A compound 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) or ionized form thereof wherein A comprises a carbohydrate or a carbohydrate derivative; R1, R2, R3, R8, R9, and R10 are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; R4 and R7 are each independently optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; R5 and R6 each independently comprises a hydrophobic moiety; Z1 and Z2 are each independently selected from the group consisting of –O–C(=O)–, –C(=O)–O–, –C(=O)–, –O–, –RaN–C(=O)–, –C(=O)–NRb–, where Ra and Rb are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; x is an integer ≥ 1; and y is an integer ≥ 0.

Description

[0001] A COMPOUND 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 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) have demonstrated successful clinical applications in delivering messenger ribonucleic acid (mRNA) for Covid-19 vaccines and therapies.

[0006] Typically, LNPs are composed of four different types of lipids, including a helper lipid such as a phospholipid, which improves LNP stability and fusogenicity; a polyethylene glycol (PEG)-ylated lipid, which prevents non-specific protein adsorption as well as particle aggregation, and helps control LNP size; an ionizable lipid, which can alternate between a neutral and a protonated state which aids in the encapsulation of mRNA via electrostatic interactions; and cholesterol which decreases the permeability of LNP and enhances its stability.

[0007] Among the aforementioned components, the ionizable lipid is a key LNP component that can trigger immune responses and long-term toxicity as it plays a major role in protecting RNAs structure and facilitating its cytosolic transport. Ionizable lipids are generally protonated at an acidic pH, which allows them to be positively charged, thereby promoting the encapsulation of RNAs into LNPs. At physiological pH, however, they remain neutral to minimize cytotoxicity. Therefore, the design and synthesis of an optimal ionizable lipid are crucial for efficient RNA delivery. However, developing optimal ionizable lipids remains a significant challenge. Specifically, the preparation of rationally designed ionizable lipid candidates is challenging due to the laborious synthesis process. Currently, clinical ionizable lipids are all synthesized through multistep routes, which in turn poses substantial obstacles for scalable manufacturing. Recent studies have shown that even the most effective delivery systems, including Food and Drug Administration (FDA)-approved LNPs, can only mediate the cytosol release with 1-4% of RNA. Thus, endosomal escape remains the primary bottleneck in achieving efficient RNA delivery. To overcome this issue, further innovation and investigation into the structural optimization of ionizable lipids are warranted.

[0008] The overall architecture of ionizable lipids typically includes three parts: the headgroup, the linker, and the tails, in which the headgroup is of high importance. Generally, the headgroup has a positive charge, and its charge density and size play a key role in entrapping the RNA, stabilizing the structure of LNP, inducing the interactions with cell membranes, and facilitating the endosomal escape. Typical headgroups of ionizable lipids mainly include amines, guanidine, and heterocyclic structures. The headgroups of ALC-0315from Pfizer-BioNTech and SM-102 from Moderna also feature a terminal hydroxyl group, which can reduce the hydration of the ionizable group and enhance the hydrogenbonding interactions with the RNA, potentially leading to improved transfection efficiency. Therefore, the rational design of headgroups is important for achieving efficient RNA delivery.

[0009] 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

[0010] In one aspect, there is provided a compound comprising a structure represented by general formula (1 ) or ionized form thereof:

[0011]

[0012] wherein

[0013] A comprises a carbohydrate or a carbohydrate derivative;

[0014] R1, R2, R3, R8, R9, and R10are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0015] R4and R7are each independently optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0016] R5and R6each independently comprises a hydrophobic moiety;

[0017] Z1and Z2are each independently selected from the group consisting of -O-C(=O)-, -C(=O)-O-, -C(=O)-, -O-, -RaN-C(=O)-, -C(=O)-NRb- where Raand Rbare each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0018] x is an integer ≥ 1; and

[0019] y is an integer ≥ 0.

[0020] In one embodiment, the ionized form of general formula (1) is R1— R4— R5

[0021] R3

[0022] R8

[0023] R1° -R7 -

[0024] R9

[0025] (1b).

[0026] In one embodiment, the hydrophobic moiety at R5and R6each independently comprises optionally substituted alkyl.

[0027] In one embodiment, A comprises pentose, hexose, fructose, ribose, mannose, glucose, galactose, deoxyribose, xylose, maltose, lactose, arabinose, cellobiose, 2-deoxy-D-ribofuranose, D-ribofuranose, D-xylofuranose, D-arabinofuranose, D-glucopyranose, D-mannopyranose, D-galactopyranose, L-rhamnopyranose, D-fructopyranose, N-acetyl-glucosamine, N-acetyl-mannosamine, N-acetyl-galactosamine, 2-amino-2-deoxy-D-glucopyranose, 2-amino-2-deoxy-D-mannopyranose, 2-amino-2-deoxy-D-galactopyranose, 1 -amino-1 -deoxy-D-fructose, 3-amino-3-deoxy-D-mannopyranose, 1 -amino-1 -deoxy-D-galactopyranose, 1 -(2-aminoethoxy)-1 -deoxy-D-glucopyranose, 1 -(2-am inoethoxy)-1 -deoxy-D-m annopyranose, 1 -(2 -am inoethoxy)-1 -deoxy-D-galactopyranose, 1 -(3-aminoethoxy)-1 -deoxy-D-glucopyranose, 1 -(3-am inoethoxy)-1 -deoxy-D-m annopyranose, 1 -(3-am inoethoxy)-1 -deoxy-D-galactopyranose, 1 -(3-aminopropoxy)-1 -deoxy-D-glucopyranose, 1 -(3-aminopropoxy)-1-deoxy-D-mannopyranose, 1 -(3-am inopropoxy)-1 -deoxy-D-galactopyranose, a derivative thereof, or combinations thereof.

[0028] In one embodiment, A comprises at least one of an -ORc or -O-C(=O)Rd group, where Rcand Rd are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl. In one embodiment, A comprises a monosaccharide and / or a derivative thereof.

[0029] In one embodiment, A is represented by general formula (2) or general formula (2A):

[0030]

[0031] (2) (2A)

[0032] wherein

[0033] R29to R51are each independently selected from -H, -ORe, or -O-C(=O)Rf, -R', -R'ORk, -NR'-C(=O)Rm, or Rn, where Reis H, Rf, R' and Rmare each independently optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl, Rkand Rlare each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl, Rjis optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene, and Rncomprises a structure represented by general formula (2A);

[0034] R52is optionally present as -O-, -OR0-, or -Rp-, where R° and Rpare each independently optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene, and wherein one of R42to R51is absent; and n is 0 or 1.

[0035] In one embodiment, the compound has a molecular weight of from about 250 g / mol to about 5,000 g / mol.

[0036] In one embodiment, the compound is selected from the group consisting of the following structures:

[0037]

[0038] ZJY-01 / ZJY-1G ZJY-02 / ZJY-2D

[0039]

[0040] ZJY-03 / ZJY-1A

[0041]

[0042] ZJY-04 / ZJY-2A / ZJY-3A / ZJY-4A ZJY-05 / ZJY-5A / ZJY-6A / ZJY-7A

[0043]

[0044] ZJY-06 ZJY-1B

[0045]

[0046] HO ZJY-1C

[0047]

[0048] ZJY-1D

[0049]

[0050] ZJY-1E ZJY-1F

[0051]

[0052] ZJY-1H

[0053]

[0054] ZJY-1I ZJY-1J ZJY-1K

[0055]

[0056] ZJY-2B ZJY-2C

[0057]

[0058] ZJY-6B

[0059]

[0060] ZJY-6C

[0061]

[0062] ZJY-6D

[0063]

[0064] ZJY-6E

[0065]

[0066] ZJY-6E ZJY-6F ZJY-6G

[0067]

[0068] ZJY-6H

[0069]

[0070] ZJY-8A

[0071]

[0072] ZJY-9A

[0073]

[0074] ZJY-10A / ZJY-1IA / ZJY-12A

[0075]

[0076] ZJY-13A / ZJY-14A / ZJY-15A

[0077]

[0078] ZJY-16A / ZJY-17A / ZJY-18A

[0079]

[0080] ZJY-17B

[0081]

[0082] ZJY-19A

[0083]

[0084] ZJY-20A

[0085]

[0086] ZJY-21A

[0087]

[0088] ZJY-25A / ZJY-26A / ZJY-27A In one aspect, there is provided a nanoparticle composition comprising: (i) a compound represented by general formula (1) or ionized form thereof disclosed herein; and

[0089] (ii) a therapeutic, prophylactic, and / or biological agent that is encapsulated by said compound or ionized form thereof.

[0090] In one embodiment, the composition further comprises:

[0091] (a) helper lipid;

[0092] (b) sterol; and

[0093] (c) polyethylene glycol (PEG)-modified lipid.

[0094] In one embodiment, the compound represented by general formula (1), helper lipid, sterol, and PEG-modified lipid are present at a molar ratio of 10 - 80: 0 - 50: 10 - 80: 0.5 -20.

[0095] In one embodiment, the helper lipid comprises a phospholipid selected from the group consisting of 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, and combinations thereof.

[0096] In one embodiment, the sterol is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, avenasterol, and combinations thereof.

[0097] In one embodiment, the PEG-modified lipid is selected from the group consisting of 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-[(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), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycolmethoxy] (sodium salt) (PEG-DMPE), PEG-DPPC, PEG-DSPE lipid, and combinations thereof.

[0098] In one embodiment, the composition comprises nanoparticles with an average particle size of from 20 nm to 400 nm and a polydispersity index (PDI) of from 0.01 to 0.7.

[0099] In one aspect, there is provided a method of preparing a compound represented by general formula (1) or ionized form thereof disclosed herein, the method comprising:

[0100] (a-i) reacting a compound comprising 1° amine represented by general formula (3) with a carbohydrate represented by general formula (4) in the presence of a reducing agent to obtain an intermediate compound represented by general formula (6): H

[0101]

[0102] (6) wherein

[0103] A comprises a carbohydrate or carbohydrate derivative;

[0104] R1, R2, and R3are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0105] R4is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0106] R5comprises a hydrophobic moiety;

[0107] Z1is selected from the group consisting of -O-C(=O)-. -C(=O)-O-, - C(= O)—, — O—, -RXN-C(=O)-, -C(=O)-NRy-, where Rxand Ryare each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0108] R11, R12, R13, R14, R15, R16, R17, R18, R19, R20, and R21are each independently selected from -H or -OH;

[0109] x is an integer ≥ 1; and

[0110] m is 0 or 1;

[0111] (a-ii) reacting the intermediate compound represented by general formula (6) with a compound comprising carbonyl represented by general formula (7) in the presence of a reducing agent to obtain a compound represented by general formula (1): R2R8

[0112]

[0113] wherein

[0114] R8, R9, and R10are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0115] R7is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0116] R6comprises a hydrophobic moiety;

[0117] Z2is selected from the group consisting of -O-C(=O)-. -C(=O)-O-, - C(=O)-, — O—, -RXN-C(=O)- -C(=O)-NRy-, where Rxand Ryare each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0118] y is an integer ≥ 0; and

[0119] (a-iii) optionally ionizing the compound represented by general formula (1) to become positively charged.

[0120] In one embodiment, the method further comprises: (b-i) reacting the compound represented by general formula (1) with an acylating agent represented by general formula (9) to replace one or more -OH group(s) in A with -O-C(=O)R9group(s):

[0121] R2R1- R4R3R8R10- R7R9

[0122]

[0123] wherein

[0124] R27and R28are each independently optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and

[0125] Rais optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

[0126] In one embodiment, the method further comprises, prior to (a-i):

[0127] (c-i) reacting a compound represented by general formula (10) with protected amine compound represented by general formula (11) to obtain an intermediate compound represented by general formula (12):

[0128]

[0129] wherein

[0130] R1, R2, and R3are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0131] R4is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0132] R5comprises a hydrophobic moiety;

[0133] Z1, Z1', and Z1” are each independently selected from the group consisting of-O-C(=O)-, -C(=O)-O-, -C(=O)-, -O-, -RXN-C(=O)-, -C(=O)-NRy-, where Rxand Ryare each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0134] R22and R23are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0135] x is an integer ≥ 1;

[0136] PG1is a protecting group; and

[0137] (c-ii) deprotecting the intermediate compound represented by general formula (12) to obtain the compound comprising 1° amine represented by general formula (3):

[0138]

[0139] In one embodiment, the method further comprises, prior to (a-ii):

[0140] (d-i) reacting a compound represented by general formula (13) with a compound represented by general formula (14) to obtain an intermediate compound represented by general formula (15):

[0141]

[0142] R8, R9, and R10are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0143] R7is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0144] R6comprises a hydrophobic moiety;

[0145] Z2, Z2’, and Z2’ are each independently selected from the group consisting of -O-C(=O)-, -C(=O)-O-, -C(=O)-, -O-, -RXN-C(=O)-, -C(=O)-NRy-, where Rxand Ryare each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0146] R24, R25, and R26are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0147] y is an integer ≥ 1; and (d-ii) subjecting the intermediate compound represented by general formula (15) to oxidation to obtain the compound comprising carbonyl represented by general formula (7):

[0148]

[0149] In one embodiment, the compound or ionized form thereof disclosed herein, or the nanoparticle composition disclosed herein is for use in medicine.

[0150] In one aspect, there is provided a method of modulating an immune response in a subject, the method comprising the step of administering to a subject a therapeutically effective amount of the nanoparticle composition disclosed herein.

[0151] In one embodiment, the nanoparticle composition disclosed herein is for use in modulating an immune response in a subject, wherein said nanoparticle composition is to be administered to the subject.

[0152] In one aspect, there is provided use of a nanoparticle composition disclosed herein in the manufacture of a medicament for modulating an immune response in a subject.

[0153] DEFINITIONS

[0154] 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 sub- particles 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.

[0155] The term “nano" as used herein is to be interpreted broadly to include dimensions in a nanoscale, i.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 200 nm, no more than about 150 nm, no more than 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.

[0156] 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.

[0157] 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, respiratory illnesses (e.g., coronavirus caused by the SARS-CoV-2 virus or flu caused by influenza virus), oncological diseases (e.g., cancer), dermatological diseases (e.g., eczema), ophthalmological diseases (e.g., age-related macular degeneration (AMD)), fibrotic diseases (e.g., fibrosis), or cardiovascular diseases. 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 titre the dosage and modify the route of administration as required to obtain the optimal therapeutic effect.

[0158] The term “subject” is intended to broadly refer to any plants or animals 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), 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). 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.

[0159] 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 -CH3 and when existing as a bridging group, the term “alkyl" having 1 carbon atom may mean -CH2- or the like.

[0160] The term "alkyl" or “alkylene” as a group or part of a group refers to a straight or branched aliphatic hydrocarbon group having 1 to 50 carbon atoms, 1 to 20 carbon atoms, 1 to 15 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, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45 or 50 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.

[0161] The term "alkenyl" or “alkenylene” 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 50 carbon atoms, 2 to 20 carbon atoms, 2 to 15 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, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45 or 50 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.

[0162] The term "alkynyl" or “alkynylene” 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 50 carbon atoms, 2 to 20 carbon atoms, 2 to 15 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, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45 or 25 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.

[0163] 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-).

[0164] The term "aryl" as a group or part of a group denotes 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.

[0165] 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 / sulphur (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, 1H-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. 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”.

[0166] 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 / sulphur (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, tetra hydrofuran, 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.

