Targeted vesicle drug prepared through erythrocyte

A technology of erythrocytes and mature erythrocytes, which is applied in the direction of drug combinations, medical preparations with non-active ingredients, medical preparations containing active ingredients, etc., can solve problems such as poor penetration, large volume, and inability to effectively administer drugs to lesions, and achieve High yield and good therapeutic effect

Active Publication Date: 2020-05-08
深圳百纳心致生命科学有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, as a drug carrier, red blood cells are slightly larger in size and have poor penetration into the lesion; they are easy to accumulate in the liver and spleen, and cannot be effectively administered to other lesions, which seriously restricts their application as a medicinal carrier.

Method used

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  • Targeted vesicle drug prepared through erythrocyte
  • Targeted vesicle drug prepared through erythrocyte
  • Targeted vesicle drug prepared through erythrocyte

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Example 1 Using erythrocyte vesicles to transport GFP protein to tumor cells

[0103] 1. Experimental method

[0104] (1) Separation of red blood cells

[0105] Anticoagulated whole blood with normal blood routine results was collected, centrifuged at 600g for 5mins, and the upper serum was removed. Add 2 times the volume of isotonic solution (150mM NaCl, 20mM HEPES, pH 7.6) to each tube, mix well, centrifuge at 600g for 5mins, remove the supernatant, and wash with isotonic solution for 3 times. After the third centrifugation at 600 g for 10 min, the supernatant was removed to obtain red blood cells.

[0106] (2) Separation of red blood cell contents

[0107] Add washed erythrocytes to hypotonic solution (5 mM KCl, 5 mM piperazine-N, N-bis-2-ethanesulfonic acid, 5 mM MgSO 4 ) at a volume ratio of 1:4, let stand at 4°C for 15 minutes, centrifuge at 600g for 5 minutes, discard the supernatant, and repeat 2 to 3 times to remove hemoglobin and other intracellular compon...

Embodiment 2

[0119] Example 2 Using erythrocyte vesicles to transport NK cells without DNA content to tumor cells

[0120] 1. NK cell culture

[0121] Take 50ml of anticoagulated peripheral blood, 800g, centrifuge at room temperature for 15min, take the centrifuged cell components, add pH 7.4 PBS to suspend cells, add human lymphocyte separation medium, mix well, 800g, and centrifuge at room temperature for 15min. Take the cell layer, add the medium alpha MEM, and add 20% FCS, 12.5% ​​horse serum, 0.2mM inositol, 0.02mM folic acid, 1.43mM β-mercaptoethanol, 600U / ml IL-2, 10U / ml penicillin and 10μg / ml ml streptomycin. When the cells proliferated to a sufficient number, the cells were collected by centrifugation.

[0122] 2. NK cell disruption and DNA removal

[0123] (1) Experimental method

[0124] Take the cells collected in the previous step, and crush NK cells with a high-pressure crusher with a power of 0.75k W, and crush them once for 25 seconds. Add 500 μl of cell homogenate and...

Embodiment 3

[0136] Example 3 Anti-tumor effect of targeted modified cell vesicles

[0137] The anti-tumor drug doxorubicin is encapsulated in erythrocyte vesicles, and targeted modification is performed to achieve targeted drug delivery to tumor cells, thereby reducing damage to normal cells.

[0138] 1. Experimental method

[0139] (1) Empty shell preparation of red blood cells

[0140] Red blood cell shells were prepared according to the method in Example 1.

[0141] (2) Doxorubicin drug loading

[0142] With hypotonic solution (5mM KCl, 5mM piperazine-N,N-bis-2-ethanesulfonic acid, 5mM MgSO 4 ) Dilute doxorubicin to 100 μg / ml, add 2 times the volume to the red blood cell shell, gently invert the centrifuge tube, mix the solution, let it stand for 5 minutes, centrifuge at 500g for 3 minutes, continue to add 2 times the volume of hypotonic solution diluted Doxorubicin, invert the centrifuge tube to mix the solution, let it stand for 10 minutes, add 1 / 10 of the low osmotic fluid volum...

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Abstract

The invention discloses a targeted vesicle drug prepared through erythrocyte. The vesicle drug wraps one or more of contents of to-be-wrapped cells without containing genetic materials or drugs. The vesicle drug utilizing the erythrocyte to perform cell preparation can wrap the bioactive substances of required cells removing nucleic acid to perform delivery, so that risks such as gene recombination and gene mutation of users caused by the containing of the genetic materials in cellular vesicles can be avoided, and therefore, the vesicle drug is safer. The vesicle drug can perform targeted delivery, so that treatment effects can be enhanced; targeted modification can be performed on the cellular vesicles, so that the drug can accurately reach disease sites, therefore, treatment effects canbe enhanced, and side effects of drugs can be reduced. Thus, cellular vesicle targeting carriers and drugs with higher safety and better treatment effects can be produced on a large scale through a method.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically, to a targeted vesicle drug prepared by using red blood cells. Background technique [0002] Cell vesicles are small vesicles secreted by cells, with different sizes and a diameter distribution range of 50-1000nm. Cell vesicles are divided into various types, which are produced by different mechanisms, including exosomes, microvesicles, etc. . Among them, exosomes are a type of small vesicles with a diameter of 50-200 nm, and most cells secrete these vesicles. In previous cognition, cell vesicles have always been considered as a way for cells to excrete metabolic waste from cells, and they do not have important physiological functions. However, with the continuous advancement of science, the transport mechanism of cell vesicles has been gradually elucidated, and it is considered to be a very important type of vesicles that transmit signals between cells, which carry abundant miRNA...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/46A61K47/54A61K31/704A61K38/16A61K35/17A61P35/00A61P35/02
CPCA61K47/46A61K47/549A61K31/704A61K38/16A61K35/17A61P35/00A61P35/02Y02A50/30
Inventor 陈瑞章成陈杰
Owner 深圳百纳心致生命科学有限公司
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