WO2023036148A1 - 一类阳离子脂质化合物及其应用 - Google Patents

一类阳离子脂质化合物及其应用 Download PDF

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WO2023036148A1
WO2023036148A1 PCT/CN2022/117373 CN2022117373W WO2023036148A1 WO 2023036148 A1 WO2023036148 A1 WO 2023036148A1 CN 2022117373 W CN2022117373 W CN 2022117373W WO 2023036148 A1 WO2023036148 A1 WO 2023036148A1
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cationic lipid
acid
class
compound
mrna
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PCT/CN2022/117373
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English (en)
French (fr)
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章雪晴
汪开振
滕以龙
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荣灿生物医药技术(上海)有限公司
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Priority to CN202280005633.4A priority Critical patent/CN115956072A/zh
Publication of WO2023036148A1 publication Critical patent/WO2023036148A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/08—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms
    • C07D295/084—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/088—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/14—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D295/145—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with the ring nitrogen atoms and the carbon atoms with three bonds to hetero atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/15—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with the ring nitrogen atoms and the carbon atoms with three bonds to hetero atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/16—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms
    • C07D295/18—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms by radicals derived from carboxylic acids, or sulfur or nitrogen analogues thereof
    • C07D295/182—Radicals derived from carboxylic acids
    • C07D295/185—Radicals derived from carboxylic acids from aliphatic carboxylic acids

Definitions

  • the invention relates to the field of nano-medicine carrier, in particular, the invention provides a cationic lipid compound and the application of using such molecule for nano-medicine delivery.
  • nucleic acids such as messenger RNA (mRNA), antisense oligonucleotides, ribozymes, DNase, plasmids, immunostimulatory nucleic acids, antagomirs, antimirs, mimics (mimics), supermirs, and aptamers
  • mRNA messenger RNA
  • antisense oligonucleotides ribozymes
  • DNase plasmid
  • immunostimulatory nucleic acids antagomirs
  • antimirs antimirs
  • mimics mimics
  • supermirs aptamers
  • Nucleic acid-based therapeutics hold great potential, but often require more efficient delivery of nucleic acids to the appropriate sites within cells or organisms to achieve this therapeutic effect. There are many challenges with regard to the delivery of nucleic acids in order to affect a desired response in a biological system.
  • the use of oligonucleotides in a therapeutic setting currently faces two problems: First, free RNA is prone to nuclease digestion in plasma. Second, cell-free RNA has limited access to intracellular compartments where associated translation machinery exists.
  • Lipid nanoparticles formed from cationic lipids with other lipid components such as neutral lipids, cholesterol, PEG, PEGylated lipids, and oligonucleotides can be used to prevent RNA degradation in plasma and promote Cellular uptake of oligonucleotides, however, there is still a need in the art to develop other cationic lipids and lipid nanoparticles that can be used to deliver oligonucleotides to provide better delivery, for example, optimized drug: lipid Compared, it protects nucleic acid from being degraded and cleared in serum, is well tolerated, provides sufficient therapeutic index, etc.
  • the purpose of the present invention is to provide a cationic lipid compound with a new structure.
  • a class of cationic lipid compounds the compound general formula is as shown in the following formula I:
  • a1 and a2 are each independently 0, 1, 2 or 3;
  • X and Y are each independently selected from N or CH, and at least one of X or Y is N;
  • R 11 and R 22 are each independently selected from the following group: substituted or unsubstituted linear or branched C 1-10 alkyl, or R 11 and R 22 together form a substituted or unsubstituted linear or branched C 1 -4 alkylene;
  • R 2 is selected from the group consisting of substituted or unsubstituted linear or branched C 1-4 alkylene
  • R 3 and R 4 are each independently selected from the following group: chemical bonds, or substituted or unsubstituted linear or branched C 2-10 alkylene groups;
  • R 5 and R 6 are each independently selected from the group consisting of chemical bonds, or substituted or unsubstituted straight or branched C 1-10 alkylene, substituted or unsubstituted straight or branched C 2-10 alkene base;
  • Each M 1 and M 2 is each independently a group selected from the group consisting of:
  • R 7 and R 8 are each independently selected from the following group: chemical bonds, or substituted or unsubstituted straight chain or branched C 1-10 alkylene, substituted or unsubstituted straight chain or branched C 2-10 alkene base;
  • R 9 and R 10 are each independently selected from the following group: substituted or unsubstituted linear or branched C 1-20 alkylene, substituted or unsubstituted linear or branched C 2-20 alkenylene;
  • Substitution means that one or more hydrogen atoms on the group are replaced by substituents selected from the following group: straight or branched C 1-10 alkyl, straight or branched C 2-4 alkenyl, OH, NH 2.
  • substituents selected from the following group: straight or branched C 1-10 alkyl, straight or branched C 2-4 alkenyl, OH, NH 2.
  • a class of cationic lipid compounds the general formula of which is shown in the following formula II:
  • X and Y are each independently selected from N or CH, and at least one of X or Y is N;
  • R 1 is selected from the group consisting of: Wherein, a, ggg, hhh, b, iii and kkk are each independently 0, 1, 2 or 3;
  • R2 is selected from the group consisting of:
  • R3 is selected from the group consisting of: Wherein, ww, xx, c, d, ff are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • R4 is selected from the group consisting of: Wherein, yy, zz, e, f, gg are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • CC, DD, A, B are substituents selected from the following group: H, CH 3 , OH, CH 2 OH, C 2 H 4 OH, NH 2 ;
  • R is selected from the group consisting of: Wherein, g, h, dd, jj and kk are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • R6 is selected from the group consisting of: Wherein, i, j, ee, mm and nn are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • R 7 is selected from the group consisting of: Wherein, k, l and m are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • R is selected from the group consisting of: Wherein, n, o and q are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • C, D, E, F, G, J are each independently selected from the following group: H, CH 3 ;
  • R9 is selected from the group consisting of: Wherein, r, s, oo, pp, qq, rr, u, v, aaa, bbb, ccc, hh are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11 or 12;
  • R 10 is selected from the group consisting of: Wherein, w, x, ss, tt, uu, vv, u, aa, ddd, eee, fff, ii are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11 or 12;
  • K, L, M and N are each independently selected from the following group: H, CH 3 ;
  • AA and BB are each independently selected from the following group: H, CH 3 , OH or NH 2 ;
  • Each independently has 2-40 carbons in the main chain (ie, the longest chain), At least one backbone has 10-40 carbons.
  • a is 0, 1 or 2.
  • b is 0, 1 or 2.
  • ggg is 0, 1 or 2.
  • hhh is 0, 1 or 2.
  • iii is 0, 1 or 2.
  • kkk is 0, 1 or 2.
  • ww is 0, 1, 2, 3, 4, 5, 6, 7 or 8.
  • xx is 0, 1, 2, 3, 4, 5, 6, 7 or 8.
  • c is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9.
  • d is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9.
  • yy is 0, 1, 2, 3, 4, 5, 6, 7 or 8.
  • zz is 0, 1, 2, 3, 4, 5, 6, 7 or 8.
  • e is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9.
  • f is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9.
  • g is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9.
  • h is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9.
  • i is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9.
  • j is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9.
  • jj is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 0, 1, 2, 3, 4, 5, 6, 7 or 8 .
  • kk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 0, 1, 2, 3, 4, 5, 6, 7 or 8 .
  • mm is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 0, 1, 2, 3, 4, 5, 6, 7 or 8 .
  • nn is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 0, 1, 2, 3, 4, 5, 6, 7 or 8 .
  • k is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 0, 1, 2, 3, 4, 5, 6, 7 or 8 .
  • l is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 0, 1, 2, 3, 4, 5, 6, 7 or 8 .
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 0, 1, 2, 3, 4, 5, 6, 7 or 8 .
  • o is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 0, 1, 2, 3, 4, 5, 6, 7 or 8 .
  • p is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • r is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 0, 1, 2, 3, 4, 5, 6, 7 or 8 .
  • s is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 0, 1, 2, 3, 4, 5, 6, 7 or 8 .
  • oo is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 0, 1, 2, 3, 4, 5, 6, 7 or 8 .
  • pp is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 0, 1, 2, 3, 4, 5, 6, 7 or 8 .
  • qq is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 0, 1, 2, 3, 4, 5, 6, 7 or 8 .
  • rr is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 0, 1, 2, 3, 4, 5, 6, 7 or 8 .
  • u is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • v is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • hh is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • aaa is 0, 1, 2, 3, 4, 5, 6, 7 or 8.
  • bbb is 0, 1, 2, 3, 4, 5, 6, 7 or 8.
  • ccc is 0, 1, 2, 3, 4, 5, 6, 7 or 8.
  • ss is 0, 1, 2, 3, 4, 5, 6, 7 or 8.
  • tt is 0, 1, 2, 3, 4, 5, 6, 7 or 8.
  • uu is 0, 1, 2, 3, 4, 5, 6, 7 or 8.
  • vv is 0, 1, 2, 3, 4, 5, 6, 7 or 8.
  • aa is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • ii is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • ddd is 0, 1, 2, 3, 4, 5, 6, 7 or 8.
  • eee is 0, 1, 2, 3, 4, 5, 6, 7 or 8.
  • fff is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • a class of cationic lipid compounds the general formula of which is shown in the following formula III:
  • X and Y are each independently selected from N or CH, and at least one of X or Y is N;
  • R 11 is selected from the group consisting of: Wherein, b2 is each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; EE is methyl or hydroxyl;
  • R 22 is selected from the group consisting of: b1 is each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; FF is methyl or hydroxyl;
  • R2 is selected from the group consisting of:
  • R3 is selected from the group consisting of: Wherein, ww, xx, c, d, ff are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • R4 is selected from the group consisting of: Wherein, yy, zz, e, f, gg are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • CC, DD, A, B are substituents selected from the following group: H, CH 3 , OH, CH 2 OH, C 2 H 4 OH, NH 2 ;
  • R is selected from the group consisting of: Wherein, g, h, dd, jj and kk are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • R6 is selected from the group consisting of: Wherein, i, j, ee, mm and nn are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • R 7 is selected from the group consisting of: Wherein, k, l and m are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • R is selected from the group consisting of: Wherein, n, o and q are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • C, D, E, F, G, J are each independently selected from the following group: H, CH 3 ;
  • R9 is selected from the group consisting of: Wherein, r, s, oo, pp, qq, rr, u, v, aaa, bbb, ccc, hh are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11 or 12;
  • R 10 is selected from the group consisting of: Wherein, w, x, ss, tt, uu, vv, u, aa, ddd, eee, fff, ii are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11 or 12;
  • K, L, M and N are each independently selected from the following group: H, CH 3 ;
  • AA and BB are each independently selected from the following group: H, CH 3 , OH or NH 2 ;
  • Each independently has 2-40 carbons in the main chain (ie the longest chain), said At least one side chain moiety has 10-25 carbons.
  • a class of cationic lipid compounds is as shown in the following formula IV:
  • R3 is selected from the group consisting of: Wherein, ww, xx, c, d, ff are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • R4 is selected from the group consisting of: Wherein, yy, zz, e, f, gg are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • a and B are substituents selected from the following group: H, CH 3 , OH, CH 2 OH, C 2 H 4 OH, NH 2 ;
  • R is selected from the group consisting of: Wherein, g, h, dd, jj and kk are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • R6 is selected from the group consisting of: Wherein, i, j, ee, mm and nn are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • R 7 is selected from the group consisting of: Wherein, k, l and m are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • R is selected from the group consisting of: Wherein, n, o and q are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • C, D, E, F, G, J are each independently selected from the following group: H, CH 3 ;
  • R9 is selected from the group consisting of: Wherein, r, s, oo, pp, qq, rr, u, v, aaa, bbb, ccc, hh are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11 or 12;
  • R 10 is selected from the group consisting of: Wherein, w, x, ss, tt, uu, vv, u, aa, ddd, eee, fff, ii are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11 or 12;
  • K, L, M and N are each independently selected from the following group: H, CH 3 ;
  • AA and BB are each independently selected from the following group: H, CH 3 , OH or NH 2 ;
  • Each independently has 2-40 carbons in the main chain (ie, the longest chain), At least one has 10-25 carbons.
  • Each independently is a structure selected from the following:
  • the aforementioned cationic lipid compound is selected from the following group:
  • compositions comprising cationic lipid compounds, stereoisomers, tautomers or pharmaceutically acceptable salts thereof. .
  • the composition containing cationic lipid compounds includes: one or a combination of carriers, loaded pharmaceutical agents, and pharmaceutical adjuvants. .
  • the carrier includes: one or more cationic lipid compounds, colipids, structured lipids, polymer conjugated lipids or amphiphilic block copolymers one or a combination of several.
  • the molar ratio of cationic lipid compounds to co-lipids is 0.5:1-10:1.
  • the molar ratio of cationic lipid compounds to structured lipids is 0.5:1-5:1.
  • the molar ratio of cationic lipid compound to polymer-conjugated lipid is 10:1-250:1.
  • the molar ratio of the cationic lipid compound to the amphiphilic block copolymer is 0.5:1-80:1.
  • the carrier is lipid nanoparticles LNP
  • the average size of lipid nanoparticles is 30-200nm
  • the polydispersity index of the preparation of lipid nanoparticles is ⁇ 0.5.
  • the contained pharmaceutical reagents include: one or more of nucleic acid molecules, small molecule compounds, polypeptides or proteins.
  • the pharmaceutical adjuvant includes: one or more of diluents, stabilizers, preservatives or lyoprotectants.
  • the cationic lipid compound of the present invention uses the nitrogen atom of piperazine and piperazine-like compounds as the charge center, and has stronger interaction with nucleic acid under the same conditions, which can better protect nucleic acid and load more nucleic acid. Double bonds are introduced into the tail of the compound, and the compounds with the same structure can be seen from experiments. The transfection efficiency of compounds without double bonds is much lower than that of compounds with double bonds, indicating that the introduction of double bonds at the tail and the compound structure of the present invention are improving the transfection effect. It has a synergistic effect; compared with the cationic lipid MC3 already used in the market, the LNP transfection efficiency is higher;
  • the introduction of degradable ester bonds into the hydrophobic tail of liposomes can change the metabolic behavior of liposomes in vivo, and at the same time increase the lipophilicity of its hydrophobic tails, thereby improving the biological safety and transfection efficiency of mRNA-LNP;
  • Modification with hydroxyl groups near the charge center can further increase the hydrophilicity of its head to further increase its transfection effect
  • the cationic lipid with the structure of the invention has high transfection efficiency, good safety, high biocompatibility, simple synthesis steps, and is suitable for the industrialization of biomedicine.
