CN115073345A - Cationic lipid molecule for mRNA delivery and preparation method and application thereof - Google Patents

Cationic lipid molecule for mRNA delivery and preparation method and application thereof Download PDF

Info

Publication number
CN115073345A
CN115073345A CN202210583271.9A CN202210583271A CN115073345A CN 115073345 A CN115073345 A CN 115073345A CN 202210583271 A CN202210583271 A CN 202210583271A CN 115073345 A CN115073345 A CN 115073345A
Authority
CN
China
Prior art keywords
mrna
amine
hours
ethyl acetate
reaction
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202210583271.9A
Other languages
Chinese (zh)
Other versions
CN115073345B (en
Inventor
燕云峰
刘晓敏
帅棋
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhejiang University of Technology ZJUT
Original Assignee
Zhejiang University of Technology ZJUT
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhejiang University of Technology ZJUT filed Critical Zhejiang University of Technology ZJUT
Priority to CN202210583271.9A priority Critical patent/CN115073345B/en
Publication of CN115073345A publication Critical patent/CN115073345A/en
Application granted granted Critical
Publication of CN115073345B publication Critical patent/CN115073345B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/08—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon radicals, substituted by hetero atoms, attached to ring carbon atoms
    • C07D207/09—Radicals substituted by nitrogen atoms, not forming part of a nitro radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0033—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being non-polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/12—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of acyclic carbon skeletons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/12—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
    • C07D295/125—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/13—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain

Abstract

The invention discloses a cationic lipid molecule for mRNA delivery, a preparation method and application thereof, wherein the preparation of the cationic lipid molecule is a product obtained by the reaction of 1, 6-hexanediol Diacrylate (DAHE) and hydrophilic amine serving as raw materialsThe intermediate is further reacted with hydrophobic amine to obtain the product, wherein the hydrophilic amine raw material is one of 2A7, 4A2, 4A3 and 4A 4;
Figure DDA0003664986730000011
the hydrophobic amine raw material is one of C12, 2C6 and 2C 8;
Figure DDA0003664986730000012
the cationic lipid molecule product structure synthesized by the invention contains a plurality of N which can be protonated into cations, so that nucleic acid with negative charges can be effectively combined, and the nucleic acid loading rate is improved; the nano-particles with high stability can be formed by carrying hydrophobic alkane chains with proper number and length, and have good application in delivering mRNA molecules.

