Novel structured lipids for lipid nanoparticle formulations

Abstract

The present disclosure provides the use of a compound of Formula I in lipid micron and nanoparticle compositions. The present disclosure also relates to microparticles or nanoparticles comprising a compound of Formula I and optionally a payload molecule, and medical uses thereof.

Description

[0001] The present invention relates to the use of compounds in lipid microparticle and nanoparticle compositions, as well as novel lipid microparticle and nanoparticle compositions per se. The compositions are suitable for therapeutic or prophylactic delivery, with higher stability and enhanced biodistribution control. In particular, the lipid microparticle and nanoparticle compositions can be used for the delivery of (modified) RNA or DNA. The invention also relates to medical and research uses of the compositions.

[0002] Nanomedicine involves the use of nanoscale materials (e.g. biocompatible nanoparticles) for diagnostic, delivery, sensing or actuation purposes. This is an increasingly important field, with the promise of providing new diagnostic, therapeutic and / or prophylactic solutions. In particular, microparticle and nanoparticle formulations are able to exploit molecular entities that are (a) unstable, (b) do not meet the rules of drug-like properties (e.g. Lipinski’s rule of five), and / or (c) have poor pharmacokinetics, including unsuitable biodistribution behaviour, thereby expanding the use of these molecular entities in terms of dosing regimens, concentrations, and resulting efficacy and / or reaching specific target cells. In these formulations, the active pharmaceutical ingredient is encapsulated, absorbed, conjugated or otherwise bound by the microparticle or nanoparticle, thereby enabling the separation of the pharmacodynamic and pharmacokinetic properties between independent molecular entities. This greatly facilitates drug design, fine-tuning of pharmacological properties, and improves the efficacy and safety of the final drug.

[0003] The use of microparticle and nanoparticle formulations is particularly beneficial in the field of DNA and RNA-based drugs. This is because these molecules have unique intracellular activity, combined with their large size and negative charge, which together hinder uptake by most cells. DNA and RNA play a central role in the biology of all forms of life, and these molecules have high levels of specificity, which provides unprecedented opportunities for finding high-value treatments and cures for a large number of diseases in humans and other species.

[0004] Several successful nanomedicines are known today, which employ various types of nanoparticle formulations. Examples in the vaccine space include the lipid nanoparticle SARS-CoV-2 targeting vaccines Comirnaty by Pfizer / BioNTech and SpikeVax by Moderna, which have had great success in both commercial and humanitarian fields. Another example is the influenza targeting inactivated virus Pandemrix (H1N1 influenza) and Prepandrix (pre-pandemic influenza) water-in-oil emulsions by GlaxoSmithKline. Examples in the siRNA space include the lipid nanoparticle Onpattro (patisiran) for hereditary transthyretin-mediated amyloidosis, Givlaari (givosiran) for acute hepatic porphyria, and Oxlumo (lumasiran) for primary hyperoxaluria type 1, all developed by Alnylam. Other examples include liposomes such as Ambisome (liposomal amphotericin) for fungal infections, Visudyne (liposomal verteporfin) for macular degeneration, Abelcet (liposomal morphine) for the treatment of pain, Caelyx (liposomal doxorubicin) for cancer, Cimzia (Rh-a / b Fab against TNF-a), nanocrystalline Lipidic (fenofibrate) for hypercholesterolemia, Rapamune (liposomal sirolimus) against organ rejection, nanosuspension Risperadol (risperidone nanosuspension) for schizophrenia, and polymer nanoparticles Renagel (sevelamer) for hyperphosphatemia, and Copaxone (glatiramer acetate) for relapsing multiple sclerosis.

[0005] Despite the increasing number of successful commercial applications of nanomedicines, there are still challenges. This is especially true for more advanced applications, such as the delivery of RNA or DNA. In such applications, it is necessary to stably encapsulate or bind the cargo to the delivery vehicle to protect the cargo and shield its negative charge, enabling it to cross the negatively charged plasma or endosomal membranes. This encapsulation and / or binding needs to be completely reversed at the intended moment of release, but not before and not after. Thus, stable and efficient delivery vehicles can typically only provide a very limited range of compositions to work with (sufficient for therapeutic use).

[0006] To date, lipid nanoparticles with a solid lipid core (commonly referred to as LNP or SNALP (stable nucleic acid lipid particles)) have shown extremely high delivery efficiency compared to other nucleic acid delivery technologies, and thus have made great progress in clinical practice and commercial applications. The SARS-CoV-2 targeting vaccines Comirnaty and SpikeVax are exactly using this technology. Such LNP is distinguished from liposomes in that it contains only a small amount of water (if any) in its core. In addition, LNP has a nearly solid lipid structure, rather than the lipid bilayer structure that defines liposomes. Furthermore, for LNP, the lipids themselves complex and neutralize the negative charge of the RNA / DNA, whereas for liposomes, the RNA / DNA must be complexed with a charge neutralizing agent (e.g., a positively charged polymer) before it can be encapsulated into the liposome.

[0007] For solid lipid nanoparticles for clinical or commercial use, the lipid composition is very similar. For example, one of the most commonly used mRNA delivery formulations contains 50 mol% ionizable lipid (e.g., DLin-MC3-DMA), 38.5 mol% structural lipid (also referred to as core lipid, e.g., cholesterol), 10 mol% phospholipid or helper lipid (e.g., DSPC or DSPE), and 1.5 mol% shielding lipid (e.g., DMG-PEG2k), and slight variations thereof. In most cases, cholesterol, cholesterol derivatives, or structurally similar stanols are used as core lipids. Cholesterol in the formulation is believed to contribute to structural integrity, facilitate endosomal uptake by binding to ApoE and ApoE receptors on the cell surface, enhance membrane fusion (e.g., Tenchov B.G., MacDonald R.C., and Siegel D.P. Biophysical J. 91, 2508-2515 (2006)), and thus also facilitate endosomal escape, primarily through its crystalline form on the surface of LNP. The importance of cholesterol in nucleic acid delivery is emphasized due to its role in viral cytosolic delivery and enhanced gene delivery that can be achieved by using specific structural variants of cholesterol (Paunovska K et al. ACS Nano (2018)), such as naturally occurring β-sitosterol, a C-24 alkyl cholesterol analog (Patel S, et al. Nat Commun 11, 983 (2020)).

[0008] Formulations and natural nanoparticles (e.g. LDL particles) that use large amounts of cholesterol as structural lipid have been shown to bind to the natural serum protein ApoE in the blood (and possibly also in other tissues). The natural role of ApoE is to bind to low density lipoprotein (LDL) and very low density lipoprotein (VLDL) particles in the blood and to mediate binding to the LDL receptor, which is highly expressed on hepatocytes. Binding to the LDL receptor leads to clathrin-mediated endocytosis of the LDL particle and endosomal cholesterol processing by NPC2 and NPC1, both of which have been shown to be involved in LNP processing (Akinc A. et al. Mol. Ther. 18, 1357-1364 (2010) and Sahay G et al. Nat. Biotechnol. 31, 653-658 (2013)).

[0009] Due to the extremely high density of LDL receptors on hepatocytes and the large volume of venous blood that flows through the liver, binding of cholesterol-based LNPs to ApoE significantly influences their biodistribution, making it significantly liver-biased. In fact, at this point, achieving maximum ApoE binding and liver targeting effect is the main rationale for further development of LNPs, on the basis that if this effect cannot be prevented, it should at least be maximized (Jayaraman M, et al. Angew Chem Int Ed Engl. 2012 Aug 20;51(34):8529-33. doi: 10.1002 / anie.201203263). LNPs administered intravenously will first interact with blood components before reaching any tissue, and therefore can be significantly liver-biased. For therapies that rely on the release of produced proteins in the blood or bile (e.g. antibody therapies), the liver can be a suitable target organ, as the liver has a high protein production capacity, which makes ApoE binding a convenient uptake route. However, for any therapeutic, diagnostic or prophylactic drug that requires high local concentrations, intracellular delivery in specific cell types or is toxic to the liver, liver-biased biodistribution will reduce efficacy and / or safety. In this case, to achieve the required local concentrations of LNP and / or expressed protein, higher doses are needed, which increase the probability and extent of side effects, and increase costs and potential bioaccumulation effects.

[0010] Conjugation of drug formulations with active targeting moieties (e.g. antibodies, antibody fragments, DARPins, small molecules, peptides, etc.) is a well-known method to induce preferential uptake by target cells that present on their surface a protein or other chemical entity that is specifically bound by the targeting moiety. Addition of active targeting moieties to the outside of the delivery vehicle generally improves biodistribution to target cells. However, the degree of this improvement depends to a large extent on the relative strength of the active targeting moiety versus all other properties (including ApoE binding etc.) that induce or facilitate uptake by other organs. Importantly, as long as the LNP formulation contains cholesterol, it is very difficult to prevent ApoE binding. For this reason, shielding lipids such as PEGylated DSPE are often included in LNP formulations to prevent or reduce unwanted cellular and molecular interactions and thus prevent or reduce inappropriate biodistribution. However, the use of large amounts of PEG-lipids, while effective in shielding, reduces delivery efficiency. Therefore, for most LNP formulations used today, a balance needs to be struck between shielding efficacy and efficient delivery, such that the cholesterol-containing LNP surface can be partially available for ApoE binding and liver is the main biodistribution area upon intravenous administration. In certain LNP compositions, DMG-PEG2000 is used, which desorbs from the LNP surface by virtue of its short single-chain lipid tail and over time makes the surface available for adsorption of serum proteins, thus enabling organ-specific uptake depending on surface properties. So far, cholesterol (or derivatives thereof) has always been included in LNP formulations due to structural stability and delivery efficiency, cholesterol is an important determinant of surface properties, and thus LNP based on cholesterol or cholesterol derivatives are best suited for local applications (e.g. intramuscular injection) and liver-targeting applications, while most other applications would benefit from a different core lipid without ApoE binding propensity in combination with active targeting moieties. An exception could be surface charge-based biodistribution approaches that target cells of the reticuloendothelial system (RES) and are taken up by scavenger receptors, as these approaches interfere / change ApoE / serum protein binding while also having extensive early-stage interactions with receptors upon intravenous injection.

[0011] In recent years, the formation and decomposition of chemical adducts of RNA when packaged in LNPs, especially during long-term storage, has been recognized as a factor affecting the efficacy and safety of oligonucleotide and polynucleotide therapeutics and prophylactic drugs (Packer M et al. Nat Commun 12, 6777 (2021)). In addition to reactive impurities produced by lipid synthesis, pure intended ingredients in LNPs can also be major contributors to the adduct formation and decomposition process. Cholesterol is prone to oxidation due to its unsaturated bond at the D5-6 position of the sterol nucleus (Maerker G, Current status. J Am Oil Chem Soc 64:387-392 (1987)). During autoxidation, hydroperoxides and oxidized cholesterol products (i.e., so-called oxysterols) are formed. Hydroperoxides can subsequently initiate oxidation of nearby cholesterol or polyunsaturated fatty acids, thereby driving and accelerating oxidation of the LNP. This process not only affects the lipid profile of the LNP, in turn affecting endosomal escape efficiency, it can also directly affect mRNA through hydrolysis. Cholesterol oxidation is known to form a variety of oxysterol products bearing a hydroxyl, ketone, or epoxide group at the 4, 5, 6, or 7 position of the A and B rings of cholesterol. In particular, epoxides are highly reactive towards a variety of nucleophiles, including the 2-amino group of guanine in RNA and DNA.

[0012] In addition to the direct effects on LNP and mRNA, oxidation products of LNP lipid components can also directly or indirectly affect target cells. Many LNP formulations cause a transient increase in pro-inflammatory cytokines within hours after injection, and the time frame of this response is attributed to the lipid components of LNP, rather than its nucleic acid load (Lutz J et al. npj Vaccines 2 (2017)). Furthermore, it was found that the lipid components of LNP enhance existing inflammatory or immunodeficient states through inflammation exacerbation (IE) (Parhiz H et al. J. of Controlled Release 344: 50-61 (2021)). Ionizable lipid components have been identified as the main drivers of the pro-inflammatory response (Ndeupen S et al. iScience 24 (2021)), however, the contribution of oxidized cholesterol cannot be ruled out based on these findings. Importantly, oxidized cholesterol is a well-known pro-inflammatory signal, for example, in atherosclerosis, it leads to arterial inflammation and subsequent plaque erosion. Thus, it can be appreciated that oxidized cholesterol and fatty acids oxidized by the hydroperoxide produced from the autoxidation of cholesterol contribute to the pro-inflammatory effects of LNP. Monocyte metabolism of oxidized cholesterol can also induce long-term inflammatory effects by reprogramming the monocyte epigenome into a foam cell (Bekkering S. et al. Arterioscler Thromb Vasc Biol. 2014 Aug;34(8):1731-8). Furthermore, the epoxides formed can form carcinogenic adducts with endogenous RNA, DNA, and proteins, affecting the viability and genomic integrity of cells.

