WO2025090023A1 - A compound for preparing lipid nanoparticles encapsulating an agent, nanoparticle composition comprising said compound, and related methods thereof - Google Patents

Abstract

There is provided a compound comprising a structure represented by general formula (1) or an ionized form thereof for preparing lipid nanoparticles encapsulating a therapeutic, prophylactic and/or biological agent: wherein R5 and each R6 are independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; R3, and each R7 are independently optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene; n is from 0 to 100; l and m are each independently 0 or 1; each A is independently selected from H, aliphatic alcohol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, an epoxy ring- opening product and/or derivatives thereof; and B is R1R2N– or R23O– where R1 and R2 are each independently H, or a hydrophobic tail with the proviso that both of R1 and R2 are not H at the same 116 time; and where R23 is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

Description

A COMPOUND FOR PREPARING LIPID NANOPARTICLES ENCAPSULATING AN AGENT, NANOPARTICLE COMPOSITION COMPRISING SAID COMPOUND, AND RELATED METHODS THEREOF

TECHNICAL FIELD

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.

BACKGROUND

Lipid nanoparticles (LNPs) are widely used in the delivery of therapeutic, prophylactic, and/or biological agents (e g., polynucleotides such as mRNA). However, a safe, stable and efficacious delivery system remains a challenge. Particularly, there have been reports of adverse health effects and cytotoxicity associated with the use of lipid nanoparticles for delivery.

Currently, there are only 2 mRNA Covid-19 vaccines from Moderna and Pfizer-BioNtech that have been approved by the United States Food and Drug Administration (US FDA) for human use. Both vaccines utilize SARS-CoV-2 mRNA as the antigen and lipids as the carrier. The LNPs consist of 4 components, ionizable lipid, PEG-lipid conjugate, helper lipid and cholesterol. The lipids assemble with mRNA or siRNAto form nanoparticles for effective in vivo delivery. There is also a lipid nanoparticle-based siRNA drug “Patisiran" that has been approved for treatment of hATTR amyloidosis. These lipids contain a single tertiary amine group that becomes ionized at low pH values (e.g., pH 4-5), allowing the positively charged lipids to condense RNA into RNA-encapsulated nanoparticles (mRNA LNPs) upon mixing for mRNA delivery. However, the currently available formulations have several disadvantages and drawbacks, and are far from desirable. Firstly, the ionizable lipids used in Moderna and Pfizer-BioNTech mRNA vaccine formulations are extremely expensive. Additionally, in the physiological environment (pH 7.4), the positive charge on the ionized tertiary amine group is neutralized, reverting the lipids to their original form, which may lead to the dissociation of mRNA from the LNPs.

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

SUMMARY

In one aspect, there is provided a compound comprising a structure represented by general formula (1 ) or an ionized form thereof for preparing lipid nanoparticles encapsulating a therapeutic, prophylactic and/or biological agent:

wherein

R⁵ and each R⁶ are independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

R³, and each R⁷ are independently optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene; n is from 0 to 100;

I and m are each independently 0 or 1 ; each A is independently selected from H, aliphatic alcohol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, an epoxy ring-opening product and/or derivatives thereof; and B is R¹ R²N- or R²³O- where R¹ and R² are each independently H, or a hydrophobic tail with the proviso that both of R¹ and R² are not H at the same time; and where R²³ is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

In one embodiment, the compound comprises three or more tertiary amines.

In one embodiment, R¹ and R² are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and combinations thereof.

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

where R⁸, R⁸’, R⁹ and R⁹’ are each independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

In one embodiment, R⁸, R⁸’ and R⁹’ are H and R⁹ is C_yH2y+i where y is from

1 to 8.

In one embodiment, the compound has a molecular weight of from 500 g/mol to 50,000 g/mol. In one embodiment, the compound comprises a structure selected from one or more of the following:

LP2 (n = 8);

LPL1 (n = 1),

LPL4 (n = 8);

In another aspect, there is provided a method of preparing a compound represented by general formula (1 ) as disclosed herein, the method comprising:

(a) reacting a lipid-b-poly(Lys) compound represented by general formula (1 b’) with a compound comprising an epoxy represented by general formula (3) to obtain the compound represented by general formula (1 ):

(1b') (3) wherein

R⁵ and each R^B are independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

R³ and each R are independently optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene; n is from 1 to 100; each R¹¹ to R¹³ are independently selected from H, aliphatic alcohol, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

R¹ and R² are each independently H, or hydrophobic tail, with the proviso that both of R¹ and R² are not H at the same time; and

R⁸ and R⁹ are each independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

In one embodiment, the method further comprises, prior to step (a):

(a-i) reacting a compound represented by general formula (4) with a protected amino acid comprising N-carboxyanhydride (NCA) represented by general formula (5) to obtain a protected lipid-b-poly(Lys) compound represented by general formula (6) via ring-opening polymerization (ROP):

wherein

R⁵ and each R⁶ are independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

R³ and each R⁷ are independently optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene; n is from 1 to 100;

R¹¹ to R¹² are each independently selected from H, aliphatic alcohol, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

R¹ and R² are each independently H, or hydrophobic tail, with the proviso that both of R¹ and R² are not H at the same time;

R¹⁰ is H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; and

PG¹ is a protecting group; and

(a-ii) deprotecting the protected lipid-b-poly(Lys) compound represented by general formula (6) obtained from step (a-i) to obtain the compound represented by general formula (1 b’):

(6) (1 b¹) wherein

R¹³ is selected from H, aliphatic alcohol, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

In one embodiment, the method further comprises, prior to step (a-i): (a-i-i) reacting a protected amino acid represented by general formula (7) with a carbonylating agent to obtain the protected amino acid comprising NCA represented by general formula (5):

(7) (5) wherein

R⁶ and R¹⁶ 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;

R¹¹ is selected from H, aliphatic alcohol, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and PG¹ is a protecting group.

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

(a-i-ii) reacting an amine compound represented by general formula (8) with a protected amino acid compound represented by general formula (9) in the presence of one or more coupling agent(s) and a base to obtain a carbamate compound represented by general formula (10)

wherein

R⁵, R¹⁴, and R¹⁵ are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

R³ is independently optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene; n is from 1 to 100;

R¹ and R² are each independently H, or hydrophobic tail, with the proviso that both of R¹ and R² are not H at the same time; and

PG² is a protecting group; and

(a-i-iii) deprotecting the carbamate compound represented by general formula (10) obtained from step (a-i-ii) to obtain the compound represented by general formula (4)

wherein

R¹⁰ is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

In another aspect, there is provided a method of preparing a compound represented by general formula (1 ) as disclosed herein, the method comprising: (b) reacting an amine compound represented by general formula (8) with a protected amino acid compound represented by general formula (11 ) in the presence of one or more coupling agent(s) and a base to obtain a dicarbamate compound represented by general formula (12)

wherein

R⁷ is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene;

R¹ and R² are each independently H, or hydrophobic tail, with the proviso that both of R¹ and R² are not H at the same time;

R¹⁴, and R¹⁷ to R¹⁹ are each independently H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; and PG⁴ and PG⁵ are each independently a protecting group;

(c) deprotecting the dicarbamate compound represented by general formula

(12) obtained from step (b) to obtain an amide compound represented by general formula (13)

(12} (13) wherein

R²⁰to R²¹ are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and

(d) reacting the amide compound represented by general formula (13) with an epoxy represented by general formula (3) to obtain the compound represented by general formula (1 ):

(13) (3)

R⁸ to R⁹ are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

In another aspect, there is provided a nanoparticle composition for delivery of a therapeutic, prophylactic, and/or biological agent, the nanoparticle composition comprising: a compound as disclosed herein; and a therapeutic, prophylactic and/or biological agent that is encapsulated in said compound as disclosed herein.

In one embodiment, the nanoparticle composition comprises nanoparticles having a N/P ratio from 1 :1 to 50:1 .

In one embodiment, the nanoparticle composition comprises nanoparticles having an average particle size of from 20 nm to 650 nm. In one embodiment, the composition further comprises:

(i) helper lipid;

(ii) cholesterol or derivatives thereof; and

(iii) polyethylene glycol (PEG)-lipid conjugate or amphiphilic lipids.

In one embodiment, the compound represented by general formula (1 ), helper lipid, cholesterol or derivatives thereof, and PEG-lipid conjugate or amphiphilic lipids are mixed at a weight ratio of 30 to 50: 5 to 50: 5 to 60: 1 to 5.

In one embodiment, the helper lipid is 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.

In one embodiment, the cholesterol or derivatives thereof is selected from cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, avenasterol, and combinations thereof. In one embodiment, the polyethylene glycol (PEG)-lipid conjugate or amphiphilic lipids is selected from PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, 2- [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), R-3-[(cu- methoxy-poly(ethylene glycol)2000)carbamoyl]-1 ,2-dimyristyloxlpropyl-3-amine (PEG-c-DOMG), 3-N-[(io-methoxypoly (ethyleneglycol)2000)carbamoyl]-1 ,2- dimyristyloxy-propylamine (PEG-S-DMG), PEG-DMPE (1 ,2-dimyristoyl-sn- glycero-3-phosphoethanolamine-N-[(polyethylene glycolj-methoxy] (sodium salt)), PEG-DPPC, PEG-DSPE lipid and combinations thereof.

In another aspect, there is provided the nanoparticle composition as disclosed herein for use in medicine.

In another aspect, there is provided the nanoparticle composition as disclosed herein for use in the treatment or prophylaxis of a disease, disorder, or condition in a subject in need thereof.

In another aspect, there is provided use of a nanoparticle composition as disclosed herein in the manufacture of a medicament for treatment or prophylaxis of a disease, disorder, or condition in a subject in need thereof.

In another aspect, there is provided a method of treating or preventing a disease, disorder, or condition in a subject in need thereof, the method comprising administering a therapeutically effective amount of the nanoparticle composition as disclosed herein to the subject.

In one embodiment, an immune response in the subject is to be induced through the administration of the nanoparticle composition thereto.

In one embodiment, the disease, disorder, or condition is mediated by a coronavirus. In one embodiment, the coronavirus is a SARS-CoV-2 coronavirus.

DEFINITIONS

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.

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

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 icron 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.

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.

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). Dosages and administration of compounds, compositions and formulations of the present disclosure may be determined by one of ordinary skill in the art of clinical pharmacology or pharmacokinetics. An effective amount of the active agent of the present disclosure to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the patient. Accordingly, it may be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect.

The term “subject” is intended to broadly refer to any animal, such as a mammal, and including humans. Exemplary subjects include but are not limited to humans and non-human primates. The term “subject” as used herein also includes patients and non-patients. The term “patient” refers to individuals suffering or are likely to suffer from a medical condition such as infectious diseases (e g., coronavirus caused by the SARS-CoV-2 virus), while “nonpatients” 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).

In the definitions of a number of substituents below, it is stated that “the group may be a terminal group or a bridging group”. This is intended to signify that the use of the term is intended to encompass the situation where the group is a terminal group/moiety as well as the situation where the group is a linker between two other portions of the molecule. Using the term “alkyl” having 1 carbon atom as an example, it will be appreciated that when existing as a terminal group, the term “alkyl” having 1 carbon atom may mean -CHs and when existing as a bridging group, the term “alkyl” having 1 carbon atom may mean -CH2- or the like.

The term "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.

The term "alkyl" as a group or part of a group refers to a straight or branched aliphatic hydrocarbon group having 1 to 20 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. Examples of suitable straight and branched alkyl substituents include methyl, ethyl, n-propyl, 2-propyl, isopropyl, n- butyl, isobutyl, sec-butyl, t-butyl, hexyl, amyl, 1 ,2-dimethylpropyl, 1 ,1 - dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1 -methylpentyl, 2- methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1 ,2- dimethylbutyl, 1 ,3-dimethylbutyl, 1 ,2,2-trimethylpropyl, 1 , 1 ,2-trimethylpropyl, 2- ethylpentyl, 3-ethylpentyl, heptyl, 1 -methylhexyl, 2,2-dimethylpentyl, 3,3- dimethylpentyl, 4,4-dimethylpentyl, 1 ,2-dimethylpentyl, 1 ,3-dimethylpentyl, 1 ,4- dimethylpentyl, 1 ,2,3-trimethylbutyl, 1 , 1 ,2-trimethylbutyl, 1 ,1 ,3-trimethylbutyl, 5- methylheptyl, 1 -methylheptyl, octyl, nonyl, decyl and the like. The group may be a terminal group or a bridging group.

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

The term "alkynyl" as a group or part of a group denotes an aliphatic hydrocarbon group containing at least one carbon-carbon triple bond and which may be straight or branched having 2 to 20 carbon atoms, 2 to 10 carbon atoms, 2 to 6 carbon atoms, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms in the chain. The group may contain a plurality of triple bonds. Exemplary alkynyl groups include, but are not limited to, acetylenyl, propynyl, 1 - butynyl, 2-butynyl, 3-butynyl, 1 -pentynyl, 2-pentynyl, 3-methyl-1 -butynyl, 4- pentynyl, 1 -hexynyl, 2-hexynyl, 5-hexynyl, 1 -heptynyl, 2-heptynyl, 6-heptynyl, 1 - octynyl, 2-octynyl, 7-octynyl, 1-nonynyl, 2-nonynyl, 8-nonynyl, 1 -decynyl, 2- decynyl, 9-decynyl and the like. The group may be a terminal group or a bridging group.

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”.

The term “heterocyclic” as used herein broadly refers to a structure where two or more different kinds of atoms are connected to form at least one ring. For example, a heterocyclic ring may be formed by carbon atoms and at least another atom (i.e. , heteroatom) selected from oxygen (O), nitrogen (N) or (NR) and sulfur (S), where R is independently a hydrogen or an organic group. The term also includes, but is not limited to, saturated and unsaturated 5-membered, and saturated and unsaturated 6-membered rings. Examples of groups having a heterocyclic structure include, but are not limited to furan, thiophene, 1 H-pyrrole, 2H-pyrrole, 1 -pyrroline, 2-pyrroline, 3-pyrroline, 1-pyrazoline, 2-pyrazoline, 3- pyrazoline, 2-imidazoline, 3-imidazoline, 4-imidazoline, pyrazole, imidazole, oxazole, isoxazole, thiazole, isothiazole, 1 ,2,3-triazole, 1 ,2,4-triazole, 1 ,2,3- oxadiazole, disubstituted 1 ,2,4-oxadiazole, 1 ,2,5-oxadiazole, 1 ,3,4-oxadiazole,

1 .2.3-thiadiazole, 1 ,2,4-thiadiazole, 1 ,2,5-thiadiazole, 1 ,3,4-thiadiazole, tetra hydrofuran, tetrahydrothiophene, pyrrolidine, 1 ,3-dioxolane, 1 ,2-oxathiolane,

1.3-oxathiolane, pyrazolidine, imidazolidine, pyridine, pyridazine, pyrimidine, pyrazine, 1 ,2-oxazine, 1 ,3-oxazine, 1 ,4-oxazine, thiazine, 1 ,2,3-triazine, 1 ,2,4- triazine, 1 ,3,5-triazine, 2H-pyran, 4H-pyran, 2-pyrone, 4-pyrone, 1 ,4-dioxin, 2H- thiopyran, 4H-thiopyran, tetrahydropyran, thiane, piperidine, 1 ,4-dioxane, 1 ,2- dithiane, 1 ,3-dithiane, 1 ,4-dithiane, 1 ,3,5-trithiane, piperazine, morpholine, thiomorpholine and the like.

