Novel ionizable lipids

Abstract

The present invention relates to a compound of formula (I). The present invention also extends to micro- or nanoparticles comprising a compound of formula (I). For example, the compound of formula (I) can be used to produce stable lipid nanoparticles (LNPs). LNPs have high encapsulation efficiency and can be used to deliver therapeutic or prophylactic agents to patients.

Description

[Technical Field]

[0001] The present invention relates to novel lipid molecules and methods for synthesizing the same. The invention extends to lipid-based nano- or microparticle compositions comprising novel lipids that are suitable for the delivery of nucleic acids, such as (modified) RNA or DNA. The invention further relates to medical and research uses of the compositions and methods for delivering nucleic acids, such as (modified) RNA or DNA, to mammalian cells, either in tissue culture or in whole organisms. [Background technology]

[0002] RNA biology is central to the functioning of most living organisms, including humans. As a result, most diseases are caused and / or manifested by changes in the presence, abundance, sequence, binding, or other properties of endogenous RNA in the cells involved. It is therefore not surprising that (personalized) solutions to these diseases are found by altering and / or correcting the presence, abundance, sequence, binding, or other properties of endogenous RNA, where correcting refers to re-establishing a non-pathological physiological state. RNA molecules, when introduced into cells, provide a solution to such changes and modifications. These RNA molecules can therefore also be used as therapeutic agents for these diseases. Examples of such RNA molecules include small interfering RNA (siRNA) and antisense RNA for suppressing the expression and / or promoting the degradation of endogenous RNA, splicing-correcting RNA for altering the splicing of pre-messenger RNA (pre-mRNA) to mature mRNA, and mRNA itself as a way to increase the expression of the encoded protein.

[0003] Recent advances in our understanding of RNA biology, including the intracellular innate immune pathways that recognize foreign (viral) RNA, have increased the commercial feasibility and interest in RNA-based therapies, as exemplified by the commercialization of the first messenger RNA and siRNA drugs in 2020 (SARS-CoV2 vaccine) and 2018 (Onpattro), respectively. Given the poor cellular uptake of such large, negatively charged, hydrophilic molecules by cells due to the negatively charged surface of the cell membrane, and the limited permeability of small, hydrophobic molecules, for which transporters and channels exist, delivery vehicles are often used to enhance the activity of RNA therapeutics and vaccines by several orders of magnitude. Therefore, interest in RNA delivery vehicles has also increased. However, the effective delivery of RNA to cells by delivery vehicles remains an ongoing scientific challenge, for which novel formulations and methods need to be developed.

[0004] These delivery vehicles often must serve multiple purposes, necessitating seemingly contradictory properties. The primary purpose of RNA delivery vehicles is to promote cell entry by packaging RNA into small (nano)particles that aid the RNA in crossing cell membranes or in inducing endocytosis and crossing the endocytic membrane. This crossing assistance requires shielding the negative charges of the RNA's phosphate backbone, interacting with and destabilizing the cellular or endocytic lipid bilayer, and subsequent dissociation and release of the RNA into the cytosol. Furthermore, delivery vehicles protect the RNA from degradative enzymes in the bloodstream, in the extracellular environment, and even during endosomal uptake by encapsulating the RNA within the vehicle. In addition, preliminary evidence suggests that encapsulation in delivery vehicles may, in some cases, protect the RNA from autocatalytic degradation by altering / reducing interactions with water molecules.

[0005] For efficient encapsulation, the vehicle must interact with RNA with sufficient affinity, while for efficient release of RNA in the cytosol, this affinity must be partially or completely reversed. Perturbation of cellular or endocytic membranes requires membrane perturbing / destabilizing activity, which may conflict with the colloidal stability of the delivery vehicle itself, for example, by using lipids as delivery vehicles with high fluidity (due to multiple unsaturated bonds in the lipid tails), such as lipid-based nanoparticles.

[0006] Delivery vehicles for RNA and DNA fall into various classes. The most important distinction can be made between polymeric delivery vehicles and lipid-based delivery vehicles. Lipid-based delivery vehicles consist of a hydrophilic head group and a hydrophobic tail and are therefore amphiphilic in nature.

[0007] Two main types of formulations based on lipid-based delivery vehicles can be distinguished: micellar formulations and lipid nanoparticles (LNPs). Micelle formulations consist of a single lipid, the delivery vehicle, which dissolves in aqueous solution, where the lipid forms micelles via its hydrophobic tail and associates with RNA or DNA via its positively charged head group. These micellar formulations, also known as lipoplexes, are typically used to deliver RNA or DNA to cells in tissue culture experiments.

[0008] LNPs consist of lipids that act as a delivery vehicle in combination with so-called helper lipids, cholesterol, and polyethylene glycol (PEG)-conjugated lipids. These LNPs are generated by mixing an ethanolic solution of lipids and an aqueous solution of RNA and are suitable for use in animals and humans. LNPs are the most commonly used type of formulation and are used in the approved RNA SARS-CoV2 vaccines and Onpattro.

[0009] The first lipid-based RNA and DNA delivery vehicles used in LNP formation were created, ensuring efficient encapsulation, shielding of the RNA negative charge, and efficient cellular interaction / uptake. All of these delivery vehicles were characterized by a constitutively positive charge. The positive charge was located in the head group, which refers to the hydrophilic portion of the lipid molecule as one of three major structural elements distinguishable in lipids. The other two are the hydrophobic hydrocarbon chain and the backbone, which connects the head group to the hydrocarbon chain. In natural phospholipids, the hydrocarbon chain exists as a carboxylic acid (fatty acid) linked to the hydroxyl group of the glycerol backbone via an ester bond. The ester bond can also be cleaved by phospholipases, releasing the fatty acid from the hydrophilic backbone and allowing further turnover of both moieties. In lipid systems, DOTAP, DOTMA, DOGS, and DOSPA are well-known examples of constitutively cationic charged species. However, as a direct consequence, intracellular release of RNA for these vehicles is small, and constitutively cationic delivery vehicles exhibit significant toxicity as well as rapid interaction with and elimination by the reticuloendothelial system (RES) (mostly particles <200 nm) and / or the mononuclear phagocyte system (mostly particles >200 nm), which determines the biodistribution of these delivery vehicles.

[0010] These problems have been primarily addressed by the use of lipids with ionizable cationic head groups. Using these compounds, the lipid head groups present on the surface of nanoparticles acquire a neutral (or near-neutral) surface charge under physiological conditions (pH approximately 7.4), e.g., while circulating in the blood. The neutral charge at physiological pH effectively prevents toxicity, and ionized lipid nanoparticles are tolerated at doses sufficient for vaccination and treatments intended to alter protein expression in whole organs, such as therapeutic siRNA therapy or protein expression. Formulation of RNA with an ionized delivery vehicle is often achieved by acidifying the pH of the aqueous solution of either the RNA, the delivery vehicle, or both during initial contact. Once charge-driven interactions between the RNA and nanoparticle components occur, the pH can be raised back to physiological levels, e.g., by dialysis, size exclusion, or the addition of excess buffer. When the ionized lipid contacts the RNA, a positive charge is maintained, while the ionized head groups on the surface of the nanoparticle are deprotonated and assume a neutral charge. Once a delivery vehicle is taken up into a cell by endocytosis, acidification of the endosomal compartment ionizes the surface lipids, reintroducing a cationic surface charge and allowing electrostatic interactions with the negatively charged endosomal lipid bilayer. This interaction is often the first step in the endosomal escape process. Alternatively, or in parallel, the ionized compound can act as a proton sponge, causing chloride ions to expand the endosomal compartment, followed by water influx and ultimately compartment rupture. Furthermore, the introduction of positive charges on the nanoparticle surface can lead to charge repulsion, thereby destabilizing the nanoparticle and promoting RNA release. Notably, the proton sponge hypothesis has been highly debated. Once the endosomal membrane is disrupted and some degree of nanoparticle destabilization occurs, the ionized compound re-encounters a neutral pH through direct contact with the cytosol, and under these conditions can finally release the exposed RNA. Well-known ionizable lipids used for RNA delivery include amine-containing lipids that can be easily protonated, such as DODAP (as the ionizable counterpart of DOTAP), DLin-DMA, and its derivatives DLin-KC2-DMA and DLin-MC3-DMA.

[0011] By reducing the exposure of the positive charge on the surface of the nanoparticles, the toxicity of (ionized) cationic delivery vehicles may be reduced through biodegradation and excretion, preventing bioaccumulation in and / or prolonged exposure of sensitive organs. Methods commonly used in the prior art include the use of compounds that are essentially water-soluble and can be excreted via urine, as well as the introduction of metabolizable groups, such as esters or protease-cleavable amides, that result in degradation products that are excreted or used as metabolites. Furthermore, disulfide bridges have been used in macromolecular conjugates, as they can be cleaved by reduction within the cytosol, leading to their degradation into smaller molecular building blocks.

[0012] Among the many ionized delivery vehicles known in the prior art, LNPs have exceptional advantages. In addition to the electrostatic interaction between the ionized lipid and RNA, particle stability is also maintained due to the hydrophobic interactions of the lipid tails. The incorporation of PEGylated lipids, which act as shielding lipids, results in colloidal stability despite the absence of a surface charge, allowing for the use of a neutral surface charge during circulation and storage. In addition, it is hypothesized that the mixture of the lipids of the delivery vehicle with those of the endosomal membrane contributes to its excellent endosomal escape activity by further disrupting the stability of the endosomal membrane. Finally, the use of multiple lipids in a single formulation allows for precise tuning of nanoparticle properties (size, stability, delivery efficiency, biodistribution) beyond what is possible or easily achieved with a single compound.

[0013] Despite years of research, the rational design of ionizable lipids (for use in lipid nanoparticles) with predictable RNA delivery properties remains elusive. As a result, the successful design of ionizable lipids always involves an element of trial and error and surprise. A consensus has emerged regarding the ideal pKa of LNPs as a complete formulation, with an apparent pKa determined by the 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) assay that ideally matches the pH of early endosomes (approximately pH 6.4) (Jayaraman et al. 2012 Angewandte Chemie, DOI:10.1002 / ang.201203263). However, even formulations with identical pKas can vary in efficacy by more than two orders of magnitude (Figure 3 in the aforementioned publication), indicating that next to pKa, the headgroup structure and / or lipid properties also significantly contribute to delivery activity, for example by influencing nanoparticle shape (Carrasco et al. 2021 Communications Biology, DOI:10.1038 / s42003-021-02441-2).

[0014] In addition to delivery activity and safety, easy and efficient synthesis of ionizable lipids is a key parameter for the commercial success of any novel ionizable lipid for RNA delivery. Many prior art ionizable lipids, including the lipid SM-102, have complex structures with branched lipid tails that induce a conical structure of the lipid layer. Such molecular structures often require multi-step synthesis using protecting groups (e.g., SM-102 in WO2017049245 and ALC-0315 in WO2018081480). The presence of by-products / incomplete conversion of precursors resulting from such multi-step procedures presents challenges during quality control. Furthermore, uncertainties exist regarding the turnover of such branched lipid structures, including SM-102 and ALC-0315, and the elimination and / or degradation of metabolites derived from such structures. Ideally, the metabolism of novel ionizable lipids would yield metabolites identical to those of normal metabolism and / or highly water-soluble metabolites, allowing for rapid excretion via urine and preventing bioaccumulation.

[0015] In summary, there remains a pressing need for novel ionizable lipids, and the combination of efficient cellular uptake, highly efficient endosomal release, predictable metabolism, and facile synthesis remains required to advance RNA-based therapeutics and vaccine administration. [Prior art documents] [Patent documents]

[0016] [Patent Document 1] WO2017049245 [Patent Document 2] WO2018081480 [Non-patent literature]

[0017] [Non-Patent Document 1] Jayaraman et al. 2012 Angewandte Chemie, DOI:10.1002 / ang.201203263 [Non-patent document 2] Carrasco et al. 2021 Communications Biology, DOI:10.1038 / s42003-021-02441-2 Summary of the Invention [Means for solving the problem]

[0018] According to a first aspect of the present invention, a compound of formula (I)

[0019] [ka]

[0020] (In the formula, R 1 and R 2 is an optionally substituted C 1~50 Alkyl, optionally substituted C 2~50 alkenyl or optionally substituted C 2~50alkynyl; L 1 and L 3 are independently absent or optionally substituted C 1~10 Alkylene, optionally substituted C 2~10 Alkenylene or optionally substituted C 2~10 is alkynylene, L 2 is absent or is NH, S or O, R 3 is -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 R 5 , -NR 4 SO2R 5 , -SO2NR 4 R 5 , -NR 4 COR 5 , -OP(O)(OH)OR 4 , optionally substituted C 3~6 Cycloalkyl, optionally substituted C 3~6 Cycloalkenyl, optionally substituted C 6~12 aryl, an optionally substituted 3- to 10-membered heterocyclic ring, or an optionally substituted 5- to 10-membered heteroaryl; R 4 ~R 6 is H, optionally substituted C 1~30 Alkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, optionally substituted C 3~6 Cycloalkyl, optionally substituted C 3~6 Cycloalkenyl, optionally substituted C 6~12aryl, an optionally substituted 3- to 10-membered heterocyclic ring, or an optionally substituted 5- to 10-membered heteroaryl; or a pharmaceutically acceptable complex, salt, solvate, tautomer or polymorph thereof is provided.

[0021] Advantageously, compounds of formula (I) can be used to produce stable lipid nanoparticles (LNPs), which have high encapsulation efficiency and can be used to deliver therapeutic or prophylactic agents to patients.

[0022] "Optional" or "optionally" means that the subsequently described phenomenon, action, or circumstance may or may not occur, and that the description includes instances where the phenomenon, action, or circumstance occurs and instances where it does not occur.

