Lipid nanoparticle with nucleic acid cargo and ionizable lipid

Abstract

Disclosed is a lipid nanoparticle (LNP) encapsulating a nucleic acid cargo preferably comprising messenger ribonucleic acid (mRNA). The LNP comprises at least an ionizable lipid fraction, and a stabilizer fraction. The stabilizer fraction preferably comprises at least one polyethylenglycol (PEG) lipid. Furthermore, the ionizable lipid fraction comprises at least one ionizable glycerol dialkyl glycerol tetraether (GDGT) lipid. Also disclosed is a pharmaceutical composition comprising the LNP, such as an mRNA vaccine. In a further aspect, the invention relates to the ionizable GDGT lipids and methods for producing them.

Description

Lipid nanoparticle with nucleic acid cargo and ionizable lipidThe field of the present invention relates to lipid nanoparticles (LNPs) with nucleic acid cargo, in particular as used in messenger ribonucleic acid (mRNA) vaccines, as well as to ionizable lipids suitable for use in LNPs.LNPs have recently come into focus because LNP-based mRNA vaccines against SARS-CoV-2 (primarily elasomeran marketed under Spikevax® by Moderna Inc., and tozinameran marketed under Comirnaty® by Biontech SE / Pfizer Inc) were administered to hundreds of millions of individuals. LNPs as delivery systems for RNA-based vaccines in general are reviewed in Aldosari et al., 2021. LNPs for mRNA delivery are discussed in detail also in Hou et al, 2021.LNPs are not only usable for vaccines but also for the delivery of other therapeutic nucleic acids. For instance, patisiran is an LNP-based drug for RNA interference therapy of Transthyretin-Mediated Amyloidosis (see Zhang et al, 2019).In general, LNPs with nucleic acid cargo (or payload) comprise a lipid layer as well as microdomains of lipid and encapsulated nucleic acid. They have a median diameter between 10 nm to 1000 nm (e.g. as determined by dynamic light scattering, DLS) and may adopt e.g. a spherical or polyhedral shape. They may be multilamellar, dependent on their specific lipid composition. The LNPs comprise ionizable lipids, in particular lipids which are protonated at endosomal pH (e.g. in the range of pH 4.5 to 6.5, preferably pH 4.5 to 6.0, especially pH 4.5 to 5.5), i.e., when in an endosome. Typically, the LNPs further comprise stabilizers such as polyethylenglycol (PEG) lipids which decrease LNP aggregation, enzymatic degradation, opsonisation and immunogenicity. In addition, LNPs usually comprise other types of lipids (often termed "helper lipids"), such as phosphatidylcholines or phosphatidylethanolamines, to improve properties such as delivery efficacy, tolerability or biodistribution. Finally, LNPs may contain cholesterol or other sterols to modulate membrane integrity and rigidity.LNPs are for instance also disclosed in US patents US 7,404,969, US 8,058,069, US 9,364,435 and US 9,404,127 as well as in US 2013 / 0245107 Al.WO 2017 / 099823 Al discloses an accelerated-blood-clearance- insensitive LNP, comprising a cationic lipid, a polyethylene glycol (PEG)-lipid, a sterol, and a helper lipid, wherein the helper lipid does not comprise a phosphatidyl choline.WO 2020 / 061284 Al and WO 2019 / 089818 Al also concern LNPs with PEG lipids.Eygeris et al, 2021, is a review on the chemistry of LNPs for RNA delivery. It discloses PEG lipids in LNP formulations.WO 2014 / 143806 Al discloses lipid particles with PEG lipids.WO 2020 / 219941 Al discloses further LNPs and formulations containing LNPs.WO 2021 / 123332 Al relates to cationic lipids and to LNPs comprising said cationic lipids useful for the delivery of nucleic acids into living cells.Despite the recent advances in the field, there is still a need for improved LNPs, in particular with regard to storage stability and / or transformation (transfection) efficiency. For instance, the LNP-based SARS-CoV-2 vaccine Comirnaty® generally has to be stored at -90°C to -60°C (Summary of product characteristics, version of 13 September 2022, EMEA / H / C / 005735 - 11 / 0143, European Medicines Agency - EMA). Furthermore, an increase in transformation efficiency would allow for lower dosage, thereby decreasing potential side effects.It is thus an object of the present invention to provide an improved LNP with nucleic acid cargo (such as mRNA, siRNA or cDNA cargo), in particular an LNP that has increased transformation efficiency and / or higher storage stability, especially at non-refrigerated temperatures (e.g., room temperature). It is a further object of the present invention to provide novel ionizable lipids suitable for use in LNPs, preferably ionizable lipids which lead to increased transformation efficiency and / or higher storage stability, especially at non-refrigerated temperatures (e.g., room temperature).The present invention provides an LNP encapsulating a nucleic acid cargo. The LNP comprises at least an ionizable lipid fraction and a stabilizer fraction (preferably comprising PEG lipids). The ionizable lipid fraction comprises at least one ionizable GDGT lipid. The LNP may also comprise neutral GDGT lipids as disclosed hereinbelow.As used herein, the ionizable lipid is a lipid which carries an overall positive charge (i.e., is cationic) at endosomal pH, e.g. a pH of 5.0-6.5, in particular at a pH of 5.0, 5.5, 6 or 6.5In another aspect, the present invention relates to a GDGT lipid comprising at least one ionizable head group S. This head group S is select from the group consisting of:wherein each occurrence of Rais independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl , alkylamine, alkyl ether, alkyl thiol, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide (preferably from alkyl, cycloalkyl, alkenyl, hydroxyalkyl and alkylamine), each occurrence of Rbis independently selected from H, alkyl,alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate, alkyl sulfonamide and alkyl thiol (preferably from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine and alkyl thiol), and each occurrence of Rcis independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide (preferably from alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide). Preferably, this GDGT lipid is suitable for use as an ionizable lipid in an LNP, such as the LNP disclosed herein.In a further aspect, the present invention provides an ether lipid fraction comprising this GDGT lipid. This ether lipid fraction is preferably obtainable by extraction from an archaeal culture, preferably a Sulfolobus culture, more preferably a Sulfolobus acldocaldarlus culture, followed by substitution (of the GDGTs present therein) with the ionizable head group S.The present invention also relates to an LNP (as defined herein) comprising the GDGT lipid or ether lipid fraction disclosed above.In yet another aspect, the present invention relates to a pharmaceutical composition (in particular a vaccine) comprising the LNP. This pharmaceutical composition typically comprises further excipients. It is preferably for use in prevention or treatment of a disease or condition in a (human) patient, in particular as a vaccine to prevent (or ameliorate) a disease such as an infectious disease or as a cancer vaccine.In yet another aspect, the present invention provides a method for producing the GDGT lipid, comprising the steps of:- obtaining a lipid fraction comprising one or more precursor GDGT lipids from an archaeal culture, preferably a Sulfolobus culture, more preferably a Sulfolobus acldocaldarlus culture;- purifying the one or more precursor GDGT lipids; and- contacting the one or more precursor GDGT lipids with one or more reagents to produce the GDGT lipid with the at least one ionizable head group S.GDGT lipids (also called "GDGTs" herein) were discovered as membrane lipids of extremophilic archaea but more recently were also observed as membrane components of some bacteria (cf. Schouten et al, 2013). Many phylogenetic groups within the Archaea synthesize GDGTs. They form a monolayer instead of a bilayer in the cell membrane. Surprisingly, both ionizable and non-ionizable GDGT lipids are exceptionally well suited to improve the properties (such as storage stability and transformation efficiency) of LNPs with nucleic acid cargo.Importantly, LNPs (as described in more detail above) are distinct from other lipid-containing delivery vehicles such as liposomes, lipo (poly)plexes or archaeosomes and present unique advantages but also challenges, especially in the context of packaging mRNA. For instance, Midoux & Pichon, 2014, reviews various lipid-based mRNA vaccine delivery systems, and distinguishes between lipoplexes, lipopolyplexes, LNPs and cationic nanoemulsions. More broadly, drug delivery for RNA therapeutics (such as small interfering RNAs and mRNAs) is enabled by lipid-based vehicles such as micelles, liposomes and LNPs (Paunovska et al, 2022).In contrast to LNPs, liposomes are spherical lipid bilayer vesicles (lipid esters) surrounding an aqueous space. They are carriers for the administration of drugs, vaccines, genes, proteins, small molecules, antibiotics and nutrients. Liposomes are made of phospholipids, mainly phosphatidylcholine, and cholesterol, but may also include other lipids, like phosphatidylethanolamine. Liposomes are generated by a large number of different methods (reviewed e.g. by van Hoogevest, 2017; Szoka et al., 1980), for example by dispersing phospholipids in aqueous medium, e.g. using mechanical treatment (e.g. in a homogenizer, preferably by high pressure homogenization) or sonication. They vary between 0.02 to 10 pm in diameter.Archaeosomes represent a special class of liposomes based on membrane lipids isolated from archaea. Archaeosomes are made oflipid ethers, namely diether structures (such as archaeols) and tetraether structures (e.g. GDGTs, such as caldarchaeols); see e.g. Kaur et al., 2016; Patel et al., 1999. Diether structures are typically composed of a glycerol moiety carrying two phytanyl chains (20-40 carbons in length) on the sn-2,3 positions. Tetraether structures typically carry two di-phytanyl chains linked to two glycerol residues in either an antiparallel manner (caldarchaeol) or a parallel manner (iso-caldarchaeol). Furthermore, one or several cyclopentane rings may occur.Benvegnu et al, 2009, relates to GDGTs with polar substituents. Vaz et al, 1985, discloses GDGTs with phosphoethanol-amine-containing substituents. Morii et al, 1994, discloses the structures of major polar lipids of M. thermoautotrophicum. Schwarzmann et al, 2015, discloses the polar GDGT caldarchaeyl-bis-amine.As used herein, "caldarchaeol" refers to the whole group of isoprenoid GDGT lipids with 0 up to 8 cyclopentane moieties. In particular, the nomenclature suggested by Schouten et al., 2013, is used herein: "GDGT-x", where x denotes the number of cyclopentane moieties, i.e. GDGT-0, GDGT-1, GDGT-2, GDGT-3, GDGT-4, GDGT-5, GDGT-6, GDGT-7 and GDGT-8. All of these belong to the group of caldarchaeols.Depending on the composition (e.g. amount of archaeols vs. caldarchaeols), the lipid layer of archaeosomes is a mono- or a bi-layer or a mixture thereof.WO 2020 / 187526 Al discloses archaeosomes comprising archaeal lipids from a Sulfolobus cell culture, mainly intended for oral acute or oral retard delivery.WO 02 / 053554 A2 relates to tetraether lipid derivatives and liposomes containing tetraether lipid derivatives and lipid agglomerates and the use thereof. In the example, the tetraether lipids were obtained from Sulfolobus acldocaldarlus. The tetraether scaffold may contain e.g. four cyclopentane and may be substituted. WO 97 / 31927 Al, WO 99 / 10337 Al, US 2010 / 316657 Al and US 6,316,260 Bl also disclose tetraether lipid derivatives. Further, WO 03 / 064360 Al and WO 2004 / 037223 A2 relate to tetraether lipid derivatives and liposomes containing tetraether lipid derivatives.Sateesh et al, 2008, concerns surface modification of medical-grade polyurethane by cyanurchloride-activated tetraether lipid, thereby providing a new approach for bacterial antiadhesion .Engelhardt et al, 2017, concerns transfection studies with colloidal systems containing highly purified bipolar tetraether lipids from Sulfolobus acidocaldarius.WO 2017 / 067642 Al discloses liposomes containing cell penetrating peptides and tetraether lipids for the oral delivery of macromolecules.Vishakarma et al, 2019, reviews various lipid-based carriers for lymphatic transportation, among them liposomes and archaeosomes .GB 2463801 A discloses liposomes comprising isoprenoid lipids.Further in relation to archaeosomes, Daswani et al, 2021, discloses that the polar lipid fraction E from Sulfolobus acidocaldarius can be used as liposomal drug stabilizing agents to reduce the leakage of the antivascular drug combretastatin a4 disodium phosphate from tetraether / diester hybrid archaeosomes.In contrast to archaeosomes, LNPs contain ionizable lipids. Such ionizable lipids are for instance discussed in Cornebise et al, 2022.Tetraether lipids and their function in membranes in general are discussed in the following publications: Weijers et al., 2010, disclose the carbon isotopic composition of branched tetraether membrane lipids in soils. Schuster et al, 1998, concern voltage clamp studies on S-layer-supported tetraether lipid membranes. EP 1777 520 Al discloses a method of coating lipid membranes which may contain tetraether lipids. WO 2002 / 066012 A2 discloses liposomes which may comprise tetraether lipids. US 2014 / 0178462 Al relates to amphoteric liposomes comprising neutral lipids which may be tetraether lipids.Typically, the ionizable GDGT lipid comprises at least one ionizable head group S. Preferably, the GDGT lipid comprises two ionizable head groups S (which are typically both the same).In a preferred embodiment of the present invention, the ionizable lipid is a lipid which carries an overall positive charge at endosomal pH, e.g. a pH between 5.0-6.5, such as pH 5.0, 5.5, 6 or 6.5, but is neutral at a higher pH such as pH 7.0. Accordingly, the ionizable head group S disclosed herein preferably carries an overall positive charge at endosomal pH, e.g. a pH between 5.0-6.5, such as pH 5.0, 5.5, 6 or 6.5, but is neutral at a higher pH such as pH 7.0.The following ionizable head groups have turned out to be particularly suitable for the present invention (in particular for use in the inventive LNP):wherein each occurrence of Rais independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl , alkylamine, alkyl ether, alkyl thiol, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide (preferably from alkyl, cycloalkyl, alkenyl, hydroxyalkyl and alkylamine), each occurrence of Rbis independently selected from H, alkyl,alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate, alkyl sulfonamide and alkyl thiol (preferably from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine and alkyl thiol), and each occurrence of Rcis independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide (preferably from alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide).It goes without saying that each occurrence of Ra, or each occurrence of Rc, may be part of the same ring structure (in particular based on alkyl, alkenyl, hydroxyalkyl alkylamine, alkyl ether, alkyl thiol, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide). Accordingly, a preferred embodiment of the head groupmay be selected from any one ofGDGT based on the first embodiment, EABM-GDGT, is particularly preferred. A representative structure (based on a GDGT-4 scaffold) is shown in the following:Further preferred embodiments of this head group are shown e.g., in Example 12 below.Preferably, the alkyl is selected from methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, pentyl, iso-pentyl, hexyl, iso-hexyl, heptyl, and iso-heptyl.Alternatively, or in addition thereto, the cycloalkyl is preferably selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.Alternatively, or in addition thereto, the alkenyl is preferably selected from prop-2-enyl, but-2-enyl, but-3-enyl, pent-2-enyl, pent-3-enyl, pent-4-enyl, hex-2-enyl, hex-3-enyl, hex-4-enyl, hex-5-enyl, hept-2-enyl, hept-3-enyl, hept-4-enyl, hept-5-enyl and hept-6-enyl.Alternatively, or in addition thereto, the hydroxyalkyl is preferably selected from hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, hydroxypentyl and hydroxyhexyl.Alternatively, or in addition thereto, the alkylamine is preferably selected from