Lipids containing an oligoglycerol group

Abstract

The present invention relates to oligoglycerol-containing lipids and in particular to cationic or cationizable oligoglycerol-containing lipids and to a method for providing these oligoglycerol-containing lipids. Further, the present invention relates to liposomes, in particular thermosensitive liposomes comprising the novel oligoglycerol-containing lipids. Further the invention relates to liposomes comprising the novel oligoglycerol-containing lipids, which liposomes have an adjustable surface charge.

Description

[0001] Lipids containing an oligoglycerol group

[0002] The present invention relates to oligoglycerol-containing lipids and in particular to cationic or cationizable oligoglycerol-containing lipids and to a method for providing these oligoglycerol-containing lipids. Further, the present invention relates to liposomes, in particular thermosensitive liposomes comprising the novel oligoglycerol-containing lipids. Further the invention relates to liposomes comprising the novel oligoglycerol-containing lipids, which liposomes have an adjustable surface charge.

[0003] Liposomes are spherical vesicles formed by a membrane bilayer composed by lipid excipients and are widely used and accepted as drug delivery nanocarriers. Today, many natural and synthetic lipids are well known excipients in liposomal formulations.

[0004] Thermosensitive liposomes (TSL) are improved liposomal drug delivery systems allowing active targeting of tumor tissue and of other diseased areas, such as inflamed tissue or joints, via heat-induced local release of the encapsulated active pharmaceutical ingredients (APIs).

[0005] Most patients with advanced tumor diseases die from progressive tumor growth despite the successes achieved with classic chemotherapy and cancer immunotherapy. Efficient drug targeting is therefore a key issue in medical research (Lindner & Hossann, 2010). Classic small-molecule API lack intrinsic affinity for diseased tissue, and considerable quantities of them are distributed throughout healthy tissue after systemic application. For API , such as those used in cancer therapy, this systemic availability is associated with severe side effects. Therefore, effective dosing of these API is limited, and the therapeutic potential of many conventional non-targeted drugs cannot be fully exploited.

[0006] With the help of innovative drug delivery systems (DDS) for targeted drug delivery, the efficiency of known drugs can be improved and the risk of occurring drug resistance can be reduced. Of the DDS developed to date, liposomes and lipid-based nanoparticles (LNPs) have achieved the greatest relevance for clinical use. Liposomes are versatile and often classified as biocompatible DDS. The pharmacodynamics of liposomally encapsulated API is determined by the lipid excipient composition, the route of administration (e.g., intravenous, intratumoral., oral), the distribution of the liposomes, the type of targeting (e.g., passive, active, targeted) and the rate at which the drug is released from the liposomes. Therefore, novel innovative DDS are needed showing affinity for the diseased tissue and limited distribution to healthy tissue.

[0007] For example, PEGylated long-circulating formulations like Caelyx® have been designed to exploit the enhanced permeability and retention (EPR) effect and passively accumulate inside tumor tissue because of the leaky tumor vasculature. Accumulation of nanoparticle in tumor tissue depends on the tumor environment, a natural barrier for DDS (Nakamura et al. 2016). Extravasation of the vesicles is the rate-limiting step and nanoparticles must circulate for days to accumulate in sufficient high concentrations. Because accumulation in tumor tissue competes with uptake by the mononuclear phagocyte system (MPS) in liver and spleen only less than 5% of injected dose accumulate in tumor (O'Brien et al. 2004). As result, Caelyx® did not show improved efficacy compared to free drug (Harrington et al 2001) due to known shortcomings of the formulation.

[0008] Wilhelm et al. surveyed relevant literature from 2005 to 2015 and concluded that only a 0.7% (median) of the injected dose of miscellaneous DDS was delivered to solid tumors (Wilhelm et al. 2016). The review article was critically debated (Torrice 2016). It was stated that the U.S. Food and Drug Administration (FDA) and nanoparticle developers usually judge delivery systems by following the drug and not the nanocarrier by pharmacokinetic (PK) parameters. Another point of criticism was, that strong-performing systems may have been statistically lost among many weaker once and the efficacy of the nanoparticle system was not compared to free drug. Nevertheless, one could conclude, that efficacy of nanoparticle delivery via EPR effect was not significantly improved in one decade. Wilhelm et al. suggested that a first step to improve DDS would be to investigate how they leave tumor vessels and / or interact with tumor tissues. Recently, it was demonstrated that up to 97% of nanoparticles enter tumors rather by an active process through endothelial cells (Sindhwani et al. 2020). Such an active process might be steered by the ability of the DDS to interact with the responsible cells and by characteristics of the DDS (e.g., absence of PEGylation, lipid composition, composition of protein corona, surface charge, size).

[0009] Surface modification of liposomal DDS with anionic oligoglycerol-containing lipid excipients is known (e.g., WO 97 / 30058, WO 2014 / 202680). It was shown that the number of glycerol moieties in anionic 1 ,2-dipalmitoyl-sn-glycero-3-phospho-oligoglycerols (DPPGX) can steer the pharmacokinetic behavior and organ distribution of liposomes (WO 97 / 30058, Schagon 1997). A TSL formulation based on the anionic 1 ,2-dipalmitoyl-sn-glycero-3-phospho- diglycerol (DPPG2) or 1 ,2-dipalmitoyl-sn-glycero-3-phospho-triglycerol (DPPG3) encapsulating the drug doxorubicin (DOX) showed superiority over clinically relevant DOX formulations in treatment of sarcoma in a rat sarcoma model (Hossann et al. 2021). Liposomes containing DPPG2 allowed a better interaction and / or uptake into cells when compared to PEGylated liposomes (Petrini et al. 2021). Furthermore, the content of DPPG2 in a formulation notably influenced the composition of the protein corona and hemocompatibility of the liposomal DDS (Lokerse et al. 2021). DPPGXwas used to replace PEGylated phospholipids in cholesterol-containing (WO 97 / 30058 A1) and cholesterol-free liposomes (Hossann et al. 2021) to prolong the circulation half-life of these nanocarriers. Alternatives like DPPGXor the cationic or cationizable oligoglycerol-containing lipids as described hereinafter are of interest as novel excipients for liposomal DDS because usage of PEGylation in human drug products was questioned in recent years for several reasons (Hoang et al. 2020).

[0010] An object of the present invention was therefore to provide novel oligoglycerol lipid excipients. These lipids should in particular allow for the formation of anionic, cationic, or cationizable liposomal DDS having an adjustable surface charge, that are covered by oligoglycerol moieties on their surface.

[0011] The present invention relates to an oligoglycerol-containing lipid of Formula (I) wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0012] A = NR3or N+R4R5;

[0013] R3, R4and R5each independently are hydrogen or a hydrocarbon group having

[0014] 1 to 5 C-atoms, and n = a number from 0 to 20, m at each occurrence independently is 0 or 1 ; y = 0 or 1 ; and z = 0 or 1.

[0015] Preferably the present invention relates to an oligoglycerol-containing lipid of Formula (I) wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0016] A = NR3;

[0017] R3is hydrogen or preferably a hydrocarbon group having 1 to 5 C-atoms; n = a number from 0 to 20; m at each occurrence independently is 0 or 1 ; y = 0 or 1 ; and z = 0 or 1.

[0018] Those compounds are also termed NLGXherein for y=1 and z=1.

[0019] Also preferably the present invention relates to an oligoglycerol-containing lipid of Formula

[0020] (I) wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0021] A = N+R4R5;

[0022] R4and R5each independently are hydrogen or preferably a hydrocarbon group having 1 to 5 C-atoms; n = a number from 0 to 20; m at each occurrence independently is 0 or 1 ; y = 0 or 1 ; and z = 0 or 1.

[0023] Those compounds are also termed CLGXherein for y=1 and z=1. In a preferred embodiment the present invention relates to an oligoglycerol-containing lipid of Formula (IA) wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0024] A = NR3or N+R4R5;

[0025] R3, R4and R5each independently are hydrogen or preferably a hydrocarbon group having 1 to 5 C-atoms, and n = a number from 0 to 20, and m at each occurrence independently is 0 or 1.

[0026] Preferably the present invention relates to an oligoglycerol-containing lipid of Formula (IA) wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0027] A = NR3;

[0028] R3is hydrogen or preferably a hydrocarbon group having 1 to 5 C-atoms; n = a number from 0 to 20; and m at each occurrence independently is 0 or 1.

[0029] Those compounds are also termed NLGXherein.

[0030] Further preferably the present invention relates to an oligoglycerol-containing lipid of Formula (IA) wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0031] A = N+R4R5;

[0032] R4and R5each independently are hydrogen or a preferably hydrocarbon group having 1 to 5 C-atoms; n = a number from 0 to 20; and m at each occurrence independently is 0 or 1.

[0033] Those compounds are also termed CLGXherein.

[0034] The present invention in particular relates to an oligoglycerol-containing lipid of Formula (IA) wherein

[0035] R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0036] A = NR3or N+R4R5;

[0037] R3, R4and R5each independently are hydrogen or preferably a hydrocarbon group having 1 to 5 C-atoms; n = a number from 0 to 20, and m at each occurrence independently is 0 or 1.

[0038] In preferred embodiments R3, R4and R5are hydrocarbon groups having 1 to 5 C-atoms.

[0039] The inventive oligogylcerol-containing lipids have at least two glycerol moieties. According to the invention n is a number from 0 to 20. Preferably, n is a number from 0 to 10, more preferably from 0 to 5. Most preferred n is 0, 1 or 2. The index m can be, at each occurrence independently 0 or 1. For m=1 a glycerol group is indicated, for m=0 an ethylene glycol group is indicated.

[0040] The residues R1and R2can be linked to the structure of the inventive oligoglycerolcontaining lipid via an ether or via an ester bond. For y = 0 or z = 0, the linkage is an ether bond, for y = 1 or z = 1 , the linkage is an ester bond. Preferably, linkage of R1and R2is via an ester bond, thus preferred y = 1 and z = 1 in the inventive oligoglycerol-containing lipids.

[0041] In particular, n is 0, 1 or 2, and m = 1 , such that the oligoglycerol-containing lipid of Formula (I) or (IA) is a diglycerol (n=0), a triglycerol (n=1) or a tetraglycerol (n=3), respectively. Even more preferably, n is 0 or 1 and most preferably n is 0.

[0042] The inventive oligoglycerol-containing lipid contains a cationizable or cationic group A. When A = NR3a cationizable oligoglycerol-containing lipid is indicated, which is also designated NLGXherein (x denotes the number of glycerol groups). When A = N+R4R5a cationic oligoglycerol-containing lipid is indicated, which is also designated CLGXherein (x denotes the number of glycerol groups). Preferred is an oligoglycerol-containing lipid, wherein A = NR3and R3= CH3or wherein A = N+R4R5and R4, R5= CH3.

[0043] In the oligoglycerol-containing lipids, residues R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms. Preferred is an oligoglycerol-containing lipid, wherein R1and R2independently are a hydrocarbon group having 13 to 19 C-atoms. The hydrocarbon groups can be saturated or mono- or polyunsaturated. The hydrocarbon groups can be linear or branched. Preferably R1and R2independently are a linear or branched saturated C13- to C19-alkyl group, more preferably a linear saturated C13- to C19-alkyl group. More preferred R1and R2independently are a linear saturated C15 or C17 alkyl group, most preferred a linear saturated C15 alkyl group.

[0044] Most preferred are NLG2 having formula (111 A) and CLG2 having formula (IVA).

[0045] The inventors of the present application succeeded in providing the oligoglycerol-containing lipid of Formula (I) and of Formula (IA) in a stereoisomeric form. Therefor the invention further relates to an oligoglycerol-containing lipid having Formula (II) wherein the three stereogenic centers *1 , *2 and *3 have a predetermined configuration and wherein *p denotes further stereogenic centers. A predetermined configuration means in particular that the stereogenic center *1 has either an (R) or an (S) configuration, stereogenic center *2 has either an (R) or an (S) configuration and stereogenic center *3 has either an (R) or an (S) configuration. Thus, a predetermined configuration means that the respective centers are determined to be either pure (R) or to be pure (S). A predetermined configuration in particular means that the respective stereogenic center is not present in a racemic configuration. Preferably each of the further stereogenic centers *p has a predetermined configuration, which means that each of the further stereogenic centers *p has either an (R) or an (S) configuration.

[0046] The invention preferably relates to a stereoisomeric oligoglycerol-containing lipid having Formula (HA) wherein the three stereogenic centers *1 , *2 and *3 have a predetermined configuration and in particular wherein stereogenic center *1 is in R-configuration, stereogenic center *2 is in S-configuration and stereogenic center *3 is in R-configuration; and wherein *p denotes further stereogenic centers. The further stereogenic centers independently may have a predetermined configuration, either R or S.

[0047] A suitable counter ion for all cationic lipids disclosed herein is Cl Br, mesylate or fumarate.

[0048] The invention further provides a method for preparing an oligoglycerol-containing lipid having Formula (I) as herein described. The method is characterized in that the synthesis route includes a central intermediate of Formula (V) or of Formula (V’) wherein PG, PG’ and PG” independently are protecting groups. PG and PG” in particular are benzyl (Bn), tetrahydropyranyl (THP), ethoxyethyl (EE), 2-methoxypropan-2-yl (MOP), silyl or para-methoxybenzyl (PMB).For the synthesis of an oligoglycerol-containing lipid of Formula (I), wherein R1and R2independently are saturated hydrocarbon group preferably PG and PG” is benzyl (Bn). For the synthesis of an oligoglycerol-containing lipid of Formula (I), wherein R1and R2independently are a mono- or polyunsaturated hydrocarbon group preferably PG and PG” is THP and silyl. PG’ in particular is CH2 (methylene), CHCH3 (ethylidene), CHPh (benzylidene), CHPhp-Ome (para-methoxybenzylidene), C(CH3)2 (isopropylidene) or SifBu2 (di-tert-butylsilylene), Si(iPr)2-O-Si(iPr)2 (tetraisopropyldisiloxanylidene), preferably PG’ is C(CH3)2.

[0049] Preferably the two stereogenic centers *2’ and *3’ have a predetermined configuration. It was found that a synthesis route including a central intermediate of Formula (V) or (V’), wherein the two stereogenic centers *2’ and *3’ have a predetermined configuration allows for preparation of oligoglycerol-containing lipids of Formula (II), wherein the three stereogenic centers *1 , *2 and *3 can be provided with a predetermined configuration.

[0050] For obtaining higher oligoglycerols, in particular tri- or tetraglycerols, the intermediate of Formula (V) or (V’) is repeatedly reacted with a glycidol to form building blocks having an increased number of glycerols:

[0051] A synthesis overview is provided in the scheme below showing a method which is in particular suitable for the synthesis of cationizable or cationic lipids wherein R1and R2are saturated alkyl groups. PG and PG” independently are protecting groups. The depicted synthesis proceeds in a stereospecific manner and not only stereoselective. With the use of starting materials being single stereoisomers or enantiomers the product obtained is a oligoglycerol-containing lipid being a single stereoisomer of Formula (II). Exemplarily the synthesis of diglycerols is shown.

[0052] A further synthesis overview is provided in the scheme below showing a method which is in particular suitable for the synthesis of cationizable or cationic lipids wherein R1and R2are unsaturated alkyl groups. PG and PG’ independently are protecting groups. The depicted synthesis proceeds in a stereospecific manner and not only stereoselective. With the use of starting materials being single stereoisomers or enantiomers the product obtained is a oligoglycerol-containing lipid being a single stereoisomer of Formula (II). Exemplarily the synthesis of diglycerols, triglycerols and tetraglycerols is shown.

[0053] A further synthesis overview is provided in the scheme below showing a method which is in particular suitable for the synthesis of anionic lipids wherein R1and R2are unsaturated or saturated alkyl groups. PG, PG’ and PG2independently are protecting groups. PG2in particular is acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl and phenoxyacetyl. The depicted synthesis proceeds without change of the configuration of the chiral centers in the starting materials. With the use of starting materials being single stereoisomers or enantiomers the product obtained is a oligoglycerol-containing lipid being a single stereoisomer of Formula (II). Exemplarily the synthesis of diglycerols, triglycerols and tetraglycerols is shown.

[0054] As reagents for phosphorylation phosphoramidite chlorides or phosphordiamidites are used, wherein R’ denotes a leaving group LG. R’ in particular is 2-cyanoethyl (CNE). R” is a 01-012 hydrocarbon group, in particular R” is methyl (Me), ethyl (Et), isopropyl (iPr) or benzyl (Bn), preferably R” is isopropyl.

[0055]

[0056] Synthesis of oligoglycerol building blocks in general is shown in the following schemes, wherein PG, PG’ and PG1independently are protecting groups. PG, PG’ and PG1may be orthogonal protecting groups. The depicted synthesis proceeds in a stereospecific manner and not only stereoselective. With the use of starting materials being single stereoisomers or enantiomers the product obtained is a oligogylcerol building block being a single stereoisomer of Formula (V) or (V’) or of Formula (VI1). Preferred synthesis for providing oligoglycerol-containing lipids having an ether linkage to residues R1and / or R2are given in the following. For preparing oligoglycerol-containing lipids having ether linkages to R1and R2, wherein R1and R2are a saturated hydrocarbon group, the steps are as follows:

[0057] A preferred synthesis route for preparing oligoglycerol-containing lipids, wherein R1and R2are linked via an ether bond and wherein R1and R2are a saturated or a mono- or polyunsaturated hydrocarbon group is as follows:

[0058] A preferred synthesis route for anionic oligoglycerol-containing lipids, in which preferably A

[0059] = PO4" having an ether linkage to R1and R2, wherein R1and R2can be a saturated or a mono- or polyunsaturated hydrocarbon group is as follows:

[0060] Synthesis of building blocks having two, three or four glycerol groups are preferably as shown below.

