CN115010681A - Cationic liposome molecule and preparation and application thereof - Google Patents

Cationic liposome molecule and preparation and application thereof Download PDF

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CN115010681A
CN115010681A CN202111045980.3A CN202111045980A CN115010681A CN 115010681 A CN115010681 A CN 115010681A CN 202111045980 A CN202111045980 A CN 202111045980A CN 115010681 A CN115010681 A CN 115010681A
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branched
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章雪晴
汪开振
滕以龙
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Anhui Rongcan Biotechnology Co ltd
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Anhui Rongcan Biotechnology Co ltd
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Priority to CN202111045980.3A priority Critical patent/CN115010681A/en
Priority to PCT/CN2022/117373 priority patent/WO2023036148A1/en
Priority to CN202280005633.4A priority patent/CN115956072A/en
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Abstract

The invention provides a cationic liposome molecule, preparation and application thereof, and particularly provides a cationic liposome molecule shown in a formula I, a preparation method thereof, and application of the cationic liposome molecule in nano-drug delivery.

Description

Cationic liposome molecule and preparation and application thereof
Technical Field
The invention relates to the field of nano-drug carriers, and particularly provides a cationic liposome molecule, a preparation method thereof and application of the cationic liposome molecule in nano-drug delivery.
Background
Therapeutic nucleic acids, such as messenger rna (mrna), antisense oligonucleotides, ribozymes, dnases, plasmids, immunostimulatory nucleic acids, antagomirs, antimirirs, mimetics (mimics), supermir, and aptamers, and the like, can be used to effect expression of specific cellular products, and thus, for treatment of, for example, diseases or conditions associated with a deficiency in a protein or enzyme. The therapeutic applications of nucleic acid drugs are extremely widespread, since constructs can be synthesized to produce any selected protein sequence, and the expression product of the nucleic acid can increase the level of protein present, replace missing or non-functional protein forms, or introduce new proteins and related functions in the cell or organism.
Nucleic acid-based therapies have great potential, but this method of treatment generally requires more efficient delivery of the nucleic acid to the appropriate site within the cell or organism to achieve this therapeutic effect. There are also challenges associated with delivering nucleic acids to affect the desired response in a biological system. The use of oligonucleotides in a therapeutic setting currently faces two problems: first, free RNA is susceptible to nuclease digestion in plasma. Second, the ability of free RNA to enter intracellular compartments where relevant translation mechanisms exist is limited.
Lipid nanoparticles formed from cationic lipids with other lipid components (e.g., 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 remains a need in the art to develop other cationic lipids and lipid nanoparticles that can be used to deliver oligonucleotides to provide better delivery effects, e.g., optimized drugs: lipid ratio, protection of nucleic acids from degradation and clearance in serum, good tolerability, providing a sufficient therapeutic index, etc.
In view of the above, there is still a need in the art to develop cationic liposome molecules with completely new structures.
Disclosure of Invention
The invention aims to provide a cationic liposome molecule with a novel structure.
In a first aspect of the present invention, there is provided a liposomal compound represented by the following formula I:
Figure BDA0003251199080000021
wherein the content of the first and second substances,
side chain moiety
Figure BDA0003251199080000022
Each independently has 1 to 40 carbons in the main chain (i.e., the longest chain);
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 Each independently selected from the group consisting of: substituted or unsubstituted straight or branched C 1-10 Alkyl, or R 11 And R 22 Together forming a substituted or unsubstituted straight or branched chain C 1-4 An alkylene group;
R 2 selected from the group consisting of: substituted or unsubstituted straight or branched C 1-4 An alkylene group;
R 3 and R 4 Each independently selected from the group consisting of: chemical bonds, or substituted or unsubstituted, straight or branched C 2-10 An alkylene group;
each R is 5 And R 6 Each independently selected from the group consisting of: chemical bond, or substituted or unsubstituted straight or branched C 1-10 Alkylene, substituted or unsubstituted straight or branched C 2-10 Alkenylene (i.e. when a1 or a2 is 2 or 3, each R 5 And R 6 May be the same or different);
each M 1 And M 2 Each independently is a group selected from:
Figure BDA0003251199080000023
R 7 and R 8 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 An alkenylene group;
R 9 and R 10 Each independently selected from the group consisting of: substituted or unsubstituted straight or branched C 1-20 Alkylene, substituted or unsubstituted straight or branched C 2-20 An alkenylene group;
wherein at least one of said side chain moieties is present but not
Figure BDA0003251199080000031
M1 or M2, and at the same time, the corresponding
Figure BDA0003251199080000032
From 10 to 40 carbons in the backbone, preferably from 12 to 30 carbons (e.g., 7, 8, 9, 10, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 carbons);
unless otherwise specified, the substitution refers to the replacement of one or more hydrogen atoms on the group by a substituent selected from the group consisting of: straight or branched C 1-10 Alkyl (e.g. straight or branched C) 1-4 Alkyl), straight or branched C 2-4 Alkenyl, OH, NH 2 Hydroxy-substituted straight or branched C 1-4 Alkyl, hydroxy substituted straight or branched C 2-4 An alkenyl group.
In another preferred embodiment, when a1 and a2 are each independently 1,2 or 3,
Figure BDA0003251199080000033
at least one of which is a segment having 2 to 10 carbons in the main chain (i.e., the longest chain); and when R is 3 、R 4 、R 5 Or R 6 When substituted, said substitution means that one or more hydrogen atoms on the group are substituted with a substituent selected from the group consisting of: straight or branched C 1-4 Alkyl, straight or branched C 2-4 Alkenyl, OH, NH 2 Hydroxy-substituted straight or branched C 1-4 Alkyl, hydroxy substituted straight or branched C 2-4 An alkenyl group.
