CN116514671A - 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 PDF

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Publication number
CN116514671A
CN116514671A CN202310045008.9A CN202310045008A CN116514671A CN 116514671 A CN116514671 A CN 116514671A CN 202310045008 A CN202310045008 A CN 202310045008A CN 116514671 A CN116514671 A CN 116514671A
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lipid
peg
cationic lipid
influenza
cationic
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严志红
王浩猛
李荩
原晋波
刘健
宇学峰
邱东旭
朱涛
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CanSino Biologics Inc
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CanSino Biologics Inc
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    • C07C219/04—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C219/16—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having at least one of the hydroxy groups esterified by an inorganic acid or a derivative thereof
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    • 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
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    • C07C233/34—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
    • C07C233/35—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
    • 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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    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/08—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
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    • C12N2760/16134—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 invention selects the lipid nanoparticle mRNA vaccine prepared by specific cationic lipid, and discovers that the lipid nanoparticle mRNA vaccine has better in vitro stability and immunogenicity compared with the prior art.

Description

Novel ionizable lipids for nucleic acid delivery and LNP compositions and vaccines thereof
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.
Influenza vaccine is used to prevent influenza caused by influenza virus, and is suitable for any healthy person possibly infected with influenza virus, and is inoculated once before epidemic season each year, and immunity can last for one year.
Influenza vaccines are one of the main measures for preventing and controlling influenza. Vaccination with influenza vaccine may reduce the chances of the vaccinator infecting influenza or reduce influenza symptoms.
The proposed component of the seasonal influenza vaccine in the northern hemisphere of 2018-2019, issued By the world health organization of 2 months of 2018, is a tetravalent influenza virus split vaccine which comprises four influenza virus antigen components of H1N1 type, H3N2 type, yamagata type B (By) and Victoria type B (Bv), and the trivalent influenza virus split vaccine comprises three influenza virus antigen components of H1N1 type A, H3N2 type and Victoria type B (Bv) and does not comprise the influenza virus antigen component of Yamagata type B (By). The tetravalent influenza virus split vaccine contains the four influenza virus antigen components of the two types A and the two types B, can cover more influenza epidemic types, and can effectively prevent and control influenza epidemic situations.
Influenza vaccines which are currently marketed are mainly whole virus inactivated vaccines, split vaccines and subunit vaccines, and no mRNA vaccine exists.
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 invention relates to a cationic lipid, which has the structure shown in the following 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.
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) a cationic lipid; (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 47:10:41.5:1.5 or 44:10:44:2.
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 lipid encapsulates the mRNA with an N/P of 2-10, preferably an N/P of 3-9, more preferably an N/P of 3, 4, 5, 6, 7, 8, 9.
Specifically, the ultrafiltrate is selected from the group consisting of: sodium salt and Tris (hydroxymethyl) aminomethane (Tris) salt, preferably the ultrafiltrate pH is 6.5 to 8.5.
The invention provides an influenza virus lipid nanoparticle mRNA vaccine, and particularly relates to an administration mode of oral administration, intramuscular injection, intravenous injection or inhalation.
In particular, the influenza virus lipid nanoparticle mRNA vaccine can be prepared into oral preparations, liquid preparations, freeze-dried powder preparations, injection or inhalation preparations, preferably intramuscular injection, intravenous injection, dry powder inhalation or aerosol inhalation.
The invention relates to an influenza virus lipid nanoparticle mRNA vaccine, which comprises the following components: (a) mRNA encoding influenza virus HA, NA, M protein and/or N protein; (b) a cationic lipid; (c) a non-cationic lipid; (d) polyethylene glycol (PEG) -lipid conjugates.
Specifically, the influenza virus lipid nanoparticle mRNA vaccine comprises: (a) mRNA encoding influenza virus HA, NA, M protein and/or N protein; (b) the cationic lipid; (c) neutral phospholipids, steroidal lipids; (d) polyethylene glycol (PEG) -lipid conjugates.
In particular, the influenza virus lipid nanoparticle mRNA vaccine or the lipid nanoparticle prepared by ALC-0315 is wrapped.
The influenza vaccine encoding antigens are derived from one or more of 4 seasonal influenza virus strains selected from influenza a virus H1N1, H3N3, influenza B virus mountain strain (Yamagata) and/or Victoria strain (Victoria).
Specifically, the amino acid sequence encoded by the mRNA comprises one or more of the sequences set forth in SEQ ID NOs 1-8. Or an amino acid sequence having 80% or more identity to the sequence shown in SEQ ID NO. 1-8, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more or 100% identity.
The amino acid sequence of the HA antigen protein of the H1N1 strain is shown in SEQ ID NO. 1;
the amino acid sequence of the HA antigen protein of the H3N2 strain A is shown as SEQ ID NO. 2;
the amino acid sequence coded by the HA gene of the Victoria strain B is shown as SEQ ID NO. 3;
the amino acid sequence of HA gene codes of the strain of the B type Yamagata is shown as SEQ ID NO. 4;
the amino acid sequence of NA antigen protein of H1N1 strain A is shown as SEQ ID NO. 5;
the NA gene of the A type H3N2 strain codes an amino acid sequence shown as SEQ ID NO. 6;
the NA gene of the strain of the B-type Yamagata strain encodes an amino acid sequence shown in SEQ ID NO. 7;
the NA gene of the Victoria strain B encodes an amino acid sequence shown in SEQ ID NO. 8.
The invention selects specific cationic lipid and combines the non-cationic lipid and polyethylene glycol (PEG) -lipid to prepare lipid nano particles, and experiments show that the lipid nano particles have good in vitro stability and mouse immune response.