[0167] 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. 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.

[0168] 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.

[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 encapsulating a therapeutic agent, prophylactic agent, and / or biological agent (e.g., a nucleic acid such as mRNA, siRNA, etc.). In various embodiments, the compound comprises a structure represented by general formula (1) or ionized form thereof:

[0178]

[0179] wherein A comprises a carbohydrate / sugar / saccharide, a polyol, a polyhydroxyl moiety / group, and / or derivatives thereof;

[0180] R1, R2, R3, R8, R9, and R10are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0181] R4and R7are each independently optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0182] R5and R6each independently comprises a hydrophobic moiety / tail / chain / group; Z1and Z2are each independently selected from the group consisting of -O-C(=O)-, -C(=O)-O-, -C(=O)-, -O-, -RaN-C(=O)-, -C(=O)-NRb- where Raand Rbare each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0183] x is an integer ≥ 1; and

[0184] y is an integer ≥ 0.

[0185] It will be appreciated that in various embodiments when y = 0, R6is a terminal group (i.e., R6is not a bridging group).

[0186] In various embodiments, A satisfies any one or more of the following conditions:

[0187] (i) A is devoid of sulfur / sulphur (S)-containing moieties (e.g., thioether bonds); (ii) A is devoid of nitrogen (N)-containing moieties (e.g., amide linkages); (iii) A is devoid of carbonyl group (i.e., -C=O-)-containing moieties (e.g., amide linkages);

[0188] (iv) A is represented by general formula ~Rh-Aawherein Aais CqH2q+1Oqwhere q > 1, q > 2, q > 3, q > 4, q > 5, q > 6, q > 7, q > 8, q > 9, q > 10, q > 11, q > 12, q > 13, q > 14, q > 15, q > 16, q > 17, q > 18, q > 19, q > 20, q > 21, q > 22, q > 23, q > 24, or q > 25, and Rhis optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene and Rhis devoid of sulfur / sulphur (S)-containing moieties and / or oxygen (O)-atom containing moieties; and / or (v) the C1atom of the carbohydrate / sugar / saccharide source / precursor of A is directly connected / linked to the nitrogen (N) atom in general formula (1 ) with no linker groups present in between.

[0189] For example, when A satisfies condition (iv), the compound may be represented by general formula (1a):

[0190]

[0191] wherein R1to R10, Z1, Z2, Rh, and Aacontain one or more features and / or share one or more properties that are similar to those described above.

[0192] In various embodiments, Rhis selected from alkylene, alkenylene, or alkynylene (e g., unsubstituted). The alkylene, alkenylene, or 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, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 carbon atoms. For example, Rhmay be CrH2r–, where r ≥ 1, r > 2, r > 3, r > 4, r > 5, r > 6, r > 7, r > 8, r > 9, r > 10, r > 11, r > 12, r > 13, r > 14, r > 15, r > 16, r > 17, r > 18, r > 19, r > 20, r > 21, r > 22, r > 23, r > 24, or r > 25. For example, Rhmay be selected from methylene, ethylene, n-propylene, 2-propylene, isopropylene, n-butylene, isobutylene, secbutylene, 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, the like, or combinations thereof.

[0193] In various embodiments, R1, R2, R3, R8, R9and R10are 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, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 carbon atoms. For example, R1, R2, R3, R8, R9and R10may be each independently CpH2p, CpH2p+1, or CpH2p-2, where p > 1, p > 2, p > 3, p > 4, p > 5, p > 6, p > 7, p > 8, p > 9, p > 10, p > 11, p > 12, p > 13, p > 14, p > 15, p > 16, p > 17, p > 18, p > 19, p > 20, p > 21, p > 22, p > 23, p > 24, or p > 25. For example, R1, R2, R3, R8, R9and R10may be 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.

[0194] In various embodiments, R4and R7are each independently selected from optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl. For example, R4and R7may be 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-m ethylheptyl, 1 -methylheptyl, octyl, nonyl, decyl, the like, or combinations thereof. In various embodiments, R4and / or R7is / are an alkyl » / vw

[0195] having 1 carbon atom, e.g.,

[0196]

[0197] . In various embodiments, R4and / or R7

[0198] «SVW H

[0199] s -c I-c I — s

[0200] I I

[0201] is / are an alkyl having 2 carbon atoms, e

[0202]

[0203] .g.,M

[0204] In various embodiments, the hydrophobic moiety / tail / chain / group at R5and R6each independently comprises optionally substituted alkyl. The alkyl may have 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, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 carbon atoms. For example, R5and R6are each independently CzH2zor CzH2z+1, where z > 3, z > 4, z > 5, z > 6, z > 7, z > 8, z > 9, z > 10, z > 11, z > 12, z > 13, z > 14, z > 15, z > 16, z > 17, z > 18, z > 19, z > 20, z > 21, z > 22, z > 23, z > 24, or z > 25.

[0205] In various embodiments, R1= R10, R2= R8, R3= R9, R4= R7, Z1= Z2, and / or R5= R6.

[0206] In various embodiments, Raand Rbare each independently selected from H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl. For example, Raand / or Rbmay be 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-m ethylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1, 3-d im ethyl butyl, 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-m ethylheptyl, 1 -methylheptyl, octyl, nonyl, decyl, the like, or combinations thereof.

[0207] In various embodiments, x = 1, x = 2, or x = 3.

[0208] In various embodiments, y = 0, y = 1, y = 2, or y = 3.

[0209] In various embodiments, the compound optionally comprises two or more, three or more, four or more, five or more, or six or more hydrophilic groups (e.g., in A). In various embodiments, the hydrophilic group (e.g., in A) optionally comprises -ORCand / or -O-C(=O)Rd, where Rcand Rdare each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

[0210] In various embodiments, the structure represented by general formula (1) is ionizable or capable of being ionized such that the composition encapsulates a therapeutic and / or prophylactic agent and / or biological agent that is coupled / bonded / linked / bound (e g., chemically coupled / bonded / linked / bound) to the composition / nanoparticle. In various embodiments, the compound comprises a lipid compound such as an ionizable lipid compound.

[0211] In various embodiments, the amine-containing groups in the structure represented by general formula (1) may be protonated and positively charged under acidic conditions (e.g., endosomal pH), thereby enhancing electrostatic interactions with negatively charged therapeutic agent and / or prophylactic agent and / or biological agent (e.g., nucleic acids). In various embodiments, these amine-containing groups may be deprotonated and neutral at physiological pH, thereby reducing cytotoxicity and improving biocompatibility.

[0212] In various embodiments, advantageously, the compound / structure represented by general formula (1) is designed / configured to be ionizable at a low pH, e.g., at a pH range of from about pH 2 to about pH 6, depending on the type or nature of the amine group, e.g., the substituent(s) attached to the amine group. In various embodiments, the compound is capable of being ionized at a pH that is lower than its pKa. In various embodiments, the compound has a pKa of from about 5.0 to about 7.0, from about 5.1 to about 7.0, from about 5.2 to about 7.0, from about 5.3 to about 7.0, from about 5.4 to about 7.0, from about 5.5 to about 7.0, from about 5.6 to about 6.9, from about 5.7 to about 6.8, from about 5.8 to about 6.7, from about 5.9 to about 6.6, from about 6.0 to about 6.5, from about 6.1 to about 6.4, from about 6.2 to about 6.3, or about 6.25. For example, the compound may be ionized at a pH range of from about 2.0 to about 6.0, from about pH 2.1 to about pH 5.9, from about pH 2.2 to about pH 5.8, from about pH 2.3 to about pH 5.7, from about pH 2.4 to about pH 5.6, from about pH 2.5 to about pH 5.5, from about pH 2.6 to about pH 5.4, from about pH 2.7 to about pH 5.3, from about pH 2.8 to about pH 5.2, from about pH 2.9 to about pH 5.1, from about pH 3.0 to about pH 5.0, from about pH 3.1 to about pH 4.9, from about pH 3.2 to about pH 4.8, from about pH 3.3 to about pH 4.7, from about pH 3.4 to about pH 4.6, from about pH 3.5 to about pH 4.5, from about pH 3.6 to about pH 4.4, from about pH 3.7 to about pH 4.3, from about pH 3.8 to about pH 4.2, from about pH 3.9 to about pH 4.1, or about pH 4.0. In various embodiments, the compound is designed / configured to be substantially neutral at physiological pH, thereby making the compound biocompatible. In various embodiments, the compound carries substantially no charge (or zero charge) at physiological pH range of from about 7.00 to about 7.80, from about 7.05 to about 7.75, from about 7.10 to about 7.70, from about 7.15 to about 7.65, from about 7.20 to about 7.60, from about 7.25 to about 7.55, from about 7.30 to about 7.50, about 7.35, about 7.36, about 7.37, about 7.38, about 7.39, about 7.40, about 7.41, about 7.42, about 7.43, about 7.44, or about 7.45. In various embodiments, the compound / structure represented by general formula (1) is ionizable or capable of being ionized to become a positively charged group / ion / cation. For example, the compound / structure may be protonated to become a quaternary ammonium compound / group / ion / cation as shown in general formula (1b) below:

[0213] R2

[0214] R1— R4— R5

[0215] R3

[0216] R8

[0217] R1° -R7 -

[0218]

[0219] R9

[0220] (1b).

[0221] In various embodiments, the carbohydrate / sugar / saccharide is selected from the group consisting of monosaccharide, disaccharide, oligosaccharide, polysaccharide, and derivatives thereof. In various embodiments, A comprises a monosaccharide and / or a derivative thereof. For example, A comprises glucose, galactose, mannose, fructose, ribose, pentose, hexose, deoxyribose, xylose, arabinose, and their derivatives such as amino, deoxy, alkoxy, or acylated sugars. For example, A comprises 2-deoxy-D-ribofuranose, D-ribofuranose, D-xylofuranose, D-arabinofuranose, D-glucopyranose, D-mannopyranose, D-galactopyranose, L-rham nopyranose, D-fructopyranose, N-acetyl-glucosamine, N-acetyl-mannosamine, N-acetyl-galactosamine, 2-am ino-2-deoxy-D-glucopyranose, 2-amino-2-deoxy-D-mannopyranose, 2-am ino-2-deoxy-D-galactopyranose, 1 -amino-1 -deoxy-D-fructose, 3-am ino-3-deoxy-D-mannopyranose, 1-amino-1-deoxy-D-galactopyranose, 1 -(2-aminoethoxy)-1 -deoxy-D-glucopyranose, 1 -(2-aminoethoxy)-1 -deoxy-D-mannopyranose, 1 -(2-aminoethoxy)-1 -deoxy-D-galactopyranose, 1 -(3-aminoethoxy)-1 -deoxy-D-glucopyranose, 1-(3-aminoethoxy)-1 -deoxy-D-mannopyranose, 1-(3-aminoethoxy)-1 -deoxy-D-galactopyranose, 1-(3-aminopropoxy)-1-deoxy-D- glucopyranose, 1 -(3-aminopropoxy)-1 -deoxy-D-mannopyranose, 1 -(3-aminopropoxy)-1-deoxy-D-galactopyranose, the like, a derivative, or combinations thereof.

[0222] In various embodiments, the carbohydrate / sugar / saccharide is in a linear form / structure, for example as a fructose, ribose, mannose, glucose, galactose, deoxyribose, xylose, sucrose, maltose, lactose, arabinose, the like, derivative(s), or combinations thereof. The carbohydrate / sugar / saccharide may comprise a pentose, hexose, or the like.

[0223] In various embodiments, the carbohydrate / sugar / saccharide comprises reducing carbohydrates / sugars / saccharides such as fructose, ribose, mannose, glucose, galactose, deoxyribose, xylose, maltose, lactose, arabinose, the like, derivative(s), or combinations thereof.

[0224] In various embodiments, the carbohydrate / sugar / saccharide is in a ring form / structure, for example as a fructose, ribose, mannose, glucose, galactose, deoxyribose, xylose, maltose, lactose, arabinose, cellobiose, 2-deoxy-D-ribofuranose, D-ribofuranose, D-xylofuranose, D-arabinofuranose, D-glucopyranose, D-mannopyranose, D-galactopyranose, L-rhamnopyranose, D-fructo pyranose, N-acetyl-glucosamine, N-acetyl-mannosamine, N -acetyl -galactosamine, 2-amino-2-deoxy-D-glucopyranose, 2-am ino-2-deoxy-D-mannopyranose, 2-amino-2-deoxy-D-galactopyranose, 1-amino-1-deoxy-D-fructose, 3-amino-3-deoxy-D- mannopyranose, 1-amino-1-deoxy-D-galactopyranose, 1 -(2-aminoethoxy)-1 -deoxy-D-glucopyranose, 1 -(2-am inoethoxy)-1 -deoxy-D-m annopyranose, 1 -(2-am inoethoxy)-1 -deoxy-D-galactopyranose, 1 -(3-aminoethoxy)-1 -deoxy-D-glucopyranose, 1 -(3-am inoethoxy)-1 -deoxy-D-m annopyranose, 1 -(3-am inoethoxy)-1 -deoxy-D-galactopyranose, 1 -(3-aminopropoxy)-1 -deoxy-D-glucopyranose, 1 -(3-aminopropoxy)-1-deoxy-D-mannopyranose, 1-(3-aminopropoxy)-1-deoxy-D-galactopyranose, the like, a derivative, or combinations thereof. The carbohydrate / sugar / saccharide may comprise a pentose, hexose, or the like. In various embodiments, the carbohydrate / sugar / saccharide comprises a structure selected from the group below, or one or more derivatives thereof derived from any of the structures selected from the group below:

[0225]

[0226] 2-deoxy- D-ribofuranose D-ribofuranose D-xylofuranose D-arabinofuranose

[0227]

[0228] D-glucopyranose D-mannopyranose D-galactopyranose L-rhamnopyranose

[0229]

[0230] D-fructopyranose N-acetyl- N-acetyl- N-acetyl- glucosamine mannosamine galactosamine

[0231]

[0232]

[0233] Lactose Maltose Cellobiose

[0234]

[0235] OH

[0236] 2-amino-2-deoxy- 2-amino-2-deoxy- 2-amino-2-deoxy- D-glucopyranose D-mannopyranose D-galactopyranose

[0237]

[0238] NH2OH

[0239] 1-amino-1-deoxy- 3-amino-3-deoxy- 1-amino-1-deoxy- D-fructose D-mannopyranose D-galactopyranose

[0240]

[0241] 1 -(2-aminoethoxy)-1 -deoxy- -(2-aminoethoxy)-1-deoxy- 1 -(2-aminoethoxy)-1 -deoxy- D-glucopyranose D-mannopyranose D-galactopyranose

[0242]

[0243] 1 -(3-aminopropoxy)-1 -deoxy- 1 -(3-aminopropoxy)-1 -deoxy- 1 -(3-aminopropoxy)-1 -deoxy- D-glucopyranose D-mannopyranose D-galactopyranose

[0244] In various embodiments, the derivatives of the carbohydrate / sugar / saccharide comprise one or more of carbonyl (e g., aldehyde or ketone) groups being converted into alkyl. For example, derivatives of the carbohydrate / sugar / saccharide may comprise -CH(=O) or -C(=O)- groups being converted into -CH2-. In various embodiments, the derivatives of the carbohydrate / sugar / saccharide optionally comprise one or more of -OH groups being converted into -ORCand / or -O-C(=O)Rd, where Rcand Rdare each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl. For example, when Rdis methyl, -O-C(=O)Rdcomprises an acetyl group (-Ac) and corresponds to -O-C(=O)-CH3 (-OAc). In various embodiments, A is represented by general formula (2) or general formula (2A):

[0245]

[0246] (2) (2A) wherein

[0247] R29to R51are each independently selected from -H, -ORe, or -O-C(=O)Rf, -Ri, -RjORk, -NRl-C(=O)Rmor Rn, where Reis H, Rf, Riand Rmare each independently optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl, Rkand Rlare each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl, Rjis optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene, and Rncomprises a structure represented by general formula (2A);

[0248] R52is optionally present as -O-, -OR0-, or -Rp-, where R° and Rpare each independently optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene, and wherein one of R42to R51is absent; and n is 0 or 1.