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

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

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

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

The term “epoxy ring-opening product” as used herein broadly refers to one or more products that is/are obtained from a ring-opening reaction of an epoxy ring. The term “epoxy” or “epoxy ring” may comprise and/or may be used interchangeably with the terms “epoxide”, “cyclic ether”, “three-membered cyclic ether”, or the like. Examples of epoxy ring-opening product include but are not limited to compounds comprising one or more, or two or more hydroxyl (-OH) groups. The epoxy ring-opening product may be a compound comprising a primary alcohol, secondary alcohol, tertiary alcohol, or a diol.

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., -CCI3, -CF3, -C(CF3)s), 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 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 "associated with", used herein when referring to two elements refers to a broad relationship between the two elements. The relationship includes, but is not limited to a physical, a chemical or a biological relationship. For example, when element A is associated with element B, elements A and B may be directly or indirectly attached to each other or element A may contain element B or vice versa.

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.

Further, in the description herein, the word “substantially” whenever used is understood to include, but not restricted to, "entirely" or “completely” and the like. In addition, terms such as "comprising", "comprise", and the like whenever used, are intended to be non-restricting descriptive language in that they broadly include elements/components recited after such terms, in addition to other components not explicitly recited. For example, when “comprising" is used, reference to a “one” feature is also intended to be a reference to “at least one” of that feature. Terms such as “consisting”, “consist”, and the like, may in the appropriate context, be considered as a subset of terms such as "comprising", "comprise", and the like. Therefore, in embodiments disclosed herein using the terms such as "comprising", "comprise", and the like, it will be appreciated that these embodiments provide teaching for corresponding embodiments using terms such as “consisting”, “consist", and the like. Further, terms such as "about", "approximately" and the like whenever used, typically means a reasonable variation, for example a variation of +/- 5% of the disclosed value, or a variance of 4% of the disclosed value, or a variance of 3% of the disclosed value, a variance of 2% of the disclosed value or a variance of 1 % of the disclosed value.

Furthermore, in the description herein, certain values may be disclosed in a range. The values showing the end points of a range are intended to illustrate a preferred range. Whenever a range has been described, it is intended that the range covers and teaches all possible sub-ranges as well as individual numerical values within that range. That is, the end points of a range should not be interpreted as inflexible limitations. For example, a description of a range of 1% to 5% is intended to have specifically disclosed sub-ranges 1 % to 2%, 1 % to 3%, 1 % to 4%, 2% to 3% etc., as well as individually, values within that range such as 1 %, 2%, 3%, 4%, and 5%. The intention of the above specific disclosure is applicable to any depth/breadth of a range.

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.

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.

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. DESCRIPTION OF EMBODIMENTS

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.

COMPOUND

There is provided a compound for preparing lipid nanoparticles. In various embodiments, the compound comprises one or more amine group(s) that is/are ionizable and/or capable of being ionized. The amine group may be selected from the group consisting of primary (1 °) amine, secondary (2°) amine, tertiary (3°) amine, and combinations thereof. In various embodiments therefore, the compound is ionizable and/or capable of being ionized and/or exists in an ionized form at e.g., physiological pH. Advantageously, the ionizable property of the compound (due to presence of ionizable amine group) allows for embodiments of the compound to be used as an encapsulation/loading agent, delivery vehicle/system and/or transfection vehicle/system. In various embodiments, the compound is designed/configured to allow loading/encapsulation of one or more types of molecules or cargoes. In various embodiments, the compound is also designed/configured to allow the loaded/encapsulated agent to be released from said compound and/or subsequently delivered to a desired target (e g., cell, cytosol, tissue, or organ). The molecules/cargoes to be loaded/encapsulated onto/into/within the compound may include but is not limited to a therapeutic agent, a prophylactic agent, a biological agent, or the like. In various embodiments, the molecules/cargoes to be loaded/encapsulated comprises a nucleic acid. For example, the molecules/cargoes to be loaded/encapsulated may be a nucleic acid selected from ribonucleic acid (RNA), microRNA (miRNA), messenger ribonucleic acid (mRNA), small interfering ribonucleic acid (siRNA), deoxyribonucleic acid (DNA), plasmid deoxyribonucleic acid (pDNA), oligonucleotides such as antisense oligonucleotide or allele-specific oligonucleotides (ASO) or the like or combinations thereof. In various embodiments, the molecules/cargoes to be loaded/encapsulated comprises therapeutics. For example, the molecules/cargoes to be loaded/encapsulated may be therapeutics selected from negatively charged therapeutics, drug molecule, vaccine (e.g., dengue vaccine, Covid-19 vaccine etc.) or the like or combinations thereof. Advantageously, the compound is suitable for use in encapsulating and/or delivering one or more therapeutic agent, prophylactic agent and/or biological agent to a desired target (e.g., subject, cell, cytosol, tissue, or organ).

Accordingly, in various embodiments, there is also provided a carrier, nanocarrier or delivery system/vehicle comprising the compound or its ionized form thereof.

Advantageously, the compound is designed/configured to be ionizable at a low pH range, e.g., at a pH range of from about 3.0 to about 6.5, from about 3.1 to about 6.4, from about 3.2 to about 6.3, from about 3.3 to about 6.2, from about 3.4 to about 6.1 , from about 3.5 to about 6.0, from about 3.6 to about 5.9, from about 3.7 to about 5.8, from about 3.8 to about 5.7, from about 3.9 to about 5.6, from about 4 to about 5.5, from about 4.1 to about 5.4, from about 4.2 to about 5.3, from about 4.3 to about 5.2, from about 4.4 to about 5.1 , from about 4.5 to about 5.0, from about 4.6 to about 4.9, from about 4.7 to about 4.8, or about 4.75, depending on the type or nature of the amine group(s). In various embodiments, the compound is designed/configured to be in an ionized form, e.g., the compound may be ionized to become positively charged.

In various embodiments, the compound comprises a structure that is represented by general formula (1 ) or an ionized form thereof:

wherein

R⁵ and each R⁶ are independently H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl;

R³ and each R⁷ are independently optionally substituted alkylene (e.g., -CH2-), optionally substituted alkenylene or optionally substituted alkynylene; n is from about 0 to about 100, from about 0 to about 50, from about 0 to about 40, from about 0 to about 30, from about 0 to about 20, from about 1 to about 19, from about 2 to about 18, from about 3 to about 17, from about 4 to about 16, from about 5 to about 15, from about 6 to about 14, from about 7 to about 13, from about 8 to about 12, from about 9 to about 11 , or about 10;

I and m are each independently 0 or 1 ; each A is independently selected from H, aliphatic alcohol, optionally substituted alkyl (e.g., alkyl alcohol), optionally substituted alkenyl (e.g., alkenyl alcohol), optionally substituted alkynyl (e.g., alkynyl alcohol), an epoxy ring-opening product and/or derivatives thereof; and

B is R¹R²N- or R²³O- where R¹ and R² are each independently H, hydrophobic tail/chain/group, or contains at least linear aliphatic, branched aliphatic, and/or cyclic hydrocarbons, with the proviso that both of R¹ and R² are not H at the same time; and where R²³ is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl. The compound may be suitable for use in preparing lipid nanoparticles.

In various embodiments, the compound represented by general formula

(1 ) comprises one or more hydrophobic tail/chain/group(s). For example, there may be one or two hydrophobic tail/chain/group(s) in general formula (1 ). In various embodiments, only one of R¹ and R² may be H. For example, in various embodiments when R¹ is H, R² is not H. For example, in various embodiments when R² is H, R¹ is not H or vice versa. In various embodiments, only one of R¹ and R² is a hydrophobic tail/chain/group. For example, when R¹ is H, R² is a hydrophobic tail/chain/group, or vice versa. In various embodiments, both R¹ and R² are hydrophobic tails/chains/groups.

In various embodiments, R¹ and R²are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and combinations thereof.

In various embodiments, the hydrophobic tail/chain/group at R¹ and R² each independently comprises optionally substituted alkyl. The alkyl may have at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, or at least 25 carbon atoms. For example, R¹ and R² are each independently CXH2X+I , where x > 3, x > 4, x > 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, or x > 25.

In various embodiments, R⁵ and each R⁶ are independently selected from

H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl. For example, R⁵ and/or R⁶ may be selected from methyl, ethyl, n-propyl, 2-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, hexyl, amyl, 1 ,2-dimethylpropyl, 1 ,1 -dimethylpropyl, pentyl, isopentyl, hexyl, 4- methylpentyl, 1 -methylpentyl, 2-m ethylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1 ,2-dimethylbutyl, 1 , 3-d im ethyl butyl, 1 ,2,2-trimethylpropyl,

I , 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.

In various embodiments, R³ and each R⁷ are independently selected from optionally substituted alkylene, optionally substituted alkenylene or optionally substituted alkynylene. For example, R³ and/or R⁷ may 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.

In various embodiments, A is represented by general formula (2):

where R⁸, R⁸’, R⁹, and R⁹’ are each independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

In various embodiments, R⁸, R⁸’, R⁹, and/or R⁹’ are each may be selected from methyl, ethyl, n-propyl, 2-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t- butyl, hexyl, amyl, 1 ,2-dimethylpropyl, 1 ,1 -dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1 -methylpentyl, 2-methylpentyl, 3-methylpentyl, 2,2- dimethylbutyl, 3,3-dimethylbutyl, 1 ,2-dim ethyl butyl, 1 ,3-dimethylbutyl, 1 ,2,2- trimethylpropyl, 1 , 1 ,2-trimethylpropyl, 2-ethylpentyl, 3-ethylpentyl, heptyl, 1 - methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl, 1 ,2- dimethylpentyl, 1 ,3-dimethylpentyl, 1 ,4-dimethylpentyl, 1 ,2,3-trimethylbutyl, 1 ,1 ,2-trimethylbutyl, 1 ,1 , 3-trimethylbutyl, 5-methylheptyl, 1 -methylheptyl, octyl, nonyl, decyl, or the like or combinations thereof.

In various embodiments, R⁸, R⁸’ and/or R⁹’ are H. In various embodiments, R⁹ is C_yH2y+i where y is from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, or from 1 to 2. In various embodiments, R⁹ is hexyl (i.e., -CeH ).

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.

In various embodiments, each A may be the same or different from one another. In various embodiments, all A are the same. In various embodiments where n > 2, A may be same or different. For example, when n = 20 and there is a total of twenty A, each of the twenty A present in the structure represented by general formula (1 ) may be same or different from each other. In various embodiments, the compound comprises three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more tertiary amine groups (e.g., ionizable tertiary amine groups). In various embodiments, the tertiary amine (3°) groups are ionizable. In various embodiments, unlike the ionizable lipids used in Moderna and Pfizer-BioNTech (ALC-0315) mRNA vaccine and Patisiran formulations which contain only a single tertiary amine group, there are multiple tertiary amine groups (e.g., up to 15) and a polypeptide in the presently disclosed lipids. In various embodiments, advantageously, the presence of multiple tertiary amine groups enhances RNA binding, providing stability during the preparation process of LNPs. In various embodiments, advantageously, the presence of multiple tertiary amine groups promotes endosomal release of RNA into cytosol for effective RNA transfection. In various embodiments, the tertiary amines are chemically conjugated to an amino acid (e.g., lysine), a polypeptide (e.g., poly-lysine) backbone, or derivatives thereof. In various embodiments, the compound is an ionizable lipid comprising of a long two-tail lipid and multiple tertiary amine group (with either 1 , 5, 8, 10, or 15 tertiary amine groups) chemically linked via a fully biodegradable polypeptide backbone (poly-lysine) Advantageously, the use of polypeptide in embodiments of the presently disclosed lipids allows for biodegradability, mitigating potential toxicity caused by long term accumulation and cellular/systemic inflammation caused by non-biodegradable ionizable lipids.

In various embodiments, the polypeptide backbone or derivatives thereof are biodegradable.

In various embodiments, -NA2 is a tertiary (3°) amine group (e.g., an ionizable tertiary amine group). In various embodiments, the compound is in an ionized form, where -NA2 has been ionized to become a positively charged group. For example, the tertiary amine group may be ionized to become a quaternary ammonium cation. In various embodiments, -NA2 is ionized/protonated at a pH range of from about 3.0 to about 6.5 to become a positively charged group/ion/cation. In various embodiments, -NA2 is protonated to become -NA2I .

In various embodiments, the compound comprises a lipid compound. The term “compound” may comprise and/or may be used interchangeably with the terms “lipid”, “lipid compound”, “ionizable lipid”, “ionizable lipid compound”, ‘cationic lipid compound", “ionizable cationic lipid compound” or the like. In various embodiments, the compound is amphiphilic/amphipathic and comprises hydrophilic and hydrophobic parts. In various embodiments, the lipid part of the compound is hydrophobic, while groups such as the amine and/or hydroxyl groups in the compound are hydrophilic. In various embodiments, the compound comprises hydrophilic part(s) at the amine groups (e.g., ionizable NA2). In various embodiments, the compound comprises hydrophobic parts/tails/chains/groups at B (e.g., R¹ and/or R²). Advantageously, in various embodiments, the presence of hydrophobic parts/tails/chains/groups in the compound aids in imparting improved cellular uptake and/or transfection, thereby leading to a higher and/or better transfection efficiency.

Advantageously, in various embodiments, the presence of multiple tertiary amine groups enhances RNA binding, providing stability during the preparation process of lipid nanoparticles (LNPs).

It will be appreciated that in various embodiments, the tertiary amine groups are ionized and positively charged at low pH values (e.g., pH 3.0 to 6.5). Advantageously, the positively charged lipids can condense RNA into RNA- encapsulated LNPs upon mixing.

