Lipid nanoparticles with nucleic acid load and ionizable lipid

Abstract

Disclosed is a lipid nanoparticle (LNP) encapsulating a nucleic acid load, preferably the nucleic acid load comprises a 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 polyethylene glycol (PEG) lipid. In addition, the ionizable lipid fraction comprises at least one ionizable glycerol dialkylglycerol tetraether (GDGT) lipid. Also disclosed is a pharmaceutical composition, such as an mRNA vaccine, comprising the LNP. In further aspects, the invention relates to ionizable GDGT lipids and methods of producing the same.

Description

Technical Field

[0001] The field of this invention relates to lipid nanoparticles (LNPs) having nucleic acid loads, particularly lipid nanoparticles (LNPs) for use in messenger ribonucleic acid (mRNA) vaccines, and to ionizable lipids suitable for use in LNPs. Background Technology

[0002] LNPs have recently come into focus because LNP-based mRNA vaccines against SARS-CoV-2 (primarily elasomeran marketed by Moderna Inc. under Spikevax® and tozinameran marketed by Biontech SE / Pfizer Inc. under Comirnaty®) have been administered to hundreds of millions of individuals. LNPs, commonly used as delivery systems for RNA-based vaccines, were reviewed in Aldosari et al. (2021). LNPs used for mRNA delivery were also discussed in detail in Hou et al. (2021).

[0003] LNPs can be used not only in vaccines but also for delivering other therapeutic nucleic acids. For example, patisiran is an LNP-based drug used for RNA interference therapy of transthyretin-mediated amyloidosis (see Zhang et al., 2019).

[0004] Typically, LNPs with nucleic acid loads (or payloads) comprise a lipid layer and microstructural domains of lipids and encapsulated nucleic acids. They have a median diameter between 10 nm and 1000 nm (determined, e.g., by dynamic light scattering (DLS)) and can take on, for example, spherical or polyhedral shapes. They can be multilayered, depending on their specific lipid composition. LNPs contain ionizable lipids, particularly at endosome pH (e.g., in the range of pH 4.5 to 6.5, preferably pH 4.5 to 6.0, and especially pH 4.5 to 5.5), i.e., lipids that are protonated when in endosomes. Typically, LNPs also contain stabilizers, such as polyethylene glycol (PEG) lipids, which reduce LNP aggregation, enzymatic degradation, opsonization, and immunogenicity. In addition, LNPs often contain other types of lipids (often referred to as “helper lipids”), such as phosphatidylcholine or phosphatidylethanolamine, to improve properties such as delivery efficacy, tolerability, or biodistribution. Finally, LNPs may contain cholesterol or other sterols to modulate membrane integrity and rigidity.

[0005] LNPs are also disclosed, for example, in U.S. Patents US 7,404,969, US 8,058,069, US 9,364,435 and US 9,404,127 and US 2013 / 0245107 A1.

[0006] WO 2017 / 099823 A1 discloses an LNP that accelerates blood clearance insensitive to lipids, comprising cationic lipids, polyethylene glycol (PEG)-lipids, sterols and auxiliary lipids, wherein the auxiliary lipids do not contain phosphatidylcholine.

[0007] WO 2020 / 061284 A1 and WO 2019 / 089818 A1 also involve LNPs with PEG lipids.

[0008] Eygeris et al. (2021) provided a review on the chemistry of LNPs for RNA delivery. They disclosed PEG lipids in LNP formulations.

[0009] WO 2014 / 143806 A1 discloses lipid particles containing PEG lipids.

[0010] WO 2020 / 219941 A1 discloses additional LNPs and formulations containing LNPs.

[0011] WO 2021 / 123332 A1 relates to cationic lipids and LNPs comprising said cationic lipids, said LNPs being used to deliver nucleic acids into living cells.

[0012] Despite recent advances in this field, improvements in LNPs remain, particularly regarding storage stability and / or conversion (transfection) efficiency. For example, the LNP-based SARS-CoV-2 vaccine Comirnaty® typically must be stored at -90°C to -60°C (Product Characteristics Summary, September 13, 2022 version, EMEA / H / C / 005735 - II / 0143, European Medicines Agency-EMA). Furthermore, improved conversion efficiency would allow for lower dosages, thereby reducing potential side effects. Summary of the Invention

[0013] Therefore, an object of the present invention is to provide improved LNPs with nucleic acid loadings (e.g., mRNA, siRNA, or cDNA loadings), particularly with improved conversion efficiency and / or higher storage stability, especially at non-refrigerated temperatures (e.g., room temperature). Another object of the present invention is to provide novel ionizable lipids suitable for use in LNPs, preferably ionizable lipids that result in improved conversion efficiency and / or higher storage stability, particularly at non-refrigerated temperatures (e.g., room temperature).

[0014] This invention provides a liquid nuclease-encapsulated polymeric nipple (LNP). The LNP comprises at least an ionizable lipid fraction and a stabilizer fraction (preferably containing PEG lipids). The ionizable lipid fraction comprises at least one ionizable GDGT lipid. The LNP may also comprise neutral GDGT lipids as disclosed below.

[0015] As used herein, ionizable lipids are lipids that carry an overall positive charge (i.e., cations) at endosome pH (e.g., pH 5.0–6.5, particularly pH 5.0, 5.5, 6, or 6.5).

[0016] In another aspect, the present invention relates to GDGT lipids comprising at least one ionizable head group S. The head group S is selected from the group consisting of:

[0017] and

[0018] Each occurrence of R a Independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkylamine, alkyl ether, alkyl thiol, alkyl ester, alkylamide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide (preferably selected from alkyl, cycloalkyl, alkenyl, hydroxyalkyl and alkylamine), each R appearing b Independently selected from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl ester, alkylamide, alkyl carbamate, alkyl sulfonate, alkyl sulfonamide and alkyl thiol (preferably selected from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine and alkyl thiol), and each R appearing c Independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkylamide, alkyl carbamate, alkyl sulfonate, and alkyl sulfonamide (preferably selected from alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkylamide, alkyl carbamate, alkyl sulfonate, and alkyl sulfonamide). Preferably, this GDGT lipid is suitable for use as an ionizable lipid in LNPs (e.g., the LNPs disclosed herein).

[0019] In another aspect, the present invention provides an ether lipid fraction comprising the GDGT lipid. This ether lipid fraction is preferably obtained by extraction from archaea cultures, preferably Sulfolobus cultures, more preferably Sulfolobus acidocaldarius cultures, followed by substitution with an ionizable head group S (wherein the GDGT is present).

[0020] The present invention also relates to (as defined herein) LNPs comprising the GDGT lipid or ether lipid fractions disclosed above.

[0021] On the other hand, the present invention relates to pharmaceutical compositions (particularly vaccines) containing LNPs. These pharmaceutical compositions typically contain additional excipients. They are preferably used for the prevention or treatment of diseases or conditions in (human) patients, particularly as vaccines for the prevention (or improvement) of diseases (e.g., infectious diseases) or as cancer vaccines.

[0022] In another aspect, the present invention provides a method for producing GDGT lipids, comprising the following steps:

[0023] - Obtain lipid fractions containing one or more precursor GDGT lipids from archaea cultures, preferably sulfur leaf culture, and more preferably acidophilic thermosulfur leaf culture;

[0024] - Purify one or more precursor GDGT lipids; and

[0025] - Contact one or more precursor GDGT lipids with one or more reagents to produce GDGT lipids having at least one ionizable head group S.

[0026] GDGT lipids (also referred to as “GDGT” in this paper) have been found to be membrane lipids of extremophilic archae, but have also recently been identified as membrane components of some bacteria (see Schouten et al., 2013). Many phylogenetic groups in archaea synthesize GDGT. They form a monolayer rather than a bilayer in the cell membrane. Surprisingly, both ionizable and non-ionizable GDGT lipids are particularly well-suited for improving the properties of LNPs loaded with nucleic acids (e.g., storage stability and transformation efficiency).

[0027] Importantly, LNPs (as described in more detail above) differ from other lipid-based delivery media (e.g., liposomes, lipopolysemblages, or archaeosomes) and present unique advantages but also challenges, particularly in the context of mRNA packaging. For example, Midoux & Pichon (2014) reviewed various lipid-based mRNA vaccine delivery systems, distinguishing between lipid complexes, lipopolysemblages, LNPs, and cationic nanoemulsions. More broadly, drug delivery of RNA therapeutics (such as small interfering RNA and mRNA) is achieved via lipid-based carriers (e.g., micelles, liposomes, and LNPs) (Paunovska et al., 2022).

[0028] In contrast to LNPs, liposomes are spherical lipid bilayer vesicles (lipoesters) surrounding an aqueous space. They are carriers used for the administration of drugs, vaccines, genes, proteins, small molecules, antibiotics, and nutrients. Liposomes are made of phospholipids (primarily phosphatidylcholine) and cholesterol, but may also include other lipids (such as phosphatidylethanolamine). Liposomes are produced by a wide variety of methods (e.g., van Hoogevest, 2017; Szoka et al., 1980), such as by dispersing phospholipids in an aqueous medium, by mechanical processing (e.g., in a homogenizer, preferably by high-pressure homogenization), or by sonication. Their diameter varies between 0.02 and 10 μm.

[0029] Archaeosomes represent a special class of liposomes based on membrane lipids isolated from archaea. Archaeosomes consist of lipid ethers, namely diether structures (e.g., dicepane diglyceride (archaeol)) and tetraether structures (e.g., GDGT, dicepane diglyceride tetraether (caldarchaeol)); see, for example, Kaur et al. (2016), Patel et al. (1999). Diether structures typically consist of a glycerol moiety carrying two phytyl chains (20-40 carbons in length) at the sn-2,3 positions. Tetraether structures typically carry two dicepane chains linked to two glycerol residues in an antiparallel (dicepane diglyceride tetraether) or parallel (isodicepane diglyceride tetraether) manner. Additionally, one or more cyclopentane rings may be present.

[0030] Benvegnu et al. (2009) addressed GDGT with polar substituents. Vaz et al. (1985) disclosed GDGT with phosphate ethanolamine substituents. Morii et al. (1994) disclosed the structure of the major polar lipid of the thermophilic methanogenic archaea (M. thermoautotrophicum). Schwarzmann et al. (2015) disclosed a polar GDGT, bis(phytane)-bis(glycerol)-tetraetheryl diamine.

[0031] As used herein, “caldarchaeol” refers to the entire group of isoprene GDGT lipids having 0 to 8 cyclopentane moieties. Specifically, this paper uses the nomenclature suggested by Schouten et al. (2013): “GDGT-x”, where x represents the number of cyclopentane moieties, namely 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 caldarchaeol group.

[0032] Depending on the composition (e.g., the amount of diphyranyl diglyceride relative to diphyranyl diglyceride tetraglyceride), the lipid layer of an archaea is a monolayer, a bilayer, or a mixture thereof.

[0033] WO 2020 / 187526 A1 discloses archaea containing archaea lipids derived from sulfur leaf fungus cell cultures, primarily intended for oral expiratory or oral sustained-release delivery.

[0034] WO 02 / 053554 A2 relates to tetraether lipid derivatives and liposomes containing tetraether lipid derivatives, as well as lipid aggregates and their uses. In the examples, the tetraether lipids are obtained from *Alternaria acidophila*. The tetraether skeleton may contain, for example, four cyclopentanes and may be substituted. WO 97 / 31927 A1, WO 99 / 10337 A1, US 2010 / 316657 A1, and US 6,316,260 B1 also disclose tetraether lipid derivatives. Furthermore, WO 03 / 064360 A1 and WO 2004 / 037223 A2 relate to tetraether lipid derivatives and liposomes containing tetraether lipid derivatives.

[0035] Sateesh et al. (2008) proposed a novel approach to surface modification of medical-grade polyurethanes using tetraether lipids activated by cyanuric acid chloride, thereby providing a new method for bacterial anti-adhesion.

[0036] Engelhardt et al. (2017) discussed transfection studies using a colloidal system containing highly purified bipolar tetraether lipids from *Leymus chinensis*.

[0037] WO 2017 / 067642 A1 discloses liposomes containing cell-penetrating peptides and tetraether lipids for oral delivery of macromolecules.

[0038] Vishakarma et al. (2019) reviewed various lipid-based carriers for lymphatic transport, including liposomes and archaea.

[0039] GB 2463801 A discloses liposomes containing isoprene-like lipids.

[0040] Furthermore, regarding archaea, Daswani et al. (2021) disclosed that polar lipid fraction E from *Leucobacterium acidophilus* can be used as a liposomal drug stabilizer to reduce leakage of the anti-angiogenic drug α4-phosphate disodium from tetraether / diester mixed archaea.

[0041] Unlike archaea, LNPs contain ionizable lipids. Such ionizable lipids are discussed, for example, by Cornebise et al. (2022).

[0042] Tetraether lipids and their functions in membranes are generally discussed in the following publications: Weijers et al. (2010) disclosed the carbon isotopic composition of branched tetraether membrane lipids in soil. Schuster et al. (1998) addressed voltage-clamp studies of S-layer supported tetraether lipid membranes. EP 1 777 520 A1 discloses a method for coating lipid membranes that may contain tetraether lipids. WO 2002 / 066012 A2 discloses liposomes that may contain tetraether lipids. US 2014 / 0178462 A1 relates to amphoteric liposomes containing neutral lipids, which may be tetraether lipids.

[0043] Typically, ionizable GDGT lipids contain at least one ionizable head group S. Preferably, GDGT lipids contain two ionizable head groups S (which are usually the same).

[0044] In a preferred embodiment of the invention, the ionizable lipid is a lipid that carries an overall positive charge at endosome pH (e.g., between pH 5.0 and 6.5, such as pH 5.0, 5.5, 6, or 6.5), but is neutral at higher pH (e.g., pH 7.0). Therefore, the ionizable head group S disclosed herein preferably carries an overall positive charge at endosome pH (e.g., between pH 5.0 and 6.5, such as pH 5.0, 5.5, 6, or 6.5), but is neutral at higher pH (e.g., pH 7.0).

[0045] The following ionizable head groups have been shown to be particularly suitable for use in this invention (especially in the LNP of this invention):

[0046] and

[0047] Each occurrence of R a Independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkylamine, alkyl ether, alkyl thiol, alkyl ester, alkylamide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide (preferably selected from alkyl, cycloalkyl, alkenyl, hydroxyalkyl and alkylamine), each R appearing b Independently selected from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl ester, alkylamide, alkyl carbamate, alkyl sulfonate, alkyl sulfonamide and alkyl thiol (preferably selected from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine and alkyl thiol), and each R appearing cIt is independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkylamide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide (preferably selected from alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkylamide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide).

[0048] It goes without saying that every occurrence of R a Or each occurrence of R c It can be part of the same ring structure (especially based on alkyl, alkenyl, hydroxyalkyl, alkylamine, alkyl ether, alkyl thiol, alkyl ester, alkylamide, alkyl carbamate, alkyl sulfonate, and alkyl sulfonamide). Therefore, the head group... The preferred implementation may be selected from any one of the following:

[0049] and .

[0050] The GDGT (EABM-GDGT) based on the first embodiment is particularly preferred. A representative structure (based on the GDGT-4 framework) is shown below:

[0051] .

[0052] A further preferred embodiment of the head group is shown, for example, in Example 12 below.

[0053] Preferably, the alkyl group is selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, isopentyl, hexyl, isohexyl, heptyl, and isohexyl.