[0061]

[0062] G3building blocks:

[0063] 30

[0064] A preferred method using sn- 1,2-diacyl-glycerol for providing lipid building blocks for oligoglycerol-containing lipids having ester linkage to R1and / or R2, which are a mono- or polyunsaturated hydrocarbon group, is shown in the following, wherein PG, PG’ and PG2independently are protecting groups. PG2in particular is acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl and phenoxyacetyl.

[0065] A preferred method using sn- 1 ,2-diacyl-glycerol for providing lipid building blocks for oligoglycerol-containing lipids having an ether linkage to residues R1and / or R2, is shown in the following, wherein R1and / or R2are a saturated or a mono- or polyunsaturated hydrocarbon group. PG, PG’ and PG3independently are protecting groups. PG3in particular is Triphenylmethyl (Trityl).

[0066] The oligoglycerol-containing lipid of the present invention is in particular suitable for preparing liposomes. Therefor the present invention also relates to a liposome comprising an oligoglycerol-containing lipid of the invention.

[0067] The cationic surface charge of drug delivery systems DDS (e.g., liposomes, LNP) is conventionally achieved by mixing neutral glycerophospholipid excipients and / or cholesterol with cationic (e.g., 1 ,2-dioleoyl-3-trimethylammonium-propane, DOTAP) or ionizable lipid-like compounds (e.g., 1 ,2-dioleoyl-3-dimethylammonium-propane, DODAP). According to the invention a cationic surface charge is provided by oligoglycerol-modified cationic or cationizable lipids and in particular by diglycerol-modified cationic or cationizable lipids. The aim of the present invention was to provide a novel class of cationizable or cationic lipid-based excipients (AXGx). The novel lipids either carry a permanent or a pH- dependent variable positive charge and an oligoglycerol in the polar head group, respectively.

[0068] The cationizable NLG2 of formula (IIIA) and the cationic CLG2 of formula (IVA) are preferred lipids from the AXGxclass. Stearic acid or palmitic acid as esterified fatty acid and a diglycerol moiety as headgroup are most preferred. NLG2 and CLG2 are a cationizable and cationic analogue to the anionic glycerophospholipid DPPG2 (1 ,2-dipalmitoyl-sn- glycero-3-phospho-diglycerol).

[0069] The present invention further relates to a liposome comprising at least one oligoglycerolcontaining lipid of Formula (I).

[0070] The inventive liposome in particular comprises at least one oligoglycerol-containing lipid of Formula (I) wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0071] A = NR3or N+R4R5;

[0072] R3, R4and R5each independently are hydrogen or preferably a hydrocarbon group having

[0073] 1 to 5 C-atoms, and n = a number from 0 to 20, m at each occurrence independently is 0 or 1 ; y = 0 or 1 ; and z = 0 or 1. The inventive liposome preferably comprises at least one oligoglycerol-containing lipid of Formula (IA) wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0074] A = NR3or N+R4R5;

[0075] R3, R4and R5each independently are hydrogen or preferably a hydrocarbon group having 1 to 5 C-atoms; n = a number from 0 to 20, and m at each occurrence independently is 0 or 1 .

[0076] It has been found that the inventive cationizable or cationic lipids are perfectly suitable lipid excipients for liposomes. By the inventive lipids a positive charge can be conferred to the liposomes.

[0077] In a preferred embodiment the inventive liposome of the invention comprises at least one oligoglycerol-containing lipid of Formula (I) wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0078] A = NR3;

[0079] R3is hydrogen or preferably a hydrocarbon group having 1 to 5 C-atoms; n = a number from 0 to 20, m at each occurrence independently is 0 or 1 and; y = 0 or 1 and z = 0 or 1 ; and at least one phosphatidyloligoglycerol of Formula (VI) wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms and wherein n is a number from 0 to 20, and m at each occurrence independently is 0 or 1 ; and y = 0 or 1 ; and z = 0 or 1 .

[0080] In a preferred embodiment the inventive liposome of the invention comprises at least one oligoglycerol-containing lipid of Formula (IA) wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0081] A = NR3;

[0082] R3is hydrogen or preferably a hydrocarbon group having 1 to 5 C-atoms; n = a number from 0 to 20, and m at each occurrence independently is 0 or 1 ; and at least one phosphatidyloligoglycerol of Formula (VIA) wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms and wherein n is a number from 0 to 20, and m at each occurrence independently is 0 or 1. Preferred meanings of R1and R2, n and m are as given herein above. The phosphatidyloligoglycerol of Formula (VIA) is also termed DPPGXherein.

[0083] Preferably the phosphatidyloligoglycerol is of Formula (VI’) wherein the three stereogenic centers *1 , *2 and *3 have a predetermined configuration; and *p denotes further stereogenic centers; and wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms; n is a number from 0 to 20; m at each occurrence independently is 0 or 1 ; y = 0 or 1 ; and z = 0 or 1 ; and in particular wherein stereogenic center *1 is in R-configuration, stereogenic center *2 is in S- configuration and stereogenic center *3 is in R-configuration and *p denotes further stereogenic centers.

[0084] Preferably the phosphatidyloligoglycerol is of Formula (VI’A) wherein the three stereogenic centers *1 , *2 and *3 have a predetermined configuration; and *p denotes further stereogenic centers; and wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms; n is a number from 0 to 20; and m at each occurrence independently is 0 or 1 and in particular wherein stereogenic center *1 is in R-configuration, stereogenic center *2 is in S- configuration and stereogenic center *3 is in R-configuration and *p denotes further stereogenic centers.

[0085] Most preferably the phosphatidyloligoglycerol has Formula (VI”)

[0086] The phosphatidyloligoglycerol of Formula (VI”) is also termed DPPG2 herein.

[0087] Anionic lipids are preferably present with Na+as counterion.

[0088] In a further preferred embodiment the liposome of the invention comprises at least one oligoglycerol-containing lipid of Formula (I) wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0089] A = N+R4R5;

[0090] R4and R5each independently are hydrogen or preferably a hydrocarbon group having 1 to 5 C-atoms; n = a number from 0 to 20, and m at each occurrence independently is 0 or 1 ; and y = 0 or 1 ; and z = 0 or 1 ; and at least one ohgoglycerol-contaming lipid of Formula (I) wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0091] A = NR3;

[0092] R3is hydrogen or preferably a hydrocarbon group having 1 to 5 C-atoms; n = a number from 0 to 20, and m at each occurrence independently is 0 or 1 ; and y = 0 or 1 ; and z = 0 or 1.

[0093] In a further preferred embodiment the liposome of the invention comprises at least one oligoglycerol-containing lipid of Formula (IA) wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0094] A = N+R4R5;

[0095] R4and R5each independently are hydrogen or preferably a hydrocarbon group having 1 to 5 C-atoms; n = a number from 0 to 20, and m at each occurrence independently is 0 or 1 ; and at least one oligoglycerol-containing lipid of Formula (IA) wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms; A = NR3;

[0096] R3is hydrogen or preferably a hydrocarbon group having 1 to 5 C-atoms; n = a number from 0 to 20, and m at each occurrence independently is 0 or 1.

[0097] Preferred inventive liposomes comprise at least one cationizable oligoglycerol-containing lipid (A=NR3) and at least one anionic phosphatidyloligoglycerol; at least one cationizable oligoglycerol-containing lipid (A=NR3) and at least one cationic oligoglycerol-containing lipid (A=N+R4R5); or at least one cationic oligoglycerol-containing lipid (A=N+R4R5) and at least one anionic phosphatidyloligoglycerol. Such a preferred inventive liposome is a drug delivery system having oligoglycerol groups on its surface, while the surface charge is adjustable due to inclusion of cationic, cationizable and / or anionic groups in varying amounts. In particular, the zeta potential can be adjusted by the relative amounts of cationic and / or cationizable oligogylcerol-containing lipids of Formula (I) or (IA) and of phosphatidyloligoglycerols of Formula (VI) or (VIA).

[0098] The application of the three oligoglycerol-containing lipid excipients allows the customdesign of liposomal DDS regarding the magnitute of their surface charge and the length of the oligoglycerol-chains covering the surface. For the first time, the surface charge (anionic or cationic) is independently adjustable by the ratio of the three types of excipients (of anionic, cationic, and cationizable) while the amount of oligoglycerol moieties on the DDS surface can be kept constant (Figure 1). Moreover, the intra- and extraliposomal surface charge is independently adjustable by the pH of the intra- and extraliposomal aqueous buffer. Finally, the liposomal DDS can be designed to disintegrate in acidic environments (e.g., tumor tissue, lysosomes) to release their payload.

[0099] In general, the membrane bilayer of liposomal formulations is composed of more than one lipid excipient, but not every combination of lipids can be mixed in liposomal formulations to the same extend. The miscibility of the used lipids is essential to form homogeneous bilayer membranes and to ensure stability and functionality. In case of incompatibility of lipid excipients, the lipids segregate and phase separation or composition fluctuations will happen. Miscibility of phospholipids with identical headgroups but chain lengths differing by two methylene groups is dependent on headgroup structure and on headgroup charge. Therefore, miscibility might be remedied by adjusting the length and degree of saturation of the acyl / alkyl chains However, in individual cases, liposomal formulation can be designed to force lipid excipient de-mixing at defined conditions, e.g., heat-induced formation of lysolipid-assisted membrane pores in TSL.

[0100] It was found that oligoglycerol-containing lipids of Formula (I) or (IA) form homogeneous bilayer membranes and to ensure stability and functionality of liposomes including these lipids. Besides miscibility of at least two oligoglycerol-containing lipids of Formula (I) or (IA) surprisingly also miscibility of oligoglycerol-containing lipids of Formula (I) or (IA) with phosphatidyloligoglycerols of Formula (VI) or (VIA) was found allowing to provide liposomes having a tailored surface charge.

[0101] The inventive liposomes may comprise further excipients.

[0102] In an embodiment the liposome according to the invention preferably comprises at least one phosphatidylcholine of formula (VII) wherein R3and R4each independently represent a hydrocarbon group having from 12 to 24 carbon atoms. R3and R4are preferably each independently of the other a saturated or mono- or poly-unsaturated, preferably linear alkyl group, in particular a saturated linear alkyl group. R3and R4are further preferably each independently a C13 to C19, in particular a C15 to C17 hydrocarbon group. Preferably, R3and R4are each independently a linear saturated C13- to C23-, in particular a C13- to C21-alkyl group. Most preferably, R3and R4are independently a linear saturated C15- or C17-alkyl group. 1 ,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC) and 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) are particularly preferred.

[0103] The liposome, and in particular the thermosensitive liposome according to the invention, preferably comprises a phosphatidylcholine of formula (VII) in the natural configuration.

[0104] In a further embodiment the liposome comprises cholesterol.

[0105] In a preferred embodiment the inventive liposome is a thermosensitive liposome. Thermosensitive liposomes (TSL) are improved liposomal drug delivery systems allowing active targeting of tumor tissue via heat-induced local release of the encapsulated active pharmaceutical ingredients (APIs). In preferred embodiments of the invention the liposome comprises (all mol.-% are based on the lipid content of the liposome):

[0106] (1) NLGX, in particular NLG2, preferably in an amount of at least 1 mol.%, more preferably at least 10 mol.-% ; and / or

[0107] (2) CLGx, in particular CLG2, preferably in an amount of at least 1 mol.%, more preferably at least 10 mol.-% ; and / or

[0108] (3) NLGX, in particular NLG2, and CLGX, in particular CLG2, preferably in an amount of at least 1 mol.% each, more preferably at least 10 mol.-% each; and / or

[0109] (4) NLGX, in particular NLG2, preferably in an amount of at least 1 mol.%, more preferably at least 10 mol.-% ; and at least one phosphatidylcholine, preferably in an amount of at least 1 mol.-%, preferably at least 10 mol.-%; and / or

[0110] (5) NLGX, in particular NLG2, preferably in an amount of at least 1 mol.%, more preferably at least 5 mol.-% and up to 80 mol.-%, more preferably up to 75 mol.-% ; and DPPC, preferably in an amount of at least 1 mol.%, more preferably at least 5 mol.-% and up to 80 mol.-%, more preferably up to 75 mol.-%; and DSPC, preferably in an amount of at least 1 mol.%, more preferably at least 10 mol.-% and up to 30 mol.-%, more preferably up to 25 mol.-%; and / or

[0111] (6) NLGX, in particular NLG2, preferably in an amount of at least 1 mol.%, more preferably at least 5 mol.-% and up to 50 mol.-%, more preferably up to 40 mol.-%; and at least one phosphatidylcholine, preferably DSPC and / or DPPC, preferably in an amount of at least 1 mol.-%, preferably at least 20 mol.-% .-% and up to 70 mol.-%, more preferably up to 60 mol.-%; and cholesterol, preferably in an amount of at least 1 mol.-%, preferably at least 30 mol.-% and up to 70 mol.-%, more preferably up to 50 mol.-%; and / or

[0112] (7) CLGx, in particular CLG2, preferably in an amount of at least 1 mol.%, more preferably at least 10 mol.-% ; and at least one phosphatidylcholine, preferably in an amount of at least 1 mol.-%, preferably at least 10 mol.-%; and / or

[0113] (8) CLGx, in particular CLG2, preferably in an amount of at least 1 mol.%, more preferably at least 5 mol.-% and up to 80 mol.-%, more preferably up to 75 mol.-% ; and DPPC, preferably in an amount of at least 1 mol.%, more preferably at least 5 mol.-% and up to 80 mol.-%, more preferably up to 75 mol.-%; and DSPC, preferably in an amount of at least 1 mol.%, more preferably at least 10 mol.-% and up to 30 mol.-%, more preferably up to 25 mol.-%; and / or

[0114] (9) CLGX, in particular CLG2, preferably in an amount of at least 1 mol.%, more preferably at least 5 mol.-% and up to 50 mol.-%, more preferably up to 40 mol.-%; and at least one phosphatidylcholine, preferably DSPC and / or DPPC, preferably in an amount of at least 1 mol.-%, preferably at least 20 mol.-% .-% and up to 70 mol.-%, more preferably up to 60 mol.-%; and cholesterol, preferably in an amount of at least 1 mol.-%, preferably at least 30 mol.-% and up to 70 mol.-%, more preferably up to 50 mol.-%; and / or

[0115] (10) NLGX, in particular NLG2, and CLGX, in particular CLG2, preferably in an amount of at least 1 mol.% each, preferably at least 10 mol.-% each; and cholesterol, preferably in an amount of at least 1 mol.-%, preferably at least 30 mol.-% and up to 70 mol.-%, more preferably up to 50 mol.-%; and / or

[0116] (11) NLGX, in particular NLG2, and CLGX, in particular CLG2, preferably in an amount of at least 1 mol.% each, preferably at least 10 mol.-% each; and at least one phosphatidylcholine, preferably in an amount of at least 1 mol.-%, preferably at least 10 mol.-%; and / or

[0117] (12) NLGX, in particular NLG2, and CLGX, in particular CLG2, preferably in an amount of at least 1 mol.% each, preferably at least 10 mol.-% each; and at least one phosphatidylcholine, preferably DSPC and / or DPPC, preferably in an amount of at least 1 mol.-%, preferably at least 20 mol.-% .-% and up to 70 mol.-%, more preferably up to 60 mol.-% and cholesterol, preferably in an amount of at least 1 mol.-%, preferably at least 30 mol.-% and up to 70 mol.-%, more preferably up to 50 mol.-%; and / or

[0118] (13) NLGX, in particular NLG2, preferably in an amount of at least 1 mol.%, more preferably at least 5 mol.-% and up to 80 mol.-%, more preferably up to 75 mol.-% ; and CLGX, in particular CLG2, preferably in an amount of at least 1 mol.%, more preferably at least 5 mol.- % and up to 80 mol.-%, more preferably up to 75 mol.-%; and DPPC, preferably in an amount of at least 1 mol.%, more preferably at least 5 mol.-% and up to 80 mol.-%, more preferably up to 75 mol.-%; and DSPC, preferably in an amount of at least 1 mol.%, more preferably at least 10 mol.-% and up to 30 mol.-%, more preferably up to 25 mol.-%; and / or (14) NLGX, in particular NLG2, preferably in an amount of at least 1 mol.%, more preferably at least 5 mol.-% and up to 99 mol.-%, more preferably up to 95 mol.-% ; and DPPGX, in particular DPPG2, preferably in an amount of at least 1 mol.%, more preferably at least 5 mol.-% and up to 99 mol.-%, more preferably up to 95 mol.-%;

[0119] (15) NLGX, in particular NLG2, and DPPGX, in particular DPPG2, preferably in an amount of at least 1 mol.% each, preferably at least 10 mol.-% each; and cholesterol, preferably in an amount of at least 1 mol.-%, preferably at least 30 mol.-% and up to 70 mol.-%, more preferably up to 50 mol.-%;

[0120] (16) NLGX, in particular NLG2, and DPPGX, in particular DPPG2, preferably in an amount of at least 1 mol.% each, preferably at least 10 mol.-% each; and at least one phosphatidylcholine, preferably in an amount of at least 1 mol.-%, preferably at least 10 mol.-%;

[0121] (17) NLGX, in particular NLG2, and DPPGX, in particular DPPG2, preferably in an amount of at least 1 mol.% each, preferably at least 10 mol.-% each; and at least one phosphatidylcholine, preferably DSPC and / or DPPC, preferably in an amount of at least 1 mol.-%, preferably at least 20 mol.-% .-% and up to 70 mol.-%, more preferably up to 60 mol.-% and cholesterol, preferably in an amount of at least 1 mol.-%, preferably at least 30 mol.-% and up to 70 mol.-%, more preferably up to 50 mol.-%; and / or

[0122] (18) NLGX, in particular NLG2, preferably in an amount of at least 1 mol.%, more preferably at least 5 mol.-% and up to 80 mol.-%, more preferably up to 75 mol.-% ; and DPPGX, in particular DPPG2, preferably in an amount of at least 1 mol.%, more preferably at least 5 mol.-% and up to 80 mol.-%, more preferably up to 75 mol.-%; and DPPC, preferably in an amount of at least 1 mol.%, more preferably at least 5 mol.-% and up to 80 mol.-%, more preferably up to 75 mol.-%; and DSPC, preferably in an amount of at least 1 mol.%, more preferably at least 10 mol.-% and up to 30 mol.-%, more preferably up to 25 mol.-%.