It will be appreciated that in various embodiments, the positive charge on the tertiary amine groups is neutralized to give rise to the original tertiary amine form in the lipids in the physiological environment (pH 7.4). Advantageously, this neutralization process may reduce the toxicity of the LNPs. It will be appreciated that in various embodiments, the presence of multiple tertiary amine groups may increase proton absorption in the endosomes which may promote endosomal release of RNA LNPs for effective RNA transfection. In various embodiments, the compound represented by general formula

(1 ) comprises a structure selected from one or more of the following: general formula

<^1a'> wherein

R⁵ and each R⁶ are independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

R³, and each R⁷ are independently optionally substituted alkylene (e.g., -CH2-), optionally substituted alkenylene or optionally substituted alkynylene; each A is independently a derivative of an epoxy ring-opening product represented by -CHR⁸-CHR⁹OH where R⁸ and R⁹ are each independently selected from H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; and

B is R¹R²N- where R¹ and R² are each independently H, hydrophobic tail/chain/group, or comprises linear aliphatic, branched aliphatic and/or cyclic hydrocarbons, with the proviso that both of R¹ and R² are not H at the same time; general formula (1 b) or (1 b’ ),

(1 b) (1b') wherein

R⁵ and each R⁶ are independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

R³, and each R⁷ are independently optionally substituted alkylene (e.g., -CH2-), optionally substituted alkenylene or optionally substituted alkynylene; n is from about 1 to about 100, from about 1 to about 50, from about 1 to about 40, from about 1 to about 30, from about 1 to about 20, from about 1 to about 19, from about 2 to about 18, from about 3 to about 17, from about 4 to about 16, from about 5 to about 15, from about 6 to about 14, from about 7 to about 13, from about 8 to about 12, from about 9 to about 11 , or about 10; each A is represented by R¹¹ to R¹³ where R¹¹ to R¹³ are each independently selected from H, aliphatic alcohol, optionally substituted alkyl (e.g., alkyl alcohol), optionally substituted alkenyl (e.g., alkenyl alcohol) or optionally substituted alkynyl (e.g., alkynyl alcohol); and B is R¹R²N- where R¹ and R² are each independently H, hydrophobic tail/chain/group, or comprises linear aliphatic, branched aliphatic and/or cyclic hydrocarbons, with the proviso that both of R¹ and R² are not H at the same time; general formula (1 c) or (1 o’),

(1c) (1c¹) wherein

R⁵ and each R⁶ are independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

R³, and each R⁷ are independently optionally substituted alkylene (e.g., -CH2-), optionally substituted alkenylene or optionally substituted alkynylene; each A is independently a derivative of an epoxy ring-opening product represented by -CHR⁸-CHR⁹OH where R⁸ and R⁹ are each independently selected from H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; and

B is R¹R²N- where R¹ and R² are each independently H, hydrophobic tail/chain/group, or comprises linear aliphatic, branched aliphatic and/or cyclic hydrocarbons, with the proviso that both of R¹ and R² are not H at the same time; or general formula (1 d) or (1 d’)

(1d) (1d') wherein

R⁵ and each R⁶ are independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

R³, and each R⁷ are independently optionally substituted alkylene (e.g., -CH2-), optionally substituted alkenylene or optionally substituted alkynylene; each A is independently a derivative of an epoxy ring-opening product represented by -CHR⁸-CHR⁹OH where R⁸ and R⁹ are each independently selected from H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; and

B is R²³O- where R²³ is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

In various embodiments, in general formula (1 ), when m = 1 , n = 1 to 100, I = 0, A is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl, and B is R¹R²N-, the compound represented by general formula (1 ) is lipid-b-poly(Lys) (LP). For example, the compound may be represented by general formula (1 b) or (1 b’). In various embodiments, in general formula (1 ), when m = 0, n = 0, I = 1 , A is an aliphatic alcohol, optionally substituted alkyl (e.g., alkyl alcohol), optionally substituted alkenyl (e.g., alkenyl alcohol), optionally substituted alkynyl (e.g., alkynyl alcohol), an epoxy ring-opening product and/or derivatives thereof, and B is R¹R²N-, the compound represented by general formula (1 ) is lipid-Lys-lipid derivative with a degree of polymerization (DP) of 1 (LPL1 ). For example, the compound may be represented by general formula (1c) or (1c’).

In various embodiments, in general formula (1 ), when m = 1 , n = 1 to 100, I = 1 , A is an aliphatic alcohol, optionally substituted alkyl (e.g., alkyl alcohol), optionally substituted alkenyl (e g., alkenyl alcohol), optionally substituted alkynyl (e.g., alkynyl alcohol), an epoxy ring-opening product and/or derivatives thereof, and B is R¹R²N-, the compound represented by general formula (1 ) is lipid-b- ionizable poly(Lys)-lipid (LPL). For example, the compound may be represented by general formula (1 a) or (1 a’).

In various embodiments, in general formula (1 ), when m = 0, n = 1 to 100, I = 1 , A is an aliphatic alcohol, optionally substituted alkyl (e.g., alkyl alcohol), optionally substituted alkenyl (e.g., alkenyl alcohol), optionally substituted alkynyl (e.g., alkynyl alcohol), an epoxy ring-opening product and/or derivatives thereof, and B is R²³O-, the compound represented by general formula (1 ) is ionizable poly( Lys)-lipid (PL). For example, the compound may be represented by general formula (1 d) or (1 d’).

In various embodiments, in general formula (1), I and m are not 0 at the same time.

In various embodiments, the compound comprises a structure selected from one or more of the following:

LPL2 (n = 15);

In various embodiments, the compound has a molecular weight of from about 500 g/mol to about 50,000 g/mol, 1000 g/mol to about 49,000 g/mol, from about 2000 g/mol to about 48,000 g/mol, from about 3000 g/mol to about 47,000 g/mol, from about 4000 g/mol to about 46,000 g/mol, from about 5000 g/mol to about 45,000 g/mol, from about 6000 g/mol to about 44,000 g/mol, from about 7000 g/mol to about 43,000 g/mol, from about 8000 g/mol to about 42,000 g/mol, from about 9000 g/mol to about 41 ,000 g/mol, from about 10,000 g/mol to about 40,000 g/mol, from about 11 ,000 g/mol to about 39,000 g/mol, from about 12,000 g/mol to about 38,000 g/mol, from about 13,000 g/mol to about 37,000 g/mol, from about 14,000 g/mol to about 36,000 g/mol, from about 15,000 g/mol to about 35,000 g/mol, from about 16,000 g/mol to about 34,000 g/mol, from about 17,000 g/mol to about 33,000 g/mol, from about 18,000 g/mol to about 32,000 g/mol, from about 19,000 g/mol to about 31 ,000 g/mol, from about 20,000 g/mol to about 30,000 g/mol, from about 21 ,000 g/mol to about 29,000 g/mol, from about 22,000 g/mol to about 28,000 g/mol, from about 23,000 g/mol to about 27,000 g/mol, from about 24,000 g/mol to about 26,000 g/mol, or about 25,000 g/mol.

METHOD OF PREPARING COMPOUND

There is provided a method of preparing a compound represented by general formula (1 ) as disclosed herein. In various embodiments, the method comprises steps of preparing a compound represented by general formula (1 a) or (1a’) which is general formula (1 ) when m = 1 , n = 1 to 100, I = 1 , A (corresponding to -CHR⁸-CHR⁹OH) is a derivative of an epoxy ring-opening product, and B is R¹R²N- (i.e., lipid-b- ionizable poly(Lys)-lipid (LPL)), the method comprising:

(a) reacting a lipid-b-poly(Lys) compound represented by general formula (1 b’) with an epoxy (e.g., 1 ,2-epoxyalkane) represented by general formula (3) to obtain the compound represented by general formula (1 a) or (1 a’):

wherein

R¹ to R³, R⁵ to R⁹, and n contain one or more features and/or share one or more properties that are similar to those described above; and R¹¹ to R¹³ are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

In various embodiments, the method further comprises, prior to step (a): (a-i) reacting/polymerizing one or more N-carboxyanhydride (NCA) monomers (e.g., protected amino acid comprising NCA such as N⁶- benzyloxycarbonyl-lysine-N-carboxyanhydride (Lys-NCA)) represented by general formula (5) with an amide compound (e.g., 2-amino-N, N- ditetradecylacetamide) represented by general formula (4) to obtain a protected lipid-b-poly(Lys) compound represented by general formula (6):

W (5) (6) wherein

R¹ to R³, R⁵ to R⁷, R¹¹, R¹² and n contain one or more features and/or share one or more properties that are similar to those described above; R¹⁰ is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and

PG¹ is a protecting group such as N-carboxybenzyl or benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), or 9-fluorenylmethyloxycarbonyl (Fmoc).

(a-ii) deprotecting the protected lipid-b-poly(Lys) compound represented by general formula (6) obtained from step (a-i) to obtain the compound represented by general formula (1 b’):

(6) (1b¹) wherein

R¹ to R³, R⁵ to R⁷, R¹¹ to R¹³, n, and PG¹ contain one or more features and/or one or more properties that are similar to those described above;

In various embodiments, the polymerizing/reacting step (a-i) comprises ring opening polymerization (ROP) of the NCA ring/group in the compound represented by general formula (5). It will be appreciated that in step (a-i), the one or more NCA monomers (i.e., represented by general formula (5)) may be added sequentially to an initiator (e g., amide compound represented by general formula (4)) to form block copolypeptides.

In various embodiments, the deprotection of the protected lipid-b-poly(Lys) compound represented by general formula (6) is carried out in the presence of one or more acids. In various embodiments, the deprotecting/deprotection step (a-ii) comprises subjecting the protected lipid-b-poly(Lys) compound to one or more of an acidic compound such as trifluoracetic acid (TFA) and hydrobromic acid (HBr) in acetic acid (AcOH). In various embodiments, the method comprises steps of preparing a compound represented by general formula (1 b) or (1 b’) which is general formula (1 ) when m = 1 , n = 1 to 100, I = 0, A (corresponding to R¹¹, R¹², R¹³) is H, optionally substituted alkyl, or optionally substituted alkenyl or optionally substituted alkynyl, and B is R¹R²N- (i.e., lipid-b-poly(Lys) (LP)), the method comprising steps (a-i) and (a-ii) as described above.

In various embodiments, the method further comprises, prior to step (a-i): (a-i-i) reacting a protected amino acid (e g., N⁶-Cbz-lysine) represented by general formula (7) with a carbonylating agent (e.g., triphosgene) to obtain the protected amino acid comprising NCA (e g., N⁶- benzyloxycarbonyl-lysine-N-carboxyanhydride (Lys-NCA)) represented by general formula (5):

(7) (5) wherein

R⁸, R⁷, R¹¹, and PG¹ contain one or more features and/or share one or more properties that are similar to those described above; and R¹⁶ is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

In various embodiments, PG¹ is N-carboxybenzyl or benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), or 9-fluorenylmethyloxycarbonyl (Fmoc).

In various embodiments, the method further comprises, prior to step (a-i): (a-i-ii) reacting an amine compound (e.g., ditetradecylamine) represented by general formula (8) with a protected amino acid compound represented by general formula (9) (e.g., Boc-glycine) in the presence of one or more coupling agent(s) (e.g., 2-(1 H-Benzotriazol-1 -yl)-1 , 1 ,3,3- tetramethylaminium hexafluorophosphate (HBTU)) and a base (e.g., N,N- Diisopropylethylamine (DIPEA)) to obtain a carbamate compound (e.g., tert-Butyl (2-(ditetradecylamino)-2-oxoethyl)carbamate) represented by general formula (10):

(8) (9) (10) wherein

R¹ to R³, and R⁵ contain one or more features and/or share one or more properties that are similar to those described above;

R¹⁴, and R¹⁵ are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and

PG² is a protecting group such as tert-butyloxycarbonyl (Boc), N- carboxybenzyl or benzyloxycarbonyl (Cbz), or 9- fluorenylmethyloxycarbonyl (Fmoc); and

(a-i-iii) deprotecting the carbamate compound represented by general formula (10) obtained from step (a-i-ii) to obtain the amide compound (e g., 2- amino-N, N-ditetradecylacetamide) represented by general formula (4):

wherein

R¹ to R³, R⁵, R¹⁰, and PG² contain one or more features and/or share one or more properties that are similar to those described above.

In various embodiments, the coupling agent(s) in step (a-i-ii) may be HBTU (2-(1 H-Benzotriazol-1 -yl)-1 ,1 ,3,3-tetramethylaminium hexafluorophosphate, hexafluorophosphate N,N-Diisopropylethylamine (DIPEA)azabenzotriazole tetramethyl uronium) (HATU), or 1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in combination with N-hydroxysuccinimide (NHS).

In various embodiments, the base in step (a-i-ii) may be N,N- Diisopropylethylamine (DIPEA), triethylamine (TEA), hydroxybenzotriazole (HOBt), or 4-dimethylaminopyridine (DMAP).

In various embodiments, the deprotection of the carbamate compound represented by general formula (10) is carried out in the presence of one or more acids. In various embodiments, the deprotecting/deprotection step (a-i-iii) comprises subjecting the carbamate compound to one or more of an acidic compound such as trifluoracetic acid (TFA), hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, formic acid, or acetic acid.

In various embodiments, the method comprises steps of preparing a compound represented by general formula (1 c) or (1c’) which is general formula (1 ) when m = 0, n = 0, I = 1 , A (corresponding to -CHR⁸-CHR⁹OH) is as defined above such as a derivative of an epoxy ring-opening product, and B is R¹R²N- (i.e., lipid-Lys-lipid with DP of 1 (LPL1 )), the method comprising:

(1c) (1c¹) (b) reacting an amine compound (e.g., ditetradecylamme) represented by general formula (8) with a protected amino acid compound represented by general formula (11 ) (e g., N², N⁶-bis(tert-butoxycarbonyl)lysine) in the presence of one or more coupling agent(s) (e.g., 2-(1 H-Benzotriazol-1-yl)- 1 ,1 ,3,3-tetramethylaminium hexafluorophosphate (HBTU)) and a base (e.g., N,N-diisopropylethylamine (DIPEA)) to obtain a dicarbamate compound (e.g., di-tert-butyl (6-(ditetradecylamino)-6-oxohexane-1 ,5- diyl)dicarbamate) represented by general formula (12):

(8) (11) (12) wherein

R¹ to R², R⁷, and R¹⁴ contain one or more features and/or share one or more properties that are similar to those described above;

R¹⁷to R¹⁹ are each independently H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; and

PG⁴ and PG⁵ are each independently a protecting group such as tertbutyloxycarbonyl (Boc), N-carboxybenzyl or benzyloxycarbonyl (Cbz), or 9-fluoreny Im ethyloxycarbonyl (Fmoc);

(c) deprotecting the dicarbamate compound represented by general formula

(12) obtained from step (b) to obtain an amide compound (e.g., 2,6- diamino-N,N-ditetradecylhexanamide) represented by general formula

(13):

(12) (13) wherein

R¹ to R², R⁷, R¹⁸ to R¹⁹, PG⁴, and PG⁵ contain one or more features and/or share one or more properties that are similar to those described above; and

R²⁰ and R²¹ are each independently H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; and

(d) reacting the amide compound represented by general formula (13) with an epoxy (e g., 1 ,2-epoxyalkane) represented by general formula (3) to obtain the compound represented by general formula (1 c) or (1 c’):

(13) (3)

(1 c) (1c') wherein

R¹ to R³, R⁵ to R⁹, and R¹⁸ to R²¹ contain one or more features and/or share one or more properties that are similar to those described above; and R²⁰ and R²¹ are each independently H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl.

In various embodiments, the coupling agent(s) in step (b) may be HBTU (2-(1 H-Benzotriazol-1 -yl)-1 ,1 ,3,3-tetramethylaminium hexafluorophosphate, hexafluorophosphate N,N-Diisopropylethylamine (DIPEA)azabenzotriazole tetramethyl uranium) (HATU), or 1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in combination with N-hydroxysuccinimide (NHS).

In various embodiments, the base in step (b) may be N,N- Diisopropylethylamine (DIPEA), triethylamine (TEA), hydroxybenzotriazole (HOBt), or 4-dimethylaminopyridine (DMAP).