[0013] Prior art solutions to prevent oxidation include light-protected, low-temperature storage of the drug substance / drug product and under a protective atmosphere, such as nitrogen or argon. These methods can prevent cholesterol oxidation, especially when only low levels of oxidized lipids are present initially. However, it can be extremely difficult to achieve such low levels of oxidized lipids by preventing oxidation, as LNP is typically formed by mixing an organic solvent containing lipid components, such as ethanol, with an excess of an aqueous solution containing oligo- or polynucleotides, and requires large volume dialysis to remove or dilute the organic solvent by tangential flow filtration (TFF). The large volume can contain a significant amount of dissolved oxygen, and removing oxygen is a technical challenge.

[0014] Adding antioxidants can be another effective strategy to prevent oxidation. However, the addition of water-soluble antioxidants has limited effect on oxidation inside the LNP lipid core. The addition of hydrophobic antioxidants, including vitamin E, can affect the function of LNP and increase the complexity of the formulation and its quality control.

[0015] The solution to reduce the pro-inflammatory effects of LNP in the prior art relies on the use of corticosteroids (e.g. dexamethasone), either as a supplementary treatment or by incorporating the corticosteroid into the LNP itself, has been shown to significantly reduce inflammation while simultaneously increasing expression of nucleic acid-encoded proteins (Zhang H et al. J Biomedical Materials Res 2022: 1-8). However, dexamethasone and other corticosteroids exhibit significant adverse effects, including abdominal discomfort, skin rash, swelling, and hot flashes (Min KH et al. Korean J Audiol. 16:65-70 (2012)). By incorporating dexamethasone into the LNP, lower doses can be used and side effects are reduced. However, the disadvantage is that another ingredient is added to the LNP composition, complicating production, quality control, and prescription.

[0016] Furthermore, cholesterol plays an important role in the stability, organization (e.g. by lipid rafts) and function of the plasma membrane. If a large amount of LNP is targeted to a specific cell, which can be necessary to achieve a specific therapeutic or prophylactic effect, a transient increase in cholesterol can occur in that cell, and the function of the plasma membrane can change. For example, it is known that in neuronal cells, the fluidity of the plasma membrane and the organization of lipid rafts control the density and location of receptors, and changes in cholesterol levels affect cognitive function (Egawa et al. J Physiol. 594(16): 4565-4579 (2016)).

[0017] Similarly, cholesterol plays a role in the structural integrity of LNP by changing the fluidity of the LNP surface (Cheng X and Lee RJ. Adv. Drug Deliv. Rev. 99: 129-137 (2016)). LNP without cholesterol and with reduced cholesterol have been shown to be less stable and less potent (Rodrigueza et al. Biochemistry 34:6208-6217 (1995) and Sato Y et al. Acta Biomater. 102:341-350 (2020)). Higher physical stability of the nanoparticles is required to maintain performance-related formulation properties, such as particle size, shape, and low polydispersity, during post-processing steps (e.g. freeze-drying and concentration), storage, and application (e.g. nebulization and spraying). However, even with the addition of cholesterol, nebulization leads to an increase in nanoparticle size and a decrease in the encapsulation efficiency of the nucleic acid load (Zhang H et al. Pharmaceutics). No clear trend between particle stability under nebulization conditions and the relative cholesterol content was observed.

[0018] Accordingly, to advance nucleic acid-based therapies, prophylaxis, and diagnostics, there is an urgent need for a novel core / structural lipid that can replace cholesterol and has the following combination of properties: maintain or enhance endosomal escape, better control of its biodistribution to organs other than the liver, reduce proinflammatory signaling upon cell contact with the LNP, and confer better chemical and / or physical stability to the LNP. The present invention stems from the inventors’ research work to identify novel core / structural lipids.

[0019] According to a first aspect of the present invention, there is provided the use of a compound of formula (I) or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof in a microparticle or nanoparticle formulation:

[0020]

[0021] (I)

[0022] wherein L 1 is , or ;

[0023] R 1 to R 10 are each independently H, OR 11 , OCOR 11 , COOR 11 , NR 11 R 12 , N + R 11 R 12 R 13 , optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 3-10 cycloalkyl, optionally substituted C 3-10 cycloalkenyl, optionally substituted 3- to 10-membered heterocycle, optionally substituted C 6-12 aryl, optionally substituted 5- to 10-membered heteroaryl, or a sugar moiety; and

[0024] R 11 to R 13 are each independently H, optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 3-10 cycloalkyl, optionally substituted C 3-10 cycloalkenyl, optionally substituted 3- to 10-membered heterocycle, optionally substituted C 6-12 aryl, or optionally substituted 5- to 10-membered heteroaryl.

[0025] Advantageously, the compounds of formula (I) can be used in improved lipid nanoparticle compositions having one or more of the following properties: enhanced delivery of the agent to target cells, increased physical stability, increased chemical stability, enhanced biodistribution when using active targeting agents, and / or showing less pro-inflammatory cytokine release upon contact with cells. The lipid structure can advantageously be used in lipid-based nanoparticle or microparticle compositions for the delivery of (modified) RNA, DNA, peptides or small molecules to mammalian cells.

[0026] Preferably, the compounds of formula (I) are used as core lipids in microparticle or nanoparticle formulations. It is understood that core lipids can also be referred to as structural lipids.

[0027] The term "alkyl", as used herein, unless otherwise indicated, refers to a saturated straight chain or branched chain hydrocarbon.

[0028] "Alkenyl" refers to an olefinically unsaturated hydrocarbon group that can be straight or branched, i.e., the hydrocarbon group contains one or more carbon-carbon double bonds. It is understood that the alkenyl group can also be partially saturated. For example, the alkenyl group can contain one or more double bonds in addition to the multiple saturated single bonds.

[0029] "Alkynyl" refers to an acetylenically unsaturated hydrocarbon group that can be straight or branched, i.e., the hydrocarbon group contains one or more carbon-carbon triple bonds. The alkynyl group can contain one or more carbon-carbon double bonds in addition to the one or more carbon-carbon triple bonds. It is understood that the alkynyl group can also be partially saturated. For example, the alkynyl group can contain one or more triple bonds in addition to the multiple saturated single bonds.

[0030] The alkyl, alkenyl and / or alkynyl group can be unsubstituted or substituted by one or more of the following groups: halogen, -NR 14 R 15 , -N + R 14 R 15 R 16 , -SR 14 , -OR 14 , -CN, -COR 14 , -COOR 14 , -OCOR 14 , -CONR 14 R 15 , -NR 14 SO2R 15 , -SO2NR 14 R 15 , -NR 14 COR 15 , -OP(O)(OH)OR 14 , oxo, optionally substituted C3-6 cycloalkyl, optionally substituted C 3-6 cycloalkenyl, optionally substituted C 6-12 aryl, optionally substituted 3- to 10-membered heterocycle, or optionally substituted 5- to 10-membered heteroaryl, and R 14 to R 16 each independently H, optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 3-10 cycloalkyl, optionally substituted C 3-10 cycloalkenyl, optionally substituted 3- to 10-membered heterocycle, optionally substituted C 6-12 aryl, or optionally substituted 5- to 10-membered heteroaryl. R 14 to R 16 may each independently be selected from H, C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 3-6 cycloalkyl, C 3-6 cycloalkenyl, C 6-12 aryl, 3- to 10-membered heterocycle, or 5- to 10-membered heteroaryl.

[0031] “Cycloalkyl” refers to a non-aromatic saturated hydrocarbon ring system. Representative examples of C3-C6cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Similarly, “cycloalkenyl” refers to a non-aromatic unsaturated 3- to 6-membered hydrocarbon ring system.

[0032] “Aryl” refers to an aromatic hydrocarbon group. The aryl group can be monocyclic, bicyclic, or polycyclic. The term “aryl” is understood to include bicyclic systems (where one ring is aromatic and one ring is unsaturated or partially saturated) and polycyclic systems (where at least one ring is aromatic and at least one ring is unsaturated or partially saturated). Representative examples of C6-C 12 Examples of aryl groups include, but are not limited to, phenyl, a-naphthyl, β-naphthyl, biphenyl, tetrahydronaphthyl, and indanyl.

[0033] “Heterocycle” or “heterocyclyl” refers to a 3- to 10-membered ring system in which at least one ring atom is a heteroatom. The heteroatom or each heteroatom can be independently selected from oxygen, sulfur, and nitrogen. The heterocycle can be saturated or partially saturated. Exemplary heterocyclyl groups include, but are not limited to, aziridine, oxirane, oxetane, 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, oxazine.

[0034] "Heteroaryl" refers to an aromatic 5- to 10-membered ring system in which at least one ring atom is a heteroatom. Heteroaryl groups can be monocyclic, bicyclic, or polycyclic. In bicyclic and polycyclic structures, the group is understood to be heteroaryl if at least one ring contains a heteroatom. The term "heteroaryl" is understood to include bicyclic ring systems in which one ring is aromatic and one ring is unsaturated or partially saturated, and polycyclic ring systems in which at least one ring is aromatic and at least one ring is unsaturated or partially saturated. The heteroatom or each heteroatom can be independently selected from 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, benzoisoxazole, benzoimidazole, N-methylbenzoimidazole, azabenzimidazole, indazol, quinazoline, quinoline, and isoquinoline. Bicyclic 5- to 10-membered heteroaryl groups include heteroaryl groups in which a phenyl, pyridine, pyrimidine, pyrazine, or pyridazine ring is fused to a 5- or 6-membered monocyclic heteroaryl ring.

[0035] Any cycloalkyl, cycloalkenyl, aryl, heterocycle, and / or heteroaryl group can be unsubstituted or substituted with one or more of the following groups: optionally substituted C1-C6alkyl, optionally substituted C1-C6alkenyl, optionally substituted C1-C6alkynyl, halogen, -NR 14 R 15 , -N + R 14 R 15 R 16 , -SR 14 , -OR 14 , -CN, -COR 14 , -COOR 14 , -OCOR 14 , -CONR 14 R 15 , -NR 14 SO2R 15 , -SO2NR 14 R 15 , -NR 14 COR 15 , -OP(O)(OH)OR 14 , oxo, optionally substituted C 3-6 cycloalkyl, optionally substituted C 3-6 cycloalkenyl, optionally substituted C 6-12 aryl, optionally substituted 3- to 10-membered heterocycle, or optionally substituted 5- to 10-membered heteroaryl, and R 14 to R16 each independently H, optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 3-10 cycloalkyl, optionally substituted C 3-10 cycloalkenyl, optionally substituted 3- to 10-membered heterocycle, optionally substituted C 6-12 aryl or optionally substituted 5- to 10-membered heteroaryl. R 14 to R 16 may each independently be selected from H, C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 3-6 cycloalkyl, C 3-6 cycloalkenyl, C 6-12 aryl, 3- to 10-membered heterocycle, or 5- to 10-membered heteroaryl.

[0036] The "sugar moiety" can be a monosaccharide, disaccharide, polysaccharide, or a derivative thereof.

[0037] The term "pharmaceutically acceptable salt" can be understood to mean any salt of a compound provided herein that retains the biological properties of the compound and that is not toxic or otherwise unacceptably deleterious to a patient. Such salts can be derived from a variety of organic and inorganic counter-ions well known in the art. Such salts include, but are not limited to: (1) acid addition salts formed with organic or inorganic acids such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, sulfamic, acetic, adepic, aspartic, trifluoroacetic, trichloroacetic, propionic, hexanoic, cyclopentylpropionic, glycolic, glutaric, pyruvic, lactic, malonic, succinic, sorbic, ascorbic, malic, maleic, fumaric, tartaric, citric, benzoic, 3-(4-hydroxybenzoyl)benzoic, picric, cinnamic, mandelic, phthalic, lauric, methanesulfonic, ethanesulfonic, 1,2-ethane-disulfonic, 2-hydroxyethanesulfonic, benzenesulfonic, 4-chlorobenzenesulfonic, 2-naphthalenesulfonic, 4-methylbenzenesulfonic, camphoric, camphorsulfonic, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic, glucoheptonic, 3-phenylpropionic, trimethylacetic, tert-butylacetic, dodecylsulfic, gluconic, benzoic, glutamic, hydroxynaphthoic, salicylic, stearic, cyclohexylsulfamic, quinic, mucic, and the like acids; or (2) base addition salts formed when an acidic proton present in the parent compound is replaced by a metal ion, for example, an alkali metal, alkaline earth metal, or aluminum ion, or an ammonium or alkylated-ammonium ion, such as sodium ion, potassium ion, calcium ion, magnesium ion, aluminum ion, lithium ion, zinc ion, and barium ion, ammonium ion, and the like, or a base metal or alkaline earth hydroxide, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, aluminum hydroxide, lithium hydroxide, zinc hydroxide, and barium hydroxide, ammonia, or an organic base, such as aliphatic, alicyclic, or aromatic organic amines, such as ammonia, methylamine, dimethylamine, diethylamine, methylpyridine, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, N-methylglucamine piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, and the like.

