2 shows the particle size distribution of the experimental group of cell vesicles;
FIG. 3 shows the yields of drug-encapsulated cell vesicles obtained by filtration with different pore sizes according to the method of the invention;
FIG. 4 shows the serum creatinine content of mice injected with drug-encapsulated cell vesicles;
FIG. 5 shows the serum glutamate pyruvate transaminase levels of mice administered drug-encapsulated cell vesicles;
FIG. 6 shows the killing effect of drug-coated cell vesicles on H22 cells extracted by two methods, respectively;
FIG. 7 is a graph of the survival of BALB/c mice inoculated with H22 cells after administration of drug-encapsulated cell vesicles;
FIG. 8 is a graph showing the survival curves of Lewis cell-inoculated C57BL/6 mice administered drug-encapsulated vesicles.
Detailed Description
The technical solution of the present invention will be further described with reference to some embodiments and the accompanying drawings, and the following embodiments do not limit the present invention.
The following are descriptions of various tumor cells, drugs, experimental animals, instrumentation and partial solution formulations used in the examples:
cell: h22 mouse liver cancer cell (H22 cell) and Lewis mouse lung cancer cell are purchased from American ATCC company or China center for type culture Collection CCTCC.
Reagents, materials and drugs: 3 μm commercial standard magnetic beads (cell vesicle relative count beads) were purchased from SIGMA-ALDRICH, USA; methotrexate (MTX) was purchased from the affiliated college of medicine (wuhan); and (3) filtering the membrane: polypropylene J100047050, SVWG04700, ptfe LSWG04700, JCWP04700 and NCG047100, nylon; a filter element: polypropylene KA3J100P1 from PALL.
Preparing a solution: cell lysate (20mM Tris-HCl, pH 7.5, 1% Triton X-100,150mM NaCl,1mM EDTA)
Others are not specifically indicated as being commercially available.
Experimental animals: BALB/C mouse, C57BL/6 mouse purchased from Hubei province medical experimental animal research center under Hubei province disease prevention and control center;
the instrument equipment comprises: a two-photon fluorescence microscope, a Nano ZS90 nanometer particle size potentiometer, a BDFACSCAntoII type flow cytometer, a UITIMate 3000 type high performance liquid chromatograph, and a SCIENTZ ultrasonic cell crusher.
The term "cell vesicle" as used herein is produced by apoptotic cells and does not encapsulate a therapeutic agent for a tumor; accordingly, a cell vesicle in which a drug has been encapsulated is referred to as a drug-encapsulated cell vesicle.
Example 1: extraction and purification of drug-encapsulating cell vesicles from cell suspensions
1. Test materials and reagents
H22 mouse hepatoma cell, adriamycin (commercially available, common clinical chemotherapeutic drug, with red fluorescence). 2. Experimental procedure
1) Culturing H22 mouse liver cancer cell in 1640 cell culture solution to make cell amount reach 4X 107Taking 4 × 107Liver cancer of H22 mouseResuspending the cells by using 10ml of culture solution, inducing apoptosis by ultraviolet irradiation, adding adriamycin until the concentration of the adriamycin in the culture solution is 100 mu g/ml, culturing for 24 hours to obtain cell suspension, and averagely dividing the cell suspension into two groups;
2) control group: centrifuging the first group of cell suspension at 500rpm, 1000rpm and 5000rpm for 10min, centrifuging at 14000g for 1min to remove cells and debris, centrifuging the centrifuged supernatant at 14000g for 1 hr, and collecting the precipitate (i.e. the cell vesicle coated with the drug);
experimental groups: the second cell suspension was filtered through a filter J100047050 (available from PALL Co.) to remove cells and debris, and the filtered supernatant was centrifuged at 14000g for 1 hour to collect a pellet.
3. Results of the experiment
And (3) resuspending the settled smears of the experimental group and the control group by using physiological saline respectively, and observing the cell vesicles of the control group and the experimental group under a two-photon fluorescence microscope.
Example 2: particle size of drug-encapsulated cell vesicles
1. Test materials and reagents
H22 mouse hepatoma cells, methotrexate.