In another preferred embodiment, the compound of formula I has the structure shown in formula II below:
Figure BDA0003251199080000034
x and Y are each independently selected from N or CH, and at least one X or Y is N;
R 1 selected from the group consisting of:
Figure BDA0003251199080000035
wherein a, ggg, hhh, b, iii, and kk are each independently 0, 1,2, or 3;
R 2 selected from the group consisting of:
Figure BDA0003251199080000036
R 3 selected from the group consisting of:
Figure BDA0003251199080000037
Figure BDA0003251199080000038
wherein ww, xx, c, d, ff are each independently 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10;
R 4 selected from the group consisting of:
Figure BDA0003251199080000041
Figure BDA0003251199080000042
wherein yy, zz, e, f, gg are each independently 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10;
in the above formulas, CC, DD, A and B are substituents selected from the group consisting of: H. CH (CH) 3 、OH、CH 2 OH、C 2 H 4 OH、NH 2 ；
R 5 Selected from the group consisting of:
Figure BDA0003251199080000043
wherein g, h, dd, jj, and kk are each independently 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10;
R 6 selected from the group consisting of:
Figure BDA0003251199080000044
wherein i, j, ee, mm, and nn are each independently 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10;
R 7 selected from the group consisting of:
Figure BDA0003251199080000045
wherein k, l and m are each independently 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10;
R 8 selected from the group consisting of:
Figure BDA0003251199080000046
wherein n, o and q are each independently 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10;
in each of the formulae above, C, D, E, F, G, J are each independently selected from the group consisting of: H. CH (CH) 3 ；
R 9 Selected from the group consisting of:
Figure BDA0003251199080000047
Figure BDA0003251199080000048
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 selected from the group consisting of:
Figure BDA0003251199080000049
Figure BDA0003251199080000051
whereinW, 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;
in each of the above formulae, K, L, M and N are each independently selected from the group consisting of: H. CH (CH) 3 (ii) a AA and BB are each independently selected from the group consisting of: H. CH (CH) 3 OH or NH 2 ；
Provided that the side chain moiety
Figure BDA0003251199080000052
Each independently having 2 to 40 carbons in the main chain (i.e., the longest chain) and 10 to 40 carbons in at least one side chain moiety.
In another preferred embodiment, a is 0, 1 or 2.
In another preferred embodiment, b is 0, 1 or 2.
In another preferred embodiment ggg is 0, 1 or 2.
In another preferred embodiment, hhh is 0, 1 or 2.
In another preferred embodiment, iii is 0, 1 or 2.
In another preferred embodiment, kkk is 0, 1 or 2.
In another preferred embodiment ww is 0, 1,2, 3, 4, 5, 6, 7 or 8.
In another preferred embodiment xx is 0, 1,2, 3, 4, 5, 6, 7 or 8.
In another preferred embodiment, c is 0, 1,2, 3, 4, 5, 6, 7, 8 or 9.
In another preferred embodiment, d is 0, 1,2, 3, 4, 5, 6, 7, 8 or 9.
In another preferred embodiment, yy is 0, 1,2, 3, 4, 5, 6, 7 or 8.
In another preferred embodiment, zz is 0, 1,2, 3, 4, 5, 6, 7, or 8.
In another preferred embodiment, e is 0, 1,2, 3, 4, 5, 6, 7, 8 or 9.
In another preferred embodiment, f is 0, 1,2, 3, 4, 5, 6, 7, 8 or 9.
In another preferred embodiment, g is 0, 1,2, 3, 4, 5, 6, 7, 8 or 9.
In another preferred embodiment, 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.
In another preferred embodiment, 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.
In another preferred embodiment, 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.
In another preferred embodiment 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.
In another preferred embodiment, 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.
In another preferred embodiment, 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.
In another preferred embodiment 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.
In another preferred embodiment, 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.
In another preferred embodiment, 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.
In another preferred embodiment, m is 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10.
In another preferred embodiment, 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.
In another preferred embodiment, 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.
In another preferred embodiment, p is 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10.
In another preferred embodiment, 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.
In another preferred embodiment, 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.
In another preferred embodiment 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.
In another preferred embodiment, 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.
In another preferred embodiment 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.
In another preferred embodiment 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.
In another preferred embodiment, u is 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10.
In another preferred embodiment, v is 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10.
In another preferred embodiment, hh is 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10.
In another preferred embodiment, aaa is 0, 1,2, 3, 4, 5, 6, 7 or 8.
In another preferred embodiment bbb is 0, 1,2, 3, 4, 5, 6, 7 or 8.
In another preferred example ccc is 0, 1,2, 3, 4, 5, 6, 7 or 8.
In another preferred embodiment, ss is 0, 1,2, 3, 4, 5, 6, 7 or 8.
In another preferred embodiment, tt is 0, 1,2, 3, 4, 5, 6, 7 or 8.
In another preferred embodiment, uu is 0, 1,2, 3, 4, 5, 6, 7 or 8.
In another preferred embodiment, vv is 0, 1,2, 3, 4, 5, 6, 7 or 8.
In another preferred embodiment, aa is 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10.
In another preferred embodiment, ii is 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10.
In another preferred embodiment, ddd is 0, 1,2, 3, 4, 5, 6, 7 or 8.
In another preferred embodiment eee is 0, 1,2, 3, 4, 5, 6, 7 or 8.
In another preferred embodiment, fff is 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10.
In another preferred embodiment, the compound of formula I has the structure shown in formula III below:
Figure BDA0003251199080000071
wherein, the first and the second end of the pipe are connected with each other,
x and Y are each independently selected from N or CH, and at least one of X or Y is N;
R 11 selected from the group consisting of:
Figure BDA0003251199080000072
wherein b1 and b2 are each independently 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10;
R 22 selected from the group consisting of:
Figure BDA0003251199080000073
R 2 selected from the group consisting of:
Figure BDA0003251199080000074
R 3 selected from the group consisting of:
Figure BDA0003251199080000075
Figure BDA0003251199080000076
wherein ww, xx, c, d, ff are each independently 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10;
R 4 selected from the group consisting of:
Figure BDA0003251199080000081
Figure BDA0003251199080000082
wherein yy, zz, e, f, gg are each independently 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10;
in the formulas, CC, DD, A and B are substituents selected from the following groups: H. CH (CH) 3 、OH、CH 2 OH、C 2 H 4 OH、NH 2 ；
R 5 Selected from the group consisting of:
Figure BDA0003251199080000083
wherein g, h, dd, jj, and kk are each independently 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10;
R 6 selected from the group consisting of:
Figure BDA0003251199080000084
wherein i, j, ee, mm and nn are each independently 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10;
R 7 selected from the group consisting of:
Figure BDA0003251199080000085
wherein k, l and m are each independently 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10;
R 8 selected from the group consisting of:
Figure BDA0003251199080000086
wherein n, o and q are each independently 0,
1.2, 3, 4, 5, 6, 7, 8, 9 or 10;
in each of the formulae above, C, D, E, F, G, J are each independently selected from the group consisting of: H. CH (CH) 3 ；
R 9 Selected from the group consisting of:
Figure BDA0003251199080000087
Figure BDA0003251199080000088
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 selected from the group consisting of:
Figure BDA0003251199080000089
Figure BDA0003251199080000091
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;
in each of the above formulae, K, L, M and N are each independently selected from the group consisting of: H. CH (CH) 3 (ii) a AA and BB are each independently selected from the group consisting of: H. CH (CH) 3 OH or NH 2 ；
Provided that the side chain moiety
Figure BDA0003251199080000092
Each independently having 2 to 40 carbons in the main chain (i.e., the longest chain) and 10 to 25 carbons in at least one side chain moiety.
