CN114874146B - Histidine lipid, histidine lipid nanoparticle, and preparation method and application thereof - Google Patents

Histidine lipid, histidine lipid nanoparticle, and preparation method and application thereof Download PDF

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CN114874146B
CN114874146B CN202210305388.0A CN202210305388A CN114874146B CN 114874146 B CN114874146 B CN 114874146B CN 202210305388 A CN202210305388 A CN 202210305388A CN 114874146 B CN114874146 B CN 114874146B
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compound
histidine
lipid
mrna
carbon atoms
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CN114874146A (en
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田伟伟
文中行
徐健
秦兵彬
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王德玲
徐军
陆阳
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Suno Biomedical Technology Suzhou Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Abstract

The invention relates to a histidine lipid, which is a compound A and/or a salt of the compound A, wherein the structural formula of the compound A isWherein R is 1 Selected from hydrocarbon groups having 1 to 24 carbon atoms, X is selected from H, COOR 2 Or COR 2 ，R 2 Selected from hydrocarbon groups having 1 to 24 carbon atoms, R 1 And/or R 2 Having a branched chain. The histidine lipid has the advantages of low toxicity and low immunogenicity, can form histidine nano-particles with high encapsulation rate and small and uniform particle size with mRNA, and can be used for effectively delivering mRNA.

Description

Histidine lipid, histidine lipid nanoparticle, and preparation method and application thereof
Technical Field
The invention relates to histidine lipid, histidine lipid nano-particles, and a preparation method and application thereof.
Background
Messenger ribonucleic acid (mRNA) is a single-stranded ribonucleic acid transcribed from DNA and carrying genetic information to guide protein synthesis, and the treatment method of mRNA is widely studied in the fields of vaccines, tumor immunotherapy and the like. In the new epidemic situation, mRNA vaccine is greatly concerned by mRNA drugs because of the characteristics of high development speed, high protection rate and high safety. LNP lipid nanoparticles (Lipid Nanoparticle, LNP) are widely studied and used in vectors for delivery of mRNA. However, the cationic or cationizable amino lipids used in the currently mainstream LNP system mainly have the problems of high biotoxicity, high immunogenicity and low escape rate of endosomes, and LNP is mainly delivered to the liver, so that the selectivity to other organs is poor.
Based on this, how to reduce the immunogenicity, biotoxicity, and increase the escape rate of endosomes of an mRNA delivery system is an urgent technical problem to be solved.
Disclosure of Invention
The invention aims to provide histidine lipid, histidine lipid nano-particles, and a preparation method and application thereof.
Aiming at the defects in the prior art, the invention adopts the following technical scheme:
a histidine lipid is compound A and/or salt of compound A, wherein the compound A has the structural formula ofWherein R is 1 Selected from hydrocarbon groups having 1 to 24 carbon atoms, X is selected from H, COOR 2 Or COR 2 ，R 2 Selected from hydrocarbon groups having 1 to 24 carbon atoms, wherein R is 1 And/or the R 2 Having a branched chain.
Preferably, said R 1 Said R is 2 Are respectively and independently selected from R 3 (R 4 ) n The method comprises the steps of carrying out a first treatment on the surface of the Wherein R is 3 Selected from alkyl, alkenyl or alkynyl with 1-24 carbon atoms, R 4 Selected from alkyl, alkenyl or alkynyl having 1 to 23 carbon atoms, R 4 And R is the product of the number of carbon atoms and n 3 The sum of the carbon numbers of (2) being less than or equal to 24 and n being selected from 0, 1, 2 or 3.
Further preferably, the R 3 Said R is 4 Respectively selected from straight-chain alkyl, alkenyl or alkynyl.
Still further preferably, when said R 1 Selected from R 3 (R 4 ) n And n is 0, said R 2 Selected from R 3 (R 4 ) n And n is selected from 1, 2 or 3; alternatively, when said R 1 Selected from R 3 (R 4 ) n And n is selected from 1, 2 or 3, said R 2 Selected from R 3 (R 4 ) n And n is selected from 0, 1, 2 or 3.
According to some preferred embodiments, when said R 1 Selected from R 3 (R 4 ) n And n is 0, said R 1 R in (a) 3 Selected from alkyl, alkenyl or alkynyl groups having 10 to 24 carbon atoms, more preferably from 15 to 21 carbon atoms.
Further preferably, the R 2 R in (a) 3 Selected from alkyl, alkenyl or alkynyl groups having 1 to 10 carbon atoms, more preferably alkyl, alkenyl or alkynyl groups having 1 to 5 carbon atoms.
Further preferably, the R 2 R in (a) 4 Selected from alkyl, alkenyl or alkynyl groups having 1 to 10 carbon atoms, more preferably alkyl, alkenyl or alkynyl groups having 1 to 5 carbon atoms.
According to some preferred embodiments, when said R 1 Selected from R 3 (R 4 ) n And n is 1, 2 or 3, said R 1 R in (a) 3 And R is 4 Each independently selected from an alkyl group, an alkenyl group or an alkynyl group having 1 to 10 carbon atoms, more preferably an alkyl group, an alkenyl group or an alkynyl group having 5 to 10 carbon atoms.
