Compounds for preparing lipid nanoparticles encapsulating agents, nanoparticle compositions comprising same, and related methods

Abstract

There is provided a compound represented by general formula (1) or an ionized form thereof for use in the preparation of lipid nanoparticles encapsulating therapeutic, prophylactic and / or biological agents: wherein NR1R2 is a group ionizable at pH from 3 to physiological pH; a comprises a linear aliphatic hydrocarbon, a branched aliphatic hydrocarbon and / or a cyclic hydrocarbon, optionally comprising one or more groups selected from the group consisting of-OH,-NR-,-O-,-O-CxH2x-O-, where x > = 1; and wherein R is H, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group; r3, R4, R5, R6 and R7 are each independently H, an optionally substituted alkyl group, an optionally substituted alkenyl group or an optionally substituted alkynyl group; and R8 and R9 are each independently a hydrophobic group.

Description

TECHNICAL FIELD

[0001] The present disclosure relates generally to compounds for use in the preparation of lipid nanoparticles encapsulating an agent and methods of preparing the compounds. The present disclosure also relates to nanoparticle compositions comprising the compounds and related methods and uses. BACKGROUND

[0002] Lipid nanoparticles are widely used for the delivery of therapeutic, prophylactic and / or biological agents (e.g., polynucleotides such as mRNA). However, safe, stable and effective delivery systems remain a challenge. In particular, there have been reports of adverse health effects and cytotoxicity associated with the use of lipid nanoparticles for delivery.

[0003] Currently, only 2 mRNA Covid-19 vaccines from Moderna and Pfizer-BioNtech have been approved by the US Food and Drug Administration (US FDA) for human use. Both vaccines use SARS-CoV-2 mRNA as an antigen and lipids as a carrier. The lipids consist of 3 different types of lipids (an ionizable lipid, a PEG-lipid conjugate, and a helper lipid) and cholesterol. The lipids are assembled with the mRNA to form nanoparticles that stimulate immune cells to mount a prophylactic response to the SARS-CoV-2 virus.

[0004] However, there are several drawbacks and deficiencies with the currently available formulations that are far from desirable. First, the ionizable lipids used in the mRNA vaccine formulations of Moderna and Pfizer-BioNTech are very expensive. Second, this formulation apparently generates anti-lipid antibodies and anti-PEG antibodies, which can cause hypersensitivity and allergic reactions in some subjects. In general, thermal stability is also a challenge for mRNA formulations that need to be stored under low temperature conditions, resulting in expensive storage and shipping costs due to cold chain logistics requirements.

[0005] In view of the above, it is necessary to address or at least ameliorate the above problems. In particular, there is a need to provide compounds and / or nanoparticle compositions for the cost-effective, substantially safe and stable, and / or effective delivery of therapeutic, prophylactic and / or biological agents. SUMMARY

[0006] According to one aspect, there is provided a compound as shown in general formula (1) or an ionized form thereof, for use in the preparation of lipid nanoparticles encapsulating a therapeutic, prophylactic and / or biological agent:

[0007]

[0008] wherein

[0009] NR 1R 2 is a group that is ionizable at a pH of 3 to physiological pH;

[0010] A comprises a linear aliphatic hydrocarbon, a branched aliphatic hydrocarbon, and / or a cyclic hydrocarbon, optionally containing one or more groups selected from -OH, -NR-, -O-, -O-C x H 2x -O- wherein x > 1; and wherein R is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0011] R 3 , R 4 , R 5 , R 6 , and R 7 are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and

[0012] R 8 , and R 9 are each independently a hydrophobic group.

[0013] In one embodiment, R 1 , and R 2 are each independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and combinations thereof.

[0014] In one embodiment, R 1 , and R 2 are each H, and -NR 1 R 2 is a primary amine group.

[0015] In one embodiment, the hydrophobic groups at R 8 , and R 9 each independently comprise an optionally substituted alkyl group.

[0016] In one embodiment, A is selected from the following general formula (2), general formula (3), general formula (4), and / or general formula (5):

[0017]

[0018]

[0019] wherein

[0020] X 1 , and X 31 are each independently selected from -H, -OH, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0021] n > 1 ; m > 1 ; p > 1 ; and q > 1.

[0022] In one embodiment, the compound is selected from the group consisting of DnO-EDEA, DD-EDEA, DTD-EDEA, HO-DnO-NH2, HO-DD-NH2, HO-DTD-NH2, DnO-NH2, DD-NH2, DTD-NH2, DMAPAPA-DTD, and combinations thereof.

[0023] In one embodiment, the compound is an ionized form of general formula (1), wherein -NR 1 R 2 has been ionized to a positively charged group.

[0024] According to another aspect, there is provided a method of preparing a compound as disclosed herein, the method comprising:

[0025] (a-i) reacting an amine compound as shown in general formula (6) with a cyclic anhydride as shown in general formula (7) to obtain a first intermediate compound comprising a carboxylate group as shown in general formula (8):

[0026]

[0027] (a-ii) reacting the first intermediate compound as shown in general formula (8) with N-hydroxysuccinimide (NHS) in the presence of a coupling agent to obtain a second intermediate compound comprising an amide group as shown in general formula (9):

[0028]

[0029] (a-iii) reacting the second intermediate compound as shown in general formula (9) with an amine compound as shown in general formula (10) to obtain a compound as shown in general formula (1):

[0030]

[0031] wherein

[0032] A comprises a linear aliphatic hydrocarbon, a branched aliphatic hydrocarbon, and / or a cyclic hydrocarbon, optionally comprising one or more groups selected from -OH, -NR-, -O-, -O-C x H 2x -O- wherein x > 1 ; and wherein R is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0033] R 1 , R 2 , R 3 , R 4 , R 5 , R6 and R 7 each independently is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and

[0034] R 8 and R 9 each independently is a hydrophobic group; and

[0035] (a-iv) optionally deprotonating -NR 1 R 2 ionizes to a positively charged group.

[0036] In one embodiment, the coupling agent includes a carbodiimide selected from the group consisting of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), and combinations thereof.

[0037] According to another aspect, there is provided a nanoparticle composition for delivering a therapeutic, prophylactic, and / or biological agent, the nanoparticle composition comprising:

[0038] a compound as disclosed herein; and

[0039] a therapeutic, prophylactic, and / or biological agent encapsulated in the compound as disclosed herein.

[0040] In one embodiment, the composition further comprises:

[0041] (a) a helper lipid;

[0042] (b) a sterol; and

[0043] (c) a polyethylene glycol (PEG)-modified lipid.

[0044] In one embodiment, the compound as shown in general formula (1), the helper lipid, the sterol, and the PEG-modified lipid are mixed in a weight ratio of 10 to 50: 2 to 20: 4 to 30: 1 to 15.

[0045] In one embodiment, the helper lipid is present in an amount of 1 mol% to 20 mol%, the sterol is present in an amount of 10 mol% to 50 mol%, and the PEG-modified lipid is present in an amount of 0.5 mol% to 10 mol%.

[0046] In one embodiment, the helper lipid is selected from 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero- phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-didodecanoyl-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- cholesteryl hemisuccinyl-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-dibehenoyl-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-dibehenoyl-sn- glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and combinations thereof.

[0047] In one embodiment, the phytosterol is selected from cholesterol, 5a-ergosta-8,24(28)- dien-3ß-ol (fecosterol), sitosterol, ergosterol, campesterol, daucosterol, brassicasterol, avenasterol, and combinations thereof.

[0048] In one embodiment, the PEG-modified lipid is selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and the like or combinations thereof. Examples of PEG-modified lipids / PEGylated lipids include, but are not limited to, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), R-3-[(omega-methoxy-poly(ethylene glycol) 2000)carbamoyl]-1,2-dimyristyloxypropyl-3-amine (PEG-c-DOMG), 3-N-[(omega-methoxy poly(ethylene glycol) 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 lipids, and combinations thereof.

[0049] In one embodiment, the nanoparticle composition comprises nanoparticles having a N / P ratio of 2:1 to 40:1.

[0050] In one embodiment, the nanoparticle composition comprises nanoparticles having an average particle size of 20 nm to 200 nm.

[0051] In one embodiment, the nanoparticle composition comprises nanoparticles having a zeta potential of -15 mV to +20 mV in phosphate buffered saline (PBS).

[0052] According to another aspect, there is provided a nanoparticle composition as disclosed herein for use in medicine.

[0053] According to another aspect, there is provided a nanoparticle composition as disclosed herein for use in the treatment or prevention of a disease, disorder, or condition in a subject in need thereof.

[0054] According to another aspect, there is provided the use of a nanoparticle composition as disclosed herein in the manufacture of a medicament for the treatment or prevention of a disease, disorder, or condition in a subject in need thereof.

[0055] According to 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 to the subject a therapeutically effective amount of a nanoparticle composition as disclosed herein.

[0056] In one embodiment, an immune response in a subject is induced by administering to the subject a nanoparticle composition.

[0057] In one embodiment, the disease, disorder, or condition is mediated by a coronavirus.

[0058] In one embodiment, the coronavirus is a SARS-CoV-2 coronavirus.

[0059] Definitions

[0060] The term "particle" as used herein is broadly intended to refer to a discrete entity or discrete body. The particles described herein can include organic particles, inorganic particles, composite particles, or biological particles. The particles used described herein can also be macroscopic particles formed from an aggregate of multiple sub-particles or an aggregate of multiple small object fragments. The particles of the present disclosure can be spherical, substantially spherical, or non-spherical, such as irregularly shaped particles or ellipsoidal shaped particles. The term "size" when used in reference to a particle is broadly intended to refer to the largest dimension of the particle. For example, the term "size" when used in the context of a nanoparticle can refer to the diameter of the nanoparticle, although it is not so limited. In various embodiments, the term "size" can refer to the diameter of the particle when the particle is substantially spherical; or the term "size" can refer to the largest length of the particle when the particle is substantially non-spherical.

[0061] The term "nanometer" as used herein is broadly construed to include dimensions on the order of nanometers, i.e., less than about 1000 nm, from about 1 nm to less than about 1000 nm, from about 1 nm to about 900 nm, from about 1 nm to about 800 nm, from about 1 nm to about 700 nm, from about 1 nm to about 600 nm, from about 1 nm to about 500 nm, from about 1 nm to about 400 nm, from about 1 nm to about 300 nm, from about 1 nm to about 200 nm, or from about 1 nm to about 100 nm. Thus, the terms "nanostructure," "nanoparticle," "nanomaterial," and the like as used herein can include structures having at least one dimension in the range of no more than the range of dimensions described. The terms "nanostructure," "nanoparticle," "nanomaterial," and the like as used herein can include structures having at least one dimension of 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.

[0062] The term "micron" as used herein is broadly interpreted to include dimensions of about 1 micron to about 1000 microns, about 1 micron to less than about 1000 microns, about 1 micron to about 900 microns, about 1 micron to about 800 microns, about 1 micron to about 700 microns, about 1 micron to about 600 microns, about 1 micron to about 500 microns, about 1 micron to about 400 microns, about 1 micron to about 300 microns, about 1 micron to about 200 microns, about 1 micron to about 100 microns, or about 1 micron to about 5 microns. In various embodiments, particles of about 5 microns or less can be used for intranasal spray administration.

[0063] The term "treat" and synonyms thereof as used herein refer to therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) a medical condition including, but not limited to, diseases, symptoms, and conditions. Medical conditions also include the body's response to a disease or condition, such as inflammation. Those in need of such treatment include those already with the medical condition as well as those prone to have the medical condition or those in whom the medical condition is to be prevented.

[0064] The term "therapeutically effective amount" of a compound, as used herein, means an amount sufficient to prevent or capable of preventing or at least slowing down (lessening) a medical condition, such as an infectious disease, a respiratory disease (e.g., coronavirus caused by the SARS-CoV-2 virus or influenza caused by the influenza virus). The dosage and administration of the compounds, compositions, and formulations of the present disclosure can be determined by one of ordinary skill in the art of clinical pharmacology or pharmacokinetics. The effective amount of the active agents of the present disclosure for treatment will depend, for example, on the therapeutic objectives, the route of administration, and the condition of the patient. It can therefore be necessary to titrate the dosage and vary the route of administration depending on the desired therapeutic effect.

[0065] The term "subject" is intended to mean any animal such as a mammal, and includes humans, broadly. Exemplary subjects include, but are not limited to, human and non-human primates. The term "subject" as used herein also includes a patient and a non-patient. The term "patient" refers to an individual having or likely to have a medical condition, such as an infectious disease (e.g., coronavirus caused by the SARS-CoV-2 virus), whereas a "non-patient" refers to an individual who does not have or is not likely to have a medical condition. "Non-patients" include healthy individuals, individuals who are not ill, and / or individuals who do not have a medical condition. The term "mammal" as used herein includes vertebrates, such as humans or large veterinary mammals (e.g., horses, cows, deer, sheep, llamas, goats, pigs).

[0066] The term "bond" refers to a linkage between atoms in a compound or molecule. A bond can be a single bond, a double bond, or a triple bond.

[0067] The term "alkyl" as a group or part of a group means a linear or branched aliphatic hydrocarbon group having from one to twenty, one to ten, one to six, or one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty carbon atoms. Examples of suitable linear alkyl substituents 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 can be terminal or bridging.

[0068] The term "alkenyl" as a group or part of a group refers to a straight-chain or branched-chain aliphatic group containing at least one carbon-carbon double bond and which can have 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. The group can contain multiple double bonds, and the orientation of each double bond is independently E or Z. Exemplary alkenyl groups include, but are not limited to, ethenyl, vinyl, allyl, 1-methylethenyl, 1-propenyl, 2-propenyl, 2-methyl-l- propenyl, 2-methyl-l-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,3-butanedienyl, 1-pentenyl, 2-pentenyl, 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-heptenyl, 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 can be terminal or bridging.

[0069] The term "alkynyl" as a group or part of a group refers to a straight-chain or branched-chain aliphatic group containing at least one carbon-carbon triple bond and which can have 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. The group can contain multiple triple bonds. Exemplary alkynyl groups include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-l-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-nonynyl, 9-decynyl, and the like. The group can be terminal or bridging.

[0070] The term "cyclic" as used herein refers broadly to a structure in which one or more series of atoms are connected to form at least one ring. The term includes, but is not limited to, saturated and unsaturated 5-membered rings, as well as 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."

[0071] The term "heterocyclic" as used herein refers broadly to a structure in which two or more different kinds of atoms are connected to form at least one ring. For example, the ring of a heterocycle can be formed from carbon atoms and at least one other atom (i.e., a heteroatom) selected from oxygen (O), nitrogen (N) or (NR), and sulfur (S), where R is independently hydrogen or an organic group. The term also includes, but is not limited to, saturated and unsaturated 5-membered rings, as well as saturated and unsaturated 6-membered rings. Examples of groups having a heterocyclic structure include, but are not limited to, furan, thiophene, 1H-pyrrole, 2H-pyrrole, 1-pyrroline, 2-pyrroline, 3-pyrroline, 1-pyrazoline, 2-pyrazoline, 3-pyrazoline, 2-imidazoline, 3-imidazoline, 4-imidazoline, pyrazole, imidazole, oxazole, isoxazole, thiazole, isothiazole, 1,2,3-triazole, 1,2,4-triazole, 1,2,3-oxadiazole, disubstituted 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,2,5-thiadiazole, 1,3,4-thiadiazole, tetrahydrofuran, tetrahydrothiophene, pyrrolidine, 1,3-dioxolane, 1,2-oxathiolane, 1,3-oxathiolane, pyrazolidine, imidazolidine, pyridine, pyridazine, pyrimidine, pyrazine, 1,2-oxazine, 1,3-oxazine, 1,4-oxazine, thiazine, 1,2,3-triazine, 1,2,4-triazine, 1,3,5-triazine, 2H-pyran, 4H-pyran, 2-pyrone, 4-pyrone, 1,4-dioxin, 2H-thiopyran, 4H-thiopyran, tetrahydropyran, thiacyclohexane, piperidine, 1,4-dioxane, 1,2-dithiane, 1,3-dithiane, 1,4-dithiane, 1,3,5-trithiane, piperazine, morpholine, thiomorpholine, and the like.