In another preferred embodiment, the compound of formula I has the structure shown in formula IV below:
Figure BDA0003251199080000093
wherein each group is as defined in any one of claims 1 to 3.
In another preferred embodiment, the side chain moiety
Figure BDA0003251199080000094
Figure BDA0003251199080000095
Each independently is a structure selected from the group consisting of:
Figure BDA0003251199080000096
Figure BDA0003251199080000101
in another preferred embodiment, the compound is selected from the group consisting of:
Figure BDA0003251199080000102
Figure BDA0003251199080000111
in a second aspect of the invention, there is provided a use of the liposomal compound as described in the first aspect of the invention for the preparation of a drug-loaded nanocomposition.
In a third aspect of the invention, there is provided a drug-loaded nanocomposition comprising a liposomal compound according to the first aspect of the invention and a therapeutic agent.
In another preferred embodiment, the therapeutic agent comprises a nucleic acid.
In another preferred embodiment, the nucleic acid is selected from the group consisting of antisense RNA and messenger RNA.
In another preferred embodiment, the composition further comprises an excipient selected from the group consisting of: neutral lipids, steroids, and polymer-conjugated lipids.
In another preferred embodiment, the neutral lipid is selected from the group consisting of: DSPC, DPPC, DMPC, DOPC, POPC, DOPE, SM, or combinations thereof; preferably, the neutral lipids are DOPE and DSPC.
In another preferred embodiment, the molar ratio of said compound to said neutral lipid in said composition is from 2:1 to 8: 1.
in another preferred embodiment, in the composition, the steroid is cholesterol.
In another preferred embodiment, the molar ratio of the compound to cholesterol in the composition is 1:1 to 2: 1.
In another preferred embodiment, in the composition, the polymer-conjugated lipid is a pegylated lipid.
In another preferred embodiment, the molar ratio of the compound to the pegylated lipid in the composition is from 100:1 to 20: 1.
In another preferred embodiment, in the composition, the pegylated lipid is selected from the group consisting of: PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer or PEG dialkoxypropylcarbamate.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Detailed Description
The inventor of the invention has long and intensive research and prepares a liposome molecule with a novel structure, and the liposome molecule and therapeutic nucleic acid can form liposome nanoparticles for encapsulating RNA together, so that the therapeutic nucleic acid is delivered to a part needing to form the drug concentration. Based on the above findings, the inventors have completed the present invention.
Therapeutic nucleic acids
As used herein, the term "therapeutic nucleic acid" refers to a nucleic acid that can be administered to a subject in need thereof in the form of an oligonucleotide or a vector expressing the corresponding oligonucleotide and produces a therapeutic effect. The therapeutic nucleic acid may be prepared according to any available technique, for example for mRNA, the main preparation method is, but not limited to, enzymatic synthesis (also known as in vitro transcription), which currently represents the most efficient method for producing long sequence-specific mRNA. In vitro transcription describes methods for template-directed synthesis of RNA molecules from engineered DNA templates comprising upstream phage promoter sequences linked to downstream sequences encoding the gene of interest (e.g., including but not limited to, from T7, T3, and SP6 escherichia coli phages). Template DNA may be prepared for in vitro transcription from a number of sources using suitable techniques well known in the art, including, but not limited to, plasmid DNA and polymerase chain reaction amplification.
Transcription of RNA occurs in vitro using a linearized DNA template in the presence of a corresponding RNA polymerase and adenosine, guanosine, uridine and cytidine ribonucleoside triphosphates (rntps) under conditions that support polymerase activity while minimizing potential degradation of the resulting mRNA transcript. In vitro transcription can be performed using various commercially available kits including, but not limited to, the RiboMax Large Scale RNA production System (Promega), the MegaScript transcription kit (Life technologies), and commercially available reagents including RNA polymerase and rNTP. methods for in vitro transcription of mRNA are well known in the art.
The desired in vitro transcribed mRNA is then purified from the undesired components of the transcription or related reaction (including unincorporated rNTP, proteinases, salts, short RNA oligonucleotides, etc.). 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 can be used include size exclusion chromatography, silica-based affinity chromatography, and polyacrylamide gel electrophoresis. Purification can be performed using various commercially available kits including, but not limited to, the SV total separation system (Promega) and the in vitro transcription cleaning and concentration kit (Norgen Biotek).
In addition, although reverse transcription can produce large quantities of mRNA, the product can contain large quantities of abnormal RNA impurities associated with undesirable polymerase activity that may need to be removed from the full-length mRNA preparation. These impurities include short RNAs resulting from failed transcription initiation and double-stranded RNAs (dsrnas) generated by RNA-dependent RNA polymerase activity, RNA-initiated transcription from RNA templates, and self-complementary 3' elongation. It has been demonstrated that these contaminants with dsRNA structures can lead to undesired immunostimulatory activity by interacting with various innate immune sensors in eukaryotic cells, which function to recognize specific nucleic acid structures and induce potent immune responses. Due to the reduced protein synthesis during the innate cellular immune response, mRNA translation may in turn be significantly reduced. Thus, additional techniques for removing these dsRNA contaminants have been developed and are known in the art, including but not limited to, tunable ratio HPLC purification. HPLC purified mRNA has been reported to be translated at much higher levels, particularly in primary cells and in vivo.
