CN111087332B - Cationic amino lipid and synthetic method and application thereof - Google Patents
Cationic amino lipid and synthetic method and application thereof Download PDFInfo
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- CN111087332B CN111087332B CN201911265892.7A CN201911265892A CN111087332B CN 111087332 B CN111087332 B CN 111087332B CN 201911265892 A CN201911265892 A CN 201911265892A CN 111087332 B CN111087332 B CN 111087332B
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C319/00—Preparation of thiols, sulfides, hydropolysulfides or polysulfides
- C07C319/14—Preparation of thiols, sulfides, hydropolysulfides or polysulfides of sulfides
- C07C319/20—Preparation of thiols, sulfides, hydropolysulfides or polysulfides of sulfides by reactions not involving the formation of sulfide groups
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C319/00—Preparation of thiols, sulfides, hydropolysulfides or polysulfides
- C07C319/14—Preparation of thiols, sulfides, hydropolysulfides or polysulfides of sulfides
- C07C319/18—Preparation of thiols, sulfides, hydropolysulfides or polysulfides of sulfides by addition of thiols to unsaturated compounds
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
- C07C323/23—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
- C07C323/24—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
- C07C323/25—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
Abstract
A cationic amino lipid and a synthesis method and application thereof, belonging to the field of cell transfection. The cationic amino lipid has the following structure:
. The invention has the advantages of mild synthesis reaction conditions, high solvent selectivity, simple and cheap raw materials and greatly reduced production cost, and greatly reduces the difficulty of the reaction and improves the yield. The cationic amino lipid molecule has the characteristics of high efficiency and low toxicity, can achieve good effect in serum, and greatly simplifies the operation steps of transfection.
Description
Technical Field
The invention belongs to the field of cell transfection, and particularly relates to cationic amino lipid and a synthetic method and application thereof.
Background
Among all types of non-viral transfection reagents at present, liposome-based transfection reagents are the most effective one that is recognized. This is because the lipofection reagents are simple and easy to use and tend to have high efficiency. Liposomes are artificially prepared spherical vesicles with a bilayer structure, which can be fused with other bilayers, such as cell membranes, etc., so as to transport the encapsulated molecules from the outside of the cell membrane to the inside of the cell. Liposomes are widely used for drug delivery due to their unique properties. Liposomes can carry both hydrophilic and hydrophobic molecules, which can bind well to biologically active molecules, such as various drugs, nucleic acids, polypeptides, etc., and can transport these substances into cells to regulate the state of the cells. This provides a new way to treat the disease.
Cationic liposomes can bind to negatively charged nucleic acids (DNA) and transport it from the outside of the cell into the nucleus where it is expressed, thereby regulating cell growth. The gene has a vital role in the research related to life science, is also used as a mode of gene therapy, and has wide application prospect. To date, there are some commercial cationic liposomes, but the number of highly effective and low toxic cationic liposomes is still limited.
The main problems of the current lipofection reagents are as follows:
1. the application range is limited. Most of the existing liposome reagents cannot be transfected efficiently in many cell lines, especially primary cells such as stem cells.
2. The synthesis is complex and the price is expensive. The current mainstream liposome transfection reagent usually has a very complex structure, and the production and development costs are high, so that the price of the liposome transfection reagent is high, and the application range of the liposome transfection reagent is greatly limited.
Thus, there is a great need in the market for efficient and inexpensive lipofection reagents.
Disclosure of Invention
The technical problem to be solved is as follows: aiming at the technical problems, the invention provides the cationic amino lipid and the synthesis method and the application thereof, and the cationic amino lipid transfected cells have the characteristics of low toxicity and high efficiency, can achieve good effect in serum, and greatly simplifies the operation steps of transfection.