[0123] By setting the amounts and proportions respectively of CLGX, NLGXand DPPGXthe surface charge of the inventive liposomes can be adjusted.

[0124] The inventive liposome further preferably comprises an active agent, in particular a cytostatic drug. Most preferred the inventive liposome comprises doxorubicin (DOX) which is a cytotoxic anthracycline. Further suitable cytostatic anthracyclines presently employed in cancer treatment include, epirubicin, idarubicin, daunorubicin and mitoxantrone. In further embodiments of the invention other cytostatics such as mitomycin C, gemcitabine, trabectedin, etc. or platinum derivatives such as cisplatin, carboplatin or oxaliplatin can be included in the liposome.

[0125] In a further preferred embodiment a liposome according to the invention, comprises an active pharmaceutical ingredient which is selected from the group consisting of anthracyclines such as doxorubicin, daunorubicin, idarubicin, epirubicin aclarubicin, amrubicin, pirarubicin, valrubicin and zorubicin; anthracenediones such as mitoxantrone and pixantrone; antineoplastic antibiotics such as mitomycin and bleomycin; vinca alkaloids such as vinblastine, vincristine and vinorelbine; alkylating agents such as cyclophosphamide and mechlorethamine hydrochloride: campthothecins such as topotecan, irinotecan (CPT-1 1), lurtotecan, 9-aminocamptothecin, 9-nitrocamptothecin and 10-hydroxycamptothecin; purine and pyrimidine derivatives such as 5-fluorouracil, gemcitabine (2,2'-difluoro-2-deoxycytidine, dFdC), floxuridine (FLIDR), cytarabine (cytosine arabinoside), 6-azauracil (6-All); oxazaphosphorines such as cyclophosphamide, ifosfamide and trofosfamide; taxanes such as paclitaxel and docetaxel; podophyllotoxin derivatives such as etopside and teniposide; platinum-based compounds such as cisplatin, carboplatin, oxaliplatin, nedaplatin; methotrexate; tyrosine kinase inhibitors such as imatinib, gefitinib, erlotinib, sunitinib, adavosertib and lapatinib; and cytarabines such as cytosine arabinoside.

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[0146] Petrini M, Lokerse WJM, Mach A, Hossann M, Merkel OM, Lindner LH. Effects of surface charge, PEGylation and functionalization with dipalmitoylphosphatidyldiglycerol on liposome-cell interactions and local drug delivery to solid tumors via thermosensitive liposomes. Int J Nanomedicine. 2021 ;16:4045-4061. DOI: 10.2147 / IJN.S305106.

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[0149] Figure 1 : Application of novel anionic, cationic, and / or cationizable lipid excipient in liposomal formulations.

[0150] Figure 2: Dispersion stability (DLS) of binary mixtures of NLG2, CLG2 and DPPG2. Dotted red lines marks quality criteria for size and PDI. Red squares indicate PDI values OOS. Numbers below the bars indicate extrusion pore size. Hydration solution.

[0151] Figure 3: Dispersion stability (DLS) of ternary mixtures of HSPC / Chol / AXGx. Z-average and PDI of (A) HSPC / CI-10I / CLG2 60-x:40:x (mol / mol) and (B) HSPC / Chol / NLG260-x:40:x (mol / mol) batches. Zeta potential of (C) HSPC / Chol / CLG2 60-x:40:x (mol / mol) and (D) HSPC / Chol / NLG2 60-x:40:x (mol / mol) batches, (x = 5 to 40). The dotted red lines mark the quality criteria for z average and PDI. Numbers below graphs (A) and (B) indicate the used extrusion membrane pore size during batch production.

[0152] Figure 4: Dispersion stability (DLS) of ternary mixtures of CLG2 / NLG2 / Chol. Investigated formulations: CLG2 / NLG2 / Chol 50:10:40 (mol / mol) and CLG2 / NLG2 / Chol 30:30:40 (mol / mol). (A) Z-average and PDI (after preparation), and (B) zeta potential. The dotted red lines mark the quality criteria for z average and PDI. Numbers below graphs indicate the used extrusion membrane pore size during batch production.

[0153] Figure 5: Effect of NLG2 content in TSL on the solid gel to liquid disordered phase transition temperature (Tm). Cooling curves from top to bottom for 5 mol% NLG2, 10 mol% NLG2, 20 mol% NLG2, 30 mol% NLG2 and heating curves from bottom to top for 5 mol% NLG2, 10 mol% NLG2, 20 mol% NLG2, 30 mol% NLG2. Refer to Table 1 for the corresponding peak temperatures of the heating and cooling phase.

[0154] Figure 6: Effect of extraliposomal pH on zeta potential of DPPC / DSPC / NLG2 liposomes. The assay was performed with (A) DPPC / DSPC / NLG2 75 / 20 / 5 (mol / mol) liposomes, (B) DPPC / DSPC / NLG2 70 / 20 / 10 (mol / mol) liposomes, (C) DPPC / DSPC / NLG2 60 / 20 / 20 (mol / mol) liposomes, and (D) DPPC / DSPC / NLG2 50 / 20 / 30 (mol / mol) liposomes.

[0155] Figure 7: Effect of extraliposomal pH on z average & PDI of DPPC / DSPC / NLG2 liposomes. The assay was performed with (A) DPPC / DSPC / NLG2 75 / 20 / 5 (mol / mol) liposomes, (B) DPPC / DSPC / NLG2 70 / 20 / 10 (mol / mol) liposomes, (C) DPPC / DSPC / NLG2 60 / 20 / 20 (mol / mol) liposomes, and (D) DPPC / DSPC / NLG2 50 / 20 / 30 (mol / mol) liposomes. Mean and S.D. of three experiments.

[0156] Figure 8: In vitro complement activation of AXGx-based liposomes. ELISA on in vitro complement activation of different liposomal formulations in human plasma. SC5b-9 was used as read-out. Zymosan: positive control. Buffer: negative control. Red dotted line indicates the SC5b-9 value induced by the control.

[0157] Figure 9: Binding of cationizable DPPC / DSPC / NLG2 liposomes to B16 cells. Live cell fluorescence microscopy: bright field (top) and RGB merged pictures (bottom). Scale bar = 50 pm.

[0158] Figure 10: Binding of neutral and anionic liposomes to B16 cells. Live cell fluorescence microscopy: bright field (top) and RGB merged pictures (bottom). Scale bar = 50 pm.

[0159] Figure 11 : CLG2-based TSL showed agglutination of THP-1 cells. (A) Cells treated with DPPC / DSPC / CLG2 70:20:10 (mol / mol) (refer also to Table 3, #6). (B) Control.

[0160] Figure 12: Vesicle size and zeta potential of DPPC / DSPC / NLG2 TSL loaded with CF. Selected DLS results: (A) Vesicle size (z-average size) and PDI. The dotted line marks vesicle size and PDI acceptance criteria. (B) Zeta potential. N=3 for batches extruded with 100 or 50 nm filters. N=1 for batches extruded with 200 nm filters.

[0161] Figure 13: Effect of vesicle size and NLG2 content on the temperature-dependent drug release (TDR) profile of DPPC / DSPC / NLG2 TSL after 5 min from 37°C to 45°C (upper graphs) and 1h for 37°C and 42°C (lower graph) with columns from left to right for 50 mM NaCI, 50 nm FCS, 100 nm NaCI, 100 nm FCS, 200 nm NaCI, 200 nm FCS for each temperature. The dotted line marks 50% CF-release.

[0162] Figure 14: Effect of CLG2 content on the temperature-dependent drug release (TDR) profile of DPPC / DSPC / CLG2 TSL. Temperature dependent CF release was measured at distinct temperatures for an incubation time of (A) 5 minutes and (B) 60 minutes. Red dashed line indicates peak release within mild-HT temperature range (41-43 °C).

[0163] Figure 15: Effect of DOXJipid ratio on heat-triggered drug release from DPPC / DSPC / CLG2 TSL. (A,C) Temperature-dependent drug release (TDR) profile for DPPC / DSPC / CLG2 75:20:5 (mol / mol), (B,D) TDR profile for DPPC / DSPC / CLG2 70:20:10 (mol / mol). Examples

[0164] Example 1 : Synthesis route of saturated cationic / cationizable lipids

[0165] Example 1.1 : Synthesis of ester linked lipids

[0166] The synthesis route for new ester linked lipids NLG2 and CLG2 is depicted below. The synthesis proceeds via a central intermediate of formula (VI’), in particular 26.1a. NLG2 is obtained in 11 synthesis steps in total and 3 steps from 26.1a. The cationic CLG2 was obtained in 2 additional steps from NLG2. The synthesis route is applicable to other AXGxlipids. The central intermediate is benzyl-protected (26.1a) and the fatty acid chloride is fully saturated and does not contain double bonds, respectively.

[0167] Synthesis route of NLG2 and CLG2

[0168] 3.8 kg (R)-(+)-glycidol are dissolved in 22.8 L NMP, then 8.02 kg PMBCI are added followed by 2.26 kg NaH at 0-5 °C. After 4 hours at RT, the reaction mixture is poured into ice water and extracted with EtOAc. The organic phase is washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The isolated residue is purified by silica gel chromatography (n-Heptane / Ethyl acetate) and 8.0 kg of 1.1a are obtained as colorless oil. It is characterized by HPLC, TLC and1H-NMR.

[0169] 1H NMR of 1.1a (400 MHz, CDCI3):

[0170] 5 ppm 7.29 (d, J = 10.8 Hz, 2H), 6.89 (d, J = 10.8 Hz, 2H), 4.53 (q, J = 11.6 Hz, 2H), 3.79 (s, 3H), 3.74 (dd, J = 3.2, 11.6 Hz, 1 H), 3.41 (dd, J = 6.0, 11.6 Hz, 1 H), 3.17 (m, 1 H), 2.79 (dd, J = 4.0, 9.2 Hz, 1 H), 2.61 (dd, J = 2.4, 4.8 Hz, 1 H).

[0171] Step 2+3:

[0172] 8.0 kg of 1.1a are dissolved in 64 L DMF, then 8.16 kg sn-1 ,2-IPG is added, followed by 6.94 kg tBuOK at 0-5 °C and the resulting reaction mixture stirred for 12 h at RT to obtain 2.1a. Characterization is done with TLC and HPLC. Subsequently, without work up or further purification additional 6.91 kg tBuOK and 12.6 kg benzyl bromide are added at 0-5 °C and the resulting reaction mixture stirred at 4 h at RT. Then, the reaction mixture is poured into ice water and extracted with EtOAc. The organic phase is washed with brine, dried over Na2SO4 and concentrated under reduced pressure. 7.0 kg of pure compound 3.1a is obtained as yellow oil after purification by silica gel chromatography (n- Heptane / Ethyl acetate). 3.1a is characterized with TLC, HPLC, and1H-NMR.

[0173] 1H NMR of 3.1a (400 MHz, CDCI3):

[0174] 5 ppm 7.39 - 7.26 (m, 8H), 6.92 (d, J = 8.4 Hz, 2H), 4.71 (s, 2H), 4.50 (s, 2H), 4.27 (d, J = 6.0 Hz, 1 H), 4.10 - 4.02 (m, 1 H), 3.85 (s, 3H), 3.83 - 3.47 (m, 9H), 1.44 (s, 3H), 1.39 (s, 3H).

[0175] Step 4:

[0176] 379 g 3.1a are dissolved in 2.65 L MeOH and then cooled to 0 °C, where 2 L 1M HCI are slowly added. The reaction mixture is stirred at 25 °C for 4 h. Then, saturated NaHCCh solution is added to adjust the mixture to pH = 7 at 0 - 5 °C, followed by extracted with EtOAc. The organic phase is washed brine, dried over Na2SO4, filtered and concentrated under reduced pressure to obtain 310 g of crude 5.1a as yellow oil.

[0177] Step 5:

[0178] 310 g of 5.1a are dissolved in 2.5 L DMF and then cooled to 0 °C, where 369 g tBuOK and 422 g BnBr are added. The reaction mixture is stirred at 25 °C for 4 h, before being poured into ice water followed by extraction with EtOAc. The organic phase is washed brine, dried over Na2SO4, filtered and concentrated under reduced pressure. After silica column chromatography (n-heptane / EtOAc) 115 g of pure 6.1a are obtained as yellow oil.1H NMR of 6.1a (400 MHz, CDCI3):

[0179] 5 ppm 7.32 - 7.10 (m, 17H), 6.81 - 6.74 (m, 2H), 4.60 (d, J = 4.0 Hz, 4H), 4.45 (s, 2H), 4.37 (s, 2H), 3.71 (s, 3H), 3.70 - 3.65 (m, 2H), 3.55 - 3.44 (m, 8H).

[0180] Step 6:

[0181] 331 g of 6.1 a are dissolved in 3 L ACN and 332 ml H2O and then cooled to 0 °C, where 652 g cerium ammonium nitrate (CAN) are added. The reaction mixture is stirred at 15 °C for 8 h, before being poured into ice water followed by extraction with EtOAc. The organic phase is washed brine, dried over Na2SC>4, filtered and concentrated under reduced pressure. After silica column chromatography (n-heptane / EtOAc) 168 g of pure 7.1a are obtained as yellow oil.

[0182] 1H NMR of 7.1a (400 MHz, CDCI3):

[0183] 5 ppm 7.32 - 7.10 (m, 17H), 6.81 - 6.74 (m, 2H), 4.60 (d, J = 4.0 Hz, 4H), 4.45 (s, 2H), 4.37 (s, 2H), 3.71 (s, 3H), 3.70 - 3.65 (m, 2H), 3.55 - 3.44 (m, 8H).

[0184] Step 7:

[0185] 153 g 7.1a are dissolved in 765 ml DCM, then 71 g triethylamine added, followed by dropwise addition of 50.1 g MsCI. The reaction mixture is stirred at 20 °C for 2 h, before being poured into water followed by extraction with DCM. The organic phase is dried over Na2SC>4, filtered and concentrated under reduced pressure to obtain 183 g of crude 25.1a as yellow oil.

[0186] Step 8:

[0187] 183 g 25.1a are dissolved in 366 ml EtOH and the resulting solution is added to 1.48 kg of a MeNH2 solution (33% wt) in EtOH. The reaction mixture is warmed to 55°C and stirred for 48 h, before evaporation to dryness. The residue is taken up in MeOH / toluene and washed with 10 % aqueous Na2COs solution, before concentration under reduced pressure to obtain 151 g of crude 26.1a as yellow oil.

[0188] 1H NMR of 26.1a (400 Hz, CDCI3):

[0189] 5 ppm 7.30 - 7.18 (m, 16H), 4.65 - 4.61 (m, 3H), 4.51 - 4.46 (m, 3H), 3.69 (td, J = 5.2, 8.0 Hz, 2H), 3.57 - 3.45 (m, 6H), 2.61 (d, J = 5.6 Hz, 2H), 2.30 (s, 3H).

[0190] Step 9:

[0191] 43.6 g 26.1a are dissolved in 109 ml toluene and then a solution of 9 g glycidol in 109 ml toluene added. The reaction mixture is warmed to 100°C and stirred for 8 h. 3 batches are run in parallel, then combined and evaporated to dryness. The residue is taken up in MTBE and adjusted to pH5 with HCI and then phases separated. The aqueous phase is adjusted to pH8 with saturated NaHCCh solution and extracted with ethyl acetate, before concentration of the organic phase under reduced pressure to obtain 161 g of crude 35.1a as yellow oil.

[0192] Step 10:

[0193] 161 g 35.1 a are dissolved in 800 ml DCM and then 62.2 g triethylamine, 11.2 g DMAP and 169 g palmitoyl chloride added subsequently. The resulting reaction mixture is stirred at 20°C for 1 h, before pouring into water. After separation the organic phase is concentrated under reduced pressure and then purified by silica column chromatography (n- heptane / EtOAc) to obtain 146 g of pure 36.1a as yellow oil.