  • Fig. 1 is the hydrogen spectrogram of P-1 cationic lipid compound of the present invention
  • Fig. 2 is the hydrogen spectrogram of P-2 cationic lipid compound of the present invention
  • Fig. 3 is the hydrogen spectrogram of P-3 cationic lipid compound of the present invention.
  • Fig. 4 is the hydrogen spectrogram of P-4 cationic lipid compound of the present invention.
  • Fig. 5 is the hydrogen spectrogram of P-5 cationic lipid compound of the present invention.
  • Fig. 6 is the hydrogen spectrogram of P-6 cationic lipid compound of the present invention.
  • Fig. 7 is the hydrogen spectrogram of P-7 cationic lipid compound of the present invention.
  • Fig. 8 is the electron micrograph of lipid nanoparticles prepared by the present invention using the compound of sample P-1;
  • Fig. 9 is a schematic diagram of the comparison experiment results of the immune effect of the LNP prepared by using the cationic lipid compound of the sample P-1 in the present invention and the LNP on the market.
  • Therapeutic nucleic acid refers to a nucleic acid substance that can be administered to a subject in need in the form of an oligonucleotide or a vector expressing the corresponding oligonucleotide and produce a therapeutic effect.
  • Said therapeutic nucleic acids can be prepared according to any available technique, for example for mRNA, the main method of preparation is, but not limited to, enzymatic synthesis (also known as in vitro transcription), which currently represents the production of long sequence-specific mRNA the most effective method.
  • In vitro transcription describes a method for template-directed synthesis of RNA molecules from engineered DNA templates comprising an upstream phage promoter sequence linked to a downstream sequence encoding a gene of interest (for example including but not limited to those from T7, T3 and SP6 coliphage).
  • Template DNA for in vitro transcription can be prepared from a variety of sources using suitable techniques well known in the art, including, but not limited to, plasmid DNA and polymerase chain reaction amplification.
  • RNA polymerase and adenosine, guanosine, uridine, and cytidine ribonucleoside triphosphates rNTPs
  • rNTPs cytidine ribonucleoside triphosphates
  • In vitro transcription can be performed using various commercially available kits and commercially available reagents including, but not limited to, the RiboMax Large-Scale RNA Production System (Promega), the MegaScript Transcription Kit (Life Technologies), and the reagents Includes RNA polymerase and rNTPs. Methods for in vitro transcription of mRNA are well known in the art.
  • the desired in vitro transcribed mRNA is then purified from undesired components of the transcription or related reactions, including unincorporated rNTPs, proteases, salts, short RNA oligonucleotides, and the like.
  • Techniques for isolation of mRNA transcripts are well known in the art. Well-known procedures include phenol/chloroform extraction or precipitation with alcohols (ethanol, isopropanol) or lithium chloride in the presence of monovalent cations. Additional non-limiting examples of purification procedures that may be used include size exclusion chromatography, silica-based affinity chromatography, and polyacrylamide gel electrophoresis. Purification can be performed using a variety of commercially available kits including, but not limited to, the SV Total Isolation System (Promega) and the In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).
  • RNA impurities associated with undesired polymerase activity, which may need to be removed from full-length mRNA preparations.
  • impurities include short RNAs resulting from failed transcription initiation as well as double-stranded RNA (dsRNA) resulting from RNA-dependent RNA polymerase activity, RNA-primed transcription from RNA templates, and self-complementary 3' elongation. It has been demonstrated that these dsRNA-structured contaminants can lead to undesired immunostimulatory activity by interacting with various innate immune sensors in eukaryotic cells that function to recognize specific nucleic acid structures and induce potent immunity answer.
  • dsRNA double-stranded RNA
  • modifications include, but are not limited to, modifications to the 5' and 3' ends of the mRNA.
  • Endogenous eukaryotic mRNA usually contains a cap structure on the 5'-end of the mature molecule, which plays an important role in mediating the binding of mRNA cap-binding protein (CBP), which in turn is responsible for enhancing mRNA stability and mRNA translation efficiency.
  • CBP mRNA cap-binding protein
  • the 5'-cap contains a 5'-5'-triphosphate linkage between the 5'-majority nucleotide and the guanine nucleotide. Conjugated guanine nucleotides are methylated at the N7 position. Additional modifications include methylation of the last and penultimate most 5'-nucleotide at the 2'-hydroxyl group.
  • cap structures can be used to generate 5'-caps for mRNAs synthesized by in vitro transcription.
  • 5'-capping of synthetic mRNA can be performed in conjunction with transcription (i.e. capping during in vitro transcription) using chemical cap analogs.
  • anti-reverse cap analog (ARCA) caps contain a 5'-5'-guanine triphosphate-guanine linkage, where one guanine contains an N7 methyl group as well as a 3'-O-methyl group.
  • the synthetic cap analogs are not identical to the 5′-cap structure of real cellular mRNAs, potentially reducing translatability and cellular stability.
  • synthetic mRNA molecules can also be capped enzymatically after transcription. This can generate a more realistic 5'-cap structure that more closely mimics, structurally or functionally, the endogenous 5'-cap with enhanced binding of cap-binding proteins, increased Half-life, reduced sensitivity to 5' endonucleases and/or reduced 5' decapping.
  • a number of synthetic 5'-cap analogs have been developed and are known in the art to enhance mRNA stability and translatability.
  • poly-A tail a long chain of adenine nucleotides
  • poly-A tail a long chain of adenine nucleotides
  • polyadenylate polymerase adds a chain of adenine nucleotides to the RNA in a process known as polyadenylation.
  • Poly A tails have been widely shown to enhance translation efficiency and mRNA stability.
  • PolyA tailing of in vitro transcribed mRNA can be achieved using various methods including, but not limited to, cloning poly(T) fragments into DNA templates, or by using poly(A) polymerase in Added after transcription.
  • the first case allows in vitro transcription of mRNAs with a poly(A) tail of defined length (depending on the size of the poly(T) fragment), but requires additional manipulation of the template.
  • the latter case involves the enzymatic addition of a poly(A) tail to in vitro transcribed mRNA using a poly(A) polymerase that catalyzes the incorporation of adenine residues into the 3' end of RNA, without the need for additional manipulation of the DNA template, whereas mRNAs with poly(A) tails of different lengths were obtained.
  • 5'-capping and 3'-poly(A) tailing can be performed using various commercially available kits including , but not limited to Poly(A) Polymerase Tailing Kit (EpiCenter), mMESSAGE mMACHINE T7Ultra Kit and Poly(A) Tailing Kit (Life Technologies).
  • RNA synthesized in vitro lacks these modifications, thus rendering it immunostimulatory and, in turn, inhibiting efficient mRNA translation as outlined above.
  • modified nucleosides into in vitro transcribed mRNA can be used to prevent the recognition and activation of RNA sensors, thereby alleviating this undesirable immunostimulatory activity and enhancing translational capacity.
  • Modified nucleosides and nucleotides for the synthesis of modified RNA can be prepared, monitored and used using general methods and procedures known in the art. A wide variety of nucleoside modifications can be used, which alone or in combination with other modified nucleosides can be incorporated to some extent into in vitro transcribed mRNA. In vitro synthesis of nucleoside-modified mRNAs has been reported to reduce the ability to activate immunosensors, with concomitant enhancement of translational ability.
  • UTRs 5' and 3' untranslated regions
  • nucleic acid payloads may also be used in the present invention.
  • methods of preparation include, but are not limited to, chemical synthesis and enzymatic, chemical cleavage of longer precursors, in vitro transcription as described above, and the like. Methods for synthesizing DNA and RNA nucleotides are widely used and well known in the art.
  • preparations used in conjunction with the present invention typically use, but are not limited to, in vitro amplification and isolation of plasmid DNA in liquid culture medium of bacteria containing the plasmid of interest.
  • the presence of genes within the plasmid of interest encoding resistance to specific antibiotics (penicillin, kanamycin, etc.) allows those bacteria containing the plasmid of interest to grow selectively in antibiotic-containing media.
  • Methods for isolating plasmid DNA are widely used and well known in the art.
  • Plasmid isolation can be performed using various commercially available kits and reagents including, but not limited to, Plasmid Plus (Qiagen), GenJET plasmad MaxiPrep (Thermo), and Pure Yield MaxiPrep (Promega) reagents box.
  • Lipid nanoparticle refers to a composite drug-loaded particle formed by self-assembly of cationic liposomes and nucleic acid drugs to be delivered. Lipid nanoparticles and compositions of the invention can be used for a variety of purposes, including delivering encapsulated or associated (e.g., complexed) therapeutic agents, such as nucleic acids, to cells in vitro and in vivo, thereby inducing expression of a desired protein Or inhibit the expression of the target gene.
  • encapsulated or associated (e.g., complexed) therapeutic agents such as nucleic acids
  • embodiments of the invention provide methods of treating or preventing diseases and disorders in a subject in need thereof by contacting the subject with lipid nanoparticles encapsulating or associated with a suitable therapeutic agent, wherein The lipid nanoparticles comprise one or more of the novel cationic lipids described herein.
  • lipid nanoparticles comprise one or more of the novel cationic lipids described herein.
  • Active Agents such as Nucleic Acids (e.g., Therapeutic Agents), to Modulate Gene and Protein Expression It is described in further detail below.
  • “Inducing expression of a desired protein” refers to the ability of a nucleic acid to increase expression of a desired protein.
  • a test sample e.g., a sample of cells in culture expressing the desired protein
  • a test mammal e.g., a mammal such as a human or such as a rodent (e.g., mouse) or non-human Longitudinal animals (eg, animal models of monkey models) are exposed to nucleic acids (eg, nucleic acids that bind lipids of the invention).
  • Expression of the desired protein in the test sample or test animal is compared to a control sample (e.g., a sample of cells in culture medium expressing the desired protein) or a control mammal (e.g., a mammal such as a human or such as Expression of the desired protein in a rodent (eg, mouse) or non-human primate (eg, monkey) model is compared.
  • a control sample or control mammal e.g., a mammal such as a human or such as Expression of the desired protein in a rodent (eg, mouse) or non-human primate (eg, monkey) model is compared.
  • Expression of the desired protein in the control sample or control mammal can be assigned a value of 1.0 when the desired protein is present in the control sample or control mammal.
  • the ratio of the desired protein expression in the test sample or test mammal to the desired protein expression level in the control sample or control mammal is greater than 1, for example, about 1.1, 1.5, 2.0, 5.0 or 10.0
  • the expression of the desired protein is induced. Inducing expression of the desired protein is achieved when any measurable level of the desired protein is detected in the test sample or test mammal when the desired protein is absent in the control sample or control mammal.
  • suitable assays to determine protein expression levels in a sample are, for example, dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function and phenotypic assays, or based on the Assays that produce fluorescent or luminescent reporter proteins.
  • the phrase "inhibiting the expression of a target gene” refers to the ability of a nucleic acid to silence, reduce or inhibit the expression of a target gene.
  • a test sample e.g., a sample of cells in culture expressing a target gene
  • a test mammal e.g., a mammal such as a human or a rodent (e.g., a mouse) or non-human Animal models of long animals (eg, monkey models) are exposed to nucleic acids that silence, reduce or inhibit expression of a target gene.
  • Expression of the target gene in the test sample or test animal is compared to a control sample (e.g., a sample of cells in a culture medium expressing the target gene) or a control mammal (e.g., a mammal such as a human or such as Expression of the target gene in a rodent (eg, mouse) or non-human primate (eg, monkey) model is compared.
  • a control sample or control mammal can be assigned a value of 100%.
  • the target gene expression level in the test sample or test mammal when the target gene expression level in the test sample or test mammal is about 95%, 90%, 85%, 80%, 75% relative to the target gene expression level in the control sample or control mammal , 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% silence achieved , Inhibit or reduce the expression of the target gene.
  • the nucleic acid is capable of silencing, reducing or inhibiting the expression of the target gene by at least about 5% in the test sample or test mammal relative to the level of target gene expression in a control sample or control mammal that has not been contacted or administered the nucleic acid , 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% %, 95% or 100%.
  • Suitable assays to determine expression levels of a target gene include, but are not limited to, assays of protein or mRNA levels using techniques known to those of skill in the art, for example, dot blots, northern blots, , in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays.
  • lipid refers to a group of organic compounds that includes, but is not limited to, esters of fatty acids, and is generally characterized by poor solubility in water, but is soluble in a wide variety of organic solvents. They generally fall into at least three categories: (1) “simple lipids”, which include fats and oils as well as waxes; (2) “compound lipids”, which include phospholipids and glycolipids; and (3) “derived lipids” , such as steroids.
  • “Cationic lipid” refers to a lipid capable of being positively charged.
  • Exemplary cationic lipids include one or more positively charged amine groups.
  • Preferred cationic lipids are ionizable so that they can exist in positively charged or neutral form depending on pH. Ionization of cationic lipids affects the surface charge of lipid nanoparticles at different pH conditions. This charge state can affect plasma protein uptake, blood clearance and tissue distribution, as well as the ability to form endosomolytic non-bilayer structures, which are critical for intracellular delivery of nucleic acids.
  • polymer-conjugated lipid refers to a molecule comprising a lipid moiety and a polymer moiety.
  • examples of polymer-conjugated lipids are pegylated lipids.
  • PEGylated lipid refers to a molecule comprising a lipid moiety and a polyethylene glycol moiety.
  • Pegylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), among others.
  • neutral lipid refers to any of a number of lipid substances that exist in uncharged or neutral zwitterionic form at a selected pH.
  • lipids include, but are not limited to, phosphatidylcholines such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl- sn-Glyceryl-3-Phosphocholine (DPPC), 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC), 1-Palmitoyl-2-oleoyl-sn-Glycerol -3-phosphocholine (POPC), 1,2-dioleoyl-sn-glyceryl-3-phosphocholine (DOPC), phosphatidylethanolamine such as 1,2-dioleoyl-sn-glyceryl- 3-Phosphoethanolamine (DOPE), sphingomyelin (SM), cer
  • DOPE 1,2-d
  • charged lipid refers to any of a variety of lipid substances that exist in a positively or negatively charged form, independent of pH within a useful physiological range, for example pH ⁇ 3 to pH ⁇ 9.
  • Charged lipids can be of synthetic or natural origin. Examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyltrimethylammonium-propane (e.g. DOTAP, DOTMA), dialkyldimethylaminopropane , ethyl phosphorylcholine, dimethylaminoethane carbamoyl sterol (eg DC-Chol).
