Preparation method and drug carrying method of escherichia coli outer membrane vesicle, and application of outer membrane vesicle in anti-tumor

A technology of Escherichia coli and outer membrane vesicles, applied in the biological field, can solve the problems of hindering the application of OMV, low diffusion efficiency, inconvenient operation, etc., and achieve the effects of inhibiting tumor cell growth, simple operation and reducing invasive ability.

Inactive Publication Date: 2017-09-08
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The 37°C incubation method relies on its own cell membrane space and concentration gradient to diffuse the drug from the outside of the OMV to the inside of the OMV, but the OMV cell space is small and has a fat-soluble membrane, so the diffusion efficiency is very low, resulting in low drug loading
Ultrasound and electroporation can increase the drug loading capacity of OMV to a certain extent, but they need to use ultrasound and electroporation, which is inconvenient to operate. These limitations hinder the application of OMV in drug carriers

Method used

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  • Preparation method and drug carrying method of escherichia coli outer membrane vesicle, and application of outer membrane vesicle in anti-tumor
  • Preparation method and drug carrying method of escherichia coli outer membrane vesicle, and application of outer membrane vesicle in anti-tumor
  • Preparation method and drug carrying method of escherichia coli outer membrane vesicle, and application of outer membrane vesicle in anti-tumor

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Preparation of E. coli Outer Membrane Vesicles

[0041] (1) Recovery and in vitro culture of Escherichia coli

[0042] Thaw the frozen storage solution of BL21 strain, use the inoculation loop to pick up the bacterial solution, inoculate it on a solid medium containing 50 μg / ml kanamycin, cultivate it in a 37°C incubator for 1 hour, and inoculate a single colony on the solid medium to LB liquid medium containing 50 μg / ml kanamycin, cultured with shaking at 37°C for 1 hour.

[0043] Then the bacterial solution was inoculated into LB liquid medium (which had been sterilized at high temperature) containing 50 μg / ml kanamycin at a ratio of 1:100 by volume, and continued shaking culture at 37°C. When the bacterial OD value (600nm) reached When 0.1, add isopropylthiogalactoside (IPTG) in LB liquid medium, make the final concentration of isopropylthiogalactoside in LB liquid medium be 1 μ g / ml, promote Escherichia coli to secrete extracorporeal Membrane vesicles were culture...

Embodiment 2

[0050] Preparation of E. coli Outer Membrane Vesicles

[0051] (1) Recovery and in vitro culture of Escherichia coli

[0052] Thaw the frozen stock solution of BL21 strain, use an inoculation loop to pick up the bacterial solution, inoculate it on a solid medium containing 50 μg / ml kanamycin, cultivate it in a 37°C incubator for 48 hours, and inoculate a single colony on the solid medium to LB liquid medium containing 50 μg / ml kanamycin, cultured with shaking at 37°C for 24 hours.

[0053] Then the bacterial solution was inoculated into LB liquid medium (which had been sterilized at high temperature) containing 50 μg / ml kanamycin at a ratio of 1:100 by volume, and continued shaking culture at 37°C. When the bacterial OD value (600nm) reached At 0.5, add isopropylthiogalactoside (IPTG) in the LB liquid medium, so that the final concentration of isopropylthiogalactoside in the LB liquid medium is 500 μg / ml, to promote Escherichia coli to secrete extracellular Membrane vesicles...

Embodiment 3

[0061] Validation of E. coli outer membrane vesicles

[0062] (1) Particle size measurement of E. coli outer membrane vesicles

[0063] First, use PBS buffer to adjust the outer membrane vesicles synthesized in Example 1 to 100 μg / ml, and measure the size of the outer membrane vesicles under the Malvern laser particle size analyzer. The results are as follows figure 1 shown.

[0064] (2) Morphological observation of outer membrane vesicles

[0065] Take the outer membrane vesicle solution of Example 1, drop it on a special copper grid, and directly observe the size and shape of the outer membrane vesicle under a scanning electron microscope, the results are as follows: figure 2 , image 3 shown.

[0066] (3) Investigate the yield of Escherichia coli outer membrane vesicles

[0067] Take the outer membrane vesicle solution of Example 1, add lysozyme (1 mg / ml), and shake at room temperature for 30 minutes. Subsequently, the BCA protein kit was used to detect the protein c...

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Abstract

The invention discloses a preparation method and a drug carrying method of an escherichia coli outer membrane vesicle, and application of the outer membrane vesicle in anti-tumor. The preparation method of the escherichia coli outer membrane vesicle comprises the following steps of culturing a culture medium using LB in vitro, culturing an escherichia coli BL21 strain, performing centrifugation, filtration and ultrafiltration treatment to obtain an upper layer culture solution free of escherichia coli, and finally centrifuging the upper layer culture solution by a supercentrifuge to prepare the escherichia coli outer membrane vesicle. The particle size of the prepared escherichia coli outer membrane vesicle is uniform, and is about 50nm. Usually, a bacterium outer membrane vesicle is low in yield and difficult in drug carrying. The invention excogitates a novel drug carrying method for the bacterium outer membrane vesicle, so that the carrying efficiency of an anti-tumor drug can be significantly improved; an inhibition effect on multiplication and invasion of a tumor cell is increased; and a good application prospect of the bacterium outer membrane vesicle serving as a novel non-virus drug carrier is presented.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the preparation and drug loading method of Escherichia coli outer membrane vesicles and its application in antitumor. Background technique [0002] Bacterial outer membrane vesicles (OMVs) are nanometer-sized, bilayer spherical proteoliposomes. Its particle size is about 50-100nm. The outer surface of the outer membrane vesicle is surrounded by a phospholipid bilayer, rich in water-soluble proteins, mRNA and microRNA and other substances. It is mainly secreted by Gram-negative bacteria and excreted from the bacteria. It is an important pathway for bacterial metabolism and an important medium for signal transduction between bacteria. Gram-negative bacteria release outer membrane vesicles spontaneously during their growth. However, the yield of bacterial outer membrane vesicles produced spontaneously is relatively low, and it often takes a large number of bacteria to extract a certai...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20A61K47/46A61K47/26A61K45/00A61P35/00C12R1/19
CPCA61K45/00A61K47/26A61K47/46C12N1/20
Inventor 彭丽华牛杰
Owner ZHEJIANG UNIV
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