[0054] Alternatively or otherwise, the cycloalkyl group is preferably selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

[0055] Alternatively or otherwise, the alkenyl group 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, hep-2-enyl, hep-3-enyl, hep-4-enyl, hep-5-enyl, and hep-6-enyl.

[0056] Alternatively or otherwise, the hydroxyalkyl group is preferably selected from hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, hydroxypentyl and hydroxyhexyl.

[0057] Alternatively or otherwise, alkylamines are preferably selected from methylamine, ethylamine, propylamine, butylamine, pentamine, hexamine and heptamine.

[0058] Alternatively or otherwise, alkyl ethers are preferably selected from methyl ether, diethyl ether, propyl ether, butyl ether, pentyl ether, hexyl ether and heptyl ether.

[0059] Alternatively or otherwise, alkyl mercaptans are preferably selected from methanethiol, ethanethiol, propanethiol, butanethiol, pentathiol, hexanethiol, and heptanethiol.

[0060] Alternatively or otherwise, the alkyl ester is preferably selected from methyl ester, ethyl ester, propyl ester, butyl ester, pentyl ester, hexyl ester and heptyl ester.

[0061] Alternatively or otherwise, alkylamides are preferably selected from formamide, acetamide, propionamide, butyramide, valerate, hexamamide and heptaamide.

[0062] Alternatively or otherwise, the alkyl carbamate is preferably selected from methyl carbamate, ethyl carbamate, propyl carbamate, butyl carbamate, pentyl carbamate, hexyl carbamate and heptyl carbamate.

[0063] Alternatively or otherwise, the alkyl sulfonate is preferably selected from methanesulfonates, ethyl sulfonates, propyl sulfonates, butyl sulfonates, pentyl sulfonates, hexyl sulfonates, and heptyl sulfonates.

[0064] Alternatively or otherwise, the alkyl sulfonamide is preferably selected from methanesulfonamide, ethylsulfonamide, propylsulfonamide, butylsulfonamide, pentylsulfonamide, hexylsulfonamide and heptaylsulfonamide.

[0065] In the context of this invention, the following head groups are particularly preferred:

[0066] and

[0067] Each occurrence of R a and R b Independently as defined above.

[0068] According to another preferred embodiment, the head group is: ,

[0069] Each occurrence of R c Independently as defined above. A particularly preferred example of this head group is disclosed in Example 11.

[0070] The following ionizable GDGT lipids (i.e., those containing an ionizable head group S as defined herein) are particularly suitable for use in the LNP of this invention:

[0071]

[0072]

[0073]

[0074]

[0075] Where S 1 It is S and S 2 It is OH or S 1 It is OH and S 2 Is it S, or S 1 and S 2 Each of them is independently S. Preferably, S 1 and S 2 It is the same head group S as defined herein.

[0076] Of the GDGT backbones described above, the following backbones 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 ether lipid fraction of the present invention) preferably contains at least two, preferably at least three, more preferably at least four, even more preferably at least five, and even more preferably at least six, particularly at least seven or even at least eight, or a mixture of all of these backbones.

[0077] Figures 1 to 1 0 describes a particularly preferred ionizable GDGT lipid. While the GDGT-4 backbone is described, other GDGT backbones (particularly GDGT-0, GDGT-1, GDGT-2, GDGT-3, GDGT-5, GDGT-6, GDGT-7, and GDGT-8) are also preferred in this respect. For substituents such as "alkyl," "cycloalkyl," etc., the preferred embodiments described above are preferably applicable (e.g., alkyl is preferably selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, isopentyl, hexyl, isohexyl, heptyl, and isohexyl; cycloalkyl is preferably selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl, etc.). Each instance of "R1" or "R2" can be independently selected from the definitions given below for the structure.

[0078] In another preferred embodiment, the ionizable lipid fraction comprises at least one additional ionizable lipid selected from the group consisting of: [(4-hydroxybutyl)azanidinediyl]bis(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-(bisdodecylamino)-N1,N1,4-tridodecyl-1-piperazineethylamine (KL10), N1-[ 2-(bis(dodecylamino)ethyl)N1,N4,N4-tridecyl-1,4-piperazindiethylamine (KL22), 14,25-bis(tetrazyl-15,18,21,24-tetraaza-octacosane (KL25), 1,2-dilinyloxy-N,N-dimethylaminopropane (Dlin-DMA), 2,2-dilinyl-4-dimethylaminomethyl-[1,3]-dioxolane (Dlin-K-DMA), heptane-6,9,28,31-tetraen-19-yl-4-(dimethylamino) Butyrate (Dlin-MC3-DMA), 2,2-dilinole-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (Dlin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8[(3β)-cholest-5-en-3-yloxy]octyl}oxy)N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA), (2R)-2-({8 8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-1-amine (octyl-CLinDMA(2R)), (2S)2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-1-amine (octyl-CLinDMA(2S)) and mixtures thereof. Alternatively or otherwise, the ionizable lipids are preferably selected from the group consisting of: (20Z,23Z)-N,N-dimethyl-20,23-diene-10-amine, (17Z,20Z)-N,N-dimethyl-17,20-diene-9-amine, (1Z,19Z)-N5N-dimethyl-16,19-diene-8-amine, (13Z,16Z)-N,N-dimethyl-13,16-diene-5-amine, (12Z,15Z)-N,N-dimethyl-12,15-diene-4-amine, (14Z,17Z)-N,N-dimethyl-14,17-Dien-6-amine, (15Z,18Z)-N,N-dimethyltetracos-15,18-dien-7-amine, (18Z,21Z)-N,N-dimethylheptadec-18,21-dien-10-amine, (15Z,18Z)-N,N-dimethyltetracos-15,18-dien-5-amine, (14Z,17Z)-N,N-dimethyltridec-14,17-dien-4-amine, (19Z,22Z)-N,N-dimethyloctadec-19,22-dien-9-amine, (18Z,21Z)-N,N-dimethylheptadec-18,21-dien-8-amine, (17Z,20Z)-N,N-dimethylhexadec-17,20 -diene-7-amine, (16Z,19Z)-N,N-dimethylpentadecano-16,19-diene-6-amine, (22Z,25Z)-N,N-dimethyltridecane-22,25-diene-10-amine, (21Z,24Z)-N,N-dimethyltridecane-21,24-diene-9-amine, (18Z)-N,N-dimethylheptadecane-18-en-10-amine, (17Z)-N,N-dimethylhexadecano-17-en-9-amine, (19Z,22Z)-N,N-dimethylhexadecano-19,22-diene-7-amine, N,N-dimethylheptadecane-10-amine, (20Z,23Z)-N-ethyl-N-methylhexadecano-20,23 -dien-10-amine, 1-[(11Z,14Z)-1-nonyleicosicos-11,14-dien-1-yl]pyrrolidine, (20Z)-N,N-dimethylheptadec-20-en-10-amine, (15Z)-N,N-dimethylheptadec-15-en-10-amine, (14Z)-N,N-dimethylheptadec-14-en-10-amine, (17Z)-N,N-dimethylheptadec-17-en-10-amine, (24Z)-N,N-dimethyltridec-24-en-10-amine, (20Z)-N,N-dimethylheptadec-20-en-10-amine, (22Z)-N,N-dimethyltridec-22-en-10-amine, (16Z) -N,N-Dimethylpentacarbon-16-en-8-amine, (12Z,15Z)-N,N-Dimethyl-2-nonyltetracos-12,15-dien-1-amine, (13Z,16Z)-N,N-Dimethyl-3-nonyltetracos-13,16-dien-1-amine, N,N-Dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecane-8-amine, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-Dimethylnonadecane-10-amine, N,N-Dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecane-10-amine, N,N-Dimethyl-21-[(1S,2R)-2-octylcyclopropyl]tetracos-10-amine, N,N-Dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine, N,N-Dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecane-8-amine, N,N-Dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradecane-5-amine, N,N-Dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecane-1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecane-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine, N,N-dimethyl-1-[( 1S,2R)-2-octylcyclopropyl]pentadecan-8-amine, RN,N-dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-(octoxy)propane-2-amine, SN,N-dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-(octoxy)propane-2-amine, 1-{2-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-1-[(octoxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy] Propane-2-amine, 1-{2-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-1-[(octoxy)methyl]ethyl}azacyclobutane, (2S)-1-(hexoxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-2-amine, (2S)-1-(heptoxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-2-amine, N,N-dimethyl-1-(nonoxy)-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-2-amine, N,N-dimethyl-1-[(9Z)- [Octadeca-9-en-1-yloxy]-3-(octoxy)propane-2-amine, (2S)-N,N-dimethyl-1-[(6Z,9Z,12Z)-octadec-6,9,12-trien-1-yloxy]-3-(octoxy)propane-2-amine, (2S)-1-[(11Z,14Z)-eicos-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentoxy)propane-2-amine, (2S)-1-(hexoxy)-3-[(11Z,14Z)-eicos-11,14-dien-1-yloxy]-N,N-dimethylpropane-2-amine, 1-[(11Z,14Z)-eicos-11,14-dien-1-yloxy]-N,N-Dimethyl-3-(octyloxy)propane-2-amine, 1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propane-2-amine, (2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropane-2-amine, (2S)-1-[(13 [(13Z)- ... H(1-formyloctyl)oxy]-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-2-amine, N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)] -2-[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propane-2-amine, N,N-dimethyl-1-{[8-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)propane-2-amine and (11E,20Z,23Z)-N,N-dimethyl-necodeca-11,20,2-trien-10-amine, and pharmaceutically acceptable salts and stereoisomers thereof, and mixtures thereof.

[0079] Other suitable ionizable lipids are disclosed, for example, in Hou et al. (2021), U.S. Patents US 7,404,969, US 8,058,069, US 9,364,435 and US 9,404,127, WO 2017 / 099823 A1, WO 2020 / 061284 A1, WO 2020 / 219941 A1 and WO 2021 / 123332 A1. These documents are incorporated herein by reference in their entirety.

[0080] Even other suitable ionizable lipids are disclosed, for example, in WO 2017 / 049245 A1, WO 2017 / 112865 A1, WO 2012 / 040184, WO 2011 / 153120 A1, WO 2011 / 149733 A1, WO 2011 / 090965 A1, WO2011 / 043913 A1, WO 2011 / 022460 A1, WO 2012 / 061259 A1, WO 2012 / 054365 A1, WO 2012 / 044638 A1, WO 2010 / 080724 A1, WO 2010 / 21865 A1, WO 2008 / 103276 A1, WO The references to these patents are 2013 / 086373A1 and WO 2013 / 086354 A1, U.S. Patents 7,893,302, 7,404,969, 8,283,333 and 8,466,122, and U.S. Patent Publications US20100036115, US20120202871, US20130064894, US20130129785, US20130150625, US20130178541 and US20130225836. These documents are incorporated herein by reference in their entirety.

[0081] The stabilizer fractions of LNPs of the present invention are generally suitable for achieving one or more of the following: reducing LNP aggregation, increasing average particle size or hydrodynamic radius, increasing the in vivo half-life of LNPs (e.g. in humans, particularly in blood circulation), and regulating zeta potential.

[0082] According to a particularly preferred embodiment, the stabilizer fraction comprises at least one PEG lipid. Suitable PEG lipids are, for example: 2-[(PEG)-2000]-N,N-bistetradecylacetamide (ALC-0159), PEGylated diacylglycerol lipid (PEG-DAG), PEGylated ceramide lipid (PEG-Cer), PEGylated phosphatidylethanolamine lipid (PEG-PE), PEGylated succinate diacylglycerol lipid (PEG-S-DAG), PEGylated dialkoxypropyl carbamate lipid, 1,2-dimethylformamide, etc. Myristoyl-rac-glycerol-3-methoxy polyethylene glycol (“PEG-DMG” or “DMG-PEG”), particularly PEG2000-DMG, 1,2-decyl-rac-glycerol-3-methoxy polyethylene glycol (Cio-diacylglycerol PEG), N-octanoyl-sphingosine-1-{succinoyl[methoxy(polyethylene glycol)2000]} (containing N-octanoyl-D-erythrosine (d18:1 / 8:0), also known as PEG-ceramide 8), or PEG lipids as disclosed in WO 2018 / 126084 A1, WO 2020 / 093061 A1 or WO2020 / 219941 A1 (all three references are incorporated herein by reference in their entirety); and any combination thereof.

[0083] Other suitable PEG lipids are disclosed, for example, in Hou et al. (2021), WO 2017 / 099823, WO 2020 / 061284A1, WO 2020 / 219941 A1 and WO 2021 / 123332 A1. All of these references are incorporated herein by reference in their entirety.

[0084] Alternatively or additionally, the stabilizer fraction may contain at least one non-PEG moiety (e.g., an XTEN peptide), which may or may not be conjugated with lipids. The XTEN peptide, due to its hydrophilicity, is able to form a hydrated shell around the LNP. This further contributes to increasing the half-life of the LNP compared to an LNP lacking (or without) the stabilizer fraction. XTEN amino acid sequences are known in the art, including, for example, those reported in U.S. Patent 9,062,299 (incorporated herein by reference in its entirety). Alternatively or additionally, in some embodiments, the stabilizer fraction may contain a non-PEG moiety, such as a PAS peptide (which may or may not be conjugated with lipids). The PAS peptide is a peptide that primarily (if not exclusively) contains proline, alanine, and serine. Like PEG and XTEN peptides, the PAS peptide is able to form a hydrated shell around the LNP. This also contributes to increasing the half-life of the LNP compared to an LNP lacking (or without) the stabilizer fraction. The amino acid sequences of PAS are known in the art, including those reported, for example, in WO 2008 / 155134 A1 (which is incorporated herein by reference in its entirety).

[0085] The LNP of the present invention preferably comprises at least one neutral (or non-ionizable, or in other words, neutral at endosome pH) GDGT lipid. This GDGT lipid may be, for example, isoprene-like GDGT lipids, such as GDGT-0, GDGT-1, GDGT-2, GDGT-3, GDGT-4, GDGT-5, GDGT-6, GDGT-7, and GDGT-8, or crenarchaeol (unsubstituted, see Schouten et al. (2013)). Figure 1 , or substituted), or branched GDGT (e.g., GDGT-I, GDGT-II, or GDGT-III), or any mixture thereof. In particular, GDGT lipids may be contained in Schouten et al. (2013) (especially... Figure 1 Any GDGT disclosed in Kaur et al. (2016) (especially Figures 2, 3 and 4) and WO 2020 / 187526 A1 (all of these documents are incorporated herein by reference in their entirety). GDGT may be substituted (e.g., substituted with a hexose moiety or a phosphatidylinositol moiety) or unsubstituted.

[0086] Diphyllane diglycerides (e.g., obtained from sulfur-bearing fungi) have proven particularly suitable for use in this invention. According to a preferred embodiment, at least one GDGT lipid (of the ether lipid fraction of LNP) thus comprises at least one neutral (or non-ionizable, or in other words, neutral at endosome pH) diphyllane diglyceride, preferably selected from the group consisting of: unsubstituted diphyllane diglycerides, phosphatidylinositol (PI)-diphyllane diglycerides, dihexose (2Hex)-diphyllane diglycerides and 2Hex-PI-diphyllane diglycerides (particularly those disclosed as in Figure 4 of WO 2020 / 187526 A1), hexose (Hex)-diphyllane diglycerides and sulfo-trihexose (3Hex)-diphyllane diglycerides, sulfo-3Hex-PI-diphyllane diglycerides, and any mixture thereof.