[0194] 1H NMR of 36.1a (400 Hz, CDCI3):

[0195] 5 7.36 - 7.15 (s, 16H), 5.20 - 5.13 (m, 1 H), 4.72 - 4.53 (m, 6H), 4.35 (dd, J = 3.2, 12.0 Hz, 1 H), 4.10 - 4.04 (m, 1 H), 3.79 - 3.70 (m, 1 H), 3.68 - 3.51 (m, 8H), 2.61 - 2.51 (m, 4H), 2.31 - 2.24 (m, 7H), 2.06 (s, 1 H), 1.64 - 1.55 (m, 6H), 1.26 (br s, 53H), 0.89 (t, J = 7.2 Hz, 6H).

[0196] 11 :

[0197] 6 g Pd / C is suspended in 300 ml AcOH and then 67 g of 36.1a are added and the reactor flushed with 30 psi H2. The resulting reaction mixture is stirred at 30°C for 24 h. 2 batches are run in parallel and worked up together. After filtration through celite, the filtrate is poured into water and the pH adjusted to 7 with saturated NaHCCh solution. After extraction with ethyl acetate, the organic phase is concentrated under reduced pressure. The residue is recrystallized from ACN, followed by purification via silica column chromatography (n- heptane / EtOAc / MeOH) to obtain 38.6 g of pure NLG2 as white solid.

[0198] 1H NMR of NLG2(400 Hz, CDCI3):

[0199] 5 5.22 (dd, J = 2.4, 4.8 Hz, 1 H), 4.30 (dd, J = 3.2, 12.0 Hz, 1 H), 4.08 (dd, J = 6.4, 12.0 Hz, 1 H), 3.91 - 3.79 (m, 2H), 3.72 - 3.44 (m, 7H), 2.67 (dd, J = 7.6, 13.6 Hz, 1 H), 2.56 - 2.47 (m, 2H), 2.43 - 2.37 (m, 1 H), 2.35 - 2.27 (m, 7H), 1.65 - 1.56 (m, 4H), 1.35 - 1.20 (m, 52H), 0.87 (t, J = 7.2 Hz, 6H).

[0200] 12:

[0201] 20 g NLG2 are suspended in 100 ml ACN and then 7.8 g methyl iodide is added. The resulting reaction mixture is stirred at 40°C for 16 h. After filtration and concentration under reduced pressure 21 g of crude 38.1a are obtained as white solid.1H NMR of 38.1a (400 Hz, DMSO)

[0202] 5 ppm 5.56 - 5.48 (m, 2H), 4.62 (d, J = 4.8 Hz, 1 H), 4.50 (br t, J = 5.4 Hz, 1 H), 4.39 (dd, J = 3.2, 12.0 Hz, 1 H), 4.22 (br s, 1 H), 4.05 (dd, J = 5.6, 12.0 Hz, 1 H), 3.82 (br d, J = 3.6 Hz, 2H), 3.63 - 3.55 (m, 1 H), 3.49 - 3.39 (m, 4H), 3.37 - 3.28 (m, 5H), 3.18 - 3.09 (m, 6H), 2.42 - 2.25 (m, 4H), 1 .57 - 1 .47 (m, 4H), 1 .23 (s, 48H), 0.88 - 0.81 (m, 6H).

[0203] Step 13:

[0204] 21 g 38.1a are added to a 315 ml 1 :1 :1 mixture of toluene, 2-propanol and a 10% aqueous NaCI solution. The resulting reaction mixture is stirred at 35°C for 48 h. After isolation, crude product is purified by recrystallization from 2-propanol, followed by multiple triturations with 2-propanol at RT for 1 h to obtain 10 g pure CLG2 as white solid. Characterization by ICP- MS confirms CLG2 as chloride salt.

[0205] 1H NMR of CLG2(400 Hz, DMSO):

[0206] 5 5.65 - 5.51 (m, 2H), 4.43 (br d, J = 2.0 Hz, 1 H), 4.41 - 4.36 (m, 1 H), 4.28 (br d, J = 4.4 Hz, 2H), 4.10 (br dd, J = 5.2, 12.0 Hz, 1 H), 3.94 - 3.79 (m, 2H), 3.62 (br s, 1 H), 3.54 - 3.34 (m, 9H), 3.19 (br d, J = 3.2 Hz, 6H), 2.39 - 2.28 (m, 4H), 1 .56 (br d, J = 6.4 Hz, 4H), 1 .26 (br s, 48H), 0.93 - 0.80 (m, 6H).

[0207] Example 1.2: Synthesis of ether linked lipids

[0208] The synthesis route for new ether linked lipids 83.1a and 85.1a is depicted below. The synthesis proceeds via a central intermediate of formula (VI’), in particular 26.1a. 83.1a is obtained in 8 synthesis steps in total and 2 steps from 26.1a. The cationic 85.1a was obtained in 2 additional steps from 83.1a. The synthesis route is applicable to other ether- linked AXGxlipids. The central intermediate is benzyl-protected (26.1a) and the fatty alcohols are fully saturated and does not contain double bonds, respectively.

[0209]

[0210] Step 1 :

[0211] Trifluoromethanesulfonic anhydride is added to DCM at 0 °C, followed by anhydrous pyridine, leading to formation of a white precipitate. The cooling bath is removed and a solution of palmityl alcohol 70.1 in DCM is added dropwise over a period of 10 min under stirring. The solution is stirred at RT until completion of the reaction. Afterward, water is added, and the mixture extracted with DCM. The combined organic layers are washed with brine, dried over MgSCU, and evaporated under reduced pressure. The resulting residue is purified by silica gel chromatography (n-hexane / diethyl ether) to obtain 71.1 as a waxy white solid.

[0212] Step 2:

[0213] 1 ,2-lsopropylidene-rac-glycerol is dissolved in NMP, then PMBCI is added and the solution cooled to 0 °C. NaH is carefully added and the resulting reaction mixture is stirred at 25 °C until completion of the reaction. The mixture is poured into ice water and extracted with EtOAc. The organic phase is washed with brine, dried over Na2SC>4 and concentrated under reduced pressure. The isolated residue is purified by silica gel chromatography (n- Heptane / Ethyl acetate) to obtain crude 31.2.

[0214] Step 3:

[0215] 31.2 is dissolved in MeOH and the solution cooled to 0 °C. Concentrated HCI is carefully added and the resulting reaction mixture is stirred at 20 °C until completion of the reaction. The mixture is quenched with saturated aqueous K2CO3 solution to adjust the pH to 7-8, followed by extraction with EtOAc. The organic is dried over Na2SC>4 and concentrated under reduced pressure to obtain crude 32.2.

[0216] Step 4:

[0217] DCM is added to a mixture of 71.1 , 32.2 and 1 ,8-bis(dimethylamino) naphthalene. The reaction mixture is stirred under reflux until completion of the reaction. DCM is removed under reduced pressure. Hexane is added and the mixture sonicated before filtration through Celite. The filtrate is evaporated under reduced pressure and the residue purified by silica gel chromatography (n-heptane / ethyl acetate) to yield 72.1.

[0218] Step 5:

[0219] 72.1 is dissolved in DCM / H2O 10:1 (vol / vol) and the mixture cooled to 0 °C. 2,3-dichloro- 5,6-dicyano-1 ,4-benzoquinone (DDQ) is added and the mixture stirred at RT until the reaction is complete. The reaction mixture is filtered and the filtrate washed with aq. NaHCO3 and water. The organic phase is dried over Na2SC>4 and the solvent removed under reduced pressure. After work up, crude residue is purified with silica gel chromatography (n-Heptane / Ethyl acetate) to obtain 73.1.

[0220] Step 6:

[0221] 73.1, triphenylphosphine, tetrabutylammonium iodide and anhydrous 1 ,2-dibromoethane are combined in a flask and heated to 60 °C. The reaction mixture is stirred at 60 °C until the reaction is complete. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was purified by silica column chromatography (n- heptane / ethyl acetate) to afford 74.1.

[0222] Step 7:

[0223] 26.1a, N,N-diisopropylethylamine and 74.1 are dissolved in acetonitrile and stirred at room temperature under nitrogen until completion of the reaction. Then the solvent is removed under reduced pressure. The residue is dissolved in DCM and washed with water. The organic fractions are dried over MgSC>4 and the solvent is removed under reduced pressure to yield the 82.1a.

[0224] Step 8:

[0225] Pd / C is suspended in AcOH and then 82.1a is added and the reactor flushed with 30 psi H2. The resulting reaction mixture is stirred at 30°C until completion of the reaction. After filtration through celite, the filtrate is poured into water and the pH adjusted to 7 with saturated NaHCCh solution. After extraction with ethyl acetate, the organic phase is concentrated under reduced pressure. The residue is recrystallized from ACN, followed by purification via silica column chromatography (n-heptane / EtOAc / MeOH) to obtain pure 83.1a as white solid. Step 9:

[0226] 83.1a is suspended in ACN and then methyl iodide is added. The resulting reaction mixture is stirred at 40°C until completion of the reaction. After filtration and concentration under reduced pressure crude 84.1a is obtained as white solid. Step 10:

[0227] 84.1a is added to a 1 :1 :1 mixture of toluene, 2-propanol and a 10% aqueous NaCI solution. The resulting reaction mixture is stirred at 35°C until completion of the reaction. After isolation, crude product is purified by recrystallization from 2-propanol, followed by multiple triturations with 2-propanol at RT for 1 h to obtain pure 85.1a as white solid. Characterization by ICP-MS confirms 85.1a as chloride salt.

[0228] Example 2: Synthesis route of unsaturated cationic / cationizable lipids

[0229] Example 2.1 : Synthesis of ester linked lipids

[0230] The synthesis route for unsaturated new lipids 46.1a and 52.1a is depicted below. The synthesis proceeds via a central intermediate of formula (V), in particular 28.1a. 46.1a is obtained in 9 synthesis steps in total and 3 steps from 28.1a. The cationic lipid 52.1a was obtained in 2 additional steps from 46.1a. The synthesis route is applicable to other AXGxlipids. The central intermediate is silyl- and acetonide-protected (28.1a) and the fatty acid chloride is monounsaturated.

[0231] Step 1 :

[0232] (R)-(+)-glycidol is added to a suspension of t-BuOK in DMF at 0 °C, followed by BnBr. The resulting reaction mixture is warmed to RT and stirred until completion of the reaction. Afterward, water is added, and the mixture extracted with MTBE. The organic layer is washed with water and brine, dried over Na2SC>4, filtered and concentrated under reduced pressure.

[0233] The resulting residue is purified by silica gel chromatography (n-Heptane / Ethyl acetate) to obtain 1.2a.

[0234] Step 2:

[0235] NaOH is added to sn-1 ,2-IPG, followed by heating up to 80 °C, where 1.2a is added and the resulting reaction mixture is stirred at 80 °C until completion of the reaction. Then, the reaction mixture is poured into water and extracted with isopropyl ethyl acetate. The organic phase is washed with brine, dried over Na2SO4 and concentrated under reduced pressure to obtain crude 2.2a. Step 3:

[0236] Triisopropylsilyl chloride is added to a solution of 2.2a and imidazole in DMF and the resulting reaction mixture is stirred at RT until completion of the reaction. Then, the reaction mixture is poured into water and extracted with EtOAc. The organic phase is washed with 10% citric acid and brine, then dried over Na2SC>4 and concentrated under reduced pressure to obtain crude 3.3a.

[0237] Step 4:

[0238] 3.3a is added to a suspension of Pd / C in EtOH / DCM, followed by degassing. The resulting mixture is stirred under H2 (0.5 MPa) at RT until completion of the reaction. The mixture is filtered and the filtrate concentrated under reduced pressure. Crude 4.3a is purified with silica gel chromatography (n-Heptane / Ethyl acetate).

[0239] Step 5:

[0240] 4.2a dissolved in DCM, then triethylamine added, followed by dropwise addition of MsCI. The reaction mixture is stirred at 20 °C until completion of the reaction. Then, the mixture is poured into water followed by extraction with DCM. The organic phase is dried over Na2SC>4, filtered and concentrated under reduced pressure to obtain crude 27.1a.

[0241] Step 6:

[0242] 27.1a is dissolved in EtOH and the resulting solution is added to a MeNH2 solution (33% wt) in EtOH. The reaction mixture is warmed to 55°C and stirred until completion of the reaction, followed by evaporation to dryness. The residue is taken up in MeOH / toluene and washed with 10 % aqueous Na2CO3 solution, before concentration under reduced pressure to obtain crude 28.1a.

[0243] Step 7:

[0244] 28.1a is dissolved in toluene and then a solution of glycidol in toluene added. The reaction mixture is warmed to 100°C and stirred until completion of the reaction, followed by evaporation to dryness. The residue is taken up in MTBE and adjusted to pH5 with HCI and then phases separated. The aqueous phase is adjusted to pH8 with saturated NaHCCh solution and extracted with ethyl acetate, before concentration of the organic phase under reduced pressure to obtain crude 40.1a.

[0245] 8: 40.1a is dissolved in DCM and then triethylamine, DMAP and oleyl chloride added subsequently. The resulting reaction mixture is stirred at 20°C until completion of the reaction. The mixture is poured into water and the phases separated. The organic phase is concentrated under reduced pressure and then purified by silica column chromatography (n-heptane / EtOAc) to obtain pure 43.1a.

[0246] Step 9:

[0247] 43.1a is dissolved in MeOH, then aqueous HCI is added at RT. The resulting reaction mixture is heated to 30-40 °C and stirred until completion of the reaction. Then, the pH is adjusted with aqueous NaHCO3 to pH 8 and the mixture extracted with DCM. The organic phase is dried over Na2SO4 and concentrated under reduced pressure. The residue is purified by preparative HPLC (n-Heptane / EtOH / THF) to obtain pure 46.1a.

[0248] Step 10:

[0249] 46.1 a is suspended in ACN and then methyl iodide is added. The resulting reaction mixture is stirred at 40°C until completion of the reaction. After filtration and concentration under reduced pressure crude 49.1a is obtained.

[0250] Step 11:

[0251] 49.1 a is added to a 1:1:1 mixture of toluene, 2-propanol and a 10% aqueous NaCI solution. The resulting reaction mixture is stirred at 35°C until completion of the reaction. After isolation, crude product is purified by recrystallization from 2-propanol, followed by multiple triturations with 2-propanol at RT for 1 h to obtain pure 52.1a. Characterization by ICP-MS confirms 52.1a as chloride salt.

[0252] Example 2.2: Synthesis of ether linked lipids

[0253] The synthesis route for unsaturated new lipids 89. cola and 95. cola is depicted below. The synthesis proceeds via a central intermediate of formula (V), in particular 28.1a. 89. cola is obtained in 6 synthesis steps in total and 2 steps from 28.1a. The cationic lipid 95. cola was obtained in 2 additional steps from 89. cola. The synthesis route is applicable to other AXGxlipids. The central intermediate is silyl- and acetonide-protected (28.1a) and the fatty acid chloride is monounsaturated.

[0254] Step 1 :

[0255] Trifluoromethanesulfonic anhydride is added to DCM at 0 °C, followed by anhydrous pyridine, leading to formation of a white precipitate. The cooling bath is removed and a solution of oleyl alcohol 70.co1 in DCM is added dropwise over a period of 10 min under stirring. The solution is stirred at RT until completion of the reaction. Afterward, water is added, and the mixture extracted with DCM. The combined organic layers are washed with brine, dried over MgSCU, and evaporated under reduced pressure. The resulting residue is purified by silica gel chromatography (n-hexane / diethyl ether) to obtain 71. col as a waxy white solid.

[0256] Step 2:

[0257] DCM is added to a mixture of 71. w1 , 32.2 and 1 ,8-bis(dimethylamino) naphthalene. The reaction mixture is stirred under reflux until completion of the reaction. DCM is removed under reduced pressure. Hexane is added and the mixture sonicated before filtration through Celite. The filtrate is evaporated under reduced pressure and the residue purified by silica gel chromatography (n-heptane / ethyl acetate) to yield 72.co1. 72.w1 is dissolved in DCM / H2O 10:1 (vol / vol) and the mixture cooled to 0 °C. 2,3-dichloro- 5,6-dicyano-1 ,4-benzoquinone (DDQ) is added and the mixture stirred at RT until the reaction is complete. The reaction mixture is filtered and the filtrate washed with aq. NaHCOs and water. The organic phase is dried over Na2SC>4 and the solvent removed under reduced pressure. After work up, crude residue is purified with silica gel chromatography (n-Heptane / Ethyl acetate) to obtain 73.OJ1.

[0258] Step 4:

[0259] 73.0)1, triphenylphosphine, tetrabutylammonium iodide and anhydrous 1 ,2-dibromoethane are combined in a flask and heated to 60 °C. The reaction mixture is stirred at 60 °C until the reaction is complete. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was purified by silica column chromatography (n- heptane / ethyl acetate) to afford 74.0)1.

[0260] Step 5:

[0261] 28.1a, N,N-diisopropylethylamine and 74.0)1 are dissolved in acetonitrile and stirred at room temperature under nitrogen until completion of the reaction. Then the solvent is removed under reduced pressure. The residue is dissolved in DCM and washed with water. The organic fractions are dried over MgSCU and the solvent is removed under reduced pressure to yield the 86.o)1a.

[0262] Step 6:

[0263] 86.u)1 a is dissolved in MeOH then aqueous HCI is added at RT. The resulting reaction mixture is heated to 30-40 °C and stirred until completion of the reaction. Then, the pH is adjusted with aqueous NaHCO3 to pH 8 and the mixture extracted with DCM. The organic phase is dried over Na2SO4 and concentrated under reduced pressure. The residue is purified by preparative HPLC (n-Heptane / EtOH / THF) to obtain pure 89.u)1a.

