WO2022207938A1

WO2022207938A1 - Ionizable lipids and lipid nanoparticles comprising said ionizable lipids for delivery of therapeutic agents

Info

Publication number: WO2022207938A1
Application number: PCT/EP2022/058841
Authority: WO
Inventors: Bruno De Geest; Emily DE LOMBAERDE; Niek Sanders; Yong Chen; Zifu ZHONG
Original assignee: Universiteit Gent
Priority date: 2021-04-02
Filing date: 2022-04-04
Publication date: 2022-10-06
Also published as: EP4313937A1

Abstract

A novel class of ionizable lipid compounds as well as lipid nanoparticles comprising said ionizable lipid compounds is provided. Advantageously, upon hydrolysis of an internal ester bond, which is likely to occur under typical in vivo conditions, a zwitterionic lipid structure is obtained, which is no longer capable of complexing a nucleic acid, which will thus be released from the lipid nanoparticle and subsequently is able to perform its therapeutic function.

Description

IONIZABLE LIPIDS AND LIPID NANOPARTICLES COMPRISING SAID IONIZABLE LIPIDS FOR DELIVERY OF THERAPEUTIC AGENTS FIELD The present application relates to ionizable lipid compounds suitable for use in lipid nanoparticles for delivery of a therapeutic agent, such as a nucleic acid, to a subject in need thereof. BACKGROUND The recent success of mRNA vaccines may in part be attributed to the development of lipid nanoparticle (LNP) delivery systems. The nanostructural properties of the mRNA LNP bear a resemblance to viral systems in terms of their size, lipid envelope and the internal genomic material that contributes to their application as delivery vehicles for vaccines and other therapeutics. Incorporation into lipid nanoparticles also protects the mRNA from enzymatic attack and enhances cell uptake. In an example of a mRNA lipid nanoparticle structure, the mRNA is bound by an ionizable lipid that occupies the central core of the LNP. Polyethylene glycol (PEG) lipid forms the surface of the LNP, along with distearoylphosphatidylcholine (DSPC) and/or 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), which is bilayer forming. Cholesterol and the ionizable lipid (in charged and uncharged forms) can be distributed throughout the LNP. Nevertheless, effective in vivo delivery of active agents such as small molecule drugs, proteins, peptides, and nucleic acids represents a continuing medical challenge. In the context of LNP delivery systems, research has focused not only on the type and concentration of the lipids, but also on LNP assembly and manufacturing. There thus remains a need for novel ionizable lipids and improved LNP delivery systems comprising said novel ionizable lipids. SUMMARY The present inventors have developed a novel class of ionizable lipids, particularly ionizable cationic lipids, which, when present in lipid nanoparticles, such as LNP encapsulating mRNA, have improved degradability, improved RNA transfection efficiency and/or reduced toxicity. In particular, the hydrolysis of an internal ester bond in the lipid structure, which is likely to occur under typical in vivo conditions, results in the formation of a zwitterionic lipid structure, which is no longer capable of complexing the nucleic acid, in particular mRNA, and, hence, the nucleic acid will be released and able to perform its in vivo therapeutic function. In addition, the ionizable lipids according to the invention comprise two linear or branched hydrocarbon chains, each connected to the ionizable amine via a C_2-20alkyl chain and a degradable ester bond. Upon hydrolysis of the degradable ester bonds, the linear or branched hydrocarbon chains are released from the zwitterionic lipid structure without impacting its charge shifting capacity. Hence, the ionizable lipids according to the invention allow degradation of the linear or branched hydrocarbon chains under in vivo conditions, thereby reducing or even avoiding accumulation of the linear or branched hydrocarbon chains. Accordingly, a first aspect the present application provides an ionizable lipid having a structure of Formula (I), or in particular of Formula (IA)

- wherein R¹ is hydrogen, C_1-12alkyl,

- wherein Y¹, Y², and Y³ are each independently C_1-12 alkyl, and wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C_1-8alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, optionally comprising O or S; - wherein R² and R³ are each independently C_2-20alkyl, preferably wherein R² and R³ are each independently C_2-6alkyl; - wherein R⁴ and R⁵ are each independently selected from C3-27alkyl or C3-27alkenyl; and - wherein R⁶ is C_2-10alkyl, preferably wherein R⁶ is C_2-6alkyl. Preferably, in an aspect, the invention provides an ionizable lipid having a structure of Formula (IA)

( ) - wherein R¹ is hydrogen, C_1-12alkyl, , whe ^{1 2}

rein Y , Y , and Y³ are each independently C_1-12 alkyl, and wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C_1-8alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, optionally comprising O or S; - wherein R² and R³ are each independently C2-20alkyl, preferably wherein R² and R³ are each independently C_2-6alkyl; more preferably wherein R² and R³ are each independently C2-3alkyl; most preferably wherein R² and R³ are ethyl; and - wherein R⁴ and R⁵ are each independently selected from C_7-25alkyl or C_13-21alkenyl; with the proviso that the ionizable lipid is not a compound having a structure of Formula (IA) wherein R¹ is methyl, R² and R³ are each ethyl, and R⁴ and R⁵ are each CH₃(CH₂)₁₆-. In embodiments, R⁴ and R⁵ may be each independently selected from CH₃(CH₂)₆-, CH₃(CH₂)8-, CH₃(CH₂)₁₀-, CH₃(CH₂)₁₂-, CH₃(CH₂)₁₄-, CH₃(CH₂)₁₆-, CH₃(CH₂)₁₈-, CH₃(CH₂)₂₀-, CH₃(CH₂)₂₂-, CH₃(CH₂)24-, [CH₃(CH₂)7][CH₃(CH₂)₅]CH-, CH₃(CH₂)3CH=CH(CH₂)7-, CH₃(CH₂)₅CH=CH(CH₂)7-, CH₃(CH₂)8CH=CH(CH₂)4-, CH₃(CH₂)7CH=CH(CH₂)7-, CH₃(CH₂)₅CH=CH(CH₂)9-, CH₃(CH₂)4CH=CHCH₂CH=CH(CH₂)7-, CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)7-, CH₃(CH₂)4CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)3-, CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)3-, CH₃(CH₂)7CH=CH(CH₂)11-, or CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)2-. Preferably, in a further aspect, the invention provides an ionizable lipid having a structure of Formula (IA), - wherein R¹ is hydrogen, C_1-12alkyl, , , or , wherein Y¹, Y², and Y³ are each independently C_1-12 alkyl, and wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C_1-8alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S, as heteroatom(s); - wherein R² and R³ are each independently C_2-20alkyl, preferably wherein R² and R³ are each independently C_2-6alkyl; more preferably wherein R² and R³ are each independently C_2-3alkyl; most preferably wherein R² and R³ are ethyl; and - wherein R⁴ and R⁵ are each independently selected from CH₃(CH₂)₆-, CH₃(CH₂)₈-,

In particular embodiments, the ionizable lipid has a structure according to formula (I) or (IA), wherein R² and R³ are each independently C2-10alkyl; preferably wherein R² and R³ are each independently C_2-6alkyl; more preferably wherein R² and R³ are each independently C2-4alkyl; even more preferably wherein R² and R³ are each independently ethyl or n-propyl. In even more particular embodiments, R² is equal to R³ and R² and R³ are each a -CH₂CH₂- or - CH₂CH₂CH₂- spacer. In certain embodiments, the ionizable lipid thus has a structure according to Formula (II) or (III), , (II) (III) wherein R¹, R⁴, and R⁵ have the same meaning as defined herein. In certain embodiments, the ionizable lipid thus has a structure according to Formula (II), wherein R¹, R⁴, and R⁵ have the same meaning as defined herein. In particular embodiments, the ionizable lipid has a structure according to formula (I), (IA), (II) or (III), wherein R¹ is hydrogen, C_1-6alkyl,

; wherein Y¹, Y², and Y³ are each independently C_1-6alkyl, preferably wherein Y¹, Y², and Y³ are each independently C_2-4alkyl; and wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C_1-6alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, optionally comprising O or S, and wherein R², R³, R⁴, and R⁵ have the same meaning as defined herein. In more particular embodiments, wherein R¹ is hydrogen, methyl, ethyl, n-propyl,

; wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C1-3alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵- N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, optionally comprising O or S, preferably wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure comprising one or two nitrogen atoms and from 2 to 8 carbon atoms, and optionally an oxygen or sulphur atom, more preferably, wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a piperazine, azepane (hexahydroazepine), pyrrolidine, imidazolidine, piperidine, morpholine, thiomorpholine, azocane, or azonane ring structure. In even more preferred embodiments, R¹ is H, CH₃, ethyl, n-propyl, , ,

or . In embodiments, R¹ is or . In particular embodiments, the ionizable lipid has a structure of Formula (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), or (XIII),

(VIII) (IX) (X) (XI) (XII) (XIII) wherein R⁴ and R⁵ have the same meaning as defined herein. In particular embodiments, the ionizable lipid has a structure of Formula (IV), (V), (VI), (VII), or (VIII), wherein R⁴ and R⁵ have the same meaning as defined herein. In particular embodiments, the ionizable lipid has a structure according to Formula (I) or (IA), (II) or (III), wherein R⁴ and R⁵ are each independently selected from C_7-25alkyl or C_13-21alkenyl; preferably R⁴ and R⁵ are each independently selected from CH₃(CH₂)₆-, CH₃(CH₂)₈-, CH₃(CH₂)₁₀-, CH₃(CH₂)₁₂-, CH₃(CH₂)₁₄-, CH₃(CH₂)₁₆-, CH₃(CH₂)₁₈-, CH₃(CH₂)₂₀-, CH₃(CH₂)₂₂-, CH₃(CH₂)₂₄-, [CH₃(CH₂)₇][CH₃(CH₂)₅]CH-, CH₃(CH₂)₃CH=CH(CH₂)₇-, CH₃(CH₂)₅CH=CH(CH₂)₇-, CH₃(CH₂)₈CH=CH(CH₂)₄-, CH₃(CH₂)₇CH=CH(CH₂)₇-, CH₃(CH₂)₅CH=CH(CH₂)₉-,

embodiments, R⁴ and R⁵ may be each independently selected from

R⁴ and R⁵ are each independently selected from [CH₃(CH₂)7][CH₃(CH₂)₅]CH-, CH₃(CH₂)3CH=CH(CH₂)7-, CH₃(CH₂)₅CH=CH(CH₂)7-, CH₃(CH₂)8CH=CH(CH₂)4-, CH₃(CH₂)7CH=CH(CH₂)7-, or CH₃(CH₂)₅CH=CH(CH₂)9-; more preferably R⁴ and R⁵ are each independently selected from [CH₃(CH₂)7][CH₃(CH₂)₅]CH- or CH₃(CH₂)7CH=CH(CH₂)7-. In particular embodiments, R⁴ and R⁵ are the same or different; preferably R⁴ and R⁵ are the same. In embodiments, R⁴ and R⁵ may be the same and may be selected from [CH₃(CH₂)7][CH₃(CH₂)₅]CH-, CH₃(CH₂)3CH=CH(CH₂)7-, CH₃(CH₂)₅CH=CH(CH₂)7-, CH₃(CH₂)8CH=CH(CH₂)4-, CH₃(CH₂)7CH=CH(CH₂)7-, or CH₃(CH₂)₅CH=CH(CH₂)9-. In embodiments, R⁴ and R⁵ may be the same and may be selected from [CH₃(CH₂)7][CH₃(CH₂)₅]CH- or CH₃(CH₂)7CH=CH(CH₂)7-. Another aspect of the present application provides a method for producing an ionizable lipid as envisaged herein, the method comprising: - reacting a diC2-20alcoholamine with a compound of formula Br-R⁶-C(=O)OCH₃, wherein R⁶ is -(CH₂)_m-, wherein m is 2 to 6, or with a methylacrylate, thereby obtaining an intermediate having a structure of Formula (XIV) or having a structure of Formula (XIVA), respectively;

( ) (XIVA) - purifying the intermediate of Formula (XIV) or Formula (XIVA); and - reacting the intermediate of Formula (XIV) or Formula (XIVA) with (i) a C_3-27alkyl acid chloride or C_3-27alkenyl acid chloride or with (ii) a C_3-27alkyl acid or C_3-27alkenyl acid in the presence of an activating agent, thereby obtaining an ionizable lipid having a structure of Formula (I.1) or Formula (IA.1), respectively,

wherein R² and R³ are each independently C2-20alkyl, preferably wherein R² and R³ are each independently C_2-6alkyl; and wherein R⁴ and R⁵ are each independently selected from C3-27alkyl or C3-27alkenyl. In certain embodiments, the method as envisaged herein further comprises: - reacting the ionizable lipid having a structure of Formula (I.1) or Formula (IA.1) in an alkaline medium or in a mixture of THF/H₂O/NaOH; and - purifying an ionizable lipid having a structure of Formula (I.2) or Formula (IA.2), respectively,

wherein R² and R³ are each independently C_2-20alkyl, preferably wherein R² and R³ are each independently C_2-6alkyl; and wherein R⁴ and R⁵ are each independently selected from C_3-27alkyl or C_3-27alkenyl. In certain embodiments, the method further comprises: - reacting the ionizable lipid having a structure of Formula (I.2) or Formula (IA.2) with oxalylchloride and dimethylformamide (DMF), thereby obtaining an acid chloride lipid intermediate having a structure of Formula (XV) or Formula (XVA), respectively; and

(XVA) - reacting the acid chloride lipid intermediate of Formula (XV) or Formula (XVA) with

, wherein Y¹, Y², and Y³ are each independently C_1-12 alkyl, Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C1-8alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, optionally comprising O or S , thereby obtaining an ionizable lipid having a structure of Formula (I.3), (I.4), (I.5), (IA.3), (IA.4) or (IA.5)

(I.3) (I.4) (I.5).