[0023] The term "alkyl," as used herein, unless otherwise specified, refers to a saturated, straight-chain or branched hydrocarbon. "Alkenyl" refers to an olefinically unsaturated hydrocarbon group that may be unbranched or branched, i.e., the hydrocarbon group contains one or more carbon-carbon double bonds. "Alkynyl" refers to an acetylenically unsaturated hydrocarbon group that may 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 contain one or more carbon-carbon double bonds.

[0024] The term "alkylene," as used herein, unless otherwise specified, refers to a divalent saturated, straight-chain or branched hydrocarbon. Similarly, the term "alkenylene," as used herein, unless otherwise specified, refers to a divalent, olefinically unsaturated, straight-chain or branched hydrocarbon. The term "alkynylene," as used herein, unless otherwise specified, refers to a divalent, acetylenically unsaturated, straight-chain or branched hydrocarbon. In addition to containing one or more carbon-carbon triple bonds, alkynylene groups may contain one or more carbon-carbon double bonds.

[0025] Any of the alkyl, alkenyl, alkynyl, alkylene, alkenylene and / or alkynylene groups may be unsubstituted and may include 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 R 5 , -NR 4 SO2R 5 , -SO2NR 4 R 5 , -NR 4 COR 5 , -OP(O)(OH)OR 4 , oxo, optionally substituted C 3~6 Cycloalkyl, optionally substituted C 3~6 Cycloalkenyl, optionally substituted C 6~12 R may be substituted with one or more of an optionally substituted aryl, an optionally substituted 3- to 10-membered heterocyclic ring, or an optionally substituted 5- to 10-membered heteroaryl. 4 ~R 6 may be as defined in relation to the first aspect. 4 ~R 6 is H, C 1~12 Alkyl, C 2~12 Alkenyl, C2~12 Alkynyl, C 3~6 Cycloalkyl, C 3~6 Cycloalkenyl, C 6~12 They may each be independently selected from the group consisting of aryl, a 3- to 10-membered heterocyclic ring, or a 5- to 10-membered heteroaryl.

[0026] "Cycloalkyl" refers to a non-aromatic saturated 3- to 6-membered ring hydrocarbon. Representative examples of C3-C6 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Similarly, "cycloalkenyl" refers to a non-aromatic unsaturated 3- to 6-membered ring hydrocarbon.

[0027] "Aryl" refers to an aromatic 6- to 12-membered hydrocarbon group. 12 Examples of aryl groups include, but are not limited to, phenyl, α-naphthyl, β-naphthyl, biphenyl, tetrahydronaphthyl, and indanyl.

[0028] "Heterocycle" or "heterocyclyl" refers to a 3- to 10-membered, monocyclic, bicyclic, or bridged molecule in which at least one ring atom is a heteroatom. Each heteroatom may be independently selected from the group consisting of oxygen, sulfur, and nitrogen. The heterocycle may be saturated or partially saturated. Exemplary heterocyclyl groups include, but are not limited to, aziridine, oxirane, oxirene, thiirane, pyrroline, pyrrolidine, dihydrofuran, tetrahydrofuran, dihydrothiophene, tetrahydrothiophene, dithiolane, piperidine, 1,2,3,6-tetrahydropyridin-1-yl, tetrahydropyran, pyran, morpholine, piperazine, thiane, thiine, piperazine, azepane, diazepane, and oxazine.

[0029] "Heteroaryl" refers to a monocyclic or bicyclic aromatic 5- to 10-membered ring system in which at least one ring atom is a heteroatom. 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-1,2,4-triazole, 1H-tetrazole, 1-methyltetrazole, benzoxazole, benzothiazole, benzofuran, benzisoxazole, benzimidazole, N-methylbenzimidazole, azabenzimidazole, indazole, quinazoline, quinoline, and isoquinoline. Bicyclic 5-10 membered heteroaryl groups include those in which a phenyl, pyridine, pyrimidine, pyrazine, or pyridazine ring is fused to a 5- or 6-membered monocyclic heteroaryl ring.

[0030] Any of the cycloalkyl, cycloalkenyl, aryl, heterocycle and / or heteroaryl groups may be unsubstituted, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkenyl, optionally substituted C1-C6 alkynyl, 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 R 5 , -NR 4 SO2R 5 , -SO2NR 4 R 5 , -NR 4 COR 5 , -OP(O)(OH)OR 4, oxo, optionally substituted C 3~6 Cycloalkyl, optionally substituted C 3~6 Cycloalkenyl, optionally substituted C 6~12 R may be substituted with one or more of an optionally substituted aryl, an optionally substituted 3- to 10-membered heterocyclic ring, or an optionally substituted 5- to 10-membered heteroaryl. 4 ~R 6 may be as defined in relation to the first aspect. 4 ~R 6 is H, C 1~12 Alkyl, C 2~12 Alkenyl, C 2~12 Alkynyl, C 3~6 Cycloalkyl, C 3~6 Cycloalkenyl, C 6~12 They may each be independently selected from the group consisting of aryl, a 3- to 10-membered heterocyclic ring, or a 5- to 10-membered heteroaryl.

[0031] The term "pharmaceutically acceptable salt" may be understood to refer to any salt of a compound provided herein that retains its biological properties and that is not toxic or otherwise undesirable for pharmaceutical use. Such salts may be derived from a variety of organic and inorganic counterions well known in the art.Such salts include, but are not limited to: (1) organic or inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, sulfamic acid, acetic acid, adepic acid, aspartic acid, trifluoroacetic acid, trichloroacetic acid, propionic acid, hexanoic acid, cyclopentylpropionic acid, glycolic acid, glutaric acid, pyruvic acid, lactic acid, malonic acid, succinic acid, sorbic acid, ascorbic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, picric acid, cinnamic acid, and mandelic acid; , phthalic acid, lauric acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonate, camphoric acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, benzoic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, Acid addition salts formed with stearic acid, cyclohexylsulfamic acid, quinic acid, muconic acid, and similar acids, or (2) salts in which the acidic protons present in the parent compound are replaced by (a) metal ions, such as alkali metal ions, alkaline earth ions, or aluminum ions, or alkali metal or alkaline earth metal hydroxides, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, aluminum hydroxide, lithium hydroxide, zinc hydroxide, and barium hydroxide, ammonia, or (b) organic bases, such as aliphatic, fatty, Base addition salts formed upon coordination with cyclic 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.

[0032] Pharmaceutically acceptable salts include sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like, and, if the compound contains a basic functionality, salts of non-toxic organic or inorganic acids, such as hydrohalide salts, e.g., hydrochloride, hydrobromide, and hydroiodide salts, carbonate or bicarbonate salts, sulfate or hydrogen sulfate salts, borates, phosphates, hydrogen phosphates, dihydrogen phosphates, pyroglutamate, saccharate, stearate, sulfamate, nitrate, orotate, oxalate, and the like. Acid salts include: acid salt, palmitate, pamoate, acetate, trifluoroacetate, trichloroacetate, propionate, hexanoate, cyclopentylpropionate, glycolate, glutarate, pyruvate, lactate, malonate, succinate, tannate, tartrate, tosylate, sorbate, ascorbate, malate, maleate, fumarate, tartrate, camsylate, citrate, cyclamate, benzoate, isethionate, esylate, formate, 3-(4-hydroxybenzoate)-2-hydroxybenzoate, 4-hydroxybenzoate, 5-hydroxybenzoate, 6-hydroxybenzoate, 7-hydroxybenzoate, 8-hydroxybenzoate, 9-hydroxybenzoate, 10-hydroxybenzoate, 11-hydroxybenzoate, 12-hydroxybenzoate, 13-hydroxybenzoate, 14-hydroxybenzoate, 15-hydroxybenzoate, 16-hydroxybenzoate, 17-hydroxybenzoate, 18-hydroxybenzoate, 19-hydroxybenzoate, 20-hydroxybenzoate, 21-hydroxybenzoate, 22-hydroxybenzoate, 23-hydroxybenzoate, 24-hydroxybenzoate, 25-hydroxybenzoate, 26-hydroxybenzoate, 27-hydroxybenzoate, 28-hydroxybenzoate, 29-hydroxybenzoate, 30-hydroxybenzoate, 31-hydroxybenzoate, 32-hydroxybenzoate, 33-hydroxybenzoate, 34-hydroxybenzoate, 35-hydroxybenzoate, 36-hydroxybenzoate, 37-hydroxybenzoate, 38-hydroxybenzoate, 39-hydroxybenzoate, 40-hydroxybenzoate, 41-hydroxybenzoate, 42-hydroxybenzoate, 43-hydroxybenzoate, 44-hydroxybenzoate, 45-hydroxybenzoate, 46-hydroxybenzoate, 47-hydroxybenzoate, 48-hydroxybenzoate, 49- Benzoylbenzoate, picrate, cinnamate, mandelate, phthalate, laurate, methanesulfonate (mesylate), methylsulfate, naphthylate, 2-napsylate, nicotinate, ethanesulfonate, 1,2-ethanedisulfonate, 2-hydroxyethanesulfonate, benzenesulfonate (besylate), 4-chlorobenzenesulfonate, 2-naphthalenesulfonate, 4-toluenesulfonate, camphorate, The salts may include benzoates, benzoates, glutamates, hydroxynaphthoates, salicylates, stearates, cyclohexylsulfamates, quinates, muconates, xinafoates, and the like.

[0033] L 1may be non-existent.

[0034] In one preferred embodiment, L 1 is an optionally substituted C 1~6 Alkylene, optionally substituted C 2~6 Alkenylene or optionally substituted C 2~6 More preferably, L is alkynylene. 1 is C 1~3 Alkylene, C 2~3 Alkenylene or C 2~3 alkynylene. Therefore, L 1 may be -CH2-, -CH2CH2- or -CH2CH2CH2-.

[0035] In some embodiments, L 2 In some embodiments, L 3 is an optionally substituted C 1~6 Alkylene, optionally substituted C 2~6 Alkenylene or optionally substituted C 2~6 L may be alkynylene. 3 is C 1~3 Alkylene, C 2~3 Alkenylene or C 2~3 alkynylene. Therefore, L 3 may be -CH2-, -CH2CH2- or -CH2CH2CH2-.

[0036] In an alternative embodiment, L 2 may be absent. 3 may be non-existent.

[0037] In some embodiments, R 3 is -NR 4 R 5 , -SR 4 , -OR 4 , an optionally substituted 3- to 10-membered heterocyclic ring, or an optionally substituted 5- to 10-membered heteroaryl. 3 is -NR 4 R 5 , -SR4 , -OR 4 , an optionally substituted 5- or 6-membered heterocyclic ring, or an optionally substituted 5- or 6-membered heteroaryl. The heterocyclic ring or heteroaryl may contain one or more nitrogen atoms. The heterocyclic ring or heteroaryl may be an N-linked heterocyclic ring or heteroaryl.

[0038] R 3 is preferably -NR 4 R 5 , -SR 4 -OR 4 R 3 is more preferably -NR 4 R 5 is.

[0039] R 4 ~R 6 is H, optionally substituted C 1~20 Alkyl, optionally substituted C 2~20 Alkenyl and optionally substituted C 2~20 alkynyl. More preferably, R 4 ~R 6 is H, optionally substituted C 1~10 Alkyl, optionally substituted C 2~10 Alkenyl and optionally substituted C 2~10 alkynyl. Most preferably, R 4 ~R 6 is C 1~5 Alkyl, C 2~5 Alkenyl and C 2~5 In some embodiments, R 4 ~R 6 are independently methyl, ethyl or propyl, more preferably methyl or ethyl.

[0040] Thus, in some embodiments, R 3 teeth,

[0041] [ka]

[0042] It may be.

[0043] In some embodiments, the compound of formula (I) can be a compound of formula (IIa), (IIb), (IIc), (IId), or (IIe):

[0044] [ka]

[0045] R 1 and R 2 is an optionally substituted C 5~30 Alkyl, optionally substituted C 5~30 alkenyl or optionally substituted C 5~30 alkynyl. More preferably, R 1 and R 2 is an optionally substituted C 10~25 Alkyl, optionally substituted C 10~25 alkenyl or optionally substituted C 10~25 alkynyl. Even more preferably, R 1 and R 2 is an 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 Both are C 15~20 Alkenyl may be a singly or doubly unsaturated alkenyl group.

[0046] In one preferred embodiment, R 2 is R 1 Thus, in some embodiments, the compound is a compound of formula (III):

[0047] [ka]

[0048] In the formula, both R 1 The base is the same.

[0049] In some embodiments, R 1 and R 2 is -(CH2)8(CH)2CH2(CH)2(CH2)4CH3 or -(CH2)8(CH)2(CH2)7CH3. More preferably, R 1 and R 2 teeth,

[0050] [ka]

[0051] In one preferred embodiment, R 1 and R 2 together

[0052] [ka]

[0053] is.

[0054] The compound may be of formula (101), (102), (103), (104), (105) or (106):

[0055] [ka]

[0056] It is understood that compounds of formula (I) have at least one stereocenter. The compounds may be S or R stereoisomers. Alternatively, the compounds may be provided as mixtures of both isomers, with variable mole fractions of R ranging from 0% to 100% and S ranging from 100% to 0%. The compounds may also be provided as racemic mixtures.

[0057] R 3 Ga-NR 4 R 5 In embodiments where the amine moiety is, the amine moiety may be unprotonated at physiological pH. Alternatively, the amine moiety may be protonated at physiological pH. Physiological pH may be understood as pH 7.5 or less at 37°C. Physiological pH may be pH 7.3-7.5 at 37°C. In some embodiments of Formula (I), the amine moiety may have a pKa greater than 7.5, which renders the lipid cationic throughout the pH range expected in the mammalian body. Such lipids are typically referred to as cationic (amino) lipids. Thus, a salt of a compound of Formula (I) may be a salt of Formula (IV):

[0058] [ka]

[0059] In the formula, X - is the counterion.