Description

Cationic lipid molecule for mRNA delivery and preparation method and application thereof
Technical Field
The invention relates to a cationic lipid molecule for mRNA delivery, a preparation method and application thereof.
Background
Messenger RNA (mRNA) has recently become a research hotspot as a novel nucleic acid drug. The synthesized mRNA coded by the specific protein is the same as natural mRNA, and can express corresponding functional protein in cells, so that the protein has wide application prospect in the fields of immunotherapy, regenerative medicine, infectious disease vaccine, protein substitution/supplement and the like. Particularly, the nano mRNA vaccine formed by the mRNA of the receptor binding domain on the spike protein or S1 subunit of the SARS-CoV-2 virus and the lipid carrier plays an important role in the prevention and treatment of the new coronavirus pneumonia COVID-19, and fully shows the great clinical application potential of mRNA-based therapeutics.
mRNA-based therapies have many potential advantages over plasmid dna (pdna) or small interfering RNA-based therapies: 1) mRNA expresses functional proteins directly in cytoplasm without entering nucleus like pDNA, so that mRNA treatment has less obstacle of crossing nuclear membrane, and can also play a role in nondividing cells or cells with slow division, such as dendritic cells; 2) mRNA does not need to be integrated into the chromosome of the cell, so that the risk of insertional gene mutation can be avoided; 3) compared with RNA interference, the process of translating target protein by mRNA is simpler and more direct, and the off-target problem similar to siRNA is avoided. Furthermore, the high charge density, flexible mRNA is loaded more efficiently in the vector than the corresponding recombinant protein; the preparation of mRNA is relatively simple and the risk of biosafety is low.
The lack of efficient and safe delivery carriers is a major bottleneck problem for promoting the wide clinical application of mRNA drugs at present. Currently, the major gene delivery vectors are divided into viral and non-viral gene vectors. The gene delivery efficiency of viral vectors is high, but the safety concerns of viral vectors (such as inserted gene mutations, canceration or immune response) greatly reduce the possibility of their direct use in humans. The wide clinical use of mRNA requires suitable non-viral vector delivery vehicles to avoid rapid degradation of mRNA in blood, reduce non-specific protein adsorption, prevent clearing by macrophages, and help these highly hydrophilic and strongly negatively charged biological macromolecules cross cell membranes. Compared with viral vectors, non-viral vectors have the advantages of low cost, simple preparation, convenience for large-scale production, high safety, controllable chemical structure and the like, and attract extensive attention of researchers.
Cationic lipid/lipoid (lipidoid) molecules and cationic polymers are currently the major mRNA non-viral delivery vehicle materials. The synthetic cationic lipid molecule and auxiliary lipid molecule (such as DOPE), cholesterol and PEG lipid form a mixed carrier, and mRNA forms Lipid Nanoparticles (LNP), which is the fastest and most effective mRNA delivery system. Research shows that cationic lipid molecules are the hydrophilic-hydrophobic water balance, the types of key amines and the composition of lipid nanoparticles are the key factors influencing the mRNA conveying efficiency.
Disclosure of Invention
The invention aims to provide a cationic lipid molecule for mRNA delivery, a preparation method and application thereof, and discloses a high-efficiency mRNA delivery carrier material which can improve the stability of mRNA and the expression efficiency of in vivo and in vitro mRNA.
The cationic lipid molecule for mRNA delivery has a structure selected from one of the following:
Figure BDA0003664986710000031
the preparation method of the cationic lipid molecule for mRNA delivery is characterized by comprising the following steps:
1) 1, 6-hexanediol Diacrylate (DAHE) and hydrophilic amine are used as raw materials, the raw materials and the hydrophilic amine are reacted together in a solvent A in the presence of a polymerization inhibitor BHT, the reaction temperature is 80-100 ℃, the reaction time is 20-30 hours, a yellow oily substance is obtained after the reaction is finished and is used as an intermediate crude product, ethyl acetate-water extraction is carried out, the intermediate crude product is extracted to an ethyl acetate phase, and the intermediate is purified by a silica gel column chromatography;