methylamine, ethylamine, propylamine, butylamine, pentylamine, hexylamine and heptylamine.Alternatively, or in addition thereto, the alkyl ether is preferably selected from methyl ether, ethyl ether, propyl ether, butyl ether, pentyl ether, hexyl ether and heptyl ether.Alternatively, or in addition thereto, the alkyl thiol is preferably selected from methyl thiol, ethyl thiol, propyl thiol, butyl thiol, pentyl thiol, hexyl thiol and heptyl thiol.Alternatively, or in addition thereto, the alkyl ester is preferably selected from methyl ester, ethyl ester, propyl ester, butyl ester, pentyl ester, hexyl ester and heptyl ester.Alternatively, or in addition thereto, the alkyl amide is preferably selected from methyl amide, ethyl amide, propyl amide, butyl amide, pentyl amide, hexyl amide and heptyl amide.Alternatively, or in addition thereto, the alkyl carbamate is preferably selected from methyl carbamate, ethyl carbamate, propyl carbamate, butyl carbamate, pentyl carbamate, hexyl carbamate and heptyl carbamate.Alternatively, or in addition thereto, the alkyl sulfonate is preferably selected from methyl sulfonate, ethyl sulfonate,propyl sulfonate, butyl sulfonate, pentyl sulfonate, hexyl sulfonate and heptyl sulfonate.Alternatively, or in addition thereto, the alkyl sulfonamide is preferably selected from methyl sulfonamide, ethyl sulfonamide, propyl sulfonamide, butyl sulfonamide, pentyl sulfonamide, hexyl sulfonamide and heptyl sulfonamide.The following head groups are particularly preferred in the context of the present invention:wherein each occurrence of Raand Rbindependently are as defined above.According to another preferred embodiment, the head groupwherein each occurrence of Rcindependently is as defined above. Particularly preferred examples of this head group are disclosed in Example 11.The following ionizable GDGT lipids (i.e. containing the ionizable head group S as defined herein) are particularly suitable for use in the inventive LNP:GDGT-X1GDGT-lllbwherein S1is S and S2is OH, or S1is OH and S2is S, or each of S1and S2independently is S. Preferably, S1and S2are the same head group S as defined herein.From the GDGT scaffolds above, the following scaffolds are particularly preferred: GDGT-0, GDGT-1, GDGT-2, GDGT-3, GDGT-4, GDGT-5, GDGT-6, GDGT-7 and / or GDGT-8. The ionizable lipid fraction of the LNP (or the inventive ether lipid fraction) preferably contains a mixture containing at least two, preferably at least three, more preferably at least four, even more preferably at least five, yet even more preferably at least six, especially at least seven or even at least eight, or all of these scaffolds.Figures 1-10 depict particularly preferred ionizable GDGT lipids. While GDGT-4 scaffolds are depicted, other GDGT scaffolds (in particular GDGT-0, GDGT-1, GDGT-2, GDGT-3, GDGT-5, GDGT-6, GDGT-7 and GDGT-8) are also preferred in this connection. For substitutions such as "alkyl", "cycloalkyl",etc., the above preferred embodiments preferably apply (e.g. that alkyl is preferably selected from methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, pentyl, iso-pentyl, hexyl, isohexyl, heptyl, and iso-heptyl; that cycloalkyl is preferably selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl, etc.). Each instance of "Rx" or "R2" may independently be selected from the definitions given directly below the structure.In another preferred embodiment, the ionizable lipid fraction comprises at least one further ionizable lipid selected from the group consisting of [(4- hydroxybutyl)azanediyl]di (hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl]amino }octanoate (SM-102),3- (didodecylamino)- N1,N1,4-tridodecyl-l-piperazineethanamine (KL10), Nl-[2- (didodecylamino)ethyl] N1,N4,N4-tridodecyl-l,4- piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24- tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N- dimethylaminopropane (Dlin-DMA), 2,2-dilinoleyl-4- dimethylaminomethyl- [1,3]-dioxolane (Dlin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4- (dimethylamino)butanoate (Dlin-MC3-DMA), 2,2-dilinoleyl-4- (2 dimethylaminoethyl)-[1,3 ]-dioxolane (Dlin-KC2-DMA), 1,2- dioleyloxy-N,N-dimethylaminopropane (DODMA), 2- ({8 [(3|3)— cholest-5-en-3-yloxy]octyl }oxy) N,N dimethyl-3-[(9Z,12Z)- octadeca-9,12-dien-l-yloxy]propan-l-amine (Octyl-CLinDMA), (2R)- 2- ({8-[(3(3)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3- [ (9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l-amine (Octyl- CLinDMA (2R)), (2S) 2- ({8-[(3(3)-cholest-5-en-3-yloxy]octyl}oxy)- N,N-dimethyl-3- [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l- amine (Octyl-CLinDMA (2S)) and mixtures thereof. Alternatively, or in addition thereto, the ionizable lipid is preferably selected from the group consisting of (20Z,23Z)—N,N- dimethylnonacosa-20,23-dien-l 0-amine, (17Z,20Z)—N,N- dimemylhexacosa-17,20-dien-9-amine, (1Z,19Z)—N5N- dimethylpentacosa-16, 19-dien-8-amine, (13Z,16Z)—N,N- dimethyldocosa-13,16-dien-5-amine, (12Z,15Z)—N,N dimethylhenicosa-12,15-dien-4-amine, (14Z,17Z)—N,N- dimethyltricosa-14,17-dien-6-amine, (15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-7-amine, (18Z,21Z)—N,N- dimethylheptacosa-18,21-dien-l 0-amine, (15Z,18Z)—N,N- dimethyltetracosa-15,18-dien-5-amine, (14Z,17Z)-N,N- dimethyltricosa-14,17-dien-4-amine, (19Z,22Z)—N,N- dimeihyloctacosa-19,22-dien-9-amine, (18Z,21 Z)-N,N- dimethylheptacosa-18,21-dien- 8-amine, (17Z,20Z)—N,N- dimethylhexacosa-17,20-dien-7-amine, (16Z,19Z)—N,N- dimethylpentacosa-16,19-dien- 6-amine, (22Z,25Z)—N,N- dimethylhentriaconta-22,25-dien-10-amine, (21 Z,24Z)-N,N- dimethyltriaconta-21,24-dien- 9-amine, (18Z)—N,N-dimetylheptacos- 18-en-l0-amine, (17Z)—N,N-dimethylhexacos-17-en-9-amine, (19Z,22Z)—N,N-dimethyloctacosa-1 9,22-dien-7-amine, N,N- dimethylheptacosan-10-amine, (20Z,23Z)—N-ethyl-N-methyInonacosa- 20,23-dien-l0-amine, 1-[(11Z,14Z)-1-nonylicosa-l1,14-dien-l- yl]pyrrolidine, (20Z)—N,N-dimethylheptacos-20-en-l0-amine, (15Z)—N,N-dimethyl eptacos-15-en-10-amine, (14Z)-N,N- dimethylnonacos-14-en-l0-amine, (17Z)—N,N-dimethylnonacos-17-en- 10-amine, (24Z)—N,N-dimethyltritriacont-24-en-l0-amine, (20Z)— N,N-dimethylnonacos-20-en-l0-amine, (22Z)—N,N- dimethylhentriacont-22-en-10-amine, (16Z)—N,N-dimethyIpentacos- 16-en-8-amine, (12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15- dien-l-amine, (13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-l- amine, N,N-dimethyl-1-[(IS,2R)-2-octylcyclopropyl] eptadecan-8- amine, 1-[(IS,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10- amine, N,N-dimethyl-1-[(IS,2R)-2-octylcyclopropyl]nonadecan-10- amine, N,N-dimethyl-21-[(IS,2R)-2-octylcyclopropyl]henicosan-10- amine,N,N-dimethyl-1- [(IS,2S)-2-{[(1R,2R)-2- pentylcyciopropyl]methyl }cyclopropyl]nonadecan-10-amine,N,N- dimethyl-1- [(1 S,2R)-2-octylcyclopropyl]hexadecan-8-amine, N,N- dimethyl- [(1R,2S)-2 undecylcyclopropyl]tetradecan-5-amine, N,N- dimethyl-3-{7- [(IS,2R)-2-octylcyclopropyl]heptyl} dodecan-1- amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9- amine, 1-[(IS,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6- amine, N,N-dimethyl-1-[(IS,2R)-2-octylcyclopropyl]pentadecan-8- amine, R—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-3- (octyloxy)propan-2-amine, S—N,N-dimethyl-1-[(9Z,12Z)-octadeca- 9,12-dien-l-yloxy]-3- (octyloxy)propan-2-amine, 1—{2—[(9Z,12Z)- octadeca-9,12-dien-l-yloxy]-1-[ (octyloxy)methyl]ethylJpyrrolidine, (2 S)—N,N-dimethyl-1-[ (9Z,12Z)-octadeca-9,12-dien-l-yloxy]-3-[(5Z)-oct-5-en-l- yloxy]propan-2-amine, 1—{2—[(9Z,12Z)-octadeca-9,12-dien-l- yloxy]-1- [(octyloxy)methyl]ethyl}azetidine, (2S)-1- (hexyloxy)- N,N-dimethyl-3- [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-2- amine, (2S)-1- (heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca- 9,12-dien-l-yloxy]propan-2-amine, N,N-dimethyl-1- (nonyloxy)-3- [ (9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-2-amine, N,N- dimethyl-1- [(9Z)-octadec-9-en-l-yloxy]-3- (octyloxy)propan-2- amine; (2S)—N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-l- yloxy]-3- (octyloxy)propan-2-amine, (2S)-1-[(11Z,14Z)-icosa- 11,14-dien-l-yloxy]-N,N-dimethyl-3- (pentyloxy)propan-2-amine, (2S)-1- (hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-l-yloxy]-N,N- dimethylpropan-2-amine, 1-[(11Z,14Z)-icosa-11,14-dien-l-yloxy]- N,N-dimethyl-3- (octyloxy)propan-2-amine, 1-[(13Z,16Z)-docosa- 13,16-dien-l-yloxy]-N,N-dimethyl-3- (octyloxy)propan-2-amine, (2S)-1- [(13Z,16Z)-docosa-13,16-dien-l-yloxy]-3- (hexyloxy)-N,N- dimethylpropan-2-amine, (2S)-1-[(13Z)-docos-13-en-l-yloxy]-3- (hexyloxy)-N,N-dimethylpropan-2-amine, 1-[(13Z)-docos-13-en-l- yloxy]-N,N-dimethyl-3- (octyloxy)propan-2-amine, 1-[(9Z)-hexadec- 9-en-l-yloxy]-N,N-dimethyl-3- (octyloxy)propan-2-amine, (2R)—N,N- dimethyl-H (1-metoyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-l- yloxy]propan-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N- dimethyl-3- [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-2-amine, N,N-dimethyl-1- (octyloxy)-3-({8-[(IS,2S)-2-[(1R,2R)-2- pentylcyclopropyl]methyl }cyclopropyl]octyl}oxy)propan-2-amine, N,N-dimethyl-1-{ [8- (2-oclylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine, and (1IE,2OZ,23Z)— N,N- dimethylnonacosa-11,20,2-trien-10-amine, and pharmaceutically acceptable salts and stereoisomers thereof, and mixtures thereof.Further suitable ionizable lipids are disclosed e.g. in Hou et al, 2021, US patents US 7,404,969, US 8,058,069, US 9,364,435 and US 9,404,127, WO 2017 / 099823 Al, WO 2020 / 061284 Al, WO 2020 / 219941 Al and WO 2021 / 123332 Al. These documents are included herein by reference in their entirety.Even further suitable ionizable lipids are disclosed e.g. in WO 2017 / 049245 Al, WO 2017 / 112865 Al, WO 2012 / 040184, WO 2011 / 153120 Al, WO 2011 / 149733 Al, WO 2011 / 090965 Al, WO 2011 / 043913 Al, WO 2011 / 022460 Al, WO 2012 / 061259 Al, WO2012 / 054365 Al, WO 2012 / 044638 Al, WO 2010 / 080724 Al, WO 2010 / 21865 Al, WO 2008 / 103276 Al, WO 2013 / 086373 Al and WO 2013 / 086354 Al, US patent nos. 7,893,302, 7,404,969, 8,283,333, and 8,466,122 and US patent publication no. US20100036115, US20120202871, US20130064894, US20130129785, US20130150625, US20130178541, and US20130225836. These documents are incorporated herein by reference in their entirety.The stabilizer fraction of the inventive LNP is typically suitable for achieving one or more of the following: decreasing LNP aggregation, increasing average particle size or hydrodynamic radius, increasing the half-life of the LNP in vivo (e.g. in a human, in particular in blood circulation) and modifying zeta potential.According to a particular preference, the stabilizer fraction comprises at least one PEG lipid. Suitable PEG lipids are for instance 2-[(polyethylene glycol)-2000]-N,N- ditetradecylacetamide (ALC-0159), pegylated diacylglycerol lipid (PEG-DAG), a pegylated ceramide lipid (PEG-Cer), a pegylated phosphatidylethanoloamine lipid (PEG-PE), a pegylated succinate diacylglycerol lipid (PEG-S-DAG), a pegylated dialkoxypropylcarbamate lipid, 1 ,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol ("PEG-DMG" or "DMG-PEG"), in particular PEG2000-DMG, 1,2-dicapryl-rac-glycero-3- methylpolyoxyethylene glycol (Cio-diacylglycerol PEG), N- octanoyl-sphingosine-1- {succinyl[methoxy (polyethylene glycol)2000]} (comprising N-octanoyl-D-erythro-sphingosine (dl8:1 / 8:0), also named PEG-Ceramide8), or a PEG lipid as disclosed in WO 2018 / 126084 Al, W0 2020 / 093061 Al, or WO 2020 / 219941 Al (all three references are incorporated by reference in their entirety); and any combination thereof.Further suitable PEG lipids are e.g. disclosed in Hou et al, 2021, WO 2017 / 099823, WO 2020 / 061284 Al, WO 2020 / 219941 Al and WO 2021 / 123332 Al. All of these documents are incorporated by reference herein in their entirety.Alternatively, or in addition thereto, the stabilizer fraction may comprise at least one non-PEG moiety such as an XTEN peptide that may or may not be conjugated to a lipid. The XTEN peptide is capable of forming a hydrated shell around theLNP due to its hydrophilic nature. It further serves to increase the half-life of the LNP, compared to an LNP lacking (or free of) a stabilizer fraction. XTEN amino acid sequences are known in the art, including for example those reported in U.S. patent no. 9,062,299 (incorporated herein by reference in its entirety). Alternatively, or in addition thereto, in some embodiments, the stabilizer fraction may comprise non-PEG moiety such as a PAS peptide (that may or may not be conjugated to a lipid). A PAS peptide is a peptide comprising primarily if not exclusively proline, alanine and serine. Like PEG and XTEN peptides, the PAS peptide is capable of forming a hydrated shell around the LNP. It too serves to increase the half-life of an LNP, compared to an LNP lacking (or free of) a stabilizer fraction. PAS amino acid sequences are known in the art, including for example, those reported in WO 2008 / 155134 Al (incorporated herein by reference in its entirety).The inventive LNP preferably comprises at least one neutral (or non-ionizable, or, in other words, neutral at endosomal pH) GDGT lipid. This GDGT lipid may e.g. be an isoprenoid GDGT lipid such as a GDGT-0, a GDGT-1, a GDGT-2, a GDGT-3, a GDGT-4, a GDGT-5, a GDGT-6, a GDGT-7 and a GDGT-8, or crenarchaeol (unsubstituted, cf. Schouten et al, 2013, Fig. 1, or substituted), or a branched GDGT such as GDGT-I, GDGT-II or GDGT-III, or any mixture thereof. In particular, the GDGT lipid may comprise any of the GDGTs disclosed in Schouten et al, 2013 (in particular Fig. 1), Kaur et al, 2016 (in particular Fig. 2, Fig. 3 and Fig. 4), and WO 2020 / 187526 Al (all of which are incorporated herein by reference in their entirety). The GDGTs may be substituted (e.g. with hexose moieties or phosphatidylinositol moieties) or unsubstituted.Caldarchaeols (as e.g. obtained from Sulfolobus) turned out to be particularly suitable for the present invention. According to a preferred embodiment, at least one GDGT lipid (of the ether lipid fraction of the LNP) thus comprises at least one neutral (or non-ionizable, or, in other words, neutral at endosomal pH) caldarchaeol, preferably selected from the group consisting of unsubstituted caldarchaeol, phosphatidylinositol (PI)- caldarchaeol, dihexose (2Hex)-caldarchaeol and 2Hex-PI- caldarchaeol (in particular as disclosed in WO 2020 / 187526 Al,Fig. 4), hexose (Hex)-caldarchaeol and sulfono-trihexose (3Hex)- caldarchaeol, sulfono-3Hex-PI-caldarchaeol, and any mixture thereof .An especially preferred Hex-caldarchaeol is:An especially preferred sulfono-3Hex-PI-caldarchaeol is:The inventive LNPs are particularly suitable for RNA cargo. Accordingly, in a preferred embodiment, the nucleic acid cargo comprises at least one (therapeutic) RNA. Examples of suitable RNA payloads are for instance disclosed in Paunovska et al, 2022 (in particular Fig. 1). In embodiments, the cargo may be a small interfering RNA (siRNA), an antisense oligonucleotide, an adenosine deaminase acting on RNA (ADAR) oligonucleotide or an mRNA.The RNA may be chemically modified, e.g. to improve its chemical stability. For instance, it may comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. In some embodiments, the RNA may comprise nucleoside analogs (e.g, 2- aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, 2-aminoadenosine, C5-bromouridine, C5- fluorouridine, C5-iodouridine, C5- propynyl-uridine, C5- propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7- deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g, methylated bases); intercalated bases; modified sugars (e.g, 2'- fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose);and / or modified phosphate groups ( e.g phosphorothioates and 5'-N- phosphoramidite linkages). Further modifications are known to the person skilled in the art. Suitable modifications are e.g. disclosed in WO 2017 / 099823 Al and WO 2020 / 061284 Al (each incorporated by reference in their entirety).In the course of the present invention, it was found that GDGT lipids improve LNP properties in particular for mRNA payload. According to an especially preferred embodiment, the nucleic acid cargo of the LNP hence comprises an mRNA. The mRNA may e.g. be a therapeutic mRNA or an mRNA encoding a vaccine antigen. The mRNA may be codon-optimized. Examples of suitable mRNAs are for instance given in WO 2017 / 099823 Al and WO 2020 / 061284 Al (each incorporated by reference in their entirety), in particular paragraphs