[0264] Step 7:

[0265] 89.0)1 a is suspended in ACN and then methyl iodide is added. The resulting reaction mixture is stirred at 40°C until completion of the reaction. After filtration and concentration under reduced pressure crude 92.o)1a is obtained.

[0266] Step 8:

[0267] 92.0)1 a is added to a 1 :1 :1 mixture of toluene, 2-propanol and a 10% aqueous NaCI solution. The resulting reaction mixture is stirred at 35°C until completion of the reaction. After isolation, crude product is purified by recrystallization from 2-propanol, followed by multiple triturations with 2-propanol at RT for 1 h to obtain pure 88.u)1a. Characterization by ICP-MS confirms 95.w1a as chloride salt.

[0268] Example 3: Synthesis route of DXPGx lipids

[0269] Example 3.1 : Synthesis of unsaturated ester linked lipids

[0270] The synthesis route depicted below is for suited mono- and polyunsaturated ester-linked

[0271] DXPGx lipids. In the example below in particular the synthesis of DOPG2 with oleyl lipid chains is shown. The synthesis proceeds via an intermediate of formula 4.2a. DOPG2 is obtained in 13 synthesis steps in total and 4 steps from 4.2a and 34.1a. Step 1 :

[0272] (R)-(+)-glycidol is added to a suspension of t-BuOK in DMF at 0 °C, followed by BnBr. The resulting reaction mixture is warmed to RT and stirred until completion of the reaction.

[0273] Afterward, water is added, and the mixture extracted with MTBE. The organic layer is washed with water and brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The resulting residue is purified by silica gel chromatography (n-Heptane / Ethyl acetate) to obtain 1.2a.

[0274] Step 2: NaOH is added to sn-1 ,2-IPG, followed by heating up to 80 °C, where 1.2a is added and the resulting reaction mixture is stirred at 80 °C until completion of the reaction. Then, the reaction mixture is poured into water and extracted with isopropyl ethyl acetate. The organic phase is washed with brine, dried over Na2SO4 and concentrated under reduced pressure to obtain crude 2.2a.

[0275] Step 3:

[0276] PPTS is added to a solution of 2.2a and dihydropyran in DCM and the resulting reaction mixture is stirred at RT until completion of the reaction. The mixture is diluted with water and extracted with DCM, followed by drying of the organic phase over Na2SC>4 and concentration under reduced pressure to obtain crude 3.2a. The resulting residue is purified by silica gel chromatography (n-Heptane / Ethyl acetate) to obtain pure 3.2a.

[0277] Step 4:

[0278] 3.2a is added to a suspension of Pd / C in EtOH, followed by degassing and heating to 50 °C. The resulting mixture is stirred under H2 (0.5 MPa) at 50 °C until completion of the reaction. The mixture is filtered over celite and the filtrate concentrated under reduced pressure. Crude 4.2a is purified by silica gel chromatography (n-Heptane / Ethyl acetate).

[0279] Step 5:

[0280] (S)-(+)-1 ,2-lsopropylideneglycerol is dissolved in DMF, then BnBr added and the solution cooled to 0 °C. tBuONa is carefully added and the resulting reaction mixture is stirred at 25 °C until completion of the reaction. The mixture is poured into ice water and the pH adjusted to 6-7 with aqueous HCI, followed by extraction with EtOAc. The organic phase is washed with brine, dried over Na2SC>4 and concentrated under reduced pressure to obtain crude 31.1a.

[0281] Step 6:

[0282] 31.1a is dissolved in MeOH and the solution cooled to 0 °C. Concentrated HCI is carefully added and the resulting reaction mixture is stirred at 20 °C until completion of the reaction. The mixture is quenched with saturated aqueous K2CO3 solution to adjust the pH to 7-8, followed by extraction with EtOAc. The organic is dried over Na2SO4 and concentrated under reduced pressure to obtain crude 32.1a.

[0283] Pyridine and DMAP are added to a solution of 32.1a in DCM, which is then cooled to 5 °C. Acetyl chloride is carefully added, and the resulting reaction mixture is stirred at 20 °C until completion of the reaction. The mixture is poured into DCM and washed with water. The organic phase is concentrated under reduced pressure to obtain crude 33.1a.

[0284] Step 8:

[0285] Pd / C is suspended in EtOH and then 33.1a and DCM are added and the reactor flushed with H2. The resulting reaction mixture is stirred under H2 (5 bar) at 25°C until completion of the reaction. The mixture is filtered and the filtrate is concentrated under reduced pressure to obtain crude 34.1a, which is recrystallized several times from n-heptane.

[0286] Step 9:

[0287] DIPEA is added to a suspension of 34.1a in MeTHF and DCM, then then mixture is cooled to -15 °C. 2-Cyanethyl-N,N-diisopropylchlorphosphoramidite (PAMCI) is dissolved in DCM and the solution cooled to -20 °C, before addition to the reaction mixture. The resulting mixture is stirred for 1 h at -15 °C, followed by 1.5 h at 0°C and 1 h at 20 °C. Then 4,5- dicyanoimidazole (DCI) and 4.2a are dissolved in MeTHF and the solution slowly added at 20 °C to the reaction mixture. If necessary, an additional amount of DCI is added as solution in DCM after stirring for 1 h, then the whole mixture is stirred overnight at 20 °C. Then aqueous 35% w / w H2O2 solution is added at 0 °C over 1 h and then stirred at 20 °C for another 1 h. The reaction mixture is diluted with 5 wt% aq. Na2S20s solution and extracted with MeTHF. The organic phase is dried over Na2SC>4 and the solvents are removed under reduced pressure. The crude intermediate 55.1a is purified by silica chromatography (DCM / acetone).

[0288] Step 10:

[0289] DBU is added to a solution of 55.1a dissolved in MeTHF and the reaction mixture stirred at RT for 2 h. Then Pd / C is added, and the obtained reaction suspension is stirred under H2 atmosphere (1.2 bar) for 1 h. The reaction mixture is filtered over celite and washed with 1M aq. citric acid solution and organic phase separated. The organic phase is washed with 5 wt%. aq. NaHCOs solution, dried over Na2SC>4 and concentrated under reduced pressure to obtain 58.1a.

[0290] Step 11 :

[0291] A mixture of 58.1a, K2CO3 and 2:1 MeOH / H2O was stirred at RT until completion of the reaction. MeOH is removed under reduced pressure, then the aqueous phase extracted with MeTHF. The organic phase is separated, dried over Na2SC>4 and concentrated under reduced to obtain crude 61.1a. Step 12:

[0292] Pyridine and DMAP are added to a solution of 61.1a in DCM, which is then cooled to 5 °C. Oleyl chloride is carefully added, and the resulting reaction mixture is stirred at 20 °C until completion of the reaction. The mixture is poured into DCM and washed with water. The organic phase is concentrated under reduced pressure to obtain crude 64.1a.

[0293] Step 13:

[0294] 64.1a is dissolved in MeOH and warmed to 40 °C, before addition of a solution of formic acid and oxalic acid in water. The resulting reaction mixture is stirred at 40 °C until completion of the reaction. The mixture is filtered, the filter cake dissolved in DCM / MeOH and the pH adjusted to 7 with NaOAc. The mixture is filtered and the filtrate concentrated under reduced pressure. The crude residue is purified by silica column chromatography (DCM / MeOH / H2O) to obtain pure 67.1a.

[0295] Example 3.2: Synthesis of unsaturated ether linked lipids

[0296] The synthesis route depicted below is for suited mono- and polyunsaturated ether linked DXPGx lipids. In the example below in particular the synthesis of 1O4.u)1a with oleyl lipid chains is shown. The synthesis proceeds via an intermediate of formula 4.2a. 1O4.u)1a is obtained in 8 synthesis steps in total and 4 steps from 4.2a and 73. cola.

[0297] Step 1 : Trifluoromethanesulfonic anhydride is added to DCM at 0 °C, followed by anhydrous pyridine, leading to formation of a white precipitate. The cooling bath is removed and a solution of oleyl alcohol 70.co1 in DCM is added dropwise over a period of 10 min under stirring. The solution is stirred at RT until completion of the reaction. Afterward, water is added, and the mixture extracted with DCM. The combined organic layers are washed with brine, dried over MgSCU, and evaporated under reduced pressure. The resulting residue is purified by silica gel chromatography (n-hexane / diethyl ether) to obtain 71. w1 as a waxy white solid.

[0298] Step 2: (S)-(+)-1 ,2-lsopropylideneglycerol is dissolved in NMP, then PMBCI is added and the solution cooled to 0 °C. NaH is carefully added and the resulting reaction mixture is stirred at 25 °C until completion of the reaction. The mixture is poured into ice water and extracted with EtOAc. The organic phase is washed with brine, dried over Na2SC>4 and concentrated under reduced pressure. The isolated residue is purified by silica gel chromatography (n- Heptane / Ethyl acetate) to obtain crude 31.2a.

[0299] Step 3:

[0300] 31.2a is dissolved in MeOH and the solution cooled to 0 °C. Concentrated HCI is carefully added and the resulting reaction mixture is stirred at 20 °C until completion of the reaction. The mixture is quenched with saturated aqueous K2CO3 solution to adjust the pH to 7-8, followed by extraction with EtOAc. The organic is dried over Na2SO4 and concentrated under reduced pressure to obtain crude 32.2a.

[0301] Step 4:

[0302] DCM is added to a mixture of 71. w1, 32.2a and 1 ,8-bis(dimethylamino) naphthalene. The reaction mixture is stirred under reflux until completion of the reaction. DCM is removed under reduced pressure. Hexane is added and the mixture sonicated before filtration through Celite. The filtrate is evaporated under reduced pressure and the residue purified by silica gel chromatography (n-heptane / ethyl acetate) to yield 72.w1a.

[0303] Step 5:

[0304] 72.w1 a is dissolved in DCM / H2O 10:1 (vol / vol) and the mixture cooled to 0 °C. 2,3-dichloro- 5,6-dicyano-1 ,4-benzoquinone (DDQ) is added and the mixture stirred at RT until the reaction is complete. The reaction mixture is filtered and the filtrate washed with aq. NaHCO3 and water. The organic phase is dried over Na2SC>4 and the solvent removed under reduced pressure. After work up, crude residue is purified with silica gel chromatography (n-Heptane / Ethyl acetate) to obtain 73.u)1a.

[0305] Step 6:

[0306] DIPEA is added to a suspension of 73.w1a in MeTHF and DCM, then then mixture is cooled to -15 °C. 2-Cyanethyl-N,N-diisopropylchlorphosphoramidite (PAMCI) is dissolved in DCM and the solution cooled to -20 °C, before addition to the reaction mixture. The resulting mixture is stirred for 1 h at -15 °C, followed by 1.5 h at 0°C and 1 h at 20 °C. Then 4,5- dicyanoimidazole (DCI) and 4.2a are dissolved in MeTHF and the solution slowly added at 20 °C to the reaction mixture. If necessary, an additional amount of DCI is added as solution in DCM after stirring for 1 h, then the whole mixture is stirred overnight at 20 °C. Then aqueous 35% w / w H2O2 solution is added at 0 °C over 1 h and then stirred at 20 °C for another 1 h. The reaction mixture is diluted with 5 wt% aq. Na2S20s solution and extracted with MeTHF. The organic phase is dried over Na2SC>4 and the solvents are removed under reduced pressure. The crude intermediate 98.u)1a is purified by silica chromatography (DCM / acetone).

[0307] Step 7:

[0308] DBU is added to a solution of 98.u)1a dissolved in MeTHF and the reaction mixture stirred at RT for 2 h. The reaction mixture is filtered over celite and washed with 1 M aq. citric acid solution and organic phase separated. The organic phase is washed with 5 wt%. aq. NaHCOs solution, dried over Na2SC>4 and concentrated under reduced pressure to obtain 101. w1a.

[0309] Step 8:

[0310] 101. w1 a is dissolved in MeOH and warmed to 40 °C, before addition of a solution of formic acid and oxalic acid in water. The resulting reaction mixture is stirred at 40 °C until completion of the reaction. The mixture is filtered, the filter cake dissolved in DCM / MeOH and the pH adjusted to 7 with NaOAc. The mixture is filtered and the filtrate concentrated under reduced pressure. The crude residue is purified by silica column chromatography (DCM / MeOH / H2O) to obtain pure 1O4.w1a.

[0311] Example 3.3: Synthesis of saturated ether lipids

[0312] The synthesis route depicted below is for suited for unsaturated, as well as mono- and polyunsaturated ether linked DXPGxlipids. In the example below in particular the synthesis of 104.1a with oleyl lipid chains is shown. The synthesis proceeds via an intermediate of formula 4.2a. 104.1a is obtained in 6 synthesis steps in total and 4 steps from 4.2a and 98.1a.

[0313] Step 1 :

[0314] Trifluoromethanesulfonic anhydride is added to DCM at 0 °C, followed by anhydrous pyridine, leading to formation of a white precipitate. The cooling bath is removed and a solution of palmityl alcohol 70.1 in DCM is added dropwise over a period of 10 min under stirring. The solution is stirred at RT until completion of the reaction. Afterward, water is added, and the mixture extracted with DCM. The combined organic layers are washed with brine, dried over MgSCU, and evaporated under reduced pressure. The resulting residue is purified by silica gel chromatography (n-hexane / diethyl ether) to obtain 71.1 as a waxy white solid.

[0315] Step 2:

[0316] DCM is added to a mixture of 71.1 , 32.1a and 1 ,8-bis(dimethylamino) naphthalene. The reaction mixture is stirred under reflux until completion of the reaction. DCM is removed under reduced pressure. Hexane is added and the mixture sonicated before filtration through Celite. The filtrate is evaporated under reduced pressure and the residue purified by silica gel chromatography (n-heptane / ethyl acetate) to yield 72.1a.

[0317] Step 3: Pd / C is suspended in EtOH and then 72.1a and DCM are added and the reactor flushed with H2. The resulting reaction mixture is stirred under H2 (5 bar) at 25°C until completion of the reaction. The mixture is filtered and the filtrate is concentrated under reduced pressure to obtain crude 73.1a, which is recrystallized several times from n-heptane.

[0318] Step 4:

[0319] DIPEA is added to a suspension of 73.1a in MeTHF and DCM, then then mixture is cooled to -15 °C. 2-Cyanethyl-N,N-diisopropylchlorphosphoramidite (PAMCI) is dissolved in DCM and the solution cooled to -20 °C, before addition to the reaction mixture. The resulting mixture is stirred for 1 h at -15 °C, followed by 1.5 h at 0°C and 1 h at 20 °C. Then 4,5- dicyanoimidazole (DCI) and 4.2a are dissolved in MeTHF and the solution slowly added at 20 °C to the reaction mixture. If necessary, an additional amount of DCI is added as solution in DCM after stirring for 1 h, then the whole mixture is stirred overnight at 20 °C. Then aqueous 35% w / w H2O2 solution is added at 0 °C over 1 h and then stirred at 20 °C for another 1 h. The reaction mixture is diluted with 5 wt% aq. Na2S20s solution and extracted with MeTHF. The organic phase is dried over Na2SC>4 and the solvents are removed under reduced pressure. The crude intermediate 98.1a is purified by silica chromatography (DCM / acetone).

[0320] Step 5:

[0321] DBU is added to a solution of 98.1a dissolved in MeTHF and the reaction mixture stirred at RT for 2 h. Then Pd / C is added, and the obtained reaction suspension is stirred under H2 atmosphere (1.2 bar) for 1 h. The reaction mixture is filtered over celite and washed with 1M aq. citric acid solution and organic phase separated. The organic phase is washed with 5 wt%. aq. NaHCOs solution, dried over Na2SC>4 and concentrated under reduced pressure to obtain 101.1a.

[0322] Step 6:

[0323] 101.1a is dissolved in MeOH and warmed to 40 °C, before addition of a solution of formic acid and oxalic acid in water. The resulting reaction mixture is stirred at 40 °C until completion of the reaction. The mixture is filtered, the filter cake dissolved in DCM / MeOH and the pH adjusted to 7 with NaOAc. The mixture is filtered and the filtrate concentrated under reduced pressure. The crude residue is purified by silica column chromatography (DCM / MeOH / H2O) to obtain pure 104.1a. Example 4: Synthesis, characterization and stability of CLG2

[0324] HPLC / CAD measurements revealed presence of only minor lipid-related impurities (8.1 Area%) in the produced batch. The batch was investigated in indicative storage stability studies using HPLC / CAD. CLG2 is stable in its powder form for at least 1 year when stored at -20°C. In contrast to NLG2, CLG2 could also be stored in organic solvents (e.g., chloroform) at temperature ranges from -20 to 2-8°C for at least 9 months.

[0325] CLG2 is readily soluble in chloroform as a clear solution with at least 10 mg / ml. Methanol and isopropanol should be avoided since both solvents affected chemical stability of NLG2. However, decomposition of CLG2 in ethanol was less pronounced.

[0326] Example 5: Preparation of liposomes

[0327] Liposomes were prepared as described elsewhere in detail (Hossann et al. 2021 , Petrini et al. 2021).