In various embodiments, the deprotection of the dicarbamate compound represented by general formula (12) is carried out in the presence of one or more acids. In various embodiments, the deprotecting/deprotection step (c) comprises subjecting the carbamate compound to one or more of an acidic compound such as trifluoracetic acid (TFA), hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, formic acid, or acetic acid.

In various embodiments, the method comprises steps of preparing a compound represented by general formula (1 d) or (1d’) which is general formula

(1 ) when m = 0, n = 1 to 100, I = 1 , A (corresponding to -CHR⁸-CHR⁹OH) is as defined above such as a derivative of an epoxy ring-opening product, and B is R²³O- (i.e., poly(Lys)-lipid derivative (PL)), the method comprising:

(e) reacting a poly-(Lys) derivative represented by general formula (14) with an epoxy (e g., 1 ,2-epoxyalkane) represented by general formula (3) to obtain the compound represented by general formula (1 d) or (1 d’):

wherein

R⁶ to R⁹, and R¹¹ contain one or more features and/or share one or more properties that are similar to those described above; and

R²²to R²⁴ are each independently H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl.

In various embodiments, the method further comprises, prior to step (e):

(e-i) reacting a protected amino acid comprising an amine group (e g., H- Lys(Boc)-Ome) represented by general formula (15) with a protected amino acid comprising N-carboxyanhydride (NCA) (e.g., N⁶- benzyloxycarbonyl-lysine-N-carboxyanhydride (Lys-NCA)) represented by general formula (5) via ring-opening polymerization (ROP) and subsequently deprotecting the resultant product to obtain the poly-(Lys) derivative represented by general formula (1 ):

(15) (5) (14) wherein

R⁶, R⁷, R¹¹, R²²to R²⁴, and PG¹ contain one or more features and/or share one or more properties that are similar to those described above; and PG⁶ is a protecting group such as tert-butyloxycarbonyl (Boc), N- carboxybenzyl or benzyloxycarbonyl (Cbz), or 9- fluorenylmethyloxycarbonyl (Fmoc).

In various embodiments, the deprotection of the ring-opening polymerization product in step (e-i) is carried out in the presence of one or more acids. In various embodiments, the deprotecting/deprotection step (a-ii) comprises subjecting the resultant product to one or more of an acidic compound such as trifluoracetic acid (TFA), hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, formic acid, or acetic acid.

In various embodiments, the method comprises optionally ionizing one or more -NA2 groups to become positively charged group(s), for e.g., -NA2H⁺ groups.

In various embodiments, the reacting/polymerizing/deprotecting step (a), (a-i), (a-ii), (a-i-i), (a-i-ii), (a-i-iii), (b), (c), (d), (e), and/or (e-i) comprises one or more of the following steps: suspending, dispersing, mixing, stirring, and/or dissolving. In various embodiments, the reacting/polymerizing/deprotecting step (a), (a-i), (a-ii), (a-i-i), (a-i-ii), (a-i-iii), (b), (c), (d), (e), and/or (e-i) is/are performed in the presence of an organic solvent. The organic solvent may be an organic solvent such as hexane, ether, methanol, ethanol, dichloromethane (DCM), tetra hydrofuran (THF), acetonitrile, chloroform, or ethyl acetate. In various embodiments, the organic solvent may be provided in a dry or anhydrous form.

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

In various embodiments, the reacting/polymerizing/deprotecting step (a), (a-i), (a-ii), (a-i-i), (a-i-ii), (a-i-iii), (b), (c), (d), (e), and/or (e-i) is/are performed over a time duration of from about 1 hour to about 200 hours, from about 10 hours to about 190 hours, from about 20 hours to about 180 hours, from about 30 hours to about 170 hours, from about 40 hours to about 160 hours, from about 50 hours to about 150 hours, from about 60 hours to about 140 hours, from about 70 hours to about 130 hours, from about 80 hours to about 120 hours, from about 90 hours to about 110 hours, or about 100 hours.

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

In various embodiments, the method further comprises: (f-i) a step of isolating the compound represented by general formula (1 a) or (1 a’) after step (a);

(f-ii) a step of isolating the compound represented by general formula (1 b) or (1 b’) after step (a-ii);

(f-iii) a step of isolating the protected amino acid comprising NCA represented by general formula (5) after step (a-i-i);

(f-iv) a step of isolating the amide compound represented by general formula (4) after step (a-i-iii);

(f-v) a step of isolating the compound represented by general formula (1c) or (1 c’) after step (d);

(f-vi) a step of isolating the compound represented by general formula (1 d) or (1 d’) after step (e); and

(f-vii) a step of isolating the poly-(Lys) derivative represented by general formula (14) after step (e-i).

In various embodiments, the isolating step(s) comprises one or more of the following steps: re-dissolving, purifying, centrifuging, washing, precipitating, recrystallizing, and/or dialysis.

In various embodiments, the dialysis medium comprises water-miscible solutions such as ethanol, methanol, or deionized water.

In various embodiments, the washing medium comprises aqueous medium/solutions such as salt solution, water, or acid. The salt solution may be chloride salt solution such as saturated sodium chlorine solution (brine). The acid may be citric acid.

In various embodiments, the salt solution comprises highly concentrated/saturated salt solution.

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

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

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

In various embodiments, the currently disclosed lipids may be synthesized in just 5 steps through an amino-epoxy reaction, whereas the synthesis of ALC- 0315 requires 6 to 7 steps, significantly reducing the manufacturing costs of the lipids.

NANOPARTICLE COMPOSITION

Advantageously, in various embodiments, the ionizable property of the compound represented by general formula (1 ) (due to presence of ionizable amine group) allows for the condensation and encapsulation/loading of molecules/cargoes into embodiments of the compound, thereby forming nanoparticles in a composition. In various embodiments, embodiments of the compound are capable of forming nanoparticles in a composition. In various embodiments, in the presence of a composition comprising molecules/cargoes (e.g., therapeutic agent, prophylactic agent and/or biological agent), the one or more amine group(s) in the compound (e g., ionized/protonated amine group(s) in the lipid compound) condenses and encapsulates/loads the molecules/cargoes into the compound to form nanoparticles (e.g., lipid nanoparticles (LNPs)) in the composition. 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.

There is provided a nanoparticle composition comprising:

(i) the compound disclosed herein (e.g., represented by general formula (1 )); and

(ii) a therapeutic and/or prophylactic agent and/or biological agent that is encapsulated/loaded/coupled/bonded/linked/bound in/to the compound disclosed herein (e.g., represented by general formula (1 )).

In various embodiments, the compound disclosed herein (e.g., represented by general formula (1 )) is capable of being ionized (e.g., protonated) at a low pH range of from 3.0 to 6.5) such that the composition encapsulates a therapeutic and/or prophylactic agent and/or biological agent that is coupled/bonded/linked/bound to the composition/nanoparticles. In various embodiments, the compound represented by general formula (1) is capable of being ionized (e.g., protonated) at a low pH such that the composition encapsulates a therapeutic and/or prophylactic agent and/or biological agent that is coupled/bonded/linked/bound to the composition/nanoparticles. The therapeutic and/or prophylactic agent and/or biological agent may be coupled/bonded/linked/bound to the composition/nanoparticles via electrostatic interaction and/or other physical interactions. In various embodiments, the therapeutic and/or prophylactic agent and/or biological agent is electrostatically and/or physically coupled/bonded/linked/bound to the composition/nanoparticle.

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

In various embodiments, the composition further comprises: (i) neutral/helper lipid;

(ii) cholesterol or derivatives thereof; and

(iii) polyethylene glycol (PEG)-lipid conjugate, or other amphiphilic lipids.

The term “polyethylene glycol (PEG)-modified lipid” may comprise and/or may be used interchangeably with the terms “PEGylated lipid” and “lipid modified with PEG”.

In various embodiments, the compound disclosed herein (e g., represented by general formula (1 )), neutral/helper lipid, cholesterol or derivatives thereof, and PEG-lipid conjugate are mixed/dissolved in an organic solvent. In various embodiments, any organic solvent that effectively serves as a medium to contain the components of the reaction mixture (e.g., reactants/substrates) may be used in embodiments of the reaction mixture disclosed herein. In various embodiments, the organic solvent is capable of substantially dissolving the components present in the mixture. In various embodiments, the organic solvent comprises ethanol, methanol, isopropanol, acetonitrile, ethyl acetate, dimethyl sulfoxide (DMSO), dimethylformamide (DMF) or the like or combinations thereof.

In various embodiments, the compound disclosed herein (e.g., represented by general formula (1 )), neutral/helper lipid, cholesterol or derivatives thereof, and PEG-lipid conjugate are mixed at a molar ratio of about 30 to 50: about 5 to 50 : about 5 to 60 : about 1 to 5.

In various embodiments, the compound disclosed herein (e.g., represented by general formula (1 )), neutral/helper lipid, cholesterol or derivatives thereof, and PEG-lipid conjugate are mixed at a molar ratio of 46:9.4:42:1.6.

In various embodiments, the compound disclosed herein (e.g., represented by general formula (1 )), neutral/helper lipid, cholesterol or derivatives thereof, and PEG-lipid conjugate are mixed at a molar ratio of 46:42:9.4:1.6.

In various embodiments, the compound disclosed herein (e g., represented by general formula (1 )), neutral/helper lipid, cholesterol or derivatives thereof, and PEG-lipid conjugate are mixed at a molar ratio of 30:12.3:55.7:2.

In various embodiments, the compound disclosed herein (e g., represented by general formula (1 )), neutral/helper lipid, cholesterol or derivatives thereof, and PEG-lipid conjugate are mixed at a molar ratio of 46.3:9.4:42.7:1.6.

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, and the like and combinations thereof. In various embodiments, the cholesterol and derivatives thereof is selected from cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, avenasterol, or the like or combinations thereof.

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

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.

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

NANOPARTICLES

There is provided nanoparticles (e.g., lipid nanoparticles) comprising:

(i) the compound disclosed herein (e.g., represented by general formula (1 )); and

(ii) a therapeutic and/or prophylactic agent and/or biological agent that is encapsulated/loaded/coupled/bonded/linked/bound in/to the compound disclosed herein (e.g., represented by general formula (1 )). In various embodiments, the nanoparticle has a N:P or N/P ratio (i.e., molar ratio of ionizable (in the physiological pH range) nitrogen atoms in a nanoparticle/compound 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 50:1 . The nanoparticles may have a N:P or N/P ratio that is from about 1 :1 to about 50:1 , about 2:1 , about 5:1 , about 10:1 about 15:1 , about 20:1 , about 25:1 , about 30:1 , about 35:1 , about 40:1 , or about 45:1.

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, miRNA, pDNA, DNA and oligonucleotides). For example, the optimal N/P ratio may be different for siRNA, mRNA, DNA and oligonucleotides. 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).

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 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 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 %, or at least about 99.0 %. In various embodiments, the composition/nanoparticle may facilitate the successful encapsulation of siRNA and mRNA into lipid nanoparticles, resulting in high transfection efficiency for both siRNA and mRNA.

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 or comparable to those using other ionizable lipids (e.g., ALC-0315). In various embodiments, advantageously, the amount of amine groups needed in the presently disclosed lipids for effective siRNA transfection is half of that in ALC-0315 LNPs (Pfizer-BioNTech mRNA vaccine formulation), indicating lower cost and better biocompatibility of the presently disclosed LNPs.

In various embodiments, the nanoparticle has a nucleic acid transfection efficiency that is less than, no less or higher than that of a corresponding nanoparticle using ALC-0315 ionizable lipid under similar conditions. For example, the mRNA transfection efficiency may be 0 time, 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 about 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, or at least 50 times higher than that of a corresponding nanoparticle using ALC-0315 ionizable lipid under similar conditions.

It will be appreciated that in various embodiments, the nanoparticle may still induce a therapeutic and/or vaccination effect even when its transfection efficiency is lower than that of ALC-0315 ionizable lipid.

In various embodiments, the nanoparticle has an average particle size (or diameter) of from about 20 nm to about 650 nm, from about 30 nm to about 640 nm, from about 40 nm to about 630 nm, from about 50 nm to about 620 nm, from about 60 nm to about 610 nm, from about 70 nm to about 600 nm, from about 80 nm to about 590 nm, from about 90 nm to about 580 nm, from about 100 nm to about 570 nm, from about 110 nm to about 560 nm, from about 120 nm to about 550 nm, from about 130 nm to about 540 nm, from about 140 nm to about 530 nm, from about 150 nm to about 520 nm, from about 160 nm to about 510 nm, from about 170 nm to about 500 nm, from about 180 nm to about 490 nm, from about 190 nm to about 480 nm, from about 200 nm to about 470 nm, from about 210 nm to about 460 nm, from about 220 nm to about 450 nm, from about 230 nm to about 440 nm, from about 240 nm to about 430 nm, from about 250 nm to about 420 nm, from about 260 nm to about 410 nm, from about 270 nm to about 400 nm, from about 280 nm to about 390 nm, from about 290 nm to about 380 nm, from about 300 nm to about 370 nm, from about 310 nm to about 360 nm, from about 320 nm to about 350 nm, from about 330 nm to about 340 nm, or about 335 nm. In various embodiments, siRNA LNPs may comprise nanoparticles with an average particle size of 70 nm and a neutral charge, while mRNA LNPs may comprise nanoparticles with an average particle size of 100 nm and a neutral charge.

In various embodiments, the composition comprising the nanoparticles has a polydispersity index (PDI) of from about 0.020 to about 0.700, from about 0.030 to about 0.690, from about 0.040 to about 0.680, from about 0.050 to about 0.670, from about 0.060 to about 0.660, from about 0.070 to about 0.650, from about 0.080 to about 0.640, from about 0.090 to about 0.630, from about 0.100 to about 0.620, from about 0.110 to about 0.610, from about 0.120 to about 0.600, from about 0.130 to about 0.590, from about 0.140 to about 0.580 nm, from about 0.150 to about 0.570 nm, from about 0.160 to about 0.560 nm, from about 0.170 to about 0.550 nm, from about 0.180 to about 0.540 nm, from about 0.190 to about 0.530 nm, from about 0.200 to about 0.520 nm, from about 0.210 to about 0.510 nm, from about 0.220 to about 0.500 nm, from about 0.230 to about 0.490 nm, from about 0.240 to about 0.480 nm, from about 0.250 to about 0.470 nm, from about 0.260 to about 0.460 nm, from about 0.270 to about 0.450 nm, from about 0.280 to about 0.440 nm, from about 0.290 to about 0.430 nm, from about 0.300 to about 0.420 nm, from about 0.310 to about 0.410 nm, from about 0.320 to about 0.400 nm, from about 0.330 to about 0.390 nm, from about 0.340 to about 0.380 nm, from about 0.350 to about 0.370 nm, or about 0.360 nm. In various embodiments, the nanoparticles have a narrow particle size distribution (PDI < 0.3), and/or the nanoparticle composition is relatively/substantially homogenous distributions and is of acceptable sizes for in vivo application.