[0087] The particularly preferred Hex-diphyrane diglyceride is:

[0088] .

[0089] The particularly preferred sulfonyl-3Hex-PI-diphyllane diglyceride is:

[0090] .

[0091] The LNP of the present invention is particularly suitable for use with RNA loads. Therefore, in a preferred embodiment, the nucleic acid load comprises at least one (therapeutic) RNA. Examples of suitable RNA payloads are, for example, in Paunovska et al. (2022) (especially...). Figure 1 The information is disclosed in [the literature]. In several embodiments, the loading material may be small interfering RNA (siRNA), antisense oligonucleotide, adenosine deaminase (ADAR) oligonucleotide acting on RNA, or mRNA.

[0092] RNA can be chemically modified, for example, to improve its chemical stability. For example, it can contain nucleoside analogs (such as analogs of chemically modified bases or sugars) and backbone modifications. In some embodiments, the RNA may comprise nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazoadenosine, 7-deazoguanosine, 8-oxoadenosine, 8-oxoguanosine, O(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., thiophosphates and 5'-N-phosphoramide bonds). Further modifications are well known to those skilled in the art. Appropriate modifications are disclosed, for example, in WO 2017 / 099823 A1 and WO 2020 / 061284 A1 (each incorporated in its entirety by reference).

[0093] In the course of this invention, it was discovered that GDGT lipids improve the properties of LNPs, particularly those related to mRNA payloads. According to a particularly preferred embodiment, the nucleic acid load of the LNP therefore comprises mRNA. The mRNA may be, for example, a therapeutic mRNA or an mRNA encoding a vaccine antigen. The mRNA may be codon-optimized. Examples of suitable mRNAs are given, for example, in WO 2017 / 099823 A1 and WO2020 / 061284 A1 (each incorporated in its entirety by reference), particularly in paragraphs

[00178] -

[00180] of the latter.

[0094] According to another preferred embodiment, the LNP comprises at least one additional (non-ionizable or neutral) ether lipid, preferably a diether lipid (particularly guilliol). Specifically, the diether lipid may comprise any diether lipid disclosed in Kaur et al. (2016), WO2017 / 212197 A1, and WO 2020 / 187526 A1 (all of which are incorporated herein by reference in their entirety). The diether lipid (particularly diphytane glycerol diether) may be substituted (e.g., substituted with a hexose moiety or a phosphatidylinositol moiety) or unsubstituted. A particularly preferred substituted diphytane glycerol diether is phosphatidylinositol-diphytane glycerol diether (PI-Arc).

[0095] According to another preferred embodiment, the LNP comprises an ether lipid fraction containing at least one (non-ionizable or neutral) GDGT lipid (particularly diphyrane diglyceride) and preferably at least one additional ether lipid (particularly diether lipid as described above).

[0096] Particularly preferred are ionizable lipid fractions (or ether lipid fractions) comprising ether lipids, which can be obtained by extraction from archaea cultures, preferably sulfur-bearing fungi cultures, more preferably acidophilic thermosulfur-bearing fungi cultures, followed by substitution with an ionizable head group S. Suitable growth conditions and extraction methods are disclosed, for example, in WO 2020 / 187526 A1 (incorporated herein by reference in its entirety), which is also disclosed as EP 3 708 151 A1. In particular, the entire ether lipid fraction can be obtained by extraction from said cultures. Suitable substituents are disclosed, for example, in the examples.

[0097] Alternatively or otherwise, it is particularly preferred that the neutral ether lipid fraction comprises ether lipids, which can be obtained by extraction from archaea cultures, preferably sulfur-bearing leaf cultures, and more preferably acidophilic thermosulfur-bearing leaf cultures. Suitable growth conditions and extraction methods are disclosed, for example, in WO 2020 / 187526 A1 (incorporated herein by reference in its entirety). In particular, the entire ether lipid fraction can be obtained by extraction from said cultures.

[0098] In various embodiments, the archaeological culture may also be, for example, a culture of the following: *S. acidocaldarius*, *M. hungatei*, *M. voltae*, *M. concilii*, *M. smithii*, *M. espanolae*, *T. acidophilum*, *M. mazei*, *M. espanole*, *T. acidophilum*, *H. salinarum*, *M. smithii*, *M. stadtmanae*, *H. halobium*, *H. morrhuae*, *M. jannaschii*, *S. Icelandic sulfurella*. islandicus, S. solfataricus, S. shibatae, S. tokodaii, S. metallicus, S. sedula, H. hispanica, or H. volcanii, or mixtures thereof (co-cultures).

[0099] For example, total archaeal lipids can be extracted from, for example, lyophilized or spray-dried biomass using an organic solvent extraction method of chloroform / methanol / water. Polar and neutral lipids can then be separated by precipitation using acetone. The resulting lipid extract can be used directly to prepare ether lipid fractions for LNP production, or can be further purified by chromatography to separate specific classes of ether lipids for LNP production. They can also be ionized into GDGT lipids by substituting head groups, such as those disclosed herein, particularly in the examples.

[0100] Methods for preparing lipids from archaea or culturing archaea are also disclosed, for example, in US 2017 / 0152533 A1, US 6,316,260 B1, EP 1 999 137 B1, EP 0 883 624 B1, EP 2 109 459 B1, Siliakus et al. (2017), WO2020 / 187526 A1, Jain et al. (2014), each of which is incorporated herein by reference in its entirety.

[0101] LNPs themselves can be produced by microfluidic mixing of LNP components (fractions) including GDGT lipids (e.g., present in a separated form or as ether lipid fractions comprising several ether lipids). Suitable methods for producing LNPs are disclosed, for example, in WO 2017 / 099823 A1, WO 2020 / 061284 A1 and WO 2021 / 123332 A1; each of which is incorporated herein by reference in its entirety. Other methods for preparing LNPs will be available to those skilled in the art.

[0102] According to another preferred embodiment, the LNP further comprises a sterol lipid fraction. Incorporation of sterol lipids into the LNP reduces the aggregation of other lipids in the LNP. The sterol lipids are preferably selected from the group consisting of: cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, rapeseed sterol, phytosterols, and mixtures thereof. According to a preferred definition, "sterol lipids" includes the entire subgroup of steroids composed of steroid alcohols.

[0103] In particular, the sterol lipid fraction includes cholesterol.

[0104] According to yet another preferred embodiment, the LNP further comprises an auxiliary lipid fraction. The auxiliary lipids that can be used in this invention comprise non-ionizable lipids (preferably composed of them). In particular, the auxiliary lipids may be phospholipids.

[0105] Preferably, the adjuvant lipid is selected from the group consisting of: distearylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DOPC), phosphatidylcholine (PC), and mixtures thereof. Other suitable auxiliary lipids include, for example: 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-diundecanoyl-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0Diether PC), 1-oleoyl-2-cholestanoyl-succinoyl-sn-glycerol-3-phosphate choline (OchemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docosahexaenoicoyl-sn-glycerol-3-phosphate choline, 1,2-diphytoyl-sn-glycerol-3-phosphate ethanolamine (ME 16.0 PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenoicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin and mixtures thereof.

[0106] Other suitable auxiliary lipids and sterol lipids are disclosed, for example, in WO 2017 / 099823 A1 and WO 2020 / 061284A1, which are incorporated herein by reference.

[0107] Certain (molar) ratios in LNP compositions have been shown to be particularly suitable for achieving improved LNPs, especially in terms of storage stability and / or conversion efficiency:

[0108] Therefore, it is preferred that the ether lipid fraction (as defined above) accounts for 1 mol% to 20 mol% of the total lipids of LNP, more preferably 2 mol% to 15 mol% of the total lipids, even more preferably 4 mol% to 12 mol% of the total lipids, particularly 6 mol% to 10 mol% of the total lipids, or even 7 mol% to 9 mol% of the total lipids.

[0109] Alternatively or otherwise, it is preferred that the molar ratio of (neutral) ether lipid fraction to auxiliary lipid fraction is 20:1 to 1:20, preferably 15:1 to 1:10, more preferably 12:1 to 1:3, even more preferably 8:1 to 4:1, and especially 7:1 to 5:1.

[0110] In addition, alternatively or otherwise, it is preferred that the molar ratio of (neutral) ether lipid fraction to sterol lipid fraction is 0.25:1 to 1:30, preferably 0.5:1 to 1:20, more preferably 1:1 to 1:10, even more preferably 1:2 to 1:6, and especially 1:3 to 1:5.

[0111] According to a particular preferred embodiment, at least 50 mol% of the ionizable lipid fraction of LNP consists of ionizable GDGT lipids, preferably at least 60 mol%, more preferably at least 70 mol%, or even at least 80 mol%, particularly at least 90 mol%.

[0112] Particularly good results were obtained using the following LNPs, which thus form another preferred embodiment of the invention (total lipids of the LNPs, i.e., 100 mol%):

[0113] (a) 40 mol% to 70 mol% of ionizable lipid fractions (which contain ionizable GDGT lipids, wherein in particular at least 50 mol% of the fraction consists of ionizable GDGT lipids, preferably at least 60 mol%, more preferably at least 70 mol%, or even at least 80 mol%, particularly at least 90 mol%).

[0114] (b) 5 mol% to 20 mol% auxiliary lipid fractions,

[0115] (c) Sterol lipid fractions ranging from 20 mol% to 40 mol%,

[0116] (d) 0.1 mol% to 4 mol% stabilizer fraction, and preferred

[0117] (e) 1 mol% to 20 mol% of neutral ether lipid fractions (especially 2 mol% to 15 mol%, even more preferably 4 mol% to 12 mol%, particularly 6 mol% to 10 mol%, or even 7 mol% to 9 mol%).

[0118] As will be apparent to those skilled in the art upon reading this application, the ratios and mol% given above can be considered as averages of the entire population of LNPs (e.g., all LNPs present in the pharmaceutical composition).

[0119] The pharmaceutical compositions of the present invention (which comprise a variety of the LNPs of the present invention) preferably contain at least one excipient. Excipients suitable for use in the pharmaceutical compositions of the present invention are known to those skilled in the art upon reading this specification, such as water (particularly water for injection), saline, Ringer's solution, glucose solution, buffer solution, Hank's solution, a 5% glucose saline solution, substances that enhance isotonicity and chemical stability, buffer solutions, and preservatives. The pharmaceutical composition can be administered (as a medicine) to patients or individuals in need (i.e., those who have or are at risk of developing a disease or condition, as referred to herein) via appropriate procedures known to those skilled in the art (upon reading this specification). The preferred route of administration of the pharmaceutical composition is parenteral administration, particularly via intraperitoneal, subcutaneous, intramuscular, and / or intravenous administration. The dosage and method of administration depend on the individual patient or the individual to be treated. The pharmaceutical composition can be administered at any suitable dose known from other biological dosing regimens or at a dose specifically evaluated and optimized for a given individual. For example, the nucleic acid loading may be present in the pharmaceutical composition in amounts from 0.1 µg to 1000 µg, preferably from 5 µg to 200 µg, and particularly from 10 µg to 100 µg. The usual dosage may also be determined based on the patient's body weight (kg), for example, a preferred dosage in the range of 0.01 µg / kg body weight to 100 µg / kg body weight, particularly 0.1 µg / kg body weight to 10 µg / kg body weight (per administration cycle). Administration may be, for example, once daily, every other day, once weekly, or once every two weeks. Alternatively, administration may be, for example, once or multiple times (e.g., weekly, monthly, or annually). Since the preferred method of administration for the pharmaceutical compositions of the present invention is parenteral administration, the pharmaceutical compositions according to the invention are preferably liquid or prepared to be dissolved in a liquid (e.g., sterile, deionized water, distilled water, or sterile isotonic phosphate-buffered saline (PBS)). Preferably, 1000 µg (dry weight) of such a composition comprises 0.1 to 990 µg, preferably 1 to 900 µg, more preferably 10 to 200 µg of a compound, and optionally 1 to 500 µg, preferably 1 to 100 µg, more preferably 5 to 15 µg (buffer) salt (preferably producing isotonic buffer in the final volume), and optionally 0.1 to 999.9 µg, preferably 100 to 999.9 µg, more preferably 200 to 999 µg of other excipients. Preferably, 100 mg of such a dried composition is dissolved in sterile deionized / distilled water or sterile isotonic phosphate-buffered saline (PBS) to produce a final volume of 0.1 to 100 mL, preferably 0.5 to 20 mL, more preferably 1 to 10 mL.

[0120] According to a particularly preferred method, the z-mean diameter of the LNP of the present invention, determined by DLS, particularly according to ISO 22412-2017, is between 10 nm and 900 nm, preferably between 20 nm and 750 nm, more preferably between 30 nm and 500 nm, particularly between 40 nm and 250 nm, or even between 50 nm and 150 nm. The z-mean diameter defined in ISO 22412-2017 is determined by the cumulant method, producing a harmonic mean particle size weighted by the intensity of scattered light. For example, Markova et al. (2022) disclose in detail how to measure the z-mean diameter of the LNP.

[0121] It will be apparent to those skilled in the art, upon reading the above disclosure, that the GDGT lipids of the present invention can also be provided as pharmaceutically acceptable salts. Therefore, the scope of the present invention should also encompass all pharmaceutically acceptable salts of the ionizable GDGT lipids of the present invention. In particular, the ionizable GDGT lipids can be provided in cationic form (especially as ammonium cations), for example as hydrochloride, sodium bisulfate, potassium bisulfate, monosodium phosphate, dipotassium phosphate, citrate, or acetate.

[0122] The present invention also relates to the following embodiments:

[0123] Implementation Method 1. Lipid nanoparticles (LNPs) encapsulating nucleic acid loads, wherein the LNPs comprise at least:

[0124] - Ionizable lipid fractions; and

[0125] - Stabilizer fraction;

[0126] The ionizable lipid fraction contains at least one ionizable dialkyl glycerol tetraether (GDGT) lipid.

[0127] Implementation Method 2. The LNP according to Implementation Method 1, wherein the nucleic acid load comprises messenger ribonucleic acid (mRNA).

[0128] Implementation Method 3. The LNP according to Implementation Method 1 or 2, wherein the stabilizer fraction comprises at least one polyethylene glycol (PEG) lipid.

[0129] Embodiment 4. The LNP according to any one of Embodiments 1 to 3, wherein the LNP further comprises a sterol lipid fraction, preferably cholesterol.

[0130] Embodiment 5. The LNP according to any one of Embodiments 1 to 4, wherein the LNP further comprises an auxiliary lipid fraction.

[0131] Embodiment 6. The LNP according to 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:

[0132] and

[0133] Each occurrence of R a The components are independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkylamine, alkyl ether, alkyl thiol, alkyl ester, alkylamide, alkyl carbamate, alkyl sulfonate, and alkyl sulfonamide, preferably selected from alkyl, cycloalkyl, alkenyl, hydroxyalkyl, and alkylamine.

[0134] Each occurrence of R b The components are independently selected from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl ester, alkylamide, alkyl carbamate, alkyl sulfonate, alkyl sulfonamide, and alkyl thiol, preferably selected from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, and alkyl thiol, and...

[0135] Each occurrence of R c Independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkylamide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide, preferably selected from alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkylamide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide.

[0136] Embodiment 7. An LNP according to any one of Embodiments 1 to 6, wherein the LNP comprises at least one additional ether lipid, preferably a tetraether lipid (preferably GDGT) or a diether lipid (especially diphytane glycerol diether).