[0060] The compounds of formula (I) may be used to form lipid nanoparticles (LNPs).

[0061] Thus, according to a second aspect of the present invention there is provided a micro- or nanoparticle comprising a compound of formula (I) as defined in the first aspect, or a pharmaceutically acceptable complex, salt, solvate, tautomer or polymorph thereof, and optionally a payload molecule.

[0062] The micro- or nanoparticles may define a dense or hollow structure. The micro- or nanoparticles may optionally consist of one or more lipid bilayers, which may optionally be cross-linked to each other and encapsulate a cavity. The micro- or nanoparticles may be lipid nanoparticles (LNPs), liposomes, lipoplexes, micelles, or lipid vesicles. In some embodiments, the micro- or nanoparticles are LNPs.

[0063] The micro- or nanoparticles may have a diameter of less than 10 μm, less than 1 μm, less than 500 nm, or less than 250 nm. More preferably, the micro- or nanoparticles 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 nanoparticles may have a diameter of 30 nm to 1 μm, 40 to 500 nm, 50 to 250 nm, 60 to 200 nm, 70 to 175 nm, 80 to 150 nm, or 90 to 125 nm. The diameter of the micro- or nanoparticles may be measured using dynamic light scattering.

[0064] In addition to the compound of formula (I) or its pharmaceutically acceptable complex, salt, solvate, tautomer or polymorph, the micro- or nanoparticles may contain one or more additional lipids.Thus, the micro- or nanoparticles may contain a lipid component comprising the compound of formula (I) or its pharmaceutically acceptable complex, salt, solvate, tautomer or polymorph, and one or more additional lipids.The one or more additional lipids may be selected from the group consisting of phospholipids, constant or ionized cationic lipids, constant or ionized anionic lipids, structural lipids, shielding lipids, functionalized lipids, and combinations thereof.

[0065] In some embodiments, the lipid component comprises a compound of formula (I), or a pharmaceutically acceptable complex, salt, solvate, tautomer, or polymorph thereof, and a structured lipid. In some embodiments, the lipid component comprises a compound of formula (I), or a pharmaceutically acceptable complex, salt, solvate, tautomer, or polymorph thereof, a structured lipid, and a phospholipid. In some embodiments, the lipid component comprises a compound of formula (I), or a pharmaceutically acceptable complex, salt, solvate, tautomer, or polymorph thereof, a structured lipid, a phospholipid, and a shielding lipid. In some embodiments, the lipid component further comprises a constant or ionized cationic lipid. In some embodiments, the lipid component further comprises a functionalized lipid.

[0066] In some embodiments, the lipid component comprises 20-80 mol%, 30-70 mol%, 40-60 mol%, or 45-55 mol% of a compound of formula (I), or a pharmaceutically acceptable complex, salt, solvate, tautomer, or polymorph thereof.

[0067] In some embodiments, the lipid component comprises 5-80 mol%, 10-60 mol%, 20-50 mol%, 30-45 mol%, or 35-40 mol% of structured lipids.

[0068] In some embodiments, the lipid component comprises 0 to 30 mol %, 2.5 to 20 mol %, 5 to 15 mol %, or 7.5 to 12.5 mol % of phospholipids.

[0069] In some embodiments, the lipid component comprises 0-15 mol%, 0.1-10 mol%, 0.5-5 mol%, 0.75-3 mol%, or 1-2 mol% of shielding lipid.

[0070] In some embodiments, the lipid component comprises 0-15 mol%, 0.1-10 mol%, 0.5-5 mol%, 0.75-3 mol%, or 1-2 mol% of a constant or ionizable cationic lipid.

[0071] The structural lipid may be a sterol. The sterol may be cholesterol or a cholesterol derivative. The cholesterol derivative may be beta-sitosterol, vitamin D2, vitamin D3, calcipotriol, stigmasterol, campesterol, fucosterol, brassicasterol, ergosterol, 9,11-dehydroergosterol, daucosterol, beta-sitosterol acetate, betutin, lupeol, ursolic acid, or oleanolic acid.

[0072] The phospholipid may be di-oleoyl-phosphatidylethanolamine (DOPE), di-oleoyl-phosphatidylcholine (DOPC), di-oleoyl-phosphatidylserine (DOPS), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) or any naturally occurring phospholipid.

[0073] Shielding lipids can be understood as functionalized lipids that prevent undesired interactions of lipid nanoparticles with other nanoparticles, extracellular compounds, and / or cell surfaces. Shielding lipids can prevent undesired interactions of lipid nanoparticles with other nanoparticles, extracellular compounds, and / or cell surfaces through steric hindrance or similar mechanisms. Shielding lipids can be lipids modified to include a shielding polymer. The shielding polymer can be a polyethylene glycol (PEG) group, a poly-sarcosine group, an oligopeptide or polypeptide, a hydroxyl-containing nonionic water-soluble polymer, polyvinylpyrrolidone (PVP), poly(2-alkyl-2-oxazoline), a zwitterionic polymer, or any other suitable shielding polymer. The oligopeptide or polypeptide can be a PAS. A PAS can be understood as an oligopeptide or polypeptide consisting of or including proline, alanine, and serine residues. The hydroxyl-containing nonionic water-soluble polymer may be poly(glycerol) (PG), poly(N(2-hydroxypropyl)methacrylamide) (pHPMA), polysarcosine, or poly(vinyl alcohol). The zwitterionic polymer may be polybetaine. In some embodiments, the shielding lipid may be a phospholipid modified to include a shielding polymer. In embodiments where the shielding lipid is a phospholipid modified to include a shielding polymer, the modified phospholipid may be a phospholipid as defined above. In some embodiments, the shielding lipid may be a DSPE, DMG, or DPPC lipid modified to include a shielding polymer. The shielding lipid may include a shielding polymer having an average molecular weight of 500-5000, 1000-3000, 1250-1750, 1500-2500, 1750-2250 Da, or 1900-2100 Da. The shielding lipid may be dimyristoyl-glycerol-PEG (DMG-PEG), PEG-DSPE, PEG-dipalmitoyl-phosphatidylcholine (DPPC), pSar-DMG, pSar-DSPE, or pSar-DPPC. In some embodiments, the shielding lipid is PEG-DSPE or pSar-DSPE.

[0074] The constant or ionizable cationic lipid may be 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 4-(dimethylamino)butanoic acid heptatriaconta-6,9,28,31-tetraen-19-yl (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), or 1,2-dioleoyl-3-trimethylammonium propane (DODAP).

[0075] The constant or ionized anionic lipid may be cholesteryl hemisuccinate, phosphatidylinositol phosphate (PIP, also known as phosphoinositides), phosphatidylserine (PS) or phosphatidic acid (PA).

[0076] The functionalized lipid may comprise one or more moieties that allow for attachment thereto, which may be independently selected from the group consisting of azide, alkyne, tetrazine, dibenzocyclooctyne (DBCO), maleimide, trans-cyclooctene (TCO), vinyl, methylcyclopropene, and succinimidyl ester.

[0077] In a preferred embodiment, the micro- or nanoparticles comprise a payload molecule, which may be a biomolecule, and / or an active pharmaceutical ingredient (API), and / or a diagnostic compound.

[0078] The API may be a hydrophobic or hydrophilic API. The API may be a polymer or a small molecule. It may be understood that a small molecule is considered to be a molecule having 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 polymer is considered to be a molecule having a molecular weight of at least 900 daltons.

[0079] Examples of APIs include anti-inflammatory compounds (e.g., non-steroidal anti-inflammatory drugs, such as aspirin, ibuprofen, naproxen, celecoxib, diclofenac, indomethacin, oxaprozin and / or piroxicam, more preferably steroidal anti-inflammatory drugs, such as prednisone, cortisone and methylprednisone, or anti-rejection drugs, such as tacrolimus, cyclosporine, mycophenolate mofetil, azathioprine, rapamycin, sirolimus), anti-cancer drugs (e.g., chemotherapy, such as alkylating agents (e.g., altretamine, bendamustine, busulfan, cyclophosphamide, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, mechlorethamine, melphalan, oxaliplatin, temozolomide, thiotepa, trabectedin), nitrosoureas (e.g., carmustine, lomustine, streptozocin), antimetabolites (e.g., azacitidine, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cladribine, clofarabine, cytarabine (Ara-C), decitabine, floxuridine, antitumor antibiotics (e.g., anthracyclines daunorubicin, doxorubicin (adriamycin), liposomal doxorubicin, epirubicin, idarubicin, valrubicin, and antitumor antibiotics bleomycin, dactinomycin, mitomycin-C, mitoxantrone), topoisomerase inhibitors (irinotecan, liposomal ibuprofen, rifampin, rifampin), rifampin-containing anti-inflammatory drugs (e.g., rifampin, rifampin-containing steroids ... rinotecan, topotecan, etoposide (VP-16), mitoxantrone, teniposide), mitotic inhibitors (e.g., the taxanes cabazitaxel, docetaxel, Nab-paclitaxel, and paclitaxel, and the vinca alkaloids vinblastine, vincristine, liposomal vincristine, and vinorelbine), and other chemotherapeutic agents (e.g., all-trans retinoic acid, arsenic trioxide, asparaginase, eribulin, hydroxyurea, ixabepilone, mitotane, omacetaxine, pegaspargase, procarbazine, romidepsin,including vorinostat), cytokine agents (including cytokines (e.g., 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-lambda 1, IFN-lambda 2, IFN-lambda 3, IFN-omega, IP-10, MIP-1alpha, TGF-beta (1-3) (recombinant forms thereof)) and anti-cytokine agents (e.g., cytokine-binding antibodies, decoy receptors, IL-1R antagonists)), growth factors ( Examples include bone morphogenetic proteins (BMPs), vascular endothelial growth factors (VEGFs), granulocyte-macrophage colony-stimulating factors (GM-CSFs), epidermal growth factors (EGFs), erythropoietin (EPO), insulin-like growth factors (IGFs), fibroblast growth factors (FGFs), hepatocyte growth factors (HGFs), platelet-derived growth factors (PDGFs), transforming growth factors (TGFs), thrombopoietin (TPOs), hormones (e.g., 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 hormones, epinephrine, norepinephrine, testosterone, melatonin, growth hormone-releasing hormone (GHRH), thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone (GnRH), corticotropin-releasing hormone (CRH), humoral factors), cardiovascular drugs (e.g., anticoagulants (e.g., apixaban, dabigatran, edoxaban, , heparin, rivaroxaban and warfarin), antiplatelet agents (examples include aspirin, clopidogrel, dipyridamole, prasugrel and ticagrelor), 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, losartan, olmesartan, telmisartan,and valsartan), beta-adrenergic blocking agents (acebutolol, atenolol, betaxolol, bisoprolol, metoprolol, nadolol, propranolol, and sotalol), calcium channel blockers (examples include amlodipine, diltiazem, felodipine, nifedipine, nimodipine, nisoldipine, and verapamil), cholesterol-lowering drugs (examples include the statin drugs atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin, niacin nicotinate, and cholesterol-lowering drugs). Drugs that may be prescribed include: steroids (e.g., steroids), ... (including cimetidine, ranitidine, famotidine, and nizatidine), prokinetics and laxatives (metoclopramide), ophthalmic medications (e.g., ophthalmic allergy medications (e.g., ketorolac, ketotifen, loteprednol, bepotastine, epinastine, emedastine, alcaftadine, azelastine, olopatadine, nedocromil, lodoxamide, and cromolyn)), topical antibiotics (e.g., besifloxacin, ciprofloxacin, moxifloxacin, ofloxacin, gatifloxacin, tobramycin, gentamicin), , polymyxin D, neomycin, bacitracin, azithromycin, and erythromycin), lipid-based artificial tears (examples include castor oil, glycerol, and mineral oil), NSAIDS and corticosteroids, glaucoma medications (examples include levobunolol, timolol, betaxolol, bimatoprost, travoprost, latanoprost, tafluprost, brimonidine, brinzolamide, and dorzolamide), and antiviral treatments (examples include acyclovir, valacyclovir, and famciclovir), pulmonary medications (e.g., antiasthma medications (e.g.,antihistamines (e.g., brompheniramine, carbinoxamine, chlorpheniramine, clemastine, diphenhydramine, hydroxyzine, triprolidine, azelastine, cetirizine, desloratadine, fexofenadine, levocetirizine, loratadine, olopatadine), antitussives (e.g., dextromethorphan and benzonatate), bronchodilators (ipratropium, theophylline, albuterol, epinephrine, levalbuterol, alfo lumoterol, formoterol, olodaterol, terbutaline, pirbuterol, metaproterenol, salmeterol, isoproterenol, indacaterol, tiotropium, umeclidinium, aclidinium, ipratropium, lebefenacin, glycopyrrolate, ipratropium, theophylline, aminophylline, and dyphylline), decongestants (examples include levomethamphetamine, naphazoline, oxymetazoline, phenylephrine, propylhexedrine, pseudoephedrine, and xylometazoline), expectorants (guaifene sucrine), leukotriene modifiers (examples include montelukast, zafirlukast, zileuton), pulmonary surfactants (examples include beractant, lucinactant, calfactant, and boractant), mucolytics (acetylcysteine), anti-infectives (examples include zanamivir, ribavirin, tobramycin, pentamidine, and colistimethate), inhaled corticosteroids (examples include fluticasone, budesonide, mometasone, beclomethasone, and ciclesonide), mast cell stabilizers (examples include cromolyn and nedocromil), and phosphatase inhibitors (examples include fluticasone, budesonide, mometasone, beclomethasone, and ciclesonide). Fodiesterase-4 inhibitors (including roflumilast), antibacterial agents (antibiotics (e.g., aminoglycosides (e.g., amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, and spectinomycin)), ansamycins (e.g., geldanamycin, herbimycin, and rifaximin), carbacephems (e.g., loracarbef), carbapenems (e.g., ertapenem, doripenem, imipenem, and meropenem), cephalosporins (e.g.,cefadroxil, cefazolin, cephradine, cephapirin, cephalothin, cephalexin, cefaclor, cefoxitin, cefotetan, cefamandole, cefmetazole, cefonicid, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, moxalactam, ceftriaxone, cefepime, ceftaroline fosamil, and ceftobiprole), glycopeptides (e.g., teicoplanin, vancomycin, teratacycline, vancin, dalbavancin, and oritavancin), lincosamides (clindamycin and lincomycin), lipopeptides (e.g., daptomycin), macrolides (azithromycin, clarithromycin, erythromycin, roxithromycin, telithromycin, spiramycin, and fidaxomicin), monobactams (e.g., aztreonam), nitrofurans (e.g., furazolidone and nitrofurantoin), oxazolidinones (e.g., linezolid, pocizolid, radezolid, and torezolid), penicillins antihistamines (e.g., amoxicillin, ampicillin, azlocillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, temocillin, and ticarcillin), polypeptides (e.g., bacitracin, colistin, and polymyxin B), quinolones (e.g., 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 sulfonamide chrysoidine), tetracyclines (examples include demeclocycline, doxycycline, methacycline, minocycline, oxytetracycline, and tetracycline),Mycobacteria-specific antibiotics (examples include clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin, rifabutin, rifapentine, and streptomycin), antifungal drugs (e.g., polyene antifungals (examples include amphotericin B, candicidin, filipin, hamycin, natamycin, nystatin, and rimocidin)), azoles (e.g., imidazoles bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, and tioconazole), triazoles vaconazole, efinaconazole, epoxiconazole, fluconazole, isavuconazole, itraconazole, posaconazole, propiconazole, ravuconazole, terconazole, and voriconazole, and thiazoles (e.g., abafungin), arylamines (e.g., butenafine, naftifine, and terbinafine), echinocandins (anidulafungin, caspofungin, and micafungin), and triterpenoids (e.g., ibrexafungelp), and antiparasitic agents (e.g., broad-spectrum Nitazoxanide, antiprotozoal drugs (examples include melarsoprol, eflornithine, metronidazole, tinidazole, and miltefosine), antinematoid drugs (examples include mebendazole, pyrantel pamoate, thiabendazole, diethylcarbamazine, and ivermectin), antitasteworm drugs (examples include niclosamide, praziquantel, and albendazole), antitrematode drugs (e.g., praziquantel), and antiamoebic drugs (e.g., rifampicin and amphotericin B), antidiabetic drugs (e.g., insulin (analogs), amylinomimetics (e.g., pramlintide), alfa glucosidase inhibitors (e.g., acarbose and miglitol), biguanides (e.g., metformin (analogs and combinations), dopamine agonists (e.g., bromocriptine), dipeptidyl peptidase-4 (DDP-4) inhibitors (e.g., alogliptin, linagliptin, saxagliptin, and sitagliptin), glucagon-like peptide-1 receptor agonists (e.g., albiglutide, dulaglutide, exenatide, liraglutide, and semaglutide), meglitinides (e.g., nateglinide and repaglinide), sodium-glucose transporter (SGLT-2) inhibitors (e.g., dapagliflozin, canagliflozin, ertugliflozin, and empagliflozin), sulfonylureas (e.g., glimepiride, gliclazide, glipizide, glyburide, chlorpropamide, tolazamide, and tolbutamide), thiazolidinediones (e.g., rosiglitazone and pioglitazone), antivirals (e.g., abacavir, acyclovir, adefovir, amantadine, ampligen, amprenavir, umifenovir, atazanavir, atriplan,Oseltamivir, zanamivir, peramivir, baloxavir, bictegravir, emtricitabine, tenofovir, boceprevir, brevirtide, cidofovir, cobicistat, daclatasvir, darunavir, delavirdine, didanosine, docosanol, dolutegravir, doravirine, edoxudine, efavirenz, emtricitabine, enfuvirtide, ensivirvir, ensitrervir, entecavir, etravirine, famciclovir, fomivirsen, fosamprenavir, foscarnet, ganciclovir, ibacitabine, ibalizumab, idoxuridine, imiquimod, inosine pranobex, indinavir, lamivudine, letervir and raltegravir, remdesivir, ribavirin, rilpivirine, rimantadine, ritonavir, saquinavir, simeprevir, sofosbuvir, stavudine, talibavirin, telaprevir, telbivudine, tenofovir, tipranavir, trifluridine, trizivir, tromantadine, truvada, umifenovir, valacyclovir, valganciclovir, vicriviroc, vidarabine, zalcitabine, zanamivir, and zidovudine), or structural or functional analogs thereof.

