A compound comprising sterol and / or a derivative(s) thereof for preparing lipid nanoparticles encapsulating an agent, nanoparticle composition comprising said compound and related methods thereof

Abstract

There is provided a compound comprising a structure represented by general formula (1) wherein A comprises a sterol and / or a derivative(s) thereof; B comprises: (i) a carbohydrate and / or a derivative(s) thereof; or (ii) an oligopeptide or a polypeptide comprising carbohydrate and / or a derivative(s) thereof; R1 and R3 are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; R2 is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene; R4 is –H or –C(=O)R, where R is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; p = 0 or 1; and q = 0 or 1.

Description

[0001] A COMPOUND COMPRISING STEROL AND / OR A DERIVATIVE(S) THEREOF FOR PREPARING LIPID NANOPARTICLES ENCAPSULATING AN AGENT, NANOPARTICLE COMPOSITION COMPRISING SAID COMPOUND AND RELATED METHODS THEREOF

[0002] TECHNICAL FIELD

[0003] The present disclosure relates broadly to a compound comprising sterol and / or a derivative(s) thereof for preparing lipid nanoparticles encapsulating an agent and a method of preparing said compound. The present disclosure also relates to a nanoparticle composition comprising said compound and related methods and uses.

[0004] BACKGROUND

[0005] Lipid nanoparticles (LNPs) are widely used for the delivery of therapeutic, prophylactic and / or biological agents. For example, lipid nanoparticles have been successfully applied in mRNA-based vaccines such as Pfizer-BioNTech’s COVID-19 vaccine as lipid nanoparticles (LNPs)-mRNA vaccines. These formulations typically contain polyethylene glycol (PEG) lipids to maintain colloidal stability of LNPs. However, a safe, stable and efficacious delivery system remains a challenge. Particularly, there have been reports of adverse health effects and cytotoxicity associated with the use of lipid nanoparticles for delivery.

[0006] Although PEGylated lipids play important roles in controlling the size distribution, biodistribution profile and storage stability of LNPs, there are concerns raised regarding their long-term safety, which stems from their poor biodegradability, and propensity to induce an anti-PEG immune response. Specifically, the PEG-specific antibodies can be induced or enhanced by PEG-based compounds, leading to accelerated clearance of systemically delivered PEGylated mRNA-LNPs and limiting their efficacy. Furthermore, polyethylene glycol (PEG) has been reported to be a high-risk allergen found hidden in drug / food items. Binding of PEG to basophils through IgE can cause release of compounds that induce allergies and individuals may develop anaphylactic conditions from PEG present in medications. PEG has also been identified as the cause of accelerated blood clearance (ABC) phenomenon. As a result, there is a need to provide PEG-free lipid nanoparticles to overcome the anti-PEG immune response from PEGylated lipids.

[0007] Sterol (e.g., cholesterol), and sterol transport proteins (e.g., cholesterol transport proteins) play important roles in the cytosolic delivery of viruses and in the endocytic retention and recycling of nanoparticles. For example, cholesterol has been demonstrated to increase the fluidity of lipid membranes, while promoting / inducing order within / to the hydrophobic lipid bilayer. The inclusion of cholesterol into nanoparticle formulations can promote endosomal escape and improve transfection efficiency, potentially through enhanced membrane fusion. Recent studies have shown that cholesterol analogues can regulate the structural properties of nanoparticles and induce the subcellular interactions, thereby enhancing the cytosolic delivery. Despite cholesterol emerging as a promising candidate due to its functional properties and beneficial effects, its practical application in lipid nanoparticles remains unexplored and unreported.

[0008] In view of the above, there is a need to address or at least ameliorate the above-mentioned problems. In particular, there is a need to provide a compound and / or nanoparticle composition for a cost efficient, substantially safe and stable, and / or efficacious delivery of therapeutic, prophylactic and / or biological agents. SUMMARY

[0009] In one aspect, there is provided a compound comprising a structure represented by general formula (1 ):

[0010] R1[R3]

[0011]

[0012] wherein

[0013] A comprises a sterol and / or a derivative(s) thereof;

[0014] B comprises:

[0015] (i) a carbohydrate and / or a derivative(s) thereof; or

[0016] (ii) an oligopeptide or a polypeptide comprising carbohydrate and / or a derivative(s) thereof;

[0017] R1and R3are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0018] R2is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;

[0019] R4is -H or -C(=O)R, where R is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0020] p = 0 or 1; and

[0021] q = 0 or 1.

[0022] In one embodiment, the sterol is selected from animal sterols, plant sterols, fungal sterols and combinations thereof. In one embodiment, A comprises a structure that is represented by general formula (2):

[0023]

[0024] (2) wherein

[0025] ring C1, C2, C3and C4each optionally contains one, two or three C=C bond(s); Ra, Ra’, Rb, Rb’, Rc, Rd, Rd’, Re, Rf, Rf’, R9, R9’, Rh, R', R Rj’, Rk, Rk, Rl, Rm, Rn, Rn’, R°, R0’, Rpand Rqare each optionally present and independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and

[0026] R' is a hydrophobic group, or contains at least linear aliphatic, branched aliphatic and / or cyclic hydrocarbons.

[0027] In one embodiment, A comprises a structure that is represented by general formula (2A):

[0028]

[0029] (2A)

[0030] wherein R' is a linear, branched, saturated and / or unsaturated hydrocarbon group. In one embodiment, B comprises a structure that is represented by general formula (3A) and / or (3B):

[0031] R5-i

[0032]

[0033] (3A) (3B)

[0034] wherein

[0035] B1comprises carbohydrate and / or a derivative(s) thereof;

[0036] B2comprises carbohydrate and / or a derivative(s) thereof;

[0037] R5is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0038] R6is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene; and

[0039] n > 1.

[0040] In one embodiment, the structure represented by general formula (1) comprises a structure that is represented by general formula (4):

[0041]

[0042] (4)

[0043] wherein

[0044] R2ais optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;

[0045] R2bis optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;

[0046] x = 0 or 1; y = 0 or 1; and

[0047] total sum of x + y = 1.

[0048] In one embodiment, the structure represented by general formula (4) comprises a structure that is represented by general formula (4A) and / or (4B):

[0049]

[0050] (4A) (4B)

[0051] In one embodiment, the carbohydrate and / or a derivative(s) thereof (in B1and / or B2) is selected from the group consisting of monosaccharide, disaccharide, oligosaccharide, polysaccharide and / or a derivative(s) thereof.

[0052] In one embodiment, the compound is substantially devoid of polyethylene glycol (PEG).

[0053] In one embodiment, B1and / or B2comprise a structure that is represented by general formula (5) having a 6-membered ring structure:

[0054] X9

[0055]

[0056] (5)

[0057] wherein

[0058] M is -O- or -S-;

[0059] X1to X7and X9to X10are each independently selected from -H, -OH, or -O-C(=O)R7, where R7is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and

[0060] X8is alkylene.

[0061] In one embodiment, the compound comprises a structure selected from one or more of the following:

[0062] cholesterol-mannose-OAc (CMO)

[0063]

[0064] cholesterol-mannose (CM),

[0065]

[0066] In another aspect, there is provided a method of preparing a compound as disclosed herein, the method comprising:

[0067] (a-i) reacting a protected alkanolamine compound represented by general formula (6) with a compound comprising B1represented by general formula (7) in the presence of a Lewis acid to obtain a first intermediate compound represented by general formula (8):

[0068] R1R1HO

[0069]

[0070] R2PG1B1—R8R2PG1(6) (7) (8) wherein R8is -H, -OH or -O-C(=O)R9, where R9is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0071] PG1is a protecting group selected from N-carboxybenzyl or benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (BOC), tert-butyl, 9- Fluorenylmethoxycarbonyl (Fmoc), 2-(4- Nitrophenylsulfonyl)ethoxycarbonyl (Nsc), 2-Fluoro-Fmoc (Fmoc(2F)), 2- Monoisooctyl-Fmoc (mio-Fmoc), 2,7-Diisooctyl-Fmoc (dio-Fmoc), or combinations thereof;

[0072] B1comprises a carbohydrate and / or a derivative(s) thereof;

[0073] R1is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0074] R2is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;

[0075] (a-ii) deprotecting the first intermediate compound represented by general formula (8) to obtain a second intermediate compound represented by general formula (9):

[0076] R1

[0077] B1

[0078] R2

[0079]

[0080] (9)

[0081] (a-iii) reacting the second intermediate compound represented by general formula (9) with a haloformate compound comprising A represented by general formula (21 ) to obtain a compound represented by general formula (4A):

[0082] R1

[0083] O

[0084] 0

[0085]

[0086] (21) (4A) wherein

[0087] X1is a halide; and

[0088] A comprises a sterol and / or a derivative(s) thereof.

[0089] In one embodiment, the method further comprises, prior to (a-i):

[0090] (b-i) reacting an alkanolamine compound represented by general formula (10) with a compound comprising PG1represented by general formula (11) in the presence of a Lewis base to obtain a protected alkanolamine compound represented by general formula (6):

[0091] R1HO.

[0092]

[0093] X — PG1R2PG1(10) (11) (6) wherein

[0094] R1is H; and

[0095] X is a halide.

[0096] In another aspect, there is provided a method of preparing a compound as disclosed herein, the method comprising:

[0097] (c-i) polymerizing one or more N-carboxyanhydride (NCA) monomers represented by general formula (12) with an initiator represented by general formula (13) to obtain a first intermediate compound represented by general formula (14):

[0098]

[0099] (12) (13)

[0100]

[0101] (14)

[0102] wherein

[0103] A comprises a sterol and / or a derivative(s) thereof;

[0104] B2comprises a carbohydrate and / or a derivative(s) thereof;

[0105] R1and R3are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0106] R2is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;

[0107] R4is -H or -C(=O)R, where R is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0108] R5is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0109] R6is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene; and

[0110] n ≥ 1;

[0111] (c-ii) reacting the first intermediate compound represented by general formula (14) with an acylating agent represented by general formula (15) to obtain a second intermediate compound represented by general formula (16):

[0112]

[0113] (15) (16) wherein R and R’ are each independently optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; and

[0114] (c-iii) deprotecting the second intermediate compound represented by general formula (16) to obtain a compound represented by general formula (4B).

[0115] In one embodiment, the method further comprises, prior to (c-i):

[0116] (d-i) reacting a protected amino acid represented by general formula (17) with a compound comprising B2represented by general formula (18) in the presence of a Lewis acid to obtain a first intermediate compound represented by general formula (19):

[0117] B2— R10

[0118]

[0119] (17) (18)

[0120] wherein

[0121] R10is -H, -OH or -O-C(=O)R11, where R11is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0122] PG2is a protecting group selected from N-carboxybenzyl or benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (BOC), tert-butyl, 9- Fluorenylmethoxycarbonyl (Fmoc), 2-(4- Nitrophenylsulfonyl)ethoxycarbonyl (Nsc), 2-Fluoro-Fmoc (Fmoc(2F)), 2- Monoisooctyl-Fmoc (mio-Fmoc), 2,7-Diisooctyl-Fmoc (dio-Fmoc), or combinations thereof;

[0123] (d-ii) deprotecting the first intermediate compound represented by general formula (19) to obtain a second intermediate compound represented by general formula (20): H H

[0124] N

[0125] O

[0126]

[0127] (20)

[0128] (d-iii) reacting the second intermediate compound represented by general formula (20) with a carbonylating agent to obtain the N-carboxyanhydride (NCA) monomers represented by general formula (12).

[0129] In another aspect, there is provided a nanoparticle composition for delivery of a therapeutic agent, prophylactic agent and / or biological agent, the nanoparticle composition comprising:

[0130] a compound as disclosed herein; and

[0131] a therapeutic agent, prophylactic agent and / or biological agent.

[0132] In one embodiment, the composition further comprises:

[0133] (i) ionizable lipid;

[0134] (ii) helper lipid;

[0135] (iii) optionally sterol; and

[0136] (iv) optionally amphiphilic lipid.

[0137] In one embodiment, the ionizable lipid, helper lipid, sterol, compound as disclosed herein, and amphiphilic lipid are mixed at a mole ratio of 25 - 75: 1 -20: 0 -60: 0.1 - 65: 0 -5.

[0138] In one embodiment, the ionizable lipid is selected from ALC-0315, SM-102, Lipid 5, DLinDMA, D-Lin-MC2-DMA, DLin-MC3-DMA, D-Lin-MC4-DMA, Dlin-KC2-DMA, YSK05, AA3-Dlin, SSPalmM, SSPalmO-Phe, Lipid A9, L319, DODMA, CL1, BP Lipid 310, ATX-001, ATX-100, Lipid 2, 80-016B, BP Lipid 309, BP Lipid 307, 93-O17S, 93-0170, NT1-O14B, 306-O12B-3, 306-O12B, 113- O16B, 3060110, 306Oi9-cis2, BAMEA-O16B, AI-28, 113-O12B, 98N12-5, Ckk-E12, OF-02, C12-200, BP Lipid 311, BP Lipid 308, BP Lipid 314, BP Lipid 312, LP01, TCL053, Lipid C24, BP Lipid 315, Lipid 29, 9A1P9, C13-112-tri-tail, C13-113-tri-tail, C13-112-tetra-tail, C13-113-tetra-tail, or combinations thereof.

[0139] In one embodiment, the helper lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1 -oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1.2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolam ine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1.2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1 -glycerol) sodium salt (DOPG), sphingomyelin, or combinations thereof.

[0140] In one embodiment, the sterol is present and is selected from cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, avenasterol, or combinations thereof.

[0141] In one embodiment, the amphiphilic lipid is present and is selected from PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, 2-[(polyethylene glycol)-2000]- N, N-ditetradecylacetamide (ALC-0159), R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DOMG), 3-N-[(ω-methoxypoly (ethyleneglycol)2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-S-DMG), PEG-DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-methoxy] (sodium salt)), PEG-DPPC, PEG-DSPE lipid, or combinations thereof.

[0142] In one embodiment, the nanoparticle composition comprises:

[0143] nanoparticles having a N / P ratio from 1:1 to 40:1; and / or nanoparticles having an average particle size of no more than 500 nm; and / or

[0144] nanoparticles having a zeta potential of from -30 mV to +30 mV.

[0145] In another aspect, there is provided a nanoparticle composition as disclosed herein for use in medicine.

[0146] In another aspect, there is provided a nanoparticle composition as disclosed herein for use in modulating an immune response in a subject, wherein said nanoparticle composition is to be administered to the subject.

[0147] In another aspect, there is provided use of a nanoparticle composition as disclosed herein in the manufacture of a medicament for modulating an immune response in a subject.

[0148] In another aspect, there is provided a method of modulating an immune response in a subject, the method comprising administering to a subject a therapeutically effective amount of the nanoparticle composition as disclosed herein. DEFINITIONS

[0149] The term “particle” as used herein broadly refers to a discrete entity or a discrete body. The particle described herein can include an organic, an inorganic, a composite particle or a biological particle. The particle used described herein may also be a macro-particle that is formed by an aggregate of a plurality of subparticles or a fragment of a small object. The particle of the present disclosure may be spherical, substantially spherical, or non-spherical, such as irregularly shaped particles or ellipsoidally shaped particles. The term “size” when used to refer to the particle broadly refers to the largest dimension of the particle. For example, the term “size” when used in the context of nanoparticle can refer to the diameter of the nanoparticle although it is not limited as such. In various embodiments, when the particle is substantially spherical, the term “size” can refer to the diameter of the particle; or when the particle is substantially non-spherical, the term “size” can refer to the largest length of the particle.

[0150] The term "nano" as used herein is to be interpreted broadly to include dimensions in a nanoscale, / '.e., less than about 1000 nm, about 1 nm to less than about 1000 nm, about 1 nm to about 900 nm, about 1 nm to about 800 nm, about 1 nm to about 700 nm, about 1 nm to about 600 nm, about 1 nm to about 500 nm, about 1 nm to about 400 nm, about 1 nm to about 300 nm, about 1 nm to about 200 nm, or from about 1 nm to about 100 nm. Accordingly, the term “nanostructures”, “nanoparticles”, “nanomaterials” and the like as used herein may include structures that have at least one dimension in the range of no more than said range. The term “nanostructures”, “nanoparticles”, “nanomaterials” and the like as used herein may include structures that have at least one dimension that is no more than about 1,000 nm, no more than about 950 nm, no more than about 900 nm, no more than about 850 nm, no more than about 800 nm, no more than about 750 nm, no more than about 700 nm, no more than about 650 nm, no more than about 600 nm, no more than about 550 nm, no more than about 500 nm, no more than about 450 nm, no more than about 400 nm, no more than about 350 nm, no more than about 300 nm, no more than about 250 nm, no more than about 200 nm, no more than about 150 nm, no more about 100 nm, no more than about 90 nm, no more than about 80 nm, no more than about 70 nm, no more than about 60 nm, no more than about 50 nm, no more than about 40 nm, no more than about 30 nm, no more than about 20 nm, or no more than about 10 nm.

[0151] The term "micro" as used herein is to be interpreted broadly to include dimensions from about 1 micron to about 1000 microns, from about 1 micron to less than about 1000 microns, from about 1 micron to about 900 microns, from about 1 micron to about 800 microns, from about 1 micron to about 700 microns, from about 1 micron to about 600 microns, from about 1 micron to about 500 microns, from about 1 micron to about 400 microns, from about 1 micron to about 300 microns, from about 1 micron to about 200 microns, from about 1 micron to about 100 microns, or from about 1 micron to about 5 microns.

[0152] The term “treatment", "treat" and “therapy”, and synonyms thereof as used herein refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) a medical condition, which includes but is not limited to diseases, symptoms and disorders. A medical condition also includes a body’s response to a disease or disorder, e.g., inflammation. Those in need of such treatment include those already with a medical condition as well as those prone to getting the medical condition or those in whom a medical condition is to be prevented.

[0153] As used herein, the term "therapeutically effective amount" of a compound is intended to refer to an amount that is sufficient or capable of preventing or at least slowing down (lessening) a medical condition, such as infectious diseases (e.g., dengue disease caused by dengue virus), respiratory illnesses (e g., coronavirus caused by the SARS-CoV-2 virus or flu caused by influenza virus), cancer, autoimmune diseases and cardiovascular diseases etc. Dosages and administration of compounds, compositions and formulations of the present disclosure may be determined by one of ordinary skill in the art of clinical pharmacology or pharmacokinetics. An effective amount of the active agent of the present disclosure to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the patient. Accordingly, it may be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect.

[0154] The term “subject" is intended to broadly refer to any animal, such as a mammal, and including humans. Exemplary subjects include but are not limited to humans and non-human primates. The term “subject" as used herein also includes patients and non-patients. The term “patient” refers to individuals suffering or are likely to suffer from a medical condition such as infectious diseases (e.g., coronavirus caused by the SARS-CoV-2 virus, dengue disease caused by dengue virus etc), while “non-patients” refer to individuals not suffering and are likely to not suffer from the medical condition. “Non-patients” include healthy individuals, non-diseased individuals and / or an individual free from the medical condition. As used herein, the term "mammal" includes vertebrate such as a human or a large veterinary mammal (e g., horses, cattle, deer, sheep, llamas, goats, pigs).

[0155] The term "bond" refers to a linkage between atoms in a compound or molecule. The bond may be a single bond, a double bond, or a triple bond.

[0156] In the definitions of a number of substituents below, it is stated that “the group may be a terminal group or a bridging group”. This is intended to signify that the use of the term is intended to encompass the situation where the group is a terminal group / moiety as well as the situation where the group is a linker between two other portions of the molecule. Using the term “alkyl” having 1 carbon atom as an example, it will be appreciated that when existing as a terminal group, the term “alkyl” having 1 carbon atom may mean -CHs and when existing as a bridging group, the term “alkyl” having 1 carbon atom may mean -CH2-or the like. The term "alkyl" as a group or part of a group refers to a straight or branched aliphatic hydrocarbon group having 1 to 20 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. Examples of suitable straight and branched alkyl substituents include methyl, ethyl, n-propyl, 2-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, hexyl, amyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1 -methylpentyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 1,2,2-trimethylpropyl, 1, 1,2-trimethylpropyl, 2-ethylpentyl, 3-ethylpentyl, heptyl, 1 -methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl, 1,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,2,3-trimethylbutyl, 1,1,2-trimethylbutyl, 1,1,3-trimethylbutyl, 5-methylheptyl, 1 -methylheptyl, octyl, nonyl, decyl and the like. The group may be a terminal group or a bridging group.

[0157] The term "alkenyl" as a group or part of a group denotes an aliphatic hydrocarbon group containing at least one carbon-carbon double bond and which may be straight or branched having 2 to 20 carbon atoms, 2 to 10 carbon atoms, 2 to 6 carbon atoms, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms in the chain. The group may contain a plurality of double bonds and the orientation about each double bond is independently E or Z. Exemplary alkenyl groups include, but are not limited to, ethenyl, vinyl, allyl, 1-methylvinyl, 1 -propenyl, 2-propenyl, 2-methyl-1 -propenyl, 2-methyl-1 -propenyl, 1-butenyl, 2-butenyl, 3-butentyl, 1,3-butadienyl, 1 -pentenyl, 2-pententyl, 3-pentenyl, 4-pentenyl, 1,3-pentadienyl, 2,4-pentadienyl, 1,4-pentadienyl, 3-methyl-2-butenyl, 1 -hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 2-methylpentenyl, 1 -heptenyl, 2-heptentyl, 3-heptenyl, 1 -octenyl, 2-octenyl, 3-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1 -decenyl, 2-decenyl, 3-decenyl and the like. The group may be a terminal group or a bridging group. The term "alkynyl" as a group or part of a group denotes an aliphatic hydrocarbon group containing at least one carbon-carbon triple bond and which may be straight or branched having 2 to 20 carbon atoms, 2 to 10 carbon atoms, 2 to 6 carbon atoms, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms in the chain. The group may contain a plurality of triple bonds. Exemplary alkynyl groups include, but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1 -pentynyl, 2-pentynyl, 3-methyl-1-butynyl, 4-pentynyl, 1 -hexynyl, 2-hexynyl, 5-hexynyl, 1 -heptynyl, 2-heptynyl, 6-heptynyl, 1-octynyl, 2-octynyl, 7-octynyl, 1-nonynyl, 2-nonynyl, 8-nonynyl, 1 -decynyl, 2-decynyl, 9-decynyl and the like. The group may be a terminal group or a bridging group.