[0038] Pharmaceutically acceptable salts can include sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like, and if the compound includes a basic functionality, salts of non-toxic organic or inorganic acids, such as hydrohalides, e.g. hydrochlorides, hydrobromides, and hydroiodides, carbonates, or bicarbonates, sulfates or hydrogen sulfates, borates, phosphates, hydrogen phosphates, dihydrogen phosphates, pyroglutamic acid, saccharate, stearate, sulfamate, nitrate, orotate, oxalate, palmitate, pamoate, acetate, trifluoroacetate, trichloroacetate, propionate, hexanoate, cyclopentylpropionate, glycolate, glutarate, pyruvate, lactate, malonate, succinate, tannate, tartrate, toluenesulfonate, sorbate, ascorbate, malate, maleate, fumarate, tartrate, camphorsulfonate, citrate, cyclamate, benzoate, isethionate, ethanesulfonate, formate, 3-(4-hydroxybenzoyl)benzoate, picrate, cinnamate, mandelate, phthalate, laurate, methanesulfonate (mesylate), methyl sulfate, naphthylate, 2-napsylate, nicotinate, ethanesulfonate, 1,2-ethanedisulfonate, 2-hydroxyethanesulfonate, benzenesulfonate (besylate), 4-chlorobenzenesulfonate, 2-naphthalenesulfonate, 4-toluenesulfonate, camphorate, camphorsulfonate, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylate, glucoheptonate, 3-phenylpropionate, trimethylacetate, t-butylacetate, dodecylsulfate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, benzoate, glutamate, hydroxynaphthoate, salicylate, stearate, cyclamates, quinate, mucate, xinofoate, and the like.

[0039] R 1 may be H.

[0040] In some embodiments, R 3 may be H.

[0041] In alternative embodiments, R 3 may be optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30alkynyl, optionally substituted C 3-10 cycloalkyl, optionally substituted C 3-10 cycloalkenyl, optionally substituted 3- to 10-membered heterocycle, optionally substituted C 6-12 aryl or optionally substituted 5- to 10-membered heteroaryl. More preferably, R 3 may be optionally substituted C 6-26 alkyl, optionally substituted C 6-26 alkenyl, optionally substituted C 6-26 alkynyl, optionally substituted C 3-6 cycloalkyl, optionally substituted C 3-6 cycloalkenyl, optionally substituted 3- to 6-membered heterocycle, optionally substituted phenyl or optionally substituted 5- or 6-membered heteroaryl. More preferably, R 3 may be optionally substituted C 12-24 alkyl, optionally substituted C 12-24 alkenyl, optionally substituted C 12-24 alkynyl, C 3-6 cycloalkyl, C 3-6 cycloalkenyl, 3- to 6-membered heterocycle, phenyl or 5- or 6-membered heteroaryl. The alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycle, aryl or heteroaryl can be unsubstituted or substituted with one or more substituents selected from the group consisting of OH, SH, NH2, CN, oxo, C 3-6 cycloalkyl, C 3-6 cycloalkenyl, C 6-12 aryl, 3- to 10-membered heterocycle or 5- to 10-membered heteroaryl. Preferably, the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycle, aryl or heteroaryl is unsubstituted or substituted with one or more substituents selected from the group consisting of OH, SH, NH2, CN, oxo, C 3-6 cycloalkyl, C 3-6 cycloalkenyl, phenyl, 5- or 6-membered heterocycle or 5- or 6-membered heteroaryl. Thus, R 3 may be or .

[0042] R 5 may be H.

[0043] In some embodiments, R 6 may be H.

[0044] In alternative embodiments, R 6 may be OR 11 , OCOR 11 , COOR 11 , NR 11 R 12 or N + R 11 R 12 R 13More preferably, R 6 is OR 11 or NR 11 R 12 . Most preferably, R 6 is OR 11 . R 11 through R 13 may independently be H, optionally substituted C 1-12 alkyl, optionally substituted C 2-12 alkenyl, optionally substituted C 2-12 alkynyl, optionally substituted C 3-10 cycloalkyl, optionally substituted C 3-10 cycloalkenyl, optionally substituted 3- to 10-membered heterocycle, optionally substituted C 6-12 aryl, or optionally substituted 5- to 10-membered heteroaryl. More preferably, R 11 through R 13 may independently be H, C 1-6 alkyl, C 2-6 alkenyl, or C 2-6 alkynyl. Most preferably, R 11 through R 13 are H. Thus, R 6 may be OH.

[0045] In some embodiments, R 7 may be H.

[0046] In alternative embodiments, R 7 may be OR 11 , OCOR 11 , COOR 11 , NR 11 R 12 , N + R 11 R 12 R 13 , optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 3-10 cycloalkyl, optionally substituted C 3-10 cycloalkenyl, optionally substituted 3- to 10-membered heterocycle, optionally substituted C 6-12 aryl, or optionally substituted 5- to 10-membered heteroaryl. More preferably, R 7 may be OR 11 , NR 11 R 12 , optionally substituted C 1-12 alkyl, optionally substituted C 2-12 alkenyl, optionally substituted C 2-12 alkynyl, optionally substituted C3-6 cycloalkyl, optionally substituted C 3-6 cycloalkenyl, optionally substituted 3-6 membered heterocycle, optionally substituted phenyl, or optionally substituted 5 or 6 membered heteroaryl. More preferably, R 7 may be OR 11 , C 3-6 alkyl, C 3-6 alkenyl, or C 3-6 alkynyl. R 11 to R 13 may independently be H, optionally substituted C 1-12 alkyl, optionally substituted C 2-12 alkenyl, optionally substituted C 2-12 alkynyl, optionally substituted C 3-10 cycloalkyl, optionally substituted C 3-10 cycloalkenyl, optionally substituted 3 to 10 membered heterocycle, optionally substituted C 6-12 aryl, or optionally substituted 5 to 10 membered heteroaryl. More preferably, R 11 to R 13 may independently be H, C 1-6 alkyl, C 2-6 alkenyl, or C 2-6 alkynyl. More preferably, R 11 to R 13 is H or methyl, most preferably methyl. Thus, R 7 may be OMe, or .

[0047] In some embodiments, R 9 may be H. In alternative embodiments, R 9 may be OR 11 , OCOR 11 , COOR 11 , NR 11 R 12 , N + R 11 R 12 R 13 , optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 3-10 cycloalkyl, optionally substituted C 3-10 cycloalkenyl, optionally substituted 3 to 10 membered heterocycle, optionally substituted C 6-12 aryl, or optionally substituted 5 to 10 membered heteroaryl. More preferably, R 9 may be OR 11 , NR 11 R 12 , optionally substituted C1-12 alkyl, optionally substituted C 2-12 alkenyl, optionally substituted C 2-12 alkynyl, optionally substituted C 3-6 cycloalkyl, optionally substituted C 3-6 cycloalkenyl, optionally substituted 3- to 6-membered heterocycle, optionally substituted phenyl, or optionally substituted 5- or 6-membered heteroaryl. More preferably, R 9 may be OR 11 , C 3-6 alkyl, C 3-6 alkenyl, or C 3-6 alkynyl. More preferably, R 11 to R 13 may independently be H, optionally substituted C 1-12 alkyl, optionally substituted C 2-12 alkenyl, optionally substituted C 2-12 alkynyl, optionally substituted C 3-10 cycloalkyl, optionally substituted C 3-10 cycloalkenyl, optionally substituted 3- to 10-membered heterocycle, optionally substituted C 6-12 aryl, or optionally substituted 5- to 10-membered heteroaryl. More preferably, R 11 to R 13 may independently be H, C 1-6 alkyl, C 2-6 alkenyl, or C 2-6 alkynyl. More preferably, R 11 to R 13 is H or methyl, most preferably methyl. Thus, R 9 may be OMe, or .

[0048] R 10 may be H.

[0049] Thus, the compound can be a compound of formula (Ia), (Ib), (Ic), or (Id):

[0050]

[0051] L 1 may be .

[0052] Thus, in some embodiments, the compound is a compound of formula (Iai), (Ibi), (Ici), or (Idi):

[0053]

[0054] or, L 1 may be .

[0055] Thus, in alternative embodiments, the compound is a compound of formula (Iaii), (Ibii), (Icii) or (Idii):

[0056]

[0057] R 2 , R 4 and R 8 may independently be H, OR 11 , OCOR 11 , COOR 11 , NR 11 R 12 , N + R 11 R 12 R 13 or a sugar moiety. More preferably, R 2 , R 4 and R 8 are independently H, OR 11 , OCOR 11 or a sugar moiety.

[0058] R 11 to R 13 are each independently H, optionally substituted C 1-20 alkyl, optionally substituted C 2-20 alkenyl, optionally substituted C 2-20 alkynyl, optionally substituted C 3-10 cycloalkyl, optionally substituted C 3-10 cycloalkenyl, optionally substituted 3- to 10-membered heterocycle, optionally substituted C 6-12 aryl, or optionally substituted 5- to 10-membered heteroaryl. More preferably, R 11 to R 13 are independently H, C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 3-6 cycloalkyl, C 3-6 cycloalkenyl, 3- to 6-membered heterocycle, phenyl, or 5- or 6-membered heteroaryl. More preferably, R 11 to R 13 are independently H, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, C 3-6 cycloalkenyl, 3- to 6-membered heterocycle, phenyl, or 5- or 6-membered heteroaryl. More preferably, R 11 to R 13 are independently H, C 1-3 alkyl, C 2-3 alkenyl, C2-3 alkynyl or 5 or 6 membered heteroaryl. Most preferably, R 11 to R 13 are independently H, methyl or 5 membered heteroaryl.

[0059] Thus, R 2 , R 4 and R 8 may independently be H, OH, OCH3or or a sugar moiety.

[0060] A sugar moiety can be understood to have the general formula:

[0061] or

[0062] where X 1 is CR 20 R 21 a bond; and

[0063] R 17 to R 25 are each independently H, OH, CH2OH, COOH, NH2, C 1-6 alkyl, halogen, or .

[0064] In some embodiments, X 1 is a bond. Preferably, X 1 is CR 20 R 21 .

[0065] In some embodiments, R 17 is H. Alternatively, R 17 may be OH, CH2OH, COOH or NH2. R 17 may be OH or CH2OH.

[0066] Preferably, R 18 , R 22 and R 24 are H. Preferably, in embodiments where it is present, R 20 is H.

[0067] Preferably, R 19 is H, OH or NH2, more preferably both are OH.

[0068] Preferably, in embodiments where it is present, R 21 is H, OH or NH2, more preferably OH.

[0069] Preferably, R 23 is OH or NH2, more preferably both are OH. Alternatively, R23 may be or R 17 to R 25 may be as defined herein.

[0070] Preferably, R 25 is CH2OH or COOH. Alternatively, R 25 may be or R 17 to R 25 may be as defined herein.

[0071] The sugar moiety can be a ribose moiety, a deoxyribose moiety, a fructose moiety, a glucose moiety, a galactose moiety, a sucrose moiety, a maltose moiety or a lactose moiety.

[0072] Thus, the sugar moiety can be , , 0, , , , or . The sugar moiety can be , , , , , , , , , , or .

[0073] In some embodiments, the sugar moiety is or . More preferably, the sugar moiety is or .

[0074] The compound of formula (I) can be:

[0075]

[0076] The compound of formula (103) can be .

[0077] The compound of formula (110) can be .

[0078] The compound of formula (115) can be .

[0079] The compound of formula (116) can be .

[0080] The compound of formula (117) can be .

[0081] According to a second aspect of the application, there is provided a microparticle or nanoparticle comprising a compound of formula (I) or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof as defined in the first aspect, and optionally a payload molecule.

[0082] The microparticle or nanoparticle can have a dense or hollow structure. The microparticle or nanoparticle can have a solid structure. The microparticle or nanoparticle can optionally be composed of one or more lipid bilayers, which can optionally be cross-linked to each other, thereby enclosing a void. The microparticle or nanoparticle can be a lipid nanoparticle (LNP), a liposome, a lipoplex, a micelle or a niosome. In some preferred embodiments, the microparticle or nanoparticle is an LNP.

[0083] The microparticle or nanoparticle can have a diameter of less than 10 pm, less than 1 pm, less than 500 nm or less than 250 nm. More preferably, the microparticle or nanoparticle can have a diameter of less than 200 nm, less than 175 nm, less than 150 nm, less than 125 nm, less than 100 nm, less than 90 nm or less than 80 nm. The microparticle or nanoparticle can have a diameter of from 25 nm to 1 pm, from 30 nm to 500 nm, from 35 nm to 250 nm, from 40 nm to 200 nm, from 45 nm to 150 nm, from 50 nm to 125 nm, from 55 nm to 100 nm, from 60 nm to 90 nm or from 65 nm to 80 nm. The diameter of the microparticle or nanoparticle can be measured using dynamic light scattering.