[0080] In a preferred embodiment, the payload molecule is a biomolecule, for example, the biomolecule may be or include an amino acid, peptide, affimer, polypeptide or protein, glycoprotein, sugar, lipid, lipopolysaccharide, antibody or fragment thereof, polymer, or nucleic acid, or a combination thereof.

[0081] The nucleic acid may be DNA, RNA, XNA (including xenonucleic acid, 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 (fluoroarabino nucleic acid), and non-locked nucleic acid), or a DNA / RNA hybrid sequence. Preferably, the nucleic acid is DNA or RNA.

[0082] Most preferably, the nucleic acid is RNA. The RNA may be single-stranded or double-stranded. The RNA may be selected from the group consisting of messenger RNA (mRNA); circular RNA (circRNA or oRNA); self-amplifying RNA (saRNA); trans-amplifying RNA (taRNA), long non-coding RNA, split replicon RNA, viral RNA, antisense RNA (AON or asRNA); RNA aptamer; interfering RNA; microRNA (miRNA); short interfering RNA (siRNA); short hairpin RNA (shRNA); and small RNA.

[0083] Preferably, the RNA is messenger RNA (mRNA).

[0084] The nucleic acid sequence, preferably RNA, may be at least 10 bases in length, at least 20 bases in length, at least 50 bases in length, at least 100 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 in length. In a preferred embodiment, the RNA is saRNA or mRNA.

[0085] The nucleic acid sequence, preferably RNA, most preferably mRNA, may be at least 100 bases in length, at least 500 bases in length, at least 1000 bases in length, at least 2000 bases in length, at least 3000 bases in length, at least 4000 bases in length, at least 5000 bases in length, at least 6000 bases in length, at least 7000 bases in length, at least 8000 bases in length, at least 9000 bases in length, at least 10000 bases in length, at least 11000 bases in length or at least 12000 bases in length.

[0086] 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 saRNA is at least 6000 bases in length.

[0087] In an alternative embodiment, the nucleic acid sequence is at least 900 bases in length. In one embodiment, the RNA is at least 900 bases in length. In a preferred embodiment, the mRNA is at least 900 bases in length.

[0088] Alternatively, the nucleic acid sequence, preferably RNA, most preferably mRNA, may be 50 to 10,000 bases in length, 100 to 9,000 bases in length, 200 to 8,000 bases in length, 300 to 7,000 bases in length, 400 to 6,000 bases in length, 500 to 6,000 bases in length, 600 to 5,000 bases in length, 700 to 4,000 bases in length, 800 to 3,000 bases in length or 900 to 2,000 bases in length.

[0089] In one embodiment, the nucleic acid sequence is 6,000 to 15,000 bases in length. The nucleic acid sequence may be 8,000 to 12,000 bases in length. The RNA may be 6,000 to 15,000 bases in length. The RNA may be 8,000 to 12,000 bases in length. Preferably, the saRNA is 6,000 to 15,000 bases in length. Preferably, the saRNA is 8,000 to 12,000 bases in length.

[0090] In an alternative embodiment, the nucleic acid sequence is 100 to 14,000, 500 to 10,000, 600 to 7,500, 700 to 5,000, 800 to 4,000, or 900 to 2,000 bases in length. The RNA may be 400 to 14,000, 500 to 10,000, 600 to 7,500, 700 to 5,000, 800 to 4,000, or 900 to 2,000 bases in length. Preferably, the mRNA is 100 to 14,000, 500 to 10,000, 600 to 7,500, 700 to 5,000, 800 to 4,000, or 900 to 2,000 bases in length.

[0091] Those skilled in the art will understand that when the nucleic acid is double-stranded, for example double-stranded RNA, "length ~ bases" refers to the length in base pairs.

[0092] The mass ratio of lipid component to payload molecule may be 1:1 to 100:1, 2:1 to 80:1, 3:1 to 70:1, 4:1 to 60:1, or 5:1 to 50:1.

[0093] In embodiments where the payload molecule is a biomolecule, the mass ratio of lipid component to payload molecule can be 6:1 to 45:1, 8:1 to 40:1, 10:1 to 35:1, or 12:1 to 30:1. In some embodiments, the mass ratio of lipid component to payload molecule can be 13:1 to 25:1, 14:1 to 20:1, or 15:1 to 17:1. In some embodiments, the mass ratio of lipid component to payload molecule can be 15:1 to 27.5:1, 20:1 to 25:1, or 22:1 to 23:1.

[0094] In embodiments where the payload molecule is a biomolecule, the N:P ratio may be 1:2 to 50:1, 1:1 to 30:1, 2:1 to 20:1, 3:1 to 15:1, or 5:1 to 12:1. In some embodiments, the N:P ratio may be 3:1 to 10:1 or 4:1 to 6:1. In alternative embodiments, the N:P ratio may be 4:1 to 10:1, 5:1 to 9:1, or 6:1 to 8:1. It may be understood that the N:P ratio is the ratio of positively chargeable polymeric amine (N) groups to negatively charged nucleic acid phosphate (P) groups.

[0095] Preferably, the micro- or nanoparticles have 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 can be understood that the encapsulation efficiency is determined by the amount of payload molecules encapsulated in the micro- or nanoparticles (i.e., not available and / or accessible to the aqueous environment outside the micro- or nanoparticles) relative to the total amount of payload molecules initially provided. The encapsulation efficiency can be determined using a RiboGreen assay, which detects solvent-accessible RNA by an increase in fluorescence upon intercalation of the water-soluble Ribogreen reagent into the RNA.

[0096] The micro- or nanoparticles may have a zeta potential at physiological pH of -50 to +50 mV, -40 to +40 mV, -30 to +30 mV, or -20 to +20 mV, more preferably -10 to +10 mV or -5 to +5 mV. It will be appreciated that the zeta potential can be measured by suspending the LNP in a conductive buffer solution having a predetermined pH. The conductive buffer solution may be PBS (phosphate buffered saline, pH 7.2).

[0097] The micro- or nanoparticles may further comprise one or more auxiliary agents, each of which may be selected from the group consisting of aluminum hydroxide, Pam2CSK4, Pam3CSK4, glucopyranosyl lipid auxiliary (GLA), LPS and its analogs, CpG oligodeoxynucleotides and other TLR-agonists, such as poly I:C and dsRNA.

[0098] The micro- or nanoparticles may further comprise one or more additional compounds. The one or more additional compounds may be selected from the group consisting of hydrophobic compounds, polymers, permeability enhancers, sugars, surface modifiers, excipients, and combinations thereof. The polymer may be polylactic-co-glycolic acid (PLGA). It may be understood that the excipient may alter the pharmacokinetic properties of the composition but not the pharmacodynamic properties of the payload.

[0099] According to a third aspect, there is provided a composition comprising a plurality of micro- or nanoparticles of the second aspect.

[0100] The micro- or nanoparticles may have an average diameter of less than 10 μm, less than 1 μm, 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 of 30 nm to 1 μm, 40 to 500 nm, 50 to 250 nm, 60 to 200 nm, 70 to 175 nm, 80 to 150 nm, or 90 to 125 nm. The average diameter of the micro- or nanoparticles can be measured using dynamic light scattering.

[0101] The micro- or nanoparticles may have a polydispersity index (PDI) of less than 0.5, less than 0.4, or less than 0.3, more preferably 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 0.001 to 0.05, 0.005 to 0.4, 0.01 to 0.3, 0.02 to 0.025, 0.03 to 0.2, 0.04 to 0.15, 0.05 to 0.1, or 0.06 to 0.08.

[0102] The composition may comprise a pharmaceutically acceptable carrier, which may improve colloidal stability, particularly under concentrated and / or refrigerated conditions (e.g., storage and / or transport at temperatures between 4°C and -80°C, e.g., 4°C, -20°C, or -70°C or -80°C). Such low temperature conditions can be used to extend the shelf life of the composition and / or, more specifically, the payload.

[0103] The composition may further comprise one or more solvents, buffers, suspending aids, fillers, flow agents, binders, salts, tonicity agents, thickeners, emulsifiers and / or preservatives.

[0104] In a fourth aspect, there is provided a micro- or nanoparticle of the second aspect, or a composition of the third aspect, for use as a medicament.

[0105] In a fifth aspect, there is provided a micro- or nanoparticle of the second aspect or a composition of the third aspect for use in the treatment and / or prevention and / or prophylaxis of a disease or disorder.

[0106] In a sixth aspect, there is provided a method for 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 nanoparticles of the second aspect, or the composition of the third aspect.

[0107] The disease or disorder may be selected from the group consisting of an inflammatory disease, an infectious disease, a proliferative disease (e.g., cancer), an autoimmune disease, an eye disease, a lung disease, a skin disease, an intestinal disease, a metabolic disease (e.g., diabetes), a vascular disease (including cardiovascular and renal vascular disease), a neurological disease (e.g., neurodegenerative disease), a disorder of the endocrine system (including disorders related to hormones, growth factors, and / or cytokines), a disorder of the reproductive system, and a rare disease.