[0158] The term “cyclic” as used herein broadly refers to a structure where one or more series of atoms are connected to form at least one ring. The term includes, but is not limited to, both saturated and unsaturated 5-membered and saturated and unsaturated 6-membered rings. Examples of groups having a cyclic structure include, but are not limited to, cyclopentane, cyclopentene, cyclohexane, cyclohexene, benzene and the like. The term “cyclic” as used herein includes “heterocyclic”.

[0159] The term “heterocyclic” as used herein broadly refers to a structure where two or more different kinds of atoms are connected to form at least one ring. For example, a heterocyclic ring may be formed by carbon atoms and at least another atom (i.e. heteroatom) selected from oxygen (O), nitrogen (N) or (NR) and sulfur (S), where R is independently a hydrogen or an organic group. The term also includes, but is not limited to, saturated and unsaturated 5-membered, and saturated and unsaturated 6-membered rings. Examples of groups having a heterocyclic structure include, but are not limited to furan, thiophene, 1 H-pyrrole, 2H-pyrrole, 1 -pyrroline, 2-pyrroline, 3-pyrroline, 1-pyrazoline, 2-pyrazoline, 3-pyrazoline, 2-imidazoline, 3-imidazoline, 4-imidazoline, pyrazole, imidazole, oxazole, isoxazole, thiazole, isothiazole, 1,2,3-triazole, 1,2,4-triazole, 1,2,3-oxadiazole, disubstituted 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, 1.2.3-thiadiazole, 1,2,4-thiadiazole, 1,2,5-thiadiazole, 1,3,4-thiadiazole, tetrahydrofuran, tetrahydrothiophene, pyrrolidine, 1,3-dioxolane, 1,2-oxathiolane, 1.3-oxathiolane, pyrazolidine, imidazolidine, pyridine, pyridazine, pyrimidine, pyrazine, 1,2-oxazine, 1,3-oxazine, 1,4-oxazine, thiazine, 1,2,3-triazine, 1,2,4-triazine, 1,3,5-triazine, 2H-pyran, 4H-pyran, 2-pyrone, 4-pyrone, 1,4-dioxin, 2H-thiopyran, 4H-thiopyran, tetrahydropyran, thiane, piperidine, 1,4-dioxane, 1,2-dithiane, 1,3-dithiane, 1,4-dithiane, 1,3,5-trithiane, piperazine, morpholine, thiomorpholine and the like.

[0160] The term "amine group" or the like is intended to broadly refer to a group containing –NR2, where R is independently a hydrogen or an organic group. The group may be a terminal group or a bridging group.

[0161] The term "amide group" or the like is intended to broadly refer to a group containing –C(=O)NR2, where R is independently a hydrogen or an organic group. The group may be a terminal group or a bridging group.

[0162] The term "aryl" as a group or part of a group denotes (i) an optionally substituted monocyclic, or fused polycyclic, aromatic carbocycle (ring structure having ring atoms that are all carbon) preferably having from 5 to 20, or 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms per ring. Examples of aryl groups include but are not limited to phenyl, tolyl, xylyl, naphthyl, anthracenyl, phenanthrenyl, fluorenyl, indenyl or indanyl and the like.

[0163] The term "heteroaryl" as a group or part of a group refers to groups containing an aromatic ring (preferably a 5- or 6- membered aromatic ring) having one or more carbon atoms (for example 1 to 6 carbon atoms) in the ring replaced by a heteroatom. Suitable heteroatoms may include nitrogen (N) or (NH), oxygen (O) and sulfur (S). Examples of heteroaryl include but are not limited to thiophene, benzothiophene, benzofuran, benzimidazole, benzoxazole, benzothiazole, benzisothiazole, naphtha[2,3-b]thiophene, furan, isoindolizine, xantholene, phenoxatine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, tetrazole, indole, isoindole, 1 H-indazole, purine, quinoline, isoquinoline, phthalazine, naphthyridine, quinoxaline, cinnoline, carbazole, phenantridine, acridine, phenazine, thiazole, isothiazole, phenothiazine, oxazole, isooxazole, furazane, phenoxazine, 2-, 3- or 4-pyridyl, 2-, 3-, 4-, 5-, or 8-quinolyl, 1-, 3-, 4-, or 5-isoquinolinyl 1-, 2-, or 3-indolyl, and 2-, or 3-thienyl and the like. The group may be a terminal group or a bridging group.

[0164] The term "halogen" represents chlorine, fluorine, bromine or iodine. The term "halide" represents chloride, fluoride, bromide or iodide.

[0165] The term “optionally substituted,” when used to describe a chemical structure or moiety, refers to the chemical structure or moiety wherein one or more of its hydrogen atoms is optionally substituted with a chemical moiety or functional group such as alcohol, alkoxy, alkanoyloxy, alkoxycarbonyl, alkenyl, alkyl (e.g., methyl, ethyl, propyl, t-butyl), alkynyl, alkylcarbonyloxy (-OC(O)alkyl), amide (-C(O)NH-alkyl- or -alkylNHC(O)alkyl), amine (such as alkylamino, arylamino, arylalkylamino), aryl, aryloxy, azo, carbamoyl (-NHC(O)O-alkyl- or -OC(O)NH-alkyl), carbamyl (e.g., CONH2, as well as CONH-alkyl, CONH-aryl, and CONH-arylalkyl), carboxyl, carboxylic acid, cyano, ester, ether (e.g., methoxy, ethoxy), halo, haloalkyl (e.g., –CCl3, –CF3, –C(CF3)3), heteroalkyl, isocyanate, isothiocyanate, nitrile, nitro, phosphodiester, sulfide, sulfonamido (e.g., SO2NH2), sulfone, sulfonyl (including alkylsulfonyl, arylsulfonyl and arylalkylsulfonyl), sulfoxide, thiol (e.g., sulfhydryl, thioether) or urea (-NHCONH-alkyl-).

[0166] The terms "coupled" or "connected" as used in this description are intended to cover both directly connected or connected through one or more intermediate means, unless otherwise stated.

[0167] The term "associated with", used herein when referring to two elements refers to a broad relationship between the two elements. The relationship includes, but is not limited to a physical, a chemical or a biological relationship. For example, when element A is associated with element B, elements A and B may be directly or indirectly attached to each other or element A may contain element B or vice versa.

[0168] The term "adjacent" used herein when referring to two elements refers to one element being in close proximity to another element and may be but is not limited to the elements contacting each other or may further include the elements being separated by one or more further elements disposed therebetween.

[0169] The term "and / or", e.g., " X and / or Y" is understood to mean either " X and Y" or " X or Y" and should be taken to provide explicit support for both meanings or for either meaning.

[0170] Further, in the description herein, the word “substantially” whenever used is understood to include, but not restricted to, "entirely" or “completely” and the like. In addition, terms such as "comprising", "comprise", and the like whenever used, are intended to be non-restricting descriptive language in that they broadly include elements / components recited after such terms, in addition to other components not explicitly recited. For example, when “comprising" is used, reference to a “one” feature is also intended to be a reference to “at least one” of that feature. Terms such as “consisting”, “consist”, and the like, may in the appropriate context, be considered as a subset of terms such as "comprising", "comprise", and the like. Therefore, in embodiments disclosed herein using the terms such as "comprising", "comprise", and the like, it will be appreciated that these embodiments provide teaching for corresponding embodiments using terms such as “consisting”, “consist", and the like. Further, terms such as "about", "approximately" and the like whenever used, typically means a reasonable variation, for example a variation of + / - 5% of the disclosed value, or a variance of 4% of the disclosed value, or a variance of 3% of the disclosed value, a variance of 2% of the disclosed value or a variance of 1 % of the disclosed value. Furthermore, in the description herein, certain values may be disclosed in a range. The values showing the end points of a range are intended to illustrate a preferred range. Whenever a range has been described, it is intended that the range covers and teaches all possible sub-ranges as well as individual numerical values within that range. That is, the end points of a range should not be interpreted as inflexible limitations. For example, a description of a range of 1% to 5% is intended to have specifically disclosed sub-ranges 1% to 2%, 1% to 3%, 1% to 4%, 2% to 3% etc., as well as individually, values within that range such as 1%, 2%, 3%, 4% and 5%. It is to be appreciated that the individual numerical values within the range also include integers, fractions and decimals. Furthermore, whenever a range has been described, it is also intended that the range covers and teaches values of up to 2 additional decimal places or significant figures (where appropriate) from the shown numerical end points. For example, a description of a range of 1% to 5% is intended to have specifically disclosed the ranges 1.00% to 5.00% and also 1.0% to 5.0% and all their intermediate values (such as 1.01%, 1.02%... 4.98%, 4.99%, 5.00% and 1.1%, 1.2%... 4.8%, 4.9%, 5.0% etc.,) spanning the ranges. The intention of the above specific disclosure is applicable to any depth / breadth of a range.

[0171] Additionally, when describing some embodiments, the disclosure may have disclosed a method and / or process as a particular sequence of steps. However, unless otherwise required, it will be appreciated that the method or process should not be limited to the particular sequence of steps disclosed. Other sequences of steps may be possible. The particular order of the steps disclosed herein should not be construed as undue limitations. Unless otherwise required, a method and / or process disclosed herein should not be limited to the steps being carried out in the order written. The sequence of steps may be varied and still remain within the scope of the disclosure.

[0172] Furthermore, it will be appreciated that while the present disclosure provides embodiments having one or more of the features / characteristics discussed herein, one or more of these features / characteristics may also be disclaimed in other alternative embodiments and the present disclosure provides support for such disclaimers and these associated alternative embodiments.

[0173] It will also be appreciated that where priority is claimed to an earlier application, the full contents of the earlier application is also taken to form part of the present disclosure and may serve as support for embodiments disclosed herein.

[0174] DESCRIPTION OF EMBODIMENTS

[0175] Exemplary, non-limiting embodiments of a compound for preparing lipid nanoparticles encapsulating an agent, a method of preparing said compound, a nanoparticle composition comprising said compound and related methods / uses thereto are disclosed hereinafter.

[0176] COMPOUND

[0177] There is provided a compound for preparing lipid nanoparticles. In various embodiments, the compound comprises one or more sterol(s) and / or a derivative(s) thereof. For example, the compound may comprise one or more sterol(s) selected from animal sterols (or zoosterols), plant sterols (or phytosterols), fungal sterols (or mycosterols), the like, or combinations thereof. In various embodiments, the compound may comprise one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more sterols and / or a derivative(s) thereof. Advantageously, the presence of sterol(s) and / or a derivative(s) thereof equips the compound with or imparts the ability to promote endosomal escape and enhance transfection efficiency. By regulating / altering the properties of endosomal membrane (e.g., fluidity and curvature) and / or promoting membrane fusion, presence of sterol(s) and / or a derivative(s) thereof modulate subcellular interactions through its effects on the membrane, which in turn enhances cytosolic delivery / transport / release of molecules or cargoes. In various embodiments, the compound comprises one or more sugar / carbohydrate / saccharide unit(s) / group(s) and / or a derivative(s) thereof. For example, the compound may comprise one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more sugar / carbohydrate / saccharide unit(s) / group(s) and / or a derivative(s) thereof. Advantageously, the presence of sugar / carbohydrate / saccharide unit(s) / group(s) and / or a derivative(s) thereof equips the compound with or imparts the ability of targeting sugar / carbohydrate / saccharide receptors found in / on cell surfaces (e g., surfaces of immune cells such as macrophages and dendritic cells, surfaces of fibroblasts and keratinocytes, and liver sinusoidal endothelial cells). Advantageously, in various embodiments therefore, the compound comprises cell-targeting ability, e.g., immune cell-targeting ability. In various embodiments, the sugar / carbohydrate / saccharide and / or a derivative(s) thereof is / are hydrophilic. Advantageously, the presence of sugar / carbohydrate / saccharide and / or a derivative(s) thereof increases the hydrophilicity of the compound, and consequently solubility of the compound.

[0178] In various embodiments, the compound comprises one or more sugar / carbohydrate / saccharide unit(s) / group(s) and / or a derivative(s) thereof that is functionalized with / conjugated with one or more peptide unit / block(s) and / or a derivative(s) thereof. For example, the compound may comprise one or more sugar / carbohydrate / saccharide unit(s) / group(s) and / or a derivative(s) thereof that is functionalized with / conjugated with one or more oligopeptides, polypeptides / poly(amino acids) and / or a derivative(s) thereof. In various embodiments, the total number of peptide units / blocks and / or a derivative(s) thereof (or total length of oligopeptides, polypeptides / poly(amino acids) and / or a derivative(s) thereof) in the compound is adjustable as desired. Advantageously, in various embodiments, the compound is designed / configured to allow the hydrophilicity / hydrophobicity balance of said compound to be customizable by the adjustment of the number of peptide units / blocks and / or a derivative(s) thereof (or length of the oligopeptides, polypeptides / poly(amino acids) and / or a derivative(s) thereof and length of lipid attached to oligopeptides, polypeptides / poly(amino acids) and / or a derivative(s) thereof).

[0179] Even more advantageously, the structure of the compound allows for embodiments of the compound to be used / formulated into nanoparticles in a composition that may be used as an encapsulation / loading agent, delivery vehicle / system and / or transfection vehicle / system. In various embodiments, the design of the compound helps prevent non-specific protein absorption, particle aggregation and controls the size of the nanoparticles formed. In various embodiments, embodiments of the compound help maintain colloidal stability (of the nanoparticles), and facilitate the condensation and encapsulating / loading of molecules / cargoes into the nanoparticle composition. In various embodiments, the compound is designed / configured to allow loading / encapsulation of one or more types of molecules or cargoes. In various embodiments, the compound is also designed / configured to allow the loaded / encapsulated agent to be released from a composition containing said compound and / or subsequently delivered to a desired target (e.g., cell, cytosol, tissue or organ). The molecules / cargoes to be loaded / encapsulated onto / into / within a composition containing the compound may include but is not limited to a therapeutic agent, a prophylactic agent, a biological agent or the like. In various embodiments, the molecules / cargoes to be loaded / encapsulated comprises a nucleic acid. For example, the molecules / cargoes to be loaded / encapsulated may be a nucleic acid selected from ribonucleic acid (RNA), messenger ribonucleic acid (mRNA), microRNA (miRNA), small interfering ribonucleic acid (siRNA), deoxyribonucleic acid (DNA), plasmid deoxyribonucleic acid (pDNA), oligonucleotides such as antisense oligonucleotide (ASO) or the like or combinations thereof. In various embodiments, the molecules / cargoes to be loaded / encapsulated comprises therapeutics, e g., negatively-charged therapeutics. For example, the molecules / cargoes to be loaded / encapsulated may be therapeutics selected from drug molecule, vaccine (e.g., dengue vaccine, Covid-19 vaccine), or the like or combinations thereof. Advantageously, the compound is suitable for use in formulating into nanoparticles for encapsulating and / or delivering one or more therapeutic agent, prophylactic agent and / or biological agent to a desired target (e.g., subject, cell, cytosol, tissue or organ).

[0180] Accordingly, in various embodiments, there is also provided a carrier, nanocarrier or delivery system / vehicle comprising the compound.

[0181] Advantageously, in various embodiments, the compound is capable of inducing subcellular interactions and / or enhancing cytosolic delivery of a therapeutic agent, prophylactic agent, and / or biological agent. In various embodiments, the compound is capable of enhancing the release of nucleic acid (e.g., messenger ribonucleic acid (mRNA), small interfering ribonucleic acid (siRNA), oligonucleotides such as antisense oligonucleotide (ASOs)) from endosomes to cytosol where the nucleic acid(s) performs its biological function(s).

[0182] In various embodiments, the compound comprises a structure that is represented by general formula (1 ):

[0183] R1[ R3

[0184]

[0185] wherein

[0186] A comprises a sterol and / or a derivative(s) thereof;

[0187] B comprises:

[0188] (i) a sugar / carbohydrate / saccharide and / or a derivative(s) thereof; or (ii) an oligopeptide / polypeptide comprising sugar / carbohydrate / saccharide and / or a derivative(s) thereof;

[0189] R1and R3are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; R2is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;

[0190] R4is -H or -C(=O)R, where R is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0191] p = 0 or 1; and

[0192] q = 0 or 1.

[0193] In various embodiments, p = q = 0. In such embodiments, the compound comprises a structure represented by general formula (1 A):

[0194] R1

[0195] A\. B

[0196] Yr2

[0197] 0

[0198] (1A)

[0199] In various embodiments, p = q = 1. In such embodiments, the compound comprises a structure represented by general formula (1 B):

[0200] R1R3

[0201] A A. I I > - B - R4

[0202] YR2

[0203]

[0204] ° (1B)

[0205] In various embodiments, A comprises a sterol and / or a derivative(s) thereof. The sterol may be selected from animal sterols (or zoosterols), plant sterols (or phytosterols), fungal sterols (or mycosterols), the like, or combinations thereof. In various embodiments, the sterol and / or a derivative(s) thereof includes, but is not limited to cholesterol, lanosterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, avenasterol, ergosterol, zymosterol, the like, derivatives thereof and combinations thereof. In various embodiments, A comprises four fused / interconnected ring structure, e g., four fused / interconnected hydrocarbon rings. In various embodiments, A comprises four fused / interconnected ring structure as its steroid backbone. For example, A may comprise a cyclopentanoperhydrophenanthrene ring structure.

[0206] In various embodiments, A comprises a structure that is represented by general formula (2):

[0207]

[0208] (2)

[0209] wherein

[0210] ring C1, C2, C3and C4each optionally contains one, two or three C=C bond(s); Ra, Ra’, Rb, Rb’, Rc, Rd, Rd’, Re, Rf, Rf’, Rg, R9’, Rh, R', Rf Rj’, Rk, Rk’, Rl, Rm, Rn, Rn’, R°, R0’, Rpand Rqare each optionally present and independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and

[0211] R' is a hydrophobic tail / chain / group, or contains at least linear aliphatic, branched aliphatic and / or cyclic hydrocarbons.

[0212] In various embodiments, ring C2contains one, two, or three C=C bonds. In various embodiments, A comprises a structure that is represented by general formula (2A):

[0213] CH3

[0214] CH

[0215]

[0216] (2A)

[0217] wherein

[0218] R' is a linear, branched, saturated and / or unsaturated hydrocarbon group / tail / chain.

[0219] In various embodiments, the hydrocarbon tail / chain / group at R' comprises optionally substituted alkyl or optionally substituted alkenyl. The alkyl or alkenyl may be linear or branched. The alkyl or alkenyl may be saturated or unsaturated. For example, R' may contain one or more C=C double bond(s). The alkyl or alkenyl may have at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 carbon atoms. For example, R' may be CaH2a+i or CaH2a, where a is an integer > 5. In various embodiments, a > 5, a > 6, a > 7, a > 8, a > 9, a > 10, a > 11, a > 12, a > 13, a > 14, a > 15, a > 16, a > 17, a > 18, a > 19, or a > 20. Advantageously, in various embodiments, the presence of hydrophobic parts / tails / chains / groups in the compound allows for ease of integration of the compound into the lipid domain of lipid nanoparticles (LNPs), presenting the sugar / carbohydrate / saccharide-functionalized polypeptide on the surface of the LNPs for stability and cell-targeting ability (e g., immune cells-targeting ability).

[0220] In various embodiments, Ra, Ra’, Rb, Rb’, Rc, Rd, Rd’, Re, Rf, Rf’, R9, R9’, Rh, R', Rj, Rj’, Rk, Rk’, R1’, Rm, Rn, Rn’, R°, R°’, Rpand R9are each independently selected from H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl. In various embodiments, Ra, Ra’, Rb, Rb’, Rc, Rd, Rd’, Re, Rf, Rf’, R9 R9’, Rh, R', Rj, R< Rk, Rk’, R1’, Rm, Rn, Rn’, R°, R°’, RP and Rqare each independently optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl. For example, Ra, Ra’, Rb, Rb’, Rc, Rd, Rd’, Re, Rf, Rf’, R9, R9’, Rb, Rj, Rj, Rj’, Rk, Rk’, R1’, Rm, Rn, Rn’, R°, R°’, RP and R” may be selected from methyl, ethyl, n-propyl, 2-propyl, isopropyl, n-butyl, isobutyl, secbutyl, f-butyl, hexyl, amyl, 1,2-dimethylpropyl, 1,1 -dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1 -methylpentyl, 2-methylpentyl, 3-methylpentyl, 2.2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 1,2,2-trimethylpropyl, 1, 1,2-trimethylpropyl, 2-ethylpentyl, 3-ethylpentyl, heptyl, 1-methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl, 1,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,2,3-trimethylbutyl, 1.1.2-trimethylbutyl, 1,1, 3-trimethylbutyl, 5-methylheptyl, 1 -methylheptyl, octyl, nonyl, decyl, or the like or combinations thereof.

[0221] In various embodiments, A comprises a derivative(s) of cholesterol. For example, A comprises a structure that is represented by general formula (2B):

[0222]

[0223] (2B) In various embodiments, A comprises a structure that is represented by general formula (2C):

[0224]

[0225] (2C)

[0226] In various embodiments, B comprises a structure that is represented by general formula (3A) and / or (3B):

[0227]

[0228] (3A)

[0229] wherein

[0230] B1comprises sugar / carbohydrate / saccharide and / or a derivative(s) thereof; B2comprises sugar / carbohydrate / saccharide and / or a derivative(s) thereof; R5is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0231] R6is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene; and

[0232] n > 1.

[0233] In various embodiments where p = q = 0, and the compound comprises a structure represented by general formula (1A), it will be appreciated that B is a terminal group and may be represented by general formula (3A).

[0234]

[0235] In various embodiments where p = q = 1, and the compound comprises a structure represented by general formula (1 B), it will be appreciated that B is a bridging group and may be represented by general formula (3B).

[0236] B - R4

[0237]

[0238] (1B)

[0239] In various embodiments, R1, R3and R5are each independently selected from H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl. The optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl may have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 carbon atoms. For example, R1, R3and R5may be each independently CbFhb, CbH2b+i, or CbH2b-2, where b is an integer > 1. In various embodiments, b > 1, b > 2, b > 3, b > 4, b > 5, b > 6, b > 7, b > 8, b > 9, b > 10, b > 11, b > 12, b > 13, b > 14, b > 15, b > 16, b > 17, b > 18, b > 19, or b > 20.

