A compound for preparing lipid nanoparticles encapsulating an agent, a nanoparticle composition comprising said compound and related methods thereof

Abstract

There is provided compound comprising a structure represented by general formula (1): wherein R1, R3, R4, R5, and R6 are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; R7 is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene; R10 is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; k is 0 or 1; n and l are each independently ≥ 1; A comprises galactose and / or a derivative(s) thereof; and B comprises a lipid and / or a derivative(s) thereof

Description

[0001] A COMPOUND FOR PREPARING LIPID NANOPARTICLES ENCAPSULATING AN AGENT, A 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] The liver is one of the most important organs in the human body and plays critical functions in metabolism, immunity, and endocrinology. In recent years, the incidence of liver disease has been rising, with more than 800 million people worldwide suffering from chronic liver problems. However, the current clinical treatments available for many liver diseases remain limited due to the challenges of achieving effective drug delivery to hepatocytes.

[0006] Specifically, although many nanomedicines have been developed to enhance systemic accumulation within the liver reticuloendothelial system, rapid sequestration of the nanomedicines by macrophages and Kupffer cells remains an obstacle as it impedes efficient drug delivery to hepatocytes, making these nanomedicines difficult to be taken up by hepatocytes.

[0007] Given the limitations of current nanomedicines in achieving efficient hepatocyte delivery, recent advances in lipid nanoparticle (LNP) technology have drawn significant attention as a potential strategy to overcome these barriers.

[0008] Particularly, LNPs have seen successful use as delivery vehicles in Moderna’s and Pfizer-BioNtech’s messenger ribonucleic acid (mRNA) Covid-19 vaccines, providing a promising platform for the delivery of cargoes involved in the treatment of liver diseases. Both vaccines utilize SARS-CoV-2 mRNA as the antigen and lipids as the carrier. Typically, LNPs are composed of three different types of lipid components (i.e., ionizable lipid, polyethylene glycol (PEG)-lipid conjugate, and helper lipid) and cholesterol, which assemble with mRNA to form nanoparticles that stimulate the immune cells responsible for the prophylactic response against the SARS-CoV-2 virus. In particular, the lipid components can encapsulate various pharmaceutical cargoes such as small molecules, proteins, and nucleic acids, protecting the cargoes from destructive enzymes and enabling transport across cell membranes.

[0009] However, the use of PEGylated lipids has raised concerns about the longterm safety of PEG-containing LNPs due to their poor biodegradability and propensity to trigger anti-PEG immune responses. Specifically, PEG has been identified as a high-risk hidden allergen in certain drugs and food products, which can bind to basophils through IgE and trigger the release of compounds that induce allergic reactions. This interaction can lead to the induction of anti-PEG antibodies, as observed in some mRNA vaccines. The formation of these antibodies may, in turn, cause anaphylactic reactions in certain individuals. Consequently, repeated exposure to PEGylated medicines can result in the accelerated blood clearance phenomenon, ultimately leading to altered vaccine immunogenicity and reactogenicity. In addition, the presence of anti-PEG antibodies in the body may reduce the plasma half-life of mRNA LNPs and compromise vaccination efficacy. Notably, to date, hepatocyte-targeting PEG-free LNPs have not been reported in existing mRNA LNP formulations.

[0010] 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 a nanoparticle composition for a cost efficient, substantially safe and stable, and / or efficacious delivery of therapeutic, prophylactic, and / or biological agents, particularly targeting hepatocytes. SUMMARY

[0011] In one aspect, there is provided a compound comprising a structure represented by general formula (1 ):

[0012]

[0013] wherein

[0014] R1, R3, R4, R5, and R6are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0015] R7is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;

[0016] R10is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0017] k is 0 or 1;

[0018] n and l are each independently ≥ 1;

[0019] A comprises galactose and / or a derivative(s) thereof; and

[0020] B comprises a lipid and / or a derivative(s) thereof.

[0021] In one embodiment, A is represented by general formula (2):

[0022]

[0023] wherein

[0024] X1to X7and X9to X10are each independently selected from -Rd, -ORe, or -0-C(=O)Rf, wherein Rd, Re, and Rfare each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0025] X8is alkylene; and

[0026] M is –O–Rg–, –S–Rh–, or –N(Ri)–Rj–, wherein Riis H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl and wherein Rg, Rh, and Rjare each independently absent or each independently present and selected from optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

[0027] In one embodiment, B is Rb-N-Rc, wherein Rband Rcare each independently a hydrophobic group or contains at least linear aliphatic, branched aliphatic, and / or cyclic hydrocarbons.

[0028] In one embodiment, B is Rb-N-Rc, wherein Rband Rcare each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and combinations thereof.

[0029] In one embodiment, B comprises sterol and / or a derivative(s) thereof.

[0030] In one embodiment, B is represented by general formula (X),

[0031]

[0032] wherein

[0033] R12is a hydrophobic group or contains at least linear aliphatic, branched aliphatic, and / or cyclic hydrocarbons.

[0034] R13-18, R20, R22, R24-29, R32-33, and R35-37are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; R19, R21, R23, R30, R31, and R34are each independently absent, H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted; and Ring Z contains one or more double bonds.

[0035] In one embodiment, the compound is substantially devoid of polyethylene glycol (PEG).

[0036] In one embodiment, the compound has an average molecular weight of from 3,000 g / mol to 50,000 g / mol.

[0037] In one embodiment, the compound is selected from the group consisting of the following structures:

[0038] N CH3

[0039] HQ

[0040]

[0041]

[0042] In one aspect, there is provided a method of preparing a compound represented by general formula (1) disclosed herein, the method comprising: (i) reacting a lipid molecule represented by general formula (3) with a compound comprising N-carboxyanhydride (NCA) represented by general formula (4) to obtain a first intermediate compound represented by general formula (5):

[0043]

[0044] wherein

[0045] R1, R3, R4, R5, R6, and R11are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; R7is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;

[0046] k is 0 or 1;

[0047] n and l are each independently ≥ 1; A comprises galactose and / or a derivative(s) thereof, and Aprepresents A protected with a protecting group; and

[0048] B comprises a lipid and / or a derivative(s) thereof;

[0049] (ii) reacting the first intermediate compound represented by general formula (5) with an acid anhydride represented by general formula (6) to obtain a second intermediate compound represented by general formula (7):

[0050]

[0051] (7)

[0052] wherein

[0053] R1, R3, R4, R5, R6, and R11are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; R7is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;

[0054] R10and R10’ are each independently optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0055] k is 0 or 1;

[0056] n and l are each independently ≥ 1; A comprises galactose and / or a derivative(s) thereof, and Aprepresents A protected with a protecting group; and

[0057] B comprises a lipid and / or a derivative(s) thereof; and

[0058] (iii) deprotecting the second intermediate compound represented by general formula (7) to obtain the compound represented by general formula (1).

[0059] In one embodiment, the method further comprises, prior to (i):

[0060] (a-i) reacting a galactose and / or a derivative(s) thereof represented by general formula (8) with an acid anhydride represented by general formula (9) to obtain a protected galactose and / or a derivative(s) thereof represented by general formula (8p), wherein one or more -OH group(s) in the galactose and / or a derivative(s) thereof represented by general formula (8) are converted into -OPG1:

[0061]

[0062] wherein X1to X7and X9to X11are each independently selected from -Rkor -OR1, wherein Rkand R1are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0063] X22to X28and X30to X32are each independently selected from -Rmor - OPG1, wherein Rmis H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0064] X8and X29are each alkylene;

[0065] R11is optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl;

[0066] M is –O–Rg–, –S–Rh–, or –N(Ri)–Rj–, wherein Riis H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl and Rg, Rh, and Rjare each independently absent or each independently present and selected from optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and PG1is -C(=O)-R13, where R13are each independently optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0067] (a-i i) reacting the protected galactose and / or a derivative(s) thereof represented by general formula (8P) obtained from (a-i) with a protected compound represented by general formula (10) in the presence of a Lewis acid to obtain a first intermediate compound comprising amide group represented by general formula (11):

[0068]

[0069] O (8p) (10) o

[0070]

[0071] (11)

[0072] wherein

[0073] X22to X28and X30to X32are each independently selected from -Rmor - OPG1, wherein Rmis H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0074] X8and X29are each alkylene;

[0075] M is -O-Rg-, -S-Rh-, or -N(R')-R’-, wherein Riis H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl and Rg, Rh, and Rfare each independently absent or each independently present and selected from optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0076] R7is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;

[0077] A comprises galactose and / or a derivative(s) thereof, and Aprepresents A protected with a protecting group; and

[0078] PG2is a protecting group;

[0079] (a-iii) deprotecting the first intermediate compound represented by general formula (11) to obtain a second intermediate represented by general formula (12):

[0080]

[0081] (11) (12) wherein R7is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;

[0082] A comprises galactose and / or a derivative(s) thereof, and Aprepresents A protected with a protecting group; and

[0083] PG2is a protecting group; and

[0084] (a-iv) reacting the second intermediate compound represented by general formula (12) with a carbonylating agent to obtain the compound comprising N-carboxyanhydride (NCA) represented by general formula (4)

[0085]

[0086] wherein

[0087] R6is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0088] R7is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene; and

[0089] A comprises galactose and / or a derivative(s) thereof, and Aprepresents A protected with a protecting group.

[0090] In one aspect, there is provided a nanoparticle composition comprising: (i) a compound represented by general formula (1 ) disclosed herein; and (ii) a therapeutic, prophylactic, and / or biological agent that is encapsulated by said compound.

[0091] In one embodiment, the composition further comprises: (a) helper lipid;

[0092] (b) cholesterol and / or a derivative(s) thereof; and

[0093] (c) ionizable lipid.

[0094] In one embodiment, the ionizable lipid, helper lipid, cholesterol and / or a derivative(s) thereof, and the compound represented by general formula (1) are mixed at a mole ratio of 20 - 50: 4 - 20: 25 - 50: 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 cholesterol and / or a derivative(s) thereof is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, avenasterol, and combinations thereof. In one embodiment, the ionizable lipid is selected from the group consisting of ALC-0315, SM-102, Lipid 5, DLinDMA, D-Lin-MC2-DMA, DLin-MC3-DMA, D-Lin-MC4-DMA, Dlin-KC2-DMA, YSK05, AA3-Dlin, SSPalmM, SSPalmO-Phe, Lipid A9, L319, DODMA, CL1, BP Lipid 310, ATX-001, ATX-100, Lipid 2, 80-O16B, BP Lipid 309, BP Lipid 307, 93-O17S, 93-O17O, NT1-O14B, 306-012B-3, 306-012B, 113-016B, 3060i10, 306Oi9-cis2, BAMEA-O16B, Al-28, 113-O12B, 98N12-5, Ckk-E12, OF-02, C12-200, BP Lipid 311, BP Lipid 308, BP Lipid 314, BP Lipid 312, LP01, TCL053, Lipid C24, BP Lipid 315, Lipid 29, 9A1P9, C13-112-tri-tail, C13-113-tri-tail, C13-112-tetra-tail, C13-113-tetra-tail, and combinations thereof.

[0097] In one embodiment, the composition comprises nanoparticles with a N / P ratio from 1:1 to 20:1, an average from 40 nm to 500 nm, a polydispersity index (PDI) of from 0.001 to 0.500, and / or a zeta potential of from -20 mV to 20 mV.

[0098] In one embodiment, the compound represented by general formula (1) disclosed herein or the nanoparticle composition disclosed herein is for use in medicine.

[0099] 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.

[0100] 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.

[0101] 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. DEFINITIONS

[0102] The term “particle” as used herein broadly refers to a discrete entity or a discrete body. The particle described herein can include an organic, an inorganic, a composite particle, or a biological particle. The particle used described herein may also be a macro-particle that is formed by an aggregate of a plurality of subparticles or a fragment of a small object. The particle of the present disclosure may be spherical, substantially spherical, or non-spherical, such as irregularly shaped particles or ellipsoidally shaped particles. The term “size” when used to refer to the particle broadly refers to the largest dimension of the particle. For example, the term “size” when used in the context of nanoparticle can refer to the diameter of the nanoparticle although it is not limited as such. In various embodiments, when the particle is substantially spherical, the term “size” can refer to the diameter of the particle; or when the particle is substantially non-spherical, the term “size" can refer to the largest length of the particle.

[0103] The term “nano" as used herein is to be interpreted broadly to include dimensions in a nanoscale, i.e., less than about 10000 nm, about 1 nm to less than about 10000 nm, about 1 nm to about 9000 nm, about 1 nm to about 8000 nm, about 1 nm to about 7000 nm, about 1 nm to about 6000 nm, about 1 nm to about 5000 nm, about 1 nm to about 4000 nm, about 1 nm to about 3000 nm, about 1 nm to about 2000 nm, from about 1 nm to 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.

[0104] The term "micro" as used herein is to be interpreted broadly to include dimensions from about 1 micron to about 1000 microns, about 1 micron to less than about 1000 microns, about 1 micron to about 900 microns, about 1 micron to about 800 microns, about 1 micron to about 700 microns, about 1 micron to about 600 microns, about 1 micron to about 500 microns, about 1 micron to about 400 microns, about 1 micron to about 300 microns, about 1 micron to about 200 microns, about 1 micron to about 100 microns, or from about 1 micron to about 5 microns. In various embodiments, particles of about 5 microns or lesser may be useful for intranasal spray delivery.

[0105] 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.

[0106] 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), cardiovascular diseases, autoimmune diseases / disorders, or liver diseases (e g., hepatitis). 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.

[0107] 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).

[0108] 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.

[0109] 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 -CH3and when existing as a bridging group, the term “alkyl” having 1 carbon atom may mean -CH2- or the like. 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.

[0110] 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.

[0111] 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.

[0112] 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-). 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.

[0113] The term "heteroaryl" as a group or part of a group refers to groups containing an aromatic ring (preferably a 5- or 6- membered aromatic ring) having one or more carbon atoms (for example 1 to 6 carbon atoms) in the ring replaced by a heteroatom. Suitable heteroatoms may include nitrogen (N) or (NH), oxygen (O) and sulfur (S). Examples of heteroaryl include but are not limited to thiophene, benzothiophene, benzofuran, benzimidazole, benzoxazole, benzothiazole, benzisothiazole, naphtha[2,3-b]thiophene, furan, isoindolizine, xantholene, phenoxatine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, tetrazole, indole, isoindole, 1 H-indazole, purine, quinoline, isoquinoline, phthalazine, naphthyridine, quinoxaline, cinnoline, carbazole, phenantridine, acridine, phenazine, thiazole, isothiazole, phenothiazine, oxazole, isooxazole, furazane, phenoxazine, 2-, 3- or 4-pyridyl, 2-, 3-, 4-, 5-, or 8-quinolyl, 1-, 3-, 4-, or 5-isoquinolinyl 1-, 2-, or 3-indolyl, and 2-, or 3-thienyl and the like. The group may be a terminal group or a bridging group.

[0114] 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”.