[0084] In addition to the compound of formula (I) or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof, the microparticle or nanoparticle can comprise one or more additional lipids. Thus, the microparticle or nanoparticle can comprise a lipid component comprising the compound of formula (I) or a pharmaceutically acceptable complex, salt, solvate, tautomeric form or polymorphic form thereof, and one or more additional lipids. The one or more additional lipids can be selected from the group consisting of a phospholipid, a permanently cationic lipid, an ionisable cationic lipid, a permanently anionic lipid, an ionisable anionic lipid, a structural lipid, a shielding lipid, a functionalised lipid, an additional core lipid and combinations thereof.

[0085] In some embodiments, the lipid component comprises a compound of Formula (I) or a pharmaceutically acceptable complex, salt, solvate, tautomeric form, or polymorphic form thereof; and a permanent or ionizable cationic lipid or mixtures thereof. In some embodiments, the lipid component of the microparticle or nanoparticle formulation comprises a compound of Formula (I) or a pharmaceutically acceptable complex, salt, solvate, tautomeric form, or polymorphic form thereof; a permanent or ionizable lipid; and a shielding lipid. In some embodiments, the lipid component of the microparticle or nanoparticle formulation further comprises a phospholipid or a helper lipid. In some embodiments, the lipid component of the microparticle or nanoparticle formulation further comprises a functionalized lipid. In some embodiments, the lipid component of the microparticle or nanoparticle formulation further comprises an additional core lipid.

[0086] The lipid component of the microparticle or nanoparticle preferably comprises a core lipid component comprising a compound of Formula (I). In some embodiments, the core lipid component consists of a compound of Formula (I). In alternative embodiments, the core lipid component comprises a compound of Formula (I) and an additional core lipid. The core lipid component can comprise at least 5 mol% of a compound of Formula (I). The core lipid component can comprise at least 10 mol%, at least 20 mol%, at least 30 mol%, at least 40 mol%, or at least 50 mol% of a compound of Formula (I). The core lipid component can comprise at least 60 mol%, at least 70 mol%, at least 80 mol%, at least 90 mol%, or 100 mol% of a compound of Formula (I). The core lipid component can comprise less than 100 mol% of a compound of Formula (I). The core lipid component can comprise less than 95 mol%, less than 90 mol%, less than 80 mol%, less than 70 mol%, less than 60 mol%, or less than 50 mol% of a compound of Formula (I). The core lipid component can comprise less than 40 mol%, less than 30 mol%, less than 20 mol%, or less than 10 mol% of a compound of Formula (I). By varying the amount of a compound of Formula (I) and the additional core lipid, the properties of the microparticle or nanoparticle can be fine-tuned.

[0087] In some embodiments, the lipid component of the microparticle or nanoparticle comprises 5 to 80 mol%, 10 to 60 mol%, 20 to 50 mol%, 30 to 45 mol%, or 35 to 40 mol% of a core lipid component.

[0088] Thus, in some embodiments, the lipid component of the microparticle or nanoparticle comprises 5 to 80 mol%, 10 to 60 mol%, 20 to 50 mol%, 30 to 45 mol%, or 35 to 40 mol% of a compound of Formula (I) or a pharmaceutically acceptable complex, salt, solvate, tautomeric form, or polymorphic form thereof.

[0089] In some embodiments, the lipid component of the microparticle or nanoparticle formulation comprises 10 to 90 mol%, 20 to 80 mol%, 30 to 70 mol%, 40 to 60 mol%, or 45 to 55 mol% of a permanently cationic lipid, an ionizable cationic lipid, or a mixture thereof.

[0090] In some embodiments, the lipid component of the microparticle or nanoparticle formulation comprises 0 to 30 mol%, 2.5 to 20 mol%, 5 to 15 mol%, or 7.5 to 12.5 mol% of a phospholipid.

[0091] In some embodiments, the lipid component of the microparticle or nanoparticle formulation comprises 0 to 15 mol%, 0.1 to 10 mol%, 0.5 to 5 mol%, 0.75 to 3 mol%, or 1 to 2 mol% of a shielding lipid.

[0092] The additional core lipid can be a sterol. The sterol can be cholesterol or a cholesterol derivative. The cholesterol derivative can be beta-sitosterol, vitamin D2, vitamin D3, Calcipotriol, Stigmasterol, Campesterol, Fucosterol, Brassicasterol, Ergosterol, 9,11-Dehydroergosterol, Daucosterol, beta-Sitosterol-Acetate, Betulin, Lupeol, Ursotic acid, or Oleanotic acid.

[0093] The phospholipid can be dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS), 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), or any naturally occurring or synthetically derived phospholipid.

[0094] A shielding lipid can be understood as a functionalized lipid that prevents undesired interactions of the lipid nanoparticle with other nanoparticles, extracellular compounds, and / or cell surfaces. A shielding lipid can prevent undesired interactions of the lipid nanoparticle with other nanoparticles, extracellular compounds, and / or cell surfaces by steric hindrance or similar mechanisms. A shielding lipid can be a lipid modified to comprise a shielding polymer. A shielding polymer can be a polyethylene glycol (PEG) group, a polysarcosine group, an oligo- or polypeptide, a hydroxyl-containing non-ionic water-soluble polymer, a polyvinylpyrrolidone (PVP), a poly(2-alkyl-2-oxazoline), or a zwitterionic polymer, or any other suitable shielding polymer. An oligo- or polypeptide can be a PAS. A PAS can be understood as an oligo- or polypeptide consisting of or comprising proline, alanine, and serine residues. A hydroxyl-containing non-ionic water-soluble polymer can be a polyglycerol (PG), a poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA), a polysarcosine, or a polyvinyl alcohol. A zwitterionic polymer can be a polybetaine. In some embodiments, a shielding lipid can be a phospholipid modified to comprise a shielding polymer. In embodiments where the shielding lipid is a phospholipid modified to comprise a shielding polymer, the modified phospholipid can be a phospholipid as defined above. In some embodiments, a shielding lipid can be a DSPE, DMG, or DPPC lipid modified to comprise a shielding polymer. A shielding lipid can comprise a shielding polymer having an average molecular weight of 500 to 5000, 1000 to 3000, 1250 to 1750, 1500 to 2500, 1750 to 2250 Da, or 1900 to 2100 Da. A shielding lipid can be di-myristoyl-glycerol-PEG (DMG-PEG), PEG-DSPE, PEG-di-palmitoyl-phosphatidyl-choline (DPPC), pSar-DMG, pSar-DSPE, or pSar-DPPC. In some embodiments, a shielding lipid is PEG-DSPE or pSar-DSPE.

[0095] A permanent or ionizable cationic lipid can be 1,2-dilinoleyl-oxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleoyl-3-trimethylammonium propane (DODAP), or a compound of Formula (II) or a pharmaceutically acceptable complex, salt, solvate, tautomeric form, or polymorphic form thereof:

[0096]

[0097] (II)

[0098] wherein:

[0099] R 26 and R 27 are independently selected from optionally substituted C 1-50 alkyl, optionally substituted C 2-50 alkenyl, or optionally substituted C 2-50 alkynyl;

[0100] L 2 and L 4 are independently absent or optionally substituted C 1-10 alkylene, optionally substituted C 2-10 alkenylene, or optionally substituted C 2-10 alkynylene;

[0101] L 3 is absent or NH, S, or O;

[0102] R 28 is selected from -NR 29 R 30 , -N + R 29 R 30 R 31 , -H, -SR 29 , -OR 29 , -CN, -COR 29 , -COOR 29 , -OCOR 29 , -CONR 29 R 30 , -NR 29 SO2R 30 , -SO2NR 29 R 30 , -NR 29 COR 30 , -OP(O)(OH)OR 29 , optionally substituted C 3-6 cycloalkyl, optionally substituted C 3-6 cycloalkenyl, optionally substituted C 6-12 aryl, optionally substituted 3- to 10-membered heterocycle, or optionally substituted 5- to 10-membered heteroaryl; and

[0103] R 29 through R 30 are independently selected from H, optionally substituted C 1-30 alkyl, optionally substituted C 2-30alkenyl, 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 heterocycle, or optionally substituted 5- to 10-membered heteroaryl.

[0104] Compounds of Formula (II) are described in UK Patent Application No. 2215200.3, which is incorporated herein by reference. Specific compounds of Formula (II) disclosed in UK Patent Application No. 2215200.3 are 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, and dioleyl 2-((2-(diethylamino)ethyl)thio)succinate maleate.

[0105] Thus, the permanent or ionizable cationic lipid can be 1,2-dilinoleyl-oxy-N,N-dimethylammoniumpropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]- dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleoyl-3- trimethylammonium propane (DOTAP), 1,2-dioleoyl-3-trimethylammonium propane (DODAP), 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-(dimethylaminopropyl)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, or dioleyl 2-((2- (diethylamino)ethyl)thio)succinate maleate.

[0106] The permanent or ionizable anionic lipid can be cholesterol hemisuccinate, phosphatidylinositol phosphate (PIP, also known as phosphoinositol), phosphatidylserine (PS), or phosphatidic acid (PA).

[0107] The functionalized lipid can comprise one or more moieties that allow for conjugation thereto. The one or more moieties can be independently selected from azide, alkyne, tetrazine, dibenzocyclooctyne (DBCO), maleimide, trans-cyclooctene (TCO), vinyl, methylcyclopropene, and succinimidyl-ester.

[0108] In a preferred embodiment, the microparticle or nanoparticle comprises a payload molecule. The payload molecule can be a biomolecule, and / or an active pharmaceutical ingredient (API), and / or a diagnostic compound.

[0109] The API can be a hydrophobic or a hydrophilic API. The API can be a macromolecule or a small molecule. It can be appreciated that a small molecule can be considered to be a molecule having a molecular weight of less than 900 Daltons. In some embodiments, the molecular weight of a small molecule can be less than 800 Daltons, less than 700 Daltons, less than 600 Daltons, less than 500 Daltons, or less than 400 Daltons. Similarly, a macromolecule can be considered to be a molecule having a molecular weight of at least 900 Daltons.

