Ionizable lipid compound and nucleic acid in-vitro cell transfection reagent

A technology of transfection reagents and compounds, applied in the field of ionizable lipid compounds and nucleic acid transfection reagents, to achieve the effects of good stability, uniform size and rich variety

Active Publication Date: 2022-03-11
WUHAN BINHUI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the existing cationic liposome transfection reagents have obvious advantages compared with viral vectors and other types of non-viral vectors in terms of encapsulation efficiency, transfection efficiency and cytotoxicity, there is still certain cytotoxicity, and the transfection Efficiency also needs to be further improved. Therefore, more in vitro cell transfection reagents with high transfection efficiency and low cytotoxicity need to be developed to provide more options for cell transfection

Method used

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  • Ionizable lipid compound and nucleic acid in-vitro cell transfection reagent
  • Ionizable lipid compound and nucleic acid in-vitro cell transfection reagent
  • Ionizable lipid compound and nucleic acid in-vitro cell transfection reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] The synthetic route of compound 1:

[0102]

[0103] Step 1: Synthesis of compound 1-1:

[0104] Add linalyl alcohol (0.267g, 1mmol) and triethylamine (0.133g, 1.3mmol) into the reaction flask in an ice-water bath, add dichloromethane (6mL), and dissolve acryloyl chloride (0.11g, 1.2mmol) in dichloromethane (2.2 mL) was slowly added dropwise into the reaction bottle, and the reaction lasted for 10 minutes. The reaction was kept below 10° C., and finally the ice bath was removed, and the reaction solution was reacted at room temperature for 2 hours. Wash with saturated brine to obtain a crude product, which is purified by chromatography (silica gel column, eluent is petroleum ether containing 0.5% EA (volume percentage)), and the pure product is evaporated to obtain light yellow oily compound 1 -1 (2-allylic acid (9Z, 12Z)-octadecadienyl ester) (0.173g, yield: 50%) The hydrogen spectrum of compound 1-1 is shown in figure 1 .

[0105] 1H NMR (400MHz, CDCl 3 )δ: 6.41(...

Embodiment 2

[0110] Synthetic route of compound 2

[0111]

[0112] Step 1: Synthesis of Compound 2-1

[0113] Dissolve 6-bromohexanoic acid (1.0g, 5.13mmol) and undecyl alcohol (1.77g, 10.25mmol) in dichloromethane (60mL), add 1-(3-dimethylaminopropyl)-3-ethane Carbodiimide hydrochloride (EDC hydrochloride, 0.98g, 5.13mmol) and DMAP (0.125g, 1.03mmol). The mixture was stirred at room temperature for 18 hours. After the reaction, dilute with DCM (200mL) and wash with saturated NaHCO 3 (100 mL) and brine (100 mL). Combine the organic layers with anhydrous Na 2 SO 4 Drying and removal of the solvent in vacuo afforded the crude product, which was purified by chromatography (silica gel column, eluent petroleum ether containing 0.5% EA (volume percent)), and the pure product was evaporated to give compound 2 as a pale yellow oil -1(undecyl 6-bromohexanoate) (0.69 g, yield 38.6%). The hydrogen spectrum of compound 2-1 is shown in Figure 5 .

[0114] 1H NMR (400MHz, CDCl 3 )δ: 4.10(...

Embodiment 3

[0119] Synthetic route of compound 3

[0120]

[0121] Step 1: Synthesis of compound 3-1

[0122] 8-Bromooctanoic acid (1.139 g, 5.13 mmol) and 3,7-dimethyloct-6-en-1-ol (citronellol, 1.599 g, 10.25 mmol) were dissolved in dichloromethane (60 mL), fully After dissolution, EDC hydrochloride (0.98 g, 5.13 mmol) and DMAP (0.125 g, 1.03 mmol) were added. The mixture was stirred at room temperature for 18 hours. After the reaction, dilute with DCM (200mL) and wash with saturated NaHCO 3 (100 mL) and brine (100 mL). Combine the organic layers with anhydrous Na 2 SO 4 Drying, removal of solvent in vacuo gave a crude product, which was purified by chromatography (silica gel column, eluent was petroleum ether containing 0.5% EA (volume percentage)), and the pure product was evaporated to give light yellow oily compound 3 -1(3,7-dimethyloct-6-enyl 6-bromohexanoate) (0.648g, 35%) The hydrogen spectrum of compound 3-1 is shown in Figure 8 .

[0123] 1H NMR (400MHz, CDCl 3 )δ:...

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Abstract

The invention provides an ionizable lipid compound and a nucleic acid in-vitro cell transfection reagent. In order to enrich the types of ionizable lipid compounds, provide more choices for transfection of deoxyribonucleic acid (DNA) to cells and provide a convenient research tool for transfection of ribonucleic acid (RNA) to cells, the invention provides an efficient and low-toxicity ionizable lipid compound. The lipidosome formed by the ionizable lipid compound can efficiently load nucleic acid, carries the nucleic acid to penetrate through cell membranes, enter various types of cells, is protonated in an acid environment of endocytosis vesicles to carry positive charges, and improves the escape ability of inclusion, so that the lipidosome formed by the ionizable lipid compound has higher transfection efficiency. Meanwhile, effective degradation can be realized, and toxic and side effects of the liposome on cells are reduced. The method is not only suitable for in-vitro cell transfection of DNA, but also can be used for in-vitro cell transfection of RNA.

Description

technical field [0001] The invention belongs to the technical field of in vitro cell transfection reagents, and in particular relates to an ionizable lipid compound and a nucleic acid transfection reagent. Background technique [0002] Cell transfection refers to the technique of introducing exogenous molecules such as DNA, RNA, etc. into eukaryotic cells. With the continuous development of molecular biology and cell biology research, transfection has become a routine tool to study and control gene function in eukaryotic cells. It is used more and more widely in biological experiments such as studying gene function, regulating gene expression, mutation analysis and protein production. [0003] In the past, due to the difficulty of making mRNA into drugs, the application of transferring mRNA into cells in vitro was very small compared to the application of DNA transfection. Nowadays, with the rapid development of mRNA drugs, the demand for in vitro transfection of mRNA into...

Claims

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Application Information

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IPC IPC(8): C07C229/16C12N15/87
CPCC07C229/16C12N15/87
Inventor 崔艳芳刘滨磊吉帅洁张宝倩
Owner WUHAN BINHUI BIOTECH CO LTD
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