(IA.3) (IA.4) (IA.5) wherein R² and R³ are each independently C_2-20alkyl, preferably wherein R² and R³ are each independently C_2-6alkyl; and wherein R⁴ and R⁵ are each independently selected from C_3-27alkyl or C_3-27alkenyl. Preferably, in an aspect, the invention provides a method for producing an ionizable lipid as envisaged herein, the method comprising: - reacting a diC_2-20alcoholamine with a compound of formula Br-R⁶-C(=O)OCH₃, wherein R⁶ is -(CH₂)_m-, wherein m is 2, or with a methylacrylate, thereby obtaining an intermediate having a structure of formula (XIVA);

- purifying the intermediate of formula (XIVA); and - reacting the intermediate of formula (XIVA) with (i) a C7-25alkyl acid chloride or C13- 21alkenyl acid chloride or (ii) a C7-25alkyl acid or C13-21alkenyl acid in the presence of an activating agent, thereby obtaining an ionizable lipid having a structure of Formula (IA.1), (IA.1) wherein R² and R³ are each independently C2-20alkyl; and wherein R⁴ and R⁵ are each independently selected from C7-25alkyl or C13-21alkenyl; with the proviso that the ionizable lipid is not a compound having a structure of Formula (IA.1) wherein R² and R³ are each ethyl, and R⁴ and R⁵ are each CH₃(CH₂)₁₆-. Preferably, in a further aspect, the invention provides a method for producing an ionizable lipid, the method comprising: - reacting a diC2-20alcoholamine with a compound of formula Br-R⁶-C(=O)OCH₃, wherein R⁶ is -(CH₂)m-, wherein m is 2, or with a methylacrylate, thereby obtaining an intermediate having a structure of formula (XIVA); - purifying the intermediate of formula (XIVA); and - reacting the intermediate of formula (XIVA) with (i) R⁴-COOCl or R⁵-COOCl, or (ii) R⁴- COOH or R⁵-COOH in the presence of an activating agent, thereby obtaining an ionizable lipid having a structure of Formula (IA.1), wherein R² and R³ are each independently C2-20alkyl; and wherein R⁴ and R⁵ are each independently selected from CH₃(CH₂)6-, CH₃(CH₂)8-, CH₃(CH₂)10- , CH₃(CH₂)12-, CH₃(CH₂)14-, CH₃(CH₂)18-, CH₃(CH₂)20-, CH₃(CH₂)22-, CH₃(CH₂)24-, [CH₃(CH₂)₇][CH₃(CH₂)₅]CH-, CH₃(CH₂)₃CH=CH(CH₂)₇-, CH₃(CH₂)₅CH=CH(CH₂)₇-, CH₃(CH₂)₈CH=CH(CH₂)₄-, CH₃(CH₂)₇CH=CH(CH₂)₇-, CH₃(CH₂)₅CH=CH(CH₂)₉-, CH₃(CH₂)₄CH=CHCH₂CH=CH(CH₂)₇-, CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)₇-, CH₃(CH₂)₄CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)₃-, CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)₃-, CH₃(CH₂)₇CH=CH(CH₂)₁₁-, or CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)₂-. In embodiments, the methods as taught herein may further comprise: - reacting the ionizable lipid having a structure of Formula (IA.1) in an alkaline medium or in a mixture of THF/H₂O/NaOH; and - purifying an ionizable lipid having a structure of Formula (IA.2) (IA.2). In embodiments, the methods as taught herein may further comprise: - reacting the ionizable lipid having a structure of Formula (IA.2) with oxalylchloride and dimethylformamide (DMF), thereby obtaining an acid chloride lipid intermediate having a structure of Formula (XVA); and

- reacting the acid chloride lipid intermediate of formula (XVA) with

, , or , wherein Y¹, Y², and Y³ are each independently C_1-12 alkyl, Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C1-8alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S, as heteroatom(s), thereby obtaining an ionizable lipid having a structure of Formula ((IA.3), (IA.4) or (IA.5)

(IA.5). Another aspect of the present application provides a lipid nanoparticle (LNP) comprising an ionizable lipid according to Formula (I) or Formula (IA) as specified herein. Preferably, in an aspect, the invention provides a lipid nanoparticle (LNP) comprising an ionizable lipid, wherein the ionizable lipid has a structure of Formula (IA), wherein R¹ is hydrogen, C_1-12alkyl,

, , or , wherein Y¹, Y², and Y³ are each independently C_1-12 alkyl, and wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C_1-8alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S, as heteroatom(s); wherein R² and R³ are each independently C_2-20alkyl; wherein R⁴ and R⁵ are each independently selected from C_7-25alkyl or C_13-21alkenyl. In particular embodiments, the LNP further comprises: - a PEGylated lipid; preferably wherein the PEGylated lipid is selected from the group consisting of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPE)-PEG, distearoyl-rac-glycerol (DSG)-PEG, or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, wherein the molecular weight of PEG ranges from 1-10 kDa; - a helper lipid; preferably wherein the helper lipid is selected from the group consisting of a sterol, particularly cholesterol, dioleoylphosphatidylethanolamine (DOPE), and 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC); and/or - a therapeutic agent, preferably a nucleic acid, such as DNA or RNA; more preferably mRNA. Another aspect of the present application provides for the use of an ionizable lipid as envisaged herein for producing a lipid nanoparticle (LNP). Another aspect of the present application provides a method of preparing lipid nanoparticles (LNP) comprising a therapeutic agent, the method comprising: - mixing an aqueous solution comprising a therapeutic agent with an organic solvent, such as an ethanolic solution, comprising an ionizable lipid according to Formula (I) or Formula (IA) as specified herein, a PEGylated lipid, and a helper lipid, particularly cholesterol; and - removing the organic solvent such as ethanol, thereby obtaining LNP comprising the therapeutic agent. Preferably, in an aspect, the invention relates to method of preparing lipid nanoparticles (LNP) comprising a therapeutic agent, the method comprising: - mixing an aqueous solution comprising a therapeutic agent with an organic solvent comprising an ionizable lipid, a PEGylated lipid, and a helper lipid, wherein the ionizable lipid has a structure of Formula (IA), wherein R¹ is hydrogen, C_1-12alkyl,

, wherein Y¹, Y², and Y³ are each independently C_1-12 alkyl, and wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C1-8alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S, as heteroatom(s); wherein R² and R³ are each independently C2-20alkyl; wherein R⁴ and R⁵ are each independently selected from C7-25alkyl or C13-21alkenyl; and - removing the organic solvent, thereby obtaining LNP comprising the therapeutic agent. In particular embodiments, the PEGylated lipid is selected from the group consisting of DMG- PEG, DSPE-PEG, DSG-PEG, or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, wherein the molecular weight of PEG ranges from 1-10 kDa; and/or the helper lipid is selected from the group consisting of a sterol, particularly cholesterol, DOPE, and DSPC. In particular embodiments, the step of removing the organic solvent such as ethanol is performed by dialysis, spin filtration, or evaporation. In embodiments, the therapeutic agent is a nucleic acid, such as DNA or RNA, preferably wherein the therapeutic agent is mRNA; and/or the therapeutic agent is a protein. A further aspect provides a lipid nanoparticle comprising a therapeutic agent, for use in human or veterinary medicine, in particular for use in a method of delivering a therapeutic agent to a subject, particularly for use in a method of delivering a nucleic acid to a subject, wherein the lipid nanoparticle comprises an ionizable lipid according to Formula (I) or Formula (IA) as specified herein. Preferably, in an aspect, the invention relates to a lipid nanoparticle comprising a therapeutic agent, for use in a method of delivering a therapeutic agent to a subject, particularly for use in a method of delivering a nucleic acid to a subject, wherein the lipid nanoparticle comprises an ionizable lipid having a structure of Formula (IA), wherein R¹ is hydrogen,

, wherein Y¹, Y², and Y³ are each independently C_1-12 alkyl, and wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C1-8alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S, as heteroatom(s); wherein R² and R³ are each independently C2-20alkyl; wherein R⁴ and R⁵ are each independently selected from C7-25alkyl or C13-21alkenyl. The present application also provides a method for delivering a therapeutic agent to a subject, wherein the method comprises administering a composition comprising a lipid nanoparticle comprising a therapeutically effective amount of the therapeutic agent to the subject, wherein the lipid nanoparticle comprises an ionizable lipid according to Formula (I) or Formula (IA) as specified herein. DESCRIPTION OF THE FIGURES Figure 1 represents an ionizable lipid according to an embodiment of the present application (structure A), which upon hydrolysis of the -C(=O)OR¹ ester bond is converted in a zwitterionic structure (structure B). Further hydrolysis of the fatty acid ester bonds releases the fatty acid lipids of the ionizable lipid and yields structure C. R¹ and R⁶ are as further specified herein. R² and R³ are carbon spacers, as further specified herein. R⁴ and R⁵ are linear or branched, and/or saturated or unsaturated alkyl chains, as further specified herein. Figure 2 represents the chemical structure (FIG.2A) and ¹H-NMR spectrum (FIG.2B) of SME. FIG. 2A shows the chemical structure with numbered atom positions. In FIG. 2B, peaks are annotated with the corresponding atom position and integrated. X-axis shows chemical shift in ppm, Y-axis shows peak intensity in arbitrary units. Figure 3 represents bar graphs illustrating the total photon of human HeLa cells transfected with LNP-formulated luciferase-encoding mRNA with control (blank) or ionizable lipids (SME, OME, BME, OC7, BC7, ODMA, or BDMA) at N:P ratio of 5:1 (FIG.3A), N:P ratio of 10:1 (FIG. 3B), or N:P ratio of 20:1 (FIG.3C) (n=4). Figure 4 represents bar graphs illustrating the total photon of murine colon adenocarcinoma MC38 cells transfected with LNP-formulated luciferase-encoding mRNA with control (blank) or ionizable lipids (SME, OME, BME, OC7, BC7, ODMA, or BDMA) at N:P ratio of 5:1 (FIG.4A), N:P ratio of 10:1 (FIG.4B), or N:P ratio of 20:1 (FIG.4C) (n=4). Figure 5 represents bar graphs illustrating the total photon of mouse dendritic DC2.4 cells transfected with LNP-formulated luciferase-encoding mRNA with control (blank) or ionizable lipids (SME, OME, BME, OC7, BC7, ODMA, or BDMA) at N:P ratio of 5:1 (FIG.5A), N:P ratio of 10:1 (FIG.5B), or N:P ratio of 20:1 (FIG.5C) (n=4). Figure 6 represents a graph illustrating the cell viability (%) in function of mRNA concentration (μg/ml) measured by MTT assay of human HeLa cells treated with LNP-formulated mRNA with ionizable lipids (SME, OC7, ODMA, or BDMA) (n=5). Figure 7 represents graphs illustrating the total photon flux (p/s) in vivo at the site of administration of LNP-formulated luciferase-encoding mRNA with ionizable lipids OC7 (FIG. 7A), ODMA (FIG. 7B), or BDMA (FIG. 7C) (n=4). Expression was monitored as a function of time (FIG.7A-C) and analysed as area under the curve (AUC) (FIG.7D). DESCRIPTION As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements, or method steps. The terms also encompass “consisting of” and “consisting essentially of”. The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. The term “about” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of and from the specified value, in particular variations of +/-10% or less, preferably +/-5% or less, more preferably +/-1% or less, and still more preferably +/-0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” refers is itself also specifically, and preferably, disclosed. Whereas the term “one or more”, such as one or more members of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any ^3, ^4, ^5, ^6 or ^7 etc. of said members, and up to all said members. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination. All documents cited in the present specification are hereby incorporated by reference in their entirety. Unless otherwise specified, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions may be included to better appreciate the teaching of the present invention. When describing the ionizable lipid compounds as taught herein, the terms used are to be construed in accordance with the following definitions, unless a context dictates otherwise. The term “alkyl”, as a group or part of a group, refers to a hydrocarbyl group of Formula CnH₂n+1 wherein n is a number of at least 1. Alkyl groups may be linear, or branched. When a subscript is used herein following a carbon atom, the subscript refers to the number of carbon atoms that the named group may contain. For example, the term “C3-27alkyl”, as a group or part of a group, refers to a hydrocarbyl group of Formula CnH₂n+1 wherein n is a number ranging from 3 to 27, i.e. 3 ≤ n ≤ 27. Thus, for example, C_3-27alkyl groups include all linear or branched alkyl groups having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 carbon atoms. The term “lower alkyl groups” as used herein refers to an alkyl group which typically comprises from 1 to 10 carbon atoms (i.e. C1-10alkyl), preferably from 1 to 6 carbon atoms (i.e. C1-6alkyl), more preferably 1, 2, 3, 4, 5 or 6 carbon atoms, which may be linear or branched. A lower alkyl group thus includes for example methyl, ethyl, n- propyl, i-propyl, 2-methyl-ethyl, butyl and its isomers (e.g. n-butyl, i-butyl and t-butyl); pentyl and its isomers, and hexyl and its isomers, and the like. The term “alkenyl” as a group or part of a group, refers to an unsaturated hydrocarbyl group, which may be linear or branched, comprising one or more carbon-carbon double bonds, such as 1, 2, 3 or 4 carbon-carbon double bonds. When a subscript is used herein following a carbon atom, the subscript refers to the number of carbon atoms that the named group may contain. Thus, for example, a C3-27alkenyl group includes all linear or branched alkyl groups having 3 to 27 carbon atoms, i.e. having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 carbon atoms, and comprising one or more, such as one, two, three or four carbon-carbon double bonds. The terms “fatty acid” or “fatty acid moiety” as used herein generally refers to carboxylic acid with a saturated or unsaturated aliphatic chain of carbon atoms. The term “fatty acid” includes saturated and unsaturated fatty acids. The fatty acids or fatty acid moieties may be naturally occurring or synthetic fatty acids or fatty acid moieties. As used herein, a fatty acid moiety typically has the formula R⁴-C(=O)O- or R⁵-C(=O)O-, wherein R⁴ and R⁵ are an alkyl or an alkenyl group as further defined herein. The term “saturated fatty acid” refers to a carboxylic acid with an aliphatic chain of carbon atoms having the Formula CH₃(CH₂)nCOOH, wherein n is an integer selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. The term “unsaturated fatty acid” refers to a carboxylic acid with an aliphatic chain of carbon atoms having one or more double bonds between carbon atoms. The term “branched fatty acids” or “branched chain fatty acids” refer to fatty acids comprising a main saturated or unsaturated aliphatic chain of carbon atoms, which is substituted with one or more lower alkyl groups, particularly substituted with a C_1-6alkyl group, such as a methyl, ethyl, n-propyl, butyl, pentyl or hexyl. Stated differently, branched fatty acids refer to carboxylic acids having a saturated or unsaturated aliphatic chain of carbon atoms, wherein said aliphatic chain of carbon atoms comprises at least one tertiary carbon atom or possibly a quaternary carbon atom, i.e. a carbon atom which is bound to three or four other carbon atoms, respectively. The term “substituted” as used in the present application is meant to indicate that one or more hydrogens on the atom indicated in the expression using “substituted” is replaced with a selection from the indicated group, provided that the indicated atom’s normal valency is not exceeded, and that the substitution results in a chemically stable compound. In the context of the present application, particular substituents include alkyl groups, more in particular lower alkyl groups. The term “saturated heterocyclic ring” or “saturated heterocyclic structure” generally refers to a ring structure, wherein the ring atoms comprise carbon atoms and one or more non-carbon or heteroatoms, preferably nitrogen, oxygen or sulphur. The term “unsaturated heterocyclic ring” or “unsaturated heterocyclic structure” generally refers to a ring structure, wherein the ring atoms comprise carbon atoms and one or more non-carbon or heteroatoms, preferably nitrogen, oxygen or sulphur, wherein the ring structure comprises at least one double bond, particularly a carbon-carbon double bond. An unsaturated heterocyclic ring may be an aromatic ring, also referred to as a heteroaryl ring. The terms “molecular weight” or “molecular mass”, as used herein, refer to the mass of a molecule. The molecular mass can be measured directly using mass spectrometry. The present disclosure generally relates to a novel class of ionizable lipids and their use in the manufacture of LNP as a delivery system for a therapeutic agent, particularly a nucleic acid, such as mRNA. In particular, the ionizable lipids according to the present invention have a charge-shifting feature. As represented in Figure 1, the ionizable lipid comprises an ionizable amine (Structure A). Upon hydrolysis of the R¹-O-C(=O) ester bond, which is likely to occur in vivo, e.g. in endosomal compartments or in the cellular cytoplasm, the cationic charge of the ionizable amine is compensated by an anionic charge of a carboxylic acid, resulting in a zwitterionic structure (Structure B), for instance at pH conditions between pH 1 and pH 14, particularly between pH 5 and pH 7.4. The latter is no longer capable of complexing a nucleic acid, such as mRNA, and hence, the nucleic acid will be released from the lipid nanoparticle and is able to perform its therapeutic function. In addition, the ionizable lipids according to the invention comprise two linear or branched hydrocarbon chains each connected to the ionizable amine via a C2-20alkyl chain and a degradable ester bond. Upon hydrolysis of the degradable ester bonds, the linear or branched hydrocarbon chains are released from the structure without impacting the charge shifting capacity (Structure C). Hence, the ionizable lipids according to the invention allow satisfactory release of the therapeutic agent such as mRNA, while at the same time reducing or even avoiding accumulation of the linear or branched hydrocarbon chains under in vivo conditions. A first aspect the present application provides an ionizable lipid having a general structure of Formula (I) (I) , - wherein R¹ is hydrogen, C_1-12alkyl,