[0240] In various embodiments, R2is selected from optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene. The optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene may have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 carbon atoms. For example, R2may be selected from methylene, ethylene, n-propylene, 2-propylene, isopropylene, n-butylene, isobutylene, sec-butylene, f-butylene, hexylene, amylene, 1,2-dimethylpropylene, 1,1 -dimethylpropylene, pentylene, isopentylene, hexylene, 4-methylpentylene, 1 -methylpentylene, 2-methylpentylene, 3-methylpentylene, 2,2-dimethylbutylene, 3,3-dimethylbutylene, 1,2-dimethylbutylene, 1,3-dimethylbutylene, 1,2,2-trimethylpropylene, 1,1,2-trimethylpropylene, 2-ethylpentylene, 3-ethylpentylene, heptylene, 1 -methylhexylene, 2,2-dimethylpentylene, 3,3-dimethylpentylene, 4,4-dimethylpentylene, 1,2-dimethylpentylene, 1,3-dimethylpentylene, 1,4-dimethylpentylene, 1,2,3-trimethylbutylene, 1,1,2-trimethylbutylene, 1,1,3-trimethylbutylene, 5-methylheptylene, 1 -methylheptylene, octylene, nonylene, decylene, or the like or combinations thereof. For example, R2may be -CH2-, — C2H4—, — C3H6—, — C4H8— or— C5H10—.

[0241] In various embodiments, R4is -C(=O)R. For example, the primary amine group of the compound may be capped with -C(=O)R, where R may be selected from methyl, ethyl, n-propyl, 2-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, hexyl, amyl, 1,2-dimethylpropyl, 1,1 -dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1 -methylpentyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dim ethyl butyl, 1,3-dimethylbutyl, 1,2,2-trimethylpropyl, 1, 1,2-trimethylpropyl, 2-ethylpentyl, 3-ethylpentyl, heptyl, 1-methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl, 1,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,2,3-trimethylbutyl, 1,1,2-trimethylbutyl, 1, 1,3-trimethylbutyl, 5-methylheptyl, 1 -methylheptyl, octyl, nonyl, decyl, the like, or combinations thereof.

[0242] In various embodiments, R6is selected from optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene. The optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene may have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 carbon atoms. For example, R6may be selected from methylene, ethylene, n-propylene, 2-propylene, isopropylene, n-butylene, isobutylene, sec-butylene, f-butylene, hexylene, amylene, 1,2-dimethylpropylene, 1,1 -dimethylpropylene, pentylene, isopentylene, hexylene, 4-methylpentylene, 1 -methylpentylene, 2-methylpentylene, 3-methylpentylene, 2,2-dimethylbutylene, 3,3-dimethylbutylene, 1,2-dimethylbutylene, 1,3-dimethylbutylene, 1,2,2-trimethylpropylene, 1,1,2-trimethylpropylene, 2-ethylpentylene, 3-ethylpentylene, heptylene, 1 -methylhexylene, 2,2-dimethylpentylene, 3,3-dimethylpentylene, 4,4-dimethylpentylene, 1,2-dimethylpentylene, 1,3-dimethylpentylene, 1,4-dimethylpentylene, 1,2,3-trimethylbutylene, 1,1,2-trimethylbutylene, 1,1,3-trimethylbutylene, 5-methylheptylene, 1 -methylheptylene, octylene, nonylene, decylene, or the like or combinations thereof. For example, R6may be -CH2-, — C2H4—, — C3H6—, — C4H8— or— C5H10—.

[0243] In various embodiments, the structure represented by general formula (1) is represented by general formula (4):

[0244]

[0245] (4) wherein

[0246] R2ais optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;

[0247] R2bis optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene; x = 0 or 1;

[0248] y = 0 or 1; and

[0249] total sum of x + y = 1.

[0250] In various embodiments, R2aand R2bcontain one or more features and / or share one or more properties that are similar to R2as described above (e.g., as defined in general formula (1)).

[0251] In various embodiments, the compound comprises B1as a terminal group (or terminal sugar / carbohydrate / saccharide unit(s) / group(s) and / or a derivative(s) thereof). In various embodiments, the compound comprises B2being functionalized with / conjugated with one or more peptide unit / block(s) and / or a derivative(s) thereof.

[0252] In various embodiments, x = 1 and y = 0. In such embodiments, the structure represented by general formula (1) may be represented by general formula (4A):

[0253] R1

[0254] o

[0255]

[0256] (4A)

[0257] In various embodiments, x = 0 and y = 1. In such embodiments, the structure represented by general formula (1) may be represented by general formula (4B):

[0258]

[0259] In various embodiments, the compound comprises a carbohydrate-functionalized polypeptide or poly(amino acid). In various embodiments, the compound comprises a monosaccharide-functionalized polypeptide such as a mannose-functionalized polypeptide where a mannose is attached onto some units (e.g., one, two, three, four, five, six, seven, eight, nine, or ten units) of the peptide in the polypeptide. In various embodiments, the compound comprises a monosaccharide-functionalized polypeptide such as a mannose-functionalized polypeptide where a mannose is attached onto each unit of the peptide in the polypeptide.

[0260] In various embodiments, the compound comprises one or more sugar / carbohydrate / saccharide unit(s) / group(s) and / or a derivative(s) thereof (e.g., B1and / or B2) that is functionalized with / conjugated with one or more peptide unit / block(s) and / or a derivative(s) thereof. For example, the compound may comprise one or more sugar / carbohydrate / saccharide unit(s) / group(s) and / or a derivative(s) thereof that is functionalized with / conjugated with one or more oligopeptides, polypeptides / poly(amino acids) and / or a derivative(s) thereof. In various embodiments, the total number of peptide units / blocks and / or a derivative(s) thereof (or total length of oligopeptides, polypeptides / poly(amino acids) and / or a derivative(s) thereof) in the compound is adjustable as desired. Advantageously, in various embodiments, the compound is designed / configured to allow the hydrophilicity / hydrophobicity balance of the compound to be adjustable / customizable / tunable by adjusting the length of the oligopeptides or polypeptides / poly(amino acids) and / or a derivative(s) thereof, that is, by adjusting / altering / tuning / controlling the degree of polymerization (i.e. value of n).

[0261] In various embodiments, n is an integer > 1. In various embodiments, n > 1, n > 2, n > 3, n > 4, n > 5, n > 6, n > 7, n > 8, n > 9, n > 10, n > 11, n > 12, n > 13, n > 14, n > 15, n > 16, n > 17, n > 18, n > 19, n > 20, n > 21, n >22, n >23, n > 24, n > 25, n > 26, n > 27, n > 28, n > 29, n > 30, n > 31, n > 32, n > 33, n > 34, n > 35, n > 36, n > 37, n > 38, n > 39, n > 40, n > 41, n > 42, n > 43, n > 44, n > 45, n > 46, n > 47, n > 48, n > 49, or n > 50. In various embodiments, n is from about 1 to about 50, from about 5 to about 45, from about 10 to about 40, from about 15 to about 35, from about 20 to about 25, or about 30.

[0262] In various embodiments, B1and / or B2is / are selected from carbohydrate / sugar / saccharide and a derivative(s) thereof. In various embodiments, the carbohydrate / sugar / saccharide and / or derivatives thereof (e.g., B1and / or B2) is selected from the group consisting of monosaccharide, disaccharide, oligosaccharide, polysaccharide and / or a derivative(s) thereof. In various embodiments, the carbohydrate / saccharide is in a cyclic form, for example as a 5-membered ring (e.g., fructose or ribose) or 6-membered ring (e.g., mannose, glucose, or galactose). Thus, in various embodiments, the carbohydrate / saccharide is a furanose or pyranose. In various embodiments, the carbohydrate / saccharide is in a linear form, for example as a linear-chain monosaccharide.

[0263] In various embodiments, the compound (or carbohydrate / sugar / saccharide and / or a derivative(s) thereof (e.g., B1and / or B2)) is substantially devoid of galactose and / or a derivative(s) thereof.

[0264] In various embodiments, the carbohydrate / sugar / saccharide and / or a derivative(s) thereof (e.g., B1and / or B2) comprises a glycosyl unit and / or a derivative(s) thereof.

[0265] In various embodiments, B1and / or B2comprise(s) a structure that is represented by general formula (5) having a 6-membered ring structure:

[0266] X9

[0267]

[0268] (5) wherein

[0269] M is -O- or -S-;

[0270] X1to X7and X9to X10are each independently selected from -H, -OH, or -O-C(=O)R7, where R7is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and

[0271] X8is alkyl / alkylene.

[0272] In various embodiments, R7is optionally substituted alkyl. In various embodiments, R7is selected from methyl, ethyl, n-propyl, 2-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, f-butyl, hexyl, amyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1 -methylpentyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 1,2,2-trimethylpropyl, 1,1,2-trimethylpropyl, 2-ethylpentyl, 3-ethylpentyl, heptyl, 1 -methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl, 1,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,2,3-trimethylbutyl, 1, 1,2-trimethylbutyl, 1,1,3-trimethylbutyl, 5-methylheptyl, 1 -methylheptyl, octyl, nonyl, decyl, the like, or combinations thereof. In various embodiments, R7is -CH3. For example, X1, X4, X6and X9may be each -O-C(=O)-CHs.

[0273] In various embodiments, X8is alkylene, e g., optionally substituted –CdH2d–, where d is an integer > 1. In various embodiments, d is from about 1 to about 20. For example, X8may be -CH2-, -C2H4-, -C3H6-, -C4H8-, -C5H10-, -C6H12-, -C7H14-, - CsHie-, -C9H18-, or -CioH2o- In various embodiments, X8is methylene (i.e., -CH2-).

[0274] In various embodiments, M is -O-. In various embodiments, general formula (5) (e.g., B1and / or B2) is / are chemically coupled / bonded to the rest of general formula (1) (or general formula (4A) and / or (4B)) via its hydroxy group. For example, B1may be connected to R2(or R2a) via a glycosidic / ether bond / linkage. For example, B2may be connected to R6via a glycosidic / ether bond / linkage. In various embodiments, B1and / or B2comprise(s) a structure that is represented by general formula (5A) and / or (5B) having a 6-membered ring structure:

[0275]

[0276] wherein

[0277] X1to X10contain one or more features and / or share one or more properties that are similar to those described above (e g., as defined in general formula (5)).

[0278] In various embodiments, B1and / or B2comprise(s) D-sugar and / or a derivative(s) thereof (e.g., represented by general formula (5A)).

[0279] In various embodiments, B1and / or B2comprise(s) L-sugar and / or a derivative(s) thereof (e.g., represented by general formula (5B)).

[0280] In various embodiments, B1and / or B2comprise(s) a mixture of two enantiomers, namely D-sugar and / or a derivative(s) thereof (e.g., represented by general formula (5A); and L-sugar and / or a derivative(s) thereof (e.g., represented by general formula (5B). In various embodiments, B1and / or B2comprises a mixture (e.g., racemic mixture of enantiomers) containing both D-sugar and L-sugar. For example, B1and / or B2may comprise D-mannose, L-mannose and / or a derivative(s) thereof.

[0281] It will be appreciated that various sugar molecules may be used as B1and / or B2in embodiments of the compound represented as general formulae (4), (4A) and / or (4B) as long as the sugar molecule is capable of providing a targeting ability and / or imparting hydrophilicity as a form of replacement of PEG. For example, B1and / or B2may be mannose (e.g., for targeting immune cells) or glucose (e.g., for imparting / increasing hydrophilicity or to be used as a hydrophilic component).

[0282] In various embodiments, the compound comprises a carbohydrate-functionalized polypeptide or carbohydrate-functional ized poly(amino acid). In various embodiments, the compound comprises a carbohydrate-functionalized polypeptide-b-lipid or carbohydrate-functionalized poly(amino acid)-b-lipid where a carbohydrate is attached onto each unit of the polypeptide. In various embodiments, the compound comprises a monosaccharide-functionalized polypeptide such as a mannose-functionalized polypeptide where a mannose is attached onto each unit of the polypeptide. In various embodiments, the compound comprises a monosaccharide-functionalized polypeptide-b-lipid such as a mannose-functionalized polypeptide-b-lipid where a mannose is attached onto each unit of the polypeptide. For example, the compound may comprise a mannose-functionalized polyserine-b-lipid wherein the mannose is attached onto each unit of the polyserine.

[0283] Advantageously, the presence of carbohydrate / sugar / saccharide and / or a derivative(s) thereof allows embodiments of the compound to be capable of targeting sugar / carbohydrate / saccharide receptors found in / on cell surfaces (e.g., immune cell surfaces, fibroblast and keratinocyte surfaces, and liver sinusoidal endothelial cell surfaces). In various embodiments, the design of the compound allows easy access of the carbohydrate to the cell (e.g., immune cell) in order to target carbohydrate receptors on the cell surface (e g., immune cell surface), thereby reducing risk of allergic reactions, prolonging the plasma halflife of nucleic acid and enhancing vaccination or treatment efficiency. In particular, in various embodiments, the design of installing / attaching a carbohydrate / sugar / saccharide group and / or a derivative(s) thereof onto each unit of the hydrophilic peptide / oligopeptide / polypeptide allows said carbohydrate / sugar / saccharide group and / or a derivative(s) thereof to be easily accessible to cells (e.g., immune cells). In various embodiments, the carbohydrate / sugar / saccharide group and / or a derivative(s) thereof is not installed / attached to cholesterol and / or a derivative(s) thereof which may otherwise prevent access of the carbohydrate / sugar / saccharide group and / or a derivative(s) thereof to cells (e.g., making it inaccessible to immune cells), e.g., when used in PEGylated LNPs or LNPs coated with another hydrophilic polymer.

[0284] Advantageously, the presence of sugar / carbohydrate / saccharide and / or a derivative(s) thereof increases the hydrophilicity of the compound, and consequently solubility of the compound. Advantageously, in various embodiments, the presence of carbohydrate / sugar / saccharide and / or a derivative(s) thereof in the compound eliminates the requirement of a hydrophilic polyethylene glycol (PEG) which is otherwise necessary in a conventional PEG-lipid conjugate for LNP formulations. In various embodiments, by eliminating the presence of polyethylene glycol (PEG) in the compound, embodiments of the carbohydrate / sugar / saccharide group and / or a derivative(s) thereof and lipid nanoparticles formed therefrom are not and / or avoid the possibility of being shielded by the long PEG chain. In various embodiments, the compound is substantially devoid of polyethylene glycol (PEG). In various embodiments, the compound is substantially devoid of polyethylene glycol (PEG)-modified lipid conjugates, polyethylene glycol (PEG)-modified lipid, PEGylated lipid, PEG-conjugated lipid, PEG-lipid conjugate, and / or lipid modified with PEG. Advantageously, in various embodiments, the design of the structure of the compound allows said compound to be used, in lieu of or as a substitute / replacement for a conventional PEG-lipid conjugate (e.g., ALC-0159).

[0285] In various embodiments, the compound comprises a lipid compound. Accordingly, in various embodiments therefore, the term “compound” may comprise and / or may be used interchangeably with the terms “sterol-functionalized carbohydrate”, “sterol-functionalized sugar’”’, “sterol-functionalized mannose”, “carbohydrate-functional ized sterol”, “sugar-functionalized sterol”, “mannose-functionalized sterol”, “sterol-b / oc -carbohydrate-functionalized polypeptide”, “sterol-b / oc -sugar-functionalized polypeptide”, “sterol-b / oc - mannose-functionalized polypeptide”, “sterol-b-carbohydrate-functionalized polypeptide”, “sterol-b-sugar-functionalized polypeptide”, “sterol-b-mannose-functionalized polypeptide” or the like.

[0286] In various embodiments, the compound has a number average molecular weight (Mn) of from about 250 g / mol to about 25,000 g / mol, from about 300 g / mol to about 24,000 g / mol, from about 350 g / mol to about 23,000 g / mol, from about 400 g / mol to about 22,000 g / mol, from about 450 g / mol to about 21,000 g / mol, from about 500 g / mol to about 20,000 g / mol, from about 550 g / mol to about 19,000 g / mol, from about 600 g / mol to about 18,000 g / mol, from about 650 g / mol to about 17,000 g / mol, from about 700 g / mol to about 16,000 g / mol, from about 750 g / mol to about 15,000 g / mol, from about 800 g / mol to about 14,000 g / mol, from about 850 g / mol to about 13,000 g / mol, from about 900 g / mol to about 12,000 g / mol, from about 950 g / mol to about 11,000 g / mol, from about 1,000 g / mol to about 10,000 g / mol, from about 1,250 g / mol to about 9,750 g / mol, from about 1,500 g / mol to about 9,500 g / mol, from about 1,750 g / mol to about 9,250 g / mol, from about 2,000 g / mol to about 9,000 g / mol, from about 2,250 g / mol to about 8,750 g / mol, from about 2,500 g / mol to about 8,500 g / mol, from about 2,750 g / mol to about 8,250 g / mol, from about 3,000 g / mol to about 8,000 g / mol, from about 3,250 g / mol to about 7,750 g / mol, from about 3,500 g / mol to about 7,500 g / mol, from about 3,750 g / mol to about 7,250 g / mol, from about 4,000 g / mol to about 7,000 g / mol, from about 4,250 g / mol to about 6,750 g / mol, from about 4,500 g / mol to about 6,500 g / mol, from about 4,750 g / mol to about 6,250 g / mol, from about 5,000 g / mol to about 6,000 g / mol, from about 5,250 g / mol to about 5,750 g / mol, or about 5,500 g / mol. In various embodiments, the compound comprises a structure selected from one or more of the following:

[0287] H

[0288] cholesterol-mannose-OAc (CMO) H

[0289]

[0290] CPSM-1 (n = 15), and

[0291]

[0292] In various embodiments, D-mannose in CMO, CM, CPSM-1 and CPSM-2 may be replaced with L-mannose. In various embodiments, both D-mannose and L-mannose may be present in CMO, CM, CPSM-1 and CPSM-2. In various embodiments, poly(Ser-D-mannose) in CPSM-1 and CPSM-2 may be replaced with poly(Ser-L-mannose). In various embodiments, poly(Ser-D, L-mannose) may be present in CPSM-1 and CPSM-2.

[0293] In various embodiments, D-mannose in CMO, CM, CPSM-1 and CPSM-2 may be replaced with furanose (e.g., ribose and fructose) and / or pyranose (e.g., glucose). In various embodiments, D-mannose in CMO, CM, CPSM-1 and CPSM-2 may be replaced with D-ribose, L-ribose, D, L-ribose, D-fructose, L-fructose, D, L-fructose, D-glucose, L-glucose and / or D, L-glucose.

[0294] In various embodiments, L-serine (or L-Ser) in CPSM-1 and CPSM-2 may be replaced with D-serine (or D-Ser). In various embodiments, both L-serine (or L-Ser) and D-serine (or D-Ser) may be present in in CPSM-1 and CPSM-2. In various embodiments, poly(L-Ser-D-mannose), poly(L-Ser-L-mannose), poly(D-Ser-D-mannose), poly(D-Ser-L-mannose), poly(L-Ser-D, L-mannose), poly(D-Ser-D, L-mannose), and / or poly(D, L-Ser-D, L-mannose) may be present in CMO, CM, CPSM-1 and CPSM-2 METHOD OF PREPARING COMPOUND

[0295] There is provided a method of preparing a compound represented by general formula (1) (e g., general formula (4A)) as disclosed herein, the method comprising:

[0296] (a-i) reacting a protected alkanolamine compound represented by general formula (6) with a compound comprising B1represented by general formula (7) in the presence of a Lewis acid to obtain a first intermediate compound represented by general formula (8):

[0297] R1R1I HO. / N. B1— R8B\ / N

[0298]

[0299] R2PG1R2TG1(6) (7) (8) wherein

[0300] R8is -H, -OH or -O-C(=O)R9, where R9is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0301] PG1is a protecting group selected from N-carboxybenzyl or benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (BOC), tert-butyl, 9- Fluorenylmethoxycarbonyl (Fmoc), 2-(4- Nitrophenylsulfonyl)ethoxycarbonyl (Nsc), 2-Fluoro-Fmoc (Fmoc(2F)), 2- Monoisooctyl-Fmoc (mio-Fmoc), 2,7-Diisooctyl-Fmoc (dio-Fmoc), the like, or combinations thereof;

[0302] B1comprises a sugar / carbohydrate / saccharide and / or a derivative(s) thereof;

[0303] R1is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0304] R2is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene; (a-ii) deprotecting the first intermediate compound represented by general formula (8) to obtain a second intermediate compound represented by general formula (9):

[0305] R1

[0306] B1. N

[0307] R2

[0308]

[0309] ; and

[0310] (a-iii) reacting the second intermediate compound represented by general formula (9) with a haloformate compound comprising A represented by general formula (21 ) to obtain a compound represented by general formula (4A):

[0311] 0

[0312]

[0313] (21) wherein

[0314] X1is a halide (e.g., F, Br, Cl, I); and

[0315] A comprises a sterol and / or a derivative(s) thereof.

[0316] In various embodiments, the method further comprises:

[0317] (a-iv) optionally deprotecting the compound represented by general formula (4A) to replace one or more -O-C(=O)R7group(s) in B1with -OH group(s).

[0318] In various embodiments, the reacting step (a-i) comprises adding the Lewis acid in a dropwise manner to a protected compound comprising B1represented by general formula (7) and / or protected alkanolamine compound represented by general formula (6) (e.g., obtained from (b-i)). Advantageously, such dropwise addition(s) ensures good control over the reaction and reduces risk of rapid, sudden and / or exothermic reactions. In various embodiments, the Lewis acid (e.g., used in (a-i) is selected from boron trifluoride diethyl etherate (BF3•OEt2), boron trifluoride, the like or combinations thereof.

[0319] In various embodiments, the deprotecting / deprotection (a-ii) of the first intermediate compound represented by general formula (8) comprises selectively deprotecting / removing the protecting group PG1from the first intermediate compound represented by general formula (8).

[0320] In various embodiments, the deprotecting / deprotection (a-ii) is carried out in the presence of catalyst (e.g., palladium on carbon).

[0321] In various embodiments, the deprotecting / deprotection (a-iv) of the compound represented by general formula (4A) comprises subjecting the compound represented by general formula (4A) to basic conditions. For example, the deprotection (a-iv) may be carried out in the presence of one or more base(s). The base may be anhydrous base such as sodium methoxide (MeONa) in methanol (MeOH). In various embodiments, the deprotection (a-iv) is carried out in the presence of one or more of the following: a nucleophilic compound comprising hydroxyl group, a metal alkoxide (e.g, sodium methoxide), and an alcohol (e.g., methanol).