[0115] 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 (0), nitrogen (N) or (NR) and sulfur (S), where R is independently a hydrogen or an organic group. The term also includes, but is not limited to, saturated and unsaturated 5-membered, and saturated and unsaturated 6-membered rings. Examples of groups having a heterocyclic structure include, but are not limited to furan, thiophene, 1 H-pyrrole, 2H-pyrrole, 1 -pyrroline, 2-pyrroline, 3-pyrroline, 1-pyrazoline, 2-pyrazoline, 3-pyrazoline, 2-imidazoline, 3-imidazoline, 4-imidazoline, pyrazole, imidazole, oxazole, isoxazole, thiazole, isothiazole, 1,2,3-triazole, 1,2,4-triazole, 1,2,3-oxadiazole, disubstituted 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, 1.2.3-thiadiazole, 1,2,4-thiadiazole, 1,2,5-thiadiazole, 1,3,4-thiadiazole, tetrahydrofuran, tetrahydrothiophene, pyrrolidine, 1,3-dioxolane, 1,2-oxathiolane, 1.3-oxathiolane, pyrazolidine, imidazolidine, pyridine, pyridazine, pyrimidine, pyrazine, 1,2-oxazine, 1,3-oxazine, 1,4-oxazine, thiazine, 1,2,3-triazine, 1,2,4-triazine, 1,3,5-triazine, 2H-pyran, 4H-pyran, 2-pyrone, 4-pyrone, 1,4-dioxin, 2H-thiopyran, 4H-thiopyran, tetrahydropyran, thiane, piperidine, 1,4-dioxane, 1,2-dithiane, 1,3-dithiane, 1,4-dithiane, 1,3,5-trithiane, piperazine, morpholine, thiomorpholine and the like.

[0116] 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.

[0117] 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.

[0118] 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. 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.

[0119] As used herein, the term “derivative thereof” or the like refers to a compound, or a portion of that compound, that is structurally based on or obtained from a referenced compound and that retains the essential structural framework of the referenced compound. Such derivatives do not materially alter the core structure or main functional identity of the referenced compound or of any broader compound in which they are incorporated. For example, in various embodiments, derivatives of the galactose and / or lipid moieties (i.e., parameters A and B) of formula (1) disclosed herein do not materially change the core structure or primary functional characteristics of the compound of formula (1). Accordingly, such derivatives still allow the compound of formula (1) to perform its intended function(s) disclosed herein, such as serving as a substitute or replacement for a conventional PEG-lipid conjugate (e.g., ALC-0159). The term “derivative thereof’ or the like further encompasses, without limitation, salts, solvates, isomers, tautomers, prodrugs, metabolites, analogues, and chemically modified forms, including substitutions, additions, deletions, or rearrangements of the referenced compound.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] DESCRIPTION OF EMBODIMENTS

[0126] 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.

[0127] COMPOUND

[0128] There is provided a compound represented by general formula (1) or ionized form thereof: R7

[0129]

[0130] (1) wherein

[0131] R1, R3, R4, R5, and R6are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0132] R7is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;

[0133] R10is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0134] k is 0 or 1;

[0135] n and l are each independently ≥ 1;

[0136] A comprises galactose and / or a derivative(s) thereof; and

[0137] B comprises a lipid and / or a derivative(s) thereof.

[0138] In various embodiments, the compound or ionized form thereof is suitable for use in a composition / nanoparticle composition for delivery of a therapeutic and / or prophylactic agent and / or biological agent (e.g., a nucleic acid such as messenger ribonucleic acid (mRNA), small interfering ribonucleic acid (siRNA), plasmid deoxyribonucleic acid (pDNA), etc.).

[0139] In various embodiments, the therapeutic agent and / or prophylactic agent and / or biological agent comprises nucleic acid (e.g., RNA, mRNA, siRNA, DNA, 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. Advantageously, in various embodiments, the design of the compound represented by general formula (1) or ionized form thereof helps prevent nonspecific protein absorption, particle aggregation and controls the size of the nanoparticles formed. In various embodiments, embodiments of the compound represented by general formula (1) or ionized form thereof helps maintain colloidal stability (of the lipid nanoparticles) and facilitate the condensation and encapsulating / loading of molecules / cargoes into the nanoparticle composition.

[0140] In various embodiments, the compound or ionized form thereof is represented by general formula (1a):

[0141]

[0142] In various embodiments, R1, R3, R4, R5and R6are each independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl. For example, R1, R3, R4, R5and R6may be selected from methyl, ethyl, n-propyl. 2-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, hexyl, amyl, 1,2-dimethylpropyl, 1,1 -dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1 -methylpentyl, 2-methylpentyl, 3-methylpentyl, 2,2- dimethylbutyl, 3,3-dimethylbutyl, 1,2-dim ethyl butyl, 1,3-dimethylbutyl, 1,2,2-trimethylpropyl, 1, 1,2-trimethylpropyl, 2-ethylpentyl, 3-ethylpentyl, heptyl, 1-methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl, 1,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,2,3-trimethylbutyl, 1,1,2-trimethylbutyl, 1,1, 3-trimethylbutyl, 5-methylheptyl, 1 -methylheptyl, octyl, nonyl, decyl, or the like or combinations thereof.

[0143] In various embodiments, R7is selected from optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene. For example, R7may be selected from methylene, ethylene, n-propylene, 2-propylene, isopropylene, n-butylene, isobutylene, sec-butylene, f-butylene, hexylene, amylene, 1,2-dimethylpropylene, 1,1 -dimethylpropylene, pentylene, isopentylene, hexylene, 4-methylpentylene, 1 -methylpentylene, 2-methylpentylene, 3-methylpentylene, 2,2-dimethylbutylene, 3,3-dimethylbutylene, 1,2-dimethylbutylene, 1,3-dimethylbutylene, 1,2,2-trimethylpropylene, 1,1,2-trimethylpropylene, 2-ethylpentylene, 3-ethylpentylene, heptylene, 1 -methylhexylene, 2,2-dimethylpentylene, 3,3-dimethylpentylene, 4,4-dimethylpentylene, 1,2-dimethylpentylene, 1,3-dimethylpentylene, 1,4-dimethylpentylene, 1,2,3-trimethylbutylene, 1,1,2-trimethylbutylene, 1,1,3-trimethylbutylene, 5-methylheptylene, 1 -methylheptylene, octylene, nonylene, decylene, or the like or combinations thereof.

[0144] In various embodiments, R7is optionally substituted –CyH2y–, where y is an integer > 1. In various embodiments, y > 1, y > 2, y > 3, y > 4, y > 5, y > 6, y > 7, y > 8, y > 9, y > 10, y > 11, y > 12, y > 13, y > 14, y > 15, y > 16, y > 17, y > 18, y > 19, or y > 20. In various embodiments, y is from about 1 to about 20, from about 2 to about 19, from about 3 to about 18, from about 4 to about 17, from about 5 to about 16, from about 6 to about 15, from about 7 to about 14, from about 8 to about 13, from about 9 to about 12, or from about 10 to about 11. For example, R7may be -CH2-, -C2H4-, -C3H6-, -C4H8- or -C5H10-. In various embodiments, R10is selected from optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl. For example, R10may be selected from methyl, ethyl, n-propyl, 2-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, hexyl, amyl, 1,2-dimethylpropyl, 1,1 -dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1 -methylpentyl, 2-methylpentyl, 3-methylpentyl, 2.2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 1,2,2-trimethylpropyl, 1, 1,2-trimethylpropyl, 2-ethylpentyl, 3-ethylpentyl, heptyl, 1-methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl, 1,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,2,3-trimethylbutyl, 1.1.2-trimethylbutyl, 1, 1,3-trimethylbutyl, 5-methylheptyl, 1 -methylheptyl, octyl, nonyl, decyl, or the like or combinations thereof.

[0145] In various embodiments, n is an integer > 1. In various embodiments, n > 1, n > 2, n > 3, n > 4, n > 5, n > 6, n > 7, n > 8, n > 9, n > 10, n > 11, n > 12, n > 13, n > 14, n > 15, n > 16, n > 17, n > 18, n > 19, n > 20, n > 21, n > 22, n > 23, n > 24, n > 25, n > 26, n > 27, n > 28, n > 29, n > 30, n > 31, n > 32, n > 33, n > 34, n > 35, n > 36, n > 37, n > 38, n > 39, n > 40, n > 41, n > 42, n > 43, n > 44, n > 45, n > 46, n > 47, n > 48, n > 49, n > 50, n > 51, n > 52, n > 53, n > 54, n > 55, n > 56, n > 57, n > 58, n > 59, n > 60, n > 61, n > 62, n > 63, n > 64, n > 65, n > 66, n > 67, n > 68, n > 69, n > 70, n > 71, n > 72, n > 73, n > 74, n > 75, n > 76, n > 77, n > 78, n > 79, n > 80, n > 81, n > 82, n > 83, n > 84, n > 85, n > 86, n > 87, n > 88, n > 89, n > 90, n > 91, n > 92, n > 93, n > 94, n > 95, n > 96, n > 97, n > 98, n > 99, or n > 100. In various embodiments, n is from about 1 to about 100, from about 10 to about 90, from about 20 to about 80, from about 30 to about 70, from about 40 to about 60, or about 50.

[0146] In various embodiments, I is an integer > 1. In various embodiments, I > 1, I > 2, I > 3, I > 4, In > 5, I > 6, I > 7, I > 8, I > 9 or I > 10. In various embodiments, I is from about 1 to about 10, from about 2 to about 9, from about 3 to about 8, from about 4 to about 7, from about 5 to about 6, or about 2.

[0147] In various embodiments, A may be hydrophilic. In various embodiment, A is in a cyclic form, for example as a 5-membered ring or 6-membered ring. In various embodiments, A is in a linear form or an open chain form.

[0148] In various embodiment, A is represented by general formula (2) having a 6-membered ring structure:

[0149] X10

[0150] 0^

[0151] X2X8I

[0152] I *6

[0153] X9

[0154]

[0155] (2)

[0156] wherein

[0157] X1to X7and X9to X10are each independently selected from -Rd, -0Re, or -0-C(=0)Rf, wherein Rd, Re, and Rfare each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0158] X8is alkyl / alkylene; and

[0159] M is -O-Rg-, -S-Rh-, or-N(R')-Rj- wherein Riis H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl and wherein Rg, Rh, and Rjare each independently absent or each independently present and selected from optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

[0160] In various embodiments, X8is optionally substituted -CaH2a-, where a is from about 1 to about 20. For example, X8may be -CH2-, -C2H4-, -CsHe-— C4H8—, — C5H10—, — CeHi2—, — C7H14—, — CsHie—, — C9H18—, or — C10H20—. In various embodiments, X8is methylene (i.e., -CH2-).

[0161] In various embodiments, M is -O-. In various embodiments, A is chemically coupled / bonded to the rest of general formula (1) or ionized form thereof via its hydroxy group. For example, A may be connected to R7via a glycosidic / ether bond / linkage.

[0162] In various embodiments, A is galactose (e.g., for targeting hepatocytes) represented by formula (2’):

[0163]

[0164] In various embodiments, A is represented by general formula (2A) and / or (2B) having a 6-membered ring structure:

[0165] X9

[0166]

[0167] (2B)

[0168] wherein X1to X10contain one or more features and / or share one or more properties that are similar to those described above (e.g., as defined in general formula (2)). In various embodiments, A comprises p-galactose and / or a derivative(s) thereof. In various embodiments, A comprises a-galactose and / or a derivative(s) thereof. In various embodiments, A comprises a mixture of a-galactose, p-galactose, and / or a derivative(s) thereof. In various embodiments, A comprises D-galactose and / or a derivative(s) thereof (e.g., represented by general formula (2A)). In various embodiments, A comprises L-galactose and / or a derivative(s) thereof (e.g., represented by general formula (2B)). In various embodiments, A comprises a mixture of two enantiomers, namely D-galactose and / or a derivative(s) thereof (e.g., represented by general formula (2A); and L-galactose and / or a derivative(s) thereof (e.g., represented by general formula (2B). In various embodiments, A comprises a mixture (e.g., racemic mixture of enantiomers) containing both D-galactose and L-galactose. For example, A may comprise D-galactose, L-galactose, and / or a derivative(s) thereof.

[0169] In various embodiments, A may comprise one or more of β-D-galactose, β-L-galactose, α-D-galactose, α-L-galactose (represented by formulae (2C), (2D), (2E), and (2F) respectively), and / or a derivative(s) thereof:

[0170]

[0171] In various embodiments, B is Rb-N-Rc, wherein Rband Rcare each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and combinations thereof.

[0172] In various embodiments, Rband Rcmay be same or different. In various embodiments, Rband Rcare the same. In various embodiments, Rbis the same as Rc. For example, Rb= Rc. In various embodiments, Rband Rcare different. In various embodiments, only one of Rband Rcis H. For example, in various embodiments, when Rbis H, Rcis selected from the group consisting of optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and combinations thereof, and vice versa.

[0173] In various embodiments, B is Rb-N-Rc, wherein Rband Rcare each independently hydrophobic part / tail / chain / group, or contains at least linear aliphatic, branched aliphatic, and / or cyclic hydrocarbons.

[0174] In various embodiments, the compound represented by general formula (1) or ionized form thereof comprises one or more hydrophobic part / tail / chain / group(s). In various embodiments, Rband Rcmay be same or different. In various embodiments, Rband Rcare the same. In various embodiments, Rbis the same as Rc. For example, Rb= Rc. In various embodiments, the compound or ionized form thereof comprises hydrophobic parts / tails / chains / groups at both Rband Rc. In various embodiments, Rband Rcare different. In various embodiments, only one of Rband Rcis a hydrophobic part / tail / chain / group. For example, in various embodiments, when Rbis a hydrophobic part / tail / chain / group, Rcis H, and vice versa.

[0175] In various embodiments, Rband Rceach independently contains at least linear aliphatic, branched aliphatic, and / or cyclic hydrocarbons. In various embodiments, the hydrophobic part / tail / chain / group at Rband Rceach independently comprises optionally substituted alkyl. In various embodiments, the hydrophobic part / tail / chain / group at Rband Rceach independently comprises unsaturated hydrocarbons such as an optionally substituted alkenyl. For example, Rband Rcmay contain one or more C=C double bond(s). The alkyl or alkenyl may have at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 carbon atoms. For example, Rband Rcmay be each independently CxH2x+1 or CxH2x, where x is an integer > 5, x > 6, x > 7, x > 8, x > 9, x > 10, x > 11, x > 12, x > 13, x > 14, x > 15, x > 16, x > 17, x > 18, x > 19, x > 20, x > 21, x > 22, x > 23, x > 24, x > 25, x > 26, x > 27, x > 28, x > 29, or x > 30. Advantageously, in various embodiments, the presence of hydrophobic parts / tails / chains / groups in the compound or ionized form thereof allows for ease of integration of the compound or ionized form thereof into the lipid domain of lipid nanoparticles (LNPs), presenting the galactose and / or a derivative(s) thereof-functionalized polypeptide on the surface of the LNPs for stability and cell-targeting ability (e.g., hepatocytes-targeting ability).

[0176] Advantageously, in various embodiments, the compound or ionized form thereof is designed / configured to allow the hydrophilicity / hydrophobicity balance of said compound or ionized form thereof to be customizable by adjusting the length or hydrophobicity of Rband Rc.

[0177] In various embodiments, B comprises sterol and / or a derivative(s) thereof.