, wherein Y¹, Y², and Y³ are each independently C_1-12 alkyl, and wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C_1-8alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure comprising N as a heteroatom, and optionally further comprising O or S as heteroatom(s); - wherein R² and R³ are each independently C_2-20alkyl; - wherein R⁴ and R⁵ are each independently selected from C_3-27alkyl or C_3-27alkenyl; and - wherein R⁶ is C_2-10alkyl, preferably wherein R⁶ is C_2-6alkyl. Preferably, in an aspect, the invention provides an ionizable lipid having a structure of Formula (IA) - wherein R¹ is hydrogen, C_1-12alkyl,

, wherein Y¹, Y², and Y³ are each independently C_1-12 alkyl, and wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C1-8alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure comprising N as a heteroatom, and optionally further comprising O or S as heteroatom(s); - wherein R² and R³ are each independently C2-20alkyl, preferably wherein R² and R³ are each independently C_2-6alkyl; more preferably wherein R² and R³ are each independently C2-3alkyl; most preferably wherein R² and R³ are ethyl; and - wherein R⁴ and R⁵ are each independently selected from C_7-25alkyl or C_13-21alkenyl; with the proviso that the ionizable lipid is not a compound having a structure of Formula (IA) wherein R¹ is methyl, R² and R³ are each ethyl, and R⁴ and R⁵ are each CH₃(CH₂)₁₆-. In the structure of Formula (I), R⁶ can be defined as -(CH₂)m-, wherein m defines the length of the carbon spacer between the ionizable amine and the -C(=O)OR¹ moiety. In particular embodiments, m is an integer ranging from 2 to 10, from 2 to 8, or from 2 to 6, more in particular m is 2, 3, 4 or 5. In particular embodiments, when m is 2, an ionizable liquid having a structure of Formula (IA) is provided,

wherein R¹ is hydrogen, C_1-12alkyl,

, wherein Y¹, Y², and Y³ are each independently C_1-12 alkyl, and wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C1-8alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S as heteroatom(s); wherein R² and R³ are each independently C2-20alkyl; and wherein R⁴ and R⁵ are each independently selected from C3-27alkyl or C3-27alkenyl. In the structure of Formula (I) or Formula (IA), R² and R³ define the length of the carbon spacer between the ionizable amine and the R⁴-C(=O)O- or R⁵-C(=O)O- residues. R² and R³ may be the same or different. In particular embodiments, R² and R³ are the same. In particular embodiments, R² and R³ are each independently a lower alkyl. More in particular, R² and R³ are each independently C_2-6alkyl. In embodiments, R² and R³ are each independently C2-3alkyl. Even more in particular, R² and R³ are each independently -CH₂CH₂- (i.e. ethyl or a C2alkyl) or -CH₂CH₂CH₂- (i.e. n-propyl or a C3alkyl). In particular embodiments, when R² and R³ are the same and are -CH₂CH₂- or when R² and R³ are the same and are -CH₂CH₂CH₂-, an ionizable liquid of Formula (II) or (III), respectively, is provided, (II) (III) wherein R¹ is hydrogen, C_1-12alkyl,

, wherein Y¹, Y², and Y³ are each independently C_1-12 alkyl, and wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C1-8alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, optionally further comprising O or S as heteroatom(s); and wherein R⁴ and R⁵ are each independently selected from C3-27alkyl or C3- 27alkenyl. In the structure of Formula (I), Formula (IA), Formula (II) and Formula (III), R¹ is H or a group that is liberated upon hydrolysis of the -C(=O)-O- R¹ ester bond. R¹ is generally a C_1-12alkyl or a residue comprising at least one nitrogen. In particular embodiments, R¹ is a lower alkyl, more in particular R¹ is a C1-6alkyl. In particularly preferred embodiments, R¹ is methyl, ethyl or n-propyl. In other particular embodiments, R¹ is a C_1-12alkyl group substituted with an amine, i.e. R¹ is a residue of formula , wherein Y¹ is a C_1-12 alkyl, and Z¹ and Z² are each independently C_1-8alkyl or hydrogen; more in particular wherein Y¹ is a C_2-4alkyl and Z¹ and Z² are each independently C_1-6alkyl or C_1-3alkyl. In particularly preferred embodiments, R¹ is -CH₂CH₂N(CH₃)₂, -CH₂CH₂CH₂N(CH₃)₂, -CH₂CH₂N(CH₂CH₃)₂ or -CH₂CH₂CH₂N(CH₂CH₃)₂. In other particular embodiments, R¹ comprises a saturated or unsaturated heterocyclic structure comprising one or two nitrogen atoms, and optionally an oxygen or sulphur atom, as the heteroatom, linked to a carbon spacer Y² or Y³. Stated differently, R¹ is a residue of formula or , wherein Y² and Y³ are a C_1-12 alkyl, and Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C1-8alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S, as heteroatom(s), more in particular wherein Y² and Y³ are a C2-4alkyl and Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C1-6alkyl or C1-3alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N- Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S, as heteroatom(s). In particularly preferred embodiments, Y² and Y³ are -CH₂CH₂- or -CH₂CH₂CH₂-. In particularly preferred embodiments, the N-Z³-Z⁴ or the N-Z⁵- N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure comprising one or two nitrogen atoms, from 2 to 8 carbon atoms and optionally an oxygen or sulphur atom. In certain embodiments, the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a heteroaryl structure comprising one or two nitrogen atoms and, optionally an oxygen or sulphur atom, as the heteroatom(s). Non limiting examples of such saturated or unsaturated heterocyclic structures N-Z³-Z⁴ or N-Z⁵-N-Z⁶ include aziridine, azirine, azetidine, azete, diazetidine, diazete, pyrrolidine, pyrroline, pyrrole, imidazolidine, imidazole, pyrazolidine, pyrazole, thiazolidine, thiazole, isothiazolidine, isothiazole, piperidine, pyridine, diazinane (e.g. piperazine, hexahydropyrimidine, hexahydropyridazine), diazine (e.g. pyrazine, pyrimidine, pyridazine), morpholine, oxazine, thiomorpholine, thiazine, azepane (hexahydroazepine), azepine, azocane, azocine, azonane, or azonine ring structures. In particular embodiments, the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated heterocyclic structure comprising one or two nitrogen atoms, from 2 to 8 carbon atoms and optionally an oxygen or sulphur atom. In embodiments, the N-Z³-Z⁴ cyclic structure is aziridine, azirine, azetidine, azete, pyrrolidine, pyrroline, pyrrole, piperidine, pyridine, azepane (hexahydroazepine), azepine, azocane, azocine, azonane, or azonine ring structure. In embodiments, the N-Z³-Z⁴ cyclic structure is azepane (hexahydroazepine). In embodiments, the N-Z⁵-N-Z⁶ cyclic structure is diazetidine, diazete, imidazolidine, imidazole, pyrazolidine, pyrazole, diazinane (e.g. piperazine, hexahydropyrimidine, hexahydropyridazine), or diazine (e.g. pyrazine, pyrimidine, pyridazine) ring structure. In embodiments, the N-Z⁵-N-Z⁶ cyclic structure is piperazine. In embodiments, Z⁷ is methyl or ethyl. In embodiments of the products or methods as taught herein, R¹ may be or

; wherein Z¹, Z², Z³, and Z⁴ are each independently C_1-3alkyl and/or wherein the N- Z³-Z⁴ cyclic structure is a saturated or unsaturated heterocyclic structure, optionally comprising

O or S. In embodiments, R¹ may be or ; wherein Z¹, Z², Z³, and Z⁴ are each independently C1-3alkyl and/or wherein the N-Z³-Z⁴ cyclic structure is a saturated or unsaturated heterocyclic structure comprising one nitrogen atom and from 2 to 8 carbon atoms, and optionally an oxygen or sulphur atom, preferably wherein the N-Z³-Z⁴ cyclic structure is an azepane (hexahydroazepine), pyrrolidine, piperidine, morpholine, thiomorpholine, azocane, or azonane ring structure. Some particularly preferred ionizable lipids include the lipids according to Formula (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), or (XIII) as shown elsewhere herein, and wherein R⁴ and R⁵ are each independently selected from C3-27alkyl or C3-27alkenyl. In the structure of Formula (I), (IA), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), and (XIII), R⁴ and R⁵ correspond to the hydrophobic hydrocarbon chains of the lipid, wherein the hydrocarbon chain can be saturated or unsaturated. R⁴ and R⁵ may be the same or different. In particular embodiments, R⁴ and R⁵ are each independently selected from C_7-25alkyl or C_13-21alkenyl, more in particular R⁴ and R⁵ are each independently selected from CH₃(CH₂)₆-,

CH₃(CH₂)₅CH=CH(CH₂)₇-, CH₃(CH₂)₈CH=CH(CH₂)₄-, CH₃(CH₂)₇CH=CH(CH₂)₇-, CH₃(CH₂)₅CH=CH(CH₂)₉-, CH₃(CH₂)₄CH=CHCH₂CH=CH(CH₂)₇-, CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)₇-, CH₃(CH₂)₄CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)₃-, CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)₃-, CH₃(CH₂)₇CH=CH(CH₂)₁₁-, or CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)₂-. In embodiments of the products or methods as taught herein, R⁴ and R⁵ may be each independently selected from [CH₃(CH₂)7][CH₃(CH₂)₅]CH-, CH₃(CH₂)3CH=CH(CH₂)7-, CH₃(CH₂)₅CH=CH(CH₂)7-, CH₃(CH₂)8CH=CH(CH₂)4-, CH₃(CH₂)7CH=CH(CH₂)7-, CH₃(CH₂)₅CH=CH(CH₂)9-, CH₃(CH₂)4CH=CHCH₂CH=CH(CH₂)7-, CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)7-, CH₃(CH₂)4CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)3-, CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)3-, CH₃(CH₂)7CH=CH(CH₂)11-, or CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)2-. For instance, R⁴ and R⁵ may be each independently selected from [CH₃(CH₂)7][CH₃(CH₂)₅]CH-, CH₃(CH₂)3CH=CH(CH₂)7-, CH₃(CH₂)₅CH=CH(CH₂)7-, CH₃(CH₂)8CH=CH(CH₂)4-, CH₃(CH₂)7CH=CH(CH₂)7-, or CH₃(CH₂)₅CH=CH(CH₂)9-; preferably R⁴ and R⁵ are each independently selected from [CH₃(CH₂)7][CH₃(CH₂)₅]CH- or CH₃(CH₂)7CH=CH(CH₂)7-. In embodiments of the products or methods as taught herein, R⁴ and R⁵ may be the same and may be selected from [CH₃(CH₂)7][CH₃(CH₂)₅]CH-, CH₃(CH₂)3CH=CH(CH₂)7-, CH₃(CH₂)₅CH=CH(CH₂)7-, CH₃(CH₂)8CH=CH(CH₂)4-, CH₃(CH₂)7CH=CH(CH₂)7-, or CH₃(CH₂)₅CH=CH(CH₂)9-. In embodiments of the products or methods as taught herein, R⁴ and R⁵ are the same and are selected from [CH₃(CH₂)7][CH₃(CH₂)₅]CH- or CH₃(CH₂)7CH=CH(CH₂)7-. As shown in the example section, such ionizable lipids advantageously allow the formation of lipid nanoparticles comprising therapeutic agents such as mRNA which provide for efficient delivery of the therapeutic agent such as the mRNA both in vitro and in vivo, while at the same time also having low cytotoxicity. It is understood that R⁴ and R⁵ as defined herein together with the C(=O)O- to which it is bound forms a fatty acid moiety with formula R⁴-C(=O)O- or R⁵-C(=O)O-. In certain embodiments said fatty acid moiety is a saturated fatty acid moiety or an unsaturated fatty acid moiety. In preferred embodiments, said fatty acid moiety is a linear or branched fatty acid moiety. A preferred saturated fatty acid moiety typically comprises from 8 to 26 carbon atoms, more preferably from 12 to 24 carbon atoms, still more preferably from 12 to 18 carbon atoms. Non-limiting examples of suitable saturated fatty acids are caprylic acid (i.e., octanoic acid), capric acid (i.e., decanoic acid), lauric acid (i.e., dodecanoic acid), myristic acid (i.e., tetradecanoic acid), palmitic acid (i.e., hexadecanoic acid), stearic acid (i.e., octadecanoic acid), arachidic acid (i.e., eicosanoic acid), behenic acid (i.e., docosanoic acid), lignoceric acid (i.e., tetracosanoic acid), and cerotic acid (i.e., hexacosanoic acid). Preferred unsaturated fatty acid moieties comprises from 12 to 22 carbon atoms, more preferably from 14 to 22 carbon atoms. Non-limiting examples of suitable unsaturated fatty acids are myristoleic acid (i.e., (Z)- Tetradec-9-enoic acid or 9-cis-tetradecenoic acid), palmitoleic acid (i.e., hexadec-9-enoic acid or 9-cis-hexadecenoic acid), sapienic acid (i.e., (Z)-6-Hexadecenoic acid or cis-6- hexadecenoic acid), oleic acid (i.e., (9Z)-Octadec-9-enoic acid or cis-9-Octadecenoic acid), elaidic acid (i.e., (E)-octadec-9-enoic acid), vaccenic acid (i.e., (E)-Octadec-11-enoic acid), linoleic acid (i.e., (9Z,12Z)-9,12-Octadecadienoic acid), linoelaidic acid (i.e., (9E,12E)- octadeca-9,12-dienoic acid or trans, trans-9,12-octadecadienoic acid), α-linolenic acid (i.e., (9Z,12Z,15Z)-9,12,15-Octadecatrienoic acid), arachidonic acid (i.e., (5Z,8Z,11Z,14Z)- 5,8,11,14-Eicosatetraenoic acid), eicosapentaenoic acid (i.e., (5Z,8Z,11Z,14Z,17Z)- 5,8,11,14,17-icosapentaenoic acid), erucic acid (i.e., Z)-Docos-13-enoic acid), and docosahexaenoic acid (i.e., (4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoic acid). Accordingly, in certain embodiments R⁴ and R⁵, or the R⁴-C(=O)O- and R⁵-C(=O)O- moiety are each independently selected from the compounds listed in Table 1. Table 1

In particularly preferred embodiments, R⁴-C(=O)O- is the same as R⁵-C(=O)O- and is a saturated or unsaturated, and/or linear or branched C₁₄, C₁₆, C₁₈ or C₂₀ fatty acid moiety. In embodiments, R⁴-C(=O)O- is the same as R⁵-C(=O)O- and is a saturated or unsaturated, and/or linear or branched C16 or C18 fatty acid moiety. In certain embodiments, R⁴ and R⁵ are each independently selected from a branched C7-25alkyl or C13-21alkenyl. More in particular, R⁴-C(=O)O- and R⁵-C(=O)O- are each independently selected from a saturated or unsaturated C8, C10, C12, C14 or C16 fatty acid moiety, substituted with one or more lower alkyl substituents, particularly substituted with one or more methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl or octyl groups. In particular embodiments, R⁴- C(=O)O- and R⁵-C(=O)O- are each a 2-hexyldecanoic acid moiety. In embodiments of the products (e.g., ionizable lipids and lipid nanoparticles) or methods as taught herein, the ionizable lipid has a structure according to Formula (IA) or (II), wherein: - R¹ is hydrogen, methyl, ethyl, n-propyl,

; wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C_1-3alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, optionally comprising O or S, preferably wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure comprising one or two nitrogen atoms and from 2 to 8 carbon atoms, and optionally an oxygen or sulphur atom, more preferably wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a piperazine, azepane (hexahydroazepine), pyrrolidine, imidazolidine, piperidine, morpholine, thiomorpholine, azocane, or azonane ring structure; preferably R¹ is H, CH₃, ethyl, n-

propyl, , , or ; more preferbaly R¹ is or ; - R² and R³ are each independently C_2-10alkyl; preferably R² and R³ are each independently C_2-6alkyl; more preferably R² and R³ are each independently C_2-3alkyl; and - R⁴ and R⁵ are the same and each independently selected from [CH₃(CH₂)7][CH₃(CH₂)₅]CH-, CH₃(CH₂)3CH=CH(CH₂)7-, CH₃(CH₂)₅CH=CH(CH₂)7-, CH₃(CH₂)8CH=CH(CH₂)4-, CH₃(CH₂)7CH=CH(CH₂)7-, CH₃(CH₂)₅CH=CH(CH₂)9-, CH₃(CH₂)4CH=CHCH₂CH=CH(CH₂)7-, CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)7-, CH₃(CH₂)4CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)3-, CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)3-, CH₃(CH₂)7CH=CH(CH₂)11-, or CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)2-; preferably R⁴ and R⁵ are the same and are selected from [CH₃(CH₂)7][CH₃(CH₂)₅]CH-, CH₃(CH₂)3CH=CH(CH₂)7-, CH₃(CH₂)₅CH=CH(CH₂)7-, CH₃(CH₂)8CH=CH(CH₂)4-, CH₃(CH₂)7CH=CH(CH₂)7-, or CH₃(CH₂)₅CH=CH(CH₂)9-; more preferably, R⁴ and R⁵ are the same and are selected from [CH₃(CH₂)7][CH₃(CH₂)₅]CH- or CH₃(CH₂)7CH=CH(CH₂)7-. In embodiments of the products or methods as taught herein, the ionizable lipid has a structure according to Formula (IA), wherein: - R¹ is H, CH₃, ethyl, n-propyl,

; - R² and R³ are each independently C_2-6alkyl; and - R⁴ and R⁵ are the same and are selected from [CH₃(CH₂)₇][CH₃(CH₂)₅]CH-, CH₃(CH₂)₃CH=CH(CH₂)₇-, CH₃(CH₂)₅CH=CH(CH₂)₇-, CH₃(CH₂)₈CH=CH(CH₂)₄-, CH₃(CH₂)₇CH=CH(CH₂)₇-, or CH₃(CH₂)₅CH=CH(CH₂)₉-. In embodiments of the products or methods as taught herein, the ionizable lipid has a structure according to Formula (IA), wherein: -

; - R² and R³ are each independently C2-3alkyl; and - R⁴ and R⁵ are the same and are selected from [CH₃(CH₂)7][CH₃(CH₂)₅]CH- or CH₃(CH₂)7CH=CH(CH₂)7-. In embodiments of the products or methods as taught herein, the ionizable lipid has a structure according to Formula (II), wherein:

- R¹ is or ; wherein Z¹, Z², Z³, and Z⁴ are each independently C1-3alkyl and/or wherein the N-Z³-Z⁴ cyclic structure is a saturated or unsaturated heterocyclic structure, optionally comprising O or S, preferably wherein the N-Z³-Z⁴ cyclic structure is a saturated or unsaturated heterocyclic structure comprising one nitrogen atom and from 2 to 8 carbon atoms, and optionally an oxygen or sulphur atom, more preferably wherein the N-Z³-Z⁴ cyclic structure is an azepane (hexahydroazepine), pyrrolidine, piperidine, morpholine, thiomorpholine, azocane, or azonane ring structure; and - R⁴ and R⁵ are the same and are selected from [CH₃(CH₂)7][CH₃(CH₂)₅]CH-, CH₃(CH₂)3CH=CH(CH₂)7-, CH₃(CH₂)₅CH=CH(CH₂)7-, CH₃(CH₂)8CH=CH(CH₂)4-, CH₃(CH₂)7CH=CH(CH₂)7-, or CH₃(CH₂)₅CH=CH(CH₂)9-. In embodiments of the products or methods as taught herein, the ionizable lipid has a structure according to Formula (II), wherein: - R¹ is H, CH₃, ethyl, n-propyl, , , or ; and - R⁴ and R⁵ are the same and are selected from [CH₃(CH₂)₇][CH₃(CH₂)₅]CH-, CH₃(CH₂)₃CH=CH(CH₂)₇-, CH₃(CH₂)₅CH=CH(CH₂)₇-, CH₃(CH₂)₈CH=CH(CH₂)₄-, CH₃(CH₂)₇CH=CH(CH₂)₇-, or CH₃(CH₂)₅CH=CH(CH₂)₉-. In embodiments of the products or methods as taught herein, the ionizable lipid has a structure according to Formula (II), wherein:

- R¹ is or ; wherein Z¹, Z², Z³, and Z⁴ are each independently C1-3alkyl and/or wherein the N-Z³-Z⁴ cyclic structure is a saturated or unsaturated heterocyclic structure, optionally comprising O or S, preferably wherein the N-Z³-Z⁴ cyclic structure is a saturated or unsaturated heterocyclic structure comprising one nitrogen atom and from 2 to 8 carbon atoms, and optionally an oxygen or sulphur atom, more preferably wherein the N-Z³-Z⁴ cyclic structure is an azepane (hexahydroazepine), pyrrolidine, piperidine, morpholine, thiomorpholine, azocane, or azonane ring structure; and - R⁴ and R⁵ are the same and are selected from [CH₃(CH₂)7][CH₃(CH₂)₅]CH- or CH₃(CH₂)7CH=CH(CH₂)7-. In embodiments of the products or methods as taught herein, the ionizable lipid has a structure according to Formula (II), wherein: - R¹ is H, CH₃, ethyl, n-propyl,

; and - R⁴ and R⁵ are the same and are selected from [CH₃(CH₂)7][CH₃(CH₂)₅]CH- or CH₃(CH₂)7CH=CH(CH₂)7-. In embodiments of the products or methods as taught herein, the ionizable lipid has a structure according to Formula (II), wherein:

- R¹ is or ; and - R⁴ and R⁵ are the same and are selected from [CH₃(CH₂)7][CH₃(CH₂)₅]CH- or CH₃(CH₂)7CH=CH(CH₂)7-. Such ionizable lipids advantageously allow the formation of lipid nanoparticles comprising therapeutic agents such as mRNA which provide for efficient delivery of the therapeutic agent such as the mRNA both in vitro and in vivo, while at the same time having low cytotoxicity to human cells. In particularly preferred embodiments, the ionizable lipid has a structure according to Formula (II) or Formula (III), wherein: - R¹ is hydrogen, methyl, ethyl, n-propyl, -CH₂CH₂N(CH₃)2, -CH₂CH₂CH₂N(CH₃)2, - CH₂CH₂N(CH₂CH₃)₂, -CH₂CH₂CH₂N(CH₂CH₃)₂, or a residue of formula

, wherein Y² and Y³ are -CH₂CH₂- or -CH₂CH₂CH₂-, Z⁷ is methyl or ethyl, and wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure comprising one or two nitrogen atoms, and optionally an oxygen or sulphur atom, as the heteroatom(s). Non limiting examples of such saturated or unsaturated heterocyclic structures N-Z³-Z⁴ or N-Z⁵-N-Z⁶ include azetidine, pyrrolidine, pyrroline, pyrrolidine, pyrrole, pyrazolidine, pyrazole, imidazolidine, imidazole, piperidine, piperazine, pyridine, pyrimidine, pyrazine, morpholine, thiomorpholine, hexahydroazepine, azocane, or azonane ring structures, and - R⁴-C(=O)O- is the same as R⁵-C(=O)O- and is a saturated or unsaturated, or linear or branched C14, C16, C18 or C20 fatty acid moiety, most preferably is a saturated or unsaturated, or linear or branched C16 or C18 fatty acid moiety. In particular, the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a pyrrolidine, imidazolidine, piperidine, piperazine, morpholine, thiomorpholine, hexahydroazepine, azocane, or azonane ring structure. Another aspect of the present application provides a method for producing an ionizable lipid as envisaged herein, the method comprising: - reacting a diC2-20alcoholamine, preferably a diC_2-6alkoholamine, with a compound of formula Br-R⁶-C(=O)OCH₃, wherein R⁶ is –(CH₂)m-, wherein m is 2 to 6, thereby obtaining an intermediate having a structure of Formula (XIV);

wherein R² and R³ are each independently C2-20alkyl, preferably wherein R² and R³ are each independently C_2-6alkyl as specified elsewhere herein; - purifying the intermediate of Formula (XIV); and - reacting the intermediate of Formula (XIV) with (i) a C3-27alkyl acid chloride or C3-27alkenyl acid chloride or with (ii) a C3-27alkyl acid or C3-27alkenyl acid in the presence of an activating agent, thereby obtaining an ionizable lipid having a structure of Formula (I.1),

wherein R² and R³ are each independently C2-20alkyl, preferably wherein R² and R³ are each independently C_2-6alkyl; and wherein R⁴ and R⁵ are each independently selected from C3-27alkyl or C3-27alkenyl, as specified elsewhere herein. In certain embodiments, the method as envisaged herein further comprises: - reacting the ionizable lipid having a structure of Formula (I.1) in an alkaline medium or in a mixture of THF/H₂O/NaOH,; and - purifying an ionizable lipid having a structure of Formula (I.2), (I.2) wherein R² and R³ are each independently C2-20alkyl, preferably wherein R² and R³ are each independently C_2-6alkyl; and wherein R⁴ and R⁵ are each independently selected from C3- 27alkyl or C3-27alkenyl, as specified elsewhere herein. In certain embodiments, the method as envisaged herein further comprises: - reacting the ionizable lipid having a structure of Formula (I.2) with oxalylchloride and dimethylformamide (DMF), thereby obtaining an acid chloride lipid intermediate having a structure of Formula (XV); and

- reacting the acid chloride lipid intermediate of formula (XV) with in

wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure comprising one or two nitrogen atoms, and optionally an oxygen or sulphur atom, as the heteroatom(s), thereby obtaining an ionizable lipid having a structure of Formula (I.3), (I.4) or (I.5)

(I.3) (I.4) (I.5). wherein R² and R³ are each independently C2-20alkyl, preferably wherein R² and R³ are each independently C_2-6alkyl; and wherein R⁴ and R⁵ are each independently selected from C3- 27alkyl or C3-27alkenyl, as specified elsewhere herein. In a particular embodiment, the method for producing an ionizable lipid as envisaged herein comprises: - reacting a diC2-20alcoholamine, preferably a diC_2-6alcoholamine, more preferably diethanolamine, in a solution of methylacrylate, thereby obtaining an intermediate an intermediate with Formula (XIVA),

(XIVA), wherein R² and R³ are each independently C_2-20alkyl, preferably wherein R² and R³ are each independently C_2-6alkyl, as specified elsewhere herein; - purifying the intermediate of Formula (XIVA); and - reacting the intermediate of Formula (XIVA) with (i) a C_3-27alkyl acid chloride or C_3-27alkenyl acid chloride or with (ii) a C_3-27alkyl acid or C_3-27alkenyl acid in the presence of an activating agent, thereby obtaining an ionizable lipid having a structure of Formula (IA.1),

(I.1A), wherein R² and R³ are each independently C2-20alkyl, preferably wherein R² and R³ are each independently C_2-6alkyl; and wherein R⁴ and R⁵ are each independently selected from C_3-27alkyl or C_3-27alkenyl, as specified elsewhere herein. In embodiments of the methods as taught herein, R⁴ and R⁵ may be each independently selected from C_7-25alkyl or C_13-21alkenyl. In embodiments of the methods as taught herein, R⁴ and R⁵ may be each independently selected from CH₃(CH₂)6-, CH₃(CH₂)8-, CH₃(CH₂)10-, CH₃(CH₂)12-, CH₃(CH₂)14-, CH₃(CH₂)₁₆-, CH₃(CH₂)18-, CH₃(CH₂)20-, CH₃(CH₂)22-, CH₃(CH₂)24-, [CH₃(CH₂)7][CH₃(CH₂)₅]CH-, CH₃(CH₂)3CH=CH(CH₂)7-, CH₃(CH₂)₅CH=CH(CH₂)7-, CH₃(CH₂)8CH=CH(CH₂)4-, CH₃(CH₂)7CH=CH(CH₂)7-, CH₃(CH₂)₅CH=CH(CH₂)9-, CH₃(CH₂)4CH=CHCH₂CH=CH(CH₂)7-, CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)7-, CH₃(CH₂)4CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)3-, CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)3-, CH₃(CH₂)7CH=CH(CH₂)11-, or CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)2-. In further preferred embodiments, the method as envisaged herein further comprises: - reacting the ionizable lipid having a structure of Formula (IA.1) in an alkaline medium or in a mixture of THF/H₂O/NaOH, and - purifying an ionizable lipid having a structure of Formula (IA.2),

(I.2A), wherein R² and R³ are each independently C_2-20alkyl, preferably wherein R² and R³ are each independently C_2-6alkyl; and wherein R⁴ and R⁵ are each independently selected from C_3-27alkyl or C_3-27alkenyl, as specified elsewhere herein, or from C_7-25alkyl or C_13-21alkenyl, or from CH₃(CH₂)₆-, CH₃(CH₂)₈-, CH₃(CH₂)₁₀-, CH₃(CH₂)₁₂-, CH₃(CH₂)₁₄-, CH₃(CH₂)₁₆-, CH₃(CH₂)₁₈-, CH₃(CH₂)₂₀-, CH₃(CH₂)₂₂-, CH₃(CH₂)₂₄-, [CH₃(CH₂)₇][CH₃(CH₂)₅]CH-, CH₃(CH₂)₃CH=CH(CH₂)₇-, CH₃(CH₂)₅CH=CH(CH₂)₇-, CH₃(CH₂)₈CH=CH(CH₂)₄-, CH₃(CH₂)₇CH=CH(CH₂)₇-, CH₃(CH₂)₅CH=CH(CH₂)₉-, CH₃(CH₂)4CH=CHCH₂CH=CH(CH₂)7-, CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)7-, CH₃(CH₂)4CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)3-, CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)3-, CH₃(CH₂)7CH=CH(CH₂)11-, or CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)2-. In further preferred embodiments, the method further comprises: - reacting the ionizable lipid having a structure of Formula (IA.2) with oxalylchloride and dimethylformamide (DMF), thereby obtaining an acid chloride lipid intermediate having a structure of Formula (XVA), (XVA); and - reacting the acid chloride lipid intermediate of formula (XVA) with

, wherein Y¹, Y², and Y³ are each independently C_1-12 alkyl, Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C_1-8alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure comprising one or two nitrogen atoms, and optionally an oxygen or sulphur atom, as the heteroatom(s)., as specified elsewhere herein, thereby obtaining an ionizable lipid having a structure of Formula (IA.3), (IA.4) or (IA.5)

