Methods and compositions for nano-delivery targeting dendritic cells

Abstract

This disclosure relates to novel compounds, methods, and cell-targeted formulations, such as lipid nanoparticles (LNPs) for targeted delivery to tissues or cell types. The compounds and formulations provided herein are designed to have formulations for incorporation into a bilayer membrane of formed lipid nanoparticles and targeting portions configured to provide selective delivery characteristics to the lipid tail.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 458,102 filed on 8 April 2023, U.S. Provisional Patent Application No. 63 / 587,231 filed on 2 October 2023, U.S. Provisional Patent Application No. 63 / 575,093 filed on 5 April 2024, and the PCT patent publication entitled “Methods and Compositions for Dendritic Cell Targeted Vaccines” filed on 8 April 2024. The entirety of the aforementioned applications is incorporated herein by reference.

[0002] This disclosure relates to novel formulations for targeted nano-delivery. Specifically, this disclosure relates to compositions and methods for novel bifunctional nanoparticle formulations capable of selectively delivering a payload to a desired region of a tissue or specific cell type, including dendritic cells. [Background technology]

[0003] Localized delivery via nanotechnology is widely used in scientific, industrial, and clinical applications. It is increasingly becoming a promising method of drug delivery, offering advantages including improved solubility and permeability of drug molecules. In a recent example of mRNA vaccines developed against the COVID-19 virus, lipid nanoparticles (LNPs) were developed to encapsulate and stabilize mRNA molecules during transport and after injection into the human body, considering the instability of mRNA molecules and the need for low-temperature storage (e.g., -70°C). Lipid nanoparticles are typically composed of several types of lipids. The ratio of these lipids requires fine-tuning, the production of lipid nanoparticles can be costly, and most importantly, lipid nanoparticles generally cannot deliver mRNA molecules with highly localized selectivity. Therefore, the need for novel nanoparticle formulations with selective delivery capabilities remains unmet. [Overview of the project]

[0004] One aspect of this disclosure relates to a bifunctional compound for forming lipid nanoparticles suitable for pharmaceutical formulations. The component is given by formula:

[0005] [ka] (In the formula, R1 includes a substituted or unsubstituted glycosyl group, X1 and X2 are independently hydrogen and C 1~30 Alkyl, C 1~30 Alkenil, C 1~30 Alkynyl, aryl, aryloxy or their substituted derivatives, The compound is -(CH2)nX4, where n is 0 to 30, and X4 is hydrogen, an aryl group, an aryloxy group, a heterocyclic group, or a substitution thereof, provided that if X4 is a heterocyclic group, the heterocyclic group contains 1 to 3 heteroatoms or combinations thereof selected from the group consisting of O, S, and N. X3 is hydrogen, C 1~6 It contains alkyl or hydroxyl compounds.

[0006] One aspect of the present disclosure relates to a formulation for forming lipid nanoparticles comprising the compound of the present disclosure, wherein the compound constitutes 1 to 10 mol% of the composition.

[0007] One aspect of the present disclosure relates to lipid nanoparticles and / or lipid nanoparticle formulations. The lipid nanoparticles and / or lipid nanoparticle formulations include a membrane defining an internal space, the membrane being formed of a plurality of lipid components including the compounds of the present disclosure.

[0008] One aspect of the present disclosure relates to a bifunctional targeted nanocarrier composition / formulation comprising lipid nanoparticles of the present disclosure.

[0009] One aspect of the present disclosure relates to a kit and / or reagent mixture for preparing lipid nanoparticles, comprising a first reagent containing a compound of the present disclosure, and a second reagent containing an ionizable lipid, a helper lipid, or a mixture thereof.

[0010] One aspect of the present disclosure relates to a method for delivering a targeted payload to a subject requiring such delivery, comprising administering to the subject an effective amount of the targeted lipid nanoparticles of the present disclosure in a pharmaceutically acceptable formulation, wherein the payload is a nucleic acid, compound, polypeptide, protein, glycan or a combination thereof, and the payload is encapsulated by lipid nanoparticles.

[0011] One aspect of the present disclosure relates to a method for preventing or treating a disease in a subject in need thereof, comprising administering to the subject an effective amount of targeted lipid nanoparticles of the present disclosure in a pharmaceutically acceptable formulation, wherein the payload is a nucleic acid, compound, polypeptide, protein, glycan or a combination thereof, the payload is encapsulated within the lipid nanoparticles, the payload is a therapeutic agent, or induces a therapeutic agent.

[0012] One aspect of the present disclosure relates to a method for boosting an adaptive immune response, comprising targeting an effective amount of targeted lipid nanoparticles of the present disclosure in a pharmaceutically acceptable formulation, wherein the payload is a nucleic acid, compound, polypeptide, protein, glycan or a combination thereof, the payload is encapsulated within the lipid nanoparticles, the payload is immunogenic, or induces an immunogenic biomolecule.

[0013] In certain embodiments of the bifunctional molecule and its formulation, Formula 1 may be a structural and / or functional analog / mimetic having cell-targeting function, and Formula 2 may be a structural / functional analog / mimetic having lipid membrane insertion / anchoring function. [Brief explanation of the drawing]

[0014] [Figure 1] This document provides a graphical representation of FACS analysis results demonstrating the effectiveness of exemplary embodiments of the present invention. Experiments demonstrated the uptake of novel nano-delivered formulations targeting BDMCs by embodiments of this disclosure compared to conventional LNPs. The FITC+ values ​​shown in the figure represent fluorescence intensity in arbitrary units (AU). [Figure 2]This disclosure provides a graphical representation of FACS analysis results demonstrating the efficacy of the exemplary novel dendritic cell-targeted formulations of this disclosure. Experiments show the uptake of the exemplary novel dendritic cell-targeted formulations of this disclosure by embodiments of this disclosure targeting dendritic cells (DCs), B cells, and T cells, compared to conventional LNPs. The FITC+ values ​​shown in the figure represent fluorescence intensity in arbitrary units (AU). [Figure 3] This disclosure provides a graphical representation of FACS analysis results demonstrating the efficacy of the exemplary novel dendritic cell targeting formulations of this disclosure. The experiment showed the uptake of the dendritic cell targeting formulations according to the embodiments of this disclosure, targeting BDMCs, compared to conventional LNPs. The FITC+ values ​​shown in the figure are fluorescence in arbitrary units (AU). 22-LNP represents a targeted formulation prepared using compound 22 of this disclosure, and the percentage in parentheses indicates the molar ratio of compound 22. Similarly, 23-LNP represents a targeted formulation prepared using compound 23 of this disclosure, and the percentage in parentheses indicates the molar ratio of compound 23. The negative control was an LNP that did not use the novel targeted compound / formulation of this disclosure (i.e., "conventional LNP" as described herein). [Figure 4] This disclosure provides a graphical representation of FACS analysis results demonstrating the efficacy of the exemplary novel dendritic cell-targeting formulations of this disclosure. The experiment demonstrated the transfection of BDMCs of the targeted formulations compared to LNPs without the compounds of this disclosure. The FITC+ values ​​shown in the figure are fluorescence in arbitrary units (AU). 22-LNP represents a targeted formulation prepared using compound 22 of this disclosure, and the percentage in parentheses indicates the molar ratio of compound 22. Similarly, 23-LNP represents a targeted formulation prepared using compound 23 of this disclosure, and the percentage in parentheses indicates the molar ratio of compound 23. The negative control was an LNP formed without using the novel targeted compound / formulation of this disclosure (i.e., a “conventional LNP” as described herein). [Figure 5]This study provides a graphical representation demonstrating the targeted efficacy and specificity of an exemplary formulation based on the distribution of targeted LNPs in an animal model. The LNPs carried mRNA configured to encode luciferase within targeted cells of test animals. The assay would produce detectable luminescence if the LNPs successfully transfected cells and the cells expressed luciferase. The results clearly demonstrated tissue-specific targeting of the spleen and lymphoid tissue by the exemplary targeted formulation, thus providing evidence for immune cell (e.g., dendritic cell) specificity. [Figure 6] The 1H NMR spectrum of compound 12 of this disclosure is shown. [Figure 7] The 13C NMR spectrum of compound 12 of this disclosure is shown. [Figure 8] A bar graph showing in vivo induction of IFNγ(A) and IL-4(B) by exemplary targeted LNP formulations according to embodiments of this disclosure is presented. Serum was collected from experimental animals 2 hours, 24 hours, and 48 hours after administration. [Figure 9] This disclosure provides a graphical representation demonstrating the neutralization inhibitory effect of exemplary LNPs compared to control LNPs. To show differences between samples, neutralization inhibition was evaluated at different dilution ratios. [Figure 10] The present disclosure provides a bar graph demonstrating that exemplary LNPs having mRNA encoding wild-type spike protein were able to induce IgG production in vivo against wild-type virus and its delta and omicron strains. [Figure 11] A comparative bar graph is shown, illustrating the induced IgG titer (A) and neutralizing ability (B) of commercially available LNPs compared to LNP formulations formulated based on and / or constructed using the exemplary compounds according to embodiments of this disclosure. Both LNPs contained mRNA encoding the wild-type spike protein. [Figure 12] The LCMS spectrum of compound 21 of this disclosure is shown. [Modes for carrying out the invention]

[0015] Nanoparticles are widely used in a variety of applications. Among them, lipid nanoparticles (e.g., liposomes) are the most established delivery system for drugs due to their biocompatibility and biodegradability. Typical liposomes have a bilayer structure formed of phospholipids due to their amphiphilic properties. The bilayer structure (i.e., the bilayer membrane) encapsulates an internal space that can contain hydrophilic molecules, while the bilayer structure itself can contain hydrophobic molecules. Lipid nanoparticles (LNPs) are also the most studied vehicle for delivering nucleic acids, such as RNA. Through encapsulation, LNPs protect nucleic acids from extracellular nucleases, thus enabling safe delivery to cells.

[0016] LNPs generally contain four lipid components in desired ratios: (i) helper lipids for cargo encapsulation, (ii) ionizable lipids for enhanced endosome escape and delivery, (iii) cholesterol for stability, and (iv) lipid-immobilized polyethylene glycol (PEG-lipid) for reduced recognition by the immune system and improved biodistribution. Cholesterol and PEG-lipids can also be classified as helper lipids. The properties of liposomes can be adjusted by selecting the desired lipid components or their ratios. Without being constrained by theory, the targeted moiety can also be conjugated with lipid components to provide selective delivery.

[0017] Targeting of lipid compounds

[0018] One aspect of the present disclosure provides compounds for forming lipid nanoparticle (LNP) formulations suitable for targeting specific cells. In certain embodiments, the compound is a bifunctional compound containing a glycan-based cell-targeting moiety and a lipid moiety, which can be incorporated into a lipid bilayer, e.g., LNPs. In some embodiments, the bifunctional compound is designed to have a targeting moiety configured to provide selective delivery functionality as part of a bifunctional molecule, and an exemplary lipid tail moiety that can be incorporated into a lipid bilayer membrane of an exemplary formulation containing lipid nanoparticles. The compounds of the present disclosure may have a bilayer structure and include two extension structures (e.g., X1 and X2 groups below). At least one of the two extension structures is configured to be incorporated into a bilayer membrane of the formed lipid nanoparticles. The term “incorporated into bilayer” as herein means that at least a portion of the extension structure is incorporated into the bilayer. In some embodiments, the entire extension structure is incorporated into the bilayer, but the term is not limited to this scenario.

[0019] In one exemplary embodiment, the compounds of the present disclosure are

[0020] [ka] (In the formula, R1 includes a substituted or unsubstituted glycosyl group, X1 and X2 are each independently hydrogen, alkyl, alkenyl, alkynyl, aryloxy or a substituted thereof, or -(CH2)nX4, where n is 0 to 50, and X4 is hydrogen, aryl, aryloxy, heterocyclic group or a substituted thereof, provided that if X4 is a heterocyclic group, the heterocyclic group includes 1 to 3 heteroatoms selected from the group consisting of O, S and N, and X3 is hydrogen, C 1~6 It contains alkyl or hydroxyl compounds.

[0021] R1 unit

[0022] Targeting function. In certain embodiments, the R1 group is configured to provide selective or targeted delivery function to exemplary LNP formulations formed by the components of the present disclosure. In some embodiments, the R1 group is configured to target antigen-presenting cells (e.g., dendritic cells). In some embodiments, the target cells may be other types of immune cells. In some other embodiments, the target may be any living cell on which the payload is designed. In certain embodiments, the R1 group is designed to have a targeting moiety, which may be a ligand for reception on the target cell. For example, the R1 group may be configured to target DC-SIGN on dendritic cells.

[0023] Without being constrained by theory, mannosides and fucosides are thought to be able to bind to dendritic cells (e.g., via binding to DC-SIGN) with specificity. Therefore, in some embodiments, the R1 group contains mannoside, fucoside, or both as the targeting moiety. The mannoside and / or fucoside can be terminal mannose or terminal fucoside of the R1 group, and these terminal mannose or terminal fucoside can increase the probability of interacting with dendritic cells.

[0024] In some other embodiments, the R1 group is configured to target Siglec-1, and thus the glycosyl group can include 9-N-(4H-thieno[3,2-c]chromene-2-carbamoyl)-Neu5Ac-α2,3-Gal-GlcNAc. In some embodiments, the R1 group is configured to target Siglec-2, and the glycosyl group can include 9-biphenylNeu5Ac-α2,6-Gal-GlcNAc. In some embodiments, the R1 group is configured to target Siglec-5 / E, and the glycosyl group can include Neu5Ac-α2,3-Gal-GlcNAc.

[0025] In some embodiments, the R1 group is of formula R2-R A -(In the formula, R2 is a substituted or unsubstituted glycosyl group, R Ais a linking group, and the linking group is aryl, alkyl, amide, alkylamide, combinations thereof, or a covalent bond.) is included. In some embodiments, the aryl includes 0 to 3 substituents (e.g., 1 to 3 substituents), and the substituents of the aryl are C 1~6 alkyl, halide, or C 1~6 haloalkyl. In some embodiments, the linking group is configured to provide structural flexibility and / or to facilitate the bond between the targeting moiety and the target. In certain embodiments, R2 is covalently conjugated to R A at the carbon of the glycosyl group to result in O-glycosylation.

[0026] Bonding under acidic conditions. In some embodiments, the bond between the glycosyl group of R1 and the target is Ca 2+ correlated, and the calcium coordination is reduced in a low pH environment, resulting in a lower binding affinity. Thus, the linking group can include an aryl group to provide a better binding affinity under acidic conditions. Without wishing to be bound by any theory, the aryl group may be involved in CH-π and hydrophobic interactions that enhance the binding under acidic conditions. The aryl group can be unsubstituted benzene or benzene substituted with halide or haloalkyl (e.g., CF3). In some embodiments, the aryl group is coupled to the targeting moiety. For example, the R1 group can include an O-arylmannoside.

[0027] Spacer. In some embodiments, the linking group of R1 includes a spacer. The spacer is configured to provide structural flexibility to R1. Without wishing to be bound by theory, this flexibility allows the glycosyl group of R1 to be mobile during the interaction between the targeting moiety and the target, thus facilitating the bond between the targeting moiety and the target.

[0028] In certain embodiments, the preferred spacer is biocompatible. In some embodiments, the starting spacer includes a saturated carbon moiety, a polyethylene glycol (PEG) moiety, or a combination thereof. For example, the spacer may be a polyethylene glycol (PEG) portion formed by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 24, 30, 36, 40, 48, 50, 55, 60, 65, or 72 (OCH2CH2) subunits, or any range defined by the aforementioned endpoints, for example, 2-72, 2-60, 2-48, 2-36, 2-24, 2-18, 2-15, 2-10, 4-72, 4-60, 4-48, 4-36, 4-24, 4-18, 4-15, 4-10, 8-72, 8-60, 8-48, 8-36, 8-24, 8-18, 8-15, or 8-10 (OCH2CH2) subunits. In some embodiments, the PEG portion can have a linear, branched, or star-shaped structure.

[0029] Structural configuration. In certain embodiments, the glycosyl group may have a linear or branched structure. In some embodiments, the glycosyl group may have multiple targeting moieties, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 targeting moieties. The multiple targeting moieties can be arranged in a linear, branched, or star-shaped configuration. For example, the glycosyl group may contain a monomannoside, dimannoside, or trimannoside, and if the glycosyl group contains a trimannoside, the trimannoside may be in a linear form or a branched structure, e.g., α-1,3-α-1,6-trimannoside. In certain embodiments, the branched configuration (e.g., a trimannoside glycan head) has been shown to exhibit excellent binding affinity to its target receptor.

[0030] In some embodiments, the R1 group is a substituted glycosyl group. The glycosyl group may contain 1 to 6 substituents, each substituent being C 1~6 Alkyl, C 1~6 Alkenyl, halogen, C 1~6 Alkyl halogens, C 1~6 Alkoxy, amine, nitro, C1~6 The substituents may be alkylamines, amides, azides, aryls, cycloalkyls, heterocycloalkyls, sulfite ester groups, or their substitutions, or combinations thereof. In certain embodiments, the substituents are directly conjugated to the carbon of the glycosyl group or conjugated to the carbon via O-yl conjugation (for example, by replacing a hydrogen of the hydroxyl group on the carbon).

[0031] In a particular embodiment, the substituent of the glycosyl group is selected from the group consisting of aryl, 5-membered cycloalkyl, 6-membered cycloalkyl, 5-membered heterocycloalkyl, and 6-membered heterocycloalkyl, as well as their substituted counterparts, where the substituted counterpart is C 1~6 Alkyl, halogen, C 1~6 Alkyl halogens, C 1~6 Alkoxy, amine, nitro, C 1~6 The molecule contains 1 to 6 substituents selected from the group consisting of alkylamines, azides, amides, carboxyls, hydroxyls, aryls, cycloalkyls, heterocycloalkyls, or substituted products thereof, or combinations thereof. In some embodiments, the heterocycloalkyl contains 1 to 3 heteroatoms selected from the group consisting of O, S, and N.

[0032] In some embodiments, the substituents on the glycosyl group are substituted or unsubstituted aryl groups, such as substituted or unsubstituted phenyl groups. In certain embodiments, the aryl group is substituted with 1 to 6 substituents, each independently of C 1~6 Alkyl, halogen, C 1~6 Alkyl halogens, C 1~6 Alkoxy, amine, nitro, C 1~6The substituents are selected from the group consisting of alkylamines, azides, amides, carboxyls, hydroxyls, aryls, cycloalkyls, heterocycloalkyls, or their substitutions, or combinations thereof. In certain embodiments, the substituent of the glycosyl group is a phenyl (benzene ring) substituted with OH, CH3, NH2, CF3, OCH3, F, Br, Cl, NO2, N3, or combinations thereof. For example, the substituted benzene ring can be a phenol group.