  • DOTAP phosphatidylglycerol
  • phosphatidylinositol sterol hemisuccinate
  • dialkyltrimethylammonium-propane e.g. DOTAP, DOTMA
  • lipid nanoparticle refers to a particle having at least one dimension on the nanometer scale (eg, 1 nm to 1,000 nm) comprising one or more compounds of structure (I) or other specific cationic lipids.
  • lipid nanoparticles are included in formulations useful for delivering active or therapeutic agents, such as nucleic acids (eg, mRNA), to target sites of interest (eg, cells, tissues, organs, tumors, etc.).
  • lipid nanoparticles of the invention comprise nucleic acids.
  • Such lipid nanoparticles generally comprise a compound of structure (I) and one or more excipients selected from neutral lipids, charged lipids, cholesterol and polymer-conjugated lipids.
  • an active or therapeutic agent such as a nucleic acid can be encapsulated within the lipid portion of a lipid nanoparticle, or in an aqueous space surrounded by some or all of the lipid portion of a lipid nanoparticle, thereby protecting It is not degraded enzymatically, or otherwise undesired effects induced by mechanisms of the host organism or cell, such as adverse immune responses.
  • Embodiments of the lipid nanoparticles of the invention are particularly useful for the delivery of nucleic acids including, for example, mRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomirs/antimirs) , Messenger-RNA-interference complementary RNA (micRNA), DNA, multivalent RNA, dicer substrate RNA, complementary DNA (cDNA), etc.
  • nucleic acids including, for example, mRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomirs/antimirs) , Messenger-RNA-interference complementary RNA (micRNA), DNA, multivalent RNA, dicer substrate RNA, complementary DNA (cDNA), etc.
  • the lipid nanoparticles and compositions of the invention can be used to induce expression of a desired protein in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more of the novel cationic lipids described herein , wherein the lipid nanoparticle encapsulates or associates a nucleic acid that is expressed to produce a desired protein (e.g., a messenger RNA or plasmid encoding a desired protein) or that is expressed to inhibit a process that terminates mRNA expression ( For example, miRNA inhibitors).
  • a desired protein e.g., a messenger RNA or plasmid encoding a desired protein
  • miRNA inhibitors e.g., miRNA inhibitors
  • the lipid nanoparticles and compositions of the invention can be used to reduce target genes and genes in vitro and in vivo by contacting cells with lipid nanoparticles comprising one or more of the novel cationic lipids described herein. Expression of the target protein, wherein the lipid nanoparticles encapsulate or associate nucleic acids (eg, antisense oligonucleotides or small interfering RNA (siRNA)) that reduce expression of the target gene.
  • nucleic acids eg, antisense oligonucleotides or small interfering RNA (siRNA)
  • Lipid nanoparticles and compositions of the present invention can also be used to co-deliver different nucleic acids (such as mRNA and plasmid DNA) alone or in combination, for example, can be used to provide different nucleic acids (such as mRNA encoding a suitable gene modification enzyme and and colocalization of DNA fragments into the host genome).
  • nucleic acids such as mRNA and plasmid DNA
  • the lipid nanoparticles used have an average diameter of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70nm to about 100nm, about 80nm to about 100nm, about 90nm to about 100nm, about 70 to about 90nm, about 80nm to about 90nm, about 70nm to about 80nm, or about 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm or 150nm and is substantially non-toxic.
  • the average diameter of about 30 n
  • lipid-encapsulated refers to lipid nanoparticles that provide complete encapsulation, partial encapsulation, or both, of an active agent, such as a nucleic acid (eg, mRNA), or a therapeutic agent.
  • an active agent such as a nucleic acid (eg, mRNA), or a therapeutic agent.
  • nucleic acids eg, mRNA
  • nucleic acids are fully encapsulated within lipid nanoparticles.
  • nucleic acid-lipid nanoparticles With respect to nucleic acid-lipid nanoparticles, "serum stable" means that the nucleotides do not degrade significantly after exposure to serum or after exposure to nuclease assays that would significantly degrade free DNA or RNA. Suitable assays include, for example, standard serum assays, DNase assays or RNase assays.
  • systemic delivery refers to the delivery of a therapeutic product that results in widespread exposure of the active agent within the organism. Some administration techniques can result in systemic delivery of some agents, but not others. Systemic delivery means that a useful amount, preferably a therapeutic amount, of an agent is exposed to most parts of the body.
  • Systemic delivery of lipid nanoparticles can be by any means known in the art, including, for example, intravenous, intraarterial, subcutaneous and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery.
  • local delivery refers to the direct delivery of an active agent to a target site within an organism.
  • the agent may be delivered locally by direct injection into a site of disease such as a tumor, other target site such as a site of inflammation, or a target organ such as liver, heart, pancreas, kidney, etc.
  • Local delivery may also involve topical application or local injection techniques, such as intramuscular, subcutaneous or intradermal injection. Local delivery does not interfere with systemic pharmacological effects.
  • Nucleic acid molecules include single-stranded DNA, double-stranded DNA, short isomers, mRNA, tRNA, rRNA, long non-coding RNA (lncRNA), micro non-coding RNA (miRNA and siRNA), telomerase RNA (Telomerase RNA Component) , small RNA (snRNA and scRNA), circular RNA (circRNA), synthetic miRNA (miRNA mimics, miRNA agomir, miRNA antagomir), antisense DNA, antisense RNA, ribozyme (ribozyme), asymmetric interfering RNA (aiRNA ), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), gRNA, sgRNA, crRNA or tracrRNA, locked nucleic acid (LNA), peptide nucleic acid (PNA), Morpholino antisense oligonucleotides, morph
  • Pharmaceutically acceptable salts refer to acid addition salts or base addition salts.
  • the acids of acid addition salts include but are not limited to: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acid phosphate, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartame Amino acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclic amic acid, dodecane Hydroxyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactobionic acid, gentisic acid, glucoheptanoic acid, gluconic acid, dextran Sugar acid,
  • the base addition salt examples include but not limited to: sodium salt, potassium salt, lithium salt, ammonium salt, calcium salt, magnesium salt, iron salt, zinc salt, copper salt, manganese salt, and aluminum salt; organic base includes but not Limited to ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dealcoholization, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine Acid, histidine, caffeine, procaine, hydrazine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucosamine , theobromine, triethanolamine, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resins; preferably, the organic base is isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclo
  • Helping lipids include: one or more combinations of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin (SM), sterols and derivatives thereof, ceramides, and charged lipids; phosphatidylcholine is a preferred Including: DSPC, DPPC, DMPC, DOPC, POPC; Phosphatidylethanolamine is preferably DOPE as a kind; Sterol is preferably cholesterol as a kind; Charged lipid is DOTAP, DOTMA, 18PA as a kind of embodiment; As long as the composition of the cationic lipid compound adopting the structure of the present invention is within the protection scope of the present invention, it is inspired by the present invention. This is not exhaustive, and the choice of co-lipids is not limited, as long as the cationic lipid compounds adopting the structure of the present invention are within the protection scope of the present invention, they are all inspired by the present invention.
  • Charged lipids refer to a class of lipid compounds that exist in a positively or negatively charged form; their charge does not depend on the pH within the physiological range, such as pH 3-9, and is not affected by pH.
  • Charged lipids can be of synthetic or natural origin. Examples of charged lipids include, but are not limited to, DOTAP, DOTMA, 18PA.
  • messenger ribonucleic acid is a type of single-stranded ribonucleic acid that is transcribed from a strand of DNA as a template, carries genetic information and can guide protein synthesis.
  • the mRNA can be either monocistronic or polycistronic.
  • the mRNA may also contain one or more functional nucleotide analogs. Examples of functional nucleotide analogs include: pseudouridine, 1-methyl-pseudouridine or 5-methylcytosine wait. The examples here are not exhaustive, and any modified mRNA or its derivatives can be used in the present invention.
  • Small molecule compounds can be active ingredients in therapeutic or prophylactic agents such as: antineoplastics, antiinfectives, local anesthetics, antidepressants, anticonvulsants, antibiotics/antibacterials, antifungals, antiparasitics Anthelmintic drugs, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, anti-glaucoma agents, anesthetics, or imaging agents are not exhaustive here.
  • Polypeptide is a compound formed by ⁇ -amino acids linked together by peptide bonds, and is an intermediate product of protein hydrolysis.
  • a protein is a substance with a certain spatial structure formed by a polypeptide chain composed of amino acids in the form of "dehydration condensation". Proteins can be interferons, protein hormones, cytokines, chemokines, or enzymes.
  • the diluent is any pharmaceutically acceptable water-soluble excipient known to those skilled in the art, including: amino acids, monosaccharides, disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, other oligosaccharides, mannitol, dextran, chlorine Sodium chloride, sorbitol, polyethylene glycol, phosphate, or its derivatives, etc.
  • the stabilizer can be any pharmaceutically acceptable excipient known to those skilled in the art: Tween-80, sodium lauryl sulfate, sodium oleate, mannitol, mannose or sodium alginate, etc.
  • the preservative can be any pharmaceutically acceptable preservative known to those skilled in the art, such as thimerosal and the like.
  • the lyoprotectant may be any pharmaceutically acceptable lyoprotectant known to those skilled in the art, such as glucose, mannitol, sucrose, lactose, trehalose, maltose and the like.
  • DSPC English name: Distearoyl Phosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphocholine; Chinese name: Distearoyl Phosphatidylcholine, CAS number: 816-94-4.
  • DPPC Chinese name: Dipalmitoylphosphatidylcholine; English name: 1,2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHOCHOLINE, CAS number: 63-89-8.
  • DMPC Chinese name: dimyristoylphosphatidylcholine; English name: 1,2-Dimyristoyl-sn-glycero-3-phosphocholine, CAS number: 18194-24-6.
  • DOPC Chinese name: 1,2-dioleoyl-sn-glycero-3-phosphocholine; English name: 1,2-dioleoyl-sn-glycero-3-phosphocholine, CAS number: 4235-95-4.
  • POPC Chinese name: 2-oleoyl-1-palmitin glycerol-3-phosphocholine; English name: 2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine, CAS number: 26853-31-6.
  • DOPE Chinese name: 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine; English name: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOETHANOLAMINE, CAS number: 4004-05-1.
  • DOTAP Chinese name: N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate; English name: 1,2-dioleoyl- 3-trimethylammonium-propane (chloride salt), CAS number: 144189-73-1; chemical structure is as follows:
  • DOTMA Chinese name: N,N,N-trimethyl-2,3-bis(octadec-9-en-1-yloxy)propan-1-ammonium chloride, CAS number: 1325214-86- 5.
  • the chemical structural formula is as follows:
  • SM Chinese name: sphingomyelin (SM); English name: sphingomyelin.
  • PEG Chinese name: polyethylene glycol; English name: Polyethylene glycol.
  • Amphiphilic block copolymers refer to: block copolymers of PEG and one or more of the following polymer components, the polymer components include: polylactic acid-polyglycolic acid copolymer (PLGA), polylactic acid (PLA) , one or more of polycaprolactone (PCL), polyorthoester, polyanhydride, poly( ⁇ -amino ester) (PBAE).
  • PLGA polylactic acid-polyglycolic acid copolymer
  • PLA polylactic acid
  • PCL polycaprolactone
  • PCL polyorthoester
  • polyanhydride poly( ⁇ -amino ester)
  • a class of liposome molecules with a novel structure prepared by the present invention the liposome molecules can form RNA-loaded liposome nanoparticles together with therapeutic nucleic acids, so as to deliver therapeutic nucleic acids to the required drug concentration parts. Based on the above findings, the inventors have accomplished the present invention.
  • the compound P-11 can also be prepared and will not be repeated here.
  • the mass ratio of liposomal nanoparticles (LNP) to mRNA is 10:1 to 30:1.
  • the obtained nanoparticles are purified by means of ultrafiltration and dialysis; filter sterilization.
  • the lipid nanoparticles of the present invention can form stable nanostructures with a narrow size distribution, and the size varies with different lipid nanoparticle structures, within the range of 30-150nm.
  • luciferase mRNA encoding luciferase (luciferase mRNA) was used as a reporter gene. Luciferase catalyzes luciferin to produce bioluminescence, and the transfection efficiency of LNP is reflected by detecting the intensity of bioluminescence per unit time.
  • luciferase mRNA purchased from ApexBio Technology
  • the mRNA-LNP obtained above was administered by intramuscular injection at a dose of 150 ⁇ g/kg mRNA.
  • mice were intraperitoneally injected with fluorescein, and 5 minutes later, the mice were placed in a small animal in vivo imager to measure the fluorescence intensity, and the final result was expressed as the average fluorescence intensity.
  • Cell viability was measured using CCK-8 (cell counting kit-8) kit.
  • A1 is the absorbance of the drug-dosed group
  • A0 is the absorbance of the blank group
  • A2 is the absorbance of the control group.
  • mice 15 six-week-old female Balb/c mice, weighing 15-20 g, were raised in an experimental environment with a temperature of 22 ⁇ 2°C and a relative humidity of 45-75%, with a light/dark cycle of 12 hours. After the mice are purchased, they must be acclimatized in the animal room for a week before conducting formal animal experiments.
  • mice 15 mice were randomly divided into 3 groups, the first group of hind leg intramuscular injection of equal volume PBS (negative control group), the second group of hind leg intramuscular injection of the mixture of commercially available contrast sample (positive control group MC3), mRNA, PBS, The third group was a mixture of hind leg intramuscular injection of sample P-1 (test group), 10 ⁇ g of mRNA, and PBS; the above mRNA was synthesized by in vitro transcription based on a self-designed template and could express the full-length Spike mRNA.
  • the experimental process was as follows: on days 0 and 14, the LNP mixture loaded with mRNA was intramuscularly injected into Balb/c mice according to the above five groups. Eye blood was collected on the 13th and 21st days, and the blood samples were incubated at 37°C for 1 hour, centrifuged at 3500rpm for 15 minutes, and the supernatant was taken for analysis. The specific antibody titer of the S1 protein of the Delta variant strain was detected by the self-made ELISA kit.
  • the specific operation process for detecting the specific antibody titer of the Delta variant strain S1 protein in the sera of the first and second immunization mice is as follows: add Spike S1 recombinant protein to a 96-well plate, add 0.25 ⁇ g to each well, and place it overnight at 4°C. The next day, the liquid in the wells was discarded and blocked with 5% BSA in PBST solution (200 ul) at 37° C. for 1 h. Afterwards, the liquid in the wells was discarded, washed 3 times with 200ul of PBST washing solution, each time for 3min, and the plate was shaken to dry.