[0249] In various embodiments, when R52is absent, R52is simply a single bond. In various embodiments, the solid line intersecting the ring structure in general formula (2A) represents a connection between any one of the carbon atoms of the ring structure and the nitrogen atom of general formula (1 ).

[0250] In various embodiments, it will be appreciated that, even though the bonds in general formulae (2) and (2A) are drawn in a non-stereochemical manner (i.e., flat or in the plane of the paper), the atom(s) to which the bonds are attached respectively may adopt any suitable types of stereochemical configurations. In various embodiments, A exists in the form of enantiomers and / or diastereoisomers, e g., as represented by general formula (2’):

[0251]

[0252] In various embodiments, A may exist in the form of L-enantiomer (e.g., L-ribose, L-mannose, deoxy-L-ribose, 2-deoxy-L-ribose, L-fructose, L-glucose, L-galactose, L-xylose, L-sucrose, L-maltose, L-lactose, L-arabinose, and / or derivatives thereof). In various embodiments, A may exist in the form of D-enantiomer (e.g., D-ribose, D-mannose, deoxy-D-ribose, 2-deoxy-D-ribose, D-fructose, D-glucose, D-galactose, D-xylose, D-sucrose, D-maltose, D-lactose, D-arabinose, and / or derivatives thereof).

[0253] In various embodiments, A exists in the form of enantiomers and / or diastereoisomers, e.g., as represented by general formula (2A’):

[0254]

[0255] (2A')

[0256] In various embodiments, R29to R51are each independently -R'. In various embodiments, R37and / or R38may be -Ri, e.g., -CwH2w+1, where w ≥ 1. In various embodiments, w is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 30. For example, R37and / or R38may be an alkyl selected from methyl (–CH3), ethyl (–CH2CH3), 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.

[0257] In various embodiments, R29to R51are each independently -RjORk. In various embodiments, Rkis H. In various embodiments, R29and / or R30may be -RjORk, e g., -CvH2vOH, where v > 1. In various embodiments, v is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 30. For example, R29and / or R30may be hydroxyalkyl selected from hydroxymethyl (–CH2OH), hydroxyethyl (–CH2CH2OH), hydroxypropyl, hydroxybutyl, hydroxypentyl, hydroxyhexyl, hydroxyheptyl, hydroxyoctyl, hydroxynonyl, hydroxydecyl, hydroxyundecyl, hydroxydodecyl, hydroxytridecyl, hydroxytetradecyl, hydroxypentadecyl, hydroxyhexadecyl, hydroxyheptadecyl, hydroxyoctadecyl, hydroxynonadecyl, the like, or combinations thereof.

[0258] In various embodiments, R29to R51are each independently -NR1-C(=O)Rm. In various embodiments, R1is H. In various embodiments, R31and / or R32may be -NR'-C(=O)Rm, e g., -NH-C(=O)CrH2r+i, where r ≥ 1. In various embodiments, r is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 30. For example, R31and / or R32may be amide selected from acetamide, propionamide, butyramide, valeronamide, caproamide, enanthamide, caprylamide, pelargonamide, capramide, undecanamide, dodecanamide, tridecanamide, tetradecanamide, pentadecanamide, hexadecanamide, heptadecanamide, octadecanamide, nonadecanamide, icosanamide, the like, or combinations thereof. In various embodiments, general formula (2A) is represented by any one of the following general formulae (2B), (2C), (2D), (2E) and (2F):

[0259]

[0260] In various embodiments, the compound is selected from the group consisting of the following structures:

[0261]

[0262] ZJY-01 / ZJY-1G ZJY-02 / ZJY-2D

[0263]

[0264] ZJY-03 / ZJY-1A

[0265]

[0266] ZJY-04 / ZJY-2A / ZJY-3A / ZJY-4A ZJY-05 / ZJY-5A / ZJY-6A / ZJY-7A

[0267]

[0268] ZJY-06 ZJY-1B

[0269]

[0270] ZJY-1C

[0271]

[0272] ZJY-1D

[0273]

[0274] ZJY-1E ZJY-1F

[0275]

[0276] ZJY-1H

[0277]

[0278] ZJY-1I ZJY-1J ZJY-1K

[0279]

[0280] ZJY-2B ZJY-2C

[0281]

[0282] ZJY-6B

[0283]

[0284] ZJY-6C

[0285]

[0286] ZJY-6D

[0287]

[0288]

[0289] ZJY-6E ZJY-6F ZJY-6G

[0290]

[0291] OH ZJY-6H

[0292]

[0293] ZJY-8A

[0294]

[0295] ZJY-9A

[0296]

[0297] ZJY-10A / ZJY-1IA / ZJY-12A

[0298]

[0299] ZJY-13A / ZJY-14A / ZJY-15A

[0300]

[0301] ZJY-16A / ZJY-17A / ZJY-18A

[0302]

[0303] ZJY-17B

[0304]

[0305] ZJY-19A

[0306]

[0307] ZJY-20A

[0308]

[0309] ZJY-21A

[0310]

[0311] ZJY-25A / ZJY-26A / ZJY-27A In various embodiments, the compound is selected from the group consisting of the following structures:

[0312]

[0313] ZJY-02

[0314]

[0315] ZJY-04 ZJY-05

[0316]

[0317] ZJY-06

[0318]

[0319] ZJY-06 ZJY-1B

[0320]

[0321] ZJY-1C

[0322]

[0323] ZJY-1D

[0324]

[0325] ZJY-1E

[0326]

[0327] ZJY-1G

[0328]

[0329] ZJY-1I

[0330]

[0331] ZJY-1K

[0332]

[0333] ZJY-2B

[0334]

[0335] ZJY-2D

[0336]

[0337] ZJY-3A ; ZJY-4A

[0338]

[0339] ZJY-5A

[0340]

[0341] ZJY-6A

[0342]

[0343] ZJY-6B

[0344]

[0345] ZJY-6C

[0346]

[0347] ZJY-6D

[0348]

[0349] ZJY-6E ZJY-6F ZJY-6G

[0350]

[0351] ZJY-6H

[0352]

[0353] ZJY-7A

[0354]

[0355] ZJY-8A

[0356]

[0357] ZJY-9A

[0358]

[0359] ZJY-10A

[0360]

[0361] ZJY-1IA

[0362]

[0363] ZJY-12A

[0364]

[0365] ZJY-13A ZJY-14A

[0366]

[0367] ZJY-15A

[0368]

[0369] ZJY-16A ZJY-17A

[0370]

[0371] ZJY-17B

[0372]

[0373] ZJY-18A

[0374]

[0375] ZJY-19A

[0376]

[0377] ZJY-20A

[0378]

[0379] ZJY-22A

[0380]

[0381] ZJY-24A ZJY-25A

[0382]

[0383] ZJY-26A; and

[0384]

[0385] ZJY-27A

[0386] In various embodiments, the compound has a molecular weight of from about 250 g / mol to about 5,000 g / mol, from about 500 g / mol to about 4,750 g / mol, from about 750 g / mol to about 4,500 g / mol, from about 1,000 g / mol to about 4,250 g / mol, from about 1,250 g / mol to about 4,000 g / mol, from about 1,500 g / mol to about 3,750 g / mol, from about 1,750 g / mol to about 3,500 g / mol, from about 2,000 g / mol to about 3,250 g / mol, from about 2,250 g / mol to about 3,000 g / mol, from about 2,500 g / mol to about 2,750 g / mol, from about 300 g / mol to about 1,000 g / mol, from about 400 g / mol to about 1,000 g / mol, from about 500 g / mol to about 1,000 g / mol, from about 600 g / mol to about 1,000 g / mol, from about 700 g / mol to about 1,000 g / mol, from about 800 g / mol to about 1,000 g / mol, from about 900 g / mol to about 1,000 g / mol, or about 950 g / mol.

[0387] 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.

[0388] In various embodiments, the compound is biocompatible, i.e., the compound 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 compound is substantially devoid of substances that elicit an adverse physiological response.

[0389] METHOD OF PREPARING COMPOUND

[0390] There is provided a method of preparing a compound represented by general formula (1) as disclosed herein, the method comprising:

[0391] (a-i) reacting a compound comprising 1° amine (e.g., 6-aminohexyl 2- hexyldecanoate) represented by general formula (3) with a carbohydrate / sugar / saccharide represented by general formula (4) (e.g., 2-deoxy-D-ribose, D-ribose, and D-ribose) in the presence of a reducing agent to obtain an intermediate compound represented by general formula (6):

[0392]

[0393] (3) (4)

[0394]

[0395] wherein

[0396] A comprises a hydrophilic moiety selected from carbohydrate / sugar / saccharide and derivatives thereof;

[0397] R1, R2, and R3are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0398] R4is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0399] R5comprises a hydrophobic moiety / tail / chain / group;

[0400] Z1is selected from the group consisting of -O-C(=O)-, –C(=O)–, -O-, -RXN-C(=O)-, -C(=O)-NRy- where Rxand Ryare each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0401] R11, R12, R13, R14, R15, R16, R17, R18, R19, R20, and R21are each independently selected from -H or -OH;

[0402] x is an integer ≥ 1; and

[0403] m is 0 or 1.

[0404] In various embodiments, (a-i) comprises a one-pot reaction or is performed in a single pot.

[0405] In various embodiments, the method comprises:

[0406] (a-ii) reacting the intermediate compound represented by general formula (6) with a compound comprising carbonyl (e g., aldehyde) represented by general formula (7) (e g., dodecanal or 6-oxohexyl 2-hexyldecanoate) in the presence of a reducing agent to obtain a compound represented by general formula (1 ):

[0407]

[0408] (1) wherein

[0409] R8, R9, and R10are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0410] R7is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0411] R6comprises a hydrophobic moiety / tail / chain / group;

[0412] Z2is selected from the group consisting of -O-C(=O)-, –C(=O)–, –O–, -RXN-C(=O)-, -C(=O)-NRy-, where Rxand Ryare each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0413] y is an integer ≥ 0.

[0414] In various embodiments, (a-ii) comprises a one-pot reaction or is performed in a single pot.

[0415] In various embodiments, the method comprises:

[0416] (a-iii) optionally ionizing the compound represented by general formula (1) to become positively charged.

[0417] In various embodiments, the method may be conducted as a two-pot reaction. For example, (a-i) and (a-ii) may be performed in two separate pots.

[0418] In various embodiments, (a-i) comprises suspending / dispersing / mixing / dissolving / reacting the compound comprising 1° amine (e.g., 6-aminohexyl 2-hexyldecanoate) represented by general formula (3) with the carbohydrate / sugar / saccharide represented by general formula (4) (e.g., 2-deoxy-D-ribose, D-ribose, and D-ribose) in a molar ratio of from about 1:1 to about 1:10, from about 1:2 to about 1:10, from about 1:3 to about 1:10, from about 1:4 to about 1:10, from about 1:5 to about 1:10, from about 1:6 to about 1:10, from about 1:7 to about 1:10, from about 1:8 to about 1:10, from about 1:9 to about 1:10, about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, about 1:5, about 1:5.5, about 1:6, about 1:6.5, about 1:7, about 1:7.5, about 1:8, about 1:8.5, about 1:9, about 1:9.5, or about 1:10.

[0419] In various embodiments, (a-ii) comprises suspending / dispersing / mixing / dissolving / reacting the intermediate compound represented by general formula (6) with a compound comprising carbonyl (e.g., aldehyde) represented by general formula (7) (e.g., dodecanal or 6-oxohexyl 2-hexyldecanoate) in a molar ratio of from about 1:1 to about 1:10, from about 1:2 to about 1:10, from about 1:3 to about 1:10, from about 1:4 to about 1:10, from about 1:5 to about 1:10, from about 1:6 to about 1:10, from about 1:7 to about 1:10, from about 1:8 to about 1:10, from about 1:9 to about 1:10, about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, about 1:5, about 1:5.5, about 1:6, about 1:6.5, about 1:7, about 1:7.5, about 1:8, about 1:8.5, about 1:9, about 1:9.5, or about 1:10.

[0420] In various embodiments, (a-iii) may be performed at a low pH, e.g., at a pH range of from about pH 2 to about pH 6, depending on the type or nature of the amine group in the compound represented by general formula (1), e.g., the substituent(s) attached to the amine group. In various embodiments, (a-iii) may be performed at a pH that is lower than the pKa of the compound represented by general formula (1). In various embodiments, the compound has a pKa of from about 5.0 to about 7.0, from about 5.1 to about 7.0, from about 5.2 to about 7.0, from about 5.3 to about 7.0, from about 5.4 to about 7.0, from about 5.5 to about 7.0, from about 5.6 to about 6.9, from about 5.7 to about 6.8, from about 5.8 to about 6.7, from about 5.9 to about 6.6, from about 6.0 to about 6.5, from about 6.1 to about 6.4, from about 6.2 to about 6.3, or about 6.25. For example, (a-iii) may be performed at a pH range of from about 2.0 to about 6.0, from about pH 2.1 to about pH 5.9, from about pH 2.2 to about pH 5.8, from about pH 2.3 to about pH 5.7, from about pH 2.4 to about pH 5.6, from about pH 2.5 to about pH 5.5, from about pH 2.6 to about pH 5.4, from about pH 2.7 to about pH 5.3, from about pH 2.8 to about pH 5.2, from about pH 2.9 to about pH 5.1, from about pH 3.0 to about pH 5.0, from about pH 3.1 to about pH 4.9, from about pH 3.2 to about pH 4.8, from about pH 3.3 to about pH 4.7, from about pH 3.4 to about pH 4.6, from about pH 3.5 to about pH 4.5, from about pH 3.6 to about pH 4.4, from about pH 3.7 to about pH 4.3, from about pH 3.8 to about pH 4.2, from about pH 3.9 to about pH 4.1, or about pH 4.0.

[0421] In various embodiments, the method further comprises:

[0422] (b-i) optionally reacting the compound represented by general formula (1) with an acylating agent (e.g., acetic anhydride) represented by general formula (9) to replace one or more -OH group(s) in A with -O-C(=O)R9group(s):

[0423]

[0424] wherein

[0425] R27and R28are each independently optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and

[0426] Rgis optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

[0427] In various embodiments, (a-i) and / or (a-ii) comprise(s) a reduction reaction carried out with a reducing agent. In various embodiments, the reducing agent comprises a metal hydride (e.g., an alkali metal hydride or an alkaline earth metal hydride). For example, the reducing agent may be sodium cyanoborohydride (NaCNBH3), sodium triacetoxyborohydride (NaBH(CH3COO)3or NaBH(OAc)3) the like, or combinations thereof.