[0033] In some embodiments, the R1 group is a monomannoside substituted with 1 to 6 substituents, each substituent being C 1~6 Alkyl, C 1~6 Alkenyl, halogen, C 1~6 Alkyl halides, amines, C 1~6 It can be an alkylamine, amide, aryl, cycloalkyl, heterocycloalkyl, sulfite ester group or a substituted thereof, or a combination thereof. In a particular embodiment, the R1 group is a monomannoside substituted with a first substitution and a second substitution, where each of the first and second substitutions is independently C 1~6 Alkyl, C 1~6 Alkenyl, halogen, C 1~6 Alkyl halides, amines, C 1~6 Selected from the group consisting of alkylamines, amides, aryls, cycloalkyls, heterocycloalkyls, and sulfite ester groups.

[0034] In some embodiments, the R1 group comprises a first mannoside and a second mannoside. Each of the first and second mannosides is independently substituted with 1 to 6 substituents, each substituent being C 1~6 Alkyl, C 1~6 Alkenyl, halogen, C 1~6 Alkyl halides, amines, C 1~6 It may be an alkylamine, amide, aryl, cycloalkyl, heterocycloalkyl, sulfite ester group, or a substituted product thereof, or a combination thereof.

[0035] Binding affinity. In some embodiments, the binding affinity between the glycosyl group of R1 and the target is the dissociation constant (K). D ) can be defined by. In some embodiments, K at pH 7.4 D The numbers are 5, 10, 15, 20, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 3000, 3250, 3500, 3750, 4000, 4250, 4500, and 47 50, 5000, 5250, 5500, 5750, 6000, 6250, 6500, 6750, 7000, 7250, 7500, 7750 or 8000 nM, or any range defined by the aforementioned endpoints, e.g., 5-8000, 5-7000, 5-6000, 5-5000, 5-4000, 5-3000, 5-25 00, 5~2000, 5~1500, 5~1250, 5~1000, 5~900, 5~800, 5~700, 5~600, 5~500, 5~400, 5~300, 5~200, 5~150, 5~100, 5~75, 5~50, 5~30, 5~20, 10~8000, 10~7000, 10~6000, 10~5000, 10~4000, 10 It can be ~3000, 10~2500, 10~2000, 10~1500, 10~1250, 10~1000, 10~900, 10~800, 10~700, 10~600, 10~500, 10~400, 10~300, 10~200, 10~150, 10~100, 10~75, 10~50, 10~30, or 10~20 nM.

[0036] In some other embodiments, K at pH 5 DThis can be 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1250, 1500, 1750 or 2000 nM, or any range defined by the aforementioned endpoints, e.g., 1-2000, 1-1500, 1-1000, 1-900, 1-800, 1-750, 1-700, 1-650, 1-600, 1-550, 1-50 It can be 0, 1~450, 1~400, 1~350, 1~300, 1~250, 1~200, 1~150, 1~100, 1~75, 1~50, 1~40, 1~30, 1~20, 1~10 or ~5, 5~2000, 5~1500, 5~1000, 5~900, 5~800, 5~750, 5~700, 5~650, 5~600, 5~550, 5~500, 5~450, 5~400, 5~350, 5~300, 5~250, 5~200, 5~150, 5~100, 5~75, 5~50, 5~40, 5~30, 5~20, 5~10 nM.

[0037] Example: In some embodiments, the R1 group is

[0038] [ka] The selected structure is one that consists of TIFF2026515588000005.tif196162 and TIFF2026515588000006.tif84161 (each of the structures shown above is independent of the others, regardless of whether they are separated from adjacent structures using semicolons).

[0039] In some embodiments, the compound of the present disclosure is of formula 3:

[0040] [ka] It has the structure shown, and the R1 group is

[0041] [ka] The structure is selected from the group consisting of the following (each of the structures shown above is independent of the others, regardless of whether they are separated from adjacent structures by semicolons).

[0042] X1 and X2

[0043] X1 and X2 are independently hydrogen and C 1~30 Alkyl, C 1~30 Alkenil, C 1~30 The compound is an alkynyl, aryl, aryloxy or a derivative thereof, or -(CH2)nX4, where n is 0 to 30, and X4 is hydrogen, aryl, aryloxy, heterocyclic group or a derivative thereof, provided that if X4 is a heterocyclic group, the heterocyclic group contains 1 to 3 heteroatoms or combinations thereof selected from the group consisting of O, S, and N. Without wishing to be constrained by theory, the present disclosure is designed to provide compounds in which at least one of the X1 and X2 groups has the desired hydrophobicity.

[0044] In some embodiments, at least one of X1 and X2 comprises a saturated hydrocarbon chain, which contains at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, or 30 carbon atoms, or any range of carbon atoms as defined by the aforementioned endpoints, e.g., 2-30, 2-28, 2-26, 2-24, 2-20, 2-18, 2-15, 2-12, 2-10, 2-8, 2-6, 2-4, 3-30, 3-28 , contains 3-26, 3-24, 3-20, 3-18, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 4-30, 4-28, 4-26, 4-24, 4-20, 4-18, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 10-30, 10-20, 15-30, 15-28, 15-26 or 15-20 carbon atoms.

[0045] In some embodiments, X1 and X2 are independently hydrogen, C 4~30 Alkyl, C 4~30 Alkenil, C 4~30 The compound is an alkynyl, aryl, aryloxy or a derivative thereof, or -(CH2)nX4, where n is 4 to 30, and X4 is hydrogen, aryl, aryloxy, heterocyclic group or a derivative thereof, provided that if X4 is a heterocyclic group, the heterocyclic group contains 1 to 3 heteroatoms or combinations thereof selected from the group consisting of O, S, and N.

[0046] In some embodiments, X1 and X2 are independently hydrogen, C 8~30 Alkyl, C 8~30 Alkenil, C 8~30 The compound is an alkynyl, aryl, aryloxy or a derivative thereof, or -(CH2)nX4, where n is 8 to 30, and X4 is hydrogen, aryl, aryloxy, heterocyclic group or a derivative thereof, provided that if X4 is a heterocyclic group, the heterocyclic group contains 1 to 3 heteroatoms or combinations thereof selected from the group consisting of O, S, and N.

[0047] In some embodiments, if one of X1 and X2 is hydrogen, the other one is not hydrogen. In some embodiments, if one of X1 and X2 is hydrogen, the other one contains at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, or 30 carbon atoms, or any range of carbon atoms as defined by the aforementioned endpoints, e.g., 2-30, 2-28, 2-26, 2-24, 2-20, 2-18, 2-15, 2-12, 2-10, 2-8, 2-6, 2-4, 3-30, 3-28, 3-26, 3-24, 3 The saturated hydrocarbon chain contains ~20, 3~18, 3~15, 3~14, 3~13, 3~12, 3~11, 3~10, 3~9, 3~8, 3~7, 3~6, ​​3~5, 4~30, 4~28, 4~26, 4~24, 4~20, 4~18, 4~15, 4~14, 4~13, 4~12, 4~11, 4~10, 4~9, 4~8, 4~7, 4~6, 6~15, 6~14, 6~13, 6~12, 6~11, 6~10, 6~9, 6~8, 10~30, 10~20, 15~30, 15~28, 15~26, or 15~20 carbon atoms. In some embodiments, one of X1 and X2 is C 15~30 One is alkyl, and the other is -(CH2)nX4 as defined above.

[0048] In some embodiments, X4 is an aryl, aryloxy, heterocyclic, cycloalkyl, heterocycloalkyl, or a combination thereof, and X4 is C 1~6 Alkyl, halogen, C 1~6 Alkyl halogens and C 1~6 It contains 0 to 6 substituents selected from the group consisting of alkoxys. In certain embodiments, X4 contains 1 to 3 substituents. The substituents can be, but are not limited to, CH3, CF3, F, or OCH3.

[0049] In some embodiments, X4 is -R3-O-R4, where R3 and R4 are independently an aryl, heterocyclic, cycloalkyl, and heterocycloalkyl group, respectively, and each is C 1~6 Alkyl, halogen, C 1~6Alkyl halogens and C 1~6 It contains 0 to 6 substituents selected from the group consisting of alkoxys.

[0050] In a particular embodiment, X4 is

[0051] [ka] It is selected from the group consisting of the following.

[0052] Exemplary Compounds of the Disclosure

[0053] This section lists some exemplary structures of the compounds of the Disclosure. However, the Disclosure is not limited to the exemplary structures listed below or within the Specification. In some embodiments, the compounds of the Disclosure do not contain glycolipid C34 or α-galactosylceramide (α-GalCer).

[0054] [ka] TIFF2026515588000011.tif204161TIFF2026515588000012.tif153167

[0055] Composition for forming lipid nanoparticles

[0056] Another aspect of this disclosure relates to compositions comprising the compounds of this disclosure. In some embodiments, the compounds constitute 1 to 10 mol% of the composition. In certain embodiments, the compounds constitute about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mol% of the composition, or any range of carbon as defined by the aforementioned endpoints, e.g., 1 to 10 mol%, 1 to 9 mol%, 1 to 8 mol%, 1 to 7 mol%, 1 to 6 mol%, 1 to 5 mol%, 1 to 4 mol%, 1 to 3 mol%, 1 to 2 mol%, 2 to 10 mol%, 2 to 9 mol%, 2 to 8 mol% of the composition. It constitutes 100%, 2-7 mol%, 2-6 mol%, 2-5 mol%, 2-4 mol%, 2-3 mol%, 3-10 mol%, 3-9 mol%, 3-8 mol%, 3-7 mol%, 3-6 mol%, 3-5 mol%, 3-4 mol%, 4-10 mol%, 4-9 mol%, 4-8 mol%, 4-7 mol%, 4-6 mol%, 4-5 mol%, 5-10 mol%, 5-9 mol%, 5-8 mol%, 5-7 mol%, or 5-6 mol%.

[0057] In some embodiments, the composition may comprise the first compound of the Disclosure and the second compound of the Disclosure. In some embodiments, the composition may comprise one, two, or more than three compounds, each independently conforming to the compounds of the Disclosure. One or two of the first compound, the second compound, or one, two, or more than three compounds may be designed to have the same targeting moiety of the R1 group, different targeting moieties of the R1 group targeting the same target, or different targeting moieties of the R1 group targeting different targets.

[0058] In some embodiments, the composition is configured to form lipid nanoparticles. The formed lipid nanoparticles are expected, in some embodiments, to carry out targeted delivery contributed by the compounds of the Disclosure. In some embodiments, the composition configured to form lipid nanoparticles may further comprise ionizable lipids, helper lipids, or mixtures thereof. In some embodiments, ionizable lipids constitute 30 to 60 mol% of the composition, helper lipids constitute 5 to 60 mol% of the composition, and the remaining percent is a carrier or solvent.

[0059] In some embodiments, the composition further includes a payload (i.e., cargo), which will be discussed further below. In certain embodiments, the payload is a first payload, and the composition further encapsulates a second payload. The first and second payloads may be the same or different.

[0060] Ionizable lipids.

[0061] In some embodiments, the ionizable lipids will be positively charged in a low pH environment. This feature facilitates the encapsulation and endosomal extrusion of introduced molecules, such as nucleic acids (e.g., RNA molecules), thereby enabling the efficient release of the cargo / payload into the cellular phenotype. In some embodiments, the ionizable lipids will be neutral in a physiological pH environment, thereby reducing their potential toxic effects on living organisms. In some embodiments, the ionizable lipids constitute 30-60 mol% of the composition. In certain embodiments, ionizable lipids constitute approximately 30, 35, 40, 45, 50, 55, or 60 mol% of the composition, or any range of carbon as defined by the aforementioned endpoints, for example, 30-60 mol%, 30-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-60 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-60 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 45-60 mol%, 45-55 mol%, 45-50 mol%, or 50-60 mol% of the composition. In some embodiments, the ionizable lipids include, but are not limited to, heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (SM-102®), (4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315®, Pfizer) or combinations thereof.

[0062] Helper lipids.

[0063] Helper lipids are lipids used to increase the particle stability and fluidity of lipid nanoparticles. Helper lipids can be, but are not limited to, phosphatidylcholine, cholesterol or its derivatives, polyethylene glycol-lipids (PEG-lipids), or mixtures thereof. Without being bound by theory, phosphatidylcholine can help increase the bilayer stability of lipid nanoparticles and reduce nonspecific binding. Cholesterol can enhance particle stability by filling gaps between lipid components forming lipid nanoparticles, thereby modulating membrane integrity and rigidity. PEG-lipids can enhance the colloidal stability and in vivo circulation time of lipid nanoparticles.

[0064] In some embodiments, helper lipids constitute 5 to 60 mol% of the composition. In certain embodiments, the helper lipids constitute approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 mol% of the composition, or any range of carbon as defined by the aforementioned endpoints, for example, 5-60 mol%, 5-55 mol%, 5-50 mol%, 5-45 mol%, 5-40 mol%, 5-35 mol%, 5-30 mol%, 5-25 mol%, 5-20 mol%, 5-15 mol%, 5-10 mol%, 10-60 mol%, 10-55 mol%, 10-50 mol%, 10-45 mol%, 10-40 mol%, 10-35 mol%, 10-30 mol%, 10-25 mol%, 10-20 mol%, 10-15 mol%, 15-60 mol%, 15-55 mol%, 15-50 mol% mol%, 15-45 mol%, 15-40 mol%, 15-35 mol%, 15-30 mol%, 15-25 mol%, 15-20 mol%, 20-60 mol%, 20-55 mol%, 20-50 mol%, 20-45 mol%, 20-40 mol%, 20-35 mol%, 20-30 mol%, 20-25 mol%, 25-60 mol%, 25-55 mol%, 25-50 mol% It constitutes 100%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25-30 mol%, 30-60 mol%, 30-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 40-60 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 50-55 mol%, or 50-60 mol%.

[0065] Phosphatidylcholine. In some embodiments, phosphatidylcholine constitutes 5 to 10 mol% of the composition. In certain embodiments, phosphatidylcholine constitutes about 5, 6, 7, 8, 9, or 10 mol% of the composition, or any range of carbon as defined by the aforementioned endpoints, for example, 5 to 10 mol%, 5 to 9 mol%, 5 to 8 mol%, 5 to 7 mol%, 5 to 6 mol%, 6 to 10 mol%, 6 to 9 mol%, 6 to 8 mol%, 6 to 7 mol%, 7 to 10 mol%, 7 to 9 mol%, 7 to 8 mol%, 8 to 10 mol%, 8 to 9 mol%, or 9 to 10 mol% of the composition. Examples of phosphatidylcholine, but not limited to these, include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DPOE), or mixtures thereof.

[0066] Cholesterol. In some embodiments, cholesterol or its derivatives constitute 30 to 40 mol% of the composition. In certain embodiments, cholesterol or its derivatives constitute about 30, 32, 34, 36, 38, or 40 mol% of the composition, or any range of carbon as defined by the aforementioned endpoints, for example, 30 to 40 mol%, 30 to 38 mol%, 30 to 36 mol%, 30 to 34 mol%, 30 to 32 mol%, 32 to 40 mol%, 32 to 38 mol%, 32 to 36 mol%, 32 to 34 mol%, 34 to 40 mol%, 34 to 38 mol%, 34 to 36 mol%, 36 to 40 mol%, 36 to 38 mol%, or 38 to 40 mol% of the composition. Examples of cholesterol or its derivatives include, but are not limited to, cholesterol, campesterol, beta-sitosterol, brassicasterol, ergosterol, dehydroergosterol, stigmasterol, fucosterol, DC-cholesterolHCl, OH-Chol, HAPC-Chol, MHAPC-Chol, DMHAPC-Chol, DMPAC-Chol, cholesteryl chloroformate, GL67, cholesteryl myristate, cholesteryl oleate, cholesteryl nervonate, LC10, cholesteryl hemysuccinate, (3β,5β)-3-hydroxycholane-24-euic acid, alkyne cholesterol, 27-alkyne cholesterol, E-cholesterol alkyne, trifluoroacetates (Dios-Arg, 2H-Cho-Arg, or Cho-Arg) or mixtures thereof.

[0067] PEG-lipids. In some embodiments, PEG-lipids constitute 1 to 10 mol% of the composition. In certain embodiments, PEG-lipids constitute about 1, 2, 4, 6, 8 or 10 mol% of the composition, or any range of carbon as defined by the aforementioned endpoints, for example, 1 to 10 mol%, 1 to 8 mol%, 1 to 6 mol%, 1 to 4 mol%, 1 to 2 mol%, 2 to 10 mol%, 2 to 8 mol%, 2 to 6 mol%, 2 to 4 mol%, 4 to 10 mol%, 4 to 8 mol%, 4 to 6 mol%, 6 to 10 mol%, 6 to 8 mol%, or 8 to 10 mol% of the composition. Examples of PEG-lipids, but not limited to these, include DMG-PEG, DSG-PEG, mPEG-DPPE, doped-PEG, mPEG-DMPE, mPEG-doped, DSPE-PEG-amine, DSPE-PEG, mPEG-DSPE, PEGPE, m-PEG-pentacosadic acid, bromoacetamide-PEG, amine-PEG, azide-PEG, or mixtures thereof. In some embodiments, the function of the PEG-lipid in the composition / lipid nanoparticles may be provided by the compounds of the present disclosure, particularly by embodiments of the present disclosure in which the R1 binding group contains a PEG moiety. In these situations, the compositions do not need to contain PEG-lipids.

[0068] For example, the following table lists some exemplary compositions as described herein. However, this disclosure is not limited to the exemplary compositions or the compounds described in the specification.

[0069] [Table 1] TIFF2026515588000014.tif192161

[0070] Lipid nanoparticles (LNPs)

[0071] Another aspect of the present disclosure relates to lipid nanoparticles (LNPs). The lipid nanoparticles of the present disclosure include a membrane that defines an internal space, and the membrane is formed of a plurality of lipid components, including the compounds of the present disclosure. The membrane of the lipid nanoparticles can be a bilayer structure, and this bilayer structure can be a single bilayer structure or a plurality of bilayer structures. In some embodiments, the plurality of lipid components may further include ionizable lipids, helper lipids, or a combination thereof. Ionizable lipids and helper lipids may be those described above. Without wishing to be bound by theory, the membrane is formed via hydrophobic interactions between the plurality of lipid components, while in some situations electrostatic interactions may also be involved in the formation of the membrane. In some embodiments, the plurality of lipid components do not include glycolipid C34 or α-galactosylceramide (α-GalCer). In some embodiments, the multiple lipid compounds include glycolipid C34 or α-galactosylceramide (α-GalCer), and glycolipid C34 or α-galactosylceramide (α-GalCer) constitutes a molar ratio of approximately 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, less than 5% of the multiple lipid compounds, or any range defined by the aforementioned endpoints. For example, these can be composed of 100%~5%, 90%~5%, 70%~5%, 70%~5%, 60%~5%, 50%~5%, 40%~5%, 30%~5%, 25%~5%, 20%~5%, 15%~5%, 10%~5%, 100%~10%, 90%~10%, 70%~10%, 70%~10%, 60%~10%, 50%~10%, 40%~10%, 30%~10%, 25%~10%, 20%~10%, or 15%~10%.

