The present invention relates to novel lipid molecules and methods of synthesising the same. The invention extends to lipid-based nano- or micro-particle compositions, including a novel lipid, suitable for delivery of a nucleic acid, such as (modified) RNA or

DNA. The invention further relates to medical and research uses of the compositions and methods of delivering a nucleic acid, such as (modified) RNA or DNA to a mammalian cell either in a tissue culture or in a whole organism. RNAbiology is central to the functioning of the majority of life forms, including humans. Consequently, most diseases are caused by and/or manifest via changes in the presence, abundance, sequence, binding, or other properties of endogenous RNA in the cells involved. Therefore, it stands to reason that (personalized) solutions for said diseases can be found by changing and/or correcting the presence, abundance, sequence, binding, or other properties of endogenous RNA where correcting relates to re-establishing the non-pathological physiological situation. RNA molecules when brought into cells provide solutions for such changes and corrections. These RNA molecules can therefore also be used as therapeutics for said diseases. Examples for such RNA molecules include small interfering RNA (siRNA) and anti-sense RNA to repress the expression and/or promote the breakdown of endogenous RNA, splice - correcting RNAs to change the splicing of pre-messenger RNA (pre-mRNA) to mature mRNA, and mRNA itself as a way to increase expression of the encoded protein.

Recent advances in our understanding of RNA biology, including the intracellular innate immunity pathways that recognize foreign (viral) RNA, have increased the commercial viability and interest in RNA-based therapies, exemplified by the commercialisation of the first messenger RNA and siRNA drugs in 2020 (SARS-C0V2 vaccines) and 2018 (Onpattro) respectively. Given the low cellular uptake of such large, negatively charged and hydrophilic molecules by the cell due to the negatively charged surface of the plasma-membrane and the restriction of permeability for small hydrophobic molecules and those for which transporters and channels exist, a delivery vehicle is often used to enhance the activity RNA therapeutics and vaccines by several orders of magnitude. Therefore, the interest in RNA delivery vehicles has also increased. However, effective delivery of RNA into cells by delivery vehicles remains an ongoing scientific challenge, for which novel formulations and methods need to be developed. These delivery vehicles often need to serve multiple purposes, requiring seemingly contradictory properties. The primary purpose of RNA delivery vehicles is to facilitate cellular entry, by packaging the RNA into a small (nano -)particle aiding the RNA in the traversal of the plasma membrane or the induction of endocytosis and traversal of the endocytic membrane. This support of traversal requires shielding of the negative charges of the phosphate backbone of the RNA, interaction with and destabilization of the plasma or endocytic lipid bilayer, and subsequent dissociation of the RNA and release in the cytosol. Furthermore, delivery vehicles protect the RNA from degrading enzymes in the bloodstream, the extracellular milieu and also during endosomal uptake via encapsulation of the RNA by the vehicle. In addition, preliminary evidence suggests that encapsulation in a delivery vehicle can in some cases protect the RNA from autocatalytic breakdown by changing/ reducing the interaction with water molecules. For efficient encapsulation, the vehicles need to interact with sufficient affinity with the RNA, whereas efficient release of the RNA in the cytosol requires this affinity to be partially or completely reversed. Disruption of the plasma- or endocytic-membrane requires membrane-disrupting/ destabilizing activity, which can be at odds with colloidal stability of the delivery vehicle itself, for example through the use of lipids as delivery vehicle with high fluidity (through multiple unsaturated bonds in the lipid tail) in lipid-based nanoparticles.

Delivery vehicles for RNA and DNA fall into different classes. The most prominent distinction can be made between polymeric delivery vehicles and lipid-based delivery vehicles. Lipid-based delivery vehicles consist out of a hydrophilic head group and a hydrophobic tail and are thus amphiphilic in nature.

Two main forms of formulations based on lipid-based delivery vehicles can be distinguished, which are micellar formulations andlipid nanoparticles (LNPs). Micellar formulations consist of one type of lipid, the delivery vehicle, which is dissolved into an aqueous solution in which the lipids form micelles through their hydrophobic tails and associate with the RNA or DNA through their positively charged head groups. These micellar formulations, also called lipoplexes are typically used to deliver RNA or DNA into cells in tissue culture experiments. LNPs consist of a lipid that serves as the delivery vehicle, in combination with a so - called helper lipid, with cholesterol, and a polyethylene glycol (PEG) -conjugated lipid. These LNPs are generated through mixing of an ethanolic solution of the lipids and an aqueous solution of RNA and are also suited for use in animals and humans. LNPs are the most frequently used type of formulation and are used in the approved RNASARS-

C0V2 vaccines and Onpattro.

The first generation of lipid-based RNA and DNA delivery vehicles used for LNP formation ensured efficient encapsulation, shielding of the RNA negative charge and high efficiency cellular interaction/ uptake. These delivery vehicles were all characterized by being constitutively positively charged. The positive charge is located in the head group, where head group refers to the hydrophilic part of the lipid molecules, as one of the three main structural elements can be distinguished in a lipid. The other two being, the hydrophobic hydrocarbon chains, and the backbone that links the head group to the hydrocarbon chains. In a natural phospholipid, the hydrocarbon chains are present as carboxylic acids (fatty acids) which are linked to the hydroxy groups of the glycerol backbone via ester bonds. The ester bond also enables cleavage through phospholipases to release the fatty acids from the hydrophilic backbone, enabling further metabolic turn-over of both parts. For the lipid systems, DOTAP, DOTMA, DOGS and DOSPA are well-known examples of constitutively cationic charged species. However, as a direct consequence, intracellular release of the RNA for these vehicles is low, and the constitutively cationic delivery vehicles exhibit significant toxicity, as well as rapid interaction with, and clearance by, the Reticuloendothelial system (RES) (mostly particles <200nm) and/ or the Mononuclear Phagocyte system (mostly particles >200nm) which determines the biodistribution of these delivery vehicles.

These issues were primarily addressed by the use of lipids with ionizable cationic head groups. Using these compounds, the lipid head groups that are present at the surface of the nanoparticle assumes a (close to) neutral surface charge under physiological (pH around 7.4) conditions, such as during circulation in the blood. The neutral charge at physiological pH effectively prevents toxicity and ionizable lipid nanoparticles are tolerated at doses sufficient for vaccination and treatments meant to change protein expression in whole organs, such as siRNA treatments or expression of proteins for therapeutic purposes. Formulation of the RNA with an ionizable delivery vehicles is often achieved by acidifying the pH of the aqueous solution of either the RNA, the delivery vehicle or both, during first contact. Once the charge-driven interaction between the RNA and the components of the nanoparticle, the pH may be raised again to physiological level via for example dialysis, size-exclusion methods or addition of excess buffer. Where the ionizable lipid is in contact with RNA, the positive charge is maintained, whereas at the surface of the nanoparticle the ionizable head group is deprotonated, assuming a neutral charge. Once the delivery vehicle is taken up by the cell via endocytosis, the acidification of the endosomal compartment ionizes the surface lipids and re-introduces the cationic surface charge, allowing electrostatic interaction with the negatively charged endosomal lipid bilayer. This interaction is often the first step of the endosomal escape process. Alternatively, or concurrently, the ionizable compounds may act as a proton sponge, resulting in swelling of the endosomal compartment due to chloride and subsequent water influx, ultimately resulting in burst of the compartment. Also, the introduction of positive charge at the surface of the nanoparticle leads to charge repulsion, thereby destabilizing the nanoparticle and promoting RNA release. Of note, the proton sponge hypothesis is heavily debated. Once the endosomal membrane has been disrupted and some destabilization of the nanoparticle occurred, the ionizable compounds may experience a neutral pH again due to direct contact with the cytosol, and under these conditions release the now exposed RNA Well-known ionizable lipids used for RNA delivery include amine- containing lipids that can be readily protonated such as DODAP (as the ionizable counterpart of DOTAP), DLin-DMAand its derivatives DLin-KC2-DMA and Dlin-MC3- DMA.

Next to reducing the exposure of positive charges at the surface of nanoparticles, the toxicity of (ionizable) cationic delivery vehicles can be reduced by biodegradation and excretion, preventing bioaccumulation in and/or prolonged exposure of sensitive organs. Frequently used strategies in the prior art include the use of compounds that are inherently water-soluble and can be excreted via the urine, as well as the introduction of metabolizable groups, such as esters or protease -cleavable amides, resulting in break-down products that are excreted or used as metabolites.

Furthermore, disulphide bridges have been used in macromolecular complexes as these are cleaved by reduction inside the cytosol leading to decomposition into smaller molecular building blocks. Of the many ionizable delivery vehicles known from the prior art, LNPs have extraordinary benefits. Besides electrostatic interactions between ionizable lipid and RNA, particle stability is also maintained by the hydrophobic interactions of the lipid tails. Incorporation of a PEGylated lipid that acts as a shielding lipid yields colloidal stability in spite of the absence of surface charge, allowing the use of a neutral surface - charge in circulation and during storage. In addition, it is hypothesized that mixing of the lipids of the delivery vehicle with the lipids ofthe endosomal membrane contributes to its superior endosomal escape activity by (further) disrupting the stability of the endosomal membrane. Finally, using multiple lipids in a single formulation, allows the precise tuning of nanoparticle properties (size, stability, delivery efficiency, bio distribution) beyond what would be possible or readily achieved with a single compound.

Despite many years of research, the rational design of ionizable lipids (for use in lipid nanoparticles) with predictable RNA delivery properties has remained elusive. As a consequence, the successful design of an ionizable lipid always entails an element of trial and error and surprise. Consensus has been reached on the ideal pKa ofthe LNP as a complete formulation, with an apparent pKa determined by the 2-(p-toluidino)-6- napthalene sulfonic acid (TNS) assay that matches the pH in the early endosome (around pH 6.4) as being ideal (Jayaraman et al. 2012 Angewandte Chemie, DOI: i0.i002/ang.20i203263). However, even for formulations with identical pKa, over 2 orders of magnitude differences in efficacy exist (figure 3 from aforementioned publication), indicating that next to pKa also the structure of the headgroup and/or lipid properties contribute significantly to delivery activity, for example by influencing nanoparticle shape (Carrasco et al. 2021 Communications Biology, DOI: 10.1038/S42003-021-02441-2).

Besides delivery activity and safety, facile and efficient synthesis of the ionizable lipid is an important parameter for the commercial success of any novel ionizable lipid for RNA delivery. Many of the ionizable lipids from the prior art, including lipid SM-102, have complicated structures, with branched lipid tails to induce a conical structure of lipid layers. Such molecular structures often require a multi-step synthesis using protecting groups (e.g., SM-102 in W02017049245 and ALC-0315 in W02018081480). The presence of side products/incomplete conversion of precursors that result from such multi-step procedures present challenges during quality control. Furthermore, uncertainties exist related to the metabolic turnover of such branched lipid structures, including SM-102 and ALC-0315, and the clearance and/ or degradation of the metabolites derived fromsuch structures. Ideally, metab olization of the novel ionizable lipid would result in metabolites identical to those of normal metabolism and/ or of high water-solubility, allowing rapid excretion via the urine, to prevent bioaccumulation. In summary, there is still an urgent need for novel ionizable lipids with a combination of a high-efficiency cellular uptake, a highly efficient endosomal release, a predictable metab olization and a facile synthesis are still needed to advance RNA-based therapy and vaccination. In accordance with a first aspect of the invention, there is provided a compound of

Formula (I): CD wherein:

R ¹ and R ² are independently selected from the group consisting of optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl or optionally substituted C2-50 alkynyl;

L ¹ and L ³ are independently absent or is an optionally substituted C1-10 alkylene, an optionally substituted C2-10 alkenylene or an optionally substituted C2-10 alkynylene;

L ² is absent or is NH, S or O;

R ³ is selected from the group consisting of -NR ⁴ R ⁵ , -N ⁺ R ⁴ R ⁵ R ⁶ , -H, -SR ⁴ , -OR ⁴ , -CN, - COR ⁴ , -COOR ⁴ , -OCOR ⁴ , -CONR ⁴ R5, -NR ⁴ SO ₂ R5, -SO ₂ NR ⁴ R5, -NR ⁴ COR ³ , - OP(O)(OH)OR ⁴ , optionally substituted C3-6 cycloalkyl, optionally substituted C3-6 cycloalkenyl, optionally substituted C6-12 aryl, optionally substituted 3 to 10 membered heterocycle or optionally substituted 5 to 10 membered heteroaryl; and

R ⁴ to R ⁶ are independently selected from the group consisting of H, optionally substituted C1-30 alkyl, optionally substituted C2-30 alkenyl, optionally substituted C2-30 alkynyl, optionally substituted C3-6 cycloalkyl, optionally substituted C3-6 cycloalkenyl, optionally substituted C6-12 aryl, optionally substituted 3 to to membered heterocycle or optionally substituted 5 to 10 membered heteroaryl; or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof.

Advantageously, compounds of formula (I) can be used to produce stable lipid nanoparticles (LNPs). The LNPs have high encapsulation efficiency and can be used to deliver a therapeutic or prophylactic agent to a patient. “Optional” or “optionally” means that the subsequently described event, operation or circumstances can or cannot occur, and that the description includes instances where the event, operation or circumstance occurs and instances where it does not.