[0428] In various embodiments, wherein the acylating agent represented by general formula (9) (used in (b-i)) comprises an acid anhydride. In various embodiments, the acylating agent is selected from acetic anhydride (Ac2O), acid halide (e g., acyl halide), N-hydroxysuccinimide (NHS) esters, imidoesters, the like, or combinations thereof.

[0429] In various embodiments, reacting in (b-i) comprises adding the acylating agent represented by general formula (9) in a dropwise manner to the compound represented by general formula (1) at a low temperature such as from about -10 °C to about 50 °C, from about -8 °C to about 48 °C, from about -6 °C to about 46 °C, from about -4 °C to about 44 °C, from about -2 °C to about 42 °C, from about 0 °C to about 40 °C, from about 2 °C to about 38 °C, from about 4 °C to about 36 °C, from about 6 °C to about 34 °C, from about 8 °C to about 32 °C, from about 10 °C to about 30 °C, from about 12 °C to about 28 °C, from about 14 °C to about 26 °C, from about 16 °C to about 24 °C, from about 18 °C to about 22 °C, or about 20 °C.

[0430] Advantageously, in various embodiments, the method is a simple synthesis process that produces high yields of the compound represented by general formula (1). In various embodiments, the yield of the compound represented by general formula (1 ) is at least about 10 %, at least about 20 %, at least about 30 %, at least about 40 %, at least about 50 %, at least about 60 %, at least about 70 %, at least about 80 %, at least about 81 %, at least about 82 %, at least about 83 %, at least about 84 %, at least about 85 %, at least about 86 %, at least about 87 %, at least about 88 %, at least about 89 %, at least about 90 %, at least about 91 %, at least about 92 %, at least about 93 %, at least about 94 %, at least about 95 %, at least about 96 %, at least about 97 %, at least about 98 %, at least about 99 %, or at least about 99.9 %.

[0431] In various embodiments, the method further comprises, prior to (a-i): (c-i) reacting a compound represented by general formula (10) (e.g., 2- hexyldecanoic acid) with protected amine compound represented by general formula (11) (e.g., tert-butyl (6-hydroxyhexyl)carbamate) to obtain an intermediate compound represented by general formula (12): H R2R1- R4- - R22R3(10) (11)

[0432] PG1

[0433]

[0434] wherein

[0435] R1, R2, and R3are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0436] R4is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0437] R5comprises a hydrophobic moiety / tail / chain / group;

[0438] Z1, Z1’, and Z1” are each independently selected from the group consisting of -O-C(=O)-, -C(=O)-O-, -C(=O)-, -O-, -RXN-C(=O)-, -C(=O)-NRy-, where Rxand Ryare each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0439] R22and R23are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0440] x is an integer > 1;

[0441] PG1is a protecting group such as N-carboxybenzyl or benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), or 9-fluorenylmethyloxycarbonyl (Fmoc); and

[0442] (c-ii) deprotecting the intermediate compound represented by general formula (12) to obtain the compound comprising 1° amine represented by general formula (3):

[0443]

[0444] In various embodiments, the deprotection / deprotecting of the intermediate compound represented by general formula (12) in (c-ii) comprises subjecting the intermediate compound to acidic conditions. For example, (c-ii) may be carried out in the presence of one or more of acids such as trifluoracetic acid (TFA), hydrochloric acid, sulfuric / sulphuric acid, phosphoric acid, hydrobromic acid, formic acid, or acetic acid.

[0445] In various embodiments, the method further comprises, prior to (a-ii): (d-i) reacting a compound represented by general formula (13) (e.g., 2- hexyldecanoic acid) with a compound represented by general formula (14) (e.g., hexane-1,6-diol) to obtain an intermediate compound represented by general formula (15):

[0446]

[0447] (15) wherein

[0448] R8, R9, and R10are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; R7is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0449] R6comprises a hydrophobic moiety / tail / chain / group;

[0450] Z2, Z2’, and Z2” are each independently selected from the group consisting of -O-C(=O)-, -C(=O)-O-, -C(=O)-, -O-, -RXN-C(=O)-, -C(=O)-NRy- where Rxand Ryare each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0451] R24, R25, and R26are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0452] y is an integer ≥ 1; and

[0453] (d-i i) subjecting the intermediate compound represented by general formula (15) to oxidation to obtain the compound comprising carbonyl (e.g., aldehyde) represented by general formula (7):

[0454]

[0455] In various embodiments, (d-ii) comprises oxidizing the intermediate compound represented by general formula (15) with an oxidizing agent. In various embodiments, the oxidizing agent is selected from pyridinium chlorochromate (PCC), Dess-Martin periodinane (DMP), oxalyl chloride, the like, or combinations thereof.

[0456] In various embodiments, (c-i) and / or (d-i) is / are performed in the presence of an activating agent (e.g, carboxyl activating agent). In various embodiments, the activating agent comprises carbodiimide. For example, the activating agent may be 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC•HCl), / V, / V’-dicyclohexylcarbodiimide (DCC), the like, or combinations thereof. In various embodiments, (c-i) and / or (d-i) is / are performed in the presence of a catalyst. In various embodiments, the catalyst comprises a nucleophilic catalyst. For example, the catalyst may be 4-dimethylaminopyridine (DMAP), / V, / V-diisopropylethylamine (DIPEA), triethylamine (TEA), the like, or combinations thereof. In various embodiments, DMAP is basic and may promote the deprotonation of general formula (10) or general formula (13) in (c-i) or (d-i).

[0457] In various embodiments, the reacting / deprotecting / oxidizing / reducing in (a-i), (a-ii), (a-iii), (b-i), (c-i), (c-ii), (d-i), and / or (d-ii) comprises one or more of the following: dispersing, mixing, stirring, dissolving, sonicating, and / or ultrasonicating.

[0458] In various embodiments, the reacting / deprotecting / oxidizing / reducing in (a-i), (a-ii), (a-iii), (b-i), (c-i), (c-ii), (d-i), and / or (d-ii) is / are performed in the presence of an organic solvent. The organic solvent may be an organic solvent such as hexane, ether, methanol, ethanol, dichloromethane (DCM), acetic acid, tetra hydrofuran (THF), acetonitrile, chloroform, or ethyl acetate. In various embodiments, the organic solvent may be provided in a dry or anhydrous form.

[0459] In various embodiments, the reacting / deprotecting / oxidizing / reducing in (a-i), (a-ii), (a-iii), (b-i), (c-i), (c-ii), (d-i), and / or (d-ii) is / are carried out under vacuum or in an inert atmosphere. For example, the dispersing, mixing, and / or stirring may be performed in the presence of an inert gas such as argon or nitrogen.

[0460] In various embodiments, the reacting / deprotecting / oxidizing / reducing in (a-i), (a-ii), (a-iii), (b-i), (c-i), (c-ii), (d-i), and / or (d-ii) is / are performed over a time duration of from about 1 hour to about 72 hours, from about 2 hours to about 60 hours, from about 3 hours to about 48 hours, from about 4 hours to about 36 hours, from about 5 hours to about 24 hours, or from about 6 hours to about 12 hours. In various embodiments, the reacting / deprotecting / oxidizing / reducing in (a-i), (a-ii), (a-iii), (b-i), (c-i), (c-ii), (d-i), and / or (d-ii) is / are optionally performed at room temperature e g., that is from about 0 °C to about 70 °C, from about 5 °C to about 65 °C, from about 10 °C to about 60 °C, from about 15 °C to about 55 °C, from about 20 °C to about 50 °C, from about 25 °C to about 45 °C, from about 30 °C to about 40 °C, or about 35 °C, or at room temperature.

[0461] In various embodiments, the method further comprises:

[0462] (e-i) isolating the intermediate compound represented by general formula (6) after (a-i):

[0463] (e-ii) isolating the compound represented by general formula (1 ) after (a-ii); (e-iii) isolating the compound represented by general formula (1) with acylated A after (b-i);

[0464] (e-iv) isolating the intermediate compound represented by general formula (12) after (c-i);

[0465] (e-v) isolating the compound comprising 1° amine represented by general formula (3) after (c-ii);

[0466] (e-vi) isolating the intermediate compound represented by general formula (15) after (d-i); and

[0467] (e-vii) isolating the compound comprising carbonyl represented by general formula (7) after (d-ii).

[0468] In various embodiments, isolating comprises one or more of the following: re-dissolving, purifying, centrifuging, quenching, washing, precipitating, and / or recrystallizing. In various embodiments, the washing medium comprises aqueous medium / solutions. In various embodiments, the washing medium comprises salt solution, deionized water or ice water. The salt solution may be bicarbonate salts such as sodium bicarbonate (NaHCOs), chloride salts such as sodium chlorine (brine) (HCI). In various embodiments, the salt solution comprises highly concentrated / saturated salt solution such as saturated brine. In various embodiments, purifying, centrifuging, quenching, 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. Advantageously, in various embodiments, the compound represented by general formula (1) can be easily purified.

[0469] In various embodiments, the method further comprises one or more of the following post reaction procedures: drying under vacuum, or in an inert atmosphere. In various embodiments, drying is performed in the presence of a drying agent such as magnesium sulfate, sodium sulfate (Na2SO4), calcium chloride, the like, or combinations thereof.

[0470] COMPOSITION

[0471] 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 ionizable lipid (e.g., ALC-0315, SM-102), in the formulation of a composition e.g., for the preparation of nanoparticles. Advantageously, in various embodiments, the ionizable property of the compound represented by general formula (1 ) (due to presence of ionizable amine group) allows for the condensation and encapsulation / loading of molecules / cargoes into embodiments of the compound, thereby forming nanoparticles. In various embodiments, embodiments of the compound are capable of forming or being formulated into a composition that is suitable for preparing nanoparticles.

[0472] In various embodiments, the composition comprises:

[0473] (i) a compound represented by general formula (1) and / or ionized form thereof disclosed herein; and

[0474] (ii) a therapeutic, prophylactic, and / or biological agent that is encapsulated / loaded / coupled / bonded / linked / bound in / to said compound. 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.

[0475] In various embodiments, the composition further comprises:

[0476] (a) neutral / helper lipid;

[0477] (b) sterol; and

[0478] (c) polyethylene glycol (PEG)-modified lipid or amphiphilic lipid.

[0479] In various embodiments, the term “polyethylene glycol (PEG)-modified lipid” may comprise and / or may be used interchangeably with the terms “PEGylated lipid”, “PEG-conjugated lipid”, “PEG-lipid conjugate” and / or “lipid modified with PEG”.

[0480] In various embodiments, the compound, neutral / helper lipid, sterol, and PEG-modified lipid / amphiphilic lipid are mixed / dissolved in an organic solvent.

[0481] In various embodiments, the compound represented by general formula (1 ), neutral / helper lipid, sterol, and PEG-modified lipid / amphiphilic lipid are mixed at a molar ratio of about 10 - 80: about 0 - 50: about 10 - 80: about 0.5 - 20.

[0482] 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.

[0483] In various embodiments, the sterol is selected from cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, avenasterol, the like, or combinations thereof.

[0484] In various embodiments, the PEG-modified lipid / amphiphilic 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-[(uj-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), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-methoxy] (sodium salt) (PEG-DMPE), PEG-DPPC, PEG-DSPE lipid, the like, or combinations thereof. In various embodiments, the organic solvent comprises hydrophilic organic solvents such as ethanol, isopropanol, acetonitrile, methanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), the like, or combinations thereof.

[0485] In various embodiments, the composition comprises from about 10 mol% to about 80 mol%, from about 12 mol% to about 80 mol%, from about 14 mol% to about 80 mol%, from about 16 mol% to about 80 mol%, from about 18 mol% to about 80 mol%, from about 20 mol% to about 78 mol%, from about 20 mol% to about 75 mol%, from about 22 mol% to about 74 mol%, from about 24 mol% to about 72 mol%, from about 26 mol% to about 70 mol%, from about 28 mol% to about 68 mol%, from about 30 mol% to about 66 mol%, from about 32 mol% to about 64 mol%, from about 34 mol% to about 62 mol%, from about 36 mol% to about 60 mol%, from about 38 mol% to about 58 mol%, from about 40 mol% to about 56 mol%, from about 42 mol% to about 54 mol%, from about 44 mol% to about 52 mol%, from about 46 mol% to about 50 mol%, from about 47 mol% to about 49 mol%, or about 48 mol% of compound represented by general formula (1).

[0486] In various embodiments, the composition comprises from about 0 mol% to about 50 mol%, from about 2 mol% to about 48 mol%, from about 4 mol% to about 46 mol%, from about 6 mol% to about 44 mol%, from about 8 mol% to about 42 mol%, from about 10 mol% to about 40 mol%, from about 12 mol% to about 38 mol%, from about 14 mol% to about 36 mol%, from about 16 mol% to about 34 mol%, from about 18 mol% to about 32 mol%, from about 20 mol% to about 30 mol%, from about 22 mol% to about 28 mol%, from about 24 mol% to about 26 mol%, or about 25 mol% of neutral / helper lipid.

[0487] In various embodiments, the composition comprises from about 10 mol% to about 80 mol%, from about 15 mol% to about 75 mol%, from about 20 mol% to about 70 mol%, from about 25 mol% to about 65 mol%, from about 30 mol% to about 60 mol%, from about 35 mol% to about 55 mol%, from about 40 mol% to about 50 mol%, or about 45 mol% of sterol. In various embodiments, the composition comprises from about 0.5 mol% to about 20 mol%, from about 0.75 mol% to about 19 mol%, from about 1 mol% to about 18 mol%, from about 1.1 mol% to about 17 mol%, from about 1.2 mol% to about 16 mol%, from about 1.3 mol% to about 15 mol%, from about 1.4 mol% to about 14 mol%, from about 1.5 mol% to about 13 mol%, from about 1.6 mol% to about 12 mol%, from about 1.7 mol% to about 11 mol%, from about 1.8 mol% to about 10 mol%, from about 1.9 mol% to about 9 mol%, from about 2 mol% to about 8 mol%, from about 3 mol% to about 7 mol%, from about 4 mol% to about 6 mol%, or about 5 mol% of PEG-modified lipid / amphiphilic lipid.

[0488] In various embodiments, the composition is a nanoparticle composition for delivery of a therapeutic, prophylactic, and / or biological agent.

[0489] In various embodiments, the composition comprises nanoparticles or are in the form of nanoparticles disclosed herein.

[0490] In various embodiments, the composition is biocompatible, i.e., the composition 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 is substantially devoid of substances that elicit an adverse physiological response.

[0491] NANOPARTICLES

[0492] The term “nanoparticles” may comprise and / or may be used interchangeably with the terms “lipid nanoparticles", “encapsulated lipid nanoparticles”, “loaded lipid nanoparticles”, “LNPs” or the like. In various embodiments, there is provided nanoparticles (e.g., lipid nanoparticle) comprising:

[0493] (i) the compound represented by general formula (1) and / or ionized form thereof disclosed herein; and

[0494] (ii) a therapeutic 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 ) or ionized form thereof.

[0495] 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.