[0110] 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 agents (e.g., chemotherapeutic agents, such as alkylating agents (examples include altretamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, mechlorethamine, melphalan, oxaliplatin, temozolomide, thiotepa, trabectedin), nitrosoureas (examples include carmustine, lomustine, streptozocin), antimetabolites (examples include azacitidine, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cladribine, clofarabine, cytarabine (Ara-C), decitabine, floxuridine, fludarabine, gemcitabine, hydroxyurea,Methotrexate, Nelarabine, Pemetrexed, Pentostatin, Pralatrexate, Thioguanine, Trifluridine / tipiracil combination), antitumor antibiotics (examples include anthracyclines: Daunorubicin, Doxorubicin (Adriamycin), Doxorubicin liposomal, Epirubicin, Idarubicin, Valrubicin, and antitumor antibiotics: Bleomycin, Dactinomycin, Mitomycin-C, Mitoxantrone), topoisomerase inhibitors: Irinotecan, Irinotecan liposomal, Topotecan, Etoposide (VP-16), Mitoxantrone, Teniposide), mitotic inhibitors (examples include taxoids: Cabazitaxel, Docetaxel, Nab-paclitaxel, and Paclitaxel, and vinca alkaloids: Vinblastine, Vincristine, Vincristine liposomal, Vinorelbine) and other chemotherapy drugs (examples include All-trans-retinoic acid, Arsenic trioxide, Asparaginase, Eribulin, Hydroxyurea, Ixabepilone, Mitotane, Omacetaxine, Pegaspargase, Procarbazine, Romidepsin, Vorinostat), cytokine drugs (including cytokines (examples include (recombinant form of) IL-1, IL-2, TNF-alpha, IL-6, IL-7, IL-10, IL-12, IL-17, IL-21, IL-22, IL-23,IFN-a, IFN-b, IFN-g, IFN-l, IFN-l, IFN-l, IFN-w, IP-10, MIP-la, TGF-b (1-3), and anti-cytokines (e.g., antibodies that bind cytokines, decoy receptors, IL-1R antagonists), growth factors (examples include bone morphogenetic proteins (BMPs), vascular endothelial growth factor (VEGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), epidermal growth factor (EGF), erythropoietin (EPO), insulin-like growth factor (IGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF), thrombopoietin (TPO)), hormones (examples include aldosterone, vasopressin, adrenocorticotropic hormone (ACTH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), oxytocin, prolactin, thyroid-stimulating hormone (TSH), renin, angiotensin, glucagon, insulin, estrogen, progesterone, parathyroid hormone (PTH), thyroid hormone, epinephrine, norepinephrine, testosterone, melatonin, growth hormone-releasing hormone (GHRH), thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone (GnRH), corticotropin-releasing hormone (CRH), humoral factors), cardiovascular drugs (e.g., anticoagulants (examples include Apibaxan, Dabigatran, Edoxaban, Heaprin, 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 statins: Atorvastatin, Fluvastatin, Lovastatin, Pitavastatin, Pravastatin, Rosuvastatin, and Simvastatin, Nitotinic acid Niacin, and cholesterol absorption inhibitors: Ezetimibe), digitalis preparations (Digoxin), diuretics (examples include Acetazolamide, Amiloride, Bumetanide, Chlorothiazide, Chlorthalidone, Furosemide, Hydro-chlorothiazide, Indapamide, Metalozone, Spironolactone, and Torsemide), and vasodilators (Isosorbidedinitrate, Isosorbide mononitrate, Hydralazine, Nitroglycerin, and Minoxidil), intestinal drugs (e.g., proton pump inhibitors (examples include Omeprazole, Lansoprazole, Rabeprazole,Esomeprazole and Pantoprazole), Histamine 2 blockers (examples include Cimetidine, Ranitidine, Famotidine, and Nizatidine), Prokinetics and laxatives (Metoclopramide), Ophthalmics (e.g., ocular antiallergics (examples include Ketorolac, Ketotifen, Loteprednol, Bepotastine, Epinastine, Emedastine, Alcaftadine, Azelastine, Olopatadine, Nedocromil, Iodoxamide, and Cromolyn), topical antibiotics (examples include Besifloxacin, Ciprofloxacin, Moxifloxacin, Ofloxacin, Gatfloxacin, Tobramycin, Gentamycin, Polymyxin D, Neomycin, Bacitracin, Azithromycin, and Erythomycin), lipid-based artificial tears (examples include castor oil, glycerin, 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., antiasthmatics (examples include dyphylline, guaifenesin, Albuterol,Levalbuterol), antihistamines (examples include Brompheniramine, Carbinoxamine, Chlorpheniramine, Clemastine, Diphenhydramine, Hydroxyzine, Tripolidine, Azelastine, Cetrizine, Desloratadine, Fexofenadine, Levocetirizine, Loratadine, Olopatadine), antitussives (examples include Dextromethorphan and Benzonatate), bronchodilators (Ipratropium, Theophylline, Albuterol, EpiNephrine, Levalbuterol, Arformoterol, Formoterol, Olodaterol, Terbutaline, Pirbuterol, Metaproterenol, Salmeterol, Isoproterenol, Indacaterol, Tiotropium, Umeclidinium, Aclidinium, Ipratropium, Revefenacin, Glycopyrrolate, Ipratropium, Theophylline, Aminopylline, and Dyphylline), decongestants (examples include Levmetamfetamine, Naphazoline, Oxymetazoline, Phenylephrine, Propylhexedrine,Pseudoephedrine and Xylometazoline, expectorants (Guaifenesin), leukotriene modulators (examples include Montelukast, Zafirlukast, Zileutron), lung surfactants (examples include Beractant, Lucinactant, Calfactant, and Poractant), mucolytics (acetylcysteine), anti-infectives (examples include Zanamivir, Ribavirin, Tobramycin, Pentamidine, and Colistimethate), inhaled corticosteroids (examples include Fluticasone, Budesone, Mometasone, Beclomethasone, and Ciclesonide), mast cell stabilizers (examples include Cromolyn and Nedocormil), phosphodiesterase-4 inhibitors (including Roflumilast), antimicrobial drugs (including antibiotics (such as aminoglycosides (examples include Amikacin, Gentamycin, Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromonmycin, Streptomycin, and Spectinomycin), ansamycins (examples include Geldanamycin, Herbimycin, and Rifaximin), carbacephems (such as Laracarbef), carbapenems (examples include Ertapenem, Doripenem, Imipenem, and Meropenem), cephalosporins (examples include Cefadroxil, Cefazolin, Cephradine, Cephapirin, Cephalothin,Cefalexin, Cefaclor, Cefoxitin, Cefotetan, Cefamandole, Cefmetazole, Cefonicid, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Maxalactam, Ceftriaxone, Cefepime, Ceftaroline fosamil, and Ceftobiprole), glycopeptides (examples include Teicoplanin, Vancomycin, Telavancin, Dalbavancin, and Oritavancin), lincosamides (Clindamycin and Lincomycin), lipopeptides (e.g. Daptomycin), macrolides (Azithromycin, Clarithromycin, Erythromycin, Roxithromycin, Telithromycin, Spiramycin, and Fidaxomicin), monobactams (e.g. Aztreonam), nitrofurans (e.g. Furazolidone and Nitrofurantoin), oxazolidinones (examples include Linezolid, Posizolid, Radezolid, and Torezolid), penicillins (examples include Amoxiciliin, Ampicillin, Azlocillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, and Temocillin), polypeptides (e.g. Bacitracin, Colistin, and Gramicidin), quinolones (e.g. Ciprofloxacin, Enoxacin, Gatifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Norfloxacin, Ofloxacin, and Perfloxacin), sulfonamides (e.g. Sulfacetamide, Sulfadiazine, Sulfamethoxazole, and Sulfisoxazole), tetracyclines (e.g. Doxycycline, Minocycline, and Tetracycline), and miscellaneous (e.g. Clofazimine, Dapsone, Metronidazole, Nitazoxanide, Paromomycin, and Trimethoprim).Oxacillin, Penicillin G, Penicillin V, Piperacillin, Temocillin, and Ticarcillin), Polypeptides (examples include Bacitracin, Cilistin, and Polymyxin B), Quinolones (examples include Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nadifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin, and Temafloxacin), Sulfonamides (examples include Mafenide, Sulfacetamide, Sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, and Sulfonamidochrysoidine), Tetracyclines (examples include Demeclocycline, Doxycycline, Metacycline, Minocycline, Oxytartacycline, and Tetracycline), Mycobacterium-specific antibiotics (examples include (Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isonioazid, Pyrazinamide, Rifampicin, Rifabutin,Rifapentine and Streptomycin), antifungal agents (e.g., polyene antifungals (examples include Amphotericin B, Candicidin, Filipin, Hamycin, Natamycin, Nystatin, and Rimocidin), azoles (examples include imidazoles: Bifonazole, Butoconazole, Clotrimazole, Econazole, Fenticonazole, Isoconazole, Ketoconazole, Luliconazole, Miconazole, Omoconazole, Oxiconazole, Sertaconazole, Sulconazole, and Tioconazole, triazoles: Albaconazole, Efinaconazole, Epoxiconazole, Fluconazole, Isavuconazole, Itraconazole, Posaconazole, Propiconazole, Ravuconazole, Terconazole, and Voriconazole, and thiazoles (e.g., Abafungin), allylamines (examples include Butenafine, Naftifine, and Terbinafine), echinocandins (Anidulafungin, Caspofungin, and Micafungin), and triterpenoids (e.g., Ibrexafungerp)), and antiparasitic agents (e.g., broad-spectrum Nitazoxanide, antiprotozoal agents (examples include Melarsoprol, Eflornithine, Metronidazole, Tinidazole, and Miltefosine), antinematodal agents (examples include Mebendazole,Pyrantel pamoate, Thiabendazole, Diethylcarbamazine and Ivermectin), Antitapeworm agents (examples include Niclosamide, Praziquantel and Albendazole), Antischistosomal agents (e.g. Praziquantel) and Antiamoebics (e.g. Rifampicin and Amphotericin B)), Anti-diabetic drugs (e.g. Insulins (analogues), Amylinomimetic drugs (e.g. Pramlintide), Alpha-glucosidase inhibitors (examples include Acarbose and miglitol), Biguanides (e.g. metformin (analogues and combinations), Dopamine agonists (e.g. Bromocriptine), Dipeptidyl peptidase-4 (DDP-4) inhibitors (examples include Alogliptin, Linagliptin, Saxagliptin and Sitagliptin), Glucagon-like peptide-1 receptor agonists (examples include Albiglutide, Dualglutaide, Exenatide, Liraglutide and Semaglutide), Meglitinides (examples include Nateglinide and Repaglinide), Sodium-glucose transporter (SGLT-2) inhibitors (examples include Dapagliflozine, Canagliflozine, Ertugliflozine and Empagliflozine), Sulfonylureas (examples include Glimepiride, Gliclazide, Glipizide, Glyburide, Chlorpropamide,Tolazamide and Tolbutamide), thiazolidinediones (e.g. Rosiglitazone and Pioglitazone), antiviral drugs (examples include Abacavir, Acyclovir, Adefovir, Amantadine, Ampligen, Amprenavir, Umifenovir, Atazanavir, Atripla, Oseltamivir, Zanamivir, Peramivir, Baloxavir, Bikctegravir, Emtricitabine, Tenofovir, Boceprevir, Bulevirtide, Cidofovir, Cobicistat, Daclatasvir, Darunavir, Delavirdine, Didanosine, Docosanol, Dolutegravir, Doravirine, Edoxudine, Efavirenz, Emtricitabine, Enfuvirtide, Ensivirtide, Ensitrelvir, Entecavir, Entravirine, Famciclovir, Fomivirsen, Fosamprenavir, Foscarnet, Ganiciclovir, Ibacitabine, Ibalizumab, Idoxuridine, Imiquimod, Insoine pranobex, Indinavir, Lamivudine, Letermovir, Lopinavir, Loviride, Maraviroc, Methisazone,Moroxydine, Nelfinavir, Nevirapine, Nitazoxanide, Norvir, Penciclovir, Pleconaril, Podophyllotoxin, Raltegravir, Remdesivir, Ribavirin, Rilpivirine, Rimantadine, Ritonavir, Saquinavir, Simeprevir, Sofosbuvir, Stavudine, Taribavirin, Telaprevir, Telbivudine, Tenofovir, Tiprenavir, Trifluridine, Trizivir, Tromantadine, Truvada, Umifenovir, Valaciclovir, Valganciclovir, Vicriviroc, Vidarabine, Zalcitabine, Zanamivir, and Zidovudine), or a structural or functional analog thereof.

[0111] In a preferred embodiment, the payload molecule is a biomolecule. For example, the biomolecule can be or comprise an amino acid, a peptide, an affimer, a polypeptide or protein, a glycoprotein, a sugar, a lipid, a lipopolysaccharide, an antibody or fragment thereof, a polymer, a nucleic acid, or a combination thereof.

[0112] The nucleic acid can be DNA, RNA, XNA (xeno nucleic acid, including 1,5-anhydrohexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), FANA (fluoroarabino nucleic acid), and unlocked nucleic acid (UNA), or a DNA / RNA hybrid sequence. Preferably, the nucleic acid is DNA or RNA.

[0113] Most preferably, the nucleic acid is RNA. The RNA can be single-stranded or double-stranded. The RNA can be selected from the group consisting of messenger RNA (mRNA); circular RNA (circRNA or oRNA); self-amplifying RNA (saRNA); reverse-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.

[0114] Preferably, the RNA is messenger RNA (mRNA).

[0115] The nucleic acid sequence, preferably the RNA, can be at least 10 bases, at least 20 bases, at least 50 bases, at least 100 bases, at least 200 bases, at least 300 bases, at least 400 bases, at least 500 bases, at least 600 bases, at least 700 bases, at least 800 bases, or at least 900 bases in length. In a preferred embodiment, the RNA is a saRNA or a mRNA.

[0116] The nucleic acid sequence, preferably the RNA, most preferably the mRNA, can be at least 100 bases, at least 500 bases, at least 1000 bases, at least 2000 bases, at least 3000 bases, at least 4000 bases, at least 5000 bases, at least 6000 bases, at least 7000 bases, at least 8000 bases, at least 9000 bases, at least 10000 bases, at least 11000 bases, or at least 12000 bases in length.

[0117] 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.

[0118] 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.

[0119] Alternatively, the nucleic acid sequence, preferably the RNA, most preferably the mRNA, can have a length of 50 to 10000 bases, 100 to 9000 bases, 200 to 8000 bases, 300 to 7000 bases, 400 to 6000 bases, 500 to 6000 bases, 600 to 5000 bases, 700 to 4000 bases, 800 to 3000 bases, or 900 to 2000 bases.

[0120] In one embodiment, the nucleic acid sequence has a length of 6000 to 15000 bases. The nucleic acid sequence can have a length of 8000 to 12000 bases. The RNA can have a length of 6000 to 15000 bases. The RNA can have a length of 8000 to 12000 bases. Preferably, the saRNA has a length of 6000 to 15000 bases. Preferably, the saRNA has a length of 8000 to 12000 bases.

[0121] In alternative embodiments, the nucleic acid sequence has a length of 100 to 14000 bases, 500 to 10000 bases, 600 to 7500 bases, 700 to 5000 bases, 800 to 4000 bases, or 900 to 2000 bases. The RNA can have a length of 400 to 14000 bases, 500 to 10000 bases, 600 to 7500 bases, 700 to 5000 bases, 800 to 4000 bases, or 900 to 2000 bases. Preferably, the mRNA has a length of 100 to 14000 bases, 500 to 10000 bases, 600 to 7500 bases, 700 to 5000 bases, 800 to 4000 bases, or 900 to 2000 bases.

[0122] The skilled person will appreciate that, when the nucleic acid is double stranded, e.g. double stranded RNA, the “base length” refers to the length of base pairs.

[0123] The weight ratio of the lipid component to the payload molecule can 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.