2) taking the purified intermediate and hydrophobic amine as raw materials, reacting in a solvent B together in the presence of a polymerization inhibitor BHT at the reaction temperature of 50-60 ℃ for 80-120 hours to obtain a yellow oily substance as a final product after the reaction is finished, and finally purifying the final product by a silica gel column chromatography to obtain the finished product; the reaction formula for preparing the cationic lipid molecules of the present invention is as follows:
Figure BDA0003664986710000041
wherein, hydrophilic amine (R) 1 -NH 2 ) The structural formula of (A) is one of 2A7, 4A2, 4A3 and 4A 4:
Figure BDA0003664986710000042
hydrophobic amines
Figure BDA0003664986710000043
The structural formula of (A) is one of C12, 2C6 and 2C 8;
Figure BDA0003664986710000044
wherein, C12 represents amine with one methyl group and one C12 alkane chain. 2C6 represents an amine with two alkane chains, each chain having 6 carbons. 2C8 represents an amine with two alkane chains, each chain having 8 carbons. It can be seen that R in the hydrophobic amine C12 2 And R 3 Methyl and C12 straight chain alkyl, R in hydrophobic amine 2C6 2 And R 3 Are all C6 straight chain alkyl.
From the above structural formula, it can be seen that the cationic lipid molecule of the present invention is prepared from which hydrophilic amine and hydrophobic amine, for example, the raw materials of hydrophilic amine and hydrophobic amine prepared from cationic lipid molecule 2A7C-2C8 are 2A7 and 2C8, respectively.
The preparation method of the cationic lipid molecule for mRNA delivery is characterized in that in the step 1), the feeding molar ratio of hydrophilic amine to 1, 6-hexanediol diacrylate is 1: 4-9, the molar ratio of hydrophilic amine to a polymerization inhibitor BHT is 2.5-20: 1, and the solvent A is DMSO.
The preparation method of the cationic lipid molecule for mRNA delivery is characterized in that in the step 1), the reaction temperature is 90 ℃, the reaction time is 24 hours, and the eluent for silica gel column chromatography purification is an ethyl acetate-methanol mixed solution with a volume ratio of 20-40: 1, or a n-hexane-ethyl acetate mixed solution with a volume ratio of 0.5-1.5: 1.
The preparation method of the cationic lipid molecule for mRNA delivery is characterized in that in the step 2), the feeding molar ratio of the purified intermediate to the hydrophobic amine is 1: 4-8, the molar ratio of the hydrophobic amine to the polymerization inhibitor BHT is 80-165: 1, and the solvent B is anhydrous THF.
The preparation method of the cationic lipid molecule for mRNA delivery is characterized in that in the step 2), the reaction temperature is 55 ℃, the reaction time is 95-100 hours, and the eluent purified by silica gel column chromatography is ethyl acetate.
The cationic lipid molecules provided by the invention have good application in delivering mRNA molecules.
Compared with the prior art, the invention has the beneficial effects that:
1) the synthesized cationic lipid molecular material comprises key amine and hydrophobic units, and researches prove that the cationic lipid molecular material is a group which must be provided by an efficient mRNA (messenger ribonucleic acid) delivery carrier. In the selection of hydrophilic amine, the invention respectively adopts 2A7, 4A2, 4A3 and 4A4, and effectively regulates the hydrophilicity, cation density and hydrophobic chain number of the material. In the aspect of hydrophobic amine, the hydrophobic amine C12, the hydrophobic amine 2C6 and the hydrophobic amine 2C8 are respectively adopted in the invention to effectively regulate the hydrophobicity of the material.
2) The cationic lipid molecule product structure synthesized by the invention contains a plurality of N which can be protonated into cations, so that nucleic acid with negative charges can be effectively combined, and the nucleic acid loading rate is improved; the nano-particle with high stability can be formed by the hydrophobic alkane chains with proper number and length.
Drawings
FIG. 1 shows 2A7C-2C8 1 H-NMR spectrum.
FIG. 2 shows 4A2C-C12 1 H-NMR spectrum.
FIG. 3 shows 4A2C-2C8 1 H-NMR spectrum.
FIG. 4 shows 4A3C-2C6 1 H-NMR spectrum.
FIG. 5 shows 4A4C-2C8 1 H-NMR spectrum.
Figure 6 shows the results of in vitro mRNA delivery efficiency testing of different delivery vehicles.
FIG. 7 shows luciferase expression in mice after tail vein injection of 2A7C-2C8 with mRNA lipid nanoparticles 6 hours (A) and 24 hours (B).
FIG. 8 shows luciferase expression in mice after tail vein injection of 4A2C-C12 with mRNA lipid nanoparticles 6 hours (A) and 24 hours (B).