[0178] -

[0180] of the latter.According to a further preferred embodiment, the LNP comprises at least one further (non-ionizable or neutral) ether lipid, preferably a diether lipid (in particular an archaeol). In particular, the diether lipid lipid may comprise any of the diether lipid disclosed in Kaur et al, 2016, WO 2017 / 212197 Al and WO 2020 / 187526 Al (all of which are incorporated herein by reference in their entirety). The diether lipid (in particular the archaeol) may be substituted (e.g. with hexose moieties or phosphatidylinositol moieties) or unsubstituted. A particularly preferred substituted archaeol is phosphatidyl inositol-archaeol (Pl-Arc).According to another preference, the LNP comprises an ether lipid fraction comprising at least one (non-ionizable or neutral) GDGT lipid (in particular a caldarchaeol) and preferably at least one further ether lipid (in particular the diether lipid as disclosed above).It is particularly preferred when the ionizable lipid fraction (or ether lipid fraction) comprises ether lipids obtainable by extraction from an archaeal culture, preferably a Sulfolobus culture, more preferably a Sulfolobus acldocaldarlus culture, followed by substitution with the ionizable head group S. Suitable growth conditions and extraction methods are e.g. disclosed in WO 2020 / 187526 Al (incorporated herein by referencein its entirety) also published as EP 3708 151 Al. In particular, the entire ether lipid fraction is obtainable by extraction from said culture. Suitable substitutions are e.g. disclosed in the Examples.Alternatively, or in addition thereto, it is particularly preferred when the neutral ether lipid fraction comprises ether lipids obtainable by extraction from an archaeal culture, preferably a Sulfolobus culture, more preferably a Sulfolobus acidocaldarius culture. Suitable growth conditions and extraction methods are e.g. disclosed in WO 2020 / 187526 Al (incorporated herein by reference in its entirety). In particular, the entire ether lipid fraction is obtainable by extraction from said culture.In embodiments, the archaeal culture may e.g. also be a culture of S. acidocaldarius, M. hungatei, M. voltae, M. concilii, M. smith!!, M. espanolae, T. acidophilum, M. maze!, M. espanole, T. acidophilum, H. salinarum, M. smith!!, M. stadtmanae, H. halobium, H. morrhuae, M. jannaschii, S. islandicus, S. solfataricus, S. shibatae, S. tokodaii, S. Metallicus, M. sedula, H. hispanica, or H.volcanii, or mixtures (co-cultures) thereof.By way of example, total archaeal lipids may be extracted from e.g. lyophilized or spray-dried biomass by organic solvent extraction using chloroform / methanol / water. Then, the polar and the neutral lipids may be separated by precipitation using acetone. The resulting lipid extracts may be used directly for the preparation of the ether lipid fraction used for LNP production or may be further purified by chromatography to isolate ether lipids of a particular class to be used for LNP production. They may also be formed into ionizable GDGT lipids by substituting head groups, e.g. as disclosed herein, in particular in the Examples.Methods for preparing lipids from archaea or for cultivating archaea are also disclosed e.g. in US 2017 / 0152533 Al, US 6,316,260 Bl, EP 1999 137 Bl, EP 0883 624 Bl, EP 2 109459 Bl, Siliakus et al., 2017, WO 2020 / 187526 Al, Jain et al., 2014; each incorporated herein by reference in its entirety.The LNPs themselves may be produced by microfluidic mixing of the LNP components (fractions), including the GDGT lipids (e.g. present in isolated form or in an ether lipid fraction comprising several ether lipids). Suitable LNP production methods are e.g. disclosed in WO 2017 / 099823 Al, WO 2020 / 061284 Al, and WO 2021 / 123332 Al; each incorporated herein by reference in its entirety. Further LNP production methods are available to the person skilled in the art.According to another preferred embodiment, the LNP further comprises a sterol lipid fraction. Incorporation of sterol lipids in the LNP mitigates aggregation of other lipids in the LNP. Sterol lipids are preferably selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, phytosterols and mixtures thereof. According to a preferable definition, "sterol lipids" comprise the entire subgroup of steroids which consists of steroid alcohols.In particular, the sterol lipid fraction comprises cholesterol .According to yet another preferred embodiment, the LNP further comprises a helper lipid fraction. Helper lipids useful in the present invention comprise (preferably consist of) non- ionizable lipids. In particular, the helper lipid may be a phospholipid .Preferably, the helper lipid is selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), Dipalmitoylphosphatidylcholine (DOPC), phosphatidylcholine (PC) and mixtures thereof. Further suitable helper lipids are e.g. 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), 1,2-di-0-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OchemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2- di1ino1enoyl-sn-glycero-3-phospho choline, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine(ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2- diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2- dioleoyl-sn-glycero-3-phospho-rac- (1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.Further suitable helper lipids and sterol lipids are e.g. disclosed in WO 2017 / 099823 Al and WO 2020 / 061284 Al which are incorporated herein by reference.Certain (molar) ratios in the LNP composition have turned out to be particularly suitable for achieving improved LNPs, in particular with regard to storage stability and / or transformation efficiency:It is thus preferred that the ether lipid fraction (as defined above) accounts for 1 moll - 20 moll of total lipids, preferably 2 moll - 15 moll of total lipids, even more preferably 4 moll - 12 moll of total lipids, especially 6 moll - 10 moll of total lipids or even 7 moll - 9 moll of total lipids of the LNP.Alternatively, or in addition thereto, it is preferred that the molar ratio of (neutral) ether lipid fraction to helper lipid fraction is from 20:1 to 1:20, preferably from 15:1 to 1:10, more preferably from 12:1 to 1:3, even more preferably from 8:1 to 4:1, especially from 7:1 to 5:1.Further, alternatively, or in addition thereto, it is preferred that the molar ratio of (neutral) ether lipid fraction to sterol lipid fraction is from 0.25:1 to 1:30, preferably from 0.5:1 to 1:20, more preferably from 1:1 to 1:10, even more preferably from 1:2 to 1:6, especially from 1:3 to 1:5.According to a particular preference, at least 50 moll of the ionizable lipid fraction of the LNP is made up by the ionizable GDGT lipids, preferably at least 60 moll, more preferably at least 70 moll or even at least 80 moll, especially at least 90moll.Particularly good results are achieved with the following LNP, which thus forms another preferred embodiment of the invention (total lipids of the LNP, i.e. 100 moll):(a) 40 moll - 70 moll ionizable lipid fraction (which comprises the ionizable GDGT lipids, wherein in particular at least 50 moll of the fraction is made up by ionizable GDGT lipids, preferably at least 60 moll, more preferably at least 70 moll or even at least 80 moll, especially at least 90moll),(b) 5 moll - 20 moll helper lipid fraction,(c) 20 moll - 40 moll sterol lipid fraction,(d) 0.1 moll - 4 moll stabilizer fraction, and preferably(e) 1 moll - 20 moll neutral ether lipid fraction (in particular 2 moll - 15 moll, even more preferably 4 moll - 12 moll, especially 6 moll - 10 moll or even 7 moll - 9 moll).As evident to the skilled person upon reading the present application, these ratios and moll given above may be observed as mean values over an entire population of LNPs (e.g. all LNPs present in a pharmaceutical composition).The pharmaceutical composition of the present invention (which comprises a plurality of the inventive LNP) is preferably provided with at least one excipient. Excipients suitable for the pharmaceutical composition of the present invention are known to the person skilled in the art, upon having read the present specification, for example water (especially water for injection), saline, Ringer's solution, dextrose solution, buffers, Hank solution, 51 dextrose in saline, substances that enhance isotonicity and chemical stability, buffers and preservatives. This pharmaceutical composition can (as a drug) be administered via appropriate procedures known to the skilled person (upon having read the present specification) to a patient or individual in need thereof (i.e. a patient or individual having or having the risk of developing the diseases or conditions mentioned herein). The preferred route of administration of said pharmaceutical composition is parenteral administration, in particular through intraperitoneal, subcutaneous, intramuscular and / or intravenous administration. The dosage and method of administration depends on the individual patient or individual to be treated. Saidpharmaceutical composition can be administered in any suitable dosage known from other biological dosage regimens or specifically evaluated and optimized for a given individual. For example, the nucleic acid cargo may be present in the pharmaceutical composition in an amount from 0.1 pg to 1000 pg, preferably 5 pg to 200 pg, in particular 10 pg to 100 pg. Usual dosages can also be determined on the basis of kg body weight of the patient, for example preferred dosages are in the range of 0.01 pg to 100 pg / kg body weight, especially 0.1 to 10 pg / kg body weight (per administration session). The administration may occur e.g. once daily, once every other day, once per week or once every two weeks. Alternatively, the administration may occur for instance a single time or multiple times (e.g. on a weekly, monthly or yearly basis). As the preferred mode of administration of the inventive pharmaceutical composition is parenteral administration, the pharmaceutical composition according to the present invention is preferably liquid or ready to be dissolved in liquid such sterile, de-ionized or distilled water or sterile isotonic phosphate-buffered saline (PBS). Preferably, 1000 pg (dry-weight) of such a composition comprises 0.1-990 pg, preferably l-900pg, more preferably 10- 200pg compound, and optionally 1-500 pg, preferably 1-100 pg, more preferably 5-15 pg (buffer) salts (preferably to yield an isotonic buffer in the final volume), and optionally 0.1-999.9 pg, preferably 100-999.9 pg, more preferably 200-999 pg other excipients. Preferably, 100 mg of such a dry composition is dissolved in sterile, de-ionized / distilled water or sterile isotonic phosphate-buffered saline (PBS) to yield a final volume of 0.1-100 ml, preferably 0.5-20 ml, more preferably 1-10 ml.According to a particular preference, the LNP of the present invention has a z-average diameter between 10 nm and 900 nm, preferably between 20 nm and 750 nm, more preferably between 30 nm and 500 nm, especially between 40 nm and 250 nm or even between 50 nm and 150 nm as determined by DLS, in particular according to ISO 22412:2017. The z-average diameter as defined in ISO 22412-2017 is determined by the cumulants method and yields a scattered light intensity-weighted harmonic mean particle diameter. For instance, Markova et al, 2022, discloses in detail how to measure the z-average diameter of LNPs.It is evident to the skilled person upon having read the above disclosure, that the inventive GDGT lipids can also be provided as pharmaceutically acceptable salts. Accordingly, the scope of the present invention shall also encompass all pharmaceutically acceptable salts of the inventive ionizable GDGT lipids. In particular, the ionizable GDGT lipids may be provided in cationic form (particularly as ammonium cations), e.g., as hydrochloride, sodium bisulfate, potassium bisulfate, monosodium phosphate, monopotassium phosphate, citrate or acetate salts.The present invention also relates to the following embodiments:Embodiment 1. A lipid nanoparticle (LNP) encapsulating a nucleic acid cargo, wherein the LNP comprises at least- an ionizable lipid fraction and- a stabilizer fraction; wherein the ionizable lipid fraction comprises at least one ionizable glycerol dialkyl glycerol tetraether (GDGT) lipid.Embodiment 2. The LNP of embodiment 1, wherein the nucleic acid cargo comprises a messenger ribonucleic acid (mRNA).Embodiment 3. The LNP of embodiment 1 or 2, wherein the stabilizer fraction comprises at least one polyethylene glycol (PEG) lipid.Embodiment 4. The LNP of any one of embodiments 1 to 3, wherein the LNP further comprises a sterol lipid fraction, preferably comprising cholesterol.Embodiment 5. The LNP of any one of embodiments 1 to 4, wherein the LNP further comprises a helper lipid fraction.Embodiment 6. The LNP of any one of embodiments 1 to 5, wherein the at least one ionizable GDGT lipid comprises at least one ionizable head group S; preferably wherein S is selected from the group consisting of:wherein each occurrence of Rais independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl , alkylamine, alkyl ether, alkyl thiol, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide, preferably from alkyl, cycloalkyl, alkenyl, hydroxyalkyl and alkylamine, each occurrence of Rbis independently selected from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate, alkyl sulfonamideand alkyl thiol, preferably from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine and alkyl thiol, and each occurrence of Rcis independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide, preferably from alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide.Embodiment 7. The LNP of any one of embodiments 1 to 6, wherein the LNP comprises at least one further ether lipid, preferably a tetraether lipid, preferably a GDGT, or a diether lipid, in particular an archaeol.Embodiment 8. The LNP of any one of embodiments 1 to 7, wherein the LNP comprises an ether lipid fraction comprising at least one further GDGT lipid (preferably at least two different GDGT lipids, more preferably at least three different GDGT lipids, especially at least four different GDGD lipids) and preferably at least one further ether lipid; in particular comprising archaeols and caldarchaeols, preferably with a composition as given in Table 1 above.Embodiment 9. The LNP of embodiment 8, wherein the ether lipid fraction comprises ether lipids obtainable by extraction from an archaeal culture, preferably a Sulfolobus culture, more preferably a Sulfolobus acldocaldarlus culture.Embodiment 10. The LNP of embodiment 9, wherein the entire ether lipid fraction is obtainable by extraction from said culture.Embodiment 11. The LNP of any one of embodiments 1 to 10, wherein the ionizable lipid fraction accounts for 40 mol% - 70 mol% of total lipids.Embodiment 12. The LNP of any one of embodiments 1 to 11, wherein the molar ratio of neutral ether lipid fraction to helper lipid fraction (in particular to DSPC, if present) is from 20:1 to 1:20, preferably from 15:1 to 1:10, more preferably from 12:1 to 1:3, even more preferably from 8:1 to 4:1, especially from 7:1 to 5:1.Embodiment 13. The LNP of any one of embodiments 1 to 12, wherein the molar ratio of neutral ether lipid fraction to sterol lipid fraction (in particular to cholesterol, if present) is from 0.25:1 to 1:30, preferably from 0.5:1 to 1:20, more preferably from 1:1 to 1:10, even more preferably from 1:2 to 1:6, especially from 1:3 to 1:5.Embodiment 14. The LNP of any one of embodiments 1 to 13, wherein the total lipids of the LNP comprise (preferably consist of):(a) 40 mol% - 70 mol% ionizable lipid fraction,(b) 5 mol% - 20 mol% helper