Novel structured lipids for lipid nanoparticle formulations

Abstract

The present disclosure provides for the use of compounds of Formula I in lipid micro- and nanoparticle compositions. The present disclosure also extends to micro- or nanoparticles comprising a compound of Formula I and an optional payload molecule, and medical uses thereof.

Description

[Technical Field]

[0001] The present invention relates to the use of compounds in lipid micro- and nanoparticle compositions, as well as to novel lipid micro- and nanoparticle compositions themselves. The compositions are suitable for therapeutic or prophylactic delivery with improved stability and enhanced control over biodistribution. In particular, the lipid micro- and nanoparticle compositions can be used to deliver (modified) RNA or DNA. The present invention further relates to medical and research uses of the compositions. [Background technology]

[0002] Nanomedicine involves the use of nanoscale materials, such as biocompatible nanoparticles, for diagnostic, delivery, sensing, or actuation purposes. This is an increasingly important field, promising to provide novel diagnostic, therapeutic, and / or preventative options. In particular, micro- and nanoparticle formulations can enable the use of molecular entities that would otherwise be (a) unstable, (b) not conform to drug-likeness rules such as Lipinski's rule of five, and / or (c) have unfavorable pharmacokinetics, including inappropriate biodistribution behavior, thereby expanding the use of these molecular entities in terms of dosing regimens, concentrations, and thus therapeutic efficacy and / or delivery to specific target cells. In these formulations, micro- or nanoparticles encapsulate, absorb, bind, or otherwise associate with active pharmaceutical ingredients, thereby enabling decoupling of pharmacodynamic and pharmacokinetic properties between independent molecular entities. This greatly facilitates drug design, fine-tuning of pharmacological properties, and improving both the efficacy and safety of resulting drugs.

[0003] The use of micro- and nanoparticle formulations has been particularly beneficial to the field of DNA- and RNA-based pharmaceuticals, as the unique intracellular activity of such molecules, combined with their large size and negative charge, prevents their uptake into most cells. The central role of both DNA and RNA in the biology of all living organisms, and the high level of specificity available with these molecules, offers unprecedented opportunities for discovering high-value therapies and therapeutics for numerous diseases in humans and other species.

[0004] Today, several examples of successful nanomedicines are known, using various types of nanoparticle formulations. Examples in the vaccine field include Comirnaty, a lipid nanoparticle SARS-CoV-2-directed vaccine marketed by Pfizer / BioNTech, and SpikeVax, marketed by Moderna, which have achieved significant commercial and humanitarian success. Another example is Pandemrix (H1N1 influenza) and Prepandrix (pre-pandemic influenza), influenza-targeting oil-in-water emulsions of inactivated viruses marketed by GlaxoSmithKline. Examples in the siRNA field include the lipid nanoparticles Onpattro (patisiran) for hereditary transthyretin-mediated amyloidosis, Givlaari (givosiran) for acute hepatic porphyria, and Oxlumo (lumasiran) for the treatment of 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 pain treatment, Caelyx (liposomal doxorubicin) for cancer, Cimzia (Rh-a / b Fab for TNF-α), nanocrystalline Lipidic (fenofibrate) for hypercholesterolemia, Rapamune (liposomal sirolimus) for organ rejection, Risperadol (risperidone nanosuspension) nanosuspension for schizophrenia, and polymeric nanoparticles Renagel (sevelamer) for hyperphosphatemia and Copaxone (glatiramer acetate) for relapsing multiple sclerosis.

[0005] Despite the increasing success of commercial applications of nanomedicines, several challenges remain. This is especially true for more advanced applications, such as delivery of RNA or DNA. Such applications require stable encapsulation or association of the cargo with the delivery vehicle to protect the cargo and shield its negative charge, allowing it to cross the negatively charged plasma or endosomal membrane. Such encapsulation and / or association must be completely reversed at the moment of intended release, but not before or after. As a result, stable yet efficient delivery vehicles often function (sufficiently for therapeutic use) within a very narrow range of compositions.

[0006] To date, lipid nanoparticles with a solid lipid core, often referred to as LNPs or SNALPs (stable nucleic acid lipid particles), have demonstrated particularly high delivery efficiencies compared to other nucleic acid delivery technologies and have therefore made the most progress in clinical practice and commercial use. This technology is used in the SARS-CoV-2-directed vaccines Comirnaty and SpikeVax. Such LNPs differ from liposomes by the fact that their core contains only small amounts, if any, of water. Furthermore, LNPs have a largely solid lipid structure instead of the lipid bilayer that defines liposomes. Furthermore, in LNPs, the lipids themselves complex and neutralize the negative charge of RNA / DNA, whereas in liposomes, RNA / DNA must be complexed with a charge-neutralizing agent, such as a positively charged polymer, before encapsulation.

[0007] In solid lipid nanoparticles used clinically or commercially, the lipid composition is remarkably similar. For example, one of the most frequently used formulations for mRNA delivery has 50 mol% ionizable lipid (e.g., DLin-MC3-DMA), 38.5 mol% structural lipid (also called 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), as well as minor variations thereof. In the majority of cases, cholesterol, cholesterol derivatives, or structurally similar sterols are used as the core lipid. Cholesterol in the formulation is thought to contribute to structural integrity, endosomal uptake via binding to ApoE and cell surface ApoE receptors, promoting membrane fusion (see, e.g., Tenchov BG, MacDonald RC, and Siegel DP, Biophysical J. 91, 2508-2515 (2006)), and ultimately improving endosomal escape, primarily via its crystalline form on the LNP surface. The importance of cholesterol's role in nucleic acid delivery is highlighted by the role of cholesterol and cholesterol transport proteins in viral cytoplasmic delivery, as well as the improved gene delivery that can be achieved 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 naturally occurring nanoparticles (e.g., LDL particles) that use significant amounts of cholesterol as a structural lipid have been shown to bind to ApoE, a naturally occurring serum protein in the bloodstream and possibly other tissues. ApoE's natural role is to bind to low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL) particles in the bloodstream and mediate binding to LDL receptors, which are highly expressed on hepatocytes. Binding to the LDL receptor leads to clathrin-mediated endocytosis of LDL particles and intraendosomal cholesterol processing via 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] Combined with the extremely high density of LDL receptors present on hepatocytes and the large volume of venous blood passing through the liver, ApoE binding of cholesterol-based LNPs significantly impacts their biodistribution, resulting in a strong liver-directed bias. Indeed, achieving maximum ApoE binding and liver targeting has been the primary rationale for further LNP development to date, following the argument that, if not 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). Intravenously administered LNPs may be significantly biased toward the liver because they interact with blood components before reaching any other tissues. For therapeutics that rely on the release of produced proteins into the blood or bile, such as antibody therapeutics, the liver may be a suitable target organ with a high protein production capacity, making ApoE binding a convenient uptake method. However, for therapeutic, diagnostic, or prophylactic methods that require high local concentrations, intracellular delivery in specific cell types, or are toxic to the liver, liver-biased biodistribution may reduce efficacy and / or safety. In such cases, higher doses may be required to achieve the desired local concentration of LNP and / or expressed protein, increasing the likelihood and severity of side effects as well as cost and potential bioaccumulation effects.

[0010] Conjugation of drug formulations with active targeting moieties, such as antibodies, antibody fragments, DARPins, small molecules, and peptides, is a well-known method for directing preferential uptake into target cells that display on their surface proteins or other chemical entities specifically bound by the targeting moiety. Adding an active targeting moiety to the outside of a delivery vehicle often improves biodistribution into target cells. However, the magnitude of such improvement depends largely on the relative strength of the active targeting moiety and the relative strength of all other properties that induce or promote uptake in other organs, including ApoE binding. Importantly, as long as the LNP formulation contains cholesterol, it is difficult to prevent ApoE binding. For this very reason, shielding lipids, such as PEGylated DSPE, are often included in LNP formulations to prevent or reduce undesired cellular and molecular interactions and, consequently, inappropriate biodistribution. However, using large amounts of PEG-lipids results in effective shielding at the expense of delivery efficiency. Therefore, most currently used LNP formulations strike a balance between shielding capacity and effective delivery, resulting in a portion of the cholesterol-containing LNP surface being available for binding by ApoE, leading to liver-preferred biodistribution upon intravenous administration. Certain LNP compositions use DMG-PEG2000, which desorbs from the LNP surface via its short, single lipid tail, making the surface available for serum protein adsorption over time and resulting in organ-specific uptake based on its surface properties. To date, cholesterol (or a derivative) has always been included in LNP formulations for structural stability and delivery efficiency, making cholesterol a key determinant of surface properties. Therefore, cholesterol- or cholesterol-derivative-based LNPs are most suitable for local application (e.g., intramuscular injection) and liver-targeted applications, but most other applications would benefit from a different core lipid that lacks ApoE-binding propensity and is combined with an active targeting moiety. Exceptions are methods of biodistribution based on surface charge, targeting LNPs to cells of the reticuloendothelial system (RES) and scavenger receptor-mediated uptake, which interfere with / modify ApoE / serum protein binding while at the same time have extensive and early interactions with receptors after intravenous injection.

[0011] Recently, chemical adduct formation and degradation of RNA packaged within LNPs have been highlighted as factors affecting the efficacy and safety of oligo- and polynucleotide therapeutics and prophylactics, especially during long-term storage (Packer M, et al. Nat Commun 12, 6777 (2021)). Besides reactive impurities arising from lipid synthesis, the pure, intended components of LNPs may be major contributors to the adduct formation and degradation process. Cholesterol is susceptible to oxidation due to the 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, so-called oxysterols, are formed. The hydroperoxides then cause the oxidation of nearby cholesterol or polyunsaturated fatty acids, which may propagate or even accelerate LNP oxidation. This process not only affects the lipid properties of LNPs and thus their endosomal escape efficiency, but may also directly affect mRNA through hydrolysis. Several oxysterol products bearing hydroxyl, ketone, or epoxide groups at positions 4, 5, 6, or 7 of the A and B rings of cholesterol are known to be formed by cholesterol oxidation. The epoxide species in particular are highly reactive toward a wide range of nucleophiles, including the 2-amino group of guanine in RNA and DNA.

[0012] In addition to direct effects on LNPs and mRNA, oxidation products from the lipid components of LNPs may affect target cells directly or indirectly. Many LNP formulations induce a transient increase in proinflammatory cytokines within hours of injection, a response timeframe that is thought to be attributable to the lipid components rather than the nucleic acid cargo of the LNPs (Lutz J et al., npj Vaccines 2 (2017)). Furthermore, lipid components of LNPs have been found to enhance existing inflammatory or immune-challenged states through exacerbated inflammation (IE) (Parhiz H et al., J. of Controlled Release 344: 50–61 (2021)). While ionized lipid components have been identified as a major driver of the proinflammatory response (Ndeupen S et al., iScience 24 (2021)), the contribution of oxidized cholesterol cannot be excluded based on these findings. Importantly, oxidized cholesterol is a well-known proinflammatory signal that contributes to arterial inflammation and subsequent plaque erosion, for example, in atherosclerosis. Therefore, it is possible that fatty acids oxidized by oxidized cholesterol and hydroperoxides derived from cholesterol autoxidation contribute to the proinflammatory effects of LNP. The metabolism of oxidized cholesterol by monocytes may also induce long-term inflammatory effects by epigenetically reprogramming monocytes into foam cells (Bekkering S. et al. Arterioscler Thromb Vasc Biol. 2014 Aug;34(8):1731-8). Furthermore, the formed epoxides may form carcinogenic adducts with endogenous RNA, DNA, and proteins, affecting cell viability and genomic integrity.

[0013] Prior art solutions to prevent oxidation include protecting the drug substance / drug from light, storing it at low temperatures, and under a protective atmosphere (e.g., nitrogen or argon). These can prevent cholesterol oxidation, especially if only low levels of oxidized lipids are present initially. However, achieving such low levels of oxidized lipids by preventing oxidation can be very difficult. This is because LNPs are typically formed by mixing an organic solvent (e.g., ethanol) containing the lipid components with an excess of an aqueous solution containing oligo- or polynucleotides, requiring large volumes of dialysis by tangential flow filtration (TFF) to remove or dilute the organic solvent. These large volumes can contain significant amounts of dissolved oxygen, making oxygen removal a technical challenge.

