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Vesicle, and preparation method and application thereof

A vesicle and functional technology, which can be used in pharmaceutical formulations, medical preparations with non-active ingredients, and medical preparations containing active ingredients, etc., and can solve problems such as lack of

Inactive Publication Date: 2020-10-13
南京启医科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] As a natural drug-loaded targeted delivery system, despite these advantages, there are still some challenges: first, exosomes will encounter phagocytosis by a large number of phagocytic cells in the body, so it is necessary to avoid drug-loaded exosomes from being phagocytized. To successfully reach the tumor lesion, it is necessary to improve and optimize the surface protein of exosomes to cope with the large amount of phagocytosis in the body; in addition, there is a lack of technical methods to load drugs or genes into exosomes in a streamlined manner to achieve mass production to meet clinical needs Case

Method used

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  • Vesicle, and preparation method and application thereof
  • Vesicle, and preparation method and application thereof
  • Vesicle, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1: Preparation of biomimetic microvesicles chimerized with specific membrane proteins

[0067] The membrane proteins on the surface of erythrocytes and BT483 cells were obtained by hypotonic lysis, mechanical membrane disruption and differential centrifugation. After confirming the acquisition of erythrocyte membrane proteins CD47 and EpCAM, Galectin3, and N-Cadherin on the surface of BT483 cell membranes by Western blot, the surface proteins of erythrocyte membranes were obtained and BT-483 cell membrane surface protein according to the quality of 1:1, mixed in PBS (pH 7.4) to obtain a mixed membrane protein.

[0068] Membrane protein separation and purification method reference: Dermot Walls and Sinéad T.Loughran (eds.), Protein Chromatography: Methods and Protocols, Methods in Molecular Biology, vol.1485, DOI 10.1007 / 978-1-4939-6412-3_21 this book Strategies for the Purification of Membrane Proteins written by Sinéad M.Smith in Chapter 21.

[0069] Artificia...

Embodiment 2

[0073] Example 2: Artificially synthesized microvesicles loaded with doxorubicin hydrochloride DOX

[0074] 1. Encapsulate DOX into microvesicles by the ammonium sulfate gradient method: Add DOX aqueous solution (5 mg / mL) to the microvesicle suspension without membrane protein and the microvesicle suspension with integral membrane protein, according to the A Mycin / lipid material = (1 / 20, w / w), obtained after incubation, wherein the lipid material contains 10.8mg 1,2-dipalmitate phosphatidylcholine, 7.2mg 1,2-distearoyl phosphatidyl Choline, 1.2 mg cholesterol (Lipoid, Germany). The obtained vesicles were then dialyzed overnight through a 1000 kDa dialysis bag to eliminate starting material and unincorporated proteins. The drug loading rate of DOX was determined by UV spectrophotometry.

[0075] 2. Determine the detection wavelength: prepare an aqueous solution of doxorubicin hydrochloride (25 μg / mL), use distilled water as a blank control, and perform ultraviolet full-wavele...

Embodiment 3

[0097] Example 3: Evaluation of BT-483 cells' uptake of integral membrane protein microvesicles and anti-macrophage phagocytosis

[0098] Based on the above-mentioned integral membrane protein microvesicles embedded with erythrocyte membrane protein CD47 and BT-482 cell mesangial surface adhesion protein, the uptake of breast cancer cell line BT-483 and its ability to resist phagocytosis by phagocytic cells were evaluated.

[0099] 1. Cell grouping and culture:

[0100] Targeted cells are BT-483 cells (( HTB-121 TM ): Add 0.01 mg / ml bovine insulin to RPMI-1640 Medium; the final concentration of fetal bovine serum is 20%. 5%CO 2 +95% air, cultured at 37°C.

[0101] The control cells were HeLa cells ( CCL-2 TM )): MEM - Minimal Essential Medium, with fetal bovine serum added to achieve a final concentration of 10%. 5%CO 2 +95% air, cultured at 37°C.

[0102] Mouse macrophage RAW264.7( SC-6003 TM ): adding fetal calf serum to DMEM medium to achieve a final concentra...

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Abstract

The invention provides a vesicle, and a preparation method and application thereof, and in particular relates to a drug loaded vesicle and application thereof in tumour treatment. The vesicle comprises a bimolecular layer, formed by amphiphilic molecules, and a functional component, wherein the functional component comprises CD47 and / or cell adhesion molecules (particularly cell adhesion moleculesthat can adhere to tumour cells). The vesicle can achieve stable mass production and resist phagocytosis in vivo, has the advantages of being steady in structure, high in drug loading capacity, customized in function, large in mass production and the like, and can further provide a better choice for clinical application of the vesicle.

Description

technical field [0001] The present invention relates to the technical field of biomedicine, in particular to a vesicle, especially a vesicle capable of resisting phagocytosis and targeting tumor cells, a preparation method and application thereof, a drug-loaded vesicle and its application in tumor treatment in the application. Background technique [0002] Exosomes (exosomes) are tiny vesicles that are actively secreted by living cells into the extracellular environment and are coated with phospholipid bilayers. Detected. Exosomes carry information related to their source cells, and their contents include proteins, mRNA, miRNA, DNA fragments, lncRNA, phospholipids, etc. In addition to common markers (such as CD9, CD63, CD81, etc.), the surface of the exosome membrane also expresses a variety of specific markers (such as GPC1, GPC3, PSMA, TMEM256, EpCAM, etc.). Generally, exosomes are considered to be important mediators of intercellular communication, and thus play a vita...

Claims

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Application Information

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IPC IPC(8): A61K9/127A61K47/42A61K47/24A61K47/28A61K45/00A61K31/704A61P35/00A61P35/02
CPCA61K9/1273A61K9/1278A61K47/42A61K47/24A61K47/28A61K45/00A61K31/704A61P35/00A61P35/02
Inventor 董鸣吴婷婷
Owner 南京启医科技有限公司
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