lipid fraction,(c) 20 mol% - 40 mol% sterol lipid fraction,(d) 0.1 mol% - 4 mol% stabilizer fraction, and / or(e) 1 mol% - 20 mol% neutral ether lipid fraction.Embodiment 15. The LNP of any one of embodiments 1 to 14, wherein the ionizable lipid fraction comprises at least one further ionizable lipid selected from the group consisting of [(4- hydroxybutyl)azanediyl]di (hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl]amino }octanoate (SM-102),3- (didodecylamino)- N1,N1,4-tridodecyl-l-piperazineethanamine (KL10), Nl-[2- (didodecylamino)ethyl] N1,N4,N4-tridodecyl-l,4- piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24- tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N- dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4- dimethylaminomethyl- [1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4- (dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4- (2 dimethylaminoethyl)-[1,3 ]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2- ({8 [(3(B)- cholest-5-en-3-yloxy]octyl }oxy) N,N dimethyl-3-[(9Z,12Z)- octadeca-9,12-dien-l-yloxy]propan-l-amine (Octyl-CLinDMA), (2R)- 2- ({8-[(3p)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3- [ (9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l-amine (Octyl- CLinDMA (2R)), (2S) 2- ({8-[(3p)-cholest-5-en-3-yloxy]octyl}oxy)- N,N-dimethyl-3- [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l- amine (Octyl-CLinDMA (2S)) and mixtures thereof; and / or wherein the ionizable lipid is selected from the group consisting of (2OZ,23Z)—N,N-dimethylnonacosa-2 0,23-dien-l0-amine, (17Z,2OZ)— N,N-dimemylhexacosa-17,20-dien- 9-amine, (1Z,19Z)—N5N- dimethylpentacosa-16, 19-dien-8-amine, (13Z,16Z)—N,N- dimethyldocosa-13,16-dien-5-amine, (12Z,15Z)—N,N dimethylhenicosa-12,15-dien-4-amine, (14Z,17Z)—N,N- dimethyltricosa-14,17-dien-6-amine, (15Z,18Z)-N,N- dimethyltetracosa-15,18-dien-7-amine, (18Z,21Z)—N,N- dimethylheptacosa-18,21-dien-l 0-amine, (15Z,18Z)—N,N- dimethyltetracosa-15,18-dien-5-amine, (14Z,17Z)-N,N- dimethyltricosa-14,17-dien-4-amine, (19Z,22Z)—N,N- dimeihyloctacosa-19,22-dien-9-amine, (18Z,21 Z)-N,N- dimethylheptacosa-18,21-dien- 8-amine, (17Z,2OZ)—N,N- dimethylhexacosa-17,20-dien-7-amine, (16Z,19Z)—N,N- dimethylpentacosa-16,19-dien- 6-amine, (22Z,25Z)—N,N- dimethylhentriaconta-22,25-dien-10-amine, (21 Z,24Z)-N,N- dimethyltriaconta-21,24-dien- 9-amine, (18Z)—N,N-dimetylheptacos- 18-en-l0-amine, (17Z)—N,N-dimethylhexacos-17-en-9-amine, (19Z,22Z)—N,N-dimethyloctacosa-1 9,22-dien-7-amine, N,N- dimethylheptacosan-10-amine, (2OZ,23Z)—N-ethyl-N-methyInonacosa- 20,23-dien-l0-amine, 1-[(11Z,14Z)-1-nonylicosa-l1,14-dien-l- yl]pyrrolidine, (20Z)—N,N-dimethylheptacos-20-en-l0-amine, (15Z)—N,N-dimethyl eptacos-15-en-10-amine, (14Z)-N,N- dimethylnonacos-14-en-l0-amine, (17Z)—N,N-dimethylnonacos-17-en- 10-amine, (24Z)—N,N-dimethyltritriacont-24-en-l0-amine, (20Z)— N,N-dimethylnonacos-20-en-l0-amine, (22Z)—N,N- dimethylhentriacont-22-en-10-amine, (16Z)—N,N-dimethyIpentacos- 16-en-8-amine, (12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15- dien-l-amine, (13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-l- amine, N,N-dimethyl-1-[(IS,2R)-2-octylcyclopropyl] eptadecan-8- amine, 1-[(IS,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine, N,N-dimethyl-1-[(IS,2R)-2-octylcyclopropyl]nonadecan-10- amine, N,N-dimethyl-21-[(IS,2R)-2-octylcyclopropyl]henicosan-10- amine,N,N-dimethyl-1- [(IS,2S)-2-{[(1R,2R)-2- pentylcyciopropyl]methyl }cyclopropyl]nonadecan-10-amine,N,N- dimethyl-1- [(1 S,2R)-2-octylcyclopropyl]hexadecan-8-amine, N,N- dimethyl- [(1R,2S)-2 undecylcyclopropyl]tetradecan-5-amine, N,N- dimethyl-3-{7- [(IS,2R)-2-octylcyclopropyl]heptyl} dodecan-1- amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9- amine, 1-[(IS,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6- amine, N,N-dimethyl-1-[(IS,2R)-2-octylcyclopropyl]pentadecan-8- amine, R—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-3- (octyloxy)propan-2-amine, S—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9.12-dien-l-yloxy]-3- (octyloxy)propan-2-amine, 1—{2—[(9Z,12Z)- octadeca-9,12-dien-l-yloxy]-1-[ (octyloxy)methyl]ethylJpyrrolidine, (2 S)—N,N-dimethyl-1- [ (9Z,12Z)-octadeca-9,12-dien-l-yloxy]-3-[(5Z)-oct-5-en-l- yloxy]propan-2-amine, 1—{2—[(9Z,12Z)-octadeca-9,12-dien-l- yloxy]-1- [(octyloxy)methyl]ethyl}azetidine, (2S)-1- (hexyloxy)- N,N-dimethyl-3- [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-2- amine, (2S)-1- (heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9.12-dien-l-yloxy]propan-2-amine, N,N-dimethyl-1- (nonyloxy)-3- [ (9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-2-amine, N,N- dimethyl-1- [(9Z)-octadec-9-en-l-yloxy]-3- (octyloxy)propan-2- amine; (2S)—N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-l- yloxy]-3- (octyloxy)propan-2-amine, (2S)-1-[(11Z,14Z)-icosa-11,14-dien-l-yloxy]-N,N-dimethyl-3- (pentyloxy)propan-2-amine, (2S)-1- (hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-l-yloxy]-N,N- dimethylpropan-2-amine, 1-[(11Z,14Z)-icosa-11,14-dien-l-yloxy]- N,N-dimethyl-3- (octyloxy)propan-2-amine, 1-[(13Z,16Z)-docosa- 13,16-dien-l-yloxy]-N,N-dimethyl-3- (octyloxy)propan-2-amine, (2S)-1- [(13Z,16Z)-docosa-13,16-dien-l-yloxy]-3- (hexyloxy)-N,N- dimethylpropan-2-amine, (2S)-1-[(13Z)-docos-13-en-l-yloxy]-3- (hexyloxy)-N,N-dimethylpropan-2-amine, 1-[(13Z)-docos-13-en-l- yloxy]-N,N-dimethyl-3- (octyloxy)propan-2-amine, 1-[(9Z)-hexadec- 9-en-l-yloxy]-N,N-dimethyl-3- (octyloxy)propan-2-amine, (2R)—N,N- dimethyl-H (1-metoyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-l- yloxy]propan-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N- dimethyl-3- [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-2-amine, N,N-dimethyl-1- (octyloxy)-3-({8-[(IS,2S)-2-[(1R,2R)-2-pentylcyclopropyl]methyl }cyclopropyl]octyl}oxy)propan-2-amine, N,N-dimethyl-1-{ [8- (2-oclylcyclopropyl)octyl]oxy}-3- (octyloxy)propan-2-amine, and (1IE,2OZ,23Z)— N,N- dimethylnonacosa-11,20,2-trien-10-amine, and pharmaceutically acceptable salts and stereoisomers thereof, and mixtures thereof.Embodiment 16. The LNP of any one of embodiments 1 to 15, wherein the helper lipid fraction comprises a helper lipid selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), Dipalmitoylphosphatidylcholine (DOPC), phosphatidylcholine (PC),1.2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), 1,2-di-0-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2- di1ino1enoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn- glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1.2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2- diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2- dioleoyl-sn-glycero-3-phospho-rac- (1-glycerol) sodium salt (DOPG), sphingomyelin and mixtures thereof.Embodiment 17. The LNP of any one of embodiments 1 to 16, wherein the LNP has a z-average diameter between 10 nm and 900 nm, preferably between 20 nm and 750 nm, more preferably between 30 nm and 500 nm, especially between 40 nm and 250 nm or even between 50 nm and 150 nm (as determined by DLS, in particular according to ISO 22412:2017).Embodiment 18.A GDGT lipid suitable for use as an ionizable lipid in an LNP, wherein the GDGT lipid comprises at least one ionizable head group S, wherein S is selected from the group consisting of:wherein each occurrence of Rais independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl , alkylamine, alkyl ether, alkyl thiol, alkyl ester, alkyl amide, alkyl carbamate,alkyl sulfonate and alkyl sulfonamide, preferably from alkyl, cycloalkyl, alkenyl, hydroxyalkyl and alkylamine, each occurrence of Rbis independently selected from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate, alkyl sulfonamide and alkyl thiol, preferably from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine and alkyl thiol, and each occurrence of Rcis independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide, preferably from alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide.Embodiment 19. The GDGT lipid of embodiment 18, wherein S is selected from the group consisting of:wherein each occurrence of Rais independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl and alkylamine, and each occurrence of Rbis independently selected from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine and alkyl thiol.Embodiment 20. The GDGT lipid of any one of embodiments 18 or 19, wherein said alkyl is selected from methyl, ethyl, propyl, preferably iso-propyl, butyl, preferably iso-butyl, pentyl, preferably iso-pentyl, hexyl, preferably iso-hexyl and heptyl, preferably iso-heptyl.Embodiment 21. The GDGT lipid of any one of embodiments 18 to 20, wherein said cycloalkyl is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.Embodiment 22. The GDGT lipid of any one of embodiments 18 to 21, wherein said alkenyl is selected from prop-2-enyl, but-2-enyl, but-3-enyl, pent-2-enyl, pent-3-enyl, pent-4-enyl, hex-2-enyl, hex-3-enyl, hex-4-enyl, hex-5-enyl, hept-2-enyl, hept-3-enyl, hept-4-enyl, hept-5-enyl and hept-6-enyl.Embodiment 23. The GDGT lipid of any one of embodiments 18 to 22, wherein said hydroxyalkyl is selected from hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, hydroxypentyl and hydroxyhexyl.Embodiment 24. The GDGT lipid of any one of embodiments 18 to 23, wherein said alkylamine is selected from methylamine, ethylamine, propylamine, butylamine, pentylamine, hexylamine and heptylamine.Embodiment 25. The GDGT lipid of any one of embodiments 18 to 24, wherein said alkyl ether is selected from methyl ether, ethyl ether, propyl ether, butyl ether, pentyl ether, hexyl ether and heptyl ether.Embodiment 26. The GDGT lipid of any one of embodiments 18 to 25, wherein said alkyl thiol is selected from methyl thiol, ethyl thiol, propyl thiol, butyl thiol, pentyl thiol, hexyl thiol and heptyl thiol.Embodiment 27. The GDGT lipid of any one of embodiments 18 to 26, wherein said alkyl ester is selected from methyl ester, ethyl ester, propyl ester, butyl ester, pentyl ester, hexyl ester and heptyl ester.Embodiment 28. The GDGT lipid of any one of embodiments 18 to 27, wherein said alkyl amide is selected from methyl amide, ethyl amide, propyl amide, butyl amide, pentyl amide, hexyl amide and heptyl amide.Embodiment 29. The GDGT lipid of any one of embodiments 18 to 28, wherein said alkyl carbamate is selected from methyl carbamate, ethyl carbamate, propyl carbamate, butyl carbamate, pentyl carbamate, hexyl carbamate and heptyl carbamate.Embodiment 30. The GDGT lipid of any one of embodiments 18 to 29, wherein said alkyl sulfonate is selected from methyl sulfonate, ethyl sulfonate, propyl sulfonate, butyl sulfonate, pentyl sulfonate, hexyl sulfonate and heptyl sulfonate.Embodiment 31. The GDGT lipid of any one of embodiments 18 to 30, wherein said alkyl sulfonamide is selected from methyl sulfonamide, ethyl sulfonamide, propyl sulfonamide, butyl sulfonamide, pentyl sulfonamide, hexyl sulfonamide and heptyl sulfonamide.Embodiment 32. The GDGT lipid of any one of embodiments 18 to 31, wherein the head group S is selected from:wherein each occurrence of R is independently selected from H and alkyl; preferably wherein the alkyl is selected from methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, pentyl, iso-pentyl, hexyl, iso-hexyl, heptyl, and iso-heptyl.Embodiment 33. The GDGT lipid of any one of embodiments 18 to 32, wherein the GDGT lipid comprises two ionizable head groups S.Embodiment 34. The GDGT lipid of any one of embodiments 18 to 33, wherein the GDGT lipid is selected from:GDGT-X1GDGT-lllbGDGT-llla wherein S1is S and S2is OH, or S1is OH and S2is S, or each ofS1and S2independently is S.Embodiment 35. The GDGT lipid of embodiment 34, wherein each of S1and S2is the same S.Embodiment 36. The GDGT lipid of any one of the previous embodiments, wherein the GDGT lipid carries an overall positive charge at endosomal pH, in particular at a pH between 5.0 and 6.5, such as pH 5.0, 5.5, 6 or 6.5; preferably wherein the GDGT lipid carries an overall neutral charge at a pH higher than endosomal pH, such as pH 7.0 or pH 7.4.Embodiment 37. The GDGT lipid of any one of embodiments 18 to 36, wherein the ionizable head group S carries an overall positive charge at endosomal pH, in particular at a pH between 5.0 and 6.5, such as pH 5.0, 5.5, 6 or 6.5; preferably wherein the ionizable head group S carries an overall neutral charge at a pH higher than endosomal pH, such as pH 7.0 or pH 7.4.Embodiment 38. The GDGT lipid of any one of embodiments 18 to 37, wherein the GDGT lipid is a caldarchaeol with the at least one ionizable head group S.Embodiment 39. The GDGT lipid of any one of embodiments 18 to 38, wherein the GDGT lipid is selected from any one of the GDGT lipids of Figs. 1-10 and stereoisomers thereof, and variants thereof with a GDGT-0, GDGT-1, GDGT-2, GDGT-3, GDGT-5, GDGT-6, GDGT-7 or GDGT-8 scaffold instead of a GDGT-4 scaffold.Embodiment 40. An ether lipid fraction comprising the GDGT lipid of any one of embodiments 18 to 39; preferably obtainable by extraction from an archaeal culture, preferably a Sulfolobus culture, more preferably a Sulfolobus acldocaldarlus culture, followed by substitution with the ionizable head group S.Embodiment 41.An LNP comprising the GDGT lipid of any one of embodiments 18 to 39 or ether lipid fraction of embodiment 40, preferably wherein the LNP is as defined in embodiments 1 to 17.Embodiment 42.A pharmaceutical composition comprising the LNP of any one of embodiments 1 to 17 and 41 and preferably at least one excipient.Embodiment 43. The pharmaceutical composition of embodiment 42, wherein the pharmaceutical composition is a vaccine, preferably an mRNA vaccine.Embodiment 44. The pharmaceutical composition of embodiment 42 or 43, for use in prevention or treatment of a disease or condition in a patient.Embodiment 45.A method for producing the GDGT lipid of any one of embodiments 18 to 39, comprising the steps of:- obtaining a lipid fraction comprising one or more precursor GDGT lipids from an archaeal culture, preferably a Sulfolobus culture, more preferably a Sulfolobus acldocaldarlus culture;- purifying the one or more precursor GDGT lipids; and- contacting the one or more precursor GDGT lipids with one or more reagents to produce the GDGT lipid with the at least one ionizable head group S.The present invention is further illustrated by the following figures and examples, without being restricted thereto .Figures 1-10 show particularly preferred ionizable GDGT lipids. The parentheses indicate that the length of the segment in parentheses may be from 0 to 6 carbon atoms (n=0-6). Each instance of "R2" or "R2" may independently be selected from the definitions given directly below the structure. Curly bonds indicate that both R and S enantiomers are preferred.ExamplesExample 1 - Purification of precursor GDGT lipidExtraction of biomass: 240 g of dried biomass of Sulfolobus acldocaldarlus were mixed with 360 g diatomaceous earth (Dionex ASE Prep DE) and the resulting 600g of solids were homogenously dispersed via shaking. 