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

[0015] Prior art solutions to reduce the pro-inflammatory effects of LNPs rely on the use of corticosteroids (e.g., dexamethasone), either as an adjunctive treatment or by incorporating corticosteroids into the LNPs themselves, which have been shown to significantly reduce inflammation and simultaneously increase the expression of nucleic acid-encoded proteins (Zhang H et al. J Biomedical Materials Res 2022:1-8). However, dexamethasone and other corticosteroids exhibit significant side effects, including abdominal discomfort, skin rash, swelling, and hot flashes (Min KH et al. Korean J Audiol. 16:65-70 (2012)). Incorporating dexamethasone into LNPs allows for the use of much lower doses, reducing side effects. However, a drawback is the addition of another component to the LNP composition, complicating manufacturing, quality control, and formulation.

[0016] Furthermore, cholesterol plays a key role in plasma membrane stability, organization (e.g., via lipid rafts), and function. When a significant amount of LNPs is targeted to a specific cell, as may be necessary for a specific therapeutic or prophylactic effect, the cell may experience a transient increase in cholesterol, resulting in altered plasma membrane function. For example, changes in cholesterol levels are known to affect cognitive function in neurons, where plasma membrane fluidity and lipid raft organization control receptor density and location (Egawa et al. J Physiol. 594(16): 4565-4579 (2016)).

[0017] Similarly, cholesterol plays a role in the structural integrity of LNPs by altering the fluidity of the LNP surface (Cheng X and Lee RJ. Adv. Drug Deliv. Rev. 99:129-137 (2016)). Cholesterol-free and cholesterol-reduced LNPs 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 nanoparticles is necessary to maintain performance-related formulation properties, such as particle size, shape, and low polydispersity, during downstream processing steps (e.g., lyophilization and concentration), storage, and application (e.g., nebulization and spraying). However, even with cholesterol, nebulization increased nanoparticle size and reduced the encapsulation efficiency of nucleic acid cargo (Zhang H et al. Pharmaceuticals). No clear trend was observed between particle stability and relative cholesterol content in the context of nebulization.

[0018] Therefore, to advance nucleic acid-based therapeutics, prophylaxis, and diagnostics, there is an urgent need for novel core / structural lipids that can replace cholesterol and have the following combination of properties: maintain or improve endosomal escape compared to cholesterol, provide better control of biodistribution to organs other than the liver, reduce pro-inflammatory signaling upon contact of LNPs with cells, and confer better chemical and / or physical stability to the LNPs. The present invention arose from the inventors' research to identify novel core / structural lipids. [Prior art documents] [Non-patent literature]

[0019] [Non-Patent Document 1] Tenchov BG, MacDonald RC, and Siegel DP Biophysical J. 91, 2508-2515 (2006) [Non-patent document 2] Paunovska K et al. ACS Nano (2018) [Non-patent document 3] Patel S et al. Nat Commun 11, 983 (2020) [Non-patent document 4] Akinc A. et al. Mol. Ther. 18, 1357-1364 (2010) [Non-patent document 5] Sahay G et al. Nat. Biotechnol. 31, 653-658 (2013) [Non-patent document 6] Jayaraman M, et al. Angew Chem Int Ed Engl. 2012 Aug 20;51(34):8529-33.doi: 10.1002 / anie.201203263 [Non-Patent Document 7] Packer M, et al. Nat Commun 12, 6777 (2021) [Non-patent document 8] Maerker G, Current status. J Am Oil Chem Soc 64: 387~392 (1987) [Non-Patent Document 9] Lutz J et al. npj Vaccines 2 (2017) [Non-Patent Document 10] Parhiz H et al. J. of Controlled Release 344: 50~61 (2021) [Non-Patent Document 11] Ndeupen S et al. iScience 24 (2021) [Non-Patent Document 12] Bekkering S. et al. Arterioscler Thromb Vasc Biol. 2014 Aug;34(8):1731-8 [Non-Patent Document 13] Zhang H et al. J Biomedical Materials Res 2022:1~8 [Non-Patent Document 14] Min KH et al. Korean J Audiol. 16:65-70 (2012) [Non-Patent Document 15] Egawa et al. J Physiol. 594(16): 4565~4579 (2016) [Non-Patent Document 16] Cheng X and Lee RJ. Adv. Drug Deliv. Rev. 99:129–137 (2016) [Non-Patent Document 17] Rodrigueza et al. Biochemistry 34: 6208-6217 (1995) [Non-Patent Document 18] Sato Y et al. Acta Biomater. 102: 341~350 (2020) [Non-Patent Document 19] Zhang H et al. Pharmaceutics Summary of the Invention

[0020] According to a first aspect of the present invention, a compound of formula (I)

[0021] [ka]

[0022] (In the formula, L 1 teeth

[0023] [ka]

[0024] and; R 1 From R 10 are 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 an aryl, an optionally substituted 5-10 membered heteroaryl, or a sugar moiety; R 11 From 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 6~12aryl or optionally substituted 5-10 membered heteroaryl or a pharmaceutically acceptable complex, salt, solvate, tautomer or polymorph thereof The present invention provides the use of the compound of formula (I) in a micro- or nanoparticle formulation.

[0025] Advantageously, compounds of formula (I) may be used in improved lipid nanoparticle compositions that exhibit one or more of the following properties: improved drug delivery to target cells, improved physical stability, improved chemical stability, improved biodistribution when using active targeting agents, and / or reduced release of pro-inflammatory cytokines upon contact with cells. The lipid structures may be beneficially used in lipid-based nano- or microparticle compositions for the delivery of (modified) RNA, DNA, peptides, or small molecules into mammalian cells.

[0026] Preferably, the compound of formula (I) is used as a core lipid in a micro- or nanoparticle formulation. It will be understood that the core lipid may also be referred to as a structural lipid.

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

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

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

[0030] The alkyl, alkenyl and / or alkynyl may be unsubstituted or may be substituted with a 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 From R 16 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 3~10Cycloalkenyl, optionally substituted 3- to 10-membered heterocycle, optionally substituted C 6~12 R is an aryl or an optionally substituted 5-10 membered heteroaryl. 14 From R 16 are independently H, C 1~12 Alkyl, C 2~12 Alkenyl, C 2~12 Alkynyl, C 3~6 Cycloalkyl, C 3~6 Cycloalkenyl, C 6~12 It may be selected from the group consisting of aryl, a 3- to 10-membered heterocycle, or a 5- to 10-membered heteroaryl.

[0031] "Cycloalkyl" refers to a non-aromatic saturated hydrocarbon ring system. Representative examples of C3-C6 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Similarly, "cycloalkenyl" refers to a non-aromatic unsaturated 3- to 6-membered hydrocarbon ring system.

[0032] "Aryl" refers to an aromatic hydrocarbon group. Aryl may be monocyclic, bicyclic, or polycyclic. The term "aryl" may be 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. C6-C 12 Examples of aryl groups include, but are not limited to, phenyl, α-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. Each heteroatom may be independently selected from the group consisting of oxygen, sulfur, and nitrogen. The heterocycle may be saturated or partially saturated. Exemplary heterocyclyl groups include, but are not limited to, aziridine, oxirane, oxirene, thiirane, pyrroline, pyrrolidine, dihydrofuran, tetrahydrofuran, dihydrothiophene, tetrahydrothiophene, dithiolane, piperidine, 1,2,3,6-tetrahydropyridin-1-yl, tetrahydropyran, pyran, morpholine, piperazine, thiane, thiine, piperazine, azepane, diazepane, and oxazine.

[0034] "Heteroaryl" refers to an aromatic 5- to 10-membered ring system in which at least one ring atom is a heteroatom. Heteroaryl may be monocyclic, bicyclic, or polycyclic. In bicyclic and polycyclic structures, if at least one of the rings contains a heteroatom, the group may be understood to be heteroaryl. The term "heteroaryl" may be understood to encompass 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. Each heteroatom may be independently selected from the group consisting of oxygen, sulfur, and nitrogen. Examples of 5- to 10-membered heteroaryl groups include furan, thiophene, indole, azaindole, oxazole, thiazole, isoxazole, isothiazole, imidazole, N-methylimidazole, pyridine, pyrimidine, pyrazine, pyrrole, N-methylpyrrole, pyrazole, N-methylpyrazole, 1,3,4-oxadiazole, 1,2,4-triazole, 1-methyl-1,2,4-triazole, 1H-tetrazole, 1-methyltetrazole, benzoxazole, benzothiazole, benzofuran, benzisoxazole, benzimidazole, N-methylbenzimidazole, azabenzimidazole, indazole, quinazoline, quinoline, and isoquinoline. Bicyclic 5- to 10-membered heteroaryl groups include those in which a phenyl, pyridine, pyrimidine, pyrazine, or pyridazine ring is fused to a 5- or 6-membered monocyclic heteroaryl ring.

[0035] Any cycloalkyl, cycloalkenyl, aryl, heterocycle and / or heteroaryl group may be unsubstituted or optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkenyl, optionally substituted C1-C6 alkynyl, 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 From R 16 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 R is an aryl or an optionally substituted 5-10 membered heteroaryl. 14 ~R 16 is H, C 1~12 Alkyl, C 2~12 Alkenyl, C 2~12 Alkynyl, C 3~6 Cycloalkyl, C 3~6 Cycloalkenyl, C 6~12 They may each be independently selected from the group consisting of aryl, a 3- to 10-membered heterocyclic ring, or a 5- to 10-membered heteroaryl.

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

[0037] The term "pharmaceutically acceptable salt" may be understood to refer to any salt of a compound provided herein that retains its biological properties and is not toxic or otherwise undesirable for pharmaceutical use. Such salts may be derived from a variety of organic and inorganic counterions well known in the art.Such salts include, but are not limited to: (1) organic or inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, sulfamic acid, acetic acid, adepic acid, aspartic acid, trifluoroacetic acid, trichloroacetic acid, propionic acid, hexanoic acid, cyclopentylpropionic acid, glycolic acid, glutaric acid, pyruvic acid, lactic acid, malonic acid, succinic acid, sorbic acid, ascorbic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, picric acid, cinnamic acid, and mandelic acid; , phthalic acid, lauric acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonate, camphoric acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, benzoic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, Acid addition salts formed with stearic acid, cyclohexylsulfamic acid, quinic acid, muconic acid, and similar acids, or (2) salts in which the acidic protons present in the parent compound are replaced by (a) metal ions, such as alkali metal ions, alkaline earth ions, or aluminum ions, or alkali metal or alkaline earth metal hydroxides, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, aluminum hydroxide, lithium hydroxide, zinc hydroxide, and barium hydroxide, ammonia, or (b) organic bases, such as aliphatic, fatty, Base addition salts formed upon coordination with cyclic or aromatic organic amines such as ammonia, methylamine, dimethylamine, diethylamine, picoline, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylene-diamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, N-methylglucamine piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, and the like.