[0072] In some embodiments, the lipid components of the LNP membrane may include the first compound and the second compound of the Disclosure. In some embodiments, the lipid components may include one, two, or more than three compounds, each independently comprising a compound of the Disclosure. One or any two of the first compound, the second compound, or one, two, or more compounds may be designed to have the same targeting moiety of the R1 group, different targeting moieties of the R1 group targeting the same target, or different targeting moieties of the R1 group targeting different targets.

[0073] In some embodiments, the compounds of the Disclosure constitute at least 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the multiple lipid components forming the LNP membrane, or any range defined by the aforementioned endpoints, e.g., 0.1% to 70%, 0.1% to 60%, 0.1% to 50%, 0.1% to 40%, 0. 1%~35%, 0.1%~30%, 0.1%~25%, 0.1%~20%, 0.1%~15%, 0.1%~10%, 0.1%~5%, 0.1%~4%, 0.1%~3%, 0.1%~2%, 0.1%~1%, 0.1%~0.5%, 0.5%~70%, 0.5%~60%, 0.5%~50%, 0.5%~40%, 0.5%~35%, 0.5%~30%, 0.5%~25%, 0.5%~20%, 0.5%~15%, 0.5%~10%, 0.5% ~5%, 0.5%~4%, 0.5%~3%, 0.5%~2%, 0.5%~1%, 1%~70%, 1%~60%, 1%~50%, 1%~40%, 1%~35%, 1%~30%, 1%~25%, 1%~20%, 1%~15%, 1%~10%, 1%~5%, 1%~4%, 1%~3%, 1%~2%, 5%~70%, 5%~60%, 5%~50%, 5%~40%, 5%~35%, 5%~30%, 5%~25%, 5%~20%, 5%~15%, 5%~ It constitutes 10%, 10%~70%, 10%~60%, 10%~50%, 10%~40%, 10%~35%, 10%~30%, 10%~25%, 10%~20%, 10~15%, 20%~70%, 20%~60%, 20%~50%, 20%~40%, 20%~30%, 30%~70%, 30%~60%, 30%~50%, 30%~40%, 50%~70%, 50%~65%, 50%~60%, 60%~70%, or 60%~65%.

[0074] LNP size.

[0075] In some embodiments, the LNPs of this disclosure have diameters of 0.01, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 microns, or any range defined by the aforementioned endpoints, for example, 0.00-5, 0.01-4, 0.00-3, 0.01-2, 0.01-1, 0.01-0.8, 0.01-0.6, 0.01-0.4, 0.01-0.2, 0.01-0.1, 0.01 They have diameters of ~0.05, 0.01~0.01, 0.05~5, 0.05~4, 0.05~3, 0.05~2, 0.05~1, 0.05~0.8, 0.05~0.6, 0.05~0.4, 0.05~0.2, 0.05~0.1, 0.1~5, 0.1~4, 0.1~3, 0.1~2, 0.1~1, 0.1~0.8, 0.1~0.6, 0.1~0.4, 0.1~0.2, 0.5~5, 0.5~4, 0.5~3, 0.5~2, 0.5~1, 0.5~0.8, 1~5, 1~4, 1~3, or 1~2 microns. The size of LNPs is not limited to these, but can be determined by using dynamic light scattering (DLS). In some embodiments, the LNP of this disclosure has a polyvariance index (PDI) of approximately 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1, or any range defined by the aforementioned endpoints, e.g., 0.01 to 1, 0.01 to 0.9, 0.01 to 0.8, 0.01 to 0.7, It has a polyvariance index (PDI) of 0.01-0.6, 0.01-0.5, 0.01-0.4, 0.01-0.3, 0.01-0.2, 0.01-0.1, 0.01-0.05, 0.1-1, 0.1-0.9, 0.1-0.8, 0.1-0.7, 0.1-0.6, 0.1-0.5, 0.1-0.4, 0.1-0.3, or 0.1-0.2.

[0076] Zeta potential and molecular weight.

[0077] Without wishing to be constrained by theory, the zeta potential and molecular weight of exemplary targeted LNPs may influence cellular uptake of exemplary targeted LNPs. In some embodiments, the exemplary targeted LNPs of this disclosure include zeta potentials of approximately -50, -40, -30, -20, -15, -10, -5, 0, +5, +10, +15, +20, +30, +40, or +50, or any range defined by the aforementioned endpoints, e.g., -50 to +50, -50 to +40, -50 to +30, -50 to +20, -50 to +15, -50 to +10, -50 to +5, -50 to -5, -50 to -10, -50 to -15, -50 Includes zeta potentials of ~-20, -20~+50, -20~+40, -20~+30, -20~+20, -20~+15, -20~+10, -20~+5, -20~-5, -20~-10, -20~-15, -15~+50, -15~+40, -15~+30, -15~+20, -15~+15, -15~+10, -15~+5, -15~-5, -15~-10, +5~+50, +5~+40, +5~+30, +5~+20, +5~+15, or +5~+10. In some embodiments, the exemplary targeted LNPs of this disclosure include molecular weights of approximately 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, and 50 kDa, or any range defined by the aforementioned endpoints, e.g., 1-50 kDa, 1-40 kDa, 1-30 kDa, 1-20 kDa, 1-15 kDa, 1-10 kDa, 1-5 kDa, 2-50 kDa, 2-40 kDa, 2-30 kDa, 2-20 kDa, 2-15 kDa, 2-1 This includes molecular weights of 0 kDa, 2-5 kDa, 5-50 kDa, 5-40 kDa, 5-30 kDa, 5-20 kDa, 5-15 kDa, 5-10 kDa, 8-50 kDa, 8-45 kDa, 8-40 kDa, 8-35 kDa, 8-30 kDa, 8-25 kDa, 8-20 kDa, 8-15 kDa, 8-10 kDa, 12-50 kDa, 12-45 kDa, 12-35 kDa, 12-25 kDa, 12-15 kDa, 25-50 kDa, 25-40 kDa, or 25-30 kDa.

[0078] payload.

[0079] In some embodiments, the membrane of the LNP defines an internal space configured for encapsulating or carrying a payload (i.e., cargo). As used herein, “encapsulating a payload” or “carrying a payload” means that the payload is held within the LNP by its membrane. The payload can be contained within the internal space defined by the LNP or embedded within the membrane (e.g., embedded within a double-layer structure). The payload can move freely within the internal space or may be covalently or noncovalently bonded to the membrane. Encapsulation may be substantial, complete, or partial and does not preclude the possibility that a portion of the payload may be exposed to the environment outside the LNP. In embodiments of partial encapsulation, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the payload is held, wrapped, or surrounded by the membrane of the LNP. In some embodiments, the payload may be a biomolecule, such as a nucleic acid, compound, polypeptide, protein, glycan head, or a combination thereof.

[0080] In some embodiments, the payload is ribonucleic acid (RNA, e.g., mRNA) or deoxyribonucleic acid (DNA, e.g., double-stranded or single-stranded DNA), which, after being delivered to target cells using the LNPs of this disclosure, can encode polypeptides or proteins in vivo. The nucleic acids, e.g., the mRNA molecules used in this disclosure, can be prepared by in vitro transcription from a reference nucleic acid. In vitro transcription can be carried out as described in PCT patent publication WO2014 / 152027, filed March 13, 2014, which incorporates its entirety by reference.

[0081] In some embodiments, the polypeptide or protein is immunogenic (e.g., antigenic) to the organism to which the exemplary targeted LNP is administered. In such embodiments, the exemplary targeted LNP of the Disclosure is used to encapsulate and carry an immunogenic protein or nucleic acid configured to encode the immunogenic protein in vivo, for example, to encapsulate and carry an mRNA molecule in an RNA vaccine. The immunogenic protein can be a protein of a pathogen of viral (e.g., severe acute respiratory syndrome coronavirus (SARS-CoV, including SARS-CoV-2), influenza (flu), respiratory syncytial virus (RSV), EBV, dengue fever, VZV, HIV, ZIKA, or NIPAH) origin, bacterial, or fungal origin. In some embodiments, the immunogenic protein can be a viral spike protein. In certain embodiments, the spike protein can be of coronavirus (CoV) origin, for example, SARS-CoV, MERS-CoV, and SARS-CoV-2. In some embodiments, examples of coronaviruses (CoVs) described herein include, but are not limited to, alpha-SARS-CoV2, beta-SARS-CoV2, gamma-SARS-CoV2, delta-SARS-CoV2, omicron-SARS-CoV2, and their variants.

[0082] In some embodiments, the payload is a nucleic acid, which can be a polynucleotide having a reading frame configured to encode a polypeptide or protein in vivo. Such a polynucleotide may be modified with a 5' end cap, which is generated during an in vitro transcription reaction using the following chemical RNA cap analogs, namely 3”-O-Me-m7G(5)ppp(5')G[ARCA cap], G(5)ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, or m7G(5')ppp(5')G (New England Bio Labs, Ipswich, Mass.). 5'-capping of the modified polynucleotide can be completed post-transcriptionally using a cowpox virus capping enzyme to generate a "CAP 0" structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, Mass.). The CAP 1 structure can be generated by producing m7G(5')ppp(5')G-2'-O-methyl using both cowpox virus capping enzyme and 2'-O-methyltransferase. The CAP 2 structure can be generated from the CAP 1 structure, followed by 2'-O-methylation of the 5'-third-to-last nucleotide using 2'-O-methyltransferase. The CAP 3 structure can be generated from the CAP 2 structure, followed by 2'-O-methylation of the 5'-fourth-to-last nucleotide using 2'-O-methyltransferase. The enzymes may be derived from recombinant sources. After transfection in mammalian cells, the modified polynucleotides have stability for 12–18 hours or longer, e.g., 24, 36, 48, 60, 72, or longer than 72 hours.

[0083] In some embodiments, nucleic acids may be modified. In some embodiments, nucleic acids may have several (more than one) modifications, which may be identical or different from one another. In some embodiments, nucleic acids may contain one, two, or more (optionally different) nucleoside or nucleotide modifications in a particular region. In some embodiments, modified nucleic acids (e.g., modified mRNA polynucleotides) exhibit reduced degradation in cells or organisms compared to unmodified nucleic acids. In some embodiments, modified nucleic acids may exhibit reduced immunogenicity in organisms (e.g., reduced innate response).

[0084] In some embodiments, modifications may include chemical modifications. In some embodiments, modifications may be naturally occurring modifications, unnaturally occurring modifications, or both. Some exemplary modifications useful in this disclosure, but not limited to, include modifications of linkages between sugars, nucleic acid bases, and nucleosides (e.g., phosphate linkages, phosphodiester linkages, or linkages to phosphodiester backbones), or combinations thereof. In some embodiments, nucleic acids (e.g., RNA) used as payloads in this disclosure are codon-optimized. For example, nucleic acids may be modified to increase their G / C content. The G / C content of nucleic acids can affect their stability. Nucleic acids with a higher amount of guanine (G) and / or cytosine (C) residues may be more functionally stable than nucleic acids with a higher amount of adenine (A) and thymine (T) or uracil (U) nucleotides. For example, WO2002 / 098443 discloses a pharmaceutical composition containing mRNA stabilized by sequence modifications within a transformed region. Due to the degenerate nature of the gene code, modifications work by substituting existing codons with codons that promote higher RNA stability, without altering the amino acids produced.

[0085] In some embodiments, the nucleic acid may further include a sequence encoding a signal peptide. The signal peptide may include three regions: (1) an N-terminal region of different lengths, which typically contains positively charged amino acids; (2) a hydrophobic region; and (3) a short carboxy-terminal peptide region. In eukaryotes, the signal peptide of a nascent precursor protein (preprotein) directs the ribosome to the rough endoplasmic reticulum (ER) membrane and initiates the trans-transport of the growing peptide chain. While the signal peptide is not typically involved in the final destination of the mature protein, this is not limited to this disclosure. The signal peptide is usually cleaved from the precursor protein by ER-resident signal peptidases. They can also remain uncleaved and function as membrane anchors. In some embodiments, the signal peptide may also be condensed with a polypeptide or protein and designed so that the payload is encoded at its C-terminus or N-terminus.

[0086] In some embodiments, the payload may be a therapeutic or prophylactic reagent for treating or preventing a disease (e.g., cancer or infection). For example, the payload may include, but is not limited to, antiviral agents such as ribavirin, penciclovir, nitazoxanide, nafamostat, chloroquine, remdesivir (GS-5734), and favipiravir (T-705), interferon, adefovir, tenofovir, acyclovir, brivudine, cidofovir, fomivirsen, foscarnet, ganciclovir, amantadine, rimantadine, zanamivir, remdesivir, mornupiravir, and pachyrovid. In other embodiments, the payload may be an anticancer agent. In certain embodiments, the payload is a nucleic acid configured to encode a therapeutic or prophylactic reagent.

[0087] In some embodiments, the N / P ratio (the ratio of positively chargeable amine groups (nitrogen atoms of ionizable lipids, N=nitrogen) to negatively charged phosphate (P) groups of nucleic acids) of exemplary targeted LNPs encapsulating nucleic acids is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 4 The range is 5 or 50, or any range defined by the aforementioned endpoints, including or excluding the endpoints, for example, 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 5-50, 5-40, 5-30, 5-20, 5-10, 10-50, 10-40, 10-30, 10-20, 8-40, 8-20, 8-12, 9-50, 9-30, or 9-15. In another embodiment, the exemplary targeted LNP encapsulating mRNA has a nanoparticle / mRNA (N / P) ratio of about 10 or about 20.

[0088] In some embodiments, where the LNP payload is a nucleic acid configured to encode a polypeptide or protein within the target cell after uptake by the target cell, the LNP encodes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 copies of the polypeptide or protein in vivo, or any range defined by the aforementioned endpoints, including or excluding the endpoints, for example. For example, the LNPs are configured to encode a range of copies 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-8, 2-6, 2-5, 2-4, 5-50, 5-40, 5-30, 5-20, 5-15, 5-10, 5-8, 4-50, 4-45, 4-35, 4-25, 4-15, 4-9, 4-6, 7-50, 7-45, 7-35, 7-25, 7-15, or 7-9. In some embodiments, after uptake by target cells, the LNPs are configured to continuously and immediately encode a polypeptide or protein in vivo until the nucleic acid (i.e., payload) is inactivated in vivo.

[0089] A kit for preparing lipid nanoparticles.

[0090] One aspect of this disclosure relates to a kit / reagent mixture for preparing the targeted LNP formulation of this disclosure. The kit comprises a first reagent and a second reagent, the first reagent comprising the compound of this disclosure, and the second reagent comprising an ionizable lipid, a helper lipid, or a mixture thereof. The ionizable lipid and helper lipid may be those described herein. In some embodiments, the second reagent comprises an ionizable lipid, and in some embodiments, the kit further comprises a third reagent comprising a helper lipid. In some embodiments, the kit further comprises a fourth reagent comprising a payload, the payload may be those described herein.

[0091] Packaging. All components of the kits of this disclosure can be individually packaged in physical containers. In some embodiments, the first reagent and the second reagent are contained in the same container, in other words, in a ready-to-use package. In some other embodiments, the first reagent and the second reagent are contained in separate containers, so that the user can decide whether and when to mix the first reagent and the second reagent.

[0092] Composition / Formulation

[0093] One aspect of this disclosure relates to a composition comprising the LNP of this disclosure. The LNP of the composition can encapsulate a payload and is configured to deliver the payload to a target region of a biological organism. The payload may include nucleic acids, compounds, peptides, proteins, glycan heads, or combinations thereof, as described herein. In some embodiments, the payload may be an immunogenic protein or nucleic acid configured to encode an immunogenic protein in vivo. In some embodiments, the formulation further comprises pharmaceutically acceptable excipients, adjuvants, or combinations thereof. In certain embodiments, the composition is a pharmaceutical composition or pharmaceutical formulation.

[0094] In some embodiments, the composition includes 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95% (w / w) of the LNPs of this disclosure, with or without the payload, or any range defined by the aforementioned endpoints, including or excluding the endpoints, for example, 0.01% to 95% (w / w), 0.01% to 90% (w / w), 0 .01%~80%(w / w), 0.01%~70%(w / w), 0.01%~60%(w / w), 0.01%~50%(w / w), 0.01%~40%(w / w), 0.01%~30%(w / w), 0.01%~20%( w / w), 0.01%~10%(w / w), 0.01%~5%(w / w), 0.01%~1%(w / w), 0.01%~0.1%(w / w), 0.1%~95%(w / w), 0.1%~90%(w / w), 0.1%~80% (w / w), 0.1%~70%(w / w), 0.1%~60%(w / w), 0.1%~50%(w / w), 0.1%~40%(w / w), 0.1%~30%(w / w), 0.1%~20%(w / w), 0.1%~10%( w / w), 0.1%~5%(w / w), 0.1%~1%(w / w), 1%~95%(w / w), 1%~90%(w / w), 1%~80%(w / w), 1%~70%(w / w), 1%~60%(w / w), 1%~50%(w / The composition includes LNPs of this disclosure in the range of 1%-40% (w / w), 1%-30% (w / w), 1%-20% (w / w), 1%-10% (w / w), 1%-5% (w / w), 5%-95% (w / w), 5%-90% (w / w), 5%-80% (w / w), 5%-70% (w / w), 5%-60% (w / w), 5%-50% (w / w), 5%-40% (w / w), 5%-30% (w / w), 5%-20% (w / w), or 5%-10% (w / w). The remainder of the composition may be excipients as described herein.

[0095] In some embodiments, the composition is an mRNA vaccine, and the LNP encapsulates mRNA configured to encode an immunogenic protein in vivo. The immunogenic protein can be the viral spike protein of a pathogen or other antigenic molecule. In certain embodiments, the composition of the present invention can be a COVID-19 mRNA vaccine.