[0108] In a seventh aspect, there is provided a vaccine composition comprising the micro- or nanoparticles of the second aspect or the composition of the third aspect.

[0109] The vaccine may contain suitable adjuvants.

[0110] In an eighth aspect, there is provided a micro- or nanoparticle of the second aspect, or a composition of the third aspect, or a vaccine of the seventh aspect, for use in stimulating an immune response in a subject.

[0111] The immune response may be stimulated against a protozoan, a bacterium, a virus, a fungus, a multicellular parasite, or a cancer, or a portion thereof.

[0112] 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 prophylactic and / or therapeutic amount of the micro- or nanoparticles of the second aspect, or the composition of the third aspect, or the vaccine of the seventh aspect.

[0113] The micro- or nanoparticles, compositions, or vaccines of the present invention may be combined into compositions having a number of different forms, depending in particular on the manner in which the compositions are to be used. Thus, for example, the compositions may be in the form of powders, tablets, capsules, liquids, ointments, creams, shampoos, gels, hydrogels, aerosols, sprays, micellar solutions, transdermal patches (including microneedles), drug depots / sustained-release formulations, (liposome) suspensions, irrigations / washes / instillations, those incorporated into biomaterials for tissue engineering, coatings on (implantable) medical devices, or any other suitable form that can be administered to humans or animals in need of treatment. It should be understood that the pharmaceutical vehicle of the present invention should be well tolerated by the subject to whom the vehicle is to be administered.

[0114] The micro- or nanoparticles, compositions or vaccines of the present invention may be incorporated into a sustained or delayed release device. Such a device may, for example, be inserted on or under the skin and may release the medication over a period of weeks or even months. The device may be placed at least adjacent to or upstream of the treatment site.

[0115] However, in a preferred embodiment, the medicament according to the invention may be administered to a subject by injection into the bloodstream, muscle, skin, or directly at the site requiring treatment. The injection may be 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).

[0116] It should be understood that the amount of micro- or nanoparticle, composition, or vaccine required will be determined by its intended therapeutic or prophylactic use and its biological activity and bioavailability, which in turn will depend on the mode of administration, payload, physiochemical properties of the micro- or nanoparticle, composition, or vaccine, and whether it is used as a monotherapy or in a combination therapy.

[0117] The frequency of administration is also affected by the half-life of the active agent and / or the half-life of the therapeutic effect in the treated subject (e.g., the half-life of a therapeutic protein translated from a therapeutic mRNA transfected by a micro- or nanoparticle composition, or the half-life of, for example, an RNA-based CRISPR gene therapy). Optimal dosages to be administered can be determined by one skilled in the art and will vary depending on the micro- or nanoparticles, composition or vaccine used, the potency of the pharmaceutical composition, the mode of administration, and the type of treatment. Dosages may need to be adjusted depending on additional factors that depend on the particular subject being treated, including the subject's age, weight, sex, diet, and time of administration.

[0118] The dosage required may depend on several factors, including but not limited to, the active agent being administered, the disease being treated and / or vaccinated against, the subject being treated, and the like.

[0119] Generally, doses of 0.001 μg / kg body weight to 10 mg / kg body weight, or 0.01 μg / kg body weight to 1 mg / kg body weight of the micro- or nanoparticles, compositions or vaccines of the present invention may be used, depending on the active agent used. Dose may be understood to relate to the amount of payload molecule delivered.

[0120] The dose may be given as a single administration (e.g., a single injection). Alternatively, the micro- or nanoparticles, composition, or vaccine may require more than one administration. By way of example, the micro- or nanoparticles, composition, or vaccine may be administered as two or more doses of 0.07 μg to 700 mg (i.e., assuming a body weight of 70 kg). Alternatively, a sustained release device may be used to provide the patient with an optimal dose of the micro- or nanoparticles, composition, or vaccine according to the invention, without the need to administer repeated doses. The route of administration may incorporate intravenous, intradermal, subcutaneous, intramuscular, intrathecal, epidural, intravitreal, or intraperitoneal routes of injection.

[0121] Known procedures, such as those conventionally used by the pharmaceutical industry (e.g., in vivo experimental methods, clinical trials, etc.), may be used to formulate the specific formulation of the micro- or nanoparticles, compositions or vaccines according to the invention, and the precise treatment regimen (e.g., drug dose and frequency of administration).

[0122] A "subject" may be a vertebrate, a mammal, or a livestock animal. Thus, the compositions and medicaments according to the present invention may be used to treat any mammal, such as livestock (e.g., horses), companion animals, and other veterinary and agricultural applications. Most preferably, however, the subject is a human.

[0123] A "therapeutically effective amount" of a micro- or nanoparticle, composition, or vaccine is any amount of the foregoing that is necessary to produce a therapeutic effect, whether partial or complete, when administered to a subject.

[0124] For example, a therapeutically effective amount of the micro- or nanoparticles, compositions or vaccines of the present invention may contain from about 0.001 μg to about 800 mg of payload molecules, preferably from about 0.01 mg to about 500 mg of payload molecules.

[0125] A "pharmaceutically acceptable vehicle," as referred to herein, is any known compound or combination of known compounds known to those skilled in the art to be useful in formulating pharmaceutical compositions.

[0126] In one embodiment, the pharmaceutically acceptable vehicle may be solid, and the composition may be in the form of a powder, capsule, or tablet. A solid pharmaceutically acceptable vehicle may contain one or more substances that may also act as flavoring agents, lubricants, solubilizers, suspending agents, dyes, fillers, flow agents, compression aids, inert binders, sweeteners, preservatives, pigments, coatings, or tablet disintegrants. The vehicle may also be an encapsulating material. In a powder, the vehicle is a finely divided solid in admixture with a finely divided active agent according to the present invention. In a tablet, the active agent (e.g., the micro- or nanoparticles of the present invention) may be mixed with a vehicle having the necessary compression properties in suitable proportions and compressed into the desired shape and size. The pharmaceutical vehicle may be a gel, and the composition may be in the form of a cream or similar.

[0127] Alternatively, the pharmaceutical vehicle may be liquid, and the pharmaceutical composition may be in the form of a solution. Liquid vehicles are used to prepare solutions, suspensions, emulsions, syrups, elixirs, and pressurized compositions. The micro- or nanoparticles according to the present invention may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle, such as water, an organic solvent, a mixture of both, or a pharmaceutically acceptable oil or fat. The liquid vehicle may contain other suitable pharmaceutical additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavorings, suspending agents, thickeners, dyes, viscosity regulators, stabilizers, or osmolality regulators. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing the above-mentioned additives, e.g., cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohols (including monohydric and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and peanut oil). For parenteral administration, the vehicle may 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 halogenated hydrocarbon or other pharmaceutically acceptable propellant.

[0128] Liquid pharmaceutical compositions that are sterile solutions or suspensions can be utilized by, for example, intramuscular, intrathecal, intravitreal, epidural, intraperitoneal, intravenous, and subcutaneous injection. The micro- or nanoparticles of the present invention can be prepared in any suitable sterile injectable medium.

[0129] The micro- or nanoparticles may be administered by inhalation, for example, the micro- or nanoparticles may be provided in the form of an aerosol.

[0130] The micro- or nanoparticles and / or compositions of the present invention may be orally administered in the form of a sterile solution or suspension containing other solutes or suspending agents (e.g., sufficient saline or glucose to make the solution isotonic), bile salts, gum arabic, gelatin, sorbitan monooleate, polysorbate 80 (oleic acid ester of sorbitol and its anhydride copolymerized with ethylene oxide), and the like. The micro- or nanoparticles and / or compositions of the present invention may be orally administered as either a liquid or solid composition form. Compositions suitable for oral administration include solid forms such as pills, capsules, granules, tablets, and powders, as well as liquid forms such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

[0131] According to a further aspect of the invention, a compound of formula (III)

[0132] [ka]

[0133] (In the formula, R 1 , R 3 and L 1 ~L 3 is as defined in relation to the first aspect, and both R 1 The base is the same) 1. A method for producing Maleic anhydride is reacted with a compound of formula (V) HO-R 1 (V) to form a compound of formula (VI)

[0134] [ka]

[0135] generating and a compound of formula (VI) with a compound of formula (VII) HS-L 1 -L2 -L 3 -R 3 (VII) thereby producing a compound of formula (III) A method is provided, comprising:

[0136] The molar ratio of maleic anhydride to the compound of formula (V) may be from 1:1 to 1:5 or from 1:1.5 to 1:3, and is preferably about 1:2.

[0137] 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 for the removal of water from the reaction under Dean-Stark conditions / by a Dean-Stark apparatus. The first organic solvent may have a boiling point of at least 70°C, at least 80°C, at least 90°C, at least 100°C, at least 110°C, at least 120°C, or at least 130°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 cycloalkane may be C 5~20 Alkane or C 5~12 The cycloalkane may be pentane, hexane, heptane, octane, nonane or cyclohexane. The nitroalkane may be C 1~3 It may be a nitroalkane or nitromethane. The ether is C 4~12 The halogenated hydrocarbon may be a chlorinated hydrocarbon. The halogenated hydrocarbon may be a C 1~3 The second solvent may be a halogenated hydrocarbon, preferably a halogenated methane. The halogenated hydrocarbon may be trichloromethane. In some embodiments, the second solvent is toluene.

[0138] The maleic anhydride and the compound of Formula (V) may be contacted in the presence of an acid. The acid may be any strong acid soluble in the first organic solvent. The acid may be p-toluenesulfonic acid (pTSA) or its hydrate, camphorsulfonic acid (CSA), methanesulfonic acid, or a sulfonic acid. The molar ratio of maleic anhydride to acid may be 1:5 to 100:1, 1:2 to 75:1, or 1:1 to 75:1. In some embodiments, the molar ratio of maleic anhydride to acid may be 5:1 to 60:1, 10:1 to 50:1, 20:1 to 45:1, or 30:1 to 40:1. In some embodiments, the molar ratio of maleic anhydride to acid may be from 1:5 to 50:1, from 1:2 to 30:1, from 1:1 to 20:1, from 3:1 to 15:1, from 5:1 to 10:1, or from 7:1 to 8:1.

[0139] The maleic anhydride and the compound of formula (V) may be contacted at an elevated temperature, which may be at least 30°C, at least 50°C, at least 70°C, at least 90°C, at least 110°C, or at least 130°C. The elevated temperature may be 30 to 500°C, 50 to 250°C, 70 to 200°C, 90 to 175°C, 110 to 150°C, or 120 to 140°C.

[0140] The molar ratio of the compound of formula (VI) to the compound of formula (VII) may be 2:1 to 1:10, 1:1 to 1:5, or 1:2 to 1:4, and is preferably about 1:3.

[0141] 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 C 1~3 The second organic solvent may be dichloromethane, dichloroethane or trichloromethane. The ether may be C 2~10 The ketone may be an ether, such as tetrahydrofuran (THF) or diethyl ether. 3~6It may be a ketone, which may be acetone. In some embodiments, the second organic solvent is dichloromethane.

[0142] The compound of formula (VI) and the compound of formula (VII) may be contacted in the presence 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 represented by the formula P(R 7 )3, wherein each R 7 are independent, C 1~10 The base is an alkyl or aryl group. Thus, the base may be triethylamine, diisopropylethylamine (DiPEA), DABCO, tributylphosphine, tripropylphosphine, triethylphosphine, trimethylphosphine, or tribenzenephosphine. The molar ratio of the compound of formula (VI) to the base may be 2:1 to 1:10, 1:1 to 1:5, or 1:2 to 1:4, and is preferably about 1:3.

[0143] The compound of formula (VI) and the compound of formula (VII) may be contacted at a second temperature, which may be 0 to 100°C, 5 to 50°C, 10 to 40°C, 15 to 30°C, or 18 to 25°C, and is preferably room temperature.

[0144] All of the features described in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be 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.

[0145] For a better understanding of the present invention, and to show how embodiments thereof may be carried into effect, reference will now be made, by way of example, to the accompanying drawings in which: FIG. [Brief explanation of the drawings]

[0146] [Figure 1] FIG. 1 shows the size distribution of LNP formulations measured by dynamic light scattering. [Figure 2] FIG. 2 shows the size distribution of LNP formulations with higher N / P ratios relative to the LNP formulation shown in FIG. 1, as measured by dynamic light scattering. [Figure 3] FIG. 1 shows the pKa of various ionized lipids determined by TNS-assay over the pH range 3-10 in 0.5 pH increments, where structures 1, 3, 5 and 6 represent L1, L3, L5 and L6, respectively. [Figure 4] [Figure 4A] Graphs showing toxicity and immunogenicity testing of L1-L6 lipids. (A) The effect of individual lipids L1-L6 (μg / well) on metabolic activity in HeLa cells was determined using a resazurin assay. 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, untreated. [Figure 4B] Graphs showing toxicity and immunogenicity testing of L1-L6 lipids. (B) The immunogenicity of individual lipids L1-L6 (μg / well) in endothelial cells was determined by measuring MCP-1 by 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, untreated. [Figure 5] 1 is a graph showing the in vitro activity of L1 (according to Formula (IIa))-containing LNPs. LNPs encapsulating nanoluciferase mRNA were generated with the ionizable lipid L1 (Formula (IIa)) or the commercially available, widely accepted lipid MC3 and added to HeLa cells. (A) Transfection efficiency was determined by measuring luciferase activity. (B) The effect on metabolic activity was assessed by a resazurin assay for toxicity. [Figure 6]

[0023] Figure 1 shows that L1-containing LNPs are immunosuppressive. Cells were transfected with L1 LNP formulations encapsulating either immunosuppressive or immunostimulatory mRNA. The effect of lipids on immunogenicity was tested 24 hours later by ELISA of the pro-inflammatory chemokine MCP-1. [Figure 7] Graphs showing the in vivo activity of L1-containing LNPs. Mice (n=5 per group) were intravenously (iv) or intraperitoneally (ip) injected with various LNP formulations encapsulating EPO mRNA (1 μg) and sacrificed 6 hours later. (A) mRNA-derived EPO protein expression in plasma was assessed by ELISA. (B) Toxicity of LNP formulations was assessed by body weight change. (C) Immunogenicity was determined by ELISA for the pro-inflammatory cytokine IL-6 in plasma. DETAILED DESCRIPTION OF THE INVENTION [Example]

[0147] (General Example) General Procedure 1

[0148] [ka]

[0149] As shown in Scheme 1 above, maleic anhydride 1a reacts with alcohol 1b (preferably a fatty acid alcohol, such as cis,cis-9,12-octadecadien-1-ol) to produce compound 1c (e.g., dioctadecadienyl maleate). Step 1 may be carried out in an organic solvent (e.g., toluene) in the presence of, for example, p-toluenesulfonic acid (pTSA) at elevated temperature (e.g., 130°C) under reflux for 18 hours. Next, maleate-di-acyl ester 1c reacts with thiol 1d (e.g., 2-(dimethylamino)ethanethiol hydrochloride) to produce compound 1e (e.g., di((9Z,12Z)-octadeca-9,12-dien-1-yl)maleate). Step 2 may be carried out in an organic solvent (e.g., dichloromethane) in the presence of triethylamine at room temperature (e.g., 25°C) for 25 hours with stirring.