[0038] Pharmaceutically acceptable salts include sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like, and, if the compound contains a basic functionality, salts of non-toxic organic or inorganic acids, such as hydrohalide salts, e.g., hydrochloride, hydrobromide, and hydroiodide salts, carbonate or bicarbonate salts, sulfate or hydrogen sulfate salts, borates, phosphates, hydrogen phosphates, dihydrogen phosphates, pyroglutamate, saccharate, stearate, sulfamate, nitrate, orotate, oxalate, and the like. Acid salts include: acid salt, palmitate, pamoate, acetate, trifluoroacetate, trichloroacetate, propionate, hexanoate, cyclopentylpropionate, glycolate, glutarate, pyruvate, lactate, malonate, succinate, tannate, tartrate, tosylate, sorbate, ascorbate, malate, maleate, fumarate, tartrate, camsylate, citrate, cyclamate, benzoate, isethionate, esylate, formate, 3-(4-hydroxybenzoate)-2-hydroxybenzoate, 4-hydroxybenzoate, 5-hydroxybenzoate, 6-hydroxybenzoate, 7-hydroxybenzoate, 8-hydroxybenzoate, 9-hydroxybenzoate, 10-hydroxybenzoate, 11-hydroxybenzoate, 12-hydroxybenzoate, 13-hydroxybenzoate, 14-hydroxybenzoate, 15-hydroxybenzoate, 16-hydroxybenzoate, 17-hydroxybenzoate, 18-hydroxybenzoate, 19-hydroxybenzoate, 20-hydroxybenzoate, 21-hydroxybenzoate, 22-hydroxybenzoate, 23-hydroxybenzoate, 24-hydroxybenzoate, 25-hydroxybenzoate, 26-hydroxybenzoate, 27-hydroxybenzoate, 28-hydroxybenzoate, 29-hydroxybenzoate, 30-hydroxybenzoate, 31-hydroxybenzoate, 32-hydroxybenzoate, 33-hydroxybenzoate, 34-hydroxybenzoate, 35-hydroxybenzoate, 36-hydroxybenzoate, 37-hydroxybenzoate, 38-hydroxybenzoate, 39-hydroxybenzoate, 40-hydroxybenzoate, 41-hydroxybenzoate, 42-hydroxybenzoate, 43-hydroxybenzoate, 44-hydroxybenzoate, 45-hydroxybenzoate, 46-hydroxybenzoate, 47-hydroxybenzoate, 48-hydroxybenzoate, 49- Benzoylbenzoate, picrate, cinnamate, mandelate, phthalate, laurate, methanesulfonate (mesylate), methylsulfate, naphthylate, 2-napsylate, nicotinate, ethanesulfonate, 1,2-ethanedisulfonate, 2-hydroxyethanesulfonate, benzenesulfonate (besylate), 4-chlorobenzenesulfonate, 2-naphthalenesulfonate, 4-toluenesulfonate, camphorate, The salts may include benzoates, benzoates, glutamates, hydroxynaphthoates, salicylates, stearates, cyclohexylsulfamates, quinates, muconates, xinafoates, and the like.

[0039] R 1may be H.

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

[0041] In an alternative embodiment, R 3 is an 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 It may be aryl or optionally substituted 5-10 membered heteroaryl. More preferably, R 3 is an 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 It may be cycloalkenyl, an optionally substituted 3- to 6-membered heterocycle, an optionally substituted phenyl, or an optionally substituted 5- or 6-membered heteroaryl. More preferably, R 3 is an 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 It may be a cycloalkenyl, a 3- to 6-membered heterocycle, phenyl, or a 5- or 6-membered heteroaryl. The alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycle, aryl, or heteroaryl may be unsubstituted or may have one or more of the following groups: OH, SH, NH, CN, oxo, C 3~6 Cycloalkyl, C 3~6 Cycloalkenyl, C 6~12It may be optionally substituted with one or more substituents selected from aryl, 3- to 10-membered heterocycle, or 5- to 10-membered heteroaryl. Preferably, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycle, aryl, or heteroaryl is unsubstituted or is selected from OH, SH, NH, CN, oxo, C 3~6 Cycloalkyl, C 3~6 cycloalkenyl, phenyl, 5- or 6-membered heterocycle, or 5- or 6-membered heteroaryl. 3 teeth

[0042] [ka]

[0043] may be.

[0044] R 5 may be H.

[0045] In some embodiments, R 6 may be H.

[0046] In an alternative embodiment, R 6 is OR 11 ,OCOR 11 , COOR 11 , N.R. 11 R 12 or N + R 11 R 12 R 13 More preferably, R 6 is OR 11 or NR 11 R 12 Most preferably, R 6 is OR 11 R 11 From R 13 are independently H, optionally substituted C 1~12 Alkyl, optionally substituted C 2~12 Alkenyl, optionally substituted C2~12 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 It may be aryl or optionally substituted 5-10 membered heteroaryl. More preferably, R 11 From R 13 are independently H, C 1~6 Alkyl, C 2~6 Alkenyl or C 2~6 It may be alkynyl. Most preferably, R 11 From R 13 is H. Therefore, R 6 may be OH.

[0047] In some embodiments, R 7 may be H.

[0048] In an alternative embodiment, R 7 is OR 11 ,OCOR 11 , COOR 11 , N.R. 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 It may be aryl or optionally substituted 5-10 membered heteroaryl. More preferably, R 7 is OR 11 , N.R. 11 R 12 , optionally substituted C 1~12 Alkyl, optionally substituted C 2~12Alkenyl, optionally substituted C 2~12 Alkynyl, optionally substituted C 3~6 Cycloalkyl, optionally substituted C 3~6 It may be cycloalkenyl, an optionally substituted 3- to 6-membered heterocycle, an optionally substituted phenyl, or an optionally substituted 5- or 6-membered heteroaryl. More preferably, R 7 is OR 11 , C 3~6 Alkyl, C 3~6 Alkenyl or C 3~6 It may be alkynyl. 11 From R 13 are independently 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 It may be aryl or optionally substituted 5-10 membered heteroaryl. More preferably, R 11 From R 13 are independently H, C 1~6 Alkyl, C 2~6 Alkenyl or C 2~6 It may be alkynyl. More preferably, R 11 From R 13 is H or methyl, most preferably methyl. Thus, R 7 OMe,

[0049] [ka]

[0050] may be.

[0051] In some embodiments, R 9 may be H. In an alternative embodiment, R 9 is OR11 ,OCOR 11 , COOR 11 , N.R. 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 It may be aryl or optionally substituted 5-10 membered heteroaryl. More preferably, R 9 is OR 11 , N.R. 11 R 12 , optionally substituted C 1~12 Alkyl, optionally substituted C 2~12 Alkenyl, optionally substituted C 2~12 Alkynyl, optionally substituted C 3~6 Cycloalkyl, optionally substituted C 3~6 It may be cycloalkenyl, an optionally substituted 3- to 6-membered heterocycle, an optionally substituted phenyl, or an optionally substituted 5- or 6-membered heteroaryl. More preferably, R 9 is OR 11 , C 3~6 Alkyl, C 3~6 Alkenyl or C 3~6 It may be alkynyl. 11 From R 13 are independently 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 C6~12 It may be aryl or optionally substituted 5-10 membered heteroaryl. More preferably, R 11 From R 13 are independently H, C 1~6 Alkyl, C 2~6 Alkenyl or C 2~6 It may be alkynyl. More preferably, R 11 From R 13 is H or methyl, most preferably methyl. Thus, R 9 OMe,

[0052] [ka]

[0053] may be.

[0054] R 10 may be H.

[0055] Thus, the compounds are compounds of formula (Ia), (Ib), (Ic) or (Id)

[0056] [ka]

[0057] may be.

[0058] L 1 teeth

[0059] [ka]

[0060] may be.

[0061] Thus, in some embodiments, the compound is a compound of formula (Iai), (Ibi), (Ici) or (Idi)

[0062] [ka]

[0063] is.

[0064] Instead, L 1 teeth

[0065] [ka]

[0066] may be.

[0067] Thus, in an alternative embodiment, the compound is a compound of formula (Iaii), (Ibii), (Icii) or (Idii)

[0068] [ka]

[0069] is.

[0070] R 2 , R 4 and R 8 are independently H, OR 11 ,OCOR 11 , COOR 11 , N.R. 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.

[0071] R 11 From R 13 are each independently H, optionally substituted C 1~20 Alkyl, optionally substituted C2~20 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 It may be aryl or optionally substituted 5-10 membered heteroaryl. More preferably, R 11 From 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 From 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 From R 13 are independently H, C 1~3 Alkyl, C 2~3 Alkenyl, C 2~3 alkynyl or 5- or 6-membered heteroaryl. Most preferably, R 11 From R 13 is independently H, methyl or 5-membered heteroaryl.

[0072] Therefore, R 2 , R 4 and R 8 are independently H, OH, OCH3,

[0073] [ka]

[0074] Or it may be a sugar moiety.

[0075] The sugar moiety has the general formula

[0076] [ka]

[0077] (In the formula, X 1 is CR 20 R 21 is a combination of; R 17 From R 25 are independently H, OH, CH2OH, COOH, NH2, and C 1~6 Alkyl, halo,

[0078] [ka]

[0079] is) It can be understood that

[0080] In some embodiments, X 1 is a bond. Preferably, X 1 is CR 20 R 21 is.

[0081] In some embodiments, R 17 is H. Instead, R 17 R may be OH, CHOH, COOH, or NH. 17 may be OH or CH2OH.

[0082] Preferably, R 18 , R 22 and R 24 is H. Preferably, in embodiments where it is present, R 20 is H.

[0083] Preferably, R 19 are H, OH or NH2, more preferably each is OH.

[0084] Preferably, in embodiments where it is present, R 21 is H, OH or NH2, more preferably OH.

[0085] Preferably, R 23 are OH or NH, more preferably each is OH. Alternatively, R 23 teeth

[0086] [ka]

[0087] The R in the additional sugar ring may be 17 From R 25 may be as defined herein.

[0088] Preferably, R 25 is CH2OH or COOH. Alternatively, R 25 teeth

[0089] [ka]

[0090] The R in the additional sugar ring may be 17 From R 25 may be as defined herein.

[0091] The sugar moiety may 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.

[0092] Therefore, the sugar moiety

[0093] [ka]

[0094] The sugar moiety may be

[0095] [ka]

[0096] may be.

[0097] In some embodiments, the sugar moiety

[0098] [ka]

[0099] More preferably, the sugar moiety is

[0100] [ka]

[0101] is.

[0102] The compound of formula (I) may be:

[0103] [ka]

[0104] [ka]

[0105] [ka]

[0106] The compound of formula (103)

[0107] [ka]

[0108] may be.

[0109] The compound of formula (110)

[0110] [ka]

[0111] may be.

[0112] The compound of formula (115)

[0113] [ka]

[0114] may be.

[0115] The compound of formula (116)

[0116] [ka]

[0117] may be.

[0118] The compound of formula (117)

[0119] [ka]

[0120] may be.

[0121] According to a second aspect of the present invention, there is provided a micro- or nanoparticle comprising a compound of formula (I) as defined in the first aspect, or a pharmaceutically acceptable complex, salt, solvate, tautomer or polymorph thereof, and optionally a payload molecule.

[0122] The micro- or nanoparticles may define a dense or hollow structure. The micro- or nanoparticles may define a solid structure. The micro- or nanoparticles may optionally consist of one or more lipid bilayers, which may optionally be cross-linked to each other and encapsulate a void. The micro- or nanoparticles may be lipid nanoparticles (LNPs), liposomes, lipoplexes, micelles or lipid vesicles. In some preferred embodiments, the micro- or nanoparticles are LNPs.

[0123] The micro- or nanoparticles may have a diameter of less than 10 μm, less than 1 μm, less than 500 nm, or less than 250 nm. More preferably, the micro- or nanoparticles may have a diameter of less than 200 nm, less than 175 nm, less than 150 nm, less than 125 nm, less than 100 nm, less than 90 nm, or less than 80 nm. The micro- or nanoparticles may have a diameter of 25 nm to 1 μm, 30 to 500 nm, 35 to 250 nm, 40 to 200 nm, 45 to 150 nm, 50 to 125 nm, 55 to 100 nm, 60 to 90 nm, or 65 to 80 nm. The diameter of the micro- or nanoparticles may be measured using dynamic light scattering.

[0124] In addition to the compound of formula (I) or its pharmaceutically acceptable complex, salt, solvate, tautomer or polymorph, the micro- or nanoparticles may contain one or more additional lipids.Thus, the micro- or nanoparticles may contain a lipid component comprising the compound of formula (I) or its pharmaceutically acceptable complex, salt, solvate, tautomer or polymorph, and one or more additional lipids.The one or more additional lipids may be selected from the group consisting of phospholipids, permanent cationic lipids, ionizable cationic lipids, permanent anionic lipids, ionizable anionic lipids, structural lipids, shielding lipids, functionalized lipids, additional core lipids, and combinations thereof.

[0125] In some embodiments, the lipid component comprises a compound of Formula (I), or a pharmaceutically acceptable complex, salt, solvate, tautomer, or polymorph thereof, and a constant or ionized cationic lipid, or a mixture thereof. In some embodiments, the lipid component of the micro- or nanoparticle formulation comprises a compound of Formula (I), or a pharmaceutically acceptable complex, salt, solvate, tautomer, or polymorph thereof, a constant or ionized lipid, and a shielding lipid. In some embodiments, the lipid component of the micro- or nanoparticle formulation further comprises a phospholipid or a helper lipid. In some embodiments, the lipid component of the micro- or nanoparticle formulation further comprises a functionalized lipid. In some embodiments, the lipid component of the micro- or nanoparticle formulation further comprises an additional core lipid.