[0137] Embodiment 8. An LNP according to any one of Embodiments 1 to 7, wherein the LNP comprises an ether lipid fraction comprising at least one additional GDGT lipid (preferably at least two different GDGT lipids, more preferably at least three different GDGT lipids, particularly at least four different GDGD lipids) and preferably at least one additional ether lipid; particularly comprising diphyrane diglyceride and diphyrane diglyceride, preferably having the composition given in Table 1 above.

[0138] Embodiment 9. The LNP according to Embodiment 8, wherein the ether lipid fraction comprises ether lipids that can be obtained by extraction from archaea cultures, preferably sulfur leaf cultures, more preferably acidophilic thermosulfur leaf cultures.

[0139] Implementation Method 10. The LNP according to Implementation Method 9, wherein all ether lipid fractions can be obtained by extraction from the culture.

[0140] Implementation 11. The LNP according to any one of Implementation 1 to 10, wherein the ionizable lipid fraction accounts for 40 mol% to 70 mol% of the total lipids.

[0141] Implementation 12. The LNP according to any one of Implementations 1 to 11, wherein the molar ratio of the neutral ether lipid fraction to the auxiliary lipid fraction (especially with DSPC, if present) is 20:1 to 1:20, preferably 15:1 to 1:10, more preferably 12:1 to 1:3, even more preferably 8:1 to 4:1, especially 7:1 to 5:1.

[0142] Embodiment 13. The LNP according to any one of Embodiments 1 to 12, wherein the molar ratio of the neutral ether lipid fraction to the sterol lipid fraction (especially with cholesterol, if present) is 0.25:1 to 1:30, preferably 0.5:1 to 1:20, more preferably 1:1 to 1:10, even more preferably 1:2 to 1:6, especially 1:3 to 1:5.

[0143] Embodiment 14. The LNP according to any one of Embodiments 1 to 13, wherein the total lipids of the LNP comprise (preferably composed of):

[0144] (a) 40 mol% to 70 mol% ionizable lipid fractions,

[0145] (b) 5 mol% to 20 mol% auxiliary lipid fractions,

[0146] (c) Sterol lipid fractions ranging from 20 mol% to 40 mol%,

[0147] (d) 0.1 mol% to 4 mol% stabilizer fraction, and / or

[0148] (e) 1 mol% to 20 mol% neutral ether lipid fraction.

[0149] Example 15. The LNP according to any one of Examples 1 to 14, wherein the ionizable lipid fraction comprises at least one additional ionizable lipid selected from the group consisting of: [(4-hydroxybutyl)azanidinediyl]bis(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-(bisdodecylamino)-N1,N1,4-tridodecyl-1-piperazine ethylamine (KL10), N1-[2-(bisdodecylamino)ethyl]N1,N4,N4-tridodecyl-1,4-piperazine diethylamine (KL22) , 14,25-ditridecyl-15,18,21,24-tetraaza-octacosane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptanetriane-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8 [(3β)-Cholester-5-en-3-yloxy]octyl}oxy)N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-Cholester-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-1-amine (octyl-CLinDMA(2R)), (2S)2-({8-[(3β)-Cholester-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-1-amine (octyl-CLinDMA(2R)), (2S)2-({8-[(3β)-Cholester-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-1-amine [-1-yloxy]propane-1-amine (octyl-CLinDMA(2S)) and mixtures thereof; and / or said ionizable lipid is selected from the group consisting of: (20Z,23Z)-N,N-dimethylnonadecano-20,23-dien-10-amine, (17Z,20Z)-N,N-dimethylhexadecano-17,20-dien-9-amine, (1Z,19Z)-N5N-dimethylpentadecano-16,19-dien-8-amine, (13Z,16Z)-N,N-dimethylhexadecano-13,16-dien-5-amine, (12Z,15Z)-N,N-dimethyltetradecano-12,15-dien-4-amine, (14Z,17Z)-N,N-Dimethyltriscar-14,17-dien-6-amine, (15Z,18Z)-N,N-dimethyltetracar-15,18-dien-7-amine, (18Z,21Z)-N,N-dimethylheptacar-18,21-dien-10-amine, (15Z,18Z)-N,N-dimethyltetracar-15,18-dien-5-amine, (14Z,17Z)-N,N-dimethyltriscar-14,17-dien-4-amine, (19Z,22Z)-N,N-dimethyloctacar-19,22-dien-9-amine, (18Z,21Z)-N,N-dimethylheptacar-18,21-dien-8-amine, (17Z,20Z)-N N-Dimethylhexadecane-17,20-diene-7-amine, (16Z,19Z)-N,N-dimethylpentadecane-16,19-diene-6-amine, (22Z,25Z)-N,N-dimethyltridecane-22,25-diene-10-amine, (21Z,24Z)-N,N-dimethyltridecane-21,24-diene-9-amine, (18Z)-N,N-dimethylhexadecane-18-en-10-amine, (17Z)-N,N-dimethylhexadecane-17-en-9-amine, (19Z,22Z)-N,N-dimethyloctadecane-19,22-diene-7-amine, N,N-dimethylhexadecane-10-amine, (20Z,23Z)- N-Ethyl-N-methyl-Nicocarb-20,23-dien-10-amine, 1-[(11Z,14Z)-1-nonyleicocarb-11,14-dien-1-yl]pyrrolidine, (20Z)-N,N-dimethyl-Heptacarb-20-en-10-amine, (15Z)-N,N-dimethyl-Heptacarb-15-en-10-amine, (14Z)-N,N-dimethyl-Nicocarb-14-en-10-amine, (17Z)-N,N-dimethyl-Nicocarb-17-en-10-amine, (24Z)-N,N-dimethyl-Thiaricarb-24-en-10-amine, (20Z)-N,N-dimethyl-Nicocarb-20-en-10-amine, (22Z)-N,N-dimethyl-N-dimethyl-Nicocarb-20-en-10-amine 2,2-en-10-amine, (16Z)-N,N-dimethylpentacarbon-16-en-8-amine, (12Z,15Z)-N,N-dimethyl-2-nonyl-2-nonyl-12,15-dien-1-amine, (13Z,16Z)-N,N-dimethyl-3-nonyl-2-nonyl-13,16-dien-1-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecane-8-amine, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecane-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecane-10-amine, N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]nonadecane-10-amine, N,N-dimethyl-21-[(1S,[2R)-2-octylcyclopropyl]eicosane-10-amine, N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecane-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecane-8-amine, N,N-dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradecane-5-amine, N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecane-1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecane-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-di Methylpentadecane-6-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecane-8-amine, RN,N-dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-(octoxy)propane-2-amine, SN,N-dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-(octoxy)propane-2-amine, 1-{2-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-1-[(octoxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]- 3-[(5Z)-oct-5-en-1-yloxy]propane-2-amine, 1-{2-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-1-[(octoxy)methyl]ethyl}azacyclobutane, (2S)-1-(hexoxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-2-amine, (2S)-1-(heptoxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-2-amine, N,N-dimethyl-1-(nonoxy)-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-2-amine N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octoxy)propane-2-amine, (2S)-N,N-dimethyl-1-[(6Z,9Z,12Z)-octadec-6,9,12-trien-1-yloxy]-3-(octoxy)propane-2-amine, (2S)-1-[(11Z,14Z)-eicos-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentoxy)propane-2-amine, (2S)-1-(hexoxy)-3-[(11Z,14Z)-eicos-11,14-dien-1-yloxy]-N,N-dimethylpropane-2-amine, 1-[(11Z,14Z)-eicos-11,[14-dien-1-yloxy]-N,N-dimethyl-3-(octoxy)propane-2-amine, 1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octoxy)propane-2-amine, (2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropane-2-amine, (2S)-1-[(13Z)-docosa-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropane-2-amine, 1-[(13Z)-docosa-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propane-2-amine, 1-[(9Z)-hexadecane-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propane-2-amine, (2R)-N [N-dimethyl-H(1-formyloctyl)oxy]-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-2-amine, N,N-dimethyl-1-(octyloxy)-3-({8-[(1S, 2S)-2-[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propane-2-amine, N,N-dimethyl-1-{[8-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)propane-2-amine and (11E,20Z,23Z)-N,N-dimethyl-necodeca-11,20,2-trien-10-amine, and pharmaceutically acceptable salts and stereoisomers thereof, and mixtures thereof.

[0150] Example 16. The LNP according to any one of Examples 1 to 15, wherein the auxiliary lipid fraction comprises auxiliary lipids selected from the group consisting of: distearylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DOPC), phosphatidylcholine (PC), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-diundecanoyl-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 DietherPC), 1-oleoyl-2-cholestyl hemisuccinyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 LysoPC), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docosahexaenoicoyl-sn-glycerol-3-phosphate choline), 1,2-diphynoyl-sn-glycerol-3-phosphate ethanolamine (ME 16.0) PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin and mixtures thereof.

[0151] Embodiment 17. The LNP according to any one of Embodiments 1 to 16, wherein the z-average diameter of the LNP is between 10 nm and 900 nm, preferably between 20 nm and 750 nm, more preferably between 30 nm and 500 nm, particularly between 40 nm and 250 nm, or even between 50 nm and 150 nm (measured by DLS, particularly according to ISO 22412:2017).

[0152] Implementation Method 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:

[0153] and

[0154] Each occurrence of R a The components are independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkylamine, alkyl ether, alkyl thiol, alkyl ester, alkylamide, alkyl carbamate, alkyl sulfonate, and alkyl sulfonamide, preferably selected from alkyl, cycloalkyl, alkenyl, hydroxyalkyl, and alkylamine.

[0155] Each occurrence of R b The components are independently selected from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl ester, alkylamide, alkyl carbamate, alkyl sulfonate, alkyl sulfonamide, and alkyl thiol, preferably selected from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, and alkyl thiol, and...

[0156] Each occurrence of R c Independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkylamide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide, preferably selected from alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkylamide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide.

[0157] Implementation Method 19. The GDGT lipid according to Implementation Method 18, wherein S is selected from the group consisting of:

[0158] and

[0159] Each occurrence of R a Independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl, and alkylamine, and each occurrence of R b It is independently selected from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine and alkyl thiol.

[0160] Example 20. GDGT lipid according to any one of Examples 18 or 19, wherein the alkyl group is selected from methyl, ethyl, propyl, preferably isopropyl, butyl, preferably isobutyl, pentyl, preferably isopentyl, hexyl, preferably isohexyl and heptyl, preferably isohexyl.

[0161] Example 21. The GDGT lipid according to any one of Examples 18 to 20, wherein the cycloalkyl group is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

[0162] Example 22. The GDGT lipid according to any one of Examples 18 to 21, wherein the alkenyl group is selected from propenyl, butenyl, butenyl, pentenyl, pentenyl, pentenyl, pentenyl, hexenyl, hexenyl, hexenyl, hexenyl, hexenyl, hexenyl, heptenyl, heptenyl, heptenyl, heptenyl, heptenyl, heptenyl, heptenyl, heptenyl, heptenyl, heptenyl, heptenyl, heptenyl, heptenyl, and heptenyl, heptenyl, heptenyl, heptenyl, heptenyl, and heptenyl, heptenyl, heptenyl, and heptenyl, heptenyl, heptenyl, and heptenyl, heptenyl.

[0163] Example 23. The GDGT lipid according to any one of Examples 18 to 22, wherein the hydroxyalkyl group is selected from hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, hydroxypentyl and hydroxyhexyl.

[0164] Example 24. The GDGT lipid according to any one of Examples 18 to 23, wherein the alkylamine is selected from methylamine, ethylamine, propylamine, butylamine, pentamine, hexamine and heptamine.

[0165] Example 25. The GDGT lipid according to any one of Examples 18 to 24, wherein the alkyl ether is selected from methyl ether, diethyl ether, propyl ether, butyl ether, pentyl ether, hexyl ether and heptyl ether.

[0166] Embodiment 26. The GDGT lipid according to any one of Embodiments 18 to 25, wherein the alkyl thiol is selected from methanethiol, ethanethiol, propanethiol, butanethiol, pentathiol, hexanethiol, and heptanethiol.

[0167] Example 27. The GDGT lipid according to any one of Examples 18 to 26, wherein the alkyl ester is selected from methyl ester, ethyl ester, propyl ester, butyl ester, pentyl ester, hexyl ester and heptyl ester.

[0168] Example 28. The GDGT lipid according to any one of Examples 18 to 27, wherein the alkylamide is selected from formamide, acetamide, propionamide, butyramide, valerate, hexamamide and heptanamide.

[0169] Example 29. The GDGT lipid according to any one of Examples 18 to 28, wherein the alkyl carbamate is selected from methyl carbamate, ethyl carbamate, propyl carbamate, butyl carbamate, pentyl carbamate, hexyl carbamate and heptyl carbamate.

[0170] Example 30. The GDGT lipid according to any one of Examples 18 to 29, wherein the alkyl sulfonate is selected from methyl sulfonate, ethyl sulfonate, propyl sulfonate, butyl sulfonate, pentyl sulfonate, hexyl sulfonate and heptyl sulfonate.

[0171] Example 31. The GDGT lipid according to any one of Examples 18 to 30, wherein the alkyl sulfonamide is selected from methyl sulfonamide, ethyl sulfonamide, propyl sulfonamide, butyl sulfonamide, pentyl sulfonamide, hexyl sulfonamide and heptayl sulfonamide.

[0172] Embodiment 32. The GDGT lipid according to any one of Embodiments 18 to 31, wherein the head group S is selected from:

[0173] and

[0174] Each R is independently selected from H and alkyl; preferably, the alkyl group is selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, isopentyl, hexyl, isohexyl, heptyl, and isohexyl.

[0175] Embodiment 33. The GDGT lipid according to any one of Embodiments 18 to 32, wherein the GDGT lipid comprises two ionizable head groups S.

[0176] Embodiment 34. The GDGT lipid according to any one of Embodiments 18 to 33, wherein the GDGT lipid is selected from:

[0177]

[0178]

[0179]

[0180]

[0181] Where S 1 It is S and S 2 It is OH or S 1 It is OH and S 2 Is it S, or S 1 and S 2 Each of them is S independently.

[0182] Embodiment 35. GDGT lipids according to Embodiment 34, wherein S 1 and S 2 Each of them is the same S.

[0183] Embodiment 36. A GDGT lipid according to any one of the foregoing embodiments, wherein the GDGT lipid carries an overall positive charge at an endosomal pH (particularly at a pH between 5.0 and 6.5, e.g., pH 5.0, 5.5, 6, or 6.5); preferably, wherein the GDGT lipid carries an overall neutral charge at a pH higher than the endosomal pH (e.g., pH 7.0 or pH 7.4).

[0184] Example 37. The GDGT lipid according to any one of Examples 18 to 36, wherein the ionizable head group S carries an overall positive charge at the endosome pH (particularly at pH between 5.0 and 6.5, e.g., 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 the endosome pH (e.g., pH 7.0 or pH 7.4).

[0185] Embodiment 38. A GDGT lipid according to any one of Embodiments 18 to 37, wherein the GDGT lipid is a bis(phytane) diglyceride having at least one ionizable head group S.

[0186] Embodiment 39. GDGT lipids according to any one of embodiments 18 to 38, wherein the GDGT lipids are selected from... Figures 1 to 1 Any of the GDGT lipids of 0 and its stereoisomers and variants, having a GDGT-0, GDGT-1, GDGT-2, GDGT-3, GDGT-5, GDGT-6, GDGT-7 or GDGT-8 backbone instead of a GDGT-4 backbone.