[0150] As shown in the specific examples below, in some embodiments, the compound of Formula 1b is cis,cis-9,12-octadecadien-1-ol. Thus, the compound of Formula 1c can be di((9Z,12Z)-octadeca-9,12-dien-1-yl)maleate.

[0151] Instead, in some alternative embodiments, as shown in the specific examples below, the compound of Formula 1b is cis-9-octadecen-1-ol. Thus, the compound of Formula 1c can be dioleyl maleate.

[0152] In some embodiments, the compound of Formula 1d is 2-(dimethylamino)ethanethiol hydrochloride, (dimethylamino)methanethiol hydrochloride, 3-(dimethylamino)-1-propanethiol hydrochloride, 2-(methylethylamino)ethanethiol hydrochloride, or (diethylamino)ethanethiol hydrochloride.

[0153] Thus, compounds of formula 1e include di((9Z,12Z)-octadeca-9,12-dien-1-yl)2-((2-(dimethylamino)ethyl)thio)succinate maleate, di((9Z,12Z)-octadeca-9,12-dien-1-yl)2-((2-(dimethylamino)methyl)thio)succinate maleate, di((9Z,12Z)-octadeca-9,12-dien-1-yl)2-((2-(dimethylamino)propyl)thio)succinate maleate, di((9Z,12Z)-octadeca-9,12-dien-1-yl)2-((2-(methylethylamino)ethyl)thio)succinate maleate, di((9Z,12Z)-octadeca-9,12-dien-1-yl)2-((2-(diethylamino)ethyl)thio)succinate maleate, di((9Z,12Z)-octadeca-9,12-dien-1-yl)2-((2-(diethylamino)ethyl)thio)succinate maleate or dioleyl 2-((2-(diethylamino)ethyl)thio)succinate maleate.

[0154] Specific Examples Example 1 Synthesis of di((9Z,12Z)-octadeca-9,12-dien-1-yl)2-((2-(dimethylamino)ethyl)thio)succinate (L1), MW: 718.17

[0155] [ka]

[0156] Step 1. Synthesis of di((9Z,12Z)-octadeca-9,12-dien-1-yl)maleate A solution of maleic anhydride (206 mg, 2.1 mmol, 1 equiv.) and cis,cis-9,12-octadecadien-1-ol (1.3 mL, 1.2 g, 4.2 mmol, 2.0 equiv.) was heated in toluene until complete dissolution. p-Toluenesulfonic acid (pTSA; 50 mg) was added, and the solution was heated under reflux at 130 °C for 18 h. The reaction mixture was then concentrated on a rotary evaporator. The residue was purified by silica column chromatography (petroleum ether, ethyl acetate 49:1) to give dioctadecadienyl maleate as a yellow oil (1.13 g, 1.85 mmol, 88%). HPLC-MS(ESI) C 40 H 68 Calculated for O4 [M+H]+: 613.51; Found: 613.7. TLC (petroleum ether, ethyl acetate 49:1), R f =0.11

[0157] Step 2. Synthesis of di((9Z,12Z)-octadeca-9,12-dien-1-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 μL, 5.55 mmol, 3.0 equiv.) were added, and the reaction mixture was stirred for 25 h at room temperature. The solution was then concentrated on a rotary evaporator. Aliquots of the crude residue (approximately 120 mg each) were purified by flash chromatography (C4-phase, water, 0.1% formic acid / acetonitrile, 46 min gradient) to give di((9Z,12Z)-octadeca-9,12-dien-1-yl)2-((2-(dimethylamino)ethyl)thio)succinate (total 110.0 mg, 0.014 mmol, 8%) as a colorless oil. HPLC-MS(ESI) calculated for C44H79NO4S[M+H]+: 718.57; found: 718.8.

[0158] Example 2 Synthesis of di((9Z,12Z)-octadeca-9,12-dien-1-yl)2-((2-(dimethylamino)methyl)thio)succinate maleate (L2), MW: 704.14

[0159] [ka]

[0160] Step 1. Synthesis of di((9Z,12Z)-octadeca-9,12-dien-1-yl)maleate A solution of maleic anhydride (206 mg, 2.1 mmol, 1 equiv.) and cis,cis-9,12-octadecadien-1-ol (1.3 mL, 1.2 g, 4.2 mmol, 2.0 equiv.) in toluene was heated until complete dissolution. p-Toluenesulfonic acid (pTSA; 50 mg) was added, and the solution was heated under reflux at 130 °C for 18 h. The reaction mixture was then concentrated on a rotary evaporator. The residue was purified by silica column chromatography (petroleum ether, ethyl acetate 49:1) to give dioctadecadienyl maleate (4) as a yellow oil (1.13 g, 1.85 mmol, 88%). HPLC-MS (ESI) calculated for C40H68O4 [M+H]+: 613.51; found: 613.7. TLC (petroleum ether, ethyl acetate 49:1), R f =0.11

[0161] Step 2. Synthesis of di((9Z,12Z)-octadeca-9,12-dien-1-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 μL, 5.55 mmol, 3.0 equiv.) were added, and the reaction mixture was stirred at room temperature for 25 h. The solution was then concentrated on a rotary evaporator. Aliquots of the crude residue (approximately 120 mg each) were purified by flash chromatography (C4-phase, water, 0.1% formic acid / acetonitrile, 46 min gradient) to give di((9Z,12Z)-octadeca-9,12-dien-1-yl)2-((2-(dimethylamino)methyl)thio)succinate maleate (110.0 mg, 0.014 mmol, 8%) as a colorless oil. HPLC-MS(ESI) calculated for C43H77NO4S[M+H]+: 704.142; found: 718.8.

[0162] Example 3 Synthesis of di((9Z,12Z)-octadeca-9,12-dien-1-yl)2-((2-(dimethylamino)propyl)thio)succinate maleate (L3), MW: 732.2

[0163] [ka]

[0164] Step 1. Synthesis of di((9Z,12Z)-octadeca-9,12-dien-1-yl)maleate A solution of maleic anhydride (206 mg, 2.1 mmol, 1 equiv.) and cis,cis-9,12-octadecadien-1-ol (1.3 mL, 1.2 g, 4.2 mmol, 2.0 equiv.) was heated in toluene until complete dissolution. p-Toluenesulfonic acid (pTSA; 50 mg) was added, and the solution was heated under reflux at 130 °C for 18 h. The reaction mixture was then concentrated on a rotary evaporator. The residue was purified by silica column chromatography (petroleum ether, ethyl acetate 49:1) to give dioctadecadienyl maleate as a yellow oil (1.13 g, 1.85 mmol, 88%). HPLC-MS(ESI) calculated for C40H68O4[M+H]+: 613.51; found: 613.7. TLC (petroleum ether, ethyl acetate 49:1), R f =0.11

[0165] Step 2. Synthesis of di((9Z,12Z)-octadeca-9,12-dien-1-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-1-thiol hydrochloride (864 mg, 5.55 mmol, 3.0 equiv.) and triethylamine (773 μL, 5.55 mmol, 3.0 equiv.) were added, and the reaction mixture was stirred for 25 h at room temperature. The solution was then concentrated on a rotary evaporator. Aliquots of the crude residue (approximately 120 mg each) were purified by flash chromatography (C4-phase, water, 0.1% formic acid / acetonitrile, 46 min gradient) to give di((9Z,12Z)-octadeca-9,12-dien-1-yl)2-((2-(dimethylamino)propyl)thio)succinate maleate (110.0 mg, 0.014 mmol, 8%) as a colorless oil. HPLC-MS(ESI) calculated for C43H77NO4S[M+H]+: 731.588; found: 718.8.

[0166] Example 4 Synthesis of di((9Z,12Z)-octadeca-9,12-dien-1-yl)2-((2-(methylethylamino)ethyl)thio)succinate maleate (L5), MW: 732.2

[0167] [ka]

[0168] Step 1. Synthesis of di((9Z,12Z)-octadeca-9,12-dien-1-yl)maleate A solution of maleic anhydride (206 mg, 2.1 mmol, 1 equiv.) and cis,cis-9,12-octadecadien-1-ol (1.3 mL, 1.2 g, 4.2 mmol, 2.0 equiv.) was heated in toluene until complete dissolution. p-Toluenesulfonic acid (pTSA; 50 mg) was added, and the solution was heated under reflux at 130 °C for 18 h. The reaction mixture was then concentrated on a rotary evaporator. The residue was purified by silica column chromatography (petroleum ether, ethyl acetate 49:1) to give dioctadecadienyl maleate as a yellow oil (1.13 g, 1.85 mmol, 88%). HPLC-MS (ESI) calculated for C40H68O4 [M+H]+: 613.51; found: 613.7. TLC (petroleum ether, ethyl acetate 49:1), R f =0.11

[0169] Step 2. Synthesis of di((9Z,12Z)-octadeca-9,12-dien-1-yl)2-((2-(methylethylamino)ethyl)thio)succinate maleate Dioctadecadienyl 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 μL, 5.55 mmol, 3.0 equiv.) were added, and the reaction mixture was stirred for 25 h at room temperature. The solution was then concentrated on a rotary evaporator. Aliquots of the crude residue (approximately 120 mg each) were purified by flash chromatography (C4-phase, water, 0.1% formic acid / acetonitrile, 46 min gradient) to give di((9Z,12Z)-octadeca-9,12-dien-1-yl)2-((2-(methylethylamino)ethyl)thio)succinate (110.0 mg, 0.014 mmol, 8%) as a colorless oil. HPLC-MS(ESI) calculated for C43H77NO4S[M+H]+: 731.588; found: 718.8.

[0170] Example 5 Synthesis of di((9Z,12Z)-octadeca-9,12-dien-1-yl)2-((2-(diethylamino)ethyl)thio)succinate maleate (L6), MW: 746.22

[0171] [ka]

[0172] Step 1. Synthesis of di((9Z,12Z)-octadeca-9,12-dien-1-yl)maleate A solution of maleic anhydride (206 mg, 2.1 mmol, 1 equiv.) and cis,cis-9,12-octadecadien-1-ol (1.3 mL, 1.2 g, 4.2 mmol, 2.0 equiv.) was heated in toluene until complete dissolution. p-Toluenesulfonic acid (pTSA; 50 mg) was added, and the solution was heated under reflux at 130 °C for 18 h. The reaction mixture was then concentrated on a rotary evaporator. The residue was purified by silica column chromatography (petroleum ether, ethyl acetate 49:1) to give dioctadecadienyl maleate as a yellow oil (1.13 g, 1.85 mmol, 88%). HPLC-MS (ESI) calculated for C40H68O4 [M+H]+: 613.51; found: 613.7. TLC (petroleum ether, ethyl acetate 49:1), Rf=0.11

[0173] Step 2. Synthesis of di((9Z,12Z)-octadeca-9,12-dien-1-yl)2-((2-(diethylamino)ethyl)thio)succinate maleate Dioctadecadienyl maleate (1.13 g, 1.85 mmol, 1 equiv.) was dissolved in dichloromethane (30 mL), 2-diethylaminoethanethiol hydrochloride (941.9 mg, 5.55 mmol, 3.0 equiv.) and triethylamine (773 μL, 5.55 mmol, 3.0 equiv.) were added, and the reaction mixture was stirred for 25 h at room temperature. The solution was then concentrated on a rotary evaporator. Aliquots of the crude residue (approximately 120 mg each) were purified by flash chromatography (C4-phase, water, 0.1% formic acid / acetonitrile, 46 min gradient) to give di((9Z,12Z)-octadeca-9,12-dien-1-yl)2-((2-(diethylamino)ethyl)thio)succinate maleate (110.0 mg, 0.014 mmol, 8%) as a colorless oil. HPLC-MS(ESI) calculated for C43H77NO4S[M+H]+: 746.222; found: 718.8.