[0124] In embodiments where the payload molecule is a biomolecule, the weight ratio of the lipid component to the 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 weight ratio of the lipid component to the payload molecule can be 13: 1 to 25: 1, 14: 1 to 20: 1, or 15: 1 to 17: 1. In some embodiments, the weight ratio of the lipid component to the payload molecule can be 15: 1 to 27.5: 1, 20: 1 to 25: 1, or 22: 1 to 23: 1.

[0125] In embodiments where the payload molecule is a biological molecule, the N:P ratio can 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 can be 3: 1 to 10: 1 or 4: 1 to 6: 1. In alternative embodiments, the N:P ratio can be 4: 1 to 10: 1, 5: 1 to 9: 1, or 6: 1 to 8: 1. It is understood that the N:P ratio is the ratio of positively charged polymeric amine (N) groups to negatively charged nucleic acid phosphate (P) groups.

[0126] Preferably, the encapsulation efficiency of the microparticles or nanoparticles is 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 is understood that the encapsulation efficiency is determined by the amount of payload molecules encapsulated in the microparticles or nanoparticles (i.e., payload molecules that are inaccessible and / or inaccessible to the aqueous environment outside the microparticles or nanoparticles) relative to the total amount of payload molecules initially provided. The encapsulation efficiency can be determined using the RiboGreen assay, which detects solvent-accessible RNA by the increase in fluorescence upon intercalation of the RNA by the water-soluble Ribogreen reagent.

[0127] The Zeta potential of the microparticles or nanoparticles at physiological pH can be -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 is understood that the Zeta potential can be measured by suspending the LNP in an electrically conductive buffer having a specific pH value. The electrically conductive buffer can be PBS (phosphate buffered saline at pH 7.2).

[0128] The microparticles or nanoparticles can further comprise one or more adjuvants. The or each adjuvant can be selected from the group consisting of aluminium hydroxide, Pam2CSK4, Pam3CSK4, Glucopyranosyl Lipid adjuvant (GLA), LPS and analogues thereof, CpG oligodeoxynucleotides, and other TLR agonists such as Poly I:C and dsRNA.

[0129] The microparticles or nanoparticles can further comprise one or more additional compounds. The one or more additional compounds can be selected from the group consisting of hydrophobic compounds, polymers, permeability enhancing molecules, carbohydrates, surface modifiers, excipients, and combinations thereof. The polymer can be polylactic glycolic acid (PLGA). It is understood that the excipient can alter the pharmacokinetic properties of the composition but not the pharmacodynamic properties of the payload.

[0130] According to a third aspect, there is provided a composition comprising a plurality of the microparticles or nanoparticles of the second aspect.

[0131] The microparticles or nanoparticles can have an average diameter of less than 10 pm, less than 1 pm, less than 500 nm, or less than 250 nm. More preferably, the microparticles or nanoparticles can have an average diameter of less than 200 nm, less than 175 nm, less than 150 nm, less than 125 nm, less than 100 nm, less than 90 nm, or less than 80 nm. The microparticles or nanoparticles can have an average diameter of from 25 nm to 1 pm, from 30 nm to 500 nm, from 35 nm to 250 nm, from 40 nm to 200 nm, from 45 nm to 150 nm, from 50 nm to 125 nm, from 55 nm to 100 nm, from 60 nm to 90 nm, or from 65 nm to 80 nm. The average diameter of the microparticles or nanoparticles can be measured using dynamic light scattering.

[0132] The microparticles or nanoparticles can have a polydispersity index (PDI) of less than 0.5, less than 0.4, or less than 0.3, more preferably the PDI can be less than 0.25, less than 0.2, less than 0.15, less than 0.13, or less than 0.11. The microparticles or nanoparticles can have a PDI of from 0.001 to 0.05, from 0.005 to 0.4, from 0.01 to 0.3, from 0.02 to 0.025, from 0.04 to 0.2, from 0.06 to 0.15, from 0.08 to 0.13, or from 0.09 to 0.11.

[0133] The composition can comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can improve colloidal stability, especially under concentrated and / or refrigerated conditions (e.g., storage and / or transport at temperatures of from 4 °C to -80 °C, for example at 4 °C, -20 °C, -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.

[0134] The composition can further comprise one or more solvents, buffers, suspension aids, fillers, glidants, binders, salts, isotonic agents, thickening agents, emulsifiers, and / or preservatives.

[0135] In a fourth aspect, there is provided the microparticles or nanoparticles of the second aspect, or the composition of the third aspect, for use as a medicament.

[0136] In a fifth aspect, there is provided the microparticles or nanoparticles of the second aspect, or the composition of the third aspect, for use in the treatment and / or prevention and / or prophylaxis of a disease or condition.

[0137] In a sixth aspect, there is provided a method of treating and / or preventing a disease or condition, the method comprising administering or having administered to a subject in need a prophylactic and / or therapeutic amount of the microparticles or nanoparticles of the second aspect or the composition of the third aspect.

[0138] The disease or condition can be selected from an inflammatory disease, an infectious disease, a proliferative disease (e.g. cancer), an autoimmune disease, an ocular disease, a pulmonary disease, a skin disease, an intestinal disease, a metabolic disease (e.g. diabetes), a vascular disease (including cardiovascular disease and renal vascular disease), a nervous system disease (e.g. a neurodegenerative disease), an endocrine system disorder (including disorders associated with hormones, growth factors and / or cytokines), a reproductive system disorder and a rare disease.

[0139] In a seventh aspect, there is provided a vaccine composition comprising the microparticles or nanoparticles of the second aspect or the composition of the third aspect.

[0140] The vaccine can comprise a suitable adjuvant.

[0141] In an eighth aspect, there is provided the microparticles or nanoparticles of the second aspect or the composition of the third aspect or the vaccine of the seventh aspect for use in stimulating an immune response in a subject.

[0142] The immune response can be stimulated against a protozoan, a bacterium, a virus, a fungus, a multicellular parasite or a cancer, or a portion thereof.

[0143] 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 a prophylactic and / or therapeutic amount of the microparticles or nanoparticles of the second aspect or the composition of the third aspect or the vaccine of the seventh aspect.

[0144] The microparticles or nanoparticles, compositions or vaccines of the application can be combined into a variety of different forms of compositions, depending on the manner in which the composition is to be used. Thus, for example, the composition can be a powder, a tablet, a capsule, a liquid, an ointment, a cream, a shampoo, a gel, a hydrogel, an aerosol, a spray, a micellar solution, a transdermal patch (including microneedles), a drug reservoir / sustained release formulation, a (liposomal) suspension, a lotion / drip, a biomaterial incorporated into regenerative medicine, a coating for a (implantable) medical device, or any other suitable form that can be administered to a human or animal in need of treatment. It will be appreciated that the carrier of the medicament of the application should be one that is well tolerated by the subject receiving it.

[0145] The microparticles or nanoparticles, compositions or vaccines of the application can also be incorporated into a sustained or delayed release device. Such a device can be inserted, for example, on or under the skin, from which the drug can be released over weeks or even months. The device can be located at least proximal to or upstream of the site of treatment.

[0146] However, in a preferred embodiment, the medicament of the present application can be administered to a subject by injection into the blood, muscle, skin, or directly into the site requiring treatment. The injection can 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).

[0147] It will be appreciated that the amount of microparticles or nanoparticles, composition or vaccine required depends on its intended therapeutic or prophylactic use and its bioactivity and bioavailability, which in turn depends on the mode of administration, the physicochemical properties of the payload, microparticles or nanoparticles, composition or vaccine, and whether it is used as monotherapy or in combination therapy.

[0148] The frequency of administration will also be influenced by the half-life of the active agent in the subject being treated and / or the half-life of the therapeutic effect (e.g. the half-life of a therapeutic protein translated from a therapeutic mRNA transfected by the microparticles or nanoparticles composition, or the half-life of e.g. an RNA-based CRISPR gene therapy). The optimal dose of administration can be determined by one skilled in the art and will vary according to the strength of the microparticles or nanoparticles, composition or vaccine, pharmaceutical composition used, the mode of administration and the type of therapy. Other factors depending on the particular subject being treated will result in a need to adjust dosages, including the age, body weight, gender, diet, and time of administration of the subject.

[0149] The required dosage can depend on a number of factors including, but not limited to, the active agent being administered, the disease being treated and / or vaccinated against, the subject being treated, etc.

[0150] Generally, the microparticles or nanoparticles, composition or vaccine of the present application can be used in a dosage of 0.001 pg / kg body weight to 10 mg / kg body weight, or 0.01 pg / kg body weight to 1 mg / kg body weight, depending on the active agent used. The dosage can be understood in relation to the number of payload molecules delivered.

[0151] The dosage can be given as a single administration (e.g. a single injection). Alternatively, the microparticles or nanoparticles, composition or vaccine can require more than one administration. For example, the microparticles or nanoparticles, composition or vaccine can be administered in two or more doses of 0.07 pg to 700 mg (i.e. assuming a body weight of 70 kg). Alternatively, a sustained release device can be used to provide the optimal dosage of the microparticles or nanoparticles, composition or vaccine of the present application to the patient without the need to administer repeated doses. The route of administration can include intravenous, intradermal, subcutaneous, intramuscular, intrathecal, epidural, intravitreal, or intraperitoneal injection routes.

[0152] Specific formulations of the microparticles or nanoparticles, compositions or vaccines of the application, as well as precise treatment regimens (e.g., dosage amounts and frequency of administration of the agent) can be formed using known procedures, such as those routinely employed by the pharmaceutical industry (e.g., in vivo experiments, clinical trials, etc.).

[0153] A "subject" can be a vertebrate, a mammal, or a domestic animal. Thus, the compositions and medicaments of the application can be used to treat any mammal, such as a livestock animal (e.g., a horse), a pet, or can be used in other veterinary and agricultural applications. Most preferably, however, the subject is a human.

[0154] A "therapeutically effective amount" of microparticles or nanoparticles, compositions or vaccines refers to any of the above amounts needed to produce a therapeutic effect (whether partial or complete) when administered to a subject.

[0155] For example, a therapeutically effective amount of microparticles or nanoparticles, compositions or vaccines of the application can comprise about 0.001 pg to about 800 mg of a payload molecule, and preferably comprises about 0.01 mg to about 500 mg of a payload molecule.

[0156] A "pharmaceutically acceptable vehicle" 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.

[0157] In one embodiment, the pharmaceutically acceptable vehicle can be a solid, and the composition can be in the form of a powder, a capsule, or a tablet. The solid pharmaceutically acceptable vehicle can include one or more substances that can also act as a flavoring agent, a lubricant, a solubilizing agent, a suspending agent, a dye, a filler, a glidant, a compression aid, an inert binder, a sweetener, a preservative, a dye, a coating agent, or a tablet disintegrant. The vehicle can also be an encapsulating material. In a powder, the vehicle is a finely divided solid, mixed with a finely divided active agent of the application. In a tablet, the active agent (e.g., microparticles or nanoparticles of the application) can be mixed with a vehicle having the necessary compression properties in the proper proportion, and compressed into the desired shape and size. The pharmaceutical vehicle can be a gel, and the composition can be in the form of a cream or the like.

[0158] Alternatively, the pharmaceutical carrier can be a liquid, and the pharmaceutical composition in the form of a solution. Liquid carriers are used in the preparation of solutions, suspensions, emulsions, syrups, elixirs, and pressurized compositions. The microparticles or nanoparticles of the present application can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, organic solvents, mixtures of both, or pharmaceutically acceptable oils or fats. The liquid carrier can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colorants, viscosity regulators, stabilizers, or osmo-regulators. Suitable examples of liquid carriers for oral and parenteral administration include water, partially

[0159] 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 microparticles or nanoparticles of the present application can be prepared in any suitable sterile injectable medium.

[0160] The microparticles or nanoparticles can be administered by inhalation. For example, the microparticles or nanoparticles can be provided in the form of an aerosol.

[0161] The microparticles or nanoparticles and / or compositions of the present application can be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitol, polysorbate 80 (oleate esters of sorbitan copolymerized with ethylene oxide) and the like. The microparticles or nanoparticles and / or compositions of the present application can also be administered in the form of liquid or solid compositions of the dosage forms appropriate for oral administration. Suitable compositions for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms suitable for parenteral administration include sterile solutions, emulsions, and suspensions.

