Ionizable lipids

Abstract

The present invention relates generally to the field of ionizable (also known as cationic) lipids, and specifically provides a novel such lipid represented by Formula (I). The present invention further provides methods for preparing such lipids and uses thereof, particularly in the preparation of nanoparticle compositions, more particularly nanoparticle compositions comprising nucleic acids. The present invention further provides vaccine formulations comprising nanoparticle compositions based on the ionizable lipids disclosed herein.

Description

Invention Field

[0001] This invention generally relates to the field of ionizable (also known as cationic) lipids, and specifically provides a novel such lipid represented by formula (I). The invention further provides methods for preparing such lipids and their uses, particularly in the preparation of nanoparticle compositions, and more particularly in nanoparticle compositions containing nucleic acids. The invention further provides vaccine formulations comprising nanoparticle compositions based on the ionizable lipids disclosed herein. Background of the Invention

[0003] Nucleic acid-based drugs are being explored in a growing number of therapeutic areas. However, the targeted delivery of nucleic acids, such as plasmid DNA, messenger RNA, short interfering RNA, single-stranded guide RNA, and microRNA, to tissues and cells poses a major challenge due to their negative charge, size, and instability. Numerous nanoparticle carrier systems have been explored for encapsulating and delivering nucleic acids. These nanoparticles require a combination of efficient and stable encapsulation of nucleic acids during storage and in the extracellular environment with maximum cellular uptake and efficient release of their payload from the endosome to the cytoplasm.

[0004] Lipid-based nanoparticles (LNPs) are used clinically for the delivery of small interfering RNA (LNP) and mRNA vaccines, representing the most advanced type of RNA delivery carrier. Lipid-based LNPs typically consist of cationic or ionizable lipids that can be protonated at acidic pH, cofactor phospholipids, PEGylated lipids, and sterols. Each component has a specific function in terms of LNP stability and activity. Sterols and PEGylated lipids are crucial for LNP structure and stability, while phospholipids contribute to stability and endosome escape. Cationic or ionizable lipids are considered key drivers of activity and tolerability by controlling mRNA encapsulation, cellular uptake, and endosome escape. While efficient nucleic acid delivery carriers, LNPs can induce dose-limiting toxicities such as complement activation-related pseudohypersensitivity, inflammatory cytokine release, and cytotoxicity caused by the accumulation of non-degradable ionizable lipids in the cell membrane. Therefore, further improvements in cationic or ionizable lipid chemistry are needed to enhance the efficacy and safety of LNP-delivered nucleic acid drugs.

[0005] Therefore, the present invention relates to a new class of ionizable lipids as defined by a set of claims of the present invention, which have improved properties compared to currently available types of ionizable lipids. Invention Overview

[0007] In a first aspect, the present invention provides a lipid, particularly an ionizable lipid represented by formula (I).

[0008]

[0009] in

[0010] R1 and R2 are each independently selected from -H and -C. 1-20 Alkyl, -C 2-20 alkenyl and -C 2-20 alkynyl group; wherein the -C 1-20 Alkyl, -C 2-20 alkenyl and -C 2-20 Each of the ynyl groups may optionally further comprise one or more heteroatoms and / or may optionally be surrounded by 1 to 3 –O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 Alkyl, -C 2-20 alkenyl and -C 2-20 Alkyne substitution; and the total number of C atoms in R1 and R2 is at least 8;

[0011] R3 and R4 are each independently -C 1-6 Alkyl group; or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocycle; said heterocycle may further optionally contain one or more additional N atoms, and / or may optionally be substituted by 1-3 substituents selected from: -C 1-6 Alkyl; and

[0012] Each R5 and R6 is independently selected from –CH2- and -O-CH2-;

[0013] Each R7 is independently selected from -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Alkyne group; wherein the -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Each of the ynyl groups may optionally further comprise one or more heteroatoms and / or may optionally be surrounded by 1 to 3 –O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Alkyne substitution;

[0014] m and n are each an independent integer selected from 1, 2, 3 and 4;

[0015] -

[0016] Each R8 and R9 is independently selected from –H, -C1-6 Alkyl and –C 3-6 cycloalkyl;

[0017] Y is selected from -NH- and -O-;

[0018] Z is -C 1-6 Alkylene-.

[0019] In a specific embodiment, the present invention provides a lipid, particularly an ionizable lipid as defined herein and represented by formula (II).

[0020]

[0021] in

[0022] R3 and R4 are each independently -C 1-6 Alkyl group; or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocycle; said heterocycle may further optionally contain one or more additional N atoms, and / or may optionally be substituted by 1-3 substituents selected from: -C 1-6 Alkyl; and

[0023] Each R5 and R6 is independently selected from –CH2- and -O-CH2-;

[0024] Each R7 is independently selected from -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Alkyne group; wherein the -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Each of the ynyl groups may optionally further comprise one or more heteroatoms and / or may optionally be surrounded by 1 to 3 –O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Alkyne substitution; and the total number of C atoms in the two R7 moieties is at least 5;

[0025] m and n are each an independent integer selected from 1, 2, 3 and 4;

[0026] -

[0027] Each R8 and R9 is independently selected from –H, -C 1-6 Alkyl and –C 3-6 cycloalkyl;

[0028] Y is selected from -NH- and -O-;

[0029] Z is -C 1-6 Alkylene-.

[0030] In yet another embodiment, the present invention provides a lipid, particularly an ionizable lipid as defined herein and represented by any one of formula (IIIa), (IIIb) or (IIIc).

[0031]

[0032] in

[0033] R3 and R4 are each independently -C 1-6 Alkyl group; or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocycle; said heterocycle may further optionally contain one or more additional N atoms, and / or may optionally be substituted by 1-3 substituents selected from: -C 1-6 Alkyl; and

[0034] Each R5 and R6 is independently selected from –CH2- and -O-CH2-;

[0035] Each R7 is independently selected from -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Alkyne group; wherein the -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Each of the ynyl groups may optionally further comprise one or more heteroatoms and / or may optionally be surrounded by 1 to 3 –O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Alkyne substitution; and the total number of C atoms in the two R7 moieties is at least 5;

[0036] m and n are each an independent integer selected from 1, 2, 3 and 4;

[0037] Each R8 and R9 is independently selected from –H, -C 1-6 Alkyl and –C 3-6 cycloalkyl;

[0038] Y is selected from -NH- and -O-;

[0039] Z is -C 1-6 Alkylene-.

[0040] The present invention further provides a lipid, particularly an ionizable lipid as defined herein and selected from the list below:

[0041]

[0042] In yet another embodiment, the present invention provides a lipid, particularly an ionizable lipid as defined herein and represented by any one of formulas (IVa), (IVb) or (IVc).

[0043]

[0044] in

[0045] R1 and R2 are each independently selected from -H and -C. 1-20 Alkyl, -C 2-20 alkenyl and -C 2-20 Alkyne group; wherein the -C 1-20 Alkyl, -C 2-20 alkenyl and -C 2-20 Each of the ynyl groups may optionally further comprise one or more heteroatoms and / or may optionally be surrounded by 1 to 3 –O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 Alkyl, -C 2-20 alkenyl and -C 2-20 Alkyne substitution; and the total number of C atoms in R1 and R2 is at least 8;

[0046] R3 and R4 are each independently -C 1-6 Alkyl group; or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocycle; said heterocycle may further optionally contain one or more additional N atoms, and / or may optionally be substituted by 1-3 substituents selected from: -C 1-6 Alkyl; and

[0047] Each R5 and R6 is independently selected from –CH2- and -O-CH2-;

[0048] Each R7 is independently selected from -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Alkyne group; wherein the -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Each of the ynyl groups may optionally further comprise one or more heteroatoms and / or may optionally be surrounded by 1 to 3 –O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20Alkyne substitution;

[0049] m and n are each an independent integer selected from 1, 2, 3 and 4;

[0050] Each R8 and R9 is independently selected from –H, -C 1-6 Alkyl and –C 3-6 cycloalkyl;

[0051] Y is selected from -NH- and -O-;

[0052] Z is -C 1-6 Alkylene-.

[0053] In another embodiment, the present invention provides a lipid, particularly an ionizable lipid as defined herein and selected from the list below:

[0054]

[0055] In yet another embodiment, the present invention provides a lipid, particularly an ionizable lipid as defined herein; wherein the total number of C atoms in R1 and R2 is at least 14.

[0056] The present invention further provides a lipid, particularly an ionizable lipid as defined herein; wherein each R5 and R6 is –CH2-.

[0057] In another embodiment, the present invention provides a lipid, particularly an ionizable lipid as defined herein; wherein m and n are the same and are integers selected from 1, 2, 3 and 4; preferably 2.

[0058] In yet another embodiment, the present invention provides a lipid, particularly an ionizable lipid as defined herein; wherein Y is -NH-.

[0059] In another aspect, the present invention provides lipid nanoparticles or lipid nanoparticle compositions comprising lipids, particularly ionizable lipids as defined herein. The nanoparticle composition may further comprise phospholipids, sterols, and PEG lipids.

[0060] In yet another embodiment of the invention, the lipid nanoparticles or lipid nanoparticle compositions as defined herein further comprise an active agent, particularly a nucleic acid, preferably mRNA.

[0061] In another aspect, the present invention provides the use of lipids, particularly ionizable lipids as defined herein, in the preparation of lipid nanoparticles or lipid nanoparticle compositions.

[0062] In a final aspect, the present invention provides a pharmaceutical composition comprising lipid nanoparticles or lipid nanoparticle compositions as defined herein and a pharmaceutically acceptable agent.

[0063] The present invention also provides pharmaceutical compositions as defined herein for use in human and / or veterinary medicines.

[0064] In yet another embodiment, the present invention provides a lipid, particularly an ionizable lipid as defined herein and represented by formula (V).

[0065]

[0066] in

[0067] R3 and R4 are each independently -C 1-6 Alkyl group; or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocycle; said heterocycle may further optionally contain one or more additional N atoms, and / or may optionally be substituted by 1-3 substituents selected from: -C 1-6 Alkyl; and

[0068] Each R5 and R6 is independently selected from –CH2- and -O-CH2-;

[0069] Each R7 is independently selected from -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Alkyne group; wherein the -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Each of the ynyl groups may optionally further comprise one or more heteroatoms and / or may optionally be surrounded by 1 to 3 –O-(C=O)-R7, -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Alkyne substitution; and the total number of C atoms in the two R7 moieties is at least 5;

[0070] m and n are each an independent integer selected from 1, 2, 3 and 4;

[0071] Y is selected from -NH- and -O-;

[0072] Z is -C 1-6 Alkylene-.

[0073] The present invention further provides a lipid, particularly an ionizable lipid as defined herein and selected from the list below:

[0074]

[0075] In yet another embodiment, the present invention provides a lipid, particularly an ionizable lipid as defined herein and represented by any one of formula (VIa) or (VIb).

[0076]

[0077] in

[0078] R3 and R4 are each independently -C 1-6 Alkyl group; or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocycle; said heterocycle may further optionally contain one or more additional N atoms, and / or may optionally be substituted by 1-3 substituents selected from: -C 1-6 Alkyl; and

[0079] Each R5 and R6 is independently selected from –CH2- and -O-CH2-;

[0080] Each R7 is independently selected from -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Alkyne group; wherein the -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Each of the ynyl groups may optionally further comprise one or more heteroatoms and / or may optionally be surrounded by 1 to 3 –O-(C=O)-R7, -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Alkyne substitution; and the total number of C atoms in the two R7 moieties is at least 5;

[0081] m and n are each an independent integer selected from 1, 2, 3 and 4;

[0082] o and p are each an integer selected independently from 1 to 10;

[0083] Y is selected from -NH- and -O-;

[0084] Z is -C 1-6 Alkylene-.

[0085] In another embodiment, the present invention provides an ionizable lipid selected from the following list:

[0086]

[0087]

[0088]

[0089]

[0090]

[0091] Brief description of the attached diagram

[0093] Referring now specifically to the accompanying drawings, it is emphasized that the details shown are by way of example and are for the purpose of illustrative discussion of different embodiments of the invention only. They are presented to provide the most useful and readily apparent description of the principles and concepts of the invention. In this respect, no attempt is made to show the structural details of the invention in more detail than necessary for a basic understanding of the invention. The description taken in conjunction with the drawings makes it apparent to those skilled in the art how several forms of the invention can be practiced in practice.

[0094] Figure 1 The relative mean fluorescence intensity of eGFP expression in HEK293T cells after incubation with the specified LNP at mRNA concentrations of 50 ng / well and 200 ng / well (measured as the fold increase in eGFP MFI compared to untreated cells).

[0095] Figure 2. Relative MFI of eGFP expression after transfection with specified LNPs into different cell types, namely HEK293T cells (A), TS / A cells (B), CT26 cells (C), and B16F10 cells (D).

[0096] Figure 3. Viability of different cell types, namely HEK293T(A) and CT26(B), after transfection with the specified LNPs.

[0097] Figure 4. Relative MFI of eGFP expression after transfection with specified LNPs into different cell types, namely HEK293T (A) and CT26 (B); and

[0098] Figure 5. Flux mRNA expression in CT26 tumors (A) or liver (B) measured by in vivo bioluminescence (photons / s / cm2 / sr) after tumor injection, and body weight of mice before and after intratumoral administration of S-Ac-Dog and MC-3 based LNPs (C).

[0099] Figure 6. Flux mRNA expression in B16F10 tumors (A) or livers (B) measured by in vivo bioluminescence (photons / s / cm2 / sr) after injection into the tumor, and the tumor / liver ratio of Flux expression after intratumoral injection of the corresponding LNP into B16F10 tumors (C).

[0100] Figure 7 The percentage of E7-specific CD8 T cells was assessed by flow cytometry after intramuscular immunization with E7 mRNA encapsulated in LNPs containing corresponding ionizable lipids.

[0101] Figure 8 The percentage of E7-specific CD8 T cells in the blood was measured by flow cytometry after intramuscular immunization of C57BL / 6 mice with mRNA LNPs containing specified ionizable lipids. All LNPs were formulated at a lipid molar ratio of 50 / 10 / 38.5 / 1.5 for ionizable lipids / DSPC / DMG-PEG2000. Mice were immunized twice, on days 1 and 7, with 5 μg of mRNA.

[0102] Figure 9 Muscle thickness at the injection site was measured before injection (d0), and on day 1 (d1) and day 4 (d4) after injection with the corresponding LNP (5 μg E7 dose). All LNPs were formulated with a lipid molar ratio of 50 / 10 / 38.5 / 1.5 for ionizable lipids / DSPC / DMG-PEG2000.

[0103] Figure 10 Anti-HA IgG1 and IgG2a antibody titers were determined after intramuscular immunization with LNPs containing specified ionizable lipids. Mice were immunized twice intramuscularly with mRNA LNPs (2 μg HA) on days 1 and 21. Blood samples were obtained on days 21 and 35 for assessing anti-HA antibody titers. LNPs containing specified ionizable lipids were formulated at a lipid molar ratio of 50 / 10 / 38.5 / 1.5 for ionizable lipids / DSPC / DMG-PEG2000.

[0104] Figure 11 The percentage of IFNg-positive CD8 T cells in the spleen following intramuscular immunization with LNPs containing specified ionizable lipids. Mice were immunized twice intramuscularly with mRNA LNPs (2 μg HA) on days 1 and 21. Spleen cells were obtained on day 35 and restimulated with or without an overlapping HA peptide library. The percentage of IFNg+ CD8 T cells was subsequently determined by flow cytometry.

[0105] Figure 12 The size of E7-specific CD8 T cell responses in blood was measured after intramuscular injection of LNPs containing S-Ac7-Dog, S-Ac7-DHDa, or MC-3 as ionizable lipids. Mice were immunized twice with 5 μg of E7 mRNA on days 1 and 21. Blood samples were analyzed by flow cytometry on days 7 and 27.

[0106] Figure 13Following in vitro stimulation with the E7-derived peptide RAHYNIVT, flow cytometry was used to assess the percentages of IFNg+, IFNg+TNFa+, IFNg+granulase b (Grnz)+, and IFNg+CD107+CD8 T cells in the spleen. Mice were immunized twice with 5 μg of E7 mRNA on days 1 and 21.

[0107] Figure 14 In C57BL / 6 mice, the percentages of Cy5-positive macrophages, dendritic cells (cDC1 and cDC2 subsets), B cells, and T cells (CD4+ and CD8+ T cell subsets) in the spleen were measured by flow cytometry 24 h after intravenous administration. Mice received 10 μg of the peptide and 10 μg of IMDQ or equivalent. n = 3.

[0108] Figure 15 In C57BL / 6 mice, 24 h after intravenous administration of the peptide, the percentage of activation marker-positive dendritic cells (cDC1 and cDC2 subsets), B cells, and T cells (CD4+ and CD8+ T cell subsets) in the spleen was measured by flow cytometry. Mice received 10 μg of the peptide and 10 μg of IMDQ or equivalent. n = 3.

[0109] Figure 16 The percentage of tetramer-positive CD8+ T cells in the blood, measured by flow cytometry, was observed one week after intravenous administration of the second dose (2 weeks between administrations) to C57BL / 6 mice. Mice received 10 μg of the peptide and 10 μg of IMDQ or equivalent. n = 5.

[0110] Figure 17 Anti-S1 spike protein IgG antibody titers were measured after intramuscular immunization with LNPs containing S-Ac7-DOg as an ionizable lipid. C57BL / 6 mice received a single injection of LNPs containing 25 μg of the TLR3 agonist polyI:C. 25 μg of S1 spike protein was incorporated or conjugated to the LNP surface via His6-Ni2+ interaction. Blood samples were obtained 7 days post-immunization and analyzed by ELISA.

[0111] Figure 18 Anti-ovalbumin (OVA) IgG antibody titers were measured after intramuscular immunization with LNP containing S-Ac7-DOg as an ionizable lipid. C57BL / 6 mice received a single injection of LNP containing 10 μg of the TLR9 agonist CpG. 50 μg of OVA was infused into LNP in blood samples obtained 7 days post-immunization and analyzed by ELISA. Invention Details

[0113] The invention will now be described further. In the following paragraphs, different aspects of the invention are defined in more detail. Unless clearly indicated to the contrary, each aspect so defined may be combined with one or more other aspects. In particular, any feature designated as preferred or advantageous may be combined with one or more other features designated as preferred or advantageous.