Further preferably, the R 2 R in (a) 3 Selected from alkyl, alkenyl or alkynyl groups having 1 to 10 carbon atoms, more preferably alkyl, alkenyl or alkynyl groups having 1 to 5 carbon atomsA base.
Further preferably, the R 2 R in (a) 4 Selected from alkyl, alkenyl or alkynyl groups having 1 to 10 carbon atoms, more preferably alkyl, alkenyl or alkynyl groups having 1 to 5 carbon atoms.
According to some specific and preferred embodiments, the R 1 Selected from the group consisting of And/or, the R 2 Selected from the group consisting of C(CH 3 ) 3 Or CH (CH) 3 。
According to some particular and preferred embodiments, the compound a is selected from
Preferably, the salt of compound A has the structural formulaWherein Y is an acid radical.
Further preferably, the acid groups are selected from CF 3 COO - 、CH 3 COO - 、Cl - 、SO 4- Or Br (Br) - 。
According to some particular and preferred embodiments, the salt of compound a is selected from
The invention also provides a preparation method of the histidine lipid, which comprises the steps of reacting a compound B with alcohol to generate the compound A, and then selectively reacting the compound A with acid to generate salt of the compound A; wherein the structural formula of the compound B isX in the compound B is the same as X in the compound a; the alcohol is R 1 OH, R in the alcohol 1 R in said compound A 1 The same; the acid is H n Y n- Y in the acid is the same as Y in the salt of the compound A.
The invention also provides the use of one or more of the histidine lipids described above or one or more of the histidine lipids produced by the production method described above for delivering one or more of mRNA, siRNA, miRNA or antisense oligonucleotides.
The present invention also provides a histidine lipid nanoparticle comprising one or more of mRNA and histidine lipid as described above or one or more of histidine lipid produced by the production method as described above.
The invention also provides the use of a histidine lipid nanoparticle as described above in the delivery of one or more of mRNA, siRNA, miRNA or antisense oligonucleotides.
The implementation of the invention has at least the following beneficial effects:
the histidine lipid has the advantages of low toxicity and low immunogenicity, can form histidine nano-particles with high encapsulation rate and small and uniform particle size with mRNA, and can be used for effectively delivering mRNA.
Drawings
FIG. 1 is a schematic representation of the preparation of mRNA/HLNP (histidine lipid nanoparticle) in an embodiment of the present invention;
FIG. 2 is an agarose gel electrophoresis diagram in an embodiment of the invention;
FIG. 3 is a fluorescence image of a mouse in an embodiment of the invention, wherein 1-5 are respectively the fluorescence images of the mouse after 24 hours of intramuscular injection of 5 mRNA/HLNP samples;
FIG. 4 is a schematic diagram showing administration and blood sampling time of mice in an embodiment of the present invention;
FIG. 5 is a schematic diagram of luciferase antibody detection in an embodiment of the invention.
Detailed Description
Among vectors for mRNA delivery, LNP is widely used, but the ionizable lipids in the current LNP are usually synthetic amino compounds, and there are mainly problems of high toxicity and high immunogenicity. In order to solve the problems, the inventor of the present invention has long studied and put forward a technical scheme of the present invention in a great deal of practice. The technical scheme, the implementation process, the principle and the like are further explained as follows.
A histidine lipid is compound A and/or salt of compound A, wherein the structural formula of compound A isWherein R is 1 Selected from hydrocarbon groups having 1 to 24 carbon atoms, X is selected from H, COOR 2 Or COR 2 ，R 2 Selected from hydrocarbon groups having 1 to 24 carbon atoms, R 1 And/or R 2 Having a branched chain. The histidine lipids in the present invention may be isomers of the above structures, for example, may be in D-configuration and/or L-configuration.
According to the invention, R 1 、R 2 Are respectively and independently selected from R 3 (R 4 ) n The method comprises the steps of carrying out a first treatment on the surface of the Wherein R is 3 Selected from alkyl, alkenyl or alkynyl with 1-24 carbon atoms, R 4 Selected from alkyl, alkenyl or alkynyl having 1 to 23 carbon atoms, R 4 And R is the product of the number of carbon atoms and n 3 The sum of the carbon numbers of (2) being less than or equal to 24 and n being selected from 0, 1, 2 or 3.
Further, R 3 And R is 4 Are all straight chain alkyl, alkenyl or alkynyl groups.
In some embodiments, when R 1 Selected from R 3 (R 4 ) n And n is 0, R 2 Selected from R 3 (R 4 ) n And n is selected from 1, 2 or 3.
Preferably, R 1 R in (a) 3 Selected from alkyl, alkenyl or alkynyl of 5-24 carbon atoms, R 3 The number of carbon atoms of (2) may be, for example, 6, 8, 10, 12, 14, 16, 17, 18, 19, 20, 22, etc.