[0126] The lipid component of the micro- 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 may comprise at least 5 mol% of the compound of formula (I). The core lipid component may comprise at least 10 mol%, at least 20 mol%, at least 30 mol%, at least 40 mol%, or at least 50 mol% of the compound of formula (I). The core lipid component may comprise at least 60 mol%, at least 70 mol%, at least 80 mol%, at least 90 mol%, or 100 mol% of the compound of formula (I). The core lipid component may comprise less than 100 mol% of the compound of formula (I). The core lipid component may 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 the compound of formula (I). The core lipid component may comprise less than 40 mol%, less than 30 mol%, less than 20 mol%, or less than 10 mol% of the compound of formula (I). By varying the amount of the compound of formula (I) and the additional core lipid, the properties of the micro- or nanoparticles can be fine-tuned.

[0127] In some embodiments, the lipid component of the micro- or nanoparticles comprises between 5-80 mol%, 10-60 mol%, 20-50 mol%, 30-45 mol%, or 35-40 mol% of the core lipid component.

[0128] Thus, in some embodiments, the lipid component of the micro- or nanoparticles comprises 5-80 mol%, 10-60 mol%, 20-50 mol%, 30-45 mol%, or 35-40 mol% of a compound of formula (I), or a pharmaceutically acceptable complex, salt, solvate, tautomer, or polymorph thereof.

[0129] In some embodiments, the lipid component of the micro- or nanoparticle formulation comprises 10-90 mol%, 20-80 mol%, 30-70 mol%, 40-60 mol%, or 45-55 mol% of permanent cationic lipids, ionizable cationic lipids, or mixtures thereof.

[0130] In some embodiments, the lipid component of the micro- or nanoparticle formulation comprises 0-30 mol%, 2.5-20 mol%, 5-15 mol%, or 7.5-12.5 mol% phospholipids.

[0131] In some embodiments, the lipid component of the micro- or nanoparticle formulation comprises 0-15 mol%, 0.1-10 mol%, 0.5-5 mol%, 0.75-3 mol%, or 1-2 mol% of a shielding lipid.

[0132] The additional core lipid may be a sterol. The sterol may be cholesterol or a cholesterol derivative. The cholesterol derivative may be beta-sitosterol, vitamin D2, vitamin D3, calcipotriol, stigmasterol, campesterol, fucosterol, brassicasterol, ergosterol, 9,11-dehydroergosterol, daucosterol, beta-sitosterol acetate, betutin, lupeol, ursolic acid, or oleanolic acid.

[0133] The phospholipid may be dioleoyl-phosphatidylethanolamine (DOPE), dioleoyl-phosphatidylcholine (DOPC), dioleoyl-phosphatidylserine (DOPS), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), or any naturally or synthetically derived phospholipid.

[0134] Shielding lipids can be understood as functionalized lipids that prevent undesired interactions of lipid nanoparticles with other nanoparticles, extracellular compounds, and / or cell surfaces. Shielding lipids can prevent undesired interactions of lipid nanoparticles with other nanoparticles, extracellular compounds, and / or cell surfaces through steric hindrance or similar mechanisms. Shielding lipids can be lipids modified to include a shielding polymer. The shielding polymer can be a polyethylene glycol (PEG) group, a poly-sarcosine group, an oligopeptide or polypeptide, a hydroxyl-containing nonionic water-soluble polymer, polyvinylpyrrolidone (PVP), poly(2-alkyl-2-oxazoline), a zwitterionic polymer, or any other suitable shielding polymer. The oligopeptide or polypeptide can be a PAS. A PAS can be understood as an oligopeptide or polypeptide consisting of or including proline, alanine, and serine residues. The hydroxyl-containing nonionic water-soluble polymer may be poly(glycerol) (PG), poly(N(2-hydroxypropyl)methacrylamide) (pHPMA), polysarcosine, or poly(vinyl alcohol). The zwitterionic polymer may be polybetaine. In some embodiments, the shielding lipid may be a phospholipid modified to include a shielding polymer. In embodiments where the shielding lipid is a phospholipid modified to include a shielding polymer, the modified phospholipid may be a phospholipid as defined above. In some embodiments, the shielding lipid may be a DSPE, DMG, or DPPC lipid modified to include a shielding polymer. The shielding lipid may include a shielding polymer having an average molecular weight of 500-5000, 1000-3000, 1250-1750, 1500-2500, 1750-2250 Da, or 1900-2100 Da. The shielding lipid may be dimyristoyl-glycerol-PEG (DMG-PEG), PEG-DSPE, PEG-dipalmitoyl-phosphatidylcholine (DPPC), pSar-DMG, pSar-DSPE, or pSar-DPPC. In some embodiments, the shielding lipid is PEG-DSPE or pSar-DSPE.

[0135] The constitutive or ionizable cationic lipid may be 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 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-trimethylammoniumpropane (DODAP), or a compound of formula (II)

[0136] [ka]

[0137] (In the formula, R 26 and R 27 are independently optionally substituted C 1~50 Alkyl, optionally substituted C 2~50 alkenyl or optionally substituted C 2~50 alkynyl; 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 is alkynylene; L 3 is absent or is NH, S or O; R 28 is -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, an optionally substituted 3- to 10-membered heterocycle, or an optionally substituted 5- to 10-membered heteroaryl; R 29 From R 30 are independently H, optionally substituted C 1~30 Alkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, optionally substituted C 3~6 Cycloalkyl, optionally substituted C 3~6 Cycloalkenyl, optionally substituted C 6~12 aryl, optionally substituted 3- to 10-membered heterocycle, or optionally substituted 5- to 10-membered heteroaryl) or a pharmaceutically acceptable complex, salt, solvate, tautomer or polymorph thereof may be.

[0138] Compounds of formula (II) are described in UK Patent Application No. 2215200.3, the contents of which are incorporated herein by reference. Specific compounds of formula (II) disclosed in UK Patent Application No. 2215200.3 include maleic acid di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-((2-(dimethylamino)ethyl)thio)succinate, maleic acid di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-((2-(dimethylamino)methyl)thio)succinate, maleic acid di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-((2-(dimethylamino)propyl)thio)succinate, maleic acid di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-((2-(methylethylamino)ethyl)thio)succinate, maleic acid di((9Z,12Z)-octadeca-9,12-dien-1-yl) 2-((2-(diethylamino)ethyl)thio)succinate, and maleic acid dioleyl 2-((2-(diethylamino)ethyl)thio)succinate.

[0139] Thus, the constitutive or ionizable cationic lipids are 1,2-dilinoleyloxy-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), maleic acid di((9Z,12Z)-octadeca-9,12-dien-1-yl)2-((2-(dimethylamino)ethyl)thio)succinate, maleic acid di((9Z,12Z)-octadeca-9, 12-dien-1-yl)2-((2-(dimethylamino)methyl)thio)succinate, maleic acid di((9Z,12Z)-octadeca-9,12-dien-1-yl)2-((2-(dimethylamino)propyl)thio)succinate, maleic acid di((9Z,12Z)-octadeca-9,12-dien-1-yl)2-((2-(methylethylamino)ethyl)thio)succinate, maleic acid di((9Z,12Z)-octadeca-9,12-dien-1-yl)2-((2-(diethylamino)ethyl)thio)succinate, or maleic acid dioleyl 2-((2-(diethylamino)ethyl)thio)succinate.

[0140] The constant or ionized anionic lipid may be cholesteryl hemisuccinate, phosphatidylinositol phosphate (PIP, also known as phosphoinositides), phosphatidylserine (PS) or phosphatidic acid (PA).

[0141] The functionalized lipid may comprise one or more moieties that allow for attachment thereto, which may be independently selected from the group consisting of azide, alkyne, tetrazine, dibenzocyclooctyne (DBCO), maleimide, trans-cyclooctene (TCO), vinyl, methylcyclopropene, and succinimidyl ester.

[0142] In a preferred embodiment, the micro- or nanoparticles comprise a payload molecule, which may be a biomolecule, and / or an active pharmaceutical ingredient (API), and / or a diagnostic compound.

[0143] The API may be a hydrophobic or hydrophilic API. The API may be a polymer or a small molecule. It may be understood that a small molecule is considered to be a molecule having a molecular weight of less than 900 daltons. In some embodiments, a small molecule may have a molecular weight of less than 800 daltons, less than 700 daltons, less than 600 daltons, less than 500 daltons, or less than 400 daltons. Similarly, a polymer is considered to be a molecule having a molecular weight of at least 900 daltons.