[0114] Unless the context otherwise indicates, this document uses an asterisk to indicate the point where a monovalent or divalent group is attached to the structure in which it relates and which group constitutes part of it.

[0115] As mentioned above, in a first aspect, the present invention provides a lipid, particularly an ionizable lipid represented by formula (I).

[0116]

[0117] in

[0118] R1 and R2 are each independently selected from -H and -C. 1-20 Alkyl, -C 2-20 alkenyl and -C 2-20 Alkyne group; wherein the -C 1-20 Alkyl, -C 2-20 alkenyl and -C 2-20 Each of the ynyl groups may optionally further comprise one or more heteroatoms and / or may optionally be surrounded by 1 to 3 –O-(C=O)-R7, -C 1-20 Alkyl, -C 2-20 alkenyl and -C 2-20 Alkyne substitution; and the total number of C atoms in R1 and R2 is at least 8;

[0119] R3 and R4 are each independently -C 1-6 Alkyl group; or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocycle; said heterocycle may further optionally contain one or more additional N atoms, and / or may optionally be substituted by 1-3 substituents selected from: -C 1-6 Alkyl; and

[0120] Each R5 and R6 is independently selected from –CH2- and -O-CH2-;

[0121] Each R7 is independently selected from -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Alkyne group; wherein the -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20Each of the ynyl groups may optionally further comprise one or more heteroatoms and / or may optionally be surrounded by 1 to 3 –O-(C=O)-R7, -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Alkyne substitution;

[0122] m and n are each an independent integer selected from 1, 2, 3 and 4;

[0123] -

[0124] Each R8 and R9 is independently selected from –H, -C 1-6 Alkyl and –C 3-6 cycloalkyl;

[0125] Y is selected from -NH- and -O-;

[0126] Z is -C 1-6 Alkylene-.

[0127] Therefore, the present invention also provides a lipid, particularly an ionizable lipid represented by formula (I).

[0128]

[0129] in

[0130] R1 and R2 are each independently selected from -H and -C. 1-20 Alkyl, -C 2-20 alkenyl and -C 2-20 Alkyne group; wherein the -C 1-20 Alkyl, -C 2-20 alkenyl and -C 2-20 Each of the ynyl groups may optionally be surrounded by 1 to 3 –O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 Alkyl, -C 2-20 alkenyl and -C 2-20 Alkyne substitution; and the total number of C atoms in R1 and R2 is at least 8;

[0131] R3 and R4 are each independently -C 1-6 Alkyl group; or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocycle; said heterocycle may further optionally contain one or more additional N atoms, and / or may optionally be substituted by 1-3 substituents selected from: -C 1-6 Alkyl; and

[0132] Each R5 and R6 is independently –CH2-;

[0133] Each R7 is independently selected from -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Alkyne group; wherein the -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Each of the ynyl groups may optionally be surrounded by 1 to 3 –O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Alkyne substitution;

[0134] m and n are each an independent integer selected from 1, 2, 3 and 4;

[0135] -

[0136] Each R8 and R9 is independently selected from –H, -C 1-6 Alkyl and –C 3-6 cycloalkyl;

[0137] Y is selected from -NH- and -O-;

[0138] Z is -C 1-6 Alkylene-.

[0139] When describing the compounds / lipids of the present invention, unless the context otherwise indicates, the terminology used shall be interpreted according to the following definitions:

[0140] The term "alkyl" itself, or as part of another substituent, refers to the formula C x H 2x+1 A fully saturated hydrocarbon, where x is a number greater than or equal to 1. Typically, the alkyl groups of this invention contain 1 to 20 carbon atoms. The alkyl groups can be straight-chain or branched and can be substituted as indicated herein. When a subscript is used hereafter after a carbon atom, the subscript indicates the number of carbon atoms that the specified group may contain. Thus, for example, C 1-4Alkyl refers to an alkyl group with one to four carbon atoms. Examples of alkyl groups are methyl, ethyl, n-propyl, isopropyl, butyl and their isomers (e.g., n-butyl, isobutyl, and tert-butyl); pentyl and its isomers, hexyl and its isomers, heptyl and its isomers, octyl and its isomers, nonyl and its isomers; decyl and its isomers, undecyl and its isomers, dodecyl and its isomers, tridecyl and its isomers, tetradecyl and its isomers, pentadecyl and its isomers, hexadecyl and its isomers, heptadecanyl and its isomers, octadecyl and its isomers, nonadecanyl and its isomers, and eicosyl and its isomers.

[0141] The term "optionally substituted alkyl" refers to an alkyl group that is optionally replaced at any available connection point by one or more substituents (e.g., 1 to 4 substituents, such as 1, 2, 3, or 4 substituents). Non-limiting examples of such substituents include esters, carboxylic acids, alkyl moieties, olefinic moieties, alkyne moieties, etc.

[0142] In the context of this invention, the alkyl, olefin, and alkyne portions as defined herein may further include one or more heteroatoms, for example, C atoms in the alkyl, olefin, or alkyne chain may be replaced by heteroatoms such as those selected from N, S, or O.

[0143] Unless otherwise stated, the term "alkenyl" or "olefin" as used herein refers to a straight-chain, cyclic, or branched hydrocarbon group containing at least one carbon-carbon double bond. Examples of alkenyl groups include vinyl, propenyl, isopropenyl, butenyl, isobutenyl, pentenyl, hexenyl, hexadienyl, etc., whether terminal or internal. Typically, the alkenyl or olefinic moiety of the present invention comprises 2 to 20 carbon atoms. Optionally substituted alkenyl refers to an alkenyl group that optionally has one or more substituents (e.g., 1, 2, 3, or 4) selected from those substituents defined above for substituted alkyl groups.

[0144] Unless otherwise stated, the terms "alkynyl" or "alkynylene" as used herein refer to a straight-chain or branched hydrocarbon group containing at least one carbon-carbon triple bond. Examples of alkynyl groups include ethynyl, E- and Z-propynyl, isopropynyl, E- and Z-butynyl, E- and Z-isobutynyl, E- and Z-pentynyl, E, Z-hexynyl, etc. Typically, the alkenyl or olefinic moiety of the present invention contains 2 to 20 carbon atoms. Optionally substituted alkynyl refers to an alkynyl group that optionally has one or more substituents (e.g., 1, 2, 3, or 4) selected from those substituents defined above for substituted alkyl groups.

[0145] The term "cycloalkyl" itself, or as part of another substituent, refers to a cyclic alkyl group, in other words, a monovalent, saturated, or unsaturated hydrocarbon group having one, two, or three rings. Cycloalkyl includes all saturated or partially saturated hydrocarbon groups containing one to three rings (containing one or two double bonds), including monocyclic, bicyclic, or polycyclic alkyl groups. A cycloalkyl group may contain three or more carbon atoms in the ring and typically contains three to 15 atoms according to the invention. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, adamantyl, and cyclodecyl, with cyclopropyl being particularly preferred. "Optionally substituted cycloalkyl" refers to a cycloalkyl group that optionally has one or more substituents (e.g., one to three substituents, such as one, two, three, or four substituents) selected from those substituents defined above for substituted alkyl groups.

[0146] When an alkyl group, as defined, is divalent, that is, has two single bonds for attaching to two other groups, it is called an "alkylene" group. Non-limiting examples of alkylene groups include methylene, ethylene, methylmethylene, trimethylene, propylene, tetramethylene, ethyl ethylene, 1,2-dimethyl ethylene, pentamethylene, and hexamethylene. Similarly, when an alkenyl group, as defined above, and an alkynyl group, as defined above, are divalent groups having single bonds for attaching to two other groups, they are called "alkenyl" and "alkynyl," respectively.

[0147] As used herein, the term "heterocycle" itself, or as part of another group, refers to a non-aromatic, fully saturated, or partially unsaturated cyclic group (e.g., a 3- to 13-membered monocyclic, 7- to 17-membered bicyclic, or 10- to 20-membered tricyclic ring system, or containing a total of 3 to 10 ring atoms) having at least one heteroatom in at least one carbon-containing ring. Each ring of a heterocyclic group containing a heteroatom may have 1, 2, 3, or 4 heteroatoms selected from nitrogen, oxygen, and / or sulfur atoms, wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. Where valence permits, the heterocyclic group may be attached to any heteroatom or carbon atom in the ring or ring system. The rings of a polycyclic heterocycle may be fused, bridged, and / or connected by one or more spiro atoms. An optionally substituted heterocycle refers to a heterocycle that optionally has one or more substituents (e.g., 1 to 4 substituents, or, for example, 1, 2, 3, or 4 substituents) selected from those substituents defined above for substituted alkyl groups. Non-limiting examples of heterocycles include: piperidinyl, nitrogen-containing heptyl, morpholinyl, etc.

[0148] As used herein, the term "aryl" (also referred to herein as an aromatic heterocycle) means a polyunsaturated aromatic hydrocarbon group having a monocyclic (i.e., phenyl) or fused together (e.g., naphthalene or anthracene) or covalently linked aromatic rings, typically containing 6 to 10 atoms; wherein at least one ring is aromatic. The aromatic ring may optionally include one to three additional rings (cycloalkyl, heterocyclic, or heteroaryl) fused thereto. Aryl is also intended to include partially hydrogenated derivatives of the carbocyclic systems listed herein. Non-limiting examples of aryl include phenyl, etc.

[0149] As defined herein, an aryl ring or heterocycle may optionally be substituted at any available junction with one or more substituents (e.g., 1 to 5 substituents, such as 1, 2, 3, or 4). Non-limiting examples of such substituents are selected from halogens, hydroxyl, oxo, nitro, amino, hydrazyl, aminocarbonyl, azide, cyano, alkyl, cycloalkyl, alkenyl, alkynyl, cycloalkylalkyl, alkylamino, alkoxy, -SO2-NH2, aryl, heteroaryl, aralkyl, haloalkyl, haloalkoxy, alkoxycarbonyl, alkylaminocarbonyl, heteroarylalkyl, alkylsulfonamide, heterocyclic, alkylcarbonylaminoalkyl, aryloxy, alkylcarbonyl, acyl, arylcarbonyl, aminocarbonyl, alkyl sulfoxide, -SO2R a alkylthio groups, carboxyl groups, etc., among which R a It is an alkyl or cycloalkyl group.

[0150] When the carbon atom in the aryl group is replaced by a heteroatom, the resulting ring is referred to in this paper as a heteroaryl ring.

[0151] As used herein, the term "heteroaryl" itself, or as part of another group, refers, but is not limited to, an aromatic ring of 5 to 12 carbon atoms or a ring system containing 1 to 3 rings fused together or covalently linked, typically containing 5 to 8 atoms; at least one of these rings is an aromatic ring, wherein one or more carbon atoms in one or more of these rings may be replaced by oxygen, nitrogen, or sulfur atoms, wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatomium may optionally be quaternized. Such rings may be fused to aryl, cycloalkyl, heteroaryl, or heterocyclic rings. Non-limiting examples of such heteroaryls include piperidinyl, azircyclic hepttrienyl, etc.

[0152] "Optionally substituted heteroaryl" means a heteroaryl group that optionally has one or more substituents (e.g., 1 to 4 substituents, such as 1, 2, 3 or 4), said substituents being selected from those substituents defined above for substituted alkyl groups.

[0153] As used in this article, the term "oxo" refers to the =O group.

[0154] As used herein, the terms "alkoxy" or "alkyloxy" refer to a compound having the formula -ORb The group, wherein R b It is an alkyl group. Preferably, the alkoxy group is C1-C. 10 Alkoxy, C1-C6 alkoxy, or C1-C4 alkoxy. Non-limiting examples of suitable alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, and hexoxy. When the oxygen atom in the alkoxy group is replaced by sulfur, the resulting group is called a thioalkoxy. A "haloalkoxy" is an alkoxy group in which one or more hydrogen atoms in the alkyl group are replaced by a halogen. Non-limiting examples of suitable haloalkoxy groups include fluoromethoxy, difluoromethoxy, trifluoromethoxy, 2,2,2-trifluoroethoxy, 1,1,2,2-tetrafluoroethoxy, 2-fluoroethoxy, 2-chloroethoxy, 2,2-difluoroethoxy, 2,2,2-trichloroethoxy; trichloromethoxy, 2-bromoethoxy, pentafluoroethyl, 3,3,3-trichloropropoxy, and 4,4,4-trichlorobutoxy.

[0155] The terms “carboxy” or “carboxyl” or “hydroxycarbonyl” refer, either on their own or as part of another substituent, to the group -CO2H. Therefore, a carboxyalkyl group is an alkyl group as defined above that has at least one substituent of -CO2H.

[0156] The term "alkoxycarbonyl" itself, or as part of another substituent, refers to a carboxyl group attached to an alkyl group, i.e., forming –C(=O)OR. e , where R e As defined above for alkyl groups.

[0157] The term "alkylcarbonyloxy" itself, or as part of another substituent, refers to –OC (=O)R e , where R e As defined above for alkyl groups.

[0158] Whenever the term “substituted” is used in this invention, it is intended to indicate that one or more hydrogen atoms on the atom specified in the use of “substituted” are selectively replaced by the specified group, provided that the replacement does not exceed the normal valence of the specified atom, and that the substitution produces a chemically stable compound, i.e., a compound sufficiently robust to withstand separation from the reaction mixture to a useful purity and formulation as a therapeutic agent.

[0159] Where a group can be optionally substituted, such a group can be substituted once or multiple times, and preferably once, twice or three times. The substituent can be selected from, for example, the group comprising halogen, hydroxyl, oxo, nitro, amide, carboxyl, amino, cyano, haloalkoxy and haloalkyl.

[0160] As used herein, terms such as “alkyl, aryl or cycloalkyl, each optionally substituted with…” or “alkyl, aryl or cycloalkyl optionally substituted with…” refer to optionally substituted alkyl, optionally substituted aryl and optionally substituted cycloalkyl.

[0161] Furthermore, when the group is divalent, i.e., has two single bonds for attaching to two other groups, its appearance can occur in either of two directions within the molecule, even if not specifically indicated in the structural formula or the definition of the R group. For example, –CH2- and –O-CH2- as part of R5 means that R5 can be represented, for example, by –O-CH2-O-CH2-, -CH2-O-CH2-O-, and –O-CH2-CH2-O-. In particular, for R5 and R6, any chemically feasible combination of the –CH2-, -O-CH2-, and –CH2-O- portions is contemplated in the context of this invention.

[0162] In the context of this invention, the term lipid refers to a chemically defined substance that is insoluble in water but particularly soluble in alcohols, ethers, and chloroform. Ionizable lipids or cationic lipids are lipids that typically consist of three parts: an amine head group, a linker moiety, and a hydrophobic tail. In the context of a compound or lipid, the term "ionizable" (or optionally cationic) means the presence of any uncharged group in said compound or lipid that is capable of generating ions (typically H+). + The ions dissociate and thus become positively charged. Alternatively, any uncharged group in the compound or lipid may generate electrons and thus become negatively charged.

[0163] In the context of this invention, the linker portion can be selected from a variety of different linkers; however, disulfide, ketal, and ether linkers are particularly preferred. Therefore, and to obtain their lipid characteristics, the compounds of this invention comprise lipid tails represented by R1 and R2, wherein the total number of carbon atoms in the two combined groups is at least 8, such as at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20. Thus, in the context of this invention, R1 can, for example, contain 3 carbon atoms, and R2 can contain 5 carbon atoms, thereby the total number of carbon atoms in the two combined groups is at least 8. This also means that R1 and R2 do not need to be the same, but in specific embodiments, they can be the same as each other.

[0164] The present invention provides two different classes of lipids: lipids in which the lipid tail is directly connected to the amide moiety (represented by formulas IVa, IVb and IVc), and lipids in which the lipid tail is connected to the amide moiety via a carboxylic acid-containing linker moiety (represented by formulas II and IIIa, IIIb and IIIc).

[0165] Therefore, in specific embodiments, the present invention provides a lipid, particularly an ionizable lipid as defined herein and represented by formula (II).

[0166]

[0167] in

[0168] R3 and R4 are each independently -C 1-6 Alkyl group; or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocycle; said heterocycle may further optionally contain one or more additional N atoms, and / or may optionally be substituted by 1-3 substituents selected from: -C 1-6 Alkyl; and

[0169] Each R5 and R6 is independently –CH2-.

[0170] Each R7 is independently selected from -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Alkyne group; wherein the -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Each of the ynyl groups may optionally be surrounded by 1 to 3 –O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Alkyne substitution; and the total number of C atoms in the two R7 moieties is at least 5;

[0171] m and n are each an independent integer selected from 1, 2, 3 and 4;

[0172] -

[0173] Each R8 and R9 is independently selected from –H, -C 1-6 Alkyl and –C 3-6 cycloalkyl;

[0174] Y is selected from -NH- and -O-;

[0175] Z is -C 1-6 Alkylene-.

[0176] In yet another embodiment, the present invention provides a lipid, particularly an ionizable lipid as defined herein and represented by any one of formula (IIIa), (IIIb) or (IIIc).

[0177]

[0178]

[0179] in

[0180] R3 and R4 are each independently -C 1-6 Alkyl group; or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocycle; said heterocycle may further optionally contain one or more additional N atoms, and / or may optionally be substituted by 1-3 substituents selected from: -C 1-6 Alkyl; and

[0181] Each R5 and R6 is independently –CH2-;

[0182] Each R7 is independently selected from -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Alkyne group; wherein the -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Each of the ynyl groups may optionally be surrounded by 1 to 3 –O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Alkyne substitution; and the total number of C atoms in the two R7 moieties is at least 5;

[0183] m and n are each an independent integer selected from 1, 2, 3 and 4;

[0184] Each R8 and R9 is independently selected from –H, -C 1-6 Alkyl and –C 3-6 cycloalkyl;

[0185] Y is selected from -NH- and -O-;

[0186] Z is -C 1-6 Alkylene-.

[0187] The present invention further provides a lipid, particularly an ionizable lipid as defined herein and selected from the list below:

[0188]

[0189] In yet another embodiment, the present invention provides a lipid, particularly an ionizable lipid as defined herein and represented by any one of formulas (IVa), (IVb) or (IVc).