[0322] In various embodiments, the method further comprises, prior to (a-i):

[0323] (b-i) reacting an alkanolamine compound represented by general formula (10) with a compound comprising PG1represented by general formula (11) in the presence of a Lewis base to obtain a protected alkanolamine compound represented by general formula (6): R1

[0324] I HO. / N

[0325] 2X— PG

[0326] R H1

[0327] (10) (11)

[0328] R1

[0329] HO. ^N.

[0330] R2^PG1

[0331]

[0332] (6)

[0333] wherein

[0334] R1is H; and

[0335] X is a halide (e.g., F, Br, Cl, I).

[0336] In various embodiments, step (b-i) comprises protecting the alkanolamine compound represented by general formula (10).

[0337] In various embodiments, the protected alkanolamine compound represented by general formula (6) is obtained by reacting an unprotected alkanolamine compound represented by general formula (10) with X-PG1represented by general formula (11) with a Lewis base, where X is a leaving group such as a halide (i.e., F, Cl, Br, or I). In various embodiments, the unprotected alkanolamine compound may be selected from a primary alkanolamine (e.g., alkanolamine containing a primary amine). Examples of primary alkanolamine include, but is not limited to ethanolamine, diglycoamine, aminomethyl propanol, or the like or combinations thereof.

[0338] In various embodiments, X-PG1is benzyl haloformate or halides of benzyloxycarbonyl (e.g., benzyl chloroformate or Cbz-CI).

[0339] In various embodiments, the Lewis base (e.g., used in (b-i)) is selected from amines such as primary amines (e.g., ammonia (NH3)), tertiary amines (e.g., triethylamine (TEA)), aromatic amines (e.g., pyridine), the like or combinations thereof.

[0340] There is provided a method of preparing a compound represented by general formula (1) (e.g., general formula (4B)) as disclosed herein, the method comprising:

[0341] (c-i) polymerizing one or more N-carboxyanhydride (NCA) monomers represented by general formula (12) with an initiator represented by general formula (13) to obtain a first intermediate compound represented by general formula (14):

[0342]

[0343] wherein

[0344] A comprises a sterol and / or a derivative(s) thereof;

[0345] B2comprises a sugar / carbohydrate / saccharide and / or a derivative(s) thereof;

[0346] R1and R3are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0347] R2is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;

[0348] R4is -H or -C(=O)R, where R is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; R5is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0349] R6is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene; and

[0350] n ≥ 1;

[0351] (c-ii) reacting the first intermediate compound represented by general formula (14) with an acylating agent represented by general formula (15) to obtain a second intermediate compound represented by general formula (16):

[0352]

[0353] wherein

[0354] R and R’ are each independently optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; and

[0355] (c-iii) deprotecting the second intermediate compound represented by general formula (16) to obtain a compound represented by general formula (4B).

[0356] In various embodiments, the reacting (c-i) comprises ring-opening and polymerization of the NCA group in the compound represented by general formula (12). Advantageously, the NCA undergoes ring-opening and polymerizes despite the bulky structure of the protected sugar group. Without being bound by theory, it is believed that such a successful cyclization (NCA formation) and polymerization is due to the specific combination of L-amino acids (e.g., L-serine) and D-sugars (e.g., D-mannose), which avoids steric hindrance. It will be appreciated that in (c-i), the one or more NCA monomers (i.e. represented by general formula (12)) may be added sequentially to the lipid initiator to polymerize and form lipid-block polypeptides or lipid-random polypeptides or lipidpolypeptides formed from both L-serine-NCA and D-serine-NCA.

[0357] In various embodiments, the acylating agent (in (c-ii)) is selected from acid anhydride (e.g., acetic anhydride (Ac2O)), acid halide (e.g., acyl halide), N-hydroxysuccinimide (NHS) esters, imidoesters or the like or combinations thereof.

[0358] In various embodiments, the deprotecting / deprotection (c-iii) of the second intermediate compound represented by general formula (16) comprises subjecting the compound represented by general formula (16) to basic conditions. For example, the deprotection (c-iii) may be carried out in the presence of one or more base(s). The base may be anhydrous base such as sodium methoxide (MeONa) in methanol (MeOH). In various embodiments, the deprotection step (c-iii) is carried out in the presence of one or more of the following: a nucleophilic compound comprising hydroxyl group, a metal alkoxide (e.g, sodium methoxide), and an alcohol (e.g., methanol).

[0359] In various embodiments, the method further comprises, prior to (c-i):

[0360] (d-i) reacting a protected amino acid represented by general formula (17) with a compound comprising B2represented by general formula (18) in the presence of a Lewis acid to obtain a first intermediate compound represented by general formula (19):

[0361] H\ / PG2H\ / PG2

[0362] B2— R10

[0363]

[0364] O (17) (18) (19)

[0365] wherein R10is -H, -OH or -O-C(=O)R11, where R11is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0366] PG2is a protecting group selected from N-carboxybenzyl or benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (BOC), tert-butyl, 9- Fluorenylmethoxycarbonyl (Fmoc), 2-(4- Nitrophenylsulfonyl)ethoxycarbonyl (Nsc), 2-Fluoro-Fmoc (Fmoc(2F)), 2- Monoisooctyl-Fmoc (mio-Fmoc), 2,7-Diisooctyl-Fmoc (dio-Fmoc), the like, or combinations thereof;

[0367] (d-ii) deprotecting the first intermediate compound represented by general formula (19) to obtain a second intermediate compound represented by general formula (20):

[0368] H\

[0369] N

[0370]

[0371] O

[0372] (20); and

[0373] (d-iii) reacting the second intermediate compound represented by general formula (20) with a carbonylating agent to obtain the N-carboxyanhydride (NCA) monomers represented by general formula (12).

[0374] It will be appreciated that, in various embodiments, the second intermediate represented by general formula (20) comprises –NH2and -COOH in order for ring cyclization to occur to obtain / make NCA prior to ringopening polymerization.

[0375] In various embodiments, the Lewis acid (e.g., used in (d-i)) is selected from boron trifluoride diethyl etherate (BF3•OEt2), boron trifluoride, the like or combinations thereof. In various embodiments, the deprotecting / deprotection (d-ii) of the first intermediate compound represented by general formula (19) comprises selectively deprotecting / removing the protecting group PG2from the first intermediate compound represented by general formula (19).

[0376] In various embodiments, the deprotecting / deprotection (d-ii) is carried out in the presence of catalyst (e.g., palladium on carbon).

[0377] In various embodiments, the carbonylating agent (used in (d-iii)) is selected from phosgene, diphosgene, triphosgene, the like, or combinations thereof.

[0378] In various embodiments, the reacting (d-iii) comprises cyclization of the second intermediate compound represented by general formula (20).

[0379] In various embodiments, the polymerizing / reacting / deprotecting (a-i), (a-ii), (a-iii), (a-iv), (b-i), (c-i), (c-ii), (c-iii), (d-i), (d-ii) and / or (d-iii) comprises one or more of the following steps: suspending, dispersing, mixing, stirring, dissolving, sonicating and / or ultrasonicating.

[0380] In various embodiments, the polymerizing / reacting / deprotecting step (a-i), (a-ii), (a-iii), (a-iv), (b-i), (c-i), (c-ii), (c-iii), (d-i), (d-ii) and / or (d-iii) is / are performed in the presence of an organic solvent. In various embodiments, any organic solvent that effectively serves as a medium to contain the components of the reaction mixture (e g., reactants / substrates) may be used in embodiments of the reaction mixture disclosed herein. In various embodiments, the organic solvent is capable of substantially dissolving the components present in the reaction mixture. The organic solvent may be an organic solvent such as dichloromethane (DCM), tetrahydrofuran (THF), dimethysulfoxide (DMSO), acetonitrile, ethyl acetate, dimethylformamide (DMF), or the like or combinations thereof. In various embodiments, the organic solvent may be provided in a dry or anhydrous form. In various embodiments, the polymerizing / reacting / deprotecting (a-i), (a-ii), (a-iii), (a-iv), (b-i), (c-i), (c-ii), (c-iii), (d-i), (d-ii) and / or (d-iii) is / are carried out under vacuum or in an inert atmosphere. For example, the step(s) of suspending, dispersing, mixing, stirring, dissolving, sonicating and / or ultrasonicating may be performed in a glove box or in the presence of an inert gas such as argon or nitrogen.

[0381] In various embodiments, the polymerizing / reacting / deprotecting (a-i), (a-ii), (a-iii), (a-iv), (b-i), (c-i), (c-ii), (c-iii), (d-i), (d-ii) and / or (d-iii) is / are performed over a time duration of from about 1 hour to about 200 hours, from about 2 hours to about 190 hours, from about 3 hours to about 180 hours, from about 4 hours to about 170 hours, from about 5 hours to about 160 hours, from about 10 hours to about 150 hours, from about 15 hours to about 140 hours, from about 20 hours to about 130 hours, from about 25 hours to about 120 hours, from about 30 hours to about 110 hours, from about 35 hours to about 100 hours, from about 40 hours to about 95 hours, from about 45 hours to about 90 hours, from about 50 hours to about 85 hours, from about 55 hours to about 80 hours, from about 60 hours to about 75 hours, or from about 65 hours to about 70 hours.

[0382] In various embodiments, the polymerizing / reacting / deprotecting of (a-i), (a-ii), (a-iii), (a-iv), (b-i), (c-i), (c-ii), (c-iii), (d-i), (d-ii) and / or (d-iii) is / are performed at a temperature that is from about -10.0°C to about 150.0°C, from about -9.0°C to about 145.0°C, from about -8.0°C to about 140.0°C, from about -7.0°C to about 135.0°C, from about -6.0°C to about 130.0°C, from about -5.0°C to about 125.0°C, from about -4.0°C to about 120.0°C, from about -3.0°C to about 115.0°C, from about -2.0°C to about 110.0°C, from about -1.0°C to about 105.0°C, from about 0°C to about 100.0°C, from about 1.0°C to about 95.0°C, from about 2.0°C to about 90.0°C, from about 3.0°C to about 85.0°C, from about 4.0°C to about 80.0°C, from about 5.0°C to about 75.0°C, from about 10.0°C to about 700°C, from about 15.0°C to about 65.0°C, from about 20.0°C to about 60.0°C, from about 25.0°C to about 55.0°C, from about 30.0°C to about 50.0°C, from about 35.0°C to about 45.0°C, or about 40.0°C.

[0383] In various embodiments, the polymerizing / reacting / deprotecting of (a-i), (a-ii), (a-iii), (a-iv), (b-i), (c-i), (c-ii), (c-iii), (d-i), (d-ii) and / or (d-iii) is / are performed at a temperature that is from about 10.0°C to about 100.0°C, from about 20.0°C to about 90.0°C, from about 30.0°C to about 80.0°C, from about 40.0°C to about 70.0°C, from about 50.0°C to about 60.0°C, or about 55.0°C. In various embodiments, the polymerizing / reacting / deprotecting of (a-i), (a-ii), (a-iii), (a-iv), (b-i), (c-i), (c-ii), (c-iii), (d-i), (d-ii) and / or (d-iii) is / are performed at room temperature e.g., that is from about 20°C to about 30°C, about 21 °C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, or about 30°C.

[0384] In various embodiments, the polymerizing / reacting / deprotecting of (a-i), (a-ii), (a-iii), (a-iv), (b-i), (c-i), (c-ii), (c-iii), (d-i), (d-ii) and / or (d-iii) is / are optionally performed at a temperature that is from about 40.0 °C to about 100.0 °C, from about 45.0 °C to about 95.0 °C, from about 50.0 °C to about 90.0 °C, from about 55.0 °C to about 85.0 °C, from about 60.0 °C to about 80.0 °C, from about 65.0 °C to about 75.0 °C, or about 70.0 °C.

[0385] In various embodiments, the polymerizing / reacting / deprotecting of (a-i), (a-ii), (a-iii), (a-iv), (b-i), (c-i), (c-ii), (c-iii), (d-i), (d-ii) and / or (d-iii) is / are optionally performed at a temperature that is from about -10°C to about 10°C, from about -9°C to about 9°C, from about -8°C to about 8°C, from about -7°C to about 7°C, from about -6°C to about 6°C, from about -5°C to about 5°C, from about -4°C to about 4°C, from about -3°C to about 3°C, from about -2°C to about 2°C, from about -1°C to about 1°C, or 0°C, e g., to control reaction kinetics. For example, the reacting step may be performed in an ice bath. In various embodiments, the method further comprises:

[0386] (e-i) a step of isolating the first intermediate compound represented by general formula (8) after (a-i);

[0387] (e-ii) a step of isolating the second intermediate compound represented by general formula (9) after (a-ii);

[0388] (e-iii) a step of isolating the compound represented by general formula (4A) after step (a-iii) and / or (a-iv);

[0389] (e-iv) a step of isolating the protected alkanolamine compound represented by general formula (6) after (b-i);

[0390] (e-v) a step of isolating the first intermediate compound represented by general formula (14) after (c-i);

[0391] (e-vi) a step of isolating the second intermediate compound represented by general formula (16) after (c-ii);

[0392] (e-vii) a step of isolating the compound represented by general formula (4B) after (c-iii);

[0393] (e-viii) a step of isolating the first intermediate compound represented by general formula (19) after (d-i);

[0394] (e-ix) a step of isolating the second intermediate compound represented by general formula (20) after (d-ii); and / or

[0395] (e-x) a step of isolating the NCA monomer represented by general formula (12) step (d-iii).

[0396] In various embodiments, the isolating step(s) comprises one or more of the following steps: re-dissolving, purifying, centrifuging, quenching, washing, precipitating, filtering, and / or recrystallizing the compounds obtained after (a-i), (a-ii), (a-iii), (a-iv), (b-i), (c-i), (c-ii), (c-iii), (d-i), (d-ii) and / or (d-iii). For example, the isolating step(s) may comprise one or more of the following steps: re-dissolving, purifying, centrifuging, quenching, washing, precipitating, filtering, and / or recrystallizing the first intermediate compound represented by general formula (8); the second intermediate compound represented by general formula (9); the compound represented by general formula (4A); the protected alkanolamine compound represented by general formula (6); the first intermediate compound represented by general formula (14); the second intermediate compound represented by general formula (16); the compound represented by general formula (4B); the first intermediate compound represented by general formula (19); the second intermediate compound represented by general formula (20); and the NCA monomer represented by general formula (12).

[0397] In various embodiments, the method further comprises one or more of the following post reaction steps: drying optionally under low temperature (e.g., freeze drying), under vacuum, and / or in an inert atmosphere.

[0398] In various embodiments, the step(s) of purifying, centrifuging, recrystallizing and / or washing is / are repeated at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times with a washing medium. In various embodiments, the step(s) of purifying, centrifuging, recrystallizing and / or washing is / are typically / usually repeated at least 3 times.

[0399] In various embodiments, the washing medium comprises aqueous medium / solutions such as salt solution, deionized water, acid or the like, or combinations thereof. The salt solution may be bicarbonate salts such as sodium bicarbonate, chloride salts such as saturated sodium chlorine (brine). The acid may be hydrochloric acid. In various embodiments, the salt solution comprises highly concentrated / saturated salt solution.

[0400] In various embodiments, the method further comprises one or more of the following post reaction steps: drying optionally under low temperature (e.g., freeze drying), under vacuum, and / or in an inert atmosphere.

[0401] In various embodiments, the step(s) of drying is performed in the presence of a drying agent such as anhydrous sodium sulfate, anhydrous magnesium sulfate, anhydrous calcium sulfate, and anhydrous calcium chloride, the like or combinations thereof.

[0402] In various embodiments, the yield of the compound represented by general formula (1) (e.g., (4A) or(4B)) is from about 20.0% to about 100.0%, from about 21.0% to about 99.0%, from about 22.0% to about 98.0%, from about 23.0% to about 97.0%, from about 24.0% to about 96.0%, from about 25.0% to about 95.0%, from about 30.0% to about 90.0%, from about 35.0% to about 85.0%, from about 40.0% to about 80.0%, from about 45.0% to about 75.0%, from about 50.0% to about 70.0%, from about 55.0% to about 60.0%, or about 65.0%. The yield of the compound represented by general formula (1 ) (e.g., (4A) or (4B)) may be from about 50.0% to about 98.0%.

[0403] Advantageously, embodiments of the method are straightforward to perform and / or have a low production / manufacturing cost (i.e. cost effective) as the reaction conditions are mild and do not require harsh and / or tedious step(s). Advantageously, embodiments of the method comprise simple purification steps (e.g., ease of isolation from by-products etc) and products are synthesized with high yields. Advantageously, embodiments of the method are scalable and / or have substantially high scalability. Advantageously, embodiments of the method disclosed herein provide a simple synthesis process, easily purifiable product with high yields and controllable degree of polymerization.

[0404] NANOPARTICLE COMPOSITION

[0405] Advantageously, in various embodiments, the design of the structure of the compound represented by general formula (1) allows said compound to be used, in lieu or in replacement / substitute of a conventional lipid-PEG conjugate (e.g., ALC-0159), in the formulation of nanoparticles in a composition. In various embodiments, embodiments of the compound are capable of being formulated into nanoparticles in a composition. Advantageously, in various embodiments, the design of the compound represented by general formula (1) helps prevent non-specific protein absorption, particle aggregation and controls the size of the nanoparticles formed. In various embodiments, embodiments of the compound represented by general formula (1) helps maintain colloidal stability (of the lipid nanoparticles), and facilitate the condensation and encapsulating / loading of molecules / cargoes into the nanoparticle composition. Advantageously, in various embodiments, embodiments of the compound represented by general formula (1 ) is capable of inducing subcellular interactions and / or enhancing cytosolic delivery of a therapeutic agent, prophylactic agent, and / or biological agent.

[0406] The term “nanoparticles” may comprise and / or may be used interchangeably with the terms “lipid nanoparticles”, “encapsulated lipid nanoparticles”, “loaded lipid nanoparticles”, “LNPs”, “cell-targeting lipid nanoparticles”, “immune cells-targeting lipid nanoparticles”, or the like.

[0407] There is provided a nanoparticle composition comprising:

[0408] (i) a compound represented by general formula (1 ) as disclosed herein; and (ii) a therapeutic agent, prophylactic agent and / or biological agent that is encapsulated / loaded / coupled / bonded / linked / bound in / to said compound.

[0409] Advantageously, the composition is suitable for use in the encapsulation, delivery and / or transfection of one or more therapeutic agent(s), prophylactic agent(s) and / or biological agent(s) e.g., to a desired target (such as subject, cell, cytosol, tissue or organ).

[0410] In various embodiments, the composition further comprises:

[0411] (i) ionizable lipid;

[0412] (ii) neutral / helper lipid;

[0413] (iii) optionally sterol; and

[0414] (iv) optionally amphiphilic lipid (e.g., polyethylene glycol (PEG)-modified lipid).

[0415] In various embodiments, the composition is substantially devoid of polyethylene glycol (PEG). In various embodiments, the composition is substantially devoid of polyethylene glycol (PEG)-modified lipid conjugates, polyethylene glycol (PEG)-modified lipid, PEGylated lipid, PEG-conjugated lipid, PEG-lipid conjugate and / or lipid modified with PEG. In various embodiments, the compound is a substitute / replacement for PEG-lipid conjugate. Advantageously, in various embodiments, the design of the structure of the compound allows said compound to be used, in lieu of or as a substitute / replacement for a conventional PEG-lipid conjugate (e.g., ALC-0159).

[0416] In various embodiments, the compound represented by general formula (1), ionizable lipid, neutral / helper lipid, optionally sterol, and optionally amphiphilic lipid / PEG-modified lipid) are mixed / dissolved in an organic solvent. In various embodiments, the formation of lipid nanoparticle comprises self-assembly of the lipid components and one or more types of molecules or cargoes. In various embodiments, any organic solvent that effectively serves as a medium to contain the lipid components may be used in embodiments of the lipid materials disclosed herein. In various embodiments, the organic solvent is capable of substantially dissolving the components present in the mixture. The organic solvent may comprise methanol, ethanol, isopropanol, acetonitrile, ethyl acetate, tetrahydrofuran, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), the like, or combinations thereof.

[0417] In various embodiments, the ionizable lipid, neutral / helper lipid, sterol, and compound represented by general formula (1) are mixed at a mole ratio of about 25 - 75: about 1 - 20: about 0 - 60: about 0.1 - 45, about 30 - 70: about 2 -19: about 10 - 55: about 0.5 - 40, about 35 - 65: about 4 - 18: about 15 - 50: about 1 - 30, about 40 - 60: about 6 - 16: about 20 - 45: about 1.5 - 25, about 45 - 55: about 8 - 14: about 25 - 40: about 2.5 - 20, or about 50: about 10 - 12: about 30 - 35: about 3- 15.

[0418] In various embodiments, the ionizable lipid, neutral / helper lipid, sterol, compound represented by general formula (1), and amphiphilic lipid / PEG-modified lipid are mixed at a mole ratio of about 25 - 75: about 1 - 20: about 0 - 60: about 0.1 - 65: about 0 -5, about 30 - 70: about 2 - 19: about 10- 55: about 0.5 - 60: about 0.25 - 4, about 35 - 65: about 4 - 18: about 15 - 50: about 1 - 55: about 0.5 - 3.5, about 40 - 60: about 6 - 16: about 20 - 45: about 10 - 50: about 0.75 - 3, about 45 - 55: about 8 - 14: about 25 - 40: about 20 - 40: about 1 - 2.5, or about 50: about 10 - 12: about 30 - 35: about 25 - 35: about 1.5 -2.

[0419] In various embodiments, the ionizable lipid is neutral. In various embodiments the ionizable lipid carries a charge (e g., charged). For example, the ionizable lipid may be protonated or positively-charged. In various embodiments, the ionizable lipid aids in the encapsulation of mRNA via electrostatic interactions. In various embodiments, the neutral / helper lipid improves LNP stability and fusogenicity. In various embodiments, the sterol and / or a derivative(s) (e.g., cholesterol) decreases permeability of lipid nanoparticles and enhances their stability. In various embodiments, the compound represented by formula (1) and / or PEG lipid prevents non-specific protein adsorption, particle aggregation and control size of the lipid nanoparticle.