[0144] Examples of APIs include anti-inflammatory compounds (e.g., non-steroidal anti-inflammatory drugs, such as aspirin, ibuprofen, naproxen, celecoxib, diclofenac, indomethacin, oxaprozin and / or piroxicam, more preferably steroidal anti-inflammatory drugs, such as prednisone, cortisone and methylprednisone, or anti-rejection drugs, such as tacrolimus, cyclosporine, mycophenolate mofetil, azathioprine, rapamycin, sirolimus), anti-cancer drugs (e.g., chemotherapy, such as alkylating agents (e.g., altretamine, bendamustine, busulfan, cyclophosphamide, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, mechlorethamine, melphalan, oxaliplatin, temozolomide, thiotepa, trabectedin), nitrosoureas (e.g., carmustine, lomustine, streptozocin), antimetabolites (e.g., azacitidine, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cladribine, clofarabine, cytarabine (Ara-C), decitabine, floxuridine, antitumor antibiotics (e.g., anthracyclines daunorubicin, doxorubicin (adriamycin), liposomal doxorubicin, epirubicin, idarubicin, valrubicin, and antitumor antibiotics bleomycin, dactinomycin, mitomycin-C, mitoxantrone), topoisomerase inhibitors (irinotecan, liposomal ibuprofen, rifampin, rifampin), rifampin-containing anti-inflammatory drugs (e.g., rifampin, rifampin-containing steroids ... rinotecan, topotecan, etoposide (VP-16), mitoxantrone, teniposide), mitotic inhibitors (e.g., the taxanes cabazitaxel, docetaxel, Nab-paclitaxel, and paclitaxel, and the vinca alkaloids vinblastine, vincristine, liposomal vincristine, and vinorelbine), and other chemotherapeutic agents (e.g., all-trans retinoic acid, arsenic trioxide, asparaginase, eribulin, hydroxyurea, ixabepilone, mitotane, omacetaxine, pegaspargase, procarbazine, romidepsin,including vorinostat), cytokine agents (including cytokines (e.g., IL-1, IL-2, TNF-alpha, IL-6, IL-7, IL-10, IL-12, IL-17, IL-21, IL-22, IL-23, IFN-alpha, IFN-beta, IFN-gamma, IFN-lambda 1, IFN-lambda 2, IFN-lambda 3, IFN-omega, IP-10, MIP-1alpha, TGF-beta (1-3) (recombinant forms thereof)) and anti-cytokine agents (e.g., cytokine-binding antibodies, decoy receptors, IL-1R antagonists)), growth factors ( Examples include bone morphogenetic proteins (BMPs), vascular endothelial growth factors (VEGFs), granulocyte-macrophage colony-stimulating factors (GM-CSFs), epidermal growth factors (EGFs), erythropoietin (EPO), insulin-like growth factors (IGFs), fibroblast growth factors (FGFs), hepatocyte growth factors (HGFs), platelet-derived growth factors (PDGFs), transforming growth factors (TGFs), thrombopoietin (TPOs), hormones (e.g., aldosterone, vasopressin, adrenocorticotropic hormone (ACTH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), Oxytocin, prolactin, thyroid-stimulating hormone (TSH), renin, angiotensin, glucagon, insulin, estrogen, progesterone, parathyroid hormone (PTH), thyroid hormones, epinephrine, norepinephrine, testosterone, melatonin, growth hormone-releasing hormone (GHRH), thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone (GnRH), corticotropin-releasing hormone (CRH), humoral factors), cardiovascular drugs (e.g., anticoagulants (e.g., apixaban, dabigatran, edoxaban, , heparin, rivaroxaban and warfarin), antiplatelet agents (examples include aspirin, clopidogrel, dipyridamole, prasugrel and ticagrelor), angiotensin-converting enzyme (ACE)-inhibitors (examples include benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril and trandolapril), angiotensin II receptor blockers (azilsartan, candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan,and valsartan), beta-adrenergic blocking agents (acebutolol, atenolol, betaxolol, bisoprolol, metoprolol, nadolol, propranolol, and sotalol), calcium channel blockers (examples include amlodipine, diltiazem, felodipine, nifedipine, nimodipine, nisoldipine, and verapamil), cholesterol-lowering drugs (examples include the statin drugs atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin, niacin nicotinate, and cholesterol-lowering drugs). Drugs that may be prescribed include: steroids (e.g., steroids), ... (including cimetidine, ranitidine, famotidine, and nizatidine), prokinetics and laxatives (metoclopramide), ophthalmic medications (e.g., ophthalmic allergy medications (e.g., ketorolac, ketotifen, loteprednol, bepotastine, epinastine, emedastine, alcaftadine, azelastine, olopatadine, nedocromil, lodoxamide, and cromolyn)), topical antibiotics (e.g., besifloxacin, ciprofloxacin, moxifloxacin, ofloxacin, gatifloxacin, tobramycin, gentamicin), , polymyxin D, neomycin, bacitracin, azithromycin, and erythromycin), lipid-based artificial tears (examples include castor oil, glycerol, and mineral oil), NSAIDS and corticosteroids, glaucoma medications (examples include levobunolol, timolol, betaxolol, bimatoprost, travoprost, latanoprost, tafluprost, brimonidine, brinzolamide, and dorzolamide), and antiviral treatments (examples include acyclovir, valacyclovir, and famciclovir), pulmonary medications (e.g., antiasthma medications (e.g.,antihistamines (e.g., brompheniramine, carbinoxamine, chlorpheniramine, clemastine, diphenhydramine, hydroxyzine, triprolidine, azelastine, cetirizine, desloratadine, fexofenadine, levocetirizine, loratadine, olopatadine), antitussives (e.g., dextromethorphan and benzonatate), bronchodilators (ipratropium, theophylline, albuterol, epinephrine, levalbuterol, alfo lumoterol, formoterol, olodaterol, terbutaline, pirbuterol, metaproterenol, salmeterol, isoproterenol, indacaterol, tiotropium, umeclidinium, aclidinium, ipratropium, lebefenacin, glycopyrrolate, ipratropium, theophylline, aminophylline, and dyphylline), decongestants (examples include levomethamphetamine, naphazoline, oxymetazoline, phenylephrine, propylhexedrine, pseudoephedrine, and xylometazoline), expectorants (guaifene sucrine), leukotriene modifiers (examples include montelukast, zafirlukast, zileuton), pulmonary surfactants (examples include beractant, lucinactant, calfactant, and boractant), mucolytics (acetylcysteine), anti-infectives (examples include zanamivir, ribavirin, tobramycin, pentamidine, and colistimethate), inhaled corticosteroids (examples include fluticasone, budesonide, mometasone, beclomethasone, and ciclesonide), mast cell stabilizers (examples include cromolyn and nedocromil), and phosphatase inhibitors (examples include fluticasone, budesonide, mometasone, beclomethasone, and ciclesonide). Fodiesterase-4 inhibitors (including roflumilast), antibacterial agents (antibiotics (e.g., aminoglycosides (e.g., amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, and spectinomycin)), ansamycins (e.g., geldanamycin, herbimycin, and rifaximin), carbacephems (e.g., loracarbef), carbapenems (e.g., ertapenem, doripenem, imipenem, and meropenem), cephalosporins (e.g.,cefadroxil, cefazolin, cephradine, cephapirin, cephalothin, cephalexin, cefaclor, cefoxitin, cefotetan, cefamandole, cefmetazole, cefonicid, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, moxalactam, ceftriaxone, cefepime, ceftaroline fosamil, and ceftobiprole), glycopeptides (e.g., teicoplanin, vancomycin, teratacycline, vancin, dalbavancin, and oritavancin), lincosamides (clindamycin and lincomycin), lipopeptides (e.g., daptomycin), macrolides (azithromycin, clarithromycin, erythromycin, roxithromycin, telithromycin, spiramycin, and fidaxomicin), monobactams (e.g., aztreonam), nitrofurans (e.g., furazolidone and nitrofurantoin), oxazolidinones (e.g., linezolid, pocizolid, radezolid, and torezolid), penicillins antihistamines (e.g., amoxicillin, ampicillin, azlocillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, temocillin, and ticarcillin), polypeptides (e.g., bacitracin, colistin, and polymyxin B), quinolones (e.g., ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nadifloxacin, nalidixic acid, norfloxacin), , ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, and temafloxacin), sulfonamides (examples include mafenide, sulfacetamide, sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilimide, sulfasalazine, sulfisoxazole, and sulfonamide chrysoidine), tetracyclines (examples include demeclocycline, doxycycline, methacycline, minocycline, oxytetracycline, and tetracycline),Mycobacteria-specific antibiotics (examples include clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin, rifabutin, rifapentine, and streptomycin), antifungal drugs (e.g., polyene antifungals (examples include amphotericin B, candicidin, filipin, hamycin, natamycin, nystatin, and rimocidin)), azoles (e.g., imidazoles bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, and tioconazole), triazoles vaconazole, efinaconazole, epoxiconazole, fluconazole, isavuconazole, itraconazole, posaconazole, propiconazole, ravuconazole, terconazole, and voriconazole, and thiazoles (e.g., abafungin), arylamines (e.g., butenafine, naftifine, and terbinafine), echinocandins (anidulafungin, caspofungin, and micafungin), and triterpenoids (e.g., ibrexafungelp), and antiparasitic agents (e.g., broad-spectrum Nitazoxanide, antiprotozoal drugs (examples include melarsoprol, eflornithine, metronidazole, tinidazole, and miltefosine), antinematoid drugs (examples include mebendazole, pyrantel pamoate, thiabendazole, diethylcarbamazine, and ivermectin), antitasteworm drugs (examples include niclosamide, praziquantel, and albendazole), antitrematode drugs (e.g., praziquantel), and antiamoebic drugs (e.g., rifampicin and amphotericin B), antidiabetic drugs (e.g., insulin (analogs), amylinomimetics (e.g., pramlintide), alfa glucosidase inhibitors (e.g., acarbose and miglitol), biguanides (e.g., metformin (analogs and combinations), dopamine agonists (e.g., bromocriptine), dipeptidyl peptidase-4 (DDP-4) inhibitors (e.g., alogliptin, linagliptin, saxagliptin, and sitagliptin), glucagon-like peptide-1 receptor agonists (e.g., albiglutide, dulaglutide, exenatide, liraglutide, and semaglutide), meglitinides (e.g., nateglinide and repaglinide), sodium-glucose transporter (SGLT-2) inhibitors (e.g., dapagliflozin, canagliflozin, ertugliflozin, and empagliflozin), sulfonylureas (e.g., glimepiride, gliclazide, glipizide, glyburide, chlorpropamide, tolazamide, and tolbutamide), thiazolidinediones (e.g., rosiglitazone and pioglitazone), antivirals (e.g., abacavir, acyclovir, adefovir, amantadine, ampligen, amprenavir, umifenovir, atazanavir, atriplan,Oseltamivir, zanamivir, peramivir, baloxavir, bictegravir, emtricitabine, tenofovir, boceprevir, brevirtide, cidofovir, cobicistat, daclatasvir, darunavir, delavirdine, didanosine, docosanol, dolutegravir, doravirine, edoxudine, efavirenz, emtricitabine, enfuvirtide, ensivirvir, ensitrervir, entecavir, etravirine, famciclovir, fomivirsen, fosamprenavir, foscarnet, ganciclovir, ibacitabine, ibalizumab, idoxuridine, imiquimod, inosine pranobex, indinavir, lamivudine, letervir and raltegravir, remdesivir, ribavirin, rilpivirine, rimantadine, ritonavir, saquinavir, simeprevir, sofosbuvir, stavudine, talibavirin, telaprevir, telbivudine, tenofovir, tipranavir, trifluridine, trizivir, tromantadine, truvada, umifenovir, valacyclovir, valganciclovir, vicriviroc, vidarabine, zalcitabine, zanamivir, and zidovudine), or structural or functional analogs thereof.

[0145] In a preferred embodiment, the payload molecule is a biomolecule, for example, the biomolecule may be or include an amino acid, peptide, affimer, polypeptide or protein, glycoprotein, sugar, lipid, lipopolysaccharide, antibody or fragment thereof, polymer, or nucleic acid, or a combination thereof.

[0146] The nucleic acid may be DNA, RNA, XNA (xenonucleic 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 (fluoroarabinonucleic acid), and unlocked nucleic acid (UNA), or a DNA / RNA hybrid sequence. Preferably, the nucleic acid is DNA or RNA.

[0147] Most preferably, the nucleic acid is RNA. The RNA may be single-stranded or double-stranded. The RNA may be selected from the group consisting of messenger RNA (mRNA); circular RNA (circRNA or oRNA); self-amplifying RNA (saRNA); trans-amplifying RNA (taRNA), long non-coding RNA, split replicon RNA, viral RNA, antisense RNA (AON or asRNA); RNA aptamer; interfering RNA; microRNA (miRNA); short interfering RNA (siRNA); short hairpin RNA (shRNA); and small RNA.

[0148] Preferably, the RNA is messenger RNA (mRNA).

[0149] The nucleic acid sequence, preferably RNA, may be at least 10 bases in length, at least 20 bases in length, at least 50 bases in length, at least 100 bases in length, at least 200 bases in length, at least 300 bases in length, at least 400 bases in length, at least 500 bases in length, at least 600 bases in length, at least 700 bases in length, at least 800 bases in length, or at least 900 bases in length. In a preferred embodiment, the RNA is saRNA or mRNA.

[0150] The nucleic acid sequence, preferably RNA, most preferably mRNA, may be at least 100 bases in length, at least 500 bases in length, at least 1000 bases in length, at least 2000 bases in length, at least 3000 bases in length, at least 4000 bases in length, at least 5000 bases in length, at least 6000 bases in length, at least 7000 bases in length, at least 8000 bases in length, at least 9000 bases in length, at least 10000 bases in length, at least 11000 bases in length or at least 12000 bases in length.

[0151] 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.

[0152] 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.

[0153] Alternatively, the nucleic acid sequence, preferably RNA, most preferably mRNA, may be 50 to 10,000 bases in length, 100 to 9,000 bases in length, 200 to 8,000 bases in length, 300 to 7,000 bases in length, 400 to 6,000 bases in length, 500 to 6,000 bases in length, 600 to 5,000 bases in length, 700 to 4,000 bases in length, 800 to 3,000 bases in length or 900 to 2,000 bases in length.

[0154] In one embodiment, the nucleic acid sequence is 6,000 to 15,000 bases in length. The nucleic acid sequence may be 8,000 to 12,000 bases in length. The RNA may be 6,000 to 15,000 bases in length. The RNA may be 8,000 to 12,000 bases in length. Preferably, the saRNA is 6,000 to 15,000 bases in length. Preferably, the saRNA is 8,000 to 12,000 bases in length.

[0155] In an alternative embodiment, the nucleic acid sequence is 100 to 14,000, 500 to 10,000, 600 to 7,500, 700 to 5,000, 800 to 4,000, or 900 to 2,000 bases in length. The RNA may be 400 to 14,000, 500 to 10,000, 600 to 7,500, 700 to 5,000, 800 to 4,000, or 900 to 2,000 bases in length. Preferably, the mRNA is 100 to 14,000, 500 to 10,000, 600 to 7,500, 700 to 5,000, 800 to 4,000, or 900 to 2,000 bases in length.

[0156] Those skilled in the art will understand that when the nucleic acid is double-stranded, for example double-stranded RNA, "length ~ bases" refers to the length in base pairs.

[0157] The mass ratio of lipid component to payload molecule may be 1:1 to 100:1, 2:1 to 80:1, 3:1 to 70:1, 4:1 to 60:1, or 5:1 to 50:1.

[0158] In embodiments where the payload molecule is a biomolecule, the mass ratio of lipid component to payload molecule can be 6:1 to 45:1, 8:1 to 40:1, 10:1 to 35:1, or 12:1 to 30:1. In some embodiments, the mass ratio of lipid component to payload molecule can be 13:1 to 25:1, 14:1 to 20:1, or 15:1 to 17:1. In some embodiments, the mass ratio of lipid component to payload molecule can be 15:1 to 27.5:1, 20:1 to 25:1, or 22:1 to 23:1.