The term “alkyl” as used herein, unless otherwise specified, refers to a saturated straight or branched hydrocarbon. “Alkenyl” refers to olefinically unsaturated hydrocarbongroups which can be unbranched or branched, i.e. the hydrocarbon group contains one or more carbon-carbon double bonds. “Alkynyl” refers to acetylenically unsaturated hydrocarbon groups which can be unbranched or branched, i.e. the hydrocarbon group contains one or more carbon-carbon triple bonds. In addition to containing one or more carbon-carbon triple bonds, an alkynyl group may also contain one or more carbon-carbon double bond.

The term “alkylene”, as used herein, unless otherwise specified, refers to a bivalent saturated straight or branched hydrocarbon. Similarly, the term “alkenylene”, as used herein, unless otherwise specified, refers to a bivalent olefinically unsaturated straight or branched hydrocarbon. The term “alkenylene”, as used herein, unless otherwise specified, refers to a bivalent acetylenically unsaturated straight or branched hydrocarbon. In addition to containing one or more carbon-carbon triple bonds, an alkynylene group may also contain one or more carbon-carbon double bonds.

Any alkyl, alkenyl, aklynyl, alkylene, alkenylene and/ or alkynylene group can be unsubstituted or substituted with one or more of halogen, -NR ⁴ R ⁵ , -N ⁺ R ⁴ R ⁵ R ⁶ , -H, -SR ⁴ , -OR ⁴ , -CN, -COR ⁴ , -COOR ⁴ , -OCOR ⁴ , -CONR ⁴ R5, -NR ⁴ SO ₂ R5, -SO ₂ NR ⁴ R5, -NR ⁴ COR5, - OP(O)(OH)OR ⁴ , oxo, optionally substituted C3-6 cycloalkyl, optionally substituted C3-6 cycloalkenyl, optionally substituted C6-12 aryl, optionally substituted 3 to 10 membered heterocycle or optionally substituted 5 to 10 membered heteroaryl . R ⁴ to R ⁶ may be as defined in relation to the first aspect. R4 to R ⁶ may each independently be selected fromthe group consisting of H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C3-6 cycloalkyl, C3-6 cycloalkenyl, C6-12 aryl, 3 to 10 membered heterocycle or 5 to 10 membered heteroaryl. “Cycloalkyl” refers to a non-aromatic, saturated, hydrocarbon 3 to 6 membered ring system. Representative examples of a C3-C6 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. Similarly, “cyclo alkenyl” refers to a non-aromatic, unsaturated, hydrocarbon 3 to 6 membered ring system. “Aryl” refersto an aromatic 6 to 12 memberedhydrocarbongroup. Examples of a C6-C12 aryl group include, but are not limited to, phenyl, a-naphthyl, p-naphthyl, biphenyl, tetrahydro naphthyl and indanyl.

“Heterocycle” or “hetero cyclyl” refers to 3 to 10 membered monocyclic, bicyclic or bridged molecules in which at least one ring atom is a heteroatom. The or each hetero atom may be independently selected from the group consisting of oxygen, sulfur and nitrogen. A heterocycle maybe saturated or partially saturated. Exemplary hetero cyclyl groups include but are not limited to aziridine, oxirane, oxirene, thiirane, pyrroline, pyrrolidine, dihydrofuran, tetrahydro furan, dihydrothiophene, tetrahydrothiophene, dithiolane, piperidine, 1,2,3,6-tetrahydropyridine-i-yl, tetrahydro pyran, pyran, morpholine, piperazine, thiane, thiine, piperazine, azepane, diazepane, oxazine.

“Heteroaryl” refers to a monocyclic or bicyclic aromatic 5 to 10 membered ring system in which at least one ring atom is a heteroatom. The or each heteroatom may be independently selected from the group consisting of oxygen, sulfur and nitrogen.

Examples of 5 to 10 membered heteroaryl groups include furan, thiophene, indole, azaindole, oxazole, thiazole, isoxazole, isothiazole, imidazole, N-methylimidazole, pyridine, pyrimidine, pyrazine, pyrrole, N-methylpyrrole, pyrazole, N-methylpyrazole, 1,3,4-oxadiazole, 1,2, 4 -triazole, 1- methyl-i,2,4-triazole, iH-tetrazole, i-methyltetrazole, benzoxazole, benzothiazole, benzofuran, benzisoxazole, benzimidazole, N- methylbenzimidazole, azabenzimidazole, indazole, quinazoline, quinoline, and isoquinoline. Bicyclic 5 to 10 membered heteroaryl groups include those where a phenyl, pyridine, pyrimidine, pyrazine or pyridazine ring is fused to a 5 or 6 -membered monocyclic hetero aryl ring. Any cycloalkyl, cycloalkenyl, aryl, heterocycle and/or heteroaryl group can be unsubstituted or substituted with one or more of optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkenyl, optionally substituted C1-C6 alkynyl, halogen, - NR4R5, -N ⁺ R4R5R6, -H, -SR4, -OR4, -CN, -COR4, -COOR4, -OCOR4, -CONR4R5, - NR4SO2R ⁵ , -SO2NR4R5, -NR4COR ⁵ , -OP(O)(OH)OR ⁴ , oxo, optionally substituted C3-6 cycloalkyl, optionally substituted C3-6 cycloalkenyl, optionally substituted C6 -12 aryl, optionally substituted 3 to 10 membered heterocycle or optionally substituted 5 to 10 membered heteroaryl. R4 to R ⁶ maybe as defined in relation to the first aspect. R4 to R ⁶ may each independently be selected from the group consisting of H, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C3-6 cycloalkyl, C3-6 cycloalkenyl, C6-12 aryl, 3 to 10 membered heterocycle or 5 to 10 membered heteroaryl.

The term “pharmaceutically acceptable salt” maybe understood to refer to any salt of a compound provided herein which retains its biological properties and which is not toxic or otherwise undesirable for pharmaceutical use. Such salts may be derived from a variety of organic and inorganic counter-ions well known in the art. Such salts include, but are not limited to: (1) acid addition salts formed with organic or inorganic acids such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, sulfamic, acetic, adepic, aspartic, trifluoro acetic, trichloroacetic, propionic, hexanoic, cyclopentylpropionic, glycolic, glutaric, pyruvic, lactic, malonic, succinic, sorbic, ascorbic, malic, maleic, fumaric, tartaric, citric, benzoic, 3-(4-hydroxybenzoyl)benzoic, picric, cinnamic, mandelic, phthalic, lauric, methanesulfonic, ethanesulfonic, 1,2-ethane-disulfonic, 2- hydr oxy ethanesulfonic, benzenesulfonic, 4-chlorobenzenesulfonic, 2- naphthalenesulfonic, 4-toluenesulfonic, camphoric, camphorsulfonic, 4- methylbicyclo [2.2.2]-oct-2-ene-i-carboxylic, glucoheptonic, 3-phenylpropionic, trimethylacetic, tert -butylacetic, lauryl sulfuric, gluconic, benzoic, glutamic, hydroxynaphthoic, salicylic, stearic, cyclohexylsulfamic, quinic, muconic acid and the like acids; or (2) base addition salts formed when an acidic protonpresent in the parent compound either (a) is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion or an aluminium ion, or alkali metal or alkaline earth metal hydroxides, such as sodium, potassium, calcium, magnesium, aluminium, lithium, zinc, and barium hydroxide, ammonia or (b) coordinates with an organic base, such as aliphatic, alicyclic, or aromatic organic amines, such as ammonia, methylamine, dimethylamine, diethylamine, picoline, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine, arginine, ornithine, choline, N,N' -dibenzylethylene-diamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, N- methylglucamine piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, and the like.

Pharmaceutically acceptable salts may include, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium and the like, and when the compound contains a basic functionality, salts of non-toxic organic or inorganic acids, such as hydrohalides, e.g. hydrochloride, hydrobromide and hydroiodide, carbonate or bicarbonate, sulfate or bisulfate, borate, phosphate, hydrogen phosphate, dihydrogen phosphate, pyroglutamate, saccharate, stearate, sulfamate, nitrate, orotate, oxalate, palmitate, pamoate, acetate, trifluoro acetate, trichloroacetate, propionate, hexanoate, cyclopentylpropionate, glycolate, glutarate, pyruvate, lactate, malonate, succinate, tannate, tartrate, tosylate, sorbate, ascorbate, malate, maleate, fumarate, tartarate, camsylate, citrate, cyclamate, benzoate, isethionate, esylate, formate, 3-(4- hydroxybenzoyl)benzoate, picrate, cinnamate, mandelate, phthalate, laurate, methanesulfonate (mesylate), methylsulphate, naphthylate, 2-napsylate, nicotinate, ethanesulfonate, 1,2-ethane-disulfonate, 2-hydroxyethanesulfonate, benzenesulfonate (besylate), 4-chlorobenzenesulfonate, 2-naphthalenesulfonate, 4 -toluenesulfonate, camphorate, camphor sulf onate, 4-methylbicyclo[2.2.2]-oct-2-ene-i-carboxylate, glucoheptonate, 3-phenylpropionate, trimethylacetate, tert -butylacetate, lauryl sulfate, gluceptate, gluconate, glucoronate, hexafluorophosphate, hibenzate, benzoate, glutamate, hydroxynaphthoate, salicylate, stearate, cyclohexylsulfamate, quinate, muconate, xinofoate and the like.

L ¹ may be absent.

In a preferred embodiment, L ¹ is an optionally substituted C1-6 alkylene, an optionally substituted C2-6 alkenylene or an optionally substituted C2-6 alkynylene. More preferably, L ¹ is C1-3 alkylene, C2-3 alkenylene or C2-3 alkynylene. Accordingly, L ¹ may be -CH2-, -CH2CH2- or -CH2CH2CH2-.

In some embodiments, L ² maybe NH. In some embodiments, L ³ maybe an optionally substituted C1-6 alkylene, an optionally substituted C2-6 alkenylene or an optionally substituted C2-6 alkynylene. L ³ is C1-3 alkylene, C2-3 alkenylene or C2-3 alkynylene. Accordingly, L ³ maybe -CH2-, -CH2CH2- or -CH2CH2CH2-.

In alternative embodiments, L ² maybe absent. L ³ maybe absent. In some embodiments, R ³ is -NR ⁴ R ⁵ , -SR ⁴ , -OR ⁴ , optionally substituted 3 to 10 membered heterocycle or optionally substituted 5 to 10 membered heteroaryl . R ³ may be -NR ⁴ R5, -SR ⁴ , -OR ⁴ , optionally substituted 5 or 6 membered heterocycle or optionally substituted 5 or 6 membered heteroaryl. The heterocycle or heteroaryl may comprise one or more nitrogen atoms. The heterocycle or heteroaryl may be an N- linked heterocycle or heteroaryl.

R ³ is preferably -NR ⁴ R ⁵ , -SR ⁴ or -OR ⁴ . R ³ is more preferably -NR ⁴ R ⁵ .

R ⁴ to R ⁶ may be independently selected from the group consisting of H, optionally substituted C1-20 alkyl, optionally substituted C2-20 alkenyl and optionally substituted C2- 20 alkynyl. More preferably, R ⁴ to R ⁶ are independently selected from the group consisting of H, optionally substituted C1-10 alkyl, optionally substituted C2-10 alkenyl and optionally substituted C2-10 alkynyl. Most preferably, R ⁴ to R ⁶ are independently selected from the group consisting of C1-5 alkyl, C2-5 alkenyl and C2-5 alkynyl. In some embodiments, R ⁴ to R ⁶ are independently methyl, ethyl or propyl, more preferably methyl or ethyl.

Accordingly, in some embodiments, R ³ maybe

In some embodiments, the compound of Formula (I) maybe a compound of Formula (Ila), (lib), (lie), (nd) or (lie):

(lib)

(Ila)

R ¹ and R ² may independently be selected from the group consisting of optionally substituted C5-30 alkyl, optionally substituted C5-30 alkenyl or optionally substituted C5-30 alkynyl. More preferably, R ¹ and R ² may independently be selected from the group consisting of optionally substituted C10-25 alkyl, optionally substituted C10-25 alkenyl or optionally substituted C10-25 alkynyl. Even more preferably, R ¹ and R ² may independently be selected from the group consisting of optionally substituted C 15-20 alkyl, optionally substituted C 15-20 alkenyl or optionally substituted C 15-20 alkynyl. In some embodiments, R ¹ and R ² are both a C15-20 alkenyl. The alkenyl may be a singly or doubly unsaturated alkenyl group.

In a preferred embodiment, R ² is the same as R ¹ . Accordingly, in some embodiments, the compound is a compound of formula (m): , wherein both R ¹ groups are the same.

In some embodiments, R ¹ and R ² are -(CH2)8(CH)2CH2(CH)2(CH2)4CH3 or -(CH2)8(CH)2(CH2) ₇ CH ₃ . More preferably, R ¹ and R ² are The compound maybe a compound of formula (to i), (102), (103), (104), (105) or (106):

It may be appreciated that the compounds of formula (I) have at least one stereocenter. The compound may be the S or the R stereoisomer. Alternatively, the compound may be provided as a mixture of both isomers with variable mole fraction of R ranging from o % to too % and S ranging from too % to o %. The compound may be provided as a racemic mixture.

