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Transfection particles

A particle and molecular technology, applied in the field of transfection particles, which can solve the problems of DNA decondensation and cytotoxicity

Inactive Publication Date: 2001-04-04
BOEHRINGER INGELHEIM INT GMBH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the solubility of detergents in water is much greater than that of lipids, and they are rapidly released into the environment, leading to DNA decondensation and detergent-associated cytotoxicity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0232] b) Synthesis of lipophilic guanidine-cysteine ​​amide detergent

[0233] according to figure 2 The shown route was used to synthesize the lipophilic compound.

[0234] step 1

[0235]Preparation of N-tert-butoxycarbonyl-pyrazole-1-carboxamidine

[0236] Add 11.3ml of N,N-diisopropylethylamine (66mmol) and 4.40g of 1H-pyrazole to a solution of di-tert-butyl carbonate (7.43g, 33mmol) in 100ml of dichloromethane - 1-Formamidine hydrochloride (30 mmol). The solution was stirred at room temperature for 3 hours and evaporated to dryness. The resulting white solid was dissolved in 200 ml of ethyl acetate, the organic phase was washed with 5% (w / w) sodium bicarbonate, dried over magnesium sulfate, and finally evaporated to dryness. The residue was dissolved with boiling hexane. The solution was stored overnight at room temperature and then stored at 4°C for 24 hours. The resulting crystals were recovered by filtration and washed in cold hexane (0°C). After drying, 5.2...

Embodiment 2

[0290] a) Synthesis of lipophilic alcohol cysteine ​​ester detergent

[0291] according to Figure 9 The synthesis was carried out according to the indicated route.

[0292] step 1

[0293] Preparation of N,S-di-tert-butoxycarbonyl-L-cysteine

[0294] 945 mg of L-cysteine ​​chlorohydrate (6 mmol) was dissolved in 10 ml of deionized water, where the gas had been removed by vacuum. To this solution was added 2.625 g of BocOBoc (12 mmol) in 10 ml of THF and 5.4 ml of triethylamine (39 mmol). After stirring for 2 days at room temperature under an argon atmosphere, the reaction medium is evaporated to a volume of slightly less than 10 ml. Then 10 ml of deionized water was added, and the liquid phase was washed with ether to remove residual unreacted BocOBoc. The aqueous phase was then acidified to pH 3 with saturated citric acid and extracted with diethyl ether (3 x 60ml). The ether phase was washed with 0.5M citric acid (2 x 100ml), dried over magnesium sulfate, and evapor...

Embodiment 3

[0317] Synthesis of lipophilic alcohol guanidino-cysteine ​​ester detergent

[0318] according to Figure 13 The synthesis was carried out according to the indicated route.

[0319] step 1

[0320] Preparation of S-tert-butoxycarbonylcysteine ​​decyl ester

[0321] 231 mg of N,S-di-tert-butoxycarbonyl-cysteine ​​decyl ester (0.5 mmol) were dissolved in 1.1 ml of 2.2N HCl / AcOEt. The solution was stirred at room temperature for 1 hour and then evaporated to dryness. The obtained residue was dissolved in 50 ml of ethyl acetate. The organic phase was washed twice with 25 ml of 5% (w / w) sodium bicarbonate, dried over magnesium sulfate and finally evaporated to dryness. The resulting oil was purified by silica gel chromatography (eluting with 0-3% methanol in dichloromethane) to give 141 mg of a yellow oil (0.39 mmol, 78% yield).

[0322] C 18 h 35 NO 4 S: 361.55g / mol

[0323] NMR 1 H(CDCl 3 ); δppm: 0.90 (t, J=7Hz, 3H, CH 3 ); 1.20-1.42 (m, 14H, CH 3 -O(CH 2 ) 7 -...

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Abstract

Transfection particles used to deliver nucleic acids into cells of higher eukaryotes in vivo and in vitro, including one or more nucleic acid molecules condensed by organic cationic molecules. The dispersed stable particles are formed by complexing the nucleic acid molecules with the same or different organic cation precursor molecules, rather than cross-linking between the nucleic acid molecules, and the precursor molecules are covalently bonded to each other on the nucleic acid template and made. For unambiguous cell orientation, the particles are loaded with orientation molecules, such as sugars. Preferred cationic precursor molecules are lipophilic detergents bonded to form lipids. The particle preferably contains only one nucleic acid molecule making it effective for gene therapy and delivery of large DNA molecules.

Description

technical field [0001] The present invention relates to the field of delivery of nucleic acids into cells of higher eukaryotes, particularly for gene therapy. The present invention provides novel transfection particles for in vitro and in vivo transfection of higher eukaryotic cells. Background technique [0002] A critical step in modern recombinant nucleic acid technology is the efficient transfection of cells with foreign genetic material both in vitro and in vivo. [0003] Typically, recombinant viruses are used to efficiently deliver foreign genetic material into cells in vitro and in vivo (Mulligan, 1993). Recombinant viruses are often specific cells with advantages or disadvantages, depending on the individual experiment. In addition to foreign material, they also carry their own genetic material and are very susceptible to mutations during the process of replicating. Another disadvantage of in vivo viruses in organisms with immune systems is that viral particles s...

Claims

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Application Information

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IPC IPC(8): A61K35/76A61K47/48A61K48/00
CPCA61K47/48884A61K47/48046B82Y5/00A61K47/543A61K47/6929A61K47/50
Inventor 让-保罗·贝尔托马斯·布辛莱厄恩斯特·瓦格纳苏珊娜·舒勒
Owner BOEHRINGER INGELHEIM INT GMBH
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