CH₃(CH₂)₄CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)₃-,

The present application further considers the use of an ionizable lipid as envisaged herein for producing a lipid nanoparticle (LNP). Accordingly, another aspect of the present application provides a lipid nanoparticle (LNP) comprising an ionizable lipid according to the present application. It is understood that the lipid nanoparticle formulation may be influenced by, but not limited to, the selection of the different lipid components, such as the ionizable lipid as envisaged herein, the PEGylated lipid and the helper lipids, the degree of lipid saturation, the nature of the PEGylation, and the like. In particular embodiments, the LNP as envisaged herein comprises: - an ionizable lipid according to Formula (I) or Formula (IA), (II) or (III) as further specified above, in particular wherein R¹, Y¹, Y², Y³, Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, Z⁷, N-Z³-Z⁴, N-Z⁵-N-Z⁶, R², R³, R⁴, and R⁵, and R⁶ are as specified above; - at least one PEGylated lipid; and - at least one helper lipid, particularly a sterol and, optionally, one other helper lipid. In particular embodiments, the LNP further comprises a therapeutic agent. The ionizable lipid as envisaged herein is particularly an ionizable cationic lipid. Such lipids may become positively charged lipids that are able to associate with nucleic acids in lipid/LNP- based delivery systems. A positive charge on the LNP also promotes association with the negatively charged cell membrane to enhance cellular uptake. The lipid component of the LNP as envisaged herein may include one or more PEGylated lipids. A PEGylated lipid is a lipid modified with polyethylene glycol. This may improve the water-solubility and stability of the LNP. A PEGylated lipid may be selected from the non-limiting group consisting of PEGylated phosphatidylethanolamines, PEGylated phosphatidic acids, PEGylated ceramides, PEGylated dialkylamines, PEGylated diacylglycerols, PEGylated dialkylglycerols, and mixtures thereof. Preferably, the PEGylated lipid is selected from the group consisting of DMG-PEG, DSPE- PEG, DSG-PEG, or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, wherein the molecular weight of PEG ranges from 1-10 kDa. In certain embodiments, the PEGylated lipid is a PEG-OH lipid. A "PEG-OH lipid", also referred to herein as "hydroxy-PEGylated lipid", is a PEGylated lipid having one or more hydroxyl (-OH) groups on the lipid and/or on the PEG chain. In certain embodiments, a PEG-OH or hydroxy-PEGylated lipid comprises a hydroxyl group at the terminus of the PEG chain. Suitable helper lipids are generally known in the art. A preferred helper lipid is a steroid or a sterol, more preferably cholesterol. Incorporation of a steroid or a sterol in the LNP may help aggregation of other lipids in the particle. As defined herein, "sterols" are a subgroup of steroids consisting of steroid alcohols. A particularly preferred sterol is cholesterol or an analogue thereof. Other examples include ergosterol and phytosterols. Other possible helper lipids include dioleoylphosphatidylethanolamine (DOPE), and 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC). A therapeutic agent as envisaged herein refers to any compound, which when administered to a subject, particularly a cell of a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect. The therapeutic agent may be a small molecule, a nucleic acid or a protein or polypeptide. The terms “protein” or “polypeptide” generally encompass proteins encoded by any open reading frame (ORF) of a genome. Where a single ORF encodes a pre-protein which is processed into one, two or more mature proteins, the term may encompass both the pre-protein and the processed mature proteins. The term “nucleic acid” as used herein means a polymer of any length composed essentially of nucleotides, e.g., deoxyribonucleotides and/or ribonucleotides. The term “nucleic acid” further preferably encompasses DNA, RNA and DNA/RNA hybrid molecules, specifically including hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g. chemically synthesised) DNA, RNA or DNA/RNA hybrids. A nucleic acid can be naturally occurring, e.g., present in or isolated from nature, can be recombinant, i.e., produced by recombinant DNA technology, and/or can be, partly or entirely, chemically or biochemically synthesised. A “nucleic acid” can be double-stranded, partly double stranded, or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear. By “encoding” is meant that a nucleic acid sequence or part(s) thereof corresponds, by virtue of the genetic code of an organism in question to a particular amino acid sequence, e.g., the amino acid sequence of one or more desired proteins or polypeptides. In preferred embodiments, the therapeutic agent is a protein or polypeptide, or a nucleic acid, such as DNA or RNA, particularly mRNA. In particular embodiments, the nucleic acid is a mRNA encoding a therapeutic protein or polypeptide. In some embodiments, the mRNA encodes multiple proteins or polypeptides. The mRNA may be designed to encode proteins or polypeptides of interest selected from any of several target categories including, but not limited to, antibodies, vaccines, therapeutic proteins or peptides, proteins associated with human disease, targeting moieties or those proteins encoded by the human genome for which no therapeutic indication has been identified but which nonetheless have utility in areas of research and discovery. "Therapeutic protein" refers to a protein that, when administered to a cell has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect. In certain embodiments, the nucleic acid is a mRNA encoding a vaccine antigen. The term “vaccine” refers to a biological preparation that improves immunity to a particular disease. In certain embodiments, the LNP as envisaged herein has a mean diameter of 10-500 nm, 20- 400 nm, 30-300 nm, or 40-200 nm, such as a mean diameter of 50-150 nm, 50-200 nm, 80- 100 nm or 80-200 nm. Particularly preferred lipid nanoparticles comprise - an ionizable lipid as envisaged herein, particularly an ionizable lipid according to Formula (I) or Formula (IA), (II) or (III) as further specified above, in particular wherein R¹, Y¹, Y², Y³, Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, Z⁷, N-Z³-Z⁴, N-Z⁵-N-Z⁶, R², R³, R⁴, R⁵, and R⁶ are as specified above; - at least one PEGylated lipid; - cholesterol, and, optionally, one other helper lipid, and - a therapeutic agent, particularly a nucleic acid, such as mRNA, encoding for a therapeutic protein or a vaccine antigen. Another aspect of the present application provides a method of preparing lipid nanoparticles (LNP) comprising a therapeutic agent. In general, the formation of lipid nanoparticles may be accomplished by methods known in the art. In particular, the method for preparing a lipid nanoparticle comprises the steps of: - mixing an aqueous solution comprising a therapeutic agent with an organic solvent, such as an ethanolic solution, comprising an ionizable lipid according to Formula (I) or Formula (IA), (II) or (III) as specified herein, a PEGylated lipid, and one or more helper lipids, particularly cholesterol; and - removing the organic solvent such as ethanol, thereby obtaining LNP comprising the therapeutic agent. In particular embodiments, the PEGylated lipid is selected from the group consisting of DMG- PEG, DSPE-PEG, DSG-PEG, or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, wherein the molecular weight of PEG ranges from 1-10 kDa; and/or the helper lipid is selected from the group consisting of a sterol, particularly cholesterol, DOPE, and DSPC. In particular embodiments, the step of removing the organic solvent such as ethanol is performed by dialysis, spin filtration, or evaporation. In embodiments, the organic solvent may be ethanol, propanol, isopropanol, or chloroform. In particular embodiments, the therapeutic agent is a nucleic acid, such as DNA or RNA, preferably wherein the therapeutic agent is mRNA; and/or the therapeutic agent is a protein. In embodiments, the ionizable lipid as taught herein may be mixed with the therapeutic agent in a ratio of 50:1 to 1:1. For instance, the ionizable lipid as taught herein may be mixed with the therapeutic agent in a ratio of 40:1 to 1:1, 30:1 to 2:1, 25:1 to 2:1, 20:1 to 2:1, 10:1 to 2:1, or 5:1 to 2:1. In embodiments, when the therapeutic agent is a nucleic acid such as mRNA, the ratio may be expressed as the N:P ratio. The wording “N:P ratio” refers to the proportion of the number of ionisable lipid molecules (expressed as “nitrogen” or “N”) to the number of nucleotides (expressed as “phosphor” or “P”). In embodiments, the ionizable lipid as taught herein may be mixed with the nucleic acid such as mRNA in a N:P ratio of 50:1 to 1:1. For instance, the ionizable lipid as taught herein may be mixed with the nucleic acid such as mRNA in a N:P ratio of 40:1 to 1:1, 30:1 to 2:1, 25:1 to 2:1, 20:1 to 2:1, 10:1 to 2:1, or 5:1 to 2:1. The present application further provides pharmaceutical compositions comprising the lipid nanoparticles as envisaged herein, particularly further comprising one or more other pharmaceutically acceptable ingredients, as known to the skilled person, including, but are not limited to polymeric excipients, for instance chitosan derivatives or salts thereof; lipid excipients, such as phospholipids; surfactants; solvents; buffering agents, and the like. In particular embodiments, in said pharmaceutical compositions, the nanoparticles are provided in a pharmaceutically-acceptable carrier. The term "pharmaceutically-acceptable carrier" means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a subject contemplated by the present application. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the lipid nanoparticles and any other optional agent(s) are combined to facilitate administration. The components of the pharmaceutical compositions are commingled in a manner that precludes interaction that would substantially impair their desired pharmaceutical efficiency. When it is desirable to deliver the pharmaceutical compositions as envisaged herein systemically, it may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form. Pharmaceutical parenteral formulations include aqueous solutions of the ingredients. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Alternatively, suspensions of ingredients may be prepared as oil-based suspensions such as are known in the art or that will be readily apparent to those of ordinary skill in the art based on this disclosure. A further aspect provides a lipid nanoparticle comprising a therapeutic agent, for use in veterinary or human medicine, in particular in a method of delivering the therapeutic agent to a subject, and more particularly for use in a method of delivering a nucleic acid to a subject, wherein the lipid nanoparticle comprises an ionizable lipid according to Formula (I) or Formula (IA) as specified herein, in particular wherein R¹, Y¹, Y², Y³, Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, Z⁷, N-Z³-Z⁴, N-Z⁵-N-Z⁶, R², R³, R⁴, R⁵, and R⁶ are as specified herein. A further embodiment provides a lipid nanoparticle or composition as defined herein comprising a therapeutic agent, in particular a nucleic acid encoding an antigen, for use as a vaccine. The vaccine may be used for inducing an immune response, in particular an immune response against a disease associated antigen or cells expressing a disease associated antigen, such as an immune response against e.g. an infectious agent or cancer. Accordingly, the vaccine may be used for prophylactic and/or therapeutic treatment of a disease involving a disease associated antigen or cells expressing a disease associated antigen. The present application also provides a method for delivering a therapeutic agent, particularly a protein or nucleic acid, wherein the method comprises administering a composition comprising a lipid nanoparticle comprising a therapeutically effective amount of the therapeutic agent to the subject, wherein the lipid nanoparticle comprises an ionizable lipid according to Formula (I) or Formula (IA), (II) or (III) as specified herein. In particular embodiments, the therapeutic agent is a protein and the method for delivering a therapeutic level of a protein of interest to a subject, comprises administering a composition to the subject in need thereof, said composition comprising a lipid nanoparticle comprising a nucleic acid encoding for the protein of interest, wherein the lipid nanoparticle comprises an ionizable lipid according to Formula (I) or Formula (IA) as specified herein. The present application further provides a method for treating a subject comprising administering to a subject in need thereof a composition comprising a lipid nanoparticle comprising a therapeutically effective amount of a therapeutic agent, particularly a protein or nucleic acid, to the subject, wherein the lipid nanoparticle comprises an ionizable lipid according to Formula (I) or Formula (IA) as specified herein, in particular wherein R¹, Y¹, Y², Y³, Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, Z⁷, R², R³, R⁴, R⁵, and R⁶ are as specified herein. In particular embodiments, the therapeutic agent is a protein or polypeptide and the method for treating a subject comprises administering a composition comprising a lipid nanoparticle comprising a nucleic acid encoding for the protein or polypeptide of interest, wherein the lipid nanoparticle comprises an ionizable lipid according to Formula (I) or Formula (IA) as specified herein, in particular wherein R¹, Y¹, Y², Y³, Z¹, Z², Z³, Z⁴, Z⁵, Z⁶, Z⁷, R², R³, R⁴, R⁵, and R⁶ are as specified herein. As used herein, the terms “subject” or “patient” are generally used interchangeably and refer to animals, preferably warm-blooded animals, more preferably vertebrates, even more preferably mammals, still more preferably primates, and specifically includes human patients and non- human mammals and primates. Preferred subjects or patients are human subjects. As used herein, a phrase such as “a subject in need of treatment” includes subjects that would benefit from treatment of a given condition, particularly proliferative diseases. Such subjects may include, without limitation, those that have been diagnosed with said condition, those prone to develop said condition and/or those in whom said condition is to be prevented. The terms “treat” or “treatment” encompass both the therapeutic treatment of an already developed disease or condition, such as the therapy of an already developed proliferative disease, as well as prophylactic or preventive measures, wherein the aim is to prevent or lessen the chances of incidence of an undesired affliction, such as to prevent occurrence, development and progression of proliferative diseases. The present application also provides aspects and embodiments as set forth in the following Statements: 1. An ionizable lipid having a structure of Formula (I),

wherein R¹ is hydrogen, C_1-12alkyl,

, wherein Y¹, Y², and Y³ are each independently C_1-12 alkyl, and wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C1-8alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S, as heteroatom(s); wherein R² and R³ are each independently C2-20alkyl; wherein R⁴ and R⁵ are each independently selected from C3-27alkyl or C3-27alkenyl; and wherein R⁶ is C2-10alkyl. 2. An ionizable lipid according to statement 1, wherein the ionizable lipid has a structure of Formula (IA), (II) or (III), (IA) (II) (III) wherein R¹, R², R³, R⁴, and R⁵ have the same meaning as defined in statement 1. 3. An ionizable lipid according to statement 1 or 2, wherein R¹ is hydrogen, C_1-6alkyl,

; wherein Y¹, Y², and Y³ are each independently C1-6alkyl, preferably wherein Y¹, Y², and Y³ are each independently C2-4alkyl, and wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C1-6alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S, as heteroatom(s); preferably wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C1-3alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, comprising one or two nitrogen atoms, 2 to 8 carbon atoms, and optionally O or S; more preferably wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a piperazine, pyrrolidine, imidazolidine, piperidine, morpholine, thiomorpholine, hexahydroazepine, azocane, or azonane ring structure. 4. An ionizable lipid according to any one of statements 1 to 3, wherein R¹ is hydrogen, methyl, ethyl, n-propyl,

. 5. An ionizable lipid according to any one of statements 1 to 4, wherein the ionizable lipid has a structure of Formula (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), or (XIII),