The technical scheme is as follows: a cationic amino lipid has a structural formula shown in formula (I),
the method for synthesizing the cationic amino lipid comprises the following steps:
adding propargylamine and dodecyl mercaptan into a tetrahydrofuran or Dichloromethane (DCM) solution according to a molar ratio of 1 (1.5-2), wherein the final solution concentration of the total amount of reactants is 30-500 mg/mL, adding a free radical photoinitiator, wherein the addition amount is 1-5% of the total mass of the reactants, introducing nitrogen to remove oxygen, and then placing under ultraviolet light for illumination for 1-3 h;
and step two, adding dimethylaminoethyl acrylate into the solution reacted in the step one, wherein the molar ratio of the dimethylaminoethyl acrylate to the propargylamine is (1-2): 1, adding an alkaline substance, wherein the molar ratio of the alkaline substance to the propargylamine is (10-50), and reacting overnight to obtain the final product, namely the cationic amino lipid.
Preferably, the radical photoinitiator in the first step is benzoin dimethyl ether.
Preferably, in the first step, the wavelength of the ultraviolet light is 250-365 nm, and the intensity is 0.5-3 mW/cm.
Preferably, the basic substance in step two is triethylamine or 1, 8-diazabicycloundec-7-ene.
The application of the cationic amino lipid in preparing lipid particles (namely liposomes).
Preferably, the lipid particle contains a bioactive agent siRNA, plasmid, miRNA or mRNA.
The application of the cationic amino lipid in preparing a cell transfection kit.
Has the advantages that: (1) the method has mild reaction conditions and high solvent selectivity, greatly reduces the difficulty of the reaction, improves the yield, has simple and cheap raw materials, and greatly reduces the generation cost;
(2) the cationic amino lipid molecule has the characteristics of high efficiency and low toxicity, can achieve good effect in serum, and greatly simplifies the operation steps of transfection.
The method of the invention is a highly generalized method, and can change the structure of the used raw materials in a large range, synthesize a large amino lipid library, and use the amino lipid library in a screening experiment. Such molecules have a long hydrophobic chain at one end and a highly hydrophilic amino group at the other end, which can form a bilayer of liposomes in water, and a positively charged amino group, which can bind to DNA and thus be used as transfection reagents for cell transfection.
Drawings
FIG. 1 is a graph showing the results of testing the transfection efficiency of four transfection reagents in example 4. In the figure, (a) is a commercially available calcium phosphate particle transfection reagent, (b) is a pioneer nanotechnology nanoparticle transfection reagent, (c) is a Lipofectamine2000 Lipofectamine from Invitrogen, and (d) is a liposome prepared in the present invention;
FIG. 2 is a graph of the effect of fluorescence microscopy on commercially available products and products of the invention of example 4, wherein (a) is a liposome of the invention, (b) is a Lipofectamine2000 product from Invitrogen, and (c) is a transfection reagent product from Pioneer nanotechnology.
Detailed Description
The invention is further illustrated by the figures and examples. The present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a process are given, but the scope of the present invention is not limited to the following embodiments.