  • the mouse serum was diluted with PBS (the dilution ratio is listed as 1:20000), or the standard was diluted with PBS to a series of concentrations (the stock solution was 1ug/ul, half-diluted, a total of 14 standard curves).
  • Goat anti-mouse IgG HRP (1:5000 dilution in PBS), 100ul per well, 37°C, 1h.
  • the liposomes of the present invention can all form stable nanostructures, and the size distribution is relatively narrow, and the size varies with different liposome structures, and the size is in the range of 30-150nm.
  • the introduction of degradable ester bonds into the hydrophobic tail of liposomes can change the metabolic behavior of liposomes in vivo, and at the same time increase the lipophilicity of the hydrophobic tail, thereby improving the biosafety and transfection efficiency of mRNA-LNP
  • piperazine and piperazine-like structures are introduced into its hydrophilic head to strengthen the interaction with nucleic acids, thereby loading more nucleic acids.
  • the structure can make the lipid molecules take a cone-shaped structure, and the cone-shaped structure can reverse the reverse hexagonal phase of the endosome membrane, destroy the endosome membrane and enhance the escape ability of the endosome, thereby increasing the transfection effect and further increasing its transfection efficiency.
  • its synthetic route is simple and easy, and the raw materials are cheap and easy to obtain, which is conducive to its industrial production.
  • the new liposome has a good application prospect.

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Abstract

本发明提供了一类阳离子脂质化合物及其应用，涉及生物医药领域，本发明如式I所示的阳离子脂质化合物制备得到的脂质纳米粒的转染效率高，安全性好，生物相容性高；且本发明的化合物合成步骤简单，适合生物医药产业化。

Description

一类阳离子脂质化合物及其应用
本申请要求向中国国家知识产权局提交的专利申请号为：2021110459803的优先权，本发明将特此引用该专利的内容。
技术领域
本发明涉及纳米药物载体领域，具体地，本发明提供了一种阳离子脂质化合物，和使用此类分子进行纳米药物递送的应用。
背景技术
治疗性核酸，如信使RNA(mRNA)、反义寡核苷酸、核酶、DNA酶、质粒、免疫刺激核酸、antagomir、antimir、模拟物(mimic)、supermir和适配子等，可以用于实现特定细胞产物的表达，从而用于治疗例如与蛋白质或酶的缺乏有关的疾病或症状。核酸类药物的治疗性应用极其广泛的，这是因为可以合成构建体以产生任何所选蛋白质序列，而核酸的表达产物可以提高蛋白质的存在水平，取代缺失的或非功能的蛋白质形式，或在细胞或生物体内引入新的蛋白质以及相关功能。
基于核酸的治疗具有巨大潜力，但这一治疗方法通常需要将核酸更有效地递送至细胞或生物体内的适当位点以实现这种治疗效果。关于递送核酸以便在生物系统中影响所期望的应答，还存在着诸多挑战。在治疗环境中使用寡核苷酸目前面临着两个问题：第一，游离的RNA易于在血浆中核酸酶消化。第二，游离RNA进入存在相关翻译机制的细胞内隔室的能力受限。
由阳离子脂质与其他脂质组分(如中性脂质、胆固醇、PEG、PEG化的脂质和寡核苷酸)形成的脂质纳米颗粒可以用于阻止RNA在血浆中的降解并促进寡核苷酸的细胞摄取，然而，本领域仍然需要开发其他能够用于递送寡核苷酸的阳离子脂质和脂质纳米颗粒，从而提供更好的递送效果，例如，优化的药物：脂质比，保护核酸不在血清中被降解和清除，良好的耐受性，提供足够的治疗指数等。
综上所述，本领域尚需要开发具有全新结构的阳离子脂质化合物。
发明内容
本发明的目的是提供一种具有全新结构的阳离子脂质化合物。
一类阳离子脂质化合物，化合物通式如以下式I所示：
Figure PCTCN2022117373-appb-000001
其中，
Figure PCTCN2022117373-appb-000002
的主链(即最长链)上各自独立地具有1-40个碳；
a1和a2各自独立地为0、1、2或3；
X、Y各自独立地选自N或CH，且X或Y至少一个为N；
R 11、R 22各自独立地选自下组：取代或未取代的直链或支链C 1-10烷基，或R 11和R 22共同构成取代或未取代的直链或支链C 1-4亚烷基；
R 2选自下组：取代或未取代的直链或支链C 1-4亚烷基；
R 3和R 4各自独立地选自下组：化学键，或取代或未取代的直链或支链C 2-10亚烷基；
R 5和R 6各自独立地选自下组：化学键，或取代或未取代的直链或支链C 1-10亚烷基、取代或未取代的直链或支链C 2-10亚烯基；
各个M 1和M 2各自独立地为选自下组的基团：
Figure PCTCN2022117373-appb-000003
R 7和R 8各自独立地选自下组：化学键，或取代或未取代的直链或支链C 1-10亚烷基、取代或未取代的直链或支链C 2-10亚烯基；
R 9和R 10各自独立地选自下组：取代或未取代的直链或支链C 1-20亚烷基、取代或未取代的直链或支链C 2-20亚烯基；
Figure PCTCN2022117373-appb-000004
的主链上具有10-40个碳；
取代指基团上的一个或多个氢原子被选自下组的取代基所取代：直链或支链C 1-10烷基、直链或支链C 2-4烯基、OH、NH 2、羟基取代的直链或支链C 1-4烷基、羟基取代的直链或 支链C 2-4烯基。
作为式I的进一步说明：式I化合物中，当a1和a2各自独立地为1、2或3时，
Figure PCTCN2022117373-appb-000005
Figure PCTCN2022117373-appb-000006
中至少一个为主链(即最长链)上具有2-10个碳的链段；且当R 3、R 4、R 5或R 6被取代时，所述的取代指基团上的一个或多个氢原子被选自下组的取代基所取代：直链或支链C 1-4烷基、直链或支链C 2-4烯基、OH、NH 2、羟基取代的直链或支链C 1-4烷基、羟基取代的直链或支链C 2-4烯基。
一类阳离子脂质化合物，化合物通式如以下式II所示：
Figure PCTCN2022117373-appb-000007
X、Y各自独立地选自N或CH，且X或Y至少一个为N；
R 1选自下组：
Figure PCTCN2022117373-appb-000008
其中，a、ggg、hhh、b、iii和kkk各自独立地为0、1、2或3；
R 2选自下组：
Figure PCTCN2022117373-appb-000009
R 3选自下组：
Figure PCTCN2022117373-appb-000010
其中，ww、xx、c、d、ff各自独立地为0、1、2、3、4、5、6、7、8、9或10；
R 4选自下组：
Figure PCTCN2022117373-appb-000011
其中，yy、zz、e、f、gg各自独立地为0、1、2、3、4、5、6、7、8、9或10；
CC、DD、A、B为选自下组的取代基：H、CH 3、OH、CH 2OH、C 2H 4OH、NH 2；
R 5选自下组：
Figure PCTCN2022117373-appb-000012
其中，g、h、dd、jj和kk各自独立地为0、1、2、3、4、5、6、7、8、9或10；
R 6选自下组：
Figure PCTCN2022117373-appb-000013
其中，i、j、ee、mm和nn各自独立地为0、1、2、3、4、5、6、7、8、9或10；
R 7选自下组：
Figure PCTCN2022117373-appb-000014
其中，k、l和m各自独立地为0、1、 2、3、4、5、6、7、8、9或10；
R 8选自下组：
Figure PCTCN2022117373-appb-000015
其中，n、o和q各自独立地为0、1、2、3、4、5、6、7、8、9或10；
C、D、E、F、G、J各自独立地选自下组：H、CH 3；
R 9选自下组：
Figure PCTCN2022117373-appb-000016
Figure PCTCN2022117373-appb-000017
其中，r、s、oo、pp、qq、rr、u、v、aaa、bbb、ccc、hh各自独立地为0、1、2、3、4、5、6、7、8、9、10、11或12；
R 10选自下组：
Figure PCTCN2022117373-appb-000018
Figure PCTCN2022117373-appb-000019
其中，w、x、ss、tt、uu、vv、u、aa、ddd、eee、fff、ii各自独立地为0、1、2、3、4、5、6、7、8、9、10、11或12；
K、L、M和N各自独立地选自下组：H、CH 3；AA和BB各自独立地选自下组：H、CH 3、OH或NH 2；
Figure PCTCN2022117373-appb-000020
的主链(即最长链)上各自独立地具有2-40个碳，
Figure PCTCN2022117373-appb-000021
至少一条的主链具有10-40个碳。
在另一优选例中，a为0、1或2。
在另一优选例中，b为0、1或2。
在另一优选例中，ggg为0、1或2。
在另一优选例中，hhh为0、1或2。
在另一优选例中，iii为0、1或2。
在另一优选例中，kkk为0、1或2。
在另一优选例中，ww为0、1、2、3、4、5、6、7或8。
在另一优选例中，xx为0、1、2、3、4、5、6、7或8。
在另一优选例中，c为0、1、2、3、4、5、6、7、8或9。
在另一优选例中，d为0、1、2、3、4、5、6、7、8或9。
在另一优选例中，yy为0、1、2、3、4、5、6、7或8。
在另一优选例中，zz为0、1、2、3、4、5、6、7或8。
在另一优选例中，e为0、1、2、3、4、5、6、7、8或9。
在另一优选例中，f为0、1、2、3、4、5、6、7、8或9。
在另一优选例中，g为0、1、2、3、4、5、6、7、8或9。
在另一优选例中，h为0、1、2、3、4、5、6、7、8、9或10，优选为0、1、2、3、4、5、6、7、8或9。
在另一优选例中，i为0、1、2、3、4、5、6、7、8、9或10，优选为0、1、2、3、4、5、6、7、8或9。
在另一优选例中，j为0、1、2、3、4、5、6、7、8、9或10，优选为0、1、2、3、4、5、6、7、8或9。
在另一优选例中，jj为0、1、2、3、4、5、6、7、8、9或10，优选为0、1、2、3、4、5、6、7或8。
在另一优选例中，kk为0、1、2、3、4、5、6、7、8、9或10，优选为0、1、2、3、4、5、6、7或8。
在另一优选例中，mm为0、1、2、3、4、5、6、7、8、9或10，优选为0、1、2、3、4、5、6、7或8。
在另一优选例中，nn为0、1、2、3、4、5、6、7、8、9或10，优选为0、1、2、3、4、5、6、7或8。
在另一优选例中，k为0、1、2、3、4、5、6、7、8、9或10，优选为0、1、2、3、4、5、6、7或8。
在另一优选例中，l为0、1、2、3、4、5、6、7、8、9或10，优选为0、1、2、3、4、5、6、7或8。
在另一优选例中，m为0、1、2、3、4、5、6、7、8、9或10。
在另一优选例中，n为0、1、2、3、4、5、6、7、8、9或10，优选为0、1、2、3、4、 5、6、7或8。
在另一优选例中，o为0、1、2、3、4、5、6、7、8、9或10，优选为0、1、2、3、4、5、6、7或8。
在另一优选例中，p为0、1、2、3、4、5、6、7、8、9或10。
在另一优选例中，r为0、1、2、3、4、5、6、7、8、9或10，优选为0、1、2、3、4、5、6、7或8。
在另一优选例中，s为0、1、2、3、4、5、6、7、8、9或10，优选为0、1、2、3、4、5、6、7或8。
在另一优选例中，oo为0、1、2、3、4、5、6、7、8、9或10，优选为0、1、2、3、4、5、6、7或8。
