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Lipid ultrasound microbubble mediated adeno-associated virus gene transfection preparation and preparation technology thereof

An ultrasonic microbubble and virus technology, which is applied in the field of biomedicine to achieve the effect of increasing permeability and promoting gene transmembrane transfection

Inactive Publication Date: 2012-12-12
钟志容
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, using virus as a carrier, although the gene transfection efficiency is relatively high, its safety and immunogenicity are issues that need to be considered

Method used

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  • Lipid ultrasound microbubble mediated adeno-associated virus gene transfection preparation and preparation technology thereof
  • Lipid ultrasound microbubble mediated adeno-associated virus gene transfection preparation and preparation technology thereof
  • Lipid ultrasound microbubble mediated adeno-associated virus gene transfection preparation and preparation technology thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0009] Precisely measure a certain proportion of AAV, phosphatidylcholine, phosphatidylethanolamine, cholesterol derivative cholesteryl hemisuccinate (CHEMS) and glycerol, dissolve in PBS, mix in water at 20°C for 10 min, and flush with perfluoropropane gas. Ultrasonication was performed with a 100W ultrasonic cleaner in a water bath at room temperature, with intervals of 10 s for 20 s, and a total of 10 cycles to prepare AAV-loaded lipid ultrasonic microbubbles.

[0010] Example 2: Morphological observation and particle size distribution detection of drug-loaded lipid microbubbles

Embodiment 2

[0011] The newly prepared lipid ultrasonic microbubbles were sealed and stored in a refrigerator at 4°C. They were taken out at 1h, 24h, 48h and 2 months respectively. After being properly diluted with PBS, they were placed under an inverted optical microscope to observe their appearance. To investigate the concentration of lipid ultrasonic microbubbles, take 1 drop of the diluent and place it on a hemocytometer, count the number of microbubbles in 8 large grids and take the average value (measured at least 3 times). And use the Malvern Sizer Nano ZS90 particle size analyzer to analyze the microbubble particle size and distribution according to the operating instructions. Observation with an inverted optical microscope showed that the lipid ultrasonic microbubbles prepared in this experiment were spherical and uniform in size. Place 1h, 24h and 48h after the lipid microbubble microscope observation result is consistent ( figure 2 ); Lipid microbubbles gathered into agglomera...

Embodiment 3

[0013] Human bronchial epithelial cells 16HBE14o-grow in DMEM / F12 (1:1) medium containing 10% fetal bovine serum, 100u / ml penicillin, 100μg / ml streptomycin, at 37°C, 5% CO 2 , cultured under saturated humidity conditions. The cryopreserved 16HBE14o- cells were cultured normally in a 150mm sterile cell culture dish after recovery.

[0014] Individually packaged Transwell chambers (clear polyester film, 0.4 μm) were placed in sterile 12-well cell culture plates. Take 16HBE14o-cells in a good growth state in the logarithmic growth phase, digest the monolayer culture cells with 0.25% trypsin for 2 min, and prepare a single cell suspension with DMEM / F12 (1:1) medium containing 10% fetal bovine serum. solution, 0.5ml per well, 3.5×10 6 Cells were inoculated in the inner chamber of Transwell, and 1.5 ml of complete medium was added to the outer chamber. Move the culture plate into CO 2 in an incubator at 37°C, 5% CO 2 Cultured under saturated humidity conditions to allow cells t...

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Abstract

The invention relates to an adeno-associated virus based and lipid ultrasound microbubble mediated novel preparation capable of obviously improving gene transfection efficiency on cells with compact structure. A structure of the novel preparation is shown as a figure 1, and consists of three parts: an adeno-associated virus, perfluoropropane, and a lipid bilayer. According to the invention, the advantage of efficient gene transfection of the adeno-associated virus vector is utilized; under the effect of ultrasound, the ''cavitation effect'' generated by the ultrasound microbubbles acts on cell membranes of the cells with compact structure to generate ''overflow pores'' and increase permeability of the cell membranes, so that adeno-associated viruses released after destruction of the microbubbles can enter blood vessel walls and even tissue spaces, thereby promoting transmembrane gene transfection. The novel preparation provided by the invention is simple for preparation and convenient for usage, and does not require special production conditions.

Description

1. Technical field [0001] The patent of the present invention relates to a new preparation based on adeno-associated virus, mediated by lipid ultrasonic microbubbles, which significantly enhances the gene transduction efficiency of cells with compact structure, and belongs to the field of biomedicine technology. [0002] 2. Background technology [0003] At present, in the study of gene therapy, the safe and effective gene delivery system is the focus of attention. Generally, gene delivery systems can be divided into viral vector systems and non-viral vector systems according to different vectors. Non-viral vectors such as liposomes, nanoparticles, microcapsules, and microspheres have the advantages of convenience, large-scale production, and non-immunogenicity, but their disadvantages are low transfection efficiency when used in vivo. Among viral vectors, adeno-associated virus vectors are widely used. The researchers found that adeno-associated virus (AAV) is stable to...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K9/127A61K47/28
Inventor 钟志容刘中兵柯发敏王述蓉李劲薇
Owner 钟志容
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