[0096] The exemplary COVID-19 mRNA vaccines described herein can be designed based on mRNA technology to remove the glycan shield of the coronavirus (e.g., SARS-CoV-2) spike protein to better expose the conserved region of the spike protein. By having a deletion of the glycosylation site in the receptor-binding domain (RBD) or subunit 2 (S2) domain, coronavirus spike protein mRNA vaccines expose highly conserved epitopes, induce antibody and CD8 T-cell responses, and provide broader protection against alpha, beta, gamma, delta, omicron, and various variants compared to unmodified mRNA. The vaccine may be a low-sugar universal vaccine (LSUV) as described in WO2022 / 221835 (mRNA containing a sequence selected from SEQ ID NOs. 1-52), WO2022 / 221837A2 (mRNA containing a sequence selected from SEQ ID NOs. 1-21), and US20200046826A1 (mRNA containing a sequence selected from SEQ ID NOs. 1-20), which are incorporated herein by reference in their entirety.

[0097] In some embodiments, the compositions / formulations of the present invention are configured to treat or prevent a disease (e.g., cancer). In such embodiments, the payload carried by the LNP may be a therapeutic reagent, a prophylactic reagent, or a nucleic acid configured to encode a therapeutic or prophylactic reagent in vivo. For example, the composition may be a personalized cancer vaccine (e.g., melanoma), a KRAS vaccine (KRAS-inducible), or a checkpoint vaccine (e.g., PD-1, PDL-1 related).

[0098] In some embodiments, the compositions / formulations of the present invention can be administered together with other compositions (e.g., vaccines or drugs). Examples of other compositions, but not limited to, include influenza (flu) vaccines, adenovirus vaccines, anthrax vaccines, cholera vaccines, diphtheria vaccines, hepatitis A or B vaccines, HPV vaccines, measles vaccines, mumps vaccines, smallpox vaccines, rotavirus vaccines, tuberculosis vaccines, pneumococcal vaccines, and Haemophilus influenzae type b vaccines.

[0099] Combination composition

[0100] In some embodiments, the composition may be a combination composition (e.g., a combo vaccine) comprising a first LNP encapsulating a first payload and a second LNP encapsulating a second payload. The first LNP and the second LNP may be LNPs described herein, but they may differ from each other in terms of the structure or properties of their copolymers. For example, the first LNP and the second LNP may differ in size, the copolymer forming their membrane, the payload encapsulated within the LNP, or a combination thereof.

[0101] For example, the first LNP contains a glycan head configured to bind to DC-SIGN, while the second LNP contains a glycan head configured to bind to CD1d. In another example, the first LNP contains a glycan configured to target antigen-presenting cells, while the second LNP contains a glycan configured to target cancer cells.

[0102] In some embodiments, the first and second payloads are different from each other. For example, the first payload may be a protein or peptide, while the second payload may be a nucleic acid. In certain embodiments, both the first and second payloads may be mRNA molecules but may encode different proteins. For example, the first payload may be an mRNA configured to encode the delta-SARS-CoV-2 spike protein, while the second payload may be an mRNA configured to encode the omicron-SARS-CoV-2 spike protein.

[0103] Additional components of the composition

[0104] In some embodiments, the disclosed compositions may further include adjuvants and / or inactive substances, such as pharmaceutically acceptable excipients. In certain embodiments, the adjuvant may be, but is not limited to, C34, Gluco-C34, 7DW8-5, C17, C23, C30, α-galactosylceramide (α-GalCer), aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), mixed aluminum salts), squalene, MF59, QS-21, Freund's complete adjuvant, Freund's incomplete adjuvant, AS03 (GlaxoSmithKline), MF59 (Seqirus), CpG1018 (Dynavax), or a combination thereof.

[0105] In certain embodiments, pharmaceutically acceptable excipients may include solvents, dispersions, diluents, dispersants, suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, polymers, peptides, proteins, cells, hyaluronidases, or mixtures thereof. Various excipients for formulating pharmaceutical compositions and techniques for preparing compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 22nd Edition, Edited by Allen, Loyd V., Jr., Pharmaceutical Press). The use of conventional excipient media may be envisioned within the scope of this disclosure, except where any conventional excipient media is incompatible with a substance or its derivative by interacting adversely with any other component(s) of the pharmaceutical composition, for example, by producing any undesirable biological effect or otherwise. The formulation of standard pharmaceutically acceptable excipients can be carried out using prescribed methods in the field of pharmacy (see Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Company, Eastern Pennsylvania, USA).

[0106] In some embodiments, the composition further comprises a phosphate conjugate. Without being bound by theory, the phosphate conjugate can increase the in vivo circulation time and / or increase the targeted delivery of the LNPs of this disclosure. Phosphate conjugates for use in this disclosure can be prepared using the methods described in PCT publication WO2013 / 033438 filed August 30, 2012, or U.S. publication US2013 / 0196948 filed June 23, 2011, the contents of which are incorporated herein by reference in their entirety. As a non-limiting example, the phosphate conjugate may contain a compound of any one of the formulas described in PCT publication WO2013 / 033438 filed August 30, 2012, the contents of which are incorporated herein by reference in their entirety.

[0107] In some embodiments, the composition further includes a conjugate for enhancing the delivery of the LNPs of this disclosure. Without wishing to be constrained by theory, the conjugate selected for use can inhibit the phagocytic clearance of the LNPs in the subject. In some examples, the conjugate may be the human membrane protein CD47 or a “self” peptide derived therefrom (e.g., the “self” particle described by Rodriguez et al. (Science 2013, 339, 971-975) which is incorporated herein by reference in its entirety).

[0108] In some embodiments, where the payload is an immunogenic agent or a nucleic acid configured to encode an immunogenic agent, the composition further comprises an immunostimulant that enhances the immune response induced by the immunogenic agent. In non-limiting examples, the composition may comprise a Th1 immunostimulant, which can enhance the Th1-based response of the immune system (see PCT publication WO2010 / 123569 and U.S. publication 2011 / 0223201, each incorporated herein by reference in whole).

[0109] In some embodiments, the composition does not contain viral components (e.g., viral capsids, viral enzymes, or other viral proteins, e.g., those required for virus-based replication) or have them packaged, encapsulated, linked, or otherwise attached within the virus or viral particles.

[0110] How to use

[0111] One aspect of the present disclosure relates to a method using the LNP or targeted formulation of the present disclosure. In particular, the method is carried out to obtain a desired effect in a subject, such as targeting and delivering a payload, preventing or treating a disease, or boosting an adaptive immune response. In some embodiments, the subject may be an animal or a human, but is not limited thereto, and the payload is designed to demonstrate its efficacy in these subjects, and there is a need for the treatment or prevention of a disease in these subjects, or for the adaptive immune response to be boosted in these subjects.

[0112] Methods for delivering targeted payloads to a target

[0113] In some embodiments, a method is provided for targeted delivery of a payload to a target, comprising administering an effective amount of LNP to the target or administering a targeted formulation of the Disclosure. The LNP and payload are as described herein, and the payload is encapsulated in the LNP. Without wishing to be bound by any theory, targeted delivery is achieved by the compounds of the Disclosure. In particular, the R1 group of the compounds of the Disclosure provides desired binding affinity / specificity to target a desired region of a target via its glycosyl group.

[0114] Methods for preventing or treating diseases in the target population.

[0115] In some embodiments, a method is provided for preventing or treating a disease in a subject, comprising administering an effective amount of LNP to the subject or administering a formulation of the present disclosure. The LNP in the present method encapsulates a payload, which is a therapeutic agent or induces a therapeutic agent configured to prevent and / or treat a disease.

[0116] Without wishing to be bound by any theory, the LNPs or formulations of the Disclosure provide targeted delivery via the compounds of the Disclosure. Therefore, by using the LNPs of the Disclosure to deliver a payload, the efficacy of the payload can be more effectively implemented. For example, in embodiments comprising a structure in which the R1 group of a compound specifically binds to DC-SIGNs on dendritic cells, an antigenic or immunogenic payload can be effectively delivered to dendritic cells to induce an immune response for the prevention of a disease of concern. This strategy is beneficial for delivering nucleic acids encoding antigens of antigens or vaccines. Several other examples include having an R1 group designed to target cancer cells so that antitumor reagents can be effectively delivered to the tumor microenvironment. This strategy can increase the efficacy of antitumor reagents and reduce therapeutic side effects.

[0117] In some embodiments, the disease is characterized by dysfunction or abnormal protein or polypeptide activity. For example, the disease is selected from the group consisting of rare diseases, infections, cancer and proliferative disorders, genetic disorders (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal diseases, and metabolic diseases.

[0118] In some embodiments, the disease may be cancer or an infectious disease. In certain embodiments, the disease may be a viral infection, but may include, without limitation, infections associated with human parainfluenza virus 3, respiratory syncytial virus (RSV), cytomegalovirus (CMV), human metapneumovirus (hMPV), or SARS-CoV-2 (COVID-19).

[0119] How to boost the adaptive immune response

[0120] In some embodiments, a method is provided for boosting an adaptive immune response, comprising administering an effective amount of the LNP of the present disclosure to a subject. The LNP of the present method encapsulates a payload within an internal space defined by the membrane of the LNP, the payload being a therapeutic agent configured to induce or induce an adaptive immune response in a subject.

[0121] Without wishing to be bound by any theory, the LNPs of this disclosure provide targeted delivery to immune cells via the compounds of this disclosure. In some embodiments, the R1 group of the compound includes a structure that binds to antigen-presenting cells with desired specificity or affinity. For example, the R1 group may include a structure that specifically binds to DC-SIGN on dendritic cells so that the LNP can specifically deliver an immunogenic payload to dendritic cells to facilitate the initiation of an adaptive immune response.

[0122] In some embodiments, the boosted adaptive immune response is against diseases, including but not limited to cancer or infectious diseases. For example, infectious diseases can be viral infections, including, but not limited to, infections associated with human parainfluenza virus 3, respiratory syncytial virus (RSV), cytomegalovirus (CMV), human metapneumovirus (hMPV), or SARS-CoV-2 (COVID-19).

[0123] Administration

[0124] With respect to the methods of the present disclosure, in some embodiments, the subject is administered a single-dose LNP or targeted formulation of the present disclosure, with or without a payload encapsulated. In some embodiments, the subject is administered an initial dose of LNP, followed by at least one booster dose, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more additional doses, with intervals of approximately 1, 2, 3, 4, 5, 6, 7 days, approximately 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks or approximately 1, The administrations are performed at intervals of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or within any range defined by the aforementioned endpoints, including or excluding the endpoints, for example, between 1 and 7 days, 1 and 5 days, 1 and 3 days, 1 and 10 weeks, 1 and 8 weeks, 1 and 6 weeks, 1 and 4 weeks, 1 and 2 weeks, 1 and 12 months, 1 and 8 months, 1 and 6 months, 1 and 4 months, 1 and 2 months, or between 6 and 12 months. In a particular embodiment, the LNP of the Disclosure containing the payload is administered twice in the same or different doses, with intervals of 1 day, 3 days, 5 days, 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, between 1 and 5 days, 1 and 2 weeks, 1 and 3 months, 1 and 6 months, 1 and 1 year, 3 and 1 year, or 6 and 1 year.

[0125] Route of administration. The LNPs or formulations described herein may be administered by any route. Suitable routes include, but are not limited to, oral, nasal, mucosal, submucosal, intravenous, intramuscular, intraperitoneal, subcutaneous, intradermal, percutaneous, and oral buccal routes. Some practical topical applications include, but are not limited to, the application of drops, sprays, aerosols, gels, or ointments to the mucosal epithelium of the eyes, nose, mouth, anus, or vagina. Other possible routes of administration are the application of sprays, aerosols, or powders by inhalation through the respiratory tract.

[0126] Effective dose. As used herein, an effective dose means an amount sufficient to produce the desired effect. In embodiments where the purpose of administering the LNP of this disclosure is to treat a disease, the effective dose means a therapeutic effective dose, while in some other embodiments where the purpose is to prevent a disease, the effective dose means a prophylactic effective dose.

[0127] In some other embodiments, where the purpose of administering LNPs with a payload is to boost an adaptive immune response, the effective dose can be determined as an amount sufficient to induce an antigen-specific immune response in the subject to which the LNPs and payload are administered. The antigen-specific immune response can be characterized by measuring the antibody titer of the anti-antigenic polypeptide (i.e., the payload or a product of the payload) produced in the subject to which the LNPs and payload are administered. In some embodiments, the measurement can be performed using enzyme-linked immunosorbent assay (ELISA).

[0128] In some embodiments, antibody titers are used to assess whether a subject has had an infection or to determine whether immunization is required. In some embodiments, antibody titers are used to determine the intensity of an autoimmune response, to determine whether booster immunization is required or whether immunization has been boosted, to determine whether a past vaccine was effective, and / or to identify any recent or previous infection.

[0129] The effective amount of the method of this disclosure can be determined based on several factors, including, but not limited to, the condition of the subject (age, sex, species, weight, health status, etc.), the progression of the disease to be treated, the route of administration, the dose and interval of administration, and the nature of the payload. With respect to the nature of the payload, for example, in embodiments in which the LNP of this disclosure is used to carry mRNA, such as in the case of an mRNA vaccine, the effective amount can be determined based on the effective amount of mRNA required to evoke a sufficient immune response in the subject. Therefore, in some embodiments where the payload is mRNA, the effective amount of the method of this disclosure is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 micrograms (μg or ug) or any range defined by the aforementioned endpoint, including or excluding the endpoint, for example, 5 micrograms to 1000 micrograms, 5 micrograms to 900 micrograms, 5 micrograms to 800 micrograms, 5 micrograms to 700 micrograms, 5 micrograms to 600 micrograms, 5 micrograms to 500 micrograms, 5 micrograms to 400 micrograms Chlorograms, 5 micrograms to 300 micrograms, 5 micrograms to 200 micrograms, 5 micrograms to 175 micrograms, 5 micrograms to 150 micrograms, 5 micrograms to 125 micrograms, 5 micrograms to 100 micrograms, 5 micrograms to 90 micrograms, 5 micrograms to 80 micrograms, 5 micrograms to 70 micrograms, 5 micrograms to 60 micrograms, 5 micrograms to 50 micrograms, 5 micrograms to 40 micrograms, 5 micrograms to 30 micrograms, 5 micrograms to 20 micrograms, 5 micrograms to 10 micrograms, 10 micrograms to 1000 micrograms, 10 micrograms to 900 micrograms, 10 micrograms to 800 micrograms, 10 micrograms to 700 micrograms,10 micrograms to 600 micrograms, 10 micrograms to 500 micrograms, 10 micrograms to 400 micrograms, 10 micrograms to 300 micrograms, 10 micrograms to 200 micrograms, 10 micrograms to 175 micrograms, 10 micrograms to 150 micrograms, 10 micrograms to 125 micrograms, 10 micrograms to 100 micrograms, 10 micrograms to 90 micrograms, 10 micrograms to 80 micrograms, 10 micrograms to 70 micrograms 10 micrograms to 60 micrograms, 10 micrograms to 50 micrograms, 10 micrograms to 40 micrograms, 10 micrograms to 30 micrograms, 10 micrograms to 20 micrograms, 50 micrograms to 1000 micrograms, 50 micrograms to 900 micrograms, 50 micrograms to 800 micrograms, 50 micrograms to 700 micrograms, 50 micrograms to 600 micrograms, 50 micrograms to 500 micrograms, 50 micrograms to 400 micrograms 50 micrograms to 300 micrograms, 50 micrograms to 200 micrograms, 50 micrograms to 175 micrograms, 50 micrograms to 150 micrograms, 50 micrograms to 125 micrograms, 50 micrograms to 100 micrograms, 50 micrograms to 90 micrograms, 50 micrograms to 80 micrograms, 50 micrograms to 70 micrograms, or 50 micrograms to 60 micrograms, 100 micrograms to 1000 micrograms, 100 micrograms to 900 micrograms Micrograms, 100 micrograms to 800 micrograms, 100 micrograms to 700 micrograms, 100 micrograms to 600 micrograms, 100 micrograms to 500 micrograms, 100 micrograms to 400 micrograms, 100 micrograms to 300 micrograms, 100 micrograms to 200 micrograms, 100 micrograms to 175 micrograms, 100 micrograms to 150 micrograms, 300 micrograms to 1000 micrograms, 300 micrograms to 900 micrograms,The ranges are 300 micrograms to 800 micrograms, 300 micrograms to 700 micrograms, 300 micrograms to 600 micrograms, 300 micrograms to 500 micrograms, 300 micrograms to 400 micrograms, 500 micrograms to 1000 micrograms, 500 micrograms to 900 micrograms, 500 micrograms to 800 micrograms, 500 micrograms to 700 micrograms, 500 micrograms to 600 micrograms, 600 micrograms to 800 micrograms, or 700 micrograms to 900 micrograms.

[0130] However, if targeted delivery is provided by the LNP of this disclosure, it can be expected that the effective amount required for the method of this disclosure may be lower than the effective amount required for other untargeted delivery methods. For example, the effective amount required for the method of this disclosure may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 95, or 99% lower than the effective amount required for other untargeted delivery methods, or any range defined by the aforementioned endpoints, including or excluding the endpoints, for example, 1-99%, 1-95%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, 1-10%, 1-5%, 5-99%, 5-95%, 5-90%, 5-80%, 5- 70%, 5-60%, 5-50%, 5-40%, 5-30%, 5-20%, 5-10%, 10-90%, 10-80%, 10-70%, 10-60%, 10-50%, 10-40%, 10-30%, 10-20%, 30-99%, 30-95%, 30-90%, 30-80%, 30-70%, 30-60 The percentage may be low in the range of %, 30-50%, 30-40%, 50-99%, 50-95%, 50-90%, 50-80%, 50-70%, 50-60%, 70-99%, 70-95%, 70-90%, 70-80%, 80-99%, 80-95%, 80-90%, 90-99%, or 95-99%.

[0131] Furthermore, in some embodiments in which the LNP of the Disclosure is used to deliver an antigenic agent or nucleic acid encoding an antigenic agent to induce an antibody against the antigenic agent, the titer of the antibody induced by the Disclosure is increased by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 log compared to the titer of the antibody induced by the untargeted delivery method, or by any range defined by the aforementioned endpoints, including or excluding the endpoints, for example, 1 to 10 log, 1 to 8 log, 1 to 6 log, 1 to 4 log, 2 to 9 log, 2 to 7 log, 2 to 5 log, 3 to 10 log, 3 to 8 log, 3 to 5 log, or 4 to 6 log.