[0187] Example 40. An ether lipid fraction comprising GDGT lipids according to any one of Examples 18 to 39; said ether lipid fraction is preferably obtainable by extraction from archaea cultures, preferably sulfur-bearing leaf cultures, more preferably acidophilic thermosulfur-bearing leaf cultures, followed by substitution with an ionizable head group S.

[0188] Embodiment 41. An LNP comprising GDGT lipids according to any one of Embodiments 18 to 39 or an ether lipid fraction according to Embodiment 40, preferably wherein the LNP is as defined in Embodiments 1 to 17.

[0189] Embodiment 42. A pharmaceutical composition comprising an LNP according to any one of Embodiments 1 to 17 and 41 and preferably at least one excipient.

[0190] Embodiment 43. The pharmaceutical composition according to Embodiment 42, wherein the pharmaceutical composition is a vaccine, preferably an mRNA vaccine.

[0191] Implementation 44. Use of the pharmaceutical composition according to Implementation 42 or 43 for the prevention or treatment of a patient’s disease or condition.

[0192] Embodiment 45. A method for producing GDGT lipids according to any one of Embodiments 18 to 39, comprising the following steps:

[0193] - Obtain lipid fractions containing one or more precursor GDGT lipids from archaea cultures, preferably sulfur leaf culture, and more preferably acidophilic thermosulfur leaf culture;

[0194] - Purify one or more precursor GDGT lipids; and

[0195] - Contact one or more precursor GDGT lipids with one or more reagents to produce GDGT lipids having at least one ionizable head group S. Attached Figure Description

[0196] The present invention is further illustrated by the following figures and embodiments, but is not limited thereto.

[0197] Figures 1 to 1 0 indicates a particularly preferred ionizable GDGT lipid. Parentheses indicate that the length of the segment within the parentheses can be 0 to 6 carbon atoms (n=0-6). Each instance of “R1” or “R2” can be independently selected from the definitions given below for the structure. A tilde indicates that both R and S enantiomers are preferred. Detailed Implementation

[0198] Example

[0199] Example 1 - Purification of precursor GDGT lipids

[0200] Biomass Extraction: 240 g of dried *Mycorrhiza pyrifolia* biomass was mixed with 360 g of diatomaceous earth (Dionex ASEPrep DE), and the resulting 600 g of solid was dispersed uniformly by shaking. Approximately equal volumes of dispersed solid (30-35 g each) were placed in 18 extraction cells (100 mL Dionex ASE 350 stainless steel) equipped with cellulose filters and placed in a Dionex ASE 350 accelerated solvent extractor. Each cell was extracted for 30 minutes at 80 °C and 1150 psi with a 3 L CHCl3:MeOH mixture (2:1). The extracted liquid fractions were combined and the mixture was concentrated (Heidolph Hei-VapCore, 50 °C, 100 mbar) to 200 mL. 50 mL of 2N HCl aqueous solution was added, and the heterogeneous two-phase mixture was heated to 65 °C for 24 hours. After cooling to room temperature, the mixture was neutralized with 50 mL of NaHCO3 to separate the phases. The aqueous phase was extracted with Et2O (3 × 90 mL). The combined organic extracts were then dried with Na2SO4, filtered, and the solvent was evaporated to obtain 4.4 g (1.8% w / w) of crude extract as a brown solid.

[0201] Separation of GDGT and major polar lipids from *Sulphurella sulfadiazine*: A brown solid was dissolved in 40 mL of dichloromethane (DCM) and subjected to column chromatography in two fractions (using a Büchi pure chromatography system equipped with a light scattering detector and an automated fraction collector). Column chromatography was performed by injecting 20 mL of the crude material liquid into a FlashPure EcoFlex 25 g SiO2 column (flow rate 32 mL / min) pre-equilibrated with light gasoline (LP). The yield was 193.4 mg (4.3%) of GDGT containing trace amounts of aromatic lipid impurities, as a brown oil. In the same chromatogram, 2.38 g (54%) of the major lipids from *Sulphurella sulfadiazine* were also separated.

[0202] Final purification of GDGT: 106.5 mg of the stated GDGT was dissolved in 1 ml of LP and separated on a 20 g Büchi FlashPure Ecoflex column using LP:DCM (DCM = 20% to 40%). All fractions that did not show a UV signal were collected and evaporated to give 92.8 mg of pure GDGT (87%) as a colorless oil.

[0203] These GDGT lipids are used as precursor GDGT lipids in the following examples to produce GDGT lipids with ionizable head groups.

[0204] Example 2 - Preparation of bis-(4-bromobutyric acid)-GDGT ester

[0205] In an 8 mL vial equipped with a diaphragm and stir bar, dimethylaminopyridine (DMAP) (5.4 mg, 44 µmol, 0.4 equivalents) was added to GDGT (142.7 mg, 110 µmol, 1 equivalent) obtained according to Example 1. The vial was evacuated and backfilled with N2 (three times), and then 2.2 mL of dry dichloromethane was added through a sleeve. Subsequently, the solution was cooled to 0 °C by an ice / water bath, and then 4-bromobutyrate chloride (45 µL, 240 µmol, 2.4 equivalents) was added dropwise. The mixture was stirred at 0 °C for 5 minutes, then warmed to room temperature and stirred for 18 hours. After the starting material was completely consumed (as verified by thin-layer chromatography), the mixture was quenched with 20 mL of saturated NaHCO3, extracted with Et2O (3 × 50 mL), washed once with brine, dried on Na2SO4, and evaporated to dryness. After column chromatography (12g SiO2), 126.5 mg (72%) of bis-(4-bromobutyric acid)-GDGT ester was separated as a white solid.

[0206] The product is a mixture of several different GDGT frameworks. Representative structures (with a GDGT-4 framework) are shown below:

[0207] .

[0208] Example 3: Preparation of bis-(4-((2-hydroxyethyl)(methyl)amino)butyric acid)-GDGT ester

[0209] In an 8 mL vial equipped with a diaphragm and a stir bar, K₂CO₃ (32.9 mg, 238 µmol, 3 equivalents) and KI (5.3 mg, 31 µmol, 0.4 equivalents) were added to bis-(4-bromobutyric acid)-GDGT ester (126.5 mg, 79 μmol, 1 equivalent) obtained according to Example 2. The vial was evacuated and backfilled with N₂ (three times), and then 2 mL of HPLC-grade MeCN was added through a sleeve, followed by N-methylaminoethanol (20 μL, 238 μmol, 3 equivalents). The turbid mixture was heated to 75 °C for 16 hours (to be verified by thin-layer chromatography to indicate complete consumption of the starting material), cooled to room temperature, and the MeCN was evaporated. The solid was dissolved in 20 mL of NaHCO₃ and extracted with 20 mL of dichloromethane (three times), washed once with brine, filtered, and evaporated to give 91.8 mg of crude yellow oil. The crude material was purified by rapid chromatography (Biotage column, 5g) to obtain 65.8 mg (52%) of bis-(4-((2-hydroxyethyl)(methyl)amino)butyric acid)-GDGT ester, which was a colorless oil.

[0210] The product is a mixture of several different substituted GDGT backbones. Representative structures (with a GDGT-4 backbone) are shown below:

[0211] .

[0212] Example 4 - Preparation of bis-(4-(diethanolamine)butyric acid)-GDGT ester

[0213] In an 8 mL vial equipped with a diaphragm and a stir bar, K₂CO₃ (3 equivalents) and KI (0.4 equivalents) were added to bis-(4-bromobutyric acid)-GDGT (1 equivalent) obtained according to Example 2. The vial was evacuated and backfilled with N₂ (three times), and then 2 mL of HPLC-grade MeCN was added through a sleeve, followed by diethanolamine (3 equivalents). The turbid mixture was heated to 75°C for 16 hours, then cooled to room temperature, and the MeCN was evaporated. The solid was dissolved in 20 mL of NaHCO₃ and extracted with 20 mL of dichloromethane (three times), washed once with brine, filtered, and evaporated. The crude material was purified by rapid chromatography to separate bis-(4-(bis-(2-hydroxyethyl)amino)butyric acid)-GDGT ester.

[0214] The products are typically mixtures of several different substituted GDGT backbones. Representative structures (with a GDGT-4 backbone) are shown below:

[0215] .

[0216] Example 5: Preparation of bis-(S)-(4-2-(hydroxymethyl)pyrrolidone-1-yl)butyric acid)-GDGT ester

[0217] In an 8 mL vial equipped with a diaphragm and a stir bar, K₂CO₃ (3 equivalents) and KI (0.4 equivalents) were added to bis-(4-bromobutyric acid)-GDGT ester (1 equivalent) obtained according to Example 2. The vial was evacuated and backfilled with N₂ (three times), and then 2 mL of HPLC-grade MeCN was added through a sleeve, followed by (S)-prolyl. The turbid mixture was heated to 75 °C for 16 hours, then cooled to room temperature, and the MeCN was evaporated. The solid was dissolved in 20 mL of NaHCO₃ and extracted with 20 mL of dichloromethane (three times), washed once with brine, filtered, and evaporated. The crude material was purified by rapid chromatography to separate bis-(4-(prolyl)butyric acid)-GDGT ester.

[0218] The products are typically mixtures of several different substituted GDGT backbones. Representative structures (with a GDGT-4 backbone) are shown below:

[0219] .

[0220] Example 6 - Preparation of bis-(4-(2-(hydroxymethyl)-1H-imidazolyl)-GDGT ester

[0221] In an 8 mL vial equipped with a diaphragm and a stir bar, K₂CO₃ (3 equivalents) and KI (0.4 equivalents) were added to bis-(4-bromobutyric acid)-GDGT ester (1 equivalent) obtained according to Example 2. The vial was evacuated and backfilled with N₂ (three times), and then 2 mL of HPLC-grade MeCN was added through a sleeve, followed by 2-(hydroxymethyl)-1H-imidazolium. The turbid mixture was heated to 75°C for 16 hours, then cooled to room temperature, and the MeCN was evaporated. The solid was dissolved in 20 mL of NaHCO₃ and extracted with 20 mL of dichloromethane (three times), washed once with brine, filtered, and evaporated. The crude material was purified by rapid chromatography to obtain bis-(4-(2-(hydroxymethyl)-1H-imidazolium)-GDGT ester.

[0222] The products are typically mixtures of several different substituted GDGT backbones. Representative structures (with a GDGT-4 backbone) are shown below:

[0223] .

[0224] Example 7 - Preparation of GDGT aldehyde

[0225] Dichloromethane was added to an 8 mL vial containing GDGT (1 equivalent) obtained according to Example 1 and trichloroisocyanuric acid (TCCA) (2.2 equivalents), and the mixture was cooled to 0°C. 2,2,6,6-Tetramethylpiperidinyloxy (TEMPO) (0.2 equivalents) was added, and the mixture was stirred at 0°C for 15 minutes. The mixture was quenched with a saturated aqueous solution of NaHCO3 and extracted with Et2O (3 × 50 mL). The combined organic extracts were washed successively with 2N HCl and brine, then dried over Na2SO4, filtered, and evaporated. This yielded crude GDGT aldehyde, which could be used without further purification.

[0226] The products are typically mixtures of several different GDGT backbones. Representative structures (with a GDGT-4 backbone) are shown below:

[0227] .

[0228] Example 8 - Preparation of bis-((methyl)amino)ethanol GDGT

[0229] In an 8 mL vial under N2 atmosphere, the GDGT aldehyde (1 equivalent) obtained according to Example 7 was dissolved in tetrahydrofuran, N-methylaminoethanol (2.2 equivalents) was added, and the mixture was stirred for 10 minutes. Sodium triacetoxyborohydride (3 equivalents) was added, and the mixture was stirred at room temperature for 24 hours. The reaction mixture was quenched by adding 3N NaOH, the product was extracted with Et2O and dried over MgSO4. The cooled ether extract was treated with diethyl ether HCl, filtered, and the product was given as bis-((methyl)amino)ethanol GDGT hydrochloride.

[0230] The products are typically mixtures of several different substituted GDGT backbones. Representative structures (with a GDGT-4 backbone) are shown below:

[0231] .

[0232] Example 9 - Synthesis of Ionizable GDGT from GDGT Acrylate

[0233] Preparation of bis-GDGT-acrylate

[0234]

[0235] The preferred GDGT (1 equivalent) obtained according to Example 1 was placed in a 20 mL vial equipped with a diaphragm and a stir bar. The vial was evacuated and backfilled with N2 (three times), and then dry CHCl3 and triethylamine (2 equivalents) were added through a sleeve. Subsequently, the solution was cooled to 0°C by an ice / water bath, and then acryloyl chloride (0.1 M, 2.2 equivalents) was added dropwise to CHCl3. The mixture was stirred at 0°C for 5 minutes, then warmed to room temperature and stirred for 5 hours. After the starting material was completely consumed (as verified by TLC), the mixture was quenched with 20 mL of saturated NaHCO3, extracted with CHCl3, washed once with brine, dried on MgSO4 and evaporated to dryness. After column chromatography (12 g SiO2), 83% of the bis-acrylate-GDGT ester was separated as a white solid.

[0236] Further steps

[0237] 1 equivalent of bis-acrylic acid-GDGT ester was added to an 8 ml vial equipped with a diaphragm and a stir bar. The vial was evacuated and backfilled with N2 (three times), followed by the addition of dry CHCl3, then amine (2.2 equivalents) and a catalytic amount of acetic acid (0.1 equivalents) through a sleeve. If the amine was solid, it was added all at once. The homogeneous mixture was heated to 40 °C for 3 hours (until the starting material was completely consumed as checked by TLC), cooled to room temperature, quenched with a saturated aqueous solution of NaHCO3, and extracted with CHCl3 (three times). The mixture was washed once with brine, dried over MgSO4, filtered, and evaporated. The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain bis-(aminopropionic acid)-GDGT ester as a colorless oil.

[0238]

[0239] The crude material obtained depends on the amine used. The product is typically a mixture of several different substituted GDGT skeletons. Representative structures (with the GDGT-4 skeleton) are shown below:

[0240] bis-(3-dimethylamino)propionic acid)-GDGT ester

[0241]

[0242] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 67% bis-(4-(dimethylamino)butyric acid)-GDGT ester, which was a colorless oil.

[0243] bis-(3-diethylamino)propionic acid)-GDGT ester

[0244]

[0245] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 63% bis-(3-(diethylamino)propionic acid)-GDGT ester, which was a colorless oil.

[0246] bis-(3-(2-hydroxyethyl)(methyl)amino)propionic acid)-GDGT ester

[0247]

[0248] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 54% bis-(3-((2-hydroxyethyl)(methyl)amino)propionic acid)-GDGT ester, which was a colorless oil.

[0249] bis-3-(bis(2-hydroxyethyl)amino)propionic acid)-GDGT ester

[0250]

[0251] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 57% bis-3-(bis(2-hydroxyethyl)amino)propionic acid)-GDGT ester, which was a colorless oil.

[0252] bis-(3-pyrrolidone-1-yl)propionic acid)-GDGT ester

[0253]

[0254] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 51% bis-(3-pyrrolidine-1-yl)propionic acid-GDGT ester, which was a colorless oil.