[0496] In various embodiments, the nanoparticle has a N: P or N / P ratio (i.e., molar ratio of ionizable (at low pH) nitrogen atoms in the ionizable lipid to phosphate groups in the therapeutic and / or prophylactic agent and / or biological agent (e.g., nucleic acid)) is from about 2:1 to about 40:1. In various embodiments, shorter nucleic acid therapeutics (e.g., siRNA) require more ionizable lipid to encapsulate them into lipid nanoparticles. In various embodiments therefore, a N / P ratio of up to about 40:1 is used to encapsulate and deliver nucleic acid therapeutics (e.g., siRNA, ASO).

[0497] In various embodiments, the concentration of the therapeutic and / or prophylactic agent and / or biological agent in the composition / nanoparticle is from about 10 µg / mL to about 200 µg / mL, from about 20 µg / mL to about 190 µg / mL, from about 30 µg / mL to about 180 µg / mL, from about 40 µg / mL to about 170 µg / mL, from about 50 µg / mL to about 165 µg / mL, from about 60 µg / mL to about 160 µg / mL, from about 70 µg / mL to about 155 µg / mL, from about 80 µg / mL to about 150 µg / mL, from about 90 µg / mL to about 145 µg / mL, from about 100 µg / mL to about 140 µg / mL, from about 105 µg / mL to about 135 µg / mL, from about 110 pg / mL to about 130 pg / mL, from about 115 pg / mL to about 125 pg / mL, or about 120 pg / mL. The concentration may be adjusted / diluted simply by adding saline (e.g., sterile phosphate-buffered saline (PBS), or Tris-buffer) to the concentrated lipid nanoparticle solutions.

[0498] In various embodiments, the total lipid concentration (i.e., the compound represented by general formula (1) or ionized forms thereof, neutral / helper lipid, sterol and / or derivatives thereof, and polyethylene glycol (PEG)-lipid conjugate or other suitable amphiphilic lipids) in the composition / nanoparticle is from about 1 mg / mL to about 5 mg / mL, 1 mg / mL to about 4.0 mg / mL, from about 1.5 mg / mL to about 4.0 mg / mL, 1.5 mg / mL to about 3.5 mg / mL, from about 1.8 mg / mL to about 3.0 mg / mL, about 2.0 mg / mL to about 3.0 mg / mL, about 2.0 mg / mL to about 2.5 mg / mL, or about 2.3 mg / mL.

[0499] 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 10 %, at least about 20 %, at least about 30 %, at least about 40 %, at least about 50 %, at least about 60 %, at least about 70 %, at least about 80 %, at least about 90 %, or at least about 99.9 %. In various embodiments, the nanoparticle has an encapsulation efficiency that is comparable to or no less or higher than that of a corresponding nanoparticle using ALC-0315 or SM-102 as the ionizable lipid under similar conditions. For example, the encapsulation efficiency may be at least about 10 %, at least about 50 %, at least about 100 %, at least about 150 %, at least about 200 %, at least about 250 %, or at least about 300 % higher than that of a corresponding nanoparticle using ALC-0315 or SM-102 as the ionizable lipid under similar conditions.

[0500] In various embodiments, the cell transfection efficiency (% of the cells that are transfected with the gene) of the composition / nanoparticle is at least about 1.0 %, at least about 5.0 %, at least about 10.0 %, 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 at least 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. In various embodiments, the nanoparticle has a cell transfection efficiency that is comparable to or no less or higher than that of a corresponding nanoparticle using ALC-0315 or SM-102 as the ionizable lipid under similar conditions. For example, the cell transfection efficiency may be at least about 0.1 time, at least about 0.2 time, at least about 0.3 time, at least about 0.4 time, at least about 0.5 time, at least about 0.6 time, at least about 0.7 time, at least about 0.8 time, at least about 0.9 time, at least about 1 time, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times higher than that of a corresponding nanoparticle using ALC-0315 or SM-102 as the ionizable lipid under similar conditions. It will be appreciated that the nanoparticle may still induce a therapeutic and / or vaccination effect even when its transfection efficiency is lower than that of ALC-0315 or SM-102 ionizable lipid.

[0501] In various embodiments, the nanoparticle has an average particle size (or diameter) of from about 20 nm to about 400 nm, from about 30 nm to about 390 nm, from about 40 nm to about 380 nm, from about 50 nm to about 370 nm, from about 60 nm to about 360 nm, from about 70 nm to about 350 nm, from about 80 nm to about 340 nm, from about 90 nm to about 330 nm, from about 100 nm to about 320 nm, from about 110 nm to about 310 nm, from about 120 nm to about 300 nm, from about 130 nm to about 290 nm, from about 140 nm to about 280 nm, from about 150 nm to about 270 nm, from about 160 nm to about 260 nm, from about 170 nm to about 250 nm, from about 180 nm to about 240 nm, from about 190 nm to about 230 nm, from about 200 nm to about 220 nm, or about 210 nm.

[0502] In various embodiments, the nanoparticle has a polydispersity index (PDI) of from about 0.01 to about 0.7, from about 0.05 to about 0.65, from about 0.1 to about 0.6, from about 0.15 to about 0.55, from about 0.2 to about 0.5, from about 0.25 to about 0.45, from about 0.3 to about 0.4, or about 0.35. In various embodiments, the nanoparticle has a narrow particle size distribution, and / or the nanoparticle composition is relatively / substantially homogenous.

[0503] In various embodiments, the nanoparticle has a zeta potential of from about -30 mV to about 30 mV, from about -30 mV to about 25 mV, from about -30 mV to about 20 mV, from about -30 mV to about 15 mV, from about -25 mV to about 14 mV, from about -24 mV to about 13 mV, from about -23 mV to about 12 mV, from about -22 mV to about 11 mV, from about -21 mV to about 10 mV, from about -20 mV to about 9 mV, from about -19 mV to about 8 mV, from about -18 mV to about 7 mV, from about -17 mV to about 6 mV, from about -16 mV to about 5 mV, from about -15 mV to about 4 mV, from about -14 mV to about 3 mV, from about -13 mV to about 2 mV, from about -12 mV to about 1 mV, from about -11 mV to about 0 mV, from about -10 mV to about -1 mV, from about -9 mV to about -2 mV, from about -8 mV to about -3 mV, from about -7 mV to about -4 mV, from about -6 mV to about -5 mV, or about -5.5 mV in saline or in a physiological environment. Advantageously, in various embodiments, the nanoparticle has a substantially neutral surface charge at physiological pH, making the nanoparticle suitable / desirable for in vivo applications.

[0504] In various embodiments, the nanoparticle is biocompatible, i.e., the 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 nanoparticle is substantially devoid of substances that elicit an adverse physiological response.

[0505] METHOD OF PREPARING NANOPARTICLES

[0506] There is provided a method of preparing a nanoparticle disclosed herein, the method comprising:

[0507] (f-i) preparing an aqueous composition comprising therapeutic and / or prophylactic agent and / or biological agent;

[0508] (f-ii) mixing the aqueous composition with a composition comprising the compound disclosed herein to obtain a nanoparticle.

[0509] 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 solutions, phosphate buffer solutions, glycine buffer solutions, the like, or combinations thereof.

[0510] In various embodiments, mixing in (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.

[0511] In various embodiments, (f-ii) comprises mixing the aqueous composition with a composition comprising the compound disclosed herein at a volume ratio of the aqueous phase to organic phase from about 6:1 to about 1:6, from about 5:1 to about 1:5, from about 4:1 to about 1:4, from about 3:1 to about 4:1, from about 2:1 to about 1:2, or about 1:1. In various embodiments, mixing the aqueous composition with the composition comprises microfluidic mixing or rapid pipetting in (f-ii).

[0512] In various embodiments, there is also provided a carrier, nanocarrier, or delivery system / vehicle comprising the composition / compound / nanoparticles as disclosed herein.

[0513] In various embodiments, there is also provided a vaccine composition comprising the composition / compound / nanoparticles as disclosed herein.

[0514] In various embodiments, there is also provided a carrier, a nanocarrier, a delivery system / vehicle, a compound or ionized form thereof, a nanoparticle composition, nanoparticle(s) (or lipid nanoparticle(s)) 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).

[0515] In various embodiments, there is also provided a carrier, a nanocarrier, a delivery system / vehicle, a compound or ionized form thereof, a nanoparticle composition, nanoparticle(s) (or lipid nanoparticle(s)) 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 or ionized form thereof, a nanoparticle composition, nanoparticle(s) (or lipid nanoparticle(s)) 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 or ionized form thereof, a nanoparticle composition, nanoparticle(s) (or lipid nanoparticle(s)) to a subject (e.g., plants or vertebrate such as a human or a large veterinary mammal (e.g., horses, cattle, deer, sheep, llamas, goats, pigs)) in need thereof. In various embodiments, the use of said carrier, a nanocarrier, a delivery system / vehicle, a compound or ionized form thereof, a nanoparticle composition, nanoparticle(s) (or lipid nanoparticle(s)) 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, comprises in vivo chimeric antigen receptor (CAR)-T cell therapy. 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, oncological diseases, dermatological diseases, ophthalmological diseases, fibrotic diseases, cardiovascular diseases, or the like, or combinations thereof. In various embodiments, the disease, disorder, or condition is cancer, eczema, age-related macular degeneration (AMD), fibrosis, or 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, H5N1). 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.

[0516] In various embodiments, there is also provided a carrier, a nanocarrier, a delivery system / vehicle, a compound or ionized form thereof, a nanoparticle composition, nanoparticle(s) (or lipid nanoparticle(s)) disclosed herein for use in encapsulating and / or delivering a therapeutic, prophylactic, and / or biological agent (e.g., pharmaceutical, drug, nucleic acid, gene, etc.) 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 or ionized form thereof, a nanoparticle composition, nanoparticle(s) (or lipid nanoparticle(s)) 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 or ionized form thereof, a nanoparticle composition, nanoparticle(s) (or lipid nanoparticle(s)) to a subject (e.g., plants or vertebrate such as a human or a large veterinary mammal (e.g., horses, cattle, deer, sheep, llamas, goats, pigs)) in need thereof, the use of said carrier, a nanocarrier, a delivery system / vehicle, a compound or ionized form thereof, a nanoparticle composition, nanoparticle(s) (or lipid nanoparticle(s)) 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), comprises in vivo CAR-T cell therapy. In various embodiments, there is provided use of the composition disclosed herein for delivery of a therapeutic, prophylactic, and / or biological agent or use of the composition disclosed herein in the manufacture of a delivery agent for delivery of a therapeutic, prophylactic, and / or biological agent to a subject in need thereof. The methods disclosed herein may be carried out in vivo or in vitro (or ex vivo). In various embodiments, the method may be in vivo CAR-T cell therapy. The biological agent disclosed herein may also be a test agent such as for testing its efficacy on a subject. Therefore, the methods disclosed herein may be encompassing delivering a test agent (e.g., a drug candidate) to determine its efficacy on a subject through in vivo or in vitro experiments.

[0517] In various embodiments, there is also provided a carrier, a nanocarrier, a delivery system / vehicle, a compound or ionized form thereof, a nanoparticle composition, nanoparticle(s) (or lipid nanoparticle(s)) disclosed herein for use in inducing / modulating an immune response in a subject (e.g., plants or 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 or ionized form thereof, a nanoparticle composition, nanoparticle(s) (or lipid nanoparticle(s)) in the manufacture of a medicament for inducing / modulating an immune response in a subject, and / or a method of inducing / modulating 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 or ionized form thereof, a nanoparticle composition, nanoparticle(s) (or lipid nanoparticle(s)) 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 or ionized form thereof, a nanoparticle composition, nanoparticle(s) (or lipid nanoparticle(s)) thereto. In various embodiments, an immune response in the subject is to be induced / modulated through in vivo CAR-T cell therapy. In various embodiments, by inducing / modulating 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, oncological diseases, dermatological diseases, ophthalmological diseases, fibrotic diseases, cardiovascular diseases, or the like, or combinations thereof as mentioned herein. The carrier, nanocarrier, delivery system / vehicle, compound or ionized form thereof, nanoparticle composition, nanoparticles may be delivered to a subject in the form of or as a component of a vaccine.

[0518] In various embodiments, the disease, disorder, or condition is cancer, eczema, age-related macular degeneration (AMD), fibrosis, or 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, H5N1. 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.

[0519] In various embodiments, the carrier, nanocarrier, delivery system / vehicle, compound or ionized form thereof, nanoparticle composition, nanoparticle(s) (or lipid nanoparticle(s)) prepared from embodiments of the method disclosed herein comprise(s) 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.

[0520] 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.

[0521] BRIEF DESCRIPTION OF FIGURES

[0522] FIG. 1 shows the1H NMR spectrum of 6-aminohexyl 2-hexyldecanoate, synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in deuterated chloroform (CDCl₃) as the solvent.

[0523] FIG. 2 shows the1H NMR spectrum of 6-hydroxyhexyl 2-hexyldecanoate, synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in CDCl₃ as the solvent.

[0524] FIG. 3 shows the1H NMR spectrum of 6-oxohexyl 2-hexyldecanoate, synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in CDCl₃ as the solvent. FIG. 4 shows the1H NMR spectrum of 6-(((3S,4R)-3,4,5-trihydroxypentyl)amino)hexyl 2-hexyldecanoate, synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in CDCl₃ as the solvent.

[0525] FIG. 5 shows the1H NMR spectrum of 6-(dodecyl((3S,4R)-3,4,5-trihydroxypentyl)amino)hexyl 2-hexyldecanoate (ZJY-01), synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in CDCl₃ as the solvent.

[0526] FIG. 6 shows the1H NMR spectrum of 6-(((2S, 3S,4R)-2, 3,4,5-tetrahydroxypentyl)amino)hexyl 2-hexyldecanoate, synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in CDCl₃ as the solvent.

[0527] FIG. 7 shows the1H NMR spectrum of 6-(dodecyl((2S, 3S,4R)-2, 3,4,5-tetrahydroxypentyl)amino)hexyl 2-hexyldecanoate (ZJY-02), synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in CDCl₃ as the solvent.

[0528] FIG. 8 shows the1H NMR spectrum of (((3S,4R)-3,4,5-trihydroxypentyl)azanediyl)bis(hexane-6, 1 -diyl) bis(2-hexyldecanoate) (ZJY-03), synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in CDCl₃ as the solvent.

[0529] FIG. 9 shows the1H NMR spectrum of (((2S, 3S,4R)-2, 3,4,5-tetrahydroxypentyl)azanediyl)bis(hexane-6, 1 -diyl) bis(2 -hexyldecanoate) (ZJY-04), synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in CDCl₃ as the solvent.

[0530] FIG. 10 shows the1H NMR spectrum of 6-(((2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)hexyl 2-hexyldecanoate, synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in CDCl₃ as the solvent.

[0531] FIG. 11 shows the1H NMR spectrum of (((2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)azanediyl)bis(hexane-6, 1 -diyl) bis(2-hexyldecanoate) (ZJY-05), synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in CDCl₃ as the solvent.

[0532] FIG. 12 shows the1H NMR spectrum of (((2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)azanediyl)bis(hexane-6, 1 -diyl) bis(2-hexyldecanoate) (ZJY-06), synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in CDCl₃ as the solvent.

[0533] FIG. 13 shows the transfection efficiency measured by relative luciferase expression (A) as well as the viability of HEK-293T cells (B) after 48 hours of incubation with mRNA LNPs formulated using ZJY-01 at N / P ratios of 3 and 6, prepared in accordance with various embodiments disclosed herein, in comparison with ALC-0315 LNPs. Statistical significance for transfection efficiency was determined using one-way ANOVA, with comparison to ALC-0315 (**** p < 0.0001).