2. Experimental procedure
1) Culturing H22 mouse liver cancer cell in 1640 cell culture solution to make cell amount reach 4X 107Taking 4 × 107Resuspending the liver cancer cells by using 10ml of culture solution, inducing apoptosis by ultraviolet irradiation, adding methotrexate until the concentration of the methotrexate in the culture solution is 1mg/ml, culturing for 24 hours to obtain cell suspension, and averagely dividing the cell suspension into two groups;
2) extracting purified cell vesicles coated with the drug from the culture solution of two groups of apoptotic hepatoma cells by the method of the experimental step 2) of example 1, and obtaining control group precipitates and experimental group precipitates respectively.
3. Results of the experiment
Detecting the control group sediment and the experimental group sediment by using a scanning electron microscope for observation and a nano-particle size potentiometer, wherein (A) is a scanning electron microscope image of the experimental group cell vesicles, and (B) is a scanning electron microscope image of the control group cell vesicles, as shown in figure 1; as shown in FIG. 2, (A) is the particle size distribution diagram of the cell vesicles of the experimental group, and (B) is the particle size distribution diagram of the cell vesicles of the control group, the results show that the particle sizes of the drug-coated cell vesicles obtained from the experimental group (i.e., the combination of microfiltration and centrifugation) and the control group are between 100 nm and 1000 nm.
Example 3: the number and the dosage of the cell vesicles wrapping the medicine
1. Test materials and reagents
H22 mouse hepatoma cells, methotrexate, cell lysate (20mM Tris-HCl, pH 7.5, 1% Triton X-100,150mM NaCl,1mM EDTA).
2. Experimental procedure
Control and experimental group pellets were obtained as in experimental steps 1) and 2) of example 2; dividing the precipitates of the experimental group and the control group into two parts, wherein one part is used for detecting the number of the cell vesicles coating the medicine, and the other part is used for detecting the content of methotrexate in the cell vesicles coating the medicine;
the detection method of the number of the cell vesicles wrapping the medicine comprises the following steps: the pellet was resuspended in physiological saline and the number of cell vesicles was then measured by flow cytometry.
The detection method of the content of the methotrexate encapsulated by the cell vesicles comprises the following steps:
A. pretreatment of cell vesicles
1) Adding 500 μ L cell lysate into the precipitate (i.e. cell vesicle) obtained in step 2), incubating on ice for 20min, pulverizing for 1min with ultrasonic cell pulverizer, centrifuging for 5min with 10000g centrifugal force, and collecting supernatant;
2) adding 2 times volume of acetonitrile into the supernatant, centrifuging for 3min at 10000g, and taking the supernatant;
3) chloroform was added to the supernatant in a volume 4 times that of the supernatant, and the mixture was centrifuged at 2000g for 10min to obtain the supernatant.
B. High performance liquid chromatograph detection
The detection conditions are as follows:
chromatographic conditions mobile phase: 0.05M potassium dihydrogen phosphate, 10% acetonitrile, pH 6.6;
flow rate: 1 ml/min;
column temperature: 40 ℃;
a detector: ultraviolet detector, 304 nm.
3. Results of the experiment
The number of the cell vesicles coated with the drug obtained from the experimental group was 5X 106The medicine content is 1.7 mu g; the number of drug-coated cell vesicles obtained in the control group was 1.5X 106The medicine content is 0.5 μ g.
Therefore, the cell vesicles coated with the drugs obtained by the experimental group method are obviously more than those obtained by the control group, and the content of the chemotherapeutic drugs coated by the cell vesicles obtained by the experimental group is far higher than that of the control group.
Example 4: the number of the cell vesicles coated with the medicine is obtained by filtering with filter membranes with different pore diameters
1. Test materials and reagents
H22 mouse hepatoma cells, methotrexate, filters with a pore size of 1 μm (JAWP04700, Millipore), filters with a pore size of 2.5 μm (J025047050, PALL), filters with a pore size of 5 μm (JMWP04700, Millipore) and filters with a pore size of 10 μm (JCWP04700, Millipore).
2. Experimental procedure
1) Culturing H22 mouse liver cancer cell in 1640 cell culture solution to make cell amount reach 4X 108Taking 4 × 108Resuspending the liver cancer cells by using 10ml of culture solution, inducing apoptosis by ultraviolet irradiation, adding methotrexate to the culture solution with the concentration of 1mg/ml of methotrexate, culturing for 24 hours to obtain cell suspension, and averagely dividing the cell suspension into four groups;
2) filtering the above four groups of cell suspensions with filter membranes with 1 μm, 2.5 μm, 5 μm and 10 μm pore size respectively to remove cells and large debris, centrifuging the filtered supernatant with 14000g centrifugal force for 1 hr, and collecting the precipitate;
3) resuspending each group of precipitates with physiological saline respectively, and detecting the number of cell vesicles wrapping the drug by using a flow cytometer;
3. results of the experiment
As shown in FIG. 3, the number of drug-coated cell vesicles obtained by filtration through filters having pore sizes of 10 μm, 5 μm, 2.5 μm and 1 μm was 2X 107、1.8×107、5×106And 1X 106. From this, it was found that the number of cell vesicles obtained by filtration through filters having pore diameters of 5 μm and 10 μm was large.