[0162] All of the features described herein (including any accompanying claims, abstract and drawings) and / or all of the steps of any method or process so disclosed can be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0163] For a better understanding of the present application, and to show how embodiments of the application can be practiced, reference will now be made, by way of example, to the accompanying drawings in which:

[0164] Figure 1 Average diameter and polydispersity index (PDI) of LNP formulations measured by dynamic light scattering (DLS) are shown, wherein the lipid component of the LNP comprises 50 mol% ionizable lipid, 38.5 mol% structural lipid, 10 mol% phospholipid, and 1.5 mol% PEG-modified lipid, and the structural lipid is resveratrol (RSV), cholesterol (chol), or a combination thereof, as shown in Figure 1 wherein the ratio of the combination is in molar ratio;

[0165] Figure 2 Average diameter and polydispersity index (PDI) of LNP formulations containing 5 mol% DOTAP as uptake promoter measured by dynamic light scattering are shown, wherein the lipid component of the LNP comprises 45 mol% ionizable lipid, 5 mol% DOTAP, 38.5 mol% structural lipid, 10 mol% phospholipid, and 1.5 mol% PEG-modified lipid, and the structural lipid is resveratrol (RSV), cholesterol (chol), or a combination thereof, as shown in Figure 1 wherein the ratio of the combination is in molar ratio;

[0166] Figure 3 pKas of various LNP formulations containing different structural lipids or mixtures thereof, all percentages in molar percentage, determined by 2-(p-Toluidinyl)naphthalene-6-sulfonic acid (TNS) assay at pH ranging from 3 to 10 in increments of 0.5 pH at 20 °C are shown;

[0167] Figure 4 Encapsulation efficiency of LNP compositions prepared with different core lipids and core lipid mixtures, all percentages in molar percentage, determined by RiboGreen assay are shown; core lipids are resveratrol (RSV), cholesterol or mixtures thereof, or resveratrol derivatives or related structures;

[0168] Figure 5 shows toxicity tests of LNP formulations containing different core lipids and core lipid mixtures determined by resazurin assay. (A) shows the effect of resveratrol and cholesterol alone and when mixed on the metabolic activity of HeLa cells, all ratios in molar ratio; (B) shows the effect of resveratrol derivatives and related structures on the toxicity of LNP formulations containing the structures as core lipids;

[0169] Figure 6 provides in vitro activity of LNP compositions containing different core lipids and mixtures of core lipids as measured by the secreted nanoluciferase assay. (A) shows the effect of resveratrol and cholesterol alone and when mixed on transfection efficiency of LNP formulations containing the structures as core lipids, all ratios are molar ratios; (B) shows the effect of resveratrol derivatives and related structures on transfection efficiency of LNP formulations containing the structures as core lipids; and

[0170] Figure 7 Figure 7 shows the lack of ApoE mediated uptake and functional delivery in HeLa cells, transfections were performed with fresh addition of different amounts of ApoE in the culture medium, followed by determination of transfection efficiency by the secreted nanoluciferase assay.

[0171] Examples

[0172] Example 1: Production of Lipid Nanoparticles (LNPs)

[0173] To determine the safety and efficacy of the lipid compositions of the present application for delivery of therapeutic and / or prophylactic molecules to cells, a series of formulations were prepared and tested.

[0174] Uniform sized nanoparticles can be reproducibly produced using an inverted herringbone microfluidic mixer. The mixer rapidly (on the order of milliseconds) mixes an aqueous fluid, typically containing a water soluble therapeutic and / or prophylactic molecule, with an organic solvent containing pre-mixed lipid components. Other types of microfluidic mixers (e.g. Y-junction, T-junction or direct high speed injection) produce similar results provided similar fluid flow mixing ratios and mixing speeds are obtained.

[0175] Lipid compositions were prepared by combining core / structural lipids (all from Sigma-Aldrich) with the ionisable lipid LI (di((9Z, 12Z)-octadeca-9, 12-dien-1-yl) 2-((2- (dimethylamino)ethyl)thio)succinate maleate salt) disclosed in UK patent application GB2215200.3, a phospholipid (e.g. DOPE, obtained from Avanti Polar Lipids), an optional cationic lipid (e.g. DOTAP, obtained from Avanti Polar Lipids), a PEG-modified lipid (e.g. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (polyethylene glycol)-2000 (also known as PEG-DSPE), obtained from Avanti Polar Lipids). Control formulations contained cholesterol (obtained from Sigma-Aldrich) as a structural lipid. Typical combined proportions of lipids were 45 mol% ionisable lipid, 5 mol% DOTAP, 38.5 mol% structural lipid, 10 mol% phospholipid and 1.5 mol% PEG-modified lipid. For lipids not containing DOTAP, the combined proportions of lipids were 50 mol% ionisable lipid, 38.5 mol% structural lipid, 10 mol% phospholipid and 1.5 mol% PEG-modified lipid.

[0176] Certain formulations exhibit low generic cellular uptake in vitro and / or in vivo, which can be overcome by the incorporation of a cellular receptor or cell membrane targeting ligand, such as a natural ligand or an antibody to a cell surface receptor. Alternatively, the zeta potential of the nanoparticle composition is increased to at least +1 mV, but preferably +10 mV, to interact generically with the plasma membrane, thereby facilitating uptake of the nanoparticle and / or cargo. In this case, the following lipid proportions are used: 40-50 mol% ionisable lipid, 0-10 mol% cationic lipid, 38.5 mol% structural lipid, 10 mol% phospholipid and 1.5 mol% PEG-modified lipid.

[0177] The lipid mixtures were diluted with ethanol to a total concentration of 12.5 mM total lipid to 50 mM total lipid. Lipids dissolved in ethanol were stored at -20 °C under argon, protected from light.

[0178] Nanoparticle compositions are prepared by mixing an acidic (pH 4 or 5) or neutral (pH 7.4) aqueous solution of a therapeutic and / or prophylactic molecule with an ethanol solution of a lipid mixture, where the weight ratio of lipid to therapeutic molecule is 5: 1 to 50: 1. To produce a defined population of nanoparticles, the aqueous solution comprising the therapeutic and / or prophylactic molecule ("aqueous solution") is rapidly mixed with the lipid solution at a volume ratio of 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.

[0179] To prepare a nanoparticle composition containing RNA, the RNA is diluted to about 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 weight ratio of RNA to lipid is typically 1: 10 to 1:30, such that the N:P ratio is 3 to 12, preferably about 5.

[0180] After mixing the aqueous solution and lipid solution, the nanoparticle composition is dialyzed to remove ethanol to less than 0.1 vol%, optionally concentrate or dilute the solution, and change the buffer to a buffer at physiological pH (e.g., pH 7.4, such as phosphate buffered saline (PBS)). Using 100 kDa MWCO dialysis tubing (e.g., Spectrum™ Spectra / Por™ Biotech Cellulose Ester (CE) Dialysis Membrane Tubing, available from Fisher Scientific), the formulation is dialyzed against at least a 100-fold excess of PBS three times. The first dialysis step is performed at room temperature for at least 2 hours, and subsequent dialysis rounds are performed at room temperature for at least 8 hours, or overnight at 4°C.

[0181] The size distribution of the LNPs is determined by dynamic light scattering (DLS) using a Zetasizer Pro (red label, Malvern) with LNP standard settings (NIBS, adaptive correlation).

[0182] LNP were formed using secNLuc (PolyA ~1000 nt) mRNA and their size was measured in lx PBS (10 mM Phosphate Buffer, 150 mM NaCl) pH 7.4 using DLS. Various LNP formulations were produced (50 mol% ionizable lipid (LI; bis((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-((2- (dimethylamino)ethyl)thio)succinate maleate), 38.5 mol% structural lipid (Resveratrol (RSV), Cholesterol or mixtures thereof), 10 mol% phospholipid (DOPE) and 1.5 mol% DSPE-PEG(2000), N / P of 5, corresponding to a lipid to oligo weight (LOW) of about 16). As shown in Figure 1 the average size of these formulations was 70 nm (65-90 nm) with an average polydispersity index (PDI) of 0.1.

[0183] Next, formulations containing 5% DOTAP were produced; (45 mol% ionizable lipid (LI; bis((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-((2-(dimethylamino)ethyl)thio)succinate maleate), 5 mol% DOTAP, 38.5 mol% structural lipid (Resveratrol, Cholesterol or mixtures thereof), 10 mol% phospholipid (DOPE) and 1.5 mol% DSPE-PEG(2000), N / P of 5, corresponding to a lipid to oligo weight (LOW) of about 16). Interestingly, the addition of DOTAP did not significantly change the size or PDI of the LNP, as shown in Figure 2

[0184] Example 2: pKa determination of lipids

[0185] ​The pKa of an ionizable lipid is known to be a major determinant of LNP content escape from endosomes upon exposure to cells. During endosomal uptake and transit through endosomal compartments, the pH of the LNP gradually decreases from physiological pH (pH 7.4, as found outside of cells) to around pH 4.5-5.0 in the lumen of the lysosome, which is the terminal stage of most endocytic vesicles. Transport to the lysosome should generally be avoided, as the degradative environment can lead to disintegration of the cargo (e.g. mRNA), and thus endosomal escape is ideally occurring at a pH higher than 5.0, thus before the endosome matures into a lysosome. The rapid increase in cationic charge of the ionizable lipid during this acidification process is thought to contribute to the interaction with the inner membrane of the endosome. Therefore, a lipid that carries a neutral or near-neutral charge at pH 7.4 and is fully ionized at pH 5.5 is considered ideal; this corresponds to a pKa of around 6.4-6.5, similar to the optimal value found by Jayaraman et al. (2012 Angewandte Chemie, DOI: 10.1002 / ang.201203263).

[0186] The local environment of an ionizable lipid (in relation to incorporation into an LNP) can influence the acquisition of charge, and thus the pKa value. It is understood that core lipids located just below the surface of the LNP can alter the surface properties, and thus the pKa. Therefore, the pKa value of the ionizable lipid LI was experimentally determined in the presence of 20 mM phosphate-citrate-ammonium citrate solutions (pH from 3 to 10 in increments of 0.5, all from Sigma-Aldrich) by adding 2-(p-Toluidinyl) naphthalene-6-sulfonic acid (TNS; from Sigma-Aldrich) to a nanoparticle composition containing the core lipids of the application or mixtures thereof. TNS is a compound that electrostatically interacts with cationic lipids to produce fluorescence. Briefly, a mixture of 50 pi 5 uM TNS, 25 pM LNP (containing 12.5 pM ionizable lipid) and 20 mM buffer was subsequently added to samples of phosphate-citrate-ammonium citrate buffer in a 384-well plate and measured on a microplate reader (iD3, Molecular Devices) at 325 nm excitation and 435 nm emission wavelength. Samples containing only LNP and buffer were used for background subtraction for each pH increment. The measured fluorescence after background correction 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 lipid composition. The pKa of each ionizable lipid was determined as the pH value at which half the maximum fluorescence was reached, Figure 3 Results are given for each LNP composition containing the core lipids of the application, cholesterol or mixtures thereof.

[0187] Notably, all core lipids did not significantly change the pKa of LNP formulations based on LI ionizable lipids. It can thus be concluded that core lipids do not affect the ionization of ionizable lipids.

[0188] Example 3: Encapsulation efficiency of LNP

[0189] High encapsulation efficiency of oligonucleotide and polynucleotide loads is essential to prevent the load from being exposed to degrading enzymes, immunostimulatory cells and receptors and to achieve high transfection efficiency. Therefore, the encapsulation efficiency was determined using the RiboGreen assay.

[0190] The nanoparticle compositions containing secreted NanoLuc (m)RNA at a concentration of about 5 pg / ml in TE buffer (10 mM Tris, HC1 pH 7.5, 1 mM EDTA, obtained from Sigma-Aldrich) or TE buffer containing 2% Triton X-100 (Sigma-Aldrich) at N / P 5:1, LOW ~ 16 were mixed 1 : 1 with an equal volume of 1 : 100 RiboGreen reagent using the QUANT-IT RIBOGREEN RNA Assay Kit (Invitrogen, obtained from Thermo Fisher Scientific). The samples were mixed well and incubated at room temperature for 5 minutes. Next, the fluorescence intensity was measured using a microplate reader (iD3, Molecular Devices) at 480 nm excitation and 520 nm emission wavelength. Blank solutions containing TE buffer with 1 :200 RiboGreen reagent or 2% Triton X-100 buffer with 1 :200 RiboGreen reagent were used as a fluorescence background control. The absolute amount of unencapsulated RNA was quantified using a standard curve of naked (m)RNA. The signal obtained with 2% Titron was used to normalize all samples, which was set to 100%. The results are listed in Table 1 and Figure 4 .

[0191] Table 1: Details of LNP compositions and mRNA encapsulation efficiency

[0192]

[0193] All LNP compositions of the present application exhibited high encapsulation efficiency and are thus suitable to protect oligonucleotides and polynucleotides from the action of degrading enzymes and to prevent any other influence of exposed oligonucleotides and polynucleotides.

[0194] Example 4: Toxicity of LNP formulations to cells

[0195] Each of the core lipids of the invention tested are considered GRAS (Generally Recognized as Safe) compounds, which are present in various food products. Therefore, the inventors investigated the impact of the core lipids on the toxicity of the complete LNP formulation. Given the well-known anti-inflammatory and antioxidant properties of the core lipids, both their toxic and protective effects are of interest. To measure the toxic effect of the core lipids, the inventors added a range of concentrations of LNP to the cells, depending on the amount of mRNA added to the cells.