In various embodiments, the nanoparticles have a zeta potential of from about -15.0 mV to about +20.0 mV, from about -14.0 mV to about +19.0 mV, from about -13.0 mV to about +18.0 mV, from about -12.0 mV to about +17.0 mV, from about -11.0 mV to about +16.0 mV, from about -10.0 mV to about +15.0 mV, from about -9.0 mV to about +1 .0 mV, from about -8.0 mV to about +13.0 mV, from about -7.0 mV to about +12.0 mV, from about -6.0 mV to about +11.0 mV, from about -5.0 mV to about +10.0 mV, from about -4.0 mV to about +9.0 mV, from about -3.0 mV to about +8.0 mV, from about -2.0 mV to about +7.0 mV, from about -1 .0 mV to about +6.0 mV, from about 0 mV to about +5.0 mV, from about +1.0 mV to about +4.0 mV, or from about +2.0 mV to about +3.0 mV in saline (e.g., phosphate-buffered saline (PBS)) or in a physiological environment. Advantageously, in various embodiments, the nanoparticles have a substantially neutral surface charge, making the nanoparticles suitable/desirable for in vivo applications.

In various embodiments, the composition/compound/nanoparticles is/are biocompatible, i.e., the composition/compound/nanoparticle is compatible with biological systems or parts of the biological systems without substantially or significantly eliciting an adverse physiological response such as a toxic reaction/response (e.g., cytotoxicity), an immune reaction/response, an injury or the like when used on the human or animal body. In various embodiments, the composition/compound/nanoparticle is substantially devoid of substances that elicit an adverse physiological response. Advantageously, the nanoparticles (e.g., lipid nanoparticles) are capable of binding therapeutic agent, prophylactic agent and/or biological agent (e.g., RNA) effectively and/or providing high transfection efficiency without causing/inducing substantial or any cytotoxicity. METHOD OF PREPARING NANOPARTICLES

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

(g) preparing an aqueous composition comprising therapeutic and/or prophylactic agent and/or biological agent;

(h) mixing the aqueous composition with the composition as described above to obtain a nanoparticle.

In various embodiments, step (g) comprises mixing therapeutic and/or prophylactic agent and/or biological agent in an aqueous buffer. The aqueous buffer may be sodium acetate buffer solution, citrate buffer solution, phosphate buffer solution, glycine buffer solution, the like or combinations thereof.

In various embodiments, mixing step (g) is performed at a pH value of from about 2.00 to about 6.00, from about 2.10 to about 5.90, from about 2.20 to about 5.80, from about 2.30 to about 5.70, from about 2.40 to about 5.60, from about 2.50 to about 5.50, from about 2.60 to about 5.40, from about 2.70 to about 5.30, from about 2.80 to about 5.20, from about 2.90 to about 5.10, from about 3.00 to about 5.00, from about 3.10 to about 4.90, from about 3.20 to about 4.80, from about 3.30 to about 4.70, from about 3.40 to about 4.60, from about 3.50 to about 4.50, from about 3.60 to about 4.40, from about 3.70 to about 4.30, from about 3.80 to about 4.20, from about 3.90 to about 4.10, or about 4.00.

In various embodiments, step (h) comprises mixing the aqueous composition with the composition as described in any one of the preceding AS at a volume ratio of from about 6:1 to about 1 :1 , from about 5:1 to about 1 :1 , from about 4:1 to about 1 :1 , from about 3:1 to about 1 :1 , or from about 2:1 to about 1 :1.

In various embodiments, step (h) of mixing the aqueous composition with the composition comprises using a microfluidic device or a pulse vortex. For example, a microfluidic device may be used to perform microfluidic mixing. The micro-mixing may be performed via passive mixing using passive micromixers such as T-shaped or Y-shaped microfluidic mixers parallel lamination, sequential, focusing enhanced mixers or droplet micromixers. The micro-mixing may also be performed via active mixing using external forces such as pressure field, electrokinetic, dielectrophoretic, electrowetting, magneto-hydrodynamic or ultrasound. Advantageously, as microfluidic mixing comprises mixing the two compositions (i.e., aqueous composition and composition disclosed herein) in a controlled manner and/or with a specif ied/fixed/control led mixing ratio, the interaction between the two compositions (e.g., between ionizable lipid and therapeutic, prophylactic and/or biological agent) is regulated, thereby producing nanoparticles with a smaller particle size and/or with a narrow size distribution or homogeneity (e.g., smaller PDI).

In various embodiments, the yield of the compound represented by general formula (1 ) 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 about60%, or about 50%.

In various embodiments, the method further comprises removing the organic phase (e.g., ethanol). For example, removing the organic phase may include dialysing the nanoparticles to remove residual organic solvents that are present. Advantageously, removal of the organic phase through dialysis may improve the encapsulation efficiency of the therapeutic and/or prophylactic agent and/or biological agent.

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

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

In various embodiments, there is also provided a carrier, a nanocarrier, a delivery system/vehicle, a compound or ionized form thereof, a nanoparticle composition, nanoparticles (or lipid nanoparticles) disclosed herein for use in the treatment or prophylaxis of a disease, disorder or condition, the use of said carrier, a nanocarrier, a delivery system/vehicle, a compound or ionized form thereof, a nanoparticle composition, nanoparticles (or lipid nanoparticles) in the manufacture of a medicament for the treatment or prophylaxis of a disease, disorder or condition and/or a method of treatment or prophylaxis of a disease, disorder or condition, comprising a step of administering (e.g., in a therapeutically effective amount of) said carrier, a nanocarrier, a delivery system/vehicle, a compound or ionized form thereof, a nanoparticle composition, nanoparticles (or lipid nanoparticles) to a subject (e.g., vertebrate such as a human or a large veterinary mammal (e.g., horses, cattle, deer, sheep, llamas, goats, pigs) in need thereof. The disease, disorder or condition may be selected from the group consisting of infectious/contagious diseases, viral infections (i.e., diseases caused by virus), bacterial infections (i.e., diseases caused by bacteria), fungal infections (i.e., diseases caused by fungi), respiratory diseases or the like, or combinations thereof. In various embodiments, the disease, disorder or condition is mediated by an influenza virus (e.g., influenza A, B, C and/or D virus). For example, the disease may be influenza A, B, C or D such as H1 N1 , H3N2). In various embodiments, the disease, disorder or condition is mediated by a coronavirus (e.g., severe acute respiratory syndrome coronavirus such as SARS- CoV-2 or SARS-CoV-1 ). For example, the disease, disorder or condition may be SARS-CoV-2 coronavirus disease. In various embodiments, there is also provided a carrier, a nanocarrier, a delivery system/vehicle, a compound or ionized form thereof, a nanoparticle composition, nanoparticles (or lipid nanoparticles) disclosed herein for use in encapsulating and/or delivering a therapeutic, prophylactic, and/or biological agent to a subject, cell, cytosol, tissue or organ (e.g., a mammalian cell, cytosol, tissue, or organ), the use of said carrier, a nanocarrier, a delivery system/vehicle, a compound or ionized form thereof, a nanoparticle composition, nanoparticles (or lipid nanoparticles) in the manufacture of a medicament for encapsulating and/or delivering a therapeutic, prophylactic and/or biological agent to a subject, cell, cytosol, tissue or organ (e.g., a mammalian cell, cytosol, tissue or organ), and/or a method of delivering a therapeutic, prophylactic and/or biological agent to a subject, cell, cytosol, tissue or organ (e.g., a mammalian cell, cytosol, tissue, or organ), comprising a step of administering (e.g., in a therapeutically effective amount of) said carrier, a nanocarrier, a delivery system/vehicle, a compound or ionized form thereof, a nanoparticle composition, nanoparticles (or lipid nanoparticles) to a subject (e.g., vertebrate such as a human or a large veterinary mammal (e.g., horses, cattle, deer, sheep, llamas, goats, pigs) in need thereof.

In various embodiments, there is also provided a carrier, a nanocarrier, a delivery system/vehicle, a compound or ionized form thereof, a nanoparticle composition, nanoparticles (or lipid nanoparticles) disclosed herein for use in inducing an immune response in a subject (e.g., vertebrate such as a human or a large veterinary mammal (e.g., horses, cattle, deer, sheep, llamas, goats, pigs), the use of said carrier, a nanocarrier, a delivery system/vehicle, a compound or ionized form thereof, a nanoparticle composition, nanoparticles (or lipid nanoparticles) in the manufacture of a medicament for inducing an immune response in a subject, and/or a method of inducing an immune response in a subject, comprising a step of administering (e g., in a therapeutically effective amount of) said carrier, a nanocarrier, a delivery system/vehicle, a compound or ionized form thereof, a nanoparticle composition, nanoparticles (or lipid nanoparticles) to a subject in need thereof. In various embodiments, an immune response in the subject is to be induced through the administration of the compound or ionized form thereof, a nanoparticle composition, nanoparticles (or lipid nanoparticles) thereto. In various embodiments, by inducing an immune response in the subject, the subject is protected against various diseases, disorders or conditions e.g., infectious/contagious diseases, viral infections (i.e., diseases caused by virus), bacterial infections (i.e. , diseases caused by bacteria), fungal infections (i.e., diseases caused by fungi), respiratory diseases or the like, or combinations thereof as mentioned herein. The carrier, nanocarrier, delivery system/vehicle, compound 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 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 carrier, nanocarrier, delivery system/vehicle, compound or ionized form thereof, nanoparticle composition, nanoparticles prepared from embodiments of the method disclosed herein comprises one or more of the following characteristics or properties: broad applicability (e.g., can be used to encapsulate, deliver and/or transfect a wide range of therapeutic, prophylactic and/or biological reagents), nanosized, substantially neutral surface charge, high encapsulation efficiency, high transfection efficiency, high stability, low toxicity (e.g., low cytotoxicity), low production/synthesis cost, therefore making them suitable for in vivo applications that require efficient cellular uptake and/or gene transfection.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows the¹H NMR spectrum of N⁶-benzyloxycarbonyl-lysine-N- carboxyanhydride (Lys-NCA), synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in deuterated dimethyl sulfoxide (DMSO-de) as the solvent.

FIG. 2 shows the¹³C NMR spectrum of N⁶-benzyloxycarbonyl-lysine-N- carboxyanhydride (Lys-NCA), synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in deuterated dimethyl sulfoxide (DMSO-de) as the solvent.

FIG. 3 shows the¹H NMR spectrum of 2-amino-/V,/\/- ditetradecylacetamide, synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in deuterated chloroform (CDCl₃) as the solvent.

FIG. 4 shows the¹H NMR spectrum of lipid-b/ock-poly(Lysine) (lipid-b- poly(Lys)) (LP) with a degree of polymerization (DP) of 5, synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in heavy water (D2O) as the solvent.

FIG. 5 shows the¹H NMR spectrum of lipid-b/ock-poly(Lysine) (lipid-b- poly(Lys)) (LP) with a degree of polymerization (DP) of 8, synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in heavy water (D2O) as the solvent.

FIG. 6 shows the¹H NMR spectrum of lipid-b/ock-poly(Lysine) (lipid-b- poly(Lys)) (LP) with a degree of polymerization (DP) of 10, synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in heavy water (D2O) as the solvent.

FIG. 7 shows the¹H NMR spectrum of lipid-b/ock-poly(Lysine) (lipid-b- poly(Lys)) (LP) with a degree of polymerization (DP) of 15, synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in deuterated dimethyl sulfoxide (DMSO-de) as the solvent. FIG. 8 shows the¹H NMR spectrum of lipid-bZock-poly(Lysine) (lipid-Jb- poly(Lys)) (LP) with a degree of polymerization (DP) of 15, synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in deuterated dimethyl sulfoxide (DMSO-de) as the solvent.

FIG. 8 shows the¹H NMR spectrum of lipid-b/ock-ionizable poly(Lysine)- lipid (lipid-b-ionizable poly(Lys)-lipid) with a degree of polymerization (DP) of 5 (LPL3), synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in deuterated chloroform (CDCh) as the solvent.

FIG. 9 shows the¹H NMR spectrum of lipid-b/ock-ionizable poly(Lysine)- lipid (lipid-b-ionizable poly(Lys)-lipid) with a degree of polymerization (DP) of 8 (LPL4), synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in deuterated chloroform (CDCh) as the solvent.

FIG. 10 shows the¹H NMR spectrum of lipid-b/ocA-ionizable poly(Lysine)- lipid (lipid-b-ionizable poly(Lys)-lipid) with a degree of polymerization (DP) of 10 (LPL5), synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in deuterated chloroform (CDCh) as the solvent.

FIG. 11 shows the¹H NMR spectrum of lipid-b/ocA-ionizable poly(Lysine)- lipid (lipid-b-ionizable poly(Lys)-lipid) with a degree of polymerization (DP) of 15 (LPL6), synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in deuterated chloroform (CDCh) as the solvent.

FIG. 12 shows the¹H NMR spectrum of lipid-b/oc/<-ionizable poly(Lysine)- lipid (lipid-b-ionizable poly(Lys)-lipid) with a degree of polymerization (DP) of 15 (LPL2) derived from 1 ,2 -epoxy propane and synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in deuterated dimethyl sulfoxide (DMSO-de) as the solvent. FIG. 13 shows the¹H NMR spectrum of lipid-Lysine-lipid (lipid-Lys-lipid) (LPL1 ), synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in deuterated chloroform (CDCh) as the solvent

FIG. 14 shows the¹H NMR spectrum of ionizable poly(Lysine)-lipid (ionizable poly(Lys)-lipid) (PL), synthesized in accordance with various embodiments disclosed herein, with the NMR analysis performed in deuterated chloroform (CDCh) as the solvent.

FIG. 15 shows the size distribution graphs of GFP-siRNA loaded lipid nanoparticles (siRNA-LNPs) comprising LDL4 at N/P ratios of 3 (FIG. 15A) and 6 (FIG. 15B), respectively, in comparison with the size distribution of ALC-0315 (FIG. 15C) LNPs prepared via microfluidics mixing in accordance with various embodiments disclosed herein.

FIG. 16 shows the GFP knockdown efficiency (FIG. 16A) and viability of HepG2 cells (FIG. 16B) after 72 hours of incubation with various siRNA-LNP formulations synthesized in accordance with various embodiments disclosed herein. Commercial transfection agent Lipofectamine 3000 was used as a positive control.

FIG. 17 shows the GFP knockdown efficiency in HepG2 cells after 72 hours of incubation with various siRNA-LNP formulations with different lipid tail lengths at N/P ratios of 3 and 6. These formulations comprise LPL3, LPL4, LPL5, or LPL6, synthesized in accordance with various embodiments disclosed herein.

FIG. 18 shows the GFP knockdown efficiency in HepG2 cells after 72 hours of incubation with various siRNA-LNP formulations with different lipid tail lengths at N/P ratios of 3 and 6. These formulations comprise LPL2 or LPL6, synthesized in accordance with various embodiments disclosed herein. FIG. 19 shows the GFP knockdown efficiency in HepG2 cells after 72 hours of incubation with various siRNA-LNP formulations at N/P ratios of 3 and 6. These formulations comprise PL or LPL6, synthesized in accordance with various embodiments disclosed herein.