[0178] In various embodiments, B is a sterol. Sterols include animal sterols, plant sterols, fungal sterols, etc. Examples of animal sterols include cholesterol and lanosterol or the like. Examples of plant sterols include p-Sitosterol, campesterol, stigmasterol, brassicasterol, or the like. Examples of fungal sterols include ergosterol, zymosterol, or the like.

[0179] In various embodiments, B is represented by general formula (X),

[0180]

[0181] wherein

[0182] R12is a hydrophobic part / tail / chain / group, or contains at least linear aliphatic, branched aliphatic, and / or cyclic hydrocarbons;

[0183] R13-18, R20, R22, R24-29, R32-33, and R35-37are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; R19, R21, R23, R30, R31, and R34are each independently absent, H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted; and Ring Z contains one or more double bonds.

[0184] In various embodiments, B has general formula (X’),

[0185]

[0186] wherein

[0187] R12is a hydrophobic part / tail / chain / group, or contains at least linear aliphatic, branched aliphatic, and / or cyclic hydrocarbons; and

[0188] Ring Z contains one or more double bonds.

[0189] In various embodiments, B is a cholesterol,

[0190]

[0191] In various embodiments, the compound represented by general formula (1) or ionized form thereof is capable of targeting asialoglycoprotein receptors (ASGP-R’s) (e.g., galactose or N-acetylglucosamine) on a cell surface such as a liver cell surface or a hepatocyte surface to enhance the delivery efficiency of mRNA. In various embodiments, ASGP-R1, which is exclusively expressed on the mammalian hepatocytes and can specifically recognize ligands containing terminal galactose, glucose, and N-acetylgalactosamine, provides an active targeting site for the compound represented by general formula (1) or ionized form thereof to enhance hepatocyte-specific drug delivery.

[0192] In various embodiments, the compound or ionized form thereof comprises a galactose and / or a derivative(s) thereof-functionalized polypeptide-b-lipid such as a galactose-functionalized polyserine / polyglutamic acid-b-lipid wherein the galactose is attached onto each unit of the polyserine / polyglutamic acid.

[0193] In various embodiments, the compound or ionized form thereof comprises a galactose and / or a derivative(s) thereof-functionalized polypeptide-b-sterol such as a galactose-functionalized polyserine / polyglutamic acid-b-cholesterol wherein the galactose is attached onto each unit of the polyserine / polyglutamic acid.

[0194] In various embodiments, the compound or ionized form thereof is substantially devoid of polyethylene glycol (PEG). For example, the compound or ionized form thereof is substantially devoid of polyethylene glycol (PEG)-modified lipid conjugates.

[0195] In various embodiments, the term “polyethylene glycol (PEG)-modified lipid” is used interchangeably with the terms “PEGylated lipid”, PEG-conjugated lipid”, “P EG-lipid conjugate”, and “lipid modified with PEG”.

[0196] Advantageously, the presence of galactose and / or a derivative(s) thereof increases the hydrophilicity of the compound or ionized form thereof, and consequently solubility of the compound or ionized form thereof. Advantageously, in various embodiments, the presence of galactose and / or a derivative(s) thereof in the compound or ionized form thereof eliminates the requirement of a hydrophilic PEG which is otherwise necessary in a conventional PEG-lipid conjugate for LNP formulations. In various embodiments, by eliminating the presence of PEG in the compound or ionized form thereof, embodiments of the galactose group and / or a derivative(s) thereof and lipid nanoparticles formed therefrom are not and / or avoid the possibility of being shielded by the long PEG chain. In various embodiments, the design of the compound or ionized form thereof without the long PEG chain allows easy access of the galactose and / or a derivative(s) thereof to the cell (e.g., immune cell) in order to target receptors of galactose and / or a derivative(s) thereof on the cell surface (e.g., immune cell surface), thereby prolonging the plasma half-life of nucleic acid and enhancing therapeutic efficiency. In various embodiments, the compound or ionized form thereof is substantially devoid of PEG. In various embodiments, the compound or ionized form thereof is substantially devoid of PEG-modified lipid conjugates, PEG-modified lipid, PEGylated lipid, PEG-conjugated lipid, PEG-lipid conjugate, and / or lipid modified with PEG. Advantageously, in various embodiments, the design of the structure of the compound or ionized form thereof allows said compound or ionized form thereof to be used, in lieu of or as a substitute / replacement for a conventional PEG-lipid conjugate (e.g., ALC-0159). Advantageously, the presence of galactose and / or a derivative(s) thereof as hepatocyte-targeting functional groups in polypeptide-based lipids mediate mRNA-LNPs to achieve improved mRNA delivery efficiency and safety in liver.

[0197] In various embodiments, the compound or ionized form thereof (e.g., lipid-b-polypeptide) has an average molecular weight or a number average molecular weight (Mn) of from about 2,000.0 g / mol to about 50,000.0 g / mol, from about 2,500.0 g / mol to about 50,000.0 g / mol, from about 3,000.0 g / mol to about 50,000.0 g / mol, from about 3,500.0 g / mol to about 50,000.0 g / mol, from about 3,500.0 g / mol to about 49,000.0 g / mol, from about 4,000.0 g / mol to about 49,000.0 g / mol, from about 5,000.0 g / mol to about 48,000.0 g / mol, from about 6,000.0 g / mol to about 47,000.0 g / mol, from about 7,000.0 g / mol to about 46,000.0 g / mol, from about 8,000.0 g / mol to about 45,000.0 g / mol, from about 9,000.0 g / mol to about 44,000.0 g / mol, from about 10,000.0 g / mol to about 43,000.0 g / mol, from about 11,000.0 g / mol to about 42,000.0 g / mol, from about 12,000.0 g / mol to about 41,000.0 g / mol, from about 13,000.0 g / mol to about 40,000.0 g / mol, from about 14,000.0 g / mol to about 39,000.0 g / mol, from about 15,000.0 g / mol to about 38,000.0 g / mol, from about 16,000.0 g / mol to about 37,000.0 g / mol, from about 17,000.0 g / mol to about 36,000.0 g / mol, from about 18,000.0 g / mol to about 35,000.0 g / mol, from about 19,000.0 g / mol to about 34,000.0 g / mol, from about 20,000.0 g / mol to about 33,000.0 g / mol, from about 21,000.0 g / mol to about 32,000.0 g / mol, from about 22,000.0 g / mol to about 31,000.0 g / mol, from about 23,000.0 g / mol to about 30,000.0 g / mol, from about 24,000.0 g / mol to about 29,000.0 g / mol, from about 25,000.0 g / mol to about 28,000.0 g / mol, from about 26,000.0 g / mol to about 27,000.0 g / mol, about 20,000.0 g / mol, about 25,000.0 g / mol, about 30,000.0 g / mol, about 35,000.0 g / mol, about 40,000.0 g / mol, or about 45,000.0 g / mol.

[0198] 1.

[0199] In various embodiments, the compound represented by general formula (1) or ionized form thereof is lipid-b-poly(Ser-D-galactose) (LPSG). In various embodiments, the compound represented by general formula (1) or ionized form thereof is cholesterol-b-poly(Ser-D-galactose) (CPSG).

[0200] In various embodiments, the compound or ionized form thereof is selected from the group consisting of the following structures:

[0201]

[0202]

[0203] In various embodiments, the compound or ionized form thereof is biocompatible, i.e., the compound or ionized form thereof 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 injury or the like when used on the human or animal body. In various embodiments, the compound or ionized form thereof is substantially devoid of substances that elicit an adverse physiological response.

[0204] METHOD OF PREPARING COMPOUND

[0205] There is provided a method of preparing a compound represented by general formula (1) or ionized form thereof as disclosed herein, the method comprising: (i) reacting a lipid molecule represented by general formula (3) with a compound comprising N-carboxyanhydride (NCA) represented by general formula (4) to obtain a first intermediate compound represented by general formula (5):

[0206]

[0207] wherein

[0208] R1, R3, R4, R5, R6, and R11are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; R7is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;

[0209] k is 0 or 1;

[0210] n and l are each independently ≥ 1;

[0211] A comprises galactose and / or a derivative(s) thereof, and Aprepresents A functionalized / protected with a protecting group; and

[0212] B comprises a lipid and / or a derivative(s) thereof.

[0213] In various embodiments, B comprises sterol and / or a derivative(s) thereof.

[0214] In various embodiments, the reacting in (i) comprises ring-opening and polymerization of the NCA group in the compound represented by general formula (4). Advantageously, the NCA undergoes ring-opening and polymerizes despite the bulky structure of the protected galactose group. Without being bound by theory, it is believed that such a successful cyclization (NCA formation) and polymerization is due to the choice of L-serine and D-galactose, which avoids steric hindrance. Advantageously, in various embodiments, the degree of polymerization may be precisely controlled.

[0215] In various embodiments, (i) comprises suspending / dispersing / mixing / dissolving / reacting the lipid molecule represented by general formula (3) with the compound comprising N-carboxyanhydride (NCA) represented by general formula (4) in a molar ratio of from about 1:20 to about 1:60, from about 1:22 to about 1:58, from about 1:24 to about 1:56, from about 1:26 to about 1:54, from about 1:28 to about 1:52, from about 1:30 to about 1:50, from about 1:32 to about 1:48, from about 1:34 to about 1:46, from about 1:36 to about 1:44, from about 1:38 to about 1:42, or about 1:40.

[0216] In various embodiments, the method further comprises:

[0217] (ii) reacting the first intermediate compound represented by general formula (5) with an acid anhydride represented by general formula (6) (e.g., acetic anhydride) to obtain a second intermediate compound represented by general formula (7):

[0218]

[0219] (7)

[0220] wherein

[0221] R1, R3, R4, R5, R6, and R11are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; R7is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;

[0222] R10and R10’ are each independently optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0223] k is 0 or 1;

[0224] n and l are each independently ≥ 1;

[0225] A comprises galactose and / or a derivative(s) thereof, and Aprepresents A functionalized / protected with a protecting group; and

[0226] B comprises a lipid and / or a derivative(s) thereof.

[0227] In various embodiments, B comprises sterol and / or a derivative(s) thereof. (iii) deprotecting the second intermediate compound represented by general formula (7) to obtain the compound represented by general formula (1) or ionized form thereof.

[0228] In various embodiments, the deprotection of the second intermediate compound represented by general formula (7) comprises subjecting the second intermediate compound to one or more of a nucleophilic compound comprising hydroxyl group, a metal alkoxide (e.g., sodium methoxide), and an alcohol (e.g., methanol). In various embodiments, the protecting group comprises -C(=O)-R, where R is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl. For example, the protecting group may be an acetyl group.

[0229] In various embodiments, the method further comprises, prior to (i):

[0230] (a-i) reacting a galactose and / or a derivative(s) thereof represented by general formula (8) with an acid anhydride represented by general formula (9) to obtain a protected galactose and / or a derivative(s) thereof represented by general formula (8P), wherein one or more -OH group(s) in the galactose and / or a derivative(s) thereof represented by general formula (8) are converted into -OPG1:

[0231]

[0232] (8) (9)

[0233]

[0234] (8p)

[0235] wherein

[0236] X1to X7and X9to X11are each independently selected from -Rkor -OR1, wherein Rkand R1are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0237] X22to X28and X30to X32are each independently selected from -Rmor - OPG1, wherein Rmis H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0238] X8and X29are each alkyl / alkylene;

[0239] R11is optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl;

[0240] M is -O-Rg-, -S-Rh-, or -N(R')-Ri-, wherein Riis H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl and Ra, Rh, and R< are each independently absent or each independently present and selected from optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and PG1is -C(=O)-R13, where R13are each independently optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

[0241] In various embodiments, X8and X29are each methylene (i.e., -CH2-).

[0242] In various embodiments, the galactose and / or a derivative(s) thereof is represented by formula (8’):

[0243]

[0244] In various embodiments, the protected galactose and / or a derivative(s) thereof is represented by general formula (8pl):

[0245]

[0246] wherein PG1is -C(=O)-R13where R13is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;.

[0247] In various embodiments, the reacting in (a-i) comprises adding the acid anhydride represented by general formula (9) in a dropwise manner to the galactose and / or a derivative(s) thereof represented by general formula (8).

[0248] In various embodiments, the addition of the acid anhydride represented by general formula (9) may be carried out at a temperature of from about -20 °C to about 50 °C, from about -15 °C to about 45 °C, from about -10 °C to about 40 °C, from about -5 °C to about 35 °C, from about 0 °C to about 30 °C, from about 5 °C to about 25 °C, from about 10 °C to about 20 °C, from about 0 °C to about 20 °C, from about 1 °C to about 19 °C, from about 2 °C to about 18 °C, from about 3 °C to about 17 °C, from about 4 °C to about 16 °C, from about 5 °C to about 15 °C, from about 6 °C to about 14 °C, from about 7 °C to about 13 °C, from about 8 °C to about 12 °C, from about 9 °C to about 11 °C, or about 10 °C.

[0249] In various embodiments, (a-i) comprises suspending / dispersing / mixing / dissolving / reacting the galactose and / or a derivative(s) thereof represented by general formula (8) with the acid anhydride represented by general formula (9) in a molar ratio of from about 1:1 to about 1:20, from about 1:2 to about 1:20, from about 1:3 to about 1:20, from about 1:4 to about 1:20, from about 1:5 to about 1:20, from about 1:6 to about 1:20, from about 1:7 to about 1:20, from about 1:8 to about 1:20, from about 1:9 to about 1:20, from about 1:10 to about 1:20, from about 1:11 to about 1:20, from about 1: 12 to about 1:20, from about 1: 13 to about 1:20, from about 1: 14 to about 1:20, from about 1:15 to about 1:20, from about 1:16 to about 1:20, from about 1:17 to about 1:20, from about 1:18 to about 1:20, from about 1:19 to about 1:20, or about 1:19.5.

[0250] In various embodiments, the method further comprises:

[0251] (a-ii) reacting the protected galactose and / or a derivative(s) thereof represented by general formula (8P) obtained from (a-i) with a protected compound represented by general formula (10) in the presence of a Lewis acid to obtain a first intermediate compound comprising amide group represented by general formula (11):

[0252]

[0253] (10) O

[0254]

[0255] (11)

[0256] wherein

[0257] X22to X28and X30to X32are each independently selected from -Rmor - OPG1, wherein Rmis H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0258] X8and X29are each alkyl / alkylene;

[0259] M is -O-Rg-, -S-Rh-, or -N(R')-R’-, wherein Riis H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl and Rg, Rh, and Rfare each independently absent or each independently present and selected from optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0260] R7is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;

[0261] A comprises galactose and / or a derivative(s) thereof, and Aprepresents A functionalized / protected with a protecting group; and

[0262] PG2is a protecting group.

[0263] In various embodiments, PG2is a protecting group such as 9-Fluorenylmethoxycarbonyl (Fmoc), 2-(4-Nitrophenylsulfonyl)ethoxycarbonyl (Nsc), 2-Fluoro-Fmoc (Fmoc(2F)), 2-Monoisooctyl-Fmoc (mio-Fmoc), 2,7-Diisooctyl-Fmoc (dio-Fmoc) and N-carboxybenzyl (Cbz), the like, or combinations thereof;

[0264] In various embodiments, the reacting in (a-ii) comprises adding the Lewis acid in a dropwise manner to the protected galactose and / or a derivative(s) thereof obtained from (a-i). In various embodiments, (a-ii) comprises suspending / dispersing / mixing / dissolving / reacting the protected galactose and / or a derivative(s) thereof represented by general formula (8P) obtained from (a-i) with the protected compound represented by general formula (10) 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.