18 extraction cells (lOOmL Dionex ASE 350 stainless steel), equipped with a cellulose filter, were loaded with roughly equal amounts of dispersed solids (30-35g each) and placed in a Dionex ASE 350 Accelerated Solvent Extractor. The cells were extracted with 3 L of CHClaiMeOH mixture (2:1) at 80°C and 1150 psi for 30 minutes each. The extracted liquid fractions were combined and the mixture was concentrated (Heidolph Hei-Vap Core, 50°C, lOOmbar) to 200 mL. 50 mL of aqueous 2N HC1 were added and the heterogenous, biphasic mixture was heated to 65°C for 24h. After cooling to room temperature, the mixture was neutralized with 50 mL NaHCOa, the phases were separated and the aqueous phase was extracted with Et2O (3x 90mL), before the combined organic extracts were dried with Na2SO2, filtered and the solvents evaporated, yielding 4.4 g (1.8% w / w) of crude extract as a brown solid.Isolation of GDGT and Sulfolobus main polar lipid: The brown solid was dissolved in 40 mL dichloromethane (DCM) and subjected in two portions to column chromatography (Btichi Pure Chromatography System equipped with a light scattering detectorand an automated fraction collector). Column chromatography was conducted by liquid injection of 20 mL crude material onto a FlashPure EcoFlex 25g SiO2column (32mL / min flowrate), prior equilibrated with light petrol (LP). The yield was 193.4 mg (4.3%) of GDGT containing minor aromatic lipid impurities as a brown oil. Alongside GDGTs, 2.38g (54%) Sulfolobus main lipid was isolated within the same chromatography.End purification of GDGT: 106.5 mg of said GDGTs were dissolved in 1 mL LP and separated on a Btichi FlashPure Ecoflex column 20g with LP:DCM (DCM=20% to 40%). All fractions displaying no UV-signal were collected and evaporated to yield 92.8 mg pure GDGTs (87%) as colorless oil.These GDGT lipids were used in the following examples as precursor GDGT lipids to produce the GDGT lipids with ionizable head groups.Example 2 - Preparation of bis- (4-bromobutanoic acid)-GDGT esterDimethylaminopyridine (DMAP) (5.4mg, 44 pmol, 0.4 equiv.) was added to GDGT as obtained according to Example 1 (142.7mg, 110 pmol, 1 equiv.) in an 8 mL vial, equipped with a septum and a stirring bar. The vial was evacuated and backfilled with N2(three times) before 2.2 mL dry dichloromethane were added via canula. Subsequently the solution was cooled to 0°C by means of an ice / water bath before 4-bromobutyric acid chloride (45 pL, 240 pmol, 2.4 equiv.) were added dropwise. The mixture was stirred for 5 minutes at 0°C and then allowed to warm to room temperature and stirred for 18h. After complete consumption of the starting material (checked via thin-layer chromatography), the mixture was quenched with 20 mL sat. NaHCOa and extracted with Et2O (3x 50 mL), once washed with brine, dried over Na2SC>4 and evaporated to dryness. After column chromatography (12g SiO2), 126.5 mg (72%) of bis- (4-bromobutanoic acid)-GDGT ester were isolated as white solid.The product was a mixture of several different GDGT scaffolds. A representative structure (with a GDGT-4 scaffold) is shown in the following:Example 3 - Preparation of bis- (4- ((2- hydroxyethyl )(methyl)amino)butanoic acid)-GDGT esterTo bis- (4-bromobutanoic acid)-GDGT ester as obtained according to Example 2 (126.5 mg, 79 pmol, 1 equiv.) were added K2CO3 (32.9mg, 238 pmol, 3 equiv.) and KI (5.3mg, 31 pmol, 0.4 equiv.) in an 8 mL vial, equipped with a septum and a stirring bar. The vial was evacuated and backfilled with N2(three times) before 2 mL HPLC-grade MeCN, followed by N-methylaminoethanol (20 pL, 238 pmol, 3 equiv.) were added via canula. The turbid mixture was heated to 75°C for 16 hours (full consumption of the starting material, checked via thin-layer chromatography), cooled to room temperature and the MeCN was evaporated. The solids were taken up in 20 mL NaHCO3 and extracted with 20mL dichloromethane (three times), once washed with brine, filtered and evaporated yielding 91.8mg of crude yellow oil. The crude material was purified via flash chromatography (Biotage column, 5g) to obtain 65.8mg (52%) of bis- (4- ((2- hydroxyethyl )(methyl)amino)butanoic acid)-GDGT ester as a colorless oil.The product was a mixture of several different substituted GDGT scaffolds. A representative structure (with a GDGT-4 scaffold) is shown in the following:Example 4 - Preparation of bis- (4- (diethanolamine)butanoic acid)-GDGT esterTo bis- (4-bromobutanoic acid)-GDGT as obtained according to Example 2 (1 equiv.) K2CO3 (3 equiv.) and KI (0.4 equiv.) are added in an 8 mL vial, equipped with a septum and a stirring bar. The vial is evacuated and backfilled with N2(three times) before 2 mL HPLC-grade MeCN, followed by addition of diethanolamine (3 equiv.) via canula. The turbid mixture is heated to 75°C for 16 hours and subsequently cooled to room temperature before the MeCN is evaporated. The solids are taken up in 20 mL NaHCO3 and extracted with 20 mL dichloromethane (three times), once washed with brine, filtered and evaporated, y. The crude material is purified via flash chromatography to isolate bis- (4- (bis- (2-hydroxyethyl)amino)butanoic acid)-GDGT ester.The product typically is a mixture of several different substituted GDGT scaffolds. A representative structure (with a GDGT-4 scaffold) is shown in the following:Example 5 - Preparation of bis- (S)- (4-2- (hydroxymethyl)pyrrolidin-l-yl)butanoic acid)-GDGT esterTo bis- (4-bromobutanoic acid)-GDGT ester as obtained according to Example 2 (1 equiv.) K2CO3 (3 equiv.) and KI (0.4 equiv.) are added in an 8 mL vial, equipped with a septum and a stirring bar. The vial is evacuated and backfilled with N2(three times) before 2 mL HPLC-grade MeCN, followed by addition of (S)- prolinol via canula. The turbid mixture is heated to 75°C for 16 hours and subsequently cooled to room temperature before the MeCN is evaporated. The solids are taken up in 20 mL NaHCOs and extracted with 20 mL dichloromethane (three times), once washedwith brine, filtered and evaporated. The crude material is purified via flash chromatography to isolate bis- (4-(prolinol)butanoic acid)-GDGT ester.The product typically is a mixture of several different substituted GDGT scaffolds. A representative structure (with a GDGT-4 scaffold) is shown in the following:Example 6 - Preparation of bis- (4- (2- (hydroxymethyl)-1H- imidazolyl)-GDGT esterTo bis- (4-bromobutanoic acid)-GDGT ester as obtained according to Example 2 (1 equiv.) K2CO3 (3 equiv.) and KI (0.4 equiv.) are added in an 8 mL vial, equipped with a septum and a stirring bar. The vial is evacuated and backfilled with N2(three times) before 2 mL HPLC-grade MeCN, followed by addition of 2- (hydroxymethyl)-IH-imidazole via canula. The turbid mixture is heated to 75°C for 16 hours and subsequently cooled to room temperature before the MeCN is evaporated. The solids are taken up in 20 mL NaHCO3 and extracted with 20 mL dichloromethane (three times), once washed with brine, filtered and evaporated. The crude material is purified via flash chromatography to bis- (4- (2- (hydroxymethyl)-IH-imidazolyl)-GDGT ester.The product typically is a mixture of several different substituted GDGT scaffolds. A representative structure (with a GDGT-4 scaffold) is shown in the following:Example 7 - Preparation of GDGT aldehydeTo an 8 mL vial containing GDGT as obtained according to Example 1 (1 equiv.) and Trichlorocyanuric acid (TCCA) (2.2 equiv) dichloromethane is added and cooled to 0°C. 2,2,6,6- Tetramethylpiperidinyloxyl (TEMPO) (0.2 equiv.) are added and the mixture is stirred at 0°C for 15 minutes. The mixture is quenched with saturated aqueous NaHCOs solution and extracted with Et2O (3x 50 mL). The combined organic extracts are once washed with 2N HC1 and brine before dried with Na2SO4, filtered and evaporated. This yields crude GDGT aldehyde, which is used without further purification.The product typically is a mixture of several different GDGT scaffolds. A representative structure (with a GDGT-4 scaffold) is shown in the following:Example 8 - Preparation of bis- ((methyl)amino)ethanol GDGTIn an 8 mL vial under N2atmosphere GDGT aldehyde as obtained according to Example 7 (1 equiv.) is dissolved in tetrahydrofuran and N-methylaminoethanol is added (2.2 eqiuv.) and stirred for 10 minutes. Sodium triacetoxyborohydride (3 equiv.) is added and the mixture is stirred at room temperature for 24h. The reaction mixture is quenched via addition of 3N NaOH and the product is extracted with Et2O and dried with MgSCg. Treatment of the cooled ethereal extract with ethereal HC1 yields bis- ((methyl)amino)ethanol GDGT hydrochloride salt after filtration.The product typically is a mixture of several different substituted GDGT scaffolds. A representative structure (with a GDGT-4 scaffold) is shown in the following:Example 9 - Synthesis of ionizable GDGTs from GDGT acrylatePreparation of bis-GDGT-acrylateGDGT as preferably obtained according to Example 1 (1 equiv.) is placed in a 20mL vial, equipped with a septum and a stirring bar. The vial is evacuated and backfilled with N2(three times) before dry CHCla and triethylamine (2 equiv.) are added via canula. Subsequently the solution is cooled to 0°C by means of an ice / water bath before acryloyl chloride in CHC13 (0.1M, 2.2 equiv.) are added dropwise. The mixture is stirred for 5 minutes at 0°C and then allowed to warm to room temperature and stirred for 5h. After complete consumption of the starting material (checked via TLC), the mixture is quenched with 20 mL sat. NaHCO3 and extracted with CHCI3, once washed with brine, dried over MgSCg and evaporated to dryness. After column chromatography (12gSiCy) 83% of the Bis-acrylic-GDGT ester is isolated as white solid .Further stepsBis-acrylic-GDGT ester (1 equiv.) is added to an 8 mL vial, equipped with a septum and a stirring bar. The vial is evacuated and backfilled with N2(three times) before dry CHCI3 was added, followed by amine (2.2 equiv.) and a catalytic amount of acetic acid (0.1 eq.) is added via canula or in one portion if the amine is a solid. The homogenous mixture is heated to 40°C for 3 hours (full consumption of the starting material, checked via TLC) , cooled to room temperature and quenched with saturated aqueous NaHCO3 and extracted with CHCI3 (three times), once washed with brine, dried with MgSCg, filtered and evaporated. The crude material is purified via flash chromatography (Biotage Sfar Amino column, LP:EE) to obtain bis- (aminopropanoic acid)- GDGT ester as a colorless oil.Crude materials obtained depend on the amine used. The products typically are mixtures of several different substituted GDGT scaffolds. Representative structures (with a GDGT-4 scaffold) are shown in the following.bis- (3-dimethylamino)propanoic acid)-GDGT esterThe crude material is purified via flash chromatography (BiotageSfar Amino column,LP:EE) to obtain 67% of bis- (4-(dimethylamino)butanoic acid)-GDGT ester as a colorless oil. bis- (3-diethylamino)propanoic acid)-GDGT esterThe crude material is purified via flash chromatography (BiotageSfar Amino column,LP:EE) to obtain 63% of bis- (3-(diethylamino)propanoic acid)-GDGT ester as a colorless oil. bis- (3- (2-hydroxyethyl)(methyl)amino)) propanoic acid)-GDGT esterThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 54% of bis- (3- (2- hydroxyethyl) (methyl)amino) propanoic acid)-GDGT ester as a colorless oil. bis 3- (bis(2-hydroxyethyl)amino)propanoic acid)-GDGT esterThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 57% 3- (bis(2- hydroxyethyl)amino)propanoic acid)-GDGT ester as a colorless oil. bis- (3-pyrrolidin-l-yl) propanoic acid)-GDGT esterThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 51% of bis- (3-pyrrolidin-l- yl) propanoic acid)-GDGT ester as a colorless oil. bis-3- ((R)- (hydroxymethyl)pyrrolidin-1-yl)propanoic acid)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 67% of bis- (3- (2 (R)- (hydroxymethyl)pyrrolidin-l-yl ) propanoic acid)-GDGT ester as a colorless oil. bis-3- ((S)- (hydroxypyrrolidin-l-yl)propanoic acid)-GDGT esterThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 62% of bis-3-((S)- (hydroxypyrrolidin-l-yl )propanoic acid)-GDGT ester as a colorless oil. bis- (3-piperidin-l-yl)) propanoic acid)-GDGT esterThe crude material is purified via flash chromatography (Biotage Sfar Amino column, LP:EE) to obtain 64% of bis- (3-piperidin-l- yl)) propanoic acid)-GDGT ester as a colorless oil. bis- (3- (4-hydroxypiperidin-l-yl)) propanoic acid)-GDGT esterThe crude material is purified via flash chromatography (Biotage Sfar Amino column, LP:EE) to obtain 61% of bis- (3- (4- hydroxypiperidin-l-yl)) propanoic acid)-GDGT ester as a colorless oil. bis- (3-morpholino)-propanoic acid)-GDGT esterThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 54% of bis- (3-morpholino) propanoic acid)-GDGT ester as a colorless oil. bis- (3- (3- (hydroxymethyl)morpholino)-propanoic acid)-GDGT esterThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 47% of a diastereomeric mixture of bis- (3- (3- (hydroxymethyl)morpholino) propanoic acid)- GDGT ester as a colorless oil. bis- (3- (2- (hydroxymethyl)-IH-imidazolyl) propanoic acid)-GDGT esterThe crude material is purified via flash chromatography (Biotage Sfar Amino column, LP:EE) to obtain 75% of bis- (3- (2- (hydroxymethyl)-IH-imidazolyl) propanoic acid)-GDGT ester as a colorless oil.bis-3- (piperazin-l-yl)-propanoic acid) GDGT esterTo bis-acrylic-GDGT ester is added 1-Boc-piperazine in an 8 mL vial, equipped with a septum and a stirring bar. The vial is evacuated and backfilled with N2(three times) before dry CHCI3 was added, followed by a catalytic amount of acetic acid (0.1 equiv.). The mixture is heated to 40°C for 4 hours (full consumption of the starting material, checked via TLC), cooled to room temperature and saturated aqueous NaHCOs solution was added. The mixture is extracted with CHCI3 (three times), once washed with brine, dried with MgSCg filtered and evaporated. The crude material is dissolved in 2 ml dry DCM and 1 ml TFA was added dropwise. The mixture is stirred at room temperature for 2 hours, cooled to 0°C and neutralized with sat. aqueous NaHCOs solution followed by extraction with DCM (3x), drying with MgSO4 and the solvents were evaporated. Purification via flash chromatography (Biotage Sfar Amino column,LP:EE) yields 42 % ofbis-3- (piperazin-l-yl)-propanoic acid)-GDGT ester as a slightly yellow oil. bis-3- (4- (2-hydroxyethyl)piperazin-l-yl)propanoic acid-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column, LP:EE) to obtain 59% of bis-3- (4- (2- hydroxyethyl)piperazin-l-yl )- propanoic aid)-GDGT ester as a yellow oil.Example 10 - Synthesis of ionizable GDGTs from bis- (4- bromobutanoic acid)-GDGT esterGeneral procedure for the preparation of bis- (4-bromobutanoic acid)-GDGT esterDMAP (0.4 equiv.) is added to GDGT as preferably obtained according to Example 1 (1 equiv.) in an 8 mL vial, equipped with a septum and a stirring bar. The vial is evacuated and backfilled with N2(three times) before dry DCM was added via canula. Subsequently the solution is cooled to 0°C by means of an ice / water bath before 4-bromobutyric acid chloride (2.4 equiv.) was added dropwise. The mixture is stirred for 5 minutes at 0°C and then allowed to warm to room temperature and stirred for 18h. After complete consumption of the starting material (checked via TLC), the mixture is quenched with sat. NaHCOa and extracted with Et2O (3x), once washed with brine, dried over Na2SC>4 and evaporated to dryness. After column chromatography (SiO2, PE:EE) 72% of bis- (4-bromobutanoic acid)-GDGT ester is isolated as white solid.Further stepsTo bis- (4-bromobutanoic acid)-GDGT ester (1 equiv.) are added Theos (3 equiv.) and KI (0.4 equiv.) in an 8 mL vial, equipped with a septum and a stirring bar. The vial is evacuated and backfilled with N2(three times) before HPLC-grade MeCN, followed by the appropriate secondary amine (3 equiv.) are added via canula or in one portion as a solid. The turbid mixture is heated to 75°C for 16 to 24 hours (full consumption of the starting material, checked via TLC), cooled to room temperature and the MeCN was evaporated. The solids are taken up in NaHCOa and extracted with DCM (three times), once washed with brine, filtered and evaporated. The crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain bis- (4-aminobutanoic acid)-GDGT ester as a colorless oil.Crude materials obtained depend on the