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Drug-loaded bacterial outer membrane vesicle,and preparation method and application thereof

A technology of outer membrane vesicles and bacteria, which can be used in antibacterial drugs, pharmaceutical formulations, microcapsules, etc., and can solve the problems of less research

Active Publication Date: 2019-11-22
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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  • Drug-loaded bacterial outer membrane vesicle,and preparation method and application thereof
  • Drug-loaded bacterial outer membrane vesicle,and preparation method and application thereof
  • Drug-loaded bacterial outer membrane vesicle,and preparation method and application thereof

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 a preparation method and application thereof, and particularly relates to a drug-loaded bacterial outer membrane vesicle prepared with the aid of liposome and a preparation method and application of the drug-loaded bacterial outer membrane vesicle. The application is in particular application in the preparation of antibacterial drugs. The invention provides the method for preparing a preparation containing the drug-loaded bacterial outer membrane vesicle, the method comprises the following steps of taking a preparationcontaining a drug-loaded liposome and a preparation containing a bacterial outer membrane vesicle as raw materials, and preparing to obtain the preparation containing the drug-loaded bacterial outermembrane vesicle. The invention further provides the method for preparing the drug-loaded bacterial outer membrane vesicle, the method comprises the following steps of taking a drug-loaded liposome and a bacterial outer membrane vesicle as raw materials, and preparing to obtain the drug-loaded bacterial outer membrane vesicle. The invention further provides the antibacterial drugs, including the preparation containing the drug-loaded bacterial outer membrane vesicle or the drug-loaded bacterial outer membrane vesicle. According to the drug-loaded bacterial outer membrane vesicle, the preparation method and application thereof, broad application prospects in the field of drug delivery and the field of antibacterial drug development achieved.

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 Applications(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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