A wide variety of modifications have been described in the art for altering specific properties of mRNA transcribed in vitro and improving its utility. These modifications include, but are not limited to, modifications to the 5 'end and 3' end of the mRNA. Endogenous eukaryotic mrnas typically contain a cap structure on the 5' -end of the mature molecule that plays an important role in mediating binding of the mRNA Cap Binding Protein (CBP), which in turn is responsible for enhancing mRNA stability and mRNA translation efficiency in the cell. Thus, the highest level of protein expression was achieved with the capped mRNA transcripts. The 5 '-cap contains a 5' -5 '-triphosphate linkage between the 5' -majority nucleotide and the guanine nucleotide. The conjugated guanine nucleotide is methylated at the N7 position. Additional modifications include methylation of the last and penultimate majority of 5 '-nucleotides at the 2' -hydroxyl.
A number of different cap structures can be used to generate the 5' -cap of the mRNA synthesized by in vitro transcription. 5' -capping of synthetic mRNA can be performed with chemical cap analogs in conjunction with transcription (i.e., capping during in vitro transcription). For example, the anti-inversion cap analogue (ARCA) cap contains a guanine-guanine linkage of 5 ' -5 ' -triphosphate, where one guanine contains N7 methyl and 3 ' -O-methyl. However, up to 20% of transcripts remain uncapped during this co-transcription process, and the synthetic cap analogs are not identical to the 5' -cap structure of real cellular mRNA, potentially reducing translatability and cellular stability. Alternatively, the synthetic mRNA molecule may also be enzymatically capped post-transcriptionally. This can result in a more authentic 5 '-cap structure that more closely mimics, structurally or functionally, an endogenous 5' -cap with enhanced binding of cap binding proteins, increased half-life, reduced sensitivity to 5 'endonucleases, and/or reduced 5' decapping. Many synthetic 5' -cap analogs have been developed and are known in the art to enhance mRNA stability and translatability.
At the 3' -end, a long chain of adenine nucleotides (poly a tail) is typically added to the mRNA molecule during RNA processing. Immediately after transcription, the 3 ' end of the transcript is cleaved to release the 3 ' hydroxyl group, and to the 3 ' end, polya polymerase adds an adenine nucleotide strand to the RNA in a process known as polyadenylation. Polya tails have been widely shown to enhance translation efficiency and mRNA stability.
Poly a tailing of in vitro transcribed mRNA can be achieved using various methods including, but not limited to, cloning of poly (T) fragments into a DNA template or post-transcriptional addition by using poly (a) polymerase. The first case allows in vitro transcription of mRNA 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 the RNA, without the need for additional manipulation of the DNA template, to give mrnas with poly (a) tails of different lengths. 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), as well as commercially available reagents, various ARCA caps, poly (A) polymerase, and the like.
In addition to 5 'cap and 3' polyadenylation, other modifications to in vitro transcripts have been reported to provide benefits related to translation efficiency and stability. It is well known in the art that pathogenic DNA and RNA can be recognized by various sensors within eukaryotic cells and elicit potent innate immune responses. Because most nucleic acids of natural origin contain modified nucleosides, the ability to discriminate between pathogenic DNA and RNA and self-DNA and RNA has been shown to be based, at least in part, on structural and nucleoside modifications. In contrast, RNA synthesized in vitro lacks these modifications, thus rendering it immunostimulatory and in turn can inhibit efficient mRNA translation as outlined above. The introduction of modified nucleosides into in vitro transcribed mRNA can be used to prevent recognition and activation of the RNA sensor, thereby alleviating this undesirable immunostimulatory activity and enhancing translational capacity. Modified nucleosides and nucleotides used to synthesize the 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 can be incorporated to some extent into in vitro transcribed mRNA, alone or in combination with other modified nucleosides. In vitro synthesis of nucleoside-modified mrnas has been reported to reduce the ability to activate immunosensors, with concomitant enhancement of translation.
Other components of an mRNA that can be modified to provide benefits in terms of translatability and stability include the 5 'and 3' untranslated regions (UTRs). Optimization of UTRs for both or alone (advantageously 5 'and 3' UTRs may be derived from cellular or viral RNA) has been shown to enhance mRNA stability and translation efficiency of in vitro transcribed mRNA.
In addition to mRNA, other nucleic acid payloads (payload) may also be used in the present invention. For oligonucleotides, 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 are well known in the art.
For plasmid DNA, preparations for use in conjunction with the present invention generally employ, but are not limited to, in vitro amplification and isolation of plasmid DNA in a liquid culture medium of a bacterium containing the plasmid of interest. The presence of genes within the plasmid of interest that encode 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 a variety of commercially available kits, as well as commercially available reagents, including, but not limited to, Plasmid Plus (Qiagen), GenJET Plasmid maxiPrep (Thermo), and Pure Yield maxiPrep (Promega) kits.
Lipid nanoparticles
As used herein, the term "lipid nanoparticle" refers to a composite drug-loaded particle formed by self-assembly using cationic liposomes with a nucleic acid drug to be delivered. The 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 inhibiting expression of a target gene. Accordingly, embodiments of the present invention provide methods of treating or preventing diseases and disorders in a subject in need thereof by contacting the subject with a lipid nanoparticle encapsulating or associated with a suitable therapeutic agent, wherein the lipid nanoparticle comprises one or more of the novel cationic lipids described herein. Various exemplary embodiments of the cationic lipids, lipid nanoparticles, and compositions comprising lipid nanoparticles of the present invention, and their use to deliver active agents (e.g., therapeutic agents) such as nucleic acids to modulate gene and protein expression are described in further detail below.
The phrase "inducing expression of a desired protein" refers to the ability of a nucleic acid to increase expression of a desired protein. To test the extent of protein expression, a test sample (e.g., a sample of cells in culture medium that expresses a desired protein) or a test mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g., mouse) or non-human primate (e.g., monkey) model) is contacted with a nucleic acid (e.g., a nucleic acid that binds to a lipid of the invention). The expression of the desired protein in the test sample or test animal is compared to the expression of the desired protein in a control sample (e.g., a sample of cells in a medium that expresses the desired protein) or a control mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g., mouse) or non-human primate (e.g., monkey) model) that has not been contacted or administered the nucleic acid. When the desired protein is present in the control sample or control mammal, the expression of the desired protein in the control sample or control mammal can be assigned a value of 1.0. In particular embodiments, inducing expression of the desired protein is achieved when 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, e.g., about 1.1, 1.5, 2.0, 5.0, or 10.0. Inducing expression of the desired protein is achieved when the desired protein is not present in the control sample or control mammal, when any measurable level of the desired protein is detected in the test sample or test mammal. One of ordinary skill in the art will appreciate that suitable assays for determining the level of protein expression in a sample are, for example, dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzymatic functional and phenotypic assays, or assays based on reporter proteins that produce fluorescence or luminescence under appropriate conditions.