(X) (XI) (XII) (XIII) wherein R⁴ and R⁵ have the same meaning as defined in statement 1. 6. An ionizable lipid according to any one of statements 1 to 5, wherein R⁴ and R⁵ are each independently selected from C7-25alkyl or C13-21alkenyl; preferably R⁴ and R⁵ are each independently selected from CH₃(CH₂)6-, CH₃(CH₂)8-, CH₃(CH₂)10-, CH₃(CH₂)12-, CH₃(CH₂)14-, CH₃(CH₂)₁₆-, CH₃(CH₂)18-, CH₃(CH₂)20-, CH₃(CH₂)22-, CH₃(CH₂)24-, [CH₃(CH₂)₇][CH₃(CH₂)₅]CH-, CH₃(CH₂)₃CH=CH(CH₂)₇-, CH₃(CH₂)₅CH=CH(CH₂)₇-, CH₃(CH₂)₈CH=CH(CH₂)₄-, CH₃(CH₂)₇CH=CH(CH₂)₇-, CH₃(CH₂)₅CH=CH(CH₂)₉-, CH₃(CH₂)₄CH=CHCH₂CH=CH(CH₂)₇-, CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)₇-, CH₃(CH₂)₄CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)₃-, CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)₃-, CH₃(CH₂)₇CH=CH(CH₂)₁₁-, or CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)₂-. 7. An ionizable lipid according to any one of statements 1 to 6, wherein R⁴ and R⁵ are the same or different; preferably wherein R⁴ and R⁵ are the same. 8. A method for producing an ionizable lipid, the method comprising: - reacting a diC_2-20alcoholamine with a compound of formula Br-R⁶-C(=O)OCH₃, wherein R⁶ is –(CH₂)_m-, wherein m is 2 to 6, or with a methylacrylate, thereby obtaining an intermediate having a structure of Formula (XIV) or having a structure of formula (XIVA); (XIV) (XIVA) - purifying the intermediate of formula (XIVA); and - reacting the intermediate of formula (XIV) or (XIVA) with (i) a C3-27alkyl acid chloride or C3-27alkenyl acid chloride or (ii) a C3-27alkyl acid or C3-27alkenyl acid in the presence of an activating agent, thereby obtaining an ionizable lipid having a structure of Formula (I.1) or (IA.1), respectively

wherein R² and R³ are each independently C_2-20alkyl; and wherein R⁴ and R⁵ are each independently selected from C_3-27alkyl or C_3-27alkenyl. 9. The method according to statement 8, further comprising: - reacting the ionizable lipid having a structure of Formula (I.1) or (IA.1) in an alkaline medium or in a mixture of THF/H₂O/NaOH; and - purifying an ionizable lipid having a structure of Formula (I.2) or (IA.2), respectively

10. The method according to statement 9, further comprising: - reacting the ionizable lipid having a structure of Formula (I.2) or (IA.2) with oxalylchloride and dimethylformamide (DMF), thereby obtaining an acid chloride lipid intermediate having a structure of Formula (XV) or (XVA), respectively; (XV) (XVA) and - reacting the acid chloride lipid intermediate of formula (XV) or (XVA) with

, , or , wherein Y¹, Y², and Y³ are each independently C_1-12 alkyl, Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C_1-8alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S, as heteroatom(s), thereby obtaining an ionizable lipid having a structure of Formula (I.3), (I.4), (I.5), (IA.3), (IA.4) or (IA.5)

(IA.5). 11. A lipid nanoparticle (LNP) comprising an ionizable lipid as defined in any one of statements 1 to 7. 12. The LNP according to statement 11, wherein the LNP further comprises: - a PEGylated lipid; preferably wherein the PEGylated lipid is selected from the group consisting of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPE)-PEG, distearoyl-rac-glycerol (DSG)-PEG, or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, wherein the molecular weight of PEG ranges from 1-10 kDa; - a helper lipid; preferably wherein the helper lipid is selected from the group consisting of cholesterol, dioleoylphosphatidylethanolamine (DOPE), and 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC); and/or - a therapeutic agent, preferably a nucleic acid, such as DNA or RNA; more preferably mRNA. 13. A method of preparing lipid nanoparticles (LNP) comprising a therapeutic agent, the method comprising: - mixing an aqueous solution comprising a therapeutic agent with an ethanolic solution comprising an ionizable lipid as defined in any one of statements 1 to 7, a PEGylated lipid, and a helper lipid; and - removing ethanol, thereby obtaining LNP comprising the therapeutic agent. 14. A method according to statement 13, wherein: - the PEGylated lipid is selected from the group consisting of DMG-PEG, DSPE-PEG, DSG-PEG, or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, wherein the molecular weight of PEG ranges from 1-10 kDa; and/or - the helper lipid is selected from the group consisting of cholesterol, DOPE, and DSPC; and/or - ethanol is removed by dialysis, spin filtration, or evaporation; and/or - the therapeutic agent is a nucleic acid, such as DNA or RNA, preferably wherein the therapeutic agent is mRNA; and/or - the therapeutic agent is a protein. 15. A lipid nanoparticle comprising a therapeutic agent, for use in a method of delivering a therapeutic agent to a subject, particularly for use in a method of delivering a nucleic acid to a subject, wherein the lipid nanoparticle comprises an ionizable lipid as defined in any one of statements 1 to 7. The above aspects and embodiments are further supported by the following non-limiting examples. EXAMPLES In the examples presented below, the present invention is illustrated by compounds wherein the R⁴-C(=O)O and the R⁵-C(=O)O moieties are a saturated, mono-unsaturated, or polyunsaturated C18 fatty acid moieties. The skilled person understands that the same principles are also valid for compounds wherein the R⁴-C(=O)O and the R⁵-C(=O)O moieties are other saturated, mono-unsaturated, or polyunsaturated fatty acid moieties as specified herein. Example 1: Synthesis of an ionizable lipid (R¹ = Me; -CH₂CH₂-; R⁴=R⁵= saturated or

unsaturated alkyl chain) Diethanolamine is dissolved in a solution of methylacrylate and refluxed overnight at 80 °C. The resulting intermediate is purified by silica column chromatography and subsequently reacted with an alkyl acid chloride. For example, reaction with stearylchloride, oleoylchloride and linoleylchloride, yields compounds 3.1, 3.2 and 3.3, respectively.

compound 3.1 compound 3.2 compound 3.3 Example 2: Synthesis of an ionizable lipid (R¹ = amine or nitrogen containing moiety; R²=R³= - CH₂CH₂-; R⁴=R⁵= saturated or unsaturated alkyl chain) Compounds 3.1, 3.2 and 3.3 are dissolved in an alkaline medium, particularly a mixture of THF/H₂O/NaOH and reacted overnight. The resulting intermediate is purified by silica column chromatography, yielding compounds 3.1a, 3.2a and 3.3a.

compound 3.1a compound 3.2a compound 3.3a Subsequently, these compounds are reacted with oxalylchloride in presence of a catalytic amount of dimethylformamide. The thus formed product is then reacted with several aminoalcohols, including dimethylethanolamine [(CH₃)2NCH₂CH₂OH], 2- (hexamethyleneimino)ethanol, and 1-(2-Hydroxyethyl)-4-methylpiperazine. Following purification, the corresponding ionizable lipids 4.1.a, 4.1.b, 4.1.c; 4.2.a, 4.2.b, 4.2.c, 4.3.a, 4.3.b, and 4.3.c are obtained.

compound 4.1.a compound 4.1.b compound 4.1.c

compound 4.2.a compound 4.2.b compound 4.2.c

compound 4.3.a compound 4.3.b compound 4.3.c Example 3: Synthesis of an ionizable lipid (R¹ = Me; -CH₂CH₂CH₂-; R⁴=R⁵= saturated

or unsaturated alkyl chain) 3,3'-azanediyldipropan-1-ol is dissolved in a solution of methylacrylate and refluxed overnight at 80 °C. The resulting intermediate is purified by silica column chromatography and subsequently reacted with an alkyl acid chloride. For example, reaction of the intermediate with stearylchloride, oleoylchloride and linoleylchloride, yields compounds 5.1, 5.2 and 5.3, respectively.

compound 5.1 compound 5.2 compound 5.3 Example 4: Synthesis of an ionizable lipid (R¹ = amine or nitrogen containing moiety; R²=R³= -CH₂CH₂CH₂-; R⁴=R⁵= saturated or unsaturated alkyl chain) Compounds 5.1, 5.2 and 5.3 are dissolved in an alkaline solution, in particular a mixture of THF/H₂O/NaOH and reacted overnight. The resulting intermediates are purified by silica column chromatography, yielding compounds 5.1a, 5.2a, 5.3a.

compound 5.1a compound 5.2a compound 5.3a Subsequently, these compounds are reacted with oxalylchloride in presence of a catalytic amount of dimethylformamide. The thus formed respective product is then reacted with several aminoalcohols, including dimethylethanolamine [(CH₃)₂NCH₂CH₂OH], 2- (hexamethyleneimino)ethanol, and 1-(2-Hydroxyethyl)-4-methylpiperazine. Following purification by silica column chromatography, the corresponding ionizable lipids 6.1.a, 6.1.b, 6.1.c, 6.2.a, 6.2.b, 6.2.c, 6.3.a, 6.3.b and 6.3.c.

compound 6.1.a compound 6.1.b compound 6.1.c

compound 6.2.a compound 6.2.b compound 6.2.c

Example 5: Synthesis of an ionizable lipid (R¹ = Me; -CH₂CH₂- or 2³ _{2 2 2} ⁴=R⁵

R=R= -CHCHCH- ; R = branched saturated alkyl chain) Diethanolamine or 3,3'-azanediyldipropan-1-ol is dissolved in a solution of methylacrylate and refluxed overnight at 80 °C. The resulting intermediate is purified by silica column chromatography and subsequently reacted with a branched alkyl acid chloride, for instance 2- hexyldecanoic acid chloride, yielding compound 7 and compound 9, respectively.

Example 6: Synthesis of an ionizable lipid (R¹ = amine or nitrogen containing moiety; R²=R³= -CH₂CH₂- or R²=R³= -CH₂CH₂CH₂- ; R⁴=R⁵= branched saturated alkyl chain) Compound 7 or compound 9 is dissolved in a mixture of THF/H₂O/NaOH and reacted overnight followed by purification by silica column chromatography, yielding compounds 7a and 9a..

Subsequently, these compounds are reacted with oxalylchloride in presence of a catalytic amount of dimethylformamide. The thus formed product is then reacted with several aminoalcohols, including dimethylethanolamine [(CH₃)2NCH₂CH₂OH], 2- (hexamethyleneimino)ethanol, and 1-(2-Hydroxyethyl)-4-methylpiperazine and purified by silica column chromatography to yield the corresponding ionizable lipids 8.1, 8.2, 8.3 and 10.1, 10.2, 10.3, respectively.