[0265] In various embodiments, the method further comprises:

[0266] (a-iii) deprotecting the first intermediate compound represented by general formula (11) to obtain a second intermediate represented by general formula (12):

[0267]

[0268] (11) (12) wherein

[0269] R7is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;

[0270] A comprises galactose and / or a derivative(s) thereof, and Aprepresents A functionalized / protected with a protecting group; and

[0271] PG2is a protecting group.

[0272] In various embodiments, PG2is a protecting group such as 9-Fluorenylmethoxycarbonyl (Fmoc), 2-(4-Nitrophenylsulfonyl)ethoxycarbonyl (Nsc), 2-Fluoro-Fmoc (Fmoc(2F)), 2-Monoisooctyl-Fmoc (mio-Fmoc), 2,7- Diisooctyl-Fmoc (dio-Fmoc) and N-carboxybenzyl (Cbz), the like, or combinations thereof;

[0273] It will be appreciated that, in various embodiments, the second intermediate represented by general formula (12) comprises -NH2 and -COOH in order for ring cyclization to occur to obtain / make NCA prior to ringopening polymerization.

[0274] In various embodiments, the deprotecting in (a-iii) comprises a reduction process such as hydrogenation.

[0275] In various embodiments, the deprotecting in (a-iii) comprises adding a catalyst (e.g., palladium on carbon (Pd / C)) to the first intermediate compound represented by general formula (11) in the presence of a reducing agent (e g., under a hydrogen (H2) atmosphere).

[0276] In various embodiments, the method further comprises:

[0277] (a-iv) reacting the second intermediate compound represented by general formula (12) with a carbonylating agent (e.g., triphosgene) to obtain the compound comprising N-carboxyanhydride (NCA) represented by general formula (4)

[0278] O

[0279]

[0280] (12) (4)

[0281] wherein R6is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0282] R7is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene; and

[0283] A comprises galactose and / or a derivative(s) thereof, and Aprepresents A functionalized / protected with a protecting group.

[0284] In various embodiments, the reacting in (a-iv) comprises cyclization of the second intermediate compound represented by general formula (12).

[0285] In various embodiments, (a-iv) comprises suspending / dispersing / mixing / dissolving / reacting the second intermediate compound represented by general formula (12) with the carbonylating agent in a molar ratio of from about 1:1 to about 10:1, from about 2:1 to about 10:1, from about 3:1 to about 10:1, from about 4:1 to about 10:1, from about 5:1 to about 10:1, from about 6: 1 to about 10:1, from about 7: 1 to about 10:1, from about 8: 1 to about 10:1, from about 9:1 to about 10:1, about 1:1, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, about 5.5:1, about 6:1, about 6.5:1, about 7:1, about 7.5:1, about 8:1, about 8.5:1, about 9:1, about 9.5:1, or about 10: 1.

[0286] In various embodiments, the reacting / deprotecting in (i), (ii), (iii), (a-i), (a-ii), (a-iii), and / or (a-iv) comprises one or more of the following: suspending, dispersing, mixing, stirring, dissolving, sonicating and / or ultrasonicating.

[0287] In various embodiments, the reacting / deprotecting in (i), (ii), (iii), (a-i), (a-ii), (a-iii), and / or (a-iv) is / are performed in the presence of an organic solvent. The organic solvent may be an organic solvent such as pyridine, dichloromethane (DCM), tetrahydrofuran (THF), methanol (MeOH), or the like or combinations thereof (e.g., 50 % THF / 50 % MeOH (v / v)). In various embodiments, the organic solvent may be provided in a dry or anhydrous form. In various embodiments, the reacting / deprotecting in (i), (ii), (iii), (a-i), (a-ii), (a-iii), and / or (a-iv) is / are carried out under vacuum or in an inert atmosphere. For example, the suspending, dispersing, mixing, stirring, dissolving, sonicating, and / or ultrasonicating may be performed in the presence of an inert gas such as argon or nitrogen.

[0288] In various embodiments, the reacting / deprotecting in (i), (ii), (iii), (a-i), (a-ii), (a-iii), and / or (a-iv) is / are performed over a time duration of from about 20 minutes to about 200 hours, from about 1 hour to about 190 hours, from about 10 hours to about 180 hours, from about 20 hours to about 170 hours, from about 30 hours to about 160 hours, from about 40 hours to about 150 hours, from about 50 hours to about 140 hours, from about 60 hours to about 130 hours, from about 70 hours to about 120 hours, from about 80 hours to about 110 hours, from about 90 hours to about 100 hours, or about 95 hours.

[0289] In various embodiments, the reacting / deprotecting in (i), (ii), (iii), (a-i), (a-ii), (a-iii), and / or (a-iv) is / are optionally performed at a temperature that is from about 0 °C to about 100 °C, from about 5 °C to about 95 °C, from about 10 °C to about 90 °C, from about 15 °C to about 85 °C, from about 20 °C to about 80 °C, from about 25 °C to about 75 °C, from about 30 °C to about 70 °C, from about 35 °C to about 65 °C, from about 40 °C to about 60 °C, from about 45 °C to about 55 °C, about 50 °C, or at room temperature.

[0290] In various embodiments, the method further comprises:

[0291] (b-i) isolating the first intermediate compound after (i);

[0292] (b-ii) isolating the second intermediate compound after (ii);

[0293] (b-iii) isolating the compound represented by general formula (1 ) or ionized form thereof after (iii);

[0294] (b-iv) isolating the protected galactose and / or a derivative(s) thereof after (a-i); (b-v) isolating the first intermediate compound after (a-ii);

[0295] (b-vi) isolating the second intermediate compound after (a-iii); and / or

[0296] (b-vii) isolating the compound comprising NCA after (a-iv). In various embodiments, the isolating comprises one or more of the following: re-dissolving, purifying, centrifuging, washing, precipitating, and / or recrystallizing.

[0297] In various embodiments, the purifying may comprise dialysis or flash silica gel column chromatography.

[0298] In various embodiments, the re-dissolving, purifying, centrifuging, washing, precipitating, and / or recrystallizing is / are performed in the presence of an organic solvent. The organic solvent may be an organic solvent such as ether, ethyl acetate, pyridine, dichloromethane (DCM), tetrahydrofuran (THF), hexane, methanol (MeOH), or the like or combinations thereof (e.g., THF / hexane mixture). In various embodiments, the organic solvent may be provided in a dry or anhydrous form (e g., glacial ether).

[0299] In various embodiments, the washing medium comprises aqueous medium / solutions such as salt solution, deionized water, ice water, or acid. The salt solution may be bicarbonate salts such as sodium bicarbonate (NaHCO3), chloride salts such as saturated sodium chlorine (brine). The acid may be hydrochloric acid. In various embodiments, the salt solution comprises highly concentrated / saturated salt solution.

[0300] In various embodiments, the purifying, centrifuging, recrystallizing, and / or washing is / are repeated at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times with a washing medium. In various embodiments, the purifying, centrifuging, recrystallizing and / or washing is / are usually repeated at least 3 times. Advantageously, in various embodiments, the compound represented by general formula (1) or ionized form thereof may be easily purified. In various embodiments, the method further comprises one or more of the following post reaction: drying optionally under low temperature (e.g., freeze drying), under vacuum, or in an inert atmosphere.

[0301] In various embodiments, the drying is performed in the presence of a drying agent such as anhydrous sodium sulfate, anhydrous magnesium sulfate, anhydrous calcium sulfate, and anhydrous calcium chloride, the like, or combinations thereof.

[0302] In various embodiments, the Lewis acid is selected from boron trifluoride diethyl etherate (BF3·OEt2), boron trifluoride, the like, or combinations thereof.

[0303] Advantageously, in various embodiments, the method is a simple synthesis process that produces high yields of the compound represented by general formula (1) or ionized form thereof. In various embodiments, the yield of the compound represented by general formula (1) or ionized form thereof is from about 50.0% to about 100.0%, from about 51.0% to about 99.0%, from about 52.0% to about 98.0%, from about 53.0% to about 97.0%, from about 54.0% to about 96.0%, from about 55.0% to about 95.0%, from about 60.0% to about 90.0%, from about 65.0% to about 85.0%, from about 70.0% to about 80.0%, or about 75.0%. The yield of the compound represented by general formula (1) or ionized form thereof may be from about 50.0% to about 98.0%.

[0304] COMPOSITION

[0305] Advantageously, in various embodiments, the design of the structure of the compound represented by general formula (1 ) or ionized form thereof allows said compound or ionized form thereof to be used, in lieu or in replacement / substitute of a conventional PEG-ylated lipid (e g., ALC-0159), in the formulation of a composition e.g., for the preparation of nanoparticles. In various embodiments, embodiments of the compound or ionized form thereof are capable of forming or being formulated into a composition that is suitable for preparing nanoparticles. In various embodiments, the composition comprises:

[0306] (i) a compound represented by general formula (1) or ionized form thereof disclosed herein; and

[0307] (ii) a therapeutic, prophylactic, and / or biological agent that is encapsulated / loaded / coupled / bonded / linked / bound in / to said compound or ionized form thereof.

[0308] In various embodiments, the therapeutic agent and / or prophylactic agent and / or biological agent comprises nucleic acid (e.g., RNA, mRNA, siRNA, DNA, pDNA, oligonucleotides such as ASO), therapeutics (e.g., negatively charged therapeutics), drug molecule, vaccine (e.g., dengue vaccine), the like, or combinations thereof.

[0309] In various embodiments, the composition comprises nanoparticles formed from the compound represented by general formula (1) or ionized form thereof.

[0310] In various embodiments, the composition further comprises:

[0311] (a) neutral / helper lipid;

[0312] (b) cholesterol and / or a derivative(s) thereof; and

[0313] (c) ionizable lipid.

[0314] In various embodiments, the composition is substantially devoid of polyethylene glycol (PEG). For example, the composition is substantially devoid of polyethylene glycol (PEG)-modified lipid conjugates.

[0315] In various embodiments, the term “polyethylene glycol (PEG)-modified lipid” is used interchangeably with the terms “PEGylated lipid”, PEG-conjugated lipid”, “P EG-lipid conjugate”, and “lipid modified with PEG”. In various embodiments, the compound or ionized form thereof, neutral / helper lipid, cholesterol and / or a derivative(s) thereof, and ionizable lipid are mixed / dissolved in an organic solvent.

[0316] In various embodiments, the formation of lipid nanoparticle comprises selfassembly of the lipid components and one or more types of molecules or cargoes. In various embodiments, any organic solvent that effectively serves as a medium to contain the lipid components may be used in embodiments of the lipid materials disclosed herein. In various embodiments, the organic solvent is capable of substantially dissolving the components present in the mixture. The organic solvent may comprise ethanol, isopropanol, acetonitrile, ethyl acetate, methanol, tetrahydrofuran, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), the like, or combinations thereof.

[0317] In various embodiments, the ionizable lipid, neutral / helper lipid, cholesterol and / or a derivative(s) thereof, and the compound represented by general formula (1) or ionized form thereof (e.g., LPSG or CPSG) are mixed at a mole ratio of about 20 - 50: about 4 - 20: about 25 - 50: about 0.5 - 20, at a mole ratio of about 24 - 46: about 6 - 18: about 28 - 47: about 1.0 — 15, at a mole ratio of about 28 - 42: about 8 - 16: about 31 - 44: about 1.5 - 10, at a mole ratio of about 30 - 38: about 10 - 14: about 34 - 41: about 2.0 - 5, at a mole ratio of about 32 - 34: about 12 - 13: about 37 - 38: about 2.5 - 3.

[0318] 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.

[0319] In various embodiments, the cholesterol and / or a derivative(s) thereof include, but are not limited to cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, avenasterol, the like, or combinations thereof.

[0320] In various embodiments, the ionizable lipid is selected from ALC-0315, SM-102, Lipid 5, DLinDMA, D-Lin-MC2-DMA, DLin-MC3-DMA, D-Lin-MC4-DMA, Dlin-KC2-DMA, YSK05, AA3-Dlin, SSPalmM, SSPalmO-Phe, Lipid A9, L319, DODMA, CL1, BP Lipid 310, ATX-001, ATX-100, Lipid 2, 80-016B, BP Lipid 309, BP Lipid 307, 93-O17S, 93-O17O, NT1-O14B, 306-O12B-3, 306-O12B, 113-O16B, 3060i10, 306Oi9-cis2, BAMEA-O16B, AI-28, 113-O12B, 98N12-5, Ckk-E12, OF-02, C12-200, BP Lipid 311, BP Lipid 308, BP Lipid 314, BP Lipid 312, LP01, TCL053, Lipid C24, BP Lipid 315, Lipid 29, 9A1P9, C13-112-tri-tail, C13-113-tri-tail, C13-112-tetra-tail, C13-113-tetra-tail, the like, or combinations thereof. It will be appreciated that any suitable ionizable lipid that effectively modulates / adjusts / changes its charge depending on the environmental pH may be used in embodiments of the composition disclosed herein.

[0321] In various embodiments, the composition comprises from 0.5 mol% to about 20.0 mol%, from about 1.0 mol% to about 19.5 mol%, from about 1.5 mol% to about 19.0 mol%, from about 2.0 mol% to about 18.5 mol%, from about 2.5 mol% to about 18.0 mol%, from about 3.0 mol% to about 17.5 mol%, from about 3.5 mol% to about 17.0 mol%, from about 4.0 mol% to about 16.5 mol%, from about 4.5 mol% to about 16.0 mol%, from about 5.0 mol% to about 15.5 mol%, from about 5.5 mol% to about 15.0 mol%, from about 6.0 mol% to about 14.5 mol%, from about 6.5 mol% to about 14.0 mol%, from about 7.0 mol% to about 13.5 mol%, from about 7.5 mol% to about 13.0 mol%, from about 8.0 mol% to about 12.5 mol%, from about 8.5 mol% to about 12.0 mol%, from about 9.0 mol% to about 11.5 mol%, from about 9.5 mol% to about 11.0 mol%, from about 10.0 mol% to about 10.5 mol%, from about 0.5 mol% to about 5.0 mol%, from about 0.6 mol% to about 4.9 mol%, from about 0.7 mol% to about 4.8 mol%, from about 0.8 mol% to about 4.7 mol%, from about 0.9 mol% to about 4.6 mol%, from about 1.0 mol% to about 4.5 mol%, from about 1.1 mol% to about 4.4 mol%, from about 1.2 mol% to about 4.3 mol%, from about 1.3 mol% to about 4.2 mol%, from about 1.4 mol% to about 4.1 mol%, from about 1.5 mol% to about 4.0 mol%, from about 1.6 mol% to about 3.9 mol%, from about 1.7 mol% to about 3.8 mol%, from about 1.8 mol% to about 3.7 mol%, from about 1.9 mol% to about 3.6 mol%, from about 2.0 mol% to about 3.5 mol%, from about 2.1 mol% to about 3.4 mol%, from about 2.2 mol% to about 3.3 mol%, from about 2.3 mol% to about 3.2 mol%, from about 2.4 mol% to about 3.1 mol%, from about 2.5 mol% to about 3.0 mol%, from about 2.6 mol% to about 2.9 mol%, from about 2.7 mol% to about 2.8 mol%, or about 2.75 mol% of compound represented by general formula (1) or ionized form thereof.