[0174] Example 6 Synthesis of dioleyl 2-((2-(diethylamino)ethyl)thio)succinate maleate (L4), MW: 722.20

[0175] [ka]

[0176] Step 1. Synthesis of dioleyl maleate A solution of maleic anhydride (206 mg, 2.1 mmol, 1 equiv.) and cis-9-octadecen-1-ol (1.3 mL, 1.13 g, 4.2 mmol, 2.0 equiv.) was heated in toluene until complete dissolution. p-Toluenesulfonic acid (pTSA; 50 mg) was added, and the solution was heated under reflux at 130 °C for 18 h. The reaction mixture was then concentrated on a rotary evaporator. The residue was purified by silica column chromatography (petroleum ether, ethyl acetate 49:1) to give dioleyl maleate as a yellow oil (1.17 g, 1.9 mmol, 90%). HPLC-MS(ESI) calculated for C40H68O4[M+H]+: 616.99; found: 613.7. TLC (petroleum ether, ethyl acetate 49:1), Rf=0.11

[0177] Step 2. Synthesis of dioleyl 2-((2-(diethylamino)ethyl)thio)succinate Dioleyl maleate (1.14 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 μL, 5.55 mmol, 3.0 equiv.) were added, and the reaction mixture was stirred for 25 h at room temperature. The solution was then concentrated on a rotary evaporator. Aliquots of the crude residue (approximately 120 mg each) were purified by flash chromatography (C4-phase, water, 0.1% formic acid / acetonitrile, 46 min gradient) to give dioleyl 2-((2-(diethylamino)ethyl)thio)succinate (total 110.0 mg, 0.014 mmol, 8%) as a colorless oil. HPLC-MS(ESI) calculated for C44H79NO4S[M+H]+: 722.2; found: 718.8.

[0178] Example 7 Generation of lipid nanoparticles (LNPs) To determine the safety and efficacy of the lipids of the present invention for the delivery of therapeutic and / or prophylactic molecules to cells, various formulations were prepared and tested. More precisely, the lipid composition of the LNPs and the ratio between said lipids were varied.

[0179] Uniformly sized nanoparticles were reproducibly produced using an inverted herringbone microfluidic mixer, which rapidly (on the order of milliseconds) mixes an aqueous fluid, typically containing water-soluble therapeutic and / or prophylactic molecules, with an organic solvent containing lipid components. Other types of microfluidic mixers (e.g., Y-junction, T-junction, or direct high-speed injection) produced similar results, as long as similar mixing ratios and mixing rates between the fluid streams were achieved.

[0180] Lipid compositions were prepared by combining an ionizable lipid of the invention (e.g., one of the lipids in Examples 1-6) with a phospholipid (e.g., DOPE, available from Avanti Polar Lipids), optionally a cationic lipid (e.g., DOTAP, available from Avanti Polar Lipids), a structured lipid (e.g., cholesterol, available from Sigma-Aldrich), and a PEG-modified lipid (e.g., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (polyethylene glycol)-2000 (also known as PEG-DSPE), available from Avanti Polar Lipids). For experiments requiring cellular uptake, the lipids were typically combined in the following ratio: 45 mol % ionizable lipid of the invention, 5 mol % DOTAP, 38.5 mol % structured lipid, 10 mol % phospholipid, and 1.5 mol % PEG-modified lipid. For experiments not requiring cellular uptake, lipids were typically combined in the following ratio: 50 mol % ionizable lipid of the present invention, 38.5 mol % structured lipid, 10 mol % phospholipid (DOPE), and 1.5 mol % PEG-modified lipid.

[0181] Certain formulations exhibit low overall cellular uptake in vitro and / or in vivo, which can be overcome by incorporating 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 +1 mV, preferably +10 mV, results in overall interaction with the cell membrane and uptake of the nanoparticles and / or cargo. In such cases, the following lipid ratios were used: 40-50 mol% ionizable lipid of the present invention, 0-10 mol% cationic lipid, 38.5 mol% structured lipid, 10 mol% phospholipid, and 1.5 mol% PEG-modified lipid.

[0182] The lipid mixture was diluted with ethanol to a final concentration of 12.5 mM to 50 mM total lipid. The lipids dissolved in ethanol were stored at -20°C under argon and protected from light.

[0183] Nanoparticle compositions were prepared by combining therapeutic and / or preventive molecules in acidic (pH 4 or 5) or neutral (pH 7.4) aqueous solutions with lipid mixtures in ethanol at lipid-to-therapeutic molecule mass ratios ranging from 5:1 to 50:1. To generate distinct nanoparticle populations, the aqueous solutions containing the therapeutic and / or preventive molecules ("aqueous solutions") and the lipid solutions were rapidly mixed in volume ratios ranging from about 2:1 (aqueous solution:lipid solution) to about 5:1 (aqueous solution:lipid solution) in an inverted herringbone microfluidic mixer at a total flow rate of 10 ml / min to 18 ml / min.

[0184] To prepare nanoparticle compositions containing RNA, the RNA was diluted to approximately 0.1 mg / ml to 3 mg / ml, preferably 0.15 mg / ml, in 100 mM sodium citrate buffer at pH 4 to 5, and then mixed with the lipid mixture. The RNA to lipid mass ratio was typically set at 1:10 to 1:30, resulting in an N:P ratio of 3 to 12, preferably 5.

[0185] After mixing the aqueous and lipid solutions, the nanoparticle composition was dialyzed to remove ethanol to less than 0.1%, optionally concentrating or diluting the solution and buffer exchanging it with a physiological pH buffer (e.g., pH 7.4, e.g., phosphate-buffered saline (PBS)). The formulation was dialyzed three times against at least a 100-fold excess of PBS using 100 kDa MWCO dialysis tubing (e.g., Spectrum™ Spectra / Por™ Biotech cellulose ester (CE) dialysis membrane tubing, available from Fisher Scientific). The first dialysis step was performed at room temperature for at least 2 hours, and subsequent dialysis steps were performed at room temperature for at least 8 hours or overnight at 4°C.

[0186] The size distribution of 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 various mRNAs (secNLuc (containing approximately 1000 nt of PolyA), eGFP, and FLuc), and their sizes were measured using DLS in 1x PBS (10 mM phosphate buffer, 150 mM NaCl) pH 7.4. Multiple LNP formulations were generated (50 mol% ionizable lipids (L1, L3, L4, L5, L6), 38.5 mol% structural lipid (cholesterol), 10 mol% phospholipid (DOPE), and 1.5 mol% DSPE-PEG(2000) at an N / P of approximately 16, where the ionizable lipids were compounds from Examples 1-6). As shown in Figure 1, these formulations clearly exhibited an average size of 120 nm (95-115 nm) with an average polydispersity index (PDI) of 0.07.

[0187] Next, we determined the effect of N / P ratio on LNP size using a larger N / P ratio (7:1 instead of 5:1, equivalent to a lipid-to-RNA mass ratio of 22.5, L1 50 mol%, DOPE 10 mol%, cholesterol 38.5 mol%, DSPE-PEG 1.5 mol%, secNLuc mRNA (containing a 1000 nt PolyA tail)) (i.e., a higher ratio of ionizable lipid to mRNA). Interestingly, as shown in Figure 2, a larger N / P ratio resulted in smaller nanoparticles, as did increasing the PEG-DSPE to total lipid ratio.

[0188] Example 8 Lipid pKa determination The pKa of ionized lipids is known to be a major factor in the endosomal escape of LNP contents upon exposure to cells. During endosomal uptake and transit through the endosomal compartment, LNPs experience a gradual decrease in pH from physiological pH (pH 7.4, similar to that present outside the cell) to approximately pH 4.5-5.0 in lysosomes, the final stage of most endocytic vesicles. Transport to lysosomes should generally be avoided because the degradative environment can lead to degradation of cargo (e.g., mRNA); therefore, endosomal escape ideally occurs at a pH above 5.0, before the endosome matures into a lysosome. The rapid increase in cationic charge of ionized lipids during this acidification step is thought to drive their interaction with the inner endosomal membrane. Therefore, a lipid that is neutral or near-neutral at pH 7.4 and fully ionized at pH 5.5 would be ideal; this corresponds to a pKa of approximately 6.4–6.5, similar to what was found to be optimal by Jayaraman et al. (2012 Angewandte Chemie, DOI: 10.1002 / ang.201203263).

[0189] The local environment of ionized lipids (relative to their incorporation in LNPs) can affect charge acquisition and thus pKa. Therefore, experimentally determined pKa values ​​of ionized lipids were measured by adding 2-(p-toluidino)naphthalene-6-sulfonic acid (TNS; obtained from Sigma-Aldrich) to representative nanoparticle compositions in the presence of 20 mM phosphate-citric acid-ammonium citrate (pH 3–10 in 0.5 increments, all obtained from Sigma-Aldrich). TNS is a compound that interacts electrostatically with cationic lipids, resulting in fluorescence. Briefly, 50 μl of a mixture of 5 μM TNS, 25 μM LNPs (containing 12.5 μM ionized lipid), and 20 mM buffer solution was added to a 384-well plate, followed by a phosphate-citric acid-ammonium citrate buffer sample, and measurements were taken at 325 nm excitation and 435 nm emission in a plate reader (iD3, Molecular Devices). A sample containing only LNP and buffer was used for background subtraction for each pH increment. The background-corrected measured fluorescence was then normalized to the difference between the maximum and minimum fluorescence obtained during the assay, and curve fitting was performed to obtain a sigmoidal curve for each ionized lipid. The pKa of each ionized 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 L1, L3, L5, and L6 (identified as structures 1, 3, 5, and 6, respectively).

[0190] It can be seen that the lipid of Example 3 was found to have a pKa of 6.35, close to the theoretical, previously determined optimum of pH 6.4. It can be seen that the lipids of Examples 5 and 6 exhibit less than 1% ionization at physiological pH, potentially making them well suited for targeted LNPs where uptake or opsonization by the RES system would be detrimental.

[0191] Example 9 Cellular toxicity and immunogenicity of lipids To measure the toxic or immunostimulatory effects of individual lipids, we added lipids in the form of highly purified micelles at various concentrations to cells. High-purity micelles were chosen to eliminate the potential toxicity and / or immunogenicity of other lipid components that may be present in the LNPs. Additionally, the inclusion of PEG-modified lipids may reduce the chances of lipids interacting with specific receptors or cell surfaces.

[0192] Briefly, unformulated (i.e., not in the form of LNPs, but as high-purity micelles) lipids from Examples 1 to 6 were added to HeLa or endothelial cells at concentrations ranging from 10 to 0.001 μg and premixed with cell culture medium in a total volume of 100 μl per well of a 96-well plate. After 24 h of incubation, metabolic activity was tested by resazurin assay. For this purpose, the culture medium was replaced with medium containing 0.1 mg / ml resazurin and incubated for 1 to 4 h at 37°C and 5% CO2. Subsequently, fluorescence was determined in the supernatant (excitation 540 / 25 nm, emission 620 / 40 nm). Prior to medium change for the resazurin assay, the medium was collected, and MCP-1 levels, as a measure of immune response induction, were tested by ELISA (R&D Systems) according to the manufacturer's protocol.

[0193] Increasing concentrations of lipids did not show significant toxic effects on endothelial cells (Figure 4A). In addition, we failed to detect induction of the endothelial cell-derived pro-inflammatory cytokine MCP-1 (Figure 4B). Results were comparable to those obtained with widely used lipids such as DODAP and DOTAP. Toll-like receptor (TLR)-2 or -4 agonists were used as positive controls and showed significant induction of MCP-1 in these cells.

[0194] Other studies in the literature have shown that some lipids can induce toxicity, which has been linked to the activation of immune responses through binding to pattern recognition receptors, such as Toll-like receptors 2 and / or 4. TLR2 and 4 recognize lipid compounds. Our results show that L1-L6 lipids are neither toxic nor immunogenic in endothelial cells by themselves.

[0195] Example 10 Activity of L1-LNP in vitro To determine whether L1-containing LNP formulations could effectively deliver mRNA to cells, we incubated cells with luciferase mRNA-containing L1-LNPs.

[0196] L1-containing LNPs were formulated with 2.5 mol% DOTAP (47.5 mol% L1, 2.5 mol% DOTAP, 10 mol% DOPE, 38.5 mol% cholesterol, 1.5 mol% DSPE-PEG(2000)) as described in Example 7 and contained secreted nanoluciferase mRNA at an N / P ratio of 5:1 (corresponding to a LOW of approximately 16). A dose range of LNPs equivalent to 100, 50, and 10 ng per well containing 100 μl of LNP was added to HeLa cells in a 96-well plate. After 24 hours, the medium was collected, and secreted nanoluciferase activity was determined using the Nano-Glo Luciferase Assay System (Promega). To investigate undesired immunostimulatory effects, MCP-1 levels were tested by ELISA according to the manufacturer's protocol (R&D Systems). To assess potential toxicity, metabolic activity was tested by resazurin assay. For this purpose, the culture medium was replaced with medium containing 0.1 mg / ml resazurin and incubated for 1 to 4 hours at 37°C and 5% CO. Subsequently, fluorescence was determined in the supernatant (excitation 540 / 25 nm, emission 620 / 40 nm).

[0197] Incubation of HeLa cells with L1-containing LNPs showed a dose-dependent induction of luciferase activity similar to that of formulations containing the widely used ionizable lipid MC3 (Figure 5A). The effect on metabolic activity, as tested by the resazurin assay, was modest and similar for both formulations (Figure 5B).

[0198] L1 has comparable efficacy to ionized lipids as the widely used DLin-MC3-DMA lipid, and also exhibits a similar lack of toxicity to cells.

[0199] Example 11 Non-immunogenicity of L1-LNP This example demonstrates that L1-containing LNP formulations are non-immunogenic in endothelial cells.