Preferably, R 2 R in (a) 3 Selected from alkyl, alkenyl or alkynyl with 1-10 carbon atoms, R 3 The number of carbon atoms of (2) may be, for example, 1, 2, 3, 4, 5, 7, 9, etc. R is R 3 Examples thereof include methyl, ethyl, n-propyl, n-butyl, n-pentyl, methyl, vinyl, ethynyl, and propenyl. R is R 2 R in (a) 4 Selected from alkyl, alkenyl or alkynyl with 1-10 carbon atoms, R 4 The number of carbon atoms of (2) may be, for example, 1, 2, 3, 4, 5, 7, 9, etc. R is R 4 Examples thereof include methyl, ethyl, n-propyl, n-butyl, n-pentyl, methyl, vinyl, ethynyl, and propenyl.
In some embodiments, when R 1 Selected from R 3 (R 4 ) n And n is selected from 1, 2 or 3, R 2 Selected from R 3 (R 4 ) n And n is selected from 0, 1, 2 or 3.
Preferably, R 1 R in (a) 3 Selected from alkyl, alkenyl or alkynyl with 1-10 carbon atoms, R 3 The number of carbon atoms of (2) may be, for example, 1, 2, 3, 4, 5, 7, 9, etc. R is R 3 Examples thereof include methyl, ethyl, n-propyl, n-butyl, n-pentyl, methyl, vinyl, ethynyl, and propenyl. R is R 1 R in (a) 4 Selected from alkyl, alkenyl or alkynyl with 1-10 carbon atoms, R 4 The number of carbon atoms of (2) may be, for example, 1, 2, 3, 4, 5, 7, 9, etc. R is R 4 Examples thereof include methyl, ethyl, n-propyl, n-butyl, n-pentyl, methyl, vinyl, ethynyl, and propenyl.
Preferably, R 2 R in (a) 3 Selected from alkyl, alkenyl or alkynyl with 1-10 carbon atoms, R 3 The number of carbon atoms of (C) may be, for example, 1,2. 3, 4, 5, 7, 9, etc. R is R 3 Examples thereof include methyl, ethyl, n-propyl, n-butyl, n-pentyl, methyl, vinyl, ethynyl, and propenyl. R is R 2 R in (a) 4 Selected from alkyl, alkenyl or alkynyl with 1-10 carbon atoms, R 4 The number of carbon atoms of (2) may be, for example, 1, 2, 3, 4, 5, 7, 9, etc. R is R 4 Examples thereof include methyl, ethyl, n-propyl, n-butyl, n-pentyl, methyl, vinyl, ethynyl, and propenyl.
In accordance with the present invention, in some specific and preferred embodiments, R 1 Selected from the group consisting of
R 2 Selected from the group consisting of C(CH 3 ) 3 Or CH (CH) 3 。
The compound A may be, for example
Or an isomer of the above structure.
According to the invention, the salt of compound A has the formulaWherein Y is an acid radical. Acid radicals are selected from CF 3 COO - 、CH 3 COO - 、Cl - 、SO 4- Or Br (Br) - 。
In some specific and preferred embodiments, the salt of compound a is selected from
Or an isomer of the above structure.
A method for producing histidine lipid, comprising reacting compound B with alcohol to produce the above compound A, and then optionally reacting compound A with acid to produce a salt of compound A; wherein the structural formula of the compound B is
X in compound B is the same as X in compound A; the alcohol is R 1 OH, R in alcohol 1 With R in compound A 1 The same; the acid is H n Y n- Y in the acid is the same as Y in the salt of Compound A.
According to the present invention, the reaction temperature of the compound B with the alcohol is controlled to be 0 to 100. For example, 0. DegreeC, 10. DegreeC, 20. DegreeC, 30. DegreeC, 40. DegreeC, 50. DegreeC, 60. DegreeC, 70. DegreeC, 80. DegreeC, 90. DegreeC, 100. DegreeC, etc. can be used.
Further, the reaction time of the compound B with the alcohol is controlled to be 1 to 50 hours, and may be, for example, 1 hour, 5 hours, 10 hours, 15 hours, 18 hours, 20 hours, 23 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours.
According to the invention, the molar ratio of compound B to alcohol fed is 1: (0.1 to 10), for example, 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, etc. are possible.
In some embodiments, compound B and the alcohol are reacted in the presence of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, 4-dimethylaminopyridine, diisopropylethylamine, and a solvent. The molar ratio of the compound B to the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is 1 (1-5), and can be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:5 and the like. The feeding mole ratio of the compound B to the 4-dimethylaminopyridine is 1: (0.1 to 1), for example, 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, etc. are possible. The feeding mole ratio of the compound B to the diisopropylethylamine is 1: (1-10), for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, etc. are possible.
In other embodiments, compound B and the alcohol are reacted in the presence of dicyclohexylcarbodiimide, 4-dimethylaminopyridine and a solvent. The feeding mole ratio of the compound B to dicyclohexylcarbodiimide is 1: (0.5 to 5), for example, 1:0.5, 1:1, 1:1.5, 1:2, 1:3, 1:4, etc. are possible. The feeding mole ratio of the compound B to the 4-dimethylaminopyridine is 1: (0.5 to 5), for example, 1:0.5, 1:1, 1:1.5, 1:2, 1:3, 1:4, etc. are possible.