In embodiments where R ³ is -NR ⁴ R ⁵ , the amine moiety may be unprotonated at a physiological pH. Alternatively, the amine moiety may be protonated at a physiological pH. A physiological pH maybe understood to be a pH of less than or equal to 7.5 at 37°C. A physiological pH may be a pH between 7.3 and 7.5 at 37°C. In some embodiments of Formula (I), the amine moiety may possess a pKa > 7.5, rendering the lipid cationic over the entire pH-range anticipated in the mammalian body. Such lipids are typically referred to as cationic (amino )lipids. Accordingly, a salt of the compound of formula (I) may be a salt of formula (IV): , wherein X- is a counterion.

A compound of formula (I) may be used to form a lipid nanoparticle (LNP). Accordingly, in accordance with a second aspect of the invention, there is provided a micro- or nanoparticle comprising a compound of formula (I), as defined by the first aspect, or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof, and an optional payload molecule. The micro- or nanoparticle may define a compact or hollow structure. The micro - or nanoparticle may optionally consist of one or more lipid bilayers, which may be optionally crosslinked to each other, encapsulating a void. The micro- or nanoparticle may be a lipid nanoparticle (LNP), a liposome, a lipoplex, a micelle or a lipid vesicle. In some embodiments, the micro- or nanoparticle is an LNP.

The micro- or nanoparticle may have a diameter less than 10 pm, less than 1 pm, less than 500 nm or less than 250 nm. More preferably, the micro - or nanoparticle may have a diameter of less than 200 nm, less than 175 nm, less than 150 nm or less than 125 nm. The micro- or nanoparticle may have a diameter between 30 nm and 1 pm, between 40 and 500 nm, between 50 and 250 nm, between 60 and 200 nm, between

70 and 175 nm, between 80 and isonm or between 90 and 125 nm. The diameter of the micro- or nanoparticle maybe measured using dynamic light scattering.

In addition to the compound of formula (I), or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof, the micro- or nanoparticle may comprise one or more further lipids. Accordingly, the micro- or nanoparticle may comprise a lipid component comprising the compound of formula (I), or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof, and one or more further lipids. The one or more further lipids may be selected from the group consisting of a phospholipid, a permanent or ionisable cationic lipid, a permanent or ionisable anionic lipid, a structural lipid, a shield lipid, a functionalised lipid and combinations thereof.

In some embodiments, the lipid component comprising a compound of formula (I), or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof, and a structural lipid. In some embodiments, the lipid component comprises a compound of formula (I), or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof, a structural lipid and a phospholipid. In some embodiments, the lipid component comprises a compound of formula (I), or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphicformthereof, a structural lipid, a phospholipid and a shield lipid. In some embodiments, the lipid component further comprises a permanent or ionisable cationic lipid. In some embodiments, the lipid component further comprises a functionalised lipid. In some embodiments, the lipid component comprises between 20 to 80 mol%, between 30 to 70 mol%, between 40 to 60 mol% or between 45 to 55 mol% of the compound of Formula (I), or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof. In some embodiments, the lipid component comprises between 5 and 80 mol%, between 10 and 60 mol %, between 20 and 50 mol%, between 30 and 45 mol% or between 35 and 40 mol% of the structural lipid.

In some embodiments, the lipid component comprises between o and 30 mol%, between 2.5 and 20 mol%, between 5 and 15 mol% or between 7.5 and 12.5 mol% of the phospholipid.

In some embodiments, the lipid component comprises between o and 15 mol%, between 0.1 and 10 mol%, between 0.5 and 5 mol%, between 0.75 and 3 mol% or between 1 and 2 mol% of the shield lipid.

In some embodiments, the lipid component comprises between o and 15 mol%, between 0.1 and 10 mol%, between 0.5 and 5 mol%, between 0.75 and 3 mol% or between 1 and 2 mol% of the permanent or ionisable cationic lipid.

The structural lipid maybe a sterol. The sterol maybe cholesterol or a cholesterol derivative. The cholesterol derivative may be beta-sitosterol, Vitamin D2, Vitamin D3, Calcipotriol, Stigmasterol, Campesterol, Fuco sterol, Brassicasterol, Ergosterol, 9,11- dehydroergosterol, Daucosterol,beta-Sitosterol-Acetate, Betutin, Lupeol, Ursoticacid, or Oleanotic acid. The phospholipid maybe di-oleoyl-phosphatidylethanolamine (DOPE), di-oleoyl- phosphatidyl choline (DOPC), Di-oleoyl-phosphatidylserine (DOPS), 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), i,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) or any naturally occurring phospholipid.

A shielding lipid may be understood to be a functionalized lipid that prevents the unwanted interaction of the lipid nanoparticle with other nanoparticles, extracellular compounds and/or cellular surfaces. The shielding lipid may prevent the unwanted interaction of the lipid nanoparticle with other nanoparticles, extracellular compounds and/ or cellular surfaces through sterical hindrance or a similar mechanism. The shield lipid may be a lipid which has been modified to comprise a shielding polymer. The shielding polymer maybe a polyethylene glycol (PEG) group, a poly-sarcosine group, an oligopeptide or a polypeptide, a hydroxyl -containing no n-ionic water-soluble polymer, polyvinylpyrrolidone (PVP), apoly(2-alkyl-2-oxazoline) or a zwitterionic polymer or any other suitable shielding polymer. The oligopeptide or polypeptide may be PAS. PAS may be understood to be an oligopeptide or polypeptide consisting or comprising proline, alanine and serine residues. The hydroxyl-containing no n-ionic water-soluble polymer may be poly(glycerol) (PG), poly(N (2 hydroxypropyl)methacrylamide) (pHPMA), polysarcosine, or poly(vinyl alcohol). The zwitterionic polymer may be a polybetaine. In some embodiments, the shield lipid may be a phospholipid which has been modified to comprise a shielding polymer. In embodiments where the shield lipid is a phospholipid which has been modified to comprise a shielding polymer, the phospholipid which is modified may be a phospholipid as defined above. In some embodiments the shield lipid may be a DSPE, DMG or DPPC lipid which has been modified to comprise a shielding polymer. The shield lipid may comprise a shielding polymer with an average molecular weight of between 500 and 5000, between 1000 and 3000, between 1250 and 1750, between 1500 and 2500, between 1750 and 2250 Da or between 1900 and 2100 Da. The shield lipid maybe di-myristoyl-glycerol-PEG (DMG-PEG), PEG-DSPE, PEG-di-palmitoyl- pho sphatidyl-cho line (DPPC), pSar-DMG, pSar-DSPE or pSar-DPPC. In some embodiments, the shield lipid is PEG-DSPE or pSar-DSPE.

The permanent or ionisable cationic lipid maybe i,2-dilinoleyloxy-N,N- dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[i,3]- dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,3i-tetraen-i9-yl 4- (dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[i,3]-dioxolane (DLin-KC2-DMA), i,2-dioleoyl-3- trimethylammonium-propane (DOTAP) or i,2-Dioleoyl-3-trimethylammonium propane (DODAP). The permanent or ionisable anionic lipid may be cholesteryl hemisuccinate, a phosphatidylinositol phosphate (PIP, also known as phosphoinositide), phosphatidylserine (PS) or phosphatidic acid (PA).

The functionalised lipid may comprise one or more moieties which allow conjunction thereto. The one or more moieties may independently be selected from the group consisting of azide, alkyne, tetrazine, dibenzocyclooctyne (DBCO), maleimide, trans- cycloctene (TCO), vinyl, methylcyclopropene and succinimidyl -ester.

In a preferred embodiment, the micro- or nanoparticle comprises a payload molecule. The payload molecule may be a bio molecule, and/ or an active pharmaceutical ingredient (API), and/or a diagnostic compound.

The API may be a hydrophobic or hydrophilic API. The API may be a macromolecule ora small molecule. It maybe appreciated that a small molecule could be consideredto be a molecule with a molecular weight of less than 900 daltons. In some embodiments, a small molecule may have a molecular weight of less than 800 daltons, less than 700 daltons, less than 600 daltons, less than 500 daltons or less than 400 daltons.

Similarly, a macromolecule maybe consideredto be a molecule with a molecular weight of at least 900 daltons.

Examples of an API include an anti-inflammatory compound (e.g. non-steroidal antiinflammatory drugs such as aspirin, ibuprofen, naproxen, celecoxib, diclofenac, indomethacin, oxaprozin and/ or piroxicam, more preferably steroidal inflammatory drugs such as prednisone, cortisone and methylprednisone, or anti -rejection drugs such as tacrolimus, cyclosporine, mycophenolate mofetil, azathioprine, rapamycin, sirolimus), an anti-cancer agent (e.g. chemotherapy such as alkylating agents (examples include Altretamine, Bendamustine, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Ifosfamide, Lomustine, Mechlorethamine, Melphalan, Oxaliplatin, Temozolomide, Thiotepa, Trabectedin), nitrosoureas (examples include carmustine, lomustine, streptozocin), antimetabolites (examples include Azacitidine, 5-fluoro uracil (5-FU), 6-mercaptopurine (6-MP), Capecitabine, Cladribine, Clofarabine, Cytarabine (Ara-C), Decitabine, Floxuridine, Fludarabine, Gemcitabine, Hydroxyurea, Methotrexate, Nelarabine, Pemetrexed, Pentostatin, Pralatrexate, Thioguanine, Trifluridine/tipiracil combination), anti-tumor antibiotics (examples include the anthracyclines Daunorubicin, Doxorubicin (Adriamycin), Doxorubicin liposomal, Epirubicin, Idarubicin, Valrubicin, and the anti -tumor antibiotics Bleomycin, Dactinomycin, Mitomycin-C, Mitoxantrone), topoisomerase inhibitors Irinotecan, Irinotecan liposomal, Topotecan, Etoposide (VP-16), Mitoxantrone, Teniposide), mitotic inhibitors (examples include the Taxanes Cabazitaxel, Docetaxel, Nab -paclitaxel and Paclitaxel, and the Vinca alkaloids Vinblastine, Vincristine, Vincristine liposomal, Vinorelbine), and other chemotherapy drugs (examples include All -trans -retinoic acid, Arsenic trioxide, Asparaginase, Eribulin, Hydroxyurea, Ixabepilone, Mitotane, Omacetaxine, Pegaspargase, Procarbazine, Romidepsin, Vorinostat)), cytokine drugs (including cytokines (examples include (recombinant versions of) IL-1, IL-2, TNF-alpha, IL-6, IL-7, IL-10, IL-12, IL-17, IL-21, IL-22, IL-23, IFN-alpha, IFN-beta, IFN-gamma, IFN-lambdai, 1FN-Iambda2,

IFN-lambda3, IFN-omega, IP-10, MIP-ialpha, TGF-beta(i-3)) and anti-cytokines (e.g. cytokine-binding antibodies, decoy receptors, IL-iR antagonist)), growth-factors (examples include bone morphogenetic proteins (BMPs), vascular endothelial growth factor (VEGF), Granulocyte -macro phage colony-stimulating factor (GM-CSF), epidermal growth factor (EGF), erythropoeitin (EPO), insulin-like growth factor (IGF), fiboblast growth factor (FGF), hepatocyte growth factor (HGF), platelet -derived growth factor (PDGF), transforming growth factor (TGF), thrombopoietin (TPO)), hormones (examples include aldosterone, vasopressin, adrenocorticotropic hormone (ACTH), luteinizing hormone(LH), follicle-stimulating hormone (FSH), oxytocin, prolactin, thyroid-stimulating hormone (TSH), renin, angiotensin, glucagon, insulin, estrogen, progesterone, parathyroid hormone (PTH), Thyroid hormone, Epinephrine, Norepinephrine, testosterone, melatonin, growth hormone releasing hormone (GHRH), Thyrotropic releasing hormone (TRH), Gonadotropic releasing hormone (GnRH), corticotropin releasing hormone (CRH), humoral factors), cardiavascular medication (e.g. anti-coagulants (examples include Apibaxan, Dabigatran, Edoxaban, Heaprin, Rivaroxaban and Warfarin), anti-platelet agents (examples include Aspirin, Clopidogrel, Dipyridamole, Prasugrel andTicagrelor), Angiotensin-Converting Enzyme (ACE)-inhibitors (examples include Benazepril, Captopril, Enalapril, Fosinopril, Lisinopril, Moexipril, Perindopril, Quinapril, Ramipril and Trandolapril), Angiotensin II receptor blockers (Azilsartan, Candesartan, Eprosartan, Irbesartan, Losasartan, Olmesartan, Telmisartan, andValsartan), beta-adrenergic blockers (Acebutolol, Atenolol, Betaxolol, Bisoprolol, MEtoprolol, Nadolol, Propranolol, and Sotalol), Calcium channel blockers (examples include Amlodipine, Diltiazem, Felodipine, Nifedipine, Nimodipine, Nisoldipine, and Verapamil), cholesterol-lowering medication (examples include Statins Atorvastatin, Fluvastatin, Lovastatin, Pitavastatin, Pravastatin, Rosuvastatin and Simvastatin, Nitotinic acid Niacin, and cholesterol absorption inhibitor Ezetimibe), digitalis preparations (Digoxin), diuretics (examples include Acetazolamide, Amiloride, Bumetanide, Chlorothiazide, Chlorthalidone, Furosemide, Hydro -chlorothiazide, Indapamide, Metalozone, Spironolactone, and Torsemide), and vasodilators (Isosorbide dinitrate, Isosorbide mononitrate, Hydralazine, Nitroglycerin and Minoxidil)), intestinal medication (e.g. Proton pump inhibitors (examples include Omeprazole, Lansoprazole, Rabeprazole, Esomeprazole, and Pantoprazole), Histamine2 blockers (examples include Cimetidine, Ranitidine, Famotidine, and Nizatidine), Pro motility agents and laxatives (Metoclopramide)), eye medication (e.g. ocular allergy medicines (examples include Ketorolac, Ketotifen, loteprednol, Bepotastine, Epinastine, Emedastine, Alcaftadine, Azelastine, Olopatadine,

