A method for extracting and purifying drug-encapsulated cell vesicles from cell suspension

A technology of cell vesicles and cell suspensions, which can be used in pharmaceutical formulations, drug combinations, anti-tumor drugs, etc., can solve the problems of low efficiency of collecting vesicles by gradient centrifugation, and achieve low loss, less vesicle loss, and reduced loss. Effect

Active Publication Date: 2021-08-06
HUBEI SOUNDNY BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the problem of low efficiency of collecting vesicles by gradient centrifugation, the purpose of the present invention is to provide a method for preparing drug-encapsulated cell vesicles from cell suspension

Method used

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  • A method for extracting and purifying drug-encapsulated cell vesicles from cell suspension
  • A method for extracting and purifying drug-encapsulated cell vesicles from cell suspension
  • A method for extracting and purifying drug-encapsulated cell vesicles from cell suspension

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Extraction and purification of drug-encapsulated cell vesicles from cell suspension

[0045] 1. Experimental materials and reagents

[0046] H22 mouse liver cancer cells, doxorubicin (commercially available, commonly used clinical chemotherapeutic drug, with red fluorescence). 2. Experimental steps

[0047] 1) Cultivate H22 mouse liver cancer cells in 1640 cell culture medium to make the cell amount reach 4×10 7 , take 4×10 7 H22 mouse liver cancer cells were resuspended in 10ml of culture medium, induced apoptosis by ultraviolet irradiation, and then added doxorubicin until the concentration of doxorubicin in the culture medium was 100 μg / ml, and the cell suspension was obtained after 24 hours of culture. equally divided into two groups;

[0048] 2) Control group: the first group of cell suspensions were centrifuged at 500rpm, 1000rpm, and 5000rpm for 10 minutes, and then centrifuged at 14000g for 1 minute to remove cells and debris. The centrifuged supe...

Embodiment 2

[0052] Example 2: Particle size of cell vesicles encapsulating drugs

[0053] 1. Experimental materials and reagents

[0054] H22 mouse hepatoma cells, methotrexate.

[0055] 2. Experimental steps

[0056] 1) Cultivate H22 mouse liver cancer cells in 1640 cell culture medium to make the cell amount reach 4×10 7 , take 4×10 7 Liver cancer cells were resuspended in 10ml of culture medium, induced apoptosis by ultraviolet irradiation, and then methotrexate was added until the concentration of methotrexate in the culture medium was 1mg / ml, and the cell suspension was obtained after culturing for 24 hours. into two groups;

[0057] 2) Purify and purify the drug-encapsulated cell vesicles from the culture medium of two groups of apoptotic liver cancer cells according to the method of the experimental step 2) of Example 1, and obtain the precipitates of the control group and the experimental group respectively.

[0058] 3. Experimental results

[0059] Use the scanning electron...

Embodiment 3

[0060] Example 3: Quantity and drug content of cell vesicles encapsulating drugs

[0061] 1. Experimental materials and reagents

[0062] H22 mouse liver cancer cells, methotrexate, cell lysate (20mM Tris-HCl, pH 7.5, 1% Triton X-100, 150mM NaCl, 1mM EDTA).

[0063] 2. Experimental steps

[0064] According to the experimental steps 1) and 2) of Example 2, the precipitation of the control group and the precipitation of the experimental group were obtained; the precipitation of the above-mentioned experimental group and the control group were divided into two parts, and one part was used to detect the number of cell vesicles wrapped in the drug , the other is used to detect the content of methotrexate in the drug-encapsulated cell vesicles;

[0065] The detection method of the number of cell vesicles encapsulating the drug: resuspend the pellet with physiological saline, and then use flow cytometry to detect the number of cell vesicles.

[0066] The method for detecting the c...

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Abstract

The invention provides a method for extracting and purifying drug-coated cell vesicles from a cell suspension, comprising: 1) filtering the cell suspension to collect the filtrate; 2) centrifuging the filtrate collected in step 1) to collect the precipitate, Obtaining drug-encapsulated cell vesicles; wherein the filtration is filtering the cell suspension through a filter membrane or filter element with a pore size of 5 μm-10 μm; the centrifugation is high-speed centrifugation with a centrifugal force of 100-100000 g; the The drug-encapsulated cell vesicles are microparticles containing tumor therapeutic drugs released by apoptotic cells, and the cell suspension is a mixed liquid containing the cells, cell fragments and cell vesicles. The process of the method of the present invention is relatively simple, the loss of the instrument is small, there is no pollution, the yield of cell vesicles is high, and it is beneficial to realize industrial production, and the cell vesicles wrapped with drugs obtained by the method have higher drug-containing quantity and better therapeutic effect.

Description

technical field [0001] The invention relates to a method for extracting and purifying drug-wrapped cell vesicles from cell suspension. Background technique [0002] Tumor is a persistent disease that seriously threatens human life. In recent years, its treatment methods mainly include radiotherapy and chemotherapy. However, these two conventional tumor treatment methods, while eliminating tumor cells, will also kill normal tissue cells and damage the patient's immune system, resulting in a defect or decline in immune function, which greatly reduces the quality of life of the patient and brings great harm to the patient. The unbearable toxic and side effects lead to the failure of radiotherapy and chemotherapy. In view of the toxic and side effects of radiotherapy and chemotherapy, people think of the method of wrapping chemotherapeutic drugs with carriers, so that the chemotherapeutic drugs can be selectively released at the tumor site, and even selectively enter the tumor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K9/127A61K47/46A61K31/519A61K31/704A61P35/00
CPCA61K9/127A61K31/519A61K31/704A61K47/46
Inventor 张一李凯
Owner HUBEI SOUNDNY BIOLOGICAL TECH
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