[0322] In various embodiments, the compound represented by general formula (1) or ionized form thereof is the major component of the composition and is present in an amount of about 0.5 mol%, about 0.6 mol%, about 0.7 mol%, about 0.8 mol%, about 0.9 mol%, about 1.0 mol%, about 1.1 mol%, about 1.2 mol%, about 1.3 mol%, about 1.4 mol%, about 1.5%, about 1.6 mol%, about 1.7 mol%, about 1.8 mol%, about 1.9 mol%, about 2.0 mol%, about 2.1 mol%, about 2.2 mol%, about 2.3 mol%, about 2.4 mol%, about 2.5 mol%, about 2.6 mol%, about 2.7 mol%, about 2.8 mol%, about 2.9 mol%, about 3.0 mol%, about 3.1 mol%, about 3.2 mol%, about 3.3 mol%, about 3.4 mol%, about 3.5 mol%, about 3.6 mol%, about 3.7 mol%, about 3.8 mol%, about 3.9 mol%, about 4.0 mol%, about 4.1 mol%, about 4.2 mol%, about 4.3 mol%, about 4.4 mol%, about 4.5 mol%, about 4.6 mol%, about 4.7 mol%, about 4.8 mol%, about 4.9 mol%, or about 5.0 mol% of the composition.

[0323] In various embodiments, the composition comprises from about 1.0 mol% to about 20.0 mol% of neutral / helper lipid.

[0324] In various embodiments, the neutral / helper lipid is present in an amount of from about 1.0 mol% to about 20.0 mol%, from about 1.5 mol% to about 19.5 mol%, from about 2.0 mol% to about 19.0 mol%, from about 2.5 mol% to about 18.5 mol%, from about 3.0 mol% to about 18.0 mol%, from about 3.5 mol% to about 17.5 mol%, from about 4.0 mol% to about 17.0 mol%, from about 4.5 mol% to about 16.5 mol%, from about 5.0 mol% to about 16.0 mol%, from about 5.5 mol% to about 15.5 mol%, from about 6.0 mol% to about 15.0 mol%, from about 6.5 mol% to about 14.5 mol%, from about 7.0 mol% to about 14.0 mol%, from about 7.5 mol% to about 13.5 mol%, from about 8.0 mol% to about 13.0 mol%, from about 8.5 mol% to about 12.5 mol%, from about 9.0 mol% to about 12.0 mol%, from about 9.5 mol% to about 11.5 mol%, from about 10.0 mol% to about 11.0 mol%, about 10.5 of neutral / helper lipid. In various embodiments, the neutral / helper lipid is present in an amount of about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, about 15 mol%, %, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, or about 20 mol% of the composition.

[0325] In various embodiments, the composition comprises from about 25.0 mol% to about 60.0 mol% of cholesterol and / or a derivative(s) thereof.

[0326] In various embodiments, the cholesterol and / or a derivative(s) thereof is / are present in an amount of about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol%, about 56 mol%, about 57 mol%, about 58 mol%, about 59 mol%, about 60 mol% of the composition.

[0327] In various embodiments, the composition comprises from about 20.0 mol% to about 60.0 mol% of ionizable lipid.

[0328] In various embodiments, the ionizable lipid is present in an amount of about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol%, about 56 mol%, about 57 mol%, about 58 mol%, about 59 mol%, about 60 mol% of the composition.

[0329] In various embodiments, the composition is a nanoparticle composition for delivery of a therapeutic, prophylactic, and / or biological agent. In various embodiments, the nanoparticle composition comprises nanoparticles formed from the compound represented by general formula (1) or ionized form thereof, or is in the form of nanoparticles disclosed herein.

[0330] 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 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.

[0331] NANOPARTICLES

[0332] 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.

[0333] In various embodiments, there is provided nanoparticles (e.g., lipid nanoparticle) comprising:

[0334] (i) the compound represented by general formula (1) or ionized form thereof disclosed herein; and

[0335] (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 disclosed herein.

[0336] In various embodiments, the therapeutic agent and / or prophylactic agent and / or biological agent comprises nucleic acid (e.g., RNA, mRNA, siRNA, DNA, pDNA, oligonucleotides such as ASO), therapeutics (e.g., negatively charged therapeutics), drug molecule, vaccine (e.g., dengue vaccine), the like, or combinations thereof.

[0337] In various embodiments, the therapeutic agent, prophylactic agent, and / or biological agent is provided in an aqueous buffer. The aqueous buffer may be sodium acetate.

[0338] In various embodiments, the nanoparticle has a N: P or N / P ratio (i.e., molar ratio of ionizable nitrogen atoms in a nanoparticle / compound or ionized form thereof to phosphate groups in the therapeutic and / or prophylactic agent and / or biological agent (e.g., nucleic acid)) is from about 1:1 to about 20:1. It will be appreciated that in various embodiments, the optimal N / P ratio is dependent on the type of therapeutic and / or prophylactic agent and / or biological agent (e.g., a nucleic acid such as mRNA, siRNA, pDNA and oligonucleotides). For example, the optimal N / P ratio may be different for siRNA, pDNA and oligonucleotides. In various embodiments, it will be appreciated that shorter nucleic acid therapeutics (e.g., siRNA) or prophylactic agents (e.g., mRNA) require more (i.e., a larger amount / concentration / volume of) ionizable lipids to encapsulate them into lipid nanoparticles. In various embodiments therefore, a N / P ratio of up to about 20:1 is used to encapsulate and deliver nucleic acid therapeutics (e.g., shorter nucleic acid therapeutics siRNA).

[0339] In various embodiments, the concentration of the therapeutic and / or prophylactic agent and / or biological agent in the composition / nanoparticle is from about 10 pg / mL to about 200 pg / mL, from about 20 pg / mL to about 190 pg / mL, from about 30 pg / mL to about 180 pg / mL, from about 40 pg / mL to about 170 pg / mL, from about 50 pg / mL to about 165 pg / mL, from about 60 pg / mL to about 160 pg / mL, from about 70 pg / mL to about 155 pg / mL, from about 80 pg / mL to about 150 pg / 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.

[0340] In various embodiments, the total lipid concentration (i.e., the compound represented by general formula (1) or ionized forms thereof, neutral / helper lipid, cholesterol and / or a derivative(s) thereof, and ionizable lipid) in the composition / nanoparticle is from about 1 mg / mL to about 5 mg / mL, from about 1.2 mg / mL to about 4.8 mg / mL, from about 1.4 mg / mL to about 4.6 mg / mL, from about 1.6 mg / mL to about 4.4 mg / mL, from about 1.8 mg / mL to about 4.2 mg / mL, from about 2.0 mg / mL to about 4.0 mg / mL, from about 2.2 mg / mL to about 3.8 mg / mL, from about 2.4 mg / mL to about 3.6 mg / mL, from about 2.6 mg / mL to about 3.4 mg / mL, from about 2.8 mg / mL to about 3.2 mg / mL, or about 3.0 mg / mL.

[0341] In various embodiments, the nanoparticle is substantially devoid of polyethylene glycol (PEG). For example, the nanoparticle is substantially devoid of polyethylene glycol (PEG)-modified lipid conjugates.

[0342] In various embodiments, the term “polyethylene glycol (PEG)-modified lipid” is used interchangeably with the terms “PEGylated lipid”, PEG-conjugated lipid”, “P EG-lipid conjugate”, and “lipid modified with PEG”.

[0343] In various embodiments, the nanoparticle has an average particle size (or diameter) in the nano meter range. In various embodiments, the nanoparticles have an average or mean particle size (or diameter) of no more than about 800 nm, no more than about 750 nm, no more than about 700 nm, no more than about 650 nm, no more than about 600 nm, no more than about 550 nm, no more than about 500 nm, no more than about 450 nm, no more than about 400 nm, no more than about 350 nm, no more than about 300 nm, no more than about 250 nm, no more than about 200 nm, no more than about 150 nm, no more about 100 nm, no more than about 90 nm, no more than about 80 nm, no more than about 70 nm, no more than about 60 nm, no more than about 50 nm, no more than about 40 nm, no more than about 30 nm, no more than about 20 nm, or no more than about 10 nm. In various embodiments, the nanoparticles have an average or mean particle size (or diameter) of from about 40.0 nm to about 500.0 nm, from about 50.0 nm to about 490.0 nm, from about 60.0 nm to about 480.0 nm, from about 70.0 nm to about 470.0 nm, from about 80.0 nm to about 460.0 nm, from about 90.0 nm to about 450.0 nm, from about 100.0 nm to about 440.0 nm, from about 110.0 nm to about 430.0 nm, from about 120.0 nm to about 420.0 nm, from about 130.0 nm to about 410.0 nm, from about 140.0 nm to about 400.0 nm, from about 150.0 nm to about 390.0 nm, from about 160.0 nm to about 380.0 nm, from about 170.0 nm to about 370.0 nm, from about 180.0 nm to about 360.0 nm, from about 190.0 nm to about 350.0 nm, from about 200.0 nm to about 340.0 nm, from about 210.0 nm to about 330.0 nm, from about 220.0 nm to about 320.0 nm, from about 230.0 nm to about 310.0 nm, from about 240.0 nm to about 300.0 nm, from about 250.0 nm to about 290.0 nm, from about 260.0 nm to about 280.0 nm, about 270.0 nm, about 200.0 nm, about 250.0 nm, about 300.0 nm, about 350.0 nm, about 400.0 nm, or about 450.0 nm.

[0344] In various embodiments, the nanoparticle has a nanosize (< 200 nm), making the nanoparticle suitable / desirable for in vivo applications.

[0345] In various embodiments, the nanoparticle has an average particle size (diameter) in the micron meter range (e.g., for nasal spray applications). For example, the average particle size (diameter) may be from about 0.10 pm to about 10.0 pm, from about 0.20 pm to about 9.5 pm, from about 0.30 pm to about 9.0 pm, from about 0.40 pm to about 8.5 pm, from about 0.50 pm to about 8.0 pm, from about 0.60 pm to about 7.5 pm, from about 0.70 pm to about 7.0 pm, from about 0.80 pm to about 6.5 pm, from about 0.90 pm to about 6.0 pm, from about 1.00 pm to about 5.5 pm, from about 1.50 pm to about 5.0 pm, from about 2.00 pm to about 4.5 pm, from about 2.50 pm to about 4.0 pm, from about 3.00 pm to about 3.5 pm,.

[0346] In various embodiments, the nanoparticle has a polydispersity index (PDI) of from about 0.001 to about 0.500, from about 0.010 to about 0.450, from about 0.020 to about 0.400, from about 0.030 to about 0.350, from about 0.040 to about 0.300, from about 0.050 to about 0.250, from about 0.060 to about 0.200, from about 0.070 to about 0.150, from about 0.080 to about 0.100, or about 0.090.

[0347] In various embodiments, the nanoparticle has a narrow particle size distribution (PDI < 0.3), and / or the nanoparticle composition is relatively / substantially homogenous, making the nanoparticle suitable / desirable for in vivo applications. In various embodiments, the nanoparticle has a zeta potential of from about -20 mV to about 20 mV, from about -19 mV to about 19 mV, from about -18 mV to about 18 mV, from about -17 mV to about 17 mV, from about -16 mV to about 16 mV, from about -15 mV to about 15 mV, from about -14 mV to about 14 mV, from about -13 mV to about 13 mV, from about -12 mV to about 12 mV, from about -11 mV to about 11 mV, from about -10 mV to about 10 mV, from about -9 mV to about 9 mV, from about -8 mV to about 8 mV, from about -7 mV to about 7 mV, from about -6 mV to about 6 mV, from about -5 mV to about 5 mV, from about -4 mV to about 4 mV, from about -3 mV to about 3 mV, from about -2 mV to about 2 mV, from about -1 mV to about 1 mV, or about 0 mV. In various embodiments, a zeta potential of about ±10 mV is desirable for in vivo applications.

[0348] Advantageously, in various embodiments, the nanoparticle has a substantially neutral surface charge (< ±10 mV), making the nanoparticle suitable / desirable for in vivo applications.

[0349] 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 5.0%, at least about 10.0%, at least about 15.0%, at least about 20.0%, at least about 25.0%, at least about 30.0%, at least about 35.0%, at least about 40.0%, at least about 45.0%, at least about 50.0%, at least about 55.0%, at least about 60.0%, at least about 65.0%, at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 90.0%, at least about 95.0%, at least about 96.0%, at least about 97.0%, at least about 98.0%, at least about 99.0%, at least about 99.5%, at least about 99.9%, or about 100%.

[0350] In various embodiments, the nanoparticle has an encapsulation efficiency that is slightly lower, comparable to, no less or is higher than that of a corresponding nanoparticle using ALC-0159 PEG-modified lipid under similar conditions. For example, the encapsulation efficiency may be at least about 0.1 % to at least about 15.0 %, at least about 1.0 % to at least about 15.0 %, at least about 2.0 % to at least about 14.0 %, at least about 3.0 % to at least about 13.0 %, at least about 4.0 % to at least about 12.0 %, at least about 5.0 % to at least about 11.0 %, at least about 6.0 % to at least about 10.0 %, at least about 7.0 % to at least about 9.0 % higher than that of a corresponding nanoparticle using ALC-0159 PEG-modified lipid under similar conditions.

[0351] In various embodiments, the cell / nucleic acid transfection efficiency (% of the cells / nucleic acids that are transfected with the gene) of the composition / nanoparticles is at least about 1.0%, at least about 5.0%, at least about 10.0%, at least about 15.0%, at least about 20.0%, at least about 25.0%, at least about 30.0%, at least about 35.0%, at least about 40.0%, at least about 45.0%, at least about 50.0%, at least about 55.0%, at least about 60.0%, at least about 65.0%, at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 90.0%, at least about 95.0%, at least about 96.0%, at least about 97.0%, at least about 98.0%, at least about 99.0%, at least about 99.5%, at least about 99.9%, or about 100%. In various embodiments, the cell transfection efficiency is not required to be 100%. For example, it will be appreciated that vaccine applications may not need / require to transfect 100% cells in order to mediate an immune response, unlike in the case of cancer therapy applications.

[0352] In various embodiments, the cell transfection efficiency of the nanoparticle may vary based on the molar concentration of the compound represented by general formula (1) or ionized form thereof, which may influence the balance between hydrophobicity and hydrophilicity.

[0353] In various embodiments, the nucleic acid transfection efficiency of the therapeutic and / or prophylactic agent and / or biological agent in the composition / nanoparticle is greater than those using PEG-conjugated lipids (e.g., ALC-0159). In various embodiments, the nanoparticle has a nucleic acid transfection efficiency that is no less or higher than that of a corresponding nanoparticle using ALC-0159 PEG-modified lipid under similar conditions. For example, the mRNA transfection efficiency may be 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 about 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 55 times, at least 60 times, at least 65 times, at least 70 times, at least 75 times, at least 80 times, at least 85 times, at least 90 times, at least 95 times, or at least 100 times higher than that of a corresponding nanoparticle using ALC-0159 PEG-modified 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-0159 PEG-modified lipid.