The phrase "inhibiting expression of a target gene" refers to the ability of a nucleic acid to silence, reduce, or inhibit expression of a target gene. To test the extent of gene silencing, a test sample (e.g., a sample of cells in culture expressing the target gene) or a test mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g., mouse) or non-human primate (e.g., monkey) model) is contacted with a nucleic acid that silences, reduces, or inhibits expression of the target gene. The expression of the target gene in the test sample or test animal is compared to the expression of the target gene in a control sample (e.g., a sample of cells in culture expressing the target gene) or a control mammal (e.g., a mammal such as a human or an animal model such as a rodent (e.g., mouse) or non-human primate (e.g., monkey) model) that has not been contacted or administered the nucleic acid. The expression of the target gene in the control sample or control mammal may be assigned a value of 100%. In particular embodiments, silencing, inhibiting, or reducing expression of the target gene is achieved when the level of expression of the target gene in the test sample or the test mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% relative to the level of expression of the target gene in the control sample or the control mammal. In other words, the nucleic acid is capable of silencing, reducing, or inhibiting at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the expression of the target gene in the test sample or test mammal relative to the level of expression of the target gene in a control sample or control mammal that has not been contacted or administered the nucleic acid. Suitable assays for determining the expression level of a target gene include, but are not limited to, assays for protein or mRNA levels using techniques known to those skilled in the art, e.g., dot blot, northern blot, in situ hybridization, ELISA, immunoprecipitation, enzymatic function, and phenotypic assays known to those skilled in the art.
The term "lipid" refers to a group of organic compounds, which include, but are not limited to, esters of fatty acids, and are generally characterized as having poor solubility in water, but being soluble in a wide variety of organic solvents. They are generally divided into at least three categories: (1) "simple lipids" which include fats and oils and waxes; (2) "compound lipids" which include phospholipids and glycolipids; and (3) "derivatized lipids", such as steroids.
"cationic lipid" refers to a lipid capable of being positively charged. Exemplary cationic lipids include one or more amine groups bearing a positive charge. Preferred cationic lipids are ionizable, so that they can be present in a positively charged or neutral form depending on the pH. Ionization of cationic lipids affects the surface charge of lipid nanoparticles under different pH conditions. This charge state can affect plasma protein absorption, blood clearance and tissue distribution, as well as the ability to form endosomolytic (endosomolytic) non-bilayer structures, and is critical for intracellular delivery of nucleic acids.
The term "polymer-conjugated lipid" refers to a molecule comprising a lipid moiety and a polymer moiety. An example of a polymer conjugated lipid is a pegylated lipid. The term "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-dimyristoyl glycerol (PEG-DMG) and the like.
The term "neutral lipid" refers to any of a number of lipid species that exist in the uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, but are not limited to, phosphatidylcholines, such as 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylethanolamines such as 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), Sphingomyelin (SM), ceramides, steroids such as sterols and derivatives thereof. Neutral lipids may be of synthetic or natural origin.
The term "charged lipid" refers to any of a variety of lipid substances that exist in a positively or negatively charged form, independent of pH in a useful physiological range, e.g., pH-3 to pH-9. The charged lipids may be of synthetic or natural origin. Examples of charged lipids include phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyl trimethylammonium-propane (e.g., DOTAP, DOTMA), dialkyl dimethylaminopropane, ethyl phosphocholine, dimethyl aminoethane carbamyl sterol (e.g., DC-Chol).
The term "lipid nanoparticle" refers to a particle having at least one dimension on the nanometer scale (e.g., 1nm to 1,000nm) that comprises one or more compounds of structure (I) or other specific cationic lipids. In some embodiments, the lipid nanoparticle is included in a formulation useful for delivering an active or therapeutic agent, such as a nucleic acid (e.g., mRNA), to a target site of interest (e.g., cell, tissue, organ, tumor, etc.). In some embodiments, the lipid nanoparticle of the present invention comprises a nucleic acid. Such lipid nanoparticles typically comprise a compound of structure (I) and one or more excipients selected from neutral lipids, charged lipids, cholesterol, and polymer-conjugated lipids. In some embodiments, an active or therapeutic agent, such as a nucleic acid, may be encapsulated in the lipid portion of a lipid nanoparticle, or in the aqueous space encapsulated by some or all of the lipid portion of a lipid nanoparticle, thereby protecting it from enzymatic degradation, or from other undesirable effects induced by the mechanisms of the host organism or cell, such as an adverse immune response.
Embodiments of the lipid nanoparticles of the present invention are particularly useful for the delivery of nucleic acids, including, for example, mRNA, antisense oligonucleotides, plasmid DNA, microrna (miRNA), miRNA inhibitors (antagomers/antigens), messenger-RNA-interfering complementary RNA (microrna), DNA, multivalent RNA, dicer substrate RNA, complementary DNA (cdna), and the like. Thus, the lipid nanoparticles and compositions of the present invention can be used to induce expression of a desired protein in vitro and in vivo by contacting a cell with a lipid nanoparticle comprising one or more of the novel cationic lipids described herein, wherein the lipid nanoparticle encapsulates or associates a nucleic acid as follows: a nucleic acid that is expressed to produce a desired protein (e.g., a messenger RNA or plasmid encoding the desired protein) or a nucleic acid that is expressed to inhibit a process that terminates mRNA expression (e.g., a miRNA inhibitor). Alternatively, the lipid nanoparticles and compositions of the invention can be used to reduce expression of target genes and target proteins in vitro and in vivo by contacting a cell with a lipid nanoparticle comprising one or more of the novel cationic lipids described herein, wherein the lipid nanoparticle encapsulates or associates a nucleic acid (e.g., an antisense oligonucleotide or small interfering rna (sirna)) that reduces expression of the target gene. The lipid nanoparticles and compositions of the invention may also be used to co-deliver different nucleic acids (e.g., mRNA and plasmid DNA), either alone or in combination, for example, may be used to provide an effect that requires co-localization of different nucleic acids (e.g., mRNA encoding suitable gene modifying enzymes and DNA fragments incorporated into the host genome).