FIG. 9 shows luciferase expression in mice after tail vein injection of 4A2C-2C8 with mRNA lipid nanoparticles 6 hours (A) and 24 hours (B).
FIG. 10 shows luciferase expression in mice after tail vein injection of 4A3C-2C6 with mRNA lipid nanoparticles 6 hours (A) and 24 hours (B).
FIG. 11 shows luciferase expression in mice after tail vein injection of 4A4C-2C8 with mRNA lipid nanoparticles 6 hours (A) and 24 hours (B).
FIG. 12 shows luciferase expression in mice after 6 hours (A) and 24 hours (B) tail vein injection of naked mRNA.
FIG. 13 shows luciferase expression in each organ of the mouse 24 hours later.
FIG. 14 shows the quantitative values of chemiluminescence intensity in each organ.
Detailed Description
The invention is further illustrated with reference to the following specific examples, without limiting the scope of the invention thereto.
Example 1: synthesis of 2A7C-2C8
(1) A50 mL round-bottomed flask was charged with 2A7(1.2690g,9.8971mmol), DAHE (9.0771g,40.1662mmol), BHT (0.4420g,2.0058mmol) as a polymerization inhibitor, DMSO (10mL), and dissolved with stirring at 90 ℃ to react for 24 hours. After the reaction was completed, the crude product was obtained as a yellow oil, which was extracted with ethyl acetate-water, and the product was dissolved in ethyl acetate. Concentrated under heating and the concentrated residue purified by column chromatography over silica gel using the eluent ethyl acetate methanol 30:1(v/v) to give the product as a yellow oily intermediate, designated 2 A7C.
(2) Purified 2A7C (1.2323g,2.1219mmol), 2C8(2.0861g,8.6395mmol), BHT (0.0234g,0.1062mmol) obtained in step (1), anhydrous THF (10mL) and reacted at 55 ℃ for 96 hours were added to a brown sample bottle. After the reaction was complete, a yellow oil was obtained, which was concentrated by heating and the concentrated residue was purified by column chromatography on silica gel eluting with ethyl acetate as eluent to remove the pure product as 2A7-2C 8. 2A7C-2C8 1 The H-NMR spectrum is shown in figure 1, and according to the spectrum characterization result of figure 1, the delta-2.8 ppm is-CH at the connecting part of 2A7 and DAHE according to the reaction of the intermediate 2A7C 2 The peak area of the hydrogen atom is 4 hydrogen atoms. Delta 2.75ppm is the CH at the junction after reaction of 2A7C with hydrophobic amine 2C8 2 And 4 hydrogen atoms with peak area.
Example 2: synthesis of 4A2C-C12
(1) A50 mL round-bottomed flask was charged with 4A2(1.3185g,9.2689mmol), DAHE (16.8283g,74.372mmol), BHT (0.8321g,3.7763mmol) as a polymerization inhibitor, DMSO (20mL), and dissolved with stirring at 90 ℃ to react for 24 hours. After the reaction was completed, the crude product was obtained as a yellow oil, which was extracted with ethyl acetate-water, and the product was dissolved in ethyl acetate. The mixture was concentrated under heating and the concentrated residue was purified by silica gel column chromatography using the eluent n-hexane and ethyl acetate 1:1.5(v/v) to purify the product as a yellow oily intermediate, note 4 A2C.
(2) Purified 4A2C (1.1298g,1.0757mmol), C12(1.7235g,8.6443mmol), BHT (0.1185g,0.0538mmol), anhydrous THF (7mL) obtained in step (1) was charged into a brown sample bottle, and the mixture was subjected to reverse reaction at 55 deg.CIt should be 96 hours. After the reaction, a yellow oily substance was obtained, which was concentrated by heating, and the concentrated residue was purified by silica gel column chromatography. The pure product was eluted with ethyl acetate as eluent, 4A 2C-C12. 4A2C-C12 1 The H-NMR spectrum is shown in figure 2, and according to the spectrum characterization result of figure 2, the delta-2.8 ppm is-CH at the connecting part of the intermediate 4A2C after the reaction of 4A2 and DAHE 2 The peak area of the hydrogen atom is 4 hydrogen atoms. Delta 2.75ppm is the CH at the junction after reaction of 4A2C with hydrophobic amine 2C8 2 The peak area of the upper hydrogen atom is 4 hydrogen.
Example 3: synthesis of 4A2C-2C8
(1) A50 mL round-bottomed flask was charged with 4A2(1.3185g,9.2689mmol), DAHE (16.8283g,74.372mmol), BHT (0.8321g,3.7763mmol) as a polymerization inhibitor, DMSO (20mL), and dissolved with stirring at 90 ℃ to react for 24 hours. After the reaction was completed, the crude product was obtained as yellow oil, which was extracted with ethyl acetate-water, and the product was dissolved in ethyl acetate. Heating for concentration, and purifying the concentrated residue by silica gel column chromatography. The product was purified with the eluent ethyl acetate 1:1.5(v/v) as a yellow oil, 4 A2C.