在另一优选例中，pp为0、1、2、3、4、5、6、7、8、9或10，优选为0、1、2、3、4、5、6、7或8。
在另一优选例中，qq为0、1、2、3、4、5、6、7、8、9或10，优选为0、1、2、3、4、5、6、7或8。
在另一优选例中，rr为0、1、2、3、4、5、6、7、8、9或10，优选为0、1、2、3、4、5、6、7或8。
在另一优选例中，u为0、1、2、3、4、5、6、7、8、9或10。
在另一优选例中，v为0、1、2、3、4、5、6、7、8、9或10。
在另一优选例中，hh为0、1、2、3、4、5、6、7、8、9或10。
在另一优选例中，aaa为0、1、2、3、4、5、6、7或8。
在另一优选例中，bbb为0、1、2、3、4、5、6、7或8。
在另一优选例中，ccc为0、1、2、3、4、5、6、7或8。
在另一优选例中，ss为0、1、2、3、4、5、6、7或8。
在另一优选例中，tt为0、1、2、3、4、5、6、7或8。
在另一优选例中，uu为0、1、2、3、4、5、6、7或8。
在另一优选例中，vv为0、1、2、3、4、5、6、7或8。
在另一优选例中，aa为0、1、2、3、4、5、6、7、8、9或10。
在另一优选例中，ii为0、1、2、3、4、5、6、7、8、9或10。
在另一优选例中，ddd为0、1、2、3、4、5、6、7或8。
在另一优选例中，eee为0、1、2、3、4、5、6、7或8。
在另一优选例中，fff为0、1、2、3、4、5、6、7、8、9或10。
一类阳离子脂质化合物，化合物通式如以下式III所示：
Figure PCTCN2022117373-appb-000022
其中，
X、Y各自独立地选自N或CH，且X或Y至少一个为N；
R 11选自下组：
Figure PCTCN2022117373-appb-000023
其中，b2各自独立地为0、1、2、3、4、5、6、7、8、9或10；EE为甲基或羟基；
R 22选自下组：
Figure PCTCN2022117373-appb-000024
b1各自独立地为0、1、2、3、4、5、6、7、8、9或10；FF为甲基或羟基；
R 2选自下组：
Figure PCTCN2022117373-appb-000025
R 3选自下组：
Figure PCTCN2022117373-appb-000026
其中，ww、xx、c、d、ff各自独立地为0、1、2、3、4、5、6、7、8、9或10；
R 4选自下组：
Figure PCTCN2022117373-appb-000027
其中，yy、zz、e、f、gg各自独立地为0、1、2、3、4、5、6、7、8、9或10；
CC、DD、A、B为选自下组的取代基：H、CH 3、OH、CH 2OH、C 2H 4OH、NH 2；
R 5选自下组：
Figure PCTCN2022117373-appb-000028
其中，g、h、dd、jj和kk各自独立地为0、1、2、3、4、5、6、7、8、9或10；
R 6选自下组：
Figure PCTCN2022117373-appb-000029
其中，i、j、ee、mm和nn各自独立地为0、1、2、3、4、5、6、7、8、9或10；
R 7选自下组：
Figure PCTCN2022117373-appb-000030
其中，k、l和m各自独立地为0、1、2、3、4、5、6、7、8、9或10；
R 8选自下组：
Figure PCTCN2022117373-appb-000031
其中，n、o和q各自独立地为0、1、2、3、4、5、6、7、8、9或10；
C、D、E、F、G、J各自独立地选自下组：H、CH 3；
R 9选自下组：
Figure PCTCN2022117373-appb-000032
Figure PCTCN2022117373-appb-000033
其中，r、s、oo、pp、qq、rr、u、v、aaa、bbb、ccc、hh各自独立地为0、1、2、3、4、5、6、7、8、9、10、11或12；
R 10选自下组：
Figure PCTCN2022117373-appb-000034
Figure PCTCN2022117373-appb-000035
其中，w、x、ss、tt、uu、vv、u、aa、ddd、eee、fff、ii各自独立地为0、1、2、3、4、5、6、7、8、9、10、11或12；
K、L、M和N各自独立地选自下组：H、CH 3；AA和BB各自独立地选自下组：H、CH 3、OH或NH 2；
Figure PCTCN2022117373-appb-000036
的主链(即最长链)上各自独立地具有2-40个碳，所述
Figure PCTCN2022117373-appb-000037
至少一条侧链部分上具有10-25个碳。
一类阳离子脂质化合物，化合物通式如以下式IV所示：
Figure PCTCN2022117373-appb-000038
R 3选自下组：
Figure PCTCN2022117373-appb-000039
其中，ww、xx、c、d、ff各自独立地为0、1、2、3、4、5、6、7、8、9或10；
R 4选自下组：
Figure PCTCN2022117373-appb-000040
其中，yy、zz、e、f、gg各自独立地为0、1、2、3、4、5、6、7、8、9或10；
A、B为选自下组的取代基：H、CH 3、OH、CH 2OH、C 2H 4OH、NH 2；
R 5选自下组：
Figure PCTCN2022117373-appb-000041
其中，g、h、dd、jj和kk各自独立地为0、1、2、3、4、5、6、7、8、9或10；
R 6选自下组：
Figure PCTCN2022117373-appb-000042
其中，i、j、ee、mm和nn各自独立地为0、1、2、3、4、5、6、7、8、9或10；
R 7选自下组：
Figure PCTCN2022117373-appb-000043
其中，k、l和m各自独立地为0、1、2、3、4、5、6、7、8、9或10；
R 8选自下组：
Figure PCTCN2022117373-appb-000044
其中，n、o和q各自独立地为0、1、2、3、4、5、6、7、8、9或10；
C、D、E、F、G、J各自独立地选自下组：H、CH 3；
R 9选自下组：
Figure PCTCN2022117373-appb-000045
Figure PCTCN2022117373-appb-000046
其中，r、s、oo、pp、qq、rr、u、v、aaa、bbb、ccc、hh各自独立地为0、1、2、3、4、5、6、7、8、9、10、11或12；
R 10选自下组：
Figure PCTCN2022117373-appb-000047
Figure PCTCN2022117373-appb-000048
其中，w、x、ss、tt、uu、vv、u、aa、ddd、eee、fff、ii各自独立地为0、1、2、3、4、5、6、7、8、9、10、11或12；
K、L、M和N各自独立地选自下组：H、CH 3；AA和BB各自独立地选自下组：H、CH 3、OH或NH 2；
Figure PCTCN2022117373-appb-000049
的主链(即最长链)上各自独立地具有2-40个碳，
Figure PCTCN2022117373-appb-000050
至少一条具有10-25个碳。
前述的一类阳离子脂质化合物，
Figure PCTCN2022117373-appb-000051
各自独立地为选自如下的结构：
Figure PCTCN2022117373-appb-000052
前述的一类阳离子脂质化合物，化合物选自下组：
Figure PCTCN2022117373-appb-000053
Figure PCTCN2022117373-appb-000054
一类阳离子脂质化合物的应用于：包含阳离子脂质化合物的组合物、其立体异构体、其互变异构体或其在药学上可接受的盐。。
作为对一类阳离子脂质化合物的应用的进一步说明，包含阳离子脂质化合物的组合物包括：载体、所载的药物试剂、药物辅助剂中的一种或几种的组合。。
作为对一类阳离子脂质化合物的应用的进一步说明，载体包括：一种或多种阳离子脂质化合物、助脂质、结构脂质、聚合物缀合脂质或两亲性嵌段共聚物中的一种或几种的组合。
作为对一类阳离子脂质化合物的应用的进一步说明，阳离子脂质化合物与助脂质的摩尔比为0.5:1-10:1。
作为对一类阳离子脂质化合物的应用的进一步说明，阳离子脂质化合物与结构脂质的摩尔比为0.5:1-5:1。
作为对一类阳离子脂质化合物的应用的进一步说明，阳离子脂质化合物与聚合物缀合脂质的摩尔比为10：1-250：1。
作为对一类阳离子脂质化合物的应用的进一步说明，阳离子脂质化合物与两亲性嵌段共聚物的摩尔比为0.5：1-80：1。
作为对一类阳离子脂质化合物的应用的进一步说明，载体为脂质纳米粒LNP，脂质纳米粒的平均尺寸为30-200nm，脂质纳米粒的制剂的多分散指数≤0.5。
作为对一类阳离子脂质化合物的应用的进一步说明，所载的药物试剂包括：核酸分子、小分子化合物、多肽或蛋白质中的一种或多种。
作为对一类阳离子脂质化合物的应用的进一步说明，药物辅助剂包括：稀释剂、稳定剂、防腐剂或冻干保护剂中的一种或多种。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
技术效果：
本发明的阳离子脂质化合物以哌嗪及类哌嗪化合物的氮原子为电荷中心，在相同条件下与核酸的相互作用更强，可以更好的保护核酸和负载更多的核酸，另外在疏水的尾部引入双键，从实验看出相同结构的化合物，未引入双键的要比引入双键的化合物转染效率低很多，说明在尾部引入双键与本发明的化合物结构在提高转染效果上具有协同作用；相比市场已在使用的阳离子脂质MC3制备得到的LNP转染效率 高；
在脂质体的疏水尾部引入可降解的酯键，可以改变脂质体在体内的代谢行为，同时增加其疏水尾部的亲脂性，进而提高mRNA-LNP的生物安全性和转染效率；
在电荷中心附近以羟基修饰可以进一步增加其头部亲水性，以进一步增加其转染效果；
本发明结构的阳离子脂质转染效率高，安全性好，生物相容性高，合成步骤简单，适合生物医药产业化。
附图说明
图1是本发明P-1阳离子脂质化合物的氢谱图；
图2是本发明P-2阳离子脂质化合物的氢谱图；
图3是本发明P-3阳离子脂质化合物的氢谱图；
图4是本发明P-4阳离子脂质化合物的氢谱图；
图5是本发明P-5阳离子脂质化合物的氢谱图；
图6是本发明P-6阳离子脂质化合物的氢谱图；
图7是本发明P-7阳离子脂质化合物的氢谱图；
图8是本发明采用样品P-1的化合物制备得到脂质纳米粒的电镜图；
图9是本发明采用样品P-1的阳离子脂质化合物制备得到的LNP和市场上的LNP的免疫效果对比实验结果示意图。
术语、英文缩写解释说明：
“治疗性核酸”指能够以寡核苷酸或表达对应寡核苷酸的载体的形式被施用于所需要的对象并产生治疗效果的核酸类物质。可以根据任何可用的技术来制备所述的治疗性核酸，例如对于mRNA，主要的制备方法为，但不限于酶促合成(也称为体外转录)，该方法目前代表着产生长序列特异的mRNA的最有效的方法。体外转录描述了由工程化的DNA模板进行RNA分子的模板导向合成的方法，该工程化的DNA模板包含与编码目标基因的下游序列连接的上游噬菌体启动子序列(例如包括但不限于来自T7、T3和SP6大肠杆菌噬菌体)。可以用本领域熟知的适当的技术由很多来源来制备模板DNA用于体外转录，所述适当的技术包括，但不限于质粒DNA和聚合酶链式反应扩增。
使用线性化DNA模板，在相应的RNA聚合酶以及腺苷、鸟苷、尿苷和胞苷核糖核苷三磷酸(rNTP)存在下，在支持聚合酶活性同时使所得的mRNA转录物潜在的降解最小化的条件下，在体外发生RNA的转录。可以使用各种商购的试剂盒以及商购的试 剂来进行体外转录，所述试剂盒包括，但不限于RiboMax大规模RNA生产系统(Promega)、MegaScript转录试剂盒(LifeTechnologies)，以及所述试剂包括RNA聚合酶和rNTP。mRNA的体外转录方法是本领域熟知的。
然后从转录或相关反应的不期望的组分(包括未并入的rNTP、蛋白质酶、盐、短RNA寡核苷酸等)中纯化期望的体外转录的mRNA。mRNA转录物的分离技术是本领域熟知的。熟知的程序包括用在单价阳离子存在下的醇(乙醇、异丙醇)或氯化锂的酚/氯仿萃取或沉淀。可以使用的纯化程序的另外非限制性实例包括尺寸排阻色谱、基于硅的亲和色谱和聚丙烯酰胺凝胶电泳。可以使用各种商购的试剂盒来进行纯化，所述试剂盒包括，但不限于SV总分离系统(Promega)以及体外转录清洁和浓缩试剂盒(Norgen Biotek)。
此外，虽然逆转录可以产生大量的mRNA，但产物可以含有大量与不期望的聚合酶活性有关的异常RNA杂质，所述杂质可能需要从全长的mRNA制备物中去除。这些杂质包括由失败的转录起始导致的短RNA以及由RNA依赖的RNA聚合酶活性、来自RNA模板的RNA引发的转录和自补3’延长生成的双链RNA(dsRNA)。已经证明这些具有dsRNA结构的污染物可以通过与真核细胞中的各种先天免疫传感器相互作用而导致不期望的免疫刺激活性，所述先天免疫传感器的作用为识别特定核酸结构并诱导强效免疫应答。由于在先天细胞免疫应答期间蛋白质合成减少，因此可以进而显著减少mRNA翻译。因此，已研发出用于去除这些dsRNA污染物的另外的技术，并且其被本领域所知晓，包括但不限于可调比例的HPLC纯化。已报导经HPLC纯化的mRNA以高得多的水平翻译，特别是在原代细胞中和体内。
本领域中已描述了很多种用于改变体外转录的mRNA的特定性质，并且改善其效用的修饰。这些修饰包括，但不限于对mRNA的5’末端和3’末端的修饰。内源性真核mRNA通常含有在成熟分子的5’-端上的帽结构，其在介导mRNA帽结合蛋白质(CBP)的结合中发挥重要作用，其进而负责增强细胞中的mRNA稳定性和mRNA翻译效率。因此，用加帽的mRNA转录物实现最高水平的蛋白质表达。5’-帽含有5’-多数核苷酸与鸟嘌呤核苷酸之间的5’-5’-三磷酸连接。缀合的鸟嘌呤核苷酸在N7位置被甲基化。另外的修饰包括在2’-羟基上的最后的和倒数第二的多数5’-核苷酸的甲基化。