[0200] L1-containing LNPs were formulated with 2.5 mol% DOTAP (47.5 mol% L1, 2.5 mol% DOTAP, 10 mol% DOPE, 38.5 mol% cholesterol, 1.5 mol% DSPE-PEG(2000)) as described in Example 7 and contained either immunosuppressive mRNA (RiboPro) or immunostimulatory mRNA (RiboPro) at an N / P ratio of 5:1 (equivalent to a LOW of approximately 16). A dose range of LNPs equivalent to 100, 50, and 10 ng per well containing a total volume of 100 μl of medium was added to endothelial cells in a 96-well plate. After 24 hours, medium was collected and MCP-1 levels were tested by ELISA according to the manufacturer's protocol (R&D Systems).

[0201] Incubation of endothelial cells with L1-containing LNPs containing immunosuppressive mRNA did not result in the production of the pro-inflammatory cytokine MCP-1 (Figure 6). In contrast, L1-containing LNPs containing mRNA with immunostimulatory properties induced high levels of MCP-1 in these cells.

[0202] In conclusion, LNPs containing ionized lipid L1 are not immunogenic per se, as LNPs carrying immunosuppressive mRNA do not show induction of the endothelial cell-derived pro-inflammatory cytokine MCP-1.

[0203] Example 12 In vivo activity of L1-LNP To determine the efficacy of in vivo delivery of mRNA using LNP formulations containing L1 lipids and to determine potential toxic or immunogenic effects, we injected L1-LNPs into mice via the intravenous and intraperitoneal routes.

[0204] L1-containing LNPs were formulated without or with DOTAP1, 2.5, or 5% (49 / 47.5 or 45 mol% L1, 2.5 or 5 mol% DOTAP1, 10 mol% DOPE, 38.5 mol% cholesterol, 1.5 mol% DSPE-PEG(2000)) as described in Example 7, and contained mEPO mRNA at an N / P ratio of 5:1 (corresponding to a LOW of approximately 16). Eight- to ten-week-old C57Bl / 6 mice received a single intravenous injection of 50 μl of L1-LNP or a single intraperitoneal injection of 100 μl of L1-LNP via the tail vein, both of which contained 1 μg of mEpo mRNA per dose. As a control, 1 μg of mEpo mRNA was formulated with TransIT (Mirus Bio) according to the manufacturer's protocol and injected within 30 minutes as previously described. After 6 hours, mice were weighed and blood was collected into EDTA tubes (BD Microtainer™ tubes with Microgard™ closures) and further processed within 1 hour by centrifugation at 500 g for 5 minutes. Plasma samples were tested for mEpo levels (R&D Systems) or IL-6 cytokine levels (R&D Systems) by ELISA according to the manufacturer's protocol.

[0205] As shown in Figure 7, injection of LNPs without DOTAP or with 2.5% or 5% DOTAP resulted in comparable mEpo activity in plasma, whereas 1% DOTAP-containing LNPs clearly demonstrated slightly greater activity. Both intravenous and intraperitoneal injection of L1-containing LNPs in mice resulted in high plasma levels of mEpo protein. Note that the endogenous mEpo background level in untreated mice was less than 500 pg / ml. The mEpo levels induced by LNPs were equal to or greater than those induced by TransIT, a transfection reagent suitable for in vivo use due to its low toxicity. Injection resulted in minimal weight change (less than 5%) in mice (Figure 7B), which tended to be smaller with LNPs compared to TransIT. Additionally, we were unable to detect significant levels of the pro-inflammatory cytokine IL-6 in the circulation (Figure 7C).

[0206] In conclusion, L1-containing LNPs can 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 injections lead to effective delivery. In addition, L1 lipids are active in LNPs without a charged surface, potentially enabling ApoE-mediated delivery and the use of positively charged lipids. Importantly, L1-LNPs show no short-term effects on mouse body weight or induction of the pro-inflammatory cytokine IL-6.

[0207] Example 13 LNP encapsulation efficiency High encapsulation efficiency of oligo- and polynucleotide cargoes is important to prevent exposure of the cargo to degradative enzymes, immune stimulatory cells, and receptors, and to achieve high transfection efficiency. Therefore, encapsulation efficiency was determined by RiboGreen assay.

[0208] Secreted NanoLuc(m)RNA-containing nanoparticle compositions (N / P 5:1, LOW approximately 16) were mixed 1:1 with QUANT-IT RIBOGREEN RNA Assay (Invitrogen, Thermo Fisher Scientific) at a concentration of approximately 5 μg / ml in TE buffer (10 mM Tris, HCl pH 7.5, 1 mM EDTA, Sigma-Aldrich) or with 2% Triton X-100 (Sigma-Aldrich) in TE buffer and an equal volume of 1:100 RiboGreen reagent. The samples were mixed thoroughly and incubated for 5 minutes at room temperature. Fluorescence intensity was then measured at 480 nm excitation and 520 nm emission on a plate reader (iD3, Molecular Devices). Blanks containing 1:200 RiboGreen reagent in TE buffer or 1:200 RiboGreen reagent in 2% Triton X-100 buffer were used as fluorescence background controls. A naked (m)RNA standard curve was used to quantify the absolute amount of unencapsulated RNA. The signal obtained with 2% Titron was used to normalize all samples to it and set it at 100%. The results are shown in Table 1.

[0209] [Table 1]

[0210] The lipids of the present invention all exhibit high encapsulation efficiency and are therefore suitable for protecting oligo- and polynucleotides from degradative enzymes and preventing any other effects of exposed oligo- and polynucleotides.

Claims

1. Compounds of formula (I) 【Chemical 1】 (In the formula, R 1 and R 2 is an optionally substituted C 1～50 Alkyl, optionally substituted C 2～50 alkenyl or optionally substituted C 2～50 alkynyl; L 1 and L 3 are independently absent or optionally substituted C 1～10 Alkylene, optionally substituted C 2～10 Alkenylene or optionally substituted C 2～10 is alkynylene, L 2 is absent or is NH, S or O, R 3 is -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 R 5 , -NR 4 SO 2 R 5 , -SO 2 NR 4 R 5 , -NR 4 COR 5 , -OP(O)(OH)OR 4 , optionally substituted C 3～6 Cycloalkyl, optionally substituted C 3～6 Cycloalkenyl, optionally substituted C 6～12 aryl, an optionally substituted 3- to 10-membered heterocyclic ring, or an optionally substituted 5- to 10-membered heteroaryl; R 4 ~R 6 is H, optionally substituted C 1～30 Alkyl, optionally substituted C 2～30 Alkenyl, optionally substituted C 2～30 Alkynyl, optionally substituted C 3～6 Cycloalkyl, optionally substituted C 3～6 Cycloalkenyl, optionally substituted C 6～12 aryl, optionally substituted 3- to 10-membered heterocyclic ring, or optionally substituted 5- to 10-membered heteroaryl; or a pharmaceutically acceptable complex, salt, solvate, tautomer or polymorph thereof.

2. L 1 optionally replaced by C 1～6 Alkylene, optionally substituted C 2～6 Alkenylene or optionally substituted C 2～6 The compound of claim 1 which is an alkynylene.

3. L 1 But C 1～3 Alkylene, C 2～3 Alkenylene or C 2～3 Alkynylene, preferably L 1 But -CH 2 -, -CH 2 CH 2 -or-CH 2 CH 2 CH 2 The compound of claim 2, wherein:

4. L 2 is non-existent and L 3 4. The compound of claim 1, wherein:

5. R 3 But, -NR 4 R 5 , -SR 4 , -OR 4 5. The compound according to claim 1, wherein the compound is an optionally substituted 3- to 10-membered heterocyclic ring or an optionally substituted 5- to 10-membered heteroaryl.

6. R 3 Ga-NR 4 R 5 6. The compound of claim 5, wherein:

7. R 4 ~R 6 is H, optionally substituted C 1～20 Alkyl, optionally substituted C 2～20 Alkenyl and optionally substituted C 2～20 7. The compound of any one of claims 1 to 6, wherein each of said compounds is independently selected from the group consisting of: alkynyl.

8. R 4 ~R 6 But C 1～5 Alkyl, C 2～5 Alkenyl and C 2～5 8. The compound of claim 7, wherein each of the alkynyl groups is independently selected from the group consisting of methyl, ethyl, or propyl.

9. R 1 and R 2 optionally replaced by C 5～30 Alkyl, optionally substituted C 5～30 alkenyl or optionally substituted C 5～30 alkynyl, preferably R 1 and R 2 optionally replaced by C 15～20 Alkyl, optionally substituted C 15～20 alkenyl or optionally substituted C 15～20 9. The compound of any one of claims 1 to 8, wherein each of said compounds is independently selected from the group consisting of: alkynyl.

10. R 1 and R 2 Both are C 15～20 10. The compound according to claim 9, wherein the alkenyl is an alkenyl group, preferably a singly or doubly unsaturated alkenyl group.

11. R 2 R 1 11. The compound of claim 1, wherein

12. 2. The compound of claim 1, which is a compound of formula (101), (102), (103), (104), (105) or (106). 【Chemistry 2】

13. 13. A micro- or nanoparticle comprising a compound of formula (I) according to any one of claims 1 to 12, or a pharmaceutically acceptable complex, salt, solvate, tautomer or polymorph thereof, and optionally an optional payload molecule.

14. 14. The micro- or nanoparticle according to claim 13, which is a lipid nanoparticle (LNP), liposome, lipoplex, micelle or lipid vesicle, preferably an LNP.

15. 15. The micro- or nanoparticles according to claim 13 or 14, comprising a compound of formula (I) or a pharmaceutically acceptable complex, salt, solvate, tautomer or polymorph thereof, and a lipid component comprising one or more additional lipids, wherein the one or more additional lipids are selected from the group consisting of phospholipids, constant or ionizable cationic lipids, constant or ionizable anionic lipids, structural lipids, shielding lipids, functionalized lipids and combinations thereof.

16. 16. The micro- or nanoparticles of claim 15, wherein the lipid component comprises 20-80 mol % of a compound of formula (I) or a pharmaceutically acceptable complex, salt, solvate, tautomer or polymorph thereof, 5-80 mol % of a structural lipid, 0-30 mol % of a phospholipid, 0-15 mol % of a shielding lipid and 0-15 mol % of a constant or ionized cationic lipid.

17. 17. Micro- or nanoparticles according to claim 15 or 16, wherein the structured lipid is a sterol.

18. 18. The micro- or nanoparticle of any one of claims 15 to 17, wherein the shielding lipid is a lipid modified to include a shielding polymer, and the shielding polymer is a polyethylene glycol (PEG) group, a polysarcosine group, an oligopeptide or polypeptide, a hydroxyl-containing non-ionic water-soluble polymer, polyvinylpyrrolidone (PVP), a poly(2-alkyl-2-oxazoline) or a zwitterionic polymer or any other suitable shielding polymer.

19. 19. The micro- or nanoparticle of any one of claims 15 to 18, wherein the mass ratio of lipid component to payload molecule is 1:1 to 100:1, 2:1 to 80:1, 3:1 to 70:1, 4:1 to 60:1 or 5:1 to 50:

1.

20. 20. The micro- or nanoparticle of any one of claims 13 to 19, wherein the payload molecule is a biomolecule, and / or an active pharmaceutical ingredient (API), and / or a diagnostic compound.

21. 21. The micro- or nanoparticle of claim 20, wherein the payload molecule is a biomolecule, and the biomolecule is or comprises an amino acid, a peptide, an affimer, a polypeptide or a protein, a glycoprotein, a sugar, a lipid, a lipopolysaccharide, an antibody or a fragment thereof, a polymer, or a nucleic acid, or a combination thereof.

22. 22. The micro- or nanoparticle of claim 21, wherein the nucleic acid is DNA or RNA, preferably RNA, more preferably messenger RNA (mRNA).

23. A composition comprising a plurality of micro- or nanoparticles according to any one of claims 13 to 22.

24. 24. The composition of claim 23, wherein the micro- or nanoparticles have an average diameter of less than 10 μm, less than 1 μm, less than 500 nm, less than 250 nm, less than 200 nm, less than 175 nm, less than 150 nm or less than 125 nm.

25. 25. The composition of claim 23 or 24, wherein the micro- or nanoparticles have a polydispersity index (PDI) of less than 0.5, less than 0.4, less than 0.3, less than 0.25, less than 0.2, less than 0.15, less than 0.1 or less than 0.

08.

26. A pharmaceutical composition comprising the micro- or nanoparticles of any one of claims 13 to 22 or the composition of any one of claims 23 to 25, and a pharmaceutically acceptable carrier.

27. 27. A micro- or nanoparticle according to any one of claims 13 to 22, a composition according to any one of claims 23 to 25, or a pharmaceutical composition according to claim 26, for use as a medicament.

28. 27. A micro- or nanoparticle according to any one of claims 13 to 22, a composition according to any one of claims 23 to 25 or a pharmaceutical composition according to claim 26 for use in the treatment and / or prevention and / or prophylaxis of a disease or disorder.

29. 27. A vaccine composition comprising the micro- or nanoparticles of any one of claims 13 to 22, the composition of any one of claims 23 to 25 or the pharmaceutical composition of claim 26.

30. 29. A micro- or nanoparticle according to any one of claims 13 to 22, a composition according to any one of claims 23 to 25, a pharmaceutical composition according to claim 26, or a vaccine according to claim 28, for use in stimulating an immune response in a subject.

31. Compound of formula (III) 【Chemistry 3】 (In the formula, R 1 , R 3 and L 1 ~L 3 is as defined in relation to the first aspect, and both R 1 The base is the same) 1. A method for producing Maleic anhydride is reacted with a compound of formula (V) HO-R 1 (V) to form a compound of formula (VI) 【Chemistry 4】 generating and a compound of formula (VI) with a compound of formula (VII) HS-L 1 -L 2 -L 3 -R 3 (VII) thereby producing a compound of formula (III) A method comprising:

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