[0190]

[0191] in

[0192] R1 and R2 are each independently selected from -H and -C. 1-20 Alkyl, -C 2-20 alkenyl and -C 2-20 Alkyne group; wherein the -C 1-20 Alkyl, -C 2-20 alkenyl and -C 2-20 Each of the ynyl groups may optionally be surrounded by 1 to 3 –O-(C=O)-R7, –O-(C=O)-R7, -C 1-20 Alkyl, -C 2-20 alkenyl and -C 2-20 Alkyne substitution; and the total number of C atoms in R1 and R2 is at least 8;

[0193] R3 and R4 are each independently -C 1-6 Alkyl group; or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocycle; said heterocycle may further optionally contain one or more additional N atoms, and / or may optionally be substituted by 1-3 substituents selected from: -C 1-6 Alkyl; and

[0194] Each R5 and R6 is independently –CH2-;

[0195] m and n are each an independent integer selected from 1, 2, 3 and 4;

[0196] Each R8 and R9 is independently selected from –H, -C 1-6 Alkyl and –C 3-6 cycloalkyl;

[0197] Y is selected from -NH- and -O-;

[0198] Z is -C 1-6 Alkylene-.

[0199] In another embodiment, the present invention provides a lipid, particularly an ionizable lipid as defined herein and selected from the list below:

[0200]

[0201] In yet another embodiment, the present invention provides a lipid, particularly an ionizable lipid as defined herein and represented by formula (V).

[0202]

[0203] in

[0204] R3 and R4 are each independently -C 1-6 Alkyl group; or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocycle; said heterocycle may further optionally contain one or more additional N atoms, and / or may optionally be substituted by 1-3 substituents selected from: -C 1-6 Alkyl; and

[0205] Each R5 and R6 is independently –CH2-;

[0206] Each R7 is independently selected from -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Alkyne group; wherein the -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Each of the ynyl groups may optionally be surrounded by 1 to 3 –O-(C=O)-R7, –O-(C=O)-R7, -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Alkyne substitution; and the total number of C atoms in the two R7 moieties is at least 5;

[0207] m and n are each an independent integer selected from 1, 2, 3 and 4;

[0208] Y is selected from -NH- and -O-;

[0209] Z is -C 1-6 Alkylene-.

[0210] The present invention further provides a lipid, particularly an ionizable lipid as defined herein and selected from the list below:

[0211]

[0212]

[0213] In yet another embodiment, the present invention provides a lipid, particularly an ionizable lipid as defined herein and represented by any one of formula (VIa) or (VIb).

[0214]

[0215] in

[0216] R3 and R4 are each independently -C 1-6 Alkyl group; or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocycle; said heterocycle may further optionally contain one or more additional N atoms, and / or may optionally be substituted by 1-3 substituents selected from: -C 1-6 Alkyl; and

[0217] Each R5 and R6 is independently –CH2-;

[0218] Each R7 is independently selected from -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Alkyne group; wherein the -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Each of the ynyl groups may optionally be surrounded by 1 to 3 –O-(C=O)-R7, –O-(C=O)-R7, -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Alkyne substitution; and the total number of C atoms in the two R7 moieties is at least 5;

[0219] m and n are each an independent integer selected from 1, 2, 3 and 4;

[0220] o and p are each an integer selected independently from 1 to 10;

[0221] Y is selected from -NH- and -O-;

[0222] Z is -C 1-6 Alkylene-.

[0223] In a very specific embodiment of the invention, one or more of the following are applicable:

[0224] R1 and R2 are each independently selected from -H and -C. 1-20 Alkyl, -C 2-20 alkenyl and -C 2-20 Alkyne group; wherein the -C 1-20 Alkyl, -C 2-20 alkenyl and -C 2-20 Each of the ynyl groups may optionally further comprise one or more heteroatoms and / or may be represented by 1 to 3 –O-(C=O)-R7, -C 1-20 Alkyl, -C 2-20 alkenyl and -C 2-20 Alkyne substitution; and the total number of C atoms in R1 and R2 is at least 8;

[0225] R1 and R2 are each independently selected from -H and -C. 1-20 Alkyl, -C 2-20 alkenyl and -C 2-20 Alkyne group; wherein the -C 1-20 Alkyl, -C 2-20 alkenyl and -C 2-20 Each of the ynyl groups may optionally be surrounded by 1 to 3 –O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 Alkyl, -C 2-20 alkenyl and -C 2-20 Alkyne substitution; and the total number of C atoms in R1 and R2 is at least 8;

[0226] R3 and R4 are each independently -C 1-6 Alkyl group; or R3 and R4 together with the N atom to which they are attached form a 5-10 membered aromatic or non-aromatic heterocycle; said heterocycle may further optionally contain one or more additional N atoms, and / or may optionally be substituted by 1-3 substituents selected from: -C 1-6 Alkyl; and

[0227] Each R5 and R6 is independently selected from -CH2- and -O-CH2-;

[0228] Each R5 and R6 is independently –CH2-;

[0229] Each R7 is independently selected from -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Alkyne group; wherein the -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Each of the ynyl groups may optionally further comprise one or more heteroatoms and / or may optionally be surrounded by 1 to 3 –O-(C=O)-R7, -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Alkyne substitution;

[0230] Each R7 is independently selected from -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Alkyne group; wherein the -C 1-20 Alkyl, -C 2-20 alkenyl, -C 2-20 Each of the ynyl groups may optionally be surrounded by 1 to 3 –O-(C=O)-R7, -(C=O)-O-R7, -C 1-20 Alkyl, -C 2-20 alkenyl, -C2-20 Alkyne substitution;

[0231] m and n are each an independent integer selected from 1, 2, 3 and 4;

[0232] -

[0233] Each R8 and R9 is independently selected from –H, -C 1-6 Alkyl and –C 3-6 cycloalkyl;

[0234] Y is selected from -NH- and -O-;

[0235] Z is -C 1-6 Alkylene-.

[0236] In another specific embodiment, the present invention provides a lipid, particularly an ionizable lipid as defined herein and selected from the list below:

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243] All lipids as defined herein can exist as different isomers / stereoisomers. In particular, lipids as defined herein can exist in trans or cis configurations, such as when they contain double bonds. In a preferred embodiment, lipids as defined herein exist in the cis configuration. In the context of this invention, the term 'cis' means that the functional groups are on the same side of the plane, while 'trans' means that they are on opposite sides.

[0244] In yet another embodiment, the present invention provides a lipid, particularly an ionizable lipid as defined herein; wherein the total number of C atoms in R1 and R2 is at least 14, such as at least 15, at least 17, at least 18, at least 19 or at least 20.

[0245] The present invention further provides a lipid, particularly an ionizable lipid as defined herein; wherein each R5 and R6 is independently –CH2-, i.e., both groups are –CH2-.

[0246] In another embodiment, the present invention provides a lipid, particularly an ionizable lipid as defined herein; wherein m and n are the same and are integers selected from 1, 2, 3, and 4; such as 1 or 2 or 3 or 4; preferably 2.

[0247] In yet another embodiment, the present invention provides a lipid, particularly an ionizable lipid as defined herein; wherein Y is -NH-.

[0248] In another aspect, the present invention provides lipid nanoparticles or lipid nanoparticle compositions comprising lipids, particularly ionizable lipids as defined herein.

[0249] In the context of this invention, the term lipid nanoparticles (LNPs), also known as solid lipid nanoparticles, refers to nanoparticles containing lipids. They are commonly used as drug delivery systems or pharmaceutical formulations. LNPs were first approved as drug delivery carriers in 2018 and are currently used in several RNA-based vaccine candidates. Lipid nanoparticles are typically spheres with an average diameter of 10 to 1000 nanometers and have a lipid core matrix capable of dissolving lipophilic molecules. The term lipid is used herein in a broader sense and includes triglycerides, diglycerides, monoglycerides, fatty acids, steroids (e.g., cholesterol), and waxes. Biomembrane lipids such as phospholipids, sphingomyelins, bile acids, and sterols are commonly used as stabilizers in LNPs.

[0250] As used herein, the term “nanoparticle” refers to any particle having a diameter that makes the particle, particularly nucleic acid, suitable for systemic, particularly intravenous administration, typically having a diameter of less than 1000 nanometers (nm), preferably less than 500 nm, and even more preferably less than 200 nm, such as, for example, a diameter of 50 nm to 200 nm; preferably a diameter of 80 nm to 160 nm.

[0251] Therefore, in the context of this invention, the nanoparticles disclosed herein further comprise one or more additional lipids, which may or may not act as stabilizers, such as phospholipids, sterols, and / or PEG lipids.

[0252] In the context of this invention, the term "PEG lipid" or alternatively "PEGylated lipid" refers to any suitable lipid modified with a PEG (polyethylene glycol) group. Particularly suitable PEG lipids in the context of this invention are characterized as C18-PEG lipids, C14-PEG lipids (e.g., DMG-PEG or DMG-PEG2000), or C16-PEG lipids.

[0253] C18-PEG lipids contain a polyethylene glycol portion defining the molecular weight of the lipid, and a fatty acid tail containing 18 C- atoms. In a specific embodiment, the C18-PEG2000 lipid is selected from the following list: (distearyl)-based PEG2000 lipids, such as DSG-PEG2000 lipid (2-distearyl-rac-glycero-3-methoxy polyethylene glycol-2000) or DSPE-PEG2000 lipid (1,2-distearyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]); or (dioleoyl)-based PEG2000 lipids, such as DOG-PEG2000 lipid (1,2-dioleoyl-rac-glycerol) or DOPE-PEG2000 lipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]).

[0254]

[0255] C14-PEG lipids contain a polyethylene glycol moiety defining the molecular weight of the lipid, and a fatty acid tail comprising 14 carbon atoms. In a specific embodiment, the C14-PEG2000 lipid is based on a myristoyl group, i.e., having two C14 tails, such as those selected from the list including: (myristoyl)-based PEG2000 lipids, such as DMG-PEG2000 lipid (1,2-myristoyl-rac-glycero-3-methoxy polyethylene glycol-2000) or 2-myristoyl-sn-glycero-3-phosphoethanolamine diol-2000 (DMPE-PEG2000).

[0256]

[0257] In the context of this invention, the term "phospholipid" refers to a lipid molecule consisting of two hydrophobic fatty acid "tails" and a hydrophilic "head" consisting of a phosphate group. These two components are most commonly linked together by glycerol molecules; therefore, the phospholipids of this invention are preferably glycerol-phospholipids. Furthermore, the phosphate group is typically modified with simple organic molecules such as choline (i.e., providing phosphocholine) or ethanolamine (i.e., providing phosphoethanolamine).

[0258] In the context of this invention, suitable phospholipids may be selected from the list including: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPC) PPC), 1,2-distearyl-sn-glycero-3-phosphocholine (DSPC), 1,2-bis(undecanoyl)-sn-glycero-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholestanoyl-semisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C 16-hemolytic PC), 1,2-dilinoyl-sn-glycero-3-phosphate choline, 1,2-disarachidoyl-sn-glycero-3-phosphate choline, 1,2-bis(docosahexaenoyl)-sn-glycero-3-phosphate choline, 1,2-diphydanoyl-sn-glycero-3-phosphate ethanolamine (ME16.0PE), 1,2-distearateyl-sn-glycero-3-phosphate ethanolamine, 1,2-di- Linoleoyl-sn-glycero-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphate ethanolamine, 1,2-diarachidoyl-sn-glycero-3-phosphate ethanolamine, 1,2-bis(docohexanoyl)-sn-glycero-3-phosphate ethanolamine, sodium salt of 1,2-dioleoyl-sn-glycero-3-phosphate-rac-(1-glycerol) (DOPG), sphingomyelin, and mixtures thereof.

[0259] In a more specific embodiment, the phospholipid is selected from the list including: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and mixtures thereof.

[0260] In the context of this invention, the term "sterol," also known as steroidal alcohols, refers to a subgroup of steroidal compounds that are naturally occurring in plants, animals, and fungi or that can be produced by certain bacteria. In the context of this invention, any suitable sterol may be used, such as those selected from the list comprising: cholesterol, ergosterol, campesterol, oxosterol, antrosterol, nicasterol, sitosterol, and stigmasterol; preferably cholesterol.

[0261] In specific embodiments of the present invention, one or more of the following are applicable:

[0262] -The LNP comprises approximately 35 mol% and 65 mol% of the ionizable lipids, and between 35 mol% and 65 mol%;

[0263] -The LNP contains approximately 5 mol% and 25 mol% of the phospholipids, and between 5 mol% and 25 mol% of the phospholipids;

[0264] - The LNP contains approximately 0.5 mol% and 3.0 mol% of the PEG lipid and between 0.5 mol% and 3.0 mol%;

[0265] The balance is the amount of the sterol.

[0266] In yet another embodiment of the invention, the lipid nanoparticles or lipid nanoparticle compositions as defined herein further comprise cargo molecules, such as pharmaceutically active agents (e.g., small molecules) or biomolecules, such as peptides, proteins, or nucleic acids. In a specific embodiment, the cargo may be a nucleic acid, such as DNA or RNA; preferably mRNA. In yet another specific embodiment, the cargo may be a TLR agonist, such as, for example, the TLR3 agonist polyI:C or the TLR9 agonist CpG.

[0267] Before being loaded into lipid nanoparticles, the cargo molecules can be further modified to induce overall polyanionic properties of the molecules. This can be done, for example, by bonding them to the Glu10 moiety illustrated in the Examples section. The Glu10 moiety is a 10-glutamic acid moiety that increases the polyanionic properties of the molecules to which it is attached.

[0268] Therefore, the lipid nanoparticles and lipid nanoparticle compositions of the present invention are particularly suitable for intracellular delivery of their cargo molecules. Thus, the present invention provides the use of lipid nanoparticles and lipid nanoparticle compositions as defined herein for intracellular delivery of cargo molecules.

[0269] In specific implementations, lipid nanoparticles or lipid nanoparticle compositions as defined herein further comprise nucleic acids, preferably mRNA.

[0270] In the context of this invention, "nucleic acid" refers to deoxyribonucleic acid (DNA) or preferably ribonucleic acid (RNA), more preferably mRNA. According to the invention, nucleic acids include genomic DNA, cDNA, mRNA, recombinant-derived molecules, and chemically synthesized molecules. According to the invention, nucleic acids can be in the form of single-stranded or double-stranded molecules that are linearly or covalently closed to form a loop. Nucleic acids (e.g., in the form of RNA that can be prepared by in vitro transcription from a DNA template) can be used for introduction into cells, i.e., transfection of cells. Furthermore, RNA can be modified prior to application by sequence stabilization, capping, and / or polyadenylation.

[0271] In the context of this invention, the term "RNA" refers to a molecule comprising ribonucleotide residues and preferably consisting entirely or substantially of ribonucleotide residues. "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2'-position of the β-D-furanose group. The term includes double-stranded RNA, single-stranded RNA, isolated RNA, such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinant RNA, and modified RNA that differs from naturally occurring RNA by adding, deleting, substituting, and / or altering one or more nucleotides. Such alterations may include the addition of non-nucleotide material, such as adding to one or more ends of the RNA, or internally, for example, adding at one or more nucleotides of the RNA. The nucleotides in the RNA molecule may also contain non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs. Nucleic acids may be contained in a vector. As used herein, the term "vector" includes any vector known to those skilled in the art, including plasmid vectors, granular vectors, phage vectors such as λ phage, viral vectors such as adenovirus vectors or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes, or analogs of naturally occurring RNA.

[0272] According to the present invention, the term "RNA" includes and preferably refers to "mRNA," which means "messenger RNA" and relates to "transcriptions" that can be produced using DNA as a template and encode peptides or proteins. mRNA typically contains a 5' untranslated region (5'-UTR), a protein or peptide coding region, and a 3' untranslated region (3'-UTR). mRNA has a limited half-life in cells and in vitro. Preferably, mRNA is produced by in vitro transcription using a DNA template. In one embodiment of the invention, RNA is obtained by in vitro transcription or chemical synthesis. In vitro transcription methods are known to those skilled in the art. For example, there are various commercially available in vitro transcription kits.

[0273] In another aspect, the present invention provides a pharmaceutical composition comprising one or more LNPs as defined herein and pharmaceutically acceptable agents, such as carriers, excipients, etc. Such pharmaceutical compositions are particularly suitable as vaccines. Therefore, the present invention also provides a vaccine comprising one or more LNPs according to the present invention.

[0274] In the context of this invention, the term "vaccine" as used herein refers to any preparation designed to provide adaptive immunity (antibody and / or T-cell response) against a disease. For this purpose, a vaccine, as referred to herein, contains at least one nucleic acid molecule, such as an mRNA molecule encoding an antigen against which an adaptive immune response is generated. This antigen may be present in an attenuated or killable form of a microorganism, a protein or peptide, or an antigen encoding a nucleic acid. In the context of this invention, an antigen refers to a protein or peptide recognized as foreign by the host's immune system, thereby stimulating the production of antibodies against it, with the aim of combating such an antigen. Vaccines can be prophylactic (e.g., preventing or mitigating the effects of future infection with any natural or "wild" pathogen) or therapeutic (e.g., actively treating or alleviating symptoms of an ongoing disease). The administration of a vaccine is referred to as vaccination.

[0275] The vaccine of the present invention can be used to induce an immune response, particularly an immune response against disease-associated antigens or cells expressing disease-associated antigens, such as an immune response against cancer. Therefore, the vaccine can be used for prophylactic and / or therapeutic treatment of diseases involving disease-associated antigens or cells expressing disease-associated antigens, such as cancer. Preferably, the immune response is a T-cell response. In one embodiment, the disease-associated antigen is a tumor antigen. The RNA-encoded antigen contained in the nanoparticles described herein is preferably a disease-associated antigen, or evokes an immune response against a disease-associated antigen or cells expressing a disease-associated antigen.

[0276] The present invention also provides LNPs, pharmaceutical compositions, and vaccines according to the invention for use in human or veterinary medicines. Use of the LNPs, pharmaceutical compositions, and vaccines according to the invention in human or veterinary medicines is also contemplated. Finally, the present invention provides a method for the prevention and treatment of disorders in humans and animals by administering the LNPs, pharmaceutical compositions, and vaccines according to the invention to a subject in need.

[0277] The present invention further provides the use of the LNP, pharmaceutical composition, or vaccine according to the invention for the immunogenic delivery of said one or more nucleic acid molecules. Therefore, the LNP, pharmaceutical composition, and vaccine of the present invention are very useful in treating certain human and animal disorders. Thus, the present invention provides the LNP, pharmaceutical composition, and vaccine of the present invention for treating cancer or infectious diseases.