Solvents include, but are not limited to, one or more of methylene chloride, chloroform, tetrahydrofuran, N-dimethylformamide.
According to the present invention, the reaction temperature of the compound A with the acid is controlled to be 0 to 50℃and may be, for example, 0℃10℃20℃30℃40℃50 ℃.
Further, the reaction time of the compound A with the acid is controlled to be 0.1 to 5 hours, and for example, 0.1 hours, 0.5 hours, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, and the like can be used.
According to the invention, the molar ratio of compound a to acid fed is 1: (0.5-100), for example, 1:1, 1:2, 1:3, 1:5, 1:10, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, etc. are possible.
According to the invention, compound a is reacted with an acid in the presence of a solvent. Acids include, but are not limited to, trifluoroacetic acid.
A histidine lipid nanoparticle comprising mRNA and histidine lipid as described above.
According to the present invention, the histidine lipid nanoparticle further comprises one or more of helper lipids, cholesterol, polyethylene glycol lipids. Wherein the auxiliary lipid comprises but is not limited to DSPC and/or DOPE, and the polyethylene glycol lipid comprises but is not limited to one or more of PEG2000-DMG, PEG2000-C-DMG, and PEG 2000-DSPE.
A method for preparing histidine lipid nanoparticles, comprising the steps of:
(1) Dissolving histidine lipids, helper lipids, cholesterol, and polyethylene glycol lipids in an organic solvent;
(2) Dissolving mRNA in a buffer;
(3) Mixing the solutions obtained in the step (1) and the step (2);
(4) Diluting the product obtained in the step (3) by using PBS buffer solution, and ultrafiltering.
According to the invention, the feeding mass ratio of histidine lipid to auxiliary lipid is 1: (0.1 to 0.5), for example, 1:0.2, 1:0.25, 1:0.3, 1:0.35, 1:0.4, and the like are possible. The feeding mass ratio of histidine lipid to cholesterol is 1: (0.3 to 0.8), for example, 1:0.4, 1:0.45, 1:0.5, 1:0.55, 1:0.6, 1:0.7, etc. are possible. The feeding mass ratio of histidine lipid to polyethylene glycol lipid is 1: (0.1 to 0.2), for example, 1:0.12, 1:0.14, 1:0.16, 1:0.18, etc. are possible. The mass ratio of histidine lipid to mRNA is (5-15): 1, for example, may be 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, etc.
According to the present invention, the histidine lipid concentration in the system of step (1) is controlled to be 1 to 10mg/mL, and may be, for example, 2mg/mL, 4mg/mL, 6mg/mL, 8mg/mL, or the like.
According to the present invention, the pH of the buffer in step (2) is controlled to 3 to 4. The mass concentration of mRNA in the system of the step (2) is controlled to be 0.1 to 0.5mg/mL, and for example, may be 0.15mg/mL, 0.2mg/mL, 0.25mg/mL, 0.3mg/mL, etc.
According to the present invention, the dilution in the control step (4) is 5 to 15 times, for example, 5, 8, 10, 13, etc.
According to the invention, the ultrafiltration in the step (4) is controlled to be carried out at 0-10 ℃ until the original volume is reached.
The following describes specific embodiments of the present invention in detail with reference to the drawings. It should be understood that the detailed description set forth herein is intended to illustrate and explain the invention, but is not intended to limit the invention in any way.
Unless otherwise indicated, the room temperature referred to in the examples below refers to 20 to 25 ℃.
The m/z values of the salts of compound a referred to in the examples below only comprise the main part and do not include acid groups.
The "yield" referred to in the examples below all refer to "molar yield".
Example 1
Synthesis of N-t-butoxycarbonyl-L-histidine-9-heptadecyl ester (Compound 1) and L-histidine-9-heptadecyl ester bistrifluoroacetate (Compound 2)
The synthetic routes for compound 1 and compound 2 are as follows:
N-t-Butoxycarbonyl-L-histidine (300 mg,1.18mmol,1 eq.) 9-heptadecanol (840 mg,3.3mmol,2.8 eq.) 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 563mg,2.95mmol,2.5 eq.) 4-dimethylaminopyridine (DMAP, 57mg,0.47mmol,0.4 eq.) were added to a 25mL reaction flask and N-terminal was used for 20 times with a three-way joint 2 The reaction flask was replaced with hot air, and the water vapor in the flask was removed. Diisopropylethylamine (DIPEA, 1.3mL,7.55mmol,6.4 eq.) and dichloromethane (DCM, 10 mL) were mixed and added together to the reaction flask. Heating in 50 ℃ water bath, tracking reaction by LC-MS, and stopping reaction after 22.5 hours. The reaction was poured into 40mL of DCM, extracted with 40mL of saturated aqueous sodium bicarbonate, the aqueous layer was washed 4 times with DCM, the combined organic layers were dried over sodium sulfate, filtered and concentrated, and the crude product obtained was purified by column chromatography on silica gel, eluent DCM/MeOH50:1-40:1-35:1, giving 144.5mg of compound 1 as pale yellow oil in 25% yield.
Characterization data for compound 1 are as follows:
ESI:m/z＝494.16[M+H]+.