[0159] In embodiments where the payload molecule is a biomolecule, the N:P ratio may be 1:2 to 50:1, 1:1 to 30:1, 2:1 to 20:1, 3:1 to 15:1, or 5:1 to 12:1. In some embodiments, the N:P ratio may be 3:1 to 10:1 or 4:1 to 6:1. In alternative embodiments, the N:P ratio may be 4:1 to 10:1, 5:1 to 9:1, or 6:1 to 8:1. It may be understood that the N:P ratio is the ratio of positively chargeable polymeric amine (N) groups to negatively charged nucleic acid phosphate (P) groups.

[0160] Preferably, the micro- or nanoparticles have an encapsulation efficiency of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%. It can be understood that the encapsulation efficiency is determined by the amount of payload molecules encapsulated in the micro- or nanoparticles (i.e., not available and / or accessible to the aqueous environment outside the micro- or nanoparticles) relative to the total amount of payload molecules initially provided. The encapsulation efficiency can be determined using a RiboGreen assay, which detects solvent-accessible RNA by an increase in fluorescence upon intercalation of the water-soluble Ribogreen reagent into the RNA.

[0161] The micro- or nanoparticles may have a zeta potential at physiological pH of -50 to +50 mV, -40 to +40 mV, -30 to +30 mV, or -20 to +20 mV, more preferably -10 to +10 mV or -5 to +5 mV. It will be appreciated that the zeta potential can be measured by suspending the LNP in a conductive buffer solution having a predetermined pH. The conductive buffer solution may be PBS (phosphate buffered saline, pH 7.2).

[0162] The micro- or nanoparticles may further comprise one or more auxiliary agents, each of which may be selected from the group consisting of aluminum hydroxide, Pam2CSK4, Pam3CSK4, glucopyranosyl lipid auxiliary (GLA), LPS and its analogs, CpG oligodeoxynucleotides and other TLR-agonists, such as poly I:C and dsRNA.

[0163] The micro- or nanoparticles may further comprise one or more additional compounds. The one or more additional compounds may be selected from the group consisting of hydrophobic compounds, polymers, permeability enhancers, sugars, surface modifiers, excipients, and combinations thereof. The polymer may be polylactic-co-glycolic acid (PLGA). It may be understood that the excipient may alter the pharmacokinetic properties of the composition but not the pharmacodynamic properties of the payload.

[0164] According to a third aspect, there is provided a composition comprising a plurality of micro- or nanoparticles of the second aspect.

[0165] The micro- or nanoparticles may have an average diameter of less than 10 μm, less than 1 μm, less than 500 nm, or less than 250 nm. More preferably, the micro- or nanoparticles may have an average diameter of less than 200 nm, less than 175 nm, less than 150 nm, less than 125 nm, less than 100 nm, less than 90 nm, or less than 80 nm. The micro- or nanoparticles may have an average diameter of 25 nm to 1 μm, 30 to 500 nm, 35 to 250 nm, 40 to 200 nm, 45 to 150 nm, 50 to 125 nm, 55 to 100 nm, 60 to 90 nm, or 65 to 80 nm. The average diameter of the micro- or nanoparticles can be measured using dynamic light scattering.

[0166] The micro- or nanoparticles may have a polydispersity index (PDI) of less than 0.5, less than 0.4, or less than 0.3, more preferably less than 0.25, less than 0.2, less than 0.15, less than 0.13, or less than 0.11. The micro- or nanoparticles may have a PDI of 0.001 to 0.05, 0.005 to 0.4, 0.01 to 0.3, 0.02 to 0.025, 0.04 to 0.2, 0.06 to 0.15, 0.08 to 0.13, or 0.09 to 0.11.

[0167] The composition may comprise a pharmaceutically acceptable carrier, which may improve colloidal stability, particularly under concentrated and / or refrigerated conditions (e.g., storage and / or transport at temperatures between 4°C and -80°C, e.g., 4°C, -20°C, or -70°C or -80°C). Such low temperature conditions can be used to extend the shelf life of the composition and / or, more specifically, the payload.

[0168] The composition may further comprise one or more solvents, buffers, suspending aids, fillers, flow agents, binders, salts, tonicity agents, thickeners, emulsifiers and / or preservatives.

[0169] In a fourth aspect, there is provided a micro- or nanoparticle of the second aspect, or a composition of the third aspect, for use as a medicament.

[0170] In a fifth aspect, there is provided a micro- or nanoparticle of the second aspect or a composition of the third aspect for use in the treatment and / or prevention and / or prophylaxis of a disease or disorder.

[0171] In a sixth aspect, there is provided a method for treating and / or preventing a disease or disorder, the method comprising administering or having administered to a subject in need thereof a prophylactic and / or therapeutic amount of the micro- or nanoparticles of the second aspect, or the composition of the third aspect.

[0172] The disease or disorder may be selected from the group consisting of an inflammatory disease, an infectious disease, a proliferative disease (e.g., cancer), an autoimmune disease, an eye disease, a lung disease, a skin disease, an intestinal disease, a metabolic disease (e.g., diabetes), a vascular disease (including cardiovascular and renal vascular disease), a neurological disease (e.g., neurodegenerative disease), a disorder of the endocrine system (including disorders related to hormones, growth factors, and / or cytokines), a disorder of the reproductive system, and a rare disease.

[0173] In a seventh aspect, there is provided a vaccine composition comprising the micro- or nanoparticles of the second aspect or the composition of the third aspect.

[0174] The vaccine may contain suitable adjuvants.

[0175] In an eighth aspect, there is provided a micro- or nanoparticle of the second aspect, or a composition of the third aspect, or a vaccine of the seventh aspect, for use in stimulating an immune response in a subject.

[0176] The immune response may be stimulated against a protozoan, a bacterium, a virus, a fungus, a multicellular parasite, or a cancer, or a portion thereof.

[0177] In a ninth aspect, there is provided a method of vaccinating a subject, the method comprising administering or having administered to a subject in need thereof a prophylactic and / or therapeutic amount of the micro- or nanoparticles of the second aspect, or the composition of the third aspect, or the vaccine of the seventh aspect.

[0178] The micro- or nanoparticles, compositions, or vaccines of the present invention may be combined into compositions having a number of different forms, depending in particular on the manner in which the compositions are to be used. Thus, for example, the compositions may be in the form of powders, tablets, capsules, liquids, ointments, creams, shampoos, gels, hydrogels, aerosols, sprays, micellar solutions, transdermal patches (including microneedles), drug depots / sustained-release formulations, (liposome) suspensions, irrigations / washes / instillations, those incorporated into biomaterials for tissue engineering, coatings on (implantable) medical devices, or any other suitable form that can be administered to humans or animals in need of treatment. It should be understood that the pharmaceutical vehicle of the present invention should be well tolerated by the subject to whom the vehicle is administered.

[0179] The micro- or nanoparticles, compositions or vaccines of the present invention may be incorporated into a sustained or delayed release device. Such a device may, for example, be inserted on or under the skin and may release the medication over a period of weeks or even months. The device may be placed at least adjacent to or upstream of the treatment site.

[0180] However, in a preferred embodiment, the medicament according to the invention may be administered to a subject by injection into the bloodstream, muscle, skin, or directly at the site requiring treatment. The injection may be intravenous (bolus or infusion), subcutaneous (bolus or infusion), intradermal (bolus or infusion), intramuscular (bolus or infusion), intrathecal (bolus or infusion), intravitreal (bolus), epidural (bolus or infusion), or intraperitoneal (bolus or infusion).

[0181] It should be understood that the amount of micro- or nanoparticle, composition, or vaccine required will be determined by its intended therapeutic or prophylactic use and its biological activity and bioavailability, which in turn will depend on the mode of administration, payload, physiochemical properties of the micro- or nanoparticle, composition, or vaccine, and whether it is used as a monotherapy or in a combination therapy.

[0182] The frequency of administration is also affected by the half-life of the active agent and / or the half-life of the therapeutic effect in the treated subject (e.g., the half-life of a therapeutic protein translated from a therapeutic mRNA transfected by a micro- or nanoparticle composition, or the half-life of, for example, an RNA-based CRISPR gene therapy). Optimal dosages to be administered can be determined by one skilled in the art and will vary depending on the micro- or nanoparticles, composition or vaccine used, the potency of the pharmaceutical composition, the mode of administration, and the type of treatment. Dosages may need to be adjusted depending on additional factors that depend on the particular subject being treated, including the subject's age, weight, sex, diet, and time of administration.

[0183] The dosage required may depend on several factors, including but not limited to, the active agent being administered, the disease being treated and / or vaccinated against, the subject being treated, and the like.

[0184] Generally, doses of 0.001 μg / kg body weight to 10 mg / kg body weight, or 0.01 μg / kg body weight to 1 mg / kg body weight of the micro- or nanoparticles, compositions or vaccines of the present invention may be used, depending on the active agent used. Dose may be understood to relate to the amount of payload molecule delivered.

[0185] The dose may be given as a single administration (e.g., a single injection). Alternatively, the micro- or nanoparticles, composition, or vaccine may require more than one administration. By way of example, the micro- or nanoparticles, composition, or vaccine may be administered as two or more doses of 0.07 μg to 700 mg (i.e., assuming a body weight of 70 kg). Alternatively, a sustained release device may be used to provide the patient with an optimal dose of the micro- or nanoparticles, composition, or vaccine according to the invention, without the need to administer repeated doses. The route of administration may incorporate intravenous, intradermal, subcutaneous, intramuscular, intrathecal, epidural, intravitreal, or intraperitoneal routes of injection.

[0186] Known procedures, such as those conventionally used by the pharmaceutical industry (e.g., in vivo experimental methods, clinical trials, etc.), may be used to formulate specific formulations of micro- or nanoparticles, compositions or vaccines according to the invention, and precise treatment regimens (e.g., drug doses and frequency of administration).

[0187] A "subject" may be a vertebrate, a mammal, or a livestock animal. Thus, the compositions and medicaments according to the present invention may be used to treat any mammal, such as livestock (e.g., horses), companion animals, and other veterinary and agricultural applications. Most preferably, however, the subject is a human.

[0188] A "therapeutically effective amount" of a micro- or nanoparticle, composition, or vaccine is any amount of the foregoing that is necessary to produce a therapeutic effect, whether partial or complete, when administered to a subject.

[0189] For example, a therapeutically effective amount of the micro- or nanoparticles, compositions or vaccines of the present invention may contain from about 0.001 μg to about 800 mg of payload molecules, preferably from about 0.01 mg to about 500 mg of payload molecules.

[0190] A "pharmaceutically acceptable vehicle," as referred to herein, is any known compound or combination of known compounds known to those skilled in the art to be useful in formulating pharmaceutical compositions.

[0191] In one embodiment, the pharmaceutically acceptable vehicle may be solid, and the composition may be in the form of a powder, capsule, or tablet. A solid pharmaceutically acceptable vehicle may contain one or more substances that may also act as flavoring agents, lubricants, solubilizers, suspending agents, dyes, fillers, flow agents, compression aids, inert binders, sweeteners, preservatives, pigments, coatings, or tablet disintegrants. The vehicle may also be an encapsulating material. In a powder, the vehicle is a finely divided solid in admixture with a finely divided active agent according to the present invention. In a tablet, the active agent (e.g., the micro- or nanoparticles of the present invention) may be mixed with a vehicle having the necessary compression properties in suitable proportions and compressed into the desired shape and size. The pharmaceutical vehicle may be a gel, and the composition may be in the form of a cream or similar.

[0192] Alternatively, the pharmaceutical vehicle may be liquid, and the pharmaceutical composition may be in the form of a solution. Liquid vehicles are used to prepare solutions, suspensions, emulsions, syrups, elixirs, and pressurized compositions. The micro- or nanoparticles according to the present invention may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle, such as water, an organic solvent, a mixture of both, or a pharmaceutically acceptable oil or fat. The liquid vehicle may contain other suitable pharmaceutical additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavorings, suspending agents, thickeners, dyes, viscosity regulators, stabilizers, or osmolality regulators. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing the above-mentioned additives, e.g., cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohols (including monohydric and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and peanut oil). For parenteral administration, the vehicle may be an oily ester, such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions can be halogenated hydrocarbon or other pharmaceutically acceptable propellant.