[0278] The lipid nanoparticles of the present invention can be prepared according to the scheme specified in the Examples section. More generally, LNPs can be prepared using methods including the following:

[0279] - Prepare a first alcohol composition comprising the ionizable lipid, the phospholipid, the sterol, the PEG lipid, and a suitable alcohol solvent;

[0280] - Prepare a second aqueous composition comprising one or more nucleic acids and an aqueous solvent;

[0281] - The first composition and the second composition are mixed in a microfluidic mixing device.

[0282] More specifically, lipid components are combined at appropriate concentrations in an alcohol medium such as ethanol. An aqueous composition containing nucleic acids is then added, and subsequently loaded into a microfluidic mixing device.

[0283] The purpose of microfluidic mixing is to achieve thorough and rapid mixing of multiple samples (i.e., lipid and nucleic acid phases) in a microdevice. Such sample mixing is typically achieved by enhancing the diffusion effect between different material flows. Several microfluidic mixing devices can be used, such as those reviewed, for example, in Lee et al., 2011. A particularly suitable microfluidic mixing device according to the invention is NanoAssemblr from Precision Nanosystems.

[0284] Other techniques suitable for preparing the LNP of the present invention include dispersing the components in a suitable dispersion medium such as an aqueous solvent and an alcohol solvent, and applying one or more of the following methods: ethanol dilution method, simple hydration method, ultrasonic treatment, heating, vortexing, ether injection method, French press method, bile acid method, Ca 2+Fusion method, freeze-thaw method, reverse evaporation method, T-joint mixing, microfluidic hydrodynamic focusing, staggered herringbone mixing, etc.

[0285] The ionizable lipids of the present invention can be prepared according to the reaction schemes provided in the examples below, but those skilled in the art will understand that these are only for illustrative purposes, and the compounds of the present invention can be prepared by any of several standard synthetic methods commonly used by those skilled in the art of organic chemistry. Example

[0286] Example 1: Preparation of lipids

[0287] 1. General Information

[0288] Unless otherwise specified, all glassware was oven-dried before use, and all reactions were performed under an argon atmosphere using standard Schlenk techniques. Drying solvents were purchased from Acros Organics or Sigma-Aldrich and used without further purification. Unless otherwise specified, all reagents were commercially available and used without further purification. Reaction progress was monitored by thin-layer chromatography (TLC) on aluminum plates coated with 0.2 mm thick Kieselgel F254. Visualization was achieved by UV light (254 nm) or by staining with potassium permanganate. Rapid column chromatography was performed using silica gel 60 (230-400 mesh, Merck ans co.). Mass spectra were obtained using Finnigan MAT 8200 (70 eV) or Agilent 5973 (70 eV) via electrospray ionization (ESI) or electron impact ionization (EI). All ¹H NMR and ¹³C NMR spectroscopy were recorded on a Bruker AV-400 in chloroform-d1 or DMSO-d6. Solvent peaks were used as internal standards (CDCl₃: 1 H = 7.26 ppm 13 C = 77.16 ppm; CD3SOCD3: 1 H = 2.50 ppm 13 Chemical shift (C = 39.52 ppm) is expressed in parts per million (ppm) with tetramethylsilane as a reference. Coupling constants are expressed in Hz. 1 H NMR splitting modes are designated as singlet (s), broad peak (brd), doublet (d), triplet (t), quartet (q), quintet (p), sextet (se), septet (sep), octet (o), or combinations thereof. Uninterpretable splitting modes are designated as multiply (m).

[0289] 2. Lipid synthesis

[0290] 2.1 The general route for synthesizing lipids represented by the structure of Formula I, and more specifically Formula IVa, is shown below.

[0291]

[0292] Synthesis of Compound 3

[0293] First, amine 2 (1.0 equivalent) and Et3N (1.5 equivalent) were dissolved in CHCl3 / hexane / THF (1 / 1 / 1). Then, the prepared solution was added dropwise to a stirred solution of compound 1 (Amano et al., 2017) (3.0 equivalent) in CH2Cl2 at 0 °C. The resulting mixture was vigorously stirred and allowed to warm to room temperature for 2 h. The solvent was then removed under vacuum, and the remaining residue was dissolved in CH2Cl2, washed with saturated citric acid (aqueous solution) and brine, dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel column chromatography (hexane / ethyl acetate = 15:1 to 10:1) to provide compound 3.

[0294]

[0295] Yield: 62%. Colorless oily substance. 1 H NMR (400MHz, chloroform-d) δ8.29 (d, J=9.2Hz, 2H, H17&H19), 7.40 (d, J=9.3Hz, 2H, H1 6&H20),4.54(t,J=6.6Hz,2H,OCH2),4.34(t,J=6.4Hz,2H,OCH2),3.18(brd, 4H,H22&H34),3.03(t,J=6.6Hz,2H,SCH2),2.97(t,J=6.4Hz,2H,SCH2),1.54 -1.48(m,4H,H23&H35),1.25(brd,36H),0.88(t,J=6.7Hz,6H,H33&H45)ppm. 13 C NMR (100MHz, chloroform-d) δ 156.0 (C=O), 155.6 (Ar-C, quaternary), 152.5 (C=O), 125.5 (ArC-H), 121.9 (ArC-H), 67.0 (C2 or C7), 62.8 (C2 or C7), 47.8 (C22 or C34), 47.2 (C22 or C34), 38.2 (C3 or C6), 36.9 (C3 or C6), 32.1, 29.81, 29.79, 29.76, 29.6, 29.5, 28.8 (C23 or C35), 28.3 (C23 or C35), 27.0, 22.8, 14.3 (C33 & C45) ppm. LRMS (ESI) (m / z): [M+H]+ (C 36 H 63 Calculated value of O7N2S2: 699.4, measured value: 699.4.

[0296]

[0297] Yield: 58%. Colorless oily substance. 1 H NMR (400MHz, chloroform-d) δ8.28 (d, J=9.2Hz, 2H, H53&H55), 7.40 (d, J=9.2Hz, 2H, H5 2&H56),4.54(t,J=6.6Hz,2H,OCH2),4.34(t,J=6.5Hz,2H,OCH2),3.21-3.15( m,4H,H16&H34),3.03(t,J=6.6Hz,2H,SCH2),2.97(t,J=6.4Hz,2H,SCH2),1.5 3-1.49(m,4H,H17&H35),1.25(brd,60H),0.88(t,J=6.7Hz,6H,H33&H51)ppm. 13 CNMR (100MHz, chloroform-d) δ 156.0 (C=O), 155.6 (Ar-C, quaternary), 152.4 (C=O), 125.5 (ArC-H), 121.9 (ArC-H), 67.0 (C2 or C7), 62.8 (C2 or C7), 47.8 (C16 or C34), 47.2 (C16 or C34), 38.2 (SCH2), 36.9 (SCH2), 32.1, 29.86, 29.83, 29.81, 29.77, 29.57, 29.51, 28.8 (C17 or C35), 28.3 (C17 or C35), 27.0, 22.8, 14.3 (C33 & C51) ppm. LRMS (ESI) (m / z): [M+H] + (C 48 H 87 Calculated value of O7N2S2: 867.6, measured value: 867.5.

[0298] Synthesis of Compound 5

[0299] Amine 4 (1.2 equivalents) was added to a stirred solution of compound 3 (1.0 equivalents) in CH2Cl2 at room temperature, followed by Et3N (1.5 equivalents). The reaction mixture was stirred vigorously at room temperature for 2 h. The organic phase was first washed with saturated Na2CO3 (aqueous solution) until it turned grayish-white, then washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel column chromatography (hexane / ethyl acetate = 3:1 to 1:1, then changed to CH2Cl2 / CH3OH = 15:1) to provide compound 5. The name of compound 5 is given below its structure.

[0300]

[0301] Yield: 75%. Colorless oily substance. 1 H NMR (400MHz, chloroform-d) δ5.48(brd,1H,H12),4.34-4.29(m,4H,OCH2),3.30(q,J=5.8Hz,2H,H13),3.21-3.14(m,4H,H20&H32),2.95-2.90(m,4H ,H5&H8),2.48(t,J=6.2Hz,2H,H14),2.28(s,6H,H18&H19),1.54-1.47(m,4H,H21&H33),1.26(s,36H),0.88(t,J=6.7Hz,6H,H31&H43)ppm. 13 C10 NMR (100MHz, chloroform-d) δ 156.0 (C2 & C11), 63.0 (OCH2), 62.6 (OCH2), 58.3 (C14), 47.8 (C20 or C32), 47.2 (C20 or C32), 45.2 (C18 & C19), 38.4 (C13), 38.2 (C5 or C8), 38.0 (C5 or C8), 32.1, 29.82, 29.79, 29.76, 29.6, 29.5, 28.8 (C21 or C33), 28.3 (C21 or C33), 27.0, 22.8, 14.3 (C31 & C43) ppm. LRMS (ESI) (m / z): [M+H] + (C 34 H 70 Calculated value of O4N3S2: 648.5, measured value: 648.4.

[0302]

[0303] Yield: 55%. Colorless oily substance. 1 ¹H NMR (400MHz, chloroform-d)δ 1¹H NMR (400MHz, chloroform-d) δ 4.33–4.28 (m, 4H, H₂ & H₇), 3.60 (brd, 2H), 3.21–3.14 (brd, 4H, H₂₄ & H₃₆), 3.07 (brd, 2H), 2.94–2.88 (m, 4H, H₃ & H₆), 1.62–1.25 (m, 5₅H), 0.88 (t, J = 6.7Hz, 6H, H₃₅ & H₄₇) ppm. 13 C10 NMR (100MHz, chloroform-d) δ 156.0 (C9 & C10), 63.0 (C2 & C7), 47.8 (C24 or C36), 47.2 (C24 or C36), 38.1 (C3 & C6), 32.1, 29.82, 29.79, 29.76, 29.6, 29.5, 28.8, 28.3, 27.0, 22.8, 14.3 (C35 & C47) ppm. LRMS (ESI) (m / z): [M+H] + (C 38 H 78 Calculated value of O4N3S2: 704.5, measured value: 704.5.

[0304]

[0305] Yield: 51%. Colorless solid. 1 H NMR (400MHz, chloroform-d) δ4.34-4.29(m,4H,H4&H9),3.32(brd,2H,H13),3.21-3.14(m,4H,H20&H32),2.95-2.91(m,4H,H5&H8),2.77-2.64(m ,6H,H14,H44&H49),1.71(brd,4H),1.62(brd,4H),1.51(t,J=7.3Hz,4H,H21&H33),1.26(brd,36H),0.88(t,J=6.7Hz,6H,H49&H55)ppm. 13C10 NMR (100MHz, chloroform-d) δ 156.0 (C2 & C11), 63.0 (OCH2), 62.7 (OCH2), 56.8 (C14), 55.4 (C44 & C49), 47.8 (C20 or C32), 47.2 (C20 or C32), 38.1 (C5 or C8 & C13), 38.0 (C5 or C8), 32.1, 29.81, 29.79, 29.76, 29.57, 29.50, 28.8 (C21 or C33), 28.3 (C21 or C33), 27.1 (C47 or C48), 27.0 (C47 or C48), 22.8, 14.3 (C31 & C43) ppm. LRMS (ESI) (m / z): [M+H] + (C 38 H 76 Calculated value of O4N3S2: 702.5, measured value: 702.5.

[0306]

[0307] Yield: 81%. Colorless oily substance. 1 H NMR (400MHz, chloroform-d) δ5.48(s,1H,H12),4.34-4.29(m,4H,H4&H9),3.30-3.28(m,2H,H13),3.21-3.14(m,4H,H20&H32),2.95-2.90(m,4H, H5&H8),2.48-2.46(m,2H,H14),2.28(s,6H,H18&H19),1.53-1.49(m,4H,H21&H33),1.25(brd,60H),0.88(t,J=6.7Hz,6H,H49&H55)ppm. 13 C10 NMR (100MHz, chloroform-d) δ 156.0 (C2 & C11), 63.0 (OCH2), 62.6 (OCH2), 58.3 (C14), 47.8 (C20 or C32), 47.2 (C20 or C32), 45.2 (C18 & C19), 38.4 (C13), 38.2 (C5 or C8), 38.0 (C5 or C8), 32.1, 29.86, 29.83, 29.81, 29.77, 29.6, 29.5, 28.8 (C21 or C33), 28.3 (C21 or C33), 27.0, 22.8, 14.3 (C49 & C55) ppm. LRMS (ESI) (m / z): [M+H] + (C 46 H 94 Calculated value of O4N3S2: 816.7, measured value: 816.6.

[0308]

[0309] Yield: 89%. Colorless oily substance. 1 H NMR(400MHz, chloroform-d)δ5.30(brd,1H,H12),4.34-4.28(m,4H,H4&H9),3.21-3.1 1(m,6H,H13,H20&H32),3.02-2.96(m,2H,H18&H19),2.95-2.90(m,4H,H5&H8 ),2.56(t,J=6.2Hz,2H,H14),1.54-1.47(m,4H,H21&H33),1.25(brd,60H),1 .00(d,J=6.6Hz,12H,H56,H57,H58&H59),0.88(t,J=6.7Hz,6H,H49&H55)ppm. 13 C NMR (100MHz, chloroform-d) δ 156.0 (C2 & C11), 63.0 (C4 or C9), 62.6 (C4 or C9), 48.1 (C18 & C19), 47.8 (C20 or C32), 47.2 (C20 or C32), 43.8 (C14), 40.3 (C13), 38.2 (C5 or C8), 38.1 (C5 or C8), 32.1, 29.86, 29.83, 29.81, 29.78, 29.58, 29.52, 28.8 (C21 or C33), 28.3 (C21 or C33), 27.0, 22.8, 20.9 (C56, C57, C58 & C59), 14.3 (C49 & C55) ppm. LRMS(ESI)(m / z): [M+H] + (C 50 H 102 Calculated value of O4N3S2: 872.7, measured value: 872.6.

[0310]

[0311] Yield: 70%. Colorless solid. 1H NMR (400MHz, chloroform-d) δ4.34-4.29(m,4H,H4&H9),3.37(brd,2H,H13),3.21-3.14(m,4H,H20&H32),2.95-2.91(m,4H,H5&H8),2.84-2.67(m,6H ,H14,H56&H61),1.75(brd,4H),1.64(brd,4H),1.50(q,J=10.8,6.5Hz,4H,H21&H33),1.25(brd,60H),0.88(t,J=6.7Hz,6H,H49&H55)ppm. 13 C NMR (100MHz, chloroform-d) δ 156.0 (C2 & C11), 63.0 (OCH2), 62.8 (OCH2), 56.9 (C14), 55.5 (C56 & C61), 47.8 (C20 or C32), 47.2 (C20 or C32), 38.1 (C5 or C8 & C13), 38.0 (C5 or C8), 32.1, 29.86, 29.83, 29.81, 29.78, 29.6, 29.5, 28.8 (C21 or C33), 28.3 (C21 or C33), 27.1 (C60 or C59), 27.0 (C60 or C59), 22.8, 14.3 (C49 & C55) ppm. LRMS(ESI)(m / z): [M+H] + (C 50 H 100 Calculated value of O4N3S2: 870.7, measured value: 870.7.

[0312] 2.2 The general route for synthesizing lipids represented by the structure of Formula I or more specifically Formula IVb is shown below.

[0313]

[0314] Synthesis of Compound 7

[0315] Compound 6 (Shenoi et al., 2012) (3.58 g, 21.8 mmol, 1.0 equivalent) and a solution of Et3N (9.1 mL, 65.4 mmol, 3.0 equivalent) in CH2Cl2 (20 mL) were added dropwise to a stirred solution of 4-nitrobenzene chloroformate (11.0 g, 54.5 mmol, 2.5 equivalent) in CH2Cl2 (80 mL). The reaction mixture was stirred vigorously and allowed to warm to room temperature over 12 h. The reaction was then quenched with saturated Na2CO3 (aqueous solution). The organic phase was separated and washed with saturated Na2CO3 (aqueous solution) until it turned grayish-white, then washed with brine, dried over Na2SO4, filtered, and concentrated to give compound 7 (6.00 g, 56% yield) as a pale yellow solid, which was used without further purification.

[0316] Synthesis of Compound 8

[0317] (1) Amine 4 (1.0 equivalent) was added dropwise to a stirred solution of compound 7 (3.0 equivalent) and Et3N (3.0 equivalent) in CH2Cl2 at 0 °C. The reaction mixture was stirred vigorously at 0 °C for 5 h. The solvent was then removed under reduced pressure. The residue was dissolved in a minimal amount of Et2O and cooled at 0 °C. After 6 h, excess compound 7 began to precipitate from the solution. The solution was then decanted and concentrated under reduced pressure. The resulting crude product was subjected to the above procedure twice more to precipitate as much compound 7 as possible from the mixture. In this way, >80% of compound 7 can be recycled and used.

[0318] (2) After three precipitations, the mixture was dissolved in CH2Cl2. Et3N (8.0 equivalents) and amine 2 pre-dissolved in CHCl3 / hexane / THF (1.5 to 3.0 equivalents, depending on the purity of the resulting mixture) were added to the solution. The reaction mixture was stirred vigorously at room temperature for 5 h.

[0319] (3) Then add Ac2O (5.0 equivalents) to the mixture and stir the solution again at room temperature for 5 hours. Note: This step is to convert any excess amine 2 into its acetamide, otherwise amine 2 will be very difficult to remove from the final product.

[0320] (4) The solvent was then removed under reduced pressure and redissolved in ethyl acetate. The organic phase was first washed with saturated Na₂CO₃ (aqueous solution) until it turned grayish-white, then washed with brine, dried over Na₂SO₄, filtered, and concentrated. The residue was purified by silica gel column chromatography (CH₂Cl₂ / CH₃OH (+0.1% Et₃N) = 30:1 to 15:1) to provide compound 8. The name of compound 8 is given below its structure.