In various embodiments, the lipid nanoparticles used have the following average diameters: about 30nm to about 150nm, about 40nm to about 150nm, about 50nm to about 150nm, about 60nm to about 130nm, about 70nm to about 110nm, 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. In certain embodiments, the nucleic acid, when present in the lipid nanoparticle, is resistant to degradation by nucleases in aqueous solution.
As used herein, "lipid-encapsulated" refers to a lipid nanoparticle that provides complete encapsulation, partial encapsulation, or both for an active or therapeutic agent, such as a nucleic acid (e.g., mRNA). In one embodiment, the nucleic acid (e.g., mRNA) is completely encapsulated in the lipid nanoparticle.
By "serum-stable" with respect to the nucleic acid-lipid nanoparticle, it is meant that the nucleotide does not significantly degrade upon exposure to serum or upon exposure to nuclease assays that significantly degrade free DNA or RNA. Suitable assays include, for example, standard serum assays, dnase assays or rnase assays.
As used herein, "systemic delivery" refers to the delivery of a therapeutic product that can result in broad exposure of the active agent in the organism. Some administration techniques may result in the systemic delivery of certain agents, but not others. Systemic delivery means that a useful, preferably therapeutic, amount of the agent is exposed to most parts of the body. Systemic delivery of the lipid nanoparticles can be by any means known in the art, including, for example, intravenous, intra-arterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of the lipid nanoparticle is by intravenous delivery.
As used herein, "local delivery" refers to the delivery of an active agent directly to a target site within an organism. For example, the agent may be delivered locally by direct injection into a disease site such as a tumor, other target sites such as sites of inflammation, or target organs such as liver, heart, pancreas, kidney, and the like. Local delivery may also include topical application or local injection techniques, such as intramuscular, subcutaneous or intradermal injection. Local delivery does not interfere with systemic pharmacological effects.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.
EXAMPLE 1 Synthesis of Compound P-1
Figure BDA0003251199080000201
Synthesis of P-1: linoleic acid (3eq) was dissolved in DCM, EDC (1.5eq), DMAP (0.4eq) and DIPEA (2eq) were added, stirred for 10min, then compound a (1eq) was added and stirred at rt overnight. After completion of the reaction monitored by TLC, the solvent was distilled off under reduced pressure. Adding ethyl acetate, washing with saturated salt water for 3 times, drying with anhydrous sodium sulfate, concentrating, and separating and purifying with column to obtain the final product.
EXAMPLE 2 Synthesis of Compound P-2
Figure BDA0003251199080000202
Synthesis of Compound a: dissolving a certain amount of linoleic acid (1eq) in a proper amount of anhydrous tetrahydrofuran, stirring for 10min in an ice bath under the protection of nitrogen, then dropwise adding a lithium aluminum hydride tetrahydrofuran solution (2.5M,1.2eq), gradually returning to room temperature after the addition is finished, and stirring overnight. After the completion of the reaction was monitored by TLC, X mL of water (X is the total mass of lithium aluminum hydride added, unit: g) was added, X mL of a 15% NaOH aqueous solution was added, 3X mL of water was added, suction filtration was conducted, and the solvent was distilled off under reduced pressure. Adding appropriate volume of ethyl acetate, washing with equal volume of 1MHCl solution for 2 times, washing with saturated salt for 1 time, drying with anhydrous sodium sulfate, and concentrating. Separating and purifying the column to obtain the product.
Synthesis of Compound b: dissolving a certain amount of the compound a (1eq) in a proper amount of anhydrous dichloromethane, adding triethylamine (2eq), stirring in an ice bath for 10min under the protection of nitrogen, then dropwise adding acryloyl chloride (1.5eq), gradually returning to room temperature after the addition is finished, and stirring overnight. After completion of the reaction monitored by TLC, the solvent was distilled off under reduced pressure. Adding appropriate volume of ethyl acetate, washing with equal volume of saturated sodium bicarbonate solution for 2 times, washing with saturated salt solution for 1 time, drying with anhydrous sodium sulfate, and concentrating. Separating and purifying the column to obtain the product.
Synthesis of P-2: dissolving a certain amount of piperazine (1eq) in a proper amount of anhydrous tetrahydrofuran, then reacting the compound b (3eq) under the protection of nitrogen, and heating at 70 ℃ for 48 hours. After completion of the reaction monitored by TLC, the solvent was distilled off under reduced pressure. Separating and purifying the column to obtain the product.
EXAMPLE 3 Synthesis of Compound P-4
Figure BDA0003251199080000211
Synthesis of Compound c: compound b (1eq) was dissolved in DCM, EDC (1.5eq), DMAP (0.4eq) and DIPEA (2eq) were added, stirred for 10min, then compound e (2eq) was added and stirred at rt overnight. After completion of the reaction monitored by TLC, the solvent was distilled off under reduced pressure. Adding ethyl acetate, washing with saturated salt water for 3 times, drying with anhydrous sodium sulfate, concentrating, and separating and purifying with column to obtain the final product.
Synthesis of Compound d: dissolve compound c (1eq) in DCM and add NaHCO 3 (8eq), after stirring for 5min, add compound dessimutan oxidant (1.6eq) and stir at room temperature for 3 h. After completion of the reaction monitored by TLC, the solvent was distilled off under reduced pressure. Adding petroleum ether, washing with saturated sodium bicarbonate solution for 3 times, washing with saturated salt solution for 1 time, drying with anhydrous sodium sulfate, concentrating, and purifying with column separation to obtain the final product.
Synthesis of Compound P-4: compound d (2.2eq) was dissolved in DCM, piperazine (1eq) was added and stirred for 10min, then sodium triacetoxyborohydride (3eq) was added and stirred at rt overnight. After completion of the reaction monitored by TLC, the solvent was distilled off under reduced pressure. Adding ethyl acetate, washing with saturated saline water for 3 times, drying with anhydrous sodium sulfate, concentrating, and purifying with column separation to obtain the final product.
EXAMPLE 4 Synthesis of Compound P-5
Figure BDA0003251199080000221
Synthesis of Compound c: compound a (1eq) was dissolved in DCM, EDC (1.5eq), DMAP (0.4eq) and DIPEA (2eq) were added, and after stirring for 10min, compound b (2eq) was added and stirred at room temperature overnight. After completion of the reaction monitored by TLC, the solvent was distilled off under reduced pressure. Adding ethyl acetate, washing with saturated saline water for 3 times, drying with anhydrous sodium sulfate, concentrating, and purifying with column separation to obtain the final product.