[0420] In various embodiments, the ionizable lipid includes, but is not limited to ALC-0315, SM-102, Lipid 5, DLinDMA, D-Lin-MC2-DMA, DLin-MC3-DMA, D-Lin-MC4-DMA, Dlin-KC2-DMA, YSK05, AA3-Dlin, SSPalmM, SSPalmO-Phe, Lipid A9, L319, DODMA, CL1, BP Lipid 310, ATX-001, ATX-100, Lipid 2, 80-016B, BP Lipid 309, BP Lipid 307, 93-O17S, 93-0170, NT1-O14B, 306-O12B-3, 306-O12B, 113-O16B, 306Oi10, 306Oi9-cis2, BAMEA-O16B, AI-28, 113-O12B, 98N12-5, Ckk-E12, OF-02, C12-200, BP Lipid 311, BP Lipid 308, BP Lipid 314, BP Lipid 312, LP01, TCL053, Lipid C24, BP Lipid 315, Lipid 29, 9A1P9, C13-112-tri-tail, C13-113-tri-tail, C13-112-tetra-tail, C13-113-tetra-tail, the like or combinations thereof. It will be appreciated that any suitable ionizable lipid (e g., commercially available ionizable lipids) that effectively modulates / adjusts / changes its charge depending on the environmental pH may be used in embodiments of the composition disclosed herein. In various embodiments, the composition comprises from about 25 mol% to about 75 mol%, from about 26 mol% to about 74 mol%, from about 27 mol% to about 73 mol%, from about 28 mol% to about 72 mol%, from about 29 mol% to about 71 mol%, from about 30 mol% to about 70 mol%, from about 31 mol% to about 69 mol%, from about 32 mol% to about 68 mol%, from about 33 mol% to about 67 mol%, from about 34 mol% to about 66 mol%, from about 35 mol% to about 65 mol%, from about 36 mol% to about 64 mol%, from about 37 mol% to about 63 mol%, from about 38 mol% to about 62 mol%, from about 39 mol% to about 61 mol%, from about 40 mol% to about 60 mol%, from about 41 mol% to about 59 mol%, from about 42 mol% to about 58 mol%, from about 43 mol% to about 57 mol%, from about 44 mol% to about 56 mol%, from about 45 mol% to about 55 mol%, from about 46 mol% to about 54 mol%, from about 47 mol% to about 53 mol%, from about 48 mol% to about 52 mol%, from about 49 mol% to about 51 mol%, or about 50 mol% of the ionizable lipid.

[0421] In various embodiments, the neutral / helper lipid comprises a phospholipid such as an unsaturated lipid. Examples of phospholipid includes, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1.2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1.2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1 -glycerol) sodium salt (DOPG), sphingomyelin, the like and combinations thereof.

[0422] In various embodiments, the composition comprises from about 1 mol% to about 20 mol%, from about 1.5 mol% to about 19.5 mol%, from about 2 mol% to about 19 mol%, from about 2.5 mol% to about 18.5 mol%, from about 3 mol% to about 18 mol%, from about 3.5 mol% to about 17.5 mol%, from about 4 mol% to about 17 mol%, from about 4.5 mol% to about 16.5 mol%, from about 5 mol% to about 16 mol%, from about 5.5 mol% to about 15.5 mol%, from about 6 mol% to about 15 mol%, from about 6.5 mol% to about 14.5 mol%, from about 7 mol% to about 14 mol%, from about 7.5 mol% to about 13.5 mol%, from about 8 mol% to about 13 mol%, from about 8.5 mol% to about 12.5 mol%, from about 9 mol% to about 12 mol%, from about 9.5 mol% to about 11.5 mol%, from about 10 mol% to about 11 mol%, or about 10.5 mol% of the neutral / helper lipid.

[0423] In various embodiments, the sterol is selected from cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, avenasterol, the like or combinations thereof.

[0424] In various embodiments, the composition comprises from about 0 mol% to about 60 mol%, from about 0.1 mol% to about 59 mol%, from about 0.25 mol% to about 58 mol%, from about 0.5 mol% to about 57 mol%, from about 0.75 mol% to about 56 mol%, from about 1 mol% to about 55 mol%, from about 1.5 mol% to about 54 mol%, from about 2 mol% to about 53 mol%, from about 3 mol% to about 52 mol%, from about 4 mol% to about 51 mol%, from about 5 mol% to about 50 mol%, from about 6 mol% to about 49 mol%, from about 7 mol% to about 48 mol%, from about 8 mol% to about 47 mol%, from about 9 mol% to about 46 mol%, from about 10 mol% to about 45 mol%, from about 11 mol% to about 44 mol%, from about 12 mol% to about 43 mol%, from about 13 mol% to about 42 mol%, from about 14 mol% to about 41 mol%, from about 15 mol% to about 40 mol%, from about 16 mol% to about 39 mol%, from about 17 mol% to about 38 mol%, from about 18 mol% to about 37 mol%, from about 19 mol% to about 36 mol%, from about 20 mol% to about 35 mol%, from about 21 mol% to about 34 mol%, from about 22 mol% to about 33 mol%, from about 23 mol% to about 32 mol%, from about 24 mol% to about 31 mol%, from about 25 mol% to about 30 mol%, from about 26 mol% to about 29 mol%, or from about 27 mol% to about 28 mol% of the sterol.

[0425] In various embodiments, the amphiphilic lipid / PEG-modified lipid is selected from PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, the like, or combinations thereof. Examples of PEG-modified / PEGylated lipid include, but is not limited to, 2-[(polyethylene glycol)-2000]-N, N-ditetradecylacetamide (ALC-0159), R-3-[(LJ-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DOMG), 3-N-[(cu-methoxypoly (ethyleneglycol)2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-S-DMG), PEG-DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [(polyethylene glycol)-methoxy] (sodium salt)), PEG-DPPC, PEG-DSPE lipid, the like and combinations thereof.

[0426] In various embodiments, the composition comprises from about 0.10 mol% to about 45.0 mol%, from about 1 mol% to about 30 mol%, from about 1.5 mol% to about 25 mol%, from about 2.5 mol% to about 20 mol%, from about 3 mol% to about 15 mol%, or about 12 mol% of the compound represented by formula (1).

[0427] In various embodiments, the composition comprises from about 0.10 mol% to about 5.0 mol%, from about 0.15 mol% to about 4.9 mol%, from about 0.2 mol% to about 4.8 mol%, from about 0.25 mol% to about 4.7 mol%, from about 0.3 mol% to about 4.6 mol%, from about 0.35 mol% to about 4.5 mol%, from about 0.4 mol% to about 4.4 mol%, from about 0.45 mol% to about 4.3 mol%, from about 0.5 mol% to about 4.2 mol%, from about 0.55 mol% to about 4.1 mol%, from about 0.6 mol% to about 4.0 mol%, from about 0.65 mol% to about 3.9 mol%, from about 0.7 mol% to about 3.8 mol%, from about 0.75 mol% to about 3.7 mol%, from about 0.8 mol% to about 3.6 mol%, from about 0.85 mol% to about 3.5 mol%, from about 0.9 mol% to about 3.4 mol%, from about 0.95 mol% to about 3.3 mol%, from about 1.0 mol% to about 3.2 mol%, from about 1.1 mol% to about 3.1 mol%, from about 1.2 mol% to about 3.0 mol%, from about 1.3 mol% to about 2.9 mol%, from about 1.4 mol% to about 2.8 mol%, from about 1.5 mol% to about 2.7 mol%, from about 1.6 mol% to about 2.6 mol%, from about 1.7 mol% to about 2.5 mol%, from about 1.8 mol% to about 2.4 mol%, from about 1.9 mol% to about 2.3 mol%, from about 2.0 mol% to about 2.2 mol%, or about 2.1 mol% of the amphiphilic lipid / PEG-modified lipid.

[0428] In various embodiments, the composition comprises from about 0.1 mol% to about 65 mol%, from about 0.2 mol% to about 64 mol%, from about 0.25 mol% to about 63 mol%, from about 0.5 mol% to about 62 mol%, from about 0.75 mol% to about 61 mol%, from about 1 mol% to about 60 mol%, from about 1.5 mol% to about 59 mol%, from about 2 mol% to about 58 mol%, from about 3 mol% to about 57 mol%, from about 4 mol% to about 56 mol%, from about 5 mol% to about 55 mol%, from about 6 mol% to about 54 mol%, from about 7 mol% to about 53 mol%, from about 8 mol% to about 52 mol%, from about 9 mol% to about 51 mol%, from about 10 mol% to about 50 mol%, from about 11 mol% to about 49 mol%, from about 12 mol% to about 48 mol%, from about 13 mol% to about 47 mol%, from about 14 mol% to about 46 mol%, from about 15 mol% to about 45 mol%, from about 16 mol% to about 44 mol%, from about 17 mol% to about 43 mol%, from about 18 mol% to about 42 mol%, from about 19 mol% to about 41 mol%, from about 20 mol% to about 40 mol%, from about 21 mol% to about 39 mol%, from about 22 mol% to about 38 mol%, from about 23 mol% to about 37 mol%, from about 24 mol% to about 36 mol%, from about 25 mol% to about 35 mol%, from about 26 mol% to about 34 mol%, from about 27 mol% to about 33 mol%, from about 28 mol% to about 32 mol%, from about 29 mol% to about 31 mol%, or about 30 mol% of the compound represented by general formula (1). In various embodiments, the therapeutic agent, prophylactic agent and / or biological agent is provided in an aqueous buffer. The aqueous buffer may be sodium acetate.

[0429] In various embodiments, the nanoparticle composition comprises nanoparticles formed from the compound represented by general formula (1).

[0430] NANOPARTICLES

[0431] There is provided nanoparticles (e.g., lipid nanoparticles) comprising: (i) the compound represented by general formula (1 ) as disclosed herein; and (ii) a therapeutic agent and / or prophylactic agent and / or biological agent that is encapsulated / loaded / coupled / bonded / linked / bound in / to the compound represented by general formula (1).

[0432] In various embodiments, the nanoparticles have a N: P or N / P ratio (i.e. molar ratio of ionizable nitrogen atoms in the ionizable lipid to phosphate groups in the therapeutic agent, prophylactic agent and / or biological agent (e.g., nucleic acid) of from about 1:1 to about 40:1. The nanoparticles may have a N: P or N / P ratio that is from about 1: 1 to about 40: 1, from about 2: 1 to about 39: 1, from about 3:1 to about 38:1, from about 4:1 to about 37:1, from about 5:1 to about 36:1, from about 6:1 to about 35:1, from about 7:1 to about 34:1, from about 8:1 to about 33:1, from about 9:1 to about 32:1, from about 10:1 to about 31:1, from about 11:1 to about 30: 1, from about 12:1 to about 29: 1, from about 13:1 to about 28:1, from about 14:1 to about 27:1, from about 15:1 to about 26:1, from about 16:1 to about 25: 1, from about 17:1 to about 24: 1, from about 18:1 to about 23: 1, from about 19:1 to about 22:1, or from about 20:1 to about 21:1.

[0433] It will be appreciated that in various embodiments, the optimal N / P ratio is dependent on the type of therapeutic and / or prophylactic agent and / or biological agent (e.g., a nucleic acid such as mRNA, siRNA, miRNA, pDNA and oligonucleotides). For example, the optimal N / P ratio may be different for siRNA, pDNA and oligonucleotides. In various embodiments, it will be appreciated that shorter nucleic acid therapeutics (e g. siRNA) or prophylactic agents (e.g., mRNA) require more (i.e. a larger amount / concentration / volume of) ionizable lipids to encapsulate them into lipid nanoparticles. In various embodiments therefore, a N / P ratio of up to about 20:1 is used to encapsulate and deliver nucleic acid therapeutics (e.g. shorter nucleic acid therapeutics siRNA).

[0434] In various embodiments, the encapsulation / loading / binding efficiency / capacity of the therapeutic and / or prophylactic agent and / or biological agent in the composition / nanoparticle is at least about 20.0%, at least about 30.0%, at least about 40.0%, at least about 50.0%, at least about 60.0%, at least about 70.0%, at least about 80.0%, at least about 90.0%, at least about 95.0%, at least about 96.0%, at least about 97.0%, at least about 98.0%, at least about 99.0%, at least about 99.5%, at least about 99.9%, or about 100%.

[0435] In various embodiments, the nanoparticles have an encapsulation efficiency that is slightly lower, comparable to, no less or is higher than that of corresponding nanoparticles using ALC-0159 as the PEG-lipid conjugate under similar conditions. For example, the encapsulation efficiency may be at least about 50% of that of a corresponding nanoparticle using ALC-0159 as the PEG-lipid conjugate under similar conditions. In another example, the encapsulation efficiency may be at least about 1% to at least about 50% higher than that of corresponding nanoparticles using ALC-0159 as the PEG-lipid conjugate under similar conditions.

[0436] In various embodiments, the cell transfection efficiency (% of the cells that are transfected with the gene) of the composition / nanoparticles is at least about 1.0%, at least about 5.0%, at least about 10.0%, at least about 15.0%, at least about 20.0%, at least about 25.0%, at least about 30.0%, at least about 35.0%, at least about 40.0%, at least about 45.0%, at least about 50.0%, at least about 55.0%, at least about 60.0%, at least about 65.0%, at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 90.0%, at least about 95.0%, at least about 96.0%, at least about 97.0%, at least about 98.0%, at least about 99.0%, at least about 99.5%, at least about 99.9%, or about 100%. In various embodiments, the cell transfection efficiency is not required to be 100%. For example, it will be appreciated that vaccine applications may not need / require to transfect 100% cells in order to mediate an immune response, unlike in the case of cancer therapy applications.

[0437] In various embodiments, the nanoparticles have a cell transfection efficiency that is comparable to, no less or is higher than that of corresponding nanoparticles using ALC-0159 as the PEG-lipid conjugate under similar conditions. For example, the cell transfection efficiency may be at least about 30% of that of a corresponding nanoparticle using ALC-0159 as the PEG-lipid conjugate under similar conditions. In another example, the cell transfection efficiency may be at least about 50% to at least about 3,000% higher than that of corresponding nanoparticles using ALC-0159 as the PEG-lipid conjugate under similar conditions (e g at least about 1.5 times to about 30 times the cell transfection efficiency of corresponding nanoparticles using ALC-0159 as the PEG-lipid conjugate under similar conditions).

[0438] In various embodiments, the transfection efficiency in certain cell lines is lower than that of corresponding nanoparticles using ALC-0159 as the PEG-lipid conjugate under similar conditions but still at the same order of magnitude. It will be appreciated that such level of gene transfection may still be applicable for gene therapy.

[0439] Advantageously, in various embodiments, the average or mean particle size (or diameter) nanoparticles are designed to be customizable / adjustable to suit a desired application. In various embodiments, the nanoparticle has an average or mean particle size (or diameter) of no more than about 500 nm, no more than about 450 nm, no more than about 400 nm, no more than about 350 nm, no more than about 300 nm, no more than about 250 nm, no more than about 200 nm, or no more than about 150 nm. In various embodiments, the nanoparticle has an average or mean particle size (or diameter) of no more than about 500 nm, no more than about 450 nm, no more than about 400 nm, no more than about 350 nm, no more than about 300 nm, no more than about 250 nm, no more than about 200 nm, or no more than about 150 nm. In various embodiments, the nanoparticles have an average or mean particle size (or diameter) of from about 40 nm to about 500 nm, from about 50 nm to about 490 nm, from about 60 nm to about 480 nm, from about 70 nm to about 470 nm, from about 80 nm to about 460 nm, from about 90 nm to about 450 nm, from about 100 nm to about 440 nm, from about 110 nm to about 430 nm, from about 120 nm to about 420 nm, from about 130 nm to about 410 nm, from about 140 nm to about 400 nm, from about 150 nm to about 390 nm, from about 160 nm to about 380 nm, from about 170 nm to about 370 nm, from about 180 nm to about 360 nm, from about 190 nm to about 350 nm, from about 200 nm to about 340 nm, from about 210 nm to about 330 nm, from about 220 nm to about 320 nm, from about 230 nm to about 310 nm, from about 240 nm to about 300 nm, from about 250 nm to about 290 nm, from about 260 nm to about 280 nm, about 270 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, or about 450 nm.

[0440] In various embodiments, the composition comprising the nanoparticles has a polydispersity index (PDI) of no more than about 0.50, no more than about 0.40, no more than about 0.30, or no more than about 0.20. For example, the composition comprising the nanoparticles has a polydispersity index (PDI) of from about 0.005 to about 0.50, from about 0.01 to about 0.40, or from about 0.05 to about 0.30. Advantageously, in various embodiments, the nanoparticle has a narrow particle size distribution, and / or the nanoparticle composition is relatively / substantially homogenous.

[0441] In various embodiments, the nanoparticle has a zeta potential of from about -30.0 mV to about +30.0 mV, from about -29.0 mV to about +29.0 mV, from about -28.0 mV to about +28.0 mV, from about -27.0 mV to about +27.0 mV, from about -26.0 mV to about +26.0 mV, from about -25.0 mV to about +25.0 mV, from about -24.0 mV to about +24.0 mV, from about -23.0 mV to about +23.0 mV, from about -22.0 mV to about +22.0 mV, from about -21.0 mV to about +21.0 mV, from about -20.0 mV to about +20.0 mV, from about -19.0 mV to about +19.0 mV, from about -18.0 mV to about +18.0 mV, from about -17.0 mV to about +17.0 mV, from about -16.0 mV to about +16.0 mV, from about -15.0 mV to about +15.0 mV, from about -14.0 mV to about +14.0 mV, from about -13.0 mV to about +13.0 mV, from about -12.0 mV to about +12.0 mV, from about -11.0 mV to about +11.0 mV, from about -10.0 mV to about +10.0 mV, from about -9.0 mV to about +9.0 mV, from about -8.0 mV to about +8.0 mV, from about -7.0 mV to about 7.0 mV, from about -6.0 mV to about 6.0 mV, from about -5.0 mV to about +5.0 mV, from about -4.0 mV to about 4.0 mV, from about -3.0 mV to about +3.0 mV, from about -2.0 mV to about +2.0 mV, from about -1.0 mV to about +1.0 mV, or about 0 mV in saline (e.g., phosphate-buffered saline (PBS)) or in a physiological environment. Advantageously, in various embodiments, the nanoparticle has a substantially neutral surface charge, making the nanoparticle suitable / desirable for in vivo applications.

[0442] In various embodiments, the cell viability of the composition / nanoparticle is at least about 20.0%, at least about 30.0%, at least about 40.0%, at least about 50.0%, at least about 60.0%, at least about 70.0%, at least about 80.0%, at least about 90.0%, at least about 95.0%, at least about 96.0%, at least about 97.0%, at least about 98.0%, at least about 99.0%, at least about 99.5%, or at least about 99.9%.

[0443] In various embodiments, the nanoparticle has a cell viability that is no less or is higher than that of a corresponding nanoparticle using ALC-0159 as the PEG-lipid conjugate under similar conditions. For example, the cell viability may be at least comparable to that of a corresponding nanoparticle using ALC-0159 as the PEG-lipid conjugate under similar conditions. Accordingly, in various embodiments, the nanoparticle comprises / possesses high cytocompatibility and / or negligible cytotoxicity. In various embodiments, the composition / compound / nanoparticle is biocompatible, i.e. the composition / compound / nanoparticle is compatible with biological systems or parts of the biological systems without substantially or significantly eliciting an adverse physiological response such as a toxic reaction / response (e.g., cytotoxicity), an immune reaction / response, an injury or the like when used on the human or animal body. In various embodiments, the composition / compound / nanoparticle is substantially devoid of substances that elicit an adverse physiological response. It will be appreciated that the composition / compound / nanoparticle may trigger / elicit an immune response (e.g., to enhance vaccination efficacy), and in such embodiments, the composition / compound / nanoparticle is still considered to be biocompatible. Advantageously, the nanoparticles (e.g., lipid nanoparticles) are capable of binding therapeutic agent, prophylactic agent and / or biological agent (e.g., RNA) effectively and / or providing high transfection efficiency without causing / inducing substantial or any cytotoxicity.

[0444] In various embodiments, the therapeutic agent and / or prophylactic agent and / or biological agent comprises nucleic acid (e.g., ribonucleic acid (RNA), messenger ribonucleic acid (mRNA), small interfering ribonucleic acid (siRNA), deoxyribonucleic acid (DNA), plasmid deoxyribonucleic acid (pDNA), oligonucleotides such as antisense oligonucleotide (ASO)), therapeutics (e.g., negatively charged therapeutics), drug molecule, vaccine (e.g., dengue vaccine), the like, or combinations thereof.

[0445] METHOD OF PREPARING NANOPARTICLES

[0446] There is provided a method of preparing nanoparticles as disclosed herein, the method comprising:

[0447] (f-i) preparing an aqueous composition comprising therapeutic agent and / or prophylactic agent and / or biological agent;

[0448] (f-ii) mixing the aqueous composition obtained from (f-i) with the composition as disclosed herein to obtain nanoparticles. In various embodiments, (f-i) comprises mixing therapeutic and / or prophylactic agent and / or biological agent in an aqueous buffer. The aqueous buffer may be sodium acetate, citrate buffer, phosphate buffer, glycine buffer solution, or the like or combinations thereof.

[0449] In various embodiments, (f-i) is performed at a pH value of from about 2.5 to about 6.5, from about 2.6 to about 6.4, from about 2.7 to about 6.3, from about 2.8 to about 6.2, from about 2.9 to about 6.1, from about 3.0 to about 6.0, from about 3.1 to about 5.9, from about 3.2 to about 5.8, from about 3.3 to about 5.7, from about 3.4 to about 5.6, from about 3.5 to about 5.5, from about 3.6 to about 5.4, from about 3.7 to about 5.3, from about 3.8 to about 5.2, from about 3.9 to about 5.1, from about 4.0 to about 5.0, from about 4.1 to about 4.9, from about 4.2 to about 4.8, from about 4.3 to about 4.7, from about 4.4 to about 4.6, or about 4.5.

[0450] In various embodiments, the composition as disclosed herein comprises organic phase (e g., ethanol). In various embodiments, the aqueous composition comprises aqueous phase. In various embodiments, (f-ii) comprises mixing the aqueous composition with the organic composition as disclosed herein at a volume ratio of the aqueous phase to organic phase from about 1: 1 to about 10: 1. For example, the aqueous phase may be mixed with the organic phase at a volume ratio of from about 11:1 to about 10:1, at about 9:1, at about 8:1, at about 7:1, at about 6:1, at about 5:1, at about 4:1, at about 3:1, or at about 2:1.