1 H NMR(400MHz,CDCl 3 )δ7.54(s,1H),6.81(s,1H),5.72(s,1H),4.85(p,J＝6.1Hz,1H),4.50(s,1H),3.15–2.99(m,2H),1.56–1.45(m,4H),1.42(s,9H),1.25(s,24H),0.87(dt,J＝6.9,1.8Hz,6H).
13 C NMR(100MHz,CDCl 3 )δ172.14,155.73,135.15,79.84,77.36,76.00,53.83,34.03,34.00,31.99,31.97,29.66,29.65,29.60,29.39,28.44,25.32,25.23,22.78,14.23.
in a 25mL reaction flask, compound 1 (124.6 mg,0.25 mmol) was charged and the flask was purged with N 2 After displacement, DCM (4 mL) was added followed by trifluoroacetic acid (TFA, 1 mL). The reaction was carried out at room temperature, followed by LC-MS, and after 2h the reaction was stopped. The reaction solution was concentrated under reduced pressure at room temperature, then 10mL of ethanol was added thereto, and after removing ethanol under reduced pressure at room temperature, then 10mL of DCM was added thereto, and DCM was removed under reduced pressure at room temperature and pumped with oil to give 156.7mg of Compound 2 as pale yellow foam in 99% yield.
Characterization data for compound 2 are as follows:
ESI:m/z＝394.38[M+H]+.
1 H NMR(400MHz,DMSO-d 6 )δ9.02(s,1H),7.47(s,1H),4.83(p,J＝6.3Hz,1H),4.43(t,J＝7.4Hz,1H),3.34–3.12(m,2H),1.54–1.36(m,4H),1.22(s,24H),0.89–0.78(m,6H).
13 C NMR(100MHz,DMSO-d 6 )δ168.07,158.95,158.62,134.55,127.09,118.12,76.56,51.01,33.12,33.08,31.30,31.28,28.91,28.85,28.82,28.71,28.69,25.55,24.52,24.48,22.11,13.94.
example 2
Synthesis of N-Boc-D-histidine-9-heptadecyl ester (Compound 3) and D-histidine-9-heptadecyl ester bistrifluoroacetate (Compound 4)
The synthetic routes for compound 3 and compound 4 are as follows:
N-t-Butoxycarbonyl-D-histidine (300 mg,1.18mmol,1 eq.) 9-heptadecanol (840 mg,3.3mmol,2.8 eq.), EDCI (563 mg,2.95mmol,2.5 eq.), DMAP (57 mg,0.47mmol,0.4 eq.) were added to a 25mL reaction flask and N was performed 20 times with a three-way joint 2 The reaction flask was replaced with hot air, and the water vapor in the flask was removed. DIPEA (1.3 mL,7.55mmol,6.4 eq.) and DCM (10 mL) were mixed and added together to the reaction flask. Heating in 50 ℃ water bath, tracking reaction by LC-MS, and stopping reaction after 17.5 hours. The reaction was poured into 40mL DCM and 40mL full was addedAnd aqueous sodium bicarbonate, the aqueous layer was washed 4 times with DCM, the organic layers were combined, dried over sodium sulfate, filtered, and concentrated, the resulting crude product was purified by column chromatography over silica gel, eluent DCM/MeOH50:1-40:1-35:1, to give 107mg of compound 3 as a pale yellow oil in 18% yield.
ESI:m/z＝494.56[M+H]+.
1 H NMR(400MHz,CDCl 3 )δ7.52(s,1H),6.79(s,1H),5.78(s,1H),4.85(p,J＝6.2Hz,1H),4.49(d,J＝7.7Hz,1H),3.10(dt,J＝13.9,6.9Hz,2H),1.56–1.45(m,4H),1.41(s,9H),1.23(s,24H),0.86(t,J＝6.6Hz,6H).
13 C NMR(100MHz,CDCl 3 )δ172.13,155.73,135.13,79.85,77.36,76.01,53.82,34.03,34.00,31.99,31.98,29.89,29.67,29.65,29.60,29.39,28.44,25.32,25.23,22.78,14.23.
In a 25mL reaction flask, compound 3 (73.75 mg,0.15 mmol) was charged, and the flask was purged with N 2 After displacement, DCM (4 mL) was added followed by trifluoroacetic acid (1 mL). The reaction was carried out at room temperature, followed by LC-MS, and after 2h the reaction was stopped. After the reaction solution was concentrated under reduced pressure at room temperature, 10mL of ethanol was further added thereto, after the ethanol was removed under reduced pressure at room temperature, 10mL of DCM was further added thereto, after the DCM was removed under reduced pressure at room temperature, 10mL of cyclohexane was further added thereto, the cyclohexane was removed under reduced pressure at room temperature and the mixture was pumped with an oil pump for about 2 hours to give 75.17mg of a white foam-like compound 4 in a yield of 81%.
ESI:m/z＝394.34[M+H]+.