[0354] 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 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.

[0355] METHOD OF PREPARING NANOPARTICLES

[0356] There is provided a method of preparing a nanoparticle disclosed herein, the method comprising:

[0357] (c-i) preparing an aqueous composition comprising therapeutic and / or prophylactic agent and / or biological agent;

[0358] (c-ii) mixing the aqueous composition with a composition comprising the compound or ionized form thereof disclosed herein to obtain a nanoparticle. In various embodiments, (c-i) comprises mixing therapeutic and / or prophylactic agent and / or biological agent in an aqueous buffer. The aqueous buffer may be sodium acetate, citrate buffer, phosphate buffer, glycine buffer solution, the like or combinations thereof.

[0359] In various embodiments, the mixing in (c-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, about 4.5, or about 4.0.

[0360] In various embodiments, the pH value at which the mixing in (c-i) is performed may vary depending on the pKa of the ionizable lipids.

[0361] In various embodiments, when the mixing in (c-i) is performed at a pH value as described above, the tertiary amine group(s) in the ionizable lipid are protonated to carry a positive charge, which condenses the therapeutic, prophylactic, and / or biological agent (e g., mRNA) into the nanoparticle through electrostatic interaction.

[0362] In various embodiments, at physiological pH (e.g., pH 7.4), the tertiary amine group(s) in the ionizable lipid are deprotonated, thereby minimizing their toxicity.

[0363] In various embodiments, (c-ii) comprises mixing the aqueous composition with a composition comprising the compound or ionized form thereof disclosed herein at a volume ratio of from about 10:1 to about 1:1, at about 9:1, at about 8:1, at about 7:1, at about 6:1, at about 5:1, at about 4:1, at about 3:1, or at about 2:1.

[0364] In various embodiments, the mixing in (c-ii) the aqueous composition with the composition comprises rapid pipetting or a microfluidic device.

[0365] In various embodiments, there is also provided a carrier, nanocarrier, or delivery system / vehicle comprising the composition / compound or ionized form thereof / nanoparticles as disclosed herein.

[0366] In various embodiments, there is also provided therapeutic / pharmaceutical composition comprising the composition / compound or ionized form thereof / nanoparticle as disclosed herein.

[0367] In various embodiments, there is also provided a vaccine composition comprising the composition / compound or ionized form thereof / nanoparticles as disclosed herein.

[0368] 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).

[0369] 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 administering step comprises delivering / administering the nanoparticle as disclosed herein to a liver cell or hepatocyte. 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 (e.g., cancer), dermatological / skin diseases, ophthalmological diseases, fibrotic diseases, cardiovascular diseases, liver diseases (e.g., hepatitis), autoimmune diseases / disorders, 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. In various embodiments, the disease, disorder, or condition is mediated by a dengue virus (e.g., DEN-1, DEN-2, DEN-3, and / or DEN-4 virus). For example, the disease may be dengue disease.

[0370] 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 (e.g., hepatocyte), cytosol, tissue (e.g., liver tissue), or organ (e.g., liver)), 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 (e g., hepatocyte), cytosol, tissue (e.g., liver tissue), or organ (e.g., liver)), 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 (e.g., hepatocyte), cytosol, tissue (e.g., liver tissue), or organ (e.g., liver)), 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 administering step comprises delivering / administering the nanoparticle as disclosed herein to a liver cell or hepatocyte. 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). 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.

[0371] 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, the administering step comprises delivering / administering the nanoparticle as disclosed herein to a liver cell or hepatocyte. 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, 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 (e.g., cancer), dermatological / skin diseases, ophthalmological diseases, fibrotic diseases, cardiovascular diseases, liver diseases (e.g., hepatitis), autoimmune diseases / disorders, the like, or combinations thereof as mentioned herein. 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. In various embodiments, the disease, disorder, or condition is mediated by a dengue virus (e.g., DEN-1, DEN-2, DEN-3, and / or DEN-4 virus). For example, the disease may be dengue disease. In various embodiments, 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. 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.

[0372] 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.

[0373] BRIEF DESCRIPTION OF FIGURES

[0374] FIG. 1 shows the1H NMR spectrum of serine(Ser)(1,2,3,4, 6-penta-O-acetyl-D-galactopyranose)-N-carboxy anhydride (NCA), synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in deuterated chloroform (CDCl3) as the solvent.

[0375] FIG. 2 shows the13C NMR spectrum of Ser(1,2,3,4, 6-penta-O-acetyl-D-galactopyranose)-NCA, synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in CDCl3as the solvent.

[0376] FIG. 3 shows the1H NMR spectrum of lipid--poly(Ser-D-galactose) with a degree of polymerization (DP) of 12 (i.e., LPSG12), synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in DMSO-d6as the solvent.

[0377] FIG. 4 shows the1H NMR spectrum of lipid--poly(Ser-D-galactose) with a DP of 15 (i.e., LPSG15), synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in DMSO-d6as the solvent.

[0378] FIG. 5 shows the1H NMR spectrum of lipid--poly(Ser-D-galactose) with a DP of 20 (i.e., LPSG20), synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in DMSO-d6as the solvent.

[0379] FIG. 6 shows the1H NMR spectrum of cholesterol--poly(Ser-D-galactose) with a DP of 15 (i.e., CPSG15), synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in DMSO-d6as the solvent.

[0380] FIG. 7 shows the viability of HepG2 cells (A) as well as the transfection efficiency measured by relative luciferase expression (B) after 48 hours of incubation with mRNA LNPs formulated using LPSG12, LPSG15, LPSG20, or CPSG15 at a molar ratio of 1.0 %, prepared in accordance with various embodiments disclosed herein, in comparison with ALC-0159 LNPs. Statistical significance for transfection efficiency was determined using Welch’s t test, with comparisons made to ALC-0159 LNPs (** p < 0.01, **** p < 0.0001 ).

[0381] FIG. 8 shows the viability of HepG2 cells (A) as well as the transfection efficiency measured by relative luciferase expression (B) after 48 hours of incubation with mRNA LNPs formulated using LPSG12, LPSG15, LPSG20, or CPSG15 at a molar ratio of 1.2 %, prepared in accordance with various embodiments disclosed herein, in comparison with ALC-0159 LNPs. Statistical significance for transfection efficiency was determined using Welch’s t test, with comparisons made to ALC-0159 LNPs (*p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

[0382] FIG. 9 shows the viability of HepG2 cells (A) as well as the transfection efficiency measured by relative luciferase expression (B) after 48 hours of incubation with mRNA LNPs formulated using LPSG12, LPSG15, LPSG20, or CPSG15 at a molar ratio of 1.4 %, prepared in accordance with various embodiments disclosed herein, in comparison with ALC-0159 LNPs. Statistical significance for transfection efficiency was determined using Welch’s t test, with comparisons made to ALC-0159 LNPs (** p < 0.01, *** p < 0.001 ).

[0383] FIG. 10 shows the viability of HepG2 cells (A) as well as the transfection efficiency measured by relative luciferase expression (B) after 48 hours of incubation with mRNA LNPs formulated using LPSG12, LPSG15, LPSG20, or CPSG15 at a molar ratio of 1.6 %, prepared in accordance with various embodiments disclosed herein, in comparison with ALC-0159 LNPs. Statistical significance for transfection efficiency was determined using Welch’s t test, with comparisons made to ALC-0159 LNPs (* p < 0.05, *** p < 0.001 ).

[0384] FIG. 11 shows the viability of DC2.4 cells (A) as well as the transfection efficiency measured by relative luciferase expression (B) after 48 hours of incubation with mRNA LNPs formulated using LPSG12, LPSG15, LPSG20, or CPSG15 at a molar ratio of 1.0 %, prepared in accordance with various embodiments disclosed herein, in comparison with ALC-0159 LNPs. Statistical significance for transfection efficiency was determined using Welch’s t test, with comparisons made to ALC-0159 LNPs (** p < 0.01, *** p < 0.001 ). FIG. 12 shows the viability of DC2.4 cells (A) as well as the transfection efficiency measured by relative luciferase expression (B) after 48 hours of incubation with mRNA LNPs formulated using LPSG12, LPSG15, LPSG20, or CPSG15 at a molar ratio of 1.2 %, prepared in accordance with various embodiments disclosed herein, in comparison with ALC-0159 LNPs. Statistical significance for transfection efficiency was determined using Welch’s t test, with comparisons made to ALC-0159 LNPs (** p < 0.01, *** p < 0.001 ).

[0385] FIG. 13 shows the viability of DC2.4 cells (A) as well as the transfection efficiency measured by relative luciferase expression (B) after 48 hours of incubation with mRNA LNPs formulated using LPSG12, LPSG15, LPSG20, or CPSG15 at a molar ratio of 1.4 %, prepared in accordance with various embodiments disclosed herein, in comparison with ALC-0159 LNPs. Statistical significance for transfection efficiency was determined using Welch’s t test, with comparisons made to ALC-0159 LNPs (** p < 0.01, **** p < 0.0001 ).

[0386] FIG. 14 shows the viability of DC2.4 cells (A) as well as the transfection efficiency measured by relative luciferase expression (B) after 48 hours of incubation with mRNA LNPs formulated using LPSG12, LPSG15, LPSG20, or CPSG15 at a molar ratio of 1.6 %, prepared in accordance with various embodiments disclosed herein, in comparison with ALC-0159 LNPs. Statistical significance for transfection efficiency was determined using Welch’s t test, with comparisons made to ALC-0159 LNPs (* p < 0.05, ** p < 0.01 ).

[0387] EXAMPLES

[0388] 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.

[0389] The following examples describe the successful syntheses of a series of novel PEG-free LNPs based on galactosylated polypeptide-based lipids, such as lipid--poly(Ser-D-galactose) (LPSG) and cholesterol-b / ock-poly(Ser-D-galactose) (CPSG), as alternatives to PEGylated lipids. Advantageously, mRNA LNPs formulated from LPSG or CPSG were found to exhibit nanosize (< 200 nm), a narrow and homogeneous particle population / distribution (PDI < 0.2), and a neutral surface (zeta potential: < ± 10 mV), making them ideal for in vivo applications. Notably, the mRNA LNPs formed from LPSG, and especially CPSG, demonstrated significantly greater mRNA transfection efficiency than the mRNA LNPs made from ALC-0159 that is used in Pfizer-BioNTech’s mRNA LNP Covid-19 vaccine in HepG2 cells, with even greater improvement observed in DC2.4 cells. Furthermore, all mRNA LNP formulations tested showed negligible cytotoxicity. These results suggest that galactosylated polypeptide-based lipids are promising as viable replacements for ALC-0159 in the delivery of mRNA and other genes to hepatocytes, potentially reducing the risk of immunogenicity caused by PEGylated lipids while enhancing delivery effectiveness.

[0390] Example 1: Materials

[0391] Chemical reagents for the synthesis of the PEG-free lipids were purchased from Sigma-Aldrich and used as received unless otherwise noted. 1,2-Distearoyl-sn-glycerol-3-phosphocholine (DSPC), cholesterol and ALC-0315 were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Sodium acetate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Triton®-X100, Tris-EDTA, ONE-Glo™ Luciferase Assay System, and VivoGlo Luciferin (In Vivo Grade) were purchased from Promega (Madison, Wl, USA). Alamar Blue was purchased from Invitrogen (Waltham, MA, USA). Other reagents used were of analytical grade. Example 2: Methods

[0392] 2.1. Synthesis of serine (Ser)(1,2,3,4,6-penta-O-acetyl-D-galactopyranose) N-carboxy anhydride (NCA)

[0393] Synthesis strategy for serine(Ser)(1,2,3,4, 6-penta-O-acetyl-D-galactopyranose) N-carboxy anhydride (NCA) is shown in Scheme 1. 1, 2, 3,4,6-penta-O-acetyl-D-galactopyranose was first synthesized. D-galactose (30.6 g, 170 mmol) was dissolved in 150 mL of pyridine under N2. Acetic anhydride (160 mL, 1.7 mol) was added dropwise to the solution at 0 °C. The reaction mixture was stirred for 24 h at room temperature. The reaction solution was slowly poured into 1000 mL of ice water and then extracted with 300 mL of ethyl acetate for 3 times. The organic phase was washed with 300 mL of saturated NaHCO3for 2 times, and then washed with 1.0 M HCI, saturated brine. The organic phase was dried over anhydrous Na2SO4and the organic solvent was evaporated in vacuo. The resulting crude product was purified by flash silica gel column chromatography (hexane / ethyl acetate 8:2, v / v). The yield of the compound was 92.0%.

[0394] Carbobenzyloxy-Ser(1,2,3,4,6-penta-O-acetyl-D-galactopyranose) was then synthesized. 26.5 mL of BF3·OEt2was added dropwise to a mixture of Cbz-Serine (6.57 g, 27.5 mmol) and 1,2,3,4,6-penta-O-acetyl-D-galactopyranose (10.8 g, 27.5 mmol) in 250 mL of CH2CI2 (DCM), and the reaction mixture was stirred under N2 for 24 h at room temperature. The solvent was evaporated in vacuo. The resulting residue was dissolved in 400 mL of ethyl acetate, washed with saturated brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The obtained crude product was purified by flash silica gel column chromatography (DCM / MeOH 30:1, v / v) to give Cbz-Ser(1,2,3,4, 6-penta-O-acetyl-D-galactopyranose) (10.2 g, 64.1%).

[0395] The Cbz group deprotection of Cbz-Ser(1,2,3,4, 6-penta-O-acetyl-D-galactopyranose) was then performed to yield Ser(1,2,3,4, 6-penta-O-acetyl-D- galactopyranose). General procedure for Ser(1,2,3,4, 6-penta-O-acetyl-D-galactopyranose): Cbz-Ser(1,2,3,4,6-penta-O-acetyl-D-galactopyranose) (10.0 g, 17.5 mmol) was dissolved in 50% THF / 50% MeOH (100 mL, v / v), followed by addition of 1.0 g of Pd / C, and the mixture was stirred for 12 h at room temperature under H2 atmosphere. After the reaction, Pd / C was removed by centrifugation. The solvent was evaporated in vacuo. The resulting crude product was purified by flash silica gel column chromatography (DCM / MeOH 10:1, v / v) to give Ser(1,2,3,4,6-penta-O-acetyl-D-galactopyranose) (6.0 g, 78.8%).

[0396] Ser(1,2,3,4, 6-penta-O-acetyl-D-galactopyranose)-NCA was finally synthesized. General procedure for synthesis of Ser(1, 2,3,4, 6-penta-O-acetyl-D-galactopyranose)-NCA: Ser(1,2,3,4,6-penta-O-acetyl-D-galactopyranose) (4.35 g, 10 mmol) was suspended in 60 mL of dry tetrahydrofuran (THF) and then triphosgene (1.4 g) was added under N2. The mixture was stirred at 50 °C under a flow of N2 for 3 h. After the reaction mixture was cooled down to room temperature, the crude product was precipitated by pouring the mixture solution into iced hexane (250 ml), collected by filtration. The resulting crude product was purified by recrystallizing with THF / hexane mixture for three times. The yield of Ser(1,2,3,4,6-penta-O-acetyl-D-galactopyranose)-NCA was 70%. The structure of Ser(1,2,3,4,6-penta-O-acetyl-D-galactopyranose)-NCA was verified by1H NMR and13C NMR spectra (FIGs. 1 and 2).