compound 8.3 compound 10.3 Example 7: Synthesis of a comparative ionizable lipid, referred to herein as “SME” Example 7 provides the synthesis of a comparative ionizable lipid, SME (Figure 2A). 2 g of distearoylamine was dissolved in 50 mL methacrylate. The mixture was stirred vigorously at 90 °C for 18 hours. The solvent was removed under vacuum and purified by silica gel column chromatography (hexane/ethylacetate 10:1) to afford the compound SME (Figure 2A) as a white solid (yield = 42 %).¹H-NMR analysis (Figure 2B) confirmed successful synthesis of the targeted structure. The peaks are annotated with the corresponding atom position (Figure 2B) and integrated. The concept of peak integration is that the area of a given peak in a 1H NMR spectrum is proportional to the number of (equivalent) protons giving rise to the peak. Example 8: Synthesis of ionizable lipid according to an embodiment of the invention, referred to herein as “BME” (R¹ = Me; R²=R³= -CH₂CH₂-; R⁴=R⁵= branched saturated alkyl chain) Example 8 provides the synthesis of the ionizable lipid BME, i.e. the compound having a structure of Formula (IA), wherein R¹ is Me; R² and R³ are each -CH₂CH₂-; R⁴ and R⁵ are each [CH3(CH2)7][CH3(CH2)5]CH- (compound 7 herein). Hexyldecanoic acid (2.4 Eq) was dissolved in 5 mL anhydrous DCM. Then Ghosez reagent (2.8 Eq) was added to reaction mixture at 0°C. The mixture was stirred at room temperature for 40 minutes. After 40 minutes, methyl 3-(bis(2-hydroxyethyl)amino)propanoate (*¹) (1 Eq) was added to the reaction mixture followed by TEA (3 Eq) and DMAP (0.2 Eq). The mixture was stirred overnight at room temperature. The solvent was removed under vacuum and was purified by silica gel column chromatography (hexane/ethylacetate 10:1) to afford the compound BME as a brown oil (yield = 11 %). ¹H-NMR analysis confirmed successful synthesis of the targeted structure (results not shown). Synthesis of (*¹) methyl 3-(bis(2-hydroxyethyl)amino)propanoate Diethanolamine (1 Eq) was dissolved in 5 mL DMF on 0 °C. Afterwards, methylbromopropionate (1 Eq) and TEA (2 Eq) were added to the reaction mixture. The reaction mixture was stirred overnight at room temperature. The solvent was removed under vacuum and was purified by silica gel column chromatography (DCM/MeOH 10:1) to afford the compound as a yellow oil (yield = 49 %). Example 9: Synthesis of ionizable lipid according to an embodiment of the invention, referred to herein as “OME” (R¹ = Me; R²=R³= -CH2CH2-; R⁴=R⁵= unsaturated alkyl chain) Example 9 provides the synthesis of the ionizable lipid OME, i.e. the compound having a structure of Formula (IA), wherein R¹ is Me; R² and R³ are each -CH2CH2-; R⁴ and R⁵ are each CH3(CH2)7CH=CH(CH2)7- (compound 3.2 herein). Methyl 3-(bis(2-hydroxyethyl)amino)propanoate (1 Eq) was dissolved in 5 mL anhydrous DCM at 0° C. Then, oleoylchloride (2.4 Eq) was added followed by dropwise addition of TEA (2.5 Eq). The reaction was stirred overnight at room temperature. The solvent was removed under vacuum and was purified by silica gel column chromatography (hexane/ethylacetate 10:1) to afford the compound OME as yellow oil (yield = 11 %).¹H-NMR analysis confirmed successful synthesis of the targeted structure (results not shown). Example 10: Synthesis of ionizable lipid according to an embodiment of the invention, referred to herein as “ODMA” (R¹ = -CH2CH2N(CH3)(CH3); R²=R³= -CH2CH2-; R⁴=R⁵= unsaturated alkyl chain) Example 10 provides the synthesis of the ionizable lipid ODMA, i.e. the compound having a structure of Formula (IA), wherein R¹ is -CH2CH2N(CH3)(CH3); R² and R³ are each -CH2CH2-; R⁴ and R⁵ are each CH3(CH2)7CH=CH(CH2)7- (compound 4.1.b herein). 2-(dimethylamino)ethyl acrylate (*²) (1 Eq) was dissolved in 5 mL anhydrous DCM. Then diethanolamine (1 Eq) and 10 mg hydroquinone were added to the reaction mixture. The reaction mixture was stirred for 24 hours at room temperature. After 24 hours, oleoylchloride (2.4 Eq) was added at 0° C followed by dropwise addition of TEA (2.5 Eq) and 2 mL anhydrous DMF. The reaction mixture was stirred overnight at room temperature. The solvent was removed under vacuum and was purified by silica gel column chromatography (DCM/methanol 30:1) to afford the compound ODMA as a yellow oil (yield = 33 %). ¹H-NMR analysis confirmed successful synthesis of the targeted structure (results not shown). Synthesis of (*²) 2-(dimethylamino)ethyl acrylate Acryloyl chloride (1.2 Eq) and dimethylaminoethanol (1 Eq) were dissolved in in anhydrous DCM at 0 °C. Then TEA (1.5 Eq) was added dropwise to reaction mixture. The reaction mixture was stirred overnight at room temperature. The solvent was removed under vacuum and was purified by silica gel column chromatography (DCM/methanol 10:1) to afford the compound as a yellow oil (41 %). Example 11: Synthesis of ionizable lipid according to an embodiment of the invention, referred to herein as “BC7” (R¹ = -CH2CH2-N(CH2)6; R²=R³= -CH2CH2-; R⁴=R⁵= branched saturated alkyl chain) Example 11 provides the synthesis of the ionizable lipid BC7, i.e. the compound having a structure of Formula (IA), wherein R¹ is -CH2CH2-N(CH2)6; R² and R³ are each -CH2CH2-; R⁴ and R⁵ are each [CH3(CH2)7][CH3(CH2)5]CH- (compound 8.2 herein). 2-(azepan-1-yl)ethyl 3-(bis(2-hydroxyethyl)amino)propanoate (*³) (1 Eq) was dissolved in 5 mL anhydrous DCM. Then diethanolamine (1 Eq) and 10 mg hydroquinone were added to the reaction mixture. The reaction mixture was stirred for 24 hours at room temperature. After 24 hours, hexyldecanoyl chloride reaction mixture (*⁴) (2.4 Eq) was added at 0° C followed by dropwise addition of TEA (2.5 Eq). The reaction mixture was stirred overnight at room temperature. The solvent was removed under vacuum, washed 5 times with deionized water and was purified by silica gel column chromatography (hexane/ethylacetate 10:1) to afford the compound BC7 as a yellow oil (yield = 13 %). ¹H-NMR analysis confirmed successful synthesis of the targeted structure (results not shown). Synthesis of (*³) 2-(azepan-1-yl)ethyl 3-(bis(2-hydroxyethyl)amino)propanoate Acryloyl chloride (1.2 Eq) and hexamethyleneiminoethanol (1 Eq) were dissolved in anhydrous DCM at 0 °C. Then TEA (1.5 Eq) was added dropwise to the reaction mixture. The reaction mixture was stirred overnight at room temperature. The solvent was removed under vacuum and was purified by silica gel column chromatography (DCM:methanol 10:1) to afford the compound as a yellow oil (yield = 60 %). Synthesis of (*⁴) hexyldecanoyl chloride Hexyldecanoic acid (2.4 Eq) was dissolved in 5 mL anhydrous DCM. Then Ghosez reagent (2.8 Eq) was added to reaction mixture at 0° C. The reaction mixture was stirred at room temperature for 40 minutes. Example 12: Synthesis of ionizable lipid according to an embodiment of the invention, referred to herein as “OC7” (R¹ = -CH₂CH₂-N(CH₂)₆; R²=R³= -CH₂CH₂-; R⁴=R⁵= unsaturated alkyl chain) Example 12 provides the synthesis of the ionizable lipid OC7, i.e. the compound having a structure of Formula (IA), wherein R¹ is -CH2CH2-N(CH2)6; R² and R³ are each -CH2CH2-; R⁴ and R⁵ are each CH3(CH2)7CH=CH(CH2)7- (compound 4.2.b herein). 2-(azepan-1-yl)ethyl 3-(bis(2-hydroxyethyl)amino)propanoate (*³, see above) (1 Eq) was dissolved in 5 mL anhydrous DCM. Then diethanolamine (1 Eq) and 10 mg hydroquinone (spatula tip) were added to the reaction mixture. The reaction mixture was stirred for 24 hours at room temperature. After 24 hours, oleoylchloride (2.4 Eq) was added at 0° C followed by dropwise addition of TEA (2.5 Eq) and 2 mL anhydrous DMF. The reaction mixture was stirred overnight at room temperature. The solvent was removed under vacuum and was purified by silica gel column chromatography (DCM:methanol 30:1) to afford the compound OC7 as a yellow oil (yield = 9 %). ¹H-NMR analysis confirmed successful synthesis of the targeted structure (results not shown). Example 13: Synthesis of ionizable lipid according to an embodiment of the invention, referred to herein as “BDMA” (R¹ = -CH2CH2N(CH3)(CH3); R²=R³= -CH2CH2-; R⁴=R⁵= branched saturated alkyl chain) Example 13 provides the synthesis of the ionizable lipid BDMA, i.e. the compound having a structure of Formula (IA), wherein R¹ is -CH2CH2N(CH3)2; R² and R³ are each -CH2CH2-; R⁴ and R⁵ are each [CH3(CH2)7][CH3(CH2)5]CH- (compound 8.1 herein). 2-(dimethylamino)ethyl acrylate (*², see above) (1 Eq) was dissolved in 5 mL anhydrous DCM. Then diethanolamine (1 Eq) and hydroquinone (spatula tip) were added to the reaction mixture. The reaction mixture was stirred for 24 hours at room temperature. After 24 hours, hexyldecanoyl chloride (*⁴, see above) reaction mixture (2.4 Eq) was added at 0° C followed by dropwise addition of TEA (2.5 Eq). The reaction mixture was stirred overnight at room temperature. The solvent was removed under vacuum, washed 5 times with desionized water and was purified by silica gel column chromatography (DCM/methanol 30:1) to afford the compound BDMA as a white oil (yield = 12 %). ¹H-NMR analysis confirmed successful synthesis of the targeted structure (results not shown). Example 14: Synthesis of lipid nanoparticles (LNP) according to embodiments of the invention LNP comprising mRNA were prepared using the comparative ionizable lipid SME (prepared according to Example 7) and the ionizable lipids illustrating the invention BME, OME, ODMA, BC7, OC7, or BDMA (prepared according to Examples 8, 9, 10, 11, 12, or 13 respectively). Synthesis of mRNA for the in vitro tests Luciferase-coding self-amplifying (sa)RNAs derived from Venezuelan Equine Encephalitis Virus (VEEV) were synthesized by in vitro transcription (IVT) from a I-SceI linearized plasmid (pV01) using the MEGAscript® kit (Thermo Fisher Scientific, Massachusetts, US). Subsequently, the saRNA was purified using silica-based columns (RNeasy Mini Kit, Qiagen, Hilden, Germany) and capped using the ScriptCapTM Cap 1 Capping System Kit (Cellscript, Wisconsin, US) according to the manufacturer’s instructions. Finally, the RNA was purified again using silica-based columns and the concentration and quality was determined spectrophotometrically (Nanodrop, Thermo Fisher Scientific, Massachusetts, US) and by gel electrophoresis, respectively. Formulation of mRNA in LNP Lipids were dissolved in absolute ethanol and mixed with DMG-PEG 2K (Avanti Polar Lipid), cholesterol and DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) in a ratio of 50: 1.5: 38.5: 10 (Lipid: DMG-PEG 2K: Cholesterol: DOPE). Sa-RNA was dissolved in RNase free NaOAC buffer (7,5mM, pH 4,5). Lipid nanoparticles (LNP) were produced by mixing ethanolic solutions comprising all lipids with aqueous solutions comprising mRNA in acetate buffer (5 mM, pH 4). Example 15: In vitro transfection using lipid nanoparticles (LNP) according to embodiments of the invention saRNA-LNP comprising the comparative ionizable lipid SME or the ionizable lipids illustrating the invention BME, OME, ODMA, BC7, OC7, or BDMA, prepared according to Example 14 were used to transfect different cell types in vitro: HeLa cells (Figure 3), MC38 cells (Figure 4), and DC2.4 cells (Figure 5) Different N:P ratios were tested namely N:P 5:1 (panel A), 10:1 (panel B) and 20:1 (panel C). The amounts of ionizable lipids and saRNA to be mixed was calculated based on the average molecular mass of nucleotides and the molecular weight of the lipids. In vitro mRNA transfection Human HeLa cells, mouse MC38 cells and mouse DC2.4 cells were seeded in a 24-well plate at a concentration of 50.000 cells/well (500 µL cell suspension/well). Cells were transfected with 10 µL saRNA-LNP (0.5 µg) per well and the plate was incubated for 24 hours in the incubator (37°C, 5% CO2, 95% humidity). After 24 hours, the medium was removed and 200 µL trypsin/EDTA was added to the cells and the plate was incubated for 5 minutes in the incubator (37°C, 5% CO2, 95% humidity). Afterwards, trypsin was neutralized with 300 µL DMEM medium. 180 µL cell suspension from each well was transferred to a black 96-well plate (one row and one column in between each cell suspension containing well) and 20 µL D- luciferin was added. The bioluminescent signal was measured using an IVIS (Perkin Elmer) imaging system. The measured total photon flux is depicted in Figure 3 (HeLa cells), Figure 4 (MC38 cells), and Figure 5 (DC2.4 cells). Data indicate that, overall, mRNA formulated in LNP comprising OC7 exhibited the highest in vitro transfection. The in vitro transfection of mRNA formulated in LNP comprising OC7 resulted in higher transfection of human HeLa cells as compared to transfection of mRNA formulated in LNP comprising the comparative ionizable lipid SME (Figure 3A, B, C). Further, mRNA formulated in LNP comprising ODMA or BDMA also showed satisfying in vitro transfection of human HeLa cells, MC38 and DC2.4 cells (Figure 3A-C, 4A-C, 5A-C). Example 16: Cytotoxicity of lipid nanoparticles (LNP) according to embodiments of the invention In order to study the viability of HeLa cells in response to treatment with LNP-formulated mRNA, MTT assays were performed using the best performing LNP illustrating the invention of Example 15, namely LNP comprising OC7, ODMA, or BDMA, and using the comparative LNP comprising SME. In vitro MTT cytotoxicity assay HeLa cells were seeded in a 96-well plate (20000 cells in 100 µL DMEM medium/well) and incubated for 24 hours at 37 °C. After 24 hours, LNPs (N:P 5:1) were made as described previously and were added to each well in following concentration range: 9 µg/mL, 3 µg/mL, 1 µg/mL, 0.33 µg/mL, 0.11 µg/mL, 0.037 µg/mL. The 96-well plate was incubated for 24 hours at 37 °C. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) stock-solution was prepared by dissolving the yellow tetrazolium salt in phosphate-buffered saline (PBS) (50 mg in 10 mL). The solution was passed through a filter with a pore diameter of 0.2 µm. Until use, the tube was wrapped in tin foil to protect the photosensitive substance from light. To each well, 20 µL MTT stock solution was added before incubating the plates for 2 hours. Thereafter, the plates were centrifuged at 300 ×g. The solution was removed by flipping the plates on absorbent paper. To dissolve the formed formazan crystals, 100 µL DMSO was added. Quantification was performed by measuring the absorbance at 590 nm using a microplate reader. Figure 6 depicts the viability of HeLa cells in response to treatment with LNP- formulated mRNA. Data indicate that mRNA formulated in LNP comprising OC7 exhibited the lowest cytotoxicity, followed by mRNA formulated in LNP comprising ODMA and by mRNA formulated in LNP comprising BDMA. mRNA formulated in LNP comprising the comparative ionizable lipid SME showed the highest cytotoxicity on human HeLa cells (Figure 6). Example 17: In vivo delivery of mRNA using LNPs according to embodiments of the invention LNP illustrating the invention, namely LNP comprising OC7, ODMA, or BDMA, were used for in vivo delivery of mRNA. mRNA synthesis for the in vivo tests Luciferase-coding self-amplifying (sa)RNAs derived from Venezuelan Equine Encephalitis Virus (VEEV) were synthesized by in vitro transcription (IVT) from a I-SceI linearized plasmid (pV01) using the MEGAscript® kit (Thermo Fisher Scientific, Massachusetts, US). For the in vivo tests the saRNA was prepared using cleancap reagent AU (TriLink Biotechnologies) and MEGAscript kit (Life Technologies, Waltham) to do transcript and capping in one step. The mRNA was purified by RNeasy Mini Kit (QIAGEN) and cellulose chromatography (Sigma- Aldrich) (doi.org/10.1016/j.ymthe.2021.01.023). Mice Female 6 weeks BALB/cJRj mice were purchased from Janvier labs (Le Genest-Saint-Isle, France) and housed in individual ventilated cages under 14 hours light and 10 hours dark cycle. Mice were accommodated for two weeks prior to the experiments. mRNA formulation and in vivo evaluation The luciferase encoding saRNA was formulated with the LNPs containing respectively OC7, ODMA and BDMA as ionizable lipids at a N:P ratio of 5:1. The saRNA-LNPs were prepared as described for the in vitro tests. Subsequently, mice were anesthetized by isoflurane and injected intramuscularly in hindlimb muscle with 1 µg saRNA formulated in the different LNPs that were dispersed in 50 µL PBS. The luciferase expression as a function of time was monitored by in vivo bioluminescence imaging. The bioluminescence signal was measured over a period of 21 days. Result are indicated as mean ± SEM of the total flux in the region of interest (ROI), i.e. the region with bioluminescence signal (n=8 for OC7-LNPs and n=4 for ODMA- and BDMA-LNPs). Results are provided in Figure 7A for OC7-LNPs, Figure 7B for ODMA-LNPs, and Figure 7C for BDMA-LNPs. The area under curve (AUC) was calculated by Prism 8.0.2 and provided in Figure 7D. The corresponding data in Figure 7 indicate that mRNA formulated in LNP containing OC7 exhibited the highest in vivo mRNA delivery.

Claims

CLAIMS 1. An ionizable lipid having a structure of Formula (IA),

wherein R¹ is hydrogen, C_1-12alkyl,

, wherein Y¹, Y², and Y³ are each independently C1-12 alkyl, and wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C1-8alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S, as heteroatom(s); wherein R² and R³ are each independently C2-20alkyl; and wherein R⁴ and R⁵ are each independently selected from C7-25alkyl or C13-21alkenyl; with the proviso that the ionizable lipid is not a compound having a structure of Formula (IA) wherein R¹ is methyl, R² and R³ are each ethyl, and R⁴ and R⁵ are each CH3(CH2)16-.

2. An ionizable lipid according to claim 1, wherein R⁴ and R⁵ are each independently selected from CH3(CH2)6-, CH3(CH2)8-, CH3(CH2)10-, CH3(CH2)12-, CH3(CH2)14-, CH3(CH2)16-, CH3(CH2)18-, CH3(CH2)20-, CH3(CH2)22-, CH3(CH2)24-, [CH3(CH2)7][CH3(CH2)5]CH-, CH3(CH2)3CH=CH(CH2)7-, CH3(CH2)5CH=CH(CH2)7-, CH3(CH2)8CH=CH(CH2)4-, CH3(CH2)7CH=CH(CH2)7-, CH3(CH2)5CH=CH(CH2)9-, CH3(CH2)4CH=CHCH2CH=CH(CH2)7-, CH3CH2CH=CHCH2CH=CHCH2CH=CH(CH2)7-, CH3(CH2)4CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)3-, CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)3-, CH3(CH2)7CH=CH(CH2)11-, or CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)2-.

3. An ionizable lipid having a structure of Formula (IA), (IA) wherein R¹ is hydrogen, C_1-12alkyl,

, wherein Y¹, Y², and Y³ are each independently C_1-12 alkyl, and wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C_1-8alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S, as heteroatom(s); wherein R² and R³ are each independently C2-20alkyl; and wherein R⁴ and R⁵ are each independently selected from CH3(CH2)6-, CH3(CH2)8-, CH3(CH2)10-, CH3(CH2)12-, CH3(CH2)14-, CH3(CH2)18-, CH3(CH2)20-, CH3(CH2)22-, CH3(CH2)24-, [CH3(CH2)7][CH3(CH2)5]CH-, CH3(CH2)3CH=CH(CH2)7-, CH3(CH2)5CH=CH(CH2)7-, CH3(CH2)8CH=CH(CH2)4-, CH3(CH2)7CH=CH(CH2)7-, CH3(CH2)5CH=CH(CH2)9-, CH3(CH2)4CH=CHCH2CH=CH(CH2)7-, CH3CH2CH=CHCH2CH=CHCH2CH=CH(CH2)7-, CH3(CH2)4CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)3-, CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)3-, CH3(CH2)7CH=CH(CH2)11-, or CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)2-.

4. An ionizable lipid according to any one of claims 1 to 3, wherein the ionizable lipid has a structure of Formula (II) or (III),

wherein R¹, R², R³, R⁴, and R⁵ have the same meaning as defined in any one of claims 1 to 3.