Nedocromil, lodoxamide, and Cromolyn), topical antibiotics (examples include Besifloxacin, Ciprofloxacin, Moxifloxacin, Ofloxacin, Gatfloxacin, Tobramycin, Gentamycin, Polymyxin D, Neomycin, Bacitracin, Azithromycin, and Erythomycin), lipid-based artificial tears (examples include castor oil, glycerol, and mineral oil), NSAIDS and corticosteroids, glaucoma drugs (examples include Levobunolol, Timolol, Betaxolol, Bimatoprost, Travoprost, Latanoprost, Tafluprost, Brimonidine, Brinzolamide, and Dorzolamide), and antiviral treatment (examples include Acyclovir, Valacyclovir, and Famciclovir)), lung medication (e.g. anti-asthmatics (examples include dyphylline, guaifenesin, Albuterol, Levalbuterol), anti -histamines (examples include Brompheniramine, Carbinoxamine, Chlorpheniramine, Clemastine, Diphenhydramine, Hydroxyzine, Tripolidine, Azelastine, Cetrizine, Desloratadine, Fexofenadine, Levo cetirizine, Loratadine, Olopatadine), Antitussives (examples include Dextromethorphan and Benzo natate), bronchodilators (Ipratropium, Theophylline, Albuterol, EpiNephrine, Levalbuterol, Arformoterol, Formoterol, Olodaterol, Terbutaline, Pirbuterol, Metaproterenol, Salmeterol, Isoproterenol, Indacaterol, Tiotropium, Umeclidinium, Aclidinium, Ipratropium, Revefenacin, Glyco pyrrolate, Ipratropium, Theophylline, Aminopylline and Dyphylline), decongestants (examples include Levmetamfetamine, Naphazoline, Oxymetazoline, Phenylephrine, Propylhexedrine, Pseudoephedrine, and Xylo metazoline), expectorants (Guaifenesin), leukotriene modifiers (examples include Montelukast, Zafirlukast, Zileutron), lung surfactants (examples include Beractant, Lucinactant, Calfactant and Poractant), mucolytics (acetylcysteine), anti-infectives (examples include Zanamivir, Ribavirin, Tobramycin, Pentamidine, and Colistimethate), inhaled corticosteroids (examples include Fluticasone, Budesone, Mometasone, Beclomethasone, and Ciclesonide), mastcell stabilizers (examples include Cromolyn andNedocormil), and phosphodiesterase-4 inhibitors(including Roflumilast)), , anti-microbial medication (including antibiotics

(e.g. aminoglycosides (examples including Amikacin, Gentamycin, Kanamycin,

Neomycin, Netilmicin, Tobramycin, Paromonmycin, Streptomycin and Spectinomycin), Ansamycins (examples include Geldanamycin, Herbimycin, and Rifaximin),

Carbacephem (for example Laracarbef), Carbapenems (examples include Ertapenem, Doripenem, Imipenem, and Meropenem), Cephalosporins (examples include

Cefadroxil, Cefazolin, Cephradine, Cephapirin, Cephalothin, Cefalexin, Cefaclor,

Cefoxitin, Cefotetan, Cefamandole, Cefmetazole, Cefonicid, Cefprozil, Cefuroxime,

Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Maxalactam, Ceftriaxone, Cefepime, Ceftaroline fosamil, and Ceftobiprole), Glycopeptides (examples include Teicoplanin, Vancomycin, Telavancin,

Dalbavancin, and Oritavancin), Lincosamides (Clindamycin and Lincomycin),

Lipopeptide (for example Daptomycin), Macrolides (Azithromycin, Clarithromycin, Erythromycin, Roxithromycin, Telithromycin, Spiramycin, and Fidaxomicin), Monobactams (for example Aztreonam), Nitrofurans (for example Furazolidone and Nitrofurantoin), Oxazolidinones (examples include Linezolid, Posizolid, Radezolid, and

Torezolid), Penicillins (examples include Amoxiciliin, Ampicillin, Azlocillin,

Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Temocillin, and Ticarcillin), Polypeptides (examples include Bacitracin, Cilistin and Polymyxin B), Quinolones (examples include Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nadifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin, and Temafloxacin), Sulfonamides (examples include Mafenide, Sulfacetamide, Sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, and Sulfo namidochrysoidine), Tetracyclines (examples include Demeclocycline, Doxycycline, Metacycline, Minocycline, Oxytartacycline, and Tetracycline), mycobacterium-specific antiobiotics (examples include (Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isonioazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine and Streptomycin)), anti- fungals (e.g. Polyene antimycotics (examples include Amphotericin B, Candicidin, Filipin, Hamycin, Natamycin, Nystatin, and Rimocidin), Azoles (examples include Imidazoles Bifonazole, Butoconazole, Clotrimazole, Econazole, Fenticonazole, Isoconazole, Ketoconazole, Luliconazole, Miconazole, Omoconazole, Oxiconazole, Sertaconazole, Sulconazole and Tioconazole, Triazoles Albaconazole, Efinaconazole, Epoxiconazole, Fluconazole, Isavuconazole, Itraconazole, Posaconazole, Propiconazole, Ravuconazole, Terconazole, and Voriconazole, and Thiazole (for example Abafungin), Allylamines (examples include Butenafine, naftifine and terbinafine), Echino candins (Anidulafungin, Caspofungin, and Micafungin), and Triterpenoids (for example Ibrexafungerp)), and anti-parasite drugs (e.g. the broad-spectrum Nitazoxanide, antiprotozoals (examples include Melarsoprol, Eflornithine, Metronidazole, Tinidazole, and Miltefosine), Antinematodes (examples include Mebendazole, Pyrantel pamoate, Thiabendazole, Diethylcarbamazine and Ivermectin), Anticestodes (examples include

Niclosamide, Praziquantel, and Albendazole), antitrematodes (for example Praziquantel), and Antiamoebics (for example Rifampicin and Amphotericin B))), antidiabetic medication (e.g. Insulin(analogues), Amylino mimetic drugs (for example Pramlintide), Alpha-glucosidase inhibitors (examples include Acarbose, and miglitol), Biguanides (e.g. metformin(analogues and combinations), Dopamine agonists (for example Bromocriptine), Dipeptidyl peptidase-4 (DDP-4) inhibitors (examples include Alogliptin, Linagliptin, Saxagliptin and Sitagliptin), Glucagon-like peptide-1 receptor agonists(examples include Albiglutide, Dualglutaide, Exenatide, Liraglutide, and Semaglutide), Meglitinides (examples include Nateglinide and Repaglinide), Sodium- glucose transporter (SGLT-2) inhibitors (examples include Dapagliflozine,

Canagliflozine, Ertugliflozine and Empagliflozine), Sulfonylureas (examples include Glimepiride, Gliclazide, Glipizide, Glyburide, Chlorpropamide, Tolazamide and Tolbutamide), Thiazolidinediones (for example Rosiglitazone and Pioglitazone)), antiviral medication (examples include Abacavir, Acyclovir, Adefovir, Amntadine, Ampligen, Amprenavir, Umifenovir, Atazanavir, Atripla, Oseltamivir, Zanamivir, Peramivir, Baloxavir, Bikctegravir, Emtricitabine, Tenofovir, Boceprevir, Bulevirtide, Cidofovir, Cobicistat, Daclatasvir, Darunavir, Delavirdine, Didanosine, Docosanol, Dolutegravir, Doravirine, Edoxudine, Efavirenz, Emtricitabine, Enfuvirtide, Ensivirtide, Ensitrelvir, Entecavir, Entravirine, Famciclovir, Fomivirsen, Fosamprenavir, Foscarnet, Ganiciclovir, Ibacitabine, Ibalizumab, Idoxuridine, Imiquimod, Insoine pranobex, Indinavir, Lamivudine, Letermovir, Lopinavir, Loviride, Maraviroc, Methisazone, Moroxydine, Nelfinavir, Nevirapine, Nitazoxanide, Norvir, Penciclovir, Pleconaril, Podophyllotoxin, Raltegravir, Remdesivir, Ribavirin, Rilpivirine, Rimantadine, Ritonavir, Saquinavir, Simeprevir, Sofosbuvir, Stavudine, Taribavirin, Telaprevir, Telbivudine, Tenofovir, Tiprenavir, Trifluridine, Trizivir, Tromantadine, Truvada, Umifenovir, Valaciclovir, Valganciclovir, Vicriviroc, Vidarabine, Zalcitabine, Zanamivir, and Zidovudine), or structural or functional analogues thereof.

In a preferred embodiment, the payload molecule is a bio molecule. For instance, the bio molecule may be or comprise an amino acid, a peptide, an affimer, a polypeptide or protein, a glycoprotein, a saccharide, a lipid, a lipopolysaccharide, an antibody or a fragment thereof, a polymer, or a nucleic acid, or a combination thereof.

The nucleic acid may be DNA, RNA, XNA (xeno nucleic acid, including 1,5- anhydrohexitol nucleic acid (HNA), Cyclohexene nucleic acid (CeNA), Threose nucleic acid (TNA), Glycol nucleic acid (GNA), Locked nucleic acid (Locked nucleic acid) Peptide nucleic acid (PNA), FANA (Fluoro Arabino nucleic acid) and unlocked nucleic acid), or a DNA/RNA hybrid sequence. Preferably, the nucleic acid is DNA or RNA. Most preferably, the nucleic acid is RNA The RNA may be single stranded or double stranded. The RNA maybe selected from the group consisting of: messenger RNA (mRNA); circular RNA (circRNA or oRNA); self-amplifying RNA (saRNA); transamplifying RNA (taRNA), long non-coding RNA, split-replicon RNA, viral RNA, antisense RNA (AON or as RNA); RNA aptamers; interference RNA; micro-RNA (miRNA); short interfering RNA (siRNA); short hairpin RNA (shRNA); and small RNA.

Preferably, the RNA is a messenger RNA (mRNA).

The nucleic acid sequence, preferably RNA, maybe at least 10 bases in length, at least 20 bases in length, at least 50 bases in length, at least too bases in length, at least 200 bases in length, at least 300 bases in length, at least 400 bases in length, at least 500 bases in length, at least 600 bases in length at least 700 bases in length, at least 800 bases in length or at least 900 bases inlength. In one preferred embodiment, the RNAis saRNA or mRNA

The nucleic acid sequence, preferably RNA, and most preferably mRNA, may be at least too bases in length, at least 500 bases in length, at least 1000 bases in length, at least 2000 bases inlength, atleastsooo bases inlength, atleast 4000 bases in length, at least 5000 bases inlength, atleast 6000 bases inlength, at least 7000 bases in length, at least 8000 bases inlength, atleast qooobases inlength at least 10000 bases in length, at least

11000 bases in length or at least 12000 bases in length. In one embodiment, the nucleic acid sequence is at least 6000 bases in length. In one embodiment, the RNA is at least 6000 bases in length. In a preferred embodiment, the saRNAis at least 6000 bases in length.

In an alternative embodiment, the nucleic acid sequence is at least 900 bases in length. In one embodiment, the RNAis at least 900 bases inlength. In a preferred embodiment, the mRNA is at least 900 bases in length. Alternatively, the nucleic acid sequence, preferably RNA, and most preferably mRNA, may be between 50 and 10000 bases in length, between 100 and 9000 bases in length, between 200 and8ooobases inlength, betweensoo and 7000 bases in length, between 400 and 6000 bases inlength, between 500 and 6000 bases in length, between 600 and 5000 bases in length, between 700 and 4000 bases in length, between 800 and 3000 bases in length or between 900 and 2000 bases in length.

In one embodiment, the nucleic acid sequence is between 6000 and 15000 bases in length. The nucleicacidsequencemaybebetween 8000 and 12000 bases inlength. The RNAmay be between 6000 and 15000 bases in length. The RNA may be between 8000 and 12000 bases in length. Preferably, the saRNA is between 6000 and 15000 bases in length. Preferably the saRNAis between 8000 and 12000 bases in length.

In an alternative embodiment, the nucleic acid sequence is between 100 and 14000, between 500 and 10000, between 600 and 7500, between 700 and 5000, between 800 and 4000 or between 900 and 2000 bases in length. The RNA may between 400 and 14000, between 500 and 10000, between 600 and 7500, between 700 and 5000, between 800 and 4000 or between 900 and 2000 bases in length. Preferably, the mRNA is between 100 and 14000, between 500 and 10000, between 600 and 7500, between 700 and 5000, between 800 and 4000 or between 900 and 2000 bases in length.