(2) To a brown sample bottle, purified 4A2C (2.8333g,2.6975mmol), 2C8(5.2896g,21.9067mmol), BHT (0.0297g,0.1349mmol) obtained in (1), dry THF (7mL) was added and reacted at 55 ℃ for 96 hours. After the reaction, a yellow oily substance was obtained, which was concentrated by heating, and the concentrated residue was purified by silica gel column chromatography. The pure product was eluted with ethyl acetate as eluent, 4A2C-2C 8. 4A2C-2C8 1 The H-NMR spectrum is shown in FIG. 3, and according to the spectrum characterization result of FIG. 3, the delta-2.8 ppm is-CH at the connecting part of the intermediate 4A2C after the reaction of 4A2 and DAHE 2 The peak area of the hydrogen atom is 4 hydrogen atoms. Delta 2.75ppm is the CH of the linker after reaction of 4A2C with hydrophobic amine C12 2 And 4 hydrogen atoms with peak area.
Example 4: synthesis of 4A3C-2C6
(1) A50 mL round-bottomed flask was charged with 4A3(1.3220g,8.9198mmol), DAHE (17.2915g,76.4197mmol), BHT (0.1021g,0.4633mmol), DMSO (10mL), dissolved with stirring at 90 ℃ and reacted for 24 hours. After the reaction was completed, the crude product was obtained as a yellow oil, which was extracted with ethyl acetate-water, and the product was dissolved in ethyl acetate. Heating for concentration, and purifying the concentrated residue by silica gel column chromatography. The product was purified with the eluent n-hexane ethyl acetate 2:1(v/v) as a yellow oil, 4 A3C.
(2) Purified 4A3C (1.0755g,1.0211mmol), 2C6(1.5123g,8.1587mmol), BHT (0.0110g,0.0499mmol) obtained in (1), dry THF (10mL) was charged into a brown sample bottle and reacted at 55 ℃ for 96 hours. After the reaction, a yellow oily substance was obtained, which was concentrated by heating, and the concentrated residue was purified by silica gel column chromatography. The pure product was eluted with ethyl acetate as eluent, 4A3C-2C 6. 4A3C-2C6 1 The H-NMR spectrum is shown in FIG. 4, and according to the spectrum characterization result of FIG. 4, the delta-2.8 ppm is-CH at the connecting part of the intermediate 4A3C after the reaction of 4A2 and DAHE 2 The peak area of the hydrogen atom is 4 hydrogen atoms. Delta-2.75 ppm is CH at the junction after reaction of 4A3C with hydrophobic amine 2C6 2 The peak area of the upper hydrogen atom is 4 hydrogen.
Example 5: synthesis of 4A4C-2C8
(1) A50 mL round-bottomed flask was charged with 4A4(1.3220g,8.9198mmol), DAHE (17.2915g,76.4197mmol), BHT (0.1021g,0.4633mmol) as a polymerization inhibitor, DMSO (10mL), and dissolved with stirring at 90 ℃ to react for 24 hours. After the reaction was completed, the crude product was obtained as a yellow oil, which was extracted with ethyl acetate-water, and the product was dissolved in ethyl acetate. Heating for concentration, and purifying the concentrated residue by silica gel column chromatography. The product was purified with the eluent n-hexane ethyl acetate 2:1(v/v) as a yellow oil, 4 A4C.
(2) Purified 4A4C (1.8693g,1.6910mmol), 2C8(3.2665g,13.5283mmol), BHT (0.0186g,0.0846mmol) obtained in (1), dry THF (8mL) was charged into a brown sample bottle and reacted at 55 ℃ for 96 hours. After the reaction, a yellow oily substance was obtained, which was concentrated by heating, and the concentrated residue was purified by silica gel column chromatography. The pure product was eluted with ethyl acetate as eluent, 4A4C-2C 8. 4A4C-2C8 1 The H-NMR spectrum is shown in FIG. 5, and according to the spectrum characterization result of FIG. 5, the delta-2.8 ppm is-CH at the connecting part of the intermediate 4A4C after the reaction of 4A4 and DAHE 2 The peak area of the hydrogen atom is 4 hydrogen atoms. Delta 2.75ppm is the CH at the junction after reaction of 4A4C with hydrophobic amine 2C8 2 Upper hydrogen atom ofThe peak area was 4 hydrogens.
1. The structure of the cationic lipid is confirmed by nuclear magnetic spectrum, and nuclear magnetic spectrograms are shown in figures 1-5.
2. In vitro mRNA delivery efficiency
The delivery vehicle was mixed with DOPE, cholesterol, PEG2000-DMG in a molar ratio of 38.5:30:30:1.5 and dissolved in anhydrous ethanol. Luciferase mrna (fluc mrna) was dissolved in sodium citrate solution (10mM, pH 4.0). The ethanol solution and the sodium citrate solution (10mM, pH 4.0) were mixed at a ratio of 1:3, the mass ratio of the delivery vehicle to luciferase mRNA (fluc mRNA) was about 20:1, and after incubation, the mixture was diluted 5-fold with PBS to obtain a composite nanoparticle solution (containing 1.67mg of luciferase mRNA per liter and 33.4mg of delivery vehicle). After IGrov1 cells were cultured in DMEM medium to an adherent state, the complex plasmid solution prepared above (added in an amount of 75. mu.L/mL of the cell solution) was added, and transfection was performed under these conditions for 48 hours. In vitro mRNA delivery efficiency was tested.