Synthesis of Compound d: dissolving a certain amount of the compound a (1eq) in a proper amount of anhydrous dichloromethane, adding m-chloroperoxybenzoic acid (1.5eq) under the condition of nitrogen protection under ice bath, gradually returning to room temperature after the addition is finished, and stirring overnight. After completion of the reaction was monitored by TLC, the reaction was quenched by addition of saturated sodium bisulfite solution and the dichloromethane was distilled off under reduced pressure. Adding ethyl acetate, washing with saturated sodium bicarbonate solution for 2 times, washing with saturated salt solution for 1 time, drying with anhydrous sodium sulfate, and concentrating. Separating and purifying the column to obtain the product.
Synthesis of P-5: dissolving a certain amount of piperazine (1eq) in a proper amount of anhydrous methanol, then carrying out a reflux reaction on a compound d (3eq) under the protection of nitrogen for 3 hours. After completion of the reaction monitored by TLC, the solvent was distilled off under reduced pressure. Separating and purifying the column to obtain the product.
In a manner analogous to that described in examples 1 to 4 above, the following compounds P3, P6-P10 were prepared, the individual compounds being characterized in the following tables:
TABLE 1 Hydrogen spectra data
Figure BDA0003251199080000222
Figure BDA0003251199080000231
EXAMPLE 5 preparation of mRNA-LNP
Cationic liposomes, DSPC or DOPE, cholesterol, and PEG-liposomes were dissolved in ethanol at a ratio of 50:40:25:2 or 50:40:38.5:1.5 and mixed well. The mass ratio of Liposome Nanoparticle (LNP) to mRNA is 10:1 to 30: 1. The mRNA was diluted to 0.2mg/mL using citrate or sodium acetate buffer (pH 3 or 5). And (3) fully and uniformly mixing the cationic liposome ethanol solution and the mRNA solution in a volume ratio of 1:5 to 1: 3. The obtained nanoparticles are purified by means of ultrafiltration and dialysis. And (5) filtering and sterilizing. The particle size of mRNA-LNP (liposome nanoparticle encapsulating mRNA) is characterized to be about 30-150 nm.
Biological test example 1 Liposomal animal test
Male ICR mice (6-8week, Shanghai Jie Si Jie laboratory animals Co., Ltd.) were bred at 22. + -. 2 ℃ and a relative humidity of 45-75% under experimental conditions with a light/dark cycle of 12 h. Luciferase-encoding mrna (luciferase mrna) was used as a reporter gene. Luciferase catalyzes luciferin to generate bioluminescence, and the transfection efficiency of LNP is reflected by detecting the bioluminescence intensity in unit time. Using luciferase mRNA (purchased from ApexBio Technology) as an example, mRNA-LNP obtained as described above was administered intramuscularly at a dose of 150. mu.g/kg of mRNA. And (3) taking a specific time point, injecting fluorescein into the abdominal cavity of the mouse, placing the mouse in a small animal living body imager to measure the fluorescence intensity after 5 minutes, and expressing the final result by the average fluorescence intensity.
TABLE 1 fluorescence intensity results after intraperitoneal injection administration of mice
Figure BDA0003251199080000232
Figure BDA0003251199080000241
*(Molar ratio:Lipid/DOPE/Cholesterol/PEG)
The results show that the liposomes of the present invention achieve better biocompatibility and higher in vivo mRNA transfection efficiency compared to the existing commercially available liposomes MC-3. The liposome of the invention can form stable nano-structure with narrow size distribution, and the size of the liposome is changed with the structure of different liposomes and is within the range of 30-150 nm. Furthermore, the liposome-formed LNP (liposomal nanoparticles) of the present invention can be stored at low temperatures for extended periods of time. Structurally, degradable ester bonds are introduced into the hydrophobic tail of the liposome, so that the metabolic behavior of the liposome in vivo can be changed, and the biological safety of mRNA-LNP is improved. Compared with MC-3, the synthetic route is simple and easy, the raw materials are cheap and easy to obtain, and the industrial production is facilitated. The new liposome has good application prospect.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (10)

1. A liposomal compound represented by the following formula I:
Figure FDA0003251199070000011
wherein the content of the first and second substances,
side chain moiety
Figure FDA0003251199070000012
Each independently has 1 to 40 carbons in the main chain (i.e., the longest chain);
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 X or Y is N;
R 11 and R 22 Each independently selected from the group consisting of: substituted or unsubstituted straight or branched C 1-10 Alkyl, or R 11 And R 22 Together forming a substituted or unsubstituted straight or branched chain C 1-4 An alkylene group;
R 2 selected from the group consisting of: substituted or unsubstituted straight or branched C 1-4 An alkylene group;
R 3 and R 4 Each independently selected from the group consisting of: chemical bonds, or substituted or unsubstituted, straight or branched C 2-10 An alkylene group;
each R is 5 And R 6 Each independently selected from the group consisting of: chemical bonds, or substituted or unsubstituted straight or branched chainsChain C 1-10 Alkylene, substituted or unsubstituted straight or branched C 2-10 Alkenylene (i.e. when a1 or a2 is 2 or 3, each R 5 And R 6 May be the same or different);
each M 1 And M 2 Each independently is a group selected from:
Figure FDA0003251199070000013
R 7 and R 8 Each independently selected from the group consisting of: chemical bond, or substituted or unsubstituted straight or branched C 1-10 Alkylene, substituted or unsubstituted straight or branched C 2-10 An alkenylene group;
R 9 and R 10 Each independently selected from the group consisting of: substituted or unsubstituted straight or branched C 1-20 Alkylene, substituted or unsubstituted straight or branched C 2-20 An alkenylene group;
wherein at least one of said side chain moieties is present but not
Figure FDA0003251199070000021
M1 or M2, and at the same time, the corresponding
Figure FDA0003251199070000022
From 10 to 40 carbons in the backbone, preferably from 12 to 30 carbons (e.g., 7, 8, 9, 10, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 carbons);
unless otherwise specified, the substitution refers to the replacement of one or more hydrogen atoms on the group by a substituent selected from the group consisting of: straight or branched C 1-10 Alkyl (e.g. straight or branched C) 1-4 Alkyl), straight or branched C 2-4 Alkenyl, OH, NH 2 Hydroxy-substituted straight or branched C 1-4 Alkyl, hydroxy substituted straight or branched C 2-4 An alkenyl group.