FIG. 20 shows the GFP knockdown efficiency (FIG. 20A) and viability of HepG2 cells (FIG. 20B) after 72 hours of incubation with siRNA-LNPs comprising LDL4 at N/P ratios of 3 and 6, respectively, in comparison with ALC-0315 LNPs prepared in accordance with various embodiments disclosed herein. The cell viability of the treated wells was expressed as a percentage relative to the negative control wells which did not receive any treatment.

FIG. 21 shows the fluorescent microscopy images and bright field images of eGFP expressing HepG2 cells transfected with siRNA-LNPs comprising LDL4 at N/P ratios of 3 and 6, respectively, in comparison with ALC-0315 LNPs prepared in accordance with various embodiments disclosed herein. The images of untreated eGFP expressing HepG2 cells were included as controls. The top row shows the fluorescence images, while the bottom row shows the bright field images.

FIG. 22 shows the transfection efficiency indicated by the relative luciferase expression (FIG. 22(A)) as well as the viability (FIG. 22(B)) of HEK- 293T cells after transfection or 48 hours of incubation with various FLuc mRNA- loaded LNP formulations (mRNA-LNPs) prepared in accordance with various embodiments disclosed herein. Commercial transfection agent Lipofectamine 3000 was used as a positive control and untreated HEK-293T cells were used as a negative control. Statistical significance was calculated using one-way ANOVA (Analysis of Variance), and data are shown as mean ± SD (ns: no significant difference, *P < 0.05, **P < 0.01 , ***P < 0.001 , ****P < 0.0001 ). EXAMPLES

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.

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.

Example 1 : Materials and Methods

1.1. Materials

All chemicals were purchased from Sigma-Aldrich, Alfa Chemistry, GL Biochem (Shanghai), and Tokyo Chemical Industry (Singapore) and used as received unless specified. Solvents purchased from VWR, or J. T. Baker, were of high-performance liquid chromatography or analytical grade and used as received. The lipids ALC-0315, 1 ,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC), cholesterol, and ALC-0159, which were used to make LNPs as control, were purchased from MedC hem Express (Monmouth Junction, NJ, USA). Fetal bovine serum (FBS) was purchased from (Corning, USA), and 0.9% w/v saline (pH 7.4) from (B Braun, Singapore). AlamarBlue Cell Viability reagents were purchased from ThermoFisher Scientific, Singapore. Phosphate-buffered saline (PBS, 10 x) was purchased from 1st BASE and diluted to 1 PBS before use. pmaxGFP siRNA VSC-1001 were purchased from Lonza as a lyophilised powder and was prepared as per manufacturer’s protocol. Lipofectamine RNAi Max and Lipofectamine 3000 were purchased from Invitrogen, Thermofisher.

1.2. Synthesis of N⁶-benzyloxycarbonyl-lysine-/V-carboxyanhydride (Lys- NCA)

Synthesis of Lys-NCA is shown in Scheme 1. General synthetic method for Lys-NCA: N⁶-benzyloxycarbonyl-lysine (7.0 g, 25 mmol) was suspended in 100 ml of dry tetrahydrofuran (THF) and then triphosgene (3.2 g) was added under N2. The mixture was stirred at 70 °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 the iced hexane (1000 mL), collected by filtration. The resulting crude product was purified by recrystallizing with THF/hexane mixture for three times. The yield of Lys-NCA was 78 %. The structure of Lys-NCA was verified by¹H NMR and¹³C NMR spectra (FIG. 1 and 2).

Scheme 1. Synthesis of N⁶-benzyloxycarbonyl-lysine-N-carboxyanhydride (Lys- NCA). 1.3. Synthesis of lipid of 2-amino-A/,/V-ditetradecylacetamide

Synthesis of the lipid of 2-amino-A/,A/-ditetradecylacetamide is shown in Scheme 2. fert-Butyl (2-(ditetradecylamino)-2-oxoethyl)carbamate was first synthesized. Boc-glycine (876 mg, 5.0 mmol) and ditetradecylamine (2.05 g, 5.0 mmol) were dissolved in dry dichloromethane (DCM) (100 mL). 2-(1 H- Benzotriazole-1 -yl)-1 , 1 ,3,3-tetramethyluronium hexafluorophosphate (HBTU) (2.38 g, 6.25 mmol) and /V-ethyl-/V-(1 -methylethyl)-2-propanamine (DIPEA) (1.62 g, 12.5 mmol) were added to the solution. The reaction mixture was stirred for 24 h at room temperature under N2. The mixture was diluted with 100 mL of DCM, and washed with 7% citric acid, brine, and H2O. The resulting organic layer was collected and dried over anhydrous Na2SC>4. DCM was evaporated in vacuo to yield crude product. The resulting crude product was purified by flash silica gel column chromatography (Hexane: Diethylether 8:2, v/v), obtaining the final product as a yellow oil. The yield of the compound was 87 %. Then, 2-amino-/V, /V-ditetradecylacetamide was synthesized. fert-Butyl (2-(ditetradecylamino)-2- oxoethyl)carbamate was dissolved in 15 mL of anhydrous DCM, to which was added 12 mL of trifluoroacetic acid (TFA). The mixture was stirred for 2 h under N2 atmosphere. The solvents were evaporated in vacuo to yield crude product. The resulting crude product was dissolved in 40 mL of DCM, and then 40 mL of NaHCOs aqueous solution (10 %) was added. The mixture was stirred for 12 h under N2 atmosphere. The organic layer was collected and dried over anhydrous Na2SO4. The DCM was evaporated in vacuo. The resulting product was dried under vacuum to obtain a white powder. The yield of the compound was 92 %. The structure of product was verified by¹H NMR spectrum (FIG. 3).

< 1) HBTU, DIPEA,

R.T. 2 h

Scheme 2. Synthesis of the lipid of 2-amino-A/,A/-ditetradecylacetamide.

1.4. Syntheses of lipid-b/ock-ionizable polylysine (lipid-b-poly(Lys)) (LP) and lipid-b/ock-ionizable polylysine lipids (lipid-b-poly(Lys)-lipids) (LPLs)

Syntheses of lipid-b/ock-ionizable polylysine and lipid-b/oc/c-ionizable polylysine lipid are shown in Scheme 3. Lipid-b-poly(N⁶-benzyloxycarbonyl-L- Lys) was first synthesized. General synthetic method for lipid-£>-poly(N⁶- benzyloxycarbonyl-L-Lys): In the glove box, 2-amino-N, N-ditetradecylacetamide (46.6 mg, 0.1 mmol) and Lys-NCA were dissolved in 40 mL of anhydrous DMF. The mixture was stirred for 24 h at room temperature in the glove box. 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 MeOH and precipitated by pouring the solution into glacial ether. The resulting product was dried under vacuum. The number of polymerization unit for lipid-b-poly(N⁶-benzyloxycarbonyl-Lys) was adjusted by varying the amount of Lys-NCA.

Scheme 3. Syntheses of lipid-b/oc/c-ionizable polylysine (lipid-b-poly(Lys)) (LP) and lipid-b/ock-ionizable polylysine lipids (lipid-b-poly(Lys)-lipids) (LPLs).

The deprotection of lipid-b-poly(N⁶-benzyloxycarbonyl-Lys) was then performed to yield lipid-b-poly(Lys). General synthetic method for lipid-b- polypeptide: 300 mg of lipid-t>-poly(N⁶-benzyloxycarbonyl-Lys) was dissolved in 6 mL of TFA, to which was added 2.0 mL of 33 % HBr in acetic acid. The mixture was stirred in ice bath for 2 h. The solvents were removed in vacuo. The resulting crude product was suspended in 15 ml of methanol and then precipitated by pouring the mixture solution into glacial ether (100 mL), collected by centrifugation. The crude product was purified by suspending it in methanol and precipitated by pouring the solution into glacial ether. The obtained product was dried under vacuum. The crude product was purified by dialysis with de-ionized (DI) water. The product was obtained by freeze-drying under vacuum. The yield of lipid-b-poly(Lys) is 65 %. The structures of lipid-b-poly(Lys) were verified by¹H NMR (FIG. 4-7).

Lipid-b-ionizable polypeptide lipid (LPL) was finally synthesized. 150 mg of lipid-b-poly(Lys) was dissolved in 6.0 mL of anhydrous ethanol, to which was added 1 .8 g of 1 ,2-epoxyoalkane. The mixture was stirred at 90 °C for 48 h under N2 atmosphere. The solvents were evaporated in vacuo to yield crude product. The resulting crude product was purified was purified by dialysis with ethanol. The solvent was removed in vacuo. The yield of the compound is 92 %. The structures of lipid-b-ionizable polypeptide lipid were verified by¹H NMR (FIG. 8- 12).

1.5. Synthesis of lipid-ionizable lysine lipid derivative (LPL1)

Synthesis of lipid-ionizable Lys derivative is shown in Scheme 4. Lipid-Lys (Boc) was first synthesized. General synthetic method for Lipid-Lys(Boc): Boc- Lys (Boc)-OH (1 .73 g, 5.0 mmol) and ditetradecylamine (2.05 g, 5.0 mmol) were dissolved in dry dichloromethane (DCM) (100 ml). 2-(1 H-Benzotriazole-1 -yl)- 1 ,1 ,3,3-tetramethyluronium hexafluorophosphate (HBTU) (2.38 g, 6.25 mmol) and N-ethyl-N-(1-methylethyl)-2-propanamine (DIPEA) (1.62 g, 12.5 mmol) were added to the solution. The reaction mixture was stirred for 24 h at room temperature under N2. The mixture was diluted with 100 mL of DCM, and washed with brine. The resulting organic layer was collected and dried over anhydrous Na2SO4. DCM was evaporated in vacuo to yield crude product. The resulting crude product was purified by flash silica gel column chromatography (Hexane:Diethylether 8:2, v/v), obtaining the final product as a yellow oil. The yield of the compound was 85 %.

Scheme 4. Synthesis of lipid-Lys-lipid derivative (LPL1).

Then, lipid-Lys was synthesized. Lipid-Boc-Lys(Boc) was dissolved in 15 mL of anhydrous DCM, to which was added 12 mL of trifluoroacetic acid (TFA). The mixture was stirred for 2 h under N2 atmosphere. The solvents were evaporated in vacuo to yield crude product. The resulting crude product was dissolved in 40 mL of DCM, and then 15 g of NaHCCh was added. The mixture was stirred for 12 h under N2 atmosphere. The organic layer was collected and dried over anhydrous Na2SC>4. The DCM was evaporated in vacuo. The resulting crude product was purified by flash silica gel column chromatography (DCM : MeOH 95:5, v/v). The yield of the compound was 80 %.

Lipid-ionizable Lys derivatives were finally synthesized. lipid-Lys was dissolved in 8.0 mL of anhydrous ethanol, to which was added 2.56 g of 1 ,2- epoxyoctane. The mixture was stirred at 90 °C for 48 h under N2 atmosphere. The solvents were evaporated in vacuo to yield crude product. The resulting crude product was purified by flash silica gel column chromatography (DCM : MeOH 95:5, v/v). The yield of Lipid-ionizable Lys derivatives is 72 %. The structure of lipid-Lys-lipid derivative was verified by¹H NMR (FIG. 13).

1.6. Synthesis of ionizable poly(Lys)-lipid derivative (PL)

Synthesis of poly(Lys)-lipid (PL) is shown in Scheme 5. poly(N⁶- benzyloxycarbonyl-Lys) was first synthesized. General synthetic method for poly(N⁶-benzyloxycarbonyl-Lys): In the glove box, H-Lys(Boc)-OMe (29.7 mg, 0.1 mmol) and Lys-NCA were dissolved in 40 mL of anhydrous DMF. The mixture was stirred for 24 h at room temperature in the glove box. 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 MeOH and precipitated by pouring the solution into glacial ether. The resulting product was dried under vacuum. The number of polymerization unit for poly(N⁶- benzyloxycarbonyl-Lys) was adjusted by varying the amount of Lys-NCA. The yield of poly(N⁶-benzyloxycarbonyl-Lys) is 82 %.

Scheme 5. Synthesis of poly(Lys)-lipid derivative (PL).

The deprotection of poly(N⁶-benzyloxycarbonyl-Lys) was then performed to yield poly(Lys). General synthetic method for poly(Lys): 500 mg of poly(N⁶- benzyloxycarbonyl-Lys) was dissolved in 8 mL of TFA, to which was added 2.7 mL of 33 % HBr in acetic acid. The mixture was stirred in ice bath for 2 h. The solvents were removed in vacuo. The resulting crude product was suspended in 30 ml of methanol and then precipitated by pouring the mixture solution into glacial ether (300 mL), collected by centrifugation. The crude product was purified by suspending it in methanol and precipitated by pouring the solution into glacial ether. The obtained product was dried under vacuum. The crude product was purified by dialysis with deionized water. The product was obtained by freeze- drying under vacuum. The yield of poly(Lys) is 85 %.

Ionizable poly(Lys)-lipid derivative was finally synthesized. 300 mg of poly(Lys) was dissolved in 8.0 mL of anhydrous ethanol, to which was added 1 .28 g of 1 ,2-epoxyoctane. The mixture was stirred at 90 °C for 48 h under N2 atmosphere. The solvents were evaporated in vacuo to yield crude product. The resulting crude product was purified was purified by dialysis with ethanol. The solvent was removed in vacuo. The yield of ionizable poly(Lys)-lipid derivative is 90 %. The structure of ionizable poly(Lys)-lipid derivative was verified by¹H NMR (FIG. 14).

Example 2: Preparation of siRNA-loaded lipid nanoparticles (siRNA LNPs) formulation by manual mixing siRNA LNPs were prepared by adding ethanol solution containing specified amount of ionisable lipid (LPL), DSPC, cholesterol and PEG-lipid quickly into sodium acetate buffer solution (pH 4, 10 mM) containing GFP-siRNA (2.5 pg) followed by 3 times of pulse vortex mixing. Molar ratios of LPL:DSPC:Cholesterol:PEG-lipid are typically 46:9.4:42:1 .6. Formulated mixture was made to stand for 30 minutes before a dilution of 1 time with 0.9 % w/v saline then filtration through a sterile filter (0.2 pm) and stored at 4 °C till usage. N/P ratios were calculated using amount of moles of nitrogen per mole of polymer to amount of phosphate of siRNA.

Example 3: Preparation of siRNA-loaded lipid nanoparticles (siRNA LNPs) formulation by using a microfluidic device siRNA LNPs were made using the NanoAssemblr Ignite system by Precision Nanosystem. The microfluidics flow rate was set at 12 mL per min, and the volume ratio of the aqueous phase to the organic phase was 3:1 . To prepare the aqueous phase, GFP-siRNA (75 pg) was diluted in sodium acetate buffer with a pH of 4 and a concentration of 10 mM. In the organic phase, LPL, cholesterol, DSPC, and PEG-lipid (ALC-0159) were dissolved in ethanol at a molar ratio of (46:9.4:42:1.6). These mole ratios were kept constant at different N/P ratios, which were calculated based on the nitrogen mole content of each LPL and P mole content of siRNA. After formulation, LNPs were diluted 15 times in 0.9 % saline solution with a pH of 7.4. Subsequently, they were concentrated using centrifugal ultrafiltration with a Vivaspin® 10k MW Cutoff centrifugal unit (Sartorius) to remove ethanol prior to characterization.