[0451] In various embodiments, (f-ii) of mixing the aqueous composition with the composition comprises rapid pipetting, microfluidic mixing, the like or combinations thereof. In various embodiments, (f-ii) of mixing the aqueous composition with the organic composition comprises injecting (e.g., direct injecting) the organic composition into the aqueous composition. In various embodiments, (f-ii) of mixing the aqueous composition with the organic composition comprises pipetting (e.g., rapid pipetting) the organic composition into the aqueous composition.

[0452] In various embodiments, mixing the aqueous composition with the composition comprises simple / straightforward T-shaped mixing. In various embodiments, (f-ii) of mixing the aqueous composition with the composition comprises micro-mixing, e.g., microfluidic mixing using a microfluidic device. The micro-mixing may be performed via passive mixing using passive micromixers such as T-shaped or Y-shaped microfluidic mixers parallel lamination, sequential, focusing enhanced mixers or droplet micromixers. The micro-mixing may also be performed via active mixing using external forces such as pressure field, electrokinetic, dielectrophoretic, electrowetting, magneto-hydrodynamic or ultrasound. Advantageously, as microfluidic mixing comprises mixing the two compositions (i.e. aqueous composition and composition disclosed herein) in a controlled manner and / or with a specified / fixed / controlled / precise mixing ratio, the interaction between the two compositions (e.g., between ionizable lipid and therapeutic, prophylactic and / or biological agent) is regulated, thereby producing nanoparticles with a smaller particle size and / or with a narrow size distribution or homogeneity (e.g, smaller PDI).

[0453] In various embodiments, the method further comprises removing the organic phase (e.g. ethanol). For example, removing the organic phase may include dialysis or filtration. Advantageously, removal of the organic phase through dialysis or filtration may improve the encapsulation efficiency of the therapeutic and / or prophylactic agent and / or biological agent.

[0454] In various embodiments, there is also provided a carrier, nanocarrier or delivery system / vehicle comprising the composition / compound / nanoparticles as disclosed herein.

[0455] In various embodiments, there is also provided a vaccine composition comprising the composition / compound / nanoparticles as disclosed herein. In various embodiments, there is also provided a carrier, a nanocarrier, a delivery system / vehicle, a compound, a nanoparticle composition, nanoparticles (or lipid nanoparticles) disclosed herein for use in medicine (e g., for the treatment or prophylaxis of one or more of the diseases, disorders or conditions mentioned herein).

[0456] In various embodiments, there is also provided a carrier, a nanocarrier, a delivery system / vehicle, a compound, a nanoparticle composition, nanoparticles (or lipid nanoparticles) disclosed herein for use in the treatment or prophylaxis of a disease, disorder or condition, the use of said carrier, a nanocarrier, a delivery system / vehicle, a compound, a nanoparticle composition, nanoparticles (or lipid nanoparticles) in the manufacture of a medicament for the treatment or prophylaxis of a disease, disorder or condition and / or a method of treatment or prophylaxis of a disease, disorder or condition, comprising a step of administering (e.g. in a therapeutically effective amount of) said carrier, a nanocarrier, a delivery system / vehicle, a compound, a nanoparticle composition, nanoparticles (or lipid nanoparticles) to a subject (e.g., vertebrate such as a human ora large veterinary mammal (e.g., horses, cattle, deer, sheep, llamas, goats, pigs) in need thereof.

[0457] The disease, disorder or condition may be selected from the group consisting of infectious / contagious diseases, viral infections (i.e. diseases caused by virus), bacterial infections (i.e. diseases caused by bacteria), fungal infections (i.e. diseases caused by fungi), respiratory diseases or the like, cancer, cardiovascular diseases, skin disease or the like, or combinations thereof. In various embodiments, the disease, disorder or condition is mediated by an influenza virus (e.g., influenza A, B, C and / or D virus). For example, the disease may be influenza A, B, C or D such as H1N1, H3N2). In various embodiments, the disease, disorder or condition is mediated by a coronavirus (e.g., severe acute respiratory syndrome coronavirus such as SARS-CoV-2 or SARS-CoV-1). For example, the disease, disorder or condition may be SARS-CoV-2 coronavirus disease. In various embodiments, the disease, disorder or condition is mediated by a dengue virus (e.g., DEN-1, DEN-2, DEN-3, and / or DEN-4 virus). For example, the disease may be dengue disease.

[0458] In various embodiments, there is also provided a carrier, a nanocarrier, a delivery system / vehicle, a compound, a nanoparticle composition, nanoparticles (or lipid nanoparticles) disclosed herein for use in encapsulating and / or delivering a therapeutic, prophylactic and / or biological agent to a subject, cell, cytosol, tissue or organ (e.g., a mammalian cell, cytosol, tissue or organ), the use of said carrier, a nanocarrier, a delivery system / vehicle, a compound, a nanoparticle composition, nanoparticles (or lipid nanoparticles) in the manufacture of a medicament for encapsulating and / or delivering a therapeutic, prophylactic and / or biological agent to a subject, cell, cytosol, tissue or organ (e.g., a mammalian cell, cytosol, tissue or organ), and / or a method of delivering a therapeutic, prophylactic and / or biological agent to a subject, cell, cytosol, tissue or organ (e.g., a mammalian cell, cytosol, tissue or organ), comprising a step of administering (e.g. in a therapeutically effective amount of) said carrier, a nanocarrier, a delivery system / vehicle, a compound, a nanoparticle composition, nanoparticles (or lipid nanoparticles) to a subject (e.g., vertebrate such as a human or a large veterinary mammal (e.g., horses, cattle, deer, sheep, llamas, goats, pigs)) in need thereof.

[0459] In various embodiments, there is also provided a carrier, a nanocarrier, a delivery system / vehicle, a compound, a nanoparticle composition, nanoparticles (or lipid nanoparticles) disclosed herein for use in inducing or modulating an immune response in a subject (e.g., vertebrate such as a human or a large veterinary mammal (e.g., horses, cattle, deer, sheep, llamas, goats, pigs)), the use of said carrier, a nanocarrier, a delivery system / vehicle, a compound, a nanoparticle composition, nanoparticles (or lipid nanoparticles) in the manufacture of a medicament for inducing or modulating an immune response in a subject, and / or a method of inducing an immune response in a subject, comprising a step of administering (e.g. in a therapeutically effective amount of) said carrier, a nanocarrier, a delivery system / vehicle, a compound, a nanoparticle composition, nanoparticles (or lipid nanoparticles) to a subject in need thereof. In various embodiments, an immune response in the subject is to be induced / modulated through the administration of the compound, a nanoparticle composition, nanoparticles (or lipid nanoparticles) thereto. In various embodiments, by inducing an immune response in the subject, the subject is protected against various diseases, disorders or conditions e.g., infectious / contagious diseases, viral infections (i.e. diseases caused by virus), bacterial infections (i.e. diseases caused by bacteria), fungal infections (i.e. diseases caused by fungi), respiratory diseases or the like, or combinations thereof as mentioned herein. The carrier, nanocarrier, delivery system / vehicle, compound, nanoparticle composition, nanoparticles may be delivered to a subject in the form of or as a component of a vaccine.

[0460] In various embodiments, the disease, disorder or condition is mediated by an influenza virus (e.g., influenza A, B, C and / or D virus). For example, the disease may be influenza A, B, C or D such as H1N1, H3N2). In various embodiments, the disease, disorder or condition is mediated by a coronavirus (e.g., severe acute respiratory syndrome coronavirus such as SARS-CoV-2 or SARS-CoV-1). For example, the disease, disorder or condition may be SARS-CoV-2 coronavirus disease.

[0461] In various embodiments, the carrier, nanocarrier, delivery system / vehicle, compound, nanoparticle composition, nanoparticles prepared from embodiments of the method disclosed herein comprises one or more of the following characteristics or properties: broad applicability (e.g., can be used to encapsulate, deliver and / or transfect a wide range of therapeutic, prophylactic, and / or biological reagents), nanosized, substantially neutral surface charge, high encapsulation efficiency, high and effective transfection efficiency both in vitro and in vivo, high stability, low toxicity (e.g., low cytotoxicity), low production / synthesis cost, therefore making them suitable for applications that require efficient cellular uptake and / or gene transfection. It will be appreciated by a person skilled in the art that other variations and / or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure as broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included, etc. or the like across different exemplary embodiments. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.

[0462] BRIEF DESCRIPTION OF FIGURES

[0463] FIG. 1 shows1H NMR of cholesterol-mannose-OAc (CMO) (solvent, CDCl3) in accordance with various embodiments disclosed herein.

[0464] FIG. 2 shows1H NMR of cholesterol-mannose (CM) (solvent, CDCl3) in accordance with various embodiments disclosed herein.

[0465] FIG. 3 shows1H NMR of Ser(1,2,3,4, 6-penta-O-acetyl-D-mannopyranose)-NCA (solvent, CDCl3) in accordance with various embodiments disclosed herein.

[0466] FIG. 4 shows13C NMR of Ser(1,2,3,4, 6-penta-O-acetyl-D-mannopyranose)-NCA (solvent, CDCl3) in accordance with various embodiments disclosed herein.

[0467] FIG. 5 shows1H NMR of cholesteryl N-(ammonioethyl)carbamate (solvent, CDCl3) in accordance with various embodiments disclosed herein.

[0468] FIG. 6 shows1H NMR of cholesterol--poly(Ser-D-mannose) with DP 15 (solvent, DMSO-d6) in accordance with various embodiments disclosed herein. FIG. 7 shows1H NMR of cholesterol--poly(Ser-D-mannose) with DP 6 (solvent, DMSO-d6) in accordance with various embodiments disclosed herein.

[0469] FIG. 8 shows transfection efficiency of mRNA LNPs at different cholesterol / CMO / CM mole ratio in HEK293T cells after 48 h of incubation with mRNA LNPs designed in accordance with various embodiments disclosed herein. Statistical significance for transfection efficiency was determined using the one-way ANOVA and comparing ALC-0159 (**** p < 0.0001 ).

[0470] FIG. 9 shows transfection efficiency of mRNA LNPs at different cholesterol / CM mole ratio in HEK293T cells after 48 h of incubation with mRNA LNPs designed in accordance with various embodiments disclosed herein. Statistical significance for transfection efficiency was determined using the oneway ANOVA and comparing ALC-0159 (**** p < 0.0001).

[0471] FIG. 10 shows transfection efficiency of PEG-free LNPs at different CPSM-1 / CM mole ratio in HEK293T cells after 48 h of incubation with PEG-free mRNA LNPs designed in accordance with various embodiments disclosed herein. Statistical significance for transfection efficiency was determined using the oneway ANOVA and comparing ALC-0159 (**** p < 0.0001).

[0472] FIG. 11 shows transfection efficiency of PEG-free LNPs at different CPSM-2 mole ratio in HEK293T cells after 48 h of incubation with PEG-free mRNA LNPs designed in accordance with various embodiments disclosed herein. Statistical significance for transfection efficiency was determined using the one-way ANOVA and comparing ALC-0159 (**** p < 0.0001).

[0473] FIG. 12 shows transfection efficiency of PEG-free LNPs at different cholesterol / CM mole ratio in HEK293T cells after 48 h of incubation with PEG-free mRNA LNPs designed in accordance with various embodiments disclosed herein. Statistical significance for transfection efficiency was determined using the one-way ANOVA and comparing ALC-0159 (**** p < 0.0001 )

[0474] FIG. 13 shows cell viability of HEK293T cells after 48 h of incubation with mRNA LNPs formulated at different cholesterol / CMO / CM mole ratios in accordance with various embodiments disclosed herein.

[0475] FIG. 14 shows cell viability of HEK293T cells after 48 h of incubation with mRNA LNPs formulated at different cholesterol / CM mole ratios in accordance with various embodiments disclosed herein.

[0476] FIG. 15 shows cell viability of HEK293T cells after 48 h of incubation with PEG-free mRNA LNPs formulated at different CPSM-1 / CM mole ratios in accordance with various embodiments disclosed herein.

[0477] FIG. 16 shows cell viability of HEK293T cells after 48 h of incubation with PEG-free mRNA LNPs formulated at different CPSM-2 mole ratios in accordance with various embodiments disclosed herein.

[0478] FIG. 17 shows cell viability of HEK293T cells after 48 h of incubation with PEG-free mRNA LNPs formulated at different cholesterol / CM mole r in accordance with various embodiments disclosed herein.

[0479] FIG. 18 shows transfection efficiency of mRNA LNPs at different cholesterol / CMO / CM mole ratio in DC2.4 cells after 48 h of incubation with mRNA LNPs designed in accordance with various embodiments disclosed herein. Statistical significance for transfection efficiency was determined using the oneway ANOVA and comparing ALC-0159 (**** p < 0.0001).

[0480] FIG. 19 shows transfection efficiency of mRNA LNPs at different cholesterol / CM mole ratio in DC2.4 cells after 48 h of incubation with mRNA LNPs designed in accordance with various embodiments disclosed herein. Statistical significance for transfection efficiency was determined using the one-way ANOVA and comparing ALC-0159 (**** p < 0.0001).

[0481] FIG. 20 shows transfection efficiency of PEG-free LNPs at different CPSM-1 / CM mole ratio in DC2.4 cells after 48 h of incubation with PEG-free mRNA LNPs designed in accordance with various embodiments disclosed herein. Statistical significance for transfection efficiency was determined using the oneway ANOVA and comparing ALC-0159 (**** p < 0.0001).

[0482] FIG. 21 shows transfection efficiency of PEG-free LNPs at different CPSM-2 mole ratio in DC2.4 cells after 48 h of incubation with PEG-free mRNA LNPs designed in accordance with various embodiments disclosed herein. Statistical significance for transfection efficiency was determined using the one-way ANOVA and comparing ALC-0159 (**** p < 0.0001).

[0483] FIG. 22 shows transfection efficiency of PEG-free LNPs at different cholesterol / CM mole ratio in DC2.4 cells after 48 h of incubation with PEG-free mRNA LNPs designed in accordance with various embodiments disclosed herein. Statistical significance for transfection efficiency was determined using the one-way ANOVA and comparing ALC-0159 (**** p < 0.0001 ).

[0484] FIG. 23 shows cell viability of DC2.4 cells after 48 h of incubation with mRNA LNPs formulated at different cholesterol / CMO / CM mole ratios in accordance with various embodiments disclosed herein.

[0485] FIG. 24 shows cell viability of DC2.4 cells after 48 h of incubation with mRNA LNPs formulated at different cholesterol / CM mole ratios in accordance with various embodiments disclosed herein.

[0486] FIG. 25 shows cell viability of DC2.4 cells after 48 h of incubation with PEG-free LNPs formulated at different CPSM-1 / CM mole ratios in accordance with various embodiments disclosed herein. FIG. 26 shows cell viability of DC2.4 cells after 48 h of incubation with PEG-free mRNA LNPs formulated at different CPSM-2 mole ratios in accordance with various embodiments disclosed herein.

[0487] FIG. 27 shows cell viability of DC2.4 cells after 48 h of incubation with PEG-free mRNA LNPs formulated at different cholesterol / CM mole ratios in accordance with various embodiments disclosed herein.

[0488] EXAMPLES

[0489] Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following examples, tables and if applicable, in conjunction with the figures. It should be appreciated that other modifications related to structural, and / or chemical changes may be made without deviating from the scope of the invention. Example embodiments are not necessarily mutually exclusive as some may be combined with one or more embodiments to form new example embodiments. The example embodiments should not be construed as limiting the scope of the disclosure.

[0490] The following examples describe the development of a series of cholesterol-derived lipids and mannopolypeptides that are useful for delivery of a therapeutic agent and / or prophylactic agent and / or biological agent (e.g., delivery of RNA, nucleic acids, DNA, negatively charged therapeutics etc). Advantageously, embodiments of the cholesterol-derived lipids and mannopolypeptides disclosed herein serve as replacement of PEGylated lipids. Cholesterol-derived lipids or mannopolypeptides are designed for tuning the structural properties of LNPs to enhance vaccination efficacy and / or RNA transfection efficiency. Specifically, mRNA LNPs formulated from cholesterolderived mannopolypeptides or cholesterol-mannose as a replacement for PEGylated lipid such as ALC-0159 show narrow size distribution (PDI < 0.2), near-neutral surface zeta potential / charge and high encapsulation efficiency. The transfection efficiencies of mRNA LNPs formulated from cholesterol-derived mannopolypeptides or cholesterol-mannose are significantly greater than that mRNA LNPs formulated from ALC-0159, which is used in Pfizer-BioNTech’s COVID-19 mRNA vaccine, in HEK 293T and DC 2.4 cells without causing cytotoxicity. Cholesterol-derived mannopolypeptides and cholesterol-mannose can serve as the replacement for ALC-0159 in the delivery of nucleic acids, potentially minimizing the risk of allergic reaction by the PEGylated lipids and the accelerated clearance of systemically delivered PEGylated nanoparticles, and enhancing vaccination / therapeutic efficacy. Furthermore, cholesterol-derived mannopolypeptides and cholesterol-mannose can induce the subcellular interactions, improving the cytosolic delivery of mRNA.

[0491] As shown in the following examples, cholesterol-derived mannopolypeptide mediates PEG-free lipid nanoparticles for efficient mRNA delivery. PEG-free LNP compositions formed from cholesterol-derived lipids or mannopolypeptidescan deliver RNA effectively with negligible cytotoxicity.

[0492] Example 1: Materials and Methods

[0493] 1.1. Materials

[0494] Chemical reagents for the synthesis of the cholesterol-derived mannopolypeptides were purchased from Sigma-Aldrich and used as received unless otherwise noted. Helper lipid 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), cholesterol and ionizable lipid ALC-0315 were purchased from MedChem Express (Monmouth Junction, NJ, USA). Sodium acetate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Triton®-X100, Tris-EDTA, and VivoGlo Luciferin (In Vivo Grade) were purchased from Promega (Madison, Wl, USA). Alamar Blue and Pierce Firefly Luciferase Glow assay kit were purchased from Invitrogen (Waltham, MA, USA). Other reagents used were of analytical grade. 1.2. Synthesis of cholesterol-mannose-OAc (CMO) and cholesterol-mannose (CM)

[0495] Synthesis strategy for cholesterol-mannose-OAc (CMO) and cholesterolmannose (CM) is shown in Scheme 1. 1,2,3,4, 6-Penta-O-acetyl-D-mannopyranose was first synthesized. D-mannose (30.6 g, 170 mmol) was dissolved in 150 mL of pyridine under N2. To the solution was added dropwise acetic anhydride (160 mL, 1.7 mol) at 0°C. The reaction mixture was stirred for 24 h at room temperature. The reaction solution was slowly poured into 1000 mL of ice water and then extracted with 300 mL of ethyl acetate for 3 times. The organic phase was washed with 300 mL of saturated NaHCO3for 2 times, and then washed with 1.0 M HCI, saturated brine. The organic phase was dried over anhydrous Na2SO4and the organic solvent was evaporated in vacuo. The resulting crude product was purified by flash silica gel column chromatography (hexane / ethyl acetate 8:2, v / v). The yield of the compound was 90.0%.

[0496] Benzyl (2-hydroxyethyl)carbamate (Cbz-ethanolamine) was then synthesized. General procedure for synthesis of Cbz-ethanolamine: To a mixture of ethanolamine (10.0 g, 163.5 mmol) and triethylamine (TEA, 27.5 mL, 197.5 mmol) in 250 mL of dry DCM was added 28 mL of benzyl carbonochloridate in ice bath, and the reaction mixture was stirred for 24 h at room temperature. The solvent was evaporated in vacuo. The resulting residue was dissolved in 400 mL of DCM, washed with saturated NaHCOs, saturated brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The obtained crude product was purified by flash silica gel column chromatography (DCM / MeOH 30:1, v / v) to give Cbz-ethanolamine (30.3 g, 95%).

[0497] Cbz-ethanolamine-1,2,3,4,6-penta-O-acetyl-manno-D-pyranose was then synthesized. General synthesis procedure for Cbz-ethanolamine-1,2, 3, 4, 6-penta-O-acetyl-manno-D-pyranose: To a mixture of Cbz-ethanolamine (5.36 g, 27.5 mmol) and 1,2,3,4,6-penta-O-acetyl-manno-D-pyranose (14.2 g, 36.4 mmol) in 200 mL of dry DCM was dropwise added 26.5 mL of BF3·OEt2, and the reaction mixture was stirred under N2for 24 h at room temperature. The solvent was evaporated in vacuo. The resulting residue was dissolved in 400 mL of ethyl acetate, washed with saturated brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The obtained crude product was purified by flash silica gel column chromatography (DCM / MeOH 30:1, v / v) to give Cbz-ethanolamine-1,2,3,4,6-penta-O-acetyl-manno-D-pyranose (9.4 g, 65%).

[0498] The Cbz group deprotection of Cbz-ethanolamine-1, 2,3,4, 6-penta-O-acetyl-manno-D-pyranose was then performed to yield ethanolamine-1, 2, 3,4,6-Penta-O-acetyl-manno-D-pyranose. General synthesis procedure for ethanolamine-1, 2, 3, 4, 6-Penta-O-acetylmanno-D-pyranose: Cbz-ethanolamine-1,2,3,4,6-Penta-O-acetyl-manno-D-pyranose (7.9 g, 15 mmol) was dissolved in 40 mL of a 1:1 mixture of THF and MeOH. Pd / C 10 w / w% (790 mg) was added into mixture under rigorous stirring. The reaction mixture was stirred under 7 mbar hydrogen atmosphere for 12 h. The resulting crude product was purified by flash silica gel column chromatography (DCM / MeOH 30:1, v / v) to give ethanolamine- 1,2,3,4,6-Penta-O-acetyl-manno-D-pyranose (5.5 g, 95%).

[0499] Cholesterol-mannose-OAc (CMO) was synthesized. To a mixture of ethanolamine-1, 2, 3, 4, 6-penta-O-acetyl-manno-D-pyranose (1.96 g, 5.0 mmol) and triethylamine (TEA, 606 mg, 6.0 mmol) in 60 mL of dry DCM was added 2.25 g of cholesteryl chloroformate in ice bath, and the reaction mixture was stirred for 24 h at room temperature under N2 atmosphere. The resulting reaction solution was washed with 1.0 M HCI aqueous solution, saturated brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The obtained crude product was purified by flash silica gel column chromatography (hexane / EA 8:2, v / v) to give CMO (3.6 g, 90%). The structure of CMO was verified by1H NMR spectra (FIG.

[0500] 1).