[0255] bis-3-((R)-(hydroxymethyl)pyrrolidone-1-yl)propionic acid)-GDGT ester

[0256]

[0257] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 67% bis-(3-(2(R)-(hydroxymethyl)pyrrolidine-1-yl)propionic acid)-GDGT ester, which was a colorless oil.

[0258] bis-3-((S)-(hydroxypyrrolidone-1-yl)propionic acid)-GDGT ester

[0259]

[0260] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 62% bis-3-((S)-(hydroxypyrrolidone-1-yl)propionic acid)-GDGT ester, which was a colorless oil.

[0261] bis-(3-piperidin-1-yl)propionic acid-GDGT ester

[0262]

[0263] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 64% bis-(3-piperidin-1-yl))propionic acid-GDGT ester, which was a colorless oil.

[0264] bis-(3-(4-hydroxypiperidin-1-yl))propionic acid)-GDGT ester

[0265]

[0266] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 61% bis-(3-(4-hydroxypiperidin-1-yl))propionic acid-GDGT ester, which was a colorless oil.

[0267] Bis-(3-morpholino)-propionic acid)-GDGT ester

[0268]

[0269] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 54% bis-(3-morpholino)propionic acid-GDGT ester, which was a colorless oil.

[0270] bis-(3-(3-(hydroxymethyl)morpholino)-propionic acid)-GDGT ester

[0271]

[0272] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain a mixture of 47% diastereomers of bis-(3-(3-(hydroxymethyl)morpholino)propionic acid)-GDGT ester, which was a colorless oil.

[0273] bis-(3-(2-(hydroxymethyl)-1H-imidazolyl)propionic acid)-GDGT ester

[0274]

[0275] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 75% bis-(3-(2-(hydroxymethyl)-1H-imidazolyl)propionic acid)-GDGT ester, which was a colorless oil.

[0276] Bis-3-(piperazin-1-yl)-propionic acid) GDGT ester

[0277]

[0278] In an 8 mL vial equipped with a diaphragm and stir bar, 1-Boc-piperazine was added to bis-acrylic acid-GDGT ester. The vial was evacuated and backfilled with N2 (three times), followed by the addition of dry CHCl3, and then a catalytic amount of acetic acid (0.1 equivalent). The mixture was heated to 40 °C for 4 hours (until the starting material was completely consumed by TLC), cooled to room temperature, and a saturated aqueous solution of NaHCO3 was added. The mixture was extracted with CHCl3 (three times), washed once with brine, dried over MgSO4, filtered, and evaporated. The crude material was dissolved in 2 mL of dry DCM, and 1 mL of TFA was added dropwise. The mixture was stirred at room temperature for 2 hours, cooled to 0 °C, neutralized with a saturated aqueous solution of NaHCO3, extracted with DCM (3x), dried over MgSO4, and the solvent was evaporated. Purification by rapid chromatography (Biotage Sfär Amino column, LP:EE) yielded 42% bis-3-(piperazin-1-yl)-propionic acid-GDGT ester as a pale yellow oil.

[0279] bis-3-(4-(2-hydroxyethyl)piperazin-1-yl)propionic acid-GDGT ester

[0280]

[0281] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 59% bis-3-(4-(2-hydroxyethyl)piperazin-1-yl)-propionic acid-GDGT ester, which was a colorless oil.

[0282] Example 10 - Synthesis of ionizable GDGT from bis-(4-bromobutyric acid)-GDGT ester

[0283] General method for preparing bis-(4-bromobutyric acid)-GDGT ester

[0284]

[0285] In an 8 mL vial equipped with a diaphragm and a stir bar, DMAP (0.4 equivalents) was preferably added to GDGT (1 equivalent) obtained according to Example 1. The vial was evacuated and backfilled with N2 (three times), and then dried DCM was added through a sleeve. Subsequently, the solution was cooled to 0°C by an ice / water bath, and then 4-bromobutyryl chloride (2.4 equivalents) was added dropwise. The mixture was stirred at 0°C for 5 minutes, then warmed to room temperature and stirred for 18 hours. After the starting material was completely consumed (as verified by TLC), the mixture was quenched with saturated NaHCO3, extracted with Et2O (3x), washed once with brine, dried on Na2SO4 and evaporated to dryness. After column chromatography (SiO2, PE:EE), 72% of bis-(4-bromobutyric acid)-GDGT ester was separated as a white solid.

[0286] Further steps

[0287] In an 8 mL vial equipped with a diaphragm and stir bar, K₂CO₃ (3 equivalents) and KI (0.4 equivalents) were added to bis-(4-bromobutyric acid)-GDGT ester (1 equivalent). The vial was evacuated and backfilled with N₂ (three times), and then HPLC-grade MeCN was added through a sleeve, followed by the appropriate secondary amine (3 equivalents), or, if the amine was solid, added all at once. The turbid mixture was heated to 75 °C for 16 to 24 hours (until the starting material was completely consumed, as checked by TLC), cooled to room temperature, and the MeCN was evaporated. The solid was dissolved in NaHCO₃ and extracted with DCM (three times), washed once with brine, filtered, and evaporated. The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain bis-(4-aminobutyric acid)-GDGT ester as a colorless oil.

[0288]

[0289] The crude material obtained depends on the amine used. The product is typically a mixture of several different substituted GDGT skeletons. Representative structures (with the GDGT-4 skeleton) are shown below:

[0290] bis-(dimethylamino)butyric acid)-GDGT ester

[0291]

[0292] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 48% bis-(4-(dimethylamino)butyric acid)-GDGT ester, which was a colorless oil.

[0293] bis-(diethylamino)butyric acid)-GDGT ester

[0294]

[0295] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 63% bis-(4-(diethylamino)butyric acid)-GDGT ester, which was a colorless oil.

[0296] bis-(4-((2-hydroxyethyl)(methyl)amino)butyric acid)GDGT ester

[0297]

[0298] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 68% bis-(4-((2-hydroxyethyl)(methyl)amino)butyric acid)GDGT ester, which was a colorless oil.

[0299] bis-4-(bis(2-hydroxyethyl)amino)butyric acid)-GDGT ester

[0300]

[0301] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 61% 4-(bis(2-hydroxyethyl)amino)butyric acid)-GDGT ester, which was a colorless oil.

[0302] bis-(4-pyrrolidone-1-yl)butyric acid)-GDGT ester

[0303]

[0304] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 53% bis-pyrrolidine-1-yl)butyric acid-GDGT ester, which was a colorless oil.

[0305] 4-(2-(hydroxymethyl)pyrrolidone-1-yl)butyric acid)-GDGT ester

[0306]

[0307] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 49% bis-(4-(2(S)-(hydroxymethyl)pyrrolidine-1-yl)butyric acid)-GDGT ester, which was a colorless oil.

[0308] bis-(4-piperidin-1-yl))butyric acid)-GDGT ester

[0309]

[0310] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 71% bis-(4-piperidin-1-yl))butyric acid-GDGT ester, which was a colorless oil.

[0311] bis-(4-(4-hydroxypiperidin-1-yl))butyric acid)-GDGT ester

[0312]

[0313] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 71% bis-(4-(4-hydroxypiperidin-1-yl))butyric acid)-GDGT ester, which was a colorless oil.

[0314] bis-(4-morpholino)butyric acid)-GDGT ester

[0315]

[0316] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 60% bis-(4-morpholino)butyric acid (GDGT) ester, which was a colorless oil.

[0317] bis-(4-(3-(hydroxymethyl)morpholino)butyric acid)-GDGT ester

[0318]

[0319] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 41% bis-(4-(3-(hydroxymethyl)morpholino)butyric acid)-GDGT ester, which was a colorless oil.

[0320] bis-(4-(2-(hydroxymethyl)-1H-imidazolyl)butyric acid)-GDGT ester

[0321]

[0322] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 73% bis-(4-(2-(hydroxymethyl)-1H-imidazolyl)butyric acid)-GDGT ester, which was a colorless oil.

[0323] bis-(4-(piperazin-1-yl)butyric acid)-GDGT ester

[0324]

[0325] In an 8 mL vial equipped with a diaphragm and stir bar, K₂CO₃ (3 equivalents), KI (0.4 equivalents), and 1-Boc-piperazine were added to bis-(4-bromobutyric acid)-GDGT ester (1 equivalent). The vial was evacuated and backfilled with N₂ (three times), then HPLC-grade MeCN was added. The turbid mixture was heated to 75 °C for 18 hours (until the starting material was completely consumed as verified by TLC), cooled to room temperature, and the MeCN was evaporated. The solid was dissolved in NaHCO₃ and extracted with DCM (three times), washed once with brine, filtered, and evaporated. The crude material was dissolved in 2 mL of dry DCM, and 1 mL of TFA was added dropwise. The mixture was stirred at room temperature for 2 hours, cooled to 0 °C, and neutralized with a saturated aqueous solution of NaHCO₃. The mixture was extracted three times with DCM, dried over MgSO₄, and the solvent was evaporated. The 45% bis-(4-(piperazin-1-yl)butyric acid)-GDGT ester was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) as a light yellow oil.

[0326] bis-(4-(4-(2-hydroxyethyl)piperazin-1-yl)butyric acid)-GDGT ester

[0327]

[0328] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 54% bis-4-(4-(2-hydroxyethyl)piperazin-1-yl)-GDGT ester, which was a colorless oil.

[0329] Example 11 - Synthesis of Ionizable GDGT from GDGT Acids

[0330] Preparation of bis-GDGT-acids

[0331]

[0332] In an 8 mL vial equipped with a diaphragm and a magnetic stir bar, GDGT (1 equivalent), 2-iodobenzoic acid (0.4 equivalent), and Oxone (2.8 equivalent), preferably obtained according to Example 1, were dissolved in MeCN / H2O = 2:1. The mixture was heated to 70°C for 6 hours and then cooled to room temperature. The high-valent iodine precipitate was removed by filtration and washed with water and DCM. The solvent mixture was extracted with DCM (3x) and dried on MgSO4. Evaporation of the solvent yielded 97% crude bis-GDGT-acid, which was ready for use without further purification.

[0333] General method for preparing bis-(4-aminobutyl)-GDGT ester

[0334]

[0335] In an 8 mL vial equipped with a diaphragm and stir bar, EDCI (2.2 equivalents) and DMAP (0.4 equivalents) were added to bis-GDGT acid (1 equivalent). The mixture was dissolved in DCM, and then 4-bromobutanol (2.8 equivalents) was added, and the mixture was stirred at room temperature for 18 hours. After quenching with a saturated aqueous solution of NaHCO3, the mixture was extracted with DCM (3x), washed with brine, dried on MgSO4, and evaporated. The crude material was purified by rapid chromatography (SiO2, LP:EE) to obtain bis-(4-bromobutane)-GDGT- ester as a colorless oil.

[0336] In an 8 mL vial equipped with a diaphragm and stir bar, K₂CO₃ (3 equivalents) and KI (0.4 equivalents) were added to bis-(4-bromobutyl)-GDGT ester (1 equivalent). The vial was evacuated and backfilled with N₂ (three times), and then HPLC-grade MeCN was added through a sleeve, followed by the appropriate secondary amine (3 equivalents), or, if the amine was solid, added all at once. The turbid mixture was heated to 75 °C for 16 to 24 hours (until the starting material was completely consumed, as checked by TLC), cooled to room temperature, and the MeCN was evaporated. The solid was dissolved in NaHCO₃ and extracted with DCM (three times), washed once with brine, filtered, and evaporated. The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to give bis-(4-aminobutyl)-GDGT ester as a colorless oil.

[0337]

[0338] The crude material obtained depends on the amine used. The product is typically a mixture of several different substituted GDGT skeletons. Representative structures (with the GDGT-4 skeleton) are shown below:

[0339] bis-(dimethylamino)butyl)-GDGT ester

[0340]

[0341] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 65% bis-(4-(dimethylamino)butyl)-GDGT ester, which was a colorless oil.

[0342] bis-(diethylamino)butyl)-GDGT ester

[0343]

[0344] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 70% bis-(4-(diethylamino)butyl)-GDGT ester, which was a colorless oil.

[0345] bis-(4-(2-hydroxyethyl)(methyl)aminobutyl)-GDGT ester

[0346]

[0347] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 68% bis-(4-(2-hydroxyethyl)(methyl)aminobutyl)-GDGT ester, which was a colorless oil.

[0348] bis-4-(bis-(2-hydroxyethyl)aminobutyl)-GDGT ester

[0349]

[0350] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 58% bis-4-(bis-(2-hydroxyethyl)aminobutyl)-GDGT ester, which was a colorless oil.

[0351] bis-(4-pyrrolidone-1-yl)butyl)-GDGT ester

[0352]

[0353] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 56% bis-pyrrolidine-1-yl)butyl)-GDGT ester, which was a colorless oil.

[0354] 4-(2(S)-(hydroxymethyl)pyrrolidone-1-yl)butyl)-GDGT ester

[0355]

[0356] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 61% bis-4-(2(S)-(hydroxymethyl)pyrrolidine-1-yl)butyl)-GDGT ester, which was a colorless oil.

[0357] bis-(4-piperidin-1-yl))butyl)-GDGT ester

[0358]

[0359] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 65% bis-(4-piperidin-1-yl))butyl)-GDGT ester, which was a colorless oil.

[0360] bis-(4-(4-hydroxypiperidin-1-yl))butyl)-GDGT ester

[0361]

[0362] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 64% bis-(4-(4-hydroxypiperidin-1-yl))butyl)-GDGT ester, which was a colorless oil.

[0363] Bis-(4-morpholino)butyl)-GDGT ester

[0364]

[0365] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 66% bis-(4-morpholino)butyl)-GDGT ester, which was a colorless oil.

[0366] bis-(4-(3-(hydroxymethyl)morpholino)butyl)-GDGT ester

[0367]

[0368] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain a mixture of 48% diastereomers of bis-(4-(3-(hydroxymethyl)morpholino)butyl)-GDGT ester, which was a colorless oil.

[0369] bis-(4-(2-(hydroxymethyl)-1H-imidazolyl)butyl)-GDGT ester

[0370]

[0371] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 63% bis-(4-(2-(hydroxymethyl)-1H-imidazolyl)butyl)-GDGT ester, which was a colorless oil.

[0372] bis-4-(piperazin-1-yl)butyl-GDGT ester

[0373]

[0374] In an 8 mL vial equipped with a diaphragm and stir bar, K₂CO₃ (3 equivalents), KI (0.4 equivalents), and 1-Boc-piperazine were added to bis-(4-bromobutyl)-GDGT ester (1 equivalent). The vial was evacuated and backfilled with N₂ (three times), then HPLC-grade MeCN was added. The turbid mixture was heated to 75 °C for 18 hours (until the starting material was completely consumed as verified by TLC), cooled to room temperature, and the MeCN was evaporated. The solid was dissolved in NaHCO₃ and extracted with DCM (three times), washed once with brine, filtered, and evaporated. The crude material was dissolved in 2 mL of dry DCM, and 1 mL of TFA was added dropwise. The mixture was stirred at room temperature for 2 hours, cooled to 0 °C, and neutralized with a saturated aqueous solution of NaHCO₃. The mixture was extracted three times with DCM, dried over MgSO₄, and the solvent was evaporated. The 48% bis-4-(piperazin-1-yl)butyl-GDGT ester was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) as a light yellow oil.

[0375] Bis-4-(4-(2-hydroxyethyl)piperazin-1-yl)butyl-GDGT ester

[0376]

[0377] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 65% bis-4-(4-(2-hydroxyethyl)piperazine-1-yl)butyl-GDGT ester, which was a colorless oil.