[0193] Liquid pharmaceutical compositions that are sterile solutions or suspensions can be utilized by, for example, intramuscular, intrathecal, intravitreal, epidural, intraperitoneal, intravenous, and subcutaneous injection. The micro- or nanoparticles of the present invention can be prepared in any suitable sterile injectable medium.

[0194] The micro- or nanoparticles may be administered by inhalation, for example, the micro- or nanoparticles may be provided in the form of an aerosol.

[0195] The micro- or nanoparticles and / or compositions of the present invention may be orally administered in the form of a sterile solution or suspension containing other solutes or suspending agents (e.g., sufficient saline or glucose to make the solution isotonic), bile salts, gum arabic, gelatin, sorbitan monooleate, polysorbate 80 (oleic acid ester of sorbitol and its anhydride copolymerized with ethylene oxide), and the like. The micro- or nanoparticles and / or compositions of the present invention may be orally administered as either a liquid or solid composition form. Compositions suitable for oral administration include solid forms such as pills, capsules, granules, tablets, and powders, as well as liquid forms such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

[0196] All of the features described in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0197] For a better understanding of the present invention, and to show how embodiments thereof may be carried into effect, reference will now be made, by way of example, to the accompanying drawings in which: FIG. [Brief explanation of the drawings]

[0198] [Figure 1] FIG. 1 shows the mean diameter and polydispersity index (PDI) of lipid nanoparticle (LNP) formulations measured by dynamic light scattering (DLS), where the lipid components of the LNPs comprise 50 mol% ionizable lipids, 38.5 mol% structural lipids, 10 mol% phospholipids, and 1.5 mol% PEG-modified lipids, and the structural lipids are either resveratrol (RSV), cholesterol (chol), or a combination thereof, as shown in FIG. 1, where the ratios of the combinations are molar ratios. [Figure 2]FIG. 1 shows the mean diameter and PDI of LNP formulations containing 5 mol% DOTAP as an uptake enhancer, as measured by dynamic light scattering. The lipid components of the LNPs include 45 mol% ionizable lipids, 5 mol% DOTAP, 38.5 mol% structural lipids, 10 mol% phospholipids, and 1.5 mol% PEG-modified lipids, where the structural lipids are either resveratrol (RSV), cholesterol (chol), or a combination thereof, as shown in FIG. 1, where the ratios of the combinations are molar ratios. [Figure 3] FIG. 1 shows the pKa of various LNP formulations determined by 2-(p-toluidino)naphthalene-6-sulfonic acid (TNS) assay at 20°C over a pH range of 3 to 10 in 0.5 pH increments, where the LNP formulations contain different structural lipids or mixtures thereof, and all percentages are molar percentages. [Figure 4] FIG. 1 shows the encapsulation efficiency of LNP compositions prepared with various core lipids and core lipid mixtures, as determined by the RiboGreen assay. The core lipid is either resveratrol (RSV), cholesterol, or a mixture thereof, or a resveratrol derivative or related structure; all percentages are molar percentages. [Figure 5] Figure 1 shows toxicity testing of LNP formulations containing different core lipids and core lipid mixtures, as determined by the resazurin assay. (A) The effect of resveratrol and cholesterol, individually and in mixtures, on the metabolic activity of HeLa cells; all ratios are molar ratios. (B) The effect of resveratrol derivatives and related structures on the toxicity of LNP formulations containing these structures as core lipids. [Figure 6]Figures 1A and 1B provide the in vitro activity of LNP compositions containing different core lipids and core lipid mixtures, as measured by secretory nanoluciferase assay. (A) shows the effect of resveratrol and cholesterol, individually and in mixture, on the transfection efficiency of LNP formulations containing these structures as core lipids; all ratios are molar. (B) shows the effect of resveratrol derivatives and related structures on the transfection efficiency of LNP formulations containing these structures as core lipids. [Figure 7] FIG. 10 demonstrates the lack of ApoE-mediated uptake and functional delivery in HeLa cells. Transfections were performed in the presence of various amounts of ApoE freshly added to the medium, and transfection efficiency was then determined by secreted nanoluciferase assay. DETAILED DESCRIPTION OF THE INVENTION [Example]

[0199] Example 1 Production of lipid nanoparticles (LNPs) To determine the safety and effectiveness of the lipid compositions of the present invention for the delivery of therapeutic and / or prophylactic molecules to cells, various formulations were prepared and tested.

[0200] Uniformly sized nanoparticles were reproducibly produced using an inverted herringbone microfluidic mixer, which rapidly (on the order of milliseconds) mixes an aqueous fluid, typically containing water-soluble therapeutic and / or prophylactic molecules, with an organic solvent containing premixed lipid components. Other types of microfluidic mixers (e.g., Y-junction, T-junction, or direct high-speed injection) produced similar results, as long as similar mixing ratios and mixing rates between the fluid streams were achieved.

[0201] The lipid compositions were prepared by combining core / structural lipids (each obtained from Sigma-Aldrich) with ionizable lipid L1 (maleic acid di((9Z,12Z)-octadeca-9,12-dien-1-yl)2-((2-(dimethylamino)ethyl)thio)succinate, as disclosed in UK Patent Application GB2215200.3), phospholipids (e.g., DOPE, obtained from Avanti Polar Lipids), optionally cationic lipids (e.g., DOTAP, obtained from Avanti Polar Lipids), and PEG-modified lipids (e.g., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (polyethylene glycol)-2000 (also known as PEG-DSPE), obtained from Avanti Polar Lipids). A control formulation contained cholesterol (obtained from Sigma-Aldrich) as the structural lipid. Typically, the lipids were combined in the following ratios: 45 mol% ionizable lipid, 5 mol% DOTAP, 38.5 mol% structural lipid, 10 mol% phospholipid, and 1.5 mol% PEG-modified lipid. For the DOTAP-free formulation, the lipids were combined in the following ratios: 50 mol% ionizable lipid, 38.5 mol% structural lipid, 10 mol% phospholipid, and 1.5 mol% PEG-modified lipid.

[0202] Certain formulations exhibit low overall cellular uptake in vitro and / or in vivo, which can be overcome by incorporating cell receptor or cell membrane targeting ligands, such as natural ligands or antibodies directed against cell surface receptors. Alternatively, increasing the zeta potential of the nanoparticle composition to at least +1 mV, preferably +10 mV, results in overall interaction with the cell membrane and uptake of the nanoparticles and / or cargo. In such cases, the following lipid ratios were used: 40-50 mol% ionizable lipid, 0-10 mol% cationic lipid, 38.5 mol% structured lipid, 10 mol% phospholipid, and 1.5 mol% PEG-modified lipid.

[0203] The lipid mixture was diluted with ethanol to a final concentration of 12.5 mM to 50 mM total lipid. The lipids dissolved in ethanol were stored at -20°C under argon and protected from light.

[0204] Nanoparticle compositions were made by combining therapeutic and / or preventive molecules in acidic (pH 4 or 5) or neutral (pH 7.4) aqueous solutions with lipid mixtures in ethanol at lipid-to-therapeutic molecule mass ratios ranging from 5:1 to 50:1. To produce well-defined nanoparticle populations, the aqueous solutions containing the therapeutic and / or preventive molecules ("aqueous solutions") and the lipid solutions were rapidly mixed in volume ratios ranging from about 2:1 (aqueous solution:lipid solution) to about 5:1 (aqueous solution:lipid solution) in an inverted herringbone microfluidic mixer at a total flow rate of 10 ml / min to 18 ml / min.

[0205] To prepare nanoparticle compositions containing RNA, the RNA was diluted to approximately 0.1 mg / ml to 3 mg / ml, preferably 0.15 mg / ml, in 100 mM sodium citrate buffer at pH 4-5, and then mixed with a lipid mixture. The RNA to lipid mass ratio was typically 1:10 to 1:30, resulting in an N:P ratio of 3-12, preferably about 5.

[0206] After mixing the aqueous and lipid solutions, the nanoparticle composition was dialyzed to remove ethanol to less than 0.1 vol%, optionally concentrating or diluting the solution and buffer exchanging it with a physiological pH buffer (e.g., pH 7.4, e.g., phosphate-buffered saline (PBS)). The formulation was dialyzed three times against at least a 100-fold excess of PBS using 100 kDa MWCO dialysis tubing (e.g., Spectrum™ Spectra / Por™ Biotech cellulose ester (CE) dialysis membrane tubing, available from Fisher Scientific). The first dialysis step was performed at room temperature for at least 2 h, and subsequent dialysis steps were performed at room temperature for at least 8 h or overnight at 4°C.

[0207] The size distribution of LNPs was determined by dynamic light scattering (DLS) using a Zetasizer Pro (red label, Malvern) with standard settings for LNPs (NIBS, adaptive correlation).

[0208] LNPs were formed using secNLuc (containing approximately 1000 nt of PolyA) mRNA, and their sizes were measured using DLS in 1x PBS (10 mM phosphate buffer, 150 mM NaCl) pH 7.4. Multiple LNP formulations were produced (50 mol% ionizable lipid (L1; maleic acid di((9Z,12Z)-octadeca-9,12-dien-1-yl)2-((2-(dimethylamino)ethyl)thio)succinate), 38.5 mol% structured lipid (resveratrol (RSV), cholesterol, or a mixture thereof), 10 mol% phospholipid (DOPE), and 1.5 mol% DSPE-PEG(2000), at a lipid-to-oligo weight (L / P) ratio of approximately 16). As shown in Figure 1, these formulations exhibited an average size of 70 nm (range, 65-90 nm) with an average polydispersity index (PDI) of 0.1.

[0209] Next, a formulation containing 5% DOTAP was produced; (ionizable lipid 45 mol% (L1; maleic acid di((9Z,12Z)-octadeca-9,12-dien-1-yl)2-((2-(dimethylamino)ethyl)thio)succinate), DOTAP 5 mol%, structural lipid 38.5 mol% (resveratrol, cholesterol, or a mixture thereof), phospholipid (DOPE) 10 mol%, and DSPE-PEG(2000) 1.5 mol%, at N / P5, corresponding to a lipid-to-oligo-weight (LOW) of approximately 16). Interestingly, as shown in Figure 2, the inclusion of DOTAP did not significantly alter the size or PDI of the LNPs.

[0210] Example 2 Lipid pKa determination The pKa of ionized lipids is known to be a major factor in the endosomal escape of LNP contents upon exposure to cells. During endosomal uptake and transit through the endosomal compartment, LNPs experience a gradual decrease in pH from physiological pH (pH 7.4, similar to that present outside the cell) to approximately pH 4.5-5.0 in lysosomes, the final stage of most endocytic vesicles. Transport to lysosomes should generally be avoided because the degradative environment can lead to degradation of cargo (e.g., mRNA); therefore, endosomal escape ideally occurs at a pH above 5.0, before the endosome matures into a lysosome. The rapid increase in cationic charge of ionized lipids during this acidification step is thought to drive their interaction with the inner endosomal membrane. Therefore, a lipid that is neutral or near-neutral at pH 7.4 and fully ionized at pH 5.5 would be ideal; this corresponds to a pKa of approximately 6.4–6.5, similar to what was found to be optimal by Jayaraman et al. (2012 Angewandte Chemie, DOI: 10.1002 / ang.201203263).

[0211] The local environment of ionized lipids (relative to their incorporation in the LNP) can affect charge acquisition and therefore pKa. It is quite possible that core lipids present just below the surface of the LNP can alter the surface properties and therefore pKa. Therefore, the experimentally determined pKa of ionized lipid L1 was measured by adding 2-(p-toluidino)naphthalene-6-sulfonic acid (TNS; obtained from Sigma-Aldrich) to nanoparticle compositions containing core lipids of the present invention, or mixtures thereof, in the presence of 20 mM phosphate-citric acid-ammonium citrate (pH 3-10 in 0.5 increments, all obtained from Sigma-Aldrich). TNS is a compound that interacts electrostatically with cationic lipids, resulting in fluorescence. Briefly, 50 μl of a mixture of 5 μM TNS, 25 μM LNPs (containing 12.5 μM ionizable lipid), and 20 mM buffer was added to a 384-well plate, followed by a phosphate-citric acid-ammonium citrate buffer sample, and measured at 325 nm excitation and 435 nm emission in a plate reader (iD3, Molecular Devices). Samples containing only LNPs and buffer were used for background subtraction for each pH increment. The background-corrected measured fluorescence was then normalized to the difference between the maximum and minimum fluorescence obtained during the assay, and curve fitting was performed to obtain a sigmoidal curve for each lipid composition. The pKa of each ionizable lipid was determined as the pH value at which half of the maximum fluorescence was reached, and the results are shown in Figure 3 for each LNP composition containing the core lipid of the present invention, cholesterol, or a mixture thereof.

