A kind of drug-loaded bacterial outer membrane vesicle and its preparation method and application

A technology of outer membrane vesicles and bacteria, applied in antibacterial drugs, pharmaceutical formulations, microcapsules, etc., can solve the problems of less research and achieve broad application prospects

Active Publication Date: 2022-02-18
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still relatively few studies using OMV as a drug carrier.
[0005] Although artificially synthesized nanomaterial carriers (such as liposomes, polymer micelles, metal nanoparticles, etc.) are considered to be efficient drug delivery carriers, these carriers cannot carry out effective cell-to-cell interactions

Method used

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  • A kind of drug-loaded bacterial outer membrane vesicle and its preparation method and application
  • A kind of drug-loaded bacterial outer membrane vesicle and its preparation method and application
  • A kind of drug-loaded bacterial outer membrane vesicle and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1, Preparation and Characterization of Bacterial Outer Membrane Vesicles (OMV)

[0080] 1. Preparation

[0081] 1. Pseudomonas aeruginosa was inoculated on a TSA medium plate and cultured at 37°C for 24 hours.

[0082] 2. After completing step 1, use an inoculation loop to pick colonies and inoculate them into 8 mL of LB broth medium, and culture them with shaking at 37°C and 80 rpm for 24 hours.

[0083] 3. After step 2 is completed, transfer the entire culture system to 40 mL LB broth medium, and shake at 37°C and 80 rpm for 180 min.

[0084] 4. After completing step 3, transfer the entire culture system to 150 mL LB broth medium, add 60 mg D-cycloserine (membrane vesicle inducer), and culture with shaking at 37° C. and 80 rpm for 24 hours.

[0085] 5. After completing step 4, divide the culture system into centrifuge tubes, centrifuge at 10,000 g for 10 minutes, discard the precipitate, collect the supernatant, combine the supernatant, filter it with a micr...

Embodiment 2

[0094] Example 2, preparation of drug-loaded ethosome OMV / drug-loaded ordinary liposome OMV / drug-loaded OMV

[0095] 1. Preparation of OMVs

[0096] Same as 1 to 5 of step 1 of embodiment 1.

[0097] 6. After completing step 5, divide into centrifuge tubes, centrifuge at 10000g for 20min, discard the supernatant, collect the precipitate, combine the precipitate, resuspend the precipitate in sterilized PBS buffer, and then transfer to MWCO 8000-14000 In the standard dialysis bag, place the dialysis bag in PBS buffer solution for dialysis for 24 hours, collect the liquid phase in the dialysis bag, adjust the protein concentration with PBS buffer solution so that the protein concentration is 1 mg / ml, which is the bacterial outer membrane vesicle solution (also known as OMV solution). Store at 4°C.

[0098] 2. Preparation of drug-loaded ethosome OMV

[0099] A method of blending drug-loaded ethosomes and OMVs through membrane extrusion was adopted.

[0100] 1. Take a round bo...

Embodiment 3

[0166] Embodiment 3, in vitro antibacterial activity test

[0167] The test substances are respectively: the clarithromycin ethosome OMV preparation prepared in Example 2 (marked as Lip-OMV-CLA), the clarithromycin OMV preparation prepared in Example 2 (marked as OMV-CLA) and clarithromycin (marked as Free CLA).

[0168] The tested bacteria were: Staphylococcus aureus (ATCC29213), Enterococcus faecalis (ATCC29212), Escherichia coli (ATCC25922), Klebsiella pneumoniae (ATCC700603). Suspend the test bacteria with MH liquid medium to obtain the test bacteria suspension.

[0169] The molten MH solid medium is naturally cooled, and when it is about to solidify, add the test substance (the test substance provides clarithromycin, and the concentration of clarithromycin in the system is 512μg / mL-1 / 8μg / mL, 2-fold gradient) , then pour flat. The test bacteria were inoculated onto the plate by point inoculation, and the inoculation amount was 10 4 CFU / point. Incubate at 37°C for 24 h...

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Abstract

The invention discloses a drug-loaded bacterial outer membrane vesicle and its preparation method and application, in particular to a drug-loaded bacterial outer membrane vesicle prepared by means of liposomes and its preparation method and application. Application in the preparation of antibacterial drugs. The invention provides a method for preparing a preparation containing drug-loaded bacterial outer membrane vesicles, comprising the following steps: using the preparation containing drug-loaded liposomes and the preparation containing bacterial outer membrane vesicles as raw materials to prepare drug-loaded liposomes containing Preparation of Bacterial Outer Membrane Vesicles. The invention also protects a method for preparing drug-loaded bacterial outer membrane vesicles, comprising the following steps: using drug-loaded liposomes and bacterial outer membrane vesicles as raw materials to prepare drug-loaded bacterial outer membrane vesicles. The invention also protects an antibacterial drug, which contains the preparation containing the drug-loaded bacterial outer membrane vesicle or the drug-loaded bacterial outer membrane vesicle. The invention has broad application prospects in the fields of drug delivery and antibacterial drug development.

Description

technical field [0001] The present invention relates to a drug-loaded bacterial outer membrane vesicle and its preparation method and application, in particular to a drug-loaded bacterial outer membrane vesicle prepared by means of liposomes and its preparation method and application. Application of antibacterial drugs. Background technique [0002] Bacterial outer membrane vesicles, which are evolutionarily conserved, are nanoscale functional vesicles released by bacteria to the outside world. They were first discovered in 1967 by Chatterjee et al. Outer membrane vesicle, OMV). The structure of OMV is similar to that of liposome, its diameter is usually between 20-300nm, the main body is phospholipid bilayer, in addition, it also includes bacterial outer membrane and periplasmic components, such as periplasmic protein, outer membrane protein, cytoplasmic protein, Peptidoglycan, lipopolysaccharide, DNA, RNA, various enzymes related to virulence, etc. All Gram-negative bac...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K9/50A61K9/127A61K47/46A61K31/7048A61K31/546A61P31/04
CPCA61K9/5068A61K9/127A61K31/7048A61K31/546A61P31/04
Inventor 李桂玲李馨儒王佳星魏潇萌刘畅牟家慧牛霞李承群张孟如
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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