The skilled person would appreciate that when the nucleic acid is double stranded, for example double stranded RNA, “bases in length” will refer to the length of base pairs.

The weight ratio ofthe lipid component to the payload molecule maybe between 1:1 and 100:1, between 2:1 and 80:1, between 3:1 and 70:1, between 4:1 and 60:1 or between 5:1 and 50:1. In embodiments where the payloadmolecule is a bio molecule, the weight ratio ofthe lipid componentto the payload molecule maybe between 6:1 and 45:1, between 8:1 and 40:1, between 10:1 and35:i or between 12:1 and3O:i. In some embodiments, the weight ratio of the lipid component to the payload molecule may be between 13:1 and 25:1 between

14:1 and 20:1 or between 15:1 and 17:1. In so me embodiments, the weight ratio of the lipid component to the payload molecule may be between 15:1 and 27.5:1, between 20:1 and 25:1 or between 22:1 and 23:1. In embodiments where the payload molecule is a biomolecule, the N:P ratio may be between 1:2 and 50:1, between 1:1 and3O:i,between2:i and 20:1 between 3:1 and 15:1 or between5:i and 12:1. In someembodiments,theN:P ratiomaybebetween3:iandio:ior between 4:1 and 6:1. In alternative embodiments, the N:P ratio maybe between 4:1 and 10 : 1, b etween 5:iand9:ior between 6 : 1 and 8 : 1. It may b e under sto 0 d that the N: P ratio is the ratio of positively-chargeable polymer amine (N) groups to negatively-charged nucleic acid phosphate (P) groups.

Preferably, the micro- or nanoparticle has an encapsulation efficiency of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95%. It may be appreciated that encapsulation efficiency is determined by the amount ofthe payload molecule that is encapsulated in the micro- or nanoparticle (i. e. , not available and/ or accessible for the aqueous environment outside the micro- or nanoparticle) relative to the total amount of the payload molecule that was initially provided. The encapsulation efficiency may be determined using a Ribo Green-assay, which detects solvent accessible RNA by the increase in fluorescence upon intercalation of the water-soluble Ribogreen reagent with the RNA.

The micro- or nanoparticle may have a zeta-potential at physiological pH between -50 and +50 mV, between -40 and +40 mV, between -30 and +30 mV or between -20 and +20 mV, more preferably between -10 and +10 mV or between -5 and +5 mV. It may be appreciated that the zeta-potential may be measured by suspending the LNPs in an electrically conducting buffer with defined pH. The electrically conducting buffer may be PBS (phosphate buffered saline PH7.2). The micro- or nanoparticle may further comprise one or more adjuvants. The or each adjuvant may be selected from the group consisting of aluminium hydroxide, Pam2CSK4, Pam3CSK4, Gluco pyr ano syl Lipid adjuvant (GLA), LPS and analogues thereof, CpG oligo deoxynucleotides and other TLR-agonists such as Poly I:C and dsRNA. The micro- or nanoparticle may further comprise one or more additional compounds. The one or more additional compounds may be selected from the group consisting of a hydrophobic compound, a polymer, a permeability enhancer molecule, a carbohydrate, a surface modifier, an excipient and combinations thereof. The polymer may be polylactic glycolic acid (PLGA). It may be appreciated that the excipient may change the pharmacokinetic properties of the composition but not the pharmacodynamic properties of the payload.

In accordance with a third aspect, there is provided a composition comprising a plurality of micro- or nanoparticles of the second aspect.

The micro- or nanoparticles may have an average diameter less than 10pm, less than 1 pm, less than 500 nm or less than 250 nm. More preferably, the micro- or nanoparticles may have an average diameter of less than 200 nm, less than 175 nm, less than 150 nm or less than 125 nm. The micro- or nanoparticles may have an average diameter between 30 nm and 1 pm, between 40 and 500 nm, between 50 and 250 nm, between 60 and 200 nm, between 70 and 175 nm, between 80 and isonrn or between 90 and 125 nm. The average diameter of the micro- or nanoparticles may be measured using dynamic light scattering. The micro- or nanoparticles may have a polydispersity index (PDI) of less than 0.5, less than 0.4 or less than 0.3, and more preferably have a PDI of less than 0.25, less than 0.2, less than 0.15, less than 0.1 or less than 0.08. The micro- or nanoparticles may have a PDI of between 0.001 and 0.05, between 0.005 and 0.4, between 0.01 and 0.3, between 0.02 and 0.025, between 0.03 and 0.2, between 0.04 and 0.15, between 0.05 and 0.1 or between 0.06 and 0.08.

The composition may comprise a pharmaceutically acceptable carrier. The pharmaceutical acceptable carrier may improve colloidal stability, especially under concentrated and/or refrigerated conditions (e.g., storage and/or shipment at a temperature between 4°C and -8o°C, for example at 4°C, -20°C, or -7O°C or -8o°C). Such low temperature conditions maybe used to extend the shelflife of the composition and/or more specifically the payload.

The composition may further comprise one or more solvents, a buffer, a suspension aid, a filler, a glidant, a binder, a salt, an isotonic agent, a thickening agent, an emulsifying agent and/or a preservative.

In a fourth aspect, there is provided the micro - or nanoparticle of the second aspect, or the composition of the third aspect, for use as a medicament.

In a fifth aspect, there is provided the micro- or nanoparticle of the second aspect, or the composition of the third aspect, for use in the treatment and/ or prevention and/or prophylaxis of a disease or disorder. In a sixth aspect, there is provided a method of treating and/or preventing a disease or disorder, the method comprising administering, or having administered, to a subject in need thereof, a prophylactic and/or therapeutic amount of the micro- or nanoparticle of the second aspect, or the composition of the third aspect. The disease or disorder may be selected from the group consisting of inflammatory diseases, infectious diseases, proliferative diseases (e.g., cancer), auto-immune diseases, eye diseases, lung diseases, skin diseases, intestinal diseases, metabolic diseases (e.g., diabetes), vascular diseases (including cardiovascular and renovascular diseases), neurological diseases (e.g., neuro degenerative diseases), disorders of the endocrine system (including disorders related to hormones, growth factors, and/ or cytokines), disorders of the reproductive system, and rare diseases.

In a seventh aspect, there is provided a vaccine composition comprising the micro- or nanoparticle of the second aspect, or the composition of the third aspect.

The vaccine may comprise a suitable adjuvant.

In an eighth aspect, there is provided the micro- or nanoparticle of the second aspect, or the composition of the third aspect or the vaccine of the seventh aspect, for use in stimulating an immune response in a subject. The immune response maybe stimulated against a protozoa, bacterium, virus, fungus, multicellular parasite, or cancer, or parts thereof.

In a ninth aspect, there is provided a method of vaccinating a subject, the method comprising administering, or having administered, to a subject in need thereof, a prophylatic and/or therapeutic amount of the micro- or nanoparticle of the second aspect, or the composition of the third aspect or the vaccine of the seventh aspect.

The micro- or nanoparticle, the composition or the vaccine of the invention maybe combinedin compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used. Thus, for example, the composition maybe in the form of a powder, tablet, capsule, liquid, ointment, cream, shampoo, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch (including microneedles), drug-depot/slow-release formulations, (liposome) suspension, wash/instillation, incorporated in a bio material for regenerative medicine, a coating of

(implantable) medical devices, or any other suitable form that may be administered to a person or animal in need of treatment. It will be appreciated that the vehicle of medicaments according to the invention should be one which is well -tolerated by the subject to whom it is given.

The micro- or nanoparticle, the composition or the vaccine of the invention may also be incorporated within a slow- or delayed-release device. Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months. The device may be located at least adjacent to, or upstream of the treatment site.

In a preferred embodiment, however, medicaments according to the invention may be administered to a subject by injection into the blood stream, muscle, skin or directly into a site requiring treatment. Injections maybe intravenous (bolus or infusion), subcutaneous (bolus or infusion), intradermal (bolus or infusion), intramuscular (bolus or infusion), intrathecal (bolus or infusion), intravitreal (bolus), epidural (bolus or infusion) or intraperitoneal (bolus or infusion).

It will be appreciated that the amount of micro- or nanoparticle, the composition or the vaccine that is required is determined by its intended therapeutic or prophylactic use and its biological activity and bio availability, which in turn depends on the mode of administration, the physio chemical properties of the payload, the micro- or nanoparticle, the composition or the vaccine and whether it is being used as a monotherapy or in a combined therapy. The frequency of administration will also be influenced by the half-life of the active agent and/or the half-life of the therapeutic effect (e.g., the half-life of the therapeutic protein translated from the therapeutic mRNA transfected with the micro- or nanoparticle composition, or e.g. the half-life of RNA-based CRISPR gene-therapy) within the subject being treated. Optimal dosages to be administered may be determined by those skilledin the art, and will vary with the micro- or nanoparticle, the composition or the vaccine in use, the strength of the pharmaceutical composition, the mode of administration, and the type of treatment. Additional factors depending on the particular subject beingtreatedwill result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.

The required dose may depend upon a number of factors including, but not limited to, the active agent being administered, the disease being treated and/or vaccinated against, the subject being treated, etc. Generally, a dose of between 0.001 pg/kg of body weight and 10 mg/kg of body weight, or between 0.01 pg/kg of body weight and 1 mg/kg of body weight, of the micro- or nanoparticle, the composition or the vaccine of the invention may be used, depending upon the active agent used. A dose may be understood to relate to the quantity of the payload molecule which is delivered.

Doses maybe given as a single administration (e.g., a single injection). Alternatively, the micro- or nanoparticle, the composition or the vaccine may require more than one administration. As an example, the micro- or nanoparticle, the composition or the vaccine maybe administered as two or more doses of between 0.07 pg and 700 mg (i.e., assuming a body weight of 70 kg). Alternatively, a slow-release device may be used to provide optimal doses of the micro- or nanoparticle, the composition or the vaccine according to the invention to a patient without the need to administer repeated doses. Routes of administration may incorporate intravenous, intradermal subcutaneous, intramuscular, intrathecal, epidural, intravitreal, or intraperitoneal routes of injection. Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g., in vivo experimentation, clinical trials, etc.), may be used to form specific formulations of the micro- or nanoparticle, the composition or the vaccine according to the invention and precise therapeutic regimes (such as doses of the agents and the frequency of administration).

A “subject” may be a vertebrate, mammal, or domestic animal. Hence, compositions and medicaments according to the invention maybe used to treat any mammal, for example livestock (e.g., a horse), pets, or may be used in other veterinary and agricultural applications. Most preferably, however, the subject is a human being.

A “therapeutically effective amount” of the micro- or nanoparticle, the composition or the vaccine is any amount which, when administered to a subject, is the amount of the aforementioned that is needed to produce a therapeutic effect, whether partial or full.

For example, a therapeutically effective amount of the micro- or nanoparticle, the compositionorthe vaccine of the inventionmay comprise from about 0.001 pgto about 800 mg of the payload molecule, and preferably from about 0.01 mg to about 500 mg of the payload molecule.

A “pharmaceutically acceptable vehicle” as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions. In one embodiment, the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder, a capsule or tablet. A solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet-disintegrating agents. The vehicle may also be an encapsulating material. In powders, the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention. In tablets, the active agent (e.g. , micro- or nanoparticle of the invention) may be mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like. Alternatively, the pharmaceutical vehicle may be a liquid, and the pharmaceutical compositionis in the form of a solution. Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The micro- or nanoparticle according to the invention may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo -regulators. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g., cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and arachis oil). For parenteral administration, the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, intravitreal, epidural, intraperitoneal, intravenous and subcutaneous injection. The micro - or nanoparticle of the invention maybe prepared as any appropriate sterile injectable medium.

The micro- or nanoparticle may be administered by inhalation. For instance, the micro- or nanoparticle maybe provided in the form of an aerosol.

The micro or nanoparticle and/or the composition of the invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like. The micro or nanoparticle and/or the composition according to the invention can also be administered orally either in liquid or solid composition form. Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions. In accordance with a further aspect of the invention, there is provided a method of producing a compound of formula (III): , wherein R ¹ , R ³ and L ¹ to L ³ are as defined in relation to the first aspect, and both R ¹ groups are the same; the method comprising contacting maleic anhydride with a compound of formula (V):

HO-R ¹

(V) to produce a compound of formula (VI): , and contacting the compound of formula (VI) with a compound of formula (VII):

HS-LW-L-R ³

(VII)

, to thereby producing a compound of formula (III).

The molar ratio of the maleic anhydride to the compound of formula (V) may be between 1:1 and 1:5 or between 1:1.5 and 1:3, and is preferably about 1:2. The maleic anhydride and the compound of formula (V) may be contacted in a solvent, preferably a first organic solvent. The first organic solvent is preferably a non-alcoholic solvent. The first organic solvent preferably allows the removal of water from the reaction under Dean-Stark conditio ns/ with a Dean-Stark setup. The first organic solvent may have a boiling point of at least 7O°C, at least 8o°C, at least 9O°C, at least ioo°C, at least no°C, at least 12O°C or at least 13O°C. The first organic solvent may be an aromatic solvent, an alkane, a cycloalkane, a nitroalkane, a halogenated hydrocarbon or an ether. The aromatic solvent may be toluene, xylene, benzene, methylbenzene, ethylbenzene or pyridine. The alkane or the cycloalkane may be a C5-20 alkane or C5-12 cycloalkane, and may be pentane, hexane, heptane, octane, nonane or cyclohexane. The nitro alkane maybe a C1-3 nitroalkane, and may be nitromethane. The ether may be a C4-12 ether, and may be dibutylether. The halogenated hydrocarbon may be a chlorinatedhydrocarbon. The halogenated hydrocarbon maybe a C1-3 halogenated hydrocarbon, preferably halogenated methane. The halogenated hydrocarbon may be trichloro methane. In some embodiments, the second solvent is toluene.