[0132] In some other embodiments in which the LNPs of this disclosure are used to deliver an antigenic agent or nucleic acid encoding an antigenic agent to induce an immune response to the antigenic agent, the antibody titer against the antigenic agent induced by this disclosure is 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher than the antibody titer of the untargeted delivery method, or within any range defined by the aforementioned endpoints, for example, 0.1 to 10, 0.1 to 9, 0.1 to 8, with or without the endpoints. The values ​​are high in the ranges of 0.1~7, 0.1~6, 0.1~5, 0.1~4, 0.1~3, 0.1~2, 0.1~1, 0.1~0.5, 0.5~10, 0.5~9, 0.5~8, 0.5~7, 0.5~6, 0.5~5, 0.5~4, 0.5~3, 0.5~2, 0.5~1, 1~10, 1~9, 1~8, 1~7, 1~6, 1~5, 1~4, 1~3, 1~2, 3~10, 3~9, 3~8, 3~7, 3~6, ​​3~5, 3~4, 5~10, 5~9, 5~8, 5~7, 5~6, 7~10, 7~9, 7~8, or 8~10 times.

[0133] In some embodiments, the LNPs of this disclosure are used to deliver an antigenic agent or nucleic acid encoding an antigenic agent to induce an immune response to the antigenic agent. In some embodiments, the immune response is induced only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 days earlier than an immune response induced by a non-targeted delivery method, or within any range defined by the aforementioned endpoints, for example, 1-20, 1-18, 1-14, 1-10, 1-6, 2-20, 2-18, 2-14, 2-10, 2-6, 5-20, 5-18, 5-14, or 5-10 days earlier.

[0134] In some embodiments, the LNP described herein is administered in a manner sufficient to achieve the desired effect in vivo, with a payload of approximately 0.0001 mg / kg to approximately 100 mg / kg, approximately 0.001 mg / kg to approximately 0.05 mg / kg, approximately 0.005 mg / kg to approximately 0.05 mg / kg, approximately 0.001 mg / kg to approximately 0.005 mg / kg, approximately 0.05 mg / kg to approximately 0.5 mg / kg, approximately 0.01 mg / kg to approximately 50 mg / kg, approximately 0.1 mg / kg to approximately 40 mg / kg, approximately 0.5 mg / kg to approximately 30 mg / kg, approximately 0.01 mg / kg to approximately 10 mg / kg, approximately 0.1 mg / kg to approximately 10 mg / kg, or approximately 1 mg / kg to approximately 25 mg / kg (body weight of one subject, per day, once or more per day).

[0135] definition

[0136] Unless otherwise specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in which this disclosure pertains. Unless otherwise stated, the technologies used or assumed herein are standard methodologies well known to those skilled in the art. Unless otherwise pointed out, the implementation of this disclosure utilizes prior art in microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, which are within the scope of the skills of those skilled in the art. Materials, methods and examples are for illustrative purposes only and are not intended to limit the scope of this disclosure. The following are presented as examples and are not intended to limit the scope of this disclosure.

[0137] It should be understood that in some cases the numbers used to describe and claim certain embodiments of this disclosure, such as the amount of raw material components, properties such as molecular weight, reaction conditions, and results, are modified with the term "approximately." Those skilled in the art will understand the meaning of the value modified by the term "approximately" in the context of the value. The numerical values ​​presented in some embodiments of this disclosure may include certain errors derived from the standard deviation in each of these test measurements. For example, the term "approximately," as used herein, refers to a measurable value such as a quantity or temporary period and is intended to include variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from a specified value, thus the variation is appropriate.

[0138] As used herein, “substantially” means sufficiently effective for the intended purpose. The term “substantially” thus allows for minor, slight variations from an absolute or perfect state, dimension, measurement, result, etc., such as variations that would be expected by a person skilled in the art but do not have a noticeable effect on the overall performance. When used in reference to numerical values, parameters, or characteristics, “substantially” means within 10 percent.

[0139] As used herein, “treat,” “treatment,” and “treating” refer to methods for obtaining beneficial or desired outcomes, such as clinical outcomes. For the purposes of this disclosure, beneficial or desired outcomes may include inhibiting or suppressing the onset or progression of an infection or disease, restoring or reducing the onset or symptoms of an infection or disease, or a combination thereof.

[0140] As used herein, “preventing” and “prevention” are used interchangeably with “preventive measures” and may mean complete prevention of an infectious disease or prevention of the onset of symptoms of such infection, delay of the onset of the disease or its symptoms, or reduction of the severity of a subsequent infectious disease or its symptoms.

[0141] As used herein, “glycan” or “glycosyl group” refers to a polysaccharide, oligosaccharide, or monosaccharide. Glycans may be monomers or polymers of sugar residues and may be linear or branched. Glycans may contain residues of natural sugars (e.g., glucose, N-acetylglucosamine, N-acetylneuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and / or modified sugars (e.g., 2'-fluororibose, 2'-deoxyribose, phosphomannose, 6'sulfo-N-acetylglucosamine, etc.).

[0142] As used herein, “alkyl” means a hydrocarbon chain that contains the indicated number of carbon atoms, which may be linear or branched, saturated or unsaturated. For example, C 1~6This indicates that the group may have 1 to 6 carbon atoms (including the values ​​at both ends). Non-limiting examples include methyl, ethyl, isopropyl, tert-butyl, and n-hexyl. A "heteroalkyl" group is an alkyl group in which at least one carbon in the chain is replaced by a heteroatom. In some embodiments, heteroalkyl groups have 1 to 20 carbon atoms. The term "alkoxy" is intended to mean partial-OR (where R is alkyl). The term "aryloxy" is intended to mean partial-OR (where R is aryl).

[0143] As used herein, “alkenyl” means a hydrocarbon chain containing at least one double bond, which may be a straight or branched chain and contains the indicated number of carbon atoms. For example, C 2~6 This indicates that the group may contain 2 to 6 carbon atoms (including the values ​​at both ends). Non-restrictive examples include ethenyl and propa-1-en-2-yl.

[0144] As used herein, “alkynyl” means a hydrocarbon chain containing at least one triple bond, which may be a straight or branched chain and contains the indicated number of carbon atoms. For example, C 2~6 This indicates that the group may contain 2 to 6 carbon atoms (including the values ​​at both ends). Non-limiting examples include ethynyl and 3,3-dimethylbuta-1-in-1-yl.

[0145] As used herein, “cycloalkyl” refers to a non-aromatic cyclic, bicyclic, condensed, or spirohydrocarbon group having 3 to 10 carbon atoms, for example, 3 to 8 carbon atoms, or for example, 3 to 7 carbon atoms, and the cycloalkyl group may be optionally substituted. Examples of cycloalkyl groups include 5-membered, 6-membered, and 7-membered rings. A cycloalkyl group may contain one or more unsaturated elements, and a cycloalkyl group containing unsaturated elements is also referred to herein as “cycloalkenyl.” Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0146] As used herein, [heterocycloalkyl] refers to a fused or spiro-based group of a non-aromatic 5- to 8-membered monocyclic, 8- to 12-membered bicyclic, or 11- to 14-membered tricyclic ring, having 1 to 3 heteroatoms in the case of a monocyclic, 1 to 6 heteroatoms in the case of a bicyclic, or 1 to 9 heteroatoms in the case of a tricyclic, wherein the heteroatoms are selected from O, N, or S (e.g., carbon atoms, and in the case of a monocyclic, bicyclic, or tricyclic, 1 to 3, 1 to 6, or 1 to 9 heteroatoms from N, O, or S, respectively), and 0, 1, 2, or 3 atoms of each ring may be substituted with substituents. Heterocycloalkyls may also include oxidized ring members, e.g., -N(O)-, -S(O)-, and -S(O)2-. Examples of heterocycloalkyls include 5-membered, 6-membered, and 7-membered heterocyclic rings. Examples include piperazinil, pyrrolidinil, dioxanil, morpholinil, and tetrahydrofuranil.

[0147] As used herein, “aryl” or “aryl group” refers to a moiety formed by the removal of one or more hydrogen atoms ("H") or deuterium atoms ("D") from an aromatic compound. An aryl group may be monocyclic (monocyclic) or may have polycyclic (two or more rings) that are fused together or covalently linked. A “carbocyclic aryl” has only carbon atoms in its aromatic ring(s). A “heteroaryl” is intended to mean an aromatic ring system containing 5 to 14 aromatic ring atoms, which may be monocyclic, two-fused rings, or three-fused rings, where at least one aromatic ring atom is a heteroatom selected from the group consisting of O, S, and N, but not limited to these. Heteroaryls may also contain oxidized ring members, e.g., -N(O)-, -S(O)-, and -S(O)2-. Examples include furanil, thienyl, pyrrolyl, imidazolyl, oxazolyl, thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridadinyl, and triazinyl. Other examples include carbazolyl, quinolidinyl, quinolinyl, isoquinolinyl, sinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, triazinyl, indolyl, isoindolyl, indazolyl, indolidinyl, prinyl, naphthylidinyl, pteridinyl, carbazolyl, acridinyl, phenadinyl, phenothiazinyl, phenoxadinyl, benzoxazolyl, benzothiazolyl, 1H-benzimidazolyl, imidazopyridinyl, benzothienyl, benzofuranil, and isobenzofuran.

[0148] As used herein, “amine” refers to a compound containing a basic nitrogen atom having an isolated pair. The term “amino” refers to a functional group or an -NH2, -NHR, or -NR2 moiety (wherein R is the same or different in each present and may be an alkyl or aryl group).

[0149] As used herein, “halogen” or “halo” means fluorine, bromine, chlorine, or iodine. In particular, this usually means fluorine or chlorine when bonded to an alkyl group, and further includes bromine or iodine when bonded to an aryl or heteroaryl group.

[0150] As used herein, the term “haloalkyl” means an alkyl group as defined herein that is substituted with one or more halo groups. Haloalkyls can be monohaloalkyls, dihaloalkyls, trihaloalkyls, or polyhaloalkyls, including perhaloalkyls. Monohaloalkyls may have one chloro or fluoro group within the alkyl group. Chloro and fluoro are commonly found as substituents on alkyl or cycloalkyl groups. Fluoro, chloro, and bromo are often found on aryl or heteroaryl groups. Dihaloalkyls and polyhaloalkyls may have two or more identical halo atoms or combinations of different halo groups on the alkyl group. Typically, polyhaloalkyls contain up to 12, 10, 8, 6, 4, 3, or 2 halo groups. Non-limiting examples of haloalkyls include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, 2,2,2-trifluoroethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl. Perhalo-alkyl refers to alkyl groups in which all hydrogen atoms are replaced by halo atoms, such as trifluoromethyl.

[0151] As used herein, unless otherwise specified, the term “heteroatom” refers to a nitrogen (N), oxygen (O), or sulfur (S) atom. [Examples]

[0152] [Example 1] Synthesis of Exemplary Compounds of the Disclosure

[0153] Chemical substances and methods

[0154] In chemical synthesis, all starting materials and commercially available reagents were purchased from Sigma-Aldrich and used untreated unless otherwise specified. All reactions were carried out in oven-dried glassware under a nitrogen atmosphere using dry solvents. 1 H and 13 The 13C NMR spectrum was recorded using a Brucker AV-600 spectrometer, with the solvent used as the reference. 1 H and 13 For C, the values ​​were as follows: CDCl3: δ 7.24 and 77.23, CD3OD: δ 3.31 and 49.2, D2O: δ 4.80, DMSO-d6: δ 2.5 and 39.51). Chemical shifts (δ) were reported in ppm using the following rules: chemical shift, multiplicity (s=singular, d=double, t=tripular, q=quadruplicate, m=multiplex), integral, and binding constant (J), J reported in Hz. High-resolution mass spectra were recorded under ESI-TOF mass spectrometry conditions. Silica gel (E, Merck) was used for flash chromatography. The IMPACT® system (Intein Mediated Purification with Affinity Chitinbinding Tag) was purchased from New England Biolabs. His-tag purification resin was purchased from Roche. HiTrap IMAC columns (5 mL) were purchased from GE Healthcare Life Sciences. Gel permeation chromatography (GPC) was performed using an Ultimate 3000 liquid chromatography system with a 101 refractive index detector and Shodex column, with THF as the eluent, at 30°C and 1 mL min. -1 Polymer products were analyzed by flow rate. Calibration was based on a narrow linear poly(styrene) Shodex standard (SM-105). Mw and dispersion of polymer products were calculated using DIONEX Chromeleon software. Transmission electron microscope (TEM) images were obtained using an FEI Tecnai G2 F20 S-Twin.

[0155] The chemicals and methods described herein apply to all examples described herein.

[0156] Synthesis and Results

[0157] The exemplary compounds described herein were synthesized according to the following synthesis schemes 1, 2, and 3. Detailed synthesis procedures are described below.

[0158] Scheme 1

[0159] [ka]

[0160] Scheme 2

[0161] [ka]

[0162] Scheme 3

[0163] [ka]

[0164] Compounds 1-5

[0165] Compounds 1-5 were synthesized and characterized according to the published protocol (ACS Nano 2021, 15, pp. 309-321).

[0166] compound 8

[0167] [ka]

[0168] (11-carboxynonyl)triphenylphosphonium bromide 6 (2.5 g, 10 mmol) was prepared by refluxing triphenylphosphine (10 mmol) and 11-bromoundecanoic acid (10 mmol). This was then dissolved in 50 ml of tetrahydrofuran (THF) and cooled to 0°C. Lithium bis(trimethylsilyl)amide (LHMDS; 1 M in THF, 20 mmol) was added to the solution to produce an orange ylide. Then, 4-(4-fluorophenoxy)benzaldehyde (12 mmol) in 20 ml of THF was added dropwise to the solution and stirred at room temperature for 4 hours. The reaction mixture was quenched with methanol and concentrated. The residue was extracted with EA and brine, and then dehydrated with MgSO4. After removal of the solvent, the mixture was subjected to silica gel chromatography (EA-Hex=1:2) to obtain unsaturated fatty acid 7. Saturated fatty acids were prepared by catalytic hydrogenation in 50 ml of methanol containing 10 mol% of 10% charcoal-supported palladium (Pd / C). The reaction mixture was stirred overnight at room temperature under H2. The hydrogenated product was filtered through Celite, and the resulting solution was concentrated and subjected to silica gel chromatography (EA-Hex=1:2) to obtain the product as a yellow solid (66%).

[0169] compound 9

[0170] [ka]

[0171] Compound 9. Compound 8 (1 mmol) in THF (10 mL) was added to EDC (1.5 mmol), HOBt (1.5 mmol), DMAP (0.1 mmol), trimethylamine (2 mmol), and phytosphingosine (1.2 mmol). The resulting solution was stirred under nitrogen at room temperature for 12 hours. The solvent was then removed by evaporation, followed by extraction with EA / H2O. The collected organic layer was washed with saturated NaHCO3 (aqueous solution), water, and brine, and dehydrated with MgSO4. The crude product was purified by column chromatography (EA / Hex 1:1) on silica gel to obtain compound 9 (74%).

[0172] compound 10

[0173] [ka]

[0174] Compound 9 (1 mmol) was added to THF (10 mL) along with 4-nitrophenyl chloroformate (2 mmol) and trimethylamine (2 mmol). The resulting solution was stirred under nitrogen at room temperature for 12 hours. The solvent was then removed by evaporation, and the crude compound was used directly in the next step without further purification.

[0175] compound 11

[0176] [ka]

[0177] Compound 5 (1 mmol) was added to THF (10 mL) along with 1 mmol of 10 and trimethylamine (2 mmol). The resulting solution was stirred under nitrogen at room temperature for 2 hours. The solvent was then removed by evaporation, followed by extraction with EA / H2O. The collected organic layer was washed with saturated NaHCO3 (aqueous solution), water, and brine, and dehydrated with MgSO4. The crude product was purified by column chromatography on silica gel (EA / Hex1:1 + 10% MeOH) to produce compound 11 (59%).

[0178] compound 12

[0179] [ka]

[0180] Compound 11 was added to NaOMe (0.2 equivalents) in MeOH, and the resulting solution was stirred under nitrogen at room temperature for 2 hours. The mixture was neutralized with IR-120, then filtered, concentrated and dried under vacuum to obtain compound 12 (quantitative).

[0181] compound 13

[0182] [ka]

[0183] Compound 4 was added to NaOMe (0.2 equivalents) in MeOH, and the resulting solution was stirred under nitrogen at room temperature for 2 hours. The mixture was neutralized with IR-120, then filtered, concentrated and dried under vacuum to obtain compound 13 (quantitative).

[0184] compound 14

[0185] [ka]

[0186] Compound 13 (1 mmol) in MeOH was added to NaOMe (0.2 equivalents), and the resulting solution was stirred under nitrogen at room temperature for 2 hours. The mixture was neutralized with IR-120, then filtered, and concentrated and dried under vacuum. This was then dissolved in anhydrous DCM (10 mL), treated with imidazole (1.5 mmol) at 0°C, followed by the addition of TBDPSCl (1.2 mmol). The mixture was stirred under a nitrogen atmosphere at room temperature for 2.5 hours. The reaction was quenched by the addition of MeOH. After stirring at room temperature for 10 minutes, the solvent was removed under reduced pressure to obtain a dry residue. This was purified by column chromatography using MeOH / DCM (1 / 10) to obtain compound 14 (82%).

[0187] compound 15

[0188] [ka]

[0189] To a solution of compound 14 (1 mmol) and a catalytic amount of CSA (0.1 mmol) in CH3CN (20 mL), trimethyl orthobenzoate (3 mmol) was added under nitrogen atmospheric pressure at room temperature. After stirring for 30 minutes, Et3N was added to quench the reaction, and the resulting mixture was dried under reduced pressure. The residue was purified by column chromatography using EA / Hex(1 / 2) to obtain compound 15 (79%).

[0190] compound 16

[0191] [ka]

[0192] Compound 15 (1 mmol) was dissolved in DCM (10 mL) and sequentially mixed with DIPEA (2 mmol), benzoic anhydride (2 mmol), and DMAP (0.1 mmol). After stirring for 2 hours, the solvent was evaporated under reduced pressure to obtain a dry residue, which was then poured into EA (20 mL) and 2N HCl (10 mL) with vigorous stirring for 30 minutes. The solvent was then removed by evaporation, followed by extraction with EA / H2O. The collected organic layer was washed with ice-cold saturated NaHCO3 (aqueous solution), water, and brine, and dried over MgSO4. The dry residue was purified by column chromatography using EA / Hex(1 / 2) to obtain compound 16 (71%).