[0534] FIG. 14 shows the transfection efficiency measured by relative luciferase expression (A) as well as the viability of HEK-293T cells (B) after 48 hours of incubation with mRNA LNPs formulated using ZJY-02 at N / P ratios of 3 and 6, prepared in accordance with various embodiments disclosed herein, in comparison with ALC-0315 LNPs. Statistical significance for transfection efficiency was determined using one-way ANOVA, with comparison to ALC-0315 (**** p < 0.0001).

[0535] FIG. 15 shows the transfection efficiency measured by relative luciferase expression (A) as well as the viability of HEK-293T cells (B) after 48 hours of incubation with mRNA LNPs formulated using ZJY-03 at N / P ratios of 3 and 6, prepared in accordance with various embodiments disclosed herein, in comparison with ALC-0315 LNPs. Statistical significance for transfection efficiency was determined using one-way ANOVA, with comparison to ALC-0315 (**** p < 0.0001).

[0536] FIG. 16 shows the transfection efficiency measured by relative luciferase expression (A) as well as the viability of HEK-293T cells (B) after 48 hours of incubation with mRNA LNPs formulated using ZJY-04 at N / P ratios of 3 and 6, prepared in accordance with various embodiments disclosed herein, in comparison with ALC-0315 LNPs. Statistical significance for transfection efficiency was determined using one-way ANOVA, with comparison to ALC-0315 (**** p < 0.0001).

[0537] FIG. 17 shows the transfection efficiency measured by relative luciferase expression (A) as well as the viability of HEK-293T cells (B) after 48 hours of incubation with mRNA LNPs formulated using ZJY-05 at N / P ratios of 3 and 6, prepared in accordance with various embodiments disclosed herein, in comparison with ALC-0315 LNPs. Statistical significance for transfection efficiency was determined using one-way ANOVA, with comparison to ALC-0315 (**** p < 0.0001).

[0538] FIG. 18 shows the transfection efficiency measured by relative luciferase expression (A) as well as the viability of HEK-293T cells (B) after 48 hours of incubation with mRNA LNPs formulated using ZJY-06 at N / P ratios of 3 and 6, prepared in accordance with various embodiments disclosed herein, in comparison with ALC-0315 LNPs. Statistical significance for transfection efficiency was determined using one-way ANOVA, with comparison to ALC-0315 (**** p < 0.0001).

[0539] FIG. 19 shows the transfection efficiency measured by relative luciferase expression (A) as well as the viability of DC2.4 cells (B) after 48 hours of incubation with mRNA LNPs formulated using ZJY-01 at N / P ratios of 3 and 6, prepared in accordance with various embodiments disclosed herein, in comparison with ALC-0315 LNPs. Statistical significance for transfection efficiency was determined using one-way ANOVA, with comparison to ALC-0315 (**** p < 0.0001).

[0540] FIG. 20 shows the transfection efficiency measured by relative luciferase expression (A) as well as the viability of DC2.4 cells (B) after 48 hours of incubation with mRNA LNPs formulated using ZJY-02 at N / P ratios of 3 and 6, prepared in accordance with various embodiments disclosed herein, in comparison with ALC-0315 LNPs. Statistical significance for transfection efficiency was determined using one-way ANOVA, with comparison to ALC-0315 (**** p < 0.0001).

[0541] FIG. 21 shows the transfection efficiency measured by relative luciferase expression (A) as well as the viability of DC2.4 cells (B) after 48 hours of incubation with mRNA LNPs formulated using ZJY-03 at N / P ratios of 3 and 6, prepared in accordance with various embodiments disclosed herein, in comparison with ALC-0315 LNPs. Statistical significance for transfection efficiency was determined using one-way ANOVA, with comparison to ALC-0315 (**** p < 0.0001).

[0542] FIG. 22 shows the transfection efficiency measured by relative luciferase expression (A) as well as the viability of DC2.4 cells (B) after 48 hours of incubation with mRNA LNPs formulated using ZJY-04 at N / P ratios of 3 and 6, prepared in accordance with various embodiments disclosed herein, in comparison with ALC-0315 LNPs. Statistical significance for transfection efficiency was determined using one-way ANOVA, with comparison to ALC-0315 (**** p < 0.0001).

[0543] FIG. 23 shows the transfection efficiency measured by relative luciferase expression (A) as well as the viability of DC2.4 cells (B) after 48 hours of incubation with mRNA LNPs formulated using ZJY-05 at N / P ratios of 3 and 6, prepared in accordance with various embodiments disclosed herein, in comparison with ALC-0315 LNPs. Statistical significance for transfection efficiency was determined using one-way ANOVA, with comparison to ALC-0315 (**** p < 0.0001).

[0544] FIG. 24 shows the transfection efficiency measured by relative luciferase expression (A) as well as the viability of DC2.4 cells (B) after 48 hours of incubation with mRNA LNPs formulated using ZJY-06 at N / P ratios of 3 and 6, prepared in accordance with various embodiments disclosed herein, in comparison with ALC-0315 LNPs. Statistical significance for transfection efficiency was determined using one-way ANOVA, with comparison to ALC-0315 (**** p < 0.0001).

[0545] EXAMPLES

[0546] 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, biological, 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.

[0547] The following examples describe the development of a series of sugar-derived ionizable lipids for mRNA-LNP formulations. This includes glycation groups that are designed for constructing the headgroup unit of ionizable lipids to enhance the delivery efficiency of mRNA. LNPs formulated from sugar-derived ionizable lipids were advantageously found to show narrow size distribution (PDI < 0.2) and high encapsulation efficiency. Specifically, the transfection efficiencies of mRNA LNPs formulated from sugar-derived ionizable lipids were significantly greater than those of mRNA LNPs formulated from ionizable lipid ALC-0315, as used in Pfizer-BioNTech’s mRNA-LNP Covid-19 vaccine. Notably, the improvement in transfection efficiency was observed in HEK293T and DC2.4 cells, with no cytotoxicity. These results suggest that sugar-derived ionizable lipids are promising in the delivery of RNAs and other genes.

[0548] Example 1: Materials

[0549] Chemical reagents for the synthesis of the sugar-derived ionizable lipids 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 and dodecanal were 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.

[0550] Example 2: Methods

[0551] 2.1. Synthesis of 6-aminohexyl 2-hexyldecanoate

[0552] Synthesis strategy for 6-aminohexyl 2-hexyldecanoate was showed in Scheme 1. 6-((tert-butoxycarbonyl)amino)hexyl 2-hexyldecanoate was first synthesized. 2-hexyldecanoic acid (10.24 g, 40 mmol), 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (11.5 g, 60 mmol) and 4-dimethylaminopyridine (2.5 g, 20 mmol) were dissolved in 200 mL of dichloromethane under N2. The reaction mixture was stirred for 15 min under N2. To the solution was added tert-butyl (6-hydroxyhexyl)carbamate (8.68 g, 40 mmol) under N2. The reaction mixture was stirred for 24 h at room temperature. The reaction solution was diluted with 200 mL of dichloromethane and then was washed with 500 mL of saturated brine for 3 times. The organic phase was dried over anhydrous Na2SC>4 and the organic solvent was evaporated in vacuo. The resulting crude product was purified by flash silica gel column chromatography (hexane / ethyl acetate 90:10, v / v). The yield of the compound was 83.0 %.

[0553] 6-aminohexyl 2-hexyldecanoate was then synthesized. 6-hydroxyhexyl 2-hexyldecanoate (9.1 g, 20 mmol) was dissolved in 60 mL of dichloromethane and then added 20 mL of trifluoroacetic acid. The reaction mixture was stirred for 2 h at room temperature. The organic solvent was evaporated in vacuo. The resulting crude product was purified by flash silica gel column chromatography (dichloromethane / methanol 95:5, v / v). The yield of 6-aminohexyl 2-hexyldecanoate was 90.0 %.

[0554]

[0555] Scheme 1. Synthesis strategy for 6-aminohexyl 2-hexyldecanoate. 2.2. Synthesis of 6-oxohexyl 2-hexyldecanoate

[0556] Synthesis strategy for 6-oxohexyl 2-hexyldecanoate was showed in Scheme 2. 6-hydroxyhexyl 2-hexyldecanoate was first synthesized. 2-hexyldecanoic acid (10.24 g, 40 mmol), 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (11.5 g, 60 mmol) and 4-dimethylaminopyridine (2.5 g, 20 mmol) were dissolved in 200 mL of dichloromethane under N2. The reaction mixture was stirred for 15 min under N2. To the solution was added hexane-1,6-diol (14.6 g, 120 mmol) under N2. The reaction mixture was stirred for 24 h at room temperature. The reaction solution was diluted with 200 mL of dichloromethane and then was washed with 500 mL of saturated brine for 3 times. 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 85:15, v / v). The yield of the compound was 85.0%.

[0557] 6-oxohexyl 2-hexyldecanoate was then synthesized. 6-hydroxyhexyl 2-hexyldecanoate (8.9 g, 25 mmol) was dissolved in 60 mL of dichloromethane and then added 6.24 g of pyridinium chlorochromate. The reaction mixture was stirred for 2 h at room temperature. The reaction solution was diluted with 200 mL of diethyl ether and the supernatant filtrated through the silica gel bed. The organic solvent was evaporated in vacuo. The resulting crude product was purified by flash silica gel column chromatography (hexane / ethyl acetate 95:5, v / v). The yield of 6-oxohexyl 2-hexyldecanoate was 80.0%.

[0558]

[0559] Scheme 2. Synthesis strategy for 6-oxohexyl 2-hexyldecanoate.

[0560] 2.3. Synthesis of 6-(dodecyl((3S,4R)-3,4,5-trihydroxypentyl)amino)hexyl 2-hexyldecanoate (ZJY-01)

[0561] Synthesis strategy for ZJY-01 was showed in Scheme 3. 6-(((3S,4R)-3,4,5-trihydroxypentyl)amino)hexyl 2-hexyldecanoate was first synthesized. 6-aminohexyl 2-hexyldecanoate (3.55 g, 10 mmol) and 2-deoxy-D-ribose (1.47 g, 11 mmol) were dissolved in 80 mL of methanol under N2. The reaction mixture was added 0.25 mL of acetic acid under N2. To the solution was added sodium cyanoborohydride (945 mg, 15 mmol) under N2. The reaction mixture was stirred for 24 h at 50 °C. The organic solvent was evaporated in vacuo. The resulting crude product was purified by flash silica gel column chromatography (dichloromethane / methanol 90:10, v / v). The yield of 6-(((3S,4R)-3,4,5-trihydroxypentyl)amino)hexyl 2-hexyldecanoate was 82.0%.

[0562] ZJY-01 was then synthesized. 6-(((3S,4R)-3,4,5-trihydroxypentyl)amino)hexyl 2-hexyldecanoate (2.37 g, 5.0 mmol) and dodecanal (1.84 g, 10 mmol) were dissolved in 50 mL of methanol under N2. The reaction mixture was added 0.12 mL of acetic acid under N2. To the solution was added sodium cyanoborohydride (630 mg, 10 mmol) under N2. The reaction mixture was stirred for 24 h at 50 °C. The organic solvent was evaporated in vacuo. The resulting crude product was purified by flash silica gel column chromatography (dichloromethane / methanol 90:10, v / v). The yield of ZJY-01 was 90.0%.

[0563]

[0564] Scheme 3. Synthesis strategy for 6-(dodecyl((3S,4R)-3,4,5- trihydroxypentyl)amino)hexyl 2-hexyldecanoate (ZJY-01).

[0565] 2.4. Synthesis of 6-(dodecyl((2S,3S,4R)-2, 3,4,5- tetrahydroxypentyl)amino)hexyl 2-hexyldecanoate (ZJY-02)

[0566] Synthesis strategy for ZJY-02 was showed in Scheme 4. 6-(((2S,3S,4R)-2,3,4,5-tetrahydroxypentyl)amino)hexyl 2-hexyldecanoate was first synthesized.

[0567] 6-aminohexyl 2-hexyldecanoate (3.55 g, 10 mmol) and D-ribose (1.65 g, 11 mmol) were dissolved in 80 mL of methanol under N₂. The reaction mixture was added 0.25 mL of acetic acid under N₂. To the solution was added sodium cyanoborohydride (945 mg, 15 mmol) under N₂. The reaction mixture was stirred for 24 h at 50 °C. The organic solvent was evaporated in vacuo. The resulting crude product was purified by flash silica gel column chromatography (dichloromethane / methanol 90:10, v / v). The yield of ZJY-02 was 84.0%.

[0568] ZJY-02 was then synthesized. (((2S, 3S,4R)-2, 3,4,5-tetrahydroxypentyl)amino)hexyl 2-hexyldecanoate (2.50 g, 5.0 mmol) and dodecanal (1.84 g, 10 mmol) were dissolved in 50 mL of methanol under N₂. The reaction mixture was added 0.12 mL of acetic acid under N₂. To the solution was added sodium cyanoborohydride (630 mg, 10 mmol) under N₂. The reaction mixture was stirred for 24 h at 50 °C. The organic solvent was evaporated in vacuo. The resulting crude product was purified by flash silica gel column chromatography (dichloromethane / methanol 90:10, v / v). The yield of 6-(dodecyl((2S,3S,4R)-2,3,4,5-tetrahydroxypentyl)amino)hexyl 2-hexyldecanoate was 89.0%. Methand, Acetic acid MaCNBHs Dodecanal 53 ‘ C 24 h

[0569]

[0570] Scheme 4. Synthesis strategy for 6-(dodecyl((2S,3S,4R)-2, 3,4,5- tetrahydroxypentyl)amino)hexyl 2-hexyldecanoate (ZJY-02).

[0571] 2.5. Synthesis of (((3S,4R)-3,4,5-trihydroxypentyl)azanediyl)bis(hexane- 6,1 -diyl) bis(2-hexyldecanoate) (ZJY-03)

[0572] Synthesis strategy for ZJY-03 was showed in Scheme 5. 6-(((3S,4R)-3,4,5-trihydroxypentyl)amino)hexyl 2-hexyldecanoate was first synthesized. 6-aminohexyl 2-hexyldecanoate (3.55 g, 10 mmol) and 2-deoxy-D-ribose (1.47 g, 11 mmol) were dissolved in 80 mL of methanol under N₂. The reaction mixture was added 0.25 mL of acetic acid under N₂. To the solution was added sodium cyanoborohydride (945 mg, 15 mmol) under N₂. The reaction mixture was stirred for 24 h at 50 °C. The organic solvent was evaporated in vacuo. The resulting crude product was purified by flash silica gel column chromatography (dichloromethane / methanol 90:10, v / v). The yield of 6-(((3S,4R)-3,4,5-trihydroxypentyl)amino)hexyl 2-hexyldecanoate was 82.0%.

[0573] ZJY-03 was then synthesized. 6-(((3S,4R)-3,4,5-trihydroxypentyl)amino)hexyl 2-hexyldecanoate (2.37 g, 5.0 mmol) and 6-oxohexyl 2-hexyldecanoate (3.54 g, 10 mmol) were dissolved in 50 mL of methanol under N₂. The reaction mixture was added 0.12 mL of acetic acid under N₂. To the solution was added sodium cyanoborohydride (630 mg, 10 mmol) under N₂. The reaction mixture was stirred for 24 h at 50 °C. The organic solvent was evaporated in vacuo. The resulting crude product was purified by flash silica gel column chromatography (dichloromethane / methanol 90:10, v / v). The yield of ZJY-03 was 90.0%.