Example 4: Preparation of mRNA-loaded lipid nanoparticles (mRNA LNPs) formulation by manual mixing mRNA LNPs were formulated by dissolving ionizable lipids, DSPC, cholesterol and PEG-lipid (ALC-0159) in ethanol at different molar ratios (30:12.3:55.7:2 and 46.3:9.4:42.7:1.6) to a final volume of 50 pL. The organic phase was then rapidly mixed with sodium acetate buffer solution (pH 4 or pH 5.25, 10 mM) containing 10 pg of FLuc mRNA (TriLink BioTechnologies, San Diego, CA) at N/P ratios of 3 or 6 to a final volume of 200 pL. The N/P ratio is calculated based on the nitrogen mol content of each ionizable lipid and the phosphate mol content of mRNA. Formulated LNPs were left to stabilize at room temperature for 2 hours prior to characterization. Example 5: Characterization of siRNA LNPs and mRNA LNPs (nanoparticle size, polydispersity (PDI), zeta potential, and encapsulation efficiency)

Nanoparticle size, polydispersity index (PDI) and zeta potential were measured using a Zetasizer (Malvern, UK). siRNA and mRNA LNPs were diluted 40 times and 20 times respectively in 0.9 % w/v saline and the average of 3 technical replicates is reported for size/zeta potential measurements. Quant-itTM RiboGreen RNA Assay Kit (Invitrogen, Waltham, MA, USA) was used to determine the encapsulation efficiencies of the formulated siRNA and mRNA LNPs. Nanoparticles were diluted to ~ 1 - 5 ng uL^-1 with nuclease-free water (pH 7). The RiboGreen RNA reagent was diluted 200 times with either Tris-EDTA buffer containing 5% Triton-X100 or Tris-EDTA (TE) buffer only. 90 pL of the buffer solution containing the RiboGreen reagent was then added to 10 pL of the siRNA and mRNA LNPs and incubated at 37 °C for 30 min in a black 96-well plate. Following that, the fluorescence intensity of the wells of the plate was read 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 calculate the encapsulation efficiencies of siRNA and mRNA in LNPs based on the equation as follows.

Example 6: Transfection studies of mRNA LNPs using HEK293T cells

The transfection efficiency of mRNA LNPs was characterized by delivering FLuc mRNA into HEK293T cells. HEK293T cell line was cultured to logarithmic growth phase in DMEM media supplemented with 10 % fetal bovine serum (FBS) (v/v) and 1 % penicillin/streptomycin (v/v). Briefly, cells were seeded into 96-well white clear-bottom plates at a seeding density of 1 x 10⁴ cells/well and were incubated at 37 °C, 5 % CO2 for 24 h. Following overnight incubation, each well was dosed with 100 pL of mRNA LNPs diluted with DMEM containing 10% FBS to a final concentration of 100 ng/well. Transfected cells were then incubated at 37 °C, 5% CO₂ for 48 h. Lipofectamine formulations were performed based on manufacturer’s protocol. After 48 h, 100 pL of ONE-Glo™ luciferase reagent (Promega) was added to each well and incubated in darkness for 3 min. Luminescence signals were measured immediately using the microplate reader (Tecan, Mannedorf, Switzerland).

Example 7: Evaluation of cytotoxicity of mRNA LNPs

The cytotoxicity of mRNA LNPs were evaluated in HEK293T cells. HEK293T cells were seeded into 96-well black clear-bottom plates at a seeding density of 1 x 10⁴ cells/well and were incubated at 37 °C, 5% CO2 for 24 h. After incubation, each well was dosed with 100 pL of mRNA LNPs diluted with DMEM containing 10 % FBS to a final concentration of 100 ng/well and then incubated at 37 °C, 5 % CO2 for 48 h. After 48 h, spent media was removed from the wells and substituted with 10% alamarBlue reagent in DMEM media. The plate was incubated at 37 °C for 2 h prior to fluorescence intensity measurement using the microplate reader (Tecan, Mannedorf, Switzerland) at an excitation wavelength of 570 nm and an emission wavelength of 600 nm. The cell viability of the treated wells was then expressed as a percentage relative to the negative control wells that did not receive any treatment.

Example 8: Transfection studies of siRNA LNPs using HepG2 cells which stably expresses eGFP

HepG2 eGFP expressing cell line was cultured in DMEM media containing 10 % fetal bovine serum (FBS) (v/v) and 1 % penicillin/streptomycin (v/v). The cells were allowed to incubate at 37 °C with 5 % CO2 in an incubator. Cells were trypsinized at confluency of 80-90 %, and were seeded at a density of 5,000 cells/well in 96-well black clear-bottom plates. They were incubated for 24 h at 37 °C, 5 % CO2, prior to transfection. On the day of transfection, the siRNA LNPs solution was mixed with DMEM with 10 % FBS to make the final volume in each well to 100 pL with a siRNA dose of 100 ng/well. The plates were then incubated for 72 h at 37 °C, 5 % CO2. Lipofectamine formulations were performed based on manufacturer’s protocol.

Example 9: Measurement of GFP knockdown efficiency of siRNA LNPs in HepG2 cells and cell viability of HepG2 cells

Total mean GFP fluorescence was measured using a Tecan Spark Control Microplate reader (Mannedorf, Switzerland) with excitation and emission wavelength at 485 and 535 nm respectively. Percentage knockdown was calculated with reference to non-treated cells using the formula (100 % - (fluorescence reading of treated cells/fluorescence reading of untreated cells)). Following 72 h of incubation, media from individual wells were removed and were replaced with 100 pL of solution containing fresh DMEM media with 10 % Alamar Blue reagent. The plate was then incubated for 2 h at 37 °C. Fluorescence intensity was then measured using the microplate reader (Tecan, Mannedorf, Switzerland) at an excitation wavelength of 570 nm and an emission wavelength of 600 nm. The cell viability of the treated wells was then expressed as a percentage relative to the negative control wells that did not receive any treatment.

Example 10: Fluorescence Microscopy Imaging

Fluorescent images were captured using an Olympus BX53 fluorescence microscope equipped with a DP80 digital camera and a FITC filter set (excitation 488 nm, emission 507 nm) suitable for GFP imaging. Images were collected using a 10X immersion objective. Image acquisition and analysis were performed using the Olympus cellSens Dimension imaging software. The settings for exposure time, gain, and offset were kept constant throughout the image acquisition process for all samples to ensure comparability. Example 11: Statistical Analysis

Statistical analysis for FIG. 17-19 was performed with Welch’s t-test and FIG. 22 was performed with one-way ANOVA and Tukey’s post hoc test using commercially available GraphPad Prism. Values marked with an asterisk are significantly different (ns, p >0.05; * p < 0.05; **, p < 0.01 ; ***, p < 0.001 ; **** p < 0.0001 ).

Example 12: Results and Discussion

12.1. Synthesis of lipid-b-ionizable polypeptide

As shown in Scheme 3, lipid-b-poly(Lys) was first synthesized via the ringopening polymerization (ROP) of N⁶-benzyloxycarbonyl-lysine-N- carboxyanhydride (Lys-NCA) (Scheme 1) using the lipid of 2-amino-N, N- ditetradecylacetamide (Scheme 2) as the initiator, followed by the deprotection of lipid-poly(N⁶-benzyloxycarbonyl-Lys) in TFA and HBr/CHsCOOH. The successful synthesis of Lys-NCA, 2-amino-N, N-ditetradecylacetamide and lipid- poly(Lys) was verified by¹H NMR and¹³C NMR spectroscopy (FIG. 1-3). As shown in FIG. 4, the disappearance of peaks (-Ph/7- and Ph-CFfe-) at 5 7.35 and 5.25 ppm, indicated successful deprotection of lipid-poly(Lys). The degree of polymerization (DP) of lysine in lipid-b-poly(Lys) was adjusted by varying the amount of Lys-NCA. The DPs of lysine in lipid-b-poly(Lys) were determined by the integration area (peak j of the polypeptide; peak a of the lipid) (FIG. 4-7). The lipid-b-poly(Lys) with DP of 5, 8, 10 and 15 were further used for the synthesis of lipid-b-ionizable poly(Lys)-lipid derivatives (LPL). The ionizable poly(Lys)-lipid derivative with DP of 15, denoted as PL, was synthesized as a control ionizable lipid (FIG. 14). Lipid-b-poly(Lys), denoted as LP, was also made as a control lipid.

To facilitate RNA encapsulation and delivery, a series of lipid-b-ionizable poly(Lys) derivatives (LPLs) was synthesized through amino-epoxy reaction. These ionizable lipids possess a long two-tail lipid, and a polypeptide backbone with different lengths of alkyl side chains. The structure of LPL was confirmed by¹H NMR spectrum. As shown in FIG. 5-12, the disappearance of the peakj (NH2- C/-/2-) at 5 2.9 ppm from lipid-b-poly(Lys) and the appearance of the peak j (-N- C/-/2-) at 6 2.6 ppm from lipid-b-ionizable poly(Lys)-lipid, indicated successful addition of primary amines with 1 ,2-epoxyoalkane. The proton signals of the epoxy alkyl chain and the integral ratios in line with the theoretical values in the¹H NMR spectra of the ionizable lipids revealed the integrity of LPLs (FIG. 8-12). Meanwhile, lipid-Lys-lipid was also synthesized as an ionizable lipid for RNA delivery. The successful synthesis of lipid-Lys-lipid was verified by¹H NMR spectroscopy (FIG. 13). Characteristics of the lipid-Lys-lipid, PL, LP and LPLs were listed in Table 1.

Table 1. Characteristics of ionizable lipids.

Repeat M_n^NMR Ionizable lipid Epoxy substrate Abbreviation unit (n) [g mol^-1]

Lipid-b-poly(Lys) - 15 2389 LP

Poly(Lys)-lipid 1 ,2-epoxyoctane 15 6442 PL

Lipid-Lys-lipid 1 ,2-epoxyoctane 1 1051 LPL1

Lipid-poly(Lys)-lipid 1 1 ,2-epoxypropane 15 4492 LPL2

Lipid-poly(Lys)-lipid 2 1 ,2-epoxyoctane 5 3031 LPL3

Lipid-poly(Lys)-lipid 3 1 ,2-epoxyoctane 8 4185 LPL4

Lipid-poly(Lys)-lipid 4 1 ,2-epoxyoctane 10 4954 LPL5

Lipid-poly(Lys)-lipid 5 1 ,2-epoxyoctane 15 6877 LPL6

12.2. Size, size distribution (PDI), zeta potential, and encapsulation efficiency of siRNA-loaded LNPs

The size, polydispersity index (PDI), zeta potentials, and encapsulation efficiencies of LNPs loaded with GFP-siRNA were assessed using a Zetasizer (Malvern, UK). These results are depicted in Tables 2-5 and FIG. 15. siRNA LNPs formed from LP containing 15 primary amines had a higher surface charge as LP is positively charged even at pH 7.4 (measured in saline). The rest lipids of tertiary amine groups formed LNPs with neutral surface at pH 7.4 (zeta potential within ± 10 mV), which is desirable for in vivo application. A majority of the LDL nanoparticles formulated were less than 100 nm in size. Despite observing a range in size distribution, this variability can be attributed to the utilization of a manual mixing formulation method. This approach was chosen for its speed and efficiency in screening different lipids and formulations, thereby aiding in the selection of the optimal mix for further investigation. The encapsulation efficiency was the lowest when N/P ratio of 2 was used. Upon identifying the best-performing polymer and N/P ratio, microfluidic mixing was conducted. The outcomes were then directly compared with the clinically approved ionizable lipid (ALC-0315), which is used Pfizer-BioNTech’s COVID-19 mRNA vaccine formulation. The best performing candidate LPL4 had comparable size, PDI, zeta potential and siRNA encapsulation efficiency as ALC-0315 (Table 5). Both types of siRNA LNPs had narrow size distribution (FIG. 15).

Table 2. Characteristics of manually formulated siRNA-loaded LNPs with varied lipid lengths.

N/P Diameter Zeta Potential Encapsulation

Formulation PDI ratio (nm) (mV) Efficiency (%)

3 71 ± 1 0.184 ± 0.050 13.63 ± 2.49 68.4 ± 9.0

LP

6 83 ± 1 0.162 ± 0.017 18.44 ± 0.61 77.5 ± 2.0

3 101 ± 2 0.198 ± 0.010 6.77 ± 1.32 56.2 ± 1.3

LPL1

6 64 ± 1 0.154 ± 0.017 10.56 ± 1.59 60.1 ± 0.6

3 53 ± 1 0.214 ± 0.002 -0.89 ± 1.08 67.8 ± 1.0

LPL5

6 58 ± 1 0.206 ± 0.015 -2.75 ± 3.29 71.9 ± 0.3 Table 3. Characteristics of manually formulated siRNA-loaded PL, LPL 2 and 6 LNPs.

N/P Diameter Zeta Potential Encapsulation

Formulation PDI ratio (nm) (mV) Efficiency (%)

3 63 ± 1 0.177±0.031 -1.19±2.14 64.9±0.1

PL

6 65 ±1 0.108 ±0.057 0.18 ±0.44 74.8 ±1.1

3 85 ±3 0.303 ±0.033 1.47 ±3.49 43.8 ± 3.7

LPL2

6 63 ±2 0.234 ±0.010 -5.94 ±0.35 55.5 ± 0.8

3 68 ±4 0.260 ±0.123 6.16 ±0.73 69.4 ± 0.6

LPL6

6 89 ±3 0.300 ±0.109 0.86 ± 1.29 54.2 ± 0.8

Table 4. Characteristics of manually formulated siRNA-loaded LPL LNPs with varied degree of polymerisation.

Diameter Zeta Potential Encapsulation

Formulation N/P ratio PDI

(nm) (mV) Efficiency (%)

3 56 ± 1 0.388 ±0.053 2.79 ±6.02 64.3 ± 0.7

LPL3 6 59 ±1 0.231 ±0.013 -2.14 ±2.06 66.0 ± 0.1

3 64 ± 4 0.218±0.011 3.71 ± 1.07 54.9±13.1

LPL4

6 57 ± 1 0.332 ±0.012 -1.88± 1.46 68.1 ± 0.3

3 40 ±1 0.173 ±0.010 -3.30 ±0.24 69.0 ± 0.7

LPL5

6 47 ± 1 0.204 ±0.038 0.77 ±0.44 76.9 ± 2.5

Table 5. Characteristics of formulated siRNA-loaded LPL4 and ALC-0315 LNPs through microfluidic mixing.

Diameter Zeta Potential Encapsulation

Formulation N/P ratio PDI

(nm) (mV) Efficiency (%)

3 74 ± 1 0.032 ±0.030 0.17 ±4.21 75.3 ±1.9

LPL4 6 77 ±1 0.091 ±0.029 -1.31 ± 1.56 70.7 ± 0.4

ALC-0315 6 80 ±1 0.015 ±0.022 0.94 ±2.11 71.6 ±0.7 12.3. In vitro knockdown efficiency of siRNA LNPs in HepG2 eGFP cells

To evaluate the efficacy of LNPs as siRNA delivery vehicles, siRNA encoding for GFP knockdown was selected as an optical reporter gene. Mean total fluorescence readings of GFP fluorescence following siRNA delivery allows for quantification of the mean knockdown of GFP protein expression. Knockdown efficiencies following treatment of cells with siRNA LNPs was compared with the commercial transfection agent Lipofectamine 3000 as a positive control.