1H NMR(400MHz,DMSO-d 6 )δ8.96(s,1H),7.45(s,1H),4.83(p,J＝6.1Hz,1H),4.42(t,J＝7.4Hz,1H),3.27(dd,J＝15.4,6.6Hz,1H),3.18(dd,J＝15.4,8.1Hz,1H),1.57–1.36(m,4H),1.23(s,24H),0.85(td,J＝6.9,2.2Hz,6H).
13 C NMR(100MHz,DMSO-d 6 )δ168.10,158.78,158.46,158.15,127.38,118.50,117.94,115.53,76.54,51.05,33.11,33.07,31.28,31.26,28.88,28.83,28.80,28.69,28.67,25.65,24.49,24.47,22.09,13.94.
Example 3
Synthesis of N-Boc-L-histidine- (2-hexyl) -decyl ester (Compound 5) and L-histidine- (2-hexyl) -decyl ester bistrifluoroacetate (Compound 6)
The synthetic routes for compound 5 and compound 6 are as follows:
N-t-Butoxycarbonyl-L-histidine (300 mg,1.18mmol,1 eq.) EDCI (563 mg,2.95mmol,2.5 eq.) DMAP (57 mg,0.47mmol,0.4 eq.) was added to a 25mL reaction flask and N was performed 20 times with a three-way joint 2 The reaction flask was replaced with hot air, and the water vapor in the flask was removed. 2-hexyl-decanol (1.7 mL,5.9mmol,5 eq.) DIPEA (1.3 mL,7.55mmol,6.4 eq.) and DCM (8 mL) were mixed and added to the reaction flask. The reaction was followed by LC-MS and stopped after 26h at room temperature. The reaction was poured into 40mL of DCM, and 40mL of saturated aqueous sodium bicarbonate was added for extraction, the aqueous layer was washed 4 times with DCM, the combined organic layers were dried over sodium sulfate, filtered, and concentrated, the crude product obtained was purified by column chromatography on silica gel, eluent DCM/MeOH 45:1-40:1-30:1, yielding 436mg of compound 5 as a colourless oil, 77% yield.
Characterization data for compound 5 are as follows:
ESI:m/z＝480.50[M+H]+.
1 H NMR(400MHz,CDCl 3 )δ7.53(d,J＝1.1Hz,1H),6.79(s,1H),5.78(s,1H),4.54(d,J＝7.9Hz,1H),3.99(d,J＝5.6Hz,2H),3.09(d,J＝5.2Hz,2H),1.64–1.55(m,1H),1.42(s,9H),1.25(s,24H),0.87(t,J＝6.7Hz,6H).
a25 mL reaction flask was charged with Compound 5 (300 mg,0.63 mmol) and the flask was purged with N 2 After displacement, DCM (6 mL) was added followed by trifluoroacetic acid (1.5 mL). The reaction was followed by LC-MS and stopped after 2.5h at room temperature. After the reaction solution was concentrated under reduced pressure at room temperature, 10mL of ethanol was further added thereto, and after the ethanol was removed under reduced pressure at room temperature, 10mL of DCM was further added thereto, and the DCM was removed under reduced pressure at room temperature and pumped with an oil pump for 1 hour, 448.6mg of Compound 6 as a white powder was obtained in 99% yield.
Characterization data for compound 6 are as follows:
ESI:m/z＝380.43[M+H]+.
1 H NMR(400MHz,CDCl 3 )δ8.46(s,1H),7.29(s,1H),4.53–4.43(m,1H),4.24–4.07(m,2H),3.64–3.29(m,2H),1.68(s,1H),1.35–1.16(m,24H),0.86(t,J＝6.7Hz,6H).
13 C NMR(100MHz,DMSO-d 6 )δ168.48,158.82,158.50,134.60,127.40,118.50,117.91,115.54,68.24,51.13,36.45,31.31,31.25,30.15,29.26,29.23,28.97,28.93,28.90,28.69,26.00,25.92,25.61,22.11,22.08,13.94.
example 4
Synthesis of N-Boc-L-histidine-oleyl ester (Compound 7) and L-histidine-oleyl ester bistrifluoroacetate (Compound 8)
The synthetic routes for compound 7 and compound 8 are as follows:
N-t-Butoxycarbonyl-L-histidine (300 mg,1.18mmol,1 eq.) in a 25mL reaction flask, oleyl alcohol (1.4 mL,3.54mmol,3.0 eq.), EDCI (676 mg,3.53mmol,3.0 eq.), DMAP (57.5 mg,0.47mmol,0.4 eq.) were added and N-well was performed 20 times with a three-way joint 2 The reaction flask was replaced with hot air, and the water vapor in the flask was removed. DIPEA (1.3 mL,7.55mmol,6.4 eq.) and DCM (8 mL) were mixed and added to the reaction flask. The reaction was followed by LC-MS at room temperature and stopped after 22 h. The reaction was poured into 40mL of DCM, extracted with 40mL of saturated aqueous sodium bicarbonate, the aqueous layer was washed 4 times with DCM, the combined organic layers were dried over sodium sulfate, filtered, and concentrated, and the resulting product was purified on a silica gel column eluting with DCM/MeOH50:1-40:1-35:1. 380mg of Compound 7 are obtained as colourless oil in 63.6% yield.