The invention relates to the use of nucleic acid-lipid nanoparticle vaccines for the preparation of vaccines for the prevention of cancer, viral infections, bacterial infections, fungal infections.
In particular, the virus infection is selected from influenza virus, and is characterized in that the influenza virus can be selected from influenza A virus and influenza B virus; preferably, the influenza a virus is selected from the group consisting of Hemagglutinin (HA) of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17 and H18; more preferably, wherein the influenza a virus is an influenza a virus of Neuraminidase (NA) selected from the group consisting of N1, N2, N3, N4, N5, N6, N7, N8, N9, N10 and N11; more preferably, the influenza a virus is selected from the group consisting of H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2, H10N8 and H10N7, preferably selected from the group consisting of H1N1, H3N2, H5N1 and H5N8.
The invention has the beneficial effects that:
the invention provides a series of novel cationic lipids, which adopts lipid nano-particles as a delivery system, has better physicochemical properties by constructing brand-new cationic lipids, has encapsulation efficiency and stability obviously superior to those of lipid nano-particle delivery systems prepared from the cationic lipids on the market,
the invention provides an influenza virus mRNA vaccine of lipid nano particles. The influenza antigen mRNA entrapped by the LNP carrier is used for preparing a vaccine, and through two times of animal immunization (intramuscular injection), high-level specific humoral and cellular immune responses are generated in the animal body, and experiments prove that the vaccine prepared by the novel cationic lipid has higher immune efficacy.
Drawings
FIG. 1 is a graph of LNP formulation stability versus average particle size data;
FIG. 2 is a graph of LNP formulation encapsulation efficiency data;
FIG. 3 is a graph showing the mRNA integrity data for LNP formulations;
FIG. 4 is a graph of LNP formulation PDI data;
FIG. 5 shows the BALB/c mouse immunization program;
FIG. 6 shows HAI antibody titers on BALB/c mouse models;
FIG. 7 shows CD4+ T cell frequency of specific secretion of TNF. Alpha., IFN. Gamma. And IL-2 by ICS method on BALB/c mouse model;
FIG. 8 shows CD8+ T cell frequency of specific secretion of TNF. Alpha., IFN. Gamma. And IL-2 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)
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 mRNA antigen of influenza Virus HA
The invention uses cationic lipid 1-14 to prepare lipid nano particle nucleic acid vaccine, 14 cationic lipid structures are shown in the table below.
Table 1: cationic lipid structural formula
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Diluting an influenza virus mRNA vaccine stock solution with 100mM sodium acetate buffer solution (pH 4.0) to a concentration of 120 mug/ml, wherein the vaccine stock solution contains a coding influenza virus HA protein, an antigen sequence is shown as SEQ ID NO.1, a target antigen is subjected to conventional modification, wherein an N end contains a 5'UTR and a cap structure, and a C end contains a 3' UTR, a 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 44:10:44:2; setting the flow rate ratio of the mRNA solution to the lipid mixed solution to 4, 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 tests were carried out under different N/P (ionizable cationic lipid to nucleotide phosphate) molar ratios (N/P molar ratios of 4, 6, 8, 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 under the same N/P conditions, the encapsulation efficiency of the samples prepared from the cationic lipids 1,2, 6, 7, 8, 9, 10, 11, 12, 13 and 14 was higher than that of the sample prepared from the cationic lipid 3, and significantly higher than that of the samples prepared from the control cationic lipids 4 and 5, and that the cationic lipids 1,2, 6, 7, 8, 9, 10, 11, 12, 13 and 14 were found to 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
LNP-mRNA prepared from 14 different cationic lipids in example 12 was placed in a constant temperature incubator at 25℃for 1,2, 3 and 4 weeks, respectively, and their stability was examined. 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 influenza virus lipid nanoparticle mRNA vaccine
Female BALB/c mice of 6-8 weeks of age were randomly divided into 14 groups of 8 mice/group and immunized by the immunization route of hind leg intramuscular injection. 1-14 samples 1-14 (prepared in example 12) were immunized on day 0 and day 14, respectively, as shown in FIG. 5, with a single immunization dose of 5. Mu.g mRNA-LNP, and serum was collected on days 14 and 28 for isolation, and antibody titer assays were performed as shown in Table 7 and FIG. 6:
TABLE 7 lipid nanoparticle mRNA vaccine antibody titres after encapsulation with different cationic lipids
The results of the antibody titer tests showed that the lipid nanoparticle mRNA vaccines prepared from cationic lipids 1,2, 6, 7, 8, 9, 10, 11, 12, 13 and 14 were able to induce higher HAI antibody titers, with No.3 cationic lipid being less effective, and the HAI titers being significantly higher than the lipid nanoparticle mRNA vaccines prepared from control cationic lipids 4 and 5. The vaccine titer of group 3 was higher than that of groups 4 and 5.
2. Evaluation of cellular immune response of influenza Virus lipid nanoparticle mRNA vaccine
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, and immunized with 5. Mu.g of mRNA-LNP on days 0 and 14 by the immunization route of hind leg intramuscular injection as shown in FIG. 5. On day 28, mice were sacrificed and spleen cells were harvested and then stimulated with influenza full-length HA overlapping peptide libraries and cytokine-producing cells were detected by the intracellular cytokine staining flow cytometry (ICS) method. The results are shown in tables 8, 9, 10, 11, 12, 13, and fig. 7 and 8.
TABLE 8 percentage of TNF- α production by HA-specific CD4+ T cells
TABLE 9 percentage of IFN-gamma production by HA-specific CD4+ T cells
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TABLE 10 percentage of IL-2 production by HA-specific CD4+ T cells
TABLE 11 percentage of TNF- α production by HA-specific CD8+ T cells
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TABLE 12 percentage of IFN-gamma production by HA-specific CD8+ T cells
TABLE 13 percentage of IL-2 production by HA-specific CD8+ T cells
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 the 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 the cationic lipids 3, 4 and 5, and the cellular immune reaction of the cationic lipid 3 is better than that of the lipid nanoparticle mRNA vaccine prepared by the cationic lipids 4 and 5.
In conclusion, the mRNA vaccine prepared by the invention has better potential for preventing influenza 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 (31)