The maleic anhydride and the compound of formula (V) may be contacted in the present of an acid. The acid may be any strong acid which is soluble in the first organic solvent. The acid may be p-toluenesulfonic acid (pTSA) or a hydrate thereof, camphor sulfonic acid (CSA), methenesulfonic acid or sulfonic acid. The molar ratio of the maleic anhydride to the acid maybe between 1:5 and 100:1, between 1:2 and 75:1 or between 1:1 and 75:1. In some embodiments, the molar ratio of the maleic anhydride to the acid may be between 5:1 and 60:1, between 10:1 and 50:1, between 20:1 and 45:1 or between 30:1 and 40:1. In some embodiments, the molar ratio of the maleic anhydride to the acid may be between 1:5 and 50:1, between 1:2 and 30:1, between 1:1 and 20:1, between 3:1 and 15:1, between 5:1 and 10:1 or between 7:1 and 8:1.

The maleic anhydride and the compound of formula (V) may be contacted at an elevated temperature. The elevated temperature maybe a temperature of atleast 3O°C, at least 5O°C, at least 7O°C, at least 9O°C, at least no°C or at least 13O°C. The elevated temperature may be between 30 and 5OO°C, between 50 and 25O°C, between 70 and 200°C, between 90 and 175°C, between 110 and 15O°C or between 120 and 14O°C.

The molar ratio of the compound of formula (VI) to the compound of formula (VII) may be between 2:1 and 1:10, between 1:1 and 1:5 or between 1:2 and 1:4, and is preferably about 1:3. The compound of formula (VI) and the compound of formula (VII) may be contacted in a solvent, preferably a second organic solvent. The second organic solvent may be a halogenated hydrocarbon, an ether or a ketone. The halogenated hydrocarbon may be a chlorinated hydrocarbon. The halogenated hydrocarbon may be a C1-3 halogenated hydrocarbon, preferably a halogenated methane. The second organic solvent may be dichloromethane, dichloroethane or trichloro methane. The ether maybe a C2-10 ether, and may be tetrahydrofuran (THF) or diethylether. The ketone may be a Cg=6 ketone, and may be acetone. In some embodiments, the second organic solvent is dichloromethane.

The compound of formula (VI) and the compound of formula (VII) may be contacted in the present of a base. The base may be a tertiary amine or a nucleophilic catalyst. The nucleophilic catalyst may be an aliphatic or aromatic phosphine. The aliphatic or aromatic phosphine may be a compound of formula P(R ⁷ )3, where each R ⁷ is independently a C1-10 alkyl or an aryl group. Accordingly, the base maybe triethylamine, di-isopropylethylamine (DiPEA), DABCO, tributylphosphine, tripropylphosphine, triethylphosphine, trimethylphosphine or tribenzenephosphine. The molar ratio of the compound of formula (VI) to the base maybe between 2:1 and 1:10, between 1:1 and 1:5 or between 1:2 and 1:4, and is preferably about 1:3.

The compound of formula (VI) and the compound of formula (VII) may be contacted at a second temperature. The seco nd temperature may be between o and ioo°C, between 5 and 5O°C, between 10 and 4O°C, between 15 and 3O°C, between 18 and 25°C and is preferably room temperature.

All of the features described herein (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, maybe combined with any of the above aspects in any combination, except combinations where at least some of such features and/ or steps are mutually exclusive.

For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:- Figure 1 shows size distribution of LNP formulations measured with dynamic light scattering; Figure 2 shows size distribution of LNP formulations with a higher N/P ratio to those shown in Figure 1 measured with dynamic light scattering;

Figure 3 provides the pKa of various ionizable lipids determined by TNS -assay over a pH range of 3-10 in 0.5 pH increments, structures 1, 3, 5 and 6 represent Li, L3, L5 and L6, respectively;

Figure 4 toxicity and immunogenicity testing of L1-L6 lipids. (A) Effect of individual lipid L1-L6 (pg/well) on the metabolic activity in HeLa cells was determined using the resazurin assay. (B) The immunogenicity of individual lipid L1-L6 (pg/well) was determined in endothelial cells was determined by measuring MCP-1 in ELISA LPS (TLR4 agonist), PAM2CSK4 (P2, TLR2/TLR6 agonist) and PAM3CSK4 (P3,

TLR2/TLR1 agonist) were used as positive controls. DODAP and DOTAP were used for comparison. NT, non-treated;

Figure 5 in vitro activity of Li (according to Formula (Ila)) -containing LNPs. LNPs encapsulating nano -luciferase mRNAwere generated with ionizable lipid Li (of Formula (Ila)) or the commercially available, widely accepted lipid MC3 and added to HeLa cells. (A) Transfection efficiency was determined by measuring luciferase activity. (B) Effect on metabolic activity was assessed by resazurin assay for toxicity;

Figure 6 Li-containing LNPs are immune-silent. Cells were transfected with LNP formulation of Li, encapsulating either immuno-silent or -active mRNA Effect of lipids on immunogenicity was tested after 24 hours by ELISA of the pro -inflammatory chemokine MCP-1; and

Figure 7 in vivo activity of Li-containing LNPs. Mice (n=5 per group) were injected with different LNP formulations encapsulating EPO mRNA (1 pg), either intravenously (i.v.) or intraperitoneally (i.p.) and sacrificed after 6 hours. (A) mRNA-derived EPO protein expression in plasma was assessed by ELISA. (B) Toxicity of the LNP formulations was assessed by weight change. (C) Immunogenicity was determined by ELISA for pro -inflammatory cytokine IL-6 in plasma.

General Examples

General Procedure i

Scheme 1: General Procedure 1

As illustrated in Scheme i above, maleic anhydride la reacts with an alcohol ib (preferably a fatty acid alcohol such as e.g. cis,cis-9,i2-octadecadien-i-ol) to afford compound ic (e.g. dioctadecadienyl maleate). Step i can take place in an organic solvent (e.g. Toluene) in the presence of, e.g. p-Toluenesulfonic acid (pTSA) at elevated temperature (e.g. 13O°C) under reflux for i8h. Next, the maleate-di-acylester ic reacts with a thiol id (e.g. 2-(dimethylamino)ethanethiol hydrochloride) to afford compound le (e.g. Di((9Z,i2Z)-octadeca-9,i2-dien-i-yl) maleate). Step 2 can take place in an organic solvent (e.g. dichloro methane) in the presence of triethylamine at room temperature (e.g. 25°C) for 25b while stirring.

As illustrated in the specific examples below, in some embodiments, the compound of formula lb is cis,cis-9,i2-octadecadien-i-ol. Accordingly, the compound of formula ic maybe di((9Z,i2Z)-octadeca-9,i2-dien-i-yl) maleate. Alternatively, as illustrated in the specific examples below, in some alternative embodiments, the compound of formula lb is cis-9-octadecen-i-ol. Accordingly, the compound of formula ic may be dioleyl maleate. In some embodiments, the compound of formula id is 2-(dimethylamino)ethanethiol hydrochloride, (dimethylamino)methanethiol hydrochloride, 3-(dimethylamino)-i- propanethiol hydrochloride, 2-(methylethylamino)ethanethiol hydrochloride or (diethylamino)ethanethiol hydrochloride.

Accordingly, the compound of formula le maybe di((9Z,i2Z)-octadeca-9,i2-dien-i-yl) 2-((2-(dimethylamino)ethyl)thio)succinate maleate, di((9Z,i2Z)-octadeca-9,i2-dien-i- yl) 2-((2-(dimethylamino)methyl)thio)succinate maleate, di((9Z,i2Z)-octadeca-9,i2- dien-i-yl) 2-((2-(dimethylamino)propyl)thio)succinate maleate, di((9Z,i2Z)-octadeca- 9,12-dien-i-yl) 2-((2-(methylethylamino)ethyl)thio)succinate maleate, di((9Z,i2Z)- octadeca-9,i2-dien-i-yl)2-((2-(diethylamino)ethyl)thio)succi nate maleate or dioleyl 2- ((2-(diethylamino)ethyl)thio)succinate maleate.

Specific Examples Example 1: Synthesis of di((9Z,i2Z)-octadeca-9,i2-dien-i-yl) 2-((2- (dimethylamino)ethyl)thio)succinate (Li), MW: 718.17)

Step 1. Synthesis of di((QZ.i2Z)-octadeca-Q.i2-dien-i-yl) maleate A solution of maleic anhydride (206 mg, 2.1 mmol, 1 equiv) and cis,cis-9,i2- octadecadien-i-ol (1.3 mL, 1.2 g, 4.2 mmol, 2.0 equiv) was heated in toluene until completely dissolved. p-Toluenesulfonic acid (pTSA; 50 mg) was added and the solution was heated under reflux at 130 °C for 18 h. Next, the reaction mixture was concentrated at the rotary evaporator. The residue was purified by silica column chromatography (petroleum ether, ethyl acetate 49:1) to yield dioctadecadienyl maleate as a yellow oil (1.13 g, 1.85 mmol, 88%).

HPLC-MS (ESI) calcd for C ₄ oH68O ₄ [M+H]+: 613.51; found: 613.7- TLC (petroleum ether, ethyl acetate 49:1), Rf = 0.11 Step 2. Synthesis of di((QZ.i2Z)-octadeca-Q.i2-dien-i-yl) 2-((2- (dimethylamino)ethyl)thio)succinate

Dioctadecadienyl maleate (1.13 g, 1.85 mmol, 1 equiv) was dissolved in dichloromethane (30 ml), 2-(dimethylamino)ethanethiol hydrochloride (786 mg, 5.55 mmol, 3.0 equiv) and triethylamine (773 LLL, 5.55 mmol, 3.0 equiv) were added and the reaction mixture was stirred for 25 h at room temperature. Next, the solution was concentrated at the rotary evaporator. Aliquots of the crude residue (ca. 120 mg each) were purified by flash chromatography (C4 -phase, water, 0.1% formic acid/ acetonitrile, gradient 46 min) to yield di((9Z,i2Z)-octadeca-9,i2-dien-i-yl) 2-((2- (dimethylamino)ethyl)thio)succinate (total 110.0 mg, 0.014 mmol, 8%) as a colorless oil.

HPLC-MS (ESI) calcd for C44H79NO4S [M+H]+: 718.57; found: 718.8.

Example 2: Synthesis of di((9Z,i2Z)-octadeca-9,i2-dien-i-yl) 2-((2- (dimethylamino)methyl)thio)succinate maleate (L2), MW: 704.14

Step 1. Synthesis of Pi((QZ.i2Z)-octadeca-Q.i2-dien-i-yl) maleate

A solution of maleic anhydride (206 mg, 2.1 mmol, 1 equiv), cis,cis-9,i2-octadecadien- i-ol (1.3 mL, 1.2 g, 4.2 mmol, 2.0 equiv) were heated in toluene until completely dissolved. p-Toluenesulfonic acid (pTSA; 50 mg) was added and the solution was heated under reflux at 130 °C for 18 h. Next, the reaction mixture was concentrated at the rotary evaporator. The residue was purified by silica column chromatography (petroleum ether, ethyl acetate 49:1) to yield dioctadecadienyl maleate (4) as a yellow oil (1.13 g, 1.85 mmol, 88%). HPLC-MS (ESI) calcd for C4OH68O4 [M+H]+: 613.51; found: 613.7- TLC (petroleum ether, ethyl acetate 49:1), Rf = 0.11

Step 2. Synthesis of Pi((QZ.i2Z)-octadeca-Q.i2-dien-i-yl) 2-((2- (dimethylamino)methyl)thio)succinate maleate Dioctadecadienyl maleate (1.13 g, 1.85 mmol, 1 equiv) was dissolved in dichloromethane (30 ml), N,N-Dimethyl(mercaptomethyl)amine (506 mg, 5.55 mmol, 3.0 equiv) and triethylamine (773 LIL, 5.55 mmol, 3.0 equiv) were added and the reaction mixture was stirred for 25 h at room temperature. Next, the solution was concentrated at the rotary evaporator. Aliquots of the crude residue (ca. 120 mg each) were purified by flash chromatography (C4 -phase, water, 0.1% formic acid/ acetonitrile, gradient 46 min) to yield Di((9Z,i2Z)-octadeca-9,i2-dien-i-yl) 2-((2- (dimethylamino)methyl)thio)succinate maleate (110.0 mg, 0.014 mmol, 8%) as a colorless oil.

HPLC-MS (ESI) calcd for C43H77NO4S [M+H]+: 704.142; found: 718.8.