[0072] The term "amine group" and the like is intended to refer broadly to a group containing -NR2, where R is independently hydrogen or an organic group. The group can be a terminal group or a bridging group.

[0073] The term "amide group" and the like is intended to refer broadly to a group containing -C(=O)NR2, where R is independently hydrogen or an organic group. The group can be a terminal group or a bridging group.

[0074] The term "aryl" as a group or part of a group refers to (i) an optionally substituted monocyclic or fused polycyclic aromatic carbocyclic ring (ring structure having all ring atoms being carbon), which preferably has 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, anthryl, phenanthryl, fluorenyl, indenyl, or indanyl, and the like.

[0075] The term "heteroaryl" as a group or part of a group refers to a group containing an aromatic ring (preferably a 5-membered or 6-membered aromatic ring) in which one or more than one carbon atom (e.g., 1 to 6 carbon atoms) in the ring is replaced by a heteroatom. Suitable heteroatoms can include nitrogen (N) or (NH), oxygen (O), and sulfur (S). Examples of heteroaryl groups include, but are not limited to, thiophene, benzothiophene, benzofuran, benzimidazole, benzoxazole, benzothiazole, benzisothiazole, naphtho[2,3-b]thiophene, furan, isoindolizine, xanthone, phenoxazine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, tetrazole, indole, isoindole, 1H-indazole, purine, quinoline, isoquinoline, phthalazine, naphthylidine, quinoxaline, cinnoline, carbazole, phenanthridine, acridine, phenoxazine, thiazole, isothiazole, phenothiazine, oxazole, isoxazole, furazan, phenoxazine, 2-pyridyl, 3-pyridyl, or 4-pyridyl, 2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, or 8-quinolyl, 1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, or 5-isoquinolyl, 1-indolyl, 2-indolyl, or 3-indolyl, and 2-thiophenyl or 3-thiophenyl, and the like. The group can be a terminal group or a bridging group.

[0076] The term "halogen" represents chlorine, fluorine, bromine, or iodine. The term "halide" represents chloride, fluoride, bromide, or iodide.

[0077] When the term "optionally substituted" is used to describe a chemical structure or moiety, it means that one or more hydrogen atoms are optionally replaced with a chemical moiety or functional group, such as an 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 (e.g., alkylamino, arylamino, arylalkylamino), aryl, aryloxy, azo, carbamoyl (-NHC(O)O-alkyl- or -OC(O)NH-alkyl), carbamyl (e.g., CONH2, and CONH-alkyl, CONH-aryl, and CONH-arylalkyl), carboxyl, carboxylic acid, cyano, ester, ether (e.g., methoxy, ethoxy), halogen, haloalkyl (e.g., -CCI3, -CF3, -C(CF3)3), heteroalkyl, isocyanate, thioisocyanate, nitrile, nitro, phosphodiester, sulfide, sulfonamido (e.g., SO2NH2), sulfone, sulfonyl (including alkylsulfonyl, arylsulfonyl, and arylalkylsulfonyl), sulfoxide, thiol (e.g., mercaptobenzyl, thioether), or urea (-NHCONH-alkyl-).

[0078] Unless otherwise indicated, the terms "coupled" or "connected" as used in the present specification intend to cover a direct connection between elements in which an intermediate medium can or can not be present between the coupled or connected elements.

[0079] The term "related to," as used herein when referring to two elements, means a broad relationship between the two elements. The relationship includes, but is not limited to, a physical relationship, a chemical relationship, or a biological relationship. For example, when element A is related to element B, element A and element B can be directly or indirectly connected to each other, or element A can contain element B, and vice versa.

[0080] The term "adjacent to," as used herein when referring to two elements, means that one element is in close proximity to the other element, and can be, but is not limited to, elements that are in contact with each other, or can further include elements that are separated by one or more other elements placed therebetween.

[0081] The term "and / or," such as "X and / or Y" is understood to allow for "X and Y" or "X or Y," and thus, is understood to cover the two meanings or one of the two meanings.

[0082] Further, in the description herein, whenever a word "substantially" is used, it is understood to include, but not limited to, "completely" or "entirely" and the like. Further, whenever terms such as "comprising" or "including," or the like, are used, it is meant as non-limiting descriptive language, as such terms encompass the elements / components recited after such terms, in addition to other components not specifically recited. For example, where "comprising" is used, reference to "one" feature is also intended to refer to "at least one" of that feature. Terms such as "consisting of," or the like, can be considered as subsets of terms such as "comprising" or "including," or the like, in the appropriate context. Thus, in embodiments disclosed herein that use terms such as "comprising" or "including," or the like, it is understood that corresponding embodiments using terms such as "consisting of," or the like, are contemplated as respective embodiments. Further, whenever terms such as "about," "approximately," or the like, are used, it is generally intended to refer to a reasonable variation that is within the bounds of manufacturing and / or other typical variations, such as + / - 5% of a disclosed value, or + / - 4% of a disclosed value, or + / - 3% of a disclosed value, + / - 2% of a disclosed value, or + / - 1% of a disclosed value.

[0083] Further, in the description herein, certain values can be disclosed using ranges. The values that show the endpoints of the range are intended to be illustrative of a preferred range. Whenever a range is described, it is intended to encompass and disclose all possible sub-ranges as well as individual numbers within that range. That is, a range of "1% to 5%" should be considered to include the end values of 1% and 5% as well as sub-ranges within the values of 1% to 5%, e.g., 1% to 2%, 1% to 3%, 1% to 4%, 2% to 3%, etc., as well as individual values within the 1% to 5% range, e.g., 1%, 2%, 3%, 4%, and 5%. The intent is to cover all possible bases of the range.

[0084] Further, in describing some embodiments, the disclosure can disclose methods and / or processes as a particular sequence of steps. However, unless otherwise required, it is understood that the methods or processes should not be limited to the specific sequence of steps disclosed. Other sequences of steps are possible. The specific sequence of steps disclosed herein should not be construed as improper limitations. Unless otherwise required, the methods and / or processes disclosed herein should not be limited to steps performed in the order as described. The order of steps can be varied and still remain within the scope of the disclosure.

[0085] Further, it is understood that while the disclosure provides embodiments having one or more features / characteristics discussed herein, in other alternative embodiments one or more of these features / characteristics can be negated, and the disclosure provides support for such negations and these related alternative embodiments.

[0086] It is also to be understood that where prior applications are referred to herein, such references are not to be construed with any dependency or relation as to allowability of any of the claims herein. It is also to be understood that the disclosure of prior applications are to be considered as being incorporated in its entirety into the disclosure of this application, and that the disclosure of prior applications can be used to support support the disclosure of the embodiments disclosed herein. DETAILED DESCRIPTION

[0087] Disclosed below are exemplary non-limiting embodiments of compounds for preparing lipid nanoparticles encapsulating an agent, methods of preparing the compounds, nanoparticle compositions comprising the compounds, and related methods / uses thereof.

[0088] Compounds

[0089] Provided are compounds for preparing lipid nanoparticles. In various embodiments, the compounds comprise one or more ionizable and / or capable of being ionized amine groups. The amine groups can be selected from primary (1°) amines, secondary (2°) amines, tertiary (3°) amines, and combinations thereof. Thus, in various embodiments, the compounds are ionizable and / or capable of being ionized and / or exist in an ionized form, e.g., at physiological pH. Advantageously, the ionizable nature of the compounds (due to the presence of ionizable amine groups) allows embodiments of the compounds to be used as encapsulating / loading agents, delivery vehicles / delivery systems, and / or transfection vehicles / transfection systems. In various embodiments, the compounds are designed / configured to allow loading / encapsulation of one or more types of molecules or cargo. In various embodiments, the compounds are also designed / configured to allow release of the loaded / encapsulated agent from the compounds and / or subsequent delivery to a desired target (e.g., a cell, cytosol, tissue, or organ). The molecules / cargo to be loaded / encapsulated onto / in the compounds can include, but are not limited to, therapeutic agents, prophylactic agents, biological agents, and the like. In various embodiments, the loaded / encapsulated molecules / cargo include nucleic acids. For example, the loaded / encapsulated molecules / cargo can be a nucleic acid selected from a ribonucleic acid (RNA), a messenger ribonucleic acid (mRNA), an interfering small ribonucleic acid (siRNA), a deoxyribonucleic acid (DNA), a plasmid deoxyribonucleic acid (pDNA), an oligonucleotide such as an antisense oligonucleotide (ASO), and the like, or combinations thereof. In various embodiments, the loaded / encapsulated molecules / cargo include therapeutic agents. For example, the loaded / encapsulated molecules / cargo can be a therapeutic agent selected from a negatively charged therapeutic agent, a pharmaceutical molecule, a vaccine (e.g., a dengue vaccine, and the like), and the like, or combinations thereof. Advantageously, the compounds are suitable for encapsulating and / or delivering one or more therapeutic agents, prophylactic agents, and / or biological agents to a desired target (e.g., a subject, a cell, a cytosol, a tissue, or an organ).

[0090] Thus, in various embodiments, also provided are vehicles, nanocarriers, or delivery systems / delivery vehicles comprising the compounds or ionized forms thereof.

[0091] Advantageously, the compound is designed / configured to be ionizable at a pH of about 3 to about physiological pH, depending on the type or nature of the amine group(s). In various embodiments, the compound is capable of being ionized at a pH of about 3.0 to about 7.8, about 3.1 to about 7.7, about 3.2 to about 7.6, about 3.3 to about 7.5, about 3.4 to about 7.4, about 3.5 to about 7.3, about 3.6 to about 7.2, about 3.7 to about 7.1, about 3.8 to about 7.0, about 3.9 to about 6.9, about 4.0 to about 6.8, about 4.1 to about 6.7, about 4.2 to about 6.6, about 4.3 to about 6.5, about 4.4 to about 6.4, about 4.5 to about 6.3, about 4.6 to about 6.2, about 4.7 to about 6.1, about 4.8 to about 6.0, about 4.9 to about 5.9, about 5.0 to about 5.8, about 5.1 to about 5.7, about 5.2 to about 5.6, about 5.3 to about 5.5, or about 5.4. In various embodiments where the compound comprises a primary amine, the compound is capable of being ionized at a physiological pH of about 7.00 to about 7.80, about 7.05 to about 7.75, about 7.10 to about 7.70, about 7.15 to about 7.65, about 7.20 to about 7.60, about 7.25 to about 7.55, about 7.30 to about 7.50, about 7.35 to about 7.45, about 7.36, about 7.37, about 7.38, about 7.39, about 7.40, about 7.41, about 7.42, about 7.43, about 7.44, or about 7.45. In various embodiments where the compound comprises a secondary and / or tertiary amine, the compound is capable of being ionized at a pH of about 3.0 to about 5.5, about 3.0 to about 5.0, about 3.1 to about 4.9, about 3.2 to about 4.8, about 3.3 to about 4.7, about 3.4 to about 4.6, about 3.5 to about 4.5, about 3.6 to about 4.4, about 3.7 to about 4.3, about 3.8 to about 4.2, about 3.9 to about 4.1, or about 4.0.

[0092] In various embodiments, the compound comprises a structure as shown in general formula (1) or an ionized form thereof:

[0093]

[0094] wherein

[0095] NR 1 R 2 is a group that is ionizable or capable of being ionized at a pH of 3 to physiological pH;

[0096] A comprises a linear aliphatic hydrocarbon, a branched aliphatic hydrocarbon, and / or a cyclic hydrocarbon, optionally comprising a moiety selected from -OH, -NR-, -O-, -O-C x H 2xone or more of the groups -O-, wherein x > 1; and wherein R is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0097] R 3 , R 4 , R 5 , R 6 , and R 7 are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and

[0098] R 8 , and R 9 are each independently a hydrophobic tail / hydrophobic chain / hydrophobic group or contain at least one group R 3 through R 7 as defined above.

[0099] In various embodiments, x is an integer > 1. In various embodiments, x > 1, x > 2, 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, or x < 20. Advantageously, when x < 20 (e.g., 1 to 20), the compound is less hydrophobic. For example, A can comprise -O-CH2-O-, -O-C2H4-O-, -O-C3H6-O-, -O-C4H8-O-, or -O-C5H 10 -O-.

[0100] In various embodiments, R 1 and R 2 are each independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl. For example, R 1 and R 2may be selected from methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, t-butyl, hexyl, amyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, pentyl, iso-pentyl, 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, or combinations thereof.

[0101] In various embodiments, R 1 and R 2 are each H. In these embodiments, -NR 1 R 2 is -NH2or a primary amine group, and the compound comprises a primary (1°) amine group (e.g., an ionizable primary amine group).

[0102] In various embodiments, R 1 or R 2 is H. In these embodiments, -NR 1 R 2 is -NHR 2 or -NR 1 H (i.e., a secondary (2°) amine group), and the compound comprises a secondary (2°) amine group (e.g., an ionizable secondary amine group).

[0103] In various embodiments, R 1 and R 2 are each other than H. In various embodiments, R 1 and R 2 are each independently selected from an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group, or combinations thereof. In these embodiments, -NR 1 R 2 is a tertiary amine group (e.g., a tertiary amine group in DMAPAPA-DTD), and the compound comprises a tertiary (3°) amine group (e.g., an ionizable tertiary amine group).

[0104] In various embodiments, the compound is in an ionized form, wherein -NR1R 2 has been ionized to a positively charged group. In various embodiments, -NR 1 R2 ionized / protonated to a positively charged group / positively charged ion / cation at a pH of about 3 to about physiological pH (or about neutral pH). In various embodiments where the compound comprises a primary amine (i.e., -NR 1 R 2 is a primary amine). In various embodiments where the compound comprises a secondary amine and / or tertiary amine (e.g., -NR 1 R 2 ionized to a positively charged group / positively charged ion / cation at physiological pH (or about neutral pH). In various embodiments where the compound comprises a secondary amine and / or tertiary amine (e.g., -NR 1 R 2 is a secondary amine and / or tertiary amine such as DMAPAPA-DTD). In various embodiments where the compound comprises a secondary amine and / or tertiary amine (e.g., -NR 1 R 2 ionized to a positively charged group / positively charged ion / cation at a pH of about 3 to about 5 or at about 4. In various embodiments, -NR 1 R 2 protonated to -NR 1 R 2 H + . For example, when R 1 and R 2 are both H, -NR 1 R 2 may be protonated to -NH3 + .

[0105] In various embodiments, the compound comprises at least 1, at least 2, or at least 3 ionizable amine groups selected from primary (1°) amines, secondary (2°) amines, tertiary (3°) amines, and combinations thereof. For example, in addition to the ionizable group at NR 1 R 2 may contain 1 or more than 1, 2 or more than 2, or 3 or more than 3 ionizable amine groups in A. The encapsulation efficiency of compounds comprising secondary (2°) amines and / or tertiary (3°) amines as one or more ionizable groups can be comparable to most compounds comprising a primary amine group as the only ionizable group.