[0321]

[0322] Yield: 28%. Colorless oily substance. 1 H NMR(400MHz, chloroform-d)δ5.60(s,1H,H12),4.20-4.15(m,4H,H2&H8),3.64-3 .60(m,4H,H3&H7),3.28-3.23(m,2H,H16),3.19-3.14(m,4H,H23&H24),2 .42-2.39(m,2H,H17),2.22(s,6H,H19&H20),1.53-1.47(m,4H,H25&H36) ,1.36(s,6H,H14&H15),1.26(brd,36H),0.89-0.86(t,6H,H35&H46)ppm. 13 C10 NMR (100MHz, chloroform-d) δ 156.5 (C9 & C11), 100.1 (C5), 64.5 (C2 or C8), 64.3 (C2 or C8), 59.6 (C3 or C7), 59.5 (C3 or C7), 58.5 (C17), 47.7 (C23 or C24), 47.1 (C23 or C24), 45.5 (C19 & C20), 38.7 (C16), 32.1, 29.80, 29.76, 29.61, 29.49, 28.8, 28.3, 27.0, 25.0 (C14 & C15), 22.8 (C34 & C45), 14.3 (C35 & C46) ppm. LRMS (ESI) (m / z): [M+H] + (C 37 H 76 Calculated value of O6N3: 658.5, measured value: 658.5

[0323]

[0324] Yield: 21%. Colorless oily substance. 1¹H NMR (400MHz, chloroform-d) δ 5.53 (s, 1H, H12), 4.20–4.15 (m, 4H, OCH2), 3.64–3.60 (m, 4H, OCH2), 3.21–3.11 (m, 6H, H23, H24 & H16), 2.99 (p, J = 6.6Hz, 2H, H20 & H19), 2.55 (t, J =6.5Hz,2H,H17),1.54-1.47(m,4H,H25&H36),1.36(s,6H,H14&H15),1.25(brd,36 H), 0.99 (d, J = 6.6Hz, 12H, H47, H48, H49 & H50), 0.88 (t, J = 6.7Hz, 6H, H49 & H55) ppm. 13 C10 NMR (100MHz, chloroform-d) δ 156.4 (C9 & C11), 100.1 (C5), 64.5 (OCH2), 64.2 (OCH2), 59.5 (OCH2), 48.3 (C19 & C20), 47.7 (C23 or C24), 47.1 (C23 or C24), 44.1 (C17), 40.7 (C16), 32.1, 29.81, 29.78, 29.62, 29.50, 28.8 (C25 or C36), 28.3 (C25 or C36), 27.0, 25.0 (C14 & C15), 22.8, 20.9 (C47, C48, C49 & C50), 14.3 (C35 & C46) ppm. LRMS(ESI)(m / z): [M+H] + (C 41 H 84 Calculated value of O6N3: 714.6, measured value: 714.6.

[0325]

[0326] Yield: 25%. Colorless oily substance. 1 H NMR (400MHz, chloroform-d) δ5.64(s,1H,H12),4.20-4.16(m,4H,OCH2),3.65-3.61(m,4H,OCH2),3.25-3.14(m,6H,H23,H24&H16),2.64(brd,6H,H17,H47 &H52),1.59(brd,8H,H48,H49,H50&H51),1.54-1.47(m,4H,H25&H36),1. 37(s,6H,H14&H15),1.25(brd,36H),0.88(t,J=6.7Hz,6H,H35&H46)ppm. 13C10 NMR (100MHz, chloroform-d) δ 156.8 (C9 or C11), 156.4 (C9 or C11), 100.2 (C5), 64.5 (OCH2), 64.2 (OCH2), 59.6 (OCH2), 56.8 (C17), 55.4 (C47 & C52), 47.8 (C23 or C24), 47.2 (C23 or C24) 38.8 (C16, inferred from HSQC), 32.1, 29.82, 29.78, 29.6, 29.5, 28.8 (C25 or C36), 28.3 (C25 or C36), 27.1 (C51 or C50), 27.0 (C51 or C50), 25.0 (C14 & C15), 22.8, 14.3 (C35 & C46) ppm. LRMS(ESI) (m / z): [M+H] + (C 41 H 82 Calculated value of O6N3: 712.6, measured value: 712.5.

[0327]

[0328] Yield: 21%. Colorless solid. 1 H NMR(400MHz, chloroform-d)δ5.60(s,1H,H12),4.20-4.15(m,4H,H2&H8),3.64-3 .60(m,4H,H3&H7),3.28-3.23(m,2H,H16),3.19-3.14(m,4H,H23&H24),2 .42-2.39(m,2H,H17),2.22(s,6H,H19&H20),1.52-1.48(m,4H,H25&H36) ,1.36(s,6H,H14&H15),1.27(brd,60H),0.89-0.86(m,6H,H52&H58)ppm. 13 C10 NMR (100MHz, chloroform-d) δ 156.5 (C9 & C11), 100.1 (C5), 64.5 (C2 or C8), 64.3 (C2 or C8), 59.6 (C3 or C7), 59.5 (C3 or C7), 58.5 (C17), 47.7 (C23 or C24), 47.1 (C23 or C24), 45.5 (C19 & C20), 38.7 (C16), 32.1, 29.86, 29.83, 29.81, 29.78, 29.6, 29.5, 28.8, 28.3, 27.0, 25.0 (C14 & C15), 22.8 (C51 & C57), 14.3 (C52 & C58) ppm. LRMS(ESI)(m / z): [M+H] +(C 49 H 100 Calculated value of O6N3: 826.7, measured value: 826.7

[0329]

[0330] Yield: 38%. Colorless oily substance. 1 ¹H NMR (400MHz, chloroform-d) δ 5.52 (s, 1H, H12), 4.20–4.15 (m, 4H, OCH2), 3.64–3.60 (m, 4H, OCH2), 3.21–3.11 (m, 6H, H23, H24 & H16), 3.02–2.95 (m, 2H, H20 & H19), 2.55 (t, J = 6 .6Hz,2H,H17),1.54-1.47(m,4H,H25&H36),1.36(s,6H,H14&H15),1.25(brd,60H ), 0.99 (d, J = 6.6Hz, 12H, H47, H48, H49 & H50), 0.88 (t, J = 6.7Hz, 6H, H49 & H55) ppm. 13 C10 NMR (100MHz, chloroform-d) δ 156.8 (C9 or C11), 156.4 (C9 or C11), 100.1 (C5), 64.5 (OCH2), 64.2 (OCH2), 59.5 (OCH2), 48.3 (C19 & C20), 47.7 (C23 or C24), 47.1 (C23 or C24), 44.1 (C17) 40.7 (C16), 32.1, 29.85, 29.82, 29.80, 29.78, 29.6, 29.5, 28.8 (C25 or C36), 28.3 (C25 or C36), 27.0, 25.0 (C14 & C15), 22.8, 20.9 (C47, C48, C49 & C50), 14.3 (C56 & C62) ppm. LRMS(ESI) (m / z): [M+H] + (C 53 H 108 Calculated value of O6N3: 882.8, measured value: 882.7.

[0331]

[0332] Yield: 22%. Colorless oily substance. 1H NMR (400MHz, chloroform-d) δ5.59 (s, 1H, H12), 4.20-4.16 (m, 4H, OCH2), 3.64-3.61 (m,4H,OCH2),3.24-3.16(m,6H,H23,H24&H16),2.63-2.57(m,6H,H17,H47 &H52),1.61-1.56(m,8H,H48,H49,H50&H51),1.54-1.47(m,4H,H25&H36), 1.37(s,6H,H14&H15),1.25(brd,60H),0.87(t,J=6.8Hz,6H,H58&H64)ppm. 13 C NMR (100MHz, chloroform-d) δ 156.7 (C9 or C11), 156.4 (C9 or C11), 100.2 (C5), 64.5 (OCH2), 64.2 (OCH2), 59.6 (OCH2), 56.7 (C17), 55.4 (C47 & C52), 47.7 (C23 or C24), 47.1 (C23 or C24), 38. 8 (C16), 32.1, 29.85, 29.82, 29.80, 29.78, 29.6, 29.5, 28.8 (C25 or C36), 28.4, 28.3 (C25 or C36), 27.1 (C51 or C50), 27.0 (C51 or C50), 25.0 (C14 & C15), 22.8, 14.3 (C58 & C64) ppm. LRMS (ESI) (m / z): [M+H] + (C 53 H 106 Calculated value of O6N3: 880.8, measured value: 880.7.

[0333] 2.3 The general route for synthesizing lipids represented by the structures of Formula I, and more specifically Formula IIIa, is shown below.

[0334]

[0335] Synthesis of Compound 12

[0336] At 0 °C, Et3N (5.3 mL, 38.1 mmol, 3.0 equivalent) was added dropwise to a stirred solution of 3-(Boc-amino)-1,2-propanediol (2.43 g, 12.7 mmol, 1.0 equivalent) and oleoyl chloride (8.6 mL, 26.0 mmol, 2.05 equivalent) in CH2Cl2 (50 mL). The reaction mixture was then covered with aluminum foil and vigorously stirred at 0 °C for another 4 h. The reaction was then quenched with saturated Na2CO3 (aqueous solution). The organic phase was separated, washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel column chromatography (hexane / ethyl acetate = 15:1 to 10:1) to provide compound 12 (4.06 g, 44%) as a colorless solid. The chromatographic data of 12 were in excellent agreement with those reported in the literature (Luo et al., 2016).

[0337] Synthesis of Compound 13

[0338] Compound 12 was then dissolved in a mixed solvent of CH2Cl2 / CF3COOH (10 mL / 10 mL). After stirring at room temperature for 30 min, the solvent was removed under reduced pressure, and the crude product 13 was further dried under vacuum without further purification before use.

[0339] Synthesis of Compound 15

[0340] Compound 13 (1.0 equivalent), compound 1 (2.0 equivalent), DMAP (0.2 equivalent), and Et3N (5.0 equivalent) were dissolved in DMF at room temperature. The resulting mixture was then covered with aluminum foil and stirred further at room temperature for 24 h, followed by the addition of amine 14 (2.5 equivalent). After 5 h, the solvent DMF was removed under reduced pressure, and the crude residue was redissolved in ethyl acetate. The organic phase was first washed with saturated Na2CO3 (aqueous solution) until it turned grayish-white, then washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel column chromatography (CH2Cl2 / CH3OH = 30:1 to 15:1) to provide compound 15. The name of compound 15 is given below its structure.

[0341]

[0342] Yield: 30%. Colorless oily substance. 1¹H NMR (400MHz, chloroform-d) δ 5.50 (brd, 1H, NH), 5.37–5.32 (m, 5H, H19, H20, H35, H36 & NH), 5.13–5.08 (m, 1H, H2), 4.33–4.26 (m, 5H, H47, H53 & H1), 4.13 (dd, J = 12.0, 5.7Hz, 1H, H1), 3.48–3.34 (m, 2H, H3), 3.32–3.28 (m, 2H, H57), 2. 92(t,J=6.4Hz,4H,H48&H52),2.50(t,J=5.9Hz,2H,H58),2.33-2.27(m,10H,H27,H28,H60&H62),2.03-1.98(m ,8H,H18,H21,H34&H37),1.63-1.58(m,4H,H12&H43),1.30-1.25(m,40H),0.88(t,J=7.0Hz,6H,H11&H44)ppm. 13 C11 NMR (100MHz, chloroform-d) δ 173.6, 173.2, 156.4, 156.3, 130.17, 130.16, 129.85, 129.83, 70.4, 62.8, 62.7, 58.3, 45.1, 41.4, 38.2, 38.0, 37.7, 34.4, 34.2, 32.0, 29.91, 29.86, 29.7, 29.47, 29.46, 29.35, 29.34, 29.3, 29.25, 29.23, 27.37, 27.32, 25.02, 24.99, 22.8, 14.3 (C11 & C44) ppm. LRMS(ESI)(m / z): [M+H] + (C 49 H 92 Calculated value of O8N3S2: 914.6, measured value: 914.5.

[0343]

[0344] Yield: 30%. Colorless oily substance. 1¹H NMR (400MHz, chloroform-d) δ 5.79 (s, ¹H, NH), 5.44–5.29 (m, 5H, H¹⁹, H²⁰, H³⁵, H³⁶, and NH), 5.13–5.08 (m, ¹H, H²), 4.33–4.26 (m, 5H, H⁴⁷, H⁵⁃, and H¹), 4.13 (dd, J = 12.0, 5.7Hz, ¹H, H¹), 3.48–3.34 (m, 4H, H³, H⁵⁷), 2.94–2.91 (m, 4H, H⁶⁁, H⁶⁶) ,2.81-2.66(m,6H,including H48,H52,H58),2.31(td,J=7.6,2.7Hz,4H,H27,H28),2.05-1.96(m,8H,H18,H21,H34,H37 ),1.73(brd,4H,H26,H29),1.64-1.56(m,8H,H62-65),1.34-1.25(m,40H),0.87(t,J=7.0Hz,6H,H11&H44)ppm. 13 C10 NMR (100 MHz, chloroform-d) δ 173.6 (ester C=O), 173.2 (ester C=O), 156.4 (carbamate C=O), 156.3 (carbamate C=O), 130.2 (olefin carbon), 129.84 (olefin carbon), 129.83 (olefin carbon), 70.4 (C2), 63.0 (C1), 62.7, 56.8, 55.4, 41 .4(C3),38.0(C57),37.6,34.4,34.2,32.0,29.90,29.86,29.66,29.47,29.45,29.3 5,29.27,29.25,29.23,27.36,27.32,27.1,25.02,24.99,22.82,14.3(C11&C44)ppm. LRMS(ESI)(m / z):[M+H] + (C 53 H 98 Calculated value of O8N3S2: 968.7, measured value: 968.6.

[0345]

[0346] Yield: 24%. Colorless oily substance. 1¹H NMR (400MHz, chloroform-d) δ 6.02 (s, 1H, NH), 5.43–5.29 (m, 5H, H19, H20, H35, H36 and NH), 5.13–5.08 (m, 1H, H2), 4.40–4.26 (m, 5H, H47, H53 and H1), 4.13 (dd, J = 12.0, 5.7Hz, 1H, H1), 3.48–3.35 (m, 2H, H3), 3.33–3.28 (m, 2H) ),3.11(brd,2H),2.96-2.88(m,6H),2.33-2.29(m,4H,H27,H28),2.05-1.96(m,8H,H18,H21,H34,H37), 1.85-1.51(m,14H),1.32-1.25(m,40H),0.98(t,J=7.4Hz,3H,H68),0.88(t,J=7.0Hz,6H,H11&H44)ppm. 13 C11 NMR (100MHz, chloroform-d) δ 173.6, 173.3, 156.7, 156.4, 130.2, 129.85, 129.83, 70.4 (C2), 62.9 (C1), 62.78, 62.74, 41.4 (C3), 37.9, 37.8, 34.4, 34.2, 32.0, 29.91, 29.87, 29.86, 29.7, 29.47, 29.46, 29.37, 29.35, 29.29, 29.28, 29.25, 29.23, 27.37, 27.32, 25.03, 25.00, 22.8, 14.3 (C11, C44), 10.4 (C68). (Due to signal cancellation, in) 13 C62 was not observed on C APT. LRMS(ESI)(m / z): [M+H] + (C 55 H 102 Calculated value of O8N3S2: 996.7, measured value: 996.6.

[0347] 2.4 The general route for synthesizing lipids represented by the structure of Formula I or more specifically Formula IIIb is shown below.

[0348]

[0349] Compound 13 (1.0 equivalent), compound 7 (2.0 equivalent), DMAP (0.2 equivalent), and Et3N (10.0 equivalent) were dissolved in DMF at room temperature. The resulting mixture was then covered with aluminum foil and stirred further at room temperature for 24 h, followed by the addition of amine 14 (2.5 equivalent). After 5 h, Ac2O (3.0 equivalent) was added, and the mixture was stirred further at room temperature for another 12 h. Note: Ac2O was added to facilitate the removal of unidentified impurities from the final product. The solvent DMF was then removed under reduced pressure, and the crude residue was redissolved in ethyl acetate. The organic phase was first washed with saturated Na2CO3 (aqueous solution) until it turned grayish-white, then washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel column chromatography (CH2Cl2 / CH3OH (+0.1% Et3N) = 30:1 to 15:1) to provide compound 16. The name of compound 16 is given below its structure.

[0350]

[0351] Yield: 20%. Colorless oily substance. 1 ¹H NMR (400MHz, chloroform-d) δ 5.72 (t, J = 5.4Hz, ¹H, NH), 5.47 (s, ¹H, NH), 5.40–5.30 (m, 4H, H19, H20, H35, H36), 5.11–5.07 (m, ¹H, H2), 4.32–4.08 (m, 6H, including H1, H47, H53), 4.37–3.61 (m, 4H, H48, H52), 3.48–3.22 (m, 4H, H3 & H61) ),2.41(t,J=5.9Hz,2H,H62),2.33-2.23(m,10H,H27,H28,H64,H65),2.04-1.96(m,8H,H18,H21,H34,H37) ,1.62-1.58(m,4H,H26&H29),1.36(s,6H,H57&H58)1.34-1.25(m,40H),0.88(t,J=7.0Hz,6H,H11&H44)ppm. 13C10 NMR (100MHz, chloroform-d) δ 173.5 (ester C=O), 173.2 (ester C=O), 156.89 (carbamate C=O), 156.86 (carbamate C=O), 130.2 (olefin carbon), 129.86 (olefin carbon), 129.84 (olefin carbon), 100.0 (C50), 70.6 (C2), 67.4, 67.3, 64.5 (C47 or C53), 64.2 (C47 or C53), 62.8 (C1), 59.5 (C48 or C52), 59.3 (C48 or C52), 58.3 (C62), 45.2 (C64, C65), 41.3 (C3), 38.4 (C61), 34.4 (C27 or C28), 34.2 (C27 or C28), 32.0, 29.92, 29.88, 29.87, 29.81, 29.7, 29.47, 29.46, 29.37, 29.35, 29.29, 29.26, 29.25, 27.37 (allyl carbon), 27.33 (allyl carbon), 25.00, 24.94 (C58, C59), 22.8, 14.3 (C11, C44) ppm. LRMS(ESI)(m / z): [M+H] + (C 52 H 98 O 10 Calculated value of N3: 924.7, measured value: 924.6.