5. An ionizable lipid according to any one of claims 1 to 4, wherein R¹ is hydrogen, C1-6alkyl,

; wherein Y¹, Y², and Y³ are each independently C_1-6alkyl, preferably wherein Y¹, Y², and Y³ are each independently C_2-4alkyl, and wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C_1-6alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S, as heteroatom(s); preferably wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C_1-3alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, comprising one or two nitrogen atoms, 2 to 8 carbon atoms, and optionally O or S; more preferably wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a piperazine, pyrrolidine, imidazolidine, piperidine, morpholine, thiomorpholine, hexahydroazepine, azocane, or azonane ring structure.

6. An ionizable lipid according to any one of claims 1 to 5, wherein R¹ is hydrogen, methyl, ethyl, n-propyl,

.

7. An ionizable lipid according to any one of claims 1 to 6, wherein the ionizable lipid has a structure of Formula (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), or (XIII),

(XII) (XIII) wherein R⁴ and R⁵ have the same meaning as defined in any one of claims 1 to 6.

8. An ionizable lipid according to any one of claims 1 to 7, wherein R⁴ and R⁵ are each independently selected from [CH3(CH2)7][CH3(CH2)5]CH-, CH3(CH2)3CH=CH(CH2)7-, CH3(CH2)5CH=CH(CH2)7-, CH3(CH2)8CH=CH(CH2)4-, CH3(CH2)7CH=CH(CH2)7-, CH3(CH2)5CH=CH(CH2)9-, CH3(CH2)4CH=CHCH2CH=CH(CH2)7-, CH3CH2CH=CHCH2CH=CHCH2CH=CH(CH2)7-, CH3(CH2)4CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)3-, CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)3-, CH3(CH2)7CH=CH(CH2)11-, or CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)2-; preferably wherein R⁴ and R⁵ are each independently selected from [CH3(CH2)7][CH3(CH2)5]CH-, CH3(CH2)3CH=CH(CH2)7-, CH3(CH2)5CH=CH(CH2)7-, CH3(CH2)8CH=CH(CH2)4-, CH3(CH2)7CH=CH(CH2)7-, or CH3(CH2)5CH=CH(CH2)9-; more preferably wherein R⁴ and R⁵ are each independently selected from [CH3(CH2)7][CH3(CH2)5]CH- or CH3(CH2)7CH=CH(CH2)7-.

9. An ionizable lipid according to any one of claims 1 to 8, wherein R⁴ and R⁵ are the same or different; preferably wherein R⁴ and R⁵ are the same.

10. An ionizable lipid according to any one of claims 1 to 9, wherein R⁴ and R⁵ are the same and are selected from [CH₃(CH₂)₇][CH₃(CH₂)₅]CH-, CH₃(CH₂)₃CH=CH(CH₂)₇-, CH₃(CH₂)₅CH=CH(CH₂)₇-, CH₃(CH₂)₈CH=CH(CH₂)₄-, CH₃(CH₂)₇CH=CH(CH₂)₇-, or CH₃(CH₂)₅CH=CH(CH₂)₉-; preferably wherein R⁴ and R⁵ are the same and are selected from [CH₃(CH₂)₇][CH₃(CH₂)₅]CH- or CH₃(CH₂)₇CH=CH(CH₂)₇-.

11. A method for producing an ionizable lipid, the method comprising: - reacting a diC_2-20alcoholamine with a compound of formula Br-R⁶-C(=O)OCH₃, wherein R⁶ is –(CH₂)_m-, wherein m is 2, or with a methylacrylate, thereby obtaining an intermediate having a structure of formula (XIVA);

- purifying the intermediate of formula (XIVA); and - reacting the intermediate of formula (XIVA) with (i) a C7-25alkyl acid chloride or C13- 21alkenyl acid chloride or (ii) a C7-25alkyl acid or C13-21alkenyl acid in the presence of an activating agent, thereby obtaining an ionizable lipid having a structure of Formula (IA.1),

(IA.1) wherein R² and R³ are each independently C_2-20alkyl; and wherein R⁴ and R⁵ are each independently selected from C_7-25alkyl or C_13-21alkenyl; with the proviso that the ionizable lipid is not a compound having a structure of Formula (IA.1) wherein R² and R³ are each ethyl, and R⁴ and R⁵ are each CH₃(CH₂)₁₆-.

12. A method for producing an ionizable lipid, the method comprising: - reacting a diC_2-20alcoholamine with a compound of formula Br-R⁶-C(=O)OCH₃, wherein R⁶ is -(CH₂)_m-, wherein m is 2, or with a methylacrylate, thereby obtaining an intermediate having a structure of formula (XIVA); - purifying the intermediate of formula (XIVA); and - reacting the intermediate of formula (XIVA) with (i) R⁴-COOCl or R⁵-COOCl, or (ii) R⁴- COOH or R⁵-COOH in the presence of an activating agent, thereby obtaining an ionizable lipid having a structure of Formula (IA.1), wherein R² and R³ are each independently C2-20alkyl; and wherein R⁴ and R⁵ are each independently selected from CH3(CH2)6-, CH3(CH2)8-, CH3(CH2)10-, CH3(CH2)12-, CH3(CH2)14-, CH3(CH2)18-, CH3(CH2)20-, CH3(CH2)22-, CH3(CH2)24-, [CH3(CH2)7][CH3(CH2)5]CH-, CH3(CH2)3CH=CH(CH2)7-, CH₃(CH₂)₅CH=CH(CH₂)₇-, CH₃(CH₂)₈CH=CH(CH₂)₄-, CH₃(CH₂)₇CH=CH(CH₂)₇-, CH₃(CH₂)₅CH=CH(CH₂)₉-, CH₃(CH₂)₄CH=CHCH₂CH=CH(CH₂)₇-, CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)₇-, CH3(CH2)4CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)3-, CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)3-, CH3(CH2)7CH=CH(CH2)11-, or CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)2-.

13. The method according to claim 11 or 12, further comprising: - reacting the ionizable lipid having a structure of Formula (IA.1) in an alkaline medium or in a mixture of THF/H2O/NaOH; and - purifying an ionizable lipid having a structure of Formula (IA.2)

(IA.2).

14. The method according to claim 13, further comprising: - reacting the ionizable lipid having a structure of Formula (IA.2) with oxalylchloride and dimethylformamide (DMF), thereby obtaining an acid chloride lipid intermediate having a structure of Formula (XVA);

and - reacting the acid chloride lipid intermediate of formula (XVA) with

, wherein Y¹, Y², and Y³ are each independently C_1-12 alkyl, Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C_1-8alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S, as heteroatom(s), thereby obtaining an ionizable lipid having a structure of Formula ((IA.3), (IA.4) or (IA.5)

15. A lipid nanoparticle (LNP) comprising an ionizable lipid, wherein the ionizable lipid has a structure of Formula (IA), wherein R¹ is hydrogen, C1-12alkyl,

, wherein Y¹, Y², and Y³ are each independently C1-12 alkyl, and wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C1-8alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S, as heteroatom(s); wherein R² and R³ are each independently C2-20alkyl; wherein R⁴ and R⁵ are each independently selected from C7-25alkyl or C13-21alkenyl.

16. The LNP according to claim 15, wherein the LNP further comprises: - a PEGylated lipid; preferably wherein the PEGylated lipid is selected from the group consisting of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPE)-PEG, distearoyl-rac-glycerol (DSG)-PEG, or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, wherein the molecular weight of PEG ranges from 1-10 kDa; - a helper lipid; preferably wherein the helper lipid is selected from the group consisting of cholesterol, dioleoylphosphatidylethanolamine (DOPE), and 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC); and/or - a therapeutic agent, preferably a nucleic acid, such as DNA or RNA; more preferably mRNA.

17. A method of preparing lipid nanoparticles (LNP) comprising a therapeutic agent, the method comprising: - mixing an aqueous solution comprising a therapeutic agent with an organic solvent comprising an ionizable lipid, a PEGylated lipid, and a helper lipid, wherein the ionizable lipid has a structure of Formula (IA), wherein R¹ is hydrogen, C_1-12alkyl,

, wherein Y¹, Y², and Y³ are each independently C_1-12 alkyl, and wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C_1-8alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵- N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S, as heteroatom(s); wherein R² and R³ are each independently C_2-20alkyl; wherein R⁴ and R⁵ are each independently selected from C_7-25alkyl or C_13-21alkenyl; and - removing the organic solvent, thereby obtaining LNP comprising the therapeutic agent.

18. A method according to claim 17, wherein: - the PEGylated lipid is selected from the group consisting of DMG-PEG, DSPE-PEG, DSG-PEG, or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, wherein the molecular weight of PEG ranges from 1-10 kDa; and/or - the helper lipid is selected from the group consisting of cholesterol, DOPE, and DSPC; and/or - the organic solvent is removed by dialysis, spin filtration, or evaporation; and/or - the therapeutic agent is a nucleic acid, such as DNA or RNA, preferably wherein the therapeutic agent is mRNA; and/or - the therapeutic agent is a protein.

19. A lipid nanoparticle comprising a therapeutic agent, for use in a method of delivering a therapeutic agent to a subject, particularly for use in a method of delivering a nucleic acid to a subject, wherein the lipid nanoparticle comprises an ionizable lipid having a structure of Formula (IA), wherein R¹ is hydrogen, C_1-12alkyl,

, wherein Y¹, Y², and Y³ are each independently C_1-12 alkyl, and wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C_1-8alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S, as heteroatom(s); wherein R² and R³ are each independently C_2-20alkyl; wherein R⁴ and R⁵ are each independently selected from C_7-25alkyl or C_13-21alkenyl.

20. The lipid nanoparticle according to claim 15 or 16, the method according to claim 17 or 18, or the lipid nanoparticle for use according to claim 19, wherein: - the ionizable lipid is as defined in any one of claims 1 to 10; - the ionizable lipid has a structure of Formula (II) or (III), wherein R¹, R⁴, and R⁵ have the same meaning as defined in claim 15; - R¹ is hydrogen, C1-6alkyl,

, , or ; wherein Y¹, Y², and Y³ are each independently C_1-6alkyl, preferably wherein Y¹, Y², and Y³ are each independently C_2-4alkyl, and wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C_1-6alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, comprising N, and optionally O or S, as heteroatom(s); preferably wherein Z¹, Z², Z³, Z⁴, Z⁵, Z⁶ and Z⁷ are each independently C1-3alkyl and/or wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a saturated or unsaturated heterocyclic structure, comprising one or two nitrogen atoms, 2 to 8 carbon atoms, and optionally O or S; more preferably wherein the N-Z³-Z⁴ or the N-Z⁵-N-Z⁶ cyclic structure is a piperazine, pyrrolidine, imidazolidine, piperidine, morpholine, thiomorpholine, hexahydroazepine, azocane, or azonane ring structure; - R¹ is hydrogen, methyl, ethyl, n-propyl,

;

- R¹ is or ; - the ionizable lipid has a structure of Formula (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), or (XIII), wherein R⁴ and R⁵ have the same meaning as defined in claim 15; - R⁴ and R⁵ are each independently selected from CH₃(CH₂)₆-, CH₃(CH₂)₈-, CH₃(CH₂)₁₀- , CH₃(CH₂)₁₂-, CH₃(CH₂)₁₄-, CH₃(CH₂)₁₆-, CH₃(CH₂)₁₈-, CH₃(CH₂)₂₀-, CH₃(CH₂)₂₂-, CH₃(CH₂)₂₄-, [CH₃(CH₂)₇][CH₃(CH₂)₅]CH-, CH₃(CH₂)₃CH=CH(CH₂)₇-, CH₃(CH₂)₅CH=CH(CH₂)₇-, CH₃(CH₂)₈CH=CH(CH₂)₄-, CH₃(CH₂)₇CH=CH(CH₂)₇-, CH₃(CH₂)₅CH=CH(CH₂)₉-, CH₃(CH₂)₄CH=CHCH₂CH=CH(CH₂)₇-, CH3CH2CH=CHCH2CH=CHCH2CH=CH(CH2)7-, CH3(CH2)4CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)3-, CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)3-, CH3(CH2)7CH=CH(CH2)11-, or CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)2-; - R⁴ and R⁵ are each independently selected from [CH₃(CH₂)₇][CH₃(CH₂)₅]CH-, CH₃(CH₂)₃CH=CH(CH₂)₇-, CH₃(CH₂)₅CH=CH(CH₂)₇-, CH₃(CH₂)₈CH=CH(CH₂)₄-, CH₃(CH₂)₇CH=CH(CH₂)₇-, CH₃(CH₂)₅CH=CH(CH₂)₉-, CH3(CH2)4CH=CHCH2CH=CH(CH2)7-, CH3CH2CH=CHCH2CH=CHCH2CH=CH(CH2)7- , CH3(CH2)4CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)3-, CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)3-, CH3(CH2)7CH=CH(CH2)11-, or CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)2-; - R⁴ and R⁵ are each independently selected from [CH3(CH2)7][CH3(CH2)5]CH-, CH3(CH2)3CH=CH(CH2)7-, CH3(CH2)5CH=CH(CH2)7-, CH3(CH2)8CH=CH(CH2)4-, CH3(CH2)7CH=CH(CH2)7-, or CH3(CH2)5CH=CH(CH2)9; - R⁴ and R⁵ are each independently selected from [CH3(CH2)7][CH3(CH2)5]CH- or CH3(CH2)7CH=CH(CH2)7-; - R⁴ and R⁵ are the same or different; - R⁴ and R⁵ are the same; - R⁴ and R⁵ are the same and are selected from CH3(CH2)6-, CH3(CH2)8-, CH3(CH2)10-, CH3(CH2)12-, CH3(CH2)14-, CH3(CH2)16-, CH3(CH2)18-, CH3(CH2)20-, CH3(CH2)22-, CH3(CH2)24-, [CH3(CH2)7][CH3(CH2)5]CH-, CH3(CH2)3CH=CH(CH2)7-, CH3(CH2)5CH=CH(CH2)7-, CH3(CH2)8CH=CH(CH2)4-, CH3(CH2)7CH=CH(CH2)7-, CH3(CH2)5CH=CH(CH2)9-, CH3(CH2)4CH=CHCH2CH=CH(CH2)7-, CH3CH2CH=CHCH2CH=CHCH2CH=CH(CH2)7-, CH3(CH2)4CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)3-, CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)3-, CH3(CH2)7CH=CH(CH2)11-, or CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)2-; - R⁴ and R⁵ are the same and are selected from [CH3(CH2)7][CH3(CH2)5]CH-, CH3(CH2)3CH=CH(CH2)7-, CH3(CH2)5CH=CH(CH2)7-, CH3(CH2)8CH=CH(CH2)4-, CH3(CH2)7CH=CH(CH2)7-, CH3(CH2)5CH=CH(CH2)9-, CH₃(CH₂)₄CH=CHCH₂CH=CH(CH₂)₇-, CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)₇- , CH₃(CH₂)₄CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)₃-, CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)₃-, CH₃(CH₂)₇CH=CH(CH₂)₁₁-, or CH₃CH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)₂-; - R⁴ and R⁵ are the same and are selected from [CH₃(CH₂)₇][CH₃(CH₂)₅]CH-, CH₃(CH₂)₃CH=CH(CH₂)₇-, CH₃(CH₂)₅CH=CH(CH₂)₇-, CH₃(CH₂)₈CH=CH(CH₂)₄-, CH₃(CH₂)₇CH=CH(CH₂)₇-, or CH₃(CH₂)₅CH=CH(CH₂)₉-; and/or - R⁴ and R⁵ are the same and are selected from [CH₃(CH₂)₇][CH₃(CH₂)₅]CH- or CH₃(CH₂)₇CH=CH(CH₂)₇-.