Characterization data for compound 7 are as follows:
ESI:m/z＝506.46[M+H]+.
1 H NMR(400MHz,CDCl 3 )δ7.53(s,1H),6.79(s,1H),5.78(s,1H),5.42–5.31(m,2H),4.51(s,1H),4.07(t,J＝6.7Hz,2H),3.09(d,J＝5.3Hz,2H),2.07–1.91(m,2H),1.66–1.52(m,2H),1.42(s,9H),1.37–1.19(m,24H),0.87(t,J＝6.7Hz,3H).
13 C NMR(100MHz,CDCl 3 )δ172.27,155.64,135.14,130.01,129.78,79.85,77.25,65.54,53.63,31.91,29.77,29.75,29.73,29.71,29.67,29.53,29.44,29.33,29.25,29.23,28.56,28.33,27.23,27.20,25.84,22.69,14.12.
a25 mL reaction flask was charged with Compound 7 (260 mg,0.51 mmol) and the flask was purged with N 2 After displacement, DCM (6 mL) was added followed by trifluoroacetic acid (1.5 mL). The reaction was followed by LC-MS and stopped after 2.5h at room temperature. The reaction solution was concentrated under reduced pressure at room temperature, then 10mL of ethanol was added thereto, and after removing ethanol under reduced pressure at room temperature, then 10mL of DCM was added thereto, and after removing DCM under reduced pressure at room temperature and pumping with oil for 1 hour, 310mg of Compound 8 was obtained as a white powder in 95% yield.
Characterization data for compound 8 are as follows:
ESI:m/z＝406.44[M+H]+.
example 5
Synthesis of N-acetyl-L-histidine-9-heptadecyl ester (Compound 9)
The synthetic route for compound 9 is as follows:
9-heptadecanol (300 mg,1.17mmol,1 eq.) and 4mL DCM were added to a 25mL reaction flask, after 10min in ice, N-acetyl-L-histidine (277 mg,1.29mmol,1.1 eq.) dispersed in 3mL DCM was added to the reaction flask, followed by 2mL of dicyclohexylcarbodiimide (DCC, 265.48mg,1.29mmol,1.1 eq.) in DCM and 2mL of DMAP (200.07 mg,1.64mmol,1.4 eq.) in DCM together, after which the reaction temperature was naturally raised to room temperature, heated in a 50℃water bath, the LC-MS followed by reaction, and the reaction was stopped after 24 h. The reaction mixture was filtered to remove the resulting white precipitate, washed with 40mL of DCM, and the resulting filtrate was washed with 40mL of 1m dilute aqueous hydrochloric acid to remove DMAP. Then adding 40mL of saturated NaHCO 3 The aqueous solution was made weakly basic, the organic layer was separated and dried over sodium sulfate, filtered, concentrated, and the resulting product was purified by column chromatography on silica gel eluting with DCM/MeOH 50:1-40:1-15:1. 137mg of compound 9 was obtained as pale yellow oil in 26% yield.
Characterization data for compound 9 are as follows:
ESI:m/z＝436.55[M+H]+.
1 H NMR(400MHz,CDCl 3 )δ7.53(s,1H),7.23(d,J＝7.7Hz,1H),6.78(s,1H),4.82(p,J＝6.2Hz,1H),4.75(dt,J＝7.7,5.5Hz,1H),3.14–3.00(m,2H),1.99(s,3H),1.57–1.39(m,3H),1.22(s,26H),0.85(t,J＝6.8Hz,6H).
13 C NMR(100MHz,CDCl 3 )δ171.54,170.42,135.10,77.36,76.09,52.75,33.93,33.90,31.95,29.62,29.60,29.58,29.55,29.43,29.36,25.28,25.20,23.25,22.75,14.20.
example 6
1. Preparation of mRNA-entrapped Histidine lipid nanoparticle [ Histidine-Lipid Nanoparticle (HLNP) ]
a) Lipid component treatment: histidine lipids (histidines-lipids) and helper lipids (including but not limited to DSPC, DOPE, etc.), cholesterol (Chol) and PEG-lipids (including but not limited to PEG2k-DMG, PEG2000-C-DMG, PEG2000-DSPE, etc.) were taken and dissolved in absolute ethanol (EtOH).
b) mRNA treatment: mRNA was dissolved in a buffer at pH 3-4.
c) mRNA/HLNP preparation: mixing the solutions obtained in the steps a and b through microfluidics.
d) Buffer substitution: after 10-fold dilution of the product from step c with PBS buffer at ph=7.4, ultrafiltration to original volume at 4 ℃ was repeated twice.
A schematic representation of the histidine lipid nanoparticle is shown in FIG. 1.
2. The mRNA/HLNP prescription (mg) is shown in Table 1 below.
TABLE 1
3. Characterization of
5 mRNA/HLNP samples were prepared using the procedure described in Table 1 above and tested for particle size, PDI and Zeta potential using a Markov laser particle sizer, with the results of the measurements shown in Table 2 below.