[0352]

[0353] Yield: 18%. Colorless oily substance. 1 ¹H NMR (400MHz, chloroform-d) δ 5.79–5.77 (m, ¹H, NH), 5.38–5.31 (m, ⁴H, H¹⁹, H²⁰, H³⁵, H³⁶), 5.13–5.09 (m, ¹H, H²), 4.32–4.10 (m, ⁶H, including H¹, H⁴⁷, H⁵⁃), 3.69–3.60 (m, ⁴H, H⁴⁸ & H⁵⁂), 3.52–3.43 (m, ¹H, H³), 3.39–3.27 (m, ³H, H³ & H⁶⁺). ),2.77-2.70(m,6H,H62,H64,H69),2.33-2.28(m,4H,H27&H28),2.04-1.96(m,8H,H18,H21,H34,H37),1.70 -1.58(m,12H,H65-68&H26,H29),1.36(s,6H,H58&H59),1.34-1.24(m,40H),0.89-0.86(m,6H,H11&H44)ppm. 13C10 NMR (100MHz, chloroform-d) δ 173.5 (ester C=O), 173.2 (ester C=O), 156.9 (carbamate C=O), 130.2 (olefin carbon), 129.85 (olefin carbon), 129.84 (olefin carbon), 100.0 (C50), 70.6 (C2), 67.6, 67.5, 67.4, 64.5, 64.2, 62.9 (C1), 59.5, 59.3, 56.5 (C62), 55.38, 55.35, 41.3 (C3), 39.6, 36.6, 34.4 (C27 or C1) 28), 34.2 (C27 or C28), 32.0, 29.91, 29.88, 29.87, 29.85, 29.7, 29.47, 29.46, 29.37, 29.35, 29.33, 29.30, 29.28, 29.25, 27.37 (allyl carbon), 27.33 (allyl carbon), 27.1, 25.00, 24.94 (C58 & C59), 22.8, 14.3 (C11 & C44) ppm. LRMS(ESI)(m / z): [M+H] + (C 56 H 104 O 10 Calculated value of N3: 978.8, measured value: 978.7.

[0354]

[0355] Yield: 15%. Colorless oily substance. 1 ¹H NMR (400MHz, chloroform-d) δ 6.05 (s, ¹H, NH), 5.73 (s, ¹H, NH), 5.38–5.31 (m, 4H, H¹⁹, H²⁰, H³⁵, H³⁶), 5.11–5.08 (m, ¹H, H²), 4.31–4.22 (m, 4H), 4.19–4.10 (m, 4H), 3.69–3.60 (m, 5H), 3.48–3.33 (m, 2H), 3.30–3.25 (m, 2H), 2.88–2.71 (m,2H),2.33-2.28(m,4H),2.20-1.97(m,8H,H18,H21,H34,H37),1.83-1.56(m,14H,H26,H29,H62,H66,H67,H6 8,H70),1.35(s,6H,H58,H59),1.29-1.26(m,40H),0.96(t,J=7.4Hz,3H,H71),0.87(t,J=7.0Hz,6H,H11&H44). 13C10 NMR (100MHz, chloroform-d) δ 173.6, 173.3, 157.2, 156.9, 130.2, 129.84, 129.83, 100.3 (C50), 100.0 (C50), 70.5 (C2), 67.4, 64.6, 64.5, 64.2, 62.9, 62.8, 59.4, 59.2, 58.97, 58.92, 58.85, 58.80, 41.32 (C3), 41.27 (C3), 34 .4 (C27 or C28), 34.2 (C27 or C28), 32.0, 29.90, 29.86, 29.7, 29.46, 29.45, 29.37, 29.35, 29.28, 29.24, 27.36 (allyl carbon), 27.32 (allyl carbon), 25.01, 24.99, 24.92 (C58, C59), 24.89 (C58, C59), 22.8, 14.3 (C11 & C44), 10.4 (C71). LRMS(ESI)(m / z): [M+H] + (C 58 H 108 O 10 Calculated value of N3: 1006.8, measured value: 1006.7.

[0356] 2.5 The general route for synthesizing lipids represented by the structure of Formula I or more specifically Formula IIIc is shown below.

[0357]

[0358] Compound 13 (1.0 equivalent), compound 17 (Yaeger et al., 2004) (2.0 equivalent), DMAP (0.2 equivalent), and Et3N (5.0 equivalent) were dissolved in DMF at room temperature. The resulting mixture was then covered with aluminum foil and stirred further at room temperature for 24 h, followed by the addition of amine 14 (2.5 equivalent). The solvent DMF was then removed under reduced pressure, and the crude residue was redissolved in ethyl acetate. The organic phase was first washed with saturated Na2CO3 (aqueous solution) until it turned grayish-white, then washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel column chromatography (CH2Cl2 / CH3OH = 30:1 to 15:1) to provide compound 18. The name of compound 18 is given below its structure.

[0359]

[0360] Yield: 24%. Colorless oily substance. 1¹H NMR (400MHz, chloroform-d) δ 5.41–5.32 (m, 5H, H19, H20, H35, H36 & NH), 5.17 (t, J = 6.2Hz, 1H, NH), 5.12–5.07 (m, 1H, H2), 4.27 (dd, J = 12.0, 4.4Hz, 1H, H1), 4.22 (t, J = 4.7Hz, 4H, H47 & H52), 4.12 (dd, J = 12.0, 5.7Hz, 1H, H1), 3.67 (t, J = 4.7Hz, 4H, H48 & H51), 3.48–3.3 4(m,2H,H3),3.30-3.25(m,2H,H56),2.44(t,J=6.1Hz,2H,H57),2.31(td,J=7.6,2.3Hz,4H,H27&H28),2.25(s,6H,H59&H61), 2.00(q,J=6.5Hz,8H,H18,H21,H34&H37),1.63-1.58(m,4H,H12&H43),1.32-1.25(m,40H),0.88(t,J=7.0Hz,6H,H11&H44)ppm. 13 C NMR (100MHz, chloroform-d) δ 173.6, 173.2, 156.6, 156.5, 130.2, 129.85, 129.84, 70.4, 69.7, 69.6, 64.3, 63.9, 62.7, 58.3, 45.2, 41.4, 38.4, 34.4, 34.2, 32.0, 29.91, 29.86, 29.7, 29.5, 29.35, 29.33, 29.27, 29.25, 29.23, 27.36, 27.32, 25.01, 24.99, 22.8, 14.3. LRMS (ESI) (m / z): [M+H] + (C 49 H 92 Calculated value of O9N3: 866.7, measured value: 866.6.

[0361]

[0362] Yield: 22%. Colorless oily substance. 1¹H NMR (400MHz, chloroform-d) δ 5.66 (s, ¹H, NH), 5.38–5.29 (m, 4H, H19, H20, H35, H36), 5.19 (s, ¹H, NH), 5.12–5.07 (m, ¹H, H2), 4.29–4.21 (m, 5H, including H1, H47, H52), 4.12 (dd, J = 12.0, 5.7Hz, ¹H, H1), 3.69–3.67 (m, 4H, H48, H51), 3.48–3.34 (m, 2H, H3), 3 .33-3.29(m,2H,H56),2.78-2.69(m,6H,H57,H60,H65),2.31(td,J=7.6,2.5Hz,4H,H27,H28),2.03-1.98(m,8H,H18,H 21, H34, H37), 1.69 (brd, 4H, H26, H29) 1.61 (brd, 8H, H62-65), 1.34-1.25 (m, 40H), 0.87 (t, J=7.0Hz, 6H, H11&H44) ppm. 13 C10 NMR (100MHz, chloroform-d) δ 173.6 (ester C=O), 173.2 (ester C=O), 156.6 (carbamate C=O), 156.5 (carbamate C=O), 130.2 (alkene carbon), 129.85 (alkene carbon), 129.83 (alkene carbon), 70.4 (C2), 69.7 (C48 or C51), 69.6 (C48 or C51), 64.3 (C47 or C52), 64.0 (C47 or C52), 62.7 (C1), 56.7 (C57) 55.4 (C60, C65), 41.4 (C3), 38.2 (C56), 34.4 (C27 or C28), 34.2 (C27 or C28), 32.0, 29.90, 29.86, 29.7, 29.46, 29.45, 29.35, 29.33, 29.27, 29.24, 29.22, 27.36 (allyl carbon), 27.32 (allyl carbon), 27.1, 25.00, 24.98, 22.8, 14.3 (C11 & C44) ppm. LRMS(ESI)(m / z): [M+H] + (C 53 H 98 Calculated value of O9N3: 920.7, measured value: 920.6.

[0363]

[0364] Yield: 16%. Colorless oily substance. 1¹H NMR (400MHz, chloroform-d) δ 5.94 (s, 1H, NH), 5.39–5.29 (m, 4H, H19, H20, H35, H36), 5.23 (s, 1H, NH), 5.10 (p, J = 5.2Hz, 1H, H2), 4.29–4.19 (m, 5H, including H1, H47, H52), 4.12 (dd, J = 12.0, 5.7Hz, 1H, H1), 3.68–3.65 (m, 4H, H48, H51), 3.48–3.34 (m, 2H, H3), 3.30–3.23 (m, 2H, 2.95 (brd, 2H, H58 or H65 or H61), 2.58-2.47 (m, 3H, H58 or H65 or H61), 2.31 (td, J=7.6, 2.7Hz, 4H, H27, H28), 2.03-1.98 (m, 8H, H18, H21, H34, H37), 1.81-1.54 (m, 14H, H26, H29, H57, H62, H63, H64, H66), 1.29-1.25 (m, 40H), 0.93-0.86 (m, 9H, H11, H44, H67) ppm. 13 C NMR (100MHz, chloroform-d) δ 173.6 (ester C=O), 173.3 (ester C=O), 156.8 (carbamate C=O), 156.2 (carbamate C=O), 70.5 (C2), 69.66 (C48 or C51), 69.60 (C48 or C51), 64.3 (C47 or C52), 63.9 (C47 or C52), 62.7 (C1), 51.0 (C58, C61, C65). Due to signal cancellation, (weaker peaks), 41.4 (C3), 34.4 (C27 or C28), 34.2 (C27 or C28), 32.0, 29.90, 29.86, 29.7, 29.46, 29.45, 29.35, 29.33, 29.27, 29.24, 29.22, 27.36 (allyl carbon), 27.31 (allyl carbon), 25.01, 24.98, 22.8, 14.3 (C11 & C44), 10.3 (C67) ppm. LRMS(ESI) (m / z): [M+H] + (C 55 H 102 Calculated value of O9N3: 948.8, measured value: 948.7.

[0365] 2.5 The general route for synthesizing lipids represented by the structure of Formula I or more specifically Formula V is shown below.

[0366]

[0367]

[0368] Synthesis of Compound 19

[0369] At 0 °C, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (13.1 g, 0.06 mol), TEA (6.42 g, 0.06 mol), and DMAP (0.66 mg, 0.005 mol) were added to a stirred solution of N-BOC-diethanolamine (5.43 g, 0.03 mol) and 2-hexyldecanoic acid (16.3 g, 0.06 mol) in CH2Cl2 (30 mL). The reaction mixture was then covered with aluminum foil and stirred vigorously at 0 °C for 1 h. The reaction was allowed to continue stirring at room temperature for 48 h. The reaction mixture was then filtered and concentrated. The residue was purified by silica gel column chromatography (hexane / ethyl acetate = 15:1) to provide compound 19 (2.84 g, 17.00%) as a brown oil.

[0370] Synthesis of Compound 20

[0371] Compound 19 was then dissolved in a mixed solvent of CH2Cl2 / CF3COOH (10 mL / 10 mL). After stirring at room temperature for 30 min, the solvent was removed under reduced pressure, and the crude product 20 was further dried under vacuum without further purification before use.

[0372] Synthesis of Compound 21

[0373] Compound 20 (1.0 equivalent), compound 1 (2.0 equivalent), DMAP (0.2 equivalent), and Et3N (5.0 equivalent) were dissolved in DMF at room temperature. The resulting mixture was then covered with aluminum foil and stirred further at room temperature for 24 h, followed by the addition of amine 14 (2.5 equivalent). After 5 h, the solvent DMF was removed under reduced pressure, and the crude residue was redissolved in ethyl acetate. The organic phase was first washed with saturated Na2CO3 (aqueous solution) until it turned grayish-white, then washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel column chromatography (CH2Cl2 / CH3OH = 30:1 to 10:1) to provide compound 21. The name of compound 21 is given below its structure.

[0374]

[0375] Yield: 16%. Colorless oily substance. 1¹H NMR (400MHz, chloroform-d) 4.39–4.28 (m, 4H, H₅₆ & H₅₈), 4.23–4.16 (m, 4H, H₂₃ & H₂₈), 3.58–3.51 (m, 4H, H₅₇ & H₅₹), 3.38–3.29 (m, 2H, H₃₃), 2.99–2.88 (m, 4H, H₂₄ & H₂₇), 2.6 1-2.51(m,2H,H34),2.41-2.27(m,8H,H4,H36,H37&H39),1.64-1.38(m,8H,H6,H7 ,H40&H41),1.32-1.20(brd,40H),0.87(t,J=6.8Hz,12H,H17,H19,H51&H53)ppm. LRMS(ESI)(m / z):[M+H] + (C 48 H 89 Calculated value of O8N3S2: 876.6, measured value: 876.5.

[0376]

[0377] Yield: 28%. Colorless oily substance. 1 H NMR(400MHz, chloroform-d)4.39-4.28(m,4H,H56&H58),4.23-4.15(m,4H,H23&H28),3.55(m,4H,H57 &H59),3.33-3.24(m,2H,H33),2.98-2.89(m,4H,H24&H27),3.63-2.54(brd,8H,H36,H37,H6 0,H62)2.51(t,2H,J=6.0Hz,H34),2.36(s,3H,H63)2.34-2.28(m,2H,H4&H39),1.65-1.37(m ,8H,H6,H7,H40&H41),1.33-1.20(brd,40H),0.87(t,J=6.8Hz,12H,H17,H19,H51&H53)ppm. LRMS(ESI)(m / z):[M+H] + (C 49 H 94 Calculated value of O8N4S2: 931.7, measured value: 931.5.

[0378]

[0379] Yield: 20%. Colorless oily substance. 1H NMR(400MHz, chloroform-d)4.40-4.27(m,4H,H54&H56),4.24-4.16(m,4H,H23&H28),3.55(m,4H ,H55&H57),3.38-3.28(m,2H,H33),3.03-2.89(m,6H,H24,H27&H63),3.03-2.28(m,2H,H 4&H37),2.18-1.68(m,10H,H34,H60,H61,H62&H64)1.65-1.38(m,8H,H6,H7,H40&H41),1 .25(brd,40H),1.01(t,J=7.4Hz,3H,H65)0.88(t,J=6.8Hz,12H,H17,H19,H51&H53)ppm. LRMS(ESI)(m / z):[M+H] + (C 52 H 99 Calculated value of O8N3S2: 958.7, measured value: 958.7.

[0380]

[0381] Yield: 68%. Colorless oily substance. 1 ¹H NMR (400MHz, chloroform-d) 4.39–4.25 (m, 4H, H₅₄, H₅₆), 4.25–4.15 (m, 4H, H₂₃, H₂₈), 3.58–3.51 (m, 6H, H₃₃, H₅₅ & H₅₇), 2.99–2.88 (m, 8H, H₂₄, H₂₇, H₅₈ & H₆₃), 2.40–2.26 (m, 2H,H4&H37),2.00-1.64(m,10H,H34,H59,H60,H61&H62),1.63-1.38(m,8H,H6,H7 ,H38&H39),1.32-1.20(brd,40H),0.87(t,J=6.8Hz,12H,H17,H19,H49&H51)ppm. LRMS(ESI)(m / z):[M+H] + (C 50 H 95 Calculated value of O8N3S2: 930.7, measured value: 930.6

[0382] 2.6 The general route for synthesizing lipids represented by the structure of Formula I or more specifically Formula VIa is shown below.

[0383]

[0384] 2.7 The general route for synthesizing lipids represented by the structure of Formula I or more specifically Formula VIb is shown below.

[0385]

[0386] Example 2: In vitro experiment

[0387] Materials and methods:

[0388] mRNA synthesis:

[0389] mRNAs encoding eGFP and firefly luciferase were prepared in vitro via T7-mediated transcription from a linearized DNA template (peTheRNAvs3 vector) containing 5' and 3' UTRs and a polyA tail. The final mRNAs utilized Cap1 and were 100% replaced with N1-methyl-pseudouridine.

[0390] LNP synthesis:

[0391] Lipid-based nanoparticles were generated by microfluidically mixing mRNA solution (100 mM, pH 4) and lipid solution in sodium acetate buffer at a 2:1 volume ratio using a NanoAssemblr Benchtop (Precision Nanosystems) at a rate of 9 mL / min. The lipid solution contained a mixture of ionizable lipids of interest, DSPC, DOPC or DOPE (Avanti), cholesterol (Sigma), and DMG-PEG2000 (Sunbright GM-020, NOF). The four lipids were mixed in six different molar ratios. LNPs were dialyzed using a slide-a-lyzer dialysis kit (20K MWCO, 3 mL, ThermoFisher) with TBS (TBS volume 10,000 times larger than LNP volume). Size, polydispersity, and zeta potential were measured using a Zetasizer Nano (Malvern). mRNA encapsulation was measured using a standard Ribogreen RNA assay (Invitrogen).

[0392] Cell line: HEK–TS / A–CT26–B16

[0393] The optimal culture conditions for each cell type, including growth medium, passage ratio, and recommended medium replacement, are summarized below. To harvest adherent cells, discard the used growth medium and wash the cells twice with phosphate-buffered saline (PBS) (Sigma), then add trypsin-EDTA (0.05%) (Gibco, Thermo Fisher Scientific) to loosen the cells. Medium replacement should be performed every 2 to 3 days whenever the cells reach approximately 70% confluence. Cell viability was measured using a Vi-Cell XR Cell viability analyzer (Beckman Coulter).

[0394] Table 1: Cell type-specific culture conditions

[0395]

[0396]

[0397] Abbreviations: DMEM: Duchenne Eagle Medium; HEPES: 4-(2-hydroxyethyl)-&-piperazine ethanesulfonic acid; RPMI: Roswell Park Memorial Institute; P / S: Penicillin / Streptomycin; FBS: Fetal Bovine Serum

[0398] transfection

[0399] Cells were seeded in 96-well plates at a density of 20–30 × 10⁴ cells / 100 μl of complete growth medium (specific for each cell type). Transfection was performed when cells reached 70%–90% confluence. The positive control Lipofectamine (MessengerMAX, Invitrogen) was diluted in OptiMEM (serum-depleted, Gibco) and incubated for 10 min. Simultaneously, eGFP mRNA and LNPs encapsulating eGFP mRNA were diluted in OptiMEM to obtain mRNA contents concentrations of 200 ng / well and 50 ng / well, respectively. The mRNA:lipid complex was incubated at a 1:1 ratio for 5 min and added quadruplicates to each condition. Cells were incubated at 37°C and 5% CO₂ for 24 h. Cells were then harvested using 1×TrypLE selectase (Gibco) and stained with the live / dead marker SYTOX blue (Life Technologies) in FACS buffer (PBS supplemented with 1% bovine serum albumin (BSA) and 0.09% azide (all from Sigma)). Cells were obtained immediately after the addition of the live / dead marker using an Attune Nxt flow cytometer (ThermoFisher Scientific). Lipofectamin-only (lipofectamin treatment without added mRNA) and blank or untreated (UT) cells were included as negative controls.