[0397]

[0398] Scheme 1. Synthesis strategy for Ser(1,2,3,4, 6-penta-O-acetyl-D-galactopyranose) N-carboxy anhydride

[0399] (NCA). 2.2. Synthesis of lipid-d / oc -polyfSer-D-galactose)

[0400] Synthesis strategy for lipid--poly(Ser-D-galactose) is shown in Scheme 2. Lipid-b / oc -poly(Ser-1,2,3,4,6-penta-O-acetyl-D-galactopyranose) was first synthesized. General synthetic method for lipid--poly(Ser-1,2,3,4,6-penta-O-acetyl-D-galactopyranose): In the glove box, 2-amino-N, N-ditetradecylacetamide (23.3 mg, 0.05 mmol) and Ser(1, 2,3,4, 6-penta-O-acetyl-D-galactopyranose)-NCA (922 mg, 2.0 mmol) were dissolved in 15 mL of anhydrous DCM. The mixture was stirred for 48 h at room temperature in the glove box. Then, 1.0 mL of acetic anhydride was added and the reaction was continued for 2 h. The crude product was precipitated by pouring the mixture solution into glacial ether (300 mL), collected by centrifugation. The resulting crude product was purified by dissolving with DCM and precipitated by pouring the solution into glacial ether. The resulting product was dried under vacuum. The number of polymerization unit for lipid--poly(Ser-1,2,3,4,6-penta-O-acetyl-D-galactopyranose) can be adjusted by varying the amount of Ser(1, 2, 3,4,6-penta-O-acetyl-D-galactopyranose)-NCA. The yield of the protected lipidpolypeptide was 70%.

[0401] The deprotection of lipid--poly(Ser-1,2,3,4,6-penta-O-acetyl-D-galactopyranose) was then performed to yield lipid--poly(Ser-D-galactose) (LPSG). General procedure for synthesis of lipid--poly(Ser-D-galactose): 600 mg of lipid-t> / oc / c-poly(Ser-1,2,3,4,6-penta-O-acetyl-D-galactopyranose) was dissolved in 15 mL of MeOH, to which was added 1.5 mL of 25% MeONa in MeOH. The mixture was stirred at room temperature for 15 mins. The crude product was purified by dialysis with deionized water for removing MeONa and MeOH. The product was obtained by freeze-drying under vacuum. The yield of the final product lipid--poly(Ser-D-galactose) was 98%. The typical structure of lipid--poly(Ser-D-galactose) was verified by1H NMR (FIGs. 3 to 5).

[0402]

[0403] Scheme 2. Synthesis strategy for lipid-b / oc / <-poly(Ser-D-galactose). 2.3. Synthesis of cholesterol-b / oc / r-poly(Ser-D-galactose)

[0404] Synthesis strategy for cholesterol-b / oc -poly(Ser-D-galactose) is shown in Scheme 3. Cholesterol-b / oc -poly(Ser-1,2,3,4,6-penta-O-acetyl-D-galactopyranose) was first synthesized. General synthetic method for cholesterol-block-poly(Ser-1,2,3,4,6-penta-O-acetyl-D-galactopyranose): In the glove box, cholesterol-NH2 (23.6 mg, 0.05 mmol) and Ser(1,2,3,4, 6-penta-O-acetyl-D-galactopyranose)-NCA (922 mg, 2.0 mmol) were dissolved in 15 mL of anhydrous DCM. The mixture was stirred for 48 h at room temperature in the glove box. Then, 1.0 mL of acetic anhydride was added and the reaction was continued for 2 h. The crude product was precipitated by pouring the mixture solution into glacial ether (300 mL), collected by centrifugation. The resulting crude product was purified by dissolving with DCM and precipitated by pouring the solution into glacial ether. The resulting product was dried under vacuum. The number of polymerization unit for cholesterol-block-poly(Ser-1, 2,3,4, 6-penta-O-acetyl-D-galactopyranose) can be adjusted by varying the amount of Ser(1,2,3,4,6-penta-O-acetyl-D-galactopyranose)-NCA. The yield of the protected cholesterol-polypeptide was 72%.

[0405] The deprotection of cholesterol-b / oc -poly(Ser-1,2,3,4,6-penta-O-acetyl-D-galactopyranose) was then performed to yield cholesterol-b / oc -poly(Ser-D-galactose) (CPSG). General procedure for cholesterol-b / ock-poly(Ser-D-galactose): 600 mg of cholesterol-b / oc -poly(Ser-1, 2,3,4, 6-penta-O-acetyl-D-galactopyranose) was dissolved in 15 mL of MeOH, to which was added 1.5 mL of 25% MeONa in MeOH. The mixture was stirred at room temperature for 15 min. The crude product was purified by dialysis with deionized water for removing MeONa and MeOH. The product was obtained by freeze-drying under vacuum. The yield of the final product cholesterol-b / oc -poly(Ser-D-galactose) was 96%. The typical structure of cholesterol-b / oc -poly(Ser-D-galactose) was verified by1H NMR (FIG. 6).

[0406]

[0407] Scheme 3. Synthesis strategy for cholesterol-d / oc / r-poly(Ser-D-galactose). 2.4. Formulation of mRNA-loaded lipid nanoparticles (mRNA LNPs)

[0408] To conduct a high-throughput screening of various hepatocyte-targeting lipids at different mole ratios, LNPs were manually formulated using LPSG and CPSG according to Tables 1 and 2, respectively, while Table 3 indicates the structure and molecular weights of the hepatocyte-targeting compounds. Furthermore, the N / P ratio between the ionizable lipids and mRNA content was standardized at a ratio of 6: 1.

[0409] Hepatocyte-targeting PEG replacement lipids were mixed with ALC-0315, a helper lipid (DSPC), and cholesterol at different mole contents to optimize mRNA LNP formulation. The difference between the mole ratio of LPSG and that of ALC-0159 (used at 1.6%) was proportionally redistributed according to the mole contents of the other lipid components.

[0410] CPSG15 was likewise mixed with ALC-0315, DSPC, and cholesterol at different mole ratio contents. However, to account for the cholesterol inherently present in CPSG15, the percentage of CPSG15 added was subtracted from the percentage of cholesterol component mixed into the LNP formulations to keep the total amount of cholesterol consistent in the formulations.

[0411] The formulation of mRNA LNPs involved two distinct phases, the organic phase and the aqueous phase. For the manual formulation process, the organic phase contained the mixture of the lipid components dissolved in ethanol to reach a final volume of 50 pL. The aqueous phase contained 10 pL of 1 mg / mL firefly luciferase mRNA (TriLink Biotechnologies) diluted in 140 pL of 10 mM sodium acetate solution at pH 4 to reach a final volume of 150 pL. The organic phase was added to the aqueous phase and mixed thoroughly through rapid pipetting. The mRNA-lipid mixture was then allowed to incubate at room temperature or 4°C for at least 30 mins to provide time for encapsulation and self-assembly of the LNPs. Table 1. Mole ratios (content) of the lipid components used in the formulation of LNPs using LPSG with a degree of polymerization of 12, 15, and 20 (i.e., LPSG12, LPSG13, and LPSG20).

[0412] Lipid ALC-0315 DSPC Cholesterol ALC-0159 LPSG Molecular Refer to 766.3 790.2 386.7 2481.0

[0413] weight (g / mol) Table 3 Mole ratio - 46.3 9.4 43.0 1.6 - ALC-0159 (%)

[0414] 46.6 9.5 43.0 - 1.0 Mole ratio - 46.5 9.4 42.9 - 1.2 LPSG (%)

[0415] 46.4 9.4 42.8 - 1.4 46.3 9.4 42.7 - 1.6 Stock

[0416] concentration 20.0 10.0 10.0 3.0 10.0 (mg / mL)

[0417]

[0418] Table 2. Mole ratios (content) of the lipid components used in the formulation of LNPs using CPSG with a degree of polymerization of 15 (i.e., CPSG15). Total mole ratio of cholesterol shown accounted for the cholesterol contributed by CPSG15 in each formulation.

[0419] Lipid ALC-0315 DSPC Cholesterol ALC-0159 LPSG Molecular Refer to 766.3 790.2 386.7 2481.0

[0420] weight (g / mol) Table 3 Mole ratio - 46.3 9.4 43.0 1.6 - ALC-0159 (%)

[0421] 46.6 9.5 43.0 - 1.0 Mole ratio - 46.5 9.4 42.9 - 1.2 CPSG15(%)

[0422] 46.4 9.4 42.8 - 1.4 46.3 9.4 42.7 - 1.6

[0423]

[0424] Lipid ALC-0315 DSPC Cholesterol ALC-0159 LPSG Stock

[0425] concentration 20.0 10.0 10.0 3.0 10.0 (mg / mL)

[0426]

[0427] Table 3. Characteristics of the hepatocyte-targeting lipids LPSG12, LPSG15, LPSG20, and CPSG15.

[0428] HepatocyteNumber of repeat Lipid-polypeptide

[0429] MnNMR[g mol-1] targeting lipids units (n) end-group

[0430] LPSG12 12 -CO-CH3 3499 LPSG15 15 -CO-CH3 4247 LPSG20 20 -CO-CH3 5493 CPSG15 15 -CO-CH3 4253

[0431]

[0432] 2.5. Assessing encapsulation efficiency of mRNA in LNPs

[0433] To determine the encapsulation efficiency of the mRNA LNPs after formulation, Quant-itTM RiboGreen RNA Assay Kit (Invitrogen, Waltham, MA, USA) was used to measure 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. ConcTrit— ConcTEEncapsulation Efficiency — - - - x 100

[0434] ConcTrit

[0435] Where Concrnt is the concentration of mRNA obtained by adding the mRNA LNPs to 0.5% Triton-X100 diluted in Tris-EDTA buffer, while ConcTEis the concentration of the respective mRNA obtained by adding the mRNA LNPs to Tris-EDTA buffer without Triton-X100.

[0436] 2.6. Physiochemical characterization of mRNA LNPs

[0437] The size, polydispersity index (PDI), and zeta potential of the mRNA LNPs were 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 1.0 mL. The samples were also measured three times at 25 °C with 20 runs per measurement.

[0438] 2.7. Cell culturing and dosing of HepG2 and DC2.4 cells with mRNA LNPs

[0439] To test the cytotoxicity and transfection efficiency of the mRNA LNPs, HepG2 and DC2.4 cells were cultured and dosed with the mRNA LNPs. The HepG2 cell line was cultured in DMEM media containing 10% Fetal Bovine Serum (FBS) (v / v) and 1% Penicillin / Streptomycin (v / v) while 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.

[0440] 2.8. In vitro viability of HepG2 and DC2.4 cells after incubation with mRNA LNPs

[0441] After 48 hours of incubation, 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 hours to allow reduction of the Alamar Blue compound by live 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.

[0442] 2.9. In vitro transfection efficiency of mRNA LNPs in HepG2 and DC2.4 cells after incubation with mRNA LNPs

[0443] The transfection efficiency of mRNA LNPs in HepG2 and DC2.4 cells was measured after 48 hours using the ONE-Glo™ Luciferase Assay System. 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.

[0444] Example 3: Results and Discussion

[0445] 3.1. Synthesis and characterization of LPSG and CPSG Lipid-b / oc / c-poly(Ser-D-galactose) (LPSG) was synthesized via the ringopening polymerization (ROP) of Ser(1,2,3,4, 6-penta-O-acetyl-D-galactopyranose)-NCA (Scheme 1) using the lipid of 2-amino-N, N-ditetradecylacetamide as the initiator, followed by the deprotection of lipid--poly(Ser-1,2,3,4,6-penta-O-acetyl-D-galactopyranose) in MeOH containing 2.5% MeONa (Scheme 2). The successful synthesis of LPSG was verified by1H NMR spectroscopy. As shown in FIGs 3 to 5, the disappearance of peaks (-CO-CH3) indicated successful deprotection of lipid-poly(Ser-1,2,3,4, 6-penta-O-acetyl-D-galactopyranose).1H NMR spectrum clearly showed the peak of n from amide groups in polypeptide backbone at 8.33 ppm, the peaks of f-m and p from Ser(1,2,3,4,6-penta-O-acetyl-D-galactopyranose) units and the peak of a (-CH3) from lipid at 0.85 ppm, suggesting successful synthesis of LPSG. The degrees of polymerization (DPs) of Ser-D-galactose in LPSG can be adjusted by varying the amount of Ser(1,2,3,4, 6-penta-O-acetyl-D-galactopyranose)-NCA. The DPs of Ser(D-galactopyranose) in lipid-poly(Ser-D-galactopyranose) were determined by the integration area (peak i of the polypeptide; peak a of the lipid) (FIGs 3 to 5). The typical LPSG with different DPs including 12, 15, and 20, were synthesized and denoted as LPSG12, LPSG15, and LPSG20, respectively.

[0446] Furthermore, cholesterol-b / oc / r-poly(Ser-D-galactose) (CPSG) was synthesized via the ROP of Ser(1, 2,3,4, 6-penta-O-acetyl-D-galactopyranose)-N-carboxyanhydride using the cholesterol-NH2as the initiator, followed by the deprotection of cholesterol-b / oc -poly(Ser-1, 2,3,4, 6-penta-O-acetyl-D-galactopyranose) in 2.5% MeONa in MeOH (Scheme 3). The successful synthesis of cholesterol-b / oc / r-poly(Ser-D-galactose) was confirmed by1H NMR spectroscopy. As shown in FIG 6, the disappearance of peaks (-CO-CH3) indicated successful deprotection of cholesterol-poly(Ser-1, 2,3,4, 6-penta-O-acetyl-D-galactopyranose).1H NMR spectrum clearly showed the peak of n from amide groups in polypeptide backbone at 8.35 ppm, the peaks of f-m and p from Ser(D-galactose) units and the peak of a (-CH3) from cholesterol at 0.65 ppm, suggesting successful synthesis of CPSG. 3.2. Size, polydispersity index (PDI), and zeta potential of mRNA LNPs

[0447] 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 physiochemical characteristics of the mRNA LNPs formulated manually are displayed in Tables 4 to 7. As observed from the results, LNPs formulated with liver-targeting compounds showed nanosize (< 200 nm) with a small polydispersity index (< 0.15) and near neutral zeta potentials (within ±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 desirable 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.

[0448] Table 4. Characteristics of manually formulated mRNA LNPs using LPSG or CPSG at a mole ratio (content) of 1.0%.

[0449] Mole ratio Diameter Zeta potential Formulation PDI

[0450] (%) (nm) (mV) LPSG12 137 ± 3 0.062 ± 0.018 4.29 ± 1.21 LPSG15 150 ± 1 0.056 ± 0.054 4.17 ± 1.40

[0451] 1.0

[0452] LPSG20 141 ± 2 0.032 ± 0.040 -4.29 ± 10.48 CPSG15 151 ± 4 0.033 ± 0.020 4.74 ± 0.56

[0453]

[0454] Table 5. Characteristics of manually formulated mRNA LNPs using LPSG or CPSG at a mole ratio (content) of 1.2%.