[0106] In various embodiments, the compound comprises a lipid compound. The term "compound" can include and / or can be used interchangeably with the terms "lipid," "lipid compound," "ionizable lipid," "ionizable lipid compound," "cationic lipid compound," "ionizable cationic lipid compound," and the like. In various embodiments, the compound is amphiphilic / amphipathic and comprises a hydrophilic portion and a hydrophobic portion. In various embodiments, the lipid portion of the compound is hydrophobic, while groups in the compound such as amine groups and / or hydroxyl groups are hydrophilic. In various embodiments, the compound comprises one or more hydrophilic moieties at the amine group (e.g., ionizable NR 1 R 2 ) of the compound. In various embodiments, the compound comprises a hydrophobic moiety / hydrophobic tail / hydrophobic chain / hydrophobic group at R 8 and R 9 . In various embodiments, the hydrophobic tail / hydrophobic chain / hydrophobic group at R 8 and R 9 each independently comprises an optionally substituted alkyl group. The alkyl group can have at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 carbon atoms. For example, R 8 and R 9 may each independently be C y H 2y+1 , where y > 5, y > 6, y > 7, y > 8, y > 9, y > 10, y > 11, y > 12, y > 13, y > 14, y > 15, y > 16, y > 17, y > 18, y > 19, or y > 20. Advantageously, in various embodiments, the presence of the hydrophobic moiety / hydrophobic tail / hydrophobic chain / hydrophobic group in the compound helps to improve cellular uptake and / or transfection, resulting in higher and / or better transfection rates.

[0107] In various embodiments, A is selected from the following general formula (2), general formula (3), general formula (4), or general formula (5):

[0108]

[0109]

[0110] wherein

[0111] X 1 to X 31 are each independently selected from -H, -OH, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group;

[0112] n≥1; m≥1; p≥1; and q≥1.

[0113] 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, or n ≤ 20.

[0114] In various embodiments, m is an integer ≥ 1. In various embodiments, m ≥ 1, m ≥ 2, m ≥ 3, m ≥ 4, m ≥ 5, m ≥ 6, m ≥ 7, m ≥ 8, m ≥ 9, m ≥ 10, m ≥ 11, m ≥ 12, m ≥ 13, m ≥ 14, m ≥ 15, m ≥ 16, m ≥ 17, m ≥ 18, m ≥ 19, or m ≤ 20.

[0115] In various embodiments, p is an integer ≥ 1. In various embodiments, p ≥ 1, p ≥ 2, p ≥ 3, p ≥ 4, p ≥ 5, p ≥ 6, p ≥ 7, p ≥ 8, p ≥ 9, p ≥ 10, p ≥ 11, p ≥ 12, p ≥ 13, p ≥ 14, p ≥ 15, p ≥ 16, p ≥ 17, p ≥ 18, p ≥ 19, or p ≤ 20.

[0116] In various embodiments, q is an integer ≥ 1. In various embodiments, q ≥ 1, q ≥ 2, q ≥ 3, q ​​≥ 4, q ≥ 5, q ≥ 6, q ≥ 7, q ≥ 8, q ≥ 9, q ≥ 10, q ≥ 11, q ≥ 12, q ≥ 13, q ≥ 14, q ≥ 15, q ≥ 16, q ≥ 17, q ≥ 18, q ≥ 19, or q ≤ 20.

[0117] In various embodiments, the compound comprises a structure selected from one or more of the following:

[0118]

[0119]

[0120]

[0121] Methods of making compounds

[0122] A method for preparing the compound disclosed herein as represented by general formula (1) is provided, the method comprising:

[0123] (ai) reacting an amine compound represented by the general formula (6) with a cyclic acid anhydride represented by the general formula (7) to obtain a first intermediate compound comprising a carboxylate group represented by the general formula (8):

[0124]

[0125] (a-ii) reacting the first intermediate compound as shown in general formula (8) with N-hydroxysuccinimide (NHS) in the presence of a coupling agent to obtain a second intermediate compound comprising an amide group as shown in general formula (9):

[0126]

[0127] (a-iii) reacting the second intermediate compound as shown in general formula (9) with an amine compound as shown in general formula (10) to obtain a compound as shown in general formula (1):

[0128]

[0129] wherein R 1 to R 9 and A comprises one or more features and / or shares one or more properties similar to those features and / or properties as described above (e.g., as defined in general formula (1)); and

[0130] (a-iv) optionally reacting the -NR 1 R 2 ionizes into a positively charged group.

[0131] Advantageously, embodiments of the method are easy to perform and have low production / manufacturing costs (i.e., cost effective) as it can simply be performed in 3 synthetic / reaction steps. It should be appreciated that currently available or known methods require at least 5 or at least 6 synthetic / reaction steps to produce ionizable lipid compounds in the art. Advantageously, embodiments of the method are scalable and / or have substantially high scalability.

[0132] In various embodiments, the cyclic anhydride as shown in general formula (7) includes succinic anhydride and the like.

[0133] In various embodiments, the coupling agent includes a carbodiimide. For example, the coupling agent can be 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N,N’-dicyclohexylcarbodiimide (DCC), N,N’-diisopropylcarbodiimide (DIC), and the like or a combination thereof.

[0134] In various embodiments, the amine compound as shown in general formula (10) includes ethylenediamine (EDA), N,N-dimethyldipropyltriamine, and the like or a combination thereof.

[0135] In various embodiments, step (a-iii) comprises reacting the amine compound as shown in Formula (10) with the second intermediate compound as shown in Formula (9) in a molar ratio of about 1 : 1 to about 5: 1. Step (a-iii) can comprise reacting the amine compound as shown in Formula (10) with the second intermediate compound as shown in Formula (9) in a molar ratio of about 1 : 1 to about 5: 1, about 1 : 1, about 2: 1, about 3: 1, about 4: 1, or about 5: 1. It will be appreciated that the amine compound as shown in Formula (10) can be provided in excess (e.g., slight excess) to ensure that the desired molar ratio between the amine compound as shown in Formula (10) and the second intermediate compound as shown in Formula (9) is achieved.

[0136] In various embodiments, reaction step (a-iii) comprises adding the second intermediate compound as shown in Formula (9) to the amine compound as shown in Formula (10) dropwise.

[0137] In various embodiments, reaction step (a-i), reaction step (a-ii), and / or reaction step (a-iii) comprises one or more than one of the following steps: dispersing, mixing, stirring, dissolving, sonicating, and / or ultrasonication.

[0138] In various embodiments, reaction step (a-i), reaction step (a-ii), and / or reaction step (a-iii) is performed in the presence of an organic solvent. In various embodiments, any organic solvent that is effective to serve as a medium for a component (e.g., reactant / substrate) contained in the reaction mixture can be used in embodiments of the reaction mixtures disclosed herein. In various embodiments, the organic solvent is capable of substantially dissolving the components present in the reaction mixture. The organic solvent can be a dry or anhydrous organic solvent, such as dry or anhydrous dichloromethane (DCM).

[0139] In various embodiments, reaction step (a-i), reaction step (a-ii), and / or reaction step (a-iii) is performed under an inert atmosphere. For example, one or more than one of the steps of dispersing, mixing, and / or stirring can be performed in the presence of an inert gas, such as argon or nitrogen, or in the absence of a reactive gas, such as oxygen (e.g., dissolved oxygen).

[0140] In various embodiments, reaction step (a-i), reaction step (a-ii), and / or reaction step (a-iii) is performed for a duration of about 1 hour to about 72 hours, about 2 hours to about 60 hours, about 3 hours to about 48 hours, about 4 hours to about 36 hours, about 5 hours to about 24 hours, or about 6 hours to about 12 hours.

[0141] In various embodiments, reaction step (a-i) and / or reaction step (a-ii) is optionally performed at room temperature, e.g., about 20 °C to about 30 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, or about 30 °C.

[0142] In various embodiments, reaction step (a-iii) is optionally performed at a temperature of about -30 °C to about -80 °C, about -35 °C to about -75 °C, about -40 °C to about -70 °C, about -45 °C to about -65 °C, about -50 °C to about -60 °C, or about -55 °C, e.g., to control reaction kinetics. For example, the reaction step can be performed in a dry ice bath.

[0143] In various embodiments, the method further comprises:

[0144] (b-i) a step of isolating the first intermediate compound after step (a-i);

[0145] (b-ii) a step of isolating the second intermediate compound after step (a-ii); and

[0146] (b-iii) a step of isolating the compound as shown in general formula (1) after step (a-iii).

[0147] In various embodiments, one or more isolation steps include one or more of the following: re-dissolving, purifying, centrifuging, quenching, washing, precipitating, and / or re-crystallizing the first intermediate compound, the second intermediate compound, and / or the compound as shown in general formula (1). One or more steps of purifying, centrifuging, quenching, and / or washing can be repeated with a wash medium at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times. In various embodiments, the isolation steps are performed to remove byproducts of the first intermediate compound, the second intermediate compound, and / or the compound as shown in general formula (1). In various embodiments, the wash medium includes an aqueous medium / solution, such as a salt solution or deionized water. The salt solution can be a bicarbonate salt such as sodium bicarbonate, a chloride salt such as sodium chloride (brine). In various embodiments, the salt solution includes a high concentration / saturated salt solution.

[0148] In various embodiments, the method further comprises one or more of the following post-reaction steps: drying the first intermediate compound, the second intermediate compound, and / or the compound as shown in general formula (1) optionally at low temperature (e.g., lyophilization), under vacuum. One or more drying steps can be performed in the presence of a drying agent such as magnesium sulfate, sodium sulfate, calcium chloride, or a combination thereof.

[0149] In various embodiments, step (a-iv) is present and is performed at a pH of about 3 to about physiological pH. In various embodiments, step (a-iv) is performed at a pH of about 3.0 to about 7.8, about 3.1 to about 7.7, about 3.2 to about 7.6, about 3.3 to about 7.5, about 3.4 to about 7.4, about 3.5 to about 7.3, about 3.6 to about 7.2, about 3.7 to about 7.1, about 3.8 to about 7.0, about 3.9 to about 6.9, about 4.0 to about 6.8, about 4.1 to about 6.7, about 4.2 to about 6.6, about 4.3 to about 6.5, about 4.4 to about 6.4, about 4.5 to about 6.3, about 4.6 to about 6.2, about 4.7 to about 6.1, about 4.8 to about 6.0, about 4.9 to about 5.9, about 5.0 to about 5.8, about 5.1 to about 5.7, about 5.2 to about 5.6, about 5.3 to about 5.5, or about 5.4.

[0150] Nanoparticle compositions

[0151] Advantageously, in various embodiments, the ionizable nature of the compound as represented by Formula (1) (due to the presence of the ionizable amine group) is able to condense and encapsulate / entrain the molecule / good into embodiments of the compound, thereby forming a nanoparticle in the composition. In various embodiments, embodiments of the compound are able to form a nanoparticle in the composition. In various embodiments, in the presence of a composition comprising a molecule / good (e.g., a therapeutic agent, a prophylactic agent, and / or a biological agent), one or more amine groups in the compound (e.g., one or more ionizable amine groups / protonated amine groups in a lipid compound) condense and encapsulate / entrain the molecule / good into the compound to form a nanoparticle (e.g., a lipid nanoparticle (LNP)) in the composition.

[0152] The term “nanoparticle” can include and / or be used interchangeably with the terms “lipid nanoparticle,” “encapsulated lipid nanoparticle,” “entrained lipid nanoparticle,” “LNP,” and the like.

[0153] Provided are nanoparticle compositions comprising:

[0154] (i) a compound as represented by Formula (1) disclosed herein or an ionized form thereof; and

[0155] (ii) a therapeutic agent, a prophylactic agent, and / or a biological agent encapsulated / entrained in the compound as represented by Formula (1) or an ionized form thereof.

[0156] In various embodiments, the compound as shown in General Formula (1) is capable of being ionized (e.g., protonated) at a pH of 3 to physiological pH (or neutral pH) such that the composition encapsulates a therapeutic agent and / or prophylactic agent and / or biological agent coupled / bonded / linked / bound to the composition / nanoparticle. In various embodiments, the compound as shown in General Formula (1) is capable of being ionized (e.g., protonated) at physiological pH (or neutral pH) such that the composition encapsulates a therapeutic agent and / or prophylactic agent and / or biological agent coupled / bonded / linked / bound to the composition / nanoparticle. The therapeutic agent and / or prophylactic agent and / or biological agent can be coupled / bonded / linked / bound to the composition / nanoparticle via electrostatic interactions and / or other physical interactions. In various embodiments, the therapeutic agent and / or prophylactic agent and / or biological agent is electrostatically and / or physically coupled / bonded / linked / bound to the composition / nanoparticle.

[0157] In various embodiments, for compounds (e.g., lipid compounds) containing primary amine groups (e.g., DnO-NH2, DD-NH2, DTD-NH2, HO-DnO-NH2, HO-DD-NH2, HO-DTD-NH2, DnO-EDEA, DD-EDEA, DTD-EDEA), these lipids readily protonate in water, resulting in the formation of positively charged molecules that condense molecules / goods (e.g., nucleic acids such as mRNA) into lipid nanoparticles (LNPs) via electrostatic interactions and / or physical interactions, forming encapsulated LNPs (e.g., mRNA LNPs). For example, after mRNA LNPs are taken up by cells via endocytosis, they can enter endolysosomes, where they fuse with the membrane of the endolysosome, resulting in the release of the encapsulated mRNA into the cytosol for transfection (e.g., gene transfection).

[0158] In various embodiments, for compounds (e.g., lipid compounds) containing secondary and / or tertiary amine groups (e.g., DMAPAPA-DTD), the lipids protonate at a pH of about 4.0, at which pH the encapsulated LNPs (e.g., mRNA LNPs) are prepared. For example, such lipids can carry a positive charge at a pH of 4.0 and condense mRNA into LNPs. The mRNA LNPs can then be taken up by cells via pinocytosis and then enter endolysosomes, where the primary and / or tertiary amine groups can absorb protons in the endolysosome, disrupt the endolysosome membrane, and release the mRNA into the cytosol for transfection (e.g., gene transfection).

[0159] Advantageously, the compositions are suitable for encapsulating, delivering, and / or transfecting one or more than one therapeutic agent, prophylactic agent, and / or biological agent to, for example, a desired target (e.g., a subject, a cell, a cytosol, a tissue, or an organ).

[0160] In various embodiments, the composition further comprises:

[0161] (a) a neutral lipid / co-lipid;

[0162] (b) a sterol; and

[0163] (c) a PEG-modified lipid.

[0164] The term "PEG-modified lipid" can include and / or be used interchangeably with the terms "PEGylated lipid" and "lipid modified with PEG."

[0165] In various embodiments, the compound or ionized form thereof, the neutral lipid / co-lipid, the sterol, and the PEG-modified lipid are mixed / dissolved in an organic solvent. In various embodiments, any organic solvent that is effective to serve as a medium for the components (e.g., reactants / substrates) of the reaction mixture can be used in the embodiments of the reaction mixture disclosed herein. In various embodiments, the organic solvent is capable of substantially dissolving the components present in the mixture. The organic solvent can include ethanol, isopropanol, acetonitrile, ethyl acetate, methanol, tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, and the like, or combinations thereof.

[0166] In various embodiments, the compound as shown in general formula (1) or ionized form thereof, the neutral lipid / co-lipid, the sterol, and the PEG-modified lipid are mixed / dissolved in a weight ratio of about 10 to about 50: about 2 to about 20: about 4 to about 30: about 1 to about 15. For example, the compound or ionized form thereof, the neutral lipid / co-lipid, the sterol, and the PEG-modified lipid can be mixed in a weight ratio of about 10 to about 50: about 2 to about 20: about 4 to about 30: about 1 to about 15, about 15 to about 45: about 4 to about 18: about 8 to about 26: about 3 to about 13, about 20 to about 40: about 6 to about 16: about 12 to about 22: about 5 to about 11, about 25 to about 35: about 8 to about 14: about 16 to about 18: about 7 to about 9, or about 30: about 11: about 17: about 8. In various embodiments, the compound as shown in general formula (1) or ionized form thereof, the neutral lipid / co-lipid, the sterol, and the PEG-modified lipid are mixed / dissolved in a weight ratio of about 28:6:13:3.