[0400] Flow cytometry:

[0401] E7-specific CD8 T cell response:

[0402] On days 14 and 25 post-tumor inoculation, blood was collected in heparin-coated tubes. Red blood cells were lysed, and the remaining white blood cells were stained with viability dye. After incubation and washing, APC-labeled E7 was added. (RAHYNIVTF) Add -dextramer (Immudex) and incubate at room temperature for 30 minutes. Wash away excess dextramer, add a mixture of antibodies against surface molecules CD3 and CD8 to the cells, and incubate at 4°C for 30 minutes.

[0403]

[0404]

[0405] eGFP expression

[0406] To assess eGFP expression, cells were stained with SYTOX blue. eGFP expression levels were measured within the gate of SYTOX blue-negative cells. The relative mean fluorescence intensity (rel MFI) was calculated as the MFI value of the expression marker divided by the MFI value of untransfected cells.

[0407] Data were acquired using the Attune Nxt cell counter and analyzed using FlowJo software. FlowJo version 10 was used to analyze the flow cytometry data.

[0408] Tumor inoculation and intratumoral injection:

[0409] For CT26 tumor inoculation, Balb / c mice were used. For B16F10 tumor inoculation, C57 / BL6 mice were used. To prepare for subcutaneous tumor inoculation, mice were anesthetized with 2.5% isoflurane, and the injection site was shaved. The injection site was typically the posterior / lateral side of the left lower ventral region. For inoculation purposes, cells needed to be cultured for approximately one week and passaged 3 to 5 times after thawing. Cold tumor cell solution was subcutaneously injected at a dose of 0.5*10e6 cells / 50μl PBS. Tumor growth was measured every 2–3 days using calipers. Tumor size was calculated using the following formula: (tumor width * tumor width * tumor length) / 2. When the tumor reached 50–100 mm... 3 At the average volume, tumors were injected with LNP containing firefly luciferase mRNA (10 μg mRNA in 20 μl TBS buffer) or with control buffer (TBS). Mice were monitored for 5–10 minutes after injection until fully awake, and no signs of pain, distress, or complications were observed.

[0410] In vivo bioluminescence:

[0411] Fluorescent mRNA expression in tumors and liver was assessed at 6 and 24 hours post-injection. Bioluminescence was measured in vivo using an IVIS spectral system via intraperitoneal injection of D-luciferin (Promega) into mice. Bioluminescence is generated through an oxidation reaction between luciferase, an enzyme derived from firefly mRNA encapsulated in LNPs, and its substrate D-luciferin. Regions of interest (ROIs) were designated in tumors and liver using Living Image software (PerkinElmer), and mean radiance (p / s / cm) was calculated within these ROIs. 2 To monitor tolerance to the different LNP types used, body weight at 6 and 24 hours post-injection was compared to baseline body weight on the day of randomization.

[0412] Data Analysis:

[0413] All raw data were analyzed using Graph Pad Prism version 7 software.

[0414] Example 2.1: Reporting the expression level of eGFP mRNA after in vitro transfection of HEK293T cells with the specified LNP composition.

[0415] LNPs were generated at a standard molar ratio of approximately 50 / 10 / 38.5 / 1.5 for ionizable lipids / DOPE / cholesterol / DMG-PEG2000. MC-3 is the ionizable lipid used in Onpattro and is considered prior art. eGFP mRNA was encapsulated in all LNPs as reporter mRNA at a molar ratio of 1 / 10 for mRNA / ionizable lipids.

[0416] Table 2:

[0417] Ionizable lipids size PDI Encapsulation % K-Adm-Dda 65,56 0,197 100 K-Adip-Dda 78,59 0,292 97 S-Adm-Dda 115,3 0,44 100 S-Adip-Dda 86,95 0,197 98 S-Ac7-Dda 74,28 0,223 95 MC3 73,14 0,103 100 S-Adm-Dsa 82,11 0,042 98 S-Adip-Dsa 70,9 0,153 101 S-Ac7-Dsa 71,05 0,188 99

[0418] As detailed in Table 2, all LNPs exhibited high encapsulation efficiency, as measured by RiboGreen assays. Size and polydispersity index were evaluated using dynamic light scattering (DLS) on a Malvern Zetasizer. MC-3 is an ionizable lipid used in Onpattro and is considered to be prior art.

[0419] Figure 1 The figure shows the relative mean fluorescence intensity (measured as the fold increase in eGFP MFI compared to untreated cells) of eGFP expression in HEK293T cells after incubation with the specified LNP at mRNA concentrations of 50 ng / well and 200 ng / well, or with MC3 as a positive control. It is clear from this figure that the LNP of the present invention generally performs as well or better than the positive control samples.

[0420] Example 2.2: Reporting eGFP mRNA expression levels after in vitro transfection of HEK293T and cancer cell lines (CT26-B16F10-TS / A) with the specified LNP composition.

[0421] LNPs were generated at a standard molar ratio of approximately 50 / 10 / 38.5 / 1.5 for ionizable lipids / phospholipids / cholesterol / DMG-PEG2000. MC-3 is the ionizable lipid used in Onpattro and is considered prior art. All LNPs were formulated with DOPE, except for the MC-3-based LNP (which was formulated with DSPC as the phospholipid). eGFP mRNA was encapsulated in all LNPs as reporter mRNA at a 1 / 10 mRNA / ionizable lipid molar ratio.

[0422] Table 3:

[0423] Ionizable lipids size PDI Encapsulation % MC3(DSPC) 73,33 0,08 94 S-Adm-DSa 91,15 0,137 100 S-Ac7-DSa 89,44 0,158 93 K-Adm-DOg 91,9 0,157 94 K-Ac7-DOg 62,26 0,108 93 K-Ac6e-DOg 75,8 0,268 95 S-Adm-DOg 68,08 0,3 98 S-Ac7-DOg 87,46 0,21 90 S-Ac6e-DOg 63,86 0,311 95 E-Adm-Dog 59,56 0,217 100 E-Ac7-Dog 93,05 0,258 100 E-Ac6e-DOg 67,3 0,241 100

[0424] Figure 2 again shows that LNPs performed as well or better than the positive control MC3 in multiple cell lines. Specifically, LNPs with ionizable lipids combining the SS linker motif with the DOg acyl chain were the most effective in transfected cells. In terms of amine groups, Ac7 was superior to Ac6e and Adm.

[0425] Example 2.3 Evaluation of the correlation between PEG lipid mol% and nitrogen / phosphate (N:P) ratio

[0426] To evaluate the effect of the molar ratio of DMG-PEG2000% and ionizable lipids to mRNA on the physicochemical properties and transfection ability of LNPs, nine mRNA LNPs were generated at three N:P ratios and with 3 mol% DMG-PEG2000. All LNPs were based on S-Ac7-Dog as an ionizable lipid. eGFP mRNA was encapsulated to enable in vitro assessment of transfection efficiency.

[0427] Table 4. Dimensions and PDI of the specified LNP compositions as measured by DLS.

[0428] LNP number Ionizable lipids PEG-lipid % N:P size PDI 1 S-Ac7-Dog 0,5 10 230,2 0,136 2 S-Ac-Dog 1,5 10 124,3 0,102 3 S-Ac-Dog 3,0 10 96,64 0,1 4 S-Ac-Dog 0,5 5 198,4 0,171 5 S-Ac-Dog 1,5 5 159,9 0,149 6 S-Ac-Dog 3,0 5 96,04 0,087 7 S-Ac-Dog 0,5 20 242,2 0,077 8 S-Ac-Dog 1,5 20 111,1 0,165 9 S-Ac7-Dog 3,0 20 84,54 0,117

[0429] Figure 3A B showed that no LNPs significantly affected the viability of transfected HEK293T cells and CT26 cells, respectively.

[0430] Reducing DMG-PEG2000% resulted in an increase in LNP size. Within the tested N:P ratio range, LNPs with 1.5% DMG-PEG2000 showed improved transfection efficiency. For transfection of HEK293T cells, an N:P ratio of 10 was the most effective. Figure 4A For transfecting CT26 tumor cells, the N:P 20 ratio was superior to the N:P 10 and N:P 5 ratios. Figure 4B ).

[0431] Example 2.4. In vivo mRNA expression of subcutaneously growing CT26 tumors after LNP injection.

[0432] Balb / c mice were subcutaneously inoculated with CT26 tumor cells. When the tumor reached 50-100 mm... 3 At the average volume, tumors were injected with the corresponding mRNA LNP (10 μg mRNA; 20 μl volume; TBS buffer) or with control buffer. Tumors were assessed via in vivo bioluminescence assays at 6 and 24 hours post-injection. Figure 5A) and liver ( Figure 5B Flux mRNA expression in the liver was assessed. Compared to the baseline LNP based on MC-3, mRNA delivery via S-Ac7-Dog-based LNP resulted in similar Flux expression levels in tumors, but showed a significant reduction in off-target expression in the liver. No weight loss was observed after mRNA delivery via S-Ac7-Dog. Figure 5C ), and delivery of MC-3 resulted in significant weight loss.

[0433] Example 2.5. In vivo mRNA expression of subcutaneously growing B16F10 tumors after LNP injection.

[0434] Balb / c mice were subcutaneously inoculated with CT26 tumor cells. When the tumor reached 100 mm... 3 At the average volume, tumors were injected with the corresponding mRNA LNP (10 μg mRNA; 20 μl volume; TBS buffer) or with control buffer. Tumors were assessed via in vivo bioluminescence assays at 6 and 24 hours post-injection. Figure 6A ) and liver ( Figure 6B Fluc mRNA expression in tumors was significantly reduced compared to baseline LNPs based on MC-3, but off-target expression in the liver was strongly reduced. Figure 6C No weight loss was observed after mRNA delivery via S-Ac-Dog, while delivery via MC-3 resulted in significant weight loss.

[0435] Example 2.6. Induction of T cell response after intramuscular vaccination

[0436] On days 1 and 8, C57BL / 6 mice were intramuscularly vaccinated with 10 μg of E7 mRNA encapsulated in LNPs, along with S-Ac7-Dog or MC-3 as ionizable lipids. LNPs were prepared at a molar ratio of S-Ac7-Dog / mRNA of 10:1. Six days after each vaccination, E7-specific CD8 T cell responses were measured by flow cytometry. Figure 7 ).

[0437] Example 3: Induction of antigen-specific CD8 T cells after intramuscular mRNA LNP vaccination

[0438] Materials and methods:

[0439] Intramuscular injection and muscle thickness assessment:

[0440] All mice were housed under specific pathogen-free conditions, and animal studies were conducted according to protocols and guidelines approved by the Ghent University Animal Care and Use Committee (ECD20 / 100). Mice were injected with mRNA LNP (50 μl volume, 5 μg mRNA) from TBS into the biceps femoris muscle. Muscle thickness at the injection site was measured using an electronic external measuring instrument (K220T, Kroeplin) on days 1 (d1) and 4 (d4) post-injection.

[0441] Flow cytometry (Assessment of E7-specific CD8 T cell responses): Anna

[0442] On day 7 (d7) after the initial immunization and day 14 (d14) after the booster immunization, 100 μL of whole blood was collected for flow cytometry staining. Following erythrocyte lysis (RBC lysis buffer, 420302 Biolegend), the whole blood cells were mixed with 0.5 μg / test mouse BD Fc Block. TM (BD, 553142) was incubated together with Zombie Aqua reactive dye (Biolegend, 423102). After incubation and washing, 5 μL / test APC-labeled E7 was added. (RAHYNIVTF) Cells were incubated with -dextramer (Immudex, JA2195) at room temperature (RT) for 30 min. After washing, cells underwent surface staining during 30 min incubation with an antibody mixture in FACS buffer (PBS, 2 mM EDTA, 1% BSA) using the following antibody mixture amounts for each test: CD3 PerCP-eFluor710 (Invitrogen, 46-0032-82), CD8-V450 (BD Biosciences, 560469), KLRG1 (Biolegend, 138408), and CD127-BV605 (Biolegend, 135041). After the next washing step, cell samples were obtained on a 3-laser AtuneNxt flow cytometer (ThermoFisher, A29003), and data were analyzed using FlowJo v10.7.1 software (BD Biosciences, FlowJo portal account).

[0443] LNP preparation

[0444] LNP formulations were prepared using a modified procedure for siRNA LNP synthesis previously described. All formulations were prepared aseptically at an N / P ratio of 10. All lipid components were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 mol% (ionizable lipids / DSPC / cholesterol / DMG-PEG2000). DSPC, cholesterol, and DMG-PEG2000 were purchased from Avanti Polar Lipids (Alabaster, Alabama, USA), while the ionizable lipids were synthesized in-house. The final lipid concentration in ethanol was fixed at 10 mg / mL. E7 mRNA was dissolved in 100 mM acetate buffer (Sigma-Aldrich, Saint-Louis, Missouri, USA) at pH 4. A microfluidic mixer was used. BenchTop (Precision Nanosystems, Vancouver, BC) was used to combine the ethanol and aqueous phases at a 2:1 (water:ethanol) ratio. Then, a Slide-A reactor with a MWCO of 20,000 Da was used. Dialysis kits (Thermo Scientific, Massachusetts, USA) were used to dialyze the formulation for 18 hours with 1X sterile TBS pH 7.4 (Sigma Aldrich, Saint-Louis, Missouri, USA). The purified formulation was then concentrated using an Amicon ultracentrifuge filter (EMD Millipore, Massachusetts, USA). Finally, the precise concentration and EE% were determined by Ribogreen assay before final dilution with TBS (100 μg / mL E7 mRNA / formulation). The size and PDI of all formulations were characterized using a dynamic light scattering Zetasizer Nano-ZS (Malvern Pananlytical Ltd., Malvern, UK) and then stored at 4°C. The physicochemical properties of each formulation are shown in Table 5.

[0445] Table 5: A list of relevant physicochemical properties of different LNP formulations. These include the type and composition of ionizable lipids, the mole fraction of each component, size and PDI as determined by dynamic light scattering, and encapsulation efficiency. Encapsulation efficiency has been measured using Ribogreen assays.

[0446]

[0447] Note: EE% is defined as the encapsulation efficiency of mRNA inside LNP nanoparticles.

[0448] result:

[0449] The corresponding mRNA LNP vaccine induced a strong E7-specific CD8 T cell response after intramuscular immunization, which was significantly influenced by the chemistry of ionizable lipids. Figure 8 Compared with LNPs formulated with MC-3 (an ionizable lipid for delivering Onpattro), LNPs formulated with the ionizable lipid of interest did not cause significant edema at the injection site (as measured by the relative increase in muscle thickness). Figure 9 ).

[0450] Example 4: Induction of anti-HA (hemagglutinin) immune response after intramuscular mRNA vaccination

[0451] Materials and methods:

[0452] Flow cytometry (ICS):

[0453] Using a concentration of 1ug / peptide / ml from HA / Puerto Rico / 8 / 1934 H1N1 (PepMix) TM2*10e6 splenocytes were collected and stimulated with a peptide library of 139 peptides from Influenza A, JPT, and PM-INFA-HAPR. Splenocytes treated with 20 ng / ml PMA (79346-1MG, Sigma) and 1 μg / ml iomycin (I0634-1MG, Sigma) served as positive controls. 0.065 μg / sample CD107a–BV711 (564348, BD) antibody was added along with the activating stimulant. Cells were stimulated for 5 h. One h after the start of stimulation, 1X GolgiPlug (555028, BD) was added to stop cytokine secretion. Cells were then incubated with a mixture of antibodies (cocktail) for surface staining: 0.125 μg / test CD4-FITC (Biolegend, 100509), 0.02 μg / test Thy1.2-Alexa700 (Biolegend, 140323), and 0.05 μg / test CD8-eFluor450 (eBioscience, 48-0081-82). Cells were fixed and permeabilized using the BD Cytofix / Cytoperm Plus Fixation / Permeabilization Solution Kit (555028, BD) according to the manufacturer's instructions. Cells were stained in permeation buffer containing the following mAbs at the following concentrations: 0.03 μg / test IFNg-PE (BD, 554412), 0.065 μg / test CD154-PerCP-eFluor710 (ebioscience, 46-1541-80), 0.62 μl / test Granz-AF647 (Biolegend, 515406), 0.125 μg / test IL2-BV605 (BD, 563911), and 0.065 μg / ml TNFα-BV785 (Biolegend, 506341). Cells were analyzed on an Atune Nxt flow cytometer (Thermo Fisher, A29003). Monocytes were gated using forward and side light scattering, then duplets were distinguished using FCS-A and FCS-H scattering, and dead cells were excluded based on fixed dead cell staining fluorescence. All additional gating was specified, and where applicable, a control was subtracted based on fluorescence. Data acquisition and analysis were performed using FlowJo v10.7.1 software (BD Biosciences).

[0454] Assessment of endpoint immunoglobulin titers in mice

[0455] On days 21 (d21) and 35 (d35), 100 μL of whole blood from mice was collected in serum gel tubes (SarsTedt). Serum was separated from the blood clot by centrifugation at 10,000 g for 10 min at 4°C.