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 ofEach 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-, orL is as follows 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 47:10:41.5:1.5 or 44:10:44:2.
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 12 to 19, comprising the step of mixing the cationic lipid, non-cationic lipid, polyethylene glycol (PEG) -lipid conjugate with mRNA after dissolution into 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 claim 21, wherein the diluent is acetate buffer, citrate buffer, phosphate buffer or tris buffer.
23. The method of preparing lipid nanoparticles according to claim 22, wherein the buffer has a pH of 3 to 6 and a concentration of 6.25 to 200mM.
24. The method of any one of claims 21 to 23, wherein the ratio of the flow rate of the lipid mixture 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 the mRNA is 1 to 5:1.
25. The method of preparing lipid nanoparticles according to any one of claims 21 to 24, wherein the lipid encapsulates the mRNA with N/P of 2 to 10, preferably with N/P of 3 to 9, more preferably with N/P of 3, 4, 5, 6, 7, 8, 9, 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 21-25, wherein the ultrafiltrate is selected from the group consisting of: sodium salt and Tris (hydroxymethyl) aminomethane (Tris) salt, preferably the ultrafiltrate pH is 6.5 to 8.5.
27. The method of preparing lipid nanoparticles according to any one of claims 21 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. An influenza lipid nanoparticle mRNA vaccine comprising: mRNA encoding influenza virus hemagglutinin proteins HA, NA, M and/or N proteins, said mRNA being encapsulated by the lipid nanoparticle of any one of claims 12-27 or by the lipid nanoparticle prepared by ALC-0315.
29. The influenza virus lipid nanoparticle mRNA vaccine of claim 28, wherein the influenza vaccine encodes antigens derived from one or more of 4 seasonal influenza virus strains selected from influenza a virus H1N1, H3N3, influenza B virus mountain strain (Yamagata) and/or Victoria strain (Victoria).
30. Use of an influenza virus lipid nanoparticle mRNA vaccine of any one of claims 28-29 in the manufacture of a medicament for preventing influenza.
31. The use according to claim 30, wherein the influenza virus is selected from influenza a virus, influenza B virus; preferably, the influenza a virus is selected from the group consisting of influenza a virus of Hemagglutinin (HA) of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17 and H18; more preferably, the influenza a virus is an influenza a virus of Neuraminidase (NA) selected from the group consisting of N1, N2, N3, N4, N5, N6, N7, N8, N9, N10 and N11; more preferably, the influenza a virus is selected from the group consisting of H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2, H10N8 and H10N7, preferably selected from the group consisting of H1N1, H3N2, H5N1 and H5N8.
CN202310045008.9A 2022-01-30 2023-01-30 Novel ionizable lipids for nucleic acid delivery and LNP compositions and vaccines thereof Pending CN116514671A (en)

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