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Organic functional molecule containing natural cholesterol and lysine lipid cations, lipidosome thereof, as well as preparation method and application for lipidosome

A technology of functional molecules and cholesterol, which is applied in the preparation of lipid-like cationic organic functional molecules and their liposomes, and the application field of small-molecule interfering RNA transfection reagents, achieving simple and efficient preparation methods, high siRNA transfection efficiency, and ease of use. The effect of low-cost large-scale preparation

Inactive Publication Date: 2013-07-24
SHANGHAI INST OF ORGANIC CHEMISTRY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the above-mentioned synthetic cholesteric lipids used as gene delivery carriers are mainly used in the cell transfection of plasmid DNA, and relatively few are used for siRNA transfection.

Method used

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  • Organic functional molecule containing natural cholesterol and lysine lipid cations, lipidosome thereof, as well as preparation method and application for lipidosome
  • Organic functional molecule containing natural cholesterol and lysine lipid cations, lipidosome thereof, as well as preparation method and application for lipidosome
  • Organic functional molecule containing natural cholesterol and lysine lipid cations, lipidosome thereof, as well as preparation method and application for lipidosome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031]

[0032] Lipid molecule Cho-5C-Lys

[0033] The first step: pentylene glycol (12.2g, 0.1mmol) is dissolved in 30ml dry dichloromethane, above-mentioned solution is slowly added dropwise to the cholesterol chloroformate (4.5 g, 0.01mol) solution, the reaction was stirred at 30°C for 12h, and then the organic solvent was distilled off. After column chromatography (eluent: ethyl acetate / petroleum ether=1 / 2v / v), the cholesterol monosubstituted pentanediol carbonate intermediate was prepared. The synthesis yield was 80%.

[0034] 1 H NMR (CDCl 3 , 300MHz): 5.32(s, 1H, C=CH, cholesterol), 4.54(t, 2H, J=6.3Hz, CH 2 OCO), 4.51(m, 1H, OCOCH), 4.36(t, 2H, J=6.3Hz, CH 2 OCO), 3.79(t, 2H, J=6.3Hz, CH 2 O), 2.30-0.95 (m, 45H, cholesterol)

[0035] ESI-MS: [M + ]=531.5m / z

[0036] The second step: Dissolve BOC-protected natural lysine (3.5g, 0.01mol) in 50ml dry chloroform, and slowly add it dropwise to the previous step dissolved in organic solvent under the catalysis of...

Embodiment 2

[0040]

[0041] Lipid molecule Cho-4C-Lys

[0042] The first step: butanediol (9.6g, 0.1mmol) was dissolved in 50ml dry dioxane, and the above solution was slowly added dropwise to cholesterol p-toluenesulfonate dissolved in 50ml dry dioxane containing 20ml pyridine (5.4g, 0.01mol) solution, stirred and reacted at 40°C for 18h, and then distilled off the organic solvent. After column chromatography (eluent: ethyl acetate / petroleum ether=1 / 2v / v), the cholesterol monosubstituted butanediol carbonate intermediate was obtained. The synthetic yield is 71%.

[0043] 1 H NMR (CDCl 3 ,300MHz): 5.32(s, 1H, C=CH, cholesterol), 3.50(t, 2H, J=6.3Hz, CH 2 OH), 3.37(t, 2H, J=5.1Hz, CHOCH 2 ), 2.30-0.95 (m, 45H, cholesterol)

[0044] ESI-MS: [M + ]=503.4m / z

[0045] Step 2: Dissolve BOC-protected natural lysine (3.5g, 0.01mol) in 50ml of dry tetrahydrofuran, and slowly add it dropwise to the previous step dissolved in 20ml of dry tetrahydrofuran under the catalysis of 0.5g of 4-di...

Embodiment 3

[0049] In a 250mL round bottom flask, weigh 1mmoL of the lipid-like cationic organic functional molecule Cho-5C-Lys synthesized above containing natural cholesterol and lysine, and then add dioleoylphosphatidylethanolamine with a molar ratio of 1:1 (DOPE) mixed with it. Then fully dissolve the mixed lipid in 100mL chromatographic grade chloroform solution, and then remove the chloroform solvent in a rotary evaporator at 40°C. After a uniform lipid film is formed at the bottom of the bottle, add 100mL deionized water, ultrasonic water After 0.5h, liposome Cho-5C-Lys-DOPE containing natural cholesterol and lysine was obtained.

[0050] Particle size of liposome Cho-5C-Lys-DOPE Surface Potential of Liposome Cho-5C-Lys-DOPE 108.6±12.3nm 23.1±2.5mV

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Abstract

The invention relates to the field of lipidosome gene transfection biological reagents, and particularly relates to an organic functional molecule containing natural cholesterol and lysine lipid cations, the lipidosome thereof, a preparation method, and an application for the lipidosome as a small interfering RNA (ribonucleic acid) (SiRNA) transfection reagent. The organic functional molecule containing natural cholesterol and lysine lipid cations provided by the invention is simple in synthesis method and high in efficiency; the lipidosome formed by compounding the organic functional molecule with auxiliary lipid dioleoyl phosphatidyl ethanolamine (DOPE) is simple and convenient in preparation method, and easy to realize large-scale preparation. As the SiRNA gene transfection reagent, the lipidosome containing natural cholesterol and lysine lipid cations provided by the invention is obviously low in cytotoxicity compared with the commercially available commercialized gene transfection reagent bPEI-25 k, relatively efficient in SiRNA gene transfection effect, and capable of being applied in SiRNA transfection as a novel lipid gene carrier functional reagent.

Description

technical field [0001] The invention relates to the field of synthetic liposome gene delivery functional carrier materials, in particular to a lipid-like cationic organic functional molecule synthesized from natural cholesterol and lysine and a method for preparing liposomes and its use as small molecule interference RNA ( SiRNA) transfection reagent application. Background technique [0002] At present, gene therapy (Gene Therapy) has become an emerging research hotspot in the field of biomedical engineering research and application. Gene defects, so as to achieve the purpose of treating diseases. These imported therapeutic genes include DNA, RNA, etc. Among them, gene therapy methods based on small interfering RNA (SiRNA) technology have aroused great interest in recent years. Compared with traditional DNA gene therapy, the advantage of this method is that SiRNA does not need to cross the nuclear membrane barrier to enter the target cell nucleus for integrated expression...

Claims

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Application Information

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IPC IPC(8): C07J41/00C12N15/88
Inventor 曹阿民盛瑞隆陈剑徐宇虹王昭田晓婧
Owner SHANGHAI INST OF ORGANIC CHEMISTRY - CHINESE ACAD OF SCI
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