多个不同的帽结构可以用于生成体外转录合成的mRNA的5’-帽。合成的mRNA的5’-加帽可以用化学帽类似物与转录共同进行(即在体外转录过程中加帽)。例如，抗反 向帽类似物(ARCA)帽含有5’-5’-三磷酸鸟嘌呤-鸟嘌呤连接，其中一个鸟嘌呤含有N7甲基以及3’-O-甲基。然而，多达20％的转录物在该共转录过程中保持未加帽，并且合成的帽类似物与真实细胞mRNA的5’-帽结构不相同，有可能降低可译性和细胞稳定性。可选地，合成的mRNA分子还可以在转录后酶促加帽。这可以生成更真实的5’-帽结构，其在结构上或功能上更接近地模拟内源性5’-帽，该内源性5’-帽具有增强的帽结合蛋白质的结合、增加的半衰期、对5’内切酶的降低的敏感性和/或减少的5’脱帽性。已研发出很多合成的5’-帽类似物且被本领域所知晓，以增强mRNA稳定性和可译性。
在3’-末端，通常在RNA加工期间将腺嘌呤核苷酸的长链(聚腺苷酸尾)加至mRNA分子。转录后，立即切割转录物的3’端以释放3’羟基，向3’端，聚腺苷酸聚合酶在被称为聚腺苷酸化的过程中将腺嘌呤核苷酸链加至RNA。聚腺苷酸尾已广泛示出了增强翻译效率和mRNA的稳定性。
可以使用各种方法来实现体外转录的mRNA的聚腺苷酸加尾，所述方法包括，但不限于，将聚(T)片段克隆至DNA模板中，或者通过使用聚(A)聚合酶在转录后添加。第一种情况允许体外转录具有限定长度(取决于聚(T)片段的尺寸)的聚(A)尾的mRNA，但需要对模板另外操作。后一种情况涉及使用催化腺嘌呤残基并入RNA的3’末端的聚(A)聚合酶，将聚(A)尾酶促添加至体外转录的mRNA，不需要对DNA模板另外操作，而得到具有不同长度的聚(A)尾的mRNA。可以使用各种商购的试剂盒以及商购的试剂、各种ARCA帽、聚(A)聚合酶等来进行5’-加帽和3’-聚(A)加尾，所述试剂盒包括，但不限于聚(A)聚合酶加尾试剂盒(EpiCenter)、mMESSAGE mMACHINE T7Ultra试剂盒和聚(A)加尾试剂盒(Life Technologies)。
除5’帽和3’聚腺苷酸化以外，还报导了对体外转录物的其他修饰以提供与翻译效率和稳定性有关的益处。本领域熟知的是，病原DNA和RNA可以通过真核细胞内的各种传感器识别，并引发强效先天免疫应答。因为天然来源的大多数核酸含有修饰的核苷，因此已显示出辨别病原DNA和RNA与自身DNA和RNA的能力至少部分基于结构和核苷修饰。相反，体外合成的RNA缺少这些修饰，因此导致其为免疫刺激的，进而可以抑制上文概述的有效的mRNA翻译。将修饰的核苷引入体外转录的mRNA可以用于阻止RNA传感器的识别和激活，由此缓解这种不期望的免疫刺激活性，并且增强翻译能力。用于合成修饰的RNA的修饰的核苷和核苷酸可以使用本领域已知的一般方法和程序来制备、监测和使用。可使用很多种核苷修饰，其可以单独或联用其他修饰的 核苷以一定程度并入体外转录的mRNA。已报导核苷修饰的mRNA的体外合成减小了激活免疫传感器的能力，伴随着增强了翻译能力。
可修饰以在可译性和稳定性方面提供益处的mRNA的其他组分包括5’和3’非翻译区(UTR)。针对两者的或单独的UTR优化(有利的5’和3’UTR可以得自细胞或病毒RNA)已显示出增强了体外转录的mRNA的mRNA稳定性和翻译效率。
除mRNA以外，其他核酸净荷(payload)也可以用于本发明。对于寡核苷酸，制备方法包括但不限于化学合成和较长前体的酶促、化学裂解，上文描述的体外转录等。合成DNA和RNA核苷酸的方法被广泛使用，并且为本领域所熟知。
对于质粒DNA，与本发明联用的制备通常使用但不限于，在含有目标质粒的细菌的液体培养基中体外扩增和分离质粒DNA。编码对特定抗生素(青霉素、卡那霉素等)的抗性的目标质粒内基因的存在允许那些含有目标质粒的细菌在含抗生素的培养基中选择性地生长。分离质粒DNA的方法被广泛使用，并且为本领域所熟知。可以使用各种可商购的试剂盒以及可商购的试剂来进行质粒分离，所述试剂盒包括，但不限于Plasmid Plus(Qiagen)、GenJET plasmid MaxiPrep(Thermo)和Pure Yield MaxiPrep(Promega)试剂盒。
“脂质纳米颗粒”指使用阳离子脂质体与需要递送的核酸药物通过自组装方式形成的复合载药颗粒。本发明的脂质纳米颗粒和组合物可以用于各种目的，包括在体外和体内将封装的或缔合的(例如，复合的)诸如核酸的治疗剂递送至细胞，从而诱导期望蛋白质的表达或者抑制靶基因的表达。因此，本发明的实施方案提供通过使有此需要的对象接触封装适合的治疗剂或与适合的治疗剂缔合的脂质纳米颗粒，来治疗或预防所述对象的疾病和病症的方法，其中所述脂质纳米颗粒包含一种或多种本文描述的新型阳离子脂质。本发明的阳离子脂质、脂质纳米颗粒和包含脂质纳米颗粒的组合物、以及它们递送诸如核酸的活性剂(例如治疗剂)，以调控基因和蛋白质表达的用途的各种示例性实施方案在下文进一步详细地描述。
“诱导期望蛋白质的表达”指核酸增加期望蛋白质表达的能力。为检验蛋白质表达的程度，使测试样品(例如，表达期望蛋白质的培养基中的细胞样品)或测试哺乳动物(例如，诸如人的哺乳动物或者诸如啮齿动物(例如，小鼠)或非人灵长类动物(例如，猴)模型的动物模型)接触核酸(例如，结合本发明脂质的核酸)。将测试样品或测试动物中的期望蛋白质的表达与未接触或未施用核酸的对照样品(例如，表达期望蛋白质 的培养基中的细胞样品)或对照哺乳动物(例如，诸如人的哺乳动物或者诸如啮齿动物(例如，小鼠)或非人灵长类动物(例如，猴)模型的动物模型)中的期望蛋白质的表达相比较。当在对照样品或对照哺乳动物中存在期望的蛋白质时，对照样品或对照哺乳动物中期望蛋白质的表达可以指定为1.0的值。在特定的实施方案中，当测试样品或测试哺乳动物中的期望蛋白质表达与对照样品或对照哺乳动物中的期望蛋白质表达水平的比例大于1时，例如，约1.1、1.5、2.0、5.0或10.0时，实现了诱导期望蛋白质的表达。当对照样品或对照哺乳动物中不存在期望的蛋白质时，当在测试样品或测试哺乳动物中检测到任何可测水平的期望蛋白质时，实现了诱导期望蛋白质的表达。本领域的普通技术人员将理解，确定样品中蛋白质表达水平的适合的测定例如，斑点印迹、northern印迹、原位杂交、ELISA、免疫沉淀、酶功能和表型测定，或者基于在适当条件下可以产生荧光或发光的报告蛋白的测定。
短语“抑制靶基因的表达”指核酸沉默、减少或抑制靶基因表达的能力。为检验基因沉默的程度，使测试样品(例如，表达靶基因的培养基中的细胞样品)或测试哺乳动物(例如，诸如人的哺乳动物或者诸如啮齿动物(例如，小鼠)或非人灵长类动物(例如，猴)模型的动物模型)接触沉默、减少或抑制靶基因表达的核酸。将测试样品或测试动物中的靶基因的表达与未接触或未施用核酸的对照样品(例如，表达靶基因的培养基中的细胞样品)或对照哺乳动物(例如，诸如人的哺乳动物或者诸如啮齿动物(例如，小鼠)或非人灵长类动物(例如，猴)模型的动物模型)中的靶基因的表达相比较。对照样品或对照哺乳动物中的靶基因的表达可以指定为100％的值。在特定的实施方案中，当测试样品或测试哺乳动物中的靶基因表达水平相对于对照样品或对照哺乳动物中的靶基因表达水平为约95％、90％、85％、80％、75％、70％、65％、60％、55％、50％、45％、40％、35％、30％、25％、20％、15％、10％、5％或0％时，实现了沉默、抑制或减少靶基因的表达。换言之，相对于未接触或未施用核酸的对照样品或对照哺乳动物中的靶基因表达水平，在测试样品或测试哺乳动物中，所述核酸能够沉默、减少或抑制靶基因表达的至少约5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或100％。确定靶基因表达水平的适合的测定包括，但不限于，使用本领域技术人员已知的技术进行的蛋白质或mRNA水平的检验，所述技术例如，本领域技术人员已知的斑点印迹、northern印迹、原位杂交、ELISA、免疫沉淀、酶功能以及表型测定。
术语“脂质”指一组有机化合物，其包括但不限于脂肪酸的酯，并且特征通常为在水中具有差的溶解性，但在很多种有机溶剂中可溶。它们通常分为至少三类：(1)“简单脂质”，其包括脂肪和油以及蜡；(2)“化合物脂质”，其包括磷脂和糖脂；以及(3)“衍生脂质”，例如类固醇。
“阳离子脂质”指能够带正电的脂质。示例性的阳离子脂质包括一种或多种带有正电荷的胺基团。优选的阳离子脂质是可电离的，以便它们可以根据pH值以带正电的形式或中性形式存在。阳离子脂质的电离影响脂质纳米颗粒在不同pH条件下的表面电荷。这种电荷状态可以影响血浆蛋白质吸收、血液清除和组织分布，以及形成核内体溶解(endosomolytic)非双层结构的能力，对于核酸的细胞内递送是至关重要的。
术语“聚合物缀合的脂质”指包含脂质部分和聚合物部分的分子。聚合物缀合的脂质的实例是聚乙二醇化脂质。术语“聚乙二醇化脂质”指包含脂质部分和聚乙二醇部分的分子。聚乙二醇化脂质为本领域已知的，并且包括1-(单甲氧基-聚乙二醇)-2,3-二肉豆蔻酰甘油(PEG-DMG)等。
术语“中性脂质”指在选定的pH下以无电荷或中性两性离子形式存在的许多脂质物质中的任一种。在生理pH下，此类脂质包括但不限于磷脂酰胆碱，例如1,2-二硬脂酰基-sn-甘油基-3-磷酸胆碱(DSPC)、1,2-二棕榈酰基-sn-甘油基-3-磷酸胆碱(DPPC)、1,2-二肉豆蔻酰基-sn-甘油基-3-磷酸胆碱(DMPC)、1-棕榈酰基-2-油酰基-sn-甘油基-3-磷酸胆碱(POPC)、1,2-二油酰基-sn-甘油基-3-磷酸胆碱(DOPC)，磷脂酰乙醇胺如1,2-二油酰基-sn-甘油基-3-磷酸乙醇胺(DOPE)，鞘磷脂(SM)，神经酰胺，类固醇如甾醇及其衍生物。中性脂质可以是合成的或天然来源的。
术语“带电脂质”指以下多种脂质物质中的任一种，其以带正电荷或带负电荷的形式存在，不依赖于在有用的生理学范围内的pH，例如pH～3至pH～9。带电脂质可以是合成的或天然来源的。带电脂质的实例包括磷脂酰丝氨酸、磷脂酸、磷脂酰甘油、磷脂酰肌醇、甾醇半琥珀酸酯、二烷基三甲铵-丙烷(例如DOTAP、DOTMA)、二烷基二甲基氨基丙烷、乙基磷酸胆碱、二甲基氨基乙烷氨基甲酰基甾醇(例如DC-Chol)。
术语“脂质纳米颗粒”指具有纳米量级上(例如，1nm至1,000nm)的至少一个维度的颗粒，其包含一种或多种结构(I)的化合物或其他特定阳离子脂质。在一些实施方案中，脂质纳米颗粒包含在可用于将诸如核酸(例如，mRNA)的活性剂或治疗剂递送至目标靶位点(例如，细胞、组织、器官、肿瘤等)的制剂中。在一些实施方案中，本发明的 脂质纳米颗粒包含核酸。这类脂质纳米颗粒通常包含结构(I)的化合物以及一种或多种选自中性脂质、带电脂质、胆固醇和聚合物缀合的脂质的赋形剂。在一些实施方案中，诸如核酸的活性剂或治疗剂可以封装于脂质纳米颗粒的脂质部分中，或者封装于由脂质纳米颗粒的一些或全部脂质部分包裹的水性空间中，从而保护其不被酶促降解，或者不受到由宿主生物体或细胞的机制诱导的其他不期望的作用，例如不良的免疫应答。
本发明的脂质纳米颗粒的实施方案对于核酸的递送是特别有用的，所述核酸包括例如mRNA、反义寡核苷酸、质粒DNA、微小RNA(miRNA)、miRNA抑制剂(antagomirs/antimirs)、信使-RNA-干扰互补RNA(micRNA)、DNA、多价RNA、dicer底物RNA、互补DNA(cDNA)等。因此，本发明的脂质纳米颗粒和组合物可以用于通过使细胞与包含一种或多种本文描述的新型阳离子脂质的脂质纳米颗粒接触，从而在体外和体内诱导所期望蛋白质的表达，其中所述脂质纳米颗粒封装或缔合如下核酸：被表达以产生所期望蛋白质的核酸(例如，编码所期望蛋白质的信使RNA或质粒)或被表达以抑制终止mRNA表达的过程的核酸(例如，miRNA抑制剂)。可选地，本发明的脂质纳米颗粒和组合物可以用于通过使细胞与包含一种或多种本文描述的新型阳离子脂质的脂质纳米颗粒接触，从而在体外和体内减少靶基因和靶蛋白质的表达，其中脂质纳米颗粒封装或缔合减少靶基因表达的核酸(例如，反义寡核苷酸或小干扰RNA(siRNA))。本发明的脂质纳米颗粒和组合物还可以用于单独或联合共递送不同的核酸(例如mRNA和质粒DNA)，例如可以用于提供需要不同核酸(例如编码适合的基因修饰酶的mRNA和并入宿主基因组的DNA片段)共同定位的作用。
在各种实施方案中，所使用的脂质纳米颗粒具有以下的平均直径：约30nm至约150nm、约40nm至约150nm、约50nm至约150nm、约60nm至约130nm、约70nm至约110nm、约70nm至约100nm、约80nm至约100nm、约90nm至约100nm、约70至约90nm、约80nm至约90nm、约70nm至约80nm、或者约30nm、35nm、40nm、45nm、50nm、55nm、60nm、65nm、70nm、75nm、80nm、85nm、90nm、95nm、100nm、105nm、110nm、115nm、120nm、125nm、130nm、135nm、140nm、145nm或150nm，并且是基本无毒的。在某些实施方案中，核酸当存在于脂质纳米颗粒时，其在水溶液中抵抗被核酸酶降解。
如本文所使用的，“脂质封装的”指为诸如核酸(例如，mRNA)的活性剂或治疗剂提供完全封装、部分封装或二者的脂质纳米颗粒。在一个实施方案中，核酸(例如， mRNA)完全封装于脂质纳米颗粒中。
就核酸-脂质纳米颗粒而言，“血清稳定的”意指核苷酸在暴露于血清后或在暴露于会显著降解游离DNA或RNA的核酸酶测定后，不会显著降解。适合的测定包括，例如，标准的血清测定、DNA酶测定或RNA酶测定。
如本文所使用的，“全身性递送”指可以导致活性剂在生物体内广泛暴露的治疗性产物的递送。一些施用技术可以导致某些试剂的全身性递送，但不会全身性递送其他试剂。全身性递送意味着有用量的，优选治疗量的试剂暴露于身体的多数部位。脂质纳米颗粒的全身性递送可以通过本领域已知的任何方式进行，包括，例如，静脉内、动脉内、皮下和腹膜内递送。在一些实施方案中，脂质纳米颗粒的全身性递送通过静脉内递送。