The optimal carbon length for achieving the best siRNA transfection was determined through initial assessment, where various lipid carbon lengths attached on poly lysine, were tested at two different N/P ratios (FIG. 16-19). Among them, the Cs moiety exhibited the highest performance (LPL6 vs. LP and LPL2). LPLs with longer carbon chain lengths were synthesised, but they were incapable of dissolving in solvents used for formulations and hence were not tested. No cytotoxicity was observed for all LPL formulations.

Degree of polymerisation (DP) of LPL LNPs was investigated to obtain the most efficient lipid while keeping the attached carbon chain length constant (from LPL3 to LPL6). Knockdowns were less efficient at low and high DPs and peaked at LPL4 and LPL5. At N/P 3, siRNA LPL4 LNPs mediated higher gene knockdown than siRNA LPL5 LNPs (FIG. 17).

Gene knockdowns of LPL2 LNP was significantly lower than LPL 6 at N/P 3 indicating the importance of having a Cs moiety over a Cs moiety while keeping the DP constant (FIG. 18). Ditetradecylamine as initiator is essential for LPL LNPs to provide efficient siRNA delivery as knockdown mediated by siRNA PL LNPs was poorer than that induced by siRNA LPL6 LNPs especially at N/P 3 (FIG. 19). siRNA LPL4 LNPs at N/P 3 and siRNA ALC-0315 LNPs induced comparable knockdown (FIG. 20). Additionally, as seen in fluorescent microscopic images, GFP expression levels for the LPL4 formulations were significantly attenuated and the efficiency was comparable with the ALC-0315 formulation. 12.4. mRNA-LNPs characterization and in vitro transfection

Beside siRNA delivery, lipids LPL1 and LPL3 were further investigated for their ability to encapsulate and deliver mRNA into cells. The N/P ratios and pH of sodium acetate buffer used to dissolve RNA were varied while the mole ratio of the ionizable lipid was fixed at 30% for mRNA-loaded LPL1 and LPL3 LNP formulations. The size, polydispersity index (PDI) and zeta potential of mRNA loaded LNPs were similarly evaluated using the Zetasizer (Malvern, UK). Although all formulations had a near neutral surface charge of ± 10mV, notable differences in size, PDI and encapsulation efficiencies were observed when the pH of sodium acetate buffer used and the N/P ratios were varied. For the initial screening, the pH of sodium acetate buffer used was kept the same as that used in Pfizer’s formulation (pH 4). At pH 4, LPL1 LNP formulations of both N/P 6 and 12 were more than 200nm in size and had a large size distribution (Table 6). Consequently, these formulations had poor encapsulation efficiencies of less than 20% (Table 6) and lower transfection efficiency depicted by poorer luminescence intensity measured in HEK293T cells when compared to Pfizer’s formulation using ALC-0315 (FIG. 22A). Unlike LPL1 LNPs, LPL3 LNPs formulated at N/P 21 and 42 had narrow size distributions (PDI < 0.3) and were of acceptable sizes for in vivo application. However, their encapsulation efficiencies were less than 50% (Table 6) and transfection efficiencies were lower than ALC-0315 (FIG. 22A), suggesting the need for further optimization.

The pH of sodium acetate buffer used was then increased to pH 5.25. At pH 5.25, LPL1 LNPs formulated at N/P 12 displayed no positive encapsulation efficiency when Ribogreen assay was performed (Table 6). This might be a result of tight binding between LPL1 lipid and FLuc mRNA, that prevents release of mRNA for accurate measurement of encapsulation efficiency. The same hypothesis could also explain the low transfection efficiency observed in vitro when LPL1 LNPs formulated at N/P 12, pH 5.25 were incubated with HEK293T cells for 48 h (FIG. 22A). However, at the same pH of 5.25, LPL1 LNPs formulated at a lower N/P ratio of 6 had a smaller size of 102nm with a good PDI of 0.14. Moreover, it had the highest encapsulation efficiency of 78.8 % among all the LNPs tested (Table 6) and significantly higher transfection efficiency that was 5 times higher than that of ALC-0315 (FIG. 22A). Although LPL3 LNPs formulated at N/P 42 resulted in a much higher encapsulation efficiency than the formulation at N/P 21 , the transfection efficiency using N/P 21 was significantly higher than that using N/P 42 (FIG. 22A). Notably, LPL3 LNPs at N/P 21 , pH 5.25 demonstrated significantly higher transfection activity than ALC-0315 (FIG. 22A).

Table 6. Characteristics of manually formulated mRNA-loaded LNPs.

12.5. Evaluation of the cytotoxicity of mRNA-loaded LNPs in HEK293T cells

The cytotoxicity of LNPs encapsulated with mRNA was assessed through a metabolic cell viability assay. The viability of HEK293T cells transfected with LPL 1 LNPs formulated at N/P 6 and 12, pH 4 and N/P 12, pH 5.25 were significantly attenuated compared to the untreated cells (FIG. 22B). The low cell viability observed could stem from free lipids-induced cytotoxicity as the encapsulation efficiencies of these formulations were low (Table 6). No cytotoxicity was observed with all LPL3 LNP formulations and LPL1 LNP formulated at N/P 6, pH 5.25. Among all the LNP formulations tested, LPL1 and LPL3 LNPs formulated at pH 5.25 with N/P ratios 6 and 21 respectively demonstrate higher transfection activity than ALC-0315 and are not toxic to cells; making them suitable candidates for both in vitro and in vivo applications (FIG. 22B)

12.6. Summary

Novel ionizable lipids were developed based on lipid-b-ionizable polypeptide, which successfully encapsulated siRNA and mRNA into LNPs. Through extensive screening of different formulations and N/P ratios, several promising candidates that exhibited not only highly efficient siRNA transfection but also mRNA transfection were identified. These candidates were compared to ALC-0315 used in Pfizer-BNT Covid19 mRNA vaccine formulation and showed sizes of around 70 - 110 nm, neutral surface charge, high siRNA and mRNA encapsulation efficiencies, and comparable knockdown efficiency of GFP. Hence, this study introduces a novel peptide-based LNP system for efficient siRNA and mRNA delivery, which holds potential for future therapeutic applications in siRNA-based and mRNA-based therapies. The LNPs may also be used to deliver other nucleic acid therapeutics.

Claims

1 . A compound comprising a structure represented by general formula (1 ) or an ionized form thereof for preparing lipid nanoparticles encapsulating a therapeutic, prophylactic and/or biological agent:

wherein

R⁵ and each R⁶ are independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

R³, and each R⁷ are independently optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene; n is from 0 to 100;

I and m are each independently 0 or 1 ; each A is independently selected from H, aliphatic alcohol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, an epoxy ring-opening product and/or derivatives thereof; and

B is R¹R²N- or R²³O- where R¹ and R² are each independently H, or a hydrophobic tail with the proviso that both of R¹ and R² are not H at the same time; and where R²³ is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

2. The compound as claimed in claim 1 , wherein the compound comprises three or more tertiary amines.

3. The compound as claimed in any one of the preceding claims, wherein R¹ and R² are each independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and combinations thereof.

4. The compound as claimed in any one of the preceding claims, wherein A is represented by general formula (2):

where R⁸, R⁸ , R⁹ and R⁹’ are each independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

5. The compound as claimed in claim 4, wherein R⁸, R⁸’ and R⁹' are H and R⁹ is C_yH2y+i where y is from 1 to 8.

6. The compound as claimed in any one of the preceding claims, wherein the compound has a molecular weight of from 500 g/mol to 50,000 g/mol.

7. The compound as claimed in any one of the preceding claims, wherein the compound comprises a structure selected from one or more of the following:

LP3 (n = 10);

LPL2 (n = 15);

10

8. A method of preparing a compound represented by general formula (1 ) as claimed in any one of claims 1 to 7, the method comprising:

(a) reacting a lipid-b-poly(Lys) compound represented by general formula (1 b’) with a compound comprising an epoxy represented by general formula (3) to obtain the compound represented by general formula (1 ):

wherein

R⁵ and each R⁶ are independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

R³ and each R are independently optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene; n is from 1 to 100; each R¹¹ to R¹³ are independently selected from H, aliphatic alcohol, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; R¹ and R² are each independently H, or hydrophobic tail, with the proviso that both of R¹ and R² are not H at the same time; and

R⁸ and R⁹ are each independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

9. The method as claimed in claim 8, wherein the method further comprises, prior to step (a):

(a-i) reacting a compound represented by general formula (4) with a protected amino acid comprising N-carboxyanhydride (NCA) represented by general formula (5) to obtain a protected lipid-b- poly(Lys) compound represented by general formula (6) via ring

wherein

R⁵ and each R⁶ are independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

R³ and each R⁷ are independently optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene; n is from 1 to 100;

R¹¹ to R¹² are each independently selected from H, aliphatic alcohol, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

R¹ and R² are each independently H, or hydrophobic tail, with the proviso that both of R¹ and R² are not H at the same time; R¹⁰ is H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; and

PG¹ is a protecting group; and

(a-ii) deprotecting the protected lipid-b-poly(Lys) compound represented by general formula (6) obtained from step (a-i) to obtain the compound represented by general formula (1 b’):

(6) (1b') wherein

R¹³ is selected from H, aliphatic alcohol, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

10. The method of claim 8 or claim 9, wherein the method further comprises, prior to step (a-i):

(a-i-i) reacting a protected amino acid represented by general formula (7) with a carbonylating agent to obtain the protected amino acid comprising NCA represented by general formula (5):

(7) (5) wherein R⁶ and R¹⁶ 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;

R¹¹ is selected from H, aliphatic alcohol, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and PG¹ is a protecting group.

11. The method of any one claims 8 to 10, wherein the method further comprises, prior to step (a-i):

(a-i-ii) reacting an amine compound represented by general formula (8) with a protected amino acid compound represented by general formula (9) in the presence of one or more coupling agent(s) and a base to obtain a carbamate compound represented by general formula (10)

wherein

R⁵, R¹⁴, and R¹⁵ are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

R³ is independently optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene; n is from 1 to 100;

R¹ and R² are each independently H, or hydrophobic tail, with the proviso that both of R¹ and R² are not H at the same time; and PG² is a protecting group; and (a-i-iii) deprotecting the carbamate compound represented by general formula (10) obtained from step (a-i-ii) to obtain the compound represented by general formula (4)

wherein

R¹⁰ is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

12. A method of preparing a compound represented by general formula (1 ) as claimed in any one of claims 1 to 7, the method comprising:

(b) reacting an amine compound represented by general formula (8) with a protected amino acid compound represented by general formula (11 ) in the presence of one or more coupling agent(s) and a base to obtain a dicarbamate compound represented by general formula (12)

(⁸) *11} (12) wherein

R⁷ is optionally substituted alkylene, optionally substituted alkenylene, or optionally substituted alkynylene; R¹ and R² are each independently H, or hydrophobic tail, with the proviso that both of R¹ and R² are not H at the same time;

R¹⁴, and R¹⁷ to R¹⁹ are each independently H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; and

PG⁴ and PG⁵ are each independently a protecting group;

(c) deprotecting the dicarbamate compound represented by general formula (12) obtained from step (b) to obtain an amide compound represented by general formula (13)

( 12; (13) wherein

R²⁰ to R²¹ are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and

(d) reacting the amide compound represented by general formula (13) with an epoxy represented by general formula (3) to obtain the compound represented by general formula (1 ):

(13) (3)

R⁸ to R⁹ are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

13. A nanoparticle composition for delivery of a therapeutic, prophylactic, and/or biological agent, the nanoparticle composition comprising: a compound as claimed in any one of claims 1 to 7; and a therapeutic, prophylactic and/or biological agent that is encapsulated in said compound as claimed in any one of claims 1 to 7.

14. The nanoparticle composition as claimed in claim 13, wherein the nanoparticle composition comprises nanoparticles having a N/P ratio from 1 :1 to 50:1.

15. The nanoparticle composition as claimed in any one of claims 13 to 14, wherein the nanoparticle composition comprises nanoparticles having an average particle size of from 20 nm to 650 nm.

16. The nanoparticle composition as claimed in any one of claims 13 to 15, wherein the composition further comprises:

(i) helper lipid;

(ii) cholesterol or derivatives thereof; and

(iii) polyethylene glycol (PEG)-lipid conjugate or amphiphilic lipids.

17. The nanoparticle composition as claimed in claim 16, wherein the compound represented by general formula (1 ), helper lipid, cholesterol or derivatives thereof, and PEG-lipid conjugate or amphiphilic lipids are mixed at a weight ratio of 30 to 50: 5 to 50: 5 to 60: 1 to 5.

18. The nanoparticle composition as claimed in claim 16, wherein the helper lipid is 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.

19. The nanoparticle composition as claimed in claim 16, wherein the cholesterol or derivatives thereof is selected from cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, avenasterol, and combinations thereof.

20. The nanoparticle composition as claimed in claim 16, wherein the polyethylene glycol (PEG)-lipid conjugate or amphiphilic lipids is selected from PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, 2- [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), R-3- [(uj-methoxy-poly(ethylene glycol)2000)carbamoyl]-1 ,2-dimyristyloxlpropyl- 3-amine (PEG-c-DOMG), 3-N-[(cu-methoxypoly

(ethyleneglycol)2000)carbamoyl]-1 ,2-dimyristyloxy-propylamine (PEG-S- DMG), PEG-DMPE (1 ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [(polyethylene glycol)-methoxy] (sodium salt)), PEG-DPPC, PEG-DSPE lipid and combinations thereof.

21. The nanoparticle composition as claimed in any one of claims 13 to 20 for use in medicine.

22. The nanoparticle composition as claimed in any one of claims 13 to 20 for use in the treatment or prophylaxis of a disease, disorder, or condition in a subject in need thereof.

23. Use of a nanoparticle composition as claimed in any one of claims 13 to 20 in the manufacture of a medicament for treatment or prophylaxis of a disease, disorder, or condition in a subject in need thereof.

24. A method of treating or preventing a disease, disorder, or condition in a subject in need thereof, the method comprising administering a therapeutically effective amount of the nanoparticle composition as claimed as claimed in any one of claims 13 to 20 to the subject.

25. The nanoparticle composition as claimed in any one of claims 13 to 20, the use as claimed in claim 23, or the method as claimed in claim 24, wherein an immune response in the subject is to be induced through the administration of the nanoparticle composition thereto.

26. The nanoparticle composition as claimed in any one of claims 13 to 20, the use as claimed in claim 23, or the method as claimed in claim 24, wherein the disease, disorder, or condition is mediated by a coronavirus.

27. The nanoparticle composition as claimed in any one of claims 13 to 20, the use as claimed in claim 23, or the method as claimed in claim 24, wherein the coronavirus is a SARS-CoV-2 coronavirus.