CN116730857A - Cationic lipid compound, and composition and application thereof - Google Patents

Cationic lipid compound, and composition and application thereof Download PDF

Info

Publication number
CN116730857A
CN116730857A CN202310623164.9A CN202310623164A CN116730857A CN 116730857 A CN116730857 A CN 116730857A CN 202310623164 A CN202310623164 A CN 202310623164A CN 116730857 A CN116730857 A CN 116730857A
Authority
CN
China
Prior art keywords
groups
lipid
alkyl
cationic lipid
compound
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.)
Pending
Application number
CN202310623164.9A
Other languages
Chinese (zh)
Inventor
刘帅
苏可欣
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 ZJU
Original Assignee
Zhejiang University ZJU
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 ZJU filed Critical Zhejiang University ZJU
Priority to CN202310623164.9A priority Critical patent/CN116730857A/en
Publication of CN116730857A publication Critical patent/CN116730857A/en
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C227/04—Formation of amino groups in compounds containing carboxyl groups
    • C07C227/06—Formation of amino groups in compounds containing carboxyl groups by addition or substitution reactions, without increasing the number of carbon atoms in the carbon skeleton of the acid
    • C07C227/08—Formation of amino groups in compounds containing carboxyl groups by addition or substitution reactions, without increasing the number of carbon atoms in the carbon skeleton of the acid by reaction of ammonia or amines with acids containing functional groups
    • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Optics & Photonics (AREA)
  • Biomedical Technology (AREA)
  • Nanotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention discloses a cationic lipid compound, a composition and application thereof, and relates to the technical field of biological medicines, wherein the cationic lipid compound has the technical key points that: the compound of the invention is a cationic lipid compound, the center is an acrylic monomer containing a degradable group, and the number and the length of peripheral hydrophobic chains are controllable. The preparation method of the cationic lipid compound has the advantages of easily available raw materials, mild reaction conditions, high reaction yield, good reaction selectivity, simple and easy operation and low requirements on instruments and equipment. The cationic lipid compound provided by the invention is screened through the characteristics of the size and the potential of lipid nano particles, in-vitro cell transfection efficiency, cytotoxicity, in-vivo delivery efficiency, lipid proportion regulation in a lipid nano particle composition and the like, and the effectiveness and the safety of nucleic acid delivery are obviously improved. The lipid nanoparticle composition can selectively deliver mRNA to different organs, and has good in vivo targeting.