amine used. The products typically are mixtures of several different substituted GDGT scaffolds. Representative structures (with a GDGT-4 scaffold) are shown in the following. bis- (dimethylamino)butanoic acid)-GDGT esterThe crude material is purified via flash chromatography (BiotageSfar Amino column,LP:EE) to obtain 48% of bis- (4-(dimethylamino)butanoic acid)-GDGT ester as a colorless oil. bis- (diethylamino)butanoic acid)-GDGT esterThe crude material is purified via flash chromatography (BiotageSfar Amino column,LP:EE) to obtain 63% of bis- (4- (diethylamino)butanoic acid)-GDGT ester as a colorless oil. bis- (4- (2-hydroxyethyl)(methyl)amino)butanoic acid)GDGT esterThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 68% of bis- (4- (2- hydroxyethyl) (methyl)amino)butanoic acid)GDGT ester as a colorless oil. bis 4- (bis(2-hydroxyethyl)amino)butanoic acid)-GDGT esterThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 61% 4- (bis(2- hydroxyethyl)amino)butanoic acid)-GDGT ester as a colorless oil. bis- (4-pyrrolidin-l-yl) butanoic acid)-GDGT esterThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 53% of bis-pyrrolidin-l-yl) butanoic acid)-GDGT ester as a colorless oil.4- (2- (hydroxymethyl)pyrrolidin-l-yl)butanoic acid)-GDGT esterThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 49% of bis- (4- (2 (S)- (hydroxymethyl)pyrrolidin-l-yl ) butanoic acid)-GDGT ester as a colorless oil.The crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 71% of bis- (4-piperidin-l- yl)) butanoic acid)-GDGT ester as a colorless oil. bis- (4- (4-hydroxypiperidin-l-yl)) butanoic acid)-GDGT esterThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 71% of bis- (4- (4- hydroxypiperidin-l-yl)) butanoic acid)-GDGT ester as a colorless oil. bis- (4-morpholino) butanoic acid)-GDGT esterThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 60% of bis- (4-morpholino) butanoic acid)-GDGT ester as a colorless oil. bis- (4- (3- (hydroxymethyl)morpholino) butanoic acid)-GDGT esterThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 41% of bis- (4- (3- (hydroxymethyl)morpholino ) butanoic acid)-GDGT ester as a colorless oil. bis- (4- (2- (hydroxymethyl)-IH-imidazolyl) butanoic acid)-GDGT esterThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 73% of bis- (4- (2- (hydroxymethyl)-IH-imidazolyl) butanoic acid)-GDGT ester as a colorless oil. bis- (4- (piperazin-l-yl) butanoic acid)-GDGT esterTo bis- (4-bromobutanoic acid)-GDGT ester (1 equiv.), K2CO3 (3 equiv.), KI (0.4 equiv.) and 1-Boc-piperazine are added in an 8 mL vial, equipped with a septum and a stirring bar. The vial is evacuated and backfilled with N2(three times) before HPLC-grade MeCN. The turbid mixture is heated to 75°C for 18 hours (full consumption of the starting material, checked via TLC), cooled to room temperature and the MeCN was evaporated. The solids are taken up in NaHCOs and extracted with DCM (three times), once washed with brine, filtered and evaporated. The crude materialis dissolved in 2 ml dry DCM and 1 ml TFA was added dropwise. The mixture is stirred at room temperature for 2 hours, cooled to 0°C and neutralized with sat. aqueous NaHCOs solution. The mixture is 3 times extracted with DCM, dried with MgSCg and the solvents evaporated. Purification via flash chromatography (Biotage Sfar Amino column,LP:EE) yields 45 % of bis- (4- (piperazin-l-yl) butanoic acid)-GDGT ester as a slightly yellow oil. bis- (4- (4- (2-hydroxyethyl)piperazin-l-yl) butanoic acid)-GDGT esterThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 54% of bis-4-(4-(2- hydroxyethyl)piperazin-l-yl )-GDGT ester as a yellow oil.Example 11 - Synthesis of ionizable GDGTs from GDGT acidPreparation of bis-GDGT-acidChemicalFormula:Ca6Hig40gMolecularWeight:1294,25MolecularWeight:1322,22GDGT as preferably obtained according to Example 1 (1 equiv.), 2-iodobenzoic acid (0.4 eq.) and Oxone (2.8 eq.) are dissolved in MeCN / H2O=2:1 in an 8 mL vial, equipped with a septum and a magnetic stirring bar. The mixture is heated to 70°C for 6 hours and then cooled to room temperature. The hypervalent iodine precipitant is removed via filtration and washed with water and DCM. The solvent mixture is extracted with DCM (3x) and dried over MgSO4. Evaporation of the solvent yields 97% of crude bis- GDGT-acid which is used without further purification.General procedure for the preparation of bis- (4-aminobutyl)-GDGT estersTo bis-GDGT acid (1 equiv.) are added EDCI (2.2 equiv.) and DMAP (0.4 equiv.) in an 8 mL vial, equipped with a septum and a stirring bar. The mixture is dissolved in DCM before 4- bromobutanol (2.8 equiv.) is added and stirred at room temperature for 18 hours. After quenching with saturated aqueous NaHCOs solution the mixture is extracted with DCM (3x), washed with brine, dried over MgSCg and evaporated. The crude material is purified via flash chromatography (SiCy, LP:EE) to obtain bis- (4-bromobutane)-GDGT-ester as a colorless oil.To bis- (4-bromobutyl)-GDGT ester (1 equiv.) were added K2CO3 (3 equiv.) and KI (0.4 equiv.) in an 8 mL vial, equipped with a septum and a stirring bar. The vial was evacuated and backfilled with N2(three times) before HPLC-grade MeCN, followed by the appropriate secondary amine (3 equiv.) were added via canula or in one portion as a solid. The turbid mixture was heated to 75°C for 16 to 24 hours (full consumption of the starting material, checked via TLC), cooled to room temperature and the MeCN was evaporated. The solids were taken up in NaHCOs and extracted with DCM (three times), once washed with brine, filtered and evaporated. The crude material is purified via flash chromatography (Biotage Sfar Amino column, LP:EE) to obtain bis- (4-aminobutyl)-GDGT ester as a colorless oil.Crude materials obtained depend on the amine used. The products typically are mixtures of several different substituted GDGT scaffolds. Representative structures (with a GDGT-4 scaffold) are shown in the following.bis- (dimethylamino)butyl)-GDGT esterChemicalFormula:C98H186N2O8MolecularWeight:1520,57The crude material is purified via flash chromatography (BiotageSfar Amino column,LP:EE) to obtain 65% of bis- (4- (dimethylamino)butyl)-GDGT ester as a colorless oil. bis- (diethylamino)butyl)-GDGT esterThe crude material is purified via flash chromatography (BiotageSfar Amino column,LP:EE) to obtain 70% of bis- (4-(diethylamino)butyl)-GDGT ester as a colorless oil. bis- (4- (2-hydroxyethyl)(methyl)aminobutyl)-GDGT esterThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 68% of bis- (4- (2- hydroxyethyl )(methyl)aminobutyl)-GDGT ester as a colorless oil.bis 4- (bis- (2-hydroxyethyl)aminobutyl)-GDGT esterThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 58% bis-4- (bis- (2- hydroxyethyl )aminobutyl)-GDGT ester as a colorless oil. bis- (4-pyrrolidin-1-yl)butyl)-GDGT esterThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 56% of bis-pyrrolidin-l-yl) butyl)-GDGT ester as a colorless oil.4- (2 (S)- (hydroxymethyl)pyrrolidin-l-yl)butyl)-GDGT esterThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 61% of bis- (4- (2 (S')- (hydroxymethyl)pyrrolidin-l-yl) butyl)-GDGT ester as a colorless oil .bis- (4-piperidin-l-yl)) butyl)-GDGT esterThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 65% of bis- (4-piperidin-l- yl)) butyl)-GDGT ester as a colorless oil. bis- (4- (4-hydroxypiperidin-l-yl)) butyl)-GDGT esterThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 64% of bis- (4- (4- hydroxypiperidin-l-yl)) butyl)-GDGT ester as a colorless oil. bis- (4-morpholino) butyl)-GDGT esterThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 66% of bis- (4-morpholino) butyl)-GDGT ester as a colorless oil.bis- (4- (3- (hydroxymethyl)morpholino) butyl)-GDGT esterThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 48% as a mixture of diastereomers of bis- (4- (3- (hydroxymethyl)morpholino) butyl)- GDGT ester as a colorless oil. bis- (4- (2- (hydroxymethyl)-IH-imidazolyl) butyl)-GDGT esterThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 63% of bis- (4- (2- (hydroxymethyl)-IH-imidazolyl ) butyl)-GDGT ester as a colorless oil.bis-4- (piperazin-l-yl) butyl-GDGT esterTo bis- (4-bromobutyl)-GDGT ester (1 equiv.), K2CO3 (3 equiv.), KI (0.4 equiv.) and 1-Boc-piperazine is added in an 8 mL vial, equipped with a septum and a stirring bar. The vial is evacuated and backfilled with N2(three times) before HPLC-grade MeCN. The turbid mixture is heated to 75°C for 18 hours (full consumption of the starting material, checked via TLC), cooled to room temperature and the MeCN was evaporated. The solids are taken up in NaHCOs and extracted with DCM (three times), once washed with brine, filtered and evaporated. The crude material is dissolved in 2 ml dry DCM and 1 ml TFA was added dropwise. The mixture is stirred at room temperature for 2 hours, cooled to 0°C and neutralized with sat. aqueous NaHCOs solution. The mixture is 3 times extracted with DCM, dried with MgSO4 and the solvents evaporated. Purification via flash chromatography (Biotage Sfar Amino column,LP:EE) yields 48 % of bis-4- (piperazin-l-yl)butyl- GDGT ester as a slightly yellow oil.bis-4- (4- (2-hydroxyethyl)piperazin-l-yl)butyl-GDGT esterThe crude material is purified via flash chromatography (BiotageSfar Amino column,LP:EE) to obtain 65% of bis-4-(4-(2- hydroxyethyl)piperazin-l-yl )butyl-GDGT ester as a yellow oil.Example 12 - Synthesis of further ionizable GDGTs from GDGT aldehydePreparation of bis-GDGT-aldehydeChemicalFormula:Ca6Hig40g MolecularWeight:1294,25MolecularWeight:1290,22GDGT as preferably obtained according to Example 1 (1 equiv.) and TCCA (2.4 equiv.) are dissolved in dry DCM in an 8 mL vial equipped with a septum and a magnetic stirring bar. The mixture is cooled to 0°C and TEMPO (0.2 eq.) is added and stirred for 10minutes. Then the yellow solution is poured into LP, filtered via celite, washed with LP and the solvents are evaporated to yield 99% of crude bis-GDGT-aldehyde which is used without further purification.General procedure for the preparation of bis-GDGT aminesBis-GDGT-aldehyde (1 equiv.) is dissolved in dry DCM and the appropriate amine (4 equiv.) is added and the mixture is stirred for 10 minutes at room temperature. Then sodium triacetoxyhydroborate (6 equiv.) is added in one portion and the heterogenous mixture was stirred for 2 hours, before being carefully quenched with 2 N NaOH. After extraction with ether (3x), washing with brine, drying over MgSCy and filtration the solvents were evaporated. Column Chromatography (Biotage Sfar Amino, LP:EE) yields GDGT amines as colorless to yellow oils.Crude materials obtained depend on the amine used. The products typically are mixtures of several different substituted GDGTscaffolds. Representative structures (with a GDGT-4 scaffold) are shown in the following. bis- (dimethylamino)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 51% of bis- (dimethylamino)- GDGT as a colorless oil. bis- (diethylamino)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 62% of bis- (diethylamino)- GDGT as a colorless oil. bis- (bis- (hydroxyethyl)amino)-GDGTThe crude material is purified via flash chromatography (BiotageSfar Amino column,LP:EE) to obtain 65% of bis- (bis (hydroxyethyl)amino)-GDGT as a colorless oil.bis- (methyl(hydroxyethyl)amino)-GDGTThe crude material is purified via flash chromatography (BiotageSfar Amino column,LP:EE) to obtain 64% of bis- (methyl (hydroxyethyl)amino)-GDGT as a colorless oil. bis- (methyl(hydroxypropyl)amino)-GDGTThe crude material is purified via flash chromatography (BiotageSfar Amino column,LP:EE) to obtain 48% of bis- (methyl (hydroxyethyl)amino)-GDGT as a colorless oil. bis- (methyl(hydroxybutyl)amino)-GDGTThe crude material is purified via flash chromatography (BiotageSfar Amino column,LP:EE) to obtain 59% of bis- (methyl (hydroxybutyl)amino)-GDGT as a colorless oil.bis- ((S)-oxetane-2-methane-N-methyl-amine)-GDGTThe crude material is purified via flash chromatography (BiotageSfar Amino column,LP:EE) to obtain 43% of bis- ((S)-oxetane-2methane-N-methyl-amine )-GDGT as a colorless oil. bis- (azetidine-3-methanoyl)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 48% of bis bis- (azetidine-3- methanoyl )-GDGT as a colorless oil. bis- (pyrrolidine)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 57% of bis- (pyrrolidine)-GDGT as a colorless oil.bis- (L-prolinol)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 55% of bis- (L-prolinol)- GDGTas a colorless oil. bis- (D-prolinol)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 55% of bis- (D-prolinol)-GDGT as a colorless oil. bis- (S-pyrrolidine-3-ol)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 60% of bis- (S-pyrrolidine-3- ol)-GDGT as a colorless oil.bis- (.R-pyrrolidine-3-ol)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 62% of bis- (A-pyrrolidine-3- ol)-GDGT as a colorless oil.Bis- ((3S,4S)-pyrrolidine-3,4-diol)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 49% of bis- ((3S,4S)- pyrrolidine-3,4-diol)-GDGT as a colorless oil.Bis- (2- (hydroxymethyl)imidazol)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 60% of bis- (2- (hydroxymethyl)imidazol )-GDGT as a colorless oil.Bis- ((1R,5S,6r)-3-azabicyclo[3.1.0]hexan-6-yl)methanol)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 61% of bis- ((1R,5S,6r)-3- azabicyclo [3.1.0]hexan-6-yl)methanol)-GDGT as a colorless oil.Bis- (piperidine)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 70% of bis- (piperidine)-GDGT as a colorless oil.Bis- (4-hydroxypiperidine)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 70% of bis- (4- hydroxypiperidine)-GDGT as a colorless oil.Bis- (2-hydroxymethylpiperidine)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 49% as a diastereomeric mixture of bis- (2-hydroxymethylpiperidine)-GDGT as a colorless oil.Bis- ((A)-piperidine-3-ol)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 58% of bis- ((A)-piperidine-3- ol)-GDGT as a colorless oil.Bis- ((S)-piperidine-3-ol)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 61% as of bis-((S)- piperidine-3-ol)-GDGT as a colorless oil.Bis- ((3R,4S)-piperidine-3,4-diol)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 43% of bis- ((3R,4S)- piperidine-3,4-diol)-GDGT as a colorless oil.Bis- (2-oxa-8-azaspiro[4.5]decane)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 71% of bis- (2-oxa-8- azaspiro [4.5]decane)-GDGT as a colorless oil.Bis- (morpholine)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 70% of bis- (morpholine)-GDGT as a colorless oil.The crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 52% as a diastereomeric mixture of bis- (morpholine-3-yl-methanol)-GDGT as a colorless oil .Bis- (piperazine)-GDGT is obtained via reductive amination with N-Boc-piperidine and subsequent Boc-cleavage with TFA. The crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 50% of bis- (piperazine)-GDGT as a colorless oil.Bis- (4- (hydroxyethal)piperazine)-GDGTThe crude material is purified via flash chromatography (BiotageSfar Amino column,LP:EE) to obtain 50% of bis- (4- hydroxyethyl )piperazine)-GDGT as a colorless oil.Bis- (azocane)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 71% of bis- (azocane)-GDGT as a colorless oil.Bis- ((R)-3-amino-l-benzylpiperidine)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 58% of bis- ((R)-3-amino-l- benzylpiperidine)-GDGT as a colorless oil.Bis- (indolin-5-yl-methanol)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 71% of bis- (indolin-5-yl- methanol)-GDGT as a colorless oil.Example 13 - Synthesis of ionizable squareamide amino GDGTs fromGDGT aldehydePreparation of 3-methoxy-4- (methylamino)cyclobut-3-ene-l,2-dione3,4-dimethoxycyclobut-3-ene-l ,2-dione (1 equiv.) is dissolved in Et2O before methylamine (2M in THE, 