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Erythrocyte membrane surface modification gene delivery system as well as preparation method and application

A red blood cell membrane and gene delivery technology, applied in gene therapy, other methods of inserting foreign genetic materials, pharmaceutical formulations, etc., can solve the problems of ineffective gene therapy, high cytotoxicity, short cycle time, etc., and achieve beneficial Effects of gene delivery and expression, high transfection efficiency, and extended cycle time

Inactive Publication Date: 2018-10-12
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to its high charge density, it also has high cytotoxicity while having high cell transfection efficiency
Moreover, due to its positively charged surface, this delivery system is very unstable in the blood, and it is easy to be cleared, the circulation time in the blood is very short, and the satisfactory effect of gene therapy cannot be achieved.
Therefore, the design of a safe, efficient and stable gene delivery system with the characteristics of long blood circulation is still facing great challenges.

Method used

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  • Erythrocyte membrane surface modification gene delivery system as well as preparation method and application
  • Erythrocyte membrane surface modification gene delivery system as well as preparation method and application
  • Erythrocyte membrane surface modification gene delivery system as well as preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The preparation method of the gene delivery system for modifying the surface of erythrocyte membrane comprises the following steps:

[0042] 1. Preparation of nano-erythrocyte membrane solution:

[0043] (1) Take 375 microliters of Kunming Kunming male mice, add EDTA to prevent blood coagulation, centrifuge at 800g and 4°C for 8 minutes, remove plasma and buffy coat, and obtain sample 1;

[0044] The ratio of said EDTA to said blood is 1.5 mg / ml;

[0045] (2) Sample 1 was washed 10 times with 1×PBS buffer solution, 0.25×PBS buffer solution equivalent to 12 times the volume of the blood was added, and placed in an ice bath for 45 minutes to rupture the red blood cell membrane and release hemoglobin. Centrifuge at 4500g for 12 minutes, collect light pink matter as sample 2;

[0046] (3) Sample 2 was washed 5 times with 1×PBS, centrifuged to obtain sample 3, added to 8 ml of PBS buffer solution with pH=7.4, and ultrasonicated for 1.5 hours to obtain sample 4;

[0047] (...

Embodiment 2

[0056] The preparation method of the gene delivery system for modifying the surface of erythrocyte membrane comprises the following steps:

[0057] 1. Preparation of nano-erythrocyte membrane solution:

[0058] (1) Take 375 microliters of Kunming male mouse blood, add EDTA to prevent its coagulation, centrifuge at 600g, 4°C for 10 minutes, remove plasma and buffy coat, and obtain sample 1;

[0059] The ratio of said EDTA to said blood is 1.5 mg / ml;

[0060] (2) Sample 1 was washed 5 times with 1×PBS buffer solution, and 0.25×PBS buffer solution equivalent to 8 times the blood volume was added, and placed in an ice bath for 30 minutes to rupture the red blood cell membrane and release hemoglobin. Centrifuge at 4000g for 15 minutes to collect light pink matter as sample 2;

[0061] (3) Sample 2 was washed 6 times with 1×PBS, centrifuged to obtain sample 3, added to 8 ml of PBS buffer solution with pH=7.4, and ultrasonicated for 1 hour to obtain sample 4;

[0062] (4) Sample 4 w...

Embodiment 3

[0071] The preparation method of the gene delivery system for modifying the surface of erythrocyte membrane comprises the following steps:

[0072] 1. Preparation of nano-erythrocyte membrane solution:

[0073] (1) Take 375 microliters of blood from Kunming male mice, add EDTA to prevent blood coagulation, centrifuge at 1000g and 4°C for 5 minutes, remove plasma and buffy coat, and obtain sample 1;

[0074] The ratio of said EDTA to said blood is 1.5 mg / ml;

[0075] (2) Sample 1 was washed 7 times with 1×PBS buffer solution, and 0.25×PBS buffer solution equivalent to 20 times the blood volume was added, and placed in an ice bath for 60 minutes to rupture the red blood cell membrane and release hemoglobin. Centrifuge at a speed of 5000g for 10 minutes, and collect the light pink substance as sample 2;

[0076] (3) Sample 2 was washed 7 times with 1×PBS, centrifuged to obtain sample 3, added to 8 ml of PBS buffer solution with pH=7.4, and ultrasonicated for 2 hours to obtain s...

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Abstract

The invention discloses an erythrocyte membrane surface modification gene delivery system as well as a preparation method and an application. The preparation method comprises the following steps: I, preparing blood, and removing plasma and a leucocytes layer, so that a sample 1 is obtained; cleaning the sample 1: adding the sample 1 to a 0.25*PBS buffer solution, keeping an obtained solution in anice bath so that erythrocyte membranes get cracked and heme is released, implementing centrifuging and collecting a sample 2; implementing cleaning and centrifuging, so that a sample 3 is obtained; adding the sample 3 to a PBS buffer solution, and implementing ultrasonic processing, so that a sample 4 is obtained; reciprocally extruding a nano polycarbonate thin film via a liposome extrusion apparatus, repeatedly conducting the operation of extruding, and collecting erythrocyte membrane vesicle liquid as a sample 5; and implementing ultrasonic processing, so as to obtain nano erythrocyte membrane liquid as a sample 6; II, preparing a gene compound; and III, dropping the sample 6 into the gene compound, so that the erythrocyte membrane surface modification gene delivery system is obtained.The system provided by the invention is high in transfer efficiency and low in cytotoxicity; the system is good in bio-compatibility, and the system, which is stable in blood, is prevented from beingrapidly removed in vivo; and in-vivo circulating time is greatly prolonged, so as to bring about benefits for gene delivery and expression.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a red blood cell membrane surface modification gene delivery system and application thereof. Background technique [0002] As the research on erythrocyte vectors becomes more and more extensive, as autologous cells, erythrocyte vectors have many unique advantages. It is the most numerous and longest-lived cell in the blood, has extremely high biocompatibility, can circulate in the body for up to 120 days, and can be completely degraded. Therefore, it is widely used as a drug carrier to treat diseases. However, there are also some defects in the direct entrapment of drugs in red blood cells, such as drug leakage and difficulty in standardized preparation, which limit its application. Therefore, researchers have isolated pure red blood cell membranes, which not only retain the advantages of red blood cells, but also are easy to prepare, and are widely used in nano-drug delivery sys...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/87A61K48/00
CPCA61K48/0025C12N15/87
Inventor 冯亚凯郝雪芳郭锦棠任相魁
Owner TIANJIN UNIV
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