[0196] Briefly, a concentration range of 10-100 ng of formulated mRNA (corresponding to 160-1600 ng of total lipid) was added to HeLa cells pre-mixed with cell culture medium in a total volume of 100 mΐ per well in a 96-well plate. After 24 hours of incubation, metabolic activity was tested by the resazurin assay. For this, the medium was replaced by medium containing 0.1 mg / ml resazurin and incubated for 1-4 hours at 37 °C and 5% C02. Subsequently, fluorescence in the supernatant was measured (excitation wavelength 540 / 25 nm, emission wavelength 620 / 40 nm).

[0197] As shown in Figure 5, an increase in lipid concentration did not result in any significant toxic effect on HeLa cells.

[0198] Example 5: In vitro activity of core lipid LNP

[0199] To determine whether alternative core lipid-containing LNP formulations are able to efficiently deliver mRNA to cells, the inventors incubated cells with LNP containing luciferase mRNA, which contained the core lipid of interest and 5% DOTAP as uptake enhancer.

[0200] Core lipid-containing LNP was formulated together with 5 mol% DOTAP (45 mol% LI, 5 mol% DOTAP, 10 mol% DOPE, 38.5 mol% resveratrol, resveratrol derivatives and / or cholesterol, and 1.5 mol% DSPE-PEG(2000)) as described in Example 1, containing secreted nanoluciferase mRNA, at an N / P of 5:1 (corresponding to LOW of about 16). A dose range of LNP (corresponding to 100, 50 and 10 ng per well, respectively, in a volume of 100 mΐ) was added to HeLa cells in a 96-well plate. After 24 hours, the medium was collected and secreted nanoluciferase activity was measured using the Nano-Glo Luciferase Assay System (Promega).

[0201] Incubation of HeLa cells with LNP containing resveratrol-lipids showed a dose-dependent induction of luciferase activity compared to LNP containing cholesterol as core lipid only. Especially when the ratio of resveratrol compared to cholesterol was high, an increase of up to 300% compared to the control (containing cholesterol as core lipid only) was observed. Interestingly, several resveratrol structural variants performed similarly, but deoxysanguiin performed worse, while polydatin performed better than the structural variants.

[0202] As Figure 6A (activity of LNP with cholesterol, resveratrol or mixtures thereof) and Figure 6B (activity of LNP with resveratrol derivatives, compared to cholesterol and resveratrol) is shown.

[0203] Example 6: Resveratrol-containing LNP do not bind to ApoE in vitro

[0204] To determine the absence of ApoE-dependent uptake, cellular uptake of resveratrol- containing LNP was tested with a range of concentrations of ApoE.

[0205] Resveratrol- or cholesterol-containing LNP containing LI were formulated as described in Example 1 with secreted nanoluciferase mRNA. In addition, resveratrol- and cholesterol- containing LNP were formulated with 5% DOTAP. LNP corresponding to 100 ng mRNA per well (volume 100 μΐ) were added to HeLa cells in a 96-well plate in the presence or absence of ApoE (R&D Systems) at concentrations ranging from 1 to 20 μg / ml. After 24 hours, the culture medium was collected and secreted nanoluciferase activity was measured using the Nano-Glo Luciferase Assay System (Promega).

[0206] Incubation of HeLa cells with resveratrol-containing LNP did not induce luciferase activity, neither in the presence nor in the absence of increasing concentrations of ApoE. In contrast, cholesterol-containing LNP showed a dose-dependent increase in luciferase activity. Resveratrol-containing LNP containing DOTAP showed similar or even increased luciferase activity compared to cholesterol-containing LNP containing DOTAP, demonstrating that resveratrol-LNP are not inherently inactive, as Figure 7 indicated.

[0207] Thus, resveratrol-containing LNP show an uptake mechanism that is independent of ApoE, which sets them apart from cholesterol-containing LNP and enables an ApoE- independent biodistribution.

Claims

1. Use of a compound of formula (I) or a pharmaceutically acceptable complex, salt, solvate, tautomer or polymorphic form thereof in micron- or nanoparticle formulations: (I) Where L 1 for , or ; R 1 To R 10 Each independently represents H and OR. 11 OCOR 11 COOR 11 NR 11 R 12 N + R 11 R 12 R 13 Optional substitution of C 1-30 Alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl group, optionally substituted C 3-10 cycloalkyl, optionally substituted C 3-10 Cycloalkenyl, optionally substituted 3- to 10-membered heterocycles, optionally substituted C 6-12 aryl, optionally substituted 5 to 10 heteroaryl or sugar moieties; and R 11 To R 13 Each independently represents H, and the C is optionally substituted. 1-30 Alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl group, optionally substituted C 3-10 cycloalkyl, optionally substituted C 3-10 Cycloalkenyl, optionally substituted 3- to 10-membered heterocycles, optionally substituted C 6-12 Aryl or optionally substituted 5 to 10 heteroaryl groups.

2. The use according to claim 1, wherein the compound of formula (I) is used as a core lipid in microparticle or nanoparticle formulations.

3. The use according to any one of the preceding claims, wherein: - R 1 It is H; - R 3 H, C with optional substitution 1-30 Alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl group, optionally substituted C 3-10 cycloalkyl, optionally substituted C 3-10 Cycloalkenyl, optionally substituted 3- to 10-membered heterocycles, optionally substituted C 6-12 Aryl or optionally substituted 5 to 10 heteroaryl groups; - R 5 It is H; - R 6 For H, OR 11 OCOR 11 COOR 11 NR 11 R 12 or N + R 11 R 12 R 13 And R 11 To R 13 H independently, or C with optional substitution 1-12 Alkyl, optionally substituted C 2-12 alkenyl, optionally substituted C 2-12 alkynyl group, optionally substituted C 3-10 cycloalkyl, optionally substituted C 3-10 Cycloalkenyl, optionally substituted 3- to 10-membered heterocycles, optionally substituted C 6-12 Aryl or optionally substituted 5 to 10 heteroaryl groups; - R 7 For H, OR 11 OCOR 11 COOR 11 NR 11 R 12 N + R 11 R 12 R 13 Optional substitution of C 1-30 Alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl group, optionally substituted C 3-10 cycloalkyl, optionally substituted C 3-10 Cycloalkenyl, optionally substituted 3- to 10-membered heterocycles, optionally substituted C 6-12 Aryl or optionally substituted 5 to 10 heteroaryl groups, and R 11 To R 13 H independently, or C with optional substitution 1-12 Alkyl, optionally substituted C 2-12 alkenyl, optionally substituted C 2-12 alkynyl group, optionally substituted C 3-10 cycloalkyl, optionally substituted C 3-10 Cycloalkenyl, optionally substituted 3- to 10-membered heterocycles, optionally substituted C 6-12 Aryl or optionally substituted 5 to 10 heteroaryl groups; - R 9 Is it H or OR? 11 OCOR 11 COOR 11 NR 11 R 12 N + R 11 R 12 R 13 Optional substitution of C 1-30 Alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl group, optionally substituted C 3-10 cycloalkyl, optionally substituted C 3-10 Cycloalkenyl, optionally substituted 3- to 10-membered heterocycles, optionally substituted C 6-12 Aryl or optionally substituted 5 to 10 heteroaryl groups, and R 11 To R 13 H independently, or C with optional substitution 1-12 Alkyl, optionally substituted C 2-12 alkenyl, optionally substituted C 2-12 alkynyl group, optionally substituted C 3-10 cycloalkyl, optionally substituted C 3-10 Cycloalkenyl, optionally substituted 3- to 10-membered heterocycles, optionally substituted C 6-12 aryl or optionally substituted 5 to 10 heteroaryl groups; and / or - R 10 It is H.

4. The use according to any one of the preceding claims, wherein the compound is a compound of formula (Ia), (Ib), (Ic) or (Id): 。 5. The use according to any one of the preceding claims, wherein L 1 yes or .

6. The use according to any one of the preceding claims, wherein R 2 R 4 and R 8 Independently for H, OR 11 OCOR 11 COOR 11 NR 11 R 12 N + R 11 R 12 R 13 Or the sugar portion, and R 11 To R 13 Each independently represents H, and the C is optionally substituted. 1-20 Alkyl, optionally substituted C 2-20 alkenyl, optionally substituted C 2-20 alkynyl group, optionally substituted C 3-10 cycloalkyl, optionally substituted C 3-10 Cycloalkenyl, optionally substituted 3- to 10-membered heterocycles, optionally substituted C 6-12 Aryl or optionally substituted 5 to 10 heteroaryl groups.

7. The use according to claim 6, wherein R 2 R 4 and R 8 Independently H, OH, OCH3 or Or the sugar portion or the following general formula: or Where X 1 For CR 20 R 21 The key; and R 17 To R 25 Each of the following is independently H, OH, CH2OH, COOH, NH2, C 1-6 Alkyl, halogen, or .

8. The use according to any one of the preceding claims, wherein the compound of formula (I) is: 。 9. A microparticle or nanoparticle comprising a compound of formula (I) or a pharmaceutically acceptable complex thereof, salt, solvate, tautomer or polymorphic form, and optionally a payload molecule: (I) Where L 1 for , or ; R 1 To R 10 Each independently represents H and OR. 11 OCOR 11 COOR 11 NR 11 R 12 N + R 11 R 12 R 13 Optional substitution of C 1-30 Alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl group, optionally substituted C 3-10 cycloalkyl, optionally substituted C 3-10 Cycloalkenyl, optionally substituted 3- to 10-membered heterocycles, optionally substituted C 6-12 aryl, optionally substituted 5 to 10 heteroaryl or sugar moieties; and R 11 To R 13 Each independently represents H, and the C is optionally substituted. 1-30 Alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl group, optionally substituted C 3-10 cycloalkyl, optionally substituted C 3-10 Cycloalkenyl, optionally substituted 3- to 10-membered heterocycles, optionally substituted C 6-12 Aryl or optionally substituted 5 to 10 heteroaryl groups.

10. The microparticles or nanoparticles according to claim 9, wherein the microparticles or nanoparticles are lipid nanoparticles (LNPs), liposomes, lipid complexes, micelles or lipid vesicles, and preferably LNPs.

11. The microparticles or nanoparticles according to claim 9 or 10, wherein the microparticles or nanoparticles comprise a lipid component, the lipid component comprising a compound of formula (I) or a pharmaceutically acceptable complex thereof, a salt, a solvate, a tautomer or a polymorphic form, and one or more other lipids selected from phospholipids, permanently cationic lipids, ionizable cationic lipids, permanently anionic lipids, ionizable anionic lipids, structural lipids, shielding lipids, functionalized lipids, other core lipids, and combinations thereof.

12. The microparticles or nanoparticles according to any one of claims 9 to 12, wherein the lipid component of the microparticles or nanoparticles comprises 5 to 80 mol%, 10 to 60 mol%, 20 to 50 mol%, 30 to 45 mol%, or 35 to 40 mol% of a compound of formula (I) or a pharmaceutically acceptable complex, salt, solvate, tautomer, or polymorphic form thereof.

13. The microparticles or nanoparticles according to any one of claims 9 to 12, wherein the microparticles or nanoparticles comprise a payload molecule, said payload molecule being a biomolecule, an active pharmaceutical ingredient (API), and / or a diagnostic compound.

14. The microparticles or nanoparticles according to any one of claims 9 to 13, wherein the payload molecule is a biomolecule and is or includes amino acids, peptides, affinity molecules, polypeptides or proteins, glycoproteins, sugars, lipids, lipopolysaccharides, antibodies or fragments thereof, polymers or nucleic acids, or combinations thereof, preferably wherein the biomolecule is or includes nucleic acids.

15. The microparticles or nanoparticles according to claim 14, wherein the nucleic acid is DNA, RNA, heteronucleotide (XNA), cyclohexene nucleic acid (CeNA), threonine nucleic acid (TNA), glycol nucleic acid (GNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), fluoroarabinonucleotide (FANA), non-locked nucleic acid (UNA), or a DNA / RNA heterosequence.

16. The microparticles or nanoparticles according to claim 15, wherein the nucleic acid is RNA, and the RNA is single-stranded or double-stranded and selected from: messenger RNA (mRNA); circular RNA (circRNA or oRNA); self-amplifying RNA (saRNA); anti-amplifying RNA (taRNA), long non-coding RNA, fissile replicon RNA, viral RNA, antisense RNA (AON or asRNA); RNA aptamers; interfering RNA; microRNA (miRNA); short interfering RNA (siRNA); short hairpin RNA (shRNA); and small RNA.

17. A composition comprising a plurality of micron-sized or nanoparticles as described in any one of claims 9 to 16.

18. The micron or nanoparticles according to any one of claims 9 to 16, or the composition according to claim 17, used as a pharmaceutical.

19. Microparticles or nanoparticles according to any one of claims 9 to 16, or the composition according to claim 17, for the treatment and / or prevention and / or relief of diseases or conditions.

20. A vaccine composition comprising micron or nanoparticles as described in any one of claims 9 to 16, or the composition of claim 17.

21. The micron or nanoparticles according to any one of claims 9 to 16, or the composition according to claim 17, or the vaccine according to claim 20, for stimulating an immune response in a subject.

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