[0574]

[0575] Scheme 5. Synthesis strategy for (((3S,4R)-3,4,5-trihydroxypentyl)azanediyl)bis(hexane-6, 1 -diyl) bis(2-hexyldecanoate) (ZJY-03). 2.6. Synthesis of (((2S, 3S,4R)-2, 3,4,5- tetrahydroxypentyl)azanediyl)bis(hexane-6,1 -diyl) bis(2- hexyldecanoate) (ZJY-04)

[0576] Synthesis strategy for ZJY-04 was showed in Scheme 6. 6-(((2S,3S,4R)-2,3,4,5-tetrahydroxypentyl)amino)hexyl 2-hexyldecanoate was first synthesized.

[0577] 6-aminohexyl 2-hexyldecanoate (3.55 g, 10 mmol) and D-ribose (1.65 g, 11 mmol) were dissolved in 80 mL of methanol under N₂. The reaction mixture was added 0.25 mL of acetic acid under N₂. To the solution was added sodium cyanoborohydride (945 mg, 15 mmol) under N₂. The reaction mixture was stirred for 24 h at 50 °C. The organic solvent was evaporated in vacuo. The resulting crude product was purified by flash silica gel column chromatography (dichloromethane / methanol 90:10, v / v). The yield of 6-(((2S, 3S,4R)-2, 3,4,5-tetrahydroxypentyl)amino)hexyl 2-hexyldecanoate was 84.0%.

[0578] ZJY-04 was then synthesized. (((2S, 3S,4R)-2, 3,4,5-tetrahydroxypentyl)amino)hexyl 2-hexyldecanoate (2.50 g, 5.0 mmol) and 6-oxohexyl 2-hexyldecanoate (3.54 g, 10 mmol) were dissolved in 50 mL of methanol under N₂. The reaction mixture was added 0.12 mL of acetic acid under N₂. To the solution was added sodium cyanoborohydride (630 mg, 10 mmol) under N₂. The reaction mixture was stirred for 24 h at 50 °C. The organic solvent was evaporated in vacuo. The resulting crude product was purified by flash silica gel column chromatography (dichloromethane / methanol 90:10, v / v). The yield of ZJY-04 was 89.0%.

[0579]

[0580] Scheme

[0581]

[0582] Synthesis

[0583]

[0584] (((2S, 3S,4R)-2, 3,4,5-tetrahydroxypentyl)azanediyl)bis(hexane-6, 1 -diyl) bis(2 -hexyldecanoate) (ZJY- 2.7. Synthesis of (((2R,3R,4R,5R)-2,3,4,5,6- pentahydroxyhexyl)azanediyl)bis(hexane-6,1 -diyl) bis(2- hexyldecanoate) (ZJY-05)

[0585] Synthesis strategy for ZJY-05 was showed in Scheme 7. 6-(((2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)hexyl 2-hexyldecanoate was first synthesized. 6-aminohexyl 2-hexyldecanoate (3.55 g, 10 mmol) and D-mannose (1.98 g, 11 mmol) were dissolved in 80 mL of methanol under N₂. The reaction mixture was added 0.25 mL of acetic acid under N₂. To the solution was added sodium cyanoborohydride (945 mg, 15 mmol) under N₂. The reaction mixture was stirred for 24 h at 50 °C. The organic solvent was evaporated in vacuo. The resulting crude product was purified by flash silica gel column chromatography (dichloromethane / methanol 90:10, v / v). The yield of

[0586] 6-(((2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)hexyl 2-hexyldecanoate was 82.0%.

[0587] ZJY-05 was then synthesized. 6-(((2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)hexyl 2-hexyldecanoate (2.60 g, 5.0 mmol) and 6-oxohexyl 2-hexyldecanoate (3.54 g, 10 mmol) were dissolved in 50 mL of methanol under N₂. The reaction mixture was added 0.12 mL of acetic acid under N₂. To the solution was added sodium cyanoborohydride (630 mg, 10 mmol) under N₂. The reaction mixture was stirred for 24 h at 50 °C. The organic solvent was evaporated in vacuo. The resulting crude product was purified by flash silica gel column chromatography (dichloromethane / methanol 90:10, v / v). The yield of ZJY-05 was 90.0%.

[0588]

[0589] Scheme 7. Synthesis strategy for (((2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)azanediyl)bis(hexane-6, 1 -diyl) bis(2-hexyldecanoate) (ZJY-05). 2.8. Synthesis of (((2R,3R,4R,5R)-2,3,4,5,6- pentahydroxyhexyl)azanediyl)bis(hexane-6,1 -diyl) bis(2- hexyldecanoate) (ZJY-06)

[0590] Synthesis strategy for ZJY-06 was showed in Scheme 8. 6-(((3S,4R)-3,4,5-trihydroxypentyl)amino)hexyl 2-hexyldecanoate was first synthesized. 6-aminohexyl 2-hexyldecanoate (3.55 g, 10 mmol) and 2-deoxy-D-ribose (1.47 g, 11 mmol) were dissolved in 80 mL of methanol under N2. The reaction mixture was added 0.25 mL of acetic acid under N2. To the solution was added sodium cyanoborohydride (945 mg, 15 mmol) under N2. The reaction mixture was stirred for 24 h at 50 °C. The organic solvent was evaporated in vacuo. The resulting crude product was purified by flash silica gel column chromatography (dichloromethane / methanol 90:10, v / v). The yield of 6-(((3S,4R)-3,4,5-trihydroxypentyl)amino)hexyl 2-hexyldecanoate was 82.0%.

[0591] (((3S,4R)-3,4,5-trihydroxypentyl)azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate) was then synthesized. 6-(((3S,4R)-3,4,5-trihydroxypentyl)amino)hexyl 2-hexyldecanoate (2.37 g, 5.0 mmol) and 6-oxohexyl 2-hexyldecanoate (3.54 g, 10 mmol) were dissolved in 50 mL of methanol under N2. The reaction mixture was added 0.12 mL of acetic acid under N2. To the solution was added sodium cyanoborohydride (630 mg, 10 mmol) under N2. The reaction mixture was stirred for 24 h at 50 °C. The organic solvent was evaporated in vacuo. The resulting crude product was purified by flash silica gel column chromatography (dichloromethane / methanol 90:10, v / v). The yield of 6-(((3S,4R)-3,4,5-trihydroxypentyl)amino)hexyl 2-hexyldecanoate was 90.0%.

[0592] ZJY-06 was finally synthesized. (((3S,4R)-3,4,5-trihydroxypentyl)azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (975 mg, 1.2 mmol) was dissolved in 3.0 mL of pyridine under N2. To the solution was added dropwise 4.0 mL of acetic anhydride at 0 °C. The reaction mixture was stirred for 24 h at room temperature. The reaction solution was slowly poured into 10 mL of ice water and then extracted with 20 mL of ethyl acetate for 3 times. The organic phase was washed with 30 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 9:1, v / v). The yield of ZJY-06 was 85.0%.

[0593]

[0594] Scheme 8. Synthesis strategy for (((2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ZJY-06). 2.9. Formulation of mRNA-loaded lipid nanoparticles (mRNA LNPs)

[0595] To conduct a high-throughput screening of various sugar-derived ionizable lipids at different N / P ratios, LNPs were manually formulated according to Tables 1 and 2. Sugar-derived ionizable lipids were mixed with a helper lipid (DSPC), ALC-0159, and cholesterol at different N / P ratios to optimize mRNA LNP formulation. The formulation of mRNA LNPs involved two distinct phases: the organic phase and the aqueous phase.

[0596] For the manual formulation process, the organic phase contained the lipid mixture dissolved in ethanol to reach a final volume of 50 pL. For the formulations with an N / P ratio of 3, the aqueous phase contained 20 pL of 1 mg / mL firefly luciferase mRNA (APExBIO) diluted in 130 pL of 10 mM sodium acetate solution at pH 4 to reach a final volume of 150 pL. For the formulations with an N / P ratio of 6, the aqueous phase contained 10 pL of 1 mg / mL firefly luciferase mRNA (APExBIO) 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 mins to provide time for mRNA encapsulation and self-assembly of LNPs.

[0597] Table 1. Characteristics of different lipid components used for mRNA LNP formulation.

[0598] Molecular weight Stock concentration

[0599] Lipid Lipid role (g / mol) (mg / mL)

[0600] DSPC 790.2 10.0 Helper lipid Cholesterol 386.7 10.0 Helper lipid ALC-0159 2481.0 3.0 PEGylated lipid ALC-0315 766.3 20.0 Ionizable lipid ZJY-01 642.1 20.0 Ionizable lipid

[0601]

[0602] Molecular weight Stock concentration

[0603] Lipid Lipid role (g / mol) (mg / mL)

[0604] ZJY-02 658.1 20.0 Ionizable lipid ZJY-03 812.3 20.0 Ionizable lipid ZJY-04 828.3 20.0 Ionizable lipid ZJY-05 858.3 20.0 Ionizable lipid ZJY-06 938.4 20.0 Ionizable lipid

[0605]

[0606] Table 2. Molar ratios (content) of lipid components used in the mRNA LNP formulations prepared with different ionizable lipids.

[0607] Lipid name Ionizable lipid DSPC Cholesterol ALC-0159 ALC-0315 46.3 9.4 42.7 1.6 ZJY-01 46.3 9.4 42.7 1.6 ZJY-02 46.3 9.4 42.7 1.6 ZJY-03 46.3 9.4 42.7 1.6 ZJY-04 46.3 9.4 42.7 1.6 ZJY-05 46.3 9.4 42.7 1.6 ZJY-06 46.3 9.4 42.7 1.6

[0608]

[0609] 2.10. Assessing encapsulation efficiency of mRNA in LNPs

[0610] To determine the encapsulation efficiency of the mRNA LNPs after formulation, Quant-itTM 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 mins 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, Mannedorf, 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.

[0611] ConcTrit— ConcTEEncapsulation Efficiency — - - - - - x 100

[0612]

[0613] Where ConcTrit is the concentration of mRNA obtained by adding the mRNA LNPs to 0.5% Triton-X100 diluted in Tris-EDTA buffer, while ConcTE is the concentration of the respective mRNA obtained by adding the mRNA LNPs to Tris-EDTA buffer without Triton-X100.

[0614] 2.11. Physiochemical characterization of mRNA LNPs

[0615] The size, polydispersity index (PDI), and zeta potential of the mRNA LNPs were also characterized using a Zetasizer (Malvern, UK). 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 per measurement and a run time of 1.68 s. 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 per measurement.

[0616] 2.12. Cell culturing and dosing of HEK293T and DC2.4 cells with mRNA LNPs

[0617] 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.

[0618] 2.13. In vitro viability of HEK293T and DC2.4 cells after incubation with mRNA LNPs

[0619] 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, Mannedorf, 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.

[0620] 2.14. In vitro transfection efficiency of mRNA LNPs in HEK293T and DC2.4 cells after incubation with mRNA LNPs

[0621] The transfection efficiency of mRNA LNPs in HEK293T and DC2.4 cells was measured after 48 hours using the ONE-Glo™ Luciferase Assay System.

[0622] 100 uL of ONE-Glo™ solution was added to each well. The samples were then incubated at 37 °C for 10 mins to allow for cell lysis and signal stabilization. Luminescence intensity was read using the microplate reader (Tecan, Mannedorf, Switzerland) at an exposure time of 1000 ms. Example 3: Results and Discussion

[0623] 3.1. Synthesis and characterization of sugar-derived ionizable lipids

[0624] The headgroups of ionizable lipids play important roles in entrapping the mRNA, stabilizing the LNP, and facilitating the endosomal escape. In FDA-approved LNP formulations, the headgroups of ALC-0315 from Pfizer and SM-102 from Moderna include a terminal hydroxyl group that can reduce the hydration of the ionizable group and improve hydrogen-bonding interactions with mRNA, potentially resulting into the improved transfection ability. For the initial screening, 3 sugar-derived headgroups (including 2-deoxy-D-ribose, D-ribose and D-mannose) were introduced into ionizable structure for tuning the hydrogenbonding interactions and the hydration of the ionizable group. Sugar-derived ionizable lipids were synthesized via the Schiff base reduction reaction of amino and aldehyde groups. Sugar-derived headgroups can be introduced by the Schiff base reduction reaction of lipids containing primary or secondary amines and reductive sugars. The successful synthesis of lipid with a primary amine group was verified by1H NMR spectroscopy (FIG. 1). The successful syntheses of lipids with an aldehyde group were confirmed by1H NMR spectroscopy (FIGs. 2 and 3). Furthermore, the successful syntheses of the sugar-derived ionizable lipids ZJY01-06 were verified by1H NMR spectroscopy (FIGs.4 to 12).

[0625] 3.2. Size, polydispersity index (PDI), and zeta potential of mRNA LNPs

[0626] The characterization of the mRNA LNPs was performed using a Zetasizer (Malvern, UK), which would elucidate the size, PDI (i.e., size distribution), and zeta potential of the respective mRNA LNPs. The values are displayed in Tables 3 to 8. As observed from the results, LNPs formulated with sugar-derived ionizable lipids ZJY01 -05 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.

[0627] Table 3. Characteristics of manually formulated mRNA LNPs using ZJY-01 at different N / P ratios.

[0628] Ionizable N / P Size Zeta potential Encapsulation PDI

[0629] lipid ratio (nm) (mV) efficiency (%) ALC-0315 6 109 ± 1 0.156 ± 0.021 -3.08 ± 2.24 80.3 ± 0.7 ZJY-01 3 144 ± 2 0.118 ± 0.026 0.92 ± 1.26 84.9 ± 0.8 ZJY-01 6 122 ± 0 0.133 ± 0.018 1.13 ± 0.93 87.8 ± 0.4

[0630]

[0631] Table 4. Characteristics of manually formulated mRNA LNPs using ZJY-02 at different N / P ratios.

[0632] Ionizable N / P Size Zeta potential Encapsulation PDI

[0633] lipid ratio (nm) (mV) efficiency (%) ALC-0315 6 109 ± 1 0.156 ± 0.021 -3.08 ± 2.24 80.3 ± 0.7 ZJY-02 3 136 ±0 0.128 ± 0.016 -0.11 ± 0.85 87.9 ± 0.2 ZJY-02 6 118 ± 2 0.125 ± 0.021 0.22 ± 0.96 88.0 ± 0.5

[0634]

[0635] Table 5. Characteristics of manually formulated mRNA LNPs using ZJY-03 at different N / P ratios.

[0636] Ionizable N / P Size Zeta potential Encapsulation PDI

[0637] lipid ratio (nm) (mV) efficiency (%) ALC-0315 6 100 ± 1 0.122 ± 0.021 -0.11 ± 0 71.6 ± 1.2 ZJY-03 3 137 ± 1 0.143 ± 0.017 -1.91 ± 1.33 83.4 ± 1.8 ZJY-03 6 114 ± 1 0.195 ± 0.009 -1.36 ± 1.62 90.2 ± 0.2

[0638]

[0639] Table 6. Characteristics of manually formulated mRNA LNPs using ZJY-04 at different N / P ratios.