[0193] compound 17

[0194] [ka]

[0195] Compound 16 (1 mmol) was added to AcOH (4 mmol) and 1 M TBAF (2.4 mmol in THF) at 0°C. The resulting mixture was gradually warmed to room temperature, stirred for a further 2 hours, and then diluted with EA. The organic layer was washed with saturated NaHCO3 (aqueous solution), water, and brine, dried over anhydrous MgSO4, and concentrated under reduced pressure. The dried residue was purified by column chromatography using EA / Hex(1 / 2) to obtain compound 47 (88%).

[0196] compound 18

[0197] [ka]

[0198] A stirred solution of 17 (1 mmol) and 4A molecular sieve (0.1 g) in anhydrous DCM (10 mL) was cooled to -40°C, and then BF3(OEt)2 (0.1 mmol) was added dropwise to the solution. Solution 3 in anhydrous DCM was added dropwise to the above mixture and stirred at -40°C for 1 hour. The reaction mixture was then gradually warmed to room temperature and stirred for another hour. The solution was quenched by adding triethylamine, then filtered, saturated aqueous solution NaHCO3 was added, and extracted with DCM. The organic layer was dried over MgSO4 and evaporated to dryness. The residue was purified by flash column chromatography using silica gel to obtain the trisaccharide product. The product was then dissolved in MeOH, NaOMe (0.2 equivalents) was added, and the resulting solution was stirred at room temperature for 2 hours. The mixture was neutralized with IR-120, then filtered, and concentrated and dried under vacuum. The deacetylated mixture was purified using Bio-Gel P-2 Gel (Biorad) with H2O as the eluent to obtain a pure trisaccharide. The compound was freeze-dried to obtain compound 18 (39%).

[0199] compound 19

[0200] [ka]

[0201] Compound 13 (1 mmol) in MeOH was added to NaOMe (0.2 equivalents), and the resulting solution was stirred under nitrogen at room temperature for 2 hours. The mixture was neutralized with IR-120, then filtered, and concentrated and dried under vacuum.

[0202] compound 20

[0203] [ka]

[0204] Compound 19 (1 mmol) was added to THF (10 mL) along with 10 (1 mmol) and trimethylamine (2 mmol). The resulting solution was stirred under nitrogen at room temperature for 2 hours. The solvent was then removed by evaporation, followed by extraction with EA / H2O. The collected organic layer was washed with saturated NaHCO3 (aqueous solution), water, and brine, and dehydrated with MgSO4. The crude product was purified by column chromatography on silica gel (EA / Hex1:1 + 10% MeOH) to obtain compound 20.

[0205] compound 21

[0206] [ka]

[0207] Compound 20 in MeOH was added to NaOMe (0.2 equivalents), and the resulting solution was stirred under nitrogen at room temperature for 2 hours. The mixture was neutralized with IR-120, then filtered, concentrated and dried under vacuum to obtain compound 21 (quantitative). Compound 21 was tested using LC-MS spectroscopy. The LCMS spectrum shows peaks at 1236.13, 1245.16, 1247.64, 1268.77, 1279.86, 1305.99, 1308.13, 1311.57, 1313.93, 1343.46, 1354.29, 1355.60, 1358.33, 1379.12, 1403.82, 1408.57, 1425.66, 1448.31, 1453.30, 1458.39, 1467.71, 1471.33, and 1491.66 (Figure 12).

[0208] compound 22

[0209] [ka]

[0210] Arylmannnoside 22 (0.1 mmol) was added to DSPE-NHS (0.1 mmol) and trimethylamine (2 mmol) in EtOH / H2O (0.5 / 0.5 mL), and the resulting solution was stirred at room temperature for 12 hours. The solvent was removed by evaporation, and the crude product was purified with Bio-Gel P-2 Gel using H2O as the eluent to produce 22 (79%).

[0211] compound 23

[0212] [ka]

[0213] Aryltrimannoside 23 (0.1 mmol) was added to DSPE-NHS (0.1 mmol) and trimethylamine (2 mmol) in EtOH / H2O (0.5 / 0.5 mL), and the resulting solution was stirred at room temperature for 12 hours. The solvent was removed by evaporation, and the crude product was purified with Bio-Gel P-2 Gel using H2O as the eluent to produce 23 (76%).

[0214] [Example 2] Preparation and Characterization of LNPs of the Present Disclosure

[0215] Preparation of LNPs

[0216] A solution of a lipid mix having a molar ratio of 50% SM-102, 10% DSPC, 38.5% cholesterol and 1.5% DMG-PEG2000 in EtOH (10 mg / ml) was prepared. An LNP formulation was prepared by mixing the compound of the present disclosure with the lipid mix solution (molar ratio: 45% SM-102, 9% DSPC, 34.5% cholesterol, 1.5% DMG-PEG2000 and 10% of the compound of the present disclosure). The LNP formulation was added to a 1.5 mL test tube. Then, an mRNA payload (10 mM, pH 4) diluted with a citrate buffer before use was added to the test tube at a final concentration of 0.18 μg / μL. Then, the aqueous mRNA solution in the test tube was quickly added to the ethanol solution and mixed well by vortexing for 1 minute. Then, the resulting solution was dialyzed against PBS at 4 °C overnight using a micro float-A-Lyzer (8-10 kD) to obtain the LNP of this example. The generated LNP can be stored at 4 °C for several days before use.

[0217] Characterization of LNP

[0218] Measurement of size. The prepared LNP was tested using dynamic light scattering (DLS) to measure its size. First, 5 μL of the LNP solution was transferred to a clean 1.5 mL test tube and diluted with 95 μL of PBS. Then, the mixture was transferred to a cuvette and the particle size of the LNP was measured using a Nano ZS instrument. The following table shows the size and polydispersity index (PDI) of the prepared LNP samples.

[0219] [Table 2]

[0220] Zeta potential and encapsulation efficiency. Next, the encapsulation efficiency of the LNPs of this disclosure was evaluated using the Quant-it Ribogreen assay. The quant-it Ribogreen reagent was prepared by diluting it 2000-fold with 1×TE (working solution). Then, RNA standard dilution series (100 μL) from 0 to 50 ng / mL were prepared to obtain a standard curve. 5 μL of the prepared LNP solution was transferred to a clean test tube and diluted to a final volume of 100 μL. Then, the working solution of the quant-it Ribogreen reagent (100 μL) was added to the LNP sample. Next, the fluorescence signal of the sample was detected using a microplate reader (ex / em485 / 535). Following the standard curve, the concentration of unencapsulated mRNA in the solution (ng / mL) was calculated using the fluorescence signal. For zeta potential measurement, 0.75 mL of the DP intermediate was introduced into a capillary cell and measured at 25°C using a Malvern Zetasizer Pro instrument.

[0221] [Table 3]

[0222] [Example 3] In vitro uptake and transfection of mRNA-LNP in dendritic cells

[0223] Experiment 3-1

[0224] Preparation of spleen cells and culture of BMDCs. In accordance with embodiments of this disclosure, this example tested the uptake of several exemplary LNPs (as shown in the table below) in bone marrow-derived dendritic cells (BMDCs) and spleen cells. To prepare spleen cells, mouse spleens were homogenized on ground-glass ends of glass slides, treated with RBC lysis buffer (Sigma) to deplete red blood cells (RBCs), and then passed through a cell strainer (BD Biosciences). Bone marrow was isolated from mouse femurs and tibias and treated with RBC lysis buffer (Sigma-Aldrich) to deplete RBCs. The cells were then cultured in RPMI-1640 containing 10% heat-inactivated FBS (Thermo Fisher Scientific), 1% penicillin / streptomycin (Thermo Fisher Scientific), 50 μM 2-mercaptoethanol (Thermo Fisher Scientific), and 20 ng / ml recombinant mouse GM-CSF (eBioscience) for 2 × 10⁶ cells. 5 Cells were cultured at a density of 100 cells / ml. On day 3, the cells were supplemented with an equal volume of complete medium (RPMI-1640, 100 U / ml Pen / Streptococcus, 55 μM 2-mercaptoethanol and 10% FBS), and on day 6, half the volume of medium was replaced with fresh medium. On day 8, the suspended cells were collected.

[0225] [Table 4]

[0226] Treatment of spleen cells and BMDCs with LNPs. Spleen cells or BMDCs were incubated with different FITC-labeled LNP preparations in RPMI-1640 at 37°C for 1 hour. Cells were blocked with an Fc receptor binding inhibitor (clone: ​​93, eBioscience) for 20 minutes. Spleen cells were stained with antibodies against CD3 (clone: ​​17A2, BV421-conjugate, Biolegend) and CD19 (clone: ​​1D3, PECy7-conjugate, BD Biosciences). BMDCs were stained with antibody against CD11c (clone N418 APC-conjugate, Biolegend). Labeled cells were analyzed using FACSC and a Flow Cytometer (BD Biosciences).

[0227] Flow cytometry. After incubation with different mRNA-LNPs, BMDC cells were washed with ice-cold FACS buffer (1% FBS in 1× DPBS containing 0.1% sodium azide), incubated with purified anti-mouse CD16 / 32 antibody (BioLegend) in FACS buffer on ice for 20 minutes, and then washed with FACS buffer. BMDCs were stained with APC anti-mouse CD11c antibody (BioLegend) at 4°C for 30 minutes and washed with FACS buffer. Finally, BMDCs were stained with propidium iodide (Sigma-Aldrich). Flow cytometry was performed using a FACS Canto® flow cytometer (BD Bioscience).

[0228] Results. The FACS results are shown in Figures 1 and 2 and in the table below. Figure 1 shows that BMDCs specifically took up LNPs prepared using the compounds of this disclosure compared to non-BMDCs. With conventional LNPs (i.e., those not using the compounds of this disclosure), BMDCs showed slightly higher uptake than non-BMDCs, but the specificity was only slightly higher than that of the LNPs of this disclosure (56.1 / 8.62 or 55.2 / 6.64 vs. 0.48 / 0.12). Similarly, in Figure 2, the specific uptake of LNPs of this disclosure shown by dendritic cells (DCs) was at least 3 times higher than that of B cells (30.6 / 10.7 and 38.3 / 11.9) and at least 6 times higher than that of T cells (30.6 / 0.5 and 38.3 / 0.68). DCs showed higher uptake than conventional LNPs, but the slope was not as significant as that of the LNPs of this disclosure.

[0229] [Table 5]

[0230] Experiment 3-2

[0231] Exemplary LNPs (as shown in the table below) prepared using different formulations according to embodiments of the present disclosure were tested in this experiment. Both uptake and transfection were tested to evaluate whether the payload delivered by the LNPs of the present disclosure could be properly expressed in targeted cells. Bone marrow-derived dendritic cells (BMDCs) were isolated from the tibia and femur of 57BL / 6 mice. Bone marrow cells were stimulated with 20 ng / mL GM-CSF for 8 days in RPMI medium (RPMI-1640, 100 U / ml Pen / Streptococcus, 55 μM 2-mercaptoethanol and 10% FBS). After 8 days of culture, 1 × 10⁶ cells were cultured. 6 Each BMDC (400g, centrifuged for 5 minutes, then the medium was replaced with 1ml of Opti-MEM) was plated into a 6-well plate, and samples of different LNPs containing mRNA were diluted in 0.25mL of Opti-MEM and incubated with the BMDC.

[0232] For uptake analysis, FITC-labeled LNPs encapsulating mRNA encoding the SARS COV2 spike protein were incubated with BMDCs at 37 °C for 2 hours. For transfection analysis, LNPs encapsulating eGFP mRNA were incubated with BMDCs at 37 °C for 4 hours. Four hours after transfection, BMDCs were supplemented with 1.25 ml of complete RPMI medium and incubated at 37 °C for 48 hours. Experiments were conducted using FACS as described above.

[0233]

Table 6

[0234] Results. The FACS results are shown in Figures 3 and 4 and the following table. All LNPs prepared using the compounds of the present disclosure at different molar ratios showed higher uptake than the negative control (conventional LNPs without using the compounds of the present disclosure). The data also confirm that the LNPs of the present disclosure can not only deliver the payload to the targeted cells, but also transfect the targeted cells and enable the expression of the payload. Since they have higher specificity for the targeted cells, the transfection signals detected from the group using the LNPs of the present disclosure were significantly higher than those detected from the conventional group. This result suggests that using the LNPs of the present disclosure makes it possible to obtain similar results even with a lower dose of the payload.

[0235]

Table 7

[0236] Experiment 3-3

[0237] To evaluate the binding of DC-SIGN to the LNPs of this disclosure, exemplary LNPs in PBS were coated onto ELISA plates overnight at 4°C. The plates were incubated with diluted DC-SIGN ECD (15–0.075 nM in HEPES buffer containing 20 mM HEPES, 150 mM NaCl, 10 mM CaCl2, and 0.1% BSA) at pH 7.4, 6.0, and 5.0 at room temperature for 1 hour. The bound DC-SIGN ECD was detected using HRP-conjugate anti-DC-SIGN(B2) IgG antibody (Santa Cruz Biotechnology). After 1 hour of incubation at room temperature, the plates were treated with tetramethylbenzidine (TMB) for 10 minutes. After adding 0.5 M sulfuric acid to the plates, the optical density was measured at 450 nm using a microplate reader. Using GraphPad Prism, the apparent Kd was calculated using a nonlinear regression curve fit for the total number of connections.

[0238] [Example 4] In vivo delivery of luciferase mRNA-LNP

[0239] This experiment tested the in vivo targeted delivery of the LNPs described herein (shown in the table below). The LNPs tested in this experiment carried mRNA encoding luciferase. Mice were intravenously injected with LNPs (200 μL) and kept in that state for 1 or 6 hours before being measured using the In vivo Imaging System (IVIS®). For IVIS measurement, the animals were first anesthetized using a rodent anesthesia system with isoflurane (2.5% (vol / vol), 0.2 L / min O2 flow rate). The animals were then intravenously injected with D-luciferin solution (dissolved in 1×PBS; 150 mg / kg body weight). Three minutes after injection, the animals were scanned using the IVIS imaging system (data not shown). After imaging, the animals were euthanized in a CO2 chamber. Animal organs (heart, lungs, liver, spleen, kidneys, and lymph nodes) were collected, luminescence was detected, and quantification was performed using the IVIS system.

[0240] [Table 8]

[0241] Results. The results (Figure 5) show that both compound 22-LNP and compound 12-LNP tended to accumulate in the spleen and lymph nodes. Compound 12-LNP also accumulated in the liver, while compound 22-LNP showed a high level of specificity targeting the spleen and lymph nodes. The results demonstrate the targeted delivery function of the lipid nanoparticle formulations of this disclosure, which is consistent with the observations of the above experiment.

[0242] [Example 6] Immunization

[0243] Animals. Balb / c mice (8 weeks old) were purchased from the National Laboratory Animal Center, Taiwan. All mice were maintained in an environment free of specific pathogens. 8-week-old Balb / c mice were immunized twice with im at a 2-week interval. Each vaccine dose contained PBS (100 μl). Ten days after the last immunization, serum collected from the immunized mice was subjected to ELISA analysis. The experimental protocol was approved by Academia Sinica's Institutional Animal Care and Utilization Committee (Approval No. 22-08-1901).

[0244] LNPs. For neutralization assays, LNPs were prepared for the experiment according to embodiments of the present disclosure. Two control LNPs were also prepared to compare the performance of the LNPs of the present disclosure. The first control LNP was formed using SM-102 and DSPC without using the compounds of the present disclosure ("L1+L2"). The second control LNP was the Moderna product against Spikevax ("LNP(M)"). All tested LNPs carried mRNA cargo encoding the SARC-CoV-2 spike protein. For IgG titer assays, the LNPs of the present disclosure were prepared to carry either mRNA encoding the wild-type SARC-CoV-2 spike protein or mRNA encoding the wild-type SARC-CoV-2 spike protein with low sugar modification.

[0245] Animal immunization. BALB / c mice (n=5) aged 6-8 weeks were immunized intramuscularly with 15 μg of LNP in phosphate-buffered saline (PBS). The animals were immunized at week 0, boosted with a second vaccination at week 2, and serum samples were collected from each mouse 2 weeks after the second immunization.

[0246] Pseudoviral Neutralization Assay. Pseudoviruses were constructed at the RNAi Core Facility of Academia Sinica using a procedure similar to that previously described. Briefly, a pseudo-lentivirus possessing the SARS-CoV-2 spike protein was generated by transiently transfecting HEK-293T cells with pCMV-ΔR8.91, pLAS2w.Fluc.Ppuro. HEK-293T cells were seeded one day prior to transfection, and the indicated plasmid was delivered to the cells using the TransITR-LT1 transfection reagent (Mirus). The medium was refreshed at 16 hours and collected at 48 and 72 hours after transfection. Cell debris was removed by centrifugation at 4,000xg for 10 minutes, and the supernatant was passed through a 0.45 μm syringe filter (Pall Corporation). The pseudo-lentiviruses were divided into equal volumes and then stored at -80°C. To predict lentiviral titer using the AlamaBlue assay (Thermo Scientific), the transduction units (TUs) of SARS-CoV-2 pseudolentivirals were predicted by using a cell viability assay responsive to limited dilutions of lentiviruses. Briefly, HEK-293T cells stably expressing the human ACE2 gene were plated into 96-well plates one day prior to lentiviral transduction. To determine the titer of the pseudolentivirals, different amounts of lentivirus were added to a medium containing polybrene (final concentration 8 μg / ml). Spin infection was performed in the 96-well plates at 1,100xg for 30 minutes at 37°C. After incubating the cells at 37°C for 16 hours, the medium containing the virus and polybrene was removed and replaced with fresh, complete DMEM containing 2.5 μg / ml puromycin. After 48 hours of treatment with puromycin, the medium was removed and cell viability was detected using 10% AlamaBlue reagent according to the manufacturer's instructions. The survival rate of uninfected cells (without puromycin treatment) was set to 100%. Viral titer (transduction units) was determined by plotting the ratio of surviving cells to diluted viral loads.For the neutralization assay, heat-inactivated serum or antibody was sequentially diluted and incubated with 1,000 TU of SARS-CoV-2 pseudolentivirus in DMEM at 37°C for 1 hour. The mixture was then inoculated into 10,000 HEK-293T cells stably expressing the human ACE2 gene in a 96-well plate. At 16 hours post-infection, the medium was replaced with fresh, complete DMEM (supplemented with 10% FBS and 100 U / mL penicillin / streptomycin), and the cells were cultured continuously for another 48 hours. Luciferase gene expression levels were determined using the Bright-Glo Luciferase Assay System (Promega). Relative light units (RLU) were detected using Tecan i-control (Infinite 500). The inhibition percentage was expressed as the ratio of the RLU reduction in the presence of diluted serum to the RLU value of the serum-free control, using the formula (RLU). control -RLU Serum Calculated using the RLU control.