如本文所使用的，“局部递送”指将活性剂直接递送至生物体内的靶位点。例如，可以通过直接注入诸如肿瘤的疾病位点，诸如炎症位点的其他靶位点，或诸如肝、心脏、胰腺、肾等靶器官中来局部递送试剂。局部递送也可以包括局部应用或局部注射技术，例如肌内、皮下或皮内注射。局部递送不妨碍全身性的药理学作用。
核酸分子包括单链DNA、双链DNA、短异构体、mRNA、tRNA、rRNA、长链非编码RNA(lncRNA)、微小非编码RNA(miRNA和siRNA)、端粒酶RNA(Telomerase RNA Component)、小分子RNA(snRNA和scRNA)、环状RNA(circRNA)、合成miRNA(miRNA mimics、miRNA agomir、miRNA antagomir)、反义DNA、反义RNA、核酶(ribozyme)、不对称干扰RNA(aiRNA)、Dicer-substrate RNA(dsRNA)、小发夹RNA(shRNA)、转移RNA(tRNA)、信使RNA(mRNA)、gRNA、sgRNA、crRNA或tracrRNA、锁核酸(LNA)、肽核酸(PNA)、吗啉反义寡核苷酸、吗啉代寡核苷酸或生物定制寡核苷酸等。这里的举例也并非穷举，只要是由核苷酸单体聚合成的都可以应用于本发明。
药物可用的盐是指酸加成盐或碱加成盐。
其中酸加成盐的酸包括但不限于：盐酸、氢溴酸、硫酸、硝酸、磷酸、酸式磷酸盐、乙酸、2,2-二氯乙酸、己二酸、海藻酸、抗坏血酸、天冬氨酸、苯磺酸、苯甲酸、4-乙酰氨基苯甲酸、樟脑酸、樟脑-10-磺酸、癸酸、己酸、辛酸、碳酸、肉桂酸，柠檬酸、环酰胺酸、十二烷基硫酸、乙烷-1,2-二磺酸、乙烷磺酸、2-羟基乙磺酸、甲酸、富马酸、半乳糖酸、龙胆酸、葡庚酸、葡糖酸、葡聚糖酸、葡糖醛酸、谷氨酸、戊二酸、2-氧代戊二酸、甘油磷酸、乙醇酸、马尿酸、异丁酸、乳酸、乳糖酸、月桂酸、马来酸、苹果酸、丙二酸、扁桃酸、甲磺酸、粘酸、萘-1,5二甲酸、萘-2-磺酸、1-羟 基-2-萘甲酸、烟酸、油酸、乳清酸、草酸、棕榈酸、棕榈酸、丙酸、焦谷氨酸、丙酮酸、水杨酸、4-氨基水杨酸、癸二酸、硬脂酸、琥珀酸、酒石酸、硫氰酸、对甲苯磺酸、三氟乙酸、季铵酸以及十一碳烯酸。
其中碱加成盐举例包括但不限于：钠盐、钾盐、锂盐、铵盐、钙盐、镁盐、铁盐，锌盐、铜盐、锰盐、以及铝盐；有机碱包括但不限于氨、异丙胺、三甲胺、二乙胺、三乙胺、三丙胺、二乙醇胺、乙醇胺、脱醇、2-二甲基氨基乙醇、2-二乙基氨基乙醇、赖氨酸、精氨酸、组氨酸、咖啡因、普鲁卡因、肼苯胺、胆碱、甜菜碱、苯那敏(benethamine)、苄星青霉素(benzathine)、乙二胺、葡糖胺、甲基葡糖胺、可可碱、三乙醇胺、嘌呤、哌嗪、哌啶、N-乙基哌啶、以及聚胺树脂；优选地，有机碱是异丙胺、二乙胺、乙醇胺、三甲胺、二环己胺、胆碱和咖啡因。
助脂质包括：磷脂酰胆碱、磷脂酰乙醇胺、鞘磷脂(SM)、甾醇及其衍生物、神经酰胺、带电脂质中的一种或几种的组合；磷脂酰胆碱作为一种优选包括：DSPC，DPPC，DMPC，DOPC，POPC；磷脂酰乙醇胺作为一种优选为DOPE；甾醇作为一种优选为胆固醇；带电脂质作为一种实施例为DOTAP、DOTMA、18PA；这里并非穷举，只要是采用本发明结构的阳离子脂质化合物的组合物均在本发明的保护范围内，均受本发明启示。这里并非穷举，助脂质的选择不受限制，只要是采用本发明结构的阳离子脂质化合物均在本发明的保护范围内，均受本发明启示。
带电脂质是指一类脂质化合物以带正电荷或带负电荷的形式存在；其所带电荷不依赖于生理学范围内的pH，例如pH 3～9，不受pH的影响。带电脂质可以是合成的或天然来源的。带电脂质的实例包括但不限于DOTAP、DOTMA、18PA。
mRNA，信使RNA，中文译名：信使核糖核酸，是由DNA的一条链作为模板转录而来的、携带遗传信息能指导蛋白质合成的一类单链核糖核酸。mRNA可以是单顺反子mRNA也可以是多顺反子mRNA。mRNA也可以包含一种或多种功能性核苷酸类似物，功能性核苷酸类似物举例包括：假尿嘧啶核苷、1-甲基-假尿嘧啶核苷或5-甲基胞嘧啶等。这里的举例也并非穷举，任何修饰的mRNA或其衍生物都可以应用于本发明。
小分子化合物可以是用于治疗或预防的试剂中的有效成分，例如：抗肿瘤药、抗感染药、局部麻醉药、抗抑郁药、抗惊厥药、抗生素/抗菌剂、抗真菌药、抗寄生虫药、激素、激素拮抗剂、免疫调节剂、神经递质拮抗剂、抗青光眼剂、麻醉剂、或成像剂等，这里并非穷举。
多肽是α－氨基酸以肽键连接在一起而形成的化合物，是蛋白质水解的中间产物。
蛋白质是由氨基酸以“脱水缩合”的方式组成的多肽链经过盘曲折叠形成的具有 一定空间结构的物质；蛋白质可以是干扰素、蛋白质激素、细胞因子、趋化因子或者酶类等。
稀释剂是本领域技术人员可知的任意可以药用的水溶性辅料，包括：氨基酸、单糖、二糖、三糖、四糖、五糖、其它寡聚糖、甘露醇、右旋糖苷、氯化钠、山梨醇、聚乙二醇、磷酸盐，或其衍生物等。
稳定剂可以是本领域技术人员可知的任意可以药用的辅料：吐温-80、十二烷基硫酸钠、油酸钠、甘露醇、甘露糖或海藻酸钠等。
防腐剂可以是本领域技术人员可知的任意可以药用的防腐剂，比如：硫柳汞等。
冻干保护剂可以是领域技术人员可知的任意可以药用的冻干保护剂，比如：葡萄糖、甘露醇、蔗糖、乳糖，海藻糖，麦芽糖等。
DSPC：英文名称：Distearoyl Phosphatidylcholine，1,2-distearoyl-sn-glycero-3-phosphocholine；中文名称：二硬脂酰基卵磷脂，CAS号:816-94-4。
DPPC：中文名称：二棕榈酸磷脂酰胆碱；英文名称：1,2-DIPALMITOYL-SN-GLYCERO-3-PHOSPHOCHOLINE，CAS号:63-89-8。
DMPC：中文名称：二肉豆蔻酰磷脂酰胆碱；英文名称：1,2-Dimyristoyl-sn-glycero-3-phosphocholine，CAS号:18194-24-6。
DOPC：中文名称：1,2-二油酰基-sn-甘油-3-磷酸胆碱；英文名称：1,2-dioleoyl-sn-glycero-3-phosphocholine，CAS号:4235-95-4。
POPC：中文名称：2-油酰-1-棕榈锡甘油-3-磷酸胆碱；英文名称：2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine，CAS号:26853-31-6。
DOPE：中文名称：1,2-二油酰-SN-甘油-3-磷酰乙醇胺；英文名称：1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOETHANOLAMINE，CAS号：4004-05-1。
DOTAP：中文名称：N-[1-(2，3-二油酰氧基)丙基]-N，N，N-三甲基铵甲基-硫酸盐；英文名称：1,2-dioleoyl-3-trimethylammonium-propane(chloride salt)，CAS号：144189-73-1；化学结构式如下所示：
Figure PCTCN2022117373-appb-000055
DOTMA：中文名称：N,N,N-三甲基-2,3-双(十八碳-9-烯-1-基氧基)丙-1-铵氯化物，CAS号:1325214-86-5，化学结构式如下所示：
Figure PCTCN2022117373-appb-000056
18PA：CAS号：108392-02-5，化学结构式如下所示：
Figure PCTCN2022117373-appb-000057
SM：中文名称：鞘磷脂(SM)；英文名称：sphingomyelin。
PEG：中文名称：聚乙二醇；英文名称：Polyethylene glycol。
两亲性嵌段共聚物指：PEG与下列一种或多种聚合物组分的嵌段共聚物，聚合物组分包括：聚乳酸-聚羟基乙酸共聚物(PLGA)、聚乳酸(PLA)、聚己内酯(PCL)、聚原酸酯、聚酸酐、聚(β-氨基酯)(PBAE)中的一种或多种。
具体实施方式
本发明制备得到的一类结构新颖的脂质体分子，所述的脂质体分子可以与治疗性核酸共同形成包载RNA的脂质体纳米粒，从而将治疗性核酸递送到需要形成药物浓度的部位。基于上述发现，发明人完成了本发明。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1 化合物P-1的合成
Figure PCTCN2022117373-appb-000058
P-1的合成：将N,N'-双(2-羟乙基)哌嗪(化合物a，1.00g，5.74mmol)，亚油酸(3.38g， 12.05mmol)，1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(2.53g，13.20mmol)，N,N'-二异丙基乙胺(1.78g，13.77mmol)，4-二甲氨基吡啶(0.14g，1.15mmol)溶于15mL二氯甲烷(DCM)中，室温搅拌反应12h。TLC监测反应完全后，使用旋转蒸发仪减压蒸馏除去溶剂。加入100mL乙酸乙酯，等体积饱和碳酸氢钠溶液洗2次，等体积饱和氯化钠溶液洗1次，无水硫酸钠干燥30min，使用旋转蒸发仪减压蒸馏除去溶剂，柱分离纯化(硅胶柱，洗脱液为DCM：MeOH＝50：1(体积比))，得3.2g无色油状液体，收率80％。 1H NMR(400MHz,Chloroform-d)δ5.45–5.23(m,8H),4.19(t,J＝5.9Hz,4H),2.79–2.71(m,4H),2.69–2.46(m,12H),2.28(t,J＝7.5Hz,4H),2.03(q,J＝6.9Hz,8H),1.59(t,J＝7.4Hz,4H),1.37–1.22(m,28H),0.91–0.82(m,6H).MS m/z(ESI)：699.64[M+H] +。
采用实施例1的方法，同样也可以制备得到化合物P-11在此不再一一赘述。
实施例2 化合物P-2的合成
Figure PCTCN2022117373-appb-000059
化合物a的合成：将亚油酸(6.00g，24.96mmol)溶于适量100mL无水四氢呋喃中，氮气保护下，冰浴搅拌10min，然后分批加入氢化铝锂(1.04g,27.46mmol)，加毕，逐渐恢复至室温，搅拌过夜。TLC监测反应完全后，冰浴条件下加入1mL水，加1mL 15％NaOH水溶液，再加3mL水，过滤，使用旋转蒸发仪减压蒸馏除去溶剂。加入200mL乙酸乙酯，等体积饱和碳酸氢钠溶液洗2次，等体积饱和氯化钠溶液洗1次，无水硫酸钠干燥30min，使用旋转蒸发仪减压蒸馏除去溶剂，柱分离纯化(硅胶柱，洗脱液为DCM)，得6.1g无色油状液体，收率92％。
化合物b的合成：：将化合物a(3.00g，11.26mmol)溶于适量50mL无水四氢呋喃中，加入三乙胺(1.12g，12.38mmol)，氮气保护下，冰浴搅拌10min，然后逐滴加入丙烯酰氯(1.37g，13.51mmol)，加毕，逐渐恢复至室温，搅拌过夜。TLC监测反应 完全后，使用旋转蒸发仪减压蒸馏除去溶剂。加入100mL乙酸乙酯，等体积饱和碳酸氢钠溶液洗3次，无水硫酸钠干燥30min，使用旋转蒸发仪减压蒸馏除去溶剂，柱分离纯化(硅胶柱，洗脱液为正己烷)，得3.1g无色油状液体，收率86％。
P-2的合成：将哌嗪(0.10g，1.16mmol)溶于10mL无水四氢呋喃中，然后化合物b(0.93g，2.90mmol)，氮气保护，70℃加热反应48h。TLC监测反应完全后，使用旋转蒸发仪减压蒸馏除去溶剂。柱分离纯化(硅胶柱，洗脱液为DCM：MeOH＝50：1(体积比))，得0.61g无色油状液体，收率72％。 1H NMR(400MHz,Chloroform-d)δ5.42–5.25(m,8H),4.05(t,J＝6.7Hz,4H),2.81–2.38(m,20H),2.03(q,J＝6.8Hz,8H),1.59(t,J＝7.1Hz,4H),1.37–1.22(m,32H),0.91–0.83(m,6H).MS m/z(ESI)：727.61[M+H] +。
实施例3 化合物P-4的合成
Figure PCTCN2022117373-appb-000060
化合物c的合成：将辛二醇(化合物a，4.00g，27.35mmol)，2-己基癸酸(3.51g，13.68mmol)，1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(2.88g，15.04mmol)，N,N'-二异丙基乙胺(2.12g，16.41mmol)，4-二甲氨基吡啶(0.33g，2.74mmol)溶于100mL二氯甲烷(DCM)中，室温搅拌反应12h。TLC监测反应完全后，使用旋转蒸发仪减压蒸馏除去溶剂。加入100mL乙酸乙酯，等体积饱和碳酸氢钠溶液洗2次，等体积饱和氯化钠溶液洗1次，无水硫酸钠干燥30min，使用旋转蒸发仪减压蒸馏除去溶剂，柱分离纯化(硅胶柱，洗脱液为PE：EA＝50：1(体积比))，得3.5g无色油状液体，收率67％。
化合物d的合成：将化合物c(2.00g，5.20mmol)溶于20mL DCM中，加入NaHCO 3(3.49g，41.60mmol)，搅拌5min后，加入化合物戴斯马丁氧化剂(3.49g，41.60mmol)室温搅拌3h。TLC监测反应完全后，使用旋转蒸发仪减压蒸馏除去溶剂。加入100mL石油醚，等体积饱和碳酸氢钠溶液洗2次，等体积饱和氯化钠溶液洗1次，无水硫酸钠 干燥30min，使用旋转蒸发仪减压蒸馏除去溶剂，柱分离纯化(硅胶柱，洗脱液为PE：EA＝200：1(体积比))，得1.5g无色油状液体，收率75％。
化合物P-4的合成：将化合物d(1.00g，2.61mmol)溶于10mL DCM中，加入哌嗪(0.10g，1.14mmol)，搅拌10min后，加三乙酰氧基硼氢化钠(0.58g，2.73mmol)，室温搅拌过夜。TLC监测反应完全后，使用旋转蒸发仪减压蒸馏除去溶剂。加入100mL乙酸乙酯，等体积饱和碳酸氢钠溶液洗2次，等体积饱和氯化钠溶液洗1次，无水硫酸钠干燥30min，使用旋转蒸发仪减压蒸馏除去溶剂，柱分离纯化(硅胶柱，洗脱液为DCM：MeOH＝50：1(体积比))，得0.6g无色油状液体，收率64％。 1H NMR(400MHz,Chloroform-d)δ4.04(t,J＝6.4Hz,4H),2.73–2.24(m,14H),1.71–1.08(m,72H),0.94–0.72(m,12H).MS m/z(ESI)：819.72[M+H] +。采用实施例3的方法，同样也可以制备得到化合物P-3在此不再一一赘述。