[0328] Choi, DPPC and DSPC were acquired from Corden Pharma Switzerland LLC (Liestal, Switzerland). HSPC, NBD-PE, and RhPE were purchased from Avanti Polar Lipids (Alabaster, AL, USA). ADU-S100 (ammonium salt) was bought from MedChemExpress (Monmouth Junction, NJ, USA). CF free acid were bought from Sigma Aldrich (Darmstadt, Germany). All other chemicals were either from Sigma Aldrich GmbH (Munchen, Germany) or Carl Roth GmbH (Karlsruhe, Germany). Unless stated, all the buffers and solutions mentioned in the study were prepared with ultra-purified deionized water from an ultrapure water system Mil liQ Advantage from Merck Millipore (Darmstadt, Germany).

[0329] Liposomes were prepared with the lipid film hydration and extrusion method. The lipid components with the desired ratios were dissolved in either pure CHCI3 or CHCh / MeOH 9: 1 (vol / vol) followed with solvent removal to obtain a homogeneous and dry lipid film. Lipid films were stored at room temperature under vacuum until being hydrated in the desired solution (e.g., HBS pH 7.4, 100 mM CF pH 7.2, 300 mM citric acid pH 4). Dispersions were manually extruded (Avestin, Ottawa, Canada) 17 times through 200, 100, or 50 nm polycarbonate filters (Cytiva, MA, USA; and Avestin, Ottawa, Canada) at 60°C. Nonencapsulated small molecules were removed by size exchange column chromatography using HBS pH 7.4 as eluent. DOX was encapsulated to preformed liposomes by active loading. For passive encapsulation of ADU-S100, the dry lipid film was hydrated with a physiological sucrose-containing buffer pH 7 with 5 mg / ml ADU-S100. Additionally, liposomes with ADU-S100 underwent freezing and thawing cycles after the extrusion. Example 6: Biophysical characterization of liposomes

[0330] Liposome batches were characterized as described elsewhere (Hossann et al. 2007, 2010, 2021). Vesicle size (hydrodynamic diameter as z-average), polydispersity index (PDI), and zeta potential were measured by dynamic light scattering (DLS) with a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). Samples were diluted 50-times in 0.2 pm filtrated 0.9% saline. The desired batch specifications were specified to be <200 nm and <0.20 for z average and PDI, respectively. The solid gel to liquid disorder phase transition temperature (Tm) was analyzed using a Mettler Toledo DSC 822e (Giessen, Germany). Liposomal dispersions were sealed in aluminum crucibles for this purpose. Temperature dependent drug release (TDR) profile and release kinetics measurements in HBS pH 7.4 or fetal calf serum (FCS, Sigma Aldrich, Darmstadt, Germany) were performed in a Cary Eclipse fluorescence spectrometer (Varian Inc., Palo Alto, CA, USA). Content of the encapsulated compound in a liposomal batch was either performed with HPLC or fluorescence spectroscopy.

[0331] Example 7: Compatibility testing of NLG2 and / or NLG2 with selected lipid excipients

[0332] Compatibility testing of NLG2 and CLG2 with selected binary and ternary lipid mixtures used for classical Chol-containing liposomes and TSL formulations was performed. Liposome preparation and characterization was performed in accordance with examples 5 and 6.

[0333] Example 7.1 : Binary mixtures of NLG2, CLG2 and DPPG2

[0334] The feasibility of NLG2, CLG2 and DPPG2 to form liposomes in binary mixtures was investigated at physiological pH (HBS pH 7.4). Extrusion was performed with either 50 nm, 100 nm, or 200 nm filter membranes to generate liposomes differing in vesicle size. Each 2-component system contained equal mol% of lipids. Additionally, feasibility of vesicle formation using pure NLG2 or CLG2 was tested. All resulting formulations were characterized by DLS after preparation and 7 days storage at 2-4°C. Results are depicted in Figure 2.

[0335] Overall, liposome formation was possible for CLG2 and CLG2 / NLG2 1 :1 (vol / vol). Vesicle size had no influence on the dispersion stability (z average / PDI) of CLG2 / NLG2 1 :1 (vol / vol) batches. Oppositely charged CLG2 and DPPG2 form preparations with suboptimal characteristics (e.g., PDI > 0.2) and tend to form coagulates for smaller vesicle sizes in the binary combination tested. CLG2 liposomes could be formed but -25% of the lipid was already hydrolyzed after preparation. After 2 months only the 100 nm extruded liposomes kept their integrity. Formation of NLG2 liposomes was not successful at pH 7.4 but using NaOAc pH 4 and ultrasound resulted in liposomes (data not shown). Pure DPPG2 was not tested since Lindner et al. previously showed that DPPG2 only formed liposomes (DPPC / DSPC matrix) when incorporated from 0 to 70 mol% (Lindner et al. 2004).

[0336] Example 7.2: Cholesterol-containing liposomes

[0337] The compatibility of NLG2 and CLG2 with a phosphatidylcholine (PC) / cholesterol (Choi) matrix was investigated. PC / Chol is commonly used in classical, non-thermosensitive formulations (e.g., Caelyx®). An overall Choi content >30 mol% is applied to suppress the phase transition of the PC by formation of liquid-ordered phase of the bilayer membrane. Distinct batches using a ternary mixture of PC / Chol / AXGx60-x:40:x (mol / mol) (x = 5, 10, 15, 30, 40, PC = DPPC, HSPC, AXGx= NLG2, CLG2) were prepared using HBS pH 7.4. Results are depicted in Figure 3.

[0338] All CLG2-containing formulations demonstrated optimal values for z average and PDI and dispersion stability was proven for at least 10 days at 2-8°C. Batches with >15 mol% of CLG2 showed no signs of sedimentation. Batches that showed sedimentation could be homogenized again by mixing without affecting z average and PDI. The zeta potential increased from 5 mV to ~25 mV with increasing content of CLG2 from 5 to 40 mol%, respectively. Comparable results were obtained independent from usage of HSPC or DPPC. Binary mixtures of Chol / CLG2 1 :1 (mol / mol) also resulted in liposomes within specification (z-average <200 nm, PDI <0.20) and maintained dispersion stability for at least 7 days.

[0339] In contrast, NLG2 exhibited only partial compatibility to ternary PC / Chol / NLG2 mixtures at physiological pH. Promising results were obtained for batches containing <15 mol% NLG2 that had been extruded with 50 or 100 nm extrusion membranes. The effect of NLG2 content on the zeta potential was less clear as observed before with CLG2 but increased from ~1 mV to ~6 mV for batches with 5 and 40 mol% NLG2, respectively. Comparable results were obtained whenever HSPC was replaced with DPPC. Batches within specification with a NLG2 content of >15% were obtained when the lipid film was hydrated with an acidic buffer (data not shown), probably due to the stabilizing effect of the cationic surface charge. This fits to the results obtained with NLG2-based TSL formulations in the pH titration assay. Binary mixtures of Chol / NLG2 1 :1 (vol / vol) formed batches when the lipid film was hydrated with HBS pH 7.4. Example 7.3: Ternary mixtures (CLG2 / NLG2 / Chol)

[0340] The compatibility to use both new excipients, NLG2 and CLG2, in Chol-containing liposomal formulations was investigated next. Mixing both in distinct ratios allows to adjust the positive zeta potential of the resulting liposomes while keeping the diglycerol content on the liposomal surface constant. Cholesterol does not contribute to the surface composition since it is incorporated in the lipophilic part of the membrane bilayer (Javanainen et al. 2017). Batches using a ternary mixture of either CLG2 / NLG2 / Chol 50:10:40 (mol / mol) or CLG2 / NLG2 / Chol 30:30:40 (mol / mol) were prepared using HBS pH 7.4. Extrusion was performed with membranes with a pore size of either 50, 100, or 200 nm, respectively. The results are depicted in Figure 4.

[0341] Both lipid compositions resulted in liposomes within specification independent from the used extrusion membrane. Presence of NLG2 induces sedimentation of the liposomes, but batches could be stored at 2-8°C for 7 days and resulted in no change in vesicle size or PDI after vortexting (data not shown). The zeta potential decreases when the CLG2 content decreased from 50 to 30 mol%.

[0342] The influence of the aqueous media on zeta potential of the formed liposomes was investigated next. CLG2 / NLG2 / Chol with a molar lipid ratio of 60-x:x:40 (mol / mol) with x = 10, 20, 30, 40, 50 mol% were prepared. The lipid film was either hydrated with 0.9% saline, HBS pH 7.4 or HBS pH 8.0, respectively. The formed multilamellar liposomes were extruded using filter membranes with 100 nm pore size.

[0343] Vesicle size, size distribution and zeta potential of the resultant batches was measured in 0.9% saline. The results are expressed as mean value of the two measurements of two independently prepared batches. Vesicle size of the batches was between 128 nm and 152 nm with a PDI of < 0.08. Zeta potential was 18.3 mV, 21.0 mV, 18.3 mV, 19.0 mV, and 13.9 mV for liposomes with 50 mol%, 40 mol%, 30 mol%, 20 mol%, and 10 mol% CLG2 prepared in 0.9% saline, respectively. For liposomes prepared with HBS pH 7.4, zeta potential was 21.3 mV, 18.0 mV, 19.3 mV, 16.9 mV, and 13.6 mV, respectively. For liposomes prepared with HBS pH 8.0, zeta potential was 20.1 mV, 19.2 mV, 15.7 mV, 16.3 mV, and 12.0 mV, respectively. In conclusion, the zeta potential of CLG2 / NLG2 / Chol 60-x:x:40 (mol / mol) was increased by increasing CLG2 content in the formulation while the surface coverage of G2 moieties was kept constant.

[0344] Example 7.4: Ternary mixtures DPPC / DSPC / AXGx

[0345] Finally, the compatibility of AXGxin the Chol-free DPPC / DSPC matrix used in TSL formulations was tested. Distinct batches of DPPC / DSPC / AXGx 80-x / 20 / x (mol / mol) x = 5, 10, 20, 30, 40, 50, 60, 70, 75 & AXGx= NLG2, CLG2 were prepared. DSPC content was fixed to 20 mol% to achieve a comparable fatty acid composition in the TSL membrane to be palmitic acid to stearic acid 80:20 (mol / mol), as done in lipid compatibility studies with the anionic DPPG2 in vitro (Lindner et al. 2004) and in vivo (Hossann et al. 2021). To exclude any potential impact of additional molecules (i.e., loading of an API), lipids had been hydrated in HBS pH 7.4 to form the intra- and extra-liposomal medium. Liposomes with <30 mol% NLG2 or <20 mol% CLG2 could be successfully formulated, yielding batches within size specification (z-average < 200 nm, PDI < 0.20). These batches were dispersion stable for at least 7 days at 2-8°C without a notable change in vesicle size or PDI.

[0346] The effect of the NLG2-content in DPPC / DSPC / NLG2 80-x / 20 / x (mol / mol) on the phase transition was investigated by DSC. The phase transition peak was broadened, and the peak maximum (Tm) was shifted towards higher temperatures with increasing NLG2 content (Figure 5, Table 1), respectively. Tmdecreased from 49.7°C to 42.1 °C if cationizable NLG2 is replaced by the anionic DPPG2 in the 30 mol% formulation.

[0347] Table 1: Phase transition temperature of NLG2-based TSL formulations. Data for the DPPG2-containing formulations from Petrini et al. 2021.

[0348] Example 8: Characterization of DPPC / DSPC / AXGx-based TSL formulations Example 8.1 : pKavalue of NLG2-based TSL

[0349] A binding assay using the anionic fluorescent dye 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) was adapted from a described protocol (Carrasco et al. 2021). Objective was to determine the pKavalue of saline-loaded DPPC / DSPC / NLG2 80-x / 20 / x (mol / mol) liposomal batches. TNS was purchased from Abeam (Cambridge, UK). Briefly, 50 l of liposomes (75 M total lipids) were mixed with 20 pl TNS stock solution (300 pM) and 1 ml buffer solutions with a pH ranging from 2 to 11 , respectively. Osmolarities of the pH buffers were adjusted to approx. 287 mOsmol / kg. The fluorescence intensity was measured immediately with a Cary Eclipse fluorescence spectrometer (Varian Inc., Palo Alto, CA, USA) in 1 ml silica glass cuvettes. The data was fitted with GraphPad using the Henderson-Hasselbalch equation to obtain the pKa.

[0350] NLG2-content had a negligible effect on pKawith 6.013, 6.095, and 5.807 for TSL with 10 mol%, 20 mol%, and 30 mol%, respectively. The measured pKavalues are comparable to LNP formulations with the cationizable lipids DODAP (5.62) and DLin-MC3-DMA (6.57) (Carrasco et al. 2021).

[0351] Example 8.2: Effect of pH on dispersion stability NLG2-containing TSL

[0352] The effect of the extraliposomal pH value on the dispersion stability of DPPC / DSPC / NLG2 80-x / 20 / x (mol / mol) (x = 5, 10, 20, 30) batches was investigated by pH titration and subsequent measurement of z-average, PDI and zeta potential by DLS. Batches differing in vesicle size had been prepared by extrusion using membranes of either 50 or 100 nm pore size. All batches showed an effect of the pH value on the zeta potential of the liposomes (Figure 6). The change in zeta potential ( 1zeta potential = zeta potentials - zeta potentialPHii) increased with increasing content of cationizable NLG2 in the formulation. Vesicle size for individual lipid compositions had a negligible effect on the zeta potential results.

[0353] All tested formulations showed a stable z average and PDI in the pH range from 3 to 8 but showed a tendency for aggregation at pH > 9 (Figure 7). All liposomal formulations lost their cationic surface charge around pH 9 where the zeta potential became neutral. Even 30 mol% NLG2 in the neutral form was not sufficient to avoid aggregation. Anionic liposomes composed of DPPC / DSPC / DPPG2 50:20:30 (mol / mol) showed a similar behavior (data not shown). Liposomes lose their strong negative zeta potential and showed substantial increase in vesicle size and PDI when the extraliposomal pH was decreased to strongly acidic conditions (pH ~2). This demonstrates that liposomes containing a diglycerol-based surface coating instead of a PEGylation need a surface charge (either anionic or cationic) to maintain dispersion stability. Therefore, electrostatic repulsion due to the surface charge is the mechanism of diglycerols to maintain dispersion stability and not providing steric hinderance as proposed for PEGylation (Lasic et al. 1992). However, the absence of a steric barrier might be beneficial for the interaction between liposomes and the target cell.

[0354] Example 8.3: In vitro complement activation of AXGx-based TSL

[0355] Complement activation, fast clearance due to serum protein adsorption and immunological recognition limits the clinical translation of charged DDS (Chonn et al. 1991 , Dezsi et al. 2014, Knudsen et al. 2015, Szebeni et al. 2012).

[0356] The in vitro complement activation of selected liposomal formulations was investigated with an enzyme-linked immunosorbent assay (ELISA) as described in Lokerse et al. 2021. Results are depicted in Figure 8. Liposomes containing the cationic CLG2 (5 mol%, 10 mol%) and the cationizable NLG2 (5 mol%, 10 mol%) showed complement activation. In contrast, cationic liposomes containing DPTAP (i.e. , 5 mol%, 10 mol%) resulted in SC5b-9 values only slightly higher than the negative control. Liposomes containing the anionic DPPG2 (5 mol%, 10 mol%) showed also complement activation comparable to the literature (Lokerse et al. 2021). Increasing the content of the anionic excipient DPPG2 from 5 to 30 mol% in DPPC / DSPC / DPPG2 TSL formulations notably reduced the in vitro complement activation, but it was still more pronounced than PEGylated TSL formulations. However, this had no negative effect on in vivo application of DPPG2-based TSL to rodent and non-rodent species (Hossann et al. 2021).

[0357] Example 8.4: Composition of the protein corona of AXGx-based TSL

[0358] Lokerse et al. 2021 demonstrated that the molar content of DPPG2 influenced the composition of the protein corona of DPPC / DSPC / DPPG2 80-x:20:x (mol / mol) (x = 5, 10, 20, 30) liposomes. Here, the effect of different AXGx-based TSL formulations was investigated and compared to standard formulations.

[0359] Preparation and characterization of liposomal batches:

[0360] The following lipid mixtures were used to prepare liposomes loaded with physiological saline. 1) Cationic CLG2-based TSL: DPPC / DSPC / CLG2 with a molar lipid ratio of 50:20:30 (mol / mol) (CLG2-TSL). 2) Non-thermosensitive liposomes with constant diglycerol moieties on the particle surface but increasing amount of cationic CLG2: CLG2 / NLG2 / Chol 60-x:x:40 (mol / mol) (CLG2-NTSL x mol%) with x = 10, 20, 30, 40. 3). Anionic DPPG2-based TSL: DPPC / DSPC / DPPG2 50:20:30 (mol / mol) (DPPG2-TSL). 4) PEGylated liposomes mimicking the lipid composition of the clinically approved liposomal formulation Caelyx®: HSPC / Chol / DSPE-PEG2000 55:40:5 (mol / mol) (PEGylated liposomes). The batches were prepared by the lipid film and hydration method and characterized as described elsewhere (Hossann et al. 2010). The results are expressed as mean value from measurements of two independently prepared batches for each formulation (Table 2).