Example 5: the number of the cell vesicles coated with the medicine is obtained by filtering with different types of filter membranes
1. Test materials and reagents
H22 mouse hepatoma cells, methotrexate, Filter J060047050(PALL Co.), Filter (cobert, PP, 5 μm), Filter (Durapore SVWG04700, Millipore Co.).
2. Experimental procedure
1) Culturing H22 mouse liver cancer cell in 1640 cell culture solution to make cell amount reach 3X 108Taking 3 × 108Resuspending the liver cancer cells by using 10ml of culture solution, inducing apoptosis by ultraviolet irradiation, adding methotrexate to the culture solution with the concentration of 1mg/ml of methotrexate, culturing for 24 hours to obtain cell suspension, and averagely dividing the cell suspension into three groups;
2) filtering the three cell suspensions with a filter J060047050, a filter (Cobetter, PP, 5 μm), and a filter Durapore SVWG04700(Millipore corporation), respectively, to remove cells and large debris, centrifuging the filtered supernatant with 14000g of centrifugal force for 1 hour, and collecting the precipitate;
3) the pellet was resuspended in saline separately for each group, and then the number of drug-encapsulated cell vesicles was measured by flow cytometry.
3. Results of the experiment
Flow cytometry results: the number of drug-coated cell vesicles obtained by filtration with filter J060047050, filter (cobtter, PP, 5 μm) and filter Durapore SVWG04700(Millipore Corp.) was 5X 107、3.2×107And 4.1X 107。
Example 6: the number of the cell vesicles coated with the medicine is obtained by filtering with filter elements of different types
1. Test materials and reagents
H22 mouse hepatoma cell, methotrexate, cartridge (KA3J100P1, PALL), cartridge (KA3J060P1, PALL).
2. Experimental procedure
1) Culturing H22 mouse liver cancer cell in 1640 cell culture solution to make cell amount reach 2X 109Resuspending the cells in 10ml of culture solution, inducing apoptosis by ultraviolet irradiation, adding methotrexate until the concentration of the methotrexate in the culture solution is 1mg/ml, culturing for 24 hours to obtain cell suspension, and averagely dividing the cell suspension into two groups;
2) filtering the above two cell suspensions with filter element (KA3J100P1, PALL) and filter element (KA3J060P1, PALL) respectively to remove cells and large debris, centrifuging the filtered supernatant with 14000g centrifugal force for 1 hr, and collecting precipitate;
3) the two groups of precipitates were resuspended in physiological saline, and then the number of drug-encapsulating cell vesicles was measured by flow cytometry.
3. Results of the experiment
Flow cytometry results: the cell vesicles coated with the drug obtained by filtration using cartridge KA3J100P1(PALL Co.) and cartridge KA3J060P1(PALL Co.) each had a number of 4.5X 108And 4.5X 108。
Example 7: cell vesicle number of encapsulated drug obtained by different centrifugal force centrifugation
1. Test materials and reagents
H22 mouse hepatoma cells, methotrexate, filter J100047050(PALL Corp.).
2. Experimental procedure
1) Culturing H22 mouse liver cancer cell in 1640 cell culture solution to make cell amount reach 3X 108The cells were resuspended in 30ml of culture medium, subjected to apoptosis induction by ultraviolet irradiation, and methotrexate was added to a concentration of 1mg/ml in the culture medium, and cultured for 24 hours to obtain cell suspensions, which were divided into three groups on average.
2) The cell suspensions were filtered through each of the above-mentioned filters J100047050(PALL Co.) to remove cells and debris, and the filtered supernatants were centrifuged at centrifugal forces of 5000g, 14000g and 30000g for 1 hour to collect precipitates.
3) The pellet was resuspended in saline and the number of vesicles was measured by flow cytometry.