[0247] Measurement of serum IgG titer. Mouse serum IgG titers were determined using ELISA. Wells of a 96-well ELISA plate (Greiner Bio-One) were coated overnight at 4°C with 100 ng of SARS-CoV-2 spike protein (ACROBiosystems, wild type, delta or omicron, respectively) in 100 mM sodium bicarbonate at pH 8.8. The wells were blocked at 37°C for 1 hour with 200 μl of 5% skim milk in 1×PBS and washed three times with 200 μl of PBST (1×PBS, 0.05% Tween 20, pH 7.4). Serially diluted mouse serum samples were added to the wells, incubated at 37°C for 2 hours, and washed six times with 200 μl of PBST. Wells were incubated with 100 μl of HRP-conjugated anti-mouse secondary antibody (1:10000, in PBS) at 37°C for 1 hour and washed six times with 200 μl of PBST. 100 μl of horseradish peroxidase substrate (1-Step® Ultra TMB-ELISA Substrate Solution) (Thermo Scientific®) was added to the wells, followed by 100 μl of 1 M H2SO4. After 30 minutes of incubation, absorbance (OD450 nm) was measured using SpectraMax M5.

[0248] Results. Figure 9 shows that all tested LNPs carrying mRNA cargo were able to deliver and express mRNA in vivo, thus inducing an immune response resulting in neutralization inhibition. However, the inhibitory effects of these tested LNPs differed as the dilution ratio increased. Only L1+L2LNP and LNP(M) showed slightly higher inhibitory effects at a 1:5000 dilution compared to the negative control, while the LNPs of this disclosure maintained inhibitory effects around 40%. The data demonstrate that the LNPs of this disclosure can induce an immune response at much lower concentrations than the other LNPs tested in this experiment.

[0249] Figure 10 demonstrates that the LNPs of this disclosure can induce antigen-specific IgG in vivo. LNPs carrying mRNA encoding wild-type spike protein induced IgG that was still able to recognize both delta and omicron variant spike proteins at good levels. The data indicate that the targeted delivery characteristics of the LNPs can at least partially overcome immune evasion due to spike protein variability between mutants.

[0250] Furthermore, LNPs carrying mRNA encoding the wild-type SARS-CoV-2 spike protein ("WT LNP") and LNPs carrying mRNA encoding the low-sugar modified spike protein ("low-sugar LNP") induced similar IgG titers against the wild-type virus. However, WT LNP exhibited lower IgG titers against the Delta and Omicron strains, suggesting immune evasion. In contrast, low-sugar LNP maintained high IgG titers against these two mutant strains. These results demonstrate that removal of glycanshield improves the immunogenicity of LNP preparations.

[0251] Figure 11 shows the results of additional experiments. In these experiments, Moderna LNPs were prepared using a proprietary Moderna formulation. In addition, LNPs were prepared using the Moderna formulation with the compound of the disclosure added, in order to test whether the compound of the disclosure improves the performance of the Moderna formulation. LNPs were administered to animals, and IgG titer assays and neutralization assays were performed as described above in this embodiment. The results demonstrate that the compound of the disclosure increased spike protein-specific IgG. Serum obtained from mice administered with the LNP compound of the disclosure also showed better neutralization. This experiment confirmed the targeted delivery characteristics of the compound of the disclosure and verified that the compound of the disclosure can be applied to commercially available LNP formulations.

[0252] Exemplary Embodiments

[0253] Embodiment 1. The component is of formula:

[0254] [ka] (In the formula, R1 includes a substituted or unsubstituted glycosyl group, and X1 and X2 are each independently hydrogen, C 1~30 Alkyl, C 1~30 Alkenil, C 1~30 The compound is an alkynyl, aryl, aryloxy or a substituted thereof, or -(CH2)nX4, where n is 0 to 30, and X4 is hydrogen, aryl, aryloxy, heterocyclic group or a substituted thereof, provided that if X4 is a heterocyclic group, the heterocyclic group contains 1 to 3 heteroatoms selected from the group consisting of O, S, and N, or a combination thereof, and X3 is hydrogen, C 1~6 Compounds for forming lipid nanoparticles, comprising alkyl or hydroxyl groups.

[0255] Embodiment 2. R1 is formula R2-R A -(In the formula, R A The compound according to Embodiment 1, wherein R2 is a binding group, R2 is a substituted or unsubstituted glycosyl group, and the binding group includes aryl, alkyl, amide, alkylamide, their substituted products, combinations thereof, or covalent bonds.

[0256] Embodiment 3.R A It contains an aryl having 0 to 3 substituents, and the substituent is C 1~6 Alkyl, halide or C 1~6 The compound according to Embodiment 2, which is an alkyl halogen.

[0257] Embodiment 4.R A The compound according to Embodiment 3, further comprising a polyethylene glycol (PEG) moiety having 2 to 72 (OCH2CH2) subunits.

[0258] Embodiment 5. The copolymer according to Embodiment 3 or Embodiment 4, wherein the PEG portion is linear.

[0259] Embodiment 6. The compound according to any one of Embodiments 1 to 5, wherein the glycosyl group comprises a mannoside, a fucoside, or a combination thereof.

[0260] Embodiment 7. The compound according to any one of Embodiments 1 to 6, wherein the glycosyl group comprises a terminal mannoside, a terminal fucoside, or both.

[0261] Embodiment 8. The compound according to any one of Embodiments 1 to 7, wherein the glycosyl group comprises a monomannoside, dimannoside, or trimannoside.

[0262] Embodiment 9. The compound according to Embodiment 8, wherein the trimannoside is a linear or branched trimannoside.

[0263] Embodiment 10. The compound according to Embodiment 9, wherein the branched trimannoside is α-1,3-α-1,6-trimannoside.

[0264] Embodiment 11. The compound according to any one of Embodiments 1 to 10, wherein R1 is a substituted glycosyl group.

[0265] Embodiment 12. The glycosyl group contains 1 to 6 substituents, and the substituent is C 1~6 Alkyl, C 1~6 Alkenyl, halogen, C 1~6 Alkyl halogens, C 1~6 Alkoxy, amine, nitro, C 1~6 The compound according to Embodiment 11, which is an alkylamine, amide, azide, aryl, cycloalkyl, heterocycloalkyl, sulfite ester, or a substituted product thereof, or a combination thereof.

[0266] Embodiment 13. The substituent of the glycosyl group is selected from the group consisting of aryl, 5-membered cycloalkyl, 6-membered cycloalkyl, 5-membered heterocycloalkyl, and 6-membered heterocycloalkyl, and their substituted products, and the substituent is C 1~6 Alkyl, halogen, C 1~6 Alkyl halogens, C 1~6 Alkoxy, amine, nitro, C1~6 The compound according to Embodiment 12, comprising 1 to 6 substituents selected from the group consisting of alkylamines, amides, azides, carboxyls, hydroxyls, aryls, cycloalkyls, heterocycloalkyls, or substituted products thereof, or combinations thereof.

[0267] Embodiment 14. The compound according to Embodiment 12 or Embodiment 13, wherein the substituent of the glycosyl group is a substituted or unsubstituted aryl, and optionally the substituent of the glycosyl group is a phenyl substituted with OH, CH3, NH2, CF3, OCH3, F, Br, Cl, NO2, N3, or a combination thereof.

[0268] Embodiment 15. The compound according to Embodiment 12 or Embodiment 13, wherein the heterocycloalkyl comprises 1 to 3 heteroatoms selected from the group consisting of O, S, and N.

[0269] Embodiment 16.R1 is,

[0270] [ka] A compound according to any one of Embodiments 1 to 15, selected from the group consisting of TIFF2026515588000043.tif180161 and TIFF2026515588000044.tif80161.

[0271] Embodiment 17. A compound according to any one of Embodiments 1 to 16, which is a compound of Formula 1.

[0272] Embodiment 18. A compound according to any one of Embodiments 1 to 16, which is a compound of Formula 2.

[0273] Embodiment 19. Formula 3:

[0274] [ka] It is a compound in which R1 is

[0275] [Chemical formula] The compound according to embodiment 18, selected from the group consisting of

[0276] Embodiment 20. The compound according to any one of embodiments 1 to 19, wherein at least one of X1 and X2 contains a saturated hydrocarbon chain containing at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28 or 30 carbon atoms.

[0277] Embodiment 21. X1 and X2 are each independently hydrogen, C 4~30 alkyl, C 4~30 alkenyl, C 4~30 alkynyl, aryl, aryloxy or a substituent thereof, or -(CH2)nX4, where n is 4 to 30 and X4 is hydrogen, aryl, aryloxy, a heterocyclic group or a substituent thereof, provided that when X4 is a heterocyclic group, the heterocyclic group contains 1 to 3 heteroatoms selected from the group consisting of O, S and N or a combination thereof. The compound according to any one of embodiments 1 to 20.

[0278] Embodiment 22. X1 and X2 are each independently hydrogen, C 8~30 alkyl, C 8~30 alkenyl, C 8~30 alkynyl, aryl, aryloxy or a substituent thereof, or -(CH2)nX4, where n is 8 to 30 and X4 is hydrogen, aryl, aryloxy, a heterocyclic group or a substituent thereof, provided that when X4 is a heterocyclic group, the heterocyclic group contains 1 to 3 heteroatoms selected from the group consisting of O, S and N or a combination thereof. The compound according to embodiment 21.

[0279] Embodiment 23. The compound according to any one of embodiments 1 to 22, wherein when one of X1 and X2 is hydrogen, the other is not hydrogen.

[0280] Embodiment 24. X4 is aryl, aryloxy, heterocyclic group, cycloalkyl, heterocycloalkyl or a combination thereof, and X4 is C 1~6 alkyl, halogen, C 1~6 alkylhalogen and C 1~6 alkoxy, and the compound according to any one of Embodiments 1 to 23, which contains 0 to 6 substituents selected from the group consisting of

[0281] Embodiment 25. The compound according to Embodiment 24, wherein the substituent is CH3, CF3, F or OCH3.

[0282] Embodiment 26. The compound according to Embodiment 24 or Embodiment 25, wherein X4 contains 1 to 3 substituents.

[0283] Embodiment 27. X4 is -R3-O-R4, and R3 and R4 are each independently aryl, heterocyclic group, cycloalkyl, heterocycloalkyl, and each contains 1~6 alkyl, halogen, C 1~6 alkylhalogen and C 1~6 alkoxy, and the compound according to any one of Embodiments 24 to 26, which contains 0 to 6 substituents selected from the group consisting of

[0284] Embodiment 28. One of X1 and X2 is C 15-30 alkyl, and the other is -(CH2)nX4, and the compound according to any one of Embodiments 1 to 27.

[0285] Embodiment 29. X4 is

[0286]

Chemical formula

[0287] Embodiment 30.

[0288]

Chemical formula

[0289] Embodiment 31. An ingredient according to any one of Embodiments 1 to 30, other than glycolipid C34 or α-galactosylceramide.

[0290] Embodiment 32. A formulation for forming lipid nanoparticles, comprising a compound described in any one of Embodiments 1 to 31, wherein the compound constitutes 1 to 10 mol% of the composition.

[0291] Embodiment 33. The formulation according to Embodiment 32, further comprising an ionizable lipid, a helper lipid, or a mixture thereof, wherein the ionizable lipid constitutes 30 to 60 mol% of the composition, the helper lipid constitutes 5 to 60 mol% of the composition, and the remaining percentage is a carrier or solvent.

[0292] Embodiment 34. The formulation according to Embodiment 33, wherein the ionizable lipid comprises heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (SM-102®), (4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315®, Pfizer) or a combination thereof.

[0293] Embodiment 35. The formulation according to Embodiment 33 or Embodiment 34, wherein the helper lipid comprises phosphatidylcholine, cholesterol or a derivative thereof, polyethylene glycol-lipid (PEG-lipid), or a mixture thereof, wherein phosphatidylcholine constitutes 5 to 10 mol% of the composition, cholesterol or a derivative thereof constitutes 30 to 40 mol% of the composition, and polyethylene glycol-lipid (PEG-lipid) constitutes 1 to 10 mol% of the composition.

[0294] Embodiment 36. The formulation according to Embodiment 35, wherein the phosphatidylcholine comprises distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylethanolamine (DPOE), or a mixture thereof.

[0295] Embodiment 37. The formulation according to Embodiment 35 or Embodiment 36, wherein cholesterol or its derivative is cholesterol, campesterol, beta-sitosterol, brassicasterol, ergosterol, dehydroergosterol, stigmasterol, fucosterol, DC-cholesterolHCl, OH-Chol, HAPC-Chol, MHAPC-Chol, DMHAPC-Chol, DMPAC-Chol, cholesteryl chloroformate, GL67, cholesteryl myristate, cholesteryl oleate, cholesteryl nervonate, LC10, cholesteryl hemysuccinate, (3β,5β)-3-hydroxycholane-24-euic acid, alkyne cholesterol, 27-alkyne cholesterol, E-cholesterol alkyne, trifluoroacetate (Dios-Arg, 2H-Cho-Arg or Cho-Arg) or a mixture thereof.

[0296] Embodiment 38. The formulation according to any one of Embodiments 35 to 37, wherein the PEG-lipid is DMG-PEG, DSG-PEG, mPEG-DPPE, dope-PEG, mPEG-DMPE, mPEG-dope, DSPE-PEG-amine, DSPE-PEG, mPEG-DSPE, PEGPE, m-PEG-pentacosadiic acid, bromoacetamide-PEG, amine-PEG, azide-PEG, or a mixture thereof.

[0297] Embodiment 39. The formulation according to any one of Embodiments 33 to 38, further comprising a payload.

[0298] Embodiment 40. The formulation according to Embodiment 39, wherein the payload is a nucleic acid, a compound, a polypeptide, a protein, a glycan, or a combination thereof.

[0299] Embodiment 41. The formulation according to Embodiment 40, wherein the nucleic acid is RNA or DNA.

[0300] Embodiment 42. The formulation according to Embodiment 41, wherein the payload encodes a polypeptide.

[0301] Embodiment 43. The formulation according to any one of Embodiments 39 to 42, wherein the payload is immunogenic, or the payload is a nucleic acid configured to encode an immunogenic polypeptide or protein.

[0302] Embodiment 44. The formulation according to any one of Embodiments 39 to 43, wherein the payload is a first payload, and the composition further encapsulates a second payload, the second payload being a nucleic acid, a compound, a polypeptide, a protein, a glycan, or a combination thereof.

[0303] Embodiment 45. The formulation according to Embodiment 44, wherein the first payload and the second payload are different.

[0304] Embodiment 46. A formulation according to any one of Embodiments 32 to 45, further comprising a pharmaceutically acceptable excipient, adjuvant, or combination thereof.

[0305] Embodiment 47. Lipid nanoparticles comprising a membrane defining an internal space, wherein the membrane is formed of a plurality of lipid components comprising a compound described in any one of Embodiments 1 to 31.

[0306] Embodiment 48. Lipid nanoparticles according to Embodiment 47, wherein the plurality of lipid components further comprise ionizable lipids, helper lipids, or combinations thereof.

[0307] Embodiment 49. Lipid nanoparticles according to Embodiment 47 or Embodiment 48, wherein the membrane is formed via hydrophobic interactions between a plurality of lipid components.

[0308] Embodiment 50. Lipid nanoparticles according to Embodiment 49, wherein the ionizable lipid comprises heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (SM-102®), (4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315®, Pfizer) or a combination thereof.

[0309] Embodiment 51. Lipid nanoparticles according to Embodiment 49 or Embodiment 50, wherein the helper lipid comprises phosphatidylcholine, cholesterol or a derivative thereof, polyethylene glycol-lipid (PEG-lipid), or a mixture thereof.

[0310] Embodiment 52. Lipid nanoparticles according to Embodiment 51, wherein the phosphatidylcholine comprises distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylethanolamine (DPOE), or a mixture thereof.

[0311] Embodiment 53. Lipid nanoparticles according to Embodiment 51 or Embodiment 52, wherein cholesterol or its derivative is cholesterol, campesterol, beta-sitosterol, brassicasterol, ergosterol, dehydroergosterol, stigmasterol, fucosterol, DC-cholesterolHCl, OH-Chol, HAPC-Chol, MHAPC-Chol, DMHAPC-Chol, DMPAC-Chol, cholesteryl chloroformate, GL67, cholesteryl myristate, cholesteryl oleate, cholesteryl nervonate, LC10, cholesteryl hemysuccinate, (3β,5β)-3-hydroxycholane-24-euic acid, alkyne cholesterol, 27-alkyne cholesterol, E-cholesterol alkyne, trifluoroacetate (Dios-Arg, 2H-Cho-Arg or Cho-Arg) or a mixture thereof.

[0312] Embodiment 54. Lipid nanoparticles according to any one of Embodiments 51 to 53, wherein the PEG-lipid is DMG-PEG, DSG-PEG, mPEG-DPPE, doped-PEG, mPEG-DMPE, mPEG-doped, DSPE-PEG-amine, DSPE-PEG, mPEG-DSPE, PEGPE, m-PEG-pentacosadiic acid, bromoacetamide-PEG, amine-PEG, azide-PEG, or a mixture thereof.

[0313] Embodiment 55. Lipid nanoparticles according to any one of Embodiments 47 to 54, wherein the membrane encapsulates the payload.

[0314] Embodiment 56. The lipid nanoparticles according to Embodiment 55, wherein the payload is a nucleic acid, a compound, a polypeptide, a protein, a glycan, or a combination thereof.

[0315] Embodiment 57. The lipid nanoparticle according to Embodiment 56, wherein the nucleic acid is RNA or DNA.

[0316] Embodiment 58. Lipid nanoparticles according to Embodiment 57, wherein the payload encodes a polypeptide.

[0317] Embodiment 59. Lipid nanoparticles according to any one of Embodiments 55 to 58, wherein the payload is immunogenic or a nucleic acid configured to encode an immunogenic polypeptide or protein.

[0318] Embodiment 60. Lipid nanoparticles according to any one of Embodiments 55 to 59, wherein the payload is a first payload and the membrane further encapsulates a second payload.

[0319] Embodiment 61. The lipid nanoparticles according to Embodiment 60, wherein the second payload is a nucleic acid, a compound, a polypeptide, a protein, a glycan, or a combination thereof.

[0320] Embodiment 62. Lipid nanoparticles according to Embodiment 60 or Embodiment 61, wherein the first payload and the second payload are different.

[0321] Embodiment 63. Lipid nanoparticles according to any one of Embodiments 47 to 62, prepared from the composition according to any one of Embodiments 29 to 43.

[0322] Embodiment 64. Lipid nanoparticles according to any one of Embodiments 47 to 63, wherein the membrane has a two-layer structure.