[0361] Formation of the protein corona:

[0362] Human plasma collected on citrate phosphate dextrose from healthy human donors was from Biowest SAS (0.2 pm filtrated) and was aliquoted and stored at -80°C. Prior to usage, thawed plasma was centrifuged (2 min / 1000 xg) to remove coagulates that may block size exclusion columns (SEC). TSL batches (25 mM of total lipid content, 50 mM for CLG2-TSL) were mixed 1 :9 with human plasma. The mixture was incubated for 30 min at 37°C in a thermoshaker (0 rpm) and subsequently loaded onto saline pre-conditioned CL-4B SEC. Fractions containing liposomes were transferred into a 1000 kDa Float-A-Lyzer dialysis chamber in physiological saline for overnight dialysis at 4°C. Afterwards, the dialyzed TSL samples were subjected to centrifugal concentration at 4°C by a single centrifugation step (1 h 4500xg) using a 10 kDa Amicon centrifugal filter and four centrifugation steps using a 1000 kDa VivaSpin centrifugal concentrator (1x 1 h 8600xg, 3x 30 min 8600xg) for washing the pellet with physiological saline. The final TSL pellet was resuspended in 100 pl physiological saline and the total protein (BCA protein assay) and lipid concentration was quantified.

[0363] Liquid-chromatography mass-spectrometry (LC-MS) analysis:

[0364] Three pg of each sample was used for protein digestion and a small fraction were injected in the LC-MS / MS. Hi3 E. coli standard (Waters) heavy peptides (6 in total) were spiked into the sample for absolute quantification purposes. Samples were processed with Preomics iST kit to obtain peptides from the protein corona. Peptides were measured by LC-MS / MS in DDA mode. Raw data was searched for human proteins (whole human proteome) plus E.Coli using MaxQuant.

[0365] Characterization of protein corona:

[0366] Protein concentration in the samples was -300 pg / ml, -150 pg / ml, -100 pg / ml, -100 pg / ml, -100 pg / ml, -250 pg / ml and -575 pg / ml for CLG2-TSL, CLG2-NTSL (50 mol%), CLG2-NTSL (40 mol%), CLG2-NTSL (30 mol%), CLG2-NTSL (20 mol%), DPPG2-TSL, and PEGylated liposomes, respectively. The composition of the protein corona (Top 10 of most abundant proteins) of each formulation is shown in Table 2. Protein corona composition of a TSL formulation was substantially changed by simply replacing the anionic DPPG2 by the cationic CLG2 while keeping the G2 surface coverage constant. Most abundant proteins were apolipoprotein A- I, C-l & C-ll for DPPG2-TSL but apolipoprotein A-ll and an immunoglobulin for CLG2-TSL, respectively. CLG2-NTSL can be applied to shift the protein corona even more to immunoglobulins. CLG2-NTSL showed the tendency to attract more immunoglobulins with increasing amounts of the cationic CLG2 in the liposome membrane. The molar immunoglobulins to apolipoprotein ratio was 7.0, 9.0, 6.3, and 4.1 for CLG2-NTSL (50 mol%), CLG2-NTSL (40 mol%), CLG2-NTSL (30 mol%), and CLG2-NTSL (20 mol%), respectively. Protein corona composition might be further modified when CLG2-NTSL are prepared in presence of buffer systems that are able to further steer the zeta potential of the liposomes (e.g., by deprotonation or protonation of NLG2). Table 2: Protein corona composition of selected formultions. List of the top 10 most abundant proteins of each formulation

[0367]

[0368] Example 8.5: Binding of NLG2-based TSL to B16 cells (live cell fluorescence microscopy)

[0369] Several liposomal formulations were investigated in vitro for their binding to B16 cells. The respective lipid film contained 0.3 mol% of the fluorescent dye 1 ,2-dipalmitoyl-sn-glycero- 3-phosphoethanolamine-N-(7-nitro-2-1 ,3 ben-zoxadiazol-4-yl) (NBD-PE, green) and was hydrated with HBS pH 7.4. Extrusion was performed with 50 nm filter membranes. Live cell fluorescence microscopy was performed as reported elsewhere (Petrini et al. 2021). Adherent mouse melanoma cells (B16 F10) were cultured in Dulbecco's Modified Eagle Medium (DM EM, ATCC, VA, USA) supplemented with 10% fetal bovine serum and 1 % penicillin-streptomicin at humidified atmosphere containing 95% air and 5% CO2 at 37 °C. Cell detachment was achieved by incubation with 0.25% trypsin at 37°C / 2 min. Cell passages were performed when reached to 70-80% confluence (every 2-3 days). 25,000 cells were seeded in each well of a chambered coverslip for the live-cell fluorescence microscopy experiments. After 24 h, the culture media was removed and replaced by 250 L of 1 mM liposome samples. Past 30 min, 50 L of 0.5 pM Lysotracker Red in FBS-free DMEM were added. The medium was removed 30 min later and, after washing the cells with FBS-free DMEM, 300 pL of Hoechst 5 pg / mL in PBS were added. 5 min later, it was removed, and the cells were washed with PBS. A Leica wide-field microscope was used to observe the cells and the images were acquired with the Leica LAS X image software (Leica, Germany). Pictures were treated on Imaged software (version 1 ,8.0_172) afterward. The results are depicted in Figure 9 (cationizable formulation) and Figure 10 (controls).

[0370] The cationizable DPPC / DSPC / NLG2 liposomes localized mainly on the tumor cell surfaces. The intensity of the signal correlated with the NLG2 content of the formulations confirming the assumption that cationizable TSL with a higher zeta potential would bind more intensely to the negatively charged surface of tumor cells. Lysotracker (red) stains acidic organelles in particular lysosomes. Orange appears upon red and green overlay, which can be interpreted as internalization of the labelled liposomes. This was seldom spotted, but relatively more noticeable for cells treated with DPPC / DSPC / NLG2 50:20:30 (mol / mol) liposomes. Co-localization with the Hoechst-stained nuclei (blue) was not observed.

[0371] Neutral DPPC / DSPC 80:20 (mol / mol) tended to aggregate due to their zeta -potential close to 0 mV. It is expected that larger particles sedimented quickly and mechanically deposited on top of the cells or on the surface of the slide. The latter could be responsible to the observed dotted pattern for this formulation.

[0372] Images of cells treated with the anionic DPPC / DSPC / DPPG2 50:20:30 (mol / mol) liposomes showed no green fluorescence, indicating that they were mostly washed away during the staining procedure. This is in good agreement with results from Petrini et al. (2021) who showed that cationic TSL (DPPC / DSPC / DPTAP / DPPG2) target tumor and endothelial cells considerably more than anionic TSL (DPPC / DSPC / DPPG2). As expected, experiments with cells incubated only with DMEM cell culture media showed no visible green NBD signal.

[0373] Overall, cationic or cationizable TSL showed superior binding to cell than neutral or anionic TSL.

[0374] Example 8.6: Binding of AXGx-based TSL to THP-1 cells (FACS)

[0375] Distinct liposomal formulations differing in their lipid composition and zeta potential were prepared for in vitro cell binding studies using a fluorescent activated cell sorter (FACS). All formulations were labelled with 0.1 mol% 1 ,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (RhPE) as fluorescent dye. The human monocytic cell line THP-1 , derived from an acute monocytic leukemia patient were seeded in a 96-well plate in an appropriate medium containing 10% FCS. 0.6 mM of a RhPE labelled liposomal sample was added to each well. FACS analysis was performed at specified time points.

[0376] Reducing the content of anionic DPPG2 from 30 mol% to 0 mol% increased the cell binding of the TSL formulation (Table 3, #1-4), probably due to a reduction of the electrostatic repulsion of the liposome and the cell. A higher content of cationic lipid (i.e. , CLG2, DPTAP) induced higher cell binding (Table 3, #5,6,9,10). Liposomes with CLG2 showed better binding than liposomes with DPTAP (Table 3, e.g., #5 & #9). Liposomes with a strong permanent cationic charge (CLG2) outperformed liposomes with a cationizable charge (NLG2) in binding of cells (Table 3, #6 & #8). Interestingly, visual inspection of incubated cells showed cell agglutination in vials with the cationic formulation DPPC / DSPC / CLG2 70:20:10 (mol / mol) (Figure 11), while all other formulations looked like the control (no agglutination).

[0377] Table 3: TH P-1 cell binding of DPPG2-, AXGx-, and DPTAP-based TSL formulations. All liposomes had been labelled with 0. 1 moi% RhPE as fluorescent dye to allow quantification of cell-liposome binding by FACS. MFI: median fluorescent intensity.

[0378] Example 9: AXGx-based TSL loaded with small molecules

[0379] The simple formulation of small molecules into AXGx-based TSL was investigated next. The fluorescent dye carboxyfluorescein (CF), the cytotoxic drug DOX, and the stimulator of IFN genes (STING) agonist ADU-S100 were used as representative small molecules, respectively.

[0380] The carboxylic acid CF is the smallest of the three used compounds. CF is commonly applied in formulation development of liposomes to evaluate release rates. Fluorescence intensity is quenched at high concentrations. It can be loaded by passive encapsulation and was extensively investigated in the anionic DPPG2-based TSL (Lindner et al. 2004, Hossann et al. 2007, 2010, 2021). At physiological pH, the molecule is negatively charged while being in its salt form. DOX belongs to the class of anthracyclines and is a weak base with fluorescent properties. Fluorescence intensity is quenched at high concentrations. The drug is water soluble at acidic pH while being positively charged, but lipophilic in its unprotonated form. The molecule can be actively loaded into preformed liposomal formulations by pH, EDTA and ammonia gradients, respectively.

[0381] ADU-S100 is the largest of the three model compounds used and is a synthetic cyclic dinucleotide that activates the STING pathway. It is a negatively charged molecule when in salt form.

[0382] The small molecules had been loaded into liposomes and the resulting batches had been characterized regarding their vesicle size, PDI and compound release properties immediately after preparation.

[0383] Example 9.1 : NLG2-based TSL passively loaded with CF

[0384] Several lipid films with the composition DPPC / DSPC / NLG2 with a molar lipid ratio of 80- x:20:x with x = 5, 10, 20, 30, 40, 50, 60, 70, 80 had been incubated with an aqueous solution of 100 mM CF pH 7.2. Hydration of the film was possible with lipid compositions <50 mol% NLG2. Hydrated films were extruded with membrane filters of either 50, 100, or 200 nm pore size, respectively, and subsequently purified with size exclusion chromatography (SEC). Only distinct formulations with a NLG2 content <30 mol% met the quality criteria for z average and PDI. Increasing the NLG2 content from 5 to 30 mol% led to an increase in the zeta potential of the formulations from 3 to 13 mV, respectively.

[0385] Vesicle size seems to affect the colloidal stability of the formulations (Figure 12). For example, using 200 nm filter membranes during extrusion did not yield monodisperse size distribution for lipid compositions of DPPC / DSPC / NLG2 75:20:5 and 70:20:10 (mol / mol), respectively, but formation of monodisperse liposomes was possible when using 50 nm and 100 nm filters. It seems that the membrane curvature has an impact on dispersion stability of the NLG2-based TSL. Since decreasing vesicle size leads to an increased membrane curvature with more packing defects and loser packing of phospholipids (Hossann et al. 2010), this seems to be important for compatibility of NLG2 to the membrane bilayer during passive loading with the anionic CF.

[0386] The temperature-dependent release (TDR) profiles of selected batches were measured either in FCS or in 0.9% saline (Figure 13), respectively. Presence of serum components was necessary to induce notable CF release from NLG2-based TSL at T>38°C during 5 min of incubation. Necessity of serum components to support CF release was previously shown also for TSL containing the anionic excipient DPPG2 (Hossann et al. 2012). The TDR profile obtained for NLG2-based TSL was increasingly less defined for higher NLG2 content. As result, with increasing NLG2 content the temperature where the maximum release occurred was slightly shifted towards higher temperatures accompanied by a slight reduction in amount of released CF during the incubation time of 5 minutes. The observation can be explained by the broadening of the phase transition with increasing NLG2 content in a formulation (see example 7.4). Maximum CF release during 5 min at 42°C was -50% and -25% for NLG2-based TSL batches extruded with 50 nm and 100 nm membranes, respectively. This is in clear contrast to the anionic DPPG2-based TSL that showed an increase in CF release when the vesicle size was decreased (Hossann et al. 2010).

[0387] Time-dependent release assay results confirmed the TDR results and further demonstrated that the steepest increase in CF release was achieved within the first seconds of the experiment (data not shown). A behavior that was also demonstrated for TSL containing the anionic excipient DPPG2 (Hossann et al. 2007). An immediate heat-triggered drug release is important for TSL formulations to be able to exploit the concept of intravascular drug release since the passage time through the heated tumor tissue is limited.

[0388] Example 9.2: CLG2-based TSL passively loaded with CF

[0389] Several lipid films with the composition DPPC / DSPC / CLG2 with a molar lipid ratio of 80- x:20:x with x = 5, 10, 20, 30 had been incubated with an aqueous solution of 100 mM CF pH 7.2. CLG2 contents >30 mol% were not tested because of the observed broadening of the phase transition with increasing NLG2 content in a formulation (see example 7.4). Hydrated films were extruded with membrane filters of either 100, or 200 nm pore size, respectively, and subsequently purified with SEC.

[0390] Acceptable values for size (z average) of < 200nm and PDI (<0.20) were observed for two batches with 5 mol% CLG2(DPPC / DSPC / CLG2 75:20:5 (mol / mol)) and 10 mol% CLG2(DPPC / DSPC / CLG2 70:20:10 (mol / mol)) and usage of 100 nm pore-size filters during extrusion. The zeta potential showed a CLG2-content dependent increase in charge from -4 mV and -12 mV for TSL with 5 mol% and 10 mol%, respectively.

[0391] An electrostatic interaction of the negatively charged CF with the positively charged CLG2 during hydration might be responsible for the failure in formation of liposomes with acceptable quality when >10 mol% CLG2 was applied. A strong interaction of drug with the lipids might interfere with the self-assembly process of the lipids when dispensed in the hydration solution.

[0392] Two batches within specification were tested for their TDR profile in either FCS or 0.9% saline (Figure 14), respectively. As shown before for NLG2-based TSL, presence of serum components was also important for CLG2- based TSL to facilitate heat-triggered CF release. Importantly for in vivo application, both formulations showed a sufficient stability at 37°C / 60 min in FCS and -80% CF release at 42°C / 60 min, respectively. Additionally, negligible CF release in physiological saline under all tested conditions suggests a promising stability of both formulations during storage. Finally, CF release kinetics of both batches was performed in FCS with real time fluorescence spectroscopy and confirmed the TDR results (data not shown).

[0393] Example 9.3: CLG2-based TSL actively loaded with DOX

[0394] Lipid compositions with 5 mol% CLG2 (DPPC / DSPC / CLG2 75:20:5 (mol / mol)) and 10 mol% CLG2 (DPPC / DSPC / CLG2 70:20:10 (mol / mol)) were selected for testing active DOX loading, based on the positive results obtained in example 9.2. Hydration of the lipid film was done with 300 mM ammonium sulfate pH 5.5 and filter membranes with 100 nm pore size were used for the subsequent extrusion. Ammonium gradient was than formed by SEC with HBS pH 7.8. DOX loading was completed after 90 min using an incubation temperature of 37°C.

[0395] Distinct molar DOXJipid ratios (0.1 , 0.2, and 0.3) were tested for active DOX loading and the TDR profile was measured. Both CLG2-based TSLs showed feasibility for active DOX loading and heat-triggered drug release (Figure 15). The amount of DOX released at elevated temperatures depended on the CLG2 content of the formulation with 10 mol% CLG2-based TSL (~40-70% / 42°C / 5min) being superior to the 5 mol% CLG2-based TSL (~20-30% / 42°C / 5min). Moreover, the molar DOXJipid ratio might also affect the amount of DOX released. However, this also impacted the TSL stability since both formulations loaded with a molar DOXJipid ratio of 0.30 showed the largest DOX leakage at 37°C / 60 min compared to the respective formulations with lower molar DOXJipid ratios. An effect of drugJipid ratio on TSL stability fits to published observations with DPPG2-based TSL actively loaded with DOX or irinotecan (W02022 / 008471), respectively. The obtained data shows that active DOX loading and subsequent heat-triggered DOX release from CLG2-based TSL is possible.

[0396] Example 9.4: CLG2-based TSL passively loaded with ADU-S100

[0397] Next, the feasibility of cationic CLG2-based TSL for stable encapsulation and heat-triggered release of the synthetic cyclic dinucleotide ADU-S100 was tested. Because of the promising results obtained with OF and DOX, lipid compositions with 5 mol% CLG2 (DPPC / DSPC / CLG2 75:20:5 (mol / mol)) and 10 mol% CLG2(DPPC / DSPC / CLG2 70:20:10 (mol / mol)) were selected for passive encapsulation. Using 5 mg / ml ADU-S100 for hydration of the lipid film yielded acceptable TSL batches. Furthermore, presence of 5% (wt / vol) saccharides (e.g., glucose, sucrose, and trehalose) were tested to act as potential stabilizers and to facilitate steric hindrance between the oppositely charged CLG2 and ADU- S100.

[0398] DPPC / DSPC / CLG2 75:20:5 (mol / mol) batches hydrated with sucrose-containing buffer containing ADU-S100 with a molar druglipid ratio of 0.14 and extruded with 100 nm pore size filters showed promising results. Vesicle size (z average) and PDI was 83 ± 9 nm and 0.07 ± 0.03, respectively. The ADU-S100 encapsulation efficacy was 8.3 ± 3.1 % and the molar druglipid ratio was 0.012 ± 0.004. The formulation showed a promising storage stability and was store-able up to 2 months at 2-8°C.