3. Results of the experiment
Flow cytometry results: the number of vesicles extracted by centrifugation at a centrifugal force of 5000g was 3.9X 107(ii) a The number of vesicles extracted by centrifugation at 14000g was 4.3X 107A plurality of; the number of vesicles extracted by centrifugation at a centrifugal force of 30000g was 4.5X 107。
Example 8: cell vesicle number of encapsulated drug obtained by centrifugation at different times
1. Test materials and reagents
H22 mouse hepatoma cells, methotrexate, filter J100047050(PALL Corp.).
2. Experimental procedure
Cell suspensions were obtained according to the method of experimental step 1) of example 7, and were divided into three groups on average, and then the three groups of cell suspensions were filtered through filters J100047050(PALL Co.), and the filtered supernatants were centrifuged at 14000g for 40 minutes, 60 minutes, and 80 minutes, respectively;
the extracted vesicles were resuspended in physiological saline and the number of vesicles was measured by flow cytometry.
3. Results of the experiment
Flow cytometry results: the number of vesicles extracted by centrifugation for 40 minutes was 4.0X 107The number of vesicles extracted by centrifugation for 60 minutes was 4.4X 107The number of vesicles extracted by centrifugation for 80 minutes was 4.5X 107。
Example 9: effect of cell vesicles coated with drugs extracted by different methods on liver and kidney functions
1. Test materials and reagents
H22 mouse hepatoma cells, methotrexate, 24 BALB/c mice.
2. Experimental procedure
1) Culturing H22 mouse liver cancer cell in 1640 cell culture solution to make cell amount reach 1 × 108。
2) Take 1X 108H22 mouse liver cancer cells were subjected to apoptosis induction by UV irradiation, and methotrexate was added to the cells at a final concentration of 1mg/ml, and the cells were cultured for 24 hours to obtain cell suspensions, which were divided into two groups on average.
3) Extracting cell vesicles wrapping the drug from the two groups of cell suspensions respectively according to two extraction methods of the experimental step 2) of the example 1 to obtain cell vesicles of an experimental group and cell vesicles of a control group;
4) blank group: BALB/c mice of 8 mice, and physiological saline is injected into tail vein;
control group: 8 mice BALB/c mice, tail vein injection by the step 3) obtained by the control group of cells vesicles;
experimental groups: injecting BALB/c mice of 8 mice into tail vein of experimental group cell vesicle obtained in step 3);
will be 5X 106The amount of drug-coated cell vesicles prepared from H22 cells was determined for 1 mouse, and the injection was continued for 14 days.
5) On day 15, venous blood was taken from each of the three groups of mice, and the contents of glutamic-pyruvic transaminase and creatinine in the serum were measured and the body weights of the mice were measured.
3. Results of the experiment
As shown in fig. 4 and 5, there was no significant change in the glutamic-pyruvic transaminase and creatinine contents of the control mice and the experimental mice compared with the blank mice injected with physiological saline. Therefore, the cell vesicles coated with the chemotherapeutic drugs extracted by the two methods have no toxic or side effect on organisms.
Example 10: in vitro apoptosis assay
1. Test materials and reagents
H22 mouse liver cancer cell, and methotrexate.
2. Experimental procedure
1) Culturing H22 mouse liver cancer cell in 1640 cell culture solution to make cell amount reach 3X 108Then, the culture broth was divided into three groups on average.
2) Collecting the first and second group of hepatocarcinoma cell culture fluid containing 1 × 10 cells respectively7H22 mouse liver cancer cell, inducing apoptosis by ultraviolet irradiation, and culturingAdding methotrexate with the final concentration of 1mg/ml, and culturing for 24 hours to obtain cell suspension; the culture solution of the liver cancer cells of the third group is not treated;
3) experimental groups: extracting the cell vesicles coated with the drug according to the experimental group method of step 2) of example 1, namely a method combining microfiltration and centrifugation;
control group: extracting the cell vesicles coated with the drug according to the control group method, namely the gradient centrifugation method in the step 2) of the example 1;
4) taking 2 parts of 5 × 10 liver cancer cell culture solution4H22 mouse liver cancer cells are respectively mixed with the cell vesicles which are extracted from the experimental group and are coated with the drugs and the cell vesicles which are extracted from the control group, the mixture is placed in 1640 cell culture solution in a 24-pore plate for co-culture for 24 hours, then PI and Annexin V dyes are respectively adopted to stain the liver cancer cells which are cultured by the 24-pore plate and are treated by the cell vesicles of the experimental group and the cell vesicles of the control group, and the death conditions of the three groups of liver cancer cells are detected by a flow cytometer.