[0501] Cholesterol-mannose (CM) was then synthesized. General synthesis procedure for cholesterol-mannose: 1.6 g of cholesterol-mannose-OAc was dissolved in 50 mL of MeOH, to which was added 5.0 mL of 25% MeONa in MeOH. The mixture was stirred at room temperature for 15 min. The crude product was precipitated with de-ionized (DI) water for removing MeONa and MeOH. The product was washed with DI water for 3 times, and obtained by freeze-drying under vacuum. The yield of the CM was 85%. The structure of CM was verified by1H NMR spectra (FIG. 2).

[0502] 1.3. Synthesis of Ser(1,2,3,4,6-penta-O-acetyl-D-mannopyranose)-NCA Synthesis strategy for the Ser(1,2,3,4, 6-penta-O-acetyl-D-mannopyranose) N-carboxy anhydride (NCA) is shown in Scheme 2.

[0503] Carbobenzyloxy-Ser(1,2,3,4,6-penta-O-acetyl-D-mannopyranose) was first synthesized. To a mixture of Cbz-Serine (6.57 g, 27.5 mmol) and 1, 2,3,4, 6-penta-O-acetyl-D-mannopyranose (10.8 g, 27.5 mmol) in 250 mL of CH2CI2 (DCM) was dropwise added 26.5 mL of BF3·OEt2, and the reaction mixture was stirred under N2for 24 h at room temperature. The solvent was evaporated in vacuo. The resulting residue was dissolved in 400 mL of ethyl acetate, washed with saturated brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The obtained crude product was purified by flash silica gel column chromatography (DCM / MeOH 30:1, v / v) to give Cbz-Ser(1,2,3,4,6-penta-O-acetyl-D-mannopyranose) (10.5 g, 65%).

[0504] The Cbz group deprotection of Cbz-Ser(1,2,3,4, 6-penta-O-acetyl-D-mannopyranose) was then performed to yield Ser(1,2,3,4, 6-penta-O-acetyl-D-mannopyranose). General synthesis procedure for Ser(1, 2,3,4, 6-penta-O-acetyl-D-mannopyranose): To Cbz-Ser(1,2,3,4,6-penta-O-acetyl-D-mannopyranose) (10.0 g, 17.5 mmol) was dissolved in 50% THF / 50% MeOH (100 mL, v / v), and the mixture was added 1.0 g of Pd / C and stirred for 12 h at room temperature under H2 atmosphere. After reaction, Pd / C was removed by centrifugation. The solvent was evaporated in vacuo. The resulting crude product was purified by flash silica gel column chromatography (DCM / MeOH 10:1, v / v) to give Ser(1,2,3,4,6-penta-O-acetyl-D-mannopyranose) (6.0 g, 78.8%).

[0505] Ser(1,2,3,4,6-penta-O-acetyl-D-mannopyranose)-NCA was then synthesized. General synthesis procedure for Ser(1,2,3,4, 6-penta-O-acetyl-D- mannopyranose)-NCA: Ser(1,2,3,4,6-penta-O-acetyl-D-mannopyranose) (4.35 g, 10 mmol) was suspended in 60 mL of dry tetrahydrofuran (THF) and then triphosgene (1.4 g) was added under N2. The mixture was stirred at 50°C under a flow of N2 for 3 h. After the reaction mixture was cooled down to room temperature, the crude product was precipitated by pouring the mixture solution into iced hexane (250 ml), collected by filtration. The resulting crude product was purified by recrystallizing with THF / hexane mixture for three times. The yield of Ser(1,2,3,4,6-penta-O-acetyl-D-mannopyranose)-NCA was 70%. The structure of Ser(1,2,3,4, 6-penta-O-acetyl-D-mannopyranose)-NCA was verified by1H NMR (FIG. 3) and13C NMR spectra (FIG. 4). \ THRMeOH \ fWC I r. I \ 12 A i

[0506] Choiestsryi chforotcrmate OCM, TEA 00 co (CMC) 2.5% IMJNa ir> MeOH R. T.

[0507] 1 S min

[0508]

[0509] Scheme 1. Synthesis strategy for cholesterol-mannose-OAc (CMO) and cholesterol-mannose (CM)

[0510]

[0511] Scheme 2. Synthesis strategy for the Ser(1,2,3,4, 6-penta-O-acetyl-D-mannopyranose)N-carboxy anhydride

[0512] 1.4. Synthesis of lipid-block-poly(L-Ser-D-Mannose)

[0513] Synthesis strategy for cholesterol-block-poly(Ser-D-mannose) is shown in Scheme 3. Cholesteryl N-(ammonioethyl)carbamate was first synthesized. Ethylene diamine (5.2 mL, 15 eq) was dissolved in 25 mL of anhydrous DCM, followed by cooling down to 0°C with ice. 2.5 g of cholesteryl chloroformate was dissolved in 25 mL of anhydrous DCM and then was dropwise added to the reaction mixture of ethylene diamine. The mixture was stirred under N2 for 24 h at room temperature. The resulting reaction solution was washed with saturated brine for 3 times, and then dried over anhydrous Na2SO4, and concentrated in vacuo. The obtained crude product was purified by flash silica gel column chromatography (DCM / MeOH 20:1, v / v) to give cholesteryl N-(ammonioethyl)carbamate (2.3 g, 88%). The structure of cholesteryl N-(ammonioethyl)carbamate was verified by1H NMR spectra (FIG. 5). Cholesterol- / oc -poly(Ser-1,2,3,4,6-penta-O-acetyl-D-mannose) was then synthesized. General synthetic method for cholesterol-block-poly(Ser-1,2,3,4,6-penta-O-acetyl-D-mannopyranose): In the glove box, cholesteryl N-(ammonioethyl)carbamate (23.6 mg, 0.05 mmol) and Ser(1,2,3,4, 6-penta-O-acetyl-D-mannopyranose)-NCA (922 mg, 2.0 mmol) were dissolved in 15 mL of anhydrous DCM. The mixture was stirred for 48 h at room temperature in the glove box. Then, 1.0 mL of acetic anhydride was added and the reaction was continued for 2 h. The crude product was precipitated by pouring the mixture solution into glacial ether (300 mL), collected by centrifugation. The resulting crude product was purified by dissolving it in DCM and precipitated by pouring the solution into glacial ether. The resulting product was dried under vacuum. The number of polymerization units for cholesterol-b / oc -poly(Ser-1, 2,3,4, 6-penta-O-acetyl-D-mannopyranose) can be adjusted by varying the amount of Ser(1,2,3,4,6-penta-O-acetyl-D-mannopyranose)-NCA. The yield of the protected lipid-polypeptide was 72%.

[0514] The deprotection of cholesterol-b / oc -poly(Ser-1,2,3,4,6-penta-O-acetyl-D-mannopyranose) was then performed to yield cholesterol-block-poly(Ser-D-mannose) (CPSM). General synthesis procedure for cholesterol-block-poly(Ser-D-mannose): 600 mg of cholesterol-b / oc -poly(Ser-1, 2,3,4, 6-penta-O-acetyl-D-mannopyranose) was dissolved in 15 mL of MeOH, to which was added 1.5 mL of 25% MeONa in MeOH. The mixture was stirred at room temperature for 15 min. The crude product was purified by dialysis with DI water for removing MeONa and MeOH. The product was obtained by freeze-drying under vacuum. The yield of the deprotected cholesterol-b / oc / <-poly(Ser-D-mannose) was 96%. The typical structures of cholesterol-block-poly(Ser-D-mannose) was verified by1H NMR (FIG. 6 and FIG. 7). CO ho

[0515]

[0516] Scheme 3. Synthesis strategy for cholesterol-£> / oc -poly(Ser-D-mannose) 1.5. Formulation of mRNA-loaded lipid nanoparticles (mRNA LNPs)

[0517] To conduct a high-throughput screening of the varying cholesterol-derived lipids at different mole ratios, LNPs were manually formulated as according to Tables 1-6. Furthermore, the N / P ratio between the ionizable lipids and mRNA content was standardized at a ratio of 6:1.

[0518] Cholesterol-derived lipids were mixed with ALC-0315, a helper lipid (DSPC), ALC-0159, and cholesterol at different mole contents to optimize mRNA LNP formulation. The difference between the mole ratio of cholesterol-derived lipids (including CMO and CM) and cholesterol was distributed relative to the mole contents of the other lipids, respectively.

[0519] PEG replacement cholesterol-derived lipids or mannopolypeptides were mixed with ALC-0315, a helper lipid (DSPC), and cholesterol at different mole contents to optimize mRNA LNP formulation. The difference between the mole ratio of CPSM and ALC-0159 (1.6%) was distributed relative to the mole contents of the other lipids, respectively.

[0520] The formulation of mRNA LNPs involved two distinct phases, the organic phase and the aqueous phase. For the manual formulation process, the organic phase contained the mixture of lipids dissolved in ethanol to reach a final volume of 50 pL. The aqueous phase contained 10 pL of 1 mg / mL firefly luciferase mRNA (TriLink Biotechnologies) diluted in 140 pL of 10 mM sodium acetate solution at pH 4 to reach a final volume of 150 pL. The organic phase was added to the aqueous phase and mixed thoroughly through rapid pipetting. The mRNA-lipid mixture was then allowed to incubate at room temperature for at least 30 min to provide time for mRNA encapsulation and self-assembly of LNPs. Table 1. Characteristics of lipids for LNP formulation

[0521] Molecular weight or „...

[0522] ,..., Stock concentration Number of Lipid. for polvmer..,....

[0523] ( ’7mol) (mg / xnL) glycosyl units (n) ALC-0315 766.3 20.0 n / a DSPC 790.2 10.0 n / a Cholesterol 386.7 10.0 n / ALC-0159 2481.0 3.0 n / a CMO 803.5 5.0 I CM 635.9 5.0 1 CPSM-1 4235 3.0 15 CPSM-2 2010 3.0 6

[0524] Table 2. Mole ratio (content) of lipids used in the LNP formulations with cholesterol analogs. Total mole ratio of cholesterol includes the mole ratio of cholesterol and cholesterol analogs in each formulation

[0525] LNP name ALC-0315 DSPC Choi CMO CM ALC-0159 Pfizer 46.3 9.4 42.7 0 0 1.6 CM 46.3 9.4 0 0 42.7 1.6 CMO 46.3 9.4 0 42.7 0 1.6 CMO 5 46.3 9.4 37.7 5 0 1.6 CMO 10 46.3 9.4 32.7 10 0 1.6 CMO 15 46.3 9.4 27.7 15 0 1.6 CMO 20 46.3 9.4 22.7 20 0 1.6

[0526] Table 3. Mole ratio (content) of lipids used in the LNP formulations with CM. Total mole ratio of cholesterol includes the mole ratio of cholesterol and CM in each formulation

[0527] LNP name ALC-0315 DSPC Choi CMO CM ALC-0159 Pfizer 46.3 9.4 42.7 0 0 1.6 CM 5 46.3 9.4 37.7 0 5 1.6 CM 10 46.3 9.4 32.7 0 10 1.6 CM 15 46.3 9.4 27.7 0 15 1.6 CM 20 46.3 9.4 22.7 0 20 1.6 Table 4. Mole ratio (content) of lipids used in the formulations for PEG-free CPSM-1 LNPs. For CPSM-1 / CM formulation, total mole ratio of cholesterol includes the mole ratio of cholesterol and CM in each formulation LNPname ALC-0315 DSPC Choi CPSM-1 CM ALC-0159 Pfizer 46.3 9.4 42.7 0 0 1.6 CPSM-1 1.0 46.58 9.46 42.96 1.0 0 0 CPSM-1 1.2 46.49 9.44 42.87 1.2 0 0 CPSM-1 1.4 46.39 9.42 42.79 1.4 0 0 CPSM-1 1.6 46.3 9.4 42.7 1.6 0 0 CPSM-1 2.0 46.11 9.36 42.53 2.0 0 0 CPSM-1 / CM 46.49 9.44 37.87 1.2 5.0 0

[0528] Table 5. Mole ratio (content) of lipids used in the formulations for PEG-free CPSM-2 LNPs

[0529] LNP name ALC-0315 DSPC Choi CPSM-2 CM ALC-0159 Pfizer 46.3 9.4 42.7 0 0 1.6 CPSM-2 1.0 46.58 9.46 42.96 1.0 0 0 CPSM-2 1.2 46.49 9.44 42.87 1.2 0 0 CPSM-2 1.4 46.39 9.42 42.79 1.4 0 0 CPSM-2 1.6 46.3 9.4 42.7 1.6 0 0 CPSM-2 2.0 46.11 9.36 42.53 2.0 0 0

[0530] Table 6. Mole ratio (content) of lipids used in the formulations for PEG-free CM LNPs. Total mole ratio of cholesterol includes the mole ratio of cholesterol and CM in each formulation

[0531] LNPname ALC-0315 DSPC Choi CPSM CM ALC-0159 Pfizer 46.3 9.4 42.7 0 0 1.6 CM 1.6 46.3 9.4 42.7 0 1.6 0 CM 6.6 46.3 9.4 37.7 0 6.6 0 CM 11.6 46.3 9.4 32.7 0 11.6 0 CM 16.6 46.3 9.4 27.7 0 16.6 0 CM 21.6 46.3 9.4 22.7 0 21.6 0 CM 44.3 46.3 9.4 0 0 44.3 0 1.6. Assessing encapsulation efficiency of mRNA in LNPs

[0532] To determine the encapsulation efficiency of the mRNA LNPs after formulation, Quant-it™ RiboGreen RNA Assay Kit (Invitrogen, Waltham, MA, USA) was used to elucidate the mRNA concentration of the mRNA LNP mixture in solutions with or without Triton-X100. The Ribogreen reagent was diluted 200 times with either Tris-EDTA buffer containing 0.5% Triton-X100 or only Tris-EDTA buffer. The buffer solution containing the diluted Ribogreen reagent was aliquoted at 90 pL into each well of a black 96-well plate and mixed with 10 pL of mRNA LNP sample. The mixtures were then incubated at room temperature for 5 min to provide time for the emulsification of mRNA LNPs and the stabilization of the signal. After incubation, the fluorescence intensity of the samples was determined using a microplate reader (Tecan, Männedorf, Switzerland) at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. The values obtained were then used to determine the encapsulation efficiency of the various mRNA LNP formulations through the following equation.

[0533] Encapsulation Efficiency = (ConcTrit- ConcTE) / ConcTrit× 100

[0534]

[0535] ConcTrit

[0536] where Concrnt is the concentration of mRNA obtained by adding the mRNA LNPs to 0.5% Triton-X100 diluted in Tris-EDTA buffer, while ConcTEis the concentration of the respective mRNA obtained by adding the mRNA LNPs to Tris-EDTA buffer without Triton-X100.

[0537] 1.7. Physiochemical characterization of mRNA LNPs

[0538] The size, polydispersity index (PDI), and zeta potential of the mRNA LNPs were also characterized using a Zetasizer (Malvern, UK). The size and PDI were obtained through dynamic light scattering (DLS) by diluting 25 pL of the mRNA LNPs sample with saline solution to achieve a final volume of 500 pL. The sample was measured three times at 25°C with 20 runs each time. The sample was also measured at 1.68 s per run. The surface zeta potential of the mRNA LNPs was measured by diluting 25 pL of the sample with saline solution to reach a final volume of 1 mL. The samples were also measured three times at 25°C with 20 runs each time when the samples were measured.

[0539] 1.8. Cell culturing and dosing of HEK293T and DC2.4 cells with mRNA LNPs To test the cytotoxicity and transfection efficiency of the mRNA LNPs, HEK293T and DC2.4 cells were cultured and dosed with the mRNA LNPs. The HEK293T cell line was cultured in DMEM media containing 10% Fetal Bovine Serum (FBS) (v / v) and 1% Penicillin / Streptomycin (v / v), whereas the DC2.4 cell line was cultured in RPMI media containing 10% FBS and 1% Penicillin / Streptomycin (v / v). The cells were allowed to incubate at 37°C with 5% CO2 in an incubator (Thermo Fisher, Waltham, MA, USA). The cells were then seeded at 10,000 cells per well in a white 96-well plate for testing mRNA transfection efficiency and in a black 96-well plate for cytotoxicity evaluation of mRNA LNPs. The amount of mRNA LNPs added to each well was standardized to a dose of 100 ng of mRNA per well. After dosing, the 96-well plates were incubated for 48 hours before assessing cell viability and transfection efficiency.

[0540] 1.9. In vitro viability of HEK293T and DC2.4 cells after incubation with mRNA LNPs

[0541] After 48 hours of incubation, the old media consisting of the mRNA LNPs was removed from the wells of the black 96-well plate. Alamar Blue reagent was then diluted 10x with fresh DMEM media or RPMI media, respectively, and 100 pL of the diluted Alamar Blue reagent was added to each well of the plate. The samples were then incubated at 37°C for 2 h to allow reduction of the Alamar Blue compound by cells and stabilization of the signal. Fluorescence intensity was measured using the microplate reader (Tecan, Männedorf, Switzerland) at an excitation wavelength of 570 nm and an emission wavelength of 600 nm. The in vitro cell viability was then taken as a percentage relative to the negative control wells that did not receive any treatment. 1.10. In vitro transfection efficiency of mRNA LNPs in HEK293T and DC2.4 cells after incubation with mRNA LNPs

[0542] The transfection efficiency of mRNA LNPs in HEK293T and DC2.4 cells was measured after 48 hours using the ONE-Glo™ Luciferase Assay System.

[0543] 100 uL of ONE-Glo™ solution was added to each well. The samples were then incubated at 37°C for 10 min to allow for cell lysis and signal stabilization. Luminescence intensity was read using the microplate reader (Tecan, Männedorf, Switzerland) at an exposure time of 1000 ms.

[0544] Example 2: Synthesis and characterization of cholesterol-derived lipids

[0545] Cholesterol-mannose (CM) was synthesized via the reaction of cholesteryl chloroformate and ethanolamine-1,2,3,4,6-penta-O-acetyl-manno-D-pyranose, followed by the deprotection of cholesterol-mannose-OAc (CMO) in MeOH containing 2.5% MeONa (Scheme 1). The successful synthesis of CM was verified by1H NMR spectroscopy (FIG. 1 and FIG. 2). As shown in FIG. 2, the disappearance of peaks (-CO-C / - / 3) indicated successful deprotection of CMO. Furthermore, the cholesterol-b / ock-poly(Ser-D-mannose) (CPSM) was synthesized via the ROP of Ser(1, 2,3,4, 6-penta-O-acetyl-D-mannopyranose)-N-carboxyanhydride (Scheme 2) using the cholesterol–NH2as the initiator, followed by the deprotection of cholesterol-b / ock-poly(Ser-1, 2,3,4, 6-penta-O-acetyl-D-mannopyranose) in 2.5% MeONa in MeOH (Scheme 3). The successful synthesis of Ser(1, 2,3,4, 6-penta-O-acetyl-D-mannopyranose)-N-carboxyanhydride was verified by1H NMR and13C NMR spectroscopy (FIG. 3 and FIG.4). The successful synthesis of the initiator cholesterol–NH2was verified by1H NMR spectroscopy (FIG.5). The successful synthesis of cholesterol-block-poly(Ser-D-mannose) was confirmed by1H NMR spectroscopy. As shown in FIG.

[0546] 6, the disappearance of peaks (-CO-C / - / 3) indicated successful deprotection of cholesterol-poly(Ser-1,2,3,4,6-penta-O-acetyl-D-mannopyranose).1H NMR spectrum clearly showed the peak of n from amide groups in polypeptide backbone at 8.35 ppm, the peaks of f-m and p from Ser(D-mannose) units and the peak of a (-CH3) from cholesterol at 0.65 ppm, suggesting successful synthesis of cholesterol-block-poly(Ser-D-mannose). The degrees of polymerization (DPs) of Ser-D-mannose in CPSM can be adjusted by varying the amount of Ser(1,2,3,4,6-penta-O-acetyl-D-mannopyranose)-NCA. The DPs of Ser(D-mannopyranose) in lipid-poly(Ser-D-mannopyranose) were determined by the integration area (peak i of the polypeptide; peak a of the lipid) (FIG. 6 and FIG. 7). The typical cholesterol-derived poly(Ser-D-mannose) with different DPs including 15 and 6, were synthesized and denoted as CPSM-1 and CPSM-2, respectively.

[0547] Example 3: Size, size distribution (PDI), and zeta potential of mRNA LNPs

[0548] The characterization of the mRNA LNPs was performed using a Zetasizer (Malvern, UK), which would elucidate the size, PDI, and zeta potential of the respective mRNA LNPs. The values are displayed in Tables 7-11. As observed from the results, LNPs formulated with cholesterol-derived lipids or mannopolypeptides showed nanosize (< 200 nm) with a small polydispersity index (< 0.2) and near neutral zeta potentials (< ±10 mV). The small size and low PDI of the formulated mRNA LNPs indicate a narrow size distribution and homogenous particle population, indicating the LNPs are viable for efficient cellular uptake and intracellular delivery of the mRNA payload. Furthermore, the near neutral zeta potential reduces undesirable non-specific interactions with proteins in the physiological environment and provides in vivo stability.

[0549] Example 4: Encapsulation efficiency of formulated mRNA LNPs

[0550] The encapsulation efficiency of the mRNA LNPs was determined through the Ribogreen Assay Kit and the values are displayed in Tables 12-16. LNPs formulated using cholesterol-derived lipids demonstrated an encapsulation efficiency comparable or greater than the encapsulation efficiency of LNPs formulated using ALC-0159 that is used in Pfizer / BioNTech’s mRNA Covid19 vaccine by modulating the mole ratio. High encapsulation efficiency of mRNA in LNPs is critical for effective mRNA delivery as it ascertains a sufficient therapeutic payload that is delivered intracellularly for mRNA expression. Taken together with the characterization of the mRNA LNPs, these results suggest that the mRNA LNPs formulated with cholesterol-derived lipids or mannopolypeptides are viable for in vivo applications.