[0456] A black flat-bottomed Maxisorp 96-well plate (437111, Life Technologies) was coated overnight at 4°C with 100 μl of 1 μg / mL recombinant H1N1 (A / Puerto Rico / 8 / 1934) HA protein (Sino Biological, 11684-V08H) in carbonate / bicarbonate buffer (0.1 M, pH 9.6). The plate was then blocked for 2 h with 100 μl of 3% BSA (05479-250g, Sigma) (w / v) in PBS. The plate was then washed three times with PBS / 0.1% Tween (10113103, Fisher Scientific). Serially diluted serum samples were added to the plate (initial 100X serum dilution for day 21, initial 1000X serum dilution for day 35; 5X dilution steps). After incubation at room temperature for 2 hours, the plate was washed 5 times and then incubated for another hour with either HRP-conjugated rabbit anti-mouse IgG1 (1:15000, Biorad, OBT1508P) or HRP-conjugated goat anti-mouse IgG2a (1:8000, STAR133P Biorad). Following the final wash, the plate was developed using fluorescent Amplex UltraRed reagent (A36006, Invitrogen) according to the manufacturer's instructions. The plate was then visualized on a Tecan Infinite200Pro using λ... ex =540nm, λ em =590nm plate reading. Serum dilutions from TBS-treated mice were used for cut-off determination, which was the mean fluorescence measured in the TBS sample plus 3 standard deviations. All points exceeding the cut-off were considered below the limit of quantitation. A 5PL curve was fitted to the dilution data, and the endpoint titer was calculated at the intersection of the modeled curve and the cut-off.

[0457] LNP preparation:

[0458] LNP formulations were prepared as described above. All formulations were prepared aseptically at an N / P ratio of 10. The size and PDI of all formulations were characterized using dynamic light scattering Zetasizer Nano-ZS (Malvern Pananlytical Ltd., Malvern, UK), and then stored at 4°C. The physicochemical properties of each formulation are shown in Table 6.

[0459] Table 6: A list of relevant physicochemical properties of different LNP formulations. These include the type of ionizable lipids, lipid composition and mole fraction of each component, size and PDI (as determined by dynamic light scattering), and encapsulation efficiency. Encapsulation efficiency has been measured using Ribogreen assays.

[0460]

[0461]

[0462] Note: EE% is defined as the encapsulation efficiency of mRNA inside LNP nanoparticles.

[0463] result:

[0464] The corresponding mRNA LNP vaccine induced anti-HA antibody titers. A significant increase in titers was observed after booster. Figure 10 In addition, mRNA LNP vaccines also elicit an IFNg+CD8 T cell response against HA, which is influenced by the chemistry of the ionizable lipids used. Figure 11 ).

[0465] Example 5. Induction of E7-specific CD8 T cell response during intramuscular immunization

[0466] Materials and methods:

[0467] Flow cytometry (assessment of E7-specific CD8 T cell responses):

[0468] On day 7 (d7) after the initial immunization and day 14 (d14) after the booster immunization, 100 μl of whole blood was collected for flow cytometry staining. Following erythrocyte lysis (RBC lysis buffer, 420302 Biolegend), the whole blood cells were mixed with 0.5 μg / sample of mouse BD Fc Block. TM (BD, 553142) was incubated together with Zombie Aqua reactive dye (Biolegend, 423102). After incubation and washing, 5 μl / sample of APC-labeled E7 was added. (RAHYNIVTF)Cells were incubated with -dextramer (Immudex, JA2195) at room temperature (RT) for 30 min. After washing, cells underwent surface staining during 30 min incubation with an antibody mixture in FACS buffer (PBS, 2 mM EDTA, 1% BSA). The antibody mixture amounts for each test were as follows: CD3 PerCP-eFluor710 (Invitrogen, 46-0032-82), CD8-V450 (BDBiosciences, 560469), KLRG1 (Biolegend, 138408), and CD127-BV605 (Biolegend, 135041). Samples were acquired on a 3-laser Atune Nxt flow cytometer (ThermoFisher, A29003), and data were analyzed using FlowJo software (FlowJo_v10.7.1, FlowJo portal account).

[0469] LNP preparation:

[0470] LNP formulations were prepared as described above. All formulations were prepared aseptically at an N / P ratio of 10. The size and PDI of all formulations were characterized using a dynamic light scattering Zetasizer Nano-ZS (Malvern Pananlytical Ltd., Malvern, UK) and then stored at 4°C. The physicochemical properties of each formulation are shown in Table 7.

[0471] Table 7: A list of relevant physicochemical properties of different LNP formulations. These include the type of ionizable lipids, lipid composition and mole fraction, size, and PDI (as determined by dynamic light scattering) of each component, as well as encapsulation efficiency. Encapsulation efficiency has been measured using Ribogreen assays.

[0472]

[0473] Note: EE% is defined as the encapsulation efficiency of mRNA inside LNP nanoparticles.

[0474] result:

[0475] The corresponding mRNA LNP vaccine induced a strong E7-specific CD8 T cell response after intramuscular immunization, which was significantly influenced by the chemistry of ionizable lipids. Figure 12 Compared to MC-3, S-Ac7-DHDa-based LNPs induced superior T cell responses. Figure 13 ).

[0476] Example 6: Intravenous immunization with LNP-co-encapsulated peptide antigen and imidazoquinoline TLR7 / 8 agonist

[0477] Materials and methods

[0478] Lipid nanoparticle (LNP) formulations

[0479] The minimal epitope amino acid sequence of E7 (RAHYNIVTF) was extended with ten glutamate residues and flanking amino acid sequences (QAEPD) and two serine residues (SS) from the native E7 protein amino acid sequence. The resulting peptide (EEEEEEEEEEESSQAEPDRAHYNIVTF) was further designated GLU10-E7. As an unformulated control, SSQAEPDRAHYNIVTF was used and further designated E7. The TLR7 / 8 agonist 1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinoline-4-amine (IMDQ) was conjugated to the peptide containing ten glutamate residues. This conjugation was further designated GLU10-IMDQ. For fluorescence-based tracking, a Cy5-labeled peptide containing ten glutamate residues was used, which was further designated GLU10-Cy5.

[0480] For LNP formulations, an aqueous phase containing GLU10-E7 (or GLU10-Cy5 for fluorescence-based tracking assays) and GLU10-IMDQ was prepared in 25 mM acetate buffer (pH 5.2) GLU10-E7 or GLU10-IMDQ. An organic phase was prepared by dissolving S-Ac7-DOG, DOPE, cholesterol, and DMG-PEG in a molar ratio of 50:10:38.5:1.5. The peptide-to-ionizable lipid ratio was fixed at an N:C ratio of 5:1 (N: ionizable amine from the ionizable lipid, C: carboxylic acid from glutamate residues). The organic and aqueous phases were mixed at a 1:3 ratio by adding the organic phase dropwise to the vigorously stirred aqueous solution. Subsequently, the formed LNPs were dialyzed against PBS for 12 h using a 3.5 kDa filtration membrane to remove ethanol.

[0481] Results and Methods

[0482] Compared to the unformulated soluble peptide (GLU10-Cy5), the LNP-formulated peptide (LNP(GLU10)) resulted in a significant increase in peptide uptake by splenic macrophages and dendritic cells (cDC1 and cDC2 subsets) after intravenous administration. Figure 14 Additionally, B cells and a slight degree of T cell association with the peptide. Co-formulation of GLU10-IMDQ in LNP further increased splenic uptake of the peptide by macrophages, cDC1 dendritic cells, B cells, and T cells (CD4+ and CD8+ T cell subsets).

[0483] LNPs containing the TLR7 / 8 agonist GLU10-IMDQ can activate dendritic cells (cDC1 and cDC2 subsets), B cells, and T cells (CD4+ and CD8+ T cell subsets) in the spleen after intravenous administration. Figure 15 ).

[0484] Compared to unformulated soluble peptide E7 antigen and soluble peptide E7 antigen adjuvanted with the TLR7 / 8 agonist IMDQ, LNPs containing GLU10-E7 peptide antigen and GLU10-IMDQ induced a significant increase in tetramer-positive CD8 T cells in the blood of immunized mice after administration twice at 2-week intervals. Administration of the antigen and TLR7 / 8 agonist within the same LNP induced a higher response than individual populations containing either the antigen or the TLR7 / 8 agonist, respectively. Figure 16 ).

[0485] Example 7: Intramuscular immunization with polyI:C LNP and protein antigen

[0486] Materials and methods

[0487] Lipid nanoparticle (LNP) formulations

[0488] LNP formulations were prepared with an N / P ratio of 10. All lipid components (S-Ac7-DOg / DOPE / cholesterol / DSG-PEG2000) were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 mol% in ethanol. Low molecular weight polyI:C was dissolved in 100 mM acetate buffer at pH 4. The ethanol and aqueous phases were mixed at a ratio of 1:3 by adding the organic phase dropwise to the vigorously stirred aqueous solution. Subsequently, the formed LNPs were dialyzed against PBS for 12 h using a 3.5 kDa filtration membrane to remove ethanol.

[0489] Results and Methods

[0490] Compared to the original polyI:C, the polyI:C formulation in LNP increases the titer of anti-S1 spike protein IgG antibodies. These titers further increase when the S1 spike protein is conjugated to the LNP surface. Figure 17 ).

[0491] Example 8: Intramuscular immunization with CpG LNP and protein antigen

[0492] Materials and methods

[0493] Lipid nanoparticle (LNP) formulations

[0494] LNP formulations were prepared at an N / P ratio of 10. All lipid components were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 mol% (S-Ac7-DOg / DOPE / cholesterol / DSG-PEG2000). CpG was dissolved in 100 mM acetate buffer, pH 4. The ethanol and aqueous phases were mixed at a ratio of 1:3 by adding the organic phase dropwise to the vigorously stirred aqueous solution. Subsequently, the formed LNPs were dialyzed against PBS for 12 h using a 3.5 kDa filtration membrane to remove ethanol.

[0495] Results and Methods

[0496] Compared to the original CpG, the formulation of CpG in LNP increases the anti-ovalbumin (OVA) IgG antibody titer. Figure 18 ).

[0497] References

[0498] 1.Amano,Y.;Umezawa,N.;Sato,S.;Watanabe,H.;Umehara,T.;Higuchi,T.Activation of lysine-specific demethylase 1inhibitor peptide by redox-controlled cleavage of a traceless linker.Bioorg.Med.Chem.2017,25,1227-1234.

[0499] 2. Shenoi, RA; Lai, BFL; Kizhakkedathu, JNSynthesis, characterization, and biocompatibility of biodegradable hyperbranchedpolyglycerols from acid-cleavable ketal group functionalized initiators. Biomacromolecules 2012, 13, 3018-3030.

[0500] 3. Luo, C.; Miao, L.; Zhao, Y.; Musetti, S.; Wang, Y.; Shi, K.; Huang, LA novelcationic lipid with intrinsic antitumor activity to facilitate gene therapy of TRAIL DNA. Biomaterials 2016, 102, 239-248.

[0501] 4. Yeager, AR; Finney, NS The first direct evaluation of the two-active site mechanism for chitin synthase. J. Org. Chem. 2004, 69, 613-618. sequence list <110> Izaen's Immunotherapy Co., Ltd. Ghent University <120> Ionizable lipids <130> ETR-094 - P179996PC00 <150> EP20216879.5 <151> 2020-12-23 <160> 4 <170> BiSSAP 1.3.6 <210> 1 <211> 9 <212> PRT <213> Artificial sequence <220> <223> E7 dextramer <400> 1 Arg Ala His Tyr Asn Ile Val Thr Phe 1 5 <210> 2 <211> 16 <212> PRT <213> Artificial sequence <220> <223> E7 peptide <400> 2 Ser Ser Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe 1 5 10 15 <210> 3 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Flanking amino acid sequences <400> 3 Gln Ala Glu Pro Asp 1 5 <210> 4 <211> 26 <212> PRT <213> Artificial sequence <220> <223> Glu10 E7 peptide <400> 4 Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Ser Ser Gln Ala Glu Pro 1 5 10 15 Asp Arg Ala His Tyr Asn Ile Val Thr Phe 20 25

Claims

1. An ionizable lipid, represented by formula (I) （I） in R1 and R2 are each independently selected from -H and -C. 1-20 Alkyl; wherein the -C 1-20 Each of the alkyl groups may optionally be substituted with 1 to 3 –O-(C=O)-R7 or –(C=O)-O-R7; and the total number of C atoms in R1 and R2 is at least 8; R3 and R4 are each independently -C 1-6 Alkyl groups; or R3 and R4 together with the N atoms to which they are attached form a 5-10 membered aromatic or non-aromatic heterocycle; said heterocycle may further optionally contain one or two additional N atoms, and / or may optionally be substituted by 1-3 substituents selected from: -C 1-6 Alkyl groups; and Each R5 and R6 is independently –CH2-; Each R7 is independently selected from -C 1-20 Alkyl, -C 2-20 alkenyl; wherein the -C 1-20 Alkyl and -C 2-20 Each of the alkenyl groups may optionally be replaced by 1 to 3 –O-(C=O)-R7 or –(C=O)-O-R7; m and n are each an independent integer selected from 1, 2, 3 and 4; X is selected from -O-, -SS-, -O-CR8R9-O-; Each R8 and R9 is independently selected from -C 1-6 alkyl; Y is selected from -NH-; Z is -C 1-6 Alkylene-.

2. The ionizable lipid as defined in claim 1, represented by formula (II). (II) in R3 and R4 are each independently -C 1-6 Alkyl groups; or R3 and R4 together with the N atoms to which they are attached form a 5-10 membered aromatic or non-aromatic heterocycle; said heterocycle may further optionally contain one or two additional N atoms, and / or may optionally be substituted by 1-3 substituents selected from: -C 1-6 alkyl; Each R5 and R6 is independently –CH2-; Each R7 is independently selected from -C 1-20 Alkyl, -C 2-20 alkenyl; wherein the -C 1-20 Alkyl and -C 2-20 Each of the alkenyl groups may optionally be replaced by 1 to 3 –O-(C=O)-R7 or –(C=O)-O-R7; and the total number of C atoms in the two R7 moieties is at least 5; m and n are each an independent integer selected from 1, 2, 3 and 4; X is selected from -O-, -SS-, -O-CR8R9-O-; Each R8 and R9 is independently selected from -C 1-6 alkyl; Y is selected from -NH-; Z is -C 1-6 Alkylene-.

3. An ionizable lipid as defined in any one of claims 1 or 2, represented by any one of formula (IIIa), (IIIb) or (IIIc). (IIIa) (IIIb) (IIIc) in R3 and R4 are each independently -C 1-6 Alkyl groups; or R3 and R4 together with the N atoms to which they are attached form a 5-10 membered aromatic or non-aromatic heterocycle; said heterocycle may further optionally contain one or two additional N atoms, and / or may optionally be substituted by 1-3 substituents selected from: -C 1-6 Alkyl groups; and Each R5 and R6 is –CH2-; Each R7 is independently selected from -C 1-20 Alkyl, -C 2-20 alkenyl; wherein the -C 1-20 Alkyl and -C 2-20 Each of the alkenyl groups may optionally be replaced by 1 to 3 –O-(C=O)-R7 or –(C=O)-O-R7; and the total number of C atoms in the two R7 moieties is at least 5; m and n are each an independent integer selected from 1, 2, 3 and 4; Each R8 and R9 is independently selected from -C 1-6 alkyl; Y is selected from -NH-; Z is -C 1-6 Alkylene-.

4. The ionizable lipid as defined in claim 1, represented by any one of formulas (IVa), (IVb), and (IVc). (IVa) (IVb) (IVc) in R1 and R2 are each independently selected from -H and -C. 1-20 Alkyl; wherein the -C 1-20 Each of the alkyl groups may optionally be substituted with 1 to 3 –O-(C=O)-R7 or –(C=O)-O-R7; and the total number of C atoms in R1 and R2 is at least 8; R3 and R4 are each independently -C 1-6 Alkyl groups; or R3 and R4 together with the N atoms to which they are attached form a 5-10 membered aromatic or non-aromatic heterocycle; said heterocycle may further optionally contain one or two additional N atoms, and / or may optionally be substituted by 1-3 substituents selected from: -C 1-6 alkyl; Each R7 is independently selected from -C 1-20 Alkyl, -C 2-20 alkenyl; wherein the -C 1-20 Alkyl and -C 2-20 Each of the alkenyl groups may optionally be replaced by 1 to 3 –O-(C=O)-R7 or –(C=O)-O-R7; Each R5 and R6 is independently –CH2-; m and n are each an independent integer selected from 1, 2, 3 and 4; Each R8 and R9 is independently selected from -C 1-6 alkyl; Y is selected from -NH-; Z is -C 1-6 Alkylene-.

5. The ionizable lipid as defined in claim 1, represented by formula (V). （V） in R3 and R4 are each independently -C 1-6 Alkyl groups; or R3 and R4 together with the N atoms to which they are attached form a 5-10 membered aromatic or non-aromatic heterocycle; said heterocycle may further optionally contain one or two additional N atoms, and / or may optionally be substituted by 1-3 substituents selected from: -C 1-6 Alkyl groups; and Each R5 and R6 is independently selected from –CH2-; Each R7 is independently selected from -C 1-20 Alkyl, -C 2-20 alkenyl; wherein the -C 1-20 Alkyl, -C 2-20 alkenyl and -C 2-20 Each of the ynyl groups may optionally be substituted by 1 to 3 –O-(C=O)-R7 groups; and the total number of C atoms in the two R7 groups is at least 5; m and n are each an independent integer selected from 1, 2, 3 and 4; Y is selected from -NH-; Z is -C 1-6 Alkylene-.

6. The ionizable lipids as defined in claim 1, selected from the list comprising: 。 7. The ionizable lipid as defined in claim 1, wherein the total number of C atoms in R1 and R2 is at least 14.

8. The ionizable lipid as defined in claim 1, wherein m and n are the same and are integers selected from 1, 2, 3 and 4.

9. The ionizable lipid as defined in claim 1, wherein m and n are 2.

10. A lipid nanoparticle or lipid nanoparticle composition comprising an ionizable lipid as defined in any one of claims 1 to 9.

11. The lipid nanoparticles or lipid nanoparticle composition according to claim 10, further comprising phospholipids, sterols and / or PEG lipids.

12. The lipid nanoparticles or lipid nanoparticle composition according to claim 10 or 11, further comprising an active agent.

13. The lipid nanoparticles or lipid nanoparticle compositions according to claim 10 or 11, further comprising nucleic acids.

14. The lipid nanoparticles or lipid nanoparticle compositions according to claim 10 or 11, further comprising mRNA.

15. Use of the ionizable lipids as defined in any one of claims 1 to 9 in the preparation of lipid nanoparticles or lipid nanoparticle compositions.

16. A pharmaceutical composition comprising lipid nanoparticles or a composition of lipid nanoparticles as defined in any one of claims 10 to 14, and a pharmaceutically acceptable agent.

17. Use of the pharmaceutical composition as defined in claim 16 in the preparation of a medicament.

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