3. Results of the experiment
As shown in fig. 6, the results show that the drug-coated cell vesicles extracted from the experimental group and the control group can effectively kill liver cancer cells, and the killing effect of the drug-coated cell vesicles in the experimental group is significantly better than that of the drug-coated cell vesicles in the control group.
Example 11: in vivo ascites model
1. Test materials and reagents
H22 mouse hepatoma cells, the chemotherapeutic drug methotrexate, BALB/c mice.
2. Experimental procedure
1) Preparing cell vesicles encapsulating drugs:
culturing H22 mouse liver cancer cells in 1640 cell culture medium, and taking two groups of 5 × 10 cells6Respectively resuspending H22 mouse liver cancer cells with 1640 cell culture solution, inducing apoptosis by ultraviolet irradiation, adding methotrexate with final concentration of 1mg/ml, and culturing for 24 hr to obtain two groups of cell suspensions;
two methods, namely the experimental group method and the control group method, according to step 2) of example 1Extracting cell vesicles containing drug from the above two cell suspensions, and mixing 5 × 106The amount of the drug-coated cell vesicles prepared from H22 cells was determined as the amount of 1 mouse.
2) Mice were inoculated with hepatoma cells: on day 1, 1X 10 injections were given per mouse5Amount of H22 mouse liver cancer cells, 1X 10 cells were extracted from H22 mouse liver cancer cell culture solution5H22 mice hepatoma cells were inoculated intraperitoneally to BALB/c mice, and 48 mice were co-injected to obtain BALB/c mice with H22 hepatoma ascites carcinoma, which were randomly divided into three groups of equal numbers.
3) Administration:
starting on day 2, feeding is carried out normally once daily for 6 times and starting on day 8 according to the following steps:
experimental groups: injecting a first group of BALB/c mice suffering from H22 hepatoma ascites carcinoma with drug-coated cell vesicles extracted by the experimental group method in the step 1);
control group: injecting a second group of BALB/c mice suffering from H22 hepatoma ascites carcinoma with drug-coated cell vesicles extracted by the control group method in the step 1);
blank group: injecting normal saline into a third group of BALB/c mice with H22 hepatoma ascites carcinoma cells;
4) the three groups of BALB/c mice obtained in step 3) were divided into two groups of equal numbers, the first group of BALB/c mice was sacrificed on day 10, ascites volume was measured, and the second group of BALB/c mice was used to observe survival time.
3. Results of the experiment
At day 10 of dissection, the abdominal water volume of the experimental mice was significantly less than that of the control mice. As shown in fig. 7, control mice began to die on day 16 and died all on day 24; mice in the experimental group began to die on day 19 and died all on day 28. The result shows that the cell vesicles coated with the methotrexate, which are derived from the liver cancer cells of the H22 mice and collected by the method of the experimental group, namely the method combining microfiltration and centrifugation, can obviously inhibit the growth of the liver cancer cells of the BALB/c mice, prolong the survival time of the mice, and have the effect of inhibiting the growth of ascites which is obviously better than that of the cell vesicles coated with the methotrexate, which is extracted by the method of the control group, namely the gradient centrifugation method.
Example 12: in vivo solid tumor model
1. Test materials and reagents
Lewis mouse lung cancer cell, chemotherapeutic medicine methotrexate, C57BL/6 mouse.
2. Experimental procedure
1) Preparing cell vesicles encapsulating drugs:
culturing Lewis mouse lung cancer cells in DMEM cell culture solution, and taking out two groups of 5X 10 cells6Respectively re-suspending Lewis mouse lung cancer cells by using a DMEM cell culture medium, inducing apoptosis by ultraviolet irradiation, then adding methotrexate with the final concentration of 1mg/ml, and culturing for 24 hours to obtain two groups of cell suspensions;
extracting cell vesicles containing the drug from the above two cell suspensions according to the two methods of step 2) of example 1, namely, the experimental group method and the control group method, and mixing 5 × 106The amount of the drug-coated cell vesicles prepared from H22 cells was determined as the amount of 1 mouse.