[0378] Example 12 - Synthesis of additional ionizable GDGT from GDGT aldehydes

[0379] Preparation of bis-GDGT-aldehyde

[0380]

[0381] In an 8 mL vial equipped with a diaphragm and a magnetic stir bar, GDGT (1 equivalent) and TCCA (2.4 equivalent), preferably obtained according to Example 1, were dissolved in dry DCM. The mixture was cooled to 0°C, TEMPO (0.2 equivalent) was added, and stirring was continued for 10 minutes. The yellow solution was then poured into a liquid silage container, filtered through diatomaceous earth, washed with the silage, and the solvent was evaporated to obtain 99% crude bis-GDGT-aldehyde, which could be used without further purification.

[0382] General methods for preparing bis-GDGT amines

[0383] Bis-GDGT-aldehyde (1 equivalent) was dissolved in dry DCM, and an appropriate amount of amine (4 equivalents) was added. The mixture was stirred at room temperature for 10 minutes. Then, sodium triacetoxyborohydride (6 equivalents) was added in one go, and the heterogeneous mixture was stirred for 2 hours, followed by careful quenching with 2N NaOH. After extraction with diethyl ether (3x), the mixture was washed with brine, dried on MgSO4, filtered, and the solvent was evaporated. Column chromatography (Biotage Sfär Amino, LP:EE) yielded GDGT amine as a colorless to yellow oil.

[0384]

[0385] The crude material obtained depends on the amine used. The product is typically a mixture of several different substituted GDGT skeletons. Representative structures (with the GDGT-4 skeleton) are shown below:

[0386] bis-(dimethylamino)-GDGT

[0387]

[0388] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 51% bis-(dimethylamino)-GDGT, which was a colorless oil.

[0389] bis-(diethylamino)-GDGT

[0390]

[0391] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 62% bis-(diethylamino)-GDGT, which was a colorless oil.

[0392] bis-(bis-(hydroxyethyl)amino)-GDGT

[0393]

[0394] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 65% bis-(bis(hydroxyethyl)amino)-GDGT, which was a colorless oil.

[0395] bis-(methyl(hydroxyethyl)amino)-GDGT

[0396]

[0397] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 64% bis-(methyl(hydroxyethyl)amino)-GDGT, which was a colorless oil.

[0398] bis-(methyl(hydroxypropyl)amino)-GDGT

[0399]

[0400] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 48% bis-(methyl(hydroxyethyl)amino)-GDGT, which was a colorless oil.

[0401] bis-(methyl(hydroxybutyl)amino)-GDGT

[0402]

[0403] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 59% bis-(methyl(hydroxybutyl)amino)-GDGT, which was a colorless oil.

[0404] bis-((S)-oxetane-2-methane-N-methyl-amine)-GDGT

[0405]

[0406] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 43% bis-((S)-oxetane-2-methane-N-methyl-amine)-GDGT, which was a colorless oil.

[0407] bis-(azacyclobutane-3-formyl)-GDGT

[0408]

[0409] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 48% bis-(azacyclobutane-3-formyl)-GDGT, which was a colorless oil.

[0410] Bis-(pyrrolidine)-GDGT

[0411]

[0412] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 57% bis-(pyrrolidine)-GDGT, which was a colorless oil.

[0413] Bis-(L-proline)-GDGT

[0414]

[0415] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 55% bis-(L-prolyl)-GDGT, which was a colorless oil.

[0416] bis-(D-proline)-GDGT

[0417]

[0418] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 55% bis-(D-proline)-GDGT, which was a colorless oil.

[0419] bis-(S-pyrrolidine-3-ol)-GDGT

[0420]

[0421] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 60% bis-(S-pyrrolidine-3-ol)-GDGT, which was a colorless oil.

[0422] bis-(R-pyrrolidine-3-ol)-GDGT

[0423]

[0424] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 62% bis-(R-pyrrolidine-3-ol)-GDGT, which was a colorless oil.

[0425] bis-((3S,4S)-pyrrolidine-3,4-diol)-GDGT

[0426]

[0427] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 49% bis-((3S,4S)-pyrrolidine-3,4-diol)-GDGT, which was a colorless oil.

[0428] Bis-(2-(hydroxymethyl)imidazole)-GDGT

[0429]

[0430] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 60% bis-(2-(hydroxymethyl)imidazole)-GDGT, which was a colorless oil.

[0431] bis-((1R,5S,6r)-3-azabicyclo[3.1.0]hexane-6-yl)methanol)-GDGT

[0432]

[0433] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 61% bis-((1R,5S,6r)-3-azabicyclo[3.1.0]hexane-6-yl)methanol)-GDGT, which was a colorless oil.

[0434] Bis-(piperidine)-GDGT

[0435]

[0436] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 70% bis-(piperidine)-GDGT, which was a colorless oil.

[0437] Bis-(4-hydroxypiperidine)-GDGT

[0438]

[0439] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 70% bis-(4-hydroxypiperidine)-GDGT, which was a colorless oil.

[0440] Bis-(2-hydroxymethylpiperidine)-GDGT

[0441]

[0442] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain a mixture of 49% diastereomers of bis-(2-hydroxymethylpiperidine)-GDGT as a colorless oil.

[0443] Bis-((R)-piperidin-3-ol)-GDGT

[0444]

[0445] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 58% bis-((R)-piperidin-3-ol)-GDGT, which was a colorless oil.

[0446] Bis-((S)-piperidin-3-ol)-GDGT

[0447]

[0448] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 61% bis-((S)-piperidin-3-ol)-GDGT, which was a colorless oil.

[0449] Bis-((3R,4S)-piperidine-3,4-diol)-GDGT

[0450]

[0451] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 43% bis-((3R,4S)-piperidine-3,4-diol)-GDGT, which was a colorless oil.

[0452] bis-(2-oxa-8-azaspiro[4.5]decane)-GDGT

[0453]

[0454] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 71% bis-(2-oxa-8-azaspiro[4.5]decane)-GDGT, which was a colorless oil.

[0455] Bis-(morpholino)-GDGT

[0456]

[0457] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 70% bis-(morpholine)-GDGT, which was a colorless oil.

[0458] bis-(morpholin-3-yl-methanol)-GDGT

[0459]

[0460] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain a mixture of 52% diastereomers of bis-(morpholino-3-yl-methanol)-GDGT as a colorless oil.

[0461] Bis-(piperazine)-GDGT

[0462]

[0463] Bis-(piperazine)-GDGT was obtained by reductive amination with N-Boc-piperidine followed by Boc cleavage with TFA. The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 50% bis-(piperazine)-GDGT as a colorless oil.

[0464] Bis-(4-(hydroxyethyl)piperazine)-GDGT

[0465]

[0466] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 50% bis-(4-hydroxyethyl)piperazine-GDGT, which was a colorless oil.

[0467] bis-(azacyclooctane)-GDGT

[0468]

[0469] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 71% bis-(azacyclooctane)-GDGT, which was a colorless oil.

[0470] bis-((R)-3-amino-1-benzylpiperidine)-GDGT

[0471]

[0472] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 58% bis-((R)-3-amino-1-benzylpiperidine)-GDGT, which was a colorless oil.

[0473] bis-(indoline-5-yl-methanol)-GDGT

[0474]

[0475] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 71% bis-(indoline-5-yl-methanol)-GDGT, which was a colorless oil.

[0476] Example 13 - Synthesis of ionizable squamamide amino GDGT from GDGT aldehydes

[0477] Preparation of 3-methoxy-4-(methylamino)cyclobut-3-ene-1,2-dione

[0478]

[0479] 3,4-Dimethoxycyclobut-3-ene-1,2-dione (1 equivalent) was dissolved in Et₂O, followed by dropwise addition of methylamine (2M in THF, 1.1 equivalent). The mixture was stirred for 24 hours, resulting in the formation of a white precipitate. The solid was removed by filtration and washed with diethyl ether. Recrystallization from ethyl acetate yielded pure 3-methoxy-4-(methylamino)cyclobut-3-ene-1,2-dione in 70% yield.

[0480] Pure 3-methoxy-4-(methylamino)cyclobut-3-ene-1,2-dione (1 equivalent) was dissolved in EtOH, and amino-NBoc-alkylamine (1.2 equivalent) was added and stirred for 22 hours. Column chromatography (Biotage Sfär Amino, LP:EE) yielded pure N-Boc-amino-squamamide as a white solid. Boc deprotection was performed by treating the N-Boc-amino-squamamide dissolved in DCM with TFA (15 equivalent) for 2 hours. Evaporation of the solvent yielded the amino-squamamide TFA-salt, which was ready for use without further purification.

[0481] General method for preparing bis-(squamamide amino)-GDGT

[0482]

[0483] Bis-GDGT-aldehyde (1 equivalent) and amino-squamousamide TFA-salt (2.2 equivalents), preferably obtained according to Examples 7 or 12, were dissolved in dry DCM. N,N-diisopropylethylamine (4.4 equivalents) was then added via syringe, and the mixture was stirred at room temperature for 10 minutes. Sodium triacetoxyborohydride (6 equivalents) was then added in a single batch, and the heterogeneous mixture was stirred for 2 hours, followed by careful quenching with 2N NaOH. After extraction with diethyl ether (3x), the mixture was washed with brine, dried over MgSO4, filtered, and the solvent was evaporated. Column chromatography (Biotage Sfär Amino, LP:EE) yielded bis-(squamousamide amino)-GDGT as a white solid.

[0484] The resulting material depends on the squaramide amine used. The product is typically a mixture of several different substituted GDGT backbones. Representative structures (with a GDGT-4 backbone) are shown below:

[0485] bis-((3-(methylamino)-4-((3-(methylamino)ethyl)amino)cyclobut-3-ene-1,2-dione)-GDGT

[0486]

[0487] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 40% bis-((3-(methylamino)-4-((3-(methylamino)ethyl)amino)cyclobut-3-ene-1,2-dione)-GDGT as a white solid.

[0488] bis-((3-(methylamino)-4-((3-(methylamino)propyl)amino)cyclobut-3-ene-1,2-dione)-GDGT

[0489]

[0490] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 41% bis-((3-(methylamino)-4-((3-(methylamino)propyl)amino)cyclobut-3-ene-1,2-dione)-GDGT as a white solid.

[0491] bis-((3-(methylamino)-4-((3-(methylamino)butyl)amino)cyclobut-3-ene-1,2-dione)-GDGT

[0492]

[0493] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 38% bis-((3-(methylamino)-4-((3-(methylamino)butyl)amino)cyclobut-3-ene-1,2-dione)-GDGT as a white solid.

[0494] bis-((3-(3-methylazacyclobutane-3-yl)amino)-4-(methylamino)cyclobut-3-ene-1,2-dione))-GDGT

[0495]

[0496] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 45% bis-((3-(3-methylazacyclobutane-3-yl)amino)-4-(methylamino)cyclobut-3-ene-1,2-dione))-GDGT as a white solid.

[0497] bis-((3-(3-methylazacyclobutane-3-yl)amino)-4-(methylamino)cyclobut-3-ene-1,2-dione))-GDGT

[0498]

[0499] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 32% bis-((3-(3-methylazacyclobutane-3-yl)amino)-4-(methylamino)cyclobut-3-ene-1,2-dione))-GDGT as a white solid.

[0500] bis-((3-(3-(methyl)amino)azacyclobutane-1-yl)-4-(methylamino)cyclobut-3-ene-1,2-dione)-GDGT

[0501]

[0502] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 28% bis-((3-(3-methylazacyclobutane-3-yl)amino)-4-(methylamino)cyclobut-3-ene-1,2-dione))-GDGT as a white solid.

[0503] Example 14 - Synthesis of Ionizable Monosubstituted GDGT

[0504] Preparation of mono-TBSO-GDGT-aldehyde

[0505]

[0506] GDGT (1 equivalent) and imidazole (2.2 equivalents) preferably obtained according to Example 1 were dissolved in dry DMF, and tert-butyldimethylchlorosilane (50% w / w in toluene, 1.2 equivalents) was added dropwise. The suspension was stirred for 20 hours, dissolved in diethyl ether, washed with 1M LiCl aqueous solution (3x), dried on MgSO4, filtered, and evaporated. The crude material was purified by column chromatography (SiO2, LP:EE) to obtain 42% mono-TBSO-GDGT as a colorless oil.

[0507] In an 8 mL vial equipped with a diaphragm and magnetic stir bar, mono-TBSO-GDGT (1 equivalent) and TCCA (1.2 equivalent) were dissolved in dry DCM. The mixture was cooled to 0 °C, TEMPO (0.1 equivalent) was added, and stirring was continued for 10 minutes. The yellow solution was then poured into a liquid precipitate (LP), filtered through diatomaceous earth, washed with LP, and the solvent was evaporated to obtain 99% crude mono-TBSO-GDGT-aldehyde, which could be used without further purification.

[0508] General methods for preparing mono-GDGT amines

[0509] Mono-TBSO-GDGT-aldehyde (1 equivalent) was dissolved in dry DCM, and an appropriate amount of amine (2 equivalents) was added. The mixture was stirred at room temperature for 10 minutes. Then, STAB (3 equivalents) was added in one go, and the heterogeneous mixture was stirred for 2 hours, followed by careful quenching with 2N NaOH. After extraction with diethyl ether (3x), the mixture was washed with brine, dried on MgSO4, filtered, and the solvent was evaporated. The crude material was dissolved in dry THF, and tetra-n-butylammonium fluoride (1M in THF, 1.5 equivalents) was added and stirred for 20 hours. The mixture was quenched by adding 1M CaCO3 aqueous solution, extracted with diethyl ether (3x), washed with brine, and dried on MgSO4. After filtering the drying agent, the solvent was removed under vacuum. Column chromatography (Biotage SfärAmino, LP:EE) yielded pure mono-GDGT amine as a colorless to yellow oil.

[0510]

[0511] The crude material obtained depends on the amine used. The product is typically a mixture of several different substituted GDGT skeletons. Representative structures (with the GDGT-4 skeleton) are shown below:

[0512] mono-(dimethylamino)-GDGT

[0513]

[0514] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 43% mono-(dimethylamino)-GDGT, which was a colorless oil.

[0515] Mono-(diethylamino)-GDGT

[0516]

[0517] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 55% mono-(diethylamino)-GDGT, which was a colorless oil.

[0518] Mono-(bis-(hydroxyethyl)amino)-GDGT

[0519]

[0520] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 54% mono-(bis(hydroxyethyl)amino)-GDGT, which was a colorless oil.

[0521] Mono-(methyl(hydroxyethyl)amino)-GDGT

[0522]

[0523] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 60% mono-(methyl(hydroxyethyl)amino)-GDGT, which was a colorless oil.

[0524] mono-(methyl(hydroxypropyl)amino)-GDGT

[0525]

[0526] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 43% mono-(methyl(hydroxyethyl)amino)-GDGT, which was a colorless oil.

[0527] mono-(methyl(hydroxybutyl)amino)-GDGT

[0528]

[0529] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 64% mono-(methyl(hydroxybutyl)amino)-GDGT, which was a colorless oil.

[0530] mono-((S)-oxetane-2-methane-N-methyl-amine)-GDGT

[0531]

[0532] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 41% mono-((S)-oxetane-2-methane-N-methyl-amine)-GDGT, which was a colorless oil.