By the above procedures, lipid molecules 2A7C-2C8, 4A2C-C12, 4A2C-2C8, 4A3C-2C6, 4A4C-2C8 synthesized in examples 1-5 of the present invention or commercial positive control reagent RNAiMax were used as delivery vehicles, the delivery vehicles were mixed with helper lipids and mRNA encoding luciferase (luciferase) to prepare nanoparticles, the nanoparticles were added to IGrov1 cells, and after 48 hours of transfection, the in vitro delivery efficiency of mRNA was evaluated by the level of luciferase expression in the cells, and the results of the in vitro mRNA delivery efficiency test for different delivery vehicles are shown in fig. 6. FIG. 6 shows that the mRNA delivery efficiency of the cationic lipid carriers synthesized in examples 1-5 of the present invention is 25-80 times higher than that of the commercial positive control reagent RNAiMax.
3. In vivo mRNA delivery Effect test
Experimental group of lipid molecules: mixing lipid molecules with DOPE, cholesterol and PEG2000-DMG at a molar ratio of 38.5:30:30:1.5, and dissolving in anhydrous ethanol. Luciferase mrna (fluc mrna) was dissolved in sodium citrate solution (10mM, pH 4.0). The ethanol solution and the sodium citrate solution (10mM, pH 4.0) were mixed at a ratio of 1:3, the mass ratio of lipid molecules to luciferase mRNA (fluc mRNA) was about 20:1, and after incubation, the mixture was diluted 5-fold with PBS to obtain a composite nanoparticle solution (containing 1.67mg of luciferase mRNA per liter and 33.4mg of lipid molecules).
Naked mRNA group: luciferase mRNA (fluc mRNA) was dissolved in a sodium citrate solution (10mM, pH 4.0), incubated, and then diluted with PBS to obtain an mRNA nanoparticle solution (containing 1.67mg of luciferase mRNA per liter of nanoparticle solution).
The nanoparticle solution prepared by the lipid molecule experimental group or the naked mRNA group is adopted, female BALB/c mice of 6 weeks old are selected, the weight of the mice is about 20g, the injection volume of each mouse is 0.2mL of the composite nanoparticle solution, and the in-vivo mRNA delivery effect is tested under the condition. Injecting a compound nanoparticle solution formed by corresponding lipid molecules and mRNA or an mRNA nanoparticle solution into a mouse body through a tail vein, and detecting the expression efficiency of luciferase in the mouse body by using a small animal imager after 6 hours and 24 hours of administration.
According to the above process, when 2A7C-2C8, 4A2C-C12, 4A2C-2C8, 4A3C-2C6 and 4A4C-2C8 were used as lipid molecules, the results of luciferase expression in mice were shown in FIGS. 7-11, respectively. From fig. 7-11, it can be seen that after injecting the composite nanoparticle solution formed by 5 lipid molecules and mRNA for 6 hours, the mice have stronger fluorescence expression in vivo, wherein 4 lipid nanoparticles still have luciferase expression 24 hours after injection. The results show that after the compound nanoparticle solution formed by lipid molecules and mRNA is injected, the luciferase expression in mice is stronger, and the related lipid carrier has excellent mRNA in-vivo delivery effect.
According to the above process, when the injected agent is mRNA nanoparticle solution, the measured luciferase expression results in the mouse are shown in fig. 12. As can be seen in FIG. 12, there was no luciferase expression after intravenous injection of naked mRNA from the tail of the mouse.
Comparison shows that the lipid molecules synthesized in the examples 1 to 5 of the invention, namely 2A7C-2C8, 4A2C-C12, 4A2C-2C8, 4A3C-2C6 and 4A4C-2C8, have good in vivo mRNA delivery efficiency.
Organ distribution of mRNA lipid nanoparticles
In vivo mRNA delivery Effect of the lipid molecule experiment group and the naked mRNA group was measured 24 hours later, organs were dissected and imaged (dissected organs were liver lever, spleen, lung, heart and kidney, respectively), the expression result of luciferase in each organ of the mouse was shown in FIG. 13, and the results of the quantitative value of chemiluminescence intensity in each organ were shown in FIG. 14. According to the results of FIGS. 13 to 14, the lipid molecules 2A7C-2C8, 4A2C-C12, 4A2C-2C8, 4A3C-2C6 and 4A4C-2C8 synthesized in examples 1 to 5 of the present invention can deliver mRNA to the liver to express a large amount of luciferase, and individual vector-delivered mRNA is also expressed in the spleen.
The statements in this specification merely set forth a list of implementations of the inventive concept and the scope of the present invention should not be construed as limited to the particular forms set forth in the examples.