[0455] Mole ratio Diameter Zeta potential Formulation PDI

[0456] (%) (nm) (mV) LPSG12 1.2 139 ± 1 0.073 ± 0.011 3.32 ± 3.05

[0457]

[0458] Mole ratio Diameter Zeta potential Formulation PDI

[0459] (%) (nm) (mV) LPSG15 138 ± 2 0.030 ± 0.016 3.88 ± 1.63 LPSG20 148 ± 2 0.067 ± 0.043 3.25 ± 1.96 CPSG15 145 ± 2 0.048 ± 0.013 5.76 ± 0.27

[0460]

[0461] Table 6. Characteristics of manually formulated mRNA LNPs using LPSG or CPSG at a mole ratio (content) of 1.4%.

[0462] Mole ratio Diameter Zeta potential Formulation PDI

[0463] (%) (nm) (mV) LPSG12 136 ± 1 0.082 ± 0.021 5.96 ± 4.92 LPSG15 154 ± 1 0.058 ± 0.039 1.52 ± 3.38

[0464] 1.4

[0465] LPSG20 133 ± 2 0.085 ± 0.028 7.61 ± 8.62 CPSG15 134 ± 1 0.088 ± 0.034 7.21 ± 8.54

[0466]

[0467] Table 7. Characteristics of manually formulated mRNA LNPs using ALC-0159, LPSG, or CPSG at a mole ratio (content) of 1.6%.

[0468] Mole ratio Diameter Zeta potential Formulation PDI

[0469] (%) (nm) (mV) ALC-0159 97 ± 1 0.106 ± 0.022 0.93 ± 0.97 LPSG12 132 ± 1 0.056 ± 0.019 4.30 ± 0.32 LPSG15 1.6 134 ± 0 0.038 ± 0.028 0.71 ± 1.15 LPSG20 146 ± 5 0.121 ± 0.008 4.99 ± 0.53 CPSG15 139 ± 0 0.071 ± 0.060 4.50 ± 2.49

[0470]

[0471] 3.3. Encapsulation efficiency of formulated mRNA LNPs

[0472] The encapsulation efficiency of the mRNA LNPs was determined through the Ribogreen Assay Kit and the values are displayed in Tables 9 to 12. All liver targeting LNPs formulated manually or through the microfluidics device demonstrated an encapsulation efficiency comparable to or greater than the encapsulation efficiency of LNPs formulated using ALC-0159, a polyethylene glycol (PEG)-ylated lipid that is used in Pfizer / BioNTech’s mRNA Covid19 vaccine. 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 characteristics of the mRNA LNPs, these results suggest that the mRNA LNPs formulated with liver targeting compounds are viable for in vivo applications.

[0473] Table 8. Encapsulation efficiency of manually formulated mRNA LNPs using LPSG or CPSG at a mole ratio of 1.0%.

[0474] Formulation Mole ratio (%) Encapsulation efficiency (%) LPSG12 68.7 ± 2.8

[0475] LPSG15 67.7 ± 0.5

[0476] 1.0

[0477] LPSG20 68.9 ± 0.7

[0478] CPSG15 68.3 ± 4.2

[0479]

[0480] Table 9. Encapsulation efficiency of manually formulated mRNA LNPs using LPSG or CPSG at a mole ratio of 1.2%.

[0481] Formulation Mole ratio (%) Encapsulation efficiency (%) LPSG12 73.4 ± 0.9

[0482] LPSG15 48.5 ± 4.8

[0483] 1.2

[0484] LPSG20 60.6 ± 1.9

[0485] CPSG15 53.5 ± 2.3

[0486]

[0487] Table 10. Encapsulation efficiency of manually formulated mRNA LNPs using LPSG or CPSG at a mole ratio of 1.4%.

[0488] Formulation Mole ratio (%) Encapsulation efficiency (%) LPSG12 50.2 ± 4.5

[0489] LPSG15 62.1 ± 2.0

[0490] 1.4

[0491] LPSG20 60.6 ± 2.1

[0492] CPSG15 67.5 ± 2.6

[0493]

[0494] Table 11. Encapsulation efficiency of manually formulated mRNA LNPs using ALC-0159, LPSG, or CPSG at a mole ratio of 1.6%.

[0495] Formulation Mole ratio (%) Encapsulation efficiency (%) ALC-0159 63.4 ± 2.4

[0496] LPSG12 70.5 ± 0.9

[0497] 1.6

[0498] LPSG15 68.6 ± 0.3

[0499] LPSG20 69.1 ± 0.5

[0500] CPSG15 55.8 ± 3.2

[0501]

[0502] 3.4. In vitro cytocompatibility and transfection efficiency of mRNA LNPs in HepG2 cells

[0503] The transfection efficiency and cytotoxicity of the mRNA LNPs formulated manually were determined by dosing the formulated LNPs in HepG2 cells, a cell line commonly used for in vitro hepatocyte testing. The results obtained are displayed in FIGs.7 to 10. 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 HepG2 cells from the luciferase assay demonstrated that the formulations formed using CPSG15 were capable of significantly improving transfection efficiency of Flue mRNA over ALC-0159. This suggests that CPSG15 compounds are not only capable of replacing PEG-conjugated lipid as a viable compound in producing nanosized LNPs, but they also enable greater transfection efficiency of mRNA by targeting hepatocytes in an in vitro setting. Variations in the transfection efficiency of the varying hepatocyte -targeting PEG-free LNPs at different mole ratios suggest that there is a hydrophobicityhydrophilicity balance that enables an optimal level of transfection efficiency.

[0504] 3.5. In vitro cytocompatibility and transfection efficiency of mRNA LNPs in DC2.4 cells

[0505] The cytocompatibility and transfection efficiency results for mRNA LNPs dosing in DC2.4 cells are detailed in FIGs. 11 to 14. Similar to HepG2 cells, analysis of the AlamarBlue assay results in DC2.4 cells revealed that the mRNA LNPs tested showed negligible cytotoxicity. The Luciferase assay however showed a different trend compared to HepG2 cells. All liver-targeting LNPs showed greater transfection efficiency compared to ALC-0159 in DC2.4 cells. Interestingly, LPSG12 at a mole ratio 1.4% yielded much greater transfection efficiency compared to both ALC-0159 and other liver targeting formulations.

[0506] 3.6. Conclusion

[0507] As shown in the examples, lipid-block-poly(Ser-D-galactose) (LPSG) and cholesterol-block-poly(Ser-D-galactose) (CPGM) were successfully synthesized. mRNA LNPs formulated using LPSG or CPSG have nanosize (< 200 nm), homogeneous particle population (PDI < 0.15), and neutral surface (zeta potential: within ± 10 mV), making them ideal for in vivo applications. The mRNA LNPs formed using LPSG and CPSG provided greater mRNA transfection efficiency than the mRNA LNPs made using ALC-0159 in HepG2 cells, and to an even greater extent in DC2.4 cells. All mRNA LNPs formulations tested showed negligible cytotoxicity. LPSG and CPSG are thus a viable replacement for ALC-0159 and other PEG-conjugated lipids as they are not only capable of fulfilling the function of PEG in maintaining nanoscale particle size, but they also provide greater mRNA transfection efficiency, especially in hepatocytes. These PEG-free LNPs are promising nanocarriers for the liver-targeted delivery of mRNA and other genes for vaccine and treatment applications.

Claims

CLAIMS1. A compound comprising a structure represented by general formula (1 ):whereinR1, R3, R4, R5, and R6are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; R7is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;R10is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;k is 0 or 1;n and l are each independently ≥ 1;A comprises galactose and / or a derivative(s) thereof; andB comprises a lipid and / or a derivative(s) thereof.

2. The compound according to claim 1, wherein A is represented by general formula (2):whereinX1to X7and X9to X10are each independently selected from -Rd, -ORe, or -0-C(=0)Rf, wherein Rd, Re, and Rfare each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;X8is alkylene; andM is -O-Rg-, -S-Rh-, or -N(R')-Ri-, wherein Riis H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl and wherein R9, Rh, and Rjare each independently absent or each independently present and selected from optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

3. The compound according to claim 1 or 2, wherein B is Rb-N-Rc, wherein Rband Rcare each independently a hydrophobic group or contains at least linear aliphatic, branched aliphatic, and / or cyclic hydrocarbons.

4. The compound according to claim 1 or 2, wherein B is Rb-N-Rc, wherein Rband Rcare each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and combinations thereof.

5. The compound according to claim 1 or 2, wherein B comprises sterol and / or a derivative(s) thereof.

6. The compound according to any one of claims 1, 2, and 5, wherein B is represented by general formula (X),whereinR12is a hydrophobic group or contains at least linear aliphatic, branched aliphatic, and / or cyclic hydrocarbons.^13-18 R2O R22 R24’29, R32’33, and R35-37are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;R19, R21, R23, R30, R31, and R34are each independently absent, H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted; andRing Z contains one or more double bonds.

7. The compound according to any one of claims 1 to 6, wherein the compound is substantially devoid of polyethylene glycol (PEG).

8. The compound according to any one of claims 1 to 7, wherein the compound has an average molecular weight of from 3,000 g / mol to 50,000 g / mol.

9. The compound according to any one of claims 1 to 8, wherein the compound is selected from the group consisting of the following structures:and10. A method of preparing a compound represented by general formula (1) according to any one of claims 1 to 9, the method comprising:(i) reacting a lipid molecule represented by general formula (3) with a compound comprising N-carboxyanhydride (NCA) represented by general formula (4) to obtain a first intermediate compound represented by general formula (5):(3)whereinR1, R3, R4, R5, R6, and R11are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;R7is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;k is 0 or 1;n and l are each independently ≥ 1;A comprises galactose and / or a derivative(s) thereof, and Aprepresents A protected with a protecting group; andB comprises a lipid and / or a derivative(s) thereof;(ii) reacting the first intermediate compound represented by general formula (5) with an acid anhydride represented by general formula (6) to obtain a second intermediate compound represented by general formula (7):(5) (6)(7)whereinR1, R3, R4, R5, R6, and R11are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;R7is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;R10and R10’ are each independently optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; k is 0 or 1;n and l are each independently ≥ 1;A comprises galactose and / or a derivative(s) thereof, and Aprepresents A protected with a protecting group; and B comprises a lipid and / or a derivative(s) thereof; and(iii) deprotecting the second intermediate compound represented by general formula (7) to obtain the compound represented by general formula (1).

11. The method according to claim 10, wherein the method further comprises, prior to (i):(a-i) reacting a galactose and / or a derivative(s) thereof represented by general formula (8) with an acid anhydride represented by general formula (9) to obtain a protected galactose and / or a derivative(s)thereof represented by general formula (8p), wherein one or more -OH group(s) in the galactose and / or a derivative(s) thereof represented by general formula (8) are converted into -OPG1:(8p) whereinX1to X7and X9to X11are each independently selected from -Rkor -OR1, wherein Rkand R1are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;X22to X28and X30to X32are each independently selected from -Rmor -OPG1, wherein Rmis H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;X8and X29are each alkylene;R11is optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl;M is -O-R3-, -S-Rh-, or - N(R')- R<- wherein Riis H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl and Rg, Rh, and Rjare each independentlyabsent or each independently present and selected from optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; andPG1is -C(=O)-R13, where R13are each independently optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;(a-ii) reacting the protected galactose and / or a derivative(s) thereof represented by general formula (8P) obtained from (a-i) with a protected compound represented by general formula (10) in the presence of a Lewis acid to obtain a first intermediate compound comprising amide group represented by general formula (11):(8p) (10)whereinX22to X28and X30to X32are each independently selected from -Rmor -OPG1, wherein Rmis H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;X8and X29are each alkylene;M is -O-Rg-, -S-Rh-, or -N(R')-Ri- wherein Riis H, optionally substituted alkyl, optionally substituted alkenyl, or optionallysubstituted alkynyl and R9, Rh, and Rjare each independently absent or each independently present and selected from optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;R7is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;A comprises galactose and / or a derivative(s) thereof, and Aprepresents A protected with a protecting group; andPG2is a protecting group;(a-iii) deprotecting the first intermediate compound represented by general formula (11) to obtain a second intermediate represented by general formula (12):(12) whereinR7is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;A comprises galactose and / or a derivative(s) thereof, and Aprepresents A protected with a protecting group; andPG2is a protecting group; and(a-iv) reacting the second intermediate compound represented by general formula (12) with a carbonylating agent to obtain the compound comprising N-carboxyanhydride (NCA) represented by general formula (4)whereinR6is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;R7is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene; and A comprises galactose and / or a derivative(s) thereof, and Aprepresents A protected with a protecting group.

12. A nanoparticle composition comprising:(i) a compound represented by general formula (1) according to any one of claims 1 to 9; and(ii) a therapeutic, prophylactic, and / or biological agent that is encapsulated by said compound.

13. The composition according to claim 12, wherein the composition further comprises:(a) helper lipid;(b) cholesterol and / or a derivative(s) thereof; and(c) ionizable lipid.

14. The composition according to claim 13, wherein the ionizable lipid, helper lipid, cholesterol and / or a derivative(s) thereof, and the compoundrepresented by general formula (1) are mixed at a mole ratio of 20 - 50: 4 -20: 25- 50: 0.5 -20.

15. The composition according to claim 13 or 14, 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 (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.

16. The composition according to any one of claims 13 to 15, wherein the cholesterol and / or a derivative(s) thereof is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, avenasterol, and combinations thereof.

17. The composition of any one of claims 15 to 16, wherein the ionizable lipid is selected from the group consisting of ALC-0315, SM-102, Lipid 5,DLinDMA, D-Lin-MC2-DMA, DLin-MC3-DMA, D-Lin-MC4-DMA, Dlin-KC2- DMA, YSK05, AA3-Dlin, SSPalmM, SSPalmO-Phe, Lipid A9, L319, DODMA, CL1, BP Lipid 310, ATX-001, ATX-100, Lipid 2, 80-O16B, BP Lipid 309, BP Lipid 307, 93-O17S, 93-O17O, NT1-O14B, 306-O12B-3, 306-012B, 113-016B, 3060i10, 306Oi9-cis2, BAMEA-O16B, AI-28, 113- 012B, 98N12-5, Ckk-E12, OF-02, C12-200, BP Lipid 311, BP Lipid 308, BP Lipid 314, BP Lipid 312, LP01, TCL053, Lipid C24, BP Lipid 315, Lipid 29, 9A1P9, C13-112-tri-tail, C13-113-tri-tail, C13-112-tetra-tail, C13-113- tetra-tail, and combinations thereof.

18. The composition according to any one of claims 12 to 17, wherein the composition comprises nanoparticles with a N / P ratio from 1:1 to 20: 1, an average from 40 nm to 500 nm, a polydispersity index (PDI) of from 0.001 to 0.500, and / or a zeta potential of from -20 mV to 20 mV.

19. The compound represented by general formula (1) according to any one of claims 1 to 9 or the nanoparticle composition according to any one of claims 12 to 18 for use in medicine.

20. 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 12 to 18.

21. The nanoparticle composition according to any one of claims 12 to 18 for use in modulating an immune response in a subject, wherein said nanoparticle composition is to be administered to the subject.

22. Use of a nanoparticle composition according to any one of claims 12 to 18 in the manufacture of a medicament for modulating an immune response in a subject.

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