2. The liposomal compound of claim 1(ii) said compound of formula I wherein, when a1 and a2 are each independently 1,2, or 3,
Figure FDA0003251199070000023
at least one of which is a segment having 2 to 10 carbons in the main chain (i.e., the longest chain); and when R is 3 、R 4 、R 5 Or R 6 When substituted, said substitution means that one or more hydrogen atoms on the group are substituted with a substituent selected from the group consisting of: straight or branched C 1-4 Alkyl, straight or branched C 2-4 Alkenyl, OH, NH 2 Hydroxy-substituted straight or branched chain C 1-4 Alkyl, hydroxy substituted straight or branched C 2-4 An alkenyl group.
3. The liposomal compound of claim 1 wherein the compound of formula I has the structure shown in formula II below:
Figure FDA0003251199070000024
x and Y are each independently selected from N or CH, and at least one X or Y is N;
R 1 selected from the group consisting of:
Figure FDA0003251199070000025
wherein a, ggg, hhh, b, iii, and kkk are each independently 0, 1,2, or 3;
R 2 selected from the group consisting of:
Figure FDA0003251199070000026
R 3 selected from the group consisting of:
Figure FDA0003251199070000027
Figure FDA0003251199070000028
wherein ww, xx, c, d, ff are each independently 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10;
R 4 selected from the group consisting of:
Figure FDA0003251199070000031
Figure FDA0003251199070000032
wherein yy, zz, e, f, gg are each independently 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10;
in the above formulas, CC, DD, A and B are substituents selected from the group consisting of: H. CH (CH) 3 、OH、CH 2 OH、C 2 H 4 OH、NH 2 ；
R 5 Selected from the group consisting of:
Figure FDA0003251199070000033
wherein g, h, dd, jj, and kk are each independently 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10;
R 6 selected from the group consisting of:
Figure FDA0003251199070000034
wherein i, j, ee, mm and nn are each independently 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10;
R 7 selected from the group consisting of:
Figure FDA0003251199070000035
wherein k, l and m are each independently 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10;
R 8 selected from the group consisting of:
Figure FDA0003251199070000036
wherein n, o and q are each independently 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10;
in each of the above formulae, C, D, E, F, G, J is independently selected from the group consisting of: H. CH (CH) 3 ；
R 9 Selected from the group consisting of:
Figure FDA0003251199070000037
Figure FDA0003251199070000038
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 selected from the group consisting of:
Figure FDA0003251199070000039
Figure FDA0003251199070000041
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;
in each of the above formulae, K, L, M and N are each independently selected from the group consisting of: H. CH (CH) 3 (ii) a AA and BB are each independently selected from the group consisting of: H. CH (CH) 3 OH or NH 2 ；
Provided that the side chain moiety
Figure FDA0003251199070000042
Each independently having 2 to 40 carbons in the main chain (i.e., the longest chain) and 10 to 40 carbons in at least one side chain moiety.
4. The liposomal compound of claim 1 wherein the compound of formula I has the structure shown in formula III below:
Figure FDA0003251199070000043
wherein the content of the first and second substances,
x and Y are each independently selected from N or CH, and at least one X or Y is N;
R 11 selected from the group consisting of:
Figure FDA0003251199070000044
wherein b1 and b2 are each independently 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10;
R 22 selected from the group consisting of:
Figure FDA0003251199070000045
R 2 selected from the group consisting of:
Figure FDA0003251199070000046
R 3 selected from the group consisting of:
Figure FDA0003251199070000047
Figure FDA0003251199070000048
wherein ww, xx, c, d, ff are each independently 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10;
R 4 selected from the group consisting of:
Figure FDA0003251199070000049
Figure FDA00032511990700000410
wherein yy, zz, e, f, gg are each independently 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10;
in the above formulas, CC, DD, A and B are substituents selected from the group consisting of: H. CH (CH) 3 、OH、CH 2 OH、C 2 H 4 OH、NH 2 ；
R 5 Selected from the group consisting of:
Figure FDA0003251199070000051
wherein g, h, dd, jj and kk are each independently 0,1.2, 3, 4, 5, 6, 7, 8, 9 or 10;
R 6 selected from the group consisting of:
Figure FDA0003251199070000052
wherein i, j, ee, mm, and nn are each independently 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10;
R 7 selected from the group consisting of:
Figure FDA0003251199070000053
wherein k, l and m are each independently 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10;
R 8 selected from the group consisting of:
Figure FDA0003251199070000054
wherein n, o and q are each independently 0, 1,2, 3, 4, 5, 6, 7, 8, 9 or 10;
in each of the above formulae, C, D, E, F, G, J is independently selected from the group consisting of: H. CH (CH) 3 ；
R 9 Selected from the group consisting of:
Figure FDA0003251199070000055
Figure FDA0003251199070000056
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 selected from the group consisting of:
Figure FDA0003251199070000057
Figure FDA0003251199070000058
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;
in each of the above formulae, K, L, M and N are each independently selected from the group consisting of: H. CH (CH) 3 (ii) a AA and BB are each independently selected from the group consisting of: H. CH (CH) 3 OH or NH 2 ；
Provided that the side chain moiety
Figure FDA0003251199070000059
Each independently having 2 to 40 carbons in the main chain (i.e., the longest chain) and 10 to 25 carbons in at least one side chain moiety.
5. The liposomal compound of claim 1 wherein the compound of formula I has the structure shown in formula IV below:
Figure FDA0003251199070000061
wherein each group is as defined in any one of claims 1 to 3.
6. The liposomal compound of claim 1 wherein the compound is selected from the group consisting of:
Figure FDA0003251199070000062
Figure FDA0003251199070000071
7. use of the liposomal compound of claim 1 for the preparation of a drug-loaded nanophase composition.
8. A drug-Nanotergerfuge composition comprising the liposomal compound of any one of claims 1-6 and a therapeutic agent.
9. The drug-Nanocarrier composition of claim 8, wherein the neutral lipid is selected from the group consisting of: DSPC, DPPC, DMPC, DOPC, POPC, DOPE, SM, or combinations thereof; preferably, the neutral lipids are DOPE and DSPC.
10. The drug-Nanocarrier composition of claim 8, wherein the polymer-conjugated lipid in the composition is a pegylated lipid.
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