[0640] Ionizable N / P Size Zeta potential Encapsulation PDI

[0641] lipid ratio (nm) (mV) efficiency (%) ALC-0315 6 100 ± 1 0.122 ± 0.021 -0.11 ± 0 71.6 ± 1.2 ZJY-04 3 141 ± 0 0.112 ± 0.02 1.63 ± 1.55 86.8 ± 0.1 ZJY-04 6 111 ± 1 0.178 ± 0.019 -0.47 ± 1.47 86.5 ± 0.7

[0642]

[0643] Table 7. Characteristics of manually formulated mRNA LNPs using ZJY-05 at different N / P ratios.

[0644] Ionizable N / P Size Zeta potential Encapsulation PDI

[0645] lipid ratio (nm) (mV) efficiency (%) ALC-0315 6 126 ± 1 0.179 ± 0.054 -4.8 ± 1.94 51.4 ± 2.7 ZJY-05 3 150 ± 1 0.191 ± 0.029 -0.19 ± 1.03 81.8 ± 0.4 ZJY-05 6 144 ± 4 0.261 ± 0.064 -0.78 ± 0 83.3 ± 1.1

[0646]

[0647] Table 8. Characteristics of manually formulated mRNA LNPs using ZJY-06 at different N / P ratios.

[0648] Ionizable N / P Size Zeta potential Encapsulation PDI

[0649] lipid ratio (nm) (mV) efficiency (%) ALC-0315 6 106 ± 2 0.139 ± 0.027 -1.75 ±3.21 56.7 ± 5 ZJY-06 3 212 ± 9 0.507 ± 0.086 -17.39 ± 1.71 17.3 ± 2.3 ZJY-06 6 133 ± 2 0.265 ± 0.01 -8.96 ± 0.78 55.2 ± 2

[0650]

[0651] 3.3. Encapsulation efficiency of formulated mRNA LNPs

[0652] The encapsulation efficiency of the mRNA LNPs was determined through the Ribogreen Assay Kit and the values are displayed in Tables 3-8. LNPs formulated using sugar-derived ionizable lipids ZJY01-05 demonstrated an encapsulation efficiency comparable to or greater than the encapsulation efficiency of LNPs formulated using ALC-0315, the ionizable lipid that is used in Pfizer / BioNTech’s mRNA Covid19 vaccine, by modulating the N / P ratio. High encapsulation efficiency of mRNA in LNPs is critical for effective mRNA delivery as it ascertains that a sufficient therapeutic payload is delivered intracellularly for mRNA expression. Taken together with the characterization of the mRNA LNPs, these results suggest that the mRNA LNPs formulated with sugar-derived ionizable lipids are viable for in vivo applications.

[0653] 3.4. In vitro cytocompatibility and transfection efficiency of mRNA LNPs in HEK293T cells

[0654] The screening process was performed to evaluate whether substitution of the ionizable lipid headgroup could lead to 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 FIGs. 13 to 18. From the cytocompatibility tests using the AlamarBlue assay, all formulations showed 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 demonstrated that the formulations formed using sugar-derived ionizable lipids were capable of significantly improving transfection efficiency of Flue mRNA over Pfizer’s formulation by changing the N / P ratio. Notably, mRNA LNP formed using ZJY-03 at an N / P ratio of 3 showed more than 25 times of enhancement in transfection efficiency compared to the ALC-0315 LNP formulation in HEK293T cells. mRNA LNP formed using ZJY-04 at an N / P ratio of 3 showed more than 10 times of enhancement in HEK293T cells in transfection efficiency compared to the ALC-0315 LNP formulation.

[0655] 3.5. In vitro cytocompatibility and transfection efficiency of mRNA LNPs in DC2.4 cells

[0656] The cytocompatibility and transfection efficiency results of mRNA LNPs in DC2.4 cells are detailed in FIGs. 19 to 24. Similar to HEK293T cells, analysis of the AlamarBlue assay results in DC2.4 cells revealed that the mRNA LNPs tested ha negligible cytotoxicity. The luciferase assay also showed significant improvement compared to Pfizer’s formulation. LNPs formulated using ZJY-03, ZJY-04, and ZJY-05 showed a greater transfection efficiency compared to Pfizer’s LNP formulated using ALC-0315 in DC2.4 cells. Specifically, mRNA LNP formed by ZJY-04 at an N / P ratio of 3 showed more than 5 times of enhancement in DC2.4 cells in transfection efficiency compared to the ALC-0315 LNP formulation.

[0657] 3.6. Conclusion

[0658] As shown in the examples, sugar-derived ionizable lipids were successfully synthesized. mRNA LNPs formulated using sugar-derived ionizable lipids ZJY-01-05 have nanosize (< 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 using sugar-derived ionizable lipids provided 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, mRNA LNP formed using ZJY-03 at an N / P ratio of 3 showed more than 25 times of enhancement in transfection efficiency compared to the ALC-0315 LNP formulation in HEK293T cells. mRNA LNP formed using ZJY-04 at an N / P ratio of 3 showed more than 10 times of enhancement in HEK293T cells and more than 5 times of enhancement in DC2.4 cells in transfection efficiency compared to the ALC-0315 LNP formulation. All mRNA LNP formulations tested showed negligible cytotoxicity. Sugar-derived ionizable lipids are thus a viable replacement for ALC-315 and other ionizable lipids as they are not only capable of condensing and stabilizing mRNA into LNPs, but also provide greater mRNA transfection efficiency. Therefore, these LNPs formulated using sugar-derived ionizable lipids are promising nanocarriers for the efficient delivery of mRNA and other genes for vaccine and treatment applications.

Claims

CLAIMS1. A compound comprising a structure represented by general formula (1) or ionized form thereof:whereinA comprises a carbohydrate or a carbohydrate derivative;R1, R2, R3, R8, R9, and R10are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; R4and R7are each independently optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;R5and R6each independently comprises a hydrophobic moiety;Z1and Z2are each independently selected from the group consisting of -O-C(=O)-, -C(=O)-O-, -C(=O)-, -O-, -RaN-C(=O)-, -C(=O)-NRb- where Raand Rbare each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;x is an integer ≥ 1; andy is an integer ≥ 0.

2. The compound of claim 1, wherein the ionized form of general formula (1 ) isR1— R4— R5R3R8R10— R7— Z2R9(1b).

3. The compound of claim 1 or 2, wherein the hydrophobic moiety at R5and R6each independently comprises optionally substituted alkyl.

4. The compound of any one of claims 1 to 3, wherein A comprises pentose, hexose, fructose, ribose, mannose, glucose, galactose, deoxyribose, xylose, maltose, lactose, arabinose, cellobiose, 2-deoxy-D-ribofuranose, D-ribofuranose, D-xylofuranose, D-arabinofuranose, D-glucopyranose, D- mannopyranose, D-galactopyranose, L-rham nopyranose, D- fructopyranose, N-acetyl-glucosamine, N-acetyl-mannosamine, N-acetyl- galactosamine, 2-amino-2-deoxy-D-glucopyranose, 2-amino-2-deoxy-D- mannopyranose, 2-amino-2-deoxy-D-galactopyranose, 1 -amino-1 -deoxy- D-fructose, 3-amino-3-deoxy-D-mannopyranose, 1 -amino-1 -deoxy-D- galactopyranose, 1 -(2-aminoethoxy)-1 -deoxy-D-glucopyranose, 1 -(2- aminoethoxy)-1 -deoxy-D-mannopyranose, 1 -(2-aminoethoxy)-1 -deoxy-D- galactopyranose, 1 -(3-aminoethoxy)-1 -deoxy-D-glucopyranose, 1 -(3- aminoethoxy)-1 -deoxy-D-mannopyranose, 1 -(3-aminoethoxy)-1 -deoxy-D- galactopyranose, 1 -(3-aminopropoxy)-1 -deoxy-D-glucopyranose, 1 -(3- aminopropoxy)-1 -deoxy-D-mannopyranose, 1 -(3-aminopropoxy)-1 - deoxy-D-galactopyranose, a derivative thereof, or combinations thereof.

5. The compound of any one of claims 1 to 4, wherein A comprises at least one of an -ORCor -O-C(=O)Rd group, where Rcand Rd are eachindependently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

6. The compound of any one of claims 1 to 5, wherein A comprises a monosaccharide and / or a derivative thereof.

7. The compound of any one of claims 1 to 6, wherein A is represented by general formula (2) or general formula (2A):(2)(2A) whereinR29to R51are each independently selected from -H, -ORe, or-O-C(=O)Rf, -R', -RjORk, -NRl-C(=O)Rm, or Rn, where Reis H, Rf, R' and Rmare each independently optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl, Rkand Rlare each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl, Rjis optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene, and Rncomprises a structure represented by general formula (2A);R52is optionally present as -O-, -OR0-, or -Rp-, where R° and Rpare each independently optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene, and wherein one of R42to R51is absent; andn is 0 or 1.

8. The compound of any one of claims 1 to 7, wherein the compound has a molecular weight of from about 250 g / mol to about 5,000 g / mol.

9. The compound of any one of claims 1 to 8, wherein the compound is selected from the group consisting of the following structures:ZJY-01 / ZJY-1GZJY-02 / ZJY-2DZJY-03 / ZJY-1AZJY-04 / ZJY-2A / ZJY-3A / ZJY-4AZJY-05 / ZJY-5A / ZJY-6A / ZJY-7AZJY-06ZJY-1BHO ZJY-1CZJY-1DZJY-1EZJY-1FZJY-1HZJY-1I ZJY-1JZJY-1KZJY-2BZJY-2CZJY-6BZJY-6CZJY-6DZJY-6E ZJY-6FZJY-6GZJY-6HZJY-8AZJY-9AZJY-10A / ZJY-1I A / ZJY-12AZJY-13A / ZJY-14A / ZJY-15AZJY-16A / ZJY-17A / ZJY-18AZJY-17BZJY-19AZJY-20AZJY-21AZJY-25A / ZJY-26A / ZJY-27A10. A nanoparticle composition comprising:(i) a compound represented by general formula (1) or ionized form thereof according to any one of claims 1 to 9; and(ii) a therapeutic, prophylactic, and / or biological agent that is encapsulated by said compound or ionized form thereof.

11. The nanoparticle composition of claim 10, wherein the composition further comprises:(a) helper lipid;(b) sterol; and(c) polyethylene glycol (PEG)-modified lipid.

12. The composition of claim 11, wherein the compound represented by general formula (1 ), helper lipid, sterol, and PEG-modified lipid are present at a molar ratio of 10 - 80: 0 - 50: 10 - 80: 0.5 - 20.

13. The composition of claim 10 or 12, wherein the helper lipid comprises a phospholipid selected from the group consisting of 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 (DUPO), 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-di linoleoyl-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, and combinations thereof.

14. The composition of any one of claims 10 to 13, wherein the sterol is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, avenasterol, and combinations thereof.

15. The composition of any one of claims 10 to 14, wherein the PEG-modified lipid is selected from the group consisting of 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-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2- dimyristyloxlpropyl-3-amine (PEG-c-DOMG), 3-N-[(cu-m ethoxy poly (ethyleneglycol)2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-S- DMG), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [(polyethylene glycol)-methoxy] (sodium salt) (PEG-DMPE), PEG-DPPC, PEG-DSPE lipid, and combinations thereof.

16. The composition of any one of claims 9 to 15, wherein the composition comprises nanoparticles with an average particle size of from 20 nm to 400 nm and a polydispersity index (PDI) of from 0.01 to 0.7.

17. A method of preparing a compound represented by general formula (1 ) or ionized form thereof according to any one of claims 1 to 9, the method comprising:(a-i) reacting a compound comprising 1° amine represented by general formula (3) with a carbohydrate represented by general formula (4) in the presence of a reducing agent to obtain an intermediate compound represented by general formula (6):I_1r2I H R13 R14D17 p18R1- R4R3Q ARH(6) whereinA comprises a carbohydrate or carbohydrate derivative;R1, R2, and R3are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;R4is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;R5comprises a hydrophobic moiety;Z1is selected from the group consisting of -O-C(=O)-, -C(=O)-O-, -C(=O)-, -O-, -RXN-C(=O)-, -C(=O)-NRy-, where Rxand Ryare each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;R11, R12, R13, R14, R15, R16, R17, R18, R19, R20, and R21are each independently selected from -H or -OH;x is an integer ≥ 1; andm is 0 or 1;(a-ii) reacting the intermediate compound represented by general formula (6) with a compound comprising carbonyl represented by general formula (7) in the presence of a reducing agent to obtain a compound represented by general formula (1):(1) whereinR8, R9, and R10are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;R7is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;R6comprises a hydrophobic moiety;Z2is selected from the group consisting of -O-C(=O)-, -C(=O)-O-, -C(=O)-, -O-, -RXN-C(=O)-, -C(=O)-NRy- where Rxand Ryare each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;y is an integer ≥ 0; and(a-iii) optionally ionizing the compound represented by general formula (1) to become positively charged.

18. The method of claim 17, wherein the method further comprises:(b-i) reacting the compound represented by general formula (1) with an acylating agent represented by general formula (9) to replace one or more -OH group(s) in A with -O-C(=O)Ragroup(s):whereinR27and R28are each independently optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and R9is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

19. The method of claim 17 or 19, wherein the method further comprises, prior to (a-i):(c-i) reacting a compound represented by general formula (10) with protected amine compound represented by general formula (11) to obtain an intermediate compound represented by general formula (12):whereinR1, R2, and R3are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; R4is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;R5comprises a hydrophobic moiety;Z1, Z1’, and Z1” are each independently selected from the group consisting of -O–C(=O)–, –C(=O)–O–, –C(=O)–, –O–, –RxN–C(=O)–, –C(=O)–NRy–, where Rxand Ryare each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;R22and R23are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; x is an integer > 1;PG1is a protecting group; and(c-ii) deprotecting the intermediate compound represented by general formula (12) to obtain the compound comprising 1° amine represented by general formula (3):

20. The method of any one of claims 17 to 19, wherein the method further comprises, prior to (a-ii):(d-i) reacting a compound represented by general formula (13) with a compound represented by general formula (14) to obtain an intermediate compound represented by general formula (15):(15) R8, R9, and R10are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;R7is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;R6comprises a hydrophobic moiety;Z2, Z2’, and Z2” are each independently selected from the group consisting of -O-C(=O)-, -C(=O)-O-, -C(=O)-, -O-, -RXN- C(=O)-, -C(=O)-NRy-, where Rxand Ryare each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;R24, R25, and R26are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;y is an integer ≥ 1; and(d-ii) subjecting the intermediate compound represented by general formula (15) to oxidation to obtain the compound comprising carbonyl represented by general formula (7):

21. The compound or ionized form thereof according to any one of claims 1 to 8 or nanoparticle composition according to any one of claims 10 to 16 for use in medicine.

22. A method of modulating an immune response in a subject, the method comprising the step of administering to a subject a therapeutically effective amount of the nanoparticle composition according to any one of claims 10 to 16.

23. The nanoparticle composition according to any one of claims 10 to 16 for use in modulating an immune response in a subject, wherein said nanoparticle composition is to be administered to the subject.

24. Use of a nanoparticle composition according to any one of claims 10 to 16 in the manufacture of a medicament for modulating an immune response in a subject.

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