[0167] In various embodiments, the composition comprises from about 10.0 mole % to about 75.0 mole %, from about 15.0 mole % to about 70.0 mole %, from about 20.0 mole % to about 65.0 mole %, from about 25.0 mole % to about 60.0 mole %, from about 30.0 mole % to about 55.0 mole %, from about 35.0 mole % to about 50.0 mole %, or from about 40.0 mole % to about 45.0 mole % of a compound as shown in Formula (1). In various embodiments, the compound as shown in Formula (1) is the major component of the composition, and the compound as shown in Formula (1) is present in an amount of no less than about 30 wt. %, no less than about 31 wt. %, no less than about 32 wt. %, no less than about 33 wt. %, no less than about 34 wt. %, no less than about 35 wt. %, no less than about 36 wt. %, no less than about 37 wt. %, no less than about 38 wt. %, no less than about 39 wt. %, no less than about 40 wt. %, no less than about 41 wt. %, no less than about 42 wt. %, no less than about 43 wt. %, no less than about 44 wt. %, no less than about 45 wt. %, no less than about 46 wt. %, no less than about 47 wt. %, no less than about 48 wt. %, no less than about 49 wt. %, no less than about 50 wt. %, no less than about 51 wt. %, no less than about 52 wt. %, no less than about 53 wt. %, no less than about 54 wt. %, no less than about 55 wt. %, or no less than about 56 wt. %, or no less than about 57 wt. %, or no less than about 58 wt. %, no less than about 59 wt. %, or no less than about 60 wt. % of the composition.

[0168] In various embodiments, the neutral lipid / auxiliary lipid includes a phospholipid such as an unsaturated lipid. Examples of phospholipids include, 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-didodecanoyl-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-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 LysoPC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-dibehenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0 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-dibehenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and the like, and combinations thereof.

[0169] In various embodiments, the composition comprises about 1.0 mole % to about 20.0 mole %, about 2.0 mole % to about 19.0 mole %, about 3.0 mole % to about 18.0 mole %, about 4.0 mole % to about 17.0 mole %, about 5.0 mole % to about 16.0 mole %, about 6.0 mole % to about 15.0 mole %, about 7.0 mole % to about 14.0 mole %, about 8.0 mole % to about 13.0 mole %, about 9.0 mole % to about 12.0 mole %, about 10.0 mole % to about 11.0 mole %, or about 10.5 mole % of a neutral lipid / auxiliary lipid. In various embodiments, the neutral lipid / auxiliary lipid is present in an amount of no less than about 3 wt. %, no less than about 4 wt. %, no less than about 5 wt. %, no less than about 6 wt. %, no less than about 7 wt. %, no less than about 8 wt. %, no less than about 9 wt. %, no less than about 10 wt. %, or no less than about 11 wt. %, or no less than about 12 wt. %, or no less than about 13 wt. %, or no less than about 14 wt. %, or no less than about 15 wt. % of the composition.

[0170] In various embodiments, the sterol is selected from the group consisting of cholesterol, 5a-ergosta-8,24(28)-dien-3b-ol, sitosterol, ergosterol, campesterol, soysterol, brassicasterol, avenasterol, and the like, or combinations thereof.

[0171] In various embodiments, the composition comprises about 10.0 mole % to about 50.0 mole %, about 15.0 mole % to about 47.5 mole %, about 17.5 mole % to about 45.0 mole %, about 20.0 mole % to about 42.5 mole %, about 22.5 mole % to about 40.0 mole %, about 25.0 mole % to about 37.5 mole %, about 27.5 mole % to about 35.0 mole %, or about 30.0 mole % to about 32.5 mole % of a sterol. In various embodiments, the sterol is present in an amount of no less than about 10 wt. %, no less than about 11 wt. %, no less than about 12 wt. %, no less than about 13 wt. %, no less than about 14 wt. %, no less than about 15 wt. %, no less than about 16 wt. %, no less than about 17 wt. %, no less than about 18 wt. %, no less than about 19 wt. %, no less than about 20 wt. %, no less than about 21 wt. %, no less than about 22 wt. %, no less than about 23 wt. %, no less than about 24 wt. %, no less than about 25 wt. %, no less than about 26 wt. %, no less than about 27 wt. %, no less than about 28 wt. %, no less than about 29 wt. %, no less than about 30 wt. %, no less than about 31 wt. %, no less than about 32 wt. %, no less than about 33 wt. %, no less than about 34 wt. %, no less than about 35 wt. %, no less than about 36 wt. %, no less than about 37 wt. %, no less than about 38 wt. %, no less than about 39 wt. %, or no less than about 40 wt. % of the composition.

[0172] In various embodiments, the PEG-modified lipid is selected from a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and the like, or combinations thereof. Examples of PEG-modified lipids / PEGylated lipids include, but are not limited to, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), R-3-[(ω-methoxy-poly(ethylene glycol) 2000)carbamoyl]-1,2-dimyristyloxypropyl-3-amine (PEG-c-DOMG), 3-N-[(ω-methoxy poly(ethylene glycol) 2000)carbamoyl]-1,2-dimyristyloxy-propanamine (PEG-S-DMG), PEG-DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)- methoxy] (sodium salt)), PEG-DPPC, PEG-DSPE lipids, and the like, or combinations thereof. In various embodiments, the composition comprises about 0.5 mole % to about 10.0 mole %, about 1.0 mole % to about 9.5 mole %, about 1.5 mole % to about 9.0 mole %, about 2.0 mole % to about 8.5 mole %, about 2.5 mole % to about 8.0 mole %, about 3.0 mole % to about 7.5 mole %, about 3.5 mole % to about 7.0 mole %, about 4.0 mole % to about 6.5 mole %, about 4.5 mole % to about 6.0 mole %, or about 5.0 mole % to about 5.5 mole % of the PEG-modified lipid. In various embodiments, the PEG-modified lipid is present in an amount of no less than about 0.5 wt.%, no less than about 0.6 wt.%, no less than about 0.7 wt.%, no less than about 0.8 wt.%, no less than about 0.9 wt.%, no less than about 1 wt.%, no less than about 2 wt.%, no less than about 3 wt.%, no less than about 4 wt.%, no less than about 5 wt.%, no less than about 6 wt.%, no less than about 7 wt.%, or no less than about 8 wt.%, or no less than about 9 wt.%, or no less than about 10 wt.%, or no less than about 11 wt.%, or no less than about 12 wt.% of the composition.

[0173] In various embodiments, the therapeutic agent, prophylactic agent, and / or biological agent is provided in an aqueous buffer. The aqueous buffer can be sodium acetate.

[0174] In various embodiments, the nanoparticle composition comprises nanoparticles formed from a compound as shown in general formula (1) or an ionized form thereof.

[0175] Nanoparticles

[0176] Provided are nanoparticles (e.g., lipid nanoparticles) comprising:

[0177] (i) a compound as disclosed herein, as represented by Formula (1) or an ionized form thereof; and

[0178] (ii) a therapeutic agent and / or prophylactic agent and / or biological agent encapsulated / loaded / coupled / bonded / linked / bound to a compound as represented by Formula (1) or an ionized form thereof.

[0179] In various embodiments, the N:P ratio or N / P ratio of the nanoparticle (i.e., the molar ratio of ionizable nitrogen atoms in the compound (e.g., ionizable lipid compound) to phosphate groups in the therapeutic agent, prophylactic agent, and / or biological agent (e.g., nucleic acid)) is about 2: 1 to about 40: 1. The N:P ratio or N / P ratio of the nanoparticle can be about 2: 1 to about 40: 1, about 3: 1 to about 39: 1, about 4: 1 to about 38: 1, about 5: 1 to about 37: 1, about 6: 1 to about 36: 1, about 7: 1 to about 35: 1, about 8: 1 to about 34: 1, about 9: 1 to about 33: 1, about 10: 1 to about 32: 1, about 11: 1 to about 31: 1, about 12: 1 to about 30: 1, about 13: 1 to about 29: 1, about 14: 1 to about 28: 1, about 15: 1 to about 27: 1, about 16: 1 to about 26: 1, about 17: 1 to about 25: 1, about 18: 1 to about 24: 1, about 19: 1 to about 23: 1, about 20: 1 to about 22: 1, or about 21: 1.

[0180] In various embodiments, it is understood that shorter nucleic acid therapeutic agents (e.g., siRNA) or prophylactic agents require more (i.e., greater amount / concentration / volume) of ionizable lipid to encapsulate them into a lipid nanoparticle. Thus, in various embodiments, an N / P ratio of up to about 40: 1 is used to encapsulate and deliver a nucleic acid therapeutic agent (e.g., shorter nucleic acid therapeutic agent siRNA) or prophylactic siRNA.

[0181] In various embodiments, the efficiency / capacity of encapsulation / loading / binding of the therapeutic agent, prophylactic agent, and / or biological agent in the composition / nanoparticle is at least about 10.0%, at least about 20.0%, at least about 30.0%, at least about 40.0%, at least about 50.0%, at least about 60.0%, at least about 70.0%, at least about 80.0%, at least about 90.0%, at least about 95.0%, at least about 96.0%, at least about 97.0%, at least about 98.0%, at least about 99.0%, at least about 99.5%, or at least about 99.9%.

[0182] In various embodiments, the encapsulation efficiency of the nanoparticles is at least about 50% of the encapsulation efficiency of the corresponding nanoparticles using ALC-0315 as the ionizable lipid under similar conditions. In another example, the encapsulation efficiency can be at least about 1% to at least about 50% higher than the encapsulation efficiency of the corresponding nanoparticles using ALC-0315 as the ionizable lipid under similar conditions.

[0183] In various embodiments, the cell transfection efficiency (% of cells transfected with the gene) of the composition / nanoparticle is at least about 5.0%, at least about 10.0%, at least about 20.0%, at least about 30.0%, at least about 40.0%, at least about 50.0%, at least about 60.0%, at least about 70.0%, at least about 80.0%, at least about 90.0%, at least about 95.0%, at least about 96.0%, at least about 97.0%, at least about 98.0%, at least about 99.0%, at least about 99.5%, at least about 99.9%, or about 100%. In various embodiments, a cell transfection efficiency of 100% is not required. For example, it should be recognized that, unlike the case for cancer treatment applications, vaccine applications can not require / transfect 100% of the cells to mediate an immune response.

[0184] In various embodiments, the cell transfection efficiency of the nanoparticles is at least about 30% of the cell transfection efficiency of the corresponding nanoparticles using ALC-0315 as the ionizable lipid under similar conditions. In another example, the cell transfection efficiency can be at least about 50% to at least about 1000% higher than the cell transfection efficiency of the corresponding nanoparticles using ALC-0315 as the ionizable lipid under similar conditions.

[0185] In various embodiments, the transfection efficiency of the nanoparticles in certain cell lines is lower than the transfection efficiency of the corresponding nanoparticles using ALC-0315 as the ionizable lipid under similar conditions, but both are still in the same order of magnitude. It should be recognized that this level of gene transfection can still be suitable for gene therapy.

[0186] In various embodiments, the nanoparticles have an average particle size (or diameter) of about 20.0 nm to about 200.0 nm, about 30.0 nm to about 190.0 nm, about 40.0 nm to about 180.0 nm, about 50.0 nm to about 170.0 nm, about 60.0 nm to about 160.0 nm, about 70.0 nm to about 150.0 nm, about 80.0 nm to about 140.0 nm, about 90.0 nm to about 130.0 nm, about 100.0 nm to about 120.0 nm, or about 110.0 nm.

[0187] In various embodiments, the composition comprises nanoparticles having a polydispersity index (PDI) of about 0.01 to about 0.50, about 0.0125 to about 0.45, about 0.015 to about 0.40, about 0.020 to about 0.35, about 0.025 to about 0.30, about 0.030 to about 0.25, about 0.035 to about 0.20, about 0.040 to about 0.15, about 0.045 to about 0.10, about 0.050 to about 0.095, about 0.055 to about 0.090, about 0.060 to about 0.085, about 0.065 to about 0.080, or about 0.070 to about 0.075. Advantageously, in various embodiments, the nanoparticles have a narrow particle size distribution and / or the nanoparticles or nanoparticle composition are relatively / substantially uniform.

[0188] In various embodiments, the nanoparticles have a zeta potential in saline (e.g., phosphate buffered saline (PBS)) or in a physiological environment of about -15.0 mV to about +20.0 mV, about -14.0 mV to about +19.0 mV, about -13.0 mV to about +18.0 mV, about -12.0 mV to about +17.0 mV, about -11.0 mV to about +16.0 mV, about -10.0 mV to about +15.0 mV, about -9.0 mV to about +14.0 mV, about -8.0 mV to about +13.0 mV, about -7.0 mV to about +12.0 mV, about -6.0 mV to about +11.0 mV, about -5.0 mV to about +10.0 mV, about -4.0 mV to about +9.0 mV, about -3.0 mV to about +8.0 mV, about -2.0 mV to about +7.0 mV, about -1.0 mV to about +6.0 mV, about 0 mV to about +5.0 mV, about +1.0 mV to about +4.0 mV, or about +2.0 mV to about +3.0 mV. Advantageously, in various embodiments, the nanoparticles have a surface charge that is substantially neutral, making the nanoparticles suitable / desirable for in vivo applications.

[0189] In various embodiments, the average molecular weight of the ionizable lipid / nanoparticle is from about 100.0 g / mole to about 2000.0 g / mole, from about 200.0 g / mole to about 1900.0 g / mole, from about 300.0 g / mole to about 1800 g / mole, from about 400.0 g / mole to about 1700.0 g / mole, from about 500.0 g / mole to about 1600.0 g / mole, from about 600.0 g / mole to about 1500.0 g / mole, from about 700.0 g / mole to about 1400.0 g / mole, from about 800.0 g / mole to about 1300.0 g / mole, from about 900.0 g / mole to about 1200.0 g / mole, or from about 1000.0 g / mole to about 1100 g / mole.

[0190] In various embodiments, the composition / compound / nanoparticle is biocompatible, i.e., the composition / compound / nanoparticle is compatible with a biological system or a portion of a biological system, and does not substantially or does not significantly cause adverse physiological reactions such as toxic reactions / responses (e.g., cytotoxicity), immune reactions / responses, damage, etc. when used in the human or animal body. In various embodiments, the composition / compound / nanoparticle is substantially free of substances that elicit adverse physiological reactions. Advantageously, the nanoparticle (e.g., lipid nanoparticle) is capable of efficiently binding a therapeutic agent, a prophylactic agent, and / or a biological agent (e.g., RNA) and / or providing a high transfection rate without causing / inducing substantial or any cytotoxicity.

[0191] Methods of making nanoparticles

[0192] Provided is a method of making a nanoparticle as disclosed herein, the method comprising:

[0193] (c-i) preparing an aqueous composition comprising a therapeutic agent and / or a prophylactic agent and / or a biological agent;

[0194] (c-ii) mixing the aqueous composition obtained from (c-i) with a composition disclosed herein to obtain a nanoparticle.

[0195] In various embodiments, step (c-i) comprises mixing the therapeutic agent and / or the prophylactic agent and / or the biological agent in an aqueous buffer. The aqueous buffer can be sodium acetate.

[0196] In various embodiments, mixing step (c-i) is performed at a pH of about 2.5 to about 6.5, about 2.6 to about 6.4, about 2.7 to about 6.3, about 2.8 to about 6.2, about 2.9 to about 6.1, about 3.0 to about 6.0, about 3.1 to about 5.9, about 3.2 to about 5.8, about 3.3 to about 5.7, about 3.4 to about 5.6, about 3.5 to about 5.5, about 3.6 to about 5.4, about 3.7 to about 5.3, about 3.8 to about 5.2, about 3.9 to about 5.1, about 4.0 to about 5.0, about 4.1 to about 4.9, about 4.2 to about 4.8, about 4.3 to about 4.7, about 4.4 to about 4.6, or about 4.5.