Example 3: Synthesis of di((9Z,i2Z)-octadeca-9,i2-dien-i-yl) 2-((2- (dimethylamino)propyl)thio)succinate maleate (L3), MW: 732.2

Step 1. Synthesis of Pi((QZ.i2Z)-octadeca-Q.i2-dien-i-yl) maleate

A solution of maleic anhydride (206 mg, 2.1 mmol, 1 equiv), cis,cis-9,i2-octadecadien- i-ol (1.3 mL, 1.2 g, 4.2 mmol, 2.0 equiv) were heated in toluene until completely dissolved. p-Toluenesulfonic acid (pTSA; 50 mg) was added and the solution was heated under reflux at 130 °C for 18 h. Next, the reaction mixture was concentrated at the rotary evaporator. The residue was purified by silica column chromatography (petroleum ether, ethyl acetate 49:1) to yield dioctadecadienyl maleate as a yellow oil (1.13 g, 1.85 mmol, 88%).

HPLC-MS (ESI) calcd for C4OH68O4 [M+H]+: 613.51; found: 613.7-

TLC (petroleum ether, ethyl acetate 49:1), Rf = 0.11

Step 2. Synthesis of Di((QZ.i2Z)-octadeca-Q.i2-dien-i-yl) 2-((2- (dimethylamino)propyl)thio)succinate maleate

Dioctadecadienyl maleate (1.13 g, 1.85 mmol, 1 equiv) was dissolved in dichloromethane (30 ml), 3-(dimethylamino)propane-i-thiol hydrochloride (864 mg, 5.55 mmol, 3.0 equiv) and triethylamine (773 LLL, 5.55 mmol, 3.0 equiv) were added and the reaction mixture was stirred for 25 h at room temperature. Next, the solution was concentrated at the rotary evaporator. Aliquots of the crude residue (ca. 120 mg each) were purified by flash chromatography (C4 -phase, water, 0.1% formic acid/ aceto nitrile, gradient 46 min) to yield Di((9Z,i2Z)-octadeca-9,i2-dien-i-yl) 2-((2- (dimethylamino)propyl)thio)succinate maleate (110.0 mg, 0.014 mmol, 8%) as a colorless oil.

HPLC-MS (ESI) calcd for C43H77NO4S [M+H]+: 731-588; found: 718.8. Example 4: Synthesis of di((9Z,i2Z)-octadeca-9,i2-dien-i-yl) 2-((2- (methylethylamino)ethyl)thio)succinate maleate (L5), MW: 732.2

Step 1. Synthesis of Pi((QZ.i2Z)-octadeca-Q.i2-dien-i-yl) maleate

A solution of maleic anhydride (206 mg, 2.1 mmol, 1 equiv), cis,cis-9,i2-octadecadien- i-ol (1.3 mL, 1.2 g, 4.2 mmol, 2.0 equiv) were heated in toluene until completely dissolved. p-Toluenesulfonic acid (pTSA; 50 mg) was added and the solution was heated under reflux at 130 °C for 18 h. Next, the reaction mixture was concentrated at the rotary evaporator. The residue was purified by silica column chromatography (petroleum ether, ethyl acetate 49:1) to yield dioctadecadienyl maleate as a yellow oil (1.13 g, 1.85 mmol, 88%).

HPLC-MS (ESI) calcd for C4OH68O4 [M+H]+: 613.51; found: 613.7-

TLC (petroleum ether, ethyl acetate 49:1), Rf = 0.11

Step 2. Synthesis of Pi((QZ.i2Z)-octadeca-Q.i2-dien-i-yl) 2-K2- (methylethylamino)ethyl)thio)succinate maleate

Pioctadecadienyl maleate (1.13 g, 1.85 mmol, 1 equiv) was dissolved in dichloromethane (30 ml), 2-(methylethylamino)ethanethiol hydrochloride (864 mg, 5.55 mmol, 3.0 equiv) and triethylamine (773 LLL, 5.55 mmol, 3.0 equiv) were added and the reaction mixture was stirred for 25 h at room temperature. Next, the solution was concentrated at the rotary evaporator. Aliquots of the crude residue (ca. 120 mg each) were purified by flash chromatography (C4 -phase, water, 0.1% formic acid/aceto nitrile, gradient 46 min) to yield di((9Z,i2Z)-octadeca-9,i2-dien-i-yl) 2-((2- (methylethylamino)ethyl)thio)succinate (110.0 mg, 0.014 mmol, 8%) as a colorless oil. HPLC-MS (ESI) calcd for C43H77NO4S [M+H]+: 731-588; found: 718.8. Example 5: Synthesis of di((9Z,i2Z)-octadeca-9,i2-dien-i-yl) 2-((2- (diethylamino)ethyl)thio)succinate maleate (L6), MW: 746.22

Step 1. Synthesis of Pi((QZ.i2Z)-octadeca-Q.i2-dien-i-yl) maleate A solution of maleic anhydride (206 mg, 2.1 mmol, 1 equiv), cis,cis-9,i2-octadecadien- i-ol (1.3 mL, 1.2 g, 4.2 mmol, 2.0 equiv) were heated in toluene until completely dissolved. p-Toluenesulfonic acid (pTSA; 50 mg) was added and the solution was heated under reflux at 130 °C for 18 h. Next, the reaction mixture was concentrated at the rotary evaporator. The residue was purified by silica column chromatography (petroleum ether, ethyl acetate 49:1) to yield dioctadecadienyl maleate as a yellow oil (1.13 g, 1.85 mmol, 88%).

HPLC-MS (ESI) calcd for C4OH68O4 [M+H]+: 613.51; found: 613.7-

TLC (petroleum ether, ethyl acetate 49:1), Rf = 0.11 Step 2. Synthesis of Pi((QZ.i2Z)-octadeca-Q.i2-dien-i-yl) 2-K2- (diethylamino)ethyl)thio)succinate maleate Pioctadecadienyl maleate (1.13 g, 1.85 mmol, 1 equiv) was dissolved in dichloromethane (30 ml), 2-Piethylaminoethanethiol hydrochloride (941.9 mg, 5.55 mmol, 3.0 equiv) and triethylamine (773 LIL, 5.55 mmol, 3.0 equiv) were added and the reaction mixture was stirred for 25 h at room temperature. Next, the solution was concentrated at the rotary evaporator. Aliquots of the crude residue (ca. 120 mg each) were purified by flash chromatography (C4 -phase, water, 0.1% formic acid/ acetonitrile, gradient 46 min) to yield Pi((9Z,i2Z)-octadeca-9,i2-dien-i-yl) 2-((2- (diethylamino)ethyl)thio)succinate maleate (110.0 mg, 0.014 mmol, 8%) as a colourless oil.

HPLC-MS (ESI) calcd for C43H77NO4S [M+H]+: 746.222; found: 718.8. Example 6: Synthesis of dioleyl 2-((2-(diethylamino)ethyl)thio)succinate maleate (L4), MW: 722.20

Step 1. Synthesis of dioleyl maleate A solution of maleic anhydride (206 mg, 2.1 mmol, 1 equiv) and cis-9-octadecen-i-ol (1.3 mL, 1.13 g, 4.2 mmol, 2.0 equiv) was heated in toluene until completely dissolved. p-Toluenesulfonic acid (pTSA; 50 mg) was added and the solution was heated under reflux at 130 °C for 18 h. Next, the reaction mixture was concentrated at the rotary evaporator. The residue was purified by silica column chromatography (petroleum ether, ethyl acetate 49:1) to yield dioleyl maleate as a yellow oil (1.17 g, 1.9 mmol, 90%). HPLC-MS (ESI) calcd for C4OH68O4 [M+H]+: 616.99; found: 613.7.

TLC (petroleum ether, ethyl acetate 49:1), Rf = 0.11

Step 2. Synthesis of dioleyl 2-((2-(diethylamino)ethyl)thio)succinate Dioleyl maleate (1.14 g, 1.85 mmol, 1 equiv) was dissolved in dichloro methane (30 ml), 2-(dimethylamino)ethanethiol hydrochloride (786 mg, 5.55 mmol, 3.0 equiv) and triethylamine (773 LIL, 5.55 mmol, 3.0 equiv) were added and the reaction mixture was stirredfor 25 h at room temperature. Next, the solution was concentrated at the rotary evaporator. Aliquots of the crude residue (ca. 120 mg each) were purified by flash chromatography (C4 -phase, water, 0.1% formic acid/ acetonitrile, gradient 46 min) to yield dioleyl 2-((2-(diethylamino)ethyl)thio)succinate (total 110.0 mg, 0.014 mmol, 8%) as a colourless oil.

HPLC-MS (ESI) calcd for C44H79NO4S [M+H]+: 722.2; found: 718.8. Example 7: Production of Lipid Nano Particles (LNPs)

To determine the safety and efficacy of the lipids of the invention for the delivery of a therapeutic and/or prophylactic molecule to cells, a range offormulations was prepared and tested. More precisely, the lipid composition of the LNPs and the ratios between said lipids was varied.

Uniformly sized nanoparticles were produced reproducibly with an inverted herringbone microfluidic mixer. Said mixer rapidly (in the order of milliseconds) mixes the aqueous fluid generally containing the water soluble therapeutic and/ or prophylactic molecule with an organic solvent containing the lipid components. Other types of microfluidic mixers (e.g. Y-junction, T-junction or direct high-speed injection) produced similar results, as long as a similar mixing ratio between fluid streams and mixing speed was obtained.

Lipid compositions were prepared by combining an ionizable lipid of the present invention (e.g. a lipid of one of examples 1 to 6) with a phospholipid (such as DOPE, obtained from Avanti Polar Lipids), optionally a cationic lipid (such as DOTAP, obtained from Avanti Polar Lipids), a structural lipid (such as cholesterol, obtained from Sigma-Aldrich), a PEG-modified lipid (such as i,2-distearoyl-sn-glycero-3- phospho ethanolamine (polyethyleneglycol)-2ooo (also known as PEG-DSPE), obtained from Avanti Polar Lipids). The lipids were typically combined in a ratio of 45 mol% ionizable lipid of the invention, 5 mol% DOTAP, 38.5 mol% structural lipid, 10 mol% phospholipid and 1.5 mol% PEG-modified lipid for experiments requiring cellular uptake. The lipids were typically combined in a ratio of 50 mol% ionizable lipid of the invention, 38.5 mol% structural lipid, 10 mol% phospholipid (DOPE) and 1.5 mol% PEG-modified lipid for experiments not requiring cellular uptake. Certain formulations display low generic cellular uptake in vitro and/ or in vivo, which can be overcome by the incorporation of cell-receptor or cell-membrane targeting ligands, such as natural ligands or antibodies directed against cell -surface receptors. Alternatively, increasing the zeta-potential of the nanoparticle composition to at least +imV, but preferably -i-iomV, results in generic interaction with the plasma- membrane, resulting in uptake of the nanoparticle and/ or cargo. In such cases, the following lipid ratio was used: 40-50 mol% ionizable lipid of the invention, 0-10 mol% cationic lipid, 38.5 mol% structural lipid, 10 mol% phospholipid and 1.5 mol% PEG- modified lipid. The lipid mixture was diluted with ethanol to a final concentration of in between i2.5mM total lipid and 50mM total lipid. Lipids dissolved in ethanol were stored at - 20°C, under argon, protected from light.

Nanoparticle compositions were made by combining a therapeutic and/ or prophylactic molecule in acidic (pH 4 or 5) or neutral (pH 7.4) aqueous solution with the lipid mix in ethanol at a lipid to therapeutic molecule weight ratio in between 5:1 and 50:1. To produce a well-defined nanoparticle population, the aqueous solution comprising the therapeutic and/or prophylactic molecule (“the aqueous solution”) and the lipid solution were rapidly mixed at a volumetric ratio of about 2:1 (aqueous solution: lipid solution) to about 5:1 (aqueous solution: lipid solution) in an inverted herringbone microfluidic mixer at total flow rates in between loml/min and 18 ml/min.

To make nanoparticle compositions containing an RNA,the RNAwas dilutedto around between o.img/ ml and 3mg/ ml, preferably o.ismg/ ml in loomM sodium citrate buffer at pH 4 to 5 and subsequently mixed with the lipid mix. The RNAto lipid weight was typically set in between 1:10 and 1:30, resulting in an N:P ratio of in between 3 and 12, preferably 5.

Subsequent to mixing the aqueous and lipid solutions, the nanoparticle composition was dialyzed to remove ethanol to below 0.1%, optionally concentrate or dilute the solution, and exchange the buffer for a buffer of physiological pH (e.g. pH 7.4, for example phosphate buffered saline (PBS)). Formulations were dialyzed three times against at least a 100-fold excess of PBS using tookDa MWCO dialysis tubing (such as Spectrum™ Spectra/Por™ Biotech Cellulose Ester (CE) Dialysis Membrane Tubing, obtained from Fisher Scientific). The first dialysis step was performed for at least 2h at room temperature, subsequent dialysis rounds were performed for at least 8h at room temperature, or overnight at 4°C.

