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Fusion mixture

A mixture and biotinylation technology, which is applied in the direction of fusion cells, cells modified by introducing foreign genetic material, liposome delivery, etc., to achieve the effect of easy preparation

Inactive Publication Date: 2016-02-03
FORSCHUNGSZENTRUM JULICH GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Thus, the disadvantages of the fusion mixtures known from the prior art are the narrow concentration range of the molecular species A, B and C and the fact that at least 3 synergistic interaction

Method used

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Examples

Experimental program
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no. 1 example

[0109] First embodiment ( figure 1 with 2 ): Fusion in cell suspension (1:0.005 to 1:0.2 mol / mol fusion mixture of molecular species A:B (DOTAP:DiR) with CHO-cells).

[0110] The first example confirms the correct molar ratio of molecular species A and B in the claimed fusion mixture.

[0111] In the case of flow cytometry measurements, the X- and Y-axis labels given in the diagram below are also used for the measurement results placed thereon.

[0112] The components of the fusion mixture, here DOTAP (molecular species A) and DiR (molecular species B) were mixed from a stock solution of 1 mg / ml in chloroform at a mixing ratio of 1:0.005 to 1:2 mol / mol to determine DiR-inducing fusion concentration range.

[0113] As a result it can be determined that starting from a molar ratio of molecular species A:B of 1:2 or greater (i.e. e.g. 1:3 mol / mol molecular species A:B), homogenization of the two substances in a fusion mixture is Difficult because these substances clump to...

no. 2 example （ pic 3）

[0123] The second embodiment ( image 3 ): Fusion with model membrane (Fusion of fusion mixture of molecular species A:B (DOTAP:DiO) with DOPC model membrane and A:B molar ratio of 1:0.1 mol / mol)

[0124]Large unilamellar vesicles composed of 1,2-dioleoyl-sn-glyceryl-3-phosphocholine swell in 250 mM sucrose solution (pH 7.4, adjusted with 4 mM imidazole / HCl) under alternating current at 1.3 V and 10 Hz . 100 μl of vesicle solution was transferred into the measurement chamber, which was previously coated with avidin and filled with 1.8 ml of 250 mM glucose solution. In addition, 100 μl of fused vesicles (DOTAP:DiO=1:0.1 mol / mol) were added. Fused vesicles were prepared as in Example 1. Cover the sample holder with a cover glass and increase the temperature to 37°C.

[0125] image 3 A shows DOPC-large vesicles (arrows) after membrane fusion has occurred. Fluorescence transfer from small microscopically indistinguishable fused vesicles to large monolayer DOPC-model membr...

no. 3 example （

[0127] The third embodiment ( Figure 4 ): fusion with cell membrane fragments - fusion of molecular species A:B (DOTAP:DiO) in a ratio of 1:0.1 mole / mole to isolated HL-1 cell plasma membrane and further fusion of this fused membrane fraction with DOPC-macrovesicles fusion.

[0128] HL1-cells (myocyte-cell line) swell osmotically in PBS-buffer with 200 mOsm. Thereafter, the cells were crushed in a homogenizer. Nuclei and mitochondria can be separated from remaining membranes by centrifugation. The remaining fractions contain cytoplasmic membranes with Dystroglykan, ER-membranes and lysosomal membranes. This fraction was dissolved in a buffer solution of 250 mM sucrose / imidazole / HCl.

[0129] It was fused with fusion liposomes of DOTAP / DiO (1:0.1 mol / mol) at a volume ratio of 1 / 10 to generate plasma membrane-containing fusion liposomes.

[0130] A second fusion is initiated between the plasma membrane-containing fused liposome and the DOPC-macrovesicle (C, D).

[0131]...

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Abstract

The invention relates to a fusion mixture for lipid-containing membrane modification of any desired target membrane, a cell membrane, a constituent part of a cell membrane or a cell membrane separated from the remaining cell constituent parts in vivo or in vitro, comprising a positively charged amphipathic molecule A and an aromatic molecule B, wherein the molecule type A and the molecule type B are present in a ratio A:B of 1:0.02 to 1:2 mol / mol.

Description

[0001] The present invention relates to fusion mixtures of lipid-containing membranes for the modification in vivo or in vitro of any lipid membrane, cell membrane, cell membrane component, or cell membrane separated from the rest of the cell, and to the modification of lipid-containing membranes by fusion with the mixture. the membrane method. Background technique [0002] For therapeutic use, there is a need for methods of introducing pharmaceutically active substances into target cells. [0003] Known by Khalil et al. (I.A. Khalil, K. Kogure, H. Akita, H. Harashima, 2006. Uptake Pathways and Subsequent Intracellular Trafficking in Nonviral Gene Delivery. PHARMACOLOGICAL REVIEWS, Vol. 58, No. 1, 32-45), a distinction can be made between endocytic and nonviral traffic for DNS Endocytosis method. According to this review article in the scientific community, the main mode of uptake of DNS is endocytosis by means of lipoplexes (also known as cationic lipid-DNA complexes). Vesi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K9/127
CPCA61K9/1272C12N5/16
Inventor B.霍夫曼A.克西斯扎尔N.赫施R.默克尔
Owner FORSCHUNGSZENTRUM JULICH GMBH
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