[0197] In various embodiments, the compositions disclosed herein comprise an organic phase (e.g., ethanol). In various embodiments, the aqueous composition comprises an aqueous phase. In various embodiments, step (c-ii) comprises mixing the aqueous composition with the composition described herein at a volume ratio of the aqueous phase to the organic phase of about 10: 1 to about 1: 1. For example, the aqueous phase and the organic phase can be mixed at a volume ratio of about 10: 1 to about 1: 1, about 9: 1, about 8: 1, about 7: 1, about 6: 1, about 5: 1, about 4: 1, about 3: 1, or about 2: 1.

[0198] In various embodiments, step (c-ii) of mixing the aqueous composition with the composition described herein comprises injecting (e.g., directly injecting) the composition described herein into the aqueous composition.

[0199] In various embodiments, step (c-ii) of mixing the aqueous composition with the composition described herein comprises micro-mixing, e.g., microfluidic mixing using a microfluidic device. Micro-mixing can be performed by passive mixing using passive microfluidic mixers such as T- or Y-shaped microfluidic mixers, parallel laminar microfluidic mixers, sequential mixers, focused enhanced mixers, or droplet micro-mixers. Micro-mixing can also be performed by active mixing using external forces such as pressure fields, electrokinetics, dielectrophoresis, electro-wetting, magnetohydrodynamics, or ultrasound. Advantageously, since microfluidic mixing involves mixing the two compositions (i.e., the aqueous composition and the composition disclosed herein) in a controlled manner and / or at a specific / fixed / controlled mixing ratio, the interaction between the two compositions (e.g., between the ionizable lipids and the therapeutic agent, prophylactic agent, and / or biological agent) is modulated, resulting in nanoparticles with smaller particle size and / or narrower or uniform particle size distribution (e.g., smaller PDI).

[0200] In various embodiments, the method further comprises removing the organic phase (e.g., ethanol). For example, removing the organic phase can comprise dialyzing the nanoparticles to remove residual organic solvent present. Advantageously, removing the organic phase by dialysis can improve the encapsulation efficiency of the therapeutic agent and / or prophylactic agent and / or biological agent.

[0201] In various embodiments, there is also provided a carrier, nanocarrier, or delivery system / delivery vehicle comprising a composition / compound / nanoparticle as disclosed herein.

[0202] In various embodiments, there is also provided a vaccine composition comprising a composition / compound / nanoparticle as disclosed herein.

[0203] In various embodiments, there is also provided a carrier, nanocarrier, delivery system / delivery vehicle, compound or ionized form thereof, nanoparticle composition, nanoparticle (or lipid nanoparticle) as disclosed herein for use in medical treatment (e.g., for use in treating or preventing one or more disease, disorder, or symptom mentioned herein).

[0204] In various embodiments, there is also provided a carrier, nanocarrier, delivery system / delivery vehicle, compound or ionized form thereof, nanoparticle composition, nanoparticle (or lipid nanoparticle) as disclosed herein for use in treating or preventing a disease, disorder, or symptom; use of a carrier, nanocarrier, delivery system / delivery vehicle, compound or ionized form thereof, nanoparticle composition, nanoparticle (or lipid nanoparticle) in the manufacture of a medicament for treating or preventing a disease, disorder, or symptom; and / or a method of treating or preventing a disease, disorder, or symptom comprising the step of administering (e.g., a therapeutically effective amount of) the carrier, nanocarrier, delivery system / delivery vehicle, compound or ionized form thereof, nanoparticle composition, nanoparticle (or lipid nanoparticle) to a subject (e.g., a vertebrate such as a human or a large veterinary mammal (e.g., a horse, a cow, a deer, a sheep, a llama, a goat, a pig)) in need thereof. The disease, disorder, or symptom can be selected from an infectious disease / contagious disease, a viral infection (i.e., a disease caused by a virus), a bacterial infection (i.e., a disease caused by a bacterium), a fungal infection (i.e., a disease caused by a fungus), a respiratory disease, and the like or a combination thereof. In various embodiments, the disease, disorder, or symptom is mediated by an influenza virus (e.g., an influenza A virus, an influenza B virus, an influenza C virus, and / or an influenza D virus). For example, the disease can be influenza A, influenza B, influenza C, or influenza D such as H1N1, H3N2. In various embodiments, the disease, disorder, or symptom is mediated by a coronavirus (e.g., a severe acute respiratory syndrome coronavirus such as SARS-CoV-2 or SARS-CoV-1). For example, the disease, disorder, or symptom can be a SARS-CoV-2 coronavirus disease.

[0205] In various embodiments, also provided are a carrier, nanocarrier, delivery system / delivery vehicle, compound or ionized form thereof, nanoparticle composition, nanoparticle (or liponanoparticle) as disclosed herein for use in encapsulating and / or delivering a therapeutic, prophylactic, and / or biologic agent to a subject, a cell, a cytosol, a tissue, or an organ (e.g., a cell, a cytosol, a tissue, or an organ of a mammal); use of the carrier, nanocarrier, delivery system / delivery vehicle, compound or ionized form thereof, nanoparticle composition, nanoparticle (or liponanoparticle) in the manufacture of a medicament for encapsulating and / or delivering a therapeutic, prophylactic, and / or biologic agent to a subject, a cell, a cytosol, a tissue, or an organ (e.g., a cell, a cytosol, a tissue, or an organ of a mammal); and / or a method of delivering a therapeutic, prophylactic, and / or biologic agent to a subject, a cell, a cytosol, a tissue, or an organ (e.g., a cell, a cytosol, a tissue, or an organ of a mammal) comprising the step of administering (e.g., a therapeutically effective amount of) the carrier, nanocarrier, delivery system / delivery vehicle, compound or ionized form thereof, nanoparticle composition, nanoparticle (or liponanoparticle) to a subject (e.g., a vertebrate such as a human or a large veterinary mammal (e.g., a horse, a cow, a deer, a sheep, a llama, a goat, a pig)) in need thereof.

[0206] In various embodiments, also provided are a carrier, nanocarrier, delivery system / delivery vehicle, compound or ionized form thereof, nanoparticle composition, nanoparticle (or liponanoparticle) as disclosed herein for use in inducing an immune response in a subject (e.g., a vertebrate such as a human or a large veterinary mammal (e.g., a horse, a cow, a deer, a sheep, a llama, a goat, a pig)); use of the carrier, nanocarrier, delivery system / delivery vehicle, compound or ionized form thereof, nanoparticle composition, nanoparticle (or liponanoparticle) 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 the step of administering (e.g., a therapeutically effective amount of) the carrier, nanocarrier, delivery system / delivery vehicle, compound or ionized form thereof, nanoparticle composition, nanoparticle (or liponanoparticle) to a subject in need thereof. In various embodiments, an immune response in a subject is induced by administering the compound or ionized form thereof, nanoparticle composition, nanoparticle (or liponanoparticle). In various embodiments, by inducing an immune response in a subject, the subject is protected from a variety of diseases, disorders, or conditions, e.g., an infectious disease / contagious disease, a viral infection (i.e., a disease caused by a virus), a bacterial infection (i.e., a disease caused by a bacterium), a fungal infection (i.e., a disease caused by a fungus), a respiratory disease, etc., or a combination thereof, as described above. The carrier, nanocarrier, delivery system / delivery vehicle, compound or ionized form thereof, nanoparticle composition, nanoparticle can be delivered to a subject in the form of a vaccine or as a component of a vaccine.

[0207] In various embodiments, the disease, disorder, or symptom is mediated by an influenza virus (e.g., an influenza A virus, an influenza B virus, an influenza C virus, and / or an influenza D virus). For example, the disease can be influenza A, influenza B, influenza C, or influenza D, such as H1N1, H3N2. In various embodiments, the disease, disorder, or symptom is mediated by a coronavirus (e.g., a severe acute respiratory syndrome coronavirus, such as SARS-CoV-2 or SARS-CoV-1). For example, the disease, disorder, or symptom can be a SARS-CoV-2 coronavirus disease.

[0208] In various embodiments, the carriers, nanocarriers, delivery systems / delivery vehicles, compounds or ionized forms thereof, nanoparticle compositions, nanoparticles prepared by embodiments of the methods disclosed herein include one or more than one of the following features or properties: broad applicability (e.g., can be used to encapsulate, deliver, and / or transfect a broad range of therapeutic, prophylactic, and / or biological agents), nanosize, substantially neutral surface charge, high encapsulation efficiency (e.g., >80%), high transfection efficiency (e.g., >80%), high stability, low toxicity (e.g., low cytotoxicity), low production / synthesis cost, thus making them suitable for in vivo applications that require efficient cellular uptake and / or gene transfection.

[0209] In various embodiments, the compounds contain only primary amine groups. Thus, in various embodiments, the compounds are substantially free of ionizable secondary (2°) and / or tertiary (3°) amine groups. Advantageously, in various embodiments, the primary amines agglomerate the molecules / goods (e.g., mRNA) into lipid nanoparticles (LNPs) via electrostatic interactions as binding groups. In various embodiments, the molecules / goods (e.g., mRNA) are released from endosomes to the cytosol via fusion of the lipids with the membrane of the endosome. This technical effect of using only primary amines is not shown or contemplated in the prior art.

[0210] In various embodiments, the ionizable lipid compounds of the present technology are different from the ionizable lipid compounds in the art that contain tertiary and / or secondary amine groups as ionizable lipids. In various embodiments, the ionizable lipid compounds of the present application are substantially free of tertiary and / or secondary amine groups. Since the presently disclosed embodiments of ionizable lipid compounds (e.g., DTD-NH2 and HO-DTD-NH2) do not contain any tertiary and / or secondary amine groups, one skilled in the art would not readily consider them as ionizable lipids for gene delivery. It should be appreciated that this is the first report of using lipids that do not contain tertiary or secondary amine groups as ionizable lipids.

[0211] In various embodiments, the ionizable lipid compounds of the present technology are different from the ionizable lipid compounds containing guanidine groups in the art (i.e., ionizable lipid compounds that do not contain primary amine groups). In addition, the inventors have found through experiments that guanidine-functionalized polycarbonates cannot transfect mRNA in cells.

[0212] In various embodiments, the ionizable lipid compounds disclosed herein are different from known ionizable lipids for siRNA delivery, specifically, known ionizable lipids containing tertiary amine groups or guanidine groups. In contrast, embodiments of the ionizable lipids disclosed herein include DTD-NH2 and / or ionizable lipids containing primary amine groups as the only ionizable groups for preparing lipid nanoparticles for nucleic acid delivery, which were previously unknown.

[0213] In various embodiments disclosed herein, DTD-NH2 and other lipids containing primary amine groups as the only ionizable groups (e.g., DD-NH2, HO-DnO-NH2, HO-DD-NH2, HO-DTD-NH2, DnO-EDEA, DD-EDEA, and DTD-EDEA) were directly used to prepare lipid nanoparticles for delivery of mRNA and DNA with promising results. This finding was unexpected because, based on common knowledge in the field of nucleic acid delivery, the precursors were not expected to work well.

[0214] It should be understood that the various embodiments of ionizable lipids disclosed herein are distinct from those known in the art involving ligands coupled to oligonucleotides for targeted delivery of oligonucleotides. BRIEF DESCRIPTION OF THE DRAWINGS

[0215] Figure 1 The DTD-NH2 prepared according to various embodiments of the methods disclosed herein is shown in FIG. 1 HNMR spectrum.

[0216] Figure 2 The results show that DMAPAPA-DTD prepared according to various embodiments of the methods disclosed herein is in CD3OD. 1 H NMR spectrum.

[0217] Figure 3Luciferase expression mediated by different mRNA LNP formulations according to various embodiments disclosed herein in HeLa cell line is shown. Cells in each well of a 96-well plate were incubated with 100 ng of each mRNA LNP formulation for 48 h. Luciferase expression was quantified by adding D-luciferin to cell lysate and measuring luminescence intensity. In HeLa cells, batch 1 ALC-0315 LNP was used. Statistical significance between ALC-0315 formulation and other LNP formulations was calculated using the Mann-Whitney test (*p<0.05).

[0218] Figure 4 Luciferase expression mediated by different mRNA LNP formulations according to various embodiments disclosed herein in HEK293 cell line is shown. Cells in each well of a 96-well plate were incubated with 100 ng of each mRNA LNP formulation for 48 h. Luciferase expression was quantified by adding D-luciferin to cell lysate and measuring luminescence intensity. In HEK293 cells, batch 2 ALC-0315 LNP was used. Statistical significance between ALC-0315 formulation and other LNP formulations was calculated using the Mann-Whitney test (*p<0.05).

[0219] Figure 5 Cell viability after incubation with various mRNA LNP formulations according to various embodiments disclosed herein for 48 h is shown. Statistical significance between ALC-0315 formulation and other LNP formulations was calculated using the Mann-Whitney test (*p<0.05, **p<0.01). In HeLa cells, batch 1 ALC-0315 LNP was used.

[0220] Figure 6 Cell viability after incubation with various mRNA LNP formulations according to various embodiments disclosed herein for 48 h is shown. Statistical significance between ALC-0315 formulation and other LNP formulations was calculated using the Mann-Whitney test (*p<0.05, **p<0.01). In HEK293 cells, batch 2 ALC-0315 LNP was used.

[0221] Figure 7 Cell viability of HeLa cells upon incubation with mRNA LNP according to various embodiments disclosed herein is shown. Statistical significance between ALC-0315 formulation and other LNP formulations was calculated using the Mann-Whitney test (*p<0.05, **p<0.01).

[0222] Figure 8Luciferase expression of HeLa cells upon incubation with mRNA LNPs according to various embodiments disclosed herein is shown. Statistical significance between ALC-0315 formulations and other LNP formulations was calculated using the Mann-Whitney test (*p<0.05, **p<0.01).

[0223] Figure 9 Cell viability of HEK293 cells upon incubation with pDNA LNPs according to various embodiments disclosed herein is shown. Statistical significance between ALC-0315 formulations and other LNP formulations was calculated using the Mann-Whitney test (**p<0.01).

[0224] Figure 10 Luciferase expression of HEK293 cells upon incubation with pDNA LNPs according to various embodiments disclosed herein is shown. Statistical significance between ALC-0315 formulations and other LNP formulations was calculated using the Mann-Whitney test (**p<0.01).

[0225] Figure 11 is a graph showing the particle size and size distribution of pDNA-loaded LNPs prepared from ALC-0315 (comparative example). The measurements were recorded on day 0.

[0226] Figure 12 is a graph showing the particle size and size distribution of pDNA-loaded LNPs prepared from DTD-NH2 according to various embodiments disclosed herein. The measurements were recorded on day 0.

[0227] Figure 13 is a graph showing the particle size and size distribution of pDNA-loaded LNPs prepared from ALC-0315 (comparative example). The measurements were recorded after 6 days of storage.

[0228] Figure 14 is a graph showing the particle size and size distribution of pDNA-loaded LNPs prepared from DTD-NH2 according to various embodiments disclosed herein. The measurements were recorded after 6 days of storage.

[0229] Figure 15 Results obtained from agarose gel electrophoresis experiments performed with pDNA LNPs prepared from DTD-NH2 according to various embodiments disclosed herein are shown. ALC0315 was used as a comparative example. Both ALC-0315 LNPs and DTD-NH2 LNPs strongly bound to pDNA. Triton was able to disassemble the LNPs to release the pDNA to measure the pDNA encapsulation efficiency.

[0230] Figure 16is a plot showing the relationship of fluorescence intensity (RFU) to GFP fluorescence for transfection efficiency of pDNA LNPs prepared from DTD-NH2 according to various embodiments disclosed herein after incubation for 48 hours in HeLa cells and HepG2 cells. ALC0315 was used as a comparative example.

[0231] Figure 17 shows confocal microscopy images of controls captured after incubation for 48 hours in HeLa cells. Scale bar = 50 pm.