Claims (7)

1. A cationic lipid molecule for mRNA delivery, the structure of which is selected from one of the following:
(I)
Figure FDA0003664986700000011
(II)
Figure FDA0003664986700000012
(III)
Figure FDA0003664986700000013
(IV)
Figure FDA0003664986700000014
(V)
Figure FDA0003664986700000015
2. the method of claim 1, comprising the steps of:
1) taking 1, 6-hexanediol diacrylate and hydrophilic amine as raw materials, reacting in a solvent A together in the presence of a polymerization inhibitor BHT at the temperature of 80-100 ℃ for 20-30 hours to obtain a yellow oily substance as an intermediate crude product after the reaction is finished, extracting with ethyl acetate-water to obtain an intermediate crude product, extracting the intermediate crude product to an ethyl acetate phase, and purifying the intermediate by silica gel column chromatography; wherein the hydrophilic amine raw material is one of 2A7, 4A2, 4A3 and 4A 4;
2A7
Figure FDA0003664986700000021
4A2
Figure FDA0003664986700000022
4A3
Figure FDA0003664986700000023
4A4
Figure FDA0003664986700000024
2) taking the purified intermediate and hydrophobic amine as raw materials, reacting in a solvent B together in the presence of a polymerization inhibitor BHT at the reaction temperature of 50-60 ℃ for 80-120 hours to obtain a yellow oily substance as a final product after the reaction is finished, and finally purifying the final product by a silica gel column chromatography to obtain the finished product; wherein the hydrophobic amine raw material is one of C12, 2C6 and 2C 8;
C12
Figure FDA0003664986700000025
2C6
Figure FDA0003664986700000026
2C8
Figure FDA0003664986700000027
3. the method of claim 2, wherein in step 1), the feeding molar ratio of the hydrophilic amine to the 1, 6-hexanediol diacrylate is 1: 4-9, the molar ratio of the hydrophilic amine to the polymerization inhibitor BHT is 2.5-20: 1, and the solvent A is DMSO.
4. The method of claim 2, wherein in the step 1), the reaction temperature is 90 ℃, the reaction time is 24 hours, and the eluent purified by silica gel column chromatography is an ethyl acetate-methanol mixture at a volume ratio of 20-40: 1, or an n-hexane-ethyl acetate mixture at a volume ratio of 0.5-1.5: 1.
5. The method of claim 2, wherein in step 2), the molar ratio of the purified intermediate to the hydrophobic amine is 1: 4-8, the molar ratio of the hydrophobic amine to the polymerization inhibitor BHT is 80-165: 1, and the solvent B is anhydrous THF.
6. The method of claim 2, wherein the reaction temperature is 55 ℃ and the reaction time is 95-100 hours, and the eluent purified by silica gel column chromatography is ethyl acetate in step 2).
7. Use of the cationic lipid molecule for mRNA delivery of claim 1 for delivering mRNA molecules.
CN202210583271.9A 2022-05-26 2022-05-26 Cationic lipid molecule for mRNA delivery and preparation method and application thereof Active CN115073345B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210583271.9A CN115073345B (en) 2022-05-26 2022-05-26 Cationic lipid molecule for mRNA delivery and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210583271.9A CN115073345B (en) 2022-05-26 2022-05-26 Cationic lipid molecule for mRNA delivery and preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN115073345A true CN115073345A (en) 2022-09-20
CN115073345B CN115073345B (en) 2023-12-05