1.1 eq.) is added dropwise. The mixture is stirred for 24h whereupon a white precipitate formed. The solid is removed via filtration and washed with ether. Pure 3-methoxy-4- (methylamino)cyclobut-3-ene-l,2-dione is obtained via recrystallisation in ethyl acetate in 70% yield.Pure 3-methoxy-4- (methylamino)cyclobut-3-ene-l,2-dione (1 equiv.) is dissolved in EtOH and amino-NBoc-alkylamine is added (1.2 equiv.) and stirred for 22 hours. Column Chromatography (Biotage Sfar Amino, LP:EE) yields pure N-Boc-amino-squareamides as white solids. Boc deprotection is performed by treating N- Boc-amino-squareamides dissolved in DCM with TFA(15 equiv.) for 2 hours. Evaporation of the solvent yields amino-squareamide TFA-salts, which are used without further purification.General procedure for the preparation of bis- (squareamide amino )-GDGTBis-GDGT-aldehyde as preferably obtained according to Example 7 or 12 (1 equiv.) and amino-squareamide TFA-salt (2.2 equiv.) were dissolved in dry DCM before N,N-Diisopropylethylamine (4.4 equiv.) is added via syringe and the mixture is stirred for 10 minutes at room temperature. Then sodium triacetoxyhydroborate (6 equiv.) is added in one portion and the heterogenous mixture was stirred for 2 hours, before being carefully quenched with 2 N NaOH. After extraction with ether (3x), washing with brine, drying over MgSCg and filtration the solvents are evaporated. Column Chromatography (Biotage Sfar Amino, LP:EE) yields bis- (squareamide amino)-GDGT as white solids.Materials obtained depend on the squareamide amine used. The products typically are mixtures of several different substituted GDGT scaffolds. Representative structures (with a GDGT-4 scaffold) are shown in the following.bis- (( 3- (methylamino)-4- ((3- (methylamino)ethyl)amino)cyclobut-3-ene-l,2-dione)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 40% of bis- (( 3- (methylamino)-4- ((3- (methylamino)ethyl)amino)cyclobut-3-ene-l,2- dione)-GDGT as a white solid. bis- (( 3- (methylamino)-4- ((3- (methylamino)propyl)amino)cyclobut-3-ene-l,2-dione)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 41% of bis- (( 3-(methylamino)-4- ((3- (methylamino)propyl)amino)cyclobut-3-ene- 1,2-dione)-GDGT as a white solid. bis- (( 3- (methylamino)-4- ((3- (methylamino)butyl)amino)cyclobut- 3-ene-l,2-dione)-GDGTThe crude material is purified via flash chromatography (BiotageSfar Amino column,LP:EE) to obtain 38% of bis- (( 3-(methylamino)-4- ((3- (methylamino)butyl)amino)cyclobut-3-ene-l,2- dione)-GDGT as a white solid. bis- ((3- (3-methylazetidin-3-yl)amino)-4- (methylamino)cyclobut-3- ene-1,2-dione))-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 45% of bis- ((3- (3- methylazetidin-3-yl)amino)-4- (methylamino)cyclobut-3-ene-l,2- dione))-GDGT as a white solid. bis- ((3- (3-methylazetidin-3-yl)amino)-4- (methylamino)cyclobut-3- ene-1,2-dione))-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 32% of bis- ((3- (3- methylazetidin-3-yl)amino)-4- (methylamino)cyclobut-3-ene-l,2- dione))-GDGT as a white solid.bis- (( 3- (3- (methyl)amino)azetidin-l-yl)-4- (methylamino)cyclobut-3-ene-l,2-dione)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 28% of bis- ((3- (3- methylazetidin-3-yl)amino)-4- (methylamino)cyclobut-3-ene-l,2- dione))-GDGT as a white solid.Example 14 - Synthesis of ionizable monosubstituted GDGTsGDGT as preferably obtained according to Example 1 (1 equiv.) and imidazole (2.2 equiv.) is dissolved in DMF dry and tert- Butyldimethylsilyl chloride ( 50%w / w in toluene, 1.2 equiv.) is added dropwise. The suspension is stirred for 20 h, taken up in diethyl ether and washed with 1 M aqueous LiCl solution (3x), dried over MgSO4, filtered and evaporated. The crude material is purified via column chromatography (SiCy, LP:EE) to yield 42% of mono-TBSO-GDGT as colorless oil.Mono-TBSO-GDGT (1 equiv.) and TCCA (1.2 equiv.) are dissolved in dry DCM in an 8 mL vial equipped with a septum and a magnetic stirring bar. The mixture is cooled to 0°C and TEMPO (0.1 eq.) is added and stirred for 10 minutes. Then the yellow solution is poured into LP, filtered via celite, washed with LP and the solvents were evaporated to yield 99% of crude mono-TBSO-GDGT- aldehyde which was used without further purification.General procedure for the preparation of mono-GDGT aminesMono-TBSO-GDGT-aldehyde (1 equiv.) is dissolved in dry DCM and the appropriate amine (2 equiv.) is added and the mixture was stirred for 10 minutes at room temperature. Then STAB (3 equiv.) is added in one portion and the heterogenous mixture is stirred for 2 hours, before being carefully quenched with 2 N NaOH. After extraction with ether (3x), washing with brine, drying over MgSCy and filtration the solvents are evaporated. The crude material was dissolved in dry THE and Tetra-n-butylammonium fluoride (1 M in THE, 1.5 equiv.) is added and stirred for 20 hours. The mixture is quenched via the addition of 1 M aqueous CaCOa solution and extracted with ether (3x), washed with brine and dried over MgSO4. After filtration of the dessicant the solvents are removed under vacuum. Column Chromatography (Biotage Sfar Amino, LP:EE) yields pure mono-GDGT amines as colorless to yellow oils.Crude materials obtained depend on the amine used. The products typically are mixtures of several different substituted GDGT scaffolds. Representative structures (with a GDGT-4 scaffold) are shown in the following. mono- (dimethylamino)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain43% of mono- (dimethylamino)- GDGT as a colorless oil. mono- (diethylamino)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 55% of mono- (diethylamino)- GDGT as a colorless oil. mono- (bis- (hydroxyethyl)amino)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 54% of mono- (bis(hydroxyethyl)amino)-GDGT as a colorless oil. mono- (methyl(hydroxyethyl)amino)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 60% of mono- (methyl(hydroxyethyl)amino)-GDGT as a colorless oil. mono- (methyl(hydroxypropyl)amino)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 43% of mono- (methyl(hydroxyethyl)amino)-GDGT as a colorless oil. mono- (methyl(hydroxybutyl)amino)-GDGTThe crude material is purified via flash chromatography (BiotageSfar Amino column,LP:EE) to obtain 64% of mono- (methyl(hydroxybutyl)amino)-GDGT as a colorless oil.Mono- ((S)-oxetane-2-methane-N-methyl-amine)-GDGTThe crude material is purified via flash chromatography (BiotageSfar Amino column,LP:EE) to obtain 41% of mono- ((S)-oxetane-2methane-N-methyl-amine )-GDGT as a colorless oil. mono- (azetidine-3-methanoyl)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 46% of mono- (azetidine-3- methanoyl)-GDGT as a colorless oil. mono- (pyrrolidine)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 71% of mono- (pyrrolidine)- GDGT as a colorless oil. mono- (L-prolinol)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 52% of mono- (L-prolinol)-GDGT as a colorless oil. mono- (D-prolinol)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 61% of mono- (D-prolinol)-GDGT as a colorless oil. mono- (S-pyrrolidine-3-ol)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 49% of mono- (S-pyrrolidine-3- ol)-GDGT as a colorless oil. mono- (.R-pyrrolidine-3-ol)-GDGTChemicalFormula:C90H171NO6ExactMass:1362,31The crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 69% of mono- (A-pyrrolidine-3- ol)-GDGT as a colorless oil. mono- ((3S,4S)-pyrrolidine-3,4-diol)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 59% of mono- ((3S,4S)- pyrrolidine-3 ,4-diol)-GDGT as a colorless oil. mono- (2- (hydroxymethyl)imidazol)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 64% of mono- (2- (hydroxymethyl)imidazol)-GDGT as a colorless oil. mono- ((lR,5S,6r)-3-azabicyclo[3.1.0]hexan-6-yl)methanol)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 55% of mono- ((1R,5S,6r)-3- azabicyclo [3.1.0]hexan-6-yl)methanol)-GDGT as a colorless oil. mono- (piperidine)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 66% of mono- (piperidine)-GDGT as a colorless oil. mono- (4-hydroxypiperidine)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 61% of mono- (4- hydroxypiperidine)-GDGT as a colorless oil. mono- (2-hydroxymethylpiperidine)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 43% as a diastereomeric mixture of mono- (2-hydroxymethylpiperidine)-GDGT as a colorless oil. mono- ((A)-piperidine-3-ol)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 48% of mono- ((A)-piperidine- 3-ol)-GDGT as a colorless oil.The crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 54% as of mono-((S)- piperidine-3-ol )-GDGT as a colorless oil.The crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 51% of mono- ((3R,4S)- piperidine-3 ,4-diol)-GDGT as a colorless oil. mono- (2-oxa-8-azaspiro [4.5]decane)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 66% of mono- (2-oxa-8- azaspiro [4.5]decane)-GDGT as a colorless oil.mono- (morpholine)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 59% of mono- (morpholine)-GDGT as a colorless oil. mono- (morpholine-3-yl-methanol)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 43% as a diastereomeric mixture of mono- (morpholine-3-yl-methanol)-GDGT as a colorless oil . mono- (piperazine)-GDGTBis- (piperazine)-GDGT is obtained via reductive amination with N-Boc-piperidine and subsequent Boc-cleavage with TEA. The crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 51% of mono- (piperazine)-GDGT as a colorless oil.mono- (4- (hydroxyethyl)piperazine)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 50% of mono- (4- hydroxyethyl)piperazine )-GDGT as a colorless oil. mono- (azocane)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 65% of mono- (azocane)-GDGT as a colorless oil. mono- ((R)-3-amino-l-benzylpiperidine)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 71% of mono- ((R)-3-amino-l- benzylpiperidine)-GDGT as a colorless oil. mono- (indolin-5-yl-methanol)-GDGTThe crude material is purified via flash chromatography (Biotage Sfar Amino column,LP:EE) to obtain 38% of bis- (indolin-5-yl- methanol)-GDGT as a colorless oil.Example 15 - Formulation of LNPsLipid stock solutions (archaeal membrane lipids obtained from Sulfolobus acidocaldarius including GDGTs (see Table 1 below; see also WO 2020 / 187526 Al), DSPC, ALC-0159, cholesterol, and ionizable GDGTs as obtained according to any one of examples 3, 4, 5, 6, and 8 (or any one of the ionizable GDGTs of examples 9, 10, 11, 12, 13 and 14) are filtered through a 0.2 pm polytetrafluoroethylene (PTFE) filter and mixed according to the desired molar ratios. A mixture of dimethyl sulfoxide (DMSO) and 2-propanol (2:3) is used as the organic solvent. The aqueous phase is prepared by dissolving the desired mRNA (e.g. the mRNA which codes for an antigen, such as SARS-CoV-2 spike protein) in a 10 mM citrate buffer (pH=4.0). LNPs are formulated using a NanoAssembleR Ignite (Precision Nanosystems) at a total flow rate of 12 mL / min and a flow rate ratio of 3:1 (aqueous phase:organic phase). Immediately after formulation, LNPs are diluted 1:2 in a 10 mM PBS (pH=7.4). Removal of organic solvent is achieved via dialysis (SpectraPor, 6-8 kDa) against 10 mM phosphate-buffered saline (PBS) buffer (pH=7.4). Physicochemical characterization of the LNPs may be performed via the Ribogreen assay (mRNA content) and Zetasizer analysis (size, polydispersity index (PDI), Zeta potential). Zetasizer is available from Malvern Panalytical Ltd, UK.A typical lipid composition for the GDGT-containing archaeal membrane lipid stock solution is given in Table 1:After formulation, LNPs are stored in 2 mL Eppendorf tubes at 25°C (room temperature), respectively. Physiochemical characterization via Ribogreen assay and ZetaSizer analysis may be conducted in certain time intervals over a period of 6 weeks.The LNPs may be used to formulate mRNA vaccines.Example 16 - Formulation and characterization of further LNPsThe primary objective was to characterize a range of ionizable GDGTs utilized LNPs. By modifying the lipid ratios of ionizable GDGTs, we several key parameters were evaluated, including size, PDI (polydispersity index) and encapsulation efficiency. Moreover, zeta potential and pKa value determinations were conducted with the objective of acquiring further insights into the physicochemical properties of the ionizable GDGTs. Finally, an in vivo study in rats was performed.BuffersThe buffer for the formulation of LNPs was a 10 mM citric acid buffer at pH 3.0. The buffer for dilution and dialysis was a 10 mM PBS at pH of 7.4LNP formulationsConventional lipids for LNPs were dissolved in ethanol to achieve a concentration of between 10 and 20 g / L, while the ionizable GDGTs were dissolved in isopropanol to reach a concentration of 30 g / L. The total lipid concentration was 10-20 mM, with 20 mM being used to determine the pKa and zeta potential value.Furthermore, an investigation was conducted into the effect of varying the N / P ratio (4 - 6 - 8 - 12). (For LNPs, the N / P ratio corresponds to the ratio between the amine groups (N) of the ionizable lipid and the phosphate group (P) of the nucleic acid cargo (1 per base for RNA, 2 for DNA). The N / P ratio of 6 yielded the best results and was chosen for further experiments.Poly (A) was used as cargo (dissolved in citric acid buffer) and encapsulated into lipid nanoparticles via microfluidic techniques, using a flow rate ratio of 3:1 (aqueous to organic phase) and a total flow rate of 12 mL / min. Furthermore, a 2:1 dilution with PBS buffer was performed to reduce the organic solvent content, followed by a dialysis step.The Zetasizer ZSP (Malvern) was used to determine the size, PDI and zeta potential of the particles, while a RiboGreen assay was employed to ascertain the encapsulation efficiency.The results shown in the following tables were obtained for the ionizable GDGT used (a mixture of several different GDGT scaffolds was used; a GDGT-4 scaffold is shown as a representative structure, respectively). The lipid ratio of the ionizable GDGTs among the conventional LNP lipid composition is given in Moll.Table 2Table 3Table 4Table 5SUBSTITUTE SHEET (RULE 26)Table 6Table 7Table 8Table 9SUBSTITUTE SHEET (RULE 26)Table 10In vivo studyThe in vivo study was conducted with some of the LNPs characterized in Tables 2 to 10 above. Formulations containing 46.3 mol-% (lipid ratio) of the respective ionizable GDGT (N / P ratio 6 or 12) and encapsulating erythropoietin (EPO) mRNA were administered intramuscularly to Wistar rats. The objective of the in vivo study was to evaluate the properties of the different ionizable GDGTs in LNP formulations and their efficiency in transporting the EPO mRNA into muscle cells. Following the administration of the LNPs, the plasma concentration of EPO (AUG levels) was monitored and quantified through the use of an ELISA assay to determine the extent of induced EPO protein.The results are shown in Table 11 below:In general, the EPO protein concentrations achieved were higher compared to a control without mRNA. For several LNPs (such as e.g., mono-OHPIPD based LNPs), the protein concentrations achieved were much higher.Taken together, these results show that ionizable GDGTs are very suitable for LNPs encapsulating mRNA cargo.Non-patent referencesAldosari, Basmah N., et al. "Lipid nanoparticles as delivery systems for RNA-based vaccines." Pharmaceutics 13.2 (2021): 206.Benvegnu, Thierry, Loic Lemiegre, and Sandrine Cammas- Marion. "New generation of liposomes called archaeosomes based on natural or synthetic archaeal lipids as innovative formulations for drug delivery." Recent patents on drug delivery & formulation 3.3 (2009): 206-220.Cornebise, Mark, et al. 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Claims