[0232] Figure 18 shows confocal microscopy images of pDNA LNPs prepared from ALC0315 (comparative example) captured after incubation for 48 hours in HeLa cells. Scale bar = 50 pm.

[0233] Figure 19 shows confocal microscopy images of pDNA LNPs prepared from DTD-NH2 according to various embodiments disclosed herein captured after incubation for 48 hours in HeLa cells. Scale bar = 50 pm.

[0234] Figure 20 shows confocal microscopy images of controls captured after incubation for 48 hours in HepG2 cells. Scale bar = 50 pm.

[0235] Figure 21 shows confocal microscopy images of pDNA LNPs prepared from ALC0315 (comparative example) captured after incubation for 48 hours in HepG2 cells. Scale bar = 50 pm.

[0236] Figure 22 shows confocal microscopy images of pDNA LNPs prepared from DTD-NH2 according to various embodiments disclosed herein captured after incubation for 48 hours in HepG2 cells. Scale bar = 50 pm.

[0237] Example

[0238] Example embodiments of the present disclosure will be better understood and become more apparent with the following examples, tables, and where applicable, in conjunction with the accompanying drawings. It is to be understood that other modifications relating to the structures and / or chemical changes can be made without departing from the scope of the present disclosure. The example embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more embodiments to form new example embodiments. The example embodiments should not be interpreted as limiting the scope of the present disclosure.

[0239] The following examples describe the development process of lipids containing primary amine groups, their compositions, the formation of RNA and DNA lipid nanoparticles (LNPs), and gene transfection. Advantageously, the present application has shown that, unlike ionizable lipids known in the art (e.g., those used in the mRNA vaccine formulations of Moderna and Pfizer-BioNTech), the primary amine groups of embodiments of the lipids disclosed herein ionize at physiological environments or neutral pH and condense mRNA into lipid nanoparticles with a >90% encapsulation efficiency. Even more advantageously, the transfection efficiency of mRNA and pDNA of LNPs designed according to various embodiments disclosed herein is significantly higher than mRNA LNP or pDNA LNP prepared from conventional ionizable lipid ALC-0315 currently used in the mRNA vaccine formulation of Pfizer-BioNTech. Furthermore, advantageously, treatment with mRNA LNP and pDNA LNP designed according to various embodiments disclosed herein does not induce any cytotoxicity. Moreover, the steps required to synthesize ionizable lipids according to various embodiments disclosed herein are 5 to 6 steps less than the preparation of ionizable lipid ALC-0315 in the art, which will significantly reduce the manufacturing cost (e.g., 3 steps).

[0240] Example 1: Materials and Methods

[0241] 1.1. Materials

[0242] Unless otherwise specified, chemical reagents used for the synthesis of lipids were purchased from Sigma-Aldrich and used as received. Ditetradecylamine (DTDA) was purchased from Ambeed, Inc (Arlington Heights, IL, USA). 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and ALC-0315 were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Sodium acetate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Agarose and Tris-acetate-EDTA were purchased from 1stBase (Singapore). GelStar nucleic acid gel dye was purchased from Lonza (Basel, Switzerland). and Tris-EDTA were purchased from Promega (Madison, WI, USA). Alamar Blue, gel loading buffer, and Pierce Firefly Luciferase Glow Assay Kit were purchased from Invitrogen (Waltham, MA, USA). Other reagents used were of analytical grade.

[0243] 1.2.Synthesis of DTD-NH2 and DMAPAPA-DTD (Scheme 1)

[0244] Scheme 1 provides an exemplary general scheme for the synthesis of compounds according to various embodiments disclosed herein, as shown in general formula (1), or an ionized form thereof.

[0245] The synthesis of DTD-NH2 is given below as a typical example, and the analogous synthesis of DMAPAP-DTA is provided below as a supplementary example.

[0246]

[0247] Synthesis of DTD-COONa: In a 500 mL three-necked round bottom flask, ditetradecylamine (DTDA, 3.44 g, 8 mmol), succinic anhydride (2.4 g, 24 mmol), and triethylamine (5.6 mL, 40 mmol) were dissolved in 300 mL of dry DCM and the reaction solution was stirred under N2atmosphere overnight. The solution was then transferred to a 1 L separation funnel and washed with a saturated solution of sodium bicarbonate (100 mL) three times. The organic phase was dried with MgS04for 4-5 h. Finally, the solution was filtered by suction, the filtrate was concentrated to dryness, and dried under vacuum to yield DTD-COONa as an off-white solid (93% yield).

[0248] 1 H NMR (400 MHz, CDC13, 22 °C): δ 3.31 (dt, 4H, -CON(CH2-)2), 2.67 (s, 4H, -CH2CH2COONa), 1.53 (m, br, 4H, -CON(CH2CH2-)2), 1.26 (s, 44H, -(CH2) 11 CH3), 0.88 (t, 6H, -CH3).

[0249] Synthesis of DTD-NHS: In a 100 mL three-necked round bottom flask, DTD-COONa (4.85 g, 9.13 mmol) and N-hydroxysuccinimide (NHS, 2.88 g, 25 mmol) were dissolved in 60 mL of dry DCM, followed by the addition of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC-HCl, 3.83 g, 20 mmol). The solution was stirred under N2atmosphere overnight. Subsequently, it was concentrated to dryness, the residue was re-dissolved in a mixture of THF and deionized (DI) water (volume / volume = 1 : 1). An additional 300 mL of DI water was added in an ice bath to precipitate the product, which was centrifuged and washed with cold DI water three times. Finally, the wet solid was freeze-dried to yield DTD-NHS as a white powder (83% yield).

[0250] 1H NMR (400 MHz, CDC13, 22 °C): δ 3.30 (dt, 4H, -CON(CH2-)2), 3.01 (t, 2H, -CH2-of NHS), 2.83 (s, 4H, -COCH2CH2CON-), 2.72 (t, 2H, -CH2-of NHS), 1.52 (m, br, 4H, -CON(CH2CH2-)2), 1.26 (s, 44H, -(CH2) 11 CH3), 0.88 (t, 6H, -CH3).

[0251] Synthesis of DTD-NH2: In a 100 mL single necked round bottom flask, ethylenediamine (EDA, 53 μL, g, 0.79 mmol) was dissolved in 15 mL of dry DCM and the solution was cooled in a dry ice bath for 30 minutes under N2atmosphere. DTD-NHS (0.455 g, 0.75 mmol) was dissolved in 15 mL of DCM and the solution was added dropwise to the EDA solution. The dry ice bath was subsequently removed and the reaction solution was stirred for an additional 2 hours. The mixture was transferred to a 250 mL separation funnel and 70 mL of DCM was added. The solution was washed with brine (20 mL) three times and dried over MgS04overnight. Finally, the mixture was filtered by suction filtration, the filtrate was concentrated to dryness and dried under vacuum to yield DTD-NH2as an off-white sticky solid (63% yield).

[0252] 1 H NMR (400 MHz, CDC13, 22 °C): δ 3.30 (dt, 4H, -CON(CH2-)2), 3.01 (t, 2H, -CH2-of NHS), 2.83 (s, 4H, -COCH2CH2CON-), 2.72 (t, 2H, -CH2-of NHS), 1.52 (m, br, 4H, -CON(CH2CH2-)2), 1.26 (s, 44H, -(CH2) 11 CH3), 0.88 (t, 6H, -CH3).

[0253] Similarly, the following ionizable lipids were synthesized:

[0254] DnO-NH2yield: 66%; 1H NMR (400 MHz, CD3OD, 22 °C): δ 2.75 (t, 2H, -CH2NH2), 2.68 (t, 2H, -NHCOCH2CH2CON-), 2.48 (t, 2H, -NHCOCH2CH2CON-), 1.62 (m, br, 2H, -CON(CH2CH2-)2), 1.52 (m, br, 2H, -CON(CH2CH2-)2), 1.30 (m, 20H, -(CH2)5CH3), 0.90 (t, 6H, -CH3).

[0255] HO-DnO-NH2 yield: 73%; 1 H NMR (400 MHz, CD3OD, 22 °C): δ 2.75 (t, 2H, -CH2NH2), 2.68 (t, 2H, -NHCOCH2CH2CON-), 2.48 (t, 2H, -NHCOCH2CH2CON-), 1.62 (m, br, 2H, -CON(CH2CH2-)2), 1.52 (m, br, 2H, -CON(CH2CH2-)2), 1.29 (m, 28H, -(CH2)7CH3), 0.90 (m, 6H, -CH3).

[0256] HO-DD-NH2 yield: 64%; 1 H NMR (400 MHz, CD3OD, 22 °C): δ 2.75 (t, 2H, -CH2NH2), 2.68 (t, 2H, -NHCOCH2CH2CON-), 2.48 (t, 2H, -NHCOCH2CH2CON-), 1.62 (m, br, 2H, -CON(CH2CH2-)2), 1.52 (m, br, 2H, -CON(CH2CH2-)2), 1.30 (m, 20H, -(CH2)5CH3), 0.90 (t, 6H, -CH3).

[0257] HO-DD-NH2 yield: 64%; 1 H NMR (400 MHz, CD3OD, 22 °C): δ 2.75 (t, 2H, -CH2NH2), 2.68 (t, 2H, -NHCOCH2CH2CON-), 2.48 (t, 2H, -NHCOCH2CH2CON-), 1.62 (m, br, 2H, -CON(CH2CH2-)2), 1.52 (m, br, 2H, -CON(CH2CH2-)2), 1.29 (m, 28H, -(CH2)7CH3), 0.90 (m, 6H, -CH3).

[0258] HO-DTD-NH2 yield: 86%; 1H NMR (400 MHz, CD3OD, 22 °C): δ 3.73 (m, 1H, -OH), 3.23 (t, 2H, -CH2NH2), 2.68 (t, 2H, -NHCOCH2CH2CON-), 2.50 (t, 2H, -NHCOCH2CH2CON-), 1.62 (m, br, 2H, -CON(CH2CH2-)2), 1.52 (m, br, 2H, -CON(CH2CH2-)2), 1.29 (m, 44H, -(CH2) 11 CH3), 0.90 (t, 6H, -CH3).

[0259] DnO-EDEA yield: 76%; 1 H NMR (400 MHz, CD3OD, 22 °C): δ 3.73 (m, 1H, -OH), 3.23 (t, 2H, -CH2NH2), 2.68 (t, 2H, -NHCOCH2CH2CON-), 2.50 (t, 2H, -NHCOCH2CH2CON-), 1.62 (m, br, 2H, -CON(CH2CH2-)2), 1.52 (m, br, 2H, -CON(CH2CH2-)2), 1.29 (m, 44H, -(CH2)

[0260] DD-EDEA yield: 72%; 1 H NMR (400 MHz, CD3OD, 22 °C): δ 3.73 (m, 1H, -OH), 3.23 (t, 2H, -CH2NH2), 2.68 (t, 2H, -NHCOCH2CH2CON-), 2.50 (t, 2H, -NHCOCH2CH2CON-), 1.62 (m, br, 2H, -CON(CH2CH2-)2), 1.52 (m, br, 2H, -CON(CH2CH2-)2), 1.29 (m, 44H, -(CH2)

[0261] DTD-EDEA yield: 59%; 1H NMR (400 MHz, CD3OD, 22 °C): δ 3.64 (m, 4H, -CH20CH2CH20CH2-), 3.58 (m, 4H, -CH20CH2CH20CH2-), 3.36 (t, 2H, -CH2NHCO-), 2.91 (t, 2H, -CH2NH2), 2.65 (t, 2H, -NHCOCH2CH2CON-), 2.49 (t, 2H, -NHCOCH2CH2CON-), 1.62 (m, br, 2H, -CON(CH2CH2-)2), 1.52 (m, br, 2H, -CON(CH2CH2-)2), 1.29 (m, 44H, -(CH2) 11 CH3), 0.90 (t, 6H, -CH3).

[0262] DMAPAPA-DTD Yield: 51 %; 1 H NMR (400 MHz, CD3OD, 22 °C): δ 2.72 (t, 4H, -CH2NHCH2-of DMAPAPA), 2.67 (t, 2H, -NHCOCH2CH2CON-), 2.47 (t, 2H, -NHCOCH2CH2CON-), 2.42 (t, 2H, -CH2N(CH3)2), 2.27 (s, 6H, -N(CH3)2), 1.74 (m, 2H, -CH2CH2N(CH3)2), 1.57 (m, br, 4H, -CON(CH2CH2-)2), 1.29 (s, 44H, -(CH2) 11 CH3), 0.90 (t, 6H, -CH3).

[0263] 1.3. Nuclear magnetic resonance spectroscopy (NMR)

[0264] The H-NMR spectra of the lipids were recorded on a Bruker Advance 400 NMR spectrometer (400 MHz) at ambient temperature, acquisition time 3.2 s, pulse repetition time 2.0 s, pulse width 30°, spectral width 5208-Hz and 32K data points. 1 The reference benchmark for the chemical shifts was the respective solvent peak (δ = 3.31 ppm for CD3OD).

[0265] 1.4. Preparation of mRNA-loaded lipid nanoparticles (mRNA LNP)

[0266] By using a microfluidic device (Labcyte Echo® 550) Ignite TMmRNA LNPs were prepared using the Lipid Nanoparticle Preparation System (Precision Nanosystem, Vancouver, CA, USA). Lipids in a weight ratio of 28:6:13:3 (ionizable lipid, DSPC, cholesterol, and ALC-0159) were dissolved in ethanol to form the organic phase. Firefly luciferase mRNA (Trilink Biotechnologies) was dissolved in 10 mM sodium acetate solution (pH = 4) to form the aqueous phase in a volume ratio of 3:1 (aqueous phase: organic phase). The aqueous phase was mixed with the organic phase at a flow ratio of 3:1 (1.5 mL:0.5 mL) to form mRNA LNPs at a total flow rate of 12 mL / min. The resulting mRNA LNPs were immediately diluted 20-fold with 0.9% physiological saline. Subsequently, the mRNA LNP suspension was concentrated using an ultracentrifuge tube with a 30,000 Da molecular weight cutoff (Sartorius, Goettingen, Germany) at 4°C, 2500 rcf, and for 30 minutes. The mRNA LNP suspension was concentrated using an ultracentrifuge tube with a 30,000 Da molecular weight cutoff (Sartorius, Goettingen, Germany). The concentrated mRNA LNPs were collected and stored at 4°C for future use.

[0267] 1.5. In vitro high-throughput screening

[0268] For high-throughput screening of candidate ionizable lipids, mRNA LNPs and pDNA LNPs were prepared by direct injection of the ethanol organic phase into the nucleic acid-containing aqueous phase. The organic phase was composed of a lipid library mixed with DSPC, cholesterol, and ALC-0159 in a weight ratio of 28:6:13:3. The aqueous phase contained firefly luciferase mRNA (Trilink Biotechnologies) or pGL4.51 luc2 / CMV / Neo vector pDNA (Promega) dissolved in 10 mM sodium acetate buffer at pH 4. The amount of mRNA and pDNA was adjusted according to each candidate lipid to achieve an N / P ratio of 6 for all formulations. The organic and aqueous phases were gently mixed and incubated at room temperature for 30 minutes to form LNPs before use for downstream analysis.