[0551] Table 7. Characteristics of manually formulated mRNA LNPs at different cholesterol / CMO / CM mole ratio

[0552] , Mole ratio (%) Size, Zeta potential LNP name - -~~j— — — —(nm)PDI

[0553]

[0554] Pfizer 42.7 0 0 99 ± 2 0.186 ± 0.025 -7.11 ± 1.88 CM 0 0 42.7 127 ± 1 0.128 ± 0.012 -6.54 ± 1.02 CMO 0 42.7 0 114 ± 1 0.209 ± 0.013 -10.25 ± 0.94 CMO 5 37.7 5.0 0 126 ± 1 0.152 ± 0.018 -10.61 ± 1.22 CMO 10 32.7 10.0 0 119 ± 1 0.182 ± 0.022 -9.90 ± 1.24 CMO 15 27.7 15.0 0 114 ± 2 0.178 ± 0.015 -10.90 ± 0.28 CMO 20 22.7 20.0 0 94 ± 1 0.148 ± 0.015 -3.39 ± 0.08

[0555] Table 8. Characteristics of manually formulated mRNA LNPs at different cholesterol / CM mole ratio

[0556] i vn Mole ratio (%), Zeta potential _ Choi CM _ _ _ (mV)

[0557]

[0558] Pfizer 42.7 0 105 ± 1 0.133 ± 0.019 -3.72 ± 0.82 CM 5 37.7 5.0 111 ± 1 0.120 ± 0.004 -3.87 ± 0.80 CM 10 32.7 10.0 111 ± 1 0.124 ± 0.001 -3.45 ± 1.19 CM 15 27.7 15.0 118 ± 1 0.150 ± 0.012 -1.06 ± 1.24 CM 20 22.7 20.0 108 ± 1 0.127 ± 0.020 -4.32 ± 1.12 Table 9. Characteristics of manually formulated mRNA LNPs at different cholesterol / CPSM-1 / CM mole ratio

[0559] Mole ratio (%) Zeta LNP name ALC- CPSM- Size PDI potential 0159 1 (nm) (mV) Pfizer 1.6 0 0 93 + 1 0.159 ± 0.018 -2.15 ± 3.00 CPSM- 1 1.0 0 1.0 0 114 ± 1 0.131 ± 0.022 -5.28 ± 1.34 CPSM- 1 1.2 0 1.2 0 102 + 1 0.128 ± 0.039 -2.08 ± 0.55 CPSM- 1 1.4 0 1.4 0 119 + 1 0.139 + 0.014 -6.54 + 0.46 CPSM- 1 1.6 0 1.6 0 121 + 0 0.142 + 0.019 -5.22 + 2.70 CPSM- 1 2.0 0 2.0 0 125 ± 1 0.235 ± 0.028 -2.30 ± 1.00 CPSM-l / CM 0 1.2 5.0 113 + 0 0.128 ± 0.051 -6.26 ± 1.89

[0560] Table 10. Characteristics of manually formulated mRNA LNPs at different CPSM- 2 mole ratio

[0561] T xrriMole ratio (%) Size Zeta potential ALC-0159 CPSM- 2 (mn) (mV) Pfizer 1.6 0 106 ± 0 0.140 ± 0.005 -5.27 ± 2.94 CPSM-2 1.0 0 1.0 126 + 1 0.159 ± 0.031 -6.67 + 1.90 CPSM-2 1.2 0 1.2 111 + 1 0.102 + 0.013 -5.14 + 0.09 CPSM-2 1.4 0 1.4 140 + 1 0.144 ± 0.014 -6.92 + 0.54 CPSM-2 1.6 0 1.6 119 + 0 0.123 ± 0.034 -6.82 + 1,00 CPSM-2 2.0 0 2.0 135 ± 0 0.155 ± 0.015 -7.58 ± 0.53

[0562] Table 11. Characteristics of manually formulated mRNA LNPs at different cholesterol / CM mole ratio Mole ratio (%) Size „,x, Zeta potential

[0563]

[0564] LdN I * -,.4-.— < T / X Lxl -\

[0565] Choi ALC-0159 CM (m) (mV) Pfizer 42.7 1.6 0 109 + 1 0.207 + 0.014 -4.64 + 2.14 CM 1.6 42.7 0 1.6 119 ± 1 0.13 ± 0.016 -9.83 ± 1.6 CM 6.6 37.7 0 6.6 114 + 2 0.116 + 0.019 -8.37 + 1.11 CM 11.6 32.7 0 11.6 105 ± 0 0.125 ± 0.008 -8.00 ± 1.24 CM 16.6 27.7 0 16.6 115 ± 1 0.141 ± 0.014 -8.11 + 1.17 CM 21.6 22.7 0 21.6 106 + 0 0.144 ± 0.016 -8.23 ± 0.98 CM 44.3 0 0 44.3 146 + 1 0.242 ± 0.029 -6.29 + 0.88 Table 12. Characteristics of manually formulated mRNA LNPs at different cholesterol / CMO / CM mole ratio

[0566] r x-n Mole ratio (%).

[0567] LNP name Encapsulation efficiency (%)

[0568] Choi CMO CM!

[0569] Pfizer 42.7 0 0 73.6 ±2.2

[0570] CM 0 0 42.7 70.6 ±0.5

[0571] CMO 0 42.7 0 56.7 ± 3.6

[0572] CMO 5 37.7 5.0 0 75.0 ± 1.0

[0573] CMO 10 32.7 10.0 0 63.2 ± 1.1

[0574] CMO 15 27.7 15.0 0 65.0 ±0.7

[0575] CMO 20 22.7 20.0 0 80.8 ± 0.1

[0576] Table 13. Characteristics of manually formulated mRNA LNPs at different cholesterol / CM mole ratio

[0577] , Mole ratio (%)..

[0578] LNP name _. Encapsulation efficiency I %) Choi CM

[0579] Pfizer 42.7 0 81.7 ± 0.1

[0580] CM 5 37.7 5.0 78.3 ± 0.8

[0581] CM 10 32.7 10.0 83.6 ±0.0

[0582] CM.15 27.7 15.0 83.0 ± 1.6

[0583] CM 20 22.7 20.0 81.8 ± 1.7

[0584] Table 14. Characteristics of manually formulated mRNA LNPs at different cholesterol / CPSM-1 / CM mole ratio

[0585] LNP name Mole ratio (%) Encapsulation efficiency (%)

[0586] ALC-0159 CPSM-1 CMF' Pfizer 1.6

[0587]

[0588] 0 0 63.4 ± 1.3 CPSM-1 1.0 0 1.0 0 52.0 ± 1.2 CPSM-1 1.2 0 1.2 0 57.1 ±0.7 CPSM-1 1.4 0 1.4 0 63.8 ± 0.5 CPSM-1 1.6 0 1.6 0 62.7 ± 1.4 CPSM-1 2.0 0 2.0 0 57.4 ±0.1 CPSM-l / CM () 1.2 5.0 71.0 ±0.9 Table 15. Characteristics of manually formulated mRNA LNPs at different CPSM- 2 mole ratio

[0589] Mole ratio (%)

[0590] LNP name ALC-0159 CPSM-2 Encapsulation efficiency (%)

[0591]

[0592] Pfizer 1.6 0 76.9 ± 2.7 CPSM-2 1.0 0 1.0 65.0 ± 0.1 CPSM-2 1.2 0

[0593]

[0594] 72.8 ± 0.6 CPSM-2 1.4 0 1.4 75.6 ± 0.0 CPSM-2 1.6 0 1.6 61.2 ± 1.2 CPSM-2 2.0 0 2.0 53.4 ± 0.5

[0595] Table 16. Characteristics of manually formulated mRNA LNPs at different cholesterol / CM mole ratio

[0596] Mole ratio (%)

[0597]

[0598] Chol ALC-0159 CM Pfizer 42.7 1.6 0 68.4 ±0.4

[0599] CM 1.6 42.7 0 1.6 77.8 ±0.1

[0600] CM 6.6 37.7 0 6.6 69.3 ± 16.8

[0601] CM 11.6 32.7 0 11.6 81.2 ± 0.4

[0602] CM 16.6 27.7 0 16.6 77.3 ± 0.3

[0603] CM 21.6 22.7 0 21.6 48.6 ± 32.5

[0604] CM 44.3 0 0 44.3 46.6 ± 16.3

[0605] Example 5: In vitro cytocompatibility and transfection efficiency of mRNA LNPs in HEK293T cells

[0606] The head of cholesterol is generally considered as a hydroxyl group. In lipid nanoparticle formulations, this hydroxyl group can interact with the aqueous phase via the polarity and hydrogen bonding. For the initial screening, 2 cholesterol-derived lipids (including CM and CMO) were designed to tune the polarity of the head of cholesterol. CM was comprised of mannose group that differ from CMO in the polarity of the head of cholesterol. The screening process was performed to evaluate whether the substitution of the head of cholesterol improved transfection efficiency. The transfection efficiency and cytotoxicity of the mRNA LNPs formulated manually were determined by dosing HEK293T cells with the formulated LNPs. The results obtained are displayed in FIG.8 - FIG. 17.

[0607] From the cytocompatibility tests using the AlamarBlue assay, all formulations show negligible cytotoxicity with a cell viability at approximately 100% relative to the negative control. Further inspection of the luminescence intensity of HEK293T cells from the luciferase assay demonstrates that the formulations formed from CM and CMO are capable of significantly improving transfection efficiency of Flue mRNA over Pfizer’s formulation by changing the mole ratio of cholesterol, CM and CMO.

[0608] LNPs formulated by using PEG replacement cholesterol-derived lipids or mannopolypeptides are capable of significantly improving transfection efficiency of Flue mRNA over Pfizer’s formulation. This suggests that CPSM-1, CPSM-2 and CM compounds not only are capable of replacing PEG-conjugated lipid as a viable compound in producing nanosized LNPs, but also enable greater transfection efficiency of mRNA in an in vitro system. Variations in the transfection efficiency of the varying cholesterol-derived PEG-free LNPs at different mole ratios suggest that there is a hydrophobicity-hydrophilicity balance that enables an optimal level of transfection efficiency. Notably, PEG-free mRNA LNP formed by CM at mole ratio 11.6% shows more than 20 times enhancement in transfection efficiency compared to Pfizer’s formulation in HEK293T cells.

[0609] Example 6: In vitro cytocompatibility and transfection efficiency of mRNA LNPs in DC2.4 cells

[0610] The cytocompatibility and transfection efficiency results of mRNA LNPs in DC2.4 cells are detailed in FIG. 18 to FIG. 27. Similar to HEK293T cells, analysis of the AlamarBlue assay results in DC2.4 cells reveal that the mRNA LNPs tested have negligible cytotoxicity. The luciferase assay also shows a significantly improving trend compared to Pfizer’s formulation. All cholesterol-derived PEG- free LNPs containing mannose groups showed a greater transfection efficiency compared to Pfizer’s LNP formulated by using ALC-0159 in DC2.4 cells. Specifically, PEG-free mRNA LNP formed by CPSM-1 at mole ratio 1.4% shows more than 15 times enhancement in transfection efficiency compared to Pfizer’s formulation in DC2.4 cells.

[0611] Example 7: Summary

[0612] As shown in the examples, a series of PEG-free LNPs based on cholesterol-derived lipids (CM and CMO) and mannopolypeptides (CPSM) are successfully made. mRNA LNPs formulated from cholesterol-derived lipids and mannopolypeptides have nano-size (< 200 nm), homogeneous particle population (PDI < 0.2) and neutral surface charge (zeta potential: < ± 10 mV), making them ideal for in vivo applications. The mRNA LNPs formed from cholesterol-derived lipids and mannopolypeptides provide greater mRNA transfection efficiency than the mRNA LNPs made from the lipids used in Pfizer / BioNTech’s mRNA COVID19 vaccine formulation in HEK293T cells and DC2.4 cells. Specifically, PEG-free mRNA LNP formed by CPSM-1 at mole ratio 1.4% shows more than 15 times enhancement in transfection efficiency compared to ALC-0159 LNP formulation in DC2.4 cells. PEG-free mRNA LNP formed by CM at mole ratio 11.6% shows more than 20 times enhancement in transfection efficiency compared to ALC-0159 LNP formulation in HEK293T cells. All mRNA LNPs formulations tested show negligible cytotoxicity. CPSM and CM are thus a viable replacement for ALC-0159 and other PEG-conjugated lipids as they are not only capable of fulfilling the function of PEG in maintaining nano-size particles, but also provide a greater mRNA transfection efficiency. These cholesterol-derived PEG-free LNPs are promising nanocarriers for the efficient delivery of mRNA and other genes as vaccine and treatment options.

[0613] It will be appreciated by a person skilled in the art that other variations and / or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure as broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included etc. or the like across different exemplary embodiments. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.

Claims

CLAIMS1. A compound comprising a structure represented by general formula (1 ):R1R3whereinA comprises a sterol and / or a derivative(s) thereof;B comprises:(i) a carbohydrate and / or a derivative(s) thereof; or(ii) an oligopeptide or a polypeptide comprising carbohydrate and / or a derivative(s) thereof;R1and R3are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;R2is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;R4is -H or -C(=O)R, where R is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;p = 0 or 1; andq = 0 or 1.

2. The compound of any one of the preceding claims, wherein the sterol is selected from animal sterols, plant sterols, fungal sterols and combinations thereof.

3. The compound of any one of the preceding claims, wherein A comprises a structure that is represented by general formula (2):(2) whereinring C1, C2, C3and C4each optionally contains one, two or three C=C bond(s); Ra, Ra’, Rb, Rb’, Rc, Rd, Rd’, Re, Rf, Rf’, R9, R9’, Rb, R', Rz, R<’, Rk, Rk’, Rl, Rm, Rn, Rn’, R°, R°’, Rpand Rqare each optionally present and independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; andRzis a hydrophobic group, or contains at least linear aliphatic, branched aliphatic and / or cyclic hydrocarbons.

4. The compound of any one of the preceding claims, wherein A comprises a structure that is represented by general formula (2A):(2A)and whereinRzis a linear, branched, saturated and / or unsaturated hydrocarbon group.

5. The compound of any one of the preceding claims, wherein B comprises a structure that is represented by general formula (3A) and / or (3B):r R5-i(3A) (3B)whereinB1comprises carbohydrate and / or a derivative(s) thereof;B2comprises carbohydrate and / or a derivative(s) thereof;R5is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;R6is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene; andn > 1.

6. The compound of any one of the preceding claims, wherein the structure represented by general formula (1) comprises a structure that is represented by general formula (4):(4)whereinR2ais optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;R2bis optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;x = 0 or 1;y = 0 or 1; andtotal sum of x + y = 1.

7. The compound of any one of the preceding claims, wherein the structure represented by general formula (4) comprises a structure that is represented by general formula (4A) and / or (4B):R1O(4B)8. The compound of any one of the preceding claims, wherein the carbohydrate and / or a derivative(s) thereof (in B1and / or B2) is selected from the group consisting of monosaccharide, disaccharide, oligosaccharide, polysaccharide and / or a derivative(s) thereof.

9. The compound according to any one of claims 1 to 8, wherein the compound is substantially devoid of polyethylene glycol (PEG).

10. The compound of any one of the preceding claims, wherein B1and / or B2comprise a structure that is represented by general formula (5) having a 6- membered ring structure:whereinM is -0- or -S-;X1to X7and X9to X10are each independently selected from -H, -OH, or -0-C(=0)R7, where R7is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; andX8is alkylene.

11. The compound of any one of the preceding claims, wherein the compound comprises a structure selected from one or more of the following:cholesterol-mannose-OAc (CMO),1012. A method of preparing a compound as claimed in any one of the preceding claims, the method comprising:(a-i) reacting a protected alkanolamine compound represented by general formula (6) with a compound comprising B1represented by general formula (7) in the presence of a Lewis acid to obtain a first intermediate compound represented by general formula (8):R1R1HCLt oB1.. N.^R2TG1 B — R^R2? G1(6) (7) (8) whereinR8is -H, -OH or -O-C(=O)R9, where R9is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;PG1is a protecting group selected from N-carboxybenzyl or benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (BOC), tert-butyl, 9- Fluorenylmethoxycarbonyl (Fmoc), 2-(4- Nitrophenylsulfonyl)ethoxycarbonyl (Nsc), 2-Fluoro-Fmoc (Fmoc(2F)), 2-Monoisooctyl-Fmoc (mio-Fmoc), 2,7-Diisooctyl-Fmoc (dio-Fmoc), or combinations thereof;B1comprises a carbohydrate and / or a derivative(s) thereof;R1is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;R2is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;(a-ii) deprotecting the first intermediate compound represented by general formula (8) to obtain a second intermediate compound represented by general formula (9):R1B1.R2(9)(a-iii) reacting the second intermediate compound represented by general formula (9) with a haloformate compound comprising A represented by general formula (21) to obtain a compound represented by general formula (4A):R1(21) (4A)whereinX1is a halide; andA comprises a sterol and / or a derivative(s) thereof.

13. The method of claim 12, wherein the method further comprises, prior to (a- i):(b-i) reacting an alkanolamine compound represented by general formula (10) with a compound comprising PG1represented by general formula (11) in the presence of a Lewis base to obtain a protected alkanolamine compound represented by general formula (6):R1R1I I HO. ' HO. / N.^R2 \|_| X — PG ^R2^PG1(10) (11) (6) whereinR1is H; andX is a halide.

14. A method of preparing a compound as claimed in any one of the preceding claims, the method comprising:(c-i) polymerizing one or more N-carboxyanhydride (NCA) monomers represented by general formula (12) with an initiator represented by general formula (13) to obtain a first intermediate compound represented by general formula (14):whereinA comprises a sterol and / or a derivative(s) thereof;B2comprises a carbohydrate and / or a derivative(s) thereof;R1and R3are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; R2is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;R4is -H or -C(=O)R, where R is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; R5is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;R6is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene; andn ≥ 1;(c-ii) reacting the first intermediate compound represented by general formula (14) with an acylating agent represented by general formula (15) to obtain a second intermediate compound represented by general formula (16):(15) (16) whereinR and R’ are each independently optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; and(c-iii) deprotecting the second intermediate compound represented by general formula (16) to obtain a compound represented by general formula (4B).

15. The method of claim 14, wherein the method further comprises, prior to (c- i):(d-i) reacting a protected amino acid represented by general formula (17) with a compound comprising B2represented by general formula (18) in the presence of a Lewis acid to obtain a first intermediate compound represented by general formula (19):B2— R10(17) (18) (19)whereinR10is -H, -OH or -O-C(=O)R11, where R11is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;PG2is a protecting group selected from N-carboxybenzyl or benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (BOC), tert-butyl, 9- Fluorenylmethoxycarbonyl (Fmoc), 2-(4- Nitrophenylsulfonyl)ethoxycarbonyl (Nsc), 2-Fluoro-Fmoc (Fmoc(2F)), 2-Monoisooctyl-Fmoc (mio-Fmoc), 2,7-Diisooctyl-Fmoc (dio-Fmoc), or combinations thereof;(d-ii) deprotecting the first intermediate compound represented by general formula (19) to obtain a second intermediate compound represented by general formula (20):H HNO(20).(d-iii) reacting the second intermediate compound represented by general formula (20) with a carbonylating agent to obtain the N- carboxyanhydride (NCA) monomers represented by general formula (12).

16. A nanoparticle composition for delivery of a therapeutic agent, prophylactic agent and / or biological agent, the nanoparticle composition comprising:a compound as claimed in any one of claims 1 to 11; and a therapeutic agent, prophylactic agent and / or biological agent.

17. The nanoparticle composition of claim 16, wherein the composition further comprises:(i) ionizable lipid;(ii) helper lipid;(iii) optionally sterol; and(iv) optionally amphiphilic lipid.

18. The nanoparticle composition of claim 17, wherein the ionizable lipid, helper lipid, sterol, compound as claimed in any one of claims 1 to 11, and amphiphilic lipid are mixed at a mole ratio of 25 - 75: 1 - 20: 0 - 60: 0.1 -65: 0 -5.

19. The nanoparticle composition of any one of claims 17 to 18, wherein the ionizable lipid is selected from ALC-0315, SM-102, Lipid 5, DLinDMA, D- Lin-MC2-DMA, DLin-MC3-DMA, D-Lin-MC4-DMA, Dlin-KC2-DMA, YSK05, AA3-Dlin, SSPalmM, SSPalmO-Phe, Lipid A9, L319, DODMA, CL1, BP Lipid 310, ATX-001, ATX-100, Lipid 2, 80-016B, BP Lipid 309, BP Lipid 307, 93-O17S, 93-0170, NT1-O14B, 306-O12B-3, 306-O12B, 113-O16B, 306Oi10, 306Oi9-cis2, BAMEA-O16B, AI-28, 113-O12B, 98N12-5, Ckk-E12, OF-02, C12-200, BP Lipid 311, BP Lipid 308, BP Lipid 314, BP Lipid 312, LP01, TCL053, Lipid C24, BP Lipid 315, Lipid 29, 9A1P9, C13-112-tri-tail, C13-113-tri-tail, C13-112-tetra-tail, C13-113-tetra- tail, or combinations thereof.

20. The nanoparticle composition of any one of claims 17 to 19, wherein the helper lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn- glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O- octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1 - hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl- sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3- phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1.2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero- 3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3- phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1 - glycerol) sodium salt (DOPG), sphingomyelin, or combinations thereof.

21. The nanoparticle composition of any one of claims 17 to 20, wherein the sterol is present and is selected from cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, avenasterol, or combinations thereof.

22. The nanoparticle composition of any one of claims 17 to 21, wherein the amphiphilic lipid is present and is selected from PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG- modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, 2-[(polyethylene glycol)- 2000]-N, N-ditetradecylacetamide (ALC-0159), R-3-[(ω-methoxypolyethylene glycol)2000)carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DOMG), 3-N-[(ω-methoxypoly (ethyleneglycol)2000)carbamoyl]- 1.2-dimyristyloxy-propylamine (PEG-S-DMG), PEG-DMPE (1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycolmethoxy] (sodium salt)), PEG-DPPC, PEG-DSPE lipid, or combinations thereof.

23. The nanoparticle composition of any one of claims 17 to 22, wherein the nanoparticle composition comprises:nanoparticles having a N / P ratio from 1:1 to 40:1; and / or nanoparticles having an average particle size of no more than 500 nm; and / ornanoparticles having a zeta potential of from -30 mV to +30 mV.

24. The nanoparticle composition as claimed in any one of claims 17 to 23 for use in medicine.

25. The nanoparticle composition as claimed in any one of claims 17 to 23 for use in modulating an immune response in a subject, wherein said nanoparticle composition is to be administered to the subject.

26. Use of a nanoparticle composition as claimed in any one of claims 17 to 23 in the manufacture of a medicament for modulating an immune response in a subject.

27. A method of modulating an immune response in a subject, the method comprising administering to a subject a therapeutically effective amount of the nanoparticle composition as claimed in any one of claims 17 to 23.

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