Example 1
A cationic amino lipid has a structural formula shown in formula (I),
the method for synthesizing the cationic amino lipid comprises the following steps:
adding propargylamine and dodecyl mercaptan into a tetrahydrofuran solution according to a molar ratio of 1:1.5, wherein the final solution concentration of the total amount of reactants is 30mg/mL, adding a free radical photoinitiator, wherein the free radical photoinitiator is benzoin dimethyl ether, the adding amount of the free radical photoinitiator is 1% of the total mass of the reactants, introducing nitrogen to remove oxygen, and then, placing under ultraviolet light for illumination for 1h, wherein the wavelength of the ultraviolet light is 250nm, and the intensity is 3mW/cm to obtain a compound (II)
Adding dimethylaminoethyl acrylate into the solution reacted in the first step, wherein the molar ratio of dimethylaminoethyl acrylate to propargylamine is 1:1, and adding an alkaline substance, wherein the alkaline substance is triethylamine, and the molar ratio of the alkaline substance to propargylamine is 1: 10, overnight to give the final product cationic amino lipid, Compound (I)
Example 2
A cationic amino lipid has a structural formula shown in formula (I),
the method for synthesizing the cationic amino lipid comprises the following steps:
adding propargylamine and dodecyl mercaptan into a Dichloromethane (DCM) solution according to a molar ratio of 1:2, wherein the concentration of the solution of the total amount of final reactants is 500mg/mL, adding a free radical photoinitiator, wherein the free radical photoinitiator is benzoin dimethyl ether, the adding amount of the free radical photoinitiator is 5% of the total mass of the reactants, introducing nitrogen to remove oxygen, and then, placing the reactants under ultraviolet light for illumination for 3h, wherein the wavelength of the ultraviolet light is 365nm, and the intensity is 0.5mW/cm to obtain a compound (II)
Step two, adding dimethylaminoethyl acrylate into the solution reacted in the step one, wherein the molar ratio of the dimethylaminoethyl acrylate to the propargylamine is 2:1, adding an alkaline substance, wherein the alkaline substance is 1, 8-diazabicycloundec-7-ene, and the molar ratio of the alkaline substance to the propargylamine is 1:50, reacting overnight to obtain a final product, namely the cationic amino lipid (I)
Example 3
A cationic amino lipid has a structural formula shown in formula (I),
the synthetic method of the cationic amino lipid comprises the following steps:
step one, synthesizing 2, 3-bis (dodecyl sulfur) -propylamine (compound (II)
) Dissolving 0.5mmol propargylamine and 1mmol dodecyl mercaptan in 3mL THF (tetrahydrofuran), adding 5mg free radical photoinitiator benzoin dimethyl ether (DMPA), transferring to a 20mL glass bottle, introducing nitrogen to remove oxygen for 5min, and irradiating the glass bottle under 365nm UV light for 1h, wherein the reaction formula is as follows:
step two, adding 1mmol of dimethylaminoethyl acrylate into the glass vial reacted in the step one, stirring uniformly, adding 0.1mmol of alkaline substance 1, 8-diazabicycloundec-7-ene, and stirring overnight. Then the solution was poured into a 25mL single-neck flask and THF (tetrahydrofuran) was evaporated to give the final product cationic amino lipid, Compound (I)
The reaction formula is as follows:
this reaction is a Michael addition reaction.
Example 4
Use of the cationic amino lipids prepared based on example 3 for the preparation of transfection reagents.
The specific application process is as follows:
(1) dissolving the synthesized cationic amino lipid molecules in ethanol solution to prepare the liposome. The specific steps are that 2mg of the cationic amino lipid prepared in the embodiment 3 is added into 1ml of acetic acid-sodium acetate buffer solution with pH value of 5 to fully hydrate the cationic amino lipid molecules, and the corresponding liposome is prepared after the cationic amino lipid molecules are incubated for 40min on a rotary evaporation instrument.
(2) 50 ng of pBL-EGFP was dissolved in 100. mu.L of PBS buffer and 50 ng of the liposome prepared in step (1) was added thereto, and the mixture was incubated at room temperature for 30min to obtain a liposome plasmid mixture.
(3) HEK293 cells were trypsinized and diluted to 10 in serum-free DMEM medium5Cell suspension per mL.
(4) And (3) adding 100 microliters of the cell suspension obtained in the step (3) into a 96-well plate, and then adding 100 microliters of the liposome plasmid mixture incubated in the step (2).
(5) The 96-well plate was then placed in a cell incubator and incubated for 24 hours before the results were detected using a fluorescent alcohol-labeling instrument. The liposome in the step one is replaced by three common commercial transfection reagents, and then the steps (2) to (5) are respectively repeated, and the result is shown in figure 1, and the liposome in the invention has higher efficiency of transfecting HEK293 cells than the products which are mainstream in the market.