[0269] 1.6. Encapsulation efficiency

[0270] Quant-it TM RiboGreen RNA Assay Kit (Invitrogen, Waltham, MA, USA) and Quant-it TMPicoGreen DNA assay kit (Invitrogen, Waltham, MA, USA) was used to determine the encapsulation efficiency of mRNA and pDNA of LNP. The mRNA LNP and pDNA LNP suspensions were diluted 5-fold with Tri-EDTA buffer. The RiboGreen and PicoGreen stock solutions were diluted 200-fold with Tri-EDTA buffer or 5% Triton-X100 in Tri-EDTA buffer solution. 90 μL of RiboGreen and PicoGreen solutions with or without Triton-X100 were added to 10 μL of diluted mRNA LNP and pDNA LNP suspensions, respectively, and incubated at 37 °C for 20 min. The fluorescence intensity was then recorded using a microplate reader (Tecan, Switzerland) at an excitation wavelength of 485 nm and an emission wavelength of 520 nm for RiboGreen and at an excitation wavelength of 480 nm and an emission wavelength of 520 nm for PicoGreen. The encapsulation efficiency of mRNA and pDNA was calculated according to the following equation:

[0271]

[0272] where C tx100 is the concentration of mRNA or pDNA measured using 5% Triton-X100 in Tri-EDTA buffer solution, C TE is the concentration of mRNA or pDNA measured using Tri-EDTA buffer.

[0273] 1.7. Dynamic light scattering (DLS)

[0274] The particle size of mRNA LNP and pDNA LNP was measured by DLS using Zetasizer (Malvern, UK). 50 μL of mRNA LNP and pDNA LNP suspensions were diluted to 1 mL with saline. The particle size of mRNA LNP and pDNA LNP was measured at 25 °C, 10 s per run, 11 measurement runs. The average particle size of each sample was obtained after 3 measurements.

[0275] 1.8. Surface zeta potential

[0276] The surface zeta potential of mRNA LNP and pDNA LNP was measured by using Zetasizer (Malvern, UK). The samples were diluted with saline. The zeta potential was measured at 25 °C. The average zeta potential was obtained after 3 measurements.

[0277] 1.9.​Analysis of mRNA binding by gel electrophoresis assay

[0278] 10 μL of mRNA LNP suspension containing 300 ng of mRNA was mixed with 2 μL of gel loading dye. The mixture was loaded onto a 1% agarose gel containing 0.02% GelStar nucleic acid gel dye. The gel was then run at 100 mV for 20 minutes in Tris-acetate-EDTA buffer. The gel was then recorded using a gel imaging system (iBright 1500, Invitrogen, Waltham, MA, USA).

[0279] 1.10. Cell culture and treatment with mRNA LNP and pDNA LNP

[0280] HELA cells and HEK293 cells were cultured in DMEM supplemented with 10% FBS (volume / volume) and 1% penicillin / streptomycin (volume / volume). All cells were maintained at 37°C and in a 5% CO2 incubator (Thermo Fisher, Waltham, MA, USA). HELA cells, HEPG2 cells, and HEK293 cells were seeded in black / white 96-well plates at a density of 10,000 cells per well. After the cells adhered overnight, the cell culture medium in the plate was replaced with 100 μL fresh culture medium containing mRNA LNPs and pDNA LNPs, wherein the final concentration of mRNA and pDNA was 100 ng per well, and the cells were incubated for 48 hours.

[0281] 1.11. In vitro cytotoxicity of mRNA LNP and pDNA LNP

[0282] After 48 hours of incubation, the culture medium was removed. 100 μL of fresh culture medium containing 10% Alamar Blue reagent was added to each well and incubated for another 2 hours. Fluorescence intensity was recorded at an excitation wavelength of 560 nm and an emission wavelength of 590 nm (Supplementary Figure 2). Cell viability was calculated based on the negative control group without treatment.

[0283] 1.12. In vitro transfection efficiency of mRNA and pDNA

[0284] After 48 hours of incubation, the culture medium was removed. 50 μL of lysis buffer and 50 μL of D-luciferin firefly luminescence detection buffer (60 μg / mL) were added to each well and incubated for 10 minutes for cell lysis and signal stabilization. The cells were analyzed using a microplate reader (Tecan, Luminescence intensity was read in a 1000 ms timer (1000 ms). Transfection efficiency was expressed as luminescence units / well.

[0285] Example 2: Preparation and characterization of primary amine-containing ionizable lipids

[0286] The preparation of lipids started with the synthesis of DTD-COONa, which was prepared by the coupling of succinic anhydride with ditetradecylamine (DTDA). DTD-COONa was then reacted with N-hydroxysuccinimide (NHS) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC-HCl) as a coupling agent to give DTD-NHS. Finally, ionizable lipids were prepared by the substitution of DTD-NHS with ethylenediamine (EDA) or N,N-dimethyldipropyltriamine (DMAPAPA) (Scheme 1). To ensure the molar ratio of amine to DTD in the lipids was 1:1, a slight excess of amine (the molar ratio of amine to DTD-NHS feed was 1.05:1) in DCM solution was cooled in a dry ice bath, followed by the dropwise addition of DTD-NHS solution. The excess amine and NHS generated in the substitution reaction were removed by washing the reaction solution with brine.

[0287] The molar ratio of amine to DTD in the DTD-NH2 lipid was determined by the integration of the resonance of the methylene protons adjacent to the amine group at 2.69 ppm relative to the hydrogen of the methyl groups at the end of the DTD chain at 0.92 ppm ( Figure 1 ). 1 The results of H NMR indicated that the molar ratio of amine to DTD in the DTD-NH2 lipid was 1:1. Similarly, the proton NMR spectrum determined the composition of the DMAPAPA-DTD lipid ( Figure 2 ), with a molar ratio of amine to DTD of 1:1.

[0288] Example 3: Preparation and characterization of mRNA-loaded lipid nanoparticles (mRNA LNP) using microfluidic mixing

[0289] mRNA LNPs were prepared using a microfluidic device, with the preparation conditions shown in Tables 1-4. The main component was an ionizable lipid. As shown in Table 5, the particle sizes of mRNA LNPs prepared using the candidate lipids were all <100 nm, except for DMAPAPA-DTD (139 nm). The polydispersities of mRNA LNPs of most candidate lipids were <0.2, except for DMAPAPA-DTD (PDI = 0.347) and DD-EDEA (PDI = 0.21). All mRNA LNP formulations had a zeta potential close to neutral. Overall, these characteristics are desirable for in vivo applications.

[0290] Table 1. Preparation conditions of ALC-0315 mRNA-LNP.

[0291]

[0292] Table 2. Preparation conditions of DTD-NH2 mRNA-LNP.

[0293]

[0294] Table 3. Preparation conditions of DMAPAPA-DTD mRNA-LNP.

[0295]

[0296] Table 4. Preparation conditions of Lipid-EDEA mRNA-LNP.

[0297]

[0298] Table 5. Particle size, size distribution and zeta potential of mRNA LNP.

[0299]

[0300] Efficient association of mRNA by ionizable lipids is an important first step in forming stable and compact mRNA LNP, which is critical for efficient cellular uptake and transfection. The mRNA association efficiency of various mRNA LNP formulations was evaluated using RiboGreen RNA assay. The encapsulation efficiency of mRNA LNP was calculated by the percentage of encapsulated mRNA over the total mRNA content (encapsulated and unencapsulated). As shown in Table 6, the mRNA LNP encapsulation efficiency prepared by candidate lipids were all >80% (except DnO-EDEA mRNA LNP), which was comparable to ALC-0315 formulation. This indicates that ionizable lipids with primary amine are able to condense mRNA into LNP formulation without affecting its particle size.

[0301] Table 6. Encapsulation efficiency of mRNA LNP.

[0302]

[0303] As Figure 3 and Figure 4The gene transfection rates of mRNA LNP prepared from DTD-NH2 were about 7-fold and about 4-fold of the gene transfection rates of mRNA LNP prepared from ALC-0315, respectively, as shown. The transfection rates induced by mRNA DMAPAPA-DTD LNP were hundreds to thousands of times lower than those of mRNA DTD-NH2 LNP. In addition, DTD-EDEA mRNA LNP exhibited comparable transfection rates to ALC0315 mRNA LNP in HeLa cells. Among mRNA LNP prepared with EDEA lipids, DTD-EDEA exhibited the best transfection rates in both cell lines, suggesting that a longer lipid tail can be beneficial for cell transfection of this lipid series. No significant cytotoxicity was observed for all mRNA LNP formulations in HeLa cells and HEK293 cells after 48 hours of incubation under the same transfection conditions Figure 5 and Figure 6 ).

[0304] Example 4: High-throughput screening of additional ionizable lipids for mRNA LNP

[0305] To high-throughput screen for subsequent ionizable lipids, mRNA LNP was prepared by directly injecting the lipid-containing organic phase into the mRNA-containing aqueous phase. Different ionizable lipids were added to the organic phase (Tables 7 and 8), and the amount of mRNA added was adjusted to achieve an N / P ratio of 6 for all formulations. This method provided higher efficiency and was more cost-effective when screening for potential candidate lipids for lipid nanoparticles.

[0306] ALC-0315 mRNA LNP prepared using the direct injection method (Table 9) had larger particle size and PDI than ALC-0315 mRNA LNP prepared using the microfluidic device (Table 5). This can be due to the difference in mixing conditions between direct injection and microfluidic mixing. During microfluidic mixing, the organic and aqueous phases are mixed in a controlled manner with a specific mixing ratio of 1:3 (organic phase:aqueous phase). This ensures a constant amount of lipids and mRNA interact, allowing efficient coacervation of mRNA with the charged ionizable lipids when forming LNP. Since the direct injection method does not control these factors, the interaction between ionizable lipids and mRNA occurs more spontaneously and can not be maximized, resulting in slightly larger particle size and PDI.

[0307] As can be seen from Table 9, the particle size of mRNA-LNPs prepared with the candidate lipids was about 120 nm to about 170 nm, with PDI < 0.2 for all. The zeta potential of all formulations was mostly close to neutral (± 10 mV). The encapsulation efficiency of all candidate lipids was significantly higher than that of ALC-0315 mRNA LNP (Table 10). The low encapsulation efficiency of ALC-0315 mRNA LNP can be related to the method of formulation. Nevertheless, ALC-0315 mRNA LNP still exhibited mRNA transfection in HeLa cells ( Figure 7 and Figure 8 ). This can be due to the mRNA loosely bound on the surface of LNP, rather than encapsulated in the internal environment of LNP, so it can still transfect cells. Among all the formulations tested, DTD-NH2 mRNA LNP and HO-DTD-NH2 mRNA LNP outperformed ALC-0315 mRNA LNP in luciferase mRNA transfection in HeLa cells ( Figure 7 and Figure 8 ). The cell viability of HeLa cells incubated with DTD-NH2 and HO-DTD-NH2 formulations also showed little or no cytotoxicity ( Figure 7 and Figure 8 ). The particle properties and cell transfection properties of DTD-NH2 mRNA LNP and HO-DTD-NH2 mRNA LNP can be further improved if microfluidic mixing is used for formulation.

[0308] Table 7. Preparation conditions of mRNA LNP for high-throughput screening.

[0309]

[0310] Table 8. Molecular weight of candidate lipids.

[0311]

[0312] Table 9. Particle size, particle size distribution, and zeta potential of mRNA LNP for high-throughput screening.

[0313]

[0314] Table 10. Encapsulation efficiency of mRNA LNP prepared using direct injection method.

[0315]

[0316] Example 5: Ionizable lipids for high-throughput screening of pDNA LNP

[0317] The high-throughput screening of ionizable lipids for pDNA LNPs was similar to that performed for mRNA LNPs. The ionizable lipids described above were added to the organic phase (Table 11), and the amount of pDNA added was adjusted to achieve an N / P ratio of 6 for all formulations.

[0318] As can be seen from Table 12, mRNA-LNPs prepared with the candidate lipids exhibited a particle size of 80 nm to 160 nm with a PDI < 0.2. The zeta potential of all formulations was mostly close to neutral (± 10 mV) except for that of HO-DnO-NH2 pDNA LNP, which was -12.87 mV. The encapsulation efficiency of all candidate lipids was higher than that of ALC-0315 pDNA LNP (Table 13). The differences in particle size, zeta potential, and encapsulation efficiency between pDNA LNPs and mRNA LNPs can be related to the different sizes of the nucleic acid cargo and their respective binding efficiencies to the ionizable lipids. Similar to the high-throughput screening of mRNA LNPs, DTD-NH2 pDNA LNP and HO-DTD-NH2 pDNA LNP were significantly superior to ALC-0315 pDNA LNP in pDNA transfection in HEK293 cells Figure 9 and Figure 10 ). The viability of HEK293 cells incubated with DTD-NH2 and HO-DTD-NH2 formulations also showed little or no cytotoxicity Figure 9 and Figure 10 ). Again, the particle properties and cell transfection properties of DTD-NH2 pDNA LNP and HO-DTD-NH2 pDNA LNP can be further improved if microfluidic mixing is used for formulation.

[0319] Table 11. Preparation conditions of pDNA LNPs for high-throughput screening.

[0320]

[0321] Table 12. Particle size, size distribution, and zeta potential of pDNA LNPs for high-throughput screening.

[0322]

[0323] Table 13. Encapsulation efficiency of pDNA LNPs prepared using direct injection method.

[0324]

[0325] Example 6: Summary

[0326] The ionizable lipid DTD-NH2 was successfully designed and prepared through 3 synthetic steps. The synthesis of the new ionizable lipid HO-DTD-NH2 was also simple. Using these lipids, mRNA and pDNA LNP with encapsulation efficiency >80%, nanosize, and neutral surface charge were formed, which are desirable properties for in vivo applications. The transfection efficiency of mRNA and pDNA LNP prepared from DTD-NH2 or HO-DTD-NH2 in the tested cell lines was significantly higher than that of mRNA and pDNA LNP prepared from the commercial lipid ALC-0315 used in the Pfizer-BioNTech mRNA vaccine formulation, without any cytotoxicity. These LNPs have great potential as nanocarriers to deliver mRNA vaccines or therapeutics and pDNA. They can also be used to deliver other nucleic acid therapeutics.

[0327] Example 7: Structures of Examples of Ionizable Lipids

[0328]

[0329] Chemical structure of DnO-EDEA (M = 471; N% = 3.0%)

[0330]

[0331] Chemical structure of DD-EDEA (M = 527; N% = 2.7%)

[0332]

[0333] Chemical structure of DTD-EDEA (M = 639; N% = 2.2%)

[0334]

[0335] Chemical structure of HO-DnO-NH2_3 (M = 413; N% = 3.4%)

[0336]

[0337] Chemical structure of HO-DD-NH2_3 (M = 469; N% = 3.0%)

[0338]

[0339] Chemical structure of HO-DTD-NH2_5 (M = 581; N% = 2.4%)

[0340]

[0341] Chemical structure of DnO-NH2_2 (M = 383; N% = 3.7%)

[0342]

[0343] Chemical structure of DnO-NH2_2 (M = 383; N% = 3.7%)

[0344]

[0345] Chemical structure of ALC-0315 shown in Table 14

[0346]

[0347] Chemical structure of DTD-NH2 shown in Table 15

[0348]

[0349] Chemical structure of DnO-EDEA shown in Table 16

[0350]

[0351] Chemical structure of DD-EDEA shown in Table 17

[0352]

[0353] Chemical structure of DTD-EDEA shown in Table 18

[0354] 8.1. Characterization of mRNA LNP: particle size, PDI, zeta potential

[0355] The particle size, PDI and zeta potential of mRNA LNPs were determined at day 0 and after 8 days of storage. The characterization results are shown in Table 19. As shown in Table 19, the particle size of mRNA LNPs prepared from DnO-EDEA, DD-EDEA or DTD-EDEA was comparable to that of mRNA LNPs prepared from DTD-NH2 or ALC-0315. mRNA LNPs prepared from DD-EDEA and DTD-EDEA were stable after 8 days of storage, which could be evidenced by comparable particle size and particle size distribution (PDI), while mRNA LNPs prepared from DnO-EDEA were not stable after 8 days of storage, which could be evidenced by the increase of particle size.