Description

Cationic lipid compound, and composition and application thereof
Technical Field
The invention relates to the technical field of biological medicines, in particular to a cationic lipid compound, a composition and application thereof.
Background
Nucleic acid drug-based therapies have proven to have good application potential in a variety of fields, including vaccines against SARS-Cov-2 virus, protein replacement therapies, gene editing, cancer immunotherapy, and the like. However, the development and use of nucleic acid therapeutic drugs still face a number of challenges, including the susceptibility of nucleic acid macromolecules to nuclease degradation in an in vivo environment, the difficulty of spontaneous entry of nucleic acids into cells, the lack of efficient and safe nucleic acid delivery vehicles, and the like. Lipid nanoparticles (Lipid nanoparticles, LNP) as a non-viral gene vector provide a reliable solution for delivery of nucleic acid drugs, a type of nucleic acid vector that is currently more successful in clinical transformation, have been successfully applied to mRNA novel crown vaccines (mRNA-1273, BNT162b2, etc.) and siRNA drugs (Onpattro).
LNP nucleic acid delivery systems are typically composed of four components, cationic lipids, phospholipids, cholesterol, and pegylated lipids, where cationic lipids directly determine the delivery efficiency, safety, targeting, etc. properties of nucleic acid drugs, which are central keys for the development of LNP delivery technologies. In order to cope with different application situations, such as different molecular weights of used nucleic acid molecules and different organs/tissues/cells to be delivered, the required LNP delivery vectors have large difference in lipid structure, so that the development of cationic lipid compounds with different chemical structures and compositions thereof for realizing safe, effective and precise nucleic acid drug delivery has important significance.
Currently, the development of novel LNP nucleic acid delivery systems is critical to nucleic acid drug development, while cationic lipids are the core of LNP systems, and the design of novel cationic lipid structures is a major concern in the art. In the prior art, cationic lipid is designed and synthesized from small molecular amine, and is prepared by connecting hydrophobic alkyl chains through a connecting group, and targeted nucleic acid delivery is difficult to achieve finally.
Accordingly, in order to obtain safer, more efficient, targeted cationic lipids and LNP nucleic acid delivery systems, the present invention aims to provide a cationic lipid compound and compositions and uses thereof, to solve the above-mentioned problems, by preparing novel cationic lipids by linking alkylamines with small molecules comprising degradable ester groups as the center; and then, the high-efficiency, safe and targeted mRNA delivery is realized by regulating and controlling the four-component proportion of the LNP.
Disclosure of Invention
The invention aims to solve the problems and provide a cationic lipid compound, a composition and application thereof aiming at the defects of the existing nucleic acid vector, enriches the types of the existing cationic lipid compound, and can select lipid compounds with specific structures as the nucleic acid vector according to the types of nucleic acid drugs and organs/tissues/cells needing to be enriched.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
the invention provides a cationic lipid compound shown in a general formula (I), or stereoisomers, tautomers, pharmaceutically acceptable salts, prodrugs or solvates thereof,
wherein R is 1 Selected from the following structures: all groups or C in the acrylate monomers other than acrylate groups 1 -C 26 Alkyl or C 1 -C 26 Alkenyl, one or more C of said alkyl or alkenyl being optionally substituted with O or N, said alkyl or alkenyl structural substituent being C 1 -C 18 One or more of alkyl, ester, carbonate, carbonyl, amide, ether, amine, hydrocarbon, alkene, carboxyl, acyl, and alkoxy, wherein C 1 -C 18 Alkyl is unsubstituted or optionally substituted with one or more of the following substituents: ester groups, carbonate groups, carbonyl groups, amide groups, ether groups, amine groups, hydrocarbon groups, olefin groups, carboxyl groups, acyl groups, alkoxy groups;
R 2 one or more selected from the following structures: H. methyl, saturated alkyl chains, unsaturated alkyl chains;
L 1 one or more selected from the following structures: -O-, -S-, -OC (=o) -, -C (=o) O-, -OC (=o) O-, -C (=o) NH-, -NHC (=o) -, -C (=s) O-, -OC (=s) O-, -C (=o) S-, -SC (=o) -, -OC (=o) S-, -C (=s) S-, -SC (=s) O-;
L 2 an alkyl or alkenyl structure comprising 1 to 18 carbon atoms that is linear or branched, saturated or unsaturated, said alkyl or alkenyl structure optionally substituted with one or more of the following substituents: c (C) 1 -C 6 Alkyl, ester, carbonate, carbonyl, amide, ether, amine, hydrocarbon, olefin, carboxyl, acyl, and alkoxy groups;
y is an integer from 0 to 6.
Further, the cationic lipid compound is one or more of the compounds shown in the following structures:
further, in formula (II): r is R 1 Selected from the following structures: all groups or C in the acrylate monomers other than acrylate groups 1 -C 26 Alkyl or C 1 -C 26 Alkenyl, one or more C of said alkyl or alkenyl being optionally substituted with O or N, the substituent of said alkyl or alkenyl structure being C 1 -C 18 One or more of alkyl, ester, carbonate, carbonyl, amide, ether, amine, hydrocarbon, alkene, carboxyl, acyl, and alkoxy, wherein C 1 -C 18 Alkyl is unsubstituted or optionally substituted with one or more of the following substituents: ester groups, carbonate groups, carbonyl groups, amide groups, ether groups, amine groups, hydrocarbon groups, olefin groups, carboxyl groups, acyl groups, alkoxy groups;
R 2 selected from the group consisting ofOne or more of the following structures: H. methyl, saturated alkyl chains, unsaturated alkyl chains;
n' is an integer from 0 to 16.
The present invention also provides a composition comprising a therapeutic or prophylactic agent and a carrier for delivering the therapeutic or prophylactic agent, the carrier comprising a cationic lipid compound comprising one or more of the general formula (I) or (II) or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt, prodrug or solvate thereof as claimed in any one of claims 1 to 3.
Further, the use of the cationic lipid with one or more auxiliary components forms a lipid nanoparticle system for delivery of therapeutic or prophylactic agents.
Further, the adjunct ingredients include sterols, charged lipids, neutral lipids, and pegylated lipids.
Further, the sterols include any one or more of cholesterol, fecal sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, lycorine, ursolic acid, alpha-tocopherol.
Further, the sterol is cholesterol.
Further, the charged lipid is any lipid molecule that exists in a positively or negatively charged form at a selected pH or range that corresponds to the pH conditions of the intended use environment of the lipid, e.g., physiological pH and endosomal lysosomal pH 4.5 to 6.5.
Further, the neutral lipids include one or more of phospholipids, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramides, sterols, and derivatives thereof.
Further, the neutral lipid is a phospholipid.
Further, the pegylated lipid is 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000.
Further, the therapeutic or prophylactic agent includes one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein.
Further, the mass ratio of the carrier to the therapeutic or prophylactic agent is 5:1 to 50:1.
Further, the nucleic acid molecule is a linear mRNA or a circular mRNA.
Further, the nucleic acid molecule is RNA, mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomir, a microrna inhibitor, a microrna activator or shRNA, DNA, an antisense nucleic acid, an aptamer, a ribozyme, an immunostimulatory nucleic acid, or PNA.
The invention also provides a lipid nanoparticle composition, which consists of 14.5 to 37.9 percent of cationic lipid compound, 14.9 to 37.9 percent of phospholipid, 22.1 to 44.5 percent of cholesterol and 1.5 to 8.2 percent of pegylated lipid
Further, the composition is composed of a cationic lipid compound, a phospholipid, cholesterol and a pegylated lipid, wherein the molar ratio of the cationic lipid compound to the phospholipid to the cholesterol to the pegylated lipid is: 15/20/25/2 or 10/10/15/1 or 10/15/20/2 or 10/20/25/3 or 10/25/30/4 or 15/10/20/3 or 15/15/15/4 or 15/20/30/1 or 15/25/25/2 or 20/10/25/4 or 20/15/30/3 or 20/20/15/2 or 20/25/20/1 or 25/10/30/2 or 25/15/25/1 or 25/20/4 or 25/25/15/3.
The invention also provides the application of the cationic lipid compound shown in the general formula (I) or a stereoisomer, a tautomer, a pharmaceutically acceptable salt, a prodrug or a solvate thereof, or the composition or the lipid nanoparticle composition in the preparation of drugs capable of delivering nucleic acid drugs to different organs or positions.
Further, the different organs or locations include spleen, liver and muscle.
The invention solves the technical problems with the following difficulties and significance:
to meet the demand for nucleic acid therapies, LNP delivery systems are suitable for delivery of a variety of nucleic acid molecules (mRNA, siRNA, sgRNA, microRNA, etc.), exhibit excellent in vivo RNA delivery properties and clinical transformation potential, and have been successfully applied to mRNA novel crown vaccines (BNT 162b2 and mRNA-1273, etc.) and siRNA drugs (onsattro). LNP nucleic acid delivery systems are typically composed of four components, cationic lipids, phospholipids, cholesterol, pegylated lipids. Among them, ionizable cationic lipids are key components of LNP, and many cationic lipids such as DLin-MC3-DMA (Onpattro core component), ALC-0315 (BNT 162b2 core component), SM-102 (mRNA-1273 core component) and the like are designed by many scientists at home and abroad in the aspect of LNP delivery system. To develop accurate nucleic acid drugs, there is still a need for safer, efficient, and accurate LNP nucleic acid delivery systems, the key to achieve this goal is to develop new cationic lipid chemical structures (new chemical structures) and optimize LNP multicomponent ratios (new LNP formulations).
Compared with the prior art, the beneficial effect of this scheme:
1. the compound provided by the scheme of the invention is a cationic lipid compound, the center of the cationic lipid compound is an acrylic monomer containing a degradable group, and the number and the length of peripheral hydrophobic chains are controllable;
2. the preparation method of the cationic lipid compound has the advantages of easily available raw materials, mild reaction conditions, high reaction yield, good reaction selectivity, simple and easy operation and low requirements on instruments and equipment;
3. the cationic lipid compound of the scheme of the invention is screened through the characteristics of the size of lipid nano particles, the potential of the lipid nano particles, the in vitro cell transfection efficiency, cytotoxicity, in vivo delivery efficiency, lipid proportion regulation in a lipid nano particle composition and the like, so that the effectiveness and the safety of nucleic acid delivery are obviously improved;
4. the lipid nanoparticle composition of the present invention can selectively deliver mRNA to different organs, and has good in vivo targeting.
Drawings
FIG. 1 is the design and preparation of cationic lipids in example 1 of the present invention;
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of cationic lipid 3Ac1-C18 in example 2 of the present invention;
FIG. 3 is a nuclear magnetic resonance hydrogen spectrum of cationic lipid 3Ac1-C212 in example 3 of the present invention;
FIG. 4 is a nuclear magnetic resonance hydrogen spectrum of cationic lipid 5Ac1-C12 in example 4 of the present invention;
FIG. 5 is a nuclear magnetic resonance hydrogen spectrum of 5 cationic lipids 6Ac1-C12 in an example of the present invention;
FIG. 6 is a nuclear magnetic resonance hydrogen spectrum of 6 cationic lipid 6Ac1-C210 in an example of the present invention;
FIG. 7 is a particle size of a representative mRNA lipid nanoparticle composition of example 8 of the present invention;
FIG. 8 is Zeta potential of representative mRNA lipid nanoparticle compositions of example 8 of the present invention;
FIG. 9 is a graph showing the cell viability of the mRNA lipid nanoparticle composition (1 Ac 1-C6-1 Ac 5-C212) of example 9 of the present invention;
FIG. 10 is a graph showing the cell viability of the mRNA lipid nanoparticle composition (2 Ac 1-C6-3 Ac 2-C212) of example 9 of the present invention;
FIG. 11 is a graph showing the cell viability of the mRNA lipid nanoparticle composition (4 Ac 1-C6-6 Ac 1-C212) of example 9 of the present invention;
FIG. 12 is the mRNA delivery efficiency of the mRNA lipid nanoparticle composition (1 Ac 1-C6-1 Ac 5-C212) of example 10 of the present invention;
FIG. 13 is the mRNA delivery efficiency of the mRNA lipid nanoparticle composition (2 Ac 1-C6-3 Ac 2-C212) of example 10 of the present invention;
FIG. 14 is the mRNA delivery efficiency of the mRNA lipid nanoparticle composition (4 Ac 1-C6-6 Ac 1-C212) of example 10 of the present invention;
FIG. 15 is an experimental design optimization of lipid ratios in mRNA lipid nanoparticle compositions using orthogonal methods for multifactorial and multifactorial levels in example 11 of the present invention ((a) cationic lipid/phospholipid/cholesterol/PEGylated lipid four component molar ratio design; effect of cationic lipid (b), phospholipid (c), cholesterol (d), PEGylated lipid (e) content on mRNA delivery efficiency);
FIG. 16 shows the optimization of lipid fraction in the mRNA lipid nanoparticle composition of example 11 of the present invention ((a) cationic lipid 6Ac1-C12 chemical structure, (b) design of four component molar ratio of 6Ac 1-C12/DOPE/cholesterol/DMG-PEG 2000, (C) delivery efficiency of lipid nanoparticle composition mRNA in different fractions, (d) cell viability);
FIG. 17 is a graph showing the effect of lipid fraction on mRNA in vivo delivery of lipid nanoparticle composition of example 12 of the present invention ((a) 6Ac 1-C12/DOPE/cholesterol/DMG-PEG 2000 four component molar ratio, 6Ac1-C12/mRNA mass ratio design; b) in vivo mRNA delivery efficiency);
FIG. 18 is the in vivo mRNA delivery efficiency of lipid nanoparticle compositions based on different cationic lipids in example 12 of the present invention;
FIG. 19 is in vivo organ selective mRNA delivery of lipid nanoparticle compositions based on different cationic lipids following intravenous administration in example 12 of the present invention;
FIG. 20 is the in vivo mRNA delivery efficiency of lipid nanoparticle compositions of different cationic lipids using intramuscular injection in example 13 of the present invention.
Detailed Description
In order that those skilled in the art will better understand the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings, wherein it is to be understood that the illustrated embodiments are merely exemplary of some, but not all, of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other. The present invention will be described in detail with reference to examples.
Example 1:
synthesis of novel cationic lipid nAcx-Cm:
acrylic ester monomer (nAcx) and amine micromolecule (Cm) are added into a dry reaction bottle, wherein the molar quantity of the acrylic ester conjugated double bond is equal to the molar quantity of the amine micromolecule secondary amine group. Stirring at 50-80deg.C for 48-72 h to obtain crude product, and purifying by silica gel column chromatography to obtain final product cationic lipid nAcx-Cm (shown in figure 1).
Example 2:
synthesis of cationic lipid Compound 3Ac 1-C18:
trimethylolpropane triacrylate (3 Ac1,0.2mmol,1.0 eq) and N-methyl-1-octadecylamine (C18, 0.6mmol,3.0 eq) were added to a dry reaction flask, the mixture was magnetically stirred at 60℃for 72h and the crude product was purified by column chromatography over silica gel to give the product 3Ac1-C18. The hydrogen spectrum of the cationic lipid 3Ac1-C18 is shown in FIG. 2.
Example 3:
synthesis of cationic lipid Compound 3Ac 1-C212:
to the dried reaction flask were added trimethylolpropane triacrylate (3 Ac1,0.2mmol,1.0 eq) and didodecylamine (C212, 0.6mmol,3.0 eq), and the mixture was magnetically stirred at 60℃for 72h, and the crude product was purified by column chromatography over silica gel to give the product 3Ac1-C212. The hydrogen spectrum of the cationic lipid 3Ac1-C212 is shown in FIG. 3.
Example 4:
synthesis of cationic lipid Compound 5Ac 1-C12:
dipentaerythritol pentaacrylate (5 Ac1,0.2mmol,1.0 eq) and N-dodecylmethylamine (C12, 1.0mmol,5.0 eq) were added to a dry reaction flask, the mixture was magnetically stirred at 60℃for 72h, and the crude product was purified by column chromatography over silica gel to give the product 5Ac1-C12. The hydrogen spectrum of the cationic lipid 5Ac1-C12 is shown in FIG. 4.
Example 5:
synthesis of cationic lipid Compound 6Ac 1-C12:
dipentaerythritol hexaacrylate (6 Ac1,0.2mmol,1.0 eq) and N-dodecylmethylamine (C12, 1.2mmol,6.0 eq) were added to a dry reaction flask, the mixture was magnetically stirred at 60℃for 72h, and the crude product was purified by column chromatography over silica gel to give the product 6Ac1-C12. The hydrogen spectrum of the cationic lipid 6Ac1-C12 is shown in FIG. 5.
Example 6:
synthesis of cationic lipid Compound 6Ac 1-C210:
dipentaerythritol hexaacrylate (6 Ac1,0.2mmol,1.0 eq) and didecylamine (C210, 1.2mmol,6.0 eq) were added to a dry reaction flask, the mixture was magnetically stirred at 60℃for 72h, and the crude product was purified by column chromatography over silica gel to give the product 6Ac1-C210. The hydrogen spectrum of the cationic lipid 6Ac1-C210 is shown in FIG. 6.
Example 7:
preparation of lipid nanoparticles (LNP formulation):
cationic lipid compound, phospholipid DOPE (purchased from Avanti,1, 2-dioleoyl-sn-glycerol-3-phosphorylethanolamine), cholesterol (purchased from Sigma), DMG-PEG2000 (purchased from Avanti,1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000) were dissolved in ethanol at a molar ratio of 15/20/25/2 or other specified molar ratio to prepare an ethanol lipid solution, and luciferase mRNA (Fluc mRNA) was diluted in 10mM citrate buffer (ph=4) to obtain an aqueous solution of mRNA. The ethanol phase and the aqueous phase solution are rapidly mixed in a volume ratio of 1:3, the mixture is stood for 10 to 30 minutes to prepare LNP, and finally 1 XPBS is added to the required volume, wherein the mass ratio of the cationic lipid to the mRNA is 5/1 to 30/1. LNP used in animal experiments was dialyzed in 1 XPBS before injection.
Example 8:
LNP particle size and Zeta potential characterization:
lipid nanoparticles LNP were prepared in a cationic lipid/DOPE/cholesterol/DMG-PEG 2000 molar ratio of 15/20/25/2, diluted to the desired volume, and the particle size and polydispersity of LNP were determined by dynamic light scattering using Malvern Zetasizer NanoZS, and the test results are shown in FIG. 7. The Zeta potential of the LNP was also measured using Malvern Zetasizer NanoZS and the test results are shown in FIG. 8.
Example 9:
cytotoxicity evaluation of lipid nanoparticle compositions:
digesting human ovarian cancer cells IGROV1 in logarithmic growth phase, re-suspending the digested and centrifuged cells with 1640 medium containing 10% fetal calf serum and 1% double antibody, inoculating into a white 96-well plate at 10000-15000 cells/well, and placing in a medium containing 5% CO 2 Incubate in a 37℃incubator for 24h. Then, LNP was added to the formulated Fluc mRNA entrapped with a cationic lipid/DOPE/cholesterol/DMG-PEG 2000 molar ratio of 15/20/25/2, 25ng per well of mRNA and incubation was continued at 37℃for 24h. Cell viability was determined by Alamarblue assay kit method, medium was discarded from a white 96-well plate, 100 μl of 1640 medium without fetal bovine serum and 10 μl of Alamarblue assay reagent were added to each well, and incubation was continued at 37 ℃ for 2h with indigo changing the color of the wells to pink. The detection is carried out by using a multifunctional enzyme-labeled instrument, the excitation light wavelength is set to be 530nm, and the emission light wavelength is set to be 590nm. The cytotoxicity results of the lipid nanoparticle compositions are shown in figures 9-11. The results show that the lipid nanoparticle composition in the embodiment of the invention has little inhibition effect on cells, and the cell survival rate can reach more than 80%.
Example 10:
in vitro mRNA delivery evaluation of lipid nanoparticle compositions:
subjecting human ovarian cancer cells IGROV1 in logarithmic growth phase toDigesting, re-suspending the digested and centrifuged cells in 1640 medium containing 10% fetal calf serum and 1% double antibody, inoculating into a white 96-well plate at a cell density of 10000-15000 cells/well, and placing in a medium containing 5% CO 2 Incubate in a 37℃incubator for 24h. Then, LNP was added to the formulated Fluc mRNA entrapped with a cationic lipid/DOPE/cholesterol/DMG-PEG 2000 molar ratio of 15/20/25/2, 25ng per well of mRNA and incubation continued at 37 ℃. After 24h, bio-Lumi was used TM Luciferase reporter gene detection kit and multifunctional enzyme-labeled instrument detect Fluc mRNA expression efficiency. The results of evaluation of the efficiency of in vitro mRNA delivery of lipid nanoparticle compositions determined by the methods described above are shown in fig. 12-14.
Example 11:
lipid component proportioning screening of lipid nanoparticle compositions:
digesting human ovarian cancer cells IGROV1 in logarithmic growth phase, re-suspending the digested and centrifuged cells with 1640 medium containing 10% fetal calf serum and 1% double antibody, inoculating into a white 96-well plate at 10000-15000 cells/well, and placing in a medium containing 5% CO 2 Incubate in a 37℃incubator for 24h. Then, LNP was added to the formulated Fluc mRNA-entrapped, and incubation was continued at 37℃with 25ng of mRNA per well. Cationic lipid/DOPE/cholesterol/DMG-PEG 2000 molar ratios were designed for multifactorial, multi-level experiments in an orthogonal manner (FIG. 15 a), 6Ac 1-C12/DOPE/cholesterol/DMG-PEG 2000 molar ratios included 10/10/15/1,10/15/20/2,10/20/25/3,10/25/30/4,15/10/20/3,15/15/15/4,15/20/30/1,15/25/25/2,20/10/25/4,20/15/30/3,20/20/15/2,20/25/20/1,25/10/30/2,25/15/25/1,25/20/20/4, 25/25/15/3. After 24h, bio-Lumi was used TM Luciferase reporter gene detection kit and multifunctional enzyme-labeled instrument detect Fluc mRNA expression efficiency. The results of in vitro mRNA delivery efficiency evaluation of lipid nanoparticle compositions of different lipid ratios are shown in fig. 15-16.
Example 12:
evaluation of mRNA delivery after intravenous injection in vivo of lipid nanoparticle composition:
female C57BL/6 mice aged around 6 weeks were selected, and the lipid nanoparticle composition LNP prepared by tail vein injection was used in an amount of 0.25mg/kg of Fluc mRNA. Cationic lipid (nAcx-Cm)/DOPE/cholesterol/DMG-PEG 2000 molar ratio of 15/20/25/2, unless otherwise specified; the cationic lipid (nAcx-Cm)/mRNA mass ratio was 10/1 or 20/1. After 6 hours, the mice were intraperitoneally injected with 100. Mu.L of D-potassium fluorescein at a concentration of 30 mg/mL. After 5 minutes, the mice were placed under a living imaging system, and the chemiluminescent intensities of the mouse body and the isolated organs were observed and photographed. As shown in fig. 17-19, the delivery efficiency of Fluc mRNA in representative lipid nanoparticle compositions was shown in vivo, and using DLin-MC3-DMA LNP as a control, DLin-MC3-DMA LNP was used in the same formulation as FDA approved on patpro drug, i.e., DLin-MC 3-DMA/DSPC/cholesterol/DMG-PEG 2000 molar ratio was 50/10/38.5/1.5. The cationic lipid has a plurality of expression intensities similar to those of DLin-MC3-DMA, and has a plurality of expression intensities obviously superior to those of DLin-MC3-DMA. Also, 2Ac3-C18 LNP can specifically deliver mRNA to spleen expression, other multiple cationic lipid LNPs can specifically deliver mRNA to liver expression (as shown in fig. 19), in fig. 19, 2Ac3-C18 LNP can deliver mRNA to spleen expression, other multiple cationic lipid LNPs deliver mRNA to liver expression, and multiple lipid nanoparticle compositions mRNA delivery efficiency is superior to DLin-MC3-DMALNP.
Example 13:
evaluation of mRNA delivery following intramuscular injection of lipid nanoparticle compositions in vivo:
female C57BL/6 mice aged around 6 weeks were selected and the prepared lipid nanoparticle composition LNP was intramuscular injected. The cationic lipid (nAcx-Cm)/DOPE/cholesterol/DMG-PEG 2000 molar ratio was 15/20/25/2. After 6 hours, the mice were intraperitoneally injected with 100. Mu.L of D-potassium fluorescein at a concentration of 30 mg/mL. After 5 minutes, the mice were placed under a living imaging system, observed and photographed to record the chemiluminescent intensity of the mice. The delivery efficiency of Fluc mRNA in vivo for representative lipid nanoparticle compositions is shown in fig. 20, with SM-102 and ALC-0315LNPs as controls. The formulation used for SM-102LNP was identical to FDA approved novel crown mRNA vaccine mRNA-1273, i.e., SM-102/DSPC/cholesterol/DMG-PEG 2000 molar ratio was 50/10/38.5/1.5; the formulation used for ALC-0315LNP was identical to FDA approved novel coronal mRNA vaccine BNT162b2, i.e., the molar ratio of ALC-0315/DSPC/cholesterol/ALC-0159 was 46.3/9.4/42.7/1.6. The cationic lipid has a plurality of expression intensities which are obviously superior to those of SM-102 and ALC-0315LNPs.
Through the above embodiment of the present invention, the compound provided by the present invention is a cationic lipid compound, wherein the center of the cationic lipid compound is an acrylic monomer containing a degradable group, and the number and the length of the peripheral hydrophobic chains are controllable. The preparation method of the cationic lipid compound has the advantages of easily available raw materials, mild reaction conditions, high reaction yield, good reaction selectivity, simple and easy operation and low requirements on instruments and equipment. The cationic lipid compound provided by the invention is screened through the characteristics of the size and the potential of lipid nano particles, in-vitro cell transfection efficiency, cytotoxicity, in-vivo delivery efficiency, lipid proportion regulation in a lipid nano particle composition and the like, and the effectiveness and the safety of nucleic acid delivery are obviously improved. The lipid nanoparticle composition can selectively deliver mRNA to different organs, and has good in vivo targeting.
The above specific embodiments are provided for illustrative purposes only and are not intended to limit the invention, and modifications, no inventive contribution, will be made to the embodiments by those skilled in the art after having read the present specification, as long as they are within the scope of the patent statutes.

Claims (13)

1. A cationic lipid compound of the general formula (I), or a stereoisomer, tautomer, pharmaceutically acceptable salt, prodrug or solvate thereof,
wherein R is 1 Selected from the following structures: all groups or C in the acrylate monomers other than acrylate groups 1 -C 26 Alkyl or C 1 -C 26 Alkenyl, one or more C of said alkyl or alkenyl optionally being replaced by O or N, said alkyl or alkenylThe substituent group of the base structure being C 1 -C 18 One or more of alkyl, ester, carbonate, carbonyl, amide, ether, amine, hydrocarbon, alkene, carboxyl, acyl, and alkoxy, wherein C 1 -C 18 Alkyl is unsubstituted or optionally substituted with one or more of the following substituents: ester groups, carbonate groups, carbonyl groups, amide groups, ether groups, amine groups, hydrocarbon groups, olefin groups, carboxyl groups, acyl groups, alkoxy groups;
R 2 one or more selected from the following structures: H. methyl, saturated alkyl chains, unsaturated alkyl chains;
L 1 one or more selected from the following structures: -O-, -S-, -OC (=o) -, -C (=o) O-, -OC (=o) O-, -C (=o) NH-, -NHC (=o) -, -C (=s) O-, -OC (=s) -, -OC (=o) S-, -SC (=o) -, -OC (=o) S-, -C (=s) S-, -SC (=s) O-;
L 2 an alkyl or alkenyl structure comprising 1 to 18 carbon atoms that is linear or branched, saturated or unsaturated, said alkyl or alkenyl structure optionally substituted with one or more of the following substituents: c (C) 1 -C 6 Alkyl, ester, carbonate, carbonyl, amide, ether, amine, hydrocarbon, olefin, carboxyl, acyl, and alkoxy groups;
y is an integer from 0 to 6.
2. A cationic lipid compound of the general formula (I), or a stereoisomer, tautomer, pharmaceutically acceptable salt, prodrug or solvate thereof, according to claim 1, wherein: the cationic lipid compound is one or more of the compounds shown in the following structures:
3. a cationic lipid compound of the general formula (I), or a stereoisomer, tautomer, pharmaceutically acceptable salt, prodrug or solvate thereof, according to claim 2, wherein:
in (a):
R 1 selected from the following structures: all groups or C in the acrylate monomers other than acrylate groups 1 -C 26 Alkyl or C 1 -C 26 Alkenyl, one or more C of said alkyl or alkenyl being optionally substituted with O or N, the substituent of said alkyl or alkenyl structure being C 1 -C 18 One or more of alkyl, ester, carbonate, carbonyl, amide, ether, amine, hydrocarbon, alkene, carboxyl, acyl, and alkoxy, wherein C 1 -C 18 Alkyl is unsubstituted or optionally substituted with one or more of the following substituents: ester groups, carbonate groups, carbonyl groups, amide groups, ether groups, amine groups, hydrocarbon groups, olefin groups, carboxyl groups, acyl groups, alkoxy groups;
R 2 one or more selected from the following structures: H. methyl, saturated alkyl chains, unsaturated alkyl chains;
n' is an integer from 0 to 16.
4. A composition characterized by: the composition comprises a therapeutic or prophylactic agent and a carrier for delivering the therapeutic or prophylactic agent, the carrier comprising a cationic lipid compound comprising one or more of the general formula (I) or (II) or stereoisomers thereof, or tautomers thereof, or pharmaceutically acceptable salts, prodrugs or solvates thereof, as claimed in any one of claims 1 to 3.
5. A composition according to claim 4, wherein: the cationic lipids are used with one or more accessory components to form a lipid nanoparticle system for delivery of therapeutic or prophylactic agents.
6. A composition according to claim 5, wherein: the auxiliary component comprises sterols, charged lipids, neutral lipids, and pegylated lipids;
preferably, the pegylated lipid is 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000;
the charged lipid is any lipid molecule that exists in a positively or negatively charged form at a selected pH or range that corresponds to the pH conditions of the intended use environment of the lipid.
7. A composition as claimed in claim 6, wherein: the sterols include any one or more of cholesterol, fecal sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassinosteroids, lycorine, ursolic acid, alpha-tocopherol;
preferably, the sterol is cholesterol.
8. A composition as claimed in claim 6, wherein: the neutral lipid comprises one or more of phospholipid, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol and derivatives thereof;
preferably, the neutral lipid is a phospholipid.
9. A composition according to claim 5, wherein: the therapeutic or prophylactic agent comprises one or more of a nucleic acid molecule, a small molecule compound, a polypeptide or a protein.
10. A composition according to claim 4, wherein: the mass ratio of the carrier to the therapeutic or prophylactic agent is 5:1-50:1.
11. A composition as claimed in claim 9, wherein: the nucleic acid molecule is a linear mRNA or a circular mRNA;
preferably, the nucleic acid molecule is RNA, mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomir, a microrna inhibitor, a microrna activator or shRNA, DNA, an antisense nucleic acid, an aptamer, a ribozyme, an immunostimulatory nucleic acid or PNA.
12. A lipid nanoparticle composition characterized by: the composition comprises 14.5-37.9 percent of cationic lipid compound, 14.9-37.9 percent of phospholipid, 22.1-44.5 percent of cholesterol and 1.5-8.2 percent of pegylated lipid;
preferably, the molar ratio of the cationic lipid compound, phospholipid, cholesterol and pegylated lipid is: 15/20/25/2 or 10/10/15/1 or 10/15/20/2 or 10/20/25/3 or 10/25/30/4 or 15/10/20/3 or 15/15/15/4 or 15/20/30/1 or 15/25/25/2 or 20/10/25/4 or 20/15/30/3 or 20/20/15/2 or 20/25/20/1 or 25/10/30/2 or 25/15/25/1 or 25/20/4 or 25/25/15/3.
13. Use of a cationic lipid compound of general formula (I) according to any one of claims 1 to 3, or a stereoisomer, tautomer, pharmaceutically acceptable salt, prodrug or solvate thereof, or a composition according to any one of claims 4 to 11, or a lipid nanoparticle composition according to claim 12, for the preparation of a medicament for the delivery of a nucleic acid drug to a different organ or location; the different organs or locations include spleen, liver and muscle.
CN202310623164.9A 2023-05-30 2023-05-30 Cationic lipid compound, and composition and application thereof Pending CN116730857A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310623164.9A CN116730857A (en) 2023-05-30 2023-05-30 Cationic lipid compound, and composition and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310623164.9A CN116730857A (en) 2023-05-30 2023-05-30 Cationic lipid compound, and composition and application thereof

