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Fusion vesicle derived from bacteria and plants as well as preparation method and application of fusion vesicle

A plant-derived, bacterial technology, applied in the field of biomedicine, can solve the problems of unsatisfactory anti-tumor effect, strong toxic and side effects, low blood drug concentration, etc. Effect

Active Publication Date: 2021-09-24
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the in-depth understanding of the tumor microenvironment and the process of tumor occurrence, development, drug resistance and metastasis, a variety of anticancer drugs have been used in clinical diagnosis and chemotherapy of tumors, but due to poor tumor targeting , poor accumulation ability in the tumor, severe damage to normal cells and tissues, strong toxic and side effects, and fast metabolism of small molecule drugs, easy to be cleared by the liver and kidney, low blood drug concentration, and unsatisfactory anti-tumor effect

Method used

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  • Fusion vesicle derived from bacteria and plants as well as preparation method and application of fusion vesicle
  • Fusion vesicle derived from bacteria and plants as well as preparation method and application of fusion vesicle
  • Fusion vesicle derived from bacteria and plants as well as preparation method and application of fusion vesicle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1: Extraction of Bacterial Outer Membrane Vesicles OMVs

[0081] Escherichia coli MG1655 was cultured in 1.5L LB medium at 37°C and 220rpm, when OD 600 When = 1, stop shaking the bacteria, centrifuge the bacterial solution at 10000g, 4°C for 10min, remove the precipitate and get the supernatant; then filter the supernatant with a 0.45μm vacuum filter bottle; use a 100kd ultrafiltration tube to centrifuge at 3000g for 10min to collect the concentrate; The concentrated solution was filtered through a 0.45 μm microporous membrane, and then ultracentrifuged at 4°C and 150,000g for 3 hours; the supernatant was discarded, and the precipitate was resuspended in 400 μL of PBS and stored at -80°C; the concentration of OMV extracted by BCA was 3.48 mg / mL.

Embodiment 2

[0082] Embodiment 2: Extraction of plant thylakoid membrane

[0083] Squeeze and homogenize 300mL BBY-1 and about 100g spinach leaves washed overnight with a juicer, filter the obtained homogenate with 10 layers of cotton gauze to remove residue; centrifuge the filtrate at 10000rpm for 10min, discard the supernatant; suspend the precipitate in In 400mL BBY-2 swelling solution, swell for 2 hours at 4°C in the dark; then centrifuge at 10,000rpm for 10min, discard the supernatant, and wash twice with HEPES (resuspend the pellet in 50mL BBY-2 each time, then centrifuge at 10,000rpm for 10min, discard the supernatant) Thylakoid membrane precipitation was suspended and homogenized in preservation solution BBY-3, and stored at -20°C. According to the above formula, the chlorophyll content measured by ultraviolet spectrophotometry is 0.77mg / ml.

Embodiment 3

[0084] Example 3: Preparation of fusion vesicle BPN

[0085] Take 2 mL of the thylakoid membrane mother solution, wash it with 20 mL of PBS, centrifuge at 10,000 rpm for 10 min, and resuspend the pellet in 5 mL of PBS; ultrasonicate the resuspended thylakoid membrane for 15 min at 4°C and 300 W; Use a manual liposome extruder to extrude through 800nm, 400nm, and 200nm polycarbonate porous membranes in sequence, each membrane corresponds to at least 21 extrusions, and quantify the chlorophyll content in thylakoids with a quartz cuvette The concentration is 102 μg / mL; take 50 μg of bacterial outer membrane vesicle mother solution and 40 μg of thylakoid membrane, dilute to 1 mL with PBS, extrude the mixed solution through a 200 nm polycarbonate porous membrane, and repeatedly extrude at least 21 times; The fusion mother solution was centrifuged at 10,000 rpm and 4°C for 10 min to remove unfused precipitates, and the supernatant was collected; the chlorophyll was quantified again ...

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Abstract

The invention provides a fusion vesicle derived from bacteria and plants as well as a preparation method and application of the fusion vesicle, and relates to the technical field of biomedicine. The fusion vesicle comprises a mixed membrane structure formed by fusing a bacterial outer membrane vesicle and a plant thylakoid membrane, wherein the bacteria are gram negative bacteria, and the plant thylakoid is a spinach thylakoid. The fusion vesicle of the present invention combines the characteristics of the bacterial outer membrane vesicle and the plant capsular membrane, the structure of the vesicle is uniform, the vesicle can efficiently and rapidly reach the tumor, the tumor targeting property is high, and the accumulation time is long; and the obvious effect is realized in the aspect of tumor treatment.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a fusion vesicle derived from bacteria and plants, a preparation method and application thereof. Background technique [0002] As a major disease that threatens human health, tumor has attracted widespread attention. With the in-depth understanding of the tumor microenvironment and the process of tumor occurrence, development, drug resistance and metastasis, a variety of anticancer drugs have been used in clinical diagnosis and chemotherapy of tumors, but due to poor tumor targeting , poor accumulation capacity in the tumor, severe damage to normal cells and tissues, strong toxic and side effects, and small molecule drug metabolism is fast, easy to be cleared by the liver and kidney, blood drug concentration is low, and the anti-tumor effect is not satisfactory. Therefore, how to efficiently deliver anti-tumor drugs to tumors, increase the accumulation of drugs in tumor site...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K41/00A61K39/39A61K9/127A61K36/185A61K47/46A61P35/00A61P37/04
CPCA61K39/39A61K9/1273A61K47/46A61K36/185A61K41/0057A61P35/00A61P37/04A61K2039/55594
Inventor 谢海燕庄婉茹王云锋黄利利聂伟东
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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