Family

ID=83250271

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210583271.9A Active CN115073345B (en) 2022-05-26 2022-05-26 Cationic lipid molecule for mRNA delivery and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN115073345B (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014066811A1 (en) * 2012-10-25 2014-05-01 The Johns Hopkins University Bioreducible poly (b-amino ester)s for sirna delivery
CN113387826A (en) * 2021-05-20 2021-09-14 广东工业大学 Water-based ultraviolet curing resin and preparation method and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014066811A1 (en) * 2012-10-25 2014-05-01 The Johns Hopkins University Bioreducible poly (b-amino ester)s for sirna delivery
CN113387826A (en) * 2021-05-20 2021-09-14 广东工业大学 Water-based ultraviolet curing resin and preparation method and application thereof

Also Published As

Publication number Publication date
CN115073345B (en) 2023-12-05

Similar Documents

Publication Publication Date Title
JP6807900B2 (en) Amine-containing transfection reagents and methods for producing and using them
CN113993839B (en) Ionizable lipid molecule, preparation method thereof and application thereof in preparation of lipid nanoparticles
CN112961065B (en) Ionizable lipid molecule, preparation method thereof and application thereof in preparation of lipid nanoparticles
RU2599449C1 (en) New oligonucleotides conjugates and use thereof
US10030243B2 (en) Nanoparticle type oligonucleotide structure having high efficiency and method for preparing same
CN114904003B (en) Use of ionizable cationic lipid analog materials as nucleic acid drug delivery vehicles or transfection reagents
CN115073345B (en) Cationic lipid molecule for mRNA delivery and preparation method and application thereof
CN110403915B (en) DNA and polymer hybrid nucleic acid drug carrier, and preparation method and application thereof
CN116354836A (en) Cationic lipid compound, preparation method and application thereof and mRNA delivery system
EP4309674A1 (en) Ionizable cationic lipid analogue material and use thereof as drug delivery carrier
CN113292616B (en) Monosaccharide ligand functionalized cationic lipid compound and preparation method and application thereof
JP4827358B2 (en) Method for producing functional polyalkyleneimine, composition containing said compound and use thereof
CN117257965B (en) Nucleic acid delivery carrier composition and application thereof
CN111053765A (en) Medicine containing paclitaxel, preparation method thereof, pharmaceutical composition and application
CN117466777B (en) Cationic lipid compound, preparation method and application thereof and mRNA delivery system
CN117466768B (en) Cationic lipid compound, preparation method and application thereof and mRNA delivery system
CN117263818A (en) Cationic lipid compound, and preparation method and application thereof
KR20130068032A (en) 'stable antisense oligonucleotide conjugate and its manufacturing method'
CN114773418B (en) Nucleic acid aptamer cluster modified nano structure and preparation method and application thereof
JP7305158B2 (en) Modified polyethyleneimine and method for producing the same
CN115429891A (en) Application of fluorine-containing polymer in intracellular delivery of mRNA
CN114349811A (en) Cationic cholesterol derivative, nano-composite, preparation method and application thereof
CN117886711A (en) Cationic lipid compound, preparation method and application thereof, and LNP composition
CN110960534A (en) Medicine containing pentafluorouracil, preparation method thereof, pharmaceutical composition and application thereof
CN117843603A (en) Ionizable lipid material and application thereof in preparation of nucleic acid delivery carrier

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant