CN116554042A - Novel ionizable lipids for nucleic acid delivery and LNP compositions and vaccines thereof - Google Patents
Novel ionizable lipids for nucleic acid delivery and LNP compositions and vaccines thereof Download PDFInfo
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- CN116554042A CN116554042A CN202310044748.0A CN202310044748A CN116554042A CN 116554042 A CN116554042 A CN 116554042A CN 202310044748 A CN202310044748 A CN 202310044748A CN 116554042 A CN116554042 A CN 116554042A
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- lipid
- peg
- cationic lipid
- cationic
- mrna
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- 150000007523 nucleic acids Chemical class 0.000 title description 3
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- XBECFEJUQZXMFE-UHFFFAOYSA-N n-(4-aminobutyl)acetamide;hydrochloride Chemical compound Cl.CC(=O)NCCCCN XBECFEJUQZXMFE-UHFFFAOYSA-N 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000001314 paroxysmal effect Effects 0.000 description 1
- 210000001184 pharyngeal muscle Anatomy 0.000 description 1
- 208000019899 phobic disease Diseases 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 229940049155 rabies serum Drugs 0.000 description 1
- 230000036647 reaction Effects 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 201000004193 respiratory failure Diseases 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 230000035807 sensation Effects 0.000 description 1
- 206010040560 shock Diseases 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
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Classifications
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- 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
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- 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
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- C07C233/36—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
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- C07C255/01—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms
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- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
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- C12N2760/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Abstract
The invention provides a novel cationic lipid, lipid nano-particles and a nucleic acid vaccine. The lipid nanoparticle mRNA vaccine prepared by the specific cationic lipid is selected, and has better in-vitro stability and immunogenicity compared with LNP prepared by the cationic lipid in the prior art.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to novel ionizable lipid for nucleic acid delivery, and LNP composition and vaccine thereof.
Background
The current clinically proven system for delivering mRNA is lipid nanoparticle (Lipid Nanoparticle, LNP), which belongs to lipid forming nanoparticles, wherein the principle comprises cationic lipid, whereas prior art studies show that mRNA expression rate is low after mRNA is delivered into cells, for example Dlin-MC3-DMA is used as cationic lipid to construct LNP, and mRNA expression level is 0.63% (Maugeri, marco et al, "Linkage between endosomal escape of LNP-mRNA and loading into EVs for transport to other cells." Nature Communications, 2019), therefore, the structure of cationic lipid is a key factor affecting mRNA expression level.
The rabies vaccine is rabies vaccine and anti-rabies serum which are inoculated after a human being is bitten by animals, and can prevent rabies infection. Rabies is a natural epidemic disease or animal-derived zoonotic acute infectious disease caused by rabies virus, has wide popularity and extremely high death rate, and causes serious threat to the life health of people.
The typical clinical manifestations of rabies are water-repellent diseases, so rabies is also called water-repellent disease, is sensitive to stimulus such as sound, light, wind and the like in the initial stage and has tight sensation on the throat, and can be expressed as extreme phobia, water-repellent, wind-repellent, paroxysmal pharyngeal muscle spasm, dyspnea and the like in the excitation stage, and finally, various paralysis occurs due to the stopping of the seizure, and can be quickly dead due to respiratory and circulatory failure. Human rabies is mainly caused by bites, scratches or mucosal infections of diseased animals, and can also be transmitted by respiratory aerosol under specific conditions. The saliva of the infected animals contains rabies virus. The infectious animals were primarily dogs (over 90%), and secondarily cats.
The rabies vaccine for human is more in variety in the past, and the cell culture vaccine is mostly used at home and abroad nowadays. The currently used refined VERO cell rabies vaccine and refined ground mouse kidney cell rabies vaccine in China, and the concentrated ground mouse kidney cell rabies vaccine is forbidden. Currently, there is no mRNA rabies vaccine on the market.
Disclosure of Invention
The term "neutral lipid" according to the present invention refers to lipid molecules that are uncharged, non-phosphoglycerides.
The term "polyethylene glycol (PEG) -lipid conjugate" in the present invention refers to a molecule comprising a lipid moiety and a polyethylene glycol moiety.
The term "lipid nanoparticle" according to the present invention refers to particles having at least one nanoscale size, comprising at least one lipid.
The term "vaccine" in accordance with the present invention refers to a composition suitable for application to animals (including humans) that induces an immune response after administration that is sufficiently strong to minimally aid in the prevention, amelioration or cure of clinical disease resulting from infection by a microorganism.
The term "delivery system" in the present invention refers to a formulation or composition that modulates the spatial, temporal and dose distribution of a biologically active ingredient within an organism.
In the term of the invention, N/P is the molar ratio of N in the cationic lipid to P in the mRNA mononucleotide.
The term "hydrocarbon group" according to the invention refers to the group remaining after the corresponding hydrocarbon has lost one hydrogen atom, in particular to aliphatic groups such as alkyl, alkenyl, alkynyl, in particular alkyl groups in the present invention.
The present invention provides cationic lipids having the structure of formula I:
wherein:
L 1 and L 2 At least one of which is-O-, -O (C=O) O- - (c=o) NRa-, -NRa (c=o) -or-NRa-,
and, in addition, the processing unit,
L 1 or L 2 is-O-, -O (c=o) O-, -NRa-, - (c=o) -, -NRa-, -O (c=o) -, - (c=o) O-, -C (=o) -, -S (O) x-, -S-, -C (=o) S-, -SC (=o) -, -NRaC (=o) NRa-, -OC (=o) NRa-or-NRaC (=o) O-;
G 1 and G 2 Each independently is unsubstituted C 1 -C 12 Alkylene or C 1 -C 12 Alkenylene;
G 3 is C 1 -C 24 Alkylene, C 1 -C 24 Alkenylene, C 3 -C 8 Cycloalkylene, C 3 -C 8 A cycloalkenyl group;
ra is H orC 1 -C 12 A hydrocarbon group;
R 1 and R is 2 Each independently is C 6 -C 24 Alkyl or C 6 -C 24 Alkenyl groups;
R 3 is H, OH OR 4 、CN、-C(=O)OR 4 、-OC(=O)R 4 or-NR 5 C(=O)R 4 ;
R 4 Is C 1 -C 12 A hydrocarbon group;
R 5 is H or C 1 -C 6 A hydrocarbon group;
x is 0, 1 or 2.
In particular, wherein the cationic lipid has L in the structure of formula I 1 And L 2 Each independently selected from-O-, -O (c=o) O-, - (c=o) NH-, -NH (c=o) -and-NH-.
Specifically, in the cationic lipid formula I structure, L 1 And L 2 Are all-O-, or L 1 And L 2 Are all-O (C=O) O-, or L 1 And L 2 Are all-NH-, or L 1 is-NH (C=O) -, L 2 Is- (c=o) NH-.
Specifically, the cationic lipid therein has the following structure (IA):
wherein:
R 6 at each occurrence independently H, OH or C 1 -C 24 A hydrocarbon group;
n is an integer from 1 to 15.
Specifically, the cationic lipid therein has the following structure (IB):
wherein y and z are each independently integers from 1 to 12.
Specifically, n in the cationic lipid structure is an integer of 2 to 12, preferably n is 2, 3, 4, 5 or 6; wherein y and z are each independently integers from 2 to 10, preferably from 4 to 9.
Specifically, R in the cationic lipid structure 1 And R is 2 Each independently has the following structure:
wherein:
R 7a and R is 7b At each occurrence independently H or C 1 -C 12 A hydrocarbon group; and a is an integer from 2 to 12, preferably a is an integer from 8 to 12;
wherein R is 7a 、R 7b And a are each selected such that R 1 And R is 2 Each independently comprising 6 to 20 carbon atoms.
Specifically, R occurs at least once in the cationic lipid structure thereof 7a Is H, preferably R 7a H at each occurrence.
Specifically, R occurs at least once in the cationic lipid structure thereof 7b Is C 1 -C 8 A hydrocarbon group; preferably, wherein C 1 -C 8 The hydrocarbyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl or n-octyl.
Specifically, R in the cationic lipid structure 1 Or R is 2 Or both have one of the following structures:
specifically, the cationic lipid compound has the following structure:
the present invention relates to a lipid nanoparticle comprising: (a) the cationic lipid described above; (b) a non-cationic lipid; (c) polyethylene glycol (PEG) -lipid conjugates. Preferably, it comprises: cationic lipids, neutral phospholipids, steroidal lipids and/or polyethylene glycol (PEG) -lipid conjugates.
Specifically, the polyethylene glycol (PEG) -lipid conjugate is selected from: 2- [ (polyethylene glycol) -2000] -N, N-tetracosylacetamide (ALC-0159), 1, 2-dimyristoyl-sn-glycerogethoxy polyethylene glycol (PEG-DMG), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ amino (polyethylene glycol) ] (PEG-DSPE), PEG-distteroylglycerol
(PEG-DSG), PEG-dipalmitoyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycerol amide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), PEG-1, 2-dimyristoyloxypropyl-3-amine (PEG-c-DMA), or DMG-PEG2000, preferably DMG-PEG2000.
Specifically, the neutral lipid is selected from one or more of 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho- (1' -rac-glycero) (DOPG), oleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE), and preferably DSPC.
Specifically, the steroid lipid is selected from oat sterol, beta-sitosterol, campesterol, ergocalcitol, campesterol, cholestanol, cholesterol, fecal sterol, dehydrocholesterol, desmosterol, dihydroergocalcitol, dihydrocholesterol, dihydroergosterol, black sea sterol, epicholesterol, ergosterol, fucosterol, hexahydrolight sterol, hydroxycholesterol and polypeptide modified cholesterol; one or more combinations of lanosterol, sitosterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid and lithocholic acid, preferably cholesterol.
Specifically, the cationic lipid content is 20-60%, the neutral phospholipid content is about 5-25%, and the steroid lipid content is about 25-55%; the molar content of the polyethylene glycol (PEG) -lipid conjugate is about 0.5% -15%,
specifically, wherein the cationic lipid: neutral phospholipids: steroid lipid: polyethylene glycol (PEG) -lipid conjugate molar ratio of 30-60:1-20:20-50:0.1-10, preferably wherein the cationic lipid: neutral phospholipids: steroid lipid: polyethylene glycol (PEG) -lipid conjugate molar ratio was 43:10:45:2 or 40:10:48:2.
Specifically, the vaccine also comprises other auxiliary materials, wherein the auxiliary materials are one or a combination of more of sodium acetate, tromethamine, monopotassium phosphate, sodium chloride, disodium hydrogen phosphate and sucrose.
In particular, wherein the nanoparticles have an average particle size of 50 to 200nm or wherein the nanoparticles have a net neutral charge at neutral pH or wherein the nanoparticles have a polydispersity of less than 0.4.
The invention relates to a preparation method of a lipid nanoparticle mRNA vaccine. Specifically, the cationic lipid, the non-cationic lipid, and the polyethylene glycol (PEG) -lipid conjugate are dissolved in a solvent and then mixed with mRNA.
Specifically, the cationic lipid, neutral phospholipid, steroid lipid and polyethylene glycol (PEG) -lipid conjugate are dissolved into ethanol, then mixed with diluted mRNA diluent, and subjected to ultrafiltration, dilution and filtration to obtain the final product; preferably, the cationic lipid, neutral phospholipid, steroid lipid and polyethylene glycol (PEG) -lipid conjugate are dissolved into ethanol, and then are mixed with diluted mRNA diluent according to a certain flow rate ratio, and are subjected to ultrafiltration, dilution and filtration to obtain the modified mRNA; preferably, the ultrafiltration mode is tangential flow filtration; more preferably, the mixing means may be turbulent mixing, laminar mixing or microfluidic mixing.
In particular, the diluent may be an acetate buffer, a citrate buffer, a phosphate buffer or a tris buffer.
Specifically, the pH of the buffer solution is 3-6, and the concentration is 6.25-200 mM.
Specifically, the ratio of the flow rate of the lipid mixed solution obtained by dissolving the cationic lipid, the non-cationic lipid and the polyethylene glycol (PEG) -lipid conjugate in the solvent to the flow rate of the solution obtained by diluting mRNA is 1-5:1.
Specifically, the N/P when the mRNA is encapsulated with the lipid is 2 to 10, preferably N/P is 3 to 8, more preferably N/P is 3, 4, 5, 6, 7, 8.
Specifically, the ultrafiltrate is selected from the group consisting of: sodium salt and Tris (hydroxymethyl) aminomethane (Tris) salt, preferably the ultrafiltrate pH is 6.0-8.0.
The invention provides a rabies virus lipid nanoparticle mRNA vaccine, and particularly relates to an administration mode of oral administration, intramuscular injection, intravenous injection or inhalation.
In particular, the rabies virus lipid nanoparticle mRNA vaccine can be prepared into oral preparations, liquid preparations, freeze-dried powder preparations, injection or inhalation preparations, and preferably intramuscular injection, intravenous injection, dry powder inhalation or aerosol inhalation.
The invention relates to a rabies virus lipid nanoparticle mRNA vaccine, which comprises the following components: (a) mRNA encoding rabies virus G protein; (b) a cationic lipid; (c) a non-cationic lipid; (d) polyethylene glycol (PEG) -lipid conjugates.
Specifically, the rabies virus lipid nanoparticle mRNA vaccine, wherein the amino acid sequence encoded by the mRNA comprises a sequence shown in SEQ ID NO. 1 (G protein of CTN-1 strain (GenBank: ACR 39382.1): SEQ ID NO. 1) or SEQ ID NO. 2 (CTN-1V-T strain G protein).
Alternatively, amino acid sequences having 80% or more identity to the sequences shown in SEQ ID NOs 1 and 2, preferably amino acid sequences having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more or 100% identity.
Specifically, the rabies virus lipid nanoparticle mRNA vaccine comprises the following components: (a) mRNA encoding rabies virus G protein; (b) a cationic lipid; (c) neutral phospholipids, steroidal lipids; (d) polyethylene glycol (PEG) -lipid conjugates.
Specifically, mRNA encoding rabies virus G protein is encapsulated by lipid nanoparticles prepared by ALC-0315.
The rabies virus lipid nanoparticle mRNA vaccine can deliver the bioactive substances through oral administration, inhalation or injection.
The invention relates to an application of rabies virus lipid nanoparticle mRNA vaccine in preparing a medicine for preventing rabies.
The beneficial effects are that:
the present invention relates to novel cationic lipids which are distinguished from the prior art. The lipid compound disclosed by the invention is used for preparing PEG-lipid/cationic lipid/neutral lipid/steroid lipid-mRNA nano particles (LNP), and shows that the lipid compound serving as mRNA LNP of cationic lipid has good stability and transfection efficiency, and can cause higher specific antibody response in experimental animals.
The invention provides a rabies virus mRNA vaccine of lipid nanoparticles, which adopts lipid nanoparticles as a delivery system, has better physicochemical properties by constructing brand-new cationic lipid, and has encapsulation efficiency obviously superior to that of the lipid nanoparticle delivery system on the market, thus obtaining the rabies virus lipid nanoparticle mRNA vaccine with stronger immunogenicity.
Drawings
FIG. 1 is a graph of LNP formulation stability versus particle size data;
FIG. 2 is a graph of LNP formulation stability versus encapsulation efficiency data;
FIG. 3 is a graph showing LNP formulation stability versus mRNA integrity data;
FIG. 4 is a graph of LNP formulation stability versus PDI data;
FIG. 5 shows the BALB/c mouse immunization program;
FIG. 6 shows serum neutralizing antibody titers on BALB/c mouse models;
FIG. 7 shows CD4+ T cell frequency of specific secretion of TNF. Alpha. And IFN. Gamma. By ICS method on BALB/c mouse model;
FIG. 8 shows CD8+ T cell frequency of specific secretion of TNFα and IFNγ by ICS method on BALB/c mouse model.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Synthesis of Compound 1
Synthesis of 6-bromohexyl (2-hexyldecyl) carbonate (1 a)
6-Bromon-hexanol (0.91 g,5.0 mmol) was dissolved in 30mL of dichloromethane, 4-dimethylaminopyridine (0.90 g,7.5 mmol) was added, phenyl p-nitrochloroformate (1.20 g,6.0 mmol) was added in portions, the reaction was stirred at room temperature for 3h, 2-hexyldecanol (1.36 g,5.6 mmol) was added to the reaction mixture, the mixture was stirred at room temperature overnight, TLC showed that the reaction was complete, 20mL of dichloromethane was added, diluted then with 30mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, column chromatography separated to give 6-bromohexyl (2-hexyldecyl) carbonate 1a (1.53 g, pale yellow oil) in 68% yield.
MS m/z(ESI):449.3[M+1]
Synthesis of Compound 1
6-bromohexyl (2-hexyldecyl) carbonate (1.12 g,2.5 mmol) was dissolved in tetrahydrofuran, acetonitrile, 4-amino-1-butanol (89.2 mg,1.0 mmol), potassium carbonate (550 mg,4.0 mmol), potassium iodide (336 mg,2.0 mmol) was added, and stirred at 83℃for 16-20h. Cooling to room temperature, filtering, washing the filter residue with dichloromethane, adding saturated sodium bicarbonate solution into the obtained filtrate, extracting with dichloromethane for 2 times, combining organic phases, drying over anhydrous sodium sulfate, filtering and concentrating, separating by column chromatography to obtain the product 1 (454 mg, pale yellow oily substance) with a yield of 55%.
MS m/z(ESI):826.9[M+1]
1 H NMR(300MHz,CDCl 3 ):δ4.13(t,4H,J=6.6Hz),4.05(d,4H,J=5.7Hz),3.56-3.55(m,2H),2.47-2.42(m,6H),1.72-1.67(m,10H),1.53-1.48(m,8H),1.45-1.28(m,52H),0.69(t,12H,J=6.2Hz)
Example 2
Synthesis of Compound 2
Synthesis of 7-Bromoheptylheptadec-9-ylcarbonate (2 a)
7-Bromoheptanol (0.98 g,5.0 mmol) was dissolved in 30mL of methylene chloride, 4-dimethylaminopyridine (1.22 g,10 mmol) was added, phenyl p-nitrochloroformate (1.11 g,5.5 mmol) was added in portions, the reaction was stirred at room temperature for 3 hours, 9-hydroxyheptadecanol (1.44 g,5.6 mmol) was added to the reaction mixture, the mixture was stirred at room temperature overnight, TLC showed that the reaction was complete, 20mL of methylene chloride was added for dilution, then washed with 30mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and column chromatography was carried out to give 7-bromoheptadec-9-ylcarbonate 2a (1.50 g, pale yellow oil) in 65% yield.
MS m/z(ESI):477.3[M+1]
Synthesis of heptadec-9-yl (7- ((2-hydroxyethyl) amino) heptyl) carbonate (2 b)
7-Bromoheptylheptadec-9-ylcarbonate (2 a) (1.38 g,3 mmol) was dissolved in 20mL of ethanol at room temperature, ethanolamine (2.75 g,45 mmol) was added, the temperature was raised to 50℃and stirred for 8h, the progress of the reaction was monitored, after the consumption of the starting material was complete, the temperature was lowered to 45℃and the ethanol was removed by spin-drying, the crude product was dissolved with dichloromethane, washed three times with saturated brine, the organic phase was dried over anhydrous sodium sulfate and concentrated to give heptadec-9-yl (7- ((2-hydroxyethyl) amino) heptyl) carbonate 2b (1.35 g, pale yellow oil).
MS m/z(ESI):458.4[M+1]
Synthesis of 5-bromopentyl undecyl carbonate (2 c)
5-Bromopentanol (0.84 g,5.0 mmol) was dissolved in 30mL of dichloromethane, 4-dimethylaminopyridine (1.22 g,10 mmol) was added, phenyl p-nitrochloroformate (1.11 g,5.5 mmol) was added in portions, the reaction was stirred at room temperature for 3h, undecanol (0.97 g,5.6 mmol) was added to the reaction mixture, the mixture was stirred at room temperature overnight, TLC showed that the reaction was complete, 20mL of dichloromethane was added, then washed with 30mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and column chromatography separated to give 5-bromopentyl undecyl carbonate 2c (1.20 g, light yellow oil) in 66% yield.
MS m/z(ESI):365.2[M+1]
Synthesis of Compound 2
Heptadec-9-yl (7- ((2-hydroxyethyl) amino) heptyl) carbonate (457 mg,1.0 mmol) was dissolved in tetrahydrofuran, acetonitrile, 5-bromopentyl undecyl carbonate (433 mg,1.2 mmol), potassium carbonate (550 mg,4.0 mmol), potassium iodide (336 mg,2.0 mmol) was added, and stirred at 83℃for 16-20h. Cooling to room temperature, filtering, washing the filter residue with dichloromethane, adding saturated sodium bicarbonate solution into the obtained filtrate, extracting with dichloromethane for 2 times, combining organic phases, drying over anhydrous sodium sulfate, filtering and concentrating, separating by column chromatography to obtain the product 2 (440 mg, pale yellow oil) in 57% yield.
MS m/z(ESI):742.8[M+1]
1 H NMR(300MHz,CDCl 3 ):δ4.71-4.68(m,1H),4.15-4.10(m,6H),3.53(t,2H,J=5.4Hz),2.94(br,1H),2.58(t,2H,J=5.4Hz),2.45(t,4H,J=5.7Hz),1.75-1.34(m,62H),0.90(t,9H,J=6.3Hz)
Example 3
Synthesis of Compound 3
Synthesis of 6-bromohexyl undecyl carbonate (3 a)
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6-Bromon-hexanol (0.91 g,5.0 mmol) was dissolved in 30mL of dichloromethane, 4-dimethylaminopyridine (0.90 g,7.5 mmol) was added, phenyl p-nitrochloroformate (1.20 g,6.0 mmol) was added in portions, the reaction was stirred at room temperature for 3h, undecanol (0.97 g,5.6 mmol) was added to the reaction mixture, the mixture was stirred at room temperature overnight, TLC showed that the reaction was complete, 20mL of dichloromethane was added to dilute, then washed with 30mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and column chromatography separated to give 6-bromohexyl undecyl carbonate 3a (1.25 g, light yellow oil) in 66% yield.
MS m/z(ESI):379.2[M+1]
Synthesis of Compound 3
6-bromohexyl undecyl carbonate (948 mg,2.5 mmol) was dissolved in tetrahydrofuran, acetonitrile, 4-amino-1-butanol (89.2 mg,1.0 mmol), potassium carbonate (550 mg,4.0 mmol), potassium iodide (336 mg,2.0 mmol) was added, and stirred at 83℃for 16-20h. Cooling to room temperature, filtering, washing the filter residue with dichloromethane, adding saturated sodium bicarbonate solution into the obtained filtrate, extracting with dichloromethane for 2 times, combining organic phases, drying over anhydrous sodium sulfate, filtering and concentrating, separating by column chromatography to obtain the product 3 (412 mg, pale yellow oil) in 60% yield.
MS m/z(ESI):686.8[M+1]
1 H NMR(300MHz,CDCl 3 ):δ4.13(t,8H,J=6.6Hz),3.58(t,2H,J=5.7Hz),2.52(t,6H,J=8.4Hz),1.74-1.64(m,12H),1.63-1.53(m,5H),1.52-1.39(m,39H),0.86(t,6H,J=6.2Hz)
Example 4
Synthesis of Compound 4
Synthesis of 6-bromohexyl heptadec-9-ylcarbonate (4 a)
6-Bromon-hexanol (0.91 g,5.0 mmol) was dissolved in 30mL of dichloromethane, 4-dimethylaminopyridine (0.90 g,7.5 mmol) was added, phenyl p-nitrochloroformate (1.20 g,6.0 mmol) was added in portions, the reaction was stirred at room temperature for 3h, 9-heptadecanol (1.44 g,5.6 mmol) was added to the reaction mixture, the mixture was stirred at room temperature overnight, TLC showed that the reaction was complete, 20mL of dichloromethane was added for dilution, then washed with 30mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and column chromatography separated to give 6-bromohexyl heptadec-9-ylcarbonate 4a (1.53 g, pale yellow oil) in 66% yield.
MS m/z(ESI):464.3[M+1]
Synthesis of Compound 4
6-bromohexylheptadec-9-ylcarbonate (1.16 g,2.5 mmol) was dissolved in tetrahydrofuran, acetonitrile, 4-amino-1-butanol (89.2 mg,1.0 mmol), potassium carbonate (550 mg,4.0 mmol), potassium iodide (336 mg,2.0 mmol) was added, and stirred at 83℃for 16-20h. Cooling to room temperature, filtering, washing the filter residue with dichloromethane, adding saturated sodium bicarbonate solution into the obtained filtrate, extracting with dichloromethane for 2 times, combining organic phases, drying over anhydrous sodium sulfate, filtering and concentrating, separating by column chromatography to obtain the product 4 (502 mg, pale yellow oil) in 59% yield.
MS m/z(ESI):855.4[M+1]
1 H NMR(300MHz,CDCl 3 ):δ4.71-4.68(m,2H),4.13(t,4H,J=6.6Hz),3.57(t,2H,J=5.4Hz),2.49-2.44(m,6H),1.74-1.28(m,76H),0.90(t,12H,J=6.3Hz)
Example 5
Synthesis of Compound 5
6-bromohexyl (2-hexyldecyl) carbonate (1.12 g,2.5 mmol) was dissolved in tetrahydrofuran, acetonitrile, ethanolamine (61.0 mg,1.0 mmol), potassium carbonate (550 mg,4.0 mmol), potassium iodide (336 mg,2.0 mmol) was added, and stirred at 83℃for 16-20 hours. Cooled to room temperature, filtered, the filter residue was washed with dichloromethane, saturated sodium bicarbonate solution was added to the resulting filtrate, extracted 2 times with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated, and separated by column chromatography to give product 5 (487 mg, pale yellow oil) in 61% yield.
MS m/z(ESI):798.9[M+1]
1 H NMR(300MHz,CDCl 3 ):δ4.14(t,4H,J=6.6Hz),4.04(d,4H,J=5.7Hz),3.54(t,2H,J=5.4Hz),2.58(t,2H,J=5.4Hz),2.46(t,4H,J=7.2Hz),1.72-1.65(m,6H),1.49-1.28(m,61H),0.69(t,12H,J=6.2Hz)
Example 6
Synthesis of Compound 6
5-bromopentyl undecyl carbonate (910 mg,2.5 mmol) was dissolved in tetrahydrofuran, acetonitrile, ethanolamine (61.0 mg,1.0 mmol), potassium carbonate (550 mg,4.0 mmol), potassium iodide (336 mg,2.0 mmol) was added, and stirred at 83℃for 16-20 hours. Cooling to room temperature, filtering, washing the filter residue with dichloromethane, adding saturated sodium bicarbonate solution into the obtained filtrate, extracting with dichloromethane for 2 times, combining organic phases, drying over anhydrous sodium sulfate, filtering and concentrating, and separating by column chromatography to obtain the product 6 (410 mg, pale yellow oil) in 65% yield.
MS m/z(ESI):630.7[M+1]
1 H NMR(300MHz,CDCl 3 ):δ4.10(t,8H,J=6.6Hz),3.52(d,2H,J=5.4Hz),2.83(br,1H),2.57(t,2H,J=5.4Hz),2.45(t,4H,J=7.2Hz),1.73-1.62(m,8H),1.52-1.39(m,40H),0.69(t,6H,J=6.2Hz)
Example 7
Synthesis of Compound 7
6-bromohexyl (2-hexyldecyl) carbonate (1.12 g,2.5 mmol) was dissolved in tetrahydrofuran, acetonitrile, 3-methoxypropylamine (89 mg,1.0 mmol), potassium carbonate (550 mg,4.0 mmol), potassium iodide (336 mg,2.0 mmol) was added, and the mixture was stirred at 83℃for 16 to 20 hours. Cooling to room temperature, filtering, washing the filter residue with dichloromethane, adding saturated sodium bicarbonate solution into the obtained filtrate, extracting with dichloromethane for 2 times, combining organic phases, drying over anhydrous sodium sulfate, filtering and concentrating, separating by column chromatography to obtain the product 7 (495 mg, pale yellow oil) in 60% yield.
MS m/z(ESI):826.7[M+1]
Example 8
Synthesis of Compound 8
6-bromohexyl (2-hexyldecyl) carbonate (1.12 g,2.5 mmol) was dissolved in tetrahydrofuran, acetonitrile, 3-aminopropionitrile (70 mg,1.0 mmol), potassium carbonate (550 mg,4.0 mmol) and potassium iodide (336 mg,2.0 mmol) were added, and stirred at 83℃for 16-20h. Cooling to room temperature, filtering, washing the filter residue with dichloromethane, adding saturated sodium bicarbonate solution to the obtained filtrate, extracting with dichloromethane for 2 times, combining organic phases, drying over anhydrous sodium sulfate, filtering and concentrating, and separating by column chromatography to obtain product 8 (469 mg, pale yellow oil) in 58% yield.
MS m/z(ESI):807.7[M+1]
Example 9
Synthesis of Compound 9
6-bromohexyl (2-hexyldecyl) carbonate (1.12 g,2.5 mmol) was dissolved in tetrahydrofuran, acetonitrile, ethyl 4-aminobutyrate hydrochloride (167 mg,1.0 mmol), potassium carbonate (550 mg,4.0 mmol) and potassium iodide (336 mg,2.0 mmol) were added and stirred at 83℃for 16-20h. Cooled to room temperature, filtered, the filter residue was washed with dichloromethane, saturated sodium bicarbonate solution was added to the resulting filtrate, extracted 2 times with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated, and separated by column chromatography to give product 9 (546 mg, pale yellow oil) in 63% yield.
MS m/z(ESI):868.8[M+1]
Example 10
Synthesis of Compound 10
6-bromohexyl (2-hexyldecyl) carbonate (1.12 g,2.5 mmol) was dissolved in tetrahydrofuran, acetonitrile, N- (4-aminobutyl) -acetamide hydrochloride (167 mg,1.0 mmol), potassium carbonate (550 mg,4.0 mmol), potassium iodide (336 mg,2.0 mmol) was added and stirred at 83℃for 16-20h. Cooling to room temperature, filtering, washing the filter residue with dichloromethane, adding saturated sodium bicarbonate solution to the obtained filtrate, extracting with dichloromethane for 2 times, combining organic phases, drying over anhydrous sodium sulfate, filtering and concentrating, and separating by column chromatography to obtain the product 10 (560 mg, pale yellow oil) in 69% yield.
MS m/z(ESI):867.8[M+1]
Example 11
Synthesis of Compound 11
Synthesis of 8-bromo-N- (heptadec-9-yl) octanamide (11 a)
8-bromooctanoic acid (1.12 g,5.0 mmol) was dissolved in 50mL of dichloromethane, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (1.05 g,5.5 mmol) was added in portions at 0deg.C, 9-aminoheptadecane (1.28 g,5.0 mmol) was added dropwise to the reaction solution after stirring for 30min, the mixture was stirred overnight at room temperature after the dropwise addition, TLC showed completion of the reaction, washed 2 times with 100mL of water, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give compound 11a (1.95 g, yellow oil) in 82% yield.
MS m/z(ESI):461.3[M+1]。
Synthesis of Compound 11b
8-bromo-N- (heptadec-9-yl) octanamide (1.15 g,2.5 mmol) was dissolved in tetrahydrofuran, acetonitrile, 4-amino-1-butanol (89.2 mg,1.0 mmol), potassium carbonate (550 mg,4.0 mmol), potassium iodide (336 mg,2.0 mmol) was added, and stirred at 83℃for 16-20h. Cooling to room temperature, filtering, washing the filter residue with dichloromethane, adding saturated sodium bicarbonate solution to the obtained filtrate, extracting with dichloromethane for 2 times, combining organic phases, drying over anhydrous sodium sulfate, filtering and concentrating, and separating by column chromatography to obtain the product 11b (534 mg, pale yellow oil) in 63% yield.
MS m/z(ESI):848.8[M+1];
1 H NMR(300MHz,CDCl 3 ):δ8.10(s,2H),4.21(s,1H),3.46-3.4(m,4H),3.02(t,6H,J=6.2Hz),2.14(t,4H,J=4.8Hz),1.57-1.47(t,14H,J=6.3Hz),1.36-1.26(m,66H),0.90(t,12H,J=6.3Hz)。
Synthesis of Compound 11
Compound 11b (1.70 g,2 mmol) was slowly added to a solution of lithium aluminum hydride (379 mg,10 mmol) in anhydrous tetrahydrofuran (10 ml) at 0deg.C and the mixture was heated to reflux for 5 hours. After the reaction is completed, the temperature is reduced, and water is added into the system to completely decompose the excessive reducing agent. The residue was filtered, washed with ethyl acetate, and the resulting filtrate was washed with water, dried over anhydrous sodium sulfate, filtered and concentrated to give compound 11 (1.45 g, yellow oil) in 90% yield.
MS m/z(ESI):820.8[M+1];
1 H NMR(300MHz,CDCl 3 ):δ4.11(s,1H),3.44(t,2H,J=4.8Hz),3.32(s,2H),3.00(t,6H,J=6.3Hz),2.52(t,4H,J=6.3Hz),2.48-2.43(m,2H),1.61-1.56(m,2H),1.36-1.26(m,82H),0.86(t,12H,J=4.8Hz)。
Example 12 lipid nanoparticle encapsulation of rabies virus G protein mRNA antigen
The invention prepares lipid nanoparticle nucleic acid vaccine by using cationic lipid I-II and control lipid III-IV respectively, and the four cationic lipid structures are shown in the table below.
Table 1: cationic lipid structural formula
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Diluting rabies virus mRNA vaccine stock solution with the concentration of 135 mug/ml by using 100mM sodium acetate buffer solution (pH 4.0), wherein the vaccine stock solution contains coding rabies virus G protein, the antigen sequence is shown as SEQ ID NO. 1, the target antigen is subjected to conventional modification, the N end contains 5'UTR and cap structure, and the C end contains 3' UTR, polyA tail and other designs; the diluted vaccine stock solution is prepared according to cationic lipid: DSPC: cholesterol: preparing a lipid mixed solution with a DMG-PEG2000 molar ratio of 43:10:45:2; setting the flow rate ratio of the mRNA solution to the lipid mixed solution to 3, wherein the total flow rate of the nano-drug manufacturing equipment is 12 ml/min: 1 and beginning encapsulation, after encapsulation, collecting the sample by ultrafiltration of the liquid by a tangential flow filtration system, and adding sucrose solution. The experiments were performed at different N/P (ionizable cationic lipid to nucleotide phosphate) molar ratios (N/P molar ratios of 3.6, 5.6, 7.6, respectively). The samples were taken to examine the encapsulation efficiency, the average particle diameter, PDI and Zeta potential, and the results are shown in the following table.
Table 2: lipid nanoparticle mRNA vaccine detection results after encapsulation of different cationic lipids
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From the above results, it can be seen that the encapsulation efficiency of the samples prepared from the cationic lipids 1,2, 6, 7, 8, 9, 10, 11, 12, 13 and 14 is higher than that of the samples prepared from the cationic lipid 3 and the control cationic lipids 4 and 5 under the same N/P condition, and thus the cationic lipids 1,2, 6, 7, 8, 9, 10, 11, 12, 13 and 14 have better encapsulation effect on mRNA antigens. The encapsulation efficiency of group 3 is slightly higher than that of groups 4 and 5.
EXAMPLE 13 investigation of the stability of LNP-mRNA prepared from different cations
The LNP-mRNA prepared in example 14 different cationic lipids were placed in a constant temperature incubator at 25℃for 1,2, 3 and 4 weeks to examine the stability, and the results are shown in FIG. 1, FIG. 2, FIG. 3, FIG. 4, table 3, table 4, table 5 and Table 6.
TABLE 3 storage stability-average particle size
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TABLE 4 storage stability-encapsulation efficiency (%)
TABLE 5 storage stability-mRNA integrity (%)
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TABLE 6 storage stability-PDI Change
The results show that the encapsulation efficiency and mRNA integrity of the samples of groups 1,2, 6, 7, 8, 9, 10, 11, 12, 13 and 14 did not significantly decrease within 4 weeks, the average particle size and PDI did not significantly increase within 4 weeks, and the average particle size and PDI of the lipid nanoparticles remained essentially unchanged within 4 weeks, exhibiting better stability compared to groups 3, 4 and 5. The stability of group 3 is better than that of groups 4 and 5.
EXAMPLE 14 immunization and detection of mice
1. Humoral immunity evaluation of rabies virus lipid nanoparticle mRNA vaccine
Female BALB/c mice of 6-8 weeks of age were randomly divided into 14 groups of 6 mice/group and immunized by the immunization route of hind leg intramuscular injection. Of these, 1-14 groups of immune samples 1-14 (prepared in example 12). As shown in FIG. 5, the single immunization dose was 5. Mu.g mRNA-LNP on day 0 and day 14, respectively. The serum was collected and isolated on days 14 and 28 of immunization, and antibody titer was measured, and the measurement results are shown in table 7 and fig. 6.
TABLE 7 lipid nanoparticle mRNA vaccine neutralizing antibody titre following encapsulation of different cationic lipids
The serum neutralization antibody titer detection result shows that the rabies virus mRNA vaccine prepared by the invention can stimulate organisms to generate higher protective neutralization antibodies in 14 days after immunization, and the protective neutralization antibody is obviously higher than 0.5IU/ml (the functional antibody response of the WHO standard is fully higher than 0.5 IU/ml); after 14d booster immunization, 28d serum neutralizing antibody titers were greatly increased, and the titers of lipid nanoparticle mRNA vaccines made with cationic lipids 1,2, 6, 7, 8, 9, 10, 11, 12, 13 and 14 were higher than the titers of vaccines made with cationic lipids 3, 4 and 5. The vaccine titer of group 3 was higher than that of groups 4 and 5.
2. Evaluation of cellular immune response on BALB/c mouse model
Samples 1-14 prepared in the examples (numbered mRNA-LNP1 to mRNA-LNP 14) were evaluated for cellular immune responses in BALB/c mouse models, respectively. Female BALB/c mice of 6-8 weeks of age were randomly divided into 14 groups at 8/group, as shown in FIG. 5, BALB/c mice were immunized with 5. Mu.g of mRNA-LNP on days 0 and 14, splenocytes were isolated from the immunized mice after blood collection on day 28, and stimulated with overlapping peptide pools of rabies virus G antigen, and cytokine-producing cells were detected by intracellular cytokine staining flow cytometry (ICS) method. The results are shown in tables 8, 9, 10, 11, and fig. 7 and 8.
TABLE 8 percentage of TNF- α production by specific CD4+ T cells
TABLE 9 percentage of IFN-gamma production by specific CD4+ T cells
TABLE 10 percentage of TNF- α production by specific CD8+ T cells
TABLE 11 percentage of IFN-gamma production by G-specific CD8+ T cells
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The prepared mRNA vaccine not only induces Th1 deflection reaction, but also can obviously activate CD8+ T cell reaction, and the cellular immune reaction of the lipid nanoparticle mRNA vaccine prepared by cationic lipids 1,2, 6, 7, 8, 9, 10, 11, 12, 13 and 14 is better than that of the lipid nanoparticle mRNA vaccine prepared by cationic lipids 3, 4 and 5, and 3 groups are slightly better than 4 and 5 groups.
In conclusion, the mRNA vaccine prepared by the invention has better potential for preventing rabies virus.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Claims (29)
1. A cationic lipid, characterized in that said cationic lipid has the structure of formula I:
wherein:
L 1 and L 2 At least one of which is-O-, -O (C=O) O- - (c=o) NRa-, -NRa (c=o) -or-NRa-,
And, in addition, the processing unit,
L 1 or L 2 is-O-, -O (c=o) O-, -NRa-, - (c=o) -, -NRa-, -O (c=o) -, - (c=o) O-, -C (=o) -, -S (O) x-, -S-, -C (=o) S-, -SC (=o) -, -NRaC (=o) NRa-, -OC (=o) NRa-or-NRaC (=o) O-;
G 1 and G 2 Each independently is unsubstituted C 1 -C 12 Alkylene or C 1 -C 12 Alkenylene;
G 3 is C 1 -C 24 Alkylene, C 1 -C 24 Alkenylene, C 3 -C 8 Cycloalkylene, C 3 -C 8 A cycloalkenyl group;
ra is H or C 1 -C 12 A hydrocarbon group;
R 1 and R is 2 Each independently is C 6 -C 24 Alkyl or C 6 -C 24 Alkenyl groups;
R 3 is H, OH OR 4 、CN、-C(=O)OR 4 、-OC(=O)R 4 or-NR 5 C(=O)R 4 ;
R 4 Is C 1 -C 12 A hydrocarbon group;
R 5 is H or C 1 -C 6 A hydrocarbon group;
x is 0, 1 or 2.
2. The cationic lipid of claim 1, wherein the cationic lipid has the structure L in formula I 1 And L 2 Each independently selected from-O-, -O (c=o) O-, - (c=o) NH-, -NH (c=o) -and-NH-.
3. The cationic lipid according to any one of claims 1-2, wherein said L in the cationic lipid formula I structure 1 And L 2 Are all-O-, or L 1 And L 2 Are all-O (C=O) O-, or L 1 And L 2 Are all-NH-, or L 1 is-NH (C=O) -, L 2 Is- (C=O)NH-。
4. A cationic lipid according to any one of claims 1 to 3, wherein the cationic lipid has the following structure (IA):
wherein:
R 6 at each occurrence independently H, OH or C 1 -C 24 A hydrocarbon group;
n is an integer from 1 to 15.
5. The cationic lipid according to any one of claims 1 to 4, wherein the cationic lipid has the following structure (IB):
wherein y and z are each independently integers from 1 to 12.
6. The cationic lipid according to any one of claims 1-5, wherein n is an integer from 2 to 12, preferably n is 2, 3, 4, 5 or 6; wherein y and z are each independently integers from 2 to 10, preferably from 4 to 9.
7. The cationic lipid according to any one of claims 1-6, wherein R in the cationic lipid structure 1 And R is 2 Each independently has the following structure:
wherein:
R 7a and R is 7b At each occurrence independently H or C 1 -C 12 A hydrocarbon group; and a is an integer from 2 to 12, preferably a is an integer from 8 to 12;
wherein R is 7a 、R 7b And a are each selected such that R 1 And R is 2 Each independently comprising 6 to 20 carbon atoms.
8. The cationic lipid according to any one of claims 1-7, wherein R occurs at least once in the cationic lipid structure 7a Is H, preferably R 7a H at each occurrence.
9. The cationic lipid according to any one of claims 1-8, wherein R occurs at least once in the cationic lipid structure 7b Is C 1 -C 8 A hydrocarbon group; preferably, wherein C 1 -C 8 The hydrocarbyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl or n-octyl.
10. The cationic lipid according to any one of claims 1-9, wherein R in the cationic lipid structure 1 Or R is 2 Or both have one of the following structures:
11. the cationic lipid according to any one of claims 1-10, wherein the cationic lipid compound has the following structure:
12. a lipid nanoparticle comprising: the cationic lipid, non-cationic lipid and/or polyethylene glycol (PEG) -lipid conjugate of any one of claims 1-11, preferably comprising: cationic lipids, neutral phospholipids, steroidal lipids and/or polyethylene glycol (PEG) -lipid conjugates.
13. The lipid nanoparticle of claim 12, wherein the polyethylene glycol (PEG) -lipid conjugate is selected from the group consisting of: 2- [ (polyethylene glycol) -2000] -N, N-tetracosylacetamide (ALC-0159), 1, 2-dimyristoyl-sn-glycerogethoxy polyethylene glycol (PEG-DMG), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ amino (polyethylene glycol) ] (PEG-DSPE, PEG-distteroylglycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycerol amide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), PEG-1, 2-dimyristoyloxypropyl-3-amine (PEG-c-DMA), or DMG-PEG2000, preferably DMG-PEG2000.
14. The lipid nanoparticle according to any one of claims 12-13, wherein the neutral lipid is selected from the group consisting of 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-di-palmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-di-oleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-di-palmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1, 2-di-myristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-di-oleoyl-sn-glycero-3-phospho- (1' -rac-glycero)
(DOPG), oleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE), preferably DSPC.
15. The lipid nanoparticle of any one of claims 12-14, wherein the steroid lipid is selected from the group consisting of oat sterol, β -sitosterol, campesterol, ergocalcitol, campesterol, cholestanol, cholesterol, fecal sterol, dehydrocholesterol, desmosterol, dihydroergocalcitol, dihydrocholesterol, dihydroergosterol, black-sea sterol, epicholesterol, ergosterol, fucosterol, hexahydro-cholesterol, hydroxycholesterol, and polypeptide-modified cholesterol; one or more combinations of lanosterol, sitosterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid and lithocholic acid, preferably cholesterol.
16. The lipid nanoparticle of any one of claims 12-15, wherein the cationic lipid comprises 20-60 mole percent of the lipid component, the neutral phospholipid comprises 5-25 mole percent of the lipid component, and the steroid lipid comprises 25-55 mole percent of the lipid component; the polyethylene glycol (PEG) -lipid conjugate accounts for 0.5-15% of the lipid component by mole percent.
17. The lipid nanoparticle of any one of claims 12-16, wherein the cationic lipid: neutral phospholipids: steroid lipid: polyethylene glycol (PEG) -lipid conjugate molar ratio of 30-60:1-20:20-50:0.1-10, preferably, the cationic lipid: neutral phospholipids: steroid lipid: polyethylene glycol (PEG) -lipid conjugate molar ratio was 43:10:45:2 or 40:10:48:2.
18. The lipid nanoparticle of any one of claims 12-17, wherein the vaccine further comprises an additional adjuvant, the adjuvant being one or more of sodium acetate, tromethamine, monobasic potassium phosphate, sodium chloride, dibasic sodium phosphate, sucrose.
19. The lipid nanoparticle according to any one of claims 12 to 18, wherein the nanoparticle has an average particle size of 50 to 200nm or has a net neutral charge at neutral pH or has a polydispersity of less than 0.4.
20. A method of preparing a lipid nanoparticle according to any one of claims 1 to 19, comprising the step of mixing the mRNA after dissolving the cationic lipid, the non-cationic lipid, the polyethylene glycol (PEG) -lipid conjugate to a solvent.
21. The method for preparing lipid nanoparticles according to claim 20, wherein the cationic lipid, neutral phospholipid, steroid lipid, polyethylene glycol (PEG) -lipid conjugate is prepared by dissolving the cationic lipid, neutral phospholipid, steroid lipid, polyethylene glycol (PEG) -lipid conjugate in ethanol, mixing the mixture with diluted mRNA diluent, and performing ultrafiltration, dilution and filtration; preferably, the cationic lipid, neutral phospholipid, steroid lipid and polyethylene glycol (PEG) -lipid conjugate are dissolved into ethanol, and then are mixed with diluted mRNA diluent according to a certain flow rate ratio, and are subjected to ultrafiltration, dilution and filtration to obtain the modified mRNA; preferably, the ultrafiltration mode is tangential flow filtration; more preferably, the mixing means may be turbulent mixing, laminar mixing or microfluidic mixing.
22. The method of preparing lipid nanoparticles according to any one of claims 20 to 21, wherein the diluent is acetate buffer, citrate buffer, phosphate buffer or tris buffer.
23. The method of preparing lipid nanoparticles according to any one of claims 20 to 22, wherein the buffer has a pH of 3 to 6 and a concentration of 6.25 to 200mM.
24. The method of preparing lipid nanoparticles according to any one of claims 20 to 23, wherein the ratio of the flow rate of the lipid mixed solution obtained by dissolving the cationic lipid, the non-cationic lipid, the polyethylene glycol (PEG) -lipid conjugate in the solvent to the flow rate of the solution obtained by diluting the mRNA is 1 to 5:1.
25. The method of preparing lipid nanoparticles according to any one of claims 20 to 24, wherein the N/P is 2 to 10, preferably 3 to 8, more preferably 3, 4, 5, 6, 7, 8 when the mRNA is encapsulated with a lipid, said N/P being the molar ratio of N in the cationic lipid to P in the mRNA mononucleotide.
26. The method of any one of claims 20-25, wherein the ultrafiltrate is selected from the group consisting of: sodium salt and Tris (hydroxymethyl) aminomethane (Tris) salt, preferably the ultrafiltrate pH is 6.0-8.0.
27. The method of preparing lipid nanoparticles according to any one of claims 20 to 26, wherein the formulation is an oral formulation, a liquid formulation, a lyophilized powder formulation, an injection or an inhalation formulation, preferably an intramuscular injection, an intravenous injection, a dry powder inhalation or an aerosol inhalation.
28. A rabies virus lipid nanoparticle mRNA vaccine comprising: mRNA encoding one or more of G protein, G delta protein, gtrimer protein, M protein, gtrimer protein M protein, the mRNA being encapsulated by the lipid nanoparticle of any one of claims 12-27 or by the lipid nanoparticle prepared by ALC-0315.
29. Use of a rabies virus lipid nanoparticle mRNA vaccine of any one of claims 1-19, 28 in the manufacture of a medicament for preventing rabies.
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