TABLE 2
4. Encapsulation efficiency (agarose gel electrophoresis)
The encapsulation efficiency of mRNA in the mRNA/HLNP samples was examined by agarose gel electrophoresis under the conditions shown in Table 3 below, and the agarose gel electrophoresis pattern is shown in FIG. 2, wherein Lane a was the positive control, lane b was the negative control, and Lane 1-5 was the mRNA/HLNP samples.
TABLE 3 Table 3
Agarose gel concentration 0.9％
EB concentration 5μL/100mL
Constant current/constant voltage Constant current 0.10A
mRNA quality in lanes (except NC) 600ng
As can be seen in FIG. 2, none of Lane 1-5 showed a clear bright band, indicating none or very little of all mRNA/HLNP samples Has the following components Free mRNA exists, and the mRNA encapsulation rate is higher.
5. Animal experiment
5.1 in vivo fluorescence imaging of mice
BABL/C mice were intramuscular injected with mRNA/HLNP samples containing 20. Mu.g mRNA, 3 mice per sample, and after 24 hours, each mouse was anesthetized with a mixed drug of sultai 50 and xylazine by intraperitoneal injection of a potassium salt solution of fluorescein for 5 minutes, after waiting for 5 minutes, the luc channel was selected using a small animal imager, and photographs were taken after exposure for 1 minute, with the results shown in FIG. 3 below.
As can be seen from fig. 3, all mice injected with mRNA/HLNP samples intramuscularly showed fluorescent expression after 24h, indicating that histidine-based HLNP can efficiently deliver Fluc mRNA into cells and translate into fluorescent proteins in the mice.
5.2 immunogenicity experiments (luciferase antibody detection ELISA)
Mice were given twice by intramuscular injection, 3 mice per sample, each with an mRNA/HLNP sample containing 20 μg mRNA per injection. After the second administration, 100-200 microliter of blood is collected from the orbital venous plexus of the mice, the mice are placed in a refrigerator at 4 ℃ for overnight, serum is separated (administration and blood collection time are shown in figure 4), luciferase is coated on an ELISA plate, after the mice are diluted at room temperature for overnight, the mice are added to the ELISA plate (an antibody of the luciferase is used as a positive control, a mixed solution of phosphate and Tween 20 is used as a negative control), the plate is washed after full reaction, an IgG-HRP antibody is added, the plate is washed after full reaction, TMB reaction solution is added, termination solution is added after full reaction, a light absorption value is read at a wavelength of 450nm on the ELISA plate, and the luciferase antibody is detected.
The results are shown (FIG. 5), where PC in FIG. 5 is a positive control, PC1:250, PC1:500 and PC1:1000 represent dilution factors, NC is negative control, NS is physiological saline, respectively. No luciferase antibodies were detected in the serum of mice after 2 doses of mRNA/HLNP prepared using His-C17 and His-C16, and no immunogenicity was shown. In contrast, antibodies were detected in the serum of mice treated with Moderna LNP and showed some immunogenicity. The main reason Moderna LNP was reported in the literature to have an immune response due to the ionizable lipids therein, but our His-C16LNP, his-C17LNP did not show significant immunogenicity, indicating that the ionizable lipids based on histidine synthesis were hardly immunogenic.
While certain embodiments of the compositions and methods have been described herein, and many details have been set forth for purposes of illustration, it will be apparent to those of ordinary skill in the art that the compositions and methods are susceptible to additional embodiments and that certain details can be varied with the practice of the invention without departing from the basic principles thereof.

Claims (8)

1. A histidine lipid, characterized by: the histidine lipid is a compound A, and the structural formula of the compound A isWherein R is 1 Selected from->
X is selected from H or COOR 2 ，R 2 Selected from C (CH) 3 ) 3 Or CH (CH) 3 。
2. The histidine lipid of claim 1, wherein: the compound A is selected from
3. A histidine lipid, characterized by: the histidine lipid is a salt of the compound A as claimed in claim 1, the salt of the compound A has the structural formulaWherein Y is n- Is an acid radical selected from CF 3 COO - 、CH 3 COO - 、Cl - Or Br (Br) - 。
4. A histidine lipid according to claim 3, characterised in that: the salt of the compound A is selected from
5. A preparation method of histidine lipid is characterized by comprising the following steps: reacting compound B with an alcohol to form compound a of claim 1 or 2, and optionally reacting compound a with an acid to form a salt of compound a of claim 3 or 4;
wherein the structural formula of the compound B isX in the compound B is the same as X in the compound a;
the alcohol is R 1 OH, R in the alcohol 1 R in said compound A 1 The same;
the acid is H n Y n- Y in the acid is the same as Y in the salt of the compound A.
6. Use of one or more of the histidine lipids of any one of claims 1 to 4 or one or more of the histidine lipids produced by the production method of claim 5 for the preparation of a medicament for the delivery of one or more of mRNA, siRNA, miRNA or antisense oligonucleotides.
7. A histidine lipid nanoparticle characterized by: the histidine lipid nanoparticle comprises one or more of mRNA and histidine lipid as claimed in any one of claims 1 to 4.
8. A histidine lipid nanoparticle characterized by: the histidine lipid nanoparticle comprises one or more of mRNA and histidine lipid produced by the method of producing as claimed in claim 5.
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