[0323] Embodiment 65. Lipid nanoparticles according to any one of Embodiments 47 to 64, having a diameter of 0.01 to 5 microns.

[0324] Embodiment 66. Lipid nanoparticles according to any one of Embodiments 47 to 65, wherein the plurality of lipid components do not contain glycolipid C34 or α-galactosylceramide (α-GalCer).

[0325] Embodiment 67. A formulation comprising lipid nanoparticles as described in any one of Embodiments 47 to 66.

[0326] The formulation according to Embodiment 67, comprising 95% (w / w) of lipid nanoparticles.

[0327] Embodiment 69. The formulation according to Embodiment 67 or Embodiment 68, wherein the lipid nanoparticles are first lipid nanoparticles, and the composition further comprises second lipid nanoparticles.

[0328] Embodiment 70. The formulation according to Embodiment 69, wherein the first lipid nanoparticles and the second lipid nanoparticles differ in size, membrane component, encapsulated payload, or combination thereof.

[0329] Embodiment 71. A formulation according to any one of Embodiments 67 to 70, further comprising an excipient, an adjuvant, or a combination thereof.

[0330] Embodiment 72. The formulation according to Embodiment 71, wherein the excipient comprises a solvent, a dispersion medium, a diluent, a dispersant, a suspension aid, a surfactant, an isotonic agent, a thickener or emulsifier, a preservative, a polymer, a peptide, a protein, a cell, hyaluronidase, or a mixture thereof.

[0331] Embodiment 73. The formulation according to Embodiment 71 or Embodiment 72, wherein the adjuvant comprises C34, Gluco-C34, 7DW8-5, C17, C23, C30, α-galactosylceramide, aluminum salt, squalene, MF59, or QS-21. Other examples of adjuvants in some vaccines that can be used in the compositions of the present disclosure are aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), mixed aluminum salts, Freund's complete adjuvant, Freund's incomplete adjuvant, AS03 (GlaxoSmithKline), MF59 (Seqirus), and CpG1018 (Dynavax), or combinations thereof.

[0332] Embodiment 74. A kit for preparing lipid nanoparticles, comprising a first reagent containing a compound described in any one of Embodiments 1 to 31, and a second reagent containing an ionizable lipid, a helper lipid, or a mixture thereof.

[0333] Embodiment 75. The kit according to Embodiment 74, wherein the second reagent contains an ionizable lipid.

[0334] Embodiment 76. The kit according to Embodiment 75, further comprising a third reagent containing a helper lipid.

[0335] Embodiment 77. A kit according to any one of Embodiments 74 to 76, wherein the ionizable lipid comprises heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (SM-102®), (4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315®, Pfizer) or a combination thereof.

[0336] Embodiment 78. The kit according to any one of Embodiments 74 to 77, wherein the helper lipid comprises phosphatidylcholine, cholesterol or a derivative thereof, polyethylene glycol-lipid (PEG-lipid), or a mixture thereof.

[0337] Embodiment 79. The kit according to Embodiment 78, wherein the phosphatidylcholine comprises distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylethanolamine (DPOE), or a mixture thereof.

[0338] Embodiment 80. The kit according to Embodiment 78 or Embodiment 79, wherein cholesterol or its derivative is cholesterol, campesterol, beta-sitosterol, brassicasterol, ergosterol, dehydroergosterol, stigmasterol, fucosterol, DC-cholesterolHCl, OH-Chol, HAPC-Chol, MHAPC-Chol, DMHAPC-Chol, DMPAC-Chol, cholesteryl chloroformate, GL67, cholesteryl myristate, cholesteryl oleate, cholesteryl nervonate, LC10, cholesteryl hemysuccinate, (3β,5β)-3-hydroxycholane-24-euic acid, alkyne cholesterol, 27-alkyne cholesterol, E-cholesterol alkyne, trifluoroacetate (Dios-Arg, 2H-Cho-Arg or Cho-Arg) or a mixture thereof.

[0339] Embodiment 81. The kit according to any one of Embodiments 78 to 80, wherein the PEG-lipid is DMG-PEG, DSG-PEG, mPEG-DPPE, doped-PEG, mPEG-DMPE, mPEG-doped, DSPE-PEG-amine, DSPE-PEG, mPEG-DSPE, PEGPE, m-PEG-pentacosadic acid, bromoacetamide-PEG, amine-PEG, azide-PEG, or a mixture thereof.

[0340] Embodiment 82. The kit according to any one of Embodiments 74 to 81, further comprising a fourth reagent containing a payload, wherein the payload is a nucleic acid, a compound, a polypeptide, a protein, a glycan, or a combination thereof.

[0341] Embodiment 83. The kit according to Embodiment 82, wherein the nucleic acid is RNA or DNA.

[0342] Embodiment 84. The kit according to Embodiment 83, wherein the payload encodes a polypeptide.

[0343] Embodiment 85. The kit according to any one of Embodiments 82 to 84, wherein the payload is immunogenic, or the payload is a nucleic acid configured to encode an immunogenic polypeptide or protein.

[0344] Embodiment 86. A method for delivering a targeted payload to a subject, comprising administering an effective amount of lipid nanoparticles described in any one of Embodiments 47 to 54 to the subject, wherein the payload is a nucleic acid, a compound, a polypeptide, a protein, a glycan, or a combination thereof, and the payload is encapsulated in lipid nanoparticles.

[0345] Embodiment 87. The method according to Embodiment 86, wherein the nucleic acid is RNA or DNA.

[0346] Embodiment 88. The method according to Embodiment 87, wherein the payload encodes a polypeptide.

[0347] Embodiment 89. The method according to any one of Embodiments 86 to 88, wherein the payload is immunogenic, or the payload is a nucleic acid configured to encode an immunogenic polypeptide or protein.

[0348] Embodiment 90. The method according to any one of Embodiments 86 to 89, wherein the payload is a first payload, the membrane further encapsulates a second payload, and the second payload is a nucleic acid, a compound, a polypeptide, a protein, a glycan, or a combination thereof.

[0349] Embodiment 91. The method according to Embodiment 90, wherein the first payload and the second payload are different.

[0350] Embodiment 92. The method according to any one of Embodiments 86 to 91, wherein lipid nanoparticles are administered together with an excipient, an adjuvant, or a combination thereof.

[0351] Embodiment 93. The method according to Embodiment 92, wherein the excipient comprises a solvent, dispersion medium, diluent, dispersion, suspension aid, surfactant, isotonic agent, thickener or emulsifier, preservative, polymer, peptide, protein, cell, hyaluronidase, or a mixture thereof.

[0352] Embodiment 94. The method according to Embodiment 92 or Embodiment 93, wherein the adjuvant comprises C34, Gluco-C34, 7DW8-5, C17, C23, C30, α-galactosylceramide, aluminum salt, squalene, MF59, or QS-21. Other examples of adjuvants in some vaccines that can be used in the compositions of the present disclosure are aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), mixed aluminum salts, Freund's complete adjuvant, Freund's incomplete adjuvant, AS03 (GlaxoSmithKline), MF59 (Seqirus), and CpG1018 (Dynavax), or combinations thereof.

[0353] Embodiment 95. A method for preventing or treating a disease in a subject, comprising administering an effective amount of lipid nanoparticles described in any one of Embodiments 47 to 54 to the subject, wherein the payload is a nucleic acid, a compound, a polypeptide, a protein, a glycan, or a combination thereof, the payload is encapsulated within the lipid nanoparticles, the payload is a therapeutic agent, or induces a therapeutic agent.

[0354] Embodiment 96. The method according to Embodiment 95, wherein the nucleic acid is RNA or DNA.

[0355] Embodiment 97. The method according to Embodiment 96, wherein the payload encodes a polypeptide.

[0356] Embodiment 98. The method according to any one of Embodiments 95 to 97, wherein the payload is immunogenic, or the elude is a nucleic acid configured to encode an immunogenic polypeptide or protein.

[0357] Embodiment 99. The method according to any one of Embodiments 95 to 98, wherein the payload is a first payload, the membrane further encapsulates a second payload, and the second payload is a nucleic acid, a compound, a polypeptide, a protein, a glycan, or a combination thereof.

[0358] Embodiment 100. The method according to Embodiment 99, wherein the first payload and the second payload are different.

[0359] Embodiment 101. The method according to any one of Embodiments 95 to 100, wherein lipid nanoparticles are administered together with an excipient, an adjuvant, or a combination thereof.

[0360] Embodiment 102. The method according to Embodiment 101, wherein the excipient comprises a solvent, dispersion medium, diluent, dispersion, suspension aid, surfactant, isotonic agent, thickener or emulsifier, preservative, polymer, peptide, protein, cell, hyaluronidase, or a mixture thereof.

[0361] Embodiment 103. The method according to Embodiment 101 or Embodiment 102, wherein the adjuvant comprises C34, Gluco-C34, 7DW8-5, C17, C23, C30, α-galactosylceramide, aluminum salt, squalene, MF59, or QS-21. Other examples of adjuvants in several vaccines that can be used in the compositions of the present disclosure are aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), mixed aluminum salts, Freund's complete adjuvant, Freund's incomplete adjuvant, AS03 (GlaxoSmithKline), MF59 (Seqirus), and CpG1018 (Dynavax), or combinations thereof.

[0362] Embodiment 104. The method according to any one of Embodiments 95 to 103, wherein lipid nanoparticles are administered in an initial dose, followed by one, two, three, four, five or more booster doses.

[0363] Embodiment 105. The method according to Embodiment 104, wherein the booster dose is administered approximately one month, two months, three months, four months, five months, six months, or thereafter, following the initial dose.

[0364] Embodiment 106. The method according to any one of Embodiments 95 to 105, wherein the effective amount is in the range of approximately 5 μg to 1000 μg.

[0365] Embodiment 107. A method for boosting an adaptive immune response, comprising administering an effective amount of lipid nanoparticles described in any one of Embodiments 47 to 54 to a target, wherein the payload is a nucleic acid, a compound, a polypeptide, a protein, a glycan or a combination thereof, the payload is encapsulated within lipid nanoparticles, the payload is immunogenic, or induces an immunogenic biomolecule.

[0366] Embodiment 108. The method according to Embodiment 107, wherein the nucleic acid is RNA or DNA.

[0367] Embodiment 109. The method according to Embodiment 108, wherein the payload encodes a polypeptide.

[0368] Embodiment 110. The method according to any one of Embodiments 107 to 109, wherein the payload is a first payload, the membrane further encapsulates a second payload, and the second payload is a nucleic acid, a compound, a polypeptide, a protein, a glycan, or a combination thereof.

[0369] Embodiment 111. The method according to Embodiment 110, wherein the first payload and the second payload are different.

[0370] Embodiment 112. The method according to any one of Embodiments 107 to 111, wherein lipid nanoparticles are administered together with an excipient, an adjuvant, or a combination thereof.

[0371] Embodiment 113. The method according to Embodiment 112, wherein the excipient comprises a solvent, dispersion medium, diluent, dispersion, suspension aid, surfactant, isotonic agent, thickener or emulsifier, preservative, polymer, peptide, protein, cell, hyaluronidase, or a mixture thereof.

[0372] Embodiment 114. The method according to Embodiment 112 or Embodiment 113, wherein the adjuvant comprises C34, Gluco-C34, 7DW8-5, C17, C23, C30, α-galactosylceramide, aluminum salt, squalene, MF59, or QS-21. Other examples of adjuvants in several vaccines that can be used in the compositions of the present disclosure are aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), mixed aluminum salts, Freund's complete adjuvant, Freund's incomplete adjuvant, AS03 (GlaxoSmithKline), MF59 (Seqirus), and CpG1018 (Dynavax), or combinations thereof.

[0373] Embodiment 115. The method according to any one of Embodiments 107 to 114, wherein lipid nanoparticles are administered in an initial dose, followed by one, two, three, four, five or more booster doses.

[0374] Embodiment 116. The method according to Embodiment 115, wherein the booster dose is administered approximately one month, two months, three months, four months, five months, or six months after the initial dose, or thereafter.

[0375] Embodiment 117. The method according to any one of Embodiments 107 to 116, wherein the effective amount is in the range of approximately 5 μg to 1000 μg.

Claims

1. A compound for forming lipid nanoparticles, wherein the component is of formula: 【Chemistry 1】 (In the formula, R １ It contains substituted or unsubstituted glycosyl groups, X １ and X ２ These are hydrogen and C, respectively, independently. １～３０ Alkyl, C １～３０ Alkenil, C １～３０ Alkynyl, aryl, aryloxy or their substituted derivatives, -(CH ２ )nX ４ where n is from 0 to 30, and X ４ is hydrogen, aryl, aryloxy, a heterocyclic group or a substituent thereof, provided that when X ４ is a heterocyclic group, the heterocyclic group contains 1 to 3 heteroatoms selected from the group consisting of O, S and N or a combination thereof. X ３ is hydrogen, C １～６ A compound containing alkyl or hydroxyl.

2. R １ However, formula R ２ -R Ａ - (wherein, R ２ R is a substituted or unsubstituted glycosyl group, Ａ The compound according to claim 1, wherein the bonded group is an aryl, alkyl, amide, alkylamide, substituted thereof, combination thereof, or covalent bond.

3. R Ａ It contains an aryl having 1 to 3 substituents, and the substituent is C １～６ Alkyl, halide or C １～６ The compound according to claim 2, which is an alkyl halogen.

4. R Ａ However, 2 to 72 (OCH ２ CH ２ The compound according to claim 3, further comprising a polyethylene glycol (PEG) portion having a subunit.

5. The compound according to claim 1, wherein the glycosyl group comprises a mannoside, a fucoside, or a combination thereof.

6. The compound according to claim 1, wherein the glycosyl group comprises a monomannoside, dimannoside, or trimannoside.

7. R １ The substituent is a substituted glycosyl group, and the glycosyl group contains 1 to 6 substituents, and the substituent is C １～６ Alkyl, C １～６ Alkenyl, halogen, C １～６ Alkyl halogens, C １～６ Alkoxy, amine, nitro, C １～６ The compound according to claim 1, which is an alkylamine, amide, azide, aryl, cycloalkyl, heterocycloalkyl, sulfite ester group, or a substituted thereof, or a combination thereof.

8. R １ but, 【Chemistry 2】 【change】 A compound according to claim 1, selected from the group consisting of the following.

9. The compound according to claim 1, which is a compound of formula 1.

10. Formula 3: 【Transformation 3】 It is a compound of R １ but, 【Chemistry 4】 A compound according to claim 1, selected from the group consisting of the following.

11. X １ and X ２ The compound according to claim 1, wherein at least one of the members comprises a saturated hydrocarbon chain containing at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, or 30 carbon atoms.

12. X １ and X ２ However, each independently, hydrogen and C ４～３０ Alkyl, C ４～３０ Alkenil, C ４～３０ Alkynyl, aryl, aryloxy or their substituted derivatives, - (CH ２ )nX ４ And n is between 4 and 30, X ４ X is hydrogen, aryl, aryloxy, heterocyclic group, or a substituted product thereof, however X ４ The compound according to claim 1, wherein, if the heterocyclic group is a heterocyclic group, the heterocyclic group comprises one to three heteroatoms or a combination thereof selected from the group consisting of O, S, and N.

13. X ４ X is an aryl, aryloxy, heterocyclic group, cycloalkyl, heterocycloalkyl, or a combination thereof. ４ C １～６ Alkyl, halogen, C １～６ Alkyl halogens and C １～６ The compound according to claim 1, comprising 0 to 6 substituents selected from the group consisting of alkoxys.

14. X ４ ga-R ３ -O-R ４ And R ３ and R ４ These are, independently, aryl, heterocyclic, cycloalkyl, and heterocycloalkyl, and each is C １～６ Alkyl, halogen, C １～６ Alkyl halogens and C １～６ The compound according to claim 13, comprising 0 to 6 substituents selected from the group consisting of alkoxys.

15. X ４ but, 【Transformation 5】 A compound according to claim 1, selected from the group consisting of the following. 【Request Item 16】 【Chemistry 6】 【change】 【change】 A compound according to claim 1, selected from the group consisting of the following.

17. Lipid nanoparticles comprising a membrane defining an internal space, wherein the membrane is formed of a plurality of lipid components comprising a compound according to any one of claims 1 to 16.

18. Lipid nanoparticles according to claim 17, wherein the plurality of lipid components further comprise ionizable lipids, helper lipids, or combinations thereof, and the helper lipids comprise phosphatidylcholine, cholesterol or derivatives thereof, polyethylene glycol-lipids (PEG-lipids), or mixtures thereof.

19. The lipid nanoparticles according to claim 17, wherein the membrane is formed via hydrophobic interactions between a plurality of lipid components.

20. Lipid nanoparticles according to claim 19, wherein the ionizable lipid comprises heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (SM-102™), (4-hydroxybutyl)azandiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315™, Pfizer) or a combination thereof.

21. The lipid nanoparticle according to claim 17, wherein the membrane encapsulates a payload, and the payload is a nucleic acid, a compound, a polypeptide, a protein, a glycan, or a combination thereof.

22. The lipid nanoparticle according to claim 21, wherein the nucleic acid is RNA or DNA encoding a polypeptide.

23. The lipid nanoparticle according to claim 21, wherein the payload is immunogenic, or the payload is a nucleic acid configured to encode an immunogenic polypeptide or protein.

24. Lipid nanoparticles according to claim 21, wherein the payload is a first payload and the membrane further encapsulates a second payload.

25. A formulation comprising lipid nanoparticles as described in claim 17.

26. The formulation according to claim 25, comprising 0.01 to 95% (w / w) of lipid nanoparticles.

27. The formulation according to claim 25, wherein the lipid nanoparticles are first lipid nanoparticles, and the composition further comprises second lipid nanoparticles.

28. The formulation according to claim 25, further comprising an excipient, an adjuvant, or a combination thereof.

29. The formulation according to claim 28, wherein the excipient comprises a solvent, dispersion medium, diluent, dispersion, suspension aid, surfactant, isotonic agent, thickener or emulsifier, preservative, polymer, peptide, protein, cell, hyaluronidase, or a mixture thereof.

30. The formulation according to claim 28, wherein the adjuvant comprises C34, Gluco-C34, 7DW8-5, C17, C23, C30, α-galactosylceramide, aluminum salt, squalene, MF59, or QS-21. Other examples of adjuvants in some vaccines that may be used in the compositions of the present disclosure include aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), mixed aluminum salts, Freund's complete adjuvant, Freund's incomplete adjuvant, AS03 (GlaxoSmithKline), MF59 (Sequirus), and CpG1018 (Dynavax), or combinations thereof.

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