The liposome plasmid mixture can also be used for the transfection of human mesenchymal stem cells HBMSC. S1, digesting HBMSC with pancreatin, and diluting HBMSC with serum-free DMEM medium to 105Cell suspension per mL; s2, adding 100 microliters of cell suspension into a 96-well plate, and then adding 100 microliters of the liposome plasmid mixture prepared in the step (2); s3, placing the 96-well plate in a cell culture box, incubating for 24 hours, detecting the result by using a fluorescent alcohol labeling instrument, and observing the effect under a fluorescent microscope. The liposome in the step (1) is replaced by two common commercial transfection reagents, and then the steps S1-S3 are respectively repeated, and the specific result chart is shown in figure 2, and the effect of the liposome in the invention is better than that of the commercial product as can be seen from figure 2.
Claims (8)
1. A cationic amino lipid is characterized in that the structural formula of the cationic amino lipid is shown as a formula (I),
2. a method for synthesizing the cationic amino lipid of claim 1, comprising the steps of:
adding propargylamine and dodecyl mercaptan into a tetrahydrofuran or dichloromethane solution according to a molar ratio of 1 (1.5-2), wherein the solution concentration of the final total amount of reactants is 30-500 mg/mL, adding a free radical photoinitiator, wherein the addition amount is 1-5% of the total mass of the reactants, introducing nitrogen to remove oxygen, and then placing under ultraviolet light for illumination for 1-3 hours;
and step two, adding dimethylaminoethyl acrylate into the solution reacted in the step one, wherein the molar ratio of the dimethylaminoethyl acrylate to the propargylamine is (1-2): 1, adding an alkaline substance, wherein the molar ratio of the alkaline substance to the propargylamine is (10-50), and reacting overnight to obtain the final product, namely the cationic amino lipid.
3. The method of claim 2, wherein the radical photoinitiator in step one is benzoin dimethyl ether.
4. The method for synthesizing the cationic amino lipid according to claim 1, wherein the ultraviolet light in the first step has a wavelength of 250-365 nm and an intensity of 0.5-3 mW/cm.
5. The method of claim 2, wherein the basic substance in step two is triethylamine or 1, 8-diazabicycloundec-7-ene.
6. Use of the cationic amino lipid of claim 1 for the preparation of lipid particles.
7. The use of claim 6, wherein the lipid particle comprises a bioactive agent siRNA, plasmid, miRNA or mRNA.
8. Use of the cationic amino lipid of claim 1 for the preparation of a cell transfection kit.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1180697A (en) * | 1996-10-22 | 1998-05-06 | 弗·哈夫曼-拉罗切有限公司 | Cationic lipids for gene therapy |
| CN101674853A (en) * | 2007-05-04 | 2010-03-17 | Mdrna有限公司 | Amino acid lipids and uses thereof |
| CN102458366A (en) * | 2009-06-15 | 2012-05-16 | 阿尔尼拉姆医药品有限公司 | Lipid formulated dsrna targeting the pcsk9 gene |
| CN103596922A (en) * | 2011-06-07 | 2014-02-19 | 因塞拉有限公司 | Amino lipids, their synthesis and uses thereof |
| CN105622473B (en) * | 2016-02-06 | 2017-07-25 | 吴国球 | A kind of cation amino lipid and its preparation method and use |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1180697A (en) * | 1996-10-22 | 1998-05-06 | 弗·哈夫曼-拉罗切有限公司 | Cationic lipids for gene therapy |
| CN101674853A (en) * | 2007-05-04 | 2010-03-17 | Mdrna有限公司 | Amino acid lipids and uses thereof |
| CN102458366A (en) * | 2009-06-15 | 2012-05-16 | 阿尔尼拉姆医药品有限公司 | Lipid formulated dsrna targeting the pcsk9 gene |
| CN103596922A (en) * | 2011-06-07 | 2014-02-19 | 因塞拉有限公司 | Amino lipids, their synthesis and uses thereof |
| CN105622473B (en) * | 2016-02-06 | 2017-07-25 | 吴国球 | A kind of cation amino lipid and its preparation method and use |
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