[0212] It can be seen that none of the core lipids significantly altered the pKa of the L1 ionizable lipid-based LNP formulations, and it can therefore be concluded that the core lipids do not affect the ionization of the ionizable lipids.

[0213] Example 3 Encapsulation efficiency of LNPs High encapsulation efficiency of oligo- and polynucleotide cargoes is important to prevent exposure of the cargo to degradative enzymes, immune stimulatory cells, and receptors, and to achieve high transfection efficiency. Therefore, encapsulation efficiency was determined by RiboGreen assay.

[0214] Secreted NanoLuc(m)RNA-containing nanoparticle compositions (N / P 5:1, LOW approximately 16) were mixed 1:1 with QUANT-IT RIBOGREEN RNA Assay (Invitrogen, Thermo Fisher Scientific) at a concentration of approximately 5 μg / ml in TE buffer (10 mM Tris, HCl pH 7.5, 1 mM EDTA, Sigma-Aldrich) or with 2% Triton X-100 (Sigma-Aldrich) in TE buffer and an equal volume of 1:100 RiboGreen reagent. The samples were mixed thoroughly and incubated for 5 minutes at room temperature. Fluorescence intensity was then measured at 480 nm excitation and 520 nm emission on a plate reader (iD3, Molecular Devices). Blanks containing 1:200 RiboGreen reagent in TE buffer or 1:200 RiboGreen reagent in 2% Triton X-100 buffer were used as fluorescence background controls. A naked (m)RNA standard curve was used to quantify the absolute amount of unencapsulated RNA. The signal obtained with 2% Titron was used to normalize all samples to it and set it at 100%. The results are shown in Table 1 and Figure 4.

[0215] [Table 1A]

[0216] [Table 1B]

[0217] All of the LNP compositions of the present invention exhibit high encapsulation efficiency and are therefore suitable for protecting oligo- and polynucleotides from degradative enzymes and preventing any other effects of exposed oligo- and polynucleotides.

[0218] Example 4 Toxicity of LNP formulations to cells Each of the tested core lipids of the present invention is considered a GRAS (generally recognized as safe) compound present in various foods. Therefore, we investigated the effect of core lipids on the toxicity of the complete LNP formulation. Given the well-known anti-inflammatory and antioxidant properties of core lipids, both toxic and protective effects were of interest. To measure the toxic effects of core lipids, we added LNPs to cells at a range of concentrations defined by the mRNA content added to the cells.

[0219] Briefly, formulated mRNA concentrations ranging from 10 to 100 ng, corresponding to 160 to 1600 ng of total lipid, were premixed with cell culture medium in a total volume of 100 μl per well of a 96-well plate and added to HeLa cells. After 24 h of incubation, metabolic activity was tested by resazurin assay. For this purpose, the culture medium was replaced with medium containing 0.1 mg / ml resazurin and incubated for 1 to 4 h at 37°C and 5% CO2. Subsequently, fluorescence was determined in the supernatant (excitation 540 / 25 nm, emission 620 / 40 nm).

[0220] As shown in Figure 5, increasing lipid concentrations did not have a significant toxic effect on HeLa cells.

[0221] Example 5 In vitro activity of core lipid LNPs To determine whether alternative core lipid-containing LNP formulations could effectively deliver mRNA into cells, we incubated cells with luciferase mRNA-containing LNPs containing the core lipid of interest and 5% DOTAP as an uptake enhancer.

[0222] Core lipid-containing LNPs were formulated with 5 mol% DOTAP (45 mol% L1, 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 and contained secreted nanoluciferase mRNA at an N / P ratio of 5:1 (corresponding to a LOW of approximately 16). A dose range of LNPs corresponding to 100, 50, and 10 ng per well containing a volume of 100 μl was added to HeLa cells in a 96-well plate. After 24 hours, the medium was collected, and secreted nanoluciferase activity was determined using the Nano-Glo Luciferase Assay System (Promega).

[0223] Incubation of HeLa cells with resveratrol-lipid-containing LNPs demonstrated a dose-dependent induction of luciferase activity compared to LNPs containing only cholesterol as the core lipid. Notably, at higher resveratrol to cholesterol ratios, an increase of up to 300% over the control (containing only cholesterol as the core lipid) was observed. Interestingly, several resveratrol structural variants performed similarly to each other, with the exception of deoxyraptogenin, which underperformed, and polydatin, which outperformed the structural variants.

[0224] As shown in Figure 6A (activity of LNPs containing cholesterol, resveratrol or a mixture thereof) and Figure 6B (activity of LNPs containing resveratrol derivatives, and cholesterol and resveratrol as a control).

[0225] Example 6 Resveratrol-containing LNPs do not bind to ApoE in vitro To determine the lack of ApoE-dependent uptake, the cellular uptake of resveratrol-containing LNPs was tested with a range of ApoE concentrations.

[0226] L1-containing LNPs were formulated with resveratrol or cholesterol as described in Example 1 and contained secreted nanoluciferase mRNA. In addition, resveratrol- and cholesterol-containing LNPs were formulated with DOTAP 5%. LNPs equivalent to 100 ng mRNA per well containing a volume of 100 μl were added to HeLa cells in a 96-well plate in the absence or presence of ApoE (R&D Systems) at concentrations ranging from 1 to 20 μg / ml. After 24 hours, the medium was collected, and secreted nanoluciferase activity was measured using the Nano-Glo Luciferase Assay System (Promega).

[0227] Incubation of HeLa cells with resveratrol-containing LNPs showed no induction of luciferase activity in the absence or presence of increasing concentrations of ApoE. In contrast, cholesterol-containing LNPs showed a dose-dependent increase in luciferase activity. Resveratrol-containing LNPs containing DOTAP showed similar or even increased luciferase activity compared to cholesterol-containing LNPs containing DOTAP, demonstrating that resveratrol LNPs are not essentially inactive, as shown in Figure 7.

[0228] Thus, resveratrol-containing LNPs demonstrate an ApoE-independent uptake mechanism that is distinct from cholesterol-containing LNPs and allows for ApoE-independent biodistribution.

Claims

1. Compounds of formula (I) 【Chemistry 1】 (In the formula, L 1 teeth, 【Chemistry 2】 and; R 1 From R 10 are 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 an aryl, an optionally substituted 5-10 membered heteroaryl, or a sugar moiety; R 11 From 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 5-10 membered heteroaryl or a pharmaceutically acceptable complex, salt, solvate, tautomer or polymorph thereof in micro- or nanoparticle formulations.

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

3. -R 1 is H; -R 3 is 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 5-10 membered heteroaryl; -R 5 is H; -R 6 H, OR 11 ,OCOR 11 , COOR 11 , N.R. 11 R 12 or N ＋ R 11 R 12 R 13 and R 11 From R 13 are independently 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 5-10 membered heteroaryl; -R 7 H, OR 11 ,OCOR 11 , COOR 11 , N.R. 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 5-10 membered heteroaryl, and R 11 From R 13 are independently 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 5-10 membered heteroaryl; -R 9 H, OR 11 ,OCOR 11 , COOR 11 , N.R. 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 5-10 membered heteroaryl, and R 11 From R 13 are independently 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 5-10 membered heteroaryl; and / or -R 10 The use according to claim 1 or 2, wherein is H.

4. The compound is a compound of formula (Ia), (Ib), (Ic) or (Id) 【Transformation 3】 4. The use according to any one of claims 1 to 3, wherein

5. L 1 but 【Chemistry 4】 5. The use according to any one of claims 1 to 4, wherein

6. R 2 , R 4 and R 8 However, independently H, OR 11 ,OCOR 11 , COOR 11 , N.R. 11 R 12 , N ＋ R 11 R 12 R 13 or sugar moiety, R 11 From 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 The use according to any one of claims 1 to 5, wherein the aryl is an aryl or an optionally substituted 5-10 membered heteroaryl.

7. R 2 , R 4 and R 8 However, independently H, OH, OCH 3 , 【Transformation 5】 or sugar moiety or general formula 【Transformation 6】 (In the formula, X 1 is CR 20 R 21 is a combination of; R 17 From R 25 are independently H, OH, CH 2 OH, COOH, NH 2 , C 1～6 Alkyl, halo, 【Transformation 7】 The use according to claim 6, wherein

8. The compound of formula (I) 【Chemical Engineering 8A】 【Chemical 8B】 【Chemical 8C】 8. The use according to any one of claims 1 to 7, wherein

9. Compounds of formula (I) 【Chemistry 9】 (In the formula, L 1 teeth 【Chemistry 10】 and; R 1 From R 10 are independently H, OR 11 ,OCOR 11 , COOR 11 , N.R. 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 an aryl, an optionally substituted 5-10 membered heteroaryl, or a sugar moiety; R 11 From 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 5-10 membered heteroaryl or a pharmaceutically acceptable complex, salt, solvate, tautomer or polymorph thereof and an optional payload molecule.

10. 10. The micro- or nanoparticle according to claim 9, which is a lipid nanoparticle (LNP), liposome, lipoplex, micelle or lipid vesicle, preferably an LNP.

11. 11. The micro- or nanoparticle of claim 9 or 10, comprising a lipid component comprising a compound of formula (I) or a pharmaceutically acceptable complex, salt, solvate, tautomer or polymorph thereof, and one or more additional lipids selected from the group consisting of phospholipids, permanent cationic lipids, ionizable cationic lipids, permanent anionic lipids, ionizable anionic lipids, structural lipids, shielding lipids, functionalized lipids, additional core lipids and combinations thereof.

12. 13. The micro- or nanoparticles according to any one of claims 9 to 12, wherein the lipid component of the micro- 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 the compound of formula (I) or a pharmaceutically acceptable complex, salt, solvate, tautomer, or polymorph thereof.

13. 13. The micro- or nanoparticle of any one of claims 9 to 12, comprising a payload molecule, which is a biomolecule, an active pharmaceutical ingredient (API) and / or a diagnostic compound.

14. 14. The micro- or nanoparticle of any one of claims 9 to 13, wherein the payload molecule is a biomolecule and is or comprises an amino acid, a peptide, an affimer, a polypeptide or a protein, a glycoprotein, a sugar, a lipid, a lipopolysaccharide, an antibody or a fragment thereof, a polymer, or a nucleic acid, or a combination thereof, preferably wherein the biomolecule is or comprises a nucleic acid.

15. 15. The micro- or nanoparticle of claim 14, wherein the nucleic acid is DNA, RNA, xenonucleic acid (XNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), fluoroarabinonucleic acid (FANA), unlocked nucleic acid (UNA), or a DNA / RNA hybrid sequence.

16. 16. The micro- or nanoparticle of claim 15, wherein the nucleic acid is RNA, the RNA is single-stranded or double-stranded and is selected from the group consisting of messenger RNA (mRNA); circular RNA (circRNA or oRNA); self-amplifying RNA (saRNA); trans-amplifying RNA (taRNA), long non-coding RNA, split replicon RNA, viral RNA, antisense RNA (AON or asRNA); RNA aptamer; interfering RNA; microRNA (miRNA); small interfering RNA (siRNA); short hairpin RNA (shRNA); and small RNA.

17. A composition comprising a plurality of micro- or nanoparticles according to any one of claims 9 to 16.

18. 18. Micro- or nanoparticles according to any one of claims 9 to 16, or a composition according to claim 17, for use as a medicament.

19. 18. Micro- or nanoparticles according to any one of claims 9 to 16, or a composition according to claim 17, for use in the treatment and / or prevention and / or prophylaxis of a disease or disorder.

20. A vaccine composition comprising the micro- or nanoparticles of any one of claims 9 to 16 or the composition of claim 17.

21. 21. A micro- or nanoparticle according to any one of claims 9 to 16, or a composition according to claim 17, or a vaccine according to claim 20, for use in stimulating an immune response in a subject.

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