[0399] ADU-S100 was released from the CLG2-based TSL in a temperature-dependent fashion. The formulation released 62.7 ± 14.0% during 5 min at 42 °C. In contrast, only traces of ADU-S100 were released at 37°C during 1 hour in FCS.

[0400] Items

[0401] Herein also the following items are disclosed.

[0402] Item 1. Oligoglycerol-containing lipid of Formula (I) wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0403] A = NR3or N+R4R5;

[0404] R3, R4and R5each independently are hydrogen or a hydrocarbon group having

[0405] 1 to 5 C-atoms, and n = a number from 0 to 20, m at each occurrence independently is 0 or 1 ; y = 0 or 1 ; and z = 0 or 1.

[0406] Item 2. Oligoglycerol-containing lipid of Formula (I) wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0407] A = NR3;

[0408] R3is hydrogen or a hydrocarbon group having 1 to 5 C-atoms; n = a number from 0 to 20; m at each occurrence independently is 0 or 1 ; y = 0 or 1 ; and z = 0 or 1.

[0409] Item 3. Oligoglycerol-containing lipid of Formula (I) wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0410] A = N+R4R5;

[0411] R4and R5each independently are hydrogen or a hydrocarbon group having 1 to 5 C-atoms; n = a number from 0 to 20; m at each occurrence independently is 0 or 1 ; y = 0 or 1 ; and z = 0 or 1.

[0412] Item 4. Oligoglycerol-containing lipid of Formula (IA) wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0413] A = NR3or N+R4R5;

[0414] R3, R4and R5each independently are hydrogen or a hydrocarbon group having

[0415] 1 to 5 C-atoms, and n = a number from 0 to 20, and m at each occurrence independently is 0 or 1. Item 5. Oligoglycerol-containing lipid of Formula (IA) wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0416] A = NR3;

[0417] R3is hydrogen or a hydrocarbon group having 1 to 5 C-atoms; n = a number from 0 to 20; and m at each occurrence independently is 0 or 1.

[0418] Those compounds are also termed NLGXherein.

[0419] Item 6. Oligoglycerol-containing lipid of Formula (IA) wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;

[0420] A = N+R4R5;

[0421] R4and R5each independently are hydrogen or a hydrocarbon group having 1 to 5 C-atoms; n = a number from 0 to 20; and m at each occurrence independently is 0 or 1.

[0422] Those compounds are also termed CLGXherein.

[0423] Item 7. Oligoglycerol-containing lipid according to any one of items 1 - 6, wherein n is a number from 0 to 10, preferably wherein n is a number from 0 to 5, more preferably wherein n is 0, 1 or 2, even more preferably wherein n is 0 or 1.

[0424] Item 8. Oligoglycerol-containing lipid according to any one of items 1 - 7, wherein n = 0.

[0425] Item 9. Oligoglycerol-containing lipid according to any one of items 1 - 8, wherein at each occurrence m = 1 .

[0426] Item 10. Oligoglycerol-containing lipid according to any one of items 1 - 9, wherein y = 1 and z = 1 .

[0427] Item 11. Oligoglycerol-containing lipid according to any one of items 1 - 10, wherein A = NR3and R3= C1-C5-alkyl, in particular R3= CH3, or wherein A = N+R4R5and R4and R5each independently are a C1-C5-alkyl, and in particular R4= R5= CH3.

[0428] Item 12. Oligoglycerol-containing lipid according to any one of items 1 - 11 , wherein R1and R2independently are a hydrocarbon group having 13 to 19 C- atoms and in particular, wherein R1and R2are a linear or branched C13- to C19-alkyl group.

[0429] Item 13. Oligoglycerol-containing lipid according to any one of items 1 - 12, wherein R1and R2independently are a mono- or polyunsaturated 013-0199 hydrocarbon group.

[0430] Item 14. Oligoglycerol-containing lipid according to any one of items 1 - 13, wherein R1and R2independently are a saturated C13-C19-alkyl group.

[0431] Item 15. Oligoglycerol-containing lipid according to any one of items 1 - 14, wherein R1and R2are a linear saturated C15 or C17 alkyl group or wherein R1and R2are a linear unsaturated C15 or 017 alkyl group.

[0432] Item 16. Oligoglycerol-containing lipid according to any one of items 1 , 2, 4, 5 or 7 - 15 having Formula (III) wherein y = 0 or 1 ; and z = 0 or 1 .

[0433] Item 17. Oligoglycerol-containing lipid according to any one of items 1 , 2, 4, 5 or 7 - 16 having Formula (I II A)

[0434] (111 A)

[0435] This compound is also termed NLG2 herein.

[0436] Item 18. Oligoglycerol-containing lipid according to any one of items 1 , 3, 4, 6, 7 - 15 having Formula (IV) wherein y = 0 or 1 ; and z = 0 or 1 .

[0437] Item 19. Oligoglycerol-containing lipid according to any one of items 1 , 3, 4, 6, 7 - 15 or

[0438] 18 having Formula (IVA)

[0439] Item 20. Oligoglycerol-containing lipid according to any one of items 1 - 19 having Formula (II) wherein the three stereogenic centers *1 , *2 and *3 have a predetermined configuration and wherein *p denotes further stereogenic centers.

[0440] Item 21. Oligoglycerol-containing lipid according to any one of items 1 - 20 having Formula (HA) wherein the three stereogenic centers *1 , *2 and *3 have a predetermined configuration and wherein *p denotes further stereogenic centers.

[0441] Item 22. Oligoglycerol-containing lipid according to item 20 or 21 having Formula (II) or having Formula (HA), wherein stereogenic center *1 is in R-configuration, stereogenic center *2 is in

[0442] S-configuration and stereogenic center *3 is in R-configuration. Item 23. Oligoglycerol-containing lipid according to any one of items 20 - 22 having Formula (II) or having Formula (HA), wherein the stereogenic centers *p have a predetermined configuration.

[0443] Item 24. Oligoglycerol-containing lipid according to any one of items 20 - 23 having Formula (II) or having Formula (HA), wherein the oligoglycerol-containing lipid is a single stereoisomer.

[0444] Item 25. A method for preparing an oligoglycerol-containing lipid according to any one of items 1 - 24, wherein the synthesis route includes an intermediate of Formula (V) or of Formula (V’) wherein PG, PG’ and PG” are independently a protecting group.

[0445] Item 26. The method for preparing an oligoglycerol-containing lipid according to item 25, wherein PG in particular is benzyl (Bn), tetrahydropyranyl (THP), ethoxyethylacetal (EE), 2-methoxypropan-2-ylacetal (MOP), silyl or paramethoxybenzyl (PMB), preferably PG is benzyl (Bn) for the synthesis of an oligoglycerol-containing lipid of Formula (I), wherein R1and R2independently are a saturated hydrocarbon group; preferably PG is THP or silyl for the synthesis of an oligoglycerol-containing lipid of Formula (I), wherein R1and R2independently are a mono- or polysaturated hydrocarbon group; and / or wherein PG’ is CH2 (methylene acetal), CHCH3 (ethylidene acetal), CHPh (benzylidene acetal), CHPhp-Ome (para-methoxybenzylidene acetal), C(CH3)2 (isopropylidene ketal) or SitBu2 (di-tert-butylsilylene), Si(iPr)2-O-Si(iPr)2 (tetraisopropyldisiloxanylidene), preferably PG’ is C(CH3)2 and / or wherein PG” in particular is benzyl (Bn), tetrahydropyranyl (THP), ethoxyethylacetal (EE), 2-methoxypropan-2-ylacetal (MOP), silyl or paramethoxybenzyl (PMB), preferably PG is benzyl (Bn). Item 27. The method for preparing an oligoglycerol-containing lipid according to item 25 or 26, comprising reacting a compound of Formula (V) or of Formula (V’) with glycidol.

[0446] Item 28. A liposome comprising at least one oligoglycerol-containing lipid according to any one of items 1-24.

[0447] Item 29. The liposome according to item 28 comprising at least 1 mol.- %, preferably at least 5 mol.-%, more preferably at least 10 mol.-% of oligoglycerol-containing lipids according to any one of items 1-24, based on the lipid content of the liposome.

[0448] Item 30. The liposome according to item 28 or 29 comprising at least 10 mol.- %, preferably at least 20 mol.-%, more preferably at least 50 mol.-% of oligoglycerol-containing lipids according to any one of items 1-24, based on the lipid content of the liposome.

[0449] Item 31. The liposome according to any one of items 28 - 30 comprising up to 100 mol.- %, preferably up to 80 mol.-%, more preferably up to 50 mol.-% of oligoglycerolcontaining lipids according to any one of items 1-24, based on the lipid content of the liposome.

[0450] Item 32. The liposome according to any one of items 28 - 31 , comprising cholesterol.

[0451] Item 33. The liposome according to any one of items 28 - 32, comprising cholesterol in an amount of from 1 mol.-%, preferably of from 30 mol.-% up to 70 mol.-%, preferably up to 60 mol.-%, based on the lipid content of the liposome.

[0452] Item 34. The liposome according to any one of items 28 - 33, further comprising at least one of phosphatidylcholine of formula (VII) wherein R3and R4each independently represent a hydrocarbon group having from 12 to 24 carbon atoms.

[0453] Item 35. The liposome according to any one of items 28 - 34, comprising at least one of phosphatidylcholine of formula (VII) wherein R3and R4are each independently a saturated or mono- or polyunsaturated, preferably linear alkyl group, in particular a saturated linear alkyl group.

[0454] Item 36. The liposome according to any one of items 28 - 35, comprising at least one of 1 ,2-dipalmitoyl-sn-glycero-3-phospho-choline (DPPC) and 1 ,2-distearoyl-sn- glycero-3-phosphocholine (DSPC).

[0455] Item 37. The liposome according to any one of items 28 - 35, comprising

[0456] (i) at least one oligoglycerol-containing lipid of Formula (I) according to item 2; and

[0457] (ii) at least one oligoglycerol-containing lipid of Formula (I) according to item 3.

[0458] Item 38. The liposome according to any one of items 28 - 35 or 37, comprising

[0459] (i) at least one oligoglycerol-containing lipid of Formula (IA) according to item 5; and

[0460] (ii) at least one oligoglycerol-containing lipid of Formula (IA) according to item 6.

[0461] Item 39. The liposome according to item 37, comprising

[0462] (i) at least one oligoglycerol-containing lipid of Formula (I) according to item 2;

[0463] (ii) at least one oligoglycerol-containing lipid of Formula (I) according to item 3; and

[0464] (iii) at least one phosphatidylcholine of Formula (VII), in particular 1 ,2- dipalmitoyl-sn-glycero-3-phospho-choline (DPPC) and / or 1 ,2-distearoyl-sn- glycero-3-phosphocholine (DSPC). Item 40. The liposome according to any one of items 37 - 39, comprising

[0465] (i) at least one oligoglycerol-containing lipid of Formula (IA) according to item 5;

[0466] (ii) at least one oligoglycerol-containing lipid of Formula (IA) according to item 6; and

[0467] (iii) at least one phosphatidylcholine of Formula (VII), in particular 1 ,2- dipalmitoyl-sn-glycero-3-phospho-choline (DPPC) and / or 1 ,2-distearoyl-sn- glycero-3-phosphocholine (DSPC).

[0468] Item 41. The liposome according to item 39 or 40, further comprising

[0469] (iv) cholesterol.

[0470] Item 42. The liposome according to any one of items 28 - 41 , comprising at least one phosphatidyloligoglycerol of Formula (VI) wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms; and wherein n is a number from 0 to 20, and m at each occurrence independently is 0 or 1 ; and y = 0 or 1 ; and z = 0 or 1 .

[0471] Item 43. The liposome according to any one of items 28 - 42, comprising at least one phosphatidyloligoglycerol of Formula (VIA) wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms and wherein n is a number from 0 to 20, and m at each occurrence independently is 0 or 1.

[0472] Item 44. The liposome according to any one of items 28 - 43, comprising at least one phosphatidyloligoglycerol of Formula (VI’) wherein the three stereogenic centers *1 , *2 and *3 have a predetermined configuration; and *p denotes further stereogenic centers; and wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms; n is a number from 0 to 20; m at each occurrence independently is 0 or 1 ; y = 0 or 1 ; and z = 0 or 1 ; and in particular wherein stereogenic center *1 is in R-configuration, stereogenic center *2 is in S-configuration and stereogenic center *3 is in R-configuration and *p denotes further stereogenic centers.

[0473] Item 45. The liposome according to any one of items 28 - 44, comprising at least one phosphatidyloligoglycerol of Formula (VI’A) wherein the three stereogenic centers *1 , *2 and *3 have a predetermined configuration; and *p denotes further stereogenic centers; and wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms; n is a number from 0 to 20; and m at each occurrence independently is 0 or 1 and in particular wherein stereogenic center *1 is in R-configuration, stereogenic center *2 is in S-configuration and stereogenic center *3 is in R-configuration and *p denotes further stereogenic centers.

[0474] Item 46. The liposome according to any one of items 28 - 45, comprising at least one phosphatidyloligoglycerol of Formula (VI”) wherein y = 0 or 1 ; and z = 0 or 1.

[0475] Item 47. The liposome according to any one of items 28 - 46, comprising at least one phosphatidyloligoglycerol of Formula (VI”A)

[0476] Item 48. The liposome according to any one of items 28 - 47, characterized in that it is a thermosensitive liposome.

[0477] Item 49. The liposome according to any one of items 28 -48, further comprising an active agent, in particular doxorubicin.

Claims

Claims1. Oligoglycerol-containing lipid of Formula (I)wherein R1and R2each independently are a hydrocarbon group having 12 to 24 C-atoms;A = NR3or N+R4R5;R3, R4and R5each independently are a hydrocarbon group having 1 to 5 C- atoms, and n = a number from 0 to 20, and m at each occurrence independently is 0 or 1 ; and y = 0 or 1 ; and z = 0 or 1.

2. Oligoglycerol-containing lipid according to claim 1 , wherein n = 0, 1 or 2 and in particular, wherein n = 0 or 1 .

3. Oligoglycerol-containing lipid according to any one of claim 1 or 2, wherein at each occurrence m = 1 ; and wherein y = 1 and z = 1.

4. Oligoglycerol-containing lipid according to any one of claims 1-3, wherein A = NR3and R3= C1-C5-alkyl, in particular R3= CH3; and / or wherein A = N+R4R5and R4and R5each independently are a C1-C5-alkyl, and in particular R4= R5= CH3.

5. Oligoglycerol-containing lipid according to any one of claims 1-4, wherein R1and R2independently are a hydrocarbon group having 13 to 19 C-atoms and in particular, wherein R1and R2are a linear or branched C13- to C19-alkyl group.

6. Oligoglycerol-containing lipid according to any one of claims 1-5, wherein R1and R2independently are a mono- or polyunsaturated C13-C19 hydrocarbon group.

7. Oligoglycerol-containing lipid according to any one of claims 1-6, wherein R1and R2independently are a saturated C13-C19-alkyl group.

8. Oligoglycerol-containing lipid according to any one of claims 1-7 having Formula (II)wherein the three stereogenic centers *1 , *2 and *3 have a predetermined configuration and in particular wherein stereogenic center *1 is in R- configuration, stereogenic center *2 is in S-configuration and stereogenic center *3 is in R-configuration.

9. A method for preparing an oligoglycerol-containing lipid according to any one of claims 1-8, wherein the synthesis route includes an intermediate of Formula (V) or of Formula (V’)wherein PG, PG’ and PG” are independently a protecting group.

10. A liposome comprising at least one oligoglycerol-containing lipid of Formula (I) according to any one of claims 1-8.11 . The liposome according to claim 10 comprising at least 1 mol.- of oligoglycerolcontaining lipids according to any one of claims 1-8, based on the lipid content of the liposome.

12. The liposome according to any one of claims 10 or 11 , further comprising cholesterol.

13. The liposome according to any one of claims 10 - 12, further comprising at least one of phosphatidylcholine of formula (VII)wherein R3and R4each independently represent a hydrocarbon group having from 12 to 24 carbon atoms; and preferably at least one of 1 ,2-dipalmitoyl-sn-glycero-3-phospho-choline (DPPC) and 1 ,2-distearoyl-sn- glycero-3-phosphocholine (DSPC).

14. The liposome according to any one of claims 10 - 13, comprising(i) at least one oligoglycerol-containing lipid of Formula (I) wherein A = NR3; and(ii) at least one oligoglycerol-containing lipid of Formula (I) wherein A = N+R4R5.

15. The liposome according to any one of claims 10 - 14, comprising at least one phosphatidyloligoglycerol of Formula (VI’)wherein the three stereogenic centers *1 , *2 and *3 have a predetermined configuration; and *p denotes further stereogenic centers; andwherein R1and R2each independently are a hydrocarbon group having 12 to24 C-atoms; n is a number from 0 to 20; and m at each occurrence independently is 0 or 1 and in particular wherein stereogenic center *1 is in R-configuration, stereogenic center *2 is in S-configuration and stereogenic center *3 is in R-configuration and *p denotes further stereogenic centers.

16. The liposome according to any one of claims 10 - 15, comprising at least one phosphatidyloligoglycerol of Formula (VI”A)17. The liposome according to any one of claims 10 - 16, characterized in that it is a thermosensitive liposome.

18. The liposome according to any one of claims 10 - 17, further comprising an active agent, in particular doxorubicin.

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