2) Mice were inoculated with Lewis mouse lung cancer cells: on day 1, 1X 10 injections were given per mouse5The amount of each Lewis mouse lung cancer cell was 1X 10 times as large as the amount of Lewis mouse lung cancer cell in the cell culture medium5The tail vein of each Lewis mouse lung cancer cell is inoculated to a C57BL/6 mouse, and 48 mice are injected in total to obtain C57BL/6 mice with Lewis mouse lung cancer, and the mice are randomly divided into three groups with equal numbers.
3) Administration:
starting on day 2, the feed was normally raised every other day for 6 times according to the following procedure:
experimental groups: injecting a first group of C57BL/6 mice with Lewis mouse lung cancer into the cell vesicles coated with the drug extracted by the experimental group method in the step 1);
control group: injecting a second group of C57BL/6 mice with Lewis mouse lung cancer into the drug-coated cell vesicles extracted by the control group method in the step 1);
blank group: in the third group, C57BL/6 mice with Lewis mouse lung cancer were injected with physiological saline.
4) The three groups of C57BL/6 mice obtained in step 3) were divided into two groups of equal numbers, the first group of C57BL/6 mice were sacrificed at day 18 and the number of lung cancer nodules was measured, and the second group of C57BL/6 mice was used to observe survival time.
3. Results of the experiment
When dissecting at day 18, the lungs of the mice in the blank group are ulcerated, and obvious nodules grow; the lung of the control group mice had nodules several times and had a small amount of lung ulceration; the number of nodules in the lungs of the mice in the experimental group was minimal. As shown in fig. 8, the survival time of the experimental group mice was significantly prolonged. The result shows that the cell vesicles, which are collected by the method of the experimental group, namely the method combining microfiltration and centrifugation and are coated with methotrexate and derived from the Lewis mouse lung cancer cells, can obviously inhibit the growth of the lung cancer cells of the C57BL/6 mouse, prolong the survival time of the mouse, and have an obviously better effect of inhibiting the growth of the lung cancer cells than the cell vesicles extracted by the method of the control group, namely the gradient centrifugation method.
According to the method for extracting and purifying the cell vesicles encapsulating the drug from the cell suspension provided by the present invention, the test results of the cell vesicles encapsulating methotrexate, which were collected using the filter membrane or the filter cartridge other than the filter membrane used in example 1, were consistent with the test results of the corresponding examples described above when the tests of examples 9 to 12 were performed.

Claims (7)

1. A method for extracting and purifying drug-encapsulating cell vesicles from a cell suspension comprising:
1) filtering the cell suspension, and collecting filtrate;
2) centrifuging the filtrate collected in the step 1), and collecting precipitates to obtain cell vesicles coated with the drug;
wherein the filtration in the step 1) is to filter the cell suspension through a filter membrane or a filter element with the pore size of 5-10 μm;
the centrifugation in the step 2) is high-speed centrifugation with the centrifugal force of 100-100000 g;
the preparation method of the cell suspension comprises the following steps:
apoptosis of cells is caused by chemical, physical or biological methods, the apoptotic cells are contacted with a tumor therapeutic agent, so that the tumor therapeutic agent is encapsulated into the apoptotic cells, the drug-encapsulated cell vesicles are microparticles containing the tumor therapeutic agent and released by the apoptotic cells, and the cell suspension is a mixed solution containing the cells, cell debris and cell vesicles.
2. The method for extracting and purifying drug-coated cell vesicles as claimed in claim 1, wherein the material of the filter membrane or the filter core is selected from one of polypropylene, polyvinylidene fluoride, polytetrafluoroethylene and nylon.
3. The method for extracting and purifying drug-coated cell vesicles according to claim 2, wherein the filter membrane is selected from the group consisting of polypropylene J100047050, polyvinylidene fluoride SVWG04700, LSWG04700, JCWP04700, and NCG 047100.
4. The method for extracting and purifying drug-encapsulating cell vesicles as claimed in claim 2, wherein the filter element is polypropylene KA3J100P1 from PALL corporation.
5. The method for extracting and purifying drug-encapsulated cell vesicles as claimed in claim 1, wherein the high speed centrifugation is performed at 100-100000g for 30-90 min.
6. The method for extracting and purifying drug-encapsulating cellular vesicles of claim 1 wherein the cells are eukaryotic cells.
7. The method for extracting and purifying drug-encapsulating cell vesicles according to claim 1, wherein the cells are tumor cells or red blood cells.
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