[0356]

[0357]

[0358]

[0359]

[0360]

[0361] Table 19. LNP Characterization

[0362]

[0363] 8.2. Encapsulation efficiency of mRNA LNP

[0364] Table 20 shows the percentage of encapsulation efficiency of mRNA LNP. As shown in Table 20, mRNA LNP prepared by DD-EDEA or DTD-EDEA as ionizable lipid has comparable encapsulation efficiency with mRNA LNP prepared by DTD-NH2. Using DD-EDEA or DTD-EDEA as ionizable lipid has higher encapsulation efficiency than using DnO-EDEA with a shorter lipid tail.

[0365] Table 20. Encapsulation efficiency (EE)

[0366] Sample Initial encapsulation efficiency (%) Final encapsulation efficiency (%) ALC-0315 65.3±0.7 88.2±0.5 DTD-NH2 89.0±0.0 96.5±0.2 DnO-EDEA 60.2±14.3 76.4±0.6 DD-EDEA 95.6±0.1 94.8±0.3 DTD-EDEA 96.3±0.1 92.7±0.4

[0367] The total volume of the aqueous phase (0.75 mL) and the organic phase (0.25 mL) is 1.0 mL

[0368] N / P ratio = 6

[0369] fLuc mRNA (Trilink) added amount = 0.06 mg

[0370] Dilute mRNA with 10 mM sodium acetate (pH 4.0)

[0371] New ionizable lipids

[0372] In Table 20, the "initial" encapsulation efficiency refers to the encapsulation efficiency before the mRNA LNPs were dialyzed to remove residual ethanol, and the "final" encapsulation efficiency refers to the encapsulation efficiency after the mRNA LNPs were dialyzed to remove residual ethanol.

[0373] 8.3. Transfection-cell viability and luminescence intensity of mRNA LNP

[0374] Table 21 shows the results of cell viability and luminescence intensity of mRNA LNPs obtained after 48 hours of incubation. As shown in Table 21, all mRNA LNPs were cell compatible and did not cause cytotoxicity to HeLa cells. Among DnO-EDEA, DD-EDEA, and DTD-EDEA, DTD-EDEA had the highest mRNA transfection efficiency in HeLa cells. Although mRNA LNPs prepared by DTD-EDEA had lower transfection efficiency, it was comparable to mRNA LNPs prepared by ALC-0351.

[0375] Table 21. Transfection in HeLa cells (48 hours)

[0376]

[0377] · Control Refers to cells without LNP transfection

[0378] (i.e., incubated with medium without LNP)

[0379] Example 9: mRNA LNP formulations prepared manually

[0380]

[0381] Chemical structure of ALC-0315 shown in Table 22

[0382]

[0383] Chemical structure of DTD-NH2 shown in Table 23

[0384]

[0385] Chemical structure of DnO-NH shown in Table 24

[0386]

[0387] Chemical structure of DD-NH2 shown in Table 25

[0388]

[0389] Chemical structure of HO-DTD-NH2 shown in Table 26

[0390]

[0391] Chemical structure of HO-DnO-NH2 shown in Table 27

[0392]

[0393] Chemical structure of HO-DD-NH2 shown in Table 28

[0394] 9.1. Characterization and encapsulation efficiency (EE) of mRNA LNP

[0395] The particle size, PDI, and zeta potential of mRNA LNPs were determined at day 0 and after storage for 1 day. Table 29 provides the results of the characterization of mRNA LNPs (i.e., particle size, PDI, and zeta potential). Table 29 also provides the encapsulation efficiency (EE) percentage of mRNA LNPs.

[0396] As shown in Table 29, most of the manually prepared lipids had higher encapsulation efficiency (about 80% to about 90%) except for ALC-0315 and HO-DnO-NH2, comparable to the encapsulation efficiency of LNP prepared by microfluidic device. The particle size of manually prepared LNP was larger than that of LNP prepared by microfluidic device. However, its particle size was still below 200 nm. The mRNA LNPs were stable after storage at 4 °C for 1 day except for DnO-NH2 LNP (increase in particle size and PDI).

[0397] 9.2. Transfection-cell viability and luminescence intensity of mRNA LNP

[0398] Table 30 shows the results of cell viability and luminescence intensity of mRNA LNPs obtained after 48 hours of incubation. As shown in Table 30, mRNA DTD-NH2 LNP prepared manually induced mRNA transfection rate in HeLa cells about 18-fold higher than mRNA ALC-0315 LNP, similar to mRNA LNP prepared using microfluidic device. mRNA HO-DTD-NH2 LNP induced transfection rate similar to DTD-NH2. mRNA LNPs prepared from the remaining ionizable lipids did not cause significant transfection rate compared to the control group.

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407] Table 30. Transfection in HeLa cells (48 hours)

[0408]

[0409] · Control Cells not transfected via LNP

[0410] (i.e. incubated with media without LNP)

[0411] Example 10: Plasmid DNA delivery

[0412]

[0413] Chemical structure of ALC-0315 shown in Table 31

[0414]

[0415] Chemical structure of DTD-NH2 shown in Table 32

[0416] 10.1. Characterization of pDNA-loaded LNP

[0417] Particle size, PDI and zeta potential of pDNA-loaded LNPs were determined at day 0 and after 6 days of storage. Table 33 provides the results of the characterization of pDNA-loaded LNPs (i.e. particle size, PDI and zeta potential). Table 33 also provides the percentage of encapsulation efficiency (EE) of pDNA-loaded LNPs.

[0418] As shown in Table 33, the average particle size of pDNA-loaded LNPs prepared from DTD-NH2 was about 80 nm. pDNA-loaded LNPs prepared from DTD-NH2 were stable after 6 days of storage at 4°C, which can be evidenced from the comparable particle size, particle size distribution (PDI) and zeta potential,

[0419] Particle size and particle size distribution of pDNA-loaded LNPs prepared from ALC-0315 and DTD-NH2, respectively, determined at day 0 are shown in Figure 11 and Figure 12 , respectively.

[0420] Particle size and particle size distribution of pDNA-loaded LNPs prepared from ALC-0315 and DTD-NH2, respectively, determined after 6 days of storage are shown in Figure 13 and Figure 14 , respectively.

[0421] 10.2. Encapsulation efficiency of pDNA LNP

[0422] Table 34 shows the particle size and particle size distribution of pDNA-loaded LNPs prepared from ALC-0315 and DTD-NH2, respectively, at day 0 and after 6 days of storage pDNA LNPThe percentage of encapsulation of pDNA in DTD-NH2 LNP was determined by measuring the absorbance of pDNA at 260 nm. The percentage of encapsulation of pDNA in DTD-NH2 LNP was determined by measuring the absorbance of pDNA at 260 nm. As shown in the table, DTD-NH2 LNP has comparable pDNA encapsulation efficiency compared to ALC-0315. The pDNA encapsulation efficiency of DTD-NH2 LNP did not change after storage at 4 °C for 6 days, indicating that pDNA-loaded DTD-NH2 LNP has stability.

[0423] 10.3. Agarose gel electrophoresis

[0424] Figure 15 The results obtained from agarose gel electrophoresis experiments performed on pDNA LNPs prepared from DTD-NH2 and ALC0315 are shown. As shown in the figure, pDNA was well encapsulated in DTD-NH2 LNP and ALC-0315 LNP, as evidenced by the absence of free pDNA.

[0425] 10.4. Transfection-cell viability and fluorescence intensity of pDNA LNP

[0426] Table 35 shows the results of cell viability and fluorescence intensity of pDNA LNPs after incubation for 48 hours in HeLa cells. Table 36 shows the results of cell viability and fluorescence intensity of pDNA LNPs after incubation for 48 hours in HEPG2 cells.

[0427] Figure 16 are graphs showing the relationship between fluorescence intensity (RFU) and GFP fluorescence of transfection efficiency of pDNA LNPs prepared from DTD-NH2 and ALC0315, respectively, after incubation for 48 hours in HeLa cells and HepG2 cells. As shown in the figures, the pDNA transfection efficiency mediated by DTD-NH2 LNP was significantly stronger than ALC-0315 LNP in both HeLa cell line and HepG2 cell line. Figure 16

[0428] Figure 17 , Figure 18 and Figure 19 show confocal microscopic images captured after incubation for 48 hours in HeLa cells. As shown in the figures, the pDNA transfection efficiency mediated by DTD-NH2 LNP was significantly stronger than ALC-0315 LNP in HeLa cells.

[0429] Figure 20 Figure 21 and Figure 22 show confocal microscopic images captured after incubation for 48 hours in HepG2 cells. As shown in the figures, the pDNA transfection efficiency mediated by DTD-NH2 LNP was significantly stronger than ALC-0315 LNP in HepG2 cells.

[0430] ​​

[0431]

[0432] Table 33. LNP characterization (pDNA)

[0433]

[0434] Table 34. Encapsulation efficiency (pDNA)

[0435] Sample Date Initial encapsulation efficiency (%) Final encapsulation efficiency (%) ALC-0315 5 / 10 / 2022 84.3±6.1 84.1±3.6 ALC-0315 11 / 10 / 2022 81.4±1.2 89.1±0.0 DTD-NH2 5 / 10 / 2022 78.8±3.3 93.1±0.0 DTD-NH2 11 / 10 / 2022 86.4±1.1 90.3±1.9

[0436] Table 35. Transfection in HeLa cells (48 hours)

[0437]

[0438] Table 36. Transfection in HEPG2 cells (48 hours)

[0439]

[0440] · Control Refers to cells not transfected with LNP (i.e. incubated with media that does not contain LNP)

[0441] It will be understood by those within the art that other variations and / or modifications of the embodiments disclosed herein can be made without departing from the spirit or scope of the disclosure as broadly described. For example, features described herein with respect to different example embodiments can be mixed, combined, interchanged, merged, adopted, modified, included, etc. across different example embodiments. Therefore, the present embodiments are to be considered in all respects as illustrative and not restrictive.

Claims

1. A compound as represented by general formula (1) or an ionized form thereof for use in preparing lipid nanoparticles for encapsulating therapeutic agents, prophylactic agents and / or biological agents: in NR 1 R 2 It is a group that is ionizable at pH 3 to physiological pH; A includes linear aliphatic hydrocarbons, branched aliphatic hydrocarbons and / or cyclic hydrocarbons, which optionally contain a group selected from -OH, -NR-, -O-, -OC x H 2x one or more than one group in -O-, wherein x ≥ 1; and wherein R is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; R 3 、R 4 、R 5 、R 6 and R 7 are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and R 8 and R 9 Each is independently a hydrophobic group.

2. The compound according to claim 1, wherein R 1 and R 2 Each is independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and combinations thereof.

3. A compound according to any one of the preceding claims, wherein R 1 and R 2 Both are H, and -NR 1 R 2 It is a primary amine group.

4. A compound according to any one of the preceding claims, wherein 8 and R 9 The hydrophobic groups of each independently comprise an optionally substituted alkyl group.

5. The compound according to any one of the preceding claims, wherein A is selected from the following general formula (2), general formula (3), general formula (4) and / or general formula (5): in X 1 To X 31 are each independently selected from -H, -OH, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; n≥1; m≥1; p≥1; and q≥1.

6. The compound of any one of the preceding claims, wherein the compound is selected from the group consisting of DnO-EDEA, DD-EDEA, DTD-EDEA, HO-DnO-NH2, HO-DD-NH2, HO-DTD-NH2, DnO-NH2, DD-NH2, DTD-NH2, DMAPAPA-DTD, and combinations thereof.

7. A compound according to any one of the preceding claims, wherein the compound is in an ionized form of the general formula (1), wherein -NR 1 R 2 Ionized into positively charged groups.

8. A method for preparing a compound according to any one of the preceding claims, comprising: (ai) reacting an amine compound represented by the general formula (6) with a cyclic acid anhydride represented by the general formula (7) to obtain a first intermediate compound comprising a carboxylate group represented by the general formula (8): (a-ii) reacting the first intermediate compound represented by the general formula (8) with N-hydroxysuccinimide (NHS) in the presence of a coupling agent to obtain a second intermediate compound comprising an amide group represented by the general formula (9): (a-iii) reacting the second intermediate compound represented by the general formula (9) with an amine compound represented by the general formula (10) to obtain a compound represented by the general formula (1): in A includes linear aliphatic hydrocarbons, branched aliphatic hydrocarbons and / or cyclic hydrocarbons, which optionally contain a group selected from -OH, -NR-, -O-, -OC x H 2x one or more than one group in -O-, wherein x ≥ 1; and wherein R is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; R 1 、R 2 、R 3 、R 4 、R 5 、R 6 and R 7 are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and R 8 and R 9 are each independently a hydrophobic group; and (a-iv) optionally adding -NR 1 R 2 Ionize to become positively charged groups.

9. The method of claim 8, wherein the coupling agent comprises a carbodiimide selected from the group consisting of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), and combinations thereof.

10. A nanoparticle composition for delivering a therapeutic, prophylactic, and / or biologic agent, the nanoparticle composition comprising: A compound according to any one of claims 1 to 7; and A therapeutic, prophylactic and / or biological agent encapsulated in a compound according to any one of the preceding claims 1 to 7.

11. The nanoparticle composition of claim 10, wherein the composition further comprises: (a) Helper lipids; (b) sterols; and (c) Lipids modified with polyethylene glycol (PEG).

12. The nanoparticle composition according to claim 11, wherein the compound represented by general formula (1), the helper lipid, the sterol and the PEG-modified lipid are mixed in a weight ratio of 10 to 50:2 to 20:4 to 30:1 to 15.

13. The nanoparticle composition of any one of claims 11 to 12, wherein the helper lipid is present in an amount of 1 to 20 mol %, the sterol is present in an amount of 10 to 50 mol %, and the PEG-modified lipid is present in an amount of 0.5 to 10 mol %.

14. The nanoparticle composition according to any one of claims 11 to 13, wherein the helper lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2 -Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diondecanoyl-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-cholestyhemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diamidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 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.

15. The nanoparticle composition of any one of claims 11 to 14, wherein the sterol is selected from the group consisting of cholesterol, 5α-ergosta-8,24(28)-dien-3β-ol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, avenasterol, and combinations thereof.

16. The nanoparticle composition according to any one of claims 11 to 15, wherein the PEG-modified lipid is selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, etc. or a combination thereof, and examples of PEG-modified lipids / PEGylated lipids include but are not limited to 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), R -3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxypropyl-3-amine (PEG-c-DOMG), 3-N-[(ω-methoxypoly(ethylene glycol)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 lipids, and combinations thereof.

17. The nanoparticle composition of any one of claims 10 to 16, wherein the nanoparticle composition comprises nanoparticles having an N / P ratio of 2:1 to 40:

1.

18. The nanoparticle composition of any one of claims 10 to 17, wherein the nanoparticle composition comprises nanoparticles having an average particle size of 20 nm to 200 nm.

19. The nanoparticle composition of any one of claims 10 to 18, wherein the nanoparticle composition comprises nanoparticles having a zeta potential of -15 mV to +20 mV in phosphate buffered saline (PBS).

20. The nanoparticle composition according to any one of claims 10 to 19, for use in medical treatment.

21. The nanoparticle composition of any one of claims 10 to 19, for use in treating or preventing a disease, disorder, or symptom in a subject in need thereof.

22. Use of the nanoparticle composition of any one of claims 10 to 19 in the preparation of a medicament for treating or preventing a disease, disorder or symptom in a subject in need thereof.

23. A method of treating or preventing a disease, disorder, or symptom in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the nanoparticle composition of any one of claims 10 to 19.

24. The nanoparticle composition of claim 21, the use of claim 22, or the method of claim 23, wherein the nanoparticle composition is administered to a subject to induce an immune response in the subject.

25. The nanoparticle composition of claim 21, the use of claim 22, or the method of claim 23, wherein the disease, disorder, or symptom is mediated by a coronavirus.

26. The nanoparticle composition, use or method of claim 25, wherein the coronavirus is the SARS-CoV-2 coronavirus.

Images