[0533] mono-(azacyclobutane-3-formyl)-GDGT

[0534]

[0535] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 46% mono-(azacyclobutane-3-formyl)-GDGT, which was a colorless oil.

[0536] Mono-(pyrrolidine)-GDGT

[0537]

[0538] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 71% mono-(pyrrolidine)-GDGT, which was a colorless oil.

[0539] mono-(L-prolyl)-GDGT

[0540]

[0541] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 52% mono-(L-proline)-GDGT, which was a colorless oil.

[0542] mono-(D-proline)-GDGT

[0543]

[0544] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 61% mono-(D-proline)-GDGT, which was a colorless oil.

[0545] mono-(S-pyrrolidine-3-ol)-GDGT

[0546]

[0547] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 49% mono-(S-pyrrolidine-3-ol)-GDGT, which was a colorless oil.

[0548] Mono-(R-pyrrolidine-3-ol)-GDGT

[0549]

[0550] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 69% mono-(R-pyrrolidine-3-ol)-GDGT, which was a colorless oil.

[0551] mono-((3S,4S)-pyrrolidine-3,4-diol)-GDGT

[0552]

[0553] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 59% mono-((3S,4S)-pyrrolidine-3,4-diol)-GDGT, which was a colorless oil.

[0554] Mono-(2-(hydroxymethyl)imidazole)-GDGT

[0555]

[0556] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 64% mono-(2-(hydroxymethyl)imidazole)-GDGT, which was a colorless oil.

[0557] Mono-((1R,5S,6r)-3-azabicyclo[3.1.0]hexane-6-yl)methanol)-GDGT

[0558]

[0559] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 55% mono-((1R,5S,6r)-3-azabicyclo[3.1.0]hexane-6-yl)methanol)-GDGT, which was a colorless oil.

[0560] Mono-(piperidine)-GDGT

[0561]

[0562] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 66% mono-(piperidine)-GDGT, which was a colorless oil.

[0563] mono-(4-hydroxypiperidine)-GDGT

[0564]

[0565] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 61% mono-(4-hydroxypiperidine)-GDGT, which was a colorless oil.

[0566] mono-(2-hydroxymethylpiperidine)-GDGT

[0567]

[0568] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 43% mono-(2-hydroxymethylpiperidine)-GDGT, which was a colorless oil.

[0569] mono-((R)-piperidin-3-ol)-GDGT

[0570]

[0571] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 48% mono-((R)-piperidin-3-ol)-GDGT, which was a colorless oil.

[0572] mono-((S)-piperidin-3-ol)-GDGT

[0573]

[0574] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 54% mono-((S)-piperidin-3-ol)-GDGT, which was a colorless oil.

[0575] mono-((3R,4S)-piperidine-3,4-diol)-GDGT

[0576]

[0577] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 51% mono-((3R,4S)-piperidine-3,4-diol)-GDGT, which was a colorless oil.

[0578] Mono-(2-oxa-8-azaspiro[4.5]decane)-GDGT

[0579]

[0580] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 66% mono-(2-oxa-8-azaspiro[4.5]decane)-GDGT, which was a colorless oil.

[0581] Mono-(morpholino)-GDGT

[0582]

[0583] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 59% mono-(morpholine)-GDGT, which was a colorless oil.

[0584] mono-(morpholino-3-yl-methanol)-GDGT

[0585]

[0586] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain a mixture of 43% diastereomers of mono-(morpholino-3-yl-methanol)-GDGT as a colorless oil.

[0587] Mono-(piperazine)-GDGT

[0588]

[0589] Mono-(piperazine)-GDGT was obtained by reductive amination with N-Boc-piperidine followed by Boc cleavage with TFA. The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 51% mono-(piperazine)-GDGT as a colorless oil.

[0590] Mono-(4-(hydroxyethyl)piperazine)-GDGT

[0591]

[0592] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 50% mono-(4-hydroxyethyl)piperazine-GDGT, which was a colorless oil.

[0593] mono-(azacyclooctane)-GDGT

[0594]

[0595] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 65% mono-(azacyclooctane)-GDGT, which was a colorless oil.

[0596] mono-((R)-3-amino-1-benzylpiperidine)-GDGT

[0597]

[0598] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 71% mono-((R)-3-amino-1-benzylpiperidine)-GDGT, which was a colorless oil.

[0599] mono-(indoline-5-yl-methanol)-GDGT

[0600]

[0601] The crude material was purified by rapid chromatography (Biotage Sfär Amino column, LP:EE) to obtain 38% bis-(indoline-5-yl-methanol)-GDGT, which was a colorless oil.

[0602] Example 15 - Preparation of LNP

[0603] The lipid stock solution (archaeal membrane lipids obtained from *Acidophilus thermosulfuricus*, comprising GDGT (see Table 1 below; also see WO 2020 / 187526 A1), DSPC, ALC-0159, cholesterol, and ionizable GDGT obtained according to any one of Examples 3, 4, 5, 6, and 8 (or ionizable GDGT according to any one of Examples 9, 10, 11, 12, 13, and 14)) was filtered through a 0.2 µm polytetrafluoroethylene (PTFE) filter and mixed according to the desired molar ratio. A mixture of dimethyl sulfoxide (DMSO) and 2-propanol (2:3) was used as the organic solvent. The aqueous phase was prepared by dissolving the desired mRNA (e.g., mRNA encoding an antigen, such as the SARS-CoV-2 spike protein) in 10 mM citrate buffer (pH=4.0). LNPs were prepared using a NanoAssemble® Ignite (Precision Nanosystems) at a total flow rate of 12 mL / min and a flow ratio of 3:1 (aqueous phase:organic phase). Immediately after preparation, the LNPs were diluted 1:2 in 10 mM PBS (pH 7.4). Removal of the organic solvent was achieved by dialysis (SpectraPor, 6 to 8 kDa) with 10 mM phosphate-buffered saline (PBS) buffer (pH 7.4). Physicochemical characterization of the LNPs was performed using Ribogreen assays (mRNA content) and Zetasizer analysis (size, polydispersity index (PDI), Zeta potential). The Zetasizer was purchased from Malvern Panalytical Ltd, UK.

[0604] Table 1 shows the typical lipid composition of archaeal membrane lipid stock solutions containing GDGT:

[0605]

[0606] After preparation, LNPs were stored in 2 mL Eppendorf tubes at 25 °C (room temperature). Physicochemical characterization was performed at 6-week intervals by Ribogreen assay and ZetaSizer analysis.

[0607] LNPs can be used to formulate mRNA vaccines.

[0608] Example 16 - Preparation and Characterization of Additional LNPs

[0609] The primary objective was to characterize a series of LNPs utilizing ionizable GDGT. By varying the lipid ratio of the ionizable GDGT, we evaluated several key parameters, including size, PDI (polydispersity index), and encapsulation efficiency. Furthermore, zeta potential and pKa values ​​were determined to further understand the physicochemical properties of the ionizable GDGT. Finally, in vivo studies were conducted in rats.

[0610] buffer solution

[0611] The buffer used to prepare LNP is 10 mM citrate buffer with a pH of 3.0. The buffer used for dilution and dialysis is 10 mM PBS with a pH of 7.4.

[0612] LNP formulations

[0613] The conventional lipids used for LNP were dissolved in ethanol to achieve a concentration between 10 and 20 g / L, while the ionizable GDGT was dissolved in isopropanol to achieve a concentration of 30 g / L. The total lipid concentration was 10 to 20 mM, of which 20 mM was used to determine the pKa and zeta potential values.

[0614] Furthermore, the effects of varying the N / P ratio (4-6-8-12) were investigated. (For LNP, the N / P ratio corresponds to the ratio between the amino groups (N) of the ionizable lipid and the phosphate groups (P) of the nucleic acid load (1 per base for RNA, 2 per base for DNA). An N / P ratio of 6 produced optimal results and was selected for further experiments.)

[0615] Poly(A) was used as a loading medium (dissolved in citrate buffer) and encapsulated into lipid nanoparticles using microfluidics at a flow ratio of 3:1 (aqueous to organic phase) and a total flow rate of 12 mL / min. Furthermore, the nanoparticles were diluted 2:1 with PBS buffer to reduce the organic solvent content, followed by a dialysis step.

[0616] Zetasizer ZSP (Malvern) was used to determine particle size, PDI, and zeta potential, while the RiboGreen assay was used to determine encapsulation efficiency.

[0617] The results shown in the table below were obtained for the ionizable GDGT used (using a mixture of several different GDGT backbones; the GDGT-4 backbone is shown as a representative structure). The lipid percentage of ionizable GDGT in the typical LNP lipid composition is given in Mol%.

[0618]

[0619]

[0620]

[0621]

[0622]

[0623]

[0624]

[0625]

[0626]

[0627] In vivo studies

[0628] In vivo studies were conducted using some of the LNPs characterized in Tables 2 to 10 above. Formulations containing 46.3 mol-% (lipid ratio) of the corresponding ionizable GDGT (N / P ratio 6 or 12) and encapsulating erythropoietin (EPO) mRNA were administered intramuscularly to Wistar rats. The aim of the in vivo studies was to evaluate the properties of the different ionizable GDGTs in the LNP formulations and their efficiency in transporting EPO mRNA into muscle cells. Following LNP administration, plasma EPO concentrations (AUC levels) were monitored and quantified using ELISA to determine the extent of EPO protein induction.

[0629] The results are shown in Table 11 below:

[0630]

[0631] Typically, higher EPO protein concentrations are achieved compared to controls without mRNA. For some LNPs (e.g., LNPs based on single-OHPIPD), the protein concentrations achieved are much higher.

[0632] In summary, these results indicate that ionizable GDGT is well-suited for use as an LNP for encapsulating mRNA loads.

[0633] Non-patent references

[0634] Aldosari, Basmah N., et al. "Lipid nanoparticles as delivery systems for RNA-based vaccines." Pharmaceutics 13.2 (2021): 206.

[0635] Benvegnu, Thierry, Loïc Lemiègre, and Sandrine Cammas-Marion. "Newgeneration of liposomes called archaeosomes based on natural or syntheticarchaeal lipids as innovative formulations for drug delivery." Recent patentson drug delivery & formulation 3.3 (2009): 206-220.

[0636] Cornebise, Mark, et al. "Discovery of a novel amino lipid thatimproves lipid nanoparticle performance through specific interactions withmRNA." Advanced Functional Materials 32.8 (2022): 2106727.

[0637] Daswani, Varsha P., et al. "The Polar Lipid Fraction E fromSulfolobus acidocaldarius Can Be Used as Liposomal Drug Stabilizing Agents toReduce the Leakage of the Antivascular Drug Combretastatin A4 DisodiumPhosphate from Tetraether / Diester Hybrid Archaeosomes." Biophysica 1.4(2021): 474-486.

[0638] Engelhardt, Konrad H., et al. "Transfection studies with colloidalsystems containing highly purified bipolar tetraether lipids from Sulfolobusacidocaldarius." Archaea 2017 (2017).

[0639] Eygeris, Yulia, et al. "Chemistry of lipid nanoparticles for RNAdelivery." Accounts of chemical research 55.1 (2021): 2-12.

[0640] Hou, Xucheng, et al. "Lipid nanoparticles for mRNA delivery." NatureReviews Materials 6.12 (2021): 1078-1094.

[0641] Jain, Samta, et al. "Biosynthesis of archaeal membrane ether lipids."Frontiers in microbiology 5 (2014): 641.

[0642] Kaur, Gurmeet, et al. "Archaeosomes: an excellent carrier for drugand cell delivery." Drug delivery 23.7 (2016): 2497-2512.

[0643] Markova, Natalia, et al. "Biophysical Characterization of Viral andLipid-Based Vectors for Vaccines and Therapeutics with Light Scattering andCalorimetric Techniques." Vaccines 10.1 (2021): 49.

[0644] Midoux, Patrick, et al. "Lipid-based mRNA vaccine delivery systems."Expert review of vaccines 14.2 (2015): 221-234.

[0645] Morii, Hiroyuki, and Yosuke Koga. "Asymmetrical topology of diether-and tetraether-type polar lipids in membranes of Methanobacteriumthermoautotrophicum cells." Journal of Biological Chemistry 269.14 (1994):10492-10497.

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Claims

1. A lipid nanoparticle (LNP) encapsulating a nucleic acid load, wherein the LNP comprises at least: - Ionizable lipid fractions, and - Stabilizer fraction; The ionizable lipid fraction contains at least one ionizable dialkyl glycerol tetraether (GDGT) lipid; At least one of the ionizable GDGT lipids comprises at least one ionizable head group S; preferably, S is selected from the group consisting of: and Each occurrence of R a The components are independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkylamine, alkyl ether, alkyl thiol, alkyl ester, alkylamide, alkyl carbamate, alkyl sulfonate, and alkyl sulfonamide, preferably selected from alkyl, cycloalkyl, alkenyl, hydroxyalkyl, and alkylamine. Each occurrence of R b The components are independently selected from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl ester, alkylamide, alkyl carbamate, alkyl sulfonate, alkyl sulfonamide, and alkyl thiol, preferably selected from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, and alkyl thiol, and... Each occurrence of R c Independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkylamide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide, preferably selected from alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkylamide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide.

2. The LNP according to claim 1, wherein the nucleic acid load comprises messenger ribonucleic acid (mRNA).

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

4. The LNP according to any one of claims 1 to 3, wherein the LNP further comprises a sterol lipid fraction, preferably 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: and Each occurrence of R a The components are independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkylamine, alkyl ether, alkyl thiol, alkyl ester, alkylamide, alkyl carbamate, alkyl sulfonate, and alkyl sulfonamide, preferably selected from alkyl, cycloalkyl, alkenyl, hydroxyalkyl, and alkylamine. Each occurrence of R b The components are independently selected from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl ester, alkylamide, alkyl carbamate, alkyl sulfonate, alkyl sulfonamide, and alkyl thiol, preferably selected from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, and alkyl thiol, and... Each occurrence of R c Independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkylamide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide, preferably selected from alkyl, cycloalkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine, alkyl thiol, alkyl ester, alkylamide, alkyl carbamate, alkyl sulfonate and alkyl sulfonamide.

6. The GDGT lipid of claim 5, wherein S is selected from the group consisting of: and Each occurrence of R a Independently selected from H, alkyl, cycloalkyl, alkenyl, hydroxyalkyl, and alkylamine, and each occurrence of R b It is independently selected from H, alkyl, alkenyl, hydroxyalkyl, alkyl ether, alkylamine and alkyl thiol.

7. The GDGT lipid according to claim 5 or 6, wherein the head group S is selected from: and Each R that appears is independently selected from H and alkyl; preferably, the alkyl group is selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, isopentyl, hexyl, isohexyl, heptyl, and isohexyl.

8. An ether lipid fraction comprising GDGT lipids according to any one of claims 5 to 7; said ether lipid fraction is preferably obtainable by extraction from archaea cultures, preferably sulfur-bearing leaf cultures, more preferably acidophilic thermosulfur-bearing leaf cultures, followed by substitution with an ionizable head group S.

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

10. A method for producing GDGT lipids according to any one of claims 5 to 7, comprising the following steps: - Obtain lipid fractions containing one or more precursor GDGT lipids from archaea cultures, preferably sulfur leaf culture, and more preferably acidophilic thermosulfur leaf culture; - Purify one or more of the precursor GDGT lipids; and - Contact one or more precursor GDGT lipids with one or more reagents to produce GDGT lipids having at least one ionizable head group S.

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