Size distribution of the LNPs was determined by dynamic light scattering (DLS) using a Zetasizer Pro (Red label, Malvern) with standard settings for LNPs (NIBS, adaptive correlation). LNPs were formed using different mRNAs (secNLuc (incl. PolyA ~iooont), eGFP, FLuc) and their size was measured using DLS in lx PBS (tomM phosphate buffer, 150 mM NaCl) pH 7.4. Multiple LNP formulations (50 mol% ionizable lipid (Li, L3, L4, L5, L6), 38.5 mol% structural lipid (cholesterol), 10 mol% phospholipid (DOPE) and 1.5 mol% DSPE-PEG(2000), at N/P 5 corresponding to a lipid to Oligo weight (LOW) of around 16), where the ionizable lipid was a compound according to Examples 1 to 6, were produced. As shown in Figure 1, these formulations revealed an average size of 120 nm (95-115 nm) with an average polydispersity index (PDI) of 0.07. Next, a higher N/P ratio (7:1 instead of 5:1, corresponding to a lipid to RNA weight ratio of 22.5, 50 mol% Li, 10 mol% DOPE, 38.5 mol% Cholesterol, 1.5 mol% DSPE-PEG, secNLuc mRNA (incl. PolyAtail looont)) was used (i.e. a higher ratio of the ionizable lipid to mRNA) to determine the effect of the N/P ratio on the size of the LNPs. Interestingly, as shown in Figure 2, a higher N/P ratio, resulted in smaller nanoparticles, similar to increasing the ratio of PEG-DSPE to total lipid.

Example 8: pKa determination of lipids

The pKa of ionizable lipid is known to be a major determinant for the endosomal escape of the contents of the LNP when exposed to cells. During endosomal uptake and passage through endosomal compartments, the LNP experiences a gradual decrease of the pH from the physiological pH (pH 7.4, as is present outside the cell) to around pH 4-5 - 5-0 in the lysosome, as the end-stage of most endocytosis vesicles. Trafficking towards the lysosome is generally to be avoided as breakdown of cargo (e.g., mRNA) may occur due to the degradative environment, thus endosomal escape ideally happens at a pH above 5.0, thus before the endosome has matured into a lysosome. A rapid increase in cationic charge of the ionizable lipid during this acidification process is thought to facilitate interaction with the inner endosomal membrane. Therefore, lipids which carry a neutral or near neutral charge at pH 7.4 and which are fully ionized at pH 5.5 are considered ideal; this corresponds to a pKa of around 64-6.5, similar what was found to be optimal by Jayaraman et al. (2012 Angewandte Chemie, DOI: i0.i002/ang.20i203263).

The local environment of the ionizable lipid (relating to the incorporation in an LNP) may influence the acquisition of charge and thus influences the pKa. Therefore, the experimentally determined pKa of the ionizable lipid was measured by the addition of 2-(p-toluidino) naphthalene-6-sulfonic acid (TNS; obtained from Sigma-Aldrich) to a representative nanoparticle composition in the presence of 20mM phosphate-citrate- ammonium citrate (pH 3-10 in 0.5 increments, all obtained from Sigma Aldrich). TNS is a compound that electrostatically interacts with the cationic lipid, resulting in fluorescence. Briefly, a mixture of 50 pl of 5uM TNS, 25pM LNPs (containing i2.5pM ionizable lipid) and 2omM buffer were added subsequently to the phosphate-citrate- ammonium citrate buffer samples in a 384 -well plate, and measured on a plate reader (iD3, Molecular Devices) at 325 nm excitation and 435 nm emission. Samples containing only LNPs and buffer were used for background subtraction for each increment in pH. The background-corrected measured fluorescence was then normalized against the difference between maximum and minimum fluorescence obtained during the assay, and a curve fit was performed to obtain an S -shaped curve for each ionizable lipid. The pKa of each ionizable lipid was determined as the pH value at which half of the maximum fluorescence was reached, and the results are given in Figure 3 for lipids Li, L3, L5 and L6 (identified as structures 1, 3, 5 and 6, respectively). It is noted that the lipid of example 3 was found to have a pKa of 6.35, close to the theoretical and previously determined optimum of pH 6.4. It is noted that the lipids of examples 5 and 6 displayed less than 1% ionization at physiological pH and may be well-suited for targeted LNPs, where uptake or opsonization by the RES-system would be detrimental.

Example 9: Toxicity and immunogenicity of lipids on cells

To measure toxic or immune-stimulatory effects of separate lipids, the inventors added a concentration range of the lipids, in the form of pure micelles, to cells. Pure micelles were chosen to exclude potential toxicity and/ or immunogenicity of other lipid components, as wouldbe present in LNPs. In addition, the inclusion of a PEG-modified lipid may reduce the opportunity of the lipids to interact with specific receptors or the cell surface.

Briefly, a concentration range of 10 to 0.001 pg for non-formulated (z.e., pure micelles, not in the form of LNPs) lipids of examples 1 to 6 were added to HeLa or endothelial cells, pre-mixed with cell-culture medium in a total volume of 100 pl per well of a 96- well plate. After 24b incubation, metabolic activity was tested by the resazurin assay. For this purpose, culture medium was replaced with medium containing 0.1 mg/ ml resazurin and incubated for 1-4 hours at 37 °C and 5% CO2. Subsequently, fluorescence was determined in the supernatant (excitation 540/25 nm, emission 620/40 nm).

Before medium renewal for the resazurin assay, medium was collected, and MCP-1 levels as a measure of immune response induction were tested in ELISA according to the manufacturer’s protocol (R&D Systems). Increasing concentrations of the lipids did not reveal any significant toxic effect on endothelial cells (Figure 4A). In addition, we could not find any induction of the endothelial cell-derived pro -inflammatory cytokine MCP-1 (Figure 4B). Results were comparable to widely used lipids like DODAP and DOTAP. Toll -like receptor (TLR)-2 or -4 agonists were used as positive control and demonstrated a significant induction of MCP-1 in these cells. Other studies in the literature have shown that some lipids can induce toxicity, which has been related to the activation of an immune response by binding to patternrecognition receptors such as toll -like receptor 2 and/ or 4. TLR2 and 4 recognize lipid compounds. Our results showthat Li to L6 lipids do not possess any toxicity or immunogenicity in endothelial cells by themselves.

Example 10: Activity of Li-LNPs in vitro

To determine if an Li-containing LNP formulation can effectively deliver mRNA into cells, the inventors incubated cells with luciferase mRNA-containing Li-LNPs.

Li-containing LNPs were formulated with 2.5 mol% DOTAP (47.5 mol% Li, 2.5 mol% DOTAP, 10 mol% DOPE, 38.5 mol% Cholesterol, 1.5 mol% DSPE-PEG(2000)) as described in example 7, containing secreted nano -luciferase mRNA at an N/P of 5:1 (corresponding to a LOW of around 16). A dose range of LNPs, corresponding to too, 50 and 10 ng per well containing a volume of too pl was added to HeLa cells in a 96- well plate. After 24 hours, medium was collected, and secreted nano -luciferase activity was determined using the Nano-Gio Luciferase Assay System (Promega). To check for unwanted immunostimulatory effects MCP-1 levels were tested by ELISA according to the manufacturer’s protocol (R&D Systems). Metabolic activity was tested by the resazurin assay to capture potential toxicity. For this purpose, culture medium was replaced with medium containing 0.1 mg/ ml resazurin and incubated for 1-4 hours at 37 °C and 5% CO2. Subsequently, fluorescence was determined in the supernatant (excitation 540/25 nm, emission 620/40 nm). Incubation of HeLa cells with Li-containing LNPs demonstrated a dose-dependent induction of luciferase activity that was similar to a formulation containing the widely used ionizable lipid MC3 (Figure 5A). The effect on metabolic activity, as tested by the resazurin assay, was not significant and similar for both formulations (Figure 5B). Li has comparable efficacy as an ionizable lipid as the widely used DLin-MC3-DMA lipid, while also showing a similar absence of toxicity on cells.

Example 11: Non -immunogenicity of Li-LNPs

This example illustrates that Li-containg LNP formulations are non-immunogenic in endothelial cells. Li-containing LNPs were formulated 2.5 mol% DOTAP (47.5 mol% Li, 2.5 mol% DOTAP, 10 mol% DOPE, 38.5 mol% Cholesterol, 1.5 mol% DSPE-PEG(2000)) as described in example 7, containing either immune-silent mRNA (RiboPro) or immune- active mRNA (Rib oPro) atanN/P of5:i (co r r espo nding to a LOW of ar 0 und 16) . A do se range of LNPs, corresponding to too, 50 and 10 ng per well containing a total volume of too pl medium, was added to endothelial cells in a 96-well plate. After 24 hours, medium was collected and MCP-1 levels were tested in ELISA according to the manufacturer’s protocol (R&D Systems). Incubation of endothelial cells with Li-containing LNPs that contained an immune- silent mRNA did not result in the production of the pro -inflammatory cytokine MCP-1 (Figure 6). In contrast, Li-containing LNPs that contained an mRNA with immune- stimulatory properties induced highlevels of MCP-i in these cells.

As a conclusion, LNPs containing the ionizable lipid Li are not immunogenic by themselves, since LNPs with immune-silent mRNA reveal no induction of the endothelial cell-derived pro -inflammatory cytokine MCP-1.

Example 12: In vivo activity of Li-LNPs

To determine the efficacy of in vivo delivery of mRNA using an LNP -formulation containing Li lipid, and determine potential toxic or immunogenic effects, the inventors injected Li-LNPs via the intravenous and intraperitoneal route into mice.

Li-containing LNPs were formulated without or with 1, 2.5 or5% of DOTAP (49/47.5 or 45 mol% Li, 1, 2.5 or 5 mol% DOTAP, 10 mol% DOPE, 38.5 mol% Cholesterol, i.5mol% DSPE-PEG(2000)) as describedin example 7 containing mEPO mRNA at an N/P of 5:1

(corresponding to a LOW of around 16). Eight to ten weeks-old C57BI/6 mice received either a single intravenous injection of 50 pl Li-LNPs in the tail vein, or a single intraperitoneal injection of too pl Li-LNPs, both containing 1 pg mEpo mRNA per dose. As a control, 1 pg mEpo mRNA was formulated with TransIT (Minis Bio) according to the manufacturer’s protocol and inj ected within 30 minutes as described above. After 6 hours, mice were weighted, and blood was collected in EDTA tubes (BD Microtainer™ Tubes with Microgard™ Closure) and further processed within 1 hour by centrifugation for 5 minutes at 500g. Plasma samples were tested in ELISA for mEpo levels (R&D Systems) or IL-6 cytokine levels (R&D Systems) according to the manufacturer’s protocol. As shown in Figure 7, injection with LNPs without DOTAP, or with 2.5% or 5% DOTAP resulted in comparable mEpo activity in plasma, while 1% DOTAP -containing LNPs revealed a slightly higher activity. Both intravenous and intraperitoneal injection of Li- containing LNPs in mice resulted in high plasma levels of mEpo protein. Note that endogenous mEpo backgroundlevels in non-treated mice were below 500 pg/ml. LNP- induced mEpo levels were equal or above those induced by TransIT, a suitable transfection reagent for in vivo use due to its low toxicity. Injections resulted in minimal weight changes (<5%) of the mice (Figure 7B), which tended to be lower for the LNPs compared to TransIT. In addition, the inventors could not detect significant levels of the pro-inflammatory cytokine IL-6 in the circulation (Figure 7C).

As a conclusion, Li-containing LNPs are able to deliver mRNA in vivo with high efficacy resulting in significant levels of the mRNA-encoded protein in the circulation of mice. Both intravenous and intraperitoneal injection lead to effective delivery. In addition, the Li lipid is active in LNPs without charged surface, which likely use ApoE- mediated delivery, and positively charged lipids. Importantly, Li-LNPs do not reveal any short-term effects on the weight of the mice or induction of the pro -inflammatory cytokine IL-6. Example 13: Encapsulation efficiency of LNPs

A high encapsulation efficiency of oligo - and poly-nucleotide cargo is important to prevent exposure of the cargo to degradative enzymes, immune -stimulatory cells and receptors, and achieving a high transfection efficiency. Therefore, the encapsulation efficiency was determined by means of a RiboGreen-assay.

Secreted Nano Luc (m)RNA-containing nanoparticle compositions (N/P 5:1, LOW ~16) were mixed 1:1 with QUANT-IT RIBOGREEN RNA assay (Invitrogen, obtained from Thermo Fisher Scientific) at a concentration of around 5pg/ ml in TE -buffer (lomM Tris, HC1 PH7.5, imM EDTA, obtained from Sigma-Aldrich) or 2% Triton X-100 (Sigma-Aldrich) in TE-buffer) with an equal volume of 1:100 RiboGreen reagent. Samples were thoroughly mixed and incubated for 5 minutes at room temperature. Next, the fluorescence intensity was measured on a plate reader (iD3, Molecular Devices), at 480 nm excitation and 520 nm emission. Blanks containing TE-buffer with 1:200 RiboGreen reagent or 2%-TritonX-ioo buffer with 1:200 RiboGreen reagent, were used as fluorescence background control. A standard curve of naked (m)RNA was used to quantify the absolute amount of non-encapsulated RNA. The signal obtained with 2% Titron was used to normalize all samples against and was set at 100%. The results are provided in Table 1.

Table 1: Details of LNP compositions and encapsulation efficiency of mRNA

All lipids of the invention displayed high encapsulation efficiency and are therefore suitable to protect oligo - and poly-nucleotides from degradative enzymes and to prevent any other effects of exposed oligo - and poly-nucleotides.