实施例4 化合物P-5的合成
Figure PCTCN2022117373-appb-000061
化合物c的合成：
将2-己基癸酸(化合物a,10.00g,39.00mmol)溶于150mL二氯甲烷(DCM)中，加入1-(3-二甲基氨基丙基)-3-乙基碳二亚胺(8.97g,46.8mmol)，4-二甲氨基吡啶(0.95g,7.8mmol)以及N,N-二异丙基乙胺(6.55g,50.7mmol)，9-癸烯-1-醇(化合物b,6.09g，39.0mmol)，室温搅拌过夜。TLC监测反应完全后，旋蒸仪减压蒸馏除去二氯甲烷。加入200mL乙酸乙酯，等体积饱和氯化钠溶液洗涤3次，有机相经无水硫酸钠干燥30min，旋蒸仪减压蒸馏除去乙酸乙酯，柱分离纯化(硅胶柱，洗脱液为PE：EA＝10：1(体积比))，得12.7g无色液体，收率83％。
化合物d的合成：
将化合物c(5.00g,12.67mmol)溶于70mL DCM中，冰浴条件下加入间氯过氧苯甲酸(3.34g,16.47mmol，质量分数85％)，搅拌15min后，撤去冰浴，搅拌过夜。TLC 监测反应完全后，加入过量的饱和亚硫酸氢钠溶液(10mL)以消耗未反应完的间氯过氧苯甲酸，旋蒸仪减压蒸馏除去二氯甲烷。加入200mL乙酸乙酯，200mL饱和碳酸氢钠溶液洗涤3次，200mL饱和氯化钠溶液洗涤1次，有机相经无水硫酸钠干燥30min，旋蒸仪减压蒸馏除去乙酸乙酯，柱分离纯化(硅胶柱，洗脱液为PE：EA＝2：1(体积比))，得6g无色液体，收率72％。
P-5的合成：
将哌嗪(0.09g,0.97mmol)溶于无水甲醇中，加入化合物d(1.00g,2.43mmol)，室温搅拌10min后，加热回流反应，反应12h。TLC监测反应完全后，旋蒸仪减压蒸馏除去溶剂。柱分离纯化(硅胶柱，洗脱液为DCM：MeOH＝200：1(体积比))，得0.65g无色液体，收率74％。2.19(m,14H),1.66–1.49(m,8H),1.47–1.15(m,70H),0.85(t,J＝6.7Hz,12H).MS m/z(ESI)：907.81[M+H] +。
采用实施例4的方法，同样也可以制备得到化合物P-6、P-7、P-8、P-9、P-12、P-14在此不再一一赘述。
实施例6 化合物P-13的合成
Figure PCTCN2022117373-appb-000062
化合物a的合成：参考实施例5中的实验方法合成。
化合物b的合成：
将哌嗪(1.52g,17.62mmol)溶于无水甲醇中，加入化合物a(2.00g,5.87mmol)，室温搅拌10min后，加热回流反应，反应12h。TLC监测反应完全后，旋蒸仪减压蒸馏除去溶剂。柱分离纯化(硅胶柱，洗脱液为DCM：MeOH＝50：1(体积比))，得1.51g无色液体，收率60％。
化合物P-13的合成：
将亚油酸(0.5g,1.78mmol)溶于5mL二氯甲烷(DCM)中，加入二环己基碳二亚胺(0.44g,2.14mmol)，4-二甲氨基吡啶(0.04g,0.36mmol)以及化合物b(0.76g，1.78mmol)，室温搅拌过夜。TLC监测反应完全后，旋蒸仪减压蒸馏除去二氯甲烷。加入100mL乙酸乙酯，等体积饱和氯化钠溶液洗涤3次，有机相经无水硫酸钠干燥30min，旋蒸仪减压蒸馏除去乙酸乙酯，柱分离纯化(硅胶柱，洗脱液为PE：EA＝10：1(体积比))，得0.81g无色液体，收率66％。MS m/z(ESI)：689.73[M+H] +。采用实施例4的方法，同样也可以制备得到化合物P-10在此不再一一赘述。
表1 氢谱数据
Figure PCTCN2022117373-appb-000063
实验一 mRNA-LNP的结构形貌表征实验：
将阳离子脂质化合物P-1，DSPC或DOPE，胆固醇，以及PEG-脂质体以50:40:25:2或者50:40:38.5:1.5的比例溶于乙醇中，并充分混匀。脂质体纳米粒(LNP)与mRNA的质量比为10:1到30:1。使用柠檬酸盐或醋酸钠缓冲液(pH＝3或5)将mRNA稀释至0.2mg/mL。将阳离子脂质体乙醇溶液与mRNA溶液以体积比为1:5到1:3的比例充分混匀。所获纳米粒通过超滤和透析的手段纯化；过滤除菌。将得到的纳米粒样品在铜网上滴上10μ制备好的样品15L，放置10min后吸干样品并晾干。醋酸双氧铀染色5min， 滤纸吸干染液后干燥过夜，利用透射电子显微镜(TEM)观察其形貌。
实验结果如图8所示，本发明的脂质纳米粒都能形成稳定的纳米结构，尺寸分布较窄，尺寸随不同的脂质纳米粒的结构有所变化，在30-150nm范围内。
实验二：转染效率实验：
雄性ICR小鼠(6-8week，上海杰思捷实验动物有限公司)饲养在22±2℃以及相对湿度为45–75％的实验条件下，光照/黑暗周期为12h。使用编码荧光素酶的mRNA(luciferase mRNA)作为报道基因。荧光素酶催化荧光素产生生物荧光，通过检测单位时间内生物荧光强度，反映LNP的转染效率。以荧光素酶mRNA(购自ApexBio Technology)为例，将上述获得的mRNA-LNP，以150μg/kg mRNA的剂量，通过肌肉注射给药。取特定的时间点，于小鼠腹腔注射荧光素，5分钟后，将小鼠置于小动物活体成像仪测定荧光强度，最后的结果以平均荧光强度表示。
表1 小鼠腹腔注射给药后荧光强度结果
Figure PCTCN2022117373-appb-000064
Figure PCTCN2022117373-appb-000065
*(Molar ratio:Lipid/DOPE/Cholesterol/PEG)
由表1可知：结果显示，相较于现有的商业可得的脂质体MC-3，本发明的脂质体具有更高的体内mRNA转染效率；
由P0和P2对比可知：相同结构的化合物，未引入双键的要比引入双键的化合物转染效率低很多，说明在尾部引入双键与本发明的化合物结构在提高转染效果上具有协同作用；双键的引入配合哌嗪及类哌嗪化合物的中心结构可以使得脂质分子呈锥形结构，锥形结构可以使内涵体膜发生反六角相反转，破坏内涵体膜增强内涵体逃逸能力，从而增加转染效果。
实验三：生物相容性实验：
使用CCK-8(cell counting kit-8)试剂盒测定细胞活力。将处于指数增长期的Hep3B细胞(100μL，细胞密度为2×10 4个/ml)悬浮液加入96孔板中，于细胞培养箱中孵育24h，然后从每个孔中除去细胞培养液，并添加100μL含mRNA 20μg/mL的LNP的新制细胞培养液，和细胞共孵育4h。随后，除去细胞上清液，加入新鲜细胞培养液，继续孵育20h。然后，除去上清液，加入含CCK-8工作溶液(10μL/mL)的新鲜的细胞培养液100μL，孵育2h，空白孔的设置：加含CCK-8工作溶液的细胞培养液。使用多功能微孔板检测仪检测每孔于450nm处的吸光度(检测过程中孔板中不能出现气泡)，将未经LNP处理的细胞作为对照组，将其细胞活力设为100％。
细胞活力(％)＝[A1-A0]/[A2-A0]×100；
A1为加药组吸光度，A0为空白组吸光度，A2为对照组吸光度。实验结果如表3所示。
表3
mRNA-LNP样品 细胞活力(％) mRNA-LNP样品 细胞活力(％)
样品P-1 96 样品P-9 93
样品P-2 95 样品P-10 96
样品P-3 97 样品P-11 95
样品P-4 96 样品P-12 97
样品P-5 94 样品P-13 95
样品P-6 96 样品P-14 96
样品P-7 97 MC-3 95
样品P-8 95    
实验结果表明在限定的LNP浓度内，大多数细胞活力大于95％，未见明显细胞毒性。
实验四：动物免疫实验：
材料准备：六周龄雌性Balb/c小鼠，体重15～20g，15只，饲养于温度22±2℃，相对湿度45-75％的实验环境中，光照/黑暗周期为12h。小鼠购入后先于动物房内适应一周后方可进行正式的动物试验。将15只小鼠随机分成3组，第一组后腿肌肉注射等体积PBS(阴性对照组)，第二组后腿肌肉注射市售对比样品(阳性对照组MC3)、mRNA、 PBS的混合物，第三组为后腿肌肉注射样品P-1(试验组)、10μg的mRNA、PBS的混合物；以上mRNA为基于自主设计的模板通过体外转录合成的可表达Spike全长的mRNA。
实验过程为：在第0和14天，按照以上五个分组将包载mRNA的LNP混合物肌肉注射到Balb/c小鼠体内。在第13和21天进行眼部取血，将血样放在37℃孵育1小时后，3500rpm离心15分钟，取上清进行分析。通过自制ELISA试剂盒检测一免和二免小鼠血清对Delta变异株S1蛋白特异性抗体滴度。
检测一免和二免小鼠血清对Delta变异株S1蛋白特异性抗体滴度的具体操作过程如下：将Spike S1重组蛋白加入96孔板，每孔加0.25μg，4℃放置过夜。第二天，弃去孔内液体，使用5％BSA的PBST溶液(200ul)在37℃条件下封闭1h。之后弃去孔内液体，用PBST洗涤液200ul洗涤3遍，每遍3min，甩板晾干。将小鼠血清用PBS稀释(稀释比列为1：20000)，或者标准品用PBS稀释为一系列浓度(母液为1ug/ul，对半稀释，共14个标曲)。将稀释后的样品和标准品100μL加入空中，37℃，孵育2h。弃去孔内液体，用PBST洗涤液200ul洗涤3遍，每遍3min，甩板晾干。加入Goat anti-mouse IgG HRP(PBS 1:5000稀释)，每孔100ul，37℃，1h。弃去孔内液体，用PBST洗涤液200ul洗涤3遍,每遍3min，甩板晾干。将TMB底物A液和B液等比例混合，每孔100μL，置37℃避光放置数分钟(3-5mins)。在650nm测吸光度，最高吸光度值在1.5附近时，即可加100ul终止液。加入终止液后15min内检测450nm处的吸光度值。根据标准曲线公式计算各组IgG含量。
实验结果如图9所示，结果显示：阳性对照组MC3和试验组两组mRNA均可产生针对S1蛋白特异性抗体，试验组的抗体滴度显著高于阳性对照组，试验组可高效递送mRNA进入细胞，表达抗原，进而激起体内免疫反应，产生相应抗体，发挥保护功能。
结果显示，相较于现有的商业可得的脂质体MC-3，本发明的脂质体均取得了更好的生物相容性以及更高的体内mRNA转染效率。本发明的脂质体都能形成稳定的纳米结构，尺寸分布较窄，尺寸随不同的脂质体的结构有所变化，在30-150nm范围内。结构上，在脂质体的疏水尾部引入可降解的酯键，可以改变脂质体在体内的代谢行为，同时增加其疏水尾部的亲脂性，进而提高mRNA-LNP的生物安全性和转染效率，另外在其亲水头部引入哌嗪及类哌嗪结构，使其与核酸的相互作用加强，进而负载更多的核酸，同时在疏水尾部引入双键，配合哌嗪及类哌嗪的中心结构可以使得脂质分子呈锥形结构，锥形结构可以使内涵体膜发生反六角相反转，破坏内涵体膜增强内涵体逃 逸能力，从而增加转染效果，从而进一步增加其转染效率。合成上，相比较MC-3，其合成路线简单易行，原料廉价易得，有利于其工业化生产。新脂质体具有良好的应用前景。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
Figure PCTCN2022117373-appb-000066
Figure PCTCN2022117373-appb-000067
Figure PCTCN2022117373-appb-000068
Figure PCTCN2022117373-appb-000069
Figure PCTCN2022117373-appb-000070
Figure PCTCN2022117373-appb-000071
Figure PCTCN2022117373-appb-000072
Figure PCTCN2022117373-appb-000073

Claims (10)

  1. 根据权利要求1-6任一项所述的一类阳离子脂质化合物的应用，其特征在于，应用于包含阳离子脂质化合物的组合物、其立体异构体、其互变异构体或其在药学上可接受的盐。。
  2. 根据权利要求8所述的一类阳离子脂质化合物的应用，其特征在于，所述包含阳离子脂质化合物的组合物包括：载体、所载的药物试剂、药物辅助剂中的一种或几种的组合。。
  3. 根据权利要求9所述的一类阳离子脂质化合物的应用，其特征在于，所述载体包括：一种或多种阳离子脂质化合物、助脂质、结构脂质、聚合物缀合脂质或两亲性嵌段共聚物中的一种或几种的组合。
  4. 根据权利要求10所述的一类阳离子脂质化合物的应用其特征在于，所述阳离子脂质化合物与助脂质的摩尔比为0.5:1-10:1。
  5. 根据权利要求10所述的一类阳离子脂质化合物的应用，其特征在于，所述阳离子脂质化合物与结构脂质的摩尔比为0.5:1-5:1。
  6. 根据权利要求10所述的一类阳离子脂质化合物的应用，其特征在于，所述阳离子脂质化合物与聚合物缀合脂质的摩尔比为10：1-250：1。
  7. 根据权利要求10所述的一类阳离子脂质化合物的应用，其特征在于，所述阳离子脂质化合物与两亲性嵌段共聚物的摩尔比为0.5：1-80：1。
  8. 根据权利要求9所述的一类阳离子脂质化合物的应用，其特征在于，所述载体为脂质纳米粒LNP，所述脂质纳米粒的平均尺寸为30-200nm，所述脂质纳米粒的制剂的多分散指数≤0.5。
  9. 根据权利要求9所述的一类阳离子脂质化合物的应用，其特征在于，所述所载的药物试剂包括：核酸分子、小分子化合物、多肽或蛋白质中的一种或多种。
  10. 根据权利要求9所述的一类阳离子脂质化合物的应用，其特征在于，所述药物辅助剂包括：稀释剂、稳定剂、防腐剂或冻干保护剂中的一种或多种。
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