Publications (1)

Publication Number Publication Date
CN116730857A true CN116730857A (en) 2023-09-12

Family

ID=87902136

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310623164.9A Pending CN116730857A (en) 2023-05-30 2023-05-30 Cationic lipid compound, and composition and application thereof

Country Status (1)

Country Link
CN (1) CN116730857A (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117257965A (en) * 2023-11-21 2023-12-22 深圳瑞吉生物科技有限公司 Nucleic acid delivery carrier composition and application thereof
CN117964577A (en) * 2024-03-29 2024-05-03 天津全和诚生物技术有限公司 Cationic lipid compound, preparation method thereof, composition containing cationic lipid compound and application of cationic lipid compound
CN118184538A (en) * 2024-05-14 2024-06-14 北京悦康科创医药科技股份有限公司 Lung high-expression cationic lipid compound, composition containing same and application

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117257965A (en) * 2023-11-21 2023-12-22 深圳瑞吉生物科技有限公司 Nucleic acid delivery carrier composition and application thereof
CN117257965B (en) * 2023-11-21 2024-02-23 深圳瑞吉生物科技有限公司 Nucleic acid delivery carrier composition and application thereof
CN117964577A (en) * 2024-03-29 2024-05-03 天津全和诚生物技术有限公司 Cationic lipid compound, preparation method thereof, composition containing cationic lipid compound and application of cationic lipid compound
CN118184538A (en) * 2024-05-14 2024-06-14 北京悦康科创医药科技股份有限公司 Lung high-expression cationic lipid compound, composition containing same and application

Similar Documents

Publication Publication Date Title
CN116730857A (en) Cationic lipid compound, and composition and application thereof
Kaczmarek et al. Systemic delivery of mRNA and DNA to the lung using polymer-lipid nanoparticles
EP3252043B1 (en) Cationic lipid
FI119738B (en) Composition and uses containing a solution of nucleic acid and a solution of a cationic polymer
Algarni et al. In vivo delivery of plasmid DNA by lipid nanoparticles: the influence of ionizable cationic lipids on organ-selective gene expression
EP4342880A1 (en) Ionizable lipid compound for nucleic acid delivery and lnp composition thereof
EP4035686A1 (en) Lipid nanoparticle lyophilized composition
CN102241790B (en) Amphipathic chitosan derivative and preparation method and application thereof
JP2002521423A (en) Lipid emulsions and solid lipid microparticles as gene or drug carriers
JP2010235618A (en) Method for delivering nucleic acid
EP3842412A1 (en) Biodegradable compound, lipid particles, composition containing lipid particles, and kit
CN114213295B (en) Cationic compound, preparation method, compound and application thereof
EP3792246A1 (en) Biodegradable compound, lipid particle, lipid particle-containing composition, and kit
CN114805212B (en) Lipid and lipid composition for delivery of therapeutic or prophylactic agents
WO2021170034A1 (en) Amino lipid compound, preparation method therefor, and application thereof
KR102560772B1 (en) Novel ionizable lipids and lipid nanoparticle compositions thereof
Neuberg et al. Design and evaluation of ionizable peptide amphiphiles for siRNA delivery
US20220249376A1 (en) Nanovesicles and its use for nucleic acid delivery
WO2023241314A1 (en) Type of novel lipid compound and use thereof
CN115872893B (en) Cationic lipid compound, and preparation method and application thereof
Reinhard et al. Sequence-Defined Cationic Lipo-Oligomers Containing Unsaturated Fatty Acids for Transfection
WO2023216232A1 (en) Lipid compound containing disulfide bond and composition thereof
US11951177B2 (en) High sterol-containing lipid nanoparticles
CN117257965B (en) Nucleic acid delivery carrier composition and application thereof
JP2018016642A (en) Manufacturing method of lipid particles, and nucleic acid delivery carrier having lipid particles

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
CB03 Change of inventor or designer information

Inventor after: Liu Shuai

Inventor after: Su Kexin

Inventor after: Ping Yuan

Inventor before: Liu Shuai

Inventor before: Su Kexin

CB03 Change of inventor or designer information