Claims1. A lipid nanoparticle (LNP) encapsulating a nucleic acid cargo, wherein the LNP comprises at least- an ionizable lipid fraction and- a stabilizer fraction; wherein the ionizable lipid fraction comprises at least one ionizable glycerol dialkyl glycerol tetraether (GDGT) lipid; wherein the at least one ionizable GDGT lipid comprises at least one ionizable head group S; preferably wherein S is selected from the group consisting of:wherein each occurrence of Rais independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkylamine, alkyl ether, alkyl thiol, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide, preferably from alkyl, cycloalkyl, alkenyl, hydroxyalkyl and alkylamine, each occurrence of Rbis independently selected from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate, alkyl sulfonamide and alkyl thiol, preferably from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine and alkyl thiol, and each occurrence of Rcis independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide, preferably from alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide.

2. The LNP of claim 1, wherein the nucleic acid cargo comprises a messenger ribonucleic acid (mRNA).

3. The LNP of claim 1 or 2, wherein the stabilizer fraction comprises at least one polyethylene glycol (PEG) lipid.

4. The LNP of any one of claims 1 to 3, wherein the LNP further comprises a sterol lipid fraction, preferably comprising cholesterol.

5. A GDGT lipid suitable for use as an ionizable lipid in an LNP, wherein the GDGT lipid comprises at least one ionizable head group S, wherein S is selected from the group consisting of:wherein each occurrence of Rais independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkylamine, alkyl ether, alkyl thiol, alkyl ester, alkyl amide, alkyl carbamate,alkyl sulfonate and alkyl sulfonamide, preferably from alkyl, cycloalkyl, alkenyl, hydroxyalkyl and alkylamine, each occurrence of Rbis independently selected from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate, alkyl sulfonamide and alkyl thiol, preferably from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine and alkyl thiol, and each occurrence of Rcis independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide, preferably from alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkyl amide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide.

6. The GDGT lipid of claim 5, wherein S is selected from the group consisting of:wherein each occurrence of Rais independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl and alkylamine, and each occurrence of Rbis independently selected from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine and alkyl thiol.

7. The GDGT lipid of claim 5 or 6, wherein the head group S is selected from:wherein each occurrence of R is independently selected from H and alkyl; preferably wherein the alkyl is selected from methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, pentyl, iso-pentyl, hexyl, iso-hexyl, heptyl, and iso-heptyl.

8. An ether lipid fraction comprising the GDGT lipid of any one of claims 5 to 7; preferably obtainable by extraction from an archaeal culture, preferably a Sulfolobus culture, more preferably a Sulfolobus acldocaldarlus culture, followed by substitution with the ionizable head group S.

9. A pharmaceutical composition comprising the LNP of any one of claims 1 to 4 and preferably at least one excipient.

10. A method for producing the GDGT lipid of any one of claims 5 to 7, comprising the steps of:- obtaining a lipid fraction comprising one or more precursor GDGT lipids from an archaeal culture, preferably aSulfolobus culture, more preferably a Sulfolobus acldocaldarlus culture;- purifying the one or more precursor GDGT lipids; and- contacting the one or more precursor GDGT lipids with one or more reagents to produce the GDGT lipid with the at least one ionizable head group S.

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